Novel 6,7-diphenyl-2,3,8,8a-tetrahydro-1H-indolizin-5-one analogues as cytotoxic agents

Article abstract of DOI:10.1016/S0960-894X(03)00263-4

A series of 6,7-diphenyl-2,3,8,8a-tetrahydro-1H-indolizin-5-one analogues were synthesized and evaluated for cytotoxic activity against eight human cancer cell lines. Compounds 18, 21, 28, 29, 30 and 31 showed cytotoxic activity with GI₅₀ value

Full text of DOI:10.1016/S0960-894X(03)00263-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1679–1682
Novel 6,7-diphenyl-2,3,8,8a-tetrahydro-1H-indolizin-5-one
y
Analogues as CytotoxicAgents
Vedula M. Sharma,a,* K. V. Adi Seshu,a C. Vamsee Krishna,a P. Prasanna,a
V. Chandra Sekhar,a A. Venkateswarlu,a Sriram Rajagopal,b R. Ajaykumar,b
Dhanvanthri S. Deevi,b N. V. S. Rao Mamidic and R. Rajagopalanb
aDiscovery Chemistry, Dr. Reddy’s Laboratories, Discovery Research, Miyapur, Hyderabad, 500 050, India
bDiscovery Biology, Dr. Reddy’s Laboratories, Discovery Research, Miyapur, Hyderabad, 500 050, India
cDrugMetabolism and Pharmacokinetics, Dr. Reddy’s Laboratories, Discovery Research, Miyapur, Hyderabad, 500 050, India
Received 3 December 2002; accepted 10 March 2003
Abstract—A series of 6,7-diphenyl-2,3,8,8a-tetrahydro-1H-indolizin-5-one analogues were synthesized and evaluated for cytotoxic
activity against eight human cancer cell lines. Compounds 18, 21, 28, 29, 30 and 31 showed cytotoxic activity with GI50 values in the
range of 2.1–8.1 mM concentration. Among these, compounds 21 and 28 exhibited good pharmacokinetic properties. These com-
pounds were further evaluated for their in vivo efficacy in modified hollow fibre assay (HFA).
# 2003 Elsevier Science Ltd. All rights reserved.
Antimitotic agents are one of the major classes of cyto-
toxic drugs for cancer treatment and have attracted
attention recently due to the clinical success of Taxol.1À4
Antimitotic agents arrest the cell division by interfering
with the normal microtubule polymerization/depoly-
merization process. Although antimitotic drugs such as
Vincristine and Paclitaxel gained wide clinical use for
the treatment of various cancers, they suffer from many
side effects, difficulty in dosing schedule and lack of
efficacy against various multidrug resistant cancer cell
lines. Therefore, there is a need to discover novel anti-
tumor agents with fewer side effects, improved pharma-
cokinetic properties and better efficacy with novel
mechanism of actions.
anti-angiogenesis effects7,8 combined with its strong
anti-tubulin activity attracted our attention and
prompted us to search for similar novel alkaloids of
plant origin with a novel ring structure as part of our
ongoing cancer research programme.9,10
Our search resulted in identification of an interesting
compound Septicine (32, Fig. 1), reported in the lit-
erature, from the Indian medicinal plant Tylophora
asthamatica.11 The prominent difference between
Septicine and Combretastatins (CA-1, CB-1 and
CA-4) is that Septicine has a novel additional indo-
lizidine ring which is absent in Combretastatins. In
this paper we describe the synthesis, cytotoxic activ-
ity and structure activity relationship of Septicine
(32) and its analogues.
