Dihydroxypyrimidine-4-carboxamides as novel potent and selective HIV integrase inhibitors

Article abstract of DOI:10.1021/jm070027u

Human immunodeficiency virus type-1 (HIV-1) integrase, one of the three constitutive viral enzymes required for replication, is a rational target for chemotherapeutic intervention in the treatment of AIDS that has also recently been confirmed in the clinical setting. We report here on the design and synthesis of N-benzyl-5,6-dihydroxypyrimidine-4-carboxamides as a class of agents which exhibits potent inhibition of the HIV-integrase-catalyzed strand transfer process. In the current study, structural modifications on these molecules were made in order to examine effects on HIV-integrase inhibitory potencies. One of the most interesting compounds for this series is 2-[1-(dimethylamino)-1-methylethyl]-N-(4-fluorobenzyl)-5,6-dihydroxypyrimidine- 4-carboxamide 38, with a CIC₉₅ of 78 nM in the cell-based assay in the presence of serum proteins. The compound has favorable pharmacokinetic properties in preclinical species (rats, dogs, and monkeys) and shows no liabilities in several counterscreening assays, highlighting its potential as a clinically useful antiviral agent.

Full text of DOI:10.1021/jm070027u

J. Med. Chem. 2007, 50, 2225-2239
2225
Dihydroxypyrimidine-4-carboxamides as Novel Potent and Selective HIV Integrase Inhibitors
Paola Pace,*,† M. Emilia Di Francesco,† Cristina Gardelli,† Steven Harper,† Ester Muraglia,† Emanuela Nizi,† Federica Orvieto,†
Alessia Petrocchi,† Marco Poma,†,§ Michael Rowley,† Rita Scarpelli,† Ralph Laufer,† Odalys Gonzalez Paz,† Edith Monteagudo,†
Fabio Bonelli,† Daria Hazuda,‡ Kara A. Stillmock,‡ and Vincenzo Summa†
Istituto Di Ricerche Di Biologia Molecolare, P. Angeletti S.p.A. (Merck Research Laboratories, Rome), Via Pontina Km 30,600,
00040 Pomezia, Italy, and Department of AntiViral Research, Merck Research Laboratories, West Point, PennsylVania 19486
ReceiVed January 9, 2007
Human immunodeficiency virus type-1 (HIV-1) integrase, one of the three constitutive viral enzymes required
for replication, is a rational target for chemotherapeutic intervention in the treatment of AIDS that has also
recently been confirmed in the clinical setting. We report here on the design and synthesis of N-benzyl-
5,6-dihydroxypyrimidine-4-carboxamides as a class of agents which exhibits potent inhibition of the HIV-
integrase-catalyzed strand transfer process. In the current study, structural modifications on these molecules
were made in order to examine effects on HIV-integrase inhibitory potencies. One of the most interesting
compounds for this series is 2-[1-(dimethylamino)-1-methylethyl]-N-(4-fluorobenzyl)-5,6-dihydroxypyri-
midine-4-carboxamide 38, with a CIC95 of 78 nM in the cell-based assay in the presence of serum proteins.
The compound has favorable pharmacokinetic properties in preclinical species (rats, dogs, and monkeys)
and shows no liabilities in several counterscreening assays, highlighting its potential as a clinically useful
antiviral agent.
Introduction
structurally and functionally distinct domains, all required for
each step of the integration process. Extensive mutagenesis
studies mapped the catalytic site to the core domain (residues
50-212), which contains the catalytic residues Asp-64, Asp-
116, and Glu-152.5 These residues are highly conserved in the
integrase superfamily and polynucleotide transferases. As with
other polynucleotidyl transferases, two metals ions (Mg2+) are
present in the active site and are required for both 3′ processing
and strand transfer and for the assembly of integrase onto
specific viral donor DNA to form a complex competent to carry
out either function.6
Human immunodeficiency virus type 1 (HIV-1) is the
causative agent of acquired immunodeficiency syndrome (AIDS),
which is one of the world’s most serious health problems. Since
1981, when it was first reported in a small number of patients,
AIDS has now become a major worldwide epidemic with more
than 38 million people infected worldwide. FDA-approved
therapies target primarily two viral enzymes, HIV reverse
transcriptase1 and HIV protease,2 to block the viral life cycle.
Multidrug cocktails consisting of a protease inhibitor (PI) or a
non-nucleoside reverse transcriptase inhibitor (NNRTI) in
combination with two nucleoside reverse transcriptase inhibitors
(HAART, highly active antiretroviral therapy) is now the
standard for treatment. In 2003 Enfuvirtide was also approved
as the first member of a new family of antiretroviral drugs that
inhibits HIV-1 replication, preventing viral entry into target
cells.3 Although current therapies are effective in reducing viral
load and morbidity and mortality, the long-lived nature of the
infection, drug toxicity, and the emergence of multidrug resistant
phenotypes underscore the need for innovative antiretroviral
strategies targeting alternative steps essential for the viral
replication cycle.
HIV-1 integrase catalyzes the insertion of the viral DNA into
the genome of the host cell,4 which is an essential step in the
viral replication cycle, and thus HIV-1 integrase is an attractive
target for novel chemotherapy. Integration is a multistep process
consisting of three biochemical steps: (i) assembly of a complex
with specific DNA sequences at the end of HIV-1 long terminal
repeat (LTR) regions, (ii) endonucleolytic processing of the viral
DNA to remove the terminal dinucleotide from each 3′ end,
and after nuclear entry, (iii) strand transfer, in which the viral
DNA 3′ ends are covalently linked to the cellular DNA. HIV-1
integrase is a 32-kDa enzyme, which is composed of three
4-Aryl-2,4-diketobutanoic acids (DKAs), the first class of
truly HIV-1 strand transfer inhibitors, followed by structurally
related 1,3-diaryl-1,3-propanediones, provided the first proof
of concept for HIV-1 integrase inhibitors as antiviral agents7 in
the cell-based assay. Because of their structure, DKAs could
coordinate one or two metal ions in the HIV integrase active
site, and mutations that engender resistance to these prototype
inhibitors map to the integrase active site proximal to the
residues that coordinate the divalent metals (Asp-64, Asp-116,
and Glu-152). Subsequently, our group reported a number of
diketoacids as potent inhibitors of NS5b HCV RNA-dependent
RNA polymerase.8 In this enzyme, the residues responsible for
the nucleotidyl transfer reaction are found in the palm domain,
which contains an Asp-(Xaa)4-Asp motif and the signature Gly-
Asp-Asp motif, common to the reverse transcriptase and other
viral polymerases. The first aspartate of each motif provides
the carboxylate side chains required for coordinating the two
metal ions involved in catalysis.9 A hypothesis to explain
inhibition by this class of compounds is that the diketoacid
fragment inhibits the RNA-dependent RNA polymerase activity
through an interaction with the catalytic metal-ions found in
the enzyme active site, as for pyrophosphate analogues such as
Foscarnet and phosphonoacetic acid. A similar metal-chelating
moiety was also found in another class of NS5b RdRP inhibitors,
namely the meconic acid derivatives.10 Both classes of com-
pounds were further developed, and a more complete under-
standing of the necessary pharmacophore for the inhibition led
* To whom correspondence should be addressed. Phone: +39 0691093313.
Fax: +39 06 91093654. E-mail: paola_pace@merck.com.
† Merck Research Laboratories, Rome.
‡ Merck Research Laboratories, West Point.
§ Present address: Industria Farmaceutica Serono, Italy.
10.1021/jm070027u CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/12/2007

2226 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Pace et al.
Table 1. Effect of Different Carboxamides on the Inhibition of HIV-1
Integrase Catalytic Strand Transfer Activity
inhibition of strand
compd
R
transfer IC50 (µM)a
1
3
4
5
6
7
8
9
CH2Ph
0.08
1.00
50.0
50.0
0.50
5.00
0.02
0.01
Ph
H
Figure 1. HIV integrase inhibitors from the HCV polymerase program.
c
CH2 hexyl
(S)CH(CH3)Ph
(R)CH(CH3)Ph
CH2 CH2Ph
to the discovery of dihydroxypyrimidine-4-carboxylic acid as
a suitable replacement of the chelating motif.11 Given the
topological resemblance between HCV polymerase and HIV
integrase, both enzymes using a carboxylate-metal ion catalytic
mechanism, the dihydroxypyrimidines derivatives and the
prototypical scaffolds from the HCV program were screened
as potential HIV integrase inhibitors. While the dihydroxypy-
rimidine carboxylic acid derivatives were almost inactive in the
HIV integrase-mediated strand transfer assay, dihydroxypyri-
midine-4-carboxamides 1 (Figure 1) were discovered as potent
reversible inhibitors of HIV integrase. Extensive structure-
activity relationships led to identification of compounds that
inhibit HIV integrase in vitro at nanomolar concentrations, block
HIV replication in cell culture, and show excellent physico-
chemical and pharmacokinetic properties.
CH2C6H4(4-F)
a Assays were performed with recombinant HIV-1 integrase (0.1 µM)
preassembled on immobilized oligonucleotides. Inhibitors were added after
assembly and washings, and IC50 is the concentration of inhibitor that
reduces HIV integrase activity by 50%. Results are the mean of at least
three independent experiments. SD was always ( 8% of the value. For
details, see ref 12.
Table 2. Effect of 2-Substituent on the Inhibition of HIV-1 Integrase
Catalytic Strand Transfer Activity
Results and Discussion
As part of our initial investigations, a collection of dihy-
droxypyrimidines that had been synthesized as potential HCV
NS5B polymerase inhibitors was screened in the HIV-1 inte-
grase assays. All compounds having free carboxylic acids or
carboxylic acid isosteres were observed to be weakly active,
while the benzyl amide derivative 1 (Figure 1) showed an IC50
of 80 nM in the strand transfer assay and in a single cycle
infectivity assay15 had an IC50 of 5.0 µM in the presence of
10% fetal bovine serum (FBS) and an IC50 of 59 µM in the
presence of 50% normal human serum (NHS). Moreover, the
benzylamide moiety marked a significant departure from the
SAR that had been observed in the NS5b polymerase project,
where the free carboxylic acid moiety is crucial for activity.11
Substitution of the thiophene with a phenyl (2, Figure 1) did
not affect the potency. In pharmacokinetic studies 1 showed
moderate oral bioavailability (15%) and low clearance (5 mL/
min/kg, Table 4). Counterscreening studies showed that 1 did
not inhibit human DNA polymerase R, â, and γ at 10 µM and
was inactive against HIV-RT and HCV polymerase at 50 µM.
These findings indicated 1 as a valid lead for the HIV-1 integrase
project.
To evaluate the dihydroxypyrimidine-benzylic amide scaffold
for development as an HIV-1 integrase inhibitor, a focused
library of more than 200 different amides was initially synthe-
sized and screened. The methodology and the scope of this
chemistry will be discussed in detail elsewhere.16 Table 1
collects results from key compounds evaluated at the beginning
of our investigation, by assessing carboxamide modifications
as an approach to improving the potency of our inhibitors. From
this set of compounds, SAR trends started to emerge. It was
immediately evident that an aromatic ring separated by at least
one sp3 atom was essential for the activity since the anilide 3
(IC50 ) 1 µM), the primary amide 4 (IC50 ) 50 µM) and the
cycloalkyl derivative 5 (IC50 ) 50 µM) lost significant activity.
inhibition of strand
compd
R
transfer IC50 (µM)a
9
10
11
12
13
14
15
16
2-thienyl
H
0.01
0.06
0.06
0.10
0.05
0.02
0.02
0.05
Me
iPr
PhCH2
p-tolyl
2-thiazolyl
2-pyridyl
a For footnote details, see Table 1.
R-Branching on the benzylic amide was also detrimental (6,
IC50 ) 0.5 µM and 7, IC50 ) 5 µM). The phenethyl amide 8
(IC50 ) 0.02 µM) showed slightly improved activity as
compared with the benzyl analogue. The 4-fluoro-substituted
benzyl amide 9 (IC50 ) 0.01 µM) was optimal for enzyme
inhibition. However, despite the increased in vitro potency,
compound 9 showed weak inhibition of the spread of HIV-1
infection in cell culture. The rat pharmacokinetic profile of 9
(Table 4) showed improved oral bioavailability (F ) 29%) and
similar plasma clearance (11 mL/min/kg) compared to that
of 1.
After optimization of the benzylamide moiety, a significant
site for exploring SAR in 9 was the thiophene ring in the
2-position of the pyrimidine core. SAR studies showed that the
thiophene ring of 9 could be removed (10) or replaced by simple
small alkyl groups (11, 12) or benzyl, aryl or heterocycles (13-
16), without loosing significant potency (Table 2). We inter-
preted the small effect that changing the 2-substituent had on
in vitro potency as evidence that this part of the molecule does
not interact strongly with the enzyme, and therefore this
appeared to be the site that would be most likely to tolerate
more dramatic changes influencing the physicochemical proper-
ties of the inhibitors aimed to develop compounds with activity

Dihydroxypyrimidine Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2227
Table 3. 2-Benzyl, 2-Aniline, and 2-Tolyl Derivatives
in the cell based assays. Compound 10 can be considered the
minimal template of this class of inhibitors capable to retain
good inhibitory potency in the enzymatic assay.
Despite the good activity in the enzymatic assay (Quick In),
all these compounds were weakly active in the spread assay.
The poor activity of our dihydroxypyrimidines in the cell based
assay was thought to be due to a suboptimal physical chemical
properties that could affect cell permeability and/or to binding
to intracellular proteins and also plasma protein present in the
cell medium (for example, compounds 9 and 12 are <1%
unbound in human plasma). The nonspecific binding of a drug
to plasma protein is an important determinant of its biological
efficacy since it modulates the availability of the drug to its
intended target. This parameter is particularly relevant in the
HIV field where it is known that compounds with very high
protein plasma protein binding show a marginal activity in the
cell based assay in the presence of human serum. In order to
assess the role of serum protein on drug availability at a
quantitative level, the inhibitor concentration required to inhibit
95% of the spread of HIV-1 infection in MT4 cell culture was
measured in the presence of 10% fetal bovine serum (FBS) and
50% normal human serum (NHS) (spread assays). Both assays
were important for compound selection, since cell-based activity
in the low serum conditions (10% FBS) reflects the ability to
cross cell membranes while the high serum conditions (50%
NHS) provided efficacy in a physiologically more relevant
environment.
To see whether polar or ionizable groups could affect the
physicochemical properties and the protein binding of our
inhibitors, therefore improving cell based activity, compounds
17-23 were synthesized and tested (Table 3). Introduction of
a methoxy group to the 2-benzyl derivative 13 in the benzylic
position resulted in compound 17 with activity in 10% FBS in
the micromolar range (CIC95 ) 2.5 µM), while two methoxy
groups on the aromatic ring had a negative impact (18, CIC95
> 10.0 µM). The installation of a basic group resulted in more
desirable physical properties, improving cell permeability as
demonstrated by compound 19 showing a very low shift between
the in vitro and the cell-based assay. This compound inhibited
the spread of the virus in low serum conditions with CIC95
values of 0.3 µM. The single enantiomers of 19 showed similar
activity in all assays suggesting that side of the molecule is not
strongly recognized from the integrase enzyme (data not shown).
The less basic aniline derivative 20 resulted in a significant loss
in potency in the spread assay with respect to 19. Introduction
of the dimethylamine on the p-tolyl position as in compound
21 also had a negative impact. Amines different from dimethy-
lamine on the 2-benzyl derivative were acceptable but did not
offer any advantage (22 and 23).
a For footnote details, see Table 1. b Spread assay: 95% cell culture
inhibitory concentrations for inhibition of the spread of HIV-1 infection in
cell culture, using HIV-1IIIb and MT-4 T-lymphoid cells, in a medium
containing 10% heat-inactivated fetal bovine serum. Results are the mean
of at least three independent experiments. SD was always <(10% of the
value. For details, see ref 13.
Table 4. Rat and Dog Pharmacokinetic Parameters, Human Plasma
Protein Binding, and Log D
T1/2
Clp
% plasma
compd species F (%)c (h)d (mL/min/kg)e protein binding log D
1
9
19
rata
rata
rata
dog
rata
dog
15
29
59
93
27
90
3
5
11
14
0.5
75
2
ndf
nd
99.0
nd
nd
2.13
1.3
1.73
6.78
0.43
6.0
27
94.4
0.79
a 2.4 mg/kg (n ) 3); vehicle iv DMSO, po 10%DMSO/90%PEG200
.
b 1.2
mg/kg iv, 1.6 mg/kg po (n ) 3); vehicle iv DMSO, po 10% DMSO/45%
H2O/45% PEG200.
c Oral bioavailability. d Terminal phase plasma half-life
Compound 19 was further characterized with in vivo and in
vitro screenings. In pharmacokinetic studies in rats (Table 4),
19 showed good oral bioavailability (F ) 59%) and low
clearance (Clp ) 14 mL/min/kg). The profile was even better
in dog with improved oral bioavailability (F ) 93%) and very
low clearance (Clp ) 0.5 mL/min/kg) and long terminal plasma
half-life (T1/2 ) 6.0 h). Counterscreening studies showed that
the compound did not inhibit cytochrome P450s 2C19, 2C9,
2D6, and 3A4.
following iv administration. e Plasma clearance. f Not determined.
weight of our inhibitors, via removal of the bulky aryl group,
would have a favorable impact on cell based potency in the
presence of 50% normal human serum. It is indeed known that
for lipophilic acids, plasma protein binding correlates to the
water/n-octanol distribution coefficient (log P). Compounds with
low lipophilicity or a basic center usually have lower protein
binding. We therefore decided to try to improve the activity of
19 by removing the phenyl group to reduce lipophilicity, but
maintaining the presence of the beneficial amino group.
A first approach was to construct analogues in which the basic
group was embedded in an aliphatic cycle. As shown in Table
5, the 2-piperidyl derivative 24 exhibited higher potency in the
in vitro enzyme assay compared to the 3- (25) and 4-piperidyl
Despite the promising cell based activity in the presence of
10% FBS, compounds in Table 3 were weakly active when
tested using 50% NHS. Our hypothesis that the 2-position
substituents were not involved in tight, specific interactions with
the enzyme but were rather mobile in nature became particularly
important as we assessed whether decreasing the molecular

