Synthesis and antitumor activity of novel 3,4-diaryl squaric acid analogs

Article abstract of DOI:10.1016/j.ejmech.2013.04.046

A series of novel 3,4-diaryl squaric acid analogs 4a-r related to combretastatin A-4 (CA4) using squaric acid as the cis-restricted linker were prepared and studied for their anticancer activity against selected human cancer cell lines. New compounds 4g,

Full text of DOI:10.1016/j.ejmech.2013.04.046

Accepted Manuscript
Synthesis and Antitumor Activity of Novel 3,4-diaryl Squaric Acid Analogues
Zong-ying Liu, Yue-ming Wang, Yan-xing Han, Ling Liu, Jie Jin, Hong Yi, Zhuo-rong
Li, Jian-dong Jiang, David W. Boykin
PII:
S0223-5234(13)00278-X
10.1016/j.ejmech.2013.04.046
EJMECH 6156
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2013
Revised Date: 19 April 2013
Accepted Date: 20 April 2013
Please cite this article as: Z.-y. Liu, Y.-m. Wang, Y.-x. Han, L. Liu, J. Jin, H. Yi, Z.-r. Li, J.-d. Jiang, D.W.
Boykin, Synthesis and Antitumor Activity of Novel 3,4-diaryl Squaric Acid Analogues, European Journal
of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.04.046.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
O
O
MeO
MeO
OH
OMe
OMe
CA4
R1
R2
4a-r
R1
R2
IC50 (CEM)
IC50=1.6 nM (CEM)
4n 3,4,5-(MeO)3 3'-OH-4'-MeO 1.7 nM
4p 3,4,5-(MeO)3 3'-NH2-4'-MeO 1.4 nM

ACCEPTED MANUSCRIPT
Synthesis and Antitumor Activity of Novel 3,4-diaryl Squaric Acid Analogues
Zong-ying Liu, †, a,b Yue-ming Wang, †, a Yan-xing Han, a Ling Liu, a Jie Jin, a Hong Yi, a Zhuo-rong Li, a,
* Jian-dong Jiang a, * and David W. Boykin b, *
aInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100050, People’s Republic of China
bDepartment of Chemistry, Georgia State University, Atlanta, GA 30303-3083, USA
* To whom correspondence should be addressed. Tel.: 404-413-5498 (D. W. B.); 86-10-63188423 (J.-d.
J.); 86-10-63027185 (Z.-r. L.). Fax: 404-413-5505 (D. W. B.); 86-10-63017302 (J.-d. J.);
86-10-63027185 (Z.-r. L.). E-mail: dboykin@gsu.edu (D. W. B.); jiang.jdong@163.com. (J.-d. J.);
l-z-r@263.net (Z.-r. L.).
† These authors made equal contribution to this work.
Abstract: A series of novel 3,4-diaryl squaric acid analogues 4a-r related to
combretastatin A-4 (CA4) using squaric acid as the cis-restricted linker were prepared and
studied for their anticancer activity against selected human cancer cell lines. New compounds
4g, 4k, 4m, 4n, 4p, 4q and 4r exhibit strong activities against human leukemia cells with IC50
values of ≤20 nM and compounds 4k, 4n, 4p, 4q and 4r showed potent activities against a
panel of human tumor cell lines. Compounds 4n and 4p arrest tumor cell cycle in G2-M
phase. Computational modeling analysis suggests that the binding mechanism of compound
4n to the colchicine binding site on the microtubules is similar to that of CA4.
Keywords: 3,4-diaryl squaric acid analogues, anticancer, cytotoxic agents
1. Introduction
Microtubules are dynamic structures that play a crucial role in cell division, and
are recognized as an important target for treating cancer [1-3]. Numerous chemically
diverse antimitotic agents, many of which are derived from natural products, such as
paclitaxel, epothilone A, vinblastine, combretastatin A-4 (CA4, Figure 1), and
colchicine, can induce cancer cell apoptosis through interference with tubulin
polymerization and depolymerization, and results in subsequent mitotic arrest. [4, 5].
Recently, it has been demonstrated that these microtubule depolymerizing anticancer
compounds represented by CA4 can also function as vascular disrupting agents (VDA)
that cause rapid and selective disruption of the established tumor vasculature [6, 7].
Since microtubule depolymerizing compounds can induce antivascular and antitumor
effects at doses that are less than one-tenth of the maximum tolerated dose and have
limited or nonexistent effects in normal tissue, the usefulness of compounds such as
combretastatin A-4P (CA4P), in combination with cytotoxic chemotherapy and
radiotherapy, is undergoing phase II trials, for the treatment of solid tumors. Another
CA4 derivative, AVE-8062, is currently under clinical evaluation as a tumor vascular
targeting agent (Fig.1). ZD6126 which is a phosphate prodrug of N-acetyl colchinol, a
derivative of colchicine, is now in phase II clinical trials [8-10].
CA4, a natural product isolated by Pettit et al. in 1989 from the South African
willowtree Combretum caffrum, displayed potent cytotoxicity against a broad
spectrum of human cancer cells including multiple drug-resistant cancer cell lines.
The structural simplicity of CA4 as well as its selective antivascular activity have
1

ACCEPTED MANUSCRIPT
drawn significant attention to examine the structure-activity relationship (SAR) of this
compound and its analogues [11-14]. SAR studies have demonstrated that the cis
orientation of double bond and the presence of a 3,4,5-trimethoxyphenyl group are
fundamental requirements for potent cytotoxitity. However, CA4 is prone to
isomerizes to the thermodynamically more stable and inactive trans-isomer, which
significantly reduces its antiproliferative activities [15, 16]. Moreover, CA4 as a
clinical antitumor agent is limited by its low aqueous solubility and low
bioavailability [17]. The goal to find even more potent and selective compounds has
encouraged many researchers to design more soluble, stable and active analogs. In
order to avoid the stability problems of CA4, suitable conformationally restricted
rings such as heterocyclic rings were used in place of the ethene bridge [15-16, 18].
Many of these molecules have been evaluated for their effects on tubulin
polymerization as well as for their antiproliferative activities, and possible
mechanisms of action have also been investigated.
Fig. 1. Chemical structures of microtubule depolymerizing compounds.
We have earlier reported series of 3,4-diarylthiazol-2(3H)-ones (imines) [15]䟍
1,5-diaryl-1H-pyrrole-2,3-diones [19], and 4-(diarylmethylene)piperidines [20] as
potential anticancer agents against human cancer cell lines. In continuation of our
efforts to search for novel anticancer agents, in this study we synthesized a variety of
3,4-diaryl squaric acid analogues using squaric acid as the cis-restricted linker and
tested their anticancer activities against a selected panel of human cancer cell lines
(Fig. 1). We also have investigated the influence of the position of the substituents
with regard to both A- and B-aromatic rings, on the cytotoxicity activities of these
analogues.
2. Results and discussion
2.1. Chemistry
We have used three different methods to synthesize the 3,4-diaryl squaric acid
2

