Pyridopyrimidinone inhibitors of HIV-1 RNase H

Article abstract of DOI:10.1016/j.ejmech.2014.06.061

Using a structure based pharmacophore design, a weak inhibitor of RNase H, identified from a small library of two metal binding HIV-1 integrase inhibitors, was optimized for potency and physicochemical properties. This manuscript describes the SAR and in vivo DMPK for the pyridopyrimidinone class of inhibitors.

Full text of DOI:10.1016/j.ejmech.2014.06.061

European Journal of Medicinal Chemistry 83 (2014) 609e616
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Pyridopyrimidinone inhibitors of HIV-1 RNase H
Emile J. Velthuisen*, Brian A. Johns, Peter Gerondelis, Yan Chen, Ming Li, Ke Mou,
Wenwen Zhang, John W. Seal, Kendra E. Hightower, Sonia R. Miranda, Kevin Brown,
Lisa Leesnitzer
GlaxoSmithKline Research & Development, Infectious Diseases Therapeutic Area Unit, Five Moore Drive, Research Triangle Park, NC 27709, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Using a structure based pharmacophore design, a weak inhibitor of RNase H, identified from a small
library of two metal binding HIV-1 integrase inhibitors, was optimized for potency and physicochemical
properties. This manuscript describes the SAR and in vivo DMPK for the pyridopyrimidinone class of
inhibitors.
Received 7 April 2014
Received in revised form
25 June 2014
Accepted 27 June 2014
Available online 28 June 2014
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
HIV
AIDS
Reverse transcriptase
Integrase
Two-metal binder
Endonuclease
1. Introduction
polymerase and a separate ribonuclease H (RNase H) region located
on the C-terminus. RNase H is a metal dependent endonuclease
The human immunodeficiency virus type 1 (HIV-1) utilizes
three virally-encoded enzymes to facilitate replication following
infection of the host: reverse transcriptase, integrase and protease.
Accordingly, these enzymes have become attractive targets for
disrupting the viral lifecycle with antiretroviral drugs. However,
despite over 30 approved agents and combination treatment op-
tions, HIV therapy remains problematic due to drug resistance and
tolerability.
HIV reverse transcriptase (RT) is a heterodimeric protein
responsible for conversion of single stranded viral RNA into double
stranded DNA. This is accomplished through the concerted function
of two active sites on the p66 domain; the N-terminus DNA
that hydrolyzes RNA as part of an RNA/DNA hybrid duplex. Several
of these RNA nicking events take place during the reverse tran-
scription process to facilitate template RNA dissociation and
negative DNA strand transfer after the initial strong stop minus
strand synthesis. Additionally, the RNase H activity facilitates the
necessary selective cleavage to free up the polypurine tract (ppt) to
act as a positive strand primer and is responsible for removal of the
original tRNA primer. Mutation studies have demonstrated that
RNase H is essential for viral replication, and despite the effort of
numerous groups, the only approved drugs targeting RT are in-
hibitors of the polymerase function. While inhibitors of the RT
polymerase function have enjoyed a rich history of successful
research, inhibitors of the RNase H functional biochemistry have
not. In fact, with the development of both HIV protease and inte-
grase inhibitors, HIV RNase H has the dubious and unique distinc-
tion of being the only virally encoded enzymatic function that is not
part of a chemotherapeutic intervention now a full 30 years into the
HIV/AIDS epidemic.
Abbreviations: ADME, absorption, distribution, metabolism, excretion; AIDS,
acquired immunodeficiency syndrome; Cl, clearance; CLND, chemi-luminescent
nitrogen detection; DIPEA, diisopropyl ethyl amine; DMPK, drug metabolism and
pharmacokinetics; DNAUC, dose-normalized area under the curve; F, oral
bioavailability; HAART, highly active antiretroviral therapy; HIV, human immuno-
deficiency virus; IN, Integrase; INST, integrase strand transfer; PFI, property forecast
index; RT, reverse transcriptase; RTST, reverse transcriptase strand transfer; TMS,
trimethylsilyl.
High resolution crystallography of RNase H complexed to the
RNA/DNA hybrid reveals two Mg2þ ions as essential in formation of
the active catalytic complex. Interestingly, a very similar two-metal
motif is required for the catalytic activity of strand-transfer inte-
grase inhibitors, and indeed, it has been demonstrated that the two
* Corresponding author.
E-mail address: emile.j.velthuisen@gsk.com (E.J. Velthuisen).
http://dx.doi.org/10.1016/j.ejmech.2014.06.061
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 83 (2014) 609e616
enzymes share numerous structural features in their active sites [1].
Because of the similarity between these two targets, we initiated a
screen of our two-metal binding library to identify novel inhibitors
in an RT strand-transfer biochemical assay. Herein, we report the
results of the screening effort and the design of highly selective and
potent RNase H inhibitors. In addition, the pharmacokinetic profile
for several of these compounds will be discussed.
2. Results and discussion
2.1. Focused metal-binder screen
Fig. 2. Incorporating the two-metal binding motif of a known RNase H inhibitor.
As part of an effort to identify novel starting points for the
development of selective inhibitors of HIV RNase H activity, a small
library of two-metal chelating HIV integrase inhibitors (consisting
of internal legacy examples as well as literature compounds) were
screened for the ability to inhibit HIV RT strand transfer activity in a
modified version of the assay described by Gabbara and Peliska [2].
Because this assay requires both RNase H and DNA polymerase
activities of RT, hits were counter screened in secondary assays
similar to those previously described to resolve specific inhibitors
of RNase H from those that inhibited the DNA polymerase activity
of RT [3,4]. This subset of compounds represents a larger collection
of 2-metal binders with an emphasis upon scaffold diversity. The
results of our screening identified several naphthyridine scaffolds
as weak inhibitors of RT strand transfer which became the starting
point for our optimization (Fig. 1).
accessibility of a pyridopyrimidinone such as 5 made this a very
attractive scaffold to initially investigate. In addition, during the
course of our studies, an N-hydroxy naphthyridinone inhibitor of
RNase H was reported, further fueling our interest in this archi-
tecture [14,15].
