N-aminoimidazole derivatives inhibiting retroviral replication via a yet unidentified mode of action

Article abstract of DOI:10.1021/jm0211117

The synthesis of a series of N-aminoimidazoles (NAIMs) with an uncommon spectrum of antiretroviral activity is described. From a group of 60 closely related molecules, we were able to subdivide the molecules in different groups based on their anti-HIV and anti-SIV activity in vitro: (i) molecules acting on a new, immediate postintegration step, (ii) molecules acting on both postintegration and HIV-1 reverse transcriptase (RT) as NNRTI, and (iii) molecules that mainly act at the HIV-1 RT according to an NNRTI-type mode of action.

Full text of DOI:10.1021/jm0211117

1546
J . Med. Chem. 2003, 46, 1546-1553
N-Am in oim id a zole Der iva tives In h ibitin g Retr ovir a l Rep lica tion via a Yet
Un id en tified Mod e of Action
Irene M. Lagoja,†,‡ Christophe Pannecouque,*,§,‡ Arthur Van Aerschot,† Myriam Witvrouw,‡ Zeger Debyser,‡
J an Balzarini,‡ Piet Herdewijn,*,† and Erik De Clercq‡
Laboratory of Medicinal Chemistry and Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research,
Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received December 10, 2002
The synthesis of a series of N-aminoimidazoles (NAIMs) with an uncommon spectrum of
antiretroviral activity is described. From a group of 60 closely related molecules, we were able
to subdivide the molecules in different groups based on their anti-HIV and anti-SIV activity
in vitro: (i) molecules acting on a new, immediate postintegration step, (ii) molecules acting
on both postintegration and HIV-1 reverse transcriptase (RT) as NNRTI, and (iii) molecules
that mainly act at the HIV-1 RT according to an NNRTI-type mode of action.
Ch a r t 1. Structure Analogy of S-1153 vs
N-Aminoimidazole Derivatives 3 and 7
In tr od u ction
Replication of human immunodeficiency virus type 1
can be reduced in HIV-infected patients using a com-
bination of antiviral drugs targeted at the reverse
transcriptase (RT) and protease.1 Unfortunately, be-
cause of the high mutation rate of HIV, treatment, even
combination therapy, is required for drug-resistant
variants.2
Shortly after the discovery of the nonnucleoside
reverse transcriptase inhibitors (NNRTIs), it became
clear that resistance to this class of compounds emerges
rapidly.3 Moreover, cross-resistance is very important.
If a patient’s virus becomes resistant to NNRTIs, no
other member of the group can be used effectively.
Capravirine (S-1153), a 1,2,4,5-substituted imidazole
derivative, is an NNRTI4 that is able to inhibit HIV-1
strains that are resistant to other NNRTIs. This ability
to cope with typical NNRTI-induced mutations is based,
at least in part, on an extensive network of hydrogen
bonds involving the main chain of residues 101, 103,
and 236 of the p66 RT subunit. Side chain mutations
are not likely to disrupt these interactions.5
This very unusual antiviral activity pattern suggests
that the antiretroviral activity of these compounds is
not based on an inhibition of the reverse transcriptase
activity as might have been expected from the structural
similarity with capravirine.
Other members of the series had an activity profile
that coincided with that of the NNRTIs (no activity
against HIV-2(ROD) and reduced activity against an
NNRTI-resistant strain), but some of these compounds
inhibited the replication of SIV(mac251) to the same
extent as wild-type HIV-1 strains, unlike the classical
NNRTIs nevirapine, delavirdine, and efavirenz.
These results prompted us to study the structure-
activity relationship and potential mode of action of
these N-aminoimidazoles (NAIMs) more thoroughly.
We synthesized a series of N-aminoimidazoles via a
one-pot synthesis. The similarity of this series of
compounds to S-1153 is depicted in Chart 1.
The in vitro antiviral evaluation of a series of 60
N-aminoimidazole and N-aminoimidazole thiones yielded
unexpected antiviral activity findings. Out of a series
of 60 compounds, 7 compounds were equipotent against
the replication of HIV-1 (strains IIIB and the molecular
clone NL4.3), an NNRTI-resistant HIV-1 strain (with
mutations K103N, Y181C in RT), HIV-2(ROD), and
SIV(mac251). The most potent of these compounds were
also active against MSV-induced transformation of C3H/
3T3 embryo murine fibroblast in vitro.
Ch em istr y
Starting from cheap, easily available compounds such
as hydrazines 1, R-bromoketones 2, and potassium thio-
cyanate, N-substituted 1-amino-2,3-dihydro-1H-imid-
azole-2-thiones 3 were formed in good yield in a one-
pot reaction (Scheme 1).6 By use of strategies of parallel
synthesis and combination of starting materials 1 and
2, a broad range of differently substituted heterocycles
3 could be obtained.
* To whom correspondence should be addressed. Phone:
+32 16 33 73 87. Fax: +32 16 33 73 40. E-mail: piet.herdewijn@
rega.kuleuven.ac.be.
This multistep reaction is considered to occur via the
conversion of an R-thiocyanatoketone (formed by nu-
cleophilic substitution reaction) to the thiocyanato-
† Laboratory of Medicinal Chemistry.
‡ Both authors contributed equally.
§ Laboratory of Virology and ChemotherapyIntroduction.
10.1021/jm0211117 CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/11/2003

N-Aminoimidazole Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1547
Sch em e 1. Synthesis of N-Aminoimidazoline-2-thiones
Ch a r t 2. Comparison between the NMR Data of
3
2-Iminothiazoles 6 and N-Aminoimidazoline-2-thiones 3
Sch em e 2. Most Likely Pathway for the Formation of
2-Iminothiazoles 6
Ch a r t 3. Structures of N-Aminoimidazoles 7 and
S-Alkylaminoimidazoles 8
for the conversion of N,N-disubstituted hydrazines 1,
obtaining the corresponding imidazoline thione 3 with
an N,N-disubstituted side chain. Further derivatization
of heterocycle 3 could be achieved by desulfurization
with hydrogen peroxide in acetic acid,8 yielding the
corresponding N-aminoimidazole derivatives 7.
Alkylation of the imidazoline-2-thiones 3 quantita-
tively formed the S-alkyl product 8 (Chart 3), even
under Mitsunobu9 conditions. Substitution patterns,
yields, melting points, Rf values and HRMS of the
imidazole derivatives 3 and 7 are shown in Table 1.
hydrazone 4. Subsequent 1,4-elimination led to the
corresponding azoalkene and thiocyanic acid. The thus
formed two intermediates can undergo a [3 + 2] cyclo-
addition reaction, and following hydrogen shift, the
1-amino-2,3-dihydro-1H-imidazole-2-thiones 3 were ob-
tained.
Biologica l Resu lts a n d Discu ssion
In the case of ortho-substituted ketones, it was found
that the reaction did not follow the pathway outlined
in Scheme 1. Instead of the cycloaddition, a 5-exo-dig
cyclization reaction (Scheme 2) took place. Likewise, in
this pathway the R-thiocyanatohydrazone 4 is the key
intermediate. In the case of substituents in the ortho
position of the former ketone 2, the 1,4-elimination
giving rise to the formation of azoalkenes, which are
planar, delocalized π-electron systems, is not favored.
