Synthesis and evaluation of 4/5-hydroxy-2,3-diaryl(substituted)-cyclopent- 2-en-1-ones as cis-restricted analogues of combretastatin A-4 as novel anticancer agents

Article abstract of DOI:10.1021/jm060938o

A new series of 2,3-diaryl-4/5-hydroxy-cyclopent-2-en-1-one analogues replacing the cis double bond of combretastatin A-4 (CA-4) by 4/5-hydroxy cyclopentenone moieties was designed and synthesized. The analogues displayed potent cytotoxic activity (IC

Full text of DOI:10.1021/jm060938o

1744
J. Med. Chem. 2007, 50, 1744-1753
Synthesis and Evaluation of 4/5-Hydroxy-2,3-diaryl(substituted)-cyclopent-2-en-1-ones as
cis-Restricted Analogues of Combretastatin A-4 as Novel Anticancer Agents
Mukund K. Gurjar,† Radhika D. Wakharkar,*,† Anu T. Singh,*,‡ Manu Jaggi,*,‡ Hanumant B. Borate,† Popat D. Shinde,†
Ritu Verma,‡ Praveen Rajendran,‡ Sarjana Dutt,‡ Gurvinder Singh,‡ Vinod K. Sanna,‡ Manoj K. Singh,‡ Sanjay K. Srivastava,‡
Vishal A. Mahajan,† Vinod H. Jadhav,† Kakali Dutta,‡ Karthik Krishnan,‡ Anika Chaudhary,‡ Shiv K. Agarwal,‡
Rama Mukherjee,‡ and Anand C. Burman‡
DiVision of Organic Chemistry: Technology, National Chemical Laboratory, Pune 411008, India, and Dabur Research Foundation,
Sahibabad, Ghaziabad 201010, India
ReceiVed August 3, 2006
A new series of 2,3-diaryl-4/5-hydroxy-cyclopent-2-en-1-one analogues replacing the cis double bond of
combretastatin A-4 (CA-4) by 4/5-hydroxy cyclopentenone moieties was designed and synthesized. The
analogues displayed potent cytotoxic activity (IC50 < 1 µg/mL) against a panel of human cancer cell lines
and endothelial cells. The most potent analogues 11 and 42 belonging to the 5-hydroxy cyclopentenone
class were further evaluated for their mechanism of action. Both of the analogues led to cell cycle arrest at
G2/M phase and induced apoptosis in endothelial cells. Antitubulin property of 42 was superior to 11 and
comparable to CA-4. The compound 42 had better aqueous solubility, metabolic stability, and pharmacokinetic
profile than CA-4 and also demonstrated significant tumor regression in the human colon xenograft model.
Our data suggests that cis-restricted analogues of CA-4 are a new class of molecules that have the potential
to be developed as novel agents for the treatment of cancer.
Introduction
Microtubules are long, filamentous tube-shaped polymers of
R and â tubulin that are essential in all eukaryotic cells. They
are crucial in the development and maintenance of cell shape,
in the transport of vesicles, mitochondria and other components
throughout the cells, in cell signaling, and in cell division or
mitosis.1 Their importance in mitosis and cell division make
microtubules an obvious and important target for anticancer
drugs.1 The dynamic process of assembly and disassembly of
microtubules to tubulin is blocked by various agents that bind
to distinct sites such as binding sites for colchicines, taxol, and
vinca alkaloids, finally leading to arrest in mitosis and cell death
by apoptosis or necrosis.2 Several microtubule targeting drugs,-
namely, Paclitaxel, Epothilones, and Dolastatin are in clinical
use or in various stages of clinical development.2
Figure 1. Structures of CA-4 phosphate (1) and CA-4 (1′). R ) H
combretastatin A-4 (CA-4, 1′), R ) PO3Na2 CA-4 disodium phosphate
ester (CA-4P, 1).
cancer revealed that the combination of 1 with either carboplatin
or paclitaxel was well tolerated.4
Preclinical studies have shown that 1 induces blood flow
reductions and subsequent tumor cell death in a variety of
preclinical models.5
The encouraging antivascular and anticancer activity of 1 has
stimulated significant interest in a number of diverse ligands
designed to mimic 1. It has been established through structure-
activityrelationship(SAR)studiesinseveralresearchlaboratories6-8a,b
that the cis-configuration of the double bond between the two
aryl rings and the 3,4,5-trimethoxy systems on the A ring are
fundamental requirements for its biological activity. However,
the main problems associated with this compound have been
the ready isomerization of the double bond to its inactive trans-
form and low water solubility.9,10 As an outcome of the
exhaustive study on 1′, it has been demonstrated11,12 that 1 could
provide the solution to overcome the problem of low water
solubility. In our laboratories we have been actively engaged
in designing several strategies to improve the potency and
overcome double bond isomerization of 1. One of the key
approaches taken has been to rigidify the olefin by a heterocyclic
compound,13-17 five-membered and six-membered rings, as also
previously shown by us.18 In our opinion, the hydroxy-
cyclopentenone moiety can be the surrogate of the cis-double
bond separating the two phenyl rings to combat with the
problems of double bond isomerization.
Combretastatin A-4 (1′; Figure 1), a natural product isolated
from the bark of the South African tree Combretum caffrum is
highly cytotoxic against a variety of human cancer cells
including multidrug resistant cancer cell lines.3 The molecule
binds at or near colchicine binding site and inhibits tubulin
polymerization ultimately leading to cell death.3 It displays
selective toxicity toward tumor vasculature by inhibiting the
blood supply to the tumor leading to cell death. Currently two
phase II clinical trials are ongoing for 1 (combretastatin A-4
phosphate) for advanced anaplastic thyroid cancer, and one
phase I study has recently begun in combination with bevaci-
zumab for subjects with advanced solid tumors (www.clinical-
trials.gov). A phase Ib trial of 1 in combination with carboplatin
or paclitaxel chemotherapy conducted in patients with advanced
* To whom correspondence should be addressed. Phone: (91) 020-
25902284 (R.D.W.); (91) 0120-4378505 (A.T.S.); (91) 0120-4378610 (M.J.).
Fax: (91) 020-25902629 (R.D.W.); (91) 0120-4376902 (A.T.S. and M.J.).