2228 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Table 5. 2-Position Cyclic Amine Analogues
Pace et al.
a For footnote details, see Table 1. b For footnote details, see Table 3.
(26) counterparts. However, 24 displayed disappointing cell
based activities, perhaps due to the presence of multiple H-bond
donors. Therefore the NH was methylated (27), giving a very
pleasing 10-fold improvement in activity in cell based assay
under both serum conditions (CIC95 ) 0.14 and 0.4 µM 10%
FBS and 50% NHS, respectively). Alkylation with higher alkyl
groups offered no advantages (28, 29). Compound 27, with a
94.4% human plasma protein binding, apparently shows a better
compromise between plasma protein binding and cell penetra-
tion, thus retaining sub-micromolar cell based activity under
high serum conditions (the resolved enantiomers of 27 both
showed equal activity to the racemate; data not shown). When
the piperidine ring was replaced by a piperazine (30) and a
pyrrolidine (32), the compounds showed cell based activity in
50% normal human serum similar to that of inhibitor 27, and
replacement with a morpholine (31) gave 2-fold improvement
in the same assay. Further characterization and evolution of
2-morpholine derivatives will be discussed in a separate
publication.17 A significant observation in this cyclic amine
series was the increase in enzyme affinity and in cell based
potency through the introduction of an additional methyl in the
benzylic position, as shown by comparison of compounds 30
and 33. On the basis of this finding, the bicyclic achiral
derivative 34, with a quaternary benzylic carbon atom, was
prepared and resulted in one of the most potent compounds in
this series in the cell based anti-HIV assays, where an IC95 of
0.12 µM was measured both in 10% FBS and in 50% NHS.
Polar heterocycles such as pyridine could also be envisaged as
alternatives to the cyclic amines, but the 2-pyridine derivative
16 (Table 2), while retaining enzymatic affinity, was completely
ineffective in the cell based assays. In addition, other less basic
and more electron-rich heterocycles (data not shown) were
suboptimal at C-2. Taken together, these data show that small,
aliphatic tertiary amines and a tetrasubstituted sp3-carbon at C-2
of the dihydroxypyrimidine core are generally preferred.
Compound 27 was evaluated in pharmacokinetic iv/po studies
in rats and dogs. In rats, it was a moderately orally bioavailable
compound, with high clearance and rather short half-life. The
pharmacokinetic profile was much better in dog showing an
outstanding oral bioavailability and very low clearance (Table 4).
A second study was done on smaller acyclic amines in the 2
position (Table 6). A dimethylaminomethyl substituent was
found to exhibit acceptable inhibitory potency (35), while further
separation of the basic amine by means of an ethyl linker as in
compound 36 reduced the activity. Mirroring the behavior
observed in the cyclic amine derivatives, a quaternary sp3-carbon
spacer afforded a significant increase in cell based potency over
a secondary and tertiary analogues (compare 38 vs 35 and 37).
Increasing the size of the N-alkyl substituent (39) reduced the
activity, and, as in the cyclized series, a free NH (40) was also
detrimental to cell based activity. Attempted substitutions of
the gem-dimethyl all had a negative impact, since residues like
trifluoromethylmethyl (41), cyclopropane (42), cyclobutane (43),
cyclopentane (44), cyclohexane (45), and tetrahydropyran (46)
led to significant loss in potency in the cell based assay carried
out in the presence of 50% normal human serum. The binding
of compound 38 to rat, dog, and human plasma was determined
by ultrafiltration method. The unbound fraction of the drug in
plasma was approximately 17% for rats, 10% for dogs, and 11%
for humans. In the cell-based antiviral assay, the addition of
50% normal human serum resulted in less than 1.5 fold reduction
in potency for 38, demonstrating that it is effective in suppress-
ing the spread of HIV-1 infection in cells in the presence of
biologically relevant proteins. Overall, 2-[1-(dimethylamino)-
1-methylethyl]-N-(4-fluorobenzyl)-5,6-dihydroxypyrimidine-4-
carboxamide 38 proved a to be very active compound with

Dihydroxypyrimidine Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2229
increased linearly (r2 ) 0.997 and 0.998 respectively) in the
rat dose proportionality study dosing the compound orally at
doses from 1 to 60 mpk (4 and 15mpk were the medium doses)
and a similar trend was observed in rhesus monkeys increasing
the oral dose from 1 to 10 mpk, confirming high levels of oral
absorption. To assess the intrinsic metabolic stability, the
compound 38 was evaluated in rat, monkey, dog, and human
liver microsomes in the presence of NADPH (no turnover, data
not shown) and UDPGA (see also Supporting Information).
Higher in vitro metabolism was observed in monkey and rat
microsomes, which correlated with the moderate in vivo
clearance, while the compound showed very low turnover in
dog liver microsomes, as mirrored by the low in vivo clearance.
Low human turnover in vitro, in combination with other data
not reported here, predicts human pharmacokinetics to be similar
to dog, where the compound had 24 h trough blood levels, with
a 2 mpk po dose, at or slightly below the CIC95 (C24h ) 67
nM). The major metabolite in urine (accounting for >90% of
the dose on the basis of 19F analysis) was characterized using
1H, 13C and 1H-13C correlation spectroscopy. These data support
the formation of the glucuronide conjugate of the hydroxyl group
in position 5 of the pyrimidine ring. The full assignment of all
1H and 13C signals ascribed to this â-glucuronide was achieved
using the crude sample of urine after solid-phase extraction.
Table 6. 2-Position Acyclic Amine Analogues
3
The same profile was found using the H material.
Further profiling of compound 38 revealed that it had no
CYP450 liabilities (1A2, 2C19, 2C9, 2D6, and 3A4 IC50 > 20
µM), did not inhibit human DNA polymerases R, â, and γ (IC50
> 100 µM) and did not bind the human IKr potassium channel
(IC50 > 130 µM) (18). The compound did not react with
glutathione after incubation at 37 °C for 24 h and the [3H]-38
after administrations in rat (25 mpk, po) did not show measur-
able covalent binding to liver, kidney, and plasma.
Conclusions
The dihydroxypyrimidine-4-carboxamides were discovered
as a new, relevant class for the development of new AIDS
therapeutics. In this report we have summarized our efforts
toward the optimization of the initial lead N-benzyl-5,6-
dihydroxy-2-(2-thienyl)pyrimidine-4-carboxamide 1. On the
hypothesis that its physical properties were the basis for its lack
of efficacy in the cell based assays, initial SAR allowed the
identification of the position where changes affecting the
physical properties could be made without compromising the
intrinsic in vitro efficacy. Introduction and optimization of amino
substituents in the 2-position of the dihydroxypyrimidine core
resulted in analogues effective in suppressing the spread of
HIV-1 infection in cells. While early analogues suffered high
protein binding, reduction of lipophilicity addressed this prob-
lem, leading to the discovery of diverse series effective for
blockade of the spread of the virus in the presence of serum
proteins. SAR optimization provided compound 38 having the
best overall potencies in the enzymatic and cellular assays. This
compound showed excellent PK properties in rat, dog, and
rhesus monkeys, was clean in an extensive panel of counter-
screening assays, and therefore may have potential for clinical
development as anti-HIV drug with a novel mechanism of
action.
a For footnote details, see Table 1. b For footnote details, see Table 3.
Table 7. Rat, Dog, and Rhesus Monkey Pharmacokinetic Parameters
for Compound 38
dosea
d
Fb
(%)
T1/2
Clp
nAUCe
(µM·h·kg/mg)
species
po
iv
(h)c
(mL/min/kg)
ratf
3
2
1
3
1
1
28
100
61
2.1
4.8
1.9
16
1.9
15
2.4
21.5
1.8
dogg
Rhesush
a mg/kg body weight; n ) 3. b Oral bioavailability. c Terminal phase
plasma half-life following iv administration. d Plasma clearance. e Area
under the curve following po administration. f Vehicle iv DMSO, po PEG200
.
g Vehicle iv 0.9% NaCl, po 1% methylcellulose. h Vehicle iv DMSO, po
0.5% methylcellulose.
virtual no shift in potency from the enzymatic assay (IC50
)
50 nM) to the spread cell based assay in the two serum
conditions [CIC95 ) 60 nM (10% FBS) and 78 nM (50% NHS)].
This agent proved to be one of the most potent compounds we
found in the cell based anti HIV assay.
The pharmacokinetic profile of 38 was determined in Spra-
gue-Dawley rats, beagle dogs and rhesus monkeys. The results
of these experiments are summarized in Table 7. In all three
species, the compound had prolonged plasma half-lives, moder-
ate to low clearance and good to excellent oral bioavailability
(from 28% in rats to 100% in dogs). AUC and Cmax were
Biology. Compounds were routinely assessed for activity
against the purified HIV-1 integrase enzyme.6 Integrase-
mediated strand transfer activity was determined as described
in ref 12. Compounds were also tested in HIV-1 replication
assays, performed in MT-4 human T-lymphoid cells as described
in ref 13. Cells were infected en masse at low multiplicity (0.01).

2230 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Pace et al.
Scheme 1. General Scheme for the Synthesis of
Scheme 3. Synthesis of Compound 21a
Pyrimidine-4-carboxamidea
a Reagents and conditions: (a) i: PhCOCl, pyridine, THF; ii: N-
bromosuccinimide, (PhCO)2O2, CCl4, 90 °C; (b) Me2NH, THF, rt; (c) 4-F-
C6H4-CH2NH2, DMF, 90 °C.
a Reagents and conditions: (a) H2NOH‚HCl, KOH, MeOH; (b) DMAD,
CHCl3, 60 °C; (c) xylene or methanol, reflux; (d) R2NH2, methanol, NMP
or DMF, 80-90 °C.
improve the solubility of the compounds and the workup from
organic solvents. Removal of the protecting groups and reaction
with p-fluorobenzylamine afforded intermediates 58. The basic
nitrogen was alkylated via reductive amination, leading to
compounds 27-29, 31, 32, 34. Piperazine derivatives 30, 33
were accessed via sequential deprotection and alkylation steps
of the two piperazine nitrogens of 59, followed by formation
of the p-fluorobenzylic amides as usual (Scheme 4).
Scheme 2. Synthesis of Compounds 19, 22, 23a
The synthesis of compounds with an acyclic amine in the
2-position is summarized in Scheme 5. Heating of the pyrim-
idines 61 with p-fluorobenzylamine produced compounds 62.
Deprotection using standard conditions followed by reductive
amination with formaldehyde yielded compounds 35, 37, 38,
41-46. In the preparation of the monomethylated derivative
40, we were not able to stop the reductive amination of 63 after
the introduction of the first methyl. Therefore, a three-step
protocol based on reductive amination with 1 equiv of benzal-
dehyde, followed by a second reductive amination with form-
aldehyde and final removal of the benzyl group via catalytic
hydrogenation, yielded compound 40. Second reductive ami-
nation with acetaldehyde afforded compound 39. Compound
36 was obtained from 65 via synthesis of the amide, removal
of the Boc, and reductive amination.
a Reagents and conditions: (a) i: PhCOCl, pyridine, THF; ii: N-
bromosuccinimide, (PhCO)2O2, CCl4, 90 °C; (b) R1R2NH, THF, rt; (c) 4-F-
C6H4-CH2NH2, DMF, 90 °C.
HIV-1 replication was assessed by p24 core antigen ELISA.
Control cultures in the absence of inhibitor were fully infected
at 4 days.
Chemistry. For the synthesis of 2-substituted methyl 5,6-
dihydroxypyrimidine-4-carboxylates, the method of Culbertson
was used14 (Scheme 1). Therefore, amidoximes 48 were
prepared from nitriles 47, either commercially available or
prepared as described in the Experimental Section, and reacted
with dimethyl acetylenedicarboxylate (DMAD). The corre-
sponding adducts 49 were not isolated, but directly cyclized in
refluxing methanol or xylene to yield the desired pyrimidine
methyl esters 50. Subsequent reaction with the appropriate amine
in methanol, N-methylpyrrolidone, or N,N-dimethylformamide
gave the required amides. Compounds 1-16, 17, 18, 20, and
24-26 (in this case, an additional deprotection step was
required) were obtained directly by this route. Schemes 2 and
3 illustrate the chemistry used for the investigation of the
2-substituent SAR in compounds 19 and 21-23. Bis-benzoy-
lation of 51, followed by bromination under standard conditions,
yielded the benzylic bromide derivative 52. Displacement of
the bromine with an amine went smoothly, with concomitant
deprotection of the 6-hydroxyl group, affording 53. Subsequent
coupling with the appropriate benzylic amine gave compounds
19, 22, and 23. Compound 21 was accessed by analogous
chemistry, starting from methyl 5,6-dihydroxy-2-(4-methylphe-
nyl)pyrimidine-4-carboxylate 54 (Scheme 3) and simultaneous
loss of the remaining COPh through amide formation.
Experimental Section
Solvents and reagents were obtained from commercial suppliers
and were used without further purification. Flash chromatography
purifications were performed on Merck silica gel (200-400 mesh)
as the stationary phase or were conducted using prepacked cartridges
on a Biotage system, eluting with petroleum ether/ethyl acetate
mixtures. HPLC-MS analyses were performed on a Waters Alliance
2795 apparatus, equipped with a diode array and a ZQ mass
spectrometer, using an X-Terra C18 column (5 µm, 4.6 × 50 mm).
The solvent system was acetonitrile-0.1% HCOOH/water-0.1%
HCOOH, gradient 10-90% over 6 min with a flow rate of 1 mL/
min. Purity of final compounds was more than 99% by area.
Preparative reversed-phase high-performance liquid chromatography
(RP-HPLC) was performed on a Shimadzu 10AV-VP operating
with a flow rate of 15 mL/min and incorporating a diode array
detector SPD10AV-VP. The stationary phases used were Waters
Symmetry C18 5 µm 19 × 100 mm, C18 7 µm 19 × 150 mm, and
C18 7 µm 19 × 300 mm. The mobile phase comprised a linear
gradient of binary mixtures of acetonitrile (containing 0.1% TFA)
and H2O (containing 0.1% TFA). Nuclear magnetic resonance
spectra 1H NMR recorded at 400 or 300 MHz, 13C NMR recorded
at 100 or 75 MHz) were obtained on Bruker AMX spectrometers
and are referenced in ppm relative to TMS. Unless indicated, spectra
were acquired at 300 K. Low-resolution mass spectra (m/z) were
recorded on a Perkin-Elmer API 100 (electrospray ionization) mass
spectrometer. High-resolution mass measurements were carried out
by electrospray ionization performed on a TSQ Quantum Ultra AM
For analogues containing a cyclic amine in the 2-position of
the dihydroxypyrimidine, synthesis of the dihydroxypyrimidine
methyl ester, with the nitrogen protected as Cbz or Boc, was
accomplished as usual. In some cases, an additional protection
of the 5-hydroxy group as its benzoate ester was required to

