Synthesis of a Potent Pan-Serotype Dengue Virus Inhibitor Having a Tetrahydrothienopyridine Core

Article abstract of DOI:10.1055/a-1323-4036

A synthesis of the first-in-class pan-serotype dengue virus inhibitor NITD-688 is presented. The Gewald reaction of N-(tert-butoxycarbonyl)-6,6-dimethylpiperidin-3-one with malononitrile and sulfur in the presence of l-proline as a catalyst gave tert-buty

Full text of DOI:10.1055/a-1323-4036

SYNLETT
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Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart
2020, 31, A–D
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Design and Chemical Synthesis of Antivirals
K. Hung et al.
Cluster
Synlett
Synthesis of a Potent Pan-Serotype Dengue Virus Inhibitor Having
a Tetrahydrothienopyridine Core
Kevin Hung*a
Yugang Liub
Oliver Simonc,1
Lei Zhanga
Peichao Lua
Bryan K. S. Yeungc
Christopher Sarkoa
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Fumiaki Yokokawa*a,c
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a Novartis Institute for Tropical Diseases, 5959 Horton
St., Emeryville, CA, 94608, USA
b Novartis Pharmaceuticals Corporation, One Health Plaza,
East Hanover, NJ, 07936, USA
c Novartis Institutes for Tropical Diseases 10 Biopolis Road,
no. 05-01, Chromos, Singapore 138670, Singapore
fumiaki.yokokawa@novartis.com
Published as part of the Cluster
Design and Chemical Synthesis of Antivirals
Corresponding Author
Received: 26.10.2020
Accepted after revision: 26.11.2020
Published online: 26.11.2020
types. However, use of this vaccine in people with no prior
infection causes a higher risk of more-severe dengue and
hospitalization, and consequently it has been approved for
use only in persons aged 9 to 45 years who have had at least
one previous documented dengue virus infection.4 To date,
no antiviral agents are available for the treatment of dengue
disease and, consequently, there is an urgent need to devel-
op an effective and safe pan-serotype dengue virus inhibi-
tor. The Novartis Institute for Tropical Diseases (NITD), in
collaboration with academia and other companies, has
been attempting to develop such an antiviral for more than
15 years.5 As one of several screening campaigns aimed at
identifying chemical starting points for medicinal-chemis-
DOI: 10.1055/a-1323-4036; Art ID: st-2020-b0566-c
Abstract A synthesis of the first-in-class pan-serotype dengue virus in-
hibitor NITD-688 is presented. The Gewald reaction of N-(tert-butoxy-
carbonyl)-6,6-dimethylpiperidin-3-one with malononitrile and sulfur in
the presence of L-proline as a catalyst gave tert-butyl 2-amino-3-cyano-
6,6-dimethyl-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-carboxylate. This
was coupled with [4-(aminosulfonyl)phenyl]acetic acid by using
propanephosphonic acid anhydride. A subsequent reductive alkylation
with cyclohexanecarboxaldehyde gave NITD-688. Preliminary results of
our attempts to control the regioselectivity of the Gewald synthesis of
the 2-amino-3-cyanothiophene core are also presented.
try optimization,6,7 we conducted
a cell-based high-
Key words medicinal chemistry, Gewald reaction, tetrahydrothieno-
pyridines, regioselectivity, dengue virus inhibitors
throughput screening (HTS) on the Novartis corporate com-
pound collection by using dengue-infected cell lines. Subse-
quent careful characterization of screening hits led to the
identification of the tetrahydrobenzothiophene 1 (Figure 1)
as a validated hit with submicromolar potency against all
four dengue serotypes. Following extensive structure–
activity relationship (SAR) studies, an optimization of physi-
cochemical properties resulted in the identification of the
N-substituted tetrahydrothienopyridine derivative NITD-
688 (2), which showed a strong potency against all four se-
rotypes (EC50 = 0.008–0.038 M) and showed excellent oral
efficacy in the DENV-2 infected AG129 mouse model, with a
1.31 log viremia reduction at a daily oral dose of 30 mg/kg
for three days. Selection of resistance mutations and bind-
ing studies with recombinant protein using NMR analysis
indicate that the molecular target of NITD-688 (2) is dengue
NS4B protein, which is a nonenzymatic transmembrane
protein and a component of the viral replication complex.
