Design and synthesis of pyrazolopyridine derivatives as sphingosine 1-phosphate receptor 2 ligands

Article abstract of DOI:10.1016/j.bmcl.2017.12.010

Eleven new sphingosine 1-phosphate receptor 2 (S1PR2) ligands were synthesized by modifying lead compound N-(2,6-dichloropyridin-4-yl)-2-(4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1-carboxamide (JTE-013) and their binding affinities toward S1PRs were determined in vitro using [³²P]S1P and cell membranes expressing recombinant human S1PRs. Among these ligands, 35a (IC₅₀ = 29.1 ± 2.6 nM) and 35b (IC₅₀ = 56.5 ± 4.0 nM) exhibit binding potency toward S1PR2 comparable to JTE-013 (IC₅₀ = 58.4 ± 7.4 nM) with good selectivity for S1PR2 over the other S1PRs (IC₅₀ > 1000 nM). Further optimization of these analogues may identify additional and more potent and selective compounds targeting S1PR2.

Full text of DOI:10.1016/j.bmcl.2017.12.010

Accepted Manuscript
Design and synthesis of pyrazolopyridine derivatives as sphingosine 1-phos-
phate receptor 2 ligands
Zonghua Luo, Xuyi Yue, Hao Yang, Hui Liu, Robyn S. Klein, Zhude Tu
PII:
S0960-894X(17)31170-8
https://doi.org/10.1016/j.bmcl.2017.12.010
BMCL 25466
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 October 2017
4 December 2017
5 December 2017
Please cite this article as: Luo, Z., Yue, X., Yang, H., Liu, H., Klein, R.S., Tu, Z., Design and synthesis of
pyrazolopyridine derivatives as sphingosine 1-phosphate receptor 2 ligands, Bioorganic & Medicinal Chemistry
Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.010
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Graphical Abstract
Design and synthesis of pyrazolopyridine
derivatives as sphingosine 1-phosphate
receptor 2 ligands
Leave this area blank for abstract info.
Zonghua Luoa, Xuyi Yuea, Hao Yanga, Hui Liua, Robyn S. Kleinb, c, d, and Zhude Tua*
2

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Design and synthesis of pyrazolopyridine derivatives as sphingosine 1-phosphate
receptor 2 ligands
Zonghua Luoa, Xuyi Yuea, Hao Yanga, Hui Liua, Robyn S. Kleinb, c, d, and Zhude Tua*
aDepartment of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA
bDepartment of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
cDepartment of Neuroscience, Washington University School of Medicine, St. Louis, MO 63110, USA
dDepartment of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Eleven new sphingosine 1-phosphate receptor 2 (S1PR2) ligands were synthesized by modifying
lead compound N-(2,6-dichloropyridin-4-yl)-2-(4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-
b]pyridin-6-yl)hydrazine-1-carboxamide (JTE-013) and their binding affinities toward S1PRs
were determined in vitro using [32P]S1P and cell membranes expressing recombinant human
S1PRs. Among these ligands, 35a (IC50 = 29.1 2.6 nM) and 35b (IC50 = 56.5 4.0 nM) exhibit
Revised
Accepted
Available online
binding potency toward S1PR2 comparable to JTE-013 (IC50 = 58.4
7.4 nM) with good
selectivity for S1PR2 over the other S1PRs (IC50 > 1,000 nM). Further optimization of these
analogues may identify additional and more potent and selective compounds targeting S1PR2.
Keywords:
Sphingosine 1-phosphate receptor 2
Multiple sclerosis
Binding affinities
Selectivity
2009 Elsevier Ltd. All rights reserved.
Sphingosine
1-phosphate
(S1P)
is
a
bioactive
with MS patients.11 Robyn Klein’s group recently demonstrated
that S1PR2 plays an important role in regulating blood–brain
barrier (BBB) function during inflammatory demyelination. An
increased expression of S1PR2 is observed in disease-susceptible
regions of both female SJL mice with experimental autoimmune
encephalomyelitis (EAE) and female patients with MS compared
with S1PR2 expression seen in their male counterparts.12
Although therapeutics have focused on S1PR1 agonists, S1PR2
could be a potential biomarker for the prognosis and treatment of
MS.
lysophospholipid that transmits signals through the family of G-
protein coupled receptors S1PR1, 2, 3, 4, and 5, which were
originally known as EDG-1, 3, 5, 6, and 8. Upon binding S1P,
these five sphingosine 1-phosphate receptors (S1PRs) regulate
diverse biological functions, from proliferation and survival to
migration and secretion, across many cell types.1-3 S1PRs are
expressed in a wide variety of tissues, with each subtype
exhibiting a different cell specificity. S1PR1, 2, and 3 are
expressed ubiquitously, whereas S1PR4 is confined to lymphoid
and hematopoietic tissues, and S1PR5 is primarily located in the
white matter of the central nervous system (CNS) and spleen.4, 5
Multiple sclerosis (MS) is an inflammatory disorder of the
brain and spinal cord in which focal lymphocytic infiltration
leads to damage of myelin and axons.6 According to World
Health Organization reports, the estimated number of people with
MS increased from 2.1 million in 2008 to 2.3 million in 2013.
MS significantly decreases life expectancy of patients by an
average of 5–10 years.7 The mechanisms that cause progression
in MS are not well understood, thus major treatment strategies
are aimed at improving neuronal function and limiting
progression of disease after initial diagnosis.8 The sphingolipid-
like immunomodulator fingolimod (FTY720) was the first oral
therapeutic agent for relapsing-remitting MS to be approved by
the US Food and Drug Administration (FDA).9 [γ-35S]GTPγS
binding assay showed that FTY720 is a potent agonist at four
S1PRs (S1PR1, 3, 4, and 5), but inactive at S1PR2.10 Several
other S1PR1 agonists have also performed well in clinical trials
There is a need to discover potent and receptor sub-type
Fig. 1. Structures of S1PR2 ligands
1

Fig. 2. Design strategy
selective ligands to better understand the biological role of these
impact on the binding potency toward S1PR2.21 Secondly,
fragment B was modified by replacing the urea linker with
heterocycle linker; this modification will reduce hydrogen bond
donor (HBD) and cause reduction of molecular total polar
surface area (tPSA) to improve the physicochemical property.22
Thirdly, fragment C was modified by replacing the 2,6-
dichloropyridine ring in JTE-013 with different fused
heterocycles or substituted pyridines. Together, eleven new
compounds were synthesized and their in vitro binding affinities
were determined using [32P]S1P and cell membranes expressing
recombinant human S1PRs.
As shown in Scheme 1, the synthesis of compound 7 was
achieved by starting with compound 1, which was synthesized as
reported.19 The pyridine N-oxide 2 was obtained using meta-
chloroperoxybenzoic acid (mCPBA) to perform an N-oxidation
reaction. The cyano group was introduced on the pyridine ring
under trimethylsilyl cyanide (TMS-CN) to give cyanide
intermediate 3, which was then converted to amide 4 by
hydration in the presence of 1 M NaOH and H2O2. The
different receptors and identify therapeutic approaches for
different pathological conditions.11 Although many S1P receptor
ligands have been reported, most are S1PR1 specific, and very
few are specific for S1PR2 (Fig. 1). JTE-013, which was first
reported in 2001, is one of the most widely-used potent and
selective S1PR2 antagonist; it inhibits specific binding of
radiolabeled S1P to cell membranes of Chinese hamster ovary
(CHO) cells stably transfected with human or rat S1PR2.13
Although CYM-5520 was reported as the first allosteric S1PR2
selective agonist, with an EC50 value of 0.48 µM, an [33P]S1P
competitive binding assay showed CYM-5520 was not
competitive with S1P.14 Recently, Takuya Seko’s group
identified several potent and selective S1PR2 antagonists in a
novel series of 1,3-bis(aryloxy)benzene derivatives.15-17 We
reported a radiolabeled JTE-013 analogue, [11C]TZ34125 and its
in vivo studies, suggesting the sexual dimorphism of S1PR2
expression in the cerebellum of cyclosporin pretreated SJL
mice.18 Here, we have continued our medicinal chemistry
exploration of JTE-013 structural analogues. We divided the
structure of JTE-013 into three fragments (A, B, and C) which
were subsequently modified as shown in Fig. 2. Firstly, on one
side fragment A was modified by reversing the pyrazole ring and
adding one more carbonyl group to check the possibility of
improving binding potency and stability, as reported;19, 20 on the
other side, fragment A was modified by replacing the isopropyl
group with a steric bioisotere group, trifluoromethyl to check its
intermediate compound
6
was generated by reacting
commercially available 2-chloro-6-methoxyisonicotinic acid 5
with diphenylphosphoryl azide. The final compound 7 was
accomplished by a two-step one-pot reaction: the azide 6 was
refluxed in toluene and converted to an isocyanate, which was
followed by treating with 7-isopropyl-1,3-dimethyl-1H-pyrazolo-
[4,3-b]pyridine-5 carbetamide
4 to afford compound 7.
