Synthesis, cytotoxic evaluation and target identification of thieno[2,3-d]pyrimidine derivatives with a dithiocarbamate side chain at C2 position

Article abstract of DOI:10.1016/j.ejmech.2018.05.028

Two series of thieno[2,3-d]pyrimidine derivatives bearing a dithiocarbamate side chain at the C2 position were synthesized and evaluated for cytotoxic activity in human lung cancer A549 and colon cancer HCT-116 cell lines. Compound 3n exhibited the most cytotoxic effect on A549 cells with an IC₅₀ value of 4.87 μM, inducing a cell cycle arrest at G2/M phase and activating the spindle assembly checkpoint (SAC). To identify the target protein(s) of 3n, we incorporated biotin with 3n through a three-carbon chain and an amide bond to synthesize probe 10. The targeted proteins were pulled down from the A549 total cell lysate by biotin-streptavidin affinity purification and analyzed by mass spectrometry. Tubulin was the only protein identified, which is related to the SAC and directly binds to probe 10 both in vivo and in vitro. Furthermore, compound 3n inhibited tubulin polymerization in vitro in a dose-dependent manner, competed with taxol in binding to tubulin, exerting cytotoxic activity toward taxol-resistant A549 cells. These results demonstrate that thieno[2,3-d]pyrimidine derivative 3n exhibits cytotoxicity in cancer cells by targeting tubulin to activate the SAC and potentially acts as a therapeutic lead compound for taxol-resistant cancers.

Full text of DOI:10.1016/j.ejmech.2018.05.028

Accepted Manuscript
Synthesis, cytotoxic evaluation and target identification of thieno[2,3-d]pyrimidine
derivatives with a dithiocarbamate side chain at C2 position
Chao-Rui Yang, Bin Peng, Sheng-Li Cao, Ting-Ting Ren, Wei Jiang, Fu-Cheng
Wang, You-Shan Li, Guo Wang, Zheng Li, Shibin Xu, Ji Liao, Hailong Wang, Jing Li,
Xingzhi Xu
PII:
S0223-5234(18)30441-0
10.1016/j.ejmech.2018.05.028
EJMECH 10439
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 March 2018
Revised Date: 30 April 2018
Accepted Date: 20 May 2018
Please cite this article as: C.-R. Yang, B. Peng, S.-L. Cao, T.-T. Ren, W. Jiang, F.-C. Wang, Y.-S. Li, G.
Wang, Z. Li, S. Xu, J. Liao, H. Wang, J. Li, X. Xu, Synthesis, cytotoxic evaluation and target identification
of thieno[2,3-d]pyrimidine derivatives with a dithiocarbamate side chain at C2 position, European Journal
of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.028.
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ACCEPTED MANUSCRIPT
Synthesis, cytotoxic evaluation and target identification of thieno[2,3-d]pyrimidine
derivatives with a dithiocarbamate side chain at C2 position
Chao-Rui Yang , Bin Peng , Sheng-Li Cao *, Ting-Ting Ren , Wei Jiang , Fu-Cheng Wang , You-Shan Li , Guo Wang , Zheng
a,1
b,1
a,
a
a
a
a
a
b,c
c
b,c
c
c
b,
Li , Shi-Bin Xu , Ji Liao , Hai-Long Wang , Jing Li , Xingzhi Xu **
a
Department of Chemistry and Beijing Key Laboratory of DNA Damage Response, Capital Normal University, Beijing 100048,
PR China
b
Shenzhen University School of Medicine and Guangdong Key Laboratory for Genome Stability & Disease Prevention, Shenzhen,
Guangdong 518060, PR China
c
College of Life Science and Beijing Key Laboratory of DNA Damage Response, Capital Normal University, Beijing 100048, PR
China

ACCEPTED MANUSCRIPT
Synthesis, cytotoxic evaluation and target identification of thieno[2,3-d]pyrimidine
derivatives with a dithiocarbamate side chain at C2 position
a,1
b,1
a,
a
a
a
a
Chao-Rui Yang , Bin Peng , Sheng-Li Cao *, Ting-Ting Ren , Wei Jiang , Fu-Cheng Wang , You-Shan Li ,
a
,c
c
b
c
c
b,
Guo Wang , Zheng Li , Shibin Xu , Ji Liao , Hailong Wang , Jing Li , Xingzhi Xu **
a
Department of Chemistry and Beijing Key Laboratory of DNA Damage Response, Capital Normal University,
Beijing 100048, PR China
b
Shenzhen University School of Medicine and Guangdong Key Laboratory for Genome Stability & Disease
Prevention, Shenzhen, Guangdong 518060, PR China
c
College of Life Science and Beijing Key Laboratory of DNA Damage Response, Capital Normal University,
Beijing 100048, PR China
ABSTRACT: Two series of thieno[2,3-d]pyrimidine derivatives bearing a dithiocarbamate side chain at the C2
position were synthesized and evaluated for cytotoxic activity in human lung cancer A549 and colon cancer
HCT-116 cell lines. Compound 3n exhibited the most cytotoxic effect on A549 cells with an IC value of 4.87 µM,
50
inducing a cell cycle arrest at G2/M phase and activating the spindle assembly checkpoint (SAC). To identify the
target protein(s) of 3n, we incorporated biotin with 3n through a three-carbon chain and an amide bond to
synthesize probe 10. The targeted proteins were pulled down from the A549 total cell lysate by biotin-streptavidin
affinity purification and analyzed by mass spectrometry. Tubulin was the only protein identified, which is related
to the SAC and directly binds to probe 10 both in vivo and in vitro. Furthermore, compound 3n inhibited tubulin
polymerization in vitro in a dose-dependent manner, competed with taxol in binding to tubulin, exerting cytotoxic
1

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activity toward taxol-resistant A549 cells. These results demonstrate that thieno[2,3-d]pyrimidine derivative 3n
exhibits cytotoxicity in cancer cells by targeting tubulin to activate the SAC and potentially acts as a therapeutic
lead compound for taxol-resistant cancers.
Keywords: Thieno[2,3-d]pyrimidine, dithiocarbamate, cytotoxicity, probe, target identification, tubulin.
Abbreviations: Boc, tert-butyloxycarbonyl; CPT, camptothecin; DMF, N,N-dimethylformamide; DMSO,
dimethyl sulphoxide; DSB, DNA double-strand break; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride; EDTA, ethylenediaminetetraacetic acid; 5-FU, 5-fluorouracil; γ-H2AX, histone H2AX
phosphorylated on serine 139; pS10 H3, phospho-histone H3 on serine 10; MTS,
3
-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PI,
propidium iodide; rt, room temperature; SAC, spindle assembly checkpoint; SDS, Sodium dodecyl sulfate; TEA,
triethylamine; TFA, trifluoroacetic acid.
*
Corresponding author.
*
*
Corresponding author.
E-mail address: slcao@cnu.edu.cn (S.-L. Cao); xingzhi.xu@szu.edu.cn (X. Xu).
1
These authors contributed equally to this work.
2

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1
. Introduction
Thieno[2,3-d]pyrimidine is an important heterocycle core structure from which various chemical derivatives
can be synthesized. These derivatives have received notable attention over the past years due to the broad
spectrum of biological activities, such as antitumor [1-3], antiviral [4], anti-inflammatory [5,6], analgesic [7] and
antibacterial activity [8,9]. As such, many antitumor agents based on thieno[2,3-d]pyrimidine have been
developed by researchers. Gangjee et al., [10,11] synthesized 2-aminothieno[2,3-d]pyrimidin-4(3H)-one
derivatives bearing a benzoyl-L-glutamic acid moiety or lipophilic side chain at the 5-position and evaluated these
derivatives as classical or non-classical antifolate antitumor agents. Of these compounds, one was an excellent
dual inhibitor of human thymidylate synthase (TS, IC = 54 nM) and dihydrofolate reductase (DHFR, IC = 19
50
50
nM) and had nanomolar GI50 values in eight human cancer cell lines (Fig. 1; I) [11]. Bugge et al., designed and
synthesized many thieno[2,3-d]pyrimidine derivatives and examined the effects of varying the 4-amino group and
6
-aryl group on its inhibitory potency against epidermal growth factor receptor tyrosine kinase (EGFR-TK),
antiproliferative and toxicity, and ADME properties. One of their compounds (Fig. 1; II) was a potential drug
candidate for EGFR-driven cancers [12,13]. A high-throughput screening (HTS) assay revealed that tricycle
tetrahydrobenzothieno[2,3-d]pyrimidine derivatives disrupt cell proliferation mediated by aberrant EGFR
signaling. Wu et al. [14] introduced a chiral side chain and a Michael acceptor group into this scaffold and the
resulting derivative showed >3 orders of magnitude enhanced HCC827 antiproliferative activity compared to the
lead compound (Fig.1; III). This compound also inhibited gefitinib-resistant EGFR double mutant (DM,
T790M/L858R) at nanomolar concentrations. Moreover, exposing a xenograft nude mouse model to this
compound shrinked tumor growth significantly [14]. Although the majority of studies have focused on
3

