Acetyl analogs of combretastatin A-4: Synthesis and biological studies

Article abstract of DOI:10.1016/j.bmc.2011.02.018

The combretastatins have received significant attention because of their simple chemical structures, excellent antitumor efficacy and novel antivascular mechanisms of action. Herein, we report the synthesis of 20 novel acetyl analogs of CA-4 (1), synthesi

Full text of DOI:10.1016/j.bmc.2011.02.018

Bioorganic & Medicinal Chemistry 19 (2011) 2359–2367
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Acetyl analogs of combretastatin A-4: Synthesis and biological studies
Balaji Babu a, Megan Lee a, Lauren Lee a, Raymond Strobel a, Olivia Brockway a, Alexis Nickols a,
Robert Sjoholm a, Samuel Tzou a, Sameer Chavda a, Dereje Desta b, Gregory Fraley b, Adam Siegfried c,
William Pennington c, Rachel M. Hartley d, Cara Westbrook d, Susan L. Mooberry d,e, Konstantinos Kiakos f,
John A. Hartley f, Moses Lee a,
⇑
a Department of Chemistry and the Division of Natural and Applied Sciences, Hope College 49423, USA
b Department of Biology, Hope College 49423, USA
c Department of Chemistry, Clemson University, SC 29634, USA
d Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
e Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
f UCL Cancer Research Institute, University College London, Paul O’ Gorman Building, 72 Huntley Street, London WC1F 6BT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The combretastatins have received significant attention because of their simple chemical structures,
excellent antitumor efficacy and novel antivascular mechanisms of action. Herein, we report the synthe-
sis of 20 novel acetyl analogs of CA-4 (1), synthesized from 3,4,5-trimethoxyphenylacetone that com-
prises the A ring of CA-4 with different aromatic aldehydes as the B ring. Molecular modeling studies
indicate that these new compounds possess a ‘twisted’ conformation similar to CA-4. The new analogs
effectively inhibit the growth of human and murine cancer cells. The most potent compounds 6k, 6s
Received 21 December 2010
Revised 9 February 2011
Accepted 11 February 2011
Available online 17 February 2011
Keywords:
and 6t, have IC50 values in the sub-
and has advantages over earlier analogs due to its enhanced water solubility (456
initiates microtubule depolymerization with an EC50 value of 1.8 M in A-10 cells. In a murine L1210 syn-
geneic tumor model 6t had antitumor activity and no apparent toxicity.
Ó 2011 Elsevier Ltd. All rights reserved.
l
M range. Analog 6t has an IC50 of 182 nM in MDA-MB-435 cells
Combretastatin A-4
Trimethoxyphenylacetone
Tubulin
Microtubule
Cytotoxicity
lM). This compound
l
Cancer
1. Introduction
relationship (SAR) studies of CA-4 analogs. Consequently, it was
found that a 3,4,5-trimethoxyphenyl as the A ring substituents
and a cis disposition of the double bond between two aryl groups
(A and B) is optimal for biological activities. Many analogs of 1 have
been developed over time that can retain the biological actions of
the parent molecule but that have improved water solubility and
The ongoing efforts of US National Cancer Institute to identify
anticancer agents of natural origin led to the identification of com-
bretastatins, which were initially isolated from the African willow
tree Combretum caffrum (Combretaceae). The most potent of these
initial combretastatins was combretastatin A-4 (CA-4 or 1, Fig. 1).1
CA-4 is an antimitotic agent that binds to the colchicine site on
tubulin causing inhibition of tubulin polymerization and in cells
microtubule depolymerization and mitotic accumulation.2 CA-4
(1) exhibits potent cytotoxicity against a broad spectrum of human
cancer lines, including those that are multidrug resistance (MDR)
positive.3 This compound also elicits irreversible vascular shut-
down in solid tumors, leaving normal vasculature intact, thereby
acting as a vascular disrupting agent.4 Major limitations of 1 are
its poor aqueous solubility and bioavailability, which significantly
impairs optimal in vivo antitumor activity.5 The relative simplicity
of the 1,2-diarylethene scaffold of the CA-4 series, along with their
biological properties, resulted in extensive structure–activity
better pharmacokinetic properties. These analogs include
a
water-soluble phosphate prodrug derivative of CA-4, CA-4P (2),
as well as AVE 8062 (3) and the amino derivative AC-7739 (4)
(Fig. 1). CA-4P (Zybrestat), CA-1 diphosphate (Oxi4503), AVE
8062 and NPI-2358 are currently being evaluated in clinical trials.
Another class of compound where the two aryl rings are bridged
through three atoms is represented by A-105972 (5a, Fig. 1), an
oxadiazoline analog, which exhibits potency towards multidrug
resistant cancer cells.6 Another cytotoxic oxadiazoline compound
5b (Fig. 1) was developed in the authors’ laboratory, and it was
found to cause microtubule depolymerization.7e
Our work has recently focused on the synthesis of novel CA-4
analogs that retain the cytotoxic and microtubule disrupting activ-
ities of the parent molecule (1a). However, we have not yet identi-
fied an analog that preserves the cytotoxic potency of CA-4.8 We
⇑
Corresponding author. Tel.: +1 616 395 7190.
