WO2010027280A1 - Nitrobenzindoles and their use in cancer therapy - Google Patents
Nitrobenzindoles and their use in cancer therapy Download PDFInfo
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- WO2010027280A1 WO2010027280A1 PCT/NZ2009/000184 NZ2009000184W WO2010027280A1 WO 2010027280 A1 WO2010027280 A1 WO 2010027280A1 NZ 2009000184 W NZ2009000184 W NZ 2009000184W WO 2010027280 A1 WO2010027280 A1 WO 2010027280A1
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- RXYPXQSKLGGKOL-UHFFFAOYSA-N CN1CCN(C)CC1 Chemical compound CN1CCN(C)CC1 RXYPXQSKLGGKOL-UHFFFAOYSA-N 0.000 description 1
- BHNHHSOHWZKFOX-UHFFFAOYSA-N Cc1cc(cccc2)c2[nH]1 Chemical compound Cc1cc(cccc2)c2[nH]1 BHNHHSOHWZKFOX-UHFFFAOYSA-N 0.000 description 1
- RHKRZQHJRZVTBP-UHFFFAOYSA-N ClCC1c(c2ccccc2cc2)c2NC1 Chemical compound ClCC1c(c2ccccc2cc2)c2NC1 RHKRZQHJRZVTBP-UHFFFAOYSA-N 0.000 description 1
- FKBYKBJFNXQINO-ZZXKWVIFSA-N OC(/C=C/C(CC1)=CC=C1OCCN1CCOCC1)=O Chemical compound OC(/C=C/C(CC1)=CC=C1OCCN1CCOCC1)=O FKBYKBJFNXQINO-ZZXKWVIFSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
- C07F9/572—Five-membered rings
- C07F9/5728—Five-membered rings condensed with carbocyclic rings or carbocyclic ring systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
Definitions
- the present invention relates generally to nitro-l,2 ⁇ dihydro-3H-benzo[ ⁇ ]indoles and related analogues, to their preparation, and to their use in cancer therapy.
- hypoxic cells have been established that many human tumors contain a significant proportion of hypoxic cells (Kennedy et al, Int. ]. Radiat. Oncol. Bio/. Phys., 1997, 37, 897-905; Brown et al., Cancer Res. 1998, 58, 1408-1416; Vaupel et al., Semin. Oncol, 2001, 28, 25-35).
- the presence of hypoxic cells arises because of chaotic growth and an inefficient microvasculature system within the tumour, so that tumours often exhibit large intercapillary distances and variable blood flow. Reduction of oxygen tension in tumors leads to radioresistance. Up to a three-fold increase in radiation dose may be required to kill anoxic tumor cells.
- the present invention provides a compound of Formula I,
- Y is selected from Cl or Br
- X is selected from SO 2 NR 1 2 or CONR 1 J , where X is located at either position 7 or 8, and where each R 1 independently represents H or a C ⁇ alkyl, optionally substituted with one or more hydroxyl groups, and wherein Z is a sidechain selected from either structure Ia or Ib
- R 2 represents a lower C 2.4 alkyl bearing a morpholine (Ic) or N-metliylpipera2ine (Id) substituent
- R 3 represents H, Q. 4 alkyl, C ⁇ alkoxy, Q ⁇ alkynyl or C 1-4 alkynyloxy and pharmaceutically acceptable salts thereof.
- R 3 represents H or C 1 . 4 alkyl.
- R 3 represents H.
- R 3 represents Q ⁇ alkynyl or C 1.4 allcynyloxy, where the alkyne functional group is a terminal alkyne.
- R is selected from the following groups:
- Y is Cl
- Y is Br
- X is located at the 7 position.
- X is7-SO 2 NH(CH 2 ) 2 OH.
- X is T-CONE ⁇ CH ⁇ OH.
- Z is selected from the group consisting of:
- R 3 is H, Y is Cl or Br, and X is 7-SO 2 NH(CBU) 2 OH or 7- SO 2 NH(CH 2 ) 2 OH.
- Z may be selected from groups A to D above.
- Z may be a group Ia, wherein R 2 is C 2 or C 3 alkyl bearing a morpholine (Ic) substituent.
- the compound of Formula I is selected from the group consisting of: l-(Chloromethyl)-N-(2-hyckoxye1 ⁇ yl)-3-[5-(2-morpholinoemoxy)-lH-indole-2-carbonyl]-5-mtro-l,2- dihydro-3fi-benzo[ ⁇ ]indole-7-sulfonamide; l-(Chlorome ⁇ yl)-N-(2-hydroxyem3d)-3-[5-(3-morpholi ⁇ opropoxy)-lH-indole-2-carbonyl]-5-nitro-
- the present invention provides a compound of Formula II, wherein Y is selected from Cl or Br, and wherein X is selected from SO 2 NR ⁇ or CONR 1 2 , where X is located at either position 7 or 8, and where each R 1 independently represents H or a C,_ 4 alkyl, optionally substituted with one or more hydroxyl groups, and wherein Z is a sidechain selected from either structure Ia or Ib
- R 2 represents a lower C 2 _ 4 alkyl bearing a morpholine (Ic) or N-methylpiperazine (Id) substituent
- R 3 represents H, a C 1-4 alkyl, C ⁇ alkoxy, Q ⁇ alkynyl or C 1-4 alkynyloxy, and pharmaceutically acceptable salts thereof.
- R 3 represents H or C 1-4 alkyl.
- R 3 represents H.
- R 3 represents C,. 4 alkynyl or C 1-4 alkynyloxy, where the alkyne functional group is a terminal alkyne.
- R 3 is selected from the following groups:
- Y is Cl
- Y is Br
- X is located at the 7-position. In certain embodiments, X is 7-SO 2 NH(CH 2 ) 2 OH.
- X is 7-CONH(CH. ⁇ !.
- Z is selected from the group consisting of:
- R 3 is H, Y is Cl or Br, and X is 7-SO 2 NH(CH 2 ) 2 OH or 7- SO 2 NH(CH 2 ) 2 OH.
- Z may be selected from groups A to C above.
- Z may be a group Ia, wherein R 2 is C 2 or C 3 alkyl bearing a morpholine (Ic) substituent.
- the compound of Formula II is selected from the group consisting of:
- the present invention provides compounds of Formula IIIa-d,
- Formula I and Formula II and the subsituents thereof are as defined above, and any compound of Formula I or Formula II bearing a free hydroxyl group is functionalised with a phosphate (Formula Ilia) or an amino acid or short polypeptide chain (Formula IHb) or a monosaccharide (Formula IHc), or wherein any compound of Formula I or Formula II bearing a free hydroxyl group or a secondary or tertiary amine is functionalised with a phosphonooxymethyl substituent (Formula IHd), wherein R 4 is selected from any substituent found in the naturally occuring amino acids, m is selected from 1, 2, 3, or 4, R 5 represents any combination of hydroxy, hydroxymethyl, carboxylic acid or other substituents found in naturally occuring monosaccharides, p represents 0 or 1, T represents O or N, and when T represents N it represents the secondary amine of the indole Ia or the tertiary amine of the morpholine Ic or either
- the compound of Formula III is a compound of Formula Ilia.
- R 3 represents H or C 1-4 alkyl.
- R 3 represents H.
- R 3 represents C 1-4 alkynyl or C 1-4 alkynyloxy, where the alkyne functional group is a terminal alkyne. In certain embodiments, R 3 is selected from the following groups:
- Y is Cl
- Y is Br
- X is 7-SO 2 NH(CH 2 ) 2 OH.
- X may be functionalized with a phosphate group to form the group 7-SO 2 NH(CH j ) 2 OPO(C)H) 2 .
- X is 7-CONH(CH 2 ) 2 OH.
- X may be functionalized with a phosphate group to form the group 7-CONH(CH 2 ) 2 OPO(OH) 2
- Z is selected from the group consisting of:
- the compound of Formula HIa is selected from the group consisting of: 2-[l-(Chloromemyl)-3-[5-(2-morpholinoethoxy)4H-indole-2-carbonyl]-5-nitro-l,2-dihydro-3H- benzo[>]indole-7-sulfonamido] ethyl dihydrogen phosphate;
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
- the present invention provides a method for the production of an anti-cancer effect in a warm-blooded animal such as a human, wherein the method comprises administering to the animal an effective amount of a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof.
- the present invention provides a method for the production of an anti-cancer effect in a cell, wherein the method comprises contacting the cell with an effective amount of a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof.
- Anti-cancer effects include, but are not limited to, anti-tumour effects, the response rate, the time to disease progression and the survival rate.
- Anti-tumour effects include but are not limited to, inhibition of tumour growth, tumour growth delay, regression of tumour, shrinkage of tumour, increased time to regrowth of tumour on cessation of treatment and slowing of disease progression.
- the present invention provides a method for the treatment of a cancer in a warm-blooded animal such as a human, which comprises administering to said animal an effective amount of a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof.
- the present invention provides the use of a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the production of an anti-cancer effect in a warm-blooded animal such as a human.
- the present invention provides the use of a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a cancer in a warm-blooded animal such as a human.
- the invention provides methods of preparing compounds of the general Formulae I, II or III, as defined above. Such methods are described below.
- Effective amount means an amount of a compound that, when administered to a subject for treating a disease state, is sufficient to effect such treatment for the disease state.
- the “effective amount” will vary depending on the compound, disease state being treated, the severity of the disease treated, the age and relative health of the subject, the route and form of administration, the judgement of the attending medical or veterinary practitioner, and other factors.
- Treating" or “treatment” of a disease state includes:
- “Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.
- “Pharmaceutically acceptable salts” of a compound means salts that are pharmaceutically acceptable, as defined herein, and that possess the desired pharmacological activity of the parent compound.
- Such salts include: acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, methanesulfonic acid, maleic acid, tartaric acid, citric acid and the like; or salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g.
- Acceptable organic bases include ethanolamine, diethanolamine, N- methylglucamine, triethanolamine and the like.
- Acceptable inorganic bases include aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.
- Warm blooded animal means any member of the mammalia class including, but not limited to humans, non-human primates such as chimpanzees and other apes and monkey species, farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
- the compound of Formula I, II or III may be in the form of a racemic mixture. In other embodiments, the compound of formula I, II or III may be in the form of a mixture in which either the S or the R enantiomer predominates, or in the form of either the S or the R enantiomer substantially free of the other enantiomer, for example containing less than about 5%, such as less than about 1 %, of the other enantiomer.
- Figure 1 shows an ORTEP representation of the crystal structure of (R)-di-/ «7'-butyl 2-[l- (chloromethy ⁇ -S-mtro-S- ⁇ ifluoroacety ⁇ -l ⁇ -dj ⁇ bydro-SH-benzo ⁇ jmdole-T-sulfonamidoJethyl phosphate (144).
- Figure 2 shows an ORTEP representation of the crystal structure of (J)-di- ⁇ ?/-/-butyl 2-[l- (chloiOmethj ⁇ - ⁇ -niiiO-S- ⁇ ifluoroaceiy ⁇ -l ⁇ -clihydiO-SH-benzof ⁇ indole-V-sulfonaiTitdoJethyl phosphate (145).
- Figure 3a is a bar graph comparing the activity in SiHa xenografts in combination with radiation of certain compounds of this invention and reference compounds, some of which are described in WO 2006/043839.
- Figure 3b is a bar graph comparing the activity in H460 xenografts in combination with radiation of certain compounds of this invention and reference compounds, some of which are described in WO 2006/043839.
- this invention relates to nitrobenzindoles and related analogues, in particular aminobenzindoles, of the Formulae I, II or III as defined above, and their use in treating cancer.
- the invention more particularly relates to the use of these compounds as prodrugs that are activated under hypoxic conditions, ie in an environment having a lower oxygen tension than that of normal tissues, to treat cancer.
- the compounds of the present invention have the same core ring structure as the compounds disclosed in WO 2006/043839. However, the compounds of the present invention differ from those disclosed in WO 2006/043839 in that they incorporate an indole or cinnamate group Z that bears an O-linked sidechain containing a morpholine or N-methylpiperazine substituent.
- the compounds of Formulae I, II and III of die present invention may be prepared using the methods described below. These methods form further aspects of the invention.
- the schemes A to M following the general descriptions of the methods below include details of the reagents used to achieve each of the synthetic steps.
- Compounds of Formula I may be prepared by first preparing an intermediate compound of Formula IV, V or VI as defined below. These compounds can be converted to desired compounds of Formula I by the methods following the description of the preparation of these intermediate compounds.
- X is defined for a compound of Formula I and J represents fe/f-butoxycarbonyl or trifluoroacetyl, can be prepared by a method including in a first step reacting a compound of Formula V
- X is defined for a compound of Formula I
- J represents / «?-butoxycarbonyl or trifluoroacetyl
- R 6 represents Q_ 4 alkyl or benzyl or benzyl substituted with C 1-4 alkyl or nitro groups
- the first step is achieved using sodium iodide in methyl ethyl ketone.
- the second step is achieved using silver mesylate in acetonitrile.
- the third step is achieved using lithium bromide in tetrahydrofuran.
- Compounds of Formula IV as defined above may also be prepared by the reaction of compounds of Formula V with an effective amount of a bromide salt in a non-nucleophilic solvent.
- Compounds of Formula I may be prepared from the above compounds of Formulae IV and V by a method which includes in a first step reacting a compound of Formula VIII
- X and Y are as defined for a compound of Formula I and J represents H, tert- butoxycarbonyl or trifluoroacetyl, with an effective amount of a nitrating agent to provide a compound of Formula IX wherein X and Y are as defined for a compound of Formula I and J represents H or trifluoroacetyl, and in a second step for the cases where J of Formula IX represents trifluoroacetyl reacting a compound of Formula IX with an effective amount of a weak base in the presence of water to provide a compound of Formula IX where J represents H, and in a third step reacting a compound of Formula IX where J represents H with a compound of Formula ZCO 2 H or ZCOCl wherein Z is as defined for a compound of Formula I, to provide a compound of Formula I.
- the first step is achieved using potassium nitrate in concentrated sulfuric acid.
- the second step is achieved using cesium carbonate in aqueous methanol.
- this step is achieved using JV-(3-dimetl ⁇ ylaminopropyl)-N'-ethyl carbodiimide or l-[3-(dimethylamino)propyl]-3- ethylcarbodiknide methiodide or other suitable coupling reagent.
- an effective amount of an acid co- reagent such as toluenesulfonic acid is also used.
- this compound is formed by the reaction of a compound of Formula ZCO 2 H with thionyl chloride or oxalyl chloride or oxalyl chloride in the presence of catalytic dimethylformamide.
- this step is achieved using at least one equivalent of non-nucleophiHc base such as di-zj ⁇ -propylethylamine.
- Compounds of the Formula ZCO 2 H can be prepared by a method which includes in a first step reacting a compound of Formula ZCO 2 R 7 wherein R 7 represents C 1-4 alkyl or phenyl or benzyl optionally substituted with one or more halide substituents, and Z is as defined for a compound of Formula I and contains the group Q wherein Q represents OH or NH 2 or CO 2 H, with a suitable derivative of a compound of Formula R 2 where R 2 is as defined for a compound of Formula I, and includes the structures Ie-In, such suitable derivatives being alcohols or halides or primary or secondary amines or acids or acid chlorides.
- Compounds of formula ZCO 2 R can be readily prepared using methods known to those skilled in the art.
- the reaction may involve alkylation with a halide derivative of R 2
- the reaction may involve a Mitsunobu reaction of an alcohol derivative of R 2
- the reaction may involve acylation using an acid chloride derivative of R 2 or amide coupling using an acid derivative of R 2
- Q represents CO 2 H the reaction may represent amide coupling with a primary or secondary amine derivative of R 2 .
- suitable examples of ZCO 2 R 7 may be formed by palladium or other metal catalysed reactions with halide derivatives of R 2 .
- the second step of the method includes reacting a compound of Formula ZCO 2 R 7 with base to provide a compound of Formula ZCO 2 H.
- the second step is achieved using potassium hydroxide or lithium hydroxide or sodium hydroxide in an aqueous alcoholic solvent.
- Compounds of Formula I wherein X represents SO 2 NR 1 , and R 1 is as defined for a compound of Formula I, may be prepared by reacting a compound of Formula I wherein X represents SO 2 Cl with a primary or secondary amine derivative of R 1 .
- reaction is conducted in the presence of at least one equivalent of a non-nucleophilic base such as di-zjo-propylethylamine or triethylamine.
- a non-nucleophilic base such as di-zjo-propylethylamine or triethylamine.
- Compounds of Formula I wherein X represents CONR' 2 and R 1 is as defined for a compound of Formula I may be prepared by reacting a compound of Formula I wherein X represents CO 2 H with a primary or secondary amine derivative of R , using an effective amount of a suitable coupling reagent.
- reaction is conducted using (benzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate.
- the reduction is achieved by hydrogenation using a suitable metal catalyst such as platinum or palladium, or by reaction with a metal such as zinc, preferably in a finely divided or powdered state, in the presence of a weak acid such as ammonium chloride.
- a suitable metal catalyst such as platinum or palladium
- a metal such as zinc
- a weak acid such as ammonium chloride
- R 8 represents C ⁇ alkyl or benzyl or substituted benzyl
- the phosphorylation reaction may be a one-step process using an effective amount of an appropriate phosphoramidite or a two- step process using phosphorus oxychloride followed by reaction of the intermediate with an alcohol R 8 OH, and in a second step reacting a compound of Formula IHe with an appropriate reagent to provide a compound of Formula Ilia, which reagent may be an acid when R 8 represents tert-butyl, or hydrogen and a metal catalyst when R 8 represents benzyl or substituted benzyl.
- R 8 represents /»V-butyl
- the first step is achieved using di-zW-butyl N,N- dietl ⁇ ylphosphoramidite or di-fe/f-butyl JV,JV-di-/jo-propylphosphoramidite.
- R 8 represents /m"-butyl
- the second step is achieved using trifiuoroacetic acid.
- Compounds of Formula IHa may also be prepared by reacting alcohol derivatives of R 1 or Formula IX using the phosphorylating conditions as described, and incorporating the so-formed phosphate ester into the general reactions as described above, to provide an alternative route to compounds of Formula IHe.
- Compounds of Formula HIb may be prepared by a method including in a first step reacting a compound of Formula I or Formula II bearing a free hydroxyl group with an amino terminal- protected amino acid or dipeptide or tripeptide or tetrapeptide using an effective amount of a peptide coupling reagent to provide a compound of Formula IHf
- R 4 and m are as defined for a compound of Formula IHb and R 9 represents a suitable amino protecting group such as / «?-butoxycarbonyl or 9-fluorenyknethoxycarbonyl, and in a second step reacting a compound of formula IHf with an appropriate reagent to provide a compound of Formula IHb, which reagent may be an acid when R 9 represents /m * -butoxycarbonyl or a base when R 9 represents 9-fiuorenylmethoxycarbonyl.
- the first step is achieved using 0-(benzotriazol-l-yl)-JV,iV,N',N'-tetramethyluroniumi hexafluorophosphate or 0-(7-azabenzotriazol-l -yl)-JV, ⁇ N ⁇ N'-tetramemyluronium hexafluorophosphate.
- R 9 represents / ⁇ n'-butoxycarbonyl
- the second step is achieved using trifiuoroacetic acid.
- R 9 represents 9-fluorenylmethoxycarbonyl
- the second step is achieved using morpholine or piperidine.
- R 4 contains a reactive functional group such as an amino group or carboxylic acid group
- suitably protected forms of these groups such as are commonly used in peptide synthesis may be employed, which protecting groups may be removed in the second step as described or in a prior or subsequent deprotection step to provide a compound of Formula IHb.
- Compounds of Formula IHb may also be prepared by reacting alcohol derivatives of R 1 or Formula IX using the peptide coupling conditions as described, and incorporating the so-formed amino terminal-protected amino acid or dipeptide or tripeptide or tetrapeptide into the general reactions as described above, to provide an alternative route to compounds of Formula IHf.
- Compounds of Formula IHc may be prepared by a method that includes in a first step reacting a compound of Formula I or Formula II bearing a free hydroxyl group with a suitably protected monosaccharide donor such as a monosaccharide bromide or monosaccharide imidate to provide a compound of Formula HIg
- R 10 represents a suitably protected form of R 5 which may represent an acetate when R 5 is hydroxy or hydroxymethyl, or a methyl ester when R 5 is a carboxylic acid, or other suitable protecting group as commonly used in the synthesis of mono- or polysaccharides, and in a second step or steps reacting a compound of Formula HIg with an appropriate reagent to provide a compound of Formula IHc, which reagent may be an aqueous base when R l ⁇ represents an acetate-protected hydroxy or hydroxymetiiyl group or a methyl ester-protected carboxylic acid.
- the first step is achieved using a monosaccharide bromide an effective amount of a silver salt such as silver triflate or silver carbonate is also used.
- a silver salt such as silver triflate or silver carbonate
- the first step is achieved using a monosaccharide imidate then a trichloroacetimidate is used, and further an effective amount of a Lewis acid such as boron trifluoride is also used.
- a Lewis acid such as boron trifluoride
- R 10 represents an acetate-protected hydroxy or hydroxymethyl group
- the second step is achieved using lithium hydroxide or potassium hydroxide in an aqueous alcoholic solvent.
- Compounds of Formula HIc may also be prepared by reacting alcohol derivatives of R 1 or Formula IX with the monosaccharide donors as described, and incorporating the so-formed suitably protected monosaccharides into the general reactions as described above, to provide an alternative route to compounds of Formula IHg.
- Compounds of Formula HId may be prepared by a method that includes in a first step reacting a compound of Formula I or Formula II bearing a free hydroxyl group or a secondary or tertiary amine group with an effective amount of a compound of Formula X wherein R 11 represents a good leaving group which may be a halide or a sulfonate such as p- toluenesulfonate or methanesulfonate or trifluoromethanesulfonate and R 12 represents a suitable phosphate protecting group which may be alkyl or allyl or benzyl or stibstituted benzyl, to provide a compound of Formula IIIh
- T, R, and q are as defined for a compound of Formula HId and R 12 is as defined for a compound of Formula X, with the understanding that where q is 2 the compound of Formula IIIh will be isolated as a salt with a counterion sufficient to balance the overall charge, and in a second step reacting a compound of Formula IIIh with a reagent suitable for deprotecting the phosphate ester, which reagent may be an acid or a base.
- the first step is achieved using a compound of Formula X wherein R 11 represents chloride and R 12 represents feti-butyl.
- the first step is achieved using a base such as sodium hydride or potassium carbonate.
- the first step is achieved using a polar aprotic solvent such as acetonitrile.
- R represents terf-hutyl
- the second step is achieved using an acid such as trifluoroacetic acid.
- Compounds of Formula IIIh may also be prepared by reacting alcohol or secondary amine or tertiary amine derivatives of R 1 or R 2 or Z or Formula IX with a compound of Formula X as described, and incorporating the so-formed suitably protected phosphonooxymethyl compound into the general reactions as described above, to provide an alternative route to compounds of Formula IIIh.
- the compounds of the Formulae I 3 II and III of the present invention can be used in the treatment of cancer of the human or animal body.
- the treatment may be of any cancer- type that includes hypoxic regions.
- the cancers treated may be solid tumors, such as ovarian, colon, brain, thyroid, pancreas, bladder, breast, prostate, lung (such as small cell lung tumor cells and large cell lung carcinoma), cervical and skin cancer.
- the cancer may be leukemia, multiple myeloma or lymphoma.
- the compounds of the invention can be administered in the form of pharmaceutical compositions, containing one or more compounds of the invention in combination with one or more pharmaceutically acceptable carriers.
- the pharmaceutically acceptable carrier(s) should be non-toxic and not interfere with the efficacy of the active ingredient.
- the precise nature of the carrier will depend on the route of administration, which can be oral, or parenteral, including intravenous, cutaneous, subcutaneous, intramuscular, intravascular or by infusion.
- compositions suitable for oral adrninistration can be in tablet, capsule, powder or liquid form.
- a tablet may comprise one or more solid carriers and/or adjuvants.
- a capsule may include a solid carrier such as gelatin.
- Liquid pharmaceutical compositions may comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
- the pharmaceutical composition may conveniently be in the form of a parenteraUy acceptable aqueous solution which is pyrogen-free and has a suitable pH, isotonicity and stability.
- a parenteraUy acceptable aqueous solution which is pyrogen-free and has a suitable pH, isotonicity and stability.
- Those of skill in the art are able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, and Lactated Ringer's injection.
- Preservatives, stabilisers, btiffers, antioxidants and/or other additives may be included as required.
- the exact dose of the compound to be administered will be at the discretion of the physician, talcing into account the condition and needs of the patient. Typical doses and administration schedules will be determined by experience in clinical trials. Total doses are expected to be in the range from about 0.1 to 200 mg/kg per subject, such as about 10 mg/kg per subject.
- the compounds of Formula I, II or III can be used as single agents or in combination with one or more other cytotoxic or other therapeutic agents or therapies, especially those that are relatively ineffective against hypoxic cells, such as radiation therapy. Where such other agents and/or radiotherapy are administered in combination with a compound of the invention, the radiation and/ or other agents may be administered before, during or after administration of the compound of Formula I, II or III.
- Pure carbamate 103 can be obtained by repeating the procedure of dissolving in petroleum ether, allowing an orange solid to separate, filtering off the solid, and evaporating the filtrate, to give 103 as an oil (100%):
- Sulfonyl chloride 106 (1.02 g, 2.48 mmol) was added to cold cone. H 2 SO 4 (10 mL) and the mixture was stirred at 0 0 C for 2 min to give a homogeneous solution. Solid KNO, (351 g, 3.47 mmol) was added portionwise over 2 min and the mixture was stirred at 0 0 C for a further 2 min. Ice-water (100 mL) was added and the mixture was stirred at 0 0 C for 2 min.
- Tetrazole (3 wt% solution in CH 3 CN, 468 mL, 159 mmol) was added gradually over 1 h to a stirred mixture of di-f ⁇ '-butyl IV,N-diisopropylphosphoramidite (95%, 53.0 mL, 159 mmol) and benzyl 2- hydiOxyethylcarbamate (109) (26.0 g, 133 mmol) in THF (500 mL) at 20 0 C under a nitrogen atmosphere. After the addition was complete the mixture was stirred at this temperature for 19 h. The mixture was cooled to 0 0 C and H 2 O 2 (30% aqueous, 48 mL, 494 mmol) was added.
- ester 128 (6.58 g, 22.6 mmol), MeOH (40 mL), water (20 mL) and KOH (3.16 g, 56.5 mmol) was stirred at 20 0 C for 21 h. The mixture was evaporated under reduced pressure at 35 0 C to remove the MeOH. The resulting alkaline mixture was washed with CH 2 Cl 2 and the aqueous layer- was filtered. The alkaline filtrate was acidified with cone. HCl at 0 0 C.
- Phosphate ester 131 (366 mg, 0.437 mmol) was stirred with TFA (5 mL) in CH 2 Cl 2 (50 mL) at 20 0 C for 3 h. The reaction solution was filtered through a cotton wool plug and the plug was washed with methanol. The filtrate was concentrated under reduced pressure until a precipitate began to form. EtOAc (150 mL) was added and the mixture was stirred at 0 0 C for 45 min. The solid was filtered off, washed with EtOAc several times, and dried in vacuum over silica gel to give 17 as a yellow solid (292 mg, 80%): mp 212 0 C (dec); 1 H NMR (C 5 D 5 N) ⁇ ca. 10.7 (v br s, 1 H), 9.81 (br s, 1 H),
- Phosphate ester 135 (1.57 g, 1.85 mmol) was dissolved in CH 2 Cl 2 (100 mL) and stirred with TFA (10 mL) at 20 0 C for 3 h. The mixture was filtered and the filter pad was washed with 10% TFA in CH 2 Cl 2 . The combined filtrates were concentrated at 25 0 C (bath temperature) under reduced pressure at to half of the original volume. EtOAC (ca. 100 mL) was added, the mixture was stirred and then left to stand at 0 0 C for 15 h.
- Example 15 ( ⁇ -l-CChloromethy ⁇ -A ⁇ Z-hydroxyethy ⁇ -S-IS- ⁇ -CZ- morpholinoethoxy)phenyl]acryloyl ⁇ -5-nitro-l,2-dihydfo-3i ⁇ -benzo[e]indole-7-carboxamide hydrochloride (6) (Scheme G).
- EDCLHCl 132 mg, 0.69 mmol
- anhydrous TsOH 6.0 mg, 0.034 mmol.
- Example 18 (5)-5-Amino-l-(chlor omethylJ-iV ⁇ -hydr oxyethyl)-3- [5-(2-morpholinoethoxy)- l ⁇ £indole-2-carbonyl] ⁇ l,2-dihydro-3i ⁇ -benzo [e] indole-7-sulfonamide hydrochloride (14) (Scheme I). To a solution of nitro compound 7 (78 mg, 0.112 mmol) in acetone (6 mL) and water (4 mL) stirred under nitrogen at 20 0 C was added ammonium chloride (312 ing, 5.8 mmol) and Zn powder (234 mg, 3.6 mmol).
- the heterogeneous mixture was stirred at 20 0 C for 1 h 30 min. Sufficient cold aqueous Na,CO 3 (2%) was added to generate pH 8-9 and then the product was extracted with CH 2 Cl 2 (15 x 10OmL). The combined CH 2 Cl 2 extracts were washed with water, dried (Na 2 SO 4 ), and concentrated under reduced pressure to ca. 10 mL in volume. HCl (1.25M in MeOH, 1.0 mL) was added followed by EtOAc (60 mL). The mixture was left to stand at 5 0 C overnight.
- Example 21 l-(Bromomethyl)-7V-(2-hydro ⁇ yethyl)-3- [5-(2-morpholinoethoxy)-lJy r -indole-2- carbonyl]-5-nitro-l,2-dihydfo-3if-benzo[e]indole-7-sulfonamide (8) (Scheme K and L).
