4,5-Dihydroxypyrimidine carboxamides and N-alkyl-5-hydroxypyrimidinone carboxamides are potent, selective HIV integrase inhibitors with good pharmacokinetic profiles in preclinical species

Article abstract of DOI:10.1021/jm060854f

The dihydroxypyrimidine carboxamide 4a was discovered as a potent and selective HIV integrase strand transfer inhibitor. The optimization of physicochemical properties, pharmacokinetic profiles, and potency led to the identification of 13 in the dihydroxypyrimidine series and 18 in the N-methylpyrimidinone series having low nanomolar activity in the cellular HIV spread assay in the presence of 50% normal human serum and very good pharmacokinetics in preclinical species.

Full text of DOI:10.1021/jm060854f

6646
J. Med. Chem. 2006, 49, 6646-6649
4,5-Dihydroxypyrimidine Carboxamides and
N-Alkyl-5-hydroxypyrimidinone Carboxamides
Are Potent, Selective HIV Integrase Inhibitors
with Good Pharmacokinetic Profiles in
Preclinical Species
Vincenzo Summa,* Alessia Petrocchi, Victor G. Matassa,†
Cristina Gardelli, Ester Muraglia, Michael Rowley,
Odalys Gonzalez Paz, Ralph Laufer, Edith Monteagudo, and
Paola Pace
Figure 1. Discovery of dihydroxypyrimidine carboxamide 4 as an HIV
integrase inhibitor.
Departments of Medicinal Chemistry and Drug Metabolism
IRBM-MRL Rome, Via Pontina, Km 30.600, 00040 Pomezia,
Rome, Italy
Table 1. Optimizing the Amide
ReceiVed July 20, 2006
Abstract: The dihydroxypyrimidine carboxamide 4a was discovered
as a potent and selective HIV integrase strand transfer inhibitor. The
optimization of physicochemical properties, pharmacokinetic profiles,
and potency led to the identification of 13 in the dihydroxypyrimidine
series and 18 in the N-methylpyrimidinone series having low nanomolar
activity in the cellular HIV spread assay in the presence of 50% normal
human serum and very good pharmacokinetics in preclinical species.
cmpd
R
IC50a (µM)
CIC95b (10% FBS/µM)
4a
5
CH2Ph
CH24F-Ph
0.085
0.010
>10
>10
a HIV strand transfer assay results are the mean of at least three
independent experiments, SD was always (8% of the value, and IC50 is
the concentration of inhibitor that reduces HIV integrase activity by 50%.
For details of the assay, see ref 143. b Spread assay results are the mean
of at least three independent experiments, SD was always <(10% of the
value, and CIC95 is the concentration of inhibitor that inhibits the HIV
replication in cell-based assays by 95%. For details of the assay, see ref
14.
Human immunodeficiency virus type 1 (HIV-1) is the
etiological agent of the acquired immunodeficiency syndrome
(AIDS). Inhibition of HIV-1 replication remains the fulcrum
for AIDS treatment, and three viral enzymes have been identified
as key targets to arrest the HIV life cycle: reverse transcriptase,
protease, and integrase. All the currently used oral drugs for
the treatment of HIV infection inhibit the first two of these
enzymes. Unfortunately, the emergence of drug-resistant virus
strains, limited oral bioavailability, and toxicity related to the
dosing regimens drastically reduce the efficacy, compliance, and
tolerability of the current treatments.
Inhibition of HIV integrase, responsible for inserting the viral
DNA into the host cellular genome, results in arrest of the HIV
life cycle and is, therefore, a very attractive therapeutic target.
