4-Hydroxy-5-pyrrolinone-3-carboxamide HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.06.056

The viral enzyme integrase is essential for the replication of HIV-1 and, after the discovery of Isentress, represents a validated target for anti-retroviral therapy. Incorporation of the dihydroxycarbonyl pharmacophore into a pyrrolinone scaffold led to the discovery of 5-pyrrolinone-3-carboxamides as a structurally diverse class of HIV-1 integrase inhibitors.

Full text of DOI:10.1016/j.bmcl.2008.06.056

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3865–3869
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Hydroxy-5-pyrrolinone-3-carboxamide HIV-1 integrase inhibitors
*
Paola Pace , Stéphane A. H. Spieser, Vincenzo Summa
Department of Medicinal Chemistry, IRBM-MRL Rome, Via Pontina Km 30,600, 00040 Pomezia, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
The viral enzyme integrase is essential for the replication of HIV-1 and, after the discovery of IsentressTM
represents a validated target for anti-retroviral therapy. Incorporation of the dihydroxycarbonyl pharma-
cophore into a pyrrolinone scaffold led to the discovery of 5-pyrrolinone-3-carboxamides as a structur-
ally diverse class of HIV-1 integrase inhibitors.
,
Article history:
Received 6 June 2008
Revised 16 June 2008
Accepted 16 June 2008
Available online 20 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
AIDS
HIV-1 integrase
Diketoacids
Pyrimidines
Pyrrolinones
A variety of anti-HIV agents rely on inhibition of key enzymes
involved with the virus life cycle. The utility of compounds direc-
ted at single targets is greatly limited by the development of resis-
tance. HAART (Highly active anti-retroviral therapy) has
demonstrated that targeting multiple enzymes provides one
means of circumventing this problem, with combination therapies
directed against both the HIV reverse transcriptase and the prote-
ase enzymes showing good results in reducing viral loads in HIV-1
infected patients.1 However, while HAART is undeniably effective,
it can fail to control HIV replication in patients due to several lim-
itations, such as lack of therapy adherence, toxicities issues, and
the emergence of drug resistant viral strains. Recently, IsentressTM
discovered in our laboratories has been approved by the FDA, an
oral drug that inhibits HIV integrase, the third enzyme encoded
by the HIV-1 genome and is expected to complement HAART offer-
ing a new treatment option and renewed hope to sufferers of this
important disease.2
Integrase catalyzes the integration of double stranded viral DNA
into the host cell’s genomic DNA.3 Integration consists of three bio-
chemical steps: the assembly of integrase on viral DNA, endonu-
cleolytic cleavage of the first two nucleotides from each 30
terminal of the viral DNA, and strand transfer of the recessed viral
DNA to the host cell DNA.4,5 1,3-Diketoacids 1 were reported to be
effective integrase inhibitors and prevent HIV-1 replication in cell
culture (Fig. 1).6 The same dihydroxycarbonyl pharmacophore
was recognized in the pyrimidines/pyrimidones 2 with tremen-
dous improvements in drug-like properties and led ultimately to
the discovery of IsentressTM.7 Continuing our work in this research
field and in an attempt to identify new scaffolds featuring a suit-
able replacement for the 1,3-diketoacid motif, we designed and
synthesized a novel series of substituted pyrrolinones 3 HIV-1
integrase inhibitors.
Other laboratories also demonstrated that the diketoacid phar-
macophore can be replaced with 2-pyrrolinones having a ketone or
an heteroaromatic substituent.8 The structural requirements for
inhibitory activity defined by our preceding studies on pyrimidine
inhibitors9 guided the preparation of this series of pyrrolinone
inhibitors having a benzylic amide substituent. The diketoacid por-
tion of the template is believed to bind to the Mg2+ cofactor located
in the active site, while the p-fluorobenzyl participates in a specific
interaction with an adjacent hydrophobic pocket. On pyrimidines/
pyrimidones it was established that in terms of potency, a variety
of substituents are tolerated in the 2-position of the heterocyclic
core, the presence of a basic amine was required to modulate phys-
ical properties and, ultimately, achieve activity in cell culture.
The initial replacement of the pyrimidine core with the pyrroli-
none, keeping fixed the p-fluorobenzyl amide moiety, resulted in 4
which inhibited the strand transfer process of HIV-1 integration
with an IC50 = 100 nM (Table 1). A dihydroxypyrimidine with a
comparable substitution pattern
2
(R0 = 4-F, R00 = 4-CH3C6H4)
showed an IC50 = 20 nM. Molecular modeling showed a good
superimposition of 4 with the above 2-arylsubstituted dihydroxy-
pyrimidine (Fig. 2) and suggested the possibility to further explore
the N-1 substituents, with the potential of changing physicochem-
ical properties of the inhibitor as with the pyrimidines/pyrimi-
dones. Conformational analysis and energy minimizations were
carried out using the MMFFs force field, as implemented in Macro-
* Corresponding author. Tel.: +39 06 919093313; fax: +39 06 910093654.
E-mail address: paola_pace@merck.com (P. Pace).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.056

