Synthesis and evaluation of C2-carbon-linked heterocyclic-5-hydroxy-6-oxo-dihydropyrimidine-4-carboxamides as HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.11.060

Integration of viral DNA into the host cell genome is an obligatory process for successful replication of HIV-1. Integrase catalyzes the insertion of viral DNA into the target DNA and is a validated target for drug discovery. Herein, we report the synthesis, antiviral activity and pharmacokinetic profiles of several C2-carbon-linked heterocyclic pyrimidinone-4-carboxamides that inhibit the strand transfer step of the integration process.

Full text of DOI:10.1016/j.bmcl.2014.11.060

Accepted Manuscript
Synthesis and evaluation of C2-carbon-linked heterocyclic-5-hydroxy-6-oxo-
dihydropyrimidine-4-carboxamides as HIV-1 integrase inhibitors
B. Narasimhulu Naidu, Margaret E. Sorenson, Manoj Patel, Yasutsugu Ueda,
Jacques Banville, Francis Beaulieu, Sagarika Bollini, Ira B. Dicker, Helen
Higley, Zeyu Lin, Lori Pajor, Dawn D. Parker, Brian J. Terry, Ming Zheng,
Alain Martel, Nicholas A. Meanwell, Mark Krystal, Michael A. Walker
PII:
S0960-894X(14)01255-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.11.060
BMCL 22215
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 October 2014
19 November 2014
21 November 2014
Please cite this article as: Narasimhulu Naidu, B., Sorenson, M.E., Patel, M., Ueda, Y., Banville, J., Beaulieu, F.,
Bollini, S., Dicker, I.B., Higley, H., Lin, Z., Pajor, L., Parker, D.D., Terry, B.J., Zheng, M., Martel, A., Meanwell,
N.A., Krystal, M., Walker, M.A., Synthesis and evaluation of C2-carbon-linked heterocyclic-5-hydroxy-6-oxo-
dihydropyrimidine-4-carboxamides as HIV-1 integrase inhibitors, Bioorganic & Medicinal Chemistry Letters
(2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.11.060
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Synthesis and evaluation of C2-carbon-
linked heterocyclic-5-hydroxy-6-oxo-
dihydropyrimidine-4-carboxamides as HIV-1
integrase inhibitors
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B. Narasimhulu Naidu,* Margaret E. Sorenson, Manoj Patel, Yasutsugu Ueda, Jacques Banville, Francis
Beaulieu, Sagarika Bollini, Ira B. Dicker, Helen Higley, Zeyu Lin, Lori Pajor, Dawn D. Parker, Brian J. Terry,
Ming Zheng, Alain Martel, Nicholas A. Meanwell, Mark Krystal, Michael A. Walker