Upon inhibition of integrase, the viral DNA is converted into a
circular DNA unable to be integrated into the host genome.1
Recently, HIV integrase has been validated as an antiviral target
in SHIV infected rhesus macaques,2 proving that in vitro
inhibitors of the strand transfer step are capable of inhibiting
viral replication in vivo. Furthermore, proof of concept has also
been achieved in man.3 Several classes of HIV integrase
inhibitors have been reported in the literature: for example,
diketo acids,4 naphthyridine ketones,5 naphthyridine carbox-
amides,6 quinolines,7 and tri-cyclic pyrroloquinolines.8 All share
a common pharmacophore capable of binding to the magnesium
ions present in the HIV integrase active site.
drug-like properties and very importantly with the aim of
maintaining the correct geometry to bind the Mg++ ions in the
active site of the HCV polymerase.11 Unfortunately, most of
these inhibitors showed a suboptimal activity in the HCV cell-
based assay.12 This property was mainly attributed to the free
carboxylic acid. Several carboxylic acid isosteres and deriva-
tives, including amides (4), were synthesized to solve this
problem.
HIV integrase and HCV polymerase share the common
feature of using two magnesium ions in the active site as a key
component of the catalytic machinery. In spite of the fact that
the two enzymes catalyze two different types of reaction, the
architecture of the amino acids within the catalytic site and the
geometry of the catalytic metals are conserved. This observation
was the basis of an integrated drug discovery program, where
we were screening compounds made as inhibitors of one viral
target using this catalytic machinery, across enzymes belonging
to the same superfamily from other viruses. While all the HCV
polymerase inhibitors having a free carboxylic acid were almost
completely inactive against HIV integrase, we were delighted
to find that the benzyl amide 4a (Table 1) was a potent,
reversible and selective HIV integrase strand transfer inhibitor
We recently reported three classes of HCV NS5b RNA-
dependent RNA polymerase inhibitors, two of which were
discovered by HTS: diketo acids9 (Figure 1, 1) and meconic
acid (2) derivatives.10 Both classes unfortunately suffer problems
such as chemical instability, irreversible covalent binding to
protein, and poor stability in plasma. The third class, dihy-
droxypyrimidine carboxylic acids (3) and N-alkyl hydroxy-
pyrimidinone carboxylic acids (3a) were designed to have more
showing nanomolar activity in the enzymatic assay (IC50
)
0.085 µM; Table 1). Compound 4a was inactive against HCV
NS5b polymerase at 50 µM. The rat pharmacokinetic profile
of 4a showed modest oral bioavailability (F ) 15%), low plasma
clearance (Clp ) 5 mL/min/kg) and good half-life (t1/2 ) 3 h;
Table 2).
Extensive structure activity relationship studies on the amide
moiety of 4a led to the identification of the para-fluorobenzyl
* To whom correspondence should be addressed. Address: Phone:
+39(0)691093680. Fax: +39(0)691093654. E-mail: Vincenzo_summa@
merck.com.
amide 5 (Table 1) as the optimal fragment, showing an IC50
0.010 µΜ in the enzymatic assay.
)
† Present address: Almirall Pharmaceutical, Barcelona, Spain.
10.1021/jm060854f CCC: $33.50 © 2006 American Chemical Society
Published on Web 10/25/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6647
Table 2. Pharmacokineticsa
e
Fb
nAUCc
Clpd
t1/2
cmpd
species
%
µM‚h‚kg/mg
mL/min/kg
h
4a
5
rat
rat
rat
dog
rat
dog
rhesus
rat
dog
rat
15
29
59
93
28
100
61
27
90
56
69
73
0.40
1.3
1.8
19.9
0.8
21.5
1.9
0.13
20.0
2.3
13.6
2.4
5
11
14
0.5
16
1.9
15
44
2.0
9
3
1.3
1.7
6.8
2
4.8
6
0.45
6
1.1
7.3
2.0
10
10
13
13
13
14
14
18
18
18
Figure 2. Strategies applied to reduce the protein binding.
