Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes

Article abstract of DOI:10.1021/acs.jmedchem.0c02274

CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934, a total of 32 compounds, composed of single-ring, 5,6-, and 6,6-fused heteroaromatic cores, were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. Compared to GSK650394 and STO-609, 13 compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge-binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs.

Full text of DOI:10.1021/acs.jmedchem.0c02274

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Article
Hinge Binder Scaffold Hopping Identifies Potent Calcium/
Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor
Chemotypes
Benjamin J. Eduful,⊥⊥ Sean N. O’Byrne,⊥⊥ Louisa Temme,⊥⊥ Christopher R. M. Asquith, Yi Liang,
Alfredo Picado, Joseph R. Pilotte, Mohammad Anwar Hossain, Carrow I. Wells, William J. Zuercher,
Carolina M. C. Catta-Preta, Priscila Zonzini Ramos, André de S. Santiago, Rafael M. Counago,
̃
Christopher G. Langendorf, Kévin Nay, Jonathan S. Oakhill, Thomas L. Pulliam, Chenchu Lin,
Dominik Awad, Timothy M. Willson, Daniel E. Frigo, John W. Scott,* and David H. Drewry*
Cite This: https://doi.org/10.1021/acs.jmedchem.0c02274
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ABSTRACT: CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple
diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify
promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding
scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole
GSK650934, a total of 32 compounds, composed of single-ring, 5,6-, and 6,6-fused heteroaromatic cores, were synthesized. The
compound set was specifically designed to probe interactions with the kinase hinge-binding residues. Compared to GSK650394 and
STO-609, 13 compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had
improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge-binding chemotypes identified several potent
and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs.
diseases. Aberrant activation and overexpression of CAMKK2
have been linked to multiple cancer types including prostate,
breast, ovarian, gastric, and hepatic cancers.8−14,17,18 Knock-
down of CAMKK2 via siRNA or pharmacological inhibition of
CAMKK2 reduced cell proliferation, migration, and invasion as
well as induced apoptosis and cell cycle arrest in numerous
cancer cell lines and tumor models.8,12−14,17,19−21 Mechanis-
tically, CAMKK2 is an important regulator of metabolic and
INTRODUCTION
■
Calcium (Ca2+)/calmodulin-dependent protein kinase kinase 2
(CAMKK2) is a serine/threonine kinase that is one of the
calmodulin (CaM)-binding proteins of the CaMK family.1−3
Ca2+ is an important second messenger that aids in the
regulation of a wide range of signaling events in part through
binding to its intracellular receptor CaM.4 Ca2+-bound CaM
modulates various cellular responses via activation of an array
of downstream proteins including CAMKK2.5 Upon activa-
tion, CAMKK2 phosphorylates and activates its substrates
including CAMK1, CAMK4, AMP-activated protein kinase,
(AMPK) and in some cases AKT. This signal transduction
results in the regulation of many important physiological and
pathological processes.6−16
Received: December 31, 2020
Due to CAMKK2’s important role in cell signaling,
dysregulation of CAMKK2 has been implicated in several
© XXXX The Authors. Published by
American Chemical Society
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inflammatory processes, which are contributory factors to its
impact on cancer growth.9,14
affinity to 14 kinases (<20% activity remaining).33 Among
these kinases were CDKL2, GRK3, and CK2, which are all
overexpressed in several cancer types.34−36 In addition to these
collateral kinase targets, STO-609 potently activates the aryl
hydrocarbon receptor.37 STO-609 is a planar molecule with
poor aqueous solubility, yet it is routinely used at high doses in
cell culture (>10 μM) and in vivo.24 Due to the liabilities of
STO-609, the field would benefit from the discovery and
development of potent and highly selective small-molecule
inhibitors of CAMKK2 as high-quality probes.
In addition, therapeutic interventions targeting CAMKK2
may be beneficial beyond oncology. For example, hepatocel-
lular carcinoma (HCC) is often preceded by non-alcoholic
fatty liver disease (NAFLD), which is driven by several risk
factors including obesity and type 2 diabetes.22−24 Inhibition of
CAMKK2 reduced food intake in mice, and Camkk2-null mice
are leaner than wild-type mice when fed a high-fat diet.25,26
Consistent with these findings, CaMKK2 was recently shown
to be inhibited by liraglutide, a glucagon-like peptide-1
receptor agonist that decreases food intake and promotes
weight loss.27 In relation to skeletal disease, CAMKK2 is
expressed in osteoblasts and osteoclasts, which play an
essential role in bone tissue maintenance.28 Inhibition of
CAMKK2 stimulated bone formation and reversed age-
associated decline in bone health by promoting osteoblast
differentiation and inhibiting osteoclast differentiation.29
Taken together, these findings suggest that inhibition of
CAMKK2 may be effective for the treatment of a variety of
diseases including various types of cancers, metabolic
disorders, and bone diseases like osteoporosis. While
CAMKK2 has emerged as an attractive therapeutic target,
there remains a shortage of high-quality CAMKK2 inhibitors,
which has impaired progress in the field.
We recently conducted an extensive literature search for
CAMKK2 inhibitors to identify starting points for medicinal
chemistry optimization.33 A promising series of potent
CAMKK2 inhibitors were recently disclosed by GlaxoSmithK-
line (GSK).25 The 7-azaindole GSK650394 (also called a
pyrrolopyridine) (Figure 1) was a potent CAMKK2 inhibitor
(IC50 = 0.63 nM). The co-crystal structure of GSK650394 with
CAMKK2 (PDB 6BKU) revealed the critical role of the
carboxylic acid and cyclopentyl moiety (Figure 2a), which was
consistent with the reported structure−activity relationship
(SAR) studies.25 The co-crystal structure reveals that the
nitrogen atoms of the pyridine and pyrrole rings act as
hydrogen bond acceptor and donor pairs, respectively, and
form interactions with the protein backbone of the ATP-
binding site. The acid functionality contributes to critical
hydrogen bonding interactions with both the protonated
amine of Lys194 and the carboxylate group of Glu236 in a
water-mediated manner.38 Additionally, CH−π interactions
(aromatic edge−face interactions) between the aromatic ring
of gatekeeper Phe267 with both the CH in the 2-position of
the 7-azaindole and the methine group in the ortho-position of
the 3-aryl moiety of the inhibitor stabilize GSK650394 in this
orientation. The pendant phenyl group in the 5-position of the
7-azaindole scaffold occupies a hydrophobic pocket which can
potentially be exploited to gain selectivity and potency and
modulate physical properties to optimize pharmacokinetic
parameters. Although a crystal structure of CAMKK2 co-
crystallized with ATP is not available, the cyclopentyl group
appears to mimic at least the position of the ribosyl moiety of
ATP based on the proximity of the cyclopentyl group to the P-
loop of CAMKK2. The 7-azaindole pharmacophore in
GSK650394 is commonly found in kinase inhibitors and is
considered a powerful but sometimes promiscuous hinge
binder.39−43 When screened against a panel of 334 kinases
(Reaction Biology Corporation), GSK650394 inhibited 29
At present, almost all CAMKK2-related pharmacological
studies rely on the use of the ATP-competitive inhibitor STO-
609 to study CAMKK2 signaling events (Figure 1).8,11,17,28−32
Figure 1. Structures of known CAMKK2 inhibitors: STO-609 and
GSK650394.
However, STO-609 is not an ideal chemical tool.33 It binds to
multiple kinases beyond CAMKK2. When screened against a
panel of over 400 wild-type human kinases (KINOMEscan,
Eurofins DiscoverX) at 1 μM, STO-609 bound with strong
Figure 2. (a) Binding mode of the co-crystallized ligand GSK650394 in the ATP-binding site of CAMKK2 (PDB-ID: 6BKU). The image was
generated with PyMOL. The protein is colored in gray. Blue-dashed lines indicate H-bond interactions; green-dashed lines display CH−π
interactions. GSK650394 is shown as purple sticks and the water molecule as a red sphere. Oxygen and nitrogen atoms are colored in red and blue,
respectively. (b) Structure of GSK650394 highlighting important sites for design of new CAMKK2 inhibitors.
B
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Figure 3. New molecules based on scaffold hopping from GSK650394 designed to interrogate several aspects of hinge binding interactions between
CAMKK2 and the proposed inhibitors. Top row: Inhibitors retaining the topology of GSK650394 (5,6-fused ring systems with the cyclopentyl-
substituted benzoic acid moiety attached to the 5-membered ring). Middle row: 5,6-fused systems with the cyclopentyl-substituted benzoic acid
moiety appended to the 6-membered ring. Bottom row: Inhibitors with ring expansion (6,6-fused ring system) or ring deletion (single 6-membered
ring as the hinge-binding core).
a
Scheme 1. Synthesis of Pinacol Boronate Esters 3 and 4
a
Reagents and conditions: (a) cyclopentylmagnesium bromide, THF, −10 to 25 °C, 5 h, then aq HCl; (b) PdCl2(dppf)·CH2Cl2, KOAc,
bis(pinacolato)diboron, 100 °C, 2 h; (c) SOCl2, MeOH, 16 h, 75 °C.
kinases by more than 90%, limiting its utility as a tool
compound for studying CAMKK2 or any other kinase.33
To improve the kinase selectivity of GSK650394, we
targeted replacement of the 7-azaindole with other hetero-
cycles that would modulate interactions with the hinge-binding
residues and in some cases reduce interactions with the hinge-
binding residues. Our compound design strategy is depicted in
Figure 2b. Our central hypothesis was based on the belief that
the 3-position aryl ring, decorated with the carboxylic acid and
cyclopentyl groups, plays a dominant role in CAMKK2
recognition and that inhibitors with enhanced selectivity
would arise from compounds with the binding contribution
from the hinge binder diminished. This led to the synthesis
and evaluation of structurally diverse novel chemotypes as
potential inhibitors of CAMKK2 (Figure 3).
Thus, we performed a scaffold-hopping exercise wherein the
hinge-binding 7-azaindole core of GSK650394 was substituted
with structurally diverse alternate hinge-binding moieties. We
retained the ortho-cyclopentyl benzoic acid moiety hypothesiz-
ing that the interactions observed in the crystal structure would
bias the new set toward CAMKK2 affinity.
attachment to the various hinge-binding pharmacophores via
Suzuki coupling reactions (Scheme 1). 4-Bromo-2-fluoroben-
zoic acid 1 underwent a Grignard reaction with cyclo-
pentylmagnesium bromide to afford the ortho-cyclopentyl-
substituted benzoic acid 2, which was then converted into the
corresponding pinacol boronate ester using Miyaura borylation
conditions yielding 3.44 The analogous methyl ester 4 was
made in a similar fashion following esterification of 2 in
methanol in the presence of thionyl chloride.
3- and 3,5-Substituted Fused 6,5-Ring Systems. Initial
analogues were fused 5,6-ring systems with 3- and 3,5-
substitution patterns that retained the topology of GSK650394
but modify the nature and/or location of heteroatoms in the
ring system and thus can alter the compound’s ability to form
H-bond interactions with the hinge region. GSK650394 is
commercially available, but its matched pair 7, truncated at the
5-position, required synthesis (Scheme 2). 3-Bromo-7-
Scheme 2. Synthesis of Azaindole 7, the Matched Pair for
a
GSK650394
RESULTS
■
Chemistry. To develop analogues of GSK650394 that
could be potent and selective CAMKK2 inhibitor scaffolds, all
our synthesized compounds incorporated the critical pharma-
cophore, ortho-cyclopentyl benzoic acid. In parallel, to
investigate the importance of the pendant phenyl ring, we
synthesized matched pairs with and without this group. We
theorized that removal of the phenyl ring could significantly
lower the cLog P and increase ligand efficiency (LE) if it was
dispensable without being detrimental to potency. To this end,
we first synthesized pinacol borate esters 3 and 4 to enable
a
Reagents and conditions: (a) SEM-Cl, NaH, DMF, 0−21 °C, 2 h;
(b) 4, Pd(OAc)2, K3PO4, P(Cy)3, PhMe/H2O, 80 °C, 16 h; (c)
CF3COOH, CH2Cl2, then NaOAc, EtOH, 21 °C, 24 h; (d) aq
NaOH, MeOH, 75 °C, 1 h, then aq HCl.
C
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a
Scheme 3. Synthesis of N-Methyl Azaindole Hinge Binders 10 and 11
a
Reagents and conditions: (a) 3, Pd2(dba)3, XPhos, Cs2CO3, dioxane, H2O, 120 °C, 16 h; (b) aq LiOH, dioxane, 100 °C, 16 h, then aq HCl; (c) 4,
PdCl2(dppf)·CH2Cl2, Cs2CO3, dioxane, H2O, rt, 16 h; (d) PhB(OH)2, Pd2(dba)3, XPhos, Cs2CO3, dioxane, H2O, 120 °C, 16 h; (e) aq LiOH,
dioxane, 100 °C, 16 h, then aq HCl.
a
Scheme 4. General Route to the Furo- and Thienopyridines
a
Reagents and conditions: (a) (EtO)3CH, PhMe, 100 °C, 2 h or H2SO4, EtOH, reflux, 24 h; (b) ethyl glycolate or ethylthioglycolate, NaH, DME,
0−75 °C, 2.5 h; (c) aq NaOH, EtOH then aq HCl, 100 °C, 2 h; (d) Tf2O, DIPEA, CH2Cl2, −10 to 25 °C, 3 h; (e) 4, Pd(PPh3)4, Na2CO3,
MeOH/CH2Cl2, 90 °C, 1 h; (f) PhB(OH)2, Pd(PPh3)4, Na2CO3, MeOH/CH2Cl2, 90 °C, 1 h or PhB(OH)2, Pd2(dba)3, XPhos, Cs2CO3, dioxane/
H2O 90 °C, 16 h; (g) aq NaOH, MeOH then aq HCl, 1 h; (h) 3, Pd(PPh3)4, Na2CO3, MeOH/CH2Cl2, 90 °C, 1 h.
azaindole 5 was converted to the silylethoxymethyl (SEM)-
protected azaindole and then subjected to Suzuki−Miyaura
conditions to afford cyclopentyl analogue 6. SEM depro-
tection, followed by base-mediated saponification, afforded the
target azaindole 7.
The N-methyl analogues of GSK650394 and 7 were
synthesized as depicted in Scheme 3. To access 10, 5-chloro-
3-iodo azaindole 8b was N-alkylated.45 Consecutive Suzuki
reactions with the ortho-cyclopentyl methyl benzoate 4 and
then phenyl boronic acid placed the key pharmacophore in the
3-position and a phenyl ring in the 5-position. Saponification
of the methyl ester proceeded smoothly affording azaindole 10.
Coupling of boronate ester 4 with 3-bromo-N-methyl
azaindole, followed by saponification of the methyl ester,
afforded target molecule 11.
The synthesis of the structurally similar furopyridine and
thienopyridine cores is described in Scheme 4. Nicotinic acid
derivatives 12a and 13a were converted into ethyl esters 12−
14b and then treated with ethyl glycolate or ethyl thioglycolate
in the presence of sodium hydride (NaH) to give the
respective furopyridines or thienopyridines 12−14c.46 A one-
pot saponification−decarboxylation of the β-keto esters
D
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a
Scheme 5. Synthesis of Pyrazolopyridine and Pyrazolopyrimidine Analogues
a
Reagents and conditions: (a) 3, Pd2(dba)3, XPhos, Cs2CO3, dioxane, H2O, 120 °C, 16 h; 17 via (b) 4, Pd2(dba)3, XPhos, Cs2CO3, dioxane, H2O,
120 °C, 16 h; 18 via (c) PdCl2(dppf)·CH2Cl2, Cs2CO3, dioxane, H2O, 80 °C, 16 h; (d) PhB(OH)2, Pd(PPh3)4, Cs2CO3, dioxane, H2O, 120 °C, 30
min, μW; (e) aq LiOH, dioxane, 100 °C, 16 h then aq HCl.
a
Scheme 6. Synthesis of the Triazolopyridazine Analogues 25 and 26
a
Reagents and conditions: (a) 3, Pd2(dba)3, XPhos, Cs2CO3, dioxane/H2O, 90 °C, 16 h; (b) 4, Pd(PPh3)4, K2CO3, dioxane/H2O, 90 °C, 16 h; (c)
Pd2(dba)3, XPhos, Cs2CO3, dioxane/H2O, 90 °C, 16 h; (d) aq NaOH, MeOH 75 °C, 1 h, then aq HCl.
a
Scheme 7. Synthesis of 2,4-Substituted N-Methyl Azaindoles
a
Reagents and conditions: (a) 4, Pd(PPh3)4, Cs2CO3, dioxane/H2O, 120 °C, 30 min; (b) aq LiOH, dioxane, 100 °C, 16 h, then aq HCl; (c)
PhB(OH)2, Pd(PPh3)4, Cs2CO3, dioxane/H2O, 120 °C, 30 min; (d) 4, Pd(PPh3)4, Cs2CO3, dioxane/H2O, 120 °C, 30 min; (e) aq LiOH, dioxane,
100 °C, 16 h, then aq HCl.
a
Scheme 8. Synthesis of the Imidazopyridine Hinge Binders
a
Reagents and conditions: (a) 4, Pd2(dba)3, XPhos, Cs2CO3, dioxane, H2O, 100 °C, 16 h; (b) CH(OMe)3, H3NSO3, MeOH, rt, 1 h; (c) aq LiOH,
dioxane, 100 °C, 16 h, then aq HCl; (d) benzoic acid, CDI, DMF, 0 °C, 30 min, then 32, 200 °C, 10 min; (e) aq LiOH, dioxane, 100 °C, 16 h,
then aq HCl.
afforded the 2-unsubstituted heterocycles 12−14d. These aryl
alcohols were converted to the corresponding triflates 12−14d,
which were able to undergo Suzuki−Miyaura reactions to yield
analogues 12f, 13g, and 14g.47 12g was obtained directly from
the commercially available 3-bromothieno[2,3-b]pyridine 15e
using Suzuki−Miyaura reaction.
The subsequent set of fused 5,6-ring derivatives synthesized
in Scheme 5 (pyrazolopyridines and pyrazolopyrimidines)
E
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a
Scheme 9. Synthesis of Thieno[3,2-d]pyrimidine and Thieno[2,3-d]pyrimidine Hinge Binders
a
Reagents and conditions: (a) 3, Pd(PPh3)4, Cs2CO3, dioxane/water, 125 °C, μW, 15 min; (b) PhB(OH)2, NaHCO3, CsF, Pd(PPh3)4, dioxane/
H2O, 95 °C, 3 h; (c) 3, Pd(PPh3)4, Cs2CO3, dioxane/water, 125 °C, 15 min; (d) 3, Pd(PPh3)4, Cs2CO3, dioxane/water, 125 °C, μW, 15 min; (e)
4, Pd(PPh3)4, Cs2CO3, dioxane/water, 145 °C, μW, 15 min; (f) PhB(OH)2, NaHCO3, CsF, Pd(PPh3)4, dioxane/H2O, 80 °C, 4 h; (g) aq LiOH,
dioxane, 100 °C, 16 h, then aq HCl.
a
Scheme 10. Synthesis of Quinoline and Quinazoline Hinge Binders
a
Reagents and conditions: (a) 3, Pd2(dba)3, XPhos, Cs2CO3, dioxane/H2O, 90 °C, 16 h; (b) 4, Pd2(dba)3, XPhos, Cs2CO3, rt, 16 h; (c)
PhB(OH)2, Pd2(dba)3, XPhos, Cs2CO3, 120 °C, 16 h; (d) aq LiOH, dioxane, 100 °C, 16 h, then aq HCl; (e) 4, Pd(PPh3)4, K2CO3, dioxane/H2O,
120 °C, 16 h; (f) aq LiOH, dioxane, 100 °C, 16 h, then aq HCl; (g) 4, Pd(PPh3)4, K2CO3, dioxane/H2O, 90 °C, 16 h; (h) (i) DIPEA, Tf2O,
CH2Cl2, 0−21 °C, 2 h, (ii) PhB(OH)2, Pd(PPh3)4, K2CO3, CH2Cl2/MeOH, 90 °C, 1 h; (i) aq NaOH, MeOH, 75 °C, 1 h, then aq HCl; (j) 4,
Pd2(dba)3, XPhos, Cs2CO3, dioxane/H2O, 90 °C, 16 h.
F
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a
Scheme 11. Synthesis of Pyrimidine and Aminopyrimidine Hinge Binders
a
Reagents and conditions: (a) 3, Pd(PPh3)4, Cs2CO3, 1,2-DME, H2O, 120 °C, 0.5 h. (b) 4, Pd(PPh3)4, Cs2CO3, 1,2-DME, H2O, 120 °C, 0.5 h; (c)
aq LiOH, dioxane, 100 °C, 16 h, then aq HCl; (d) PhBr, Pd2(dba)3, XantPhos, NaOtBu, PhMe, 80 °C, 19 h.
dramatically altered the placement of the heteroatoms based
on the parent compound. Analogues 19 and 20 were obtained
by stepwise Suzuki couplings, first with 4 and then with
phenylboronic acid, followed by methyl ester hydrolysis. The
monosubstituted complementary matched pairs were synthe-
sized from commercially available 3-bromopyrazolo[1,5-a]-
pyridine or -pyrimidine via Suzuki couplings with 4 to afford
21 and 22. The same set of reaction conditions was used to
afford the triazolopyridazine analogues 25 and 26 (Scheme 6).
2- and 2,4-Substituted Fused 5,6-Ring Systems. The
first scaffolds in this category were N-methyl azaindoles and are
described in Scheme 7. The disubstituted azaindole 29 was
obtained from coupling phenylboronic acid at the more
reactive 2-position of 2-iodo-4-chloro-N-methyl azaindole,
followed by coupling with intermediate 4 at the 4-position.
Saponification of the methyl ester afforded acid 29. The
monosubstituted compound 30 was synthesized from
commercially available 4-chloro-N-methyl-7-azaindole with a
Suzuki coupling reaction, followed by saponification of the
methyl ester.
Imidazopyridine analogues were obtained from initial
coupling of 2,3-diamino-4-chloropyridine with 4 to afford the
aryl-substituted pyridine 32 (Scheme 8). Imidazopyridine 33
was synthesized by condensation of diamine 32 with trimethyl
orthoformate in methanol, followed by saponification of the
ester to afford the desired carboxylic acid. The 2-aryl analogue
34 was obtained by converting benzoic acid into an activated
ester with 1,1′-carbonyldiimidazole (CDI) and addition of
diamine 32 to form a putative amide intermediate that
underwent a ring closure under thermal conditions resulting in
the imidazopyridine intermediate. Ester hydrolysis yielded the
desired imidazopyridine 34.
mediate 36, which was then coupled with 3 to yield the
disubstituted thieno[3,2-d]pyrimidine. The reaction sequence
was reversed in the synthesis of the disubstituted thieno[2,3-
d]pyrimidine 41 since the chlorine substituent in 39b was
found to be more reactive than the thiophene bromine.