E-mail: rd.wakharkar@ncl.res.in (R.D.W.); singhat@dabur.com (A.T.S.);
jaggim@dabur.com (M.J.).
A family of 2,3-diaryl-4/5-hydroxy-cyclopent-2-en-1-one
analogues replacing the cis-double bond by 4/5-hydroxy cy-
clopentenone moieties was designed and synthesized to study
† National Chemical Laboratory.
‡ Dabur Research Foundation.
10.1021/jm060938o CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/21/2007

Combretastatin A-4 Analogues as Anticancer Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1745
Table 1. 2,3-Diaryl-4-hydroxycyclopent-2-en-1-one Analogues of 1′
Sr. No
cmpd
R
R1
R2
R3
R4
R5
R6
OMe
OMe
H
OMe
H
OMe
H
R7
X
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
OH
OH
OH
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
H
H
H
OMe
H
H
H
OMe
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
Me
H
Me
OMe
H
OTBS
OTBS
OTBS
OTBS
OH
OAc
OTBS
OTBS
OTBS
OTBS
OAc
OAc
OTBS
OH
OAc
OTBS
OH
OTBS
OTBS
OH
H
H
H
Me
H
Me
OMe
H
H
OMe
H
H
H
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
H
N-OH
N-OH
N-OH
O
H
COOMe
OMe
Me
Me
OMe
H
OMe
OMe
OMe
OMe
OMe
H
O
O
O
N-OH
N-OH
N-OH
N-OH
O
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMOM
OH
OMOM
OH
OH
OH
H
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
H
O
N-OH
O
N-OH
N-OH
O
OAc
OH
H
26
27
39
40
OH
OAc
OMe
OMe
OMe
OMe
OMe
OMe
H
H
COONa
H
OMe
OMe
H
H
O
N-OH
their potential cytotoxicity. Introduction of hydroxy cyclopen-
tenone moiety indeed proved to be advantageous with respect
to the stability, being a cis-restricted structural modification.
The hydroxy group at the 4- or 5-position in the cyclopentenone
ring provided a handle to design several stable analogues. The
synthetic methods developed allowed us to generate 28 ana-
logues each of 4-hydroxy and 5-hydroxy cyclopentenone
compounds. The analogues were screened for cytotoxicity using
nine human cell lines. They were selected on the basis of in
vitro cytotoxicity data and were then evaluated for their potential
to inhibit tubulin polymerization, cause cell cycle arrest, and
induce apoptosis. Additionally, the aqueous solubility, metabolic
stability, pharmacokinetics, and in vivo efficacy in the tumor
xenograft model were also evaluated for the selected analogues.
In the present manuscript, it is shown that the replacement of
the cis-double bond by the hydroxy-cyclopentenone moiety
produced several stable cytotoxic compounds. The SAR and
pharmacodynamic profiles of the analogues is also described
here.
Protection of the 4-hydroxy group in 3 with tert-butyldimeth-
ylsilyl chloride gave the corresponding derivatives, which were
subjected to the Heck reaction using palladium acetate and
aryl halide in the presence of base to obtain the desired
analogues 4.
This methodology provided 28 analogues (27 compounds in
Table 1 and compound 10 in Scheme 3) by employing various
benzaldehydes, aryl halides, and further deprotection, followed
by derivatization. This series included the synthesis of com-
bretastatin A-2 analogue 5 (Scheme 1), for which 5-methoxy
piperonaldehyde was treated with furyllithium, and the Heck
reaction was performed using 4-iodo-1-methoxy-2-methoxym-
ethyloxybenzene in our synthetic strategy described above. It
was observed that oximation of the products thus obtained led
to the more soluble substances.
The second group of compounds possessing a hydroxy group
at the 5-position in the cyclopentenone ring was accomplished
from the intermediate 6. The intermediate 6 was treated (Scheme
2) with the desired aryl halide (bromide or iodide) in the
presence of magnesium or n-butyllithium, which reacted to give
a 1,2-addition product as the tertiary alcohol, in good to excellent
yields. Additionally, reaction of the cyclopentenol 7 performed
with pyridinium dichromate (PDC) in dichloromethane at 0 °C
to room temperature afforded the corresponding 2,3-diaryl-5-
tert-butyldimethylsilyloxycyclopentenones 8. The desired 5-hy-
droxy analogues were obtained by deprotection of the corre-
sponding 2,3-diaryl-5-tert-butyldimethylsilyloxycyclopentenones
Chemistry
The 4-hydroxy-2,3-diaryl-cyclopentenone analogues 4 were
synthesized starting from condensation of furyl lithium with
corresponding substituted benzaldehydes. A simple and efficient
one step method18 was developed for the preparation of 2-aryl-
4-hydroxy-cyclopentenone 3 from 2-furylmethanol 2 in excellent
yield using zinc chloride as a weak Lewis acid at 110 °C.

1746 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gurjar et al.
Scheme 1a
acylation, (3) oximation of the ketone group, and so on. Other
derivatizations leading to analogues having a molecular weight
of more than 500 were avoided to select better druglike
molecules. The noteworthy observations based on the screening
data (Table 3) of 28 samples are discussed below.
Some of the samples were purposely synthesized with absence
of a methoxy group at 4-position in the B-ring, such samples,
for example, the compounds 16, 18, and 20, showed reduced
cytotoxicity, which confirmed that the presence of a methoxy
group at position 4 in the ring-B is mandatory for the biological
activity.
In addition to having a methoxy functional group on B-ring,
a carboxylic acid group was also attached to the B-ring to
enhance the solubility of the analogues. These analogues were
also amenable to salt formation. We synthesized a compound
with -COOH at the 3-position of the B-ring, that is, the
compound 39. This option was not found beneficial as it lowered
the cytotoxic activity. Similarly, the methoxy group at the
4-position in the ring B was replaced by carboxymethyloxy
(-OCH2COOH) group (Scheme 3) and its water-soluble sodium
salt (compound 10), which was less active.
a Reagents and conditions: (a) (i) n-BuLi, THF; (ii) substituted benzal-
dehyde; (b) ZnCl2, dioxan/water, reflux, 24 h; (c) TBDMSCl, DMAP, DCM,
Et3N, 3 h; (d) Pd(OAc)2 (12 mol %), K2CO3 (2 equiv), Bu4NBr, CH3CN.
