4-Aminopyrimidines as novel HIV-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.09.047

A series of 4-aminopyrimidines (1) was identified as novel HIV inhibitors of unknown molecular target. Structural modifications were carried out to establish its SAR and identify the linking site for target identification. A number of analogs were found to possess single digit inhibitory activity for HIV replication. Several analogs with various potential linkers, including a biotinated analog, also exhibited excellent potency, and could serve as tools for the identification of novel anti-HIV targets.

Full text of DOI:10.1016/j.bmcl.2006.09.047

Bioorganic & Medicinal Chemistry Letters 17 (2007) 260–265
4-Aminopyrimidines as novel HIV-1 inhibitors
Venkat R. Gadhachanda,a Baogen Wu,a Zhiwei Wang,a Kelli L. Kuhen,a
Jeremy Caldwell,a Helmut Zondler,b Harald Walter,c Mark Havenhandb and Yun Hea,*
aGenomics Institute of the Novartis Research Foundation (GNF), 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA
bNovartis Pharma AG, Postfach, CH-4002 Basel, Switzerland
cSyngenta Crop Protection AG, Research Chemistry, CH-4002 Base, Switzerland
Received 1 August 2006; revised 15 September 2006; accepted 18 September 2006
Available online 10 October 2006
Abstract—A series of 4-aminopyrimidines (1) was identified as novel HIV inhibitors of unknown molecular target. Structural mod-
ifications were carried out to establish its SAR and identify the linking site for target identification. A number of analogs were found
to possess single digit inhibitory activity for HIV replication. Several analogs with various potential linkers, including a biotinated
analog, also exhibited excellent potency, and could serve as tools for the identification of novel anti-HIV targets.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The introduction of highly active antiretroviral therapy
(HAART) has significantly decreased morbidity and
mortality among patients infected with human immuno-
deficiency virus type 1 (HIV-1).1,2 However, HIV-1 can
acquire resistance against all currently available antiret-
roviral drugs. Significant resistance has been generated
against the currently marketed viral reverse transcrip-
tase inhibitors (RTI) and protease inhibitors (PI).3,4
Resistance has also been observed for the newly ap-
proved entry inhibitor Enfuvirtide (T-20).5 Moreover,
in a growing number of patients, the development of
multidrug-resistant viruses compromises HAART effica-
cy and limits therapeutic options.6 There is an urgent
need of new drugs that could combat the current resis-
tance. New classes of antiviral drugs, especially against
novel targets, will provide the only hope of treatment
for an increasing number of patients.7,8
tity potential novel targets for HIV inhibition. Biochem-
ical inhibition studies suggested that these compounds
are neither HIV NRTI’s nor integrase inhibitors. As
HIV-1 protease is not part of the recombinant HIV cell
model used for screening, it can be excluded as a target
of these inhibitors. We were intrigued by the novel struc-
tural features of these HIV inhibitors, and believed that
these compounds could serve as a molecular probe to
identify novel targets for HIV inhibition. Affinity chro-
matography is one of the most commonly used tech-
niques in target identification.19,20 It requires the
attachment of the ligand of unknown target to a solid
support, in a manner as to maintain the affinity for the
potential target after the ligand is attached to the sup-
port. We wish to report in this communication our effort
to establish the preliminary SAR of these pyrimidines as
novel HIV inhibitors and the identification of the
attachment site for target identification.
During our effort to develop new anti-HIV drugs,9–13 we
discovered a series of benzylaminopyrimidines (Fig. 1)
that exhibited a range of inhibitory activities against
HIV replication. Although 4-aminopyrimidines related
to compound 1 have been extensively studied as pesti-
cides,14–18 no antiviral activity for these pyrimidines
has been reported to date. One of our main goals for
our cell-based anti-HIV screening platform was to iden-
A representative synthesis of these amino pyrimidines is
shown in Scheme 1, which calls for the preparation of a
dichloro intermediate 4. Pyrimidine 4 could be easily
prepared from methyl 2-chloro-3-oxo-pentanoate (2)
by sequential pyrimidine ring formation with formamid-
inium chloride and chlorination of the resulting
hydroxypyrimidine 3 with phosphoryl chloride.21 In
parallel, nitrobenzylamine 5 was first protected with
Boc2O and the nitro group was then reduced to give ani-
line 6. Acetylation of the aniline followed by the removal
of the Boc group with trifluoroacetic acid provided
benzylamine 8 for the final coupling with pyrimidine
Keywords: Aminopyrimidine; HIV-1 inhibitor.
*
Corresponding author. Tel.: +1 858 332 4706; fax: +1 858 332
4513; e-mail: yhe@gnf.org
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.047