- a solution of NaI (7.19 g, 47.9 mmol) in 2-butanone (30 mL) was heated at 85 0 C for 1 h.
- Indoline 105 5.0 g, 16.0 mmol
- EtOAc and water were added and the organic layer was separated.
- EDCLHBr 230 mg, 0.98 mmol
- anhydrous TsOH 6 mg, 0.033 mmol
- acid 163 121 mg, 0.33 mmol
- indoline 160 70 mg, 0.16 mmol
- CH 2 Cl 2 15 mL
- DMA 2 mL
- EDCLHBr 230 mg, 0.98 mmol
- TsOH 20 mg, 0.12 mmol
- Example 22 2-[l-(Bromomethyl)-3-[5-(2-motpholinoethoxy)-li ⁇ indole-2-carbonyl]-5-nitro- l,2-dihydro-3i ⁇ benzo[e]indole-7-sulfonamido] ethyl dihydrogen phosphate trifluoroacetate (23) (Scheme L).
- EDCLHBr prepared as described in Example 21, 3.74 g, 15.8 mmol
- anhydrous TsOH 109 mg, 0.63 mmol
- acid 163 1.30 g, 3.48 mmol
- indoline 162 1.97 g, 3.17 mmol
- CH 2 Cl 2 55 mL
- DMSO DMSO
- anhydrous TsOH 200 mg, 1.16 mmol
- indoline 162 600 mg, 1.61 mmol
- EDCLHBr prepared as described in Example 21, 329 mg, 1.40 mmol
- anhydrous TsOH 8 mg, 0.047 mmol
- acid 165 166 mg, 0.47 mmol
- EDCLHBr 329 mg, 1.40 mmol
- anhydrous TsOH 8 mg, 0.047 mmol
- Example 24 ⁇ E)-2- [l-(Bromomethyl)-3- ⁇ 3- [4-(2-morpholinoethoxy)phenyl] actyloyl ⁇ -5-nitro- l,2-dihydro-3i ⁇ benzo[e]indole-7-sulfonamido] ethyl dihydrogen phosphate ttifluoroacetate (24) (Scheme M).
- EDCLHBt prepared as described in Example 21, 820 mg, 3.47 rnmol
- anhydrous TsOH (20 mg, 0.12 rnmol)
- acid 165 410 mg, 1.16 mmol
- indoline 162 360 mg, 0.58 mmol
- DMA 1 ml
- the mixture was poured into a mixture of cold water and cold EtOAc.
- the organic layer was washed with cold water, cold brine and then dried (Na 2 SO 4 ) and evaporated.
- the residue was dissolved in a mixed solvent of CH 2 Cl 2 and MeOH and the solution was slowly concentrated to induce precipitation.
- mice The ability of compounds of Formula III to eliminate colony forming cells of human tumor xenografts grown in nude mice (CD1-Foxnl nu ) was determined using an excision assay. Tumors were grown subcutaneously in the flank by inoculating cells grown in tissue culture (10 7 cells in 100 ⁇ L). When tumors reached treatment size (500-700 mm 3 ) mice were randomised to treatment groups (3 mice per group for radiation or drug alone, 5 mice per group for compound plus radiation). Compounds were dissolved in phosphate buffered saline containing 2-4 equivalents of NaHCO 3 and given as single intravenous doses alone or 5 min after whole body irradiation ( 60 Co source).
- Clonogens per gram of tumor tissue were calculated relative to controls: for compound alone relative to no treatment, and for compound plus radiation relative to radiation alone.
- the compounds were administeied at the following doses: 42 ⁇ imol/kg for SiHa, 56 ⁇ mol/kg for
- This assay provides evidence of hypoxia selective cell killing in vivo.
- a whole body radiation dose of 15 Gy is sufficient to steiilize well oxygenated tumor cells, and provides 1-2 logs of cell loll in these xenograft models. Additional cell kill when radiation is combined with the compounds of Formula III (15-24) shows that these compounds aie eliminating hypoxic tumor cells.
- the compounds of Formula III aie significantly moie active in this regard than the reference compounds of WO 2006/043839 (Rl-4).
- the compounds of Foimula III are also significantly more active than two ieference compounds R5 and R6, which both incorporate a morphokne in the sidechain, but not one which is attached to an indole (Formula Ia) or cinnamate (Formula Ib) sidechain.
- the difference between the vaiious refeience compounds and the examples of this invention is illustrated in Figures 3a and 3b, respectively, for SiHa and H460 xenografts. The difference is also notable for the Hl 299 and HCTIl 6 xenografts in Table 4. Table 4 also shows evidence of single agent activity.
- the superior in vivo activity of the compounds of Formula III is unexpected since the structures of the reference compounds Rl-4 of WO 2006/043839 are very similar to the structures of the examples of Formula III (15-24 using the numbering in Table 3).
- the superior in vivo activity of the compounds of Formula III is particularly unexpected since reference compounds which include a morpholine in the sidechain (R5 and R6), but not one which is attached to an indole or cinnamate sidechain, show very little activity in vivo.
Abstract
The invention provides novel nitro-l,2-dihydro-3.H-benzo[e]indole compounds and related analogues that are useful in cancer treatment, pharmaceutical compositions containing such compounds and methods of treating cancer comprising administering the compounds. The compounds of the invention include compounds of the Formula (I), wherein Y is selected from Cl or Br, and wherein X is selected from SO2NR1 2 or CONR1 2, where X is located at either position 7 or 8, and where each R1 independently represents H or a C1-4alkyl, optionally substituted with one or more hydroxyl groups, and wherein Z is a side chain selected from either structure (Ia) or (Ib), wherein R2 represents a lower C2-4 alkyl bearing a morpholine (Ic) or N-methylpiperazine (Id) substituent, and R3 represents H, C1-4 alkyl, C1-4alkoxy, C1-4alkynyl or C1-4alkynyloxy and pharmaceutically acceptable salts thereof. Also provided are compounds of Formula (II), where the nitro group of Formula (I) is replaced with an amino group, and compounds of Formula (III), where a free hydroxyl group of a compound of Formula (I) or (II) is functionalised with a phosphate, amino acid, short polypeptide chain or monosaccharide, or a free hydroxyl or secondary or tertiary amine group of a compound of Formula (I) or (II) is functionalised with a phosphonooxymethyl substituent.
Description
NITROBENZINDOLES AND THEIR USE IN CANCER THERAPY
TECHNICAL FIELD
The present invention relates generally to nitro-l,2~dihydro-3H-benzo[ϊ]indoles and related analogues, to their preparation, and to their use in cancer therapy.
BACKGROUND TO THE INVENTION
It has been established that many human tumors contain a significant proportion of hypoxic cells (Kennedy et al, Int. ]. Radiat. Oncol. Bio/. Phys., 1997, 37, 897-905; Brown et al., Cancer Res. 1998, 58, 1408-1416; Vaupel et al., Semin. Oncol, 2001, 28, 25-35). The presence of hypoxic cells arises because of chaotic growth and an inefficient microvasculature system within the tumour, so that tumours often exhibit large intercapillary distances and variable blood flow. Reduction of oxygen tension in tumors leads to radioresistance. Up to a three-fold increase in radiation dose may be required to kill anoxic tumor cells. A link has been identified between the presence of tumor hypoxia and failure of local control by radiation therapy (Nordsmark et al., Radiother. Oncol., 1996, 41, 31-39; Brizel et al., Radiother. Oncol, 1999, 53, 113-117; Movsas et al., Urology, 2002, 60, 634-639; Koukourakis et al.,/. CHn. Oncol, 2006, 24, 727-735). Hypoxia also contributes to chemoresistance by mechanisms which include limitation of the delivery of drugs to hypoxic regions of tumors (Minchinton & Tannock, Nat. Rep. Cancer, 2006, 6, 583-592; Hicks et al.J. Natl. Cancer Inst., 2006, 98, 11184128). The phenomenon of tumor hypoxia has been exploited in the development of a class of anticancer agents termed liypoxia-activated prodrugs' which are also sometimes referred to as 'bioreductive drugs' although the latter term also encompasses prodrugs activated by reduction under oxic conditions (Brown et al., Semin. Radiat. Oncol, 1966, 6, 22-36; Denny et al., Br. J. Cancer, 1996, 74 (Suppl. XXVII) 32-38; Stratford & W otlmxnn, Anti-Cancer Dmg Des., 1998, 13, 519-528; Brown et al., Nat. Rep. Cancer 2004, 4, 437; Brown et al., Frontiers in Bioscience, 2007, 12, 3483).
Various nitro(hetero)aromatic compounds have been reported as hypoxia-activated prodrugs. These include
• the nitroimidazole (i), which is proposed to undergo fragmentation following nitro group reduction by endogenous cellular nitroreductase enzymes (McClelland et al., Biochem.
Pharmacol., 1984, 33, 303-309),
the dinitrobenzamide mustard (ii) and analogues, where similar reduction of the nitro group activates the mustard (Palmer et al.,/. Med. Chetn. 1996, 39, 2518; Helsby et al., Chem. Res. Toxicol., 2003, 16, 469-478; Wilson et al., Radiation Res. 2007, 167, 625; Patterson et al., Clin. Cancer Res. 2007, 13, 3922; Denny et al., NZ Patent 529249), and the nitrobenzofήindole (iii) and analogues have been reported as potential bioreductive drugs activated by the E. rø/z NTR enzyme (Denny et al., PCT Int. Appl. WO 98/11101 A2, 1998; Atwell et al.J. Org. Chem. 1998, 63, 9414-9420; Atwell et al., Bioorg. Med. Chem. Lett. 1997, 7, 1493-1496.)
• Certain nitrobenzo[e]indoles and analogues thereof, and their use in cancer therapy, are also disclosed in PCT international application PCT/NZ2005/000278 (WO 2006/043839).
It is an object of the present invention to provide a specific class of nitro-1 ,2-dihydro-3H- benzo[e]indoles, that are useful in cancer treatment, or to at least provide the public with a useful alternative.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a compound of Formula I,
wherein R2 represents a lower C2.4 alkyl bearing a morpholine (Ic) or N-metliylpipera2ine (Id) substituent, and R3 represents H, Q.4 alkyl, C^alkoxy, Q^alkynyl or C1-4 alkynyloxy and pharmaceutically acceptable salts thereof.
In certain embodiments, R3 represents H or C1.4 alkyl.
In certain embodiments, R3 represents H.
In certain embodiments, R3 represents Q^alkynyl or C1.4allcynyloxy, where the alkyne functional group is a terminal alkyne.
In certain embodiments, R is selected from the following groups:
In certain embodiments, Y is Cl.
In certain embodiments, Y is Br.
In certain embodiments, X is located at the 7 position.
In certain embodiments, X is7-SO2NH(CH2)2OH.
In certain embodiments, X is T-CONE^CH^OH.
In certain embodiments, Z is selected from the group consisting of:
In certain embodiments, R3 is H, Y is Cl or Br, and X is 7-SO2NH(CBU)2OH or 7- SO2NH(CH2)2OH. In such embodiments, Z may be selected from groups A to D above. Alternatively, Z may be a group Ia, wherein R2 is C2 or C3 alkyl bearing a morpholine (Ic) substituent.
In certain embodiments, the compound of Formula I is selected from the group consisting of: l-(Chloromethyl)-N-(2-hyckoxye1±ιyl)-3-[5-(2-morpholinoemoxy)-lH-indole-2-carbonyl]-5-mtro-l,2- dihydro-3fi-benzo[^]indole-7-sulfonamide; l-(Chloromeώyl)-N-(2-hydroxyem3d)-3-[5-(3-morpholiαopropoxy)-lH-indole-2-carbonyl]-5-nitro-
1 ,2-dihydiO-3 H-benzo [tf]indole-7-sulfonamide;
(^-l-(Chloromemyl)-JV-(2-hydroxyetliyl)-3-{3-[4-(2-rnorpholinoethoxy)phenyl]acryloyl}-5-nitro-
1 ,2-clϋiydro-3H-benzo [φndole-7-sulfonamide; l-(Chlorornethyl)-JV-(2-hydroxyethyl)-3-{4-[2-(4-mLethylpiperazin-l-yl)etlαoxy]benzoyl}-5-nitro-l,2- dihydro-3H-benzo[ά]indole-7-sulfonamide; l-(Chlorornethyl)-iV-(2-hydroxyeiiiyl)-3-[5-(2-morpholinoethoxy)-lH-indole-2-carbonyl]-5-nitro-l,2- dihydro-3H-benzo[άi]indole-7-carboxamide;
(^-l-(chloromethyl)-N-(2-hydiOxyethyl)-3-{3-[4-(2-morpholinoethoxy)phenyl]acryloyl}-5--nitro-l,2- dihydro-3J:J-benzo[e]indole-7-carboxamide;
(S)-I- (Chloromethyl) -N- (2-hydroxy ethyl) -3- [5- (2-morpholinoethoxy) -1 H-indole-2-carbonyl] -5-nitro- l,2-dihydro-3H-benzo[(?]indole-7-sulfonamide; l-(Bromomediyl)-N-(2-hydroxyeώyl)-3-[5-(2-moi^ho]Jiioemoxy)4H-indole-2-carbonyl]-5-mtt dihydro-3f"J-benzo [<?]indole-7-sulfonamide; and
(iB)-l-(Brom.omemyl)-JV-(2-hydiOxyethyl)-3-{3-[4-(2-rnoiphoHnoemoxy)phenyl]aciyloyl}-5-nitro-
1 ,2-diliydro-3f/-benzo [ήindole-7-sulfonamide.
In a second aspect, the present invention provides a compound of Formula II,
wherein Y is selected from Cl or Br, and wherein X is selected from SO2NR^ or CONR1 2, where X is located at either position 7 or 8, and where each R1 independently represents H or a C,_4alkyl, optionally substituted with one or more hydroxyl groups, and wherein Z is a sidechain selected from either structure Ia or Ib
wherein R2 represents a lower C2_4 alkyl bearing a morpholine (Ic) or N-methylpiperazine (Id) substituent, and R3 represents H, a C1-4 alkyl, C^alkoxy, Q^alkynyl or C1-4 alkynyloxy, and pharmaceutically acceptable salts thereof.
In certain embodiments, R3 represents H or C1-4 alkyl.
In certain embodiments, R3 represents H.
In certain embodiments, R3 represents C,.4alkynyl or C1-4 alkynyloxy, where the alkyne functional group is a terminal alkyne.
In certain embodiments, R3 is selected from the following groups:
In certain embodiments, Y is Cl.
In certain embodiments, Y is Br.
In certain embodiments, X is located at the 7-position.
In certain embodiments, X is 7-SO2NH(CH2)2OH.
In other embodiments, X is 7-CONH(CH.^!!.
In certain embodiments, Z is selected from the group consisting of:
In certain embodiments, R3 is H, Y is Cl or Br, and X is 7-SO2NH(CH2)2OH or 7- SO2NH(CH2)2OH. In such embodiments, Z may be selected from groups A to C above. Alternatively, Z may be a group Ia, wherein R2 is C2 or C3 alkyl bearing a morpholine (Ic) substituent.
In certain embodiments, the compound of Formula II is selected from the group consisting of:
5-Amino-l-(chloromethyl)-JV-(2-hydroxyeώyl)-3-[5-(2-rnorpholinoeώoxy)-lH-indole-2-carbonyl]-
1 ,2-dihydro-3H-benzo [ήindole-7-sulfonamide;
5-Aiiiino-l-(chloromethyl)-JV-(2-hyckoxyemyl)-3-[5-(3-morpholinopropoxy)-lH-indole-2-carbonyl]-
1 ,2-dihydro-3H-benzo [>]indole-7-sulfonamide;
(E)-5-Arrdno-l-(chloronaeώyl)-JV-(2-hyckoxyethyl)-3-{3-[4-(2-ixιoipholinoethoxy)phenyl]acryloyl}-
1 ,2-dihydiO-3H-benzo [e]indole-7-sulfonamide;
5-Amino-l-(chloromeώyl)-JV-(2-hycUOxyeώ^
1 ,2-dihydiO-3H-benzo [tf]indole-7-carboxamide; and
(i)-5-Amiiio4-(chloromediyl)-JV-(24iydiOxyeώyl)-3-[5-(2-moiphoHnoethoxy)-lH-indole-2- carbonyl]-l,2-dihydro-3H-benzo[ά]indole-7-sulfonamide.
In a third aspect, the present invention provides compounds of Formula IIIa-d,
In certain embodiments, the compound of Formula III is a compound of Formula Ilia.
In certain embodiments, R3 represents H or C1-4 alkyl.
In certain embodiments, R3 represents H.
In certain embodiments, R3 represents C1-4 alkynyl or C1-4 alkynyloxy, where the alkyne functional group is a terminal alkyne.
In certain embodiments, R3 is selected from the following groups:
In certain embodiments, Y is Cl.
In certain embodiments, Y is Br.
In certain embodiments, X is 7-SO2NH(CH2)2OH. In such embodiments X may be functionalized with a phosphate group to form the group 7-SO2NH(CHj)2OPO(C)H)2 .
In other embodiments, X is 7-CONH(CH2)2OH. In these embodiments, X may be functionalized with a phosphate group to form the group 7-CONH(CH2)2OPO(OH)2
In certain embodiments, Z is selected from the group consisting of:
In certain embodiments, the compound of Formula HIa is selected from the group consisting of: 2-[l-(Chloromemyl)-3-[5-(2-morpholinoethoxy)4H-indole-2-carbonyl]-5-nitro-l,2-dihydro-3H- benzo[>]indole-7-sulfonamido] ethyl dihydrogen phosphate;
2-[l-(Chloromemyl)-3-[5-(3-moφho]inopropoxy)-lH-indole-2-carbonyl]-5-nitiO-l,2-diliydro-3H- benzo[e]indole-7-sulfonamido]ethyl dihydrogen phosphate;
(Ej-2-(l-(chloromethyl)-3-{3-[4-(2-morphoHnoethoxy)phenyl]aci7loyl}-5-nitro-l,2-dihydro-3H- benzo[^]indole-7-sulfonamido)ethyl dihydrogen phosphate;
2-[l-(Chloromemyl)-3-{4-[2-(4-methylpiperazin-l-yl)eii-ioxy]benzoyl}-5-nitiO-l,2-diliydiO-3H- benzo[tf]indole-7-sulfonamido]ethyl dihydrogen phosphate;
2-{l-(ChloiOmethyl)-3-[5-(2-moi^holboethoxy)-lH-indole-2-carbonyl]-5-nitro-l,2-dQαydro-3H- benzo[^]indole-7-carboxamido} ethyl dihydrogen phosphate;
(E)-2-[l-(Chloromethyl)-3-{3-[4-(2-moipho benzo[£]indole-7-carboxamido] ethyl dihydiOgen phosphate;
(i)-2-[l-(Chloromeώyl)-3-[5-(2-moipholm^ benzo[tf]indole-7-sulfonamido]ethyl dihydiOgen phosphate;
(^jB)-2-[l-(CUorome%l)-3-{3-[4-(2-morpholin^ benzo[<?]indole-7-sulfonamido] ethyl dihydrogen phosphate;
2-[l-(Bromomeώyl)-3-[5-(2-moipholinoemoxy)-lH-m^ benzo[«]indole-7-sulfonamido] ethyl dihydrogen phosphate; and
(E)-2-[l-(3romome%l)-3-{3-[4-(2-moφholinoethoxy)phenyl]aci7loyl}-5-nitto4,2-dihyd£o-3H- benzo[£]indole-7-sulfonamido] ethyl dihydrogen phosphate.
In a further aspect, the present invention provides a pharmaceutical composition comprising a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
In a further aspect, the present invention provides a method for the production of an anti-cancer effect in a warm-blooded animal such as a human, wherein the method comprises administering to the animal an effective amount of a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof.
In a further aspect, the present invention provides a method for the production of an anti-cancer effect in a cell, wherein the method comprises contacting the cell with an effective amount of a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof. Anti-cancer effects include, but are not limited to, anti-tumour effects, the response rate, the time to disease progression and the survival rate. Anti-tumour effects include but are not limited to, inhibition of tumour growth, tumour growth delay, regression of tumour, shrinkage of tumour, increased time to regrowth of tumour on cessation of treatment and slowing of disease progression.
In a further aspect, the present invention provides a method for the treatment of a cancer in a warm-blooded animal such as a human, which comprises administering to said animal an effective amount of a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof.
In a further aspect, the present invention provides the use of a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the production of an anti-cancer effect in a warm-blooded animal such as a human.
In a further aspect, the present invention provides the use of a compound of Formula I, II or III as defined above or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a cancer in a warm-blooded animal such as a human.
In further aspects, the invention provides methods of preparing compounds of the general Formulae I, II or III, as defined above. Such methods are described below.
"Effective amount" means an amount of a compound that, when administered to a subject for treating a disease state, is sufficient to effect such treatment for the disease state. The "effective amount" will vary depending on the compound, disease state being treated, the severity of the disease treated, the age and relative health of the subject, the route and form of administration, the judgement of the attending medical or veterinary practitioner, and other factors.
"Treating" or "treatment" of a disease state includes:
(i) preventing the disease state, i.e. causing the clinical symptoms of the disease state not to develop in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state,
(if) inhibiting the disease state, or
(iϋ) relieving the disease state, i.e. causing temporary or permanent regression of the disease state or its clinical symptoms.
"Pharmaceutically acceptable" means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.
"Pharmaceutically acceptable salts" of a compound means salts that are pharmaceutically acceptable, as defined herein, and that possess the desired pharmacological activity of the parent compound. Such salts include:
acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, methanesulfonic acid, maleic acid, tartaric acid, citric acid and the like; or salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g. an alkali metal ion, an alkaline earth ion, or an aluminium ion; or coordinates with an organic or inorganic base. Acceptable organic bases include ethanolamine, diethanolamine, N- methylglucamine, triethanolamine and the like. Acceptable inorganic bases include aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.
"Warm blooded animal" means any member of the mammalia class including, but not limited to humans, non-human primates such as chimpanzees and other apes and monkey species, farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
Compounds of Formulae I, II or III as defined above may exist in different enantiomeric and/or diastereomeric forms. In such cases it is to be understood that Formulae I, II and III include any possible enantiomeric or diastereomeric forms, and mixtures of such forms, and also any pharmaceutically acceptable salts thereof.
In certain embodiments, the compound of Formula I, II or III may be in the form of a racemic mixture. In other embodiments, the compound of formula I, II or III may be in the form of a mixture in which either the S or the R enantiomer predominates, or in the form of either the S or the R enantiomer substantially free of the other enantiomer, for example containing less than about 5%, such as less than about 1 %, of the other enantiomer.
While the invention is broadly as defined above, further aspects of the invention will become apparent with reference to the following description, reaction schemes and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows an ORTEP representation of the crystal structure of (R)-di-/«7'-butyl 2-[l- (chloromethy^-S-mtro-S-^ifluoroacety^-l^-djϋbydro-SH-benzo^jmdole-T-sulfonamidoJethyl phosphate (144).
Figure 2 shows an ORTEP representation of the crystal structure of (J)-di-Λ?/-/-butyl 2-[l- (chloiOmethj^-δ-niiiO-S-^ifluoroaceiy^-l^-clihydiO-SH-benzof^indole-V-sulfonaiTitdoJethyl phosphate (145).
Figure 3a is a bar graph comparing the activity in SiHa xenografts in combination with radiation of certain compounds of this invention and reference compounds, some of which are described in WO 2006/043839.
Figure 3b is a bar graph comparing the activity in H460 xenografts in combination with radiation of certain compounds of this invention and reference compounds, some of which are described in WO 2006/043839.
DETAILED DESCRIPTION OF THE INVENTION
Compounds of the invention
As defined above, this invention relates to nitrobenzindoles and related analogues, in particular aminobenzindoles, of the Formulae I, II or III as defined above, and their use in treating cancer. The invention more particularly relates to the use of these compounds as prodrugs that are activated under hypoxic conditions, ie in an environment having a lower oxygen tension than that of normal tissues, to treat cancer.
The compounds of the present invention have the same core ring structure as the compounds disclosed in WO 2006/043839. However, the compounds of the present invention differ from those disclosed in WO 2006/043839 in that they incorporate an indole or cinnamate group Z that bears an O-linked sidechain containing a morpholine or N-methylpiperazine substituent.
Examples of compounds of the invention of the Formulae I, II and III, respectively, are set out in Tables 1 to 3 below.
Methods of preparation of compounds of the invention
The compounds of Formulae I, II and III of die present invention may be prepared using the methods described below. These methods form further aspects of the invention. The schemes A to M following the general descriptions of the methods below include details of the reagents used to achieve each of the synthetic steps.
Preparation of compounds of Formula I
Compounds of Formula I may be prepared by first preparing an intermediate compound of Formula IV, V or VI as defined below. These compounds can be converted to desired compounds of Formula I by the methods following the description of the preparation of these intermediate compounds.
Compounds of Formula IV
wherein X is defined for a compound of Formula I and J represents Λrf-butoxycarbonyl or trifluoroacetyl, with an effective amount of an iodide salt in a non-nucleophilic solvent, to provide a compound of Formula VI
Preferably the first step is achieved using sodium iodide in methyl ethyl ketone.
Preferably the second step is achieved using silver mesylate in acetonitrile.
Preferably the third step is achieved using lithium bromide in tetrahydrofuran.
Compounds of Formula IV as defined above may also be prepared by the reaction of compounds of Formula V with an effective amount of a bromide salt in a non-nucleophilic solvent.
Compounds of Formula V as defined above can be prepared by the methods described in WO 2006/043839, the full content of which is incorporated herein by reference.
Compounds of Formula I may be prepared from the above compounds of Formulae IV and V by a method which includes in a first step reacting a compound of Formula VIII
Preferably the first step is achieved using potassium nitrate in concentrated sulfuric acid. Preferably the second step is achieved using cesium carbonate in aqueous methanol.
Preferably when a compound of Formula ZCO2H is used in the third step then this step is achieved using JV-(3-dimetlαylaminopropyl)-N'-ethyl carbodiimide or l-[3-(dimethylamino)propyl]-3- ethylcarbodiknide methiodide or other suitable coupling reagent.
Preferably when iV-(3-dimethylaminopropyl)-JV'-ethyl carbodiimide or l-[3-(dimethylamino)propyl]- 3-ethylcarbodϋmide methiodide is used in the third step then an effective amount of an acid co- reagent such as toluenesulfonic acid is also used.
Preferably when a compound of Formula ZCOCl is used in the third step then this compound is formed by the reaction of a compound of Formula ZCO2H with thionyl chloride or oxalyl chloride or oxalyl chloride in the presence of catalytic dimethylformamide.
Preferably when a compound of Formula ZCOCl is used in the third step then this step is achieved using at least one equivalent of non-nucleophiHc base such as di-zjø-propylethylamine. -
Compounds of the Formula ZCO2H can be prepared by a method which includes in a first step reacting a compound of Formula ZCO2R7 wherein R7 represents C1-4 alkyl or phenyl or benzyl optionally substituted with one or more halide substituents, and Z is as defined for a compound of Formula I and contains the group Q wherein Q represents OH or NH2 or CO2H, with a suitable derivative of a compound of Formula R2 where R2 is as defined for a compound of Formula I, and
includes the structures Ie-In, such suitable derivatives being alcohols or halides or primary or secondary amines or acids or acid chlorides. Compounds of formula ZCO2R can be readily prepared using methods known to those skilled in the art.
When Q represents OH or NH2 the reaction may involve alkylation with a halide derivative of R2, when Q represents OH or an aromatic ring the reaction may involve a Mitsunobu reaction of an alcohol derivative of R2, when Q represents NH2 the reaction may involve acylation using an acid chloride derivative of R2 or amide coupling using an acid derivative of R2, when Q represents CO2H the reaction may represent amide coupling with a primary or secondary amine derivative of R2.
Additionally, when Q represents a halogen, suitable examples of ZCO2R7 may be formed by palladium or other metal catalysed reactions with halide derivatives of R2. The second step of the method includes reacting a compound of Formula ZCO2R7 with base to provide a compound of Formula ZCO2H.
Preferably the second step is achieved using potassium hydroxide or lithium hydroxide or sodium hydroxide in an aqueous alcoholic solvent.
Compounds of Formula I wherein X represents SO2NR1, and R1 is as defined for a compound of Formula I, may be prepared by reacting a compound of Formula I wherein X represents SO2Cl with a primary or secondary amine derivative of R1.
Preferably the reaction is conducted in the presence of at least one equivalent of a non-nucleophilic base such as di-zjo-propylethylamine or triethylamine.
Compounds of Formula I wherein X represents CONR'2 and R1 is as defined for a compound of Formula I, may be prepared by reacting a compound of Formula I wherein X represents CO2H with a primary or secondary amine derivative of R , using an effective amount of a suitable coupling reagent.
Preferably the reaction is conducted using (benzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate.
Preparation of compounds of Formula II
Compounds of the Formula II may be prepared by reducing a compound of Formula I under suitable conditions.
Preferably the reduction is achieved by hydrogenation using a suitable metal catalyst such as platinum or palladium, or by reaction with a metal such as zinc, preferably in a finely divided or powdered state, in the presence of a weak acid such as ammonium chloride.
Preparation of compounds of Formula III
Compounds of Formula Ilia may be prepared by a method that includes in a first step reacting a compound of Formula I or Formula II bearing a free hydroxyl group with an effective amount of a phosphorylating agent to provide a compound of Formula IHe
Preferably when R8 represents /»V-butyl the first step is achieved using di-zW-butyl N,N- dietlαylphosphoramidite or di-fe/f-butyl JV,JV-di-/jo-propylphosphoramidite.
Preferably when R8 represents /m"-butyl the second step is achieved using trifiuoroacetic acid.
Compounds of Formula IHa may also be prepared by reacting alcohol derivatives of R1 or Formula IX using the phosphorylating conditions as described, and incorporating the so-formed phosphate ester into the general reactions as described above, to provide an alternative route to compounds of Formula IHe.
