Effect of stereochemistry on the anti-HIV activity of chiral thiourea compounds

Article abstract of DOI:10.1016/j.bmc.2004.04.050

Chiral derivatives of several substituted halopyridyl and thiazolyl PETT compounds were synthesized as non-nucleoside inhibitors of the reverse transcriptase (RT) enzyme (NNRTI) of the human immunodeficiency virus (HIV-1). Molecular modeling studies indic

Full text of DOI:10.1016/j.bmc.2004.04.050

Bioorganic & Medicinal Chemistry 12 (2004) 4275–4284
Effect of stereochemistry on the anti-HIV activity of
chiral thiourea compounds
T. K. Venkatachalam,a C. Maob and Fatih. M. Uckunc,d,
*
aDepartment of Chemistry, Parker Hughes Institute, 2699 Patton Road, Roseville, MN 55113, USA
bDepartment of Structural Biology, Parker Hughes Institute, 2699 Patton Road, Roseville, MN 55113, USA
cDepartment of Immunology, Parker Hughes Institute, 2699 Patton Road, Roseville, MN 55113, USA
dDepartment of Virology, Parker Hughes Institute, 2699 Patton Road, Roseville, MN 55113, USA
Received 8 May 2003; accepted 5 April 2004
Abstract—Chiral derivatives of several substituted halopyridyl and thiazolyl PETT compounds were synthesized as non-nucleoside
inhibitors of the reverse transcriptase (RT) enzyme (NNRTI) of the human immunodeficiency virus (HIV-1). Molecular modeling
studies indicated that because of the asymmetric geometry of the NNRTI binding pocket, the R stereoisomers would fit the NNRTI
binding pocket of the HIV-1 RT much better than the corresponding S stereoisomers, as reflected by their 104-fold lower Ki values.
The R stereoisomers of several PETT derivatives inhibited recombinant RT in vitro with lower IC50 values than their enantiomers.
The active compounds were further evaluated for their ability to inhibit HIV-1 replication in human peripheral blood mononuclear
cells (PBMC). All the R isomers once again showed potent anti-HIV activity and inhibited the replication of the HIV-1 strain
HTLVIIIB in peripheral blood mononuclear cells (PBMC) at nanomolar concentrations whereas their enantiomers were sub-
stantially less potent. The lead compounds in the respective groups were further tested against the NNRTI-resistant HIV strains,
A17 (Y181C mutant), and A17Var (Y181C+K103N mutant) and RT MDR (V106N). The results showed that the lead compounds
were several logs more potent than the standard NNRTI nevirapine. Structure–activity relationship studies also revealed a pref-
erence for the pyridyl unit with halo substitutions primarily at 5-position demonstrating the importance of regiochemistry. Our data
provides experimental evidence that the stereochemistry as well as regiochemistry of NNRTI can profoundly affect their anti-HIV
activity.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
certain structure–activity relationships among various
substituents in their structure. However, there is no
published report on the role of stereochemistry for the
anti-HIV activity of PETT derivatives. Recently, we
have identified by rational drug design several struc-
turally distinct thiourea compounds as potent NNRTIs
for HIV-RT14–18 and examined their structure–activity
relationships. Here, we report the previously unknown
impact of stereochemistry on the anti-HIV activity of
thiourea compounds with special emphasis on the anti-
HIV potency of several lead chiral thiourea compounds.
Human immunodeficiency virus type-1 (HIV-1) is the
causative agent for the transmission and development of
the acquired immunodeficiency syndrome (AIDS).1 The
main targets in contemporary drug discovery efforts
against HIV-1 is RT, a vital enzyme that is responsible
for the reverse transcription of retroviral RNA to pro-
viral DNA.2–9 Non-nucleoside reverse transcriptase
inhibitors (NNRTI’s) inhibit HIV RT by altering either
of the conformation or mobility of RT by binding to a
specific allosteric site near the polymerase site, thereby
resulting in noncompetitive inhibition of the enzyme.10;11
In a systematic search for potent anti-AIDS drugs, Bell
et al.12;13 discovered phenethylthiazolylthiourea (PETT)
compounds as potent inhibitors of HIV-1 and disclosed
2. Results and discussion
For the synthesis of the chiral thiourea compounds, we
followed the general procedure shown in Scheme 1 as
previously reported.13;14 The chiral phenethyl thiazolyl
as well as halopyridyl thioureas in the current study
* Corresponding author. Tel.: +1-651-697-9228; fax: +1-651-697-1042;
e-mail: fatih_uckun@ih.org
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.050

4276
T. K. Venkatachalam et al. / Bioorg. Med. Chem. 12 (2004) 4275–4284
X
DMF, 100 oC, 15h
X
H
N
H
N
acetonitrile, rt, 12h
S
X
N
Y
NH2
S
N
N
S
N
S
N
N
NH2
H
N
N
N
N
X= H, Cl, Br, Me
Y
H
N
DMF, 100 oC, 15h
H
N
N
acetonitrile, rt, 12h
N
S
N
S
N
S
NH2
S
N
N
NH2
S
H
N
N
N
N
Z
S
H
N
S
H
N
Z
DMF, 100 oC, 15h
acetonitrile, rt, 12h
N
S
N
N
N
S
N
N
H
S
NH2
S
NH2
N
N
N
N
Z
Scheme 1.
were prepared by treating an appropriately substituted
aminopyridine or aminothiazole/benzothiazole and
thiocarbonyldiimidazole in acetonitrile medium. The
resultant intermediate was condensed with various chi-
ral amines in anhydrous dimethyl formamide and heated
to 100 ꢁC for 18 h over an oil bath and worked up to
yield thioureas. The purity and identity of these com-
pounds were confirmed by standard analytical tech-
niques.
more active than nevirapine, 2-fold more active than
trovirdine and 190-fold more potent than delavirdine,
against A17. Compound 3 was >200-fold more potent
than nevirapine, trovirdine or delavirdine against A17V.
We also confirmed the superior activity of compound 3
against the multi-drug resistant RT-MDR strain (Table
1). Based on the above experimental results, we postu-
late that b-MPT compounds may be useful candidates
for further development as anti-HIV agents especially
since they show remarkable activity against mutant
strains of HIV-1.
Overall, the R stereoisomers of all compounds inhibited
recombinant RT in cell free assays with lower IC50
values than their enantiomers. Among the 10 b-methyl
phenylethyl pyridyl thiourea (b-MPT) compounds, the R
stereoisomers of compounds 1, 3, and 5 with halogen
(Br, Cl) or methyl substitutions, respectively, at the 5-
position of their pyridyl unit were the most potent RT
inhibitors (Table 1). These compounds displayed
nanomolar IC50 values against recombinant RT in vitro.
In contrast, the S stereoisomers showed significantly
lower potency with micromolar IC50 values. Similarly,
only the R stereoisomers of the b-methyl phenylethyl
thiazolyl thioureas (compounds 11 and 15) exhibited
anti-HIV activity (Table 1).
We next focused our attention towards the synthesis of
chiral a-methyl benzyl thioureas (a-MBT) and accord-
ingly we followed the general procedure shown in
Scheme 2.
The purity and identity of these compounds was con-
firmed by usual analytical techniques and are reported
under physical constant section. Table 2 shows the anti-
HIV activity observed for all the isomers of chiral a-
MBT derivatives towards recombinant RT. The alpha
methyl groups were maintained constant and the pyridyl
group was replaced by thiozolyl and benzothiazolyl
groups. In addition, several substituents were intro-
duced in those rings in order to extend our study and
have some understanding about structure–activity rela-
tionships among these derivatives. The results in Table 2
show that all the R isomers were active where as their
enantiomers (S isomers) were inactive.