E-mail address: lee@hope.edu (M. Lee).
recently reported the synthesis and cytotoxicity of a,b-unsaturated
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.018

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B. Babu et al. / Bioorg. Med. Chem. 19 (2011) 2359–2367
Ac
N
N
OCH3
NH2
H3CO
H3CO
R
H3CO
O
H3CO
OCH3
OCH3
5a, A-105972
H3CO
1, R = OH, combretastatin A4
2, R = phosphate
3, R = NH2, AC-7739
4, R = NHCOCHNH2CH2OH, AVE-8062
CH3
O
Ac
R1
N
N
H3CO
H3CO
R2
H3CO
R5
O
R3
H3CO
OCH3
R4
OCH3
5b
H3CO
6a-t
6a, R1=R2=R4=R5=H; R3=NO2
6b, R1=R3=R4=R5=H; R2=NO2
6c, R1=R2=R4=R5=H; R3=Cl
6d, R1=R3=R4=R5=H; R2=Br
6e, R1=R2=R4=R5=H; R3=Br
6f, R1=R2=R4=R5=H; R3=CN
6g, R1=R2=R4=R5=H; R3=F
6h, R1=R2=R4=R5=H; R3=OMe
6i, R1=OMe;R2=R3=R4=R5=H
6j, R1=R3=R4=R5=H; R2=OMe
6k, R1=R2=R4=R5=H, R3=Me
6l, R1=R2=R3=R4=R5=H
6m, R1=R4=R5=H; R2=R3=Cl
6n, R1=R4=R5=H; R2=NO2; R3=OMe
6o, R1=R4=OMe; R2=R3=R5=H
6p, R1=R4=R5=H; R2=R3=OMe
6q, R1=R3=R5=OMe; R2=R4=H
6r, ; R1=R5=H;R2=R3=R4=OMe
6s, R1=R4=R5=H; R2=OTBS; R3=OMe
6t, R1=R4=R5=H; R2=OH; R3=OMe
Figure 1. Structures of combretastatin A-4 (CA-4, 1), CA-4 phosphate derivative, 2, amino acid analog of CA-4, 3 (AVE-8062), amino analog of CA-4, 4 (AC-7739), oxadiazoline
analogs A-105972 (5a), 5b and novel acetyl combretastatin analogs reported herein 6a–6t.
ketone analogs of CA-4 from simple phenylacetone, which showed
modest activity against L1210 and B16 cancer cells.7d However,
those compounds did not contain the 3,4,5-trimethoxyphenyl A
ring normally found in combretastatin analogs. The goal of this
study was to design novel structures that contained a 3,4,5-trime-
thoxyphenyl group. Accordingly, 20 novel acetyl-CA-4 analogs
6a–t (Fig. 1) containing the 3,4,5-trimethoxyphenyl group A ring
and a variety of aromatic aldehydes as the B ring were synthesized.
Compounds 6a–t were tested for their cytotoxicity against L1210
and B16 cell lines and for effects on cellular microtubules. The
in vivo antitumor activities were evaluated for the most potent
compounds 6k and 6t.
correlates with literature precedence,7d thereby, providing support
for the cis geometry. The conformation of compounds 6a and 6t
were also examined by molecular modeling studies using the suite
of programs in MacSpartan, version ’04. Upon optimization of the
structure using molecular mechanics (MMFF) and molecular
dynamics (equilibrium conformer search option and molecular
mechanics), the structure was energy optimized using Hartree–
Fock (3-21G) calculations, followed by density function theory
(B3LYP and 6-31G⁄) calculations. The conformation of CA-4 (1)
has been previously reported using the same protocol,10 and was
used for comparison purposes in this study. The determined con-
formations of the compounds 6a, 6t and 1, depicted in Figure 3,
are remarkably similar to the X-ray structure of 6a. It is evident
from these results that due to the steric hindrance in the cis-stil-
bene framework the molecule adopts a slightly ‘twisted’ conforma-
tion. According to the X-ray crystal structure and the molecular
models of compound 6a and 6t, the 4-nitrophenyl-3-buten-2-one
sub-structure is held relatively planar (ꢀ7°) and the trimethoxy-
phenyl group rotates by ꢀ60° about the alkene to minimize steric
hindrance between the two aromatic units. The conformation is
similar to that observed in the X-ray structure11 and molecular
model of CA-4 suggesting these compounds are capable of exhibit-
ing a similar tubulin-targeting mechanism of action.
2. Results and discussion
2.1. Chemistry
Compounds 6a–t were prepared through the Claisen–Schmidt
condensation of 3,4,5-trimethoxyphenylacetone with the appro-
priate aldehydes using piperidine and benzoic acid as catalysts
(Scheme 1). The starting material 3,4,5-trimethoxyphenyl acetone
was prepared as described previously.9 In general, the phenolic
group was protected using TBDMSCl (tert-butyldimethylsilyl chlo-
ride) during the reaction and was regenerated by using 1 M TBAF
in THF after the reaction. The yields range from 15% to 96% after
purification by silica gel column chromatography.
2.3. Solubility assay
To test our assumption that adding an acetyl group increases
water solubility, a solubility assay was conducted. The aqueous
solubility of 6h, 6k and 6t were measured using a modified version
of the Multiscreen Solubility filter plate protocol developed by Mil-
2.2. Conformation
Confirmation of the cis configuration about the double bond be-
tween the A and B rings was achieved with a single X-ray crystal-
lographic analysis of the nitro compound 6a (Fig. 2). The chemical
shift of the b-vinylic proton in the 1H NMR spectra at 7.58 ppm
lipore.7e The maximum aqueous solubility for this assay is 500
l
M.
Two independent experiments were conducted and the data indi-
cate that the aqueous solubility of 6h is 319 M and 6k is
l

B. Babu et al. / Bioorg. Med. Chem. 19 (2011) 2359–2367
2361
R1
R2
O
H
O
O
H3C
H3C
R3
MeO
MeO
R1
R3
R5
R4
MeO
MeO
piperidine, benzoic acid
toluene, reflux, 4h
R5
R2
OMe
OMe
R4
6
6a, R1=R2=R4=R5=H; R3=NO2
6b, R1=R3=R4=R5=H; R2=NO2
6c, R1=R2=R4=R5=H; R3=Cl
6d, R1=R3=R4=R5=H; R2=Br
6e, R1=R2=R4=R5=H; R3=Br
6f, R1=R2=R4=R5=H; R3=CN
6g, R1=R2=R4=R5=H; R3=F
6h, R1=R2=R4=R5=H; R3=OMe
6i, R1=OMe;R2=R3=R4=R5=H
6j, R1=R3=R4=R5=H; R2=OMe
6k, R1=R2=R4=R5=H, R3=Me
6l, R1=R2=R3=R4=R5=H
6m, R1=R4=R5=H; R2=R3=Cl
6n, R1=R4=R5=H; R2=NO2; R3=OMe
6o, R1=R4=OMe; R2=R3=R5=H
6p, R1=R4=R5=H; R2=R3=OMe
6q, R1=R3=R5=OMe; R2=R4=H
6r, ; R1=R5=H;R2=R3=R4=OMe
6s, R1=R4=R5=H; R2=OTBS; R3=OMe
6t, R1=R4=R5=H; R2=OH; R3=OMe
Scheme 1. Synthesis of acetyl-combretastatin analogs 6a–t.