Compounds of Formula HIb may be prepared by a method including in a first step reacting a compound of Formula I or Formula II bearing a free hydroxyl group with an amino terminal-
protected amino acid or dipeptide or tripeptide or tetrapeptide using an effective amount of a peptide coupling reagent to provide a compound of Formula IHf
Preferably the first step is achieved using 0-(benzotriazol-l-yl)-JV,iV,N',N'-tetramethyluroniumi hexafluorophosphate or 0-(7-azabenzotriazol-l -yl)-JV,Λ^N\N'-tetramemyluronium hexafluorophosphate.
Preferably when R9 represents /øn'-butoxycarbonyl the second step is achieved using trifiuoroacetic acid.
Preferably when R9 represents 9-fluorenylmethoxycarbonyl the second step is achieved using morpholine or piperidine.
For those examples of compounds of Formula IHb where R4 contains a reactive functional group such as an amino group or carboxylic acid group, then suitably protected forms of these groups such as are commonly used in peptide synthesis may be employed, which protecting groups may be removed in the second step as described or in a prior or subsequent deprotection step to provide a compound of Formula IHb.
Compounds of Formula IHb may also be prepared by reacting alcohol derivatives of R1 or Formula IX using the peptide coupling conditions as described, and incorporating the so-formed amino terminal-protected amino acid or dipeptide or tripeptide or tetrapeptide into the general reactions as described above, to provide an alternative route to compounds of Formula IHf.
Compounds of Formula IHc may be prepared by a method that includes in a first step reacting a compound of Formula I or Formula II bearing a free hydroxyl group with a suitably protected
monosaccharide donor such as a monosaccharide bromide or monosaccharide imidate to provide a compound of Formula HIg
Preferably when the first step is achieved using a monosaccharide bromide an effective amount of a silver salt such as silver triflate or silver carbonate is also used.
Preferably when the first step is achieved using a monosaccharide imidate then a trichloroacetimidate is used, and further an effective amount of a Lewis acid such as boron trifluoride is also used.
Preferably when R10 represents an acetate-protected hydroxy or hydroxymethyl group the second step is achieved using lithium hydroxide or potassium hydroxide in an aqueous alcoholic solvent.
Compounds of Formula HIc may also be prepared by reacting alcohol derivatives of R1 or Formula IX with the monosaccharide donors as described, and incorporating the so-formed suitably protected monosaccharides into the general reactions as described above, to provide an alternative route to compounds of Formula IHg.
Compounds of Formula HId may be prepared by a method that includes in a first step reacting a compound of Formula I or Formula II bearing a free hydroxyl group or a secondary or tertiary amine group with an effective amount of a compound of Formula X
wherein R11 represents a good leaving group which may be a halide or a sulfonate such as p- toluenesulfonate or methanesulfonate or trifluoromethanesulfonate and R12 represents a suitable phosphate protecting group which may be alkyl or allyl or benzyl or stibstituted benzyl, to provide a compound of Formula IIIh
Preferably the first step is achieved using a compound of Formula X wherein R11 represents chloride and R12 represents feti-butyl.
Preferably when T is O or T is N and q is 1 the first step is achieved using a base such as sodium hydride or potassium carbonate.
Preferably when T is N and q is 2 the first step is achieved using a polar aprotic solvent such as acetonitrile.
Preferably when R represents terf-hutyl the second step is achieved using an acid such as trifluoroacetic acid.
Compounds of Formula IIIh may also be prepared by reacting alcohol or secondary amine or tertiary amine derivatives of R1 or R2 or Z or Formula IX with a compound of Formula X as described, and incorporating the so-formed suitably protected phosphonooxymethyl compound into the general reactions as described above, to provide an alternative route to compounds of Formula IIIh.
The compounds of the Formulae I3 II and III of the present invention can be used in the treatment of cancer of the human or animal body. In particular embodiments, the treatment may be of any cancer- type that includes hypoxic regions. For example, the cancers treated may be solid tumors, such as ovarian, colon, brain, thyroid, pancreas, bladder, breast, prostate, lung (such as small cell lung tumor cells and large cell lung carcinoma), cervical and skin cancer. Alternatively, the cancer may be leukemia, multiple myeloma or lymphoma.
The compounds of the invention can be administered in the form of pharmaceutical compositions, containing one or more compounds of the invention in combination with one or more pharmaceutically acceptable carriers.
The pharmaceutically acceptable carrier(s) should be non-toxic and not interfere with the efficacy of the active ingredient. The precise nature of the carrier will depend on the route of administration, which can be oral, or parenteral, including intravenous, cutaneous, subcutaneous, intramuscular, intravascular or by infusion.
Pharmaceutical compositions suitable for oral adrninistration can be in tablet, capsule, powder or liquid form. A tablet may comprise one or more solid carriers and/or adjuvants. A capsule may include a solid carrier such as gelatin. Liquid pharmaceutical compositions may comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For parenteral injection, the pharmaceutical composition may conveniently be in the form of a parenteraUy acceptable aqueous solution which is pyrogen-free and has a suitable pH, isotonicity and stability. Those of skill in the art are able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, and Lactated Ringer's injection. Preservatives, stabilisers, btiffers, antioxidants and/or other additives may be included as required.
The exact dose of the compound to be administered will be at the discretion of the physician, talcing into account the condition and needs of the patient. Typical doses and administration schedules will be determined by experience in clinical trials. Total doses are expected to be in the range from about 0.1 to 200 mg/kg per subject, such as about 10 mg/kg per subject.
The compounds of Formula I, II or III can be used as single agents or in combination with one or more other cytotoxic or other therapeutic agents or therapies, especially those that are relatively ineffective against hypoxic cells, such as radiation therapy. Where such other agents and/or radiotherapy are administered in combination with a compound of the invention, the radiation and/ or other agents may be administered before, during or after administration of the compound of Formula I, II or III.
Without wishing to be bound by theory, it is believed that compounds of Formula III, upon administration to a subject, are hydrolysed in vivo to form compounds of Formula I, and that compounds of Formula I are reduced selectively under hypoxic conditions to form cytotoxic amino compounds of Formula II.
EXAMPLES
The following examples are representative of the invention, and provide detailed methods for preparing the compounds of the invention. In these examples, elemental analyses were carried out in the Microchemical Laboratory, University of Otago, Dunedin, NZ. Melting points were determined on an Electrothermal 2300 Melting Point Apparatus. NMR spectra were obtained on a Bruker Avance-400 spectrometer at 400 MHz for 1H and 100 MHz for 13C spectra, referenced to Me4Si. Column chromatography was carried out on silica gel unless otherwise stated.
Example 1. l-(Chloromethyl)- JV-(2-hydroxyethyl)-3- [5-(2-mofpholinoethoxy)-liJ-indole-2- carbonyl]-5-nitro-l,2-dihydfo-3Jftf'ben2o[e]mdole-7-sulfonamide (1) (Scheme A and B). Et3N
(89.2 mL, 638 mmol) was added to solid 2-naphthoic acid 100 (100 g, 581 mmol) and the mixture was stirred at 20 0C for 10 min. After this time the mixture solidified and was allowed to stand at 20 0C for a further 20 min. Diphenyl phosphoryl azide (131.6 mL, 609 mmol) was added, followed by dry /m"-BuOH (277.2 mL, 2.90 moles), causing the internal temperature to rise to 35 0C. The reaction flask was placed in an oil-bath and the bath temperature was raised from 20 0C to 86-87 0C over 1 h 20 min, then held at this temperature for 2 h. The reaction mixture was cooled briefly and poured into ice-water (ca.2.5 L) with vigorous stirring at 20 0C causing a sticky solid mass to separate. The mixture was left to stand at 20 0C overnight (15 h) to give a loose suspended solid. The mixture was stirred again for 5 h. The fine solid was filtered off, washed with water (4 x 300 mL) and dried in vacuum oven at 40-45 0C to constant weight to give tert-butyl 2-naphthylcarbamate
(101) as a beige solid (140 g, 99%): mp 85-87 0C, suitable (1H NMR) for the following reactions. A portion was reciystallised from CH2Cl2/petroleum ether to give a cream solid: 1H NMR (CDCl3) δ 7.98 (s, 1 H), 7.75 (dj = 8.6 Hz, 3 H), 7.43 (dtj = 7.5, 1.1 Hz, 1 H), 7.40-7.30 (m, 2 H), 6.61 (s, 1 H), 1.55 (s, 9 H).
A stirred solution of carbamate 101 (92 g, 0.38 mol) in MeCN (680 mL) at 0 0C was treated portionwise over 2 h with solid N-bromosuccinimide (81 g, 0.45 mol). After the addition was complete the reaction mixture was stirred for a further 45 min at 0 0C. To the mixture was added a cold solution of aqueous NaHCO3 (0.2N, 2 L) and the mixture was stirred at 0 0C for 1 h. The solid was filtered off and washed with water (5 X 200 mL), then dried in vacuum over solid KOH overnight to give tert-hutyl l-bromo-2-naphthylcarbamate (102) as a beige solid (121 g, 100%): mp 88-89 0C; 1H NMR [(CD3)2SO] δ 8.82 (s, 1 H), 8.15 (dj = 8.5 Hz, 1 H), 7.96 (d J = 9.6 Hz, 1 H), 7.93 (d,/ = 9.3 Hz, 1 H), 7.71 (dj = 8.8 Hz, 1 H), 7.66 (t,/ = 7.7 Hz, 1 H), 7.56 (t,/ = 7.4 Hz, 1 H), 1.49 (s, 9 H).
A mixture of carbamate 102 (53.7 g, 167 mmol) and dry K2CO3 (55.2 g, 400 mmol) in dry DMF (500 mL) was stirred at 20 0C for 5 min, then 1,3-dichloropropene (23.1 mL, 251 mmol, mixed isomers) was added. The mixture was stirred at 70-80 0C (bath temperature) under a dry atmosphere for 4 h. The mixture was cooled and partitioned between petroleum ether (500 mL) and water (500 mL). The petroleum ether layer was separated and washed with water (3 x 20OmL), then dried (Na2SO4) and allowed to stand at 5 0C overnight. The precipitated orange solid was filtered off and the filtrate was evaporated to give /«?-butyl l-bromo-2-naphthyl-(3-chloro-2-propen-l-yl)carbamate (103) as an amber oil (67.5 g; 102%), suitable (1H NMR) for the following reactions. Pure carbamate 103 can be obtained by repeating the procedure of dissolving in petroleum ether, allowing an orange solid to separate, filtering off the solid, and evaporating the filtrate, to give 103 as an oil (100%): 1H NMR [(CD3)2SO] (mixture of rotamers and E and Z forms) δ 8.23 (dj = 8.4 Hz, 1 H), 8.07-7.94 (m, 2 H), 7.71 (tj = 7.5 Hz, 1 H), 7.65 (tj= 7.4 Hz, 1 H), 7.51, 7.45 (2 dj = 8.6 Hz, 1 H), 6.44-6.26 (m, 1 H), 6.21-5.99 (m, 1 H), 4.58-4.46, 4.44-4.17, 4.14-3.96 (3 m, 2 H), 1.50, 1.26 (2 s, 9 H).
A solution of bromide 103 (48.8 g, 123 mmol) in toluene (150 mL) was stirred at 20 0C under nitrogen for 20 min, then Bu3SnH (34.2 mL, 123 mmol) and AIBN (60.7 mg, 0.37 mmol) were added. The reaction mixture was stirred at 85-90 0C (bath temperature) under nitrogen for 1 h and then cooled to 20 0C. Powdered anhydrous KF (35.7 g, 615 mmol) was added and the mixture was stirred at 20 0C for 14 h. EtOAc (150 mL) and petroleum ether (300 mL) were added and the mixture stirred for a further 1 h 30 min. The mixture was filtered through a neutral alumina pad (300
g), washing with EtOAc/petroleum ether (1:2) (10 x 50 mL). The combined filtrates were washed successively with water (300 tnL), aqueous Na2CO3 (5%, 300 mL) and water (300 mL), and then dried (Na9SO4) and evaporated to give a yellow solid (74 g). 1H NMR analysis showed this material contained about 10% tin residues. The solid was dissolved in MeOH (150 mL) and HCl gas was bubbled into the solution at room temperature over 25 min. The acidic mixture was stkred for a further 10 min, then diluted with petroleum ether (300 mL). The mixture was stkred at 20 0C for 15 min, then the petroleum ether layer was separated and discarded. The petroleum ether procedure was repeated three more times. The acidic MeOH solution was then evaporated under reduced pressure at 35-40 0C (bath temperature) to give l-(chloromeώyl)-l,2-dihydiO-3iϊ-benzo[V[indole hydrochloride (104) as a greenish foamy solid (30.1 g, 96%): mp 180 0C (dec); 1H NMR [(CD3)2SO] δ 10.3 (v br, 1 H), 8.07-7.94 (m, 3 H), 7.67-7.59 (m, 1 H), 7.57-7.49 (m, 1 H), 7.45 (d J = 8.7 Hz, 1 H), 4.45-4.36 (m, 1 H), 4.08 (ddj = 11.0, 3.5 Hz, 1 H), 4.00-3.90 (m, 2 H), 3.84 (ddj = 11.5, 2.5 Hz, 1 H). This material contained about 1% tin residues. On a smaller scale (3.0 g) repeated washes with petroleum ether (4 x 100 mL) were sufficient to remove all traces of tin residues to give an analytically pure product: Anal. (C13H13C12NO.4H2O) CaIc: C, 59.74; H, 5.32; N, 5.36. Found: C, 59.71; H, 5.36; N, 5.32.
To a stirred mixture of indoline hydrochloride 104 (30.1 g, 118 mmol) in p-dioxane (100 mL) at 0 0C was added trifluoroacetic anhydride (33.4 mL, 236 mmol), followed by Et3N (49.2 mL, 354 mmol). The mixture was stitred at 0 0C for 10 min, at 20 0C for another 50 min, and then poured into ice- water (500 mL). Cone. HCl (40 mL) was added and the mixture was stirred at 20 0C for 20 min. The precipitated solid was filtered off, washed successively with dilute HCl (IN, 4 x 20 mL), water (10 x 5OmL), petroleum ether (5 x 25 mL), and dried to give l-(chloromethyl)-3-(trifluoroacetyl)-l,2- dihydro-3H-benz[«]indole (105) as a pale yellow solid (37.7 g, 102%) suitable for the following reactions: 1H NMR [(CD3J2SO] δ 8.32 (dj = 9.0 Hz, 1 H), 8.07-7.96 (m, 3 H), 7.62 (ddd,/ = 8.2, 6.9, 1.2 Hz, 1 H), 7.53 (dddj = 8.1, 6.9, 1.1 Hz, 1 H), 4.61-4.52 (m, 1 H), 4.51-4.39 (m, 2 H), 4.15 (ddj = 11.3, 3.0 Hz, 1 H), 4.04 (ddj = 11.3, 5.9 Hz, 1 H). This material was free of tin residues (1H NMR) but contained a small impurity Of Et3N that can be removed by further washing with HCl.
A solution of chlorosulfonic acid (1.77 mL, 26.5 mmol) in dry CH2Cl2 (20 mL) at 20 0C was added dropwise over 1 h to a stirred mixture of indoline 105 (6.92 g, 22.1 mmol) and CH2Cl2 (60 mL) in an ice bath, maintaining the internal temperature at 3-4 0C throughout. The mixture was stirred for a further 2 h 30 min at the same temperature. Another batch of chlorosulfonic acid (1.77 mL, 26.5 mmol) in dry CH2Cl2 (10 mL) was added dropwise over 45 min and the reaction was stirred in the
ice bath for a further 1 h 30 min. Dry DMF (10 mL) was added followed by oxalyl chloride (5.55 ml, 63.6 mmol). The reaction mixture was stirred in the ice bath for 30 min and then left to stand at 0-5 0C overnight. The CH2Cl2 was evaporated under reduced pressure at 20 0C to give an amber solid. Cold water (100 mL) was added and the mixture was stirred at 0 0C (bath temperature) for 15 min. The solid was filtered off, washed with cold water (5 x 20 mL), and dried to give 1- (chloromethyl)-3-(trifluoroacetyl)-l,2-dihydro-3H-benzo[^]indole-7-sulfonyl chloride (106) as a pale yellow solid (8.8 g, 97%): mp 187-190 0C; 1H NMR (CDCl3) δ 8.70-8.64 (m, 2 H), 8.13-8.08 (m, 2 H), 8.00 (d,/ = 9.0 Hz, 1 H), 4.72-4.68 (m, 1 H), 4.55-4.48 (m, 1 H), 4.33-4.25 (m, 1 H), 3.98-3.93 (m, 1 H), 3.68-3.61 (m, 1 H). The 1H NMR spectrum was identical to that of a sample recrystallised from EtOAc/petroleum ether, mp 189-192 0C.
Sulfonyl chloride 106 (1.02 g, 2.48 mmol) was added to cold cone. H2SO4 (10 mL) and the mixture was stirred at 0 0C for 2 min to give a homogeneous solution. Solid KNO, (351 g, 3.47 mmol) was added portionwise over 2 min and the mixture was stirred at 0 0C for a further 2 min. Ice-water (100 mL) was added and the mixture was stirred at 0 0C for 2 min. The precipitated solid was filtered off, washed with cold water (5 x 2OmL), and dried in vacuum over silica gel to give l-(chloromethyl)-5- nitro-3-(trifluoroacetyl)-l,2-dihydro-3H-benzo[(?]indole-7-sulfonyl chloride 107 as a light brown solid (1.09 g, 97%) suitable (1H NMR) for the following reactions. The product can be further purified by dissolving in
CH2Cl2, diluting with /-Pr2O (2 volumes), allowing the solution to stand at 5 0C overnight, and filtering off the precipitate as a light brown solid (71%). The mother liquor left at -10 0C gives an additional crop (0.48 g, 2%). A recrystallised sample has mp (EtOAc/petroleum ether) 184-189 0C; 1H NMR (CDCl3) δ 9.34 (s, 1 H), 9.28 (dj = 1.8 Hz, 1 H), 8.22 (ddj = 9.0, 1.9 Hz, 1 H), 8.11 (dj = 9.0 Hz, 1 H), 4.77-4.71 (m, 1 H), 4.58 (ddj = 11.5, 8.8 Hz, 1 H), 4.42-4.33 (m, 1 H), 3.95 (dd,/ = 11.7, 3.5 Hz, 1 H), 3.73 (ddj = 11.7, 7.7 Hz, 1 H).
Sulfonyl chloride 107 was converted to alcohol 108 as previously described (formation of compound 121 of WO 2006/043839).
To a mixture of benzyl 2-hydroxyethylcarbamate (109) (3.90 g, 20.0 mmol) and tert- butylchlorodimefhylsilane (4.52 g, 30.0 mmol) in DMF (50 mL) stirred at 20 0C was added όi-iso- propylethylamine (DIPEA) (12.0 mL, 68.8 mmol). The reaction mixture was stirred for a further 20 min and then partitioned between EtOAc and water. The EtOAc layer was separated, washed with dilute aqueous Na2CO3, dried (Na2SO4), and evaporated to give benzyl 2-(tert- butyldimethylsilyloxy)ethylcarbamate (UO) as a pale amber oil (6.2 g, 100%): 1H NMR (CDCl3) δ
7.38-7.28 (m, 5 H), 5.11 (s, 2 H), 5.06 (br s, 1 H), 3.68 (t,/ = 5.3 Hz, 2 H), 3.31 (q,/ = 5.3 Hz, 2 H), 0.89 (s, 9 H), 0.05 (s, 6 H). HRMS (FAB) calcd. for C16H28NO3Si (MH+) W^ 310.1838, found 310.1838.
A solution of carbamate 110 (1.5 g, 4.85 mmol) in THF (100 mL) was hydrogenated over Pd/C (5%) at 40 psi for 2 h 30 min. The mixture was filtered through Celite and the Celite was washed with THF (5 X 20 mL). To the filtrate containing amine Ul was added sulfonyl chloride 107 (1.84 g, 4.03 mmol) and DIPEA (0.84 mL, 4.85 mmol). The mixture was stirred at 0 0C for 40 min. Cesium carbonate (1.31 g, 4.03 mmol) and MeOH (50 mL) were added and the mixture was stirred at 20 0C for 45 min, then allowed to stand at 5 0C for 14 h. The mixture was concentrated under reduced pressure at 25 0C to remove most of the THF. Ice-water was added and stirred at 0 0C for Ih 30 min. The resulting solid was filtered off, washed with cold water several times, and dried in vacuum over silica-gel to give iV-[2-(Λ/f-butyldinaethylsilyloxy)ethyl]-l-(chloiOmethyl)-5-nitro-l,2-dihydro- 3H-benzo[<?]indole-7-sulfonamide (112) as a reddish orange solid (2.0 g, 100%): mp 127-129 0C; 1H NMR (CDCl3) δ 8.92 (dj = 1.6 FIz, 1 H), 7.90 (ddj = 8.9, 1.6 Hz, 1 H), 7.79 (d,/ = 8.9 Hz, 1 H),
7.73 (s, 1 H), 4.88 (t,J = 5.8 Hz, 1 H), 4.40 (br s, 1 H), 4.17-4.08 (m, 1 H), 4.05-3.95 (m, 2 H), 3.83-
3.74 (m, 1 H), 3.68 (t,J = 5.2 Hz, 2 H), 3.60 (ddj = 11.1, 10.0 Hz, 1 H), 3.18-3.11 (m, 2 H), 0.84 (s, 9 H), 0.00 (s, 6 H). Anal. (C21H30ClN3O5SSi) CaIc: C, 50.44; H, 6.05; N, 8.40. Found: C, 50.71; H, 6.35; N, 8.32.
Tetrazole (3 wt% solution in CH3CN, 468 mL, 159 mmol) was added gradually over 1 h to a stirred mixture of di-føπ'-butyl IV,N-diisopropylphosphoramidite (95%, 53.0 mL, 159 mmol) and benzyl 2- hydiOxyethylcarbamate (109) (26.0 g, 133 mmol) in THF (500 mL) at 20 0C under a nitrogen atmosphere. After the addition was complete the mixture was stirred at this temperature for 19 h. The mixture was cooled to 0 0C and H2O2 (30% aqueous, 48 mL, 494 mmol) was added. After 1.5 h at 0 0C the mixture was poured into ice-water and the product was extracted into EtOAc. The EtOAc layer was washed successively with cold aqueous Na2CO3 and water (x 2) and then dried (Na2SO4). The organic solvents were removed under reduced pressure to give a colourless oil. A seed crystal was added and the mixture was allowed to stand at 20 0C for 1 h, then petroleum ether was added and the mixture was left at 5 0C for crystallisation to complete. The resulting solid was filtered off, washed with petroleum ether, and dried to give benzyl 2-{[di(tert- butoxy)phosphoryl]oxy}ethylcarbamate (113) (24.9 g, 49%) as a colourless solid: mp 43-45 0C; 1H NMR (CDCl3) δ 7.37-7.28 (m, 5 H), 5.43 (br s, 1 H), 5.11 (s, 2 H), 4.07-4.00 (m, 2 H), 3.46 (qj = 5.1 Hz, 2 H), 1.47 (s, 18 H). HRMS (FAB) calc for C18H31NO6P (MH+) m/z 388.1889, found 388.1889. The mother liquor was left at 5 0C to give a second crop (12.9 g, 25%).
A solution of benzyl carbamate 113 (2.32 g, 5.99 mmol) in MeOH (100 mL) with Pd/ C (5%, 0.46 g) was hydrogenated at 40 psi for 2.5 h. The mixture was filtered through Celite, washing with MeOH, and the filtrate was evaporated to give 2-aminoethyl di(Λ?/y-butyl) phosphate (114) (1.52 g, 100%) as a colourless oil: 1H NMR (CDCl3) δ 4.01-3.94 (m, 2 H), 2.96-2.90 (m, 2 H), 1.58 (br s, 2 H), 1.49 (s, 18 H). HRMS (FAB) calc for C10H25NO4P (MH+) /»/£254.1521, found 254.1519.
A cold solution of amine 114 (1.98g, 7.8 mmol) and DIPEA (1.22 mL, 6.8 mmol) in THF (10 mL) was added to solution of sulfonyl chloride 107 (2.98 g, 6.5 mmol) in THF (30 mL) at 0 0C. The mixture was stirred at 0 0C for a further 20 min, then solid Cs2CO3 (4.04 g, 12.4 mmol) and cold MeOFI (20 mL) were added. The mixture was stirred at 0 0C for a further 10 min and then partitioned between EtOAc (400 mL) and ice-water (400 mL). The organic layer was separated and the aqueous layer was further extracted with EtOAc. The combined EtOAc extracts were washed again with water, then dried (Na2SO4), and evaporated under reduced pressure at 25 0C (bath temperature). The residue was dissolved in CH2Cl9 and the solution was filtered through a short silica column, eluting with CH2Cl2. The product-containing fractions were evaporated to give όi(tert- butyl) 2-[l-(chlororneώyl)-5-nitro-l,2-dmydxo-3H-benzo[φndole-7-sulfonamido]ethyl phosphate (115) as a red-orange foam (3.60 g, 95%): 1H NMR [(CD3)2SO] δ 8.59 (d,J = 1.6 Hz, 1 H), 8.04 (dj = 8.9 Hz, 1 H), 7.95 (br s, 1 H), 7.79 (ddj= 8.9, 1.8 Hz, 1 H), 7.77 (s, 1 FI), 6.74 (s, 1 H), 4.28-4.20 (m, 1 H), 3.95-3.86 (m, 2 H), 3.83-3.72 (tn, 4 H), 2.99 (t,J = 6.0 Hz, 2 H), 1.35 (s, 18 H). HRMS (FAB) calc for C23H33 35ClN3O8PS (M+) »/- 577.1415, found 577.1412.
To a stirred mixture of 4-nitrophenol (116) (42 g, 0.30 mol) and 4-(2-chloroethyl)morpholine hydrochloride (56 g, 0.30 mol) in anhydrous DMF (400 mL) was added dry K2CO3 (104 g, 0.75 mol). The mixture was stirred under a nitrogen atmosphere at 80-90 0C (bath temperature) for 3 h, then at 20 0C overnight (15 h). The mixture was poured into ice-water (1.5 L) and stirred at 00C for 1 h. The precipitated solid was filtered off, washed with water (6 x 100 mL) until the washes were colourless, then dried to give 4-[2-(4-nitrophenoxy)ethyl]morpholine (117) as a pale yellow solid (68.4 g, 90%): mp 79-81 0C; 1H NMR (CDCl3) 6 8.24-8.18 (m, 2 H), 7.01-6.93 (m, 2 H), 4.20 (tj = 5.7 Hz, 2 H), 3.73 (tj = 4.7 Hz, 4 H), 2.84 (tj = 5.7 Hz, 2 H), 2.58 (t J = 4.7 Hz, 4 H). Anal. (C12H16N2O4) Calc: C, 57.13; H, 6.39; N, 11.10. Found: C, 57.36; H, 6.44; N, 11.25.
A solution of nitro compound 117 (33.3 g, 132 mmol) in MeOH (170 mL) and TFIF (50 mL) with Pd/C (5%, ca. 3 g) was hydrogenated at 40 psi overnight (17 h). The mixture was filtered through Celite and the Celite plug was washed with CH2Cl2:MeOH (5:1) several times. The combined
filtrates were evaporated to give 4-(2-morpholinoethoxy)aniline (118) as a pink solid (29.0 g, 99%): mp 39-40 0C; 1H NMR (CDCl3) δ 6.78-6.73 (m, 2 H), 6.66-6.61 (m, 2 H), 4.04 (tj = 5.8 Hz, 2 H), 3.73 (t, / = 4.7 Hz, 4 H), 3.41 (br s, 2 H), 2.76 (t, J = 5.8 Hz, 2 H), 2.57 (t, / = 4.7Hz, 4 H). Anal. (C12H18N2O2) CaIc: C, 64.84; H, 8.16; N, 12.60. Found: C, 64.94; H, 8.08; N3 12.66.
A solution of sodium nitrite (9.95 g, 144 mmol) in water (27 mL) was added to a mixture of aniline 118 (29.0 g, 131 mmol), water (223 mL) and cone. HCl (65.7 mL, 657 mmol) at 0 0C. The mixture was stirred for a further 10 min at 0 0C, then added to a stirred mixture of ethyl 2-methyl-3- oxobutanoate (19.8 g, 138 mmol), sodium acetate (112 g, 1.37 mol), EtOH (163 mL), and ice (133 g, freshly added just before mixing) at 0 0C (bath temperature). The final reaction mixture was stirred at 0 0C for 5 min, then at 20 0C for 1 h 40 min. Solid Na2CO3 was added to give a pH of approximately 7-8. The mixture was extracted with CH2Cl2 (2 x 300 mL) and the extracts were washed with cold water (400 mL), then dried (Na2SO4) and evaporated to give a red oil. To the oil was added EtOH (60 mL) and HCl-saturated EtOH (100 mL). The mixture was stirred at reflux for 30 min, then cooled and evaporated under reduced pressure to give a dark solid. Water (200 mL) was added and the aqueous mixture was basified at 0 0C with 10% aqueous Na2CO3 solution to a pH of approximately 7-8. The resulting pale orange solid was filtered off, washed with water (5 x 80 mL), and dried to give ethyl 5-(2-morpholinoethoxy)-lH-indole-2-carboxylate (119) as a yellow solid (35.9 g, 86%): mp 130-132 0C; 1H NMR [(CD3)2SO] δ 11.69 (s, 1 H), 7.34 (d, / = 9.0 Hz, 1 H), 7.12 (dj = 2.3 Hz, 1 H), 7.03 (s, 1 H), 6.91 (dd,/ = 8.9, 2.5 Hz, 1 H), 4.32 (q,/ = 7.1 Hz, 2 H), 4.07 (t,/ = 5.8 Hz, 2 H), 3.61-3.56 (m, 4 H), 2.71 (tj = 5.8 Hz, 2 H), 2.50-2.46 (m, 4 H, partially obscured by DMSO peak), 1.33 (t, J - 7.1 Hz, 3 H). Anal. (C17H22N2O4) CaIc: C, 64.14; H, 6.97; N, 8.80. Found: C, 63.82; H, 7.23; N, 8.91.
