Serendipitous discovery of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine derivatives as novel HIV-1 replication inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.10.007

We identified a novel class of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine compounds as potent HIV-1 replication inhibitors serendipitously during the process of evaluation of triazolothienopyrimidine (TTPM) compounds. Herein, we report synthesis and biological evaluation of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine compounds using a cell-based full replication assay to identify thienopyrimidines 6 and 30, which could be further utilized as viable lead compounds.

Full text of DOI:10.1016/j.bmcl.2014.10.007

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5473–5477
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Serendipitous discovery of 2-((phenylsulfonyl)methyl)-thieno
[
3,2-d]pyrimidine derivatives as novel HIV-1 replication inhibitors
Junwon Kim, Jeongjin Kwon, Doohyun Lee, Suyeon Jo, Dong-Sik Park, Jihyun Choi, Eunjung Park,
Jong Yeon Hwang, Yoonae Ko, Inhee Choi, Moon Kyeong Ju, JiYe Ahn, Junghwan Kim, Sung-Jun Han,
⇑
Tae-Hee Kim, Jonathan Cechetto, Jiyoun Nam, Sujin Ahn, Peter Sommer, Michel Liuzzi, Jinhwa Lee
Institut Pasteur Korea, 16, Daewangpangyo-ro 712 beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We identified a novel class of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine compounds as potent
Received 11 August 2014
Revised 1 October 2014
Accepted 2 October 2014
Available online 23 October 2014
HIV-1 replication inhibitors serendipitously during the process of evaluation of triazolothienopyrimidine
(TTPM) compounds. Herein, we report synthesis and biological evaluation of 2-((phenylsulfonyl)methyl)-
thieno[3,2-d]pyrimidine compounds using a cell-based full replication assay to identify thienopyrimi-
dines 6 and 30, which could be further utilized as viable lead compounds.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
HIV-1
Thienopyrimidine
Structure–activity relationship
Cell-based assay
Inhibitor
The human immunodeficiency virus (HIV) outbreak still
remains a major public health attention since greater than 30 mil-
lion people around the world live with HIV and 2.7 million people
recently to exhibit the structure–activity relationship as a novel
class of HIV-1 replication inhibitors.
7
d
During the course of synthesis of triazolothienopyrimidine
derivatives, we encountered surprisingly interesting reaction as
shown in Figure 2. Thus, when chlorothienotriazolopyrimidine 4
was treated with various aryl boronic acids such as (6-methoxy-
pyridin-3-yl)boronic acid (5) under microwave-assisted Suzuki
1
get newly infected with HIV each year. The discovery and imple-
mentation of highly active antiretroviral therapy (HAART) in 1995
provided fundamental improvements in the treatment of HIV
2
infection, resulting in significant reductions in mortality rates.
8
HAART is a combination of a few antiretroviral drugs from different
drug groups, but yet eradication of HIV has not been achieved
coupling conditions to provide the anticipated product 7, the
resulting product turned out to be bicyclic thienopyrimidine 6
3
because of the rapid emergence of drug-resistant HIV mutants.
instead. The difference between current conditions and prior con-
7
d
Also, undesired side effects of combination therapy have limited
their clinical effectiveness.4 As a result, there is a clear need to
discover novel anti-HIV agents with unique resistance profiles. In
ditions reported recently should be longer duration of reaction at
,5
higher temperature under microwave irradiation (130 °C, 30 min
1
6
vs 110 °C, 5 min).
6
the past several years, high-throughput screening (HTS) assays
This type of reaction has been used for rhodium-catalyzed
9
for the discovery of novel antiretrovirals evaluated more than
transannulation and denitrogenative hydration or hydrocarbon
1
0
2
00,000 compounds using a cell-based full replication assay based
insertion , but as far as we are concerned, this is the first time
in that heating without transition metal such as rhodium as a cat-
alyst on triazolothienopyrimidine extrudes nitrogen to directly
form 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine ring
system. Since we obtained more attractive and concise compound
6, we decided to test its inhibitory activity against HIV replication.
We were then delighted to find out that bicyclic thienopyrimidine
6 showed excellent potency against anti-HIV assay and no cytotox-
on reporter cells harboring an EGFP expression cassette under the
control of HIV promoter at Institut Pasteur Korea. We identified hit
compounds containing 3,4-dihydropyrimidin-2(1H)-one 1, triazine
scaffold 2, and triazolothienopyrimidine 3, respectively (Fig 1).
