AU762914B2 - N-protected amines and their use as prodrugs - Google Patents
N-protected amines and their use as prodrugs Download PDFInfo
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- AU762914B2 AU762914B2 AU44211/00A AU4421100A AU762914B2 AU 762914 B2 AU762914 B2 AU 762914B2 AU 44211/00 A AU44211/00 A AU 44211/00A AU 4421100 A AU4421100 A AU 4421100A AU 762914 B2 AU762914 B2 AU 762914B2
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- C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
- C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
- C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D333/42—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms with nitro or nitroso radicals directly attached to ring carbon atoms
- C07D333/44—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms with nitro or nitroso radicals directly attached to ring carbon atoms attached in position 5
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- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
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- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/26—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring
- C07C271/28—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring to a carbon atom of a non-condensed six-membered aromatic ring
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- C07D209/56—Ring systems containing three or more rings
- C07D209/58—[b]- or [c]-condensed
- C07D209/60—Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
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- C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
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- C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
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- C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
- C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D231/16—Halogen atoms or nitro radicals
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- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D233/91—Nitro radicals
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- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D233/91—Nitro radicals
- C07D233/92—Nitro radicals attached in position 4 or 5
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- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
- C07D277/66—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2 with aromatic rings or ring systems directly attached in position 2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/70—Nitro radicals
- C07D307/71—Nitro radicals attached in position 5
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/22—Ortho- or ortho- and peri-condensed systems containing three rings containing only six-membered rings
- C07C2603/24—Anthracenes; Hydrogenated anthracenes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Description
PCT/GBO 0 16 1 -1- N-PROTECTED AMINES AND THEIR USE AS PRODRUGS The present invention relates to methods and compounds for providing amines with N-protecting groups. It further relates to the protected amines themselves and their use as prodrugs.
The amines are protected as nitroaromatic carbamates (where "aromatic" includes "heteroaromatic"). They include nitro groups which are susceptible to reduction, leading to loss of the protecting group and liberation of the amine. The amine is desirably biologically active, the activity being substantially suppressed by N-protection. Thus it may be an amine-based cytotoxin, e.g actinomycin D, an anthracycline, an aniline mustard or an enediyne.
Thus suitable protected amines may be valuable as anticancer agents, and/or may be useful as prodrugs for antibody-directed enzyme prodrug therapy (ADEPT) or genedirected enzyme prodrug therapy (GDEPT), in conjunction with nitroreductase enzymes.
BACKGROUND TO THE INVENTION The use of prodrugs (relatively inactive compounds which can be converted into more active compounds in ivo) represents a clinically very valuable concept, particularly in cancer therapy. For example a prodrug may be converted into an anti-tumour agent under the influence of an enzyme that is linkable to a monoclonal antibody that will bind to a tumour associated antigen. The combination of such a prodrug with such an enzyme monoclonal/antibody conjugate represents a very powerful clinical agent. This approach to cancer therapy, often referred to as "antibody directed enzyme/prodrug therapy" (ADEPT), is disclosed in W088/07378.
A further therapeutic approach termed "virus-directed enzyme prodrug therapy" (VDEPT) has been proposed as a method for treating tumour cells in patients using prodrugs.
Tumour cells are targeted with a viral vector carrying a gene encoding an enzyme capable of activating a prodrug. The gene may be transcriptionally regulated by tissue specific promoter or enhancer sequences. The viral vector enters tumour cells and expresses the enzyme, in order that a prodrug is converted to an active drug within the tumour cells (Huber et al., Proc.
Natl. Acad. Sci. USA (1991) 88, 8039). Alternatively, non-viral methods for the delivery of genes have been used. Such methods include calcium phosphate co-precipitation, microinjection, liposomes, direct DNA uptake, and receptor-mediated DNA transfer. These ruinU U u 6 1 -2are reviewed in Morgan French, Annu. Rev. Biochem., 1993, 62;191. The term "GDEPT" (gene-directed enzyme prodrug therapy) is used to include both viral and non-viral delivery systems.
4-Nitrobenzyl carbamates undergo multi-electron reduction to produce amines.
The mechanism probably involves the formation of electron-donating 4-hydroxylamine (B;Q=OH) or 4-amine species, which then fragment to generate a quinoneimine methide and an amine Carl, P.K. Charkravarty, and J.A. Katzenellenbogen, J.
Med. Chem., 1981, 24,479].
NO
2
NHQ
I C02 RNH2 0 0
CH
2 C NHR ONHR
C
A B Despite a low reduction potential (ca. -490 mV) Wardman, Environ. Health Perspect., 1985, 64, 309] the 4-nitrobenzyl carbamate moiety undergoes facile reduction by the E. coliNR enzyme, and has been used as a prodrug "trigger" to deactivate highly cytotoxic amine "effectors" Hay and W.A. Denny, Drugs Future,1996, 21, 917]. The E.
coli enzyme has been shown to activate 4-nitrobenzyl carbamate derivatives of a limited number of amine-based cytotoxins, including actinomycin D and anthracyclines [A.B.
Mauger, P.J. Burke, H.H. Somani, F. Friedlos and RJ. Knox, J. Med. Chem., 1994,37, 3452], aniline mustards Mauger, P.J. Burke, H.H. Somani, F. Friedlos and R.J. Knox, J. Med. Chem., 1994, 37, 3452; M. Lee, J.E. Simpson Jnr, S. Woo, C. Kaenzig, G.M.
Anlezark, E. Eno-Amooquaye, and P.J. Burke, Bioorg. Med. Chem. Lett., 1997, 7, 1065] and enediynes Hay, W.R. Wilson, and W.A. Denny, Bioorg. Med. Chem. Lett., 1995,5, 2829]. All of these studies have used the otherwise unsubstituted 4-nitrobenzyl carbamate moiety.
To be fully effective, such prodrugs must be activated efficiently by the enzyme, and the resulting reduced species must fragment rapidly to release the cytotoxic amine effector.
Kinetic structure-activity relationships (SAR) have been extensively studied for the oneelectron reduction of nitrobenzyl halides Kirkpatrick, K.E. Johnson, and A.C.
Sartorelli, J. Med. Chem., 1986, 29, 2048] and quaternary salts Tercel, W.R. Wilson, WO 00/64864 PCT/GBOO/01612 -3- R.F. Anderson, and W.A. Denny, J. Med. Chem., 1996, 39, 1084 and refs therein], but not for 4-nitrobenzyl carbamates. We have found that suitable substituents on the 4-nitrobenzyl ring and/or alpha-carbon result in more rapid fragmentation of the 4-hydroxylamine intermediates, and can also serve as sites for attaching solubilising functionalities.
For a series of substituted 4-nitrobenzyl carbamate model compounds fragmentation rates of the corresponding 4-hydroxylamines to release amines (Z) correlated with electron-donating properties of the substituent, as shown in Table 1. The maximum half-lives (Mta) of the hydroxylamine derivatives were measured by HPLC, following 4-fold stoichiometry radiolytic reduction of the corresponding substituted 4nitrobenzyl carbamates. Assuming first order conditions, the half-life (tn) of species R is calculated from the equation t(ln2/t). The ratio was taken as the fraction of nitrobenzyl carbamate which had not released the amine after 4-fold reduction. This method yields a maximum value for the half-life of fragmentation.
NHOH
C
-N
X(E NO 2 Y (E NHOH) Table 1 Half-lives for fragmentation (Mt, and percent of amine released for substituted 4-hydroxylaminobenzyl carbamates (derived from the corresponding 4nitrobenzyl carbamates by radiolytic reduction).
WO 00/64864 PCT/GBOO/01612 -4-
I
Mt. (min~ td7'~/o) n A Mt., (min) t. I 2-NO, H 0.78 88 18 3-NO, H 0.71 65 22 3-COMe H 0.37 20 44 3-OMe H 0.12 17 37 H H 0.0 16 2-OMe H -0.27 12 48 2-NHMe H -0.84 7 H Me 0.0 9.5 Table 1 shows that the unsubstituted hydroxylaminobenzyl carbamate normally used as a trigger has a half-life of 16 minutes. This is relatively long and, under biological conditions, may result in substantial loss of material by side reactions not involving (activating) amine release. The half-life can be lowered significantly by the use of electron-donating substituents, and/or by the use of a-substituents DISCLOSURE OF THE INVENTION In a first aspect, the invention provides a method of providing an amine with a protecting group comprising providing a plurality of different compounds selected from compounds of formulae and (II) 002 z NO2Z° o o Ez L n
O
-E
Ii wherein: X represents H, C,4 alkyl or C1_ alkoxy, said alkyl or alkoxy being optionally substituted with one or more of the following groups: hydroxy ether amino mono-substituted amino (NRH), di-substituted amino (NR'R, 2 cyclic Ci.s alkylamino, imidazolyl, C,1 alkylpiperazinyl, morpholino, thiol thioether (SR), tetrazole, carboxy (COOH), carboxylate (COOR), sulphoxy 2 0H), sulphonate 3NSOOCID: <WO 0064864A1 I> WO 00/64864 PCT/GB00/01612 2 OR), sulphonyl 2 Rx, sulphixy sulphinate sulphinyl phosphonooxy (OP(=O)(OH) 2 and phosphate (OP(=O)(OR) 2 where Rx, Rx' and
R
2 are selected from a C,6 alkyl group, a C 3 2 0 heterocyclyl group or a C5-2 0 aryl group, preferably a C, 4 alkyl group; a is 0,1,2,3 or 4; Y represents H or C, 4 alkyl; 1, 2 or 3 of the members Z of the 5-membered aromatic ring are independently selected from or NR-, (where R is H or C, 4 alkyl optionally substituted with one or more of the following groups: hydroxy ether amino mono-substituted amino (NRpH), disubstituted amino (NRR'Ra 2
C,.
5 cyclic amino, imidazolyl, alkylpiperazinyl, morpholino, thiol thioether tetrazole, carboxy (COOH), carboxylate (COOR,), sulphoxy sulphonate 2 ORR), sulphonyl 2 sulphixy sulphinate sulphinyl phosphonooxy (OP(=O)(OH) 2 and phosphate where RR, RR' and RR 2 are selected from a C, 4 alkyl group, a C 320 heterocyclyl group or a C 5 2 o aryl group, preferably a C, 4 alkyl group), the other ring atoms being C; n is 0 or 1; and E represents a moiety such that EH is an amine; (ii) measuring the rates of fragmentation of the compounds to release EH when the nitro group is reduced and selecting a compound having a desired rate of decomposition; and (iii) providing the amine to be protected with a protecting group corresponding to that in the selected compound.
In this aspect, the step of selecting the compound is preferably carried out in order to provide a protecting group with a faster rate of fragmentation than unsubstituted 4-nitrobenzyl carbamate. However selecting a compound bearing a protecting group with a slower rate of fragmentation than 4-nitrobenzyl carbamate may be preferred. This particularly applies in situations in which it is desired to provide a prodrug which can diffuse away from the site of actuation by the appropriate enzyme, and thus kill tumour cells further away from the site of actuation (the "bystander" effect).
In a second aspect, the present invention relates to a compound represented by the general formula or (II) as shown above, wherein X, Y, Z, E, a and n are as defined above; provided that in formula if a 0 then Y H.
EH is preferably a cytotoxic amine. E may be selected from formulae (III-XIII).
In (m-XIII), R, represents H or C 14 alkyl, being optionally substituted with one or more of the following groups: one or more of.the following groups: hydroxy ether (ORE), amino (NH2), mono-substituted amino (NREH), di-substituted amino (NRE'RE 2 cyclic WO 00/64864 PCT/GBOO/01612 -6alkylamino, imidazolyl, alkylpiperazinyl, morpholino, thiol thioether (SRE), tetmzoe, carboxy (COOH), carboxylate (COORE), suiphoxy 2 0H), suiphonate 2 0RE), suiphonyl (S(7=O) 2 RE), suiphixy suiphinate (SQ=O)ORE), suiphinyl phosphonooxy (OP(=O)(OH) 2 and phosphate where RE, RE' and RE2 are selected from a ailkyl group, a C 320 heterocyclyl group or a C,- 2 aryl group, More preferably from a C, 4 6 alkyl group R 2 represents H, C,-6 alkyl, C,- 6 alkoxy, OH, halogen,
NO
2
NH
2 NHMe, NNe 2
SO
2 Me, CF 3 CN, CONI{ 2 or CONHMe; each R 3 is independently selected from Cl, Br, I and OMS; and R 4 is selected from -C&=O)Me and -C(=O)CH 2 OH; Q represents substituted indole, substituted benzofuran or substituted cinnamoyl; in (IX) and each n is independently from 2-4, and each m is independently from 2-4, and p 0 or 1.
3NSDOCID: <WO 0064864A1 I>
I)
(mV (V) (VI~a) (VIIb) 0 R2 0
NP
0 N (I (X) where L= H 2
H
2 P H 2
(X)
NH
2
N-
Me 0
NH
I (il)
(XII)
WO 00/64864 PCT/GB00/01612 -8- Compounds of formula V are described in EP 0 938 474, which is incorporated herein by reference. Compounds of formula VI are described in EP 0 850 220, which is incorporated herein by reference.
A compound of formula or may be basic or acidic and may thus form pharmaceutically acceptable salts with both organic and inorganic acids and bases. These are included within the scope of the second aspect.
In a first type of preferred embodiment, X represents C.
6 alkyl or C,6 alkoxy, said alkyl or alkoxy being optionally substituted with one or more of the following groups: hydroxy, ether amino, alkylamino (NRH), dialkylamino (NR.'RX 2 cyclic Ci.
alkylamino, imidazolyl, C,6 alkylpiperazinyl, morpholino, thiol, alkylthioether (SRJ, tetrazole and -COzX' where X' is selected from the possibilities listed for X and R, 1 and ar 2 e selected from C, 4 alkyl; a is 0,1,2,3 or 4; Y represents H or lower alkyl; 1, 2 or 3 of the members Z of the 5-membered aromatic ring are independently selected from or NR-, (where R is H or lower alkyl optionally substituted with one or more of the following groups: hydroxy, ether amino, alkylamino (NRRH), dialkylamino (NRRI'R'), cyclic alkylamino, imidazolyl, C 1 alkylpiperazinyl, morpholino, thiol, alkylthioether (SRI), tetrazole and -COzR' where R' is selected from the possibilities listed for R and RR, and RZ are selected from C, alkyl); and E represents Ri represents H or Ci. alkyl, being optionally substituted with one or more of the following groups hydroxy, ether (ORE), amino, alkylamino (NRH), dialkylamino (NRERE 2 cyclic alkylamino, imidazolyl, C, 1 alkylpiperazinyl, morpholino, thiol, alkylthioether (SRE), tetrazole and -CO 2 X' where X' is selected from the possibilities listed for X and R, E' and RE 2 are selected from C.6 alkyl.
In a second type of preferred embodiment, the compound is of formula where a is 1. It is further preferred in this embodiment that X represents optionally substituted C,6 alkoxy and more preferably in the 2 position. The alkoxy group is preferably selected from methyl, ethyl and n-propyl, and the substituents from hydroxy, methoxy, phosphonoxy, NMe2, Nmorph, OCO,-tBu, and OCOzH. In more preferred embodiments the ethyl or n-propyl group is singly substituted, most preferably with hydroxy, whereas the Me group is unsubstituted.
In this type of embodiment Y is preferably selected from H or Me. E is preferably selected from HI XIII, most preferably V or XIII. IfE is selected from XIII, n is preferably 1.
In a third preferred type of embodiment, the compound is of formula with one Z BNSOOCID: <WO 0064864A1 I WO 00/64864 PCT/GB00/01612 -9being and another Z being R being preferably Me or Et, the other members of the heterocyclic ring being C. If R is Et, it is preferably substituted with hydroxy. A is preferably 0. More preferably the and -NR- are not adjacent in the heterocyclic ring. The most preferred arrangement is 2 and 5 with the NO 2 at the 3 position. In this type of embodiment, Y is preferably selected from H or Me. E is preferably selected from V or XIII. IfE is selected from XIII, than n is preferably 1.
In a fourth preferred type of embodiment, the compound is of formula a is 0, and Z is either O or S. It is further preferred that the O or S is in the 2 position in the ring, and the NO, is attached to the 3 position. In this type of embodiment, Y is preferably selected from H or Me.
In a fifth preferred embodiment, the compound is of formula and Z is NR, preferably NMe. There may be one further substituent or there may be no further substituents on the ring The further substituent is preferably CO 2 Et. In this type of embodiment, Y is preferably selected from H or Me.
In a third aspect the invention provides a compound according to the second aspect for pharmaceutical use.
In a fourth aspect the invention provides the use of a compound according to the second aspect for the manufacture of a composition for use in the treatment of a hyperproliferative disease, particularly a neoplastic disease. The composition may also include activating means for simultaneous or separate administration, the activating means typically comprising an enzyme or means for providing an enzyme, for performing ADEPT or VDEPT therapy. The activating means typically leads to liberation of the amine EH.
In a fifth aspect the invention provides a compound of the formula XVI or XVII where X, n, Z and Y are as defined for the second aspect and T is OH or an activated alcohol functionality (such as -O.CO.L where L is a leaving group such as Cl) suitable for reaction with an amine EH to produce a compound according to the second aspect.
In a sixth aspect the invention provides the use of a compound of formula (XVI) or N02 N02 Xa
Z
0N Xa Z Y o NH O NH XVI T 0
T
xvi XVII WO 00/64864 PCT/GB00/01612 (XVII) in protecting an amine. This may include activation of an alcohol (XVI or XVII where T is OH) with a reagent such as phosgene, diphosgene or triphosgene or a chloroformate, e.g. 4-nitrophenylchloroformate or pentafluorophenylchloroformate, optionally in conjunction with HOBT(l-hydroxybenzotriazole).
In a further aspect, the present invention relates to a method of preparing compounds of the general formula examples of the methods are outlined in Schemes 1-24.
Thus (Scheme reaction of the amine 7 Shi, T. D. Bradshaw, S. Wrigley, C. J.
McCall, P. Lelieveld, I. Fitchner, M. F. G. Stevens. J. Med. Chem., 1996, 39, 3375] with 4nitrobenzylchloroformate gave carbamnate 8.
Scheme I N02 7 0 S Reagents: 4-nitrobenzlchbmformate, pyridine. 8 Reaction of the 1,4-difluoro-5,8-dihydroxyanthracene-9, I0-dione 22 with amine 15 in pyridine gave the monocarbamate 23 and biscarbamate 24 (Scheme Further reaction of 23 with 2-(2aminoethylamino)ethanol gave carbamate 26.
In another example (Scheme 4-nitrosalicylic acid (27) was methylated using a solution of diazomethane in ether and the methyl ester 28 reduced with DBALH in THF to give the nitrobenzyl alcohol 29. Activation of the alcohol 29 with triphosgene (or alternatively phosgene or diphosgene) in the presence of pyridine, and reaction with N',N'-bis(2hydroxyethyl)-1,4-benzenediamine (57) gives the carbamate 31, which was elaborated to the mustard 32 using standard methods.
BNSDOOGIt <WO 0064864AI I PCTGBOOIOI 612 WO 00/64864 -1I1I- Scheme 2
-OR
2
R,
27R, =CO 2 ,P.2 H) 28 R, C 2 Me R 2 Me-(i 29 R, CH 2 OH. R 2 Me Reagents: CH 2
N
2 ,Et2OI. DIBALH, THF; (ui) trijhosgefle. pyndie, THF, 57 (Nv) MsCI, prIidine; LCI, OMF.
N02(v
N
OV IN owe ow 31 32 Similarly (Scheme activation of alcohol 29 with triphosgene in the presence of pyridine and reaction with amaine 33 Tercel and W. A. Denny. J. Chemn. Soc. Perkin Trans.
1, 1998,509] or amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] gave carbamnates 34 and 35, respectively.
Scheme 3 a -r -Oh I R 35 R CO2C 2 2 'OM8)h.N 33R=H(I 34 R COCH 2 (2.oM8)Ph-4-NO 2 Reagents: (Q tnphosgene. 29, pyiid he Similarly (Scheme activation of alcohol 29 with triphosgene in the presence of* pyridine and reaction with amine 7 Shi, T. D. Bradshaw, S. Wrigley, C. J. McCall, P.
Lelieveld, I. Fitchner, M. F. G. Stevens. J. Med Chem., 1996, 39, 3375] ]gave carbamnate 36.
WO 00/64864 PCT/GB00/01612 -12- Scheme 4
RHN-/-
7R=H 36 R C02 CH2 (2-OMB)Ph-4 NO2 3 Reagents: triphosgene, 29, pyridhe.
In another example (Scheme activation of alcohol 29 with 4nitrophenylchloroformate gave the carbonate 37 which was reacted with doxorubicin (13) and triethylanmine in DMF to give the doxorubicin carbamate 38.
0 OH 0 Scheme 5 i( 2 OH
'OH
29 OMe OM O 37 z Reagents: 4nitrophenyichlroformate, pyrkine; 38 13, Et 3 N. DMF.
O
2 N OMe In another example (Scheme carbonate 37 was coupled to amine 9 using 1hydroxybenzotriazole (HOBT), 4A molecular sieves and triethylamine to give protected carbamate 39. Removal of the TBDMS protecting group with aqueous acid gave the alcohol which was activated with 4-nitrophenylchloroformate to give the carbonate 41. Reaction of 41 with doxorubicin (13) and triethylamine in DMF gave the carbamate 42.
INSDOCID:<WO 0064864A1 I> PCT/GBOO/01612 WO 00/64864 13 Scheme 6 N2 37 aTBDMS N0 2 Dox Is
U
HO 4ROH 41 42 Reagents: HOBT. moL sieves, Et 3 N. THF; (ii)HlIt.q. MeOH: cii) NO 2 PhOCOCI, Et 3 N. TIF; (iv) DOX. Et 3 N. DMIF.
in another example (Scheme reaction of alcohol 29 with triphosgene and triethylamine, and coupling to 2,22trfluoro-N-[2-(mehy1aino)ehy]aceamde trifluoroacetate gave the trifluoroacetainide 43 which was deprotected under basic conditions to give amine 44. Activation of the 5-ehl9-x-, 0dh o-acinecar)xylic acid (16) with thionyl chloride and coupling of the intermediate 9-chloroacridinyl acid chloride with amine 44 gave amidde 45 which was converted to carbamnate 46 using ammonia in phenol.
Schemie7 0NO 2
NO
2
R
q OMe OMe. M Me 16 0 ~~NH0 M e Me 43R~cocF,Ci 45 R=ci ~44R=H o 4RNH Reagents: tuiphosgene, CF 3 CONHCI-2CKNHWe OIEA; (Ii)cs 2 Co 3 (ii) S0012, DIV; PhOH, NH 3 WO 00/64864 WO 0064864PCT/GBOO/01612 -14in another example (Scheme the bistrifluoroacetamide 47 was coupled to alcohol 29 to give bisamide 48 which was deprotected under basic conditions to give the amine 49.
The amine 49 was coupled to 4-(IH-imidazol-1-ylcarbonyl)-5-methylacridine (50) A.
Gamage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, J Med. Chem., 1999, 42, 2383-2393] to give the carbamate 51.
Scheme 8 NK NH 2 0i) 0 48 R= COCF 3 49R p(ii) Reagents: (0 EtOCOCF3, H 2 0, MeCN; triphosgene, 29, DIEA, DCM (Bi) Cs 2
CO
3
H
2 0, MeOHK (mv) 50. THE.
Reaction of the 1,4-difluoro-5,8-dihydroxyantracele-9,10-diofle 22 with amnine 44 gave the monocarbamate 52 and biscarbamnate 53 (Scheme Further reaction of 52 with 2-(2aminoethylarnino)ethanol gave carbamnate 55. 3NSOCID <WO 0064864AI I PCT/GBOO/01612 WO 00/64864 15 Schemne 9
(I)
52 v Reagets:Q) 44. pyridine; NH 2 (Cti 2 2 NH (CH2)20H K pridne.
In another example (Scheme 10), reaction of 4-nitrophenylethafl-lI-ol (56) with triphosgene and pyridine, with the subsequent addition of N',N'-bis(2-hydroxyethyI)-l
A-
benzenediamne, (57) gave the carbamate 58 which was elaborated to the dichloride 59.
WO 00/64864 WO 0064864PCT/GBOO/01 612 -16- Scheme
NO
2 me 'OH 56 HO N 1H 57 00i 58 Reagents: tdphosgeneTHF; (0i) MsCI.
In another example (Scheme 11), coupling of the alcohol 56 with amnine 1 J.
Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem Lett., 1997, 7, 1483] using triphosgene and pyridine in THF gave the carbaniate Schem 11 cl al OMe /0 N Me (0 OMe so e N H HNeO
NH
2 e 6 Reageits- (0 tdphwguee pyrldrw. THF. N102 Oxidation of alcohol 29 with pyridinium ch-lorochromate (PCC) in DCM gave the aldehyde 61 (Scheme 12). Reaction of aldehyde 61 with methyl magnesium bromide in THF gave the alcohol 62 which was coupled to amine 57 to give carbamate dial 63. The diol 63 was elaborated to the di chloride 64 using standard methods.
BNSDOCID: <WO 0064864AI PCT/GBOOIOI 612 WO 00/64864 17 Schemne 12
NOP
R
61R =CHO 62 R =C(O N! 02 (I)H O M
CNH
IgBr. THF. (11) Of4 IvM Reagentds: 0I POC. DCM; (iD MeN triphosgefle, yrine. 57. THF; (I C!- 64 In another example (Scheme 13), alkylation of methyl 2-hydroxy-4-nitrobelzoate 66 with bromide 65 under basic conditions gave the ester 67 which was reduced to alcohol 68 using DIBALH in THF. Activation of amine 1 JI Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 68 using catalytic dibutyltin diacetate to give carbamnate 69. Deprotection of 69 under acidic conditions gave Scheine 13 Co 2 Nb 00 2 W O 66 67 68 sr
DMS
Reagents: 65, K 2 00 3 DMF; (fl) IBAI.HTHF;(00)1. triphosgene% Et 3
N,
DCM. then 68, nBu 2 Sn(OAr-) 2 HCI. MeOH.
69 TBCMS; (IV) 70R=H WO 00/64864 WO 0064864PCTGBOO/01612 18 Similarly (Scheme 14), reaction of phenol 66 with 2-bromoethyl methyl ether gave ester 71 which was reduced to alcohol 72. Activation of amine 1 I Atwell,.W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 14831 with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 72 using catalytic dibutyltin diacetate to give carbamate 73.
Scheme 14 N2aOMe OMe 66 -10-OMe- OMe 0OMe cO 2 ~OH 11fO 71 72 Reagens: BCH 2
CH
2 O0 K 2
C
3 DMF; (iH) DIBALH. TIF; 1. I m triphosgene. 8 3 N, DCM. then 72. nBU 2 Sn(OAc) 2
NO
2 73 In another example (Scheme 15), phenol 66 was alkylated with 3-iodopropyl tetahydropyranyl ether under basic conditions to give ester 74 which was reduced to alcohol 75 using DIBALH in THE. Activation of the alcohol 75 with triphosgene and triethylamine (or pyridine, or another organic base) in THF and subsequent reaction with amine 57 gave the carbamate diol 76. The diol 76 was converted to the dichloride 77 using standard methods and the tetrahydropyranyl ether deprotected under acidic conditions to give carbamate 78.
Scheme 15 C2N) 66 0-I -b- O-,N.-OT-P O'"'OT-P ''OR
CO
2 F& OH 0 Reagents: @i BrCH 2
CH
2
CH
2 OTHP, K 2 C0 3 DMF; (iO DIBALH, THF; (Hi) triphosgene, Et3K~ THF, then 57; (wv) Msa, pyridne; f N LiCL DMF; (vi) HCL MeOH. TR 76 R I=-OH, R 2 THP. ('4I (V) 77R a, R 2 =THP (i 78R, a.R2 H BNSOOCIO: <V40 0064864Al I WO 00/64864 PCT/GB00101612 -19- In another example (Scheme 16), alcohol 75 was activated using triphosgene and triethylamine (or pyridine, or another organic base) and coupled to amine 33 Tercel and W. A. Denny. J. Chem. Soc. Perkin Trans. 1, 1998, 509] to give carbamnate 79 which was deprotected under acidic conditions to give Scheme 16 OMe 0- OTHP 0 OMe OH HN 0 Reagents: @I 33. tnphosgene, pyridine, THF; 79R=THP HCL MeOH.
NO
2 60 R=H In another example (Scheme 17), alcylation of phenol 66 with 3-bromopropyl tertbutyl(dimethyl)silyl ether under basic conditions gave the ester 81 which was reduced to alcohol 82 using DIBALH in THE. Activation of amine 1 J. Atwell, W. R. Wilson, W. A.
Denny, Bioorg. Med Chein. Lett., 1997, 7, 1483] with triphosgene and triethylaniine gave an intermediate isocyanate which was coupled with alcohol 82 using catalytic dibutyltin diacetate to give carbamate 83. Deprotection of 83 under acidic conditions gave carbamate 84.
Scheme 17N0 a Ohm 66 0~ OOwOBDM
M
00WOH -'0 61 82 83 R =TBDMS (iv 0-- 84R=H Reagerts: BrCH 2
CH
2
CH
2 0TBDMS, K 2 C0 3 DMF; (1)1 OIBALJ-, THF; 1, thiphosgetle, Et 3 N. DCM, then 82, nBuj2Sn(OA) 2 (t4) HCI.
MeOH.
WO 00/64864 WO 0064864PCT/GBOO/01612 In another example (Scheme 18), activation of alcohol 75 with 4nitrophenyichioroformate gave the carbonate 37 which was deprotected under acidic conditions to give carbonate 86. Reaction of 86 with doxorubicin (13) and triethylamine in DMF gave the doxorubicin carbamnate 87.
Scheme 18 NO 2 0 OH 0 OP
OH
0'-"OR
*OH
0 ~OMeO OHO0 of-;I--O -N2MeT 0 Ho~rH 86R=H;00 Reagerts: NOPhOCOGI. DIEA, THF HCI 87.
THF;(NI)13, Et 3 N.DWF. 02N4 0'---OH In another example (Scheme 19) alcohol 75 was activated with triphosgene and coupled to amine 47 to give trifluoroacetamnide 88 which was deprotected under basic conditions to give bis-anine 89. Coupling of the bis-amine 89 with the imidazolide 50 A.
Gamage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, J Med Chem., 1999, 42, 2383-2393] gave the carbamnate 90 which was deprotected under acidic conditions to give carbamnate 91.
Scheme 19 NO 2
N
q5 10 1 OTHP_ 00. 0 0 QHR]0 I) 22 Reagents: @i 47. DIEA, DCM CS 2
CO
3 aq. MeOH; (di) 60, THP; (Iv) HCI. 9RH (i is aq.MeOH.
BNSDOCID: <WO 0064864A1 I> WO 00/64864 PCT/GBOO/01 612 -21in another example (Scheme 20), the alcohol 84 was reacted with di-tert-butyl dietbyiphosphoranlidite and tetrazole inTHF and the intermediate oxidised with MCPBA to give ester 92. Deprotection of 92 with trifluoroacetic, acid (TFAA) gave the phosphate 93.
Scheme 20 a3 OMe NC I OMe 84R=H H 0 93R=P(O)(OH) 2 2 (i Reagents: @t Et; 2 NP(OtBu) 2 tetrazole, 1WF;
I.
MCPBA, DCM QUi)TFAA. DCM NO 2 In another example (Scheme 2 phenol 66 was alkylated with epichiorohydrin under basic conditions to give epoxide 94. Hydrolysis of 94 with perchioric acid gave diol. which was protected as the acetonide 96. Reduction of 96 with DLBALH in THF gave the alcohol 97. Activation of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, BioorgMe Chem. Lett., 1997, 7, 1483] with triphosgene and triethylam-ine gave an intermediate isocyanate which was coupled with alcohol 97 using catalytic dibutyltin diacetate to give carbarnate 98. Deprotection of 98 under acidic conditions gave carbamnate 99.
WO 00/64864 WO 0064864PCTGBOO/01 612 22 Scheme 21 N02 0 I (0i) C02W Co 2 Nt OH 94 95 N%02 96 M )2 98 Reagents: @I epichbrohydiin. K 2 C0 3 DMF;* H010 4 .THF; (HQi dinethoxyacetone, PPTS, DMF; (iv) DIBALH, THF; 1, tdpjhosgene. Et 3 N. DCM, then 97, nBu 2 Sn(OAc} (vi) HCI, sq. THF.
In another example (Scheme 22), phenol 66 was alkylated with N-(3-chloropropyl)- N,N-dimethylamine under basic conditions to give amine 100. Reduction of 100 with DIBALH in THF gave the alcohol 101. Activation of amine 1 JI Atwell, W. R. Wilson, W.
A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 101 using catalytic dibutyltin diacetate to give carbaniate 102.
Similarly (Scheme 22), phenol 66 was alkylated with 4-(3-chloropropyl)morpholine under basic conditions to give amine 103. Reduction of 103 with D1BALH in TI{F gave the alcohol 104. Activation of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 104 using catalytic dibutyltin diacetate to give carbamnate 105.
3NSDOCID: <WO 0064864AI I PCT/GBOOIO1612 WO 00/64864 23 Scheme 22
W
CO
2 Me 100 R= Ie 2 103 R rop (ii) 0 101 R= Wle 2 104 R t~motph Reagents: @i BrCH 2
CH
2
CH
2 NRR, K 2 C0 3 OMF; DIBALH, TH-F; (hi) 1, triphosgene, Et 3 N. DCM. then 101 or 104, nBU 2 Sn(OAc) 2 T 102 R We 2
NO
2 IO5R~tknoiPh In another example (Scheme 23), reaction of alcohol 91 with methanesuiphonyl chloride gave the mesylate 106 which was reacted with morpholine to give carbamate 107.
Seme 23 N0 2 /9i 91 R =OH Sch 0106 R =OMs N 0 107 R =I orph~ i Me 2Reagents: MsCI. DOCA; (Hi) morpholine.
In another example (Scheme 24), phenol 66 was alkylated with tert-butyl 4bromobutanoate under basic conditions to give ester 108. Hydrolysis of 108 under basic conditions gave acid 109 which was reduced with borane.dimethylsufide in THF to give alcohol 110. Activation of alcohol 110 with triphosgene and diisopropylethylamine and subsequent coupling with Nj-ip(-ehlciie4croaiopoy~mn (111) [S.
A. Ganiage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, J Med WO 00/64864 WO 0064864PCT/GBOOIOI 612 24 Chem., 1999, 42, 2383-2393] gave carbamate 112. Carbamnate 112 was deprotected under acidic conditions to give acid 113.
Scheme 24
NO
2
NO
2
NO
2 66 10-_ 10 1. (ii FI 6 0- C2 C 2 uAOC 2 tBu
N
C0 2 R K O K N Z 108RM i 110 Me 0N 0 R L 2 Reagents:Q BCH 2
CH
2
CH
2
CO
2 tBu. K 2 C0 3 DMF; (Hi) MaOH, aq. MeOH 112 R C 2 tBU (hi) BH 3 .DMS. THF; (iv)tnphasgene. DIEA. DCM. then 111: HCI. MeOH. 113 R 00 2 H w In a further aspect, the present invention relates to a method of preparing compounds of the general formula examples of the methods are outlined in Schemes Thus (Scheme 25), (2-nitro-lH-imidazol-5-yI)methanol (115) is obtained from the known ethyl 2-nitro-lH-imidazol-5-ylcarboxylate (113) Cavalleri, R. Ballotta, G.C Lancini. J1 Heterocyclic Chem. 1972, 9, 979.] by basic hydrolysis to the acid 114 and reduction of an intermediate imidazolide with sodium borohydride. This procedure is a major improvement upon the above published methods. Reaction of 115 with 4-nitrophenyl chioroformate gives the activated carbonate 116 which reacts with NNA-bis-(2chloroethyl)aniine to give carbaniate 117.
Sceme 26 Reagents: (Q NaOH. MOH; (Hi) CDI, NaBH4, THP 00i NO 2 PhOCOCI. pydne, THF; H N(CH 2 0H 2
CO
2 p)Iidne.
BNSDOCID: <WO 0064864AI I>~ WO 00/64864 PCTGBOOIOI 612 Similarly (Scheme 26), reaction of 116 with the protected phenyldiamine diol 120, derived from the nitrophenylamino diol 118, gives carbamnate 121. Deprotection of the bis-silyl alcohol 121 with TB3AF gives the diol 122 which can be converted to the dichloride 123 under standard conditions.
Scheme 26
N
2 Me121 R OTBDMS O N 0\ 0 122 R= OH R0>H I,-N 123 R =a )V) RO RT8DMSOf OTBWMS 118 R H 119 ROTBDMS 120
\R
Reagents: (Q TBtDMSCI, imldazde (Hi) PdIC, H 2 EtO- (ii) 116. pyridine; (iv) TBAF, THF, MCI, pyidine.
R
LKCI, DMF.
In another example (Scheme 27), activation of amine 1 J. Atwell, W. K. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine (or pyridine, or another organic base) gave an intermediate isocyanate which was coupled with alcohol 115 using catalytic dibutyltin diacetate to give carbamnate 124.
Scheme 27 a1 0 OH oNb N f N 115 N tJNNO, Reagents: (Q 1, tdphosgene. Et 3 N. DCM12 Mie then 115, n& Sl(oAc 2 .14 BNSOOCID: <WO 0064864A1 I WO 00/64864 WO 0064864PCT/GBOO/01 612 26 In another example (Scheme 28), reaction of the carbonate 116 with doxorubicin (13) and triethylamine in DMF gave the carbamnate 125.
0 OH 0 Schemie28 1 HOH 116 OMeC0 OH 0 13. Et 3 N. DMF. 2N ,1 125 Similarly (Scheme 29), reaction of carbonate 116 with amnine 9 using HOBT, molecular sieves, and triethylamine gave the silyl ether 126. Deprotection of silyl ether 126 'under acidic conditions gave alcohol 127 which was reacted with 4-nitrophenyl chioroformate to give carbonate 128. Reaction of the carbonate 128 with doxorubicin (13) and triethylamine in DMP gave the carbamnate 129.
Scheme 29 NO 2 OH 0 J,
OH
116 OWv) 2 OMeO0 OH HN-- -OMe 0 0 HO
H
OR H 127 RH =OBIS 1 2
N
4 I 129 126 R =TSM H (I) 128 Co 2 PttJO 2 (iii) Reagerts:(@ 9. HOST, Et 3 N, nmil. sieves, TIF; (ii) HCI. aq. MeOH; (Ui 4.NO 2 PhOCOC1,THF; (vi) 13. Et 3 K~ DMF.
In another example (Scheme 30), ozonolysis of the styrene 130 C. Baker, S. R.
Putt, H. D. H. Showalter, J Heterocyclic Chem., 1983, 30, 629-634.] gave the alcohol 131.
BNSOID: <MO 0064864A1 I WO 00/64864 PCT/GB00101612 27 Activation of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 131 using catalytic dibutyltin diacetate to give carbamnate 132.
Scheme 30 a1 N HN OWe 0 2 N ON~ OH HN0Ohf 130 /131 p7 132 N0 2 Reagents: (0 0, DCM, MeOH 01) NaBH 4 EtOH; (HO~ 1. tripthosgene, Et 3 N, DCM. then 131, nBu 2 Sn(oAc) 2 In another example (Scheme 3 treatment of (N-methyl-5-nitro-1IH-imidazol-2yl)methanol (133) Rufer, H. J. Kessler, E. Schroder. J. Med Chem. 1971, 14, 94.] with 4nitrophenyichioroformate gave the carbonate 134, which was displaced with NN-bis(2chloroethyl)amine to give the carbamnate 135.
Scheme 31 ~OH Oc IcNO 2 N0 2
N~
4 Uv 0 133 13N2N W2e Reagerds: @i NO 2 PhOCOCI, pyridneTHF;
NO
HN(CH
2 CH2CI)2, pyr~inie.13N0 Similarly (Scheme 32), activation of 133 with diphosgene and subsequent reaction with MM-bis(2-chloroethyl)-1,4-benzenedianinfe hydrochloride (136) gave the carbarnate 137.
WO 00/64864 WO 0064864PCT/GBOO/01 612 -28 Scheme 32
OH
N We N0 2 N02
MI
13 137 Reagents: (Idphosgene. Et 3 N. THF: 138, pidne.
In another example (Scheme 33), activation of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 133 using catalytic dibutyltin diacetate to give carbamate 138.
Scheme 33
NO
2 N' NMe i) OH HN 0 118 0 Reagents: 1. triphosgene. Et 3 N, DCM. 124 then 115, nBu 2 -Sn(OAC) 2 .14 In another example (Scheme 34), reaction of carbonate 134 with amine 9 using HOBT, molecular sieves, and triethylaniine gave the silyl ether 139. Deprotection of silyl ether 139 under acidic conditions gave alcohol 140 which was reacted with 4-nitrophenyl chioroformate to carbonate 141. Reaction of the carbonate 141 with doxorubicin (13) and triethylamine in DMF gave the carbamate 142.
BNSDOCID: <WO OD64864AI I WO 00/64864 PCT/GBOD/01612 -29- Scheme 34 NO 2
H
(ArS I O OH Me)_ OH__OO O M1H 0
HH
OR0 139 R =TBDMS (i)14 141 CO 2 PhNO 2
(I
Reagents: 9, HOBT, Et 3 N, irni. sieves, THF; HOI. aq. MeOH-; 4-NO 2 PhOCOCI, THF; Mv) 13, Et 3 N. DMF.
in another example (Scheme 35), condensation of metronidazole (143) and benzaldehyde gave the styrene 144 which was protected with TBDMS triflate to give 145.
Ozonolysis of styrene 145 gave alcohol 146. Activation of amine 1 I. Atwell, W. R.
Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylaniine gave an intermediate isocyanate which was coupled with alcohol 146 using catalytic dibutyltin diacetate to give carbamate 147. Deprotection under acidic conditions gave the earbaniate 148.
Scheme 36 c OH -me 4 'O -\OR -OTBOMS
NOM.
N N% NO 2
NO
2 143 144~y) 146
O
146~~~4 R TBDMS 1) -0 Reagerts: NaOMe, PhCHO, DM60; TBDMSTf, pridne, DCM; 147 R =HTDS (i (11) DCI4 MeOH; (vi) NaBH 4 EtOH; 1, triphosgene. Et 3 N, DCK 14 R= in then 146, n&u 2 Sn(OAc)i. (V~i HCI. a. MeOH.
WO 00164864 WO 0064864PCT/GBOO/D 1612 Similarly (Scheme 36), activation of amine 1 J. Atwell, W. R. Wilson, W. A.
Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 149 C. Baker, S.R. Putt, H. H.
Showalter, J1 Heterocyclic Chem., 1983, 20, 629-634.] using catalytic dibutyltin diacetate to give carbamnate 150.
cI Scheme 36 H- NO 2 Owe HN. 0 149 Reagents: (D1.tnphosgene. Et 3 N, DCM,NO t he n 149, n Bu 2 S n(OAc) 2 IO 0 In another example (Scheme 37), reaction of the 5-nitrofuran-l-methaol (151) P3. MA Berry, C. Y. Watson, W. J. D. Whish, and M. D. Threadgill, J Chem. Soc. Perkin Trans. I, 1997, 1147.] with 4-nitrophenyichioroformate gave carbonate 152, which was displaced with N',N-bis(2-hydroxyethyl)-1,4-benzenediamnine (57) to give the carbamnate diol 153. The diol 153 was converted to the dichloride 154 using standard methods.
Similarly (Scheme 37), reaction of (5-nitrothien-2-yl)methanol (156) J.
Narcombe, R. K. Norris. Aust. J Chem. 1979,32,2647] with 4-nitrophenyichioroformate gave carbonate 157, which was displaced with N',N-bis(2-hydroxyethyl)-l,4-benzenediamine (57) to give the carbamnate diol 158. The diol 158 was converted to the dichloride 159 using -standard methods. The same technique was used on 5-nitrofiun-1I-methanol (151) [P.M.
Berry, C.Y. Watson, W.J. Whish, and M.D. Threadgill, J1 Chem. Soc. Perkin Trans. 1, 1997, 1147].
