U.S. patent application number 10/881729 was filed with the patent office on 2006-01-05 for pyrrolo[2,1-c][1,4]benzodiazepine-napthalimide conjugates linked through piperazine moiety and process for preparation thereof.
This patent application is currently assigned to COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH. Invention is credited to Ahmed Kamal, Gollpalli Bhasker Ramesh Khanna, Ramu Rondla.
Application Number | 20060003994 10/881729 |
Document ID | / |
Family ID | 35482538 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003994 |
Kind Code |
A1 |
Kamal; Ahmed ; et
al. |
January 5, 2006 |
PYRROLO[2,1-C][1,4]BENZODIAZEPINE-NAPTHALIMIDE CONJUGATES LINKED
THROUGH PIPERAZINE MOIETY AND PROCESS FOR PREPARATION THEREOF
Abstract
The present invention relates to novel
pyrrolo[2,1-c][1,4]benzodiazepine-napthalimide hybrids linked
through piperazine moiety as potential antitumour agents. The
present invention also relates to a process for the preparation of
novel pyrrolo[2,1-c][1,4]benzodiazepine-napthalimide hybrids linked
through piperazine moiety useful as potential antitumour
agents.
Inventors: |
Kamal; Ahmed; (Hyderabad,
IN) ; Rondla; Ramu; (Hyderabad, IN) ; Khanna;
Gollpalli Bhasker Ramesh; (Hyderabad, IN) |
Correspondence
Address: |
William R. Evans;Ladas & Parry
26 West 61 Street
New York
NY
10023
US
|
Assignee: |
COUNCIL OF SCIENTIFIC AND
INDUSTRIAL RESEARCH
|
Family ID: |
35482538 |
Appl. No.: |
10/881729 |
Filed: |
June 30, 2004 |
Current U.S.
Class: |
514/221 ;
540/496 |
Current CPC
Class: |
C07D 403/14 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
514/221 ;
540/496 |
International
Class: |
A61K 31/551 20060101
A61K031/551; C07D 487/04 20060101 C07D487/04 |
Claims
1. A compound of formula VIII where n.sub.1 is 2, 3, or 4, and
n.sub.2 is 2, 3, or 4 ##STR6##
2.
7-Methoxy-8-{2-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)alkyl]piperaz-
in-1-yl]alkyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of formula VIII where n.sub.1 is 2,
3 or 4 and n.sub.2 is 2, 3 or 4 such that the two alkyl groups are
the same or different and are selected from ethyl, propyl, or butyl
##STR7##
3.
7-Methoxy-8-{2-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]piperaz-
in-1-yl]ethyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the structural formula
##STR8##
4.
7-Methoxy-8-{3-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]piperaz-
in-1-yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the structural formula
##STR9##
5.
7-Methoxy-8-{4-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]piperaz-
in-1-yl]butyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the structural formula
##STR10##
6.
7-Methoxy-8-{3-[4-[3-(1,3-dioxo-benz[de]isoquinolin-2-yl)propyl]pipera-
zin-1-yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the structural formula
##STR11##
7.
7-Methoxy-8-{4-[4-[4-(1,3-dioxo-benz[de]isoquinolin-2-yl)butyl]piperaz-
in-1-yl]butyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the structural formula
##STR12##
8.
7-Methoxy-8-{4-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)propyl]pipera-
zin-1-yl]butyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the structural formula
##STR13##
9.
7-Methoxy-8-{3-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)butyl]piperaz-
in-1-yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the structural formula
##STR14##
10.
7-Methoxy-8-{3-[4-[3-(1,3-dioxo-benz[de]isoquinolin-2-yl)propyl]piper-
azin-1-yl]ethyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the structural formula
##STR15##
11. A process for the preparation of a compound of formula VIII
##STR16## where n.sub.1 is 2, 3, or 4, and n.sub.2 is 2, 3, or 4,
which comprises the steps of: a) reacting
(2S)--N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV ##STR17## with a dibromoalkane in
an aprotic water miscible organic solvent in the presence of a mild
inorganic base at reflux temperature for a period of 48hours, b)
isolating 2S--N-[4-(n-bromo
alkoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carbaxaldehyde
diethyl thioacetal of formula V; ##STR18## reacting the compound of
formula V with piperazine attached naphthalimide in presence of a
mild inorganic base; ##STR19## and reducing it with
SnCl.sub.2.2H.sub.2O in the presence of organic solvent at a reflux
temperature to obtain a compound of formula VIII ##STR20## and d)
reacting the compound of formula VII with a deprotecting agent to
obtain a compound of formula VIII wherein n is as defined
above.
