N-(4-Fluorobenzyl)-3-hydroxy-9,9-dimethyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrazino[1,2-a]pyrimidine-2-carboxamides a novel class of potent HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.05.098

A novel class of tetrahydro-pyrazinopyrimidine-2-carboxamides have been identified as HIV-1 integrase inhibitors. Optimization of the initial lead culminated in the discovery of a series of compounds with high potency on the enzyme and an antiviral cell-b

Full text of DOI:10.1016/j.bmcl.2009.05.098

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4245–4249
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-(4-Fluorobenzyl)-3-hydroxy-9,9-dimethyl-4-oxo-6,7,8,9-tetrahydro-4H-
pyrazino[1,2-a]pyrimidine-2-carboxamides a novel class of potent HIV-1
integrase inhibitors
*
Alessia Petrocchi , Philip Jones, Michael Rowley, Fabrizio Fiore, Vincenzo Summa
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 novel class of tetrahydro-pyrazinopyrimidine-2-carboxamides have been identified as HIV-1 integrase
inhibitors. Optimization of the initial lead culminated in the discovery of a series of compounds with high
potency on the enzyme and an antiviral cell-based activity equivalent to that showed by Raltegravir, the
first in class HIV-1 integrase inhibitor.
Received 4 May 2009
Revised 21 May 2009
Accepted 22 May 2009
Available online 29 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
HIV integrasee
HIV integrase is one of the three enzymes encoded by the HIV
genome and is essential for HIV replication. Consequently,
integrase inhibitors are emerging as an exciting class of novel
therapies for the management of HIV infection. In our laborato-
ries, we discovered Raltegravir (1), which is the first in class HIV
O
N
O-K+
F
O
N
H
N
H
N
N
N
IC50 = 15 nM
CIC9 (10% FBS) =19 nM
CIC95 (50% NHS) = 31 nM
O
O
1 (Raltegravir)
integrase inhibitor and represents
a
new and improved
treatment of AIDS.1 We also conducted a wide structure activity
relationship exploration around the pyrimidone core focused on
constraining the N-methyl group to the pyrimidone C-2 substitu-
O
O
OH
F
( )n
OH
F
N
X
N
N
H
H
ent. These efforts resulted in
a series of potent bicyclic
N
N
N
R
N
compounds bearing pendent amino group located at the
a
N
O
O
pseudo-benzylic position (e.g., compound 2, HIV strand transfer
2
A
inhibition QUICKIN IC50 = 69 nM).2,3
Figure 1.
Herein, an alternative cyclization strategy is described which
capitalizes on closing the N-methyl group of the pyrimidone core
directly to the basic nitrogen of the side chain to provide the gen-
eral structure A (Fig. 1).
The gem dimethyl group of compound 1 (Raltegravir) was
maintained in order to eliminate the chiral center and to block po-
tential oxidative metabolism. Preliminary exploration on this new
class of compounds was directed to modify the size of the aliphatic
ring and the functional groups linked to the nitrogen within it
(Table 1).
they showed comparatively low antiviral activity. The corre-
sponding bicyclic amine 7 proved to have not only a good
activity on the enzyme but also significant efficacy in the cell
based assay under both serum conditions. A tertiary amine
was also tolerated as observed for compound 8 which was
found nearly equipotent to 7. A further ring expansion to give
the corresponding seven-membered ring analog 9 did not im-
prove the cellular potency.
The lack of a chiral center, the potency displayed in the viral
spread assay and the excellent rat PK profile (Clp = 10 mL/min/
kg, F = 57%) of compound 8 led to the investigation of further
structural modifications of this new class of [6,6]-fused
derivatives.
The closure to a six-membered lactam 5 or a cyclic sulfon-
amide 6 yielded more potent enzyme inhibitors with respect
to the five-membered derivatives (compounds 3 and 4), but
* Corresponding author. Tel.: +39 06 91093500; fax: +39 06 91093654.
E-mail address: alessia_petrocchi@merck.com (A. Petrocchi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.098