Dihydroxypyrimidine Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2231
Scheme 4. Synthesis of Compounds 27-34a
a Reagents and conditions: (a) i: H2, Pd/C, MeOH or CF3COOH/CH2Cl2; ii: 4-F-C6H4CH2NH2,MeOH, NMP or DMF, 80-90 °C; (b) RCHO, NaCNBH3,
CH3COONa (or CH3COOH, Et3N), MeOH; (c) i: H2, Pd/C, MeOH; ii: HCHO, NaCNBH3, CH3COONa, MeOH; (d) i: CF3COOH/CH2Cl2 (9:1); ii: HCHO,
NaCNBH3, CH3COONa, CH3OH; iii: 4-F-C6H4CH2NH2, MeOH or NMP, 80-90 °C.
Scheme 5. Synthesis of Compounds 35-46a
a Reagents and conditions: (a) 4-F-C6H4CH2NH2, CH3OH, 80 °C; (b) H2, Pd/C, CH3OH or CF3COOH/CH2Cl2; (c) HCHO, NaCNBH3, CH3COOH, Et3N,
CH3OH; (d) i: PhCHO, NaCNBH3, CH3COOH, Et3N, CH3OH; ii: HCHO, NaCNBH3, CH3COOH, Et3N, CH3OH; (e) H2, Pd/C, CH3OH; (f) CH3CHO,
NaCNBH3, CH3COOH, Et3N, CH3OH; (g) i: 4-F-C6H4CH2NH2, CH3OH, 80 °C; ii: CF3COOH/CH2Cl2; iii: HCHO, NaCNBH3, CH3COOH, Et3N, CH3OH.
operating in enhanced mass resolution mode. The accurate mass
measurements were carried out on a chromatographic time scale
by means of flow injection of 5 µL of each working solution (20
µg/mL concentration) in acetonitrile on a Waters C18 Sunfire (20
× 2.1 mm, 5 µm) column using an isocratic elution (solvent A:
solvent B, 50:50) of solvent A: water + 0.1% formic acid and
solvent B: acetonitrile + 0.1% formic acid at a flow rate of 1000
µL/min.
The following compounds were prepared according to literature
procedures: 1,3-thiazole-2-carbonitrile,19 1-[(benzyloxy)carbonyl]-
4-(tert-butoxycarbonyl)piperazine-2-carboxylic acid,20 7-benzyl 1-
tert-butyl 7-azabicyclo[2.1.1]heptane-1,7-dicarboxylate,21 2-amino-
2-methylpropanenitrile,22 tert-butyl-3-cyanopiperidine-1-carboxy-
late,23 tert-butyl-4-cyanopiperidine-1-carboxylate,24 tert-butyl-
2-cyanopyrrolidine-1-carboxylate,25 tert-butyl-(1-cyanoethyl)car-
bamate.26
bertson.14 In a subsequent step, the corresponding 4-carboxamides
were obtained by heating the 2-substituted methyl 5,6-dihydroxy-
pyrimidine-4-carboxylate with the appropriate amines in N,N-
dimethylformamide, in N-methylpyrrolidone, or in methanol.
Amidoximes 48 were prepared from the corresponding nitriles by
use of known procedures. Unless differently specified, 1 equiv of
potassium hydroxide dissolved in methanol was added to a solution
of hydroxylamine hydrochloride (1 equiv) in methanol. The
precipitated potassium chloride was removed by filtration, and to
the above solution was added the appropriate aryl nitrile (1 equiv).
The reaction mixture was stirred at 60 °C for the appropriate time
(3-6 h, TLC monitoring). After cooling, the solvent was removed
under vacuum, and the residue was triturated with diethyl ether.
The precipitate was collected and eventually recrystallized from
an appropriate solvent, furnishing the desired amidoxime in 60-
70% yield. Usually amidoximes were used without further purifica-
tion in the reaction with dimethyl acetylenedicarboxylate. Michael
adducts 49 were not isolated.
General Procedure for the Synthesis of 2-Substituted Methyl-
5,6-dihydroxypyrimidine-4-carboxamide. 2-Substituted methyl
5,6-dihydroxypyrimidine-4-carboxylates 50 were prepared from the
appropriate amidoximes following the method described by Cul-
Synthesis of N-(4-fluorobenzyl)-5,6-dihydroxy-2-thien-2-ylpy-
rimidine-4-carboxamide 9 is a representative example.

2232 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Pace et al.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-thien-2-ylpyrimidine-4-
carboxamide (9). Step 1: N-Hydroxythiophene-2-carboximidamide
(1.0 g, 7.0 mmol) was suspended in chloroform (20 mL) and
refluxed overnight in the presence of 1.0 equiv of dimethyl
acetylenedicarboxylate (0.87 mL, 7.0 mmol). After being cooled
to room temperature, volatiles were evaporated and the residue was
refluxed in xylene (25 mL) for 3 h. The mixture was cooled to
room temperature to allow the formation of a precipitate. Methyl
5,6-dihydroxy-2-thien-2-ylpyrimidine-4-carboxylate was collected
by filtration and washed with diethyl ether (0.265 g, 15% yield).
1H NMR (DMSO-d6, 300 MHz) δ 13.0 (bs, 1 H), 8.08 (d, J ) 3.2
Hz, 1 H), 7.85 (d, J ) 4.4 Hz, 1 H), 7.25 (dd, J ) 4.9 Hz, 3.9 Hz,
1 H), 3.93 (s, 3 H). Step 2: The solid from above (0.265 g, 1.05
mmol) was dissolved in DMF (3 mL) and 2.0 equiv of 4-fluo-
robenzylamine (0.24 mL, 2.1 mmol) was added to the stirred
solution. The reaction mixture was left overnight at 90 °C. After
the mixture was cooled to room temperature, 1 N HCl (2 mL) was
added and a solid precipitated from the mixture. This solid was
collected by filtration, washed with ethyl ether, and dried under
J ) 4.92, 3.69 Hz, 1 H), 3.19 (t, J ) 6.64 Hz, 2 H), 1.70-1.60 (m,
6 H), 1.21-1.12 (m, 3 H), 0.99-0.91 (m, 2 H); MS m/z 334 (M +
H)+. HRMS Calcd for C16H20N3O3S (M + H)+: 334.12199.
Found: 334.12042.
5,6-Dihydroxy-N-[(1S)1-phenylethyl]-2-(2-thienyl)pyrimidine-
4-carboxamide (6). As for the preparation of 9, replacing in step
1
2 4-fluorobenzylamine with (1S)-1-phenylethanamine. H NMR
(DMSO-d6, 400 MHz) δ 13.07 (bs, 1 H), 12.25 (s, 1 H), 8.60 (m,
1 H), 8.03 (d, J ) 3.7 Hz, 1 H), 7.80 (d, J ) 4.92 Hz, 1 H), 7.44
(d, J ) 7.63 Hz, 2 H), 7.37 (t, J ) 7.38 Hz, 2 H), 7.28 (t, J ) 7.14
Hz, 1 H), 7.18 (t, J ) 4.30 Hz, 1 H), 5.17 (q, J ) 7.63 Hz, 1 H),
1.57 (d, J ) 7.14 Hz, 3 H); MS m/z 342 (M + H)+. HRMS Calcd
for C17H16N3O3S (M + H)+: 342.09069. Found: 342.08968.
5,6-Dihydroxy-N-[(1R)1-phenylethyl]-2-(2-thienyl)pyrimidine-
4-carboxamide (7). As for the preparation of 9, replacing in step
1
2 4-fluorobenzylamine with (1R)-1-phenylethanamine. H NMR
(DMSO-d6, 400 MHz) δ 13.07 (bs, 1 H), 12.25 (s, 1 H), 8.60 (m,
1 H), 8.03 (d, J ) 3.7 Hz, 1 H), 7.80 (d, J ) 4.92 Hz, 1 H), 7.44
(d, J ) 7.63 Hz, 2 H), 7.37 (t, J ) 7.38 Hz, 2 H), 7.28 (t, J ) 7.14
Hz, 1 H), 7.18 (t, J ) 4.30 Hz, 1 H), 5.17 (q, J ) 7.63 Hz, 1 H),
1.57 (d, J ) 7.14 Hz, 3 H); MS m/z 342 (M + H)+. HRMS Calcd
for C17H16N3O3S (M + H)+: 342.09069. Found: 342.09023.
5,6-Dihydroxy-N-(2-phenylethyl)-2-(2-thienyl)pyrimidine-4-
carboxamide (8). As for the preparation of 9, replacing in step 2
4-fluorobenzylamine with 2-phenylethanamine. 1H NMR (DMSO-
d6, 300 MHz) δ 13.08 (Bs, 1 H), 12.58 (bs, 1 H), 8.61 (bs, 1 H),
8.07 (dd, J ) 3.6, 0.8 Hz, 1 H, 7.87 (d, J ) 4.9 Hz, 1 H), 7.40-
7.21 (m, 6 H), 2.48 (bq, J ) 7.1 Hz, 2 H), 2.94 (t, J ) 7.4 Hz, 2
H);); MS m/z 342 (M + H)+. HRMS Calcd for C17H16N3O3S (M
+ H)+: 342.09069. Found: 342.08935.
1
vacuum to afford title compound (0.127 g, 35% yield). H NMR
(DMSO-d6, 300 MHz) δ 13.06 (s, 1 H), 12.38 (s, 1 H), 8.70 (bs,
1 H), 8.03 (d, J ) 3.2 Hz, 1 H), 7.81 (d, J ) 4.8 Hz, 1 H), 7.39
(dd, J ) 5.7, 8.4 Hz, 2 H), 7.20-7.18 (m, 3 H), 4.51 (d, J ) 6.3
Hz, 2 H); MS m/z 346 (M + H)+. HRMS Calcd for C16H13FN3O3S
(M + H)+: 346.06562. Found: 346.06515.
Unless otherwise specified, the following compounds were
prepared according to the above procedure and isolated with similar
yields.
N-Benzyl-5,6-dihydroxy-2-thien-2-ylpyrimidine-4-carboxam-
ide (1). As for the preparation of 9, replacing in step 2 4-fluo-
robenzylamine with benzylamine. 1H NMR (DMSO-d6, 300 MHz),
δ 13.06 (bs, 1 H), 12.53 (bs, 1 H), 9.19 (t, J ) 6.2 Hz, 1 H), 8.06
(d, J ) 3.5 Hz, 1 H), 7.82 (d, J ) 4.2 Hz, 1 H), 7.44-7.24 (m, 5
H), 7.19 (dd, J ) 4.2, 3.5 Hz, 1 H), 4.56 (d, J ) 6.2 Hz, 2 H); MS
(ES+) m/z 328 (M + H)+. HRMS Calcd for C16H14N3O3S (M +
H)+: 328.07504. Found: 328.07608.
N-(4-Fluorobenzyl)-5,6-dihydroxypyrimidine-4-carboxam-
ide (10). Step 1: 2-Ethoxycarbonyl-4,5-dihydroxy-6-(methoxycar-
bonyl)pyrimidine (0.5 g, 2.06 mmol, prepared according to the
procedure described in step 1, compound 9, by using ethyl amino-
(hydroxyimino)ethanoate27 in place of N-hydroxythiophene-2-
carboximidamide) was suspended in dioxane/THF 2:1 (10 mL) and
treated with 1 equiv of 1 N NaOH. After 20 min the mixture was
acidified with 1 N HCl and concentrated, and 4,5-dihydroxy-6-
(methoxycarbonyl)pyrimidine-2-carboxylic acid was collected by
N-Benzyl-5,6-dihydroxy-2-phenylpyrimidine-4-carboxam-
ide (2). As for the preparation of 9, replacing in step 1 N-hydrox-
ythiophene-2-carboximidamide with N-hydroxybenzenecarboxim-
1
1
idamide and in step 2 4-fluorobenzylamine with benzylamine. H
filtration (0.42 g, 95% yield). H NMR (DMSO-d6, 400 MHz) δ
NMR (DMSO-d6, 400 MHz) δ 12.97 (bs, 1 H), 12.58 (s, 1 H),
9.52 (bs, 1 H), 8.23 (d, J ) 8.8 Hz, 2 H), 7.53-7.42 (m, 3 H),
7.38-7.20 (m, 5 H), 4.55 (d, J ) 7.5 Hz, 2 H); MS m/z 322 (M +
H)+. HRMS Calcd for C18H16N3O3S (M + H)+: 322.11862.
Found: 322.11790.
5,6-Dihydroxy-N-phenyl-2-(2-thienyl)pyrimidine-4-carboxa-
mide (3). As for the preparation of 9, replacing in step 2
4-fluorobenzylamine with aniline. 1H NMR (DMSO-d6, 400 MHz)
δ 13.16 (bs, 1 H), 11.70 (bs, 1 H), 10.13 (s, 1 H), 8.08 (d, J ) 3.2
Hz, 1 H), 7.83 (d, J ) 4.93 Hz, 1 H), 7.73 (d, J ) 7.89 Hz, 2 H),
7.41 (t, J ) 7.62 Hz, 2 H), 7.22-7.20 (m, 3 H); MS m/z 314 (M
+ H)+. HRMS Calcd for C15H12N3O3S (M + H)+: 314.05939.
Found: 314.05885.
5,6-Dihydroxy-2-(2-thienyl)pyrimidine-4-carboxamide (4). To
a suspension of 0.200 g (0.79 mmol) of methyl 5,6-dihydroxy-2-
(2-thienyl)pyrimidine-4-carboxylate (prepared according to the
procedure described for compound 9, step 1) in MeOH (2 mL)
was added ammonia (2 N solution in MeOH, 20 equiv) and the
resulting reaction mixture was stirred in a sealed vessel at 40 °C
for 72 h. The volatiles were removed under reduced pressure, and
the solid residue was triturated with Et2O to give the title compound
as a solid product (0.085 g, 45% yield). 1H NMR (DMSO-d6, 400
MHz) δ 8.46 (bs, 1H), 7.84 (bs, 1H), 7.81 (d, J ) 3.6 Hz, 1H),
7.57 (d, J ) 4.9 Hz, 1H), 7.08 (t, J ) 3.9 Hz, 1H); MS m/z 238 (M
+ H)+. HRMS Calcd for C9H8N3O3S (M + H)+: 238.02809.
Found: 238.02734.
13.1 (bs, 1 H), 11.1 (bs, 1 H), 3.82 (s, 3 H); MS m/z 213 (M +
H)+. Step 2: The solid from above (0.42 g, 1.98 mmol) was taken
up in 1 N HCl (15 mL) and stirred for 6 h at 90 °C. After being
cooled to room temperature, the mixture was filtered and the solid
was washed with cold 1 N HCl. Evaporation of the filtrate afforded
methyl 5,6-dihydroxypyrimidine-4-carboxylate as a solid (0.33 g,
100% yield). 1H NMR (DMSO-d6, 400 MHz) δ 7.75 (s, 1 H), 3.82
(s, 3 H). Step 3: Solid from above was dissolved in DMF (4 mL),
and 4-fluorobenzylamine (0.45 mL, 3.96 mmol) was added. After
2 h at 90 °C the mixture was evaporated. Compound 10 was isolated
in 56% yield (0.28 g) by preparative HPLC (C18, 5 µm, gradient
of CH3CN/H2O both containing 0.1% trifluoroacetic acid). 1H NMR
(DMSO-d6, 400 MHz) δ 12.72 (bs, 1 H), 12.54 (bs, 1 H), 9.48 (bs,
1 H), 7.77 (s, 1 H), 7.36 (t, J ) 8.0 Hz, 2 H), 7.14 (t, J ) 8.8 Hz,
2 H), 4.43 (d, J ) 6.3 Hz, 2 H); MS m/z 264 (M + H)+. HRMS
Calcd for C12H11FN3O3 (M + H)+: 264.07790. Found: 264.07816.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-methylpyrimidine-4-car-
boxamide (11). As for the preparation of 9, replacing in step 1
N-hydroxythiophene-2-carboximidamide with N-hydroxyethanimi-
damide.28 1H NMR (DMSO-d6, 400 MHz) δ 12.58 (bs, 1 H), 12.28
(bs, 1 H), 9.33 (bs, 1 H), 7.36 (dd, J ) 8.6 and 5.8 Hz, 2 H), 7.15
(t, J ) 8.9 Hz, 2 H), 4.43 (d, J ) 6.36 Hz, 2 H), 2.24 (s, 3 H); MS
m/z 278 (M + H)+. HRMS Calcd for C13H13FN3O3 (M + H)+:
278.09355. Found: 278.09460.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-(1-methylethyl)pyrimi-
dine-4-carboxamide (12). As for the preparation of 9, replacing
in step 1 N-hydroxythiophene-2-carboximidamide with N-hydroxy-
2-methylpropanimidamide. 1H NMR (DMSO-d6, 300 MHz) δ 12.53
(bs, 1 H), 12.28 (bs, 1 H), 9.23 (t, J ) 6.4 Hz, 1 H), 7.36 (dd, J )
8.1, 5.0 Hz, 2 H), 7.17 (t, J ) 8.9 Hz, 2 H), 4.48 (d, J ) 6.4 Hz,
2 H), 2.84-2.70 (m, 1 H), 1.19 (d, J ) 7.3 Hz, 6 H); MS m/z 306
N-(Cyclohexylmethyl)-5,6-dihydroxy-2-(2-thienyl)pyrimidine-
4-carboxamide (5). As for the preparation of 9, replacing in step
1
2 4-fluorobenzylamine with cyclohexylamine. H NMR (DMSO-
d6, 400 MHz) δ 13.03 (bs, 1 H), 12.62 (bs, 1 H), 8.54 (bs, 1 H),
8.02 (d, J ) 3.69 Hz, 1 H), 7.81 (d, J ) 4.43 Hz, 1 H), 7.18 (dd,

