Thieno[2,3-d]pyrimidinedione derivatives as antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2012.02.035

Several thieno[2,3-d]pyrimidinediones have been synthesized and examined for antibacterial activity against a range of Gram-positive and Gram-negative pathogens. Two compounds displayed potent activity (2-16 mg/L) against multi-drug resistant Gram-positiv

Full text of DOI:10.1016/j.ejmech.2012.02.035

European Journal of Medicinal Chemistry 51 (2012) 145e153
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Thieno[2,3-d]pyrimidinedione derivatives as antibacterial agents
,
Mahender B. Dewal a, Amit S. Wani a, Celine Vidaillac b 1, David Oupický a, Michael J. Rybak b,
,
Steven M. Firestine a
*
a Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA
b Department of Pharmacy Practice, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Several thieno[2,3-d]pyrimidinediones have been synthesized and examined for antibacterial activity
against a range of Gram-positive and Gram-negative pathogens. Two compounds displayed potent
activity (2e16 mg/L) against multi-drug resistant Gram-positive organisms, including methicillin resis-
tant, vancomycin-intermediate, vancomycin-resistant Staphylococcus aureus (MRSA, VISA, VRSA) and
vancomycin-resistant enterococci (VRE). Only one of these agents possessed moderate activity (16
e32 mg/L) against Gram-negative strains. An examination of the cytotoxicity of these agents revealed
that they displayed low toxicity (40e50 mg/L) against mammalian cells and very low hemolytic activity
(2e7%). Taken together, these studies suggest that thieno[2,3-d]pyrimidinediones are interesting scaf-
folds for the development of novel Gram-positive antibacterial agents.
Received 22 November 2011
Received in revised form
10 February 2012
Accepted 16 February 2012
Available online 25 February 2012
Keywords:
Thieno[2,3-d]pyrimidinediones
Antibacterial
Ó 2012 Elsevier Masson SAS. All rights reserved.
MRSA
Antibiotic-resistant bacteria
Heterocycle synthesis
1. Introduction
antidiabetic [11] and anticancer [12,13] agents [14e16]. Despite the
breadth of biological activities displayed by these agents, the anti-
The increasing prevalence of pathogenic bacteria that are
resistant to currently available antibiotics represents an alarming
threat to public health. The most commonly encountered
antibiotic-resistant bacteria, methicillin-resistant Staphylococcus
aureus (MRSA), has had a major impact on infections in both the
hospital and community setting [1,2]. While vancomycin continues
to be the standard treatment option for antibiotic-resistant infec-
tions, the isolation of vancomycin-resistant Staphylococcus (VRSA)
and Enterococci (VRE) foreshadows a day in which the utilization of
vancomycin may become limited [3]. Unfortunately, as antibiotic-
resistant organisms have become more commonplace, the pipe-
line for the discovery of new antimicrobial agents has decreased [4].
Thus, there is a pressing need for new antimicrobial agents that are
capable of treating resistant bacterial strains.
bacterial activity of this class of compounds has been underexplored.
El-Sherbeny and colleagues examined the antimicrobial and anti-
viral activity of cyclopenteno and cyclohexeno [b]thieno[2,3-d]-3,4-
dihydropyrimidine-4-one derivatives (Fig. 1a) [8]. These agents
displayed reasonable activity (MIC values 6.25e25 mg/L) against
both Gram-positive and Gram-negative bacteria; however, these
agents were significantly more potent against herpes simplex virus
[8]. Furanyl-thieno[2,3-d]pyrimidin-4-ones (Fig. 1b) were examined
by Bahekar et al. for their antibacterial activity [7]. These agents
displayed MIC values in the range of 4e100 mg/L against a collection
of Gram-positive and Gram-negative microbes. Interestingly, these
compounds also displayed antimycobaterial activity [7]. The anti-
bacterial activity of thieno[2,3-d]pyrimidindiones (Fig. 1c) has not
been reported in the literature; however, these compounds have
been examined for antiviral activity [15].
Recently, during a study on thieno[2,3-d]pyrimidinones, we
discovered a set of compounds that possessed antibacterial activity
(Fig. 1d). These agents are structurally unrelated to any clinically
used antibiotic and display discreet structural overlap with thieno
[2,3-d]pyrimidines that have been reported in the literature. In this
report, we discuss the synthesis of thieno[2,3-d]pyrimidinediones
and their antibacterial and cytotoxic activities.
Thienopyrimidines are interesting heterocyclic compounds and
a number of derivatives of these compounds display therapeutic
activity as antimicrobial [5e7], antiviral [8,9], antiinflammatory [10]
* Corresponding author. Tel.: þ1 313 577 0455.
E-mail address: sfirestine@wayne.edu (S.M. Firestine).
Present address: UMR 7565, CNRS, Nancy University and ABC Plateforme,
1
Nancy University, Nancy, France.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.035

146
M.B. Dewal et al. / European Journal of Medicinal Chemistry 51 (2012) 145e153
Fig. 1. Thieno[2,3-d]pyrimidineone derivatives.
2. Results and discussion
3 (Scheme 1) [16]. The unconstrained compounds 2 and 4 were
prepared using similar methodology starting from the ethyl 3-oxo-
5-phenylpentanoate, 13 (Scheme 2).
2.1. Chemistry
The amines, 5 and 6, were prepared, as shown in the Scheme 3,
from commercially available compounds (17 and 21) using proce-
dures published for related compounds [20e26]. Amine 5 was
prepared from p-hydroxycinnamic acid (17) by first protecting the
phenol as the silyl ether followed by conversion of the acid into the
azide (20). Selective reduction of the azide to 5 was accomplished
using catalytic hydrogenation in the presence of Lindlar’s catalyst
(Pd/CaCO3). Amine 6 was synthesized from p-iodoanisole (21) using
a Heck reaction with acrylonitrile to generate E-cinnamonitrile
(22). Reduction of the nitrile to the amine was accomplished with
LiAlH4.
For a project on the development of antiviral therapeutics, we
required the synthesis of two constrained (1 and 3) and two
unconstrained (2 and 4) thieno[2,3-d]pyrimidine-2,4-dione deriv-
atives (Fig. 1d), neither of which had been described in the litera-
ture. A retrosynthetic analysis of these agents suggested that an
amino thiophene ester ring would be prepared first using the
standard Gewald reaction [17,18]. Once the thiophene was in hand,
the pyrimidine ring could be prepared by converting the amine into
a urea followed by cyclizing with the ester under basic conditions.
The synthesis of compounds 1e4 is shown in schemes 1e3. The
synthesis of the constrained derivatives 1 and 3 starts from the
commercially available ethyl-2-cyanoacetate and racemic benzy-
lated isopropyl cyclohexanone (8, prepared from 7). These were
reacted with sulfur to obtain the amino thiophene ester, 9, in
decent yields. Activation of 9 with p-nitrophenyl chloroformate
generated the unstable intermediate 10, which upon reaction with
amines 5 or 6 produced urido compounds 11a and 11b [19].