The synthesis of 5-amino substituted pyridopyrimidinone ana-
logs is shown in Scheme 1. Two slightly different protecting group
strategies were utilized in the synthesis of these analogs and both
are described. Starting from commercially available 2-
fluoronicotinic acid, nucleophilic addition of O-benzyl hydroxyl-
amine under microwave irradiation conditions provided pyridine 7.
The carboxylic acid was then esterified using TMS-diazomethane
and the O-benzyl hydroxylamine moiety was acylated with 4-
methoxybenzylisocyanate. Cyclization to form the imide was ach-
ieved using sodium methoxide and the 4-methyoxybenzyl group
was then oxidatively cleaved using ceric ammonium nitrate. In a
similar manner, synthesis of the tert-butyl protected analogs star-
ted with addition of O-tert-butyl hydroxylamine into 2-
fluoronicotinonitrile 10. The nitrile was hydrolyzed under basic
conditions and the resulting carboxylic acid was esterified using
TMS-diazomethane. Aniline 11 was treated with trichloroacetyl
isocyanate and the resulting urea 12 was then cyclized and
concomitantly deprotected using sodium methoxide. Treatment of
the imide 9 or 13 with phosphorous oxychloride afforded chloride
14 or 15 which underwent nucleophilic addition with a variety of
amines. The protecting group was then removed under acidic
conditions to afford examples 18e29 (Table 1).
2.2. Chemistry
In designing more potent and selective RNase H inhibitors, it is
important to consider which structural features of the naphthyr-
idine hits are part of the integrase strand transfer pharmacophore
[5,6] and which features can be appended to the molecule to better
mimic the reverse transcriptase strand transfer pharmacophore
[7,8]. In general, integrase strand transfer inhibitors contain three
domains: a hydrophobic region, a flexible linker, and a two-metal
binding region [9]. As can be seen in Fig. 1, the 4-chloro benzyl-
amine is crucial for the integrase strand transfer activity [5,10] (2
INSTIC50 ¼ 0.135
m
M; 3 INSTIC50 ¼ 11
mM) and removal of this group is
likely beneficial for designing a more selective RT strand transfer
inhibitor. In addition, the 5-amino substituent of naphthyridine 1
[11] appears to provide a slight boost in potency in the RT-ST assay
2.3. Structure activity relationship
(1 RTSTIC50 ¼ 10
m
M; 3 RTSTIC50 ¼ 11
mM) and could serve as a useful
To establish whether these compounds conveyed an antiviral
effect, a single round of infection assay was used as previously
described [16]. This assay has been employed to resolve antiviral
activity for separate NNRTI and integrase inhibitor development
programs and has been engineered to allow for the mechanistic
resolution of susceptibility changes against site-directed mutants
specific for these types of inhibitors. All of the compounds reported
in Table 1 were tested for activity against RT-polymerase but
functional handle for future SAR exploration. There have been
numerous examples reported in the literature of N-hydroxyimides
as potent endonuclease inhibitors [12]. Our interest in this scaffold
was to take a well established two-metal binding motif (i.e. 4) and
incorporate this onto naphthyridine 3 (Fig. 2). Although the oxygen
of the carbonyl imide of 4 is likely better match for the hard metal
properties of the divalent magnesium cofactor [13], the synthetic
Fig. 1. Initial hits from a RT strand transfer screen.

E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 83 (2014) 609e616
611
Scheme 1. Synthesis of pyridopyrimidinone analogs. Reagents and conditions: (i) NH2OBn, DIPEA (69%); (ii) TMS-CH2N2, Et2O (96%); (iii) 1-isocyanatomethyl-4-methoxybenzene,
1,2-DCE (73%); (iv) NaOMe, MeOH (74%); (v) cerium (IV) ammonium nitrate, CH3CN/H2O (79%); (vi) NH2OtBu$HCl, DIPEA (96%); (vii) NaOH, H2O (79%); (viii) TMS-CH2N2, Et2O (97%);
(ix) trichloroacetyl isocyanate, 1,2-DCE; (x) NaOMe, MeOH (75% over 2-steps); (xi) POCl3, DIPEA (97%); (xii) POCl3, DIPEA (97%); (xiii) 4-fluorobenzyl amine, DMF (85%); (xiv) 4,4-
difluoropiperidine, DIPEA, DMF (81%); (xv) HBr, AcOH, 80 ꢁC; (xvi) TFA, DCM (65%).
demonstrated no inhibition up to a concentration of 10
m
M. Dose-
To explore the antiviral effect of additional aromatic groups
appended to the lead 18, biphenyl aniline 21 was synthesized. This
analog was significantly more potent than 18 in both the antiviral
dependent cytotoxicity measures were determined in parallel
with the antiviral measures by the use of cells that were pre-
infected and constitutively express the fire-fly luciferase reporter.
Since the virus employed to create these cells is replication defec-
tive, inhibition of this signal would reflect a mechanism that is
independent of viral reverse transcription and integration and
hence resolve a correlate (CC50) for the resolution of selectivity
versus the antiviral effect (IC50). In addition to optimization of
antiviral potency, the optimization of physicochemical properties
was also taken into account. There have been numerous studies in
the literature that demonstrate a negative impact upon compound
developability with an increasing number of aromatic rings and
high lipophilicity [17,18]. A simple metric to track this is known as
the property forecast index (PFI) which is the sum of the chro-
matographic log DpH7.4 plus the aromatic ring count [19]. A recent
retrospective analysis indicates that 75% of orally available mar-
keted drugs have a PFI of ꢀ6 [20]. To measure some of the devel-
opability criteria for analogs synthesized, both the permeability and
solubility for select compounds are reported (Table 2).
(
(
PHIVIC50
¼
0.10
m
M) and biochemical RNase
H
assay
RTSTIC50 ¼ 0.032
mM). Interestingly, there was also modest activity
in the IN strand transfer assay (INSTIC50 ¼ 0.6
mM) and measurable
cellular cytotoxicity at a concentration of 2.5
mM. Installation of
biphenyl benzyl amine 22 afforded a similar antiviral profile as
biphenyl aniline 21, albeit less potent in the single round infectivity
assay (PHIVIC50 ¼ 0.20
mM) and significantly more potent in the
RNase H assay (RTSTIC50 ¼ 0.010
mM). As with the biphenyl analog,
there was measurable activity in the IN strand transfer assay
(
INSTIC50 ¼ 0.8
m
M) and slightly less cytotoxicity (CC50 ¼ 4
mM).