Table 1, show several interesting trends based upon the substitu-
tion pattern of the aromatic ring B. The compound 6l (derived from
benzaldehyde), yielded IC50 values of 25 and 5.4 lM against B16
and L1210 cells, respectively. For the B ring monosubstituted com-
pounds (Table 1), a nitro group in the meta-position (6b) yielded
good potency against L1210 and B16 cells (IC50 3.8 and 3.3 lM,
respectively). Moving this nitro group to the para-position (6a)
lowered the potency 4 to 5-fold against both cell lines in compar-
ison to 6b, suggesting that the electronic influence (due to induc-
tive effects) of the meta-nitro group enhances the interaction of
6b with it is cellular targets. In contrast, the compounds with hal-
ogens in the meta- and para-position (6c–6e and 6g) showed mod-
est to poor activity (IC50 values in the range of 7.4–65 lM), with 6c
and 6e (containing para-chloro and para-bromo substituents,
respectively) exhibiting the highest potency against one or both
cell lines. Interestingly, incorporation of the slightly less polar
and larger bromo substituent at the meta-position in 6d (in com-
parison to 6c) resulted in additional loss in cytotoxic potency in
both cell lines (IC50 values of 39 and 35 lM in B16 and L1210 cells,
respectively). Similar trends (based upon substitution pattern)
seem apparent for compounds 6h–6k (Table 1). Electron-donating
methoxy and methyl substituents in the para-position (6h and 6k)
increased potency against cell lines and gave IC50 values for L1210
Figure 2. X-ray structure of compound 6a. Complete X-ray structural data for 6a
were deposited with the Cambridge Crystallographic Data Centre (CCDC deposition
number: 811797).
cells in the sub-lM range; 0.38 and 0.36 lM, respectively. Only
443 lM. The analog 6t has an aqueous solubility of 456 lM, indi-
modest levels of cytotoxicity were observed for 6i and 6j. This sug-
gests that substituent size is a factor contributing to the potency of
these compounds, as could substituent polarizability in the para-
position. Based upon the information obtained from the study of
the monosubstituted compounds, we were curious to ascertain
whether some of the trends observed for monosubstituted com-
pounds could be enhanced via incorporation of two substituents
into the aromatic ring. In comparison to 6b, 6c and 6h, similar
cating that the addition an acetyl group on the ethenyl moiety in-
creases the aqueous solubility of the compound when compared to
CA-4 itself and acetyl oxadiazoline 5b.7e
2.4. Cytotoxicity
The compounds were evaluated for cytotoxic activity against
murine B16 melanoma and L1210 leukemia cells. The results,
Figure 3. Molecular models of compounds 6a, 6t, and CA-4, 1.

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B. Babu et al. / Bioorg. Med. Chem. 19 (2011) 2359–2367
Table 1
Cytotoxicity of the compounds 6a–6t on L1210 and B16 cancer cell lines
Compound
IC50 (lM)
O
H3C
MeO
MeO
B16
L1210
B16
L1210
0.36
Ar
OMe
Ar =
CH3
18 7.8
18 9.2
2.9
6k
6l
NO2
6a
NO2
3.8 0.3
7.4 2.5
39 1.4
3.3 0.2
5.0 0.1
35 6.4
25 4.2
6.4 1.2
3.8 0.2
5.4 0.2
6b
6c
Cl
4.0
Cl
Br
Cl
6m
6n
NO2
OMe
3.1
6d
6e
OMe
Br
19 9.2
4.0 1.5
4.0 0.1
3.7 0.01
OMe
6o
OMe
OMe
29 12
8.0
4
42 4.9
37 11.3
CN
F
6f
6p
OMe
65 7.1
43 4.2
>100
3.7 0.9
6g
MeO
OMe
6q
OMe
OMe
OMe
7.3 1.7
0.38
23 2.8
7.2
2
6h
OMe
6r
OMe
OTBS
OMe
41 13.4
31 0.7
40
5.1 2.9
3.5 1.4
0.18
6s
6t
6i
6j
OMe
OH
39 0.7
0.45 0.1
OMe
IC50 values are expressed in lM.