Formation of the pyrimidinedione ring was accomplished with
refluxing sodium methoxide to provide the final compounds 1 and
2.2. In vitro antibacterial activity
The initial examination of compounds 1e4 failed to detect
antiviral activity. As part of a further investigation of the biological
activities of these compounds, we examined the antibacterial
activity of compounds 1e4 and intermediates 11a, 11b, 16a and 16b
against a panel of five Gram-positive and four Gram-negative
bacteria (Table 1). Compounds 1 and 2 demonstrated significant
Scheme 1. a) nBuLi, BnBr, THF, ꢀ78 ꢁC to r.t.,18 h, 80% b) ethyl 2-cyanoacetate, S8, morpholine, EtOH,
77% d) pyridine, DMAP, THF, r.t. 12e24 h, 69e79% e) NaOMe, MeOH, reflux, 3 h, 76e77%.
D, 24e48 h., 39% c) p-nitrophenyl chloroformate, pyridine, CH2Cl2, r.t. 6e12 h,

M.B. Dewal et al. / European Journal of Medicinal Chemistry 51 (2012) 145e153
147
Scheme 2. a) nBuLi, BnBr, THF, 0e30 ꢁC,18 h, 66% b) ethyl 2-cyanoacetate, S8, morpholine, EtOH,
pyridine, DMAP, THF, r.t. 12e24 h, 80e85% e) NaOMe, MeOH, reflux, 3 h, 88e90%.
D, 24e48 h, 38% c) p-nitrophenyl chloroformate, pyridine, CH2Cl2, r.t. 6e12 h, 77% d)
antibacterial activity with MIC values in the 2e16 mg/L range
against Gram-positive bacteria such as MRSA, VRSA, VISA, VRE and
Streptococcus pneumoniae. The Gram-negative activity of 1 and 2
was weak with MIC values in the range of 16 to over 32 mg/L.
Compounds 3 and 4 displayed almost no antibacterial activity with
the exception being the moderate activity (8 mg/L) 3 displayed
against Enterobacter aerogenes. Compounds 11a, 16a and 16b
showed no activity against any bacterial strain; compound 11b was
insoluble in media, water and DMSO and thus could not be tested.
Taken together, the data support the conclusion that 1 and 2
possess Gram-positive antibacterial activity, while compounds 3, 4,
11a, 16a and 16b are essentially inactive.
2.3. In vitro cytotoxicity
We examined the cytotoxic activity of compounds 1e4 against
mammalian cells (NIH-3T3) using the MTS assay. As shown in Fig. 2,
all compounds displayed moderate levels of toxicity at 100 mg/L
(<50% of cell viability). The LD50 of each compound was deter-
mined from a doseeresponse curve of cytotoxicity versus concen-
tration (Table 1). As shown in the table, the LD50 (w52 mg/L) of the
most potent compound, 2, is 25 times higher than its MIC value
indicating that 2 is selectively toxic to bacteria. In contrast, 1 dis-
played a therapeutic index of only 5 suggesting that the toxicity of
the compound may have also played a role in its antibacterial
activity.
2.4. In vitro hemolytic activity
Another common measure of the toxicity of an agent is lysis of
red blood cells. We examined the ability of compounds 1 and 2 to
lyse Sheep erythrocytes. As shown in Fig. 3, compound 1 displayed
approximately 7% hemolysis at a concentration 25 times its MIC
value. In contrast, 2, which is the most selective antibacterial agent
described here, showed only 2% hemolysis at 50 times its MIC value.
These results indicate that the antibacterial activity observed for
compounds 1 and 2 is not due to non-specific membrane damage.
This would suggest that there are specific antibacterial targets for
these agents in bacteria.
2.5. Discussion
In this report we have explored thieno[2,3-d]pyrimidinedione
derivatives as antibacterial agents. While thieno[2,3-d]pyrimidines
have been extensively explored for their varied biological activity,
the compounds reported here are unique and have not been
prepared in the literature. Among the compounds synthesized,
compound 2 showed selective antibacterial activity against Gram-
positive antibiotic-resistant bacterial strains such as MRSA, VRSA,
VISA and VRE. Unfortunately, these compounds were inactive
against Gram-negative pathogens.
Scheme 3. a) TBDMSCl, imidazole/DMF, r.t. 3 h, 98% b) DIBAL/CH2Cl2, ꢀ78 ꢁC, 8 h, 59%
c) MsCl, Et3N/THF d) NaN3/DMF, 82% e) Pd/CaCO3, H2(g)/EtOH, r.t.,93% f) LiAlH4/Et2O,
r.t. 15 min, 61% g) Acrylonitrile, Pd(OAc)2, Bu4NBr, NaHCO3/H2O, 48%.

148
M.B. Dewal et al. / European Journal of Medicinal Chemistry 51 (2012) 145e153
Table 1
MIC and LD50 values for compounds 1e4, 11a, 11b, 16a, and 16b.
MIC (mg/L)
LD50
Therapeutic
(mg/L) Indexa
S. aureus
S. aureus
S. aureus
S. pneumoniae E. faecium
E. coli
P. aeruginosa K. pneumoniae E. aerogenes
(VISA Mu50) (MRSA 494) (VRSA MI) (ATCC 49619) (VRE 7303) (ATCC 25922) (ATCC 27853) (CEF 2324)
(CEF 3978)
1
2
3
4
11a
11bb
16a
16b
16
2
16
2
8
2
32
8
32
>32
>32
nd
>32
>32
0.5
8
4
32
16
32
32
32
32
16
32
8
>32
nd
>32
>32
nd
42
52
120
98
nd
nd
nd
nd
nd
5
26
4
>32
>32
>32
>32
nd
>32
>32
nd
>32
>32
>32
nd
>32
>32
>32
>32
>32
nd
>32
>32
0.5
>32
>32
>32
nd
>32
>32
nd
>32
>32
>32
nd
>32
>32
>32
>32
>32
>32
nd
>32
>32
>32
>32
>32
>32
nd
>32
>32
nd
3
nd
nd
nd
nd
nd
Vancomycin nd
a
Therapeutic index was calculated as the ratio of LD50/MIC for VRSA.
Not determined due to insolubility of compound in media.
b
A complete structureeactivity relationship cannot be deter-
of action of these thieno[2,3-d]pyrimidinediones and hope to
mined from the limited set of compounds prepared here. However,
inactivity of urido compounds (11a, 16a and 16b) clearly demon-
strates the importance of the pyrimidine ring for the activity and
our data also suggests that the presence of a phenol is critical for
activity (compare compound 2 to compound 4). Furthermore, the
presence of a methoxy group increases the cytotoxicity by 2-fold
report on their mechanism in due course.
3. Conclusion
We have synthesized constrained and unconstrained thieno
[2,3-d]pyrimidinedione derivatives and examined their antibacte-
rial, cytotoxicity and hemolytic activity. Only one compound, 2,
displayed potent antibacterial activity against a wide range of
antibiotic-resistant bacteria including MRSA, VRSA, VISA and VRE.
This compound was minimally cytotoxic against mammalian cells
and possessed essentially no hemolytic activity. While the mech-
anism of action of this compound is unknown, efforts to determine
the reason for its antibacterial activity are ongoing and will be
reported in due course.
when compared to the phenol.
A well-known method for
enhancing potency of compounds is to lock flexible portions of the
molecule into a fixed conformation. Tethering the side chains
located on the thiophene results in the racemic compound 1. While
1 displayed antibacterial activity against Gram-positive pathogens,
the MIC values were 2e8-fold higher for the unconstrained analog
2. Thus, the flexibility seen in compound 2 appear to be necessary
for the compound to adopt a biologically active conformation.