Of the 19 b-MPT compounds whose ‘R’ stereoisomers
exhibited nanomolar IC50 values against recombinant
RT, five were further evaluated for their ability to in-
hibit HIV-1 replication in human peripheral blood
mononuclear cells (PBMC). All five R stereoisomers
were active anti-HIV agents and inhibited the replica-
tion of the HIV-1 strain HTLVIIIB (NNRTI-sensitive) at
nanomolar concentrations. When examined for their
ability to inhibit the replication of the NNRTI-resistant
HIV-1 strain, A17 (Y181C mutant) and A17Var
(Y181C+K103N mutant), the majority of the R isomers
showed activity at nanomolar to micromolar concen-
trations. The R stereoisomers of b-MPT compounds
were less potent against NNRTI-resistant HIV-strains
than they were against HTLVIIIB. Nevertheless, all
compounds were markedly more potent against the
NNRTI-resistant HIV-1 strains than the standard
NNRTI nevirapine. In addition, the lead b-MPT com-
pound 3, the 5-chloropyridyl R isomer, was 380-fold
This is in accordance with the results obtained with
chiral b-MPT compounds as discussed previously (see
Table 1). Substitution on the pyridyl ring at 5-position
was essential to retain the activity of the compounds as
evidenced from the table values. Among the substituents
at 5-position, halo as well as alkyl substitutions seem to
yield active derivatives. Among the methyl substituted
derivatives, the 5-methyl compound was more active
than the 6-methyl substituted compound in these assays.
It has been found that changing the pyridyl ring to
thiazole ring did not affect the activity of the compound.
Also methyl substitution at 4-position of the thiazolyl
ring was found to retain the activity of the compound in

T. K. Venkatachalam et al. / Bioorg. Med. Chem. 12 (2004) 4275–4284
4277
Table 1. Effect of stereochemistry on anti-HIV activity of b-methyl phenylethyl substituted thiourea compounds
H
H
H
H
S
Y
S
N
N
H
H
N
N
C
S
C
S
N
N
N
C
S
N
X
N
Compd
Isomer
X
Y
rRT (lM)
HTLVIIIB
(lM)
A17 (Y181C) A17V (Y181C)
(lM)
RT-MDR
(V106N)
(K103N) (lM)
(lM)
1
R
S
5Br
5Br
5Cl
5Cl
5Me
5Me
6Me
6Me
H
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
4-Me
4-Me
0.4
1.9
0.003
0.030
<0.001
0.04
0.003
ND
0.7
1.2
1.1
1.2
0.02
0.2
2
3
R
S
0.1
1.4
0.3
0.5
0.5
>100
1.8
0.02
0.2
4
5
R
S
0.5
7.7
1.1
0.05
ND
0.1
6
ND
1.4
ND
1.2
7
R
S
16.7
0.04
ND
8
>100
ND
2.3
ND
0.5
ND
0.2
9
R
S
8.4
3.5
5.1
0.03
ND
10
11
12
13
H
ND
6.9
ND
1.1
ND
ND
ND
ND
R
S
NA
NA
NA
0.06
ND
>100
ND
ND
ND
ND
R
4-Methylene- >100
carbo
ND
14
S
NA
4-Methylene- >100
carbo
ND
ND
ND
ND
15
16
R
S
NA
NA
H
H
10.0
ND
ND
ND
ND
ND
ND
ND
>100.0
0.5
ND
ND
ND
ND
ND
ND
ND
1.4
>100
>100
>100
17
18
R
R
R
––
––
4-Methylbenzo
Benzothiazolyl
ND
ND
ND
ND
19
Nevirapine
Trovirdine
4,6 Me
––
––
NA
––
38.8
ND
ND
23.0
0.8
0.03
0.007
0.009
>100.0
>100.0
>100.0
––
––
ND
0.4
Delavirdine ––
––
1.5
50.0
The anti-HIV activity was measured by determining the inhibition of the HIV-1 strain HTLVIIIB in human PBMC as previously described in
detail.19;20 The cell-free RT inhibition assays using recombinant RT(rRT) and the Quan-RT assay kit (Amersham, Arlington heights-IL) were
performed as reported.20;21 Results are presented as the mean IC50 values.
DMF, 100 o C, 15h
S
acetonitrile, rt, 12h
S
R1NH2
R1
N
S
N
N
H
R1NH
NHR2
R2NH2
N
N
N
N
Me
N
S
R1=
N
S
R2 =
S
N
Et
N
MeO
Scheme 2.
our assay. On the other hand, benzothiazolyl com-
pounds, showed a considerable decrease in activity
implying that this substitution is not beneficial among
the thioureas. In order to circumvent this problem we
introduced a few groups on the benzothiazolyl structure
of the compounds 28 and 32, however we observed no
improvement in their anti-HIV activity.
(PBMCs). As shown in Table 2, severalof the MBT
derivatives inhibited HIV-1 replication at nanomolar
concentrations. Once again the R isomers were more
active compared to their enantiomers. The lead com-
pounds 20, 21, and 24 exhibited potent activity against
the NNRTI resistant HIV-1 strains A17, 17-Variant as
well as the multi-drug resistant HIV strain RT-MDR
(Table 2).
We next examined the ability of these novel thiourea
compounds to inhibit the replication of the HIV-1 strain
HTLVIIIB in human peripheral blood mononuclear cells
Our molecular modeling studies indicated that the R
stereoisomers of chiral halopyrdiyl as well as chiral

4278
Table 2. Effect of stereochemistry on anti-HIV activity of alpha-methyl benzyl substituted thiourea compounds
Me
T. K. Venkatachalam et al. / Bioorg. Med. Chem. 12 (2004) 4275–4284
S
X
N
N
H
H
Compd
Isomer
X
rRT (lM)
HTLVIIIB
(lM)
A17 (lM)
A17V (lM)
RTMDR
(lM)
Br
Cl
20
21
22
23
R
R
S
S
1.6
1.2
<0.01
<0.01
>1
0.010
0.200
ND
2.7
0.005
0.010
ND
N
10.20
ND
ND
N
N
Br
Cl
>100
>100
ND
ND
ND
N
CH3
N
24
25
R
S
0.8
<0.001
ND
0.164
ND
0.196
ND
0.001
ND
S
CH3
N
>100
S
26
27
S
>100
1.5
ND
ND
ND
ND
ND
ND
ND
ND
N
CH3
CH3
R
N
H3C
28
R
8.7
ND
ND
ND
ND
N
N
S
OCH3
29
30
31
S
S
R
>100
>100
1.7
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
S
CH3
ND
N
0.025
N
CH3
OCH3
N
32
R
>100
ND
ND
ND
ND
S
Nevirapine
Trovirdine
Delavirdine
HI-240
NA
NA
NA
NA
NA
NA
NA
NA
23
0.034
0.007
0.009
0.001
100
0.50
50
100
100
100
41.0
1.4
0.8
1.5
0.6
ND
0.40
ND
0.20
The anti-HIV activity was measured by determining the inhibition of the HIV-1 strain HTLVIIIB in human PBMC as previously described in detail.20 The
cell-free RT inhibition assays using recombinant RT (rRT) and the Quan-RT assay kit (Amersham, Arlington heights-Il) were performed as reported.20

T. K. Venkatachalam et al. / Bioorg. Med. Chem. 12 (2004) 4275–4284
4279
thiazolyl thiourea compounds would fit the target
NNRTI binding pocket on HIV-RT much better than
their enantiomers. Unfavorable interactions with the
NNRTI binding pocket residues near the Y181 side
chain would impair the binding of the S stereoisomers in
their lower energy ‘staggered’ conformation. This steric
hindrance could be relieved if an S stereoisomer would
adopt an energetically unfavorable ‘eclipsed’ confor-
mation. In either case, the estimated binding energies
would be significantly higher for S compounds com-
pared to R isomers. Figure 1 shows a representative
picture of a phenylethyl pyridylthiourea molecule
docked into the NNRTI binding site. Substitutions at
the R and S positions would lead to different confor-
mations of the phenethyl group in the Wing 2 region.
The R stereoisomer is energetically more favored than
the S stereoisomer, consistent with the better-fit and
thus stronger binding with the NNRTI binding pocket.