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A. Petrocchi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4245–4249
Table 1
Table 3
Bicyclic pyrimidones
Introduction of an amide group: reducing basicity
O
N
O
( )n
OH
F
OH
F
X
N
N
N
H
H
N
R3
N
N
R1
N
O
O
R1
QUICKINa
IC50 (nM)
Spread CIC95 (nM)
(10% FBS) (50% NHS)
Compd
R3
QUICKINa
IC50 (nM)
Spread CIC95 (nM)
(10% FBS)(50% NHS)
b
b
Compd
n
X
3
4
5
6
7
8
9
0
0
1
1
1
1
2
CH2
CO
CO
SO2
CH2
CH2
CH2
CH3
H
H
H
H
30
32
5
7
16
6
500
1000
>1000
>1000
>10,000
166
15
16
17
18
19
20
21
22
23
24
25
CH2N(CH3)2
CH2N(CHCH3)2
CH2-morpholine
CONH2
CONHCH3
CON(CH3)2
CO-morpholine
CO-pyrrolidine
CO-(4-CH3-piperazine)
COOH
CN
62
76
3
5
21
8
7
7
21
8
5
1250
625
500
125
62
62
62
31
62
1250
625
1000
250
125
125
250
250
125
1000
125
>1000
>1000
5000
125
125
500
CH3
H
250
250
20
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. 4.
500
31
a,b
For footnote details, see Table 1.
b
Spread assay: 95% inhibitory concentration for inhibition of the spread of HIV-1
infection in cell culture (HIV-1IIIB and MT-4 human T-lymphoid cells) in the
presence of 10% heat-inactivated fetal bovine serum or 50% normal human serum.
SD was always 10% of the value. For details see Ref. 5.
The trend was more evident with 20 and 21 which displayed an
eightfold improvement in Quickin activity and a 20-fold in the
Spread assay (10% FBS) if compared to the amine analog 15. Most
noticeably, 22 displayed CIC95 (10% FBS) = 31 nM, but as detected
for all the amides, exhibited decreased activity in presence of
50% NHS. This was probably due to their higher affinity for serum
proteins, which reduced their effective concentrations toward the
viral enzyme.
Table 2
SAR on the nitrogen
O
OH
F
N
H
N
N
R2
N
O
Table 4
Heterocyclic derivatives
Compd
R2
QUICKINa
IC50 (nM)
Spreadb (10% FBS)
CIC95 (nM)
O
OH
F
N
10
11
12
13
14
COCH3
COCON(Me)2
SO2CH3
CONHCH2CH3
SO2N(CH3)2
5
6
10
15
8
1000
1000
1000
500
H
R4
N
N
N
O
1000
R4
QUICKINa
IC50 (nM)
SpreadB CIC95
(nM) (10% FBS) (50% NHS)
a,b
Compd
For footnote details, see Table 1.
26
Ph
5
125
250
Capping the amine with different polar functionalities led to po-
tent integrase inhibitors such as the acetamide 10, the oxalamide
11, the sulfonamide 12, the urea 13 and the sulfamide 14. How-
ever, these compounds exhibited significantly reduced antiviral
activities (Table 2).
Subsequently, side chain substitution and the insertion of a
further basic group were undertaken to see if a boost in activity
could be achieved by the installation of a polar moiety maintain-
ing the amino group on the ring (Table 3). The introduction of a
second basic moiety resulted in little or no improvement; that is,
15 and 16, which displayed a suboptimal activity both against
the enzyme and in the cell based assay and the morpholine
derivative 17 which exhibited an improved potency with modest
cellular activity.
27
28
29
30
31
9
31
125
N
12
6
250
31
1000
125
N
N
N
H
N
5
62
250
N
N
N
7
31
125
In contrast, the addition of a carboxamide group was tolerated
and improved the efficacy in cell culture as shown for the amides
18 and 19.
(continued on next page)

