Dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.07.092

A series of potent novel dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 6 is active against replication of HIV

Full text of DOI:10.1016/j.bmcl.2007.07.092

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5595–5599
Dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors
John S. Wai,a,* Boyoung Kim,a Thorsten E. Fisher,a Linghang Zhuang,a
Mark W. Embrey,a Peter D. Williams,a Donnette D. Staas,a Chris Culberson,a
Terry A. Lyle,a Joseph P. Vacca,a Daria J. Hazuda,b Peter J. Felock,b
William A. Schleif,c Lori J. Gabryelski,c Lixia Jin,d I-Wu Chen,d
Joan D. Ellis,d Rama Mallaie and Steven D. Younga
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Vaccine and Biologics Research, Merck Research Laboratories, West Point, PA 19486, USA
dDepartment of Drug Metabolism and Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA
eDepartment of Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 29 June 2007; revised 26 July 2007; accepted 27 July 2007
Available online 22 August 2007
Abstract—A series of potent novel dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors was identified. These compounds
inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 6 is active against replication of
HIV with a CIC95 of 0.31 lM and exhibits no shift in potency in the presence of 50% normal human serum. It displays a good phar-
macokinetic profile when dosed in rats and no covalent binding with microsomal proteins in both in vitro and in vivo models.
Ó 2007 Elsevier Ltd. All rights reserved.
Human immunodeficiency virus-type 1 (HIV-1) is the
etiological agent of acquired immunodeficiency syn-
drome (AIDS). The unique nature of the replicative cy-
cle of HIV-1 provides many potential targets for
chemotherapeutic intervention. One of these, the viral
enzyme integrase, catalyzes the insertion of the proviral
DNA into the genome of host cells. Integration is a mul-
tistep process which includes three different biochemical
steps: assembly of the proviral DNA on integrase, endo-
nucleolytic processing of the proviral DNA, and strand
transfer of the proviral DNA to host cell DNA.1
ery, SAR, computer modeling, pharmacokinetic profile,
and synthesis of a novel series of dihydroxypyridopyr-
azine-1,6-diones HIV-1 integrase inhibitors.
Incorporation of the dihydroxycarbonyl pharmaco-
phore (in red) into a pyridinone scaffold led to the
dihydroxypyridinone5 moiety on the right-hand portion
of the bicyclic system 4. Addition of the benzyl amide
functionality (in blue) and imposing restrictions on the
rotation of the amide side chain via a cyclic constraint
(in magenta) led to the novel dihydroxypyridinone-car-
boxylic derivatives depicted as 4. Literature searches
revealed no precedent for the preparation of such struc-
ture (Fig. 1).
1,3-Diketoacids 1 were reported to be effective integrase
inhibitors and prevent HIV-1 replication in cell culture.2
Recently, novel naphthyridines 2 and 3 were identified
to be suitable replacements for the diketoacid pharma-
cophore.3 Furthermore, naphthyridine carboxamide 3
was found to be efficacious in rhesus macaques infected
with the simian-human immunodeficiency virus (SHIV)
89.6P.4 In this communication, we describe the discov-
Among the three different ring constrained compounds
prepared, compounds 6 and 7 were found to be signifi-
cantly more active against HIV integrase than com-
pound 5 (Table 1). Molecular modeling (MMFF)7 of
the bicyclic core suggests that the amide carbonyl group
in the constrained seven-membered ring compound 5 is
not coplanar relative to the remaining pharmacophore
(Fig. 2), while in both 6-membered ring constrained ana-
logs 6 and 7, the amide groups are coplanar with respect
to the dihydroxypyridinone core.
Keywords: HIV; Integrase; Strand transfer inhibitors; Dihydroxypyr-
idopyrazine-1, 6-dione.
*
Corresponding author. Tel.: +1 215 652 3020; e-mail:
john_wai@merck.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.092

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J. S. Wai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5595–5599
R
R
O
N
R'
R'
OH
N
O
OH
O
OH
2
1
N
O
O
O
N
R"
N
N
R
H
R'
N
N
OH
N
O
OH
O
OH
3
4
Figure 1. Conception of dihydroxypyridopyrazine-1,6-diones inhibitors.