ACCEPTED MANUSCRIPT
analogues. Compounds 4a-e that contain electron-donating groups on the aromatic
rings were prepared using sequential Friedel-Crafts procedures as outlined in Scheme
1. Squaric acid 1 in thionyl dichloride with DMF as catalyst was heated under reflux
to afford 3,4-dichloro-3-cyclobutene-1,2-dione 2 [21] which on treatment with
substituted
benzenes
under
Friedel-Crafts
conditions
yielded
3-chloro-4-R2Ph-3-cyclobutene-1,2-dione 3. Treatment of 3 with substituted benzenes
under Friedel-Crafts conditions afforded the desired compounds 4a-e in good yield.
Although derivatives 4f-k also contained electron-donating groups on the
aromatic ring, the Friedel-Crafts procedure was not successful using
1,2,3-trimethoxybenzene. As a result, we employed an alternate synthesis that is
shown in Scheme 2. Derivatives 4f-k was easily obtained under Stille cross-coupling
[22] conditions by treating 3 with (3,4,5-trimethoxyphenyl)tri-n-butylstannane
[23-24].
O
O
O
O
O
O
O
O
4a: R1 = 4-MeO,
R2 = 4'-MeO
a
c
b
4b: R1 = 2,4-(MeO)2, R2 = 4'-MeO
4c: R1 = 3,4-(MeO)2, R2 = 4'-MeO
4d: R1 = 2,4,6-(MeO)3, R2 = 4'-MeO
4e: R1 = 2,3,4-(MeO)3, R2 = 4'-MeO
Cl
Cl
Cl
HO
OH
R1
R2
R2
1
2
3
4a-e
Scheme 1. Reagents and conditions: a. SOCl2, DMF, reflux, 3h; b. AlCl3, Ph-R2, 0oC, 1.5h; c.
AlCl3, Ph-R1, 0oC, 1.5h
O
O
O
O
4f: R2 = H
4g: R2 = 4'-MeO
a
4h: R2 = 3',4'-(MeO)2
4i: R2 = 2',4'-(MeO)2
4j: R2 = 3'-Cl-4'-MeO
4k: R2 = 3'-F-4'-MeO
Cl
MeO
MeO
R2
R2
OMe
4f-k
3
Scheme 2. Reagents and conditions: a. 3,4,5-(MeO)3-Ph-Sn(n-Bu)3, (CH3CN)2PdCl2, rt, 3h
3

ACCEPTED MANUSCRIPT
O
O
S
O
O
S
O
O
a
b
Cl
Cl
Cl
MeO
MeO
OMe
OMe
2
5
6
OMe
O
O
4l: R2 = 3',4',5'-(MeO)3
4m: R2 = 3'-AcO-4'-MeO
4n: R2 = 3'-OH-4'-MeO
4o: R2 = 3'-NO2-4'-MeO
4p: R2 = 3'-NH2-4'-MeO
4q: 1H-indol-5'substituted composed by R2 and
the benzene ring
4r: 1H-indol-6'-substituted composed by R2 and
the benzene ring
c
NaHCO3
H2/Pd
MeO
R2
OMe
4l-r
MeO
Scheme 3. Reagents and conditions: a. Et3N, 4-OMe-Ph-SH, 0ꢀ, 5h; b. 3,4,5-(MeO)3-Ph-
Sn(n-Bu)3, (CH3CN)2PdCl2, rt, 3h; c. R2-Ar-B(OH)2, Pd2bda3 (1%), TFP (3%) and CuTC, 50 , 20
h
For compounds 4l-m, 4o, and 4q-r that contain electron-poor aromatic rings,
both Stille cross-coupling [22] and Liebeskind-Srogl cross-coupling [22, 25] were
used to prepare them (Scheme 3). 3-Chloro-4-(4-methoxyphenylthio)-3-cyclobutene-
1,2-dione 5 was prepared in 90% yield by treating 2 with 0.5 equiv of
p-methoxybenzenethiol with triethylamine as catalyst. Treatment of 5 with
(3,4,5-trimethoxyphenyl)tri-n-butylstannane [23-24] under Stille cross-coupling
conditions yielded the corresponding compound 6. Reaction of 6 with various
arylboronic acids [25-27] under Liebeskind-Srogl cross-coupling conditions gave the
corresponding compounds 4l-m, 4o, 4q-r. Compound 4n was obtained from
compound 4m in the presence of NaHCO3 in refluxing methanol [28]. Reduction of
the nitro group of compound 4o with Pd-C/H2 in ethanol and EtOAc afforded the
desired amino-substituted compound 4p.
2.2. Biological Evaluations
CA4, delivered by its prodrug CA4P, is an important vascular disrupting agent.
Our main objectives were to determine the cytotoxic activities and to attempt to gain
some understanding of the antiproliferative mechanism of these new CA4 analogues.
Therefore, this series of 3,4-diaryl squaric acid analogues were evaluated for their in
vitro anticancer activities against several cell lines and preliminary mode of action
studies were performed for compound 4n.
2.2.1. In vitro cytotoxic activities and SAR
4

ACCEPTED MANUSCRIPT
Since human CEM Leukemia cells are known for their rapid proliferation and
high sensitivity to standard anticancer agents, the cytotoxic activities of the
synthesized compounds were first evaluated against human CEM Leukemia cells
(Table 1). Many compounds exhibited strong activities against human leukemia cells
with IC50 values of ≤20 nM (4g, 4k, 4m, 4n, 4p, 4q, 4r).
Similar to the well-known SAR of CA4, compound 4n, with a
3,4,5-trimethoxyphenyl ring and 3’- hydroxy-4’-methoxyphenyl ring, showed strong
cytotoxic activity against human leukemia cells, as reflected by the IC50 value of 1.7
nM, essentially the same as that for CA4 (IC50 of 1.6 nM). This cytotoxic activity
demonstrated that squaric acid ring system can be used as the cis-restricted linker in
this series of compounds related to CA4.
In order to investigate the influence of the position of the substituents with
regard to A- and B-aromatic rings and as the p-methoxy group in the B-ring of CA4 is
important for its activity, we first fixed the B-ring of this series of compounds with the
4’-methoxyphenyl ring and examined variations of the A-ring substituents.
Comparing the A-ring derivatives 4a, 4b, 4c, 4g, the order of activity for the
substituent(s) on the A-ring was 4-methoxy (4a) < 2,4-dimethoxy (4b), 3,4-dimethoxy
(4c)< 3,4,5-trimethoxy (4g). For these four compounds, IC50 values were >20 uM for
4a, 1.51 uM for 4b and 12.74 uM for 4c, while for 4g the IC50 was 16 nM, much
better than that of 4a, 4b and 4c. These results are consistent with that found for CA4
analogues, where it is well known that three methoxy groups on A ring are essential
for potent cytotoxicity.
By keeping the 3,4,5-trimethoxyphenyl as a constant for the A-ring, we
evaluated the antiproliferative effect of varying substitution on the B-ring for the
squaric acid series. The number and position of methoxy substituents of the B-ring
had a major influence on antiproliferative activity. The 3’,4’,5’-trimethoxy (4l)
derivative was the least active compound in the series, with an IC50 value of over 20
ꢀM. Replacement of the 3’,4’,5’- trimethoxy (4l) with the 3’,4’-dimethoxy (4h) and
2’,4’-dimethoxy (4i) resulted in increased activity. Replacement of the 3’,4’,5’-
trimethoxy (4l) with the 4’-methoxy (4g) resulted in a marked increase in activity
with IC50 value of 16 nM. However, replacement of the the 4’-methoxy(4g) with H (4f)
resulted in significant loss of bioactivity. In an effort to increase potency, the
3’-hydroxy and 3’-amino substituted compounds 4n and 4p were prepared to mimic
analogues of CA4. Both compounds 4n and 4p showed potent cytotoxic activity
against human leukemia cells similar to that of CA4. The insertion of a 3’-chloro
substituent on the B ring to yield 4j gave a 10-fold less potent compound than that of
4g. However, the insertion of a 3’-fluoro substituent on the B ring to yield 4k
maintained the cytotoxicity compared with compound 4g. The indole compounds 4q
and 4r, exhibited considerable cytotoxicity against human leukemia cells with IC50
values of 15.4 nM and 6.6 nM, respectively. This result suggested that the indole core
is well tolerated as a replacement of the 4-methoxyphenyl B ring of CA4 with the
retention of potent cytotoxic activity.
Table 1
Cytotoxicity of synthesized compounds and CA4 in CEM leukemia cells
5

