Synthesis and biological evaluation of pyridazine derivatives as novel HIV-1 NNRTIs

Article abstract of DOI:10.1016/j.bmc.2012.12.049

In continuation of our efforts toward identification and optimization of novel non-nucleoside reverse transcriptase inhibitors (NNRTIs), we have employed a structure-based bioisosterism strategy, with which a new series of diarylpyridazine (DAPD) derivatives were synthesized and evaluated for their anti-HIV-1 (human immunodeficiency virus type 1) activity. Most of the title compounds displayed excellent anti-HIV-1 activity at submicromolar concentrations ranging from 34 nM to 5.08 μM. The most promising compound 8g inhibited HIV-1 IIIB in MT-4 cells at a low EC₅₀ value (0.034 μM), which was lower than the reference drug nevirapine and delavirdine. The structure activity relationships (SARs) were discussed and rationalized by docking simulations.

Full text of DOI:10.1016/j.bmc.2012.12.049

Accepted Manuscript
Synthesis and biological evaluation of pyridazine derivatives as novel HIV-1
NNRTIs
Dongyue Li, Peng Zhan, Huiqing Liu, Christophe Pannecouque, Jan Balzarini,
Erik De Clercq, Xinyong Liu
PII:
S0968-0896(13)00026-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2012.12.049
BMC 10517
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
22 October 2012
25 December 2012
27 December 2012
Please cite this article as: Li, D., Zhan, P., Liu, H., Pannecouque, C., Balzarini, J., De Clercq, E., Liu, X., Synthesis
and biological evaluation of pyridazine derivatives as novel HIV-1 NNRTIs, Bioorganic & Medicinal Chemistry
(2013), doi: http://dx.doi.org/10.1016/j.bmc.2012.12.049
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Synthesis and biological evaluation of pyridazine derivatives as novel
HIV-1 NNRTIs
Dongyue Li a, Peng Zhan a, Huiqing Liua, Christophe Pannecouqueb, Jan Balzarini b,
Erik De Clercq b and Xinyong Liu a,*
,
a
Department of Medicinal Chemistry, Key laboratory of Chemical Biology, Ministry
of Education, School of Pharmaceutical Sciences, Shandong University, 44, West
Culture Road, 250012, Jinan, Shandong, P.R.China
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven,
Belgium
Abstract: In continuation of our efforts toward identification and optimization for
novel non-nucleoside reverse transcriptase inhibitors (NNRTIs) project, we have
employed a structure-based bioisosterism strategy, with which, a new series of
diarylpyridazine (DAPD) derivatives were synthesized and evaluated for their
anti-HIV-1 (human immunodeficiency virus type 1) activity. Most of the title
compounds displayed excellent anti-HIV-1 activitiy at submicromolar concentrations
ranged from 34nM to 5.08μM. The most promising compound 8g inhibited HIV-1
IIIB in MT-4 cells with low EC50 value (0.034 μM)，which was higher than the
reference drugs nevirapine and delavirdine. The structure activity relationships (SARs)
were discussed and rationalized by docking simulations.
Keywords: HIV-1, AIDS, NNRTIs, Pyridazine derivatives, Anti-HIV-1 activity
1. Introduction
Aquired Immune Deficiency Syndrome (AIDS), caused by human immunodeficiency
virus (HIV), is still prevalent worldwide. The most efficient and standard treatment
regimen for HIV-1 infection is highly active antiretroviral therapy (HAART), which
commonly involves HIV-1 reverse transcriptase (RT) inhibitors and HIV protease
*Corresponding authors. Tel: +86 531 88380270; fax: +86 531 88382731; E-mail address:
xinyongl@sdu.edu.cn

inhibitors. The RT inhibitors can be grouped into two classes, i.e. nucleoside reverse
transcriptase inhibitors (NRTIs) and the non-nucleoside reverse transcriptase
inhibitors (NNRTIs). NNRTIs have advantages of higher specificity and lower
toxicity than NRTIs, playing an important role in current AIDS therapy.1,2 However,
during the clinical usage of first generation NNRTIs (nevirapine, delavirdine and
efavirenz), the rapid emergency of drug resistance dramatically reduced their potency,
and eventually compromised the patient’s clinical compliance. Alternatively, the
second generation of NNRTIs (etravirine and most recently rilpivirine, both belonging
diarylpyrimidine (DAPY) derivatives) possesses a high genetic barrier to resist
various clinically relevant mutations.3 Encouraged by the efficient clinically used
drugs, currently the investigation of new DAPY analogues have become a hotspot in
NNRTI research.4
According to the results obtained from molecular modeling and structure activity
relationship(SAR), the central pyrimidine ring of etravirine is relatively tolerant. Thus
replacement of the central pyrimidine ring with other functional groups had led to the
discovery of novel series of potent NNRTIs, such as pyridine derivative 15,
4-nitrobenzene-1-amine
compound
26,
pyrazin-2(1H)-one analogue
37,
pyridazin-3(2H)-one 48 and quinazoline 59 derivatives. Their antiviral potency differs
greatly attributing to the different central rings (Table 1). In view of the
above-mentioned and in order to explore the influence on antiviral activity of other
heterocycle rings, a novel series of pyridazine and pyridazinone derivatives were
designed according to the bioisosterism principle of medicinal chemistry strategy
(Figure 1). Herein we report the results of synthesis, antiviral activity and SAR study ,
which elicit a novel series of HIV-1 NNRTIs.