Table 1. Effect of different constraints
O
X
F
N
N
OH
O
OH
Compound
X
Inhibition
of strand transfer
Figure 4. Two low energy conformers of compound 6.
Table 2. Effect of substitutions on benzyl group
a
IC50 (lM)
5
6
7
CH2CH2CH2
CH2CH2
1.04 ( 0.05)
0.10 ( 0.06)
0.04 ( 0.02)
O
CH@CH
R
a Assays were performed with recombinant HIV-integrase (0.1 lM)
preassembled on immobilized oliogonucleotides.6 Values are means
of three experiments, standard deviation is given in parentheses.
4
N
N
3
OH
2
O
OH
Compound
R
Inhibition Antiviral activity
of strand transfer in cell culture,
a
b
IC50 (lM)
CIC95 (lM)
6
8
4-F
H
0.10 ( 0.06)
0.23 ( 0.06)
0.37 ( 0.10)
0.04 ( 0.01)
0.38 ( 0.07)
0.31 ( 0.08)
2.50 ( 0.12)
>1.00
9
2-Cl
3-Cl
4-Cl
10
11
12
0.50 ( 0.11)
1.00 ( 0.18)
0.25 ( 0.03)
4-F, 3-Cl 0.04 ( 0.02)
a Assays were performed with recombinant HIV-integrase (0.1 lM)
preassembled on immobilized oliogonucleotides.6 Values are means
of three experiments, standard deviation is given in parentheses.
b Cell culture inhibitory concentrations (CIC95) are defined as those
which inhibited by > 95% the spread of HIV-1 infection in MT-4
human T-lymphoid cells maintained in RPMI 1640 medium con-
taining 10% heat-inactivated fetal bovine serum.8 Cytotoxicity is not
observed in cell culture at concentrations up to 20 lM.
Figure 2. Molecular modeling of bicyclic core of inhibitors 5–7.
O
F
N
N
(bond 2). The two lower energy conformers differ only
by rotation about bond 1 and are depicted in Figure. 4.
This suggests that the bicyclic dihydroxypyrido-pyra-
zine-1,6-dione significantly limited conformation flexi-
bility of the 4-fluorobenzyl side chain in compound 6.
OH
2
1
O
OH
Figure 3. Torsional search on compound 6.
It was anticipated that the cyclic constraint would also
bias the orientation of the benzyl side chain through ste-
ric interaction. Torsional search on compound 6 (vary-
ing the two bonds identified as 1 and 2 in Fig. 3)
revealed four low energy regions. Two of the minima
correspond to rotations about the 4-fluorophenyl ring
Compound 6 inhibits 95% of the replication of HIV-1 in
cell culture at 0.31 lM (Table 2). There is no shift in
potency when the compound is assayed in the presence of
50% normal human serum.8 Removal of the 4-fluoro
substitution in compound 6 leads to a significant drop in

J. S. Wai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5595–5599
5597
potency (cf. compound 8). A chloro substitution walk
around the benzyl group shows that a 3-chloro substituent
improves integrase inhibition relative to the 2- and 4-posi-
tion isomers (compounds 10 vs 9 and 11). However, com-
bining both the 4-fluoro and 3-chloro substituents only
leads to a modest improvement in inhibitory potency in
the cell based assay (compound 12). These compounds
do not exhibit cytotoxicity in cell culture at concentrations
up to 20 lM.
ylcellulose (Chart 1). Plasma concentrations of com-
pound 6 were maintained between 0.64 and 0.50 lM
from the second to the twenty-fourth hour. This is well
above the concentration (0.31 lM) required for >95%
inhibition of HIV-1 replication in cell culture.