In designing analogs that improve the physicochemical prop-
erties of this series, an emphasis was placed upon incorporation of
heteroaromatic and heterocyclic groups. An example of this is
replacement of the distal phenyl ring of 21 with a pyridine such as
in compound 23. Although this initial analog was less potent in
both biochemical assays (RTSTIC50 ¼ 0.079
m
M; INSTIC50 ¼ 0.4
mM)
mM), there
and the single round infectivity assay (PHIVIC50 ¼ 0.26
As a starting point for our optimization, 4-fluorobenzyl amine 18
was synthesized. This analog had modest potency in the single
was no measurable cytotoxicity up to a maximum concentration of
50
m
M, and the PFI was significantly lower, as expected (¼5).
round infectivity assay (pHIVIC50 ¼ 0.50
m
M) and decent potency in
Interestingly, the benzyl amine version of this analog (24) was
the RNase H biochemical assay (RTSTIC50 ¼ 0.158
mM). In addition,
nearly equipotent in the antiviral assay (PHIVIC50 ¼ 0.30
mM) albeit
this analog was weakly active in the IN-strand transfer assay
more potent in the RNase H biochemical assay (RTSTIC50 ¼ 0.016
mM)
(
INSTIC50 ¼ 3.16
m
M) and did not exhibit any cytotoxicity up to a
and similar in potency in the IN strand transfer activity
maximum concentration of 39
mM. Although a promising lead in
(
INSTIC50 ¼ 1.26
mM).
terms of potency and selectivity for RNase H over INST activity, the
PFI (¼7.2) for this compound was higher than desired. Unfortu-
nately, removal of the 4-fluorobenzyl amine and installation of non-
aromatic containing side chains (i.e. 19 and 20) had a deleterious
To further improve upon the physicochemical properties of this
series, we investigated replacement of the distal aromatic ring of 21
with morpholine. The 4-morpholino phenyl analog 25 was slightly
less potent than biphenyl 21 in the antiviral assay
effect upon both the antiviral (19 PHIVIC50
¼
1.91
mM; 20
(
PHIVIC50 ¼ 0.25
mM), however it had similar potency in both
PHIVIC50 ¼ 0.71
m
M) and the RNase H activity (19 RTSTIC50 ¼ 0.501
mM;
biochemical assays (RTSTIC50 ¼ 0.050
m
M; INSTIC50 ¼ 0.5
mM). As
20 RTSTIC50 ¼ 0.398
(19 ¼ 6.3; 20 ¼ 2.6).
m
M) although it did offer lower PFI values
expected, the PFI of this compound (¼4) was significantly lower
than the corresponding biphenyl analog 21 (PFI ¼ 7.2).

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E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 83 (2014) 609e616
Table 1
Antiviral profile of pyridopyrimidinone analogs.
Cmpd
Structure
pHIV
RT-RNase H
IC50 M)
IN-ST
IC50
Fold selectivity
RT/IN-ST
Cytotoxicity
CC50 (mM)
PFI
7.2
IC50
(
m
M)
(
m
(m
M)
18
0.50
0.158
3.16
20
>39
19
20
1.91
0.71
0.501
0.398
7.94
3.9
16
10
>50
>39
6.3
2.6
21
22
0.10
0.20
0.032
0.010
0.79
0.63
25
63
2.5
7.2
7.0
4.0
23
0.26
0.079
1.99
25
>50
5.0
24
25
26
0.30
0.25
0.47
0.016
0.050
0.100
1.26
0.50
1.58
78
10
16
>50
>50
>50
5
4
5
27
0.16
0.200
1.3
7
>50
3.6
28
29
0.38
0.30
0.200
0.398
1.99
10
40
>30
>50
4.4
6.5
15.9
Having identified a suitable replacement for the distal phenyl
ring of 21, our attention turned to optimizing the positioning of the
morpholine group. The objective was to increase the antiviral po-
tency of these compounds while maintaining a good selectivity of
RNase H over IN strand transfer. The benzyl amine analog 26 did
improve the desired selectivity in the biochemical assays (25, 10ꢂ;
26; 16ꢂ) albeit at the cost of antiviral potency (25,
PHIVIC50 ¼ 0.25
m
M; 26, PHIVIC50 ¼ 0.47
mM). Interestingly, synthesis
of the 4-(morpholinomethyl)aniline 27, where the extra methylene
linker is now connected to the morpholine, provided an analog
with increased potency in the antiviral (PHIVIC50 ¼ 0.16
mM) and IN
mM), although the activity in
strand transfer assays (INSTIC50 ¼ 1.3

E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 83 (2014) 609e616
613
Table 2
alternative approach would be to build-in potency for both enzyme
functions in a single molecule. Our efforts in this area resulting in
highly potent dual inhibitors, as well as details on the mechanism
of action, will be the subject of future publications.
In vivo and developability profiles of pyridopyrimidinone analogs.
Cmpd Cl (mL/
min/kg)
t1/2 (h) i.v. DNAUC
F (%) Permeability CLND (mM
(ng h/mL/mg)
(nm/s)
solubility)
21
23
24
25
26
27
5.9
12
16
45
55
70
7.4
10
0.5
2.4
4.5
0.4
2934
1942
1000
314
713
226
0
0
ND
ND
ND
ND
172
13
16
7
14
5
3
3
16
161
293
503
4. Experimental section
4.1. Chemistry
All commercially available reagents were used without further
purification. Column chromatography was carried out on silica gel
(70e230 mesh). TLC was conducted on silica gel 250 micron, F254
plates. 1H NMR spectra were recorded at 300 or 400 MHz. Melting
points are uncorrected. Purities of test compounds were estab-
lished by analytical HPLC (C-18 column, 5.0 micron, 0 / 100%
CH3CN (or MeOH)/water with 0.05e0.1% HCOOH (or TFA)) and UV
detection with or without mass spectrometer detection. All test
compounds showed >95% purity (AUCs by UV detection).
the RNase H biochemical assay was now slightly decreased
(
RTSTIC50 ¼ 0.200
mM). Moving the distal morpholine from a para to
meta position (26e28), also slightly improved the antiviral potency
(
PHIVIC50 ¼ 0.38
mM) while decreasing the inhibitory effect in both
biochemical assays (RTSTIC50 ¼ 0.200
m
M; INSTIC50 ¼ 1.99
mM). And
finally, placing an extra methylene spacer between the phenyl ring
and the morpholine (29) greatly reduced the IN-strand transfer
activity (INSTIC50 ¼ 15.9
m
M) while further improving the antiviral
4.1.1. Procedure A (OBn protecting group)
potency (PHIVIC50 ¼ 0.30
mM).