3866
P. Pace et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3865–3869
R"
R'
R'
H,Me
O
N
N
H
N
OH
O
O
OH
O
OH
2
1
O
N
O
N
NH
R
N
F
F
N
N
H
H
N
N
O
O
O
O
OH
OH
IsentressTM
3
Figure 1. Conception of 4-hydroxy-5-pyrrolinone-3-carboxamide inhibitors.
Table 1
Inhibition of HIV-1 integrase catalytic activities and HIV-1 replication in cells by compounds 4–12
a
b
Compound
Structure
Inhibition of strand transfer IC50
(
lM)
Antiviral activity CIC95 (lM)
O
OH
nac
N
4
H
0.1
F
N
O
O
OH
N
5
6
H
0.006
0.004
nac
F
N
O
O
OH
OMe
nac
N
H
F
N
O
O
OH
N
H
F
7
0.005
0.094
>1.0
nac
nac
nac
nac
N
O
O
OH
N
8
H
F
N
N
O
O
OH
N
9
H
F
N
N
N
O
O
OH
NH
10
>1.0
F
O

P. Pace et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3865–3869
3867
Table 1 (continued)
a
b
Compound
Structure
Inhibition of strand transfer IC50
(l
M)
Antiviral activity CIC95 (lM)
O
O
O
N
N
OH
11
>1.0
nac
H
N
F
NH
O
F
O
N
OH
12
0.018
35% @ 25 lM
NH
O
F
a
Assays were performed with recombinant HIV-1 integrase (0.1 l
M) preassembled on immobilized oligonucleotides.12 Values are means of three experiments.
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
b
RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum.13 Cytotoxicity is not observed in cell culture at concentrations up to 20
l
M.
c
Not active at 25 lM.
we prepared the pyrrolinone analog 12, which rescued the in vitro
activity to low nanomolar values. Furthermore, with this com-
pound for the first time we started to see some inhibition in the
cell-based assay, although weak. This more rigid bicyclic core
superimposes well with the corresponding pyrimidinone thus
offering additional opportunities for SAR exploration (Fig. 3).
None of the novel pyrrolinones showed cellular toxicity at a
maximum tested concentration of 20 lM.
The target N-substituted-4-hydroxy-5-oxo-2,5-dihydro-1H-
pyrrole-3-carboxamides 4–10 were prepared with minimal modi-
fications of a literature procedure.14 Michael addition of the appro-
priate amine 13 on N-(4-fluorobenzyl)acrylamide 14 gives the
b-aminopropionate 4-fluorobenzylamide 15 which is then con-
densed with diethyloxalate in the presence of sodium ethoxide.
The synthetic sequence employed is shown in Scheme 1. Synthesis
of compound 11 was achieved by a different reaction sequence,
based on the alkylation of 2-(4-morpholinyl)ethanamine 16 with
diethyl (2Z)-2-butenedioate 17, base promoted condensation with
diethyl oxalate and final installation of the 4-fluorobenzylamide by
reaction with p-fluorobenzylamine in ethanol, in the presence of
NaOEt (Scheme 2). For the synthesis of 12, we used an imine-addi-
tion method based on condensation of imine 19 with ethyl oxalac-
Figure 2. Superimposition of 4 with a dihydroxypyrimidine.
Model v9.0. Schrodinger’s maestro front end was used to build ini-
tial conformers and to superimpose molecular structures.10
A significant increase in affinity was observed upon introduc-
tion of a methyl on the benzyl substituent (compound 5) or by
increasing the length of the carbon chain between the aryl ring
and the pyrrolinone nitrogen (compounds 6 and 7), with IC50s in
the low nanomolar range.
Reasoning that perhaps the lack of cellular activity was due to
suboptimal physical chemical properties that could affect cell per-
meability and/or binding to intracellular proteins and also plasma
proteins present in the cell medium, we tried to include polar or
ionizable groups into the molecules.
etate.15 As reported in the literature, imine 19 ( I-piperidine) is an
D
elusive species, therefore was prepared by dehydrohalogenation of
N-chloropiperidine and refluxed with a benzene solution of dieth-
yloxalacetate. The p-fluorobenzylamide moiety was installed upon
microwave irradiation in a N-methylpyrrolidone solution (Scheme
3).
However, this strategy was not successful since we did not ob-
serve any improvement in cellular activity. Moreover, while the
pyridyl-substituted compound 8 retained potency with respect to
its phenyl counterpart (IC50 = 94 nM), the piperidine-derivative 9
did not show significant inhibitory activity. Protein binding of 8
was very high indeed since the compound was ꢀ1% unbound in
human plasma.
Apart from N-1 substitution, we went on to explore substitu-
ents at the 2-position. However, as illustrated by comparison of
compounds 10 and 4, moving the phenyl ring to the 2-position
was detrimental, indicating that there is limited tolerance for sub-
stitution at this site. The more functionalized compound 11 was
inactive too. The introduction of a substituent in this position re-
sults in the loss of oxygen atoms co-planarity, which is required
for Mg2+ chelation. As a bicyclic tetrahydropyridopyrimidone was
found to be an important scaffold for HIV-1 integrase activity,11
Figure 3. Superimposition of 12 with a bicyclic-pyrimidinone.

