CA2150711A1 - Antitumour agents - Google Patents
Antitumour agentsInfo
- Publication number
- CA2150711A1 CA2150711A1 CA002150711A CA2150711A CA2150711A1 CA 2150711 A1 CA2150711 A1 CA 2150711A1 CA 002150711 A CA002150711 A CA 002150711A CA 2150711 A CA2150711 A CA 2150711A CA 2150711 A1 CA2150711 A1 CA 2150711A1
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- Prior art keywords
- compound
- formula
- chh
- mixture
- indole
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
Novel mitosenes useful as antitumour agents are compounds of formula (II) in which R1 and R2 together with the nitrogen to which they are attached represent a cyclic or acylic secondary amine, R3 is hydrogen or an alkyl group, R4 and R5 are each independently hydrogen or an alkyl group and Z is a leaving group, or a salt thereof.
Description
PCTIGB93/0 '~33 WO 94/1367~ ' 2 1 5 0 7 1 ~
ANTITUHOUR AGENTS
This invention relates to novel cyclopropapyrroloindolequin-ones suitable for use as antitumour agents.
Certain aziridinopyrroloindolequinones, known as mitomycins, have been known for some time as potent antitumour antibiotics.
Examples are mitomycin C and mitomycin A of the formula I below X 7 o OCONH2 ~ OMe (I) Me ~ N ~ NH
where X is -NH2 (mitomycin C) or -OMe (mitomycin A). It has long been known that mitomycins require reductive activation before they can react covalently with DNA and hence exert their cytotoxic action. Recently details of this activation process have begun to emerge. It appears from the work of McGuinness et al, J. Org. Chem., (1991), 56, 4826-4829 that activation involves opening of the aziridine ring and bonding of the DNA bases via the 2 position of the pyrrole ring. Other work in understanding the mechanism of the activation process has involved the synthesis of the cyclopropane analogue of mitomycin A (ie formula I where X is OMe and the aziridine ring has been replaced by cyclopropane). The intention of this synthesis was to provide a 2~ compound of less electrophilicity at C-l so as to focus attention on the role of C-10 in the DNA activation process (see Jones et al, J. Chem. Soc. Chem. Commun., (1989), 186 to 187 and J. Chem.
Soc. Perkin Trans. 1 (1989), 2449 to 2462).
This cyclopropane analogue of the mitosene derived from mitomycin A and the corresponding compound lacking a methyl at C-6 have been found to also exhibit antitumour activity. The latter compound has been reported as 10 to 20 times less active than mitomycin C when tested under normal oxic conditions against 2 ~
several human tumour cell lines, while an in vitro test of the compound against Chinese hamster V79 cells under aerobic and anaerobic conditions showed an almost 35 fold differential cytotoxicity (IC50(air): IC50(nitrogen)) as opposed to a value of 2 for mitomycin C.
~ hile it was somewhat unexpected to observe that cyclopropane analogues of mitomycin retained anti tumour activity, no significant benefits were apparent. It is against this background that a group of new cyclopropapyrroloindolequinones have been found that exhibit considerably enhanced anti tumour activity when compared with the known mitomycins and cyclopropyl analogue of mitomycin A described above and which demonstrate this cytotoxicity under both aerobic and hypoxic conditions.
Accordingly, the present invention provides compounds of the formula II
Rl ~ R5 (Il) in which Rl and R2 together with the nitrogen to which they are attached represent a cyclic or acylic secondary amine, R3 is hydrogen or an alkyl group, R4 and R~ are each independently hydrogen or an alkyl group and Z is a leaving group, or a salt thereof. Suitable salts include acid addition salts of one or both nitrogen atoms with an acid, such as hydrochloric acid, methane sulphonic acid, tartaric acid or other, especially physiologically acceptable, acids.
Preferably Z is selected from carbamate, amide, ester, ether and halogen substituents. Preferably Z is -OCONH2.
WO 94/13674 PCT/GB93/Ot533 215~
Rl and R2 are preferably linked together with the carbon atom to which they are attached to form a heterocyclic ring. The heterocyclic ring may suitably contain 3 to 6 members. A
ANTITUHOUR AGENTS
This invention relates to novel cyclopropapyrroloindolequin-ones suitable for use as antitumour agents.
Certain aziridinopyrroloindolequinones, known as mitomycins, have been known for some time as potent antitumour antibiotics.
Examples are mitomycin C and mitomycin A of the formula I below X 7 o OCONH2 ~ OMe (I) Me ~ N ~ NH
where X is -NH2 (mitomycin C) or -OMe (mitomycin A). It has long been known that mitomycins require reductive activation before they can react covalently with DNA and hence exert their cytotoxic action. Recently details of this activation process have begun to emerge. It appears from the work of McGuinness et al, J. Org. Chem., (1991), 56, 4826-4829 that activation involves opening of the aziridine ring and bonding of the DNA bases via the 2 position of the pyrrole ring. Other work in understanding the mechanism of the activation process has involved the synthesis of the cyclopropane analogue of mitomycin A (ie formula I where X is OMe and the aziridine ring has been replaced by cyclopropane). The intention of this synthesis was to provide a 2~ compound of less electrophilicity at C-l so as to focus attention on the role of C-10 in the DNA activation process (see Jones et al, J. Chem. Soc. Chem. Commun., (1989), 186 to 187 and J. Chem.
Soc. Perkin Trans. 1 (1989), 2449 to 2462).
This cyclopropane analogue of the mitosene derived from mitomycin A and the corresponding compound lacking a methyl at C-6 have been found to also exhibit antitumour activity. The latter compound has been reported as 10 to 20 times less active than mitomycin C when tested under normal oxic conditions against 2 ~
several human tumour cell lines, while an in vitro test of the compound against Chinese hamster V79 cells under aerobic and anaerobic conditions showed an almost 35 fold differential cytotoxicity (IC50(air): IC50(nitrogen)) as opposed to a value of 2 for mitomycin C.
~ hile it was somewhat unexpected to observe that cyclopropane analogues of mitomycin retained anti tumour activity, no significant benefits were apparent. It is against this background that a group of new cyclopropapyrroloindolequinones have been found that exhibit considerably enhanced anti tumour activity when compared with the known mitomycins and cyclopropyl analogue of mitomycin A described above and which demonstrate this cytotoxicity under both aerobic and hypoxic conditions.
Accordingly, the present invention provides compounds of the formula II
Rl ~ R5 (Il) in which Rl and R2 together with the nitrogen to which they are attached represent a cyclic or acylic secondary amine, R3 is hydrogen or an alkyl group, R4 and R~ are each independently hydrogen or an alkyl group and Z is a leaving group, or a salt thereof. Suitable salts include acid addition salts of one or both nitrogen atoms with an acid, such as hydrochloric acid, methane sulphonic acid, tartaric acid or other, especially physiologically acceptable, acids.
Preferably Z is selected from carbamate, amide, ester, ether and halogen substituents. Preferably Z is -OCONH2.
WO 94/13674 PCT/GB93/Ot533 215~
Rl and R2 are preferably linked together with the carbon atom to which they are attached to form a heterocyclic ring. The heterocyclic ring may suitably contain 3 to 6 members. A
3-membered (aziridine) ring, which may be substituted, is preferred. R3 is preferably hydrogen. R4 and R5 may each independently be an alkyl group such as methyl but preferably R4 and R5 are hydrogen.
A preferred compound of formula II is that in which Rl and R2 together with the adjacent nitrogen form an aziridine ring, R2, R3 and R4 are hydrogen and Z is -OCONH2.
Compounds of formula II where Z is an amide or ester group may be made in accordance with the reaction scheme as described below. Compounds where Z is other than amide or ester, eg halide, may be prepared by modification of the desired scheme using methods well known to the skilled chemist, for example modification of the penultimate step in the following scheme:
A preferred compound of formula II is that in which Rl and R2 together with the adjacent nitrogen form an aziridine ring, R2, R3 and R4 are hydrogen and Z is -OCONH2.
Compounds of formula II where Z is an amide or ester group may be made in accordance with the reaction scheme as described below. Compounds where Z is other than amide or ester, eg halide, may be prepared by modification of the desired scheme using methods well known to the skilled chemist, for example modification of the penultimate step in the following scheme:
4 PCT/GB93/02!;33 ~ 215~
~ I =~j I
G
~ o _ O O
IL ,~ ~ ~ C
~ CD Z, a /
s~ t ~ t t -D 0~ CV ~ ~
'~ O G
O
O
c~ G /
0=~
, 0~ G
O ~-~ O ~ Z
c~ ~ cV Ir ~
W o 94/13674 21 5 ~1 1 PCT/GB93/0253~
In the above reaction scheme, Rl to R5 have the meanings defined above for formula II and R6 is -COCH3 or -CONH2. When R6 is -COCH3, it may be introduced by reaction of the alcohol with acetic anhydride in pyridine. When R6 is -CONH2 it may be introduced by reaction of the alcohol with phenyl chloroformate followed by reaction wirh ammonia. TsNHNH2 is 4-toluenesulphonyl hydrazide and Fremy's salt is potassium nitrosodisulphonate.
The invention therefore includes a method of preparation of compounds of formula II comprising reaction of a compound of formula III
MeO ~
l5 0 N ~ RR5 (lll) where R3, R4, R5 and Z are as defined above, with a secondary amine of formula RlR2NH where Rl and R2 are as defined above in the presence of a suitable solvent, for example, dimethylformamide.
Preferably Z in the compound of formula III is -CONH2 and the compound of formula III has been prepared by reaction of a compound of formula IV
MeO~R~ ~IV) 21~ 6-where R3, R4 and R5 are as defined above, with phenyl chloroformate followed by reaction with ammonia.
Compounds of formula II and salts thereof may find application as antitumour agents. The invention therefore further includes pharmaceutical compositions comprising at least one compound of formula II, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.
Suitable carriers will depend on the way in which the composition is to be administered. The for example, the compounds of formula II may be used in a composition, for example an aqueous, oily or emulsified composition, incorporating a liquid diluent which may be intended to be employed in injectable form for parenteral administration and is therefore sterile and pyrogen free.
Alternatively the composition may be suitable for oral administration, in which case the carrier is conventionally a solid material such as starch, lactose, dextrin or magnesium stearate. Such solid compositions may conveniently be formed into tablets or capsules. Other forms of administration may be used, for example suppositaries or pessaries.
The dosage level employed will depend on the mode of formulation and intended use. Thus, for example, if the composition is to be administered intravenously, a suitable dosage may lie in the range of 0.001 to 0.5mg/kg of active ingredient at intervals which may vary from 24 hours to several weeks.
Preparation of compounds in accordance with the invention will now be described by way of example.
ExamDle 1 7-(Aziridin-l-yl)-9-hYdroxvmethyl-1.2-dihYdro-3H-1.2-cycloproDapyrrolo [1.2-a]indole-5.8-dione carbamate 3a ~ OCONH2 W o 94/13674 2 1 5 ~ 7 1 1 PCT/GB93/02533 The title compound was prepared starting from Q-vanillin by steps a) to n) as described below:
a) 2-Benzyloxy-3-methoxybenzaldehyde Potassium hydroxide pellets (40.6g, 724mmol) were added to a stirred solution of Q-vanillin (lOO.Og, 657mmol) in ethanol (99.7%, 590cm3), followed by benzyl chloride (83.45cm3, 723mmol).
The stirred mixture was refluxed for 12 h, then water (lOOcm3) was added, and the mixture extracted with ether (3x750cm3). The ethereal extracts were washed with water (3x200cm3), potassium 10 hydroxide solution (2M, 5x500cm3), again with water (2xlSOcm3), then brine (lSOcm3). The organic layer was dried (MgS04), then condensed in vacuo, to give the title compound (9.2g, 62%) as a colourless solid on trituration wlth light petroleum; vmax (Nujol) 1697, 1584, 1367, 1265, 1247, and 1221cm~l; ~H(270MHz, 15 CDC13) 10.24 (lH, s, CHO), 7.41-7.14 (8H, m, Ar-H), 5.18 (2H, s, OCH2Ph), and 3.95 (3H, s, OMe); mL~ 243 (MH+, 11%), 242 (M+, 62), 213(27), and 91 (100).
b) Methyl 2-Azido-3-(2-benzyloxv-3-methoxyphenyl)propenoate Sodium metal (12.54g, 545 mmol) was added to dry methanol 20 (300cm3). The solution was cooled to -15C, and a solution of methyl azidoacetate (61.00g, 530mmol) and 2-benzyloxy-3-methoxybenzaldehyde (33.079, 137mmol) in dry methanol (25cm3) was added dropwise by syringe. The mixture was stirred at -10C for 3 h then at +4C for 12 h. Water (lOOcm3) was cautiously added to the mixture, which was then extracted with ethyl acetate (3xSOOcm3). The combined extracts were washed with water (3xSOOcm3), brine (300cm3), and dried (MgS04). Removal of the solvent in vacuo gave a pale yellow residue, which was triturated with ether to give the title com~ound (38.03~, 82%) as 30 pale yellow crystals; vmaX (Film)2121, 1712, 1456, 1378, 1260, and 1218cm~1; ~H (270MHz; CDC13) 7.77 (lH, dd, J 1.4, 8.0Hz Ar 6-H), 7.44-7.27 (SH, m, Bn-H), 7.26 (lH, s, CH=), 7.11 (lH, t, J
8.0 Hz, 4/5-H), 6.95 (1 H, dd, J 1.5, 8.0 Hz, 5/4-H), 5.00 (2 H, s, OCH2Ph), 3.90 (3H, s, C02Me), and 3.85 (3 H, s, ArOMe).
c) Methyl 4-Benzyloxy-5-methoxyindole-2-carboxylate A solution of methyl 2-azido-3-(2-benzyloxy-3-methoxyphenyl) propenoate (2.2089, 6.513mmol), in dry xylene (48cm3) ~as introduced, dropwise, by means of a pressure e~ualising dropping funnel, to refluxing dry xylene (200cm3). After the addition was complete (ca. 30min), the solution was refluxed for a further 45 min. Removal of solvent in vacuo gave a yellow solid residue, which was recrystallised from ether to give the title compound (1.323g, 65%) as a colourless solid; ''max (CHC13) 3464, 3013, 1707, 1533, 1453, and 1253cm~l;~H (400MHz; CDC13) 9.36 (lH, s, NH), 7.54 (2H, d, J 7.1 Hz, Bn-H), 7.53-7.29 (4H, m, 3Bn-H+3-H), 7.09 (lH, s, 6+7-H), 5.29 (2H, s, OCH2Ph), 3.94 (3H, s, MeO/C02Me), and 3.89 (3H, s, C02Me/OMe).
d) 4-Benzyloxy-5-methoxyindole-2-methanol A solution of methyl 4-benzyloxy-5-methoxyindole-2-carboxylate (6.975g, 22.4mmol) in dry THF (200cm3) was added dropwise to a stirred suspension of lithium aluminium hydride (1.709g, 44.8mmol) in dry THF (85cm3), such that the mixture achieved gentle reflux. After 30min, water (1.7cm3), 15% sodium hydroxide (1.7cm3), and water again (5.1cm3), were added to the mixture, and the resultant precipitate removed by flltration (through a bed of Celite). The filtrate was dried (MgS04), then condensed 1n vacuo to give the title com~ound (5.8169, 92%) as a colourless solid; vmax (Nujol) 3476, 3284, 1504, 1324, 1284, 1244, 1090, 1016, and 702cm~l; ~H (250MHz; CDC13+TMS) 8.25 (lH, br s, NH), 7.52 (2H, m, Bn-H), 7.41-7.30 (3H, m, Bn-H), 7.00 (1 H, d J 8.4 Hz, 7/6-H), 6.91 (1 H, d, J 8.6 Hz, 6/7-H) 6.40 (1 H, s, 3-H), 5.20 (2 H,s, CH20Ph), 4.75 (2H, s, CH20H), and 3.88 (3H, s, OMe).
