Novel diarylpyridinones, diarylpyridazinones and diarylphthalazinones as potential HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs)

Article abstract of DOI:10.1016/j.bmc.2011.08.060

In this Letter, we report on diarylpyridinone, diarylpyridazinone and diarylphthalazinone analogs as potential inhibitors of HIV-1 nonnucleoside reverse transcriptase (NNRTIs). The most promising compounds in these series are three diarylpyridazinones 25a

Full text of DOI:10.1016/j.bmc.2011.08.060

Bioorganic & Medicinal Chemistry 19 (2011) 5924–5934
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel diarylpyridinones, diarylpyridazinones and diarylphthalazinones
as potential HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs)
Muthusamy Venkatraj a, Kevin K. Ariën b, Jan Heeres a, Bertrand Dirié a, Jurgen Joossens a,
Sebastiaan Van Goethem a, Pieter Van der Veken a, Johan Michiels b, Christophe M.L. Vande Velde c,
Guido Vanham b,d,e, Paul J. Lewi a, Koen Augustyns a,
⇑
a Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
b Virology Unit, Division of Microbiology, Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium
c Karel de Grote University College, Department of Applied Engineering, X-ray Crystallography Lab, Salesianenlaan 30, B-2660 Antwerp, Belgium
d Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
e Faculty of Medicine and Pharmacy, Free University of Brussels, Laarbeeklaan 103, B-1090 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 June 2011
Revised 19 August 2011
Accepted 28 August 2011
Available online 1 September 2011
In this Letter, we report on diarylpyridinone, diarylpyridazinone and diarylphthalazinone analogs as
potential inhibitors of HIV-1 nonnucleoside reverse transcriptase (NNRTIs). The most promising
compounds in these series are three diarylpyridazinones 25a, 25l and 25n which demonstrated
submicromolar activity against wild-type HIV-1 and moderate activity against the single mutant strain
Ba-L V106A.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
HIV-1 reverse transcriptase
NNRTIs
Diarylpyridinones
Diarylpyridazinones
Diarylphthalazinones
1. Introduction
strains. It exhibits high potency against wild-type and a number
of mutated viral strains with nanomolar EC50 values and has dem-
According to the most recent UNAIDS report, worldwide 60 mil-
lion people have been infected by the human immunodeficiency
virus (HIV) and about 25 million patients died of AIDS.1 HIV-1
reverse transcriptase (RT) is one of the most important viral
enzymes and plays a vital role in the HIV-1 life cycle. There are
two known drug-target sites, the substrate catalytic site and an
allosteric site that is distinct from but located closely to the sub-
strate site.2–4 Nonnucleoside reverse transcriptase inhibitors
(NNRTIs) interact with the allosteric site in a noncompetitive man-
ner to distort the enzyme’s active conformation and hence disrupt
the function of the enzyme. The first-generation NNRTIs such as
nevirapine (1),5 delavirdine (2)6 and efavirenz (3)7 exhibit very
potent anti-HIV-1 activity and low toxicity. However, rapid drug-
resistance emergence due to single point mutations8 in the NNRTI
binding site, compromises their clinical utility. Etravirine (TMC125,
4),9 a diarylpyrimidine (DAPY) was recently approved as a next-
generation NNRTI for the treatment of HIV-1 infection in treat-
ment-experienced patients who show evidence of HIV-1 resistant
onstrated a higher genetic barrier10 to the emergence of drug-
resistance. The most advanced NNRTI (approved recently by FDA)
is another DAPY derivative rilpivirine (TMC278, 5)11, which shows
a better potency and safety profile than 4. It allows once daily
administration and will be prescribed for treatment of HIV-1 infec-
tion in treatment-naive patients.12 Dapivirine, also known as
TMC120 (6)13a is currently under development for HIV-microbici-
dal applications (Fig. 1).13b
The excellent pharmacological profiles of the DAPYs 4–6 have
encouraged several research groups to explore next-generation
NNRTI agents.14 In this Letter, we report on the synthesis and
antiviral activity of diarylpyridinone, diarylpyridazinone and
diarylphthalazinone analogs (Series A–C).
2. Results and discussion
2.1. Compound design and molecular modeling studies
On the basis of SAR data from the DAPY series, our strategy was
to synthesize novel series of NNRTIs by replacing the central
pyrimidine ring. The new compounds designed in this study have
⇑
Corresponding author. Tel.: +32 3 265 27 17; fax: +32 3 265 27 39.
E-mail address: Koen.Augustyns@ua.ac.be (K. Augustyns).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.060

M. Venkatraj et al. / Bioorg. Med. Chem. 19 (2011) 5924–5934
5925
Figure 1. Structures of reported HIV-1 NNRTI compounds.
Figure 2. Design of diarylpyridinones, diarylpyridazinones and diarylphthalazinones based on the interactions of TMC 120 (6) with its binding site on reverse transcriptase.
a central pyridinone, pyridazinone and phthalazinone which retain
the two phenyl rings in the Western and Eastern wing. In TMC120
(6) the NH linker connecting the central pyrimidine ring and the
eastern wing serves as hydrogen donor with the backbone car-
bonyl of Lys101 and the pyrimidine nitrogen at position 1 serves
model reveals that the designed compounds have the potential
to bind to HIV-1 reverse transcriptase in a similar horseshoe
conformation.9b
2.2. Chemistry
as hydrogen bond acceptor with the backbone
a-amino group of
Lys101. In our newly designed compounds the NH linker attached
to a nitrogen of the central ring is maintained whereas the car-
bonyl group of the central ring serves as hydrogen bond acceptor.
The presence of the central ring will assure a similar molecular
topology and flexibility, and hence we expected the new com-
pounds would have binding orientations and conformations simi-
lar to those of the DAPYs (Fig. 2).
The synthesis of compounds belonging to Series A–C is outlined
in Schemes 1–3. Scheme 1 shows the synthesis of diarylpyridinon-
es 15a–g (Series A). Selective O-benzylation of bromopyridin-2-
one 7 provided bromopyridine 8.15 Palladium-catalyzed amination
of bromopyridine
8 with amines 9a–c gave the respective
amino-substituted pyridines 10a–c.15 Catalytic debenzylation of
aminopyridines 10a–c was easily achieved to afford the corre-
sponding pyridin-2-ones 11a–c. Phosphine-based amino transfer
reagent O-(diphenylphosphinyl)hydroxyl amine (12) was synthe-
sized according to a reported procedure.16 N-amination of the
pyridin-2-ones 11a–c using 12 yielded the N-aminopyridin-2-ones
Figure
3 shows the superposition of low-energy docking
conformations of our compounds 15a, 25a and 30a as the repre-
sentatives of the pyridinone, pyridazinone and phthalazinone
series, respectively, along with TMC125 (4) and TMC120 (6). The

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M. Venkatraj et al. / Bioorg. Med. Chem. 19 (2011) 5924–5934
more potent. Replacement of the para-methyl group with chlorine
(25l) and bromine (25n) resulted in similar anti-HIV-1 activity.
Moreover, compound 25n demonstrated less cytotoxicity, result-
ing in a more favorable selectivity index. Replacement of the
para-cyano or its shift to the meta position was detrimental for
potency.
Similar conclusions can be formulated for the phthalazinones
30a–c (Table 3). In summary, replacement of the pyrimidine core
of the DAPYs with pyridinone, pyridazinone and phthalazinone re-
sulted in compounds with lower anti-HIV-1 activity in comparison
with TMC120, but with similar structure–activity relationship
around the two aryl substituents.
2.4. Antiviral activity against mutant strains of HIV-1
Three submicromolar compounds 25a, 25l and 25n were tested
against single mutant strains, Ba-L V106A and VI829 Y181C in
comparison with 6 (TMC120). These three compounds exhibited
moderate activity against the Ba-L V106A with EC50 values lower
than 5 lM but lost the potency against VI829 Y181C (Table 4).