Dengue is a mosquito-borne viral disease that is wide-
spread in tropical and subtropical countries. Climate
change in nonendemic regions is increasing the incidence
of dengue transmission, and more than half the world’s
population is currently exposed to the risk of dengue infec-
tion.2 The dengue virus is a single-stranded RNA flavivirus
that can be divided into four distinct but closely related se-
rotypes, all of which can cause the disease. Infection with
the any of four serotypes is believed to induce protective
immunity against that particular serotype; cross-immunity
to the other serotypes is only partial and temporary. Sec-
ondary infection with a different serotype is known to in-
crease the risk of developing severe dengue disease, which
is a major cause of hospitalization and death.3 One dengue
vaccine, Dengvaxia, has been licensed and indicated for the
prevention of dengue disease caused by all four viral sero-
© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–D
Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany

B
K. Hung et al.
Cluster
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NITD-688 (2) is currently under preclinical development as
a first-in-class pan-serotype dengue virus inhibitor.7,8 Here,
we present our medicinal-chemistry synthesis route to
NITD-688 (2), together with preliminary results of our at-
tempts to control the regioselectivity of the synthesis of the
2-amino-3-cyanothiophene core of 2.
Alkylation of glycine ethyl ester (5) with the bromide 4,12
gave amine 6 in 60% yield. Subsequent benzylation with
benzyl bromide gave the N-benzylglycine derivative 7 in
41% yield. To form the piperidine core, we conducted an in-
tramolecular Claisen cyclization of 7 with potassium tert-
butoxide to afford the piperidine-3,5-dione 8 in 58% yield.13
Treatment of this intermediate with trimethyl orthofor-
mate under acidic conditions gave a 78:22 mixture of the
regioisomeric methyl enol ethers 9 and 9′ in 84% combined
yield. Subsequent 1,2-reduction of the ketone moiety in 9
and 9′ with lithium aluminum hydride, followed by acidic
hydrolysis of the methyl enol ether group, gave enones 10
and 10′ as an 83:17 regioisomeric mixture in 55% yield.12
This regioisomeric mixture was further purified by recrys-
tallization from 5% ethyl acetate in hexane to give the single
isomer 10. Hydrogenolysis of the N-benzyl protecting
group in 10, with reduction of the enone moiety, followed
by concomitant protection of the secondary amine gave the
pure ketone 11 in 71% yield. With ketone 11, the stage was
set for the formation of the 2-amino-3-cyanothiophene
core through a Gewald pathway. This was accomplished by
treating ketone 11 with malononitrile and elemental sulfur
with L-proline as a catalyst at 60 °C.9 The desired regioiso-
mer 12 was separated at this stage by column chromatogra-
phy to give an isolated yield of 24%. Moving forward, simple
T3P-mediated coupling of 12 with acid 13 furnished amide
14 in 42% yield. In the final stretch of the synthesis, the sec-
ondary amine in 15 was obtained by removal of the tert-
butoxycarbonyl (Boc) protecting group of 14 in quantitative
yield through the action of trifluoroacetic acid. Finally, a re-
ductive amination reaction with picoline–borane complex
was used to couple aldehyde 16 and amine 15 to furnish
NITD-688 (2) in 53% yield.13 Overall, NITD-688 (2) was as-
sembled from simple industrial feedstocks in 11 linear
steps with a serviceable yield.