4.1.1.1. 2-{[(Phenylmethyl)oxy]amino}-3-pyridinecarboxylic acid (7).
A solution of 2-fluoro-3-pyridinecarboxylic acid (3.0 g, 21.2 mmol),
O-(phenylmethyl)hydroxylamine (3.4 g, 27.6 mmol) and diisopro-
pylethylamine (7.4 mL, 42.5 mmol) was irradiated in the microwave
at 120 ꢁC. After 40 min, the reaction mixture was concentrated in
vacuo and the residue purified by silica gel chromatography (0e10%
MeOHedichloromethane) to afford the title compound (3.58 g,
During the course of our optimization, it became challenging to
correlate closely between the antiviral and biochemical assays. One
factor believed to contribute to this was poor cell permeability. As
can be seen in Table 2, compound 21 has moderate cell perme-
ability (172 nm/s), and there is a good correlation between the
biochemical and antiviral assays (Table 1). However, many of the
other analogs have low cell permeability (MDCK <16 nm/s) and also
lack a reliable correlation between the antiviral and biochemical
assays. Additional mechanism of action studies around these in-
hibitors will be published in due course.
69%) as a yellow solid: 1H NMR (400 MHz, DMSO-d6)
d ppm 8.26 (br.
s., 1H), 8.07 (dd, J ¼ 7.62, 1.37 Hz, 1H), 7.42e7.50 (m, 2H), 7.28e7.44
(m, 3H), 6.66e6.85 (m, 1H), 4.99 (s, 2H).
4 .1. 1. 2 . M e t h y l 2 - { [ ( p h e n y l m e t h y l ) o x y ] a m i n o } - 3 -
pyridinecarboxylate. A solution of 2-{[(phenylmethyl)oxy]amino}-
3-pyridinecarboxylic acid (3.6 g, 14.7 mmol) in diethyl ether
(37 mL) and methanol (37 mL) was treated with TMS-
diazomethane (14.66 mL, 29.3 mmol, 2.0 M in hexane). After
5 min, AcOH was added dropwise until gas evolution ceased. The
reaction mixture was concentrated in vacuo to afford the title
compound (3.6 g, 96%) as an orange oil: 1H NMR (400 MHz,
2.4. In vivo DMPK
To evaluate metabolic stability, a number of analogs were dosed
both intravenously and orally in SpragueeDawley rats. In general,
incorporation of the morpholine moiety (Table 2; 25, 26, 27)
resulted in higher clearance values relative to the hepatic blood
flow in rats. Examples with distal aromatic or heteroaromatic
groups (Table 2; 21, 23, 24) had lower clearance values but no oral
exposure was observed. Despite a modest permeability for 21, poor
solubility is likely limiting the oral absorption for this compound.
Although the solubility for the more hydrophilic analogs (i.e. 25, 26,
27) was greatly improved, both low permeability and high clear-
ance values prevented progression of these compounds.
CHLOROFORM-d)
d
ppm 10.04 (s, 1H), 8.51 (dd, J ¼ 1.8, 4.8 Hz, 1H),
8.17 (dd, J ¼ 1.9, 7.8 Hz, 1H), 7.52e7.47 (m, 2H), 7.44e7.33 (m, 3H),
6.81 (dd, J ¼ 4.8, 7.7 Hz, 1H), 5.09 (s, 2H), 3.85 (s, 3H); ES þ MS: m/
z ¼ 259 (Mþ1).
4.1.1.3. Methyl 2-{[({[4-(methyloxy)phenyl]methyl}amino)carbonyl]
[(phenylmethyl)oxy]amino}-3-pyridinecarboxylate (8). A solution of
3. Conclusion
methyl
2-{[(phenylmethyl)oxy]amino}-3-pyridinecarboxylate
(3.1 g, 12 mmol) and 1-(isocyanatomethyl)-4-(methyloxy)benzene
(5.1 mL, 36 mmol) in 1,2-dichloroethane (24 mL) was irradiated in
microwave at 120 ꢁC. After 90 min, the reaction mixture was
concentrated in vacuo and the residue purified by silica gel chro-
matography (0e100% ethyl acetateehexanes) to afford the title
compound (3.7 g, 73%) as a white solid: 1H NMR (400 MHz,
In conclusion, a number of HIV-1 RT RNase H inhibitors were
synthesized. These compounds had good potency in the RNase H
biochemical assay, were highly selective over HIV-1 RT polymerase,
and were partially selectivity over HIV-1 Integrase. In addition to
potency, the physicochemical properties of these compounds were
optimized using the property forecast index (PFI). Because of this,
several analogs had both improved solubility and good potency in
the biochemical assay. Unfortunately, one of the challenges for this
class of molecule was low permeability that resulted in poor in vivo
oral absorption. Additionally, high clearance was observed when
dosed intravenously in rats, possibly due to conjugation of the
central hydroxamic acid moiety as a sulfate or glucuronide. None-
theless, the ability to differentiate between two-metal utilizing
endonuclease active sites through our initial structural modifica-
tions and demonstrate clear biochemical inhibition was very
encouraging. This report focused on our attempts to design selec-
tive inhibitors of RNase H biochemistry over integrase. The
CHLOROFORM-d)
d
ppm 8.58 (dd, J ¼ 4.88, 1.76 Hz, 1H), 8.10 (dd,
J ¼ 7.80, 1.76 Hz, 1H), 7.37e7.45 (m, 2H), 7.27e7.33 (m, 3H),
7.18e7.24 (m, 1H), 7.06 (d, J ¼ 8.59 Hz, 2H), 6.76e6.89 (m, 2H),
4.95e5.04 (m, 2H), 4.26 (d, J ¼ 5.85 Hz, 2H), 3.73e3.81 (m, 6H);
ES þ MS: m/z ¼ 422 (Mþ1).
