8-Hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.09.086

A series of potent novel 8-hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 12 is active agains

Full text of DOI:10.1016/j.bmcl.2007.09.086

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6511–6515
8-Hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one HIV-1
integrase inhibitors
Thorsten E. Fisher,a,* Boyoung Kim,a Donnette D. Staas,a Terry A. Lyle,a
Steven D. Young,a Joseph P. Vacca,a Matthew M. Zrada,a Daria J. Hazuda,b
Peter J. Felock,b William A. Schleif,c Lori J. Gabryelski,c M. Reza Anari,d
Christopher J. Kochanskyd and John S. Waia
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, Merck Research Laboratories, West Point, PA 19486, USA
Received 10 September 2007; accepted 25 September 2007
Available online 29 September 2007
Abstract—A series of potent novel 8-hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one HIV-1 integrase inhibitors was identi-
fied. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 12 is active
against replication of HIV-1 in cell culture with a CIC95 of 0.31 lM. Further SAR exploration led to the preparation of pseudosym-
metrical tricyclic pyrrolopyrazine inhibitors 23 and 24 with further improvement in antiviral activity.
Ó 2007 Elsevier Ltd. All rights reserved.
Human immunodeficiency virus-type 1 (HIV-1) is the eti-
ological agent of acquired immunodeficiency syndrome
(AIDS). The unique nature of the replicative cycle of
HIV-1 provides many potential targets for chemothera-
peutic intervention. One of these, the viral enzyme integr-
ase, catalyzes the insertion of the proviral DNA into the
genome of host cells. Integration is a multistep process
which includes three different biochemical steps: the
assembly of proviral DNA on integrase, endonucleolytic
processing of the proviral DNA, andstrand transfer of the
proviral DNA to host cell DNA.1
scaffold for the diketoacid pharmacophore.5a In this com-
munication we describe the discovery, structure–activity
relationships (SAR),and synthesis of a series of novel
8-hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one
HIV-1 integrase inhibitors.
Contraction of the six membered pyridinone ring in
dihydroxypyridopyrazine-1,6-dione 3 to a five mem-
bered pyrrole ring led to a 8-hydroxy-3,4-dihydropyrrol-
o[1,2-a]pyrazine-1(2H)-one system 4. The addition of an
exocyclic carboxyl group, indicated in the overlay in
Figure 1, provided a new template that mimics the
diketoacid pharmacophore. Although electron-rich 3-
hydroxypyrroles were known to be unstable towards
air oxidation, they are rendered air stable with the addi-
tion of electron withdrawing groups such as carboxylic
esters.6 These structural requirements for chemical sta-
bility coincide with those required for activities against
HIV-1 integrase. No precedent for the preparation of
these structures is evident in a survey of the literature.
We have previously reported from our laboratories that
1,3-diketoacids 1 are potent integrase inhibitors and pre-
vent HIV-1 replication in cell culture.2 In addition, we
have shown that the diketoacid pharmacophore is effec-
tively replaced with a novel series of napthyridines 2,3
and were found to be efficacious in rhesus macaques
infected with the simian-human immunodeficiency virus
(SHIV) 89.6P.4 Recently, we described the discovery of
dihydroxypyridopyrazine-1,6-diones 3 as an alternative
The effect of varying ring size was first investigated and
found to be consistent with those observed in the dihydr-
oxypyridopyrazine-1,6-dione integrase inhibitors.5a
Analog 5, with a 7-membered ring constraint, is
significantly less active against HIV integrase than the
Keywords: HIV; Integrase inhibitor; Antiviral.
*
Corresponding author. Tel.: +1 215 652 3634; fax: +1 215 652
3971; e-mail: thor_fisher@merck.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.086