3NSDOCID: cMO 0064864A1 I WO 00/64864 WO OO648~PCTGBOOIOI 612 -31 Scheme 37 2 2 0 2 N x OH fx 1 151 X=o 0OO'N 156 X= S[IIJ 152 X=O0 f 157 X= S
R
153 X= 0, R -OH Reagenlts: N2PhOCOCI. pyridnfe, THF. 16'4X=O. R~a is 57, pyridine, (Hs) sCI. pyridine, 158X= S R OH (b)lI,ODMF. 159X=S.R=CI in another example (Scheme 38), activation of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7,1483] with triphosgene and triethylainine gave an intermediate isocyanate which was coupled with alcohol 151 M. Berry, C. Y.
Watson, W. J. D. Whish, and M. D. Threadgill, J Chem. Soc. Perkin Trans. 1997, 1147.] using catalytic dibutyltin diacetate to give carbamate 155.
Similarly (Scheme 38), activation of amine 1 L. Atwell, W. R. Wilson, W. A.
Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 156 I. Narcombe, R. K Norris.
Aust. J Chem. 1979, 32,2647] using catalytic dibutyltin diacetate to give carbamnate 160.
Scheme 38a 0 0 2 .N X OH N OMO
OFM
151 X= 0 0 156 X= s Reagernts: tdphosgene, Et 3 N, DCM. L-1X N then alcohol. nlu 2 Sn(OAc) 2 160 x S WO 00/64864 PCT/GBOO/01612 32- In another example (Scheme 39), l-methyl-5-nitro-H-pyrazole4-caboxylic acid (161) C. Cheng, J Heterocyclic Chem. 1968,5, 195-197] was reduced with borane.dimethyl sulfide to give alcohol 162. Activation of amine 1 J. Atwell, W. R.
Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 162 using catalytic dibutyltin diacetate to give carbamate 163.
Scheme 39 Me Me C1i N 0)O N/7N 0 2 N0 0 N~ C0 2 H -OH N HN- n e 161 162 COMe HNO 0 Reagents: BH 3 ,DMS, THF; (i i) 1, triphosgene, Et 3 N, 0,1 \NMe DCM. then 162, nBU 2 Sn(OAC) 2 163 0 2
N
in another example (Scheme 40), ethyl (164) Fomnari, M. Farnier, C. Fournier, Bull. Soc. Chim. Fr. 1972, 283-29 1] was alkylated with dimethyl sulfate to give pyrrole 165. Reduction of 165 with sodium borohydride gave the alcohol 166. Activation of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med.
Chem. Left., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyariate which was coupled with alcohol 166 using catalytic dibutyltin diacetate to give carbamate 167.
In another example (Scheme 40), hydrolysis of ester 166 followed by decarboxylation with copper in quinoline gave alcohol 168. Activation of amnine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 168 using catalytic dibutyltin diacetate to give carbamnate 169.
BNSDOCIDY. <WO 0064864AI I> WO 00/64864 WO 0064864PCT/GBOO/01 612 -33-
CI
Scheme 0 2 N CO 2 Et 0 N- e R N /N~ 164 R H f 166 R 00Et\ 1 165 R W 168R =H (I I NV)e Reagents: Ma 2
SO
4
K
2 (fl 3 DM80: (ii NaBH 4 BtO Ft 0 2
N
trkhosgene, Et 3 N. DCM. then alcoihol, nBu 2 S n(QAc 2 167 R H 0E EtOK (V)Cu,quinoline.16R=H Similarly (Scheme 41), ethyl 5-formyl-4-nitro- 1H-pyrrole-2-carboxylate (170) [P.
Fornari, M. Farnier, C. Fournier, Bull. Soc. Chim. Fr. 1972, 283-29 1] was alkcylated with dimethyl sulfate to give pyrrole 171. Reduction of 171 with sodium borohydride gave the alcohol 172. Activation of amine 1 J. Atwell, W. R Wilson, W. A. Denny, Bioorg. Med Chemn. Lett., 1997, 7, 1483) with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 172 using catalytic dibutyltin diacetate to give carbamate 173.
In another example (Scheme 41), hydrolysis of ester 172 followed by decarboxylation with copper in quinoline gave alcohol 174. Activation of amnine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem Lent., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 174 using catalytic dibutyltin diacetate to give carbamate 175.
CI
Scheme 41 H e a~M 0j HN Oe 0 2 N 0 2 N W~e 170R =H 172 R =C 2 EtN 171 R =M pb14R= v Reagents: @i)Me 2
SO
4 K2003, DM5IO; (10 NaBH4I. Et0K 0 2
N
(Hi) 1. triphosgene, Et 3 N. 0CM. then alcohol, nBu 2 Sn(OAc 2 173 R 0 2 Et Na0H. eq. EtOK Cu. quinolie. 175 R =H OktCfltflfl. .%htfl flflOAOOA A 4 I WO 00/64864 PCT/GB00/01612 34- In another example (Scheme 42), 1-methyl-5-nitro-lH-pyrrole-2-carbaldehyde (176) P. Fournari, Bull. Soc. Chim. Fr. 1963, 488-491] was reduced with sodium borohydride to give alcohol 177. Activation of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg.
Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 177 using catalytic dibutyltin diacetate to give carbamate 178.
ci Scheme 42 e e OH N 1- OM OMe HN 0 176 177 r Reagents:(i) NaBH 4 EtOH; (ii) 1, trphosgene, 178 NO, EtN., DCM. then 177,nBu 2 Sn(OAc) 2 Me In a further preferred aspect, the present invention relates to the use of a compound of formula or (II) as defined in the second aspect of the invention, in conjunction with a 10 nitroreductase enzyme (for example, isolated from E. coli) in a method of ADEPT or GDEPT therapy. A drug produced by the action of the nitroreductase enzyme on a compound of formula or (II) may be used for the selective killing ofoxic and hypoxic tumour cells in methods of treatment of cancers, for example leukemias and particularly solid cancers including breast, bowel and lung tumours, including small cell lung carcinoma.
15 The invention also provides a pharmaceutical composition comprising a compound of the formula or (II) as defined in the second aspect of the invention together with a pharmaceutically acceptable carrier or diluent.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Any description of prior art documents herein is not an admission that the documents form part of the common general knowledge of the relevant art in Australia.
34A Detailed Description of the Invention
GDEPT
Vector systems In general, the vector for use in GDEPT therapies may be any suitable DNA or RNA vector.
Suitable viral vectors include those which are based upon a retrovirus. Such vectors are widely available in the art. Huber et al. (ibid) report the use of amphotropic retroviruses for the transformation of hepatoma, breast, colon or skin cells. Culver et al. (Science(1992) *o*ee.
*e coco- WO 00/64864 PCT/GB00/01612 256; 1550-1552) also describe the use ofretroviral vectors in GDEPT. Such vectors or vectors derived from them may also be used. Other retroviruses may also be used to make vectors suitable for use in the present invention. Such retroviruses include rous sarcoma virus (RSV).
Englehardt et al. (Nature Genetics (1993) 4; 27-34) describe the use of adenovirus based vectors in the delivery of the cystic fibrosis transmembrane conductance product (CFTR) into cells, and such adenovirus based vectors may also be used. Vectors utilising adenovirus promoter and other control sequences may be of use in delivering a system according to the invention to cells in the lung, and hence useful in treating lung tumours.
Other vector systems including vectors based on the Molony murine leukaemia virus are known (Ram, Z et al. Cancer Research (1993) 53; 83-88; Dalton Treisman, Cell (1992) 68; 597-612). These vectors contain the Murine Leukaemia virus (MLV) enhancer cloned upstream at a p-globin minimal promoter. The P-globin 5' untranslated region up to the initiation ATG is supplied to direct efficient translation of the enzyme.
Suitable promoters which may be used in vectors described above, include MLV, CMV, RSV and adenovirus promoters. Preferred adenovirus promoters are the adenovirus early gene promoters. Strong mammalian promoters may also be suitable. An example of such a promoter is the EF-1 a promoter which may be obtained by reference to Mizushima and Nagata ((1990), Nucl. Acids Res. 18; 5322). Variants of such promoters retaining substantially similar transcriptional activities may also be used.
Nitroreductase Compounds of the formula or can be activated by reduction of one (or more) of the available nitro groups by nitroreductase.
Preferably, the enzyme is a non-mammalian nitroreductase enzyme, such as a bacterial nitroreductase. An E.coli nitroreductase as disclosed in WO93/08288 is particularly preferred.
The enzyme may be modified by standard recombinant DNA techniques, e.g. by cloning the enzyme, determining its gene sequence and altering the gene sequence by methods such as truncation, substitution, deletion or insertion of sequences for example by site-directed mutagenesis. Reference may be made to "Molecular Cloning" by Sambrook et al. (1989, Cold Spring Harbor) for discussion of standard recombinant DNA techniques. The modification made may be any which still leaves the enzyme with the ability to reduce the nitro group in formula I or II but alters other properties of the enzyme, for example its rate of reaction or selectivity.
WO 00/64864 PCT/GBOO/01612 -36- In addition, small truncations in the N- and/or C-terminal sequence may occur as a result of the manipulations required to produce a vector in which a nucleic acid sequence encoding the enzyme is linked to the various other vector sequences.
ADEPT
For applications in ADEPT systems, an antibody directed against a tumour specific marker is linked to the nitroreductase enzyme, which may be modified as described above.
The antibody may be monoclonal or polyclonal. For the purposes of the present invention, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a tumour target antigen. Such fragments include Fv, F(ab') and F(ab') fragments, as well as single chain antibodies. Furthermore, the antibodies and fragments thereof may be humanised antibodies, e.g. as described in EP-A-239400.
The antibodies may be produced by conventional hybridoma techniques or, in the case of modified antibodies or fragments, by recombinant DNA technology, eg by the expression in a suitable host vector of a DNA construct encoding the modified antibody or fragment operably linked to a promoter. Suitable host cells include bacterial (eg. E.coli), yeast, insect and mammalian. When the antibody is produced by such recombinant techniques the enzyme may be produced by linking a nucleic acid sequence encoding the enzyme (optionally modified as described above) to the 3' or 5' end of the sequence of the construct encoding the antibody or fragment thereof.
Applications of the invention Compounds of the invention can be used in vitro or in vivo for a range of applications.
For example, a number of vector systems for the expression of nitroreductase in a cell have been developed. The further development of such systems the development of promoters suitable for specific cell types) requires suitable candidate prodrugs capable of killing cells when activated by nitroreductase. Prodrug compounds of the present invention may be used in such model systems. The model systems may be in vitro model systems or xenograft model systems comprising for example human tumour cells implanted in nude mice.
Compounds of the invention which are not activatable by an enzyme may be tested in vitro against panels of different tumour cells types to determine efficacy against such tumour cells. The efficacy of compounds of the invention against a range of tumour cell types may be used as points of reference for the development of further antitumour compounds.
Compounds of the present invention may also be tested in combination with additional anti- BNSDOCID: <WO 0064864A1 I WO 00/64864 PCT/GB00/01612 -37cancer compounds to determine potential combination drug systems, for example combinations which are synergistic.
The compounds of the invention may also be used in a method of treatment of the human or animal body. Such treatment includes a method of treating the growth of neoplastic cells in a patient with neoplastic disease which comprises administering to a patient in need of treatment a compound of formula or (II) of the invention as part of an ADEPT or GDEPT therapy system. Neoplastic diseases include leukaemia and solid tumours such as breast, bowel and lung tumours including small cell lung carcinoma.
It will be understood that where treatment of tumours is concerned, treatment includes any measure taken by the physician to alleviate the effect of the tumour on a patient. Thus, although complete remission of the tumour is a desirable goal, effective treatment will also include any measures capable of achieving partial remission of the tumour as well as a slowing down in the rate of growth of a tumour including metastases. Such measures can be effective in prolonging and/or enhancing the quality of life and relieving the symptoms of the disease.
ADEPT therapy The antibody/enzyme conjugate for ADEPT can be administered simultaneously but it is often found preferable, in clinical practice, to administer the enzyme/agent conjugate before the prodrug, e.g. up to 72 hours or even 1 week before, in order to give the enzyme/agent conjugate an opportunity to localise in the region of the tumour target. By operating in this way, when the prodrug is administered, conversion of the prodrug to the cytotoxic agent tends to be confined to the regions where the enzyme/agent conjugate is localised, i.e. the region of the target tumour, and the premature release of the compound produced by the action of the nitroreductase on the compound of formula or (II) is minimised.
In ADEPT the degree of localisation of the enzyme/agent conjugate (in terms of the ratio of localized to freely circulating active conjugate) can be further enhanced using the clearance and/or inactivation systems described in W089/10140. This involves, usually following administration of the conjugate and before administration of the prodrug, the administration of a component (a "second component") which is able to bind to part of the conjugate so as to inactivate the enzyme and/or accelerate the clearance of the conjugate from the blood. Such a component may include an antibody to the enzyme component of the system which is capable of inactivating the enzyme.
The second component may be linked to a macromolecule such as dextran, a liposome, PCT/GBOO/01612 WO 00/64864 -38albumin, macroglobulin or a blood group O erythrocyte so that the second component is restrained from leaving the vascular compartment. In addition or as an alternative, the second component may include a sufficient number ofcovalently bound galactose residues, or residues of other sugars such as lactose or mannose, so that it can bind the conjugate in plasma but be removed together with the conjugate from plasma by receptors for galactose or other sugars in the liver. The second component should be administered and designed for use such that it will not, to any appreciable extent, enter the extravascular space of the tumour where it could inactivate localised conjugate prior to and during administration of the prodrug.
In ADEPT systems, the dose of the prodrug and conjugate will ultimately be at the discretion of the physician, who will take into account such factors as the age, weight and condition of the patient. Suitable doses of prodrug and conjugate are given in Bagshawe et al.
Antibody, Immunoconjugates, and Radiopharmaceuticals (1991), 4, 915-922. A suitable dose of conjugate may be from 500 to 200,000 enzyme units/m 2 20,000 enzyme units/m 2 and a suitable dose of prodrug may be from about 0.1 to 200 mg/Kg, preferably about from 10 to 100 mg/Kg per patient per day.
In order to secure maximum concentration of the conjugate at the site of desired treatment, it is normally desirable to space apart administration of the two components by at least 4 hours. The exact regime will be influenced by various factors including the nature of the tumour to be targeted and the nature of the prodrug, but usually there will be an adequate concentration of the conjugate at the site of desired treatment within 48 hours.
The ADEPT system when used with nitroreductase also preferably comprises a suitable cofactor for the enzyme. Suitable cofactors include a riboside or ribotide of nicotinic acid or nicotinamide.
The antibody/enzyme conjugate may be administered by any suitable route usually used in ADEPT therapy. This includes parenteral administration of the antibody in a manner and in formulations similar to that described below.
GDEPT therapy For use of the vectors in therapy, the vectors will usually be packaged into viral particles and the particles delivered to the site of the tumour, as described in for example Ram et al. (ibid). The viral particles may be modified to include an antibody, fragment thereof (including a single chain) or tumour-directed ligand to enhance targeting of the tumour.
Alternatively the vectors may be packaged into liposomes. The liposomes may be targeted to BNSDOCIIM WO 0064864A1 I> WO 00/64864 PCT/GB00/01612 -39a particular tumour. This can be achieved by attaching a tumour-directed antibody to the liposome. Viral particles may also be incorporated into liposomes. The particles may be delivered to the tumour by any suitable means at the disposal of the physician. Preferably, the viral particles will be capable of selectively infecting the tumour cells. By "selectively infecting" it is meant that the viral particles will primarily infect tumour cells and that the proportion of non-tumour cells infected is such that the damage to non-tumour cells by administration of a prodrug will be acceptably low, given the nature of the disease being treated. Ultimately, this will be determined by the physician.
One suitable route of administration is by injection of the particles in a sterile solution.
Viruses, for example isolated from packaging cell lines may also be administered by regional perfusion or direct intratumoral direction, or direct injection into a body cavity (intracaviterial administration), for example by intra-peritoneum injection.
The exact dosage regime for GDEPT will, of course, need to be determined by individual clinicians for individual patients and this, in turn, will be controlled by the exact nature of the prodrug and the cytotoxic agent to be released from the prodrug but some general guidance can be given. Chemotherapy of this type will normally involve parenteral administration of modified virus and administration by the intravenous route is frequently found to be the most practical.
In GDEPT systems the amount of virus or other vector delivered will be such as to provide a similar cellular concentration of enzyme as in the ADEPT system mentioned above.
Typically, the vector will be administered to the patient and then the uptake of the vector by transfected or infected (in the case of viral vectors) cells monitored, for example by recovery and analysis of a biopsy sample of targeted tissue. This may be determined by clinical trials which involve administering a range of trial doses to a patient and measuring the degree of infection or transfection of a target cell or tumour. The amount of prodrug required will be similar to or greater than that for ADEPT systems.
In using a GDEPT system the prodrug will usually be administered following administration of the vector encoding an enzyme. Suitable doses of prodrug are from about 0.1 to 200 mg/Kg, preferably about from 10 to 100 mg/Kg per patient per day.
Administration of prodrug While it is possible for a compound of formula or to be administered alone it is preferable to present it as a pharmaceutical formulation. Suitable formulations comprise the WO 00/64864 PCT/GB00/01612 compounds, together with one or more acceptable carriers thereof and optionally other therapeutic ingredients. The carrier or carriers must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipients thereof, for example, liposomes. Suitable liposomes include, for example, those comprising the positively charged lipid (N[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA), those comprising dioleoylphosphatidylethanolamine (DOPE), and those comprising 33[N-(N'N-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol).
Formulations suitable for parenteral or intramuscular administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, bactericidal antibiotics and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injections, immediately prior to use. Injection solutions and suspensions may be prepared extemporaneously from sterile powders, granules and tablets of the kind previously described.
It should be understood that in addition to the ingredients particularly mentioned above the formulations may include other agents conventional in the art having regard to the type of formulation in question. Of the possible formulations, sterile pyrogen-free aqueous and nonaqueous solutions are preferred.
The doses may be administered sequentially, eg. at daily, weekly or monthly intervals, or in response to a specific need of a patient. Preferred routes of administration are oral delivery and injection, typically parenteral or intramuscular injection or intratumoral injection.
The exact dosage regime will, of course, need to be determined by individual clinicians for individual patients and this, in turn, will be controlled by the exact nature of the compound of formula or (II) but some general guidance can be given. Typical dosage ranges generally will be those described above which may be administered in single or multiple doses. Other doses may be used according to the condition of the patient and other factors at the discretion of the physician.
BNSOOCID: <M 006484AI I> WO 00/64864 PCT/GB00/01612 -41- The following Examples illustrate the invention.
General procedures Analyses were carried out in the Microchemical Laboratory, University of Otago, Dunedin, NZ. Melting points were determined on an Electrothermal 2300 Melting Point Apparatus. IR spectra were recorded on a Midac FT-IR as KBr discs, unless otherwise stated. NMR spectra were obtained on a Bruker AM-400 spectrometer at 400 MHz for 'H and 100 MHz for 1 3
C
spectra. Spectra were obtained in CDCI 3 unless otherwise specified, and are referenced to Me 4 Si. Chemical shifts and coupling constants were recorded in units ofppm and Hz, respectively. Assignments were determined by APT, COSY, HSQC, and HMBC experiments. Mass spectra were determined on a VG-70SE mass spectrometer using an ionizing potential of 70 eV at a nominal resolution of 1000. High resolution spectra were obtained at nominal resolutions of 3000, 5000, or 10000 as appropriate. All spectra were obtained as electron impact (EI) using PFK as the reference unless otherwise stated. Solutions in organic solvents were dried with anhydrous Na 2
SO
4 Solvents were evaporated under reduced pressure on a Buchi rotary evaporator. Thin-layer chromatography was carried out on aluminium-backed silica gel plates (Merck 60 F2) with visualisation of components by UV light (254 nm) or exposure to 12. Column chromatography was carried out on silica gel, (Merck 230-400 mesh). All compounds designated for biological testing were analyzed at >99% purity by reverse phase HPLC using a Philips PU4100 liquid chromatograph, a Phenomenex BondClone 10-C18 stainless steel column (300mm x 3.9 mm i.d.)and a Philips PU4120 diode array detector. Chromatograms were run using various gradients of aqueous (1 M NaHPO 4 0.75 M heptanesulfonic acid, 0.5 M dibutylammonium phosphate, and MilliQ water in a 1:1:1:97 ratio) and organic (80% MeOH/MilliQ water) phases. DCM refers to dichloromethane; DIEA refers to diisopropylethylamine, DMF refers to dry dimethyl formamide; DMSO refers to dimethylsulphoxide; ether refers to diethyl ether, EtOAc refers to ethyl acetate; EtOH refers to ethanol; iPr 2 O refers to diisopropyl ether; light petroleum refers to petroleum ether, boiling range 40-60 MeOH refers to methanol; THF refers to tetrahydrofuran dried over sodium benzophenone ketyl. All solvents were freshly distilled.
Example 1. Preparation of 4-nitrobenzyl 4-(1,3-benzothiazol-2-yl)phenylcarbamate 4-Nitrophenyl chloroformate (0.15 g, 0.46 mmol) was added to a stirred solution of 2- BNSDOCID: WO 0064Al I PCT/GB00/01612 WO 00/64864 -42- (4-aminophenyl)benzthiazole Shi, T.D. Bradshaw, S. Wrigley, CJ. McCall, P.
Lelieveld, I. Fitchner, M.F.G.Stevens. J. Med. Chem., 1996, 39, 3375] in pyridine (5 mL) and the solution stirred at 20 *C for 2 h. The solution was dilute with water (10 mL) and the mixture stirred for 40 min, filtered and the solid triturated with hot EtOH to give 8 (157 mg, 87%) as a pale green powder, mp 232-234 'H NMR [(CD 3 ),SO]S 10.31 1 H, OCONH), 8.29 (ddd, J= 8.7, 3.2, 2.2 Hz, 2 H, H H 8.11 8.3 Hz, 1 H, H 4), 8.04 (br d, J= 8.7 Hz, 2 H, H H 8.02 J 8.3 Hz, 1 H, H 7.72 (br d, J= 8.7 Hz, 2 H, H H 7.68 (br d, J= Hz, 2 H, H H 7.51-7.55 1 H, H 7.40-7.46 1 H, H 5.32 2 H, CH20); Anal. (C 21 HsN 3 0 4 S) C, H, N.
Example 2A. Preparation of 2-methoxy-4-nitrobenzyl 4-[bis(2chloroethyl)amino]phenylcarbamate (32).
Methyl 2-methoxy-4-nitrobenzoate An ethereal solution ofdiazomethane (CAUTION) was added to a solution of 4-nitrosalicylic acid (27) (1.0 g, 5.46 mmol) in ether (50 mL) until a yellow colour persisted and the solution stood at 20 *C for 4 h. The reaction was quenched with glacial acetic acid (2 mL), poured into sat. aq. NaHCO 3 solution and extracted with ether (2 x 50 mL). The combined organic fractions were dried and the solvent evaporated to give 28 (1.11 g, 96%) as white needles, mp (ether) 89-90 *C; IR N 1740, 1526, 1252, and 1086 'H NMR 6 7.89 J= 8.3 Hz 1 H, H 7.82-7.85 2 H, H 3, H 4.01 3 H, OCH 3 and 3.94 3 H, OCH 3 3 C NMR 165.2 (CO 2 159.2 (C 150.7 (C 132.0 (C 126.0 (C 115.0 (C 106.9 (C 56.6 (OCH 3 and 52.6 (OCH 3 Anal. (CH9NOS) C, H, N.
2-Methoxy-4-nitrobenzyl alcohol A solution of 28 (0.9 g, 4.26 mmol) in THF mL) was added dropwise to a stirred solution of DIBALH (1 M solution in toluene, 13.4 mL, 13.4 mmol) in THF (20 mL) at 2 oC and the solution stirred at 2 *C for 15 min. The solvent was evaporated and residue partitioned between EtOAc (100 mL) and water (100 mL). The aqueous fraction was extracted with EtOAc (2 x 50 mL) and the combined organic fraction dried and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 29 (0.74 g, 93%) as cream needles, mp (EtOAc/light petroleum) 103-104 IR n 3310, 1523, 1250, and 1036 'H NMR 7.86 (dd, J= 8.3, 2.1 Hz, 1 H, H 7.71 J= 2.1 Hz, 1 H, H 7.52 (d, BNSDOCID: WO 0064864A1 I> WO 00/64864 PCT/GB00/01612 -43- J= 8.3 Hz, 1 H, H 4.76 J= 5.5 Hz, 2 H, CHO), 3.96 3 H, OCH 3 and 2.27 (br s, 1 H, OH); 3 C NMR 5 157.1, 148.3, 136.6, 127.9, 116.0, 105.0, 60.7, and 55.9; Anal.
(CH
9 NO0 4 C, H, N.
2-Methoxy-4-nitrobenzyl 4-[bis(2-hydroxyethyl)amino]phenylcarbamate Pyridine (91 uL, 1.13 mmol) was added dropwise to a stirred solution of 29 (207 mg, 1.13 mmol) and triphosgene (117 mg, 0.40 mmol) in THF (10 mL) at 5 *C and the suspension stirred at "C for 1 h. A solution of N',N-bis(2-hydroxyethyl)-1,4-benzenediamine (57) [prepared by catalytic hydrogenation of NN-bis-(2-hydroxyethyl) 4-nitroaniline (244 mg, 1.24 mmol) with Pd/C under H 2 (60 psi) in EtOH) in THF (10 mL) and DMF (10 mL) was added and the mixture stirred at 20 *C for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc to give 31 (251 mg, 55%) as orange prisms, mp (EtOAc) 153-154 'H NMR J 9.45 (br s, 1 H, OCONH), 7.90 (dd, J= 8.3, Hz, 1 H, H 7.80 J= 2.0 Hz, 1 H, H 7.60 (br d, J= 8.3 Hz, 1 H, H 7.21 (brd, J=9.0Hz, 2H, H2, H6), 6 6 1 (d,
J
9.0Hz, 2 H, H 3, H 5.17 2 4.71 J= 5.4 Hz, 2 H, 2 x OH), 3.97 3 H, OCH,), 3.48-3.53 4 H, 2 x CH20), 3.32- 3.37 4 H, 2 x CH 2 3 C NMR 6 157.4, 153.7, 148.6, 144.6, 133.2, 129.0, 127.8, 120.7 116.0, 111.9 105.9, 60.6, 58.7 56.7, 53.9 Anal. (C, 9 H23N 3 O) C, H, N.
2-Methoxy-4-nitrobenzyl 4-[bis(2-chloroethyl)amino]phenylcarbamate (32).
Methanesulphonyl chloride (129 zL, 1.67 mmol) was added dropwise to a stirred solution of 31 (226 mg, 0.55 mmol) in pyridine (10 mL) at 20 *C and the solution stirred for 1 h.
The solvent was evaporated and the residue partitioned between DCM/water (100 mL). The aqueous fraction was extracted with DCM (2 x 50 mL) and the combined organic fraction washed with brine (50 mL), dried and the solvent evaporated. The residue was dissolved in DMF (10 mL), LiC (0.15 g, 3.34 mmol) added, and the mixture stirred at 80 °C for 2 h.
The solvent was evaporated and the residue partitioned between EtOAc/water (100 mL).
The aqueous fraction was extracted with EtOAc (2 x 25 mL). The combined organic fraction was washed with brine (30 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 25% EtOAc/light petroleum, to give 32 (230 mg, 93%) as pale green needles, mp (EtOAc/light petroleum) 129-130 'H NMR 8 7.84 (dd, J= 8.3, 2.1 Hz, 1 H, H 7.72 J 2.1 Hz, 1 H, H 7.51 8.3 Hz, 1 H, H RNnrlin~l C n UWA NiAi4A1 I WO 00/64864 PCT/GBOO/01612 -44 7.27 (br d,J =9.0Hz, 2 H, H 2, H 6.65 (ddd,J 3.5, 2.1 Hz, 2 H, H 3, H 5.29, 2 H, CH 2 3.96 3 H, OCH 3 3.68-3.72 (in, 4 H, 2 x CH 2 3.58-3.63 (mn, 4 H, 2 x CH 2 Cl); 3 C NMR 5 157.2, 153.4, 148.6, 142.9, 132.2, 128.5, 128.1, 121.4 115.7, 112.7 105.3, 61.3, 56.0, 53.7 40.5 Anal. (C 19
H
21 C1 2
N
3 0 5 C, H, N.
Example 2B. Preparation of 2-methoxy-4-litrobelzyl 3-(chloromethyl)-1-[5,6,7triinethoxy-1H-indo-2yl)carboylY2,3dihydro1HindolI 6 ylcarbam ate (34).
Pyridine (20 gL, 0.25 mmol) was added dropwise to a stirred solution of 2- methoxy-4nitrobenzyl alcohol (29) (45 mng, 0.25 rmol) and triphosgene (26 ing, 0.09 mmol) in THF (10 mL) at 5 'C and the suspension stirred at 5 *C for 1 h. A solution of 6-amino-3- (chioromethyl)- 1 -[(5,6,7-triinethoxyildo-2-y1)carboflyl]indoline (33) [Mv. Tercel and W. A.
Denny. J. Chem. Soc. Perkin Trans. 1, 1998, 509] (102 mg, 0.25 mmol) in THF (10 niL) was added and the mixture stirred at 20 *C for 16 h. The suspension was filtered and the solvent evaporated. The residue was purified by chromatography, eluting with 40%EtOAc/DCM, to give 34 (102 ing, 65%) as a tan powder, mp (DOM/pet. ether) 144- 150 1H NMR 5 9.74 I H, indole-NH), 8.26 J =0.8 Hz, 1 H, H 7.62-7.68 (in, 3 H, H 5, H H 7.58 (br s, 1 H, OCONH), 7.35 (br d, J =8.1 Hz, 1 H, H 7.20 J 8.3 Hz, I H, H 6.91 J= 2.1 Hz, 1 H, H 6.83 1 H, H 5.21 2 H,
CH
2 4.5 8 (dd, J =10.6, 8.9 Hz, I H, H 4.41 J =10.6, 4.3 Hz, 1 H, 14 4.03, 3 H, OCH 3 3.92 3 H, OCH 3 3.90 3 H, OCH 3 3.89 3 H, OCH 3 3.76-3.80 (in, 2 H, H 3, CH 2 CI), 3.51 (dd, J 11.7, 10.6 Hz, 1 H, CH 2 C1); 1 3 C NMvR 8 160.5, 157.1, 153. 1, 150.2, 148.4, 144.0, 140.5, 138.8, 138.7, 132.0, 129.5, 128.3, 126.0, 125.6, 124.5, 123.5, 115.6, 115.5, 108.8, 106.7, 105.5, 97.5, 61.4, 61.3, 61.1, 56.2, 55.9, 54.8, 46.9, 43.2; MIS (FAB-) m/z 627 (MW, 625 (MW, 12), 234 149 (100); HRMS (FAB~) caic. for
C
30
H
30 35 C1N 4 0 10 (MW) m/z 625.1701, found 625.1690; C 30
H
30 37
CIN
4 01 0 MW) m/z 627.1672, found 627.1623; Anal. (C 30
H
2 qC1N 4 0 9 C, H, N.
Example 2C. Preparation of 2-methoxy-4-nitrolbezyl 1-(chloromethyl)-3-[(5, 6 7 trmtoyl-nol2y Ibnl-23dhdol- enzleindol-5-ylcarbamate Phosgene (300 giL, 0.3 iniol, IM in toluene) was added to a stirred solution of 2methoxy-4-nitrobenzyl alcohol (29) (20 mg, 0. 11 mniol) in THE (10 inL) and stirred at BNSOOCID; <WO 0064864Al WO 00/64864 WO 0064864PCT/GBOO/01612 45 *C for 16 h. The solvent was evaporated, the residue dissolved in THIF (10 mL), a solution of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (50 mg, 0. 11 mmol) in THIF 10 mL) was added and the solution stirred at 20 OC for 4 days. The solvent was evaporated and the residue purified by chromatography, eluting with 50% EtOAc/light petroleum to give 35 (31 mg, 43%) as a tan solid, mp (EtOAcf light petroleum) 162-165 'H NMR 8 9.52 1 H, indole-NH), 8.90 I H, OCONH), 7.90 J= 8.7 Hz, 1 H, H 7.80 J= 8.7 Hz, 1 H, H 7.77 J= 8.4 Hz, 1 H, H 9), 7.70 (br s, 1 H, H 7.50-7.57 (in, 2 H, H 8, H 7.42-7.47 (mn, 1 H, H 7.25 (br s, 1 H, H4), 6.99 J =2.2 Hz, 1 H, H 6.87 1 H, H 5.34 J =1.9 Hz, 2 H,
CH
2 4.78 (dd, J= 10.7, 1.6 Hz, 1 H, H 4.64 (dd, J= 10.7, 8.8 Hz, 1 H, H 4.07- 4.17 5H, H1, CH 2
CIOCH
3 .95 3 H, OCH 3 3.94 3H, OCH 3 3.91 3 H,
OCH
3 3.45 10.9 Hz, 1 H, CH 2 CI); 3 C NMR 8 160.3, 157.2, 154.0, 150.2, 148.6, 141.6, 140.6, 138.9, 133.9, 132.0, 129.7, 129.6, 128.8, 127.4, 127.2, 125.6, 125.4, 125.0, 123.6, 123.1, 123.0, 121.8, 122.4, 115.7, 106.5, 105.1, 97.6, 61.8, 61.5, 61.1, 56.2, 56.0, 54.9, 45.8, 43.4; MIS (FAB') m/z 675 (MH 4 677 659 639 517 234 HRMS (FAB) caic. for C 35 H 32 3 'C1N 4 0 9 (MWf) mlz 675.1858, found 674.1832; calc for C 35
H
32 VC1N 4 0 9 m/z 677.1828, found 677.1834; Anal. (C34H 3 1 C1N 4
O
9
.H
2 O) C, H,
N.
Example 2D. Preparation of 2-methoxy-4-nitrobenzyl 4-(1,3-benzothiazol-2yl)phenylcarbamate Pyridine (36 mL, 0.44 mmol) was added dropwise to a stirred solution of alcohol of 2-methoxy-4-nitrobenzyl alcohol (29) (81 mg, 0.44 inmol) and triphosgene (66 mg, 0.22 mng) in DCM (10 mL) and the mixture was stirred at 20*C for min. A solution of 2-(4-aminophenyl)benzthiazole Shi, T. D. Bradshaw, S.
Wrigley, C. J. McCall, P. Lelieveld, I. Fitchner, M. F. G. Stevens. J Med Chem., 1996, 39, 3375] (100 ing, 0.44 iniol) in DCM (5 mL) and the mixture stirred at 20 OC for 4 h. The mixture was partitioned between EtOAc (100 mL) and sat. aq. KHCO 3 solution (50 mL), the organic fraction dried and the solvent evaporated. The residue was slurried in warmn EtOAcIMeOH 1, 20 mL), filtered and the solvent evaporated to give 36 (123 ing, 64%) as a pale green powder mp (EtOH) 213-2 14 C; 'H NMR [(CD 3 2 S0] 8 10.31 1 H, OCONH), 8.11 J= 7.8 Hz, I H, H 8.01-8.06 (in, 3 H, H 7, H H 7.92 (dd, J 8.3, 2.2 Hz, 1 H, H 7.81 J= 2.2 Hz, 1 H, H 7.65-7.69 (in, 3 H, H H 5' H BNSDOCID: <WO 00ORRA1I I WO 00/64864 WO 0064864PCT/GBOO/01 612 -46 1-7.5 5 (in, 1 H, H 7.40-7.46 (mn, 1 H, H 5.27 2 H, CH 2 O) 3.9 8 3 H,
OCH
3 3 C NMR [(CD 3 2 S01 8 166.9, 157.1, 153.6, 152.9, 148.3, 141.8, 134.2, 132.0, 129.0 128.1, 127.0, 126.5, 125.1, 122.4, 122.1, 118.3 115.5, 105.5, 60.7, 56.2; Anal. (CnH 7
N
3
O
5 S) C, H, N.
Example 2E. Preparation of 2-methoxy-4-nitrobelzyl doxorubicin carbamate (38).
2-Methoxy-4-nitrobelzyl 4-nitrophenyl carbonate A solution of 4-nitrophenyl chloroformate (1.00 g, 4.97 inmol) in. pyridine (4 inL) was added dropwise to a stirred solution of 2-inethoxy-4-nitrobelzyl alcohol (29) (617 mg, 3.31 mmol) in pyridine (15 niL at 20 *C and the solution stirred for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (20-50%) EtOAc/light petroleum, to give 37 (928 mg, 80%) as pale yellow solid, mp (EtOAc/light petroleum) 105-106 'H NMvR 8 8.28 (ddd, J 3.1, 2.1 Hz, 2 H, H 7.89 (dd, J 2.1 Hz, 1 H, H 7.77 J= 2.1 Hz, 1 H, H 7.58 J= 8.3 Hz, I H, H 7.40 (ddd, J 3.1, 2.1 Hz, 2 H, H 5.41 2 H, CH 2 4.00 3 H, OCH 3 3 C NMR 8 157.6 (C 155.4 (0C0 2 152.3 (C 149.2 (C 145.5 (C 129.8 (C 129.3 (C 125.3 (C 121.7 (C 115.8 (C 105.5 (C 65.3 (CH 2 56.2 (OCH 3 Anal. (C,,H 12
N
2 C, H, N.
2-Methoxy-4-nitrobelzyl doxorubicin carbamate A solution of carbonate 37 (23 ing, 66 mmol) in DMff (2 mL) was added to a solution of doxorubicin (13) (30 mg, mmol) and Et 3 N (9 mL 66 inmol) in DMff (5 niL) at 20 *C and the solution stirred for 16 h.
The solvent was evaporated and the residue purified by chromatography, eluting with a gradient of MeOHIDCM, to give 38 (37 mng, 88%) as a red solid, mp (DCM) 159- 161 'H NMR 8 13.97 I H, 6-OH), 13.22 1 H, 11I-OH), 8.02 (dd, J 1.0 Hz, 1 H, H 7.77-7.81 (mn,2 H, H2, H 5"),7.66 (br s, 1 H, 7.41 J =8.0OHz, 1 H, H 7.39 (dd, J 1.0Hz, 1 H, H3), 5.52 (br d, J =3.3 Hz, I H, H 1V), 5.29 (br s, I H, H 5.25 J =8.7 'Hz, I H, OCONH), 5.13 (2 d, J =14.0 Hz, 2 H, CH 2 4.75 2 H, H 14), 4.51 1 H, 9-OH), 4.13-4.17 (in, 1 H, H 4.08 3 H, 4-OCH 3 3.90 3 H, 2" -OCH), 3.84-3.88 (in, I H, H 3.69 1 H, H 3.24 (dd, J= 18.9,1.3 Hz, 1 H, H 10), 3.03 I H, 14-OH), 3.01 J =18.9 Hz, 1 H, H 10), 2.34 (br d,J 14.7 Hz, 1 H, H 2.18 (dd, J =14.7, 4.0 Hz, 1 H, H 2.02 (br s, 1 H, 1.90 (dd, J= 13.2, 4.7 Hz, 1 H, H 1.79 (dd, J =13.2, 3.3 Hz, 1 H, H 1.30 J =6.5 Hz, 3 H, H 1 3 C NMR QKIQnrw,.Ir%. AAAn rfi"AdVAAAI I WO 00/64864 WO 0064864PCTGBOOIOI 612 -47- 8213.7(C 13), 187.1 (C 186.7 (C 12), 161.0(C 157.0(C 156.1 (C 155.6(C 11), 155.2 (OCONH), 148.5 (C 135.8 (C 135.5 (Cl2a), 133.5 (C 6a), 133.4 (C 132.5 (C 128.4 (C 6'j, 120.8 (C 4a), 119.9 (C 118.5 (C 115.7 (C 111.6 (C 111.4 (C 11 la), 105.1 (C 100.7 (C 76.6 (C 69.8 (C 69.6 (C 67.2 (C 5f), 65.5 (C 14), 61.1 (CH 2 56.7 (4-OCH 3 56.0 (2'-OCH 3 47.1 (C 35.6 (C 34.0 (C 10), 30.2 (C 16.8 (C MS (FAB") m/z 753 HRMS (FAB-) caic. for
C
36
H
37
N
2 0 16 m/z 753.2143, found 753.2100; Anal (C 36
H
36
N
2 0 1 6 C, H, N.
Example 2F. Preparation of 4-([[(2-methoxy-4nlfrobenzyl)oxyJcarbonyl)amilo)belzyl doxorubicin carbamate (42).
2-Methoxy-4-flitrobelzyl 4-({jtert-butyl(dimethyl)sil] loxy)methyl)phenylcarbamate Et 3 N (0.40 mL, 2.84 mmol) was added to a stirred suspension of carbonate 37 (0.90 g, 2.58 minol), {[tert-butyl(dimethyl)silyll oxy}methyl)aniline (0.64 g, 2.71 mnmol), HOJ3T (0.35 g, 2.58 mmol), and 4 A molecular sieves (900 mg) in THE (80 mnL) and the mixture stirred at 20 *C for 16 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The organic fraction was washed with 1 M HCl (2 x 40 mL), water (100 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 39 (0.89 g, 77%) as a white solid, mp (EtOAc/light petroleum) 120-122 'H NMR 8 7.84 (dd, J= 8.3, 2.1 Hz, I H, H 7.72 2.1 Hz, 1 H, H 7.51 J =8.3 Hz, 1 H, H 7.3 5 J= 8.3 Hz, 2 H, H 2, H 7.26 J =8.3 Hz, 2 H, H 3, H 6.76 (br s, 1 H, OCONH), 5.30 2 H, CH 2 4.69 2 H, CH 2 OSi), 3.93 3 H, OCH 3 0.92 9 H, SiC(CH 3 3 0.09 6 H, Si(CH 3 2 1 3 C NMR 8 157.3 (C 153.0 (OCONH), 148.7 (C 137.0 (C 136.4 (C 132.1 (C 128.7 (C 126.9 (C 3, C 118.6 (C 2, C 6), 115.7 (C 105.2 (C 64.6 (CH 2 61.4 (CH 2 56.0 (OCH 3 26.9 (SiC(-CH 3 3 18.4 (SiK(CH 3 3 -5.2 (Si(CH 3 2 Anal. (C22H 30
N
2 0 6 Si) C, H, N.
2-Metlioxy-4-nitrobenzyl 4-(hydroxymethyI)phenyIcarbamfate 1 M HCI (4 mL, 4 mmol) was added to a stirred solution of silyl ether 39 (0.89 g, 0.2 mmol) in MeOH mL) and stirred at 20 *C for 1 h. The solution was poured into brine (50 mE) and extracted with EtOAc (3 x 50 mE). The combined organic fraction was washed with water (50 ML), dried, and the solvent evaporated. The residue was purified by chromatography, eluting OklOflflt'Sfl. Utfl flflCAOOA*4 I WO 00/64864 WO 0064864PCT/GBOO/01 612 -48with a gradient (20-50%) EtOAc/light petroleum, to give 40 (628 mg, 95%) as a white solid, mp (EtOAc/light petroleum) 164-165 'H NMR [(CD 3 2 S0] 8 9.83 (br s, 1 H, OCONH), 7.90 (dd, J 2.1 Hz, I H, H 7.80 J =2.1 Hz, 1 H, H 7.63 J 8.3 Hz, 1 H, H 7.41 J 8.4 Hz, 2 H, H 2, H 7.22 J =8.4 Hz, 2 H, H 3, H 5.21 2 H, CH 2 5.07 J= 5.6 Hz, 1 H, OH), 4.41 J =5.6 Hz, 2 H, CH 2 3.97 (s, 3 H, OCH 3 1 3 C NMR [(CD 3 2 S0] 8 157.0 (C 153.0 (OCONH), 148.2 (C 137.4 (C 136.7 (C 132.3 (C 128.8 (C 6f), 127.0 (C 3, C 117.9 (C 2, C 115.5 (C 105.4 (C 62.5 (CH 2 60.4 (CH 2 56.0 (OCH 3 Anal. (C, 6
H,
6
N
2 0 6 C, H, N.