Intermediate 4 was coupled to the 4-position of 39b affording
40. Subsequent Suzuki coupling with phenylboronic acid,
followed by saponification of the ester, yielded disubstituted
analogue 41.
Synthesis of Substituted Fused 6,6-Ring Hinge
Binders. This set of hinge binders moves from 5,6-fused
ring systems to 6,6-fused systems. The disubstituted quinoline
analogue 45 was synthesized in a straightforward manner from
commercially available 4,6-dichloroquinoline 43b via sequen-
tial Suzuki couplings with 4 and phenylboronic acid, followed
by saponification of the methyl ester. The monosubstituted
quinoline 46 was synthesized in one step via Suzuki coupling
between the commercially available 43a and 3.
The quinazoline hinge binder derivatives were obtained via
similar routes utilized in the synthesis of the quinolines
(Scheme 10) with the exception of compound 49. The
disubstituted quinazoline 49 was prepared from 4-chloroqui-
nazolin-6-ol 47b with initial installation of boronate 4 and
subsequent conversion of the hydroxy group of 48 into a
triflate which readily underwent Suzuki coupling with phenyl-
boronic acid. Saponification then afforded the target
compound. 4-Aryl-2-methylquinoline 52 and 1,6-naphthyr-
idine 54 analogues were synthesized in one step via Suzuki
coupling with 3 (Scheme 10c).
Synthesis of Pyrimidine Hinge Binders. The final set of
hinge binders we explored consisted of substituted pyrimi-
dines. The monosubstituted pyrimidine 56 was obtained via
Suzuki coupling of 55 with 3 (Scheme 11a). 2-Phenyl-4-aryl
pyrimidine 58 was synthesized from 2-phenyl-4-chloropyr-
imidine 57 by Suzuki coupling with 3. The synthesis of
aminopyrimidines 61 and 62 is described in Scheme 11b.
Starting from commercially available 4-chloropyrimidin-2-
amine 59, Suzuki coupling with 4 afforded 60. Saponification
of the resulting methyl ester afforded 61. 60 was utilized in a
Mono- and disubstituted thienopyrimidine analogues of the
[3,2-d] and [2,3-d] core scaffolds were synthesized from
commercially available starting materials (Scheme 9). The
monosubstituted analogues 38 and 42 were easily accessed in
one step via Suzuki coupling using commercially available 35a
and 39a, respectively. 37 was synthesized by first coupling 35b
with phenylboronic acid to afford 2-aryl-substituted inter-
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a
Table 1. CAMKK2 Enzyme Inhibition Data for Compounds with 3- and 3,5-Substituted Fused 5,6-Ring Systems
a
R = ortho-cyclopentyl benzoic acid moiety; PoC (percent of control = percent of enzyme activity remaining, when compared with the control);
IC50 (half-maximal inhibitory concentration); NG (not generated); IC50 values were generated in an 8-point full dose response assay.
Buchwald−Hartwig coupling with bromobenzene, and sub-
sequent ester hydrolysis yielded 2-phenylaminopyrimidine 62.
Assays Used for In Vitro Compound Affinity Evalua-
tion. DSF Assay. We used a thermal-shift assay (differential
scanning fluorimetry, DSF) to detect protein−ligand inter-
actions.48 For many proteins, including kinases, this is a useful
high-throughput method to identify compounds that bind to a
protein of interest, and in many cases, the melting temperature
correlates with binding affinity. CAMKK1 and CAMKK2 are
∼60% identical at the amino acid sequence level.49 We
measured DSF ΔTm for all analogues against both CAMKK2
and CAMKK1 using the purified kinase domains of these two
proteins. As in the end we did not utilize the DSF data to drive
the project, all DSF data are reported in the Supporting
Information.
the assay to ensure that the assay performed correctly and
CAMKK2 inhibition was observed.
In Vitro Compound Screening Results. The initial set of
compounds evaluated was focused on those with similar
topology to GSK650394. Data in Table 1 depict compounds,
the enzyme CAMKK2 inhibition data (PoC) at three
concentrations, and CAMKK2 enzyme inhibition IC50 data
for selected compounds. Although we collected thermal shift
(DSF) data for our inhibitors with both CAMKK1 and
CAMKK2, we found that they did not provide enough
information to drive chemistry decisions and did not always
correlate with the enzymatic activity. The DSF data are
provided in the Supporting Information for reference. We thus
relied on the CAMKK2 enzyme inhibition data as our primary
assay.
In Vitro CAMKK1 and CAMKK2 Enzyme Inhibition Assays.
We used purified recombinant CAMKK1 and CAMKK2 in an
assay that measured the transfer of radiolabeled phosphate
from [γ-32P]-ATP to a synthetic CAMKK2 substrate
(CAMKKtide). Initially, we evaluated CAMKK2 inhibitory
activity of all synthesized compounds at three different
concentrations (10, 100, and 1000 nM) to rank compounds
and provide preliminary dose response information. In the
percent of control (PoC) experiments, the assay with no
inhibitor present serves as the control. Half-maximal inhibitory
concentrations (IC50) were then determined for analogues
with PoC values <10 at the 1 μM screening concentration. For
a subset of CAMKK2 inhibitors, we also determined half-
maximal inhibitory concentration (IC50) values. STO-609 and
GSK650394 were routinely used as reference compounds in
Removal of the phenyl group from the 5-position of
GSK650394 led to a roughly 3-fold decrease in enzyme
inhibitory activity (IC50 = 26 vs 3 nM), implying that the
phenyl group or an appropriately adorned phenyl group may
be useful to enhance CAMKK2 enzyme affinity and/or activity.
Similar pairs of analogues for the different hinge binders
allowed for direct comparison (Table 1).
The goal of this project was to discover alternate scaffolds
for CAMKK2 with sufficient potency and suitable kinome
selectivity that can be used as starting points for medicinal
chemistry optimization programs. We hypothesized that
carefully modulating the hydrogen bonding interactions at
the hinge-binding region via a variety of heterocyclic scaffolds
could identify a new series that maintained CAMKK2 affinity
and improved selectivity profiles. Kinase inhibitors with
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a
Table 2. CAMKK2 Enzyme Inhibition Data for Compounds with 2- and 2,4-Substituted Fused 6,5-Ring Systems
a
R = ortho-cyclopentyl benzoic acid moiety; PoC (percent of control = percent of enzyme activity remaining, when compared with control); IC50
(half-maximal inhibitory concentration); IC50 values were generated in an 8-point full dose response assay.
multiple hinge-binding interactions can suffer from off-target
effects owing to their potential for poor selectivity across the
kinome.39,41,50 However, careful modification of even pro-
miscuous starting points can in some cases lead to desired
levels of selectivity. There are examples demonstrating that
modifications to the hydrogen bonding interactions between a
kinase inhibitor and the hinge region of the kinase can improve
selectivity without severely impacting potency.41 N-Methyl
azaindoles 10 and 11 (Table 1) offer a particularly drastic
change in hinge binding by blocking the H-bonding donor
property of the pyrrole ring. This resulted in a complete loss in
enzyme inhibition potency in vitro. This suggested that the
steric bulk of the N-methyl group is not accommodated within
the binding pocket of CAMKK2 when the cyclopentyl benzoic
acid moiety is attached at the 3-position of the azaindole
scaffold.
Further structural modifications to the hinge-binding moiety
that maintained the fused 5,6-ring system are outlined here.
Compounds 13g and 12f replaced the pyrrole ring with a furan
ring. Compounds 14g and 12g replaced the pyrrole ring of
GSK650394 with a thiophene ring. The pyridine nitrogen that
makes a key hydrogen bond with the hinge is maintained, but
the NH hydrogen bonding opportunity has been removed.
Compounds 19, 20, 21, 22, 25, and 26 all maintain the 5,6-
fused core but have key differences from GSK650394. They do
not have a nitrogen in a position analogous to the nitrogen in
the 7-position of GSK650394, thus necessitating different
hinge-binding interactions. They also incorporate nitrogen
atoms in other locations within the 5,6-ring system.
point out that an analogue of compound 13g is listed as a
CAMKK2 chemical probe (https://www.thesgc.org/chemical-
probes/SGC-CAMKK2-1) with the name SGC-CAMKK2-1.
The kinome-wide selectivity profiles and cellular potencies of
these two close analogues are comparable (vide infra and the
SGC probe link above). In addition, SGC-CAMKK2-1 is
currently commercially available from Sigma-Aldrich. The
results depicted in Table 1 demonstrate that the choice of a
hinge binder plays a role in the type of substitutions that are
tolerated. Both versions of the triazolopyridazine hinge binder
(25, 26) showed no activity. We hypothesize that this is due to
repulsion between the lone pair on the nitrogen in the 2-
position and a backbone protein carbonyl (carbonyl from E268
in CAMKK2). In many kinase/inhibitor co-crystal structures,
this carbonyl is engaged in hydrogen bonding with a NH from
the inhibitor or at least pointing toward a polarized CH on the
inhibitor. In summary, four scaffolds (furopyridine 13g,
thienopyridine 14g, pyrazolopyridine 19 and 21, and
pyrazolopyrimidine 20) in this set exhibited robust
CAMKK2 enzyme inhibition (IC50 values < 150 nM), with
14g (IC50 = 5 nM) exhibiting comparable enzymatic inhibitory
activity to GSK650394 (IC50 = 3 nM).
To further evaluate fused 5,6-ring structures as CAMKK2
inhibitors, we switched from 3,5- to 2,4-ring substitutions as
depicted in Table 2. N-methyl azaindoles 29 and 30 were the
first pair of analogues synthesized. Interestingly, these two N-
methyl-substituted azaindole analogues were well tolerated,
displaying good CAMKK2 enzyme inhibitory activity (29 IC50
= 120 nM, 30 IC50 = 56 nM). This result is in stark contrast to
3,5-N-methyl azaindoles 10 and 11 in Table 1. The truncated
analogue 30 was significantly more potent than its correspond-
ing phenyl analogue 29.
Nearly all the phenyl-substituted analogues of active
CAMKK2 inhibitor scaffolds showed greater enzyme potency
than their corresponding truncated analogues. An exception
was compound 19 (phenyl version), which had an IC50 = 145
nM compared to an IC50 = 44 nM for compound 21
(truncated version). Replacement of the pyrrole ring in the
azaindole with either a furan or thiophene ring retained
potency in the phenyl-substituted analogues (13g IC50 = 65
nM, 14g IC50 = 5 nM), but their corresponding truncated
analogues (12f, 12g) were relatively inactive. We would like to
The 2,4-substituted analogues of Table 2 are all inhibitors of
CAMKK2. Incorporation of an extra nitrogen into the pyrrole
ring to create imidazopyridine analogues 33 and 34 was well
tolerated, with the phenyl-substituted analogue demonstrating
higher potency (34 IC50 = 23 nM) than the corresponding
non-phenyl version (33 IC50 = 193 nM). Similar to N-methyl
azaindole 30, non-phenyl-substituted thienopyrimidines (38
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a
Table 3. CAMKK2 Enzyme Inhibition Data for Compounds with Fused 6,6-Ring Systems
a
R = ortho-cyclopentyl benzoic acid moiety; PoC (percent of control = percent of enzyme activity remaining, when compared with the control);
IC50 (half-maximal inhibitory concentration); NG (not generated); IC50 values were generated in an 8-point full dose response assay.
a
Table 4. CAMKK2 Enzyme Inhibition Data for Substituted Pyrimidines and 4-Aminopyrimidines
a
R = ortho-cyclopentyl benzoic acid moiety; PoC (percent of control = percent of enzyme activity remaining, when compared with the control);
IC50 (half-maximal inhibitory concentration); NG (not generated); IC50 values were generated in an 8-point full dose response assay.
IC50 = 24 nM, 42 IC50 = 31 nM) were more potent than their
corresponding phenyl-substituted analogues (37 IC50 = 169
nM, 41 IC50 = 232 nM). Both [3,2-d] analogues 37 and 38
(with the sulfur oriented away from the hinge-binding region)
and [2,3-d] analogues 41 and 42 (with the sulfur oriented
toward the hinge) inhibit CAMKK2. For both these isomeric
thienopyrimidines, removal of the pendant phenyl actually
increases CAMKK2 potency (37 with phenyl IC50 = 169 nM
38 without phenyl IC50 = 24 nM; 41 with phenyl IC50 = 232
nM 42 without phenyl IC50 = 31 nM).
Next, we explored replacement of the 5,6-bicyclic cores with
6,6-bicyclic structures. Quinazolines and quinolines are
frequently used as kinase hinge-binding heterocycles and
were identified as active scaffolds for CAMKK2 inhibition
(Table 3). Both quinoline 45, with a phenyl in the 6-position,
and 46, unsubstituted in the 6-position, inhibited CAMKK2
(45 IC50 = 137 nM, IC50 46 = 13 nM). The potency of the
low-molecular-weight compound 46 suggests that further
exploration may be fruitful. Similarly, the two quinazolines
49 (IC50 = 12 nM) and 50 (IC50 = 96 nM) were also potent
CAMKK2 inhibitors.
activity. The steric bulk of the 2′ methyl group is not tolerated
and likely hinders the ability of the quinoline nitrogen to
effectively participate in hydrogen bonding with the hinge
region. Naphthyridine 54 showed poor CAMKK2 enzymatic
activity.
Finally, we evaluated a small set of pyrimidines, well-known
kinase ATP-competitive inhibitor scaffolds, for their CAMKK2
inhibitory activity (Table 4). Pyrimidine 56, with a phenyl in
the 2-position, had a CAMKK2 IC50 = 21 nM. 2-Anilino-
pyrimidine 62 was also potent with CAMKK2 IC50 = 51 nM.
Pyrimidine 58, unsubstituted in the 2-position, lost consid-
erable activity relative to 56. 2-Amino-pyrimidine 61 retained
CAMKK2 potency (IC50 = 108 nM).
X-ray Crystallography and In Silico Docking Studies.
In order to more fully understand the binding modes and to
plan optimization studies on these scaffolds, we turned to X-
ray crystallography and in silico docking analysis of key
molecules. We previously reported the crystal structure of 7-
azaindole GSK650394 bound to CAMKK2 (PDB ID
6BKU).51 A crystal structure of the closely related 7-azaindole
GSK650393 is also available (PDB ID 6CMJ).25 These two
structures clearly demonstrate a hydrogen bond interaction
between the NH of the pyrrole in the 7-azaindole to the
As expected, introduction of a methyl group at the 2-
position of 52 resulted in a complete loss of CAMKK2 enzyme
J
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Table 5. Crystallization Conditions and Data Collection Statistics
ligand
13g
UNC10244803
data collection
X-ray source
wavelength (Å)
space group
APS 24-ID-C
0.9791
P43212
DLS I03
0.9763
P43212
cell dimensions
a, b, c (Å)
α, β, γ (deg)
resolution (Å)
no. of unique reflections
Rmerge (%)
73.3, 73.3, 122.0
90.0, 90.0, 90.0
19.74−1.70 (1.73−1.70)
37,374 (1,767)
14.7 (183.7)
73.2, 73.2, 120.3
90.0, 90.0, 90.0
19.61−1.60 (1.63−1.60)
43,864 (3,161)
12.2 (31.5)
mean I/σI
14.2 (2.0)
21.3 (10.9)
mean CC(1/2)
completeness (%)
redundancy
1.0 (0.75)
99.9 (100)
21.7 (22.1)
1.0 (0.99)
99.9 (100)
25.8 (25.0)
refinement
resolution (Å)
19.75−1.70 (1.75−1.70)
19.61−1.60 (1.64−1.60)
R
cryst/Rfree (%)
16.0/18.2
15.3/17.1
no. of non-hydrogen atoms/mean
B-factor (Å)
protein atoms
solvent atoms
ligand atoms
rmsd bond lengths (Å)
rmsd bond angles (deg)
Ramachandran statistics (%)
favored
2230/28.1
370/44.3
29/19.8
0.010
2229/21.3
338/33.6
26/12.5
0.01
1.05
1.04
98.2
1.8
98.2
1.8
allowed
PDB ID
5UY6
5UYJ
crystallization conditions
25% PEG 3350, 0.2 M ammonium sulfate, 0.1 M bis−tris 20% PEG 3350, 0.02 M ammonium sulfate, 0.1 M CHC buffer
buffer, pH 6.5
system, pH 8.5
carbonyl group of Glu268 at the CAMKK2 hinge region. The
nitrogen atom at the 7-position forms an interaction with the
hinge via the hydrogen bond with the NH of Val270. We were
able to obtain a crystal structure of furopyridine 13g (PDB ID
5UY6). Data collection statistics and crystallization conditions
are shown in Table 5.
Furopyridine 13g displays a similar binding mode to
GSK650394. The 5,6-fused ring systems are oriented in the
same way in the CAMKK2 active site, and the pyridine
moieties of the two heterocycles form comparable hydrogen
bond interactions with the NH group of Val270, and the furan
oxygen atom and pyrrole NH group are in the same region of
space. Although we have no crystal structure of thienopyridine
14g, we hypothesize that it would likely bind in a similar
fashion.
the original pose from the crystal structures. The root-mean-
square deviation (rmsd) value was lower than 1 Å, indicating a
reliable docking procedure. The predicted binding modes of
active compounds (Figure 3) show conserved H-bonds or
close proximity between the compound’s carboxylate group
and both the protonated amine of Lys194 and the carboxylate
group of Glu236 in a water-mediated manner, analogous to
what was observed in the crystal structures. The overall
orientation of the tested compounds and their interactions
with the hinge region is also of interest.
As expected, the predicted binding mode of thienopyridine
14g overlaid almost perfectly with the pose of furopyridine 13g
in the X-ray structure (Figure 4a). The backbone NH group of
Val270 and the thienopyridine N-atom are well positioned for
a hydrogen bond interaction. Interestingly, there appears to be
an additional favorable interaction between the sulfur atom of
14g and the carbonyl group of Glu268. Inter- and intra-
molecular interactions between sulfur and oxygen atoms are
relatively common and can be used to the medicinal chemists’
advantage.53,54
We were also able to obtain a crystal structure of quinoline
UNC10244803 (PDB ID: 5UYJ). The quinoline nitrogen
atom is positioned to form a hydrogen bond with the NH
group of Val270. The predicted binding pose of 46 (Figure 4b)
is highly comparable to the binding mode of the co-crystallized
ligand UNC10244803. Similar to quinoline 46, 2-phenyl-
pyrimidine 56 has only one heteroatom that is capable of
forming hydrogen bonds with the hinge (Figure 4c). The N-
atom of pyridine in the 1-position of 56 shows a H-bond with
the NH group of Val270. Together with the presumed H-bond
To assess the binding modes of 14g and other compounds,
we performed in silico docking studies with the docking
52
̈
software Glide version 2014 (Schrodinger). For our docking
studies, we utilized three of the X-ray crystal structures of
CAMKK2 (PDB-ID: 6BKU, 5UY6, and 5UYJ). We based our
choice of the CAMKK2 PDB protein structure to use for our
modeling on the structural similarity between the co-
crystallized ligand and the compound we planned to use in
the in silico docking. In order to ensure reliability of the
docking protocol and the ensuing results for hypothesis
generation, the co-crystallized ligands GSK650394, 13g, and
UNC10244803 [2-cyclopentyl-4-(7-methoxyquinolin-4-yl)-
benzoic acid, CAMKK2 enzyme IC50 = 33 nM] were removed
from the binding site and the ligands were re-docked into the
binding pocket. The docking poses were then compared with
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Figure 4. X-ray structures and in silico docking. In silico docking was performed with Glide, and images were generated with PyMOL. The protein
is colored in gray. Blue-dashed lines indicate H-bond interactions, while green-dashed lines display CH−π interactions and orange-dashed lines
refer to sulfur−σ hole bonding. Docked ligands are shown as yellow sticks or orange sticks, co-crystallized ligands as purple sticks, and the water
molecule as a red sphere. Oxygen and nitrogen atoms are colored in red and blue, respectively. (a) Predicted binding mode of 14g (yellow) using
the protein structure from PDB-ID 5UY6 compared to the co-crystallized ligand 13g (purple). (b) Predicted binding modes of 46 (yellow) using
the protein structure from PDB-ID 5UYJ and the co-crystallized ligand UNC10244803 (purple). (c) Predicted binding mode of 56 using the
protein structure from PDB-ID 5UYJ. (d) Predicted binding mode of 22 using the protein structure from PDB-ID 6BKU. (e) Predicted binding
modes of 10 (orange) and 29 (yellow) using the protein structure from PDB-ID 6BKU (light gray) and 5UYJ (dark gray), respectively. (f)
Predicted binding mode of 38 using the protein structure from PDB-ID 5UYJ.
formed between the protonated N-atom of Lys194 and the
carboxylate group of 56, these two interactions anchor 56 in
the active site. In this orientation, the phenyl ring in the 2-
position is tolerated by the binding pocket. 2-Aryl-pyrimidines
are much less common as kinase inhibitors than 2-
anilinopyrimidines and so warrant further exploration. The 2-
position phenyl ring of 56 is adjacent to Leu269 of CAMKK2.
We speculate that kinases incorporating amino acids with
larger side chains such as Phe or Tyr in this location at the
hinge region will be less tolerant of this scaffold, perhaps
offering a path to enhance selectivity.
4f) to those of 56. Additionally, the orientation of 38 is further
stabilized by an interaction that is formed between the O-atom
of the carbonyl moiety of Ile171 and the S-atom of the
thienopyrimidine scaffold.
Compound Selectivity. In order to begin to understand
kinome-wide selectivity for these new compounds, we profiled
9 exemplars in a panel of over 400 wild-type human kinases
using the Eurofins DiscoverX’s KINOMEscan technology50
(Freemont, CA USA). A summary of the kinome selectivity
results is depicted in Table 6, and the complete KINOMEscan
data sets are available in the Supporting Information. Table 6
contains the structures of the compounds profiled, the PoC
values for CAMKK1 and CAMKK2 at the 1 μM screening
concentration, and a list of all the kinases in the panel that
bound with a PoC < 10. The S-score is a quantitative measure
of a compound’s selectivity at a particular screening
concentration, calculated by dividing the number of kinases
that a particular compound binds to at a chosen threshold by
the total number of distinct kinases tested. All the compounds
we tested from this scaffold-hopping effort have an S10 (1 μM)
< 0.05, which implies that they bind to fewer than 5% of the
wild-type kinases tested in this panel with a PoC of 10 or less,
thus offering promising selectivity for a starting point for
further optimization.