Table 1. Inhibition of HIV-1 (EC50 a, μM)5,6,7,8,9
Compd
LAIb
0.0014
0.003
0.006
1.75
L100I / K103N
K103N/Y181C
1
2
3
4
5
-
0.39
0.231
0.061
-
-
0.158
-
-
0.0018
0.06
aEC50: concentration of compound required to achieve 50% protection of MT-4 cell against
HIV-1-induced cytotoxicity, as determined by the MTT method.
bLAI: the strain of wild-type HIV-1.
Figure 1. Bioisosterism based design of pyridazine and pyridazinone derivatives.
2. Results and discussion
2.1. Chemistry
The title compounds of chloropyridazine series (8a-l) and pyridazinone series (9a-n)
were synthesized as depicted in Scheme 1 referring to the reported literatures.10,11 The
synthetic work was started with the commercially available 3,4,6-trichloropyridazine
(6), which was undergone two nucleophilic substitution reactions with different
substituted phenols and anilines under conditions of dimethyl formamide/NaH and
ethanol/HCl, respectively, affording chloropyridazine derivatives (8a-l). Pyridazinone
derivatives (9a-n) were synthesized by hydrolysis of the corresponding
chloropyridazines in acetic acid and then by methylation of 9 with iodomethane.

Scheme 1. Reagents and conditions: (i) ArOH/NaH/DMF; (ii) ArNH2/HCl/EtOH; (iii)
AcOH/100oC; (iv) CH3I/K2CO3/100oC.
2.2. Anti-HIV evaluation
The newly synthesized chloropyridazine derivatives (8a-l) and pyridazinone
derivatives (9a-n) were evaluated for anti-HIV-1 (IIIB and RES056) activity in MT-4
cells using the MTT method.10,11 The antiviral assay was used nevirapine (NVP),
delaviridine (DLV), efavirenz (EFV) and Etravirine (ETV) as reference drugs.
As shown in Table 2, most of the tested compounds inhibited wild type HIV-1
replication at low concentrations with EC50 values ranging from 34nM to 5.08μM.
The most potent HIV-1 inhibitor was 8g (EC50 = 34nM, CC50 = 19.2μM, SI = 565),
which was more potent than the reference drugs NVP (EC50 = 0.199μM) and DLV
(EC50 = 0.042μM), but was still inferior to the reference drug EFV (EC50 = 0.007μM)
and ETV (EC50 = 0.003μM).
Table 2 Anti-HIV activities, cytotoxicities and selectivity indices of chloropyridazine derivatives
(8a-l) and pyridazinone derivatives (9a-n)

EC50(M)a
Compd
R1
R2
R3 R4
SI(IIIB)c
CC50(M)b
HIV-1 WT
(IIIB)
HIV-1(RES056) HIV-2
(K103N / Y181C)
(ROD)
8a
8b
8c
8d
8e
8f
2,4,6-triMe
2,4,6-triMe
2,4,6-triMe
2,4,6-triMe
2,4,6-triCl
2,4,6-triBr
p-Cl
0.139±0.050
0.201±0.043
>0.94
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
210±10.4
244±22.8
0.94±0.07
36.3±2.23
18.6±5.89
22.3±1.02
19.2±3.05
84.1±19.1
2.90±0.48
93.1±45.2
53.2±3.37
290±10.1
5.98±0.27
185±37.6
37.1±2.44
5.88±0.29
47.0±2.43
35.0±2.88
1511
1214
<1
p-Me
p-NO2
p-CN
p-CN
p-CN
0.079±0.015
0.155±0.042
0.18±0.06
0.034±0.012
0.40±0.15
>2.90
461
120
124
565
210
<1
8g
8h
8i
2,6-diBr-4-Me p-CN
2,6-diMe-4-Br p-CN
2,6-diMeO
2,6-diCl
p-CN
p-CN
8j
0.22±0.051
0.144±0.029
0.54±0.21
0.78±0.17
1.55±0.32
5.08±1.00
1.59±0.21
0.26±0.04
423
369
537
8
8k
8l
4-CN-2,6-diMe p-CN
2,6-diMe
p-CN
p-Cl
9a
9b
9c
9d
9e
2,4,6-triMe
2,4,6-triMe
2,4,6-triMe
2,4,6-triMe
2,4,6-triMe
2,4,6-triCl
H
H
H
H
H
H
H
H
H
H
H
H
p-Me
p-NO2
p-OMe
p-CN
p-CN
119
7
4
180
≤4
9f
≥9.20
9g
9h
2,4,6-triBr
p-CN
H
H
H
H
H
H
H
H
0.63±0.20
0.21±0.03
-
-
139±23.4
154±20.1
19.5±3.57
119±21.3
205±13.2
272±14.5
143±12.3
≥246
221
733
16
2,6-diBr-4-Me p-CN
2,6-diMe-4-Br p-CN
H
-
-
9i
H
1.19±0.24
-
-
2,4,6-triMe
2,6-diMe
m-Cl
H
14.4±4.21
-
-
8
9j
9k
p-CN
H
1.90±0.31
-
-
108
96
9l
4-CN-2,6-diMe p-CN
H
2.83±0.54
-
-
9m
2,4,6-triMe
2,4,6-triMe
p-CN
p-CN
Me
1.16±0.25
-
-
123
≥73
>75
>1034
>12
>1537
9n
Me Me
3.36±0.99
-
-
-
NVP
DEV
EFV
ETVd,12
0.199±0.028
0.042±0.003
0.007±0.0003
0.003±0.0002
-
>14.9
-
-
0.02
-
>43.4
0.59
0.026
>2.00
>4.6
EC50a: concentration of compound required to achieve 50% protection of MT-4 cell against
HIV-1-induced cytotoxicity, as determined by the MTT method.