~5~7~1 e) 4-Benzvloxy-5-methoxyindole-2-carboxaldehyde Manganese dioxide (17.40g, 200.00mmol) was added, in portions to a stirred solution of 4-benzyloxy-5-methoxy-4-methylindole-2-methanol (5.829, 20.56mmol) in dry dichloromethane (650cm3). The suspension was then refluxed for 12h. The mixture was filtered, and the residue washed with hot dichloromethane (3x300cm3). The combined filtrate and washings were evaporated to give an oil which was purified by column chromatography to give the title compound (3.56g, 62X) as a pale yellow; vmax (Nujol) 3458, 3015, 2837, 1663, 1531, and 1143cm~l; ~H (400MHz; CDC13) 9.76 (lH, s, CHO), 9.25 (lH, s, NH), 7.49 (2H, m, Bn-H), 7.39-7.30 (3H, m, Bn-H), 7.24 (lH, dd, J 0.8, 2.2Hz, 3-H), 7.17 (lH, d, J 8.9Hz, 6-H), 7.12 (lH, dd, J 0.8, 8.9Hz, 7-H), 5.28 (2H, s, CH20Ph), and 3.91 (3H, s, OMe).
f) 1-Allvl-4-benzyloxy-5-methoxyindole-2-carboxaldehYde To a flask charged with sodium hydride (80%, 0.475g, 15.84mmol) was added dry light petroleum (10cm3). The mixture was stirred for 10 min, the petroleum removed by syringe, and the flask contents dried under vacuum. 4-Benzyloxy-5-methoxyindole-2-carboxaldehyde (3.562g, 12.68mmol) in DMF (260cm3) was added dropwise, and the mixture was stirred at room temperature for 3G
min. Allyl bromide (1.907g, 15.76mmol) was added, and the mixture was stirred at room temperature. After lh, water (150cm3) was cautiously added, and the mixture was extracted with ethyl acetate (4x250cm3). The combined extracts were washed with water (8x200cm3), brine (200cm3), dried (MgS04), and evaporated to give the title compound (4.070g, 99%) as a brown solid; vmax (Nujol) 1670, 1490, 1406, 1250, and 1140cm~l; ~H (250MHz; CDC13+TMS) 9.79 (lH, s, CH0), 7.51 (2H, m, Bn-H), 7.38 (3H, m, Bn-H), 7.25 (lH.
s, 3-H), 7.19 (lH, d, J 9Hz, 7/6-H), 7.04 (lH, d, J 9Hz, 6/7-H), 5.96 (lH, m, CH2CH=CH2), 5.28 (2H, s, CH20Ph), 5.13 (3H, m, CH2CH=CH2 and CH2CH=CHH-cis), 4.89 (lH, dd, J 1.0, 17.1Hz, CH2CH=CHH-trans), and 3.92 (3H, s, OMe).
2~
g) l-Allyl-4-benzyloxy-5-methoxyindole-2-carboxaldehyde tosylhydrazone l-Allyl-4-benzyloxy-5-methoxyindole-2-carboxaldehyde (3.095g, 9.642mmol) was added to a stirred solution of 4-toluenesulphonyl hydrazide (2.334g, 12.539mmol) in methanol (25cm3). After stirring at 40C for lh, the solvent was removed in vacuo, and the residue purified by chromatography, eluting with ether, to give the title compound (4.694g,99%) as a pale yellow solid; vmax (CHC13)2956, 1718, 1492, 1456, 1358, and 1166cm~l; ~H (250MHz; CDC13+TMS) 7.83 (2H, m, Ar-H [AA'BB']), 7.73 (2H, m, NH+CH~N), 7.47 (2H, m, Bn-H), 7.36-7.28 (5H, m, 3Bn-H+2 Ar-H [AA'BB']), 7.03 (lH, d, J 8.8Hz, 6/7-H), 6.94 (lH, d, J8.8Hz, 716-H), 6.67 (lH, s, 3-H), 5.85 (lH, m, -CH'CH2), 5.20 (2H, s, OCH2Ph), 5.04 (3H, m, CH2,CH-CH2 + CH2CH-CHH-cis), 4.85 (lH, dd, Jl.2, 17.2Hz, CH2CH.CHH-trans), 3.88 (3H, s, OMe), and 2.41 (3H, s, Ar-Me).
h) 8-Benzyloxy-7-methoxy-1,2-dihydro-3H-1,2-CYcloDroDapyrrolo [1,2-a] indole Sodium hydride (80~, 0.237g, 7.907mmol) was added to a stirred solution of the tosylhydrazone of g) above (2.602g, 5.321mmol) in dry THF(80cm3). After 25 min, the solution was filtered, and the filtrate evaporated. The residue was dissolved in dry chlorobenzene (800cm3), and the solution refluxed for 3 h. The sovent was evaporated, and the residue purified by chromatography to give the title compound (1.530g, 94X) as a colourless oil;
vmax (Film) 1560, 1492, 1258, 1232, and 744cm~l; ~H (250MHz;
CDC13+TMS) 7.55 (2H, m, Bn-H), 7.42-7.29 (3H, m, Bn-H), 6.81 (2H, 2d, J 8.6Hz, 5+6-H), 6.22 (lH, s, 9-H), 5.20 (2H, s, 0CH2Ph), 4.07 (2H, m, 3-CH2), 3.89 (3H, s, OMe), 2.37 (2H, m, 1+2-H), 1.2S
(lH, m, la-CHH), and 0.64 (lH, m la-CHH). Noting 2d=2 overlapping doublets.
~ WO 94113674 215 0711 PCT/GB93102533 i) 8-Benzyloxy-7-methoxy-1,2-dihydro-3H-l,Z-cyclopropapyrrolo[1,2 -a]indole-9-carboxaldehyde N-Methylformanilide (1.222g, 9.037mmol) and phosphorus oxychloride (1.386g, 9.037mmol) were stirred under a calcium oxide drying tube for 20 min. The resulting precipitate was cooled to 0C, and 1,2-dichloroethane (34cm3) was added.
8-Benzyloxy-7-methoxy-1,2- dihydro-3H-1,2-cyclopropapyrrolo [1,2-a]indole (2.297g, 7.S31mmol) was added, and the mixture was refluxed for 1.25h. Sodium acetate (lM; 49cm3) was added, and the mixture was extracted with ethyl acetate (2x200cm3). The combined extracts were washed with water (3x250cm3), brine (200cm3), dried (MgS04), and evaporated. Purification of the residue by column chromatography gave the title com~ound (1.902g, 76X); vmax (Nujol) 1638, 1604, lS22, and 742, cm~l; ~H (250MHz; CDC13+TMS) 10.32 (lH, s, CHO), 7.49 (2H, m, Bn-H), 7.41-7.26 (3H, m, Bn-H), 6.88 (2H, 2d, J 8.6Hz, 5+6-H), 5.16 (2H, s, OCH2Ph), 4.13 (2H, m, 3-CH2), 3.92 (3H, s, OMe), 3.00 (lH, m, l-H), 2.48 (lH, m, 2-H), 1.49 (lH, m, la-CHH), and 0.72 (lH, m, la-CHH).
j) 8-Hydroxy-7-methoxy-1,2-dihydro-3H-1,2-cyclopropapyrrolo [1,2-a] indole-9-carboxaldehyde To a solution of 8-benzyloxy-7-methoxy-1,2-dihydro-3H-1,2-cyclopropapyrrolo[1,2-a]indole-9-carboxaldehyde (0.5009, 1.501mmol) in ethyl acetate (250cm3) was added 10% palladium on carbon catalyst (O.lOOg), and dllute sulphuric acid (8drops). The mixture was stirred under an atmosphere of hydrogen for 2h. After this time, the suspension was filtered and washed with DCM. The filtrate and washings were extracted with water (3xlOOcm3), brine (75cm3), and dried (MgS04). The organic layer was evaporated to dryness to afford a brown solid residue. Purification of the residue by column chromatography gave the title compound (0.288g, 79%) as a colourless solid; vmax (Nujol) 1606, 1304, 1252, and 824cm~l; ~H (250MHz; CDC13+TMS) 10.87 (lH, s, CHO), 9.58 (lH, s, OH), 6.88 (lH, d, J 8.5Hz, 6/5-H), 6.52 (lH, d, J8.5Hz, 5/6-H), 4.13 (2H, m, 3-CH2), 3.91 (3H, s, OMe), 2.72 (lH, m, l-H), 2.57 (lH, m, 2-H), 1.51 (lH, m, la-CHH), and 0.81 (lH, m, la-CHH).
WO 94tl3674 2~ PCT/GB93/02533 k) 9-Formyl-7-methoxy-1,2-dihydro-3H-1,2-cyclo~ropapyrrolo[1,2-a]
indole-~,8-dione Potassium nitrosodisulphonate (1.407g, 5.243mmol) in water (70cm3), was added to a stirred solution of 8-hydroxy-7-methoxy-1,2-d~hydro-3H-1,2-cyclopropapyrrolo[1,2-a]ind ole-9-carboxaldehyde (0.5799, 2.383mmol) in acetone (232cm3). The mixture was buffered with sodium dihydrogen phosphate solution (0.167M; 70cm3). After stirring at room temperature for 12h, the mixture was concentrated in vacuo, filtered, and the residue recrystalised from dichloromethane-light petroleum to give the title compound (0.581g, 95%) as orange crystals; vmax (Nujol) 1680, 1662, 1638, 1586, 1500, 1244, and 1212cm~l; ~H (250MHz;
CDC13+TMS) 10.37 (lH, s, CH0), 5.68 (lH, s, 6-H), 4.32 (2H, m, 3-CH2), 3.85 (3H, s, OMe), 2.86 (lH, m, l-H), 2.47 (lH, m, 2-H), 1.47 (lH, m, la-CHH), and 0.65 (lH, m, la-CHH).
1) 9-Hvdroxymethyl-7-methoxy-1,2-dihydro-3H-1,2-cycloDropapyrrolo [1,2-a]indole-5-8-dione Sodium borohydride (0.189g, 4.996mmol) was added to a stirred solution of 9-formyl-7-methoxy-1,2-dihydro-3H-1,2-cyclopropapyrrolo[l,2-a]indole-5,8-dione (0.176g, 0.685mmol) in methanol (132cm3) and the solution rigorously degassed with nitrogen. After stirring for 4h at room temperature, air was blown through the solution rapidly, and the mixture was transferred to a separating funnel, and extracted with dichloromethane (3x200cm3). The combined extracts were washed w~th water (2x200cm3), brine (300cm3), and dried (MgS04). The solvent was evaporated, and the residue pur~fied by column chromatography to give the title comDound (0.159g, 90%) as a red solid; vmax (Nujol) 3312, 1668, 1630, 1586, 1586, and 722cm~1;
~H (250MHz; CDC13+TMS) 5.61 (lH, s, 6-H), 4.68 (2H, m, 10-CH2), 4.26 (2H, m, 3-CH2), 3.89 (lH, t, J 7.1Hz), 3.82 (3H, s, OMe), 2.36 (2H, m, 1 2-H), 1.30 (lH, m, la-CHH), and 0.60 (lH, m, la-CHH).
O WO 94/13674 PCT/GB93/02~;33 ~ ~i50711 m) 9-Hydroxymethyl-7-methoxy-1,2-dihydro-3H-1,2-cYclopropa~yrrolo [1,2-a]indole-5,8-dione carbamate Phenyl chloroformate (0.08cm3, 0.618mmol) was added dropwise to a stirred, ice cold solution of the alcohol of 1) above (O.lOOg, 0.386mmol) in dry pyridine (25cm3). The mixture was stirred at room temperature for 2h, then water (lOcm3) was added. The mixture was extracted with dichloromethane (3x50cm3), and the combined extracts were washed with water (3xSOcm3), brine (lOOcm3), and dried (MgS04). The solvent was evaporated, and the residue purified by column chromatography to give an orange gummy solid.
A solution of this phenyl carbonate in dry dichloromethane (55cm3), was cooled to -78C. Ammonia gas was bubbled into the solution for approximately 45 min (monitored the reaction by TLC), after which time the contents were allowed to warm to room temperature, and the solvent removed in vacuo. Recrystallisation of the residue from dlchloromethane-light petroleum gave the title compound (0.097g, 83%) as a red crystalline solid, m.p.