Pyrazolopyrimidine 22 (Figure 4d) forms one hinge-binding
interaction between the N-atom in position 1 and the
backbone NH group of Val270. The distance between the
+
NH3 group of Lys194 and the carboxylate group of 22 is
greater compared to the predicted binding modes of the other
compounds (Figure 4b,e). The N-methyl group of compound
10 (Figure 4e) appears to preclude binding of this compound
in the same orientation as GSK650394. This compound is
inactive in the enzyme assay, and the docking predicts a flipped
orientation of the compound. N-methyl compound 29,
however, retains activity, and the in silico docking result
suggests a conformation that allows the pyridyl nitrogen atom
to interact with the NH group of Val270. The methyl group in
this orientation is tolerated, perhaps analogously to the 2-
phenyl of compound 56. This flipped binding mode is possibly
due to the shift of the attachment position of the cyclopentyl
benzoic acid moiety from the 5-position of 10 to the 4-position
in 29. The hinge-binding interactions shown in the predicted
binding mode of thienopyrimidine 38 are comparable (Figure
Figure 5 is a visual representation of the KINOMEscan data
for two of the compounds, azaindole compound 7 and 2-
anilino-pyrimidine compound 62. All kinases bound with a
PoC < 10 are depicted as red dots. These kinases are listed in
Table 5, and the full data sets are available in the Supporting
Information. For compound 7, 12 kinases have a PoC less than
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Table 6. Selectivity Screening Summary Results for Nine Different Exemplars
or equal to 10, including CAMKK2 and CAMKK1. For
compound 7, S10 (1 μM) = 0.03 implies that 3% of the kinases
tested had a PoC < 10 at an inhibitor screening concentration
of 1 μM. For compound 62, only two kinases (CAMKK1 and
CAMKK2) have a PoC < 10. S10 (1 μM) = 0.005 implies that
0.5% of the kinases tested had a PoC < 10 at an inhibitor
screening concentration of 1 μM. Next steps will need to
include testing of the compounds in enzyme inhibition assays
and cell-based assays for the identified kinases for each scaffold.
CAMKK1 activity. CAMKK1 is a closely related and also
understudied kinase. In order to learn if selectivity between
CAMKK1 and CAMKK2 may be attainable in these series, we
determined IC50 values for CAMKK1 inhibition for the 18
compounds with CAMKK2 IC50 < 150 nM, with the results
depicted in Table 7. STO-609 is reported to have some
selectivity for CAMKK2 over CAMKK1, and this is
recapitulated in our assays.55 In general, the compounds are
more potent inhibitors of CAMKK2 than CAMKK1.
M
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Figure 5. In vitro kinase selectivity profile of compound 7 (5a) and compound 62 (5b) at 1 μM (Eurofins DiscoverX KINOMEscan). TREEspot
interaction maps for 7 and 62 profiled against >400 human kinase targets. In this assay, primary screen binding interactions are reported as PoC,
where lower values are an indication of stronger hits. Red dots depict the kinases for which PoC at 1 μM < 10. The size of the dot is based on the
PoC values, with larger dots having smaller PoC values (more potent binders). PoC values for all kinases can be found in the Supporting
Information.
Table 7. CAMKK1 and CAMKK2 Enzyme Inhibition Data, CAMKK2 NanoBRET in Cell Target Engagement, LE Metrics, and
a
cLog P and Solubility Data for Potent CAMKK2 Inhibitors
ID
CAMKK1 enzyme IC50 [nM] CAMKK2 enzyme IC50 [nM] CAMKK2 NB IC50 [nM] cLog P
LE
LLE
solubility [μg/mL]
STO-609
GSK650394
7
408 22
33 6.9
58 5.7
0.4
26 6.1
65 15
NG
<3
NG
700 9.3
210 24
530 95
190 19
200 11
3.59
5.67
3.78
5.68
6.32
6.01
5.17
4.13
6.06
3.97
5.42
3.99
3.78
6.59
4.7
0.41
0.40
0.45
0.34
0.39
0.32
0.36
0.44
0.32
0.42
0.36
0.45
0.45
0.32
0.45
0.36
0.40
0.41
0.46
0.37
3.61
2.83
3.82
1.52
1.98
0.79
2.53
3.27
0.84
3.33
2.18
3.61
3.72
0.31
3.2
60
2
3
195 25
961 132
384 53
1480 120
238 32
2013 174
>10,000
13g
14g
19
20
21
29
30
34
38
42
45
46
49
50
56
61
62
77
1.5
42
50.5
48.3
5
1.1
145 30
21 1.3
44 9.1
120 21
56 14
23 4.2
24 1.1
31 4.3
137 12
13 1.0
12 3.9
96 14
21 2.6
108 6.9
51 13
240
7
1345 222
27 4.5
470 44
8.1 0.8
170 18
1400 250
540 62
140 12
890 71
1900 200
290 23
950 110
<3
40
55.1
55.4
62.6
38
64.4
70.4
407 25
7280 216
1838 348
239 20
2614 349
5481 130
>10,000
5.86
3.97
4.68
2.57
5.38
2.04
3.03
3.02
4.43
1.92
19.4
10.6
>10,000
31 6.5
a
NB = data from the NanoBRET in the cell target engagement assay; NG = data not generated.
Compound 34 (CAMKK1 IC50 = 27 nM, CAMKK2 IC50 = 23
nM) and compound 62 (CAMKK1 IC50 = 31 nM, CAMKK2
IC50 = 51 nM) are exceptions, being equipotent or slightly
more potent on CAMKK1. For STO-609, a switch in a residue
near the inhibitor hinge-binding group from a valine in
CAMKK2 (Val269) to a leucine in CAMKK1 (Leu233) has
been hypothesized as the source of selectivity.56 Further
experiments will need to be done to understand and exploit the
differences in CAMKK1 and CAMKK2 activity observed for
the inhibitors described here.
Compound Properties. A number of key physicochemical
screens and calculations are considered in early-stage drug
discovery, including lipophilicity, pKa, solubility, permeability,
and stability. Solubility is one of the most critical
physicochemical properties as poor solubility can lead to an
underestimation of activity, inaccurate SAR, inaccurate in vitro
ADMET test results, and downstream compound development
N
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issues.57,58 Highlighting the critical importance of solubility, it
has been shown that 87% of commercial drugs have solubility
≥65 μg/mL, but only 7% had solubility ≤20 μg/mL.59
Therefore, solubility data can identify a series liability that
needs to be fixed and highlight promising series with enhanced
solubility. This information can help guide optimization
strategies. LE metrics are also a commonly used tool that
can guide hit-to-lead optimization for drug candidates.60,61 To
this end, we calculated LE and lipophilic ligand efficiency
(LLE) and collected kinetic solubility data for compounds with
IC50 values <150 nM in the CAMKK2 enzyme assay (Table
7).62
LE values are influenced by molecular size; hence, it was
unsurprising to see two clear ranges depending on the presence
or absence of the phenyl ring. The hinge binders without the
phenyl ring had LE values ranging from 0.41 to 0.46. These are
smaller compounds with molecular weights between 300 and
325, and generally, LE values above 0.3 are acceptable for this
MW range.60 The LLE values for these compounds were
between 3.02 and 4.43. Aminopyridine 61 had the highest LE
and LLE values of 0.46 and 4.43, respectively. Both 7 and 38
had comparable LE values of 0.45 but significantly lower LLE
values of 3.82 and 3.61, respectively.
Both the LE and LLE metrics of the phenyl-substituted
analogues have lower values. This is reflective of the increased
atom count and higher cLog P values due to the additional
lipophilicity of the phenyl ring. The LE values are between
0.32 and 0.40 for these compounds. GSK650394 has the
highest LE and LLE values of 0.40 and 2.83, respectively. The
analogous thienopyridine 14g has the next highest LE value of
0.39 but a lower LLE value of 1.98. Quinoline 45 had the
lowest LE and LLE values of 0.32 and 0.31, respectively. Solely
relying on LE metrics to select compounds is unadvisable as
many other factors, such as solubility, underpin successful drug
development. A future direction could be to focus on polar
substituents on the pendant phenyl to lower log P, enhance
solubility, and mitigate the metabolic liability that may be seen
in high-logP compounds. Similarly, non-aromatic substituents
could be explored to access this same region of the active site.
Of the phenyl-substituted analogues, GSK650394 and 14g
were the most potent, with IC50 values of 3 and 5 nM,
respectively, and had the best LE and LLE values. Although
promising in this regard, these compounds have extremely
poor solubility values of 2 and 1.5 μg/mL, respectively.
Optimization of these compounds will thus require extensive
structure−property-relationship studies in parallel to SAR
studies to develop them into useful tools with adequate
solubility for further study. Interestingly, furopyridine 13g had
the highest measured solubility value of 77 μg/mL.
Substitution of the azaindole’s NH with an oxygen greatly
improved solubility. The effect was reversed when a more
lipophilic sulfur atom is incorporated into the same position,
14g. Pyrimidine analogues 56 and 62 are also only modestly
soluble (10.6 and 19.4 μg/mL). Optimization to tool
compounds will likely require the introduction of solubilizing
functional groups. A number of the compounds tested had
reasonable solubility ranging from 38 to 55 μg/mL. Four
compounds had a promising solubility above that recorded for
STO-609, 60 μg/mL. These were the thienopyrimidine 42
(62.6 μg/mL) and quinoline 46 (64.4 μg/mL), both without
the pendant phenyl ring. The phenyl-substituted quinazoline
49 has a solubility of 70.4 μg/mL, second only to furopyridine
13g. Overall, the solubility of several potent compounds was
encouraging but will need to be monitored as optimization
proceeds.
CAMKK2 NanoBRET Cellular Target Engagement.
Having demonstrated potent CAMKK2 inhibition and
promising selectivity profiles for these compounds we tested
in kinome-wide profiling, we turned our attention to the
cellular activity of the compounds (Table 7). To this end, we
developed a CAMKK2 NanoBRET target engagement assay.63
This assay uses bioluminescence resonance energy transfer
(BRET) between nanoluciferase (NL) fused to the kinase
domain of CAMKK2 (BRET donor) and a tracer molecule
that binds to the ATP-binding site of the kinase (BRET
acceptor). The addition of cell penetrant CAMKK2 inhibitors
leads to displacement of the tracer molecule and a quantifiable
reduction in BRET signal. This assay is conducted in living
cells, and the observation of binding not only depends on
affinity between the compound and CAMKK2 but also on
compound cell penetrance. The parent molecule, GSK650394,
was very potent in this assay (NB IC50 < 3 nM). Several
additional key compounds were evaluated in this assay (Table
7 and the Supporting Information). A key takeaway here is that
all the compounds that advanced to this assay (those with
CAMKK2 enzyme IC50 < 150 nM) demonstrated measurable
CAMKK2 target engagement in cells. The potency was below
500 nM for thienopyridine 14g (210 nM), pyrazolopyrimi-
dines 20 and 21 (190 and 200 nM), N-methyl azaindoles 29
and 30 (240 and 470 nM), imidazopyridine 34 (8 nM),
thienopyrimidine 38 (170 nM), quinoline 46 (140 nM), 2-
phenylpyrimidine 56 (290 nM), and 2-amino-pyrimidine 62
(<3 nM). The results for exemplar compounds from five
different scaffolds (13g, 45, 46, 56, and 62) are depicted in
Figure 6.
Figure 6. CAMKK2 NanoBRET dose−response curves for
compounds 13g, 45, 46, 56, and 62.
On-Target Cellular Effect (Phosphorylation Inhibition
Assay). We also assessed the impact of our inhibitors on
phosphorylation downstream from CAMKK2 using Western
blot analysis with C4-2 prostate cancer cells to provide
evidence of a phenotypic and on-target effect of our CAMKK2
inhibitors in intact cells. AMPK (Thr172) was chosen because
in intact cells, CAMKK2, but not the related CAMKK1, can
readily phosphorylate this substrate.64 We performed prelimi-
nary Western blot analysis at a single inhibitor concentration of
1 μM for the 18 compounds with CAMKK2 enzyme IC50
<
150 nM along with STO-609. These results are shown in
Figure 7.
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of these compounds, but not all, have some selectivity over the
closely related enzyme CAMKK1 in enzyme assays. Further
work will be needed to understand selectivity determinants, to
optimize this selectivity, and to determine if the observed
selectivity holds in a cellular context. Kinome-wide profiling
revealed that the nine exemplars we screened in the Eurofins
DiscoverX KINOMEscan platform are not broadly promiscu-
ous, with S10 (1 μM) < 0.05. In addition, we have
demonstrated that exemplars have cellular activity in two
orthogonal assays, a CAMKK2 NanoBRET in cell target
engagement assay, and Western blot experiments looking at
inhibition of the production of p-AMPK. Our work has shown
that kinase inhibitors with weakened (non-ideal) hinge-binding
interactions can show highly selective kinase inhibition and still
have useful on-target enzyme potency and cellular potency.
This work has led to the identification of several CAMKK2
scaffolds with alternate hinge-binding moieties that hold
promise as starting points for the discovery of potent, selective,
and cell-active CAMKK2 inhibitors.
Figure 7. Western blots of the 18 compounds with IC50 < 150 nM in
the CAMKK2 enzyme assay, together with STO-609, screened at a
single inhibitor concentration = 1 μM.
Based on these results from screening at a single
concentration, compounds 7, 20, 29, 34, 38, 46, 56, and 62
showed the most robust reduction of the p-AMPK band and
were advanced into full dose response experiments in this same
assay format to provide IC50 estimates for inhibition of
phosphorylation of AMPK at Thr172. The IC50 values were
determined from dose response experiments using 0−10 μM
of the compound. The Western blot dose response experi-
ments for the most potent compound, 62, are depicted in
Figure 8a. The Western blot dose response data for
EXPERIMENTAL SECTION
■
Biology. Protein Expression and Purification/DSF Assay. Small-
molecule screening by DSF was performed as described previ-
ously.48,65 Briefly, the DSF assay was performed in the 96-well format.
Purified CAMKK1 or CAMKK2 was diluted to 2 μM kinase in 100
mM potassium phosphate pH 7.5, 150 mM NaCl, and 10% glycerol
supplemented with 5 × SYPRO Orange (Invitrogen, Carlsbad, CA,
USA). All assay experiments used 19.5 μL of 2 μM kinase and SYPRO
Orange mixture. Compounds solubilized in dimethyl sulfoxide
(DMSO) were used at a 12.5 μM final concentration, with a 2.5%
concentration of DMSO per well. PCR plates were sealed using
optically clear films and transferred to a C1000 thermal cycler with
CFX-96 RT-PCR head (BioRad, Hercules, CA, USA). The
fluorescence intensity was measured over a temperature gradient
from 25 to 95 °C at a constant rate of 0.05 °C/s. Curve fitting and
protein melting temperatures were calculated based on a Boltzmann
function fitting to experimental data (GraphPad Prism 8). Protein
with the addition of 2.5% DMSO was used as a reference. All
experiments were carried out in triplicate, and the mean of the ΔTm is
reported. Compounds that provided negative values are presented as
having a ΔTm of 0 °C.
CAMKK1 and CAMKK2 Enzyme Assays. CAMKK1 and CAMKK2
activity was determined by measuring the transfer of radiolabeled
phosphate from [γ-32P]-ATP to a synthetic peptide substrate
(CaMKKtide) as previously described.66 Briefly, purified recombinant
CAMKK1 or CAMKK2 (100 pM) was incubated in the assay buffer
[50 mM HEPES (pH 7.4), 1 mM dithiothreitol, 0.02% (v/v) Brij-35]
containing 200 μM CaMKKtide (Genscript), 100 μM CaCl2, 1 μM
CaM (Sigma-Aldrich, Castle Hill, NSW, Australia), 200 μM [γ-32P]-
ATP (Perkin Elmer, Boston, MA, USA), 5 mM MgCl2 (Sigma-
Aldrich, Castle Hill, NSW, Australia), and various concentrations of
inhibitors (0−1 μM) in a standard 30 μL assay for 10 min at 30 °C.
Reactions were terminated by spotting 15 μl onto P81 phosphocellu-
lose paper (GE Lifesciences, Paramatta, NSW, Australia) and washing
extensively in 1% phosphoric acid (Sigma-Aldrich, Castle Hill, NSW,
Australia). Radioactivity was quantified by liquid scintillation
counting.
Figure 8. (a) Dose response for inhibition of phosphorylation of
AMPK at Thr172 in C4-2 prostate cancer cells by compound 62. (b).
Calculated IC50 values for inhibition of phosphorylation of AMPK at
Thr172 in C4-2 prostate cancer cells for the top compounds from the
single concentration experiment depicted in Figure 7.
compounds 7, 20, 29, 34, 38, 46, and 56 can be found in
the Supporting Information. In addition, dose response
experiments for compound 62 utilizing lower inhibitor
concentrations can be found in the Supporting Information.
The calculated IC50 values for the dose response experiments
for all eight compounds are shown in Figure 8b. Compounds
62 (IC50 = 10 nM), 46 (IC50 = 390 nM), 56 (IC50 = 790 nM),
20 (IC50 = 900 nM), and 29 (IC50 980 nM) all have IC50
values for inhibition of phosphorylation of AMPK at Thr172
below 1 μM, suggesting that optimization into compounds
with potent cellular activity will be achievable for these series.
DISCUSSION AND CONCLUSIONS
■
Through a hinge-binder scaffold hopping strategy, we have
designed, synthesized, and biologically evaluated as CAMKK2
inhibitors a series of 32 compounds that utilize 5,6-bicyclic,
6,6-bicyclic, and single-ring hinge-binding moieties that are
based on the 7-azaindole GSK650394. These inhibitors were
designed with the aim of retaining CAMKK2 potency and
improving the kinase selectivity of the promiscuous azaindole
by changing the strong H-bond interactions that the parent
azaindole makes with the kinase hinge-binding residues.
Additionally, we sought to identify inhibitors with improved
physicochemical properties, drug likeness, and selectivity all
while retaining CAMKK2 inhibitory potency. Several com-
pounds with similar or better potency than GSK650394 and
STO-609 have been identified, some of which also showed
improved physicochemical properties. We found that a number
CAMKK2 NanoBRET Assay. To quantify the cellular activity of
these inhibitors, we developed a CAMKK2 NanoBRET target
engagement assay.63 Briefly, this assay utilizes an NL fused to the
kinase domain of CAMKK2. This NL kinase fusion is then transiently
transfected into HEK293 cells, and after 24 h, the tracer is added to
the cells. When the tracer and the NL-CAMKK2 fusion come into
proximity, they create a BRET signal that can be competed in a dose-
dependent manner by the addition of cell-penetrant CAMKK2
inhibitors.
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Western Blot Analysis. C4-2 cells were plated in 6-well plates in
the IMEM medium containing 0.5% fetal bovine serum. After 72 h,
the cells were then treated with the compounds for 24 h before the
media were aspirated and wells were washed twice in ice-cold
phosphate-buffered saline. Cells were lysed using RIPA buffer
containing phosphatase and the protease inhibitor cocktail while
rotating for 30 min at 4 °C. In each lane, 30 μg/well of protein lysate
was loaded into a 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis gel and run for 1 h and 30 min. Gels were then
transferred overnight in a TRIS-glycine/methanol transfer buffer onto
a poly(vinylidene difluoride) membrane at 4 °C. Membranes were
blocked, incubated with primary overnight at 4 °C, washed, incubated
with secondary at room temperature (rt) for 1 h, washed, and then
developed on an Azure Biosystems C-600 imager.
slowly added to the solution, followed by EtOAc (40.0 mL). The
organic phase was separated, concentrated, and purified using column
chromatography (10% EtOAc/hexane) to afford 4-bromo-2-cyclo-
pentylbenzoic acid 2 (1.70 g, 71%) as a pure white solid. The NMR
data for this compound match those previously reported.38
1H NMR (400 MHz, DMSO-d6): δ 13.07 (s, 1H), 7.60−7.56 (m,
2H), 7.45 (dd, J = 8.3, 2.1 Hz, 1H), 3.68 (tt, J = 9.5, 7.5 Hz, 1H),
2.03−1.94 (m, 2H), 1.82−1.72 (m, 2H), 1.67−1.46 (m, 4H). 13C
NMR (100 MHz, DMSO-d6): δ 168.9, 148.6, 131.2, 131.1, 129.5,
128.6, 125.1, 41.2, 34.2, 25.2; LCMS: [M + H]+ m/z, 269.0.
2-Cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
benzoic Acid (3). To a stirred solution of 4-bromo-2-cyclo-
pentylbenzoic acid 2 (2.50 g, 9.30 mmol) and bis(pinacolato)diboron
(2.80 g, 11.0 mmol) in dioxane (50.0 mL) were added PdCl2(dppf)·
CH2Cl2 (0.76 g, 0.96 mmol) and KOAc (3.60 g, 37.0 mmol). The
solution was heated to 95 °C for 2 h. Once cooled to rt, the solution
was diluted with EtOAc and filtered through a celite pad. The solution
was concentrated, and the crude product was purified by column
chromatography (10% EtOAc/hexane) to afford 2-cyclopentyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 3 (2.30 g,
78%) as an off-white solid.
[Cell signaling: phospho-AMPKα (Thr172) (40H9) rabbit mAb:
Cat#: 2535; AMPKα (D5A2) Rabbit mAb Cat#: 5831; BD
Bioscience: CAMKK mouse mAb Cat# 610544; Sigma: GAPDH
rabbit pAb: Cat# G9545; secondary antibody: goat anti-rabbit IgG (H
+ L)-HRP conjugate was from Bio-Rad (Cat#:1706515)].
Crystallization, Data Collection, and Structure Determination.
Crystallization of the CAMKK2-kinase domain bound to 13g or
UNC10244803 followed a previously established protocol.38 Briefly,
the inhibitor (dissolved in 100% DMSO) was added to the protein in
3-fold molar excess and incubated on ice for approximately 30 min.
The mixture was centrifuged at 21,000g for 10 minutes at 4 °C before
setting up 150 nL volume sitting drops at three ratios (2:1, 1:1, or 1:2
protein−inhibitor complex to reservoir solution). Crystallization
experiments were performed at 20 °C. Crystal optimization used
Newman’s buffer system.67 Crystals were cryoprotected in the mother
liquor supplemented with 25−30% glycerol before flash cooling in
liquid nitrogen for data collection. Diffraction data were collected at
100 K at the Advanced Photon Source 24ID-C or at the Diamond
Light Source (DLS) I03 beamline.