There was a general concern about the biological liability
of a dihydroxypyridinone core and its potential metabo-
14
lites. C Labeled compound 6 was prepared9 to assess
whether this series of compounds would irreversibly inter-
act with liver microsomal proteins. Tables 4 and 5 depict
the findings. There is only a low level of covalent interac-
tion for radiolabeled 6 with liver microsomes derived from
rat and human, well within the <50 pmol equiv/mg/60 min
criteria (Table 4).10 Furthermore, there is no difference in
the results obtained with or without addition of NADPH.
Results obtained in vivo in rat are also consistent with the
observation made in vitro (Table 5).
In our attempt to attenuate the acidity of the 8-hydroxyl
group to mimic diketoacid 1, electron-withdrawing sub-
stituents were incorporated adjacent to the 8-hydroxyl
group on the bicyclic system. This led to a significant
improvement in their intrinsic potency (Table 3, com-
pounds 13, 14, 15 vs 6). However, this did not translate
into improvements in antiviral activity. One possible
explanation is that the equilibrium between neutral and
ionized inhibitor has shifted to an extent which adversely
affects the cell permeability of these analogs.
The synthesis of compound 6 is depicted in Scheme 1.
Reductive alkylation of dimethoxyethylamine with 4-
fluorobenzaldehyde 16 in the presence of sodium boro-
hydride provided the corresponding benzylamine, which
was treated with a mixture of N-acetylglycine, EDC, and
HOBt in DMF to provide the bis amide 17. Acid cata-
lyzed cyclization11 of 17, followed by hydrogenation of
the resultant product 18 in the presence of 5% Pd/C in
ethanol, provided the acetyl piperazinone 19. Treatment
of 19 in anhydrous DMF with LiHMDS, followed by
addition of diethyl oxalate, led to three oxalation prod-
ucts, one major and two minor ones, as indicated by LC-
MS analysis. Upon addition of more base, product 6
was generated from one of the minor oxalation prod-
ucts. The other two resisted cyclization and decomposed
upon further exposure to base. The crude product mix-
ture was purified by C-18 reverse phase HPLC eluting
with a water–acetonitrile gradient.
The bioavailability of compound 6 was 69% when dosed
orally in rats at 10 mg/Kg as a solution in 1% aq meth-
Table 3. Effect of substitutions on pyridopyrazine-1,6-dione core
O
6
F
R'
N
8
N
1
OH
O
OH
Compound
R0
Inhibition
of strand transfer
Antiviral activity
in cell culture,
a
b
IC50 (lM)
CIC95 (lM)
6
13
14
15
H
0.10 ( 0.06)
0.02 ( 0.06)
0.03 ( 0.01)
0.02 ( 0.02)
0.31 ( 0.08)
>1.00
CN
Br
I
1.00 ( 0.16)
>1.00
Table 4. In vitro covalent binding of [14C] compound 6 with liver
microsomes
a Assays were performed with recombinant HIV-integrase (0.1 lM)
preassembled on immobilized oliogonucleotides.6 Values are means
of three experiments, standard deviation is given in parentheses.
b Cell culture inhibitory concentrations (CIC95) are defined as those
which inhibited by > 95% the spread of HIV-1 infection in MT-4
human T-lymphoid cells maintained in RPMI 1640 medium con-
taining 10% heat-inactivated fetal bovine serum.8 Cytotoxicity is not
observed in cell culture at concentrations up to 20 lM.
O
F
N
N
*
OH
*
O
OH
Species
Irreversibly bound radioactivity
(pmol equiv/mg/60 min)
ꢀNADPH
24.7
15.2
+NADPH
Compound 6
Rat
Human
24.2
19.6
100
0010
0011
0012
10
1
Average
Table 5. In vivo covalent binding of [14C] Compound 6 in rats
Time (h)
Irreversibly bound radioactivity
(pmol equiv/mg protein)
Plasma
Liver
0.1
0
10
15
20
25
30
5
2
6
<5
<5
<5
<5
<5
<5
Time (Hours)
24
Chart 1. Oral pharmacokinetic profile of compound 6 in rat.