Dihydroxypyrimidine Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2233
(M + H)+. HRMS Calcd for C15H17FN3O3 (M + H)+: 306.12485.
Found: 306.12512.
mmol) in chloroform (20 mL), and the reaction mixture was
refluxed for 1 h. After complete conversion (TLC monitoring),
volatiles were evaporated, p-xylenes (20 mL) were added to the
residue, and the solution was refluxed for 6 h. Methyl-2-benzyl-
5,6-dihydroxypyrimidine-4-carboxylate 51 precipitated from the
solution after cooling to room temperature and was collected by
2-Benzyl-N-(4-fluorobenzyl)-5,6-dihydroxypyrimidine-4-car-
boxamide (13). As for the preparation of 9, replacing in step 1
N-hydroxythiophene-2-carboximidamide with N-hydroxy-2-phe-
nylethanimidamide. 1H NMR (DMSO-d6, 400 MHz) δ 12.81 (s, 1
H), 12.32 (s, 1 H), 9.37 (t, J ) 6.2 Hz, 1 H), 7.42-7.32 (m, 6 H),
7.25 (t, J ) 8.2 Hz, 1 H), 7.14 (t, J ) 8.8 Hz, 2 H), 4.46 (d, J )
6.2 Hz, 2 H), 3.84 (s, 2 H); MS m/z 354 (M + H)+. HRMS Calcd
for C19H17FN3O3 (M + H)+: 354.12485. Found: 354.12573.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-(4-methylphenyl)pyrimi-
dine-4-carboxamide (14). As for the preparation of 9, replacing
in step 1 N-hydroxythiophene-2-carboximidamide with N-hydroxy-
4-methylbenzenecarboximidamide. 1H NMR (DMSO-d6, 300 MHz)
δ 12.90 (bs, 1 H), 12.30 (bs, 1 H), 9.78 (t, J ) 5.3 Hz, 1 H), 8.24
(d, J ) 8.8 Hz, 2 H), 7.49 (dd, J ) 5.6 Hz, 8.7 Hz, 2 H), 7.41 (d,
J ) 8.8 Hz, 2 H), 7.27 (t, J ) 8.8 Hz, 2 H), 4.64 (d, J ) 5.3 Hz,
2 H), 2.48 (s, 3 H); MS m/z 354 (M + H)+. HRMS Calcd for
C19H17FN3O3: 354.12485. Found: 354.12500.
1
filtration and dried (0.35 g, 20% yield). H NMR (DMSO-d6) δ
12.9 (bs, 1 H), 10.2 (bs, 1 H), 7.40-7.20 (m, 5 H), 3.82 (s, 2 H),
3.80 (s, 3 H); MS m/z 261 (M + H)+. Step 2: To a stirred solution
of methyl 2-benzyl-5,6-dihydroxypyrimidine-4-carboxylate (51, 0.35
g, 1.35 mmol) in anhydrous pyridine (3 mL) was added benzoyl
chloride (0.78 mL, 6.75 mmol) dropwise with external cooling, and
the reaction was stirred overnight at room temperature. The mixture
was poured into 1 N HCl and extracted with ethyl acetate. The
organic phase was washed with a saturated solution of NaHCO3
and with brine, dried (Na2SO4), filtered, and concentrated under
vacuum. The residue was purified by flash column chromatography
(SiO2, 80/20 V/V petroleum ether/ethyl acetate as eluent), yielding
methyl 5,6-bis(benzoyloxy)-2-benzylpyrimidine-4-carboxylate as a
1
colorless oil (0.47 g, 75% yield). H NMR (CDCl3, 400 MHz) δ
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-(1,3-thiazol-2-yl)pyrimi-
dine-4-carboxamide (15). As for the preparation of 9, replacing
in step 1 N-hydroxythiophene-2-carboximidamide with N-hydroxy-
8.07 (t, J ) 9.0 Hz, 4 H), 7.62-7.57 (m, 2 H), 7.48-7-40 (m, 6
H), 7.31 (t, J ) 8.9 Hz, 2 H), 7.28 (d, J ) 8.9 Hz, 1 H), 4.41 (s,
2 H), 3.91 (s, 3 H). Step 3: A solution of methyl 5,6-bis-
(benzoyloxy)2-benzylpyrimidine-4-carboxylate (0.47 g, 1.0 mmol,
1.0 equiv) in carbon tetrachloride (25 mL) was heated up to 90 °C
under nitrogen. N-Bromosuccinimide (0.18 g, 1.0 mmol) and
benzoyl peroxide (0.026 g, 0.1 mmol) were added as dry powder,
and the mixture was refluxed for 3 h. Succinimide was removed
by filtration, and the filtrate was concentrated and purified by flash
column chromatography (SiO2, 85/15 V/V petroleum ether/ethyl
acetate as eluent), yielding methyl 5,6-bis(benzoyloxy)-2-[bromo-
(phenyl)methyl]pyrimidine-4-carboxylate 52 as a white solid (0.47
g, 87% yield). 1H NMR (CDCl3, 400 MHz) δ 8.11 (d, J ) 8.6 Hz,
2 H), 8.05 (d, J ) 8.6 Hz, 2 H), 7.79 (d, J ) 8.9 Hz, 2 H), 7.56-
7.49 (m, 2 H), 7.50-7.30 (m, 7 H), 6.30 (s, 1 H), 3.90 (s, 3 H).
Step 4: Compound from above (0.47 g, 0.87 mmol) was added to
a 2.0 M solution of dimethylamine in THF (2 mL). After the mixture
was stirred for 10 min at room temperature, volatiles were
evaporated under a stream of nitrogen and the residue was taken
up in N,N-dimethylformamide (2 mL) and treated with 4 equiv of
p-fluorobenzylamine (0.40 mL, 3.48 mmol). The reaction mixture
was stirred at 90 °C for 1 h. After the mixture was cooled to room
temperature, compound 19 was isolated in 35% yield (0.15 g) as
the trifluoroacetate salt by RP-HPLC (C18, acetonitrile/water
containing 0.1% trifluoroacetic acid as eluent). 1H NMR (DMSO-
d6, 600 MHz) δ 13.42 (bs, 1 H), 12.34 (s, 1 H), 10.06 (bs, 1 H),
9.64 (t, J ) 5.9 Hz, 1 H), 7.52 (s, 5 H), 7.43 (dd, J ) 8.4 Hz, 5.6
Hz, 2 H), 7.23 (t, J ) 8.8 Hz, 2 H), 5.28 (s, 1 H), 4.67 (dd, J )
15.4 Hz, 6.6 Hz, 1 H), 4.59 (dd, J ) 15.5 Hz, 6.0 Hz, 1 H), 3.02
(s, 3 H), 2.06 (s, 3 H); 13C NMR (DMSO-d6, 600 MHz) δ 168.25,
161.32 (d, J ) 242.9 Hz), 148.41, 144.29, 134.30, 130.89, 130.61,
129.40, 129.24, 129.00 (d, J ) 8.2 Hz), 125.95, 115.56 (d, J )
21.4 Hz), 68.90, 43.30, 41.20, 40.80; MS m/z 397 (M + H)+.
HRMS Calcd for C21H22FN4O3 (M + H)+: 397.16705. Found:
397.16830.
1
1,3-thiazole-2-carboximidamide. H NMR (DMSO-d6, 300 MHz)
δ 13.24 (bs, 1 H), 12.99 (bs, 1 H), 9.58 (t, J ) 6.7 Hz, 1 H), 8.28-
8.20 (m, 2 H), 7.59 (dd, J ) 8.0, 5.5 Hz, 2 H), 7.37 (t, J ) 8.0 Hz,
2 H), 4.72 (d, J ) 6.7 Hz, 2 H); MS m/z 347 (M + H)+. HRMS
Calcd for C15H12FN4O3S (M + H)+: 347.06087. Found: 347.06015.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-pyridin-2-ylpyrimidine-
4-carboxamide (16). As for the preparation of 9, replacing in step
1 N-hydroxythiophene-2-carboximidamide with N-hydroxypyridine-
1
2-carboximidamide. H NMR (DMSO-d6, 400 MHz) δ 12.98 (bs,
1 H), 12.50 (bs, 1 H), 9.71 (t, J ) 7.4 Hz, 1 H), 8.72-8.65 (m, 2
H), 8.02 (t, J ) 6.2 Hz, 1 H), 7.60-7.52 (m, 1 H), 7.38 (dd, J )
8.3, 6.5 Hz, 2 H), 7.18 (t, J ) 8.8 Hz, 2 H), 4.52 (d, J ) 7.4 Hz,
2 H); MS m/z 341 (M + H)+. HRMS Calcd for C17H14FN4O3 (M
+ H)+: 341.10445. Found: 341.10366.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-[methoxy(phenyl)methyl]-
pyrimidine-4-carboxamide (17). As for the preparation of 9,
replacing in step 1 N-hydroxythiophene-2-carboximidamide with
N-hydroxy-2-methoxy-2-phenylethanimidamide. 1H NMR (DMSO-
d6, 400 MHz) δ 12.75 (bs, 1 H), 12.40 (bs, 1 H), 9.27 (bs, 1 H),
7.52 (d, J ) 7.1 Hz, 2 H), 7.39-7.28 (m, 5 H), 7.18-7.12 (m, 2
H), 5.14 (s, 1 H), 4.52-4.41(m, 2 H), 3.34 (s, 3 H); MS m/z 384
(M + H)+. HRMS Calcd for C20H19FN3O4 (M + H)+: 384.135410.
Found: 384.13498.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-[methyl(phenyl)amino]-
pyrimidine-4-carboxamide (18). As for the preparation of 9,
replacing in step 1 N-hydroxythiophene-2-carboximidamide with
N”-hydroxy-N-methyl-N-phenylguanidine. 1H NMR (DMSO-d6,
400 MHz) δ 11.6 (bs, 1 H), 9.09 (bs, 1 H), 7.41-7.35 (m, 4 H),
7.26-7.22 (m, 3 H), 7.17 (t, J ) 8.8 Hz, 2 H), 4.47 (d, J ) 6.4
Hz, 2 H), 3.36 (s, 3 H); MS m/z 369 (M + H)+. HRMS Calcd for
C19H18FN4O3 (M + H)+: 369.13629. Found: 369.13692.
2-(3,4-Dimethoxybenzyl)-N-(4-fluorobenzyl)-5,6-dihydroxy-
2-methylpyrimidine-4-carboxamide (20). As for the preparation
of 9, replacing in step 1 N-hydroxythiophene-2-carboximidamide
with 2-(3,4-dimethoxyphenyl)-N-hydroxyethanimidamide. 1H NMR
(DMSO-d6, 400 MHz) δ 12.85 (bs, 1 H), 12.12 (bs, 1 H), 9.39 (t,
J ) 5.8 Hz, 1 H), 7.40-7.33 (m, 2 H), 7.17 (t, J ) 8.7 Hz, 2 H),
7.04 (s, 1 H), 6.88 (t, J ) 8.8 Hz, 2 H), 4.46 (d, J ) 5.8 Hz, 2 H),
3.74 (s, 2 H), 3.70 (s, 6 H); MS m/z 414 (M + H)+; HRMS Calcd
for C21H21FN3O5 (M + H)+: 414.14598. Found: 414.14526.
Synthesis of Compounds 19, 22, and 23. Synthesis of 2-[(dim-
ethylamino)phenylmethyl]-N-(4-fluorobenzyl)-5,6-dihydroxypyri-
midine-4-carboxamide 19 is a representative example. Compounds
22 and 23 were isolated as trifluoroacetate salts by preparative RP
HPLC in similar yields to 19.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-[morpholin-4-yl(phenyl)-
methyl]pyrimidine-4-carboxamide (22). As for the preparation
1
of 19, replacing in step 4 dimethylamine with morpholine. H
NMR (DMSO-d6, 400 MHz) δ 12.30 (bs, 1 H), 9.90 (bs, 1 H),
9.34 (bs, 1 H), 7.63-7.58 (m, 2 H), 7.52-7.32 (m, 5 H), 7.18 (t,
J ) 8.8 Hz, 1 H), 5.39 (bs, 1 H), 4.60-4.39 (m, 2 H), 3.9-3.3
(m, 8 H, partially obscured by water); MS m/z 439 (M + H)+;
HRMS Calcd for C23H24FN4O4 (M + H)+: 439.17816. Found:
439.17854.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-[phenyl(piperidin-1-yl)-
methyl]pyrimidine-4-carboxamide (23). As for the preparation
of 19, replacing in step 4 dimethylamine with piperidine. 1H NMR
(DMSO-d6, 300 MHz) δ 13.38 (bs, 1 H), 12.31 (s, 1 H), 9.90-
9.70 (bs, 2 H), 7.58-7.40 (m, 7 H), 7.28 (t, J ) 8.9 Hz, 2 H), 5.28
(bs, 1 H), 4.76-4.63 (m, 1 H), 4.58-4.48 (m, 1 H), 3.80-3.60
(m, 2 H), 3.20-2.80 (m, 2 H), 1.90-1.50 (m, 6 H); MS m/z 437
2-[(Dimethylamino)phenylmethyl]-N-(4-fluorobenzyl)-5,6-di-
hydroxypyrimidine-4-carboxamide (19). Step 1: Dimethyl acety-
lenedicarboxylate (0.82 mL, 6.7 mmol) was added to a stirred
suspension of N′-hydroxy-2-phenylethanimidamide (1.0 g, 6.7