In 1987, Petit et al.,5 reported the isolation of Com-
bretastatin A-1 (CA-1) and B-1 (CB-1) from the South
African willow tree Combretum caffrum (Com-
bretaceae). Both natural products were shown to be
significant cancer cell growth inhibitors and antimitotic
agents. Later it was found that monophenol derivative
of CA-1, Combretastatin A-4 (CA-4) is much more
potent in its phosphate prodrug.6 Current phase-II
clinical trials and the uncovering of its very promising
Literature search revealed that Septicine has been syn-
thesized earlier by different routes.12À18 We employed
the method reported by Bhakuni et al.17,18 with a few
modifications to synthesize analogues as shown in
Scheme 1. Appropriately substituted 3-oxo-3-phenyl-
propionicacid methyl esters (1) were hydrolyzed to cor-
responding 3-oxo-3-phenyl-propionicacids (2), these
were condensed with 1-pyrroline 319 to give 1-phenyl-2-
pyrrolidin-2-yl-ethanones (4). Reaction of 4 with differ-
ent substituted phenylacetylchlorides (5) in the presence
of triethylamine yielded 1-phenyl-2-(1-phenylace-
*Corresponding author. Tel.: +91-40-23045439; fax: +91-40-
23045438; e-mail: sarmavm@drreddys.com
yDRL Publication No. 274.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00263-4

1680
V. M. Sharma et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1679–1682
Scheme 2. (a) BH3:DMS, THF, rt, 6–12 h, 40–45%.
Reduction of lactum was achieved by using BH3:DMS
in THF as shown in Scheme 2.
Cytotoxic activities of the synthesized compounds were
tested against Breast (MCF/ADR), CNS (U251), Colon
(SW620), Lung (H-522), Melanoma (M14), Ovarian
(SKOV3), Prostate (DU145) and Renal (A498) human
cancer cell lines. The results are shown in Table 1. For
comparison purposes, the average cytotoxic activities of
compounds in all cell lines were calculated and the
results of this computation are included in Table 1. We
started our SAR from septicine (32) expecting a mini-
mum activity due to structural similarity with Com-
bretastatin. But to our surprise the compound did not
show cytotoxic activity. Then we screened compound 7,
which is a precursor for 32, and 9 which has similar
methoxy/hydroxylation pattern of CA-4, against a panel
Figure 1. Structures of Combretastatin A-1(CA-1), Combretastatin B-
1(CB-1), Combretastatin A-4 (CA-4) and Septicine (32).
tylpyrrolidin-2-yl-)-ethanones (6). These 1-phenyl-2-(1-
phenylacetylpyrrolidin-2-yl-)-ethanones
(6)
were
cyclized in alcoholic potassium hydroxide to obtain 6,7-
diphenyl-2,3,8,8a-tetrahydro-1H-indolizin-5-ones (7–31)
in good yields.
Scheme 1. (a) 2–5% aq KOH, rt, 24 h, 65–70%; (b) phosphate buffer (pH 7.0), rt, 60 h, 55–60%; (c) Et3N, CH2Cl2, rt, 2–4 h, 48–55%; (d) 1–4%
ethanolic KOH, reflux, 2–4 h, 75–80%; (e) concd HCl/MeOH, reflux, 10 h, 85%; (f) concd HCl/MeOH, reflux, 10 h, 87%; (g) N,N0-Carbonyldiimi-
dazole (1 equiv), N-Boc-glycine (1 equiv), CH2Cl2, rt, 12 h, 62%; (h) N,N0-carbonyldimidazole (1 equiv), N-Boc-(l)-proline (1 equiv), CH2Cl2, rt,
12 h, 58%; (i) concd HCl/MeOH, reflux, 12 h, 85%; (j) 47% aq HBr, reflux, 2 h, 40%; (k) 47% aq HBr, reflux, 2 h, 85%; (l) Chloro-
ethylmorpholine.HCl (1.5 equiv), K2CO3 (2 equiv), acetone, reflux, 12 h, 75%; (m) epichlorohydrin (1.5 equiv), NaH (2 equiv), benzene, reflux, 12 h,
87%.