Scheme 1. Synthesis of analogue 7. Reagents and conditions: (a) mCPBA, CHCl3, 70 °C, 79%; (b) TMS-CN, triethylamine, CH3CN, 110
°C, 75%; (c) 1 M NaOH, H2O2, methanol, rt, 90%; (d) diphenylphosphoryl azide, triethylamine, 1,4-dioxane, 0 °C-rt, 55%; (e) toluene,
reflux, THF, 50 °C, 65%.
2

CF3
N
CF3
N
CF3
N
CF3
N
a
b
c
d
N
NH2
N
N
N
N
N
R1
NH2
N
N
N
N
OH
Br
CN
CF3
N
R1
9a: R1 = H
: R1 = CH3
R1
R1
R1
O
8a: R1 = H
: R1 = CH3
10a: R1 = H
: R1 = CH3
11a: R1 = H
12a: R1 = H
H
H
f
N
R2
N
N
: R1 = CH3
: R1 = CH3
N
8b
9b
10b
11b
12b
R1
O
O
N
COOH
CON3
e
13a: R1 = H; R2 = OCH3
13b: R1 = H; R2 = Cl
13c: R1 = CH3; R2 = OCH3
13d: R1 = CH3; R2 = Cl
Cl
N
R2
Cl
N
R2
5: R2 = OCH3
5a: R2 = Cl
6: R2 = OCH3
6a: R2 = Cl
Scheme 2. Syntheses of analogues 13a-d. Reagents and conditions: (a) ethyl 4,4,4-trifluoro-acetoacetate, propionic acid, 140 °C, 61% for 9a, 75%
for 9b; (b) POBr3, anisole, 140 °C, 78% for 10a, 70% for 10b; (c) CuCN, DMF, 110 °C, 60% for 11a, 75% for 11b; (d) 1 M NaOH, H2O2,
methanol, rt, 60% for 12a, 82% for 12b; (e) diphenylphosphoryl azide, triethylamine, 1,4-dioxane, 0 °C-rt, 60%; (f) toluene, reflux, THF, rt, 53%
for 13a, 48% for 13b, 64% for 13c, 67% for 13d.
Scheme 3. Syntheses of analogues 22 and 23. Reagents and conditions: (a) ethyl isobutyrylacetate, acetic acid, reflux, 10%; (b) POBr3, anisole,
reflux, 98%; (c) CuCN, DMF, 110 °C, 60%; (d) hydroxylamine hydrochloride, NaHCO3, methanol, reflux, 81%; (e) NaOCH3, methanol, rt,
NH4Cl, 70 °C, 50%; (f) (i) 2-chloro-6-methoxyisonicotinic acid 19, DIPEA, PyBOP, DMF (ii) dioxane, 90 °C, 40%; (g) (i) oxalyl chloride, DMF,
CH2Cl2, rt (ii) tert-butyl carbazate, triethylamine, CH2Cl2, 0 ºC-rt, 99%; (h) CF3COOH, CH2Cl2, 0 °C-rt, 25%; (i) ethanol, reflux, 63%.
Scheme 4. Syntheses of analogues 33, 34, 35a, and 35b. Reagents and conditions: (a) cyanogen bromide, ethanol, reflux, 64%; (b) triphosgene,
triethylamine, toluene, rt, 63%; (c) 2-bromo-1,1-dimethoxyethane,48% HBr, ethanol, reflux, 79%; (d) diphenylphosphoryl azide, triethylamine,
DMF, 55%; (e) diphenylphosphoryl azide, triethylamine, 1,4-dioxane, 0 °C-rt, 64% for 31a, 69% for 31b; (f) hydrazine, ethanol, reflux, 65%; (g)
THF, rt, 20%; (h) toluene, reflux, THF, rt, 57% for 34, 67% for 35a, and 83% for 35b.
To generate trifluoromethyl substituted compounds 13a-d,
Scheme 2 was followed. Starting with 3-methyl-1H-pyrazol-5-
3

Table 1
Compd.
The IC50 values (mean SD)a of new compounds toward S1PR2
Structure
S1PR2 IC50 (nM)
Compd.
Structure
S1PR2 IC50 (nM)
58.4 7.4
> 1,000
JTE-013
22
> 1,000
> 1,000
> 1,000
> 1,000
> 1,000
> 1,000
> 1,000
29.1 2.6
7
23
33
CF3
H
N
H
N
N
O
N
H
13a
13b
13c
N
O
O
N
Cl
34
CF3
N
H
N
H
N
N
O
N
35a
O
O
N
Cl
> 1,000
56.5 4.0
13d
35b
a IC50 values were determined at least two independent experiments, each run was performed in duplicate; for compounds with IC50 < 100 nM, at
least three independent experiments were performed, each run was performed in duplicate.
amine 8a or 1,3-dimethyl-1H-pyrazol-5-amine 8b, cyclization
was accomplished by reacting with ethyl 4, 4, 4-trifluoro-
acetoacetate in propionic acid at 140 °C to give hydroxyheter-
oarenes 9a or 9b, respectively. Bromination of compounds 9a or
9b using POBr3 afforded bromoheteroarenes 10a or 10b in high
yields, which were subjected to react with CuCN to afford
cyanide intermediates 11a or 11b. After hydration, the amide
intermediates 12a and 12b were obtained. Compounds 13a-d
were prepared using a two-step one-pot reaction procedure
similar to the procedure of making compound 7.
Finally, fragment C was modified as shown in Scheme 4. Key
intermediate 32 was first prepared by treating intermediate 15
with hydrazine in ethanol. Cyanogen bromide treatment of 2,6-
dichloropyridine-3,4-diamine 24, synthesized as reported,20 gave
4,6-dichloro-1H-imidazo[4,5-c]pyridin-2-amine 25. Compound
25 was reacted with triphosgene under triethylamine to afford the
isocyanate 26, which was coupled with intermediate 32 to give
compound 33. Treatment of commercially available 2-amino-6-
chloroisonicotinic acid 27 using 2-bromo-1,1-dimethoxyethane in
the presence of 48% HBr in ethanol afforded 5-
chloroimidazo[1,2-a]pyridine-7-carboxylic acid 28. Compound
28 was reacted with dipenylphosphorylazide under triethylamine
in DMF to afford 5-chloroimidazo [1,2-a]pyridine-7-carbonyl
azide 29, followed by coupling with intermediate 32 afforded
compound 34. Compounds 35a and 35b were obtained using a
procedure similar to that used to synthesize 34, starting with the
corresponding substituted isonicotinic acid.
The in vitro binding potency of newly synthesized analogues
and reference compound JTE-013 was evaluated using
competitive radioligand binding assay with cell membranes
following published protocol.23 As shown in Table 1, compound
7, having a reversed pyrazole ring and one more carbonyl group
showed decreased binding potency compared to JTE-013;
compounds 13a-d, containing trifluoromethyl group used as
bioisotere of isopropyl also exhibited poor binding potency (IC50
> 1,000 nM). Modification of fragment A did not improve
binding potency. Modification of fragment B gave compounds 22
and 23, with oxadiazole and triazole rings instead of urea linker.
The IC50 values of 22 and 23 are > 1,000 nM, indicating that the
heterocyclic linker reduced binding potency. Modification of
fragment C gave compounds 33, 34, and 35a-b. Compounds 33
and 34, possessing imidazo[4,5-c]pyridine and imidazo[1,2-a]-
To investigate the impact of replacing urea linker (fragment
B) with a heterocycle linker, compounds 22 and 23 were
synthesized by following Scheme 3. The commercially available
1,3-dimethyl-1H-pyrazol-5-amine 8b was cyclized with ethyl
isobutyrylacetate to afford 4-isopropyl-1,3-dimethyl-1H-
pyrazolo[3,4-b]pyridin-6-ol 14. Under similar conditions as
Scheme 2, the key cyanide intermediate 4-isopropyl-1,3-
dimethyl-1H-pyrazolo[3,4-b]pyridin-e-6-carbonitrile 16 was
obtained. Next, the cyanide 16 was treated with hydroxylamine
hydrochloride to afford N-hydroxy-4-isopropyl-1,3-dimethyl-1H-
pyrazolo[3,4-b]pyridine-6-carboximidamide 17. Meanwhile, the
cyanide 16 can also be converted to 4-isopropyl-1,3-dimethyl-
1H-pyrazolo[3,4-b]pyridine-6-carboximidamide 18 by reacting
with NH4Cl in the presence of sodium methoxide. The
oxadiazole compound 22 was obtained by a two-step reaction.