ACCEPTED MANUSCRIPT
O
O
A
B
R1
R2
Compound
R1
R2
Cytotoxicity
IC50, uMa
>20
4a
4b
4c
4d
4e
4f
4-MeO
4’-MeO
4’-MeO
2,4-(MeO)2
3,4-(MeO)2
1.51
4’-MeO
12.74
0.85
2,4,6-(MeO)3
2,3,4-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
4’-MeO
4’-MeO
2.48
H
16.1
4g
4h
4i
4’-MeO
0.016
0.21
3’,4’-(MeO)2
2’,4’-(MeO)2
3’-Cl-4’-MeO
3’-F-4’-MeO
3’,4’,5’-(MeO)3
3’-AcO-4’-MeO
3’-OH-4’-MeO
3’-NO2-4’-MeO
3’-NH2-4’-MeO
1.77
4j
0.136
0.0115
>20
4k
4l
4m
4n
4o
4p
4q
0.0018
0.0017
12.17
0.0014
0.0154
1H-indol-5’-substituted
composed by R2 and the
benzene ring
4r
3,4,5-(MeO)3
1H-indol-6’-substituted
composed by R2 and the
benzene ring
0.0066
0.0016
CA4
aIC50 values, concentration required to inhibit 50% of human tumor cells proliferation after 72 h
treatment.
2.2.2. In vitro anticancer activity of 4k, 4n, 4p, 4q and 4r against several cancer cell
lines
Since compounds 4k, 4n, 4p, 4q and 4r showed potent activity against the
non-solid human CEM cell line, they were further evaluated against a panel of human
tumor cell lines including human liver cancer cells Bel-7402, HepG2, SMMC-7221,
human breast cancer cells MCF-7, human pancreatic cancer cells SW-1990, human
colon adenocarcinoma HCT116 and human leukemia cells CEM with CA4 employed
as a positive control (Table 2).
6

ACCEPTED MANUSCRIPT
Compounds 4n and 4p showed potent cytotoxicity against almost all seven cell
lines studied. The highest cytotoxicity (IC50 < 2 nM for both compound 4n and 4p)
was observed against leukemia cells. The least sensitive cell line for compound 4n
was human liver cancer cells SMMC-7221, and for compound 4p was human
pancreatic cancer cells SW-1990. For compound 4n and 4p, the cytotoxicity of human
liver cancer cell HepG2 were also high, with IC50 values of less than 14 nM, similar to
the positive control CA4. Moreover, the cytotoxicity of compound 4n against human
liver cancer cells Bel-7402 and human breast cancer cells MCF-7 was 5- to 6-fold
stronger than that of compound CA4, and the cytotoxicity of compound 4p against
human liver cancer cells Bel-7402 was 122-fold stronger than that of compound CA4.
However, the cytotoxicity of compound 4p against human breast cancer cells MCF-7
and human pancreatic cancer cells SW-1990 were 5- to 7-fold less active than that of
compound CA4.
With the goal of obtaining more SAR information, we also examined the
antiproliferative activities of compounds 4q, 4r and 4k compared to compound 4n
against 7 cancer cell lines. From these results, it can be seen that antiproliferative
activities of the indole compounds 4q and 4r against leukemia cells and human liver
cancer cells HepG2, are quite similar to that of 4n. However, the cytotoxicities of
compounds 4q and 4r against human liver cancer cells Bel-7402 were 5- to 12-fold
less potent than compound 4n. Interestingly, introduction of a 3’-fluoro substituent on
the B ring (4k) displayed strong growth inhibitory activity, with IC50 values lower
than 200 nM against all 7 cell lines. These SAR results further demonstrate that
B-ring was amenable to modifications to yield potent compounds and the 3’-hydroxy
substituent of the B-ring is not indispensable for potent biological activities in this
3,4-diaryl squaric acid analogues series.
Table 2
The IC50 values (µM) of compounds 4n, 4p, 4q, 4r, 4k, and CA4 versus various cancer cell lines.
IC50, µM a
Cell line
4n
4p
4q
4r
4k
CA4
0.0016
1.718
0.006
0.289
CEM
0.0017
0.309
0.014
1.517
0.0014
0.014
0.005
0.190
0.0154
1.496
0.006
0.0066
3.621
0.006
2.544
0.0115
0.013
0.105
0.176
Bel-7402
HepG2
SMMC-7721
0.646
7

ACCEPTED MANUSCRIPT
MCF-7
0.006
0.245
0.005
0.164
1.768
0.005
NTb
NTb
NTb
NTb
NTb
NTb
0.005
0.027
0.081
0.029
0.250
0.006
SW-1990
HCT116
aIC50 values, concentration required to inhibit 50% of human tumor cells proliferation after 72 h
treatment.
bNT = not tested
2.2.3. Mode of action of 4n
Compound 4n has shown potent in vitro activities, and initiation of mode of
action studies seemed warranted. Flow cytometric analysis with MCF-7 cells (Figure
3) was performed using CA4 as the positive control. The results suggest that the
compound 4n blocks the cell cycle in the G2-M phase.
control
4n
CA4
Fig. 2. Cell cycle analysis. MCF-7 cells untreated or treated with CA4 and 4n were analyzed for
cell cycle distribution using flow cytometry.
2.2.4. Molecular modeling study
To further expand the understanding of our experimental findings, molecular
modeling studies for 4n and CA4 were carried out with the use of CDOCKER within
the Discovery Studio 3.0 program package. Compounds 4n and CA4 were docked
into the colchicine binding site of tubulin (PDB 1SA0). The binding mode observed
of 4n was similar to that of CA4. The trimethoxyphenyl group ring occupied the same
position as the corresponding moiety of CA4 and showed nonpolar interactions with
Cys241, Leu248, Ala250 and Leu255. The B-rings of both compounds also occupied
the same pocket, established one hydrogen-bond interaction with Met259 and made
hydrophobic interactions with Ala180, Met259 and Lys352. Thus, results of the
computational modeling analysis are consistent with our experimental studies (Fig. 3).
8

ACCEPTED MANUSCRIPT
Fig. 3. Docking conformation of 4n (yellow) and CA4 (cyan) in the colchicine binding site of
tubulin (PDB 1SA0) using CDOCKER program in Discovery Studio 3.0.
3. Conclusion
In this study, synthetic and activity studies for the 3,4-diaryl squaric acid
analogues as a new class of potent anticancer agents were performed. New
compounds 4g, 4k, 4m, 4n, 4p, 4q and 4r exhibit strong activities against human
leukemia cells and compounds 4k, 4n, 4p, 4q and 4r showed potent activities against
a panel of human tumor cell lines. This cytotoxic activity showed that the squaric acid
ring system can be used for the cis-restricted linker in this series of compounds related
to CA4. The SAR information revealed that all three methoxy groups of A ring are
essential for potent cytotoxicity. Replacement of the B ring of CA4 with the indole
core and replacement of the 3’-hydroxy substituent of the B-ring with 3’-amino or
3’-fluoro is tolerated with retention of the potent cytotoxic activity. Preliminary mode
of action studies demonstrated that compound 4n arrests the tumor cell cycle in the
G2-M phase. All these results are important for the design of structurally-related
tubulin inhibitors or combretastatin analogs in the future.
4. Experimental section
4.1. Chemistry Melting points were recorded using a Mel-Temp 3.0 capillary melting
1
point apparatus and are uncorrected. H and 13C NMR spectra were recorded
employing a Bruker 400 UltrashieldTM instrument; chemical shifts (δ) are in ppm
relative to internal TMS. Mass spectra were recorded on a VG analytical 70-SE
spectrometer. Elemental analyses were obtained from Atlantic Microlab, Inc.
(Norcross, GA).
4.1.1. 3,4-bis(4-methoxyphenyl)cyclobut-3-ene-1,2-dione (4a)
A mixture of squaric acid (0.55 g, 4.8 mmol), thionyl dichloride (0.88 ml, 9.7
mmol) and DMF (5 drops) was heated at reflux for 3 h. The resultant mixture was
9