4.1.1.4. 1-[(Phenylmethyl)oxy]pyrido[2,3-d]pyrimidine-2,4(1H, 3H)-
dione (9). An ice-cold solution of methyl 2-{[({[4-(methyloxy)
phenyl]methyl}amino)carbonyl][(phenylmethyl)oxy]amino}-3-
pyridinecarboxylate (1.0 g, 2.49 mmol) in methanol (25 mL) was
treated with sodium methoxide (0.062 mL, 0.249 mmol, 25% in
MeOH). After 5 min, the white precipitate was filtered and washed

614
E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 83 (2014) 609e616
with MeOH to afford the title compound (0.72 g, 74%): 1H NMR
(400 MHz, DMSO-d6) ppm 8.74e8.92 (m, 1H), 8.33e8.53 (m, 1H),
1H NMR (400 MHz, CDCl3)
d
ppm 8.34 (dd, J ¼ 1.6, 5.2 Hz, 1H), 8.82
d
(dd, J ¼ 1.6, 7.6 Hz, 1H), 7.44 (s, 1H), 6.85 (dd, J ¼ 5.2, 7.6 Hz, 1H), 1.39
7.59e7.65 (m, 2H), 7.42 (d, J ¼ 1.95 Hz, 5H), 7.29e7.34 (m, 2H),
6.85e6.91 (m, 1H), 5.21 (s, 2H), 5.06 (s, 2H), 3.72 (s, 3H); ES þ MS:
m/z ¼ 399 (Mþ1).
(s, 9H); ES þ MS: m/z ¼ 192.3 (Mþ1).
4.1.2.2. 2-(tert-Butoxyamino)-3-pyridinecarboxylic acid. To a solu-
tion of 2-(tert-butoxyamino)nicotinonitrile (9.00 g, 47.1 mmol) in
methanol was added a solution of NaOH (8.69 g, 141.0 mmol) in
water (50.0 mL), and the mixture was heated at 100 ꢁC. After 13 h,
the reaction mixture was cooled to ambient temperature and
acidified to pH ¼ 3e4 with 1 M HCl. The mixture was concentrated
in vacuo and subsequently azeotroped with acetonitrile 3ꢂ. The
residue was purified by silica gel chromatography (0e20% MeOH in
DCM) to afford 2-(tert-butoxyamino)-3-pyridinecarboxylic acid
(8.76 g, 79% yield) as a yellow solid: 1H NMR (400 MHz, CDCl3)
A suspension of methyl 2-{[({[4-(methyloxy)phenyl]methyl}
amino)carbonyl][(phenylmethyl)oxy]amino}-3-
pyridinecarboxylate (0.71 g, 1.83 mmol) in Acetonitrile (16 mL)/
Water (1.6 mL) was treated with cerium (IV) ammonium nitrate
(5.0 g, 9.13 mmol) and heated to reflux. After 30 min, the reaction
mixture was cooled, diluted with water and extracted with ethyl
acetate. The combined organic layers were washed with NaHCO3,
brine, dried (MgSO4), filtered and concentrated. The residue was
purified by silica gel chromatography (0e10% MeOH/dichloro-
methane) to afford the title compound (0.39 g, 1.4 mmol, 79% yield)
d
ppm 8.50 (d, J ¼ 6.8 Hz, 1H), 8.32 (d, J ¼ 6.8 Hz, 1H), 6.72 (t,
as a yellow solid: 1H NMR (400 MHz, DMSO-d6)
d
ppm 11.92 (s, 1H),
J ¼ 6.8 Hz, 1H), 1.41 (s, 9H); ES þ MS: m/z ¼ 211.2 (Mþ1).
8.79 (dd, J ¼ 1.7, 4.9 Hz, 1H), 8.35 (dd, J ¼ 1.7, 7.8 Hz, 1H), 7.62 (dd,
J ¼ 1.6, 7.6 Hz, 2H), 7.47e7.36 (m, 5H), 5.17 (s, 2H); ES þ MS: m/
z ¼ 270 (Mþ1).
4.1.2.3. Methyl 2-(tert-butoxyamino)-3-pyridinecarboxylate (11).
A
solution of 2-(tert-butoxyamino)-3-pyridinecarboxylic acid
(4.06 g, 19.31 mmol) in diethyl ether (54.6 mL) and methanol
(54.6 mL), was treated with TMS-diazomethane (2 M in hexanes)
(19.31 mL, 38.62 mmol) dropwise. After 5 min, excess TMS-
diazomethane was quenched by the dropwise addition of AcOH
until gas evolution ceased. The reaction mixture was concentrated
in vacuo and the residue was purified by silica gel chromatography
(25% EtOAc in hexanes) to afford methyl 2-(tert-butoxyamino)-3-
pyridinecarboxylate (4.18 g, 97% yield) as yellow oil: 1H NMR
4.1.1.5. 4-Chloro-1-[(phenylmethyl)oxy]pyrido[2,3-d]pyrimidin-
2(1H)-one (14). A solution of 1-[(phenylmethyl)oxy]pyrido[2,3-d]
pyrimidine-2,4(1H, 3H)-dione (60 mg, 0.223 mmol) in diisopro-
pylethylamine (195
us(V)oxychloride (104
m
L, 1.114 mmol) was treated with phosphor-
m
L, 1.114 mmol) and heated to 100 ꢁC. After
45 min, the reaction mixture was concentrated in vacuo and the
residue (62 mg, 97%) was used immediately without further puri-
fication: 1H NMR (400 MHz, DMSO-d6)
d
ppm 9.00 (dd, J ¼ 4.62,
(400 MHz, CDCl3)
d
ppm 9.85 (s, 1H), 8.42 (dd, J ¼ 2.0, 4.8 Hz, 1H),
1.61 Hz,1H), 8.50 (dd, J ¼ 7.95,1.72 Hz,1H) 7.66 (dd, J ¼ 7.52,1.72 Hz,
2H), 7.56 (dd, J ¼ 8.17, 4.73 Hz, 1H), 7.42e7.46 (m, 3H), 5.22 (s, 2H);
ES þ MS: m/z ¼ 288 (Mþ1).