First, intermediate 17 and 2-chloro-6-methoxyisonicotinic acid
19 were condensed in the presence of PyBOP, DIPEA, and DMF,
then the coupled product was cyclized in 1,4-dioxane at 90 °C to
give compound 22. The triazole compound 23 was obtained by
refluxing the intermediate 18 with 2-chloro-6-methoxy-
isonicotinohydrazide 21 in ethanol.
4

S1PR2. Our future work will focus on the continued exploration
of structure modifications of fragment C building on this SAR
information.
Experimental methods and characterization data for
intermediates and target compounds are provided in the Notes
along with the binding assay methods.24
In summary, a series of pyrazolopyridine derivatives were
synthesized and evaluated for their S1PR2 binding potency and
selectivity. The in vitro data indicated compounds 35a and 35b
have comparable binding affinities for S1PR2 with IC50 values of
29.1 2.6, 56.5 4.0 nM, respectively compared to compound
JTE-013 (IC50 = 58.4
7.4 nM). Further evaluation of the
selectivity of S1PR2 over other S1PRs (1, 3, 4, and 5) indicated
that both 35a and 35b are selective for S1PR2. The initial SAR
analysis suggest 1H-pyrazolo[3,4-b]pyridine in fragment A and
urea linker in fragment B are essential pharmacophores, but the
2,6-dichloropyridine in fragment C can be modified. Further
optimization of these analogues is needed to identify additional
and more potent and selective compounds targeting S1PR2.
Fig. 3. Competitive binding curves of compounds 35a, 35b, and JTE-013 for
S1PR2. A CHO cell membrane containing recombinant human S1PR2 was
used in a [32P]S1P competitive binding assay to measure the binding
affinities.
Acknowledgments
This study was supported by National Multiple Sclerosis
Society grant RG150705331 and USA Department of Energy
pyridine, respectively, were also not potent (IC50 > 1,000 nM);
Compounds
isopropoxypyridine
35a
and
and
35b,
2-chloro-6-cyclopropoxypyridine,
possessing
2-chloro-6-
(DOE)-Training
Grants
DOE,
DESC0008432
and
DESC0012737. It was partially supported by National Institutes
of Health (NIH) through the National Institute of Neurological
Disorders and Stroke NS075527 and the National Institute of
Mental Health MH092797. Mass spectrometry was generated
from the Washington University Mass Spectrometry facility that
is supported by NIH National Institute of Health Research
Resource RR0954.
respectively, had modest improved binding potency (35a, IC50
29.1 2.6 nM; 35b, IC50 = 56.5 4.0 nM) compared to JTE-013
(IC50 = 58.4 7.4 nM). The competitive binding curves of
=
compounds 35a, 35b, and JTE-013 for S1PR2 were presented in
Fig 3. These results indicated that replacement of 2,6-
dichloropyridine moiety with imidazo[4,5-c]pyridine and
imidazo[1,2-a]pyridine had a negative impact on S1PR2 binding
potency, and the replacement of a chloro on 2,6-dichloropyridine
ring with isopropoxyl and cyclopropoxyl resulted in a modest
improvement in the binding potency. Although most of
compounds exhibited poor binding potency with IC50 > 1,000
nM, useful structure-activity relationship (SAR) information of
these new analogues was generated. The 1H-pyrazolo[3,4-
b]pyridine in fragment A and urea linker in fragment B are
important pharmacophores, modifications of either fragment
resulted in loss of binding activity. The modification of 2,6-
dichloropyridine in the fragment C is able to remain or improve
the binding potency toward S1PR2 when the different substituted
groups were introduced. Because compounds 35a and 35b
showed good binding affinity for S1PR2, their binding potency
toward other S1P receptor subtypes were determined to evaluate
their selectivity for S1PR2. As shown in Table 2, compounds 35a
and 35b exhibited high selectivity toward S1PR2 over S1PR1, 3,
4, and 5, indicating these two new ligands are selective for
References and notes
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2.
3.
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Chun J, Goetzl EJ, Hla T, et al. Pharmacol Rev.
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Brinkmann V, Davis MD, Heise CE, et al. J Biol Chem.
2002;277:21453.
Table 2
The IC50 values (mean SD)a of 35a and 35b binding toward
other S1PRs
11.
12.
Abbasi T, Garcia JG. Handb Exp Pharmacol. 2013:201.
Cruz-Orengo L, Daniels BP, Dorsey D, et al. J Clin Invest.
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Chem Lett. 2016;26:1209.
IC50 (nM)
Compd.
S1PR1
> 1,000
1.4 0.3
> 1,000
> 1,000
S1PR2
S1PR3
> 1,000
0.4 0.2
> 1,000
> 1,000
S1PR4
ND
S1PR5
ND
58.4 7.4
3.6 0.5
29.1 2.6
56.5 4.0
JTE-013
S1P
151 82
> 1,000
> 1,000
3.1 1.1
> 1,000
> 1,000
35a
35b
a IC50 values were determined at least two independent experiments, each run
was performed in duplicate; for compounds with IC50 < 100 nM, at least three
independent experiments were performed, each run was performed in
duplicate, ND-no determined.
Yue X, Jin H, Liu H, Rosenberg AJ, Klein RS, Tu Z. Org
Biomol Chem. 2015;13:7928.
5

19.
20.
21.
Fang WK, Wang L, Corpuz EG, Chow K, Im WB. PCT
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CONH2), 5.43 (br, 1H, CONH2), 4.18 (s, 3H, -N-CH3), 3.63 (hept, J
= 7.0 Hz, 1H, -CH-), 2.58 (s, 3H, -C-CH3), 1.38 (d , J = 7.0 Hz, 6H, -
CH-CH3).
2-Chloro-6-methoxyisonicotinoyl azide (6). To a round-bottomed
flask equipped with a magnetic stir bar was added 2-chloro-6-
methoxyisonicotinic
acid
(5)
(1.88
g,
10.0
mmol),
22.
23.
Rankovic Z. J Med Chem. 2017;60:5943.
Rosenberg AJ, Liu H, Tu Z. Appl Radiat Isot. 2015;102:5.
diphenylphosphorylazide (3.03 g, 11.0 mmol), and 1,4-dioxane (20
mL). Triethylamine (2.53 g, 25.0 mmol) was added slowly through
syringe under nitrogen at 0 °C. The reaction was warmed and stirred
at room temperature for 5 h. Then, the mixture was diluted with
water and extracted with ethyl acetate. The organic layer was washed
with saturated brine and dried over anhydrous MgSO4. After
filtration and concentration, the crude product was purified by flash
chromatography with hexane/ethyl acetate (30/1, v/v) to afford 6
(1.12 g, 55%). 1H NMR (400 MHz, Acetone-d6) d = 7.21 (s, 1H, Ar-
H), 6.99 (s, 1H, Ar-H), 3.82 (s, 3H, -OCH3).
24. A) General: Commercially available starting materials,
reagents, and solvents were used as received. Unless otherwise
indicated, all reactions were conducted in oven dried glassware.
Reactions were monitored by thin-layer chromatography (TLC)
carried out on pre-coated glass plates of silica gel 60 F254 from EMD
Chemicals Inc. Visualization was accomplished under ultraviolet
light (UV 254 nm). Flash column chromatography was performed
using 230–400 mesh silica gel purchased from Silicycle. Yields refer
to isolated yield by chromatography, unless otherwise stated.
Melting points were determined on a MEL-TEMP 3.0 apparatus and
N-((2-Chloro-6-methoxypyridin-4-yl)carbamoyl)-7-isopropyl-1,3-
a
dimethyl-1H-pyrazolo[4,3-b]pyridine-5-carboxamide (7). To
are uncorrected. H NMR and 13C NMR spectra were recorded on
1
round-bottomed flask equipped with a magnetic stir bar was added 2-
chloro-6-methoxyisonicotinoyl azide (6) (54 mg, 0.25 mmol) and
toluene (10 mL). The mixture was refluxed for 2 h and cooled to 50
ºC before 7-isopropyl-1, 3-dimethyl-1H-pyrazolo[4,3-b]pyridine-5-
carboxamide (4) (50 mg, 0.21 mmol) in THF (10 mL) was added.
The reaction was stirred for another 10 h. The reaction then was
diluted with water and extracted with ethyl acetate. The organic layer
was washed with saturated brine and dried over anhydrous MgSO4.
After filtration and concentration, the crude product was purified by
flash chromatography with hexane/ethyl acetate (1/1, v/v) to give 7
as a white solid (57 mg, 65%). MP: 117-119 °C. 1H NMR (400 MHz,
CDCl3) δ 10.90 (s, 1H, CONHCO), 10.16 (s, 1H, CONH), 8.10 (s,
1H, Ar-H), 7.17 (d, J = 1.0 Hz, 1H, Ar-H), 6.94 (d, J = 1.0 Hz, 1H,
Ar-H), 4.27 (s, 3H, -N-CH3), 3.92 (s, 3H, -OCH3), 3.78 – 3.67 (m,
1H, -CH-), 2.64 (s, 3H, -C-CH3), 1.45 (d, J = 6.8 Hz, 6H, -CH-CH3).