As shown in Scheme 2, a cyclization reaction yielding a
2-iminothiazole 6 took place instead. Attempts to con-
vert the 2-iminothiazole 6 to the desired imidazoline-
2-thione derivative 3 by way of a Dimroth rearrange-
ment7 failed.
The two isomers can be easily distinguished by NMR
methods. The signal corresponding to the carbon 2 of
the imidazoline-2-thione derivative 3 was found in the
range δ 160-162 ppm, whereas the corresponding
signal of the 2-iminothiazole 6 was found in the range
δ 169-171 ppm.
In the case of isomer 3, saturation of the 3-NH causes
a 10% nuclear Overhauser enhancement (NOE) at the
4-substituent, whereas irradiation of the imino NH
showed an NOE enhancement of 5% relative to the
exocyclic NH. The results of structure elucidation are
summarized in Chart 2.
Besides the restriction of having a substituent in the
ortho position of ketone 2, the method of generating
N-aminoimidazoline-2-thiones 3 in a one-pot-reaction
starting from hydrazines 1, ketones 2, and potassium
thiocyanate is suitable for all substituents, R1, R2, and
R3 being either aryl or alkyl. The method is also suitable
In h ibition of HIV a n d SIV Rep lica tion in Cell
Cu ltu r e by N-Am in oim id a zoles 3 a n d 7. We tested
a series of N-arylaminoimidazoline-2-thione derivatives
3 and N-arylaminoimidazoles 7 for their potential to
inhibit the replication of HIV and SIV in a cell culture
model for acute infection. The cytotoxicity of the com-
pounds was determined in parallel. The antiviral activ-
ity and cytotoxicity data are presented in Table 2. From
a series of 60 compounds, 26 compounds were active
against the replication of at least one of the included
virus strains. Seven compounds (3.01, 3.05, 3.07, 3.39,
7.01, 7.02, and 7.03) were equally potent inhibitors of
the HIV-1, HIV-2, and SIV replication in MT-4 cells.
Similar results were obtained in peripheral blood mono-
nuclear cells (PBMCs) (data not shown).
Some molecules showed reduced activity against a
clinically relevant NNRTI-resistant strain (mutations
in RT K103N and Y181C), but unlike established
NNRTIs as nevirapine, delavirdine, and efavirenz, they
remained potent inhibitors of SIV(mac251) replication
in vitro. Moreover, the most potent broad-spectrum
N-aminoimidazole derivatives 3 and 7 inhibited MSV-
induced transformation of C3H/3TC embryo murine
fibroblasts in vitro (Table 3).
Str u ctu r e-Activity Rela tion sh ip (SAR) for N-
Am in oim id a zole Der iva tives 3 a n d 7. All the anti-
retroviral-active N-aminoimidazoles 3 and 7 have in
common the basic molecule depicted in Chart 4. No
marked difference in antiviral activity and cytotoxicity
was observed between the imidazoles 7 and their 2-
thiono homologue 3 series. However, alkylation (methyl-
ation or benzylation) of the sulfur led to complete

1548 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Lagoja et al.
Ta ble 1. Analytical Data of N-Aminoimidazole-2-thiones 3 (Written as Thiole Tautomer with R4 ) SH) and N-Aminoimidazoles 7 (R4
) H)a
yield
(%)
mp
(MeOH, °C)
b
compd
R1
R2
R3
CH3
Rf
exact mass
3.011
3-ClC6H4
C6H5
C6H5
C6H5
C6H5
82
86
68
85
86
84
85
82
88
83
79
92
62
52
32
60
48
36
58
87
78
85
46
68
72
56
68
52
75
82
80
84
88
90
80
70
61
73
47
58
59
56
88
52
66
76
86
67
82
86
75
79
81
81
66
68
75
70
84
70
65
77
54
0.55
0.54
0.53
0.54
0.52
0.52
0.52
0.52
0.53
0.53
0.52
0.49
0.53
0.52
0.52
0.54
0.49
0.53
0.53
0.54
0.53
0.54
0.50
0.52
0.53
0.57
0.49
0.30
0.45
0.53
0.53
0.53
0.53
0.54
0.52
0.34
0.33
0.35
0.56
0.50
0.48
0.62
0.82
0.85
0.83
0.76
0.84
0.76
0.77
0.78
0.84
0.76
0.85
0.85
0.86
0.84
0.84
0.84
0.67
0.66
0.80
0.63
0.82
215-217
220-221
228-231
212-214
196-200
220-222
204-207
225-226
226-229
238-240
148-151
224-226
228-230
190-192
190-193
266-270
214-116
226-229
206-207
234-236
217-219
230-232
220-222
116-118
202-204
178-180
194-196
166-168
160-162
240-242
184-186
168-170
160-162
170-172
164-166
170-172
166-168
164-166
194-196
180-182
215-217
223-225
216-218
164-166
168-170
179-180
165-167
176-178
146-148
144-146
159-160
153-155
158-160
138-140
154-156
156-158
140-142
138-140
228-230
220-222
172-174
176-178
110-112
Calcd: 316.0675 [M + H]+. Found: 316.0660
Calcd: 316.0675 [M + H]+. Found: 316.0703
Calcd: 300.0971 [M + H]+. Found: 300.0996
Calcd: 316.0675 [M + H]+. Found: 316.0662
Calcd: 393.9790 [M + H]+. Found: 393.9839
Calcd: 393.9790 [M + H]+. Found: 393.9779
Calcd: 350.0855 [M + H]+. Found: 350.0271
Calcd: 350.0855 [M + H]+. Found: 350.0356
Calcd: 346.0781 [M + H]+. Found: 350.0831
Calcd: 316.0675 [M + H]+. Found: 316.0684
Calcd: 280.0675 [M + H]+. Found: 280.0733
Calcd: 282.1065 [M + H]+. Found: 282.1037
Calcd: 310.1378 [M + H]+. Found: 310.1373
Calcd: 360.0170 [M + H]+. Found: 360.0168
Calcd: 330.0832 [M + H]+. Found: 330.0834
Calcd: 350.0285 [M + H]+. Found: 350.0248
Calcd: 327.0916 [M + H]+. Found: 327.0928
Calcd: 300.0971 [M + H]+. Found: 300.0933
Calcd: 296.1221 [M + H]+. Found: 296.1208
Calcd: 254.0519 [M + H]+. Found: 254.0521
Calcd: 220.0908 [M + H]+. Found: 220.0844
Calcd: 234.1065 [M + H]+. Found: 234.1078
Calcd: 324.1534 [M + H]+. Found: 324.1524
Calcd: 330.0831 [M + H]+. Found: 330.0833
Calcd: 310.1377 [M + H]+. Found: 310.1375
Calcd: 378.0832 [M + H]+. Found: 378.0863
Calcd: 298.0417 [M + H]+. Found: 298.0421
Calcd: 284.0261 [M + H]+. Found: 284.0261
Calcd: 298.0417 [M + H]+. Found: 298.0410
Calcd: 310.1378 [M + H]+. Found: 310.1394
Calcd: 312.1171 [M + H]+. Found: 312.1188
Calcd: 341.0628 [M + H]+. Found: 341.0675
Calcd: 321.1174 [M + H]+. Found: 321.1152
Calcd: 374.0730 [M + H]+. Found: 374.0757
Calcd: 354.1276 [M + H]+. Found: 354.1305
Calcd: 360.0573 [M + H]+. Found: 360.0608
Calcd: 340.1119 [M + H]+. Found: 340.1147
Calcd: 359.0733 [M + H]+. Found: 359.0702
Calcd: 332.1143 [M + H]+. Found: 332.1224
Calcd: 310.1378 [M + H]+. Found: 310.1361
Calcd: 328.0942 [M + H]+. Found: 328.0928
Calcd: 296.1221 [M + H]+. Found: 296.1205
Calcd: 284.0945 [M + H]+. Found: 284.0955
Calcd: 362.0059 [M + H]+. Found: 362.0062
Calcd: 318.0565 [M + H]+. Found: 318.0616
Calcd: 248.0954 [M + H]+. Found: 248.0953
Calcd: 250.1344 [M + H]+. Found: 250.1322
Calcd: 188.1188 [M + H]+. Found: 188.1153
Calcd: 222.0798 [M + H]+. Found: 222.0793.