4-({I(2-Methoxy4-fitrobelzY)oxyi carbonyllano)belzyI 4-nitrophenyl carbonate A solution of 4-nitrophenylchloroformate (205 mg, 1.02 mmol) in THF (5 mL) was added dropwise to a stirred solution of alcohol 40 (282 mg, 0.85 mmol) and Et 3 N (142 gtL, 1.02 mmol) in THF/DMF 30 mL) the solution stirred at 20 *C for 16 h. The solvent was evaporated and the residue was purified by chromatography, eluting with EtOAcIDCM, to give 41 (238 mg, 56 as a white powder mp (EtOAcJDCM) 144-146 OC; 'H NMvR[(CD 3 2 S0] 8 10.01 1 H, OCONH), 8.31 (ddd, J=9.1, 3.4,2.2 Hz 2 H, H 3, H 7.91 (dd, J= 8.3, 2.2 Hz, 1 H, H 7.81 J =2.2 Hz, 1 H, H 7.64 J= 8.3 Hz, 1 H, H 7.56 (ddd, J= 9.1, 3.4,2.2 Hz, 2 H, H 2, H 7.53 (br d, J= 8.6 Hz, 2 H, H H 7.41 (br d, J 8.6 Hz, 2 H, H H 5.24 4 H, 2 x CH 2 3.98 3 H, OCH 3 3 C NMR [(CD 3 2 S0] 8 157.0 (C 155.2 (0C0 2 153.0 (OCONH), 151.9 (C 148.2 (C 145.1 (C 139.4 (C 132.2 (C 129.6 (C C 128.9 (C 128.5 (C 125.3 (C 2, C 122.6 (C 3, C 118.0 (C C 115.5 (C 105.5 (C 70.2 (CH 2 60.5 (CH 2 56.2 (OCH 3 MS m/z 498
FIRMS
(FAR*) caic. for C-2H 20
N
3
O
10 (M)m/z 498.1149, found 498.115 1. Anal. (C H 19
N
3
O,
0
C,
H, N.
4-({I(2-Methoxy4-nifrobenzyI)oxyI carbonyl~amiflo)beflzyl doxorubicin carbamnate A solution of carbonate 41 (52 mg, 103 ptmol) in DMF (2 m.L) was added dropwise to a stirred solution of doxorubicin (13) (45 mg, 86 Aunol) and Et 3 N (15 4L, 103 Atmol) in DMF (5 mL) at 20 0 C and the solution stirred at 20 *C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient of MeOH/DCM, to give 42 (61 mg, 80%) as a red solid, mp (DCM) 128-131 0 C; 'H NMR BNSDOCIO- <Wa 0064864AI I> WO 00/64864 WO 0064864PCT/GBOO/OI 612 -49
[(CD
3 2 S0] 8 14.01 I H, 6-OH), 13.25 I H, I11-OH), 9.88 1 H, OCONH), 7.87- 7.90 (in, 3 H, H 1, H 2, H 7.79 2.2 Hz, 1 H, H 7.59-7.63 (in, 2 HL H 3, H 7.41 J =8.3 Hz, 2 H, H H 7.22 J =8.3 Hz, 2 H, H H 6.81 J 8.0 Hz, I H, OCONH), 5.44 1 H, H 5.21 J =3.0 Hz, 1 H, H 5.19 2 H,
CH
2 4.91-4.94 (in, 1 H, 9-OH), 4.87 2 H, CH 2 4.83 (dd, J 6.3, 5.9 Hz, 1 H, 14- OH), 4.69 J= 5.7 Hz, 1 H, 4-OH), 4.58 J= 6.0 Hz, 2 H, H 14), 4.12-4.18 (mn, 1 H, H 3.97 3 H, OCH 3 3.95 3 H, OCH 3 3.68-3.75 (in, 1 H, H 3.43-3.47 (in, 1 H, H 4')2.99 J= 18.4 Hz, 1 H, H 10), 2.92 J =18.4 Hz, 1 H, H 10), 2.20 (br d, J= 14.1 Hz, 1 H, H8), 2.12 (dd, J =14.1 Hz, 1 H, H8), 1.85 (dt, J= 12.8, 3.7 Hz, 1 H,H 2%) 1.47 (dd, J= 12.8, 4.1 Hz, 1 H, H 1. 13 J =6.5 Hz, 3 H, H 13 C NMR [(CD 3 2 S0] 8 213.7 (C 13), 186.4 (C 186.3 (C 12), 160.7 (C 157.0 (C 156.0 (C 155.2 (C 11), 154.4 (OCONII), 152.9 (OCONII), 148.2 (C 138.4 (C 136.1 (C 135.4 (C 12a), 134.6 (C 6a), 134.0 (C 10a), 132.2 (C 131.0 (C 128.9 (C 128.6 (C 2", C 119.9 (C 4a), 119.6 (C 118.9 (C 117.9 (C C 115.4 (C 110.7 (C 5a), 110.6 (C I 105.4 (C 100.2 (C 74.9 (C 69.8 (C 67.9 (C 66.6 (C 64.8 (C 14), 63.6 (CH 2 60.4 (CH 2 56.5 (OCH 3 56.2 (OCH 3 47.0 (C 36.5 (C 32.0 (C 10), 29.7 (C 16.9 (C MS (FAB~) m/z 902 Anal.
(C4H 43
N
3
O
1 8-H 2 O) C, H. N, caic 4.6, found 5.6%.
Example 2G. Preparation of 2-methoxy-4-nitrobenzyl 2-f I(9-amino-5-methyl4.
acridinyl)carbonylJ amino) ethyl(methyl)carb amate (46).
2-Methoxy-4-nitrobenzyl methyl{2-[(trifluoroacetyl)amliflolethyl~carbamate
A
solution of 2-inethoxy-4-nitrobenzyl alcohol (29) (183 ing, 1.0 mmol) and DIEA (0.19 mL, 1.2 mmol) in DCM (2 niL) was added dropwise to a solution of triphosgene (104 mng, 0.3 rnmol) in DCM (1.5 mL) over 30 min at The reaction was stirred at 5 *C for I h, then a solution of 2,2,2-trifluoro-N-[2-(methylaniino)ethyl]acetainide trifluoroacetate (282 ing, inuol) and DIEA (0.38 niL, 2.4 mmol) in DCM (2 mL) was added and the solution stirred at 20 'C for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAc/petroleuin ether, to give 43 (371 ing, 94%) as a white solid, mp 107-109 0 C; 'H NMR 57.86 (dd, J=8.4,2.2 Hz, 1H, H5), 7.72 J= 2.2 HzI H, H3), 7.56 (br s, 1 H, CONH), 7.44 J =8.4 Hz, I H, H 5.23 2 H, CH 2 3.95 3 H,
OCH
3 3.58 (br s, 4H, 2 CH 2 3.03 3H, NCH 3 'C NMR 5157.7 (C 157.4 J WO 00/64864 WO 0064864PCT/GBOOIOI 612 -37 Hz, COCF 3 ),157.1 (OCON), 148.6 (C 4)3, 132.2 (C 128.1 (C 115.7 (C 115.7 J 288 Hz, CF 3 105.2 (C 62.3 (CH 2 O0), 56.0 (OCH 3 47.9 (CH 2 39.5 (CH 2 N),o 35.0 (NCH 3 Anal. (C, 4
H,
6
F
3
N
3 0 6 C, N.
2-Methoxy-4-nitrobenzyl 2-aminoethyl(methyl)carbamate A solution of carbamnate 43 (948 mg, 2.5 MMol), Cs 2
CO
3 (4.0 go 12.0 mmol) and water (5 mL) in methanol (20 mL) was stirred at 20 'C for 8 h. The pH was adjusted to 10 with 1 M HC1, water (50 mL) was added, and the solution was extracted with DCM (3 50 niL). The combined organic phase was dried and the solvent was evaporated to give 44 (578 mg, 83%) as a colorless oil which was used directly, 'H NMR 8 7.85 (dd, J= 2.0, 8.2 Hz, I H, H 7.72 J= 2.0 Hz, I H, H 7.47 J 8.2 Hz, I H, H 5.23 2 H, CH 2 3.95 3 H, OCHA) 3.3 7-3 (in, 2 H, CHO), 2.99-3.02 (in, 3 H, CH 3 2.87-2.90 (in, 2 H, CHO), NH 2 not observed 3
C
NMR 8 153.0, 148.5, 132.9, 128.3, 127.8, 115.7, 105.1, 61.7, 56.0, 52.2, 40.1, 35.2.
2-Methoxy-4-nitrobenzyl 2-1I(9-chloro-5-methyl-4acridinyl)carbonyl] amino} ethyl(methyl)carbamate A stirred suspension of methyl-9-oxo-9. I0-dihydro-4-acridinecarboxylic acid (16) (5 07 mg, 2.0 mniol) in SOCI 2 mL) containing DMF (2 drops) was heated gently under reflux until homogeneous and then for a further 45 min. The solution was evaporated below 40 and the residue azeotroped with benzene. The residue was dissolved in DCM (30 niL), cooled to 5 *C, DIEA (1 mL, 6 inmol) and 44 added, and the solution stirred at 20 C for 3 0 min. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc/light petroleum, to give 45 (255 mg. 50 as a yellow solid, mp (EtOAc/light petroleum) 60-65'C; 'H NM'lR [(CD 3 2 S0] 8 11.92-11.96 (in, 1 H, 8.91-9.00 (in, 1 H), 8.53-8.61 (mn, I 8.26-8.29 (in, I 7.66-7.75 mn, 2 7.5 1-7.60 (in, 2 7.17-7.30 (mn, 2 5.15 2 H, CH 2 3.89 3 H, OCH 3 3.72-3.75 2 H, CH 2 3.62-3.65 (in, 2 H, CHO), 3.09 3 H, CH 3 2.75 3 H, CH 3 1 3 C NMR [(CD 3 2 S0] 8 165.9, 156.3, 146.6, 145.0, 143.3, 135.9, 135.6,,132.4, 131.6, 128.8, 128.7, 128.4, 128.2,,127.7, 127.5, 126.5, 124.2, 123.8, 123.0, 115.5, 104.3, 61.5, 55.8, 49.0, 38.0,35.2, 18.9.
2-Methoxy-4-nitrobenzyl [(9-amino-5-methyl-4acridinyl)carbonyllamino~ethyl(methyl)carbamate A solution of chloride 45 (100 BNSDOCID. <WO 006644-A1 i> WO 00/64864 WO 0064864PCT/GBOO/O1 612 -51 mg, 0. 17 mmol) in dry phenol (1.2 g, 13 mmol) was heated at 50 OC. Dry ammonia was bubbled through the solution while the temperature was raised from 50 to 120, 0 C. Addition of ammonia was continued for 15 min, then the mixture was cooled and diluted with excess aqueous NaOH. Prolonged cooling gave a solid that was purified by chromatography, eluting with 20%MeOHIDCM, to give 46 (80 mg, 92 as a yellow solid, mp (MeOH/DCM) 245-248 0 C; 1 H NMR [(CD 3 2 S0] 8 12.72-12.74 (in, 1 H, NH), 8.55-8.66 (mn, 2 8.28-8.24 (in, 1 8.10-8.14 (in, 2 7.66-7.09 (in, 4 5.00 2 H, CH 2
O),
3.73 3 H, OCH- 3 .65 (in, 4 H, 2 CH 2 2.95 3 H, CHAN, 2.51 3 H, CHO); 1 3
C
NMR [(CD 3 2 S0] 8 165.9, 155.7, 155.2, 152.2, 147.1, 146.2, 145.4, 134.3, 134.0, 132.2, 130.9, 127.2, 126.8, 126.6, 121.7, 121.1,120.4,114.7,113.0,11l.7, 104.4,60.8,55.7,48.4, 3 6.7, 34.3, 18.5. Anal. (C 27 H I 7
N
5 0 6 C, H, N.
Example 2H. Preparation of 2-methoxy-4-nitrobenzyl bis(3-([(5-methyl-4acridinyl)carbonyl~ainino~propyl)carbainate (51).
2,2,2-Trifiuoro-N-[3-({3-[(trifluoroacetyl)aminolpropyl} amino)propylj acetamide trifluoroacetate Water (1.2 mL, 70 inmol) was added to a stirred solution of N-3aniinopropyl)-1,3-propanediamine (4.0 g, 30.5 inmol) and ethyl trifluoroacetate (15.0 g, 105 minol) in MeCN (60 mL) and the solution heated at reflux. for 3 h. The solution was cooled, the solvent evaporated, and the residue was triturated with DCM (100 int). The suspension was filtered to give 47 (1.20 g, 90%) as white solid, mp (DCM) 175-178 11H NMR [(CD 3 2 S0] 8 9.55 2 H, NH 2 8.45 (br s, 2 H, 2 CONH), 3.24-3.28 (mn, 4 H, 2 CH1 2 2.90-2.94 (in, 4 H, 2 CH 2 1.76-1.84 (mn, 4 H, 2 CH 2 Anal. (C, 2
H
26 F9N 3 0 4
C,
H, N.
2-Methoxy-4-nitrobenzyl 3-I(trifluoroacetyl)aminol propyl(6,6,6-trifluoro-5oxohexyl)carbamate A solution of 2-methoxy-4-nitrobenzyl alcohol (29) (183 mng, minol) and DIEA 19 mL, 1.2 mmol) in DCM (2 mL) was added dropwise to a solution of triphosgene (104 mng, 0.35 inmol) in DCM (1.5 inL) over 30 minutes at 5 *C and the solution stirred for I h. The solution was added dropwise to a suspension of bistrifluoroacetamide 47 (437 mg, 1.0 mrnol) and DIEA (0.38 inL, 2.4 minol) in DCM (2 niL) and the solution stiffed at 20 'C for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAc/light petroleum, to give 48 (373 mg, Rmnr.J wn nlArAAA I WO 00/64864 PCT/GBOO/O1 612 52as a white solid, mp (lEtOAc/light petroleum) 133-135*C; 'H NMR [(CD 3 2 S0] &9.41 2H, 2 CONH), 7.85 (ddJ=8.0, 2.0Hz, 1H, H5), 7.78 J=2.0 Hz,1H, H3), 7.50 J =8.0 Hz, I H, H 5.12 2 H, CH 2 3.95 3 H, OCH 3 3.15-3.35 (in, 8 H, 4
CH
2 1.74 (in, 4 H, 2 CH 2 Anal. (C, 9
H,
2
F
6 N,0 7 C, H, N.
2-Methoxy4-itrobenflzY bis(3-1I(5-methyl-4acridinyl)carbonyllaino) propyl)carbamate A solution of carbamate 48 (107 mg, 0.2 nunol), CS 2
CO
3 (1.0 g, 3.0 mniol) and water (1 mL) in methanol (4 niL) was stirred at 0 C for 8 hrs. The pH was adjusted to 10 with I M HCI, water (5 0 mL) added, and the solution was extracted with DCM (3 50 niL). The combined organic fraction was dried, the solvent was evaporated to give crude 2-inethoxy-4-flitrobenzyl bis(3an-dnopropyl)carbamate 49). 4-(1H-Imidazol- 1-ylcarbonyl)-5-inethylacridine-(50)
A.
Gamage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, J. Med.
Chem., 1999, 42, 2383-2393] (104 mg, 0.36 minol) was added to a solution of the crude carbamate 49 in THF (10 niL) at 5 0 C and the reaction mixture was stirred at 20 0 C for 8 hrs. The solvent was evaporated, and the residue was purified by chromatography on alumina-9O, eluting with 2%MeOH/45%EtOAc/DCM, to give 51 (85 ing, 64%) as a yellow solid, mp (EtOAcIDCM) 88-90 'H NMR 8 11.87 1 H, NH), 11.81 1 H, NH), 8.90 2 8.70 1 8.67 1 8.03 (in, 2 7.78 (in, 2 7.58 (in, 4 7.40 (in, 2 7.28 J =2.0 Hz, I H, H 3 7.05 J 8.4 Hz, I H, H 6.97 (dd, J 8.4, Hz, 1 H, H 5.01 2 HL CH 2 3.71 (in, 7 3.58 (in, 4 2.81 3 H, CH 3 2.70 3 H, CH 3 2.11 (in, 4 3 C NMIR 8 166.1, 156.3, 155.6, 147.8, 146.9 146.7, 145.2, 145.1, 137.8, 135.7 135.3, 135.1 132.3 132.2 130.9, 128.2, 127.9, 126.9 126.5, 126.3, 126.2, 126.1 125.7 125.3 115.1, 104.2, 61.6, 55.6, 45.8, 45. 1, 37.7, 37.1, 29.2, 28.5, 18.9, 18.7; Anal. (C 4 5
H
4 2
N
6 0 7 .V2H 2 0) C, H, N.
Example 21. Preparation of 2-inethoxy-4-flitrobelzyl 2+[5,8-dihydroxy-U2 (methyill(2-inethoxy4-itrobenzy)oxy] carbonyl)amilo)ethyllamliflo}.
9 9 ,lO-dihydro-l-afthracenyl)aminIoethyl(iethyl)carbamate A solution of 1,4diloo58dhdoynhaee9 -in (22) (1.0 g, 3.6 minol) and 2-methoxy-4nitrobenzyl 2-arninoethyl(methyi)carbamate (44) (0.8 g, 2.7 mmnol) in pyridine (20 niL) was stirred at 20 'C for 48 h. The solvent was evaporated and residue was purified by BNSDOCID: <WO ODS4864AI1 i> WO 00/64864 WO 0064864PCTIGBOO/01612 53 column chromatography, eluting with a gradient (50-85%) of EtOAc/DCM, to give: starting material (22) (0.15 g, and: (i i) 2-methoxy-4-nitrobenzyl 2-[(4-fluoro-5 ,8-dihydroxy-9, 1 0-dioxo-9, I 0-dihydro- 1 anthracenyl)amino]ethyl(methyl)carbamfate (52) (0.54 g, 37%) as a purple solid mp (EtOAc/DCM) 122-125 0 C; 'H NMR 8 13.05-13.77 (in, 2 H, 2 x OH), 9.84-10.07 (br d, I H, NH), 6.99-7.83 (in, 7 5.27 2 H, CH 2 3.95 3 H, OCH 3 3.63 (in, 4 3.09 3 H, NCH 3 HRMS (FAB~) caic. for C 26 H22FN 3
O
9 (Mi) m/z 539.1340, found 539.133 1; and: (iii) 53 (120 mng, as a blue solid, 'H NMR 8 13.15-13.36 (in, 2 H, 2 x OH), 10.29- 10.42 (in, 2 H, 2 x NH), 6.97-7.82 (in, 10 5.26 2 H, CH 2 5.12 2 H, CH 2
O),
3.95 3 H, OCH 3 2.78 3 H, OCH 3 3.50 (br, 8 3.05 6 H, 2 x NWHO; HRMS (FAB') caic. for C 38
H
38 N,0 1 4 (Mt) m/z 802.2446, found 802.2446.
Example 2J. Preparation of 2-methoxy-4-nitrobenzyl 2-{[5,8-dihydroxy-4-({2-[(2hydroxyethyl)aminolethyl)amilo)-9,10-dioxo-9,1O-dihydro-l anthracenyl] amino) ethyl(methyl)carbamate A solution of fluoride 52 (0.54 g, 1.0 inmol) and 2-(2-azninoethylamino)ethanol (2.0 g, 19 mmol) in pyridine (20 niL) was stirred at 20 *C for 54 h. The solvent was evaporated and residue was purified by column chromatography, eluting with a gradient (50-85%) of EtOAc/light petroleum followed by MeOH/EtOAc, to give: 2-methoxy4-nitrobenzyl 2- 8,1 1 -dihydroxy-4-(2-hydroxyethyl)-7, 12-dioxo- 1,2,3,4,7,1 2-hexahydronaphtho[2,3-Aquinoxalin-6-yl]amfilo}ethyl(inethyl)carbainate (54) 1 g, 16%) as a blue solid; mp (DCM/light petroleum) 221-224 1 H NUMR 8 13.43- 14.25 (mn, 2 H, 2 x OH), 10.89-11.29 (in, 2 H, 2 x NH), 6.96-7.77 (in, 6 6.20 1 H), 4.80 2 H, CH 2 3.94 3 H, OCH 3 3.65 (in, 12 2.97 3 H, NCH 3 and: (ii) 55 (0.45 g, 72%) as a blue oil, 'H NMR 8 13.26-13.51 (in, 2 H, 2 x OH), 10.44-10.50 (in, 2 H, 2 x NH), 7.02-7.88 (mn, 7 5.28 2 H, CH 2 3.96 3 H, OCH 3 3.39-3.65 (in, 12 3.07 3 H, NCH 3 NH, OH not observed; FIRMS (FAB) calc. for
C
30
H
34
N
5 01 0 (MW) m/z 624.2306, found 624.2297..
Example 3A. Preparation of 1-(4-nitrophenyl)ethyl 4-Ibis(2chloroethyl)aminolphenylcarbamate (59).
BNSDOCID:. 0064864AI I WO 00/64864 PCT/GBOO/01612 -54- 1-(4-Nitrophenyl)ethyl 4-[bis(2-hydroxyethyl)amino]phenylcarbamate Pyridine (320 mL, 3.95 mmol) was added dropwise to a stirred solution of 1-(4-nitrophenyl)ethanol 56 (660 mg, 3.95 mmol) and triphosgene (410 mg, 1.38 mmol) in THF (50 mL) at 5 *C and the suspension stirred at 5 *C for 1 h. A solution of N,N-bis(2-hydroxyethyl)-1,4benzenediamine (57) (prepared by catalytic hydrogenation ofNN-bis-(2-hydroxyethyl)-4nitroaniline (30) (0.85 g, 4.34 mmol) with Pd/C under H z (60 psi) in EtOH) in THF mL) and DMF (20 mL) was added and the mixture stirred at 20 *C for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc to give 58 (1.01 g, 66 as a brown oil, 'H NMR 8 8.19 J= 8.8 Hz, 2 H, H H 7.52 (br d, J =8.8Hz, 2 H, H H 7.16(d,J=9.0 Hz, 2 H, H 2, H 6.69(brs, 1 H,OCONH), 6.62 J= 9.0 Hz, 2 H, H 3, H 5.90 J= 6.6 Hz, 1 H, CHO), 3.76-3.80 4 H, 2 x
CH
2 3.55 (br s, 2 H, 2 x OH), 3.48-3.51 4 H, 2 x CH 2 1.58 J= 6.6 Hz, 3 H,
CH
3 3 C NMR 8 153.1, 149.4, 147.4, 144.9, 127.3, 126.6 123.8 121.4 113.3 71.9, 60.6 55.3 22.5; MS (FAB m/z 390 (MH HRMS (FAB+) calc.
for C 1 9
H
24
N
3 0 6 (MH m/z 390.1665, found 390.1656.
1-(4-nitrophenyl)ethyl 4-[bis(2-chloroethyl)amino]phenylcarbamate Methanesulphonyl chloride (600 pL, 7.7 mmol) was added dropwise to a stirred solution of 58 g, 2.57 mmol) in pyridine (20 mL) at 20 *C and the solution stirred for I h. The solvent was evaporated and the residue partitioned between DCM/water (100 mL). The aqueous fraction was extracted with DCM (2 x 50 mL) and the combined organic fraction washed with brine (50 mL), dried and the solvent evaporated. The residue was dissolved in DMF mL), LiCI (0.65 g, 15.4 mmol) added, and the mixture stirred at 80 *C for 3 h. The solvent was evaporated and the residue partitioned between EtOAc/water (200 mL). The aqueous fraction was extracted with EtOAc (2 x 50 mL). The combined organic fraction was washed with brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 25% EtOAc/light petroleum, to give 59 (1.0 g, 92 as a tan oil, 'H NMR 8 8.20 (ddd, J= 8.9, 2.2,1.9 Hz, 2 H, H H 7.53 (br d, J= 8.9 Hz, 2 H, H H 7.23 (br d, J= 9.0 Hz, 2 H, H 2, H 6.64 (ddd, J= 9.0, 3.4, 2.1 Hz, 2 H, H 3, H 6.61 (br s, 1 H, OCONH), 5.92 J= 6.6 Hz, 1 H, CHO), 3.67-3.71 4 H, 2 x CH 2 3.58-3.62 4 H, 2 x CH 2 CI), 1.59 J= 6.6 Hz, 3 H, CH 3
C
NMR 8 152.9, 149.3, 147.4, 142.9, 128.0, 126.6 123.8 121.4 112.7 71.9, OktCPtrtrI -t A6,J«4A I WO 00164864 PCT/GBOO/01612 53.6 40.4 22.4; MS (FAB') m/z 429 427 425 HRMS (FAB~) cai~c. for C 19
H
2 1 35 C1 2
N
3 0 4 (Ml) m/z 425.0909, found 425.0901; caic. for C 19
H
2 3 5c1 37 C1N 3 0 4 m/z 427.0880, found 427.0882; calc. for C 1 9
H
2 1 37 C1 2
N
3 0 4 (Mtr) m/z 429.0850, found 429.0868.
Example 3B. Preparation of 1-(4-nitrophelyl)ethyl 1-(chloromethyl)-3-I5,6,7trimethoxy- 1H-indoI-2yl)carbonll2,3dihydro.1Hf-benzo lei A solution of of 1-(4-nitrophenyl)ethanol (56) (18 mg, 0.11 mmol) in DCM (2 mL) was added dropwise to a stirred solution of triphosgene (16 mg, 0.054 mmol) and pyridine (9 gsL, 0.11 mmol) in DCM (2 mL) at 20 The mixture was stirred at 20 0 C for 2 h, the solvent evaporated and the residue dissolved in THF (5 ML). A solution of 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (50 mg, 0. 11 mmol) in THF 5 mL) was added and the solution stirred at 20 C for 16 h. The mixture was partitioned between EtOAc (50 mL) and sat. aq. KHCO 3 solution, the organic fraction dried and the solvent evaporated. The residue was purified by chromatography, eluting with EtOAc/light petroleum to give: 60 (23 mg, 32%) as a tan solid mp (EtOAc/light petroleum) 175-178 1H NIR 8 9.49 1 H, indole-NH), 8.88 1 H, OCONH), 8.18 J= 7.6 Hz, 2 H, H H 7.88 J =8.3 Hz, 1 H, H 7.78 J 8.3 Hz, 1 H, H 7.52-7.58 (in, 3 H, H 8, H H 7.45 (dd, J= 7.8, 7.5 Hz, 1 H, H 7.16 (br s, 1 H, H 7.00 J 1.90 Hz, 1 H, H 6.87 1 H, H 6.00 J 6.6 Hz, 1 H, CHO), 4.80 (dd,J =10.7, 1.2 Hz, 1 H, H2), 4.65 (dd, J =10.7, 8.8 Hz, 1 H, H2), 4.11-4.17 1 H, CH 2 CI), 4.08 3 H, OCH 3 3.93-3.97 (in, 4 H, OCH 3
CH
2 CI), 3.91 3 H, OCH 3 3.45 (dt, J =10.7, 3.3 Hz, 1 H, H 1.65 (br d,J =6.6 Hz, 3 H, CH 3 3 CNMRS 160.3, 153.4, 150.2, 149.0, 147.5, 141.6, 140.6, 138.9, 133.8, 130.4, 129.7, 129.6, 127.4, 126.8 125.6, 125.0, 123.9, 123.8 123.6, 123.1, 122.3, 121.7, 106.5, 97.6, 72.6, 61.5, 6 1.1, 6.3, 54.9, 45.8, 43.4, 22.6; MS (FAB4) m/z 659 (MW, 65 8 510 234 HRMS calc. for C 34
H
3 3 C1N 4 0 8 (MW) m/z 659.1909, found 659.1881; calc for
C
34
H
3 23 7 C1N 4 OB (MWH) m/lz 661-1879, found 661.1882; Anal. (C34H 31 ClN 4
O
8 C, H, N: and (ii) starting material (30 mng, Example 3C. Preparation of 1-(2-methoxy-4-nitrophelyl)ethyI 4-[bis(2chloroethyl)amlil phefllcarbafl ate (64).
WO 00/64864 PCT/GB00/01612 -56- 2-Methoxy-4-nitrobenzaldehyde PCC (0.76 g, 3.52 mmol) and 4A molecular sieves g) were added to a stirred solution of 2-methoxy-4-nitrobenzyl alcohol 29 (0.43 g, 2.35 mmol) in DCM (100 mL) and the suspension stirred at 20 *C for 1 h. The suspension was diluted with diethyl ether (150 mL) and the suspension filtered through Celite, washed with diethyl ether (2 x 20 mL). The combined organic fraction was evaporated and the residue purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 61 (0.42 g, 98%) as white crystals, mp (EtOAc/light petroleum) 117-119 'H NMR 8 10.52 1 H, CHO), 7.98 (br d, J= 8.2 Hz, 1 H, H 7.85-7.89 2 H, H 3, H 4.07 3 H,
OCH
3 3 C NMR 8 188.2 (CHO), 161.8 (C 152.2 (C 129.5 (C 128.6 (C 115.6 (C 107.2 (C 56.4 (OCH 3 Anal. (CHNO 4 C, H, N.
1-(2-Methoxy-4-nitrophenyl)ethanol A solution of MeMgBr (3 M in diethyl ether: 3.64 mL, 10.9 mmol) was added dropwise to a stirred solution of 61 (1.80 g, 9.93 mmol) in THF (100 mL) at -78 *C and the solution stirred at -78 *C for 20 min. The solution was quenched with sat. aq NH 4 CI solution (5 mL) and allowed to warm to 20 The solvent was evaporated and the residue purified by chromatography, eluting with 20% EtOAc/light petroleum, to give starting material (0.38 g, 21%) and (ii) 62 (0.88 g, 45%) as a white solid, mp (EtOAc/light petroleum) 63-65 'H NMR 8 7.86 (dd, J= 8.4,2.1 Hz, 1 H, H 7.70 J= 2.1 Hz, 1 H, H 7.58 J= 8.4 Hz, 1 H, H 5.19 (dq, J= 6.4,4.7 Hz, 1 H, CHOCO), 3.96 3 H, OCH 3 2.34 J= 4.7 Hz, 1 H, OH), 1.48 J= 6.4 Hz, 3 H,
CH
3 "C NMR 8 156.4 (C 148.0 (C 141.3 (C 126.3 (C 116.2 (C 105.3 (C 65.5 (CHOCO), 55.9 (OCH 3 23.1 (CH 3 Anal. (CgH,INO 4 C, H, N.
1-(2-Methoxy-4-nitrophenyl)ethyl 4-[bis(2-hydroxyethyl)amino]phenylcarbamate Pyridine (178 zL, 2.20 mmol) was added dropwise to a stirred solution of ethanol 62 (430 mg, 2.20 mmol) and triphosgene (229 mg, 0.77 mmol) in THF (50 mL) at 5 *C and the suspension stirred at 5 "C for 1 h. A solution of N',N-bis(2-hydroxyethyl)-1,4benzenediamine 57 (476 mg, 2.42 mmol) with Pd/C under H 2 (60 psi) in EtOH) in THF mL) and DMF (10 mL) was added and the mixture stirred at 20 *C for 10 days. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc to give 63 (860 mg, 93%) as a tan foam, 'H NMR 8 9.42 (br s, 1 H, OCONH), 7.91 (dd, J= 8.4,2.1 Hz, 1 H, H 7.79 J= 2.1 Hz, 1 H, H 7.60 J= 8.4 Hz, 1 H, H 7.18 J= BNSDOCID: V4 0064864A1 I> WO 00/64864 PCT/GBOO/0 1612 57 Hz, 2 H, H H 6.59 J =9.0 Hz, 2 H, H 3',H 6.00 J= 6.5 Hz, I H, CHOCO), 4.69 (br s, 2 H, 2 x OH), 3.98 3 H, OCH 3 3.48-3.53 (in, 4 H, 2 x CH 2
O),
3.32-3.37 (in, 4 H, 2 x CH 2 1.46 J =6.5 Hz, 3 H, CH 3 3 C NMR 8 15 5.7 (C 2), 152.6 (OCONH), 147.4 (C 144.0 (C 138.7 (C 127.3 (C 125.7 (C 120.1 (C C 115.8 (C 111.4 (C C 105.7 (C 66.0 (&HOCO), 59.7 (2 x CH 2
O),
58.2 (OCH 3 53.4 (2 x CH 2 20.8 (CH 3 MS (FAB~) m/z 420
HRMS
(FAB') caic. for C2JH 26
N
3
O
7 m/z 420.1771, found 420.1761.
1-(2-Methoxy-4-nitrophel)ethyl 4-[bis(2-chloroethyl)amilphenylcarbamate (64).
MethanesuipholYl chloride (404 psL, 5.2 inmol) was added dropwise to a stirred solution of 63 (0.73 g, 1.74 mmol) in pyridine (20 mL) at 20 *C and the solution stirred for 1 h. The solvent was evaporated and the residue partitioned between DCMlwater (100 ML). The aqueous fraction was extracted with DCM (2 x 50 mL) and the combined organic fraction washed with brine (50 mL), dried and the solvent evaporated. The residue was dissolved in DMFT (20 mL), LiCI (0.44 g, 10.4 mmol) added, and the mixture stirred at 80 'C for 3 h.
The solvent was evaporated and the residue partitioned between EtOAc/water (200 mL).
The aqueous fraction was extracted with EtOAc (2 x 50 xnL). The combined organic fraction was washed with brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 64 (0.53 g, 67 as atan foami, 'H NMR 87.84 (dd,J 1.9 Hz, 1 H, H 7.71 J= 1.9 Hz, 1 H, H 7.50 J= 8.4 Hz, 1 H, H 7.24 (br d,J =9.0 Hz, 2 H, H H (d,J Hz, 2 H, H H 6.62 (br s, 1 H, OCONH), 6.19 J= 6.5 Hz, 1 H, CHOCO), 3.95 3 H, OCH 3 3.69 (dd, J 6.4 Hz, 4 H, 2 x CH 2 3.60 (dd, J 6.4 Hz, 4 H, 2 x CH 2 CI, 1.52 J =6.5 Hz, 3 H, CH 3 1 3 C NMR a 156.0 (C 152.8 (OCONH), 148.1 (C 142.9 (C 138.5 (C 1V), 128.3 (C 125.8 (C 121.3 (C C 116.0 (C 112.8 (C C 105.5 (C 67.6 (CHOCO), 56.0 (OCH 3 53.7 (2 x CH 2 40.5 (2 x
CH
2 CI), 21.2 (CH 3 MS m/z 459 457 12), 455 16), 276 231 (100); HRMS calc. for C 20 Hi 3 3C1 2
N
3
O
5 m/z 455.1015, found 455.1017; cale. for C20H2 3 3 5 C1 3 1CN 3
O
5 m/z 457.0985, found 457.0990; caic. for C2OH 23 37 C1 2
N
3
O
5
M/Z
459.0956, found 459.0972.
Example 4A. Preparation of 2-(2-hydroxyethoxy)-4-litrobelzyI 1-(chloromethyl)-3- WO 00/64864 PCT/GBOO/O1 612 58 [(,,-rmtoyl-no--lcabnl-,-iyr Hbnoein ylcarbamnate 2-Bromoethyl tert-butyl(dimethyl)silyI ether TBDMS triflate (5.0 mL, 21.8 inmol) was added dropwise to a stirred solution of bromoethanol (1.40 mL, 19.8 mmol) and pyridine (2.4 mL, 29.7 minol) in DCM (50 mL) at 5 TC and the solution stirred at 20 0 C for 16 h. MeOH (5 mL) was carefully added, the solution stirred for 5 min and poured into sat.
aq. KHCO 3 (150 mL). The mixture was extracted with DCM (3 x 80 mL), the combined organic fraction dried, and the solvent evaporated. Chromatography of the residue, eluting with 10% EtOAc/light petroleum, gave 65 (3.95 g, 83%) as a colourless oil, 'H NMR 8 3.88 J= 6.6 Hz, 2 H, CH 2 3.40 J= 6.6 Hz, 2 H, CH 2 Br), 0.90 9 H, SiC(CH 3 3 0.08 6 H, Si(CH 3 2 1 3 C NMR 8 63.5 (CH 2 33.3 (CH.Br), 25.8 (SiC(0H 3 3 )I 18.3 (SiC(CH 3 3 -5.3 (Si(CH 3 2 MS (CI, NH 3 m/z 241 239 225 223 183 181 139 (100); HRMS (CI, NH 3 calc for CsH, 0 79 BrOSi ?n/z 239.0467; found 239.0460; calc for CgH 20 BrOSi (MH) m/z 241.0446; found 241.0450.
Methyl 2-(2-{[terl-butyl(dimethyl)silyIJoxy)ethoxy)4fitrobenzoate A mixture of methyl 2-hydroxy-4-nitrobenzoate (66) (0.55 g, 2.79 minol) and K 2 C0 3 (0.58 g, 4.19 mmol) in DMF (15 mL) was stirred at 20 for 30 min. A solution of 65 (1.00 g, 4.19 mmol) in DMff (5 mL) was added and the mixture was stirred at 100 for 4 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mnL) and water (100 niL). The organic fraction was washed with water (2 x 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatographyeluting with EtOAc/light petroleum, to give 67 (0.76 g, 77%) as a white solid, mp (EtOAc/light petroleum) 47-48 'H NMR 867.89 J= 1.3 Hz, 1 H, H 7.81-7.85 (mn, 2 H, H 5, H 4.24 J =5.0 Hz, 2 H, CH 2 4.03 J =5.0 Hz, 2 H, CH 2 3.92 3 H, OCH 3 0.88 9 H, SiC(CH 3 3 0.08 6 H, Si(CH 3 2 1 3 C NM (CDCI 3 8 165.5 (C0 2 158.6 (C 150.4 (C 131.8 (C 126.7 (C 115.0 (C 108.3 (C 7 1.1 (CH 2 61.7
(CH
2 52.5 (OCH 3 25.7 (SiC(&H 3 3 18.3 (SiC(CH 3 3 -5.5 (Si(CH 3 2 Anal.
(CI
6 H2SNO 6 Si) C, H, N.
12(-fetBtldmty~illoy toy4ntohnl methanol DLBALH (I M in DOM, 6.7 mL, 6.7 inmol) was added dropwise to a stirred solution of ester 67 (0.72 BNSDOCID: <WO 0064864AI I> WO 00/64864 PC'r/GBOO/01612 59g, 2.02 minol) in THF (50 mL) at 5 0 C and the solution stirred at 5 'C for 1 h. The solution was poured into a solution of potassiumi sodium tartrate (1 M, 50 mL) and stirred vigorously for 30 min. The mixture was extracted with EtOAc (3 x 50 mL), the combined organic fraction washed with water (50 mL), brine (50 mL), dried and the solvent was evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 68 (0.64 g, 97%) as a white solid, mp (EtOAc/light petroleum) 89-90 OC; 'H NMR 8 7.85 (dd, J= 8.2, 2.1 Hz, 1 H, H 7.74 J= 2.1 Hz, I H, H 7.46 J 8.2 Hz, I H, H 4.75 2 H, CH 2 4.21 (dd, J 4.4 Hz, 2 H, CH 2 4.01 dd, J 4.9, 4.4 Hz, 2 H, CH 2 2.84 (br s, I H, OH), 0.90 9 H, SiC(CH 3 3 0.06 6 H, Si(CH 3 2 3 C NM4R 8 157.0 (C 148.3 (C 137.1 (C 128.4 (C 116.3 (C 106.8 (C 70.5 (CH 2 61.6 (CH 2 61.3 (CH 2 25.8 (SiC(CHO) 3 18.3 (SiC(CH 3 3 -5.4 (Si(CH 3 2 Anal. (C 15
H
25
NO
5 Si) C, H, N.
2-21tr-uy~iehlslloyehx)-irbny 1-(chloromethyD-3+I5,6,7trimethoxy-lH-ifldol2-yl)carbonyl2dihydro1H-benzo[elindol.S-ylcarbamate A solution of triphosgene (24 mg, 80 gmol) in DCM (2 mL) was added dropwise to a stirred solution of amine I [G.1J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem.
Lett., 1997, 7, 1483] (107 mg, 230 jimol) and Et 3 N (64 460 p.mol) in DCM (20 mL) and stirred at 20 *C for 2 h. A solution of {[tert-butyl(dimethyl)silyl]oxy} ethoxy)4 nitrophenyllmethanol (68) (83 mg, 253 Mmol) in DCM (5 mL) was added, followed by nBu.Sn(OAc)2 (2 drops) and the solution stirred at 20 C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (0-20%) EtOAcIDCM, to give 69 (177 mg, 94%) as a white solid, mp (EtOAc/light petroleum) 182- 185 0 C; 'H NMR 8 9.45 1 H, indole-NH), 8.93 1 H, OCONH), 7.92 J =8.5 Hz, 1 H, H 7.76-7.83 (in, 3 H, H 9, H H 7.53-7.60 (in, 2 H, H 8, H 7.47 (ddd, J= 7.1, 0.8 Hz, I H, H 7.13 (br s, 1 H, H 7.01 J 2.2 Hz, 1 H, H 6.88 1 H, H 41),5.39 2 H, CH 2 4.81 (dd, J =10.7, 1.8 Hz, I H, H 4.67 (dd, J =10.7, 8.7 Hz, 1 H, H 4.21 (br dd, J= 5.0,4.8 Hz, 2 H, CH 2 4.15-4.18 (in, 1 H, H 4.09 3 H, OCH 3 4.02 (br d, J= 5.0,4.8 Hz, 2 H, CH 2 3.97 J =11.5, 3.1 Hz, I H, CH 2 Cl), 3.95 3 H, OCH 3 3.92 3 H, 0C11 3 3.48 (dd, J 11.5, 10.9 Hz, 1 H, CH 2 Cl, 0.90 (s, 9 H, SiC(CH 3 0. 10 6 H, Si(CH 3 2 1 3 C NMR 8 160.3 156.7 (C 154.0 (OCONH), 150.2 (C 148.5 (C 141.7 (C 3a), 140.6 (C 138.9 (C 133.8 (C WO 00/64864 WO 0064864PCT/GBOO/01612 60 132.3 (C 129.7 (C 129.6 (C 9a), 128.7 (C 127.5 (C 125.6 (C 125.0 (C 123.6 (C 123. 1 (C 122.4 (C6, C9b), 121.8 (C 5a), 115.8 (C 112.8 (C 106.5 (C C 97.7 (C 70.7 (CH 2 61.8 (CH 2 61.7 (CH 2 61.5 (OCH 3 6 1.1 (OCH 3 56.3 (OCH 3 54.9 (C 45.8 (CHPC), 43.5 (C 25.8 (SiC(CH 3 3 18.3 (SiC(CH 3 3 -5.4 (Si(CH 3 2 MS (FAB~) in/z 819 821 (NMW, 12); HRMS (FAB') caicci for C 41
H
48 3 'C1N 4 OSi (MWH) m/z 819.2828, found 819.2804; caic. for
C
41
H
48 37 C1N 4 OSi m/z 821.2799, found 821.2803; Anal. (C 4 jH 47 C1N 4 OSi) C, H, N.