ACCEPTED MANUSCRIPT
cooled to 0ꢀ. Anhydrous methylene chloride (6 ml) and anhydrous AlCl3 (0.64 g, 4.8
mmol) were added to the reaction mixture. Then anisole (0.52 g, 4.8 mmol) was
added to the mixture dropwise. The mixture was stirred at 0ꢀ for 1.5 h. The resultant
mixture was poured onto crushed ice and extracted with CH2Cl2 (2×15 ml), the
combined organic layer was washed with water (15 ml) followed by brine (15 ml) and
concentrated under reduced pressure to give crude product, which was further purified
by column chromatography on silica gel using hexane/EtOAc (5:1) as eluent to afford
1
title compound as yellow solid (1.02 g, 72%). mp 183-184ꢀ; H NMR (400 MHz,
CDCl3): δ 3.94 (s, 6H), 7.07 (dd, J = 2.0, 6.8 Hz, 4H), 8.14 (dd, J = 2.0, 6.8 Hz, 4H);
13C NMR (CDCl3): δ 55.6, 114.8, 121.3, 130.4, 163.5, 184.4, 196.3 ; Anal. Calcd. for
C18H14O4: C, 73.46; H, 4.79. Found: C, 73.69; H, 4.81.
4.1.2. 3-(2,4-dimethoxyphenyl)-4-(4’-methoxyphenyl)cyclobut-3-ene-1,2-dione (4b)
A mixture of squaric acid (0.55 g, 4.8 mmol), thionyl dichloride (0.88 ml, 9.7
mmol) and DMF (5 drops) was heated at reflux for 3 h. The resultant mixture was
cooled to 0ꢀ. Anhydrous methylene chloride (6 ml) and anhydrous AlCl3 (0.32 g, 2.4
mmol) were added to the reaction mixture. Then anisole (0.13 g, 1.2 mmol) was
added to the mixture dropwise. The mixture was stirred at 0ꢀ for 1.5 h. The resultant
mixture was poured onto crushed ice and extracted with CH2Cl2 (2×15 ml), the
combined organic layer was washed with water (15 ml) followed by brine (15 ml) and
concentrated under reduced pressure to give crude product, which was further
recrystalized from CH2Cl2/ hexane to give 3-chloro-4-(4-methoxyphenyl)cyclobut
-3-ene-1,2-dione (3, 0.54 g, 50%) and was used in the next step without further
purification.
3 (0.54 g, 2.4 mmol) was dissolved in anhydrous methylene chloride (6 ml). The
resultant mixture was cooled to 0ꢀ. Anhydrous AlCl3 (0.32 g, 2.4 mmol) was added
to the reaction mixture. Then 1,3-dimethoxybenzene (0.33 g, 2.4 mmol) was added
(dropwise) to the mixture. The mixture was stirred at 0ꢀ for 1.5 h. The resultant
mixture was poured onto crushed ice and extracted with CH2Cl2 (2×15 ml), the
combined organic layer was washed with water (15 ml) followed by brine (15 ml) and
concentrated under reduced pressure to give crude product, which was further purified
by column chromatography on silica gel using hexane/EtOAc (5:1) as eluent to afford
1
title compound as yellow solid (0.54 g, 69%). mp 182-183ꢀ; H NMR (400 MHz,
CDCl3): δ 3.69 (s, 3H), 3.90 (s, 3H), 3.92 (s, 3H), 6.55 (d, J = 2.4 Hz, 1H), 6.68 (dd, J
= 2.4, 8.4 Hz, 1H), 6.97 (dd, J = 2.0, 6.8 Hz, 2H), 7.85 (dd, J = 2.0, 6.8 Hz, 2H), 7.97
13
(d, J = 8.4 Hz, 1H); C NMR (400 MHz, CDCl3): δ 55.0, 55.5, 55.8, 98.7, 105.6,
111.3, 113.7, 122.6, 131.5, 131.8, 158.4, 163.2, 165.0, 182.2, 185.2, 196.5, 196.9;
10

ACCEPTED MANUSCRIPT
Anal. Calcd. for C19H16O5: C, 70.36; H, 4.97. Found: C, 70.58; H, 5.01.
4.1.3. 3-(3,4-dimethoxyphenyl)-4-(4’-methoxyphenyl)cyclobut-3-ene-1,2-dione (4c)
The title compound was obtained as a yellow solid in 72% yield from
1,2-dimethoxybenzene using a procedure similar to that for compound 4b. mp
1
165-166ꢀ; H NMR (400 MHz, CDCl3): δ 3.92 (s, 3H), 3.94 (s, 3H), 4.00 (s, 3H),
7.02 (d, J = 8.4 Hz, 1H), 7.05 (dd, J = 2.0, 7.2 Hz, 2H), 7.63 (d, J = 2.0 Hz, 1H), 7.81
(dd, J = 2.0, 8.4 Hz, 1H), 8.14 (dd, J = 2.0, 7.2 Hz, 2H); 13C NMR (400 MHz, CDCl3):
δ 55.6, 56.1, 56.2, 110.5, 111.3, 114.7, 121.2, 121.3, 122.9, 130.5, 149.2, 153.2, 163.6,
184.4, 196.2, 196.2; Anal. Calcd. for C19H16O5: C, 70.36; H, 4.97. Found: C, 70.19; H,
4.96.
4.1.4. 3-(4’-methoxyphenyl)-4-(2,4,6-trimethoxyphenyl)cyclobut-3-ene-1,2-dione (4d)
The title compound was obtained as a yellow solid in 80% yield from
1,3,5-trimethoxybenzene using a procedure similar to that for compound 4b. mp
1
185-187ꢀ; H NMR (400 MHz, CDCl3): δ 3.77 (s, 6H), 3.90 (s, 3H), 3.92 (s, 3H),
13
6.23 (s, 2H), 6.96 (dd, J = 2.0, 6.8 Hz, 2H), 7.92 (dd, J = 2.0, 6.8 Hz, 2H); C NMR
(CDCl3): δ 55.5, 55.6, 55.8, 90.8, 100.7, 113.9, 122.6, 131.5, 158.8, 163.3, 165.1,
184.6, 186.9, 195.9, 198.0; Anal. Calcd. for C20H18O6: C, 67.79; H, 5.12. Found: C,
67.74; H, 5.14.
4.1.5. 3-(4’-methoxyphenyl)-4-(2,3,4-trimethoxyphenyl)cyclobut-3-ene-1,2-dione (4e)
The title compound was obtained as a yellow solid in 62% yield from
1,2,3-trimethoxybenzene using a procedure similar to that for compound 4b. mp
115-116ꢀ; 1H NMR (400 MHz, CDCl3): δ 3.78 (s, 3H), 3.92 (s, 3H), 3.94 (s, 3H),
3.99 (s, 3H), 6.84 (d, J = 8.8 Hz, 1H), 6.99 (dd, J = 2.0, 6.8 Hz, 2H), 7.58 (d, J = 8.8
13
Hz, 1H), 8.01 (dd, J = 2.0, 6.8 Hz, 2H); C NMR (400 MHz, CDCl3): δ 55.5, 56.3,
61.2, 61.4, 107.3, 114.1, 115.7, 121.8, 124.6, 131.6, 142.2, 151.8, 157.5, 163.7, 183.6,
186.3, 195.9, 197.2; Anal. Calcd. for C20H18O6: C, 67.79; H, 5.12. Found: C, 67.40; H,
5.16.
4.1.6. 3-phenyl-4-(3,4,5-trimethoxyphenyl)cyclobut-3-ene-1,2-dione (4f)
A mixture of squaric acid (0.55 g, 4.8 mmol), thionyl dichloride (0.88 ml, 9.7
mmol) and DMF (5 drops) was refluxed for 3 h. The resultant mixture was cooled to
0ꢀ. Anhydrous methylene chloride (6 ml) and anhydrous AlCl3 (0.32 g, 2.4 mmol)
were added to the reaction mixture. Then benzene (0.09g, 1.2mmol) was added to the
mixture dropwise. The mixture was stirred at room temperature for 1.5 h. The
resultant mixture was poured onto crushed ice and extracted with CH2Cl2 (2×15 ml),
the combined organic layer was washed with water (15 ml) followed by brine (15 ml)
and concentrated under reduced pressure to give crude product, which was further
recrystalized from CH2Cl2/ hexane to give 3-chloro-4-(4-phenyl)cyclobut-
3-ene-1,2-dione (0.51 g, 55%) and was used in the next step without further
purification.
3-Chloro-4-(4-phenyl)cyclobut-3-ene-1,2-dione(0.46 g, 2.4 mmol) and
11

