From dihydroxypyrimidine carboxylic acids to carboxamide HIV-1 integrase inhibitors: SAR around the amide moiety

Article abstract of DOI:10.1016/j.bmcl.2006.10.054

4,5-Dihyroxypyrimidine carboxamides, which evolved from a related series of HCV NS5b polymerase inhibitors, have been optimized to provide selective HIV integrase strand transfer inhibitors. Extensive SAR around the benzylamide moiety led to the identification of the p-fluorobenzylamide as optimal in the enzymatic assay.

Full text of DOI:10.1016/j.bmcl.2006.10.054

Bioorganic & Medicinal Chemistry Letters 17 (2007) 350–353
From dihydroxypyrimidine carboxylic acids to carboxamide
HIV-1 integrase inhibitors: SAR around the amide moiety
Alessia Petrocchi,a,* Uwe Koch,a Victor G. Matassa, Barbara Pacini,a
Kara A. Stillmockb and Vincenzo Summaa
aDepartment of Medicinal Chemistry and Drug Metabolism, IRBM-MRL Rome, Via Pontina, Km 30.600,
00040 Pomezia, Rome, Italy
bDepartment of Antiviral Research, MRL West Point, Sumneytown Pike, PO Box 4, 19486, PA, USA
Received 21 September 2006; revised 20 October 2006; accepted 20 October 2006
Available online 25 October 2006
Abstract—4,5-Dihyroxypyrimidine carboxamides, which evolved from a related series of HCV NS5b polymerase inhibitors, have
been optimized to provide selective HIV integrase strand transfer inhibitors. Extensive SAR around the benzylamide moiety led
to the identification of the p-fluorobenzylamide as optimal in the enzymatic assay.
Ó 2006 Elsevier Ltd. All rights reserved.
Human immunodeficiency virus type 1 (HIV-1) is the
etiological agent of the acquired immunodeficiency syn-
drome (AIDS). AIDS1 is a major epidemic with more
than 38 million infected people worldwide. FDA-ap-
proved therapies target three steps of the HIV life cycle:
reverse transcription (NRTI, nucleoside reverse trans-
criptase inhibitor and NNRTI, non-nucleoside reverse
transcriptase inhibitor), proteolytic maturation, and fu-
sion. Triple therapy, commonly referred to as highly ac-
tive antiretroviral therapy (HAART), is now the
standard for treatment and consists of a protease inhib-
itor or an NNRTI in combination with two NRTIs.
HAART, however, is often not well tolerated, requires
stringent compliance, and leads to multidrug resistance.
Therefore, novel therapies are needed. Integrase is an
enzyme encoded by the HIV genome and represents a
potential alternative target. It catalyzes the insertion
and the integration of the proviral DNA into the gen-
ome of the host cell in two steps: 30-processing, the
endonucleolytic sequence-specific hydrolysis of 30-ends
of the viral cDNA and strand transfer, the ligation of
the viral 30-OH cDNA ends to the phosphate backbone
of the host DNA acceptor.
The first generation integrase inhibitors reported
approximately 12 years ago2,3 blocked the whole assem-
bly process. More recently, 1,3-diketoacids have been
described which specifically inhibit the strand transfer
reaction catalyzed by HIV integrase: their evolution to
a molecule with more drug-like properties led to the
naphthyridine series.4,5
HIV integrase together with the related enzyme hepatitis
C virus (HCV) NS5b polymerase share a common
mechanism of action with catalytic activity that is med-
iated through Mg2+ ions believed present in their active
sites. Recently, we reported the discovery of 1,3-diketo-
acids6 (e.g., 1) and meconic acid derivatives7 (e.g., 2) as
potent inhibitors of HCV NS5b polymerase (Fig. 1).
These classes lacked drug-like characteristics: meconic
acid derivatives were almost completely decarboxylated
in acidic media, while DKAs showed a high level of irre-
versible covalent binding to proteins.4 Dihydroxypyrim-
idine carboxylic acid (3), a designed hybrid of these two
series,8 provided a novel class of inhibitors of HCV
NS5b polymerase with improved chemical and biologi-
cal stability. Despite their potent activity against HCV
NS5b polymerase, and a mechanism of action that in-
volves direct interaction with the active site metal ions,
dihydroxypyrimidine carboxylic acids 3 showed no
activity against the HIV integrase enzyme.
Keyword: HIV integrase inhibitors.
*
Corresponding author. Tel.: +39 06 91093500; fax: +39 06
91093654; e-mail: alessia_petrocchi@merck.com
Present address: Almirall SA, Calle Treball 2-4, Sant Just Desvern,
Barcelona 08960, Spain.
However, as part of the optimization work to identify
viable replacements for the carboxylic acid 3,9 carboxa-
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.054