cytotoxic potency for the disubstituted counterparts 6m (contain-
ing chlorine atoms in both the meta- and para-positions) and 6n
(meta-nitro and para-methoxy) were observed, confirming the
trends seen with the mono substituted compounds. The molecules
6q and 6r which contain trisubstituted methoxy groups in the 2,4,6
and 3,4,5 positions (Table 1) showed no significant activity against
either cell line, suggesting that the steric congestion resulting from
multiple methoxy groups prevents optimal orientation within the
binding site as compared to the analogs 6h and 6k. The synthe-
sized exact acetyl analogs of CA-4 1 (6s and 6t) showed significant
activity (IC50 in 0.18–3.5 lM range, Table 1). Finally, the enhanced
potency of some of these molecules towards leukemia cells
(L1210) over solid tumor cells (B16) is, in general, consistent with
studies on other combretastatin analogs from our laboratory.7,12
2.5. NCI data
Compound 6t was evaluated at the National Cancer Institute
against a panel of 60 human cancer cell lines.13 Inhibition of cell
proliferation after 48 h continuous exposure was determined using

B. Babu et al. / Bioorg. Med. Chem. 19 (2011) 2359–2367
2363
the sulforhodamine B assay. The GI50 values (the concentration for
cell growth inhibition by 50%) for compound 6t against the panel
are shown in Figure 4. Bars extending to the right indicate cell lines
that are more sensitive than average to 6t, whereas bars extending
to the left indicate less sensitive cell lines. Interesting observations
were made on these results. First, 6t exhibited selectivity against
certain leukemia, ovarian, colon, melanoma and renal cancer cell
lines. It showed extremely high activity against the MDA-MB 435
melanoma cell line. Second, the TGI (the concentration for total cell
growth inhibition) indicates that it is highly active against some
non-small lung cancer, colon cancer, CNS cancer, ovarian cancer,
renal cancer, prostate cancer, breast cancer and the most potent
against MDA-MB-435. In a separate study using a previously pub-
lished procedure,7e the IC50 values for compounds 6h, 6k, and 6t
against MDA-MB-435 were found to be 0.35, 0.095, and
evaluated in A-10 cells, a non-transformed cell line that arrests
in interphase in response to microtubule targeting agents.7e,14 Only
6h, 6k and 6t showed significant microtubule depolymerization
activity at concentrations below 30 lM (see Fig. 5). A range of con-
centrations was evaluated for compounds 6h, 6k, 6t and the EC50
values, the concentration required to cause 50% loss of cellular
microtubules, as visualized by microscopy was calculated and the
values were found to be 18.6, 5.6 and 1.8 lM, respectively. The
data were further analyzed by calculating ratios of these EC50 val-
ues and IC50 against MDA-MB-435 cells. In this analysis, the link-
age of microtubule depolymerizing and cytotoxic effects can be
compared among large groups of compounds. Tight linkage is seen
with CA-4, which gives a ratio of 2–3 in these assays.7e The ratios
were found to be 53, 59, and 10 for 6h, 6k and 6t, respectively. This
ratio suggests that the acetyl-analog 6t of this series has a mecha-
nism of action closest to the CA-4. In contrast, the significantly
higher ratios or compounds 6h and 6k suggest microtubule disrup-
tion is not the only mechanism of cytotoxicity. This is not surpris-
ing since analogs of combretastatins have been found to bind to
other biological targets, such as the kinesin spindle protein
(KSP)15 and DNA.16
0.182 lM, respectively, indicating that acetyl-CA4 analogs are via-
ble cytotoxic agents worthy of further studies.
2.6. Microtubule depolymerization activity
To evaluate the cellular mechanism of action, the effects of 6b,
6c, 6h, 6k, 6n, and 6t on interphase cellular microtubules were
Figure 4. NCI 60-cell line screen data (GI50 and TGI values) for 6t against a panel of human cancer cell lines. GI50 is the concentration needed to inhibit the growth of cancer
cells by 50%. TGI is total growth inhibition.

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B. Babu et al. / Bioorg. Med. Chem. 19 (2011) 2359–2367
Figure 5. Effects of 6t on cellular microtubules. A-10 cells were treated for 18 h with vehicle (A) or 4.5
lM 6t (B) and cellular microtubule structures visualized with indirect
immunofluorescence techniques. Panel A shows the normal interphase microtubules are visible. Panel B shows the 6t-induced loss of interphase microtubules.
2.7. In vivo studies of 6k and 6t against DBA2 mice
vehicle used to formulate the samples was identical to that used in
the toxicity experiment, as was the ip route and volume of injec-
tion. The animals were treated at a dose of 50 mg/kg of compound
6t once a day, starting on day 1 and continued on days 3, 5, 7, 9, 11,
13, 15, and 17. This dose was chosen with the purpose of keeping
the total mass of the compound close to that used in the toxicity
study. Another group of four mice were administered with only
the vehicle and that provided a negative control group for the
study. On day 23, the flank tumors on animals treated with 6t were
on average reduced by 35% compared than those on the control
animals, indicating that compound 6t has some in vivo antitumor
activity.
Compounds 6k and 6t were evaluated for toxicity against fe-
male DBA2 mice using a dose of 75 mg/kg. Five mice were used
on each test group. The compounds were administered via intra-
peritonial (ip) injections on days 1, 5, 9, 13, and 17. The body
weights of the mice over the course of the experiment are shown
in Figure 6 and indicate that similar to the control vehicle-treated
mice neither compound caused significant body weight loss, which
is indicative of negligible systemic toxicity over 19 days. At the end
of the experiment (day 23), all the animals were euthanized and
the blood and vital organs (kidney, liver, and spleen) were exam-
ined. Compared to the vehicle-treated controls, the vital organs ap-
pear the same in terms of color, weight, and size, indicating
negligible toxicity to the animals. Blood chemistry including
hematocrit, white blood count, granulocyte count and platelet
count was evaluated. Again, comparing to the vehicle control, no
significant difference was observed for the drug-treated animals.
Collectively, these results provided preliminary evidence that com-
pounds 6k and 6t, formulated and dosed as described above do not
cause systemic toxicity of a 23-day period.
3. Conclusion
Twenty novel acetyl-combretastatin analogs were designed and
synthesized with good yields. Incorporation of an acetyl function-
ality enhanced water solubility as compared to CA-4 and ease of
synthesis. Acetyl-containing compounds possess good cytotoxic
potency and 6t appears to retain the microtubule disrupting mech-
anism of action of CA-4. Compound 6t at a dose of 75 mg/kg given
five times, over 19 days was found to be non-toxic in mice, and
according to a preliminary study, it produced some antitumor ef-
fect on the growth of L1210 grown on the right flank in mice. Fur-
A preliminary in vivo antitumor study on compound 6t was ini-
tiated using four female DBA2 mice per test group. The mice were
inoculated on day 0 with 2 Â 105 L1210 cells on the right flank. The
Figure 6. Toxicity studies. Change in body weight of mice treated with vehicle control (solid square) and 75 mg/kg dose of 6k (open circle) or 6t (solid triangle) given five
times over 19 days as indicated by the arrows.