8. The mechanism19 of this rearrangement involves initial
formation of the chromate ester A from the tertiary alcohol,
followed by an allylic rearrangement of the chromate ester of
the secondary alcohol. Typical fragmentation of the resultant
chromate ester B delivered the ketone. In this series we have
mainly synthesized the B-ring modified analogues of cis-
restricted 1′ analogues in which the hydroxy group at 3-position
of B-ring was replaced with substituents like H, Cl, F,
O-isopropyl, O-allyl, NHAc, NHCOPh, NMe2, NHCHO, and
NMeCHO (Table 2).
To synthesize some water soluble analogues in this series of
compounds and also to confirm the role of methoxy group at
the 4-position in the B-ring, we considered replacing 4-methoxy
in the B-ring of the 1′ analogue by employing 4-iodophenoxy-
acetic acid ethyl ester in the Heck reaction step, which furnished
the intermediate 9 (Scheme 3). Alkaline hydrolysis of the ethyl
ester 9 and sequential treatment with sodium hydroxide in
distilled water gave the water-soluble sodium salt 10.
A highly water soluble analogue of 5-hydroxy-2-(3,4,5-
trimethoxyphenyl)-3-(4-methoxyphenyl)-cyclopent-2-ene-1-
one 11 was accomplished as the disodium salt of its phosphate
analogue 13. The compound 11 was reacted with di-tert-butyl-
N,N-diethylphosphoramidite and tetrazole in THF to get the
corresponding phosphate analogue 12. Further hydrolytic cleav-
age of tert-butyl groups with TFA in DCM followed by
treatment with sodium hydroxide successfully formed the water-
soluble analogue 13. All compounds synthesized possessing 4/5-
hydroxy functionality and their derivatives were characterized
by spectroscopic methods, and purity was confirmed by analyti-
cal methods (high performance liquid chromatography (HPLC)
> 95%).
Biological Activity Results and SAR. The synthesized
cyclopentenone analogues were assessed for cytotoxicity in the
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide) cytotoxicity assay. 2,3-Diaryl-4-hydroxycyclopent-2-en-
1-one analogues (Table 1) were synthesized and screened for
cytotoxicity. In the designed analogues, ring A possessed 3,4,5-
trimethoxy substitution, which was considered to be fundamental
for the tubulin binding activity, whereas variations were made
in the ring B. For every variation in the B-ring, we achieved
4-6 samples for SAR study by simple organic transformations
like (1) removal of tert-butyldimethylsilyl (-OTBDMS) pro-
tecting group, (2) derivatization of this free hydroxy group by
The 1′ mimic, that is, the compound 33 and its analogues
35-37, displayed comparable or even better cancer cell growth
inhibition against a number of cancer cell lines such as HT29
(colon), DU145 (prostate), KB (oral), L132 (lung), Hep2
(larynx), and PA1 (ovary). In most of the examples studied,
oximation of the ketone in the cyclopentenone ring did not
influence the IC50 values significantly, for example, 21, 28-
31, and 34, while compounds 22, 23, 36, and 37 exhibited good
cytotoxicity. The compound 40 was found to be the most active
among the oximes studied, as shown in Table 3. The oxime
analogues were preferred in some cases due to their higher
solubility and served as prodrugs. The TBDMS protection of
the hydroxy group did not influence the cytotoxicity.
Table 2 exhibits the 5-hydroxy cyclopentenone analogues
screened for the cytotoxic activity, and the IC50 values are
depicted in Table 4. Some of the compounds from this class,
for example, 11, 41, 42, 46-48, and 66 showed strong cytotoxic
activity. One of the additional features studied in this group of
analogues was the halogenated B-ring compounds. The 3-chloro-
and 3-fluoro-substituted B-ring compounds 53-57, 59, and 60
displayed marked tumor growth suppression (IC50 < 1 µg/mL)
selectively against HT29, L132, and Hep2 cell lines.
On the contrary, the free hydroxy group as well as the
methoxy group at the 3-position of the B-ring decreased the
cytotoxic activity. Other alkyl ethers at the 3-position of ring B
like allyl ether and isopropyl ether also abolished the activity,
for example, compounds 44, 45, 58, 63, and 64. Potent efficacy
has been attributed to the replacement of the phenolic OH of 1′
with an amino group in the literature.8b We investigated the
compounds with the NHAc, the NMeCHO, and the NHCHO
group in place of the phenolic OH at 3-position in the B-ring.
These groups were well tolerated, and the compound 66 was
highly toxic to the tumor cells. This indicated that the nature
of the substituent in the phenyl ring located at position 3 in
5-hydroxy-cyclopentenone analogues was crucial.
By interchanging the substitution pattern of rings A and B,
we synthesized the compounds 49-51. It was noteworthy that
this pattern destroyed the efficacy of the compound to inhibit
the growth of cancer cells, especially in the case of the
compounds with an electron-withdrawing (nitro) group in place
of the phenolic OH (in ring A in this example). The presence
of a primary amine instead of a phenolic OH in the ring A (51)

Combretastatin A-4 Analogues as Anticancer Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1747
Scheme 2a
a Reagents and conditions: (a) Mg, THF or n-BuLi, THF; (b) PDC, DCM, 0 °C-rt.
Scheme 3a
a Reagents and conditions: (a) Pd(OAc)2 (12 mol %), K2CO3 (2 equiv), Bu4NBr, CH3CN, 36 h; (b) TBAF (1 M soln.), THF, 0 °C-rt, 1 h; (c) 10% NaOH,
MeOH; H+; (d) NaOH, dist. water.
Table 2. 2,3-Diaryl-5-hydroxycyclopent-2-en-1-one Analogues of 1′
was effective for inhibition (IC50 < 1 µg/mL) of at least two
cell lines, namely, HT-29 and KB.
Finally, the 4-methoxy group in the B-ring was replaced by
a 4-thiomethyl group, and it was observed that compound 48
still retained the cytotoxicity. However, this activity was lost
to some extent when the TBDMS protection was removed
(compound 52). The role of the TBDMS group under biological
systems has not been studied, however, it is noteworthy that,
in some of the analogues possessing TBDMS instead of the
free hydroxyl group in the cyclopentenone ring, enhanced
cytotoxicity was observed, for example, compounds 23 vs 16;
24 vs 30; 47 vs 44; and 45 and 48 vs 52.
Sr.
