WO2006070380A1 - Pyrrolo [2, 1-c] [1, 4] benzodiazepine-anthraquinone conjugates useful as antitumour agents - Google Patents

Pyrrolo [2, 1-c] [1, 4] benzodiazepine-anthraquinone conjugates useful as antitumour agents Download PDF

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WO2006070380A1
WO2006070380A1 PCT/IN2004/000412 IN2004000412W WO2006070380A1 WO 2006070380 A1 WO2006070380 A1 WO 2006070380A1 IN 2004000412 W IN2004000412 W IN 2004000412W WO 2006070380 A1 WO2006070380 A1 WO 2006070380A1
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formula
pyrrolo
anthracenyl
dioxo
dihydro
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PCT/IN2004/000412
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French (fr)
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Ahmed Kamal
Gollapalli Bhasker Ramesh Khanna
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Council Of Scientific And Industrial Research
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Priority to JP2007547794A priority Critical patent/JP2008525429A/en
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Priority to GB0710111A priority patent/GB2434795A/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present invention relates to pyrrolo[2,l-c][l,4]benzodiazepine-anthraquinone hybrids useful as potential antitumour agents.
  • the present invention particularly relates to the synthesis of pyrrolo[2,l-c][l,4]benzodiazepine-anthraquinone hybrids as useful anticancer agents.
  • the present invention also relates to a process a process for the preparation of such hybrids and to the use thereof as anti-tumour agents.
  • Pyrrolo[2, l-c][l,4]benzodiazepines are a family of DNA interactive antitumour antibiotics derived from streptomyces species.
  • Examples of naturally occurring pyrrolo[2, 1- c][l,4] benzodiazepines include anthramycin, tomaymycin, sibiromycin and DC-81. These compounds show their biological activity through covalent binding via their NlO-CI l imine/carbinol amine moiety to the C2-amine position of a guanine residue within the minor groove of DNA giving rise to the preference for pu-G-pu sequences.
  • the molecules have a right- handed twist, when viewed from the C-ring towards the A-ring. This enables the PBD to mirror the curvature of B-form DNA and maintain isohelical contact with the walls and floor of the minor groove.
  • the main object of the invention is to provide new pyrrolo[2,l-c] [1,4] benzodiazepines useful as anticancer agents.
  • Another object of the invention is to provide a process for preparing novel pyrrolo[2,l-c][l,4]benzodiazepines useful as antitumor agents.
  • the compound of formula V is selected from the group consisting of
  • the present invention also provides a process for the preparation of pyrrolo[2,l- c][l, ⁇ benzodiazepines of formula V wherein n is 3-4 and R is H, OH,
  • the compound of formula I is reacted with the compound of formula II at refluxing temperature and for a period of 48h.
  • the thioacetal of formula III is reduced using
  • the organic solvent in step (a) comprises acetone.
  • the base in step (a) comprises K 2 C ⁇ 3 .
  • step (b) is carried out in methanol solvent.
  • the present invention also provides a method for the treatment of tumours in a subject, comprising administering to the subject a pharmaceutically effective amount of a pyrrolo[2,l-c][l,4]benzodiazepines of formula V wherein n is 3-4 and R is H, OH,
  • the subject is a mammal.
  • the subject is a human being.
  • the tumour is a human cancer cell line selected from the group consisting of HT-29, HCT- 15, A-549, HOP-62 and SiHA.
  • HT-29 human cancer cell line selected from the group consisting of HT-29, HCT- 15, A-549, HOP-62 and SiHA.
  • Detailed description of the invention The present invention provides a novel pyrrolo[2,l-c][l,4]benzodiazepine of formula
  • the thioacetal of formula III is reduced with with SnCl 2 .2H 2 O in presence of organic solvent and isolating 2S-N- ⁇ 4-[N-(9,10-dihydro-9,10-dioxo-l-anthracenyl)- alkane-3- carboxamide]-oxy-5-methoxy-2-aminobenzoyl ⁇ pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula IV so obtained.
  • the compound of formula I is reacted with the compound of formula II at refluxing temperature and for a period of 48h.
  • the thioacetal of formula III is reduced using SnCl 2 .2H 2 O and in presence of an organic solvent and at reflux temperature.
  • the organic solvent in step (a) of the process is preferably acetone and the base comprises K2CO 3 .