These hit compounds were utilized independently to generate
novel and potent leads for further development.7 Among them,
triazolothienopyrimidine derivatives have been reported only
1
7
icity , respectively (EC50 = 0.025 lM, CC50 >50 lM). To further
explore the structure–activity relationship of this bicyclic thieno-
⇑
Corresponding author.
pyrimidine system, we first synthesized the target compounds to
http://dx.doi.org/10.1016/j.bmcl.2014.10.007
960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
0

5
474
J. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5473–5477
N
N
N
O
HO
O
S
N
N
O
N
S
N
N
H
HN
O
HN
N
NH
O
N
H
1
2
3
EC50 = 0.53 uM
CC50 >10 uM
EC50 = 0.27 uM
CC50 >10 uM
EC50 = 9.84 uM
Figure 1. Hit compounds from cell-based HIV-1 replication assay.
(
HO)
2
B
N
N
N
O
S
N
O
5
N
O
N
N
N
O
O
S
S
Cl
O
O
O
N
N
Pd(dppf)Cl
2
, Na
2
CO
3
S
N
S
not
Cl
N
S
DME/H O (3:1)
2
Cl
MW, 130 oC, 30 min, 55%
N2
Cl
N
-
O
4
7
6
Figure 2. Denitrogenative formation of bicyclic thienopyrimidine.
2
evaluate the effect of aryl groups or amines as described in
Scheme 1. Thus, the bicyclic intermediate 2-(chloromethyl)thie-
no[3,2-d]pyrimidin-4(3H)-one (9) was prepared in 95% yield by
cyclization of 3-amino-thiophene-2-carboxylic acid methyl ester
6-methoxypyridin-3-yl group 6 at R position with EC50 = 0.025 -
M. The activity strongly depends on the nature of the substituents
and ranges from EC50 = 0.025 M to EC50 = 6.0 M. The activity
l
l
l
changes in 240-fold. Interestingly, among derivatives with the
3-pyridinyl groups, substituents at para-position of pyridine influ-
ence activity in a dramatic fashion. Thus, 3-pyridinyl 15 without
substituent reduced antiviral activity in 20-fold (EC50 = 0.485
Halogens at para-position even further decreased anti-HIV activity
in 128-fold and 240-fold, respectively (18, EC50 = 3.2 M; 19,
EC50 = 6.0 M), suggesting that electron-donating group is favor-
(8) with malononitrile in the presence of 4 N HCl under mild heat-
ing conditions. The requisite sodium phenylsulfinates 11 were pre-
pared by irradiation in a microwave on a water solution of sodium
sulfite, sodium bicarbonate and phenylsulfonyl chloride 10. An
ethanolic solution of chloride 9 was then added to sodium phenyl-
sulfinates 11 and potassium iodide and stirred under reflux for an
additional hour to give sulfone 12.11 The lactam 12 was then acti-
lM).
l
l
able at para-position on pyridinyl substituents. Also 4-pyridinyl
vated with POCl
3
to provide 4-chloro-thienopyrimidine 13. The key
groups appeared to be tolerated for this region, showing only
intermediate 13 was treated with a variety of aryl boronic acids
minor reduction in anti-HIV activity (16, EC50 = 0.189
EC50 = 0.27 M). Other heteroaryl groups including pyridinone
20, thiophene 22, furan 23, or morpholine 24 also turned out to
assume decent anti-HIV activity, respectively (20, EC50 = 0.915 M;
22, EC50 = 0.14 M; 23, EC50 = 0.775 M, 24, EC50 = 0.59 M), indi-
cating that any rings that can occupy suitable space are tolerable
for the region. Along the line, 4-methoxyphenyl at the position
provided the second best anti-HIV activity (21, EC50 = 0.103 lM)
in Table 1, while a bit longer 4-methoxyphenylamino 25 and
lM; 17,
under microwave-assisted Suzuki coupling condition to produce
l
7
d
the compounds 15–23 or with amines or phenol to afford deriv-
12
atives 24, 25, and 26, respectively.