No cmpd
Compound 11 displayed a promising cytotoxicity profile in
R
X
R1
R2
R3
R5
R6
R7
a number of cancer cell lines. In addition to 11, the correspond-
ing oxime 42 also exhibited strong cytotoxicity against a number
of cancer cell lines such as HT29, KB, L132, MiaPaCa2, Hep
2, PA-1, and ECV 304. The overall biological activity studies
of the compounds in this group indicated that the compound
with the A-ring possessing 3,4,5-trimethoxy systems and the
B-ring with a 4-methoxy substitution was a very potent cytotoxic
compound. A water-soluble disodium phosphate derivative (13)
of analogue retained the activity shown by the parent molecule
(11).
Based on the in vitro cytotoxicity data, two cyclopentenone
analogues 11 and 42 were selected. These analogues were
assessed for their effects on cell cycle, ability to induce
programmed cell death, and in vitro tubulin polymerization
inhibition potential. The reported antivascular properties of 1′
prompted us to select endothelial cells (ECV304) as the model
for investigating the mechanism of anticancer/antivascular action
of cyclopentenone analogues.
Tubulin Polymerization. The cyclopentenone analogues 11
and 42 were assessed for their ability to inhibit tubulin
polymerization. IC50 values for inhibition of tubulin polymer-
ization for 1′, 11 and 42 were found to be 0.98, 5.34 and 1.75
µM respectively (Table 5). The comparison of IC50 values of
1′ and 42 indicate that 42 was comparable to 1′ and may act as
a cytotoxic agent through inhibition of tubulin polymerization.
The antitubulin potential of 42 was evidently superior to 11.
1
2
3
4
5
6
7
8
9
11 OH
41 OAc
42 OH
O
O
OMe OMe OMe
OMe OMe OMe
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
SMe
H
H
H
H
H
H
H
H
H
N-OH OMe OMe OMe
43 OAc N-OH OMe OMe OMe
44 OH
45 OH
46 OTBS
47 OTBS
48 OTBS
O
OMe OMe OMe OiPr
N-OH OMe OMe OMe OiPr
O
O
O
O
O
O
OMe OMe OMe
OMe OMe OMe OiPr
OMe OMe OMe
NO2 OMe
NO2 OMe
NH2 OMe
H
H
10 49 OTBS
11 50 OH
12 51 OH
13 52 OH
14 53 OH
15 54 OH
16 55 OAc
17 56 OH
18 57 OH
19 58 OAc
20 59 OAc
H
H
H
OMe
OMe
OMe
H
OMe OMe
OMe OMe
OMe OMe
N-OH OMe OMe OMe
N-OH OMe OMe OMe Cl
SMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
OMe OMe OMe
OMe OMe OMe
F
F
F
N-OH OMe OMe OMe
O
O
O
OMe OMe OMe Cl
OMe OMe OMe OiPr
OMe OMe OMe Cl
21 60 OAc N-OH OMe OMe OMe Cl
22 61 OAc
23 62 OH
24 63 OH
25 64 OH
26 65 OH
27 66 OAc
O
OMe OMe OMe OH
N-OH OMe OMe OMe OH
OMe OMe OMe O-allyl
O
N-OH OMe OMe OMe O-allyl
O
O
OMe OMe OMe NHCOPh OMe
OMe OMe OMe NHCHO OMe
Hence, based on the superior inhibition of tubulin polymeriza-
tion, 42 was selected for further preclinical studies.

1748 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gurjar et al.
Table 3. Cytotoxicity Data of Cyclopentenone Analogues from 2,3-Diaryl-4-hydroxycyclopent-2-en-1-one Seriesa
IC50 (µg/mL)
cmpd of
formula
Sr. No.
No.
HT29
DU145
KB
<1
L132
MiaPaCa-2
Hep2
PA1
ECV 304
293
1′
10
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
5
ND
15
NA
NA
NA
NA
10
5
15
1.5
50
1
NA
NA
NA
NA
20
NA
NA
43
57
<1
ND
10
NA
NA
NA
NA
NA
100
11
<1
NA
NA
NA
NA
NA
NA
ND
NA
NA
1.0
1
5
<1
ND
3.38
NA
NA
NA
NA
32
<1
ND
NA
NA
NA
NA
NA
15
6.5
NA
4
6
4
NA
NA
NA
NA
36
NA
NA
40
NA
16.5
30
NA
NA
NA
NA
60
5.7
NA
10
NA
NA
NA
NA
29
9.5
NA
32
7
5
NA
NA
NA
NA
70
NA
92.3
NA
NA
18
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
NA
18.7
NA
NA
NA
NA
NA
100
34.25
20
NA
1.5
NA
NA
NA
NA
60
NA
NA
34
8
30
75
23
23
NA
ND
NA
NA
NA
NA
40
10
ND
5
NA
ND
NA
NA
NA
NA
15
NA
9
33
NA
NA
NA
NA
NA
NA
26
19
NA
3
17
2.35
20
20
NA
40
7
4
8.5
1
1.61
NA
NA
NA
NA
ND
NA
NA
13.5
<1
8
14
3.7
<1
NA
NA
<1.0
NA
NA
NA
NA
60
NA
NA
32
NA
NA
NA
NA
15
NA
4.5
50
NA
6
55
NA
<1.0
NA
NA
<1.0
NA
NA
NA
NA
32
NA
NA
12
<1
13.7
14.5
<1
NA
NA
NA
<1.0
<1
<1
38
<1
27
<1
28
6.5
10
<1.0
NA
NA
<1.0
<1
8
<1.0
NA
NA
<1.0
42
9
NA
6.06
NA
NA
22
<1
NA
NA
<1
NA
NA
21
a NA ) not active, that is, the IC50 is greater than the maximal concentration tested (100 µg/mL). ND ) not done.
Table 4. Cytotoxicity Data of Cyclopentenone Analogues from 2,3-Diaryl-5-hydroxycyclopent-2-en-1-one Seriesa
IC50 (µg/mL)
cmpd of
formula
Sr. No.
No.
