Synthesis and characterization of thiophene-derived amido bis-nitrogen mustard and its antimicrobial and anticancer activities

Article abstract of DOI:10.1002/cjoc.201100668

The thiophene-derived amido bis-nitrogen mustard N²,N ²,N⁵,N⁵-tetrakis(2-chloroethyl)-3,4- dimethylthiophene-2,5-dicarboxamide was designed and synthesized via five-step reactions from commercially available 2-chloroacetonitrile. This target compound was confirmed by ¹H NMR, ¹³C NMR, MS, IR spectra and elemental analyses, and its structure was further characterized by X-ray single-crystal analysis. The biological activities for the title compound and some intermediates were evaluated in vitro for their antibacterial, antifungal and cytotoxic activities. The preliminary results showed that the title compound could inhibit efficiently the growth of the tested microorganisms including drug-resistant bacteria MRSA to some extent. Moreover, the target compound was found to be effective against prostatic carcinoma cell line (PC-3), breast carcinoma cell line (MCF-7), colon carcinoma (LoVo) and lung cancer (A549). Especially, it gave selective antitumor efficacy against prostatic carcinoma cell line (PC-3) at a low dose.

Full text of DOI:10.1002/cjoc.201100668

FULL PAPER
DOI: 10.1002/cjoc.201100668
Synthesis and Characterization of Thiophene-derived Amido
Bis-nitrogen Mustard and Its Antimicrobial and Anticancer
Activities
Tang, Yidana(唐一丹)
Zhang, Jingqingb(张景勍)
Zhang, Shaolina(张少林)
Geng, Rongxia*,a(耿蓉霞)
Zhou, Chenghe*,a(周成合)
a Laboratory of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest
University, Chongqing 400715, China
b Chongqing Key Laboratory of Biochemical & Molecular Pharmacology, Medicine Engineering Research Center,
Chongqing Medical University, Chongqing 400016, China
The thiophene-derived amido bis-nitrogen mustard N2,N2,N5,N5-tetrakis(2-chloroethyl)-3,4-dimethylthio-
phene-2,5-dicarboxamide was designed and synthesized via five-step reactions from commercially available
1
2-chloroacetonitrile. This target compound was confirmed by H NMR, 13C NMR, MS, IR spectra and elemental
analyses, and its structure was further characterized by X-ray single-crystal analysis. The biological activities for
the title compound and some intermediates were evaluated in vitro for their antibacterial, antifungal and cytotoxic
activities. The preliminary results showed that the title compound could inhibit efficiently the growth of the tested
microorganisms including drug-resistant bacteria MRSA to some extent. Moreover, the target compound was found
to be effective against prostatic carcinoma cell line (PC-3), breast carcinoma cell line (MCF-7), colon carcinoma
(LoVo) and lung cancer (A549). Especially, it gave selective antitumor efficacy against prostatic carcinoma cell line
(PC-3) at a low dose.
Keywords nitrogen mustard, thiophene, antibacterial, antifungal, cytotoxicity
Introduction
tramustine Phosphate which contains an amido
N-mustard moiety has been used for the clinical treat-
ment of advanced prostatic carcinoma with low toxicity.
Nitrogen mustard (N-mustard) compounds with spe-
cial N(CH2CH2Cl)2 moiety have been paid considerable
attention in medicinal chemistry due to their extensively
potential bioactivities in anticancer,[1,2] antibacterial,[3]
antifungal[4] and agrochemical field,[5] as well as being
important intermediates for the syntheses of azole com-
pounds[6-8] which as predominant antimicrobial drugs
have been extensively used in the treatment of various
types of microbial infections in current prevalence of
pathogens.[9-18] Especially in cancer chemotherapy, they
have been playing important roles since the introduction
of Mechlorethamine 1 more than sixty years ago. Its
higher therapeutic index as clinical drug encouraged
much effort toward the research and development of
nitrogen mustard compounds including alkyl, aralkyl,
aryl, amide and metal supramolecular complex ones. So
far more than 70 nitrogen mustards such as aryl
N-mustards Chlorambucil 2, amide-type N-mustard
Cyclophosphamide 3, Estramustine Phosphate 4 and so
on (Figure 1) have been employed in clinical treatment
of various types of cancer diseases.[19] Especially, Es-
Cl
Cl
N
H3C N
COOH
Cl
2
1
Cl
H
H
Cl
Cl
O
H
O
N
N P NH
O
O
O
H
Cl
Cl
HO P O
OH
3
4
Figure 1 Some clinically used nitrogen mustard agents.
Though N-mustards as anticancer drugs acquired
significant therapeutic efficacy against malignant hu-
man tumors,[20] they exhibited toxicity to normal tissue,
lack of drug-specific affinity to tumor cells and drug
resistance in patients with relapsed cancers.[21-23] On the
other hand, in recent years a drastic increase in cancer
*
E-mail: zhouch@swu.edu.cn, geng0712@swu.edu.cn; Tel.: 0086-023-68254967; Fax: 0086-023-68254967
Received December 6, 2011; accepted June 13, 2012.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201200668 or from the author.
Chin. J. Chem. 2012, 30, 1831—1840
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Tang et al.
FULL PAPER
diseases has become a serious problem worldwide,
which caused the horrendous death of millions of people
at a horribly high rate. In spite of the fact that a large
number of innovative agents including the metal su-
pramolecular drugs[24-26] are available, these therapeu-
tics are still largely limited for clinical requirement be-
cause the survival rate of the malignant tumors is still
very low. Therefore, nitrogen mustards have still been
attracting great interest due to easy synthesis, lower cost,
high inherent chemical reactivity to tumor cells, clini-
cally extensive use with definite mechanism and good
therapeutic efficacy. A number of researches were con-
tinuously directed toward the development of nitrogen
mustards with low toxicity and better curative effects
ranging from earlier aliphatic,[27-28] aromatic[29] and
phosphamide[30] ones to recent DNA-targeting pyr-
roloamido alkylating agents.[31-33]
due to the dispersion of N atom electron atmosphere
density, which diminished alkylation ability through the
attenuation of the nitrogen alkality. Formylmerphalan
and Cyclophosphamide are one of the typical represen-
tatives of this class of compounds. Their successful
clinical use in the treatment of diverse types of human
cancers encouraged numerous medicinal chemists to
focus on the researches and developments of aromatic
and/or amide nitrogen mustards.[38-40] So far, various
kinds of aromatic and amido N-mustards as anticancer
drugs have been used in clinic, and played important
roles during the past decades in the unending struggles
against morbidity and mortality caused by cancer.