3868
P. Pace et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3865–3869
R1
HN
F
F
H
H
R1
NH2
13
R2
N
a
+
N
R2
O
15
O
14
O
OEt
EtO
O
b
O
R1
N
OH
R2
NH
O
1-10
F
Scheme 1. Reagents and conditions: (a) EtOH, 60 °C, 12 h; (b) NaOEt (1.2 equiv), EtOH, reflux, 2 h.
O
O
O
OEt
H
N
b
NH2
OEt
OEt
a
N
+
N
O
O
O
OEt
16
17
(CO2Et)2
O
O
O
O
O
N
N
O
OH
NH
c
N
N
H
N
OH
F
EtO
F
OEt
O
18
11
O
Scheme 2. Reagents and conditions: (a) EtOH, 60 °C, 12 h; (b) NaOEt (1.2 equiv), EtOH, reflux, 2 h (20%, 2 steps); (c) 4-F-C6H4CH2NH2 (2.2 equiv), NaOEt (2 equiv), EtOH, refl.
(45%).
O
O
OEt
EtO
H
N
N
O
O
N
OH
a
b
OEt
20
O
19
c
O
N
OH
NH
12
O
F
Scheme 3. Reagents and conditions: (a) i—N-chlorosuccinimide (1.8 equiv), Et2O; ii—KOH (1 equiv), EtOH, 0 °C; (b) benzene, reflux, 3.5 h (30%, 2 steps); (c) 4-F-C6H4CH2NH2
(3 equiv), NMP, 200 °C, 300ss (60%).
In conclusion, we have identified a series of 4-hydroxy-5-pyrr-
olinone-3-carboxamide HIV-1 integrase inhibitors incorporating
the co-planar diketoacid pharmacophore as a novel class of com-
pounds reasonably having the same mode of binding to the en-
zyme as the dihydroxypyrimidines/pyrimidones. A limited SAR
around the pyrrolinone core resulted in the discovery of com-
pounds with inhibitory activity in the low nanomolar range. Fur-
ther chemical optimization of the substituents would better
define the potential of this template for the development of inhib-
itors which may have different profiles than other known
inhibitors.
Acknowledgments
We thank Kara A. Stillmock, Daniel J. DiStefano, Peter J. Felock,
and William A. Schlief for HIV biological testing, and Michael Row-
ley for discussion.

