Lead optimization of spiropyrazolopyridones: A new and potent class of dengue virus inhibitors

Article abstract of DOI:10.1021/ml500521r

Spiropyrazolopyridone 1 was identified, as a novel dengue virus (DENV) inhibitor, from a DENV serotype 2 (DENV-2) high-throughput phenotypic screen. As a general trend within this chemical class, chiral resolution of the racemate revealed that R enantiomer was significantly more potent than the S. Cell-based lead optimization of the spiropyrazolopyridones focusing on improving the physicochemical properties is described. As a result, an optimal compound 14a, with balanced in vitro potency and pharmacokinetic profile, achieved about 1.9 log viremia reduction at 3 × 50 mg/kg (bid) or 3 × 100 mg/kg (QD) oral doses in the dengue in vivo mouse efficacy model.

Full text of DOI:10.1021/ml500521r

Letter
pubs.acs.org/acsmedchemlett
Lead Optimization of Spiropyrazolopyridones: A New and Potent
Class of Dengue Virus Inhibitors
Bin Zou,*,† Wai Ling Chan,† Mei Ding,† Seh Yong Leong,† Shahul Nilar,† Peck Gee Seah,† Wei Liu,†
Ratna Karuna,† Francesca Blasco,† Andy Yip,† Alex Chao,† Agatha Susila,† Hongping Dong,†
Qing Yin Wang,† Hao Ying Xu,† Katherine Chan,† Kah Fei Wan,† Feng Gu,† Thierry T. Diagana,†
Trixie Wagner,‡ Ina Dix,‡ Pei-Yong Shi,† and Paul W. Smith†
†Novartis Institute for Tropical Diseases, 10 Biopolis Road #05-01 Chromos, Singapore 138670, Singapore
‡Novartis Institute for Biomedical Research, Basel CH-4056, Switzerland
S
* Supporting Information
ABSTRACT: Spiropyrazolopyridone 1 was identified, as a
novel dengue virus (DENV) inhibitor, from a DENV serotype
2 (DENV-2) high-throughput phenotypic screen. As a general
trend within this chemical class, chiral resolution of the
racemate revealed that R enantiomer was significantly more
potent than the S. Cell-based lead optimization of the
spiropyrazolopyridones focusing on improving the physico-
chemical properties is described. As a result, an optimal
compound 14a, with balanced in vitro potency and
pharmacokinetic profile, achieved about 1.9 log viremia
reduction at 3 × 50 mg/kg (bid) or 3 × 100 mg/kg (QD) oral doses in the dengue in vivo mouse efficacy model.
KEYWORDS: Dengue virus (DENV), spiropyrazolopyridones, structure−activity relationship, lead optimization
engue fever is a febrile disease caused by dengue virus
D(DENV), which is transmitted by Aedes aegypti, a
mosquito that feeds on humans.1 DENV threatens up to 2.5
billion people in more than 100 endemic countries. According
to a World Health Organization (WHO) report,2 there are 50−
100 million infections annually (and yet this number could be
far underestimated),3 with approximately 500,000 cases of
dengue hemorrhagic fever and 22,000 deaths globally.4,5
Despite the clear unmet medical need, currently there is no
Figure 1. DENV-2 in vitro potency of spiropyrazolopyridone 1 and its
enantiomers.
clinically approved vaccine or antiviral therapy available for
treatment of dengue.6−10 Therefore, it is urgent to develop safe
and effective therapeutics.
High-throughput phenotypic screening is a powerful tool to
identify compounds that are active against DENV by inhibiting
either host pathways and/or viral proteins, which are essential
for viral replication.11−16 As a result of DENV-2 screening on
Novartis compound library, a new chemical class spiropyr-
azolopyridone 1 was identified to be an interesting starting
point for further optimization (Figure 1). As compound 1 was a
racemate, two enantiomers were subsequently separated by
chiral high-performance purification chromatography (HPLC).
The structure of R enantiomer 1a was unambiguously
determined by X-ray crystallography (Figure 2). Interestingly,
when tested in the DENV-2 in vitro assay, these two individual
enantiomers displayed remarkably different potency where R
enantiomer 1a was greater than 200-fold more potent than the
S enantiomer 1b (Figure 1). Very recently, genetic analysis
demonstrated that mutations in dengue viral NS4B conferred
the resistance of compound 1a, suggesting that this class of
compounds inhibited DENV replication by targeting NS4B
protein.11,17−19 In this report, lead optimization efforts to
explore the structure−activity relationships (SARs) and
improve the physicochemical properties are described.