8.13 (dd, J ¼ 2.0, 7.6 Hz,1H), 6.68 (dd, J ¼ 4.8, 7.6 Hz,1H), 3.87 (s, 3H),
1.34 (s, 9H); ESþ MS: m/z ¼ 225.3 (Mþ1).
4.1.2.4. 1-(tert-Butoxy)pyrido[2,3-d]pyrimidine-2,4(1H,
3H)-dione
4.1.1.6. 1-(Benzyloxy)-4-((4-fluorobenzyl)amino)pyrido[2,3-d]pyr-
imidin-2(1H)-one (16). A solution of 4-chloro-1-[(phenylmethyl)
oxy]pyrido[2,3-d]pyrimidin-2(1H)-one (40 mg, 0.139 mmol) in
DMF (1.4 mL) was treated with [(4-fluorophenyl)methyl]amine
(174 mg, 1.390 mmol) and stirred at ambient temperature. After 2 h,
the reaction mixture was concentrated in vacuo and purified by
reverse phase HPLC to afford the title compound (32 mg, 61.2%) as a
white solid: ES þ MS: m/z ¼ 377 (Mþ1).
(13). A solution of methyl 2-(tert-butoxyamino)-3-
pyridinecarboxylate (4.18 g, 18.64 mmol) in 1,2-dichloroethane
(240 mL) was treated with trichloroacetyl isocyanate (4.93 mL,
41.6 mmol) dropwise. After 2 h, the reaction mixture was concen-
trated in vacuo and treated with methanol (240 mL) and sodium
methoxide (20.14 mL, 93 mmol) (25% by weight in MeOH). After
45 min, the reaction mixture was concentrated in vacuo and was
treated with 1 N HCl (42 mL) and the 0.1 N HCl (85 mL). The layers
were partitioned and the organic phase was washed with brine,
dried over Na2SO4, filtered and concentrated. The residue was pu-
rified by silica gel chromatography (0e30% EtOAc in hexanes) to
afford 1-(tert-butoxy)pyrido[2,3-d]pyrimidine-2,4(1H, 3H)-dione
(3.27 g, 74.6% yield) as a light yellow solid: 1H NMR (400 MHz,
4.1.1.7. 4-{[(4-Fluorophenyl)methyl]amino}-1-hydroxypyrido[2,3-d]
pyrimidin-2(1H)-one hydrobromide (18). A solution of 4-{[(4-
fluorophenyl)methyl]amino}-1-[(phenylmethyl)oxy]pyrido[2,3-d]
pyrimidin-2(1H)-one (41 mg, 0.109 mmol) in acetic acid (1.3 mL)
was treated with HBr (179
m
L, 1.089 mmol, 33% in acetic acid), and
CDCl3)
d
ppm 9.29 (s, 1H), 8.71 (dd, J ¼ 2.0, 4.8 Hz, 1H), 8.44 (dd,
warmed to 80 ꢁC. After 6 h, the reaction mixture was concentrated
in vacuo and purified by reverse phase HPLC to afford the title
compound as a white solid (34 mg, 85%): 1H NMR (400 MHz,
J ¼ 2.0, 7.6 Hz, 1H), 7.25 (dd, J ¼ 4.8, 7.6 Hz,1H), 1.48 (s, 9H); ESþ MS:
m/z ¼ 236.4 (Mþ1).
DMSO-d6)
d
ppm 10.37 (s, 1H), 9.05 (s, 1H), 8.69 (dd, J ¼ 1.5, 4.8 Hz,
4.1.2.5. 4-(4,4-Difluoro-1-piperidinyl)-1-[(1,1-dimethylethyl)oxy]pyr-
ido[2,3-d]pyrimidin-2(1H)-one (17). A solution of 1-(tert-butoxy)
pyrido[2,3-d]pyrimidine-2,4(1H, 3H)-dione (42 mg, 0.177 mmol) in
toluene (3 mL) was treated with DIPEA (0.16 mL, 0.89 mmol) and
POCl3 (0.04 mL, 0.44 mmol) and the mixture was heated at 100 ꢁC.
After 1 h, the solvent was removed in vacuo to afford 4-chloro-1-
(tert-butoxy)pyrido[2,3-d]pyrimidin-2(1H)-one (15) that was used
immediately without further purification.
A solution of 4-chloro-1-(tert-butoxy)pyrido[2,3-d]pyrimidin-
2(1H)-one (45 mg, 0.177 mmol) in N,N-dimethylformamide (5 mL)
and DIPEA (2.0 mL, 11.45 mmol) was treated with 4,4-
difluoropiperidine (335 mg, 2.129 mmol) and stirred at ambient
temperature. After 15 min, the mixture was diluted by EtOAc, and
washed with water and brine, dried over Na2SO4 and concentrated
in vacuo. The residue was purified by silica gel chromatography
(0e5% MeOH in DCM) to provide 4-(4,4-difluoro-1-piperidinyl)-1-
1H), 8.54 (dd, J ¼ 1.5, 8.0 Hz, 1H), 7.41 (dd, J ¼ 5.6, 8.4 Hz, 2H), 7.28
(dd, J ¼ 4.8, 8.0 Hz, 1H), 7.17 (t, J ¼ 8.9 Hz, 2H), 4.68 (d, J ¼ 5.5 Hz,
2H); ES þ MS: m/z ¼ 287 (Mþ1).
4.1.2. Procedure B (OtBu protecting group)
4.1.2.1. 2-(tert-Butoxyamino)nicotinonitrile. A mixture of 3-cyano-
2-fluoropyridine (9.0 g, 73.71 mmol), O-tert-butyl hydroxylamine
hydrochloride (10.17 g, 81.0 mmol) and DIPEA (28.32 mL,
162.3 mmol) was heated in a microwave at 120 ꢁC. After 9 h, the
reaction mixture was cooled to ambient temperature and parti-
tioned between EtOAc and water. The organics were washed with
brine, and the aqueous layer was extracted 3ꢂ with EtOAc. The
organic phases were combined, dried (Na2SO4), and concentrated
in vacuo to provide crude 2-(tert-butoxyamino)nicotinonitrile
(13.11 g, 93% yield) as dark red oil which solidified upon standing.