However, the two-carbon linker between the phenyl
group and the thiourea group is adjustable and more
forgiving. The energetic difference is minimized by
adopting alternative conformation to maintain the
snugly fit with the binding pocket for both R and S
stereoisomers, in comparison with those of the com-
pounds with one carbon spacing. The compounds with
one-carbon spacing are more rigid and their S stereo-
isomers have to overcome greater energetic barrier to be
accommodated by the binding pocket. Based on this
rationale we expect that R stereoisomers of b-methyl
phenylethyl halopyridylthioureas may show much more
potency compared to S stereoisomers against reverse
transcriptase, which is consistent with our experimental
results. It is interesting to note that aza analogs of chiral
2-phosphonomethoxy (propyl]guanines of R stereoiso-
meric configuration were found to show potent anti-
HIV activity as compared to the S isomers19 a result,
which is consistent with our experimental data on chiral
thiourea compounds.
compounds, which differ only by a carbon atom, which
can fit into the NNRTI binding pocket as discussed in
the previous paragraphs. The experimental data also
provide evidence that R stereoisomers of a-MBT com-
pounds were active as compared to their enantiomers.
3. Experimental
All chemicals were purchased from Aldrich (Milwaukee,
WI) and were used without further purification. Unless
otherwise noted, each reaction vessel was secured with a
rubber septa, and the reaction was performed under
nitrogen atmosphere. 1H, 13C NMR were obtained on a
Varian Mercury 300 instrument at ambient temperature
in DMSO-d6 Chemical shifts are reported as d values in
parts per million down field from tetramethylsilane
(d ¼ 0 ppm) as an internal standard or from the residual
dimethylsulfoxide signal (d ¼ 2:49 ppm for 1H NMR or
d ¼ 39:7 ppm for 13C NMR). Splitting patterns are
designated as follows: s, singlet; d, doublet; t, triplet; m,
multiplet; br, broad peak. FT-IR spectra were recorded
on a Nicolet Protege 460 spectrometer. Mass spectra
were performed on a Hewlett Packard MALDI-TOF
spectrometer (Model G2025A LD-TOF). Melting points
were determined using a Melt John’s apparatus and are
uncorrected. HPLC’s were done using a Hewlett Pack-
ard 1100 series instrument consisting of a automatic
sampler, a electronic degasser, a thermostatic control
unit, a diode array detector in conjunction with a
chemstation software assembly.22,23 The column used
was an analytical RP-18 Lichrosphere column, 5u
(4.6 · 150 mm) and eluent was 35:65, H2O (0.1%
ACOH): ACN. The flow rate was maintained at 1.0 mL/
min and the detection wavelength was set at 275 nm. The
column was maintained at room temperature through-
out the analysis. Column chromatography was per-
formed using silica gel obtained from J.T. Baker
Company. The solvents used for elution varied
depending on the compound and included either one or
a combination of the following: ethylacetate, methanol,
chloroform, hexane, methylene chloride, THF, and
ether. The optical rotation of the thioureas were deter-
mined using a polarimeter. However no effort was made
to determine the optical purity of these chiral thiourea
derivatives. Elemental analysis was performed at
Atlanta Micro Labs, for all the novel thiourea com-
pounds.
Based on the results obtained in the case of b-MPT
compounds from molecular modeling studies, we
rationalize that a similar trend is anticipated for a-MBT
Compounds were made by condensing the respective
chiral amines and thiocarbonylimidazole derivatives of 5
and 60-substituted amino pyridines in anhydrous dim-
ethylformamide (Scheme 1). The chiral amines were
purchased from Aldrich Chemical Company and were
used without further purification. Thiazolyl and benzo-
thiazolyl substituted thioureas were prepared in a simi-
lar fashion using thiocarbaimidazole derivatives of
either substituted amino thiazoles or benzothiazoles,
respectively.
Figure 1. Molecular model of a phenethyl pyridyl thiourea molecule
that is docked into the NNRTI binding pocket. Green background
indicates the phenyl group attached through a two carbon linker
marked as B and A in the figure. The chiral center in the molecule is
labeled as ‘B’. The pyridyl group is represented on the left hand side of
the figure and is connected through thiourea unit.
In brief, Scheme 1 involved the addition of thiocar-
bonyldimidazole and 2-amino-5-halo pyridine to

4280
T. K. Venkatachalam et al. / Bioorg. Med. Chem. 12 (2004) 4275–4284
100 mL of dry acetonitrile under nitrogen atmosphere.
The contents were stirred at room temperature for 12–
15 h. The precipitate obtained was filtered, washed with
cold acetonitrile, and dried thoroughly under vacuum to
yield the thiocarbonyl intermediate. In the subsequent
step, the intermediates was taken up in a dry flask under
nitrogen, 50 mL of anhydrous dimethylformamide was
added, and the contents were stirred for 30 min at room
temperature. To this mixture, appropriate chiral amines
dissolved in 10 mL of dry dimethylformamide was added
and the reaction mixture was heated to 110 ꢁC over an
oil bath for 15 h. After this period, the reaction mixture
was cooled to room temperature and poured into cru-
shed ice/water mixture and the contents were stirred for
an additional hour. The precipitate was filtered, washed
with cold water, and dried under vacuum. The dried
precipitate was dissolved in chloroform and washed with
brine, water, and the organic layer was dried over
anhydrous magnesium sulfate. Filtration and evapora-
tion of the solvent yielded the target thiourea com-
pound. Compounds were further purified using silica gel
column chromatography and finally recrystallized using
ethanol as solvent. A similar procedure was followed for
other substituted compounds using either thiazolyl or
benzothiazolyl precursors.
which utilizes a murine mAb to HIV core protein coated
onto microwell strips to which the antigen present in the
test culture supernatant samples binds. Percent viral
inhibition was calculated by comparing the p24 values
from untreated infected cells (i.e., virus controls). In
addition, the IC50 values measuring the activity of
compounds against recombinant HIV-1 RT (rRT) were
determined using the Quant-RT assay system (Amer-
sham, Arlington Heights, Il, USA), which utilizes the
scintillation proximity assay principle.20;21
3.2. Physical constants of compounds
3.2.1. N-[(2R)-b-Methylphenylethyl]-N0-[2-(5-bromopyr-
1
idyl)]thiourea (1). Melting point 152–153 ꢁC; H NMR
(DMSO-d6) d 11.14 (s, 1H), 10.68 (s, 1H), 7.97 (s, 1H),
7.91 (m, 1H), 7.32 (s, 4H), 7.23 (m, 1H), 7.07 (d, 1H,
J ¼ 8:7 Hz), 3.87 (m, 1H), 3.67 (m, 1H), 3.12 (m, 1H),
1.26 (d, 3H, J ¼ 5:1 Hz); 13C NMR (DMSO-d6) d 179.6,
152.9, 146.1, 144.7, 141.9, 129.1, 127.9, 127.2, 115.1,
112.4, 52.7, 20.1; IR m 3519, 3222, 3027, 2962, 1592,
t
1548, 1475, 1309, 1176, 823 cmꢀ1; ½aꢁ +4.5 (CHCl3);
D
MALDI-TOF m=z 352.1 (C15H16BrN3S+2Hþ); HPLC
Rt: 3.64 min. Anal. Calcd for C15H16BrN3S: C, 51.44; H,
4.60; N, 12.00. Found: C, 49.62; H, 4.30; N, 12.16.
Stock HTLVIIIB virus: The HIV-1 strain HTLVIIIB
,
which was propagated in CCRF-CEM cells, was used in
in vitro assays of the anti-HIV-1 activity of the synthe-
sized thiourea derivatives. Cell-free supernatants of
HTLVIIIB-infected CCRF-CEM cells were harvested,
dispensed into 1 mL aliquots, and frozen at )70 ꢁC.
Periodic titration of stock virus was performed by
examining its cytopathic effects in MT-2 cells.20;21 The
HIV-1 strains HTLVIIIB (wild-type RT, NRTI-sensi-
tive, NNRTI-sensitive), A17 (Y181C mutant, NNRTI-
resistant), A17-variant (Y181C+K103N mutant,
NNRTI-resistant), and RT-MDR (M41L, V106N,
Y181C, T215Y; NRTI-resistant, NNRTI-resistant) were
also obtained from the AIDS Research and Reference
Reagent Program, NIAID.