2.5. Anti-reverse transcriptase activity
The compounds 15a, 20a, 25a and 30a were further evaluated
Figure 3. Superposition of low-energy docking binding conformations of newly
designed compounds 15a (yellow), 25a (red), 30a (orange) and DAPYs 4 (blue) and
6 (green).
for anti-reverse transcriptase activity in comparison with
6
(TMC120) and efavirenz in an enzymatic recombinant HIV-1 RT-
activity assay and the results are compared with anti-HIV-1 activ-
ity (Table 5). All compounds are consistently much more active
against the whole virus compared to their inhibitory activity
against the reverse transcriptase (150–1250 times). The reasons
for this discrepancy remain unclear.
13a–c.17 Coupling18 of arylboronic acids 14a–d with N-aminopyri-
din-2-ones 13a–c furnished the desired compounds 15a–g (Series
A).
Scheme 2 shows the synthesis of diarylpyridazinones 20a–c
and 25a–o (Series B). Fusion of 3,6-dichloropyridazine (16) with
2,4,6-trimethylaniline (9a) yielded 6-chloro-N-mesitylpyridazin-
3-amine (17). Reaction of 16 with sodium phenoxide derivatives
21a–e afforded the corresponding 3-aryloxy-6-chloropyridazines
22a–e. Hydrolysis of 17 and 22a–e in glacial acetic acid furnished
2.6. Crystal structures
In order to confirm the position of amination at nitrogen instead
of oxygen with the aminating reagent 12, we determined the struc-
tures of one of the intermediates 24c and one of the target com-
pounds 25o by X-ray analysis. Their structures are shown in
Figures 4 and 5, respectively.20
the
corresponding
6-(mesitylamino)pyridazin-3(2H)-one
(18)14h and 6-aryloxypyridazin-3(2H)-ones 23a–e, respectively.
N-amination of the pyridazin-3-one 18 and 23a–e with amine 12
yielded N-aminopyridazin-3-one 1919 and 24a–e, respectively.
Coupling of 19 and 24a–e with arylboronic acids 14a–f delivered
the desired compounds 20a–c and 25a–o (Scheme 2).
3. Conclusion
In summary, we designed and synthesized three novel series of
pyridinones 15a–g (Series A), pyridazinones 20a–c and 25a–o
(Series B) and phthalazinones 30a–c (Series C) and evaluated their
activity against wild-type HIV-1 in TZM-bl cells. The most promis-
ing compounds in these series are three diarylpyridazinones 25a,
25l and 25n which showed submicromolar activity. Furthermore,
25a, 25l and 25n were tested against single mutant strains, Ba-L
V106A and VI829 Y181C. The activities against mutated strains of
HIV-1 indicated that the pyridazinones 25a, 25l and 25n showed
moderate activity against Ba-L V106A with EC50 values lower than
Scheme 3 shows the synthesis of diarylphthalazinones 30a–c
(Series C). Reaction of 26 with phenols 21a and 21d afforded 1-
chloro-4-aryloxyphthalazines 27a and 27b which were hydrolyzed
to furnish 4-aryloxyphthalazin-1(2H)-ones 28a and 28b, respec-
tively. N-amination of 28a–b gave 29a–b which were coupled with
arylboronic acids 14a and 14e to yield the desired compounds
30a–c.
2.3. Antiviral activity against wild-type HIV-1
5 lM and no activity against VI829 Y181C. In addition, enzyme
inhibitory assays were performed with selected compounds
against HIV-1 wtRT.
The compounds 15a–g, 20a–c, 25a–o and 30a–c were evaluated
for their anti-HIV-1 activity and cytotoxicity in TZM-bl cells in
comparison with TMC120 (6). The results, expressed as EC50 (50%
effective concentration), CC50 (50% cytotoxic concentration) and
SI (selectivity index given by the CC50/EC50 ratio) values are sum-
marized in Tables 1–3. The compounds 15a–g showed no activity
(Table 1).
4. Experimental section
4.1. Chemistry
Reagents were supplied by Sigma–Aldrich and Acros. Character-
ization of all compounds was done with 1H NMR and mass spec-
trometry. 1H NMR spectra were recorded on a Bruker Avance
DRX-400 spectrometer (400 MHz) and coupling constants (J) are
reported in Hz. Single crystal X-ray diffraction data were recorded
Table 2 shows the anti-HIV-1 activity of pyridazinones 20a–c
and 25a–o. Compound 20a (R1@H and R2@CN) showed micromolar
activity (EC50 = 1.37
EC50 value of the corresponding pyrimidine compound
(TMC120). Compound 25a, the oxygen analog of 20a, was slightly
lM). This is about 700 times higher than the
6

M. Venkatraj et al. / Bioorg. Med. Chem. 19 (2011) 5924–5934
5927
Scheme 1. Synthetic route to target compounds 15a–g. Reagents and conditions: (a) C6H5CH2Br, Ag2CO3, dry THF, reflux; (b) Pd2(dba)3, 2-dicyclohexylphosphino-20-(N,N-
dimethylamino)biphenyl, NaOtBu, dry toluene, 85 °C; (c) H2, Pd/C, MeOH/EtOAc; (d) (diphenylphosphinyl)hydroxyl amine, Cs2CO3, dry DMF, 50 °C; (e) Cu(OAc)2, CH2Cl2, Et3N,
rt.
at 100 K on a Bruker Smart 1000 system with Mo K
a
radiation. Col-
4.8 mmol) and subsequently refluxed in the dark overnight. The
reaction mixture was filtered through celite and concentrated to
provide brown solid which was flash chromatographed on silica
gel eluting with 10% EtOAc in hexanes to provide 1.8 g (85%) of a
white solid;1H NMR (CDCl3) d (ppm) 5.34 (s, 2H), 6.71 (d, 1H,
J = 8.7 Hz), 7.25–7.44 (m, 5H), 7.64 (dd, 1H, J = 8.8 Hz), 8.20 (d,
1H, J = 2.5 Hz); MS (ESI) m/z 289 (M+Na)+.
umn chromatography was performed on a Flashmaster II instru-
ment (Jones Chromatography) with Isolute columns pre-packed
with silica gel. Electrospray ionization (ESI) mass spectra were ac-
quired on an ion trap mass spectrometer (Bruker Daltonics
Esquire™ 3000 plus). LC–MS spectra were recorded on an Agilent
1100 Series LC system equipped with
a
C18 column
(2.1 ꢀ 50 mm, 5 mm, Supelco, Sigma–Aldrich) coupled to the mass
spectrometer; solvent A: H2O + 0.1% formic acid, solvent B:
CH3CN + 0.1% formic acid; gradient: 5% B ? 100% B over 23 min
at 0.2 mL minꢁ1. HPLC was performed on a Gilson instrument
equipped with a C18 column (4.6 mm ꢀ 25 cm, 5 mm, Ultra-
sphere™ ODS), with standard UV detection at k 214 nm; solvent
A: H2O + 0.1% trifluoroacetic acid (TFA), solvent B: CH3CN + 0.1%
TFA; gradient: 10% B ? 100% B over 36 min at 1 mL minꢁ1. The
synthesis of compounds 8, 17, 18, 22a and 23a has been reported
elsewhere14h,15 and O-(diphenylphosphinyl)hydroxyl amine (12)
was synthesized according to a reported procedure.16
4.2.1. General procedure for the synthesis of 10a–c
Compound
8
(7.5 mmol), 9a–c (10.5 mmol), Pd2dba3
(0.19 mmol), 20-(dicyclohexyl phosphino)-N,N-dimethylbiphenyl-
2-amine (0.75 mmol) and NaOtBu (10.5 mmol) were weighed in
a 50 mL RB flask and dry toluene (20 mL) was added and flushed
with argon for 15 min. Subsequently the reaction mixture was stir-
red at 85 °C overnight, cooled to room temperature, diluted with
EtOAc and filtered through a pad of celite and concentrated to ob-
tain a dark brown solid which was flash chromatographed on silica
gel eluting with 30% EtOAc in hexanes.