dog
rhesus
2.2
14
(CIC95 ) 0.3 µM 10% FBS), showing virtually no shift with
respect to enzymatic activity (IC50 ) 0.2 µM). Unfortunately,
a great potency shift was observed in the cell-based assay in
the presence of 50% NHS (CIC95 > 10 µM). The PK profile
was improved with respect to 4 and 5, with very good oral
bioavailability (F ) 59% rat, 93% dog) and low plasma
clearance (Clp ) 14 mL/min/kg rat, 0.5 mL/min/kg, dog, Table
2). The high protein binding in human plasma (hPPB ) 99.9%)
explained the large shift in potency between low and high serum
conditions in the cell-based assays. A reduction of hPPB was,
therefore, mandatory to gain activity in the more physiological
conditions. Given the well-established correlation between
binding of lipophilic acids to albumin and log P, the strategy
focused on reducing the lipophilicity of 10 in either acyclic or
cyclized analogues (Figure 2.)
The first approach (Figure 2, A) was to remove the phenyl
ring, generating compounds as 11, where a simple methyl group
replaces the phenyl moiety. This modification was beneficial
in reducing the plasma protein binding (hPPB ) 92.5%), and
the compound showed only a 4-fold shift between low and high
serum conditions (CIC95 ) 0.125 µM 10% FBS, 0.50 µM 50%
NHS). The methylene analogue (12) and the gem dimethyl
derivative (13) were synthesized to remove the chiral center of
11 (Table 3). No improvement in activity was achieved with
12. However, 13 is an extremely interesting compound, being
equipotent on the enzyme and in the cell-based assay in low
serum conditions with a marginal shift in high serum concentra-
a See Supporting Information for PK details. b F ) oral bioavailability.
c nAUC ) dose normalized area under the curve. d Clp ) plasma clearance.
e t1/2 ) plasma half-life.
The rat pharmacokinetic profile of 5 showed improved oral
bioavailability (F ) 39%) and similar plasma clearance (Clp
) 11 mL/min/kg) compared to that of 4a. Unfortunately, in
spite of the very good potency in the enzymatic assay, these
molecules did not show either activity or toxicity in the cellular
assay (Spread) under two serum conditions [10% fetal bovine
serum (FBS) and 50% normal human serum (NHS)]. This
finding can probably be explained by a combination of different
factors such as poor solubility, poor cell permeability, and high
protein binding. To improve these parameters, effort was
devoted to the replacement of the thiophene at the 2-position
of the dihydroxypyrimidine carboxamide. Removal of the
thiophene moiety gave the unsubstituted compound 6 (Table
3), which retained nanomolar activity in the enzymatic assay
(IC50 ) 0.06 µM), thus indicating that the substituent at the
2-position is not involved in a strong interaction with the
enzyme. Therefore, 6 represents the minimal scaffold of this
new class of HIV integrase inhibitors. Replacement of thiophene
with methyl 7, phenyl 8, and benzyl 9 generated compounds
having the same level of potency as the lead 4a (IC50 ) 0.05-
0.07 µM). In spite of the similar potency on the enzyme, 9
showed an evident gain of activity in the cell-based assay (CIC95
) 5.8 µM in 10% FBS). To optimize further this compound, it
was decided to introduce a basic amine, which could improve
solubility and which potentially could also balance the negative
charge of the deprotonated hydroxyl group at the 5 position of
the scaffold. This strategy led to 10 (racemic), one of the first
compounds with submicromolar activity in the spread assay
tions due to the low hPPB ) 88.7% (IC50 ) 0.05 µM; CIC95
0.05 µM 10% FBS; 0.11 µM 50% NHS).