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substitutions at the C4, C5, and C6 positions of thieno[2,3-d]pyrimidine, there have been increasing reports of C2
substitutions on antitumor activity [15-17].
Natural and synthetic dithiocarbamates have excellent biological activities. Brassinin (Fig. 1) is a
phytoalexin that can induce apoptosis in human prostate cancer cells by blocking the PI3K/Akt/mTOR/S6K1
signaling pathways [18]. Brassinin and its synthetic analogue 5-bromobrassinin exert anticancer effects by
inhibiting indoleamine 2,3-dioxygenase, a pro-toleragenic enzyme that drives immune escape in cancer [19]. Li et
al. [20] synthesized a compound with potent antiproliferative activity against four selected human cancer cell
lines (HL-60, Bel-7402, SK-BR-3 and MDA-MB-468) (Fig. 1; IV). This compound induced apoptosis and G2/M
cell-cycle arrest by disrupting spindle assembly during mitotic progression. We incorporated the dithiocarbamate
moiety into the 6-position of 2-methylquinazlin-4(3H)-one to generate a set of compounds, several of which
inhibited proliferation of human cancer cell lines with IC values at micromolar concentration (Fig. 1; V and VI)
50
[
21-23]. Recently, we transferred the dithiocarbamate moiety from the 6-position to 2-position of
quinazlin-4(3H)-one to synthesize another series of compounds, most of which markedly inhibited the
proliferation of A549, MCF-7, HeLa, HT29 and HCT-116 cell lines . In particular, one compound induced G2/M
arrest in HT-29 cells by promoting tubulin polymerization in vitro and subsequently activating the spindle
assembly checkpoint (Fig. 1; VII) [24]. The dithiocarbamate motif is also a suitable linkage region for combining
different biologically active scaffolds and thus to design and synthesize new chemical entities [25].
<
Fig. 1 >
In this study, compounds 3a−s were synthesized as isosteric analogues of compound VII, in which the
thieno[2,3-d]pyrimidin-4(3H)-one ring is connected to the sulfur atom of dithiocarbamate via a methylene group
4

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(
Scheme 1). For comparison purposes, we also synthesized compounds 6a−l in which the
thieno[2,3-d]pyrimidin-4(3H)-one ring is connected to the nitrogen atom of dithiocarbamate (Scheme 2). We
evaluated the cytotoxic activity of the resulting compounds in cultured lung cancer A549 and colon HCT-116 cells.
Of these compounds, 3n was most potent towards A549 cells, which was carried forward to investigate the
mechanisms underlying its cytotoxicity.
2
. Results and discussion
2
.1. Synthesis of compounds 3a−s and 6a−l
Scheme 1 outlines the synthetic pathway of compounds 3a−s in which the thieno[2,3-d]pyrimidin-4(3H)-one
ring is connected to the sulfur atom of dithiocarbamate via a methylene group. Reacting ethyl 2-cyanoacetate and
propionaldehyde with elemental sulfur in the presence of triethylamine (TEA) gave ethyl
2
2
2
-amino-5-methylthiophene-3-carboxylate (intermediate 1) [10]. This product was reacted with excess
-chloroacetonitrile
in
the
presence
of
hydrogen
chloride
gas
to
yield
-(chloromethyl)-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (intermediate 2) [26]. Intermediate 2 reacted with
anhydrous potassium phosphate, carbon disulfide and the appropriate benzylamine, substituted benzylamine, or
1
heterocyclylmethylamine to generate compounds 3a−s (for R groups, see Scheme 1).
<
Scheme 1 >
Scheme 2 outlines the synthetic pathway of compounds 6a−l in which the thieno[2,3-d]pyrimidin-4(3H)-one
ring is linked to the nitrogen atom of dithiocarbamate. Reacting intermediate 2 with hexamethylenetetramine in
tetrahydrofuran (THF) generated quaternary ammonium (4), which was treated with concentrated hydrochloric
acid in methanol to give 2-(aminomethyl)-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (5), as previously
5

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described [23]. Reaction of intermediate 5, anhydrous potassium phosphate and carbon disulfide with the
2
appropriate benzyl bromide, or substituted benzyl bromide, generated compounds 6a−l (for R groups, see
Scheme 2).
<
Scheme 2 >
2
.2. Synthesis of probe 10 (biotinylated compound 3n) and control probe 14
Cell proliferation assays indicated that compound 3n had the most cytotoxic effect on A549 cells (IC , 4.87
50
µM) compared to all the other thieno[2,3-d]pyrimidine derivatives. To identify the target protein, we synthesized a
biotinylated 3n probe (10) for affinity pull-down assays. Here, 4-cyanobenzylbromide reacted with
mono-protected propane-1,3-diamine to give intermediate
7
(Scheme 3). Reaction of
7
with
2
-chloromethyl-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (2), carbon disulfide and potassium phosphate
generated 8, which was deprotected with trifluoroacetic acid (TFA) to give intermediate 9. Finally, coupling 9
with D-(+)-biotin in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI)
yielded the probe (10).
<
Scheme 3 >
We then prepared compound 11 by reacting intermediate 2 with 4-cyanobenzylamine, from which the
carbodithionate moiety was removed (Scheme 4). This analogue of 3n had no cytotoxicity towards A549 and
HCT-116 cells, indicating that the carbodithionate moiety is critical to cytotoxic activity. Therefore, we introduced
biotin into compound 11 via an amide bond and a three-carbon chain to synthesize compound 14, which served as
a control probe. Reaction of intermediate 2 with 7 gave compound 12 (Scheme 4). Removal of the Boc group in 12
with TFA, followed by the EDCI-mediated coupling of 13 with D-(+)-biotin produced compound 14.
6

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<
Scheme 4 >
2
.3. Structures of the target compounds
1
13
The structures of target compounds 3a−s and 6a−l were characterized by H NMR, C NMR and ESI-HRMS.
l
In the H NMR spectra of compounds 3a−s, the signals of CH3 groups at the C6 position of the
thieno[2,3-d]pyrimidin-4(3H)-one ring appear at δ 2.48−2.50 ppm as doublets (J = 1.2 Hz), due to a long-range
coupling of the C5-hydrogen to one of the methyl protons. Accordingly, the signals of C5-hydrogens appear at δ
7
.05−7.07 ppm as doublets with the same J value (1.2 Hz). The singlets at δ 4.45−4.50 ppm are assigned to the
CH groups connected to the sulfur atoms, and the doublets at δ 4.65−4.99 ppm belong to the CH groups linked to
2
2
the NH groups in the dithiocarbamate moiety. The triplets at δ 10.29−10.70 ppm are assigned to the NH groups
adjacent to the CH groups, and the singlets at δ 12.50−12.54 ppm are attributed to the NH groups in the
2
thieno[2,3-d]pyrimidin-4(3H)-one ring. In addition to the signals as discussed above, the signals ranging from δ
1
3
6
.36 to 8.53 ppm belong to CH groups in phenyl, substituted phenyl or heterocyclic rings. The C NMR spectra of
compounds 3a−s exhibit characteristic signals at δ 15.48−16.42 for methyl carbons, δ 36.89−37.83 and
3.62−50.87 for S-methylene and N-methylene carbons, δ 162.93−163.45 and 194.74−196.54 for carbonyl and
4
l
13
thiocarbonyl carbons, and other expected signals as well. The H NMR and C NMR spectra of compounds 6a−l
are very similar to those of compounds 3a−s. The ESI-HRMS data of all the target compounds is in good
agreement with their calculated values.
The dithiocarbamate N–C(=S) single bonds (Fig. 2, shown in red) have partial double bond character. For
compounds 3a−s and 6a−l, rotations around the N–C(=S) bonds are free due to a small hydrogen atom attached to
1
each nitrogen atom of the dithiocarbamates; doubling of signals was not observed in their H NMR spectra. In
intermediates 8, 9 and probe 10, however, the hydrogen atom attached to the nitrogen atom of dithiocarbamate is
substituted by a (tert-butyloxycarbonyl)aminopropyl (8), 3-aminopropyl (9) or biotinylaminopropyl group (10),
respectively, and a 4-cyanobenzyl group is attached to the nitrogen atom. Rotations around the C–N(=S) bonds in
these molecules (8−10) are restricted, and they can exist as rotamers (rotational isomers). The protons of
7