2-(2-Hydroxyethoxy)-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-2yI)carbonylj-2,3-dihydro-1H-benzoie~indol-5-ylcarbamate 1 M HCl (0.4 mL, 400 /2mol) was added to a stirred solution of silyl ether 69 (157 mg, 192 AsmoI) in MeOH mL) and the solution stirred at 20 *C for 1 h. The solvent was evaporated and the residue partitoned between EtOAc (50 niL) and water (50 mL). The organic fraction was washed with water (50 niL), brine (25 niL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (20-50%) of EtOAc/light petroleum, to give 70 (119 mig, 88%) as a hygroscopic white solid, 'H NMvR 8 9.72 1 H, indole-NH), 8.80 1 H, OCONH), 7.86 (di, J= 8.5 Hz, 1 H, H 7.79 (br d, J= 8.1 Hz, 1 H, H 7.67-7.73 (in, 2 H, H 9, H 7.47-7.53 (in, 3 H, H 4, H 8, H 7.37 (ddd, J 8.5, 7. 1, 0.8 Hz, 1 H, H 6.97 J 2.2 Hz, 1 H, H 6.87 1 H, H 4D), 5.40 2 H, CH 2
O),
4.73 (dd, J= 10.7, 1.6 Hiz, 1 H, H 4.59 (dci, J= 10.7, 8.7 Hz, 1 H, H 4.21. (br dcl, J= 4.6,4.0 Hz, 2 H, CH 2 4.07-4.11 (in, 4 H, H 1, OCH 3 ),4.00-4.04 (in, 2 H, CH 2 3.95 3 H, OCH 3 3.92 3 H, OCH 3 3.85 (ci, J= 11.3, 3.0 Hz, I H, CH.C1), 3.39 (br s, 1IH OH), 3.28 J= 11.3, 10.9 Hz, 1 H, CH 2 CI); 1 3 CNMR 8 160.5 157.2 (C 154.4 (OCONH), 150.2 (C 148.9 (C 141.4 (C 3a), 140.6 (C 6f), 138.9 (C 133.8 (C 131.9 (C 130.3 (C 129.6 (C C 9a), 127.4 (C 125.8 (C 125.0 (C 7), 123.6 (C 123.0 (C 122.5 (C 6, C 9b), 121.9 (C 5a), 115.9 (C 1 12.8 (C 4), 106.7 (C 106.6 (C 97.7 (C 70.7 (CH 2 62.0 (CH 2 61.5 (OCH 3 61.1 (OCHA) 60.9 (CH 2 56.3 (OCHA) 55.1 (C 45.6 (CH 2 C1), 43.3 (C MS (FAB~) m/z 707 705 WMH, 14); 1-fRMS (FAB~) caicci for C 35
H
34 35 1 4 1 (MW) mlz 705.1964, found 705.1919; caic. for C 3
,H
34 37
CIN
4 O1 0 m/z 707.1934, found 707.1931; Anal. (C 35
H
33
CIN
4 0 10 C, H, N.
BNSDOCID: <WO 006464A i i WO 00/64864 PCT/GBOO/01612 -61- Example 4B. Preparation of 2-(2-niethoxyethoxy)-4-nitrobenzyl 1-(chloromethyl)-3- 1(,,-rmtoylHidl2y [bnyl23dhdol- eJ ylcarbamnate (73).
Methyl 2-(2-methoxyethoxy)-4-nitrobenzoate A mixture of methyl 2-hydroxy-4nitrobenzoate (66) (1.0 g, 5.07 mmol) and K 2 C0 3 (1.05 g, 7.61 mmol) in DMF (25 mL) was stirred at 20 00 for 30 min. A solution of 2-bromoethyl methyl ether (0.72 mL, 7.61 mmol) in DMF (3 mL) was added and the mixture was stiffed at 100 *00 for 4 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The organic fraction was washed with water (2 x 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography,eluting with 30% EtOAc/light petroleum, to give 71 (1.27 g, 98%) as a white solid, mp (EtOAc/light petroleum) 45-46 OC; 'H NMR 8 7.90 J =8.3 Hz, I H, H 7.82-7.86 (in, 2 H, H 3, H 4.28-4.30 (in, 2 H, 01120), 3.93 3 H, O003)3.82-3.87 (in, 2 H, CH 2 3.48 3 H, OCH 3 13C NMR 8 165.1 (002), 15 8.6 (C 150.6 (C 132.1 (C 126.4 (C 115.2 (C 108.4 (C 3), 70.5 (01120), 69.4 (CH 2 59.4 (OCH- 3 52.5 (OCH 3 Anal. 3 N0 6 C, H, N.
2-I2-(Methoxy)ethoxyJ-4-nitrobenzyl alcohol DIBALH (1 M in DCM, 16.4 mL, 16.4 inmol) was added dropwise to a stirred solution of ester 71 (1.27 g, 4.97 nimol) in THfF (100 mL) at 5 00 and the solution stirred at 5 00C for 1 h. The solution was poured into a solution of potassium sodium tartrate (I M, 100 mnL) and stirred vigorously for 30 min.
The mixture was extracted with EtOAc (2 x 100 inL), the combined organic fraction washed with water (50 mL), brine (50 mL), dried and the solvent was evaporated. The residue was purified by chromatography, eluting with a gradient (30-50%) of EtOAc/light petroleum, to give 72 (1.03 g, 91%) as a white solid, mp (EtOAc/light petroleum) 89-90.5 00; 'H NMR 8 7.86 (dd, J= 8.2, 2.1 Hz, 1 H, H 7.72 J= 2.1 Hz, 111,11 7.47 J 8.2 Hz, I H, H 4.74 (br s, 2 H, CH 2 4.26-4.29 (in, 2 H, CH 2 3.78-3.80 (in, 2 H,
CH
2 3.45 3 H, OCH 3 3. 10 (br s, 1 H, OH); 13C NMR 8 156.8 (C 148.2 (0 4), 137.5 (C 128.6 (C 116.5 (C 107.0 (C 70.5 (CH 2 68.4 (CH 2 61.1 (CH 2 59.1 (00113); Anal. (C, 0
H
13 N0 5 C, H, N.
2-(2-Methoxyethoxy)-4-nitrobenzylI -(chloromethyl)-3-(5,6,7-trimethoxy-1H-ifldol-2- QWd~flfI.Ir- %A tdMAd"A I, PCTGBOOIOI 612 WO 00/64864 62 y1)carbonyl1-2,3-dihydro-lH-befl~ellfldol-5-ylcarbamate A solution of triphosgene (12 mg, 40 psmol) in DGM (2 mL) was added dropwise to a stirred solution of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (53 mg, 114 g.mol) and Et 3 N (32 ML, 228 g2mol) in DGM (10 mL) and stirred at 0 G for 2 h. A solution of 2-[2-(methoxy)ethoxy]-4-nitrobenzy1 alcohol 72 (28 mg, 125 /2smol) in DCM (2 mL) was added, followed by TiBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 0 G for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 20% EtOAcIDGM, to give 73 (75 mg, 9 as a tan gum, 'H NMR 869.49 1. H, indole-NH), 8.19 1 H, OGONH), 7.92 J 8.5 Hz, 1 H, H 6), 7.80-7.82 (in, I H, H 7.78 J= 8.3 Hz, 1 H, H 7.71 J= 1.8 Hz, 1 H, H 7.56 (ddd, J= 8.3, 7.1, 0.8 Hz, 1 H, H 7.49-7.54 (in, 1 H, H 7.45 (ddd, J= 8.5, 7.1, 0.8 Hz, 1 H, H 7.27 (br s, I H, H 7.00 J= 2.3 Hz, 1 H, H 6.87 I H, H 4%) 5.39 2 H, CH 2 4.79 (dd, J= 10.7, 1.7 Hz, 1 H, H 4.66 (dd, J= 10.7, 8.7 Hz, 1 H, H 4.23 (dd, J= 4.6,4.4 Hz, 2 H, CH 2 4.15-4.20 (mn, 1 H, H 4.08 3 H, OGH 3 3.94-3.98 (in, 4 H, OGH 3 C11 2 G0), 3.91 3 H, 0GB 3 3.80 (dd, J 4.6, 4.4 Hz, 2 H,
CH
2 3.47 J 10.9 Hz, I H, GH 2 C1), 3.44 3 H, 0GB 3 3 C NME. 6 160.3 (GO), 156.5 (C 2"j, 154.0 (OGONH), 150.2 (G 148.5 (C 141.7 (C 3a), 140.6 (G 6%) 138.9 (C 133.9 (G 132.4 (G 129.7 (C 129.6 (C 9a), 128.7 (C 127.5 (C 125.6 (C 125.0 (C 123.6 (C 123.1 (G 122.5 (C 6, G 9b), 121.8 (C 116.0 (C 112.7 (G 106.5 (C 106.3 (C 97.7 (G 70.7 (GH 2 68.5
(GH
2 61.9 (GH 2 61.5 (0GB 3 6 1.1 (0GB 3 59.3 (0GB 3 56.3 (0GB1 3 54.9 (G 2), 45.9 (GH 2 G1), 43.1 (G MIS (FAB~) m/z 721 WMH, 719 WMH, HERMS (FAB') caic. for G 36
H
36 37
GN
4 0 1 0 (MB"r) m/z 721.209 1, found 721.213 1; calc. for
G
36
H
3 3GIN 4
O,
0 (MHW) m/z 719.2120, found 719.2133; Anal. (C 36
H
35 G1N 4 0, 0 C, H, N.
Example 4C. Preparation of 1- [2-(3-hydroxypropoxy)-4-litrophell ethyl 4-[bis(2chloroethyl)aminolphenylcarbamate (78).
Methyl 4-nitro-2-[3-(tetrahydro-2H-pyra--yloxy)propoxylbeflzoate A mixture of methyl 4-nitrosalicylate (66) (2.3 g, 11.7 inmol) and K 2 C0 3 (2.42 g, 17.5 iniol) in DMF (25 mL) was stirred at 20 0 G for 20 min. A solution of 3-iodopropyl tetraliydropyranyl ether (4.7 g, 17.5 nunol) in DMF (5 mL) was added and the mixture stirred at 100 0 G for 2 h. The mixture was poured into water, extracted with EtOAc: (3 x 100 inL, the combined WO 00164864 PCTIGBOO/0I612 63 organic extracts washed with water (2 x50 mL), brine (50 mL), dried and the solvent evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 74 (3.66 g, 92 as a colourless oil, 'H NMR 8 7.89 J 8.5 Hz, I H, H 7.80-7.84 (in, 2 H, H 3, H 4.60-4.62 (in, 1 H, OGHO), 4.27 J 6.2 Hz, 2 H,
CH
2 3.95-4.00 (in, 1 H CH 2 3.94 3 H, OCH 3 3.79-3.86 (mn, 1 H, CH 2 3.59- 3.66 (in, 1 H, CH 2 3.47-3.52 (in, 1 H, CH 2 2.13-2.17 H, CH 2 1.78-1.84 (in, I H, CH 2 1.68-1.75 (in, 1 H, CH 2 1.47-1.62 (in, 4 H, 2 CH 2 1 3 C NMR 8 164.5, 15 8.6, 150.7, 132.0, 126.2, 114.8, 107.9, 99.0, 66.5, 63.4, 62.4, 52.5, 30.6, 29.3, 25.4, 19.6; MS m/z 339 322 239 222(40), 85 (100); HRMS calc. for C, 6
H
21 N0 7 m/z 339.1318, found 339.1317.
4 -Nitro-23(tetrahydro-2H-pyran-2yloxy)propoxyI phenyl) methanol
DIBALH
(1 M in DGM, 34 mL, 34 mmol) was added dropwise to a solution of 74 (3.46 g, 10.2 mmol) in THF (100 mL) at 5 *C and the solution stirred at 5 *C for 1 h. The solution was poured into a solution of sodium potassium tartrate (1 M, 100 inL) and stirred for 30 min.
The mixture was extracted with EtOAc (3 x 100 inL), the combined organic fraction washed with water (100 mQL, brine (50 inL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50%EtOAcllight petroleum, to give 75 (3.11 g, 98 as a pale yellow solid, mp (EtOAc/light petroleum) 64-65.5 'H NMR 8 7.84 (dd, J 2.1 Hz, 1 H, H 7.72 J= 2.1 Hz, 1 H, H 7.5 0 J =8.2 Hz, 1 H, H 4.74 (dd, J 14.8, 4.2 Hz, 2 H, CH 2 4.5 8-4.6 1 (in, I H, OCHO), 4.24 J 6.1 Hz, 2 H, CH 2 3.96 (dt, J 10.0, 5.8 Hz, 1 H, CH 2 3.80-3.86 (mn, 1 H, CH 2 3.62 (dt, 10.0, 5.8 Hz, 1 H, CH 2 3.46-3.5 1 (in, 1 H, CH 2 2.3 0 (br s, I H, OH), 2.08-2.11 (in, 2 H,
CH
2 1.79-1.85 (in, 1 H, CH 2 1.69-1.77 (in, 1 H, CH 2 1.48-1.62 (in, 4 H, 2 x CHO); 1 3
C
NMRS 156.5, 148.2, 136.8, 128.1, 115.9, 105.8, 99.3, 65.9, 63.9, 62.8, 60.8, 30.6, 29.3, 25.3, 19.7; MIS (CI, NH 3 m/lz 312 (MW, 294 245 227(30), 85 (100); HRMS (CI, NH 3 caic. for C, 5 H22NO 6 (MW)i m/z 312.1447, found 312.1438. Anal.
(C
15
H
21 N0 6 C, H, N.
4-ir--3(erhdo2-prn2yoypooybny 4-Ibis(2hydroxyetbyl)aminolpheflelarbamate Pyridine (135 AZL, 1.67 innol) was added dropwise to a stirred solution of alcohol 75 (521 ing, 1.67 minol) and triphosgene (174 mg, WO 00/64864 PCT/GB00/01612 -64- 0.59 mmol) in THF (20 mL) at 5 °C and the suspension stirred at 5 *C for 1 h. A solution of N,N-bis(2-hydroxyethyl)-1,4-benzenediamine 57 [prepared by catalytic hydrogenation of NN-bis-(2-hydroxyethyl) 4-nitroaniline (360 mg, 1.84 mmol) with Pd/C under H 2 psi) in EtOH] in THF (10 mL) and DMF (10 mL) was added and the mixture stirred at *C for 16 h. The solvent was evaporated and the residue partitioned between EtOAc/water (100 mL). The aqueous fraction was extracted with EtOAc (2 x 50 mL) and the combined organic fraction washed with brine (50 mL), dried and the solvent evaporated. The residue was purified by chromatography, eluting with 80-100% EtOAc/light petroleum to give 76 (220 mg, 25 as a colourless oil, 'H NMR [(CD 3 2 SO] 5 9.42 1 H, OCONH), 7.88 (dd, J= 8.3, 2.0 Hz, 1 H, H 7.79 J= 2.0 Hz, 1 H, H 7.61 (br d, J= 8.3 Hz, 1 H, H 7.72 (br d, J= 9.1 Hz, 2 H, H 2, H 6.61 d, J= 9.1 Hz, 2 H, H 3, H 5.19 2 H, CH0O), 4.73 J= 5.4 Hz, 1 H, CH20), 4.54-4.58 1 H, OCHO), 4.23 J= 6.1 Hz, 2 H, CH20), 3.80 (dt, J= 9.9, 6.4 Hz, 1 H, CH20), 3.67-3.71 1 H, CH20), 3.55 J= 5.4 Hz, 1 H, CH20), 3.50 J= 6.0 Hz, 4 H, 2 x CH20), 3.32-3.35 4 H, 2 x CH 2 1.99- 2.06 2 H, CH 2 1.67-1.73 1 H, CH 2 1.56-1.62 1 H, CH), 1.40-1.49 4 H, 2 x CH 2 C NMR [(CD 3 2 SO] 8 156.2, 153.2, 148.1, 144.1, 132.9, 128.6, 127.4, 120.2 115.4, 111.4 106.0, 98.0, 65.7, 63.0, 61.3, 60.1, 58.1 53.4 30.2, 28.8, 25.0, 19.1; MS (FAB') m/z 533 20 HRMS (FAB') calc. for C 2 6
H
35 N,0 9 (M m/z 533.2373, found 533.2365.
2-(3-Hydroxypropoxy)-4-nitrobenzyl 4-[bis(2-chloroethyl)amino]phenylcarbamate Methanesulphonyl chloride (85 juL, 1.1 mmol) was added dropwise to a stirred solution of 76 (195 mg, 0.36 mmol) in pyridine (10 mL) at 20 °C and the solution stirred for 1 h. The solvent was evaporated and the residue partitioned between DCM/water (100 mL). The aqueous fraction was extracted with DCM (2 x 50 mL) and the combined organic fraction washed with brine (50 mL), dried and the solvent evaporated. The residue was dissolved in DMF (10 mL), LiCI (93 mg, 2.2 mmol) added, and the mixture stirred at 80 °C for 3 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The aqueous fraction was extracted with EtOAc (2 x 50 mL). The combined organic fraction was washed with brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 25% EtOAc/light petroleum, to give 4-nitro-2-[3-(tetrahydro-2H-pyran-2-yloxy)propoxy]benzyl 4-[bis(2- BNSDOCID: <WO 00648641 1> WO 00/64864 WO 0064864PCT/GBOO/01612 chloroethyl)aminolphenylcarbamate (77) (46 mg, 22 as a colourless oil, 'H NMR 8 7.82 (dd, J 8.3, 2.1 Hz, 1 H, H 5 D, 7.73 J 2.1 Hz, 1 H, H 3Y), 7. 5 0 (br d, J 8.3 Hz, 1 H, H 6f), 7.26-7.29 (in, 3 H, OCONH, H 2, H 6.65 (ddd,J 9.1, 3.4, 1.9 Hz, 2 H, H 3, H 5.25 2 H, CH 2 4.67 (br s, 1 H, OCHO), 4.20-4.27 (in, 2 H, CH 2 4.01 (dt, J= 9.7, 6.1 Hz, 1 H, CH 2 3.65-3.75 (in, 6 H, CH 2 O, 2 x CH 2 3.58-3.63 (in, 4 H, 2 x
CH
2 CI), 3.44-3.50 (in, 1 H, CH 2 2.11-2.15 (in, 2 H, CH 2 1.66-1.72 (in, 1 H, CH 2 1.55- 1.62 (in, I H, CH 2 1.40-1.50 (in, 4 H, 2 x CH 2 3 C NMR 8 157.2, 153.6, 148.8, 142.6, 132.0, 129.8, 128.7, 121.3 115.5, 112.8 105.9, 98.4, 65.3, 63.0, 61.7 53.7 40.5 30.5, 29.3,25.4, 19.0; MS (FAB') m/z 569 HRMS (FAB') caic for
C
26
H
33 3 5 C1 2
N
3 0 7 OW~) m/z 569.1696, found 569.1689; caic. for C 26
H
33 3 1C1 37 C1N 3 0 7
(M
4 m/z 571.1666, found 569.1680; caic. for C 2
,H
33 37 C1 2
N
3 0 7 m/z 573.1637, found 569.1654.
Further elution gave 78 (99 mg, 57 as a white powder, mp (DCM/pet. ether) 104-105 OC; 'H 8 7.84 (ddJ= 8.3, 2.0 Hz, 1 H, H 7.74 J= 2.0 Hz, 1 H, H 7.51 (br ciJ= 8.3 Hz, 1 H, H 6f), 7.24 (br d, J =9.0 Hz, 2 H, H 2, H 6.87 (br s, 1 H, OCONH), 6.64 (d, J 9.0 Hz, 2 H, H 3, H 5.27 2 H, CH 2 4.27 J 5.8 Hz, 2 H, CH 2 O0), 3.89 J 5.7 Hz, 2 H, CH 2 3.67-3.72 (in, 4 H, 2 x CH 2 3.58-3.64 (in, 4 H, 2 x CH 2 C1, 2.08- 2.12 (in, 2 H, CH 2 1 3 C NMR 8 156.8, 153.4, 148.7, 145.6, 132.0, 129.4, 128.2, 123.4 115.6, 112.7 106.1, 66.6, 61.5, 59.9, 53.6 40.5 31.6; Anal. (C 2 ,H25C1 2
N
3
O
6
C,
H, N, C1.
Example 4D. Preparation of 2-(3-hydroxypropoxy)-4-nitrobenzyI 3-(chloromethyl) -1- [(5,6,7-trimethoxy-1H-indol-2-y)carbonylj-2,3-dihydro-I-indol-6-ylcarbamate Pyridine (3 5 iiL, 0.44 iniol) was added dropwise to a stirred solution of alcohol 75 mg, 0.25 mmol) and triphosgene (45 mg, 0.15 minol) in THF (10,mL) at 5 0 C and the suspension stirred at 5 C for 1 h. A solution of 3 -(chloroinethyl)-1I-[(5,6,7-trimethoxy-1IHindol-2-yl)carbonyl]-2,3-dibydro-1H-indol-6-ylamine (33) Tercel and W. A. Denny. J Chem. Soc. Perkin Trans. 1, 1998, 509] (199 ing, 0.48 minol) in THF (10 mL) was added and the mixture stirred at 20 *C for 16 h. The suspension was filtered and the solvent evaporated. The residue was purified by chromatography, eluting with 40% EtOAcIDCM, to give 4-nitro-2-13-(tetrahydro-2H-pyran-2-yloxy)propoxyjbenzyI 3-(chloromethyl)- 1- [(5,6,7-trimethoxy- 1H-indol-2-yl)carbonyl]-2,3-dihydro- 1H-indol-6-ylcarbainate 79 (116 BNSOOCID: <VVO 00640.64A1I i WO 00/64864 PCT/GBOO/01612 66mg, 35%) as an oil, 1H NMR 869.54 1 H, NH), 8.24 1 H, H 7.84 1 H, H 7.77 (dd, J 2.0 Hz, 1 H, H 7.71 d, J 2.0 Hz, 1 H, H 7.52 (br s, 1 H, OCONH), 7.48 J= 8.3 Hz, 1 H, 7.22 (d,J =8.3 Hz, I H, H4), 6.93 (d,J =2.2 Hz, 1 H, H 6.85 1 H, H 5.27 2 H, CH 2 4.69 (br s, 1 H, OCHO), 4.62 (dd, J= 10.6, 9.4 Hz, 1 H, H 4.45 (dd, J =10.6, 3.8 Hz, 1 H, H 4.20-4.24 (in, 2 H, CH 2 4.08 3 H,
OCH
3 3.94-3.98 (in, 1 H, CH 2 3.93 3 H, OCH 3 3.91 3 H, OCH 3 3.71-3.82 (in, 3 H, H 3, CH 2 O, CH 2 Cl), 3.49-3.61 (mn, 3 H, CH 2 C1, CH 2 2.10-2.15 (in, 2 H, CH 2 1.65- 1.78 (in, 2 H, CH 2 1.45-1.60 (in, 4 H, 2 x 3 C NMR 8 160.3, 157.2, 153.3, 150.2, 148.8, 144.1, 140.5, 138.9, 138.8, 131.8, 129.6, 129.5, 125.9, 125.5, 124.5, 123.6, 115.5, 114.6, 108.7, 107.9, 105.9, 98.4,97.6, 67.6, 65.4, 63.0, 61.8, 61.4, 61.1, 56.2, 54.7,46.9, 43.2, 30.6, 29.2, 25.4, 19.0; MS (FAB') m/z 752 (Mt, 669 234 HERMS (FAB') caic. for C 37
H
4 35 C1N 4 011 m/z 752.2460, found 752.2455; caic. for
C
37
H
41 37
CN
4 011 m/z 754.243 1, found 754.2424.
Further elution gave 80 (44 mg, 15%) as a tan solid, mp (EtOAc/light petroleum) 166-168 OC; 'H NMR 6 9.47 1 H, indole-NH), 8.24 J= 1.8 Hz, 1 H, H 7.79 (dd, J= 8.3, 2.1 Hz, 1 H,H 7.71 J =2.1 Hz, 1 H, H 7.48 J =8.3 Hz, 1 H, 7.42 (br s, 1 H, OCONH), 7.33 1 H, H 7.21 J =8.2 Hz, 1 H, H 6.93 J 2.3 Hz, I H, H 6.85 1 H, H 5.29 2 H, CH 2 4.62 (dd, J =10.8, 9.8 Hz, 1 H, H 4.46 (dd, J= 10.8, 4.5 Hz, 1 H, H 4.25 J =5.8 Hz, 2 H, CH 2 4.07 3 H, OCH 3 3.94 3 H, OCH 3 3.88-3.93 (mn, 5 H, OCH 3
CH
2 3.79-3.85 (in, 2 H, H 3, CH 2 Cl), 3.55 (dd, J= 12.1, 10.3 Hz, 1 H, CH.Cl), 2.28 (br t, J =4.9 Hz, 1 H, OH), 2.09-2.14 2H,
CL!
2 1 3 CNMR 8 160.5, 156.8, 153.1, 150.1,148.5,144.0,140.5,138.7,138.6,131.7, 13 0.8, 129.5, 129.3, 126.0, 125.8, 124.5, 123.5, 115.5, 114.6, 108.5, 106.7, 106.0, 66.0, 61.7, 61.5, 61.1, 59:4, 5 6.1, 54.7, 46.9, 43.2, 31.6; MS (FAB~) m/lz 671 669 (MW, 391 149 (100); HRMS (FABV) caic. for C 3 2 H34 5 ClN 4
O
0 (MW) m/lz 669.1964, found 669.192 1; caic. for C 3 2 H 34 3 'C1N 4 0, 0 m/z 669.1934, found 671.1875; Anal. (C 32
H
33 C1N 4 01 0
VA
2 2) C, H, N.
Compound 80 was also prepared by treating a solution of 79 (96 mg, 0. 13 inmol) in MeOL! mL) with 0. 1 M HCI (2 inL) and stirring at 20 'C for 16 h. The solvent was evaporated and the residue partitioned between DCM (50 mL) and water (50 inL). The organic fraction WO 00/64864 PCT/GB00/01612 -67was washed with water (10 mL), brine (10 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 40% EtOAc/DCM, to give 80 (69 mg, 79%) as a tan solid, spectroscopically identical with the sample prepared above.
Example 4E. Preparation of 2-(3-hydroxypropoxy)-4-nitrobenzyl 1-(chloromethyl)-3- [(5,6,7-trimethoxy-lH-indol-2-yl)carbonyl]-2,3-dihydro-H-benzo[e]indol-5ylcarbamate (84).
Methyl 2-(3-{[tert-butyl(dimethyl)silylloxy}propoxy)-4-nitrobenzoate A mixture of methyl 2-hydroxy-4-nitrobenzoate (66) (1.82 g, 9.23 mmol) and K 2
CO
3 (1.91 g, 13.85 mmol) in DMF (30 mL) was stirred at 20 *C for 30 min. A solution of 3-bromopropyl tertbutyl(dimethyl)silyl ether (3.50 g, 13.85 mmol) in DMF (10 mL) was added and the mixture stirred at 100 *C for 3 h. The mixture was poured into water (300 mL), extracted with EtOAc (3 x 100 mL) and the combined organic extract washed with water (2 x 100 mL), brine (50 mL), dried and the solvent evaporated. The residue was purified by chromatography, eluting with 10% EtOAc/light petroleum, to give 81 (3.19 g, 93%) as a pale yellow solid, mp (EtOAc) 36.5-37 'H NMR 6 7.88 J= 8.9 Hz, 1 H, H 7.80- 7.84 2 H, H 3, H 4.24 J= 6.0 Hz, 2 H, CH20), 3.92 3 H, OCH 3 3.85 J= 5.9 Hz, 2 H, CH20), 2.04-2.09 2 H, 0.88 9 H, OSi(CH 3 3 0.04 6 H, OSi(CH 3 2 3 C NMR 5 165.4 (C0 2 158.7 (C 150.7 (C 132.0 (C 126.1 (C 1), 114.8 (C 107.7 (C 64.0 (CH20), 59.0 (CH20), 52.5 (OCH 3 32.0 (CH 2 25.9 (SiC(CH 3 3 18.3 (SiC(CH 3 3 -5.5 (Si(CH 3 2 Anal. (C, 7
H
27
NO
6 Si) C, H, N.
[2-(3-{[tert-Butyl(dimethyl)silylloxy}propoxy)-4-nitrophenyl]methanol
DIBALH
(1 M in DCM, 16.5 mL, 16.5 mmol) was added to a stirred solution of ester 81 (1.85 g, mmol) in THF (100 mL) at 5 *C and the solution stirred at 5 *C for 1 h. The solution was poured into a solution of potassium sodium tartrate (1 M, 100 mL) and the mixture stirred vigorously for 20 min. The mixture was extracted with EtOAc (3 x 100 mL), the combined organic fraction washed with water (100 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 82 (1.64 g, 94%) as a pale yellow solid, mp (EtOAc/light petroleum) 48- 49 'H NMR 6 7.84 (dd, J= 8.3, 2.1 Hz, 1 H, H 7.71 J= 2.1 Hz, 1 H, H 7.51 J= 8.3 Hz, 1 H, H 4.76 J= 6.3 Hz, 2 H, CH20), 4.21 J= 6.1 Hz, 2 H, lmrtfm.ti^ .uw^h fUtfjnj« t WO 00/64864 WO 0064864PCT/GBOOIOI 612 68 3.82 J 5.9 Hz, 2 H, CH 2 OSi), 2.40 J 6.3 Hz, 1 H, OH), 2.02-2.08 (in, 2 H, CHA) 0.89 9 H, OSiC(CH 3 3 0.06 6 H, OSi(CH 3 2 1 3 C NMR 8 156.5 (C 148.2 (C 4), 136.7 (C 127.8 (C 115.9 (C 105.8 (C 65.5 (CH 2 60.8 (CH 2 59.3 (CH 2
O),
32.0 (CH 2 25.9 (OSi(CH 3 3 18.3 (OSiC(CH 3 3 -5.4 (OSi(CH 3 2 Anal. (C 1 6
H
27
NO
5 Si) C, H, N.
2-(3-{[tert-Butyl(dimethyl)silylI oxy) prop oxy)-4-nitrobeiizyl 1-(chloromethyI)-3-I5,6,7trimethoxy-1H-indol-2-yl)Carbofyly1-2,3dihydro-lH-benzo A solution of triphosgene (15 mg, 5 1 zmol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem.
Lett., 1997, 7, 1483] (60 mg, 129 gimol) and Et 3 N (40 A.L, 289 pmol) in DCM (10 mL) and stirred at 20 *C for 2 h. A solution of alcohol 82 (54 mg, 159 Armol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 *C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient MeOHIEtOAc, to give 83 (72 mg, 67%) as a yellow solid mnp (MeOH) 149-151 OC; 1H NMR 8 9.42 1 H, indole-NH), 8.96 1 H, OCONH), 7.91 J= 8.4 Hz, I H, H 7.78-7.85 (in, 2 H, H 9, H 7.75 J, 1.7 Hz, 1 H, H 7.53-7.59 (mn, 2 H, H 8, H 7.47 (ddd, J= 8.4, 7.4,0.8 Hz, 1 H, H 7.08 (br s, 1 H, H 7.02 J= 2.2 Hz, 1 H, H 6.89 I H, H 5.38 2 H, CH 2 4.82 (dd, J 10.7, 1.7 Hz, 1 H, H 4.69 (dd, J= 10.7, 8.7 Hz, 1 H, H2), 4.21 J =6.0OHz, 2H, CH 2 4.17-4.20 I HK
CH
2 Cl), 4.09 3 H, OCH 3 3.99 (dd, J =11.3, 2.9 Hz, 1 H, H 3.95 3 H, OCH 3 3.92 3 H, OCH 3 ),3.83 J =5.9 Hz, 2 H, CH 2 3.49 J 11.0 Hz, 1 H, CH 2 C1), 2.02-2.08 (in, 2 H, CH 2 )P 0.88 9 H, OSiC(CH 3 3 0.04 6 H, OSi(CH 3 2 MS (FAB+) milz 833 835 12), 775 599 HRMS (FAB~) calc. for
C
42
H.
50 35 C1N 4
Q
10 Si (MWf) m/z 833.2985, found 833.3008; calc. for C- 42
H
50 37 C1N 4 0OSi (MWf) mlz 835.2955, found 835.2982; Anal. (C 42
H
49 C1N 4
O
0 Si) C, H, N.
2-(3-Hydroxypropoxy)4-nitrobeIzyI 1-(chloromethyl)-3-[K5,6,7-trimethoxy-1H-indoI- 2-lcroyl23dhdol-ezl~no--labmt 1 M HCl (0.2 niL, 200 jiniol) was added to a stirred solution of silyl ether 83 (64 mg, 77 Armol) in MeOH mL) and the solution stirred at 20 *C for 30 min. The solvent was evaporated and the residue dissolved in EtOAc (50 niL), washed with water (2 x 50 mL), brine (25 inL), dried BNSDOCIO: <WO oomsw4A i WO 00/64864 WO 0064864PCT/GBOO/01612 -69and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (50-100%) of EtOAc/light petroleum, to give 84 (52 mg, 94%) as a tan solid, mp (EtOAc) 122-126 00; 'H NMR 8 9.51 I H, indole-NH), 8.90 I H, OCONH), 7.92 (d, J= 8.5 Hz, 1 H, H 7.80 J =8.2 Hz, 1 H, H 7.77 J =8.3 Hz, 1 H, H 7.73 J, 1.8 Hz, 1 H, H 7.50-7.57 (in, 2 H, H 8, H 7.40-7.46 (mn, 2 H, H 4, H 6.99 J =2.2 Hz, 1 H, H 6.87 1 H, H 5.37 J =13.1 Hz, I H, CH 2 5.32 J 13.1 Hz, 1 H, CH 2 4.77 (dd, J= 10.8, 1.6 Hz, 1 H, H 4.64 (dd, J 10.8, 8.6 Hz, I H, H 4.27 J= 5.7 Hz, 2 H, CH 2 4.1 14.18 (in, 1 H, CH 2 C1), 4.09 3 H, 00H 3 3.96 3 H, 00HA) 3.91-3.95 (in, 3 H, H 1, 01120), 3.90 3 H, 00H 3 3.44 J= 10.9 Hz, 1 H, CH 2 C1), 2.75 (br s, I H, OH), 2.12-2.18 (in, 2 H, C1H 2 13C NMR 160.4 157.2 (C 153.8 (OCONH), 150.2 (C 148.9 (C 141.6 (0 3a), 140.6 (C 138.9 (C 7') 134.0 131.6(C 130.1 (0 129.7(C02'), 129.6 (C 9a), 127.5 125.7 (0 125.0 123.6 (C3a), 123.1(C09), 122A4(C06, C9b), 121.6 (C5a), 115.7 (C5"1), 112.2 (0 106.6 (C 106.1 (0 97.7 (C 66.8 (01120), 62.2 (CH 2 61.5 (00HA) 61.1 (OCH 3 ),60.1 (01120), 56.3 (00H1 3 55.0 (C 45.8 (CH 2 C1), 43.4 (0 1), 31.6 (OH 2 MS (FAB~) m/z 721 WMH, 719 WMH, HRMS (FAB~) calc. for
C
36 H 35 3 1N 4 01 0 (MMH~ m/z 719.2120, found 719.2107; caic. for C 3 13.
37
CIN
4 0 10 (4' ,n/z 721.209 1, found 721.2093; Anal. (C 36
H
35 C1N 4 0 10 C, H, N.
Example 4F. Preparation of 2-(3-hydroxypropoxy)-4-litrobenzyI doxorubicin carbamate (87).
4-Nitrophenyl 4-nitro-2-[3-(tetrahydro-2-pyra-2-yoxy)propoxylbelzyI carbonate A solution of 4-nitrophenylchloroformate (0.43 g, 2.1 inmol) in THF (10 inL) was added dropwise to a stirred solution of alcohol 75 (0.44 g, 1.4 minol) and DIEA (0.49 mL, 2.8 mmnol) in THF (40 mL) and the mixture stirred at 20 00 for 48 h. The solution was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 inL). The organic fraction was washed with water (3 x 50 mL), brine (50 inL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (10-50%) EtOAc/light petroleum, to give starting material (176 ing, and (ii) 85 (0.3 8 g, 56%) as a pale yellow oil, 'H NMR 8 8.30 (ddd, J= 9.2,3.1, 2.1 Hz, 2 H, H 3, H 7.87 (dd, J= 8.4, 2.1lHz, I H, H 5%)7.79 J=2.1 Hz, 1 H, 7.57 (d,J =8.4 Hz, 1 H, H 7.41 (ddd, J= 9.2, 3.1, 2.1 Hz, 2 H, H 2, H 5.42 2 H, 0H 2 4.58-4.61 (in, 1 H, WO 00/64864 PCT/GBOO/01 612 OCHO), 4.28 J =6.3 Hz, 2 H, CH 2 3.96 (dt, J =10.0, 6.0 Hz, 1 H, CH 2 3.78-3.83 (in, 1 H, CH 2 3.59 (dt,J= 10.0, 6.0 Hz, 1 H, CH 2 3.45-3.52(in, 1 H, CH 2 2.13- 2.18 (in, 2 H, CH 2 1.79-1.86 (mn, 1 H, CH 2 1.67-1.76 (mn, 1 H, CH 2 1.48-1.60 (mn, 4 H, 2 x CH1 2 1 3 C NMR 8 157.0 (C 155.4 (C 153.4 (OCONH), 149.2 (C 145.5 (C 129.9 (C 129.2 (C 125.3 (C 3, C 121.7 (C 2, C 115.6 (C 106.3 (C 6%) 99.1 (OCO), 66.1 (CH 2 65.3 (CH 2 63.5 (CH 2 60.4 (CH 2 30.6 (Cl- 2 29.4
(CH
2 25.4 (CH 2 19.7 (CH 2 MS m/z 476 459 392 210(30), 85 (100); HIRMS caic. for C 22 H1 24
N
2 0 10 m/z 476.143 1, found 476.1425.
2-(3-Hydroxypropoxy)-4-litrobelzyI 4-nitrophenyl carbonate A solution of ether (207 mg, 0.47 mmol) in THE (20 mL) and 1 M HC1 (5 mL) was stirred at 20 *C for 16 h. The solvent was evaporated and the residue partitioned between EtOAc (50 mL) and water (50 niL. The organic fraction was dried, the solvent evaporated, and the residue purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 86 (125 mig, 68%) as a white solid, mp (EtOAc/light petroleum) 116-117 'H1NMR 8 8.29 (ddd,J 9.1, 3.2, 2.1 Hz, 2 H, H 3, H 7.88 (dd, J 2.1 Hz, 1 H, H 7.80 J 2.1 Hz, 1 H, H 7.5 8 J =8.3 Hz, 1 H, H 7.40 (ddd, J 3.2, 2.1 Hz, 2 H, H 2, H 6), 5.41 2 H, CH 2 4.3 0 (t,J 6.0 Hz, 2 H, CH 2 3.90 (dt, J 4.6 Hz, 2 H, CH 2
O),
2.10-2.15 (in, 2 H, CH 2 1.65 (br s, I H, OH); 3 C NMR 8 157.0 (C 155.3 (C 152.3 (OCONH), 149.3 (C 145.5 (C 129.8 (C 129.6 (C 125.4 (C 2, C 121.7 (C 3, C5), 115.8 (C 106.4 (C 66.2 (CH 2 65.3 (CH 2 59.5 (CH 2 31.7 (CH 2 Anal. (C, 7
H,
6
N
2 0 9 C, H, N.
2-(3-Hydroxypropoxy)4-lirobelzyl doxorubicin carbamiate A solution of carbonate 86 (41 mg, 104 psmol) in DWvi (2 mL) was added dropwise to a stirred solution of doxorabicin 13 (46 mg, 86 Mmol) and Et 3 N (15 ptL, 104 Mmol) in DMF (5 mL) at 20 C and the solution stirred for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient of MeOHIDCM, to give 87 (69 mg, 84%) as a red solid, mp (DCM) 154-160 'H NMR [(CD 3 2 S0] 8 14.00 1 H, 6-OH), 13.24 1 H, 1 1 7.85-7.89 (in, 2 H, H 1, H 7.80 (dd, J 8.3, 1.8 Hz, 1 H, H 7.71 (d, J =1.8 Hz, 1 H, H 7.62 (dd, J 2.8 Hz, 1 H, H 7.50 J= 8.3 Hz, 1 H, H 7.07 J= 8.0 Hz, 1 H, OCONH), 5.42 1 H, 5.14 (br s, 1 H, H 5.05 J= BNSDOCIO: <MO 0064184AI I WO 00/64864 PCT/GBOO/01 612 -71- 18.4 Hz, 1 H, CH 2 4.99 J= 18.4 Hz, 1 H, CH 2 4.92 (br s, 1 H, H 4.85 J= Hz, 1 H, 14-OH), 4.74 J 5.8 Hz, 1 H, 4.58 J =6.0 Hz, 2 H, H 14), 4.55 J =5.3 Hz, 1 H, H 4.14-4.20 (in, 2 H, CH 2 3.97 3 H, 4-OCH 3 3.69-3.76 (in, I H, H 3.54 (dt, J 5.7 Hz, 2 H, CH 2 3.48 (br s, 1 H, H 3.30 (br s, I H, OH), 2.98 J =18.2 Hz, 1 H, H 10), 2.90 J =18.2 Hz, 1 H, H 10), 2.22 (br d, J =14.4 Hz, 1 H, H 2.09 (dd, J =14.4, 5.5 Hz, I H, H 1.88-1.92 (mn, 1 H, H 1.82-1.87 (in, 2 H, CH1 2 1.50 (dd, J 12.4, 3.7 Hz, 1 H, H 1, 13 J =6.4 Hz, 3 H, H 'IC NM
((CD
3 2 S0] 8 213.7 (C 13), 186.4 (C 186.3 (C 12), 160.7 (C 156.0 (C 155.9 (C 154.9 (C 11), 154.4 (OCONH), 147.8 (C 136.1 (C 135.4 (C 12a), 134.5 (C 6a), 134.0 (C 10a), 133.2 (C 127.8 (C 119.9 (C 4a), 119.6 (C 118.9 (C 115.3 (C 110.6 (C 5a), 110.5 (C I 105.8 (C 100.2 (C 74.8 (C 69.8 (C 67.9 (C 66.6 (C 65.6 (CH 2 63.6 (C 14), 59.8 (CH 2 57.0 (CH 2 56.5 (4-OCH 3 47.2 (C 36.5 (C 32.0 (C 10), 31.7 (CL! 2 29.7 (C 16.9 (C MS (FAB~) m/z 797 HEMS (FAB~) caic. for C 3 gH 40
N
2 01 7 WI[H) m/z 797.2405, found 797.2953; Anal. (C 38
H
4
N
2 0 17 1
A
2 0) C, H, N.
Example 4G. Preparation of 2-(3-hydroxypropoxy)-4-fitrobelzyI bis(3-{[(5-methyl-4acridinyl)carboflyllamilpropyl)carbamate (91).
4-ir--3(erhdo2-prn2yoypooybny bis{3- [(trifluoroacetyl)amliflorpyl)carbamate A solution of alcohol 75 (623 mg, minol) and DIEA (0.40 mL, 2.4 minol) in DCM (8 mL) was added dropwise to -a solution of triphosgene (208 mg, 0.70 mmnol) in DCM (6 inL) over 30 minutes at 5 *C and stirred for 1 b. The reaction mixture was added dropwise to a suspension of bistrifluoroacetaifide 47 (880 mg, 2.0 minol) and DLEA (0.76 mL, 4.8 nimol) in DCM (8 mL) and the solution stirred at 20 *C for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with 50% EtOAc/petroleumf ether, to give 88 (804 mng, 61%) as a colorless oil, 1 H NMR [(CD 3 2 S0] 8 7.85 (dd, J 2.0 Hz, 1 H, H 7.80 (br s, 1 H, CONH), 7.76 J 2.0 Hz, 1 H, H 7.44 J =8.0 Hz, I H, H 6.81 (br s, 1 H, CONIH), 5.25 2 H, CH 2 4.58-4.61 (in, I 4.22-4.26 (in, 2 3.9 1-3.98 (in, I H), 3.79-3.86 (in, 1 3.56-3.63 (in, 1 3.46-3.53 (in, 1 3.28-3.40 (in, 8 2.04-2.16 (in, 2 1.72-1.87 (in, 6 1.50-1.63 (in, 4 3 C NM [(CD 3 2 S0] 8 157.0, 156.9, 148.9, 131.5, 129.4, 115.7, 105.3, 99.2, 65.9, 63.7, 62.6, 62.5, 44.3 37.4, 36.1, 30.7, WO 00/64864 WO 0064864PCT/GBOO/0I 612 72 29.4, 28.1, 27.1, 25.3, 19.6, 2 x CF 3 CO not observed; HRMS (FAB') caic. for
C
26
H
34
F
6
N
4 0 9 m/z 660.2230; found 660.2234 4-Nitro-2-I3-(tetrahydro-2H-pyran-3-yloxy)propoxylbelzyI bis(3- ([(5-methyl-4acridinyl)carbonyllamino~propyl)carbamate A solution of carbamnate 88 (165 mg, 0.25 mmol), CS 2
CO
3 (1.0 g, 3.0 mmol) and water (I mL) in methanol (4 mL) was stirred at *C for 8 h. The pH was adjusted to 10, water (50 mL) added, the solution was extracted with DCM (3 x 50 mL). The combined organic fraction was dried, and the solvent was evaporated to give crude 4-nitro-2-[3-(tetrahydro-2H-pyran-2-yloxy)propoxy]benzyl bis(3aminopropyl)carbamate 4-(lH-Imidazol-l-ylcarbonyl)-5-methylacridine (50) A.