4.2. 2-(benzyloxy)-5-bromopyridine (8)
4.2.1.1. 6-(Benzyloxy)-N-mesitylpyridin-3-amine (10a).
Yield
50%; 1H NMR (CDCl3) d (ppm) 2.17 (s, 6H), 2.30 (s, 3H), 5.3 (s, 2H),
6.65 (d, 1H, J = 8.8 Hz), 6.82 (dd, 1H, J = 8.8 Hz), 6.93 (s, 2H),
To a solution of 1 (1.4 g, 8 mmol) in dry THF (80 mL) was added
benzyl bromide (1.14 mL, 9.6 mmol) and Ag2CO3 (1.32 g,

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M. Venkatraj et al. / Bioorg. Med. Chem. 19 (2011) 5924–5934
Scheme 2. Synthetic route to target compounds 20a–c and 25a–o. Reagents and conditions: (a) Fusion, 150 °C; (b) NaH, DMF, rt; (c) glacial acetic acid, reflux; (d)
(diphenylphosphinyl)hydroxyl amine, Cs2CO3, dry dioxane, 60 °C; (e) Cu(OAc)2, CH2Cl2, Et3N, rt.
Scheme 3. Synthetic route to target compounds 30a–c. Reagents and conditions: (a) NaH, DMF, rt; (b) glacial acetic acid, reflux; (c) (diphenylphosphinyl)hydroxyl amine,
NaH, dry dioxane, 100 °C; (d) Cu(OAc)2, CH2Cl2, Et3N, rt.

M. Venkatraj et al. / Bioorg. Med. Chem. 19 (2011) 5924–5934
5929
Table 1
Table 3
Anti-HIV-1 activity of 15a–g in TZM-bl cells
Anti-HIV-1 activity of 30a–c in TZM-bl cells
R3
R5
R1
R2
R4
R1
R2
HN
NH
O
N
NH
O
N
N
O
Compd
R1
R2
R3
R4
R5
EC50
(l
M)
CC50
(
lM)
SI
15a
15b
15c
15d
15e
15f
15g
6
Me
Me
Me
Me
H
Me
Me
Me
Me
H
Me
Me
Me
Me
H
H
H
CN
H
H
CN
CF3
H
H
CN
H
>10
>10
>10
>10
>10
>10
>10
nda
nda
nda
nda
nda
nda
nda
2.88
—
—
—
—
—
—
—
1440
Compd
R1
R2
EC50
(l
M)
CC50
(
lM)
SI
30a
30b
30c
6
Me
Cl
Cl
CN
CN
Cl
1.39
3.60
>10
60.40
51.23
nda
43.3
14.2
—
0.002
2.88
1440
H
H
H
H
H
CN
H
H
CN
a
nd, not determined.
0.002
a
nd, not determined when EC50 >10 lM.
Table 4
Activity of 25a, 25l and 25n against mutated strains of HIV-1
Table 2
Anti-HIV-1 activity of 20a–c and 25a–o in TZM-bl cells
Compd
WT Ba-L
WT VI829
Ba-L V106A
VI829 Y181C
EC50
(
lM)
EC50
(l
M)
EC50
(lM)
FC
EC50
(lM)
FC
R1
R3
25a
25l
25n
6
0.69
0.60
0.64
0.16
0.50
0.34
1.12
4.55
2.97
2
8
5
1.3
>10
>10
>10
>64
>20
>29
5.4
R2
0.002
0.002
0.0025
0.0108
X
N
NH
O
N
Table 5
HIV-1 RT inhibitory activity in comparison with anti HIV-1 activity
Compd
X
R1
R2
R3
EC50
(
lM)
CC50
(l
M)
SI
Compd
WT Ba-L
WT RT
IC50 (lM)
20a
20b
20c
25a
25b
25c
25d
25e
25f
25g
25h
25i
25j
25k
25l
25m
25n
25o
6
NH
NH
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
CN
CN
Cl
Cl
Br
H
H
CN
H
H
H
H
CN
H
H
CN
H
H
CN
H
CN
H
CN
CF3
H
CN
CF3
F
Cl
H
CN
F
H
H
CN
H
CN
H
CN
Cl
1.37
>10
>10
0.69
10
9.64
3.95
>10
7.63
>10
>10
>10
1.75
>10
0.60
>5
>100
nda
>69
—
EC50
(l
M)
5.04
19.95
21.15
23.53
22.27
nda
< 1
29
2.1
2.4
5.6
—
>13
—
—
—
14
—
23
—
>193
20
1440
15a
20a
25a
30a
>10
760
547
100
928
2.5
1.37
0.69
1.39
0.002
0.002
6
Efavirenz
1.52
>100
nda
nda
nda
24.74
4.2.2. General procedure for synthesis of 11a–c
nda
To a solution of 10a–c (3 mmol) in MeOH (20 mL) and EtOAc
(10 mL) was added 10% Pd/C (1.5 mmol). The reaction mixture
was stirred for 3 h under hydrogen atmosphere and filtered
through a celite pad. After removal of solvent the crude mixture
was chromatographed on silica using 10% MeOH in DCM.
14.10
nda
>100
63.46
2.88
0.64
3.19
0.002
Br
H
a
nd, not determined.
4.2.2.1. 5-(Mesitylamino)pyridin-2(1H)-one (11a).
Yield 79%;
1H NMR (MeOD) d (ppm) 2.15 (s, 6H), 2.25 (s, 3H), 6.17 (dd, 1H,
J = 3.0 Hz), 6.53 (dd, 1H, J = 9.6 Hz), 6.91 (s, 2H), 7.36 (dd, 1H,
J = 9.6 Hz); MS (ESI) m/z 229 (M+H)+.
7.30–7.39 (m, 3H), 7.44 (d, 2H, J = 8.2 Hz), 7.52 (d, 1H, J = 3.0 Hz); MS
(ESI) m/z 320 (M+H)+.
4.2.1.2. 6-(Benzyloxy)-N-phenylpyridin-3-amine (10b).
Yield
4.2.2.2. 5-(Phenylamino)pyridin-2(1H)-one (11b).
Yield 73%;
56%; 1H NMR (CDCl3)
d
(ppm) 5.36 (s, 2H), 6.78 (d, 1H,
1H NMR (MeOD) d (ppm) 6.57 (dd, 1H, J = 9.6 Hz), 6.75–6.81 (m,
3H), 7.14–7.19 (m, 2H), 7.23 (d, 1H, J = 2.6 Hz), 7.55 (dd, 1H,
J = 9.6 Hz); MS (ESI) m/z 187 (M+H)+.
J = 8.2 Hz), 6.84–6.88 (m, 3H), 7.20–7.48 (m, 8H), 8.00 (d, 1H,
J = 2.8 Hz); MS (ESI) m/z 278 (M+H)+.
4.2.1.3. 4-(6-(Benzyloxy)pyridin-3-ylamino)benzonitrile (10c).
Yield 42%; 1H NMR (CDCl3) d (ppm) 5.40 (s, 2H), 6.78 (d, 2H,
J = 8.4 Hz), 6.86 (d, 1H, J = 8.8 Hz), 7.27–7.51 (m, 8H), 8.07 (d, 1H,
J = 2.7 Hz); MS (ESI) m/z 324 (M+Na)+.
4.2.2.3.
(11c).