  • Reduction in step (b) is carried out in methanol solvent.
  • the present invention also provides a method for the treatment of tumours in a subject, comprising administering to the subject a pharmaceutically effective amount of a pyrrolo[2,l-c][l, ⁇ benzodiazepines of formula V wherein n is 3-4 and R is H, OH,
  • the subject is a mammal such as a human being.
  • the tumour is a human cancer cell line selected from the group consisting of HT-29, HCT- 15, A-549, HOP-62 and SiHA.
  • the ether linkage is at C-8 position of DC-81 intermediates with Anthraquinone moiety.
  • reaction mixture is refluxed for a period of 24-48h
  • C-8 linked PBD hybrids are synthesised. Purification is effected by column chromatography using different solvents like ethyl acetate, hexane, dichloromethane and methanol.
  • Cells were grown in tissue culture flasks in complete growth medium (RPMI- 1640 medium with 2 mM glutamine, 100 ⁇ g/ml streptomycin, pH 7.4, sterilized by filtration and supplemented with 10% fetal calf serum and 100 units/ml penicillin before use) at 37 0 C in an atmosphere of 5% CO 2 and 90% relative humidity in a carbon dioxide incubator.
  • the cells at subconfluent stage were harvested from the flask by treatment with trypsin (0.5% in PBS containing 0.02% EDTA) for determination of cytotoxicity. Cells with viability of more than 98% as determined by trypan blue exclusion were used for assay.
  • Cell suspension of the required cell density were prepared in complete growth medium with gentamycin (50 ⁇ g/ml) for determination of cytotoxicity.
  • 96-well tissue culture plates 100 ⁇ l of cell suspension was added to each well of the 96-well tissue culture plate. The cells were incubated for 24 hours. Test materials in complete growth medium (lOO ⁇ l) were added after 24 hours incubation to the wells containing cell suspension. The plates were further incubated for 48 hours (at 37 0 C in an atmosphere of 5% and 90% relative humidity in a carbon dioxide incubator) after addition of test material and then the cell growth was stopped by gently layering trichloroacetic acid (TCA, 50 ⁇ l, 50%) on top of the medium in all the wells. The plates were incubated at 4 0 C for one hour to fix the cells attached to the bottom of the wells.
  • TCA trichloroacetic acid
  • the cell growth was calculated by subtracting mean OD value of respective blank from the mean OD value of experimental set. Percent growth in presence of test material was calculated considering the growth in absence of any test material as 100% and in turn percent growth inhibition in presence of test material will be calculated.

Abstract

The present invention relates to synthesis of pyrrolo[2,1-c][1,4]benzodiazepine-anthraquinone hybrids (V) wherein n=3,4; R=H, OH and to their use as antitumour agents.

Description

PYRROLO [2, 1 C] [1, 4] BENZODIAZEPEVE-ANTHRAQUEVONE CONJUGATES USEFUL AS ANTITUMOUR AGENTS
FIELD OF THE INVENTION
The present invention relates to pyrrolo[2,l-c][l,4]benzodiazepine-anthraquinone hybrids useful as potential antitumour agents. The present invention particularly relates to the synthesis of pyrrolo[2,l-c][l,4]benzodiazepine-anthraquinone hybrids as useful anticancer agents. The structural formula of novel pyrrolo[2,l-c][l,4]benzodiazepine-anthraquinone hybrids (V) is as follows, wherein n = 3,4; R = H, OH.
Figure imgf000002_0001
V
The present invention also relates to a process a process for the preparation of such hybrids and to the use thereof as anti-tumour agents. BACKGROUND OF THE INVENTION
Pyrrolo[2, l-c][l,4]benzodiazepines are a family of DNA interactive antitumour antibiotics derived from streptomyces species. Examples of naturally occurring pyrrolo[2, 1- c][l,4] benzodiazepines include anthramycin, tomaymycin, sibiromycin and DC-81. These compounds show their biological activity through covalent binding via their NlO-CI l imine/carbinol amine moiety to the C2-amine position of a guanine residue within the minor groove of DNA giving rise to the preference for pu-G-pu sequences. (Kunimoto, S.; Masuda, T.; Kanbayashi, N.; Hamada, M.; Naganawa, H.; Miyamoto, M.; Takeuchi, T and Unezawa, H. J. Antibiot, 1980, 33, 665.; Kohn, K. W. and Speous, C. L. J. MoI. Biol., 1970, 91, 551.; Hurley, L. H.; Gairpla, C. and Zmijewski, M. Biochem. Biophy. Acta., 1977, 475, 521.; Kaplan, D. J. and Hurley, L. H. Biochemistry, 1981, 20, 7572.) The molecules have a right- handed twist, when viewed from the C-ring towards the A-ring. This enables the PBD to mirror the curvature of B-form DNA and maintain isohelical contact with the walls and floor of the minor groove.