)
The oral bioavailability of 13 was good in rat (28%) and
excellent both in dog (100%) and in rhesus (61%). Plasma
clearance was low for dog and medium in rat and rhesus, with
good plasma half-life (Clp ) 16, 2, and 15 mL/min/kg; t1/2
)
Table 3. Optimizing the Dihydroxypyrimidines
a
b
b
IC50
CIC95
CIC95
cmpd
R1
thiophene
H
Me
Ph
CH2Ph
CH(Ph)NMe2
CH(Me)NMe2
CH2NMe2
C(Me)2NMe2
2-NMe pip
2-NMe morpholine
R2
(µM)
(10% FBS/µM)
(50% NHS/µM)
5
6
7
8
9
10
11
12
13
14
15
CH24F-Ph
CH24F-Ph
CH24F-Ph
CH2Ph
CH24F-Ph
CH24F-Ph
CH24F-Ph
CH24F-Ph
CH24F-Ph
CH24F-Ph
CH24F-Ph
0.01
0.06
0.06
0.07
0.05
0.20
0.08
0.20
0.05
0.20
>10
>10
10
>10
>10
>10
>10
>10
>10
0.50
>1.0
0.11
0.40
0.24
>10
5.80
0.30
0.125
>1.0
0.05
0.15
0.03
a For footnote details, see Table 1. b For footnote details, see Table 1.

6648 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Table 4. N-Methylpyrimidones
Letters
Scheme 1. Synthesis of Compounds 4-10a
b
b
CIC95
CIC95
cmpd
X
IC50a (µM) (10% FBS/µM) (50% NHS/µM)
16
17
18
19
CH2(+/-)
O (+/-)
O (+)
0.21
0.06
0.020
0.025
0.840
0.065
0.040
0.09
1.10
0.100
0.065
0.19
O (-)
a For footnote details, see Table 1. b For footnote details, see Table 1.
a Reagents and conditions: (a) R2NH2 (3 equiv), MeOH, DMF, or NMP,
90 °C; (b) PhCOCl (5 equiv), pyr, rt; (c) NBS (1.1 equiv), (PhCO)2O2 (0.1
equiv), CCl4, reflux; (d) 2 M Me2NH in THF; (e) 4-F-C6H4CH2NH2 (3
equiv), DMF, 90 °C.
2, 5, and 6 h for rat dog and rhesus, respectively). Furthermore,
13 was found to be inactive against HIV RT, HCV NS5B
polymerase and human R, â, and γ polymerases (IC50 > 10µM).
The second approach (Figure 2, B) to reduce the lipophilicity
and increase solubility of 10 was the simultaneous deletion of
the phenyl moiety and cyclization of the amine to form N-methyl
piperidine ring derivative 14 (Table 3). This again reduced the
binding to human plasma protein (hPPB, 94.8%), and conse-
quently, the shift between the low and the high serum conditions
was only 3-fold in the spread assays (CIC95 ) 0.14 µM 10%
FBS, 0.40 µM 50% NHS). The PK profile was again very good,
especially in dog, displaying high oral bioavailability (90%) and
low plasma clearance (2 mL/min/kg). Replacing the piperidine
moiety of 14 with morpholine in 15 led to a further improvement
in potency in the spread assay (CIC95 ) 0.03 µM 10% FBS,
0.24 µM 50% NHS). Capitalizing on the experience gained in
the HCV polymerase inhibitor program, where the N-methyl-
pyrimidinone 3a showed similar or improved activity and
pharmacokinetic profile with respect to the dihydroxypyrimidine,
the N-methyl pyrimidinones 16 and 17 (Table 4) were prepared
as racemates.