Gamage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, J. Med.
Chem., 1999, 42, 23 83-2393] (144 mg, 0.50 mmol) was added to a solution of carbamate (89) in THE (10 mL) at 5 *C and the reaction mixture was stirred at 20 0 C for 8 h. The solvent was evaporated, and the residue was purified by chromatography on eluting with 1%MeOH-/55%EtOAcIDCM, to give 90 (183 mg, 88%) as a yellow solid, mp (EtOAc/DCM) 80-81 'H NMR 8 11.90 1 H, NIH), 11 .83 1 H, NH), 8.88-8.92 (in, 2 8.62-8.72 (in, 2 8.02-8.05 (mn, 2 7.74-7.83 (in, 2 7.50-7.63 (in, 4 7.36- 7.45 (in, 2 7.30 J= 2.0 Hz, 1 H, H 7.02 J =8.4 Hz, 1 H, H 6.90 (dd, J 2.0 Hz, 1 H, H 5.01 2 H, CH 2 4.56 1 4.00-3.50 (in, 14 2.83 (s, 3 H, CHO), 2.71 3 H, CHO), 2.14-1.50 (in, 12 1 3 C NMR 8 166.1, 155.6, 155.5, 147.5 147.0 145.2 137.9 135.8, 135.4, 135.2 132.6 132.3 131.0 128.3, 128.0, 126.5, 126.4, 126.2, 126.1 125.7 125.3 114.9, 104.9, 99.1, 65.6, 63.6, 62.5, 61.6, 45.9, 45.1, 3 7.7, 37.1, 30.7, 29.3, 29.4, 28.6, 25.4, 19.7, 18.9, 18.8, 14.2; Anal. (CS 2 H54N 6
O
9
.MA
2 O) C, H, N.
2-(3-Hydroxypropoxy)-4-nitrobenzyI bis(3-fJ(5-methyl-4acridinyl)carbonyll amino) propyl) carbainate dihydrochloride A solution of ether.
(51 mg, 56 Asmol) and HCl (1 M, 1.5 mL) in MeOH (10 niL) was stirred at 20 CC for 4 hrs. The solvent was evaporated and the residue was recrystallized to give 91 (46 mg, 92%) as a yellow solid, mp (MeOHIEtOAcflight petroleum) 143-145'C; 'H NMR [(CD 3 2 S0] 8 11.23 2 H, 2 x NH), 9.17 1 9.11I 1 8.70 (br s, 2 8.24 (br s, 2 7.96 (br s, 2 7.66 (br s, 4 7.51 (br s, 2 7.32 J =2.0 Hz, 1 H, H 3 7.02 J =8.4 8NSDOCjD: <WO oomwi4A i WO 00/64864 PCT/GBOO/01612 73 Hz, I H, H 6.97 (dd, J= 8.4, 2.0 Hz, I H, H 4.80 2 H, CH 2 3.92-3.96 (in, 2 3.50-3.53 (mn, 10 2.72 3 H, CO 3 2.63 3 H, CH 3 2.01-2.04 (mn, 4 H, 2 x
CH
2 1.75-1.82 (in, 2 HRMS (FAB+) caic. for (C, 7
H
46
N
6 0 8 m/z 823.3455, found 823.3467; Anal. (C 47
H
46
N
6 0s.2HCI.2'/aH2 O) C, H, N.
Example 4H. Preparation of 4-nitro-2-[3-(phosphoflooxy)propoxy~beflzyI
I-
(chloromethyl)-3(5,6,7trimethoxyHifdol2y)carbonyIF- 2 3 dihydro-IHf (93).
2-(3-flDi(ter-butoxy)phosphoryloxy) propoxy)-4-nitrobenzyl 1-(chloromethyl)-3ylcarbamate Tetrazole (40 mng, 567 jusiol) was added to a stirred solution of alcohol 84 (Example 4E) 136 mig, 189 gmioI) and di-tert-butyl diethylphosphorainidite (68 g.L, 227 iniol) in THF (10 inL) under N 2 and the solution stirred at 20 00 for 4 h. The solution was cooled to -40 00 and a dried (Na 2
SO
4 solution of MCPBA (70 65 ing, 265 Amol) in DCM (3 niL) added. The solution was stirred at -40 00 for 10 min and a solution of NaHSO 4 (1 0 mL) added and the mixture stirred for 10 min. The mixture was extracted with diethyl ether (80 inL), the organic fraction washed with 10% aq. Na]HSO 4 (10 iL), sat. aq.
KHCO
3 (10 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (10-50%) EtOAc/light petroleum, to give 92 (160 mg, 93%) as an oil,'11 NMR 8 9.42 1 H1, indole-NH), 8.89 1 H, OCONII), 8.15 J 8.5 Hz, 1 H, H 7.83 (dd,J= 8.2,2.1 Hz, I H, H 7.79 1 H, H 7.75 J= 8.2 Hz, 1 H, H 7.73 J= 2.1 Hz, I H, H 7.60 J= 8.3 Hz, I H, H 7.53 (ddd, J 8.3, 7.1, 0.8 Hz, 1 H, H 7.39 (ddd, J 7.1, 0.8 Hz, 1 H, H 7.01 J= 2.1 Hz, 1 H, H 6.89 1 H, H 5.34 2 H, CH 2 4.81 (dd, J 10.7, 1.8 Hz, I H, H 2), 4.64 (dd, J =10.7, 8.6 Hz, I H, H 4.3 5 (dt, J 5.5 Hz, 211, 01120), 4.29 J 5.6 Hz, 211, CH 2 4.15-4.20 (mn, I H, H 4. 10 3 H, OCH2), 3.98 (dd, J 11.2, 2.9 Hz, 1 H, 01120), 3.95(s, 3 H,00113), 3.92 3 11, OCH 3 3.45 (dd, J =10.9, 10.8 Hz, I H, 01120), 2.19-2.25 (in, 211, 0112), 1.35 (2s, 1811, 2 x OC(CH 3 3 MS (FABW) m/z 913 (MWH, 911 HRMS (FAB+) caic. for C4HS 3 3 1C'N 4 0, 3 P M/Z 911.3035, found 911.3003; calc. for C4H 5 3 37 ClN 4 0 1 3 p (MW) m/z 913.3006, found 913.3002.
ouonnr'In. ~%AJr% nnaaooA*. I WO 00/64864 WO 0064864PCT/GBOO/01 612 74- 4-Nitro-2-13-(phosphonooxy)propoxyJ benzylI -(chloromethyl)-3-I(5,6,7-trimethoxy- IH-indo1-2.yl)carbony1I -2,3-dihydro-1H-belzo e ifldol-5-ylcarba mate (93).
Trifluoroacetic acid (130 ILzL, 1.64 Ismol) was added to a stirred solution of ester 92 (150 mg, 165 gmol) in DCM (5 mL) and the solution stirred at 20 *C for I h. The solvent was evaporated, and the residue azeotroped with benzene (3 x 1 mL) to give 93 (88 mg, 66%) as a gum, 'H NMR 8 11.47 1 H, indole-NH), 9.94 I H, OCONH), 8.57 (br s, 3 H, H 4,2 x OH), 8.11 J= 8.5 Hz, 1 H, H 7.98 J= 8.3 Hz, 1 H, H 7.91 J= 8.3 Hz, 1 H, 7.81 1.8 Hz, 1 H, H 7.69 J= 8.3 Hz, 1 H, 7.58 (ddd, J 7.2, 0.7 Hz, 1 H, H 7.47 (ddd, J= 8.5, 7.2, 0.7 Hz, 1 H, H 7. 10 J =2.2 Hz, 1 H, H 6.97 1 H, H 5.29 2 H, CH 2 4.80 (dd, J 10.8, 9.4 Hz, 1 H, H 2), 4.53 (dd, J =10.8, 1.7 Hz, 1 H, H 4.31 -4.37 (in, 1 H, H 4.27 (t J 6.1 Hz, 2 HK
CH
2 4.07 (dd,J= 11.2 Hz, 1 H, CH 2 CI), 4.03 (dt, J= 7.1, 6.2 Hz, 2 H, CH 2 3.91-3.95 (in, 4 H, OCH 3 CH1 2 3.82 3 H, OCH 3 3.80 3 H, OCH 3 2.02-2. 10 (in, 2 H, CH 2 1 3 C NMR 8 160.2 156.2 (C 154.4 (OCONH), 149.1 (C 148.1 (C 141.4 (C 3a), 139.9 (C 139.0 (C 134.4 (C 132.8 (C 130.8 (C 9a), 129.4 (C 128.5 (C 127.1 (C 125.4 (C 5a, C 124.3 (C 123.8 (C 123.2 (C 123.1 (C 122.0 (C 9b), 115.4 (C 113.0 (C 106.2 (C 106.0 (C 98.0 (C 65.1
(CH
2 61.3 (CH 2 6 1.0 (OCH 3 60.9 (OCH 3 60.7 (CH7O), 5 5.9 (OCH 3 54.9 (C 2), 47.5 (CH 2 C1), 41.2 (C 29.6 (CH 2 MS (FAB~) m/z 801 (MW, 799 0.8); HEMS (FAB") caic. for C 36
H
37 3 'C1N 4 01 3 P (MW) m/zk 799.1783, found 799.1757; caic. for
C
3
H
37 3 C1N 4 0 1 3 P (MW) m/z 801.1754, found 801.1730.
Example 41. Preparation of 2-(2,3-dihydroxypropoxy)4-litrobelzyI 1-(chloromethyl)ylcarbamate (99).
Methyl 4-nitro-2-(2-oxiranylmethoxy)belzoate A mixture of methyl 4nitrosalicylate (66) (0.99 g, 5.02 nimol) and K 2 C0 3 (1.04 g, 7.53 mxnol) in DMF (25 niL was stirred at 20 0 C for 20 min. Epichlorohydrin (0.59 mL, 7.53 mmol) was added and the mixture stirred at 100 0 C for 2 h. The mixture was poured into water, extracted with EtOAc (3 x 100 rnL), the combined organic extracts washed with water (2 x 50 inL), brine mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (20-50%) EtOAcflight petroleum, to give starting material 18 FLRnnr~fIfl WO (YrRdAdA1 I PCT/GB00/01612 WO 00/64864 g, 18%) and (ii) 94 (0.75 g, 59 as a colourless solid, mp (EtOAc/light petroleum) 62-63 'H NMR 8 7.91 (dd, J= 7.7, 1.0 Hz, 1 H, H 7.84-7.86 2 H, H 3, H 4.49 (dd, J= 11.2, 2.4 Hz, 1 H, H 4.14 (dd, J= 11.2, 5.2 Hz, 1 H, H 3.94 3 H, OCH 3 3.40-3.44 1 H, H 2.91-2.97 2 H, H "C NMR 8 165.0 158.1 (C 2), 150.6 (C 132.3 (C 126.1 (C 115.6 (C 108.4 (C 69.6 (OCH 3 52.6 49.7 (CH20), 44.3 (C MS (CI, NH 3 m/z 295 (M+CH 3 CN 259 (MH 100%); Anal. (C,,H,,NO 6 C, H, N.
Methyl 2-(2,3-dihydroxypropoxy)-4-nitrobenzoate Perchloric acid (1 mL) and water (3 mL) was added to a stirred solution of 94 (205 mg, 0.81 mmol) in THF (20 mL) and the solution stirred at 20 *C for 16 h. The solvent was evaporated and the residue partitioned between EtOAc (50 mL) and water (50 mL). The organic fraction was washed with water (50 mL), brine (25 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 70% EtOAc/light petroleum, to give 95 (172 mg, 78%) as an oil which solidified on standing, mp 60-65 'H NMR 8 8.02 J= 8.5 Hz, 1 H, H 7.87 (dd, J= 8.5, 2.0 Hz, 1 H, H 7.84 J= 2.0 Hz, 1 H, H 4.38 (dd, J= 9.3, 5.4 Hz, 1 H, H 4.23 (dd, J= 9.3, 5.4 Hz, 1 H, H 4.10-4.14 1 H, H 3.95 3 H, OCH 3 3.88 (br d,J= 4.1 Hz, 2 H,H 3.05 (brs, 1 H, OH), 1.95 (brs, 1 H, OH); 3 C NMR 8 164.8 (CO 2 159.1 (C 151.0 (C 132.8 (C 124.9 (C 115.6 (C 108.8 (C 73.0 (CH20), 69.2 (C 63.2 (CH20), 52.8 (OCH 3 MS (CI, NH 3 m/z 272 (MWH, 240 165 (100); HRMS (CI, NH 3 calc. for C,,IH 4 NO (MH m/z 272.0770, found 272.0766. Anal. 3
NO
7 C, H, N.
Methyl 2-[(2,2-dimethyl-l,3-dioxolan-4-yl)methoxy]-4-nitrobenzoate 2,2- Dimethoxypropane (0.91 mL, 7.37 mmol) was added dropwise to a stirred solution of diol (400 mg, 1.47 mmol) and PPTS (37 mg, 0.15 mmol) in DMF (20 mL) under N 2 and stirred at 20 *C for 24 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The organic fraction was washed with water mL), brine (25 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 30% EtOAc/light petroleum, to give 96 (458 mg, 100%) as a yellow oil, 'H NMR 8 7.90 J 8.2 Hz, 1 H, H 7.84-7.88 2 H, H 3, H 4.49- 4.54 1 H, H 4.25 (dd, J= 9.6, 4.6 Hz, 1 H, H 4.19 (dd, J= 8.5, 6.4 Hz, 1 H, H 4.15 (dd, J= 9.6, 4.6 Hz, 1 H, H 4.03 (dd, J= 8.5, 5.8 Hz, 1 H, H 3.94 3 H,
OCH
3 1.46 3 H, CH 3 1.41 3 H, CH 3 "C NMR 8 165.1 (CO 2 158.2 (C 150.6 WO 00/64864 PCT/GBOO/01612 76- (C 132.2 (C 126.4 (C 115.5 (C 109.9 (C 108.4 (C 73.6 (C 69.8
(CH
2 66.5 (CH 2 52.6 (OCH 3 26.6 (CHO), 25.3 (CH 3 MS (CI, NH 3 m/z 312 (MH~, 296 101 (95),171 (100); HRMS (CI, NH 3 caic. for C1 4 HsN0 7 (MH) m/z 312.1083, found 312.1092.
12122Dmty-,-ixln-lmtoy4ntohnl methanol DIBALH (1 M in DCM, 5.1 mL, 5.1 mmol) was added to a stirred solution of ester 96 (457 mg, 1.47 mmol) in THF (50 mL) at 5 *C and the solution stirred at 5 'C for 1 h. The solution was poured into a solution of potassium sodium tartrate (1 M, 100 mL) and the mixture stirred vigorously for 20 min. The mixture was extracted with EtOAc (3 x 50 mL), the combined organic fraction washed with water (50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 97 (385 mg, 92%) as a white solid, mp (EtOAc/light petroleum) 90-92 CC; 'H NMR 6 7.87 (dd, J= 8.2, 2.1 Hz, I H, H 7.72 J= 2.1 Hz, 1 H, H 7.50 J 8.2 Hz, 1 H, H 4.82 J= 14.1 Hz, 1 H, CH 2 4.70 J= 14.1 Hz, I H, CH 2
O),
4.51-4.57 (in, I H, H 4.23 (dd, J 4.0 Hz, 1 H, H 4.19 (dd, J 5.4 Hiz, 1 H, H 4.11 (dd,J 9.8, 5.4 Hz, 1 H, H 3.95 (dd, J 8.7, 5.4 Hz, 1 H, H 3.25 (br s, 1 H, OH), 1.48 3 H, CH1 3 1.41 3 H, OH 3 13 C NMR 8 156.5 (0 148.2 (C 4), 137.2 (C 128.6 (C 116.6 (C 110.0 (C 106.4 (C 73.7 (01120), 69.7 (CH 2
O),
66.0 (0120), 61.0 (01120), 26.6 (OH 3 25.0 (CHO); MS m/z 283 (Mt, 268 225 101 (100); FIRMS calc for C 13 Hj 7 N0 6 m/z 283.1056, found 283.1055; Anal.
(C,
3
H,
7 N0 6 C, H1, N.
2-(,-iehl13dooa4y~ehx]-irbny 1-(chloromethyl)-3-I5,6,7trimethoxy-lH-indol-2-yl)carbonylY2,3dihydrolHbeInzo[eindol.S-ylcarbamate A solution of triphosgene (22 mg, 75 gmol) in DOM (3 mL) was added dropwise to a stirred solution of amin 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem.
Lett., 1997, 7, 1483] (100 mg, 215 /LmoI) and Et 3 N (60 guL, 429 /2mo1) in DOM (10 mL) and stirred at 00 for 2 h. A solution of alcohol 97 (73 mg, 256 Imiol) in DOM (3 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 00 for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with to give 98 (160 mg, 96%) as a gum; 'H NMR 6 9.44 1 H, indole- NH), 8.94 I H, OCONH), 7.92 J =8.5 Hz, I H, H 7.87 (dd, J 8.2, 2.1 Hz, 1 H, BNSDOCID:. <WO 0064864AI I WO 00/64864 PCT/GBOO/01612 77 H511), 7.81 J=8.2 Hz, 1 H, H9), 7.73 J=2.1 Hz, 1 H, HF3), 7.58 (ddcd, J=8.2, 7.3, 0.7 Hz, 1 H, H 7.45-7.51 (mn, 2 H, H 7, H 7.13 (br s, 1 H, H 7.02 J 2.2 Hz, 1 H, H 6.89 1 H, H 5.38 2 H, CH 2 4.83 (dci, J 10.8, 1.7 Hz, 1 H, H 2), 4.69 (dd, J =10.8, 8.7 Hz, 1 H, H- 4.50-4.56 (in, I H, H 4.23 (dd, J= 9.8, 4.0 Hz, 1 H, H 4.15-4.20 (in, 2 H, H 1, H 4.09-4.14 (in, 4 H, OCH 3 H 3.95-4.00 (in, H, OCH 3
CH
2 C1, H 3.92 3 H, OCH 3 3.50 (dd, J =10.9, 10.8 Hz, 1 H, CH 2 ClI 1.45 3 H, CH 3 1.39 3 H, CH 3 MS (FAB~) m/z 777 775 HRMvS (FAB") caic. for C 39
H
4 35 C1N 4 01 1 m/z 775.2381, found 777.2379; caic. for
C
39
H
40 37 C1N 4 0 1 1 m/z 777.2535, found 777.2354.
2 2 ,3-Dilhydroxypropoxy)4nitrobenzyI 1-(chloromethy)3[(5,6,7trimethoxyindoI- 2 -y)cabofyll2,3-dihydrolHbhenzoelindoS5-ylcarbamate 1 M HCI (1 niL) was added to a stirred suspension of 98 (160 ing, 206 g~mol) in THF (20 mL) and the mixture stirred at 20 0 C for 16 h. The mixture was evaporated and the residue partitioned between DCM (50 mL) and water (50 mQL. The organic fraction was washed with water mL), brine (30 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50%EtOAcIDCM, to give 99 (87 mg, 56%) as a white solid, mnp (MeOH~iPr 2 O) 147-149 'H NMR [(CD 3 2 S0] 11.46 I H, indole-NH), 9.90 (s, I H, OCONH), 8.56 1 H, H 8.12 (di, J= 8.5 Hz, 1 H, H 7.98 J= 8.3 Hz, 1 H, H 7.92 (dd, J 1.9 Hz, 1 H, H 7.83 (di, J =1.9 Hz, 1 H, H 7.69 J= 8.3 Hz, 1 H, H 7.59 (dd, J 8.2, 7.6 Hz, 1 H, H 7.49 (dd, J 7.6 Hz, I H, H 7.09 (di, J 2.1 Hz, 1 H, H 6.98 1 H, H 5.3 3 2 H, CH 2 5.07 J =5.2 Hz, 1 H, OH), 4.81 (dd, J= 11.0, 9.7 Hz, 1 H, H2), 4.7 3 J =5.7 Hz, 1 H, H 4.53 (dd,J 11.0, 3.5 Hz, 1 HL H 4.32-4.37 (mn, 1 H, H 4.24 (dd, J =10.0, 3.9 Hz, 1 H, CH 2
CI),
4.09-4.13 (mn, 1 H, H 4.024.06 (in, 1 H, H 3.93-3.96 (in, 4 H, OCH 3
.CH
2 C1), 3.84-3.89 (in, I H, OH), 3.83 3 H, OCH 3 3.81 3 H, OCH 3 3.51 J= 5.7 Hz, 2 H, H 1 3 C NMR [(CD 3 2 S0] 8 160.2 156.2 (C 154.3 (OCONH), 149.2 (C 148.0 (C 141.5 (C 3a), 139.9 (C 13 9.0 (C 134.3 (C 133.0 (C 130.7 (C 9a), 129.4 (C 128.0 (C 127.1 (C 125.4 (C 5a, C 7a), 124.3 (C 123.9 (C 9), 123.3 (C 123.1 (C 122.0 (C 9b), 115.4 (C 113.0 (C 106.3 (C 106.2 (C 98.0 (C 70.7 (CH 2 69.7 (CHOH) 62.4 (CH 2 61.0 (OCH 3 60.9 (OCH 3 60.7
(CH
2 55.9 (OCH 3 54.9 (C 47.5 (CH 2 Cl), 41.4 (C MS (FAB~) m/z 737 (MH~, 735 HRMS (FAB') caic. for C 36 H 6 35
CN
4 011 (MH) mz 735.2069, found WO 00/64864 WO 0064864PCT/GBOO/01612 78 735.2050; calc. for C 36
H
36 1 7 C1N 4 0 1 1 (MW) m/z 737.2040, found 737.2000; Anal.
(C
36
H
35 C1N 4 0 1 I CH 3 OH) C, H, N.
Example 4J. Preparation of 2-[3-(dimethylamino)propoxy1-4-litrobelZYl 1- (clrmty)3[567tiehx-Hidl2y~abnl-,-iyr-H (102).
Methyl 2-I3-(dimethylamino)propyloxyI-4-flitrobelzoate (100). A mixture of methyl 2hydroxy-4-nitrobenzoate (66) (1.03 g, 5.22 mmol) and K 2 C0 3 (2.17 g, 15.67 mmol) in DMF (30 mL) was stirred at 20 0 C for 30 min. A solution of N-(3-chloropropyl)-NNdimethylamine (1.24 g, 7.83 mmol) in DMF (10 mL) was added and the mixture stirred at 100 0 C for 3 h. The mixture was poured into water (300 mL), extracted with EtOAc (3 x 100 mL) and the combined organic extract washed with water (2 x 100 mL), brine (50 mL), dried and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (20-50%) of EtOAc/light petroleum, to give 100 (1.05 g, 7 as a pale yellow oil which was stored as the HC1 salt, mp (EtOAc) 175-177 'H NMR [(CD 3 2 S0] 8 10.90 (br s, 1 H, NHCI), 7.87-7.93 (in, 3 H, H 3, H 5, H 4.32 J 6.0 Hz, 2 H, CH 2
O),
3.89 3 H, OCH 3 3.18-3.23 (in, 2 H, CH 2 2.77 J 4.8 Hz, 6 H, N(CH 3 2 2.18- 2.24 (in, 2 H, CH 2 1 3 C NMR [(CD 3 2 S0] 8 165.0 (C0 2 157.3 (C 150.2 (C 131.6 (C 126.0 (C 115.3 (C 108.4 (C 66.5 (CH 2 53.7 (CH 2 52.6 (OCH 3 42.0
(N(CH
3 2 23.3 (CH 2 Anal. (C, 3
H,
9 C1N 2 0 5 C, H, N, Cl.
(2-I3-(Dimethylamino)propoxy1]4-fitrophefllmethanoI (101). DIBALH (1 M, in DCM, 13.0 inL, 13.0 nimol) was added to a stirred solution of ester 100 (1.05 g, 3.72 inmol) in THE (50 inL) at 5 0 C and the solution stirred at 5 *C for I h. The solution was poured into a solution of potassium sodium tartrate (1 M, 100 niL) and the mixture stirred vigorously for 20 min. The mixture was extracted with EtOAc (3 x 100 mL), the combined organic fraction washed with water (100 inL), brine (5 0 inL, dried, and the solvent evaporated. The residue was purified by chromatography on alumina, eluting with a gradient of MeOH/EtOAc, to give 101 (0.81 g, 86%) as a pale yellow solid, mp (EtOAc) 104-105 'H NMR 8 7.87 (dd, J 8.3, 2.1 Hz, 1 H, H 7.69 J 2.1 Hz, 1 H, H 7.64 J 8.3 Hz, I H, H 5.43 (br s, I H, OH), 4.5 8 2 H, CH 2 4.14 J 6.5 Hz, 2 H, CH 2 2.36 J 7.0 Hz, 2 H, CH 2 2.15 6 H, N(CH 3 2 1.85-1.91 (in, 2 H, C11 2 1 3 C NMR 8 15*5.2 (C 147.0 (C 13 8.9 (C 126.7 (C 115.3 (C BNSDOCID: .<WO 0064864A1 I WO 00/64864 PCT/GBOO/01612 79 105.2 (C 66.5 (CH 2 57.6 (CH 2 55.5 (NCH 2 45.1 (N(CH 3 2 26.5 (CHO); Anal.
(C 1 2
H,
8
N
2 0 4 C, H, N.
2 -13-(Dimethylamino)propoxy1]4flitrobenzyI 1 -(chloromethyl)-3-I(5,6,7-rimfethoxy- HidS--lcr onl-,-iy H- eliindol-5-ylcarbamfate (102). A solution of triphosgene (17 mg, 55 /Lmol) in DCM (2 mL) was added dropwise to a stirred solution of amine I J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (65 mg, 140 jimol) and Et 3 N (44 gL, 313 humol) in DCM (10 mL) and stirred at 20 0 C for 2 h. A solution of alcohol 101 (44 mg, 172 g.mol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 *C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient MeOH/EtOAc, to give 102 (57 mg, 55%) as a yellow solid which was converted to the hydrochloride salt, mp (MeOH) 176-180 'H NMvR [(CD 3 2 S0 8 11.43 1I H, indole-NH), 10.47 (br s, I H, NH+ClV), 9.92 1 H, OCONH), 8.58 I HL H 8.10 J 8.5 Hz, 1 H, H 7.97 J =8.3 Hz, 1 H, H 7.93 (dd, J =8.4,2.0 Hz, 1 H, H 7.82 J 2.0 Hz, I H, H 7.71 (br d, J =8.4 Hz, 1 H, H 7.56-7.61 (in, 1 H, H 8), 7.46-7.51 (mn, 1 H, H 7. 10 J 2.1 Hz, I H, H 6.97 1 H, H 5.34 2 H,
CH
2 O),4.81 (dd, J =10.8, 9.5 Hz, 1 H, H2), 4.5 3 (dd, J= 10.8, 1.7 Hz, 1 H, H2), 4.33- 4.38 (in, 1 H, H 4.30 5.9 Hz, 2 H, CH 2 4.07 (dd, J =11.1, 2.9 Hz, 1 H, CH 2 Cl), 3.94-3.97 (mn, 4 H, CH 2 ClOCH 3 3.83 3 H, OCH 3 3.81 3 H, OCH 3 3.23-3.27
J
Hz, 2 H, CH 2 2.75 6 H, N(CH 3 2 2.17-2.23 (in, 2 H, CHO); 13 c NivIR
[(CD
3 2 S0] 8 160.2 155.8 (C 154.3 (OICONH), 149.2 (C 148.0 (C 141.5 (C 3a), 139.9 (C 138.9 (C 134.3 (C 132.9 (C 130.7 (C 129.5 (C 9a), 128.5 (C 127.2 (C 125.4 (C 124.4 (C 123.7 (C 123.3 (C 123.1 (C 122.1 (C 9b), 121.2 (C 5a), 115.7 (C 113.0 (C 106.3 (C C 98.0 (C 4') 66.8 (CH 2 61.0 (OCH 3 60.9 (OCH 3 60.7 (CH 2 55.9 (OCH 3 54.9 (C 53.7
(CH
2 47.5 (CH 2 Cl), 42.0 (N(CH 3 h2), 41.1 (C 23.6 (CHO); Anal. (C 38
H
4 jC1N_,0q.2HCl): C, H,N.
Example 4K. Preparation of 2-[3-(4-morpholiflyl)propoxyIAInitrobenzyI 1- (clrmty)31567tiehx-Hidl2y~abnl-,-iyr-H (105).
Methyl 2-3(-opoiy~rooy--irbnot (103). A mixture of methyl 2- WO 00164864 WO 0064864PCT/GBOO/01 612 80 hydroxy-4-nitrobenzoate (1.0 g, 5.12 mmol) and K 2 C01.06 g, 7.68 mmol) in DMF mL) was stirred at 20 *C for 30 min. A solution of 4-(3-chloropropyl)morpholine (0.98 g, 7.68 inmol) in DM1F (5 mL) was added and the mixture stirred at 100 *C for 6 h. The mixture was cooled to 20 'C and poured into water (300 mL) and extracted with EtOAc (3 x 100 mL). The combined organic fraction was washed with water (2 x 50 mL), brine mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with EtOAc, to give 103 (1.3 3 g, 80%) as an oil, 'H NMR 8 7.88 J= 9.1 Hz, I H, H 7.81 (in, 2 H, H 3, H 4.21 J 6.3 Hz, 2 H, CH 2 3.93 3 H, OCH 3 3.69-3.74 (in, 4 H, 2 x CH 2 2.57 J 7.0 Hz, 2 H, CH 2 2.47-2.5 1 (in, 4 H, 2 x
CH
2 2.02-2.07 (in, 2 H, CH 2 1 3 CNMR 8 165.3 (C0 2 15 8.6 (C 150.6 (C 132.0 (C 126.2 (C 114.8 (C 107.8 (C 67.6 (CH 2 66.9 (2 x CH 2 55.0 (CH 2
N),
53.7 (2 x 52.5 (OCH 3 25.9 (CL! 2 Compound 103 was conveniently stored as the hydrochloride salt, mp (EtOAc) 160-163 Anal. (C1 5
H
2
ON
2 0 6 .HCl) C, H, Cl. N, calc. 7.8, found 9. 1%.
(2-[3-(4-Morpholinyl)propoxyJ-4-nitrophenyl) methanol (104). A solution of ester 103 (1.33 g, 4. 10 minol) in THE (100 mL) was added dropwise to a stirred solution of DLBALH (1 M in DCM, 13.5 mL, 13.5 mmnol) at 5 0 C and the solution stirred at 5 *C for I h. The solution was carefully poured into I M HCI (50 mL) and stirred for 10 min. The solution was concentrated under reduced pressure, neutralised and extracted with EtOAc 3 x 100 mL). The combined organic fraction was dried and the solvent evaporated. The -residue was purified by chromatography on alumina, eluting with a gradient MeOHIEtOAc, to give 104 (1.07 g, 88%) as a tan solid, mnp (EtOAc) 105-106 'H NMvR 8 7.83 (dd, J= 8.2, 2.1 Hz, I H, H 7.70 J= 2.1 H z, 1 H, H 7.46 J= 8.2 Hz, 1 H, H 4.71 2 H, CH 2 4.18 J =6.0 Hz, 2 H, CH 2 3.74-3.77 (in, 4 H, 2 x CH 2 2.56 (dd, J 6.7, 6.5 Hz, 2 H, CH 2 2.45-2.49 (mn, 4 H, 2 x CH 2 2.01-2.06 (in, 2 H, CL! 2 1 3
C
NMR 8 156.7 (C 148.2 (C 137.2 (C 128.2 (C 116.1 (C 106.2 (C 67.7
(CH
2 66.5 (2 x CH 2 60.7 (CH 2 56.3 (CH 2 53.9 (2 x CH 2 N) 25.5 (CH 2 Anal.
(C
14
H
2
ON
2 05) C, H, N.
2-13-(4-Morpholinyl)propoxyl-4-nitrobenzyl 1 -(chloromethyl)-3-I(5,6,7-triinethoxy- IH-indol-2-ylocarbonyll-2,3-dihydro-IH-benzolelindol-5-ylcarbamate (105). A solution of triphosgene (15.5 mg, 52 pmioI) in DCM (2 mL) was added dropwise to a stirred BNSD0010: <WO 0064864A1 I WO 00/64864 PTGO/11 -81solution of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (52 mg, 133 psmol) and Et 3 N (42 gL, 299 /.tiol) in DCM (10 mL) and stirred at 0 C for 2 h. A solution of alcohol 104 (49 mg, 164 humol) in DCM (2 ml) was added, followed by nBu 2 Sn(OAC) 2 (2 drops) and the solution stirred at 20 *C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient MeOH/EtOAc, to give 105 (96 mng, 92%) as a tan powder mp (EtOAc) 102-107 CC; 'H NMR 8 9.48 1 H, indole-NH), 8.93 I H, OCONH), 7.91 J 8.5 Hz, I H, H 7.78-7.84 (in, 2 H, H 9, H 7.73 J =1.8 Hz, 1 H, H 7.57 (ddd, J= 8.2, Hz, 1 H, H 7.50-7.53 (in, 1 H, H 7.45 (ddd, J 7.0, 1.0 Hz, I H, H 7.28 (br s, 1 H, H4), 7.00 (d,J =2.2Hz, I H, H 3%)6.88( s, 1 H, H 4%)5.36 2H, CH 2
O),
4.81 (dd, J =10.8, 1.8 Hz, 1 HL H 4.68 (dd, J =10.8, 8.7 Hz, 1 H, H 4.16-4.22 (in, 3 H, CH 2 O, CH 2 Cl), 4.08 3 H, OCH 3 3.93-3.99 (in, 4 H, H 1, OCH 3 3.92 3 H,
OCH
3 3.69 J 4.6 Hz, 4 HL 2 x CH 2 3.49 J =10.9 Hz, 1 H, CH 2 Cl), 2.55 J= 7.1 Hz, 2 H, CH 2 2.43-2.49 (mn, 4 H, 2 x CH 2 2.00-2.08 (mn, 2 H, CH 2 13C NMR 8 160.4 156.7 (C 154.0 (OCONH), 150.2 (C 148.6 C 141.7 (C 3a), 140.6 (C 138.9 (C 133.9 (C 132.2 (C 129.8 (C 129.6 (C 9a), 128.8 (C 6"1), 127.5 (C 125.7 (C 125.0 (C 123.6 (C 123.2 (C 122.4 (C 6, C 9b), 121.6 (C 5a), 115.7 (C 112.2 (C 106.5 (C 106.1 (C 97.7 (CV4), 67.1 (CH 2 66.8 (2 x CH 2 61.9 (CH 2 61.5 (OCH 3 6 1.1 (OCH 3 56.3 (OCH 3 5 5.2 (C 54.9
(CH
2 53.6 (2 x CH 2 45.8 (CH 2 Cl), 43.4 (C 26.1 (CH 2 MS (FAB~) m/z 788 IH, 790 WMH, HRMS (FAB~) calc. for C40H 43 35 C1N 5
O,
0 (lIW) in/z 788.2699, found 7 88.272 1; caic. for C0H 43 37 C1N 5 O1 0 (1IH) mnk 790.2699, found 790.2728; Anal.
(C
40
H
42 C1N 5 0 10
'A
2 O) C, H, N.
Example 4L. Preparation of 2-3(-opoiy~rpx]-irbny bis(3-1I(5methy1l4-acridinyl)carbonyliaiino)propyl)carbamate (107). DIEA (0.3 inL, 3 mmol) was added to a suspension of carbaniate dihydrochioride 91 (44 ing, 0.5 iniol) in DCM niL) at 5 'C and the mixture stirred for 10 min. Methanesulfonyl chloride 1 niL), was added and the mixture stirred for 30 min. MeOH (2 mL) was added, the mixture stirred for 10 min, and the solvent evaporated. The residue was purified by chromatography on aluinina-9O, eluting with 1 %MeOH/40%EtOAc/DCM to give the crude mesylate 106 (42 ing, 95%) as a yellow solid, 'H NMR 8 11.94 1 H, NH), 11.85 1 H, NH), 8.94 (mn, 2 8.77 (in, 2 8.07 (in, 2 7.81 (in, 2 7.62 (in, 4 7.43 (in, 2 7.30 J= WO 00/64864 WO 0064864PCIIGBOOIOI 612 82 Hz, I H, H 3 7.05 J 8.4 Hz, I H, H 6 6.95 (dd, J 2.0 Hz, 1 H, H 5.02 2 H, CH 2 4.3 9 2 H, CH 2 3.98 2 3.73 -3.5 8 (in, 8 3.02 3 H,
CH
3
SO
2 2.85 3 H, GH 3 2.76 3 H, CH 3 2.20-2.05 (mn, 6 H).
A solution of mesylate 106 (42 mg, 0.047 nimol) in morpholine (0.5 mL) was stirred at *C for 20 hrs. The reaction mixture was purified by chromatography on alumina-90, eluting with I1%MeOH/40%/EtOAcJDGM to give 107 (3 8 mg, 9 as a yellow solid, 'H NMR 11.94 1 H, NH), 11.86 I1H, N 8.89-8.9 1 (in, 2 8.73-8.77 (mn, 2 8.06-8.08 (mn, 2 7.80-7.84 (in, 2 7.59-7.64 (in, 4 7.42-7.46 (in, 2 7.32 J 2.0 Hz, 1 H, H 3 7.06 J =8.4 Hz, 1 H, H 6.95 (dd, J= 8.4, 2.0 Hz, I H, H 5.03 2 H, CH4 2 00,.92 2 3.75-3.67 (in, 8 H, 4 x GH 2 3.59 (in, 4 H, 2 x GH 2 2.86 3 H, C14 3 2.74 3 H, CH 3 2.46 (br s, 6 H, CH1 2 2.18-2.04 (mn, 4 1.93 (br s, 2 H); HRMS (FAB*) caic. for (C 51
H
54
N
7 0 8 (Mff) ,n/z 892.4034, found 892.4055.
Example 4M. Preparation of 4-(2+Ij[bis(3-fl(5-methyl-4acridinyl)carbonyll amino propyl)a milcarboflyl) nitrophenoxy~butanoic acid (111).
Methyl 2-(4-tert-butoxy-4-oxobutoxy)-4-nItrobelzoate (108). A mixture of methyl 2hydroxy-4-nitrobenzoate (66) (0.61 g, 3.09 mmol) and K 2 C0 3 (0.64 g, 4.64 mmol) in DMF rnL) was stirred at 20 0 C for 30 min. A solution of tert-butyl 4-bromobutanoate (1.04 g, 4.64 minol) in DMF (5 mL) was added and the mixture stirred at 100 C for 6 h. The mixture was cooled to 20 0 C, poured into water (300 mnL) and extracted with EtOAc (3 100 rnL). The combined organic fraction was washed with water (2 50 mL), brine mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 20%EtOAcllight petroleum, to give 108 (0.86 g, 82%) as an oil, IR N 2978, 1728, 1532, 1350, 1285, 1250, 1155 cm-1; 'H NNM 8 7.89 J =8.5 Hz, 1 H, H 7.85 (dd, J 2.0OHz, 1 H, H5), 7.79 J =2.0OHz, I H, H 4.17 J =6.2Rz, 2 H,
CH-
2 3.93 3 H, OCH 3 2.47 J =7.3 Hz, 2 H, CH 2 CO), 2.11-2.16 (mn, 2 H, GHi) 1.49 9 H, CHO); 3 C NMRS8172.3 (GO 2 165.4 (C0 2 158.5 (C 150.7 (C 132.1 (C 126.2 (C 115.0 (C 107.9 (C 80.6 (OC(CH 3 3 68.4 (CH 2 52.5 (OCH 3 31.5 (CH 2 28.1 (OC(-CH 3 3 24.4 (CH 2 MSmlnz 3 39 308 197 87 57 (100); HRMS caic. for C,6,H 21 NO, m/z 339.1318, found 339.1314.
2-(4-ter1-Butoxy-4-oxobutoxy)4-litrobelzoic acid (109). A mixture of ester 108 (0.61 g, BNSOiD: <WO 0064864AI I> WO 00164864 PTG0111 83 1.80 mmol) and NaOH (I M, 9 mL, 9 mmol) in MeOH (20 mL) was stirred at 20 0 C for 2 h. The mixture was extracted with diethyl ether (20 mL) and the pH adjusted to 4 with 1 M HC1. The mixture was extracted with EtOAc (3 50 mL), the combined organic fraction washed with brine (50 mL), dried and the solvent evaporated. The residue was crystallized to give 109 as a white solid, mp (EtOAc/light petroleum) 76-77 JR N 1723, 1678, 1532, 1350, 1260, 1154 'H NMR [(CD 3 2 S0] 8 13.0 (br s, 1 H, CO 2 7.80-7.86 (in, 3 H, H 3, H 5, H 4.20 J =6.2 Hz, 2 H, CH 2 2.40-2.46 (in, 2 H, CH 2 CO), 1.92-1.99 (in, 2 H, CHO), 1.40 9 H, CH 3 1 3 C NMR [(CD 3 2 S0] 8 174.0 (COD., 171.8 166.2 (C 157.0 (C 13 0.9 (C 128.3 (C 115.1 (C 107.9 (C 79.7 (OC(CH 3 3 68.0 (CH 2 3 1.0 (CHO), 27.7 (OC(C-H 3 3 24.0 (CO 2 Anal. (C,,H,gN0 7 .1/H 2 0) C, H, N.
tert-ButyI 4
-I
2 -(hydroxymethy1)-5-flitrophefloxylbutanoate (110). BH 3 .DMS (0.28 mL, 2.8 ramol) was added to a stirred solution of acid 109 and trimethyl borate (0.64 mL, 5.66 nimol) in THF (50 mL) and the solution heated at reflux temperature under N 2 for 3 h. The solution was cooled to 20 MeOH (2 mL) added carefully and the mixture stirred for min. Water (2 niL was carefully added, the solution stirred for 5 min, I M aq. citric acid solution added and the mixture stirred for 3 0 min. The mixture was extracted with EtOAc (3 60 mL), the combined organic fraction washed with water (50 niL), brine (30 mL), dried, and the solvent evaporated. The residue was purified by chromatography, elutiing with a gradient (3 0-50%) of EtOAc/light petroleum, to give 110 (0.30 g, 68%) as a white solid, mp (EtOAc/light petroleum) 42-43 IR N 3434, 1726, 1524, 1346, 1248, 115 5 cmf I H NMR 8 7.85 (dd, J= 8.2,2.1 Hz, 1 H, H 7.68 J= 2.1 Hz, 1 H, H 7.53 J= 8.2 Hz, 1 H, H 4.76 J =4.6 Hz, 2 H, CH 2 4.12 J= 6.2 Hz, 2 H, CH 2 2.54 (br t, J= 5.0 Hz, 1 H, OH), 2.45 J= 7.2 Hz, 2 H, CH 2 CO), 2.1 1-2.18 (mn, 2 H, CH 2 1.50 (s, 9 H, 0113); 3 C NMR 8 172.3 (COO), 156.2 (C 148.1 (C 136.8 (C 127.0 (C 6), 1160 5) 15.7(C (O(C 3 3 67.7 (CH 2 60.6 (CH 2 31.9 (CH 2 28.1 (OC(C-H 3 3 24.3 (CH 2 MS (CI, NH 3 m/z 329 (M*+NH 3 312 273 (100); HRMS (CL, NH 3 calc. for C 1 ,HNO 6 (MHRI m/z 312.1447, found 312.1448. Anal.
(C,
5
HNO
6 C, H, N.
4-(2+R{[Bis(3-fI(5-methyl- 4 acridinyl)carbofll amino) propyl)ainfol carbonl) oxy) nitrophenoxy)bitaloic acid (113). A solution of alcohol 110 (19 mng, 0.06 inmol) and WO 00/64864 WO 0064864PCT/GBOO/01612 84- DIFA (25 pL, 140 jimol) in DCM (2 mL) was added dropwise to a solution of triphosgene mg, 35 4smol) in DCM (1.5 mL) over 30 minutes at 5 *C and the solution stirred for I hr. A solution of NN-bis[3-(5-methylacridine-4-carboxamido)propyl]amine (111) A.
Gamage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, J. Med.