13C NMR (101 MHz, CDCl3) δ 165.75, 164.65, 149.97, 149.07,
148.24, 143.63, 142.17, 141.68, 140.32, 133.71, 115.87, 107.57,
98.22, 54.12, 39.51, 28.22, 23.11, 10.90. HRMS (ESI) calcd. for
C19H21ClN6NaO3 [M + Na]+, 439.1256, found 439.1247. HPLC
purity: 98%, tR = 10.1 min.
Varian 400 MHz instrument. Chemical shifts are reported in parts
per million (ppm) and are calibrated using residual undeuterated
solvent as an internal reference (CDCl3: δ 7.26 ppm; CD3OD: δ 3.31
ppm; DMSO-d6: δ 2.50 ppm; Acetone-d6: δ 2.05 ppm). High
resolution positive ion mass was acquired by a Bruker MaXis 4G Q-
TOF mass spectrometer with electrospray ionization source.
Chemical purities of finial compounds were determined by reversed
phase HPLC (Agilent C-18 column; mobile phase: 80% Acetonitrile
in 0.1 M Ammonium formate, pH 4.5; flow rate: 1.0 mL/min; UV:
254 nm).
7-Isopropyl-1,3-dimethyl-1H-pyrazolo[4,3-b]pyridine 4-oxide (2).
To
a solution of 7-isopropyl-1,3-dimethyl-1H-pyrazolo [4,3-b]
pyridine (1) (0.66 g, 3.5 mmol) in 50 mL of chloroform was added
77% 3-chloroperbenzoic acid (mCPBA) (1.2 g, 7.0 mmol) at 0 °C.
The resulting reaction mixture was stirred at 90 °C for 3 h. After
cooling to room temperature, the mixture was neutralized with
aqueous NaHCO3 and extracted with dichloromethane (3 × 100 mL).
The combined organic phases were washed with water, saturated
brine, and concentrated under reduced pressure. The crude was
purified by flash chromatography with hexane/ethyl acetate (2/1, v/v)
to give 2 (0.57 g, 79%). 1H NMR (400 MHz, CDCl3): δ 8.01 (d, J =
6.3 Hz, 1H, Ar-H), 6.78 (d, J = 6.3 Hz, 1H, Ar-H),. 4.21 (s, 3H, -N-
CH3), 3.60 (hept, J = 6.7 Hz, 1H, -CH-), 2.83 (s, 3H, -C-CH3), 1.37
(d, J = 6.7 Hz, 6H, -CH-CH3).
3-Methyl-4-(trifluoromethyl)-1,7-dihydro-6H-pyrazolo[3,4-b]-
pyridin-6-one (9a). To a round-bottomed flask equipped with a
magnetic stir bar was added 3-methyl-1H-pyrazol-5-amine (8a) (1.0
g, 10 mmol), ethyl 4, 4, 4-trifluoroacetoacetate (2.0 mL, 13 mmol),
and propionic acid (50 mL). The mixture was heated at 140 °C for 12
h. After cooling, the mixture was concentrated under reduced
pressure and the residue was treated with ethyl acetate. The
precipitate was filtered and dried as product 9a (1.3 g, 61%). 1H
NMR (400 MHz, CD3OD) δ 6.47 (s, 1H, Ar-H), 2.42 (s, 3H, -C-
CH3).
7-Isopropyl-1, 3-dimethyl-1H-pyrazolo [4, 3-b] pyridine-5-
carbonitrile (3). To a solution of 7- isopropyl-1,3-dimethyl-1H-
pyrazolo[4,3-b]pyridine N-oxide (2) (0.72 g, 3.5 mmol) and triethyl
amine (0.71 g, 7.0 mmol) in 20 mL acetonitrile was added
trimethylsilyl cyanide (TMS-CN) (0.53 g, 5.2 mmol). The resulting
solution was stirred at reflux for 18 h. Then another portion of
trimethylsilyl cyanide (0.26 g, 2.6 mmol) was added and the reaction
was continued to stir for another 7 h. The solvent was removed
under reduced pressure and the residue was purified by flash
chromatography with hexane/ethyl acetate (3/1, v/v) to give 3 (562
1,3-Dimethyl-4-(trifluoromethyl)-1,7-dihydro-6H-pyrazolo[3,4-
b]pyridin-6-one (9b). To a round-bottomed flask equipped with a
magnetic stir bar was added 1,3-dimethyl-1H-pyrazol-5-amine (8b)
(10.0 g, 90 mmol), ethyl 4, 4, 4-trifluoroacetoacetate (17 mL, 110
mmol), and propionic acid (100 mL). The mixture was heated at 140
°C for 12 h. After cooling, the mixture was concentrated under
reduced pressure and the residue was treated with ethyl acetate. The
1
mg, 75%). H NMR (400 MHz, CDCl3): δ 7.46 (s, 1H, Ar-H), 4.26
(s, 3H, -N-CH3), 3.69 (hept, J = 6.7 Hz, 1H, -CH-), 2.64 (s, 3H, -C-
CH3), 1.40 (d, J = 6.7 Hz, 6H, -CH-CH3).
1
precipitate was filtered and dried as product 9b (15.5 g, 75 %). H
NMR (400 MHz, CD3OD) δ 6.60 (s, 1H, Ar-H), 3.87 (s, 3H, -N-
CH3), 2.41 (s, 3H, -C-CH3).
7-Isopropyl-1, 3-dimethyl-1H-pyrazolo[4,3-b]pyridine-5-carbox-
amide (4). To a solution of 7-isopropyl-1, 3-dimethyl-1H-pyrazolo
[4,3-b] pyridine-5-carbonitrile (3) (81 mg, 0.38 mmol) in methanol
(5 mL) was added 1 M NaOH (7 mL) and 35% H2O2 (1.3 mL). The
reaction mixture was stirred at room temperature for 2 h. Then, the
reaction mixture was diluted with dichloromethane. The organic
layer was washed with saturated brine and dried over anhydrous
MgSO4. After filtration and concentration, the crude product 4 was
obtained as a white solid without further purification (79 mg, 90%).
1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H, Ar-H), 7.84 (br, 1H,
6-Bromo-3-methyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-b]-
pyridine (10a). A suspension of 3-methyl-4-(trifluoromethyl)-1,7-
dihydro-6H-pyrazolo[3,4-b]pyridin-6-one (9a) (1.7 g, 7.7 mmol) and
POBr3 (2.9 g, 10 mmol) in anisole (100 mL) was heated at 140 °C
for 3 h under nitrogen. The reaction mixture was diluted with toluene
(50 mL). The organic layer was then washed with water (3 × 50 mL)
and dried over anhydrous MgSO4. After filtration and concentration,
the crude product was purified by flash chromatography with
6

1
hexane/ethyl acetate (3/1, v/v) to give 10a (1.7 g, 78%). H NMR
General procedure for the synthesis of 13a-d. To a round-
bottomed flask equipped with a magnetic stir bar was added 6 or 6a
(1.2 eq) and toluene (10 mL). The mixture was refluxed for 2 h and
cooled to 50 ºC before 12a or 12b (1.0 eq) in THF (10 mL) was
added. The reaction was stirred for another 10 h. Then, the reaction
was diluted with water and extracted with ethyl acetate. The organic
layer was washed with saturated brine and dried over anhydrous
MgSO4. After filtration and concentration, the crude product was
purified by flash chromatography with hexane/ethyl acetate to give
13a-d as a white solid.
(400 MHz, CDCl3) δ 7.53 (s, 1H, Ar-H), 2.65 (s, 3H, -C-CH3).
6-Bromo-1,3-dimethyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-b]-
pyridine (10b). A suspension of 1,3-dimethyl-4-(trifluoromethyl)-
1,7-dihydro-6H-pyrazolo[3,4-b]pyridin-6-one (9b) (800 mg, 2.7
mmol) and POBr3 (1.3 g, 4.5 mmol) in anisole (100 mL) was heated
with stirring at 140 °C for 3 h under nitrogen. The reaction mixture
was diluted with toluene (50 mL). The organic layer was then
washed with water (3 x 50 mL) and dried over anhydrous MgSO4.
