CA1127153A - Bridged catharanthus alkaloid dimers - Google Patents

Abstract

Abstract of the Disclosure Novel bridged vinca dimers in which two indole-dihydroindole C-3 carboxamides are linked through the nitrogen atom of a carboxamide group are described herein. These compounds are prepared by reacting the corresponding azide of an indole-dihydroindole alkaloid with an appropriate diamine or triamine. The compounds are useful as oncolytic agents.

Description

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X-4956~ -1-Bridged Catharanthus Alkaloid Dimers I
Several naturally-occurring alkaloids obtainable from Vinca rosea (Catharanthus roseus don. - Catharanthus or Vinca alkaloids) have been found active in the treatment of experimental ma-lignancies in animals~ Among these are leurosine (U.S. Patent No. 3,370,057), vincaleukoblastine ~vinblastine) to be referred to hereinafter as VLB
~U.S. Patent No. 3,097,137), leuroformine ~Belgian Patent 811,110); leurosidine (vinrosidine) and leurocristine (to be referred to hereafter as vincristine) (both in U.S. Patent No. 3,205,220): !
deoxy VLB "A" and ~B~, Tetrahedron Letters, 783 ~1958); 4-desacetoxyvinblastine (U.S. Patent No.
3,954,773; 4-desacetoxy-3'-hydroxyvinblastine (U.S. -~
Patent No. 3,944,554); leurocolombine (~.S. Patent No. 3,890,325) and vincadioline (U.S. Patent No.
3,887,565). Two of these alkaloids, VLB and vin-cristine, are now marketed as drugs for the treat-ment of malignancies, particularly the leukemias and related diseases in humans. The two marketed ~lkaloids are customarily adm,inistered by the i.v.
route.
Chemical modification of the Vinca alkaloids has been rather limited. In the first place, the molecular structures involved are ex-tremely complex, and chemical reactions which modify one specific functional group of the molecule without affecting other groups are difficult to .

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develop. Secondly, dimeric alkaloids lacking desirable chemotherapeutic properties have been recovered or produced from vinca rosea fractions or alkaloids, and a determination of their structures has led to the conclusion that these compounds are closely related to the active alkaloids, frequently differing only as to stereochemistry at a single carbon. Thus, anti-neoplastic activity seems to be limited to very specific basic structures, and the chances of obtaining more active drugs by modifica-tion of these structures would seem to be corre-spondingly slight. Among the successful modifica-tions of physiologically-active al~aloids has been the preparation of 6,7-dihydro VLB (U.S. Patent No.
3,352,868) and the replacement of the acetyl group at C-4 (carbon no. 4 of the VLB ring system-see the numbered structure below) with higher alkanoyl group or with unrelated acyl groups. (See U.S.
Patent No. 3,392,173.) Several of these C-4 deriva-tives are capable of prolong~ng the life of mice inoculated with P1534 leukemia. One of the C-4 derivatives in which a chloracetyl group replaces the C-4 acetyl group of VL~ is also a useful inter-mediate for the preparation of structurally modified VLB compounds in which an N,N-dialkylglycyl group replaces the C-4 acetyl group of VLB (See U.S.
Patent No. 3,3B7,001). C-3 car~oxamide and carbox-hydrazide derivatives of VLB, vincristine, vincadio-line and other compounds have also been prepared and found to be active anti-tumor agents (see Belgian .. ,, . . . . .. _ . , _ _ ... . .. .

11271~3 .