176-178C, vmax (Nujol) 3408, 3212, 1764, 1668, 1620, 1584, 1350, 1306, and 1242cm~l; ~H (250 MHz; CDC13+TMS) 5.59 (lH, s, 6-H), 5.29 (2H, m, 10-CH2), 4.60 (2H, br, NH2), 4.27 (2H, m, 3-CH2), 3.80 (3H, s, OMe), 2.54 (lH, m, l-H), 2.33 (lH, m, 2-H), 1.31 (lH, m, la-CHH), and 0.58 (lH, m, la-CHH); ~C (100.6MHz; CDC13) 177.68 (CO, 8-C), 177.20 (CO, 5-C), 160.44 (C/CO, 7-C/urethane), 156.77 (CO/C, 7-C/urethane), 146.31 (C, 4a-C), 129.24 (C, 8a/9a-C), 123.90 (C, 9a/8a-C), 111.33 (C, 9-C), 105.35 (CH, 6-C), 57.87 (CH2, 10-C), 56.39 (CH3, OMe), 50.00 (CH2, 3-C), 20.63 (CH, l-C), 16.17 (CH2, la-C), 14.69 (CH, 2-C).
Lf~O ~
n) 7-(Aziridin-l-yl)-9-hydroxymethyl-1,2-dihydro-3H-1,2-cyclo-~ropapyrrolo[1,2-a]indole-5,8-dione carbamate A solution of 9-hydroxymethyl-7-methoxy-1,2-dihydro-3H-1,2-cyclopropapyrrolotl,2-a]indole-5,8-dione carbamate (0.015g, 0.050mmol) in dry DMF (2.5cm3) was treated with an excess of distilled aziridine (0.5cm3), and th~s mixture was stirred at room temperature under nitrogen for 15h. After this time, water (Scm3) was added, and extracted with dichloromethane (3x25cm3). The dichloromethane extracts were washed with water (6x25cm3), brine (30cm3), and dried (MgSO4). Removal of solvent in vacuo gave a red residue, which was recrystallised form dichloromethane-light petroleum to afford the title compound (0.013g, 84%) as a red solid, mp 184C (decomp), (Found: M+, 313.1064. C16H15N3O4 requires M, 313.1063); ~max (MeOH) 239 (log F 4.45), 308 (4.30), and 490nm (3.39); vmax (CHC13) 3532,3424, 1724, 1662, 1632, and 1582cm~l; ~H (400MHz, CDC13) 5.72 (lH, s, 6-H) 5.27 (2H, AB, J 12.5Hz, 10-CH2), 4.62 (2H, br s, NH2), 4.24 (2H, m, 3-CH2), 2.53 (lH, m, l-H), 2.34 (lH, m, 2-H), 2.18 (4H, s, 2xCH2-N [aziridine]), 1.29 (lH, m, la-CHH), and 0.55 (lH, m, la-CHH); ~C (100.6MHz, CDC13) 179.08 (CO, 8-C), 177.48 (CO, 5-C), 157.73 (C/CO, 7-C/urethane), 156.77 (CO/C, urethane/7-C), 146.16 (C, 4a-C), 127.40 (C, 9a/8a-C), 126.50 (C, 8a/9a-C), 115.57 (CH, 6-C), 111.20 (C, 9-C), 57.85 (CH2, 10-C), 49.90 (CH2, 3-C), 27.51 (CH2, CH2-N [aziridine]), 20.59 (CH, l-C), 16.16 (CH2, la-C), and 14.65 (CH, 2-C); m/z 313 (M+, 16X), 270 (27), 251 (17), 77 (9), and 41 (100).
WO 94/13674 PCT/GB93102~33 ~ 2i ~0~11 Example 2 Me /~N~NH2 9-Hydroxymethyl-7-(2-methylaziridin-1-yl)-1,2-dihydro-3H-1,2-cycloPro-papyrrolo-tl~2-a]indole-5~8-dione carbamate A solution of 9-hydroxymethyl-7-methoxy-1,2-dihydro-3H-l, 2-cyclopropapyrrolotl,2-a]indole-5,8-dione carbamate (prepared as described in Example 1 steps a) to m)) (O.OlOg, 0.033mmol) in dry DMF (1.5cm3) was treated with an excess of 2-methylaziridine (0.25cm3 initially + 0.25cm3 24h later), and this mixture was stirred at room temperature under nitrogen for 48 h. After this time, water (5cm3) was added, and extracted with dichloromethane (3x25cm3). The dichloromethane extracts were washed with water (5x25cm3), brine (30cm3), and dried (MgS04). Removal of solvent in vacuo gave a red residue, which was purified by column chromatography (eluting with ethyl acetate) to give a red solid.
Recrystallisation of the red residue from dichloromethane-light petroleum gave the title comDound (0.00759, 68%) as a red solid, m.p. 176-178C; (Found: _+, 327.1219. C17H17N304 requires M, 327.1219); ~max (MeOH) 240 (log ~ 4.34), 314 (4.17), and 499nm (3.2g); vmax (CHC13) 3544, 3424, 1724, 1662, 1630, 1580, 1496, 1338, and 1256cm~l; ~H (250MHz, CDC13+TMS) 5.70 (lH, s, 6-H) 5.29 (2H, m, 10-CH2), 4.64 (2H, br s, NH2), 4.25 (2H, m, 3-CH2), 2.52 (lH, m, l-H), 2.27 (2H, m, 2-H+MeCH-N), 2.08 (2H, m, CH2-N
taziridine]), 1.42 (3H, d, J 5.5Hz, Me), 1.30 (lH, m, la-CHH), and 0.55 (lH, m, la-CHH); m/Z 328 (MH+, 20%) 327, (M+, 100).
Example 3 ~N~$NH2 9-Hydroxymethyl-7-(pyrrolidin-1-yl)-1,2-dihydro-3H-1,2-cyclopr~pa-pyrrolo-[1,2-a]ind~le-5,8-dione carbamate A solution of 9-hydroxymethyl-7-methoxy-1,2-dihydro-3H-1,2-cyclopropapyrrolo[l,2-a]indole-5,8-dione carbamate (prepared as described in Example 1 steps a) to m)) (O.OlOg, 0.033mmol) in dry DMF (1.5cm3) was treated with distilled pyrrolidine (0.008cm3, O.O99mmol), and this mixture was stirred at room temperature under nitrogen for 15h. After this time, water was added (5cm3), and extracted with dichloromethane (3x25cm3). The dichloromethane extracts were washed with water (5x25cm3), brine (30cm3), and dried (MgS04) Removal of solvent in vacuo gave a purple residue, which was recrystallised from dichloromethane-light petroleum to afford the title compound (0.008g, 73Z) as a purple solid, m.p.
210C (decomp); (Found: M+, 341.1376. C18HlgN304 requires M, 341.1376); ~max (MeOH) (qualitative) 247, 328, and 549nm; vmax (CHC13) 3612, 3408, 1726, 1686, 1608, 1544, 1498, and 1228cm~l;
~H (250MHz, CDC13+TMS-@ 45C) 5.31 (2H, m, 10-CH2) 5.19 (lH, s, 6-H), 4.55 (2H, br s, NH2), 4.29 (2H, m, 3-CH2), 3.64~(4H, br m, 2 x CH2 [pyrrolidine ring]), 2.49 (lH, m, l-H), 2.30 (lH, m, 2-H), 1.95 (4H, m, 2 x CH2[pyrrolidine ring]), 1.27 (lH, m, la-CHH), and 0.55 (lH, m, la-CHH~; ~C (100.6 MHz, CDC13) 180.25 (CO, 8-C), 176.63 (CO, 5-C), 159.47 (C/CO, 7-C/urethane), 156.6Z
(COIC, urethane/7-C), 144.49 (C, 4a-C), 128.43 (C, 9a/8a-C), 122.91 (8a/9a-C), 109.75 (9-C), 100.16 (CH, 6-C), 58.36 (CH2, 10-C), 51.21 (CH2, 2 x CH2N [pyrrolidine ring]), 49.73 (CH2,3-C), 29.60 (CH2, 2xCH2-pyrrolidine ring), 20.68 (l-C), 16.24 (CH2, la-C), and 14.40 (CH, 2-C); m/Z 341 (_+, 4%), 279 (52), 91 (30), and 44 (100).
-PCT/GB93/02~33 ~ WO 94/13674 2 ~ ~ d 711 [~Heated this compound to 45C in CDC13. The peak at 3.64ppm went from a broad peak to a sharper multiple at this temperature.]
Example 4 ~ -(Aziridin-l-yl)-9-hydroxymethyl-1.2-dihydro-la.la-dimethyl-3H-l, 2-cvclonropapyrrolorl.Z-a]indole-5.8-dione carbama~e ~ N ~ OCONH~
~N~D< Me The title compound was prepared starting from o-vanillin by repeating steps a) to e) of Example 1 followed by the following further steps f) to n) f) 4-Benzyloxy-5-methoxy-1-(2-methyl-2-butenyl)indole-2-carboxalde-hyde To a flask charged with sodium hydride (80%, 0.3009, lO.OOOmmol) was added dry light petroleum (5cm3). The mixture was stirred for 10 min, the petroleum removed by syringe, and the flask contents dried under vacuum. 4-Benzyloxy-5-methoxyindole-2-carboxaldehyde (prepared as described in steps a) to e) of Example 1) (2.3409, 8.327mmol) in DMF (170cm3) was added dropwise, and the mixture was stirred at room temperature for 45 min.
4-Bromo-2-methylbutene (1.15cm3, lO.OOOmmol) was added, and the mixture was stirred at room temperature. After 15h, water (20cm3) was cautiously added, and the mixture was extracted with ethyl acetate (3xl50cm3). The combined extracts were washed with water (8xl50cm3), brine (150cm3), dried (MgS04), and evaporated to dryness. The residue was purified by column chromatography (eluting with 50% ether-50%1ight petroleum) to give the title compound (1.8949, 65Z) as a pale yellow solid, m.p. 55-57C, WO 94/13674 21~ ~ 7 ~1 PCT/GB93/02533 (Found: C, 75.4; H, 6.6; N, 3.9. C22H23N03 requires C, 75.6; H, 6.6; N, 4.0%); vmax. (CHC13) 1666, 1516, 1488, 1460, 1290, 1248, and 1140cm~l; ~H (250MHz; CDC13+TMS) 9.79 (lH, s, CHO), 7.51-7.00 (8H, m, 5 Ar-H+3, 6, 7-H), 5.27 (2H, s, OCH2Ph), 5.18 (3H, m, NCH2CH.CMe2 and CH2CH~CMe2), 3.91 (3H, s, OMe), 1.86 (3H, s, Me), and 1.68 (3H, s, Me); m/Z 350 (MH+, 7%), 349 (M+, 28), 258 (41), 190 (100), 91 (60), 69 (93), and 41 (71).
g) 4-Benzyloxy-5-methoxy-1-(2-methyl-2-butenyl)indole-2-carboxaldehyde tosylhydrazone 4-Toluenesulphonylhydrazide (97%, 1.516g, 8.141mmol) ~n dry methanol (ZOcm3) was added to a stirred solution of 4-benzyloxy-5-methoxy-1-(2-methyl-2-butenyl)indole-2-carboxaldehyd e (1.894g, 5.427mmol) in dry methanol (30cm3). After stirring at 40C for 15h, the solvent was removed in vacuo, and the residue was purified by chromatography (70% ether-30% light petroleum) to give a cream foam. Recrystallisation of this foam from dichloromethane-light petroleum gave the tltle compound (1.681g, 60%) as a colourless solid, m.p. 77-79C, (Found: C, 67.0; H, 6-0;N, 8-2- C2gH31N3O45 requires C, 67.3; H, 6.0; N, 8.1%); vmax (CHC13) 3184, 1608, 1598, 1516, 1490, 1454, 1344, and 1166cm~l;
~H (250MHz; CDC13+TMS) 7.82 (2H, m, Ar-H[Ts-AA'BB']), 7.74 (2H, br s, NH and -CH-N-), 7.47 (2H, m, Bn-H), 7.36-7.29 (5H, m, 3 Bn-H+2 Ar-H [Ts-AA'BB']), 7.03 (lH, d, J8.8Hz, 7/6-H), 6.94 (lH, d, J8.8Hz, 6/7-H), 6.66 (lH, s, 3-H), 5.19 (2H, s, OCH2Ph), 5.05 (3H, m, NCH2CH=CMe2+-CH2CH=CMe2), 3.88 (3H, s, OMe), 2.40 (3H, s, Ar-Me), 1.84 (3H, s, Me), and 1.69 (3H, s, Me); m/Z (FAB, 3-NBA
Matrix) 518 (MH+, 55%), 426 (100), and 242 (20).
WO 94/13674 ~15 ~ 7 1 1 PCT/GB93/02533 h) 8-~enzyloxy-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-l, Z-cyclopro,~aDyrrolo-[1,2-a]indole Sodium hydride (80%, 0.032g, 1.066mmol) was added to a stirred solution of the tosylhydrazone of g) above (0.3699, 0.711mmol) in dry THF (llcm3). After 20 min, the solution was filtered, and the filtrate evaporated. The residue was dissolved in dry chlorobenzene (105cm3), and the solution refluxed for 3.5 h. The solvent was evaporated, and the residue purified by chromatography (gradient elution-started with 100% light petroleum and going down in 5% intervals to 60% 1ight petroleum-40% ether) to give the title compound (0.1439, 60%) as a brown oil, (Found: Mt, 333.1729. C22H23N02 requires M, 33.1729); vmax (CHC13) 1570, 1490, 1452, 1432, 1262, and 1232cm~l, ~H (250MHz; CDC13+TMS) 7.56-7.53 (2H, m, Bn-H), 15 7.38-7.26 (3H, m, Bn-H), 6.85-6.78 (2H, 2d, J8.6Hz, 5+6-H), 6.21 (lH, s, 9-H), 5.22 (2H, s, OCH2Ph), 4.13 (lH, dd, J6.1, 10.8Hz, 3-CHH), 3.90 (3H, s, OMe), 3.87 (lH, m, 3-CHH), 2.28 (lH, dd, JO.5, 6.8Hz, l-H), 2.09 (lH, t, J7.0Hz, 2-H), 1.19 (3H, s, Me), and 0.67 (3H, s, Me); m/Z 334 (MH+, 8~), 333 (M+, 31), 242 (100), 20 200 (13), 91 (17).
~) 8-Benzyloxy-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclop ro-papyrrolo-~1,2-a]indole-9-carboxaldehyde N-Methylformanilide (0.5cm3), and phosphorus oxychloride (0.38cm3), were stirred under a calcium oxide tube for 10 min.