5598
J. S. Wai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5595–5599
OMe
HN
identified. The lead compound 6 inhibits replication of
O
MeO
N
F
HIV-1 in cell culture at CIC95 = 0.31 lM, and exhibits
no significant shift in potency when assayed with addi-
tion of 50% normal human serum. No cytotoxicity is ob-
served in cell culture at concentrations up to 20 lM.
When compound 6 was dosed orally in rat at 10 mg/kg
as a solution in 1% methylcellulose, it displayed good
pharmacokinetic profile and maintained excellent expo-
sure through 24 h of the study. No covalent binding of
compound 6 with microsomal proteins was observed
in both vitro and in vivo models. Further exploration
of this dihydroxypyridopyrazine-1,6-dione template
and analogous bicyclic systems is in progress.
F
a
CHO
17
O
b
16
O
O
F
F
N
c
N
N
O
N
18
O
19
d
O
References and notes
F
N
N
O
1. For a recent review on the structure and function of HIV-1
integrase, see: (a) Davies, D. R.; Chiu, T. K. Curr. Top.
Med. Chem. 2004, 4, 965; (b) Pommier, Y.; Johnson, A.
A.; Marchand, C. Nat. Rev. Drug Discov. 2005, 4, 236; For
a recent review on HIV-1 integrase inhibitors, see:
Gordon, C. P.; Griffith, R.; Keller, P. A. Med. Chem.
2007, 3, 199; Deng, J.; Dayam, R.; Al-Mawsawi, L. Q.;
Neamati, N. Curr. Pharm. Des. 2007, 13, 129; Anthony,
N. J. Curr. Top. Med. Chem. 2004, 4, 979.
OH
OH
6
Scheme 1. Synthesis of compound 6. Reagents: (a) i—
H2NCH2CH(OMe)2, NaBH4, MeOH (82%); ii—N-Acetyl-Gly, EDC,
HOBt, i-Pr2NEt, DMF (95%); (b) MsOH, CH2Cl2 (62%); (c) H2, 5%
Pd/C, EtOH (95%); (d) i—LiHMDS, diethyl oxalate, DMF; ii—excess
LiHMDS (15–20%).
NMR studies of the purified oxalation products led to the
identification of the major component as 20, and one of
the minor oxalation products as 22. Treatment of each
one of them independently with LiHMDS in DMF did
not lead to cyclization product 6. The third oxalation
product readily cyclized to compound 6 upon workup.
However, 20 was readily converted to the third oxalation
product when exposed to a mixture of acetonitrile, water,
and trifluoroacetic acid. NMR data of this material are
consistent with those of structure 21. Treatment of 21 in
DMF with LiHMDS led to the cyclization product 6.
2. 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., 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. J. Med. Chem. 2000,
43, 4923.
3. For monocyclic pyrimidinone integrase inhibitors, see (a)
Zhuang, L.; Wai, J. S.; Embrey, M. W.; Fisher, T. E.;
Egbertson, M. S.; Payne, L. S.; Guare, J. P., Jr.; Vacca, J.
P.; Hazuda, D. J.; Felock, P. J.; Wolfe, A. L.; Stillmock,
K. A.; Witmer, M. V.; Moyer, G.; Schleif, W. A.;
Gabryelski, L. J.; Leonard, Y. M.; Lynch, J. J., Jr.;
Michelson, S. R.; Young, S. D. J. Med. Chem. 2003, 46,
453; (b) Hazuda, D. J.; Anthony, N. J.; Gomez, R. P.;
Jolly, S. M.; Wai, J. S.; Zhuang, L.; Fisher, T. E.; Embrey,
M. W.; Guare, J. P., Jr.; Egbertson, M. S.; Vacca, J. P.;
Huff, J. R.; Felock, P. J.; Witmer, M. V.; Stillmock, K. A.;
Danovich, R.; Grobler, J.; Miller, M. D.; Espeseth, A. S.;
Jin, L.; Chen, I.-W.; Lin, J.; Kassahun, K.; Ellis, J. D.;
Wong, B. K.; Xu, W.; Pearson, P. G.; Schleif, W. A.;
Cortese, R.; Emini, E.; Summa, V.; Holloway, M. K.;
Young, S. D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
11233.