O
O
S
Me
DENV-1 EC50 0.89 μM
DENV-2 EC50 0.21 μM
DENV-3 EC50 0.18 μM
DENV-4 EC50 0.09 μM
CN
NH
O
S
Me
HTS hit (1)
medicinal
chemistry
optimization
O
O
S
NH2
CN
DENV-1 EC50 0.038 μM
DENV-2 EC50 0.008 μM
DENV-3 EC50 0.008 μM
DENV-4 EC50 0.012 μM
N
NH
O
Me
Me
S
>1 log viremia reduction
at 3 x 30 mg/kg (QD) oral
doses in the dengue mouse
model.
NITD-688 (2)
under preclinical development
Figure 1 Chemical structures of the dengue cell-based HTS hit (1) and
the advanced lead compound, NITD-688 (2), and their antidengue ac-
tivities
In our medicinal-chemistry optimization of the screen-
ing hit compound 1, the 2-amino-3-cyanothiophene cores
were synthesized by a Gewald reaction of malononitrile, the
appropriate ketone, and sulfur with L-proline as a catalyst
(Scheme 1).9 The resulting 2-amino-3-cyanothiophenes
were coupled with various carboxylic acids by using propa-
nephosphonic acid anhydride (T3P)10 to provide rapid ac-
cess to a collection of 3-cyanothiophene analogues for SAR
studies of antidengue activity.
Our early to mid-stage medicinal-chemistry investiga-
tions were adequately supported by the successful imple-
mentation of the aforementioned synthetic route to NITD-
688 (2). However, as the program matured, the need for
greater quantities of NITD-688 (2) of higher quality and
with improved synthetic efficiency became increasingly ap-
parent. Hence, the synthesis was scrutinized for areas that
could be further optimized. Specifically, the formation of
the desired methyl enol ether 9 from the 1,3-diketone 8
CN
O
CN
CN
L
-proline
+
S8
+
NH2
DMF
60 °C
S
CN
R-COOH
T3P
i-Pr2NEt
NH
R
S
DMF
O
Scheme 1 General synthetic route to 3-cyanothiophene-2-amide ana-
showed
a low regioselectivity. Furthermore, material
logue dengue inhibitors
throughput of the 2-amino-3-cyanothiophene core 12 from
the ketone 11 through the Gewald pathway was subopti-
mal, and screening efforts were unable to improve on the
low <30% isolated yield, due to poor regioselectivity. The
lack of robust regiocontrol in the coupling was also of con-
cern from a cost-of-goods standpoint, because separation
of the isomers required extensive column chromatography.
Hence, a proof-of-concept campaign was launched in an at-
tempt to address the regioselectivity of the critical thio-
phene-formation step. A review of the literature revealed
Once NITD-688 (2) was identified as a candidate for pre-
clinical development, our original synthetic route11 was
modified to permit access to sufficient amounts of this ma-
terial to support formulation and in vivo safety studies
(Scheme 2). Large-scale synthesis of the corresponding ke-
tone intermediate for NITD-688 (2) commenced with bro-
mination of methyl isopropyl ketone (3) with bromine to
provide 3-bromo-3-methylbutan-2-one (4) in 50% yield. N-
© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–D

C
K. Hung et al.
Cluster
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K2CO3, KI
PhCH2Br, DMF
41%
Na2CO3, THF
Bn
O
Bn
5, 60 oC
Br2, AcOH
50%
t-BuOK, THF
O
O
O
N
COOEt
Me
N
Me
Br
H
58%
Me
Me
60%
Me
Me
Me
EtOOC
N
Me
Me
O
Me
Me
Me
Me Me
O
7
3
4
6
8
O
mixture of
regioisomers
(9/9' = 78:22)
CH(OMe)3
H2SO4, MeOH
84%
NH2
EtO
5
Bn
N
OMe
Bn
O
N
H2N
O
O
LAH, THF then
concd HCl, acetone
55%
Me
Me
CH2(CN)2,
L
-proline
Me
Me
Pd/C, Boc2O
S
CN
S8, DMF, 60 o
24%
C
H2, MeOH, 60 o
71%
C
Boc
O
Boc
N
O
10
N
+
NH2
Me
Me
9
Me
Me
O
Bn
mixture of
regioisomers
(10/10' = 83:17)
S
N
Bn
O
N
Me
Me
13
12
11
HO
Me
Me
O
OMe
9'
T3P, DMF
42%
10'
O
16
H2N
H2N
H2N
O
O
O
O
S
S
S
Et3N, picoline–BH3
16, AcOH, i-PrOH
53%
O
O
TFA, CH2Cl2
quant.