l
2
The first series of R modification with various aryl groups or
amines while maintaining 3-chlorophenyl at R1 was evaluated
for their inhibitory activity against HIV-1 replication in a cell-based
assay, and nevirapine was used as a positive control.13 The assay
results of compounds 15–26 are shown in Table 1. Among the com-
pounds tested in Table 1, none of them are more active than
l
l
l
4-methoxyphenoxy 26 derivatives deteriorated anti-HIV activity
NH2
N
O
O
S
(a)
Cl
O O
(
b)
OMe
S
NH
(d)
R1
R1
S
Cl
NaO
S
O
10
11
9
8
N
R1
N
O
R1
N
R1
(c)
S
S
S
(e)
9
11
O
O
O
O
O
O
N
NH
N
S
S
S
R2
Cl
12
13
14
Scheme 1. Synthesis of thienopyrimidines. Reagents and conditions: (a) malononitrile, 4 N HCl, 50 °C, 10 h, 95%; (b) Na
2
SO
3
, NaHCO
3
, H
2
O, microwave, 100 °C, 40 min, ꢀ90%;
2
(
c) KI, EtOH, 90 °C, 1 h, ꢀ95%; (d) POCl
3 2 2 2 3 2
, pyridine, reflux, 2 h, ꢀ80%; (e) (HO) B-R , Pd(dppf)Cl , Na CO , DME/H O (3:1 v/v), microwave, 130 °C, 30 min.

J. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5473–5477
5475
Table 1
in 31-fold and 8-fold, respectively (25, EC50 = 3.2
EC50 = 0.831 M).
l
M; 26,
2
Cell-based antiviral activity of thienopyrimidine derivatives with R modifications
against HIV-1
l
The next series of modifications to be explored was linker
1
replacement while maintaining 3-chlorophenyl ring at R and
2
N
6-methoxypyridin-3-yl ring at R in 2-((phenylsulfonyl)methyl)-
S
Cl
thieno[3,2-d]pyrimidine derivatives and the results were summa-
rized in Table 2. The first exploration task was regarding the effect
of homologous compounds. Thus, it was found that phenylsulfo-
nyl-thienopyrimidine 27 with one less methylene group from com-
O
O
N
S
R2
R2
EC50a
CC50b
(lM)
TIc
Compound
(l
M)
pound 6 reduced anti-HIV activity in 13-fold (EC50 = 0.322
And also one more methylene unit from compound 6 even further
decreased anti-HIV activity in 216-fold (28, EC50 = 5.4 M), mean-
lM).
l
6
N
0.025
>10
>400
ing that linker length-wise, current linker length in 2-((phenylsul-
fonyl)methyl)-thieno[3,2-d]pyrimidine appears to be optimal.
Next, the effect of sulfonyl functional group in thienopyrimidine
6 was evaluated by reducing to the corresponding sulfoxide 29
or sulfide 30. Surprisingly, replacement of sulfone 6 to sulfide
OMe
1
1
5
6
0.485
0.189
>50
>50
>103
>264
N
3
0 exhibited enhanced inhibitory activity in 1.8-fold (EC50
0.014 M), whereas sulfoxide 29 decreased anti-HIV activity in
1-fold (EC50 = 0.532 M) compared with sulfone 6. We next
=
l
2
l
N
N
explored the effect of sulfonyl group by replacing sulfonyl group
with simple methylene unit or unsaturated carbon linker. The
anti-HIV activity of simple phenethyl compound 32 decreased in
1
1
7
8
0.27
3.2
63
70
233
21
Cl
6
4-fold (EC50 = 1.6
ished anti-HIV activity (EC50 = 8.0
gen bond acceptor at the particular position in the linker (Y@SO
l
M), and olefinic styryl compound 31 demol-
lM), suggesting that the hydro-
2
,
N
Table 2) is critical for anti-HIV activity. We then moved to explore
the possibility of replacing sulfonyl group with divalent atoms or
groups. Interestingly, carbonyl group 33 reduced anti-HIV activity
Cl
in eight fold (EC50 = 0.20 lM) although it has similar motif to
1
2
9
0
N
F
6
40
6.6
Table 2
Cell-based antiviral activity of thienopyrimidines with R1 modifications against HIV-1
NH
0.915
>50
>54
N
X
O
Y
Cl
N
N
S
2
1
0.103
69
669
OMe
S
OMe
2
2