The serendipitous discovery of antibacterial activity for these
agents unfortunately means that the mechanism of action is
unknown. While there have been reports of antibacterial activity
for structurally unrelated thieno-pyrimidinones (see Introduction),
the mechanism of action of those agents has never been reported.
Thieno-pyrimidines and thieno-pyrimidinediones have been
reported as inhibitors of proteases [27], kinases [28e30] and folate
utilizing enzymes [8,9,31] suggesting that the mechanism of action
of our agent could be due to inhibition of a specific protein. The
inhibition of folate utilizing enzymes (i.e. dihydrofolate reductase,
etc.) is especially interesting given the fact that antibacterial agents
specifically targeting bacterial folate enzymes are well known. Of
course, numerous other pathways could be responsible for the
antibacterial activity of the compounds, including non-specific
membrane effects. We are currently investigating the mechanism
4. Experimental
4.1. Materials and instruments
All chemicals were purchased from Acros, SigmaeAldrich,
Matrix Scientific, EMD or Frinton labs and used without further
purification. 1H NMR and 13C NMR spectra were recorded on Varian
DRX400. The mass spectra of respective final compounds were
recorded on Waters-Micromass ZQ quadupole located in the
central instrumentation facility at Wayne State University, Detroit,
MI. For the MTS and hemolysis assays, absorbance was collected
Fig. 2. Cytotoxicity of compounds 1e4 obtained my MTS assay against mammalian
Fig. 3. Hemolytic activity of compounds 1 and 2 against Sheep erythrocytes. Hemo-
cells (NIH-3T3), 5% DMSO is used as control.
lysis was calculated using 1% Triton-X as positive control which set to 100% lysis.

M.B. Dewal et al. / European Journal of Medicinal Chemistry 51 (2012) 145e153
149
using Synergy 2 multi-mode plate reader from Biotek Instruments
Inc. Winooski, VT.
4.1.4. (E)-Ethyl 4-benzyl-2-(3-(3-(4-(tert-butyldimethylsilyl)
phenyl)allyl)ureido)-6-isopropyl-4,5,-6,7-tetrahydrobenzo[b]
thiophene-3-carboxylate (11a)
4.1.1. 2-Benzyl-4-isopropyl cyclohexanone (8)
Compound 5 (0.250 g, 0.95 mmols) was dissolved in 20 mL of
dry THF followed by the addition of pyridine (0.090 g, 1.14 mmols)
and DMAP (0.017 g, 0.14 mmols). The resulting solution was stirred
for 20 min. A solution of 10 (0.496 g, 0.95 mmols) in 15 mL of dry
THF was added drop wise to the reaction mixture and the reaction
was stirred for an additional 12e24 h. The reaction was then
evaporated to dryness in vacuo and the residue was purified by
flash silica column chromatography (2:8 ethyl acetate:hexanes) to
yield 0.49 g (79%) of the expected products. 1H NMR (400 MHz,
To a cooled (ꢀ20 ꢁC) solution of diisopropylamine (0.160 g,
1.57 mmols) in dry toluene, n-butyl lithium (1.6 M in hexanes,
0.95 mL, 1.5 mmols) was added and stirred for 15 min. To the
solution, isopropyl cyclohexanone (7) (0.200 g, 1.43 mmols), dis-
solved in 5 mL of dry toluene, was added drop wise. The reaction
was stirred at ꢀ20 ꢁC for 30 min and then cooled to ꢀ78 ꢁC. To the
chilled solution, benzyl bromide (0.270 g, 1.57 mmols), dissolved in
5 mL dry toluene, was added drop wise and the reaction mixture
was warmed to ꢀ45 ꢁC and stirred at this temperature for 18 h. The
reaction was quenched with 0.5 N HCl (10 mL) and extracted with
ether (150 mL). The combined organic layers were washed with
brine, H2O and then dried over anhydrous Na2SO4 before being
concentrated in vacuo. The crude product was purified by flash
silica column chromatography (1:9 ethyl acetate:hexanes) to yield
CD2Cl2)
d
0.86 (d, J ¼ 6.8 Hz, 3H, CH3), 0.91 (d, J ¼ 6.8 Hz, 3H,
CH3),1.46 (t, J ¼ 7.6 Hz, 3H, CH3), 1.83e1.88 (m, 2H, CH, CH),
2.50e2.82 (m, 4H, CH2, CH2), 3.14e3.17 (m, 2H, CH2), 3.59e3.62 (m,
1H, CH), 4.47 (q, J ¼ 7.2 Hz, 2H, CH2), 7.16e7.27 (m, 5H), 7.48 (d,
J ¼ 8.0 Hz, 2H, Ph), 8.32 (d, J ¼ 8.0 Hz, 2H, Ph), 11.09 (s, 1H, NH); 13C
NMR (100 MHz, CD2Cl2)
d 14.67, 19.62, 19.99, 28.32, 28.54, 32.36,
pure 8 (0.26, 80%). 1H NMR (400 MHz, CD2Cl2)
d
0.85 (d, J ¼ 2.8 Hz,
35.19, 40.85, 61.25, 111.58, 122.21, 125.37, 126.12, 127.81, 128.35,
129.30, 135.87, 141.30, 145.48, 148.67, 150.12, 155.50, 166.09. HRMS
could not be obtained because of decomposition in the mass
spectrometer.
3H, CH3), 0.86 (d, J ¼ 2.4 Hz, 3H, CH3), 1.44e1.59 (m, 4H, CH2, CH2),
2.31e2.37 (m, 4H, CH2, CH2), 2.38e2.65 (m, 2H, CH, CH), 3.2 (dd,
Ja ¼ 14 Hz, Jb ¼ 5.2 Hz, 1H, CH), 7.15e7.28 (m, 5H, Ph); 13C NMR
(100 MHz, CD2Cl2)
d 19.47, 20.00, 30.61, 32.22, 35.64, 37.13, 41.71,
43.16, 51.50, 125.96, 128.34, 129.29, 141.02, 212.38.