1H NMR (400 MHz, DMSO-d6): δ 13.00 (s, 1H), 7.69 (d, J = 1.1
Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.53 (dd, J = 7.6, 1.1 Hz, 1H), 3.62
(tt, J = 9.8, 7.4 Hz, 1H), 2.05−1.95 (m, 2H), 1.83−1.73 (m, 2H),
1.69−1.45 (m, 4H), 1.29 (s, 12H); 13C NMR (100 MHz, DMSO-d6):
δ 169.7, 144.4, 134.9, 132.1, 131.5, 128.2, 83.9, 41.2, 34.4, 25.2, 24.7;
LCMS: [M + H]+ m/z, 317.2.
Methyl 2-Cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)benzoate (4). To a stirred solution of methyl 4-bromo-2-
cyclopentylbenzoate SI1 (1.50 g, 5.29 mmol) and bis(pinacolato)-
diboron (1.88 g, 7.42 mmol) in dioxane (35.0 mL) were added
Pd(dppf)Cl2 (194 mg, 0.26 mmol) and KOAc (1.56 g, 15.9 mmol).
The solution was heated to 100 °C for 2 h. Once cooled to rt, the
solution was filtered through a pad of Celite and washed with 100 mL
of EtOAc. The volatiles were removed in vacuo, and the crude residue
was purified via column chromatography (0−5% EtOAc/hexane) to
afford methyl 2-cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)benzoate 4 (1.37 g, 78%) as oil which slowly solidified into
a white solid under vacuum.
Diffraction data were integrated with XDS68 and scaled using
AIMLESS from the CCP4 software suite.69 The structure was solved
by molecular replacement using Phaser70 and the kinase domain of
CAMKK2 as the search model (PDB ID 2ZV2).71 Refinement was
performed using REFMAC5,72 and Coot73 was used for model
building. Structure validation was performed using MolProbity.74
Structure factors and coordinates for the structure were deposited at
the PDB (PDB ID 5UY6).
1H NMR (400 MHz, DMSO-d6): δ 7.70 (d, J = 1.1 Hz, 1H), 7.60
(d, J = 7.7 Hz, 1H), 7.56 (d, J = 1.1 Hz, 1H), 3.83 (s, 3H), 3.50 (tt, J
= 9.9, 7.5 Hz, 1H), 2.04−1.93 (m, 2H), 1.85−1.70 (m, 2H), 1.70−
1.44 (m, 4H), 1.30 (s, 12H); 13C NMR (100 MHz, DMSO-d6): δ
168.3, 144.6, 133.6, 132.2, 131.6, 128.3, 84.0, 52.2, 41.3, 34.3, 25.2,
24.7; LCMS: [M + H]+ m/z, 330.2.
Chemistry. General Chemistry Information. All reagents and
solvents, unless specifically stated, were used as obtained from their
commercial sources without further purification. Solvents were
degassed with nitrogen for cross-coupling reactions. Air- and
moisture-sensitive reactions were performed under an inert
atmosphere using nitrogen in a previously oven-dried or flame-dried
reaction flask, and addition of reagents was done using a syringe. All
microwave (μW) reactions were carried out in a Biotage Initiator EXP
US 400W microwave synthesizer. Thin-layer chromatography (TLC)
analyses were performed using 200 μm pre-coated sorbtech
fluorescent TLC plates, and spots were visualized using UV light.
High-resolution mass spectrometry samples were analyzed with a
ThermoFisher Q Exactive HF-X (ThermoFisher, Bremen, Germany)
mass spectrometer coupled with a Waters Acquity H-class liquid
chromatograph system. All high-resolution mass spectrometry
(HRMS) spectra were recorded via electrospray ionization (ESI).
Column chromatography was undertaken with a Biotage Isolera One
or Prime instrument. Nuclear magnetic resonance (NMR) spectrom-
etry was run on a Varian Inova 400 MHz or Bruker AVANCE III 700
MHz spectrometer equipped with a TCI H-C/N-D 5 mm cryoprobe,
and data were processed using the MestReNova processor. Chemical
shifts are reported in ppm with residual solvent peaks referenced as
the internal standard. All compounds were >95% pure by analytical
LC.
Methyl 2-Cyclopentyl-4-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-
pyrrolo[2,3-b]pyridin-3-yl)benzoate (6). To a stirred solution of 3-
bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]-
pyridine (450 mg, 1.37 mmol) and methyl 2-cyclopentyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 4 (681 mg, 2.06 mmol)
in PhMe (8.0 mL) and H2O (1.00 mL) were added Pd(OAc)2 (15.4
mg, 68.7 μmol), K3PO4 (934 mg, 4.40 mmol), and P(Cy)3 (38.6 mg,
137 μmol). The reaction mixture was heated to 100 °C for 18 h and
allowed to cool to rt, filtered through a pad of Celite, and washed with
EtOAc (10 mL). The filtrate was concentrated in vacuo, and the crude
was purified by column chromatography eluting with 0−30% EtOAc/
hexane to afford methyl 2-cyclopentyl-4-(1-((2-(trimethylsilyl)-
ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoate (545 mg,
88%) as a light-yellow solid (LCMS: [M + H]+ m/z, 327.3). A
solution of the above material (“SEM-protected 6”), 2-cyclopentyl-4-
(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-3-
yl)benzoate (500 mg, 1.11 mmol), in CH2Cl2 (10 mL) was treated
with trifluoroacetyl (3.50 mL, 45.5 mmol). The solution was stirred at
25 °C for 2 h and then neutralized with sat. NaHCO3 (50 mL). The
solution was extracted with EtOAc (3 × 100 mL), and the combined
organic phases were washed with sat. NaCl solution, dried (Na2SO4),
filtered, and concentrated in vacuo. The residue was dissolved in
ethanol (40.0 mL), and NaOAc (1.82 mg, 22.2 mmol) was added.
The mixture was stirred at 50 °C for 20 h and allowed to cool to rt.
The solution was diluted with H2O (50 mL) and EtOAc (75.0 mL).
4-Bromo-2-cyclopentylbenzoic Acid (2). 4-Bromo-2-fluorobenzoic
acid (2.00 g, 9.00 mmol) was dissolved in tetrahydrofuran (THF)
(20.0 mL) and cooled to 0 °C. Cyclopentylmagnesium bromide
solution (16.0 mL of a 2 M solution, 32.0 mmol) was added dropwise.
The reaction was stirred at 0 °C for 4 h. 2 M HCl (25.0 mL) was then
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The layers were separated, and the aqueous phase was extracted with
EtOAc (2 × 75 mL). The combined organics were washed with a
saturated NaCl solution (50 mL), dried (Na2SO4), filtered, and
concentrated in vacuo. The crude material was purified by column
chromatography (0−30% EtOAc/hexane) to afford methyl 2-
cyclopentyl-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)benzoate 6 (245 mg,
69%) as a deep-yellow solid.
LiOH (1.00 mL, 1 M) and dioxane (1.00 mL) at 100 °C for 16 h. The
solution was cooled to rt, diluted with water (2 mL), and then
acidified with aq HCl (1 M) until pH 4. The solution was extracted
with EtOAc (2 × 3.00 mL). The combined organics were dried
(Na2SO4), filtered, and concentrated in vacuo. The solid was washed
with cold water (5.00 mL), followed by hexane (10.0 mL), and dried
to afford 2-cyclopentyl-4-(1-methyl-5-phenyl-1H-pyrrolo[2,3-b]-
pyridin-3-yl)benzoic acid 10 (77.0 mg, 72%) as an off-white solid.
1H NMR (400 MHz, DMSO-d6): δ 12.75 (br s, 1H), 8.63 (d, J =
2.1 Hz, 1H), 8.43 (d, J = 2.1 Hz, 1H), 8.15 (s, 1H), 7.83−7.67 (m,
5H), 7.50 (dd, J = 8.4, 7.0 Hz, 2H), 7.43−7.34 (m, 1H), 3.94−3.85
(m, 1H), 3.92 (s, 3H), 2.12−1.99 (m, 2H), 1.88−1.76 (m, 2H),
1.75−1.58 (m, 4H); 13C NMR (100 MHz, DMSO-d6): δ 169.7,
148.0, 147.7, 142.4, 139.2, 138.2, 131.0, 130.0, 129.6, 129.5, 128.9,
127.6, 126.1, 124.5, 123.6, 117.9, 113.3, 41.4, 34.8, 31.6, 25.8; HRMS:
calcd for C26H25N2O2 [M + H]+ m/z, 397.1916; found m/z,
397.1911; mp range 199−201 °C.
2-Cyclopentyl-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic
Acid (11). 3-Bromo-1-methyl-1H-pyrrolo[2,3-b]pyridine (100 mg,
0.47 mmol), methyl 2-cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)benzoate (180 mg, 0.54 mmol), Pd2(dba)3 (22.0 mg, 24
μmol), XPhos (23.0 mg, 48.0 μmol), and Cs2CO3 (540 mg, 1.66
mmol) were loaded into a microwave vial. A 3:1 mixture of dioxane/
water (2.00 mL) was added, and the vial was flushed with nitrogen
and capped. The solution was heated to 120 °C for 16 h. Once
cooled, the solution was diluted with EtOAc (2.0 mL) and water
(2.00 mL). The layers were separated, and the aqueous phase was
extracted with EtOAc (2 × 3.00 mL). The combined organics were
dried (Na2SO4), filtered, and concentrated in vacuo. The crude was
purified via column chromatography (0−20% EtOAc/hexane) to
afford the methyl ester intermediate. The methyl ester was saponified
in a solution of aq LiOH (2.00 mL, 1.00 M) and dioxane (2.00 mL) at
100 °C for 16 h. The solution was cooled to rt, diluted with water
(2.00 mL), and then acidified with aq HCl (1.00 M) until pH 4. The
solution was extracted with EtOAc (2 × 3.00 mL). The combined
organics were dried (Na2SO4), filtered, and concentrated in vacuo.
The solid was washed with cold water (5.00 mL), followed by hexane
(10.0 mL), and dried to afford 11 (113 mg, 75%) as an off-white
solid.
1H NMR (400 MHz, DMSO-d6): δ 12.10−12.05 (m, 1H), 8.32−
8.24 (m, 2H), 8.05 (d, J = 2.7 Hz, 1H), 7.78−7.72 (m, 2H), 7.63 (dd,
J = 8.1, 1.7 Hz, 1H), 7.20 (dd, J = 8.0, 4.6 Hz, 1H), 3.84 (s, 3H),
3.79−3.67 (m, 1H), 2.11−1.98 (m, 2H), 1.88−1.77 (m, 2H), 1.74−
1.58 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ 167.9, 149.2,
147.2, 143.1, 138.7, 130.3, 127.4, 127.2, 125.1, 124.1, 123.2, 117.2,
116.4, 113.4, 51.9, 41.3, 34.3, 25.2; LCMS: [M + H]+ m/z, 320.4.
2-Cyclopentyl-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic Acid (7).
Methyl 2-cyclopentyl-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)benzoate
(100 mg, 0.312 mmol) was dissolved in methanol (8.00 mL),
followed by the addition of 50% NaOH solution (170 μL, 3.12
mmol). The mixture was heated to 75 °C for 1 h, allowed to cool, and
extracted with diethyl ether (20.0 mL). The aqueous layer was then
acidified to pH 5 with 1 M HCl and extracted with CH2Cl2 (15 mL).
The organic layer was then dried (Na2SO4), filtered, and concentrated
in vacuo to afford 2-cyclopentyl-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)-
benzoic acid 7 (85.0 mg, 89%) as a pale-yellow solid.
1H NMR (400 MHz, DMSO-d6): δ 12.64 (s, 1H), 12.12−12.08
(m, 1H), 8.32−8.27 (m, 2H), 8.03 (d, J = 2.6 Hz, 1H), 7.78−7.71 (m,
2H), 7.60 (dd, J = 8.1, 1.8 Hz, 1H), 7.22 (dd, J = 8.0, 4.7 Hz, 1H),
3.92−3.81 (m, 1H), 2.07 (d, J = 14.0 Hz, 2H), 1.82 (t, J = 5.8 Hz,
2H), 1.73−1.61 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ 169.3,
148.3, 147.1, 142.3, 138.1, 130.4, 128.6, 128.2, 125.2, 124.2, 123.2,
117.7, 116.4, 113.8, 41.2, 34.4, 25.3; HRMS: calcd for C19H18N2O2
[M + H]+ m/z, 307.1447; found, 307.1426; mp range 238−242 °C.
Methyl 4-(5-Chloro-1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-
cyclopentylbenzoate (9). 5-Chloro-3-iodo-1-methyl-1H-pyrrolo[2,3-
b]pyridine (100 mg, 0.34 mmol), methyl 2-cyclopentyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (113 mg, 0.34 mmol),
Pd(dppf)Cl2·CH2Cl2 (28.0 mg, 34.0 μmol), and Cs2CO3 (334 mg,
1.00 mmol) were loaded into a microwave vial. A 3:1 mixture of
dioxane/water (2 mL) was added before the vial was flushed with
nitrogen and capped. The solution was stirred at rt for 16 h. Once
cooled, the solution was diluted with EtOAc (2.00 mL) and water
(2.00 mL). The layers were separated, and the aqueous phase was
extracted with EtOAc (2 × 3.00 mL). The combined organics were
dried (Na2SO4), filtered, and concentrated in vacuo. The crude was
purified via column chromatography (5−20% EtOAc/hexane) to
afford methyl 4-(5-chloro-1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-
cyclopentylbenzoate 9 (217 mg, 69%) as a white solid.
1H NMR (400 MHz, DMSO-d6): δ 8.33 (d, J = 2.2 Hz, 1H), 8.29
(d, J = 2.2 Hz, 1H), 8.20 (s, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.65 (d, J =
1.8 Hz, 1H), 7.59 (dd, J = 8.1, 1.8 Hz, 1H), 3.91−3.80 (m, 1H) 3.87
(s, 3H), 2.12−1.96 (m, 2H), 1.82 (m, 2H), 1.71−159 (m, 4H); 13C
NMR (100 MHz, DMSO-d6): δ 169.3, 147.0, 146.3, 141.0, 136.9,
130.7, 130.5, 129.1, 126.8, 124.0, 123.4, 123.1, 118.2, 112.3, 41.1,
34.3, 31.3, 25.3; HRMS: calcd for C20H21N2O2 [M + H]+ m/z,
321.1603; found m/z, 321.1596; mp range 181−184 °C.
1H NMR (400 MHz, CDCl3): δ 8.34 (d, J = 2.2 Hz, 1H), 8.15 (d, J
= 2.0 Hz, 1H), 7.86 (d, J = 8.1 Hz, 1H), 7.60 (d, J = 1.6 Hz, 1H), 7.49
(s, 1H), 7.42 (dd, J = 8.1, 1.7 Hz, 1H), 3.97 (s, 3H), 3.93 (s, 3H),
3.92−3.84 (m, 1H), 2.31−2.06 (m, 2H), 1.92−1.80 (m, 2H), 1.80−
1.71 (m, 2H), 1.71−1.59 (m, 4H); 13C NMR (100 MHz, CDCl3): δ
168.5, 148.6, 146.3, 141.7, 137.6, 130.8, 128.5, 128.3, 127.6, 125.0,
124.4, 123.6, 119.3, 114.2, 52.0, 41.7, 34.9, 31.8, 25.7; HRMS: calcd
for C21H22N2O2Cl [M + H]+ m/z, 369.1369; found: m/z, 369.1348;
mp range 144−148 °C.
2-Cyclopentyl-4-(1-methyl-5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-
yl)benzoic Acid (10). Methyl 4-(5-chloro-1-methyl-1H-pyrrolo[2,3-
b]pyridin-3-yl)-2-cyclopentylbenzoate 9 (100 mg, 0.27 mmol),
phenylboronic acid (40.0 mg, 0.32 mmol), Pd2(dba)3 (12.0 mg,
13.0 μmol), XPhos (13.0 mg, 27.0 μmol), and Cs2CO3 (265 mg, 0.81
mmol) were loaded into a microwave vial. A 3:1 mixture of dioxane/
water (2 ml) was added, and the vial was flushed with nitrogen and
capped. The solution was heated to 120 °C for 16 h. Once cooled, the
solution was diluted with EtOAc (2 mL) and water (2.00 mL). The
layers were separated, and the aqueous phase was extracted with
EtOAc (2 × 3.00 mL). The combined organics were dried (Na2SO4),
filtered, and concentrated in vacuo. The crude was purified via column
chromatography (5−10% EtOAc/hexane) to afford the methyl ester
intermediate. The methyl ester was saponified in a solution of aq
Ethyl 2-Chloronicotinate (12b). To a solution of 2-chloronicotinic
acid (5.00 g, 31.7 mmol) in toluene (35.0 mL) was added dropwise
with stirring triethyl orthoacetate (17.5 mL, 95.2 mmol). The mixture
was heated to reflux for 16 h and allowed to cool to rt, and the
resultant solution was washed with sat. NaHCO3 (50.0 mL). The
organic phase was dried with MgSO4, and the solvent was removed in
vacuo to afford the title compound, ethyl 2-chloronicotinate 12b (5.40
g, 92%), as a clear oil.
1H NMR (400 MHz, DMSO-d6): δ 8.58 (dd, J = 4.8, 2.0 Hz, 1H),
8.24 (dd, J = 7.7, 2.0 Hz, 1H), 7.57 (dd, J = 7.7, 4.8 Hz, 1H), 4.35 (q,
J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz,
DMSO-d6): δ 164.1, 152.2, 147.8, 140.2, 127.1, 123.2, 61.8, 13.9;
LCMS: [M + H]+ m/z, 186.0.
Ethyl 5-Bromo-2-chloronicotinate (13b). To a solution of 5-
bromo-2-chloronicotinic acid (10.0 g, 42.0 mmol) in ethanol (60.0
mL) was added slowly H2SO4 (9.10 g, 5.0 mL, 93.0 mmol). The
reaction mixture was heated to reflux for 16 h. The mixture was
concentrated in vacuo, re-dissolved in aq NaHCO3 (150 mL), and
extracted with EtOAc (2 × 300 mL). The combined organic layers
were dried over Na2SO4 and concentrated to give the desired product,
13b (10.5 g, 92%), as a light-yellowish oil.
1H NMR (400 MHz, DMSO-d6): δ 8.76 (d, J = 2.5 Hz, 1H), 8.47
(d, J = 2.5 Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H);
R
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13C NMR (100 MHz, DMSO-d6): δ 162.88, 152.79, 146.65, 142.12,
128.34, 118.85, 62.24, 13.83; LCMS: [M + H]+ m/z, 186.0.
Ethyl 2,5-Dichloronicotinate (14b). Prepared following the
procedure for 12b using 2,5-dichloronicotinic acid (10.0 g, 42.0
mmol). After purification, ethyl 2,5-dichloronicotinate 14b (11.0 g,
99%) was afforded as a clear oil.
5-bromofuro[2,3-b]pyridin-3(2H)-one 13d (6.30 g, 84%) was
afforded as a brown solid.
1H NMR (400 MHz, DMSO-d6): δ 8.71 (d, J = 2.5 Hz, 1H), 8.42
(d, J = 2.5 Hz, 1H), 4.96 (s, 2H); 13C NMR (100 MHz, DMSO): δ
196.4, 175.6, 156.9, 136.3, 115.5, 113.2, 75.8; LCMS: [M + H]+ m/z,
213.4.
1H NMR (400 MHz, DMSO-d6): δ 8.69 (d, J = 2.6 Hz, 1H), 8.38
(d, J = 2.6 Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H).
13C NMR (100 MHz, DMSO-d6): δ 162.9, 150.6, 146.1, 139.5, 130.3,
5-Chlorothieno[2,3-b]pyridin-3(2H)-one (14d). Prepared follow-
ing the procedure for 12d using ethyl 5-chloro-3-hydroxythieno[2,3-
b]pyridine-2-carboxylate 14c (3.0g, 12 mmol). After purification, 5-
chlorothieno[2,3-b]pyridin-3(2H)-one 14d (1.0 g, 50%) was isolated
as an orange solid composed of keto- and enol-isomers.
128.0, 62.3, 13.8; LCMS: [M + H]+ m/z, 220.1.
Ethyl 3-Hydroxyfuro[2,3-b]pyridine-2-carboxylate (12c). To a
suspension of sodium hydride, 60% dispersed in mineral oil, (5.60 g,
0.14 mol) in 1,2-dimethoxyethane (60.0 mL) was added ethyl 2-
hydroxyacetate (13.0 mL, 0.13 mol) under ice cooling and stirring.
The ice bath was removed, and the solution was allowed warm to rt.
After 30 min, a solution of ethyl 2-chloronicotinate (10.0 g, 54.0
mmol) in 100 mL of 1,2-dimethoxyethane was slowly added and the
mixture was heated to 75 °C for 2 h. The solvent was removed in
vacuo, and the residual solid was re-dissolved in aq NaHCO3 solution
(150 mL) and EtOAc (250 mL). The aq layer was acidified with
AcOH (pH 4) and extracted with CH2Cl2 (3 × 200 mL). The
combined organic phases were dried over Na2SO4 and concentrated
in vacuo. Purification by flash column chromatography (10% EtOAc/
hexane) afforded ethyl 3-hydroxyfuro[2,3-b]pyridine-2-carboxylate
12c (8.5 g, 76%) as a pale-white solid.
1H NMR (400 MHz, DMSO-d6): δ 10.46 (s, 1H), 8.77 (d, J = 2.5
Hz, 1H), 8.56 (dd, J = 2.3, 0.5 Hz, 1H), 8.16 (d, J = 2.4 Hz, 1H), 8.11
(d, J = 2.5 Hz, 1H), 6.70 (d, J = 0.5 Hz, 1H), 4.18 (s, 2H); 13C NMR
(101 MHz, DMSO-d6): δ 196.8, 172.1, 156.5, 154.7, 145.4, 145.3,
133.2, 127.9, 127.8, 126.9, 126.0, 100.9, 40.8; LCMS: [M + H]+ m/z,
186.4.
Furo[2,3-b]pyridin-3-yl Trifluoromethanesulfonate (12e). A
solution of 12d (450 mg, 3.33 mmol) in CH2Cl2 (8.00 mL) was
cooled to 0 °C. DIPEA (0.70 mL, 4.00 mmol) was added, followed by
dropwise addition of trifluoromethanesulfonic anhydride (788 μL,
4.66 mmol). The reaction mixture was slowly warmed to rt and stirred
for 2 h and then quenched with water. The aqueous phase was
extracted with CH2Cl2, and the combined organic extracts were dried,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography (10% EtOAc/hexane) to afford furo[2,3-
b]pyridin-3-yl trifluoromethanesulfonate 12e (640 mg, 72%) as a
brown oil.