A. Petrocchi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 350–353
OH
351
O
OH
OH
N
OH
N
O
OH
OH
OH
NHR
1
N
N
Ar
Ar
O
O
O
OH
OH
Ar= 2-thienyl
EtO
3
4 R= Bn
HCV not active
HIV IC50 = 85 nM
O
HCV IC50 = 2.6 μM
O
O
HIV not active
2
Figure 1. From dihydroxypyrimidine carboxylic acid to carboxamide.
mides were found to be inactive against HCV NS5b
polymerase but selective inhibitors of the HIV integrase
enzyme.
moiety produced the following results: elongating by an
additional methylene unit (9) provided 4-fold gain in
potency compared to the benzyl analog, whereas short-
ening the linker distance produced the weakly active ani-
lide 10 (IC50 = 1 lM).
Here, we report our initial exploration of this series
which focused exclusively on structure–activity relation-
ships (SAR) at the benzylamide portion of the inhibitor.
Insertion of the 1-naphthalene ring (11) increased poten-
cy 3-fold with respect to the lead. Similar enhancement
was observed when the benzylic methylene was substi-
tuted with a methyl group, but only for the S-enantio-
mer (12, IC50 = 40 nM). The corresponding 1-naphthyl
N-methylated (tertiary) amide 14 provided 7-fold drop
in activity with respect to 11 that emphasizes the impor-
tance of the secondary amide moiety in the molecule.
Scheme 1 describes a general procedure10 for the synthe-
sis of the dihydroxypyrimidine carboxamide 7 contained
in this study. N0-Hydroxythiophene-2-carboximida-
mide, generated from thiophene-2-carbonitrile, was
reacted with dimethyl acetylendicarboxylate in chloro-
form at 90 °C, to produce the Michael adduct as a mix-
ture of cis–trans isomers. Heating in refluxing xylenes
the foregoing mixture afforded the methyl ester 6. The
final carboxamide 7 was easily obtained by reacting 6
with the proper amine in N,N-dimethylformamide at
90 °C (or in refluxing methanol) and isolating the final
product by trituration with diethyl ether, without any
further purification.
Indane chiral derivatives (15, S, IC50 = 20 nM; 16, R,
IC50 = 610 nM) displayed different inhibitory activity
indicating again that stereochemistry at the benzylic po-
sition is vital for potency of branched compounds.
Efforts aimed at replacing the phenyl ring with a hetero-
cycle are shown in Table 2. Compounds bearing polar
heterocycles were completely inactive (17, 18, and 19).
Substitution of the phenyl ring with thiophene (20) or
with thiazoles (21 and 22) produced less active com-
pounds. Potency was improved 10-fold and 2-fold,
respectively, for benzothiophenes 23 and 24, and 5-fold
for indole 25 confirming the presence of enough space
The preliminary investigation of SAR at the carboxam-
ide 7 is shown in Table 1. Replacement of the benzyl
group with a cyclohexylmethyl substituent completely
abolished activity (8) underlining the importance of
the aromatic moiety in this part of the molecule. Vary-
ing the distance between the amide nitrogen and the aryl
CO2Me
N O
NH2
S
N OH
NH2
S
S
DMAD
NH2OH HCl
CN
CO2Me
MeOH
40˚C
(65%)
CHCl3
reflux
5
xylenes
140 ˚C
(15% for 2 steps)
OH
OH
OH
OH
N
N
RR1NH
NR1R
OMe
N
S
S
N
DMF, 90
or MeOH reflux
˚C
O
O
7
6
(35%)
Scheme 1. Synthetic procedure.

352
A. Petrocchi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 350–353
Table 1. Preliminary SAR at the carboxamide
5
4
OH
OH
N
R
N
S
N
R1
3
O
Compound
R
R1
QUICKIN
a
IC50 (nM)
2
4
8
CH2Ph
H
85
50,000
20
1
CH2-cyclohexane
CH2CH2Ph
Ph
H
9
H
10
11
12
13
14
15
16
H
1000
30
0
1-Naphthalene
H
(S)-CH(CH3)-2-naphthyl
(R)-CH(CH3)-2-naphthyl
1-Naphthalene
H
40
-1
H
610
210
20
10.00
100.00
1000.00 10000.00 100000.00
CH3
H
(1S)-1-Indane
(1R)-1-Indane
Quickin IC50 [nM]
H
610
Figure 2. logP versus QUICKIN IC50
.
a HIV strand transfer assay, see Ref. 4.
Table 3. SAR at the benzylamide
Table 2. SAR at the heteroaryl amide
OH
OH
OH
N
R3
H
N
OH
N
S
H
N
O
N
R2
S
N
O
a
QUICKIN IC50 (nM)
Compound
R3
R2
4-Pyridine
a
QUICKIN IC50 (nM)
Compound
4
26
27
28
29
30
31
32
33
34
35
36
37
38
39
H
85
90
17
18
19
20
21
22
23
24
25
16,600
50,000
17,000
100
2-OMe
3-OMe
4-OMe
2-Me
3-Me
4-Me
2-Ph
3-Ph
4-Ph
4-Cl
1H-1,2,4-Triazole
1H-Imidazole
2-Thiophene
50
550
110
40
2-(1,3-Thiazole)
5-(1,3-Thiazole)
2-Benzothiophene
3-Benzothiophene
3-1H-Indole
200
2500
10
70
130
50
50
20
50,000
20
a HIV strand transfer assay, see Ref. 4.
2-F
3-F
160
70
4-F
3,4-Cl
10
10
around the benzyl amide, crucial for additional lipophil-
ic interactions with the enzyme.
a HIV strand transfer assay, see Ref. 4.
A rationale behind the observed potency trends for the
heterocyclic analogs was found by correlating IC50 data
versus calculated logP (Fig. 2).11 Less polar heterocycles
placed in the benzylic position yielded compounds with
increased inhibitory activity suggesting that these bind
in an apolar environment.
tolerated. When halogen atoms were located in para (35
and 38) inhibitory activity was significantly enhanced,
and the p-fluorine derivative 38 emerged as the optimal
substituent displaying an 8-fold improvement in potency
compared to 4.
A range of di-substituted compounds were investigated
(e.g., 39), but they provided no further gain over com-
pound 38. Best compounds (IC50 < 1 lM) were tested
in cellular assay (Spread) under two serum conditions
(10% fetal bovine serum and 50% normal human serum)
showing no significant activity in the micromolar range.
In order to further investigate the space available
around the aromatic portion of benzylamides an exten-
sive variety of substituents were explored (Table 3). Sub-
stituents in the ortho and meta positions were generally
tolerated but did not lead to significantly improved com-
pounds (26–27, 29–30, and 32–33 all have potency with-
in 2-fold of 4). Tighter SAR was found around the para
position where bulky groups dramatically diminished
the inhibitory activity (e.g., 28 and 34). Apparently the
space available for enzyme interaction is reduced at this
position and only smaller groups like a methyl (31) were
Compound 38 was selected for further evaluation and
interestingly displayed acceptable rat pharmacokinetics
showing 39% oral bioavailability (F) and low plasma
clearance (11 ml/min/kg). Counterscreening studies