4.1.5. (E)-Ethyl 4-benzyl-6-isopropyl-2-(3-(3-(4-methoxyphenyl)
allyl)ureido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate
(11b)
Compound 6 (0.097 g, 0.60 mmols) was dissolved in 15 mL of
dry THF followed by the addition of pyridine (0.052 g,
0.65 mmols) and DMAP (0.008 g, 0.06 mmols). To the reaction,
4.1.2. Ethyl 2-amino-4-benzyl-6-isopropyl-4,5,6,7-tetrahydrobenzo
[b]thiophene-3-carboxylate (9)
To a 50 mL round bottom flask, 8 (0.341 g, 1.48 mmols), sulfur
(0.048 g, 1.48 mmols), ethylcyanoacetate (0.167 g, 1.48 mmols) and
morpholine (0.129 g, 1.48 mmols) were added along with 15 mL of
absolute ethanol. The resulting solution was refluxed for 48 h,
cooled and then evaporated to dryness in vacuo. The resulting crude
product was purified by flash silica column chromatography (1:9
ethyl acetate:hexanes) to generate 0.21 g (39%) of 9. 1H NMR
which was stirred for 20 min,
a solution of 10 (0.310 g,
0.60 mmols) in 10 mL of dry THF was added drop wise. The
reaction was stirred at room temperature for 12e24 h and
evaporated to dryness in vacuo. The expected product (0.16 g,
69%) was obtained from the residue after purification by flash
silica column chromatography (3:7 ethyl acetate:hexanes). 1H
(400 MHz, CD2Cl2)
d
0.85 (d, J ¼ 7.2 Hz, 3H, CH3), 0.89 (d, J ¼ 6.8 Hz,
3H, CH3), 1.35 (t, J ¼ 5.2 Hz, 3H, CH3), 1.78e1.89 (m, 2H, CH, CH),
2.45 (t, J ¼ 2.8 Hz, 2H, CH2), 2.56e2.62 (m, 2H, CH2, CH2), 3.15 (dd,
Ja ¼ 13.6 Hz, Jb ¼ 2.8 Hz,1H, CH), 4.41 (q, J ¼ 8.4 Hz, 2H, CH2), 6.03 (s,
2H, NH2), 7.20e7.32 (m, 5H, Ph); 13C NMR (100 MHz, CD2Cl2)
NMR (400 MHz, CD2Cl2)
d
0.85 (d, J ¼ 6.8 Hz, 3H, CH3), 0.90 (d,
J ¼ 6.8 Hz, 3H, CH3), 1.19e1.26 (m, 1H, CH), 1.37 (t, J ¼ 7.6 Hz, 3H,
CH3), 1.41e1.48 (m, 1H, CH), 1.79e1.85 (m, 1H, CH), 2.28 (t,
J ¼ 5.6 Hz, 2H, CH2), 2.55 (d, J ¼ 6.2 Hz, 2H), 3.13 (d, J ¼ 6.0 Hz,
2H, CH2), 3.76 (s, 3H, CH3), 4.05 (t, J ¼ 6.0 Hz, 2H, CH2), 4.41 (q,
J ¼ 6.8 Hz, 2H, CH2), 5.71 (t, J ¼ 4.8 Hz, 1H, NH), 6.08e6.17
(sextet, J ¼ 15.6 Hz, 1H, CH]CH), 6.53 (d, J ¼ 15.6 Hz, 1H,
CH]CH), 6.84 (d, J ¼ 8.4 Hz, 2H, Ph), 7.19e7.33 (m, 7H, Ph), 10.80
d
14.93, 19.56, 20.10, 30.64, 32.27, 40.54, 41.75, 43.19, 51.55, 59.74,
118.50, 126.03, 128.41, 129.35, 136.61, 141.05, 141.94, 162.80, 165.63.
HRMS (ESþ) m/z (M þ H) calcd for C21H27NO2S 358.1839, found
358.1837 (M þ H).
(s, 1H, NH); 13C NMR (100 MHz, CD2Cl2)
d 14.77, 19.66, 19.81,
4.1.3. Ethyl 4-benzyl-6-isopropyl-2-((4-nitrophenoxy)
carbonylamino)-4,5,6,7-tetrahydrobenzo[b]-thiophene-3-
carboxylate (10)
28.48, 32.83, 35.23, 36.72, 40.87, 42.96, 55.41, 60.68, 108.39,
114.15, 123.76, 125.72, 126.00, 127.75, 128.40, 129.16, 129.44,
131.52, 134.52, 141.62, 152.25, 153.85, 159.60, 166.76. HRMS (ESþ)
m/z (M þ H) calcd for C32H38N2O4S 547.2632, found 547.2631
(M þ H)
A solution of dry pyridine (0.16 g, 2.0 mmols) and 9 (0.358 g,
1.0 mmols) in 20 mL of dry CH2Cl2 was stirred at room temperature
for 30 min. To the reaction, a solution of p-nitrophenyl chlor-
oformate (0.303 g, 1.5 mmols), in 5 mL of dry CH2Cl2, was added
drop wise over a period of 15 min. The reaction was stirred at room
temperature for 12 h and then evaporated in vacuo to yield the
crude product which was purified by flash silica column chroma-
tography (3:7 ethyl acetate:hexanes) to give 0.41 g (77%) of the
4.1.6. (E)-Ethyl 4-benzyl-6-isopropyl-2-(3-(3-(4-methoxyphenyl)
allyl)theino[2,3-d]pyrimidine-2,4-dione (3)
Compound 11b (0.050 g, 0.09 mmols) was refluxed in sodium
methoxide NaOCH3 (0.005 g, 0.10 mmols) and 10 mL of methanol
for 3 h. The reaction was cooled and neutralized with Dowex
50 Hþ resin which resulted in a white precipitate. The precipitate
was dissolved in hot methanol and filtered through a sintered
glass funnel. The resulting filtrate was dried and purified using
flash silica column chromatography (1:1 ethyl acetate:hexane) to
yield 0.035 g (76%) of the final product. 1H NMR (400 MHz,
desired product. 1H NMR (400 MHz, CD2Cl2)
d
0.86 (d, J ¼ 6.8 Hz, 3H,
CH3), 0.91 (d, J ¼ 6.8 Hz, 3H, CH3), 1.46 (t, J ¼ 7.6 Hz, 3H, CH3),
1.83e1.88 (m, 2H, CH, CH), 2.50e2.82 (m, 4H, CH2, CH2), 3.14e3.17
(m, 2H, CH2), 3.59e3.62 (m, 1H, CH), 4.47 (q, J ¼ 7.2 Hz, 2H, CH2),
7.16e7.27 (m, 5H), 7.48 (d, J ¼ 8.0 Hz, 2H, Ph), 8.32 (d, J ¼ 8.0 Hz, 2H,
Ph), 11.09 (s, 1H, NH); 13C NMR (100 MHz, CD2Cl2)
d
14.67, 19.62,
DMSO)
d
0.86 (d, J ¼ 6.8 Hz, 3H, CH3), 0.91 (d, J ¼ 6.4 Hz, 3H,
19.99, 28.32, 28.54, 32.36, 35.19, 40.85, 61.25, 111.58, 122.21, 125.37,
126.12, 127.81, 128.35, 129.30, 135.87, 141.30, 145.48, 148.67, 150.12,
155.50, 166.09. HRMS could not be obtained because of decompo-
sition in the mass spectrometer.
CH3), 1.46e1.58 (m, 1H, CH), 1.81e2.12 (m, 1H, CH), 2.26e2.43 (m,
4H, CH2, CH2), 2.73 (dd, Ja ¼ 11.6 Hz, Jb ¼ 4.8 Hz, 1H, CH), 3.18 (d,
J ¼ 12.0 Hz, 2H, CH2), 3.73 (s, 3H, CH3), 4.61 (d, J ¼ 8.0 Hz,
2H, CH2), 6.15e6.19 (sextet, J ¼ 16 Hz, 1H, CH]CH), 6.48

150
M.B. Dewal et al. / European Journal of Medicinal Chemistry 51 (2012) 145e153
(d, J ¼ 16 Hz, 1H, CH]CH), 6.85 (d, J ¼ 8.8 Hz, 2H, Ph), 7.18e7.38
4.1.10. Diethyl 5-((4-nitrophenoxy)carbonylamino)-3-
phenethylthiophene-2,4-dicarboxylate (15)
(m, 7H, Ph), 12.18 (s, 1H, NH); 13C NMR (100 MHz, CD2Cl2)
d
20.25, 20.41, 27.31, 28.84, 32.61, 35.65, 37.46, 42.09, 55.75,
A mixture of dry pyridine (0.12 g, 1.5 mmols) and 14 (0.300 g,
0.86 mmols) in 20 mL of dry CH2Cl2 was placed under argon and
stirred for 30 min at room temperature. To the solution, p-nitro-
phenyl chloroformate (0.186 g, 0.93 mmols) dissolved in 10 mL of
dry CH2Cl2 was added drop wise over a period of 15 min and the
resulting mixture was stirred at room temperature for 18 h. The
solvent was evaporated in vacuo and the residue was purified by
flash silica column chromatography (2:2:6 ethyl aceta-
te:CH2Cl2:hexanes) to give 0.34 g (77%) of the final, pure product.