6512
T. E. Fisher et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6511–6515
R
R
O
R'
N
H
R'
OH
N
N
O
OH
2
O
OH
1
O
R"
R"'
N
O
N
R
R
N
N
O
OH
OH
OH
OH
OO
O
OH
3
4
Figure 1. Evolution of hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one inhibitors.
Table 2. Effect of substitutions on benzyl group
6-membered ring compound6 (Table 1). Molecular mod-
eling suggested that only the latter compound 6 presents
the key pharmacophore in a coplanar manner.5a It was
also anticipated that the cyclic constraint would bias the
orientation of the benzyl side chain through steric interac-
tion in a manner similar to those observed for 3.5a
R
OEt
4
3
N
N
O
OH
O
Compound
R
Inhibition of strand Antiviral activity
a
transfer IC50 (lM) in cell culture,
Halogen substitution on the benzyl group showed signif-
icant potency effects on the inhibitory activity against
HIV-1 integrase and viral replication in cell culture. Com-
pound 6 inhibits 95% of the replication of HIV-1 in cell
culture at 3.23 lM (Table 2). When the 4-fluoro substitu-
ent is replaced by a 4-chloro group, intrinsic integrase
inhibition potency is maintained and the antiviral activity
improves by 3 fold (compound 8 versus 6). While migra-
tion of the fluoro substituent from the 4- to the 3-position
results in a slight loss in antiviral potency (compare com-
pounds 6 and 9), the 4-chloro and 3-chloro analogs are
equipotent with CIC95 values ꢀ 1 lM (compounds 8
and 10). All of these mono-halogenated analogs are
significantly more potent than the des- halo inhibitor 7.
Combination of chloro and fluoro substituents at both
the 3- and 4-positions leads to further improvements in
potency in the cell based assay with sub-micromolar
CIC95 (compounds 11 and 12). These compounds do
not exhibit cytotoxicity in cell culture at concentrations
up to 20 lM.
b
CIC95 (lM)
6
4-F
H
0.04 ( 0.02)
0.22 ( 0.03)
0.04 ( 0.02)
0.11 ( 0.03)
0.03 ( 0.02)
3.23 ( 1.72)
20.00 ( 5.55)
1.02 ( 0.33)
9.38 ( 4.42)
1.25 ( 0.40)
0.94 ( 0.40)
0.31 ( 0.10)
7
8
4-Cl
3-F
3-Cl
9
10
11
12
4-F, 3-Cl 0.02 ( 0.01)
3,4-di-Cl 0.01 ( 0.01)
a See footnotes under Table 1.
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.13 Cytotoxicity is not
observed in cell culture at concentrations up to 20 lM.
N
O
O
HN
N
O
H
F
13
Table 1. Effect of different constraints
The pharmacokinetic profile of compound 12 was
examined in rat. When dosed intravenously as a solution
in DMSO (2 mg/Kg), a relatively high clearance of
30 6 mL/min/Kg and a short half life of 0.5 h were
observed. The ethyl ester was found to be rapidly
hydrolyzed in vivo. It was surmised that, unlike other
carboxylic acids which are ionized at physiological pH
and are apparently cell impermeable, the corresponding
acids from these esters would have pKa values similar to
that of the quinoline antibiotic ciprofloxacin 13 (pKa
6.0).7 This relatively high pKa is attributed to the elec-
tron donating effect of the aromatic nitrogen to the car-
bonyl carbon and the hydrogen bonding interaction of
X
F
OEt
O
N
N
OH
O
Compound
X
Inhibition of strand
a
transfer IC50 (lM)
5
6
CH2CH2CH2
CH2CH2
>5.00
0.04 ( 0.02)
a Assays were performed with recombinant HIV-integrase (0.1 lM)
preassembled on immobilized oliogonucleotides.12 Values are means
of three experiments, standard deviation is given in parentheses.