HT29
DU145
KB
L132
MiaPaCa-2
Hep2
PA1
ECV 304
293
1′
11
13
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
5
57
<1
<1
72
72
<1
69
<1
<1
ND
NA
<1
ND
NA
75
9
7
<1
NA
NA
54
NA
<1
<1
<1
<1
<1
NA
<1
<1
6
<1
<1
<1
<1
<1
<1
NA
98
<1
NA
<1
NA
NA
NA
40
5
<1
<1
ND
<1
<1
<1
NA
61
<1
<1
46
5.7
6.3
ND
ND
4.2
25.6
NA
36.77
NA
NA
NA
NA
NA
NA
27
50
NA
59
NA
NA
NA
60
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
<1
ND
<1
<1
1
NA
69
5
NA
7.5
NA
NA
NA
NA
81
<1
<1
18
<1
100
NA
40
18
<1
NA
NA
96
NA
95
7
7.5
10
NA
NA
<1
15
9.8
4.5
33
<1
2.5
NA
64
50
35
90
NA
NA
NA
8
28
16
7
16
9
6.3
NA
NA
NA
41
2.5
16
27
16
10
40
NA
64
17
NA
10
NA
NA
NA
NA
7.1
NA
NA
NA
16
<1
<1
<1
<1
<1
<1
NA
<1
<1
NA
20
7.1
<1
NA
NA
20
25.3
NA
NA
NA
NA
NA
NA
NA
37.5
6
NA
NA
NA
NA
NA
57
18.5
19.5
41
<1
NA
NA
<1
36
<1
<1
<1
<1
<1
NA
<1
<1
<1
69
33
64
39.5
94
NA
NA
24
43
20
NA
3.5
8
NA
NA
NA
NA
NA
ND
10
32
92
18.5
NA
NA
NA
NA
2
81
26
NA
NA
NA
NA
4.16
NA
NA
NA
<1
NA
NA
NA
2
NA
NA
NA
3.27
NA
NA
NA
5.0
a NA ) not active, that is, the IC50 is greater than the maximal concentration tested (100 µg/mL). ND ) not done.
Arrest of Endothelial Cells in G2/M and Inhibition of
Mitosis. The ability of cyclopentenone analogues 11 and 42 to
modulate endothelial cell cycle progression was evaluated using
asynchronous proliferating endothelial cultures (ECV304) ex-
posed to the drug, which were then stained with propidium
iodide (PI) and analyzed by flow cytometry. Cells were treated

Combretastatin A-4 Analogues as Anticancer Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1749
Figure 2. Effect on the cell cycle as determined by flow cytometry.
Untreated cells were taken as control (2a). Cells were treated with
analogues 11 (2b), 42 (2c), and 1′ (2d) at the concentration of 1 µM.
Table 5. In Vitro Tubulin Polymerization Inhibition Potential of 1′, 11,
and 42
Figure 3. Evaluation of apoptosis by DNA fragmentation in ECV-
304 cells. (3a and 3b) Depiction of the DNA fragmentation pattern
observed with treatment of cells with 11 and (3c and 3d) depiction of
the DNA fragmentation pattern observed with treatment of cells with
42 in strongly adherent and loosely adherent cells, respectively, at
concentrations of 10, 100, and 1000 nM. 1′ was included as a reference
standard at the concentration of 100 nM. Lane 1, 100 bp DNA marker;
Lane 2, control cells; Lanes 3-5, cells treated with 10, 100, and 1000
nM of analogues; and Lane 6, 100 nM 1′. Lanes 3, 4, 5, and 6 of 3b
and 3d depict discrete DNA fragments, which are characteristic
morphological marker of apoptosis.
IC50
(µM)
cmpd
1′
11
42
0.98
5.34
1.75
Table 6. Physical Properties of 1′and 42
solubility
(µM)
metabolic stability
(% compound remaining)
cmpd
1′
42
122.8
359.6
84.8
96.4
evident DNA fragmentation in the loosely adherent cells at all
the concentrations studied. (Figure 3). Similar apoptotic effects
of 1 (100 nM) were previously reported28 in human umbilical
vein endothelial cells.
DAPI Staining for Visualization of Apoptosis. CA-4 (1′)
and analogues 11 and 42 were found to cause apoptosis as seen
by staining micronuclei with DAPI (4′-6-diamidino-2-phenylin-
dole) in loosely adherent ECV304 cells, in a dose-dependent
manner at concentrations ranging from 10 nM to 1 µM
(Figure 4).
Multiple damaged, fragmented, and unevenly displayed
micronuclei were observed in a significant number of cells.
Hence, the cyclopentenone analogues 11 and 42 may possess
similar apoptotic effects in endothelial cells that were compa-
rable to that observed for 1′.
with 1′, 11, and 42 at the concentration of 1 µM for 24 h. There
was an accumulation of 41, 54, and 59% of cells in the G2/M
phase of the cell cycle observed with 1′, 11, and 42 as compared
to that of the untreated control (Figure 2) in which only 18%
accumulation was observed. Hence, both the derivatives 11 and
42 at a concentration of 1 µM led to enhanced mitotic arrest in
G2/M phase in endothelial cell cultures.
DNA Fragmentation Analysis. Proliferating endothelial cells
were treated with 1′, 11, and 42 at concentrations ranging from
10 nM to 1 µM for 24 h. After the treatment, many rounded,
loosely adherent cells accumulated in the cultures. Evaluation
of apoptosis by DNA fragmentation was carried out in both
strongly and loosely adherent cells. No DNA fragmentation was
observed in strongly adherent cells at any of the concentrations
studied. However, the treatment with 1′, 11, and 42 led to
Physical Properties. Based on better inhibition of tubulin,
42 was selected for studying physical properties such as
Table 7. Pharmacokinetic Parameters of 1′ and 42
maximum
volume of
distribution,
Vd
mean
residence
time, MRT
(h)
plasma
concn., Cmax
(µg/mL)
area under
curve, AUC
(h‚µg/mL)
half-life, t1/2
clearance, Cl
(mL/h)
cmpd
(h)
(mL)
1′
42
0.19 ( 0.08
0.47 ( 0.1
3.83 ( 1.0
7.05 ( 1.2
0.92 ( 0.2
3.84 ( 0.3
80.82 ( 47.8
46.56 ( 14.05
291.36 ( 65.5
68.94 ( 3.2
0.15 ( 0.04
0.53 ( 0.09

1750 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gurjar et al.