V. M. Sharma et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1679–1682
Table 1. In vitro cytotoxic activities of indolizidinone derivatives
1681
Compd
GI50 values in mM
Breast
MCF/ADR
CNS
U 251
Colon
SW620
Lung
H-522
Melanoma
M14
Ovarian
SKOV3
Prostate
DU145
Renal
A498
Average
GI50 (mM)
7
9
>100
>100
5.0
>100
>100
5.0
>100
>100
20.0
20.0
10.0
9.0
>100
>100
6.0
>100
>100
nt
>100
>100
>100
20.0
10.0
8.0
>100
>100
6.0
>100
>100
nt
>100
>100
23.6
11.5
6.0
11
12
13
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
CA-4
6.0
4.0
8.0
6.0
0.4
0.8
0.8
0.5
6.0
2.0
8.0
0.8
0.09
15.0
10.0
0.2
7.0
5.0
7.0
0.7
0.1
0.2
0.3
0.5
2.0
60.0
0.8
nt
nt
9.0
3.0
0.2
0.3
0.8
0.8
5.0
nt
4.0
10.0
5.0
8.0
0.9
0.9
0.5
0.5
0.4
nt
nt
5.0
0.6
0.4
0.1
0.3
0.4
8.0
nt
0.8
7.75
2.5
2.1
2.1
1.7
1.1
3.6
5.0
0.5
0.2
0.7
0.6
3.0
50.0
3.0
3.0
15.0
0.1
0.3
6.0
9.0
0.6
80.0
5.0
0.3
30.0
2.0
1.5
60.0
0.9
80.0
3.0
>100
9.0
60
2.4
>100
11.5
2.6
>100
9.0
>100
>100
9.0
>100
>100
20.0
0.09
9.0
>100
20.0
0.5
>100
9.0
0.5
9.0
0.08
7.0
0.4
0.8
0.09
5.0
7.0
0.03
7.0
0.9
0.8
0.25
10.0
8.0
0.6
0.8
0.09
7.0
3.0
0.8
0.5
7.0
10.0
9.0
9.0
15.0
30.0
2.0
9.0
0.6
0.7
0.5
7.0
4.0
20.0
0.1
7.8
3.0
6.3
4.1
6.0
12.0
0.6
0.8
8.0
>100
>100
0.09
15.0
>100
>100
0.007
5.0
8.0
15.0
>100
>100
8.1
>100
>100
0.006
>100
>100
0.005
>100
>100
0.0001
>100
>100
<0.0001
>100
>100
nt
>100
>100
0.103
0.61
GI50 mM—Concentration that produces 50% inhibition.
nt, Not tested.
of eight human cancer cell lines. Compounds 7 and 9
were also found inactive. A very slight improvement in
activity with an average GI50 of 23.6 mM concentration
was seen when all the methoxy groups in ring A of
compound 9 were replaced with 4-thiomethyl sub-
stituent (11). However the corresponding reduced com-
pound (33) did not show any activity. This proves the
importance of the 4-thiomethyl substituent in ring A
and the presence of lactum carbonyl in central indolizi-
dine ring for the activity. Further improvement in
activity was observed for ester derivatives 12 and 13
with 11.5 and 6.0 average GI50 values, respectively.
Compound 15, a positional isomer of 11, has shown
better activity (7.75 mM). Compound 21,21 where the
hydroxy group of compound 11 was replaced with
methoxy, exhibited potent activity in various cell lines
ranging from 0.3–9.0 mM. It is significant to note that
activity at less than 1 mM concentration in many of the
tested cell lines was achieved for the first time in this series
with compounds, where one of the methoxy group in ring
B of compound 21 was replaced with bromo (16) and
methyl (17) substituents. Comparison of cytotoxic activ-
ities of compounds 11, 15, 16, 17, 21 and compounds 18,
19, 20 revealed that methoxy substituents in ring B seem to
be not very important for these compounds for their cyto-
toxicity. Compounds 18, 19 and 20 where both methoxy
groups in ring B of compound 21 were replaced by fluoro
and/or methyl substituents resulted in marked increase in
antiproliferative activities. However, compound 22, a
dihydroxy derivative was found to be inactive.
biological activity or one is sufficient. To test this, com-
pound 23 was synthesized with 4-methoxy substituent in
ring B. The compound displayed similar activity to that
of its counterpart 21. Compounds with 4-bromo (27),
4-methyl (2822), 4-trifluoromethyl (29), 4-fluoro (30) and
4-thiomethyl (31) substituents in ring B showed better
activity in the increasing order of SCH3 <Br <F<CF3
<CH3. Compound 25, where morpholinoethyl group
was introduced in the place of methyl group of 23 by
aiming to improve the solubility and in vitro potency
has shown less activity, whereas 26 with (2,3-epoxy)-
propyl substituent has shown excellent activity with
nanomolar GI50 values in three out of eight cell lines.
