Pharmacophore-based design, synthesis, and biological evaluation of novel chloro-pyridazine piperazines as human rhinovirus (HRV-3) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.12.001

A series of chloro-pyridazine piperazines were developed based on the structure of human rhinovirus (HRV) capsid-binding inhibitors with proven activity using a pharmacophore model. A preliminary evaluation demonstrated potent activity against HRV-3 with low cytotoxicity. A docking analysis indicated that 8a could fit into, and form tight interactions (e.g., H-bonds, σ-π effect) with the active site in VP1.

Full text of DOI:10.1016/j.bmcl.2010.12.001

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1057–1059
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pharmacophore-based design, synthesis, and biological evaluation of novel
chloro-pyridazine piperazines as human rhinovirus (HRV-3) inhibitors
Hongliang Wang a,b, Junhai Xiao b, , Dapeng Gao a,b, Xian Zhang a,b, Hui Yan b, Zehui Gong b, Tinmin Sun a,
⇑
⇑
,
Song Li b
a Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang
110016, PR China
b Laboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of chloro-pyridazine piperazines were developed based on the structure of human rhinovirus
(HRV) capsid-binding inhibitors with proven activity using a pharmacophore model. A preliminary eval-
uation demonstrated potent activity against HRV-3 with low cytotoxicity. A docking analysis indicated
Received 8 September 2010
Revised 2 November 2010
Accepted 1 December 2010
Available online 7 December 2010
that 8a could fit into, and form tight interactions (e.g., H-bonds,
r–p effect) with the active site in VP1.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Pharmacophore
Chloro-pyridazine piperazines
Anti-HRV activity
Human rhinoviruses (HRV), a genus of Picornaviridae family, is
most often associated with common colds. Although rhinovirus
infection is self-limited, complications could still occur in patients
with asthma, congestive heart failure, bronchiectasis and cystic
fibrosis.1,2 The fact that the US FDA has not approved a single
antiviral agent for the treatment or prevention of HRV infection3
clearly indicates a need to develop novel antiviral agents against
HRV.
Based on the features of the aforementioned compounds, we
built a four-point pharmacophore model using the Common Fea-
ture Pharmacophore Generation within Accelrys Discovery Studio
2.5.7 The optimal pharmacophore Hypo1 contained an aromatic
ring (A), a hydrogen bond acceptor (HBA) and two hydrophobic
moieties (HY). All features of Hypo1 model were nicely mapped
with BTA-188 (Fig. 2A). Search our in-house chemical database
which contains nearly 10,000 compounds using Ligand Pharmaco-
phore Mapping within Accelrys Discovery Studio 2.5 with such a
pharmacophore identified one hit (compound 1) with a chloro-pyr-
idazine piperazine skeleton (Fig. 2C), and the mapping picture was
shown in Figure 2B.
Compound 1 had significant anti-HRV activity against HRV-3.
The EC50 of this lead compound in cell culture cytopathic effect
(CPE) assay8–11 was 25 ng/ml. Using compound 1 as a lead
compound, a number of novel compounds were synthesized.
Modification included replacing of the methoxy group with other
hydrophobic moieties (e.g., ethoxyl, methyl and methoxycarbonyl
groups) and varying the number of carbon atoms between the piper-
azine and benzene ring. Screening of 30 candidate compounds
yielded seven compounds (8a–g) with promising profiles.
The capsids of HRV are composed of four structural viral
proteins, namely, VP1, VP2, VP3, and VP4.3 Pleconaril, an antiviral
agent, has been shown to have a broad spectrum of activities
against rhinovirus by binding to the hydrophobic pocket in VP1,
resulting in the inhibition of the virus attachment to the cells
and uncoating of viral RNA.4,5 Subsequent search, spurred by the
encouraging results with pleconaril, yielded additional compounds
that bind to VP1, including pirodavir, BTA-188, 13 and 5f (Fig. 1).
Pirodavir could inhibit 80% of all HRV strains.2 BTA-188 has been
shown to inhibits 87 of 100 HRV serotypes, and the activity against
HRV-14 (EC50: 1.0 ng/ml) was found to be superior to both
pleconaril (EC50
: 30 ng/ml) and pirodavir (EC50:
3.2 ng/ml).3
Compound 13 displayed efficient activity against 16 HRV strains
and the average EC50 is 3.88 ng/ml.6 Compound 5f was developed
in our lab with the equal excellent anti-virus activity against
HRV-2 and 14 (EC50: 32 ng/ml).2
The synthetic route of these compounds (8a–g) is illustrated in
Scheme 1. The common intermediate piperazin-1-yl-alkylalcohol
4a–b was synthesized in a two-step reaction by alkylating ethyl
piperazine-1-carboxylate 2 with 3a–b in the presence of K2CO3 in
acetonitrile and deprotecting the ethoxycarbonyl group in the
presence of aq NaOH in ethanol. The 3-chloro group of 3,6-
dichloropyridazine 5 was substituted with 4a–b in the presence
⇑
Corresponding author.
E-mail address: xiaojunhai@139.com (J. Xiao).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.001