CN
CN
CN
Boc
Me
N
HN
N
NH
NH
O
NH
O
Me
Me
Me
S
S
S
Me
Me
O
14
15
NITD-688 (2)
Scheme 2 A medicinal-chemistry synthetic route to NITD-688 (2)
that introduction of an electrophilic leaving group in the
position  to the ketone was critical in controlling the
regioselectivity of the formation of the heterocycle.14 Thus,
enone 10 was envisioned as a suitable intermediate to
leverage regioselective halide installation through the
enone moiety (Scheme 3). Conjugate reduction of this scaf-
fold was unknown, to the best of our knowledge, and vari-
ous hydride sources gave clean and exclusive 1,2-reduc-
tions.15 However, Wilkinson’s catalyst and triethylsilane
smoothly afforded the silyl enol ether 17 in 72% isolated
yield under buffered purification conditions.16 Next, bromi-
nation and concomitant desilylation of 17 with pyridinium
tribromide gave the -oxo bromide 18.17 The unstable bro-
mide 18 was then immediately subjected to Gewald condi-
tions, followed by a silica-gel mediated cyclization of the
putative Knoevenagel intermediate, to give the cyanothio-
phene core 19 as a single regioisomer in 20% isolated yield.
Although there is room for improving the efficiency of the
thiophene-formation step by further optimization and
side-product profiling, we have nevertheless demonstrated
a regioselective synthesis of the cyanothiophene core of
NITD-688 (2) on a discovery scale by way of these currently
unoptimized reaction and purification conditions.
tial adaptations to large-scale synthetic access were validat-
ed by exploratory chemistry. The feasibility of the modified
route and its impact on the cost-of-goods is currently being
evaluated at a process scale. We are actively investigating
further ways to improve the synthesis, and the results will
be reported in subsequent publications.
Funding Information
We thank the Wellcome Trust Innovator Award (218216/Z/19/Z) for
£500,000 in support of this research.Welco
m
e
Trust(2
1
8
2
1
6
/Z/1
9
/Z)
Me Me
N
Me Me
N
RhCl(PPh3)3
TES-H
Bn
Bn
72%
O
OTES
10
17
pyr Br3
Me Me
NaSH, CH2(CN)2
CN
Bn
L
-proline then
Bn
N
SiO2
N
NH2
Me
Me
Br
20%
over 2 steps
S
In conclusion, we have disclosed the first-in-class pan-
serotype dengue virus inhibitor NITD-688 (2). A medicinal
chemistry route to 2, which was used to support preclinical
development, has been described.18,19 Furthermore, poten-
O
18
19
Scheme 3 Exploratory chemistry to develop a regiocontrolled synthe-
sis of the 2-amino-3-cyanothiophene core of NITD-688 (2)
© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–D

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K. Hung et al.
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Acknowledgment
(16) Ojima, I.; Nihonyanagi, M.; Kogure, T.; Kumagai, M.; Horiuchi,
S.; Nakatsugawa, K.; Nagai, Y. J. Organomet. Chem. 1975, 94, 449.
(17) N-Bromosuccinimide was found to be deleterious to the reac-
tion yield.
(18) tert-Butyl 2-Amino-3-cyano-6,6-dimethyl-6,7-dihydrothieno-
[3,2-c]pyridine-5(4H)-carboxylate (12)
We thank Feng Zhao and his colleagues at WuXi AppTec, and Kondaji
Gajulapati and his colleagues at GVK Biosciences for their technical
assistance. We thank Shengtian Yang for his NMR analysis and
Weiping Jia for his high-resolution mass analysis. We thank NITD Alli-
ance Management and Partnering, Legal, and Finance Team (Thomas
Krucker, Mark Hopkins, Marcus Hall, and Jean Claude Poilevey) for
their legal and financial support.