Following the identification of compound 1, which was
subsequently found to be poorly soluble in aqueous media, we
initiated a lead optimization effort in an attempt to improve the
solubility while maintaining the potency. The general synthetic
route to the spiropyrazolopyridone derivatives is outlined in
Scheme 1. The synthesis, featuring a three component
condensation of aminopyrazoles, isatin derivatives, and Mel-
Received: December 16, 2014
Accepted: February 2, 2015
© XXXX American Chemical Society
A
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Letter
Table 1. Structure−Activity Relationship and Cytotoxicity
Figure 2. X-ray structure of the R enantiomer 1a.
a
Scheme 1. Synthetic Route to the Spiropyrazolopyridones
a
Reagents and conditions: (a) N2H4·H2O, EtOH, room temperature
(rt); (b) i. di-tert-butylcarbazate, PPh3, THF, 0 °C; ii. HCl in dioxane,
rt; (c) (E)-ethyl 2-cyano-3-ethoxyacrylate 5, EtOH, 80 °C; (d) CH3I,
NaH, DMF, 0 °C to rt; (e) i. NaOH, EtOH/H2O, reflux; ii. AcOH,
reflux; (f) BH3·Me2S, THF, rt.
drum’s acid, provides the access to a series of spiropyrazolopyr-
idone analogues for SAR investigation.20,21 Since isatin
derivatives (9) and Meldrum’s acid (10) are largely
commercially available, our main effort to diversify the class
was to make aminopyrazole derivatives (6 and 7).
Briefly, substituted hydrazine 4 was made either by
substitution of chloride 2 with hydrazine or Mitsunobu reaction
of the alcohol 3 with di-tert-butylcarbazate followed by
deprotection in the presence of acid. Then, condensation of
hydrazine 4 with (E)-ethyl 2-cyano-3-ethoxyacrylate 5 in
ethanol afforded aminocarboxylate 6,22 which could be
methylated to deliver compound 7. Lastly, hydrolysis of the
esters 6 or 7 in the presence of NaOH gave the corresponding
sodium salts, which were directly used for the three component
condensation with substituted isatins 8 and Meldrum’s acid 9 in
acetic acid at reflux to afford the spiropyrazolopyridones 1, 10−
19, and 22−26. Reduction of the compound 1 in the presence
of borane−dimethyl sulfide gave a mixture of compounds 20
and 21, which were separated by chromatography. Finally,
those enantiomers mentioned in this report were obtained by
chiral HPLC separation of their corresponding racemates.
The structure−activity relationship (SAR) of this spiropyr-
azolopyridone class is summarized in Table 1. For all the chiral
separated pairs (compounds 13a/b, 14a/b, and 23a/b),
B
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Table 1. continued
Figure 3. DENV-2 in vivo mouse efficacy of compound 14a.
So we believe that this phenomenon is a general trend within
this class of compounds.23 As we explored the SAR around the
R1 substitution, we found the para-chlorobenzyl was preferred,
while meta- or ortho-chlorobenzyl (10 and 11) resulted in a loss
of activity on DENV-2. In addition, for the para-benzyl
substitutions, electron withdrawing group (13) was superior to
electron donating group (12). Interestingly, 3-pyridyl (14) was
tolerated, and the R enantiomer 14a maintained a similar level
of potency in the secondary viral-titer reduction assay7 (EC50
=
42 nM) with lower lipophilicity (measured LogP = 2.8).
However, the other R1 substitutions, such as 2-pyridyl (15),
3,5-pyrimidine (16), cyclohexyl (17), phenyl (18), or
homobenzyl (19), resulted in significant loss of potency,
suggesting a narrow range of substitutions are tolerated at the
R1-position. Next, we started to investigate the SAR around the
core modification. Both of the compounds 20 and 21 resulted
in a loss of potency, indicating both of the amide carbonyl
groups are important for the activity. Furthermore, compounds
22 and 23 with methyl on either of the amides maintained the
same level of potency, indicating that neither of the NH is
critical for the potency. In fact, R enantiomer 23a was the most
potent compound identified so far; however, it suffered with
high LogP and poor metabolic stability (Table 2). Finally, SAR
around the R4 substitutions was investigated. Though bromo
(24) was well tolerated, both electron-withdrawing (25) and
electron-donating groups (26) diminished the potency,
suggesting again a tight range of tolerated substitutions at
this position.