Calcd: 202.1344 [M + H]+. Found: 202.1356.
Calcd: 264.1500 [M + H]+. Found: 264.1536.
Calcd: 268.1250 [M + H]+. Found: 268.1237
Calcd: 278.1657 [M + H]+. Found: 278.1641
Calcd: 278.1657 [M + H]+. Found: 278.1641
Calcd: 278.1657 [M + H]+. Found: 278.1636.
Calcd: 280.1450 [M + H]+. Found: 280.1449.
Calcd: 309.0907 [M + H]+. Found: 309.0911
Calcd: 289.1453 [M + H]+. Found: 289.1456
Calcd: 327.1013 [M + H]+. Found: 327.1017
Calcd: 307.1556 [M + H]+. Found: 307.1544
Calcd: 342.1009 [M + H]+. Found: 342.1015
Calcd: 328.0853 [M + H]+. Found: 328.0854
Calcd: 264.1501 [M + H]+. Found: 264.1517
3.0211 2-ClC6H4
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
3.03
3.046
3.05
3.06
3.07
3.08
3.09
3.10
3.11
3.126
3.13
3.14
3.15
3.16
3.17
3.18
3.19
3.20
3.21
3.22
3.23
3.24
3.25
3.26
3.27
3.28
3.29
3.30
3.31
3.32
3.33
3.34
3.35
3.36
3.37
3.38
3.39
3.40
3.41
3.428
7.01
7.02
7.03
7.04
7.05
7.06
7.07
7.08
7.09
7.10
7.11
7.12
7.13
7.14
7.15
7.16
7.17
7.18
7.19
7.20
7.218
4-FC6H4
4-ClC6H4
3-ClC6H4
3-ClC6H4
3-ClC6H4
3-ClC6H4
3-ClC6H4
3-ClC6H4
3-ClC6H4
C6H5
3-BrC6H4
4-BrC6H4
3-ClC6H4
4-ClC6H4
4-CH3OC6H4
CH3
-(CH2)4-
C6H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
3-CH3-4-CH3-C6H3 C6H5
3-BrC6H4
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
CH3
CH3
CH3
C6H5
C6H5
C6H5
3-Cl-4-CH3C6H3
2-Cl-5-ClC6H3
3-NO2C6H4
3-FC6H4
3-CH3C6H4
3-ClC6H4
CH3
CH3
CH3
C6H5
3-CH3C6H4
3-CH3C6H4
3-ClC6H4
3-CH3C6H4
3-ClC6H4
(CH3)2CH
CH3CH2
CH3CH2
C6H5
CH3
CH3
CH3OCO
CH3
C6H5
3-ClC6H4
3-ClC6H4
3-ClC6H4
CH3OCO
COOH
CH3
3-CH3-5-CH3C6H3
3-CH3OC6H4
3-ClC6H4
3-MeC6H4
3-ClC6H4
3-CH3C6H4
3-ClC6H4
3-CH3C6H4
3-ClC6H4
R-naphthyl
4-CH3CH2C6H4
4-CH3SC6H4
C6H5, CH3
3-ClC6H4
C6H5
C6H5
CH3
CH3
CH3
3-CNC6H4
3-CNC6H4
3-MeOCOC6H4 CH3
3-MeOCOC6H4 CH3
3-HOCOC6H4
3-HOCOC6H4
3-NH2COC6H4 CH3
CH3
CH3
C6H5
C6H5
C6H5
C6H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
3-ClC6H4
3-ClC6H4
3-ClC6H4
C6H5
C6H5
3-ClC6H4
3-BrC6H4
3-ClC6H4
-(CH2)4-
C6H5
CH3
CH3
CH3
C6H5
C6H5
C6H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3CH2
CH3
CH3
CH3
CH3
CH3
3-CH3C6H4
3-CH3C6H4
4-FC6H4
3-CH3C6H4
3-ClC6H4
3-CH3-5-CH3C6H3
3-CH3OC6H4
3-ClC6H4
3-MeC6H4
3-ClC6H4
CH3OCO
C6H5
C6H5
3-CNC6H4
3-CNC6H4
3-NH2COC6H4 CH3
3-NH2COC6H4 CH3
3-MeOCOC6H4 CH3
3-MeC6H4
3-ClC6H4
3-ClC6H4
3-HOCOC6H4
C6H5
CH3
CH3
C6H5, CH3
a
b
The results of elemental analyses (C, H, N) of all active compounds were within (0.4% of the theoretical values. Solvent system for
N-aminoimidazoline-2-thiones 3: CH2Cl2/MeOH, 99:1. Solvent system for N-aminoimidazoles 7: CH2Cl2/MeOH, 9:1.

