Design and synthesis of substituted 4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-2-carboxamides, novel HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.11.049

A series of 4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-2-carboxamides was synthesized and tested for their inhibition of HIV-1 integrase catalytic activity and HIV-1 replication in cells. Structure-activity studies around lead compound 5 indicated that a coplanar relationship of metal-binding heteroatoms provides optimal binding to the integrase active site. Identification of potency-enhancing substituents and adjustments in lipophilicity provided 17b which inhibits integrase-catalyzed strand transfer with an IC₅₀ value of 74 nM and inhibits HIV-1 replication in cell culture in the presence of 50% normal human serum with an IC₉₅ value of 63 nM.

Full text of DOI:10.1016/j.bmcl.2007.11.049

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 721–725
Design and synthesis of substituted 4-oxo-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrazine-2-carboxamides,
novel HIV-1 integrase inhibitors
H. Marie Langford,a,* Peter D. Williams,a Carl F. Homnick,a Joseph P. Vacca,a
Peter J. Felock,b Kara A. Stillmock,b Marc V. Witmer,b Daria J. Hazuda,b
Lori J. Gabryelskic and William A. Schleifc
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
Received 29 October 2007; revised 12 November 2007; accepted 14 November 2007
Available online 19 November 2007
Abstract—A series of 4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-2-carboxamides was synthesized and tested for their inhibi-
tion of HIV-1 integrase catalytic activity and HIV-1 replication in cells. Structure–activity studies around lead compound 5 indi-
cated that a coplanar relationship of metal-binding heteroatoms provides optimal binding to the integrase active site.
Identification of potency-enhancing substituents and adjustments in lipophilicity provided 17b which inhibits integrase-catalyzed
strand transfer with an IC50 value of 74 nM and inhibits HIV-1 replication in cell culture in the presence of 50% normal human
serum with an IC95 value of 63 nM.
Ó 2007 Elsevier Ltd. All rights reserved.
The causative agent of acquired immune deficiency syn-
drome (AIDS) is the human immunodeficiency virus
type 1 (HIV-1). Currently marketed therapies for treat-
ing HIV-1 infection include drugs which target two of
the three viral enzymes required for replication, reverse
transcriptase and protease. Resistance to these drugs is
increasing at an alarming rate, and thus there is a need
to develop new agents which work by different mecha-
nisms. The third viral enzyme, integrase, is used by the
virus to insert double stranded proviral DNA into host
chromosomal DNA. The three-step integration process
includes assembly of integrase and proviral DNA, endo-
nucleolytic processing of proviral DNA catalyzed by
integrase, and strand transfer of proviral DNA into
the host cell DNA catalyzed by integrase.1 Several
integrase inhibitors which work by inhibiting the strand
transfer step have progressed to the stage of clinical test-
ing in patients infected with HIV-1 and have shown very
promising results.2
A recent report from our laboratories3 described pyrrol-
opyrazinone 1 as a novel mimetic of the metal-binding
diketoacid portion4 of diketoacid integrase inhibitors
(Fig. 1). Consistent with previous observations in
another structural series,5 modifications of 1 which rein-
force a coplanar relationship between important metal-
binding functionalities, for example, cyclization to
lactam 2, were found to improve potency in biochemical
and cell-based assays. Another way of facilitating a
coplanar arrangement between metal-binding function-
alities in 1 was envisioned with pyrazolopyrazinone 3.
H
H
N
OH
N
N
R
O
O
O
OH
O
OH
Diketoacid
1
2
H
N N
N
N
N
N
N
O
O
O
O
OH
OH
Keywords: HIV-1; Integrase inhibitor; Antiviral.