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1
intermediates 8 and 9, all exhibited two sets of peaks in the H NMR spectra (in a nearly 3:2, or 1:1 ratio,
respectively) corresponding to the two rotamers (Fig. 2). For probe 10, protons (except those in the biotin moiety)
1
also exhibited major and minor signals (in a nearly 3:2 ratio) in the H NMR spectrum. As intermediate 8 or probe
1
0 has a more bulky tert-butyloxycarbonyl (Boc) or biotinyl group than a proton in 9, the E-rotamers are more
13
stable than the Z-rotamers, and the former accounts for the larger proportion in the mixture (~60%). The C NMR
spectrum of probe 10 showed a similar signal doubling pattern for the carbon atoms except for those in the biotin
moiety and the methyl group at the C5-position of thieno[2,3-d]pyrimidine.
<
Fig. 2 >
2
.4. Cytotoxic activity
Compounds 3a−s and 6a−l were initially evaluated by MTS assay at a single concentration of 30 µM for their
cytotoxicity towards A549 (lung cancer), HeLa (cervical carcinoma), MCF-7 and MDA-MB-231 (breast
adenocarcinoma), HCT-116 and HT-29 (colorectal cancer) cells. Both A549 and HCT-116 cells were more
sensitive than all other cells to compounds 3a−s. However, hardly any cancer cells tested were sensitive to
compounds 6a−l at 30 µM (data not shown). These results indicated that connection of the
thieno[2,3-d]pyrimidine ring to the sulfur atom of the dithiocarbamate moiety is essential for cytotoxicity.
The compounds in the 3a−s series that induced ≥50% inhibition compared to vehicle were considered
“active”. We therefore analyzed the effects of these active compounds across a range of concentrations to
determine the IC values for 50% inhibition of cellular proliferation (Tables 1). 5-Fluorouracil (5-FU) was used
5
0
as a positive control. Most of the compounds 3a−s exhibited a cytotoxic effect on cultured A549 and HCT-116
cells (IC < 30 µM), and they were generally more active in A549 than HCT-116 cells (Table 1). Compound 3a
5
0
8

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(
without a substituent on the benzene ring) had an IC value of 29.53 µM against A549 cells. Compounds with a
50
methyl (3b) or methoxy (3c) group at the 4-position of benzene ring were comparable to the parent compound 3a,
whereas compound 3d (with a methoxy group at the 2-position of benzene ring) was more potent towards A549
cells than 3a. Compound 3e containing a 2,4-dimethoxy phenyl group exhibited weak cytotoxic activity.
Compounds containing 3,4,5-trimethoxyphenyl (3f) or 3,4-methylenedioxyphenyl group (3g) were more active
than compound 3a. Compounds containing a moderate electron-withdrawing group at the 4-position of the
benzene ring, namely, 3h (4-bromo), 3i (4-chloro) or 3k (4-fluoro), but not 3l (2-fluoro), were more cytotoxic than
the parent compound 3a. Compound 3j (containing a 2,4-dichlorophenyl group) had equivalent activity as 3a,
whereas 3m (containing a 2,4-difluorophenyl group) was more active than 3a. Notably, compounds bearing a
strong electron-withdrawing group at the 4-position of benzene ring, 3n (4-cyano) and 3o (4-nitro), were more
potent than 3a, with 3n being the most potent against A549 cells (IC , 4.87 µM).
50
Replacement of the phenyl group with an electron-rich heterocycle in compound 3a resulted in compounds
1
1
3
p (R = thiophen-2-yl) and 3q (R = furan-2-yl), which were comparable to or less potent than 3a, respectively.
1
1
However, compounds containing an electron-deficient pyridinyl groups, namely, 3r (R = pyridin-3-yl) and 3s (R
=
pyridin-4-yl), were more potent than 3a. These results demonstrate that aryl groups with an
electron-withdrawing group instead of an electron-donating group are favorable for cytotoxicity, suggesting that
the density of the electron cloud on aryl or heteroaryl groups is critical for cytotoxicity.
Most compounds in the 3a−s series had IC values >20 µM in HCT-116 cells. Only four compounds, namely,
50
3
d, 3f, 3r and 3s, had IC values <20 µM, but they were less potent than 5-FU. By contrast, compound 3n had a
50
comparable IC50 value to 5-FU (4.78 versus 3.52 µM) towards A549 cells. Therefore, compound 3n was taken
9

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forward to further investigate the mechanisms underlying cytotoxicity of cancer cells.
<
Table 1 >
2
.5. Compound 3n induced G2/M arrest in A549 cells
We first examined the effects of compound 3n on cell-cycle progression. A549 cells were exposed to 3n at
various concentrations (0.5×, 1.0×, or 2.0× the IC ), 5-FU (IC ), or taxol (0.10 µM) for 24 h, and analyzed for
50
50
DNA content by flow cytometry. Three independent experiments were performed (Table S1 and Fig. 3A), and the
results from one representative experiment are shown in Fig. 3B. In agreement with previous reports, A549 cells
treated with 5-FU or taxol for 24 h arrested in S-phase or G2/M phase, respectively; we observed an obvious
G2/M phase arrest in 3n-treated A549 cells. Moreover, we observed a dose-dependent accumulation of G2/M
cells when using concentrations from half to twice of the 3n IC value.
50
<
Fig. 3 >
2
.6. Compound 3n did not induce DNA double strand breaks in A549 cells
As compound 3n induces G2/M arrest in A549 cells, we reasoned that 3n might induce DNAdamage. To test
this hypothesis, we treated A549 cells with camptothecin (CPT, 2 µM) for 2 h or compound 3n for 12 h.
Immunofluorescence staining and immunoblotting using an antibody specific for the DSB (DNA double-strands
break) marker γ-H2AX (histone H2AX phosphorylated on serine 139) was performed to examine whether these
compounds could induce DSBs in cultured cancer cells. CPT is an inhibitor of topoisomerase I and induces
replication-dependent DNA damage. Here, we found that treatment with CPT, but not compound 3n, resulted in
distinct γ-H2AX foci formation (Fig. 4A) and an increase in phosphorylation of H2AX and CHK1 (in response to
DNA damage, ATR phosphorylates CHK1 at Ser345) (Fig. 4B). These results demonstrated that compound 3n
1
0

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cannot induce DNA damage under these conditions.
<
Fig. 4 >
2
.7. Compound 3n activated the spindle assembly checkpoint
Both DNA damage-induced G2/M checkpoint activation and tubulin poison-induced spindle-assembly
checkpoint activation can result in G2/M arrest. As compound 3n failed to induce DNA damage, we speculated
that compound 3n might activate the spindle assembly checkpoint to elicit G2/M arrest. To this end, we
determined the mitotic indexes of A549 cells treated with nocodazole or compound 3n for different durations. The
mitotic index was determined by the percentage of cells positive for phospho-histone H3 on serine 10 (pS10 H3).
S10 phosphorylation starts at prophase and quickly disappears when cells move from metaphase to anaphase
[
27,28]. Therefore, cells in G2 or M phase can be distinguished by immunostaining with a pS10 H3 antibody, and
the percentage of M phase cells can be determined by flow cytometry. Nocodazole elicits spindle-assembly
checkpoint activation mainly through binding tubulin, which destabilizes tubulin polymerization and arrests cells
in prometaphase. Nocodazole treatment led to a continuing increase of mitotic indexes from 2 to 16 hours (Table
2
5
3
). Compound 3n treatment resulted in a similar increase in the mitotic indexes, but to a less extent (Table 2, Fig.
, A and B). Immunoblotting analysis confirmed that H3 phosphorylation at S10 increased following compound
n exposure from 12 h up to 24 h (Fig. 5C). BubR1 (MAD-3 like) protein is an essential kinase for kinetochores to
establish microtubule attachments and the spindle assembly checkpoint. BubR1 is hyperphosphorylated by PLK1
during mitosis, which stabilizes the kinetochore attachments, leading to spindle checkpoint activation [29,30].
A549 cells treated with compound 3n resulted in an increase of slow migration form of BubR1, which suggests a
phosphorylation modification (Fig. 5C). Taken together, these results are consistent with compound 3n activating
1
1

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the spindle assembly checkpoint.
<
Table 2 >
<
Fig. 5 >
2
.8. Affinity purification and identification of compound 3n-binding proteins
To identify the target protein(s) of compound 3n, we incubated probe 10 (biotinylated compound 3n) with
A549 total cell lysate. Streptavidin-coated magnetic beads were then used to pull down the biotinylated probe and
associated binding proteins, which were resolved by SDS−PAGE and visualized by silver staining (Fig. 6). Both
biotin alone (left lane) and the control probe (right lane) only pulled down a single band at the size of about 55 kDa,
while probe 10 (middle lane) pulled down numerous bands. The binding proteins were eluted from the
streptavidin-biotin probes and digested in situ with trypsin, the resulting peptide mixture was analyzed by mass
spectrometry (Q Exactive HF LC/MS). The protein identities were determined by the molecular weight of the
peptide mass fingerprint using pFind software (version 2.8, Chinese Academy of Sciences) searching from the
UniProt database (Table 3). Among the major proteins pulled down by probe 10 (Table 3), both Tubulin α and
Tubulin β functionally link to the SAC.
<
Fig. 6 >
<
Table 3 >
We then sought to determine if compound 3n directly binds to tubulin. We found by immunoblotting analysis
that α-tubulin was pulled down by the probe 10, but not by biotin or the control probe 14 (Fig. 7A). As the
pull-down of α-tubulin by the probe may be indirect, we carried out streptavidin-pulldown assays using purified
porcine neuronal tubulin and the probes. It was found that α-tubulin was present in the protein complex pulled
1
2