The key physicochemical properties of the representative
spiropyrazolopyridones are summarized in Table 2. Compound
13a displayed similar profile to compound 1a with marginal
improvement of solubility, while compound 23a suffered from
poor metabolic stability with high mouse hepatic extraction
ratio and short half-life. Gratifying, compound 14a with lower
LogP showed improved solubility, though the microsomal
stability was slightly compromised. In order to determine if the
a
EC50 values are determined in the DENV-2 CFI assay (see
b
Supporting Information). Determined in the DENV-2 viral-titer
reduction assay.7
Table 2. Summary of the Key in Vitro Properties
mouse microsomal stability
a
cmpd
solubility (μM, pH 6.8)
LogP
ER (%)
T1/2 (min)
1a
11
4.9
4.1
4.6
2.8
66
49
91
71
31
62
5.8
24
13a
23a
14a
27
161
504
a
Hepatic extraction ratio.
similarly to what has been described for 1a and 1b, R
enantiomers were markedly more potent than S enantiomers.
a
Table 3. Pharmacokinetic Parameters Following 25 mg/kg Oral and 5 mg/kg Intravenous Dosing in Rats
b
c
oral PK parameters
intravenous PK parameters
cmpd
Cmax (μg/mL)
Tmax (h)
AUC (h·μg/mL)
F (%)
Vss (L/kg)
CL (mL/min/kg)
T1/2 (h)
1a
0.78
3.86
2.0
9.5
33
63
4.3
3.0
15.1
18.8
3.8
1.3
14a
0.17
13.8
a
Cmax, maximum concentration of drug in plasma; Tmax, time to maximum concentration of drug in plasma; AUC, area under the curve (t = 0 to 24
b
h); Vss, volume of distribution at steady state; CL, plasma clearance; T1/2, terminal half-life; F, absolute oral bioavailability. Formulation for oral
dose: suspension of 0.5% Tween80 and 0.5% methylcellulose in phosphate buffer, pH 6.8. Formulation for intravenous dose: 10% NMP and 90%
c
rat plasma.
C
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(4) Guzman, M. G.; Kouri, G. Dengue: an update. Lancet Infect. Dis.
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improved solubility would translate to a better in vivo PK
profile, compounds 1a and 14a were evaluated in rats by oral
(p.o.) and intravenous (i.v.) routes at 25 and 5 mg/kg,
respectively (Table 3). Indeed, compared to compound 1a,
optimized compound 14a showed higher Cmax and exposure
(AUC), as well as increased oral bioavailability (63%).
Next, in order to determine the in vivo efficacy of this class of
compounds, compound 14a was tested in a DENV-2 viremia
mouse model (Figure 3).24 When compound 14a was
administrated orally at 50 mg/kg twice daily (bid) for 3 days,
compared to vehicle control group, significant viremia
reduction (about 1.9 log, P value <0.05) was achieved. Similar
efficacy was observed when compound 14a was orally dosed at
100 mg/kg once daily (QD) for 3 days. These results were
similar if not better than the positive control group, which was
treated with nucleoside analogue NITD008 (25 mg/kg bid for
3 days).25 In summary, this class of compounds clearly
demonstrated a good in vivo mouse efficacy and proved that
DENV NS4B protein is a druggable target for dengue drug
discovery.
In conclusion, a novel spiropyrazolopyridone scaffold was
identified from dengue phenotypic screening. Chiral HPLC
separation of the racemates revealed that R enantiomers were
significantly more potent than S enantiomers. After exploring
the SARs, we identified compound 14a with optimal in vitro
potency and physicochemical properties, which translated into
good in vivo PK and efficacy. However, the main drawback for
this class of compounds is the relatively weak potency in
DENV-1 and -4, though the potency of DENV-3 tracks well
with the DENV-2 activity.17 Further optimization to acquire
potency of all four serotypes will be reported in due course.
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ASSOCIATED CONTENT
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■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: +65-67222921. Fax: +65-67222918. E-mail: bin.zou@
novartis.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank all the other NITD colleagues for their
support during the course of this study. We thank Ms. Joefina
Lim for high-resolution mass spectrometry (HRMS) measure-
ment.
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