CC50b: concentration required to reduce the viability of mock-infected cells by 50%, as determined
by the MTT method.
SIc: selectivity index (CC50/EC50).
ETVd: Etravirine. The data were obtained from the same laboratory (Prof. Erik De Clercq, Rega
Institute for Medical Research, K.U. Leuven, Belgium).
Comparative study of the anti-HIV-1(WT IIIB) activities showed that,
chloropyridazine series (8) displayed higher potency than corresponding pyridazinone
series(9) when they have the same functionalities on left and right rings, probably
because chlorine atom had a preferable space to form less hydrophobic interaction
with the RT was than a carbonyl group. Methylated pyridazinone derivatives seemed
to be not active than the corresponding unmethylated pyridazinone derivatives.
With regard to pyridazine and pyridazinone derivatives, it was interesting to note that
2,4,6-Trisubstituted phenoxyl derivatives were more active than 2,6-disubstituted
phenoxyl derivatives. The potency of 2,6-dimethoxy substituted compound 8i was the
lowest in pyridazine series attributing to a high steric hindrance effect. Optimal
anti-HIV-1 activity was obtained with compounds having a 4-CN or 4-Cl substituted
aniline derivatives, which had the activity prior to 4-NO2 and 3-substituted
compounds.
As the double mutant HIV-1 strain (K103N/Y181C) appears frequently in the clinical
treatment of AIDS, the activity of title compounds against this mutant strain was also
tested. Unfortunately, effective inhibition by the compounds tested was not observed
for the replication of HIV-1 (K103N/Y181C) (data not shown). In addition, all the
derivatives tested did not show effective inhibition for HIV-2 (ROD) replication in
MT-4 cells, which might suggest that this new series of DAPDs was specific for
HIV-1 and probably belonged to typical NNRTIs.
2.3 Inhibition of HIV-1 RT
In order to further confirm the binding target, compound 8g was selected to conduct
an HIV-1 RT inhibitory assay that used poly(rA)-oligo(dT) as template primer.13,14 As
shown in Table 3, compound 8g exhibited inhibition activity against HIV-1 RT with

an IC50 value of 3.18 μM comparable to those of reference drugs NVP (IC50
=
1.20μM) and ETV (IC50 = 0.55μM). It is reasonable that the newly synthesized
pyridazine and pyridazinone NNRTIs may target HIV-1 RT.
Table 3 Activity of pyridazine NNRTI 8g against HIV-1 RT.
Compd
IC50
NVP
ETV
8g
3.18 μM
1.20 μM
0.55 μM
aIC50: Inhibitory concentration required to inhibit biotin deoxyuridine triphosphate (biotin-dUTP)
incorporation into the HIV-1 RT by 50% (RT kit, Roche).
2.4 Molecular modeling analysis
Molecular modeling (docking) was carried out to investigate the binding mode of the
title compounds. Compound 8g was selected and docked into the NNRTIs binding
pocket (NNIBP) of HIV-1 RT by the aid of SYBYL-X (Tripos, St. Louis, MO, USA).
Three-dimensional coordinates of the HIV-1 RT/etravirine complex (PDB entry
3MEC) were used as the input structure for docking. The results showed that 8g
exerted favorable interactions with HIV-1 RT compared to etravirine.
As shown in figure 2, compound 8g had nearly the same binding mode as etravirine.15
The left phenyl ring of 8g was oriented in a hydrophobic/π-electron region comprised
of Tyr181, Tyr188, Leu234, Phe227, and Trp229. The right phenyl ring of 8g was
oriented in a hydrophobic/π-electron pocket comprised of Leu234, Pro236 and
Tyr318. The NH group of compound 8g formed a key hydrogen bond with the
mainchain carbonyl of Lys101. The existence of O and NH group allowed 8g to adapt
to changes that caused by amino acid mutation through repositioning and reorienting
itself in the binding pocket. The antiviral potency of 8g was retained owing to its
nearly the same binding mode as etravirine.
However, the potency of 8g against both wild-type and mutant HIV-1 was inferior to
the marketed drug etravirine, which could be explained by molecular modeling results.
As shown in figure 3, the small binding pocket of NNIBP that acted with the aniline
part of 8g displayed hydrophobicity (colors near the top of the colorized strip

represented more hydrophobicity). The aniline part of etravirine (light green) was
hydrophobic and could form interactions with the small hydrophobic pocket. Whereas
the aniline part of 8g (blue) was hydrophilic and had low binding affinity with the
hydrophobic pocket. In addition, the region below the pyridazine N atom of 8g was
hydrophilic (light blue). Etravirine that had an extended amino group formed extra
interactions with the NNIBP than 8g.
Firure 2. Model of 8g docked into the HIV-1 NNIBP (PDB entry 3MEC) using SYBYL-X, shown
by use of PyMOL. 8g was displayed by green sticks. Etravirine was displayed by white sticks. The
left phenyl ring of 8g and etravirine were oriented in a hydrophobic/π-electron region comprised
of Tyr181, Tyr188, Leu234, Phe227, and Trp229. The right phenyl ring of 8g and etravirine were
oriented in a hydrophobic/π-electron pocket comprised of Leu234, Pro236 and Tyr318. The NH
group of compound 8g and etravirine formed key hydrogen bonds with the mainchain carbonyl of
Lys101.
Firure 3. Binding analysis of 8g and etravirine with the HIV-1 NNIBP using SYBYL-X. Colors