112.18, 114.66, 122.72, 126.57, 126.82, 128.19, 128.84, 129.65,
129.78, 131.73, 135.63, 141.86, 150.87, 151.07, 158.99, 159.50;
HRMS (ESþ) m/z (M þ H) calcd for C30H32N2O3S 501.2212, found
501.2210 (M þ H).
4.1.7. (E)-Ethyl 4-benzyl-2-(3-(3-(4-(hydroxy)phenyl)allyl) theino
[2,3-d]pyrimidine-2,4-dione (1)
Compound 11a (0.100 g, 0.15 mmols) was refluxed in sodium
methoxide NaOCH3 (0.010 g, 0.19 mmols) and 15 mL of methanol
for 3 h. The reaction was cooled and neutralized with Dowex 50 Hþ
resin which resulted in a white precipitate. The precipitate was
dissolved in hot methanol and filtered through a sintered glass
funnel. The resulting filtrate was dried and purified using flash
silica column chromatography (1:1 ethyl acetate:hexane) to yield
1H NMR (400 MHz, CD2Cl2)
d
1.35 (t, J ¼ 6.8 Hz, 3H, CH3), 1.43 (t,
J ¼ 7.2 Hz, 3H, CH3), 2.86 (t, J ¼ 8.4 Hz, 2H, CH2), 3.61 (t, J ¼ 8.4 Hz,
2H, CH2), 4.3 (q, J ¼ 7.2 Hz, 2H, CH2), 4.42 (q, J ¼ 7.2 Hz, 2H, CH2),
7.19e7.34 (m, 5H, Ph), 7.47 (d, J ¼ 9.2 Hz, 2H, Ph), 8.31 (d, J ¼ 9.2 Hz,
2H, Ph),11.36 (s,1H, NH); 13C NMR (100 MHz, CD2Cl2)
d 14.33, 30.63,
36.85, 61.2, 61.86, 114.15, 118.61, 122.19, 125.48, 126.1, 128.46,
128.59, 142.17, 145.73, 148.85, 150.24, 153.67, 155.17, 162.25, 166.15.
HRMS could not be obtained because of decomposition in the mass
spectrometer.
0.058 g (77%) of the final product. 1H NMR (400 MHz, DMSO)
d 0.86
(d, J ¼ 6.8 Hz, 3H, CH3), 0.90 (d, J ¼ 6.8 Hz, 3H, CH3), 1.44e1.60 (m,
2H, CH, CH), 2.25e2.42 (m, 4H, CH2, CH2), 2.70e2.75 (m, 1H, CH),
3.16 (d, J ¼ 7.8 Hz, 2H, CH2), 4.59 (d, J ¼ 7.0 Hz, 2H, CH2), 6.04e6.14
(sextet, J ¼ 16 Hz, 1H, CH]CH), 6.44 (d, J ¼ 16 Hz, 1H, CH]CH), 6.67
(d, J ¼ 8.8 Hz, 2H, Ph), 7.18e7.38 (m, 7H, Ph), 9.47 (s, 1H, OH), 12.18
4.1.11. (E)-Diethyl 3-phenethyl-5-(3-(3-(4-(trimethylsilyloxy)
phenyl)allyl)ureido)thiophene-2,4-dicarboxylate (16a)
(s, 1H, NH); 13C NMR (100 MHz, DMSO)
d
20.26, 20.42, 27.31, 28.85,
Compound 5 (0.170 g, 0.65 mmols) was dissolved in 15 mL of
dry THF and to the resulting solution, pyridine (0.061 g,
0.78 mmols) and DMAP (0.012 g, 0.10 mmols) were added. The
amine solution was stirred at room temperature for 20 min fol-
lowed by a drop wise addition of a solution of 15 (0.331 g,
0.65 mmols) in 15 mL of dry THF. The reaction was stirred for an
additional 12 h and then evaporated in vacuo. The residue was
purified by flash silica column chromatography (2:8 ethyl aceta-
te:hexanes) to yield 0.33 g (80%) of pure 16a. 1H NMR (400 MHz,
32.62, 35.66, 37.47, 42.15, 60.44, 112.21, 116.04, 121.49, 126.58,
126.86,128.04,128.25,128.86,129.79,132.29,135.65,141.86,150.82,
150.90, 157.78, 158.97; HRMS (ESþ) m/z (M þ H) calcd for
C29H30N2O3S 487.2055, found 487.2046 (M þ H).
4.1.8. Ethyl 3-oxo-5-phenylpentanoate (13)
Sodium ethylacetoacetate (12) (5 g, 32.87 mmols) was dissolved
in 50 mL of dry THF and cooled to 0 ꢁC under argon. Once cooled,
a solution (1.6 M in hexane) of n-butyl lithium (21.88 mL,
34.51 mmols) was added drop wise and the reaction was stirred for
30 min at 0 ꢁC to generate the enolate. Benzyl bromide (5.62 g,
32.87 mmols) was then added and the reaction continued stirring
for another 90 min at 0 ꢁC. Upon completion, the reaction was
poured into a cold solution of saturated potassium hydrogen
phosphate and the resulting aqueous solution was extracted with
ethyl ether (3 ꢂ 100 mL). The combined organic layers were washed
with water, dried over anhydrous Na2SO4 and concentrated in
vacuo. The resulting material was purified by flash silica column
chromatography (1:1:8 CH2Cl2:ethyl acetate:hexanes) to obtain
CD2Cl2) d 0.19 (s, 6H, CH3), 0.98 (s, 9H, tBu) 1.30e1.39 (m, 6H, CH3),
2.82 (t, J ¼ 8.0 Hz, 2H, CH2), 3.57 (t, J ¼ 8.0 Hz, 2H, CH2), 4.07 (t,
J ¼ 5.8 Hz, 2H, CH2), 4.26e4.37 (m, 4H, CH2, CH2), 5.49 (t,
J ¼ 5.2 Hz, 1H, NH), 6.12 (sextet, J ¼ 15.6 Hz, 1H, CH]CH), 6.55 (d,
J ¼ 16.4 Hz, 1H, CH]CH), 6.80 (d, J ¼ 8.8 Hz, 2H, Ph), 7.25e7.30 (m,
7H, Ph), 11.04 (s, 1H, NH); 13C NMR (100 MHz, CD2Cl2)
d
ꢀ4.49,
14.44, 25.66, 30.69, 31.91, 32.44, 36.87, 53.67, 60.86, 61.35, 120.15,
120.44, 123.54, 126.02, 127.75, 128.45, 128.59, 129.42,130.56,
131.89, 142.50, 148.59, 153.86, 155.77, 162.88, 166.78. HRMS (ESþ)
m/z (M þ H) calcd for C34H44N2O6SSi 637.2755, found 637.2715
(M þ H).