The resulting precipitate was cooled to 0C. This yellow precipitate (0.150g, N-methylformanilide 0.070g, 0.515mmol, and phosphorus oxychloride 0.080g, 0.0515mmol) was added to 8-benzyloxy-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclopropa pyrrolo[l,2-a]indole (0.143g, 0.430mmol) in 1,2 dichloroethane 30 (2cm3). The mixture was refluxed for 1.25 h. Sodium acetate (lM, 9cm3) was added, and the mixture extracted with ethyl acetate (3x50cm3). The combined extracts were washed with water (2x50cm3), brine (50cm3), dried (MgS04~, and evaporated.
Purification of the residue by column chromatography (eluting W 0 94t13674 ~ ~ PCT/GB93/0~533 with ether) gave the title compound (0.118g, 76%) as a yellow oil, (Found: M+, 361.1678. C23H23N03 requires M, 361.1678); vmax (CHC13) 1666, 1596, 1526, 149Z, 1452, and 696cm~l, ~H (250MHz, CDC13+TMS) 10 28 (lH, s, CHO), 7.49-7.15 (5H, m, Bn-H), 6.87 (2H, 2d, J 8.6 Hz, 5+6H), 5.18 (2H, s, OCH2Ph), 4.13 (lH, dd, J 6.4, 11.8 Hz, 3-CHH), 3.91 (3H, s, OMe), 3.86 (lH, m, 3-CHH), 2.86 (lH, d, J 6.3Hz, l-H), 2.20 (lH, t, J 6.3Hz, 2-H), 1.26 (3H, s, Me) and 0.68 (3H, s, Me); ml~ 362 (MH+, 12%), 361 (_+, 28%), 270 (100), 228 (32), and 91 (45).
~) 8-HydroxY-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-l, 2-cycloproDaDyrrolo-[1,2-a]indole-9-carboxaldehyde.
To a solution of 8-benzyloxy-7-methoxy-1,2-dihydro-la, la-dimethyl-3H-1,2-cyclopropapyr-rolo[1,2-a]indole-9-carboxaldehyd e (0.300g, 0.831mmol) in ethyl acetate (160cm3) was added 10%
palladium on carbon catalyst (0.065g), dilute sulphuric acid (10 drops), and the mixture was stirred under an atmosphere of hydrogen. After 2.5 h the sùspension was filtered and washed with dichloromethane (ca 150cm3). The organic layer was extracted with water (3x50cm3), brine (50cm3), dried (MgS04), and evaporated.
Purification of the residue by column chromatography (gradient elution:-started with 507. light petroleum: 50% ether-100~0 ether, in 10% intervals) gave the title com~ound (0.123g, 557.) as a colourless solid, m.p. 119-121C, (Found: _+, 271.1208. C16H17N03 requires M, 271.1208); vmax (CHC13) 1606 (br), 1300, and 1252cm~l, ~H (250MHz, CDC13+TMS) 10.90 (lH, s, CHO), 9.54 (lH, s, ~H), 6.89 (lH, d, J8.5Hz, 6.5-H), 6.55 (lH, d, J8.5Hz, 5.6-H), 4.18 (lH, dd, J6.35, 12.3Hz, 3-CHH), 3.91 (3H, s, OMe), 3.86 (lH, m, 3-CHH), 2.60 (lH, dd, Jl.4, 6.6Hz, l-H), 2.34 (lH, t, J6.5Hz, 2-H), 1.29 (3H, s, Me), and 0.77 (3H, s, Me); _/z Z73 (MH2+, 1770), 272 (MH+, 57), 271 (M+, 100).
W 0 94/13674 ~ 1 5 ~ 7 1 1 PCT/GB93/02533 k) 9-FormYl-7-methoxY-1,2-dihydro-la,la-dimethYl-3H-1~2-cvclopropapyrrolo[l,2-a]indole-5,~-dione Potassium nitrosodisulphonate (0.2689, 0.998mmol) in water (13cm3) was added to a stirred solution of 8-hydroxy-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclopropapy rrolo[l,2-a]indole-9-carboxaldehyde (0.123g, 0.454mmol) in acetone (44cm3). The mixture was buffered with sodium dihydrogen phosphate solution (0.167M, 13cm3). After stirring at room temperature for 12 h, the mixture was concentrated in vacuo, filtered, and the residue recrystallised from dichloromethane-light petroleum to give the title compound (O.lllg, 86%), as orange crystals, m.p. 246-248C, (Found: M+, 285-0999- C16HlsN04 requires M, 285-1001); vmax (CHC13 1678,1640, 1596, 1506, 1238, and 1136cm~l; ~H(250MHz, CDC13+TMS) 10.37 (lH, s, CHO), 5.69 (lH, s, 6-H), 4.32 (lH, dd, J 6.4, 14.1Hz, 3-CHH), 4.18 (lH, d, J 14.2Hz, 3-CHH), 3.85 (3H, s, OMe), 2.71 (lH, dd, Jl.5, 6.5Hz, l-H), 2.22 (lH, m, 2-H), 1.26 3H, s, Me), and 0.72 (3H, s, Me); m/z 286 (MH+, 20Z), 285 (_+, 100), and 270 (69).
1) 9-Hydroxymethyl-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclopropaDyrrolo-[1,2-a]indole-5,8-dione Sodium borohydride (0.0719, 1861mmol) was added to a stirred solution of 9-formyl-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclopropapyrrolo[l,2-a] indole-5,8-dione (0.078g, 0.274mmol), in methanol (53cm3), and the solution rigorously degassed with nitrogen. After stirring for 4 h at room temperature under nitrogen, air was blown rapidly through the solution for 5 min.
The mixture was transferred to a separating funnel, and extracted with dichloromethane (3xlOOcm3), washed with brine (lOOcm3), dried (MgS04), and evaporated. Purification of the residue b column chromatography (gradient elution:-started with 50h ethvl acetate: 50% light petroleum-100/O ethyl acetate) gave the title compound (0.0549, 69%), as a red solid, m.p. 162-164C, (Found:
WO 9~/13674 ~, ~ 5 ~ 7 11 PCT/GB93/0~533 M+, 287.1158. C16H17N04 requires M, 287.1158); vmax (CHC13) 3468, 1658, 1636, and 1594cm~l; ~H (250MHz, CDC13+TMS) 5.61 (lH, s, 6-H), 4.64 (2H, m, 10-CH2), 4.28 (lH, dd, J 6.3, 14.0Hz, 3-CHH), 4.08 (lH, d, J 14.0Hz, 3-CHH), 3.82 (3H, s, OMe), 2.20 (lH, d, J 6.9Hz, l-H), 2.08 (lH, t, J 6.5Hz, 2-H), 1.19 (3H, s, Me), and 0.72 (3H, s, Me); mlz 289 (MH2+, 8%), 288 (MH+, 19), and 287 (M+, 100).
m) 9-Hydroxymethyl-7-methoxY-1,2-d.hydro-la,la-dimethyl-3H-1,2-cyclopropapyrrolo-[1,2-a]indole-5,8-dione carbamate Phenyl chloroformate (0.04cm3, 0.28mmol), was added dropwise to a stirred, ice cold solution of the alcohol of step 1) above (O.OSOg, 0.17mmol) in distilled pyridine (llcm3). The mixture was stirred at room temperature for 2 h, then water (5cm3) was added.
The mixture was extracted with dichloromethane (3x50cm3), and the combined extracts were washed with water (3x50cm3), brine (lOOcm3), and dr~.ed (MgS04). The solvent was evaporated, and the residue pur.fied by column chromatography (eluting with ether) to give an orange gummy solid.
A solution of this phenyl carbonate in dry dichloromethane (30cm3), was cooled to -78C. Ammonia gas was bubbled into the solution for 30 min (calOOcm3), after which the contents were allowed to warm to room temperature, and the solvent was evaporated. Recrystallisation of the residue from dichloromethane-light petroleum gave the title com~ound (0.040g, 10~.) as an orange crystalline solid, m.p. 204-2060C, (Found: _+, 330.1216. C17H18N2C5 requires M, 330.1216); ~max (MeOH) 240 (log 4.32), 292 (4.21), 348 (3.57), and 474nm (3.24); vmax (CHC13) 3540, 3428, 1724, 1670, 1636, 1594, 1496, and 1232cm~1; ~H
(250MHz, CDC13+TMS) 5.59 (lH, s, 6-H), 5.28 (2H, m, 10-CH2), 4.59 (2H, br s, NH2), 4.28 (lH, dd, J6.3, 14.0Hz7 3-CHH), 4.08 (lH, d, J14.0Hz, 3-CHH), 3.80 (3H, s, OMe), 2.37 (lH, d, J6.9Hz, l-H), 2.09 (lH, t, J6.0Hz, 2-H), 1.19 (3H, s, Me), and 0.69 (3H, s, Me); mtz 330 (M+, 15%), Z87 (100), 273 (83), 254 (33), 228 (18), and 51 (14).
n) 7-(Aziridin-l-yl)-9-hydroxymethyl-1,2-dihydro-la,la-dimethyl -3H-1,2-cyclopropapyrrolo[1,2-a]indoie-dione carbamate.
A solution of 9-hydroxymethyl-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclopropa-pyrrolo[1,2-a]indole-5,8-dione carbamate (O.OlOg, 0.030mmol) in distilled DMF (1.5cm3) was treated with distilled aziridine (0.0049, O.O91mmol), and the mixture was stirred at room temperature under nitrogen for 48 h.
After this time, water (5cm3) was added, and the mixture extracted with dichloromethane (3x25cm3). The dichloromethane extracts were washed with water (8x25cm3), brine (30cm3), and dried (MgS04). Removal of solvent in vacuo gave a red residue, which was recrystallised from dichloromethane-light petroleum to afford the title compound (0.0075g, 75%), as a red solid, m.p.
169-171C, (Found: M+, 341.1376. C18HlgN304 requires M, 341.1376); ~max (MeOH) (qualitative) 241, 313, 495nm; vmax (CHC13) 3540, 3430, 1725, 1665, 1630, 1580, 1335, and 1255cm~l;
~H (250MHz, CDC13+TMS) 5.73 (lH, s, 6-H), 5.27 (2H, m, 10-CH2), 4.69 (2H, s, NH2), 4.26 (lH, dd, J 6.3, 14.0Hz, 3-CHH), 4.06 (lH, d, J14.0Hz, 3-CHH), 2.37 (lH, dd, J 1.2, 6.9Hz, l-H), 2.19 (4H, s, 2 x CH2-N [aziridine ring]), 2.09 (lH, t, J 6.5Hz, 2-H), 1.19 (3H, s, la-CCH3CH3), and, 0.68 (3H, s, la-CCH3CH3); _/~ 341 (M+, 14X), 298 (22), 265 (14), 91 (7), and 44 (100).
Example 5 ~ CO H2 W O 9~/13674 21~ 0711 PCT/GB93/0~53~ -9-Hydroxymethyl-7-(pyrrolidin-1-YI)-1.2-dihydro-la,la-dimethYl-3H-1.2-cyclopropapyrrolo[1.2-a]indole-5.8-dione carbamate A solution of 9-hydroxymethyl-7-methoxy-1,2-dihydro-la, la-dimethyl-3H-1,2-cyclopro-papyrrolo[1,2-a]indole-5,8-dione carbamate (prepared as described in Example 1 steps a) to e) and Example 2 steps f) to m)) (O.OlOg, 0.030 mmol) in distilled DMF) 1.5cm3) was treated with pyrrolidine ~0.008cm3, 0.091 mmol), and the mixture was stirred at room temperature under nitrogen for 15 h. After this time, water (5cm3) was added, and extracted with dichloromethane (3x25cm3). The dichloromethane extracts were washed with water (8x25cm3), brine (30cm3), and dried (MgS04) Removal of solvent in vacuo gave a purple residue, which was purified by column chromatography (gradient elution: 50% ethyl acetate: 50/0 light petroleum-100% ethyl acetate) to give a purple residue. Recrystallisation of the purple residue from dichloromethane-light petroleum gave the title compound (0.008g, 73%), as a purple solid, m.p. 202C (decomp), (Found: M+, 369.1688. C20H23N304 requires M, 369.1699); ~max. (MeOH) 254 (log~ 4.40), 330 (4.06), and 549nm (3.52); vmaX (CHC13) 3540, 3424, 1724, 1662, 160Z, 1544, 1498, and 1266cm~1; ~H (250MHz~
CDC13+TMS) 5.28 (2H, m, 10-CH2), 5.17 (lH, 5, 6-H), 4.59 (2H, s.
NH2), 4.29 (lH, dd, J 6.3, 14.0Hz, 3-CHH), 4.10 (lH, d, J 14.0Hz.
3-CHH), 3.59 (4H, br, 2xCH2-N[pyrrolidine ring]*), 2.33 (lH, d, J
6.9, l-H), 2.04(1H, t, J 5.9Hz, 2-H), 1.94 (4H, m, 2xCH2[pyrrolidine ring]), 1.17 (3H, s, la-CCH3CH3), and 0.67 (3H, s, CCH3CH3); m/~ 369 (M+, 9/.), 308 (lOO),and 293 (81).
Noting-*lHNMR (250MHz, CDC13+TMS). In a second experiment, heated title compound to 45C in CDC13+TMS. The peak at 3.59ppm went from a broad band to a sharper multiple at this higher temperature.
WO 9~/13671 PCT/GB93/0'5~
2t3~7l1 Example 6 - Bioloqical Testinq A) The aerobic toxicity of the compound of Example 1 was demonstrated in V79 cells and compared with that shown by mitomycin C - comparison compound a) ("Comp a") and by S 9-hydroxymethyl-7-methoxy-1,2-dihydro-3H-1,2,-cyclopropapyrrolo[l, 2-a]-indole-5,3-dione carbamate (prepared as described in steps a) to m) of Example 1) - comparision compound b) ("Comp b").
The toxicity was determined by exposure to cells for 3 hours at 37C in air followed by MTT assay as described by Stratford et al, Int. J. Radiat. Oncol. Biol. Phys. (1989), 16, 973.
The results are as shown in Figure 1 of the drawings.
B) The effect of adding the enzyme inhibitor dicoumarol to th~
cells during hypoxic exposure to the compound of Example 1 and to comparison compound b) was measured as follows and the results lS are shown in Figures 2 and 3 and show that substantial protection is observed, thus implying that DT-diaphorase is important with regard to the activation of the compound of Example 1 in air.