12. (canceled)
13. A method for the treatment of lung, colon or cervical cancer in
a human comprising administering to the human a compound of formula
VIII ##STR21## where n.sub.1 is 2, 3 or 4 and n.sub.2 is 2, 3 or 4
or a pharmaceutically acceptable derivative or salt thereof; and
along with a pharmaceutically acceptable additive.
14. A pharmaceutical composition comprising a pharmaceutically
effective amount of a compound of formula VIII where n.sub.1 is 2,
3 or 4, and n.sub.2 is 2, 3, or 4 and a pharmaceutically acceptable
excipient ##STR22##
15. A pharmaceutical composition comprising a pharmaceutically
effective amount of
7-Methoxy-8-{2-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)alkyl]-
piperazin-1-yl]alkyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of formula VIII were n.sub.1 is 2,
3 or 4 and n.sub.2 is 2, 3 or 4 such that the two alkyl groups are
the same or different and are selected from ethyl, propyl, or butyl
and a pharmaceutically acceptable excipient ##STR23##
16. A pharmaceutical composition comprising a pharmaceutically
effective amount of
7-Methoxy-8-{2-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]-
piperazin-1-yl]ethyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the formula ##STR24## and a
pharmaceutically acceptable excipient.
17. A pharmaceutical composition comprising a pharmaceutically
effective amount of
7-Methoxy-8-{3-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]-
piperazin-1-yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the formula ##STR25## and a
pharmaceutically acceptable excipient.
18. A pharmaceutical composition comprising a pharmaceutically
effective amount of
7-Methoxy-8-{4-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]-
piperazin-1-yl]butyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the formula ##STR26## and a
pharmaceutically acceptable excipient.
19. A pharmaceutical composition comprising a pharmaceutically
effective amount of
7-Methoxy-8-{3-[4-[3-(1,3-dioxo-benz[de]isoquinolin-2-yl)propyl-
]piperazin-1-yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the formula ##STR27## and a
pharmaceutically acceptable excipient.
20. A pharmaceutical composition comprising a pharmaceutically
effective amount of
7-Methoxy-8-{4-[4-[4-(1,3-dioxo-benz[de]isoquinolin-2-yl)butyl]-
piperazin-1-yl]butyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the formula ##STR28## and a
pharmaceutically acceptable excipient.
21. A pharmaceutical composition comprising a pharmaceutically
effective amount of
7-Methoxy-8-{4-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)propyl-
]piperazin-1-yl]butyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the formula ##STR29## and a
pharmaceutically acceptable excipient.
22. A pharmaceutical composition comprising a pharmaceutically
effective amount of
7-Methoxy-8-{3-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)butyl]-
piperazin-1-yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the formula ##STR30## and a
pharmaceutically acceptable excipient.
23. A pharmaceutical composition comprising a pharmaceutically
effective amount of
7-Methoxy-8-{3-[4-[3-(1,3-dioxo-benz[de]isoquinolin-2-yl)propyl-
]piperazin-1-yl]ethyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one of the formula ##STR31## and a
pharmaceutically acceptable excipient.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel
pyrrolo[2,1-c][1,4]benzodiazepine-napthalimide hybrids linked
through piperazine moiety as potential antitumour agents. The
present invention also relates to a process for the preparation of
novel pyrrolo[2,1-c][1,4]benzodiazepine-napthalimide hybrids linked
through piperazine moiety useful as potential antitumour
agents.
[0002] The present invention particularly relates to the synthesis
of pyrrolo[2,1-c][1,4]benzodiazepine-napthalimide hybrids linked
through piperazine moiety as possible anticancer agents. The
structural formula of novel pyrrolo[2,1
-c][1,4]benzodiazepine-napthalimide hybrids (VIII) is as follows,
wherein n.sub.1=2, 3, 4, n.sub.2=2, 3, 4. ##STR1##
BACKGROUND OF THE INVENTION
[0003] Pyrrolo[2,1-c][1,4]benzodiazepines are a family of DNA
interactive antitumour antibiotics derived from Streptomyces
species. Examples of naturally occurring
pyrrolo[2,1-c][1,4]benzodiazepines include anthramycin, tomaymycin,
sibiromycin and DC-81. These compounds show their biological
activity through covalent binding via their N10-C11 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. Mol.