Chiem., 1999, 42, 2383-2393] (24 mg, 42 /2mol) and DIEA (4 j2L, 240 pmol) in DCM (2 mL) was added and the solution stirred at 20 *C for 16 h. The solvent was evaporated and the residue was purified by chromatography on alumina-90, eluting with l%MeOHI6O%EtOAc/DCM to give crude ester 112 (34 mg, 89%) as a yellow foam, IH NMvlR 5 11.90 1 H, NH), 11.86 I H, NH), 8.85-8.95 (in, 2 8.66-8.76 (in, 2 H), 8.02-8.08 (in, 2 7.78-7.87 (in, 2 7.50-7.64 (in, 4 7.3 8-7.46 (in, 2 7.25 J Hz, 1 H, H 7.01 J =8.3 Hz, 1 H, H 6.89 (dd, J 2.0 Hz, 1 H, H 4.77 2 H, CH 2 3.84-3.88 (in, 2 3.58-3.73 (mn, 8 2.83 3 H, CH 3 2.73 3 H, CH 3 2.42-3.46 (in, 2 2.10-2.17 (in, 4 2.00-2.02 (in, 2 1.45 9 3
C
NMR 8 172.2, 166.2, 156.2, 155.6, 155.3, 147.4, 147.0 145.2 137.9 136.9, 13 5.8, 135.4 135.2, 132.6, 132.2 131.0 127.9 126.5, 126.4, 126.2, 126.1 125.8 125.3, 116.0, 115.1, 105.7, 104.8, 67.7, 61.6, 60.5, 45.9, 45.1, 37.7, 37.0, 31.9, 31.6, 29.2, 28.6, 28.1, 24.3, 24.3, 18.9, 18.8.
Ester 112 (80 mg, 88 !inmol) was added to a HCI saturated solution of MeOH (20 mL) and DCM (20 mL) at 5 0 C and the solution stood for 3 days at 5 The solvent was evaporated and the residue crystallized to give 113 (45 mng, 58%) as a yellow solid, mp (MeOHfEtOAc/light petroleum) 128-130'C; 'H NMR [(CD 3 2 50] 8 11.39 2 H, 2 CONH), 9.15 1 9.08 1 8.68 (br s, 2 8.25 (br s, 2 7.95 (br s, 2 7.65 (br s, 41H), 7.49 (br s, 2 7.32 J =2.0 Hz, 1 7.00 J =8.3 Hz, 1 6.93 (dd, J 8.3, 2.0 Hz, 1 4.77 2 H, CH 2 3.88-3.9 1 (in, 2 3.50-3.58 (in, 8 2.77 3 H, CH 3 .66 3 H, CH 3 2.01 (br s, 4 1.87-1.93 (in, 2 Anal.
(C
48 H46N 6
O
9 .2HC1.2H 2 O) C, H, N.
Example 5A. Preparation of (1-methyl-2-nitro-lH-iinidazol-5-yI)m ethyl bis(2chloroethyl)carbamate (117).
I-Methyl-2-nitro-1H-imdazole-5-carboxylic acid (114). Sodium hydroxide solution (1 M, 125 ml, 125 innol) was added slowly to a stirred suspension of ethyl I -methyl-2-nitro- -carboxylate (114) Cavalleri, R. Ballotta, G.C. Lancini. J. Heterocyclic Chem. 1972, 9, 979] (5.0 g, 25.1 minol) in water (50 mL) and the mixture stirred at 20 'C BNSDOCID: <WO 0064860A1 1 WO 00/64864 PCT/GB0/01612 until complete dissolution occurred. The pH of the solution was adjusted to 3 with 5 N HCI and the mixture extracted with EtOAc (3 x 100 mL). The combined organic fractions were dried and the solvent evaporated to give 114 (4.29 g, 100%), as white crystals, mp 160-161 *C (lit. Cavalleri, R. Ballotta, V. Arioli, G.C. Lancini, J. Med. Chem. 1973, 16, 557) (EtOAc) 161-163 'H NMR [(CD 3 2 SO] 8 13.60 (br s, 1 H, CO 2 7.37 1 H, H 4), 4.20 3 H, NCH 3 "C NMR [(CD 3 8 160.3, 147.2, 133.7, 127.0, 35.0.
(1-methyl-2-nitro-1H-imidazol-5-yl)methanol (115). A solution of CDI (7.0 g, 43.1 mmol) and 114 was stirred at 20 *C for 30 min and then added to a stirred solution of NaBH 4 (4.07 g, 108 mmol) in EtOH (10 mL) and the mixture stirred at 20 °C for 1 h. 5 M HCI (20 mL) was added carefully and the mixture stirred for 30 min. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc, to give 115 (2.23 g, 68%) as a white solid, mp 138-140 "C (lit. Cavalleri; R. Ballotta; V. Arioli; G.C. Lancini, J. Med. Chem. 1973, 16, 557] 142-144 'H NMR 2 SO] 87.12 1 H, H 5.49 (br s, 1 H, OH), 4.55 2 H, CH20), 3.92 3 H, NCH 3 "C NMR
[(CD
3 2 SO] 8 145.6, 138.6, 126.5, 52.9, 34.0.
(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl 4-nitrophenyl carbonate (116). A solution of 4-nitrophenylchloroformate (0.67 g, 3.34 mmol) in THF (5 mL) was added to a stirred solution of alcohol 115 (0.50 g, 3.18 mmol) and pyridine (283 yL, 3.50 mmol) in THF mL) at 20 OC under N 2 The solution was stirred at 20 *C for 16 h, the solvent evaporated and the residue dissolved in EtOAc (100mL). The solution was washed with water (2 x mL), brine (50 mL), dried and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 116 (0.87 g, 84%) as a white solid, mp (EtOAc) 156.5-1 57 5 IR N 1771, 1537, 1359 cm'; 'H NMR
[(CD
3 2 SO] 8 8.33 (ddd, J= 9.1, 3.2, 2.1 Hz, 2 H, H 3, H 7.59 (ddd, J= 9.1, 3.2, 2.1 Hz, 2 H, H 2, H 7.37 1 H, H 5.48 2 H, CH20), 4.00 3 H, NCH 3 "C NMR
[(CD
3 2 SO] 8 155.1, 151.4, 146.3, 145.2, 131.5, 129.6, 125.4 122.5 59.4, 34.3; Anal. (C, 2
HN
4 0 7 C, H. N calc. 17.4, found 16.7%.
(1-Methyl-2-nitro-H-imidazol-5-yl)methyl bis(2-chloroethyl)carbamate (117). A solution of carbonate 116 (0.68 g, 2.11 mmol) in pyridine (3 mL) was added to a solution ofbis-(2-chloroethyl)amine hydrochloride (0.75 g, 4.22 mmol) in pyridine (30 mL) under WO 00/64864 WO 0064864PCT/GBOO/01612 86
N
2 The solution was stirred at 20 'C for 16 h and the solvent evaporated. The residue was dissolved in DCM (100 mL) and washed with 2% citric acid solution (2 x 50 mL), water mL), brine (50 mL), dried and the. solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 117 (0.519g, 74%) as white crystals, mp (EtOAc) 100- 10 1 IR N 1703, 1489, 1344 cm'; 'H NM~R 6 7.23 I H, H 5.21 2 H, CH 2 4.05 3 H, NCH 3 3.58-3.70 (in, 8 H, 2 x CH 2 N, 2 x
CH
2 Cl; 3 C NMvR 6 154.8, 144.5, 132.1, 129.7, 56.2, 50.8 41.6 34.3; Anal.
(C,
0 H1 4 C1 2
N
4 0 4 C, H, N.
Example 5B. Preparation of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl 4-[bis(2chloroethyl)aminolphenylcarbamate (123).
N,N-Bis(2- I[tert-butyl(dimetbyl)silyll oxy) ethyl) 4-nitroaniline (119). A solution of TBDMSC1 (4.20 g, 27.9 minol) in DMF (15 mL) was added to a stiffed solution of NNbis(2-hydroxyethyl)-4-nitroaniline (118) (3.0 g, 13.26 inmol) and imidazole (3.79 g, 55.7 minol) in DMF (50 mL) and the solution stirred at 20 *C for 48 h. The solvent was evaporated and the residue partitioned between EtOAc (150 mL) and water (150 mL). The organic fraction was washed with water (2 x 200 mL), dried, and the solvent evaporated.The residue was purified by chromatography, eluting with 10% EtOAc/light petroleum, to give 119 (5.72 g, 95%) as a white solid, mp (pet. ether) 48-49 IR N 1597, 1520, 1300, 1202, 1107 cm'; 'H NMvR 6 8.07 (ddd,J= 9.5, 3.5, 2.1 Hz, 2 H, H 3, H 6.67 (ddd, J 9.5, 3.5, 2.1 Hz, 2 H, H 2, H 3.80 (dd, J 6.0, 5.7 Hz, 4 H, 2 x CH 2
N),
3.63 (dd, J= 5.9, 5.7 Hz, 4 H, 2 x CH 2 0.86 18 H, 2 x SiC(CH 3 3 0.01 12 H, 2 x Si(CH 3 1 3 C NMR 8 153.0, 13 8.6, 126.2 110.4 60.2 53.6 25.8 18.2 -5.5 MS (DEI) m/z 454 439 397 309 (100); HRMS (DEI) calc. for C22H 42
N
2
O
4 Si 2 m/z 454.2683, found 454.2668; Anal. (C 22
H
42
N
2
O
4 Si 2 C, H,
N.
N
1 ,'-Bis(2-{[tert-butyl(dimethyl)silyl] oxy~ethyl)-1,4-benzenediamine (120). A mixture of 119 (1.54 g, 3.39 inmol) and Pd/C (50 mg) in EtOAc/EtOH (50 mL) was stirred under hydrogen (60 psi) for 30 minutes, filtered through celite, washed with EtOH (2 x mL) and the solvent evaporated to give crude benzenediamine (120) as an oil that was used directly without further purification or characterization.
BNSDOCID: <WO 0064B64AI I> WO 00/64864 PCT/GBOO/01612 87 butyl(dimethyl)siIylloxy)ethyl)aminol phenylcarbamate (121). A solution of carbonate 116 (0.87 g, 2.68 mmol), 120 (3.39 mmol), and pyridine (217 AL, 2.68 mmol) in THF mL) was stirred at 20 'C for 48 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The organic fraction was washed with water (2 x 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (20-50%) of EtOAcflight petroleum to give 121 (1.37 g, 84%) as a white solid, mp 143-144 IR N 3258, 1721, 1539, 1257, 1103 cm'; 1H NMR [(CD 3 2 .S0] 7.23 1 H, H 7.15 (br d, J= 8.9 Hz, 2 H, H 3, H 6.63 J= 8.9 Hz, 2 H, H 2, H 6.52 (br s, 1 H, OCONIH), 5.20 2 H, CH 2 4.06 (s, 3 H, NCH 3 3.73 (dd, J 6.3 Hz, 4 H, 2 x CH 2 3.47 (dd, J 6.3 Hz, 4 H, 2 x
CH
2 0.88 18 H, 2 x Si(C(CH 3 3 0.02 12 H, 2 x Si(CH 3 2 3 C NMR [(CD 3 2 S0] 8 152.9, 146.1, 145.4, 132.5, 129.6, 125.4, 121.7 111.8 60.3 55.2, 53.6 34.3, 26.0 18.2 -5.4 Anal. (C 2 gH~qN 5
O
6 Si 2 C, H, N.
hydroxyethyl)amilphefllcarbamate (122). TBAF (1 M in THE, 3.9 xnL, 3.9 mmol) was added to dropwise to a stirred solution of 121 (1.07 g, 1.76 mmol) in THF (30 mL) at The solution was stirred for 30 minutes and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient 10%) of MeOHIEtOAc, to give 122 (0.59 g, 88%) as a white solid, mp (MeOH) 171-174 IR N 3445, 3329, 3266, 1717, 1609, 1549, 1491, 13 75, 1248 cm- 1 'H NMR [(CD 3 2 S01 8 9.37 1 H, OCONH), 7.28 (s, 1 H,I-H 7.19 (br d, J =9.1 Hz, 2 H, H 3, H 6.61 J= 9.1 Hz, 2 H, H 2, H 5.23 2 H, CH 2 4.71 J 5.4 Hz, 2 H, 2 x OH), 3.96 3 H, NCH 3 3.51 (dd, J 6.4, 5.9 2 5 Hz, 4 H, 2 x CH 2 3.34 (dd, J 5.9 Hz, 4 H, 2 x CH 2 13C NMR f(CD 3 2 S0) 8 152.8, 146.0, 144.2, 13 3.6, 128.6, 127.1, 120.3 111.4 5 9.7, 5 8.2 53.4 34.2; MS (FAB-) m/z 380 348 HRMS (FABV) m/z calc. for CI 6 H22N 5
O
6
(MW)
380.1570, found 380.1579; Anal. (C, 6
H
2
,N
5 0 6 C, H, N.
(1Mty--ir-Hiiao--lmty 4-Ibis(2chloroethyl)amillphefllcarbamate (123). Methanesulfonyl chloride (191 AtL, 2.47 mmol) was added dropwise to a stirred solution of diol 122 (312 mg, 0.82 mmol) in pyridine (10 mL) at 5 0 C and the solution stirred at 20 0 C for 1 h. The solvent was WO 00/64864 PCT/GB00/01612 -88evaporated and the residue partitioned between DCM (100 mL) and water (100 mL). The aqueous fraction was washed with DCM (2 x 50 mL), the combined organic extracts dried, and the solvent evaporated. The residue was dissolved in DMF (10 mL), LiCI (210 mg, 4.9 mmol) added and the mixture stirred at 80 *C for 3 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The aqueous fraction was extracted with EtOAc (2 x 50 mL), the combined extracts dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 123 (227 mg, 66%) as a white solid, mp (MeOH) 156-157 IR N 3408, 3246, 1725, 1531, 1354, 1221 'H NMR [(CD 3 2 SO] 5 9.49 1 H, OCONH), 7.26-7.29 3 H, H H 3, H 6.70 J= 9.1 Hz, 2 H, H 2, H 5.24 2 H, 3.96 3 H, NCH 3 3.64-3.70 8 H, 2 x CH 2 N, 2 x CH 2 CI); "C NMR [(CD 3 2
SO]
152.8, 146.0, 142.3, 133.5, 128.7, 128.6, 120.3 112.3 54.9, 52.2 41.1 34.1.
Anal. (C, 6
H,
9
CI
2
N
5 0 4 C, H, N, Cl.
Example 5C. Preparartion of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl 1- (chloromethyl)-3-[(5,6,7-trimethoxy-lH-indol-l-yl)carbonyl]-2,3-dihydro-lH- (124). A solution oftriphosgene (14.5 mg, 49 Amol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 J. Atwell, W. R.
Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (58 mg, 124 gmol) and Et 3 N (39 mL, 280 pmol) in DCM (10 mL) and stirred at 20 °C for 2 h. A solution of alcohol 115 24 mg, 154 panol) in DCM (2 mL) was added, followed by nBuzSn(OAc) 2 (2 drops) and the solution stirred at 20 *C for 48 h. The solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAc/light petroleum, to give 124 mg, 68%) as a tan powder, mp (EtOAc) 202-204 'H NMR 8 9.49 (br s, 1 H, indole- NH), 8.81 (br s, 1 H, H d, J= 8.5 Hz, 1 H, H 7.78 J= 8.3 Hz, 1 H, H 7.57 (m, 1 H, H 7.43 1 H, H 7.25 1 H, H 7.21 (br s, 1 H, OCONH), 7.00 J= 1.6 Hz, 1 H H 6.87 1 H, H 5.31 J= 13.6 Hz, 1 H, CH20), 5.25 J= 13.6 Hz, 1 H, CH20), 4.80 (dd, J= 10.5, 1.6 Hz, 1 H, H 4.65 (dd, J= 10.5, 8.7 Hz, 1 H, H 4.13- 4.20 1 H, H 4.11 3 H, OCH 3 4.01 (br s, 3 H, NCH 3 3.94-3.98 4 H, CH 2
CI,
OCH
3 3.92 3 H, OCH 3 3.47 (dd, J= 10.8, 10.8 Hz, 1 H, CH 2 3 C NMR 8 160.4 153.5 (OCONH), 150.2 (C 146.4 (C 141.6 (C 3a), 140.7 (C 138.9 (C 133.3 (C 132.1 (C 129.8 (C 129.7 (C 9a), 129.5 (C 127.6 (C 125.7 (C 125.1 (C 7, C 5a), 123.6 (C 123.2 (C 122.3 (C 6, C 9b), 113.0 (C 106.6 8NSDOCID: <WO 0064864A1 I> WO 00/64864 PCT/GBOO/01612 89 (C 97.6 (C 61.5 (OCHA) 61.2 (OCH 3 56.3 (OCH 3 55.8 (CH 2 O0), 54.9 (C 45.8
(CH
2 CI), 43.4 (C 34.3 (NCH 3 MS (FAB') m/z 649 HRMS (FAB3') caic. for
C
31 H4 30 35 C1N 6 0 8 m/z 649.1814, found 649.1767; caic. for C 31
H
30 37 C1N 6 0 8 (Mr) m/z 651.1784, found 651.1819; Anal (C 3
,H
29 C1N 6 0 8 .'/2H 2 0) C, H, N.
Alternative preparation of (1-methyl-2fitroIHimidazol1SyI)methyI 1- (chloromethyl)-3(5,6,7trimethoxylHindo-11y1)carbonyl]- 2 3 dihydroIHbenzole] indol-5-y1carbamate (124). A solution of alcohol 115 (17 mg, 0. 11 mmol) in DCM (2 mL) was added dropwise to a stirred solution of triphosgene (12 mg, 0.04 mmol) and pyridine (9 kzL, 0.11 mnmol) in DCM (2 mnL) at 20 The mixture was stirred at 20 0
C
for 2 h, the solvent evaporated and the residue dissolved in THE (5 mL). A solution of amine 1 J. Atwell, W. R Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (50 mg, 0. 11 mmol) in THF 5 mnt was added and the solution stirred at 20 0 C for 16 h. The mixture was partitioned between EtOAc (50 mL) and sat. aq. KHCO 3 solution, the organic fraction dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (50-100%) of EtOAc/light petroleum to give 124 (23 mg, 33%) as a tan solid, mnp 200-205 *C (dee); spectroscopically identical with an authentic sample prepared above.
Example 5D. Preparation of (1mty--fr-Hiiao--lmty doxorubicin carbamnate (125). A solution of (1mty--ir-Hiiao--lmty 4-nitrophenyl carbonate (116) (33 mg, 104 pumol) in DMF (2 mL) was added dropwise to a stirred solution of doxorubicin (13) (46 mg, 86 Mmol) and Et 3 N (15 pL, 104 prnol) in DMF (5 mL) at 20 0 C and the solution stirred for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient of MeOH/DCM, to give 125 (44 mg, 70%) as a red solid, mnp (DCM) 162-166 C; 'H NMR [(CD 3 2 S0] &13.96 1 H, 6-OH), 13.21 1 H, 11I-OH), 7.82-7.87 (in, 2 H, H 1, H 7.58 (dd, J= 7.5, 2.1 Hz, 1 H, H 7.18 I H, H 7.02 J =7.9 Hiz, I H, OCONH), 5.42 1 H, 9-OH1), 5.21 J 2.6 Hz, 1 H, H 5.07 2 H, CH 2 4.86-4.91 (mn, 2 H, H 7, 14-OH1), 4.73 J 5.9 Hz, 1 H, 4-OH), 4.58 J 5.9 Hz, 2 H, H 14), 4.13-4.17 (in, I H, H 3.96 3 H, 4- OCHA) 3.88 3 H, NCH 3 3.66-3.74 (in, 1 H, H 3.4 1-3.46 (in, 1 H, H 2.97 J 18.3 Hz, 1 H, H 10), 2.87 J 18.3 Hz, 1 H, H 10), 2.21 J =14.0 Hz, I H, H 8), WO 00/64864 WO 0064864PCT/GBOO/01 612 90 2.17 (cd, J= 14.0, 5.41Hz, 1 H, H 1.84 (dt, J 12.8, 3.5 Hz, 1 H, 1.57 (dd, J= 12.8, 3.8 Hz, 1 H, H 1. 12 J 6.4 Hz, 3 H, H 6 );1 3 C NMR [(CD 3 2 S0] a 213.7 (C 13), 186.3 (C 186.2 (C 12), 160.7 (C 156.0 154.6 (C 11), 154.4 (OCONH), 145.8 (C 136.1 (C 135.4 (C 12a), 134.5 (C 6a), 134.0 (C 1lOa), 133.8 (C 128.3 (C 119.8 (C 4a), 119.6 (C 118.9 (C 110.6 (C 5a), 110.5 (C I 100.3 (C 74.9 (C 69.8 (C 67.8 (C 66.6 (C 63.7 (C 14), 56.5 (4-OCH 3 54.7 (CH 2 47.3 (C 38.4 (C 34.1 (NCHA) 32.0 (C 10), 29.7 (C 17.0 (C MS (FAB') m/z 727 (MW, HRMS (FAB) caic. for C 33 H34N 4
O,
5 (MW) m/z 727.2099, found 727.2075; Anal (C 33 H34N 4
O,
1
H
2 O) C, H, N.
Example 5E. Preparation of 4-(f(1-methyl-2-nitro-1H-imidazol-5yl)methoxyl carbonyl~amino)benzyI doxorubicin carbamnate (129).
butyl(dimethyl)silylloxy~methyI)pheflylcarbamate (126). Et 3 N (0.26 mL, 1.86 imnol) was added to a stirred suspension of (1-methyl-2-nitro-IH-imidazol-5-yl)methyI 4nitrophenyl carbonate (116) (0.50 g, 1.55 mmol), 4-({jtertbutyl(dimethyl)silyl]oxy~methyl)afliline (0.40 g, 1.71 nimol), HOBT (0.21 g, 1.55 nimol), and 4 A molecular sieves (500 mig) in THF (80 niL) and the mixture stirred at *C for 16 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 niL). The organic fraction was washed with 1 M HCI (2 x 40 niL), water (100 niL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 40% EtOAc/light petroleum, to give 126 (0.43 mg, 66%) as a white solid, mp (EtOAc/light petroleum) 13 1-132 0 C; 'H NMR 8 7.3 3 (br di, J =8.8 Hz, 2 H, H 3, H 7.27 (di, J =8.8 Hz, 2 H, H 2, H 7.23 1 H, H 6.83 (br s, 1 H, OCONH), 5.22 2 H, CH 2 4.69 2 H, CH 2 4.05 3 H, NCH 3 0.93 9 H, SiC(CH 3 3 0.09 6 H, Si(CH 3 2 1 3 C NMVR 8 152.3 (OCONH), 146.1 (C 137.4 (C 1), 135.8 (C 132.5 (C 129.6 (C 126.9 (C 2, C 118.8 (C 3, C 64.5 (CH 2
O),
55.4 (CH 2 34.3 (NCH 3 25.9 (SiC(CH 3 3 18.4 (SiC(CH 3 3 -5.3 (Si(CH 3 2 Anal.
(CIPHUNSNOC, H, N.
(1.Methy1-2-nitro-1H-imidazo1-5-yI)methyI 4-(hydroxymethyl)phenylcarbamate (127).
1 M HCI (2 niL, 2 mmol) was added to a stirred solution of silyl ether 126 (0.39 g, 0.93 nimol) in MeOH (10 mL) and stirred at 20 *C for 1 h. The solution was poured into brine BNSOOCID: <WO 0064864AI I WO 00/64864 PCT/GB00/01612 -91mL) and extracted with EtOAc (3 x 50 mL). The combined organic fraction was washed with water (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (50-100%) EtOAc/light petroleum, to give 127 (247 mg, 87%) as a pale yellow solid, mp (EtOAc) 180-181 OC; 'H NMR [(CD 3 8 9.77 (br s, 1 H, OCONH), 7.40 J= 8.5 Hz, 2 H, H 3, H 7.31 1 H, H 7.22 J 8.5 Hz, 2 H, H 2, H 5.27 2 H, CHO2), 5.08 J= 5.6 Hz, 1 H, OH), 4.42 J= 5.6 Hz, 2 H, CH20), 3.97 3 H, NCH 3 3 C NMR [(CD 3 2 SO] 8 152.6 (OCONH), 146.0 (C 137.2 (C 136.8 (C 133.3 (C 128.7 (C 127.0 (C 2, C 118.0 (C 3, C 62.4 (CH0O), 55.0 (CH2O), 34.2 (NCH 3 Anal. (C,H, 4
N
4 0s) C, H, N.
4 -({[(l-methyl-2-nitro-1H-imidazol-5-yl)methoxylcarbonyl}amino)benzyl 4nitrophenyl carbonate (128). A solution of 4-nitrophenylchloroformate (216 mg, 1.07 mmol) in THF (5 mL) was added dropwise to a stirred solution of alcohol 127 (219 mg, 0.72 mmol) in THF (40 mL) and the solution stirred at 20 *C for 96 h. The solvent was evaporated and the residue partitioned between EtOAc (100 ml) and water (100 mL). The organic fraction was washed with water (2 x 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 128 (62 mg, 18%) as a white solid; 'H NMR [(CD) 2 SO] 5 9.96 1 H, OCONH), 8.31 (ddd J= 9.2, 3.3, 2.2 Hz, 2 H, H H 7.56 (ddd, J= 9.2, 3.3, 2.2 Hz, 2 H, H H 7.51 J= 8.6 Hz, 2 H, H 3, H 7.40 J= 8.6 Hz, 2 H, H 2, H 7.31 1 H, H 5.33 2 H, CH20), 5.24 2 H, CH20), 3.98 3 H, NCH 3 'C NMR
[(CD
3 2 SO] 5 155.2 (OC02), 152.6 (OCONH), 151.9 (C 145.1 (C 139.2 (C 1), 133.2 (C 129.6 (C C 128.8 (C 128.7 (C 125.6 (C 2, C 122.2 (C C 118.8 (C 2, C 70.2 (CH20), 55.2 (CH20), 34.2 (NCH 3 MS (FAB+) m/z 472 443 HRMS (FAB") calc. for C, 2
H,,N
5 0 (MHI) m/z 472.1105, found 472.1106.
4-(([(1-Methyl-2-nitro-H-imidazol-5-yl)methoxylcarbonyl}amino)benzyl doxorubicin carbamate (129). A solution of carbonate 128 (81 mg, 172 /mol) in DMF (2 mL) was added dropwise to a stirred solution of doxorubicin (45 mg, 86 imol) and Et 3 N (15 AL, 103 fimol) in DMF (5 mL) at 20 OC and the solution stirred for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 5% MeOH/DCM, to give 129) (57 mg, 75%) as a red solid, mp (DCM) 160-162 'H NMR [(CD 3 2
SO]
WO 00/64864 PCT/GB00/01612 -92- 13.99 1 H, 6-OH), 13.24 1 H, I 1-OH), 9.82 1 H, OCONH), 7.84-7.89 2 H, H 1, H 7.60-7.63 1 H, H 7.40 J= 8.3 Hz, 2 H, H H 7.29 1 H, H 7.23 J= 8.3 Hz, 2 H, H H 6.81 J= 8.0 Hz, 1 H, OCONH), 5.43 1 H, H 7), 5.25 2 H, CH20), 5.21 J= 2.9 Hz, 1 H, H 4.89-4.91 1 H, 9-OH), 4.87 2 H, CH20), 4.84 (dd, J= 6.3, 5.8 Hz, 1 H, 14-OH), 4.69 J= 5.7 Hz, 1 H, 4-OH), 4.58 J= Hz, 2 H, H 14), 4.13-4.17 1 H, H 3.97 3 H, OCH 3 3.95 3 H, NCH 3 3.68-3.75 1 H, H 3.43-3.46 1 H, H 2.99 J= 18.3 Hz, I H, H 10), 2.91 18.3 Hz, 1 H,H 10), 2.21 (br d, J= 14.1 Hz, 1 H, H 2.10 (dd, J= 14.1 Hz, 1 H, H 1.84 (dt, J= 12.9, 3.6 Hz, 1 H, H 1.47 (dd, J= 12.9, 3.8 Hz, 1 H, H 1.13 J 6.5 Hz, 3 H, H 3 C NMR [(CD 3 2 SO] 5 213.7 (C 13), 186.4 (C 186.3 (C 12), 160.7 (C 156.0 (C 155.2 (C 11), 154.4 (OCONH), 152.6 (OCONH), 146.0 (C 138.2 (C 136.1 (C 135.4 (C 12a), 134.6 (C 6a), 134.0 (C 10a), 13.2 (C 131.2 (C 128.7 (C 128.6 (C C 119.9 (C 4a), 119.6 (C 118.9 (C 118.0 (C C 110.7 (C 5a), 110.5 (C la), 100.2 (C 74.9 (C 69.8 (C 67.9 (C 66.6 (C 64.8 (C 14), 63.6 (CH20), 56.5 (OCH 3 55.1 (CH20), 47.0 (C 36.5 (C 34.1
(NCH
3 32.0 (C 10), 29.8 (C 16.9 (C MS (FAB') m/z 876 (MW, Anal.
(C
4
,H
4 1 NsO0 7
.H
2 0) C, H, N.
Example 5F. Preparation of (1-methyl-4-nitro-1H-imidazol-5-yl)methyl 1- (chloromethyl)-3-[(5,6,7-trimethoxy-lH-indol-l-yl)carbonyll-2,3-dihydro-1H- (132).
(1-Methyl-4-nitro-lH-imidazol-5-yl)methanol (131). Ozone was bubled through a solution of 1-methyl-4-nitro-5-[(E)-2-phenylethenyl]-lH-imidazole (130) D. C. Baker, S.
R Putt, H. D. H. Showalter, J. Heterocyclic Chem., 1983, 30, 629-634.] (1.0 g, 4.36 mmol) in DCM/MeOH 120 mL) at -78 *C until a blue colour persisted. The solution was warmed to -40 *C with a nitrogen purge to remove excess ozone. A solution ofNaBH 4 (165 mg, 4.36 mmol) in EtOH (10 mL) was added dropwise over 15 min and the mixture stirred for 30 min. Acetic acid (0.5 mL) was added and the solvent evaporated. The residue was partitioned between water (50 mL) and light petroleum (50 mL). The aqueous fraction was evaporated and the residue triturated with hot acetone (60 mL). The mixture was filtered and the solution concentrated to give 131 (523 mg, 78%) as a tan powder, mp (acetone) 135-137 'H NMR 8 7.78 1 H, H 5.48 J 5.6 Hz, 1 H, OH), 4.85 (d, J= 5.6 Hz, 2 H, CH20), 3.75 3 H, NCH 3 "C NMR 8 143.5 (C 136.8 (C 133.2 (C BNSDOCID: <WO 0064864A1 I> WO 00/64864 PCT/GBOO/01612 -93 51.4 (CH 2 32.5 (NCH 3 Anal. (C 5
H
7
N
3 0 3 C, H, N.
(1-Methy14-fitro-Iimidazol5-yl)methyl I -(chloromethy1)-3-[(5,6,7-trimethoxy-1Hindol-l-yl)carbonyl2,3dihydroIHhbenzolelindolS5ylcarbamate (132). A solution of triphosgene (13 mg, 43 jumol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (51 mg, 109 ptmol) and Et 3 N (34 pL, 246 gmol) in DCM (10 mL) and stirred at 0 C for 2 h. A solution of (l-methyl4-nitro-1HimidazolS5yl)methanoI 131 (23 mng, 147 pgmol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAC) 2 (2 drops) and the solution stiffed at 20 0 C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc, to give 132 (40 mng, 56%) as a white powder, mp (EtOAc/light petroleum) 229-231 'H NMR 8 9.48 1 H, indole-NH), 8.87 1 H, OCONH), 7.87 J =8.5 Hz, 1 HK H 7.76 J =8.3 Hz, I H, H 7.55 (ddd, J 8.3, 7.4, 0.7 Hz, 1 H, H 7.44 (ddd, J 7.4, 0.7 Hz, 1 H, H 7.40 1 H, H 7.33 (br s''IH, H4), 7.00 J =2.
3 Hz, 1 H, H3Y), 6.87( s, 1 H, H 4% 5.61 2H, CH 2
O),
4.78 (dd, J =10.7, 1.7 Hz, 1 H, H 4.65 (dd, J =10.7, 8.7 Hz, 1 H, H 4.15-4.20 (in, 1 H, H 4.05-4. 10 (in, 4 H, OCH 3 CHPC), 3.95 3 H, OCH 3 .92 3 H, OCH 3 3.83 (br s, 3 H, NCH 3 .44 (dd, J= 10.9, 10.7 Hz, 1 H, CH 2 Cl); 1 3 C NMR 8 160.4 153.8 (OCONH), 150.2 (C 146.3 (C 141.5 (C 3a), 140.6 (C 138.8 (C 136.3 (C 133.4 (C 129.7 (C 9a), 129.5 (C 127.6 (C 126.9 (C 125.7 (C 125.2 (C 125.2 (C 5a), 123.6 (C 3a7), 123.1 (C 122.3 (C 121.9 (C 9b), 112.5 106.6 (C 97.7 (C 61.5 (OCH 3 61.1 (OCH- 3 56.3 (OCH 3 54.9 (C 54.4 (CH 2 45.8
(CH
2 Cl), 43.4 (C 33.2 (NCH 3 MS (FAB~) m/z 651 649
J{RMS
(FAB*) calc. for C 31
H
30 35 C1N 6 0 8 (M7W) mlz 649.1814, found 649.1818; calc. for
C
3
,H
30 37 C1N 6 0 8 (Wu) m/z 651.1784, found 651.1805; Anal. (C 3
,H
29 C1N 6 C, H, N.
Example 5G. Preparation of (1mty--ir-Hiiao--lmty bis(2chloroethyl)carbamate (135).
(l-MethyI-5fitrolHimidazo12-yI)methanoI (133). A mixture of 1-methyl-5-nitro- 1Himidazole Hazeldine, F.L. Pyman, J. Winchester. J Chem. Soc. 1924, 143 1] (1.0 g, 7.9 mmol) and paraformaldehyde (1.4 g, 15.7 mmol) in DMSO (10 mL) was heated in a sealed tube at 100 *C for 24 h. The mixture was cooled to 20 EtOH (50 mL) was added, and the suspension was filtered. The filtrate was concentrated and the residue was purified WO 00/64864 WO 0064864PCTGBOO/01 612 94 by chromatography on alumina, eluting with a gradient of MeOH/CHCl 3 to give 133 (0.71 g, 57%) as a white solid, mp (CHCl 3 116-118 *C (lit. Rufer, H. J. Kessler, E.
Schroder. J Med Chem. 1971, 14, 94] mp I1 I 0 'H NMR [(CD 3 2 S0] 6 7.90 1 H, H 5.62 J =5.8 Hz, I H, OH), 4.62 J =5.8 H-z, 2 H, CH 2 3.99 3 H, NCH 3 1 3
C
NMR [(CD 3 2 S0] 6 151.8, 138.8, 130.9, 56.1, 32.9.
(1-Methyl-5-nitro-1H-imidazol-2-yl)methyl 4-nitrophenyl carbonate (134). A solution of 4-nitrophenyl chioroformate (1.48 g, 7.4 mmol) in THF (8 mL) was added slowly to a stirred solution of alcohol 133 10 g, 7.0 mmol) and pyridine (0.62 mL, 7.7 mnxol) in THF (50 niL) at 20 *C under N 2 The mixture was stirred at 20 *C for 16 h, then partitioned between EtOAcJH 2 O. The organic fraction was washed with saturated aqueous NaHCO 3 mL), and the solvent evaporated to give 134 (2.04g, 94%) as a tan solid, mp (EtOAc/light petroleum) 168-171 0 C; 'H NMR [(CD 3 2 S0] 8.33 (ddd, J 9.1, 3.2, 2.2 Hz, 2 H, H3, H 8.11 1 H, H 7.5 9 (ddd, J 3.2, 2.2 Hz 2 H, H2, H6), 5.48 2 H, CH 2 3.97 3 H, NCH 3 1 3 C [(CD 3 2 S0] 155.0, 151.3, 145.9, 145.2, 139.5, 131.6, 125.3 122.4 61.7, 33.5.
(1-Metliyl-5-nitro-lH-imidazol-2-y)methyl bis(2-chloroethyl)carbamate (135). A solution of carbonate 134 (2.0 g, 6.5 nimol) in pyridine (5 m.L) was added to a stir-red solution of NN-bis-(2-chloroethyl)amine hydrochloride (1.5 g, 8.4 nimol) in pyridine niL) at 0 0 C. The solution was stirred at 20 0 C for 16 h, then solvent was evaporated and the residue was partitioned between DCM (100 niL) and 10% aqueous citric acid (100 niL).
The organic fraction was dried, the solvent evaporated, and the residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 135 (2.0 g, 95%) as an oil; 'H NMvIR 6 7.99 I H, H 5.27 2 H, CH 2 4.03 3 H, NCH 3 3.60-3.7 1 (in, 8 H, 2 x CH 2 N, 2 x CH1 2 1 3 C NNR 6 154.8, 146.9, 139.6, 132.2, 58.7, 51.1 41.8 39.7; HRMS caic. for C,,H, 4 C1 2
N
4 0 4 (M)m/z 324.1392; found 324.1381.
Example 5H. Preparation of (I-methyl-5-nitro-lH-imidazol-2-yl)methyI 4-[bis(2chloroethyl) amino] pbenylcarbamfate (137). Diphosgene (85mL, 0.7 nimol) was added dropwise to a stirred solution of (I-methyl-5 -nitro- IH-imidazol-2-yl)methanol (133) (0.2 g, 1.27 mmol) and Et 3 N (98 mL, 0.7 mmol) in THF (10 niL) at 5 TC. The suspension was stirred at 5 TC for 30 min and a mixture of A',M'-bis(2-chloroethyl)- 1,4-benzenedianiine BNSDOCIO <WO 0064864AI I WO 00/64864 PCT/GBOO/O1 612 hydrochloride (136) L. Everett, W. C. J. Ross. J Chem. Soc. 1949, 1972]1(0.38 g, 1.40 mmol) and Et 3 N (195 mL, 1.40 mniol) in THIF (4 mL) was added dropwise to the above suspension. The mixture was stirred at 20 0 C for 4 h, the solvent evaporated and the residue purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 137 (0.19 g, mp (CHCI 3 /pet ether) 164-164.5 IR n 3250, 3185, 3127, 1723, 1603, 1547, 1516, 1381 cm- 1; 'H NMR ([(CD 3 2 S0] 8 9.57 (br s, I H, OCONH), 8.08 I H, HW4), 7.27 (br d, J =8.0 Hz, 2 H, H 3, H 6.70 J =9.1 Hz, 2 H, H 2, H 5.26 2 H, CH 2
O),
3.95 3 H, NCH 3 3.65-3.72 (in, 8 H, 2 x CH 2 N, 2 x CH 2 Cl); 1 3 C NMR 2 S0] 8 152.7, 148.0, 142.3, 139.3, 131.7, 128.7, 120.3 112.3 57.4, 52.2 41.1 33.4; MS (DEL) m/lz 415 366 316 258 211 209 (100); HRMS (DEL) m/z caic. for C, 6
H,
9 35 C1 2
N
5 0 4 415.0814, found 415.0808; calc. for C,,H, 9 35 C1 37 C1N 5 0 4 417.0785, found 417.0781; calc. for C, 6
H,
9 37 C1 2
N
5 0 4 419.0755, found 419.0769; Anal. (C, 6
H,
9 C1 2
N
5 0 4 C, H, N.
Example 51. Preparation of (1mty--ir-Hiiao--lmty 1- (clrmty)3[567tiehx-Hidl2y~abnl-,-iyr-H (138). A solution of triphosgene (12 mg, 41 gimo1) in DCM (2 niL) was added dropwise to a stirred solution of amine 1 3. Atwell, W. R. Wilson, W.
A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (48 mg, 103 Mimo1) and Et 3 N (32 gsL, 231 jismol) in DCM (10 niL) and stirred at 20 'C for 2 h. A solution of lH-imidazo1-2-yl)methanol (133) (20 mg, 127 gsmol) in DCM (2 niL) was added, followed by nBu 2 Sn(OAc), 2 (2 drops) and the solution stirred at 20 0 C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAcIDCM, to give 138 (23 mg, 34%) as a tan powder mp (EtOAc) 169-170 1 H NMR 8 9.47 1 H, indole-NH), 8.85 1 H, OCONH), 7.98 1 H, H 7.86 J =8.5 Hz, 1 H, H 7.77 J 8.3 Hz, 1 H, H 7.57 (br dd, J 7.4 Hz, 1 H, H 7.44 (ddd, J 8.5, 7.4, 0.7 Hz, I H, H 7.37 (br s, 1 H, H 6.99 J =2.3 Hz, 1 H, H 6.87 s, 1 H, H 5.3 8 J 13.4 Hz, 1 H, CH 2 5.34 J =13.4 Hz, 1 H, CH 2 4.79 (dd, J 10.7, 1.6 Hz, I H, H 4.65 (dd, J 10.7, 8.7 Hz, 1 H, H 4.15-4.19 (in, 1 H, H 4.09 3 H, OCH 3 4.02 (br s, 3 H, NCH 3 3.95 3 H, OCH 3 3.92-3.94 (in, 4 H, OCH 3
CH
2 CI), 3.45 (dd, J 10.9, 10.7 Hz, I H, CH 2 CI); 1 3 C NMiR 8 160.4 153.4 (OCONH), 150.2 (C 146.9 (C 141.6 (C 3a), 140.7 (C 139.6 (C 138.9 (C 133.3 (C 132.1 (C 4"1), 129.7 (C 9a), 129.6 (C 127.6 (C 125.7 (C 125.2 (C 7 and C 5a), 123.6 (C 3a'), WO 00/64864 WO 0064864PCTGBOOIOI 612 -96- 123.2 (C 122.3 (C 122.2 (C 9b), 112.8 (C 106.6 (C 97.7 (C 61.5 (OCH 3 61.2 (OCH 3 58.4 (CH 2 56.3 (OCH 3 54.9 (C 45.8 (CH 2 CI), 43.4 (C 33.8
(NCH
3 MS (FAB') m/z 649 651 HRMS (FAB+) caic. for
C
31
H
30 3 1C1N 6 0 8 mzlz 649.1814, found 649.1797; calc. for C 31
H
30 3 1C1N 6 0 8 (MHW) m/z 651.1784, found 651.1802; Anal. (C 3
,H
29 C1N 6 0g) C, H, N.
Example 5J. Preparation of 4-((1lmethyl-5-nitro-1H-imidazoI-2yl)methoxylcarbonyl)amilo)beIzyI doxorubicin carbamnate (142).
1 -Methyl-5-nitro-1H-imidazol-2-yI 4-({[tertbutyl(dimethyl)silyljoxy~methyl)phefllcarbamate (139). Et 3 N (1.10 mL, 7.87 mniol) was added to a stirred suspension of (I-methyl-5-nitro- IH-imidazol-2-yI)methyl 4nitrophenyl carbonate (134) (2.31 g, 7.17 mmol), 4-({[tertbutyl(dimethyl)silyl]oxy~metbyl)aniline (120) (1.79 g, 7.87 mmol), HOBT (0.97 g, 7.17 mmol), and 4 A molecular sieves (2.5 g) in THE (100 niL) and the mixture stirred at 20 C for 48 h. The solvent was evaporated and the residue partitioned between EtOAc (100 niL) and water (100 niL). The organic fraction was washed with 1 M HC1 (2 x 40 mL), water (100 niL), brine (5 0 niL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 139 (2.57 g, 85%) as a white solid, mp (EtOAc/light petroleum) 145-146 'H NMR 5 7.99 I H, H 7.32 (br d, J =8.1Hz, 2H, H3, H5), 7.27 J =8.1 Hz, 2H, H2, H6), 6.96 (br s, I1H, OCONH), 5.30 2 H, CH 2 4.69 2 H, CH 2 4.05 3 H, NCH 3 0.93.(s,9 H, SiC(CH 3 3 0.09 6 H, Si(CH 3 2 13C NMVR 8 152.3 (OCONH), 147.0 (C 139.6 (C 137.4 (C 135.8 (C 129.6 (C 126.9 (C 2, C 118.8 (C 3, C 64.5 (CH 2
O),
8.0 (CH 2 33.7 25.9 (SiC(C-H3)3), 18.4 (SiK(CHA)), -5.3 (Si(CH,) 2 Anal.