1H NMR (400 MHz, CDCl3): δ 8.53 (dd, J = 4.8, 1.8 Hz, 1H),
8.11 (dd, J = 7.8, 1.8 Hz, 1H), 7.30 (dd, J = 7.8, 4.8 Hz, 1H), 4.48 (q,
J = 7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H).
1H NMR (400 MHz, CDCl3): δ 8.55−8.41 (m, 1H), 8.03 (dd, J =
7.8, 1.7 Hz, 1H), 7.91 (s, 1H), 7.43−7.35 (m, 1H); 13C NMR (101
MHz, CDCl3): δ 158.81, 146.35, 135.28, 132.54, 129.95, 128.17,
120.11, 118.71 (q, J = 321.6 Hz); LCMS: [M + H]+ m/z, 268.2.
5-Bromofuro[2,3-b]pyridin-3-yl Trifluoromethanesulfonate
(13e). Prepared following the procedure for 12e using 5-bromofuro-
[2,3-b]pyridin-3(2H)-one 13d (10.0 g, 47.0 mmol). After purification,
5-bromofuro[2,3-b]pyridin-3-yl trifluoromethanesulfonate 13e (12.8
g, 79%) was isolated as a light-brown oil which solidified upon
standing.
LCMS: [M + H]+ m/z, 208.1.
Ethyl 5-Bromo-3-hydroxyfuro[2,3-b]pyridine-2-carboxylate
(13c). Prepared following the procedure for 12c using ethyl 5-
bromo-2-chloronicotinate 13b (18.0 g, 68.0 mmol). After purification,
ethyl 5-bromo-3-hydroxyfuro[2,3-b]pyridine-2-carboxylate 13c (19.0
g, 76%) was afforded as a light-brown solid.
1H NMR (400 MHz, DMSO-d6): δ 11.34−11.30 (m, 1H), 8.58 (d,
J = 2.3 Hz, 1H), 8.53 (d, J = 2.3 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H),
1.31 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ 158.7,
156.7, 148.8, 144.7, 133.5, 127.2, 115.9, 114.3, 60.4, 14.3; LCMS: [M
+ H]+ m/z, 286.0.
1H NMR (400 MHz, CDCl3): δ 8.50 (dd, J = 2.2, 0.4 Hz, 1H),
8.14 (d, J = 2.2 Hz, 1H), 7.92 (s, 1H); LCMS: [M + H]+ m/z, 347.1.
5-Chlorothieno[2,3-b]pyridin-3-yl Trifluoromethanesulfonate
(14e). Prepared following the procedure for 12e using 5-chlorothieno-
[2,3-b]pyridin-3(2H)-one 14d (800 mg, 4.31 mmol). After
purification, 5-Chlorothieno[2,3-b]pyridin-3-yl trifluoromethanesulfo-
nate 14e (1.00 g, 74%) was isolated as a brown oil.
Ethyl 5-Chloro-3-hydroxythieno[2,3-b]pyridine-2-carboxylate
(14c). Prepared following the procedure for 12c using ethyl 2,5-
dichloronicotinate 14b (15.0 g, 68.0 mmol) and ethyl 2-
mercaptoacetate (15.0 mL, 0.14 mmol). After purification, ethyl 5-
chloro-3-hydroxythieno[2,3-b]pyridine-2-carboxylate 14c (12.4 g,
71%) was afforded as a light-orange solid.
1H NMR (400 MHz, CDCl3): δ 8.60 (d, J = 2.3 Hz, 1H), 8.02 (d, J
= 2.3 Hz, 1H), 7.55 (d, J = 0.5 Hz, 1H); 13C NMR (101 MHz,
CDCl3): δ 155.34, 147.62, 134.57, 129.60, 127.41, 125.23, 118.67 (q,
J = 321.2 Hz), 118.13; LCMS: [M + H]+ m/z, 318.4.
1H NMR (400 MHz, DMSO-d6): δ 11.04 (s, 1H), 8.73 (d, J = 2.4
Hz, 1H), 8.41 (d, J = 2.4 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 1.31 (t, J
= 7.1 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ 162.6, 155.7,
152.2, 149.4, 130.3, 127.7, 126.7, 105.5, 61.1, 14.2; LCMS: [M + H]+
m/z, 258.0.
2-Cyclopentyl-4-(furo[2,3-b]pyridin-3-yl)benzoic Acid (12f). A
microwave vial was loaded with furo[2,3-b]pyridin-3-yl trifluorome-
thanesulfonate 12e (250 mg, 936 μmol), 2-cyclopentyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 3 (444 mg, 1.40
mmol), Pd(PPh3)4 (11.0 mg, 9.50 μmol), sodium carbonate (397 mg,
3.74 mmol), MeOH (2.00 mL), and CH2Cl2 (0.50 mL). The vial was
sealed and heated to 90 °C for 2 h. The solution was neutralized with
aq HCl (10 mL) and then extracted with ethyl acetate (2 × 20.0 mL).
The organic phases were combined and dried (Na2SO4), filtered, and
concentrated in vacuo. The crude was purified via column
chromatography (30% EtOAc/hexane) to afford 12f (217 mg, 76%)
as an off-white solid.
Furo[2,3-b]pyridin-3(2H)-one (12d). A mixture of ethyl 3-
hydroxyfuro[2,3-b]pyridine-2-carboxylate 12c (6.00 g, 30.0 mmol)
in ethanol (100 mL) and sodium hydroxide (5.00 g, 100 mmol)
dissolved in 15.0 mL of water was refluxed for 1 h. After evaporation
of the solvent, the yellow-orange crystalline mass was dissolved in
water (100 mL), acidified to pH 2−3 with conc. HCl, and heated to
reflux for 45 min. Once cooled, the solution was neutralized with
sodium bicarbonate and extracted with chloroform. The organic phase
was dried (Na2SO4) and filtered, and the solvent was removed in
vacuo to afford furo[2,3-b]pyridin-3(2H)-one 12d (2.70 g, 67%) as a
light-brown solid. The title compound was isolated as a mixture of
keto−enol tautomers (9:1).
1H NMR (400 MHz, DMSO-d6): δ 12.94 (s, 1H), 8.67 (s, 1H),
8.41 (d, J = 6.3 Hz, 2H), 7.81−7.75 (m, 2H), 7.67 (dd, J = 8.0, 1.8
Hz, 1H), 7.52−7.46 (m, 1H), 3.84−3.73 (m, 1H), 2.07−2.03 (m,
2H), 1.86−1.81 (m, 2H), 1.73−1.62 (m, 4H); 13C NMR (101 MHz,
DMSO-d6): δ 169.2, 161.9, 147.0, 144.5, 143.0, 133.7, 131.0, 130.2,
124.8, 123.9, 120.2, 119.9, 117.4, 41.3, 34.3, 25.2; HRMS calcd for
C19H17NO3: [M + H]+ m/z, 308.1287; found, 308.1266; mp range
164−168 °C.
1H NMR (400 MHz, DMSO-d6): δ 9.71 (s, 0H), 8.60 (dd, J = 4.9,
2.0 Hz, 1H), 8.15 (dd, J = 7.5, 1.9 Hz, 1H), 7.27−7.22 (m, 1H), 4.89
(s, 2H); 13C NMR (101 MHz, DMSO-d6): δ 197.7, 177.1, 156.9,
134.3, 118.8, 113.6, 74.6; LCMS: [M + H]+ m/z, 136.1.
5-Bromofuro[2,3-b]pyridin-3(2H)-one (13d). Prepared following
the procedure for 12d using ethyl 5-bromo-3-hydroxyfuro[2,3-
b]pyridine-2-carboxylate 13c (10.0 g, 35.0 mmol). After purification,
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Methyl 4-(5-Bromofuro[2,3-b]pyridin-3-yl)-2-cyclopentylben-
zoate (13f). Prepared following the procedure for 12f using 5-
bromofuro[2,3-b]pyridin-3-yl trifluoromethanesulfonate 13e (100 mg,
0.29 mmol) and 4 (95.4 mg, 0.29 mmol). After purification, methyl 4-
(5-bromofuro[2,3-b]pyridin-3-yl)-2-cyclopentylbenzoate 13f (68 mg,
59%) was isolated as an off-white solid.
1H NMR (400 MHz, DMSO-d6): δ 12.94 (s, 1H), 8.96 (d, J = 2.1
Hz, 1H), 8.38 (d, J = 2.2 Hz, 1H), 8.17 (s, 1H), 7.83−7.77 (m, 3H),
7.72 (d, J = 1.7 Hz, 1H), 7.62 (dd, J = 8.0, 1.7 Hz, 1H), 7.55−7.50
(m, 2H), 7.47−7.41 (m, 1H), 3.87−3.78 (m, 1H), 2.09−2.04 (m,
2H), 1.83−1.78 (m, 2H), 1.71−1.63 (m, 4H); 13C NMR (100 MHz,
DMSO-d6): δ 169.4, 160.6, 147.0, 145.8, 137.3, 137.3, 134.4, 132.5,
131.2, 130.4, 130.1, 129.2, 128.1, 128.1, 127.3, 126.5, 126.2, 125.3,
41.2, 34.5, 25.3; HRMS: calcd for C25H21NO2S [M + H]+ m/z,
400.1371; found, 400.1365; mp range 269−273 °C.
2-Cyclopentyl-4-(thieno[2,3-b]pyridin-3-yl)benzoic Acid (12g).
Prepared following the procedure for 12f using 3-bromothieno[2,3-
b]pyridine 15e (50.0 mg, 0.23 mmol). After purification, 2-
cyclopentyl-4-(thieno[2,3-b]pyridin-3-yl)benzoic acid 12g (66.0 mg,
88%) was afforded as a light-brown solid.
1H NMR (400 MHz, DMSO-d6): δ 8.74 (s, 1H), 8.56 (d, J = 1.8
Hz, 1H), 8.48 (d, J = 1.6 Hz, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.69 (dd,
J = 7.9, 1.7 Hz, 1H), 3.85 (s, 3H), 3.71−3.58 (m, 1H), 2.08−1.96 (m,
2H), 1.87−1.76 (m, 2H), 1.73−1.61 (m, 4H); 13C NMR (101 MHz,
DMSO-d6): δ 167.9, 160.5, 147.0, 144.9, 144.8, 133.4, 132.3, 130.2,
130.0, 125.0, 124.1, 119.6, 119.5, 115.3, 52.2, 41.4, 34.2, 25.2.
Methyl 4-(5-Chlorothieno[2,3-b]pyridin-3-yl)-2-cyclopentylben-
zoate (14f). Prepared following the procedure for 12f using 5-
chlorothieno[2,3-b]pyridin-3-yl trifluoromethanesulfonate 14e (950
mg, 2.99 mmol) and 4 (988 mg, 2.99 mmol). After purification,
methyl 4-(5-chlorothieno[2,3-b]pyridin-3-yl)-2-cyclopentylbenzoate
14f (870 mg, 78%) was isolated as a near-white solid.
1H NMR (400 MHz, DMSO-d6): δ 12.99 (s, 1H), 8.65 (dd, J =
4.6, 1.6 Hz, 1H), 8.27 (dd, J = 8.2, 1.6 Hz, 1H), 8.10 (s, 1H), 7.80 (d,
J = 8.0 Hz, 1H), 7.64 (d, J = 1.8 Hz, 1H), 7.56−7.50 (m, 2H), 3.85−
3.76 (m, 1H), 2.08−2.03 (m, 2H), 1.82−1.78 (m, 2H), 1.72−1.58
(m, 4H); 13C NMR (176 MHz, DMSO-d6): δ 172.6, 164.7, 150.1,
149.9, 140.4, 137.4, 134.5, 133.7, 133.4, 133.1, 129.5, 128.4, 128.4,
123.4, 44.4, 37.5, 28.4; HRMS: calcd for C19H17NO2S [M + H]+ m/z,
324.1058; found, 324.1054; mp range 194−197 °C.
Methyl 4-(5-Bromopyrazolo[1,5-a]pyridin-3-yl)-2-cyclopentyl-
benzoate (17). 5-Bromo-3-iodopyrazolo[1,5-a]pyridine (200 mg,
0.62 mmol) and methyl 2-cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzoate 4 (194 mg, 0.60 mmol) were dissolved in
a 3:1 solution of dioxane/water (4.00 mL). Pd2(dba)3 (28 mg, 31
μmol), XPhos (30.0 mg, 62.0 μmol), and Cs2CO3 (605 mg, 1.90
mmol) were added. The mixture was stirred at rt for 16 h. The
aqueous phase was removed, and the solution was concentrated in
vacuo. The crude was purified via column chromatography (0−20%
EtOAc/hexane) to afford ethyl 4-(5-bromopyrazolo[1,5-a]pyridin-3-
yl)-2-cyclopentylbenzoate 17 (189 mg, 76%) as a mustard yellow
solid.
1H NMR (400 MHz, DMSO-d6): δ 8.69 (d, J = 2.3 Hz, 1H), 8.27−
8.24 (m, 2H), 7.79 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 1.8 Hz, 1H), 7.57
(dd, J = 8.0, 1.7 Hz, 1H), 3.87 (s, 3H), 3.71−3.61 (m, 1H), 2.08−
2.01 (m, 2H), 1.85−1.75 (m, 2H), 1.69−1.62 (m, 4H); 13C NMR
(100 MHz, DMSO-d6): δ 168.0, 159.5, 147.1, 145.4, 137.1, 133.6,
131.3, 130.2, 130.0, 129.7, 128.1, 128.0, 126.6, 125.3, 52.2, 41.4, 34.3,
25.2.
2-Cyclopentyl-4-(5-phenylfuro[2,3-b]pyridin-3-yl)benzoic Acid
(13g). 4-(5-Bromofuro[2,3-b]pyridin-3-yl)-2-cyclopentylbenzoic
acid, 13f (48.0 mg, 0.12 mmol), phenylboronic acid (30.0 mg, 0.25
mmol), Na2CO3 (40.0 mg, 0.37 mmol), and Pd(PPh3)4 (14.0 mg, 12
μmol) were dissolved in a 4:1 mixture of dioxane (2.5 mL) and
heated to 90 °C under nitrogen for 16 h. The mixture was neutralized
to pH 6−7 with aq HCl and extracted with ethyl acetate (5.00 mL).
The combined organics were dried (Na2SO4), filtered, and
concentrated in vacuo. The crude residue was purified using flash
column chromatography (30% EtOAc/hexane) to obtain the methyl
ester intermediate which was immediately dissolved in MeOH (3.0
mL), added aq NaOH (small excess), and heated to 70 °C for 1 h.
After cooling, H2O (4.0 mL) was added and the mixture was extracted
with diethyl ether (2 × 4.0 mL). The aqueous phase was acidified to
pH 6 with aq HCl, extracted with CH2Cl2 (6.00 mL), and
concentrated to afford 2-cyclopentyl-4-(5-phenylfuro[2,3-b]pyridin-
3-yl)benzoic acid 13g (39 mg, 82%) as a white solid.
1H NMR (400 MHz, CDCl3): δ 8.36 (dd, J = 7.3, 0.8 Hz, 1H),
8.16 (s, 1H), 7.94 (dd, J = 2.1, 0.8 Hz, 1H), 7.86 (dd, J = 8.1, 0.4 Hz,
1H), 7.57 (d, J = 1.8 Hz, 1H), 7.40 (dd, J = 8.1, 1.8 Hz, 1H), 6.91
(ddd, J = 7.3, 2.1, 0.4 Hz, 1H), 3.93 (s, 3H), 3.91−3.83 (m, 1H),
2.24−2.11 (m, 2H), 1.93−1.81 (m, 2H), 1.81−1.72 (m, 2H), 1.71−
1.61 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 168.5, 148.7, 141.6,
137.6, 135.8, 130.9, 129.7, 128.4, 125.3, 123.7, 119.7, 118.7, 116.0,
112.4, 52.0, 41.7, 34.8, 25.7; HRMS: calcd for C20H20N2O2Br [M +
H]+ m/z, 399.0708; found m/z, 399.0705; mp range 182−184 °C.
Methyl 4-(5-Chloropyrazolo[1,5-a]pyrimidin-3-yl)-2-cyclopentyl-
benzoate (18). 5-Chloro-3-iodopyrazolo[1,5-a]pyrimidine (500 mg,
1.79 mmol) and methyl 2-cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzoate 4 (591 mg) were dissolved in a 10:1
mixture of dioxane/water (10.0 mL). Pd(dppf)Cl2·CH2Cl2 (73.0 mg,
0.9 mmol) and Cs2CO3 (1.75 g, 5.37 mmol) were added, and the
solution was heated to 80 °C for 16 h. The solution was allowed to
cool to rt, the aqueous phase was removed, and the organic phase was
concentrated in vacuo. The crude was purified via column
chromatography (10−20% EtOAc/hexane) to afford methyl 4-(5-
chloropyrazolo[1,5-a]pyrimidin-3-yl)-2-cyclopentylbenzoate 18 (270
mg, 44%) as a bright-yellow solid.
1H NMR (400 MHz, DMSO-d6): δ 12.95 (s, 1H), 8.72 (s, 1H),
8.67 (d, J = 2.2 Hz, 1H), 8.53 (d, J = 2.2 Hz, 1H), 7.82 (d, J = 1.5 Hz,
1H), 7.81−7.79 (m, 4H), 7.56−7.50 (m, 2H), 7.47−7.41 (m, 1H),
3.86−3.77 (m, 1H), 2.11−2.01 (m, 2H), 1.87−1.79 (m, 2H), 1.73−
1.62 (m, 4H); 13C NMR (101 MHz, DMSO-d6): δ 169.3, 161.6,
147.0, 143.8, 143.2, 137.5, 133.6, 132.9, 131.0, 130.3, 129.1, 128.2,
127.8, 127.5, 125.0, 124.1, 120.2, 117.6, 41.2, 34.4, 25.3; HRMS:
calcd for C25H21NO3 [M + H]+ m/z, 384.1600; found, 384.1593; mp
range 259−262 °C.
2-Cyclopentyl-4-(5-phenylthieno[2,3-b]pyridin-3-yl)benzoic Acid
(14g). Methyl 4-(5-chlorothieno[2,3-b]pyridin-3-yl)-2-cyclopentyl-
benzoate 14f (150 mg, 403 μmol), phenylboronic acid (73.8 mg,
605 μmol), Pd2(dba)3 (18.5 mg, 20.2 μmol), dicyclohexyl(2′,4′,6′-
triisopropyl-[1,1′-biphenyl]-2-yl)phosphane (28.8 mg, 60.5 μmol),
and Cs2CO3 (394 mg, 1.21 mmol) were dissolved in dioxane (3.00
mL) and H2O (0.8 mL). The solution was heated to 90 °C for 16 h.
The reaction mixture was allowed to cool to rt, neutralized to pH 7
with aq HCl, and extracted with ethyl acetate (15 mL). The combined
organic phases were dried (Na2SO4), filtered, and concentrated in
vacuo. The crude was purified by column chromatography (25%
EtOAc/hexane) to afford the methyl ester intermediate. This material
was dissolved in MeOH (6 mL), added aq NaOH (small excess), and
heated to 70 °C for 1 h. After cooling, H2O (8 mL) was added and
the mixture was extracted with diethyl ether (2 × 10.0 mL). The
aqueous phase was acidified to pH 6, extracted with CH2Cl2 (10 mL),
and concentrated to afford 2-cyclopentyl-4-(5-phenylthieno[2,3-
b]pyridin-3-yl)benzoic acid 14g (116 mg, 72%) as a yellow solid.
1H NMR (400 MHz, CDCl3): δ 8.59 (d, J = 7.2 Hz, 1H), 8.48 (s,
1H), 8.12 (d, J = 1.5 Hz, 1H), 7.92−7.78 (m, 2H), 6.86 (d, J = 7.2
Hz, 1H), 3.99−3.86 (m, 4H), 2.28−2.04 (m, 2H), 1.97−1.84 (m,
2H), 1.84−1.61 (m, 4H); 13C NMR (100 MHz, CDCl3): δ 168.6,
150.8, 148.6, 143.9, 136.5, 134.4, 130.6, 128.3, 124.5, 122.8, 110.1,
109.4, 51.9, 41.7, 34.8, 25.8; HRMS: calcd for C19H19N3O2Cl [M +
H]+ m/z, 356.1165; found m/z, 356.1161; mp range 163−166 °C.
2-Cyclopentyl-4-(5-phenylpyrazolo[1,5-a]pyridin-3-yl)benzoic
Acid (19). Methyl 4-(5-bromopyrazolo[1,5-a]pyridin-3-yl)-2-cyclo-
pentylbenzoate 17 (100 mg, 0.25 mmol), phenylboronic acid (37.0
mg, 0.30 mmol), Pd(PPh3)4 (29.0 mg, 25.0 μmol), and Cs2CO3 (245
mg, 0.75 mmol) were loaded into a microwave vial. A 3:1 mixture of
dioxane/water (1.50 mL) was added, and the vial was flushed with
nitrogen and sealed. The mixture was irradiated at 120 °C for 30 min.
Once cooled, the solution was diluted with EtOAc (2.00 mL) and
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water (2.00 mL). The layers were separated, and the aqueous phase
was extracted with EtOAc (2 × 3.00 mL). The combined organics
were dried (Na2SO4), filtered, and concentrated in vacuo. The crude
was purified via column chromatography (5−10% EtOAc/hexane) to
afford the methyl ester intermediate. The methyl ester was saponified
in a solution of aq LiOH (1.00 mL, 1.00 M) and dioxane (1.00 mL) at
100 °C for 16 h. The solution was cooled to rt, diluted with water
(2.00 mL), and then acidified with aq HCl (1 M) until pH 4. The
solution was extracted with EtOAc (2 × 3.00 mL). The combined
organics were dried (Na2SO4), filtered, and concentrated in vacuo.
The solid was washed with cold water (5.00 mL), followed by hexane
(10.0 mL), and dried to afford the acid (68.0 mg, 71%) as a yellow
solid.
1H NMR (400 MHz, DMSO-d6): δ 12.78 (br s, 1H), 8.76 (d, J =
7.0 Hz, 1H), 8.47 (s, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 8.1
Hz, 1H), 7.67 (d, J = 1.6 Hz, 1H), 7.57 (dd, J = 8.1, 1.7 Hz, 1H), 7.39
(ddd, J = 8.9, 6.7, 1.0 Hz, 1H), 6.99 (td, J = 6.9, 1.1 Hz, 1H), 3.90−
3.79 (m, 1H), 2.09−1.97 (m, 2H), 1.88−1.76 (m, 2H), 1.73−1.58
(m, 4H); 13C NMR (101 MHz, DMSO-d6): δ 169.2, 147.1, 140.8,
136.3, 135.8, 130.5, 129.6, 128.7, 125.4, 124.2, 123.2, 117.1, 112.7,
110.9, 41.2, 34.3, 25.2; HRMS (ESI): calcd for C19H19N2O2 [M +
H]+ m/z, 307.1446; found m/z, 307.1439; mp range 167−169 °C.