3.2.2. N-[(2S)-b-Methylphenylethyl]-N0-[2-(5-bromopyr-
idyl)thiourea (2). Melting point 157–158 ꢁC; H NMR
(DMSO-d6) d 11.13 (t, 1H), 10.67 (s, 1H), 7.98 (d, 1H,
J ¼ 2:7 Hz), 7.92 (dd, 1H, J ¼ 2:4, 8.7 Hz), 7.32 (s, 4H),
7.24 (m, 1H), 7.07 (d, 1H, J ¼ 8:7 Hz), 3.85 (m, 1H),
3.68 (m, 1H), 3.11 (m, 1H), 1.26 (d, 3H, J ¼ 6:9 Hz); 13C
NMR (DMSO-d6) d 179.0, 152.3, 145.5, 144.1, 141.4,
128.5, 127.3, 126.2, 114.5, 111.7, 52.1, 19.5; IR m 3441,
1
3226, 3024, 2968, 1597, 1541, 1472, 1304, 1175,
t
827 cmꢀ1; ½aꢁ )4.4 (CHCl3); MALDI-TOF m=z 352.7
D
(C15H16BrN3S+2Hþ); HPLC Rt: 3.64 min. Anal. Calcd
for C15H16BrN3S: C, 51.44; H, 4.60; N, 12.00. Found: C,
51.67; H, 4.63; N, 11.86.
3.1. In vitro assays of anti-HIV-1 activity
3.2.3. N-[(2R)-b-Methylphenylethyl]-N0-[2-(5-chloropyr-
idyl)]thiourea (3). Melting point 167–169 ꢁC; H NMR
1
Normal human peripheral blood mononuclear cells
(PBMC) from HIV-negative donors were cultured 72 h
in RPMI 1640 supplemented with 20% (v/v) heat-inac-
tivated fetal bovine serum (FBS), 3% interleukin-2,
(DMSO-d6) d 11.14 (s. 1H), 10.67 (s, 1H), 7.90 (s, 1H),
7.80 (m, 1H), 7.32 (s, 4H), 7.23 (m, 1H), 7.12 (m, 1H),
3.86 (m, 1H), 3.67 (m, 1H), 3.10 (m, 1H), 1.23 (m, 3H);
13C NMR (DMSO-d6) d 179.1, 152.1, 144.2, 143.3,
138.9, 128.6, 127.3, 126.6, 123.7, 114.1, 52.1, 38.5, 19.5;
2 mM
L-glutamine, 25 mM HEPES, 2 g/L NaHCO3,
50 lg/mL gentamicin, and 4 lg/mL phytohemagglutinin
prior to exposure to HIV-1 at a multiplicity of infection
(MOI) of 0.1 during a 1 h adsorption period at 37 ꢁC in a
humidified 5% CO2 atmosphere. Subsequently, cells
were cultured in 96-well microtiter plates (100 lL/well;
2 · 106 cells/mL) in the presence of various concentra-
tions of thiourea analogues or nevirapine, trovirdine or
AZT and aliquots of culture supernatants were removed
from the wells on the seventh day after infection for p24
antigen assays, as previously described.20;21 The applied
p24 enzyme immunoassay (EIA) was the unmodified
kinetic assay commercially available from Coulter
Corporation/Immunotech, Inc. (Westbrooke, ME),
IR m 3507, 3220, 3027, 2966, 1600, 1533, 1477, 1311,
t
1230, 1178, 1109, 825, 698 cmꢀ1; ½aꢁ +3.1 (CHCl3);
D
MALDI-TOF m=z 312.4 (C15H16ClN3S+4Hþ); HPLC
Rt: 11.20 min. Anal. Calcd for C15H16ClN3S: C, 58.91;
H, 5,27; N, 13.74. Found: C, 56.81; H, 4.90; N, 13.74.
3.2.4. N-[(2S)-b-Methylphenylethyl]-N0-[2-(5-chloropyr-
1
idyl)]thiourea (4). Melting point 171–173 ꢁC; H NMR
(DMSO-d6) d 11.14 (s. 1H), 10.67 (s, 1H), 7.91 (d, 1H,
J ¼ 2:4 Hz), 7.81 (dd, 1H, J ¼ 2:4, 9 Hz), 7.32 (s, 4H),
7.23 (m, 1H), 7.12 (d, 1H, J ¼ 9:3 Hz), 3.85 (m, 1H),
3.67 (m, 1H), 3.11 (q, 1H), 1.26 (d, 3H, J ¼ 6:9 Hz); 13C

T. K. Venkatachalam et al. / Bioorg. Med. Chem. 12 (2004) 4275–4284
4281
NMR (DMSO-d6) d 179.1, 152.1, 144.2, 143.3, 138.9,
128.6, 127.3, 126.6, 123.7, 114.1, 52.1, 38.5, 19.5; IR m
3500, 3220, 3027, 2966, 1598, 1556, 1477, 1311, 1230,
3.2.9. N-[(2R)-b-Methylphenylethyl]-N0-[2-(pyridyl)]thio-
urea (9). Melting point 130–131 ꢁC; H NMR (DMSO-
d6) d 11.65 (s, 1H), 10.53 (d, 1H, J ¼ 8:7 Hz), 7.90 (q,
1H), 7.70 (q, 1H), 7.32 (m, 4H), 7.22 (m, 1H), 7.09 (t,
1H), 6.95 (m, 1H), 3.88 (m, 1 h), 3.69 (m, 1H), 3.12 (m,
1H), 1.28 (q, 3H); 13C NMR (DMSO-d6) d 179.3, 153.7,
145.2, 144.3, 138.9, 128.5, 127.3, 126.6, 117.7, 112.5,
1
t
1178 cmꢀ1; ½aꢁ )3.1 (CHCl3); MALDI-TOF m=z 307.4
D
(C15H16ClN3S); HPLC Rt: 11.20 min. Anal. Calcd for
C15H16ClN3S: C, 58.91; H, 5.27; N, 13.74. Found: C,
59.07; H, 5.28; N, 13.74.
52.1, 38.6, 19.5; IR m 3463, 3228, 3023, 2966, 1602, 1560,
t
1540, 1481, 1317, 769 cmꢀ1; ½aꢁ +2.4 (CHCl3); MALDI-
D
TOF m=z 272.9 (C15H17N3S+2Hþ); HPLC Rt: 7.26 min.
Anal. Calcd for C15H17N3S: C, 66.39; H, 6.31; N, 15.48.
Found: C, 66.13; H, 6.20; N, 15.57.
3.2.5. N-[(2R)-b-Methylphenylethyl]-N0-[2-(5-methylpyr-
idyl)]thiourea (5). Melting point 131–132 ꢁC; H NMR
(DMSO-d6) d 11.56 (s, 1H), 10.45 (s, 1H), 7.75 (s, 1H),
7.55 (m, 1H), 7.33 (s, 4H), 7.23 (m, 1H), 7.01 (m, 1H),
3.85 (m, 1H), 3.70 (m, 1 h), 3.10 (m, 1H), 2.17 (s, 3H),
1.27 (t, 3H); 13C NMR (DMSO-d6) d 179.1, 151.7, 144.4,
144.2, 139.6, 128.5, 127.2, 126.5, 1112.0, 52.0, 38.6, 19.5,
1
3.2.10.
N-[(2S)-b-Methylphenylethyl]-N0-[2-(pyridyl)]-
thiourea (10). Melting point 118–119 ꢁC; 1H NMR
(DMSO-d6) d 11.63 (t, 1H), 10.51 (s, 1H), 7.88 (dd, 1H,
J ¼ 1:8, 5.1 Hz), 7.68 (m, 1H), 7.32 (d, 3H, J ¼ 4:5 Hz),
7.21 (m, 2H), 7.07 (d, 1H, J ¼ 8:7 Hz), 6.93 (dd, 1H,
J ¼ 5:4, 7.5 Hz), 3.87 (m, 1H), 3.68 (m, 1H), 3.10 (q,
1H), 1.26 (d, 3H, J ¼ 6:6 Hz); 13C NMR (DMSO-d6) d
179.3, 153.6, 145.1, 144.2, 138.9, 128.5, 128.4, 127.3,
127.1, 126.5, 117.6, 112.4, 52.0, 38.16, 19.5; IR m 3228,
17.4; IR m 3519, 3228, 3022, 2968, 1610, 1556, 1533,
t
1492, 1303, 1188, 702 cmꢀ1; ½aꢁ +2.9 (CHCl3); MALDI-
D
TOF m=z 287.2 (C16H19N3S+2Hþ); HPLC Rt: 9.72 min.