4-(6-Oxo-1,6-dihydropyridin-3-ylamino)benzonitrile
Yield 76%; 1H NMR (MeOD) d (ppm) 6.60 (dd, 1H,
J = 9.6 Hz), 6.78–6.80 (m, 2H), 7.33 (d, 1H, J = 2.9 Hz), 7.45–7.48
(m, 2H), 7.54 (dd, 1H, J = 9.6 Hz); MS (ESI) m/z 212 (M+H)+.

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M. Venkatraj et al. / Bioorg. Med. Chem. 19 (2011) 5924–5934
Figure 4. Crystal structure of compound 24c.
4.2.3.2.
1-Amino-5-(phenylamino)pyridin-2(1H)-one (13b).
Yield 58%; 1H NMR (MeOD) d (ppm) 6.60 (d, 1H, J = 9.6 Hz),
6.75–6.82 (m, 3H), 7.15–7.19 (m, 2H), 7.42 (dd, 1H, J = 9.4 Hz),
7.57 (d, 1H, J = 2.8 Hz); MS (ESI) m/z 202 (M+H)+.
4.2.3.3. 4-(1-Amino-6-oxo-1,6-dihydropyridin-3-ylamino)ben-
zonitrile (13c).
Yield 64%; 1H NMR (MeOD) d (ppm) 6.65 (d,
1H, J = 9.4 Hz), 6.81 (d, 2H, J = 8.3 Hz), 7.43 (br s, 1H), 7.47 (d, 2H,
J = 8.1 Hz), 7.69 (br s, 1H); MS (ESI) m/z 227 (M+H)+.
4.2.4. General procedure for the synthesis of 15a–g
To a solution of 13a–c (0.38 mmol) in dichloromethane (7 mL)
was added diacetoxycopper hydrate (0.38 mmol), Et3N
(0.45 mmol) and 14a–d (0.45 mmol) in dichloromethane (0.5 mL)
was added dropwise, allowed to stir at room temperature for 2 h.
Then the reaction mixture was concentrated and chromatographed
on silica using 10% MeOH in DCM. The resulting fine sticky pow-
ders/oily materials were lyophilized to powders.
4.2.4.1.
benzonitrile (15a).
4-(5-(Mesitylamino)-2-oxopyridin-1(2H)-ylamino)-
Yield 23%; 1H NMR (CDCl3) d (ppm) 2.19
(s, 6H), 2.26 (s, 3H), 6.41 (br s, 1H), 6.63 (d, 2H, J = 8.4 Hz), 6.74
(br s, 1H), 6.90 (br s, 2H), 7.20–7.30 (m, 2H), 7.45 (d, 2H,
J = 8.4 Hz); MS (ESI) m/z 345 (M+H)+; HPLC (214 nm) tr 23.8 min,
98%; LC–MS (214 nm) tr 17.1 min, 100%.
Figure 5. Crystal structure of compound 25o.
4.2.4.2. 5-(Mesitylamino)-1-(4-(trifluoromethyl)phenylamino)-
4.2.3. General procedure for the synthesis of 13a–c
To solution of 11a–c (1.8 mmol) in DMF (20 mL) was
pyridin-2(1H)-one (15b).
Yield 26%; 1H NMR (MeOD) d (ppm)
a
2.19 (s, 6H), 2.24 (s, 3H), 6.67 (d, 2H, J = 7.8 Hz), 6.89 (d, 2H,
J = 8.7 Hz), 7.46 (br s, 2H), 7.61 (br s, 2H), 7.77 (br s, 1H); MS
(ESI) m/z 388 (M+H)+; HPLC (214 nm) tr 27.4 min, 85%.
added cesium carbonate (2.16 mmol) and allowed to stir for
15 min. Then 12 (2.16 mmol) was added and the reaction mix-
ture was stirred at room temperature for 36 h and subse-
quently at 50 °C for 12 h. Water was added to the reaction
mixture, extracted with EtOAc (3 ꢀ 100 mL) and the combined
organic layers were washed with water and evaporated. The
crude mixture was chromatographed on silica using 10% MeOH
in DCM.
4.2.4.3.
benzonitrile (15c).
3-(5-(Mesitylamino)-2-oxopyridin-1(2H)-ylamino)-
Yield 22%; 1H NMR (MeOD) d (ppm) 2.20
(s, 6H), 2.23 (s, 3H), 6.20 (br s, 1H), 6.69 (d, 1H, J = 9.6 Hz), 6.80
(br s, 1H), 6.86 (d, 1H, J = 7.8 Hz), 6.91 (br s, 2H), 7.20 (d, 1H,
J = 7.3 Hz), 7.36 (t, 1H, J = 7.9 Hz), 7.45 (d, 1H, J = 8.9 Hz); MS 346
(M+H)+; HPLC (214 nm) tr 23.5 min, 95%.
4.2.3.1. 1-Amino-5-(mesitylamino)pyridin-2(1H)-one (13a).
Yield 41%; 1H NMR (MeOD) d (ppm) 2.16 (s, 6H), 2.26 (s, 3H),
6.49 (d, 1H, J = 3.0 Hz), 6.55 (d, 1H, J = 9.6 Hz), 6.92 (s, 2H), 7.22
(dd, 1H, J = 9.5 Hz); MS (ESI) m/z 244 (M+H)+.
4.2.4.4.
(15d).
5-(Mesitylamino)-1-(phenylamino)pyridin-2(1H)-one
Yield 25%; 1H NMR (MeOD) d (ppm) 2.15 (s, 6H), 2.84
(s, 3H), 6.30 (br s, 1H), 6.58 (d, 2H, J = 7.0 Hz), 6.66 (d, 1H,

M. Venkatraj et al. / Bioorg. Med. Chem. 19 (2011) 5924–5934
5931
J = 9.9 Hz), 6.90 (br s, 1H), 7.18 (t, 2H, J = 7.6 Hz), 7.34 (d, 2H,
J = 8.7 Hz), 7.59 (d, 1H, J = 7.0 Hz); MS (ESI) m/z 342 (M+Na)+; HPLC
(214 nm) tr 24.8 min, 77%.
(d, 1H, J = 9.6 Hz), 7.22 (d, 1H, J = 9.0 Hz), 7.50 (d, 2H, J = 8.0 Hz);
MS (ESI) m/z 346 (M+H)+; HPLC (214 nm) tr 20.4 min, 100%.
4.2.5.2. 6-(Mesitylamino)-2-(4-(trifluoromethyl)phenylamino)-
4.2.4.5. 4-(2-Oxo-5-(phenylamino)pyridin-1(2H)-ylamino)ben-
zonitrile (15e).
pyridazin-3(2H)-one (20b).
Yield 26%; 1H NMR (MeOD) d
Yield 39%; 1H NMR (MeOD) d (ppm) 6.71 (d,
(ppm) 2.15 (s, 6H), 2.20 (s, 3H), 6.70 (br s, 2H), 6.84 (br s, 2H),
7.04 (d, 1H, J = 9.7 Hz), 7.21 (br s, 1H), 7.46 (d, 2H, J = 7.5 Hz); MS
(ESI) m/z 389 (M+H)+; HPLC (214 nm) tr 24.1 min, 97%.
1H, J = 8.70 Hz), 6.73–6.78 (m, 1H), 6.80–6.82 (m, 1H), 6.85–6.87
(m, 2H), 7.17–7.21 (m, 2H), 7.43 (d, 1H, J = 2.8 Hz), 7.54–7.57 (m,
2H), 7.59 (d, 1H, J = 3.0 Hz), 7.61 (d, 1H, J = 3.0 Hz); MS (ESI) m/z
303 (M+H)+; HPLC (214 nm) tr 19.4 min, 100%; LC–MS (214 nm)
tr 14.8 min, 100%.