2234 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Pace et al.
(M + H)+; HRMS Calcd for C24H26FN4O3 (M + H)+: 437.19835.
Found: 437.19714.
1H NMR (DMSO-d6 + TFA, 400 MHz) δ 13.1 (bs, 1 H), 12.2 (s,
1 H), 9.38 (t, J ) 6.4 Hz, 1 H), 9.20-9.10 (m, 1 H), 8.80-8.65
(m, 1 H), 7.41 (dd, J ) 5.6 Hz, 8.4 Hz, 2 H), 7.17 (t, J ) 8.8 Hz,
2 H), 4.65-4.43 (m, 2 H), 4.15-4.05 (m, 1 H), 3.49-3.38 (m, 1
H), 3.12-3.00 (m, 1 H), 2.30-2.20 (m, 1 H), 1.88-1.50 (m, 5 H);
MS m/z 347 (M + H)+. HRMS Calcd for C17H20FN4O3 (M +
H)+: 347.15140. Found: 347.15009.
2-{4-[(Dimethylamino)methyl]phenyl}-N-(4-fluorobenzyl)-
5,6-dihydroxypyimidine-4-carboxamide (21). Step 1: Dimethyl
acetylenedicarboxylate (0.82 mL, 6.7 mmol) was added to a stirred
solution of N′-hydroxy-4-methylbenzenecarboximidamide (1.0 g,
6.7 mmol) in chloroform (20 mL), and reaction mixture was
refluxed for 1 h. After complete conversion (TLC monitoring),
volatiles were evaporated, p-xylenes (20 mL) were added to the
residue, and the solution was refluxed for 6 h. Methyl 5,6-
dihydroxy-2-(4-methylphenyl)pyrimidine-4-carboxylate (54) pre-
cipitated from the solution after cooling to room temperature and
was collected by filtration and dried (0.50 g, 29% yield). MS m/z
261 (M + H)+. Step 2: A mixture of methyl 5,6-dihydroxy-2-(4-
methylphenyl)pyrimidine-4-carboxylate from above (54, 0.50 g, 1.9
mmol), benzoyl chloride (0.88 mL, 7.7 mmol), and dry pyridine
(1.2 g, 15.2 mmol, 8 equiv) was stirred in dry dichloromethane (3
mL) at room temperature overnight. The reaction mixture was
diluted with ethyl acetate, and the organic layer was washed twice
with 1 N HCl and once with brine. The organic layer was dried
over anhydrous sodium sulfate, filtered, and concentrated in
vacuum. The solid residue was triturated with diethyl ether to give
methyl 5,6-bis(benzoyloxy)-2-(4-methylphenyl)pyrimidine-4-car-
boxylate (0.81 g, 91% yield). 1H NMR (CDCl3, 400 MHz) δ 8.38
(d, J ) 8.2 Hz, 2 H), 8.14 (d, J ) 7.44 z, 2 H), 8.10 (d, J ) 7.44
Hz, 2 H), 7.62 (m, 2 H), 7.45 (m, 4 H), 7.30 (d, J ) 8.06 Hz, 2 H),
3.93 (s, 3 H), 2.44 (s, 3 H). Step 3: A suspension of methyl ester
from above (0.81 g, 1.7 mmol), an equimolar amount of N-
bromosuccinimide (0.30 g, 1.7 mmol), and 5% of dibenzoyl
hydroperoxide (0.022 g, 0.08 mmol) in carbon tetrachloride (50
mL) was heated at 95 °C. The reaction mixture was refluxed for 2
h and then cooled to room temperature. Succinimide was filtered
off, and volatiles were removed in vacuum, yielding 0.80 g of
methyl 5,6-bis(benzoyloxy)-2-[4-(bromomethyl)phenyl]pyrimidine-
4-carboxylate (55, 85% yield). 1H NMR (CDCl3, 400 MHz) δ 8.47
(d, J ) 8.25 Hz, 2 H), 8.14 (d, J ) 7.56 Hz, 2 H), 8.10 (d, J )
7.50 Hz, 2 H), 7.63 (m, 2 H), 7.53 (d, J ) 8.23, 2 H), 7.46 (m, 4
H), 4.56 (s, 2 H), 3.94 (s, 3 H). Step 4: A mixture of the benzyl
bromide from above (55, 0.80 g, 1.5 mmol) and dimethylamine (3
mL as 2 M solution in THF) was allowed to stir at room temperature
overnight. Volatiles were then removed in vacuum, the residue was
taken up in DMF (4 mL), and after addition of 4-fluorobenzylamine
(0.34 mL, 3.0 mmol) the reaction mixture was stirred at 90 °C
overnight. After being cooled to room temperature, the mixture was
acidified with 1 N HCl and compound 21 was purified by
preparative RP-HPLC (C18, acetonitrile/water containing 0.1%
trifluoroacetic acid as eluent) and isolated in 44% yield (0.34 g) as
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-piperidin-3-ylpyrimidine-
4-carboxamide (25). Step 1: Identical procedure used for the
preparation of 9, replacing in step 1 N-hydroxythiophene-2-
carboximidamide with tert-butyl 3-[amino(hydroxyimino)methyl]-
piperidine-1-carboxylate, afforded tert-butyl 3-(4-{[(4-fluorobenzyl)-
amino]carbonyl}-5,6-dihydroxypyrimidin-2-yl)piperidine-1-
carboxylate (23% yield). MS m/z 447 (M + H)+. Step 2: The crude
compound from the previous step (0.050 g, 0.11 mmol) was stirred
in 2 mL of CH2Cl2:TFA (8:2) for 1 h at room temperature.
Evaporation of volatiles and purification by preparative RP-HPLC
(C18, acetonitrile/water containing 0.1% trifluoroacetic acid as
eluent) yielded the title compound as its trifluoroacetate salt (0.034
g, 67% yield). 1H NMR (DMSO-d6, 400 MHz) δ 12.83 (bs, 1 H),
12.38 (s, 1 H), 9.32 (t, J ) 5.6 Hz, 1 H), 8.69 (bs, 1 H), 8.50 (bs,
1 H), 7.38 (dd, J ) 8.8, 5.7 Hz, 2 H), 7.29 (t, J ) 8.8 Hz, 2 H),
4.58-4.43 (m, 2 H), 3.29-3.17 (m, 3 H), 2.98-2.89 (m, 1 H),
2.88-2.76 (m, 1 H), 2.10-2.00 (m, 1 H), 1.90-1.79 (m, 1 H),
1.72-1.58 (m, 2 H); MS m/z 347 (M + H)+. HRMS Calcd for
C17H20FN4O3 (M + H)+: 347.15140. Found: 347.15161.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-piperidin-4-ylpyrimidine-
4-carboxamide (26). Step 1: Identical procedure used for the
preparation of 9, replacing in step 1 N-hydroxythiophene-2-
carboximidamide with tert-butyl 4-[amino(hydroxyimino)methyl]-
piperidine-1-carboxylate, afforded tert-butyl-4-(4-{[(4-fluorobenzyl)-
amino]carbonyl}-5,6-dihydroxypyrimidin-2-yl)piperidine-1-
carboxylate (27% yield). MS m/z 447 (M + H)+. Step 2: The crude
compound from the previous step (0.050 g, 0.11 mmol) was stirred
in 2 mL of CH2Cl2:TFA (8:2) for 1 h at room temperature.
Evaporation of volatiles and purification by preparative RP-HPLC
(C18, acetonitrile/water containing 0.1% trifluoroacetic acid as
eluent) yielded the title compound as its trifluoroacetate salt (0.029
g, 58% yield). 1H NMR (DMSO-d6, 400 MHz) δ 12.68 (bs, 1 H),
12.36 (s, 1 H), 9.19 (d, J ) 5.8 Hz, 1 H), 8.58 (bs, 1 H), 8.29 (bs,
1 H), 7.38 (dd, J ) 8.8, 5.7 Hz, 2 H), 7.18 (t, J ) 8.8 Hz, 2 H),
4.49 (d, J ) 5.8 Hz, 2 H), 3.37-3.28 (m, 2 H), 3.01-2.88 (m, 2
H), 2.82-2.75 (m, 1 H), 2.10-2.02 (m, 2 H), 1.99-1.86 (m, 2 H);
MS m/z 347 (M + H)+. HRMS Calcd for C17H20FN4O3 (M +
H)+: 347.15140. Found: 347.15256.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-(1-methylpiperidin-2-yl)-
pyrimidine-4-carboxamide (27). To a solution of compound 24
(0.070 g, 0.15 mmol) in methanol (2 mL) were added formaldehyde
(37 wt % in H2O, 0.065 mL), NaBH3CN (0.050 g, 0.78 mmol),
and NaOAc (0.070 g, 0.87 mmol). The mixture was stirred at room
temperature under nitrogen for 12 h and then concentrated and
purified by preparative RP-HPLC purification (C18, water/aceto-
nitrile containing 0.1% trifluoroacetic acid as eluent) to isolate title
1
the trifluoroacetate salt. H NMR (DMSO-d6, 400 MHz) δ 13.00
(bs, 1 H), 12.59 (s, 1 H), 9.69 (bs, 1 H), 9.66 (t, J ) 5.8 Hz, 1 H),
8.37 (d, J ) 9.7 Hz, 2 H), 7.62 (d, J ) 9.7 Hz, 2 H), 7.41 (dd, J
) 8.3, 5.4 Hz, 2 H), 7.19 (t, J ) 8.4 Hz, 2 H), 4.54 (d, J ) 5.8 Hz,
2 H), 4.34 (s, 2 H), 2.77 (s, 6 H); MS m/z 397 (M + H)+. HRMS
Calcd for C21H22FN4O3 (M + H)+: 397.16705. Found: 397.16830.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-piperidin-2-ylpyrimidine-
4-carboxamide (24). Step 1: Identical procedure used for the
preparation of 9, replacing in step 1 N-hydroxythiophene-2-
carboximidamide with benzyl 2-[amino(hydroxyimino)methyl]-
piperidine-1-carboxylate, afforded benzyl 2-(4-{[(4-fluorobenzyl)-
amino]carbonyl}-5,6-dihydroxypyrimidin-2-yl)piperidine-1-
carboxylate (18% yield). 1H NMR (DMSO-d6, 400 MHz) δ 12.67
(bs, 1 H), 12.29 (bs, 1 H), 8.68 (bs, 1 H), 7.35 (dd, J ) 8.4, 5.6
Hz, 2 H), 7.27 (bs, 5 H), 7.17 (t, J ) 8.8 Hz, 2 H), 5.12 (d, J )
12.2 Hz, 1 H), 5.00-4.93 (m, 2 H), 4.49 (d, J ) 5.8 Hz, 2 H),
3.93-3.87 (m, 1 H), 3.30-3.27 (m, 1 H), 2.29-2.18 (m, 1 H),
1.88-1.32 (m, 5 H); MS m/z 481 (M + H)+. Step 2: The compound
from the previous step (0.05 g, 0.1 mmol) was taken up in methanol
(5 mL) and hydrogenated in the presence of 10% Pd/C at room
temperature from 1 h. Catalyst was filtered off, and the filtrate was
concentrated and purified by preparative RP-HPLC (C18, acetoni-
trile/water containing 0.1% trifluoroacetic acid as eluent), giving
title compound in 60% yield (0.029 g) as its trifluoroacetate salt.
1
compound in 55% yield (0.039 g) as its trifluoroacetate salt. H
NMR (DMSO-d6, 400 MHz) δ 13.1 (bs, 1 H), 12.2 (s, 1 H), 9.45
(bs, 1 H), 9.34 (t, J ) 6.4 Hz, 1 H), 7.37 (dd, J ) 5.6 Hz, 8.4 Hz,
2 H), 7.18 (t, J ) 8.8 Hz, 2 H), 4.57 (d, J ) 6.4 Hz, 2 H), 4.05 (bs,
1 H), 3.61 (bd, J ) 12.4 Hz, 1 H), 3.52-3.50 (m, 1 H), 2.78 (bs,
3 H), 2.16 (d, J ) 13.6 Hz, 1 H), 1.92-1.80 (m, 2 H), 1.65-1.46
(m, 3 H); MS m/z 361 (M + H)+. HRMS Calcd for C18H22FN4O3
(M + H)+: 361.16705. Found: 361.16583.
2-(1-Ethylpiperidin-2-yl)-N-(4-fluorobenzyl)-5,6-dihydroxy-
pyrimidine-4-carboxamide (28). As for the preparation of 27,
replacing formaldehyde with 1.5 equiv of acetaldehyde (43% yield).
1H NMR (DMSO-d6, 400 MHz, 330 K) δ 13.11 (bs, 1 H), 12.19
(s, 1 H), 9.29 (bs, 1 H), 8.97 (bs, 1 H), 7.38 (dd, J ) 8.4, 5.6 Hz,
2 H), 7.19 (t, J ) 8.8 Hz, 2 H), 4.72-4.63 (m, 1 H), 4.58-4.50
(m, 1 H), 4.11-4.06 (m, 1 H), 3.79-3.71 (m, 1 H), 3.42-3.30
(m, 2 H), 3.09-3.00 (m, 1 H), 2.22-2.14 (m, 1 H), 1.98-1.50
(m, 5 H), 1.16 (t, J ) 6.6 Hz, 3 H); MS m/z 375 (M + H)+. HRMS
Calcd for C19H24FN4O3 (M + H)+: 375.18270. Found: 375.18088.

Dihydroxypyrimidine Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2235
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-(1-isobutylpiperidin-2-
yl)pyrimidine-4-carboxamide (29). As for the preparation of 27,
replacing formaldehyde with 1.5 equiv of 2-methylpropanal (22%
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-(4-methylmorpholin-3-
yl)pyrimidine-4-carboxamide (31). Step 1: A solution of tert-
butyl-3-[amino(hydroxyimino)methyl]morpholine-4-carboxylate (2.0
g, 7.3 mmol) in chloroform (25 mL) was treated with dimethyl
acetylenedicarboxylate (1.08 mL, 8.76 mmol) and refluxed for 3
h. The reaction mixture was cooled to room temperature and
concentrated. The residue was taken up in xylene (25 mL) and
refluxed for 24 h. The solvent was removed in vacuum, and the
crude residue was treated with 20 mL of a mixture of trifluoroacetic
acid:dichloromethane:water (65:35:10) at room temperature for 15
min. The reaction mixture was concentrated, and the residue was
taken up in Et2O and evaporated. A solid residue corresponding to
the TFA salt of methyl 5,6-dihydroxy-2-morpholin-3-ylpyrimidine-
4-carboxylate was obtained after trituration with Et2O (1.35 g, 50%
1
yield). H NMR (DMSO-d6, 400 MHz, 330 K) δ 12.21 (bs, 1 H),
9.19 (bs, 1 H), 8.47 (bs, 1 H), 7.38 (dd, J ) 8.5, 5.7 Hz, 2 H), 7.26
(t, J ) 8.9 Hz, 2 H), 4.78-4.68 (m, 1 H), 4.48-4.39 (m, 1 H),
4.08-3.97 (m,1 H), 3.37-3.28 (m, 1 H), 2.92-2.83 (m, 2 H),
2.20-1.40 (m, 8 H), 0.86-0.75 (m, 6 H); MS m/z 403 (M + H)+.
HRMS Calcd for C21H28FN4O3 (M + H)+: 403.21400. Found:
403.21561.
2-(1,4-Dimethylpiperazin-2-yl)-N-(4-fluorobenzyl)-5,6-dihy-
droxypyrimidine-4-carboxamide (30). Step 1: A solution of
1-benzyl 4-tert-butyl 2-[amino(hydroxyimino)methyl]piperazine-
1,4-dicarboxylate (2 g, 5.3 mmol) in chloroform (30 mL) was
treated with dimethyl acetylenedicarboxylate (0.78 mL, 6.36 mmol)
and refluxed for 3 h. The reaction mixture was cooled to room
temperature and concentrated. The residue was taken up in xylene
(30 mL) and refluxed for 24 h. The solvent was removed in vacuum,
and the residue was triturated with a 1:1 mixture of Et2O:n-hexane.
The solid was filtered off and washed further with Et2O:n-hexane.
The crude product was taken up in pyridine (8 mL), and to the
stirred solution was added benzoic anhydride (1.8 g, 7.9 mmol).
The reaction mixture was stirred at room temperature until the
starting material was consumed as determined by MS analysis. The
reaction mixture was concentrated, and the oily residue was diluted
with EtOAc and washed with 1 N HCl, NaHCO3 (sat. soln), and
brine. The organic phase was dried (Na2SO4), filtered, and
concentrated, yielding 0.69 g of crude 1-benzyl-4-tert-butyl-2-[5-
(benzoyloxy)-4-hydroxy-6-(methoxycarbonyl)pyrimidin-2-yl]pip-
erazine-1,4-dicarboxylate (LC-MS analysis, about 22% yield). MS
m/z 593 (M + H)+. Step 2: 1-Benzyl-4-tert-butyl-2-[5-(benzoy-
loxy)-4-hydroxy-6-(methoxycarbonyl)pyrimidin-2-yl]piperazine-
1,4-dicarboxylate from the previous step was treated with TFA/
CH2Cl2 1:1 (10 mL). After being stirred 1 h at room temperature,
the solution was evaporated to the crude methyl 5-(benzoyloxy)-
2-{1-[(benzyloxy)carbonyl]piperazin-2-yl}-6-hydroxypyrimidine-
4-carboxylate, which was further reacted without further purifica-
tion. MS m/z 493 (M + H)+. Step 3: The crude product from step
2 was dissolved in methanol (5 mL) and treated with NaCNBH3
(0.10 g, 1.62 mmol), NaOAc (0.150 g, 1.86 mmol), and formal-
dehyde (37 wt % in H2O, 0.2 mL). After being stirred 1 h at room
temperature, the mixture was evaporated and the crude residue
containing (LC-MS analysis) methyl 5-(benzoyloxy)-2-{1-[(ben-
zyloxy)carbonyl]-4-methyl-piperazin-2-yl}-6-hydroxypyrimidine-
4-carboxylate was further reacted in the next step. MS m/z 507 (M
+ H)+. Step 4: Crude material from the previous step was dissolved
in methanol (10 mL) and hydrogenated at atmospheric pressure in
the presence of 10% Pd/C overnight. Crude methyl 5,6-dihydroxy-
2-(4-methyl-piperazin-2-yl)pyrimidine-4-carboxylate was obtained
by filtration and evaporation of the filtrate. MS m/z 269 (M + H)+.
Step 5: Crude product from step 4 was dissolved in methanol (10
mL) and treated with NaCNBH3 (0.10 g, 1.62 mmol), NaOAc (0.15
g, 1.86 mmol), and formaldehyde (37 wt % in H2O, 0.2 mL), and
the resulting mixture was stirred at room temperature for 2.5 h.
After evaporation, the crude residue containing (LC-MS analysis)
methyl 2-(1,4-dimethylpiperazin-2-yl)-5,6-dihydroxypyrimidine-4-
carboxylate was taken on without purification. MS m/z 283 (M +
H)+. Step 6: Crude product from the previous step was dissolved
in N-methylpyrrolidone (6 mL/mmol) and treated with 4-fluoroben-
zylamine (0.40 mL, 3.48 mmol). The mixture was stirred at 90 °C
overnight and then cooled to room temperature, and an aliquot of
the crude reaction mixture was purified by preparative RP-HPLC
(C18, acetonitrile/water containing 0.1% trifluoroacetic acid as
eluent) to obtain 0.015 g of 2-(1,4-dimethylpiperazin-2-yl)-N-(4-
fluorobenzyl)-5,6-dihydroxypyrimidine-4-carboxamide (30) as its
trifluoroacetate salt. 1H NMR (DMSO-d6 + TFA, 400 MHz) δ 12.5
(bs, 1 H), 7.38 (dd, J ) 5.8, 8.8 Hz, 2 H), 7.17 (t, J ) 8.8 Hz, 2
H), 4.58-4.40 (m, 2 H), 3.66 (bs, 1 H), 3.55-3.35 (m, 3 H), 3.20
(d, J ) 13.3 Hz, 1 H), 3.03 (t, J ) 11.7 Hz, 1 H), 2.79 (s, 3 H),
2.85-2.70 (m, 1 H), 2.33 (bs, 3 H); MS m/z 376 (M + H)+. HRMS
Calcd for C18H23FN5O3 (M + H)+: 376.17794. Found: 376.17925.
1
yield). H NMR (DMSO-d6, 400 MHz) δ 13.24 (bs, 1 H), 10.54
(bs, 1 H), 9.54 (bs, 2 H), 4.34 (d, J ) 6.9 Hz, 1 H), 4.24 (dd, J )
12.2, 3.2 Hz,1 H), 3.93 (d, J ) 11.2 Hz, 1 H), 3.84 (s, 3 H), 3.75
(t, J ) 10.3 Hz, 1 H), 3.58 (t, J ) 10.5 Hz, 1 H), 3.32 (d, J ) 12.8
Hz, 1 H), 3.20 (td, J ) 11.0, 3.7 Hz, 1 H); MS m/z 256 (M + H)+.
Step 2: The compound from step 1 was dissolved in dry methanol
(20 mL) and treated with 4-fluorobenzyl amine (0.83 mL, 7.30
mmol) at 90 °C for 1 h. After being cooled to room temperature,
the reaction mixture was concentrated and the residue triturated
with Et2O. N-(4-Fluorobenzyl)-5,6-dihydroxy-2-morpholin-3-ylpy-
rimidine-4-carboxamide was isolated in 90% yield (1.5 g) by
preparative RP-HPLC (C18, acetonitrile/water containing 0.1%
1
trifluoroacetic acid as eluent) as its trifluoroacetate salt. H NMR
(DMSO-d6+TFA, 400 MHz) δ 9.63 (bs, 1 H), 9.4 (t, J ) 6.07 Hz,
1 H), 9.2 (bs, 1 H), 7.39 (dd, J ) 8.31, 5.76 Hz, 2 H), 7.18 (t, J )
8.84 Hz, 2 H), 4.59 (dd, J ) 15.53, 6.80 Hz, 2 H), 4.53 (dd, J )
15.36, 6.24 Hz, 1 H), 4.37 (bs, 1 H), 4.24 (dd, J ) 12.41, 3.24 Hz,
1 H), 3.99 (d, J ) 12.04 Hz, 1 H), 3.74 - 3.62 (m, 2 H), 3.41 (d,
J ) 13.09 Hz, 1 H), 3.40 (bs, 1 H); MS m/z 349 (M + H)+. Step
3: To a methanolic solution of compound from above (0.200 g,
0.43 mmol) were added formaldehyde (37 wt % in H2O, 0.2 mL),
NaBH3CN (0.140 g, 2.24 mmol), and NaOAc (0.20 g, 2.49 mmol).
The mixture was stirred at room temperature under nitrogen for 12
h and then concentrated and purified by preparative RP-HPLC
purification (C18, water acetonitrile containing 0.1% trifluoroacetic
acid as eluent), yielding N-(4-fluorobenzyl)-5,6-dihydroxy-2-(4-
methylmorpholin-3-yl)pyrimidine-4-carboxamide (31) as a its tri-
fluoroacetate salt (0.14 g, 67% yield). 1H NMR (DMSO-d6+TFA,
400 MHz) δ 9.2 (bt, 1 H), 7.40 (dd, J 0 8.38, 5.75 Hz, 2 H), 7.16
(t, J ) 8.84 Hz, 2 H), 4.57 (d, J ) 6.34 Hz, 2 H), 4.27 (dd, J )
10.03, 3.55 Hz, 1 H), 4.22 (dd, J ) 12.82, 3.22 Hz, 1 H), 4.10 (d,
J ) 13.73 Hz, 1 H), 3.77 (t, J ) 11.84 Hz, 1 H), 3.65-3.60 (m, 2
H), 3.41 (td, J ) 12.54, 3.67 Hz, 1 H), 2.87 (s, 3 H); MS m/z 363
(M + H)+. HRMS Calcd for C17H20FN4O4 (M + H)+: 363.14631.
Found: 363.14565.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-(1-methylpyrrolidin-2-
yl)pyrimidine-4-carboxamide (32). Step 1: A solution of tert-
butyl-2-[amino(hydroxyimino)methyl]pyrrolidine-1-carboxylate (2.0
g, 8.7 mmol) in chloroform (25 mL) was treated with dimethyl
acetylenedicarboxylate (1.28 mL, 10.44 mmol) and refluxed for 3
h. The reaction mixture was cooled to room temperature and
concentrated. The residue was taken up in xylene (30 mL) and
refluxed for 24 h. The solvent was removed in vacuum, and the
crude residue was taken up in pyridine (10 mL). To the solution
was added benzoic anhydride (2.9 g, 13 mmol, 1.5 equiv), and the
reaction mixture was stirred at room temperature until the starting
material was consumed as determined by MS analysis. The reaction
mixture was concentrated, and the oily residue was diluted with
EtOAc and washed with 1 N HCl, NaHCO3 (sat. soln), and brine.
The organic phase was dried (Na2SO4), filtered, and concentrated,
and the residue was purified by flash column chromatography (SiO2,
petroleum ether/ethyl acetate 60/40 V/v as eluent), to obtain the
methyl 5-(benzoyloxy)-2-[1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-
6-hydroxypyrimidine-4-carboxylate as a yellow solid (0.73 g, 19%
1
yield). H NMR (CDCl3, 400 MHz) δ 12.08 (bs, 1 H), 8.18 (d, J
) 7.6 Hz, 2 H), 7.64 (t, J ) 7.6 Hz, 1 H), 7.50 (t, J ) 7.6 Hz, 2
H), 4.80-4.60 (m, 1 H), 3.82 (s, 3 H), 3.60-3.50 (m, 1 H), 3.40-