T. E. Fisher et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6511–6515
6513
Table 4. Integrase and HIV replication inhibition activities of amides
the acidic proton with the neighboring hydroxy group in
both systems. In silico pKa estimation using ACD Labs
software suggests that these pyrrolocarboxylic acids
should have pKa values ca. 5.9.8 This result was encour-
aging in that, similar to ciprofloxacin 13, these pyrrolo-
carboxylic acid inhibitors might be cell permeable.
R
HN
4
N
N
3
O
OH
O
Compound
R
Inhibition of strand Antiviral activity
a
transfer IC50 (lM) in cell culture,
Analogs 7 and 9 (Table 2) were hydrolyzed to the corre-
sponding acids 14 and 15 (Table 3). They were found to
be significantly more active against HIV integrase in the
enzyme assay than their ester precursors. However, this
did not translate into improvements in antiviral activity.
In fact, no antiviral activities were observed with these
inhibitors up to 20 lM.
a
CIC95 (lM)
19
20
21
4-F
4-Cl
0.08 ( 0.02)
0.06 ( 0.02)
10.00 ( 2.11)
2.50 ( 0.55)
1.25 ( 0.20)
3,4-di-Cl 0.04 ( 0.01)
a See footnotes under Table 2.
carboxamide at 180 degrees from the desired conforma-
tion for binding (Fig. 2),11 which would negatively im-
pact the inhibitory activities of compounds 19–21
against HIV-1 integrase.
pKa
4.20
R
H
OH
OH
O
N
17
18
N
O
OH 2.96
OH
R
O
16
To circumvent this unfavorable conformational bias, an
additional cyclic constraint was introduced between the
central pyrrole ring and the exocyclic carboxamide
group. The pseudosymmetry of the scaffold permits
the pharmacophore to be examined in both a forward
and reverse spatial configuration or mode (Table 5).
Analogs were prepared in both modes and tricyclic
inhibitors 22 and 23 exhibited enzyme inhibition below
the lower limit of the assay (0.01 lM) and enhancement
in their antiviral activities. Furthermore, preparation of
the highly pseudo-symmetrical bis-4-fluorobenzyl inhib-
itor 24 led to even greater activity against HIV replica-
tion in cell culture.
Compound 16, a water soluble N-methylated analog of
these carboxylic acids, was determined to have a pKa va-
lue of 3.76.9 Examination of pKa values reported for
benzoic acid (17) and o-salicyclic acid (18) indicates that
an ortho-hydroxy substitution in benzoic acid greatly
enhances its acidity.10 The ortho-hydroxy substituent
seems to have a more dominant effect in influencing
the acidity observed with our hydroxy-pyrrolocarboxy-
lic acids. The algorithm employed by the ACD Lab
pKa prediction software appears to have underestimated
the effect of this substitution. At physiological pH, it is
very likely that inhibitors 14 and 15, with pKa’s of
approximately 3.8 (based on compound 16) exist
primarily as the negatively charged carboxylate. This
would likely compromise their ability to penetrate cells.
The pseudosymmetrical nature of compounds 22, 23,
and 24, discussed above, showcases the characteristic
1,3-diketoacid pharmacophore of HIV-1 integrase
It was reasoned that the more metabolically stable and
cell permeable amide analogs might resolve the liabilities
encountered with the ester and acid inhibitors. Further-
more, the amide inhibitors, with higher electron density
at the exocyclic carbonyl oxygen, were expected to be
more potent than the ester inhibitors. However, the
methyl amide inhibitors 19, 20, & 21 (Table 4) were
found to be slightly less active than the corresponding
esters (Table 2, compounds 6, 8, & 12) in both the en-
zyme and HIV replication inhibition assays. Molecular
modeling (PM3 semi-empirical calculation) suggests
that there may be a bias for orientation of the exocyclic
O
N
HN
O
N
N
N
N
H
O
O
O
O
H
H
-94.9 kcal/mol
-98.1 kcal/mol
Figure 2. Conformational bias of pyrrolopyrazine carboxamide.
Table 5. Integrase and HIV replication inhibition activities
N
N
N
R"
R'
Table 3. Integrase and HIV replication inhibition activities of acids
O
O
R
OH
O
4
N
C2
N
3
O
H
Compound R0
R00
Inhibition
of strand
transfer
Antiviral
activity in cell
culture, CIC95
O
O
H
a
a
Compound
R
H
Inhibition of strand Antiviral activity in cell
a
transfer IC50 (lM) culture, CIC95 (lM)
IC50 (lM) (lM)
a
22
23
24
4-F-Bn CH3
CH3 4-F-Bn <0.01
4-F-Bn 4-F-Bn <0.01
<0.01
0.39 ( 0.12)
0.16 ( 0.11)
0.07 ( 0.02)
14
15
0.08 ( 0.01)
>20.00
>20.00
3-Cl 0.01 ( 0.01)
a See footnotes under Table 2.
a See footnotes under Table 2.