V. R. Gadhachanda et al. / Bioorg. Med. Chem. Lett. 17 (2007) 260–265
261
number of heterocycles in this region, and the majority
of these modifications maintained good anti-HIV activ-
ity. In particular, compounds 11a, 11c, 11f, and 11g
showed double digit nM IC50s. These results again sug-
gest that this region of the molecule might be a suitable
position for linker attachment for target identification.
Figure 1.
The anti-HIV activity of 12a–12c suggested that a small
hydrophobic functionality like a methyl group is favored
at the benzylic position, as it is almost twenty-fold more
potent than the unsubstituted analog 12a, and about
twofold more potent than the corresponding ethyl
analog 12c.
chloride 4. Under the influence of potassium carbonate,
pyrimidine 9 was conveniently obtained in a moderate
yield.
Using similar chemistry, a large number of aminopyrim-
idines could be easily synthesized for SAR studies
(Fig. 2). These compounds were tested in the cell-based
HIV inhibition assay and their inhibitory activities are
summarized in Table 1. It was clear that the anti-HIV
activities of the pyrimidines are sensitive to structural
perturbations. Compounds 10a–10e have the common
benzylaminopyrimidine core but with varying R groups
at the 4 position of the phenyl ring. The antiviral activ-
ities of these analogs are shown in Table 1. Data indicate
that a hydrophobic R moiety is preferred, and the biphe-
nyl analog 10d exhibited a 17 nM inhibitory activity.
The quinolinoxy analog 10e also had an IC50 of
48 nM. This is very useful information, as it suggests
that a large space is available to accommodate substitu-
tion at the 4 position of the phenyl ring. On the other
hand, small and polar substitutions at the 4 position
resulted in the reduction in potency, and 10a and 10b
have IC50s of 2.659 lM and 341 nM, respectively.
To explore the SAR in the pyrimidine region of this
scaffold, a number of analogs with various substitutions
on the pyrimidine ring were examined (Fig. 2 and Table
1). For the purpose of this study, the 4-fluorophenoxy
benzyl moiety was held constant. Our studies suggest
that a slightly larger propyl group at position 6 (R2 in
13) is preferred over the methyl group for anti-HIV
activity (13a vs. 13b). The methoxymethyl group (13c)
leads to a further improvement in potency. The combi-
nation of a bromo R1 and an ethyl R2 gave an extremely
potent HIV inhibitor (13d) with an IC50 of 3 nM. How-
ever, a bromo R1 and a propyl R2 are not compatible,
resulting in only a moderately potent analog 13e
(IC50 = 760 nM vs. 96 and 3 nM). These data suggest
that space around positions 5 and 6 is limited, and sen-
sitive to the size of the substituent. Further substitution
of 13d with a methyl group at position 2 also gave a po-
tent analog (13f) with an IC50 of 18 nM. More interest-
ingly, the 5-amino analog 13g also exhibited potent anti-
viral activity (18 nM). The combined data of 13c and
13g suggest that the 5 and 6 positions of the pyrimidines
ring are not very sensitive to polarity perturbations.
To explore the SAR in this region, various heterocycles
(11a–11j) were introduced to replace the phenyl moiety
in 10. Data suggested that this scaffold tolerates a
Scheme 1. Synthesis of benzylaminopyrimidine 9. Reagents and conditions: (a) H2NCH@NHÆHCl, CH3ONa, MeOH; (b) POCl3, toluene, 77%; (c)
BOC2O, NaHCO3, CH2Cl2/H2O, 25 ꢁC, 12 h, 92%; (d) Ni, H2, N2H4, EtOH, 25 ꢁC, 24 h, 50%; (e) Ac2O, Py, DMAP, CH2Cl2, 0–25 ꢁC, 3 h, 88%; (f)
CF3COOH, CH2Cl2, 25 ꢁC, 3 h, 88%; (g) 4, DMF, K2CO3, 80 ꢁC, 5 h, 45%.

