Discovery of 3,3′-Spiro[Azetidine]-2-oxo-indoline Derivatives as Fusion Inhibitors for Treatment of RSV Infection

Article abstract of DOI:10.1021/acsmedchemlett.7b00418

A new series of 3,3′-spirocyclic-2-oxo-indoline derivatives was synthesized and evaluated against respiratory syncytial virus (RSV) in a cell-based assay and animal model. Extensive structure-activity relationship study led to a lead compound 14h, which e

Full text of DOI:10.1021/acsmedchemlett.7b00418

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Letter
The Discovery of 3,3’-spiro[azetidine]-2-oxo-indoline
Derivatives as Fusion Inhibitors for Treatment of RSV Infection
Weihua Shi, Zhigan Jiang, Haiying He, Fubiao Xiao, Fusen Lin, Ya Sun,
Lijuan Hou, Liang Shen, Lixia Han, Minggao Zeng, Kunmin Lai, Zhengxian
Gu, Xinsheng Chen, Tao Zhao, Li Guo, Chun Yang, Jian Li, and Shuhui Chen
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00418 • Publication Date (Web): 10 Jan 2018
Downloaded from http://pubs.acs.org on January 11, 2018
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The Discovery of 3,3’-spiro[azetidine]-2-oxo-indoline Derivatives as
Fusion Inhibitors for Treatment of RSV Infection
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Weihua Shi1, Zhigan Jiang1, Haiying He1*, Fubiao Xiao1, Fusen Lin1, Ya Sun1, Lijuan Hou1, Liang
Shen1, Lixia Han1, Minggao Zeng1, Kunmin Lai1, Zhengxian Gu1, Xinsheng Chen1, Tao Zhao2, Li Guo2,
Chun Yang2, Jian Li1, Shuhui Chen1.
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1 WuXi AppTec (Shanghai) Co., Ltd, 288 FuTe Zhong Road, Shanghai 200131, P. R. China
2 Buchang Pharmaceutical, 50 Gaoxin Road, Gaoxin District, Xi'an 710075, Shanxi Province, P. R. China
KEYWORDS RSV, Fusion inhibitor, Benzimidazole
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ABSTRACT: A new series of 3,3’-spirocyclic-2-oxo-indoline derivatives were synthesized and evaluated against Respirato-
ry Syncytial Virus (RSV) in the cell-based assay and animal model. Extensive structure-activity relationship study led to a
lead compound 14h, which exhibited excellent in vitro potency with an EC50 value of 0.8 nM and demonstrated 71% oral
bioavailability in mice. In a mouse challenge model of RVS infection, 14h demonstrated superior efficacy with a 3.9log
RSV virus load reduction in the lung following an oral dose of 50 mg/kg.
Introduction
Figure 1. RSV inhibitors explored in early stage clini-
cal studies
Respiratory syncytial virus (RSV) is a major cause of
pneumonia and bronchiolitis in young children, immuno-
compromised adults and the elderly, resulting in ~125,000
hospitalizations annually in the United States1,2. However,
the treatment of RSV lower respiratory tract infection
(LRTI) is limited3. Synagis (Palivizumab), a marketed hu-
manized monoclonal antibody targeting fusion glycopro-
tein (F protein) of RSV, was licensed for passive prophy-
laxis intervention in high-risk infants. However, its high
cost of treatment and relatively low protection to high-
risk children limit its usage. An aerosol formulation of
Ribavirin (Virazole) is the only chemotherapeutic agent
on the market. Virazole is not a specific protection for
young children and is rarely used due to its variable effi-
cacy and toxicity.
Results and Discussion
In order to investigate a strategy of pyridinoimidazo-
lone replacements on BMS-433771, a small library contain-
ing nineteen scaffold hopping derivatives were designed
and synthesized (Scheme 1). The key intermediate 4 was
prepared from starting material 1 via displacement reac-
tion, hydrogenation reaction followed by cyclization reac-
tion with excellent overall yields (68.4%). The target
compounds 5a-s were obtained by alkylation reaction of
intermediate 4 in the presence of K2CO3 in CH3CN or
NaH in DMF.