This suggests that the compound can find application in treating cell types with high levels of DT diaphorase.
C) The effect of the compound of Example 1 in killing both aerobic and hypoxic cells in contrast to comparison compound b) was measured as follows and the results are shown in Figures and 5 and demonstrate a marked change in behaviour between the two compounds.
D) The toxicity of the compound of Example 1 was compared with that of comparison compound a) (mitomycin C) for a range of cell lines chosen for their differing repair capabilities.
The results are shown in Table 1 below.
W O 91/13674215 ~7 11 PCT/GB93/0~53~ -Table 1 Cell lineConcentration (~M) to kill 90X cells follo~ing 3 hours in air Comp. a) Ex.l V79 (wild type) 1 0.02 IRS-l (DNA strand break repair 0.02 <0.002 deficient: radiation sensitive) CH0-Kl (wild type) 1.5 0.2 CH0-MMCR 25 0.8 (low in P450 reductase) AA8 (wild type) 1 0.06 UV41 (cross link repair 0.02 0.003 deficient) As can be seen from Table 1, the compound of Example 1 shows collateral sensitivity to Mitomycin C in both the IRS-l and UV41 cells, indicating the nature of the molecular damage caused by these drugs may be similar. Although the Example 1 compound is always more potent, it is least effective in killing MMCR cells but the degree of resistance (MMCR vs CH0-Kl) is much less than that observed for mitomycin C and probably indicates that it is less dependent on P450 reductase for activation.
E) The in vitro toxicity of the compound of Example 1 and mitomycin C to murine bone marrow cells was investigated and the results are shown in Table 2 below:
Table 2 Drug IC50 (yM)~
Mitomycin C 1-0 Example 1 compound 0.3 ~150711 * Concentration required to reduce survival by 50% following exposure for 3hrs at 37C in air The results show that bone marrow toxicity is dose limiting for the use of Mitomycin C. However, in vitro the difference in S toxicity between the compound of Example 1 and Mitomycin C is only a factor of 3, which is far less than the difference in potency seen for the other cell types in Table 1. This is a positive advantage in favour of the compound of Example 1.
-
~ I =~j I
G
~ o _ O O
IL ,~ ~ ~ C
~ CD Z, a /
s~ t ~ t t -D 0~ CV ~ ~
'~ O G
O
O
c~ G /
0=~
, 0~ G
O ~-~ O ~ Z
c~ ~ cV Ir ~
W o 94/13674 21 5 ~1 1 PCT/GB93/0253~
In the above reaction scheme, Rl to R5 have the meanings defined above for formula II and R6 is -COCH3 or -CONH2. When R6 is -COCH3, it may be introduced by reaction of the alcohol with acetic anhydride in pyridine. When R6 is -CONH2 it may be introduced by reaction of the alcohol with phenyl chloroformate followed by reaction wirh ammonia. TsNHNH2 is 4-toluenesulphonyl hydrazide and Fremy's salt is potassium nitrosodisulphonate.
The invention therefore includes a method of preparation of compounds of formula II comprising reaction of a compound of formula III
MeO ~
l5 0 N ~ RR5 (lll) where R3, R4, R5 and Z are as defined above, with a secondary amine of formula RlR2NH where Rl and R2 are as defined above in the presence of a suitable solvent, for example, dimethylformamide.
Preferably Z in the compound of formula III is -CONH2 and the compound of formula III has been prepared by reaction of a compound of formula IV
MeO~R~ ~IV) 21~ 6-where R3, R4 and R5 are as defined above, with phenyl chloroformate followed by reaction with ammonia.
Compounds of formula II and salts thereof may find application as antitumour agents. The invention therefore further includes pharmaceutical compositions comprising at least one compound of formula II, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.
Suitable carriers will depend on the way in which the composition is to be administered. The for example, the compounds of formula II may be used in a composition, for example an aqueous, oily or emulsified composition, incorporating a liquid diluent which may be intended to be employed in injectable form for parenteral administration and is therefore sterile and pyrogen free.
Alternatively the composition may be suitable for oral administration, in which case the carrier is conventionally a solid material such as starch, lactose, dextrin or magnesium stearate. Such solid compositions may conveniently be formed into tablets or capsules. Other forms of administration may be used, for example suppositaries or pessaries.
The dosage level employed will depend on the mode of formulation and intended use. Thus, for example, if the composition is to be administered intravenously, a suitable dosage may lie in the range of 0.001 to 0.5mg/kg of active ingredient at intervals which may vary from 24 hours to several weeks.
Preparation of compounds in accordance with the invention will now be described by way of example.
ExamDle 1 7-(Aziridin-l-yl)-9-hYdroxvmethyl-1.2-dihYdro-3H-1.2-cycloproDapyrrolo [1.2-a]indole-5.8-dione carbamate 3a ~ OCONH2 W o 94/13674 2 1 5 ~ 7 1 1 PCT/GB93/02533 The title compound was prepared starting from Q-vanillin by steps a) to n) as described below:
a) 2-Benzyloxy-3-methoxybenzaldehyde Potassium hydroxide pellets (40.6g, 724mmol) were added to a stirred solution of Q-vanillin (lOO.Og, 657mmol) in ethanol (99.7%, 590cm3), followed by benzyl chloride (83.45cm3, 723mmol).
The stirred mixture was refluxed for 12 h, then water (lOOcm3) was added, and the mixture extracted with ether (3x750cm3). The ethereal extracts were washed with water (3x200cm3), potassium 10 hydroxide solution (2M, 5x500cm3), again with water (2xlSOcm3), then brine (lSOcm3). The organic layer was dried (MgS04), then condensed in vacuo, to give the title compound (9.2g, 62%) as a colourless solid on trituration wlth light petroleum; vmax (Nujol) 1697, 1584, 1367, 1265, 1247, and 1221cm~l; ~H(270MHz, 15 CDC13) 10.24 (lH, s, CHO), 7.41-7.14 (8H, m, Ar-H), 5.18 (2H, s, OCH2Ph), and 3.95 (3H, s, OMe); mL~ 243 (MH+, 11%), 242 (M+, 62), 213(27), and 91 (100).
b) Methyl 2-Azido-3-(2-benzyloxv-3-methoxyphenyl)propenoate Sodium metal (12.54g, 545 mmol) was added to dry methanol 20 (300cm3). The solution was cooled to -15C, and a solution of methyl azidoacetate (61.00g, 530mmol) and 2-benzyloxy-3-methoxybenzaldehyde (33.079, 137mmol) in dry methanol (25cm3) was added dropwise by syringe. The mixture was stirred at -10C for 3 h then at +4C for 12 h. Water (lOOcm3) was cautiously added to the mixture, which was then extracted with ethyl acetate (3xSOOcm3). The combined extracts were washed with water (3xSOOcm3), brine (300cm3), and dried (MgS04). Removal of the solvent in vacuo gave a pale yellow residue, which was triturated with ether to give the title com~ound (38.03~, 82%) as 30 pale yellow crystals; vmaX (Film)2121, 1712, 1456, 1378, 1260, and 1218cm~1; ~H (270MHz; CDC13) 7.77 (lH, dd, J 1.4, 8.0Hz Ar 6-H), 7.44-7.27 (SH, m, Bn-H), 7.26 (lH, s, CH=), 7.11 (lH, t, J
8.0 Hz, 4/5-H), 6.95 (1 H, dd, J 1.5, 8.0 Hz, 5/4-H), 5.00 (2 H, s, OCH2Ph), 3.90 (3H, s, C02Me), and 3.85 (3 H, s, ArOMe).
c) Methyl 4-Benzyloxy-5-methoxyindole-2-carboxylate A solution of methyl 2-azido-3-(2-benzyloxy-3-methoxyphenyl) propenoate (2.2089, 6.513mmol), in dry xylene (48cm3) ~as introduced, dropwise, by means of a pressure e~ualising dropping funnel, to refluxing dry xylene (200cm3). After the addition was complete (ca. 30min), the solution was refluxed for a further 45 min. Removal of solvent in vacuo gave a yellow solid residue, which was recrystallised from ether to give the title compound (1.323g, 65%) as a colourless solid; ''max (CHC13) 3464, 3013, 1707, 1533, 1453, and 1253cm~l;~H (400MHz; CDC13) 9.36 (lH, s, NH), 7.54 (2H, d, J 7.1 Hz, Bn-H), 7.53-7.29 (4H, m, 3Bn-H+3-H), 7.09 (lH, s, 6+7-H), 5.29 (2H, s, OCH2Ph), 3.94 (3H, s, MeO/C02Me), and 3.89 (3H, s, C02Me/OMe).
d) 4-Benzyloxy-5-methoxyindole-2-methanol A solution of methyl 4-benzyloxy-5-methoxyindole-2-carboxylate (6.975g, 22.4mmol) in dry THF (200cm3) was added dropwise to a stirred suspension of lithium aluminium hydride (1.709g, 44.8mmol) in dry THF (85cm3), such that the mixture achieved gentle reflux. After 30min, water (1.7cm3), 15% sodium hydroxide (1.7cm3), and water again (5.1cm3), were added to the mixture, and the resultant precipitate removed by flltration (through a bed of Celite). The filtrate was dried (MgS04), then condensed 1n vacuo to give the title com~ound (5.8169, 92%) as a colourless solid; vmax (Nujol) 3476, 3284, 1504, 1324, 1284, 1244, 1090, 1016, and 702cm~l; ~H (250MHz; CDC13+TMS) 8.25 (lH, br s, NH), 7.52 (2H, m, Bn-H), 7.41-7.30 (3H, m, Bn-H), 7.00 (1 H, d J 8.4 Hz, 7/6-H), 6.91 (1 H, d, J 8.6 Hz, 6/7-H) 6.40 (1 H, s, 3-H), 5.20 (2 H,s, CH20Ph), 4.75 (2H, s, CH20H), and 3.88 (3H, s, OMe).
~5~7~1 e) 4-Benzvloxy-5-methoxyindole-2-carboxaldehyde Manganese dioxide (17.40g, 200.00mmol) was added, in portions to a stirred solution of 4-benzyloxy-5-methoxy-4-methylindole-2-methanol (5.829, 20.56mmol) in dry dichloromethane (650cm3). The suspension was then refluxed for 12h. The mixture was filtered, and the residue washed with hot dichloromethane (3x300cm3). The combined filtrate and washings were evaporated to give an oil which was purified by column chromatography to give the title compound (3.56g, 62X) as a pale yellow; vmax (Nujol) 3458, 3015, 2837, 1663, 1531, and 1143cm~l; ~H (400MHz; CDC13) 9.76 (lH, s, CHO), 9.25 (lH, s, NH), 7.49 (2H, m, Bn-H), 7.39-7.30 (3H, m, Bn-H), 7.24 (lH, dd, J 0.8, 2.2Hz, 3-H), 7.17 (lH, d, J 8.9Hz, 6-H), 7.12 (lH, dd, J 0.8, 8.9Hz, 7-H), 5.28 (2H, s, CH20Ph), and 3.91 (3H, s, OMe).
f) 1-Allvl-4-benzyloxy-5-methoxyindole-2-carboxaldehYde To a flask charged with sodium hydride (80%, 0.475g, 15.84mmol) was added dry light petroleum (10cm3). The mixture was stirred for 10 min, the petroleum removed by syringe, and the flask contents dried under vacuum. 4-Benzyloxy-5-methoxyindole-2-carboxaldehyde (3.562g, 12.68mmol) in DMF (260cm3) was added dropwise, and the mixture was stirred at room temperature for 3G
min. Allyl bromide (1.907g, 15.76mmol) was added, and the mixture was stirred at room temperature. After lh, water (150cm3) was cautiously added, and the mixture was extracted with ethyl acetate (4x250cm3). The combined extracts were washed with water (8x200cm3), brine (200cm3), dried (MgS04), and evaporated to give the title compound (4.070g, 99%) as a brown solid; vmax (Nujol) 1670, 1490, 1406, 1250, and 1140cm~l; ~H (250MHz; CDC13+TMS) 9.79 (lH, s, CH0), 7.51 (2H, m, Bn-H), 7.38 (3H, m, Bn-H), 7.25 (lH.
s, 3-H), 7.19 (lH, d, J 9Hz, 7/6-H), 7.04 (lH, d, J 9Hz, 6/7-H), 5.96 (lH, m, CH2CH=CH2), 5.28 (2H, s, CH20Ph), 5.13 (3H, m, CH2CH=CH2 and CH2CH=CHH-cis), 4.89 (lH, dd, J 1.0, 17.1Hz, CH2CH=CHH-trans), and 3.92 (3H, s, OMe).
2~
g) l-Allyl-4-benzyloxy-5-methoxyindole-2-carboxaldehyde tosylhydrazone l-Allyl-4-benzyloxy-5-methoxyindole-2-carboxaldehyde (3.095g, 9.642mmol) was added to a stirred solution of 4-toluenesulphonyl hydrazide (2.334g, 12.539mmol) in methanol (25cm3). After stirring at 40C for lh, the solvent was removed in vacuo, and the residue purified by chromatography, eluting with ether, to give the title compound (4.694g,99%) as a pale yellow solid; vmax (CHC13)2956, 1718, 1492, 1456, 1358, and 1166cm~l; ~H (250MHz; CDC13+TMS) 7.83 (2H, m, Ar-H [AA'BB']), 7.73 (2H, m, NH+CH~N), 7.47 (2H, m, Bn-H), 7.36-7.28 (5H, m, 3Bn-H+2 Ar-H [AA'BB']), 7.03 (lH, d, J 8.8Hz, 6/7-H), 6.94 (lH, d, J8.8Hz, 716-H), 6.67 (lH, s, 3-H), 5.85 (lH, m, -CH'CH2), 5.20 (2H, s, OCH2Ph), 5.04 (3H, m, CH2,CH-CH2 + CH2CH-CHH-cis), 4.85 (lH, dd, Jl.2, 17.2Hz, CH2CH.CHH-trans), 3.88 (3H, s, OMe), and 2.41 (3H, s, Ar-Me).
h) 8-Benzyloxy-7-methoxy-1,2-dihydro-3H-1,2-CYcloDroDapyrrolo [1,2-a] indole Sodium hydride (80~, 0.237g, 7.907mmol) was added to a stirred solution of the tosylhydrazone of g) above (2.602g, 5.321mmol) in dry THF(80cm3). After 25 min, the solution was filtered, and the filtrate evaporated. The residue was dissolved in dry chlorobenzene (800cm3), and the solution refluxed for 3 h. The sovent was evaporated, and the residue purified by chromatography to give the title compound (1.530g, 94X) as a colourless oil;
vmax (Film) 1560, 1492, 1258, 1232, and 744cm~l; ~H (250MHz;
CDC13+TMS) 7.55 (2H, m, Bn-H), 7.42-7.29 (3H, m, Bn-H), 6.81 (2H, 2d, J 8.6Hz, 5+6-H), 6.22 (lH, s, 9-H), 5.20 (2H, s, 0CH2Ph), 4.07 (2H, m, 3-CH2), 3.89 (3H, s, OMe), 2.37 (2H, m, 1+2-H), 1.2S
(lH, m, la-CHH), and 0.64 (lH, m la-CHH). Noting 2d=2 overlapping doublets.