2236 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Pace et al.
3.20 (m, 1 H), 2.50-2.10 (m, 2 H), 2.00-1.70 (m, 2 H), 1.50 (s,
9 H); MS m/z 444 (M + H)+. Step 2: Methyl 5-(benzoyloxy)-2-
[1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-6-hydroxypyrimidine-4-car-
boxylate from above (0.73 g, 1.64 mmol) was treated with 20 mL
of CH2Cl2:TFA (7:3) at 0 °C. The solution was allowed to warm
to room temperature and stirred for 1 h. Volatiles were removed
under reduced pressure, the residue was triturated with Et2O, and
methyl 5-(benzoyloxy)-6-hydroxy-2-pyrrolidin-2-yl-pyrimidine-4-
carboxylate was collected by filtration (0.54 g, 96% yield). 1H NMR
(CDCl3, 400 MHz) δ 8.14 (d, J ) 7.5 Hz, 2 H), 7.67 (t, J ) 7.6
Hz, 1 H), 7.50 (dd, J ) 7.6, 7.6 Hz, 2 H), 4.99 (dd, J ) 14.9, 7.3
Hz, 1 H), 3.78 (s, 3 H), 3.60-3.40 (m, 2 H), 2.60-2.45 (m, 1 H),
2.40-2.30 (m, 1 H), 2.20-2.10 (m, 2 H); MS m/z 344 (M + H)+.
Step 3: A solution of compound from step 2 (0.10 g, 0.29 mmol)
in MeOH (3 mL) was treated with 4-fluorobenzylamine (0.10 mL,
0.87 mmol), and the solution was refluxed for 5 h. After being
cooled to room temperature, the reaction mixture was concentrated
under reduced pressure, and the product N-(4-fluorobenzyl)-5,6-
dihydroxy-2-pyrrolidin-2-ylpyrimidine-4-carboxamide that precipi-
7.66-7.58 (m, 2 H), 4.22 (d, J ) 14.4 Hz, 1 H), 3.80 (s, 3 H),
3.75-3.67 (m, 2 H), 3.63-3.44 (m, 2 H), 3.32-3.24 (m, 1 H),
1.68 (s, 3 H), 1.38 (s, 9 H); MS m/z 473 (M + H)+. Step 3: Crude
material from above (0.29 g, 0.61 mmol) was dissolved in methanol
(10 mL), and to the stirred mixture were added NaCNBH3 (0.054
g, 0.85 mmol), NaOAc (0.158 g, 1.95 mmol), and 37% water
solution of formaldehyde (0.1 mL). The reaction mixture was stirred
for 0.5 h at room temperature, volatiles were evaporated, and the
residue containing methyl 2-[4-(tert-butoxycarbonyl)-1,2-dimeth-
ylpiperazin-2-yl]-5,6-dihydroxypirimidine-4-carboxylate (LC-MS
analysis) was triturated with Et2O. MS m/z 383 (M + H)+. Step 4:
Crude material obtained in the previous step (assuming quantitative
yield) was dissolved in methanol (15 mL) and treated with an excess
of 4-fluorobenzylamine (0.21 mL, 1.8 mmol, about 3 equiv). The
mixture was refluxed overnight, cooled to room temperature, and
concentrated, allowing the precipitation of crude tert-butyl 3(4-{-
[(4-fluorobenzyl)amino]carbonyl}-5,6-dihydropyrimidin-2-yl)-3,4-
dimethylpiperazine-1-carboxylate as a solid, which was filtered off
and washed with Et2O (0.150 g). MS m/z 476 (M + H)+. Step 5:
Crude material from the previous step was stirred in 10 mL of CH2-
Cl2/TFA (1:1) for 1 h. Evaporation of the solvent afforded 2-(1,2-
dimethylpiperazin-2-yl)-N-(4-fluorobenzyl)-5,6-dihydroxypyrimidine-
4-carboxamide as a crude compound (0.11 g). MS m/z 376 (M +
H)+. Step 6: Crude material obtained in step 5 was dissolved in
methanol (5 mL), and triethylamine (0.19 mL, 1.3 mmol), NaC-
NBH3 (0.054 g, 0.85 mmol), NaOAc (0.158 g, 1.95 mmol), and
37% aqueous HCHO (0.15 mL) were added to the solution under
stirring. The reaction mixture was stirred at room temperature
overnight and then evaporated under reduced pressure to an oily
residue. An aliquot of this residue was purified by preparative RP-
HPLC (C18, acetonitrile/water containing 0.1% trifluoroacetic acid
as eluent), yielding 0.014 g of N-(4-fluorobenzyl)-5,6-dihydroxy-
2-(1,2,4-trimethylpiperazin-2-yl)pyrimidine-4-carboxamide (33) as
its trifluoroacetate salt. 1H NMR (CD3CN + TFA, 600 MHz, 280
K) δ 7.50-7.38 (m, 2 H), 7.13-7.07 (m, 2 H), 4.66-4.51 (m, 2
H), 4.00-3.72 (m, 4.3 H), 3.60-3.56 (t, J ) 12.7 Hz. 0.7 H), 3.49-
3.44 (t, J ) 15.5 Hz, 1 H), 3.04 (s, 2 H), 2.91 (s, 1 H), 2.73 (s, 1
H), 2.69 (bs, 2 H), 2.05 (s, 1 H), 1.95 (2 H, partially obscured by
solvent); MS m/z 390 (M + H)+. HRMS Calcd for C19H25FN5O3
(M + H)+: 390.19359. Found: 390.19353.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-(7-methyl-7-azabicyclo-
[2.1.1]hept-1-yl)pyrimidine-4-carboxamide (34). Step 1: A solu-
tion of benzyl 1-[amino(hydroxyimino)methyl]-7-azabicyclo[2.2.1]-
heptane-7-carboxylate (3 g, 10.4 mmol) in chloroform (45 mL) was
treated with dimethyl acetylenedicarboxylate (1.52 mL, 12.4 mmol)
and refluxed for 5 h. The reaction mixture was cooled to room
temperature and concentrated. The residue was taken up in xylene
(75 mL) and refluxed overnight. The solvent was removed in
vacuum, and the residue was dissolved in pyridine (10 mL) and
treated with 2 equiv of benzoic anhydride (4.7 g, 20.7 mmol). The
reaction mixture was stirred at room temperature for 3 h and then
concentrated under reduced pressure, and the residue was taken up
in ethyl acetate and washed with 1 N HCl and saturated NaHCO3
aqueous solution. The organic phase was dried over Na2SO4,
filtered, and concentrated. Benzyl 1-[5-(benzoyloxy)-4-hydroxy-
6-(methoxycarbonyl)pyrimidin-2-yl]-7-azabicyclo[2.2.1]heptane-7-
carboxylate was purified by flash column chromatography (SiO2,
60/40 petroleum ether/ethyl acetate as eluent) and obtained in 20%
yield (1.04 g, 2.1 mmol). 1H NMR (DMSO-d6, 300 MHz) δ 13.38
(s, 1 H), 8.09 (d, J ) 7.5 Hz, 2 H), 7.80 (t, J ) 7.5 Hz, 1 H), 7.64
(t, J ) 7.5 Hz, 2 H), 7.40-7.22 (m, 5 H), 5.00 (s, 2 H), 4.40 (t, J
) 4.3 Hz, 1 H), 3.76 (s, 3 H), 2.32-2.11 (m, 2 H), 1.95-1.79 (m,
4 H), 1.66-1.51 (m, 2 H); MS m/z 504 (M + H)+. Step 2: The
compound from the previous step (0.200 g, 0.40 mmol) was taken
up in methanol (5 mL) and hydrogenated under H2 atmosphere in
the presence of Pd/C 10% (10% w/w) at room temperature for 2 h.
Catalyst was filtered off, and filtrate was evaporated under reduced
pressure. Residue was dissolved in methanol (5 mL) and treated
with about 3.5 equiv of p-fluorobenzylamine (0.16 mL, 1.40 mmol).
The reaction mixture was refluxed overnight and then cooled to
room temperature, allowing the precipitation of a solid, which was
1
tated was collected by filtration (0.046 g, 48% yield). H NMR
(DMSO-d6, 400 MHz) δ 9.55 (bs, 1 H), 7.35 (dd, J ) 13.7, 7.8
Hz, 2 H), 7.16 (dd, J ) 17.5, 8.8 Hz, 2 H), 4.60-4.40 (m, 2 H),
4.06 (dd, J ) 14.0, 6.9 Hz, 1 H), 3.15-3.10 (m, 1 H), 3.00-2.90
(m, 1 H), 2.20-2.10 (m, 1 H), 1.90-1.70 (m, 3 H); MS m/z 333
(M + H)+. Step 4: To a stirred solution of the above compound
(0.046 g, 0.14 mmol) in MeOH (2 mL) were added Et3N (0.02
mL, 0.14 mmol), NaOAc (0.018 g, 0.22 mmol), AcOH glacial
(0.015 mL), formaldehyde (37 wt % in H2O, 0.03 mL), and NaBH-
(AcO)3 (0.046 g, 0.22 mmol). The reaction mixture was stirred at
room temperature overnight and then poured into aqueous NaHCO3
and extracted with EtOAc. The organic layer was collected, dried
(Na2SO4), filtered, and concentrated under reduced pressure. The
residue was purified by preparative RP-HPLC (C18, water/aceto-
nitrile with 0.1% trifluoroacetic acid as eluent) to give the N-(4-
fluorobenzyl)-5,6-dihydroxy-2-(1-methylpyrrolidin-2-yl)pyrimidine-
4-carboxamide (32) as its trifluoroacetate salt (0.037 g, 57% yield).
1H NMR (DMSO-d6, 400 MHz) δ 13.20 (bs, 1 H), 12.50 (bs, 1
H), 10.0-9.70 (m, 1 H), 9.63 (bs, 1 H), 7.35 (dd, J ) 13.8, 8.2
Hz, 2 H), 7.18 (dd, J ) 17.5, 8.8 Hz, 2 H), 4.54 (m, 2 H), 4.40
(dd, J ) 15.7, 7.7 Hz, 1 H), 3.82-3.70 (m, 1 H), 3.40-3.20 (m,
1 H), 2.94 (s, 3 H), 2.60-2.50 (m, 1 H), 2.20-1.80 (m, 3 H); MS
m/z 347 (M + H)+. HRMS Calcd for C17H20FN4O3 (M + H)+:
347.15140. Found: 347.15021.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-(1,2,4-trimethylpiperazin-
2-yl)pyrimidine-4-carboxamide (33). Step 1: A solution of
1-benzyl 4-tert-butyl-2-[amino(hydroximino)methyl]-2-methylpip-
erazine-1,4-dicarboxylate (2.0 g, 5.10 mmol) in chloroform (30 mL)
was treated with dimethyl acetylenedicarboxylate (0.75 mL, 6.12
mmol) and refluxed for 3 h. The reaction mixture was cooled to
room temperature and concentrated. The residue was taken up in
xylene (30 mL) and refluxed for 24 h. The solvent was removed
in vacuum, and the crude residue was dissolved in pyridine (8 mL)
and treated with benzoic anhydride (1.73 g, 7.65 mmol). The
reaction mixture was stirred at room temperature overnight and then
concentrated under reduced pressure and partitioned between EtOAc
and 1 N HCl. The organic layer was dried (Na2SO4), filtered, and
evaporated, and the 1-benzyl 4-tert-butyl 2-[5-(benzoyloxy)-4-
hydroxy-6-(methoxycarbonyl)pyrimidin-2-yl]-2-methylpiperazine-
1,4-dicarboxylate was purified by flash chromatography on silica
gel eluting with petroleum ether/ethyl acetate 70:30 V/V (0.37 g,
1
12% yield). H NMR (DMSO-d6, 400 MHz, 340 K) δ 12.96 (bs,
1 H), 8.11-8.04 (m, 2 H), 7.79-7.73 (m, 1 H), 7.66-7.58 (m, 2
H), 7.37-7.22 (m, 5 H), 5.03 (s, 2 H), 4.00-3.91 (m, 1 H), 3.80-
3.52 (m, 7 H), 3.47-3.40 (m, 1 H), 1.65 (s, 3 H), 1.35 (s, 9 H);
MS m/z 607 (M + H)+. Step 2: ompound from above (0.37 g,
0.61 mmol) was dissolved in ethyl acetate (15 mL) and hydroge-
nated at 1 atm on 10% w/w Pd/C overnight. Catalyst was filtered
off and solvent evaporated to methyl 5-(benzoyloxy)-2-[4-(tert-
butoxycarbonyl)-2-methylpiperazin-2-yl]-6-hydroxypyrimidine-4-
1
carboxylate (0.29 g, 100% yield). H NMR (DMSO-d6 + TFA,
400 MHz, 340 K) δ 8.11-8.04 (m, 2 H), 7.81-7.74 (m, 1 H),