2-Cyclopentyl-4-(pyrazolo[1,5-a]pyrimidin-3-yl)benzoic Acid
(22). 3-Bromopyrazolo[1,5-a]pyrimidine (150 mg, 0.75 mmol), 2-
cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic
acid 3 (287 mg, 0.90 mmol), Pd(PPh3)4 (87.0 mg, 75.0 μmol), and
Cs2CO3 (864 mg, 2.65 mmol) were loaded into a microwave vial. A
3:1 mixture of dioxane/water (2.00 mL) was added, and the vial was
flushed with nitrogen and capped. The solution was irradiated at 120
°C for 30 min. Once cooled, the solution was diluted with EtOAc
(2.00 mL) and water (2.00 mL). The mixture was acidified to pH 4
with 1 M aq HCl solution. The layers were separated, and the
aqueous phase was extracted with EtOAc (2 × 3.00 mL). The
combined organics were dried (Na2SO4), filtered, and concentrated in
vacuo. The crude was purified via column chromatography (50−100%
EtOAc/hexane) to afford 2-cyclopentyl-4-(pyrazolo[1,5-a]pyrimidin-
3-yl)benzoic acid 22 (137 mg, 59%) as a bright-yellow solid.
1H NMR (700 MHz, DMSO-d6): δ 12.74 (br s, 1H), 9.24−9.12 (d,
J = 6.0 Hz, 1H), 8.87 (d, J = 2.9 Hz, 1H), 8.72 (s, 1H), 8.25 (s, 1H),
8.03 (d, J = 8.2 Hz, 1H), 7.75 (dd, J = 8.1, 2.8 Hz, 1H), 7.14 (dt, J =
7.4, 3.5 Hz, 1H), 3.89−3.83 (m, 1H), 2.08−2.01 (m, 2H), 1.88−1.82
(m, 2H), 1.71−1.63 (m, 4H); 13C NMR (176 MHz, DMSO-d6): δ
169.3, 150.9, 146.9, 144.5, 143.2, 136.6, 135.0, 130.1, 128.6, 123.6,
122.5, 109.0, 108.2, 41.2, 34.3, 25.2; HRMS: calcd for C18H18N3O2
[M + H]+ m/z, 308.1399; found m/z, 308.1393; mp range 196−199
°C.
1H NMR (700 MHz, DMSO-d6): δ 8.83 (d, J = 6.9 Hz, 1H), 8.50
(s, 1H), 8.10 (s, 1H), 8.02 (s, 1H), 7.85 (d, J = 7.1 Hz, 2H), 7.79 (d, J
= 7.8 Hz, 1H), 7.72 (s, 1H), 7.67 (d, J = 7.6 Hz, 1H), 7.53 (t, J = 6.9
Hz, 2H), 7.45 (t, J = 6.8 Hz, 1H), 7.33 (d, J = 6.8 Hz, 1H), 3.91−3.85
(m, 1H), 2.10−2.01 (m, 2H), 1.88−1.78 (m, 2H), 1.73−1.60 (m,
4H); 13C NMR (176 MHz, DMSO-d6): δ 169.2, 147.2, 141.4, 137.6,
136.9, 136.5, 135.7, 131.5, 131.4, 130.5, 129.7, 129.3, 129.1, 128.9,
128.7, 128.5, 126.7, 124.5, 123.4, 113.5, 111.9, 111.7, 41.1, 34.3, 25.3;
HRMS: calcd for C25H23N2O2 [M + H]+ m/z, 383.1759; found m/z,
383.1754; mp range 153−155 °C.
2-Cyclopentyl-4-(5-phenylpyrazolo[1,5-a]pyrimidin-3-yl)benzoic
Acid (20). Methyl 4-(5-chloropyrazolo[1,5-a]pyrimidin-3-yl)-2-cyclo-
pentylbenzoate 18 (100 mg, 0.28 mmol), phenylboronic acid (41.0
mg, 0.33 mmol), Pd(PPh3)4 (32.0 mg, 28.0 μmol), and Cs2CO3 (275
mg, 0.84 mmol) were loaded into a microwave vial. A 3:1 mixture of
dioxane/water (2.00 mL) was added, and the vial was flushed with
nitrogen and capped. The solution was irradiated at 120 °C for 30
min. Once cooled, the solution was diluted with EtOAc (2.00 mL)
and water (2.00 mL). The layers were separated, and the aqueous
phase was extracted with EtOAc (2 × 3.00 mL). The combined
organics were dried (Na2SO4), filtered, and concentrated in vacuo.
The crude was purified via column chromatography (5−10% EtOAc/
hexane) to afford the methyl ester intermediate. The methyl ester was
saponified in a solution of aq LiOH (1.00 mL, 1.00 M) and dioxane
(1.00 mL) at 100 °C for 16 h. The solution was cooled to rt, diluted
with water (2.00 mL), and then acidified with aq HCl (1.00 M) until
pH 4. The solution was extracted with EtOAc (2 × 3.00 mL). The
combined organics were dried (Na2SO4), filtered, and concentrated
in vacuo. The solid was washed with cold water (5.00 mL), followed
by hexane (10.0 mL), and dried to afford 2-cyclopentyl-4-(5-
phenylpyrazolo[1,5-a]pyrimidin-3-yl)benzoic acid 20 (87.0 mg,
81%) as a bright-yellow solid.
Methyl 4-(6-Chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)-2-cyclo-
pentylbenzoate (24). 3-Bromo-6-chloro-[1,2,4]triazolo[4,3-b]-
pyridazine (924 mg, 3.96 mmol), methyl 2-cyclopentyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 4 (1.31 g, 3.96 mmol),
K2CO3 (1.64 g, 11.9 mmol), and Pd(PPh3)4 (320 mg, 0.28 mmol) in
a mixture of dioxane (12.0 mL) and water (3.00 mL) were degassed
and put under a N2 atmosphere. The reaction mixture was heated to
90 °C and stirred for 16 h. The reaction mixture was diluted with aq
HCl (50.0 mL) to pH 6 and extracted with EtOAc (2 × 75.0 mL),
and the combined organic phases were concentrated in vacuo. The
crude residue was purified via column chromatography (35% EtOAc/
hexane) to afford methyl 4-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-
1H NMR (700 MHz, DMSO-d6): δ 12.94 (s, 1H), 9.24 (dd, J =
7.6, 2.8 Hz, 1H), 8.87 (s, 1H), 8.65 (s, 1H), 8.39−8.28 (m, 2H), 7.95
(d, J = 8.1 Hz, 1H), 7.84−7.72 (m, 2H), 7.61−7.55 (m, 3H), 3.98−
3.91 (m, 1H), 2.15−2.09 (m, 2H), 1.93−1.86 (m, 2H), 1.77−1.68
(m, 4H); 13C NMR (176 MHz, DMSO-d6): 169.2, 155.9, 147.3,
144.3, 143.6, 137.0, 136.6, 135.3, 131.0, 130.1, 129.0 (2 × ArCH),
128.4, 127.2 (2 × ArCH), 123.8, 122.1, 108.2, 105.9, 41.0, 34.7, 25.6;
HRMS: calcd for C24H22N3O2 [M + H]+ m/z, 384.1712; found, m/z
384.1707; mp range 210−214 °C.
2-Cyclopentyl-4-(pyrazolo[1,5-a]pyridin-3-yl)benzoic Acid (21).
3-Bromopyrazolo[1,5-a]pyridine (100 mg, 0.50 mmol), 2-cyclo-
pentyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 3
(193 mg, 0.61 mmol), Pd2(dba)3 (23.0 mg, 25.0 μmol), XPhos (24.0
mg, 51.0 μmol), and Cs2CO3 (579 mg, 1.80 mmol) were loaded into
a microwave vial. A 3:1 mixture of dioxane/water (3.00 mL) was
added, and the vial was flushed with nitrogen and sealed. The mixture
was heated to 120 °C for 16 h. Once cooled, the solution was diluted
with EtOAc (2.00 mL) and water (2.00 mL). The mixture was
acidified to pH 4 with 1.00 M aq HCl solution. The layers were
separated, and the aqueous phase was extracted with EtOAc (2 × 3.00
mL). The combined organics were dried (Na2SO4), filtered, and
concentrated in vacuo. The crude was purified via column
chromatography (50−100% EtOAc/hexane) to afford 2-cyclopentyl-
4-(pyrazolo[1,5-a]pyridin-3-yl)benzoic acid 21 (64.0 mg, 41%) as a
yellow solid.
1
3-yl)-2-cyclopentylbenzoate 24 (1.20 g, 85%) as a grayish solid. H
NMR (400 MHz, DMSO-d6): δ 8.58 (d, J = 9.7 Hz, 1H), 8.47 (d, J =
1.7 Hz, 1H), 8.21 (dd, J = 8.2, 1.7 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H),
7.59 (d, J = 2.8 Hz, 1H), 3.88 (s, 3H), 3.77−3.66 (m, 1H), 2.14−2.03
(m, 2H), 1.91−1.79 (m, 2H), 1.75−1.58 (m, 4H). LCMS: [M + H]+
m/z, 356.2.
2-Cyclopentyl-4-(6-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)-
benzoic Acid (25). Following the procedure for 14g using 4-(6-
chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)-2-cyclopentylbenzoate 24
(220 mg, 0.62 mmol). After purification, cyclopentyl-4-(6-phenyl-
[1,2,4]triazolo[4,3-b]pyridazin-3-yl)benzoic acid 25 (200 mg, 84%)
was afforded as a near-yellow solid.
1H NMR (400 MHz, DMSO-d6): δ 13.14 (s, 1H), 8.74 (d, J = 1.7
Hz, 1H), 8.58 (d, J = 9.8 Hz, 1H), 8.32 (dd, J = 8.2, 1.7 Hz, 1H),
8.21−8.18 (m, 2H), 8.07 (d, J = 9.8 Hz, 1H), 7.89 (d, J = 8.2 Hz,
1H), 7.65−7.59 (m, 3H), 3.91−3.82 (m, 1H), 2.15−2.11 (m, 2H),
1.88−1.83 (m, 2H), 1.72−1.66 (m, 4H); 13C NMR (100 MHz,
DMSO-d6): δ 169.2, 153.5, 146.6, 146.4, 144.5, 134.1, 133.0, 131.1,
129.7, 129.1, 128.7, 127.4, 125.6, 125.2, 124.2, 119.9, 41.1, 34.7, 25.6;
HRMS: calcd for C23H20N4O2 [M + H]+ m/z, 385.1665; found,
385.1659; mp range 228−231 °C.
4-([1,2,4]Triazolo[4,3-b]pyridazin-3-yl)-2-cyclopentylbenzoic
Acid (26). 3-Bromo-[1,2,4]triazolo[4,3-b]pyridazine (150 mg, 754
μmol), methyl 2-cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)benzoate 4 (373 mg, 1.13 mmol), XPhos (53.9 mg, 113
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μmol), Cs2CO3 (516 mg, 1.58 mmol), and Pd2(dba)3 (34.5 mg, 0.04
mmol) in a 3:1 mixture of dioxane/water (4.00 mL) were heated to
90 °C under a N2 atmosphere for 16 h. The reaction mixture was
diluted with aq HCl (25.0 mL, pH 6) and extracted with EtOAc (2 ×
50.0 mL); the organic phases were combined, concentrated, and
purified using flash column chromatography (40% EtOAc/hexane) to
afford 4-([1,2,4]triazolo[4,3-b]pyridazin-3-yl)-2-cyclopentylbenzoic
acid 26 (178.0 mg, 77%) as an off-white solid.
0.45 mmol), methyl 2-cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)benzoate 4 (180 mg, 0.54 mmol), Pd(PPh3)4 (52.0 mg,
45.0 μmol), and Cs2CO3 (440 mg, 1.40 mmol) were loaded into a
microwave vial. A 3:1 mixture of dioxane/water (3.00 mL) was added,
and the vial was flushed with nitrogen and capped. The solution was
irradiated at 120 °C for 30 min. Once cooled, the solution was diluted
with EtOAc (2.00 mL) and water (2.00 mL). The layers were
separated, and the aqueous phase was extracted with EtOAc (2 × 3.00
mL). The combined organics were dried (Na2SO4), filtered, and
concentrated in vacuo. The crude was purified via column
chromatography (5−10% EtOAc/hexane) to afford the methyl ester
intermediate. The methyl ester was saponified in a solution of aq
LiOH (1.00 mL, 1.00 M) and dioxane (1.00 mL) at 100 °C for 16 h.
The solution was cooled to rt, diluted with water (2.00 mL), and then
acidified with aq HCl (1.00 M) until pH 4. The solution was extracted
with EtOAc (2 × 3.00 mL). The combined organics were dried
(Na2SO4), filtered, and concentrated in vacuo. The solid was washed
with cold water (5.00 mL), followed by hexane (10.0 mL), and dried
to afford 2-cyclopentyl-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-
benzoic acid 30 (97.0 mg, 67%) as a pale-yellow solid.
1H NMR (400 MHz, DMSO-d6): δ 13.14 (s, 1H), 8.79 (dd, J =
4.3, 1.5 Hz, 1H), 8.50−8.46 (m, 2H), 8.29 (dd, J = 8.2, 1.7 Hz, 1H),
7.86 (d, J = 8.2 Hz, 1H), 7.44 (dd, J = 9.4, 4.3 Hz, 1H), 3.86−3.75
(m, 1H), 2.14−2.04 (m, 2H), 1.89−1.79 (m, 2H), 1.74−1.58 (m,
4H); 13C NMR (100 MHz, DMSO-d6): δ 169.2, 146.8, 146.4, 146.3,
145.1, 133.1, 129.6, 128.6, 125.4, 125.3, 124.1, 120.9, 41.3, 34.4, 25.3;
HRMS: calcd for C17H16N4O2 [M + H]+ m/z, 309.1352; found,
309.1346; mp range 208−211 °C.
4-Chloro-1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridine (28). 4-
Chloro-2-iodo-1-methyl-1H-pyrrolo[2,3-b]pyridine (75.0 mg, 0.25
mmol), phenylboronic acid (31.0 mg, 0.25 mmol), Cs2CO3 (251
mg, 0.77 mmol), and Pd(PPh3)4 (30.0 mg, 0.02 mmol) were loaded
into a microwave vial. A 3:1 mixture of dioxane/water (2.00 mL) was
added, and the vial was flushed with nitrogen and capped. The
solution was irradiated at 120 °C for 30 min. Once cooled, the
solution was diluted with EtOAc (2.00 mL) and water (2.00 mL).
The layers were separated, and the aqueous phase was extracted with
EtOAc (2 × 3.00 mL). The combined organics were dried (Na2SO4),
filtered, and concentrated in vacuo. The crude was purified via column
chromatography (10% EtOAc/hexane) to afford 4-chloro-1-methyl-2-
phenyl-1H-pyrrolo[2,3-b]pyridine 28 (58.0 mg, 93%) as a white-gray
solid.
1H NMR (700 MHz, DMSO-d6): δ 13.01 (s, 1H), 8.36 (s, 1H),
7.81 (d, J = 7.6 Hz, 1H), 7.76 (s, 1H), 7.67−7.61 (m, 2H), 7.27 (s,
1H), 6.57 (s, 1H), 3.87 (s, 3H), 3.80 (dd, J = 19.7, 12.5 Hz, 1H),
2.13−2.00 (m, 2H), 1.87−1.74 (m, 2H), 1.71−1.56 (m, 4H); 13C
NMR (100 MHz, DMSO-d6): δ 169.3, 147.9, 146.6, 142.6, 140.8,
139.9, 131.7, 131.0, 129.9, 126.5, 125.4, 117.5, 114.3, 97.8, 41.1, 34.4,
31.2, 25.3; HRMS: calcd for C20H21N2O2 [M + H]+ m/z, 321.1603;
found m/z, 321.1595; mp range 201−203 °C.
Methyl 2-Cyclopentyl-4-(2,3-diaminopyridin-4-yl)benzoate (32).
4-Chloropyridine-2,3-diamine (103 mg, 0.71 mmol) and methyl 2-
cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 4
(250 mg, 0.75 mmol) were dissolved in a 10:1 mixture of dioxane/
H2O (10.0 mL). Pd2(dba)3 (69.0 mg, 75 μmol), XPhos (72.0 mg, 150
μmol), and Cs2CO3 (740 mg, 2.30 mmol) were added. The solution
was heated to 100 °C for 16 h. Once cooled to rt, the aqueous phase
was removed and the organic phase was concentrated in vacuo. The
crude was purified via column chromatography (25−75% EtOAc/
hexane) to afford methyl 2-cyclopentyl-4-(2,3-diaminopyridin-4-
yl)benzoate 32 (169 mg, 71%) as a dark-brown solid.
1H NMR (400 MHz, CDCl3): δ 8.23 (d, J = 5.2 Hz, 1H), 7.63−
7.40 (m, 5H), 7.12 (d, J = 5.2 Hz, 1H), 6.63 (s, 1H), 3.89 (s, 3H);
13C NMR (101 MHz, CDCl3): δ 149.6, 142.6, 142.5, 135.3, 131.7,
129.1 (2 × ArCH), 128.7 (2 × ArCH), 128.6, 119.9, 116.2, 97.9, 30.3;
mp range 113−116 °C.
2-Cyclopentyl-4-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-4-
yl)benzoic Acid (29). 4-Chloro-1-methyl-2-phenyl-1H-pyrrolo[2,3-
b]pyridine 28 (60.0 mg, 0.25 mmol), methyl 2-cyclopentyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 4 (98.0 mg, 0.30
mmol), Pd(PPh3)4 (29.0 mg, 25.0 μmol), and Cs2CO3 (242 mg,
0.74 mmol) were loaded into a microwave vial. A 3:1 mixture of
dioxane/water (2.00 mL) was added, and the vial was flushed with
nitrogen and capped. The solution was irradiated at 120 °C for 30
min. Once cooled, the solution was diluted with EtOAc (2.00 mL)
and water (2.00 mL). The layers were separated, and the aqueous
phase was extracted with EtOAc (2 × 3.00 mL). The combined
organics were dried (Na2SO4), filtered, and concentrated in vacuo.
The crude was purified via column chromatography (5−10% EtOAc/
hexane) to afford the methyl-substituted ester intermediate. The
methyl ester was saponified in a solution of aq LiOH (1.00 mL, 1.00
M) and dioxane (1.00 mL) at 100 °C for 16 h. The solution was
cooled to rt, diluted with water (2.00 mL), and then acidified with aq
HCl (1.00 M) until pH 4. The solution was extracted with EtOAc (2
× 3.00 mL). The combined organics were dried (Na2SO4), filtered,
and concentrated in vacuo. The solid was washed with cold water
(5.00 mL), followed by hexane (10.0 mL), and dried to afford 2-
cyclopentyl-4-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-
benzoic acid 29 (79.0 mg, 80%) as a brown-yellow solid.
1H NMR (400 MHz, CDCl3): δ 7.82 (d, J = 8.0 Hz, 1H), 7.66 (d, J
= 5.3 Hz, 1H), 7.47 (d, J = 1.8 Hz, 1H), 7.31−7.23 (dd, 1H), 6.61 (d,
J = 5.3 Hz, 1H), 4.63 (br s, 2H), 3.93 (s, 3H), 3.86−3.71 (m, 1H),
3.61 (br s, 2H), 2.19−2.07 (m, 2H), 1.90−1.73 (m, 2H), 1.75−1.65
(m, 2H), 1.65−1.50 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 168.5,
149.1, 148.4, 140.6, 136.8, 133.8, 130.4, 130.4, 127.2, 126.6, 125.5,
116.3, 52.2, 41.7, 35.0, 25.7; HRMS: calcd for C18H22N3O2 [M + H]+
m/z, 312.1712; found m/z, 312.1704; mp range 125−129 °C.
2-Cyclopentyl-4-(3H-imidazo[4,5-b]pyridin-7-yl)benzoic Acid
(33). Methyl 2-cyclopentyl-4-(2,3-diaminopyridin-4-yl)benzoate 32
(100 mg, 0.32 mmol) was dissolved in MeOH (1 mL). Sulfamic acid
(3.00 mg, 32.0 μmol) and trimethyl orthoformate (85.0 mg, 0.80
mmol) were added, and the mixture was stirred at rt for 1 h. The
solvent was removed in vacuo, and the residue was dissolved in EtOAc
(10.0 mL) and water (10.0 mL). The phases were separated, and the
aqueous phase was extracted with EtOAc (2 × 10.0 mL). The
combined organics were dried (Na2SO4), filtered, and concentrated in
vacuo. The crude residue was purified by column chromatography
(50−100% EtOAc/hexane). The isolated methyl ester was dissolved
in dioxane (1.00 mL) and aq LiOH (1.00 mL of a 1.00 M solution).
The solution was heated to 100 °C for 16 h, cooled, and diluted with
EtOAc (3.00 mL) and water (3.00 mL). The mixture was acidified to
pH 4 with 1.00 M aq HCl solution. The layers were separated, and the
aqueous phase was extracted with EtOAc (3 × 3.00 mL). The
combined organics were dried (Na2SO4), filtered, and concentrated in
vacuo to afford 2-cyclopentyl-4-(3H-imidazo[4,5-b]pyridin-7-yl)-
benzoic acid 33 (45 mg, 46%) as a white solid.
1H NMR (700 MHz, DMSO-d6): δ 8.40 (d, J = 4.9 Hz, 1H), 7.82
(d, J = 8.0 Hz, 1H), 7.80 (d, J = 1.7 Hz, 1H), 7.71 (t, J = 7.7 Hz, 3H),
7.56 (t, J = 7.5 Hz, 2H), 7.50 (t, J = 7.4 Hz, 1H), 7.33 (d, J = 4.9 Hz,
1H), 6.73 (s, 1H), 3.89 (s, 3H), 3.86−3.76 (m, 1H), 2.12−2.04 (m,
2H), 1.84−1.77 (m, 2H), 1.70−1.59 (m, 4H); 13C NMR (176 MHz,
DMSO-d6): δ 169.3, 149.5, 146.7, 143.0, 142.2, 140.8, 139.4, 131.8,
131.6, 130.0, 128.9 (2 × ArCH), 128.8 (2 × ArCH), 128.6, 126.5,
125.5, 117.5, 115.1, 98.1, 41.2, 34.4, 29.9, 25.3; HRMS: calcd for
C26H25N2O2 [M + H]+ m/z, 397.1916; found m/z, 397.1908; mp
range 187−189 °C.