*
3
Corresponding author. Tel.: +1 215 652 8116; fax: +1 215 652
3971; e-mail: hmarie_langford@merck.com
Figure 1.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.049

722
H. M. Langford et al. / Bioorg. Med. Chem. Lett. 18 (2008) 721–725
This modification replaces a non-bonded interaction be-
tween amide and pyrrole protons with a potentially
attractive hydrogen bonding interaction between the
amide proton and pyrazole nitrogen. Herein we describe
the synthesis of novel pyrazolopyrazinone HIV-1 integr-
ase inhibitors and structure–activity relationships which
led to the discovery of analogs with nanomolar potency
in biochemical and cell-based assays.
zole nitrogen was alkylated with BocNHCH2CH(OMs)-
CO2Et7 to give 23. The Boc group was removed under
acidic conditions and basic work-up induced closure of
the lactam ring. The pyrazinone nitrogen was alkylated
and the ethyl ester was selectively hydrolyzed using one
equivalent of NaOH to give 24. Formation of the acid
chloride, followed by treatment with diazomethane
and then HCl, gave an a-chloroketone which was cyc-
lized to an aminothiazole. Straightforward manipula-
tions gave 14. Route 4 differed from the three previous
routes in that the amide bond was formed first, followed
by ring closure onto the pyrazole nitrogen. Mono acid
25 was produced in high yield using dimethylhydrazine.8
Peptide coupling of 25 to an amino alcohol derivative,
R2NHCH2CHR3OH, provided 26 which then under-
went intramolecular Mitsunobu reaction to give bicycle
27. Straightforward manipulations provided 7, 8, 12, 13,
15, and 16. Asymmetric syntheses of 17a and 17b were
achieved using the S and R isomers of N-methyl 2-hy-
droxy-2-phenylethylamine,9 respectively. Enantiomeri-
cally pure 17a and 17b were obtained by final
purification on a chiral column to remove traces of the
other enantiomer (ChiralPak AS column, 80:10:10 hex-
ane/MeOH/EtOH eluant with 0.1% TFA modifier,
retention time 17a = 13.96 min, 17b = 11.09 min).
All of the inhibitors were synthesized starting with di-
4-(benzyloxy)-1H-pyrazole-3,5-dicarboxylate6
methyl
(see Scheme 1). In Route 1, the pyrazole nitrogen was
alkylated using 1,2-dibromoethane or 1,3-dibromopro-
pane followed by displacement of the bromine using so-
dium azide to give 18. Staudinger reduction of the azide
was accompanied by ring closure to 19, and straightfor-
ward manipulations led to 5, 6, and 11. In Route 2, the
pyrazole nitrogen was alkylated using chloroacetone or
2-chloroacetophenone to give 20. When R4 was methyl,
reductive amination of the ketone using sodium triacet-
oxyborohydride and methylamine proceeded smoothly
and was accompanied by lactam ring closure to 21.
When R4 was phenyl, the reductive amination/ring clo-
sure was problematic, and it was necessary to first form
a mono-amide and then close the ring by heating in the
presence of toluene sulfonic acid to form enamide 22.
The double bond was then reduced and straightforward
manipulations provided 9 and 10. In Route 3, the pyra-
To address the question of the metal-binding pharmaco-
phore geometry and binding to the integrase active site,
N3
n
O
n
R2
n
NH
N
N
d, e, f
N
N
N
N
R1HN
c
N
O
O
a, b
O
O
O
O
O
O
OH
OBn
OBn
18
19
5, 6, 11
1
e
t
R4
u
N
N
R4
o
N
O
h
d, e, f
l, m
g
R
R4 = CH3
2
O
O
N
N
N
N
R1HN
21
22
O
O
e
N
t
OBn
O
u
Ph
o
i, j, k