L-Proline (12.66 g, 110 mmol, 1.0 equiv) and sulfur (5.27 g, 165
mmol, 1.5 equiv) were added sequentially to a solution of 11
(25 g, 110 mmol) in DMF (100 mL) at rt, and the mixture was
stirred at rt for 10–15 min. Malononitrile (8 g, 121 mmol, 1.1
equiv) was added, and mixture was stirred at 55–60 °C for 10–
12 h. When the reaction was complete, the mixture was washed
with hexane (3 × 50 mL). The bottom DMF layer was slowly
added to cold H2O (250 mL), and the precipitated solids were
collected by filtration, washed with H2O (50 mL), and dried. The
wet product was purified by column chromatography (silica gel,
CH2Cl2–MeOH) to give 12 as a pale brown color solid; yield: 8 g
(26 mmol, 24%) (96.1% pure; 96.22:0.75 isomeric mixture based
on HPLC).
Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/a-1323-4036.
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References and Notes
(1) Current address: Novartis (Singapore) Pte Ltd, 20 Pasir Panjang
Road #10-25/28 Mapletree Business City, Singapore 117439.
(2) (a) Wilder-Smith, A.; Ooi, E.-E.; Horstick, O.; Wills, B. Lancet
2019, 393, 350. (b) Dengue and Severe Dengue; World Health
Organization: Geneva, 2020, https://www.who.int/news-
room/fact-sheets/detail/dengue-and-severe-dengue (accessed
Dec 15, 2020).
(3) Halstead, S. B. Hum. Vaccines Immunother. 2018, 14, 2158.
(4) World Health Organization: Vaccine; 2017, 35 1200.
(5) Lim, S. P.; Wang, Q.-Y.; Noble, C. G.; Chen, Y.-L.; Dong, H.; Zou,
B.; Yokokawa, F.; Nilar, S.; Smith, P.; Beer, D.; Lescar, J.; Shi, P.-Y.
Antiviral Res. 2013, 100, 500.
1H NMR (400 MHz, DMSO-d6):  = 17.13 (br s, 2 H), 4.18 (s, 2 H),
3.31 (s, 2 H), 2.54 (s, 2 H), 1.40 (s, 9 H), 1.38 (s, 6 H). 13C NMR
(100 MHz, DMSO-d6):  = 1163.71, 154.76, 128.71, 115.51,
114.31, 80.68, 79.24, 55.22, 42.66, 37.85, 28.06, 27.40. HRMS
(ESI): m/z [M – H]– calcd for C15H20N3O2S: 306.1271; found:
306.1281.
(19) 2-Amino-5-benzyl-6,6-dimethyl-4,5,6,7-tetrahydro-
thieno[3,2-c]pyridine-3-carbonitrile (19): Regiocontrolled
Synthesis
Pyridinium tribromide (382 mg, 1.194 mmol, 1.2 equiv) was
added to a 0.1 M solution of silyl enol ether 17 (330 mg, 0.995
mmol, 1.0 equiv) in CH2Cl2 (10 mL) at 23 °C, and the mixture
was stirred for 15 min. The reaction was then quenched with
sat. aq Na2S2O3 and extracted with CH2Cl2 (×3). The combined
organic layers were washed with brine, dried (Na2SO4), and con-
centrated in vacuo to provide bromide 18, which was used
immediately in the next step without further purification.