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down by probe 10, but not by biotin or the control probe 14, through immunoblotting analysis (Fig. 7B). These
results indicate that α-tubulin can directly bind compound 3n.
<
Fig. 7 >
2
.9. Compound 3n interferes with tubulin polymerization in vitro
Given that compound 3n directly binds tubulin, we sought to determine if the compound would interfere with
tubulin function. In vitro tubulin polymerization assays were conducted with 3n at various concentrations (5, 10
and 20 µM). Nocodazole (a well-known inhibitor of tubulin polymerization) and taxol (a stabilizer of tubulin
polymerization) were used as controls. Here, we found that compound 3n compromised tubulin polymerization in
a dose-dependent manner, with less effect at 20 µM when compared to nocodazole treatment (Fig. 8).
<
Fig. 8 >
2
.10. Compound 3n competes with taxol in binding to tubulin
The data thus far prove that compound 3n targets tubulin, likely activating the SAC through destabilizing
tubulin polymerization. There are three distinct and well-documented domains in tubulin for binding to paclitaxel,
colchicine and vinblastine [31,32]. To determine if compound 3n competitively binds to any of these three
domains, we preincubated tubulin with excess taxol, colchicine, vinblastine, or nocodazole whose binding region
in tubulin overlaps with that of colchicines [33], for 2 h and then repeated the binding experiment. We found that
preincubation of tubulin with colchicine, vinblastine or nocodazole did not affect the binding of the probe with
tubulin, while the probe bound less tubulin when preincubated with taxol (Fig. 9A, 9B). Next, we performed
binding experiments using purified tubulin and probe 10. The bead-bound probe-tubulin complex was then
incubated with increasing concentrations of nocodazole, taxol, vinblastine, or colchicine before SDS-PAGE and
1
3

ACCEPTED MANUSCRIPT
immunoblotting. Again, we found that the increasing doses of nocodazole, vinblastine, or colchicine did not
reduce the amount of bound tubulin with the probe (Fig. 9D, 9E, 9F), while less tubulin bound the probe after
incubation with taxol (Fig. 9C). These results demonstrated that compound 3n competes with taxol in binding to
tubulin.
<
Fig. 9 >
Competitive binding assays uncovered that compound 3n may share the same binding site as taxol in tubulin.
However, in contrast to taxol stabilizing tubulin polymerization, 3n inhibits tubulin polymerization. We therefore
exposed taxol-resistant A549 cells to compound 3n or taxol for 72 h, and then assessed cell proliferation by MTS
assay. Taxol was ineffective in taxol-resistant A549 cells (IC50 > 30 µM), whereas compound 3n markedly
inhibited the proliferation of taxol-resistant A549 cells (IC = 7.31 ± 0.11 µM). Although 3n was less potent to
50
taxol-resistant A549 cells than A549 cells, it may be a potential lead for the development of anticancer agents
treating taxol-resistant cancers.
3
. Conclusion
This study designed and synthesized two series of compounds by incorporating a dithiocarbamate moiety
into the C2 position of thieno[2,3-d]pyrimidine scaffold. These compounds (3a−s and 6a−l) were evaluated as
antitumor agents in cultured lung cancer A549 and colon HCT-116 cells. Most compounds in the series 3a−s (in
which thieno[2,3-d]pyrimidine is linked to the sulfur atom of dithiocarbamate) were cytotoxic towards A549 and
HCT-116 cells. However, compounds 6a−l (in which thieno[2,3-d]pyrimidine is linked to the nitrogen atom of
dithiocarbamate) were inactive against the two cancer cell lines. Flow cytometry analysis demonstrated that the
representive compound 3n arrests A549 cells in G2/M phase and activates the spindle assembly checkpoint. In
1
4

ACCEPTED MANUSCRIPT
vitro tubulin polymerization assays revealed that compound 3n inhibited tubulin polymerization in a
dose-dependent manner. LC-MS/MS mass spectrometry analysis and both in vitro and in vivo binding assays
uncovered that tubulin directly binds to the biotinylated conjugating 3n (probe 10). Unexpectedly, compound 3n
competes with taxol, a stabilizer of tubulin polymerization, in binding to tubulin, and thus inhibits proliferation of
taxol-resistant A549 cells. Taken together, thieno[2,3-d]pyrimidine derivatives bearing a dithiocarbamate side
chain at the C2-position interfere with tubulin polymerization to activate the spindle assembly checkpoint, arrest
the cell cycle at G2/M phase and exert cytotoxicity toward taxol-resistant A549 cells, potentially acting as a novel
therapeutic lead compound for taxol-resistant cancers.
4
. Experimental
4
.1. Chemistry
1
Melting points were determined on an XT5B microscopic melting point apparatus and are uncorrected. H
NMR spectra were recorded on a Varian VNMRS-600 spectrometer at 600 MHz using tetramethylsilane (TMS) as
1
3
an internal standard. C NMR spectra were recorded on a Varian VNMRS-600 spectrometer at 150 MHz using
tetramethylsilane (TMS) as internal standard. High-resolution electrospray ionization (HR-ESI) mass spectra
were recorded on a Thermo Scientific LTQ Orbitrap Discovery (Bremen, Germany) mass spectrometer. Column
chromatography was carried out on a silica gel (200−300 mesh). High performance liquid chromatography
(
(
HPLC) analyses were performed on an Agilent 1200 Series HPLC instrument with a G1314B VWD
variable-wavelength detector) (λ = 256 nm, mobile phase: MeOH:H O = 7:3 (v/v)), and the data are presented in
2
the Supplementary data. The purity of all compounds used in the biological studies was ≥ 95%. Arylmethylamines,
heterocyclylmethylamines and arylmethylbromides were obtained commercially and used without further
1
5

ACCEPTED MANUSCRIPT
purification.
4
.1.1. Preparation of ethyl 2-amino-5-methylthiophene-3-carboxylate (intermediate 1)
A mixture of ethyl cyanoacetate (2.26 g, 20 mmol), triethylamine (1.02 g, 10 mmol) and sulfur (0.64 g, 20
mmol) in N,N-dimethylformamide (DMF, 20 mL) was stirred at 50 °C for 30 min. Propionaldehyde (1.16 g, 20
mmol) was added dropwise, and the reaction mixture was stirred at room temperature for 2 h. The mixture was
poured into ice-cold water and extracted three times with ethyl acetate (15 mL, each). The combined extracts were
washed with brine, dried over anhydrous sodium sulfate, and filtered and concentrated in vacuo. The residue was
purified by column chromatography on a silica gel (petroleum/ethyl acetate = 9:1) to afford intermediate 1 as a
1
pale yellow solid (2.32 g, 63%), mp 46−47°C (Lit [10] mp 45−47 °C). H NMR (600 MHz, DMSO-d )
δ
: 1.22 (t,
6
J = 7.2 Hz, 3H, CH CH ), 2.18 (d, J = 0.6 Hz, 3H, CH ), 4.14 (q, J = 7.2 Hz, 2H, CH CH ), 6.49 (s, 1H, thiophene
3
2
3
3
2
+
5
-H), 7.08 (s, 2H, NH ). ESI-HRMS m/z: calcd for C H NO S ([M+H] ): 186.0589; found:186.0584.
2 8 12 2
4
.1.2. Preparation of 2-(chloromethyl)-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (intermediate 2)
A solution of ethyl 2-amino-5-methylthiophene-3-carboxylate (1) (32.4 g, 175 mmol) and chloroacetonitrile
(
15.8 g, 210 mmol) in 1,4-dioxane (300 mL) was stirred at room temperature for 6 h, while hydrogen chloride gas
was simultaneously passed through the solution. The resulting solid was collected by filtration and washed with
1
,4-dioxane. The solid was suspended in water (50 mL) and the mixture was adjusted with 25% aqueous ammonia
to pH 7. After stirring for 2 h, the precipitate was collected by filtration and air-dried, and purified by
1
re-crystallization from 1,4-dioxane to give intermediate 2 as a white solid (32.3 g, 86%), mp 211−215 °C. H
NMR (600 MHz, DMSO-d6) δ: 2.52 (d, J = 1.2 Hz, 3H, CH ), 4.56 (s, 2H, ClCH ), 7.11 (d, J = 1.2 Hz, 1H,
3 2
+
thieno[2,3-d]pyrimidine 5-H), 12.74 (s, 1H, NH). ESI-HRMS m/z: calcd for C H ClN OS ([M+H] ): 215.0046;
8
8
2
1
6