A. Petrocchi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4245–4249
4247
Table 4 (continued)
As before, the insertion of an additional basic group, to gen-
erate the piperazine derivative 23, did not gain any further
improvement in activity although it displayed a smaller serum
shift. When the carboxamide moiety was replaced by carboxylic
acid functionality (24), even if a potent integrase inhibitor was
identified, it displayed low cellular activity demonstrating the
desirability for a neutral moiety on the side chain region. This
observation was confirmed by the excellent potency displayed
by the nitrile 25 in both assays.
Given the encouraging results for 19, 20 and 25, heterocyclic
amide isosteres were explored with a view to improve its biologi-
cal activity and generate a more drug-like molecule.
Overall results highlighted that heterocyclic replacements for
the amide bond usually displayed the improved potency in cell
culture in the presence of low and high serum conditions
(Table 4).
Generally, best results were obtained by five-membered
heterocycles; although the six-membered counterparts such as
compounds 26, 27 and 29 maintained enzyme inhibitory activity,
they exhibited a high shift between the in vitro and the cell based
assay. No benefit came from increasing the distance between
the aromatic moiety and the basic nitrogen as observed for
pyridine 28.
Several trends can be drawn from a closer analysis of the
data. Pyrazoles (30, 31 and 32) displayed similar inhibition of
the spread of HIV-1 infection: acceptable cell permeability and
a moderate shift in the presence of 50% of normal human ser-
um. Interestingly, N-methyl substituted heterocycles usually
showed improved cellular activity compared to those with NH
substitution (as observed for 31 and 32 compared to 30; 45
and 46 compared to 33; 48 and 49 compared to 47). Oxazoles
(35, 37 and 41) were found active on the enzyme displaying
single digit nanomolar value. The triazole 38 showed virtually
no shift between the in vitro and the cell based assay. In con-
trast, thiazole 36 exhibited similar enzymatic potency, but
exhibited eightfold serum shift between low and high serum
conditions. Oxadiazoles like 39, 40, 43 and 44, while not dis-
playing outstanding enzymatic activity, gave compounds with
excellent cellular potency, with all four analogs showing
CIC95 = 62 nM in high serum.
Compd
R4
QUICKINa
IC50 (nM)
SpreadB CIC95
(nM) (10% FBS) (50% NHS)
N
N
32
9
6
7
3
31
31
62
31
125
125
1000
93
H
N
N
N
33
34
35
HN
N
N
N
O
O
S
36
9
15
125
N
N
37
38
39
40
41
42
43
4
25
21
5
31
31
15
7
125
62
N
N
O
N
N
62
O
62
N
N
O
N
8
15
31
15
62
N
O
O
62
13
125
62
N
N
Specific individual compounds of interest, including the
c-methyltriazole 45, were identified as exceptionally potent
enzyme inhibitors with IC50 = 2 nM with more than acceptable
cell penetration. The 2-methyl tetrazole 49 was the most potent
inhibitor from this series displaying CIC95 = 31 nM in high
serum.
In conclusion, tethering the N-methyl group of the pyrimidones
onto the amino gem-dimethyl group was a successful strategy that
led to a novel potent series of bicyclic HIV integrase inhibitors.
These compounds have excellent antiviral activity equivalent to
that showed by Raltegravir.
O
N
N
N
44
45
6
2
15
31
62
62
N
N
N
N
N
N
46
47
48
N
N
6
2
5
6
15
500
15
125
1000
62
The synthetic route used for the advanced intermediate 50 is
fully described in a separate publication.6
In Scheme 1 the synthetic routes for the assembly of the differ-
ent sized bicycles are reported. A diverse approach to cyclize the
[6,6]-fused ring, starting from the already known6 hydrochloride
salt 51, is described in Scheme 2.
Furthermore, the preparation of the derivatives listed in Tables
2 and 3 is illustrated in both Schemes 3 and 4 as well as the prep-
aration of the secondary amine 52, which represents the common
intermediate to provide almost all the heterocyclic compounds re-
ported in Table 4.7
N
H
N
N
N
N
N
N
N
49
N
7
31
a,b
For footnote details, see Table 1.