Figure 6. In vivo antitumor activity of 42 in human colon (PTC)
xenograft model. Treatment was initiated on day 6 (tumor volume,
approx. 500 mm3). Values represent mean ( SD, n ) 3. (v) Represents
initiation of treatment, (V) represents withdrawal of treatment, and (*)
represents p < 0.05.
Figure 4. Visualization of micronuclei in ECV 304 cells by DAPI
staining. Untreated cells were taken as control (4a). Cells were treated
with 1′ (4b, 4c, 4d), 11 (4e, 4f, 4g), and 42 (4h, 4i, 4j) at the
concentrations of 10 nM, 100 nM, and 1 µM, respectively.
values for 42 were 3.84 h‚µg/mL, 46.56 mL, 68.94 mL/h, and
0.53 h, respectively, and for 1′ were 0.92 h‚µg/mL, 80.82 mL,
291.36 mL/h, and 0.15 h, respectively. The pharmacokinetic
parameters in mice show that 42 stays in plasma for a longer
time, as shown by a marginally higher half-life and mean
residence time and provides a higher drug exposure as compared
to 1′, as shown by a higher Cmax and AUC and a lower clearance
from plasma.
In Vivo Activity in Xenograft Model. Previous studies have
shown that 1 and 1′ were inactive in colon SW1222,20 colon
26,21 and HT-2922 tumor xenografts in nude mice, suggesting
that these compounds may not be endowed with anticancer
activity specific to colon cancers. The promising anticancer
activities of our analogues in colon cancer cell lines prompted
us to explore their potential in vivo. Based on the in vitro
cytotoxicity (IC50 ) 271 ng/mL) of 42 in primary tumor colon
cells (PTCa) obtained from patients with advanced metastatic
colon cancer, we tested the in vivo antitumor activity of 42 in
PTC xenografted in nude mice.
Different treatment regimes have been reported for 1 and 1′,
varying from single dose,21 daily doses,23 once in two days,24
and weekly.25 It has also been suggested that continuous dosing
with 1 gives better response compared to single dose, probably
by preventing revascularization and repopulation of the tumor
that is initially necrosed by drug treatment.23 Based on the above
studies, we have determined the antitumor activity of 42 at the
maximum tolerated dose of 20 mg/kg by daily administration
for 14 days. Figure 6 shows the tumor kinetics up to day 22 in
the treated and untreated animals. Daily treatment with 42 by
an intravenous route induced tumor regression in the xenografts
leading to significantly decreased tumor volume (P < 0.05) on
days 19 and 22 and T/C% of 45.8% on day 22 post tumor
inoculation as compared to untreated controls.
Figure 5. Plasma concentration profile of 1′ and 42 upon intravenous
administration in Swiss albino mice (each point represents mean (
SD, n ) 3).
solubility and metabolic stability. The data is summarized in
Table 6. The aqueous solubility of 42 and 1′ was 359.6 µM
and 122.8 µM, respectively, in phosphate buffer at pH 7.4, as
estimated using the DMSO precipitation method. In terms of
stability, 42 appeared to have better metabolic stability (96.4%
of parent compound remaining) as compared to 1′ (84.8% of
parent compound remaining) after incubation for 60 min at
37 °C with human liver microsomes. These results show that
cyclopentenone analogue 42 has better aqueous solubility and
metabolic stability as compared to 1′.
Pharmacokinetics. The plasma concentration curve of
compounds 1′ and 42 in mice is shown in Figure 5. Pharma-
cokinetic parameters are given in Table 7. The compounds were
rapidly eliminated in mice following i.v. administration with a
short terminal half-life of 0.19 h (1′) and 0.47 h (42). The Cmax
coincides with the first sample collection point at 3 min and is
low for both 42 (7.05 µg/mL) and 1′ (3.83 µg/mL). The AUC,
volume of distribution, clearance, and mean residence time
Conclusion
Microtubules are the key components of cytoskeleton and
are acted upon by a large number of chemically diverse
a Abbreviation: PTC, primary tumor colon cells.

Combretastatin A-4 Analogues as Anticancer Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1751
Experimental Section
compounds, many of which are derived from natural products
that inhibit the cell proliferation in mitosis.2 Hence, microtubules
are an attractive target for the treatment of cancer.1 Combret-
astatin A-4 (1′), a natural product isolated from Combretum
caffrum has potent anticancer and antivascular properties. It
destabilizes microtubules and disrupts rapidly the vascular
network of tumors leading to secondary tumor cell death. The
two main problems associated with 1′ are the ready isomeriza-
tion of the cis double bond to its inactive trans form and low
water solubility.9,10 To combat some of these problems, we have
reported here the development of successful protocols to
synthesize 2,3-diaryl (substituted)-4 or 5-hydroxy-cyclopent-2-
en-1-ones, which provided cis-restricted combretastatin ana-
logues. A common intermediate 3 was synthesized starting from
bench chemicals in three steps. Heck reaction using appropriate
aryl halide on this intermediate accomplished the synthesis of
4-hydroxy analogues, whereas 1,2-addition of aryl lithium or
the corresponding Grignard reagent followed by double rear-
rangement in single step employing PDC resulted into the
5-hydroxy analogues. The protocols efficiently produced 56 new
chemical entities, which were assessed for cytotoxicity and other
biological parameters such as antitubulin and apoptotic proper-
ties. Rigidifying 1′ with the hydroxycyclopentenone system did
not alter the reported SAR; on the contrary, it was in full
agreement and exhibited enhanced broad-spectrum cytotoxicity.
In vitro cytotoxicity data prompted us to select two promising
analogues 11 and 42 belonging to 2,3-diaryl-5-hydroxy-cyclo-
pent-2-en-1-one series that were highly cytotoxic (IC50 < 1 µg/
mL) in a panel of human cancer cell lines comprising of HT29
(colon), KB (oral), L132 (lung), MiaPaCa-2 (pancreas), Hep 2
(larynx), PA1 (ovary), and endothelial cell line ECV304.