B. Babu et al. / Bioorg. Med. Chem. 19 (2011) 2359–2367
2365
ther investigations are underway to continue to improve 6t in rela-
tion to increasing potency and aqueous solubility, and maintaining
the anti-tubulin mechanism of action.
1529, 1462, 1455, 1412, 1350, 1307, 1287, 1237, 1198, 1182,
1125, 1006, 920, 876, 809, 774, 735, 675; 1H NMR (400 MHz,
CDCl3): d 8.05–8.09 (m, 1H), 7.97 (s, 1H), 7.61 (s, 1H), 7.39
(m, 2H), 6.38 (s, 2H), 3.86 (s, 3H), 3.79 (s, 6H), 2.39 (s, 3H); LRMS:
EI+ 357 (100% M+); HRMS: obs = 357.1211; calcd = 357.1212.
4. Experimental
4.1. Synthesis
4.1.5. Compound 6c
Color: off white solid (43%); Rf = 0.62 (50% ethyl acetate/hex-
ane); mp 99–101 °C; IR (KBr disc)/cmÀ1 2996, 2959, 2936, 2829,
1721, 1667, 1580, 1558, 1538, 1503, 1490, 1462, 1452, 1410,
1353, 1316, 1300, 1275, 1235, 1195, 1181, 1126, 1090, 1011,
974, 876, 818, 772, 745, 679; 1H NMR (400 MHz, CDCl3): d 7.54
(s, 1H), 7.16 (d, J = 8.0 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 6.35
(s, 2H), 3.90 (s, 3H), 3.77 (s, 6H), 2.34 (s, 3H); LRMS: EI+ 347
(100% M+).
4.1.1. General
Solvents and organic reagents were purchased from Aldrich or
Fischer, and were used without further purification. Melting points
(mp) were performed using a Mel-temp instrument and the results
were not corrected. Infrared (IR) spectra were recorded using a
Midac M1700 FT-IR instrument as films on KBr discs unless stated
otherwise. Proton NMR spectra were recorded on a Varian INOVA
400 MHz Fourier transform spectrometer using an internal deute-
rium lock. Chemical shifts are quoted in parts per million down-
field from tetramethylsilane. High-resolution mass spectra
(HRMS) and low-resolution mass spectra (LRMS) were provided
by the Mass Spectrometry Laboratory, University of South Carolina,
Columbia, SC. The single crystal X-ray diffraction studies on com-
pound 6a were performed at Clemson University, Clemson, SC, in
the laboratory of Professor William Pennington. Reactions were
monitored using thin-layer chromatography (TLC) using commer-
cially available pre-coated plates (Merck Kieselgel 60 F254 silica).
Visualization was achieved with UV light at 254 nm, I2 vapor stain-
ing and ninhydrin spray.
4.1.6. Compound 6d
Color: orange oil (98%); Rf = 0.32 (50% ethyl acetate/hexane); IR
(KBr disc)/cmÀ1 3063, 2997, 2939, 2837, 1960, 1710, 1669, 1590,
1505, 1462, 1432, 1354, 1341, 1312, 1273, 1183, 1154, 1127,
1075, 1040, 1007, 923, 881, 834, 781, 752, 688, 669; 1H NMR
(400 MHz, CDCl3): d 7.50 (s, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.24
(s, 1H), 7.05 (t, J = 8.0 Hz, 1H); 6.92 (d, J = 8.0 Hz, 1H), 6.35 (s,
2H), 3.90 (s, 3H), 3.80 (s, 6H), 2.35 (s, 3H); LRMS: EI+ 392 (M+1,
30%).
4.1.7. Compound 6e
Color: yellow solid (61%); Rf = 0.64 (50% ethyl acetate/hexane);
mp 107–109 °C; IR (KBr disc)/cmÀ1 2936, 2827, 1866, 1841, 1789,
1737, 1721, 1710, 1680, 1666, 1660, 1651, 1644, 1632, 1613, 1581,
1557, 1537, 1531, 1503, 1486, 1462, 1416, 1351, 1272, 1238, 1125,
980; 1H NMR (400 MHz, CDCl3): d 7.52 (s, 1H), 7.32 (d, J = 8.0 Hz,
2H), 6.95 (d, J = 8.0 Hz, 2H), 6.35 (s, 2H), 3.84 (s, 3H), 3.78 (s, 6H),
2.35 (s, 3H); LRMS: EI+ 390 (100%, M+).
4.1.2. General procedure for the synthesis of 6a–t
To a solution of 3,4,5-trimethoxyphenyl acetone (1 equiv) and
aromatic aldehyde (1 equiv) in toluene was added benzoic acid
(1 equiv) followed by piperidine (0.02 equiv). The solution was re-
fluxed for 4 h. TLC showed disappearance of starting materials. The
solvent was removed under vacuum using rotary evaporator. The
residue was purified by column chromatography using hexane
(100:0) and ethyl acetate (0:100).
4.1.8. Compound 6f
Color: colorless oil (91%); Rf = 0.22 (50% ethyl acetate/hexane);
IR (KBr disc)/cmÀ1 3404, 3087, 3062, 3000, 2939, 2837, 1959,
1939, 1710, 1672, 1588, 1537, 1505, 1462, 1455, 1414, 1355,
1325, 1276, 1237, 1198, 1183, 1126, 1041, 1005, 976, 956, 913,
881, 830, 754, 680, 666; 1H NMR (400 MHz, CDCl3): d 7.54
(s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.34
(s, 2H), 3.92 (s, 3H), 3.78 (s, 6H), 2.36 (s, 3H); LRMS: EI+ 337
(100% M+).