R4 = Ph
R
O
O
O
O
N
OH
9, 10
N
OBn
N
O
20
N
NH
O
O
O
O
OBn
O
NH2
N
O
O
OBn
OEt
S
N
OH
O
3
e
t
u
NH-Boc
O
N
N
N
o
N
N
R1HN
o, p, q
n
N
N
O
r, s, t, u, d, e, f
O
R
O
4
e
O
O
O
O
O
t
OH
OBn
OBn
u
o
14
23
24
R
R3
v
R3
R3
HO
N
NH
w
x
d, e, f
N
NH
N
N
N
N
O
OH
O
N
R2
N
R2
N
O
O
R1HN
R2
O
O
O
O
O
O
O
OBn
OBn
OBn
OH
25
27
26
7, 8, 12, 13, 15, 16, 17a, 17b
Scheme 1. Reagents and conditions: (a) BrCH2(CH2)nBr (n = 1 or 2), Cs2CO3, DMF; (b) NaN3, DMF; (c) PPh3, DMF, 0 °C, H2O, 90 °C; (d) NaOH,
MeOH; (e) R1NH2, EDC, HOBT, DMF; (f) H2, 10% Pd/C, MeOH; (g) R4COCH2Cl, Cs2CO3, DMF; (h) NaBH(OAc)3, MeNH2, CH2Cl2; (i) NaOH,
MeOH/THF, 50 °C; (j) MeNH2, EDC, HOBT, DMF; (k) TsOH, toluene, 125 °C; (l) PtO2, H2, MeOH; (m) R1NH2, MeOH, reflux; (n)
BOCNHCH2CH(OMs)CO2Et, Cs2CO3, CH3CN; (o) TFA, CH2Cl2, basic work-up; (p) MeI, NaH, DMF; (q) NaOH, MeOH/THF; (r) oxalyl
chloride, DMF, CH2Cl2, 0 °C; (s) CH2N2, CH2Cl2, 0 °C; (t) HCl in ether, CH2Cl2; (u) thiourea, MeOH, reflux; (v) Me2NNH2, reflux; (w)
R2NHCH2CHR3OH, EDC, HOBT, DMF; (x) DEAD, PPh3, THF.

H. M. Langford et al. / Bioorg. Med. Chem. Lett. 18 (2008) 721–725
723
Table 1. Calculated conformations and inhibition of strand transfer
activity for selected compounds
Table 2. Inhibition of strand transfer activity for selected compounds
R3
N
R1
F
F
R2
F
H
N
H
N
N
N
N
N
N
( )n
CH3
CH3
H
N
X
N
N
O
O
CH3
O
O
3'
OH
5, 7-10
OH
3
2
2'
1
O4'
4O
11, 12
OH
Compound
R1
R2
R3
Strand transfer
a
IC50 (nM)
Compound
X
n
Dihedral
anglea
Strand transfer
b
IC50 (nM)
5
H
H
—
—
—
—
—
H
610b
13
A
B
7
CH3
Ph
H
H
8
H
610b
40
4
5
6
CH
N
1
1
2
20°
3°
2°
5°
4°
23°
400
610c
470
9
CH3
Ph
—
—
N
10
11
12
H
110
40
a Conformations from MMFF calculations in Spartan, dihedral
A = C1–C2–C3–O4, dihedral B = C1–C20–C30–O40.
b For assay details, see Ref. 11.
—
—
Ph
130
a For assay details, see Ref. 11.
b Lower limit of assay is 10 nM.
c Lower limit of assay is 10 nM.
the conformational properties of pyrrolopiperazinone 4,
pyrazolopiperazinone 5, and ring-expanded pyrazolo-
diazepinone 6 were investigated using molecular mechan-
ics force field calculations contained in the Spartan molec-
ular modeling program.10 As shown in Table 1 with
dihedral angle measurements A and B, pyrazolopyrazi-
none 5 adopts a low energy conformation in which both
carbonyl groups are essentially coplanar with the central
heterocycle (dihedral angles A and B are both near 0 de-
grees), whereas significant deviation from coplanarity is
seen with the exocyclic carbonyl group in pyrrolopyrazi-
none4andtheendocycliccarbonylgroupinpyrazolodiaz-
epinone 6 (dihedral angles A and B are approximately 20
degrees,respectively).Usinganintegrase-catalyzedstrand
transfer assay,11 it is seen that 5 is greater than 40-fold
more potent than 4 and 6, thus providing additional evi-
dence that optimal binding to integrase active site metals4
is achieved with an inhibitor containing a coplanar
arrangement of ligating heteroatoms.
(see Table 3). The lesser potency of ‘reversed analogs’ 11
and 12 compared to 8 in the strand transfer assay was sim-
ilarly manifested in the viral replication assay. Both 5 and
8 showed a 5-fold shift to lower potency in the antiviral as-
say conducted in the presence of higher concentrations of
serum. We therefore sought analogs of 8 with modifica-
tions which might reduce binding to serum proteins.