2
3
0.140
0.775
>50
>50
>357
>64
Compound
X
Y
EC50a
(
lm)
CC50b
(
lm)
TIc
6
CH
—
(CH )
2 2
CH
CH
–CH–
CH
CH
CH
CH
CH
2
–SO
–SO
–SO
2
–
2
–
2
–
0.025
0.322
5.4
0.532
0.014
8.0
>10
>50
>50
>50
>10
53
>400
>155
>9.2
>93
>714
6.6
2
2
2
7
8
9
O
N
2
–SO–
–S–
–CH–
CH
–(CO)–
–CH(OH)–
–NH–
30
2
2
2
4
5
0.59
3.2
>50
60
>84
31
32
33
34
2
2
2
2
2
2
1.6
0.20
9.0
>50
30
31
>31
150
3.4
O
HN
35
0.672
0.384
>50
>50
>74
>130
18.8
36
–O–
OMe
OMe
3
7
8
–SO
2
–
0.069
>50
>724
3
39
4
4
NVP
–CH(CH
–CHF–
3
)–
–SO
–SO
–SO
–SO
2
2
2
2
–
–
–
–
0.084
0.087
0.237
2.3
>50
>50
>50
>50
>10
>595
>574
>210
>21
O
2
6
0.831
0.15
>50
>10
>60
>66
0
1
–CF
3 2
–C(CH ) –
2
NVPd
d
0.105
>66
a
EC50 is the concentration of compound that inhibits HIV-1 replication by 50%.
a
EC50 is the concentration of compound that inhibits HIV-1 replication by 50%.
For compounds 15–26, the values are the geometric mean of two determinations;
For compounds 27–41, the values are the geometric mean of two determinations;
all individual values are within 25% of the mean.
all individual values are within 25% of the mean.
b
CC50 is the cytotoxic concentration of compound that reduces viability of
b
CC50 is the cytotoxic concentration of compound that reduces viability of
uninfected cells by 50%.
uninfected cells by 50%.
c
Therapeutic index (TI) is defined by CC50/EC50
.
c
Therapeutic index (TI) is defined by CC50/EC50
.
d
Nevirapine (NVP) was used as a positive control.
d
Nevirapine (NVP) was used as a positive control.

5
476
J. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5473–5477
Table 3
Cell-based antiviral activity of sulfides with linker modifications against HIV-1
moiety is not favorable due to possible steric repulsion. So far
the thienopyrimidine series of compounds do not have particular
cytotoxic issues as shown in Tables 1 and 2.
N
R1
S
1
We next investigated the effect of R moiety in thienopyrimi-
N
N
2
S
dine series with 6-methoxy-3-pyridinyl group at R position using
1
4
sulfide linker (Table 3), since sulfide 30 previously showed
enhanced anti-HIV activity, albeit in only 1.8-fold. Compounds
with heteroaryl such as CF -pyridinyl 48 or chloro-benzyl 49
3
showed loss of anti-HIV activity in three orders of magnitude, indi-
cating that a phenyl group at R is essential to display antiviral
activity. Substitution patterns on the phenyl ring also affected sig-
nificant variations in potency. Di-substitution of chlorides on the
phenyl ring reduced anti-HIV activity in approximately 50-fold
and 350-fold, respectively (42, EC50 = 0.728 lM; 43, EC50 = 5.0 lM).
Replacement of chlorine with trifluoromethyl group 44 also
decreased activity in 19-fold, while bromide 45 decreased activity
in only 11-fold (44, EC50 = 0.262 lM; 45, EC50 = 0.158 lM), mean-
O
1
Compound
R1
EC50a
(lm)
CC50b
>10
(lm)
TIc
3
0
2
0.014
0.728
>714
Cl
Cl
4
>50
>50
>68
>10
Cl
ing that mono-substitution on meta-position with halogen charac-
ter might be tolerable. Di-substitution of fluorine or methyl group
on the phenyl ring again suffered from loss of activity as shown in
derivatives 46 and 47, implying that multi-substitution on the phe-
4
3
5.0
Cl
Cl
1
nyl ring at R region is not tolerable. From this brief survey, the
1
4
4
4
5
0.262
0.158
>50
>50
>190
>316
optimal substitution for R phenyl ring is the mono-substitution
CF3
with a chlorine atom at meta-position (30, EC50 = 0.014 lM).