Anal. Calcd for C16H19N3S: C, 67.33; H, 6.71; N, 14.72.
Found: C, 67.39; H, 6.64; N, 14.78.
3170, 3023, 2966, 1602, 1560, 1540, 1450, 1317,
3.2.6. N-[(2S)-b-Methylphenylethyl]-N0-[2-(5-methylpyr-
idyl)]thiourea (6). Melting point 130–131 ꢁC; H NMR
(DMSO-d6) d 11.57 (t, 1H), 10.44 (s, 1H), 7.73 (s, 1H),
7.53 (dd, 1H, J ¼ 2:4, 8.4 Hz), 7.32 (s, 4H), 7.23 (m, 1H),
7.00 (m, 1H), 3.84 (m, 1H), 3.69 (m, 1H), 3.10 (q, 1H),
2.16 (s, 3H), 1.26 (d, 3H, J ¼ 7:2 Hz); 13C NMR
(DMSO-d6) d 179.1, 151.7, 144.4, 144.2, 139.6, 128.5,
127.2, 126.5, 112.0, 52.0, 38.6, 19.5, 17.4; IR m 3506,
t
769 cmꢀ1; ½aꢁ )3.1 (CHCl3); MALDI-TOF m=z 292.2
1
D
(C15H17N3S+Na-2Hþ); HPLC Rt: 7.21 min. Anal. Calcd
for C15H17N3S: C, 66.39; H, 6.31; N, 15.48. Found: C,
66.79; H, 6.34; N, 15.43.
3.2.11.
N-[(2R)-b-Methylphenylethyl]-N0-[2-(4-methyl-
thiazolyl)]thiourea (11). Melting point 163–164 ꢁC; 1H
NMR (DMSO-d6) d 11.49 (s, 1H), 10.04 (s, 1H), 7.31 9s,
4H), 7.22 (s, 1H), 6.61 (s, 1H), 3.79 (m, 1H), 3.69 (m,
1H), 3.09 (t, 1H), 2.09 (s, 3H), 1.25 (t, 3H); 13C NMR
(DMSO-d6) d 177.8, 161.1, 144.1, 128.5, 127.2, 126.5,
3228, 3022, 2968, 1610, 1533, 1492, 1303, 1234,
t
703 cmꢀ1; ½aꢁ )3.4 (CHCl3); MALDI-TOF m=z 287.1
D
(C16H19N3S+2Hþ); HPLC Rt: 9.74 min. Anal. Calcd for
C16H19N3S: C, 67.33; H, 6.71; N, 14.72. Found: C,
67.37; H, 6.69; N, 14.87.
106.2, 51.4, 38.5, 19.7, 16.9; IR m 3500, 3170, 3025, 2977,
t
1567, 1531, 1504, 1209, 754, 713 cmꢀ1; ½aꢁ +3.4
D
(CHCl3); MALDI-TOF m=z 292.9 (C14H17N3S2+2Hþ);
HPLC Rt: 8.84 min. Anal. Calcd for C14H17N3S2: C,
57.70; H, 5.88; N, 14.42. Found: C, 57.72; H, 5.84; N,
14.46.
3.2.7. N-[(2R)-b-Methylphenylethyl]-N0-[2-(6-methylpyr-
1
idyl)]thiourea (7). Melting point 125–126 ꢁC; H NMR
(DMSO-d6) d 11.77 (t, 1H), 10.46 (s, 1H), 7.58 (t, 1H),
7.30 (d, 4H, J ¼ 4:2 Hz), 7.21 (m, 1H), 6.89 (d, 1H,
J ¼ 8:4 Hz), 6.79 (d, 1H, J ¼ 7:2 Hz), 3.90 (m, 1H), 3.79
(m, 1H), 3.11 (q, 1H), 2.11 (s, 3H), 1.26 (d, 3H,
J ¼ 6:9 Hz); 13C NMR (DMSO-d6) d 179.5, 154.3, 153.2,
144.4, 139.1, 128.6, 127.2, 126.1, 116.8, 109.3, 51.4, 38.7,
3.2.12.
N-[(2S)-b-Methylphenylethyl]-N0-[2-(4-methyl-
thiazolyl)]thiourea (12). Melting point 163–164 ꢁC; 1H
NMR (DMSO-d6) d 11.49 (s, 1H), 10.03 (s, 1H), 7.30 (s,
4H), 7.22 (m, 1H), 6.60 (s, 1H), 3.79 (m, 1H), 3.68 (m,
1H), 3.08 (q, 1H), 2.09 (s, 3H), 1.24 (d, 3H, J ¼ 6:9 Hz);
13C NMR (DMSO-d6) d 177.6, 160.9, 146.5, 144.1,
128.5, 127.2, 126.5, 106.1, 51.4, 38.5, 19.7, 16.9; IR m
23.5, 20.2; IR m 3500, 3228, 3027, 2962, 1608, 1537, 1450,
t
1236, 788 cmꢀ1; ½aꢁ +2.9 (CHCl3); MALDI-TOF m=z
D
286.9 (C16H19N3S+2Hþ); HPLC Rt: 10.13 min.
3430, 3170, 3025, 2919, 1567, 1531, 1504, 1209 cmꢀ1
;
t
D
3.2.8. N-[(2S)-b-Methylphenylethyl]-N0-[2-(6-methylpyr-
½aꢁ
)2.5 (CHCl3); MALDI-TOF m=z 293.0
1
idyl)]thiourea (8). Melting point 123–124 ꢁC; H NMR
(C14H17N3S2+2Hþ); HPLC Rt: 8.87 min.
(DMSO-d6) d 11.76 (s, 1H), 10.46 (s, 1H), 7.56 (t, 1H),
7.29 (d, 4H, J ¼ 4:2 Hz), 7.19 (m, 1H), 6.88 (d, 1H,
J ¼ 8:1 Hz), 6.78 (d, 1H, J ¼ 8:1 Hz), 3.89 (m, 1H), 3.78
(m, 1H), 3.11 (q, 1H), 2.10 (s, 3H), 1.25 (d, 3H,
J ¼ 6:9 Hz); 13C NMR (DMSO-d6) d 179.5, 154.3, 153.2,
144.4, 139.1, 128.6, 127.1, 126.5, 116.8, 109.3, 51.4, 38.7,
3.2.13.
N-[(2R)-b-Methylphenylethyl]-N0-[2-(4-methyl-
enecarboethoxy thioazolyl)]thiourea (DDE 13). Melting
point 106–108 ꢁC; 1H NMR (DMSO-d6) d 11.56 (s, 1H),
9.56 (s, 1H), 7.28 (s, 4H), 7.20 (m, 1H), 6.84 (s, 1H), 4.07
(q, 2H), 3.70 (q, 2H), 3.56 (s, 2H), 3.07 (q, 2H), 1.23 (d,
3H, J ¼ 6:9 Hz), 1.17 (t, 3H); 13C NMR (DMSO-d6) d
177.5, 169.7, 160.8, 144.1, 143.3, 128.5, 127.1, 126.5,
23.5, 20.2; IR m 3480, 3228, 3027, 29634, 1606, 1548,
t
1450, 1236, 810 cmꢀ1; ½aꢁ )2.7 (CHCl3); MALDI-TOF
D
m=z 287.1 (C16H19N3S+2Hþ); HPLC Rt: 10.05 min.

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T. K. Venkatachalam et al. / Bioorg. Med. Chem. 12 (2004) 4275–4284
t
D
109.2, 60.5, 51.3, 38.5, 36.5, 19.6, 14.3; IR m 3448, 3170,
1195, 754 cmꢀ1; ½aꢁ +2.5 (CHCl3); MALDI-TOF m=z
t
3025, 2977, 1733, 1562, 1506, 1182, 696 cmꢀ1; ½aꢁ +2.5
329.2 (C17H17N3S2+2Hþ); HPLC Rt: 12.66 min.