Modifying the potency-enhancing phenyl group in 8 by
making it smaller and less lipophilic (16) did not signifi-
cantly alter the antiviral potency or the serum shift. Mod-
ifications to the phenyl group in 8 which involved the
inclusion of heteroatoms to make it more polar (13–15)
did in fact reduce the magnitude of the serum shift, how-
ever, these modifications also reduced the antiviral
potency relative to 8, presumably by making the com-
pounds less cell-penetrant. In a related series of naphthy-
ridinone integrase inhibitors, inclusion of a hydrogen
bond acceptor group at the ortho position of the fluorob-
enzyl amide was found to reduce plasma protein binding
and improve antiviral potency in cell-based assays.13 This
tactic produced excellent results in the present series.
Asymmetric syntheses of the pyrazolopyrazinone core
of 8 were developed starting with the individual enantio-
mers of styrene epoxide, and final products were
elaborated with the inclusion of an ortho-N-methylcarb-
oxamido group to give R and S enantiomers, 17a and
17b, respectively. The two enantiomers did not differenti-
ate from one another in the strand transfer assay, and
both were greater than 5-fold less potent than the racemic
des-carboxamido analog 8. The antiviral potencies of 17a
and 17b showed no serum shift, and the S isomer 17b was
4-fold more potent than 17a. The reduced serum shift for
17a and 17b relative to 8 in the antiviral assay may be re-
lated to their 10-fold greater free fraction as measured in a
human plasma protein binding assay13 (12%, 12%, 1.1%
unbound, respectively). The greater free fraction of 17a
and 17b relative to 8 likely contributes to their mainte-
nance of antiviral potency despite being less potent than
8 in the strand transfer assay.
Compound 5 was used as a starting point for further
structure–activity studies (Table 2). In comparing the
potencies of 5, 7, and 8 in the strand transfer assay, it
was seen that methyl and phenyl substituents at the R1
position on the pyrazinone ring are well tolerated. Sim-
ilar substitutions at the adjacent R2 position on the pyr-
azinone ring reduced potency (compare 5, 9, and 10).
Because the pyrazolopyrazinone scaffold is pseudo-sym-
metric about the axis containing the central C–OH
bond, we also investigated ‘reverse analogs’ in which
the fluorobenzyl group is attached to the pyrazinone lac-
tam nitrogen. Potencies of the ‘reverse analogs’ 11 and
12 in the strand transfer assay were less than their iso-
meric counterparts 5 and 8, respectively, and therefore
additional structure–activity studies focused on the iso-
mer series related to 5.
In an assay which measures antiviral activity in MT4 cells
conducted in the presence of 10% fetal bovine serum
(FBS) or 50% normal human serum (NHS),12 phenyl-
substituted pyrazinone 8 proved to be 10-fold more po-
tent than the unsubstituted analog 5, thus demonstrating
a beneficial role for substitution on the pyrazinone ring
The antiviral IC95 value of 63 nM in the presence of 50%
NHS for 17b compares favorably with the potency of

724
H. M. Langford et al. / Bioorg. Med. Chem. Lett. 18 (2008) 721–725
Table 3. Inhibition of strand transfer activity and viral replication for selected compounds
NH2
S
N
(ii) =
(i) =
N
R2
F
H
N
N
O
N
N
CH3
R1
N
H
(iii) =
O
O
OH
N
O
(iv) =
H
Compound
R1
R2
Strand transfera IC50 (nM)
Antiviral activityb IC95 (nM)
(10% FBS) (50% NHS)
5
H
H
H
Ph
610d
610d
40
630
50
2500
310
8
11c
12c
13
14
15
16
17a
17b
5000
10,000
250
500
1000
63
>10,000
>10,000
500
130
610d
610d
610d
20
H
(i)
H
(ii)
(iii)
i-Pr
1000
1000
250
H
H
(iv)
(iv)
Ph(R)
Ph(S)
62
74
250
63
250
63
a For assay details, see Ref. 11.
b Viral replication in MT4 cells, for assay details, see Ref. 12, FBS, fetal bovine serum; NHS, normal human serum.
c See Table 2 for structure.
d Lower limit of assay is 10 nM.