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis and evaluation of C2-carbon-linked heterocyclic-5-hydroxy-6-oxo-
dihydropyrimidine-4-carboxamides as HIV-1 integrase inhibitors
B. Narasimhulu Naidu,*a Margaret E. Sorenson,a Manoj Patel,a Yasutsugu Ueda,a Jacques Banville,a
Francis Beaulieu,a Sagarika Bollini,b Ira B. Dicker,b Helen Higley,b Zeyu Lin,b Lori Pajor,c Dawn D.
Parker,c Brian J. Terry,b Ming Zheng,c Alain Martel,a Nicholas A. Meanwell,a Mark Krystal,b Michael A.
Walkera
Department of Discovery Chemistrya, Virologyb and Pharmaceutical Candidate Optimization,c Bristol-Myers Squibb Research and Development, 5 Research
Parkway, Wallingford, CT-06492, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Integration of viral DNA into the host cell genome is an obligatory process for successful
replication of HIV-1. Integrase catalyzes the insertion of viral DNA into the target DNA and is
a validated target for drug discovery. Herein, we report the synthesis, antiviral activity and
pharmacokinetic profiles of several C2-carbon-linked heterocyclic pyrimidinone-4-
carboxamides that inhibit the strand transfer step of the integration process.
2009 Elsevier Ltd. All rights reserved.
Keywords:
HIV-1
Integrase
Strand transfer
Inhibitor
Pyrimidine-4-carboxamide
The HIV-1 integrase enzyme, one of three virus-encoded
enzymes, plays a critical role in retroviral replication. After
reverse transcription of viral RNA into a double stranded DNA,
the integrase enzyme, as part of a pre-integration complex,
migrates into the nucleus and catalyzes the insertion of the viral
genome into the host chromosome via a multi-step process. The
two enzymatic steps in this insertion process are: (1) cleavage of
a dinucleotide from the 3’-ends of the viral DNA and (2) the
subsequent insertion of these processed ends into the host DNA
(strand transfer).1 Although interrupting either of these steps
could, in principle, lead to inhibition of viral replication,
compounds that inhibit the strand transfer reaction were found to
potently and specifically exhibit antiviral activity, leading to
FDA approval of three strand transfer inhibitors, raltegravir,2
elvitegravir3 and dolutegravir4 (Figure 1). We have examined
pyrimidinone-4-carboxamides as inhibitors of HIV-1 integrase
Figure 1. FDA approved strand transfer inhibitors.
The desired pyrimidinone carboxamides were prepared in a
short sequence as outlined in Scheme 1. Treatment of nitriles 1
with N-methylhydroxylamine provided the hydroxyamidine
adducts 2 which upon exposure to diethyl acetylenedicarboxylate
afforded the oxadiazolines 3.5 Pyrolysis of 3 in xylenes furnished
the desired pyrimidinone esters 4,6 which upon heating with the
appropriate benzylamine in the presence of triethylamine
provided the final pyrimidinone-4-carboxamides 5.
and
a systematic survey of C2-substitutents led to the
identification of tetrahydrofuran and tetrahydropyran substituents
at C-2 as optimal moieties that combined high antiviral potency
with good pharmacokinetic profiles in preclinical species. In this
article, we disclose the synthesis, antiviral activity and
pharmacokinetic properties of a series of C2-carbon linked
heterocyclic-5-hydroxy-6-oxo-dihydropyrimidine-4-
carboxamides as novel HIV-1 integrase strand transfer inhibitors.

14
10
49
301

O
aCompounds 8-14 were prepared and screened as racemic mixtures, bEC50
values were determined in the presence of 10% FBS, cHSA-EC50 values were
determined in the presence of 15 mg/mL of human serum albumin and 10%
FBS.
Interestingly, the nature of the substituent on the THF ring and
the position of attachment of the THF moiety to the pyrimidinone
core exert a significant influence on the antiviral activity of this
series of pyrimidinone carboxamides. For example, the polar 4-
hydroxy-THF derivative 9 maintained the potent enzymatic
inhibitory activity associated with its progenitor 8 but exhibited
diminished antiviral activity in the cell culture. On the contrary,
the enzymatic and antiviral activities of the 4-thiomethyl-THF
analog 10 are comparable with those of 8. In addition, 10 is
subject to a lower effect of HSA than that observed with 8
despite being more lipophilic. Interestingly, tetrahydrofuran 11
and tetrahydrothiophene 12 display comparable enzymatic and
cell culture inhibitory activities to 8 but suffer from a higher
HSA-shift. The tetrahydropyran-4-yl (THP) analog 13 exhibited
excellent enzymatic and cell culture activities whereas the C2-
tetrahydropyran-2-yl derivative 14 possesses diminished
activities in both assays. Furthermore, 14 exhibited substantially
reduced antiviral activity in the presence of HSA when compared
with 13. The data for compounds 8, 10-14 suggest that the
oxygen atom in the tetrahydrofuran-3-yl and tetrahydropyran-4-
yl moieties increases the hydrophilic character of the molecule
which, in turn, lowers the effect of HSA. In addition, it is
possible that the oxygen atom in the tetrahydropyran-4-yl moiety
may be engaging additional binding interaction with integrase,
leading to improved antiviral activity for 13 when compared with
14.
Scheme 1. Synthesis of pyrimidinone-4-carboxamides.
The initial structure-activity relationship (SAR) study focused
on identifying suitable C2-substituents and a variety of five- and
six-membered carbon-linked heterocycles were surveyed with the
results summarized in Table 1. Both the furan and the 2,5-
dihydrofuran analogs 6-7 exhibited good enzymatic inhibition in
a biochemical strand transfer assay7 but showed significantly
reduced antiviral activity in a cell culture virus replication assay.
In addition, compounds 6 and 7 displayed a large shift in antiviral
activity in the presence of human serum albumin (HSA). For this
inhibitor class, it was shown that HSA can substitute for human
serum (HS), with 15 mg/mL mimicking results observed when
40% HS is added, while the addition of 45 mg/mL represents
activity in 100% HS.7
tetrahydrofuran-3-yl (THF) analog
On the other hand, the C2-
exhibited excellent
8
inhibitory activity in both the strand transfer and antiviral cell
culture assays. Furthermore, compound 8 is significantly more
potent than the unsaturated compounds 6 and 7 in the presence of
HSA, suggesting lower protein binding.
Table 1. SAR of C2-heterocyclic substituents in a series of HIV-
1 integrase inhibitors
Select compounds were evaluated for pharmacokinetic (PK)
evaluation; results are summarized in Table 2. In a rat IV study,
8, 10 and 13 are characterized as low clearance compounds with
values of 4.9, 3.6 and 2.6 mL/min/kg, respectively. All three
Compounda
R
Inhibition of
strand transfer
IC50 (nM)
Antiviral EC50 in
cell culture (nM)
compounds have short half-lives and
a low volume of
FBSb
HSAc
distribution. In the rat oral PK study, excellent oral exposure and
good bioavailability was observed for 8 and 10.
Table 2. Rata pharmacokinetic data for 8, 10 and 13
6
21
19
4
424
14,490
Compound
IV dose (mg/kg)b
CL (mL/min/kg)
t1/2 (h)
8
10
13
7
206
9
1,783
60