4.75 g (66%) of the final product. 1H NMR (400 MHz, CD2Cl2)
d 1.25
(t, J ¼ 7.2 Hz, 3H, CH3), 2.87e2.92 (m, 4H, CH2, CH2), 3.42 (s, 2H,
4.1.12. (E)-Diethyl 5-(3-(3-(4-methoxyphenyl)allyl)ureido)-3-
phenethylthiophene-2,4-dicarboxylate (16b)
CH2) 4.15 (q, J ¼ 6.8 Hz, 2H, CH2), 7.19e7.31 (m, 5H, Ph); 13C NMR
(100 MHz, CD2Cl2)
d
14.1, 29.52, 44.56, 49.58, 61.46, 126.31, 128.5,
To a solution of 6 (0.173 g, 1.06 mmols), dissolved in 20 mL of
dry THF, pyridine (0.092 g, 1.17 mmols) and DMAP (0.013 g,
0.106 mmols) were added and the resulting solution was stirred
at room temperature for 20 min. To the amine solution, a solu-
tion of 15 (0.543 g, 1.06 mmols) in 15 mL of dry THF was added
drop wise and the resulting reaction was allowed to stir for an
additional 12 h. Upon completion, the solvent was evaporated in
vacuo and the resulting mixture was purified by flash silica
column chromatography (3:7 ethyl acetate:hexanes) to generate
128.64, 141.05, 167.26, 202.11.
4.1.9. Diethyl 5-amino-3-phenethylthiophene-2,4-dicarboxylate (14)
Compound 13 (3.04 g, 13.8 mmols), sulfur (0.443 g,
13.8 mmols), ethylcyanoacetate (1.56 g, 13.8 mmols) and mor-
pholine (1.2 g, 13.8 mmols) were refluxed in 60 mL of absolute
ethanol for 48 h. Upon completion, the solvent was evaporated in
vacuo and the resulting material was purified by flash silica
column chromatography (1:2:7 ethyl acetate:CH2Cl2:hexanes) to
yield 1.78 g (38%) of the final product. 1H NMR (400 MHz, CD2Cl2)
0.49 g (85%) of 16b. 1H NMR (400 MHz, CD2Cl2)
d 1.32 (t,
J ¼ 7.2 Hz, 3H, CH3), 1.37 (t, J ¼ 7.2 Hz, 3H, CH3), 2.83 (t,
J ¼ 8.4 Hz, 2H, CH2) 3.60 (t, J ¼ 8.4 Hz, 2H, CH2), 3.8 (s, 3H, CH3),
4.10 (t, J ¼ 5.6 Hz, 2H, CH2), 4.28 (q, J ¼ 7.2 Hz, 2H, CH2), 4.37 (q,
J ¼ 7.6 Hz, 2H, CH2), 5.57 (t, J ¼ 5.2 Hz, 1H, NH), 6.07e6.14 (sextet,
J ¼ 15.6 Hz, 1H, CH]CH), 6.56 (d, J ¼ 15.6 Hz, 1H, CH]CH), 6.84
(d, J ¼ 8.4 Hz, 2H, Ph), 6.95 (d, J ¼ 8.8 Hz, 2H, Ph), 7.17e7.31 (m,
3H, Ph), 8.13 (d, J ¼ 9.2, 2H, Ph), 11.14 (s, 1H); 13C NMR (100 MHz,
d
1.31 (t, J ¼ 7.2 Hz, 3H, CH3), 1.37 (t, J ¼ 7.2 Hz, 3H, CH3), 2.82 (t,
J ¼ 8.4 Hz, 2H, CH2), 3.52 (t, J ¼ 8.4 Hz, 2H, CH2), 4.24 (q,
J ¼ 7.2 Hz, 2H, CH2), 4.34 (q, J ¼ 7.2 Hz, 2H, CH2), 6.58 (s, 2H, br,
NH2), 7.19 (q, J ¼ 4.8 Hz, 1H, Ph), 7.29 (d, J ¼ 4.4 Hz, 4H, Ph); 13C
NMR (100 MHz, CD2Cl2)
d 14.47, 14.54, 31.25, 36.73, 60.44, 60.73,
107.79, 109.02, 125.96, 128.42, 128.63, 142.64, 151.57, 162.51,
165.91, 166.83. HRMS (ESþ) m/z (M þ Na) calcd for C18H21NO4SNa
370.1079, found 370.1083 (M þ Na).
CD2Cl2)
d 14.35, 30.58, 36.82, 55.47, 61.26, 61.57, 114.20, 115.94,
126.07, 126.32, 127.79, 128.46, 128.55,129.31, 142.27, 148.87,

M.B. Dewal et al. / European Journal of Medicinal Chemistry 51 (2012) 145e153
151
154.18, 156.30, 159.70, 162.61, 163.32, 166.82. HRMS (ESþ) m/z
4.1.16. (E)-3-(4-(tert-Butyldimethylsilyloxy)phenyl)prop-2-en-1-ol
(19)
(M þ Na) calcd for C29H32N2O6SNa 559.1869, found 559.1872
(M þ Na).
Compound 18 (5.0 g, 17.98 mmols) was dissolved in 25 mL of dry
CH2Cl2 and the solution was cooled to ꢀ78 ꢁC. To the chilled solu-
tion, DIBAL (20% solution in toluene, 61 mL, 71.91 mmol) was added
drop wise and the reaction was stirred at ꢀ78 ꢁC for 8 h. Upon
completion, 75 mL of ether was added to the reaction mixture and
the mixture was slowly warmed to 0 ꢁC. Once the solution was
equilibrated to 0 ꢁC, 2.5 mL of H2O was added drop wise, followed
by 5 mL of 1 N NaOH and an additional 5 mL of H2O. The solution
was then was warmed to room temperature and stirred for an
additional 15 min and anhydrous MgSO4 was added slowly with
stirring another 15 min until no hydrated MgSO4 aggregates
formed. The salts were filtered and filtrate evaporated to obtain the
pure product in 2.43 g (59%) yield. 1H NMR (400 MHz, CD2Cl2)
4.1.13. (E)-Ethyl 3-(3-(4-methoxyphenyl)allyl)-2,4-dioxo-5-
phenethyl-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-6-carboxylate
(4)
Compound 16b (0.385 g, 0.72 mmols) was refluxed in sodium
methoxide NaOCH3 (0.043 g, 0.79 mmols) and 30 mL methanol for
3 h. The reaction was cooled and neutralized with Dowex 50 Hþ
resin which resulted in a white precipitate. The precipitate was
dissolved in hot methanol and filtered through a sintered glass
funnel. The resulting filtrate was dried and purified using flash
silica column chromatography (3:7 ethyl acetate:hexane) to yield
0.31 g (88%) of the final product. 1H NMR (400 MHz, DMSO-d6)
d
1.28 (t, J ¼ 6.8 Hz, 3H, CH3), 2.78 (t, J ¼ 7.6 Hz, 2H, CH2) 3.55 (t,
d 0.22 (s, 6H, CH3), 1.01 (s, 9H, tBu) 2.13 (s, br, 1H, OH), 4.25 (t,
J ¼ 8.0 Hz, 2H, CH2), 3.73 (s, 3H, CH3), 4.25 (q, J ¼ 7.2 Hz, 2H, CH2),
4.60 (d, J ¼ 6.0 Hz, 2H, CH2), 6.11e6.18 (sextet, J ¼ 16 Hz, 1H, CH]
CH), 6.52 (d, J ¼ 16 Hz, 1H, CH]CH), 6.87 (d, J ¼ 8.8 Hz, 2H, Ph), 7.19
(t, J ¼ 6.8 Hz, 1H, Ph), 7.25e7.32 (m, 4H, Ph), 7.36 (d, J ¼ 8.8 Hz, 2H,
J ¼ 4.8 Hz, 2H, CH2), 6.24 (quintet, J ¼ 16 Hz, 1H, CH]CH), 6.54 (d,
J ¼ 16 Hz, 1H, CH]CH), 6.81 (d, J ¼ 8.8 Hz, 2H, Ph), 7.28 (d,
J ¼ 8.4 Hz, 2H, Ph); 13C NMR (100 MHz, CD2Cl2)
d
ꢀ4.48, 18.34,
25.67, 63.74, 120.43, 127.14, 127.77, 130.44, 130.51, 155.64.