Harvey, C. M.; Isaacs, R. D. Lancet 2007, 369, 1261; (b)
Cahn, P.; Sued, O. Lancet 2007, 369, 1235; (c) Miller, M.;
Witmer, M.; Stillmock, K.; Felock, P.; Ecto, L.; Flynn, J.;
Schleif, W.; Donradula, G.; Danovich, R.; Hazuda, D.
International AIDS Conference, 2006, Toronto, Canada,
Abstract No. ThAa0302.
other integrase inhibitors measured in this assay that have
progressed to clinical studies and demonstrated efficacy
for reducing viral load in HIV-infected patients (e.g.,
MK-05182 IC95 = 33 nM, L-87081014 IC95 = 100 nM).
Reported herein is the design and synthesis of a novel
series of pyrazolopyrazinone HIV-1 integrase inhibitors.
Structural features which facilitate a coplanar relation-
ship between the central heterocycle and flanking car-
bonyl groups provided inhibitors with high potency in
an in vitro biochemical assay (strand transfer IC50 is less
than or equal to 10 nM). Inclusion of a lipophilic substi-
tuent on the pyrazinone ring improved antiviral activity
in a cell-based assay, and addition of a polar group at
the ortho position of the fluorobenzyl amide reduced
plasma protein binding and improved antiviral potency
in the presence of human serum in a cell-based assay. An
optimal compound from this work, 17b, exhibited anti-
viral activity in cell culture in the presence of human ser-
um (IC95 = 63 nM) that is similar to integrase inhibitors
which have demonstrated efficacy for reducing viral load
in HIV-infected patients. Further manipulation around
this series may aid in the development of next generation
anti-HIV drugs.
3. Fisher, T. E.; Kim, B.; Staas, D. D.; Lyle, T. A.; Young,
S. D.; Vacca, J. P.; Zrada, M. M.; Hazuda, D. J.;
Felock, P. A.; Schleif, W. A.; Gabryelski, L. J.; Anari,
M. R.; Kochansky, C. J.; Wai, J. S. Bioorg. Med. Chem.
Lett. 2007, 17 6511.
4. Grobler, J. A.; Stillmock, K. A.; Hu, B.; Witmer, M.;
Felock, P.; Espeseth, A. S.; Wolfe, A.; Egbertson, M.;
Bourgeois, M.; Melamed, J.; Wai, J. S.; Young, S. D.;
Vacca, J. P.; Hazuda, D. J. Proc. Natl. Acad. Sci. U.S.A.
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5. 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., Jr.; Michelson, S. R.;
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¨
278; (b) Rodriguez-Franco, M. I.; San Lorenzo, P.;
Martinez, A.; Navarro, P. Tetrahedron 1999, 55, 2763.
7. Prepared in two steps (hydrogenation in the presence of
di-tert-butyldicarbonate, mesylation) from ethyl 3-azido-
2-hydroxypropionate. See: McCort, G. A.; Pascal, J. C.
Tetrahedron Lett. 1992, 33, 4443.
8. Nematollahi, J.; Kasina, S. J. Chem. Soc., Chem. Com-
mun. 1974, 19, 775.
9. Prepared by addition of methylamine to 98% ee R- or S-
styrene oxide (Aldrich). See: Gurjar, M. K.; Krishna, L.
M.; Sarma, B. V. N. B. S.; Chorghade, M. S. Org. Process
Res. Dev. 1998, 2, 422.
10. Spartan is a molecular modeling software package avail-
able from Wavefunction, Inc., 18401 Von Karman Ave-
nue, Suite 370, Irvine, CA 92612, USA.
11. Hazuda, D. J.; Felock, P.; Hastings, J. C.; Pramanik, B.;
Wolfe, A. J. Virol. 1997, 71, 7005.
Acknowledgment
Special thanks to Matthew M. Zrada for plasma protein
binding data.
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