After filtration and concentration, the crude product was purified by
flash chromatography with hexane/ethyl acetate (3/1, v/v) to give
N-((2-Chloro-6-methoxypyridin-4-yl)carbamoyl)-3-methyl-4-
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridine-6-carboxamide
(13a). Yield: 53%. MP: 91-94 °C. 1H NMR (400 MHz, DMSO-d6) δ
9.65 (s, 1H, CONH), 8.08 (s, 1H, Ar-H), 6.21 (s, 1H, Ar-H), 5.78 (s,
1H, Ar-H), 3.70 (s, 3H, -OCH3), 2.58 (s, 3H, -C-CH3). 13C NMR
(101 MHz, DMSO-d6) δ 165.54, 164.66, 159.09, 152.83, 151.85,
150.74, 150.27, 148.15, (d, J = 22.2 Hz), 139.62, 123.11 (q, J =
274.7 Hz), 110.89, 106.66, 103.30, 96.73, 53.64, 14.45. HRMS (ESI)
calcd. for C16H12ClF3N6NaO3 [M + Na]+, 451.0504, found 451.0536.
HPLC purity: 96%, tR = 4.0 min.
1
10b (556 mg, 70%). H NMR (400 MHz, CDCl3) δ 7.41 (s, 1H, Ar-
H), 4.03 (s, 3H, -N-CH3), 2.54 (s, 3H, -C-CH3).
3-Methyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridine-6-
A
carbonitrile (11a).
mixture of 6-bromo-3-methyl-4-
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridine (10a) (1.1 g, 4.0
mmol), CuCN (0.7 g, 8.0 mmol), and anhydrous DMF (20 mL) was
heated at 110 °C for 7 hours. After cooling, the mixture was diluted
and filtered. The filtrate was concentrated and purified by flash
chromatography with hexane/ethyl acetate (3/1, v/v) to give 11a (542
mg, 60%). 1H NMR (400 MHz, CDCl3) δ 7.88 (s, 1H, Ar-H), 2.66 (s,
3H, -C-CH3).
N-((2,6-Dichloropyridin-4-yl)carbamoyl)-3-methyl-4-(trifluoro-
methyl)-1H-pyrazolo[3,4-b]pyridine-6-carboxamide (15). Yield:
1
1,3-Dimethyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridine-6-
48%. MP: 141-145 °C. H NMR (400 MHz, DMSO-d6) δ 10.01 (s,
carbonitrile (11b).
A
mixture of 6-bromo-1,3-dimethyl-4-
1H, CONH), 8.17 (s, 1H, Ar-H), 6.49 (s, 2H, Ar-H), 2.58 (s, 3H, -C-
CH3). 13C NMR (101 MHz, DMSO-d6) δ 165.56, 158.98, 152.79,
150.28, 150.11, 149.60, 139.81, 123.14, (q, J = 279.8 Hz), 111.25,
110.83, 106.94, 14.58. HRMS (ESI) calcd. for C15H9Cl2F3N6NaO2
[M + Na]+, 455.0008, found 451.0047. HPLC purity: 97%, tR = 3.9
min.
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridine (10b) (1.5 g, 5.1
mmol), CuCN (1.37 g, 15.5 mmol), and anhydrous DMF (100 mL)
was heated at 110 °C for 7 h. After cooling, the mixture was diluted
and filtered. The filtrate was concentrated and purified by flash
chromatography with hexane/ethyl acetate (3/1, v/v) to give 11b
(919 mg, 75 %). 1H NMR (400 MHz, CDCl3) δ 7.61 (s, 1H, Ar-H),
4.10 (s, 3H, -N-CH3), 2.60 (s, 3H, -C-CH3).
N-((2-Chloro-6-methoxypyridin-4-yl)carbamoyl)-1,3-dimethyl-4-
pyrazolo[3,4-b]pyridine-6-carboxamide
(trifluoromethyl)-1H
1
3-Methyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridine-6-
carboxamide (12a). To a solution of 3-methyl-4-(trifluoromethyl)-
1H-pyrazolo[3,4-b]pyridine-6-carbonitrile (11a) (750 mg, 3.3 mmol)
in methanol (5 mL) was added 1 M NaOH (6.5 mL) and 35% H2O2
(1.2 mL). The reaction mixture was stirred at room temperature for 2
h. The reaction mixture was diluted with dichloromethane. Then, the
organic layer was washed with saturated brine and dried over
anhydrous MgSO4. After filtration and concentration, the crude
product 12a was obtained without further purification (483 mg,
60%). 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H, Ar-H), 2.58 (s, 3H,
-C-CH3).
(13c). Yield: 64%. MP: 103-106 °C. H NMR (400 MHz, CDCl3) δ
10.74 (s, 1H, CONHCO), 9.93 (s, 1H, CONH), 8.30 (s, 1H, Ar-H),
7.19 (s, 1H, Ar-H), 6.97 (s, 1H, Ar-H), 4.22 (s, 3H, -N-CH3), 3.93 (s,
3H, -OCH3), 2.68 (s, 3H, -C-CH3). 13C NMR (101 MHz, CDCl3) δ
164.71, 164.11, 150.44, 149.44, 149.25, 147.85, 145.30, 140.16,
133.46 (d, J = 36.4 Hz), 122.15 (q, J = 275.7 Hz), 112.61, 111.15,
107.65, 98.46, 54.20, 34.47, 14.18. HRMS (ESI) calcd. for
C17H14ClF3N6NaO3 [M + Na]+, 465.0660, found 465.0674. HPLC
purity: 95%, tR = 10.0 min.
N-((2,6-Dichloropyridin-4-yl)carbamoyl)-1,3-dimethyl-4-
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridine-6-carboxamide
(13d). Yield: 67%. MP: 133-135 °C. 1H NMR (400 MHz, CDCl3) δ
10.95 (s, 1H, CONHCO), 9.99 (s, 1H, CONH), 8.31 (s, 1H, Ar-H),
1,3-Dimethyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridine-6-
carboxamide (12b). To
(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridine-6-carbonitrile
a
solution of 1, 3-dimethyl-4-
(11b)
7.60 (s, 2H, Ar-H), 4.24 (s, 3H, -N-CH3), 2.70 (s, 3H, -C-CH3). 13
C
(670 mg, 2.8 mmol) in methanol (5 mL) was added 1 M NaOH (7
mL) and 35% H2O2 (1.3 mL). The reaction mixture was stirred at
room temperature for 2 h. The reaction mixture was diluted with
dichloromethane. Then, the organic layer was washed with brine and
dried over anhydrous MgSO4. After filtration and concentration, the
crude product 12b was obtained without further purification (593
mg, 82%). 1H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H, Ar-H), 4.15 (s,
3H, -N-CH3), 2.65 (s, 3H, -C-CH3).
NMR (101 MHz, CDCl3) δ 164.30, 151.33, 150.45, 149.37, 147.94,
145.01, 140.25, 133.56 (d, J = 32.3 Hz), 122.11 (q, J = 274.7 Hz),
112.79, 111.26, 111.21, 34.53, 14.20. HRMS (ESI) calcd. for
C16H11Cl2F3N6NaO2 [M + Na]+, 469.0165, found 469.0191. HPLC
purity: 99%, tR = 9.0 min.
4-Isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridin-6-ol (14). To
a round-bottomed flask equipped with a condenser and magnetic stir
bar was added commercial 1,3-dimethyl-1H-pyrazol-5-amine (8b)
(5.56 g, 50 mmol), ethyl isobutyrylacetate (8.70 g, 55 mmol), and
acetic acid (40 mL). The reaction vessel was immersed in a 120 oC
preheated oil bath for 24 h. After cooling, the mixture was
concentrated and the resulting oil was triturated with ethyl acetate
(20 mL). The resulting solid was filtered and dried under vacuum to
2,6-Dichloroisonicotinoyl azide (6a). To a round-bottomed flask
equipped with a magnetic stir bar was added 2,6-dichloroisonicotinic
acid (5a) (1.92 g, 10.0 mmol), diphenylphosphorylazide (3.03 g, 11.0
mmol), and 1,4-dioxane (20 mL). Triethyamine (2.53 g, 25.0 mmol)
was added slowly through syringe under nitrogen at 0 °C. The
reaction was warmed and stirred at room temperature for 5 h. The
mixture then was diluted with water and extracted with ethyl acetate.
The organic layer was washed with saturated brine, dried over
anhydrous MgSO4. After filtration and concentration, the crude
product was purified by flash chromatography with hexane/ethyl
1
afford 14 (1.06 g, 10.3%). H NMR (400 MHz, CDCl3) δ 13.85 (s,
1H, -OH), 6.16 (d, J = 0.6 Hz, 1H, Ar-H), 3.99 (s, 3H, -N-CH3), 3.37
– 3.25 (m, 1H,-CH-), 2.51 (s, 3H, -C-CH3), 1.31 (d, J = 6.8 Hz, 6H, -
CH-CH3).