6514
T. E. Fisher et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6511–6515
inhibitors. Together with the observation that both 4-
fluorobenzyl groups on 24 contributes to binding, it is
very likely that the mono-benzyl inhibitors (compounds
6–23) may bind to HIV integrase in more than one
mode. This observation represents the first structural
evidence for the hypothesis presented in a recent com-
munication from our laboratories that there is poten-
tially more than one mode of binding for HIV-1
integrase inhibitors.3b It is our contention that these
analogs provide a unique scaffold to be optimized for
binding in multiple modes. Potentially if one binding
mode is rendered less effective by mutation of the integr-
ase enzyme, the inhibitors may be able to maintain
inhibitory activity against replication of the mutant
viruses by binding in a different mode. Further progress
towards optimization of this series of tricyclic hydroxy-
pyrrole integrase inhibitors with a higher genetic barrier
to mutation has been recently reported.5b
the amide 26. Acid catalyzed cyclization14 of 26, fol-
lowed by a one pot stepwise saturation of the resultant
product 27 in the presence of 5% Pt on charcoal and
cleavage of the CBz group with 5% Pd on charcoal pro-
vided the piperazinone 28. Compound 28 and diethyl
ethoxymethylenemalonate were heated in toluene at
80 °C overnight to provide the adduct 29 and its subse-
quent addition to a refluxing solution of lithium hexa-
methyldisilylamide in THF afforded the cyclization
product 6. Ester 6 was then hydrolyzed to the corre-
sponding acid with aqueous sodium hydroxide in etha-
nol in a sealed tube at 100 °C overnight. Alternatively,
treatment of the ester 6 with a suspension of AlCl3 in
anhydrous chloroform purged with methylamine gas at
70 °C overnight provided the corresponding methyl
amide 19.
Tricyclic inhibitors such as 22 and 24 were prepared by
heating a mixture of the piperazinone 28 and an appro-
priate lactam 3015 in ethylene glycol or 1,2-dichloroben-
zene in a sealed tube at 250 °C in a microwave reactor
for 20 minutes. The crude product mixture was purified
by C-18 reverse phase HPLC eluting with a water-aceto-
nitrile gradient.
The synthesis of compound 6 is depicted in Scheme 1.
Reductive alkylation of dimethoxyethylamine with 4-
fluorobenzaldehyde 25 in the presence of sodium boro-
hydride provided the corresponding benzylamine, which
was treated with N-CBz-glycine under a standard cou-
pling protocol (EDC and HOBt in DMF) to provide
In summary, a series of potent 8-hydroxy-3,4-dihydro-
HIV-1
pyrrolo[1,2-a]pyrazine-1(2H)-one
integrase
inhibitors which inhibit replication of HIV-1 in cell cul-
ture has been established. Further exploration revealed
the pseudosymmetrical nature of the integrase inhibitor
pharmacophore. Efforts are ongoing to identify the
potential of the tricyclic pyrrolopyrazine template and
analogous bicyclic systems.
OMe
O
MeO
N
F
F
a
HN
OBn
26
CHO
O
25
b
O
References and notes
F
c
F
NH
N
OBn
N
O
N
1. For recent reviews 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
recent reviews on HIV-1 integrase inhibitors, see: (c)
Gordon, C. P.; Griffith, R.; Keller, P. A. Med. Chem.
2007, 3, 199; (d) Deng, J.; Dayam, R.; Al-Mawsawi, L. Q.;
Neamati, N. Curr. Pharm. Des. 2007, 13, 129; (e)
Anthony, N. J. Curr. Top. Med. Chem. 2004, 4, 979.
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. (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.
27
O
28
N
O
R
MeO
d
EtO2C
30
h
F
CO2Et
N
N
R
F
N
N
O
CO2Et
N
O
OH
O
29
R = CH3 22
e
4-F-Bn 24
F
F
Y
N
N
f or g
CO2Et
N
O
N
O
OH
OH
O
6
19
Y = NHMe
Scheme 1. Synthesis of Compounds 6, 19, 22, and 24. Reagents and
conditions: (a) i—H2NCH2CH(OMe)2, NaBH4, MeOH, rt (82%);
ii—N-Cbz-Gly, EDC, HOBt, i-Pr2NEt, DMF, rt, overnight (95%); (b)
TsOH, toluene, 80 °C, 5 days (62%); (c) H2, 10% Pt/C, EtOH; H2, 20%
Pd(OH)2/C, EtOH (95%); (d) diethyl ethoxymethylenemalonate,
toluene, 100 °C, 4 h; (e) LiHMDS, THF, 80 °C (78%); (f) for Y = OH,
NaOH, EtOH, 100 °C, overnight; (g) AlCl3, MeNH2, CHCl3, 70 °C
(85%); (h) Ethylene glycol, microwave at 250 °C for 20 min (25%).

T. E. Fisher et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6511–6515
6515
G.; Schleif, W. A.; Cortese, R.; Emini, E.; Summa, V.;
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12. 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
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.
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,
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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.; 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, 95% Cell culture inhibitory
concentrations (CIC95) are defined as those which inhib-
ited by P 95% the spread of HIV-1 infection in susceptible
cell culture. MT-4 human T-lymphoid cells were main-
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 two-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.
5. (a) Wai, J. S.; Kim, B. Y.; Fisher, T. E.; Zhuang, L. H.;
Embrey, M. W.; Williams, P. D.; Staas, D. D.; Culber-
son, C.; Lyle, T. A.; Vacca, J. P.; Hazuda, D. J.; Felock,
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Embrey, M.; Egbertson, M.; Vacca, J.; Hazuda, D.;
Miller, M.; Witmer, M.; Gabryelski, L.; and Lyle, T.
Next Generation HIV-1 Integrase Strand Transfer Inhib-
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(CROI), Feb 25–28, 2007.
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Ed.; John Wiley & Sons Inc: New York, 1992, Part 2,
Chapter 4, p 525.
7. Dollery, C., Ed.; Churchill Living-stone: London, 1991;
p 247.
8. pKa estimation for Ciprofloxacin 13, 6.04; pyrrolo car-
boxylic acid 14, 5.97. ACD/Lab Extension version 8.0,
pKa estimation for CS ChemDraw.
9. Franz, Robert G. AAPS Pharmsci. 2001, 3(2), article 10
and those references cited therein.
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11. PM3-semi-empirical calculation; Spartan’04 Windows ES
2.0.0. 2003.
15. The dihydropyridinones were prepared by treatment of
appropriate substituted b-aminoesters with alkyl 3-chloro-
3-oxopropionates, followed by a based catalyzed cycliza-
tion, see Yang, Y.; Decken, A.; Deslongchamps, G.
Tetrahedron 1998, 54, 9043, The hydroxypyridinones were
readily O-methylated with diazomethane.