Herein, the conjugation of aryl/carbonyl moiety with
N-mustards was employed as an effective strategy for
the development of new N-mustards with potentially
high efficacy and low toxicity.
Aliphatic nitrogen mustards possess high chemical
reactivity toward tumor cells and thus exhibit good
anticancer activities, so a lot of researches focused on
this class of compounds including aralkyl N-mustards.
Recently, some aliphatic bis-nitrogen mustards which
have two sets of N-mustard groups N(CH2CH2Cl)2 and
four potential covalent sites bonding to DNA per mole-
cule, were designed and synthesized, and were found to
display superior therapeutic index to mono-nitrogen
mustards (Figure 2). Compounds 5a and 5b with two
N-mustard groups of equal size and locating in a bi-
sected half had proper log P and PSA constants which
could result in a high lipid solubility and cell membrane
permeability, and thus displayed more effective penetra-
tion of the blood-barrier and better cytotoxicity than
Mechlorethamine and Cyclophosphamide.[34] Recently,
some aralkyl bis-nitrogen mustards were also synthe-
sized,[35-37] and exhibited inhibition potential toward
microorganism and tumor cell. In this work, we would
like to investigate such bis-nitrogen mustard as target
compound and evaluate its biological activity.
Recently, much work showed that incorporation of
heterocycle with amido group into aromatic N-mustards
could effectively increase anticancer activities. Espe-
cially, the pyrroloamido group as typical example was
introduced into phenyl N-mustards to be capable of tar-
geting effectively DNA and to show good anticancer
activity in murine cancer.[41,42] Thiophene ring, as the
most prominent bioisostere of pyrrole in medicinal
chemistry, having been receiving great interest, was
often employed as an important building block and ex-
tensively introduced into target compounds in modern
drug design, and could significantly enhance biological
activities.[43] Especially, some of those derivatives con-
taining amido group possessed good inhibition potency
to tumor cell.[44] Several thiophene compounds such as
Teniposide and Raltitrexed have shown excellent anti-
tumor efficacy which were being used effectively in
clinical therapy. In view of this, we would like to in-
corporate thiophene ring, instead of the pyrrolyl moiety,
in combination with amido group into nitrogen mustards
to prepare the first thiophene-derived amido
bis-nitrogen mustard 12, and investigate its anticancer
activity.
Although nitrogen mustards as anticancer drugs have
been widely used in clinic and played important roles in
chemotherapy of various cancer diseases, as antimicro-
bial agents they have also been exploited in recent years
along with the drastic emergence of multiple-drug-re-
sistant organisms which evoked urgent need for new
antimicrobial drugs. It was found that some nitrogen
mustards with chloroethyl amine moiety showed
broad-spectrum antibacterial and antifungal activities
with significantly inhibitory efficiency.[6-8,38,45] Some
N-mustard derivatives including bis(2-bromoethyl)-
amine and their azole-substituted compounds such as
triazole, imidazole, benzimidazole ones and so on ex-
hibited good biological activities. For example, com-
pound 6 (Figure 3) with bromoethyl amine moiety dis-
played excellent activities toward the tested bacterial
strains including MRSA with MIC values ranging from
0.25 to 4 μg/mL, which were equivalent or even better
H3C
CH3
CH3
5c, n = 1, R =
Cl
Cl
Cl
Cl
H3C
N
R
N
n
5
5a, n = 0, R = CH2
5b, n = 4, R = CH2
5d, n = 1, R =
Figure 2 Aliphatic alkyl and aralkyl bis-nitrogen mustards.
Aliphatic nitrogen mustards showed good antitumor
activities accompanied with relatively high toxicity,
thus much effort has been directed toward the decrease
of their toxicities to normal cell. Extensive research
provided evidence that the introduction of some conju-
gated system such as aryl and/or carbonyl moiety into
nitrogen mustards could helpfully decrease the toxicity
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Chin. J. Chem. 2012, 30, 1831—1840

Synthesis and Characterization of Thiophene-derived Amido Bis-nitrogen Mustard
potency in comparison with the clinical drug Norflox-
acin. More importantly, this compound also exhibited
significant antifungal activities against Candida albi-
cans ATCC 76615 with MIC values of 0.25—2 μg/mL,
which were comparable to Fluconazole (MIC＝0.25
μg/mL). These results showed that nitrogen mustards
possessed large probability as new type of potential an-
timicrobial agents. Reasonably, we are interested in in-
vestigating the antimicrobial activity of the prepared
thiophene amido bis-nitrogen mustard 12 containing
four chloroethyl amine moieties.
(MCF-7), colon carcinoma (LoVo) and lung cancer
(A549), and screened its antibacterial and antifungal
activities in vitro. Furthermore, our previously obtained
results showed that antimicrobial activity strongly de-
pended on the water solubility[9-18] and it was found that
imidazole hydrochloride and triazolium as well as imi-
dazolium possessed good water solubility and resulted
in an increase of antimicrobial efficiency. Therefore, we
prepared the hydrochloride 13 and nitrate 14 from
compound 12 and investigated their biological activi-
ties.
X2
X1
Experimental
Br
Br
N
X3
Reagents and measurements
X1, X2, X3 = F, Cl
6
Melting points were determined using an X-6 melt-
ing point apparatus and are uncorrected. Infrared (IR)
spectra were recorded on a Bio-Rad FTS-85 (Bio-Rad,
Cambridge, MA, USA) using KBr disks in the 4000—
Figure 3 Analogs of nitrogen mustards.
400 cm－ region. NMR spectra were recorded using
1
The target thiophene-derived bis-nitrogen mustard
12 was conveniently synthesized through etherization of
chloroacetonitrile, cyclization by base catalysis, hy-
drolysis of nitrile, halogenation of dicarboxylic acid and
amidation reaction. The synthetic route is outlined in
Scheme 1. According to the achievements of above lit-
eratures, such target molecule with bis-nitrogen mustard
containing four bioactive chloroethylamino groups
might give stronger antitumor and antimicrobial activi-
ties. The conjugated system of thiophene ring with the
amido groups should be helpful for dispersing the N
atom electron density, and thereby make the title com-
pound 12 low toxic to normal cells and high cytotoxic to
tumor cells. Naturally, we evaluated its anticancer effi-
cacy against four tumor cells including prostatic
carcinoma cell line (PC-3), breast carcinoma cell line
TMS as an internal reference standard on a Bruker AV
300 spectrometer. The mass spectra were recorded on
LCMS-010A. Elemental analyses were carried out on a
Carlo Erba model EA 1106 elemental analyzer. TLC
analyses were done using silica gel plates; Column
chromatography was performed on silica gel (300—400
mesh) column. All chemicals and solvents were of AR
grade and, when necessary, were purified by standard
methods.