Biological Assays. MTT, PI, DAPI, guanosine 5′-triphosphate
(GTP), and PIPES were obtained from Sigma, U.S.A. DMEM
(Dulbecco’s modified eagles medium) and fetal bovine serum (FBS)
were procured from Gibco BRL, U.S.A. DMSO was from Merck,
India, and antibiotic solution (containing penicillin and streptomy-
cin) was obtained from Hyclone, U.S.A. Purified bovine brain
tubulin (>99% pure) was procured from Cytoskeleton and NP-40
was purchased from Calbiochem. Combretastatin A-4 was synthe-
sized at the National Chemical Laboratory, Pune. Human cancer
cell lines HT29 (colon), DU145 (prostate), KB (oral), L132 (lung),
MiaPaCa-2 (pancreas), Hep2 (larynx), PA-1 (human ovary), and
293 (kidney) were procured from NCCS, Pune, India. The ECV304
(endothelial) cell line was generously gifted by Dr. Takahashi
(Tokyo University, Tokyo, Japan). Cell lines were grown in DMEM,
containing L-glutamine and 25 mM HEPES, and supplemented with
10% FBS, penicillin (100 units/mL), streptomycin (100 µg/mL),
and amphotericin B (0.25 mg/mL) at 37 °C, 5% CO2, and 100%
humidity.
Cytotoxicity Assay. Various concentrations of cyclopentenone
analogues were tested for in vitro cytotoxic activity on human
cancer cell lines representing colon (HT29), prostate (DU145), oral
(KB), lung (L132), pancreas (MiaPaCa-2), larynx (Hep2), ovary
(PA1), kidney (293), and normal endothelial cells (ECV304).
Cytotoxicity was measured by MTT assay, which is based on the
principle of uptake of MTT by the metabolically active cells, where
it is metabolized by active mitochondria into a blue-colored
formazan product that is read spectrophotometrically.26 Briefly,
tumor cells were seeded (5000-10 000 cells/well) in 96-well culture
plates and incubated at 37 °C in a CO2 incubator with various
concentrations of cyclopentenone analogues ranging from 1 to 100
µg/mL, with relevant controls in triplicate wells. After 72 h, the
assay was terminated by the addition of 25 µL of MTT solution (5
mg/mL) in each well. Percentage cytotoxicity was calculated as
given below. The IC50 values were determined by nonlinear
regression using Prism software v 4.01.
Both 11 and 42 led to cell cycle arrest in proliferating
endothelial cells. At the concentration of 1 µM, the analogues
displayed better activity than the parent compound 1′. The
analogues also induced apoptosis in ECV304 cells as shown
by DNA fragmentation and DAPI staining of micronuclei of
proliferating ECV304 cells, thereby indicating that the analogues
may have similar apoptotic activity as that of 1′.
percentage cytotoxicity ) 100 × [1 - (X/R1)]
where X ) absorbance of treated sample at 540 nm and R1 )
absorbance of control sample at 540 nm.
In Vitro Tubulin Polymerization Assay. The tubulin assembly
reaction was carried out spectrophotometrically at 37 °C in 1 mL
of buffer containing 80 mM PIPES (pH 6.9), 0.5 mM EGTA, 1.0
mM GTP, 2 mM MgCl2, and 20% glycerol by the procedure
modified from the method of Sampath et al.27 Highly purified
bovine brain tubulin was used at the concentration of 1 mg/mL in
the presence or absence of 1′ and the cyclopentenone analogues
11 and 42 were used at different concentrations. The compounds
were dissolved in 100% DMSO, and the final concentration of
DMSO was kept less than 0.4%. Tubulin polymerization was
followed by measuring the increase in absorbance of the solution
at 340 nm every 60 s. The amount of tubulin polymerized is directly
proportional to the AUC, which was calculated using the prism
software v 4.01. AUC for the control was set to 100% polymeri-
zation, and the concentration of the compound that inhibited tubulin
polymerization by 50% (IC50) was determined (Table 5).
Analysis of Cell Cycle by Flow Cytometry. Cell cycle of human
endothelial cancer cells (ECV304) treated with drugs was analyzed
by flow cytometry.28 Cells were plated at a density of 1.5 × 106
cells in 90 mm tissue culture dishes in DMEM medium supple-
mented with 10% FCS and incubated overnight. Subsequently, the
medium was replaced with fresh medium and cells were treated
with 1′ and cyclopentenone analogues 11 and 42 at the concentration
of 1 µM for 24 h. Cells were trypsinized, washed in phosphate
buffered saline (PBS), and fixed with ice cold 70% ethanol. Fixed
samples were extensively washed in wash buffer (3.5 mM trisodium
citrate, 0.1% NP-40, 0.5 mM tris chloride, and 1.5 mM spermine
tetrahydrochloride pH 7.6). The pellet was resuspended in 250 µL
of solution A (30 µg/mL trypsin prepared in wash buffer), mixed
When assessed for their ability to inhibit tubulin polymeri-
zation, there was a marked difference between 11 and 42 for
inhibition of tubulin polymerization. The compound 42 was
evidently a more potent antitubulin than 11, as evident from its
low IC50 value of 1.75 µM. Also the antitubulin activity of 42
was found to be comparable with 1′, which may indicate a
similarity in the mechanism of action with combretastatin A-4.
Hence, 42 was selected for further evaluation of solubility,
stability, pharmacokinetics, and in vivo efficacy studies.
Previous studies had indicated that 1′ and 1 do not cause
significant tumor regression of human colon tumor xenografts
in mice,20-22 while 42 displayed good cytotoxicity in colon
cancer cells. This prompted us to evaluate the anticancer
potential of selected analogue 42 in colon carcinoma. PTC of
human colon adenocarcinoma implanted tumor xenografts in
nude mice were employed. The compound 42 caused significant
(p < 0.05) tumor regression in colon tumors and delayed tumor
growth as compared to untreated controls. The antitumor activity
further confirmed the potency seen in the in vitro cytotoxicity
assay. It may be concluded that cis-restricted analogues of 1′,
particularly 42, possess promising anticancer activity combined
with good aqueous solubility, stability, and acceptable phar-
macokinetic profile. This compound is now undergoing further
lead optimization in our laboratories for development as an
anticancer agent.

1752 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gurjar et al.
by tapping, and incubated at RT for 10-15 min. After 15 min,
200 µL of solution B (500 µg/mL trypsin inhibitor and 100 µg/mL
RNase prepared in wash buffer) was added to the cell suspension
and further incubated for 15 min. Subsequently, 200 µL of ice-
cold solution C (420 µg/mL PI, 3.33 mM spermine tetrahydro-
chloride) was added to the suspension and incubated in the dark at
4-10 °C for 1 h. Stained cells were acquired on a FACS calibur
flow cytometer (Becton Dickinson). The low-level gate was set at
10% of the value of the G1 peak and the percentages of cells within
the G1 and G2/M phases of the cell cycle were determined by
analysis with Modfit software (Becton Dickinson; Figure 2).