P. Pace et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3865–3869
3869
9. (a) Pace, P.; Di Francesco, M. E.; Gardelli, C.; Harper, S.; Muraglia, E.; Nizi, E.;
Orvieto, F.; Petrocchi, A.; Scarpelli, R.; Laufer, R.; Gonzalez Paz, O.; Monteagudo,
E.; Bonelli, F.; Hazuda, D. J.; Stillmock, K.; Summa, V. J. Med. Chem. 2007, 50,
2225; (b) Gardelli, C.; Nizi, E.; Muraglia, E.; Crescenzi, B.; Ferrara, M.; Orvieto,
F.; Pace, P.; Pescatore, G.; Poma, M.; Rico Ferreira, M.; Scarpelli, R.; Homnick, C.
F.; Ikemoto, N.; Alfieri, A.; Verdirame, M.; Bonelli, F.; Gonzalez Paz, O.; Taliani,
M.; Monteagudo, E.; Pesci, S.; Laufer, R.; Felock, P.; Stillmock, K.; Hazuda, D. J.;
Rowley, M.; Summa, V. J. Med. Chem. 2007, 50, 4953.
10. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield,
C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comp. Chem. 1990, 11, 440.
11. Muraglia, E.; Kinzel, O.; Gardelli, C. ; Crescenzi, B.; Donghi, M.; Ferrara, M.; Nizi,
E.; Orvieto, F.; Pescatore, G.; Laufer, R.; Gonzalez Paz, O.; Di Marco, A.; Fiore, F.;
Monteagudo, E.; Fonsi, M.; Felock, P. J.; Rowley, M.; Summa, V. J. Med. Chem.
2008, 51, 861.
References and notes
1. Laurent, C.; Gueye, N. F. N.; Ndour, C. T.; Gueye, P. M.; Diouf, M.; Diakhatè, N.;
Kane, N. C. T.; Lanièce, I.; Ndir, A.; Vergne, L.; Ndoye, I.; Mboup, S.; Sow, P. S.;
Delaporte, E. J. Acq. Imm. Defic. Syndr. 2005, 38, 14.
2. Summa, V. J. Med. Chem. Soc. 2008, XX, XXX.
3. Esposito, D.; Craigie, R. Adv. Virus Res. 1999, 52, 351.
4. (a) Chiu, T. K.; Davies, D. R. Curr. Top. Med. Chem. 2004, 4, 965; (b) Pommier, Y.;
Johnson, A. A.; Marchand, C. Nat. Rev. Drug. Disc. 2005, 4, 236.
5. (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. Science 2000, 287,
646; (b) Gordon, C. P.; Griffith, R.; Keller, P. A. Med. Chem. 2007, 3, 199; (c)
Anthony, N. J. Curr. Top. Med. Chem. 2004, 4, 979.
6. (a) 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; (b) 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. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6661.
7. Summa, V.; Petrocchi, A.; Matassa, V. G.; Gardelli, C.; Muraglia, E.; Rowley, M.;
Gonzalez Paz, O.; Laufer, R.; Monteagudo, E.; Pace, P. J. Med. Chem. 2006, 49,
6646.
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. J. Med. Chem.
2003, 46, 453.
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. J.; Condra, J. H.; Godib, L.; Holloway, M. K.; Lin, J.; Chen, I.; Vastag,
K.; Ostovic, D.; Anderson, P. S.; Emini, E. A.; Huff, J. R. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 4096.
8. (a) Kawasuji, T.; Fuji, M.; Yoshinaga, T.; Sato, A.; Fujiwara, T.; Kiyama, R. Bioorg.
Med. Chem. Lett. 2007, 15, 5487; (b) Davam, R.; Al-Mawsawi, L. Q.; Neamati, N.
Bioorg. Med. Chem. Lett. 2007, 17, 6155.
14. Southwick, P. L.; Crouch, R. T. J. Am. Chem. Soc. 1953, 75, 3413.
15. Bender, D. R.; Bjeldanes, L. F.; Knapp, D. R.; Rapoport, H. J. Org. Chem. 1975, 40,
1264.