Synthesis of bis(cyanomethyl)sulfide (7)
A mixture of sodium sulfide nonahydrate (24.0 g,
0.1 mol), benzyltrimethylammonium chloride (1.0 g)
and chloroacetonitile (15.1 g, 0.2 mol) in the solvent of
water/dichloromethane (100 mL, 1/1, V/V) was stirred at
Scheme 1 Synthetic route of thiophene-derived amido bis-nitrogen mustards
H3C
O
CH3
O
H3C
CH3
(b)
(a)
(c)
(d)
Cl
NC
S
CN
Cl
HO
OH
NH2
N
S
S
7
N
O
9
8
H3C
CH3
O
(e)
H3C
CH3
O
Cl
Cl
Cl
S
(g)
N
N
O
10
S
H
H
O
N
Cl
Cl
N
(f)
12
HO
OH
Cl
Cl
11
(h)
(i)
H3C
CH3
Cl
Cl
H3C
CH3
Cl
Cl
N
N
2HCl
Cl
N
2HNO3
Cl
N
S
S
O
O
Cl
O
O
13
Cl
14
Reagents and conditions: (a) Na2S, CH2Cl2, H2O, benzyltrimethylammonium chloride, 0—5 ℃; (b) butane-2,3-dione, NaOCH3, 0—5 ℃; (c)
NaOH aqueous solution (8 mol/L), 100 °C, (d) hydrochloric acid (4 mol/L); (e) SOCl2; (f) SOCl2; (g) Et3N, CH2Cl2, 0—5 ℃; (h) hydrochloric
acid (4 mol/L), methanol; (i) nitric acid (4 mol/L), ethyl ether
Chin. J. Chem. 2012, 30, 1831—1840
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Tang et al.
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Synthesis of N2,N2,N5,N5-tetrakis(2-chloroethyl)-3,4-
0—5 ℃ under N2 protection. After 30 min the reac-
tants were stirred for 3 h at room temperature, and then
the organic layer was separated and dried over anhy-
drous sodium sulfate. Evaporation of the solvent and
recrystallization from methanol (5 mL) gave desired
bis(cyanomethyl)sulfide 7 as colorless crystal (8.0 g).
Yield 71.4%; m.p. 45—47 ℃ [lit.[46] 45—47 ℃].
dimethylthiophene-2,5-dicarboxamide (12)
To a suspension of thiophene-2,5-dicarbonyl dichlo-
ride 10 (1.2 g, 5.0 mmol) and bis(2-chloroethyl)amine
(1.8 g, 10.0 mmol) in dry dichloromethane (20 mL) was
added dropwise slowly triethylamine (5 mL) under stir-
ring at 0—5 ℃. The resultant mixture was warmed
gradually to room temperature, stirred for 30 h, and then
treated with ethyl acetate (50 mL). After filtration, the
filtrate was evaporated under reduced pressure and the
resulting residue was purified by silica gel column
chromatography using ethyl acetate/petroleum ether
(1/1, V/V) as eluent to afford the target compound 12 as
colorless crystal (1.2 g). Yield 53.6%; m.p. 104—105;
1H NMR (300 MHz, DMSO-d6) δ: 3.86—3.82 (t, J＝6
Hz, 8H, CH2Cl), 3.71 (bs, 8H, CONCH2), 2.17 (s, 6H,
CH3); 13C NMR (75 MHz, DMSO-d6) δ: 161.70 (C＝O),
146.76 (thienyl 2-C), 129.90 (thienyl 3-C), 48.60
(NCH2), 45.80 (CH2Cl); IR (KBr) ν: 2977, 2918, 2863
Synthesis of 5-cyano-3,4-dimeth-ylthiophene-2-car-
boxamide (8)
To a solution of bis(cyanomethyl)sulfide 7 (11.2 g,
0.1 mol) and butane-2,3-dione (17.2 g, 0.2 mol) in
methanol (50 mL) was added quickly 1 mol/L sodium
methoxide solution in methanol (200 mL, 0.2 mol). The
mixture was stirred for 15 min at 0—5 ℃, then water
(20 mL) was added to quench the reaction, and metha-
nol was removed by evaporation under vacuum to give
a brown solid. The crude product was washed with wa-
ter to afford the desired compound 8 as white solid
(16.9 g). Yield 94.2%; m.p. 192—194 ℃ [lit.[46] 192—
193 ℃].
－
1
(CH3, CH2), 1628 (C＝O), 1216 (C—N), 1152 cm ;
MS m/z: 449 [M＋H]＋. Anal. calcd for C16H22Cl4N2O2S:
C 42.87, H 4.95, N 6.25; found C 42.90, H 4.91, N 6.28.
Synthesis of 3,4-dimethyl thiophene-2,5-dicarboxylic
acid (9)
Synthesis of N2,N2,N5,N5-tetrakis(2-chloroethyl)-3,4-
dimethylthiophene-2,5-dicarboxamide hydrochloride
(13)
Compound 8 (18.0 g, 0.1 mol) in aqueous sodium
hydroxide (100 mL, 8 mol/L) was refluxed for 48 h,
then the reaction mixture was cooled and the pH value
was adjusted to 3—4 with 0.1 mol/L hydrochloric acid.
The resulting precipitate was collected, washed with
water and dried in oven at 120 ℃ to afford product 9
as white solid (19.6 g). Yield 98.0%; m.p. 325—326 ℃
To a stirring solution of compound 12 (90 mg, 0.2
mmol) in methanol (2 mL) was added hydrochloric acid
(4 mol/L, 2 mL) dropwise at room temperature. The
reactants were stirred for 24 h at 40 ℃. After the reac-
tion came to the end (monitored by TLC, eluent, ethyl
acetate/petroleum ether, 1/2, V/V), the excess hydro-
chloric acid was distilled off under reduced pressure and
the residue was recrystallized from acetone to afford
compound 13 (91 mg) as colorless crystal. Yield 86.4%;
1
[CAS NO. 19799-13-4: 327—328 ℃]; H NMR (300
MHz, DMSO-d6) δ: 9.43 (s, 2H, COOH), 2.26 (s, 6H,
CH3); IR (KBr) ν: 2927, 1747 (C＝O), 1419, 1375,
－
＋
＋
1
1174 cm ; MS m/z: 200 [M] , 155 [M－45] .