A representative compound 6 was further evaluated for effects of
15
CYP inhibition. Compound 6 showed no appreciable inhibition
Br
F
against CYP1A2, CYP2D6, showing IC50 >10
ate inhibitor against CYP3A4 (IC50 = 6.71
.25 M).
In summary, we investigated a series of 2-((phenylsulfo-
l
M, while 6 is a moder-
l
M), CYP2C9 (IC50
=
5
l
4
4
6
7
5.0
>50
>10
F
Me
nyl)methyl)-thieno[3,2-d]pyrimidine derivatives for the treatment
of HIV-1 infection. These novel bicyclic thienopyrimidine struc-
tures were discovered serendipitously during the course of synthe-
sis of triazolothienopyrimidine derivatives. From preliminary SAR
in a cell-based full replication assay, we discovered that thienopyr-
imidine derivatives 6 and 30 exhibit significant inhibitory activity
against HIV-1 replication. Subsequently, CYP inhibition was
assessed for compound 6, which showed decent CYP inhibition
profiles. Mode of action and further optimization of this novel ser-
ies of anti-HIV compounds will be reported in due course.
0.774
18.0
>50
>50
>64
Me
CF3
N
4
4
8
9
>2.7
Cl
23.0
>50
>10
>2.2
>66
NVd
0.150
Acknowledgments
a
EC50 is the concentration of compound that inhibits HIV-1 replication by 50%.
For compounds 42-49, the values are the geometric mean of two determinations;
all individual values are within 25% of the mean.
In memory of Dr. Junwon Kim. This work was supported by the
National Research Foundation of Korea (NRF) Grant funded by the
Korea government (MSIP) (No. 2012-00011), Gyeonggi-do and
KISTI.
b
CC50 is the cytotoxic concentration of compound that reduces viability of
uninfected cells by 50%.
c
Therapeutic index (TI) is defined by CC50/EC50
Nevirapine (NVP) was used as a positive control.
.
d
References and notes
sulfonyl group in 6. In a similar study on thienopyrimidine analogs,
moderate anti-HIV activities were found for Y@O, and NH (36, EC50
1
2
.
.
http://www.who.int/hiv/pub/progress_report2011/en/index.html.
http://www.who.int/hiv/pub/arv/adult2010/en/index.html.
=
0.384
lM; 35, EC50 = 0.672
lM), while much weaker activity was
shown for Y@CH(OH) (34, EC50 = 9.0
lM), suggesting that Y@O or
3. Menedez-Arias, L. Antiviral Res. 2010, 85, 210.
4
5
6
.
.
.
Esplugues, J. V.; Blas-Garcia, A.; Apostolova, N. Curr. Med. Chem. 2011, 18, 2186.
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Brodin, P.; Christophe, T. Curr. Opin. Chem. Biol. 2011, 15, 1.
NH can contribute to hydrogen bond character to some degree,
but Y@CH(OH) may not form adequate hydrogen bond at all. We
next investigated the effect of substitution on methylene unit (X)
flanked by phenylsulfonyl group on the east and thienopyrimidine
ring on the west. We also hoped that these substituents would be
useful for rendering an ostensibly sensitive carbon atom resistant
to metabolic attack. It was found that only slight reductions in
potency were observed for spiro-cyclopropyl (37), mono-methyl
7. (a) Kim, J.; Park, C.; Ok, T.; So, W.; Jo, M.; Seo, M.; Kim, Y.; Sohn, J.-H.; Park, Y.;
Ju, M. K.; Kim, J.; Han, S.-J.; Kim, T.-H.; Cechettos, J.; Nam, J.; Sommer, P.; No, Z.
Bioorg. Med. Chem. Lett. 2012, 22, 2119; (b) Kim, J.; Ok, T.; Park, C.; So, W.; Jo,
M.; Kim, Y.; Seo, M.; Lee, D.; Jo, S.; Ko, Y.; Choi, I.; Park, Y.; Yoon, J.; Ju, M. K.;
Ahn, J.; Kim, J.; Han, S.-J.; Kim, T.-H.; Cechetto, J.; Nam, J.; Liuzzi, M.; Sommer,
P.; No, Z. Bioorg. Med. Chem. Lett. 2012, 22, 2522; (c) Kim, J.; Lee, D.; Park, C.; So,
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Choi, I.; Park, Y.; Yoon, J.; Ju, M. K.; Kim, J.; Han, S.-J.; Kim, T.-H.; Cechetto, J.;
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Ahn, S.; Sommer, P.; Liuzzi, M.; No, Z.; Lee, J. Bioorg. Med. Chem. Lett. 2013, 23,
(38), and mono-fluoro (39) as indicated by EC50 = 0.069
lM for
3
7, EC50 = 0.084 M for 38, and EC50 = 0.087 M for 39. However,
l
l
gem-difluoro 40 and gem-dimethyl 41 reduced anti-HIV activity
in approximately 10-fold and 92-fold, respectively, compared with
no substitution 6, suggesting that considerable bulkiness at this
153.