Ph), 12.56 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
d 14.91, 30.51,
36.47, 42.17, 55.76, 61.33, 114.68, 116.51, 123.02, 126.53, 126.88,
128.17, 128.91,128.97, 129.77, 131.79, 140.27, 142.27, 149.31, 159.48,
160.03, 162.55, 164.69; HRMS (ESþ) m/z (M þ Na) calcd for
C27H26N2O5SNa 513.1460, found 513.1453 (M þ Na).
4.1.17. (E)-(4-(3-Azidoprop-1-enyl)phenoxy)(tert-butyl)
dimethylsilane (20)
Compound 19 (0.300 g, 1.13 mmols) was dissolved in 10 mL of
dry THF followed by the addition of triethylamine (0.172 g,
1.7 mmol). Methanesulfonyl chloride (0.195 g, 1.7 mmols) was
added drop wise to the reaction and the reaction was stirred at
room temperature for 3 h. The reaction was then quenched by the
addition of 20 mL H2O and the resulting solution was extracted
with ethyl acetate (3 ꢂ 25 mL). The combined organic layers were
then washed with brine and dried over anhydrous Na2SO4. The
organic layer was evaporated in vacuo to obtain the crude product
which was used immediately in the next reaction.
The crude mesylated compound was dissolved in dry DMF and
sodium azide (0.067 g, 1.64 mmol) was added. The reaction was
stirred at room temperature for 5 h and then poured into 50 mL
H2O to generate a white, cloudy solution. The solution was
extracted with ethyl acetate (3 ꢂ 50 mL) and the combined organic
layers were washed with brine and dried over anhydrous Na2SO4.
The organic layer was evaporated in vacuo to dryness and the crude
product was purified by flash silica column chromatography (1:9
ethyl acetate:hexanes) to give 0.25 g (82%) of the final product. 1H
4.1.14. (E)-Ethyl 3-(3-(4-hydroxyphenyl)allyl)-2,4-dioxo-5-
phenethyl-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-6-
carboxylate (2)
Compound 16a (0.100 g, 0.16 mmols) was refluxed in sodium
methoxide NaOCH3 (0.009 g, 0.17 mmols) in 30 mL methanol for
3 h. The reaction was cooled and neutralized with Dowex 50 Hþ
resin which resulted in a white precipitate. The precipitate was
dissolved in hot methanol and filtered through a sintered glass
funnel. The resulting filtrate was dried and purified using flash
silica column chromatography (3:7 ethyl acetate:hexane) to yield
0.083 g (90%) of the final product. 1H NMR (400 MHz, DMSO-d6)
d
1.28 (t, J ¼ 7.2 Hz, 3H, CH3), 2.78 (t, J ¼ 8.0 Hz, 2H, CH2) 3.55 (t,
J ¼ 7.8 Hz, 2H, CH2), 4.26 (q, J ¼ 6.8 Hz, 2H, CH2), 4.58 (d,
J ¼ 6.0 Hz, 2H, CH2), 6.06 (sextet, J ¼ 15.6 Hz, 1H, CH]CH), 6.47
(d, J ¼ 16 Hz, 1H, CH]CH), 6.69 (d, J ¼ 8.4 Hz, 2H, Ph), 7.17e7.32
(m, 7H, Ph), 9.48 (s, 1H, OH), 12.55 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6)
d
14.62, 30.30, 36.33, 42.31, 61.60, 114.01, 115.89, 117.27,
NMR (400 MHz, CDCl3) d 0.22 (s, 6H, CH3), 1.01 (s, 9H, tBu) 3.93 (d,
120.99, 126.50, 128.05, 128.20,128.83, 128.90, 132.69, 141.99,
148.87, 150.47, 154.81, 157.67, 159.20, 162.20. HRMS HRMS (ESþ)
m/z (M þ Na) calcd for C26H24N2NaO4S 499.1304, found 499.1306
(M þ Na)
J ¼ 6.8 Hz, 2H, CH2), 6.12 (quintet, J ¼ 16 Hz, 1H, CH]CH), 6.60 (d,
J ¼ 16 Hz, 1H, CH]CH), 6.83 (d, J ¼ 8.4 Hz, 2H, Ph), 7.30 (d,
J ¼ 8.4 Hz, 2H, Ph); 13C NMR (100 MHz, CDCl3)
ꢀ4.17, 18.47, 53.45,
d
120.40, 120.52, 128.08, 129.53, 134.58, 156.12.
4.1.15. (E)-3-(4-(tert-Butyldimethylsilyloxy)phenyl)acrylic acid (18)
p-Hydroxycinnmic acid (17) (5.0 g, 30.46 mmols) and imidazole
(5.2 g, 76.15 mmols) were dissolved in 25 mL of dry DMF and the
solution was cooled to 0 ꢁC. To the chilled solution, t-butyldime-
thylsilylchloride (11.5 g, 76.15 mmols) was added and the reaction
was stirred at room temperature for 3 h. The resulting reaction
mixture was poured into 125 mL of water. The white, cloudy
solution was extracted with ethyl acetate (3 ꢂ 100 mL) and the
combined organic layers were washed with brine and dried with
sodium sulfate. The organic layer was evaporated to dryness and
the residue was purified by flash silica column chromatography
(3:7 ethyl acetate:hexanes) to yield 8.32 g (98%) of the final
4.1.18. (E)-3-(4-(tert-Butyldimethylsilyloxy)phenyl)prop-2-en-1-
amine (5)
Compound 20 (0.200 g, 0.69 mmols) was dissolved in 8 mL of
ethanol and Lindlar’s catalyst (Pd/CaCO3, 0.043 g, 0.21 mmol) was
added to the solution. The reaction was stirred under 1 atm of H2
for 5 h, filtered through celite and evaporated to dryness in vacuo.