4.2.5.3.
benzonitrile (20c).
3-(3-(Mesitylamino)-6-oxopyridazin-1(6H)-ylamino)-
Yield 16%; 1H NMR (MeOD) d (ppm) 2.15
(s, 6H), 2.19 (s, 3H), 6.83 (br s, 4H), 7.03 (d, 1H, J = 9.7 Hz), 7.24–
7.33 (m, 3H); MS (ESI) m/z 346 (M+H)+; HPLC (214 nm) tr
20.5 min, 100%.
4.2.4.6. 1,5-Bis(phenylamino)pyridin-2(1H)-one (15f).
Yield
26%; 1H NMR (MeOD) d (ppm) 6.70–6.73 (m, 2H), 6.77–6.81 (m,
1H), 6.84 (dd, 2H, J = 8.6 Hz) 6.90–6.94 (m, 1H), 7.14–7.25 (m,
4H), 7.30–7.38 (m, 1H), 7.50 (d, 1H, J = 2.7 Hz), 7.60 (dd, 1H,
J = 7.0.0 Hz); MS (ESI) m/z 300 (M+Na)+; HPLC (214 nm) tr
20.9 min, 77%.
4.2.6. General procedure for the synthesis of 22a–e
To a solution of 16 (10 mmol) and 21a–e (10 mmol) in dry DMF
(20 mL) was added NaH (11 mmol) portionwise (1 h) and the reac-
tion mixture was stirred at room temperature for 3 h, poured on
ice-cold water (40 mL), the precipitated solid was filtered and
dried under high vacuum.
4.2.4.7. 4-((5-((4-Cyanophenyl)amino)-2-oxopyridin-1(2H)-yl)-
amino)benzonitrile (15g).
Yield 37%; 1H NMR (MeOD) d (ppm)
6.75–6.80 (m, 3H), 6.85–6.87 (m, 2H), 7.48–7.50 (m, 2H), 7.56–7.59
(m, 2H), 7.62 (dd, 2H, J = 7.3 Hz); MS (ESI) m/z 328 (M+H)+; HPLC
(214 nm) tr 17.6 min, 100%; LC–MS (214 nm) tr 14.2 min, 100%.
4.2.6.1. 3-Chloro-6-(mesityloxy)pyridazine (22a).
Yield 80%;
1H NMR (CDCl3) d (ppm) 2.10 (s, 6H), 2.31 (s, 3H), 6.93 (s, 2H), 7.12
(d, 1H, J = 9.1 Hz), 7.47 (d, 1H, J = 9.1 Hz); MS (ESI) m/z 273 (M+Na)+.
4.2.4.8. 6-Chloro-N-mesitylpyridazin-3-amine (17).
Com-
pound 16 (7.45 g, 50 mmol) and 9a (7.75 mL, 55 mmol) were
mixed together and stirred at 150 °C for 3 h. The crude mixture
was chromatographed on silica using 10% MeOH in DCM to provide
1.8 g (40%) of a white solid. 1H NMR (CDCl3) d (ppm) 2.17 (br s, 6H),
2.30 (s, 3H), 6.28 (d, 1H, J = 9.1 Hz), 6.94 (s, 2H), 7.13 (d, 1H,
J = 9.4 Hz); MS (ESI) m/z 248 (M+H)+.
4.2.6.2.
3-Chloro-6-(2,6-dimethylphenoxy)pyridazine (22b).
Yield 78%; 1H NMR (DMSO-d6) d (ppm) 2.05 (s, 6H), 7.10–7.18
(m, 3H), 7.62 (d, 1H, J = 9.2 Hz), 7.96 (d, 1H, J = 9.1 Hz); MS (ESI)
m/z 259 (M+Na)+.
4.2.6.3. 4-(6-Chloropyridazin-3-yloxy)-3,5-dimethylbenzonitrile
(22c).
Yield 80%; 1H NMR (DMSO-d6) d (ppm) 2.10 (s, 6H), 7.20
4.2.4.9. 6-(Mesitylamino)pyridazin-3(2H)-one (18).
Com-
(s, 2H), 7.42 (d, 1H, J = 9.1 Hz), 7.60 (d, 1H, J = 9.1 Hz); MS (ESI) m/z
pound 17 (1.98 g, 8 mmol) was refluxed in glacial acetic acid
(25 mL) at 120 °C for 6 h. The solvent was removed under reduced
pressure, water (50 mL) was added and the solid product obtained
was collected by filtration. Purification of the crude solid by col-
umn chromatography using 8% MeOH in DCM afforded 0.14 g
(51%) white solid. 1H NMR (CDCl3) d (ppm) 2.16 (s, 6H), 2.25 (s,
3H), 6.85 (d, 1H, J = 9.9 Hz), 6.88 (s, 2H), 7.12 (d, 1H, J = 9.9 Hz);
MS (ESI) m/z 228 (MꢁH)ꢁ.
261 (M+Na)+.
4.2.6.4. 3-Chloro-6-(4-chloro-2,6-dimethylphenoxy)pyridazine
(22d).
Yield 71%; 1H NMR (DMSO-d6) d (ppm) 2.05 (s, 6H),
7.28 (s, 2H), 7.67 (d, 1H, J = 9.1 Hz), 7.98 (d, 1H, J = 9.2 Hz); MS
(ESI) m/z 293 (M+Na)+.
4.2.6.5. 3-(4-Bromo-2,6-dimethylphenoxy)-6-chloropyridazine
(22e).
Yield 87%; 1H NMR (DMSO-d6) d (ppm) 2.05 (s, 6H), 7.40
4.2.4.10. 2-Amino-6-(mesitylamino)pyridazin-3(2H)-one (19).
To a solution of 18 (0.46 g, 2 mmol) in dry dioxane (12 mL) was
added NaH (90 mg, 2.2 mmol). The mixture was stirred at 60 °C
for 1 h and then cooled to room temperature, 12 (0.52 g, 2.2 mmol)
was added and allowed to stir at 60° for 24 h. Water was added to
the reaction mixture, extracted with EtOAc (3 ꢀ 75 mL) and the
combined organic layers were washed with water. Then the
evaporation of solvent afforded a light brown solid which was used
in next step without further purification. MS (ESI) m/z 267
(M+Na)+.
(s, 2H), 7.67 (d, 1H, J = 9.2 Hz), 7.99 (d, 1H, J = 9.2 Hz); MS (ESI) m/z
337 (M+Na)+.
4.2.7. General procedure for the synthesis of 23a–e
Compounds 22a–e (4 mmol) were refluxed in glacial acetic acid
(25 mL) at 120 °C for 6 h. The solvent was removed under reduced
pressure, water (50 mL) was added and the solid products formed
were collected by filtration.
4.2.7.1. 6-(Mesityloxy)pyridazin-3(2H)-one (23a).
Yield 79%;
1H NMR (CDCl3) d (ppm) 2.13 (s, 6H), 2.31 (s, 3H), 6.92 (s, 2H),
7.04 (d, 1H, J = 9.9 Hz), 7.28 (d, 1H, J = 9.9 Hz), 9.94 (s, 1H); MS
(ESI) m/z 231 (M+H)+.
4.2.5. General procedure for the synthesis of 20a–c
To a solution of 19 (0.6 mmol) in dichloromethane (9 mL) was
added diacetoxycopper hydrate (0.6 mmol), Et3N (0.72 mmol)
and 14a–c (0.72 mmol) in dichloromethane (1 mL) was added
dropwise and allowed to stir at room temperature for 2 h. The
reaction mixture was then concentrated and chromatographed
on silica using 10% MeOH in DCM. The resulting oily materials
were lyophilized to powders.
4.2.7.2.
6-(2,6-Dimethylphenoxy)pyridazin-3(2H)-one (23b).