In the last few years a growing interest has been shown in the development of new pyrrolo[2, l-c][l,4]benzodiazepine.hybrids. Many PBD conjugates have been synthesized and investigated for their anticancer activity. (Thurston, D. E.; Morris, S. J.; Hartley, J. A. Chem. Commun. 1996, 563.; Damayanthi, Y.; Reddy, B. S. P.; Lown, J. W. J. Org. Chem. 1999, 64, 290.; Kamal, A.; Reddy, B. S. N.; Reddy, G. S. K.; Ramesh, G Bioorg. Med. Chem. Lett. 2002, 12, 1933). Recently C-8 linked PBD dimers with C2/C2 exounsaturation have been designed and synthesized (Gregson, S. J.; Howard, P. W.; Hartley, J. A.; Brooks, N. A.; Adam, L. J.; Jenkins, T. C; Kelland, L. R. and Thurston, D. E., J. Med. Chem. 2001, 44, 737).
Recently, a non cross-linking mixed imine-amide PBD dimers have been synthesized that have significant DNA binding ability and potent antitumor activity (Kamal, A.; Ramesh, G.; Laxman, N.; Ramulu, P.; Srinivas, O.; Neelima, K.; Kondapi, A. K.; Srinu, V. B.; Nagarajaram, H. M. J. Med. Chem. 2002, 45, 4679).
Figure imgf000003_0001
Figure imgf000003_0002
SJG-136
Figure imgf000003_0003
imine-amide PBD dimers
Objects of the invention
The main object of the invention is to provide new pyrrolo[2,l-c] [1,4] benzodiazepines useful as anticancer agents.
Another object of the invention is to provide a process for preparing novel pyrrolo[2,l-c][l,4]benzodiazepines useful as antitumor agents. Summary of the invention
Accordingly, the present invention provides novel pyrrolo[2,l-c][l,4]benzodiazepine of formula V where n = 3, 4; R = H, OH
Figure imgf000004_0001
In one embodiment of the invention, the compound of formula V is selected from the group consisting of
(a) 7-Methoxy-8-[N-(9,10-dihydro-9,10-dioxo-l-anthracenyl)-propane-3-carboxamide] -oxy- (1 IaS)-1, 2,3,1 Ia tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-one;
(b) 7-Methoxy-8-[N-(9, 10-dihydro-9, 10-dioxo- 1 -anthracenyl)-propane-3 -carboxamide] -oxy- (4R)-hydroxy-(l IaS)- 1,2,3, 1 latetrahydro-5H-pyrrolo[2, 1-C][1, 4] benzodiazepine -5-one;
(c) 7-Methoxy-8-[N-(9, 10-dihydro-9, 10-dioxo- 1 -anthracenyl)-butane-4-carboxamide]-oxy- (1 laS)-l,2,3,l Ia tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-one; and
(d) 7-Methoxy-8-[N-(9, 10-dihydro-9, 10-dioxo- 1 -anthracenyl)-butane-4-carboxamide]-oxy- (4R)-hydroxy-(l IaS)- 1,2,3, 1 Ia tetrahydro-5H-pyrrolo[2,l -C][1, 4]benzodiazepine -5-one.