Scheme 2. Synthesis of Compounds 11-17a
The human protein binding was drastically reduced for both
compounds (hPPB 16 ) 48%, 17 ) 70%), and in the cell-based
assays, 16 had reduced potency (CIC95 ) 0.840 µM 10% FBS,
1.1 µM 50% NHS). However, 17 proved to be very active in
the cell-based assays (CIC95 ) 0.065 µM 10% FBS, 0.100 µM
50% NHS). Therefore, 17 was resolved into the single enanti-
omers 18 and 19. The intrinsic activity on the enzyme was the
same (IC50 ) 0.020 and 0.025 µM for 18 and 19), further
confirmation that the moiety at the 2-position is not involved
in enzyme recognition. In the cell-based assay, 18 and 19
showed a 2-fold shift under the two serum conditions (18, CIC95
) 0.04 µM 10% FBS, 0.065 µM 50% NHS; 19, CIC95 ) 0.09
µM 10% FBS, 0.19 µM 50% NHS). Along with its very high
potency, 18 had excellent pharmacokinetics in all preclinical
species. It showed high oral bioavailability and exposure after
oral dosing in rat dog and rhesus (F ) 56, 69, and 73%; nAUC
) 2.3, 13.6, and 2.4 µM‚h‚kg/mg for rat, dog, and rhesus,
respectively) with low plasma clearance in all species [Clp )
9 (rat), 2.2 (dog), and 14 (rhesus) mL/min/kg] and moderate to
very good plasma half-lives (t1/2 ) 1.1 (rat), 7.3 (dog), and 2.0
(rhesus) h). Furthermore, 18 was inactive against HIV RT, HCV
NS5B polymerase, and human R, â, and γ polymerases (IC50
> 10 µM).
a Reagents and conditions: (a) 4-F-C6H4CH2NH2 (3 equiv), MeOH, 90
°C; (b) CH2Cl2/TFA (8:2), rt or H2, Pd/C (10%), MeOH, rt; (c) 37% HCHO
(5 equiv), NaCNBH3 (1.5 equiv), AcOH (10 equiv), MeOH, rt; or 37%
HCHO (6 equiv), NaCNBH3 (5 equiv), NaOAc (6 equiv), MeOH, rt; (d)
(PhCO)2O (2 equiv), pyr; (e) Me2SO4 (1.5 equiv), LiH (1.2 equiv) or Cs2CO3
(1.5 equiv), dioxane, 60 °C; (f) chiral HPLC.
sized from the appropriately substituted amidoximes, either
commercially available or prepared from nitriles, following the
literature procedure.15 Compound 10 was prepared from 20e
via bis-benzoylation, followed by bromination under standard
conditions. Treatment of the resulting benzyl bromide with
dimethylamine in THF gave displacement of the bromine with
concomitant deprotection of the 6-hydroxyl group. Subsequent
coupling with p-fluorobenzylamine in DMF gave compound 10.
Compounds 11-15 were prepared from the corresponding
2-substituted pyrimidine methyl esters 20f-l, with the nitrogen
of the 2-substitutent protected as Boc or Cbz (Scheme 2).
Synthesis of the benzylic amide was performed as previously
described and followed by removal of the protecting group to
give intermediates 21f-l. Alkylation of the nitrogen was
achieved via reductive amination. A similar reaction sequence
was applied to the N-methylpyrimidones 22i-l prepared by
Schemes 1 and 2 depict the general procedures for the
synthesis of compounds. 2-Substituted-5,6-dihydroxypyrimidine-
4-carboxamides 4-10 were accessed by treatment of the
corresponding methyl esters with the desired amine in solvents
like methanol, DMF, or NMP (Scheme 1). 2-Substituted methyl-
5,6-dihydroxypyrimidine-4-carboxylates 20a-e were synthe-

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6649
Schleif, W. A.; Gabryelski, L. J.; Leonard, Y. M.; Lynch, J. J., Jr.;
Michelson, S. R.; Young, S. D. Design and synthesis of 8-hydroxy-
[1,6]naphthyridines as novel inhibitors of HIV-1 integrase in vitro
and in infected cells. J. Med. Chem. 2003, 46, 453-456.
(6) 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. A naph-
thyridine carboxamide provides evidence for discordant resistance
between mechanistically identical inhibitors of HIV-1 integrase. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 11233-11238.
N-methylation of the monobenzoylated methyl ester, yielding
compounds 16 and 17. Resolution of 17 by chiral HPLC gave
the single enantiomers 18 and 19.