near the top of the colorized strip (located in the left of A) represented more hydrophobicity. (A)
The hydrophobic/hydrophilic properties of etravirine. The right aniline part of etravirine in the
white circle (green) represented more hydrophobic. The amino part of etravirine in yellow circle
(blue) represented more hydrophilic; (B) The hydrophobic/hydrophilic properties of 8g. The right
aniline part of 8g in the white circle (blue) represented more hydrophilic. The region below the
pyridazine N atom of 8g (blue) represented more hydrophilic; (C) The hydrophobic/hydrophilic
properties of NNIBP. 8g was docked into the HIV-1 NNIBP (PDB entry 3MEC) using SYBYL-X
as previous mentioned. The surface of NNIBP was displayed by MOLCAD module of SYBYL-X
to show hydrophobic/hydrophilic properties. The part of NNIBP (brown and green) in white circle
represented more hydrophobic. And the part of NNIBP in yellow circle (light blue) represented
more hydrophilic.
In summary, the molecular modeling results revealed the binding mode of our newly
synthesized compounds and explained the reasons why 8g possessed lower potency
than etravirine. Further optimization of pyridazine derivatives will be guided by these
molecular modeling results.
4. Conclusion
In summary, the newly pyridazine and pyridazinone derivatives were synthesized and
evaluated for their antiviral potency. Most derivatives proved to be highly effective in
inhibiting HIV-1 replication at low micromolar ranges. Among them, compound 8g
was found to be the most potent inhibitor with an EC50 value of 34nM and a SI value
of 563 against wild type HIV-1. 8g showed higher antiretroviral activity in MT-4 cells
than the reference drugs nevirapine and delavirdine, but lower potency than efavirenz
and etravirine, requiring further studies to improve antiviral activity, especially to
improve the activity against K103N/Y181C mutant HIV-1.
4. Experimental
4.1. Chemistry
All melting points were determined on a micromelting point apparatus and are
uncorrected. Infrared spectra (IR) were recorded with a Nexus 470FT-IR
Spectrometer. NMR spectra were obtained on
a
Bruker Avance-400

NMR-spectrometer in the indicated solvents. Chemical shifts are expressed in d units
and TMS as internal reference. Mass spectra were taken on a LC Autosampler Device:
Standard G1313A instrument. TLC was performed on Silica Gel GF254 for TLC
(Merck) and spots were visualized by iodine vapors or by irradiation with UV light
(254 nm). Solvents were reagent grade and, when necessary, were purified and dried
by standard methods. Concentration of the reaction solutions involved the use of
rotary evaporator at reduced pressure.
4.2. General procedure for the synthesis of pyridazine derivatives 8a-l and
pyridazinone derivatives 9a-n.
To a stirred solution of substituted phenol (5 mmol) in dimethyl formamide (20 mL)
was added 60% sodium hydride (5 mmol) under ice-water bath. After further stirring
for 30 min, 3,4,6-trichloropyridazine (6, 5 mmol) was added and reacted at room
temperature for 1-24h. The reaction solution was poured into cold water, then the
formed solid was filtered, washed with water and dried to give intermediate 7, which
didn’t need any further purification. Intermediate 7 (5 mmol) with substituted aniline
(5 mmol) and a few drops of 12 M hydrochloric acid was added and boiled with 50 ml
ethanol for 12-48h under reflux. The reaction solution was poured into cold water,
then alkalized to pH 8 with 4 M NaOH solution. Then the solid was filtered, washed
with water, dried and recrystallized from DMF/H2O to obtain pyridazine derivatives
8a-l.
Pyridazine derivatives (2 mmol) was boiled with 10 mL acetic acid under reflux
overnight. The reaction solution was poured into cold water, then alkalized to pH 8
with 4 M NaOH solution. Then the solid was filtered, washed with water, dried and
recrystallized from DMF/H2O to obtain pyridazinone derivatives 9a-l. To a mixture of
9e (2 mmol) in DMF (10 mL) was added absolute potassium carbonate (4 mmol)
followed by iodomethane (2 mmol). The solution was refluxed overnight, then poured
into cold water, filtered, washed with water, dried and recrystallized from DMF/H2O
to obtain 9m. To a mixture of 9e (2 mmol) in DMF (10 mL) was added absolute
potassium carbonate (8 mmol) followed by iodomethane (4 mmol). The solution was
refluxed overnight, then poured into cold water, filtered, washed with water, dried and