Pd/C (5%) was added to a solution of ethyl 5-(benzyloxy)-lH-indole-2-carboxylate (120) (5.10 g, 17.3 mmol) in EtOAc (40 mL), THF (40 mL), and EtOH (30 mL). The mixture was hydrogenated at 50 psi for 17 h, then filtered through Celite. The filtrate was evaporated to give crude ethyl 5- hydiOxy-lH-indole-2-carboxylate as a grey solid. A portion of this crude material (5.8 mmol) was dissolved in THF (40 mL) with triphenylphosphine (2.26 g, 8.7 mmol) and 4-(2- hydroxyetiiyl)morpholine (1.06 mL, 8.7 mmol). Diethyl azodicarboxylate (1.50 g, 8.7 mmol) was added dropwise and die mixture was stirred at room temperature for 2 days. The THF was evaporated and the residue was partitioned between EtOAc and water. The EtOAc layer was washed with water (x2) and extracted with aqueous HCl (2N, X2). The combined acid extracts were cooled to 0 0C, basified with cone, aqueous NH3, and extracted with EtOAc (χ2). The combined organic extracts were dried (MgSO4) and evaporated to give a light brown oil which crystallised on
standing. ReciystalHsation from EtOH gave indole 119 as pale yellow crystals (0.80 g, 44%): mp 130- 132 0C; 1H NMR [(CD3)2SO] identical to that described above.
KOH (19.0 g, 339 mmol) was added to a mixture of ester 119 (35.9 g, 113 mmol), MeOH (250 mL) and water (125 mL) and the mixture was stirred at 20 0C for 1 h. The MeOH was evaporated under reduced pressure at 40 0C (bath temperature). The basic aqueous mixture was washed with ether (3 x 200 mL), filtered, and the filtrate acidified with cone. HCl at 0 0C to pH 5-6. The precipitated solid was filtered off, washed with water many times (until the washings were no longer red), and dried in a vacuum oven at 50-60 0C for 7 h to give 5-(2-morphoHnoethoxy)-lH-indole-2-carboxylic acid
(121) (31.3 g, 96%) as a pale yellow solid; 1H NMR [(CD3)2SO] δ 12.80 (br s, 1 H), 11.56 (s, 1 H), 7.32 (<_,/ = 8.9 Hz, 1 H), 7.12 (d, / = 2.4 Hz, 1 H), 6.99-6.96 (m, 1 H), 6.89 (ddj = 8.9, 2.4 Hz, 1 H), 4.07 (t, / = 5.8 Hz, 2 H), 3.59 (t, / = 4.6 Hz, 4 H), 2.71 (t, / = 5.8 Hz, 2 H), (4H partially obscured by DMSO peak).
Dioxane (300 mL) and HCl in dioxane (4M, 40.5 mL, 162 mmol) were added to acid 121 (31.3 g, 108 mmol). The mixture was stirred at 20 0C for 15 h. The solid was filtered off, washed with EtOAc several times, and dried to give 5-(2-morpholinoethoxy)-l H-indole-2-carboxylic acid hydrochloride
(122) as a grey-white solid (32 g, 91%): mp 251-2540C; 1H NMR [(CD3)2SO] δ 12.86 (br s, 1 H), 11.66 (s, 1 H), 10.97 (br s, 1 H), 7.36 (d,/= 8.9 Hz, 1 H), 7.20 (d,/= 2.2 Hz, 1 H), 7.01 (d,/ = 1.4 Hz, 1 H), 6.97 (ddj = 8.9, 2.4 Hz, 1 H), 4.44-4.37 (m, 2 H), 4.03-3.92 (m, 2 H), 3.88-3.75 (m, 2 H), 3.60-3.45 (m, 4 H), 3.26-3.15 (m, 2 H). Anal. (C15H18N2O4HCl) CaIc: C, 55.13; H, 5.86; N, 8.57. Found: C, 55.02; H, 5.89; N, 8.50.
A mixture of indoline 108, acid 122 (104 mg, 0.32 mmol), EDCLHCl (175 mg, 0.92 mmol), and anhydrous TsOH (7.9 mg, 0.05 mmol) in DMA (2.5 mL) was stirred at room temperature for 2.5 h then cooled in an ice bath. Cold aqueous NaHCO3 (5%) was added and the precipitated solid was filtered off, washed with water, and dried to give 1 as a yellow solid (150 mg, 100%): mp 131-135 0C; 1H NMR [(CD3)2SO] δ 11.73 (d,/= 1.7 Hz, 1 H), 9.29 (s, 1 H), 8.85 (d,/= 1.7 Hz, 1 H), 8.43 (d,J = 8.9 Hz, 1 H), 8.03 (ddj = 8.9, 1.7 Hz, 1 H), 7.91 (t,/ = 5.6 Hz, 1 H), 7.41 (d, / = 8.9 Hz, 1 H), 7.21 (d,/ = 1.8 Hz, 1 H), 7.19 (dj = 2.3 Hz, 1 H), 6.96 (ddj = 8.9, 2.4 Hz, 1 H), 5.01-4.94 (m, 1 H), 4.73 (ddj = 10.9, 2.4 Hz, 1 H), 4.68-4.62 (m, 1 H), 4.67 (tj = 5.6 Hz, 1 H), 4.20-4.10 (m, 4 H), 3.64-3.58 (m, 4 H), 3.39 (q, / = 6.0 Hz, 2 H), 2.87 (q, / = 6.2 Hz, 2 H), 2.73 (t, / = 5.7 Hz, 2 H), ca. 2.52-2.46 (m, 4 H, partially obscured by DMSO peak). Anal. (C30H32ClN5O8S-3AH2O) CaIc: C, 53.65; H, 5.03; N, 10.43. Found: C, 53.79; H, 4.85; N, 10.37.
Example 2. 5-Amino-l-(chlofomethyl)-ΛL(2-hydfoxyethyl)-3-[5-(2-morpholinoethoxy)-liϊ: indole-2-carbonyl]-l,2-dihydro-3i^benzo[e]indole-7-sulfonamide (10) (Scheme B). A solution of nitro compound 1 (49.6 mg, 0.075 mmol) in THF (20 mL) with PtO2 (59 mg) was hydϊogenated at 50 psi for 70 min. The catalyst was filtered off through Celite, the filtrate was evaporated, and the residue was triturated with EtOAc to give 10 as a pale green solid (35 mg, 74%): mp 220-225 0C (dec); 1H NMR [(CD3)2SO] δ 11.57 (s, 1 H), 8.54 (d,/ = 1.6 Hz, 1 H), 7.94 (dj = 8.9 Hz, 1 H), 7.82 (s, 1 H), 7.74 (ddj = 8.8, 1.7 Hz, 1 H), 7.45 (br s, 1 H), 7.40 (dj = 8.9 Hz, 1 H), 7.18 (dj= 2.3 Hz, 1 H), 7.09 (dj= 1.2 Hz, 1 H), 6.93 (ddj = 8.9, 2.4 Hz, 1 H), 6.29 (s, 2 H), 4.76 (ddj= 10.8, 9.0 Hz, 1 H), 4.65 (t,J = 5.4 Hz, 1 H), 4.53 (dd,/= 10.9, 1.8 Hz, 1 H), 4.21-4.14 (m, 1 H), 4.12 (tj = 5.8 Hz, 2 H), 4.01 (ddj = 11.0, 3.1 Hz, 1 H), 3.80 (dd, / = 11.0, 7.7 Hz, 1 H), 3.63- 3.58 (m, 4 H), 3.40 (qj = 6.0 Hz, 2 H), 2.84 (br t,J = 6.2 Hz, 2 H), 2.72 (tj = 5.8 Hz, 2 H), ca. 2.52-2.46 (m, 4 H, partially obscured by DMSO peak). Anal. (C30H34ClN5O6S) CaIc: C, 57.37; H, 5.46; N, 11.15. Found: C, 57.12; H, 5.36; N, 11.00.
Example 3. 2-{l-(Chloromethyl)-3-[5-(2-moφholinoethoxy)-liϊ-indole-2-catbonyl]-5-nitro- l,2-dihydro-3i^benzo[e]indole-7-sulfonamido} ethyl dihydtogen phosphate trifluoroacetate (15) (Scheme B). A mixture of indoline 115 (182 mg, 0.31 mmol), acid 122 (134 mg, 0.40 mmol), EDCLHCl (241 mg, 1.24 mmol), and anhydrous TsOH (11 mg, 0.06 mmol) in DMA (2 mL) was stirred at room temperature for 2 h then cooled in an ice bath. Cold aqueous NaHCO3 (5%) was added and the precipitated solid was filtered off, washed with water, and dried. The resulting solid was triturated with acetone to give di-tert-butyl 2-{l-(chlorometliyl)-3-[5-(2-morpholinoethoxy)-lH- indole-2-carbonyl]-5-mtto-l,2-dihydro-3H-benzo[^]indole-7-sulfonamido}ethyl phosphate (123) as a yellow solid (176 mg, 66%): mp 200-204 0C (dec); 1H NMR [(CD3)2SO] δ 11.77 (d,/= 1.6 Hz, 1 H), 9.30 (s, 1 H), 8.86 (d, / = 1.7 Hz, 1 H), 8.45 (dj = 8.9 Hz, 1 H), 8.20 (tj = 5.9 Hz, 1 H), 8.00 (dd,/= 8.9, 1.7 Hz, 1 H), 7.39 (d,/ = 8.9 Hz, 1 H), 7.21 (d,/ = 1.7 Hz, 1 H), 7.08 (d,/ = 2.3 Hz, 1 H), 6.95 (ddLJ = 8.9, 2.4 Hz, 1 H), 5.01-4.94 (m, 1 H), 4.71 (ddj = 10.9, 2.2 Hz, 1 H), 4.69-4.63 (m, 1 H), 4.20-4.09 (m, 4 H), 3.83 (qj = 6.3 Hz, 2 H), 3.63-3.58 (m, 4 H), 3.06 (qj = 5.8 Hz, 2 H), 2.73 (tj = 5.7 Hz, 2 H), ca. 2.52-2.46 (m, 4 H, partially obscured by DMSO peak), 1.35 (s, 18 H). Anal. (C38H49ClN5OnPS) CaIc: C, 53.68; H, 5.81; N, 8.24. Found: C, 53.59; H, 5.61; N, 8.19.
TFA (0.15 mL, 1.9 mmol) was added to a suspension of the phosphate ester 123 (164 mg, 0.19 mmol) in CH2Cl2 (5 mL) and the resulting solution was stirred at room temperature for 16 h, during which time a precipitate separated. The mixture was evaporated, the residue was resuspended in CH2Cl2 and evaporated once more, and the residue was triturated with EtOAc to give 15 as a yellow solid (158 mg, 98%): mp 166-169 0C; 1H NMR [(CD3)2SO] δ 11.84 (s, 1 H), ca. 10.7 (v br s, 1 H),
9.29 (s, 1 H), 8.87 (d,/ = 1.7 Hz, 1 H), 8.45 (dj = 8.9 Hz, 1 H), 8.27-8.20 (m, 1 H), 8.03 (ddj = 8.9, 1.7 Hz, 1 H), 7.44 (dj = 8.9 Hz, 1 H), 7.26 (dj = 2.2 Hz, 1 H), 7.24 (dj = 1.8 Hz, 1 H), 7.02 (ddj = 8.9, 2.3 Hz, 1 H), 5.01-4.94 (m, 1 H), 4.71 (ddj = 10.8, 2.1 Hz, 1 H), 4.68-4.62 (m, 1 H), 4.34-4.29 (m, 2 H), 4.19-4.09 (m, 2 H), 3.87-3.76 (m, 6 H), 3.26-3.13 (m, 4 H), 3.06-3.00 (m, 2 H), (2 H not observed, obscured by water peak δ 3.5-3.3). Anal. (C311H33ClN5O11PS-CF3CO2H) CaIc: C, 45.11; H, 4.02; N, 8.22. Found: C, 44.72; H, 4.36; N, 8.57.
Example 4. l-(Chloromethyl)-Λr-(2-hydroxyethyl)-3-[5-(3-morpholmopropoxy)-li3T-indole-2- carbonyl]-5-nitfo-l,2-dihydro-3iϊbenzo[e]indole-7-sulfonamide (2) (Scheme C). Crude ethyl 5-hydiOxy-lH-indole-2-carboxylate as prepared in Example 1 (5.51 mmol) was dissolved in THF (40 mL) with triphenylphosphine (2.17 g, 8.3 mmol) and 4-(3-hydroxyproρyl)morpholine (1.20 g, 8.3 mmol). Diethyl azodicarboxylate (1.44 g, 8.3 mmol) was added dropwise and the mixture was stirred at room temperature for 2.5 h. The THF was evaporated and the residue was partitioned between EtOAc and water. The EtOAc layer was washed with brine (X3) and extracted with aqueous HCl (2N, X2). The combined acid extracts were cooled to 0 0C, basifϊed with cone, aqueous NH3, and extracted with EtOAc (x3). The combined organic extracts were dried (MgSO4) and evaporated to give a tan solid. Recrystallisation from EtOPI gave ethyl 5-(3-morpholinopropoxy)-l H-indole-2- carboxylate (124) as pale yellow crystals (0.86 g, 47%): mp 152-155 0C; 1H NMR [(CD3)2SO] δ 11.68 (s, 1 H), 7.33 (d, / = 8.9 Hz, 1 H), 7.10 (d, / = 2.3 Hz, 1 H), 7.03 (s, 1 H), 6.91 (dd,/ = 8.9, 2.4 Hz, 1 H), 4.33 (qj = 7.1 Hz, 2 H), 3.99 (tj = 6.4 Hz, 2 H), 3.60-3.56 (m, 4 H), 2.43 (tj = 7.2 Hz, 2 H), 2.39-2.35 (m, 4 H), 1.93-1.84 (m, 2 H), 1.33 (t,/ = 7.1 Hz, 3 H). Anal. (C18H24N2O4) CaIc: C, 65.04; H, 7.28; N, 8.43. Found: C, 64.70; H, 7.58; N, 8.56.
A solution of KOH (651 mg, 11.6 mmol) in water (10 mL) was added to a suspension of the ester 124 (811 mg, 2.44 mmol) in EtOH (20 mL). The mixture was stirred at reflux for 10 min then cooled to room temperature. The EtOH was evaporated and the aqueous residue was neutralised by the addition of aqueous HCl (2N, 5.80 mL, 11.6 mmol). An oil separated that solidified on standing. The solid was filtered off and dried, then suspended in dioxane (30 mL) and treated with HCl- saturated dioxane (10 mL). After stirring at room temperature for 3 h the solid was filtered off and dried to give 5-(3-morpholinopropoxy)-lH-indole-2-carboxylic acid hydrochloride (125) as a white solid (824 mg, 99%): mp 246-249 0C; 1H NMR [(CD3)2SO] δ 12.81 (br s, 1 H), 11.61 (s, 1 H), 10.69 (br s, 1 H), 7.34 (dj = 8.9 Hz, 1 H), 7.13 (dj = 2.3 Hz, 1 H), 6.99 (ddj = 2.0, 0.6 Hz, 1 H), 6.91 (ddj = 8.9, 2.4 Hz, 1 H), 4.06 (tj = 6.0 Hz, 2 H), 4.01-3.92 (m, 2 H), 3.85-3.74 (m, 2 H), 3.53-3.43 (m, 2 H), 3.16-3.03 (m, 2 H), 2.24-2.14 (m, 2 H), (2 H obscured by water peak observed after D2O
exchange). Anal. (C16H20N2O4-HCrV2H2O) CaIc: C, 54.94; H, 6.34; N, 8.01. Found: C, 55.00; H, 6.62; N, 7.64.
A mixture of indoline 108 (87 mg, 0.23 mmol), acid 125 (123 mg, 0.36 mmol), EDCI.HC1 (173 mg, 0.92 mmol), and anhydrous TsOH (7.8 mg, 0.05 mmol) in DMA (2.5 mL) was stirred at toom temperature for 3 h then cooled in an ice bath. Cold aqueous NaHCO3 (5%) was added and the precipitated solid was filtered off, washed with water, and dried to give 2 as a yellow solid (153 mg, 100%): mp 130-1340C; 1H NMR [(CD3)2SO] δ 11.72 (d J = 1.7 Hz, 1 H), 9.29 (s, 1 H), 8.85 (dj = 1.6 Hz, 1 H), 8.44 (dj = 8.9 Hz, 1 H), 8.04 (dd,/ = 8.9, 1.7 Hz, 1 H), 7.91 (t, / = 5.9 Hz, 1 H), 7.42 (dj = 8.9 Hz, 1 H), 7.22 (dj = 1.8 Hz, 1 H), 7.16 (dj = 2.3 Hz, 1 H), 6.95 (ddj = 8.9, 2.4 Hz, 1 H), 5.01-4.94 (m, 1 H), 4.72 (ddj = 10.9, 2.4 Hz, 1 H), 4.69-4.63 (m, 1 H), 4.67 (tj = 5.6 Hz, 1 H), 4.21-4.10 (m, 2 H), 4.04 (t,J = 6.4 Hz, 2 H), 3.63-3.57 (m, 4 H), 3.39 (qj = 6.0 Hz, 2 H), 2.88 (qj = 6.1 Hz, 2 H), 2.47 (t,/ = 7.2 Hz, 2 H), 2.43-2.36 (m, 4 H), 1.96-1.88 (m, 2 H). Anal. (C31H34ClN5O8S-H2O) CaIc: C, 53.95; H, 5.26; N, 10.15. Found: C, 53.97; H, 5.21; N, 10.29.
Example 5. 5-Ammo-l-(chloromethyl)-/Λ£(2-hydroxyethyl)-3- [5-(3-morpholinopropoxy)-l/£- indole-2-carbonyl]-l,2-dihydro-3//:'benzo[e]indole-7-sulfonamide (11) (Scheme C). A solution of nitro compound 2 (52.6 mg, 0.078 mmol) in THF (20 mL) with PtO2 (53 mg) was hydrogenated at 50 psi for 2 h. The catalyst was filtered off through Celite, the filtrate was evaporated, and die residue was triturated with EtOAc to give 11 as a pale green solid (38 mg, 76%): mp 220-225 0C (dec); 1H NMR [(CD3)2SO] δ 11.56 (dj = 1.5 Hz, 1 H), 8.54 (dj = 1.6 Hz, 1 H), 7.94 (dj = 8.9 Hz, 1 H), 7.81 (s, 1 H), 7.74 (ddj = 8.9, 1.7 Hz, 1 H), 7.49-7.44 (m, 1 H), 7.40 (dj = 8.9 Hz, 1 H), 7.16 (d,/ = 2.3 Hz, 1 H), 7.09 (dj = 1.5 Hz, 1 H), 6.92 (ddj = 8.9, 2.4 Hz, 1 H), 6.30 (s, 2 H), 4.76 (ddj = 10.7, 9.0 Hz, 1 H), 4.65 (t, J = 5.4 Hz, 1 H), 4.53 (ddj = 10.9, 1.7 Hz, 1 H), 4.21-4.15 (m, 1 H), 4.06-3.98 (m, 3 H), 3.82 (ddj = 11.0, 7.7 Hz, 1 H), 3.63-3.56 (m, 4 H), 3.39 (q, /= 6.0 Hz, 2 H), 2.88-2.81 (m, 2 H), 2.46 (t,/= 7.2 Hz, 2 H), 2.42-2.36 (m, 4 H), 1.95-1.87 (m, 2 H). Anal. (C31H36ON5O6S) CaIc: C, 57.98; H, 5.65; N, 10.91. Found: C, 57.70; H, 5.70; N, 11.10.
Example 6. 2-{l-(Chlofomethyl)-3-[5-(3-morpholinopfopoxy)-l//:indole-2-catbonyl]-5-nitro- l,2-dihydro-3ϋ/-benzo[e]indole-7-sulfonamido}ethyl dihydfogen phosphate trifluoroacetate (16) (Scheme C). A mixture of indoline 115 (176 mg, 0.30 mmol), acid 125 (156 mg, 0.45 mmol), EDCI.HC1 (233 mg, 1.2 mmol), and anhydrous TsOH (10.5 mg, 0.06 mmol) in DMA (2 mL) was stirred at room temperature for 2 h then cooled in an ice bath. Cold aqueous NaHCO3 (5%) was added and the precipitated solid was filtered off, washed with water, and dried. The resulting solid was triturated with acetone to give di-tert-bvΛyl 2-{l-(chloromethyl)-3-[5-(3-morpholinopropoxy)-
lH-indole-2-carbonyl]-5-nitro-l ,2-dihychO-3H-benzo[e]indole-7-sulfonamido} ethyl phosphate (126) as a pale yellow solid (175 mg, 66 %): mp 200-203 0C (dec); 1H NMR [(CD3J2SOl δ H-76 (d,/ = 2.0 Hz, 1 H), 9.30 (s, 1 H), 8.87 (d,/ = 1.7 Hz, 1 H), 8.46 (dj= 8.9 Hz, 1 H), 8.20 (t,/ = 5.9 Hz, 1 H), 8.02 (dd,/ = 8.9, 1.7 Hz, 1 H), 7.42 (d,/ = 8.9 Hz, 1 H), 7.23 (dj = 1.7 Hz, 1 H), 7.16 (dj = 2.3 Hz, 1 H), 6.95 (ddj = 8.9, 2.4 Hz, 1 H), 5.02-4.95 (m, 1 H), 4.74 (dd J = 10.9, 2.3 Hz, 1 H), 4.70- 4.63 (m, 1 H), 4.20-4.11 (m, 2 H), 4.03 (tj = 6.3 Hz, 2 H), 3.84 (qj = 6.3 Hz, 2 H), 3.62-3.56 (m, 4 H), 3.05 (qj = 5.8 Hz, 2 H), 2.46 (tj - 7.2 Hz, 2 H), 2.41-2.36 (m, 4 H), 1.35 (s, 18 H). Anal. (C39H51ClN5O11PS) CaIc: C, 54.20; H, 5.95; N, 8.10. Found: C, 54.13; H, 5.91; N, 7.82.
TFA (0.14 mL, 1.9 mmol) was added to a suspension of die phosphate ester 126 (162 mg, 0.19 nitnol) in CH2Cl2 (6 mL) and the resulting solution was stirred at room temperature for 16 h. The mixture was evaporated, the residue was resuspended in CH2Cl2 and evaporated once more, and the residue was triturated with EtOAc to give 16 as a pale yellow powder (165 mg, 100%): mp 165-168 0C; 1H NMR [(CD3)2SO] δ 11.82 (d,/ = 1.6 Hz, 1 H), ca. 9.7 (v br s, 1 H), 9.30 (s, 1 H), 8.87 (dj = 1.6 Hz, 1 H), 8.44 (dj= 8.9 Hz, 1 H), 8.20 (t,/ = 5.9 Hz, 1 H), 8.02 (ddj= 8.9, 1.7 Hz, 1 H), 7.44 (dj = 8.9 Hz, 1 H), 7.23 (dj = 1.7 Hz, 1 H), 7.20 (dj = 2.2 Hz, 1 H), 6.97 (dd,/ - 8.9, 2.4 Hz, 1 H), 5.02-4.93 (m, 1 H), 4.71 (ddj= 10.9, 2.2 Hz, 1 H), 4.68-4.62 (m, 1 H), 4.20-3.97 (m, 6 H), 3.81 (qj = 6.5 Hz, 2 H), 3.34-3.28 (m, 2 H), 3.20-3.08 (m, 2 H), 3.02 (qj = 5.9 Hz, 2 H), 2.21-2.10 (m, 2 H), (4 H not observed, obscured by water peak). Anal. (C31H35ClN5O11PS CF3CO2H-H2O) CaIc: C, 44.83; H, 4.33; N, 7.92. Found: C, 44.96; H, 4.41; N, 8.20.
Example 7. (£)-l-(Chloromethyl)--7V-(2-hydroxyethyl)-3-{3-[4-(2- tnotpholinoethoxy)phenyl] aciyloyl} -5-nitto-l,2-dihydro-3Jf-ben2o [ e] indole-7 -sulfonamide (3) (Scheme D). A mixture of 4-(2-chloroediyl)morpholine hydrochloride (6.42 g, 34.5 mmol), NaOH (1.52 g, 38.1 mmol), water (24 mL) and toluene (26 mL) was stirred at 0 0C and saturated with solid NaCl. The toluene layer was separated and the alkaline aqueous solution was further extracted widi toluene (4 x 15 mL). The combined toluene extracts were dried over KOH and used for the following reaction.
To a solution of (H)-methyl 3-(4-hydroxyphenyl)acrylate (127) (5.43 g, 30.5 mmol) in anhydrous ether (100 mL) stirred under nitrogen at 20 0C was added portionwise NaH (60% dispersion in oil, 1.28 g, 32.0 mmol). After die addition was complete the mixture was stirred for a further 2 h. Most of the ether was evaporated, petroleum ether (100 mL) was added, and the mixture was stirred for 5 min. The petroleum ether was decanted and the process was repeated once more. To the residue was added toluene (26 mL) followed by the toluene solution of 4-(2-chloroethyl)morpholine prepared above. The heterogeneous mixture was stirred at reflux temperature for 92 h. The mixture
was cooled and filtered through a short Al2O3 (neutral) column eluting with hot toluene. The combined eluates were washed successively with cold aqueous NaHCO3 and then with water, then dried (NTa2SO4) and evaporated to give (E)-methyl 3-[4-(2-tnorpholinoethoxy)phenyl]acrylate (128) as a colourless solid (6.66 g, 75%): mp 61-62 0C; 1H NMR (CDCl3) δ 7.64 (d J = 16.0 Hz, 1 H), 7.51- 7.45 (m, 2 H), 6.93-6.88 (m, 2 H), 6.31 (dj= 16.0 Hz, 1 H), 4.14 (tj = 5.7 Hz, 2 H), 3.79 (s, 3 H), 3.77-3.70 (m, 4 H), 2.81 (t J = 5.7 Hz, 2 H), 2.61-2.55 (m, 4 H). Anal. (C16H21NO4) CaIc: C, 65.96; H, 7.27; N, 4.81. Found: C, 65.98; H, 7.33; N, 4.82.
A fiiixture of ester 128 (6.58 g, 22.6 mmol), MeOH (40 mL), water (20 mL) and KOH (3.16 g, 56.5 mmol) was stirred at 20 0C for 21 h. The mixture was evaporated under reduced pressure at 35 0C to remove the MeOH. The resulting alkaline mixture was washed with CH2Cl2 and the aqueous layer- was filtered. The alkaline filtrate was acidified with cone. HCl at 0 0C. The resulting precipitate was filtered off, washed with 6N HCl and dried over silica gel under vacuum to give (E)~3-[4-(2- morpholinoethoxy)phenyl]acrylic acid hydrochloride (129) as a colourless solid (5.90 g, 83%): mp 252-2540C; 1H NMR [(CD3)2SO] δ 12.22 (br s, 1 H), 10.90 (br s, 1 H), 7.69 (d,/= 8.8 Hz, 2 H), 7.56 (dj = 16.0 Hz, 1 H), 7.05 (d,/ = 8.8 Hz, 2 H), 6.41 (dj = 16.0 Hz, 1 H), 4.46 (br s, 2 H), 4.06-3.73 (m, 4 H), 3.67-3.42 (m, 4 H), 3.28-3.14 (m, 2 H). Anal. (C15H20ClNO4-O-SH2O) CaIc: C, 56.45; H, 6.51; N, 4.39. Found: C, 56.49; H, 6.46; N, 4.42.
A mixture of acid 129 (68.1 mg, 0.24 mmol), DMF (one drop) and thionyl chloride (2 mL) was stirred at reflux for 10 min. The mixture was cooled and evaporated under reduced pressure to dryness. The reaction flask was immersed in an ice-bath and solid indoline 112 (108 mg, 0.215 mmol) was added, followed by a mixture of DMA (2 mL) and DIPEA (0.041 mL, 0.237 mmol). The resulting mixture was stirred at 0 0C for 2 h. Dilute aqueous NaHCO3 (5%, 7 mL) was added, followed by water (7 mL), and the mixture was stirred at 0 0C for 10 min. The precipitated solid was filtered off, washed with cold water several times, then dried and triturated with EtOAc/petroleum ether (1:10) to give (E)-N-[2-(/^-butyldimetliylsilyloxy)ethyl]-l-(chlorometliyl)-3-{3-[4-(2- moi-ρholinoethoxy)phenyl]aciyloyl}-5-nitro-l,2-dihydro-3H-benzo[ά']indole-7-sulfonamide (130) as a yellowish orange solid (128 mg, 79%): mp 203-205 0C; 1H NMR (CDCl3) δ 9.42 (br s, 1 H), 8.99 (dj = 1.6 Hz, 1 H), 8.03 (ddj = 8.9, 1.6 Hz, 1 H), 7.95 (dj = 8.9 Hz, 1 H), 7.90 (d,/ = 15.2 Hz, 1 H), 7.58 (dj = 8.8 Hz, 2 H), 6.96 (d,/ = 8.8 Hz, 2 H), 6.74 (d,J = 15.2 Hz, 1 H), 4.96 (t,/ = 5.9, 1 H), 4.64 (ddj = 10.7, 2.4 Hz, 1 H), 4.56 (br tj = 9.7 Hz, 1 H), 4.35-4.26 (m, 1 H), 4.18 (tj = 5.7, 2 H), 3.95 (br dd, / = 11.4, 3.3 Hz, 1 H), 3.80-3.72 (m, 4 H), 3.68 (t, / = 5.1 Hz, 2 H), 3.63 (br dd, / = 11.4, 9.5 Hz, 1 H), 3.22-3.13 (m, 2 H), 2.84 (t,J = 5.7 Hz, 2 H), 2.65-2.55 (m, 4 H), 0.833 (s, 9 H), -
0.008 (s, 6 H, obscured by TMS). Anal. (C36H47ClN4O8SSi) CaIc: C, 56.94; H, 6.24; N, 7.38. Found: C, 57.09; H, 6.14; N, 7.27.