1
1
1
O
4.9
1.63
0.14
5
3.6
1.08
0.15
5
2.6
0.58
0.12
ndc
8
Vss (L/kg)
O
9
6
48
7
126
33
PO dose (mg/kg)b

OH
S
Cmax (M)
10.45
0.67
33.89
68
8.04
1.67
39.45
67
O
Tmax (M)
10
3

AUC (M*h)
F (%)
O
11
12
13
5
5
5
10
15
6
120
153
28


aMale Sprauge-Dawley rats, bVehicle: PEG-400/Ethanol (90:10), cnd = not
done
O
Although the tetrahydrofuran derivative 8 exhibited excellent
antiviral activity and a good rat PK profile, it displayed greater
than an 18-fold reduction in antiviral activity when tested in the
presence of 45 mg/mL of HSA. Consequently, in order to reduce
the HSA effect and improve overall drug-like properties, we
S

O
synthesized and evaluated
a variety of ortho-substituted

23
24
16
10
2
2
22
22
t-Bu
O
benzylamides based on this C2-THF substituted template and the
results are summarized in Table 3. These data reveal that a
variety of polar groups are well tolerated at the ortho-position
and these moieties contributed to improved antiviral activity in
N
S
O
N

cell culture with a lower HSA-shift.
For example, the
N
O
methylsulfonyl analog 15 and two sulfonamide derivatives 16
and 17 exhibited excellent antiviral activities8 and displayed
significantly reduced HSA-shifts compared to 8. Similarly, the
ortho-heterocyclic analogs 18-26 exhibited very potent antiviral
activity and they are 5- to 15-fold more potent than 8 in the
presence of 45 mg/mL HSA. The unsubstituted triazole
compound 18 is equipotent with 8 both in the strand transfer and
the FBS assays. Interestingly, the antiviral activity of the methyl
triazole analogue 19 in HSA was similar to that of 18, despite the
fact that this compound exhibited reduced activity in both the
strand transfer and the cell culture (FBS) assays. The lack of a
shift in 45 mg/mL HSA for 19 may partly be due to the lower
protein binding effected by the methyltriazole moiety. On the
other hand, the 1,2,3-triazole analog 20 exhibited similar
potencies both in the enzymatic and the FBS assays as 19, but it
displayed about a 2-fold shift in the HSA assay. Between the
two carbon-linked methyl triazole derivatives 21 and 22, the 1-
methyl-1,2,4-triazole 21 is more potent than the 4-methyl-1,2,4-
triazole 22 in all three assays. The thiadiazolidine analogs 23-24
and the imidazolidine compounds 25-26 displayed potent cellular
activity in the FBS cell culture assay but, unfortunately, these
compounds suffer from higher shifts in the presence of HSA (4 to
11-fold loss of activity).
S
O
N

