Development of 2-pyrrolidinyl-N-methyl pyrimidones as potent and orally bioavailable HIV integrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.07.042

A series of 2-pyrrolidinyl-N-methyl pyrimidones HIV integrase inhibitors has been explored leading to the identification of derivative 13, which showed high activity at inhibiting viral replication in cell culture, favorable pharmacokinetic profile in two

Full text of DOI:10.1016/j.bmcl.2010.07.042

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5031–5034
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of 2-pyrrolidinyl-N-methyl pyrimidones as potent and orally
bioavailable HIV integrase inhibitors
*
Marco Ferrara , Fabrizio Fiore, Vincenzo Summa, Cristina Gardelli
Departments of Medicinal Chemistry and Drug Metabolism IRBM-MRL, Via Pontina, Km 30.600, 00040 Pomezia (RM), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-pyrrolidinyl-N-methyl pyrimidones HIV integrase inhibitors has been explored leading to
the identification of derivative 13, which showed high activity at inhibiting viral replication in cell cul-
ture, favorable pharmacokinetic profile in two preclinical species, and an attractive profile against a panel
of HIV-integrase mutants.
Received 20 June 2010
Accepted 11 July 2010
Available online 15 July 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
HIV integrase
N-Methyl pyrimidones
Table 1
HIV/AIDS continues to be a major threat with global estimates
for 2008 of two million deaths, 33 million people infected world-
wide with HIV and three million new infections, thus rendering
the discovery of new drugs imperative. HIV integrase is one of
the three enzymes encoded by the HIV genome and, being vital
for the replication of the virus,1 represents an attractive target
for the management of HIV infection. We have reported the devel-
opment of potent and orally bioavailable N-methyl pyrimidones,2
which has also led to the discovery of Raltegravir3 (brand name
Isentress™), a first in class inhibitor of HIV integrase approved in
both the US and Europe at the end of 2007. Within this effort, 2-
pyrrolidinyl-N-methyl pyrimidones emerged as especially interest-
ing and although the structure–activity relationship surrounding
the pyrrolidine ring revealed a certain degree of tolerance, a
(2S,4S)-4-fluoro-pyrrolidine 1 stood out as having strong enzymatic
activity and efficacy at inhibiting viral spread in the cell-based
assay (Table 1). The N-methyl pyrrolidine 1 was further evaluated
in vivo (Table 2), where it showed low/moderate plasma clear-
ance (21 and 4.4 mL/min/kg in rat and dog, respectively) and very
high oral bioavailability (95% and 116% in rat and dog, respec-
tively). Based on the attractive profile of 1 we sought to further
optimize its antiviral activity, with our initial approach focusing
on an evaluation of substituents other than a methyl group on
the pyrrolidine N-atom (Table 1). Alternative N-alkyl substituents
(e.g., 2 and 3) consistently retained the intrinsic activity of the ref-
erence compound but cell-based antiviral activity was negatively
Pyrrolidine N-substitution SAR
O
N
OH
F
N
H
N
F
N
O
R
QUICKINa IC50 (nM)
Spread CIC95 (nM)
b
Compd
R
10% FBS
50% NHS
1
2
3
4
5
6
7
8
9
Me
c-Pr
23
20
10
15
10
10
15
9
63
63
156
14
31
125
31
31
16
16
125
250
625
17
CH2(3-isoxazole)
COMe
CO2Me
CONMe2
SO2Me
SO2NMe2
CO-pyrazine
COCONMe2
63
125
125
125
16
10
21
10
16
a
Results are the mean of at least three independent experiments. SD was always
8% of the value. Assays were performed with recombinant HIV-1 integrase pre-
assembled on immobilized oligonucleotides. Inhibitors were added after assembly
and washings, and IC50 is the concentration of inhibitor that reduces HIV integrase
activity by 50%. For details see Ref. 8.
b
Spread assay: 95% cell culture inhibitory concentrations for inhibition of the
spread of HIV-1 infection in cell culture, using HIV-1IIIb and MT-4 T-lymphoid cells,
in a medium containing 10% fetal bovine serum (FBS) or 50% normal human serum
(NHS). Results are the mean of at least three independent experiments, SD was
always < 10% of the value. For details see Ref. 9.
* Corresponding author. Address: Chemistry Research Centre, Boehringer
Ingelheim S.p.A., via Lorenzini 8, I-20139 Milano, Italy. Tel.: +39 02 5355685; fax:
+39 025355283.