(Cj, 9
HN
4
O
5 Si) C, H, N.
(1-Methyl-5-nitro-1H-imidazol-2-yl)methyI 4-(hydroxymethyl)phenylcarbamate (140).
1 M HCl (16 niL, 16 ninol) was added to a stirred solution of silyl ether 139 (1.36 g, 3.22 nimol) in MeOH (50 niL) and stirred at 20 'C for 1 h. The solution was poured into brine (50 niL) and extracted with EtOAc (3 x 50 mL). The combined organic fraction was washed with water (50 niL), dried, and the solvent evaporated. The residue was crystallized from EtOAc/light petroleum, to give 140 (0.86g, 47%) as a white solid, mp (EtOAc/light petroleum) 18 1-183 'H NMR [(CD3) 2 S01 8 9.85 (br s, 1 H, OCONH), 8.09 1 H, H BNSDOCIO; <WO 0064864Ai 1> WO 00/64864 PCTIGBOO/01612 -97- 7.40 (di, J =8.5 Hz, 2 H, H 3, H 7.22 J =8.5 Hz, 2 H, 112, H 5.29 2 H,
CH
2 4.42 2 H, CH 2 3.96 3 H, NCH 3 3.79 (br s, I H, OH); 3 C NMR
[(CD
3 2 S0] 8 152.5 (OCON7H), 147.8 (C 139.3 (C 137.4 (C 136.8 (C 131.7 (C 2, C 127.0 (C 3, C 118.0 (C 62.4 (CH 2 57.5 (CH 2 33.4 (NCH 3 Anal.
(C,
3
H,
4
N
4 0 5 C, H, N.
4-((-ehl5ntol-mdzl2y~ehxlabnlaiobny 4nitrophenyl carbonate (141). A solution of 4-nitrophenylchlorofoflnate (0.72 g, 3.55 mmol) in THF (10 mL) was added dropwise to a stirred solution of alcohol 140 (0.73 g, 2.37 mmol) and Et 3 N (0.66 mL,, 4.73 mmol) in THF (40 mL) and the solution stirred at *C for 16 h. The solvent was evaporated and the residue was purified by chromatography, eluting with 20% EtOAcIDCM, to give 141 (0.71 g, 63%) as a white solid; 'H NMvIR
I(CD
3 2 S0] 8 10.02 I HL OCONH), 8.31 (ddd J= 9.1, 3.3, 2.1 Hz, 2 HL H 3, H 8.08 1 H, H 7.57 (ddd,J= 9.1, 3.3, 2.1 Hz, 2 H, H 2, H 7.51 J =8.5 Hz, 2 H, H 3', H 7.40 (di, J =8.5 Hz, 2 H, H H 5.32 2 H, CH 2 4.70 2 H, CH 2 3.97 3 H, NCH 3 MS (FAB~) mlz 472 (MW, HRMS (FAB~) caic. for C 20
H
18
N
5 m1z 4 7 2.1105, found 472.1108.
4-((-ebl5ntol-mdzl2y~ehxlabnlaiobny doxorubicin carbamnate (142). A solution of carbonate 141 (61 mg, 129 /imo1) in DMF (2 niL) was added dropwise to a stirred solution of doxorubicin (13) (45 mg, 86 Atmol) and. Et 3 N (18 MiL, 129 ktmo1) in DMF (5 niL) at 20 *C and the solution stirred for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 5% MeOH/DCM, to give 142 (50 mg, 66%) as a red solid, mp (DCM) 170-173 'H NMvR [(CD 3 h2SO] 8 14.00 I H, 6-OH), 13.24 1 H, 11I-OH), 9.91 1 H, OCONH), 8.07 1 H, H 7.86-7.90 (in, 2 Hl H 1, H 7.60-7.63 (in, 1 H, H 7.40 (di, J 8.4 Hz, 2 H, H H 51"), 7.23 (di, J 8.4 Hz, 2 H, H H 6.81 (di, J =8.0 Hz, 1 H, OCONH), 5.43 I H, H 7), 5.27 2 H, CH 2 5.21 (di, J =2.9 Hz, 1 H, H 4.92-4.95 (mn, 1 H, 9-OH), 4.87 2 H,
CH
2 4.84 (dd, J 5.9HRz, 111, 14-OH), 4.69 J =5.7 Hz, 1 H, 4-OH), 4.5 8 J 6.0 Hz, 2 H, H 14), 4.12-4.18 (in, 1 H, H 3.97 3 H, OCHA) 3.95 3 H, NCH 3 3.68-3.75 (in, I H, H 3.43-3.46 (in, 1 1,114'), 2.98 J =18.3 Hz, 1 H, H 10), 2.91 J= 18.3 Hz, I H, H 1O), 2.20 (br d,J =14.1 Hz, 1 H, H8), 2.11 (dd, J= 14.1 Hz, 1 H, H 1.84 (dt, J =12.9, 3.7 Hz, 111,11 1.47 (dd, J =12.9, 4.0 Hz, I11,11 1. 12 J WO 00/64864 WO 0064864PCT/GBOO/OI 612 98 Hz, 3 H, H 13 C NMR [(CD 3 2 S0] 8 213.7 (C 13), 186.4 (C 186.3 (C 12), 160.7 (C 156.0 (C 155.2 (C 11), 154.4 (OCONI-), 152.4 (OCONH), 147.8 (C 139.3 (C 138.2 (C 136.1 (C 135.4 (C 12a), 134.6 (C 6a), 134.0 (C 10a), 131.7 (C 131.2 (C 128.6 (C C 119.9 (C 4a), 119.6 (C 118.9 (C 118.0 (C 3", C 110.7 (C 5a), 110.5 (C 1 la), 100.2 (C 74.9 (C 69.8 (C 67.9 (C 66.6 (C 64.8 (C 14), 63.6 (CH 2 57.6 (CH 2 56.5 (OCH 3 47.0 (C 36.5 (C 33.4
(NCH
3 32.0 (C 10), 29.7 (C 16.9 (C MS (FAB 4 m/z 876 HRMS (FAB caic. for C 41
H
42
N
5 01 7 (Ill) m/z 876.2576, found 876.2573; Anal.
(C
4 1 4
IN
5 0 17 .H1 2 0) C, H, N.
Example 5K. Preparation of l-(2-hydroxyethyl)-5-nitro-1H-imidazol-2-yImethyI 1- (chloromethyl)-3-[(5,6,7-trimethoxy-1H-ifldol-2-yl)carboflI-2,3-dihydro-lH- (148).
2-{5-Nitro-2-[(E)-2-phenylethenyII-1H-imidazol-1 -yl)ethanlol (144). A solution of Na (2.0 g, 87.6 mmol) in dry MeOH (30 mL) was added in one portion to a stirred solution of metronidazole (143) (10.0 g, 58.4 mmol) and benzaldehyde (7.1 mL, 70 mnmol) in DMS0 mL) at 20 The mixture was stood at 20 *C for 24 h. Water (80 mL) was added and the resulting solid filtered. The solid was dissolved in EtOAc (100 mL) dried, and the solvent evaporated. The residue was purified by chromatography, eluted with EtOAc/light petroleum, to give 144 (4.0 g, 26%) as a yellow powder, mp 155 *C (lit mp 156-157 *C W. J. Ross, W. B. Jamieson, and M. C. McCowen, J Med Chem.; 1972,15S, 1035-1039]); 'H NMR 8 8.06 1 H, H 7.83 J= 15.8 Hz, 1 H, 7.52-7.58 (in, 2 H, H H 7.33-7.38 (in, 3 H, H H H 7.05 J= 15.8 Hz, 1 H, CH=), 4.64 (dd, J= 5.1, 5.0 Hz, 2 H, H 4.07 (dd, J 5.0 Hz, 2 H, H 2.42 (br s, 1 H, OH); 3 C NMR 8 150.9 (C 140.0 138.5 (C 135.3 (C 134.6 (C 4') 129.7 (C 128.9 (C C 127.6 (C C 112.1 61.8 (C 47.7 (C 2).
tert-Butyl(dimethyl)silyl 2-(-ir--[E--hnlthnl-Hiiao--l ty ether (145). TBDMS triflate (2.7 mL, 11.75 mmol) was added dropwise to a stirred 'solution of alcohol 144 (2.77 g, 10.7 mmol) and pyridine (1.3 mL, 16.0 nimol) in DCM (100 niL) at -S *C and the solution stirred at 20 *C for 16 h. The reaction was quenched with MeOH (5 niL) and poured in sat. aq. KHCO 3 (100 niL). The mixture was extracted with DCM (3 x 50 niL), the combined organic fraction dried and the solvent evaporated.
BNSOID: <WO 0064864AI I WO 00/64864 PCT/GBOO/01612 99 The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 145 (4.00 g, 100%) as a yellow solid, mp (EtOAc/liglit petroleum) 99-100.5
'H
NMR 858.13 1 H, H 7.87 J 15.8 Hz, I H, 7.57 J 6.8 Hz, 2 K~ H 2", H 7.34-7.41 (in, 3 H, H H H 7.06 J =15.8 Hz, 1 H, 4.62 (dd, J= 5.0, 4.8 Hz, 2 HK H 4.00 (dd, J 5.0, 4.8 Hz, 2 H, H 0.77 9 H, SiC(CH 3 3 0. 6 H, Si(CH 3 2 1 3 C NMR 8 151.2 (C 139.3 138.4 (C 135.5 (C 134.8 (C 129.6 (C 128.9 (C C 127.5 (C C 112.9 62.3 (C 47.8 (C 25.7 (SiC(QH 3 3 18.1 (SiC(CH 3 3 -5.8 (Si(CH 3 2 Anal. (C, 9
H
2
,N
3
O
3 Si) C, H, N.
l-(-IlertButl~dmetyl~ilyloy~ehyl-5-itr-IHimiazo-2-lImethanol (146).
Ozone was bubbled into a solution of imidazole 145 (1.3 g, 3.48 mmol) in DCMIMeOH 120 mL) at -78 *C until a blue colour persisted. The solution was warmed to -40 *C with a N 2 purge to remove excess ozone. A solution of NaBH 4 (132 mg, 3.48 mmol) in EtOH (10 niL was added dropwise over 15 min and the mixture stirred for 30 min. The mixture was treated with acetic acid (0.5 niL), stirred for 10 min and the solvent evaporated. The residue was partitioned between EtOAc (100 niL) and water (100 mQL.
The organic fraction was washed with water (50 mL), brine (25 niL), dried, and the sol vent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 146 (0.92 g, 88%) as a white solid, mp 104-105 0 C; 'H NMR 8 7.97 1 H, H 4.79 2 H, CH 2 4.62 J =4.8 Hz, 2 H, CH 2 3.97 J =4.8 Hz, 2 H,
CH
2 3.80 (br s, 1 H, OH), 0.81 9 H, SiC(CH 3 3 0. 10 6 H, Si(CH 3 2 1.
3 C NMvR 8 157.2 (C 138.8 (C 132.3 (C 62.0 (CH 2 57.2 (CH 2 48.3 (CH 2 25.7 (SiC(-CH3) 3 18.2 (Si!(CH 3 3 -5.8 (Si(CH 3 2 Anal. (Cl 2
H
23
N
3
O
4 Si) C, H, N.
Il-(2-{Itert-Butyl(dimethyi)silyIloxy~ethyD-5nitro 1H imidazoi-2yl mtyI 1- (chloromethyl)-3(5,6,7trimethoxylHindo-2-yI)carbonylI- 2 ,3-ihydr-H (147). A solution of triphosgene (25 mng, 84 Mmol) in DCM (3 niL) was added dropwise to a stirred solution of amine 1 J. Atwell, W. R. Wilson,
W.
A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (112 mg, 240 Asmol) and Et 3 N (67 AL, 481 M~mol) in DCM (10 mL) and stirred at 20 *C for 2 h. A solution of alcohol 146 (78 mng, 264p~moI) in DCM (3 mL) was added, followed by nBU 2 Sn(OAC) 2 (2 drops) and the solution stirred at 20 *C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 20% EtOAc/DCM, to give 147 (93 mg, 49%) as a colourless WO 00/64864 WO 0064864PCT/GBOO/01612 100 oil, IIH NMR 5 9.41 I H, indole-N-H), 8.88 1 H, OCONH), 8.06 1 H, H 7.87 J 8.5 Hz, 1 H, H 7.79 J =8.3 Hz, I H, H 7.57 (dd, J 7.5 Hz, 1 H, H 7.46 (dd, J 7.5 Hz, I H, H 7.17 (br s, 1 H, H 7.00 J 2.2 Hz, I H, H 31), 6.88 s, I H, H 5.44 J 13.5 Hz, 1 H, CH 2 5.3 9 J =13.5 Hz, 1 H, CH 2
O),
4.81 (dd, J 10.7, 1.5 Hz, 1 H, H 4.65-4.74 (mn, 3 H, H 2, CH 2 4.16-4.22 (in, 1 H, H 4. 10 3 H, OCH 3 3.95-3.99 (in, 4 H, CH1 2 CI, OCH 3 3.89-3.93 (in, 5 H, OCH 3 CH1 2 3.48 J 10.9 Hz, 1 H, CH 2 CI) 0.81 9 H, SiC(CH 3 3 -0.08 6 H Si(CH 3 2 MS (FAB~) m/z 795 793 HRMS (FAB+) caic. for
C
3 gH 46 35 C1N 6 OqSi mlz 793.2784, found 793.2762; caic. for C 3 ,1H 46 37 C1N 6 OqSi(M) m/z 795.2755, found 795.2751.
ll(-yrxehl--ir-Hiiao--lmty 1-(chloromethy)-3-[5,6,7- (148). 1M HCI (0.23 mL, 230 /.mo1) was added to a stirred solution of silyl ether 147 (91 mg, 115 ,umol) in MeOH (5 mL) and the solution stirred at 20 'C for 4 h. The solvent was evaporated and the residue partitioned between EtOAc (40 mnL) and water (40 inL). The organic fr-action was washed with water (25 rnL), brine (20 mnL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (0- 10%MeOHI4O%EtOAcJDCM), to give 148 as a white solid, mp (EtOAc/light petroleum) 148-150 'H NNM 8 [(CD 3 2 S0] 11 .45 1 H, indole-NH), 9.92 1 H, OCONH), 8.45 1 H, H 8.16 1 H, H 8.06 J =8.5 Hz, 1 HL H 7.97 J 8.3 Hz, 1 H, H 7.58 (ddd, J 8.3, 7.2, 0.8 Hz, 1 H, H 7.46 (ddd, J 8.5, 7.2, 0.8 Hz, 1 H, H 7.09 J =2.0 Hz, I H, H 6.97 1 H, H 5.37 2 H, CH 2 5.12 J= 5.4 Hz, I H, OH), 4.80 (dd, J 10.7, 9.4 Hz, I H, H 4.56-4.60 (in, 3 H, H 2, CH 2 4.32- 4.38 (in, 1 H, H 4.06 (dd, J= 11.1, 3.2 Hz, 1 H, C11 2 3.91-3.95 (in, 4 H, OCH 3
CH
2 Cl), 3.83 3 H, OCH 3 3.81 3 H, OCH 3 3.70-3.75 (in, 2 H, CH 2 1 3 C NMIR 8
[(CD
3 2 S0] 160.1 154.0 (OCONH), 149.1 (C 148.7 (C 141.4 (C 3a), 139.9 (C 139.0 (C 138.9 (C 134.1 (C 132.5 (C 130.7 (C 9a), 129.4 (C 2') 127.1 (C 125.4 (C 125.3 (C 5a), 124.3 (C 123.8 (C 123.3 (C 123.1 (C 122.2 (C 9b), 113.2 (C 106.2 (C 98.0 (C 6 1.0 (OCH 3 60.9 (OCH 3 59.7
(CH
2 58.1 (CH 2 55.9 (OCHA) 54.9 (C 48.2 (CH 2 47.8 (CH 2 CI) 41.1 (C MS (FAB m/z 681 679 HRMSB (FAB~) calc. for C 32
H
32 35
CIN
6 0 9 BNSDOCID: <MO 0064864AI I WO 00/64864 PCT/GBOO/01612 101 m/z 679.1919, found 679.1797; calc. for C 32
H
3 2 CIN 6 0 9 m/z 681.1890, found 681.1892; Anal. (C 32
H
31 C1N 6 0 9 C, H, N.
Example 5L. Preparation of (1-methyl-5fitrolHimidazol4-yI)methyI 1- (chloromethyl)3(5,6,7trimethoxylHindo-1-yI)carbonyl]1, 3 dihydrolH- (150). A solution of triphosgene (21 mg, 70 M.mol) in DOM (2 mL) was added dropwise to a stirred solution of amine 1 J. Atwell, W. R. Wilson, W.
A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (93 mg, 200 jsmol) and Et 3 N (55 AzL, 400 pimol) in DCM (10 mL) and stirred at 20 0 C for 2 h. A solution of (1 IH-imidazol-4-yl)Tfethaflol 149 D. C. Baker, S.R. Putt, H. D. H. Showalter,
J.
Heterocyclic Chem., 1983, 20, 629-634.] (3 7 mg, 240 jsmol) in DCM (3 mL) was added, followed by nBU 2 Sn(OAC) 2 (2 drops) and the solution stirred at 20 0 C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with MeOHIEtOAc, to give 150 (40 mg, 56%) as a tan powder, mp (EtOAc) 2 19-220 OC; 1H NMR [(CD 3 2 S0] 611.47 1 H, indole-NH), 9.80 I H, OCONH), 8.54 (br s, 1 H, H 4), 8.08-8.11 (in, 2 H, H 6, H 7.97 J =8.4 Hz, 1 H, H 7.57 (ddd, J= 8.4, 7.2, 0.7 Hz, 1 H, H 7.45 (ddd, J 7.2, 0.7 Hz, I H, H 7.09 J =2.0 Hz, I H, H 6.97 s, I H, H 5.40 2 H, CH 2 4.79 (dd, J =10.8, 1.4 Hz, 1 H, H 4.52 (dd, J 11.0, 1.9 Hz, I HL H 4.31-4.36 (in, I H, H 4.07 (dd, J= 11. 1, 3.0 Hz, I H, CH 2
CI),
3.89-3.95 (in, 7 H, OCH 3
CH
2 C1, NCH 3 3.83 3 HK OCHA) 3.81 3 H, OCH 3 1 3
C
NMR [(CD 3 2 S0] 6160.1 154.3 (OCONH), 154.2 (C 149.1 (C 141.4 (C 2"1), 141.2 (C 3a), 139.9 (C 139.0 (C 135.1 (C 134.4 (C 130.8 (C 9a), 129.4 (C 127.0 (C 125.5 (C 5a), 125.4 (C 124.2 (C 7) 123.9 (C 123.2 (C 123.1 (C 122.0 (C 9b), 113.1 (C 106.2 (C 31), 98.0 (C 6 1.0 (OCH 3 60.9 (OCH 3 59.6
(CH
2 55.9 (OCH 3 54.8 (C 47.5 (CH 2 C1), 41.1 (C 35.1 (NCH 3 MS (FAB~) m/z 651 WMH, 649 (MHr, HRMS (FAB*) caic. for C 3
,H
30 MC1N 6 0g (MWi) m/z 649.1814, found 649.1802; calc. for C 31
H
30 3 1C1N 6 08 (MHW) m/z 651.1784, found 651.1761; Anal. (C 3 ,H29C1N 6 H, N.
Example 6A. Preparation of (5-nitro-2-furyi)methyl 4-[bis(2chloroethyl)aminolphenlelarbam ate (154).
(5-Nitro-2-furyl)methyl 4-nitropheflyl carbonate (152). A solution of 4-nitrophenyl chloroformate (4.17 g, 20.7 inmol) in dry THF (50 mL) was added slowly to a stirred PCT/GBOO/01612 WO 00/64864 -102solution of (5-nitrofuran-2-yl)methanol (151) M. Berry, C. Y. Watson, W. J. D. Whish, and M. D. Threadgill. J. Chem. Soc. Perkin Trans. 1, 1997, 1147] (2.69 g, 18.7 mmol) and pyridine (1.67 mL, 20.7 mmol) in THF (100 mL) at 20 OC under N 2 The mixture was stirred at 20 OC for 16 h, then partitioned between EtOAc (100 mL) and H 2 0 (100 mL). The organic layer was washed with saturated aqueous NaHCO 3 (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (25-50%) of EtOAc/light petroleum to give 152 (4.79 g, 83%) as a white powder, mp (EtOAc/light petroleum) 93-94 OC; IR N 1775, 1526, 1352, 1215 'H NMR [(CD 3 2 SO] 8 8.34 (ddd, J= 9.2, 3.2, 2.1 Hz, 2 H, H 3, H 7.72 J= 3.8 Hz, 1 H, H 7.61 (ddd, J= 9.2, 3.2, 2.1 Hz, 2 H, H 2, H 7.07 J= 3.8 Hz, 1 H, H 5.43 2 H, CH 20 3 C [(CD 3 2
SO]
8 155.0, 154.1, 151.8, 151.5, 145.2, 125.4(2), 122.5(2), 115.1, 113.3, 61.5; Anal.
(C,
2
HN
2 C, H, N.
(5-Nitro-2-furyl)methyl 4-[bis(2-hydroxyethyl)amino]phenylcarbamate (153). A solution of carbonate 152 (1.00 g, 3.24 mmol), Nt',N-bis(2-hydroxyethyl)-l,4benzenediamine (57) (3.24 mmol), and pyridine (260 3.24 mmol) in THF (80 mL) was stirred at 20 *C for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient of MeOH/EtOAc, to give 153 (0.74 g, 63%) as an oil; 'H NMR [(CD 3 2 SO] 8 9.44 1 H, OCONH), 7.68 J= 3.7 Hz, 1 H, H 7.20 (br d, J= 9.1 Hz, 2 H, H2, H 6.93 J= 3.7 Hz, 1 H, H 6.62 J= 9.1 Hz, 2 H, H 3, H 5.19 2 H, CH20), 4.71 J= 5.4 Hz, 2 H, 2 x OH), 3.48-3.54 4 H, 2 x CH 2 3.33-3.38 4 H, 2 x CH 2 13 C NMR [(CD 3 8 154.0, 152.7, 151.5, 144.2, 127.0, 120.4 113.8, 113.6, 111.4 58.1 57.1, 53.4 MS (DEI) m/z 365 334 222 196 191 (100); HRMS (DEI) calc. for C, 6 m/z 365.1223, found 365.1218.
(5-Nitro-2-furyl)methyl 4-[bis(2-chloroethyl)aminolphenylcarbamate (154). Methanesulphonyl chloride (460 /4L, 6.0 mmol) was added dropwise to a stirred solution of diol 153 (0.73 g, 2.0 mmol) in pyridine (30 mL) at 5 *C and the solution stirred at 20 *C for 2 h.
The solvent was evaporated and the residue partitioned between DCM (100 mL) and water (100 mL). The aqueous fraction was washed with DCM (2 x 50 mL), the combined organic extracts dried, and the solvent evaporated. The residue was dissolved in DMF (20 mL), LiCI (0.51 g, 12.0 mmol) added and the mixture stirred at 80 *C for 3 h. The solvent was BNSDOCID: WO 0064864A1 I WO 00/64864 PCT/GBOD/01612 103 evaporated and the residue partitioned between EtOAc (150 mL) and water (150 mL). The aqueous fraction was extracted with EtOAc (2 x 80 mL), the combined extracts dried, and the -solvent evaporated. The residue was purified by chromatography, eluting with EtOAc/light petroleum, to give 154 (0.62 g, as an oil; 'H NMR 5 9.57 (br s, 1 H, OCONH), 7.69 J =3.7 Hz, 1 H, H 7.28 (br d, J= 9.1 Hz, 2 H, H 2, H 6.95 J 3.7 Hz, 1 H, H 6.70 J =9.1 Hz, 2 H, H 3, H 5.21 2 H, CH 2 3.63-3.72 (in, 8 H, 2 x CH 2 N, 2 x CH 2 Cl); 1 3 C NMR (CDCI 3 8 153.9,152.7, 151.5, 1142.3, 128.7, 120.3 113.8, 113.6, 112.3 57.2, 52.3 41.1 MS (DEI) m/z 401 (M 4 403 405 (10) 354 352 (100); HIRMS (DEI) caic. for C, 6
H
7 3 1C1 2
N
3 0 5 m/z 401.0545, found 401.0546; caic. for C, 6
H,
7 3 1C11 7 C1N 3 0, (M t mlz 403.0516, found 403.0521; caic. for C, 6 H1 7 37 C1 2
N
3 0 5 m/z 405.0486, found 405.0498.
Example 6B. Preparation of (5-nitro-2-furyl)methyl 1-(chloromethyl)-3-[(5,6,7trimethoxy-lH-indlO1-y)carboylY2,dihydrolHbenzokelindolIS.ylcarbamate (155). A solution of triphosgene (14 mng, 48 Amio1) in DCM (2 mQL was added dropwise to a stirred solution of amine 1 JI Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (57 mg, 122 A~mol) and Et 3 N (38 /LSL, 275 Amol) in DCM inL and stirred at 20 0 C for 2 h. A solution of (5-nitrofurafl-2-yl)mfethanol (151) P. M.
Berry, C. Y. Watson, W. J. D. Whish, and M. D. Threadgill. J Chem. Soc. Perkin Trans. I, 1997, 1147] (24 ing, 165 ~Inol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 2 drops) and the solution stirred at 20 OC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 30% EtOAcIDCM, to give 155 (65 mg, 84%) as a white solid, mp (EtOAc/light petroleum) 185-187 'H NMR 8 11.46 1 H, indole-NH), 9.92 I H, OCONH), 8.54 1 H, H 8.04 J =8.5 Hz, I H, H 7.98 (d,J 8.3 Hz, 1 H, H 7.72 J =3.8 Hz, 1 H, H 7.5 8 (ddd, J 7.2, 0.7 Hz, I H, H 7.45 (ddd, J 7.2, 0.7 Hz, 1 H, H 7.09 J 2.1 Hz, I H, H 6.98-7.00 (in, 2 H, H H 5.30 2 H, CH 2 4.80 (dd, J 10.8, 9.5 Hz, 1 H, H 4.53 (dd, J 10.8, 1.9 Hz, 1 H, H 4.32-4.37 (in, 1 H, H 4.07 (dd, J 11. 1, 3.0 Hz, 1 H, CH 2 Cl), 3.91-3.96 (in, 4 H, OCH 3 CH1 2 3.83 3 H, OCH 3 ),3.80 3 H, OCH 3 3 C NMR 8 160.1 153.9 (OCONH), 153.8 (C 15 1.1 (C 149.2 (C 141.4 (C 3a), 139.8 (C 139.0 (C 134.0 (C 130.7 (C 9a), 129.4 (C 127.1 (C 125.4 (C 125.3 (C 124.3 (C 123.7 (C 123.3 (C 123.1 (C 3a), 122.2 (C 9b), 113.9 (C 113.6 (C 113.1 (C 106.2 (C 98.0 (C 6 1.0 60.9 (OCH 3 57.7 WO 00/64864 WO 0064864PCTGBDO/O1 612 -104-
(CH
2 55.9 (OCH 3 54 .8 (C 47.5 (CH 2 C1), 41.1 (C MS (FAB+) m/z 635 (MH, 637 HRMS (FAB') calcd for C 3
,H
28 35
CIN
4 0 9 m/z 635.1545, found 635.1552; calcd for C 3
,H
28 37 C1N 4 0 9 (MHV) m/z 637.1515, found 637.1514; Axial.
(C
3
H
27 C1N 4 0 9 Example 7A. Preparation of (5-nitro-2-thienyl)methyl 4-Ibis(2chloroethyl)aminolphenylcarbamate (159).
4-Nitrophenyl (5-nitro-2-thienyl)methyl carbonate (157). 4-Nitrophenyl chioroforinate (2.58 g, 12.8 mniol) in dry THF (20 mL) was added slowly to a stirred solution of nitrothien-2-yl)methanol (156) J. Narcombe, R. K. Norris. Aust. J. Chem. 1979, 32, 2647] (1.85 g, 11.6 mmol) and pyridine (1.03 mL, 12.8 mmol) in THF (50 mL) at 20 OC under N 2 The mixture was stirred at 20 *C for 16 h, then partitioned between EtOAc (100 mL) and H 2 0 (100 mL). The organic layer was washed with saturated aqueous NaHCO3 mL), and the solvent evaporated to give 157 (1.86g, mp (EtOAc/light petroleum) 121-122 IR N 1763, 1522,1345,1231 cm';'H NMR [(CD 3 2 ,S0] 8 8.33 (ddd, J= 9.2, 3.4, 2.2 Hz, 2 H, H H 8.08 J= 4.2 Hz, 1 H, H 7.60 (ddd, J 9.2, 3.4, 2.2 Hz, 2 H, H H 7.4 J 4.2 Hz7, 1 H, H 5.56 2 H, CH 2 1 3 C NNM [(CD 3 2 S0] 8 155.0, 151.6, 148.2, 145.2, 144.8, 129.5, 129.0, 125.4 122.6 64.4; Anal.
(CI
2
HSN
2 0 7 S) C, H, N.
(5-Nitro-2-thienyl)methyl 4-Ibis(2-hydroxyethyl)aminojphenylcarbamate (158). A solution of 157 (0.75 g, 2.3 mmol), N',N-bis(2-hydroxyethyl)-l ,4-benzenediamine (57) mmol), and pyridine (206 .sL, 2.5 mmol) in THF (50 mL) was stirred at 20 *C for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc to give 158 (0.56 g, mp (EtOAc/light petroleum) 139-140.5 IR N 3360, 3208, 1730, 1530, 1337, 1215 cm'; 'H NMR [(CD 3 2 S0] 8 9.46 I H, OCONH), 8.02 (d, J =4.2 Hz, I H, H 7.29 J= 4.2 Hz, 1 H, H 7.21 (br d, J= 9.1 Hz, 2 H, H 2, H 6), 6.62 J 9.1 Hz, 2 H, H 3, H 5.3 3 2 H, CH 2 4.72 J 5.5 Hz, 2 H, 2 x OH), 3.49-3.56 (in, 4 H, 2 x CH 2 3.36 J 6.2 Hz, 4 H, 2 x CH 2 1 3 C NMR [(CD 3 2 S0] 8 153.0, 150.8, 148.1, 144.3, 129.6, 127.5, 126.9, 120.5 111.4 60.1,58.2(2), 53.4 Anal. (CI 6
H,
9
N
3 0 6 S) C, H; N, calc 11.0, found 10.5%.
(5-Nitro-2-thienyl)methyl 4-[bis(2-chloroethyl)aminoJ phenylcarbamate (159).
INSOOCID: <WO 0064864A1 I> WO 00/6484 PCT/GBOO/01612 105 Methane-suiphoflyl chloride (260 jiL, 3.4 mmol) was added dropwise to a stirred solution of diol 158 (0.43 g, 1 .1 mmol) in pyridine (10 mL) at 5 0 C and the solution stirred at 20 00 for 2 h. The solvent was evaporated and the residue partitioned between DCM (50 mL) and water (50 mL). The aqueous fraction was washed with DCM (2 x 50 mL), the combined organic extracts dried, and the solvent evaporated. The residue was dissolved in DMF nit), LiCI (0.29 g, 6.8 mmol) added and the mixture stirred at 80 00 for 3 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL).
The aqueous fraction was extracted with EtOAc (2 x 50 mL), the combined extracts dried, and the solvent evaporated. The residue was purified by chromatography, eluting with EtOAc/light petroleum, to give 159 (0.35 g, 69%) as pale green needles, mp (EtOAc/light petroleum) 99-100 IR N 3353, 1723, 1547, 1530, 1339, 1219 cm'; 'H NMR
[(CD
3 2 S0] 8 9.5 8 (br s, 1 H, OCONH), 8.04 J= 4.2 Hz, 1 H, H 41), 7.28-7.30 (in, 3 H, H H 3, H 6.71 J =9.1 Hz, 2 H, H 2, H 5.34 2 H, CH 2 3.65-3.72 (mn, 8 H, 2 x CH 2 N, 2 x CH 2 CI); '3C NMR [(CD 3 2 S011 8 153.0, 150.8, 148.2, 142.4, 129.5, 128.6, 127.5, 120.4 112.3 60.1, 52.2 41.1 Anal. (C, 6
H,
9 C1 2
N
5 0 4 C, H, N, Cl.
Example 7B. Preparation of (5-nitro-2-thienyI)mlethyl 1-(chloromethyl)-3+[5, 6 .7trhnethoxy-lH-ifldol-1y1)carbony1F-2,3dihydro-1HbenzoleindolS5ylcarbamate (160). A solution of triphosgene (15 mg, 51 jimol) in DCM (2 mL) was added dropwise to a stirred solution of amnine 1 [1G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med.
Chem. Lett., 1997, 7, 1483] (61 mng, 131 /.inol) and Et 3 N (41 AiL, 294 Amiol) in.DCM niL) and stirred at 20 00 for 2 h. A solution of (5-nitro-2-thienyl)nmethanol (156) J.
Narcoinbe, R. K. Norris. Aust. J. Chem. 1979, 32, 2647] (28 mg, 176 Asmol) in DCM (2 niL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 00 for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc/DCM, to give 160 (76 mng, 89%) as a white solid, mp (EtOAc/light petroleum) 218-219 'H NMvR [(CD 3 2 S0] 8 11.48 (br s, 1 H, indole-NH), 9.34 (br s, 1 H, OCONH), 8.55 (br s, 1 H, H 8.08 J =4.1 Hz, 1 H, H 8.04 J 8.5 Hz, 1 H, H 7.99 J= 8.3 Hz, 1 H, H 7.58 (dd, J= 8.3, 7.4 Hz, 1 H, H 7.47 (dd, J= 8.5, 7.4 Hz, 1 H, H 7.33 (d,J =4.1 Hz, 1 H, H 7. 10 J =1.9 Hz, 1 H, H 6.97 1 H, H 5.43 2 H, CH 2 4.80 (dd, J =11.0, 9.5 Hz, 1 H, H 4.54 (dd, J =11.0, 1.8 Hz, 1 H, H 4.32-4.36 (in, 1 H, H 4.07 (dd, J =11.0, 3.0 Hz, I H, CH 2 Cl, 3.92-3.96 (in, 4 H, C11 2 0, OCH 3 3.92 (in, 3 H, OCH 3 3.81 3 H, OCH 3 '3 C NMR [(CD 3 2 S0] 8 160.3 WO 00/64864 PCT/GBOO/01612 106 154.4 (OCONHl), 15 1.1 (C 149.3 (C 148.2 (C 141.6 (C 3a), 140.0 (C 139.1 (C 134.1 (C 130.8 (C 9a), 129.7 (C 129.6 (C 127.8 (C 127.4 (C 125.6 (C 5 125.5 (C 124.6 (C 123.8 (C 123.3 (C 123.2 (C 3 122.5 (C 9b), 113.5 (C 106.4 (C 98.1 (C 61.2 (0C11 3 6 1.0 (OCH 3 60.8
(CH
2 56.0 (OCH 3 55.0 (C 47.7 (CHPC), 41.2 (C MS (FAB+) mlz 653 (MHW,3 651 HRMS (FAB+) calc. for C 31
H
28 35 C1N 4 0 8 S (MHW) m/z 651.1316, found 651.1311; calc. for C 3
H
28 37
CN
4 0 8 S (MH 4 m/z 653.1287, found 653.1307; Anal.
(C
3
,H
27 C1N 4 0 8 S) C, H, N.
Example 8. Preparation of (1-methyl-5-nitro-1H-pyrazol-4-yl)methyl 1- (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-1-y)carboflyl]-2,3-dihydro-ILH- (163).
(1-methyl-5-nitro-lH-pyrazol4-yl)lethalol (162). Borane dimethylsulfide (2 M solution in THF, 4.2 mL, 8.4 mmol) was added to a solution of pyrazole-4-carboxylic acid (161) Cheng, J. Heterocyclic Chem. 1968, 5, 195-197] (1.11 g, 6.5 mniol) in THfF (50 rnL) under N 2 and the mixture stirred at reflux temperature for 80 min, then cooled. MeOH (5 niL), then water (5 niL), then 2 M HC1 (5 nQL were added, the TE was evaporated, and the residue was diluted with water and extracted with EtOAc (3 x 50 niL). The combined organic extract was dried, the solvent evaporated, and the residue purified by chromatography, eluting with 50% EtOAc/petroleum ether, to give 162 (0.52 g, 51%) as a white solid, mp (benzene) 78-80 'H NMR 8 7.58 I H, H 3), 4.82 J= 3.4 Hz, 2 H, CH 2 4.25 3 H, NCH 3 ),2.39 (br s, 1 H, OH); Anal.
(C
5 H1 7
N
3 0 3 C, H, N.
(1-Methyl-5-nitro-1H-pyrazol-4-yl)methyI 1 -(chloromethyl)-3-[(5,6,7-trimethoxy-1HindoI-1-yI~carbonyII-2,3-dihydro-1H-benzole1 indol-5-ylcarbamate (163). A solution of triphosgene (14.3 mg, 48 gsmol) in DCM (2 niL) was added dropwise to a stirred solution of amine I J. Atwell, W. R. Wilson, W. A. Denny. Bioorg. Med. Chem. Left., 1997, 7, 1483] (57 mg, 122 /.zmol) and Et 3 N (38 12L, 275 Aumol) in DCM (10 mL) and stirred at *C for 2 h. A solution of alcohol 162 (26 mg, 165 ismol) in DCM (2 niL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 *C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 20 EtOAc/DCM, to give 163 (41 mg, 52%) as a white solid, mp (EtOAc/light petroleum) 201 BNSDOCID: -MO 0064864AI I> WO 00/64864 PCT/GBOO/01 612 -107- 202 'H NMR [(CD 3 2 S03 8 11.47 (br s, I H, indole-NH), 9.80 (br s, 1 H, OCONH), 8.56 (br s, 1 H, H 8.06 J =8.5 Hz, I H, H 7.98 J= 8.3 Hz, 1 H, H 7.74 (br s, 1 H, H 7.58 (dd, J 7.4 Hz, 1 H, H 7.47 (dd, J 8.5, 7.4 Hz, 1 H, H 7.45 J= 1.6 Hz, 1 H, H 6.98 I H, H 5.33 2 H, CH 2 4.80 (dd, J= 11.0, 9.4 Hz, 1 H, H2), 4.53 (dd, J= 11.0, 1.8 Hz, 1 H, H2), 4.32-4.3 8(m, I H, H 4.17 3 H,
NCH
3 4.07 (dd, J= 11 3.1 Hz, 1 H, CH 2 CI), 3.91-3.96 (in, 4 H, OCH 3
CH
2 CI), 3.82 (s, 3 H, OCH 3 3.80 3 H, OCH 3 1 3 C NM4R [(CD 3 2 S0] 8 160.2 154.3 (OCONH), 149.1 (C 142.5 (C 141.5 (C 3a), 139.9 (C 139.0 (C 137.5 (C 134.2 (C 130.7 (C 9a), 129.4 (C 127.1 (C 125.4 (C 5a, C 7a), 124.3 (C 123.7 (C 9), 123.3 (C 123.1 (C 122.0 (C 9b), 117.4 (C 113.0 (C 106.2 (C 98.0 (C 61.0 (OCH 3 60.9 (OCH 3 57.2 (CH 2 55.9 (OCH 3 54.8 (C 47.5 (CH 2 C1, 41.1 (C 40.8 (NCH 3 MS (FAB~) m/z 650 648 HRMS (FAB~) caic.
for C 3
,H
3 3 1C1N 6 0 8 (MHW) m/z 649.1814, found 649.1803; caic. for C 3
,H
3 37
CIN
6 0 8
(W
m/z 651.1784, found 651.1796; Anal. (C 3
,H
29 C1N 6 C, H, N.
Example 9A. Preparation of ethyl 4-(fl({11-(chloromethyl)-3-[(5,6,7-trimethoxy-lHlno--lcroyl23dhdol-ez~eidl5y~mn~abnloymty) (167).
Ethyl 4-omllmty--ir-l-yrl--abxlt (165). Dimethyl sulfate (0.31 niL, 3.2 mmol) was added to a mixture of ethyl 4-formy1-5 -nitro-1IH-pyrTole- 2 carboxylate (164) Fornari, M. Farnier, C. Fournier, Bull. Soc. Chim. Fr. 1972, 283-29 1] (0.57 g, 2.7 inmol) and K 2 C0 3 (0.56 g, 4.0 nimol) in DMSO (4 niL) and the brown suspension was stirred at 20 'C for 1 h. The mixture was diluted with water (50 niL), acidified with HCl (2 and extracted with EtOAc (2 x 50 niL). The combined extract was dried, the solvent evaporated. The residue was chromatograped, eluting with petroleum, to give 165 (0.53 g, 86%) as a pale green solid, mp (benzene/light petroleum) 59-60.5 'H NMR S 10.32 1 H, CHO), 7.42 1 H, H 3), 4.37 7.1 Hz, 2 H, CH 2 4.33 3 H, NCH 3 .39 J= 7.1 Hz, 3 H, CH 3 Anal.
(C9H 10
N
2 0, 5 C, H, N.
Ethyl 4-(hydroxymethyl)-l-mty -ir-Hpyrl--abxlt (166). NaBH, (0.33 g, 8.7 inmol) was added in portions to a solution of aldehyde 165 (3.96 g, 17.5 nimol) in EtOH (100 mL) and the mixture was stirred at 20 'C for 20 min. Water (5 mL) was WO 00164864 PCVGBOO/01612 -108slowly added, the EtOH was evaporated, and the residue was diluted with aq. NaCI and extracted with EtOAc (2 50 mL). The combined extract was washed with aq. NaCI mL), dried, and the solvent evaporated. The residue was recystallized to give 166 (1.40 g, as white needles, mp, (benzene) 95.5-96.5 'H NMvR 8 7.01 1 H, H 4.80 (br s, 2 H, CH 2 4.36 J 7.1 Hz, 2 H, CH 2 4.31 3 H, NCH 3 2.49 (br s, I H, OH), 1.38 J= 7.1 Hz, 3 H, CH 3 Anal. (C 9
H,
2
N
2 0 5 C, H, N. The mother liquor was evaporated and purified by chromatography 10% EtOAcflight petroleum to give more 166 (1.46 g, 37%).
Ethyl 4-((I(1-(chloromethyl)-3-(5,6,7-trimethoxy-1H-ildol-1-yl)carbofll2,3dihydro-1H-benzolel indol-5-yl)amino)carbonylloxy} pyrrole-2-carboxylate (167). A solution of triphosgene (13.8 mg, 46 /jimol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 J. Atwell, W. R. Wilson, W.