ACCEPTED MANUSCRIPT
found:215.0042.
4
.1.3. General preparation procedure for compounds 3a−s
A suspension of amine (2 mmol), carbon disulfide (0.8 mL, 13.3 mmol), and finely powdered potassium
phosphate (0.52 g, 2.45 mmol) in DMF (20 mL) was stirred at room temperature for 0.5 h. After adding
2
-(chloromethyl)-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (2) (0.43 g, 2 mmol), stirring was continued
overnight. The mixture was poured into water (100 mL) and the separated solid was collected by filtration and
air-dried. The crude product was purified by column chromatography on a silica gel using the eluent indicated
below to give compounds 3a−s.
4
.1.3.1. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl benzylcarbamodithioate (3a).
Yield: 86%, white solid, mp 258−260 °C (eluent: dichloromethane/methanol = 98:2), HPLC retention time
1
(
rt) = 6.644 min. H NMR (600 MHz, DMSO-d )
δ
: 2.49 (s, 3H, CH ), 4.48 (s, 2H, SCH ), 4.84 (d, J = 5.4 Hz, 2H,
6
3
2
CH NH), 7.06 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.28 (m, 3H, Ph 2′-H, 4′-H, 6′-H), 7.34 (t, J = 7.2
2
13
Hz, 2H, Ph 3′-H, 5′-H), 10.58 (t, J = 5.4 Hz, 1H, CH NH), 12.52 (s, 1H, NH). C NMR (150 MHz, DMSO-d ) δ:
2
6
1
6
5.95 (6-CH ), 37.33 (SCH ), 50.29 (CH NH), 119.42 (4a-C), 123.62 (5-C), 127.70 (4′-C), 128.08 (2C, 2′-C,
3 2 2
′-C), 128.83 (2C, 3′-C, 5′-C), 137.39 (6-C), 137.42 (1′-C), 153.73 (7a-C), 157.83 (2-C), 163.44 (4-C), 195.52
+
(
C=S). ESI-HRMS m/z: calcd for C H N OS ([M+H] ): 362.0455; found: 362.0442.
1
6
16
3
3
4
.1.3.2. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 4-methylbenzylcarbamodithioate (3b).
Yield: 37%, light yellow solid, mp 168−171 °C (eluent: dichloromethane/methanol = 99:1), HPLC rt =
1
1
0.314 min. H NMR (600 MHz, DMSO-d ) δ: 2.28 (s, 3H, 4′-CH ), 2.49 (d, J = 1.2 Hz, 3H, 6-CH ), 4.47 (s, 2H,
6
3
3
SCH ), 4.77 (d, J = 5.4 Hz, 2H, CH NH), 7.06 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.14 (d, J = 7.8
2
2
1
7

ACCEPTED MANUSCRIPT
Hz, 2H, Ph 3′-H, 5′-H), 7.18 (d, J = 7.8 Hz, 2H, Ph 2′-H, 6′-H), 10.53 (t, J = 5.4 Hz, 1H, CH NH), 12.51 (s, 1H,
2
1
3
NH). C NMR (150 MHz, DMSO-d ) δ: 15.94 (6-CH ), 21.15 (4′-CH ), 37.33 (SCH ), 50.12 (CH NH), 119.42
6
3
3
2
2
(
4a-C), 123.62 (5-C), 128.13 (2C, 2′-C, 6′-C), 129.37 (2C, 3′-C, 5′-C), 134.35 (1′-C), 136.87 (4′-C), 137.39 (6-C),
+
1
3
4
53.75 (7a-C), 157.82 (2-C), 163.42 (4-C), 195.26 (C=S). ESI-HRMS m/z: calcd for C H N OS ([M+H] ):
17
18
3
3
76.0612; found: 376.0611.
.1.3.3. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 4-methoxybenzylcarbamodithioate (3c)
Yield: 45%, light yellow solid, mp 195−198 °C (eluent: dichloromethane/methanol = 98:2), HPLC rt =
1
7
.163 min. H NMR (600 MHz, DMSO-d ) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 3.73 (s, 3H, OCH ), 4.47 (s, 2H,
6
3
3
SCH ), 4.75 (d, J = 5.4 Hz, 2H, CH NH), 6.89 (d, J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 7.06 (d, J = 1.2 Hz, 1H,
2
2
thieno[2,3-d]pyrimidine 5-H), 7.23 (d, J = 8.4 Hz, 2H, Ph 2′-H, 6′-H), 10.50 (t, J = 5.4 Hz, 1H, CH NH), 12.51 (s,
2
1
3
1
H, NH). C NMR (150 MHz, DMSO-d ) δ: 15.94 (6-CH ), 37.32 (SCH ), 49.90 (CH NH), 55.52 (4′-OCH ),
6 3 2 2 3
114.24 (2C, 3′-C, 5′-C), 119.42 (4a-C), 123.62 (5-C), 129.29 (1′-C), 129.64 (2C, 2′-C, 6′-C), 137.39 (6-C),
1
53.77 (7a-C), 157.82 (2-C), 159.02 (4′-C), 163.40 (4-C), 195.02 (C=S). ESI-HRMS m/z: calcd for
+
C H N O S ([M+H] ): 392.0561; found: 392.0562.
1
7
18
3
2 3
4
.1.3.4. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 2-methoxybenzylcarbamodithioate
(3d)
Yield: 47%, light yellow solid, mp 251−253 °C (eluent: dichloromethane/methanol = 99.5:0.5), HPLC rt =
1
8
.382 min. H NMR (600 MHz, DMSO-d ) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 3.81 (s, 3H, OCH ), 4.47 (s, 2H,
6
3
3
SCH ), 4.75 (d, J = 5.4 Hz, 2H, CH NH), 6.91 (t, J = 7.2 Hz, 1H, Ph 5′-H), 7.01 (d, J = 7.2 Hz, 1H, Ph 3′-H), 7.06
2
2
(
d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.17 (d, J = 7.2 Hz, 1H, Ph 6′-H), 7.28 (t, J = 7.2 Hz, 1H, Ph
1
8

ACCEPTED MANUSCRIPT
1
3
4
3
1
′-H), 10.40 (t, J = 5.4 Hz, 1H, CH NH), 12.52 (s, 1H, NH). C NMR (150 MHz, DMSO-d ) δ: 15.95 (6-CH ),
2 6 3
7.36 (SCH ), 46.02 (CH NH), 55.83 (2′-OCH ), 111.15 (3′-C), 119.43 (4a-C), 120.56 (5′-C), 123.63 (5-C),
2
2
3
24.60 (4′-C), 128.78 (6′-C), 129.15 (1′-C), 137.39 (6-C), 153.82 (7a-C), 157.29 (2′-C), 157.83 (2-C), 163.42
+
(
4-C), 195.31 (C=S). ESI-HRMS m/z: calcd for C H N O S ([M+H] ): 392.0561; found:392.0545.
17
18
3
2 3
4
.1.3.5. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 2,4-dimethoxybenzylcarbamodithioate
(3e)
Yield: 61%, yellow solid, mp 170−173 °C (eluent: dichloromethane/methanol = 95:5), HPLC rt = 6.805 min.
1
H NMR (600 MHz, DMSO-d ) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 3.75 (s, 3H, OCH ), 3.79 (s, 3H, OCH ), 4.45 (s,
6
3
3
3
2
3
1
H, SCH ), 4.65 (d, J = 5.4 Hz, 2H, CH NH), 6.47 (dd, J = 7.8, 2.4 Hz, 1H, Ph 5′-H), 6.57 (d, J = 2.4 Hz, 1H, Ph
2 2
′-H), 7.06 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.10 (d, J = 7.8 Hz, 1H, Ph 6′-H), 10.29 (t, J = 5.4 Hz,
1
3
H, CH NH), 12.50 (s, 1H, NH). C NMR (150 MHz, DMSO-d ) δ: 15.93 (6-CH ), 37.35 (SCH ), 45.95
2
6
3
2
(
CH NH), 55.68 (OCH ), 55.93 (OCH ), 98.80 (3′-C), 104.87 (5′-C), 116.72 (1′-C), 119.42 (4a-C), 123.62 (5-C),
2 3 3
1
30.16 (6′-C), 137.39 (6-C), 153.90 (7a-C), 157.81 (2-C), 158.47 (4′-C), 160.69 (2′-C), 163.37 (4-C), 194.74
+
(
C=S). ESI-HRMS m/z: calcd for C H N O S ([M+H] ): 422.0667; found: 422.0663.
1
8
20
3
3 3
4
3
.1.3.6. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl
,4,5-trimethoxybenzylcarbamodithioate (3f)
Yield: 51%, light yellow solid, mp 193−195 °C (eluent: dichloromethane/methanol = 98:2 to 95 :5), HPLC rt
1
=
4.342 min. H NMR (600 MHz, DMSO-d ) δ: 2.48 (s, 3H, CH ), 3.64 (s, 3H, OCH ), 3.73 (s, 6H, 2OCH ), 4.49
6
3
3
3
(
s, 2H, SCH ), 4.75 (d, J = 5.4 Hz, 2H, CH NH), 6.44 (s, 2H, Ph 2′-H, 6′-H), 7.05 (s, 1H, thieno[2,3-d]pyrimidine
2 2
13
5
-H), 10.50 (t, J = 5.4 Hz, 1H, CH NH), 12.51 (s, 1H, NH). C NMR (150 MHz, DMSO-d ) δ: 15.91 (6-CH ),
2 6 3
1
9