E-mail address: marco.ferrara@boehringer-ingelheim.com (M. Ferrara).
Present address: Medicinal Chemistry Department R&D, AstraZeneca, Scheelevä-
gen 2, 22187 Lund, Sweden.
impacted; 3 was fourfold less effective than 1 despite a twofold
improvement in enzyme inhibition. In contrast evaluation of a
variety of electron withdrawing groups on the pyrrolidine nitrogen
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.042

5032
M. Ferrara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5031–5034
Table 2
Table 5
Pharmacokinetic profile of 1 in rata and dogb
Pharmacokinetic profiles of pyrrolidine amides and carbamates in rata and doga
e
a
Species
Clc (mL/min/kg)
Fd (%)
t1/2 (h)
AUCf (
lM h)
Compd
Species
Cla (mL/min/kg)
Fa (%)
t1/2 (h)
AUCa
(lM h)
Rat
Dog
21
4.4
94
116
1.7
3.7
4.5
23.1
11
11
12
13
13
14
15
Rat
Dog
Rat
Rat
Dog
Rat
Rat
43
9.3
39
29
9.7
9.9
21
20
34
17
94
80
10
25
0.7
7.1
1.4
3.0
1.2
7.4
1.5
0.5
2.8
0.5
3.7
6.4
1.2
1.3
a
Dose: iv = po, 3 mg/kg. Formulation: iv, DMSO/PEG400/H2O (20–40–40%); po,
1% methylcellulose.
Dose: iv, 1 mg/kg; po, 2 mg/kg. Formulation: iv, DMSO/PEG400/H2O (20–40–
40%); po, 1% methylcellulose.
b
c
Terminal phase plasma half life following iv administration.
Oral bioavailability.
Terminal phase plasma half life following iv administration.
Area under the curve following po administration.
d
a
For footnote details see Table 2.
e
f
compound unbound in the presence of plasma proteins,4 as indeed
confirmed by human plasma protein binding measurements (79%
for 4 vs 94% for 15). In light of the promising activity displayed
by acetamide 4, extensive screening of pyrrolidine N-amides was
conducted. Alternative alkyl or arylamides typically had unaccept-
able shifts from intrinsic enzymatic activity to the cell-based po-
tency (data not shown) but heteroaryl amides (e.g., 9) and
oxalamides (e.g., 10) maintained the potency profile of 4, confirm-
ing them as privileged substituents for pyrimidone-based HIV-
integrase inhibitors.3,4 The most interesting compounds from the
potency screening were evaluated in rat pharmacokinetics, where
all derivatives displayed moderate to very high plasma clearance
and moderate bioavailability (Table 3). Carbamate 5 and acetamide
4 exhibited the best profile amongst these compounds, but in all
cases inferior PK properties with respect to the lead compound 1
were observed. The metabolic stability in rat liver microsomes of
4 was studied in the presence of NADPH and UDPGA to evaluate
the metabolism rate by oxidation and glucuronidation: modest sta-
bility was observed in both cases with 40% and 43% of initial
remaining after 1 h, respectively.6 These findings, together with
the low value of rat plasma protein binding, 63%,5 were consistent
with the high plasma clearance observed in vivo.
Table 3
Pharmacokinetic profile of pyrrolidine N-substituted analogs in rata
Compd
Clb (mL/min/kg)
Fb (%)
t1/2 (h)
AUCb
(lM h)
b
4
5
8
9
10
64
46
64
77
138
31
44
16
18
19
0.3
0.6
ndc
0.2
1.6
1.2
1.2
ndc
2.3
0.2
a
Dose: iv = po, 3 mg/kg. Formulation: iv, DMSO/PEG400/H2O (20–40–40%); po,
1% methylcellulose.
b
For footnote details see Table 2.
Not determined.
c
gave pleasing results. For compounds 4–10 not only was intrinsic
activity improved, but antiviral activity was at least maintained
and usually enhanced. Amide analog 4 and carbamate 5 led to
superior antiviral activity compared to the urea 6, the sulfonamide
7, or the sulfamide 8. N-Acetyl analog 4 displayed a low shift both
from the intrinsic enzymatic activity to the activity in the low ser-
um conditions (suggesting it has good cell penetration) and also
between the low and high serum cell-based assay conditions. This
latter result is probably a consequence of the significant fraction of
In order to align the attractive activity profile of our optimized
pyrrolidine-N-amide and pyrrolidine-N-carbamoyl analogs we
Table 4
Benzylamide SAR on pyrrolidine amides and carbamates
O
N
OH
H
N
N
R2
F
N
O
R1
a
Compd
R1
R2
QUICKINa IC50 (nM)
Spread CIC95 (nM)
10% FBS
3
50% NHS
17
F
11
12
13
14
COMe
COMe
COMe
CO2Me
24
27
32
20
Cl
F
16
5
31
46
63
Cl
16
Cl
F
15
CO2Me
19
16
31
Cl
a
For footnote details see Table 1.

