10-Hydroxy-7,8-dihydropyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrid azine-1,9(2H,6H)-diones: Potent, orally bioavailable HIV-1 integrase strand-transfer inhibitors with activity against integrase mutants

Article abstract of DOI:10.1016/j.bmcl.2008.07.037

A series of 10-hydroxy-7,8-dihydropyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrid azine-1,9(2H,6H)-diones was synthesized and tested for their inhibition of HIV-1 replication in cell culture. Structure-activity studies indicated that high antiviral potency against wild-type virus as well as viruses containing integrase mutations that confer resistance to three different structural classes of integrase inhibitors could be achieved by incorporation of small aliphatic groups at certain positions on the core template. An optimal compound from this study, 16, inhibits integrase strand-transfer activity with an IC₅₀ value of ≤10 nM, inhibits HIV-1 replication in cell culture with an IC₉₅ value of 35 nM in the presence of 50% normal human serum, and displays modest pharmacokinetic properties in rats (iv t_1/2 = 5.3 h, F = 17%).

Full text of DOI:10.1016/j.bmcl.2008.07.037

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4581–4583
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
10-Hydroxy-7,8-dihydropyrazino[10,20:1,5]pyrrolo[2,3-d]pyridazine-1,9(2H,6H)-
diones: Potent, orally bioavailable HIV-1 integrase strand-transfer
inhibitors with activity against integrase mutants
a
a
a
a
Catherine M. Wiscount a, , Peter D. Williams , Lekhanh O. Tran , Mark W. Embrey , Thorsten E. Fisher ,
Vanessa Sherman a, Carl F. Homnick a, D. Donnette Staas a, Terry A. Lyle a, John S. Wai a, Joseph P. Vacca a,
ZiQiang Wang a, Peter J. Felock b, Kara A. Stillmock b, Marc V. Witmer b, Michael D. Miller b, Daria J. Hazuda b,
Alysha M. Day b, Lori J. Gabryelski c, Linda T. Ecto c, William A. Schleif c, Daniel J. DiStefano c,
Christopher J. Kochansky d, M. Reza Anari d
*
a Department of Medicinal Chemistry, WP14-3, Merck Research Laboratories, West Point, PA 19486, USA
b Department of Antiviral Research, Merck Research Laboratories, West Point, PA 19486, USA
c Department of Vaccine and Biologics Research, Merck Research Laboratories, West Point, PA 19486, USA
d Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
A series of 10-hydroxy-7,8-dihydropyrazino[10,20:1,5]pyrrolo[2,3-d]pyridazine-1,9(2H,6H)-diones was
synthesized and tested for their inhibition of HIV-1 replication in cell culture. Structure–activity studies
indicated that high antiviral potency against wild-type virus as well as viruses containing integrase muta-
tions that confer resistance to three different structural classes of integrase inhibitors could be achieved
by incorporation of small aliphatic groups at certain positions on the core template. An optimal com-
pound from this study, 16, inhibits integrase strand-transfer activity with an IC50 value of 610 nM, inhib-
its HIV-1 replication in cell culture with an IC95 value of 35 nM in the presence of 50% normal human
serum, and displays modest pharmacokinetic properties in rats (iv t1/2 = 5.3 h, F = 17%).
Ó 2008 Elsevier Ltd. All rights reserved.
Article history:
Received 14 May 2008
Accepted 10 July 2008
Available online 15 July 2008
Keywords:
HIV integrase
Antiviral
The causative agent of acquired immune deficiency syndrome
(AIDS) is the human immunodeficiency virus type 1 (HIV-1). Resis-
tance to marketed anti-HIV drugs is increasing at an alarming rate
and thus there is a need to improve existing agents and develop
new agents which work by different mechanisms. The viral en-
zyme, integrase, is used by the virus to insert double-stranded pro-
viral DNA into host chromosomal DNA. Several integrase inhibitors
which work by inhibiting the strand-transfer step have progressed
to the stage of clinical testing in patients infected with HIV-1, and
one of these, RaltegravirTM, has recently received FDA approval.1 We
have been working toward second-generation integrase strand-
transfer inhibitors with the goal of identifying inhibitors with
activity against viruses that contain integrase mutations. A recent
report from our laboratories2 described tricyclic hydroxypyrrole
1 as a novel mimetic of the metal binding pharmacophore3 of dike-
toacid integrase inhibitors (Fig. 1). Scaffold 1 offers unique oppor-
tunities to maintain activity against integrase mutants because it
may take advantage of multiple binding modes due to its pseu-
do-C2 symmetry.2 However, in follow-up investigations we found
that alkali metal salts of 1, which were desired for oral dosing in
pharmacokinetic experiments, were susceptible to air oxidation.