ACCEPTED MANUSCRIPT
3
7.37 (SCH ), 50.87 (CH NH), 56.25 (2C, 3′-OCH , 5′-OCH ), 60.41 (4′-OCH ), 105.84 (2C, 2′-C, 6′-C), 119.40
2
2
3
3
3
(
4a-C), 123.60 (5-C), 132.79 (1′-C), 137.24 (4′-C), 137.38 (6-C), 153.27 (2C, 3′-C, 5′-C), 153.84 (7a-C), 157.81
+
(
2-C), 163.37 (4-C), 195.34 (C=S). ESI-HRMS m/z: calcd for C H N O S ([M+H] ): 452.0772; found:
19
22
3
4 3
4
4
52.0770.
.1.3.7. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl
(3,4-methylenedioxy)benzylcarbamodithioate (3g)
Yield: 33%, pale brown solid, mp 183−187 °C (eluent: dichloromethane/methanol = 98 :2 to 95 :5), HPLC rt
1
=
5.478 min. H NMR (600 MHz, DMSO-d ) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 4.47 (s, 2H, SCH ), 4.72 (d, J = 5.4
6
3
2
Hz, 2H, CH NH), 6.00 (s, 2H, OCH O), 6.79 (dd, J = 7.8, 1.8 Hz, 1H, Ph 6′-H), 6.86 (d, J = 7.8 Hz, 1H, Ph 5′-H),
2
2
6
.87 (d, J = 1.8 Hz, 1H, Ph 2′-H), 7.05 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 10.49 (t, J = 5.4 Hz, 1H,
13
CH NH), 12.51 (s, 1H, NH). C NMR (150 MHz, DMSO-d ) δ: 15.48 (6-CH ), 36.89 (SCH ), 49.73 (CH NH),
2
6
3
2
2
1
00.94 (OCH O), 108.11 (2′-C), 108.39 (5′-C), 118.96 (4a-C), 121.25 (6′-C), 123.17 (5-C), 130.62 (1′-C), 136.96
2
(
6-C), 146.49 (3′-C), 147.24 (4′-C), 153.30 (7a-C), 157.37 (2-C), 162.93 (4-C), 194.74 (C=S). ESI-HRMS m/z:
+
calcd for C H N O S ([M+H] ): 406.0354; found: 406.0339.
17
16
3
3 3
4
.1.3.8. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 4-bromobenzylcarbamodithioate (3h)
Yield: 31%, light yellow solid, mp 198−202 °C (eluent: dichloromethane/methanol = 97:3), HPLC rt =
1
1
2.545 min. H NMR (600 MHz, DMSO-d ) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 4.48 (s, 2H, SCH ), 4.80 (d, J = 5.4
6
3
2
Hz, 2H, CH NH), 7.06 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.25 (d, J = 8.4 Hz, 2H, Ph 2′-H, 6′-H),
2
13
7
.52 (d, J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 10.58 (t, J = 5.4 Hz, 1H, CH NH), 12.52 (s, 1H, NH). C NMR (150 MHz,
2
DMSO-d ) δ: 15.96 (6-CH ), 37.36 (SCH ), 49.53 (CH NH), 119.43 (4a-C), 120.79 (4′-C), 123.63 (5-C), 130.29
6
3
2
2
2
0

ACCEPTED MANUSCRIPT
(
2C, 2′-C, 6′-C), 131.70 (2C, 3′-C, 5′-C), 136.90 (1′-C), 137.40 (6-C), 153.65 (7a-C), 157.82 (2-C), 163.40 (4-C),
+
1
4
4
95.76 (C=S). ESI-HRMS m/z: calcd for C H BrN OS ([M+H] ): 439.9561, 441.9540; found: 439.9552,
16
15
3
3
41.9532.
.1.3.9. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 4-chlorobenzylcarbamodithioate (3i)
Yield: 31%, light yellow solid, mp 163−166 °C (eluent: dichloromethane/methanol = 99:1 to 98 :2), HPLC rt
1
=
10.519 min. H NMR (600 MHz, DMSO-d ) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 4.48 (s, 2H, SCH ), 4.82 (d, J =
6
3
2
6
6
.0 Hz, 2H, CH NH), 7.06 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.31 (d, J = 8.4 Hz, 2H, Ph 2′-H,
2
13
′-H), 7.39 (d, J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 10.58 (t, J = 6.0 Hz, 1H, CH NH), 12.52 (s, 1H, NH). C NMR (150
2
MHz, DMSO-d ) δ: 16.42 (6-CH ), 37.83 (SCH ), 49.96 (CH NH), 119.90 (4a-C), 124.10 (5-C), 129.26 (2C,
6
3
2
2
3
1
4
′-C, 5′-C), 130.42 (2C, 2′-C, 6′-C), 132.77 (4′-C), 136.95 (1′-C), 137.88 (6-C), 154.13 (7a-C), 158.30 (2-C),
+
63.89 (4-C), 196.23 (C=S). ESI-HRMS m/z: calcd for C H ClN OS ([M+H] ): 396.0066; found: 396.0062.
16
13
3
3
.1.3.10. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 2,4-dichlorobenzylcarbamodithioate
(3j)
Yield: 14%, light yellow solid, mp 182−186 °C (eluent: dichloromethane/methanol = 98:2), HPLC rt =
1
1
9.537 min. H NMR (600 MHz, DMSO-d ) δ: 2.50 (d, J = 1.2 Hz, 3H, CH ), 4.49 (s, 2H, SCH ), 4.83 (d, J = 5.4
6
3
2
Hz, 2H, CH NH), 7.06 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.32 (d, J = 8.4 Hz, 1H, Ph 6′-H), 7.41
2
(
dd, J = 8.4, 2.4 Hz, 1H, Ph 5′-H), 7.64 (d, J = 2.4 Hz, Ph 3′-H), 10.56 (t, J = 5.4 Hz, 1H, CH NH), 12.53 (s, 1H,
2
1
3
NH). C NMR (150 MHz, DMSO-d ) δ: 15.95 (6-CH ), 37.43 (SCH ), 47.77 (CH NH), 119.44 (4a-C), 123.64
6
3
2
2
(
5-C), 127.75 (5′-C), 129.22 (3′-C), 131.06 (6′-C), 133.22 (4′-C), 133.68 (1′-C), 133.76 (2′-C), 137.40 (6-C),
+
1
53.60 (7a-C), 157.82 (2-C), 163.40 (4-C), 196.26 (C=S). ESI-HRMS m/z: calcd for C H Cl N OS ([M+H] ):
16
14
2
3
3
2
1

ACCEPTED MANUSCRIPT
4
29.9676, 431.9647; found: 429.9665, 431.9625.
4
.1.3.11. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 4-fluorobenzylcarbamodithioate (3k)
Yield: 63%, light yellow solid, mp 155−158 °C (eluent: dichloromethane/methanol = 98:2), HPLC rt = 6.526
1
min. H NMR (600 MHz, DMSO-d ) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 4.48 (s, 2H, SCH ), 4.81 (d, J = 5.4 Hz, 2H,
6
3
2
CH NH), 7.06 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.16 (t, J = 9.0 Hz, 2H, Ph 3′-H, 5′-H), 7.34 (dd,
2
13
J = 9.0, 5.4 Hz, 2H, Ph 2′-H, 6′-H), 10.57 (t, J = 5.4 Hz, 1H, CH NH), 12.52 (s, 1H, NH). C NMR (150 MHz,
2
DMSO-d ) δ: 15.93 (6-CH ), 37.34 (SCH ), 49.52 (CH NH), 115.58 (d, J = 21.3 Hz, 2C, 3′-C, 5′-C), 119.41
6
3
2
2
(
(
4a-C), 123.62 (5-C), 130.20 (d, J = 8.25 Hz, 2C, 2′-C, 6′-C), 133.61 (d, J = 3.0 Hz, 1′-C), 137.39 (6-C), 153.68
7a-C), 157.82 (2-C), 161.85 (d, J = 241.65 Hz, 4′-C), 163.41 (4-C), 195.55 (C=S). ESI-HRMS m/z: calcd for
+
C H FN OS ([M+H] ): 380.0361; found: 380.0354.
1
6
15
3
3
4
.1.3.12. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 2-fluorobenzylcarbamodithioate (3l)
Yield: 76%, yellow solid, mp 153−158 °C (eluent: dichloromethane/methanol = 98:2), HPLC rt = 6.285 min.
1
H NMR (600 MHz, DMSO-d6) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 4.48 (s, 2H, SCH ), 4.84 (d, J = 5.4 Hz, 2H,
3 2
CH NH), 7.06 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.16-7.22 (m, 2H, Ph-H), 7.33-7.37 (m, 2H,
2
13
Ph-H), 10.56 (t, J = 5.4 Hz, 1H, CH NH), 12.52 (s, 1H, NH). C NMR (150 MHz, DMSO-d ) δ: 15.94 (6-CH ),
2
6
3
3
7.38 (SCH ), 44.35 (d, J = 4.5 Hz, CH NH), 115.70 (d, J = 21.15 Hz, 3′-C), 119.43 (4a-C), 123.63 (5-C), 124.03
2 2
(
d, J = 14.55 Hz, 1′-C), 124.79 (d, J = 3.45 Hz, 5′-C), 129.95 (d, J = 8.1 Hz, 4′-C), 130.40 (d, J = 4.35 Hz, 6′-C),
1
37.39 (6-C), 153.69 (7a-C), 157.82 (2-C), 160.59 (d, J = 244.05 Hz, 2′-C), 163.43 (4-C), 195.88 (C=S).
+
ESI-HRMS m/z: calcd for C H FN OS ([M+H] ): 380.0361; found: 380.0357.
1
6
15
3
3
2
2