recrystallized from DMF/H2O to obtain 9n.
4.2.1. 6-chloro-N-(4-chlorophenyl)-5-(mesityloxy)pyridazin-3-amine (8a)
1
White solid, yield: 30%. Mp: 281-283°C. H-NMR (DMSO-d6, ppm) δ: 9.34 (s, 1H,
Ph-NH), 7.68 (d, 2H, J=7.8Hz, Ph-H), 7.33 (d, 2H, J=7.8Hz, Ph-H), 7.08 (s, 2H,
OPh-H), 6.12 (s, 1H, pyridazine-H), 2.29 (s, 3H, CH3), 2.05 (s, 6H, CH3). IR (KBr,
cm-1): 3300 (NH). ESI-MS: m/z 374 (M+1)，376 (M+3). C19H17Cl2N3O (373.1).
4.2.2. 6-chloro-5-(mesityloxy)-N-(p-tolyl)pyridazin-3-amine (8b)
1
White solid, yield: 32%. Mp: 273-277°C. H-NMR (DMSO-d6, ppm) δ: 9.14 (s, 1H,
Ph-NH), 7.53 (d, 2H, J=7.8Hz, Ph-H), 7.09 (d, 2H, J=7.8Hz, Ph-H), 7.07 (s, 2H,
OPh-H), 6.11 (s, 1H, pyridazine-H), 2.29 (s, 3H, CH3), 2.24 (s, 3H, CH3), 2.06 (s, 6H,
CH3). IR (KBr, cm-1): 3299 (NH). ESI-MS: m/z 354.3 (M+1). C20H20ClN3O (353.1).
4.2.3. 6-chloro-5-(mesityloxy)-N-(4-nitrophenyl)pyridazin-3-amine (8c)
Yellow solid, yield: 28%. Mp: 226-227°C. 1H-NMR (DMSO-d6, ppm) δ: 9.97 (s, 1H,
Ph-NH), 8.21 (d, 2H, J=9.28Hz, Ph-H), 7.89 (d, 2H, J=9.28Hz, Ph-H), 7.03 (s, 2H,
OPh-H), 6.25 (s, 1H, pyridazine-H), 2.30 (s, 3H, CH3), 2.06 (s, 6H, CH3). IR (KBr,
cm-1): 3362 (NH); 1503, 1325 (NO2)．ESI-MS: m/z 385.3 (M+1). C19H17ClN4O3
(384.1).
4.2.4. 4-((6-chloro-5-(mesityloxy)pyridazin-3-yl)amino)benzonitrile (8d)
1
White solid, yield: 31%. Mp: 285-287°C. H-NMR (DMSO-d6, ppm) δ: 9.72 (s, 1H,
Ph-NH), 7.84 (d, 2H, J=8.88Hz, Ph-H), 7.73 (d, 2H, J=8.88Hz, Ph-H), 7.08 (s, 2H,
OPh-H), 6.21 (s, 1H, pyridazine-H), 2.30 (s, 3H, CH3), 2.05 (s, 6H, CH3). IR (KBr,
cm-1): 3298 (NH); 2223 (CN)．ESI-MS: m/z 365.3 (M+1). C20H17ClN4O (364.1).
4.2.5. 4-((6-chloro-5-(2,4,6-trichlorophenoxy)pyridazin-3-yl)amino)benzonitrile (8e)
Yellow solid, yield: 25%. Mp: 283-284°C. 1H-NMR (DMSO-d6, ppm) δ: 9.73 (s, 1H,
Ph-NH), 8.08 (s, 2H, OPh-H), 7.83 (d, 2H, J=8.88Hz, Ph-H), 7.77 (d, 2H, J=8.88Hz,
Ph-H), 6.43 (s, 1H, pyridazine-H). IR (KBr, cm-1): 3232 (NH); 2219 (CN). ESI-MS:
m/z 425.2 (M+1)，427.1 (M+3)，429.2 (M+5). C17H8Cl4N4O (423.9).
4.2.6. 4-((6-chloro-5-(2,4,6-tribromophenoxy)pyridazin-3-yl)amino)benzonitrile (8f)

1
White solid, yield: 28%. Mp: 305-307°C. H-NMR (DMSO-d6, ppm) δ: 9.73 (s, 1H,
Ph-NH), 8.28 (s, 2H, OPh-H), 7.83 (d, 2H, J=8.88Hz, Ph-H), 7.76 (d, 2H, J=8.88Hz,
Ph-H), 6.39 (s, 1H, pyridazine-H). IR (KBr, cm-1): 3319 (NH); 2213 (CN)．ESI-MS:
m/z 559.1 (M+3)，561.1(M+5)，563.0(M+7). C17H8Br3ClN4O (555.8).
4.2.7.
4-((6-chloro-5-(2,6-dibromo-4-methylphenoxy)pyridazin-3-yl)amino)
benzonitrile (8g)
1
White solid, yield: 20%. Mp: 299-300°C. H-NMR (DMSO-d6, ppm) δ: 9.78 (s, 1H,
Ph-NH), 7.83 (s, 2H, OPh-H), 7.84 (d, 2H, J=8.88Hz, Ph-H), 7.76 (d, 2H, J=8.88Hz,
Ph-H), 6.34 (s, 1H, pyridazine-H), 3.04 (s, 3H, CH3). IR (KBr, cm-1): 3227 (NH);
2218 (CN)．ESI-MS: m/z 493.1 (M+1)，494.9 (M+3)，497.1 (M+5). C18H11Br2ClN4O
(491.9).
4.2.8.
4-((5-(4-bromo-2,6-dimethylphenoxy)-6-chloropyridazin-3-yl)amino)
benzonitrile (8h)
1
White solid, yield: 23%. Mp: 281-283°C. H-NMR (DMSO-d6, ppm) δ: 9.70 (s, 1H,
Ph-NH), 7.84 (d, 2H, J=8.88Hz, Ph-H), 7.74 (d, 2H, J=8.88Hz, Ph-H), 7.55 (s, 2H,
OPh-H), 6.22 (s, 1H, pyridazine-H), 2.10 (s, 3H, CH3). IR (KBr, cm-1): 3310 (NH);
2222 (CN)．ESI-MS: m/z 429.1 (M+1)，431.5 (M+3)，433.6 (M+5). C19H14BrClN4O
(428.0).
4.2.9. 4-((6-chloro-5-(2,6-dimethoxyphenoxy)pyridazin-3-yl)amino)benzonitrile (8i)
1
White solid, yield: 21%. Mp: 292-295°C. H-NMR (DMSO-d6, ppm) δ: 9.32 (s, 1H,
Ph-NH), 8.18 (d, 2H, J=8.88Hz, Ph-H), 7.74 (d, 2H, J=8.88Hz, Ph-H), 7.87-7.33 (m,
3H, OPh-H), 6.21 (s, 1H, pyridazine-H), 3.78 (s, 6H, CH3). IR (KBr, cm-1): 3205
(NH); 2220 (CN)．ESI-MS: m/z 383.4 (M+1). C19H15ClN4O3 (382.1).
4.2.10. 4-((6-chloro-5-(2,6-dichlorophenoxy)pyridazin-3-yl)amino)benzonitrile (8j)
1
White solid, yield: 35%. Mp: 298-300°C. H-NMR (DMSO-d6, ppm) δ: 9.78 (s, 1H,
Ph-NH), 7.84-7.75 (m, 6H, Ph-H), 7.54 (t, 1H, Ph-H), 7.74 (d, 2H, J=8.88Hz, Ph-H),
6.37 (s, 1H, pyridazine-H). IR (KBr, cm-1): 3305 (NH); 2220 (CN)．ESI-MS: m/z
391.1 (M+1), 393.1 (M+3), 395.0 (M+5). C17H9Cl3N4O (390.0)