4248
A. Petrocchi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4245–4249
3
CH2O, AcONa
NaBH3CN, MeOH
Cl
O
S
OH
OH
O
Cl
OH
N
F
O
O
ClSO2CH2Cl
TEA , DCM
N
phosgene
TEA, THF
O
F
H
S
N
H2N
HCl
N
4
H
N
HN
N
O
O
50
1)
OHCCH2CH2OBn
NaCNBH3, MeOH
Cs2CO3
dioxane, 110°C
2) H2, Pd/C
MeOH, 6N NCl
6
OH
N
OH
F
Ph3P, DE A D, TH F
N
H
N
H
N
9
HO
O
Scheme 1.
OH
O
N
OBz
O
N
O
OBz
OMe
BrCOCH2Br
Cs2CO3
Fluorobenzylamine, MeOH
reflux
N
H2N
HCl
5
N
HN
O
O
dioxane, 110ºC
51
Scheme 2.
26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37
13
28
EtNCO, TEA,
THF
1) Ac2O, TEA, DMAP
CHCl3, reflux
N
RCHO, AcOH,
AcOH, MeOH,
60°C
10
11
NaBH3CN, MeOH
R=Het
2) NaOH, MeOH, 40°C
OH
H
Cl
OH
F
1) ClCOCO2Me
pyridine, 50°C
N
H
O
Cs2CO3, dioxane
100 °C
H
50
N
N
N
Cl
7
NaBH3CN, MeOH
2) (Me)2NH, THF
O
1) MeSO2Cl, pyridine,
1) BocNHCH2CHO, AcOH
NaBH3CN, MeOH
2) DCM/TFA
CH2O
12
14
2) NaOH, MeOH, 40°C
1) MsCl, pyridine, 65°C
NaBH3CN, MeOH
Ph3P, DEAD,
benzyl alcohol,
THF
3) CH2O, AcOH
NaBH3CN, MeOH
2) NaOH, MeOH, 40°C
8
O
15
OBn
H
N
F
N
N
H
N
O
52
1) R1X or R2X
acetone/2-butanone, 75°C
2) H2, Pd/C, MeOH
R1=CH2CON(Me)2, X=Cl, 20
R2=CH2CH2N(Et)2, X=Br, 16
Scheme 3.

A. Petrocchi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4245–4249
4249
45, 46
43
1) K2CO3
acetone/2-butanone
38
1) CH3SiCH2N3,
toluene, reflux
reflux
NH2
Cl
N
N
HO
N
EtONa, EtOH
reflux
2) TBAF, -78ºC to RT
N
N
O
2) H2, Pd/C, MeOH
O
N
Br
OBn
F
OBn
F
O
BrCH2CO2tBu
52
H
H
N
N
N
N
N
O
N
DMF, K2CO3
45ºC
DMF, K2CO3
45ºC
O
O
1)EtNO2, PhNCO
TEA, toluene
BrCH2CN
DMF, K2CO3
BrCH2CO2Me
DMF, K2CO3
65°C
reflux
1) NH2OH HCl
KOH, MeOH
O
2) H2, Pd/C, MeOH
45°C
OBn
F
N
H
44
NC
N
N
42
N
2) Ac2O,
O
toluene, reflux
O
OBn
F
18
24
N
O
3) H2, Pd/C, MeOH
H
N
N
O
N
NaN3, NH4Cl
DMF, 120ºC
1) NH3/H2O
2) H2, Pd/C, MeOH
O
H2, Pd/C,
MeOH
1)
NH2NH2 H2O,
MeOH, reflux
47, 25
Bz2O
LiOH,
O
H2O/THF
2) CR(OCH3)3, TsOH
toluene, 110ºC
OBn
F
DMAP, TEA, THF
O
N
H
N
N
HO
N
3) H2 , Pd/C, MeOH
R=H, CH3
O
O
OBz
F
HN
N
N
N
1)
H
1) EDC, HOBT, CHCl
39, 40
N
N
3
NH2
N
N
amine
EDCI, HOBT
2) Hg(OAc)2,
O
2) H2, Pd/C, MeOH
1) MeI, K2CO3, MeCN
2) 1N NaOH, MeCN
AcOH, 100ºC
3) H2, Pd/C, MeOH
48, 49
41
19, 21, 22, 23
Scheme 4.
3. Kinzel, O. D.; Ball, R. G.; Donghi, M.; Maguire, C. K.; Muraglia, E.; Pesci, S.;
Rowley, M.; Summa, V. Tetrahedron. Lett. 2008, 49, 6556.
Acknowledgments
4. 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.
We thank Silvia Pesci for NMR analysis. This work was sup-
ported in part by a grant from MIUR.
5. 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.;
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1994, 91, 4096.
6. Pace, P.; Di Francesco, M. E.; Gardelli, C.; Harper, S.; Muraglia, E.; Nizi, E.;
Orvieto, F.; Petrocchi, A.; Poma, M.; Rowley, M.; Scarpelli, R.; Laufer, R.; Gonzalez
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References and notes
1. 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, R.;
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