D
(CHCl3); MALDI-TOF m=z 365.3 (C17H21N3O2S2+
2Hþ); HPLC Rt: 7.09 min.
3.2.19. N-[(2R)-b-Methylphenylethyl]-N0-[2-(4,6-dimeth-
ylpyridyl)]thiourea (19). Melting point 123–124 ꢁC; H
1
3.2.14. N-[(2S)-b-Methylphenylethyl]-N0-[2-(4-methylene-
carboethoxy thioazolyl)]thiourea (DDE 14). Melting
point 104–106 ꢁC; 1H NMR (DMSO-d6) d 11.57 (s, 1H),
9.56 (s, 1H), 7.28 (m, 4H), 7.21 (m, 1H), 6.84 (s, 1H), 4.07
(q, 2H), 3.71 (q, 2H), 3.56 (s, 2H), 3.07 (q, 1H), 1.24 (d,
3H, J ¼ 6:9 Hz), 1.17 (t, 3H); 13C NMR (DMSO-d6) d
177.5, 169.7, 160.8, 144.1, 143.3, 128.5, 127.1, 126.5,
NMR (DMSO-d6) d 12.60 (d, 1H, J ¼ 8:1 Hz), 10.46 (s,
1H), 7.36 (m, 4H), 7.28 (m, 1H), 6.76 (d, 2H,
J ¼ 5:1 Hz), 5.47 (m, 1H), 2.33 (s, 3H), 2.21 (s, 3H), 1.53
(d, 3H, J ¼ 6:9 Hz); 13C NMR (DMSO-d6) d 178.6,
153.8, 153.5, 150.1, 143.3, 128.6, 127.2, 126.2, 118.2,
109.5,. 54.2, 23.4, 22.9, 21.0; IR m 3484, 3224, 3029,
2964, 1623, 1592, 1540, 1211, 698 cmꢀ1; HPLC Rt:
13.46 min.
109.2, 60.5, 51.3, 38.5, 36.5, 19.6, 14.3; IR m 3448, 3172,
t
3025, 2977, 1733, 1562, 1506, 1182, 696 cmꢀ1; ½aꢁ )2.5
D
(CHCl3); MALDI-TOF m=z 365.1 (C17H21N3O2S2+
2Hþ); HPLC Rt: 7.10 min.
3.2.20.
N-[1-(1R)-a-Methylbenzyl]-N0-[2-(5-bromopyr-
idyl)]thiourea (20). Melting point 170–172 ꢁC; 1H
NMR (DMSO-d6) d 11.79 (m, 1H), 10.76 (s, 1H), 8.39
(d, 1H, J ¼ 2:1 Hz), 7.98 (dd, 1H, J ¼ 9:0, 9.0 Hz), 7.35
(d, 4H, J ¼ 3:9 Hz), 7.28–7.23 (m, 1H), 7.15 (d, 1H,
J ¼ 9 Hz), 5.55 (q, 1H), 1.51 (m, 3H); 13C NMR
(DMSO-d6) d 178.4, 152.5, 146.2, 143.2, 141.5, 128.6,
127.0, 126.1, 114.7, 112.0, 54.0, 22.7; IR m 3245, 3027,
3.2.15. N-[(2R)-b-Methylphenylethyl]-N0-[2-(thioazolyl)]-
thiourea (15). 1H NMR (DMSO-d6) d 11.55 (s, 1H), 9.65
(s, 1H), 7.29 (t, 5H), 7.21 (m, 1H), 7.06 (t, 1H), 3.71 (m,
2), 3.11 (q, 1H), 1.23 (t, 3H); 13C NMR (DMSO-d6) d
177.9, 161.7, 144.1, 136.8, 128.5, 127.2, 126.5, 112.1,
2979, 2925, 1594, 1525, 1475, 1188, 704 cmꢀ1; UV
51.4, 38.4, 19.5; IR m 3178, 3023, 2950, 1558, 1508, 1467,
t
t
(MeOH) kmax 211, 257, 275, 278 nm; ½aꢁ )21.5 (CHCl3);
1178, 698 cmꢀ1; ½aꢁ +3.8 (CHCl3); MALDI-TOF m=z
D
D
MALDI-TOF MS m=z 337.7 (C14H14BrN3S+Hþ). Anal.
Calcd for C14H14BrN3S: C, 50.01; H, 4.20; N, 12.50.
Found: C, 50.27; H, 4.16; N, 12.53.
280.1 (C13H15N3S2+2Hþ); HPLC Rt: 6.23 min.
3.2.16. N-[(2S)-b-Methylphenylethyl]-N0-[2-(thioazolyl)]-
thiourea (16). Melting point 128–130 ꢁC; 1H NMR
(DMSO-d6) d 11.56 (s, 1H), 9.66 (s, 1H), 7.27 (t, 4H),
7.21 (m, 2H), 7.04 (d, 1H, J ¼ 3:6 Hz), 3.71 (m, 2H),
3.10 (q, 1H), 1.23 (d, 3H, J ¼ 7:2 Hz); 13C NMR
(DMSO-d6) d 177.9, 161.7, 144.0, 136.8, 128.5, 127.2,
3.2.21.
N-[1-(1-(1R)-a-Methylbenzyl]-N0-[2-(5-chloro-
1
pyridiyl)]thiourea (21). Melting point 185–187.5 ꢁC; H
NMR (CDCl3) d 11.77 (m, 1H), 9.47 (s, 1H), 8.12 (d,
1H, J ¼ 2:1 Hz), 7.53 (d, 1H, J ¼ 8:7 Hz), 7.40–7.26 (m,
H), 6.85 (d, 1H, J ¼ 8:7 Hz), 5.73–5.64 (m, 1H), 1.65 (d,
3H, J ¼ 6:9 Hz); 13C NMR (CDCl3) d 178.4. 151.8,
142.8, 144.4, 138.8, 128.9, 127.5, 126.4, 125.4, 113.6,
126.5, 112.1, 51.4, 38.4, 19.4; IR m 3444, 3174, 3025,
t
1576, 1515, 1176, 678 cmꢀ1; ½aꢁ )4.0 (CHCl3); MALDI-
D
TOF m=z 280.3 (C13H15N3S2+2Hþ); HPLC Rt: 6.52 min.
55.3, 22.7; IR m 3247, 3169, 3087, 2978, 1600, 1529, 1483,
t
D
1189, 1034, 822, 761, 694 cmꢀ1; ½aꢁ )20.9 (CHCl3); UV
(MeOH) kmax 204, 255, 275, 305 nm; MALDI-TOF MS
m=z 293.7 (C14H14ClN3S+2Hþ). Anal. Calcd for
C14H14ClN3S: C, 57.63; H, 4.84; N, 14.40. Found: C,
57.71; H, 4.84; N, 14.38.
3.2.17.
N-[(2R)-b-Methylphenylethyl]-N0-[2-(4-methyl
benzothiazolyl)]thiourea (17). Melting point 203–205 ꢁC;
1H NMR (DMSO-d6) d 11.87 (s, 1H), 10.16 (s, 1H), 7.67
(d, 1H, J ¼ 7:2 Hz), 7.27 (d, 4H, J ¼ 4:2 Hz), 7.18 (dd,
1H, J ¼ 3:9, 12.6 Hz), 7.13 (s, 1H), 7.11 (s, 1H), 3.81 (t,
2H), 3.14 (q, 1H), 2.34 (s, 3H), 1.26 (d, 3H, J ¼ 6:9 Hz);
13C NMR (DMSO-d6) d 177.9, 159.8, 147.8, 144.0,
129.7, 129.2, 128.5, 127.1, 126.7, 126.5, 123.7, 118.9,
51.2, 38.4, 19.9, 17.9; IR m 3443, 3172, 3027, 2966, 1556,
3.2.22.