Patent 813,168). These compounds are extremely interesting because, for example, the 3-carboxamides of VLB are more active against Ridgeway osteogenic sarcoma and Gardner lymphosarcoma than is VLB, the basic alkaloid from which they are derived. Certain of these amide derivatives actually approach the activity of vincristine against these tumors. In particular, 4-desacetyl VLB C-3 carboxamide (vindesine) is currently on clinical trial in humans, where it appears to have less neurotoxicity than does vin-cristine and to be effective against leukemias in-cluding vincristine-resistant leukemias.
Belgian Patent 813,168 discloses and claims bridged bis vinca dimers which can be rep-resented by the formula R-NH-CH2-(CH2)n-S-S-CH2-(CH2)n-NH-R wherein R is a residue of a dimeric C-3 carbonyl-containing vinca alkaloid with oncolytic activity and n is l-S. Compounds of this structure were found to be the chief product of the reaction of a vinca C-3 carboxazide (R-N3) and NH2-CH2-CH2-SH
~nd are believed to arise by air oxidation of the C-3 carboxamidoethyl mercaptan group, as in the cysteine--cystine system. This oxidation is, of course, limited to the formation of a disulfide link from a mercaptoalkylamide, and is not generally appllcable to the synthesis of other bridged bis Catharanthus dimers.
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r Non-toxic acids useful for forming pharma- I
c~utically-acceptable acid ~ddition salts of the 3 compounds of formula IIA below includP salts derived from inorganic acids such as: hydrochloric acid, nitric 20 acid, phosphoric acid, sulfuric acid, hydrobromic ~ ~cid, hydriodic acid, nitrous acid, and phosphorous ; acid, as well as salts of non-toxic or~anic acids including aliphatic mono and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic 25 and alkandioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids~ Such pharmaceutically- i acceptable salts thus include, for example, the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydro- ;
30 genphosphate, metaphosphate, pyrophosphate, chloride, . , , ,-~ lZ~5~
x-4956 - 5 -bromide, iodide, acetate, propionate, decanoate,caprylate, acylate, formate, isobutyrate, caprate, heptanoate, propiolate,.oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, tere-phthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenyl-acetate, phenylpropionate, phenylbutyrate, citrate, lactate, 2-hydroxybutyrate, glycollate, malate, , tartrate, methanesulfonate, propanes~lfonate, naphthalene-l-sulfonate, and naphthalene-2-sulfonate alts.

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~ 11~7153 x-4956 - 6 -The first step in the preparation of the 5 compounds of formula IIA below is carried out by mixingthe azide, R6-N3, in a suitable inert solvent, customarily methylenedichloride, with the diamine or triamine NH2-CH2-~CH2)n~Q~(CH2)n CH2 2 has the same meaning as hereinabove, in the ratio of

2 moles of azide per mole of diamine or 3 moles of azide per mole of triamine. The diamine or triamine is also dissolved in an inert solvent, usually methylene dichloride, prior to addition. As pointed out below, since the azide ordinarily is not purified but used as isolated from the reaction mixture, that solvent in which it is isolated is conveniently employed for the reaction with the diamine or triamine. The bis or tris vinca amide thus produced is isolated by standard techniques and purified by chromatography.
The compounds of formula IIA below are pre-pared by the following synthetic procedure in which the above novel process is the final step. Using VLB as exemplary of the synthetic procedure, VL~ is reacted with anhydrous hydrazine in a mutual inert solvent such as a lower alkanol in a ~ealed reaction .~ .
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vessel. The reaction mixture is customarily heated in the range 40-100C. for periods of from 12 to 48 hours during which time the reaction is substan-tially complete. The product of the reaction is 4-desacetyl VLB C-3 carboxhydrazide since the reaction of hydrazine not only serves to convert the ester group at C-3 to a hydrazide group, but also, under the basic reaction condition, ser~es to hydrolyze the acetyl at C-4 to yield a hydroxyl.
Other ester groups in the molecule are not affected by the abcve reaction conditions, except that a quantity of the 18'-descarbomethoxy derivative is ordinarily produced. However, the main product of the reaction is 4-desacetyl VLi C-3 carbo~hydrazide.
The 4-desacetyl C-3 carboxhydrazide derivatives of deoxy VLB ~A" and "B", of leurosidine and of leuro-sine are produced similarly. The carboxhydrazides thus produced are next converted to the correspond-ing azides by treatment with sodium nitrite in acid.
The product of this reaction is ordinarily used as isolated from the reaction since it has been found - that it is not necessary to purify the azide in order to obtain proper yields of the desired bisamide.
Reaction of two moles of the 4-desacetyl C-3 carboxazides of VLB, leurosidine, deoxy VLB "A"
- and ~B", leurosine and other alkaloids of the structure R6-OCH3 wherein R6 is represented by ; formula IIA(except that R2 is CH3 or H only), with the diamine NH2-CH2-(CH2) -Q-(CH2) -CH2-NH2 ; . .