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[2,1-c][1,4]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, Kamal, A.; Reddy, B. S. N.; Reddy Indian patent application
No.209/DEL/2000). 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). Also, 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). During
earlier studies in this laboratory PBDs have been linked to
naphthalimides through alkane chain which have shown promising
anticancer activity (Kamal, A.; Reddy, B. S. N.; Reddy, G. S. K.;
Ramesh, G Bioorg. Med. Chem. Lett. 2002, 12, 1933, Kamal, A.;
Reddy, B. S. N.; Reddy Indian patent application No.209/DEL/2000).
However, in the present invention the PBD and naphthalimide
moieties have been linked through piperazine moiety with alkyl side
arms, instead of simple alkane chain spacers. By incorporation of a
piperazine moiety in the spacer these new hybrids not only exhibit
enhanced in vitro anticancer activity but remarkable DNA binding
affinity for a number of this type of hybrids as illustrated in
Table 1 and 2. ##STR2##
OBJECTS OF THE INVENTION
[0004] The main objective of the present invention is to provide
new pyrrolo[2,1-c][1,4]benzodiazepines useful as anticancer agents.
Another objective of the present invention is to provide a process
for the preparation of novel pyrrolo[2,1-c][1,4]benzodiazepines
useful as antitumor agents.
SUMMARY OF THE INVENTION
[0005] Accordingly, the present invention provides a novel
pyrrolo[2,1-c][1,4]benzodiazepines of formula VIII where n.sub.1=2,
3, 4, n.sub.2=2, 3, 4. ##STR3##
[0006] The present invention also provides a process for the
preparation of pyrrolo[2,1-c][1,4]benzodiazepines of formula VIII
shown above where n.sub.1=2, 3, 4, n.sub.2=2, 3, 4, which comprises
of reacting
(2S)--N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV with dibromoalkanes in an aprotic
water miscible organic solvent in the presence of a mild inorganic
base at refluxing temperature for a period of 48 h, isolating
2S--N-[4-(n-bromo
alkoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carbaxaldehyde
diethyl thioacetal of formula V, reacting the compound of formula V
with piperazine attached naphthalimide in presence of mild
inorganic bases isolating compound of formula VI, reducing it with
SnCl.sub.2.2H.sub.2O in presence of organic solvent at a reflux
temperature, reacting the above amino compound of formula VII with
known deprotecting agents in a conventional manner to give novel
pyrrol[2,1-c][1,4]benzodiazepine of formula VIII wherein n is as
stated above.
DETAILED DESCRIPTION OF THE INVENTION
[0007] The precursor,
(2S)--N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethylthioacetal of formula IV (Thurston, D. E.; Murthy, V. S.;
Langley, D. R.; Jones, G.; B. Synthesis, 1990, 81) has been
prepared by literature methods.
[0008] Some representative compounds of formula VIII of present
invention are given below: 1.
7-Methoxy-8-{2-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]piperazin--
1yl]ethyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one 2.
7-Methoxy-8-{3-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]piperazin--
1-yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one 3.
7-Methoxy-8-{4-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]piperazin
1-yl]butyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one 4.
7-Methoxy-8-{3-[4-[3-(1,3-dioxo-benz[de]isoquinolin-2-yl)propyl]piperazin-
- 1yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one 5.
7-Methoxy-8-{4-[4-[4-(1,3-dioxo-benz[de]isoquinolin-2-yl)butyl]piperazin-
1yl]butyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one
[0009] These new analogues of pyrrolo[2,1-c][1,4]benzodiazepine
hybrids have shown promising anticancer activity in in selected
human cancer cell lines of colon (HT-29, HCT-15), lung (A-549,
HOP-62), cervix (SiHa) origin. 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 illustrated in Scheme-I which comprises of 1. The
ether linkage at C-8 position of DC-81 intermediates with
napthalimide moiety. 2. Refluxing the reaction mixture for 24-48 h.