M. Ferrara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5031–5034
5033
Table 6
Resistance profiles with HIV-1 containing site-directed integrase mutationsa
Compd
T66I
V151I
F121Y
T125K
T66I*b M154I
T66I*b S153Y
N155S*b
T125K*b F121Y
T66I*/L74Mb V151I
Raltegravir
4
11
13
1
1
3
1
1
2
3
0.8
1
5
3
0.8
10
15
31
8
8
15
17
3.5
6
10
31
ndc
2
ndc
1
ndc
4
ndc
1
1
1
1.5
1
2.5
a
Shift in IC50 relative to wild type HIV-1Àsingle cycle infectivity assay.
Viruses that exhibit >50% reduction in specific infectivity.
Not determined.
b*
c
screened substituted benzylamides as alternatives to the 4-fluor-
obenzylamide fragment in the compounds 4 and 5. The benzyla-
mide moiety was known from previous work on the project to
modulate the physicochemical properties of our inhibitors.7
After evaluating N-acetylpyrrolidine-based inhibitors contain-
ing an extensive variety of different benzylamides (results not
shown), meta-para-di-substituted benzylamides 11, 12, and 13
emerged as the most interesting derivatives (Table 4). These com-
pounds displayed an improvement in rat pharmacokinetics in
terms of decreased plasma clearance when compared to the refer-
ence compound 4 and retained a similar level of cell-based potency
(Tables 4 and 5).
Encouraged by these results, the benzylamide screening was ex-
tended to pyrrolidine carbamates leading to the identification of 14
and 15. Similarly to what was observed for 5 when compared to 4,
these carbamates displayed a somewhat reduced antiviral potency
when compared to the corresponding acetamides, but are charac-
terized by a higher stability in rat pharmacokinetics being elimi-
nated less rapidly (Tables 4 and 5). The improved stability in rat
pharmacokinetic of 13 in comparison to the reference compound
4 may be attributed to its significantly larger fraction of compound
bound to the plasma proteins (97.8% vs 63% bound, respectively),5
and hence not available to be metabolized, which may exceed its
reduced stability in liver microsomes (% of initial after 1 h: 8 vs
43 with UDPGA and 0.7 vs 40 with NADPH, respectively),6 the
net result being a reduced in vivo clearance.
Compounds 11 and 13 were selected for further evaluation,
based on the best overall results in terms of potency and rat PK.
Compounds 11 and 13 were then evaluated in dog pharmacokinet-
ics study and both compounds were found to have moderate plas-
ma clearance (9.3 and 9.7 mL/min/kg, respectively). In terms of
oral bioavailability, compound 13 was however much superior to
11 (80% vs 34%, respectively) (Table 5). Compound 13 excelled also
for its profile against a panel of HIV-integrase mutants selected in
2
3
OSiMe3
1
CHO
5
OEt
HCOH, NaCNBH3
MeOH
N
NaCNBH3, AcOH
O
MeOH, reflux
MeOCOCl
NEt3
MeCN, H2O
NaCNBH3
MeOH
O
O
1. 4F-BnNH2
MeOH, reflux
OBz
OH
H
F
Me2NCOCl
N
N
6
N
NEt3
N
CO2Me
N
2. H2, Pd/C
TFA, MeOH
F
F
MeCN, H2O
N
N
O
Cbz
H
MeSO2Cl
NEt3, DCM
CF3CO2H
A
B
1. ClCOCO2Me
NEt3, CHCl3
Me2NSO2Cl
NEt3, DCM
7
N
CO2H
2. NHMe2
THF
N
8
EDC, HOBT
i-Pr2EtN, CHCl3
10
F
9
4, R =
F
1. H2, Pd/C
TFA, MeOH
1. H2, Pd/C
14, R =
15, R =
Cl
F
Ac2O, AcOEt
11, R =
12, R =
13, R =
Cl
F
A
2. RCH2NH2
MeOH, reflux
2. MeOCOCl, NEt3
MeCN, H2O
Cl
3. RCH2NH2
MeOH, reflux
Cl
Scheme 1.