Yield 88%; 1H NMR (DMSO-d6) d (ppm) 2.10 (s, 6H), 7.01–7.08
(m, 2H), 7.11 (br s, 1H), 7.13 (br s, 1H), 7.48 (d, 1H, J = 9.9 Hz),
12.1 (s, 1H); MS (ESI) m/z 239 (M+Na)+.
4.2.7.3. 3,5-Dimethyl-4-(6-oxo-1,6-dihydropyridazin-3-yloxy)-
4.2.5.1.
benzonitrile (20a).
(s, 6H), 2.21 (s, 3H), 6.67 (d, 2H, J = 7.6 Hz), 6.84 (br s, 2H), 7.03
4-(3-(Mesitylamino)-6-oxopyridazin-1(6H)-ylamino)-
benzonitrile (23c).
Yield 54%; 1H NMR (DMSO-d6) d (ppm)
Yield 17%; 1H NMR (CDCl3) d (ppm) 2.15
2.13 (s, 6H), 7.06 (d, 1H, J = 9.9 Hz), 7.53 (d, 1H, J = 9.9 Hz), 7.67
(s, 2H), 12.17 (s, 1H); MS (ESI) m/z 264 (M+Na)+.

5932
M. Venkatraj et al. / Bioorg. Med. Chem. 19 (2011) 5924–5934
4.2.7.4. 6-(4-Chloro-2,6-dimethylphenoxy)pyridazin-3(2H)-one
(23d).
Yield 97%; 1H NMR (DMSO-d6) d (ppm) 2.08 (s, 6H), 7.03
4.2.9.7. 6-(2,6-Dimethylphenoxy)-2-(4-fluorophenylamino)pyri-
dazin-3(2H)-one (25g).
Yield 14%; 1H NMR (CDCl3) d (ppm)
(d, 1H, J = 9.9 Hz), 7.20 (s, 2H), 7.49 (d, 1H, J = 9.9 Hz), 12.12 (s, 1H);
2.18 (s, 6H), 6.79–6.82 (m, 2H), 6.89–6.93 (m, 2H), 7.10 (s, 2H),
7.18 (d, 1H, J = 9.8 Hz), 7.28 (d, 1H, J = 4.0 Hz), 7.57 (br s, 1H); MS
(ESI) m/z 348 (M+Na)+; HPLC (214 nm) tr 22.1 min, 98%.
MS (ESI) m/z 275 (M+Na)+.
4.2.7.5. 6-(4-Bromo-2,6-dimethylphenoxy)pyridazin-3(2H)-one
(23e).
Yield 91%; 1H NMR (DMSO-d6) d (ppm) 2.08 (s, 6H), 7.03
4.2.9.8. 3-(3-(2,6-Dimethylphenoxy)-6-oxopyridazin-1(6H)-yla-
mino)benzonitrile (25h).
2.18 (s, 6H), 6.86 (s, 1H), 6.92 (d, 1H, J = 9.8 Hz), 7.00–7.11 (m,
3H), 7.18–7.39 (m, 3H), 7.81 (br s, 1H); MS (ESI) m/z 355
(M+Na)+; HPLC (214 nm) tr 20.8 min, 99%.
Yield 15%; 1H NMR (CDCl3) d (ppm)
(d, 1H, J = 9.9 Hz), 7.36 (s, 2H), 7.49 (d, 1H, J = 9.9 Hz), 12.12 (s, 1H);
MS (ESI) m/z 319 (M+Na)+.
4.2.8. General procedure for the synthesis of 24a–e
To a solution of 23a–e (2 mmol) in dry dioxane (12 mL) was
added NaH (2.2 mmol). The mixture was stirred at 60 °C for 1 h
and then cooled to room temperature, 12 (2.2 mmol) was added
and allowed to stir at 60 °C for 24 h. Water was added to the reac-
tion mixture and extracted with EtOAc (3 ꢀ 75 mL). The combined
organic layers were washed with water and evaporation of solvent
afforded light brown solids which were used in next step without
further purification.
4.2.9.9.
3(2H)-one (25i).
6H), 6.77–6.81 (m, 2H), 6.89–7.03 (m, 1H), 7.07 (br s, 2H), 7.19–
7.24 (m, 3H), 7.29 (d, 1H, J = 9.8 Hz), 7.53 (br s, 1H); MS (ESI) m/z
330 (M+Na)+; HPLC (214 nm) tr 21.7 min, 97%.
6-(2,6-Dimethylphenoxy)-2-(phenylamino)pyridazin-
Yield 12%; 1H NMR (CDCl3) d (ppm) 2.19 (s,
4.2.9.10. 4-(1-(4-Cyanophenylamino)-6-oxo-1,6-dihydropyrida-
zin-3-yloxy)-3,5-dimethylbenzonitrile (25j).
Yield 22%; 1H
NMR (CDCl3) d (ppm) 2.21 (s, 6H), 6.65 (d, 2H, J = 8.6 Hz), 7.27–7.30
(m, 1H), 7.38 (t, 3H, J = 9.2 Hz), 7.49 (d, 1H, J = 8.6 Hz), 7.59 (br s,
1H); MS (ESI) m/z 380 (M+Na)+; LC–MS (214 nm) tr 15.8 min, 92%.
4.2.9. General procedure for the synthesis of 25a–o
To a solution of 24a–e (0.8 mmol) in dichloromethane (7 mL)
was added diacetoxycopper hydrate (0.8 mmol), Et3N (0.96 mmol)
and 14a–f (0.96 mmol) in dichloromethane (1.5 mL) was added
dropwise and allowed to stir at room temperature for 2 h. The
reaction mixture was concentrated and chromatographed on silica
using 10% MeOH in DCM. The resulting fine sticky powders/oily
materials were lyophilized to powders.
4.2.9.11. 4-(1-(3-Cyanophenylamino)-6-oxo-1,6-dihydropyrida-
zin-3-yloxy)-3,5-dimethylbenzonitrile (25k).
Yield 20%; 1H
NMR (CDCl3) d (ppm) 2.23 (s, 6H), 6.86 (br s, 1H), 6.97 (br s, 1H),
7.28–7.37 (m, 2H), 7.40–7.49 (m, 3H), 7.67 (br s, 1H); MS (ESI)
m/z 380 (M+Na)+; LC–MS (214 nm) tr 16.1 min, 100%
4.2.9.1.4-(3-(Mesityloxy)-6-oxopyridazin-1(6H)-ylamino)benzo-
nitrile (25a).
2.23 (s, 3H), 6.60 (br s, 2H), 6.85 (s, 2H), 7.35 (br s, 1H), 7.45 (br
s, 2H), 7.78 (br s, 1H); MS (ESI) m/z 347 (M+H)+; HPLC (214 nm)
tr 22.3 min, 100%.
4.2.9.12. 4-(3-(4-Chloro-2,6-dimethylphenoxy)-6-oxopyridazin-
1(6H)-ylamino)benzonitrile (25l).
d (ppm) 2.13 (s, 6H), 6.61 (d, 1H, J = 9.3 Hz), 7.04 (s, 2H), 7.22–7.26
(m, 1H), 7.56–7.64 (m, 4H); MS (ESI) m/z 389 (M+Na)+; HPLC
(214 nm) tr 22.69 min, 98%
Yield 11%; 1H NMR (CDCl3) d (ppm) 2.13 (s, 6H),
Yield 14%; 1H NMR (CDCl3)
4.2.9.2. 6-(Mesityloxy)-2-(4-(trifluoromethyl)phenylamino)pyri-
dazin-3(2H)-one (25b).