The present invention also provides a process for the preparation of pyrrolo[2,l- c][l, ^benzodiazepines of formula V wherein n is 3-4 and R is H, OH,
Figure imgf000004_0002
the process comprising: reacting N-(9,10-dihydro-9,10-dioxo-l-anthracenyl)-l-bromo-alkanamide of formula I with (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula II in an aprotic water miscible organic solvent in the presence of a mild inorganic base
Figure imgf000004_0003
Figure imgf000005_0001
and isolating 2S - N - {4 - [N - (9, 10 - dihydro - 9, 10 - dioxo - 1 - anthracenyl) - alkane - 3 - carboxamide] - oxy - 5 - methoxy - 2 - nitrobenzoyl} pyrrolidine - 2 - carbaxaldehyde diethyl thioacetal of formula III so obtained;
Figure imgf000005_0002
(b) reducing the thioacetal of formula III in presence of organic solvent and isolating 2S- N- {4-[N-(9, 10-dihydro-9, 10-dioxo- 1 -anthracenyl)-alkane-3-carboxamide]-oxy-5- methoxy-2-aminobenzoyl}pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula IV so obtained;
Figure imgf000005_0003
, IV
(c) reacting the amino thioacetal of formula IV with a deprotecting agent to give the pyrrol[2,l-c][l,4]benzodiazepine of formula V wherein n and R are as stated above.
In one embodiment of the invention, the compound of formula I is reacted with the compound of formula II at refluxing temperature and for a period of 48h. In another embodiment of the invention, the thioacetal of formula III is reduced using
SnCl2.2H2O and in presence of an organic solvent and at reflux temperature.
In another embodiment of the invention, the organic solvent in step (a) comprises acetone.
In yet another embodiment of the invention, the base in step (a) comprises K2Cθ3. In another embodiment of the invention, step (b) is carried out in methanol solvent. The present invention also provides a method for the treatment of tumours in a subject, comprising administering to the subject a pharmaceutically effective amount of a pyrrolo[2,l-c][l,4]benzodiazepines of formula V wherein n is 3-4 and R is H, OH,
Figure imgf000006_0001
V In one embodiment of the invention, the subject is a mammal.
In another embodiment of the invention, the subject is a human being. In another embodiment of the invention, the tumour is a human cancer cell line selected from the group consisting of HT-29, HCT- 15, A-549, HOP-62 and SiHA. Detailed description of the invention The present invention provides a novel pyrrolo[2,l-c][l,4]benzodiazepine of formula
V wherein n is 3-4 and R is H, OH,
Figure imgf000006_0002
V
The precursors, N-(9,10-dihydro-9,10-dioxo-l-anthracenyl)-l-bromo-alkanamide of formula I (Collier, D. A.; Neidle, S.; J. Med. Chem., 1988, 847) and (2S)-N-[4-hydroxy-5- methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thio-acetal of formula II
(Thurston, D. E.; Murthy, V. S.; Langley, D. R.; Jones, G.; B. Synthesis, 1990, 81) have been prepared by literature methods.
Some representative compounds of formula V of present invention are given below:
1. 7-Methoxy-8-[N-(9, 10-dihydro-9, 10-dioxo- 1 -anthracenyl)-propane-3-carboxamide] -oxy- (11 aS)- 1 ,2,3, 11 a tetrahydro-5H-pyrrolo [2, 1 -c] [ 1 ,4]benzodiazepin-5-one
2. 7-Methoxy-8-[N-(9, 10-dihydro-9, 10-dioxo- 1 -anthracenyl)-propane-3-carboxamide] -oxy- (4R)-hydroxy-(l IaS)- 1,2,3, 1 latetrahydro-5H-pyrrolo[2,l -C] [1,4] benzodiazepine -5-one
3. 7-Methoxy-8-[N-(9, 10-dihydro-9, 10-dioxo- 1 -anthracenyl)-butane-4-carboxamide]-oxy- (1 IaS)-1, 2,3, 1 Ia tetrahydro-5H-pyrrolo [2, l-c][l,4]benzodiazepin-5-one 4. 7-Methoxy-8-[N-(9, 10-dihydro-9, 10-dioxo-l-anthracenyl)-butane-4-carboxamide]-oxy-
(4R)-hydroxy-(l laS)-l,2,3,l Ia tetrahydro-5H-pyrrolo[2,l-C][l,4] benzodiazepine -5-one The pyrrolo[2,l-c][l,4]benzodiazepines of formula V wherein n is 3-4 and R is H, OH are prepared by,
Figure imgf000007_0001
reacting N-(9,10-dihydro-9,10-dioxo-l-anthracenyl)-l-bromo-alkanamide of formula I with (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula II in an aprotic water miscible organic solvent in the presence of a mild inorganic base
Figure imgf000007_0002
Figure imgf000007_0003
and isolating 2S - N - {4 - [N - (9, 10 - dihydro - 9, 10 - dioxo - 1 - anthracenyl) - alkane - 3 - carboxamide] - oxy - 5 - methoxy - 2 - nitrobenzoyl} pyrrolidine - 2 - carbaxaldehyde diethyl thioacetal of formula III so obtained.