5034
M. Ferrara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5031–5034
R.; Monteagudo, E.; Muraglia, E.; Nizi, E.; Orvieto, F.; Pace, P.; Pescatore, G.;
Scarpelli, R.; Stillmock, K. A.; Witmer, M. V.; Rowley, M. J. Med. Chem. 2008, 51,
5843.
our laboratories employing different classes of inhibitors (Table 6).
In particular, 13 compared favorably on all the mutants to its ana-
logs 11 and 4 and to the marketed drug Raltegravir.
4. Binding to plasma proteins reduces the effective concentration of drug at the
target. See, for example: 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. and references cited
therein.
5. The test compound was dissolved in DMSO and incubated with human plasma
at 37 °C for 1 h. The mixture was then spun in an ultrafiltration device and the
filtrate was assayed for free drug by analytical LC/MS/MS detection.
The synthetic route used for 1, comprising the advanced inter-
mediate A, is fully described in a separate publication.2 In Scheme
1 the synthetic routes for the assembly of the different derivatives
are reported,10 consisting of manipulation of A by benzylamide for-
mation, pyrrolidine nitrogen deprotection, and functionalisation
carried out in this or in an alternative order made using routine
procedures that have been described in our previous work.2
In summary, optimization of 1 resulted in the identification of
13, characterized by high potency in the cell-based assay, favorable
PK profile when dosed orally to preclinical species and excellent
mutant profile. Compound 13 thus represents a promising agent
against HIV-1 and is the subject of further studies.
6. The concentration of the test compound was 1 lM and microsomes 1 mg/mL.
The degradation of the substrate was measured by LC/MS/MS after a 1 h
incubation.
7. Petrocchi, A.; Koch, U.; Matassa, V. G.; Pacini, B.; Stillmock, K.; Summa, V.
Bioorg. Med. Chem. Lett. 2007, 17, 350.
8. 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.
9. 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. I.; Condra, J. H.; Gotlib, 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.
Acknowledgments
We thank Silvia Pesci for NMR analysis; Marina Taliani, Massi-
miliano Fonsi, and Francesca Naimo for plasma protein binding
determination and microsome stability studies; Kara A. Stillmock,
Peter J. Felock, and William A. Schlief for HIV biological testing;
Steven Harper for proofreading the manuscript.
10. Synthesis of 13: A (102 mg, 0.20 mmol) dissolved in ethyl acetate (8 mL) was
treated with Pd/C 10% (10 mg) and acetic anhydride (19 lL, 0.20 mmol) and
submitted under H2 atmosphere at room temperature. The reaction mixture
was stirred at room temperature for 18 h and then was filtered through Celite.
The filtrate was evaporated under reduced pressure, dissolved in MeOH
(2.4 mL), treated with 3-Cl-4-Me-benzylamine (93 mg, 0.60 mmol) and
refluxed for 48 h, then it was cooled down. The solvent was evaporated and
the residue purified by RP-HPLC (stationary phase: column symmetry C18,
References and notes
1. 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.
2. 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.; Laupher, R.; Felock, P.; Stillmock, K.; Hazuda, D. J.;
Rowley, M.; Summa, V. J. Med. Chem. 2007, 50, 4953.
5
lm, 19 Â 100 mm. Mobile phase: acetonitrile/H2O buffered with 0.1% TFA).
Fractions containing the pure compound were combined and freeze dried to
afford 13 as a white powder (69.2 mg, 79% over two steps). 1H NMR (DMSO-d6,
300 MHz, 300 K) d 12.06 (bs, 1H), 8.62 (t, J = 6.3 Hz, major rotamer) + 8.42 (t,
J = 6.3 Hz, minor rotamer) (1H), 7.43–7.35 (m, 2H), 7.24–7.18 (m, 1H), 5.60–
5.23 (m, 2H), 4.58–4.49 (m, 2H), 4.33–3.83 (m, 2H), 3.58 (s, 3H), 2.75–2.32 (m,
2H), 2.37 (s, 3H), 2.10 (s, major rotamer) + 1.91 (s, minor rotamer) (3H). MS: m/
z 437 (M+H)+.
3. Summa, V.; Petrocchi, A.; Bonelli, F.; Crescenzi, B.; Donghi, M.; Ferrara, M.;
Fiore, F.; Gardelli, C.; Gonzalez Paz, O.; Hazuda, D. J.; Jones, P.; Kinzel, O.; Laufer,