~ WO 94113674 215 0711 PCT/GB93102533 i) 8-Benzyloxy-7-methoxy-1,2-dihydro-3H-l,Z-cyclopropapyrrolo[1,2 -a]indole-9-carboxaldehyde N-Methylformanilide (1.222g, 9.037mmol) and phosphorus oxychloride (1.386g, 9.037mmol) were stirred under a calcium oxide drying tube for 20 min. The resulting precipitate was cooled to 0C, and 1,2-dichloroethane (34cm3) was added.
8-Benzyloxy-7-methoxy-1,2- dihydro-3H-1,2-cyclopropapyrrolo [1,2-a]indole (2.297g, 7.S31mmol) was added, and the mixture was refluxed for 1.25h. Sodium acetate (lM; 49cm3) was added, and the mixture was extracted with ethyl acetate (2x200cm3). The combined extracts were washed with water (3x250cm3), brine (200cm3), dried (MgS04), and evaporated. Purification of the residue by column chromatography gave the title com~ound (1.902g, 76X); vmax (Nujol) 1638, 1604, lS22, and 742, cm~l; ~H (250MHz; CDC13+TMS) 10.32 (lH, s, CHO), 7.49 (2H, m, Bn-H), 7.41-7.26 (3H, m, Bn-H), 6.88 (2H, 2d, J 8.6Hz, 5+6-H), 5.16 (2H, s, OCH2Ph), 4.13 (2H, m, 3-CH2), 3.92 (3H, s, OMe), 3.00 (lH, m, l-H), 2.48 (lH, m, 2-H), 1.49 (lH, m, la-CHH), and 0.72 (lH, m, la-CHH).
j) 8-Hydroxy-7-methoxy-1,2-dihydro-3H-1,2-cyclopropapyrrolo [1,2-a] indole-9-carboxaldehyde To a solution of 8-benzyloxy-7-methoxy-1,2-dihydro-3H-1,2-cyclopropapyrrolo[1,2-a]indole-9-carboxaldehyde (0.5009, 1.501mmol) in ethyl acetate (250cm3) was added 10% palladium on carbon catalyst (O.lOOg), and dllute sulphuric acid (8drops). The mixture was stirred under an atmosphere of hydrogen for 2h. After this time, the suspension was filtered and washed with DCM. The filtrate and washings were extracted with water (3xlOOcm3), brine (75cm3), and dried (MgS04). The organic layer was evaporated to dryness to afford a brown solid residue. Purification of the residue by column chromatography gave the title compound (0.288g, 79%) as a colourless solid; vmax (Nujol) 1606, 1304, 1252, and 824cm~l; ~H (250MHz; CDC13+TMS) 10.87 (lH, s, CHO), 9.58 (lH, s, OH), 6.88 (lH, d, J 8.5Hz, 6/5-H), 6.52 (lH, d, J8.5Hz, 5/6-H), 4.13 (2H, m, 3-CH2), 3.91 (3H, s, OMe), 2.72 (lH, m, l-H), 2.57 (lH, m, 2-H), 1.51 (lH, m, la-CHH), and 0.81 (lH, m, la-CHH).
WO 94tl3674 2~ PCT/GB93/02533 k) 9-Formyl-7-methoxy-1,2-dihydro-3H-1,2-cyclo~ropapyrrolo[1,2-a]
indole-~,8-dione Potassium nitrosodisulphonate (1.407g, 5.243mmol) in water (70cm3), was added to a stirred solution of 8-hydroxy-7-methoxy-1,2-d~hydro-3H-1,2-cyclopropapyrrolo[1,2-a]ind ole-9-carboxaldehyde (0.5799, 2.383mmol) in acetone (232cm3). The mixture was buffered with sodium dihydrogen phosphate solution (0.167M; 70cm3). After stirring at room temperature for 12h, the mixture was concentrated in vacuo, filtered, and the residue recrystalised from dichloromethane-light petroleum to give the title compound (0.581g, 95%) as orange crystals; vmax (Nujol) 1680, 1662, 1638, 1586, 1500, 1244, and 1212cm~l; ~H (250MHz;
CDC13+TMS) 10.37 (lH, s, CH0), 5.68 (lH, s, 6-H), 4.32 (2H, m, 3-CH2), 3.85 (3H, s, OMe), 2.86 (lH, m, l-H), 2.47 (lH, m, 2-H), 1.47 (lH, m, la-CHH), and 0.65 (lH, m, la-CHH).
1) 9-Hvdroxymethyl-7-methoxy-1,2-dihydro-3H-1,2-cycloDropapyrrolo [1,2-a]indole-5-8-dione Sodium borohydride (0.189g, 4.996mmol) was added to a stirred solution of 9-formyl-7-methoxy-1,2-dihydro-3H-1,2-cyclopropapyrrolo[l,2-a]indole-5,8-dione (0.176g, 0.685mmol) in methanol (132cm3) and the solution rigorously degassed with nitrogen. After stirring for 4h at room temperature, air was blown through the solution rapidly, and the mixture was transferred to a separating funnel, and extracted with dichloromethane (3x200cm3). The combined extracts were washed w~th water (2x200cm3), brine (300cm3), and dried (MgS04). The solvent was evaporated, and the residue pur~fied by column chromatography to give the title comDound (0.159g, 90%) as a red solid; vmax (Nujol) 3312, 1668, 1630, 1586, 1586, and 722cm~1;
~H (250MHz; CDC13+TMS) 5.61 (lH, s, 6-H), 4.68 (2H, m, 10-CH2), 4.26 (2H, m, 3-CH2), 3.89 (lH, t, J 7.1Hz), 3.82 (3H, s, OMe), 2.36 (2H, m, 1 2-H), 1.30 (lH, m, la-CHH), and 0.60 (lH, m, la-CHH).
O WO 94/13674 PCT/GB93/02~;33 ~ ~i50711 m) 9-Hydroxymethyl-7-methoxy-1,2-dihydro-3H-1,2-cYclopropa~yrrolo [1,2-a]indole-5,8-dione carbamate Phenyl chloroformate (0.08cm3, 0.618mmol) was added dropwise to a stirred, ice cold solution of the alcohol of 1) above (O.lOOg, 0.386mmol) in dry pyridine (25cm3). The mixture was stirred at room temperature for 2h, then water (lOcm3) was added. The mixture was extracted with dichloromethane (3x50cm3), and the combined extracts were washed with water (3xSOcm3), brine (lOOcm3), and dried (MgS04). The solvent was evaporated, and the residue purified by column chromatography to give an orange gummy solid.
A solution of this phenyl carbonate in dry dichloromethane (55cm3), was cooled to -78C. Ammonia gas was bubbled into the solution for approximately 45 min (monitored the reaction by TLC), after which time the contents were allowed to warm to room temperature, and the solvent removed in vacuo. Recrystallisation of the residue from dlchloromethane-light petroleum gave the title compound (0.097g, 83%) as a red crystalline solid, m.p.
176-178C, vmax (Nujol) 3408, 3212, 1764, 1668, 1620, 1584, 1350, 1306, and 1242cm~l; ~H (250 MHz; CDC13+TMS) 5.59 (lH, s, 6-H), 5.29 (2H, m, 10-CH2), 4.60 (2H, br, NH2), 4.27 (2H, m, 3-CH2), 3.80 (3H, s, OMe), 2.54 (lH, m, l-H), 2.33 (lH, m, 2-H), 1.31 (lH, m, la-CHH), and 0.58 (lH, m, la-CHH); ~C (100.6MHz; CDC13) 177.68 (CO, 8-C), 177.20 (CO, 5-C), 160.44 (C/CO, 7-C/urethane), 156.77 (CO/C, 7-C/urethane), 146.31 (C, 4a-C), 129.24 (C, 8a/9a-C), 123.90 (C, 9a/8a-C), 111.33 (C, 9-C), 105.35 (CH, 6-C), 57.87 (CH2, 10-C), 56.39 (CH3, OMe), 50.00 (CH2, 3-C), 20.63 (CH, l-C), 16.17 (CH2, la-C), 14.69 (CH, 2-C).
Lf~O ~
n) 7-(Aziridin-l-yl)-9-hydroxymethyl-1,2-dihydro-3H-1,2-cyclo-~ropapyrrolo[1,2-a]indole-5,8-dione carbamate A solution of 9-hydroxymethyl-7-methoxy-1,2-dihydro-3H-1,2-cyclopropapyrrolotl,2-a]indole-5,8-dione carbamate (0.015g, 0.050mmol) in dry DMF (2.5cm3) was treated with an excess of distilled aziridine (0.5cm3), and th~s mixture was stirred at room temperature under nitrogen for 15h. After this time, water (Scm3) was added, and extracted with dichloromethane (3x25cm3). The dichloromethane extracts were washed with water (6x25cm3), brine (30cm3), and dried (MgSO4). Removal of solvent in vacuo gave a red residue, which was recrystallised form dichloromethane-light petroleum to afford the title compound (0.013g, 84%) as a red solid, mp 184C (decomp), (Found: M+, 313.1064. C16H15N3O4 requires M, 313.1063); ~max (MeOH) 239 (log F 4.45), 308 (4.30), and 490nm (3.39); vmax (CHC13) 3532,3424, 1724, 1662, 1632, and 1582cm~l; ~H (400MHz, CDC13) 5.72 (lH, s, 6-H) 5.27 (2H, AB, J 12.5Hz, 10-CH2), 4.62 (2H, br s, NH2), 4.24 (2H, m, 3-CH2), 2.53 (lH, m, l-H), 2.34 (lH, m, 2-H), 2.18 (4H, s, 2xCH2-N [aziridine]), 1.29 (lH, m, la-CHH), and 0.55 (lH, m, la-CHH); ~C (100.6MHz, CDC13) 179.08 (CO, 8-C), 177.48 (CO, 5-C), 157.73 (C/CO, 7-C/urethane), 156.77 (CO/C, urethane/7-C), 146.16 (C, 4a-C), 127.40 (C, 9a/8a-C), 126.50 (C, 8a/9a-C), 115.57 (CH, 6-C), 111.20 (C, 9-C), 57.85 (CH2, 10-C), 49.90 (CH2, 3-C), 27.51 (CH2, CH2-N [aziridine]), 20.59 (CH, l-C), 16.16 (CH2, la-C), and 14.65 (CH, 2-C); m/z 313 (M+, 16X), 270 (27), 251 (17), 77 (9), and 41 (100).
WO 94/13674 PCT/GB93102~33 ~ 2i ~0~11 Example 2 Me /~N~NH2 9-Hydroxymethyl-7-(2-methylaziridin-1-yl)-1,2-dihydro-3H-1,2-cycloPro-papyrrolo-tl~2-a]indole-5~8-dione carbamate A solution of 9-hydroxymethyl-7-methoxy-1,2-dihydro-3H-l, 2-cyclopropapyrrolotl,2-a]indole-5,8-dione carbamate (prepared as described in Example 1 steps a) to m)) (O.OlOg, 0.033mmol) in dry DMF (1.5cm3) was treated with an excess of 2-methylaziridine (0.25cm3 initially + 0.25cm3 24h later), and this mixture was stirred at room temperature under nitrogen for 48 h. After this time, water (5cm3) was added, and extracted with dichloromethane (3x25cm3). The dichloromethane extracts were washed with water (5x25cm3), brine (30cm3), and dried (MgS04). Removal of solvent in vacuo gave a red residue, which was purified by column chromatography (eluting with ethyl acetate) to give a red solid.
Recrystallisation of the red residue from dichloromethane-light petroleum gave the title comDound (0.00759, 68%) as a red solid, m.p. 176-178C; (Found: _+, 327.1219. C17H17N304 requires M, 327.1219); ~max (MeOH) 240 (log ~ 4.34), 314 (4.17), and 499nm (3.2g); vmax (CHC13) 3544, 3424, 1724, 1662, 1630, 1580, 1496, 1338, and 1256cm~l; ~H (250MHz, CDC13+TMS) 5.70 (lH, s, 6-H) 5.29 (2H, m, 10-CH2), 4.64 (2H, br s, NH2), 4.25 (2H, m, 3-CH2), 2.52 (lH, m, l-H), 2.27 (2H, m, 2-H+MeCH-N), 2.08 (2H, m, CH2-N
taziridine]), 1.42 (3H, d, J 5.5Hz, Me), 1.30 (lH, m, la-CHH), and 0.55 (lH, m, la-CHH); m/Z 328 (MH+, 20%) 327, (M+, 100).
Example 3 ~N~$NH2 9-Hydroxymethyl-7-(pyrrolidin-1-yl)-1,2-dihydro-3H-1,2-cyclopr~pa-pyrrolo-[1,2-a]ind~le-5,8-dione carbamate A solution of 9-hydroxymethyl-7-methoxy-1,2-dihydro-3H-1,2-cyclopropapyrrolo[l,2-a]indole-5,8-dione carbamate (prepared as described in Example 1 steps a) to m)) (O.OlOg, 0.033mmol) in dry DMF (1.5cm3) was treated with distilled pyrrolidine (0.008cm3, O.O99mmol), and this mixture was stirred at room temperature under nitrogen for 15h. After this time, water was added (5cm3), and extracted with dichloromethane (3x25cm3). The dichloromethane extracts were washed with water (5x25cm3), brine (30cm3), and dried (MgS04) Removal of solvent in vacuo gave a purple residue, which was recrystallised from dichloromethane-light petroleum to afford the title compound (0.008g, 73Z) as a purple solid, m.p.