ACCEPTED MANUSCRIPT
(3,4,5-trimethoxyphenyl)tri-n-butylstannane (1.43 g, 3.1 mmol) were dissolved in
unhydrous CH3CN (6 ml) under N2. Then, the mixture was purged with N2 for 5 min
whereupon (CH3CN)2PdCl2 (0.03 g, 0.12 mmol) was added and the reaction mixture
was stirred at room temperature for 3 h. The mixture was filtered and purified by
column chromatography on silica gel using hexane/EtOAc (5:1) as eluent to afford
1
title compound as yellow solid (0.51 g, 66%). mp 161-162ꢀ; H NMR (400 MHz,
13
CDCl3): δ 3.90 (s, 6H), 4.00 (s, 3H), 7.42 (s, 2H), 7.60 (m, 3H), 8.12 (m, 2H); C
NMR (400 MHz, CDCl3): δ 56.4, 61.0, 106.3, 123.0, 128.1, 128.5, 129.1, 132.9,
143.3, 153.7, 186.3, 186.7, 195.5, 196.2; Anal. Calcd. for C19H16O5: C, 70.36; H, 4.97.
Found: C, 70.43; H, 4.88.
4.1.7. 3-(4’-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)cyclobut-3-ene-1,2-dione (4g)
The title compound was obtained as a yellow solid in 35% yield from anisole
1
using a procedure similar to that for compound 4f. mp 154-155ꢀ; H NMR (400
MHz, CDCl3): δ 3.91 (s, 6H), 3.95 (s, 3H), 3.99 (s, 3H), 7.08 (dd, J = 2.0, 6.8 Hz, 2H),
13
7.37 (s, 2H), 8.20 (dd, J = 2.0, 6.8 Hz, 2H); C NMR (400 MHz, CDCl3): δ 55.7,
56.4, 61.1, 105.6, 114.7, 121.0, 123.5, 130.7, 142.2, 153.6, 163.8, 184.6, 185.4, 195.8,
196.4; Anal. Calcd. for C20H18O6: C, 67.79; H, 5.12. Found: C, 67.71; H, 5.18.
4.1. 8. 3-(3,’4’-dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)cyclobut-3-ene-1,2-dione
(4h)
The title compound was obtained as a yellow solid in 38% yield from
1,2-dimethoxybenzene using a procedure similar to that for compound 4f. mp
1
186-187ꢀ; H NMR (400 MHz, CDCl3): δ 3.91 (s, 6H), 3.94 (s, 3H), 3.99 (s, 3H),
4.02 (s, 3H), 7.03 (d, J = 8.4 Hz, 1H), 7.38 (s, 2H), 7.69 (d, J = 2.0 Hz, 1H), 7.88 (dd,
13
J = 2.0, 8.4 Hz, 1H); C NMR (400 MHz, CDCl3): δ 56.2, 56.3, 56.4, 61.1, 105.6,
110.8, 111.2, 121.1, 123.1, 123.5, 142.3, 149.3, 153.5, 153.6, 184.7, 185.4, 195.8,
196.3; Anal. Calcd. for C21H20O7+0.9H2O: C, 62.96; H, 5.49. Found: C, 63.00; H,
5.18.
4.1.9. 3-(2’,4’-dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)cyclobut-3-ene-1,2-dione
(4i)
The title compound was obtained as a yellow solid in 42% yield from
1,3-dimethoxybenzene using a procedure similar to that for compound 4f. mp
1
152-153ꢀ; H NMR (400 MHz, CDCl3): δ 3.71(s, 3H), 3.85 (s, 6H), 3.93 (s, 3H),
3.96 (s, 3H), 6.56 (d, J = 2.0 Hz, 1H), 6.69 (dd, J = 2.0, 8.8 Hz, 1H), 7.16 (s, 2H) ,
7.97 (d, J = 8.8 Hz, 1H); 13C NMR (400 MHz, CDCl3): δ 55.2, 55.8, 56.3, 61.1, 98.6,
105.6, 106.9, 111.0, 124.7, 131.6, 142.0, 152.8, 158.6, 165.3, 183.7, 185.4, 196.4,
196.6; Anal. Calcd. for C21H20O7: C, 65.62; H, 5.24. Found: C, 65.65; H, 5.28.
4.1.10. 3-(3’-chloro-4’-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)cyclobut-3-ene-1,2
-dione (4j)
The title compound was obtained as a yellow solid in 46% yield from
1-chloro-2-methoxybenzene using a procedure similar to that for compound 4f. mp
12

ACCEPTED MANUSCRIPT
1
212-214ꢀ; H NMR (400 MHz, CDCl3): δ 3.93(s, 6H), 4.01 (s, 3H), 4.05 (s, 3H),
7.12 (d, J = 8.4 Hz, 1H), 7.38 (s, 2H), 8.20 (dd, J = 2.0, 8.4 Hz, 1H), 8.24 (d, J = 2.0
Hz, 1H); 13C NMR (400 MHz, CDCl3): δ 56.4, 56.5, 61.2, 105.7, 112.2, 121.6, 123.1,
123.7, 129.3, 129.8, 142.6, 153.6, 158.9, 183.8, 185.2, 195.7, 195.8; Anal. Calcd. for
C20H17ClO6: C, 61.78; H, 4.41. Found: C, 62.07; H, 4.35.
4.1.11. 3-(3’-fluoro-4’-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)cyclobut-3-ene-1,2
-dione (4k)
The title compound was obtained as a yellow solid in 45% yield from
1-fluoro-2-methoxybenzene using a procedure similar to that for compound 4f. mp
1
179-180ꢀ; H NMR (400 MHz, CDCl3): δ 3.92(s, 6H), 4.00 (s, 3H), 4.04 (s, 3H),
7.14 (t, J = 8.4 Hz, 1H), 7.36 (s, 2H), 7.94 (dd, J = 2.0, 8.4 Hz, 1H), 8.07 (dt, J = 2.0,
13
8.4 Hz, 1H); C NMR (400 MHz, CDCl3): δ 56.3, 56.4, 61.2, 105.7, 113.3, 113.4,
115.4, 115.6, 120.9, 121.0, 123.1, 126.2, 126.3, 142.6, 150.9, 152.0, 152.1, 153.4,
153.6, 184.1, 185.2, 195.7, 195.8; Anal. Calcd. for C20H17FO6: C, 64.51; H, 4.60.
Found: C, 64.46; H, 4.61.
4.1.12. 3-(4’-methoxyphenylthio)-4-(3,4,5-trimethoxyphenyl)cyclobut-3-ene-1,2-
dione (6)
A mixture of squaric acid (5.7 g, 50 mmol), thionyl dichloride (9.0 ml, 99 mmol)
and DMF (15 drops) was heated at reflux for 3 h. The mixture was cooled to room
temperature, hexane (50 ml) was added to the mixture, filtered, concentration and
after cooling was filtered under nitrogen, to give yellow crystalls of
3,4-dichlorocyclobut-3-ene-1,2-dione (2, 4.38 g, 58%) which was used in the next
step without further purification.
2 (2.0g, 13.2 mmol) was dissolved in anhydrous THF (100 ml) under N2 and the
temperature was lowered to 0ꢀ. p-Methoxybenzenethiol (0.9 ml, 6.6 mmol) and
triethylamine (0.8 ml, 6.6 mmol) were sequentially added. The mixture was stirred at
0ꢀ for 5 h whereupon the solvent was removed under reduced pressure. The
remaining solid was extracted with EtOAc (2×150 ml), the organic extracts was
passed through a aid pad and then the solvent was removed under reduced pressure,
and further recrystalized from CH2Cl2/ hexane to give a pale yellow solid
3-chloro-4-(4-methoxyphenylthio)cyclobut-3-ene-1,2-dione (5, 2.86 g, 85%) which
was used in the next step without further purification.
5 (2.55 g, 10 mmol) and (3,4,5-trimethoxyphenyl)tri-n-butylstannane (5.94 g, 13
mmol) were dissolved in anhydrous CH3CN (30 ml) under N2. Then, the mixture was
purged with N2 for 5 min whereupon (CH3CN)2PdCl2 (0.13 g, 0.5 mmol) was added
and the reaction mixture was stirred at room temperature for 3 h. The mixture then
was filtered and purified by column chromatography on silica gel using
hexane/EtOAc (5:1) as eluent to afford the title compound as a yellow solid (3.09 g,
80%). mp 184-185ꢀ; 1H NMR (400 MHz, CDCl3): δ 3.89 (s, 3H), 3.98 (s, 6H), 3.99
13