3. Synthesis of C-8 linked PBD hybrids. 4. Purification by column
chromatography using different solvents like ethyl acetate, hexane,
dichloromethane and methanol. ##STR4## ##STR5##
[0010] 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
[0011] To a solution of
(2S)--N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were
added anhydrous K.sub.2CO.sub.3 (829 mg, 6 mmol) and 1,2 dibromo
ethane (940 mg, 5 mmol) and the mixture was refluxed for 48 h.
After completion of reaction K.sub.2CO.sub.3 was removed by
filtration and the solvent was evaporated under redused pressure,
purification by column chromatography afforded compound V. .sup.1H
NMR (CDCl.sub.3) 1.30-1.45 (m, 6H), 1.70-2.35 (m, 4H), 2.70-2.85
(m, 4H), 3.12-3.30 (m, 2H), 3.70 (t, 2H, J=6.3 Hz), 3.95 (s, 3H),
4.40 (t, 2H, J=6 Hz), 4.60-4.75 (m, 1H), 4.82 (d, 1H, J=4.3 Hz),
6.80 (s, 1H), 7.65 (s, 1H).
[0012] To a solution of 2S--N-[4-(2-bromo
ethoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carbaxaldehyde
diethyl thioacetal of formula V (507 mg, 1 mmol), piperazine
attached naphthalimide (340 mg, 1.1 mmol) in acetone was added
anhydrous K.sub.2CO.sub.3 (415 mg, 3 mmol) and the mixture was
refluxed for 24 h. After completion of reaction K.sub.2CO.sub.3 was
removed by filtration and the solvent was evaporated under reduced
pressure, purification by column chromatography afforded compound
VI. .sup.1H NMR (CDCl.sub.3, 200 MHz) 1.22-1.40 (m, 6H), 1.70-2.35
(m, 4H), 2.55-2.95 (m, 16H), 3.15-3.32 (m, 2H), 3.92 (s, 3H),
4.15-4.35 (m, 4H), 4.57-4.72 (m, 1H), 4.8 (d, 1H, J=4.3 Hz), 6.77
(s, 1H), 7.60-7.80 (m, 3H), 8.30 (t, 2H, J=8 Hz), 8.55 (d, 2H,
J=7.6 Hz).
[0013] To a solution of
2S--N-{4-[2-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]piperazin-1-y-
l]ethyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carbaxaldehyde
diethyl thioacetal of formula VI (736 mg, 1 mmol) in methanol was
added SnCl.sub.2.2H.sub.2O (1.12 gr, 5 mmol) and the mixture was
refluxed until the TLC indicated the completion of reaction.
Methanol was evaporated and 10% NaHCO.sub.3 solution was added.
Aqueous layer was extracted with ethyl acetate, combined organic
phases were dried over Na.sub.2SO.sub.4 and evaporated under vacuum
to afford amino thioacetal (VII) and directly used in the next
step.
[0014] A solution of VII (706 mg, 1 mmol) HgCl.sub.2 (624 mg, 2.3
mmol) and CaCO.sub.3 (250 mg, 2.5 mmol) in CH.sub.3CN--H.sub.2O
(4:1) was stirred at room temperature until the TLC indicated
complete consumption 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 VIII. .sup.1H NMR (CDCl.sub.3,
200 MHz) 1.90-2.40 (m, 4H), 2.45-2.92 (m, 12H), 3.55-3.82 (m, 3H),
3.92 (s, 3H), 4.05-4.40 (m, 4H), 6.77 (s, 1H), 7.45 (s, 1H), 7.62
(d, 1H, J=4.39 Hz), 7.76 (t, 2H, J=7.69 Hz), 8,20 (d, 2H, J=8.2
Hz), 8.60 (d, 2H, J=7.32 Hz).