To a mixture of silyl ether 130 (142 mg, 0.187 mmol), dioxane (6 mL) and MeOH (3 roL) stirred at 20 0C was added HCl (1.25M in MeOH, 1.0 mL). The mixture was stirred at this temperature for a further 30 min. EtOAc was added and the mixture was stirred for another 30 min. The resulting solid was filtered off, washed with EtOAc several times, and dried to give 3 as a yellowish orange solid (127 mg, 99%): mp 280-282 0C (dec); 1H NMR [(CDj)2SO] δ 10.75 (br s, 1 H), 9.35 (s, 1 H), 8.83 (dj = 1.6 Hz, 1 H), 8.38 (dj = 8.9 Hz, 1 H), 8.02 (dd,/ = 8.9, 1.6 Hz, 1 H), 7.92 (t,/ = 6.0 Hz, 1 H), 7.84 (dj= 8.7 Hz, 2 H), 7.75 (dj= 15.3 Hz, 1 H), 7.14 (d,J = 15.3 Hz, 1 H), 7.09 (dj = 8.7 Hz, 2 H), 4.72-4.60 (m, 3 H), 4.49 (br s, 2 H), 4.10 (br dj = 4.1 Hz, 2 H), 3.99 (bi d, J- 13.8, 2 H), 3.80 (br t,/ = 11.7 Hz, 2 H), 3.63-3.49 (m, 4 H), 3.39 (tj = 6.2 Hz, 2 H, partially obscured by water peak), 3.30-3.16 (m, 2 H, partially obscured by water peak), 2.87 (q, / = 6.0 Hz, 2 H). HRMS (FAB) calc for C30H34 35ClN4O8S (MH+) m/~ 645.1785, found 645.1787. CaIc for C30H34 37ClN4O8S (MH+) m/z 647.1756, found 647.1770. Anal. (C30H34Cl2N4O8S-H2O-V2EtOAc) Calc: C, 51.68; H, 5.42; N, 7.53. Found: C, 51.41; H, 5.31; N, 7.50.
Example 8. (£)-5-Amino-l-(chlot omethyl)-iV-(2-hydroxyethyl)-3-{3- [4-(2- morpholinoethoxy)phenyl]acryloyl}-l,2-dihydro-3i^benzo[e]indole-7-sulfonamide (12) (Scheme D). Saturated aqueous ammonium chloride (4 mL) and then Zn powder (450 mg) were added to a stirred solution of nitro compound 130 (107 mg, 0.141mmol) in acetone (8 mL) and water (4 mL). The mixture was stirred at room temperature for 1 h, then filtered through Celite and the Celite pad was rinsed with acetone. The combined filtrates were concentrated to remove acetone and the aqueous residue was extracted with dichlorome thane (x 2). The organic extracts were washed with water and then brine, and then dried (Na2SO4) and evaporated. The residue was dissolved in dioxane (1.5 mL) and MeOH (0.75 mL), and HCl (4M in MeOH, 1 mL) was added. The resulting mixture was stirred at room temperature for 30 min, concentrated under reduce pressure, and diluted with CH2Cl2 (2 mL). The mixture was cooled at 0 0C and ice-cold aqueous NaHCO3 (10%, 10 mL) was added, followed by water (10 mL). The mixture was stirred for 30 min then extracted with CH2Cl2 (x 2). The organic extracts were washed with water and then brine, and then dried (Na2SO4) and evaporated. The residue was dissolved in the minimum quantity of MeOH, cooled to 0 0C, and diluted with Et2O. After stirring at 0 0C for 30 min the resulting precipitate was filtered off and washed with water and then Et2O to provide 12 as a yellow powder (38 mg, 44%): mp 225-230 0C (dec); 1H NMR [(CD3)2SO] δ 8.51 (d,/ = 1.5 Hz, 1 H), 7.89-7.84 (m, 2 H), 7.76-7.68 (m, 3 H), 7.63 (dj = 15.3 Hz, 1 H), 7.41 (br s, 1 H), 7.06 (dj = 15.6 Hz, 1 H), 7.01 (dj = 8.8 Hz,
2 H), 6.25 (s, 2 H), 4.63 (tj = 5.6 Hz, 1 H), 4.51-4.38 (m, 2 H), 4.16-4.02 (m, 3 H), 3.97-3.92 (ddj = 10.9, 2.9 Hz, 1 H), 3.81-3.76 (dd J = 11, 8 Hz, 1 H), 3.61-3.56 (m, 4 H), 3.38 (q J = 12, 6.3 Hz, 2 H), 2.83 (m, 2 H), 2.71 (tj = 5.8 Hz, 2 H), 2.53-2.46 (4 H obscured by DMSO peak). Anal. (C30H35ClN4O6S -0.2H2O) CaIc: C, 58.24; H, 5.77; N, 9.06. Found: C, 58.31; H, 5.63; N, 8.74.
Example 9. (£)-2-(l-(Chloromethyl)-3-{3-[4-(2-tnorpholinoethoxy)phenyl]acryloyl}-5-nitfo- l,2-dihydro-3i£benzo[e]mdole-7-sulfonamido) ethyl dihydrogen phosphate trifluoroacetate (17) (Scheme D). To a stirred mixture of indoHne 115 (290 mg, 0.50 mmol) and acid 129 (198 mg, 0.60 mmol) in DMA (10 mL) was added EDCLHCl (385 mg, 2.00 mmol) and anhydrous TsOH (17.5 mg, 0.10 mmol). The mixture was stirred at 20 0C for 4 h and another batch of acid 129 (198 mg, 0.60 mmol), EDCI.HC1 (385 mg, 2.00 mmol), and anhydrous TsOH (17.5 mg, 0.10 mmol) were added. After a total reaction time of 24 h the reaction flask was cooled in an ice-bath and cold aqueous NaHCO3 (5%, 20 mL) was added, followed by water (20 mL). After stirring at 0 0C for 10 min the precipitated solid was filtered off, washed with cold water several times, and dried to give (E)-di-tert-butyl 2-(l-(chloromethyl)-3-{3-[4-(2-morpholinoethoxy)phenyl]acryloy}-5-nitro-l,2- dihydiO-3i-f-benzo[έ?]indole-7-sulfonamido) ethyl phosphate (131) as a yellowish orange solid (395 mg, 94%): mp 245-248 0C (dec); 1H NMR (CDCl3) δ 9.41 (br s, 1 H), 8.97 (d J = 1.6 Hz, 1 H), 8.50 (ddj = 8.9, 1.6 Hz, 1 H), 7.96 (dj = 8.9 Hz, 1 H), 7.91 (dj = 15.2 Hz, 1 H), 7.58 (dj = 8.7 Hz, 2 H), 6.96 (dj = 8.7 Hz, 2 H), 6.74 (dj = 15.2 Hz, 1 H), 5.95 (tj = 5.7 Hz, 1 H), 4.63 (br ddj = 10.8, 2.5 Hz, 1 H), 4.56 (br tj = 9.8 Hz, 1 H), 4.34-4.26 (m, 1 H), 4.18 (tj = 5.6, 2 H), 4.12-4.04 (m, 2 H), 3.96 (br ddj = 11.5, 3.2 Hz, 1 H), 3.75 (br tj = 4.6 Hz, 4 H), 3.62 (br ddj = 11.4, 9.9 Hz, 1 H), 3.33 (br qj = 5.4 Hz, 2 H), 2.85 (br tj = 5.6 Hz, 2 H), 2.61 (unresolved t, 4 H), 1.46 and
1.45 (2 s, 18 H). Anal. (C38H511ClN4O11PS-V2H2O) CaIc: C, 53.93; H, 6.07; N, 6.62. Found: C, 53.83; H, 5.99; N, 6.53.
Phosphate ester 131 (366 mg, 0.437 mmol) was stirred with TFA (5 mL) in CH2Cl2 (50 mL) at 20 0C for 3 h. The reaction solution was filtered through a cotton wool plug and the plug was washed with methanol. The filtrate was concentrated under reduced pressure until a precipitate began to form. EtOAc (150 mL) was added and the mixture was stirred at 0 0C for 45 min. The solid was filtered off, washed with EtOAc several times, and dried in vacuum over silica gel to give 17 as a yellow solid (292 mg, 80%): mp 212 0C (dec); 1H NMR (C5D5N) δ ca. 10.7 (v br s, 1 H), 9.81 (br s, 1 H),
9.46 (dj = 1.6 Hz, 1 H), 8.39 (ddj= 8.9, 1.6 Hz, 1 H), 8.22 (dj= 15.2 Hz, 1 H), 8.14 (dj= 8.9 Hz, 1 FI), 7.74 (dj = 8.8 Hz, 2 H), 7.16 (dj= 15.2 Hz, 2 H), 7.10 (dj = 8.8 Hz, 2 H), 4.80 (br dd, / = 10.8, 2.6 Hz, 1 H), 4.65 (br tj = 10.0 Hz, 1 H), 4.59-4.51 (m, 2 H), 4.50-4.42 (m, 1 H), 4.21-4.13 (m, 3 H), 4.01 (br dd J = 11.3, 8.3 Hz, 1 H), 3.73 (br t J = 4.6 Hz, 6 H), 2.76 (t, 7 = 5.7 Hz, 2 H),
2.53 (t, / = 4.6 Hz, 4 H). Anal. (C3nH34ClN4O11PS-1Z2CF3CO2H) CaIc. C, 47.61; H, 4.45; N, 7.16. Found: C, 47.69; H, 4.65; N, 6.97.
Example 10. l-(Chlotomethyl)-iV-(2-hydroxyethyl)-3-{4-[2-(4-inethylpiperazin-l- yl)ethoxy]benzoyl}-5-nitto-l,2-dihydro-3i^benzo[eIindole-7-sulfonamide hydrochloride (4) (Scheme E). To a solution of (E) -methyl 3-(4-hydroxyphenyl)acrylate (127) (5.4 g, 30.5 mmol) in anhydrous Et2O (100 mL) was added NaH (0.77 g, 32.0 mmol) and the mixture was stirred under nitrogen at 20 0C for 6 h. The Et2O was evaporated and DMA (20 mL) was added, followed by 1,2- dibromoethane (13.2 mL, 153 mmol). The reaction mixture was stirred at 130-135 0C (bath temperature) for 21 h. The mixture was cooled to 20 0C and more NaH (0.77 g, 32.0 mmol) and 1,2-dibromoethane (13.2 mL, 153 mmol) were added. The mixture stirred again at 150-160 0C (bath temperature) for 5 h. The mixture was cooled a little and toluene (100 mL) was added. The mixture was filtered and the filter pad was washed with hot toluene several times. The combined filtrates were washed successively with cold water, cold aqueous NaHCO3, and again with water. The organic layer was dried (Na2SO4) and evaporated to give an oil which was dissolved in DMF (30 mL) and stirred with 1-methylpiperazine (14.7 mL, 133 mmol) at 20 0C for 3 h. The mixture was diluted with EtOAc (ca. 200 mL) and then stirred with aqueous Na2CO3 (2%) at 0 0C for 5-10 min. The organic layer was separated, washed with water (x 2), and then dried (Na2SO4) and evaporated. The resulting oil was dissolved in EtOAc and the solution was treated with HCl (4M in p-dioxane, 17 mL, 68 mmol). The mixture was stirred and then left to stand at 5 0C over-night. The solid was filtered off, washed with EtOAc several times, and dried to give (J3)-metbyl 3-{4-[2-(4- methylpiperazin-l-yl)ethoxy]phenyl}acrylate hydrochloride (132) as beige solid (5.47g, 48%): mp 235-237 0C (dec); 1H NMR [(CD3),SO] δ 11.78 (br s, 2 H), 7.71 (dj = 8.8 Hz, 2 H), 7.64 (dj = 16.0 Hz, 1 H), 7.05 (dj = 8.8 Hz, 2 H), 6.52 (dj = 16.0 Hz, 1 H), 4.44 (poorly resolved t resolved after D2O exchange, / = 4.9 Hz, 2 H), 3.71 (s, 3 H), 3.52-3.31 (m, partially obscured by water peak, revealed after D2O exchange, 10 H), 2.81 (s, 3 H). Anal. (C17H26Cl2N2O3 1Z2H2O) CaIc: C, 52.86; H, 7.05; N, 7.25. Found: C, 52.79; H, 6.98; N, 6.94.
Ester
132 (5.18 g, 13.7 mmol) was treated with KOH (3.5 g, 62 mmol) in a mixture of MeOH (30 mL) and water (15 mL) at 20 0C for 29 h. The mixture was evaporated under reduced pressure at 35 0C to remove the MeOH. The alkaline remainder was washed with CH2Cl2 and filtered. The filtrate was acidified with cone. HCl at 0 0C and left to stand at 5 0C overnight. The resulting precipitate was filtered off, washed with cold 6N HCl (3 x 1 mL), and dried over silica gel in vacuum overnight to give (£)-3-{4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl}acrylic acid hydrochloride (133) as a beige solid (2.97 g, 60%): mp 264-267 0C (dec); 1H NMR [(CD3)2SO] 8 11.89 (br s, 3 H), 7.67 (dj = 8.8
Hz, 2 H), 7.56 (d J = 16.0 Hz, 1 H), 7.05 (d, / = 8.8 Hz, 2 H), 6.41 (dj = 16.0 Hz, 1 H), 4.46 (unresolved t resolved after D2O exchange,/ = 4.8 Hz, 2 H), 3.71 (s, 3 H), 3.57-3.30 (m, partially obscured by water peak, revealed after D2O exchange, 10 H), 2.82 (s, 3 H). Anal. (C16H24Cl2N2O3V2H2O) CaIc: C, 51.62; H, 6.77; N, 7.53. Found: C, 51.77; H, 6.63; N, 7.17.
To a stirred mixture of indoline 112 (90 mg, 0.18 mmol) and acid 133 (78 mg, 0.22 mmol) in DMA (10 mL) were added EDCLHCl (138 mg, 0.72 mmol) and anhydrous TsOH (6.3 mg, 0.04 mmol). The mixture was stirred at 20 0C for 3 h then another batch of EDCLHCl (138 mg, 0.72 mmol) and anhydrous TsOH (6.3 mg, 0.04 mmol) were added and the mixture was stirred for a further 16 h. The reaction flask was immersed in an ice-bath and cold aqueous NaHCO3 (5%, 10 mL) was added, followed by water (30 mL). The mixture was stirred at 0 0C for ca. 10 min. The solid was filtered off, washed with cold water several times, and dried to give iV-p-^//- butyldirnethylsilyloxy)ethyl]-l-(chloromethyl)-3-{4-[2-(4-rnethylpiperazin-l-yl)ethoxy]benzoyl}-5- riitro-l,2-dihydro-3H-benzo[tf]indole-7-sulfonamide (134) as a yellowish orange solid (118 mg, 85%): mp 202 0C (dec); 1H NMR (CDCl3) δ 9.42 (br s, 1 H), 8.98 (poorly resolved dj = 1.6 Hz, 1 H), 8.03 (dd,/ = 8.8, 1.6 Hz, 1 H), 7.95 (dj = 8.8 Hz, 1 H), 7.91 (dj = 15.2 Hz, 1 H), 7.57 (dj = 8.7 Hz, 2 H), 6.95 (dj = 8.7 Hz, 2 H), 6.73 (dj = 15.2 Hz, 1 H), 4.99 (tj = 5.8 Hz, 1 H), 4.63 (ddj = 10.8, 2.5 Hz, 1 H), 4.45 (br t,J = 9.7 Hz, 1 H), 4.34-4.26 (m, 1 H), 4.17 (tj = 5.8 Hz, 2 H), 3.94 (dd, / = 11.4, 3.3 Hz, 1 H), 3.69 (tj = 5.0 Hz, 2 H), 3.62 (ddj = 11.4, 9.5 Hz, 1 H), 3.22-3.13 (m, 2 H), 2.85 (tj = 5.8 Hz, 2 H), 2.73-2.42 (m, 8 H), 2.30 (s, 3 H), 0.84 (s, 9 H), -0.008 (s, 6 H, obscured by TMS). Anal. (C37H50ClN5O7SSi) CaIc: C, 57.53; H, 6.52; N, 9.07. Found: C, 57.51; H, 6.52; N, 8.86.
HCl (1.25M in MeOH, 1 mL) was added to a solution of silyl ether 134 (98 mg, 0.13 mmol) in CH2Cl2 (15 mL) and the mixture was stirred at 20 0C for 2 h 20 min. EtOAc (80 mL) was added and the mixture was stirred at 0 0C for 2 h. The resulting solid was filtered off, washed with EtOAc, and dried to give 4 as a yellowish orange solid (93 mg, 100%): mp 225 0C (dec); 1H NMR [(CD3)2SO] δ 11.10 (v br s, exchangeable with D20, 1 H), 9.35 (br s, 1 H), 8.83 (poorly resolved dj = 1.6 Hz, 1 H), 8.39 (d J = 8.9 Hz, 1 H), 8.02 (ddj = 8.9, 1.6 Hz, 1 H), 7.92 (t J = 5.9 Hz, exchangeable with D2O, 1 H), 7.83 (dj = 8.7 Hz, 2 H), 7.74 (dj = 15.3 Hz, 1 H), 7.13 (dj = 15.3 Hz, 1 H), 7.08 (dj = 8.7 Hz, 2 H), 4.72-4.59 (m, 3 H), 4.39 (br s, 2 H), 4.08 (poorly resolved dj = 4.0 Hz, 2 H), 3.38 (t, /= 6.2 Hz, 2 H), 3.35-2.91 (m, revealed after D2O exchange, 10 H), 2.93-2.75 (m, 5 H). Anal. (C31H38Cl3N5O7S O^H2O) CaIc: C, 49.83; H, 5.37; N, 9.37. Found: C, 50.14; H, 5.32; N, 9.03.
Example 11. 2-[l-(Chloromethyl)-3-{4-[2-(4-methylpiperazin-l-yl)ethoxy]benzoyl}-5-nitro- l,2-dihydro-3i^benzo[e]indole-7-sulfonamido]ethyl dihydrogen phosphate ttifluoroacetate
(18) (Scheme E). To a stirred mixture of indoline 115 (1.16 g, 2.00 mmol) and acid 133 (0.87 g, 2.40 mmol) in DiNlA (45 mL) was added EDCLHCl (1.54 g, 8.00 mmol) and anhydrous TsOH (70.4 mg, 0.40 mmol). The mixture was stirred at 20 0C for 4 h and then another batch of acid 133 (0.87 g, 2.40 mmol), EDCI.HC1 (1.54 g, 8.00 mmol) and anhydrous TsOH (70.4 mg, 0.40 mmol) were added and the mixture was stirred for a further 18 h. The reaction flask was immersed in an ice-bath and cold aqueous NaHCO3 (5%, 90 mL) was added, followed by water (90 mL). The mixture was stirred at O0C for 10 min. The precipitated solid was filtered off, washed with cold water several times, and dried to give di-/»?-butyl 2-[l-(chloromethyl)-3-{4-[2-(4-methylpipera2in-l-yl)ethoxy]benzoyl}-5- nitro-l,2-dihydro-3Η-ben2o[ήindole-7-sulfonamido]ethyl phosphate (135) as a yellowish orange solid (1.58 g, 93%): mp 118-120 0C (dec); 1H NMR (CDCl3) δ 9.41 (br s, 1 H), 8.98 (poorly resolved d,/ = 1.3 Hz, 1 H), 8.04 (ddj= 8.8, 1.4 Hz, 1 H), 7.95 (dj= 8.8 Hz, 1 H), 7.90 (dj= 15.2 Hz, 1 H), 7.57 (d, / = 8.6 Hz, 2 H), 6.94 (dj = 8.6 Hz, 2 H), 6.73 (dj = 15.2 Hz, 1 H), 5.96 (br s, 1 H), 4.63 (dd,/ = 10.7, 2.3 Hz, 1 H), 4.55 (br t,/ = 9.7 Hz, 1 H), 4.32-4.23 (m, 1 H), 4.17 (t,J = 5.8 Hz, 2 H), 4.12-4.02 (m, 2 H), 3.95 (ddj = 11.4, 3.2 Hz, 1 H), 3.62 (ddj = 11.3, 9.5 Hz, 1 H), 3.32 (br s, 2 H), 2.85 (t,/ = 5.8 Hz, 2 H), 2.66 (br s, 4 H), 2.52 (br s, 4 H), 2.32 (s, 3 H), 1.46 and 1.45 (2 s, 18 H). Anal. (C39H53ClN5O10S-V2H2O) CaIc: C, 54.51; H, 6.33; N, 8.15. Found: C, 54.52; H, 6.34; N, 7.91.
Phosphate ester 135 (1.57 g, 1.85 mmol) was dissolved in CH2Cl2 (100 mL) and stirred with TFA (10 mL) at 20 0C for 3 h. The mixture was filtered and the filter pad was washed with 10% TFA in CH2Cl2. The combined filtrates were concentrated at 25 0C (bath temperature) under reduced pressure at to half of the original volume. EtOAC (ca. 100 mL) was added, the mixture was stirred and then left to stand at 00C for 15 h. The precipitated solid was filtered off, washed with EtOAc several times, and dried to give 18 as a yellowish orange solid (1.37 g, 87%): mp 230 0C (dec); 1H NMR [(CD3)2SO] δ 9.35 (br s, 1 H), 9.35 (br s, 1 H), 8.85 (poorly resolved d,J = 1.6 Hz, 1 H), 8.38 (dj= 8.9 Hz, 1 H), 8.13 (t,J = 5.9 Hz, exchangeable with D2O, 1 H), 8.02 (ddj = 8.9, 1.6 Hz, 1 H), 7.82 (d,J - 8.8 Hz, 2 H), 7.74 (dj = 15.2 Hz, 1 H), 7.11 (dj = 15.2 Hz, 1 H), 7.04 (d,/ = 8.8 Hz, 2 H), 4.12-4.58 (m, 3 H), 4.21 (t, / = 5.2 Hz, 2 H), 4.09 (poorly resolved ά,J - 4.9 Hz, 2 H), 3.82 (q,/ = 6.2 Hz, 2 H), 3.51-2.85 (m, partially obscured by water peak, revealed after D2O exchange, 12 H), 2.77 (s, 3 H), 2 Hs not observed. Anal. (C31H37ClN5O10Pl.5CF3COOH-EtOAc) CaIc: C, 45.77; H, 4.70; N, 7.02. Found: C, 45.72; H, 4.67; N, 7.09.
Example 12. l-(Chloromethyl)-iV-(2-hydroxyethyl)-3- [5-(2-morpholinoethoxy)-Lfif-indole-2- carbonyl]-5-nitro~l,2-dihydro-3i£benzo[e]indole-7-carboxamide (5) (Scheme F). A solution of compound 110 (193 mg, 0.623 mmol) in THF (15 mL) was hydrogenated over Pd/C (5%) at 40
psi for 2 h 30 min. The mixture was filtered through Celite and the Celite was washed with THF (5 X lO mL). The combined THF filtrate (containing amine 111) was added to an ice-cold mixture of
1 -(chloiOmethyl)-5-nitro-3-(2,2,2-trifluoroacetyl)-l ,2-dihydro-3i-J-benzo [>]indole~7-carboxylic acid (136) (209 mg, 0.519 mmol) and DIPEA (0.27 mL, 1.56 mmol) in THF (3 mL), followed by the addition of pyBOP (benzotriazol-l-yloxy)tiipyriOlidinophosphonium hexafluorophosphate) (352 mg, 0.675 mmol). The mixture was stirred at 0 0C for a further 1 h. Cesium carbonate (339 mg, 1.04 mmol) and MeOH (10 mL) were added and the mixture was stirred at 20 0C for 2 h, then allowed to stand at 5 0C overnight. The mixture was concentrated under reduced pressure at 25 0C to remove most of the THF. Ice-water was added and the mixture was stirred at 0 0C for 5 h 30 min. The resulting solid was filtered off, washed with cold water several times, and dried in vacuum over silica-gel to give a crude product. This was purified by column chromatography (eluting with CH2Cl2:EtOAc 5:1) to give pure iV-(2-(/«7'-butyldimethyls£yloxy)ethyl)-l-(chloromethyl)-5-nitro-l,2- dihydiO-3H-benzo[«]indole-7-carboxamide (137) as a red solid (212 mg, 88%): mp 156-158 0C; 1H NMR (CDCl3) δ 8.75 (poorly resolved d,/= 1.4 Hz, 1 H), 8.02 (dd,/= 8.8, 1.7 Hz, 1 H), 7.77 (dj = 8.8 Hz, 1 H), 7.71 (s, 1 H), 6.67 (br s, 1 H), 4.26 (s, 1 H), 4.14-4.07 (m, 1 H), 4.01-3.91 (m, 2 H), 3.87-3.77 (m, 3 H), 3.67-3.55 (m, 3 H), 0.92 (s, 9 H), 0.11 (s, 6 H). Anal. (C22H30ClN3O4Si) CaIc: C, 56.94; H, 6.52; N, 9.06. Found: C, 57.07; H, 6.67; N, 9.20.
To a stirred mixture of 137 (126 mg, 0.27 mmol) and acid 122 (107 mg, 0.33 mmol) in DMA (5 mL) was added EDCI.HC1 (210 mg, 1.09 mmol), and anhydrous TsOH (9.5 mg, 0.054 mmol). The mixture was stirred at room temperature for 2.5 h then cooled in an ice bath. Cold aqueous NaHCO3 (5%, 10 mL) was added, followed by cold water (10 mL). The precipitated solid was filtered off, washed with water, and dried to give JV- f2-(/"«7'-butyldimethylsilyloxy) ethyl] -1- (chloromediyl)-3-[5-(2-tnoi^holinoethoxy)-lH-indole-2-carbonyl]-5-iiitro-l,2-dihydro-3H- benzo|Y|indole-7-carboxamide (138) as a yellow solid (193 mg, 97%): mp 1240C; 1H NMR (CDCl3) δ 9.38 (br s, 1 H), 9.28 (s, 1 H), 8.79 (poorly resolved d,/ = 1.3 Hz, 1 H), 8.15 (ddj= 8.8, 1.6 Hz, 1 H), 7.94 (dj = 8.8 Hz, 1 H), 7.38 (dj = 8.9 Hz, 1 H), 7.14 (poorly resolved dj = 2.3 Hz, 1 H), 7.09-7.04 (m, 2 H), 6.79 (t,J - 5.2 Hz, 1 H), 4.92 (ddj= 11.3, 2.1 Hz, 1 H), 4.81 (br tj= 9.8 Hz, 1 H), 4.39-4.29 (m, 1 H), 4.18 (t,/ = 5.7 Hz, 1 H), 3.99 (ddj = 11.4, 3.2 Hz, 1 H), 3.86 (t, / = 5.2 Hz,
2 H), 3.82-3.74 (m, 4 H), 3.71-3.59 (m, 3 H), 2.86 (tj = 5.7 Hz, 2 H), 2.67-2.58 (m, 4 H), 0.94 (s, 9 H), 0.12 (s, 6 H). Anal. (C37H4^ClN5O7Si 0.3H2O) CaIc: C, 59.91; H, 6.33; N, 9.44. Found: C, 59.76; H, 6.41; N, 9.58.
To a filtered solution of 138 (83 mg, 0.113 mmol) in CH2Cl2 (10 mL) was added HCl (1.25M in MeOH, 1.0 mL) and the mixture was stirred at 20 0C for 2 h. The mixture was diluted with EtOAc
(60 mL) and the precipitated solid was filtered off, washed with EtOAc several times, and dried to give 5 as a yellowish orange hydrochloride salt (69 mg, 93%): mp 214-217 0C; 1H NMR [(CD3)2SO] δ 11.80 (s, exchangeable with D2O, 1 H), 10.70 (s, exchangeable with D2O, 1 H), 9.16 (s, 1 H), 8.82 (poorly resolved dj = 1.3 Hz, 1 H), 8.78 (tj = 5.6 Hz, exchangeable with D2O, 1 H), 8.30 (dj = 8.8 Hz, 1 H), 8.16 (ddj= 8.8, 1.5 Hz, 1 H), 7.46 (dj = 8.9 Hz, 1 H), 7.28 (br s, 1 H), 7.22 (poorly resolved dj = 1.6 Hz, 1 H), 7.04 (poorly resolved ddj = 8.9, 2.0 Hz, 1 H), 4.96 (br tj = 10.1 Hz,
1 H), 4.70 (poorly resolved ddj = 10.9, 2.3 Hz, 1 H), 4.68-4.59 (m, 1 H), 4.43 (s, 2 H), 4.19-4.07 (m,
2 H), 4.05-3.71 (m, 4 H), 3.69-3.48 (m, 5 H, 1 H exchangeable with D2O), 3.40 (q, collapses to t after D2O exchange,/ = 5.9 Hz, 2 H), 3.36 (m, partially obscured by water peak, revealed after D2O exchange, 4 H). Anal. (C31H32C]N5O7-HCrV2H2O) CaIc: C, 55.78; H, 5.13; N, 10.49. Found: C, 55.65; H, 5.12; N, 10.43.