In summary, the knowledge of the common active site
architecture of the HCV polymerase and HIV integrase and the
fact that both enzymes use Mg++ ions in their catalytic
machinery led us to test designed HCV polymerase inhibitors
against HIV integrase. The screening led to the identification
of a new potent and selective class of HIV integrase strand
transfer inhibitors represented by 5. Optimization of potency,
physical chemical properties, and pharmacokinetic profiles in
preclinical species culminated in the identification of 13 and
18. Compounds 13 and 18 showed CIC95 equal or superior to
the HIV drugs on the market and very good pharmacokinetic
profiles in preclinical species. Further work in these promising
series will be reported in due course.
(7) Sato, M.; Motomura, T.; Aramaki, H.; Matsuda, T.; Yamashita, M.;
Ito, Y.; Kawakami, H.; Matsuzaki, Y.; Watanabe, W.; Yamataka,
K.; Ikeda, S.; Kodama, E.; Matsuoka, M.; Shinkai, H. Novel HIV-1
integrase inhibitors derived from quinolone antibiotics. J. Med. Chem.
2006, 49, 1506-1508.
(8) Jin, H.; Cai, R. Z.; Schacherer, L.; Jabri, S.; Tsiang, M.; Fardis, M.;
Chen, X.; Chen, J. M.; Kim, C. U. Design, synthesis, and SAR studies
of novel and highly active tri-cyclic HIV integrase inhibitors. Bioorg.
Med. Chem. Lett. 2006, 16, 3989-3992.
(9) Summa, V.; Petrocchi, A.; Pace, P.; Matassa, V. G.; De Francesco,
R.; Altamura, S.; Tomei, L.; Koch, U.; Neuner, P. Discovery of R,γ-
diketo acids as potent selective and reversible inhibitors of hepatitis
C virus NS5b RNA-dependent RNA polymerase. J. Med. Chem.
2004, 47, 14-17.
(10) Pace, P.; Nizi, E.; Pacini, B.; Pesci, S.; Matassa, V.; De Francesco,
R.; Altamura, S.; Summa, V. The monoethyl ester of meconic acid
is an active site inhibitor of HCV NS5B RNA-dependent RNA
polymerase. Bioorg. Med. Chem. Lett. 2004, 14, 3257-3261.
(11) Summa, V.; Petrocchi, A.; Matassa, V. G.; Taliani, M.; Laufer, R.;
De Francesco, R.; Altamura, S.; Pace, P. HCV NS5b RNA-dependent
RNA polymerase inhibitors: from R,γ-diketoacids to 4,5-dihydroxy-
pyrimidine- or 3-methyl-5-hydroxypyrimidinonecarboxylic acids.
Design and synthesis. J. Med. Chem. 2004, 47, 5336-5339.
(12) Koch, U.; Attenni, B.; Malancona, S.; Colarusso, S.; Conte, I.; Di
Filippo, M.; Harper, S.; Pacini, B.; Giomini, C.; Thomas, S.; Incitti,
I.; Tomei, L.; De Francesco, R.; Altamura, S.; Matassa, V. G.; Narjes,
F. 2-(2-Thienyl)-5,6-dihydroxy-4-carboxypyrimidines as inhibitors
of the hepatitis C virus NS5B polymerase: discovery, SAR, modeling,
and mutagenesis. J. Med. Chem. 2006, 49, 1693-1705.
Acknowledgment. The authors thank Kara A. Stillmock and
Peter Felock for the IC50 values of the HIV integrase strand
transfer assay; William A. Schleif, Greg Moyer, and Lory
Gabryelski for the CIC95 values for the HIV spread assay; Fabio
Bonelli, Francesca Naimo, and Anna Alfieri for the determi-
nation of the physical chemical properties of the compounds
and HRMS; and Daria J. Hazuda and Steven D. Young for
fruitful discussion. This work was supported in part by a grant
from the MIUR.
Supporting Information Available: Synthetic procedures,
NMR, MS, and PK procedures are reported in detail. This material
is available free of charge via the Internet at http://pubs.acs.org
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JM060854F