2.13 (s, 6H), 2.24 (s, 3H), 6.70 (d, 2H, J = 8.4 Hz), 6.85 (br s, 2H),
7.20 (d, 1H, J = 9.8 Hz), 7.31 (d, 1H, J = 9.2 Hz), 7.42 (d, 1H,
J = 8.4 Hz), 7.55 (br s, 1H); MS (ESI) m/z 390 (M+H)+; LC–MS
(214 nm) tr 16.8 min, 100%.
4.2.9.13. 3-(3-(4-Chloro-2,6-dimethylphenoxy)-6-oxopyridazin-
1(6H)-ylamino)benzonitrile (25m).
(CDCl3) d (ppm) 2.16 (s, 6H), 6.87 (s, 1H), 6.95 (d, 1H, J = 9.8 Hz),
7.07 (br s, 2H), 7.22–7.37 (m, 4H), 7.66 (br s, 1H); MS (ESI) m/z
389 (M+Na)+; HPLC (214 nm) tr 22.9 min, 90%.
Yield 20%; 1H NMR (CDCl3) d (ppm)
Yield 21%; 1H NMR
4.2.9.14. 4-(3-(4-Bromo-2,6-dimethylphenoxy)-6-oxopyridazin-
1(6H)-ylamino)benzonitrile (25n).
d (ppm) 2.14 (s, 6H), 6.65 (d, 2H, J = 7.0 Hz), 7.20 (d, 2H, J = 9.2 Hz),
7.24 (s, 1H), 7.35 (d, 1H, J = 9.8 Hz), 7.47 (d, 2H, J = 7.0 Hz), 7.60 (s,
1H); MS (ESI) m/z 413 (M+H)+; LC–MS (214 nm) tr 17.2 min, 100%.
4.2.9.3.
3(2H)-one (25c).
6H), 2.29 (s, 3H), 6.77 (br s, 2H), 6.89 (s, 2H), 6.94 (br s, 2H), 7.20
(d, 1H, J = 9.4 Hz), 7.55 (br s, 1H); MS (ESI) m/z 362 (M+Na)+; LC–
MS (214 nm) tr 14.6 min, 100%.
2-(4-Fluorophenylamino)-6-(mesityloxy)pyridazin-
Yield 15%; 1H NMR (CDCl3) d (ppm) 2.12 (s,
Yield 17%; 1H NMR (CDCl3)
4.2.9.15. 6-(4-Bromo-2,6-dimethylphenoxy)-2-(4-chlorophenyl-
amino)pyridazin-3(2H)-one (25o).
d (ppm) 2.12 (s, 6H), 6.65 (d, 2H, J = 6.8 Hz), 7.12–7.16 (m, 3H), 7.27
(d, 2H, J = 9.8 Hz), 7.56 (s, 1H); MS (ESI) m/z 422 (M+H)+; LC–MS
(214 nm) tr 19.1 min, 100%.
4.2.9.4.
3(2H)-one (25d).
6H), 2.29 (s, 3H), 6.88 (br s, 2H), 6.89 (d, 2H, J = 9.4 Hz), 7.15–
7.22 (m, 2H), 7.28–7.33 (m, 1H), 7.47–7.54 (m, 1H); MS (ESI) m/z
380 (M+Na)+; LC–MS (214 nm) tr 16.8 min, 100%.
2-(4-Chlorophenylamino)-6-(mesityloxy)pyridazin-
Yield 17%; 1H NMR (CDCl3) d (ppm) 2.13 (s,
Yield 16%; 1H NMR (CDCl3)
4.2.10. General procedure for the synthesis of 27a–b
To a solution of 26 (1 mmol) and 21a and 21d (1 mmol) in dry
DMF (5 mL) was added NaH (1.1 mmol) portionwise and the reac-
tion mixture was allowed to stir at room temperature for 3 h,
poured on ice-cold water (20 mL) and the solid precipitated out
was filtered and dried under high vacuum.
4.2.9.5.3-(3-(Mesityloxy)-6-oxopyridazin-1(6H)-ylamino)benzo-
nitrile (25e).
2.23 (s, 3H), 6.79 (s, 1H), 6.85 (s, 3H), 7.19–7.28 (m, 2H), 7.34 (d,
1H, J = 9.8 Hz), 7.97 (br s, 1H); MS (ESI) m/z 369 (M+Na)+; HPLC
(214 nm) tr 22.9 min, 100%.
Yield 11%; 1H NMR (CDCl3) d (ppm) 2.13 (s, 6H),
4.2.9.6. 4-(3-(2,6-Dimethylphenoxy)-6-oxopyridazin-1(6H)-yla-
mino)benzonitrile (25f).
Yield 15%; 1H NMR (CDCl3) d (ppm)
4.2.10.1. 1-Chloro-4-(mesityloxy)phthalazine (27a).
Yield
2.17 (s, 6H), 6.68 (d, 1H, J = 8.8 Hz), 7.05 (s, 3H), 7.21 (d, 1H,
J = 9.8 Hz), 7.34 (d, 1H, J = 9.8 Hz), 7.45–7.49 (m, 3H); MS (ESI) m/
z 355 (M+Na)+; HPLC (214 nm) tr 20.5 min, 99%.
97%; 1H NMR (DMSO-d6) d (ppm) 2.04 (s, 6H), 2.31 (s, 3H), 7.0 (s,
2H), 8.22–8.26 (m, 2H), 8.29 (br s, 1H), 8.54–8.56 (m, 1H); MS
(ESI) m/z 299 (M+H)+.

M. Venkatraj et al. / Bioorg. Med. Chem. 19 (2011) 5924–5934
5933
4.2.10.2.
azine (27b).
1-Chloro-4-(4-chloro-2,6-dimethylphenoxy)phthal-
coordinates of the NNBS were taken from the crystal structure
of the RT/4-(4-(mesitylamino)pyrimidin-2-ylamino)benzonitrile
(TMC120) complex (pdb code: 1S6Q).9b Throughout the docking
process, the program standard settings were employed, unless sta-
ted otherwise. Prior to the docking, the X-ray structure was pro-
tonated with the Protonate 3D algorithm. First, selected target
compounds were drawn in MOE, protonated, minimized and
merged in a database. Then, this database was used to generate
all possible conformations of these compounds using the Conf-
search algorithm (Low mode). All these low energy conformations
were withheld for docking. Finally, an automated docking protocol
was employed to dock all conformations in the active site of HIV-1-
RT, using TMC-120 as a template ligand. The first pose, sorted on
docking score, of each docked compound was used as such in
Figure 3.
Yield 98%; 1H NMR (DMSO-d6) d (ppm) 2.13 (s,
6H), 7.3 (s, 2H), 8.21–8.29 (m, 2H), 8.30–8.33 (m, 1H), 8.53–8.57
(m, 1H); MS (ESI) m/z 320 (M+H)+.
4.2.11. General procedure for the synthesis of 28a–b
Compounds 27a–b (4 mmol) were refluxed in glacial acetic acid
(25 mL) at 120 °C for 6 h. The solvent was removed under reduced
pressure, water (50 mL) was added and the solid products obtained
were collected by filtration.
4.2.11.1. 4-(Mesityloxy)phthalazin-1(2H)-one (28a).
Yield
87%; 1H NMR (DMSO-d6) d (ppm) 2.07 (s, 6H), 2.26 (s, 3H), 6.95
(s, 2H), 7.96–8.07 (m, 2H), 8.24–8.30 (m, 2H), 11.8 (s, 1H); MS
(ESI) m/z 303 (M+Na)+.
4.2.11.2. 4-(4-Chloro-2,6-dimethylphenoxy)phthalazin-1(2H)-
one (28b).