1~27153 wherein Q is defined as above except for the grouping N-(CH2)m-CH2-NH2 under the standard azide-amine reaction conditions of Belgian patent 813,168 for the preparation of monoamides of the same vinca .5 alkaloids, yields the desired products R6-NH-CH2-2~n Q (CH2)n-cH2-NH-R

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When a tetramine reactant NH2-CH2-(CH2)n-: . 15 Q-(CH2)n-CH2-NH2 wherein Q is N-~(CH2)m-CH2-NH2]
~ is employed, three moles of azide are employed per ; mole of tetramine and the final product is a tris-~mide of the structure [R6-NH-CH2-~CH2)n]2 N-~CH2 ) m-CH2-NH- R6, . 20 Reaction of vincristine, leuroformine or other alkaloids represented by R6-OCH3 wherein R2 is ~HO, ~ith hydrazine results not only in hydrolysis ~ of the C-4 acetoxy to give a C-4 hydroxyl but also : in deformylation at N-l. The product of this reaction, 1-desformyl-4-desacetyl vincristine C-3 carboxhydrazide for example, can be transformed to .~ I the azide and two or three moles of the azide reacted with one mole of NH2-CH2-(CH2)n-Q-(CH2)n-CH2-NH2 wherein Q and n hav~ their previous meaning to yield a compound of the formula R6-NH-CH2-(CH2)n-,X-4956 - 9 -.
Q-(CH2)n-CH2-NH-R6, in which R6 has its previous meaning except that R is ~ only. ~ r~

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lS Compounds according to formula IIA below in which R represents two different moieties are pre-pared as follows: one m~le of azide R6-N3 is reacted with one mole of diamine NH2-CH2-(CH2)n-Q-(CH2)n-CH2-NH2 to yield a half amide, R -NH-CH2~(CH2)n~Q~(CH2)n CH2 2 This mvention relates to a prefenx~ group of intenE~iate half-amides which are oompounds of the fonN~a R6~NH~CH2~(CH2)n~Q (CH2)n 2 2 wherein R6 is an indole-dihydroindole part structure of the formula .
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7' ~,~ ~ R3 ~1 ' S 4 4 ' ~ R4 t8 1 ~ ~ R5 H ~ 0 CH10--~CH/