N-Aminoimidazole Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1549
Ta ble 2. Inhibition of Viral Replication in MT-4 Cells by N-Aminoimidazole Derivatives 3 and 7
a
IC50 (µM)
HIV-1
compd
IIIB
NL4.3
NNRTI(res)b
HIV-2 ROD
SIV mac251
CC50 (µM)c
3.01
3.05
3.07
3.39
7.01
7.02
7.03
3.13
3.14
3.17
3.19
3.25
3.30
3.31
7.05
7.09
7.15
3.02
3.08
3.09
3.13
3.18
3.23
3.24
3.32
7.13
3.03
3.04
3.06
3.10
3.11
3.12
3.16
3.20
3.21
3.22
3.26
3.27
3.28
3.29
3.33
3.34
3.36
3.37
3.38
3.40
3.41
7.04
7.06
7.07
7.08
7.10
7.11
7.12
7.14
7.16
7.17
7.18
7.19
7.20
8.01
8.02
6.39 ( 0.94
8.54 ( 1.06
5.00 ( 1.17
5.55 ( 2.14
1.59 ( 0.32
0.58 ( 0.08
0.72 ( 0.59
9.30 ( 0.55
9.96 ( 1.61
7.29 ( 1.53
1.56 ( 0.61
17.64 ( 13.02
1.03 ( 0.58
4.78 ( 1.38
g11.67
3.26 ( 0.91
6.96 ( 2.00
>161.81
>40.80
>38.69
>44.29
8.99 ( 6.14
5.60 ( 4.81
2.48 ( 0.91
6.58 ( 4.80
1.90 ( 0.60
1.30 ( 0.77
0.63 ( 0.03
3.54 ( 0.81
4.52 ( 3.80
11.25 ( 4.57
2.17 ( 0.88
12.80 ( 3.39
0.87 ( 0.52
9.79 ( 0.22
20.98 ( 2.64
5.20 ( 0.04
8.91 ( 3.01
23.27 ( 8.49
>40.80
9.72 ( 4.99
3.00 ( 1.20
1.86 ( 1.20
11.89 ( 5.31
1.73 ( 0.74
0.97 ( 0.14
0.38 ( 0.28
>55.58
5.77 ( 0.28
42.18 ( 2.39
26.10 ( 3.96
g53.55
>194.21
>38.02
>52.18
3.99 ( 0.04
22.57 ( 5.05
>161.81
>40.80
>38.69
>44.29
g43.42
>42.63
>38.96
>99.40
>20.44
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
>36.31
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
>67.29
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
>10
8.11 ( 0.38
10.46 ( 6.71
3.91 ( 0.65
5.10 ( 3.77
1.65 ( 1.55
0.91 ( 0.19
0.72 ( 0.03
>55.58
8.20 ( 4.62
1.75 ( 0.20
1.22 ( 0.77
4.68 ( 0.30
2.15 ( 0.42
0.66 ( 0.25
0.31 ( 0.13
10.31 ( 3.59
2.72 ( 0.17
7.54 ( 1.59
6.50 ( 4.60
>53.55
66.65 ( 19.00
43.98 ( 20.60
30.95 ( 11.93
32.05 ( 9.17
17.48 ( 7.58
16.41 ( 7.94
11.94 ( 9.93
55.58 ( 18.68
44.24 ( 14.15
48.26 ( 22.49
88.25 ( 35.71
53.55 ( 28.73
194.21 ( 132.79d
38.02 ( 5.34
52.18 ( 11.52
27.04 ( 11.81
36.79 ( 5.15
>44.24
>48.26
>35.71
>53.55
>194.21
>38.02
>194.21
>38.02
>52.18
>52.18
>27.04
3.08 ( 0.41
7.22 ( 0.027
>161.81
7.71 ( 3.88
10.12 ( 11.65
10.88 ( 8.45
33.80 ( 20.36
>42.63
>36.79
>161.81
>40.80
161.81 ( 52.84
40.80 ( 13.90
38.69 ( 13.01
44.29 ( 14.34
108.23 ( 71.38
42.63 ( 16.79
38.96 ( 15.86
99.40 ( 42.13
20.44 ( 9.55
220.13 ( 60.29
134.41 ( 33.82
33.24 ( 14.31
49.96 ( 27.07
126.42 ( 40.14
191.27 ( 72.25
147.12 ( 78.80
259.42 ( 25.69
180.79 ( 29.78
359.86 ( 77.44
36.31 ( 14.87
190.31 ( 41.98
265.56 ( 41.94
48.80 ( 28.14
45.63 ( 36.45
67.36 ( 59.70
120.03 ( 40.10
187.13 ( 45.74
42.16 ( 12.49
34.45 ( 2.37
53.04 ( 14.87
67.29 ( 16.95
351.42 ( 53.09
213.68 ( 55.17
312.12 ( 29.36
30.82 ( 9.39
27.87 ( 13.45
106.70 ( 70.83
34.44 ( 10.81
44.70 ( 9.68
260.50 ( 62.76
162.31 ( 56.76
33.15 ( 3.83
160.57 ( 21.14
42.11 ( 17.19
39.66 ( 17.29
>10
>38.69
>38.69
>44.29
10.85 ( 0.82
18.44 ( 9.39
g42.63
>108.23
>42.63
9.06 ( 1.55
g99.40
>108.23
>42.63
>38.96
>38.96
3.88 ( 3.27
>99.40
>20.44
41.96 ( 2.08
10.99 ( 2.85
>220.13
>134.41
>33.24
>99.40
>20.44
>20.44
>220.13
>134.41
>33.24
>220.13
>134.41
>33.24
>220.13
>134.41
>33.24
>49.96
>49.96
>49.96
>49.96
>126.42
>191.27
>147.12
>259.42
>180.79
>359.86
>36.31
>126.42
>191.27
>147.12
>259.42
>180.79
>359.86
>36.31
>126.42
>191.27
>147.12
>259.42
>180.79
>359.86
>36.31
>126.42
>191.27
>147.12
>259.42
>180.79
>359.86
>36.31
>190.31
>265.56
>48.80
>45.63
>67.36
>190.31
>265.56
>48.80
>45.63
>67.36
>190.31
>265.56
>48.80
>45.63
>67.36
>190.31
>265.56
>48.80
>45.63
>67.36
>120.03
>187.13
>42.16
>120.03
>187.13
>42.16
>120.03
>187.13
>42.16
>120.03
>187.13
>42.16
>34.45
>34.45
>34.45
>34.45
>53.04
>53.04
>53.04
>53.04
>67.29
>67.29
>67.29
>67.29
>351.42
>213.68
>312.12
>30.82
>351.42
>213.68
>312.12
>30.82
>351.42
>213.68
>312.12
>30.82
>351.42
>213.68
>312.12
>30.82
>27.87
>27.87
>27.87
>27.87
>106.70
>34.44
>106.70
>34.44
>106.70
>34.44
>106.70
>34.44
>44.70
>44.70
>44.70
>44.70
>260.50
>162.31
>33.15
>260.50
>162.31
>33.15
>260.50
>162.31
>33.15
>260.50
>162.31
>33.15
>160.57
>42.11
>39.66
0.014
>160.57
>42.11
>39.66
0.006
>160.57
>42.11
>39.66
>10
>160.57
>42.11
>39.66
>10
R89439 (R-APA)
a
50% inhibitory concentration, or concentration required to inhibit the viral cytopathic effect by 50% in MT-4 cells. Data represent
average values ( SD for at least two independent experiments. S0561945. c 50% cytotoxic concentration, or concentration that reduced
the MT-4 cell viability by 50%. Crystals observed at 16.15 µM.
b
d
abolishment of the antiviral activity (8.01, 8.02 vs 3.01,
3.19). Comparison of the antiviral activity of a series of
compounds bearing methyl, ethyl, propyl, or phenyl at
position 4 of the imidazole moiety revealed that the
smaller the substituent, the more pronounced was the
antiviral activity. The nature of the different substitu-
ents at position 5 of the imidazole ring suggested the
need of an aromatic group with certain limitations to

1550 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Lagoja et al.
Ta ble 3. Inhibitory effects of N-Aminoimidazoles 3 and 7 on
MSV-Induced Transformation of C3H/3T3 Embryo Murine
Fibroblast in Vitro
a
b
compd
IC50 (µM)
MIC (µM)
3.01
3.02
3.05
3.07
3.13
3.19
3.24
3.39
7.01
7.02
7.03
7.09
38.0 ( 14.2
>12.7
>63.3 (316.6)
g12.7
>10.1
>10.1 (50.7)
>11.4 (57.1)
>64.6 (323.2)
>67.7 (338.5)
>12.13 (60.63)
>12.07 (60.35)
>14.1 (70.5)
>11.03 (55.15)
g12.6
>11.4
>64.6
>67.7
>12.13
>12.07
6.9 ( 3.8
6.89 ( 1.10
5.37 ( 1.04
13.37 ( 2.05
>15.2 (75.95)
a
b
50% effective dose. Minimal inhibitory concentration.