4.1.3. Compound 6a
Color: orange yellow solid (62%); Rf = 0.2 (50% ethyl acetate/
hexane); mp 145–147 °C; IR (KBr disc)/cmÀ1 2940, 2838, 1709,
1592, 1552, 1518, 1508.56, 1456, 1415, 1378, 1344, 1238, 1126,
1068, 1005, 861, 834, 722, 669; 1H NMR (400 MHz, CDCl3): d 8.07
(d, J = 8.0 Hz, 2H), 7.58 (s, 1H), 7.24 (d, 2H), 6.35 (s, 2H), 3.92 (s,
3H), 3.77 (s, 6H), 2.37 (s, 3H); LRMS: EI+ 357 (100% M+).
Crystallographic data for 6a: C19H19NO6, M = 357.35, mono-
clinic, C2/c (#15), a = 30.6394(12), b = 7.2842(4), c = 18.4845(7) Å,
4.1.9. Compound 6g
Color: light yellow solid (31%); Rf = 0.5 (50% ethyl acetate/hex-
ane); mp 88–90 °C; IR (KBr disc)/cmÀ1 2997, 2940, 2836, 1709,
1682, 1504, 1462, 1422, 1354, 1334, 1312, 1281, 1238.82, 1181,
b = 124.045(9)°, V = 3418.3(3) Å3, Z = 8, Dc = 1.39 g cmÀ3
,
l
(Mo
Ka
) = 0.104 mmÀ1
,
light yellow parallelepiped
crystal,
1156, 1126, 1042, 1007; 1H-NMR (400 MHz, CDCl3):
d 7.57
0.22 Â 0.27 Â 0.31 mm, F0 0 0 = 1504. Data collection at 163(2) K
on a Rigaku Mercury/AFC8 diffractometer yielded 15555 reflec-
tions (2.21 < h < 26.35°) of which 3480 were unique (Rint = 0.025)
(s, 1H), 7.08 (dd, J = 4.0 Hz, 8.0 Hz, 2H), 6.90 (t, J = 8.0 Hz, 2H),
6.37 (s, 2H), 3.92 (s, 3H), 3.79 (s, 6H), 2.36 (s, 3H); LRMS: EI+ 330
(100%, M+).
and 3021 were observed (I > 2r(I). Structure solution and refine-
ment were performed with SHELXTL (version 6.10). Final residuals:
R1 = 0.043, wR2 = 0.107, R1(all data) = 0.050, wR2(all data) = 0.114,
S = 1.06. The crystals were grown by slow evaporation of an ethyl
acetate/hexane solution (50:50). The molecules are linked by sev-
eral weak C–HÁ Á ÁO interactions with a CÁ Á ÁO distance range of
3.179(2)–3.6393(18) Å (average of 3.42(14) Å). CCDC deposition
number: 811797. This data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.a-
c.uk/data_request/cif.
4.1.10. Compound 6h
Color: yellow oil (55%); Rf = 0.6 (50% ethyl acetate/hexane); IR
(KBr disc)/cmÀ1 3070, 3001, 2936, 2836, 1681, 1661, 1598, 1580,
1509, 1461, 1410, 1353, 1298, 1237, 1174, 1125, 1026, 975, 952,
921, 880, 830, 758, 698, 681, 666; 1H NMR (400 MHz, CDCl3): d
7.59 (s, 1H), 7.04 (d, J = 8.0 Hz, 2H), 6.73 (d, J = 8.0 Hz, 2H), 6.39
(s, 2H), 3.92 (s, 3H), 3.79 (s, 6H), 3.78 (s, 3H), 2.35 (s, 3H); LRMS:
EI+ 342 (100% M+); HRMS: obs = 342.1471; calcd = 342.1467.
4.1.4. Compound 6b
4.1.11. Compound 6i
Color: yellow oil (15%); Rf = 0.32 (50% ethyl acetate/hexane); IR
Color: yellow oil (27%); Rf = 0.26 (50% ethyl acetate/hexane); IR
(KBr disc)/cmÀ1 2996, 2961, 2938, 2832, 1708, 1687, 1671, 1581,
(KBr disc)/cmÀ1 2999, 2938, 2837, 1681, 1667, 1595, 1581, 1538,

2366
B. Babu et al. / Bioorg. Med. Chem. 19 (2011) 2359–2367
1504, 1485, 1463, 1438, 1411, 1353, 1315, 1297, 1238, 1196, 1179,
1163, 1125, 1048, 1024, 1007, 880, 824, 776, 755, 696; 1H NMR
(400 MHz, CDCl3): d 7.98 (s, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.86 (d,
J = 8.0 Hz, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.62 (t, J = 8.0 Hz, 1H), 3.88
(s, 3H), 3.87 (s, 3H), 3.72 (s, 6H), 2.43 (s, 3H); LRMS: EI+ 342
(100% M+).
809, 752, 667, 680; 1H NMR (400 MHz, CDCl3): d 7.59 (s, 1H),
6.91–6.88 (dd, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.52 (s, 1H), 6.45
(s, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.81 (s, 6H), 3.49 (s, 3H), 2.37
(s, 3H); LRMS: EI+ 372 (100% M+).
4.1.19. Compound 6q
Color: yellow oil (72%); Rf = 0.61 (50% ethyl acetate/hexane); IR
(KBr disc)/cmÀ1 2998, 2938, 2838, 1955, 1709, 1667, 1601, 1504,
1461, 1414, 1351, 1335, 1298, 1234, 1207, 1183, 1156, 1127,
1058, 1033, 1006, 951, 815, 751, 733, 685; 1H NMR (400 MHz,
CDCl3): d 7.59 (s, 1H), 6.28 (s, 2H), 5.98 (s, 2H), 3.80 (s, 3 H), 3.77
(s, 3H), 3.65 (s, 6H), 3.51 (s, 6H), 2.50 (s, 3H); LRMS: EI+ 402
(100% M+).