1H NMR (700 MHz, DMSO-d6): δ 13.23 (s, 1H), 8.51 (s, 1H),
8.45 (s, 1H), 8.41 (d, J = 4.7 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.79
(d, J = 8.1 Hz, 1H), 7.59 (d, J = 4.7 Hz, 1H), 3.80 (t, J = 8.7 Hz, 1H),
2.10−2.03 (br m, 2H), 1.87−1.73 (br m, 2H), 1.67 (m, 4H); 13C
2-Cyclopentyl-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)benzoic
Acid (30). 4-Chloro-1-methyl-1H-pyrrolo[2,3-b]pyridine (75.0 mg,
V
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NMR (176 MHz, DMSO-d6): δ 171.9, 169.4, 148.8, 146.1, 144.2,
143.8, 138.2, 136.5, 132.1, 131.8, 129.3, 127.6, 125.8, 115.3, 41.3,
34.4, 25.2; HRMS: calcd for C18H18N3O2 [M + H]+ m/z, 308.1399;
found, m/z 308.1392; mp range 183−186 °C.
vial was sealed, and the reaction mixture was irradiated at 125 °C for
15 min. The volatiles were removed in vacuo, and remaining solids
were suspended in ice/water before the pH was adjusted to 2−3 with
2 M HCl. After an initial purification over silica gel, the compound
was further purified by recrystallization from diethyl ether, affording
2-cyclopentyl-4-(6-phenylthieno[3,2-d]pyrimidin-4-yl)benzoic acid
37 (12.0 mg, 15%) as a white solid.
2-Cyclopentyl-4-(2-phenyl-3H-imidazo[4,5-b]pyridin-7-yl)-
benzoic Acid (34). Benzoic acid (47.0 mg, 0.38 mmol) and CDI (63.0
mg, 0.38 mmol) were loaded into a microwave vial and dissolved in
dimethylformamide (DMF) (1.0 mL). The solution was cooled to 0
°C and stirred for 30 min. Methyl 2-cyclopentyl-4-(2,3-diaminopyr-
idin-4-yl)benzoate 4 (100 mg, 0.32 mmol) was added, and the vial
was sealed. The mixture was heated to 200 °C for 10 min and allowed
to cool to rt, and water (3.00 mL) was added, causing a precipitate to
form. The precipitate was filtered and washed with cold water (10.0
mL) and hexane (10.0 mL). The precipitate was dissolved in dioxane
(1.00 mL) and aq LiOH (1.00 mL of a 1.00 M solution). The
solution was heated to 100 °C for 16 h and then diluted with EtOAc
(3.00 mL) and water (3.00 mL). The mixture was acidified to pH 4
with 1 M aq HCl solution. The layers were separated, and the
aqueous phase was extracted with EtOAc (3 × 3.00 mL). The
combined organics were dried (Na2SO4), filtered, and concentrated in
vacuo. The crude was purified via column chromatography (50−100%
EtOAc/hexane) to afford 2-cyclopentyl-4-(2-phenyl-3H-imidazo[4,5-
b]pyridin-7-yl)benzoic acid 34 (93.0 mg, 76%) as a white solid.
1H NMR (700 MHz, DMSO-d6): δ 8.55−8.44 (m, 2H), 8.33 (d, J
= 6.3 Hz, 2H), 8.05 (d, J = 6.7 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.67
(s, 1H), 7.61 (d, J = 6.6 Hz, 3H), 3.93−3.79 (m, 1H), 2.18−2.05 (m,
2H), 1.93−1.83 (m, 2H), 1.82−1.66 (m, 4H); 13C NMR (176 MHz,
DMSO-d6): δ 169.4, 150.2, 146.6, 137.7, 132.4, 131.1, 129.5, 129.1,
128.0, 127.1, 125.6, 116.2, 41.1, 39.5, 34.5, 25.4; HRMS: calcd for
C24H22N3O2 [M + H]+ m/z, 384.1712; found m/z, 384.1704; mp
range 196−199 °C.
1H NMR (400 MHz, DMSO-d6): δ 13.25 (s, 1H), 9.30 (s, 1H),
8.23 (s, 2H), 8.07 (dd, J = 8.1, 1.8 Hz, 1H), 8.00 (dd, J = 7.1, 2.5 Hz,
2H), 7.89 (d, J = 8.1 Hz, 1H), 7.62−7.52 (m, 3H), 3.80 (p, J = 8.3
Hz, 1H), 2.18−2.06 (m, 2H), 1.92−1.78 (m, 2H), 1.75−1.60 (m,
4H); 13C NMR (176 MHz, DMSO-d6): δ 169.2, 162.98, 157.7, 155.2,
153.6, 146.6, 138.9, 134.4, 131.9, 130.6, 129.8, 129.5, 127.4, 126.8,
126.6, 125.4, 120.3, 41.3, 34.5, 25.3; HRMS calcd for C24H20N2O2S
[M + H]+, 401.1324; found, 401.1329; mp range >250 °C.
2-Cyclopentyl-4-(thieno[3,2-d]pyrimidin-4-yl)benzoic Acid (38).
A 5 mL microwave vial was loaded with 4-chlorothieno[3,2-
d]pyrimidine 35a (0.41 mmol, 70.0 mg), 2-cyclopentyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 3 (0.41 mmol, 130
mg), Cs2CO3 (0.82 mmol, 270 mg), and Pd(Ph3)4 (5 mol %, 25.0
mg). The solids were dissolved in a solution of 1,4-dioxane/water
(3:1, 1.50 mL). The vial was sealed, and the reaction mixture was
irradiated at 125 °C for 15 min. The volatiles were removed in vacuo,
and the remaining solids were suspended with ice/water before the
pH was adjusted to 2−3 with 2 M HCl(aq). The crude was purified
via column chromatography (CH2Cl2/MeOH/HOAc, 95:4:1−
90:9:1), affording 2-cyclopentyl-4-(thieno[3,2-d]pyrimidin-4-yl)-
benzoic acid 38 (70.0 mg, 48%) as a white solid.
1H NMR (400 MHz, DMSO-d6): δ 13.24 (s, 1H), 9.31 (s, 1H),
8.60 (d, J = 5.5 Hz, 1H), 8.22 (d, J = 1.7 Hz, 1H), 8.04 (dd, J = 8.1,
1.7 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.76 (d, J = 5.5 Hz, 1H), 3.80
(p, J = 8.4, 7.9 Hz, 1H), 2.16−2.06 (m, 2H), 1.89−1.79 (m, 2H),
1.74−1.60 (m, 4H); 13C NMR (176 MHz, DMSO-d6): δ 169.2,
162.1, 158.3, 154.7, 146.6, 139.0, 138.9, 134.4, 129.8, 127.6, 126.6,
125.5, 124.5, 41.2, 34.5, 25.4; HRMS calcd for C18H16N2O2S [M +
H]+, 325.1011; found, 325.1011; mp range 205−209 °C.
Methyl 4-(6-Bromothieno[2,3-d]pyrimidin-4-yl)-2-cyclopentyl-
benzoate (40). A 5 mL microwave vial was loaded with 6-bromo-4-
chlorothieno[2,3-d]pyrimidine 39b (0.40 mmol, 100 mg), methyl 2-
cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 4
(0.36 mmol, 120 mg), Cs2CO3 (0.60 mmol, 195 mg), and Pd(Ph3)4
(5 mol %, 23.0 mg). The solids were dissolved in a solution of 1,4-
dioxane/water (3:1, 1.5 mL). The vial was sealed, and the reaction
mixture was irradiated at 145 °C for 15 min. The volatiles were
removed in vacuo, and the crude was purified via column
chromatography (0−5% hexane/EtOAc) to afford methyl 4-(6-
bromothieno[2,3-d]pyrimidin-4-yl)-2-cyclopentylbenzoate 40 (50.0
mg, 30%) as a white solid.
4-Chloro-6-iodothieno[3,2-d]pyrimidine (35b). An oven-dried
250 mL three-neck round-bottom flask provided with a pressure-
equalizing addition funnel was cooled under N2. 4-Chlorothieno[3,2-
d]pyrimidine 35a (3.40 g, 20.0 mmol) was added under positive N2
pressure, followed by the addition of anhydrous THF (65.0 mL). The
solution was cooled down to −78 °C; LDA (12.0 mL, 24.0 mmol)
was added dropwise over 5 min, and the mixture was stirred at −78
°C for 20 min. A solution of I2 (6.08 g, 24.0 mmol) in anhydrous
THF (15.0 mL) was added dropwise over 10 min using the equalizing
addition funnel. The resulting mixture was stirred at −78 °C for 30
min, allowed to warm up to rt over 1 h, and then stirred for 16 h. The
reaction was quenched with saturated NH4Cl/ice and diluted with
CHCl3 (200 mL). The layers were separated, and the aqueous layer
was extracted with CHCl3 (2 × 50.0 mL). The combined organics
were rinsed with aq Na2S2O3 (2 × 25.0 mL) and sat. NaCl solution (2
× 25.0 mL), dried (Na2SO4), filtered, and concentrated in vacuo. The
crude was purified via column chromatography (0−100% hexane/
EtOAc), affording 4-chloro-6-iodothieno[3,2-d]pyrimidine 35b (4.60
g, 68%) as a clear brown solid.
1H NMR (400 MHz, DMSO-d6): δ 8.95 (s, 1H), 8.22 (s, 1H), 7.98
(d, J = 1.9 Hz, 1H), 7.83−7.76 (m, 2H), 3.86 (s, 3H), 3.63−3.57 (m,
1H), 2.08−2.00 (m, 2H), 1.90−1.81 (m, 2H), 1.75−1.63 (m, 4H).
2-Cyclopentyl-4-(6-phenylthieno[2,3-d]pyrimidin-4-yl)benzoic
Acid (41). Methyl 4-(6-bromothieno[2,3-d]pyrimidin-4-yl)-2-cyclo-
pentylbenzoate 40 (0.11 mmol, 45.0 mg), phenylboronic acid (0.12
mmol, 15.0 mg), Cs2CO3 (0.38 mmol, 52.0 mg), and Pd(Ph3)4 (5
mol %, 6.00 mg) were dissolved in a solution of 1,4-dioxane/water
(3:1, 2.00 mL). The reaction mixture was stirred at reflux for 3 h. The
volatiles were removed in vacuo, and the crude was purified via
column chromatography (0−10% EtOAc/hexane). The material was
further purified using preparative TLC silica plates, affording methyl
2-cyclopentyl-4-(6-phenylthieno[2,3-d]pyrimidin-4-yl)benzoate (25
mg, 55%) as a white solid. The methyl ester was saponified as
follows: methyl 2-cyclopentyl-4-(6-phenylthieno[2,3-d]pyrimidin-4-
yl)benzoate (0.06 mmol, 25.0 mg) was dissolved in 1,4-dioxane (2.50
mL). LiOH (0.30 mmol, 7.00 mg) was dissolved in H2O (0.50 mL)
and added to the solution. This mixture was stirred at 80 °C for 4 h.
Solvents were removed in vacuo; the material was suspended in ice/
water, and the pH was adjusted to 2−3 with 2 M HCl. The resulting
solid was filtrated, thoroughly rinsed with water, and air- and vacuum-
1H NMR (400 MHz, CDCl3): δ 8.90 (s, 1H), 7.84 (s, 1H); 13C
NMR (176 MHz, DMSO-d6): δ 162.6, 154.9, 154.8, 151.7, 134.8,
134.2, 97.4.
4-Chloro-6-phenylthieno[3,2-d]pyrimidine (36). 4-Chloro-6-
iodothieno[3,2-d]pyrimidine 35b (1.01 mmol, 300 mg), phenyl-
boronic acid (1.01 mmol, 125 mg), NaHCO3 (1.01 mmol, 85.0 mg),
CsF (1.01 mmol, 155 mg), and Pd(Ph3)4 (35.0 mg, 0.03 mmol) were
dissolved in a 3:1 mixture of dioxane/water (7.00 mL). The reaction
mixture was stirred at reflux for 3 h. The volatiles were removed under
reduced pressure, and the crude was purified via column
chromatography (hexane/EtOAc 0−30%) to afford 4-chloro-6-
phenylthieno[3,2-d]pyrimidine 36 (175 mg, 70%) as a white solid.
1H NMR (400 MHz, DMSO-d6): δ 9.04 (s, 1H), 8.26 (s, 1H),
8.05−7.97 (m, 2H), 7.62−7.52 (m, 3H).
2-Cyclopentyl-4-(6-phenylthieno[3,2-d]pyrimidin-4-yl)benzoic
Acid (37). A 5 mL microwave vial was loaded with pyrimidine 36
(55.0 mg, 0.22 mmol) and 2-cyclopentyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzoic acid 3 (70.0 mg, 0.22 mmol), Cs2CO3
(0.45 mmol, 150 mg), and Pd(Ph3)4 (5 mol %, 13.0 mg). The solids
were dissolved in a solution of 1,4-dioxane/water (3:1, 1.50 mL). The
W
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dried, affording 2-cyclopentyl-4-(6-phenylthieno[2,3-d]pyrimidin-4-
yl)benzoic acid 41 (17.0 mg, 70%) as a white solid.
145.2, 139.8, 138.9, 138.8, 132.8, 130.7, 129.6, 129.3, 129.2, 128.3,
128.1, 127.1, 126.8, 126.2, 123.0, 122.3, 119.4, 99.5, 41.2, 34.5, 25.3;
HRMS: calcd for C27H24NO2 [M + H]+ m/z, 394.1807; found m/z,
394.1786; mp range 164−167 °C.
2-Cyclopentyl-4-(quinolin-4-yl)benzoic Acid (46). Prepared fol-
lowing the procedure for 21 using 4-chloroquinoline (100 mg, 611
μmol). After purification, 2-cyclopentyl-4-(quinolin-4-yl)benzoic acid
46 (l60 mg, 83%) was afforded as a white solid.
1H NMR (400 MHz, DMSO-d6): δ 13.11 (s, 1H), 9.16 (s, 1H),
8.16 (s, 1H), 8.13−8.06 (m, 2H), 7.83 (dd, J = 4.3, 2.4 Hz, 2H),
7.78−7.74 (m, 1H), 7.68−7.63 (m, 3H), 3.68−3.62 (m, 1H), 2.10−
1.98 (m, 2H), 1.87−1.79 (m, 2H), 1.70−1.62 (m, 4H); 13C NMR
(176 MHz, DMSO-d6): δ 169.1, 168.8, 159.9, 153.4, 146.8, 142.8,
136.9, 134.9, 130.7, 130.2, 129.4, 129.0, 128.9, 126.7, 124.9, 124.4,
117.7, 41.4, 34.2, 25.1; HRMS (HESI) calcd for C24H20N2O2S [M +
H]+, 401.1324; found, 401.1330; mp range >250 °C.
1H NMR (400 MHz, DMSO-d6): δ 13.09 (s, 1H), 8.97 (d, J = 4.4
Hz, 1H), 8.14−8.11 (m, 1H), 7.8−7.78 (m, 3H), 7.62 (ddd, J = 8.3,
6.9, 1.3 Hz, 1H), 7.56 (d, J = 1.7 Hz, 1H), 7.50 (d, J = 4.4 Hz, 1H),
7.44 (dd, J = 7.9, 1.7 Hz, 1H), 3.85−3.73 (m, 1H), 2.11−2.00 (m,
2H), 1.81−1.71 (m, 2H), 1.70−1.54 (m, 4H); 13C NMR (100 MHz,
DMSO-d6): δ 169.4, 150.2, 148.1, 146.8, 146.6, 140.0, 132.1, 129.6,
129.6, 129.5, 127.8, 127.2, 126.7, 125.7, 125.2, 121.5, 41.2, 34.5, 25.3;
HRMS: calcd for C21H19NO2 [M − H]− m/z, 316.1338; found,
316.1339; mp range 259−263 °C.
Methyl 2-Cyclopentyl-4-(6-hydroxyquinazolin-4-yl)benzoate
(48). Prepared following the procedure for 32 using 4-chloroquina-
zolin-6-ol 47b (50.0 mg, 0.28 mmol). After purification, methyl 2-
cyclopentyl-4-(6-hydroxyquinazolin-4-yl)benzoate 48 (63.0 mg, 65%)
was afforded as a thick oil.
2-Cyclopentyl-4-(thieno[2,3-d]pyrimidin-4-yl)benzoic Acid (42).
Prepared following the procedure for 38 using 4-chlorothieno[2,3-
d]pyrimidine 35a (70.0 mg, 0.41 mmol) and 2-cyclopentyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 3 (130 mg, 0.41
mmol). After purification, 2-cyclopentyl-4-(thieno[2,3-d]pyrimidin-4-
yl)benzoic acid 42 (72.0 mg, 55%) was afforded as a white solid.
1H NMR (400 MHz, DMSO-d6): δ 9.19 (s, 1H), 8.09 (d, J = 6.1
Hz, 1H), 7.98 (d, J = 1.6 Hz, 1H), 7.87−7.79 (m, 2H), 7.68 (d, J =
6.1 Hz, 1H), 3.83−3.70 (m, 1H), 2.12−2.02 (m, 2H), 1.86−1.74 (m,
2H), 1.65−1.56 (m, 4H); 13C NMR (176 MHz, DMSO-d6): δ 169.3,
169.3, 159.2, 153.1, 146.2, 139.4, 133.9, 129.5, 129.3, 127.4, 127.4,
126.4, 120.7, 41.3, 34.4, 25.3; HRMS (HESI) calcd for C18H16N2O2S
[M + H]+, 325.1011; found, 325.1013; mp range 146−149.5 °C.
Methyl 4-(6-Chloroquinolin-4-yl)-2-cyclopentylbenzoate (44).
4,6-Dichloroquinoline (1.00 g, 5.05 mmol) and 2-cyclopentyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 4 (1.58 g, 4.80
mmol) were dissolved in a 10:1 solution of dioxane/water (27.5 mL).
Pd2(dba)3 (231 mg, 0.25 mmol), XPhos (240 mg, 0.51 mmol), and
Cs2CO3 (4.94 g, 15.2 mmol) were added, and the mixture was stirred
for 16 h at 25 °C. The solution was diluted with EtOAc (20.0 mL)
and water (25.0 mL). The layers were separated, and the aqueous
phase was extracted with EtOAc (3 × 20.0 mL). The combined
organics were dried (Na2SO4), filtered, and concentrated in vacuo.
The crude was purified via column chromatography (10−30%
EtOAc/hexane) to afford methyl 4-(6-chloroquinolin-4-yl)-2-cyclo-
pentylbenzoate 44 (1.54 g, 83%) as a white solid.
1H NMR (400 MHz, DMSO-d6): δ 9.42 (s, 1H), 8.16 (d, J = 9.0
Hz, 1H), 8.10 (dd, J = 9.0, 2.3 Hz, 1H), 7.99−7.98 (m, 1H), 7.88−
7.84 (m, 3H), 7.71 (dd, J = 7.9, 1.7 Hz, 1H), 3.70−3.62 (m, 1H),
2.13−2.01 (m, 2H), 1.81−1.75 (m, 2H), 1.72−1.57 (m, 4H); 13C
NMR (101 MHz, DMSO-d6): δ 167.9, 166.2, 154.7, 149.0, 146.4,
138.9, 134.8, 132.6, 132.3, 130.8, 129.4, 128.4, 127.1, 125.2, 122.9,
52.4, 41.3, 34.4, 25.2.
2-Cyclopentyl-4-(6-phenylquinazolin-4-yl)benzoic Acid (49).
Methyl 2-cyclopentyl-4-(6-phenylquinazolin-4-yl)benzoate SI4 (170
mg, 0.41 mmol) was dissolved in MeOH (3.5 mL) and aq NaOH (1
mL). The resulting solution was heated to 70 °C for 1 h and allowed
to cool to rt, added water (4 mL), and extracted with ether (2 × 10
mL). The aqueous phase was acidified with 1 N HCl (pH 6) and then
extracted with EtOAc. The organic phase was dried (Na2CO3) and
concentrated to afford 2-cyclopentyl-4-(6-phenylquinazolin-4-yl)-
benzoic acid 49 (150 mg, 91%) as a light-yellow solid.
1H NMR (400 MHz, CDCl3): δ 8.96 (d, J = 4.4 Hz, 1H), 8.15 (d, J
= 9.0 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 2.3 Hz, 1H), 7.69
(dd, J = 9.0, 2.3 Hz, 1H), 7.51 (d, J = 1.6 Hz, 1H), 7.42−7.33 (m,
2H), 3.97 (s, 3H), 3.89−3.78 (m, 1H), 2.23−2.12 (m, 2H), 1.86−
1.69 (m, 4H), 1.63 (ddd, J = 16.2, 12.9, 8.8 Hz, 2H); 13C NMR (100
MHz, CDCl3): δ 168.5, 145.0, 148.1, 147.2, 146.9, 140.4, 133.0,
131.5, 131.1, 130.5, 130.0, 128.1, 127.2, 126.4, 124.5, 121.8, 77.0,
52.3, 41.8, 35.9, 25.7; HRMS: calcd for C22H21ClNO2 [M + H]+ m/z,
366.1260; found m/z, 366.1246; mp range 137−139 °C.
1H NMR (400 MHz, DMSO-d6): δ 13.19 (s, 1H), 9.39 (s, 1H),
8.39 (dd, J = 8.8, 2.1 Hz, 1H), 8.24−8.19 (m, 2H), 7.92 (d, J = 1.7
Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.78−7.73 (m, 3H), 7.51 (t, J = 7.4
Hz, 2H), 7.45 (d, J = 7.3 Hz, 1H), 3.86−3.75 (m, 1H), 2.12−2.04 (m,
2H), 1.80−1.74 (m, 2H), 1.69−1.60 (m, 4H); 13C NMR (101 MHz,
DMSO-d6): δ 169.4, 167.0, 154.4, 149.9, 146.1, 139.8, 139.0, 138.8,
133.5, 133.4, 129.3, 129.2, 129.2, 128.5, 128.3, 127.2, 127.1, 123.7,
122.5, 41.2, 34.5, 25.3; HRMS: calcd for C26H22N2O2 [M − H]− m/z,
393.1607; found, 393.1603; mp range 265−269 °C.
2-Cyclopentyl-4-(quinazolin-4-yl)benzoic Acid (50). 4-Chloroqui-
nazoline (100 mg, 0.60 mmol), 2-cyclopentyl-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzoate 4 (240 mg, 0.72 mmol), Pd(PPh3)4
(70.0 mg, 60.0 μmol), and Cs2CO3 (0.60 g, 1.80 mmol) were loaded
into a microwave vial. A solution of dioxane/water (6.0 mL of a 10:1
mix) was added, and the vial was flushed with nitrogen. The mixture
was irradiated at 100 °C for 30 min. Once cooled, the aqueous layer
was removed and the remaining volatiles were removed in vacuo. The
crude was purified via column chromatography, and the intermediate
ester was isolated. The ester was dissolved in dioxane (1 mL) and aq
LiOH (1 mL of a 1 M solution). The solution was heated to 100 °C
for 16 h and then acidified to pH 4 with 1 M aq HCl, causing a
precipitate to form. The solid was collected by filtration, washed with
cold H2O, and then dried under vacuum to afford the quinazoline
(128 mg, 67%) as a pale-white solid.