F igu r e 1. Time of addition experiment. MT-4 cells were
infected with HIV-1(IIIB) at a multiplicity of infection of 0.5,
and the test compounds were added at different times post-
infection. Viral p24 Ag production was determined at 31 h
postinfection and is expressed as the log 10 of the p24 Ag
content in pg/mL. Symbols: (]) control; (9) dextran sulfate;
(2) AZT; ([) L-708,906; (/), 7.03; (0) ritonavir.
Ch a r t 4. General Structural Requirements of the
N-Aminoimidazole Derivatives 3 and 7
Addition of these compounds can be delayed for 0, 4,
7, and 18-19 h (Figure 1). Addition of 3.19 or 7.03 can
be postponed for up to 7 h postinfection, suggesting a
possible interaction at the moment of integration. These
results should be interpreted with care, not excluding
the possibility of an interaction with earlier processes
in the HIV replication cycle because TOA experiments
only reveal the last step targeted by the inhibitor.
its substitution pattern. Chloro or bromo being well
accepted in the meta position, the same halogens
introduced at the para position reduced the antiviral
properties of the molecules.
R ever se Tr a n scr ip t a se. Several compounds from
the 3 and 7 series have been evaluated against recom-
binant HIV-1 reverse transcriptase). 3.19 was inhibiting
at 125 µM using polyrA.oligodT as the template primer
and at 105 µM when using polyrC.oligodG. This com-
pound was, however, inactive against Maloney murine
leukaemia virus RT using endogenous RNA as the
template at 1000 µM.
Because the in vitro inhibition of the HIV-1 RT
activity was observed for 3.19 and typical NNRTI-
resistance mutations were observed when genotypically
analyzing the HIV-1(IIIB) strain selected in the presence
of increasing concentrations of 3.19, it is clear that 3.19
acts as an NNRTI in addition to its action at a later
process in the viral replication cycle.
The nature of the substitutions on the anilino sub-
stitutent at position 1 of the imidazole has serious
repercussions on the antiviral activity of the N-amino-
imidazoles and 2-thiones (NAIMs) 3 and 7. Not only do
these substitutions influence the potency, more impor-
tantly they do influence the activity spectrum of the
molecules. The most active molecules were found among
meta-substituted anilino derivatives. The active mol-
ecules bearing a chlorine at position 3 of the anilino
phenyl ring had a broader activity spectrum [HIV-1
(strains IIIB and NL4.3), HIV-1 (NNRTI-resistant strain
with mutations K103N, Y181C in the RT), HIV-2(ROD),
SIV(mac251), and MSV(Moloney)]. The activity of these
molecules is mainly due to a yet unidentified mode of
action taking place immediately after the integration
is finished. Replacement of the m-chloro on the aniline
moiety by a methyl increases the NNRTI action of the
molecules (see 3.01 vs 3.19).
In h ibition of HIV-1 P r od u ction fr om Ch r on i-
ca lly In fected Cells. When 7.03 was evaluated for its
influence on the production of virus from chronically
HIV-1(NL4.3)-infected HUT-78 cells, we noticed an
inhibitory effect. From TOA experiments, in vitro RT
assays suggested the possibility of an action at the
reverse transcription, integration, or later step in the
replicative cycle of HIV. AZT, ritonavir, and L-708,906
were also included in the experiment. For molecules
acting before the termination of the integration process,
AZT and L-708,906, no inhibitory effect on release of
virus could be observed (Figure 2).
Tim e of In ter ven tion . A time of addition (TOA)
experiment was carried out to investigate which step
of the replicative cycle was inhibited by different N-
aminoimidazoles 3 and 7. Briefly, this experiment
determines how long the addition of an anti-HIV
compound can be postponed before losing its antiviral
activity in the viral replication cycle. Virus was added
at a high multiplicity of infection (moi ) 0.5) to
synchronize all steps of the viral replication. Reference
compounds with a known mode of action were included.
Dextran sulfate, a polyanion, is known to interfere with
the binding of the virus to the cell. The nucleoside
analogue AZT inhibits the reverse transcription process.
L-708,906 is an established inhibitor of the strand
transfer step of the integration process.10 Ritonavir is
an inhibitor of the proteolytic cleavage.
As expected, for ritonavir an inhibition of the virus
production was observed. 7.03 inhibited virus produc-
tion up to the level of ritonavir. This fact reveals that
although the unknown target of molecule 7.03 is situ-
ated around the moment of integration, it must coincide
with a process taking place immediately after integra-
tion.

N-Aminoimidazole Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1551
in acetic acid (10 mL) for 2 days. The white precipitate formed
was filtered off (0.4 g). Water (50 mL) was added, and the
additional precipitate formed was likewise filtered off. Recrys-
tallization from MeOH/water (1:1) yielded 1.5 g (56%) of 3.26.
Con ver sion of Meth oxyca r bon yl Gr ou p s in to th e F r ee
Acid s. Gen er a l P r oced u r e. The corresponding methoxy-
carbonyl-substituted imidazoline-2-thione derivative (1.33 mmol)
(3.27, 3.34, 3.35) was stirred in a 10% solution of NaOH (25
mL) for 6 h. After washing with CH2Cl2, the aqueous layer
was acidified. The precipitate formed was filtered off and dried.
Syn t h esis of 5-(3-Ca r b oxa m id op h en yl)-1-(3-ch lor o-
p h e n yla m in o)-2,3-d ih yd r o-4-m e t h yl-1H -im id a zole -2-
th ion e (3.38). A mixture of the corresponding methoxycar-
bonyl-substituted imidazoline-2-thione 3.34 (1.33 mmol) was
stirred in NH3/MeOH fore 2 days. After removal of the solvent,
the residue was recrystallized from MeOH/water (1:3) to yield
3.38 in 73% yield.
P r ep a r a tion of 1-(3-Ch lor op h en yla m in o)-4-m eth yl-2-
m eth ylsu lfa n yl-5-p h en yl-1H-im id a zole (8.01). A mixture
of 1-(3-chlorophenylamino)-2,3-dihydro-4-methyl-5-phenyl-1H-
imidazoline-2-thione (3.01) (0.1 g, 0.37 mmol) and methyl
iodide (1.19 g, 8.40 mmol) in dichloromethane (5 mL) was
heated under reflux for 1 h. Excess of methyl iodide and
solvent were removed in vacuo. Further purification was
achieved by recrystallization from CH2Cl2/n-hexane, 1:1.
P r ep a r a t ion of 2-Ben zylsu lfa n yl-1-(3-ch lor op h en yl-
a m in o)-4-m eth yl-5-p h en yl-1H-im id a zole (8.02). A mixture
of 1-(3-chlorophenylamino)-2,3-dihydro-4-methyl-5-phenyl-1H-
imidazoline-2-thione (3.01) (0.20 g, 0.62 mmol) and benzyl
chloride (0.08 g, 0.62 mmol) in pyridine (5 mL) was heated
under reflux for 3 h. After removal of the solvent, the crude
product was purified by column chromatography (silica gel,
ethyl acetate/hexane ) 2:1.