We reasoned that introduction of an electron withdrawing group
into the ring system, such as contained in pyridazinone analog 2,
would stabilize the electron-rich hydroxypyrrole moiety and
attenuate oxidation. Indeed, alkali metal salts of scaffold 2 were
found to possess markedly improved chemical stability. Herein
OH
N
N
N
R
O
O
O
O
OH
1
OH
diketoacid
N
N
N
N
O
O
2
OH
* Corresponding author. Tel.: +1 215 652 7812; fax: +1 215 652 3971.
E-mail address: cathy_wiscount@merck.com (C.M. Wiscount).
Figure 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.037

4582
C. M. Wiscount et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4581–4583
Table 1
Antiviral potency of pyrazino-pyrrolopyridazines in cell culture assays
F
R2
R3b
R3a
N
R1
N
R4
N
N
O
O
OH
Compound
R1
R2
R3a
R3b
S:Ra
R4
Viral replication assays
Single cyclec
Multi-cycleb
IC95 (nM)
IC50 (nM)
WT
Fold shift vs WT
T66I S153Y
10% FBS
50% NHS
F121Y
N155S
11
12
13
14
15
16
17
18
19
20
21
22
H
H
H
H
H
H
H
H
H
H
CI
CI
H
H
H
H
H
H
Me
H
H
H
Me
Me
H
—
—
Me
Me
Me
Me
Et
i-Pr
Et
Et
120
43
10
250
17
16
24
250
10
13
31
380
130
31
1000
40
35
46
500
30
26
72
21
10
66
14
10
16
52
19
19
19
28
11
10
4
9
1
1
5
8
10
2
7
8
2
8
1
1
1
9
3
1
1
1
53
48
20
91
6
Me
Me
Me
Me
Me
H
H
H
H
H
Me
H
Me
Me
Me
H
Me
Me
Me
Me
99:1
1:99
97:3
95:5
99:1
1:99
—
95:5
97:3
95:5
2
27
10
27
6
7
3
Et
i-Pr
Et
H
H
160
220
2
1
H
i-Pr
30
a
Ratio of S and R enantiomers as analyzed on a Chiracel OD column.
Antiviral activites in MT-4 cells cultured in the presence of 10% fetal bovine serum (FBS) or 50% normal human serum (NHS) and infected with an HIV-1 H9/IIB virus. All
b
values reported are means of three or more independent determinations.
c
Antiviral activities in P4/5 cells cultured in the presence of 10% FBS and infected with an HIV-1 IIIB virus. Fold-shift values are defined as IC50 (mutant)/IC50 (wild type).
we present SAR findings with scaffold 2 that demonstrate a path-
way to potent, orally bioavailable integrase inhibitors with activity
against a selected panel of integrase mutants.
Chemical synthesis (Scheme 1): Key early intermediates for syn-
theses of the compounds in Table 1 were piperazinones 7. Conver-
R3a
O
R3a
R3b
O
R3b
a
H
b, c, d
RO
N
RO
N
R3a R3b
HN
H
H
HN
O
R4
3
5
4
N
sion to enamides
8 and base-induced ring closure gave
CH3
R4
f
pyrrolopiperazines 9. The hydroxyl group was methylated, the pyr-
role brominated, and the bromide underwent Stille- or Heck-type
couplings to give acyl pyrroles 10. Ring closure to the tricyclic core
was achieved by treating 10 with hydrazine hydrate. N-Benzyla-
tion followed by demethylation gave inhibitors 11–22. Enantiomer
pairs 13/14 and 17/18 were obtained by separation of the race-
mates on a Chiracel OD column. The racemic piperazinones 7 uti-
lized in these syntheses were obtained by hydrogenation of
pyrazinones 6.4 The absolute stereochemistry was established by
asymmetric syntheses starting with S-aldehyde 35 and using a
four-step sequence6 to obtain enantiomerically enriched piperazi-
nones 7. Enantiomeric purity was assessed using a Chiracel OD col-
umn at the final product stage and was found to be ꢀ95:5 S:R, see
Table 1 for specific ratios. An analogous piperazinone synthesis
starting with N-Cbz-dimethylglycinal provided the achiral inhibi-
tor 19. Syntheses of 11 and 12 began with commercially available
1-methyl-2-piperazinone.