1
m.p. 213—216 ℃; H NMR (300 MHz, DMSO-d6) δ:
Synthesis of 3,4-dimethyl thiophene-2,5-dicarbonyl
dichloride (10)
4.59 (bs, 8H, CH2Cl), 4.01—3.97 (t, J＝6 Hz, 8H,
CONCH2), 2.53 (s, 6H, CH3); 13C NMR (75 MHz,
DMSO-d6) δ: 165.39 (C＝O), 146.27 (thienyl 2-C),
130.96 (thienyl 3-C), 48.53 (NCH2), 45.90 (CH2Cl); IR
(KBr) ν: 2957, 2853 (CH3, CH2), 1723 (C＝O), 1250
(C—N), 1154 cm ; MS m/z: 449 [M＋H－2HCl] .
Anal. calcd for C16H24Cl6N2O2S: C 36.87, H 4.64, N
5.38; found C 36.74, H 4.68, N 5.35.
Compound 3 (2.0 g, 10.0 mol) in thionyl chloride
(10 mL) was refluxed for 6 h and the surplus thionyl
chloride was distilled off. Recrystallization of the crude
product from petroleum ether (60—90 ℃) gave acyl
chloride 10 as white solid (1.5 g). Yield 64.7%; m.p. 78
－
＋
1
—79 ℃; IR (KBr) ν: 2959, 2853 (CH3), 1754 (C＝O),
－
1
1
Synthesis of N2,N2,N5,N5-tetrakis(2-chloroethyl)-3,4-
dimethylthiophene-2,5-dicarboxamide nitrate (14)
1203, 1171, 917 cm ; H NMR (300 MHz, DMSO-d6)
δ: 2.42 (s, 6H, CH3); MS m/z: 237 [M]＋.
To a stirring solution of compound 12 (90 mg, 0.2
mmol) in ethyl ether (5 mL) and chloroform (5 mL) was
added nitric acid (4 mol/L, 1 mL) dropwise at room
temperature. The reactant mixture was stirred for 24 h at
30 ℃. After the reaction came to the end (monitored by
TLC, eluent, ethyl acetate/petroleum ether, 1/2, V/V),
the excess nitric acid was distilled off under reduced
pressure and the residue was recrystallized from acetone
to afford compound 14 (79 mg) as colorless crystal.
Synthesis of bis(2-chloro ethyl)amine hydrochloride
(11)
To a stirring solution of diethanolamine (21.0 g, 0.2
mol) in chloroform (20 mL) was added thionyl chloride
(25 mL) dropwise in ice-bath. The reactants were stirred
for 1 h at room temperature and then refluxed overnight.
The excess thionyl chloride was distilled off to afford
crude product. The further recrystallization of the crude
product from acetone gave bis(2-chloroethyl)amine hy-
drochloride 11 as colorless crystal (23.2 g). Yield 81.7%;
m.p. 216—218 ℃ [lit.[46] 216—217 ℃].
1
Yield 69.3%; m.p. 216—218 ℃; H NMR (300 MHz,
DMSO-d6) δ: 4.55 (bs, 8H, CH2Cl), 3.80 (bs, 8H,
CONCH2), 2.52 (s, 6H, CH3); 13C NMR (75 MHz,
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Synthesis and Characterization of Thiophene-derived Amido Bis-nitrogen Mustard
DMSO-d6) δ: 165.42 (C＝O), 146.27 (thienyl 2-C),
130.95 (thienyl 3-C), 48.59 (NCH2), 45.84 (CH2Cl); IR
(KBr) ν: 2964, 2877 (CH3, CH2), 1713 (C＝O), 1255
(C—N), 1157 cm ; MS m/z: 449 [M＋H－2HNO3] .
Anal. calcd for C16H24Cl4N4O8S: C 33.46, H 4.21, N
9.76; found C 33.52, H 4.18, N 9.72.
rial growth, a control test was performed with test me-
dium supplemented with DMSO at the same dilutions as
used in the experiment. The growth was monitored
visually and spectrophotometrically. The lowest con-
centration (highest dilution) required to arrest the
growth of bacteria was regarded as MIC. The MICs
were summarized in Table 5.
－
＋
1
X-ray crystallography
The compounds were evaluated for their antifungal
activities against C. albicans and S. cerevisiae. A spore
suspension in sterile distilled water was prepared from 1
day old culture of the fungi growing on Sabouraud agar
(SA) media. The final spore concentration was 1—5×
A single crystal of the title compound with dimen-
sions of 0.25 mm×0.22 mm×0.20 mm was chosen for
X-ray diffraction study. The data were collected on
Bruker APEXII diffractometer equipped with graphite-
monochromated Mo Kα (λ＝0.71073 Å) radiation by
using a ω-2θ scan mode in the range of 1.9°＜θ＜26.0°
at 293(2) K. A total of 13412 reflections were collected
with 4008 independent ones (Rint＝0.018), of which
3342 observed reflections with I＞2σ(I) were used in
the structure solution and refinement. The final R＝
－
1
103 spore•mL . From the stock solutions of the tested
compounds and reference antifungal drug Fluconazole,
dilutions in sterile RPMI 1640 medium (Neuronbc La-
boraton Technology Co., Ltd, Beijing, China) were
made resulting in eleven wanted concentrations of each
tested compounds. 100 μL of each fungus culture with
equal volume of each concentration of tested com-
pounds dilutions were added to each well. These dilu-
tions were inoculated and incubated at 35 ℃ for 24 h.
The growth was monitored visually and spectrophoto-
metrically. The lowest concentration (highest dilution)
required to arrest the growth of fungi was regarded as
MIC. The MICs were summarized in Table 5.