Syn th esis of 1-Ar yla m in o-1H-im id a zoles 7. Gen er a l
P r oced u r e. A suspension of the respective 1-amino-2,3-
dihydro-1H-imidazole-2-thione (3) (2 mmol) in glacial acetic
acid (10 mL) was stirred in an ice bath. Upon dropwise
addition of 30% hydrogen peroxide (1 mL, 9.76 mmol), the
reaction mixture cleared up, resulting in a clear-brown solu-
tion. After stirring for another 15 min, the reaction mixture
was made alkaline (pH 8-9) with 10% NaOH. The resulting
precipitate was filtered off, washed with cold water, and
recrystallized from water.
Experimental data concerning the syntheses of the starting
hydrazines 1 and R-bromo compounds 2, the NMR data of all
NAIMs (N-aminoimidazole-2-thiones 3, N-aminoimidazoles 7,
and imidazole derivatives 8-10), and elemental analyses are
available as Supporting Information.
Refer en ce Com p ou n d s. Dextran sulfate with an average
molecular weight of 5000 was purchased from Sigma. AZT was
synthesized as previously described.11 Loviride (R-APA, R89439)
was obtained from J anssen Research Foundation (Beerse,
Belgium). The diketo acid L-708,90613a was kindly provided
by Drs. N. Neamati and T. R. Burke (NIH, Bethesda, MD).
Ritonavir (ABT538) was kindly provided by J . M. Leonard,
Abbott Laboratories (Abbott Park, IL).
Vir u ses a n d Cells. The origin of HIV-1(IIIB) virus stock
has been described.12 The HIV-1(NL4.3) strain13 is a molec-
ular clone obtained from the National Institutes of Health
(Bethesda, MD). HIV-1 and HIV-2(ROD)14 stocks were ob-
tained from the culture supernatant of HIV-1- or HIV-2-
infected MT-4 cells, respectively. Simian immunodeficiency
virus, SIV(mac251), was originally isolated by Daniel et al.15
and was obtained from C. Bruck (Smith Kline-RIT, Rixensart,
Belgium). SIV(mac251) stocks were prepared from the super-
natant of SIV-infected MT-4 cells.16 S0561945 is an HIV-1(IIIB)
strain possessing the K103N and Y181C mutations in its
reverse transcriptase gene, resulting in resistance toward
nonnucleoside reverse transcriptase inhibitors. MSV was
prepared from tumors induced following intramuscular in-
oculation of 3-day-old NMRI mice with MSV, as described
previously.17
F igu r e 2. Percentage of virus (HIV-1 NL4.3) produced from
chronically infected HUT78 cells in the presence of varying
concentrations of compounds. After incubation for 44 h, virus
p24 Ag production was determined. Symbols: (2) AZT; ([)
L-708,906; (+) 3.19; (/) 7.03; (0) ritonavir.
Con clu sion
From a study to discover new NNRTIs with anti-HIV
activity against clinically relevant mutants, a group of
molecules with a new antiretroviral mode of action was
identified. Although the exact molecular target remains
to be resolved, the experiments clearly show that some
of the N-aminoimidazole derivatives interfere with an
(immediate) postintegrational event occurring after
integration of the viral DNA into the host cell genome.
On the basis of a limited structure-activity relation-
ship, it was possible to identify some structural require-
ments that confer antiretroviral activity via a new mode
of action to these molecules.
Exp er im en ta l Section
Ma ter ia ls a n d Gen er a l Ch em ica l Meth od s. NMR spec-
tra were recorded on a Varian, Gemini 200 spectrometer (for
1
1H (200 MHz) and 13C). 13C and H are referenced to TMS. All
NH and OH protons were assigned by exchange with D2O.
Exact mass measurements were performed on a quadrupole
time-of-flight mass spectrometer (Q-Tof-2, Micromass, Man-
chester, U.K.) equipped with a standard electrospray ionization
(ESI) interface. Samples were infused in a 2-propanol/water
(1:1) mixture at 3 µL/min. TLC was performed with TLC
aluminum sheets (Merck, silica gel 60 F254), and silica (200-
425 mesh) was used for column chromatography. Melting
points (mp [°C]) were determined with
a Bu¨chi-SMP-20
capillary melting apparatus. For all reactions, analytical grade
solvents were used.
Syn t h eses of 1-Am in o-2,3-d ih yd r o-1H -im id a zole-2-
th ion es 3. Gen er a l P r oced u r e. To a stirred solution of the
R-haloketone 2 (2.5 mmol) in acetic acid (10 mL), potassium
thiocyanate (0.37 g, 3.8 mmol) was added at ambient temper-
ature. After 30 min the hydrazine 1 (2.5 mmol) or hydrazine
hydrochloride was added. Following stirring for 4 h at 30 °C,
the reaction mixture was evaluated by TLC (CH2Cl2/MeOH )
99 :1). In case no product formation could be observed, the
mixture was warmed to 80 °C for 2 h. After addition of water
(30 mL), the precipitate was filtered off and washed with
water. In general, recrystallization from methanol afforded
pure products. Only in some cases, further purification by
column chromatography (silica, CH2Cl2/MeOH ) 99 :1) was
necessary. When N,N-methylphenylhydrazine was used as a
starting material, 2,3-dihydro-4-methyl-1-(N-methyl-N-phenyl-
amino)-5-phenyl-1H-imidazole-2-thione 3.42 could be obtained
in an analogous way.
Syn th esis of 1-(3-Ch lor op h en yla m in o)-2,3-d ih yd r o-4,5-
d ip h en yl-1H-im id a zole-2-th ion e (3.26). A mixture of ben-
zoine (1.0 g, 7.1 mmol), KSCN (0.68 g, 7.1 mmol), and
3-chlorophenylhydrazine‚HCl (1.26 g, 7.1 mmol) was stirred
An tivir a l Activity a n d Cytotoxicity Assa ys. The inhibi-
tory effects of the N-aminoimidazole derivatives on HIV-1,

1552 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Lagoja et al.
HIV-2, and SIV replication were monitored by measuring the
viability of MT-4 cells 5 days after infection.18 Cytotoxicity of
the compounds was determined in parallel by measuring the
viability of mock-infected cells on day 5. The number of viable
cells was quantified semiautomatically by a tetrazolium-based
colorimetric method using 3-(4,5-dimethylthiazolyl-2-yl)-2,5-
diphenyltetrazolium (MTT), as described by Pauwels et al.20
Peripheral blood mononuclear cells (PBMCs) from healthy
donors were isolated by density centrifugation (Lymphoprep,
Nycomed Pharma AS, Diagnostics, Oslo, Norway) and stimu-
lated with phytohemagglutinin (PHA, 2 µg/mL final concentra-
tion) (Sigma Chemical Co., Bornem, Belgium) for 3 days. The
activated cells (PHA-stimulated blasts) were washed three
times with PBS, and viral infections were done as described
by the ACTG protocols.19 HIV-infected or mock-infected PHA-
stimulated blasts were cultured in the presence of 25 U/mL of
IL-2 and varying concentrations of drugs. The supernatant was
collected at day 7, and HIV-1 core antigen (p24 Ag) in the
supernatant was analyzed by enzyme linked immunosorbent
assay (ELISA) (NEN, Brussels, Belgium).
deoxynucleotide substrate (INT1/INT2), and 230 nM His-tag
IN in a volume of 10 µL. Inhibitors were incubated briefly with
the reaction components before the addition of IN. Reactions
were started by addition of the enzyme. All the reactions were
stopped by addition of formamide loading dye, and the
products were separated in a 15% denaturating polyacryl-
amide/urea gel. Autoradiography was performed by exposing
the wet gel to X-ray film (CURIX RP1, Agfa). Quantification
of the results was performed using the Cyclone (Canberra
Packard, Zellik, Belgium).