ACCEPTED MANUSCRIPT
4
.1.3.13. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 2,4-difluorobenzylcarbamodithioate
(3m)
Yield: 66%, pale brown solid, mp 153−158 °C (eluent: dichloromethane/methanol = 98:2), HPLC rt = 8.675
1
min. H NMR (600 MHz, DMSO-d ) δ: 2.49 (s, 3H, CH ), 4.48 (s, 2H, SCH ), 4.79 (d, J = 5.4 Hz, 2H, CH NH),
6
3
2
2
7
5
.06 (s, 1H, thieno[2,3-d]pyrimidine 5-H), 7.07 (td, J = 8.4, 2.4 Hz, 1H, Ph 3′-H), 7.25 (td, J = 8.4, 2.4 Hz, 1H, Ph
13
′-H), 7.41 (m, 1H, Ph 6′-H), 10.54 (t, J = 5.4 Hz, 1H, CH NH), 12.52 (s, 1H, NH). C NMR (150 MHz,
2
DMSO-d ) δ: 15.94 (6-CH ), 37.38 (SCH ), 43.96 (d, J = 3.0 Hz, CH NH), 104.31 (t, J = 25.65 Hz, 3′-C), 111.80
6
3
2
2
(
dd, J = 21.15, 3.6 Hz, 5′-C), 119.42 (4a-C), 120.44 (dd, J = 15.0, 3.75 Hz, 1′-C), 123.62 (5-C), 131.79 (dd, J =
9
.75, 5.7 Hz, 6′-C), 137.40 (6-C), 153.66 (7a-C), 157.81 (2-C), 160.69 (dd, J = 246.75, 12.3 Hz, 2′-C), 162.19 (dd,
+
J = 244.65, 12.0 Hz, 4′-C), 163.41 (4-C), 195.87 (C=S). ESI-HRMS m/z: calcd for C H F N OS ([M+H] ):
16
14
2
3
3
3
98.0267; found: 398.0263.
4
.1.3.14. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 4-cyanobenzylcarbamodithioate (3n)
Yield: 41%, light yellow solid, mp 195−198 °C (eluent: dichloromethane/methanol = 99.5:0.5 to 98:2),
1
HPLC rt = 4.484 min. H NMR (600 MHz, DMSO-d ) δ: 2.50 (d, J = 1.2 Hz, 3H, CH ), 4.49 (s, 2H, SCH ), 4.92
6
3
2
(
d, J = 6.0 Hz, 2H, CH NH), 7.06 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.46 (d, J = 8.4 Hz, 2H, Ph
2
13
2
′-H, 6′-H), 7.80 (d, J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 10.65 (t, J = 6.0 Hz, 1H, CH NH), 12.53 (s, 1H, NH). C NMR
2
(
150 MHz, DMSO-d ) δ: 15.95 (6-CH ), 37.39 (SCH ), 49.68 (CH NH), 110.40 (4′-C), 119.21 (CN), 119.43
6 3 2 2
(
4a-C), 123.63 (5-C), 128.73 (2C, 2′-C, 6′-C), 132.77 (2C, 3′-C, 5′-C), 137.42 (6-C), 143.34 (1′-C), 153.56
+
(
7a-C), 157.83 (2-C), 163.42 (4-C), 196.33 (C=S). ESI-HRMS m/z: calcd for C H N OS ([M+H] ): 387.0408;
17
15
4
3
found: 387.0398.
2
3

ACCEPTED MANUSCRIPT
4
.1.3.15. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl 4-nitrobenzylcarbamodithioate (3o)
Yield: 46%, yellow solid, mp 183−188 °C (eluent: dichloromethane/methanol = 99:1 to 98:2) , HPLC rt =
1
5
.650 min. H NMR (600 MHz, DMSO-d ) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 4.50 (s, 2H, SCH ), 4.96 (d, J = 5.4
6
3
2
Hz, 2H, CH NH), 7.06 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.53 (d, J = 8.4 Hz, 2H, Ph 2′-H, 6′-H),
2
13
8
.19 (d, J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 10.70 (t, J = 5.4 Hz, 1H, CH NH), 12.54 (s, 1H, NH). C NMR (150 MHz,
2
DMSO-d ) δ: 15.48 (6-CH ), 36.96 (SCH ), 48.99 (CH NH), 119.00 (4a-C), 123.18 (5-C), 123.53 (2C, 3′-C,
6
3
2
2
5
′-C), 128.46 (2C, 2′-C, 6′-C), 136.94 (6-C), 145.04 (1′-C), 146.63 (4′-C), 153.12 (7a-C), 157.38 (2-C), 162.97
+
(
4-C), 195.99 (C=S). ESI-HRMS m/z: calcd for C H N O S ([M+H] ): 407.0306; found: 437.0308.
16
15
4
3 3
4
.1.3.16. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl
(thiophen-2-ylmethyl)carbamodithioate (3p)
Yield: 24%, off-white solid, mp 192−195 °C (eluent: dichloromethane/methanol = 97:3), HPLC rt = 5.617
1
min. H NMR (600 MHz, DMSO-d ) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 4.48 (s, 2H, SCH ), 4.99 (d, J = 5.4 Hz, 2H,
6
3
2
CH NH), 6.98 (dd, J = 5.4, 3.6 Hz, 1H, thiophene 4′-H ), 7.05 (d, J = 1.2 Hz, thieno[2,3-d]pyrimidine 5-H), 7.07
2
(
dd, J = 3.6, 1.2 Hz, 1H, thiophene 3′-H), 7.43 (dd, J = 4.8, 1.2 Hz, 1H, thiophene 5′-H), 10.62 (t, J = 5.4 Hz, 1H,
13
CH NH), 12.51 (s, 1H, NH). C NMR (150 MHz, DMSO-d ) δ: 15.94 (6-CH ), 37.35 (SCH ), 45.07 (CH NH),
2
6
3
2
2
119.40 (4a-C), 123.61 (5-C), 126.28 (3′-C), 127.04 (5′-C), 127.42 (4′-C), 137.39 (6-C), 139.48 (2′-C), 153.62
+
(
7a-C), 157.81 (2-C), 163.42 (4-C), 195.36 (C=S). ESI-HRMS m/z: calcd for C H N OS ([M+H] ): 368.0020;
14 14 3 4
found: 368.0018.
2
4

ACCEPTED MANUSCRIPT
4
.1.3.17. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl (furan-2-ylmethyl)carbamodithioate
(3q)
Yield: 25%, off-white solid, mp 181−184 °C (eluent: dichloromethane/methanol = 99:1 to 98:2), HPLC rt =
1
4
.493 min. H NMR (600 MHz, DMSO-d ) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 4.47 (s, 2H, SCH ), 4.80 (d, J = 4.8
6
3
2
Hz, 2H, CH NH), 6.36 (d, J = 3.0 Hz, 1H, furan 3′-H), 6.42 (dd, J = 3.0, 1.8 Hz, 1H, furan 4′-H), 7.06 (d, J = 1.2
2
Hz, thieno[2,3-d]pyrimidine 5-H), 7.62 (dd, J = 1.8, 0.6 Hz, 1H, furan 5′-H), 10.54 (t, J = 4.8 Hz, 1H, CH NH),
2
1
3
1
2.51 (s, 1H, NH). C NMR (150 MHz, DMSO-d ) δ: 15.94 (6-CH ), 37.36 (SCH ), 43.62 (CH NH), 109.08
6 3 2 2
(
3′-C), 111.00 (4′-C), 119.41 (4a-C), 123.61 (5-C), 137.39 (6-C), 143.12 (5′-C), 150.07 (2′-C), 153.71 (7a-C),
+
1
3
4
57.81 (2-C), 163.40 (4-C), 195.60 (C=S). ESI-HRMS m/z: calcd for C H N O S ([M+H] ): 352.0248; found:
14
14
3
2 3
52.0247.
.1.3.18. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl
(pyridin-3-ylmethyl)carbamodithioate (3r)
Yield: 89%, white solid, mp 256−260 °C (eluent: dichloromethane/methanol = 90:10), HPLC rt = 2.656 min.
1
H NMR (600 MHz, DMSO-d ) δ: 2.49 (d, J = 1.2 Hz, 3H, CH ), 4.49 (s, 2H, SCH ), 4.85 (s, 2H, CH NH), 7.06 (d,
6
3
2
2
J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.37 (dd. J = 7.8, 4.8 Hz, 1H, pyridine 5′-H), 7.70 (dt, J = 7.8, 1.8
Hz, 1H, pyridine 4′-H), 8.49 (dd, J = 4.8, 1.2 Hz, 1H, pyridine 6′-H), 8.53 (d, J = 1.8 Hz, 1H, pyridine 2′-H), 10.62
1
3
(
(
s, 1H, CH NH), 12.53 (s, 1H, NH). C NMR (150 MHz, DMSO-d ) δ: 15.94 (6-CH ), 37.37 (SCH ), 47.88
2 6 3 2
CH NH), 119.42 (4a-C), 123.62 (5-C), 123.95 (5′-C), 133.07 (3′-C), 135.91 (4′-C), 137.40 (6-C), 148.93 (6′-C),
2
1
49.53 (2′-C), 153.63 (7a-C), 157.82 (2-C), 163.42 (4-C), 195.92 (C=S). ESI-HRMS m/z: calcd for C H N OS
15 15 4 3
+
(
[M+H] ): 363.0408; found: 363.0404.
2
5