NH2
O
O
e
N
H
N
7
R4
N
O
R4
g
6
R3a R3b
O
R3a R3b
EtO
O
h
EtO
N
N
N
N
EtO
O
R4
R4
i, j, k
HO
O
O
9
8
F
R2
R2
R3a
N
R3b
O
R3a
R3b
N
R1
N
N
EtO
O
N
l, m, n
R4
N
R4
O
O
MeO
O
OH
Biological methods: The compounds in Table 1 were tested for
antiviral efficacy in two cell-based assays. No evidence of com-
pound-related cytotoxicity was observed in either assay. Multi-cy-
cle viral replication assays7 were performed in MT-4 human T
lymphoid cells cultured in media containing 10% fetal bovine ser-
um (FBS) or 50% normal human serum (NHS) and infected with
HIV-1 H9/IIIB virus. Inhibition of viral growth was assessed by
measuring P24 levels with an ELISA. Single-cycle viral replication
assays8 were performed in P4/R5 cells (HeLa cells with a stably
integrated LTR-LacZ reporter gene) cultured in a medium contain-
ing 10% FBS and infected with HIV-1 IIIB virus. Viral growth was
measured using a b-galactosidase readout. In single-cycle infectiv-
10
11-22
Scheme 1. Reagents and conditions: (a) R4NH2, NaBH(OAc)3, AcOH, THF or CH2Cl2
(81–89%); (b) BrCH2CO2H, DCC, CH2Cl2 or BrCH2COBr, Et3N, THF (84–91%); (c) NaH,
THF or DMF (61–85%); (d) HCl, EtOAc (R = t-Bu) or H2, Pd–C, MeOH (R = CH2Ph) (91–
98%); (e) pyruvaldehyde, NaOH, H2O, 100 °C (84%); (f) H2, PtO2, EtOAc (92%); (g)
diethyl ethoxymethylidene-malonate, EtN(i-Pr)2, toluene, 80 °C (85–89%); (h)
LiN(TMS)2, THF, 50 °C (50–65%); (i) MeI, K2CO3, DMF (88–93%); (j) Br2, NaHCO3,
CH2Cl2 (82–90%); (k) R2 = H:Bu3SnCH@CH2, (t-Bu3P)2Pd, toluene, microwave 100 °C,
then OsO4, NaIO4, THF:H2O (71–79%); (k) R2 = CH3:n-BuOCH@CH2, (t-Bu3P)2Pd,
toluene, microwave 100 °C, then AcOH, THF:H2O; or Bu3SnC(OMe)@CH2,
PdCl2(PPh3)2, DMF, microwave 100 °C (78–87%); (l) N2H2ꢁH2O, microwave 100 °C
(78–95%); (m) 4-F- or 4-F, 3-Cl-benzyl bromide, Cs2CO3, DMF (82–91%); (n) BBr3,
CH2Cl2 (80–95%).

C. M. Wiscount et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4581–4583
4583
Table 2
gests that they are more highly bound to serum proteins than the
des-chloro analogs and their antiviral potencies in the presence of
human serum are less optimal (IC95 >100 nM). Overall, the two N-
isopropyl analogs 16 and 20 exhibited a favorable combination of
properties from the standpoint of antiviral potency in the presence
of 50% NHS, potency against the three mutants, and pharmacoki-
netic properties in rats. Both compounds were additionally charac-
terized in an integrase-catalyzed strand-transfer assay9 and
exhibited potent inhibition with IC50 values 610 nM (lower limit
of the assay). In a single-cycle replication assay using virus that
incorporates a Q148K mutation in integrase10, a mutation which
confers ꢀ50-fold resistance to RaltegravirTM, 16 and 20 showed dif-
ferential activity with shifts versus wild-type virus of 3- and 45-
fold, respectively. In pharmacokinetic experiments in male beagle
dogs, poor oral bioavailability (<5%), moderate clearance
(ꢀ10 mL/min/kg), and short intravenous half-lives (ꢀ1 h) were ob-
served for both 16 and 20.