Analysis of Apoptosis by DNA Fragmentation. DNA frag-
mentation was taken as an index of apoptosis.29 Human endothelial
cancer cells (ECV304) were plated at a density of 2 × 106 cells in
90 mm tissue culture dishes in DMEM medium supplemented with
10% FCS. The cells were incubated for 5-6 h to allow complete
reattachment to the dishes. Subsequently, the medium was changed
to fresh medium supplemented with 10% FCS and cells were treated
with analogues 11 and 42 at concentrations ranging from 10 nM
to 1 µM for 24 h. The compound 1′ (100 nM) was used as a positive
control for the experiment. After the treatment, both the strongly
adherent and the loosely adherent cells were harvested, pelleted at
2500 rpm, and lysed in buffer containing 1 mM Tris-Cl, 20 mM
EDTA (pH 8), and 1% NP-40. The fragmented DNA was separated
from intact chromatin using 5 M NaCl and isopropanol. The mixture
was centrifuged at 12 000 rpm for 20 min to obtain the DNA pellet.
The pellet was washed with 70% ethanol and analyzed by gel
electrophoresis using 1.75% agarose gel (Figure 3).
Visualization of Apoptotic Bodies by DAPI Staining. Apop-
totic bodies were visualized by staining the nuclei with DAPI.28
ECV 304 cells were plated at a density of 1 × 106 cells per well
in a six-well plate in DMEM medium and incubated in a CO2
incubator at 37 °C for 6 h. Cells were treated in the presence or
absence of 1′ and cyclopentenone analogues 11 and 42 at
concentrations ranging from 10 nM to 1 µM for 24 h. The
compound 1′ and analogues were dissolved in 100% DMSO, and
the final concentration of DMSO was kept less than 0.1%. The
loosely adherent cells were harvested, washed with PBS, and fixed
in 70% ice-cold ethanol for 2 h. DAPI stock of 5 mg/mL was
prepared in water and stored at -20 °C. The fixed cells were stained
with 500 µL of 10 µg/mL DAPI prepared in methanol and incubated
at 37 °C for 20 min. Subsequently, the cells were washed and
resuspended in 50 µL of PBS. A 10 µL amount of suspension was
taken on a slide and observed under the NIKON fluorescence
microscope (emission wavelength of 344 nm and excitation
wavelength of 450 nm) for visualization of multiple damaged,
fragmented, and unevenly displayed micronuclei (Figure 4).
Physical Properties. The physical properties were determined
by following standard methods and measured using a previously
standardized HPLC method (as described in Pharmacokinetics in
the Experimental Section). Briefly, solubility of the analogues was
determined using the DMSO precipitation method.30 Test com-
pounds were dissolved in DMSO and kept with shaking for 1.5 h
in phosphate buffer (pH 7.4). The soluble portion was filtered and
measured by HPLC. The metabolic stability of the analogues was
determined as described previously31 by calculating the percentage
of test substance remaining unmetabolized using HPLC analysis,
following incubation for 60 min in pooled human liver microsomes.
Animal Studies. All animal experiments were carried out as per
the guidelines of the Institutional Animal Ethics Committee (IAEC),
Dabur Research Foundation.
Treatment with compound 42 was initiated when the average tumor
volumes, as measured using a vernier caliper, were around 500
mm3. The test compound was formulated using a cosolvent
formulation, diluted in 5% dextrose, and administered intravenously
to the treatment group of tumor-bearing animals at a dose of 20
mg/kg once daily for a period of 14 days. Control animals received
an equivalent volume of vehicle alone for the same period. The
antitumor activity of the compound was monitored by measuring
tumor volumes every fourth day using the formula W × W × L ×
0.4 (W ) smaller diameter, L ) larger diameter). Tumor regression
was calculated using the formula (1 - tumor volume(treated)/tumor
volume(control)) × 100. Additionally, tumor volumes of different
groups were compared with the multiple comparison procedure
using LSMEANS, with ADJUST ) Dunnett in PROC GLM of
SAS 9.1.3. A value of p < 0.05 was considered significant.
Pharmacokinetics. Compounds were formulated using a cosol-
vent formulation, diluted in 5% dextrose, and administered intra-
venously in Swiss albino mice, age 6-8 weeks, weighing around
25 g, and maintained at the small animal facility, Dabur Research
Foundation, India. After the dose administration, blood samples
(200 µL) were collected by orbital bleeding under mild ether
anesthesia at time points of 3, 10, 30 min, 1, 2, 4, 6, 8, and 24 h
in EDTA-containing tubes. Not more than five bleeds were collected
from a single mouse. The plasma was separated, extracted using
organic solvents, and centrifuged, and the supernatant was evapo-
rated to dryness. It was reconstituted with 200 µL in diluent (45:
55 acetonitrile/water) and analyzed using HPLC (L2010, Shimadzu,
Japan) to determine plasma concentrations. The mobile phase
consisted of 0.005 M phosphate buffer (55%) and acetonitrile
(45%). The HPLC column used was C18, 250 × 4.6 mm, 5 µm,
YMC-Pack ODS-A. The flow rate was adjusted to 1 mL/min. The
retention time for 1′ and 42 was 15.4 and 7.5 min, respectively.
The concentration of the test compound was determined by
calculating the ratio of its peak to the internal standard peak and
then comparing the ratio to a simultaneously performed standard
curve. The limit of quantitation for both 1′ and 42 was 0.15 µg/
mL (75 µL injection). The pharmacokinetic parameters were
determined with WinNonlin v 5.0.1 software by performing
noncompartmental analysis.
Acknowledgment. We are thankful to the Department of
Science and Technology (DST), India, and Dabur Research
Foundation for the financial support for this research. Popat D.
Shinde and Vishal A. Mahajan are thankful to the Council of
Scientific and Industrial Research (CSIR) for the research
fellowship (SRF).
Supporting Information Available: Synthetic procedures,
characterization (mp, IR, and 1H and 13C NMR data) and elemental
analyses of all new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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nude mice, age 6-8 weeks, weighing around 20 g were obtained
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