ACCEPTED MANUSCRIPT
(s, 3H), 6.99 (dd, J = 2.0, 6.8 Hz, 2H), 7.34 (s, 2H), 7.58 (dd, J = 2.0, 6.8 Hz, 2H); 13C
NMR (400 MHz, CDCl3): δ 55.4, 56.4, 61.2, 106.2, 115.0, 115.2, 123.7, 135.5, 142.3,
153.6, 161.6, 180.6, 188.7, 190.8, 192.3; Anal. Calcd. for C20H18O6S: C, 62.16; H,
4.70. Found: C, 61.86; H, 4.59.
4.1.13. 3,4-bis(3,4,5-trimethoxyphenyl)cyclobut-3-ene-1,2-dione (4l)
6 (0.39 g, 1 mmol) and 3,4,5-trimethoxyphenylboronic acid (0.53 g, 2.5 mmol)
were dissolved in dry THF (6 ml) under N2. Then, the mixture was purged with N2 for
10 min whereupon, Pd2bda3 (9.16 mg, 1%), TFP (6.96 mg, 3%) and CuTC (0.57g, 3
mmol) were added. The reaction mixture was heated at 50ꢀ for 20 h, cooled to room
temperature, and then quenched with an aqueous NH4Cl solution. Then, it was
extracted with EtOAc (2×30 ml), dried over MgSO4, filtered and the solvent was
removed under reduced pressure. The product was purified by column
chromatography on silica gel using hexane/EtOAc (3:1) as eluent to afford title
compound as yellow solid (0.29 g, 70%). mp 161-163ꢀ; 1H NMR (400 MHz, CDCl3):
δ 3.92 (s, 12H), 4.00 (s, 6H), 7.41 (s, 4H); 13C NMR (400 MHz, CDCl3): δ 56.5, 61.2,
105.9, 123.3, 142.7, 153.6, 185.6, 195.9; Anal. Calcd. for C22H22O8: C, 63.76; H, 5.35.
Found: C, 63.67; H, 5.31.
4.1.14.
acetate (4m)
The title compound was obtained as a yellow solid in 63% yield from
5-(3,4-dioxo-2-(3,4,5-trimethoxyphenyl)cyclobut-1-enyl)-2-methoxyphenyl
3-acetoxy-4-methoxyphenylboronic acid using a procedure similar to that of
1
compound 4l. mp 159-160ꢀ; H NMR (400 MHz, CDCl3): δ 2.36 (s, 3H), 3.91 (s,
6H), 3.97 (s, 3H), 4.00 (s, 3H), 7.14 (d, J = 8.8 Hz, 1H), 7.35 (s, 2H), 7.89 (d, J = 2.4
13
Hz, 1H), 8.17 (dd, J = 2.4, 8.8 Hz, 1H); C NMR (400 MHz, CDCl3): δ 20.6, 56.3,
56.4, 61.2, 105.5, 112.6, 121.0, 122.9, 123.2, 128.6, 104.1, 142.4, 153.6, 155.4, 168.7,
184.4, 185.1, 195.7, 195.9; Anal. Calcd. for C22H20O8: C, 64.07; H, 4.89. Found: C,
64.17; H, 4.83.
4.1.15. 3-(3’-hydroxy-4’-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)cyclobut-3-
ene-1,2-dione (4n)
A solution of 4m (0.5 g, 1.2 mmol ), 10% NaHCO3 (3 ml, 1.8 mmol), and MeOH
(2 ml) was allowed to reflux for 1.5 h. Cooled to room temperature and filtered and
washed with water to give title compound as yellow solid (0.38 g, 85%). mp
1
223-224ꢀ; H NMR (400 MHz, CDCl3): δ 3.92 (s, 6H), 4.00 (s, 3H), 4.04 (s, 3H),
5.76 (s, 1H), 7.03 (d, J = 8.4 Hz, 1H), 7.40 (s, 2H), 7.73 (d, J = 2.0 Hz, 1H), 7.88 (dd,
J = 2.0, 8.4 Hz, 1H); 13C NMR (CDCl3): δ 56.3, 56.4, 60.8, 105.7, 112.8, 114.5, 121.0,
122.1, 123.7, 141.7, 147.5, 153.1, 153.6, 183.9, 185.3, 196.0, 196.9; Anal. Calcd. for
C20H18O7: C, 64.86; H, 4.90. Found: C, 64.95; H, 4.85.
4.1.16. 3-(4’-methoxy-3’-nitrophenyl)-4-(3,4,5-trimethoxyphenyl)cyclobut-3-ene-
1,2-dione (4o)
The title compound was obtained as a yellow solid in 65% yield from
14