210C (decomp); (Found: M+, 341.1376. C18HlgN304 requires M, 341.1376); ~max (MeOH) (qualitative) 247, 328, and 549nm; vmax (CHC13) 3612, 3408, 1726, 1686, 1608, 1544, 1498, and 1228cm~l;
~H (250MHz, CDC13+TMS-@ 45C) 5.31 (2H, m, 10-CH2) 5.19 (lH, s, 6-H), 4.55 (2H, br s, NH2), 4.29 (2H, m, 3-CH2), 3.64~(4H, br m, 2 x CH2 [pyrrolidine ring]), 2.49 (lH, m, l-H), 2.30 (lH, m, 2-H), 1.95 (4H, m, 2 x CH2[pyrrolidine ring]), 1.27 (lH, m, la-CHH), and 0.55 (lH, m, la-CHH~; ~C (100.6 MHz, CDC13) 180.25 (CO, 8-C), 176.63 (CO, 5-C), 159.47 (C/CO, 7-C/urethane), 156.6Z
(COIC, urethane/7-C), 144.49 (C, 4a-C), 128.43 (C, 9a/8a-C), 122.91 (8a/9a-C), 109.75 (9-C), 100.16 (CH, 6-C), 58.36 (CH2, 10-C), 51.21 (CH2, 2 x CH2N [pyrrolidine ring]), 49.73 (CH2,3-C), 29.60 (CH2, 2xCH2-pyrrolidine ring), 20.68 (l-C), 16.24 (CH2, la-C), and 14.40 (CH, 2-C); m/Z 341 (_+, 4%), 279 (52), 91 (30), and 44 (100).
-PCT/GB93/02~33 ~ WO 94/13674 2 ~ ~ d 711 [~Heated this compound to 45C in CDC13. The peak at 3.64ppm went from a broad peak to a sharper multiple at this temperature.]
Example 4 ~ -(Aziridin-l-yl)-9-hydroxymethyl-1.2-dihydro-la.la-dimethyl-3H-l, 2-cvclonropapyrrolorl.Z-a]indole-5.8-dione carbama~e ~ N ~ OCONH~
~N~D< Me The title compound was prepared starting from o-vanillin by repeating steps a) to e) of Example 1 followed by the following further steps f) to n) f) 4-Benzyloxy-5-methoxy-1-(2-methyl-2-butenyl)indole-2-carboxalde-hyde To a flask charged with sodium hydride (80%, 0.3009, lO.OOOmmol) was added dry light petroleum (5cm3). The mixture was stirred for 10 min, the petroleum removed by syringe, and the flask contents dried under vacuum. 4-Benzyloxy-5-methoxyindole-2-carboxaldehyde (prepared as described in steps a) to e) of Example 1) (2.3409, 8.327mmol) in DMF (170cm3) was added dropwise, and the mixture was stirred at room temperature for 45 min.
4-Bromo-2-methylbutene (1.15cm3, lO.OOOmmol) was added, and the mixture was stirred at room temperature. After 15h, water (20cm3) was cautiously added, and the mixture was extracted with ethyl acetate (3xl50cm3). The combined extracts were washed with water (8xl50cm3), brine (150cm3), dried (MgS04), and evaporated to dryness. The residue was purified by column chromatography (eluting with 50% ether-50%1ight petroleum) to give the title compound (1.8949, 65Z) as a pale yellow solid, m.p. 55-57C, WO 94/13674 21~ ~ 7 ~1 PCT/GB93/02533 (Found: C, 75.4; H, 6.6; N, 3.9. C22H23N03 requires C, 75.6; H, 6.6; N, 4.0%); vmax. (CHC13) 1666, 1516, 1488, 1460, 1290, 1248, and 1140cm~l; ~H (250MHz; CDC13+TMS) 9.79 (lH, s, CHO), 7.51-7.00 (8H, m, 5 Ar-H+3, 6, 7-H), 5.27 (2H, s, OCH2Ph), 5.18 (3H, m, NCH2CH.CMe2 and CH2CH~CMe2), 3.91 (3H, s, OMe), 1.86 (3H, s, Me), and 1.68 (3H, s, Me); m/Z 350 (MH+, 7%), 349 (M+, 28), 258 (41), 190 (100), 91 (60), 69 (93), and 41 (71).
g) 4-Benzyloxy-5-methoxy-1-(2-methyl-2-butenyl)indole-2-carboxaldehyde tosylhydrazone 4-Toluenesulphonylhydrazide (97%, 1.516g, 8.141mmol) ~n dry methanol (ZOcm3) was added to a stirred solution of 4-benzyloxy-5-methoxy-1-(2-methyl-2-butenyl)indole-2-carboxaldehyd e (1.894g, 5.427mmol) in dry methanol (30cm3). After stirring at 40C for 15h, the solvent was removed in vacuo, and the residue was purified by chromatography (70% ether-30% light petroleum) to give a cream foam. Recrystallisation of this foam from dichloromethane-light petroleum gave the tltle compound (1.681g, 60%) as a colourless solid, m.p. 77-79C, (Found: C, 67.0; H, 6-0;N, 8-2- C2gH31N3O45 requires C, 67.3; H, 6.0; N, 8.1%); vmax (CHC13) 3184, 1608, 1598, 1516, 1490, 1454, 1344, and 1166cm~l;
~H (250MHz; CDC13+TMS) 7.82 (2H, m, Ar-H[Ts-AA'BB']), 7.74 (2H, br s, NH and -CH-N-), 7.47 (2H, m, Bn-H), 7.36-7.29 (5H, m, 3 Bn-H+2 Ar-H [Ts-AA'BB']), 7.03 (lH, d, J8.8Hz, 7/6-H), 6.94 (lH, d, J8.8Hz, 6/7-H), 6.66 (lH, s, 3-H), 5.19 (2H, s, OCH2Ph), 5.05 (3H, m, NCH2CH=CMe2+-CH2CH=CMe2), 3.88 (3H, s, OMe), 2.40 (3H, s, Ar-Me), 1.84 (3H, s, Me), and 1.69 (3H, s, Me); m/Z (FAB, 3-NBA
Matrix) 518 (MH+, 55%), 426 (100), and 242 (20).
WO 94/13674 ~15 ~ 7 1 1 PCT/GB93/02533 h) 8-~enzyloxy-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-l, Z-cyclopro,~aDyrrolo-[1,2-a]indole Sodium hydride (80%, 0.032g, 1.066mmol) was added to a stirred solution of the tosylhydrazone of g) above (0.3699, 0.711mmol) in dry THF (llcm3). After 20 min, the solution was filtered, and the filtrate evaporated. The residue was dissolved in dry chlorobenzene (105cm3), and the solution refluxed for 3.5 h. The solvent was evaporated, and the residue purified by chromatography (gradient elution-started with 100% light petroleum and going down in 5% intervals to 60% 1ight petroleum-40% ether) to give the title compound (0.1439, 60%) as a brown oil, (Found: Mt, 333.1729. C22H23N02 requires M, 33.1729); vmax (CHC13) 1570, 1490, 1452, 1432, 1262, and 1232cm~l, ~H (250MHz; CDC13+TMS) 7.56-7.53 (2H, m, Bn-H), 15 7.38-7.26 (3H, m, Bn-H), 6.85-6.78 (2H, 2d, J8.6Hz, 5+6-H), 6.21 (lH, s, 9-H), 5.22 (2H, s, OCH2Ph), 4.13 (lH, dd, J6.1, 10.8Hz, 3-CHH), 3.90 (3H, s, OMe), 3.87 (lH, m, 3-CHH), 2.28 (lH, dd, JO.5, 6.8Hz, l-H), 2.09 (lH, t, J7.0Hz, 2-H), 1.19 (3H, s, Me), and 0.67 (3H, s, Me); m/Z 334 (MH+, 8~), 333 (M+, 31), 242 (100), 20 200 (13), 91 (17).
~) 8-Benzyloxy-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclop ro-papyrrolo-~1,2-a]indole-9-carboxaldehyde N-Methylformanilide (0.5cm3), and phosphorus oxychloride (0.38cm3), were stirred under a calcium oxide tube for 10 min.
The resulting precipitate was cooled to 0C. This yellow precipitate (0.150g, N-methylformanilide 0.070g, 0.515mmol, and phosphorus oxychloride 0.080g, 0.0515mmol) was added to 8-benzyloxy-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclopropa pyrrolo[l,2-a]indole (0.143g, 0.430mmol) in 1,2 dichloroethane 30 (2cm3). The mixture was refluxed for 1.25 h. Sodium acetate (lM, 9cm3) was added, and the mixture extracted with ethyl acetate (3x50cm3). The combined extracts were washed with water (2x50cm3), brine (50cm3), dried (MgS04~, and evaporated.
Purification of the residue by column chromatography (eluting W 0 94t13674 ~ ~ PCT/GB93/0~533 with ether) gave the title compound (0.118g, 76%) as a yellow oil, (Found: M+, 361.1678. C23H23N03 requires M, 361.1678); vmax (CHC13) 1666, 1596, 1526, 149Z, 1452, and 696cm~l, ~H (250MHz, CDC13+TMS) 10 28 (lH, s, CHO), 7.49-7.15 (5H, m, Bn-H), 6.87 (2H, 2d, J 8.6 Hz, 5+6H), 5.18 (2H, s, OCH2Ph), 4.13 (lH, dd, J 6.4, 11.8 Hz, 3-CHH), 3.91 (3H, s, OMe), 3.86 (lH, m, 3-CHH), 2.86 (lH, d, J 6.3Hz, l-H), 2.20 (lH, t, J 6.3Hz, 2-H), 1.26 (3H, s, Me) and 0.68 (3H, s, Me); ml~ 362 (MH+, 12%), 361 (_+, 28%), 270 (100), 228 (32), and 91 (45).
~) 8-HydroxY-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-l, 2-cycloproDaDyrrolo-[1,2-a]indole-9-carboxaldehyde.
To a solution of 8-benzyloxy-7-methoxy-1,2-dihydro-la, la-dimethyl-3H-1,2-cyclopropapyr-rolo[1,2-a]indole-9-carboxaldehyd e (0.300g, 0.831mmol) in ethyl acetate (160cm3) was added 10%
palladium on carbon catalyst (0.065g), dilute sulphuric acid (10 drops), and the mixture was stirred under an atmosphere of hydrogen. After 2.5 h the sùspension was filtered and washed with dichloromethane (ca 150cm3). The organic layer was extracted with water (3x50cm3), brine (50cm3), dried (MgS04), and evaporated.
Purification of the residue by column chromatography (gradient elution:-started with 507. light petroleum: 50% ether-100~0 ether, in 10% intervals) gave the title com~ound (0.123g, 557.) as a colourless solid, m.p. 119-121C, (Found: _+, 271.1208. C16H17N03 requires M, 271.1208); vmax (CHC13) 1606 (br), 1300, and 1252cm~l, ~H (250MHz, CDC13+TMS) 10.90 (lH, s, CHO), 9.54 (lH, s, ~H), 6.89 (lH, d, J8.5Hz, 6.5-H), 6.55 (lH, d, J8.5Hz, 5.6-H), 4.18 (lH, dd, J6.35, 12.3Hz, 3-CHH), 3.91 (3H, s, OMe), 3.86 (lH, m, 3-CHH), 2.60 (lH, dd, Jl.4, 6.6Hz, l-H), 2.34 (lH, t, J6.5Hz, 2-H), 1.29 (3H, s, Me), and 0.77 (3H, s, Me); _/z Z73 (MH2+, 1770), 272 (MH+, 57), 271 (M+, 100).
W 0 94/13674 ~ 1 5 ~ 7 1 1 PCT/GB93/02533 k) 9-FormYl-7-methoxY-1,2-dihydro-la,la-dimethYl-3H-1~2-cvclopropapyrrolo[l,2-a]indole-5,~-dione Potassium nitrosodisulphonate (0.2689, 0.998mmol) in water (13cm3) was added to a stirred solution of 8-hydroxy-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclopropapy rrolo[l,2-a]indole-9-carboxaldehyde (0.123g, 0.454mmol) in acetone (44cm3). The mixture was buffered with sodium dihydrogen phosphate solution (0.167M, 13cm3). After stirring at room temperature for 12 h, the mixture was concentrated in vacuo, filtered, and the residue recrystallised from dichloromethane-light petroleum to give the title compound (O.lllg, 86%), as orange crystals, m.p. 246-248C, (Found: M+, 285-0999- C16HlsN04 requires M, 285-1001); vmax (CHC13 1678,1640, 1596, 1506, 1238, and 1136cm~l; ~H(250MHz, CDC13+TMS) 10.37 (lH, s, CHO), 5.69 (lH, s, 6-H), 4.32 (lH, dd, J 6.4, 14.1Hz, 3-CHH), 4.18 (lH, d, J 14.2Hz, 3-CHH), 3.85 (3H, s, OMe), 2.71 (lH, dd, Jl.5, 6.5Hz, l-H), 2.22 (lH, m, 2-H), 1.26 3H, s, Me), and 0.72 (3H, s, Me); m/z 286 (MH+, 20Z), 285 (_+, 100), and 270 (69).
1) 9-Hydroxymethyl-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclopropaDyrrolo-[1,2-a]indole-5,8-dione Sodium borohydride (0.0719, 1861mmol) was added to a stirred solution of 9-formyl-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclopropapyrrolo[l,2-a] indole-5,8-dione (0.078g, 0.274mmol), in methanol (53cm3), and the solution rigorously degassed with nitrogen. After stirring for 4 h at room temperature under nitrogen, air was blown rapidly through the solution for 5 min.