Dihydroxypyrimidine Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2237
recovered by filtration and washed with Et2O. The solid was taken
up in methanol (5 mL) and treated with NaCNBH3 (0.035 g, 0.56
mmol), NaOAc (0.052 g, 0.64 mmol), and 37% aqueous solution
of formaldehyde (0.04 mL). The reaction mixture was stirred at
room temperature overnight. The cooled reaction mixture was
purified by preparative RP-HPLC (C18, acetonitrile/water containing
0.1% trifluoroacetic acid as eluent), and the product N-(4-fluo-
robenzyl)-5,6-dihydroxy-2-(7-methyl-7-azabicyclo[2.2.1]hept-1-yl)-
pyrimidine-4-carboxamide (34) was isolated as its trifluoroacetate
salt in 25% yield (0.048 g). 1H NMR (DMSO-d6, 400 MHz) δ 12.9
(bs, 1 H), 12.2 (s, 1 H), 10.95 (bs, 1 H), 9.66 (bs, 1 H), 7.47-7.40
(m, 2 H), 7.21-7.12 (m, 2 H), 4.50 (d, J ) 6.0 Hz, 2 H), 4.17 (bs,
1 H), 2.67 (s, 3 H), 2.45-2.1 (m, 6 H), 1.95-1.80 (m, 2 H); MS
m/z 373 (M + H)+. HRMS Calcd for C19H22FN4O3 (M + H)+:
373.16705. Found: 373.16714.
2-[(Dimethylamino)methyl]-N-(4-fluorobenzyl)-5,6-dihydrox-
ypyrimidine-4-carboxamide (35). As for the preparation of 38,
replacing in step 1 benzyl [2-amino-2-(hydroxyimino)-1,1-dimeth-
ylethyl]carbamate with benzyl [2-amino-2-(hydroxyimino)ethyl]-
carbamate. 1H NMR (DMSO-d6, 400 MHz) δ 13.15 (bs, 1 H), 12.33
(bs, 1 H), 9.46 (t, J ) 7.9 Hz, 1 H), 7.34 (dd, J ) 8.8, 5.6 Hz, 2
H), 7.19 (t, J ) 8.9 Hz, 2 H), 4.56 (d, J ) 7.9 Hz, 2 H), 4.27 (s,
2 H), 2.93 (s, 6 H); MS m/z 321 (M + H)+. HRMS Calcd for
C15H18FN4O3 (M + H)+: 321.13575. Found: 321.13562.
2-[1-(Dimethylamino)ethyl]-N-(4-fluorobenzyl)-5,6-dihydrox-
ypyrimidine-4-carboxamide (37). As for the preparation of 38,
replacing in step 1 benzyl [2-amino-2-(hydroxyimino)-1,1-dimeth-
ylethyl]carbamate with tert-butyl [2-amino-2-(hydroxyimino)1-
methylethyl]carbamate, and in step 3 removal of the Boc protecting
group was obtained by stirring in a CH2Cl2:TFA 8:2 solution at
room temperature for 1 h. 1H NMR (DMSO-d6, 400 MHz) δ 13.05
(bs, 1 H), 12.27 (bs, 1 H), 9.51 (t, J ) 6.21 Hz, 1 H), 7.37 (dd, J
) 8.41, 5.56 Hz, 2 H), 7.19 (t, J ) 8.79 Hz, 2 H), 4.59-4.48 (m,
2 H), 4.22 (bs, 1 H), 2.73 (s, 6 H), 1.46 (d, J ) 6.73 Hz, 3 H); MS
m/z 335(M + H)+. HRMS Calcd for C16H20FN4O3 (M + H)+:
335.15140. Found: 335.15030.
Synthesis of Compounds 35, 37, 38, 41-46. Synthesis of 2-[1-
(dimethylamino)-1-methylethyl]-N-(4-fluorobenzyl)-5,6-dihydroxy-
pyrimidine-4-carboxamide (38) is a representative example. Com-
pounds 35, 37, 41-46 were isolated as trifluoroacetate salts by
preparative RP HPLC in yields similar to 38.
2-[1-(Dimethylamino)-1-methylethyl]-N-(4-fluorobenzyl)-5,6-
dihydroxypyrimidine-4-carboxamide (38). Step 1: A solution of
benzyl [2-amino-2-(hydroxyimino)-1,1-dimethylethyl]carbamate (10
g, 40 mmol) in chloroform (250 mL) was treated with dimethyl
acetylenedicarboxylate (5.90 mL, 48 mmol) and stirred at 60 °C
for 3 h. The mixture was cooled to room temperature and
concentrated. The residue was taken up in xylene (0.300 mL) and
refluxed overnight. After the mixture was cooled to room temper-
ature, methyl 2-(1-{[(benzyloxy)carbonyl]amino}-1-methylethyl)-
5,6-dihydroxypyrimidine-4-carboxylate precipitated as a brown solid
and was collected by filtration and washed with diethyl ether (5.92
g, 41% yield). 1H NMR (DMSO-d6, 400 MHz) δ 12.58 (bs, 1 H),
10.21 (bs, 1 H), 7.45-7.25 (m, 5 H), 4.98 (s, 2 H), 3.81 (s, 3 H),
1.48 (s, 6 H); MS m/z 362 (M + H)+. Step 2: A solution of the
above compound (2.0 g, 5.54 mmol) in methanol (75 mL) was
treated with 2 equiv of 4-fluorobenzylamine (1.26 mL, 11.08 mmol)
and refluxed overnight. After evaporation of the solvent, the residue
was poured in EtOAc and washed with 1 N HCl and brine. The
organic phase was dried over Na2SO4, filtered, and concentrated
under vacuum. The solid residue was triturated with diethyl ether
to give benzyl 1-(4-{[(4-fluorobenzyl)amino]carbonyl}-5,6-dihy-
droxypyrimidin-2-yl)-1-methylethylcarbamate (2.16 g, 86% yield).
1H NMR (DMSO-d6, 400 MHz) δ 12.4 (bs, 1 H), 12.3 (bs, 1 H),
9.20 (bs, 1 H), 7.5-7.25 (m, 7 H), 7.16 (t, J ) 8.8 Hz, 2 H), 4.97
(s, 2 H), 4.48 (d, J ) 6.4 Hz, 2 H), 1.51 (s, 6 H); MS m/z 455 (M
+ H)+. Step 3: To a solution of compound from above (2.16 g,
4.76 mmol) in methanol (100 mL) was added 10% Pd/C (10% by
weight). The flask was evacuated, filled with hydrogen, and stirred
under hydrogen atmosphere at room temperature for 1 h. The
catalyst was filtered off and washed with cold methanol, the filtrate
was evaporated to dryness, and the residue was triturated with Et2O
to obtain 2-(1-amino-1-methylethyl)-N-(4-fluorobenzyl)-5,6-dihy-
droxypyrimidine-4-carboxamide as a pale yellow solid in quantita-
tive yield (1.52 g). 1H NMR (DMSO-d6, 400 MHz) δ 7.36 (t, J )
8.2 Hz, 2 H), 7.16 (t, J ) 8.8 Hz, 2 H), 4.46 (d, J ) 6.2 Hz, 2 H),
1.43 (s, 6 H); MS m/z 321 (M + H)+. Step 4: To a stirred solution
of compound from step 3 (1.52 g, 4.76 mmol) in methanol (250
mL) was added an excess (about 11 equiv) of acetic acid and
subsequently NaBH3CN (2.39 g, 38.08 mmol) and formaldehyde
(37 wt % in H2O, 0.9 mL). The mixture was stirred at room
temperature for 5 days, concentrated by rotary evaporation, and
purified by preparative RP-HPLC (C18, water/acetonitrile with 0.1%
trifluoroacetic acid as eluent). Collection and lyophilization of
appropriate fractions afforded compound 38 as its trifluoroacetate
salt in about 90% yield (1.98 g). The white powder was dissolved
in 1 N HCl and lyophilized again to be converted into the
corresponding hydrochloride salt. Mp: 218 °C. 1H NMR (DMSO-
d6, 300 MHz) δ 12.4 (s, 1 H), 10.2 (bs, 2 H), 7.41 (dd, J ) 8.3 Hz,
6.9 Hz, 2 H), 7.16 (t, J ) 8.3 Hz, 2 H), 4.50 (d, J ) 5.9 Hz, 2 H),
2.73 (s, 6 H), 1.60 (s, 6 H); MS m/z 349 (M + H)+. HRMS Calcd
for C17H22FN4O3 (M + H)+: 349.16705. Found: 349.16675.
2-[1-(Dimethylamino)-2,2,2-trifluoro-1-methylethyl]-N-(4-
fluorobenzyl)-5,6-dihydroxypyrimidine-4-carboxamide (41). As
for the preparation of 38, replacing in step 1 benzyl [2-amino-2-
(hydroxyimino)-1,1-dimethylethyl]carbamate with tert-butyl{1-
[amino(hydroxyimino)methyl]-2,2,2-trifluoro-1-methylethyl}-
carbamate, and in step 3 removal of the Boc protecting group was
obtained by stirring in a CH2Cl2:TFA 8:2 solution at room
1
temperature for 1 h. H NMR (DMSO-d6, 600 MHz) δ 12.46 (bs,
1 H), 12.26 (bs, 1 H), 8.73 (bs, 1 H), 7.39 (dd, J ) 8.4, 6.0 Hz, 2
H), 7.17 (t, J ) 9.0 Hz, 2 H), 4.52-4.48 (m, 2 H), 2.28 (s, 6 H),
1.57 (s, 3 H); MS m/z 403 (M + H)+.
2-[1-(Dimethylamino)cyclopropyl]-N-(4-fluorobenzyl)-5,6-di-
hydroxypyrimidine-4-carboxamide (42). As for the preparation
of 38, replacing in step 1 benzyl [2-amino-2-(hydroxyimino)-1,1-
dimethylethyl]carbamate with benzyl {1-[amino(hydroxyimino)-
methyl]cyclopropyl}carbamate. 1H NMR (DMSO-d6, 400 MHz) δ
12.48 (bs, 1 H), 9.40 (bs, 1 H), 7.41 (dd, J ) 8.3 Hz, 6.9 Hz, 2 H),
7.16 (t, J ) 8.3 Hz, 2 H), 4.53 (d, J ) 6.2 Hz, 2 H), 2.75 (bs, 6 H),
1.40 (bs, 4 H); MS m/z 347 (M + H)+. HRMS Calcd for C17H20-
FN4O3 (M + H)+: 347.15140. Found: 347.15079.
2-[1-(Dimethylamino)cyclobutyl]-N-(4-fluorobenzyl)-5,6-dihy-
droxypyrimidine-4-carboxamide (43). As for the preparation of
38, replacing in step 1 benzyl [2-amino-2-(hydroxyimino)-1,1-
dimethylethyl]carbamate with benzyl {1-[amino(hydroxyimino)-
1
methyl]cyclobutyl}carbamate. H NMR (DMSO-d6, 300 MHz) δ
12.90 (bs, 1 H), 12.65 (bs, 1 H), 10.35 (bs, 1 H), 9.43 (bs, 1 H),
7.37 (dd, J ) 8.4, 5.5 Hz, 2 H), 7.16 (t, J ) 8.85 Hz, 2 H), 4.49
(d, J ) 5.97 Hz, 2 H), 2.86 (bs, 2H), 2.63 (s, 6 H), 2.61-2.49 (m,
2 H, partially obscured by solvent), 1.70-1.63 (m, 2 H); MS m/z
361 (M + H)+. HRMS Calcd for C18H22FN4O3 (M + H)+:
361.16705. Found: 361.16815.
2-[1-(Dimethylamino)cyclopentyl]-N-(4-fluorobenzyl)-5,6-di-
hydroxypyrimidine-4-carboxamide (44). As for the preparation
of 38, replacing in step 1 benzyl [2-amino-2-(hydroxyimino)-1,1-
dimethylethyl]carbamate with benzyl {1-[amino(hydroxyimino)-
methyl]cyclopentyl}carbamate. 1H NMR (DMSO-d6, 400 MHz) δ
12.80-12.40 (bs, 2 H), 10.89 (bs, 1 H), 9.85 (bs, 1 H), 7.48 (dd,
J ) 8.4, 6.0 Hz, 2 H), 7.12 (t, J ) 8.8 Hz, 2 H), 4.49 (d, J ) 8.0
Hz, 2 H), 2.80-2.64 (s, 6 H + m, 2 H), 2.22-2.05 (m, 2 H), 1.88-
1.70 (m, 2 H), 1.68-1.50 (m, 2 H);); MS m/z 375 (M + H)+.
HRMS Calcd for C19H24FN4O3 (M + H)+: 375.18270. Found:
375.18109.
2-[1-(Dimethylamino)cyclohexyl]-N-(4-fluorobenzyl)-5,6-dihy-
droxypyrimidine-4-carboxamide (45). As for the preparation of
38, replacing in step 1 benzyl [2-amino-2-(hydroxyimino)-1,1-
dimethylethyl]carbamate with benzyl {1-[amino(hydroxyimino)-
methyl]cyclohexyl}carbamate.1H NMR (DMSO-d6, 300 MHz) δ
12.62 (bs, 1 H), 9.82 (bs, 1 H), 9.41 (bs, 1 H), 7.39 (dd, J ) 8.3
Hz, 6.9 Hz, 2 H), 7.15 (t, J ) 8.3 Hz, 2 H), 4.51 (d, J ) 6.0 Hz,
2 H), 3.09 (bd, 2 H), 2.68 (s, 6 H), 1.80-1.47 (m, 5 H), 1.20-