Clearly, there is an unmet need for a new drug to pre-
vent and treat RSV infection. In the past two decades,
several chemical series of RSV fusion protein inhibitors
have been reported, including the most advanced com-
pound GS-5806 which is currently in Phase 2 study4,5. An-
other Phase 2 compound is MDT-637 (formerly VP-14637)
and is being developed as a dry powder inhaled product
due to its low oral bioavailability. Both BMS-4337716 and
TMC-3531217 were progressed into late preclinical / early
clinical development but discontinued, mainly due to the
unfavorable pharmaceutical properties or early safety
findings. The two RSV F protein inhibitors contain the
same benzimidazole heterocycle. Therefore, we intend to
address the drug-like properties of benzimidazole RSV
fusion inhibitors especially improving activity in vitro,
pharmacokinetics, and pharmacodynamics. Herein we
describe the identification of a promising candidate iso-
Scheme 1. General synthesis of pyridinoimidazolone
replacements analogs
propyl
1'-((5-bromo-1-(4-hydroxybutyl)-1H-
benzo[d]imidazol-2-yl)methyl)-2'-oxospiro[azetidine-3,3'-
indoline]-1-carboxylate (14h) that was effective in a mouse
challenge model of RSV infection. Meanwhile, the exten-
sive structure-activity relationship (SAR) of this 3,3’-
spirocyclic-2-oxo-indoline derivatives was revealed herein.
Cyclopropyl indolin-2-one 5a was first recorded in ReViral
ltd’s patent for RSV treatment, but no antiviral activity
was reported8. Hoffmann-La Roche discolsed spiro azain-
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dolin-2-one analogs, however only spirocylicpropanes
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on 5-cyclohexyl substituent (6c) possibly due to increased
steric bulk, electronic withdrawing groups 5-CN (6e) and
5-CF3 (6f) attenuated the activities by more than 10-fold.
Single-digital nanomolar activity was obtained when
bromine was introduced and 6h demonstrated 7-fold po-
tency improvement in comparison to 5a. Given that the
introduction of 5-bromo had a significant impact on anti-
were introduced9-11. In order to perform pyridinoimidazo-
lone replacement strategy, 5a-c were synthesized and
evaluated by the CPE assay. The antiviral activities were
listed in Table 1. The importance of carbonyl group for
the activities against RSV was demonstrated when its re-
placement with a hydrogen (5e and 5f, with EC50 of 0.20
and 0.18 μM, respectively) resulted in a moderate activity
in comparison to BMS-433771. The presence of small ali-
phatic substituent at ortho-position of carbonyl was also
found to be desirable (5g-j). Attempt to replace the fused
phenyl ring with saturated ring resulted in a 100-fold less
active compound 5k with an EC50 of 12 μM versus 5f. Oth-
er pyridinoimidazolone replacements (5m-s) showed
negative impact on activities. In summary, though the
SAR for pyridinoimidazolone replacement strategy is kind
of rigid, the ring system 3,3’-spiro[cyclopropane]-2-oxo-
indoline (5a-c) was identified to gain a superior level of
activity.
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RSV
activity,
we
decided
to
pursue
5-
bromobenzimidazole moiety for further SAR optimiza-
tions.
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Table 2 Antiviral activity of compound 6a-i
Table 1 Antiviral activity of a small library of pyridi-
noimidazolone replacements
aRSV CPE assay in Hep-2 cells, data was generated from
two or more determinations. CC50 was performed on the
b
same cells and method.
Furthermore, 3,3’-spirocylic-2-oxo-indolines are found
in compounds that exert a range of pharmacological ac-
tivities. Analog 3,3’-spiro[succinimide]-2-oxo-indoline
antagonized chemoattractant receptor-homologous ex-
pressed on Th2 lymphocytes (CRTH2 or DP2)14, and 3,3’-
spiro[piperidine]-2-oxo-indolines were in development
for cathepsin K inhibitors15. Therefore, the following study
focused on optimizing 3,3’-spirocylic-2-oxo-indoline moi-
ety of 6h.
Scheme 2. Synthetic route to produce key intermedi-
ate 12
aRSV CPE assay in Hep-2 cells, data was generated from
In order to explore more potent 3,3-spirocylic-2-oxo-
indoline derivatives, a small library of spiro hetero cyclic
analogs were successfully designed and synthesized. The
synthesis of intermediate 12, as shown in Scheme 2, was
generally commenced with commercially available 2-
bromo aniline, which was condensed with the acid 8 via
T3P to generate the amide 9, followed by alkylation with
PBMCl to yield intermediates 10. This intermediate was
then subjected to an intramolecular palladium-catalyzed
b
two or more determinations. CC50 was performed on the
same cells and method.