E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 83 (2014) 609e616
615
[(1,1-dimethylethyl)oxy]pyrido[2,3-d]pyrimidin-2(1H)-one
prepared using procedure B to provide the title compound as a TFA
salt: 1H NMR (400 MHz, DMSO-d6)
ppm 9.04 (s, 1H), 8.67 (dd,
(48.9 mg, 0.145 mmol, 81% yield) as a brown oil: 1H NMR (400 MHz,
d
CDCl3)
d
ppm 8.60 (d, J ¼ 4.8 Hz,1H), 7.85 (d, J ¼ 8.0 Hz,1H), 7.10 (dd,
J ¼ 1.5, 4.7 Hz, 1H), 8.55 (dd, J ¼ 1.5, 8.0 Hz, 1H), 7.28e7.19 (m, 3H),
6.92 (d, J ¼ 8.7 Hz, 2H), 4.60 (d, J ¼ 5.5 Hz, 2H), 3.77e3.68 (m, 4H),
3.12e3.03 (m, 3H); ESþ MS: 354.0 (Mþ1).
J ¼ 4.8, 8.0 Hz, 1H), 8.88 (m, 4H), 2.12 (m, 4H), 1.42 (s, 9H); ESþ MS:
m/z ¼ 339.4 (Mþ1).
4.1.2.6. 4-(4,4-Difluoropiperidin-1-yl)-1-hydroxypyrido[2,3-d]pyr-
imidin-2(1H)-one (19). A solution of 4-(4,4-difluoro-1-piperidinyl)-
4.1.2.14. 1-Hydroxy-4-((4-(morpholinomethyl)phenyl)amino)pyrido
[2,3-d]pyrimidin-2(1H)-one (27). The title compound was prepared
using procedure B to provide the title compound as a TFA salt: 1H
1-(tert-butoxy)pyrido[2,3-d]pyrimidin-2(1H)-one
(48.9
mg,
0.145 mmol) in dichloromethane (10 mL), was treated with tri-
fluoroacetic acid (1.0 mL). After 4 h, the reaction mixture was
concentrated in vacuo. The residue was crystallized from MeOH/
DCM to afford 4-(4,4-difluoro-1-piperidinyl)-1-hydroxypyrido[2,3-
d]pyrimidin-2(1H)-one (37.8 mg, 65.3% yield) as a brown solid: 1H
NMR (300 MHz, DMSO-d6) d ppm 10.63 (s, 1H), 10.05 (s, 1H),
8.84e8.77 (m, 2H), 7.96 (d, 2H), 7.55 (s, 2H), 7.40 (dd, 1H), 4.37 (s,
2H), 4.01e3.98 (m, 2H), 3.69e3.61 (m, 2H), 3.40 (m, 2H), 3.29e3.18
(m, 2H); ESþ MS: 354.1 (Mþ1).
NMR (400 MHz, METHANOL-d4)
d
ppm 8.70 (dd, J ¼ 1.6, 4.7 Hz,1H),
4.1.2.15. 1-Hydroxy-4-({[3-(4-morpholinyl)phenyl]methyl}amino)
pyrido[2,3-d]pyrimidin-2(1H)-one (28). The title compound was
prepared using procedure B to provide the title compound as a TFA
salt: 1H NMR (400 MHz, METHANOL-d4) Shift ¼ 8.67 (d, J ¼ 4.5 Hz,
1H), 8.51 (br. s., 1H), 7.49e7.17 (m, 5H), 4.76 (br. s., 2H), 3.92 (br. s.,
4H), 3.41 (br. s., 4H); ESþ MS: 354.0 (Mþ1).
8.34 (s, 1H), 7.37e7.30 (m, 1H), 4.01e3.91 (m, 4H), 2.19 (s, 4H); ESþ
MS: m/z ¼ 282.9 (Mþ1).
4.1.2.7. 4-((Cyclopropylmethyl)amino)-1-hydroxypyrido[2,3-d]pyr-
imidin-2(1H)-one (20). The title compound was prepared using
procedure A to provide a white solid (6.12 mg, 90%) as a TFA salt. 1H
NMR (400 MHz, DMSO-d6)
d
ppm 8.73 (br. s., 1H), 8.67 (dd, J ¼ 1.5,
4.1.2.16. 1-Hydroxy-4-({[3-(4-morpholinylmethyl)phenyl]methyl}
amino)pyrido[2,3-d]pyrimidin-2(1H)-one (29). The title compound
was prepared using procedure A to provide the title compound as a
TFA salt following purification by reverse phase HPLC: 1H NMR
(400 MHz, METHANOL-d4) Shift ¼ 8.65 (d, J ¼ 4.6 Hz, 1H), 8.45 (d,
J ¼ 7.2 Hz, 1H), 7.62 (br. s., 1H), 7.52e7.24 (m, 4H), 4.74 (s, 2H), 4.32
(br. s., 2H), 4.05e3.94 (m, 2H), 3.68 (t, J ¼ 11.5 Hz, 2H), 3.31 (s, 2H),
3.15 (d, J ¼ 9.6 Hz, 2H); ES þ MS: 368 (Mþ1).
4.7 Hz,1H), 8.55 (dd, J ¼ 1.5, 8.0 Hz,1H), 7.27 (dd, J ¼ 4.7, 8.0 Hz,1H),
3.36e3.27 (m, 2H), 1.13 (d, J ¼ 17.3 Hz, 1H), 0.52e0.43 (m, 2H),
0.33e0.24 (m, 2H); ES þ MS: 233 (Mþ1).
4.1.2.8. 4-([1,10-Biphenyl]-4-ylamino)-1-hydroxypyrido[2,3-d]pyr-
imidin-2(1H)-one (21). The title compound was prepared using
procedure A to provide the title compound as a TFA salt following
purification by reverse phase HPLC: 1H NMR (400 MHz, DMSO-d6)
d
ppm 10.05e9.94 (m, 1H), 8.85e8.72 (m, 2H), 7.94 (d, J ¼ 8.4 Hz,
4.2. Virology
2H), 7.72 (dd, J ¼ 8.2, 10.9 Hz, 4H), 7.48 (t, J ¼ 7.7 Hz, 2H), 7.41e7.32
(m, 2H); ES þ MS: 331 (Mþ1).