2
0.042 and wR＝0.126 (w＝1/[σ2(Fo )＋(0.0694P)2＋
2
1.0128P], where P＝(Fo ＋2Fc2)/3, S＝1.04, (Δ/σ)max＜
－
3
0.001, Δρmax＝0.88 and Δρmin＝－0.63 e/Å ). Cell re-
finement and data reduction were carried out using
SAINT software (Bruker AXS Inc., Madison, Wiscon-
sin, USA). The structure of the target compound was
solved by directed methods using the program
SHELXS97 and refined on F2 by full-matrix
least-squares techniques (SHELXL97).[48] The hydrogen
atoms were placed by calculation and refined using rid-
ing model. Details of crystal data including selected
bond lengths and bond angles were given in Table 4,
hydrogen bonds were given in Table 3, the ORTEP
drawing of the molecule was shown in Figure 5 and the
molecular packing was shown in Figure 6.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra zolium
bromide (MTT) assay for cytotoxicity
The human prostatic carcinoma cell line (PC-3),
breast carcinoma cell line (MCF-7), colon carcinoma
(LoVo) and lung cancer (A549) were purchased from
the Chongqing Medical University, China. Ham’s F12
Nutrient Mixture, enriched with 10% heat inactivated
foetal bovine serum (FBS) and 1% of penicillin-strepto-
mycin was used for cell cultivation and to perform the
tests. The cytotoxicity was investigated using the MTT
assay. Stock solution (100 mmol/L) of test compound
12 was prepared in DMSO and stored at －20 ℃ prior
to dilution into each tested concentration for the bio-
logical assay. Cell suspensions were diluted to 105
cells/mL, suitably prepared and distributed in plates of
culture with 96 wells (100 μL in each well), 100 μL of
each tested concentration was added to each well. The
plate was incubated at 37 ℃ for 48 h. Then, 25 μL of
MTT solution (5 mg/mL) was added to each well, and
the mixture was incubated at 37 ℃ for another 4 h. At
the end of this period, the culture medium with the MTT
excess was aspirated and after that, 100 μL of DMSO
was added to each well to dissolve the formazan crystals.
The optical density (OD) of the wells was measured at
490 nm. Results were evaluated by comparing the ab-
sorbance of the wells containing compound treated cells
with the absorbance of wells containing 0.1% DMSO
alone (solvent control). Conventionally, cell growth
inhibition can be defined as
Antibacterial and antifungal test
The in vitro minimal inhibitory concentrations
(MICs) of tested compounds were determined by the
micro-broth dilution method in 96-well microtest
plates[49,50] according to the method recommended by
the National Committee for Clinical Laboratory Stan-
dards (NCCLS). Chloramphenicol and Fluconazole
were used as standard drugs. Initially the tested com-
pounds 7, 8, 9, 12, 13 and 14 were dissolved in DMSO
to prepare the stock solution, and then the test com-
pounds and reference drugs were prepared in Mueller-
Hinton broth (Guangdong Huaikai Microbial Sci. &
Tech Co., Ltd, Guangzhou, Guangdong, China) by
twofold serial dilution to give the required concentra-
tions.
The prepared compounds were evaluated for their
antibacterial activity against S. aureus, MRSA, B. sub-
tilis, M. luteus as Gram-positive bacteria, E. coli, P.
vulgaris, S. typhi, P. aeruginosa as Gram-negative bac-
teria. The bacterial suspension was adjusted with sterile
saline to a concentration of 1×105 CFU/mL. 100 μL of
each bacterial culture with equal volume of each con-
centration of tested compounds dilutions were added to
each well. These dilutions were inoculated at 37 ℃ for
24 h. To ensure that the solvent had no effect on bacte-
Cell growth inhibition ＝ (ODcell
－
negative control
ODcompound control)/(ODcell negative control－ODblank)×100%
All assays were performed in triplicate and mean ±
Chin. J. Chem. 2012, 30, 1831—1840
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
1835

Tang et al.
FULL PAPER
SD value was used to estimate cell growth inhibition
rate. The cell growth inhibition rates were summarized
in Table 6 and Figure 7.
MS, IR and NMR spectra. The spectral data were con-
sistent with the assigned structures and listed in the ex-
perimental section. The MS spectra of the compounds
showed [M＋H－2HCl]＋, [M＋H－2HNO3]＋, [M＋H]＋
or [M]＋ peaks, in agreement with their corresponding
molecular formulas.
Results and Discussion
Synthesis
In IR spectra, intermediate 9 gave broad absorption
bands at 3000—2500 cm−1 which suggested the pres-
ence of carboxyl group. Another strong absorption at
1747 cm−1 should be attributed to the stretching vibra-
tion of C＝O group. The disappearance of the charac-
teristic COOH absorption in compound 10 suggested
that the carboxyl group underwent chlorination with
thionyl chloride. Moreover, the C＝O group in com-
pound 10 appeared with strong absorption at higher
frequency in comparison with carboxyl one owing to the
electrophilic inductive effect of chlorine moiety. How-
ever, the vibration frequency of C＝O group in target
compound 12 was greatly shifted to lower wave number
due to the presence of strong electron-donating effect of
N(CH2CH2Cl)2 moiety which resulted in the increase of
carbonyl electron atmosphere density. Moreover, it was
found that the carbonyl vibration frequency in hydro-
chloride 13 and nitrate 14 was shifted to a higher wave
number in contrast to compound 12, which was mainly
responsible for inductive effects of the positive charge
on N atom in amide moiety. The characteristic absorp-
tion of C＝O group was given in Table 1.
The target thiophene bis-nitrogen mustard 12 was
prepared starting from commercial chloroacetonitrile.
Chloroacetonitrile easily reacted with sodium sulfide in
the mixture solvent of dichloromethane and water using
benzyltrimethylammonium chloride as catalyst to pro-
duce bis-cyanomethyl sulfide 7 as white solid in good
yield via a slightly improved procedure that is to per-
form this reaction under N2 protection which could ef-
fectively avoid the oxidation of thioether 7. The yield
for compound 7 in our experiment was up to 72% which
is much higher than the literature yield 54%.[46] The
cyclization of thioether 7 with butane-2,3-dione in the
presence of sodium methoxide formed the intermediate
5-cyano-3,4-dimethylthiophene-2-carboxamide 8, which
was prepared according to a known procedure with a
little improvement. The difference between the litera-
ture[47] and this improved method is to add some water
into the cyclization reaction mixture in the organic
phase before evaporating the solvent, this could effi-
ciently avoid the formation of byproducts at a higher
temperature, and produce pure product as white solid
without further recrystallization. Compound 8 was hy-
drolyzed by aqueous solution of sodium hydrate, acidi-
fied by hydrochloric acid, washed with water and
methanol consecutively, and then dried in oven to yield
3,4-dimethylthiophene-2,5-dicarboxylic acid 9 as pow-
der. For the hydrolysis of compound 8, a suitable con-
centration of the sodium hydrate should be 20%—30%,
neither higher nor lower concentration was favorable for
the formation of compound 9. The treatment of com-
pound 9 with fresh thionyl chloride conveniently and
efficiently produced the 3,4-dimethylthiophene-2,5-
dicarbonyl dichloride 10 with high purity after recrys-
tallization from petroleum ether. The thiophene acyl
chloride 10 was unstable in moist air because it is very
sensitive to water due to hydrolysis of acyl chloride, so
it is better to be used immediately for the next step reac-
tion. The amidation of thiophene dicarbonyl dichloride
10 with bis(2-chloroethyl)amine hydrochloride 11 sus-
pended in methylene chloride using triethylamine as
catalyst at 0 ℃ readily produced the target compound
12 in moderate yield after chromatographic purification
on silica gel. It is better to control the reactant ratio for
compound 10 and bis(2-chloroethyl)amine at 1∶2 be-
cause the excess bis(2-chloroethyl)amine would react
with itself in the alkalinity condition. Additionally, this
reaction should be performed under N2 protection be-
cause of the sensitivity of reactants 10 and 11 to mois-
ture which resulted in low yield of title compound.