Figure imgf000007_0004
The thioacetal of formula III is reduced with with SnCl2.2H2O in presence of organic solvent and isolating 2S-N-{4-[N-(9,10-dihydro-9,10-dioxo-l-anthracenyl)- alkane-3- carboxamide]-oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula IV so obtained.
Figure imgf000008_0001
, IV
The amino thioacetal of formula IV is reacted with a known deprotecting agent in a conventional manner to give the pyrrol[2,l-c][l, ^benzodiazepine of formula V wherein n and R are as stated above.
In the process, the compound of formula I is reacted with the compound of formula II at refluxing temperature and for a period of 48h. The thioacetal of formula III is reduced using SnCl2.2H2O and in presence of an organic solvent and at reflux temperature. The organic solvent in step (a) of the process is preferably acetone and the base comprises K2CO3.
Reduction in step (b) is carried out in methanol solvent.
The present invention also provides a method for the treatment of tumours in a subject, comprising administering to the subject a pharmaceutically effective amount of a pyrrolo[2,l-c][l, ^benzodiazepines of formula V wherein n is 3-4 and R is H, OH,
Figure imgf000008_0002
The subject is a mammal such as a human being. The tumour is a human cancer cell line selected from the group consisting of HT-29, HCT- 15, A-549, HOP-62 and SiHA.
These new analogues of pyrrlo [2,l-c][l,4]benzodiazepine hybrids have shown promising anticancer activity in various cell lines. The molecules synthesized are of immense biological significance with potential sequence selective DNA-binding property. This resulted in design and synthesis of new congeners as shown in scheme-I below wherein:
The ether linkage is at C-8 position of DC-81 intermediates with Anthraquinone moiety.
The reaction mixture is refluxed for a period of 24-48h
C-8 linked PBD hybrids are synthesised. Purification is effected by column chromatography using different solvents like ethyl acetate, hexane, dichloromethane and methanol.
Figure imgf000009_0001
V
Scheme I The following examples are given by way of illustration and therefore should not be construed to the present limit of the scope of invention. Example 1
To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2- carboxaldehyde diethyl thioacetal (400 mg, 1 m.mol) of formula II in acetone, anhydrous K2CO3 (553 mg, 4 m.mol) and N-(9,10-dihydro-9,10-dioxo-l-anthracenyl)-l-bromo- propanamide (372 mg, 1 m.mg) of formula I were added and mixture refluxed for 48h. After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under reduced pressure, and purified by column chromatography to provide compound III. 1HNMR (CDCl3) 1.21-1.38 (m, 6H), 1.53-2.42 (m, 6H), 2.62-2.81 (m, 6H), 3.10-3.28 (m, 2H), 3.91 (s, 3H), 4.25 (m, 2H), 4.65 (m, IH), 4.80 (d, IH), 6.74 (s, IH), 7.68 (s, IH), 7.71- 7.85 (m, 3H), 8.0 (d, IH), 8.20-8.30 (m, 2H), 9.15 (d, IH), 12.38 (bs, IH).