Example 2
[0015] To a solution of
(2S)--N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were
added anhydrous K.sub.2CO.sub.3 (828 mg, 6 mmol) and 1,3 dibromo
propane (1 gr, 5 mmol) and the mixture was refluxed for 48 h. After
completion of reaction K.sub.2CO.sub.3 was removed by filtration
and the solvent was evaporated under reduced pressure, purification
by column chromatography afforded compound V. .sup.1H NMR
(CDCl.sub.3, 200 MHz) 1.25-1.40 (m, 6H), 1.72-2.42 (m, 6H),
2.70-2.8 (m, 4H), 3.15-3.30 (m, 2H), 3.60 (t, 2H, J=6.20 Hz), 3.95
(s, 3H), 4.20 (t, 2H, J=4.96 Hz), 4.60-4.75 (m, 1H), 4.82 (d, 1H,
J=4.33 Hz), 6.78 (s, 1H), 7.68 (s, 1H).
[0016] To a solution of 2S--N-[4-(3-bromo
propoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carbaxaldehyde
diethyl thioacetal of formula V (521 mg, 1 mmol), piperazine
attached naphthalimide (340 mg, 1.1 mmol) in acetone was added
anhydrous K.sub.2CO.sub.3 (415 mg, 3 mmol) and the mixture was
refluxed for 24 h. After completion of reaction K.sub.2CO.sub.3 was
removed by filtration and the solvent was evaporated under reduced
pressure, purification by column chromatography afforded compound
VI. .sup.1H NMR (CDCl.sub.3, 200 MHz) 1.30-1.42 (m, 6H), 1.70-2.30
(m, 6H), 2.40-2.82 (m, 16H), 3.15-3.30 (m, 2H), 3.92 (s, 3H), 4.15
(m, 2H), 4.30 (m, 2H), 4.60-4.70 (m, 1H), 4.82 (d, 1H, J=4.25 Hz),
6.75 (s, 1H), 7.65 (s, 1H), 7.75 (t, 2H, J=7.4 Hz), 8.2 (d, 2H, J=8
Hz), 8.6 (d, 2H, J=7.6 Hz).
[0017] To a solution of
2S--N-{4-[3-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]piperazin-1-y-
l]propyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carbaxaldehyde
diethyl thioacetal of formula VI (750 mg, 1 mmol) .in methanol was
added SnCl.sub.2.2H.sub.2O (1.12 gr, 5 mmol) and the mixture was
refluxed until the TLC indicated the completion of reaction.
Methanol was evaporated and 10% NaHCO.sub.3 solution was added.
Aqueous layer was extracted with ethyl acetate. Combined organic
phases were dried over Na.sub.2SO.sub.4 and evaporated under vacuum
to afford amino thioacetal (VII) and directly used in the next
step.
[0018] A solution of VII (720 mg, 1 mmol) HgCl.sub.2 (624 mg, 2.3
mmol) and CaCO.sub.3 (250 mg, 2.5 mmol) in CH.sub.3CN--H.sub.2O
(4:1) was stirred at room temperature until the TLC indicated
complete consumption 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 VIII. .sup.1H NMR (CDCl.sub.3,
200 MHz) 1.60-2.16 (m, 6H), 2.25-2.80 (m, 12H), 3.50-3.82 (m, 3H),
3.95 (s, 3H), 4.05-4.40 (m, 4H), 6.80 (s, 1H), 7.45 (s, 1H), 7.62
(d, 1H, J=3.33 Hz), 7.79 (t, 2H, J=7.32 Hz), 8.20 (d, 2H, J=8.05
Hz), 8.60 (d, 2H, J=7.32 Hz).
Example 3
[0019] To a solution of
(2S)--N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula (800 mg, 2 mmol) in acetone were
added anhydrous K.sub.2CO.sub.3 (829 mg, 6 mmol) and 1,4 dibromo
butane (1.07 gr, 5 mmol) and the mixture was refluxed for 48 h.
After completion of reaction K.sub.2CO.sub.3 was removed by
filtration and the solvent was evaporated under redused pressure,
purification by column chromatography afforded compound V. .sup.1H
NMR (CDCl.sub.3, 300 MHz) 1.30-1.40 (m, 6H), 1.75-2.40 (m, 8H),
2.70-2.85 (m, 4H), 3.15-3.30 (m, 2H), 3.50 (t, 2H, J=6.25 Hz), 3.95
(s, 3H), 4.10 (m, 2H), 4.60-4.70 (m, 1H), 4.82 (d, 1H, J=4.3 Hz),
6.75 (s, 1H), 7.62 (s, 1H).