The resulting product (0.17 g, 93%) was used without any additional
purification. 1H NMR (400 MHz, CD2Cl2)
d 0.19 (s, 6H, CH3), 0.98 (s,
9H, tBu), 1.19 (br, 2H, NH2), 3.40 (d, J ¼ 5.6 Hz, 2H, CH2), 6.19
(quintet, J ¼ 15.6 Hz, 1H, CH]CH), 6.42 (d, J ¼ 16 Hz, 1H, CH]CH),
6.78 (d, J ¼ 8.4 Hz, 2H, Ph), 7.25 (d, J ¼ 8.4 Hz, 2H, Ph); 13C NMR
(100 MHz, CDCl3)
128.68, 130.12, 130.99, 155.27.
d
ꢀ4.54, 18.29, 25.63, 44.50, 120.35, 127.41,
product. 1H NMR (400 MHz, CDCl3)
d
0.23 (s, 6H, CH3), 0.99 (s, 9H,
tBu) 6.33 (d, J ¼ 16 Hz, 1H, CH]CH), 6.85 (d, J ¼ 8.4 Hz, 2H, Ph), 7.46
(d, J ¼ 8.4 Hz, 2H, Ph), 7.74 (d, J ¼ 16 Hz, 1H, CH]CH); 13C NMR
4.1.19. (E)-3-(4-Methoxyphenyl)acrylonitrile (22)
A mixture of 4-iodo anisole (21) (20 g, 85.46 mmols), acryloni-
trile (18.14 g, 342 mmols), Pd(OAc)2 (1.92 g, 8.55 mmols), Bu4NBr
(CDCl3)
d
ꢀ4.16, 18.46, 25.83, 114.77, 120.80, 127.59, 130.24, 146.94,
158.49, 171.23.

152
M.B. Dewal et al. / European Journal of Medicinal Chemistry 51 (2012) 145e153
(5.51 g, 17.1 mmols) and NaHCO3 (14.36 g, 171 mmols) were
vigorously stirred in 80 mL of distilled water at 80 ꢁC for 10 h under
argon. The reaction mixture was cooled to room temperature and
extracted with diethyl ether (3 ꢂ 150 mL). The combined organic
layer was washed with distilled water, brine and then dried over
anhydrous Na2SO4. The organic layer was evaporated in vacuo and
the resulting material purified by silica column chromatography
(7:3 CH2Cl2:hexane) yield 4.89 g (48%) of the product as a white
each well, 120
mL of the 1% red blood cell stock was treated with
increasing concentrations of 1 or 2, dissolved in DMSO) and the
final volume was adjusted to 150 mL by the addition of TBS. The
plate was incubated at 37 ꢁC for 1 h. After incubation, the plate was
centrifuged at 3800 rpm for 5 min to collect the red blood cells and
the supernantant was analyzed by diluting 20
with TBS buffer until the final volume reached 120
bance at 414 nm was measure to determine the release of hemo-
globin. The absorbance of negative control solution was set to 0%
hemolysis while the absorbance of the 1% Triton-X was set to 100%
hemolysis.
mL of the supernatent
mL. The absor-
solid. 1H NMR (400 MHz, CD2Cl2)
d 3.83 (s, 3H, CH3), 5.75 (d,
J ¼ 16.8 Hz, 1H, CH]CH), 6.92 (d, J ¼ 8.8 Hz, 2H, Ph), 7.35 (d,
J ¼ 16.4 Hz, 1H, CH]CH), 7.42 (d, J ¼ 8.4 Hz, 2H, Ph); 13C NMR
(100 MHz, CD2Cl2)
150.43, 162.64.
d 30.24, 93.91, 114.97, 119.2, 126.93, 129.61,
4.4. Antibacterial activity
4.1.20. (E)-3-(4-Methoxyphenyl)prop-2-en-1-amine (6)
Eight bacterial strains (five Gram-positive and 3 Gram-negative
pathogens) were selected from the Anti-Infective Research Labo-
ratory collection. Tested isolates included 3 S. aureus (VISA Mu50,
MRSA 494, and VRSA MI) strains, S. pneumoniae (ATCC 49619),
Enterococcus faecium (VRE 7303), Escherichia coli (ATCC 25922),
Pseudomonas aeruginosa (ATCC 27853), E. aerogenes (CEF 3978) and
Klebsiella pneumoniae (CEF 3978). Antibacterial activity of the
thieno[2,3-d]pyrimidinedione compounds 1e4, 11a, 16a, and 16b
was evaluated by determining minimum inhibitory concentration
(MIC) values using the broth microdilution method [32] Briefly,
strains were grown overnight at 35 ꢁC on Tryptic soy agar plates
(TSA, Difco, Detroit, MI). Bacterial inocula (0.5 Mac Farland, i.e.
108 CFU/mL) were prepared by suspending few colonies grown
onto a TSA plate in sterile normal saline solution. Resulted bacterial
suspensions were diluted in MuellereHinton broth (MHB, Difco,
Detroit, MI) in order to reach an inoculum of 1e2 ꢂ 106 CFU/mL. A
Lithium aluminum hydride (0.751 g, 19.79 mmols) was added to
25 mL of anhydrous ethyl ether and the resulting solution was
stirred at room temperature for 20 min. To a solution of the
reductant, 22 (3.15 g, 19.79 mmols), dissolved in 15 mL of anhy-
drous ethyl ether, was added drop wise over a 10 min period. The
reaction was stirred at room temperature for an additional 30 min
and then was quenched by the addition of (2.5 mL) of15% NaOH.
The reaction was extracted with ethyl ether (3 X 100 mL) and the
combined organic layers were washed with brine before being
dried with anhydrous Na2SO4. The organic layer was evaporated in
vacuo and the resulting crude residue was purified by flash silica
column chromatography (1:10:89 NH4OH: CH3OH: CH2Cl2)to
generate the desired pure product (1.95 g, 61%) in good yield. 1H
NMR (400 MHz, CDCl3)
d 1.40 (s, 2H, br, NH2), 3.47 (s, 2H, br, CH2),
3.81 (s, 3H, CH3) 6.17e6.22 (quintet, J ¼ 20 Hz, 1H, CH]CH), 6.45 (d,
J ¼ 16 Hz, 1H, CH]CH), 6.85 (d, J ¼ 8.4 Hz, 2H, Ph), 7.31 (d,
volume of 100
added to 100
m
L of this inoculum (final titer 5 ꢂ 105 CFU/mL) was
J ¼ 8.4 Hz, 2H, Ph); 13C NMR (100 MHz, CDCl3)
d 29.93, 44.64, 55.51,
mL of twofold serial dilutions of compounds 1e4 (final
114.2, 127.58, 129.16, 130.2, 159.22.
concentrations of 0.06e32 mg/L) in MuellereHinton broth. Inocu-
lated plates were incubated at 35 ꢁC for 18e24 h, after which the
MIC values were visually read as the lowest concentration of
compound that prevented bacterial growth.
4.2. MTS assay
The MTS assay was performed using the Cell Titer 96 aqueous
cell proliferation assay (Promega). Fibroblast cells derived from
a NIH Swiss mouse embryo (NIH-3T3) were obtained from ATCC
and maintained in Dulbecco’s Modified Eagles Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS). The cells were
grown to 3 passages and approximately 10,000 cells were seeded
into each well of a 96-well plate and allowed to incubate overnight
to allow cells to attach to the substrate. Individual wells were
treated with various concentrations of compounds 1e4 (10, 25, 50
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