To a solution of 2S-N-{4-[N-(9,10-dihydro-9,10-dioxo-l-anthracenyl)-propane-3- carboxamide]-oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carbaxaldehyde diethyl thioacetal (692 mg, 1 m.mol) of formula III in methanol SnCl2.2H2O (1128 mg, 5 m.mol) was added and mixture was refluxed till TLC indicated completion of reaction. Methanol was evaporated and 10% NaHCO3 solution was added. The aqueous layer was extracted with ethyl acetate, the combined organic phases were dried over Na2SO4 and evaporated under vacuum to provide a amino thioacetal (IV) and directly used in the next step. A solution of compound IV (662 mg, 1 m.mol) HgCl2 (624 mg, 2.3 m.mol) and
CaCO3 (250 mg, 2.5 mg) in CH3CN-H2O (4: 1) was stirred at room temperature till TLC indicated complete consumption of starting material. Reaction mixture was diluted with ethyl acetate and filtered through a celite bed. Organic layer was concentrated, dried and purified by column chromatography to give the compound V. 1HNMR (CDCl3) 2.05 (m, 2H), 2.20-2.40 (m, 4H), 2.81 (m, 2H), 3.50-3.81 (m, 3H), 3.91 (s, 3H), 4.15-4.26 (m, 2H), 6.76 (s, IH), 7.42 (s, IH), 7.55 (d, IH), 7.80 (m, 3H), 8.0 (d, IH), 8.2-8.3 (m, 2H), 9.15 (d, IH), 12.38 (bs, IH). Example 2
To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2- carboxaldehyde diethyl thioacetal (400 mg, 1 m.mol) of formula II in acetone were added anhydrous K2CO3 (553 mg, 4 m.mol) and N-(9,10-dihydro-9,10-dioxo-l-anthracenyl)-l- bromo-butanamide (386 mg, 1 m.mg) of formula I and the mixture was refluxed for 48h. K2CO3 was removed by filtration and then the solvent was evaporated under reduced pressure, purification by column chromatography afforded compound III. 1HNMR (CDCl3) 1.21-1.42 (m, 6H), 1.60-2.40 (m, 8H), 2.62-2.85 (m, 6H), 3.15-3.30 (m, 2H), 3.95 (s, 3H), 4.10-4.25 (m, 2H), 4.65 (m, IH), 4.84 (d, IH), 6.78 (s, IH), 7.68 (s, IH), 7.75-7.90 (m, 3H), 8.05 (d, IH), 8.20-8.35 (m, 2H), 9.15 (d, IH), 12.38 (bs, IH).
To a solution of 2S-N-{4-[N-(9,10-dihydro-9,10-dioxo-l-anthracenyl)-butane-3- carboxamide]-oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carbaxaldehyde diethyl thioacetal (706mg, 1 mmol) of formula III in methanol, SnCl2.2H2O (1128mg, 5 mmol) was added. The mixture was refluxed till TLC indicated completion of reaction. Methanol was evaporated and 10% NaHCO3 solution was added. Aqueous layer was extracted with ethyl acetate. Combined organic phases was dried over Na2SO4 and evaporated under vacuum to obtain amino thioacetal (IV) which was directly used in the next step.
A solution of IV (676 mg, 1 mmol) HgCl2 (624 mg, 2.3 mmol) and CaCO3 (250 mg, 2.5 mg) in CH3CN-H2O (4: 1) was stirred at room temperature until the TLC indicated complete loss of the starting material. The reaction mixture was diluted with ethyl acetate and filtered through a celite bed. The organic layer was concentrated, dried and purified by column chromatography to give the compound V.
1HNMR (CDCl3) 1.85-2.40 (m, 8H), 2.60-2.78 (m, 2H), 3.51-3.80 (m, 3H), 3.93 (s, 3H), 4.15-4.20 (m, 2H), 6.78 (s, IH), 7.42 (s, IH), 7.60 (d, IH), 7.65-7.83 (m, 3H), 8.0 (d, IH), 8.2-8.25 (m, 2H), 9.15 (d, IH), 12.38 (bs, IH). Biological activity In vitro cytotoxicity against human cancer cell lines: The human cancer cell lines procured from National Cancer Institute, Frederick, U. S. A or National Center for Cell Science; Pune, India, were used in present study. Cells were grown in tissue culture flasks in complete growth medium (RPMI- 1640 medium with 2 mM glutamine, 100 μg/ml streptomycin, pH 7.4, sterilized by filtration and supplemented with 10% fetal calf serum and 100 units/ml penicillin before use) at 370C in an atmosphere of 5% CO2 and 90% relative humidity in a carbon dioxide incubator. The cells at subconfluent stage were harvested from the flask by treatment with trypsin (0.5% in PBS containing 0.02% EDTA) for determination of cytotoxicity. Cells with viability of more than 98% as determined by trypan blue exclusion were used for assay. Cell suspension of the required cell density were prepared in complete growth medium with gentamycin (50μg/ml) for determination of cytotoxicity.