[0020] To a solution of 2S--N-[4-(4-bromo
butoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carbaxaldehyde
diethyl thioacetal of formula V (535 mg, 1 mmol), piperazine
attached naphthalimide (340 mg, 1.1 mmol) in acetone was added
anhydrous K.sub.2CO.sub.3 (415 mg, 3 mmol) and the mixture was
refluxed for 24 h. After completion of reaction K.sub.2CO.sub.3 was
removed by filtration and the solvent was evaporated under redused
pressure, purification by column chromatography afforded compound
VI. .sup.1H NMR (CDCl.sub.3, 200 MHz) 1.25-1.40 (m, 6H), 1.60-2.15
(m, 8H), 2.35-2.85 (m, 16H), 3.15-3.30 (m, 2H), 3.92 (s, 3H), 4.12
(m, 1H), 4.30 (m, 2H), 4.60-4.72 (m, 1H), 4.80 (d, 1H, J=4.23 Hz),
6.75 (s, 1H), 7.60 (s, 1H), 7.75 (t, 2H, J=7.45 Hz), 8.2 (d, 2H,
J=8.25 Hz), 8.56 (d, 2H, J=7.62 Hz).
[0021] To a solution of
2S--N-{4-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]piperazin-1-yl]b-
utyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carbaxaldehyde
diethyl thioacetal (764 mg, 1 mmol) of formula VI .in methanol was
added SnCl.sub.2.2H.sub.2O (1.12 gr, 5 mmol) and the mixture was
refluxed until the TLC indicated the completion of reaction. The
methanol was evaporated and 10% NaHCO.sub.3 solution was added. The
aqueous layer was extracted with ethyl acetate, the combined
organic phases were dried over Na.sub.2SO.sub.4 and evaporated
under vacuum to afford the amino thioacetal (VII) and directly used
in the next step.
[0022] A solution of VII (734 mg, 1 mmol) HgCl.sub.2 (624 mg, 2.3
mmol) and CaCO.sub.3 (250 mg, 2.5 mmol) in CH.sub.3CN--H.sub.2O
(4:1) was stirred at room temperature until the TLC indicated
complete consumption 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 VIII. .sup.1H NMR (CDCl.sub.3,
200 MHz) 1.56-2.15 (m, 8H), 2.25-2.80 (m, 16H), 3.45-3.82 (m, 3H),
3.92 (s, 3H), 4.0-4.15 (m, 2H), 4.22-4.37 (t, 2H), 6.70 (s, 1H),
7.42 (s, 1H), 7.60 (d, 1H, J=4.25 Hz), 7.72 (t, 2H, J=7.4 Hz), 8.16
(d, 2H, J=8.1 Hz), 8.56 (d, 2H, J=7.42 Hz).
Example 4
[0023] To a solution of
(2S)--N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were
added anhydrous K.sub.2CO.sub.3 (829 mg, 6 mmol) and 1,3 dibromo
propane (1 gr, 5 mmol) and the mixture was refluxed for 48 h. After
completion of reaction K.sub.2CO.sub.3 was removed by filtration
and the solvent was evaporated under redused pressure, purification
by column chromatography afforded compound V. .sup.1H NMR
(CDCl.sub.3, 200 MHz) 1.25-1.40 (m, 6H), 1.72-2.42 (m, 6H),
2.70-2.8 (m, 4H), 3.15-3.30 (m, 2H), 3.60 (t, 2H, J=6.20 Hz), 3.95
(s, 3H), 4.20 (t, 2H, J=4.96 Hz), 4.60-4.75 (m, 1H), 4.82 (d, 1H,
J=4.33 Hz), 6.78 (s, 1H), 7.68 (s, 1H).
[0024] To a solution of 2S--N-[4-(3-bromo
propoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carbaxaldehyde
diethyl thioacetal of formula V (521 mg, 1 mmol), piperazine
attached naphthalimide (324 mg, 1 mmol) in acetone were added
anhydrous K.sub.2CO.sub.3 (415 mg, 3 mmol) and the mixture was
refluxed for 24 h. After completion of reaction K.sub.2CO.sub.3 was
removed by filtration and the solvent was evaporated under reduced
pressure, purification by column chromatography afforded compound
VI. .sup.1H NMR (CDCl.sub.3, 200 MHz) 1.25-1.42 (m, 6H), 1.70-2.40
(m, 8H), 2.60-3.30 (m, 18H), 3.92 (s, 3H), 4.05 (m, 4H), 4.70-4.80
(m, 1H), 4.82 (d, 1H, J=4.25 Hz), 6.77 (s, 1H), 7.60 (s, 1H), 7.75
(t, 2H, J=7.35 Hz), 8.18 (d, 2H, J=8 Hz), 8.55 (d, 7.55 Hz).