Table 1 Some typical spectral data for compounds 9—10 and
12—14
IR/cm－
1H NMR δ
13C NMR δ
C＝O
—
1
Compd.
C＝O Thiophene-CH3 CH2Cl NCH2
9
1747
1754
1628
1723
1713
2.26
2.42
2.17
2.53
2.52
—
—
—
—
10
12
13
14
—
3.84 3.71
4.59 3.99
4.55 3.90
161.70
165.39
165.22
The 13C NMR spectra showed peaks at δ 161.70—
165.39 which were assigned to the C＝O groups in
compounds 12 —14. An obviously downfield shift
(δ 3.52—3.69) for the carbon signal of amide groups in
compounds 13 and 14 was observed in contrast to com-
pound 12 as a result of the strong electron withdrawing
character of positively charged amide group, as seen in
Table 1.
The CH3 protons in compounds 9—10 and 12—14
1
all gave downfield H NMR signals as singlet varying
from δ 2.17 to 2.53 due to the formation of large conju-
gation system between thienyl ring and C＝O moiety
which resulted in the enhancement of deshielding for
methyl protons. The chemical shifts of CH3 groups in-
creased in the order 12＜9＜10＜13≈14 with the in-
crease of electron-withdrawing inductive effects of car-
bonyl moieties in the order CONR1R2＜COOH＜COCl
Analysis of spectra
The synthesized compounds were characterized by
1836
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 1831—1840

Synthesis and Characterization of Thiophene-derived Amido Bis-nitrogen Mustard
＜(CONR1R2).
amide groups adopted trans-conformation arrangement
compared with the central thienyl ring so the four ter-
minal 2-chloroethyl arms are oriented in the different
orientation. As indicated in Figure 6, in the solid state,
these molecules are bonded together with Cl…H—C
hydrogen bonds into an H-bonding-driven three-dimen-
sional network, corresponding O(7)…H(3 A), O(7)…
O(3), and O(7)…H(3A)—O(3) data are 2.33 Å, 3.19 Å
and 145.1°, respectively. The H-bonding data showed
that the O and Cl atoms could form week H-bonding
effects among the molecules which could play impor-
tant roles in the chemical and biological activity.
O1
Cl4
Cd
Ha'
Ca
Hd'
Cl1
Hd
Ha
Hb
S
N
Hc
N
Cc
Cb
Cl3
Hc'
Hb'
Cl2
O2
Figure 4 The molecular structure of compound 12.
The methylene protons in the hydrochloride 13 and
nitrate 14 displayed downfield shifts in the range of
δ 0.20—0.77 in comparison with the corresponding
compound 12 due to the positive charge on the amido
group which could obviously affect on the chemical
shifts of the neighboring groups. Surprisingly, the me-
thylene protons of compounds 12—14 gave abnormal
splits instead of normal triplet at the integration ratio of
1∶2∶1. This fact would be partially attributed to the
concentrations of tested compounds. As seen in Figure 4,
the C—N bond in amide group possessed partially dou-
ble bond property and thus was fixed. The chemical
environment of Ca atom was not equal to Cb atom.
Therefore, the electrophilic inductive effects of O and
Cl atoms to the Ha and Hb atoms were different, result-
ing from the different distances between them. In order
to further clarify the steric effect of the target compound,
the minimize energy calculation of compound 12 was
carried out and it showed that O atom has different dis-
tances with the four protons. For O1—Ha and O2—Hc
the distance was 2.421—2.734 Å, and for O1—Hb and
O2—Hd the distance was 3.769—3.941 Å. But the Cl—
H distance was in a wide range because the CH2Cl
group could revolve freely, for this reason and in order
to confirm the structure, the target compound was crys-
tallized to afford single crystal and further characterized
by X-ray diffraction analysis. The calculative and actual
bond lengths were listed in Table 2.
Figure 5 ORTEP of compound 12 at 50% probability.
Figure 6 Packing of compound 12 along the a axis. The dashed
lines represent the intermolecular hydrogen bonds.
Table 2 The calculative length and actual length of atom O on
the four methylene groups
Bond
Calculative length/Å
Actual length/Å
2.516
Table 3 Hydrogen-bond geometry [Å and (°)]
O(1)—H(a)
O(1)—H(b)
O(2)—H(c)
O(2)—H(d)
2.734
3.874
2.421
3.941
D—H…A
D—H H…A D…A D—H…A
C(14)—H(14)B…Oi
C(14)—H(14)B…Clii
C(6)—H(6)B…O1iii
C(5)—H(5)B…O1iv
0.97 2.45 3.257(3)
0.97 2.80 3.632(3)
0.96 2.54 3.474(3)
0.96 2.54 3.477(3)
141
145
166
165
3.769
2.533
3.800
Single-crystal structure of target compound 12
Symmetry codes: (i) x, －y＋1/2; z－1/2; (ii) x－1, y, z－1; (iii)
－x＋1, －y, －z＋1; (iv) x＋1, y, z.