Stock solutions of (2 X 10-2 M) of test material were prepared in DMSO (Dimethyl sulphoxide). The stock solutions were serially diluted with complete growth medium containing 50μg/ml of gentamycin to obtain working test solutions of required concentrations. In vitro cytotoxicity against human cancer cell lines was determined (Monks, A., Scudiero,D., Skehan,P., Shoemaker R., Paull, K., Vistica,D., Hose, C.,Langley, j., Cronise, P., Vaigro-WolfF, A., Gray-Goodrich, M., Campbell,H., Mayo, J and Boyd m.J. Natl. Cancer Inst., 1991, 83, 757-766) using 96-well tissue culture plates. 100 μl of cell suspension was added to each well of the 96-well tissue culture plate. The cells were incubated for 24 hours. Test materials in complete growth medium (lOOμl) were added after 24 hours incubation to the wells containing cell suspension. The plates were further incubated for 48 hours (at 370C in an atmosphere of 5% and 90% relative humidity in a carbon dioxide incubator) after addition of test material and then the cell growth was stopped by gently layering trichloroacetic acid (TCA, 50μl, 50%) on top of the medium in all the wells. The plates were incubated at 40C for one hour to fix the cells attached to the bottom of the wells. The liquid of all the wells was gently pipetted out and discarded. The plates were washed five times with distilled water to remove TCA, growth medium low molecular weight metabolites, serum proteins etc and air-dried. Cell growth was measured by staining with sulforhodamine B dye (Skehan et al., 1990). The adsorbed dye was dissolved in Tris-Buffer (100 m 1, 0.01M, pH 10.4) and plates were gently stirred for 5 minutes on a mechanical stirrer. The optical density was recorded on ELISA reader at 540 nm.
The cell growth was calculated by subtracting mean OD value of respective blank from the mean OD value of experimental set. Percent growth in presence of test material was calculated considering the growth in absence of any test material as 100% and in turn percent growth inhibition in presence of test material will be calculated.
Cytotoxicity: Compounds Va and Vc were evaluated for the primary anticancer activity Table 1. The percentage growth inhibition data for compound Va
Concentration Cell lines
(mol/L) HT-29 HCT- 15 A-549 HOP-62 SiHa
10-6 68 59 47 74 41 10-5 86 66 27 90 52 10-4 93 at. 93 n.t. 57
Table 2. The percentage growth inhibition data for compound Vc
Concentration Cell lines
(mol/L) HT-29 HCT- 15 A-549 HOP-62 SiHa
10-6 73 61 56 73 40 10-5 87 82 10 97 49 10-4 93 n.t. 93 n.t. 73 n.t. not tested

Claims

We claim:
1. Pyrrolo[2, l-c][l, ^benzodiazepine of formula V
Figure imgf000013_0001
where n = 3, 4; R = H, OH 2. 7-Methoxy-8-[N-(9, 10-dihydro-9, 10-dioxo- 1 -anthracenyl)-propane-3-carboxamide]-oxy- (l laS)-l,
2,3,l la tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-one of the formula shown below:
Figure imgf000013_0002
3. 7-Methoxy-8-pSf-(9,10-dihydro-9,10-dioxo-l-anthracenyl)-propane-3-carboxamide]-oxy- (4R)-hydroxy-(l IaS)- 1,2,3,1 Ia tetrahydro-5H-pyrrolo[2,l-C][l,4] benzodiazepine-5-one of the formula shown below:
Figure imgf000013_0003
4. 7-Methoxy-8-[N-(9, 10-dihydro-9, 10-dioxo-l-anthracenyl)-butane-4-carboxamide]-oxy-
(HaS)-1, 2,3, 11a tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-one of the formula shown below:
Figure imgf000013_0004
5. 7-Methoxy-8-[N-(9, 10-dihydro-9, 10-dioxo- 1 -anthracenyl)-butane-4-carboxamide]-oxy- (4R)-hydroxy-(l laS)-l,2,3,l Ia tetrahydro-5H-pyrrolo[2,l-C][l,4] benzodiazepine-5-one of the formula shown below:
Figure imgf000014_0001
6. A process for the preparation of pyrrolo[2, 1 -c] [ 1 ^benzodiazepines of formula V wherein n is 3-4 and R is H, OH,
Figure imgf000014_0002
the process comprising: (a) reacting N-(9,10-dihydro-9,10-dioxo-l-anthracenyl)-l-bromo-alkanamide of formula I with (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula II in an aprotic water miscible organic solvent in the presence of a mild inorganic base
Figure imgf000014_0003
Figure imgf000014_0004
and isolating 2S - N - {4 - [N - (9, 10 - dihydro - 9, 10 - dioxo - 1 - anthracenyl) - alkane - 3 - carboxamide] - oxy - 5 - methoxy - 2 - nitrobenzoyl) pyrrolidine - 2 - carbaxaldehyde diethyl thioacetal of formula III so obtained;
Figure imgf000015_0001
(b) reducing the thioacetal of formula III in presence of organic solvent and isolating 2S- N-{4-pSf-(9,10-dihydro-9,10-dioxo-l-anthracenyl)-alkane-3-carboxamide]-oxy-5- methoxy-2-aminobenzoyl}pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula IV so obtained;
Figure imgf000015_0002
(c) reacting the amino thioacetal of formula IV with a deprotecting agent to give the pyrrol[2,l-c][l,4]benzodiazepine of formula V wherein n and R are as stated above.