[0025] To a solution of
2S--N-{4-[3-[3-[4-[3-(1,3-dioxo-benz[de]isoquinolin-2-yl)propyl]piperazin-
-1-yl]propyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carbaxaldehyde
diethyl thioacetal of formula VI (764 mg, 1 mmol) .in methanol was
added SnCl.sub.2.2H.sub.2O (1.12 gr, 5 mmol) and the mixture was
refluxed until the TLC indicated the completion of reaction. The
methanol was evaporated and 10% NaHCO.sub.3 solution was added. The
aqueous layer was extracted with ethyl acetate, the combined
organic phases were dried over Na.sub.2SO.sub.4 and evaporated
under vacuum to afford the amino thioacetal (VII) and directly used
in the next step.
[0026] A solution of VII (734 mg, 1 mmol) HgCl.sub.2 (624 mg, 2.3
mmol) and CaCO.sub.3 (250 mg, 2.5 mmol) in CH.sub.3CN--H.sub.2O
(4:1) was stirred at room temperature until the TLC indicated
complete consumption 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 VIII. .sup.1H NMR (CDCl.sub.3,
200 MHz) 1.75-2.18 (m, 8H), 2.22-2.80 (m, 16H), 3.45-4.30 (m, 10H),
6.75 (s, 1H), 7.45 (s, 1H), 7.60 (d, 1H, J=4.2 Hz), 7.72 (t, 2H,
J=7.4 Hz), 8.20 (d, 2H, J=8.1 Hz), 8.58 (d, 2H, J=7.35 Hz).
Example 5
[0027] To a solution of
(2S)--N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were
added anhydrous K.sub.2CO.sub.3 (829 mg, 6 mmol) and 1,4 dibromo
butane (1 gr, 5 mmol) and the mixture was refluxed for 48 h. After
completion of reaction K.sub.2CO.sub.3 was removed by filtration
and the solvent was evaporated under redused pressure, purification
by column chromatography afforded compound V. .sup.1H NMR
(CDCl.sub.3, 300 MHz) 1.30-1.40 (m, 6H), 1.75-2.40 (m, 8H),
2.70-2.85 (m, 4H), 3.15-3.30 (m, 2H), 3.50 (t, 2H, J=6.25 Hz), 3.95
(s, 3H), 4.10 (m, 2H), 4.60-4,70 (m, 1H), 4.82 (d, 1H, J=4.3 Hz),
6.75 (s, 1H), 7.62 (s, 1H).
[0028] To a solution of 2S--N-[4-(4-bromo
butoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carbaxaldehyde
diethyl thioacetal of formula V (538 mg, 1 mmol), piperazine
attached naphthalimide (337 mg, 1 mmol) in acetone were added
anhydrous K.sub.2CO.sub.3 (415 mg, 3 mmol) and the mixture was
refluxed for 24 h. After completion of reaction K.sub.2CO.sub.3 was
removed by filtration and the solvent was evaporated under reduced
pressure, purification by column chromatography afforded compound
VI. .sup.1H NMR (CDCl.sub.3, 200 MHz) 1.60-2.33 (m, 12H), 2.52-3.0
(m, 16H), 3.12-3.30 (m, 2H), 3.95 (s, 1H), 4.02-4.25 (m, 4H),
4.60-4.72 (m, 1H), 4.80 (d, 1H, J=4.3 Hz), 6.75 (s, 1H), 7.60 (s,
1H), 7.75 (t, 2H, J=7.45 Hz), 8.18 (d, 2H, J=8.2 Hz), 8.56 (d, 2H,
J=7.6 Hz).