4.2.11.
4-((3-chloro-6-((4-cyanophenyl)amino)pyridazin-4-yl)oxy)-3,5-dimethyl-
benzonitrile (8k)
1
White solid, yield: 22%. Mp: 287-290°C. H-NMR (DMSO-d6, ppm) δ: 9.68 (s, 1H,
Ph-NH), 7.87 (s, 2H, OPh-H), 7.83 (d, 2H, J=8.88Hz, Ph-H), 7.74 (d, 2H, J=8.88Hz,
Ph-H), 6.19 (s, 1H, pyridazine-H), 2.15 (s, 6H, CH3). IR (KBr, cm-1): 3329 (NH);
2226 (CN)．ESI-MS: m/z 376.4 (M+1). C20H14ClN5O (375.1)
4.2.12. 4-((6-chloro-5-(2,6-dimethylphenoxy)pyridazin-3-yl)amino)benzonitrile (8l)
Deep yellow solid, yield: 34%. Mp: 275-276°C. 1H-NMR (DMSO-d6, ppm) δ: 9.74 (s,
1H, Ph-NH), 7.84 (d, 2H, J=8.88Hz, Ph-H), 7.73 (d, 2H, J=8.88Hz, Ph-H), 7.30-7.25
(m, 3H, OPh-H), 6.20 (s, 1H, pyridazine-H), 2.10 (s, 6H, CH3). IR (KBr, cm-1): 3304
(NH); 2220 (CN)．ESI-MS: m/z 351.2 (M+1). C19H15ClN4O (350.1)
4.2.13. 6-((4-chlorophenyl)amino)-4-(mesityloxy)pyridazin-3(2H)-one (9a)
White solid, yield: 60%. Mp: 280-283°C. 1H-NMR (DMSO-d6, ppm) δ: 12.28 (s, 1H,
N-NH), 8.78(s, 1H, Ph-NH), 7.46 (d, 2H, J=8.88Hz, Ph-H), 7.25 (d, 2H, J=8.88Hz,
Ph-H), 7.04 (s, 2H, OPh-H), 5.94 (s, 1H, pyridazine-H), 2.28 (s, 3H, CH3), 2.05 (s, 6H,
CH3). IR (KBr, cm-1): 3311, 3203 (NH); 1679 (C=O)．ESI-MS: m/z 356.4 (M+1).
C19H18ClN3O2 (355.1)
4.2.14. 4-(mesityloxy)-6-(p-tolylamino)pyridazin-3(2H)-one (9b)
White solid, yield: 72%. Mp: 284-285°C. 1H-NMR (DMSO-d6, ppm) δ: 12.17 (s, 1H,
N-NH), 8.51 (s, 1H, Ph-NH), 7.33 (d, 2H, J=8.88Hz, Ph-H), 7.00 (d, 2H, J=8.88Hz,
Ph-H), 7.04 (s, 2H, OPh-H), 5.94 (s, 1H, pyridazine-H), 2.28 (s, 3H, CH3), 2.20 (s, 3H,
CH3), 2.05 (s, 6H, CH3). IR (KBr, cm-1): 3324, 3206 (NH); 1671 (C=O)．ESI-MS: m/z
336.5 (M+1). C20H21N3O2 (335.2)
4.2.15. 4-(mesityloxy)-6-((4-nitrophenyl)amino)pyridazin-3(2H)-one (9c)
Yellow solid, yield: 65%. Mp: 320-323°C. 1H-NMR (DMSO-d6, ppm) δ: 12.57 (s, 1H,
N-NH), 9.49 (s, 1H, Ph-NH), 8.12 (d, 2H, J=9.32Hz, Ph-H), 7.61 (d, 2H, J=9.32Hz,
Ph-H), 7.05 (s, 2H, OPh-H), 6.02 (s, 1H, pyridazine-H), 2.29 (s, 3H, CH3), 2.06 (s, 6H,
CH3). IR (KBr, cm-1): 3317, 3153 (NH); 1668 (C=O); 1503, 1327 (NO2)．ESI-MS:

m/z 367.3 (M+1). C19H18N4O4 (366.1)
4.2.16. 4-(mesityloxy)-6-((4-methoxyphenyl)amino)pyridazin-3(2H)-one (9d)
Yellow solid, yield: 15%. Mp: 257-259°C. 1H-NMR (DMSO-d6, ppm) δ: 12.11 (s, 1H,
N-NH), 8.41 (s, 1H, Ph-NH), 7.36 (d, 2H, J=8.88Hz, Ph-H), 6.80 (d, 2H, J=8.88Hz,
Ph-H), 7.04 (s, 2H, OPh-H), 5.91 (s, 1H, pyridazine-H), 3.68 (s, 3H, OCH3), 2.10 (s,
3H, CH3), 2.05 (s, 6H, CH3). IR (KBr, cm-1): 3326 (NH); 1671 (C=O)．ESI-MS: m/z
352.3 (M+1). C20H21N3O3 (351.2)
4.2.17. 4-((5-(mesityloxy)-6-oxo-1,6-dihydropyridazin-3-yl)amino)benzonitrile (9e)
White solid, yield: 61%. Mp: 315-317°C. 1H-NMR (DMSO-d6, ppm) δ: 12.46 (s, 1H,
N-NH), 9.21 (s, 1H, Ph-NH), 7.66 (d, 2H, J=8.88Hz, Ph-H), 7.57 (d, 2H, J=8.88Hz,
Ph-H), 7.05 (s, 2H, OPh-H), 5.98 (s, 1H, pyridazine-H), 2.28 (s, 3H, CH3), 2.05 (s, 6H,
CH3). IR (KBr, cm-1): 3195(NH); 1671 (C=O); 2221 (CN)．ESI-MS: m/z 347.3 (M+1).
C20H18N4O2 (346.1)
4.2.18.
4-((6-oxo-5-(2,4,6-trichlorophenoxy)-1,6-dihydropyridazin-3-yl)amino)
benzonitrile (9f)
1
Deep yellow solid, yield: 78%. Mp: 260-261°C. H-NMR (DMSO-d6, ppm) δ: 12.66
(s, 1H, N-NH), 9.20 (s, 1H, Ph-NH), 7.93 (s, 2H, OPh-H), 7.69 (d, 2H, J=8.88Hz,
Ph-H), 7.56 (d, 2H, J=8.88Hz, Ph-H), 6.25 (s, 1H, pyridazine-H). IR (KBr, cm-1):
3431, 3201 (NH); 1674 (C=O); 2224 (CN). ESI-MS: m/z 407.5 (M+1)，409.5(M+3).
C17H9Cl3N4O2 (406.0)
4.2.19.
4-((6-oxo-5-(2,4,6-tribromophenoxy)-1,6-dihydropyridazin-3-yl)amino)
benzonitrile (9g)
White solid, yield: 59%. Mp: 310-312°C. 1H-NMR (DMSO-d6, ppm) δ: 12.65 (s, 1H,
N-NH), 9.21 (s, 1H, Ph-NH), 8.22 (s, 2H, OPh-H), 7.69 (d, 2H, J=8.88Hz, Ph-H),
7.57 (d, 2H, J=8.88Hz, Ph-H), 6.19 (s, 1H, pyridazine-H). IR (KBr, cm-1): 3439, 3193
(NH); 1676 (C=O); 2213 (CN) ． ESI-MS: m/z 541.1 (M+3) ， 543.1( M+5).
C17H9Br3N4O2 (537.8)
4.2.20. 4-((5-(2,6-dibromo-4-methylphenoxy)-6-oxo-1,6-dihydropyridazin-3-yl)amino)

benzonitrile (9h)
1
Light red solid, yield: 81%. Mp: 300-302°C. H-NMR (DMSO-d6, ppm) δ: 12.61 (s,
1H, N-NH), 9.25 (s, 1H, Ph-NH), 7.74 (s, 2H, OPh-H), 7.68 (d, 2H, J=8.88Hz, Ph-H),
7.57 (d, 2H, J=8.88Hz, Ph-H), 6.12 (s, 1H, pyridazine-H), 2.37 (s, 6H, CH3). IR (KBr,
cm-1): 3426, 3188 (NH); 1677 (C=O); 2221 (CN)．ESI-MS: m/z 475.2 (M+1)，477.2
(M+3)，479.2 (M+5). C18H12Br2N4O2 (472.9)
4.2.21. 4-((5-(4-bromo-2,6-dimethylphenoxy)-6-oxo-1,6-dihydropyridazin-3-yl)amino)
benzonitrile (9i)
White solid, yield: 72%. Mp: 301-303°C. 1H-NMR (DMSO-d6, ppm) δ: 12.51 (s, 1H,
N-NH), 9.19 (s, 1H, Ph-NH), 7.69 (d, 2H, J=8.88Hz, Ph-H), 7.57 (d, 2H, J=8.88Hz,
Ph-H), 7.51 (s, 2H, OPh-H), 6.01 (s, 1H, pyridazine-H), 2.10 (s, 6H, CH3). IR (KBr,
cm-1): 3412, 3195 (NH); 1670 (C=O); 2220 (CN). ESI-MS: m/z 411.4 (M+1)，
413.5(M+3). C19H15BrN4O2 (410.0)
4.2.22. 6-((3-chlorophenyl)amino)-4-(mesityloxy)pyridazin-3(2H)-one (9j)
White solid, yield: 20%. Mp: 285-286°C. 1H-NMR (DMSO-d6, ppm) δ: 12.32 (s, 1H,
N-NH), 8.87 (s, 1H, Ph-NH), 7.76-6.87 (m, 4H, Ph-H), 7.05 (s, 2H, OPh-H), 5.94 (s,
1H, pyridazine-H), 2.28 (s, 3H, CH3), 2.06 (s, 6H, CH3). IR (KBr, cm-1): 3302, 3187
(NH); 1670 (C=O)．ESI-MS: m/z 356.3 (M+1). C19H18ClN3O2 (355.1)
4.2.23.
4-((5-(2,6-dimethylphenoxy)-6-oxo-1,6-dihydropyridazin-3-yl)amino)
benzonitrile (9k)
White solid, yield: 80%. Mp: 296°-297C. 1H-NMR (DMSO-d6, ppm) δ: 12.49 (s, 1H,
N-NH), 9.23 (s, 1H, Ph-NH), 7.66 (d, 2H, J=8.88Hz, Ph-H), 7.57 (d, 2H, J=8.88Hz,
Ph-H), 7.26-7.18 (m, 3H, OPh-H), 5.98 (s, 1H, pyridazine-H), 2.10 (s, 6H, CH3). IR
(KBr, cm-1): 3402, 3317, 3196 (NH); 1669 (C=O); 2221 (CN)．ESI-MS: m/z 333.5
(M+1). C19H16N4O2 (332.1)
4.2.24.
4-((6-((4-cyanophenyl)amino)-3-oxo-2,3-dihydropyridazin-4-yl)oxy)-3,5-
dimethylbenzonitrile (9l)
White solid, yield: 68%. Mp: 330-334°C. 1H-NMR (DMSO-d6, ppm) δ: 12.56 (s, 1H,