25
26
6
3
7
5
30
27
N
O
N

N
O
O
N

a Compounds were evaluated as racemic mixtures, bEC50 valued were
determined in the presence of 10% FBS, cHSA-EC50 values were determined
in the presence of 45 mg/mL human serum albumin and 10% FBS.
In parallel, the ortho-substituted benzylamide analogs 27–35
based on the C2-THP substituted core were prepared and
evaluated, and the results are summarized in Table 4. Similar to
the C2-THF series, several C2-THP derivatives showed
improved antiviral activity in cell culture and displayed a lower
HSA effect with less than a 4-fold loss of antiviral activity in the
presence of 45 mg/mL HSA. Although, the C2-THP compounds
27-35 displayed comparable activities to those of the
corresponding C2-THF derivatives, both in the strand transfer
and cell culture (FBS) assays, the C2-THP analogs diverged from
the C2-THF derivatives in the HSA assay. The majority of the
C2-THP analogs exhibited 2-4 fold improved potency in
presence of HSA than the corresponding C2-THF derivatives.
Furthermore, the C2-THP compounds displayed reduced HSA
shifts when compared with the corresponding C2-THF analogs.
Table 3. SAR associated with N-(arylmethyl)-5-hydroxy-1-
methyl-6-oxo-2-(tetrahydrofuran-3-yl)-1,6-dihydropyrimidine-4-
carboxamide derivatives
Table 4. SAR associated with N-(arylmethyl)-5-hydroxy-1-
methyl-6-oxo-2-(tetrahydro-2-pyran-4-yl)-1,6-
dihydropyrimidine-4-carboxamide derivatives
Compounda
R
Inhibition of
strand transfer
IC50 (nM)
Antiviral EC50 in
cell culture (nM)
FBSb
HSAc
168
33
8
H
4
9
15
16
17
SO2Me
SO2NMe2
9
14
4
Compound
R
Inhibition of
strand transfer
IC50 (nM)
Antiviral EC50 in
cell culture (nM)
10
9
25
FBSb
HSAc
Ndc
13
5
19
O
S
O
N
13
27
28
29
H
5
6
6
4
4

SO2Me
SO2NMe2
8
18
19
4
7
18
18
N
N
N 
12
11
15
N
N
N
12
14
11
N 
O
S
O
N

20
21
22
10
6
13
5
28
11
27
N
30
31
6
8
10
9
18
14
N
N
N 
N 
N
N
N
N
N
N
N
N 

N
N
32
33
13
9
8
4
18
7
N
18
28
N 

N
N

N

34
35
17
6
2
2
7
7
t-Bu
O
Several ortho-substituted benzylamides were evaluated for
their PK properties in rats and the results are compiled in Table
5. The cyclic sulfonamide 17 and methylsulfone 27 exhibited
low clearance with short half-lives and low volumes of
distribution. Compound 17 also demonstrated very good oral PK
properties with high oral exposure and good bioavailability. On
the other hand, in the rat IV screen, compounds 18, 19, 21, 23, 26
and 34 displayed moderate to high clearance, short half-lives and
low volumes of distribution.
N
S
O
N

N
O
S
O
N

a Compounds 15-26 were evaluated as racemic mixtures, bEC50 valued were
determined in the presence of 10% FBS, cHSA-EC50 values were determined
in the presence of 45 mg/mL human serum albumin and 10% FBS, cnd = not
determined.