4.1.12. Compound 6j
Color: yellow oil (41%); Rf = 0.28 (50% ethyl acetate/hexane); IR
(KBr disc)/cmÀ1 3051, 2998, 2936, 2833, 1666, 1596, 1581, 1558,
1537, 1531, 1503, 1488, 1462, 1432, 1410, 1352, 1313, 1260,
1237, 1158, 1125, 1042, 1006, 911, 876, 783, 751, 694, 666; 1H
NMR (400 MHz, CDCl3): d 7.59 (s, 1H), 7.14 (t, J = 8.0 Hz, 1H),
6.60–6.80 (m, 1H), 6.59 (s, 1H), 6.40 (s, 2H), 3.90 (s, 3H), 3.79
(s, 6H), 3.54 (s, 3H), 2.38 (s, 3H); LRMS: EI+ 342 (100% M+).
4.1.20. Compound 6r
Color: yellow oil (96%); Rf = 0.4 (50% ethyl acetate/hexane); IR
(KBr disc)/cmÀ1 2997, 2937, 2835, 1707, 1681, 1663, 1578, 1502,
1459, 1419, 1330, 1237, 1184, 1157, 1125, 1039, 1003, 953, 924,
880, 833, 753, 695, 670; 1H NMR (400 MHz, CDCl3): d 7.54 (s,
1H), 6.45 (s, 2H), 6.40 (s, 2H), 3.87 (s, 3H), 3.83 (s, 3H), 3.82 (s,
6H), 3.61 (s, 6H), 2.37 (s, 3H); LRMS: EI+ 402 (M+, 100%).
4.1.13. Compound 6k
Color: yellow solid (48%); Rf = 0.6 (50% ethyl acetate/hexane);
mp 102–104 °C; IR (KBr disc)/cmÀ1 2997, 2928, 2835, 1667,
1581, 1505, 1456, 1411, 1348, 1317, 1236, 1183, 1123, 1008,
813; 1H NMR (400 MHz, CDCl3): d 7.60 (s, 1H), 7.02 (d, J = 8.0 Hz,
2H), 6.99 (d, J = 8.0 Hz, 2H), 6.38 (s, 3H), 3.92 (s, 3H); 3.78
(s, 6H); 2.37 (s, 3H); 2.30 (s, 3H); LRMS: EI+ 326 (100% M+); HRMS:
obs = 326.1515; calcd = 326.1518.
4.1.21. Compound 6s
Color: brown oil (42%); Rf = 0.5 (50% ethyl acetate/hexane); IR
(KBr disc)/cmÀ1 2996, 2938, 2848, 1710, 1662, 1584, 1507, 1456,
1418, 1238, 1127, 1005, 844, 782, 762, 717; 1H NMR (400 MHz,
CDCl3): d 7.53 (s, 1H), 6.83 (d, J = 8 Hz, 1H), 6.72 (d, J = 8 Hz, 1H),
6.56 (s, 1H), 3.90 (s, 3H), 3.78 (s, 6H), 3.77 (s, 3H) 2.30 (s, 3H),
0.89 (s, 9H), 0.07 (s, 6H); LRMS: EI+ 472 (M+, 30%).
4.1.14. Compound 6l
Color: light yellow oil (29%); Rf = 0.3 (50% ethyl acetate/hex-
ane); IR (KBr disc)/cmÀ1 3059, 3022, 2998, 2937, 2831, 2592,
2469, 2426, 2360, 2247, 2147, 1964, 1841, 1789, 1737, 1681,
1666, 1580, 1537, 1503, 1462, 1448, 1410, 1386, 1354, 1327,
1303, 1286, 1235, 1197, 1182, 1125, 1077, 1020, 1005, 953, 773,
757, 679; 1H NMR (400 MHz, CDCl3): d 7.60 (s, 1H), 7.25–7.16
(m, 3H), 7.09 (d, J = 4.0 Hz, 2H), 6.38 (s, 2H), 3.90 (s, 3H), 3.75
(s, 6H), 2.37 (s, 3H); LRMS: EI+ 312 (100% M+).
4.1.22. Compound 6t
Color: light yellow solid (48%); Rf = 0.25; (50% ethyl acetate/
hexane); mp 113–115 °C; IR (KBr disc)/cmÀ1 3014, 2937, 2843,
1661, 1583, 1508, 1457, 1411, 1350, 1277, 1237, 1126, 1018,
758, 668; 1H NMR (400 MHz, CDCl3): d 7.51 (s, 1H), 6.62–6.67
(m, 3H), 6.37 (s, 2H), 5.46 (s, 1H), 3.91 (s, 3H), 3.86 (s, 3H), 3.79
(s, 6H), 2.32 (s, 3H); LRMS: EI+ 358 (M+, 100%); HRMS:
obs = 358.1423; calcd = 358.1416.
4.1.15. Compound 6m
Color: yellow oil (90%); Rf = 0.44 (50% ethyl acetate/hexane); IR
(KBr disc)/cmÀ1 2359, 1658, 1652, 1581, 1557, 1538, 1504, 1462,
1455, 1410, 1378, 1353, 1316, 1277, 1236, 1126, 1026, 1003,
916, 883, 819, 698, 695; 1H NMR (400 MHz, CDCl3): d 7.47
(s, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 4 Hz 1H), 6.87–90 (dd,
1H), 6.38 (s, 2H), 3.91 (s, 3 H), 3.79 (s, 6H), 2.35 (s, 3H); LRMS:
EI+ 380 (100% M+).