Summary and conclusion: Modification of a previously reported
pyrazino-pyrrolopyrazine integrase inhibitor by changing one of
the pyrazinone rings to a pyridazinone improved chemical proper-
ties such that stable alkali metal salts could be prepared and used
for oral dosing in pharmacokinetic experiments. Addition of small
aliphatic groups to the pyrazinopyrrolopyridazine core provided a
means to obtain potent antiviral activity in cell culture against
wild-type virus as well as viruses which contain mutations in
integrase that confer resistance to integrase inhibitors from three
different structural classes. From these studies, 16 was found to
possess a number of favorable attributes: antiviral IC95 = 35 nM
in the presence of 50% NHS against HIV-1 in cell culture; 1- to 3-
fold shift in potency against viruses that contain resistance-confer-
ring mutations to diketoacid, naphthyridine, and pyrimidine clas-
ses of integrase inhibitors; and modest oral bioavailability and
plasma half-life in rats (F = 17%, intravenous t1/2 = 5.3 h). Com-
pound 16 thus represents a promising lead for further optimization
studies toward second-generation integrase strand-transfer
inhibitors.
Pharmacokinetic parameters of selected compounds in male Sprague–Dawley ratsa
Compound
CL (mL/min/kg)
Vd (L/kg)
t1/2 (h)
%F
15
16
17
19
20
21
22
17
3.0
2.6
0.3
0.2
0.3
0.2
0.3
0.2
6.2
5.3
6.0
5.2
12
7
17
31
13
21
13
14
0.50
0.68
0.17
0.27
0.17
9.5
13
a
Compounds were dosed orally as sodium salts at a dose of 10 mg/kg in 0.5%
methocel suspension and intravenously as parent phenols at a dose of 2 mg/kg in
1:2 DMSO:H2O solution. Clearance, volume of distribution, and half-life values were
calculated from the intravenous experiments.
ity experiments, wild-type virus as well as viruses containing
integrase mutations that confer resistance to diketoacid and naph-
thyridine strand-transfer inhibitors8 were used to assess potency
of test compounds. Selected compounds were tested for pharmaco-
kinetic behavior in male Sprague–Dawley rats. Compounds were
dosed orally as sodium salts in 0.5% methocel suspension at
10 mg/kg and intravenously as parent phenols in 1:2 DMSO:H2O
solution at 2 mg/kg, using three animals per dose group. Plasma
drug levels were measured at 10 time points between 0.2 and
24 h in the intravenous experiments and at eight time points be-
tween 0.2 and 24 h in the oral experiments.
Results and discussion: The parent compound in this series, 11,
demonstrated that this scaffold is capable of producing good anti-
viral activity in cell culture with minimal shift in potency in the
presence of 50% normal human serum (NHS). However, 11 suffers
a 10- to 50-fold loss in potency against three integrase mutants,
F121Y, N155S, and T66I/S153Y. Addition of a methyl group at R2
on the pyridazinone ring (12) improved antiviral potency by 3-fold
but did not improve potency against the mutants. Addition of an-
other methyl group at R3 on the piperazinone ring had a significant
effect on potency: compared to 12 in the multi-cycle assay, the S
isomer (13) gained 4-fold in potency whereas the R isomer (14)
lost 5-fold in potency. A similarly large difference in potency favor-
ing the S-isomer was observed with enantiomers 17 and 18, and
we therefore focused our attention exclusively on analogs in the
S-series for additional structure-activity studies. Two analogs of
13 with larger piperazinone N-substituents at R4, 15 and 16, main-
tained antiviral potency and showed significantly improved po-
tency against the three integrase mutants. In pharmacokinetic
experiments (see Table 2), 15 and 16 exhibited modest oral bio-
availability with moderate to low clearance, and half-lives in the
range of 5–6 h. Compounds 15 and 16 thus demonstrated that this
series was capable of delivering orally bioavailable analogs with
high antiviral potency and excellent activity against integrase mu-
tants. In the related series with R2 = H, the antiviral potency of the
achiral gem-dimethyl analog 19 compares more closely to that of
the S-monomethyl analog 17 than the R-monomethyl analog 18.