7. A process as claimed in claim 6 wherein the compound of formula I is reacted with the compound of formula II at refluxing temperature and for a period of 48h.
8. A process as claimed in claim 6 wherein the thioacetal of formula III is reduced using SnCl2.2H2O and in presence of an organic solvent and at reflux temperature.
9. A process as claimed in claim 6 wherein the organic solvent in step (a) comprises acetone.
10. A process as claimed in claim 6 wherein the base in step (a) comprises K2CO3.
11. A process as claimed in claim 6 wherein step (b) is carried out in methanol solvent.
12. A method for the treatment of tumours in a subject, comprising administering to the subject a pharmaceutically effective amount of a pyrrolo[2,l-c][l,4]benzodiazepines of formula V wherein n is 3-4 and R is H, OH,
Figure imgf000016_0001
13. A method as claimed in claim 12 wherein the subject is a mammal.
14. A method as claimed in claim 12 wherein the subject is a human being.
15. A method as claimed in claim 12 wherein the tumour is a human cancer cell line selected from the group consisting of HT-29, HCT-15, A-549, HOP-62 and SiHA.
PCT/IN2004/000412 2004-12-27 2004-12-27 Pyrrolo [2, 1-c] [1, 4] benzodiazepine-anthraquinone conjugates useful as antitumour agents WO2006070380A1 (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008020455A2 (en) * 2006-08-14 2008-02-21 Council Of Scientific & Industrial Research Pyrrolo[2,1-c][1,4]benzodiazepine hybrids and a process for the preparation thereof
WO2009118748A1 (en) * 2008-03-26 2009-10-01 Council Of Scientific & Industrial Research Isoxazoline linked pyrrolo[2,1-c[1,4]benzodiazepine hybrids as potential anticancer agents and the process for preparation thereof

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GB9818731D0 (en) * 1998-08-27 1998-10-21 Univ Portsmouth Compounds

Non-Patent Citations (1)

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Title
KAMAL A ET AL: "Pyrrolo'2,1-c!'1,4]benzodiazepine-anthraquinone conjugates. Synthesis, DNA binding and cytotoxicity", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, OXFORD, GB, vol. 14, no. 19, 4 October 2004 (2004-10-04), pages 4907 - 4909, XP004548786, ISSN: 0960-894X *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008020455A2 (en) * 2006-08-14 2008-02-21 Council Of Scientific & Industrial Research Pyrrolo[2,1-c][1,4]benzodiazepine hybrids and a process for the preparation thereof
WO2008020455A3 (en) * 2006-08-14 2008-04-17 Council Scient Ind Res Pyrrolo[2,1-c][1,4]benzodiazepine hybrids and a process for the preparation thereof
WO2009118748A1 (en) * 2008-03-26 2009-10-01 Council Of Scientific & Industrial Research Isoxazoline linked pyrrolo[2,1-c[1,4]benzodiazepine hybrids as potential anticancer agents and the process for preparation thereof
US8372831B2 (en) 2008-03-26 2013-02-12 Council Of Scientific & Industrial Research Isoxazoline linked pyrrolo[2,1-c][1,4]benzodiazepine hybrids as potential anticancer agents and the process for preparation thereof

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