A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (55 mg, 118 Mmol) and EtN (37 4
LL,
265 giol) in DCM (10 mL) and stirred at 20 *C for 2 h. A solution of ethyl 4- 1H-pyrrole-2-carboxylate (166) (36 mg, 159 ,inol) in DCM (2 niL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at *C for 24 h. Thbe solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAcIDCM, to give 167 (26 mng, 31%) as a white solid, mp (EtOAc/light petroleum) 248-250 'H NMR [(CD 3 2 S0] 11.45 1 H, indole-NH), 9.86 1 H, OCONH), 8.56 (br s, 1 H, H 8.09 J 8.5 Hz, 1 H, H 7.99 J= 8.3 Hz, 1 H, H 7.59 (dd, J 7.7 Hz, 1 H, H 7.48 (dd, J 8.5, 7.7 Hz, 1 H, H 7.10 J =2.0 Hz, 1 H, H 3 7.06 (br s, 1 H, H 6.97 1 H, H 5.38 2 H,
CH
2 4.80 (dd, J 11.0, 9.6 Hz, I H, H 4.53 (dd, J 11.0, 2.0 Hz, 1 H, H 4.3 4.40 (in, 1 H, H 1 4.29 (q,J 7.1 Hz, 2 H, H 4.19 3 H, NCH 3 4.07 (dd, J 11.0, Hz, I H, CH 2 CI), 3.92-3.96 (in, 4 H, CH 2 CI OCH 3 3.83 (in, 3 H, OCH 3 3.81 3 H,
OCH
3 1.31 J 7.1 Hz, 3 H, H 1 3 C NMvR [(CD 3 2 S0] 6 160.2 159.4 (CC) 2 154.3 (OCONH), 149.1 (C 141.5 (C 139.9 (C 139.0 (C 137.1 (C 134.2 (C 133.9 (C 130.7 (C 129.5 (C 127.1 (C 125.6 (C 125.5 (C 125.4 (C 124.3 (C 123.7 (C 123.3 (C 123.1 (C 122.1 (C 114.6 (C 113.0 (C 106.2 (C 98.0 (C 61.2 (C 6 1.0 (OCH 3 60.9
(OCH
3 59.6 (CH 2 55.9 (OCH 3 54.9 (C 47.5 (CH 2 Cl), 41.1 (C 1V), 35.2 (NCH 3 14.0 (C MS (FAB~) m/z 722 720 HRMS (FAB') caic. for BNSDOCID: <WO 0064864AI I> WO 00/64864 PCT/GBOO/01612 109-
C
35
H
35 35
CN
5 01 0 m/z 720.2073, found 720.2059; calc. for C 35
H
35 37 C1N 5 01 0
(MH+)
m/z 722.2043, found 722.203 1; Anal. (C 35
H
34 C1N 5 0 1 0 C, H, N.
Example 9B. Preparation of (1-methy1-2-flitro-lH-pyrrol-3-yl)methy1 1- (clrmty)31567tiehx-Hidl1y~abnl-,-iyr-H (169).
(1mty--ir-Hpro--lmtao (168). A solution of NaOH (2.7 g, 68 mmol) in water (15 mL) was added to a solution of ester 166 (1.02 g, 4.47 minol) in EtOH and the mixture was stirred at 20 0 C for 1 h. The EtOH was evaporated, and the aqueous phase washed with EtOAc (20 mL) and then acidified (HCI). The aqueous mixture was extracted with EtOAc (3 50 inL), the combined organic extract was dried and the solvent evaporated to give crude 4-(hydroxymethyl)-1-methyl-5-nitropyrrole- 2 -caboxylic acid (0.84 g, 96%) as a red-brown solid.
The acid was suspended in quinoline (6 mL) with Cu powder (0.44 g) and the mixture was heated at 170-180 OC for 50 min. The cooled mixture was diluted with HC1 extracted with EtOAc (3 50 mL), the combined extract was dried, and the solvent evaporated. The residue was purified by chromatography, eluting 40% EtOAc/light petroleum, to give 168 as a pink solid (0.45 g, mp (benzene) 79-80.5 'H NMR 8 6.78 J 2.8 Hz, 1 H, H 6.27 (di, J 2.5 Hz, I H, H 4.80 J =6.8 Hz, 2 H, CH 2 4.00 3 H,
NCH
3 2.75 J= 6.8 Hz, 1 H, OH); 3 C NMR 8 131.6 (C 129.1 (C 109.0 (C 58.8 (CH 2 38.6 (NCH 3 Anal. (C 6 HsN 2 0 3 C, H, N.
(1Mty--ir-Hpro--lmty 1-(chloromethyl)-3-I(5,6,7-trimethoxyl1Hino--lcroyl2,-iyr Hbnoclno--labmt(169). A solution of triphosgene (13.3 mg, 45 Mmiol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 J. Atwell, W. R. Wilson, W. A. Denny. Bzoorg. Med Chem. Lett., 1997, 7, 1483] (53 mg, 114 /jtmol) and Et 3 N (36 IzL, 256 jismol) in DCM (10 mL) and stirred at *C for 2 h. A solution of(-ehl2ntoIHpro--lmtao (168) (24 mg, 153 /.tmol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 *C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (10-20%) of EtOAc/DCM, to give 169 (21 mg, 28%) as a white solid, mp (EtOAc) 218-220 0 C; 'H NMR [(CD 3 2 S0] 8 11.46 (br s, 1 H, Rw~rrlflIf .Wnl rAAAl I WO 00/64864 PCT/GBOO/OI612 110indole-NH), 9.80 (br S, 1 H, OCONH), 8.55 (br s, 1 H, H 8.09 J 8.5 Hz, I H, H 6), 7.98 J 8.3 Hz, 1 H, H 7.5 8 (dd, J 8.3, 7.3 Hz, 1 H, H 7.48 (dd, J 8.5, 7.3 Hz' I H, H7), 7.3 3 (d,J =2.6 Hz, 1 H, 7.09 J= 1.9 Hz, 1 H, 6.98 1 H, H 6.37 (br s, 1 H, H 5.37 2 H, CH 2 4.80 (dd, J 11.0, 9.3 Hz, 1 H, H 4.52 (dd, J =11.0, 1.9 Hz, 1 H, H2), 4.31-4.37 I H, H 4.07 (dd, J =11. 1, 3.0 Hz, 1 H,
CH
2 CI), 3.96 3 H, NCH 3 3.9 1-3.94 (in, 4 H, CHPC, OCH1 3 3.82 3 H, OCH 3 3.80 3 H, OCH 3 1 3 C NMR f(CD 3 2 S0] 8 160.2 154.4 (OCONH), 149.1 (C 141.5 (C 3a), 139.9 (C 139.0 (C 134.3 (C 133.2 (C 130.7 (C 9a), 129.4 (C 2') 127.1 (C 125.4 (C 5a), 125.3 (C 124.3 (C 123.8 (C 123.3 (C 123.1 (C 122.0 (C 9b), 113.0 (C 108.1 (C 106.1 (C 98.0 (C 61.0 (QCH 3 60.9
(OCH
3 60.2 (CH 2 55.9 (OCH 3 54.8 (C 47.5 (CH 2 CI), 41.1 (C 37.9 (NCH 3
MS
(FAB-) m/z 650 (MWr, 648 (IvH~, HRMS (FAB~) caic. for C 32 H1 3 35 c1N 5 0 8 MW) mlz 648.186 1, found 648.1844; caic. for C 32
H
31 37
CIN
5 0 8
(MN
4 rn/z 650.1832, found 650.1826; Anal. (C 32
H
30 C1N 5 0s) C, H, N.
Example 9C. Preparation of ethyl 5-({[(1-(choromethyl)3(5,6,7rimethoxy-lH indol-1-ylocarbonyl-2,3-dihydro-1H-belzo~e1lindol-5-yllanmino)carboflloxy~methyl)- 1-ehl-ir-Hpyrl--abxlt (173).
Ethyl 5-omllmty4ntol-yrl--abxlt (171). Dimethyl sulfate (0.31 mL, 3.2 nimol) was added to a mixture of ethyl 5-formy1-4-nitro-1H-pyrrole- 2 carboxylate (170) Fornari, M. Famnier, C. Fournier, Bull. Soc. Chim. Fr. 1972, 283-291] (0.57 g, 2.7 nimol) and K 2 C0 3 (0.56 g, 4.0 nimol) in DMSO (4 mL) and the brown suspension was stirred at 20 *C for 1 h. The mixture was diluted with water (50 niL), acidified with HC1 (2 and extracted with EtOAc (2 x 50 niL). The combined extract was dried, the solvent evaporated. The residue was chromatograped, eluting with petroleum, to give 171 as a pale green solid mp (benzene/light petroleum) 70.5-7 1.5 'H NMR 8 10.55 1 H, CHO), 7.49 1 H, H 4.38 J= 7.2 Hz, 2 H, CH 2 4.33 3 H, NCH 3 1.40 J= 7.2 Hz, 3 H, CHO); Anal. (C91H, 0
N
2 0 5 C, H, N.
Ethyl 5(hydroxymetbyl)-lmethyl fitrolHpyrrole 2 carboxylate (172). NaBH 4 (0.33 g, 8.7 inmol) was added in portions to a solution of aldehyde 171 (3.96 g, 17.5 inmol) in EtOH (100 mL) and the mixture was stiffed at 20 *C for 20 min. Water (5 mL) was BNSDOCID: <WO 0064864AI I WO 00/64864 PCTIGB0OO612 -111Islowly added, the EtOH was evaporated, and the residue was diluted with brine and extracted with EtOAc (2 50 mL). The combined extract was washed with brine (50 mL), dried, and the solvent evaporated. The residue was recystallized to give 172 as a cream solid (8 1 mp (benzene) 119-120.5 'H NMR 867.48 1 H, H 4.97 6.8 Hz, 2 H, CH 2 4.32 J 7.1 Hz, 2 H, CH 2 4.06 3 H, NCH 3 2.72 J =7.1 Hz, I H, OH), 1.37 J= 7.1 Hz, 3 H, CH 3 Anal. (C 9
H
12
N
2 0 5 C, H, N.
Ethyl 5-((1(hooety)3[567timtoy -no--yl)carbonylj-2,3pyrrole-2-carboxylate (173). A solution of triphosgene (13.5 mg, 46 iAsmol) in DCM (2 mL) was added dropwise to a stirred solution of amnine 1 J. Atwell, W. R. Wilson, W.
A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (54 mg, 116 Psmol) and Et 3 N (36ALS, 260 Asmol) in DCM (10 miL) and stirred at 20 for 2 h. A solution of ethyl (hdoyehl--ehl4ntol-yrl--abxlt (172) (36 mg, 156 pAmol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 0 C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAcIDCM, to give 173 (52 mg, 62%) as a white solid, mp (EtOAc/light petroleumn) 227-229 'H NMR 1(CD 3 2 S0] 8 11.46 I H, indole-NH), 9.83 1 H, OCONH), 8.56 (br s, 1 H, H 8.02 J =8.5 Hz, 1 H, H 7.97 J= 8.3 Hz, 1 H, H 7.57 (dcl, J 7.4 Hz, I H, H 7.46 (dd, J 8.5, 7.4 Hz, 1 H, H 7.43 I H, H 7. 10 J 2.0 Hz, 1 H, H 6.98 I H, H 5.63 2 H,
CH
2 4.80 (cd, J 11.0, 9.4 Hz, 1 H, H 4.53 (dcl, J =11.0, 1.9 Hz, 1 H, H 4.33- 4.37 (in, 1 H, H 4.29 J =7.1 Hz, 2 H, H 4.00-4.08 (in, 4 H, CH 2 CI, NOCl 3 3.91-3.95 (in, 4 H, 01120, OCHA) 3.83 (in, 3 H, OCH 3 .80 3 H, OCH 3 1.31 J 7.1 Hz, 3 H, H 3 C. NMR [(CD 3 2 S0 8 160.1 159.3 154.0 (OCONH), 149.1 (C 141.4 (C 139.9 (C 139.0 (C 134.1 (C 133.6 (C 133.4 (C 130.7 129.4 (C 127.1 (C 125.4 125.3 (C 124.3 (C 123.7 (C 123.3 6")2 123.1 (C 122.4 (C 122.1 113.0 (C 111.2 (C 106.2 (C 98.0 (C 4 6 1.0 (OCH 3 60.9 (OCH 3 60.8 (CH 2 55.9 (OCHA) 54.8 (C 54.6 (C 47.5 (CHPC), 4 1.1 (C 3 3.6 (NCH 3 13.9 (C MS (FAB-) m/z 722 720 HRMS (FAB~) calc. for C 35
H
35 35
CIN
5 0 10 (MHW) miz 720.2073, found 720.2045; calc. for C 35
H
35 37 C1N 5 01 0 (MHW) m/z 722.2043, A~~lf f 1" ~i I WO 00/64864 PCT/GBOO01612 -112found 722.2039; Anal. (C 35
H
34 CINsO, 0 C, H, N.
Example 9D. Preparation of (1-methyl-3-nitro-1H-pyrrol-2-yl)methyl 1- (chloromethyl)-3-l(5,6,7-trimethoxy-lH-indol-l-yl)carbonyl]-23-dihydro-lHbenzolelindol-5-ylcarbamate (175).
2-(Hydroxymethyl)-l-methyl-3-nitropyrrole (174). A solution of NaOH (1 M, 16.7 mL, 16.7 mmol) was added to a solution of ester 172 (0.76 g, 3.34 mmol) in EtOH (50 mL), and the mixture was stirred at 20 "C for 1 h. The EtOH was evaporated, water (50 mL) added and the aqueous phase washed with diethyl ether (50 mL). The pH of the aqueous phase was adjusted to 2 with 1 M HCI. The aqueous mixture was extracted with EtOAc (3 mL), the combined organic extract was dried and the solvent evaporated to give crude 4- 2 -carboxylic acid (0.65 g, 97%) as a red-brown solid.
The acid was suspended in quinoline (10 mL) with Cu powder (0.50 g) and the mixture was heated at 180-190 OC for 50 min under N 2 The cooled mixture was diluted with EtOAc mL) and 1 M HCI (50 mL), extracted with EtOAc (3 50 mL), the combined extract was dried, and the solvent evaporated. The residue was purified by chromatography, eluting EtOAc/light petroleum, to give 174 as a pale yellow solid (0.29 g, mp 63-64 °C (benzene); 'H NMR 6 6.73 J= 3.4 Hz, 1 H, H 6.50 J= 3.4 Hz, 1 H, H 4.89 (d, J= 7.2 Hz, 2 H, CH20), 3.73 3 H, NCH 3 2.83 J= 7.2 Hz, 1 H, OH); 3 C NMR 8 134.9 (C 133.5 (C 122.0 (C 105.7 (C 53.8 (CH20), 34.8 (NCH 3 Anal.
(C
6 HsN 2 0 3 C, H, N.
(1-Methyl-3-nitro-1H-pyrrol-2-yl)methyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1Hindol--yl)carbonyl]-2,3-dihydro-lH-benzo[e]indol-5-ylcarbamate (175). A solution of triphosgene (15 mg, 50 /mol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (59 mg, 127 pmol) and Et 3 N (40 pL, 284 /imol) in DCM (10 mL) and stirred at °C for 2 h. A solution of alcohol 174 (27 mg, 171 Amol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 °C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient of EtOAc/DCM, to give 175 (11 mg, 13%) as a white solid, mp (EtOAc) 218-220 *C; 'H NMR 8 9.42 1 H, indole-NH), 8.94 1 H, OCONH), 7.87 J= 8.4 Hz, 1 H, H 6), ONSDOCIID.<WO 0064864A1 I PCT/GBOO/01612 WO 00/64864 113 7.79 J 8.2 Hz, 1 H, H 7.57 (ddd, J 7.4, 0.9 Hz, 1 H, H 7.47 (ddd, J 8.4, 7.4,0.9 Hz, 1 H, H7), 7.08 (br s, 1 H, H4), 7.01 J =2.2 Hz, I H, H 3%)6.89 I H, H 6.80 J 3.3 Hz, I H, H 6.57 J 3.3 Hz, I H, H 5.65 2 H, CH 2
O),
4.81 (dd, J =10.7, 91.7 Hz, 1 H, H 4.67 (dcl, J 10.7, 8.6 Hz, I H, H 4.15-4.20 (in, 1 H, H 4.10 3 H, OCH 3 3.95-3.99 (in, 4 H, OCH 3
CH
2 CI, 3.92 3 H, OCH 3 3.80 (br s, 3 H, NCH 3 .48 (dd, J= 11.0, 10.7 Hz, 1 H, CH4 2 CI; "C NMR 6 160.4 153.9 (OCONH), 150.2 (C 141.6 (C 3a), 140.6 (C 138.9 (C 133.7 (C 130.9 (C 129.7 (C 9a), 129.6 (C 128.8 (C 127.5 (C 125.6 (C 125.1 (C 7, C 123.6 (C 123.1 (C 122.6 (C 122.2 (C 121.6 (C 9b), 113.0 (C 106.5 (C 106.1 (C 97.7 (C 61.5 (OCH 3 61.1 (OCH 3 56.3 (OCH 3 55.3 (CH 2 54.9 (C 45.8 (CH- 2 CI), 43.4 (C 35.3 (NCH 3 MS (FAB 4 m/z 650 (MW, 648 (MH'r, HIRMS (FAB') caic. for C 32
H
31 3 3 5 C1N 5 0 8 (M7H+) m/z 648.1861, found 648.1850; caic. for C3 2
H
31 37
CIN
5 0 8 m/z 650.1832, found 650.1841; Anal. (C 32
H
30 C1N 5 0g) C, H,
N.
Example 9E. Preparation of (1-methyl-5-nitro-lH-pyrrol-2-yl)methyl 1- (chloromethyl)3(5,6,7trimethoxylHindol1l)carbonylk-2,-dihydrolH- (178).
(IMty--ir-Hpro--lmtao (177). NaBH 4 19 g, 5.03 inmol) was added to a stirred solution of 1-methy1-5-nitro-1H-pyrTole-2-carbaldehyde (176) P.
Fournari, Bull. Soc. Chim. Fr. 1963,488-491] (0.78 g, 5.07 inmol) in MeOH (40 niL) at room temperature under N 2 After addition was complete, the reaction mixture was stirred for a further 20 min, then water (40 mL) was added and the mixture was saturated with solid K 2 C0 3 The mixture was extracted with EtOAc (3 50 mL), the combined organic fraction dried, and the solvent evaporated to give 177 (0.77 g, 97%)as a white solid, mp (EtOAc/light petroleum) 76-77 0 C; 'H NMR 8 7.16 J =4.3 Hz, 1 H, H 6.17 J 4.3 Hz, I H, H 4.68 2 H, CH 2 4.02 3 H, CHO); Anal. (C 6
HN
2 0 3 C, H, N.
(1-Metbyl-5-nitro-1H-pyrrol2-yl)methyI 1-(chloromethyl)-3-I(5,6,7-trimethoxylHino--lcroyl23dhdol-ezlln l5ycrsmt (178). A solution of triphosgene (16 mg, 52 m.mol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (62 mng, 133 psmoI) and Et 3 N (42 juL, 299 humol) in DCM (10 mQL and stirred at WO 00/64864 PCT/GBOO/01612 114- 'C for 2 h. A solution of alcohol 177 (28 mg, 179 gumol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 *C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAcIDCM, to give 178 (54 mg, 63%) as a white solid, mp (EtOAc) 212-214 'H NMR [(CD 3 2 S0] a 11.45 (br s, 1 H, indole-NH), 9.82 (br s, 1 H, OCONH), 8.55 (br s, 1 H, H 8.04 J =8.5 Hz, 1 H, H 7.89 J 8.3 Hz, 1 H, H 7.57 (dd, J= 8.3, 7.2 Hz, I H, H 7.45 (dd, J 7.2 Hz, 1 H, H 7.24 J 4.4 Hz, 1 H, H 7.09 (d, J 2.0 Hz, I H, H 6.98 1 H, H 6.45 J 4.4 Hz, I H, H 5.30 2 H,
CH
2 4.80 (dd, J =11.0, 9.4 Hz, 1 H, H2), 4.53 (dd, J =11.0, 1.8 Hz, 1 H, H2), 4.32- 4.37 (in, 1 H, H 4.07 (dd, J =11. 1,3.1 Hz, I H, CH1 2 CI, 3.91-3.96 (m,7 H, CH 2
CI,
NCH
3
OCH
3 3.83 3 H, OCH 3 3.81 3 H, OCH 3 1 3 C NMR [(CD 3 2 S0] 8 160.2 154.0 (OCONH), 149.1 (C 142.1 (C 141.4 (C 3a), 139.9 (C 138.2 (C 136.1 (C 134.2 (C 130.7 (C 9a), 129.4 (C 127.1 (C 125.3 (C 5a), 125.2 (C 124.3 (C 123.7 (C 123.3 (C 123.1 (C 122.1 (C 9b), 113.1 (C 113.0 (C 110.6 (C 106.2 (C 98.0 (C 61.0 (OCH 3 60.9 (OCH 3 57.6
(CH
2 55.9 (0C11 3 54.8 (C 47.5 (CH 2 C1), 41.1 (C 33.9 (NCH 3 MS (FAB') mnk 650 (ME, 648 IHRIS (FAB~) caic. for C 32
H
31 3 3 5 C1NS0 8 m/Z 648.1861, found 648.1852; caic. for C 32
H
3 1 37 C1N 5 0 8 1 (ME'r) mnk 650.1832, found.650.1836; Anal. (C 32
H
30 C1N 5 0 8 C, H, N.
Elemental analysis data No Formula Calculated ()Found 1 8 C2,HsN,0 4 S C, 62.2; K, 3.7; N, 10.4 C, 62.3; H, 3.6; N, 10.7 2A 132 C,AH,C1,N,0 C, 5 1.6: H, 4.8: N, 9.5 C, 5 1.8; H, 4.9;N, 2B -134 CIJ,H,CIN.OQ C, 57.65; H, N, 9.0 C, 57.45: H, 4.7; N, 8.7 2C 135 C,,H,,C[NAOQ.H,O C, 59. 1; K, 4.5; 8.1 59.1; H, 4.2; N, 7.9 2D 136 1C.H., 7 N,S C, 60.7; H, 3.9; N, 9.7 C, 60.6; H, 4.0; N. 9.8 2EF 38- cJIno, 57.4; H, 4.8; N, 3.7 C, 57.2; H, 5.1; N, 3.9 2F 142-j- ,N,O,t.H2O C, 57.45; H, 4.9 C, 57.4; K, 5.1 2G 146 1C, 7 H.,NsOfi C, 62.7; K 5.3; N, 13.5 C. 62. 1:H. 5.5; N, 13.4 2H 151 C 41
H
4 ,NII07.'/,H, C, 68.6; H, 5.5; N, 10.7 C, 68.4; H, 5.6; N, 10.65 21 153 H,,N,0 4 M)802.2446 802.2446 21 155 C.JI.NO, M' 624.2306 624.2297 3A 59 3 C1,N 04 (Mi 425.0909 425.0901 3B 60 C,,H,,CNA, C, 62.0; H, 4.7; N, 8.5 C, 61.9; H, 4.8; N, 8.3 3C 64 C1N,O M*i 455.1015 455.1017 4A 70 C,,HlCINdO,, C, 59.6; H, 4.7; N, 7.95 C, 59.4; H, 4.9; N, 7.65 4B r73 C,H,,ClN 4
O
0 a C, 60.1; H, 4.9; N, 7.8 C, 60.3; H, 5. 1; N, 4C 78 C2HsCNO C, 51.9; H, 5.2; N, 8.6; Ch, 14.6 51.95; H, 5.2; N, 1.
4D 80 IC,H OCN O 0 4 C, 56.7; H, 5.05; N, 8.3 C, 56.9: H, 4.8; N, 8.1 4E 184 1 C. 60.1. H, 4.9; N. 7.8 C 60.2; H. 5.2; N, 7.6 BNSOID: <WO 006486,4A I> PCTGBOOIOI 612 WO 00164864 115 56.7; H, 5.1;N, 4F 4G 4H 41 4J 4K 4L 4M
SA
SC
SE
SF
51 6A 6B 7A 7B 8 9A 9B 9C 9D 9E 102 15i 107 113 117 123 124- 125 129 132 138 -142 148 150 154 155 159 160 163 -167 169 173 175 178 C4,H 4 6N 0..2HCI.2V2H, IC, 60.0; H, 5.7; N. 8.9 C16HI7 31
CIN
4 1 P 799.1783
C
1 H,,C1N4011.CHOH C, 57.9; H, 5.1; N, 7.3 -CIftH 4 1CINIOQ.2HCI C, 55.7; H, 5.2; N, 8.5: C,,H,CINO,p'/ 2HO C, 60.3; H, 5.4; N, 8.8 CjH54N 7 9J 892.4034 C,,tH~fN 6 0 0 ,.2HCI. H,O C 60. 1; H, 5.45; N, 8.
CtnH, 4 C1,N 4 0 4 C, 36.9; H, 4.3; N, 17.
C,AH1 9 CI, C 46.2; H, 4.6; N. 16.
C,
1
H,
0 CINO,.'/2H, C, 56.6; H, 4.6; N, 12.
C,,H~tN40,j.V21iO C, 53.9; H, 4.8; N, 7.6
C
4
,H
4 INqOI7.H, C, 55. 1; H, 4.85; N, 7.
C
11 H2QC1N 1 0f C, 57.4; H, 4.5; N, 12.
CI!.JCIN
1 O0 (M 324.1392
C,
6 HQoClNOa C, 46.2; H, 4.6; H, 16.
C,%H,
4 C1N#;Ot C, 57.4; H, 4.5; N, 12 C4AH.,N5O-,HO C, 55.1; H, 4.85; N,
C,,H
11 CIN,99 C, 56.6; H, 4.6; N, 12 C,,H,,CIN&OR C 57.4; H, N, 12 E 3 C I N 0'.M 4 401.0545 C,,H27CN40o C, 58.6; N. 8.1 0 C1,N~O~ C, 45.9; H -Cl,HCINORS C, 57.2; H, 4.2; N, 8.4 C,,H22C1NO. C, 57.4;1 H, 4.5; N. 12 CI,HuCIN!IO,o C, 58A4; H, 4.8; N, 9.'
CAXH
0 CNS0 2 C, 593; H, 4.7; N, 10
C
1
,HCINO
16 C, 58.4; H, 4.8; N, 9.
C,,H-mCINjOa C, 59.3; H. 4. N, 10 -C,,HX~INsO 1 C, 59.3; H, 4.7 N, 10 3.
C.99.7,5 9;N 79C.757:.4.:.72 C, 56.1; K, 4.8;N, 8.6 5 C6.; H, 5.5; N 8.6 60.3: H. 5.5; N, 1892.4055 8C, 37.4; H, 5.6; N, 1.2; 2.
2; CI 21.8 63 H, 4.1; N. 17.2; CI, 217.8 8.C,1.
8 .8 C, 46.6; H, 4.4; N, 12.5;C,1.
C- 53.7: H. 4.8: N. 7.3 IC. 57.7; H, 4.5; N, 1.9 3245.38145;N 1.
C.24.18146 N 1.
r. 57.5: H. 4.6; N. 12.9 .95 .8 A4 C. 57.5- H. 4.6: N, 7.9 C, 56.5; H, 49; N. 12.1 K 401.05469 40.085: 4.3 r. 46.2: H. 4.0: N. 9.9;C,1.
.1;CI, 17.0 1, 7.
C. 57.4;. H, 4.0; N, 8.4;C,1.
-I
C, 57.5: H. 4.6; N, 1.1 C. 57.5- H. N. 13.1 .95 7. C, 59. 4 94.; N,1.
7 1.8 1.8 C, 58.6;_H, 4.9; N, 9.89 C. 59.1:, -H.4.9;N,91.9 C. 59.4; H, 4.8; N, 10.7 N, 10.7 Example Biological activity Selected compounds were evaluated for cytotoxicity (measured as IC 50 values in .uM following and 18 h drug exposure) in pairs of mammalian cell lines, and the results are given in Table 2. The human ovarian carcinoma line (SKOV3) is wild-type, while the SC3.2 line is the NR+ transfectant. The human colon carcinoma line NR- line (WIDR) is wild-type, while the WCI14.l10 line is the NR+ transfectant The murine mammal)' carcinoma (EMT6-V) is wild-type, while the EN2A is the NR+ transfectant. Ratios (NRprovide a major measure of efficacy of action.
Table 3. Biological activity for selected compounds.
WO 00/64864 PCT/GBOO/01612 -116- 3B 60 0.157 5.6 0.15 6.6 4A 70 0.224 84 0.30 147 0.106 104 4B 73 0.105 67 0.109 45 0.033 78 4E 84 0.145 58 0.19 91 0.47 91 4G 91 9.4 >36 6.6 22 1.6 15.6 41 99 0.114 40 0.13 25 0.44 13.4 4K 105 0.088 6.4 0.097 9 0.024 6.1 123 2.3 28 1.7 6.7 SC 124 0.075 21 0.075 40 5D 125 3.74 14.5 3.59 10.3 0.46 6.4 129 >1.5 >12 >1.5 >5.6 0.24 4.95 51 138 0.15 50 0.23 99 0.078 71 53 142 >3 >9.0 2.72 1.64 0.23 >1.22 148 0.23 30 0.38 <3 8 0.067 <7 5L 150 0.061 5.6 0.024 3.4 0.063 7B 160 0.01 7.0 0.018 13.5 0.004 16.5 8 163 0.032 30 0.011 13.5 0.039 87 9A 167 0.097 9.6 0.094 13.2 0.028 11.6 BNSDOCID: <WO 0064864AI I>
Claims (23)
1. A compound of formula (II): wherein: E 0 11 X represents H, C 1 6 alkyl or C 1 6 alkoxy, said alkyl or alkoxy being optionally substituted with one or more of the following groups: hydroxy ether amino (NH 2 mono-substituted amino (NRXH), di-substituted amino 2 cyclic C 1 5 alkylamino, imidazolyl, C 1 6 alkylpiperazinyl, morpholino, thiol thioether tetrazole, carboxy (COOH), carboxylate suiphoxy 2 0H), suiphonate 2 suiphonyl 2 suiphixy suiphinate suiphinyl phosphonooxy (OP(=O)(OH) 2 and phosphate 2 where and are selected from a C 1 6 alkyl group, a C 3 20 heterocyclyl group or a C 5 20 aryl group; a is 0,1,2,3 or 4; Y represents H or C 14 6 ailkyl; 1, 2 or 3 of the members Z of the aromatic ring are independently selected from or where R is H or C 1 6 alkyl optionally substituted with one or more of the following groups: hydroxy ether (ORR), amino (NH 2 mono-substituted amino (NRRH), di-substituted amino (NRR'RR 2 cyclic C 1 5 alkylamino, imidazolyl, alkylpipbrazinyl, morpholino, thiol thioether (SRR), tetrazole, carboxy (COOH), carboxylate (COORR), sulphoxy 2 OH), sulphonate (S(=O)2ORR), sulphonyl suiphixy suiphinate sulphinyl phosphonooxy (OP(0)(OH) 2 and phosphate where RR, RR I and RR 2 are selected from a C 1 6 alkyl group, a C 3 20 heterocyclyl group or a C 5 20 aryl group, the other ring atoms being C; n is 0 or 1; and Printed:29-05-2001.Aede lam Amended claims avc iCTI GBOO/O1.61 2 ~LS~ M P. E is selected from formulae (1II-XIII); wherein, R, represents H or Ci- 6 alkyl, being optionally substituted with one or more of the following groups: one or more of the following groups: hydroxy ether (ORE), amino (NIH 2 mono- substituted amino (NREH), di-substituted amino (NRE'RE 2 cyclic C 1 5 alkylamino, imidazolyl, 6 alkylpiperazinyl, morpholino, thiol thioether (SRE), tetrazole, carboxy (COOH), carboxylate (COORE), suiphoxy 2 0H), sulphonate (S(=0)2ORE), sulphonyl (S=O)2RE), suiphixy sulphinate (S(0)ORE), sulphinyl phosphonooxy (OP(=O)(OH) 2 and phosphate (OP(=O)(ORE) 2 where RE, RE'I and RE 2 are selected from a 6 alkyl group, a C 3 20 heterocyclyl group or a C 52 o aryl group; R 2 represents H, C,. 6 alkyl, CI. 6 alkoxy, OH, halogen, NO 2 NH 2 NHMe, NMe 2 S0 2 Me, CF 3 CN, CONH 2 or CONIHMe; each R 3 is independently selected from Cl, Br, I and OMS; and R 4 is selected from -C(0)Me and -C(=O)CH 2 OH; Q represents substituted indole, substituted benzofuran or substituted cinnamoyl; in (IX) and each n is independently from 2-4, and each mn is independently from 2-4, and p =0 or 1. IFPrnted:29 05-2001 Amended claims R 3 fR (111 (V)(VI) OH 0 H R OH 0 HN -A (VIla) N, 0 HN (VIIb) 0 R 2 0 (I1)9) N 0 N- whereL= C+ N+C* N-C H, H, tm j i (MI) NH, N Me 0 NH (Xii) 120
2. A compound according to claim 1, wherein Rx, Rx, Rx 2 RR, RR 1 and RR 2 are independently Ci-6 alkyl groups.
3. A compound according to either claims 1 or claim 2, wherein the compound is of formula (II) with two Z, one Z being and the other Z being -NR-.
4. A compound according to claim 3, wherein R is either Me or Et.
A compound according to claim 4, wherein R is Et substituted with hydroxy.
6. A compound according to any one of claims 3 to 5, wherein a is 0.
7. A compound according to any one of claims 3 to 6, wherein the and -NR- are not adjacent in the heterocyclic ring.
8. A compound according to any one of claims 3 to 7, wherein E is of formula V.
9. A compound according to any one of claims 3 to 7, wherein E is of formula XIII.
10. A compound according to claim 9, wherein n 1.
11. A compound according to claim 1, wherein the compound is of formula a is 0, and Z is S.
12. A compound according to claiml, wherein the compound is of formula Z is NR, and a is either 0 or 1.
13. A compound according to claim 12, wherein Z is NMe.
14. A compound according to either claim 12 or 13, wherein a is 1 and X is C02Et.
A compound according to any one of the preceding claims for pharmaceutical use.
16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier or diluent.
17. A two component system for the treatment of neoplastic disease which comprises: a vector encoding and capable of expressing a nitroreductase enzyme in a tumour cell; and (ii) a compound as defined in any one of claims 1 to 14.
18. A two component system for the treatment of neoplastic disease which comprises: a tumour directed antibody linked to a nitroreductase enzyme; and (ii) a compound as defined in any one of claims 1 to 14.
19. A compound according to any one of claims 1 to 14, a composition according to claim 16, or a system according to claims 17 or 18 for use in a method of medical treatment.
20. A method of treating neoplastic disease which comprises administering to a patient in need of treatment an effective amount of a compound according to any one of claims 1 to 14, a composition according to claim 16, or a system according to claims 17 or 18.
21. The use of a compound according to any one of claims 1 to 14 for the manufacture of a composition for use in the treatment of a hyper-proliferative disease.
22. A method of providing an amine with a protecting group comprising: 25 providing a plurality of different compounds selected from compounds of formula (II) Z 2 ^koo E O 3CP04 204' JPWG1806 046.1 2 122 wherein: X represents H, C,. 6 alkyl or C 1 6 alkoxy, said alkyl or alkoxy being optionally substituted with one or more of the following groups: hydroxy ether amino (Nl 2 mono-substituted amino (NRXH), di-substituted amino (NRX'Rx 2 cyclic C 1 5 aikylamino, S imidazolyl, C 6 alkylpiperazinyl, morpholino, thiol thioether tetrazole, carboxy (COOH), carboxylate (COOR,), sulphoxy 2 0H), suiphonate 2 OR,), suiphonyl 2 suiphixy suiphinate suiphinyl phosphonooxy (OP(=O)(OH) 2 and phosphate (OP(=O)(OR) 2 where and R, 2 are selected from a CI. 6 alkyl group, a C 3 20 heterocyclyl group or a CS- 2 0 aryl group; a is 0,1,2,3 or 4; Y represents H or CI- 6 alkyl; 1, 2 or 3 of the members Z of the aromatic ring are independently selected from or where R is H or CI- 6 alkyl optionally substituted with one or more of the following groups: hydroxy ether (ORR), amino (NH 2 mono-substituted amino (NRRH), di-substituted amino (NRR'RR 2 cyclic C.. 5 alkylamino, imidazolyl, alkylpiperazinyl, morpholino, thiol thioether (SRR), tetrazole, carboxy (COOH), carboxylate (COORR), suiphoxy 2 OH), sulphonate 2 ORR), suiphonyl 2 RR), suiphixy sulphinate suiphinyl phosphonooxy (OP(=O)(OH) 2 and phosphate where RR, RR' and RR 2 are selected from a C,. 6 alkyl group, a C 3 2 0 heterocyclyl group or a Cs- 2 aryl group, the other ring atoms being C; n is 0 or 1; and E is selected from formulae (III-XIII); wherein, R, represents H or C,. 6 alkyl, being optionally substituted with one or more of the following groups: one or more of the following groups: hydroxy ether (ORE), amino (NH 2 mono-substituted amino (NREH), di-substituted amino (NRE'RE 2 Cyclic C 1 5 alkylamino, imidazolyl, C1-6 alkylpiperazinyl, morpholino, thiol thioether (SRE), tetrazole, carboxy (COOH), carboxylate (COORE), sulphoxy 2 0H), sulphonate (S(=O)2ORE), suiphonyl sulphixy suiphinate suiphinyl phosphonooxy (OP(=O)(OH) 2 and phosphate where RE, RE' and RE 2 are selected from a C,. 6 alkyl group, a C 3 20 heterocyclyl group or a C 5 20 aryl group; R 2 represents H, C.. 6 alkyl, C1- 6 alkoxy, OH, halogen, NO 2 NH 2 NHMe, NMe 2 SO 2 Me, CF 3 CN, CONH 2 or CONHMe; each R 3 is independently selected from Cl, Br, I and OMS; and R 4 is selected from -C(=O)Me and -C(=O)CH 2 OH; Q represents substituted indole, substituted benzofiran or substituted cinnamoyl; in (IX) and each n is independently rinted:29-5-20 I Amended claims from 2-4, and each m is independently from 2-4, and p 0 or 1 ~NR, c1 fN -N R3R 3 R 3 R 3 RF (VI) N S R2- N R (Vill) OH 0 HN~~~ (VIIb) (VIle) N 0 N 0(1)) HhrL H 2 H 2 1 P H 2 (MI) NH 2 N Me 0 NH HO, RIN I (XlI) 124 (ii) measuring the rates of fragmentation of the compounds to release EH when the nitro group is reduced and selecting a compound having a desired rate of decomposition; and (iii) providing the amine to be protected with a protecting group corresponding to that in the selected compound.
23. A compound as hereinbefore defined with reference to any one of Examples 2-9. DATED this eighth day of November 2001 Patent Attorneys for the Applicant: F.B. RICE CO.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9909612.5A GB9909612D0 (en) | 1999-04-26 | 1999-04-26 | N-protected amines and their use as prodrugs |
| GB9909612 | 1999-04-26 | ||
| PCT/GB2000/001612 WO2000064864A1 (en) | 1999-04-26 | 2000-04-26 | N-protected amines and their use as prodrugs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU4421100A AU4421100A (en) | 2000-11-10 |
| AU762914B2 true AU762914B2 (en) | 2003-07-10 |
Family
ID=10852308
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU44211/00A Ceased AU762914B2 (en) | 1999-04-26 | 2000-04-26 | N-protected amines and their use as prodrugs |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1173414A1 (en) |
| JP (1) | JP2002543059A (en) |
| AU (1) | AU762914B2 (en) |
| CA (1) | CA2368205A1 (en) |
| GB (1) | GB9909612D0 (en) |
| NZ (1) | NZ515044A (en) |
| WO (1) | WO2000064864A1 (en) |
Families Citing this family (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9818730D0 (en) | 1998-08-27 | 1998-10-21 | Univ Portsmouth | Collections of compounds |
| ATE320436T1 (en) | 1998-08-27 | 2006-04-15 | Spirogen Ltd | DIMERIC PYRROLOBENZODIAZEPINES |
| TW200410951A (en) * | 2002-08-06 | 2004-07-01 | Glaxo Group Ltd | M3 muscarinic acetylcholine receptor antagonists |
| EP1560599A1 (en) | 2002-11-14 | 2005-08-10 | Syntarga B.V. | Prodrugs built as multiple self-elimination-release spacers |
| GB0226593D0 (en) | 2002-11-14 | 2002-12-24 | Consultants Ltd | Compounds |
| ZA200507752B (en) * | 2003-03-28 | 2007-01-31 | Threshold Pharmaceuticals Inc | Compositions and methods for treating cancer |
| GB0321295D0 (en) | 2003-09-11 | 2003-10-15 | Spirogen Ltd | Synthesis of protected pyrrolobenzodiazepines |
| EP2336141B1 (en) | 2005-06-29 | 2016-03-30 | Threshold Pharmaceuticals, Inc. | Phosphoramidate alkylator prodrugs |
| RU2489423C2 (en) * | 2006-02-02 | 2013-08-10 | Синтарга Б.В. | Water-soluble analogues cc-1065 and their conjugates |
| US8552048B2 (en) | 2006-12-26 | 2013-10-08 | Threshold Pharmaceuticals, Inc. | Phosphoramidate alkylator prodrugs for the treatment of cancer |
| ES2884674T3 (en) | 2008-10-21 | 2021-12-10 | Immunogenesis Inc | Cancer treatment with the hypoxia-activated prodrug TH-302 in combination with docetaxel or pemetrexed |
| CA2803675A1 (en) | 2010-07-12 | 2012-01-19 | Threshold Pharmaceuticals, Inc. | Administration of hypoxia activated prodrugs and antiangiogenic agents for the treatment of cancer |
| WO2013096687A1 (en) | 2011-12-22 | 2013-06-27 | Threshold Pharmaceuticals, Inc. | Administration of hypoxia activated prodrugs in combination with chk1 inhibitors for treating cancer |
| EP2816036B1 (en) | 2012-02-13 | 2019-04-17 | University of Tsukuba | Prodrug using nitroimidazole |
| CN103450091A (en) * | 2012-05-29 | 2013-12-18 | 南京大学 | Imidazole derivatives, preparation method and applications thereof |
| WO2014062856A1 (en) | 2012-10-16 | 2014-04-24 | Halozyme, Inc. | Hypoxia and hyaluronan and markers thereof for diagnosis and monitoring of diseases and conditions and related methods |
| CN103910681A (en) * | 2013-01-09 | 2014-07-09 | 南京大学 | Metronidazole chalcone derivatives, and preparation method thereof and application utilizing antibacterial activity thereof |
| US20160158253A1 (en) | 2013-07-26 | 2016-06-09 | Threshold Pharmaceuticals, Inc. | Treatment of pancreatic cancer with a combination of a hypoxia-activated prodrug and a taxane |
| ES2785551T3 (en) | 2014-06-30 | 2020-10-07 | Glykos Finland Oy | Saccharide derivative of a toxic payload and its conjugates with antibodies |
| CN105801434A (en) * | 2016-04-18 | 2016-07-27 | 宜兴市新宇化工有限公司 | Production process of N,N-bis(beta-ethoxy)p-phenylenediamine sulfate |
| CN107266407B (en) * | 2017-06-08 | 2020-08-21 | 浙江工业大学 | Photosensitive targeted anti-tumor prodrug capable of killing tumor cells in response to nitroreductase and preparation method and application thereof |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL6717045A (en) * | 1967-01-06 | 1968-07-08 | ||
| US3450710A (en) * | 1967-01-06 | 1969-06-17 | Merck & Co Inc | Process for the preparation of nitroimidazole carbamates |
| ES2279506T3 (en) * | 1991-10-23 | 2007-08-16 | Cancer Research Technology Limited | BACTERIAL NITRORREDUCTASE FOR THE REDUCTION OF CB1954 AND ANALOGS OF THE SAME TO A CYTOTOXIC FORM. |
| NZ334344A (en) * | 1996-09-12 | 2000-08-25 | Cancer Res Campaign Tech | benzo[e]indole and pyrrolo[3,2-e]indole compounds and their use as prodrugs |
| GB9625913D0 (en) * | 1996-12-13 | 1997-01-29 | Cancer Soc Auckland Div Nz Inc | Novel cyclopropylindoles and their seco precursors,and their use as prodrugs |
-
1999
- 1999-04-26 GB GBGB9909612.5A patent/GB9909612D0/en not_active Ceased
-
2000
- 2000-04-26 AU AU44211/00A patent/AU762914B2/en not_active Ceased
- 2000-04-26 WO PCT/GB2000/001612 patent/WO2000064864A1/en not_active Application Discontinuation
- 2000-04-26 CA CA002368205A patent/CA2368205A1/en not_active Abandoned
- 2000-04-26 EP EP00925489A patent/EP1173414A1/en not_active Withdrawn
- 2000-04-26 JP JP2000613817A patent/JP2002543059A/en active Pending
- 2000-04-26 NZ NZ515044A patent/NZ515044A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO2000064864A9 (en) | 2001-12-27 |
| GB9909612D0 (en) | 1999-06-23 |
| CA2368205A1 (en) | 2000-11-02 |
| EP1173414A1 (en) | 2002-01-23 |
| NZ515044A (en) | 2003-05-30 |
| JP2002543059A (en) | 2002-12-17 |
| WO2000064864A1 (en) | 2000-11-02 |
| AU4421100A (en) | 2000-11-10 |
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