ACCEPTED MANUSCRIPT
4-methoxy-3-nitrophenylboronic acid using a procedure similar to that of compound
4l. mp 233-234ꢀ; 1H NMR (400 MHz, CDCl3): δ 3.95 (s, 6H), 4.02 (s, 3H), 4.12 (s,
3H), 7.32 (d, J = 8.8 Hz, 1H), 7.38 (s, 2H), 8.51 (dd, J = 2.0, 8.8 Hz, 1H), 8.67 (d, J =
13
2.0 Hz, 1H); C NMR (400 MHz, CDCl3): δ 56.7, 57.9, 60.9, 106.5, 116.1, 120.6,
123.2, 125.0, 134.6, 139.9, 142.9, 153.8, 155.8, 183.1, 185.6, 195.7, 196.0; Anal.
Calcd. for C20H17NO8: C, 60.15; H, 4.29; N, 3.51. Found: C, 60.09; H, 4.24; N, 3.69.
4.1.17. 3-(3’-amino-4’-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)cyclobut-3-
ene-1,2-dione (4p)
A solution of 4o (0.2 g, 0.5 mmol) in ethyl acetate (30 mL) and EtOH (20 ml) was
hydrogenated (40 psi) at room temperature over 10% Pd/C (0.02 mg) for 1.5 h. The
black suspension was filtered through a filter aid pad and the filtrate was concentrated
under reduced pressure to give the amino compound as yellow solid (0.15 g, 83%). mp
1
198-200ꢀ; H NMR (400 MHz, CDCl3): δ 3.92 (s, 6H), 3.99 (s, 6H), 4.02 (s, 2H),
6.93 (d, J = 8.4 Hz, 1H), 7.41 (s, 2H), 7.54 (d, J = 2.0 Hz, 1H), 7.67 (dd, J = 2.0, 8.4
Hz, 1H); 13C NMR (400 MHz, CDCl3): δ 56.1, 56.4, 60.8, 105.7, 111.0, 112.0, 119.1,
121.2, 123.8, 139.0, 141.7, 151.4, 153.5, 183.5, 186.0, 196.2, 197.1; Anal. Calcd. for
C20H19NO6 + 0.8 H2O: C, 63.73; H, 5.25; N, 3.54. Found: C, 63.61; H, 5.14; N, 3.70.
4.1.18. 3-(1H-indol-5’-yl)-4-(3,4,5-trimethoxyphenyl)cyclobut-3-ene-1,2-dione (4q)
The title compound was obtained as a yellow solid in 66% yield from
1H-indol-5-ylboronic acid using a procedure similar to that of compound 4l. mp
228-230ꢀ; 1H NMR (400 MHz, CDCl3): δ 3.91 (s, 6H), 4.00 (s, 3H), 6.73 (t, J = 2.4
Hz, 1H), 7.36 (t, J = 2.4 Hz, 1H), 7.47 (s, 2H), 7.56 (d, J = 8.4 Hz, 1H), 8.02 (dd, J =
1.6, 8.4 Hz, 1H), 8.51 (s, 1H), 8.66 (s, 1H); 13C NMR (400 MHz, CDCl3): δ 56.4, 60.8,
103.7, 105.6, 113.0, 119.5, 121.6, 122.8, 124.0, 128.3, 139.2, 141.7, 153.6, 183.5,
187.2, 196.0, 197.4; Anal. Calcd. for C21H17NO5 + 0.3 H2O: C, 68.40; H, 4.81; N,
3.80. Found: C, 68.26; H, 4.70; N, 3.73.
4.1.19. 3-(1H-indol-6’-yl)-4-(3,4,5-trimethoxyphenyl)cyclobut-3-ene-1,2-dione (4r)
The title compound was obtained as a yellow solid in 53% yield from
1H-indol-6-ylboronic acid using a procedure similar to that of compound 4l. mp
193-195ꢀ; 1H NMR (400 MHz, CDCl3): δ 3.92 (s, 6H), 4.00 (s, 3H), 6.91 (t, J = 2.4
Hz, 1H), 7.47 (s, 2H), 7.51 (t, J = 2.4 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.87 (dd, J =
1.6, 8.4 Hz, 1H), 8.50 (s, 1H), 8.72 (s, 1H); 13C NMR (400 MHz, CDCl3): δ 56.5, 60.8,
102.9, 105.6, 113.2, 119.2, 120.8, 121.3, 124.0, 131.3, 132.4, 135.9, 141.6, 153.6,
183.8, 186.8, 195.9, 197.3; Anal. Calcd. for C21H17NO5 + 0.6 H2O: C, 67.41; H,
4.90; N, 3.74. Found: C, 67.56; H, 4.77; N, 3.86.
4.2. Biology
4.2.1. cell lines
Human tumor cell lines CEM (leukemia), HepG2(liver cancer), MCF-7 (breast
cancer), SW-1990(pancreatic cancer) and HCT116 (colon adenocarcinoma) were from
the American Type Culture Collection. The human hepatoma cells Bel-7402 and
15

ACCEPTED MANUSCRIPT
SMMC-7721 were provided by the Cancer Institute of Chinese Academy of Medical
Sciences (Beijing, China). The cell lines were cultured in RPMI 1640 supplemented
with 10% heat inactivated fetal bovine serum, 100 units/mL penicillin, and 100
Ag/mL streptomycin. Cells (cultured in 5% CO2 at 37°C) in exponential growth were
used in all experiments.
4.2.2. Cytotoxicity assay
Cell viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay as described previously [29] . Briefly, cells were
seeded into the 96-well culture plates at 10000 per well followed by the treatment of
test compounds at 11 increasing concentrations between 0-10 ꢀM at 37 °C. After 72 h,
20 ꢀL of MTT PBS solution (5 mg/mL) was added into each well. After additional 4 h
incubation at 37 °C, the supernatants were removed gently and 150 ꢀL of DMSO was
added into each well. The plate was then incubated at room temperature for 10 min
with 300 rpm shaking and instantly read with a microplate reader (Molecular Device,
US) at wavelength of 590 nm with a reference filter of 620 nm. The IC50 values were
defined as 50% inhibition of cells growth compared with untreated controls,
calculated by regression analysis, and were determined in duplicates.
4.2.3. Cell cycle analysis
Cell cycle analysis was conducted followed a method reported previously [29].
Briefly, MCF-7 cells were plated in 6-well plates in growth medium at a density of
500,000 cells per well overnight, followed by treatment with 0.08 ꢀM of compound
CA4 and 0.02 ꢀM of compound 4n. After 24 h, cells were fixed in 70% ethanol
overnight and then treated with 50 ꢀg/mL propidium iodide containing RNase A for
0.5 h at room temperature. Stained cell samples were immediately subjected to an
analysis in a flow cytometer (Beckman Coulter). For each sample, 10,000 cells were
counted and the cell cycle phase distribution was calculated using EPICS software
(version 2.0).
Acknowledgments
We thank the National Science Foundation of the P. R. China (30901840) and
the Georgia State University Center of Biotechnology and Drug Design, Technology
Development Program for support of this research. The sponsors had no role in study
design; in collection, analysis and interpretation of data; in the writing of this report;
nor in the decision to publish.
References
[1] F. Pellegrini, D.R. Budman, Review: tubulin function, action of antitubulin drugs, and new
drug development, Cancer Invest. 23 (2005) 264-273.
[2] R. Romagnoli, P.G. Baraldi, A. Brancale, A. Ricci, E. Hamel, R. Bortolozzi, G. Basso, G. Viola,
Convergent synthesis and biological evaluation of 2-amino-4-(3',4',5'-trimethoxyphenyl)-5-
16

ACCEPTED MANUSCRIPT
aryl thiazoles as microtubule targeting agents, J. Med. Chem. 54 (2011) 5144-5153.
[3] R. Romagnoli, P.G. Baraldi, M.D. Carrion, C.L. Cara, O. Cruz-Lopez, M. Tolomeo, S.
Grimaudo, A. Di Cristina, M.R. Pipitone, J. Balzarini, N. Zonta, A. Brancale, E. Hamel,
Design, synthesis and structure-activity relationship of 2-(3',4',5'-trimethoxybenzoyl)-
benzo[b]furan derivatives as a novel class of inhibitors of tubulin polymerization, Bioorg.
Med. Chem. 17 (2009) 6862-6871.
[4] J.P. Liou, J.Y. Chang, C.W. Chang, C.Y. Chang, N. Mahindroo, F.M. Kuo, H.P. Hsieh,
Synthesis and structure-activity relationships of 3-aminobenzophenones as antimitotic agents,
J. Med. Chem. 47 ( 2004) 2897-2905.
[5] M.J. Lai, J.Y. Chang, H.Y. Lee, C.C. Kuo, M.H. Lin, H.P. Hsieh, C.Y. Chang, J.S. Wu, S.Y.
Wu, K.S. Shey, J.P. Liou, Synthesis and biological evaluation of 1-(4'-indolyl and
6'-quinolinyl) indoles as a new class of potent anticancer agents, Eur. J. Med. Chem. 46 (2011)
3623-3629.
[6] M. Sriram, J.J. Hall, N.C. Grohmann, T.E. Strecker, T. Wootton, A. Franken, M.L. Trawick,
K.G. Pinney, Design, synthesis and biological evaluation of dihydronaphthalene and
benzosuberene analogs of the combretastatins as inhibitors of tubulin polymerization in
cancer chemotherapy, Bioorg. Med. Chem. 16 (2008) 8161-8171.
[7] J. Kaffy, R. Pontikis, D. Carrez, A. Croisy, C. Monneret, J.C. Florent, Isoxazole-type
derivatives related to combretastatin A-4, synthesis and biological evaluation, Bioorg. Med.
Chem. 14 (2006) 4067-4077.
[8] C.Y. Nien, Y.C. Chen, C.C. Kuo, H.P. Hsieh, C.Y. Chang, J.S Wu, S.Y. Wu, J.P. Liou, J.Y.
Chang, 5-Amino-2-aroylquinolines as highly potent tubulin polymerization inhibitors, J. Med.
Chem. 53 (2010) 2309-2313.
[9] M. Marrelli, F. Conforti, G.A. Statti, X. Cachet, S. Michel, F. Tillequin, F. Menichini,
Biological potential and structure-activity relationships of most recently developed vascular
disrupting agents: an overview of new derivatives of natural combretastatin A-4, Curr. Med.
Chem. 18 (2011) 3035-3081.
[10]P.M. LoRusso, S.M. Gadgeel, A. Wozniak, A.J. Barge, H.K. Jones, Z.S. DelProposto, P.A.
DeLuca, J.L. Evelhoch, S.A. Boerner, C. Wheeler, Phase I clinical evaluation of ZD6126, a
novel vascular-targeting agent, in patients with solid tumors, Invest. New Drugs 26 (2008)
159-167.
[11]S. Ducki, D. Rennison, M. Woo, A. Kendall, J.F. Chabert, A.T. McGown, N.J. Lawrence,
Combretastatin-like chalcones as inhibitors of microtubule polymerization. Part 1: synthesis
and biological evaluation of antivascular activity, Bioorg. Med. Chem. 17 (2009) 7698-7710.
[12]R. Romagnoli, P.G. Baraldi, M.D. Carrion, O. Cruz-Lopez, M. Tolomeo, S. Grimaudo, A. Di
Cristina, M.R. Pipitone, J. Balzarini, A. Brancale, E. Hamel, Substituted
2-(3',4',5'-trimethoxybenzoyl)-benzo[b]thiophene derivatives as potent tubulin polymerization
inhibitors, Bioorg. Med. Chem. 18 (2010) 5114-5122.
[13]R. Romagnoli, P.G. Baraldi, M.K. Jung, M.A. Iaconinoto, M.D. Carrion, V. Remusat, D. Preti,
M.A. Tabrizi, F. Francesca, E. De Clercq, J. Balzarini, E. Hamel, Synthesis and preliminary
biological evaluation of new anti-tubulin agents containing different benzoheterocycles,
Bioorg. Med. Chem. Lett. 15 (2005) 4048-4052.
[14]R. Romagnoli, P.G. Baraldi, T. Sarkar, M.D. Carrion, O. Cruz-Lopez, C. Lopez Cara, M.
Tolomeo, S. Grimaudo, A. Di Cristina, M.R. Pipitone, J. Balzarini, R. Gambari, L. Ilaria, R.
17