4. Hazuda, D. J.; Young, S. D.; Guare, J. P., Jr.; Anthony,
N. J.; Gomez, R. P.; Wai, J. S.; Vacca, J. P.; Handt, L.;
Motzel, S. L.; Klein, H. J.; Dornadula, G.; Danovich, R.
M.; Witmer, M. V.; Wilson, K. A. A.; Tussey, L.; Schleif,
W. A.; Gabryelski, L. S.; Lin, J.; Miller, M. D.; Casimiro,
D. R.; Emini, E. A.; Shiver, J. W. Science 2004, 305, 528–
532.
5. (a) Summa, V.; Petrocchi, A.; Matassa, V.; Gardelli, C.;
Muraglia, E.; Rowley, M.; Paz, O. G.; Laufer, R.;
Monteagudo, E.; Pace, P. J. Med. Chem 2006, 49, 6646;
(b) Petrocchi, A.; Koch, U.; Matassa, V. G.; Pacini, B.;
Stillmock, K. A.; Summa, V. Biorg. Med. Chem. Lett.
2007, 17, 350.
O
F
N
N
O
19
LiHMDS, DMF
diethyl oxalate
O
O
O
OH
CO2R
N
N
N
+
+
N
CO2R
OH
N
OH
CO2R
20 (major)
N
O
O
O
22 (minor)
21 (minor)
O
N
N
OH
O
OH
6
6. Hazuda, D. J.; Felock, P.; Hastings, J. C.; Pramanik, B.;
Wolfe, A. J. Virol. 1997, 71, 7005, Assays were performed
with recombinant HIV-1 integrase (0.1 lM) preassembled
on immobilized oligonucleotides. Inhibitors were either
In summary, a series of potent dihydroxypyrido-pyra-
zine-1,6-dione HIV-1 integrase inhibitors which inhib-
ited replication of HIV-1 in cell culture has been

J. S. Wai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5595–5599
5599
added during assembly without washing or subsequent to
assembly and washings. Inhibition was determined in
relation to the integrase control reaction (without inhib-
itor) performed in quadruplicate and averaged. All sam-
ples were background subtracted.
tained in RPMI 1640 medium containing 10% heat
inactivated fetal bovine serum. Cells were infected en
masse at low multiplicity (0.01) using HIV-1 strain IIIb
and were incubated for 24 h. At this time, cells were
washed and distributed into 96-well microtiter dishes.
Serial 2-fold dilutions of inhibitor were added to the wells
and the cultures were maintained for three additional
days. Virus spread was assessed by HIV-1 p24 core antigen
ELISA. Control cultures in the absence of inhibitor were
fully infected at 4 days.
7. Spartan ’04 Windows ES 2.0.0. 2003.
8. Vacca, J. P.; Dorsey, B. D.; Schleif, W. A.; Levin, R. B.;
McDaniel, S. L.; Darke, P. L.; Zugay, J.; Quintero, J. C.;
Blahy, O. M.; Roth, E.; Sairdana, V. V.; Schlabac, A. J.;
Graham, P. I.; Condra, J. H.; Gotlib, L.; Holloway, M.
K.; Lin, J.; Chen, I.-W.; Vastag, K.; Ostovic, D.; Ander-
son, P. S.; Emini, E. E.; Huff, J. R. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 4096–4100, 95% Cell culture inhibitory
concentrations (CIC95) are defined as those which inhib-
ited by P95% the spread of HIV-1 infection in susceptible
cell culture. MT-4 human T-lymphoid cells were main-
9. 14C-labeled compound 6 was prepared as described in
Scheme 1 using 14C-labeled diethyl oxalate.
10. Evans, D. C.; Watt, A. P.; Nicoll-Griffith, D. A.; Baillie,
T. A. Chem. Res. Toxicol. 2004, 17, 3–16.
11. Kim, J. H.; Lee, Y. S.; Kim, S. S. Heterocycles 1998, 48,
2279–2286.