N-[1-(1S)-a-Methylbenzyl]-N0-[2-(5-bromopyr-
1
idyl)]thiourea (22). Melting point 170–172 ꢁC; H NMR
(CDCl3) d 11.73 (m, 1H), 8.95 (s, 1H), 8.23 (d, 1H,
J ¼ 2:1 Hz), 7.69 (d, 1H, J ¼ 8:7 Hz), 7.40–7.28 (m, 5H),
6.73 (d, 1H, J ¼ 8:7 Hz), 5.72–5.63 (m, 1H), 1.65 (d, 3H,
J ¼ 6:8 Hz); 13C NMR (CDCl3) d 178.8, 151.9, 146.8,
142.8, 141.5, 128.9, 127.5, 126.4, 113.8, 113.0, 55.4, 22.6; IR
1521, 1216, 696 cmꢀ1
;
MALDI-TOF m=z 343.3
(C18H19N3S2+2Hþ); HPLC Rt: 18.99 min.
m 3461, 3243, 3089, 2979, 1594, 1525, 1473, 1186, 825,
t
3.2.18. N-[(2R)-b-Methylphenylethyl]-N0-[2-(benzothiaz-
olyl)]thiourea (18). Melting point 181–183 ꢁC; 1H
NMR (DMSO-d6) d 11.86 (s, 1H), 10.14 (s, 1H), 7.85 (d,
1H, J ¼ 7:8 Hz), 7.47 (d, 1H, J ¼ 8:1 Hz), 7.38 (d, 1H,
J ¼ 7:2 Hz), 7.32 (d, 4H, J ¼ 4:5 Hz), 7.22 (m, 2H), 3.86
(m, 1H), 3.72 (m, 1H), 3.15 (q, 1H), 1.26 (d, 3H,
J ¼ 6:9 Hz); 13C NMR (DMSO-d6) d 178.6, 161.1, 148.2,
129.7, 128.6, 127.2, 126.6, 126.2, 123.7, 121.7, 119.5,
51.6, 38.4, 19.7; IR m 3444, 3160, 3025, 2958, 1556, 1525,
703 cmꢀ1; ½aꢁ +21.3 (CHCl3); UV (MeOH) kmax 209, 257,
D
306 nm;
276,
MALDI-TOF
MS
m=z
337.0
(C14H14BrN3S+Hþ). Anal. Calcd for C14H14ClN3S: C,
57.63; H, 4.84; N, 14.40. Found: C, 57.69; H, 4.80; N, 14.24.
3.2.23.
idyl)]thiourea (23). Melting point 185–187 ꢁC; 1H
NMR (CDCl3) d 11.75 (m, 1H), 9.12 (s, 1H), 8.13 (d,
N-[1-(1S)-a-Methylbenzyl]-N0-[2-(5-chloropyr-

T. K. Venkatachalam et al. / Bioorg. Med. Chem. 12 (2004) 4275–4284
4283
1H, J ¼ 2:4), 7.55 (d, 1H, J ¼ 8:7 Hz), 7.41–7.25 (m,
6H), 6.79 (d, 1H, J ¼ 8:7 Hz), 5.73–5.63 (m, 1H), 1.65
(d, 3H, J ¼ 6:9 Hz); 13C NMR (CDCl3) 178.5, 151.7,
144.5, 142.8, 138.9, 128.9, 127.5, 126.4, 125.4, 113.4,
55.3, 22.6; IR m 3236, 2973, 1601, 1524, 1483, 1189, 828,
3.2.28. N-[1-(1R)-a-Methylbenzyl]-N0-[2-(4-methylbenzo-
thiazolyl)]thiourea (28). Melting point 166–171 ꢁC; 1H
NMR (DMSO-d6) d 11.88 (s, 1H), 10.92 (s, 1H), 7.70
(m, 1H), 7.42–7.34 (m, 5H), 7.30–7.26 (m, 1H), 7.18–
7.14 (m, 1H), 5.51–5.39 (m, 1H), 2.45 (m, 3H), 1.56 (m,
3H); 13C NMR (DMSO-d6) 177.1, 160.4, 147.8, 142.8,
129.6, 129.2, 128.8, 127.5, 127.0, 126.3, 124.0, 119.3,
54.2, 22.5, 18.1; IR m 3170, 3023, 1565, 1527, 1203, 740,
702 cmꢀ1; MALDI-TOF m=z 329.4 (C17H17N3S2+2Hþ).
761 cmꢀ1; IR m 3461, 3243, 3089, 2979, 1594, 1525, 1473,
t
1186, 825, 703 cmꢀ1; ½aꢁ +21.6 (CHCl3); UV (MeOH)
D
kmax 207, 256, 276 nm; MALDI-TOF MS m=z 293.8
(C14H14ClN3S+2Hþ).
3.2.29.
N-[1-(1S)-a-Methylbenzyl]-N0-[2-(6-methoxy-
3.2.24. N-[1-(1R)-a-Methylbenzyl]-N0-[2-(4-methylthiaz-
olyl)]thiourea (24). Melting point 120–121 ꢁC; 1H
NMR (DMSO-d6) d 11.50 (s, 1H), 10.24 (s, 1H), 7.35
(m, 4H), 7.26 (m, 1H), 6.63 (s, 1H), 5.44 (m, 1H), 2.21 (s,
3H), 1.49 (m, 3H); 13C NMR (DMSO-d6) d 177.3, 143.2,
benzothiazolyl)]thiourea (29). Melting point 181–182 ꢁC;
1H NMR (DMSO-d6) d 11.74 (s, 1H), 10.42 (s, 1H),
7.55 (d, 1H, J ¼ 9 Hz), 7.50 (s, 1H), 7.40–7.33 (m, 4H),
7.25 (t, 1H), 6.98 (dd, 1H, J ¼ 1:5, 8.7 Hz), 5.54–
5.46 (m, 1H), 1.54 (m, 3H); 13C NMR (DMSO-d6) 177.5,
159.6, 156.2, 149.0, 131.1, 128.6, 128.3, 127.2, 126.2,
128.6, 127.1, 126.1, 106.2, 53.6, 22.5, 16.7; IR m 3170,
t
3014, 2961, 1579, 1504, 1207, 700 cmꢀ1; ½aꢁ )8.4
D
120.1, 114.6, 105.2, 55.8, 53.8, 22.4; IR m 3170, 3031,
(CHCl3); UV (MeOH) kmax 210, 259, 293 nm; MALDI-
TOF MS m=z 279.1 (C13H15N3S2+2Hþ). Anal. Calcd for
C13H15BrN3S2: C, 56.29; H, 5.45; N, 15.15. Found: C,
56.33; H, 5.41; N, 15.24.
t
D
1558, 1527, 1465, 1218, 694 cmꢀ1; ½aꢁ +18.9 (CHCl3);
MALDI-TOF m=z 344.8 (C17H17N3OS2+2Hþ). Anal.
Calcd for C17H17N3OS2: C, 59.45; H, 4.99; N, 12.23.
Found: C, 59.42; H, 4.95; N, 12.20.
3.2.25. N-[1-(1S)-a-Methylbenzyl]-N0-[2-(4-methylthiaz-
olyl)]thiourea (25). Melting point 120–121 ꢁC; 1H
NMR (DMSO-d6) d 11.50 (s, 1H), 10.23 (s, 1H), 7.34
(m, 4H), 7.26 (m, 1H), 6.63 (s, 1H), 5.44 (m, 1H), 2.21 (s,
3H), 1.48 (m, 3H); 13C NMR (DMSO-d6) d 177.6, 157.5,
3.2.30.
N-[1-(1S)-a-Methylbenzyl]-N0-[2-(5-methylpyr-
idyl)]thiourea (30). Melting point 161–162 ꢁC; 1H
NMR (DMSO-d6) d 12.21 (d, 1H), 10.54 (s, 1H), 8.04 (t,
1H), 7.58 (dd, 1H, J ¼ 2:1, 8.4 Hz), 7.35 (d, 4H,
J ¼ 3:6 Hz), 7.27–7.23 (m, 1H), 7.08 (d, 1H, J ¼ 8:7 Hz),
5.61–5.52 (m, 1H), 2.19 (s, 3H), 1.51 (m, 3H); 13C NMR
(DMSO-d6) d 178.5, 152.0, 144.9, 143.4, 139.8, 128.6,
144.9, 143.2, 128.6, 127.1, 126.1, 106.2, 53.7, 22.5, 16.7;
t
D
IR m 3170, 3014, 2919, 1589, 1502, 1207, 700 cmꢀ1; ½aꢁ
+8.8 (CHCl3); UV (MeOH) kmax 207, 212, 260, 294 nm;
MALDI-TOF MS m=z 279.4 (C13H15N3S2+2Hþ).