4.2. Biological studies
4.2.1. Cytotoxicity studies on L1210 murine lymphoma and
B16-F0 melanoma cell lines
The L1210 and B16-F0 cell lines were obtained from American
Type Tissue Culture Collection (ATCC) and the MMT based cytotox-
icity studies were conducted exactly according to a recently pub-
lished procedure.7,17
4.1.16. Compound 6n
Color: orange solid (85%); Rf = 0.25 (50% ethyl acetate/hexane);
mp 88–90 °C; IR (KBr disc)/cmÀ1 2998, 2940, 2840, 1709, 1666,
1613, 1581, 1530, 1504.0, 1461, 1412, 1353, 1281.49, 1236,
1198, 1184, 1159, 1123, 1088, 1010, 820, 755, 733, 619; 1H NMR
(400 MHz, CDCl3): d 7.62 (s, 1H), 7.51 (s, 1H), 7.22–7.20 (dd, 1H),
6.90 (d, J = 8.0 Hz, 1H), 6.38 (s, 2H), 3.94 (s, 3H), 3.93 (s, 3H), 3.81
(s, 3H), 2.35 (s, 3H); LRMS: EI+ 387 (100% M+); HRMS:
obs = 387.1311, calcd = 387.1318.
4.2.2. NCI in vitro cytotoxicity screen
Compound 6t was also tested against a panel of 60 human can-
cer cell lines at the National Cancer Institute, Bethesda, MD.13a The
cytotoxicity studies were conducted using a 48 h exposure proto-
col using the sulforhodamine B assay.13b Dose–response curves
were used to generate the GI50 (concentration of drug needed to in-
hibit the growth by 50%), TGI (total growth inhibition), and LC50
values (concentration needed to inhibit the growth of cells by 50%).
4.1.17. Compound 6o
Color: yellow oil (81%); Rf = 0.33 (50% ethyl acetate/hexane); IR
(KBr disc)/cmÀ1 3000, 2938, 2831, 1684, 1666, 1598, 1582, 1506,
1462, 1454, 1413, 1385, 1354, 1318, 1302, 1287, 1235, 1195; 1H
NMR (400 MHz, CDCl3): d 8.01 (s, 1H), 6.80 (d, J = 4.0 Hz, 2H),
6.40 (s, 2H), 6.36 (d, J = 4.0 Hz, 1H), 3.86 (s, 6H), 3.77 (s, 6H), 3.34
(s, 3H), 2.45 (s, 3H); LRMS: EI+ 372 (75%, M+).
4.2.3. Toxicity studies of compounds 6k and 6t on healthy
female DBA2 mice
Compound 6k and 6t was formulated by mixing the appropriate
amount of the compound with 1.5 mL of PET (polyethylene glycol
400, absolute ethanol, and Tween 80 in 6:3:1 portions), 1.2 mL of
5% glucose in water, and 0.3 mL of DMSO for 6t and 0.6 mL of
DMSO for 6k. The final concentration of ethanol was 15% and
DMSO 10%. In each case clear, colorless solutions were obtained.
4.1.18. Compound 6p
Color: yellow oil (91%); Rf = 0.66 (50% ethyl acetate/hexane); IR
(KBr disc)/cmÀ1 3001, 2935, 2835, 1712, 1659, 1581, 1512, 1462,
1415, 1353, 1334, 1306, 1268, 1239, 1162, 1125, 1023, 879, 858,
Using an average weight of a mouse of 20 g, a 100 lL (for 6t) and

B. Babu et al. / Bioorg. Med. Chem. 19 (2011) 2359–2367
2367
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C. C. Bioorg. Med. Chem. Lett. 2003, 13, 101; (b) Pettit, G. R.; Singh, S. B.; Boyd, M.
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110 lL (for 6k) injection of this solution provided a dose of 75 mg/
kg. The solution was stored at 20 °C and the compound’s stability
was monitored by TLC analysis.
Week (5–7) old female DBA2 mice obtained from the Jackson
Laboratory were treated via an intraperitoneal (IP) route using
the abovementioned formulation. Five mice were included in each
group. Injections were made on days 1, 5, 9, 13 and 17. The control
animals received only the vehicle (100 lL). Body weights were
5. Galbraith, S. M.; Maxwel, R. J.; Lodge, M. A.; Tozer, G. M.; Wilson, J.; Taylor, N. J.;
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measured every other day and at the end of the experiment (day
23) the animals were sacrificed. The blood was analyzed by Kent-
wood Veterinary Hospital, Grand Rapids, MI and the appearance
and weights of the internal organs (liver, kidney, spleen) were also
examined.18
4.2.4. In vivo antitumor activity of 6k and 6t against L1210
murine lymphocytic leukemia18
Week (5–7) old female DBA2 mice were used in this study.
Groups of four mice were inoculated with L1210 cells that have
been passaged through a mouse. The cells were washed three
times with PBS and resuspended in PBS at a concentration of
4.0 Â 106 cells/mL. The cell suspensions (100
lL) were delivered
subcutaneously (sc) to the left flank at day 0. At this concentration,
each mouse received 200,000 tumor cells. The length and width of
the tumors were measured almost every other day using a digital
calipers and the tumor volume was calculated as: tumor volume
(mm3) = [length (mm) Â (width (mm)]2/2.
The drug solution was formulated as described. Each mouse re-
ceived 100 lL (for 6t) and 110 lL (for 6k) of the drug solution via
an intraperitoneal (ip) administration route on days 1, 3, 5, 7, 9, 11,
13, 15 and 17. Each injection delivered 50 mg/kg of compound 6k
and 6t. Animal weights were taken every other day. The vehicle
control group of mice was treated on a similar schedule with
13. (a) Boyd, R. B. The NCI In Vitro Anticancer Drug Discovery Screen. In Anticancer
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3, 1048.
100 lL of the vehicle.
Acknowledgments
15. (a) Cox, C.; Breslin, M.; Mariano, B.; Coleman, P.; Buser, C.; Walsh, E.; Hamilton,
K.; Huber, H.; Kohl, N.; Torrent, M.; Yan, Y.; Kuo, L.; Hartman, G. Bioorg. Med.
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D.; Fernandes, C.; Lobell, R. B.; Tao, W.; South, V. J.; Yan, Y.; Kuo, L. C.;
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The authors thank Conjura Pharmaceuticals, LLC for supporting
this project (M.L.). Support from the President’s Council Excellence
award is acknowledged (S.L.M.).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.02.018.
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