The gem-dimethyl analog 19 did not provide any advantage for po-
tency against the mutants. In comparing the potencies of the
R4 = ethyl and isopropyl analogs in the two series with R2 = H or
methyl (17 and 20 vs 15 and 16), it is seen that that there is a slight
advantage for the series with R2 = methyl in terms of antiviral po-
tency and activity against mutants. However, 17 and 20 in the
R2 = H series exhibited lower clearance in pharmacokinetic exper-
iments in rats. Additionally, 20 exhibited a long plasma half-life of
12 h after intravenous administration. The chloro analogs, 21 and
22, demonstrated an upper limit for lipophilicity: in the multi-cy-
cle viral replication assay these two compounds exhibited a 5- to
6-fold shift when comparing potencies in the 10% FBS and 50%
NHS experiments, whereas the des-chloro analogs in Table 1 exhib-
ited a shift of only 2- to 3-fold. The greater shift for 21 and 22 sug-
References
1. Anthony, N. J. Curr. Top. Med. Chem. 2004, 4, 979; Egbertson, M. S. Curr. Top.
Med. Chem. 2007, 7, 1251.
2. 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. J.; Schleif, W. A.; Gabryelski, L. J.; Anari, M. R.;
Kochansky, C. J.; Wai, J. Bioorg. Med. Chem. Lett. 2007, 17, 6511.
3. Grobler, J. A.; Stillmock, K.; 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. 2002, 99, 6661.
4. Armarego, W. L. F.; Waring, P.; Williams, J. W. J. Chem. Soc. Chem. Commun.
1980, 8, 334.
5. Fehrentz, J. A.; Castro, B. Synthesis 1983, 8, 676.
6. Williams, T. M.; Bergman, J. M.; Brashear, K.; Breslin, M. J.; Dinsmore, C. J.;
Hutchinson, J. H.; MacTough, S. C.; Stump, C. A.; Wei, D. D.; Zartman, C. B.;
Bogusky, M. J.; Culberson, J. C.; Buser-Doepner, C.; Davide, J.; Greenberg, I. B.;
Hamilton, K. A.; Koblan, K. S.; Kohl, N. E.; Liu, D.; Lobell, R. B.; Mosser, S. D.;
O’Neill, T. J.; Rands, E.; Schaber, M. D.; Wilson, F.; Senderak, E.; Motzel, S. L.;
Gibb, J. B.; Graham, S. L.; Heimbrook, D. C.; Hartman, G. D.; Oliff, A. I.; Huff, J. R.
J. Med. Chem. 1999, 42, 3779.
7. 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.; Sardana, V. V.; Schlabac, A. J.;
Graham, P. I.; Condra, J. H.; Gotlib, L.; Holloway, M. K.; Lim, J.; Chen, I.-W.;
Vastag, K.; Ostovic, D.; Anderson, P. S.; Emini, E. E.; Huff, J. R. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 4096.
8. Hazuda, D. J.; Anthony, N. J.; Gomez, R. P.; Jolly, S. M.; Wai, J. S.; Zhuang, L.;
Fisher, T. E.; Embrey, M.; Guare, J. P.; Egbertson, M. S.; Vacca, J. P.; Huff, J. R.;
Felock, P. J.; Witmer, M. V.; Stillmock, K. A.; Danovich, R.; Grobler, J.; Miller, M.
D.; Espeseth, A. S.; Jin, L.; Chen, I.-W.; Lin, J. H.; Kassahun, K.; Ellis, J. D.; Wong,
B. K.; Xu, W.; Pearson, P. G.; Schleif, W. A.; Cortese, R.; Emini, E.; Summa, V.;
Holloway, M. K.; Young, S. D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11233.
9. Hazuda, D. J.; Felock, P.; Hastings, J. C.; Pramanik, B.; Wolfe, A. J. Virol. 1997, 71,
7005.
10. Ke, Y; Danovich, R. M.; personal communication. See also Cooper, D., Gatell, J.;
Rockstroh, J.; Katlama, C.; Yeni, P.; Lazzarin, A.; Chen, J.; Isaacs, R.; Teppler, H.;
Nguyen, B.; 14th Conference on Retroviruses and Opportunistic Infections,
February 25–28, 2007.