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V. R. Gadhachanda et al. / Bioorg. Med. Chem. Lett. 17 (2007) 260–265
Figure 2. Structures of aminopyrimidines 10–13.
Table 1. Anti-HIV activities of 4-aminopyridines22
23
EC50 (lM)
24
CC50 (lM)
Compound
Compound
EC50 (lM)
CC50 (lM)
Efavirenz
Nevirapine
9
0.0005
0.050
1.188
2.659
0.341
0.091
0.017
0.048
0.095
0.139
0.042
0.170
0.778
0.029
0.035
0.691
0.816
0.353
0.331
0.016
ꢀ10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
12c
13a
13b
13c
13d
13e
13f
13g
16a
16b
16c
17a
17b
18a
18b
18c
18d
19a
19b
21
0.028
0.324
0.096
0.031
0.003
0.760
0.018
0.015
0.004
0.004
0.327
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
>10
11f
11g
0.190
0.005
0.005
0.190
3.020
4.889
0.622
11h
11i
11j
12a
12b
These preliminary SAR studies clearly indicate that the
phenyl moiety in 1 could tolerate a large variety of struc-
tural modifications and is the most promising position
for attaching a linker for the purpose of target identifi-
cation. To test this hypothesis, a number of analogs with
potential linkers were prepared (16–19, Scheme 2).

V. R. Gadhachanda et al. / Bioorg. Med. Chem. Lett. 17 (2007) 260–265
263
Scheme 2. Synthesis of benzylaminopyrimidines 16–19. Reagents and conditions: (a) NH(CH3)R, CH2Cl2, 25 ꢁC, 30 min, 92%; (b) TFA, CH2Cl2,
25 ꢁC, 3 h, 84%; (c) 4, K2CO3, DMF, 25 ꢁC, 4 h, 70%; (d) MeI, K2CO3, DMF, 38 ꢁC, 12 h, 90–95%.
Reaction of the activated 4-nitrophenoxyl carbonate 14
with various amines gave the carbamates, which were
easily converted to the corresponding benzyl amines 15
after the removal of the Boc group. Selective displace-
ment of the chlorine at the 4 position in 4 with amines
15a–15c furnished the amino pyrimidines 16a–16c. We
were pleased to find out that both the simple dimethyl-
amine (16a) and the extended amine analog (16b) exhib-
ited an IC50 of 4 nM. Interestingly, the other extended
analog 16c has significantly reduced activity, suggesting
some sensitivity in the linker region in the maintenance
of the antiviral potency. Treatment of 16a and 16b with
methyl iodide in the presence of potassium carbonate
furnished the corresponding N-methylated analogs 17a
and 17b in excellent yields (Scheme 2). As suspected,
both 17a and 17b lost all anti-viral activity, suggesting
the critical role of the N–H hydrogen in 1 in the interac-
tion with the target. More importantly, the N-methylat-
ed analogs like 17b could serve as a negative control for
target identification.
creased water solubility compared to the corresponding
N-methyl analogs (16a–16c). To our delight, the removal
of the N-methyl groups in 16b had no negative impact on
the anti-viral activity. Similar to 16c, the 1,3-diamine
analog 18a showed sub-micromolar activity. On the
other hand, analog 18b fully maintained the potent anti-
viral activity when compared to its counterpart 16b. The
slightly extended analog 18c is still a potent HIV inhibi-
tor. Although the further extended analog 18d lost some
antiviral activity, it still had an IC50 of 190 nM, which
could be sufficient as a ligand for affinity chromatogra-
phy. Two urea analogs 19a and 19b were also prepared
for SAR analysis (Scheme 2), and both compounds
exhibited only micromolar activity, confirming that a
carbamate linker is superior for antiviral activity.
A biotinated analog 21 was designed to evaluate its po-
tential as a probe for affinity chromatography (Scheme
3). It was easily synthesized from the Boc-protected ani-
line 6 by (1) standard amide coupling; (2) removal of the
Boc protecting group; and (3) selective displacement of
dichloropyrimidine (4) with the resulting amine. Our
data demonstrate that 21 is still a potential HIV inhibi-
tor with an IC50 of 622 nM, confirming that these com-
pounds could serve as suitable probes for identifying the
molecular target of this series of novel HIV inhibitors.
To further explore the effect of various potential linkers
on anti-HIV activity, additional analogs 18a–18d, 19a,
and 19b were synthesized using similar chemistry
(Scheme 2). All these compounds incorporate an NH in
the carbamate linker, which could potentially result in in-