A crystal of compound 12 suitable for X-ray analysis
was grown from a mixture solution of ethyl acetate and
petroleum ether by slow evaporation at room tempera-
ture. An ORTEP drawing of 12 was given in Figure 5
while Figure 6 depicted the unit cell packing. The
structure belongs to monoclinic system with space
group P21/c: a＝7.9238 (4) Å, b＝21.1712 (11) Å, c＝
12.6186 (7) Å, β＝99.2380 (10)° and Z＝4. The struc-
tural arrangement of compound 12 shows that the two
Pharmacology
Compounds 7—9 and 12—14 were evaluated for
their antibacterial activities against Gram-positive and
negative bacteria, including S. aureus, MRSA, B. sub-
tilis, M. luteus, E. coli, P. vulgaris, P. aeruginosa, S.
typh as well as antifungal activities against C. albicans,
S. cerevisiae by two fold serial dilution method. The
Chin. J. Chem. 2012, 30, 1831—1840
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1837

Tang et al.
FULL PAPER
activities of the synthesized compounds were compared
with standard drugs Chloramphenicol and Fluconazole.
The antimicrobial activities of the tested compounds
were reported as MICs (mmol/L), and were shown in
Table 5.
to compound 12 with lower MIC value of 0.07 mmol/L
which was comparable to the clinical drug Chloram-
phenicol (MIC 0.012 mmol/L) to some extent.
Unexpectedly, although compounds 13 and 14 had a
better water solubility than compound 12, they were less
active against the tested microorganisms especially the
Gram-negative one. This result was different from our
previous work.[9-18] Probably, this was due to the fact
that penetration of hydrochloride and nitrate into the cell
was more difficult for microorganisms which have a
more lipophilic cell wall.[51]
Cytotoxicity The cytotoxicity of the title com-
pound was determined in vitro on a panel against four
human cancer cell lines derived from different cancer
types including human prostatic carcinoma cell line
(PC-3), colon carcinoma (LoVo), lung cancer (A549)
and breast carcinoma cell line (MCF-7), using
Fluorouracil as standard drugs. The results were shown
in Table 6 and Figure 7.
As shown in Figure 7, the preliminary cytotoxicities
showed that the target compound 12 demonstrated poor
to moderate antiproliferative effect on all tested cell
lines. Figure 7 clearly demonstrated that human
prostatic carcinoma cell line (PC-3) was more sensitive
to the title compound 12 than other three tested tumor
cells: lung cancer (A549), colon carcinoma (LoVo) and
breast carcinoma cell line (MCF-7). Among all tested
cells, compound 12 toward human prostatic carcinoma
cell line (PC-3) was the most active, while the lowest
inhibitory efficiency was against breast carcinoma cell
Antibacterial and antifungal activities The an-
timicrobial assay revealed that the target compound 12
could inhibit effectively the growth of tested bacteria
and fungi including the drug-resistant strain MRSA,
whereas the intermediates 7 and 9 were not sensitive to
all the tested microorganisms even at higher concentra-
tions. Surprisingly, compound 8 with amido and cyano
groups showed better antibacterial and antifungal effi-
ciency than the other two intermediates and gave a
lower MIC value of 1.75 mmol/L against the tested
bacteria strains that is 2—3 folds active than compounds
7 and 9. It was noteworthy that the biological activities
decreased in the order of the target compounds 12＞
hydrochloride 13 and nitrate 14＞amdio thiophene 8＞
carboxyl thiophene 9. Thioether 7 with no thiophene
ring was the most inactive one. These facts suggested
that the amido residue, heterocyclic thienyl ring and
chloroethylamino moiety exert the synergistic effect on
biological activity.
Compared with the referenced drugs chlorampheni-
col and fluconazole in clinic, Table 5 showed that the
target compound 12 exhibited poor to moderate inhibi-
tory activities in vitro against most of the tested
Gram-positive and Gram-negative bacteria as well as
fungi. Notably, Proteus vulgaris was the most sensitive
Table 4 Selected bond lengths (Å) and bond angles (°) for the target compound 12
Length Bond Length Bond
1.717(2) C(15)—N(1) 1.469(3) C(2)—C(7)
Bond
Length
1.494(3)
1.440(3)
1.497(3)
1.504(3)
1.505(4)
1.490(4)
1.498(3)
1.514(4)
C(1)—S(1)
C(2)—S(1)
C(7)—O(1)
C(8)—O(2)
C(7)—N(1)
C(8)—N(2)
C(9)—N(2)
C(11)—N(2)
C(13)—N(1)
1.724(2)
1.231(3)
1.227(3)
1.350(3)
1.350(3)
1.465(3)
1.468(3)
1.460(3)
C(10)—Cl(2)
C(12)—Cl(1)
C(14)—Cl(4)
C(16)—Cl(3)
C(1)—C(3)
C(1)—C(8)
C(2)—C(4)
1.784(3)
1.766(4)
1.786(3)
1.777(3)
1.366(3)
1.502(3)
1.365(3)
C(3)—C(4)
C(3)—C(5)
C(4)—C(6)
C(9)—C(10)
C(11)—C(12)
C(13)—C(14)
C(15)—C(16)
Bond
Angel
121.0(2)
119.5(2)
119.6(2)
122.0(2)
120.3(2)
117.7(2)
110.34(19)
110.00(19)
113.7(2)
112.64(19)
110.22(18)
Bond
Angel
Bond
Angel
112.36(17)
116.12(17)
111.6(2)
124.5(2)
123.9(2)
112.1(2)
125.2(2)
122.7(2)
112.3(3)
114.0(2)
110.80(15)
O(1)-C(7)-N(1)
O(1)-C(7)-C(2)
N(1)-C(7)-C(2)
O(2)-C(8)-N(2)
O(2)-C(8-C(1)
N(2)-C(8)-C(1)
N(2)-C(9)-C(10)
C(9)-C(10)-Cl(2)
N(2)-C(11)-C(12)
N(1)-C(15)-C(16)
C(15)-C(16)-Cl(3)
C(7)-N(1)-C(13)
C(7)-N(1)-C(15)
C(13)-N(1)-C(15)
C(8)-N(2)-C(9)
C(8)-N(2)-C(11)
C(9)-N(2)-C(11)
C(1)-S(1)-C(2)
C(3)-C(1)-C(8)
C(3)-C(1)-S(1)
C(8)-C(1)-S(1)
C(4)-C(2)-C(7)
124.28(19)
117.58(19)
117.75(17)
123.80(19)
118.4(2)
C(4)-C(2)-S(1)
C(7)-C(2)-S(1)
C(1)-C(3)-C(4)
C(1)-C(3)-C(5)
C(4)-C(3)-C(5)
C(2)-C(4)-C(3)
C(2)-C(4)-C(6)
C(3)-C(4)-C(6)
C(11)-C(12)-Cl(1)
N(1)-C(13)-C(14)
C(13)-C(14)-Cl(4)
117.63(19)
91.11(11)
127.6(2)
112.81(17)
119.44(16)
131.1(2)
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Chin. J. Chem. 2012, 30, 1831—1840

Synthesis and Characterization of Thiophene-derived Amido Bis-nitrogen Mustard
Table 5 Antibacterial and antifungal activities in vitro of thiophene derivatives expressed as MIC (mmol/L)a,b
Gram-positive bacteria
Gram-negtive bacteria
Fungi
Compd.