N-NH), 9.16 (s, 1H, Ph-NH), 7.83 (s, 2H, OPh-H), 7.67 (d, 2H, J=8.88Hz, Ph-H),
7.56 (d, 2H, J=8.88Hz, Ph-H), 5.99 (s, 1H, pyridazine-H), 2.15 (s, 6H, CH3). IR (KBr,
cm-1): 3355 (NH); 1671 (C=O); 2219 (CN). ESI-MS: m/z 358.4 (M+1). C20H15N5O2
(357.1)
4.2.25. 6-((4-chlorophenyl)amino)-4-(mesityloxy)-2-methylpyridazin-3(2H)-one (9m)
Light yellow solid, yield: 60%. Mp: 298-300°C. 1H-NMR (DMSO-d6, ppm) δ: 8.84 (s,
1H, Ph-NH), 7.50 (d, 2H, J=8.88Hz, Ph-H), 7.25 (d, 2H, J=8.88Hz, Ph-H), 7.04 (s, 2H,
OPh-H), 5.94 (s, 1H, pyridazine-H), 3.64 (s, 3H, NCH3), 2.28 (s, 3H, CH3), 2.05 (s,
6H, CH3). IR (KBr, cm-1): 3310 (NH); 1660 (C=O)．ESI-MS: m/z 370.4 (M+1).
C20H20ClN3O2 (369.1)
4.2.26. 4-((5-(mesityloxy)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)(methyl)amino)
benzonitrile (9n)
Light yellow solid, yield: 52%. Mp: 268-270°C. 1H-NMR (DMSO-d6, ppm) δ: 7.61 (d,
2H, J=8.88Hz, Ph-H), 7.03 (d, 2H, J=8.88Hz, Ph-H), 6.93 (s, 2H, OPh-H), 5.76 (s, 1H,
pyridazine-H), 3.68 (s, 3H, PhNCH3), 3.24 (s, 3H, NCH3), 2.19 (s, 1H, CH3) 2.05 (s,
6H, CH3). IR (KBr, cm-1): 1668 (C=O); 2215 (CN)．ESI-MS: m/z 375.5 (M+1).
C22H22N4O2 (374.2)
4.6. Anti-HIV activity assays
Evaluation of the antiviral activity of the compounds against HIV-1 strain IIIB,
RES056 and HIV-2 strain (ROD) in MT-4 cells was performed using the MTT assay
as previously described.16 Stock solutions (10×final concentration) of test compounds
were added in 25-μl volumes to two series of triplicate wells so as to allow
simultaneous evaluation of their effects on mock- and HIV-infected cells at the
beginning of each experiment. Serial fivefold dilutions of test compounds were made
directly in flat-bottomed 96-well microtiter trays using a Biomek 3000 robot
(Beckman instruments, Fullerton, CA). Untreated control HIV- and mock-infected
cell samples were included for each sample.
HIV-117 or HIV-2 (ROD)18 stock (50 μL) at 100–300 CCID50 (cell culture infectious

dose) or culture medium was added to either the infected or mock-infected wells of
the microtiter tray. Mock-infected cells were used to evaluate the effect of test
compound on uninfected cells in order to assess the cytotoxicity of the test compound.
Exponentially growing MT-4 cells25 were centrifuged for 5 min at 1000 rpm and the
supernatant was discarded. The MT-4 cells were resuspended at 6×105 cells/mL, and
50-μL volumes were transferred to the microtiter tray wells. Five days after infection,
the viability of mock- and HIV-infected cells was examined spectrophotometrically
by the MTT assay.
The MTT assay is based on the reduction of yellow colored
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (Acros
Organics, Geel, Belgium) by mitochondrial dehydrogenase of metabolically active
cells to a blue-purple formazan that can be measured spectrophotometrically. The
absorbances were read in an eight-channel computer-controlled photometer
(Multiscan Ascent Reader, Labsystems, Helsinki, Finland), at two wavelengths (540
and 690 nm). All data were calculated using the median OD (optical density) value of
tree wells. The 50% cytotoxic concentration (CC50) was defined as the concentration
of the test compound that reduced the absorbance (OD540) of the mock-infected
control sample by 50%. The concentration achieving 50% protection from the
cytopathic effect of the virus in infected cells was defined as the 50% effective
concentration (EC50).
4.7 HIV-1 RT inhibition assay
Inhibition of HIV-1 RT assay was performed by using homopolymer template/primer
linked to microtiter plate, biotin-dUTP and RT with detection of ELISA for
quantifying expression. The incorporated quantities of the biotin-dUTP into the
template represented the activity of HIV-1 RT. IC50 values corresponded to the
concentration of the piperidine-substituted triazine derivatives required to inhibit
biotin-dUTP incorporation by 50%.
Acknowledgment
The financial support from the National Natural Science Foundation of China (NSFC

No.81102320, No.30873133, No.30772629 and No.30371686), Key Project of NSFC
for International Cooperation (No.30910103908), Research Fund for the Doctoral
Program of Higher Education of China (No.20110131130005, 20110131120037 and
No.070422083), Independent Innovation Foundation of Shandong University
(IIFSDU, No.2010GN044), Shandong Postdoctoral Innovation Science Research
Special Program (No.201002023) and China Postdoctoral Science Foundation funded
project (No.20100481282) is gratefully acknowledged.
We thank K. Erven, K. Uyttersprot and C. Heens for technical assistance with the
HIV assays.
The authors have declared no conflict of interest.
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*Graphical Abstract (for review)
Figure 1. Bioisosterism based design of pyridazine and pyridazinone derivatives.