2238 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Pace et al.
1.02 (m, 3 H); MS m/z 389 (M + H)+. HRMS Calcd for C20H26-
FN4O3 (M + H)+: 389.19835. Found: 389.19928.
m/z 425 (M + H)+. Step 2: A solution of compound from the
previous step (0.21 g, 0.39 mmol) in methanol (5 mL) was treated
with 10% Pd/C, and the mixture was stirred for 2 h under H2
atmosphere. Catalyst was filtered off, and filtrate was evaporated
under vacuum. Compound 40 was isolated as its TFA salt by
2-[4-(Dimethylamino)tetrahydro-2H-pyran-4-yl]-N-(4-fluo-
robenzyl)-5,6-dihydroxypyrimidine-4-carboxamide (46). As the
procedure used for the preparation of 38, replacing in step 1 benzyl
[2-amino-2-(hydroxyimino)-1,1-dimethylethyl]carbamate with tert-
butyl{4-[amino(hydroxyimino)methyl]tetrahydro-2H-pyran-4-yl}-
carbamate, and in step 3 removal of the Boc protecting group was
obtained by stirring in a CH2Cl2:TFA 8:2 solution at room
temperature for 1 h. 1H NMR (DMSO-d6+TFA, 300 MHz) δ 9.85
(bs, 1 H), 9.51 (t, J ) 6.26 Hz, 1 H), 7.37 (dd, J ) 8.54, 5.71 Hz,
2 H), 7.17 (t, J ) 8.85 Hz, 2 H), 4.51 (d, J ) 6.26 Hz, 2 H),
3.93-3.90 (m, 2 H), 3.19-3.11 (m, 4 H), 2.71 (s, 6 H), 1.89-
1.82 (m, 2 H); MS m/z 391 (M + H)+. HRMS Calcd for C19H24-
FN4O4 (M + H)+: 391.17761. Found: 391.17679.
1
preparative RP HPLC (0.15 g, 85% yield). H NMR (DMSO-d6,
400 MHz) δ 12.83 (bs, 1 H), 12.31 (bs, 1 H, 9.46 (t, J ) 6.27 Hz,
1 H), 9.02 (bs, 2 H), 7.37 (dd, J ) 8.48, 5.59 Hz, 2 H), 7.18 (t, J
) 8.82 Hz, 2 H), 4.54 (d, J ) 6.28 Hz, 2 H), 2.48 (s, 3 H), 1.58
(s, 6 H);); MS m/z 335 (M + H)+. HRMS Calcd for C16H20FN4O3
(M + H)+: 335.15140. Found: 335.15222.
2-{1-[Ethyl(methyl)amino]-1-methylethyl}-N-(4-fluorobenzyl)-
5,6-dihydroxypyrimidine-4-carboxamide (39). A solution of 40
(20 mg, 0.04 mmol) in methanol (1 mL) was treated with
acetaldehyde (0.03 mL, 0.05 mmol), Et3N (0.011 mL, 0.08 mmol),
AcOH (0.05 mL, 0.08 mmol), and NaBH3CN (0.007 g, 0.12 mmol).
The resulting mixture was stirred 5 h at room temperature. After
evaporation of volatiles, compound 65 was isolated as its TFA salt
by preparative RP-HPLC (0.010 g, 54% yield). 1H NMR (DMSO-
d6, 400 MHz) δ 12.78 (bs, 1 H), 12.30 (bs, 1 H), 10.49 (bs, 1 H),
10.18 (bs, 1 H),), 7.44 (dd, J ) 8.48 Hz, 5.58 Hz, 2 H), 7.16 (t, J
) 8.80 Hz, 2 H), 4.62-4.51 (m, 1 H), 4.42-4.32 (m, 1 H), 3.21-
3.11 (m, 1 H), 2.98-2.96 (m, 1 H), 2.78 (s, 3 H), 1.66 (s, 3 H),
1.58 (s, 3 H), 1.27 (t, J ) 5.6 Hz, 3 H); MS m/z 362 (M + H)+.
HRMS Calcd for C18H24FN4O3 (M + H)+: 363.18270. Found:
363.18353.
2-{2-[Benzyl(methyl)amino]ethyl}-N-(4-fluorobenzyl)-5,6-di-
hydroxypyrimidine-4-carboxamide (36). Step 1: A solution of
tert-butyl [3-amino-3-(hydroxyimino)propyl]benzylcarbamate (1 g,
3.4 mmol) in chloroform (30 mL) was treated with dimethyl
acetylenedicarboxylate (0.5 mL, 4.09 mmol) and stirred at 60 °C
for 3 h. The reaction mixture was cooled to room temperature and
concentrated. The residue was taken up in xylene (50 mL) and
refluxed overnight. The solvent was removed in vacuum, and a
brown solid was formed upon trituration with CH2Cl2:Et2O 50:50.
The solid was collected by filtration, taken up in methanol (30 mL),
treated with an excess of p-fluorobenzylamine (1.2 mL, 10.2 mmol),
and refluxed overnight. After evaporation of the solvent, the residue
was poured in EtOAc and washed with 1 N HCl and brine. The
organic phase was dried over Na2SO4, filtered, and concentrated
under vacuum. The crude residue was stirred in a 80:20 mixture of
CH2Cl2:TFA (20 mL) for 0.5 h. After evaporation of volatiles, the
residue was purified by preparative RP HPLC, yielding 2-[2-
(benzylamino)ethyl]-N-(4-fluorobenzyl)5,6-dihydroxypyrimidine-
Acknowledgment. We thank Barbara Pacini for the syn-
thesis of compound 1, Anna Alfieri and Francesca Naimo for
plasma protein binding determination and for analytical chem-
istry, Silvia Pesci for NMR spectrometry, and Vincenzo Pucci
for accurate mass measurements.
1
4-carboxamide as its trifluoroacetate salt (0.087 g, 5% yield). H
Supporting Information Available: Synthesis and analytical
data for the preparation of nitriles and amidoximes intermediates.
This material is available free of charge via the Internet at http://
pubs.acs.org.
NMR (DMSO-d6, 400 MHz) δ 12.73 (bs, 1 H), 12.38 (bs, 1 H),
9.34 (bs, 1 H), 8.71 (bs, 1 H), 7.47 (s, 5 H), 7.37 (dd, J ) 8.5, 5.4
Hz, 2 H), 7.28 (t, J ) 8.9 Hz, 2 H), 4.51 (d, J ) 6.3 Hz, 2 H), 4.29
(bs, 2 H), 3.48 (bs, 2 H), 2.93-2.88 (m, 2 H); MS m/z 397 (M +
H)+. Step 2: The compound from the previous step (0.087 g, 0.17
mmol) was taken up in methanol (2 mL) and treated with NaCNBH3
(0.013 g, 0.20 mmol), NaOAc (0.022 g, 0.27 mmol), and 37%
aqueous solution of formaldehyde (0.02 mL). The mixture was
stirred at room temperature for 1 h, concentrated, and purified by
preparative RP HPLC (C18, acetonitrile/water containing 0.1%
trifluoroacetic acid as eluent) to isolate compound 36 as its
trifluoroacetate salt (0.022 g, 25% yield). 1H NMR (DMSO-d6, 400
MHz) δ 12.71 (bs, 1 H), 12.32 (bs, 1 H), 9.40 (bs, 1 H), 9.30 (bs,
1 H), 7.48 (s, 5 H), 7.36 (dd, J ) 8.3 Hz, 6.9 Hz, 2 H), 7.16 (t, J
) 8.3 Hz, 2 H), 4.59-4.49 (m, 3 H), 4.23 (bs, 1 H), 3.78 (bs, 2
H), 3.03 (bs, 2 H), 2.68 (bs, 3 H); MS m/z 411 (M + H)+. HRMS
Calcd for C22H24FN4O3 (M + H)+: 411.18270. Found: 411.18300.
N-(4-Fluorobenzyl)-5,6-dihydroxy-2-[1-methyl-1-(methylami-
no)ethyl]pyrimidine-4-carboxamide (40). Step 1: To a stirred
solution of 0.250 g (0.78 mmol) of 2-(1-amino-1-methylethyl)-N-
(4-fluorobenzyl)-5,6-dihydroxypyrimidine-4-carboxamide (prepared
as described in the synthesis of compound 38, steps 1-3) in
methanol (5 mL), benzaldehyde (0.08 mL g, 0.78 mmol), triethy-
lamine (0.11 mL, 0.78 mmol), acetic acid (0.78 mmol), and NaBH3-
CN (0.147 g, 2.34 mmol) were added. The reaction mixture was
stirred 4 h at room temperature, until disappearance of starting
material (LC-MS monitoring). Formaldehyde (0.06 mL) (37 wt %
in H2O) was added to the reaction, and resulting mixture was stirred
at room temperature overnight. After evaporation of volatiles, 2-{1-
[benzyl(methyl)amino]-1-methylethyl}-N-(4-fluorobenzyl)-5,6-di-
hydroxypyri midine-4-carboxamide was isolated in 50% yield (0.21
g) as its TFA salt by preparative RP HPLC (C18, gradient
acetonitrile/water 0.1% TFA as eluent, from 80/20 to 20/80 in 18
References
(1) Recent reviews on HIV-1 reverse transcriptase inhibitors: (a)
Hogberg, M.; Morrison, I. HIV-1 non nucleoside reverse transcriptase
inhibitors. Expert Opin. Ther. Pat. 2000, 10, 1189-1199. (b)
Jonckheere, H.; Anne, J.; De Clercq, E. The HIV-1 reverse transcrip-
tion (RT) as target for RT inhibitors. Med. Res. ReV. 2000, 20, 129-
154.
(2) Recent reviews on HIV-1 protease inhibitors: (a) Lebon, F.; Ledecq,
M. Approaches to the design of effective HIV-1 protease inhibitors.
Curr. Med. Chem. 2000, 7, 455-477. (b) Vacca, J. P.; Condra, J. H.
Clinically effective HIV-1 protease inhibitors. Drug DiscoVery Today
1997, 2, 261-272.
(3) Cervia, J. S.; Smith, M. A. Enfuvirtide (T-20): a novel human
immunodeficiency virus type 1 fusion inhibitor. Clin. Infect. Diseases
2003, 37, 1102-1106.
(4) For reviews on biology of HIV-1 integrase, see: (a) Esposito, D.;
Craigie, R. HIV Integrase Structure and Function. AdV. Virus Res.
1999, 52, 319-333. (b) Asante-Appiah, E.; Skalka, A. M. HIV-1
Integrase: Structural organization, conformational changes, and
catalysis. AdV. Virus Res. 1999, 52, 351-369.
(5) (a) Engelman, A.; Craigie, R. Identification of conserved amino acid
residues critical for human immunodeficiency virus type 1 integrase
function in vitro. J. Virol. 1992, 66, 6361-6369. (b) Kulkosky, J.;
Jones, K. S.; Katz, R. A.; Mack, J. P.; Skalka, A. M. Residues critical
for retroviral integrative recombination in a region that is highly
conserved among retroviral/retrotransposon integrases and bacterial
insertion sequence transposases. Mol. Cell Biol. 1992, 12, 2331-
2338.
(6) Hazuda, D. J.; Felock, P. J.; Hastings, J. C.; Pramanik, B.; Wolfe,
A. L. Differential divalent cation requirements uncouple the assembly
and catalytic reaction of human immunodeficiency virus type 1
integrase. J. Virol. 1997, 71, 7005-7011.
(7) (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) Wai, J. S.; Egbertson, M. S.; Payne, L. S.; Fisher, T. E.; Embrey,
M. W.; Tran, L. O.; Melamed, J. Y.; Langford, H. M.; Guare, J. P.,
1
min). H NMR (DMSO-d6, 400 MHz) δ 12.80 (bs, 1 H), 12.18
(bs, 1 H), 9.75 (bs, 1 H), 9.28 (bs, 1 H), 7.45-7.35 (m, 5 H), 7.25-
7.19 (m, 4 H), 4.62 (d, J ) 6.5 Hz, 2 H), 4.40-4.32 (m, 1 H),
4.08-3.95 (m, 1 H), 2.81 (s, 3 H), 1.83 (s, 3 H), 1.58 (s, 3 H); MS

Dihydroxypyrimidine Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2239
Jr.; Zhuang, L.; Grey, V. E.; Vacca, J. P.; Holloway, M. K.; Naylor-
Olsen, A. M.; Hazuda, D. J.; Felock, P. J.; Wolfe, A. L.; Stillmock,
K. A.; Schleif, W. A.; Gabryelski, L. J.; Young, S. D. 4-Aryl-2,4-
dioxobutanoic acid inhibitors of HIV-1 integrase and viral replication
in cells. J. Med. Chem. 2000, 43, 4923-4926. (c) Grobler, J. A.;
Stillmock, K.; Hu, B.; Witmer, M.; Felock, P.; Espeseth, A.; Wolfe,
A.; Egbertson, M. S.; Bourgeois, M.; Melamed, J. Y.; Wai, J. S.;
Young, S. D.; Vacca, J. P.; Hazuda, D. J. Diketoacids inhibitor
mechanism and HIV-1 integrase: Implication for metal binding in
the active site of phosphotransferase enzymes. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 6661-6666.
(16) Petrocchi, A.; Koch, U.; Matassa, V. G.; Pacini, B.; Stillmock, K.
A.; Summa, V. From dihydroxypyrimidine carboxylic acids to
carboxamide HIV integrase inhibitors: SAR around the amide
moiety. Bioorg. Med. Chem. Lett. 2007, 17, 350-353.
(17) Gardelli, C. Manuscript in preparation.
(18) Bell, I. M.; Gallicchio, S. N.; Abrams, M.; Beshore, D. C.; Buser,
C. A.; Culbertson, J. C.; Davide, J.; Ellis-Hutchings, M.; Fernandes,
C.; Gibbs, J. B.; Graham, S. L.; Hartman, G. D.; Heimbrook, D. C.;
Homnick, C. F.; Huff, J. R.; Kassaun, K.; Koblan, K. S.; Kohl, N.
E.; Lobell, R. B.; Lynch, J. J.; Miller, P. A.; Omer, C. A.; Rodrigues,
A. D.; Walsh, E. S.; Williams, T. M. Design and biological activity
of (S)-4-(5-{[1-(3-chlorobenzyl)-2-oxopyrrolidin-3-ylamino]methyl}-
imidazol-1-ylmethyl)benzonitrile, a 3-aminopyrrolidinone farnesyl-
transferase inhibitor with excellent potency. J. Med. Chem. 2001,
44, 2933-2949.
(19) Henry, D. W. Chemotherapeutic nitroheterocycles. Derivatives of
5-nitrothiazole-2-carboxaldehyde and 5-nitrothiazole-2-carboxylic
acid. J. Med. Chem. 1969, 12, 303-306.
(20) Bigge, C. F.; Hays, S. J.; Novak, P. M.; Drummond, J. T.; Johnson,
G.; Bobovski, T. P. New preparations of the N-methyl-D-aspartate
receptor antagonist, 4-(3-phosphonopropyl)-2-piperazinecarboxylic
acid (CPP). Tetrahedron Lett. 1989, 30, 5193-5196.
(21) Krow, G. R.; Herzon, S. B.; Lin, G.; Qiu, F.; Sonnet, P. E. Complex
induced proximity effects. Temperature dependent regiochemical
diversity in lithiation-electrophilic substitution reactions of N-Boc-
2-azabicyclo[2.1.1]hexane. 2,4- and 3,5-methanoprolines. Org. Lett.
2002, 4, 3151-3154.
(22) Organic Synthesis; Wiley: New York, Coll. Vol. II, p 29.
(23) Nakajima, N.; Saito, M.; Ubukata, M. Activated dimethyl sulfoxide
dehydration of amide and its application to one-pot preparation of
benzyl-type perfluoroimidates. Tetrahedron 2002, 58, 3561-3577.
(24) Orjales, A.; Mosquera, R.; Toledo, A.; Pumar, C. M.; Garc´ıa, N.;
Cortizo, L.; Labeaga, L.; Innera´riti, A. Syntheses and binding studies
of new [(aryl)(aryloxy)methyl]piperidine derivatives and related
compounds as potential antidepressant drugs with high affinity for
serotonin(5-HT) and norepinefrine (NE) transporters. J. Med. Chem.
2003, 46, 5512-5532.
(25) Shono, T.; Terauchi, J.; Kitajiama, K.; Takeshima, Y.; Matsumura,
Y. Electroorganic chemistry 139. Electroreductive decyanation of
nitriles and its application to synthesis of R-alkylamines. Tetrahedron
1992, 48, 8253-8262.
(26) Kohrt, A.; Hartke, K. Dithio and thiono esters, 53. Synthesis of
N-protected R-amino dithioesters from R-amino nitriles and R-amino
acids. Liebigs Ann. Chem. 1992, 6, 595-606.
(8) Summa, V.; Petrocchi, A.; Pace, P.; Matassa, V. G.; De Francesco,
R.; Altamura, S.; Tomei, L.; Koch, U.; Neuner, P. Discovery of R,γ-
diketoacids as potent selective and reversible inhibitors of hepatitis
C virus NS5b RNA-dependent RNA polymerase. J. Med. Chem.
2004, 47, 14-17.
(9) De Francesco, R.; Tomei, L.; Altamura, S.; Summa, V.; Migliaccio,
G. Approaching a new era for hepatitis C virus therapy: inhibitors
of the NS3-4A serine protease and the NS5B RNA-dependent RNA
polymerase. AntiViral Res. 2003, 58, 1-16.
(10) Pace, P.; Nizi, E.; Pacini, B.; Pesci, S.; Matassa, V. G.; 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) (a) 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-
dihydroxypyrimidine or 3-methyl-5-hydroxypyrimidinonecarboxylic
acids. Design and synthesis. J. Med. Chem. 2004, 47, 5336-5339;
(b) 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 Franceso, 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, ASAP.
(12) Zhuang, L.; Wai, J. S.; Embrey, M. W.; Fisher, T. E.; Egbertson, M.
S.; Payne, L. S.; Guare, J. P.; 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.; 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.
(13) Vacca, J. P.; Dorsey, B. D.; Schleif, W. A.; Levin, R. B.; McDaniels,
S. L.; Darke, P. L.; Zugay, J.; Quintero, J.; Blahy, O. M.; Roth, E.;
Sardana, V. V.; Schlabach, A. J.; Graham, P. I.; Condra, J. H.; Gotlib,
L.; Holloway, M. K.; Lin, J.; Chen, I.; Vastag, K.; Ostovic, D.;
Anderson, P. S.; Emini, E. A.; Huff, J. R. L-735,524: an orally
bioavailable human immunodeficiency virus type I protease inhibitor.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4096-4100.
(14) Culbertson, T. P. Synthesis of methyl 5,6-dihydroxy-2-phenyl-4-
pyrimidinecarboxylate: a corrected structure. J. Heterocycl. Chem.
1979, 16, 1423-1424.
(15) Hazuda, D. J.; Felock, P.; Witmar, 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.
(27) Branco, P. S.; Prabhakar, S.; Lobo, A. M.; Williams, D. J. Reaction
of hydroxylamines with ethyl cyanoformate. Preparation of amino-
nitrones and their synthetic applications. Tetrahedron 1992, 48,
6335-6360.
(28) Hamze, A.; Hernandez, J. F.; Fulcrand, P.; Martinez, J. Synthesis of
various 3-substituted 1,2,4-oxadiazole-containing chiral â3- and
R-amino acids from Fmoc-protected aspartic acid. J. Org. Chem.
2003, 68, 7316-7321.
JM070027U