6-Bromo-4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine
(15). To a round-bottomed flask equipped with a magnetic stir bar
was added 4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridin-6-ol
(14) (1.03 g, 4.89 mmol), POBr3 (2.10 g, 7.33 mmol), and anisole
1
acetate (50/1, v/v) to afford 6a (1.30 g, 60%). H NMR (400 MHz,
CDCl3) δ 7.75 (s, 2H, Ar-H).
7

(10 mL). The reaction vessel was immersed in a 120oC preheated oil
bath for 3 h. After cooling, saturated NaHCO3 was added to quench
the mixture. The organic layer was washed with saturated brine and
dried over anhydrous MgSO4. After filtration and concentration, the
crude product was purified by flash chromatography with
10.50 (s, 1H-OCONH), 9.08 (s, 1H, CONH), 7.42 (s, 1H, Ar-H), 7.19
(s, 1H, Ar-H), 3.90 (s, 3H, -O-CH3), 1.43 (s, 9H, -C-CH3).
3-Chloro-5-methoxybenzohydrazide (21). To a solution of tert-
butyl 2-(2-chloro-6-methoxyisonicotinoyl)hydrazine-1-carboxylate
(20) (300 mg, 1.0 mmol) in CH2Cl2 (5 mL) was added CF3COOH
1
o
hexane/ethyl acetate to (10/1, v/v) to afford 15 (1.28 g, 98%). H
(1.0 mL) at 0 C and the mixture was stirred at room temperature
NMR (400 MHz, CDCl3) δ 7.06 (s, 1H, Ar-H), 4.02 (s, 3H, -N-CH3),
3.71 – 3.58 (m, 1H, -CH-), 2.66 (s, 3H, -C-CH3), 1.35 (d, J = 6.9 Hz,
6H, -CH-CH3).
until the reaction was completed as determined by TLC. The mixture
was washed with saturated NaHCO3 solution, saturated brine, and
dried over anhydrous MgSO4. After filtration and concentration, the
crude product was purified by flash chromatography with
4-Isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine-6-
carbonitrile (16). To a round-bottomed flask equipped with a
magnetic stir bar were added 6-bromo-4-isopropyl-1,3-dimethyl-1H-
pyrazolo[3,4-b]pyridine (15) (1.34 g, 5.0 mmol), CuCN (1.80 g, 20.0
mmol), and DMF (15.0 mL). The reaction vessel was immersed in a
110 oC preheated oil bath for 12 h until the reaction was completed
as determined by TLC. After cooling, the reaction mixture was
diluted with water and extracted with ethyl acetate. The ethyl acetate
layer was washed with saturated brine and dried over anhydrous
MgSO4. After filtration and concentration, the crude product was
purified by flash chromatography with hexane/ethyl acetate (5/1) to
afford 16 (0.64 g, 60%). 1H NMR (400 MHz, CDCl3) δ 7.29 (s, 1H,
Ar-H), 4.07 (s, 3H, -N-CH3), 3.72 – 3.60 (m, 1H, -CH-), 2.71 (s, 3H,
-C-CH3), 1.38 (d, J = 6.9 Hz, 6H, -CH-CH3).
1
hexane/ethyl acetate (1/3, v/v) to afford 21 (52 mg, 25%). H NMR
(400 MHz, DMSO-d6) δ 10.10 (s, 1H, CONH), 7.40 (s, 1H, Ar-H),
7.16 (s, 1H, Ar-H), 4.65 (s, 2H, NH2), 3.89 (s, 3H, -O-CH3).
5-(2-Chloro-6-methoxypyridin-4-yl)-3-(4-isopropyl-1,3-dimethyl-
1H-pyrazolo[3,4-b]pyridin-6-yl)-1,2,4-oxadiazole (22). To
a
solution of 2-chloro-6-methoxyisonicotinic acid (19) (132 mg, 0.70
mmol) and DIPEA (136 mg, 1.05 mmol) in DMF (5 mL) was added
PyBOP (406 mg, 0.78 mmol) at 0 oC. After stirring for 15 min, (Z)-
N'-hydroxy-4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine-6-
carboximidamide (17) (90 mg, 0.36 mmol) was added into the
mixture, the reaction was stirring at room temperature monitored by
TLC. Then, the reaction mixture was diluted with water and
extracted with ethyl acetate. The ethyl acetate layer was washed with
saturated brine and dried over anhydrous MgSO4. After filtration and
concentration, the residue was dissolved with 1,4-dioxane (10 mL)
and stirred at 100 oC for 6 h. After cooling, the reaction mixture was
evaporated with silica gel and purified by flash chromatography with
hexane/ethyl acetate (2/1, v/v) to afford 22 (56 mg, 40%). MP: 212-
(Z)-N'-Hydroxy-4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]-
pyridine-6-carboximidamide (17). To
a
suspension of
hydroxylamine hydrochloride (0.42 g, 6.0 mmol) in methanol (10
mL) was added NaHCO3 (0.55 g, 6.6 mmol), followed by adding 4-
isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine-6-carbonitrile
(16) (0.64 g, 3.0 mmol). The reaction vessel was immersed in 80 oC
preheated oil bath for 12 h until the reaction was completed as
determined by TLC. After cooling, the solvent was removed under
vacuum, the residue was diluted with water and extracted with ethyl
acetate. The ethyl acetate layer was washed with saturated brine and
dried over anhydrous MgSO4. After filtration and concentration, the
o
1
215 C. H NMR (400 MHz, CDCl3) δ 7.87 (s, 1H, Ar-H), 7.75 (s,
1H, Ar-H), 7.50 (s, 1H, Ar-H), 4.18 (s, 3H, -N-CH3), 4.04 (s, 3H, -O-
CH3), 3.76 – 3.69 (m, 1H, -CH-), 2.74 (s, 3H, -C-CH3), 1.45 (d, J =
6.9 Hz, 6H, -CH-CH3). HRMS (ESI) calcd. for C19H21ClN6O2 [M +
H]+ 399.1332, found 399.1340. HPLC purity: 98%, tR = 13.4 min.
6-(5-(2-Chloro-6-methoxypyridin-4-yl)-4H-1,2,4-triazol-3-yl)-4-
1
crude product 17 was obtained. (0.6 g, 81%). H NMR (400 MHz,
isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine (23). To a
DMSO-d6) δ 10.04 (s, 1H, -OH), 7.56 (s, 1H, Ar-H), 5.95 (s, 2H, -
NH2), 4.00 (s, 3H, -N-CH3), 3.65 – 3.50 (m, 1H, -CH-), 2.60 (s, 3H, -
C-CH3), 1.30 (d, J = 6.8 Hz, 6H, -CH-CH3).
round-bottomed flask equipped with a magnetic stir bar were added
4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine-6-
carboximidamide hydrochloride (18) (66 mg, 0.25 mmol), 3-chloro-
5-methoxybenzohydrazide (21) (50 mg, 0.25 mmol), and ethanol
(3.0 mL). The reaction vessel was immersed in a 100 oC preheated
oil bath for 12 h until the reaction was completed as determined by
TLC. After cooling, the reaction mixture was diluted with water and
extracted with ethyl acetate. The ethyl acetate layer was washed with
saturated NaHCO3, saturated brine, and dried over anhydrous
MgSO4. After filtration and concentration, the crude product was
purified by flash chromatography with hexane/ethyl acetate (3/2, v/v)
4-Isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine-6-
carboximidamide hydrochloride (18). To a solution of NaOCH3
(0.18 g, 3.3 mmol) in methanol (5.0 mL) was added 4-isopropyl-1,3-
dimethyl-1H-pyrazolo[3,4-b]pyridine-6-carbonitrile (16) (0.41 g,
0.78 mmol). The mixture was stirred at room temperature and
monitored by TLC. After the 16 disappeared, NH4Cl (0.17 g, 3.2
mmol) was added to the mixture and stirred at 70 oC until the
reaction was completed as determined by TLC. After cooling, the
solvent was removed under vacuum and ethyl acetate was added into
the residue, the suspension was stirred at room temperature for 3 h.