127.0, 126.8, 126.0, 112.3, 53.8, 22.8, 17.4; IR m 3317,
t
3139, 2977, 1589, 1558, 1403, 1180, 825 cmꢀ1; ½aꢁ +19.7
D
(CHCl3); MALDI-TOF m=z 273.4 (C15H17N3S+2Hþ).
Anal. Calcd for C15H17N3S: C, 66.39; H, 6.31; N, 15.48.
Found: C, 66.56; H, 6.27; N, 15.49.
3.2.26.
N-[1-(1S)-a-Methylbenzyl]-N0-[2-(6-methylpyr-
idyl)]thiourea (26). Melting point 134–135 ꢁC; 1H
NMR (DMSO-d6) d 12.52 (d, 1H, J ¼ 7:8 Hz), 10.52 (s,
1H), 7.61 (t, 1H), 7.40–7.31 (m, 4H), 7.27–7.25 (m, 1H),
6.93 (d, 1H, J ¼ 8:4 Hz), 6.84 (d, 1H, J ¼ 7:2 Hz), 5.51–
5.42 (m, 1H), 2.34 (s, 3H), 1.52 (d, 3H, J ¼ 6:6 Hz); 13C
NMR (DMSO-d6) d 178.6, 154.3, 153.5, 143.4, 139.4,
128.8, 127.3, 126.3, 117.1, 109.6, 54.4, 23.7, 22.9; IR m
3.2.31. N-[1-(1R)-a-Methylbenzyl]-N0-[2-(6-methylpyr-
idyl)]thiourea (31). Melting point 136–137 ꢁC; 1H
NMR (DMSO-d6) d 12.52 (m, 1H), 10.55 (s, 1H), 4.61 (t,
1H), 7.41–7.33 (m, 4H), 7.28–7.25 (m, 1H), 6.95 (d, 1H,
J ¼ 8:1 Hz), 6.84 (d, 1H, J ¼ 7:2 Hz), 5.53–5.44 (m, 1H),
2.35 (s, 3H), 1.54 (d, 3H, J ¼ 6:9 Hz); 13C NMR
(DMSO-d6) d 178.5, 154.2, 153.4, 143.3, 139.3, 128.6,
127.2, 126.2, 116.9, 109.5, 54.2, 23.6, 22.8; IR m 3193,
3193, 3031, 2923, 1604, 1535, 1450, 1226, 1195,
t
786 cmꢀ1; ½aꢁ +14.6 (CHCl3); MALDI-TOF m=z 272.9
D
(C15H17N3S+2Hþ). Anal. Calcd for C15H17N3S: C,
66.39; H, 6.31; N, 15.48. Found: C, 66.39, 66.27; H,
6.25, 6.36; N, 15.55, 15.44.
3031, 2923, 1604, 1565, 1535, 1450, 1226, 1195,
t
D
786 cmꢀ1; ½aꢁ )16.1 (CHCl3); MALDI-TOF m=z 273.1
(C15H17N3S+2Hþ). Anal. Calcd for C15H17N3S: C,
66.39; H, 6.31; N, 15.48. Found: C, 66.41; H, 6.27; N,
15.52.
3.2.27. N-[1-(1R)-a-Methylbenzyl]-N0-[2-(5-methylpyr-
idyl)]thiourea (27). Melting point 161–162 ꢁC; 1H
NMR (DMSO-d6) d 12.21 (d, 1H, J ¼ 7:8 Hz), 10.55 (s,
1H), 8.04 (s, 1H), 7.57 (dd, 1H, J ¼ 8:4, 8.7 Hz), 7.34 (t,
4H), 7.26–7.22 (m, 1H), 7.09 (d, 1H, J ¼ 8:4 Hz), 5.62–
5.52 (m, 1H), 2.18 (s, 3H), 1.51 (d, 3H, J ¼ 6:9 Hz); 13C
NMR (DMSO-d6) d 179.1, 152.6, 145.5, 144.0, 140.4,
3.2.32.
N-[1-(1R)-a-Methylbenzyl]-N0-[2-(6-methoxy-
benzothiazolyl)]thiourea (32). Melting point 182–183 ꢁC;
1H NMR (DMSO-d6) d 11.71 (s, 1H), 10.41 (s, 1H), 7.55
(d, 1H, J ¼ 8:7 Hz), 7.50 (s, 1H), 7.38 (s, 4H), 7.27 (d,
1H, J ¼ 5:7 Hz), 6.79 (d, 1H, J ¼ 8:7 Hz), 5.49 (t, 1H),
3.77 (s, 3H), 1.54 (m, 3H); 13C NMR (DMSO-d6) d
177.4, 159.6, 156.2, 143.0, 131.1, 128.6, 127.2, 126.2,
129.2, 127.6, 127.4, 126.7, 112.9, 54.4, 23.4, 18.0; IR m
t
3317, 3139, 2954, 1558, 1488, 1180, 640 cmꢀ1; ½aꢁ )19.6
D
(CHCl3); MALDI-TOF m=z 273.2 (C15H17N3S+2Hþ).
Anal. Calcd for C15H17N3S: C, 66.39; H, 6.31; N, 15.48.
Found: C, 66.38; H, 6.30; N, 15.48.
120.2, 114.6, 105.3, 55.8, 53.8, 22.4; IR m 3170, 3031,
t
D
1565, 1527, 1465, 1218, 694 cmꢀ1; ½aꢁ )16.4 (CHCl3);

4284
T. K. Venkatachalam et al. / Bioorg. Med. Chem. 12 (2004) 4275–4284
MALDI-TOF m=z 345.4 (C17H17N3OS2+Hþ). Anal.
Calcd for C17H17N3OS2: C, 59.45; H, 4.99; N, 12.23.
Found: C, 59.51; H, 4.95; N, 12.24.
11. Ding, J.; Das, K.; Moereels, H.; Koymans, L.; Andries,
K.; Janssen, P. A.; Hughes, S. H.; Arnold, E. Nat. Struct.
Biol. 1995, 2, 407.
12. Bell, F. W.; Cantrell, A. S.; Hogberg, M.; Jaskunas, S. R.;
Johansson, N. G.; Jordan, C. L.; Kinnick, M. D.; Lind, P.;
Morin, J. M., Jr; Noreen, R.; Oberg, B.; Palkowitz, J. A.;
Parrish, C. A.; Pranc, P.; Sahlberg, C.; Ternansky, R. T.;
Vasileff, R. T.; Vrang, L.; West, S. J.; Zhang, H.; Zhou, X.
X. J. Med. Chem. 1995, 38, 4929.
13. Cantrell, A. S.; Engelhardt, P.; Hogberg, M.; Jaskunas, S.
R.; Johansson, N. G.; Jordan, C. L.; Kangasmetsa, J.;
Kinnick, M. D.; Lind, P.; Morin, J. M., Jr; Muesing, M.
A.; Noreen, R.; Oberg, B.; Pranc, P.; Sahlberg, C.;
Ternansky, R. J.; Vasileff, R. T.; Vrang, L.; West, S. J.;
Zhang, H. J. Med. Chem. 1996, 39, 4261.
14. Mao, C.; Vig, R.; Venkatachalam, T. K.; Sudbeck, E. A.;
Uckun, F. M. Bioorg. Med. Chem. Lett. 1998, 8, 2213.
15. Mao, C.; Sudbeck, E. A.; Venkatachalam, T. K.; Uckun,
F. M. Bioorg. Med. Chem. Lett. 1999, 9, 1593.
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4. Conclusion
In summary, our data provide unprecedented evidence
that the stereochemistry is a key determinant of thiourea
compounds as NNRTI. Molecular modeling studies indi-
cated that the two carbon linker between the phenyl group
and the thiourea groups is adjustable and more forgiving.
In addition, the energetic difference is minimized by
adopting alternative conformation to maintain the snugly
fit with the binding pocket. Furthermore, we have identi-
fied b-methyl phenyl ethyl thioureas as a promising new
class of NNRTI with potent anti-HIV activity.
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