A. Petrocchi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 350–353
353
4. Zhuang, L.; Wai, J. S.; Embrey, M. W.; Fisher, T. S.;
Egbertson, M. S.; Payne, L. P.; Guare, J. P., Jr.; Vacca, J. P.;
Hazuda, D. J.; Felock, P. J.; Wolfe, A. L.; Stillmock, K. A.;
Witmer, M. V.; Moyer, G.; Schleif, W. A.; Gabryelski, L. J.;
Leonard, Y. M.; Lynch, J. J., Jr.; Michelson, S. R.; Young,
S. D. J. Med. Chem. 2003, 46, 453.
5. Hazuda, D. J.; Anthony, N. J.; Gomez, R. P.; Jolly, S. M.;
Wai, J. S.; Zhuang, L.; Fisher, T. E.; Embrey, M.; Guare,
J. P., Jr.; Egbertson, M. S.; Vacca, J. P.; Huff, J. R.;
Felock, P. J.; Witmer, M. V.; Stillmock, K. A.; Danovich,
R.; Grobler, J.; Miller, M. D.; Espeseth, A. S.; Jin, L.;
Chen, I-W.; Lin, J. H.; Kassahun, K.; Ellis, J. D.; Wong,
B. K.; Xu, W.; Pearson, P. G.; Schleif, W. A.; Cortese, R.;
Emini, E.; Summa, V.; Holloway, M. K.; Young, S. D.
Proc. Natl. Acad. Sci. 2004, 101, 11233.
proved that 38 did not inhibit human DNA polymerase
a, b, and c or HIV-RT. The efforts made to improve the
potency of this novel class of HIV integrase inhibitors in
cell-based assay will be reported elsewhere.12
In conclusion, dihydroxypyrimidine carboxamides were
identified as a novel and selective class of HIV-1 inte-
grase inhibitors. SAR around the carboxamide moiety
generated low nanomolar inhibitors which have poten-
tial for future development as anti-HIV agents.
Acknowledgments
6. Summa, V.; Petrocchi, A.; Pace, P.; Matassa, V.; De
Francesco, R.; Altamura, S.; Tomei, L.; Koch, U.;
Neuner, P. J. Med. Chem. 2004, 47, 14.
7. Pace, P.; Nizi, E.; Pacini, B.; Pesci, S.; Matassa, V.; De
Francesco, R.; Altamura, S.; Summa, V. Bioorg. Med.
Chem. Lett. 2004, 14, 3257.
We thank Odalys Paz Gonzalez and Ralph Laufer for
PK studies; William B. Schleif and Peter J. Felock for
biological testing; Steven Harper and Michael Rowley
for their helpful support and suggestions. This work
was supported in part by a grant from the MIUR.
8. Summa, V.; Petrocchi, A.; Matassa, V.; Taliani, M.;
Laufer, R.; De Francesco, R.; Altamura, S.; Pace, P. J.
Med. Chem. 2004, 47, 5336.
References and notes
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10. Culbertson, T. P. J. Heterocycl. Chem. 1979, 16, 1423.
11. logP values were calculated using the ACD PhysChem
program suite, version 8.00.
2. Fesen, M. R.; Kohn, K. W.; Leteurtre, F.; Pommier, Y.
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 2399.
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12. Summa, V., et al. J. Med. Chem., in press.