The mixture was filtered and the filter cake was collected as product
hydrochloride salt. 1H NMR (400 MHz, CD3OD) 7.97 (s, 1H, Ar-H),
4.06 (s, 3H, -N-CH3), 3.76 – 3.64 (m, 1H, -CH-), 2.67 (s, 3H, -C-
CH3), 1.36 (d, J = 6.8 Hz, 6H, -CH-CH3).
o
1
to afford 23 (63 mg, 63%). MP: 267-269 C. H NMR (400 MHz,
DMSO-d6) δ 7.82 (s, 1H, Ar-H), 7.65 (s, 1H, Ar-H), 7.38 (s, 1H, Ar-
H), 4.06 (s, 3H, -N-CH3), 3.93 (s, 3H, -O-CH3), 3.74 – 3.60 (m, 1H, -
CH-), 2.65 (s, 3H, -C-CH3), 1.39 (d, J = 6.8 Hz, 6H, -CH-CH3). 13C
NMR (101 MHz, DMSO) δ 164.51, 158.67, 155.54, 151.03, 148.57,
145.23, 144.02, 139.89, 135.87, 113.81, 113.50, 110.02, 105.78,
54.70, 33.72, 29.61, 23.26, 15.39. HRMS (ESI) calcd. for
C19H21ClN7O [M + H]+, 398.1491, found 398.1487. HPLC purity:
99%, tR = 9.2 min.
tert-Butyl
2-(2-chloro-6-methoxyisonicotinoyl)hydrazine-1-
carboxylate (20). To a solution of 2-chloro-6-methoxyisonicotinic
acid (19) (188 mg, 1.0 mmol) in dichloromethane (10 mL) was
added oxalyl chloride (381 mg, 3.0 mmol) and 3 drops of DMF as
catalyst. The mixture was stirred at room temperature for 1 h and the
solvent and excess oxalyl chloride was removed under vacuum. The
residue was dissolved in the dichloromethane. To the solution was
added tert-butyl hydrazinecarboxylate (132 mg, 1.0 mmol) and
triethylamine (303 mg, 3.0 mmol) at 0 oC. The mixture was stirred at
room temperature until the reaction was completed as determined by
TLC. Then, the mixture was washed with water, saturated brine, and
dried over anhydrous MgSO4. After filtration and concentration, the
crude product was collected. 1H NMR (400 MHz, DMSO-d6) δ
4,6-Dichloro-1H-imidazo[4,5-c]pyridin-2-amine (25). To a round-
bottomed flask equipped with a magnetic stir bar were added 2,6-
dichloropyridine-3,4-diamine (24) (0.18 g, 1.0 mmol), cyanogen
bromide (0.16 g, 1.5 mmol), and ethanol (3.0 mL). The reaction
vessel was immersed in a 100 oC preheated oil bath for 12 h until the
reaction was completed as determined by TLC. After cooling,
ammonia solution was added and the precipitated was filtered as
1
gray product 25 (0.13 g, 64%). H NMR (400 MHz, DMSO-d6) δ
11.12 (s, 1H, -NH-), 7.12 (s, 1H, Ar-H), 6.92 (s, 2H, -NH2). 13C
NMR (101 MHz, DMSO) δ 158.62, 146.97, 137.38, 135.12, 130.41,
105.96.
8

4,6-Dichloro-2-isocyanato-1H-imidazo[4,5-c]pyridine (26). To a
round-bottomed flask equipped with a magnetic stir bar were added
4,6-dichloro-1H-imidazo[4,5-c]pyridin-2-amine (25) (0.12 g, 0.6
mmol), triphosgene (89 mg, 0.3 mmol), and toluene (5.0 mL). After
cooling to 0 oC, triethyamine was added to the mixture dropwise and
the reaction mixture was stirred at room temperature for 3 h until the
reaction was completed as determined by TLC. Then, the mixture
was quenched with water and extracted with ethyl acetate. The ethyl
acetate layer was washed with saturated brine and dried over
anhydrous MgSO4. After filtration and concentration, the crude
product was obtained which was used directly for the next step (87
mg, 63%).
was filtered, washed with cold ethanol to afford 32 (0.68 g, 65%) as
a white solid. 1H NMR (400 MHz, CDCl3) δ 6.23 (s, 1H, Ar-H), 5.87
(s, 1H, -NH-), 4.00 (br, 2H, -NH2), 3.92 (d, J = 1.5 Hz, 3H, -N-CH3),
3.51 – 3.36 (m, 1H, -CH-), 2.58 (s, 3H, -C-CH3), 1.36 (d, J = 6.8 Hz,
6H, -CH-CH3).
N-(4,6-dichloro-1H-imidazo[4,5-c]pyridin-2-yl)-2-(4-isopropyl-
1,3-dimethyl-1H-pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1-
carboxamide (33). To a round-bottomed flask equipped with a
magnetic stir bar was added 4,6-dichloro-2-isocyanato-1H-
imidazo[4,5-c]pyridine (26) (0.12 g, 0.6 mmol), 6-hydrazinyl-4-
isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine (32) (0.11 g,
0.48 mmol) and THF (3.0 mL). The reaction mixture was stirred at
room temperature for 12 h until the reaction was completed as
determined by TLC. Then, the mixture was concentrated and purified
by flash chromatography with dichloromethane/methanol (10/1, v/v)
(54 mg, 20%). MP: 143-147 oC. 1H NMR (400 MHz, CD3OD) δ 7.38
(s, 1H, Ar-H), 6.51 (s, 1H, Ar-H), 3.81 (s, 3H, -N-CH3), 3.54 – 3.46
(m, 1H, -CH-), 2.55 (s, 3H, -C-CH3), 1.32 (d, J = 6.8 Hz, 6H, -CH-
CH3). 13C NMR (101 MHz, Acetone-d6) δ 164.33, 154.42, 150.75,
146.59, 146.06, 143.38, 138.99, 120.04, 109.96, 106.29, 103.55,
103.26, 98.20, 32.22, 29.49, 22.31, 14.41. HRMS (ESI) calcd. for
C19H20Cl2N9O [M + H]+, 448.1164, found 448.1175. HPLC purity:
96%, tR = 2.9 min.
5-Chloroimidazo[1,2-a]pyridine-7-carboxylic acid (28). To
a
round-bottomed flask equipped with a magnetic stir bar was added 2-
amino-6-chloropyridine-4-carboxylic acid (27) (0.5 g, 2.9 mmol), 2-
bromo-1-dimethoxyethane (0.61 g, 3.6 mmol) and ethanol (25 mL).
To the mixture was added 48 % HBr (0.33 mL, 2.0 mmol) slowly
through syringe under nitrogen. The reaction vessel was immersed in
a 100 oC preheated oil bath for 3 h. After cooling to room
temperature, the precipitate was filtered to afford 28 (0.45 g, 79%).
1H NMR (400 MHz, DMSO-d6) δ 8.51 (d, J = 1.8 Hz, 1H, Ar-H),
8.34 (s, 1H, Ar-H), 8.31 (d, J = 0.7 Hz, 1H, Ar-H), 7.88 (s, 1H, Ar-
H).
5-Chloroimidazo[1,2-a]pyridine-7-carbonyl azide (29). To
a
N-(5-Chloroimidazo[1,2-a]pyridin-7-yl)-2-(4-isopropyl-1,3-
dimethyl-1H-pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1-carbox-
amide (34). To a round-bottomed flask equipped with a magnetic stir
bar was added 5-chloroimidazo[1,2-a]pyridine-7-carbonyl azide (29)
(0.28 g, 1.26 mmol) and toluene (10 mL). The mixture was refluxed
for 2 h and cooled to 50 ºC before 6-hydrazinyl-4-isopropyl-1,3-
dimethyl-1H-pyrazolo[3,4-b]pyridine (32) (0.27 g, 1.26 mmol) in
THF (10 mL) was added. The reaction was stirred for another 10 h
until the reaction was completed as determined by TLC. Then, the
reaction was diluted with water, extracted with ethyl acetate. The
organic layer was washed with saturated brine and dried over
anhydrous MgSO4. After filtration and concentration, the crude
product was purified by flash chromatography with
dichloromethane/methanol (15/1, v/v) to afford 34 (0.3 g, 57%). MP:
160-163 oC. 1H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H, Ar-
NHCO-), 8.64 (s, 1H, Ar-NH-), 8.50 (s, 1H, -CONH-), 7.88 – 7.72
(m, 2H, Ar-H), 7.59 – 7.40 (m, 2H, Ar-H), 6.37 (s, 1H, Ar-H), 3.71
(s, 3H, -N-CH3), 2.58 – 2.36 (m, 4H, -CH-, -C-CH3), 1.32 – 1.17 (m,
6H, -CH-CH3). 13C NMR (101 MHz, DMSO-d6) δ 160.08, 154.38,
151.23, 146.42, 145.26, 139.13, 137.82, 134.08, 128.64, 125.38,
110.73, 107.82, 107.25, 98.73, 33.20, 29.36, 23.18, 15.26. HRMS
(ESI) calcd. for C19H22ClN8O [M + H]+,413.1600, found 413.1598.