4.4. Antiviral assay
Yield 90%; 1H NMR (DMSO-d6)
d (ppm) 2.11
(s, 6H), 7.26 (s, 2H), 7.97–8.07 (m, 2H), 8.24–8.30 (m, 2H), 11.8
4.4.1. Cells
(s, 1H); MS (ESI) m/z 324 (M+Na)+
The JC53-BL cell line, also known as the TZM-bl cell line (NIH
AIDS Research and Reference Reagent Program, Germantown,
USA), was used for the evaluation of anti-HIV-1 activity of the
diarylpyridinones, diarylpyridazinones and diarylphthalazinones.
TZM-bl cells were cultured in Dulbecco’s Minimum Essential Med-
ium (DMEM) (Lonza) containing 10% heat-inactivated FBS and
4.2.12. General procedure for the synthesis of 29a–b
To a solution of 28a–b (2.2 mmol) in dry dioxane (12 mL) was
added NaH (3.3 mmol). The mixture was stirred at 100 °C for 1 h
and then cooled to room temperature, 12 (2.86 mmol) was added
and allowed to stir at 100 °C for 24 h. Water was added to the reac-
tion mixture and extracted with EtOAc (3 ꢀ 75 mL). The combined
organic layers were washed with water and evaporation of solvent
afforded light brown solids which were used in the next step
without further purification.
50 lg gentamycin/mL at 37 °C in a humidified 5% CO2, 95% air envi-
ronment. Twice a week the cells were treated with 0.25% trypsin –
1 mM EDTA (Lonza) for 10 minutes. The resulting cell suspension
was washed with an equivalent amount of TZM-bl medium and
subsequently seeded in a T75 culture flask (Greiner Bio-One, Ger-
many) at 106 cells in 20 mL medium.
4.2.13. General procedure for the synthesis of 30a–c
To a solution of 29a–b (0.75 mmol) in dichloromethane (7 mL)
was added diacetoxycopper hydrate (0.75 mmol), Et3N (0.9 mmol)
and 14a/14e (0.9 mmol) in dichloromethane (1.5 mL) was added
dropwise and allowed to stir at room temperature for 2 h. The
reaction mixture was concentrated and chromatographed on silica
using 5% MeOH in DCM. The resulting oily materials were lyophi-
lized to powders.
4.4.2. TZM-bl virus inhibition assay
The antiviral activity of the newly designed compounds was
measured by pre-incubating ten thousand TZM-bl cells (at 105
cells/mL in culture medium supplemented with 30 lg/mL DEAE
dextran) in a 96-well plates for 30 min at 37 °C, 5% CO2 in the
presence or absence of serial dilutions of the respective compound.
Subsequently, 200 TCID50 of HIV-1 BaL or mutant virus (V106A or
Y181C) was added to each well and cultures were incubated for
48 h before quantifying luciferase activity, using a TriStar LB941
luminometer (Berthold Technologies GmbH & Co., KG, Bad Wild-
bad, Germany). Each condition was evaluated in triplicate wells
and in at least two independent experiments. The antiviral activity
of the compound was expressed as the percentage of viral inhibi-
tion compared to the untreated controls and subsequently plotted
against the compound concentration. Nonlinear regression analy-
4.2.13.1.
benzonitrile (30a).
4-(4-(Mesityloxy)-1-oxophthalazin-2(1H)-ylamino)-
Yield 40%; 1H NMR (CDCl3) d (ppm) 2.16
(s, 6H), 2.25 (s, 3H), 6.55 (d, 2H, J = 8.80 Hz), 6.83 (s, 2H), 7.35 (d,
2H, J = 7.0 Hz), 7.69 (s, 1H), 7.88–7.99 (m, 2H), 8.31 (d, 1H,
J = 8.0 Hz), 8.49 (d, 1H, J = 8.0 Hz); MS (ESI) m/z 397 (M+H)+; LC–
MS (214 nm) tr 20.1 min, 100%.
4.2.13.2. 4-(4-(4-Chloro-2,6-dimethylphenoxy)-1-oxophthala-
sis was used to calculate the 50% effective concentration (EC50
)
zin-2(1H)-ylamino)benzonitrile (30b).
Yield 18%; 1H NMR
based on at least two independent measurements and using
GraphPad Prism version 5.03 for Windows (GraphPad Software,
San Diego, CA, USA).
(CDCl3) d (ppm) 2.15 (s, 6H), 6.65 (d, 2H, J = 7.0 Hz), 7.05 (s, 2H),
7.43 (d, 2H, J = 7.4 Hz), 7.55 (s, 1H), 7.9–8.01 (m, 2H), 8.30 (d, 1H,
J = 7.4 Hz), 8.48 (d, 1H, J = 7.4 Hz); MS (ESI) m/z 418 (M+H)+; LC–MS
(214 nm) tr 19.2 min, 100%.
4.4.3. Reverse transcriptase activity assay
In order to asses the activity of a selected number of compounds
on recombinant HIV-1 reverse transcriptase, the Lenti RT activity
kit from CAVIDI (Uppsala, Sweden) was used according to the
manufacturer’s protocol. In brief, a serial dilution series of each
4.2.13.3. 4-(4-Chloro-2,6-dimethylphenoxy)-2-(4-chlorophenyl-
amino)phthalazin-1(2H)-one (30c).
(CDCl3) d (ppm) 2.16 (s, 6H), 6.68 (d, 2H, J = 7.0 Hz), 7.07 (s, 2H),
7.13–7.21 (m, 3H), 7.9–8.0 (m, 2H), 8.3 (d, 1H, J = 7.1 Hz), 8.48 (d,
1H, J = 7.0 Hz); MS (ESI) m/z 427 (M+H)+; LC–MS (214 nm) tr
21.6 min, 100%.
Yield 24%; 1H NMR
compound was prepared starting at 103 M down to 10ꢁ4
l lM.
TMC-120 and efavirenz, two NNRTIs with low nM anti-HIV activity,
were used as controls. Each compound was tested against 25 and
2.5 pg of recombinant HIV-1 RT, in three independent experiments.
The anti-RT activity of each compound was expressed as the
percentage of residual RT activity compared to ‘no-compound’
controls and subsequently plotted against the compound concen-
tration. IC50 values were calculated by nonlinear regression
4.3. Modeling
The molecular modeling study was performed using MOE
2009.10 software (Chemical Computing Group) to investigate the
binding mode of our compounds with NNBS of HIV-1 RT. The

5934
M. Venkatraj et al. / Bioorg. Med. Chem. 19 (2011) 5924–5934
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The Water Soluble Tetrazolium-1 (WST-1) Cell Proliferation
Assay is a colorimetric assay for the measurement of cell prolifer-
ation and viability. The assay is based on the cleavage of the
tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-
2H-5-tetrazolio]-1,3-benzene disulfonate) to a formazan dye by a
complex cellular mechanism. This bioreduction is largely depen-
dent on the glycolytic production of NAD(P)H in viable cells. There-
fore, the amount of formazan dye formed correlates directly to the
number of viable cells in the culture, and can be quantified by mea-
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greater the number of viable cells, the greater the amount of for-
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Acknowledgments
The research leading to these results has received funding from
the European Community’s Seventh Framework programme (FP7/
2007–2013) under Grant Agreement No. 242135 (CHAARM). This
work was supported by BOF GOA (Research Council of University
of Antwerp), IOF-POC (Industrial Research Fund-Proof of Concept)
and Hercules Foundation. The authors thank Dr. Matthias Zeller
(STaRBURSTT Cyberdiffraction Consortium, Youngstown State Uni-
versity, OH, USA) for collecting the X-ray datasets. P.V.V., K.K.A. and
G.V. are indebted to the Flemish Fund for Scientific research (FWO-
Vlaanderen) for financial support. The Laboratory of Medicinal
Chemistry belongs to the Department of Pharmaceutical Sciences
and is also a partner of the Antwerp Drug Discovery Network
(ADDN). The excellent technical assistance of W. Bollaert is greatly
appreciated.
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