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- (IIA) o wherein 2 . R is CH3 or H;
: when taken singly, one of R3 and R4 is OH
or H and the other is C H5 and RS is H; and when taXen together R4 and R~ form an -epoxide ring and R3 is C2H5; and Q' i8 S, S-S, O, or Se-Se, wherein n is O, 1, 2, 3 or 4.
These half-amides are readily separable from any bis-amide concomitantly prepared and can be purified and characterized by conventional means.
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11;~7~53 In fact, a half-amide fraction is customarily obtained in addition to the bisamide as a by-product even when two moles of an azide are xeacted with one mole of diamine. One mole of the half-amide is then reacted with one mole of a secondazide R6-N3, in which the R6 group is different from the one already present in the half-amide, R6-NH-2 ( 2)n Q (CH2)n C~2~NH2 to yield an unsymmet-rically substituted the bis amide. The unsymmet-rical amides containing two different R groups whichare not directly synt~hesizeable by the above procedures can be prepared by acetylating at C-4 or f~rmylating at N-~, using conventional procedures, an unsymmetrical bis amide prepared as above lacking lS one or both of those features. The chief utility of these half-amides is as intermediates.
Starting materials useful in this syn-thetic procedure are pr~pared according to the following procedures.
Preparation of Starting Materials 4-Desacètyl VLB C-3 Carboxhydrazide VLB was heated in anhydrous ethanol with an excess of anhydrous hydrazine in a sealed reaction vessel at about 60C. for about 18 hours. The reaction vessel was cooled, and opened, the contents removed, and the volatile constituents evaporated therefrom in vacuo. The resulting residue, com-prising 4-desacetyl VLB C-3 carboxhydrazide, was taken up in methylene dichloride, the methylene . . . . .
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dichloride solution washed with water, separated and dried, and the methylene dichloride removed by evaporation in vacuo. The resulting residue was dissolved in a 1:1 chloroform:benzene solvent S mixture and chromatographed over silica gel. A
benzene-chloroform-triethylamine eluant was employed to develop the chromatogram. The initial chromatog-raphic fractions contained unreacted starting materials plus 4-desacetyl VLB produced as a by-product of the reaction. Further fractions werefound to contain 4-desacetyl 18'-descarbomethoxy VLB
C-3 carboxhydrazide previously described by Neuss et al., Tetrahedron Letters, 1~68, ~83. The succeeding ; fractions, found to contain 4-desacetyl VLB C-3 carboxhydrazide by thin layer chromatography, were combined, and the solvents evaporated therefrom in vacuo. ~he resulting solid melted at about 219-220C.
with decomposition.
Following the above procedure, 4-desacetyl VLB also can be reacted with hydrazine to form 4-desacetyl VLB C-3 carboxhydrazide and leurocristine or 4-desacetyl leurocristine (available from ~argrove U.S. Patent 3,392,173) can be re~cted with anhydrous hydrazine in anhydrous ethanol to yield 4-desacetyl l-desformyl leurocristine C-3 carboxhydrazide, isolated as an amorphous powder. 4-Desacetyl C-3 carboxhydrazides of the deoxy VLB's, of leurosidine and of leurosine are prepared in analogous fashion.
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~127153 4-Desacetyl VLB C-3 Carboxazide A solution of 678 mg. of 4-desacetyl VLB
~-3 carboxhydrazide was prepared in 15 ml. of anhydrous methanol. About 50 ml. of 1 N aqueous hydrochloric acid were added, and the resulting solution cooled to about 0C. Approximately 140 mg.
of sodium nitrite were then added, and the resulting reaction mixture stirred for 10 minutes while maintaining the temperature at about 0C. The solution turned dark red-brown upon the addition of the sodium nitrite. The reaction mixture was next made basic by the addition of an e,xcess of cold 5 percent aqueous sodium bicarbonate. The aqueous solution was extracted three times with methylene lS dichloride. 4-Desacetyl VLB C-3 carboxazide formed in the above reaction passed into the methylene dichloride which layer w~s separated. The methylene dichloride solution of 4-desacetyl vinblastine C-3 carboxazide thus obtained is ordinarily used without further purification.
4-Desacetyl C-3 carboxazides of l-des-formylvincristine, leurosidine, 4'-deoxy VLB "A" and ~B~ and leurosine are prepared in analogous fashion.
This invention is further illustrated by the following specific examples:
Example 1 Preparation of Bis[~-(4-desacetyl VLB C-3 carboxamido~ethyl]disulfide Eight grams of 4-desacetyl VLB C-3 carbox-hydrazide in aqueous hydrochloric acid were reacted .