S. aureus MRSA B. subtilis M. luteus E. coli P. vulgaris S. typhi P. aeruginosa C. albicans S. cerevisiae
7
＞4.5c
＞4.5
＞4.5
＞1.75
2.56
＞4.5
1.75
2.56
0.14
＞4.5
1.75
2.56
0.07
＞4.5
1.75
2.56
0.56
0.99
0.89
0.012
—
＞4.5
＞1.75
＞2.56
0.14
＞4.5
1.75
＞4.5
＞1.75
＞2.56
0.14
＞4.5
＞1.75
＞2.56
0.14
1.75
1.75
8
＞2.56 ＞2.56
2.56
9
0.28
0.49
0.44
0.012
—
0.14
＞0.99
0.89
0.56
0.28
12
13
0.12
＞0.99 ＞0.99
＞0.99
＞0.89
0.024
—
＞0.99
0.89
＞0.99
＞0.89
—
＞0.99
0.89
0.44
0.89
0.024
—
＞0.89
0.012
—
14
Chloramphenicol
0.096
0.096
0.048
—
Fluconazole
—
—
—
0.0033
0.013
a Minimum inhibitory concentrations were determined by micro broth dilution method. b S. aureus, Staphylococcus aureus (ATCC 25923);
MRSA, Staphylococcus aureus (N315); B. subtilis, Bacillus subtilis (ATCC 6633); M. luteus, Micrococcus luteus (ATCC 4698); E. coli,
Escherichia coli (ATCC 25922); P. vulgaris, Proteus vulgaris (ATCC 6896); S. typhi, Salmonella typhi (ATCC 9484); P. aeruginosa,
Pseudomonas aeruginosa; C. albicans, Candida albicans (ATCC 76615); S. cerevisiae, Saccharomyces cerevisiae (ATCC 9763).
36.2% and 38.6%, respectively. Meanwhile, for lower
tested concentrations, 3.12 μmol/L (1.4 μg/mL), 6.25
μmol/L (2.8 μg/mL) and 12.5 μmol/L (5.6 μg/mL), the
average growth inhibition activities against the tumor
cells were 31.9%, 31.2% and 32.3% respectively. It is
clearly observed that these tumor cell growth inhibition
rates were slightly changed for each tested concentra-
tion and compound 12 did not show obviously
dose-dependent antiproliferative property. However, as
shown in Table 6, the other three tested tumor cells,
colon carcinoma (LoVo), lung cancer (A549) and breast
carcinoma cell line (MCF-7) were not sensitive to the
target mustard in comparison to the clinically used
Fluorouracil and the cell growth inhibition percentages
were 11.07%, 11.14% and 3.92% to each cell line at 25
μmol/L. The cell growth inhibition rate did not change
obviously along with different dose used as shown in
Figure 7.
Table 6 In vitro cytotoxic activities of compound 12 compared
with Fluorouacil
Percentage growth inhibition (%)
Compd.
at 25 μmol/L in different cell lines
PC-3
33.61*, #
50.17*
LoVo
11.07#
51.63*
A549
15.03*, #
34.44*
MCF-7
6.09#
12
5-Fluorouracil
47.67*
p ＜0.01 means statistically significantly difference between
treatment and control as evaluated by Student's t-test. * p＜0.01,
compared with the cell negative group (negative control); # p＜
0.01, compared with the cell group treated with fluorouacil (posi-
tive control).
In conclusion, the thiophene-derived amido
bis-nitrogen mustard exhibited poor to moderate cyto-
toxic activities against all the tested tumor cell lines. In
particular, PC-3 cell line was the most sensitive to the
target mustard among the four tested tumor cells. Com-
pound 12 exerted a higher cytotoxic activity against
PC-3 tumor cell at a lower dose which indicated that
compound 12 with a thiophene core and two amide ni-
trogen mustard moieties could be a new and potential
scaffold for the development of novel anticancer agents.
Furthermore, whether or not the target mustard 12
show dose-dependent response pattern, this compound
might act as DNA groove binder due to its special
structure making the cell DNA a dominant target for
antiproliferative activity as other literatures described. It
would be worth for pharmacological scientists to con-
firm by other experiments.[52]
Figure 7 Cell growth inhibition of compound 12.
line (MCF-7). Compound 12 could selectively inhibit
the cell growth of PC-3 at micromolar concentration,
however, there was no concentration-dependent re-
sponse pattern. As noticed in Figure 7, for higher tested
concentrations, 25 μmol/L (11.2 μg/mL), 50 μmol/L
(22.4 μg/mL) and 100 μmol/L (44.8 μg/mL), the aver-
age growth inhibition rates of PC-3 cell line were 35.2%,
Conclusions
The thiophene-derived amido bis-nitrogen mustard
Chin. J. Chem. 2012, 30, 1831—1840
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
1839

Tang et al.
FULL PAPER
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12 was designed and synthesized via five-step reactions
starting from commercially available 2-chloroaceto-
nitrile, and characterized by NMR, MS, IR spectra and
elemental analyses. Its single crystal was successfully
cultivated from a mixture solvent of ethyl acetate and
petroleum ether, and further analyzed by X-ray crystal
diffraction on Bruker APEXII diffractometer. Antimi-
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exhibited broad inhibitory efficacy and better bioactivi-
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amide and nitrile moieties are helpful for the title mus-
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selective inhibitory activities against PC-3 tumor cell
line, which made the thiophene bis-amido nitrogen
mustard a potential and promising antitumor scaffold
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Acknowledgements
This work was partially supported by National
Natural Science Foundation of China (No. 21172181),
the key program from Natural Science Foundation of
Chongqing (CSTC2012jjB10026), the Specialized Re-
search Fund for the Doctoral Program of Higher Educa-
tion of China (SRFDP 20110182110007) and the Re-
search
Funds
for
the
Central Universities
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