1058
H. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1057–1059
Figure 1. Structure of training set and their biological activity data.
Scheme 1. Synthesis of compound 8a–g. Reagents and conditions: (i) K2CO3, MeCN,
reflux; (ii) 10% NaOH(aq), EtOH, reflux; (iii) Na2CO3, DMA, rt; (iv) Ph3P, DEAD, THF.
Figure 2. Pharmacophore model of HRV capsid-binding inhibitors generated by
Hiphop. (A) Hypo1 mapping with BTA-188. (B) Hypo1 mapping with compound 1.
Pharmacophore features are color-coded with light-blue for hydrophobic feature,
orange for ring aromatic feature and green for hydrogen-bond acceptor. (C) The
chemical structure of compound 1.
Table 1
The anti-HRV activity and cytotoxicity of compounds 1, 5f and 8a–g on HRV-3
Compound
EC50 (ng/ml)
CC50
(lg/ml)
SI
1
<25
50
<3.2
25
15.6
7.8
624
156
>4875
624
624
>1248
2496
624
5f
8a
8b
8c
8d
8e
8f
8g
of Na2CO3 in DMA to yield 6a–b. Finally, 6a–b were coupled with
4-substituted phenol 7a–g to produce compounds 8a–g by
Mitsunobu reaction.12
15.6
15.6
15.6
15.6
15.6
7.8
25
<12.5
6.25
12.5
6.25
The anti-HRV activity (EC50 in CPE assay) of these compounds is
presented in Table 1. Relative to compound 5f (EC50: 50 ng/ml),
these compounds were 2–15 times more potent against HRV-3
(EC50: 3.2–25 ng/ml). Among these, 8a was the most potent, with
an EC50 at <3.2 ng/ml. These compounds also had much higher ra-
tio of antiviral potency versus cytotoxicity (CC50/EC50: 4875-624)
than 5f (CC50/EC50: 156).
A docking analysis with LigandFit modular of Discovery Studio
2.5 against crystal structure of HRV-3 VP1 (PDB code: 1RHI) indi-
cated that 8a could fit into the active site of VP1 in a fashion similar
to 5f (Fig. 3).
15.6
>2496
SI (selectivity index) = CC50 (toxicity)/EC50 (antiviral activity).
phenolic hydroxyl group of Tyr197, respectively, also indicating
formation of H-bonds. The benzene ring is 3.77 Å from the methy-
lene group of Tyr152, suggesting potential
r–p effect.
In compound 8a, the benzene ring performs a similar
r–p effect
with the methylene group of Tyr152 to 5f at a distance of 3.64 Å.
The ethoxyl group is 3.03 Å from the backbone NH of Ser175, indi-
cating similar H-bond interaction to 5f (H-bond between oxygen
atom and NH of Val176). N1 and N2 of the 3-chloropyridazine
In 5f, the oxygen atom in the carbonyl group is 3.10 Å from the
carbamate NH of Val176, indicating H-bond interaction. The N2 of
the pyridazine ring and Cl atom are 2.16 and 1.60 Å away from the

H. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1057–1059
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Similar types of interactions with the active site of HRV-3 VP1
are noted for compounds 8b–g. The hydrophobic pocket has suffi-
cient space to accommodate a chain of 3–5 carbon atoms between
the piperazine and benzene.
In conclusion, the chloro-pyridazine piperazine derivatives
developed in this study interacted with the active site of HRV
VP1 in a manner similar to 5f, a compound with reported activity
against HRV. Among these, compounds 8a and 8g had excellent
anti-HRV activity and low cytotoxicity, and represent promising
candidates as anti-HRV agents.
Acknowledgments
This study was supported by the National High-Tech Research
and Development Program of China (863 Program; No.
2006AA020601) and Natural Science Foundation (No. 30472093).
Figure 3. Compounds 5f (blue) and 8a (pink) docked in the active site with similar
fashion in the structure of HRV-3 VP1.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.001.
References and notes
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Figure 4. Compounds 5f (blue) and 8a (pink) docked in the active site: some
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important amino acid residues in hydrophobic pocket, H-bonds (blue) and
effect (yellow) are present.
r–p
group are 2.25 and 2.03 Å from the phenolic hydroxyl group of
Tyr197, respectively, also indicating similar H-bonds interaction
to 5f. The Cl atom is also located in hydrophobic pocket by forming
a hydrogen-bond with Asp125 in the distance of 2.99 Å, this addi-
tional H-bond interaction may result in the higher activity of 8a
than 5f (Fig. 4).
11. Andries, K.; Dewindt, B.; Snoeks, J.; Wouters, L.; Moereels, H.; Lewi, P. J.;
Janssen, P. A. J. J. Virol. 1990, 64, 1117.
12. Selected data for compound 8a: 1H NMR (400 MHz, CD3Cl) d = 7.21 (d, 1H,
J = 9.6 Hz); 6.90 (d, 1H, J = 9.6 Hz); 6.83 (s, 4H); 3.98 (m, 4H); 3.67 (br, 4H); 2.61
(br, 6H); 2.00 (m, 2H); 1.39 (t, 3H, J = 7.2 Hz). MS (EI) [M+1]+: m/z = 376.2. Mp
122–124 °C.