; , ~ ., ~ith sodium nitrite to form 4-desacetyl VLB C-3 carboxazide. The azide product of the reaction was isolated as a solution in 400 ml. of methylene dichloride.
Cystamine dihydrochloride (22.5 g.) was added to 500 ml. of methanol, containing dissolved therein 10.8 g. of sodium methoxide. The reaction mixture was stirred for about 4 hours after which time the solvent was removed by evaporation. The residue was stirred with ether for about 1 hour and then filtered. m e ethereal filtrate was evaporated to dryness in vacuo yielding about 10 g. of cystamine free base as an oily residue.
Cystamine free base (760 mg.) in methylene dichloride was added to the solution of the 4-desacetyl VLB C-3 carboxazide in methylene dichloride.
75 ml. of tetrahydrofuran were added, and the ; reaction stirred in the`dark for about 16 hours at ambient temperature. The reaction mixture was then filtered and thé filtrate evaporated to dryness in vacuo. The resulting residue containing bis~-t4-desacetyl VLB C-3 carboxamido)ethyl]disulfide was dissolved in methylene dichloride. The methylene dichloride solution was washed with water and then dried. Evaporation of the methylene dichloride yielded a residue which was shown to contain 2 major products by TLC ~ 1 methylene dichloride-methanol-ethyl acetate). The residue was then chromatographed over silica using the same solvent 30 system as the eluant. Fractions shown by TLC to , _ . ., ~ . .

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X-495~ - 15 -contain bis~-(4-desacetyl VLB C-3 carboxamido)-ethyl]disulfide were combined and the solvents evaporated therefrom. The other major product of the reaction was also separated and purified and proved to be the reaction product of 1 mole of 4-desacetyl VLB C-3 carboxazide and cystamine, 4-desacetyl VLB C-3 N-2-(2-aminoethyldithio)ethyl carboxamide. Higher yields of this 1:1 product are obtained if one mole of azide is reacted with one lp mole of cystamine. Bis[~-(4-desacetyl VLB C-3 carboxamido)ethyl]disulfide thus prepared was identical to that obtained ~y the procedure de-scribed in Belgian ~atent 813,168.
The disulfate salt of bis[~-(4-desacetyl VLB C-3 carboxamido)ethylldisulfide was prepared in ethanol by the stoichiometric addition of 2 percent (V/V) ethanolic sulfuric acid. The disulfate was also prepared by titrating a solution of the base with 2 percent ethanolic sulfuric acid to the point where an aliquot of the solution diluted with 5 volumes of water had a pH in the range 3.0-3.5.
Either method of preparation yielded an off-white amorphous salt after evaporation of the ethanol.

Claims (2)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a novel intermediate compound of the formula R6-NH-CH2-(CH2)n-Q'-(CH2)n-CH2-NH2 wherein R6 is an indole-dihydroindole part structure of the formula (IIA) wherein R2 is CH3 or H;
when taken singly, one of R3 and R4 is OH
or H and the other is C2H5 and R5 is H; and when taken together R4 and R5 form an .alpha.-epoxide ring and R3 is C2H5; and Q' is S, S-S, O, or Se-Se, wherein n is 0, 1, 2, 3 or 4, and pharmaceuti-cally-acceptable acid addition salts thereof, which comprises reacting an azide of the formula R6-N3 wherein R6 is the residue of an indole-dihydroindole alkaloid of the formula:

(II) wherein R2 is CH3 or H;
when taken singly, one of R3 and R4 is OH
or H and the other is C2H5 and R5 is H; and when R4 and R5 are taken together, they form an .alpha.-epoxide ring and R3 is C2H5; with a diamine of the formula NH2-CH2-(CH2)n-Q'-(CH2)n-CH2-NH2 wherein Q' and n are defined as above, in the ratio of one mole of R6-N3 per mole of diamine when Q' is S, S-S, Se-Se or O.

2. An intermediate compound of the formula R6-NH-CH2-(CH2)n-Q'-(CH2)n-CH2-NH2 wherein R6 is an indole-dihydroindole part structure of the formula (IIA) wherein R2 is CH3 or H;
when taken singly, one of R3 and R4 is OH
or H and the other is C2H5 and R5 is H; and when taken together R4 and R5 form an .alpha.-epoxide ring and R3 is C2H5; and Q' is S, S-S, O; or Se-Se, wherein n is 0, 1, 2, 3 or 4, whenever prepared by the process of claim 1 or by an obvious chemical equivalent thereof.