Example 13. 5-Amino-l-(chloromethyl)-/Λt(2-hydroxyethyl)-3-[5-(2-morpholinoethoxy)-liϊ indole-2-carbonyl]-l,2-dihydro-3iϊ-benzo[e]indole-7-carboxamide (13) (Scheme F). A solution of nitro compound 138 (39.4 mg, 0.054 mmol) in THF (20 mL) with PtO2 (60 mg) was hydrogenated at 40 psi for 15 min. The catalyst was filtered off through Celite and the filtrate was stirred with HCl (1.25M in MeOH, 0.5 mL) at 20 0C for 1 h. The mixture was evaporated and the residue was triturated with EtOAc to give 13 as a pale green hydrochloride salt (30 mg, 89%): mp >300 0C; 1H NMR (C5D5N) δ 12.98 (s, 1 H), ca. 11.0 (v br s, 1 H), 9.59 (poorly resolved dj = 1.3 Hz, 1 H), 9.35 (t,J = 5.4 Hz, 1 H), 8.55 (br s, 1 H), 8.43 (poorly resolved ddj = 8.7, 1.5 Hz, 1 H), 7.86 (dj = 8.7 Hz, 1 H), 7.68 (dj = 8.8 Hz, 1 H), 7.41 (d J = 2.3 Hz, 1 H), 7.34-7.24 (m, 2 H), 4.93 (poorly resolved ddj = 10.8, 1.7 Hz, 1 H), 4.73 (br t J = 8.8 Hz, 1 H), 4.36 (tj = 5.7 Hz, 2 H), 4.23-4.12 (m, 3 H), 4.10-3.96 (m, 3 H), 3.87-3.75 (m, 4 H), 3.65 (ddj = 10.9, 9.5 Hz, 1 H), 2.93 (tj = 5.7 Hz, 2 H), 2.73-2.63 (m, 4 H), 3 H not observed (obscured by water peak). HRMS (FAB) calc for C31H35 35ClN5O5 (MH+) ml ^ 592.2327, found 592.2318. For C31H35 37ClN5O5 (MH+) ml ^ 594.2297, found 594.2310.
Example 14. 2-{l-(Chloromethyl)-3- [5-(2-morphoϋnoethoxy)-ljyr-indole-2-carbonyl] -5-nitro- l,2-dihydro-3i£benzo[e]indole-7-carboxamido} ethyl dihydrogen phosphate hydrochloride (19) (Scheme F). A solution of amine 114 (218 mg, 0.86 mmol) in THF (3 mL) was added to an ice-cold mixture of acid 136 (290 mg, 0.72 mmol), DIPEA (0.38 mL, 2.16 mmol) and THF (7 mL), followed by the addition of pyBOP (486 mg, 0.93 mmol). The resulting mixture was stirred at 0 0C for a further 50 min. Cesium carbonate (470 mg, 1.44 mmol) and MeOH (4 mL) were added and the mixture was stirred at 0 0C for 30 min, then allowed to stand at 5 0C overnight. The mixture was partitioned between CH2Cl2 and cold water. The organic layer was separated and the aqueous
portion was further exacted with CH2Cl2 twice. The combined CH2Cl2 layers were dried (Na2SO4) and evaporated under reduced pressure at 25 0C (bath temperature) to give a crude product. This was purified by column chromatography (eluting with CH2Cl2:EtOAc 2:1) to give pure di-Λ?/7"-butyl 2-[l-(chloiOmethyl)-5-nitro-l,2-dihydro-3H-benzo[β]indole-7-carboxamido]ethyl phosphate (139) as a red solid (194 mg, 50%): 1H NMR [(CD3)2SO] δ 8.76 (tj = 5.5 Hz, 1 H), 8.58 (poorly resolved dj = 0.78 Hz, 1 H), 7.93 (ddj = 8.9, 1.5 Hz, 1 H), 7.91 (dj = 8.7 Hz, 1 H), 7.65 (s, 1 H), 4.26-4.18 (m, 1 H), 4.00 (qj = 6.1 Hz, 2 H), 3.91 (ddj = 11.0, 3.8 Hz, 1 H), 3.85 (ddj = 9.9, 2.2 Hz, 1 H), 3.77 (ddj = 11.0, 8.6 Hz, 1 H), 3.72 (ddj = 10.3, 2.9 Hz, 1 H), 3.52 (qj = 5.6 Hz, 2 H), 1.39 (s, 18 H). HRMS (FAB) calc for C24H33 35ClN3O7P (MH+) m/Z 541.1745, found 541.1743. CaIc for C24H33 37ClN3O7P (MH+) »/- 543.1715, found 543.1731.
To a stirred mixture of indoline 139 (143 mg, 0.26 mmol) and acid 122 (104 mg, 0.32 mmol) in DMA (5 mL) was added EDCLHCl (204 mg, 1.06 mmol), and anhydrous TsOH (9.3 mg, 0.053 mmol). The mixture was stirred at room temperature for 3 h then cooled in an ice bath. Cold aqueous NaHCO3 (5%, 10 mL) was added, followed by cold water (10 mL). The precipitated solid was filtered off, washed with water, and dried to give di-/«f-butyl 2-[l-(chloiOmethyl)-3-[5-(2- rnorpho]inoethoxy)-lH-indole-2-carbonyl]-5-nitro-l,2-dihydro-3H-benzo[^]indole-7- carboxamido] ethyl phosphate (140) as a yellowish orange solid (213 mg, 99%): mp 130-132 0C; 1H NMR (CDCl3) δ 9.34 (br s, 1 H), 9.24 (s, 1 H), 8.93 (poorly resolved dj = 1.3 Hz, 1 H), 8.20 (ddj = 8.8, 1.6 Hz, 1 H), 8.06 (tj = 4.9 Hz, 1 H), 7.92 (d,/ = 8.8 Hz, 1 H), 7.38 (dj = 8.9 Hz, 1 H), 7.14 (poorly resolved d,/ = 2.3 Hz, 1 H), 7.10-6.99 (m, 2 H), 4.91 (dd,/ = 10.8, 2.1 Hz, 1 H), 4.80 (br t,J = 9.7 Hz, 1 H), 4.38-4.13 (m, 5 H), 3.98 (ddj= 11.5, 3.3 Hz, 1 H), 3.86-3.72 (m, 6 H), 3.61 (dd,/ = 11.4, 9.5 Hz, 1 H), 2.87 (t,J = 5.6 Hz, 2 H), 2.70-2.58 (m, 4 H), 1.503 and 1.502 (2 s, 18 H). Anal. (C39H49ClN5O111POJH2O) Calc: C, 57.15; H, 6.10; N, 8.54. Found: C, 57.02; H, 6.06; N, 8.44.
To a stirred solution of 140 (197 mg, 0.242 mmol) in CH2Cl2 (10 mL) was added HCl (saturated solution in CH2Cl2, 1.0 mL). The mixture was stirred at 20 0C for 22 h. The mixture was concentrated under reduced pressure and EtOAc (50 mL) was added. The mixture was stirred at 0 0C for a further 2 h. The resulting solid was filtered off, washed with EtOAc several times, and dried to give crude 19 (172 mg, 96%). LC-MS analysis showed the crude product contained 85% 19 and 13% N-(2-cMoroethyl)-l-(chloromethyl)-3-[5-(2-moiphormoethoxy)-lH-indole-2-carbonyl]-5-nitro- l,2-dihydiO-3H-benzo[tf]mdole-7-carboxamide. The crude product was dissolved in the minimum amount of DMSO (3 mL) and CH2Cl2 (20 mL) was added slowly. The resulting mixture was stirred at 20 0C for 1 h. The precipitated solid was filtered off, washed repeatedly with CH2Cl2 and dried to give pure 19 (153 mg, 85%) as a yellowish orange hydrochloride salt (98.1% pure by HPLC), mp 225
0C (dec); 1H NMR [(CD3),SO] δ 11.80 (poorly resolved dj = 1.6 Hz, exchangeable with D2O, 1 H), ca. 12.0-9.7 (v br s, exchangeable with D2O, ca. 3 H), 9.17 (s, 1 H), 8.97 (tj = 5.5 Hz, exchangeable with D20, 1 H), 8.84 (poorly resolved dj = 1.3 Hz, 1 H), 8.32 (dj = 8.8 Hz, 1 H), 8.16 (ddj = 8.8, 1.5 Hz, 1 H), 7.46 (d,/ = 8.9 Hz, 1 H), 7.27 (poorly resolved d,J = 2.3 Hz, 1 H), 7.22 (poorly resolved d,J = 1.6 Hz, collapses to s after D2O exchange, 1 H), 4.96 (br t,J = 10.2 Hz, 1 H), 4.75- 4.58 (m, 2 H), 4.41 (tj = 4.9 Hz, 2 H), 4.20-3.77 (m, 8 H), 3.64-3.12 (m, partially obscured by water peak, revealed after D2O exchange, 8 H). Anal. (C31H33ClN5O10-HClH2O) CaIc: C, 49.22; H, 4.80; N, 9.26. Found: C, 49.14; H, 4.83; N, 9.17.
Example 15. (^-l-CChloromethy^-A^^Z-hydroxyethy^-S-IS-^-CZ- morpholinoethoxy)phenyl]acryloyl}-5-nitro-l,2-dihydfo-3iϊ-benzo[e]indole-7-carboxamide hydrochloride (6) (Scheme G). To a stirred mixture of indoline 137 (80 mg, 0.17 mmol) and acid 129 (65 mg, 0.21 mmol) in DMA (4 mL) was added EDCLHCl (132 mg, 0.69 mmol) and anhydrous TsOH (6.0 mg, 0.034 mmol). The mixture was stirred at 20 0C for 5 h and another batch of acid 129 (65 mg, 0.21 mmol), EDCLHCl (132 mg, 0.69 mmol) and anhydrous TsOH (6.0 mg, 0.034 mmol) were added. After a total reaction time of 21 h the reaction flask was cooled in an ice-bath and cold aqueous NaHCO3 (5%, 8 mL) was added, followed by water (8 mL). After stirring at 0 0C for 10 min the precipitated solid was filtered off, washed with cold water several times, and dried to give (£)-N-[2-(/«f-butyldimetliylsilyloxy)ethyl]-l-(chloromediyl)-3-{3-[4-(2- morpholmoethoxy)phenyl]acryloyl}-5-m1iO-l,2-dihydro-3H-benzo[ήindole-7-carboxamide (141) as a yellowish orange solid (121 mg, 97%): mp 164-165 0C; 1H NMR (CDCl3) δ 9.35 (br s, 1 H), 8.75 (poorly resolved dj = 1.3 Hz, 1 H), 8.12 (ddj = 8.8, 1.6 Hz, 1 H), 7.93-7.86 (m, 2 H), 7.58 (dj = 8.8 Hz, 2 H), 6.95 (d, / = 8.8 Hz, 2 H), 6.78-6.69 (m, 2 H), 4.63 (ddj = 10.8, 2.4 Hz, 1 H), 4.53 (t,/ = 9.7 Hz, 1 H), 4.33-4.25 (m, 1 H), 4.17 (t,/ = 5.7 Hz, 2 H), 3.97 (ddj= 10.4, 3.2 Hz, 1 H), 3.85 (t, / = 5.2 Hz, 2 H), 3.75 (poorly resolved tj = 4.7 Hz, 4 H), 3.69-3.56 (m, 3 H), 2.83 (tj = 5.7 Hz, 2 H), 2.59 (poorly resolved t, / = 4.7 Hz, 4 H), 0.93 (s, 9 H), 0.11 (s, 6 H). Anal. (C37H47ClN4O7Si) CaIc: C, 61.44; H, 6.55; N, 7.75. Found: C, 61.31; H, 6.66; N, 7.99.
To a stirred solution of silyl ether 141 (97 mg, 0.134 mmol) in CH2Cl2 (12 mL) was added HCl (1.25M in MeOH, 1.0 mL). The mixture was stirred at 20 0C for a further 2 h. EtOAc was added and the mixture was stirred for another 30 min. The resulting solid was filtered off, washed with EtOAc several times, and dried to give 6 as a yellowish orange solid (87 mg, 100%): mp 239-241 0C (dec); 1H NMR [(CD3)2SO] δ 10.81 (br s, 1 H), 9.22 (s, 1 H), 8.82-8.74 (m, 2 H), 8.24 (dj = 8.8 Hz, 1 H), 8.13 (ddj = 8.8, 1.5 Hz, 1 H), 7.84 (dj = 8.7 Hz, 2 H), 7.74 (dj= 15.3 Hz, 1 H), 7.18-7.05 (m, 3 H), 4.72-4.58 (m, 3 H), 4.48 (br s, 2 H), 4.09 (br d,J= 4.2 Hz, 2 H), 3.98 (br dj = 12.0, 2 H),
3.81 (br 1, / = 11.7 Hz, 2 H), 3.66-3.47 (m, 5 H), 3.40 (q, / = 5.9 Hz, 2 H, partially obscured by water peak), 3.26 (br s, 4 H, partially obscured by water peak, revealed on D2O exchange). Anal. (C31H33ClN4O7-HCrH2O) CaIc: C, 56.11; H, 5.47; N, 8.44. Found: C, 56.08; H, 5.42; N, 8.44.
Example 16. (E)-2-[l-(Chlorotnethyl)-3-{3-[4-(2-morpholinoethoxy)phenyl]actyloyl}-5-nitro- l,2-dihydro-3i£benzo[e]indole-7-carboxamido] ethyl dihydrogen phosphate hydrochloride (20) (Scheme G). To a stirred mixture of indoline 139 (176 mg, 0.33 mmol) and acid 129 (123 rag, 0.39 mmol) in DMA (4 raL) was added EDCLHCl (250 mg, 1.30 mmol) and anhydrous TsOH (10.5 mg, 0.06 mmol). The mixture was stirred at 20 0C for 4 h and another batch of acid 129 (123 mg, 0.39 mmol), EDCLHCl (250 mg, 1.30 mmol) and anhydrous TsOH (10.5 mg, 0.06 mmol) were added. After a total reaction time of 21 h the reaction flask was cooled in an ice-bath and cold aqueous NaHCO3 (5%, 12 mL) was added, followed by water (12 mL). After stirring at 0 0C for 10 min the precipitated solid was filtered off, washed with cold water several times, and dried to give (E)-di-^/f-butyl 2-[l-(chloromemyl)-3-{3-[4-(2-morpholinoethoxy)phenyl]acryloyl}-5-nitro-l,2- dihydro-3H-benzo[φndole-7-carboxamido]ethyl phosphate (142) as a yellowish orange solid (211 mg, 81%): mp 128-131 0C; 1H NMR (CDCl3) δ 9.30 (br s, 1 H), 8.90 (poorly resolved dj = 1.4 Hz, 1 H), 8.17 (ddj = 8.7, 1.4 Hz, 1 H), 8.01 (t, / = 4.6 Hz, 1 H), 7.94-7.86 (m, 2 H), 7.58 (dj = 8.7 Hz, 2 H), 6.96 (dj = 8.7 Hz, 2 H), 6.76 (dj = 15.2 Hz, 1 H), 4.62 (ddj = 10.8, 2.1 Hz, 1 H), 4.52 (br tj = 9.7 Hz, 1 H), 4.32-4.20 (m, 3 H), 4.17 (t,/= 5.7 Hz, 2 H), 3.96 (ddj = 11.4, 3.1 Hz, 1 H), 3.81 (qj = 4.6 Hz, 2 H), 3.74 (poorly resolved t,J - 4.6 Hz, 4 H), 3.59 (br tj = 10.6 Hz, 1 H), 2.84 (tj = 5.7 Hz, 2 H), 2.60 (poorly resolved tj = 4.5 Hz, 4 H), 1.50 (s, 18 H). Anal. (C39H511CIN4O111Py2H2O) CaIc: C, 57.81; H, 6.34; N, 6.91. Found: C, 57.51; H, 6.10; N, 6.95.
To a stirred solution of phosphate ester 142 (190 mg, 0.237 mmol) in CH2Cl2 (10 mL) was added HCl (saturated solution in CH2Cl2, 10 mL). The mixture was stirred at 20 0C for a further 18 h. The mixture was concentrated under reduced pressure and EtOAc was added. The mixture was stirred at 0 0C for another 2 h. The resulting solid was filtered off, washed with EtOAc several times, and dried to give 20 as a yellowish orange solid (147 mg, 86%): mp 229 0C (dec); 1H NMR [(CD3)2SO] δ ca.10.6 (v br s, 1 H), 9.23 (br s, 1 H), 8.97 (poorly resolved t,J = 4.7 Hz, 1 H), 8.82 (poorly resolved dj = 0.9 Hz, 1 H), 8.26 (dj = 8.8 Hz, 1 H), 8.14 (br dj= 8.7 Hz, 1 H), 7.83 (dj = 8.5 Hz, 2 H), 7.73 (d,/ = 15.3 Hz, 1 H), 7.12 (d,/ = 15.3 Hz, 1 H)5 7.08 (d,/ = 8.5 Hz, 2 H), 4.72-4.56 (m, 3 H), 4.39 (br s, 2 H), 4.12-3.95 (m, 4 H), 3.80 (br s, 4 H), 3.55 (qj = 5.6 Hz, 2 H), 3.37 (br s, 2 H, partially obscured by water peak, revealed on D2O exchange), 3.15 (br s, 4 H, partially obscured by water peak, revealed on D2O exchange). Anal. (C31H34ClN4O10P-HCrV4H2O) CaIc: C, 51.07; H, 4.89; N, 7.68. Found: C, 51.00; H, 4.90; N, 7.68.
Example 17. (5)-l-(Chloromethyl)-iV-(2-hydroxyethyl)-3- [5-(2-motpholinoethoxy)-liyr- indole-2-catbonyl]-5-nitro-l,2-dihydro-3iϊ-benzo[e]indole-7-sulfonaniide hydrochloride (7) (Scheme H and I). An ice-cold solution of amine 114 (3.39 g) 13.4 mmol) and DIPEA (2.10 mL, 11.8 mmol) in THF (17 mL) was added to solution of sulfonyl chloride 107 (5.12 g, 11.2 mmol) in THF (50 mL) at 0 0C. After the addition was complete the mixture was stirred at 0 0C for a further 20 min. Ice-water (400 mL) was added and the mixture was stirred for 15 min. The precipitated solid was filtered off, washed with water, and dried to give di(/^-butyl) 2-[l-(chloromethyl)-5-nitro-3- (trifluoroacetyl)-l,2-dihydro-3Jf-benzo[f]indole-7-sulfonamido] ethyl phosphate (143) as a yellow solid (7.19 g, 95%): mp 162 0C (dec); 1H NMR (CDCl3) δ 9.23 (s, 1 H), 9.01 (dj = 1.5 Hz, 1 H), 8.10 (ddj = 8.8, 1.5 H2, 1 H), 8.00 (dj = 8.8 Hz, 1 H), 6.10 (tj = 5.5 Hz, 1 H), 4.72 (dj = 11.5 Hz, 1 H), 4.57 (ddj = 11.4, 8.7 Hz, 1 H), 4.41-4.31 (m, 1 H), 4.13-4.03 (m, 2 H), 3.94 (ddj = 11.6, 3.4 Hz, 1 H), 3.71 (ddj = 11.6, 7.9 Hz, 1 H), 3.33 (qj = 5.5 Hz, 2 FI), 1.45 and 1.46 (2 s, 18 H). Anal. (C25H32ClF3N3O9PS-V4THF) CaIc: C, 45.13; H, 4.95; N, 6.07. Found: C, 45.19; H, 4.97; N, 6.21.
Compound 143 (3.68 g) was resolved by preparative chiral HPLC (Daicel Chiralpak IA 250 x 21 mm column, EtOH:hexane 40:60, 7 mL/min, run time 27 min, α 1.30). The sample was dissolved in a mixture of MeOH and EtOH and then diluted with hexane, and 55-60 mg portions were resolved per injection. The product-containing fractions were combined and evaporated to give (B.)-di-/«f- butyl 2-(l-(chloromethyl)-5-nitro-3-(trifluoiOacetyl)-l,2-dihydro-3H-benzo[^]indole-7- sulfonamido)ethyl phosphate (144) as a yellowish orange solid (1.425 g, 38.7%): mp 153-154 0C (EtOAc/petroleum ether); [oc]D 20 +28° (c 1.0, CFICl3); ee 100% (Daicel Chiralpak IA 150 x 4.6 mm column, EtOH:hexane 40:60, 0.5 mL/min, R1- 13.54 min); 1H NMR (CDCl3) identical to that described for 143. Anal. (C25H32ClF3N3O9PSO.1 hexane) CaIc: C, 45.04; H, 4.93; N, 6.16. Found: C, 45.09; H, 4.94; N, 6.28. The structure of phosphate 144 was confirmed by X-ray crystallography, see Figure 1. and
(^-di-Λπ^buty^-fl-^hloromethy^-S-nitxo-S^trifluoroacety^-l^-clihydro-SH-benzo^jindole-?- sulfonamido] ethyl phosphate (145) as a yellowish orange solid (1.423 g, 38.7%): mp 154-155 0C (EtOAc/petroleum ether); [α]D 20 -28° (c 1.0, CHCl3); ee 100% (Daicel Chiralpak IA 150 x 4.6 mm column, EtOH:hexane 40:60, 0.5 mL/min, R1. 17.47 min); 1H NMR (CDCl3) identical to that described for 143. Anal. (C25H32ClF3N3O9PS) CaIc: C, 44.55; H, 4.79; N, 6.23. Found: C, 44.49; H, 4.93; N, 6.19. The structure of phosphate 145 was confirmed by X-ray crystallography, see Figure 2.
Cold THF (32 tnL) was added to solid trifluroacetamide 144 (1.38 g, 2.05 tnmol) in a flask immersed in an ice-bath. To this stirred cold mixture was added solid cesium carbonate (1.33 g, 4.1 mmol) followed by slow addition of cold MeOH (8 mL). The mixture was stirred for 10 min and then partitioned between EtOAc and ice-water. The EtOAc layer was separated and the aqueous layer was extracted with EtOAc once more. The combined EtOAc extracts was dried (Na2SO4) and evaporated to give (R)-di-te/i-butyl 2-[l-(chloiOmethyl)-5-nitro-l,2-dihydiO-3H-benzo[έ']indole-7- sulfonamido] ethyl phosphate (146) as a red foamy solid (1.19 g, 100%): mp 94-96 0C (EtOAc); [α]D 2υ -27° (c 1.0, CHCl3); ee 100% (Daicel Chiralpak IA 150 x 4.6 mm column, EtOH:hexane 40:60, 0.5 mL/min, Rτ 14.92 min); 1H NMR (CDCl3) δ 8.92 (poorly resolved d J = 1.5 Hz, 1 H), 7.91 (dd, / = 8.9, 1.8 Hz, 1 H), 7.79 (ddj = 8.9, 0.33 Hz, 1 H), 7.74 (s, 1 H), 5.71 (t,/ = 5.8 Hz, 1 H)3 4.40 (s, 1 H), 4.18-3.91 (m, 5 H), 3.78 (ddj = 11.2, 3.5 Hz, 1 H), 3.59 (dd, / = 11.1, 10.0 Hz, 1 H), 3.35-3.24 (m, 2 H), 1.46 and 1.45 (2 s, 18 H). Anal. (C23H33ClN3O8PS) CaIc: C, 47.79; H, 5.75; N, 7.27. Found: C, 47.59; H, 5.79; N, 6.96.
Trifluoroacetamide 145 (1.38 g, 2.05 tnmol) was deprotected to the corresponding indoline by the same procedure to give (J)-di-/OTf-butyl 2-[l-(chloromethyl)-5-nitiO-l,2-dihydro-3f/-benzo[^]indole- 7-sulfonamido] ethyl phosphate (147) as a red foamy solid (1.19 g, 100%): mp 89-92 0C (EtOAc); [α]D 2u +29° (c 1.0, CHCl3); ee 100% (Daicel Chiralpak IA 150 x 4.6 mm column, EtOH:hexane 40:60, 0.5 mL/min, Rx 17.78 min); 1H NMR (CDCl3) identical to that described for 165. Anal. (C23H33ClN3O8PS) CaIc: C, 47.79; H, 5.75; N, 7.27. Found: C, 48.23; H, 5.79; N, 7.01.
Compound 115 (4.26 g) was resolved by preparative chiral HPLC (Daicel Chiralpak IA 250 x 21 mm column, EtOH:hexane 40:60, 7 mL/min, run time 24 min, α 1.18). The sample was dissolved in EtOH:hexane (40:60) and 80 mg portions were resolved per injection. The product-containing fractions were combined and evaporated to give R-enantiomer 146 (1.91 g, 44.8%), ee 100% (analytical HPLC conditions as described above); and ^-enantiomer 147 (1.93 g, 45.3%), ee 100% (analytical HPLC conditions as described above).
A solution of 147 (174 mg, 0.30 mmol) in CH2Cl2 (15 mL) was treated with TFA (1.5 mL) at 20 0C for 30 min. The mixture was evaporated under reduced pressure to give a thick yellow oil. To the oil was added water (225 mL) and acid phosphatase (wheat germ, 0.5 units/mg, 300 mg). The mixture (pH ca. 5.5) was stirred at 20 0C for 23 h and then extracted with EtOAc (2 x 300 mL). The combined extracts were washed with water, dried (Na2SO4), and evaporated under reduced pressure at 35 0C (bath temperature) to give (J)-l-(cliloromethyl)-JV-(2-hydroxyethyl)-5-nitro-l,2-dihydro- 3H-benzo[<?]indole-7-sulfonamide (148) as a red solid (105 mg, 91%): mp 170-171 0C
(EtOAc/MeOH); [oc]D 2" +90.5° (c 0.22, EtOAc); ee 100% (Daicel Chiralpak IA 150 x 4.6 mm column, EtOH:hexane 60:40, 0.5 mL/min, Rx 20.88 min); 1H NMR [(CD3)2SO] δ 8.57 (poorly resolved dj = 1.6 Hz, 1 H), 8.03 (d,/ = 8.9 Hz, 1 H), 7.80 (dd,/ = 8.9, 1.8 Hz, 1 H), 7.77 (s, 1 H), 7.69 (tj = 5.9 Hz, 1 H), 6.73 (s, 1 H), 4.63 (tj = 5.6 Hz, 1 H), 4.29-4.20 (m, 1 H), 3.96-3.71 (m, 4 H), 3.37 (br qj = 6.1 Hz, 2 H), 2.81 (br q J = 6.2 Hz, 2 H). Anal. (C15H16ClN3O5S) CaIc: C, 46.70; H, 4.18; N, 10.89. Found: C, 46.88; H, 4.27; N, 10.64.
To a stirred solution of 148 (107 mg, 0.32 mmol) in DMF (5 mL) was added &/?-butyldimethylsilyl chloride (84 mg, 0.55 mmol), followed by DIPEA (0.10 mL, 0.58 mmol). The mixture was stirred at 20 0C for 45 min and then partitioned between EtOAc and cold water. The ethyl acetate layer was separated and washed with cold 2% Na2CO3 and water. The organic layer was dried (Na2SO4) evaporated to give (J)-JV-[2-(/CTf-butyldirnei±ιylsπyloxy)ethyl]-l-(chloroniethyl)-5-nitro-l,2-diliydro- 3H-benzo[<?]indole-7-sulfonamide (149) as a red solid (134 mg, 100%): 1H NMR (CDCl3) identical to that described for 112.
To a stirred mixture of 149 (134 mg, 0.27 mmol) and acid 122 (132 mg, 0.40 mmol) in DMA (5 mL) was added EDCLHCl (308 mg, 1.61 mmol) and anhydrous TsOH (14.0 mg, 0.08 mmol). The mixture was stirred at room temperature for 2 h and another batch of acid 122 (28 mg, 0.084 mmol), EDCLHCl (51 mg, 0.27 mmol), and anhydrous TsOH (2.3 mg, 0.013 mmol) was added. The reaction mixture was stirred for a further 1 h 30 min, then cooled in an ice bath. Cold aqueous NaHCO3 (5%, 10 mL) was added, followed by cold water (10 mL). The precipitated solid was filtered off, washed with water then hexane, and dried to give (J)-N-[2-(/^- butyldinieiiiylsilyloxy)emyl]-l-(chlorometiiyl)-3-[5-(2-iταoiphoHnoemoxy)-lH-indole-2-carbonyl]-5- nitro-l,2-dihydro-3H-benzo[^indole-7-sulfonamide (150) as a yellowish orange solid (198 mg, 95%): mp 179-181 0C; [oc]D 20 +22.5° (c 0.71, CHCl3); 1H NMR [(CDCl3] δ 9.36 (s, 1 H), 9.33 (br s, 1 H), 9.02 (poorly resolved d,J = 1.4 Hz, 1 H), 8.05 (ddj = 8.8, 1.6 Hz, 1 H), 8.00 (dj = 8.8 Hz, 1 H), 7.39 (d,/ = 8.9 Hz, 1 H), 7.14 (poorly resolved dj = 2.0 Hz, 1 H), 7.12-7.03 (m, 2 H), 5.01 (t,/ = 5.8 Hz, 1 H), 4.93 (ddj = 10.8, 2.1 Hz, 1 H), 4.83 (br tj = 9.8 Hz, 1 H), 4.41-4.30 (m, 1 H), 4.18 (t, / = 5.7 Hz, 1 H), 3.96 (ddj = 11.5, 3.2 Hz, 1 H), 3.83-3.60 (m, 7 H), 3.23-3.14 (m, 2 H), 2.86 (tj = 5.7 Hz, 2 H), 2.68-2.56 (m, 4 H), 0.84 (s, 9 H), 0.005 and 0.002 (2 s, partially obscured by TMS, 6 H). Anal. (C36H46CIN5O8SSiO.3hexane) CaIc: C, 56.88; H, 6.34; N, 8.77. Found: C, 56.71; H, 6.47; N, 8.66.