To evaluate inhibition of 3′-processing, the reaction was
allowed to proceed at 37 °C for 7 min. Inhibition of formation
of the -2 band was determined. In the overall integration
assay, the reaction was allowed to proceed for 60 min. Both
inhibition of formation of the -2 band and DNA strand
transfer products were scored. The DNA strand transfer was
assayed in the following way. DNA substrate (INT1/INT2, 30
nM) was preincubated with 230 nM IN at 37 °C for 5 min to
allow the cleavage reaction to occur. The composition of the
reaction mixture was identical to that in the processing assay.
After 5 min, 1 µL of excess target DNA (SK70/T35, at a final
concentration of 250 nM) was added with or without inhibitor
and the samples were incubated at 37 °C for 1 h. The excess
of unspecific target DNA competitively blocked further binding
of IN to the viral substrate so that inhibition of DNA strand
transfer in the preformed complexes can be monitored inde-
pendently of DNA binding and 3′-processing.
C3H/3T3 cells were seeded at 20 000 cells per milliliter into
wells of tissue culture cluster plates (48 wells per plate).
Following a 24 h incubation period, cell cultures were infected
with 80 focus-forming units of MSV for 120 min, whereafter
the culture medium was replaced by 1 mL of fresh medium
containing appropriate concentrations of the test compound.
After 6 days, transformation of the cells was examined
microscopically.20
Tim e-of-Ad d ition Exp er im en t. MT-4 cells were infected
with HIV-1(IIIB) at a multiplicity of infection (moi) of 0.5. The
test compounds were added at different times after infection.21
Viral p24 Ag production was determined at 31 h postinfection
by ELISA (NEN, Brussels, Belgium). The reference compounds
were added at 100 times their 50% inhibitory concentration
(IC50) obtained in the MT-4/MTT assay.
In h ibition of HIV-1 P r od u ction fr om Ch r on ica lly
In fected Cells. Persistently infected (NL4.3) HUT-78 cells
were washed three times in order to remove cell-free virus.
The test compounds at the required concentrations were
transferred into the cups of a 48-well plate. To each cup
200 000 cells were added (final volume of 1 mL). The cultures
were allowed to grow for 44 h. The supernatant was collected,
and viral p24 Ag production was determined by ELISA (NEN,
Brussels, Belgium).
Ack n ow led gm en t. We are grateful to Liesbet De
Dier, Kristien Erven, Cindy Heens, Martine Michiels,
Lizette van Berckelaer, and Be´ne´dicte Van Maele for
their excellent technical assistance. We thank Prof. J ef
Rozenski for the mass spectrometry, and Carolien
Bonroy, Sara Vijgen, and Ding Zhou for their assistance
in the chemical synthesis. Elemental analyses were
obtained from the “Microanalytical Labor, Fakulta¨t fu¨r
Chemie, Universita¨t Konstanz”. I. Lagoja is a research
associate of the Rega Foundation. Results of this
research are the subject of a patent application (Lagoja,
I. M.; Pannecouque, C.; Van Aerschot, A.; Herdewijn,
P.; De Clercq, E. Patent Application PCT/EPOI/02140,
24.02, 2001).
HIV-1 RT Assa y. Poly(rC).oligo(dG) and [3H]dGTP and
poly(rA).oligo(dT) and [3H]dTTP were used as a template
primer and radiolabeled substrate, respectively. The final
[3H]dGTP and [3H]dTTP concentrations in the reaction mix-
ture were both 2.5 µM.
Su ppor tin g In for m ation Available: Syntheses and NMR
data of NAIM derivatives. This material is available free of
charge via the Internet at http://pubs.acs.org.
Selection of 3.19- a n d 7.03-Resista n t Vir u s Str a in s.
HIV-1(IIIB) was subjected to passages in MT-4 cells (4 × 105
cells/2 mL) in the presence of increasing concentrations of the
compounds. Passages were performed every 3-4 days.
Deter m in a tion of th e Am in o Acid Sequ en ce of th e
3.19- a n d 7.03-Resista n t HIV-1 Str a in s RT. The procedures
for the MT-4 cells infection with resistant HIV-1, preparation
of the samples for the PCR assays, amplification of the proviral
DNA, and sequencing of the 727-bp fragment covering amino
acid residues 50-270 have been reported elsewhere.22
HIV In tegr a se Assa ys. Wild type His-tagged recombinant
HIV-1 integrase and the substrate and target DNA were as
previously described.23 The following high-performance-
liquid-chromatography-purified deoxynucleotides were pur-
chased from Amersham-Pharmacia Biotech: INT1, 5′-TGTG-
GAAAATCTCTAGCAGT; INT2, 5′-ACTGCTAGAGATTTTC-
CACA; T35, 5′-ACTATACCAGACAATAATTGTCTGGCCTG-
TACCGT; SK70, 5′-ACGGTACAGGCCAGACAATTATTGTCTG-
GTATAGT. The oligodeoxynucleotides INT1 and INT2 cor-
respond to the U5 end of the HIV-1 LTR. The 3′-processing,
overall integration, and strand-transfer assay were slightly
modified from published procedures. The final reaction mixture
of the 3′-processing assays contained 20 mM HEPES, pH 7.5,
5 mM dithiothreitol, 10 mM MgCl2, 75 mM NaCl, 5% (v/v) poly-
(ethylene glycol) 8000, 15% dimethyl sulfoxide, 30 nM oligo-
Refer en ces
(1) De Clercq, E. Toward improved anti-HIV chemotherapy: Thera-
peutic strategies for intervention with HIV infections. J . Med.
Chem. 1995, 38, 2491-2517.
(2) (a) Coffin, J . M. HIV population dynamics in vivo: implications
for genetic variation, pathogenesis, and therapy. Science 1995,
267, 483-489. (b) Vandamme, A.-M.; Van Vaerenbergh, K.; De
Clercq, E. Anti-human immunodeficiency virus drug combination
strategies. Antiviral Chem. Chemother. 1998, 9, 187-203.
(3) Richman, D. D.; Havlir, D.; Corbeil, J .; Looney, D.; Ignacio, C.;
Spector, S. A.; Sullivan, J .; Cheeseman, S.; Barringer, K.;
Pauletti, D.; Shih, C.-K.; Myers, M.; Griffin, J . Nevirapine
resistance mutations of human immunodeficiency virus type 1
selected during therapy. J . Virol. 1994, 68, 1660-1666.
(4) Fujiwara, T.; Sato, A.; El-Farrash, M.; Miki, S.; Abe, K.; Isaka,
Y.; Kodama, M.; Wu, Y.; Chen, L. B.; Harada, H.; Sugimoto, H.;
Hatanaka, M.; Hinuma, Y. S-1153 inhibits replication of known
drug-resistant strains of human immunodeficiency virus type
1. Antimicrob. Agents Chemother. 1998, 42, 1340-1345.
(5) Ren, J .; Nichols, C.; Bird, L. E.; Fujiwara, T.; Sugimoto, H.;
Stuart, D. I.; Stammers, D. K. Binding of the second generation
non-nucleoside inhibitor S-1153 to HIV-1 reverse transcriptase
involves extensive main chain hydrogen bonding. J . Biol. Chem.