4.2.1. Reagents
Reverse transcriptase and integrase were prepared as previously
described [21]. Deoxynucleotide triphosphates (dNTPs) and
protease-free Bovine Serum Albumin (BSA; Fraction V) were ac-
quired from Roche Applied Science (Indianapolis, IN). [8-3H(N)]-
deoxyguanosine 50-triphosphate (dGTP) and streptavidin-coated
polystyrene SPA imaging beads were acquired from PerkinElmer
(Waltham, MA). 1,4-dithiothreitol (DTT), CHAPS and dimethyl
sulfoxide (DMSO) were acquired from SigmaeAldrich (St. Louis,
MO). NP-40 was acquired from Thermo Scientific (Rockford, IL).
Nuclease-free dH2O, Tris pH 8.0, NaCl, KCl, MgCl, and EDTA were
acquired from Life Technologies (Grand Island, NY). All compounds
were prepared as 10 mM stocks in DMSO and stored at ꢃ20 ꢁC.
The following oligonucleotides were used and were acquired
from Midland Certified Reagent Company (Midland, TX) and Inte-
grated DNA Technologies (Coralville, IA).
4.1.2.9. 4-(([1,10-Biphenyl]-4-ylmethyl)amino)-1-hydroxypyrido[2,3-
d]pyrimidin-2(1H)-one (22). The title compound was prepared us-
ing a procedure B to provide the title compound as a TFA salt: 1H
NMR (400 MHz, DMSO-d6)
d ppm 10.21e10.09 (m, 1H), 8.90 (d,
J ¼ 6.6 Hz, 2H), 8.84 (d, J ¼ 8.1 Hz, 1H), 8.78 (dd, J ¼ 1.4, 4.6 Hz, 1H),
8.33 (d, J ¼ 6.6 Hz, 2H), 8.18e8.10 (m, 4H), 7.40 (dd, J ¼ 4.7, 8.1 Hz,
1H); ESþ MS: 344.9 (Mþ1).
4.1.2.10. 1-Hydroxy-4-((4-(pyridin-4-yl)phenyl)amino)pyrido[2,3-d]
pyrimidin-2(1H)-one (23). The title compound was prepared using
procedure A to provide the title compound as a TFA salt: 1H NMR
(300 MHz, DMSO-d6) d ppm 10.16 (s, 1H), 8.87 (m, 4H), 8.35 (m, 2H),
8.15 (m, 4H), 7.41 (m, 1H), 3.17 (s, 1H); ESþ MS: 332 (Mþ1).
4.1.2.11. 1-Hydroxy-4-({[4-(4-pyridinyl)phenyl]methyl}amino)pyrido
[2,3-d]pyrimidin-2(1H)-one (24). The title compound was prepared
using procedure B to provide the title compound as a TFA salt: 1H
Oligo 1, 50-AGGUG AGUGA GAUGA UAACA AAUUU GCGAG
CCCCA GAUGC-30
Oligo 2, 50-Biotin-GCATC TGGGG CTCGC AAATT TG-30
Oligo 3, 50-CCCCC CCCCC CCCCC AGGTG AGTGA GATGA TAAC-
cordycepin-30
NMR (300 MHz, DMSO-d6) d ppm 9.17 (m, 1H), 8.80 (m, 1H), 8.70 (d,
1H), 8.57 (d, 1H), 8.07 (d, 2H), 7.91 (d, 2H), 7.56 (d, 2H), 7.30 (m, 1H),
4.78 (d, 2H); ESþ MS: 346.0 (Mþ1).
All lyophilized oligonucleotides were reconstituted to 200 mM in
10 mM Tris pH 8.0, 10 mM NaCl and stored at ꢃ20 ꢁC in small ali-
4.1.2.12. 1-Hydroxy-4-{[4-(4-morpholinyl)phenyl]amino}pyrido[2,3-
d]pyrimidin-2(1H)-one (25). The title compound was prepared us-
ing procedure B to provide the title compound as a TFA salt
following purification by reverse phase HPLC: 1H NMR (400 MHz,
quots prior to use.
Substrate A was prepared by equilibrating 20 mM of Oligo 1 and
20 m
M of Oligo 2 in 10 mM Tris pH 8.0, 10 mM NaCl at 95 ꢁC, fol-
lowed by a gradual decrease to 80 ꢁC (0.1 ꢁC decrease/6 s) and then
to 4 ꢁC (0.1C/20 s) with a Dyad DNA Engine thermal cycler (BioRad;
Hercules, CA).
DMSO-d6)
d
ppm 9.81 (s, 1H), 8.80e8.66 (m, 2H), 7.63 (d, J ¼ 8.9 Hz,
2H), 7.32 (dd, J ¼ 4.7, 8.0 Hz, 1H), 6.99 (d, J ¼ 9.1 Hz, 2H), 3.82e3.71
(m, 4H), 3.16e3.06 (m, 4H); ESþ MS: 339.9 (Mþ1).
4.2.2. Reverse transcriptase RNase H strand transfer activity
4.1.2.13. 1-Hydroxy-4-({[4-(4-morpholinyl)phenyl]methyl}amino)
Test compounds were serially diluted in DMSO and plated
pyrido[2,3-d]pyrimidin-2(1H)-one (26). The title compound was
(0.1 mL) in low volume, white 384-well plates. High and low

616
E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 83 (2014) 609e616
control wells contained 0.1
mL of DMSO alone. To start the assay,
Appendix A. Supplementary data
5
m
L of 2ꢂ enzyme solution (40 nM RT in assay buffer (50 mM Tris
pH 8.0, 80 mM KCl, 10 mM MgCl2, 0.02% NP-40, 5 mM DTT,
Nuclease-free dH2O)) were dispensed to all but the low control
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.061.
wells, which received 5
was allowed to incubate with the test compounds for 30 min at
room temperature. The assay was initiated by the addition of 5
mL of assay buffer only, and the enzyme
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buffer) to each well. The final assay composition was 50 mM Tris
pH 8.0, 80 mM KCl, 10 mM MgCl2, 0.02% NP-40, 5 mM DTT, 200 nM
dATP, 200 nM dCTP, 200 nM dTTP, 27 nM [8-3H(N)]-dGTP, 10 nM
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Acknowledgments
The authors would like to acknowledge the contributions of
Derek Parks, Iris Paulus, and Rob Ferris.