Combretastatin A-4 when tested for its cytotoxic activ-
ity in the above human cancer cell lines showed 100-fold
potency in comparison to compounds 21 and 28.
The pharmacokinetic properties in mice, for selected
indolizidinone analogues (18, 21, 28, 29, 30, 31) were
studied at 200 mg/kg ip dose. The results are summarized
in Table 2. The pharmacokinetic profiles for compounds
Table 2. Pharmacokinetic parameters in mice
Parameters
18
21
28
200
38.54
8.02
0.25
6.24
29
200
10.72
3.79
0.25
2.66
30
200
3.88
0.99
1.50
2.59
31
200
2.50
0.86
1.00
#
Dose mg/kg, ip
AUC (0-t) mM. h.
Cmax mM
100 200
6.92 102.37
3.61
0.29
#
22.99
0.25
7.29
Tmax
h
t1=2ꢀð2;4;6;8
h
hÞ
# Not possible to calculate because of detection limits of the analytical
method and limited data points available in the elimination phase.
Each value is meanꢀS.D. of n=3–4 observations.
At this juncture, we shifted our focus to know whether
both the substituents in ring B are required for the

1682
V. M. Sharma et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1679–1682
21 and 28 are encouraging and are characterized by
reasonable Cmax and t1=2 values. On the basis of trends
we observed in vitro followed by pharmacokinetic pro-
files, we chose to examine the preliminary in vivo effi-
cacy of compounds 21 and 28 in modified hollow fiber
assay (HFA). Each compound was tested at two differ-
ent doses 200 and 400 mg/kg intraperitoneally in QDx4
schedule by following the procedure described by
Melinda et. al.20 The actively growing different types of
cancer cells in hollow fibre capsules were implanted in
SAM (Swiss Albino Mice) aseptically under light ether
anesthesia in to the subcutaneous (sc) and intraper-
itonial (ip) compartments. The anticancer activity in this
model was assessed based on percent net growth in both
the compartments. Compounds 21 and 28 were tested
against all the eight cell lines. To simplify evaluation, a
points system has been adopted which allows rapid
viewing of the activity of a given compound by using
HFA criteria, that is a %T/C of 50 or less in 10 of the
32 possible test concentrations (eight cell lines ꢁ two
sites ꢁ two compound doses). For this a value of two is
assigned for each compound dose which results in a
50% or greater reduction in a viable cell mass. The ip
and sc samples were scored separately.
5. Pettit, G. R.; Singh, S. B.; Niven, M. L.; Hamel, E.;
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21. Note 1. Compound 21: mp 150–151 ꢃC; IR: 2926.4,
1
1631.2, 1514.1 cmÀ1; HNMR (CDCl3) d 1.5–2.0 (2H, m), 2.1
References and Notes
(1H, m), 2.3 (1H, m), 2.45 (3H, s), 2.7–2.85 (2H, m), 3.5–4.0
(3H, m), 3.65 (3H, s), 3.85 (3H, s), 6.6–6.7 (3H, m), 6.9–7.1
(4H, dd, J=8.4 Hz); EIMS m/z 395 (M+, 100%).
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22. Note 2. Compound 28: mp 105–106 ꢃC; IR: 1640.6,
1434.8, 1363.0 cmÀ1 1HNMR (CDCl3) d 1.6–2.0 (2H,m),
;
2.1(1H, m), 2.2 (1H, m), 2.45 (3H, s), 2.5 (3H, s), 2.8 (2H, m),
3.7 (2H, m), 4.0 (1H, m), 7.0 (8H, m); EIMS m/z 349 (M+,
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