ACCEPTED MANUSCRIPT
4
.1.3.19. (6-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl
(pyridin-4-ylmethyl)carbamodithioate (3s)
Yield: 58%, off-white solid, mp 260−263 °C (eluent: dichloromethane/methanol = 90:10), HPLC rt = 2.651
1
min. H NMR (600 MHz, DMSO-d ) δ: 2.50 (s, 3H, CH ), 4.50 (s, 2H, SCH ), 4.87 (s, 2H, CH NH), 7.07 (d, J =
6
3
2
2
0
.6 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.26 (d, J = 5.4 Hz, 2H, pyridine 3′-H, 5′-H), 8.51 (d, J = 5.4 Hz, 2H,
13
pyridine 2′-H, 6′-H), 10.65 (s, 1H, CH NH), 12.54 (s, 1H, NH). C NMR (150 MHz, DMSO-d ) δ: 15.90 (6-CH ),
2
6
3
3
1
7.36 (SCH ), 48.93 (CH NH), 119.43 (4a-C), 122.67 (2C, 3′-C, 5′-C), 123.62 (5-C), 137.41 (6-C), 146.46 (4′-C),
2 2
50.03 (2C, 2′-C, 6′-C), 153.58 (7a-C), 157.84 (2-C), 163.45 (4-C), 196.54 (C=S). ESI-HRMS m/z: calcd for
+
C H N OS ([M+H] ): 363.0408; found: 363.0404.
1
5
15
4
3
4
.1.4. Preparation of 2-(aminomethyl)-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (intermediate 5)
A solution of 2-(chloromethyl)-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (2) (4.3 g, 20 mmol) and
hexamethylenetetramine (3.6 g, 26 mmol) in anhydrous tetrahydrofuran (THF, 100 mL) was stirred under reflux
for 12 h. The mixture was allowed to cool to room temperature and the precipitate was collected by filtration,
washed with 1,4-dioxane and air-dried to give intermediate 4 as an off-white solid, which was used directly in the
next step without purification.
A mixture of intermediate 4 (6.98 g, 19.7 mmol), concentrated hydrochloric acid (50 mL) and methanol (50
mL) was stirred under reflux for 8 h. After cooling to room temperature, the seperated solid was collected by
filtration, and washed with methanol. The solid was suspended in water and the mixture was adjusted with 25%
aqueous ammonia to pH 10. The precipitate was collected by filtration and washed with a small volume of cold
water. The air-dried crude product was purified by column chromatography on a silica gel (eluent:
2
6

ACCEPTED MANUSCRIPT
dichloromethane/methanol = 8:2) to give intermediate 5 as a pale brown solid (1.5 g, 38%, overall yield of two
1
steps), mp 223−228 °C. H NMR (600 MHz, DMSO-d )
δ
: 2.51 (s, 3H, CH ), 4.08 (s, 2H, CH ), 7.11 (d, J = 1.2
6
3
2
+
Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 9.58 (br s, 2H, NH ). ESI-HRMS m/z: calcd for C H N OS ([M+H] ):
2
8
10
3
1
96.0545; found:196.0542.
4
.1.5. General preparation procedure for compounds 6a−l
A suspension of 2-(aminomethyl)-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (5) (2 mmol), carbon disulfide
(
0.8 mL, 13.3 mmol), and finely powdered potassium phosphate (0.52 g, 2.45 mmol) in DMF (20 mL) was stirred
at room temperature for 0.5 h. After adding the appropriate benzyl bromide (2 mmol), the mixture was stirred at
room temperature overnight. The mixture was poured into water (20 mL) and the resulting precipitate was
collected by filtration and air-dried. The crude product was purified by column chromatography on a silica gel
using the eluent indicated below to give compounds 6a−l.
4
.1.5.1. Benzyl ((6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl)carbamodithioate (6a)
Yield: 35%, white solid, mp 223−225 °C (eluent: dichloromethane/methanol = 98:2), HPLC rt = 8.827 min.
1
H NMR (600 MHz, DMSO-d6) δ: 2.50 (s, 3H, CH ), 4.51 (s, 2H, SCH ), 4.75 (d, J = 4.8 Hz, 2H, CH NH), 7.06 (d,
3 2 2
J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.26 (t, J = 7.2 Hz, 1H, Ph 4′-H), 7.32 (t, J = 7.2 Hz, 2H, Ph 3′-H,
13
5
′-H), 7.38 (d, J = 7.2 Hz, 2H, Ph 2′-H, 6′-H) 10.42 (t, J = 4.8 Hz, 1H, CH NH), 12.51 (s, 1H, NH). C NMR (150
2
MHz, DMSO-d ) δ: 15.95 (6-CH ), 38.98 (CH S), 48.32 (NHCH ), 119.42 (4a-C), 123.77 (5-C), 127.67 (4′-C),
6
3
2
2
1
1
28.89 (2C, 3′-C, 5′-C), 129.37 (2C, 2′-C, 6′-C), 137.13 (1′-C), 137.41 (6-C), 153.39 (7a-C), 157.90 (2-C),
+
63.57 (4-C), 198.13 (C=S). ESI-HRMS m/z: calcd for C H N OS ([M+H] ): 362.0455; found: 362.0456.
16
16
3
3
2
7

ACCEPTED MANUSCRIPT
4
.1.5.2. 4-Methylbenzyl ((6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl)carbamodithioate (6b)
Yield: 43%, white solid, mp 190−192 °C (eluent: dichloromethane/methanol = 98:2), HPLC rt = 13.388 min.
1
H NMR (600 MHz, DMSO-d6) δ: 2.27 (s, 3H, 4′-CH ), 2.49 (d, J = 1.2 Hz, 3H, 6-CH ), 4.46 (s, 2H, SCH ), 4.75
3 3 2
(
d, J = 5.4 Hz, 2H, CH NH), 7.06 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.12 (d, J = 7.8 Hz, 2H, Ph
2
13
3
′-H, 5′-H), 7.26 (d, J = 7.8 Hz, 2H, Ph 2′-H, 6′-H), 10.38 (t, J = 5.4 Hz, 1H, CH NH), 12.50 (s, 1H, NH). C NMR
2
(
(
150 MHz, DMSO-d ) δ: 15.94 (6-CH ), 21.14 (4′-CH ), 38.83 (CH S), 48.28 (NHCH ), 119.42 (4a-C), 123.77
6 3 3 2 2
5-C), 129.30 (2C, 3′-C, 5′-C), 129.45 (2C, 2′-C, 6′-C), 134.12 (1′-C), 136.88 (4′-C), 137.12 (6-C), 153.40 (7a-C),
+
1
3
4
57.89 (2-C), 163.56 (4-C), 198.21 (C=S). ESI-HRMS m/z: calcd for C H N OS ([M+H] ): 376.0612; found:
17
18
3
3
76.0613.
.1.5.3. 4-Methoxybenzyl ((6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl)methyl)carbamodithioate
(6c)
Yield: 39%, white solid, mp 209−212 °C (eluent: dichloromethane/methanol = 99.5:0.5 to 98:2), HPLC rt =
1
7
.163 min. H NMR (600 MHz, DMSO-d )
δ
: 2.49 (d, J = 1.2 Hz, 3H, CH ), 3.73 (s, 3H, OCH ), 4.44 (s, 2H,
6
3
3
SCH ), 4.74 (d, J = 5.4 Hz, 2H, CH NH), 6.88 (d, J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 7.06 (d, J = 1.2 Hz, 1H,
2
2
thieno[2,3-d]pyrimidine 5-H), 7.30 (d, J = 8.4 Hz, 2H, Ph 2′-H, 6′-H), 10.36 (t, J = 5.4 Hz, 1H, CH NH), 12.50 (s,
2
1
3
1
H, NH). C NMR (150 MHz, DMSO-d ) δ: 15.94 (6-CH ), 38.62 (CH S), 48.24 (NHCH ), 55.51 (4′-OCH ),
6 3 2 2 3
114.32 (2C, 3′-C, 5′-C), 119.42 (4a-C), 123.78 (5-C), 128.92 (1′-C), 130.60 (2C, 2′-C, 6′-C), 137.12 (6-C),
1
53.42 (7a-C), 157.90 (2-C), 158.93 (4′-C), 163.57 (4-C), 198.29 (C=S). ESI-HRMS m/z: calcd for C H N OS
17 18 3 3
+
(
[M+H] ): 392.0561; found: 392.0561.
2
8