To a filtered solution of 150 (189 mg, 0.245 mmol) in CH2Cl2 (15 mL) was added HCl (1.25M in MeOH, 1.5 mL) and the mixture was stirred at 20 0C for 1 h 15 min. The mixture was diluted with
EtOAc (100 mL) and the precipitated solid was filtered off, washed with EtOAc several times, and dried to give 7 as a yellowish orange hydrochloride salt (144 mg, 85%): mp 220 0C (dec); [oc]D 2" . +32.0° (c 0.17, MeOH); 1H NMR [(CD3)2SO] δ 11.82 (br s, exchangeable with D2O, 1 H), 10.63 (br s, exchangeable with D2O, 1 H), 9.28 (s, 1 H), 8.86 (poorly resolved dj = 1.6 Hz, 1 H), 8.44 (dj = 8.9 Hz, 1 H), 8.03 (ddj = 8.9, 1.7 Hz, 1 H), 7.94 (t,/ = 5.9 Hz, exchangeable with D2O, 1 H), 7.47 (dj = 8.9 Hz, 1 H), 7.28 (poorly resolved d,J = 2.2 Hz, 1 H), 7.24 (poorly resolved dj = 1.7 Hz, collapses to s after D2O exchange, 1 H), 7.05 (dd, / = 8.9, 2.4 Hz, 1 H), 4.97 (br t,J = 10.2 Hz, 1 H), 4.77-4.61 (m, 2 H), 4.49-4.39 (m, 2 H), 4.22-4.08 (m, 2H), 4.06-3.49 (m, 8 H), 3.47-3.17 (m, partially obscured by water peak, revealed after D2O exchange, 4 H), 2.87 (q,/ = 6.1 Hz, collapses to t after D2O exchange, 2 H), one H not observed. Anal. (C30H32ClN5O8S HQH2O) CaIc: C, 50.57; H, 4.95; N, 9.83. Found: C, 50.55; H, 5.19; N, 9.98.
Example 18. (5)-5-Amino-l-(chlor omethylJ-iV^-hydr oxyethyl)-3- [5-(2-morpholinoethoxy)- lϋ£indole-2-carbonyl] ~l,2-dihydro-3iϊ-benzo [e] indole-7-sulfonamide hydrochloride (14) (Scheme I). To a solution of nitro compound 7 (78 mg, 0.112 mmol) in acetone (6 mL) and water (4 mL) stirred under nitrogen at 20 0C was added ammonium chloride (312 ing, 5.8 mmol) and Zn powder (234 mg, 3.6 mmol). The heterogeneous mixture was stirred at 20 0C for 1 h 30 min. Sufficient cold aqueous Na,CO3 (2%) was added to generate pH 8-9 and then the product was extracted with CH2Cl2 (15 x 10OmL). The combined CH2Cl2 extracts were washed with water, dried (Na2SO4), and concentrated under reduced pressure to ca. 10 mL in volume. HCl (1.25M in MeOH, 1.0 mL) was added followed by EtOAc (60 mL). The mixture was left to stand at 5 0C overnight. The precipitated solid was filtered off, washed with EtOAc, and dried to give 14 as greenish beige solid (60 mg, 81%): mp 230 0C (dec); [α]D 20 +17.6° (c 0.17, MeOH); 1H NMR [(CD3)2SO] δ 11.67 (poorly resolved d,J — 1.7 Hz, exchangeable with D2O, 1 H), 10.70 (br s, exchangeable with D2O, 1 H), 8.55 (poorly resolved d J = 1.6 Hz, 1 H), 7.95 (d, / = 8.9 Hz, 1 H), 7.86 (s, 1 H), 7.76 (dd, / = 8.9, 1.7 Hz, 1 H), 7.49 (poorly resolved tj = 5.7 Hz, exchangeable with D2O, 1 H), 7.45 (dj = 8.9 Hz, 1 H), 7.27 (poorly resolved d, / = 2.3 Hz, 1 H), 7.12 (poorly resolved d,/ = 1.7 Hz, collapses to s after D2O exchange, 1 H), 7.01 (ddj = 8.9, 2.4 Hz, 1 H), 4.77 (ddj = 10.7, 9.1 Hz, 1 H), 4.53 (dd, /= 10.9, 1.7 Hz, 1 H), 4.43 (poorly resolved tj = 4.9 Hz, 2 H), 4.24-4.16 (m, 1 H), 4.06-3.61 (m, partially obscured by water peak, revealed after D2O exchange, 6 H), 3.58-3.06 (m, partially obscured by water peak, revealed after D2O exchange, 8 H), 2.84 (br q, / = 6.0 Hz, collapses to t after D2O exchange,/ = 6.0 Hz, 2 H). HRMS (FAB) calc for C30H35 35ClN5O6S (MH+) m/z 628.1997, found 628.1996. For C30H35 37ClN5O6S (MH+) m/% 630.1967, found 630.1974.
Example 19. (5)-2-[l-(Chloromethyl)-3-[5-(2-morpholinoethoxy)-liy-indole-2-catbonyl]-5- nitt o-l,2-dihydro-3//-benzo [e] indole-7-sulfonamido] ethyl dihydrogen phosphate trifluoroacetate (21) (Scheme I). To a stirred mixture of 147 (1.93 g, 3.35 mmol) and acid 122 (1.38 g, 4.22 mmol) in DMA (70 mL) was added EDCLHCl (1.29 g, 6.70 mmol) and anhydrous TsOH (117 mg, 0.67 mmol). The mixture was stirred at 20 0C for 2 h and another batch of EDCI.HC1 (1.29 g, 6.70 mmol) and anhydrous TsOH (59 mg, 0.34 mmol) was added. The reaction mixture was stirred for a further 3 h, then cooled in an ice bath. Cold aqueous NaHCO3 (5%, 140 mL) was added, followed by cold water (140 mL). The precipitated solid was filtered off, washed with water, and dried to give (S)-όi-fert-butyl 2-[l-(chloromethyl)-3-[5-(2-morpholinoethoxy)-lH- indole-2-carbonyl]-5-mtro-l,2-dihydro-3H-benzo[g]indole-7-sulfonamido]ethyl phosphate (151) as a yellowish orange solid (2.70 g, 95%): mp 219 0C; [oc]D 20 +20.1° (c 0.70, CHCl3); 1H NMR [CDCl3] δ 9.37 (br s, 1 H), 9.33 (s, 1 H), 8.99 (poorly resolved dj = 1.5 Hz, 1 H), 8.05 (ddj = 8.8, 1.7 H2, 1 H), 7.98 (dj = 8.8 Hz, 1 H), 7.38 (dj = 8.9 Hz, 1 H), 7.14 (poorly resolved d,J = 2.2 Hz5 1 H), 7.10-7.06 (m, 2 H), 6.10-5.97 (m, 1 H), 4.92 (ddj = 10.8, 2.2 Hz, 1 H), 4.82 (br t,J- 9.8 Hz, 1 H), 4.39-4.30 (m, 1 H), 4.18 (t,/ = 5.7 Hz, 2 H), 4.13-4.03 (m, 2 H), 3.96 (ddj = 11.5, 3.3 Hz, 1 H), 3.76 (poorly resolved t,/ = 4.7 Hz, 4 H), 3.66 (dd,/ = 11.5, 9.1 Hz, 1 H), 3.39-3.29 (m, 2 H), 2.86 (t, / = 5.7 Hz, 2 H), 2.62 (poorly resolved t, / = 4.5 Hz, 4 H), 1.47 and 1.46 (2 s, 18 H). Anal. (C38H49ClN5OnPS V2H2O) CaIc: C, 53.11; H, 5.87; N, 8.15. Found: C, 53.03; H, 5.90; N, 8.20.
TFA (5 mL, 63 mmol) was added to a filtered solution of phosphate ester 151 (2.70 g, 3.18 mmol) in CH2Cl2 (50 mL) and the resulting solution was stirred at 20 0C for 3 h. EtOAc (500 mL) was added, the mixture was stirred for a further 3 h, and then left to stand at 5 0C overnight. The precipitated solid was filtered off, washed with EtOAc several times and dried to give 21 as a yellowish orange solid (2.63 g, 97%): mp 210 0C (dec); [α]D 20 +19.7° (c 0.20, DMSO); 1H NMR [(CD3),SO] δ 11.75 (br s, 1 H), 9.27 (s, 1 H), 8.86 (dj = 1.5 Hz, 1 H), 8.42 (dj = 8.9 Hz, 1 H), 8.23 (br s, 1 H), 8.02 (ddj = 8.9, 1.5 Hz, 1 H), 7.41 (dj = 8.9 FIz, 1 H), 7.21 (br s, 2 H), 6.96 (ddj = 8.9, 1.3 Hz, 1 H), 4.94 (br t,J = 10.1 Hz, 1 H), 4.76-4.57 (m, 2 H)3 4.22 (poorly resolved tj = 5.2 Hz, 2 H), 4.19-4.05 (m, 2 H), 3.86-3.66 (m, partially obscured by water peak, 6 H), 3.14-2.78 (m, partially obscured by water peak, 8 H), 3 H not observed, obscured by water peak δ 3.5-3.3. Anal.
(C30H33ClN5O11PSO7CF3CO,H-1/2H2O) CaIc: C, 45.61; FI, 4.23; N, 8.47; Cl, 4.29; F, 4.83. Found: C, 46.00; H, 4.35; N, 8.04; Cl, 4.20; F, 4.67.
Example 20. (S,E)-2- [l-(Chloromethyl)-3-{3- [4-(2-morpholinoethoxy)phenyl] acryloyl}-5- nitro-l,2-dihydro-3i£benzo[e] indole-7-sulfonamido] ethyl dihydrogen phosphate trifluofoacetate (22) (Scheme J). To a stirred mixture of indoline 147 (116 mg, 0.20 mmol) and
acid 129 (79.0 mg, 0.25 mmol) in DMA (4 mL) was added EDCLHCl (154 mg, 0.80 mmol) and anhydrous TsOH (7.0 mg, 0.04 mmol). The mixture was stirred at 20 0C for 1 h 30 min and another batch of acid 129 (79.0 mg, 0.25 mmol), EDCI.HC1 (154 mg, 0.80 mmol) and anhydrous TsOH (7.0 mg, 0.04 mmol) were added. After a total reaction time of 18 h, the reaction flask was immersed in an ice-bath and cold aqueous NaHCO3 (5%, 8 mL) was added, followed by water (8 mL). The mixture was stirred at 0 0C for 10 min, then the precipitated solid was filtered off, washed with water then petroleum ether, and dried to give (S,E)-di-ϊert-butyl 2-[l-(chloromethyl)-3-{3-[4-(2- morpholinoethoxy)phenyl] acryloyl} -5-nitro-l ,2-dihydro-3H-benzo [e]indole-7-sulfonamido] ethyl phosphate (152) as a yellowish orange solid (155 mg, 93%): mp 255 0C (dec); [α]D 2u -6.5° (c 0.62, CHCl3); ee 100% (Daicel Chiralpak IA 150 x 4.6 mm column, EtOH:acetonitrile:dietlτ.ylamine 35:65:0.1, 0.6 mL/min, Rτ 13.82 min); 1H NMR (CDCl3) δ 9.41 (br s, 1 H), 8.97 (poorly resolved d,J = 1.5 Hz, 1 H), 8.04 (ddj = 8.9, 1.6 Hz, 1 H), 7.94 (dj = 8.9 Hz, 1 H), 7.90 (dj= 15.2 Hz, 1 H), 7.57 (dj = 8.7 Hz, 2 H), 6.96 (d,/ = 8.7 Hz, 2 H), 6.74 (d,/ = 15.2 Hz, 1 H), 5.92 (t,/ = 5.4 Hz, 1 H), 4.63 (br ddj= 10.7, 2.3 Hz, 1 H), 4.55 (br t,J= 9.7 Hz, 1 H), 4.34-4.24 (m, 1 H), 4.17 (t,J = 5.7 Hz, 2 H), 4.13-4.02 (m, 2 H), 3.95 (br ddj = 11.4, 3.2 Hz, 1 H), 3.75 (poorly resolved tj = 4.7 Hz, 4 H), 3.62 (ddj = 11.4, 9.4 Hz, 1 H), 3.32 (poorly resolved qj = 5.0 Hz, 2 H), 2.83 (tj = 5.7 Hz, 2 H), 2.59 (poorly resolved tj = 4.6 Hz, 4 H), 1.46 and 1.45 (2 s, 18 H). Anal. (C38H5nClN4O11PS) CaIc: C, 54.51; H, 6.02; N, 6.69. Found: C, 54.29; H, 5.97; N, 6.71.
TFA (2 mL, 25 mmol) was added to a filtered solution of phosphate ester 152 (149 mg, 0.18 mmol) in CH2Cl2 (20 mL) and the resulting solution was stirred at 20 0C for 1 h 30 min. EtOAc (80 mL) was added, the mixture was stirred for a further 2 h, and then left to stand at 5 0C overnight. The precipitated solid was filtered off, washed with EtOAc several times, and dried to give 22 as a yellowish orange solid (105 g, 71%): mp 213 0C (dec); [α]D 2n +38.5° (c 0.21, DMSO); 1H NMR (C5D5N) δ ca. 10.6 (v br s, 1 H), 9.81 (br s, 1 H), 9.45 (poorly resolved dj = 1.4 Hz, 1 H), 8.40 (poorly resolved ddj = 8.8, 1.5 Hz, 1 H), 8.21 (dj = 15.2 Hz, 1 H), 8.14 (dj = 8.8 Hz, 1 H), 7.73 (dj = 8.7 Hz, 2 H), 7.16 (dj - 15.2 Hz, 1 H), 7.10 (dj = 8.7 Hz, 2 H), 4.80 (poorly resolved ddj = 10.8, 2.5 Hz, 1 H), 4.65 (br
10.0 Hz, 1 H), 4.58-4.42 (m, 3 H), 4.22-4.12 (m, 3H), 4.00 (br dd,/= 11.3, 8.3 Hz, 1 H), 3.78-3.66 (m, 6 H), 2.75
5.7 Hz, 2 H), 2.57-2.48 (m, 4 H), 3 H not observed, obscured by water peak. Anal. (C30H34CIN4O11PSUSCF3CO2H) CaIc: C, 46.50; H, 4.30; N, 6.86. Found: C, 46.50; H, 4.64; N, 7.00.
Example 21. l-(Bromomethyl)-7V-(2-hydroχyethyl)-3- [5-(2-morpholinoethoxy)-lJyr-indole-2- carbonyl]-5-nitro-l,2-dihydfo-3if-benzo[e]indole-7-sulfonamide (8) (Scheme K and L). A solution of NaI (7.19 g, 47.9 mmol) in 2-butanone (30 mL) was heated at 85 0C for 1 h. Indoline 105
(5.0 g, 16.0 mmol) was added and the mixture was heated at 85 0C for a further 17 h. EtOAc and water were added and the organic layer was separated. The organic layer was washed successively with aqueous sodium disulfite (10%), water, and brine, and then dried (Na2SO4) and evaporated. The residue was dissolved in EtOAc and the solution was diluted with petroleum ether to give 1- (iodomethyl)-3-(trifluoroacetyl)-l,2-clmydro-3H-benzo[?]indole (153) as a cream powder (4.91 g, 76%): mp 132-135 0C; 1H NMR (CDCl3) δ 8.42 (dj = 9.0 Hz, 1 H), 7.92 (dj = 7.9 Hz, 1 H), 7.89 (d,/ = 8.9 Hz, I H), 7.77 (dJ = 8.4 Hz, 1 H), 7.58 (td J = 8.1, 1.1 Hz, 1 H), 7.48 (tdJ = 8.1, 1.1 Hz, 1 H), 4.49 (dj = 11.5 Hz, 1 H), 4.43 (ddj = 11.5, 8.2 Hz, 1 H), 4.21 (ttj = 9.8, 2.3 Hz, 1 H), 3.65 (ddj = 10.5, 2.3 Hz, 1 H), 3.23 (tj = 10.2 Hz, 1 H). Anal. (C15H11F3INO) CaIc: C, 44.47; H, 2.74; N, 3.46. Found: C, 44.74; H, 3.04; N, 3.41.
A solution of iodide 153 (3.6 g, 8.89 mmol) and AgOMs (10.8 g, 53.3 mmol) in CH3CN (50 mL) was stirred in the dark for 24 h. The CH3CN was removed under reduced pressure, EtOAc was added to the residue, and the resulting mixture was filtered through Celite. The filtrate was evaporated and the crude product was recrystallised (EtOAc/petroleum ether) to give (3-trifluoroacetyl-l,2-dihydro-3H- benzo[<?]indol-l-yl)methyl methanesulfonate 154 as colourless crystals (3.15 g, 95%): mp 122-1240C; 1H NMR (CDCl3) δ 8.43 (dj = 9.0 Hz, 1 H), 7.91 (dj = 7.8 Hz, 1 H), 7.88 (dj = 8.8 Hz, 1 H), 7.83 (ddj = 8.3, 0.5 Hz, 1 H), 7.61 (tdj = 8.2, 1.2 Hz, 1 H), 7.51 (tdj = 8.1, 1.1 Hz, 1 H), 4.65 (ddj = 3.8, 0.6 Hz, 1 H), 4.59 (dtj = 11.5, 1.4 Hz, 1 H), 4.42 (ddj = 11.5, 8.2 Hz, 1 H), 4.29 (tdj = 8.2, 3.4 Hz, 1 H), 4.14 (ddj = 10.6, 9.1 Hz, 1 H), 2.95 (s, 3 H). Anal. (C16H14F3NO4S) CaIc: C, 51.47; H, 3.78; N, 3.75. Found: C, 51.77; H, 3.82; N, 3.86.
A solution of mesylate 154 (8.9 g, 23.9 mmol) and LiBr (41.5 g, 477 mmol) in THF (100 mL) was stirred in the dark for 36 h. EtOAc was added and the solution was washed with water and brine and then dried (Na2SO4) and evaporated. The crude product was recrystallised (MeOH/water) to give l-(biOmomethyl)-3-(trifluoroacetyl)-l,2-dihydro-3H-benzo[^]indole (155) as colourless crystals (8.1 g, 95%): mp 152-154 0C; 1H NMR (CDCl3) δ 8.43 (dj= 9.0 Hz, 1 H), 7.92 (dj= 9.1 Hz, 1 H), 7.88 (dj = 9.3 Hz, 1 H), 7.79 (dj = 8.3 Hz, 1 H), 7.58 (tdj = 8.1, 1.2 Hz, 1 H), 7.49 (tdj = 8.1, 1.1 Hz, 1 H), 4.62 (dtj = 11.5, 1.4 Hz, 1 H), 4.43 (ddj = 11.4, 8.5 Hz, 1 H), 4.25 (tj = 9.1 Hz, 1 H), 3.84 (dddj = 10.7, 3.2, 0.8 Hz, 1 H), 3.39 (t J = 10.2 Hz, 1 H). Anal. (C15H11BrF3NO) CaIc: C, 50.30; H, 3.10; N, 3.91. Found: C, 50.58; H, 3.07; N, 3.92.
A solution of 105 (500 mg, 1.60 mmol) and LiBr (2.19 g, 26.39 mmol) was heated at 85 0C in 2- butanone (15 mL) for 9 days. EtOAc and water were added and the organic layer was separated. The organic layer was washed with water and brine and then dried (Na2SO4) and evaporated. The residue
was purified by flash column chromatography (eluting with petroleum etherrEtOAc 100:0 then 99.5:0.5) to give 155 (333 mg, 55%) contaminated with 105 (37 mg, 10%). Attempted recrystallisation (EtOAc/petroleum ether) failed to remove 105.
A solution of mesylate 154 (1.40 g, 3.75 mmol) in CH2Cl2 (5 mL) was added dropwise to a solution Of ClSO3H (1.74 g, 15.0 mmol) in CH2Cl2 (20 mL) at -78 0C. The stirred mixture was allowed to warm to 15 0C over 12 h producing a grey precipitate. The mixture was then cooled to 0 0C and the minimum quantity of DMF was slowly added to dissolve the precipitate. Oxalyl chloride (1.5 mL) was added dropwise, the mixture was stirred at 0 0C for 1 h, and then poured into ice water. The mixture was extracted with cold EtOAc and the organic layer was washed with cold water, cold brine, and then dried (Na2SO4). The solution was filtered through a plug of Celite/silica gel and the filtrate was evaporated. The crude product was dissolved in CH2Cl2 and the solution was diluted with /-Pr2O to precipitate ^-(chlorosulfony^-S-^fluoroacety^-l^-dihydro-SH-benzo^Jindol-l-ylJrnethyl methanesulfonate (156) as a white powder (1.64 g, 94%): 1H NMR [(CD3)2SO] δ 8.32 (dj = 8.9 Hz, 1 H), 8.21 (dj = 1.4 Hz, 1 H), 8.06 (d,/= 9.0 Hz, 1 H), 8.00 (dj = 8.7 Hz, 1 H), 7.81 (ddj = 8.7, 1.6 Hz, 1 H), 4.58 (ddj = 10.0, 3.2 Hz, 1 H), 4.58-4.54 (m, 1 H), 4.49-4.36 (m, 3 H), 3.05 (s, 3 H). HRMS (FAB) calc for C16H13 35ClF3NO6S2 (MH+) w/ξ 470.9825, found 470.9824. For C16H13 37ClF3NO6S2 (MH+) ml ^ 472.9795, found 472.9801.
A solution of indoline 155 (7.50 g, 21.0 mmol) in CH2Cl2 (40 mL) was added dropwise to a solution of ClSO3H (9.72 g, 83.8 mmol) in CH2Cl2 (40 mL) at -78 0C. The stirred mixture was allowed to warm to 5 0C over 7 h producing a grey precipitate. The mixture was kept at 5 0C overnight and then cooled to 0 0C. The minimum quantity of DMF was slowly added to dissolve the precipitate and then oxalyl chloride (4 mL) was added dropwise. After stirring at 0 0C for 1 h the mixture was poured into ice water and extracted with cold EtOAc. The organic layer was washed with cold water, cold brine, and then dried (Na2SO4). The solution was filtered through a plug of Celite/silica gel and the filtrate was evaporated. The crude product was dissolved in CH2Cl2 and the solution was diluted with /-Pr2O to precipitate l-(bromomethyl)-3-(trifluoroacetyl)-l,2-dihydro-3H- benzo|>]indole-7-sulfonyl chloride (157) as a white powder (8.88 g, 93%): mp 194-196 0C; 1H NMR (CDCl3) δ 8.65 (dj = 7.5 Hz, 1 H), 8.63 (dj = 2.0 Hz, 1 H), 8.11 (dj= 9.0 Hz, 1 H), 8.09 (ddj = 9.0, 2.0 Hz, 1 H), 7.99 (dj = 9.0 Hz, 1 H), 4.67 (dtj = 11.7, 1.6 Hz, 1 H), 4.52 (dd,/ = 11.4, 8.7 Hz, 1 H), 4.32 (ttj = 8.7, 2.6 Hz, 1 H), 3.80 (ddj = 10.8, 3.3 Hz, 1 H), 3.47 (ddj = 10.8, 9.1 Hz, 1 H). Anal. (C15H10BrClF3NO3SO-Ii-Pr2O) Calc: C, 40.13; H, 2.46; N, 3.00. Found: C, 40.17; H, 2.27; N, 2.97.
A solution of mesylate 156 (80 mg, 0.17 mmol) and LiBt (400 mg, 4.60 mmol) in THF (8 mL) was stirred at 20 0C for 4 days. EtOAc and water were added and the organic layer was separated and then washed with water and brine. The organic layer was dried (Na2SO4) and evaporated and the residue was purified by flash column chromatography (eluting with petroleum etherrEtOAc 100:0 to 4:1) to give 157 (36 mg, 47%).
An ice-cold solution of IdSfO3 (117 mg, 1.16 mmol) in 98% H2SO4 (1 mL) was added dropwise to a solution of 157 (440 mg, 0.97 mmol) in 98% H2SO4 (40 mL) at -12 0C. After 15 min the mixture was poured into ice water and extracted with cold EtOAc. The organic layer was washed with cold water, cold brine, and then dried (Na2SO4) and evaporated. The residue was purified by flash column chromatography (petroleum ether:EtOAc 100:0 to 7:3) to give l-(bromomethyl)-5-nitro-3- (tafluoroacetyl)-l,2-dihydro-3H-benzo[>]indole-7-sulfonyl chloride (158) as a white powder (287 mg, 59%). A portion was recrystallised from CH2Cl2//-Pr2O as colourless crystals: mp 217-219 0C; 1H NMR (CDCl3) δ 9.35 (s, 1 H), 9.29 (dj = 1.7 Hz, 1 H), 8.23 (dd J = 9.0, 1.9 Hz, 1 H), 8.11 (d J = 9.1 Hz), 4.72 (br dj = 11.5 Hz, 1 H), 4.61 (ddj = 11.5, 8.9 Hz, 1 H), 4.43 (ttj = 8.6, 3.0 Hz, 1 H), 3.81 (ddj = 11.0, 3.2 Hz, 1 H), 3.58 (dd, / = 11.0, 7.8 Hz, 1 H). Anal. (C15H9BrClF3N2O5S) CaIc: C, 35.91; H, 1.81; N, 5.58. Found: C, 36.00; H, 1.77; N, 5.27. A second product isolated from the column was l-(brornomethyl)-9-nitiO-3-(trifluoroacetyl)-l,2-diliydro-3H-benzo[^]indole-7-sulfonyl chloride (159) (10 mg, 2%). A portion was recrystallised from CH2Cl2/z'~Pr2O as colourless crystals: mp 238-243 0C; 1H NMR (CDCl3) δ 8.91 (dj = 9.1 Hz, 1 H), 8.81 (dj = 1.9 Hz, 1 H), 8.36 (dj = 2.0 Hz, 1 H), 8.26 (dj = 9.1 Hz, 1 H), 4.58-4.45 (m, 2 H), 4.18-4.11 (m, 1 H), 3.47 (ddj = 8.1, 3.2 Hz, 1 H), 3.18 (ddj = 11.0, 7.3 Hz, 1 H). Anal. (C15H9BrClF3N2O5SO.2/-Pr2O) CaIc: C, 37.27; H, 2.28; N, 5.37. Found: C, 37.10; H, 2.11; N, 5.18.
The reaction was repeated on a larger scale with 157 (870 mg, 1.91 mmol) and the crude product purified by crystallisation (CH2Cl2/i-Pr2O) to give 158 in higher yield (724 mg, 75%) and free of 159.
A solution of ethanolamine (18 mg, 0.30 mmol) in CH2Cl2 (0.5 mL) was added dropwise to a solution of 158 (50 mg, 0.10 mmol) in CH2Cl2 (10 mL) at 0 0C. After 15 min Cs2CO3 (67 mg, 0.20 mmol), MeOH (3 mL), and water (2 drops) were added. After a further 15 min ice was added and the mixture was poured into a mixture of cold water and cold EtOAc. The organic layer was separated and washed with cold water and cold brine. The organic layer was dried (Na2SO4) and evaporated, and the residue was recrystallised from EtOAc//-Pr2O to give l-(bromomethyl)-JV-(2- hydroxyethyl)-5-nitLO-l,2-dihydro-3H-benzo[(?]indole-7-sulfonamide (160) as red crystals (43 mg, 100%): mp 144-147 0C; 1H NMR [(CD3)2SO] δ 8.59 (dj = 1.7 Hz, 1 H), 8.02 (dj = 8.9 Hz, 1 H), 7.79 (ddj = 8.9, 1.8 Hz, 1 H), 7.69 (tj = 5.9 Hz, 1 H), 6.73 (s, 1 H), 4.63 (t,J= 5.6 Hz, 1 H), 4.33-
4.25 (m, 1 H), 3.89 (td,/ = 10.4, 1.9 Hz, 1 H), 3.82 (dd,/ = 10.2, 3.4 Hz, 1 H), 3.74-3.66 (m, 2 H), 3.43-3.30 (m, 3 H), 2.81 (qj = 6.1 Hz, 2 H). Anal. (C15H16BrN3O5SO-Iz-Pr2O) CaIc: C, 42.54; H, 3.98; N, 9.54. Found: C, 42.73; H, 3.92; N, 9.30.
A solution of /wV-butylchlorodimethylsilane (21 mg, 0.14 mmol) and DIPEA (30 mg, 0.23 mmol) in DMF (0.5 mL) was added dropwise to a solution of 160 (50 mg, 0.12 mmol) in DMF (2 mL) at 0 °C and the mixture was allowed to warm to room temperature over 1 h. A further portion of tert- butylchlorodimethylsilane (42 mg, 0.28 mmol) and DIPEA (30 mg, 0.23 mmol) were added and after 15 min the mixture was poured into a mixture of cold water and cold EtOAc. The organic layer was separated and washed with cold water and cold brine. The organic layer was dried (Na2SO4) and evaporated, and the residue was triturated with petroleum ether and Et2O to give l-(bromometliyl)- N-[2-(&/f-butyldim.eiiylsπyloxy)ethyl]-5-mtto-l,2-dihydro-3H-benzo[^indole-7-sulfonamide (161) as a red powder (60 mg, 95%): mp 69-72 0C (dec); 1H NMR [(CD3)2SO] δ 8.58 (s, 1 H), 8.02 (dj = 9.0 Hz, 1 H), 7.80 (ddj = 8.9, 1.6 Hz, 1 H), 7.78-7.72 (m, 2 H), 6.74 (s, 1 H), 4.34-4.24 (m, 1 H), 3.88 (t,J = 10.4 Hz, 1 H), 3.81 (ddj = 10.3, 3.4 Hz, 1 H), 3.74-3.65 (m, 2 H), 3.52 (t, / = 6.3 Hz, 2 H), 2.85 (qj = 6.0 Hz, 2 H), 0.79 (s, 9 H), -0.05 (s, 6 H). Anal. (C21H30BrN3O5SSiOJH2O) CaIc: C, 45.87; H, 5.61; N, 7.64. Found: C, 45.55; H, 5.28; N, 8.04.