Table 5. Rata pharmacokinetic data for C2-tetrahydrofuran-3-yl and C2-tetrahydropyran-4-yl pyrimidinone benzylamides
Compound
IV dose (mg/kg)b
CL (mL/min/kg)
t1/2 (h)
17
18
19
21
23
26
27
34
1
1
1
1
1
1
1
1
6
47
37
12
31
15
3.9
0.58
0.13
ndc
41
0.54
0.2
5
0.42
0.98
ndc
0.23
0.76
ndc
0.5
0.36
ndc
0.24
0.36
ndc
0.46
0.44
ndc
0.22
0.61
ndc
Vss (L/kg)
PO dose (mg/kg)b
Cmax (M)
3.51
0.63
12.35
60
Tmax (M)
AUC (M*h)
F (%)
aMale Sprauge-Dawley rats, bVehicle: PEG-400/Ethanol (90:10), cnd = not done
2. Summa, V.; Petrocchi, A.; Bonelli, F.; Crescenzi, B.; Donghi, M.;
Ferrara, M.; Fiore, F.; Gardelli, C.; Paz, O. G.; Hazuda, D. J.;
Jones, P.; Kinzel, O.; Laufer, R.; Monteaguda, E.; Muraglia, E.;
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Stillmock, K.; Witmer, M. V.; Rowley, M. J. Med. Chem. 2008,
51, 5843.
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T.; Yamashita, M.; Ito, Y.; Matsuzaki, Y.; Yamataka, K.; Ikeda,
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Temelkoff, D. P.; Yoshida, H.; Akiyama, T.; Taoda, Y.; Murai,
H.; Kiyama, R.; Fuji, M.; Tanimoto, N.; Jefferey, J.; Foster, S. A.;
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Garvey, E. P.; Fujiwara, T. J. Med. Chem. 2013, 56, 5901.
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G. O.; Reamer, R. A.; Houk, K. N.; Askin, D. Angew. Chem. Int.
Ed. 2008, 47, 4134.
In conclusion, we have identified tetrahydrofuran-3-yl and
tetrahydropyran-4-yl moieties as optimal C2-substituents in a
series of pyrimidinone HIV-1 integrase inhibitors by systematic
investigation of various carbon-linked five and six membered
heterocycles at the C2-position of the heterocycle core. In
addition, in order to lower the effect of human serum albumin on
the antiviral activity of the compound, we surveyed the effect of
polar substituents at the benzylamide moiety using C2-THF and
C2-THP derivatives as the vehicles. A variety of polar
substituents at the ortho-position are well tolerated that exhibit
improved antiviral activity and significantly lowered HSA shifts.
Furthermore, several compounds were evaluated for their
pharmacokinetic properties in rats, and compounds 8, 10 and 17
displayed low clearance, high oral exposure and good
bioavailability.
7. Dicker, I.B., Walker, M.A., Lin, Z., Terry, B., Pajor, L., Zheng,
M., Naidu, B.N., Banville, J., Meanwell, N.A., Krystal, M. A
Simple and accurate in vitro method for predicting serum protein
binding of HIV integrase strand transfer inhibitors. In HIV-1
Integrase; Mechanism and Inhibitor Design; Neamati, N., Ed.;
John Wiley & Sons: Hoboken, New Jersey, 2011, pp275-286.
8. The individual epimers of 17 were evaluated for their antiviral
activity and both epimers exhibited similar activities in strand
transfer and cell culture assays.
Acknowledgment
We are grateful to Mr. Henry Wong and Mr. Ashok Trehan of
Department of Discover Synthesis for the contributions to the
scale up of intermediates (4-fluoro-2-(1H-1,2,4-triazol-1-
yl)phenyl)methanamine and (4-fluoro-2-(5-methyl-1H-1,2,4-
triazol-1-yl)phenyl)methanamine. We also thank Mr. Fukang
Yang for the scale up of (4-fluoro-2-(4-methyl-4H-1,2,4-triazol-
3-yl)phenyl)methanamine.
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References and notes
1. Craigie, R. Future Virol., 2012, 7, 679.