HPLC purity: 95%, tR = 3.3 min.
round-bottomed flask equipped with a magnetic stir bar was added 5-
chloroimidazo[1,2-a]pyridine-7-carboxylic acid (28) (0.45 g, 2.3
mmol), diphenylphosphorylazide (0.63 g, 2.3 mmol) and DMF (10
o
mL). After cooling to 0 C, triethyamine (0.70 g, 6.9 mmol) was
added slowly through syringe under nitrogen. The reaction was
stirred at room temperature for 5 h. Then, the mixture was diluted
with water, extracted with ethyl acetate. The organic layer was
washed with saturated brine and dried over anhydrous MgSO4. After
filtration and concentration, the crude product was purified by flash
chromatography with hexane/ethyl acetate (3/2, v/v) to afford 29
1
(0.28 g, 55%). H NMR (400 MHz, CDCl3) δ 8.39 (s, 1H, Ar-H),
7.92 (d, J = 1.4 Hz, 2H, Ar-H), 7.55 (d, J = 1.4 Hz, 1H, Ar-H).
2-Chloro-6-isopropoxyisonicotinoyl azide (31a). To a round-
bottomed flask equipped with a magnetic stir bar was added 2-
chloro-6-isopropoxyisonicotinic acid (30a) (0.56 g, 2.6 mmol),
diphenylphosphorylazide (1.0 g, 3.6 mmol), and 1,4-dioxane (10
mL). triethylamine (0.37 g, 3.6 mmol) was added slowly through
syringe under nitrogen at 0 °C. The reaction was warmed and stirred
at room temperature for 5 h. The mixture then was diluted with water
and extracted with ethyl acetate. The organic layer was washed with
saturated brine, dried over anhydrous MgSO4. After filtration and
concentration, the crude product was purified by flash
chromatography with hexane/ethyl acetate (20/1, v/v) to afford 31a
(0.43 g, 69%).
N-(2-Chloro-6-isopropoxypyridin-4-yl)-2-(4-isopropyl-1,3-di-
methyl-1H-pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1-carboxamide
(35a). To a round-bottomed flask equipped with a magnetic stir bar
was added 2-chloro-6-isopropoxyisonicotinoyl azide (31a) (48 mg,
0.2 mmol) and toluene (5 mL). The mixture was refluxed for 2 h and
cooled to 50 ºC before 6-hydrazinyl-4-isopropyl-1,3-dimethyl-1H-
pyrazolo[3,4-b]pyridine (32) (34 mg, 0.15 mmol) in THF (5 mL) was
added. The reaction was stirred for another 10 h until the reaction
was completed as determined by TLC. Then, the reaction was diluted
with water, extracted with ethyl acetate. The organic layer was
washed with saturated brine and dried over anhydrous MgSO4. After
filtration and concentration, the crude product was purified by flash
chromatography with dichloromethane/methanol (30/1, v/v) to afford
2-Chloro-6-cyclopropoxyisonicotinoyl azide (31b). To a round-
bottomed flask equipped with a magnetic stir bar was added 2-
chloro-6-cyclopropoxyisonicotinic acid (30a) (0.43 g, 1.9 mmol),
diphenylphosphorylazide (0.73 g, 2.6 mmol), and 1,4-dioxane (10
mL). triethylamine (0.26 g, 2.6 mmol) was added slowly through
syringe under nitrogen at 0 °C. The reaction was warmed and stirred
at room temperature for 5 h. Then, the mixture was diluted with
water and extracted with ethyl acetate. The organic layer was washed
with saturated brine, dried over anhydrous MgSO4. After filtration
and concentration, the crude product was purified by flash
chromatography with hexane/ethyl acetate (20/1, v/v) to afford 31b
(0.31 g, 65%).
1
35a (45 mg, 67%). MP: 110-112 oC. H NMR (400 MHz, Acetone-
6-Hydrazinyl-4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]-
pyridine (32). To a round-bottomed flask equipped with a magnetic
stir bar was added 6-bromo-4-isopropyl-1,3-dimethyl-1H-
pyrazolo[3,4-b]pyridine (15) (1.29 g, 4.8 mmol), hydrazine (4.62 g,
144 mmol) and ethanol (10 mL). The reaction vessel was immersed
in a 100 oC preheated oil bath for 18 h. After cooling, the mixture
d6) δ 8.94 (br, 1H, Ar-NHCO-), 7.78 (br, 2H, -NHCONH), 7.17 (s,
1H, Ar-H), 6.90 (s, 1H, Ar-H), 6.38 (s, 1H, Ar-H), 5.10 – 5.02 (m,
1H, O-CH-), 3.66 (s, 3H, -N-CH3), 3.40 – 3.30 (m, 1H, -CH-), 2.38
(s, 3H, -C-CH3), 1.15 (d, J = 6.8 Hz, 6H, O-CH-CH3), 1.13 (d, J =
6.0 Hz, 6H, Ar-CH-CH3); 13C NMR (101 MHz, Acetone-d6) δ 163.8,
159.5, 155.8, 155.1, 151.0, 148.2, 139.1, 108.0, 106.0, 97.9, 97.8,
9

97.2, 68.4, 54.1, 32.3, 22.3, 21.2, 14.4. HRMS (ESI) calcd. for
C20H26ClN7NaO2 [M + Na]+, 454.1729, found 454.1726. HPLC
purity: 98%, tR = 4.2 min.
N-(2-Chloro-6-(cyclopropylmethoxy)pyridin-4-yl)-2-(4-
isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridin-6-
yl)hydrazine-1-carboxamide (35b). To a round-bottomed flask
equipped with
a magnetic stir bar was added 2-Chloro-6-
cyclopropoxyisonicotinoyl azide (31b) (76 mg, 0.30 mmol) and
toluene (5 mL). The mixture was refluxed for 2 h and cooled to 50
ºC before 6-hydrazinyl-4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-
b]pyridine (32) (51 mg, 0.23 mmol) in THF (5 mL) was added. The
reaction was stirred for another 10 h until the reaction was completed
as determined by TLC. Then, the reaction was diluted with water,
extracted with ethyl acetate. The organic layer was washed with
saturated brine and dried over anhydrous MgSO4. After filtration and
concentration, the crude product was purified by flash
chromatography with dichloromethane/methanol (30/1, v/v) to afford
1
35b (86 mg, 83%). MP: 189-191 oC. H NMR (400 MHz, Acetone-
d6) δ 8.80 (br, 1H, Ar-NHCO-), 7.78 (s, 1H, ArNHCO), 7.63 (s, 1H,
CONH), 7.62 (s, 1H, Ar-H), 7.03 (s, 1H, Ar-H), 6.22 (s, 1H, Ar-H),
3.74 (d, J = 7.2 Hz, 2H, -O-CH2), 3.50 (s, 3H, -N-CH3), 3.23 – 3.08
(m, 1H, Ar-CH-), 2.21 (s, 3H, -C-CH3), 1.76 (t, J = 32.8 Hz, 1H,-
CH-), 0.98 (d, J = 7.2 Hz, 6H, Ar-CH-CH3), 0.25 – 0.22 (m, 2H, -
CH2CH2), 0.04 – 0.01 (m, 2H, -CH2CH2); 13C NMR (101 MHz,
Acetone) δ 161.7, 156.9, 153.7, 152.6, 148.5, 148.4, 145.6, 136.5,
105.8, 103.7, 95.3, 94.1, 68.2, 52.1, 29.7, 19.7, 11.8, 7.2, 0.0. HRMS
(ESI) calcd. for C21H26ClN7NaO2 [M + Na]+, 466.1729, found
466.1723. HPLC purity: 98%, tR = 4.2 min.
B) Sphingosine-1-phosphate receptor binding assay. 32P Labeled
S1P was prepared by incubating sphingosine and [γ-32P]ATP with
sphingosine kinase 1 as previously reported. [32P]S1P was dissolved
in DMSO, and then diluted in the assay buffer [50 mM HEPES-Na
(pH 7.5), 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid-free BSA].
Test compounds in DMSO were pre-incubated with commercial cell
membranes expressing recombinant human S1PRs (1, 2, 3, 4, and 5)
in the assay buffer for 30 min at room temperature. [32P]S1P
solutions were added to give a final volume of 150 µL, 0.1 nM [32P]-
S1P and 1 µg membrane protein per well. Competitive binding was
performed for 60 min at room temperature and terminated by
collecting the membranes onto 96-well glass fiber (GF/B) filtration
plates (Millipore, Billerica, MA). Each filter was washed with 200
µL of assay buffer for a total of five times. The filter bound
radionuclide was measured by a Beckman LS 3801 scintillation
counter using Cherenkov counting. Specific binding was calculated
by subtracting radioactivity that remained in the presence of 1000-
fold excess of non-labeled S1P (1 µM) diluted with binding buffer
from a 1 mM S1P stock solution in DMSO. The reported IC50 values
were calculated using the 4 parameter equation, least-square non-
linear regression curve-fit, with GraphPad Prism software (GraphPad
Software, Inc). Each assay was repeated three times with duplicate
wells for each sample; the reported values (mean SD, nM) are
calculated from average of all assays. Assays for compounds which
showed no activity (IC50 > 1000 nM) were only repeated twice.
10