[0029] To a solution of
2S--N-{4-[4-[4-[4-(1,3-dioxo-benz[de]isoquinolin-2-yl)butyl]piperazin-1-y-
l]butyl]-oxy-5-methoxy-2-nitrobenzoyl }pyrrolidine-2-carbaxaldehyde
diethyl thioacetal (792 mg, 1 mmol) of formula VI .in methanol was
added SnCl.sub.2.2H.sub.2O (1.12 gr, 5 mmol) and the mixture was
refluxed until the TLC indicated the completion of reaction. The
methanol was evaporated and 10% NaHCO.sub.3 solution was added. The
aqueous layer was extracted with ethyl acetate, the combined
organic phases were dried over Na.sub.2SO.sub.4 and evaporated
under vacuum to afford amino thioacetal (VII) and directly used in
the next step.
[0030] A solution of VII (762 mg, 1 mmol) HgCl.sub.2 (624 mg, 2.3
mmol) and CaCO.sub.3 (250 mg, 2.5 mmol) in CH.sub.3CN--H.sub.2O
(4:1) was stirred at room temperature until the TLC indicated
complete consumption 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 VIII. .sup.1H NMR (CDCl.sub.3,
300 MHz) 1.50-2.10 (m, 12H), 2.25-2.80 (m, 16H), 3.45-4.30 (m,
10H), 6.75 (s, 1H), 7.45 (s, 1H), 7.62 (d, 1H, J=4.2 Hz), 7.75 (t,
2H, J=7.3 Hz), 8.20 (d, 2H, J=8.1 Hz), 8.56 (d, 2H, J=7.40 Hz).
Biological Activity:
[0031] 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 .mu.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.degree. C. in an atmosphere
of 5% CO.sub.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. The cell suspension of the required cell density were
prepared in complete growth medium with gentamycin (50 .mu.g/ml)
for determination of cytotoxicity.
[0032] A stock solutions of (2.times.10.sup.-2 M) of test material
were prepared in DMSO. The stock solutions were serially diluted
with complete growth medium containing 50 .mu.g/ml of gentamycin to
obtain working test solutions of required concentrations.
[0033] 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. The 100 .mu.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 (100 .mu.l )
were added after 24 hours incubation to the wells containing cell
suspension. The plates were further incubated for 48 hours (at
37.degree. 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 .mu.l, 50%) on top of the medium in all the wells. The
plates were incubated at 4.degree. C. 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 ml, 0.01 M, 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.
[0034] 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:
[0035] Compounds were evaluated for the primary anticancer
activity. The cytotoxicity data for some representative compounds
has shown in Table 1. TABLE-US-00001 TABLE 1 The percentage growth
inhibition data for napthalimide-PBD hybrids Cancer cell lines
Compd HT-29 HCT-15 A-549 HOP-62 SiHa (mol/L) 10.sup.-4 10.sup.-5
10.sup.-6 10.sup.-4 10.sup.-5 10.sup.-6 10.sup.-4 10.sup.-5
10.sup.-6 10.sup.-4 10.sup.-5 10.sup.-6 10.sup.-4 10.sup.-5
10.sup.-6 VIIIb 88 85 91 a 59 58 88 a a a 86 72 a 44 54 VIIIc 94 86
80 a 69 60 95 a a a 93 86 56 39 a VIIId 95 62 63 a a a 95 30 51 78
a a 85 65 31 VIIIe 92 78 67 a 62 59 93 a a a 93 80 42 40 44 a: not
tested
[0036] TABLE-US-00002 TABLE 2 DNA Thermal Denaturation Studies:
Induced .quadrature.T.sub.m .degree. C. after incubation at
37.degree. C. Compound 0 h 18 h VIIIa 21.9 22.7 VIIIb 25.8 26.7
VIIIc 23.4 24.2 VIIId 13.1 14.3 VIIIe 20.9 21.7 DC-81 0.3 0.7
[0037] For CT-DNA alone at pH 7.00.+-.0.01, T.sub.m =69.8 .degree.
C..+-.0.01 (mean value from 10 separate determinations), all
.DELTA.T.sub.m values are.+-.0.1-0.2.degree. C. For a 1:5 molar
ratio of [PBD]/[DNA], where CT-DNA concentration=100 .mu.M and
ligand concentration=20 .mu.M in aqueous sodium phosphate buffer
[10 mM sodium phosphate+1 mM EDTA, pH 7.00.+-.0.01].
* * * * *