8. Lared, M.; Moberg, C.; Hallberg, A. Acc. Chem. Res. 2002, 35, 717.

J. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5473–5477
5477
9
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1
5. Compound 6 has been tested with recombinant CYP enzymes to confirm its
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working solution in the absence of presence of 98 L of cofactor solutions for
0 min for CYP1A2 and CYP2C9, 20 min for CYP2D6, and 3 min for CYP3A4. The
lL)
4
1
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l
1
1
1
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final incubations were terminated by 200 lL of cold IS-fortified (100 ng/mL
tolbutamide) stop solution, and the samples were analyzed by LC-MS/MS. The
reaction mixtures (200 L final volume) contained approximately 100 mM
potassium phosphate buffer (pH 7.4), 3.3 mM MgCl 1 mM NADPH, and
8
80.
l
2. Ivachtchenko, A. V.; Golovina, E. S.; Kadieva, M. G.; Koryakova, A. G.;
Kovalenko, S. M.; Mitkin, O. D.; Okun, I. M.; Ravnyeyko, I. M.; Tkachenko, S.
E.; Zaremba, O. V. Bioorg. Med. Chem. 2010, 18, 5282.
2
,
5
pmol/mL recombinant CYP enzymes.
1
1
6. Use of Pd(dppf)Cl
2
produced bicyclic compound 6 as a single product. By
yielded a mixture of bicyclic compound
and tricyclic compound 7 under the same conditions.
7. Cytotoxicity of the selected hits was determined using the same cell lines
CEMx174-LTR-GFP CG8 cells and HeLa-LTR-GFP cells) plated in Evotec 384-
well plates with pre-dispensed compounds but without viral infection. After
the 5th day of incubation, cell viability was evaluated using 100 g/mL of
resazurin (Sigma, St. Louis, MO). After 2 h of incubation at 37 °C, 5% CO , the
fluorescence of resorufin/resazurin was measured at 544 nm/595 nm
excitation/emission) on a PE Victor 3 plate reader.
1
3. HIV full replication assay: CEMx174-LTR-GFP cells (clone CG8) were seeded
with a microplate dispenser (WellMate; Thermo Scientific Matrix; USA) at a
density of 4000 cells/well into 384-well glass plates (Evotec, Hamburg,
3 4
comparison, employment of Pd(PPh )
6
Germany) pre-dispensed with 10
incubated for 1 h at 37 °C, 5% CO . Then cells were infected with HIV-1LAI at a
multiplicity of infection (MOI) of 3 and incubated for 5 days at 37 °C, 5% CO
Fluorescence intensities were then measured using a multilabel plate reader
Victor3; PerkinElmer, Inc.; USA). And see Sommer, P.; Vartanian, J. P.;
Wachsmuth, M.; Henry, M.; Guetard, D.; Wain-Hobson, S. J. Mol. Biol. 2004,
44, 11.
4. Synthesis of sulfide compounds 30, 42–49 was conducted as follows:
compound 9 was treated with POCl in the presence of pyridine to generate
dichloride 50, which was subjected to boronic acid 5 under microwave-
assisted Suzuki cross-coupling conditions [Pd(PPh CsF, DME/H O,
lL of compound diluted in DMSO and
(
2
2
.
l
2
(
(
3
1
3
3
4
) ,
2
microwave, 150 °C, 20–30 min] yielding chloride 51. Finally, treatment of 51
with a variety of thiols in the presence of NaH produced the target compounds
in Table 3.
N
N
SR
Cl
N
N
N
N
RSH
NaH
S
N
S
POCl
pyr
3
Cl
5
9
N
S
Suzuki
Cl
OMe
OMe
50
3
0, 42-49
51