Anhyd DMF (3.32 mL, 0.1 M) was added to the crude bromide
18 (1.0 equiv), NaSH (27.9 mg, 0.498 mmol, 1.5 equiv), malono-
nitrile (24.1 mg, 0.365 mmol, 1.1 equiv), and L-proline (38.2 mg,
0.332 mmol, 1.0 equiv) under an inert atmosphere at 23 °C, and
the mixture was stirred at 23 °C for 2 h. When the reaction was
complete it was quenched with sat. aq Na2S2O3 and sat. aq
NaHCO3, and the mixture was extracted with EtOAc (×3). The
combined organic layers were washed sequentially with H2O
and brine, dried (Na2SO4), and concentrated in vacuo. The resid-
ual crude mixture was redissolved in CH2Cl2 at 23 °C, silica gel
(3 g) was added, and the mixture was stirred for 4 h. The solvent
was then removed in vacuo and the crude residue was purified
by flash column chromatography on an ISCO machine [silica gel,
heptane–EtOAc (0–50%)] to give the cyano derivative 19 as a
yellow solid; yield: 21 mg (20%, 0.071).
(6) Lim, S. P. Antiviral Res. 2019, 163, 156.
(7) Yokokawa, F. RSC Med. Chem. 2020, 11, 541.
(8) Moquin, S. A.; Simon, O.; Karuna, R.; Lakshminarayana, S. B.;
Yokokawa, F.; Wang, F.; Saravanan, C.; Zhang, J.; Day, C. W.;
Chan, K.; Wang, Q.-Y.; Lu, S.; Dong, H.; Wan, K. F.; Lim, S. P.; Liu,
W.; Seh, C. C.; Chen, Y.-L.; Xu, H.; Barkan, D. T.; Kounde, C. S.;
Sim, W. L. S.; Wang, G.; Yeo, H.-Q.; Zou, B.; Chan, W. L.; Ding, M.;
Song, J.-G.; Li, M.; Osborne, C.; Blasco, F.; Sarko, C.; Beer, D.;
Bonamy, G. M. C.; Sasseville, V. G.; Shi, P.-Y.; Diagana, T. T.;
Yeung, B. K. S.; Gu, F. Sci. Transl. Med. 2020, in press.
(9) Wang, T.; Huang, X.-G.; Liu, J.; Li, B.; Wu, J.-J.; Chen, K.-X.; Zhu,
W.-L.; Xu, X.-Y.; Zeng, B.-B. Synlett 2010, 1351.
(10) Waghmare, A. A.; Hindupur, R. M.; Pati, H. N. Rev. J. Chem. 2014,
4, 53.
(11) Kounde, C.; Sim, W. L. S.; Simon, O.; Wang, G.; Yeo, H. Q.; Yeung,
B. K. S.; Yokokawa, F.; Zou, B. WO2019244047, 2019.
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Acta 1998, 81, 303.
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(14) (a) Buchstaller, H.-P.; Siebert, C. D.; Lyssy, R. H.; Frank, I.; Duran,
A.; Gottschlich, R.; Noe, C. R. Monatsh. Chem. 2001, 132, 279.
(b) Hawksley, D.; Griffin, D. A.; Leeper, F. J. J. Chem. Soc., Perkin
Trans. 1 2001, 144. (c) Madding, G. D.; Thompson, M. D. J. Het-
erocycl. Chem. 1987, 24, 581.
(15) Hydride sources such as L-Selectride, lithium aluminum
hydride, diisobutylaluminum hydride, or tetramethylammo-
nium borohydride gave 1,2-reduction exclusively.
1H NMR (500 MHz, CDCl3):  = 17.36–7.28 (m, 4 H), 7.30–7.22
(m, 1 H), 4.59 (s, 2 H), 3.66 (s, 2 H), 3.39 (t, J = 2.0 Hz, 2 H), 2.51
(d, J = 2.2 Hz, 2 H), 1.23 (s, 6 H). 13C NMR (126 MHz, CDCl3):  =
160.56, 139.92, 129.78, 128.52 (2 C), 128.36 (2 C), 126.91,
118.51, 100.45, 86.83, 53.94, 53.05, 46.93, 37.46, 24.21 (2 C).
HRMS (ESI): m/z [M + H]+ calcd for C17H20N3S: 298.1372; found:
298.1374.
© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–D