As a new chemical starting point, chloride (6a) and me-
thyl group (6b) were introduced at 5-position of the cor-
responding benzimidazole moiety following Feng’s work
on imidazolepyrimidines12,13. Unfortunately, both of these
two compounds did not boost the antiviral activities,
demonstrating 10 nM and 16 nM respectively versus 15 nM
of 5a. In the meantime, an entire attrition was observed
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α-arylation reaction16. After removal of PMB via in CAN
or TfOH acid conditions, intermediate 12 was generated
in moderate to good yield (30-75%). As shown in Table 3,
14a and 14b displayed good potency, with EC50 of 6 nM
and 8 nM. Sulfide 14c, sulfone 14d exhibited 5 nM and 21
nM antiviral activities, respectively. Encouraged by the
tolerances of hetero atoms, nitrogen was introduced. 5-
Membered analogs 14e, 14f and 14g attenuated 4-5 fold
anti-RSV activities compared with 6h. However, 4- mem-
bered azetidine spiro analogs, isopropyl carbamate 14h,
improved the antiviral activity into subnanomolar level of
0.8 nM, 3 fold more potent compared to that of 6h, and
isobutyl amide 14i and methanesulfonyl amide 14j exhib-
ited comparable activities of 3 nM. Other approaches of
aliphatic amine (14k-l) and steric bulky 6 membered spiro
rings (14m-p) attenuated the activities.
pound 14h showed lower clearance and volume of distri-
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bution (Vd), but higher AUC with similar half-life (T1/2).
However, compound 14h had much better oral profile
than that of BMS433771, as evidenced by oral exposure
(8076 nM*h versus 1170 nM*h) and absolute oral bioavail-
ability (71% versus 13%).
Table 4 Pharmacokinetic parameters after single in-
travenous and oral administration of 14h and
BMS433771 to female Balb/c mice.
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Table 3 Antiviral activity of 3,3-spirocyclic-2-oxo-
indoline derivatives 14a-p
(A) Data reported are mean values from the dosing co-
horts (Female Balb/c-Mouse, fasted, n = 3/dose). (B) Dos-
ages are 1 mg/kg i.v. (1 mg/mL in DMSO: PEG400: water =
5:40:55, clear solution) and 10 mg/kg p.o. (2 mg/mL in
DMSO: PEG400: water = 5:40:55, clear solution). (C) CL
stands for plasma clearance. T1/2 is the plasma half-life of
the compounds. Vd means volume of distribution. Cmax
represents the highest observed plasma concentration
and Tmax is time to reach Cmax. AUC is the area under the
plasma concentration-time curve. %F represents absolute
oral bioavailability.
Pharmacology Results and Discussion
The in vivo efficacy was evaluated in the Balb/c mouse
RSV infection model. As shown in table 5, compound 14h
showed a clear dose-dependent effect on virus load reduc-
tion from 5 mg/kg to 50 mg/kg. In addition, the 5 mg/kg
group of 14h reduced the lung tissue virus load Δlog10 to
1.6 which was superior to BMS-433771 1.2 Δlog10 at 50
mg/kg dose. Moreover, the 50 mg/kg group of 14h re-
duced virus load of all the experimental animals to below
the detection limit, which demonstrated that compound
14h exhibited excellent anti-RSV efficacy and significantly
superior to BMS433771 in this RSV mouse challenge mod-
el.
Table 5 In vivo antiviral activity in a mouse model of
RSV infection
aRSV CPE assay in Hep-2 cells, data was generated from
b
two or more determinations. CC50 was performed on the
same cells and method.
PK Results and Discussion
Plasma pharmacokinetics after 1 mg/kg intravenous
and 10 mg/kg oral administration of compound 14h and
BMS4337716 to female balb/c mice were characterized. PK
parameters were listed in table 4, indicating that com-
Conclusion
In summary, we have explored a series of 3,3’-
spiro[azetidine]-2-oxo-indoline derivatives proved to be
potent inhibitors of RSV in the CPE assay. The investiga-
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5806 activity in a respiratory syncytial virus challenge
study. New. Engl. J. Med. 2014, 371(8), 711-722.
tion led to the identification of 14h as a compound
demonstrating additional efficacy in the Balb/c mouse
model of RSV infection after oral dosing. Further safety
assessment will be conducted in due course.
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ASSOCIATED CONTENT
Supporting Information
Synthetic procedures, analytical data, assay protocol are
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AUTHOR INFORMATION
Corresponding Author
* E-mail: he_haiying@wuxiapptec.com
ABBREVIATIONS
8.
9.
PK, pharmacokinetics; PO, per oral; Cmax, peak concentra-
tion; Tmax, time to peak; CL, clearance; T1/2, half-life; Vd,
volume of distribution; AUC, area under curve; mpk,
mg/kg;; %F, oral bioavailability.
Feng, S; Gao, L; Guo, L; Huang, M; Liang, C; Wang, B;
Wang, L; Wu, G; Yun, H; Zhang, W; Zheng, X; Zhu, W.
Aza-oxo-indoles for the treatment and prophylaxis of res-
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prophylaxis of respiratory syncytial virus infection.
WO2015/22263
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