264
V. R. Gadhachanda et al. / Bioorg. Med. Chem. Lett. 17 (2007) 260–265
Scheme 3. Synthesis of biotin conjugated benzylaminopyrimidine 21. Reagents and conditions: (a) (+)-biotin, HATU, DIEA, THF, 25 ꢁC, 24 h,
58%; (b) CF3COOH, CH2Cl2, 25 ꢁC, 3 h, 82%; (c) 4, DMF, K2CO3, 80 ꢁC, 4 h, 45%.
9. Jiang, T.; Kuhen, K. L.; Wolff, K.; Yin, H.; Bieza, K.;
Caldwell, J.; Bursulaya, B.; Wu, T. Y.-H.; He, Y. Bioorg.
Med. Chem. Lett. 2006, 16, 2105.
10. Jiang, T.; Kuhen, K. L.; Wolff, K.; Yin, H.; Bieza, K.;
Caldwell, J.; Bursulaya, B.; Tuntland, T.; Zhang, K.;
Karanewsky, D.; He, Y. Bioorg. Med. Chem. Lett. 2006,
16, 2109.
11. Wu, B.; Kuhen, K.; Nguyen, T. N.; Ellis, D.; Anaclerio,
B.; He, X.; Yang, K.; Karanewsky, D.; Yin, H.; Wolff, K.;
Bieza, K.; Caldwell, J.; He, Y. Bioorg. Med. Chem. Lett.
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12. Wang, Z.; Wu, B.; Kuhen, K. L.; Bursulaya, B.; Nguyen,
T. N.; Nguyen, D. G.; He, Y. Bioorg. Med. Chem. Lett.
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13. Ellis, D.; Kuhen, K. L.; Anaclerio, B.; Wu, B.; Wolff, K.;
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14. Yamanaka, Y.; Moritomo, M.; Fujii, K.; Tanaka, T.;
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Yoshioka, H. Pest. Sci. 1992, 34, 133.
In summary, we have identified a series of 4-aminopyr-
imidines as novel HIV inhibitors through high-through-
put screening and systematic structural modifications. A
number of these pyrimidines possessed single digit nano-
molar potency. These pyrimidines were believed to act
on unknown anti-HIV target(s). Analog synthesis was
directed toward establishing a SAR, with a particular
focus to identify a handle on the molecule for linking
to the solid support for target identification. Our studies
suggest that the phenyl ring in 1 is very amenable for
chemical modifications and that various potential link-
ers could be attached to the 4 position of the phenyl ring
while maintaining potent anti-HIV activity. (+)-Biotin
was also linked via an amide bond at this position,
and the linked molecule 21 showed submicromolar
inhibitory activity in the cell-based assay. The synthesis
and utilization of optimal molecular probes in identify-
ing the unknown target are on-going, and the results will
be reported in due course.
16. Walter, H. WO9501975, 1995.
17. Walter, H.; Havenhand, M. EP530149, 1993.
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22. All compounds except 21 are racemic. Compound 21 is a
mixture of two diastereoisomer.
Acknowledgments
The authors thank Karen Wolff and Hong Yin for
performing the HIV inhibition assays.
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(5 lL/well, Aquamax) followed by plate reading on the
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265
CLIPR apparatus (Molecular Devices) using a 20 s shuttle
speed.
The treated and uninfected cells are cultured for an
additional 48 h at 37 ꢁC. Cell viability is assessed by
addition of 1 lL of Alamar Blue (Promega, Cat. No.
00-100) diluted 1:1 in DMEM. Cells are further incubated
for 4 h at room temperature and subsequent fluorescence
intensity is read using an Acquest (TREK systems) with a
50/50 beam splitter.
24. 293T cells are seeded in the 1536-well format at 700 cells/
well (5 lL volume) using an Aquamax (Molecular Devic-
es) liquid dispenser. Cells are cultured at 37 ꢁC under 5%
CO2 for 24 h. Fifty nanoliter of each compound (serially
diluted in DMSO) is transferred using the PinTool (GNF).