The mixture was transferred to a separating funnel, and extracted with dichloromethane (3xlOOcm3), washed with brine (lOOcm3), dried (MgS04), and evaporated. Purification of the residue b column chromatography (gradient elution:-started with 50h ethvl acetate: 50% light petroleum-100/O ethyl acetate) gave the title compound (0.0549, 69%), as a red solid, m.p. 162-164C, (Found:
WO 9~/13674 ~, ~ 5 ~ 7 11 PCT/GB93/0~533 M+, 287.1158. C16H17N04 requires M, 287.1158); vmax (CHC13) 3468, 1658, 1636, and 1594cm~l; ~H (250MHz, CDC13+TMS) 5.61 (lH, s, 6-H), 4.64 (2H, m, 10-CH2), 4.28 (lH, dd, J 6.3, 14.0Hz, 3-CHH), 4.08 (lH, d, J 14.0Hz, 3-CHH), 3.82 (3H, s, OMe), 2.20 (lH, d, J 6.9Hz, l-H), 2.08 (lH, t, J 6.5Hz, 2-H), 1.19 (3H, s, Me), and 0.72 (3H, s, Me); mlz 289 (MH2+, 8%), 288 (MH+, 19), and 287 (M+, 100).
m) 9-Hydroxymethyl-7-methoxY-1,2-d.hydro-la,la-dimethyl-3H-1,2-cyclopropapyrrolo-[1,2-a]indole-5,8-dione carbamate Phenyl chloroformate (0.04cm3, 0.28mmol), was added dropwise to a stirred, ice cold solution of the alcohol of step 1) above (O.OSOg, 0.17mmol) in distilled pyridine (llcm3). The mixture was stirred at room temperature for 2 h, then water (5cm3) was added.
The mixture was extracted with dichloromethane (3x50cm3), and the combined extracts were washed with water (3x50cm3), brine (lOOcm3), and dr~.ed (MgS04). The solvent was evaporated, and the residue pur.fied by column chromatography (eluting with ether) to give an orange gummy solid.
A solution of this phenyl carbonate in dry dichloromethane (30cm3), was cooled to -78C. Ammonia gas was bubbled into the solution for 30 min (calOOcm3), after which the contents were allowed to warm to room temperature, and the solvent was evaporated. Recrystallisation of the residue from dichloromethane-light petroleum gave the title com~ound (0.040g, 10~.) as an orange crystalline solid, m.p. 204-2060C, (Found: _+, 330.1216. C17H18N2C5 requires M, 330.1216); ~max (MeOH) 240 (log 4.32), 292 (4.21), 348 (3.57), and 474nm (3.24); vmax (CHC13) 3540, 3428, 1724, 1670, 1636, 1594, 1496, and 1232cm~1; ~H
(250MHz, CDC13+TMS) 5.59 (lH, s, 6-H), 5.28 (2H, m, 10-CH2), 4.59 (2H, br s, NH2), 4.28 (lH, dd, J6.3, 14.0Hz7 3-CHH), 4.08 (lH, d, J14.0Hz, 3-CHH), 3.80 (3H, s, OMe), 2.37 (lH, d, J6.9Hz, l-H), 2.09 (lH, t, J6.0Hz, 2-H), 1.19 (3H, s, Me), and 0.69 (3H, s, Me); mtz 330 (M+, 15%), Z87 (100), 273 (83), 254 (33), 228 (18), and 51 (14).
n) 7-(Aziridin-l-yl)-9-hydroxymethyl-1,2-dihydro-la,la-dimethyl -3H-1,2-cyclopropapyrrolo[1,2-a]indoie-dione carbamate.
A solution of 9-hydroxymethyl-7-methoxy-1,2-dihydro-la,la-dimethyl-3H-1,2-cyclopropa-pyrrolo[1,2-a]indole-5,8-dione carbamate (O.OlOg, 0.030mmol) in distilled DMF (1.5cm3) was treated with distilled aziridine (0.0049, O.O91mmol), and the mixture was stirred at room temperature under nitrogen for 48 h.
After this time, water (5cm3) was added, and the mixture extracted with dichloromethane (3x25cm3). The dichloromethane extracts were washed with water (8x25cm3), brine (30cm3), and dried (MgS04). Removal of solvent in vacuo gave a red residue, which was recrystallised from dichloromethane-light petroleum to afford the title compound (0.0075g, 75%), as a red solid, m.p.
169-171C, (Found: M+, 341.1376. C18HlgN304 requires M, 341.1376); ~max (MeOH) (qualitative) 241, 313, 495nm; vmax (CHC13) 3540, 3430, 1725, 1665, 1630, 1580, 1335, and 1255cm~l;
~H (250MHz, CDC13+TMS) 5.73 (lH, s, 6-H), 5.27 (2H, m, 10-CH2), 4.69 (2H, s, NH2), 4.26 (lH, dd, J 6.3, 14.0Hz, 3-CHH), 4.06 (lH, d, J14.0Hz, 3-CHH), 2.37 (lH, dd, J 1.2, 6.9Hz, l-H), 2.19 (4H, s, 2 x CH2-N [aziridine ring]), 2.09 (lH, t, J 6.5Hz, 2-H), 1.19 (3H, s, la-CCH3CH3), and, 0.68 (3H, s, la-CCH3CH3); _/~ 341 (M+, 14X), 298 (22), 265 (14), 91 (7), and 44 (100).
Example 5 ~ CO H2 W O 9~/13674 21~ 0711 PCT/GB93/0~53~ -9-Hydroxymethyl-7-(pyrrolidin-1-YI)-1.2-dihydro-la,la-dimethYl-3H-1.2-cyclopropapyrrolo[1.2-a]indole-5.8-dione carbamate A solution of 9-hydroxymethyl-7-methoxy-1,2-dihydro-la, la-dimethyl-3H-1,2-cyclopro-papyrrolo[1,2-a]indole-5,8-dione carbamate (prepared as described in Example 1 steps a) to e) and Example 2 steps f) to m)) (O.OlOg, 0.030 mmol) in distilled DMF) 1.5cm3) was treated with pyrrolidine ~0.008cm3, 0.091 mmol), and the mixture was stirred at room temperature under nitrogen for 15 h. After this time, water (5cm3) was added, and extracted with dichloromethane (3x25cm3). The dichloromethane extracts were washed with water (8x25cm3), brine (30cm3), and dried (MgS04) Removal of solvent in vacuo gave a purple residue, which was purified by column chromatography (gradient elution: 50% ethyl acetate: 50/0 light petroleum-100% ethyl acetate) to give a purple residue. Recrystallisation of the purple residue from dichloromethane-light petroleum gave the title compound (0.008g, 73%), as a purple solid, m.p. 202C (decomp), (Found: M+, 369.1688. C20H23N304 requires M, 369.1699); ~max. (MeOH) 254 (log~ 4.40), 330 (4.06), and 549nm (3.52); vmaX (CHC13) 3540, 3424, 1724, 1662, 160Z, 1544, 1498, and 1266cm~1; ~H (250MHz~
CDC13+TMS) 5.28 (2H, m, 10-CH2), 5.17 (lH, 5, 6-H), 4.59 (2H, s.
NH2), 4.29 (lH, dd, J 6.3, 14.0Hz, 3-CHH), 4.10 (lH, d, J 14.0Hz.
3-CHH), 3.59 (4H, br, 2xCH2-N[pyrrolidine ring]*), 2.33 (lH, d, J
6.9, l-H), 2.04(1H, t, J 5.9Hz, 2-H), 1.94 (4H, m, 2xCH2[pyrrolidine ring]), 1.17 (3H, s, la-CCH3CH3), and 0.67 (3H, s, CCH3CH3); m/~ 369 (M+, 9/.), 308 (lOO),and 293 (81).
Noting-*lHNMR (250MHz, CDC13+TMS). In a second experiment, heated title compound to 45C in CDC13+TMS. The peak at 3.59ppm went from a broad band to a sharper multiple at this higher temperature.
WO 9~/13671 PCT/GB93/0'5~
2t3~7l1 Example 6 - Bioloqical Testinq A) The aerobic toxicity of the compound of Example 1 was demonstrated in V79 cells and compared with that shown by mitomycin C - comparison compound a) ("Comp a") and by S 9-hydroxymethyl-7-methoxy-1,2-dihydro-3H-1,2,-cyclopropapyrrolo[l, 2-a]-indole-5,3-dione carbamate (prepared as described in steps a) to m) of Example 1) - comparision compound b) ("Comp b").
The toxicity was determined by exposure to cells for 3 hours at 37C in air followed by MTT assay as described by Stratford et al, Int. J. Radiat. Oncol. Biol. Phys. (1989), 16, 973.
The results are as shown in Figure 1 of the drawings.
B) The effect of adding the enzyme inhibitor dicoumarol to th~
cells during hypoxic exposure to the compound of Example 1 and to comparison compound b) was measured as follows and the results lS are shown in Figures 2 and 3 and show that substantial protection is observed, thus implying that DT-diaphorase is important with regard to the activation of the compound of Example 1 in air.
This suggests that the compound can find application in treating cell types with high levels of DT diaphorase.
C) The effect of the compound of Example 1 in killing both aerobic and hypoxic cells in contrast to comparison compound b) was measured as follows and the results are shown in Figures and 5 and demonstrate a marked change in behaviour between the two compounds.
D) The toxicity of the compound of Example 1 was compared with that of comparison compound a) (mitomycin C) for a range of cell lines chosen for their differing repair capabilities.
The results are shown in Table 1 below.
W O 91/13674215 ~7 11 PCT/GB93/0~53~ -Table 1 Cell lineConcentration (~M) to kill 90X cells follo~ing 3 hours in air Comp. a) Ex.l V79 (wild type) 1 0.02 IRS-l (DNA strand break repair 0.02 <0.002 deficient: radiation sensitive) CH0-Kl (wild type) 1.5 0.2 CH0-MMCR 25 0.8 (low in P450 reductase) AA8 (wild type) 1 0.06 UV41 (cross link repair 0.02 0.003 deficient) As can be seen from Table 1, the compound of Example 1 shows collateral sensitivity to Mitomycin C in both the IRS-l and UV41 cells, indicating the nature of the molecular damage caused by these drugs may be similar. Although the Example 1 compound is always more potent, it is least effective in killing MMCR cells but the degree of resistance (MMCR vs CH0-Kl) is much less than that observed for mitomycin C and probably indicates that it is less dependent on P450 reductase for activation.
E) The in vitro toxicity of the compound of Example 1 and mitomycin C to murine bone marrow cells was investigated and the results are shown in Table 2 below:
Table 2 Drug IC50 (yM)~
Mitomycin C 1-0 Example 1 compound 0.3 ~150711 * Concentration required to reduce survival by 50% following exposure for 3hrs at 37C in air The results show that bone marrow toxicity is dose limiting for the use of Mitomycin C. However, in vitro the difference in S toxicity between the compound of Example 1 and Mitomycin C is only a factor of 3, which is far less than the difference in potency seen for the other cell types in Table 1. This is a positive advantage in favour of the compound of Example 1.
-
Claims (11)
1. A compound of the formula II
(II) in which R1 and R2 together with the nitrogen to which they are attached represent a cyclic or acylic secondary amine, R3 is hydrogen or an alkyl group, R4 and R5 are each independently hydrogen or an alkyl group and Z is a leaving group, or a salt thereof.
(II) in which R1 and R2 together with the nitrogen to which they are attached represent a cyclic or acylic secondary amine, R3 is hydrogen or an alkyl group, R4 and R5 are each independently hydrogen or an alkyl group and Z is a leaving group, or a salt thereof.
2. A compound according to claim 1 wherein Z is selected from carbamate, amide, ester, ether and halogen substituents.
3. A compound according to claim 2 wherein Z is -OCONH2.
4. A compound according to any one of c1aims 1 to 3 wherein R1 and R2 are linked together with the carbon atom to which they are attached to form a heterocyclic ring..
5. A compound according to claim 4 wherein R1 and R2 together with the carbon atom to which they are attached form an aziridine ring.
6. A compound according to any one of claims 1 to 5 wherein R3 is hydrogen.
7. A compound according to any one of claims 1 to 5 wherein R4 and R5 are hydrogen.
8. A method of preparation of a compound of formula II as defined in any one of claims 1 to 7 comprising reaction of a compound of formula III.
(III) where R3, R4, R5 and Z are as defined in any one of claims 1 to 7 with a secondary amine of formula R1R2NH where R1 and R2 are as defined in any one of claims 1 to 7 in the presence of a suitable solvent.
(III) where R3, R4, R5 and Z are as defined in any one of claims 1 to 7 with a secondary amine of formula R1R2NH where R1 and R2 are as defined in any one of claims 1 to 7 in the presence of a suitable solvent.
9. A method according to claim 8 wherein Z in the compound of formula II is -CONH2 and the compound of formula III has been prepared by reaction of a compound of formula IV
(IV) where R3, R4 and R5 are as defined in any one of claims 1 to 7 with phenyl chloroformate followed by reduction with ammonia.
(IV) where R3, R4 and R5 are as defined in any one of claims 1 to 7 with phenyl chloroformate followed by reduction with ammonia.
10. A pharmaceutical composition comprising a compound of formula II as defined in any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.
11. A compound of formula II as defined in any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof for use as an antitumour agent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB929225799A GB9225799D0 (en) | 1992-12-10 | 1992-12-10 | Antitumour agents |
| GB9225799.7 | 1992-12-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2150711A1 true CA2150711A1 (en) | 1994-06-23 |
Family
ID=10726401
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002150711A Abandoned CA2150711A1 (en) | 1992-12-10 | 1993-12-10 | Antitumour agents |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0673379A1 (en) |
| JP (1) | JPH08504434A (en) |
| CA (1) | CA2150711A1 (en) |
| GB (2) | GB9225799D0 (en) |
| WO (1) | WO1994013674A1 (en) |
-
1992
- 1992-12-10 GB GB929225799A patent/GB9225799D0/en active Pending
-
1993
- 1993-12-10 EP EP94902063A patent/EP0673379A1/en not_active Withdrawn
- 1993-12-10 JP JP6513949A patent/JPH08504434A/en active Pending
- 1993-12-10 CA CA002150711A patent/CA2150711A1/en not_active Abandoned
- 1993-12-10 WO PCT/GB1993/002533 patent/WO1994013674A1/en not_active Application Discontinuation
- 1993-12-10 GB GB9325324A patent/GB2273500B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| GB2273500A (en) | 1994-06-22 |
| EP0673379A1 (en) | 1995-09-27 |
| GB9325324D0 (en) | 1994-02-16 |
| GB9225799D0 (en) | 1993-02-03 |
| JPH08504434A (en) | 1996-05-14 |
| GB2273500B (en) | 1996-12-18 |
| WO1994013674A1 (en) | 1994-06-23 |
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