A solution of DIPEA (155 mg, 1.20 mmol) and amine 114 (300 mg, 1.20 mmol) in CH2Cl2 (1 mL) was added dropwise to a solution of 158 (500 mg, 1.00 mmol) in CH2Cl2 (10 mL) at -50 0C. After 30 min Cs2CO3 (650 mg, 2.0 mmol), MeOH (5 mL), and water (2 mL) were added. The mixture was stirred for 15 min at -10 0C and then poured into a mixture of cold water and cold EtOAc. The organic layer was separated and washed with cold water and cold brine. The organic layer was dried (Na2SO4) and filtered through a plug of silica gel (eluting with EtOAc:petroleum ether: 9:1), and the filtrates were evaporated to give di(/W-butyl) 2-[l-(bromomethyl)-5-nitro-l,2-diliydro-3H- benzo[φndole-7-sulfonarnido] ethyl phosphate (162) suitable for the following reactions (500 mg, 81%). A portion was recrystallised from CH2Cl2A-Pt2O as red crystals: mp 76 0C (dec); 1H NMR [(CD3),SO] δ 8.59 (d,/ = 1.6 Hz, 1 H), 8.04 (dj = 8.9 Hz, 1 H), 7.94 (t, / = 5.6 Hz, 1 H), 7.80 (ddj = 9.0, 1.8 Hz, 1 H), 7.75 (s, 1 H), 6.75 (s, 1 H), 4.33-4.25 (m, 1 H), 3.89 (tdj= 10.5, 1.9 Hz, 1 H), 3.84-3.76 (m, 3 H), 3.74-3.65 (m, 2 H), 3.01 (qj = 5.6 Hz, 2 H), 1.35 (s, 18 H). Anal. (C23H33BrN3O8PS) CaIc: C, 44.38; H, 5.34; N, 6.75. Found: C, 44.48; H, 5.20; N, 6.65.
A solution of EDCLHCl (9.0 g, 58.1 mmol) in CH2Cl2 (20 mL) was washed with a solution of aqueous 40% K2CO3. The organic layer was treated with pyridinium.HBr (7.5 g, 46.5 mmol). Slow
addition of Et2O to the resulting solution caused a precipitate to form. The solid was filtered off, washed with Et2O, and dried to give EDCLHBr (8.63 g, 79%) as a white powder. Biorad AG 1-X4 resin (chloride form, 45 g) was converted to the bromide form by washing with a solution of NaBr (45 g) in water (450 mL). The resin was then washed successively with water (450 mL) and MeOH (450 mL). A solution of acid hydrochloride 122 (1.5 g) in MeOH (20 mL) was passed through the resin and the eluate was evaporated to give acid hydrobromide 163 as a cream powder (1.33 g, 78%).
EDCLHBr (230 mg, 0.98 mmol), anhydrous TsOH (6 mg, 0.033 mmol), and acid 163 (121 mg, 0.33 mmol) were added to a solution of indoline 160 (70 mg, 0.16 mmol) in CH2Cl2 (15 mL) and DMA (2 mL). After 30 min further portions of EDCLHBr (230 mg, 0.98 mmol) and TsOH (20 mg, 0.12 mmol) were added. After 1.5 h a further portion of acid 163 (60 mg, 0.16 mmol) was added. After a further 5 h the mixture was poured into a mixture of cold water and cold EtOAc. The organic layer was washed with cold water, cold brine, and then dried (Na2SO4) and evaporated. The residue was dissolved in a mixed solvent of CH2Cl2 and MeOH and the solution was slowly concentrated to induce precipitation. The solid was filtered off and washed with MeOH to give 8 as a yellow powder (65 mg, 57%): mp 210-216 0C (dec); 1H NMR [(CD3)2SO] δ 11.74 (s, 1 H), 9.29 (s, 1 H), 8.85 (dj = 1.5 Hz, 1 H), 8.43 (d,/ = 8.9 Hz, 1 H), 8.02 (dd,/ = 8.9, 1.6 Hz, 1 H), 7.91 (t,J = 5.9 Hz, 1 H), 7.42 (dj= 8.9 Hz, 1 H), 7.22-7.15 (m, 2 H), 6.96 (ddj = 8.9, 2.3 Hz, 1 H), 4.97 (t,/ = 10.5 Hz, 1 H), 4.73-4.62 (m, 3 H), 4.13 (t,J = 5.8 Hz, 2 H), 4.08-3.98 (m, 2 H), 3.63-3.56 (m, 4 H), 3.39 (qj = 6.1 Hz, 2 H), 2.88 (q,/ = 6.1 Hz, 2 H), 2.73 (t,/ = 5.7 Hz, 2 H), 4 Hs not observed. Anal. (C30H32BrN5O8S-V2H2O) CaIc: C, 50.64; H, 4.67; N, 9.84. Found: C, 50.47; H, 4.60; N, 9.67.
Example 22. 2-[l-(Bromomethyl)-3-[5-(2-motpholinoethoxy)-li^indole-2-carbonyl]-5-nitro- l,2-dihydro-3i^benzo[e]indole-7-sulfonamido] ethyl dihydrogen phosphate trifluoroacetate (23) (Scheme L). EDCLHBr (prepared as described in Example 21, 3.74 g, 15.8 mmol), anhydrous TsOH (109 mg, 0.63 mmol) and acid 163 (1.30 g, 3.48 mmol) were added to a solution of indoline 162 (1.97 g, 3.17 mmol) in CH2Cl2 (55 mL) and DMSO (5 mL). After 1 h further portions of EDCLHBr (3.0 g, 12.7 mmol), anhydrous TsOH (200 mg, 1.16 mmol) and indoline 162 (600 mg, 1.61 mmol) were added. After a further 3 h the mixture was poured into a mixture of cold water and cold EtOAc. The organic layer was washed with cold water, cold brine and then dried (Na2SO4) and concentrated to a small volume to precipitate the crude product. The solid was filtered off and triturated with acetone to give βi-tert-butyl 2-{l-(biOmomemyl)-3-[5-(2-morpholinoethoxy)-lfJ- indole-2-carbonyl]-5-nitro-l,2-dihydro-3H-benzo[ήindole-7-sulfonamido}ethyl phosphate (188) as a yellow powder (1.08 g, 38%): mp 228-233 0C (dec); 1H NMR [(CD3)2SO] 5 11.73 (s, 1 H), 9.30 (s, 1 H), 8.87 (dj = 1.7 Hz, 1 H), 8.45 (d,J - 8.9 Hz, 1 H), 8.18 (t,/ = 5.9 Hz, 1 H), 8.02 (dd,/ = 8.9,
1.7 Hz, 1 H), 7.42 (dj = 8.9 Hz, 1 H), 7.23-7.17 (m, 2 H), 6.96 (ddj = 8.9, 2.4 Hz, 1 H), 4.97 (t J = 10.4 Hz, 1 H), 4.73-4.63 (m, 2 H), 4.13 (tj = 5.7 Hz, 2 H), 4.08-3.99 (m, 2 H), 3.83 (qj = 6.0 Hz, 2 H), 3.60 (t,/ = 4.5 Hz, 4 H), 3.07 (qj = 5.8 Hz, 2 H), 2.74 (t,/ = 5.4 Hz, 2 H), 1.36 (s, 18 H), 4 Hs not observed. Anal. (C38H49BrN5OnPS-1Z2H2O) CaIc: C, 50.50; H, 5.58; N, 7.75. Found: C, 50.68; H, 5.57; N, 7.82.
A solution of 164 (140 mg, 0.16 mmol) and TFA (178 mg, 1.56 mmol) in CH2Cl2 (5 mL) was stirred at room temperature for 20 h. Removal of solvents gave 23 as a yellow powder (146 mg, 100%): mp 206-210 0C (dec); 1H NMR [(CD3)2SO] δ 11.78 (s, 1 H), 9.28 (s, 1 H), 8.87 (dj = 1.5 Hz, 1 H), 8.42 (dj = 8.9 Hz, 1 H), 8.19 (br s, 1 H), 8.01 (dd,/ = 8.9, 1.5 Hz, 1 H), 7.43 (dj = 8.9 Hz, 1 H), 7.24 (dj = 2.0 Hz, 1 H), 7.21 (d,J = 1.4 H2, 1 H), 6.99 (ddj = 8.9, 2.3 Hz, 1 H), 4.97 (tj - 10.5 Hz, 1 H), 4.72-4.63 (m, 2 H), 4.27 (tj = 5.9 Hz, 2 H), 4.08-3.97 (m, 2 H), 3.82 (qj = 6.1 Hz, 2 H), 3.87- 3.70 (m, 2 H), 3.09-2.95 (m, 4 H), 9 Hs not observed, obscured by water peak. Anal. (C32H34BrF3N5O13PS) CaIc: C, 42.87; H, 3.82; N, 7.81. Found: C, 43.21; H, 4.11; N, 7.73.
Example 23. (JE)-l-(Bromomethyl)-7V-(2-hydfoxyethyl)-3-{3-[4-(2- mofpholinoethoxy)phenyl]acryloyl}-5-nitfo-l,2-dihydro-3iϊ-benzo[e]indole-7-sulfonamide (9) (Scheme M). Biorad AG 1-X4 resin (chloride form, 45 g) was converted to the bromide form by washing with a solution of NaBr (45 g) in water (450 mL). The resin was then washed successively with water (450 mL) and MeOH (450 mL). A solution of acid hydrochloride 129 (1.0 g) in MeOH (20 mL) was passed through the resin and the eluate was evaporated to give acid hydrobromide 165 as a cream powder (1.0 g, 88%).
EDCLHBr (prepared as described in Example 21, 329 mg, 1.40 mmol), anhydrous TsOH (8 mg, 0.047 mmol) and acid 165 (166 mg, 0.47 mmol) were added to a solution of indoline 160 (100 mg, 0.23 mmol) in CH2Cl2 (8 mL) and DMA (1 mL). After 2 h further portions of acid 165 (166 mg, 0.47 mmol), EDCLHBr (329 mg, 1.40 mmol) and anhydrous TsOH (8 mg, 0.047 mmol) were added. After a further 2 h the mixture was poured into a mixture of cold water and cold EtOAc. The organic layer was washed with cold water, cold brine and then dried (Na2SO4) and evaporated. The residue was dissolved in a mixed solvent of CH2Cl2 and MeOH and the solution was slowly concentrated to induce precipitation. The solid was filtered off and washed with MeOH to give 9 as a yellow powder (91 mg, 57%): mp 185-190 0C (dec); 1H NMR [(CD3)2SO] δ 9.34 (s, 1 H), 8.83 (s, 1 H), 8.35 (dj= 8.8 Hz, 1 H), 8.01 (dj = 8.9 Hz, 1 H), 7.90 (t,J = 5.8 Hz, 1 H), 7.79 (dj = 8.7 Hz, 2 H), 7.73 (dj = 15.3 Hz, 1 H), 7.09 (dj = 15.4 Hz, 1 H), 7.03 (d,J = 8.7 Hz, 2 H), 4.73-4.63 (m, 3 H), 4.59-4.51 (m, 1 H), 4.16 (t,/ = 5.7 Hz, 3 H), 4.05-3.92 (m, 3 H), 3.63-3.55 (m, 4 H), 3.54-3.47
(m, 3 H), 2.86 (qj = 6.0 Hz, 3 H), 2.72 (tj = 5.7 Hz, 2 H). Anal. (C30H33Bi-N4O8S-CH3OH) CaIc: C, 51.60; H, 5.17; N5 7.76. Found: C, 51.89; H, 5.07; N, 7.60.
Example 24. {E)-2- [l-(Bromomethyl)-3-{3- [4-(2-morpholinoethoxy)phenyl] actyloyl}-5-nitro- l,2-dihydro-3i^benzo[e]indole-7-sulfonamido] ethyl dihydrogen phosphate ttifluoroacetate (24) (Scheme M). EDCLHBt (prepared as described in Example 21, 820 mg, 3.47 rnmol), anhydrous TsOH (20 mg, 0.12 rnmol) and acid 165 (410 mg, 1.16 mmol) were added to a solution of indoline 162 (360 mg, 0.58 mmol) in CH2Cl2 (15 mL) and DMA (1 ml). After 3 h the mixture was poured into a mixture of cold water and cold EtOAc. The organic layer was washed with cold water, cold brine and then dried (Na2SO4) and evaporated. The residue was dissolved in a mixed solvent of CH2Cl2 and MeOH and the solution was slowly concentrated to induce precipitation. The solid was filtered off and washed with MeOH to give (E)-di-tert-hxAy\ 2-[l-(bromomethyl)-3-{3-[4-(2- rnorphoHnoethoxy)phenyl]acryloy}-5-nitro-l,2-dihydro-3H-benzo[^]indole-7-sulfonamido]ethyl phosphate (166) as a yellow powder (250 mg, 50%): mp 177-182 0C (dec); 1H NMR [(CD3)2SO] δ 9.35 (s, 1 H), 8.86 (dj = 1.7 Hz, 1 H), 8.39 (dj = 8.9 Hz, 1 H), 8.16 (t,J - 5.9 Hz, 1 H), 8.00 (ddj = 8.9, 1.7 Hz, 1 H), 7.79 (dj = 8.8 Hz, 2 H), 7.73 (dj = 15.3 Hz, 1 H), 7.10 (dj = 15.3 Hz, 1 H), 7.03 (d,J = 8.8 Hz, 2 H), 4.71-4.63 (m, 2 H), 4.61-4.53 (m, 1 H), 4.17 (t,/ = 5.7 Hz, 2 H), 4.04-3.93 (m, 2 H), 3.82 (q, J = 6.0 Hz, 2 H), 3.59 (t, / = 4.6 Hz, 4 H), 3.06 (q, / = 5.8 Hz, 2 H), 2.72 (t, / = 5.7 Hz, 2 H), 1.35 (s, 18 H), 4 Hs not observed. Anal. (C38H50BrN4O11PS) CaIc: C, 51.76; H, 5.72; N, 6.35. Found: C, 51.80; H, 5.91; N, 6.21.
A solution of 166 (250 mg, 0.28 mmol) and TFA (320 mg, 2.84 mmol) in CH2Cl2 (12 mL) was stirred at room temperature for 20 h. Removal of solvents and trituration of the residue with Et2O gave 24 as a yellow powder (250 mg, 100%): mp 178-182 0C (dec); 1H NMR [(CD3)2SO] δ 9.35 (s, 1 H), 8.85 (s, 1 H), 8.38 (d,J - 8.8 Hz, 1 H), 8.18-8.08 (m, 1 H), 8.01 (dj = 8.4 Hz, 1 H), 7.86 (dj = 8.4 Hz, 2 H), 7.76 (dj = 15.2 Hz, 1 H), 7.14 (dj = 15.2 Hz, 1 H), 7.11 (dj = 8.4 Hz, 2 H), 4.73-4.63 (m, 2 H), 4.60-4.52 (m, 1 H), 4.46-4.36 (m, 2 H), 4.07-3.94 (m, 2 H), 3.89-3.74 (m, 4 H), 3.09-2.98 (m, 2 H), 11 Hs not observed, obscured by water peak. Anal. (C32H35BrF3N4O13PS-H2O) CaIc: C, 42.63; H, 4.14; N, 6.21. Found: C, 42.93; H, 4.25; N, 6.02.
In Vivo Activity of Compounds of Formula III.
The ability of compounds of Formula III to eliminate colony forming cells of human tumor xenografts grown in nude mice (CD1-Foxnlnu) was determined using an excision assay. Tumors were grown subcutaneously in the flank by inoculating cells grown in tissue culture (107 cells in 100 μL). When tumors reached treatment size (500-700 mm3) mice were randomised to treatment groups (3 mice per group for radiation or drug alone, 5 mice per group for compound plus radiation). Compounds were dissolved in phosphate buffered saline containing 2-4 equivalents of NaHCO3 and given as single intravenous doses alone or 5 min after whole body irradiation (60Co source). Eighteen hours after treatment tumors were excised, weighed, minced, dissociated enzymatically, and plated to determine clonogenicity as described (Hicks et al.,/. Natl. Cancer Inst., 2006, 98, 1118-1128). Clonogens per gram of tumor tissue were calculated relative to controls: for compound alone relative to no treatment, and for compound plus radiation relative to radiation alone.
HCTl 16, and 75 μmol/kg foi H460 and Hl 299, with the exception of R3 dosed at 24 μmol/kg in
StHa and H460, R5 dosed at 13 μmol/kg in H460, 17 dosed at 56 μmol/kg in H460, and 21, 22, and
24 dosed at 30 μmol/kg m SiHa. bRadiation alone provides following log cell loll- 2.04 (SiHa), 1.43 (H460), 1.26 (Hl 299), 2.21
(HCTl 16). cStructures of ieference compounds (Rl-4 from WO 2006/043839) shown below.
This assay provides evidence of hypoxia selective cell killing in vivo. A whole body radiation dose of 15 Gy is sufficient to steiilize well oxygenated tumor cells, and provides 1-2 logs of cell loll in these xenograft models. Additional cell kill when radiation is combined with the compounds of Formula III (15-24) shows that these compounds aie eliminating hypoxic tumor cells. The compounds of Formula III aie significantly moie active in this regard than the reference compounds of WO 2006/043839 (Rl-4). The compounds of Foimula III are also significantly more active than two ieference compounds R5 and R6, which both incorporate a morphokne in the sidechain, but not one which is attached to an indole (Formula Ia) or cinnamate (Formula Ib) sidechain. The difference between the vaiious refeience compounds and the examples of this invention is illustrated in Figures 3a and 3b, respectively, for SiHa and H460 xenografts. The difference is also
notable for the Hl 299 and HCTIl 6 xenografts in Table 4. Table 4 also shows evidence of single agent activity. This may be as a consequence of a bystander effect where the compounds of Formula III are activated in hypoxic regions of tumors and the reduced metabolites diffuse to and kill surrounding better oxygenated tumor cells. The compounds of Formula III are generally superior to the reference compounds of WO 2006/043839 in single agent activity.
The superior in vivo activity of the compounds of Formula III is unexpected since the structures of the reference compounds Rl-4 of WO 2006/043839 are very similar to the structures of the examples of Formula III (15-24 using the numbering in Table 3). The superior in vivo activity of the compounds of Formula III is particularly unexpected since reference compounds which include a morpholine in the sidechain (R5 and R6), but not one which is attached to an indole or cinnamate sidechain, show very little activity in vivo.
Although this invention has been described with reference to certain embodiments and examples, those persons skilled in the art will appreciate that the specific description is illustrative only and that variations may be made without departing from the scope of the invention.
Claims
1. A compound of Formula I:
wherein R" represents a lower C,.4 alkyl bearing a morpholine (Ic) or N-methylpiperazine (Id) substituent, and R3 represents H, C1-4 alkyl, CMalkoxy, Q^alkynyl or C,.4alkynyloxy, and pharmaceutically acceptable salts thereof.
2. A compound of Formula I:
wherein R2 represents a lower C2.4 alkyl bearing a morpholine (Ic) or N-methylpiperazine (Id) substituent, and R3 represents H or CMalkyl, and pharmaceutically acceptable salts thereof.
3. A compound according to claim 2, wherein R3 represents H.
4. A compound according to claim 3, wherein Y is Cl.
5. A compound according to claim 3, wherein Y is Br.
6. A compound according to claim 4 or 5, wherein, X is 7-SO2NH(CH2)2OH.
7. A compound according to claim 4 or 5, wherein X is 7-CONH(CH2)2OH.
8. A compound according to any one of claims 2 to 7, wherein Z is selected from the group consisting of:
9. A compound of Formula I according to claim 2, selected from the group consisting of: l-(Chloromemyl)-N-(2-hydroxyeiiiyl)-3-[5-(2-morpholinoeώoxy)-lH-indole-2-carbonyl]-5-nitLO-l,2- dihydiO-3H-benzo[^]indole-7-sulfonamide; l-(CMoromemyl)-N^(2-hydiOxyetliyl)-3-[5-(3-moipholinopropoxy)-lH"-indole-2-carbonyl]-5-nitLO- 1 ,2-dihydro-3H-benzo [^]indole-7-sulfonamide; (E)-l-(ChloiOmethyl)-N-(2-hydroxyethyl)-3-{3-[4-(2-rnorpholinoetlioxy)phenyl]aci7loyl}-5-nitro- l,2-diliydiO-3H-benzo[(?]indole-7-sulfonamide; l-(Chloromeώyl)-iV-(2-hydiOxyethyl)-3-{4-[2-(4-meώ^ dihydro-3H-benzo[e]indole-7-sulfonamide; l-(ChloiOmethyl)-iV-(2-hydroxyethyl)-3-[5-(2-moiphoHnoetiιoxy)-1H-indole-2-carbonyl]-5-nitiO-l,2- dihydro-3Iϊ-benzo[6]indole-7-carboxamide;
(E)-l-(chloiOmeώyl)-N-(2-hydroxyethyl)-3-{3-[4-(2-moφhoHnoe1±ιoxy)phenyl]aci7loyl}-5-nitro-l,2- dihydro-3H-benzo[e]indole-7-carboxamide;
(S)-l-(Chloromemyl)-lV-(24iydroxyetdiyl)-3-[5-(2-morphor^
1 ,2-dihydro-3H-benzo [tf]indole-7-sulfonamide;
1 - (Bromomethyl) -JV- (2-hydroxyethyl) -3- [5- (2-morpholinoethoxy) - 1 H-indole-2-carbonyl]-5-nitro-l,2- dihydiO-3i-f-benzo [e]indole-7-sulfonaffiide; and
(E)-l-(Εromomethyl)-N-(2-hydtoxyethyl)-3-{3-[4-(2-morpholinoeώoxy)phenyl]acryloyl}-5-nitiO- l,2-dihydro-3H-benzo[tf]mdole-7-sulfonamide; and pharmaceutically acceptable salts thereof.
10. A compound of Formula II:
wherein R2 represents a lower C2.4 aLkyl bearing a morpholine (Ic) or N-methylpiperazine (Id) substituent, and R3 represents H, a C1-4 alkyl, C1-4 alkoxy, C1-4 alkynyl or C1-4 alkynyloxy, and pharmaceutically acceptable salts thereof.
11. A compound of Formula II,
wherein R2 represents a lower C,_4 alkyl bearing a morpholine (Ic) or N-methylpiperazine (Id) substituent, and R3 represents H or C1-4 alkyl, and pharmaceutically acceptable salts thereof.
12. A compound according to claim 11, wherein R3 represents H.
13. A compound according to claim 12, wherein Y is Cl.
14. A compound according to claim 12, wherein Y is Br.
15. A compound according to claim 13 or 14, wherein X is 7-SO2NH(CH2)2OH.
16. A compound according to claim 13 or 14, wherein X is 7-CONH(CH2)2OH.
17. A compound according to any one of claims 11 to 16, wherein Z is selected from the group consisting of:
18. A compound of Formula II according to claim 11, selected from the group consisting of: 5-Arrjdno-l-(cMoromemyl)-N-(2-hydroxyethyl)-3-[5-(2-morpholinoemoxy)-lH-indole-2-carbo 1 ,2-dihydro-3 H-benzo [<?]indole-7-sulfonamide; 5-Amino-l-(chloromeώyl)-JV-(2-hydiOxyemyl)-3-[5-(3-m 1 ,2-dihydro-3H-benzo [<?]indole-7-sulfonamide; (B)-5-Amino-l-(cUoromethyl)-IV-(2-hyclroxye 1 ,2-dihydro-3H-benzo [<?]indole-7-sulfonamide;
5-Amino- 1 - (chloromethyl) -N- (2-hydroxyethyl) -3 - [5- (2-morpholinoethoxy)- 1 H-indole-2-carbonyl]- 1 ,2-diliydro-3 J-f-benzo [<?]indole-7~carboxamide; and
(i)-5-Amino-l-(chloromemyl)-N-(2-hydroxyetiiyl)-3-[5-(2-mor^holinoeώoxy)-lH-indole-2- carbonyl]~l,2-dihycko-3H-benzo[e]indole-7-sulfonamide; and pharmaceutically acceptable salts thereof.
19. A compound of Formula IHa, IHb, IHc or IHd:
20. A compound of Formula IHa, IHb, IHc or IHd:
21. A compound according to claim 20 wherein R3 represents H.
22. A compound according to claim 21 wherein Y is Cl.
23. A compound according to claim 21 wherein Y is Br.
24. A compound according to any one of claims 21 to 23 wherein X is 7-SO2NH(CH2)2OH, and is functionalized with a phosphate, amino acid, short polypeptide chain, monosaccharide or phosphonooxymethyl substituent as defined in claim 20.
25. A compound according to any one of claims 21 to 23 wherein X is 7-CONH(CHj)2OH, and is functionalized with a phosphate, amino acid, short polypeptide chain, monosaccharide or phosphonooxymethyl substituent as defined in claim 20.
26. A compound according to any one of claims 20 to 23 which is a compound of Formula IlIa.
27. A compound according to claim 26 wherein X is functionalized with a phosphate group to form the group 7-SO2NH(CH2)2OPO(OH)2 .
28. A compound according to claim 26 wherein X is functionalized with a phosphate group to form the group 7-CONH(CHJ)2OPO(OH),
29. A compound according to any one of claims 20 to 28, wherein Z is selected from the group consisting of: 
30. A compound of Formula Ilia according to claim 20, selected from the group consisting of: 2-[l-(Chlorometh7l)-3-[5-(2-moipholinoethoxy)-lH-indole-2-carbonyl]-5-nitro-l,2-dihydro-3H- benzo [φndole-7-sulfonamido] ethyl dihydcogen phosphate;
2-[l-(ChloromeUiyl)-3-[5-(3-morpholinopropoxy)-lH-indole-2-carbonyl]-5-nitiO-l,2-dihydro-3i:J- benzo (Vjindole- 7-sulfonamido] ethyl dihydrogen phosphate;
(Ηj-2-(l-(chloromethyl)-3-{3-[4-(2-moipholinoethoxy)phenyl]acryloyl}-5-nitiO-l,2-dihydro-3H- benzo[(?]indole-7-sulfonamido)ethyl dihydrogen phosphate;
2-[l -(Chloromethyl)-3- {4-[2-(4-methylpiperazin-l -yl)ethoxy]benzoyl} -5-nitro-l ,2-dihydro-3ϋ- benzo[tf]indole-7-sulfonarnido]ethyl dihydrogen phosphate;
2- { 1 -(Chloromethyl)-3-[5-(2-morpholinoethoxy)-l H-indole-2-carbonyl]-5-nitro-l ,2-dihydro-3H- benzo[φndole-7-carboxamido}ethyl dihydrogen phosphate;
(E)-2- [1 -(Chloromethyl)-3- {3- [4-(2-morpholinoethoxy)phenyl] acryloyl} -5-nitro-l ,2-dihydro-3H- benzo[<?]indole-7-carboxamido] ethyl dihydrogen phosphate;
(J)-2-[l-(Chloromeώyl)-3-[5-(2-moiphoHnoetiboxy)-lH-indole-2-carbonyl]-5-nitiO-l,2-dihydiO-3H- benzo[tf]mdole-7-sulfonamido] ethyl dihydrogen phosphate;
(i',jΞ)-2-[l-(Clilorometiιyl)-3-{3-[4-(2-moipholinoethoxy)phenyl]acryloyl}-5-rύtiO-l,2-dihydro-3^ benzo [6>]indole-7-sulfonamido] ethyl dihydrogen phosphate;
2-[l-(Bromomethyl)-3-[5-(2-morpholinoetiιoxy)-lH-indole-2-carbonyl]-5-nitiO-l,2-dihydro-3J1J- benzo [<?]indole-7-sulfonamido] ethyl dihydrogen phosphate; and
(jE^-2-[l-(Bromomethyl)-3-{3-[4-(2-moφhoLmoethoxy)phenyl]acryloyl}-5-nitiO-l,2-dihydro-3H- benzo [ι?]indole-7-sulfonamido] ethyl dihydrogen phosphate.
31. A pharmaceutical composition comprising a compound of Formula I, as defined in claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
32. A pharmaceutical composition comprising a compound of Formula II, as defined in claim 10, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
33. A pharmaceutical composition comprising a compound of Formula IHa, IHb, IHc or IHd, as defined in claim 19, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
34. A method for the treatment of a cancer in a warm-blooded animal such as a human, which comprises administering to said animal an effective amount of a compound of Formula I as defined in claim 1 or a pharmaceutically acceptable salt thereof.
35. A method for the treatment of a cancer in a warm-blooded animal such as a human, which comprises administering to said animal an effective amount of a compound of Formula II as defined in claim 10 or a pharmaceutically acceptable salt thereof.
36. A method for the treatment of a cancer in a warm-blooded animal such as a human, which comprises administering to said animal an effective amount of a compound of Formula IHa, IHb, IHc or IHd as defined in claim 19 or a pharmaceutically acceptable salt thereof.
37. The use of a compound of Formula I, II or HI as defined above or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the production of an anticancer effect in a warm-blooded animal such as a human.
38. The use of a compound of Formula I as defined in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a cancer in a warm-blooded animal such as a human.
39. The use of a compound of Formula II as defined in claim 10 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a cancer in a warmblooded animal such as a human.
40. The use of a compound of Formula IHa, IHb, HIc or HId as defined in claim 19 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a cancer in a warm-blooded animal such as a human.
41. A method or a use according to any one of claims 34 to 40, wherein the cancer is selected from the group consisting of cervical cancer, non-small cell lung carcinoma, large cell lung carcinoma, colon cancer and prostate cancer.
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| 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 |
| US9421278B2 (en) | 2014-01-10 | 2016-08-23 | Synthon Biopharmaceuticals B.V. | Duocarmycin ADCS showing improved in vivo antitumor activity |
| US9427480B2 (en) | 2014-01-10 | 2016-08-30 | Synthon Biopharmaceuticals B.V. | Duocarmycin ADCs for use in treatment of endometrial cancer |
| US9815784B2 (en) | 2008-11-03 | 2017-11-14 | Syntarga B.V. | CC-1065 analogs and their conjugates |
| US9901567B2 (en) | 2007-08-01 | 2018-02-27 | Syntarga B.V. | Substituted CC-1065 analogs and their conjugates |
| US10266606B2 (en) | 2014-01-10 | 2019-04-23 | Synthon Biopharmaceuticals B.V. | Method for purifying Cys-linked antibody-drug conjugates |
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