ACCEPTED MANUSCRIPT
Saletti, A. Brancale, E. Hamel, Synthesis and biological evaluation of
2-(3',4',5'-trimethoxybenzoyl)-3-N,N-dimethylamino benzo[b]furan derivatives as inhibitors
of tubulin polymerization, Bioorg. Med. Chem. 16 (2008) 8419-8426.
[15]Z.Y. Liu, Y.M. Wang, Z.R. Li, J.D. Jiang, D.W. Boykin, Synthesis and anticancer activity of
novel 3,4-diarylthiazol-2(3H)-ones (imines), Bioorg. Med. Chem. Lett. 19 (2009) 5661-5664.
[16]M.K. Gurjar, R.D. Wakharkar, A.T. Singh, M. Jaggi, H.B. Borate, P.D. Shinde, R. Verma, P.
Rajendran, S. Dutt, G. Singh, V.K. Sanna, M.K. Singh, S.K. Srivastava, V.A. Mahajan, V.H.
Jadhav, K. Dutta, K. Krishnan, A. Chaudhary, S.K. Agarwal, R. Mukherjee, A.C. Burman,
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, J. Med. Chem. 50
(2007) 1744-1753.
[17]L.X. Hu, J.D. Jiang, J. Qu, Y. Li, J. Jin, Z.R. Li, D.W. Boykin, Novel potent antimitotic
heterocyclic ketones: synthesis, antiproliferative activity, and structure-activity relationships,
Bioorg. Med. Chem. Lett. 17 (2007) 3613-3617.
[18]M. Wu, Q. Sun, C. Yang, D. Chen, J. Ding, Y. Chen, L. Lin, Y. Xie, Synthesis and activity of
Combretastatin A-4 analogues: 1,2,3-thiadiazoles as potent antitumor agents, Bioorg. Med.
Chem. Lett. 17 (2007) 869-873.
[19]Z.Y. Liu, Z.R. Li, Y.X Li, G.F. Wang, J.D. Jiang, D.W. Boykin. Synthesis of Novel
1,5-Diaryl-1H-Pyrrole-2,3-Diones. Heterocycl. Commun. 15 (2009) 189-193.
[20]Z.Y. Liu, Z.R. Li, J.D. Jiang, D.W. Boykin, Synthesis of novel 4-(diarylmethylene)piperidines,
Heterocycl. Commun. 15 (2009) 401-405.
[21]S.A. Ivanovsky, M.V. Dorogov, D.V. Kravchenko, A.V. Ivachtchenko, Synthesis of the
substituted 3-cyclobutene-1,2-diones, Synthetic Commun. 37 (2007) 2527-2542.
[22]A.
Aguilar-Aguilar,
E.
Pena-Cabrera,
Selective
cross-couplings.
Sequential
Stille-Liebeskind/Srogl reactions of 3-chloro-4-arylthiocyclobutene-1,2-dione, Org. Lett. 9
(2007) 4163-4166.
[23]S. Combes, J.P. Finet, D. Siri, On the optical activity of the 3-aryl-4-hydroxycoumarin
isolated from Millettia griffoniana: molecular modelling and total synthesis, J. Chem. Soc.,
Perkin Trans. 1 1 (2002) 38-44.
[24]H. Suginome, M. Ishikawa, K. Yorita, N. Shimoyama, T. Sasaki, K.Orito, Photoinduced
molecular transformations. 157.
A new stereo- and regioselective synthesis of
2,6-diaryl-3,7-dioxabicyclo[3.3.0]octane lignans involving a. beta.-scission of alkoxyl radicals
as the key step. New total syntheses of (.+-.)-Sesamin, (.+-.)-Eudesmin, and (.+-.)-Yangambin,
J. Org. Chem. 60 (1995) 3052-3064.
[25]A. Aguilar-Aguilar, L.S. Liebeskind, E. Pena-Cabrera, Pd-catalyzed, Cu(I)-mediated
cross-couplings of bisarylthiocyclobutenediones with boronic acids and organostannanes, J.
Org. Chem. 72 (2007) 8539-8542.
[26]T. Ishiyama, M. Murata, N. Miyaura, Palladium(0)-catalyzed cross-coupling reaction of
alkoxydiboron with haloarenes: a direct procedure for arylboronic esters, J. Org. Chem. 60
(1995) 7508-7510.
[27]S. Yu, J. Saenz, J.K. Srirangam, Facile synthesis of N-aryl pyrroles via Cu(II)-mediated cross
coupling of electron deficient pyrroles and arylboronic acids, J. Org. Chem. 67 (2002)
1699-1702.
[28]A. S. Paraskar, A. Sudalai, Co-catalyzed reductive cyclization of azido and cyano substituted
18

ACCEPTED MANUSCRIPT
a,b-unsaturated esters with NaBH4:enantioselective synthesis of (R)-baclofen and
(R)-rolipram, Tetrahedron 62 (2006) 4907-4916.
[29]Y.M. Wang, L.X. Hu, Z.M. Liu, X.F. You, S.H. Zhang, J.R. Qu, Z.R. Li, Y. Li, W.J. Kong, H.W. He,
R.G. Shao, L.R. Zhang, Z.G. Peng, D.W. Boykin, J-D. Jiang, N-(2,6-dimethoxypyridine-3-yl)-
9-methylcarbazole-3-sulfonamide as a novel tubulin ligand against human cancer, Clin. Cancer Res.
14 (2008) 6218-6227.
19

ACCEPTED MANUSCRIPT
► Some novel 3,4-diaryl squaric acid analogues were synthesized. ► Compounds were tested for
cytotoxic activities against human tumour cell lines. ► Compounds
,
,
,
,
,
and
4r
4g 4k 4m 4n 4p 4q
exhibit strong activities against human leukemia cells with IC50 values of ≤20 nM. ► Compounds
,
4k
,
,
and showed potent activities against a panel of human tumor cell lines.
4r
4n 4p 4q