2000, 275, 14316-14320.
(6) Schantl, J . G.; Lagoja, I. Direct synthetic approach to N-
substituted 1-amino-2,3-dihydro-1H-imidazole-2-thiones. Hetero-
cycles 1997, 45, 691-700.

N-Aminoimidazole Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1553
(7) Sitte, A.; Wessel, R.; Paul, H. Umwandlung von 1,3,4-Thia-
diazolinen in 1,2,4-Triazole durch Dimroth-Umlagerung.
Monatsh. Chem. 1975, 106, 1291-1296.
(18) Pauwels, R.; Balzarini, J .; Baba, M.; Snoeck, R.; Schols, D.;
Herdewijn, P.; Desmyter, J .; De Clercq, E. Rapid and Automated
Tetrazolium-Based Colorimetric Assay for Detection of anti-HIV
Compounds. J . Virol. Methods 1988, 20, 309-321.
(8) Schantl, J . G.; Lagoja, I. M. Oxidative Reduction of Cyclic
Thiosemicarbazides: Conversion of 1-Arylamino-2,3-dihydro-1H-
imidazole-2-thiones into 1-Arylamino-1H-imidazoles. Hetero-
cycles 1998, 48 (5), 929-938.
(19) J apour, A. J .; Mayers, D. L.; J ohnson, V. A.; Kuritzkes, D. R.;
Beckett, L. A.; Arduino, J . M.; Lane, J .; Black, R. J .; Reichelder-
fer, P. S.; D’Aquila, R. T.; Crumpacker, C. S. Standardized
peripheral blood mononuclear cell culture assay for determina-
tion of drug susceptibilities of clinical human immunodeficiency
virus type 1 isolates. The RV-43 Study Group, the AIDS Clinical
Trials Group Virology Committee Resistance Working Group.
Antimicrob. Agents Chemother. 1993, 37, 1095-1101.
(20) Balzarini, J .; Holy´, A.; J indrich, J .; Naesens, L.; Snoeck, R.;
Schols, D.; De Clercq, E. Differential antiherpesvrius and
antiretrovirus effects of the (S) and (R) enantiomers of acyclic
nucleoside phosphonates: potent and selective in vitro and in
vivo antiretrovirus activities of (R)-9-52-phosphonomethoxy-
propyl)-2,6-diaminopurine. Antimicrob. Agents Chemother. 1993,
37, 332-338.
(21) Pauwels, R.; Andries, K.; Desmyter, J .; Schols, D.; Kukla, M. J .;
Breslin, H. J .; Raeymaeckers, A.; Van Gelder, J .; Woestenborghs,
R.; Heykants, J . Potent and selective inhibition of HIV-1
replication by a novel series of TIBO derivatives. Nature 1990,
343, 470-474.
(22) (a) Balzarini, J .; Karlsson, A.; Pe´rez-Pe´rez, M.-J .; Vrang, L.;
Walbers, J .; Zhang, H.; O¨ berg, B.; Vandamme, A.-M.; Camarasa,
M.-J .; De Clercq, E. HIV-1-specific reverse transcriptase inhibi-
tors show differential activity against HIV-1 mutant strains
containing different amino acids substitutions in the reverse
transcriptase. Virology 1993, 192, 246-253. (b) Balzarini, J .;
Karlsson, A.; De Clercq, E. Human immunodeficiency virus type
1 drug resistance patterns with different 1-(2-hydroxyethoxy)-
methyl-6-(phenylthio)thymine derivatives. Mol. Pharmacol. 1993,
44, 694-701.
(9) Dodd, D. S.; Kozikowski, Conversion of Alcohols to Protected
Guanidines Using the Mitsunobu Protocol. Tetrahedron Lett.
1994, 35 (7), 977-980.
(10) (a) Hazuda, D. J .; Felock, P.; Witmer, M.; Wolfe, A.; Stillmock,
K.; Grobler, J . A.; Espeseth, A.; Gabryelski, L.; Schleif, W.; Blau,
C.; Miller, M. D. Inhibitors of strand transfer that prevent
integration and inhibit HIV-1 replication in cells. Science 2000,
287, 646-650. (b) Pluymers, W.; Pais, G.; Van Maele, B.;
Pannecouque, C.; Fikkert, V.; Burke, T.; De Clercq, E.; Witvrouw,
M.; Neamati, N.; Debyser, Z. Inhibition of HIV-1 integrase
activity and replication by a series of diketo derivatives. Anti-
microb. Agents Chemother., submitted.
(11) Horwitz, J . P.; Chua, J .; Noel, M. Nucleoside V, the mono-
mesylates of 1-(2′-deoxy-â-D-lyxyfuranosyl)thymine. J . Org. Chem.
1964, 29, 2076.
(12) Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C.
Detection, Isolation and Continuous Production of Cytopathic
Retroviruses (HTLV-III) from patients with AIDS or pre-AIDS.
Science 1984, 224, 497-500.
(13) Adachi, A.; Gendelman, H.; Koenig, S.; Folks, T.; Willey, R.;
Rabson, A.; Martin, M. Production of acquired immunodeficiency
syndrome-associated retrovirus in human and nonhuman cells
transfected with an infectious molecular clone. J . Virol. 1986,
59, 284-291.
(14) Barre´-Sinoussi, F.; Chermann, J . C.; Nugeyre, M. T.; Chamaret,
S.; Gruest, J .; Dauguet, C.; Axler-Blin, C.; Ve´zinet-Brun, F.;
Rouzioux, C.; Rozenbaum, W.; Montagnier, L. Isolation of a
T-Lymphotropic Retrovirus from a Patient at Risk for AIDS.
Science 1983, 220, 868-871.
(15) Daniel, M. D.; Letvin, N. L.; Sehgal, P. K.; Hunsmann, G.;
Schmidt, D. K.; King, N. W.; Desrosiers, R. C. Long-term persist-
ent infection of macaque monkeys with the simian immuno-
deficiency virus. J . Gen. Virol. 1987, 68, 3183-3189.
(16) Harada, S.; Koyanagi, Y.; Yamamoto, N. Infection of HTLV-III/
LAV in HTLV-I carrying cells MT-2 and MT-4: an application
in a plaque assay. Science 1985, 229, 563-566.
(17) De Clercq, E.; Merigan, T. C. Moloney sarcoma virus-induced
tumors in mice: inhibition or stimulation by (polyrI):poly(rC).
Proc. Soc. Exp. Biol. Med. 1971, 137, 590-594.
(23) (a) Cherepanov, P.; Este´, J . A.; Rando, R. F.; Ojwang, J . O.;
Reekmans, G.; Steinfeld, R.; David, G.; De Clercq, E.; Debyser,
Z. Mode of action of G-quartets with the integrase of human
immunodeficiency virus type 1. Mol. Pharmacol. 1997, 52, 771-
780. (b) Debyser, Z.; Cherepanov, P.; Pluymers, W.; De Clercq,
E. Assays for the evaluation of HIV-1 integrase inhibitors. In
Methods in Molecular Biology. Nucleases. Humana Press: Clif-
ton, NJ , 2001.
J M0211117