2-Cyclopentyl-4-(6-phenylquinolin-4-yl)benzoic Acid (45). Meth-
yl 4-(6-chloroquinolin-4-yl)-2-cyclopentylbenzoate 44 (100 mg, 0.27
mmol) and phenylboronic acid (66.0 mg, 0.32 mmol) were dissolved
in a 10:1 solution of dioxane/water (12 mL). Pd2(dba)3 (25 mg, 0.03
mmol), XPhos (29.0 mg, 0.06 mmol), and Cs2CO3 (264 mg, 0.81
mmol) were added, and the solution was heated to 80 °C for 16 h.
Once cooled, the solution was diluted with EtOAc (10 mL) and water
(10 mL). The layers were separated, and the aqueous phase was
extracted with EtOAc (3 × 10 mL). The combined organics were
dried (Na2SO4), filtered, and concentrated in vacuo. The crude was
purified via column chromatography (10−30% EtOAc/hexane) to
afford methyl 2-cyclopentyl-4-(6-phenylquinolin-4-yl)benzoate as an
intermediate. The intermediate methyl ester was dissolved in dioxane
(1 mL) and aq LiOH (1 mL of a 1 M solution). The solution was
heated to 100 °C for 16 h and then acidified to pH 4 with 1 M aq
HCl, causing a precipitate to form. The solid was collected by
filtration, washed with cold water (4 mL) and Et2O (10 mL), and
dried under vacuum to afford 2-cyclopentyl-4-(6-phenylquinolin-4-
yl)benzoic acid 45 (81 mg, 76%) as an off-white solid.
1H NMR (400 MHz, DMSO-d6): δ 9.38 (s, 1H), 8.15−8.03 (m,
3H), 7.85 (d, J = 7.9 Hz, 1H), 7.78 (ddd, J = 9.7, 6.8, 1.4 Hz, 3H),
7.66 (dd, J = 7.9, 1.7 Hz, 1H), 3.83−3.73 (m, 1H), 2.11−2.02 (m,
2H), 1.81−1.71 (m, 2H), 1.70−1.57 (m, 4H); 13C NMR (101 MHz,
DMSO-d6): δ 169.3, 167.0, 154.3, 150.3, 146.0, 139.0, 134.4, 133.5,
129.1, 128.6, 128.4, 128.2, 127.08, 126.6, 122.3, 41.3, 34.4, 25.2;
HRMS: calcd for C20H19N2O2 [M + H]+ m/z, 319.1446; found, m/z
319.1427; mp range 120−122 °C.
1H NMR (400 MHz, DMSO-d6): δ 9.15 (d, J = 4.8 Hz, 1H), 8.39
(d, J = 8.6 Hz, 1H), 8.32 (d, J = 8.7 Hz, 1H), 8.13 (d, J = 1.7 Hz, 1H),
7.87 (d, J = 7.9 Hz, 1H), 7.84−7.79 (m, 1H), 7.74−7.69 (m, 3H),
7.59 (dd, J = 8.0, 1.6 Hz, 1H), 7.51 (t, J = 7.4 Hz, 2H), 7.47−7.41 (m,
1H), 3.88−3.72 (m, 1H), 2.14−2.01 (m, 2H), 1.84−1.71 (m, 2H),
1.71−1.50 (m, 4H); 13C NMR (176 MHz, DMSO): δ 169.3, 146.4,
X
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Article
2-Cyclopentyl-4-(2-methylquinolin-4-yl)benzoic Acid (52). Pre-
pared following the procedure for 21 using 4-chloro-2-methylquino-
line 51 (200 mg, 1.13 mmol). After purification, 2-cyclopentyl-4-(2-
methylquinolin-4-yl)benzoic acid 52 (290 mg, 77%) was afforded as
an off-white solid.
mmol) were added, and the solution was irradiated at 120 °C for 30
min. The resulting suspension was filtered through Celite under
vacuum. The filter cake was washed with ethyl acetate, and the filtrate
was dried (Na2SO4), filtered, and concentrated in vacuo. The crude
product was purified by column chromatography (20% EtOAc/
hexane) to afford methyl 4-(2-aminopyrimidin-4-yl)-2-cyclopentyl-
benzoate 60 (523 mg, 45%) as a white solid.
1H NMR (400 MHz, DMSO-d6): δ 13.08 (s, 1H), 8.01 (dd, J =
8.5, 1.5 Hz, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.78−7.72 (m, 2H), 7.56−
7.52 (m, 2H), 7.43−7.39 (m, 2H), 3.86−3.74 (m, 1H), 2.70 (s, 3H),
2.12−2.00 (m, 2H), 1.78−1.74 (m, 2H), 1.67−1.56 (m, 4H); 13C
NMR (176 MHz, DMSO-d6): δ 172.6, 161.6, 151.0, 150.0, 149.3,
143.2, 135.4, 132.6, 132.5, 132.0, 130.8, 129.7, 129.3, 128.1, 127.2,
125.3, 44.4, 37.6, 28.4, 27.9; HRMS: calcd for C22H21NO2 [M − H]−
m/z, 332.1651; found, 332.1642; mp range 271−275 °C.
1H NMR (400 MHz, DMSO-d6): δ 8.34 (d, J = 5.2 Hz, 1H), 8.13
(d, J = 1.7 Hz, 1H), 7.93 (dd, J = 8.1, 1.7 Hz, 1H), 7.73 (d, J = 8.1 Hz,
1H), 7.19 (d, J = 5.2 Hz, 1H), 6.74 (s, 2H), 3.85 (s, 3H), 3.69−3.54
(m, 1H), 2.11−1.96 (m, 2H), 1.90−1.75 (m, 2H), 1.73−1.55 (m,
3H); 13C NMR (100 MHz, DMSO-d6): δ 168.00, 163.81, 162.68,
159.31, 146.28, 139.90, 132.37, 129.51, 124.83, 124.03, 106.29, 52.23,
41.48, 34.34, 25.20; mp range 178 °C.
2-Cyclopentyl-4-(1,6-naphthyridin-4-yl)benzoic Acid (54). Pre-
pared following the procedure for 21 using 4-chloro-1,6-naphthyr-
idine 53 (80.0 mg, 0.49 mmol). After purification, 2-cyclopentyl-4-
(1,6-naphthyridin-4-yl)benzoic acid 54 (110 mg, 54%) was afforded
as a yellow solid.
4-(2-Aminopyrimidin-4-yl)-2-cyclopentylbenzoic Acid Hydro-
chloride (61·HCl). Methyl 4-(2-aminopyrimidin-4-yl)-2-cyclopentyl-
benzoate 60 (40.0 mg, 0.13 mmol) was dissolved in 1,4-dioxane (1.00
mL). 1 M LiOH (1.00 mL) was added, and the solution was stirred at
100 °C for 17 h. HCl (6.00 M, 6.00 mL) was added, resulting in
precipitate formation. The precipitate was filtered and washed with
HCl (6.00 M). The precipitate was dried under reduced pressure to
afford 4-(2-aminopyrimidin-4-yl)-2-cyclopentylbenzoic acid hydro-
chloride 61·HCl (35.0 mg, 85%) as a white solid.
1H NMR (400 MHz, DMSO-d6): δ 13.14 (s, 1H), 9.24−9.15 (m,
2H), 8.79 (d, J = 5.8 Hz, 1H), 8.01 (dd, J = 5.9, 0.9 Hz, 1H), 7.84 (d,
J = 7.9 Hz, 1H), 7.71−7.65 (m, 2H), 7.54 (dd, J = 7.9, 1.8 Hz, 1H),
3.84−3.73 (m, 1H), 2.09−2.04 (m, 2H), 1.80−1.71 (m, 2H), 1.71−
1.56 (m, 4H); 13C NMR (176 MHz, DMSO-d6): δ 169.88, 155.32,
150.85, 150.45, 147.84, 146.88, 146.7, 138.4, 133.1, 129.9, 128.4,
127.3, 123.5, 122.3, 121.5, 41.6, 34.8, 25.6; HRMS calcd for
C20H18N2O2 [M − H]− m/z, 319.1447; found, 319.1427; mp range
195−200 °C.
1H NMR (400 MHz, DMSO-d6): δ 8.54−8.14 (br s, 1H) 8.53 (d, J
= 6.3 Hz, 1H), 8.22 (d, J = 1.8 Hz, 1H), 8.05 (dd, J = 8.2, 1.8 Hz,
1H), 7.77 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 6.3 Hz, 1H), 3.73−3.60
(m, 1H), 2.10−1.98 (m, 2H), 1.90−1.76 (m, 2H), 1.73−1.57 (m,
4H); 13C NMR (100 MHz, DMSO-d6): δ 169.2, 168.3, 157.1, 150.3,
146.1, 136.7, 136.0, 129.5, 126.1, 125.3, 106.5, 41.5, 34.4, 25.2;
HRMS: calcd for C16H18N3O2 [M + H]+ m/z, 284.1394; found,
284.1394; mp range >250 °C.
2-Cyclopentyl-4-(2-(phenylamino)pyrimidin-4-yl)benzoic Acid
Hydrochloride (62·HCl). Methyl 2-cyclopentyl-4-(2-(phenylamino)-
pyrimidin-4-yl)benzoate SI5 (20 mg, 54 μmol) was dissolved in 1,4-
dioxane (1.0 mL). 1 M LiOH (1.0 mL) was added, and the solution
was stirred for at 100 °C for 16 h. HCl (6.0 M, 6.0 mL) was added,
resulting in precipitate formation. The precipitate was filtered and
washed with HCl (6.0 M). The precipitate was dried under reduced
pressure to afford 2-cyclopentyl-4-(2-(phenylamino)pyrimidin-4-yl)-
benzoic acid hydrochloride (62·HCl) (12 mg, 63%) as a yellow solid.
1H NMR (700 MHz, DMSO-d6): δ 13.10 (br s, 1H), 9.74 (s, 1H),
8.61−8.55 (m, 1H), 8.29 (s, 1H), 8.00 (d, J = 7.4 Hz, 1H), 7.86 (d, J
= 7.3 Hz, 2H), 7.77 (d, J = 7.4 Hz, 1H), 7.50−7.46 (m, 1H), 7.33−
7.27 (m, 2H), 7.01−6.96 (m, 1H), 3.77 (p, J = 9.6 Hz, 1H), 2.13−
2.03 (m, 2H), 1.89−1.80 (m, 2H), 1.73−1.61 (m, 4H); 13C NMR
(176 MHz, DMSO-d6): δ 169.3, 162.6, 160.1, 159.3, 146.4, 140.5,
139.0, 134.0, 129.6, 128.4, 125.1, 124.0, 121.5, 119.0, 108.3, 41.2,
34.5, 25.4; HRMS: calcd for C22H22N3O2 [M + H]+ m/z, 360.4365;
found, 360.1712; mp range >250 °C.
2-Cyclopentyl-4-(pyrimidin-4-yl)benzoic Acid (56). 4-Chloropyr-
imidine (162 mg, 1.40 mmol) and Cs2CO3 (1.32 g, 4.00 mmol) were
dissolved in 1,2-DME (4.00 mL) and H2O (0.70 mL). Pd(PPh3)4 (78
mg, 68 μmol) and 3 (503 mg, 1.60 mmol) were added, and the
solution was stirred at 120 °C for 30 min. The resulting suspension
was filtered through Celite under vacuum. The filter cake was washed
with ethyl acetate, and the filtrate was dried (Na2SO4), filtered, and
concentrated in vacuo. The crude product was purified by column
chromatography [0−50% EtOAc(0.01% acetic acid)/hexane] to
afford 2-cyclopentyl-4-(pyrimidin-4-yl)benzoic acid 56 (29 mg, 8%)
as a white solid.
1H NMR (700 MHz, DMSO-d6): δ 13.14 (br s, 1H), 9.28 (s, 1H),
8.92−8.87 (m, 1H), 8.25 (s, 1H), 8.20−8.16 (m, 1H), 8.06 (d, J = 7.7
Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 3.72 (p, J = 8.0 Hz, 1H), 2.09−
2.01 (m, 2H), 1.87−1.79 (m, 2H), 1.73−1.59 (m, 4H); 13C NMR
(176 MHz, DMSO-d6): δ 169.3, 158.8, 158.8, 158.3, 146.4, 138.3,
134.5, 129.7, 125.1, 124.2, 117.7, 41.4, 34.4, 25.3; HRMS: calcd for
C16H15N2O2 [M − H]− m/z, 267.1138; found, 267.1139; mp range
245 °C.
2-Cyclopentyl-4-(2-phenylpyrimidin-4-yl)benzoic Acid (58). 4-
Chloro-2-phenylpyrimidine (155 mg, 0.81 mmol) and Cs2CO3 (775
mg, 2.40 mmol) were dissolved in 1,2-DME (2.40 mL) and H2O
(0.40 mL). Pd(PPh3)4 (48.0 mg, 42.0 μmol) and 3 (307 mg, 0.97
mmol) were added, and the solution was stirred at 120 °C for 30 min.
The resulting suspension was filtered through Celite under vacuum.
The filter cake was washed with ethyl acetate, and the filtrate was
dried (Na2SO4), filtered, and concentrated in vacuo. The crude
product was purified by column chromatography [0−10% EtOAc-
(0.01% acetic acid)/hexane] to afford 2-cyclopentyl-4-(2-phenyl-
pyrimidin-4-yl)benzoic acid 58 (21 mg, 8%) as a white solid.
1H NMR (700 MHz, DMSO-d6): δ 13.16 (br s, 1H), 9.04−8.95
(m, 1H), 8.52 (d, J = 6.0 Hz, 2H), 8.33 (s, 1H), 8.21 (d, J = 7.6 Hz,
1H), 8.15−8.07 (m, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.63−7.53 (m,
3H), 3.76 (p, J = 8.8 Hz, 1H), 2.12−2.03 (m, 2H), 1.90−1.82 (m,
2H), 1.75−1.65 (m, 4H); 13C NMR (176 MHz, DMSO-d6): δ 169.4,
163.4, 162.3, 158.9, 146.4, 138.5, 137.2, 134.5, 131.0, 129.7, 128.8,
127.8, 125.3, 124.3, 115.7, 41.4, 34.4, 25.3; HRMS: calcd for
C22H21N2O2 [M + H]+ m/z, 345.1598; found, 345.1600; mp range
>250 °C.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02274.
■
sı
Mass spectrometry methods, compound characterization
spectra, CAMKK2 in cell target engagement NanoBRET
data, p-AMPK Western blot images, details on docking
studies, and treespot images of kinase selectivity data
(PDF)
Compound selectivity data (CSV)
Compound molecular formula strings (XLSX)
AUTHOR INFORMATION
Corresponding Authors
■
Methyl 4-(2-Aminopyrimidin-4-yl)-2-cyclopentylbenzoate (60).
4-Chloropyrimidin-2-amine (501 mg, 3.90 mmol) and Cs2CO3 (3.81
mg, 12.0 mmol) were dissolved in 1,2-DME (8.00 mL) and H2O
(2.00 mL). Pd(PPh3)4 (238 mg, 0.21 mmol) and 4 (1.50 g, 4.50
John W. Scott − St Vincent’s Institute and Department of
Medicine, The University of Melbourne, Fitzroy 3065,
Australia; Mary MacKillop Institute for Health Research,
Y
https://doi.org/10.1021/acs.jmedchem.0c02274
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Australian Catholic University, Melbourne 3000, Australia;
The Florey Institute of Neuroscience and Mental Health,
Parkville 3052, Australia; Phone: +61-3-9231-3510;
Email: jscott@svi.edu.au
David H. Drewry − Structural Genomics Consortium and
Division of Chemical Biology and Medicinal Chemistry, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599, United
States; UNC Lineberger Comprehensive Cancer Center, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599, United
States; orcid.org/0000-0001-5973-5798; Phone: +1-
919-602-1327; Email: david.drewry@unc.edu
William J. Zuercher − Structural Genomics Consortium and
Division of Chemical Biology and Medicinal Chemistry, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599, United
States
Carolina M. C. Catta-Preta − Centro de Química Medicinal
(CQMED), Centro de Biologia Molecular e Engenharia
Genética (CBMEG), Universidade Estadual de Campinas
(UNICAMP), Campinas, Sao
Structural Genomics Consortium, Departamento de Genética
e Evoluca , Instituto de Biologia, UNICAMP, Campinas, Sao
̃
Paulo 13083-875, Brazil;
̧
o
̃
̃
Paulo 13083-886, Brazil
Priscila Zonzini Ramos − Centro de Química Medicinal
(CQMED), Centro de Biologia Molecular e Engenharia
Genética (CBMEG), Universidade Estadual de Campinas
Authors
(UNICAMP), Campinas, Sao
Structural Genomics Consortium, Departamento de Genética
e Evoluca , Instituto de Biologia, UNICAMP, Campinas, Sao
̃
Paulo 13083-875, Brazil;
Benjamin J. Eduful − Structural Genomics Consortium and
Division of Chemical Biology and Medicinal Chemistry, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599, United
States
Sean N. O’Byrne − Structural Genomics Consortium and
Division of Chemical Biology and Medicinal Chemistry, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599, United
States
Louisa Temme − Structural Genomics Consortium and
Division of Chemical Biology and Medicinal Chemistry, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599, United
States
Christopher R. M. Asquith − Structural Genomics
Consortium and Division of Chemical Biology and Medicinal
Chemistry, UNC Eshelman School of Pharmacy, University
of North Carolina at Chapel Hill, Chapel Hill, North
Carolina 27599, United States; Department of
Pharmacology, School of Medicine, University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,
United States; orcid.org/0000-0001-5871-3458
Yi Liang − Structural Genomics Consortium and Division of
Chemical Biology and Medicinal Chemistry, UNC Eshelman
School of Pharmacy, University of North Carolina at Chapel
Hill, Chapel Hill, North Carolina 27599, United States
Alfredo Picado − Structural Genomics Consortium and
Division of Chemical Biology and Medicinal Chemistry, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599, United
States
Joseph R. Pilotte − Structural Genomics Consortium and
Division of Chemical Biology and Medicinal Chemistry, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599, United
States
Mohammad Anwar Hossain − Structural Genomics
Consortium and Division of Chemical Biology and Medicinal
Chemistry, UNC Eshelman School of Pharmacy, University
of North Carolina at Chapel Hill, Chapel Hill, North
Carolina 27599, United States; orcid.org/0000-0001-
8684-8755
Carrow I. Wells − Structural Genomics Consortium and
Division of Chemical Biology and Medicinal Chemistry, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599, United
States; orcid.org/0000-0003-4799-6792
̧
o
̃
̃
Paulo 13083-886, Brazil
André de S. Santiago − Centro de Química Medicinal
(CQMED), Centro de Biologia Molecular e Engenharia
Genética (CBMEG), Universidade Estadual de Campinas
(UNICAMP), Campinas, Sao
Structural Genomics Consortium, Departamento de Genética
e Evoluca , Instituto de Biologia, UNICAMP, Campinas, Sao
Paulo 13083-886, Brazil
Rafael M. Coun
̃
Paulo 13083-875, Brazil;
̧
o
̃
̃
̃
ago − Centro de Química Medicinal
(CQMED), Centro de Biologia Molecular e Engenharia
Genética (CBMEG), Universidade Estadual de Campinas
(UNICAMP), Campinas, Sao
Structural Genomics Consortium, Departamento de Genética
e Evoluca , Instituto de Biologia, UNICAMP, Campinas, Sao
̃
Paulo 13083-875, Brazil;
̧
o
̃
̃
Paulo 13083-886, Brazil
Christopher G. Langendorf − St Vincent’s Institute and
Department of Medicine, The University of Melbourne,
Fitzroy 3065, Australia
Kévin Nay − St Vincent’s Institute and Department of
Medicine, The University of Melbourne, Fitzroy 3065,
Australia; Mary MacKillop Institute for Health Research,
Australian Catholic University, Melbourne 3000, Australia
Jonathan S. Oakhill − St Vincent’s Institute and Department
of Medicine, The University of Melbourne, Fitzroy 3065,
Australia; Mary MacKillop Institute for Health Research,
Australian Catholic University, Melbourne 3000, Australia
Thomas L. Pulliam − Department of Cancer Systems Imaging,
University of Texas MD Anderson Cancer Center, Houston,
Texas 77054, United States; Center for Nuclear Receptors
and Cell Signaling and Department of Biology and
Biochemistry, University of Houston, Houston, Texas 77204,
United States
Chenchu Lin − Department of Cancer Systems Imaging,
University of Texas MD Anderson Cancer Center, Houston,
Texas 77054, United States; The University of Texas MD
Anderson Cancer Center UTHealth Graduate School of
Biomedical Sciences, Houston, Texas 77030, United States
Dominik Awad − Department of Cancer Systems Imaging,
University of Texas MD Anderson Cancer Center, Houston,
Texas 77054, United States; The University of Texas MD
Anderson Cancer Center UTHealth Graduate School of
Biomedical Sciences, Houston, Texas 77030, United States
Timothy M. Willson − Structural Genomics Consortium and
Division of Chemical Biology and Medicinal Chemistry, UNC
Eshelman School of Pharmacy, University of North Carolina
Z
https://doi.org/10.1021/acs.jmedchem.0c02274
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
at Chapel Hill, Chapel Hill, North Carolina 27599, United
States; orcid.org/0000-0003-4181-8223
PDB ID codes: 5UY6; 5UYJ. Authors will release the atomic
coordinates upon article publication.
Daniel E. Frigo − Department of Cancer Systems Imaging,
University of Texas MD Anderson Cancer Center, Houston,
Texas 77054, United States; Center for Nuclear Receptors
and Cell Signaling and Department of Biology and
Biochemistry, University of Houston, Houston, Texas 77204,
United States; Department of Genitourinary Medical
Oncology, University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States; The Methodist
Hospital Research Institute, Houston, Texas 77030, United
States; orcid.org/0000-0002-0713-471X
ACKNOWLEDGMENTS
■
We are grateful for LC−MS/HRMS support provided by Dr.
Brandie Ehrmann and Diane E. Wallace in the Mass
Spectrometry Core Laboratory at the University of North
Carolina at Chapel Hill. The core is supported by the National
Science Foundation under grant no. CHE-1726291.
ABBREVIATIONS
■
CAMKK2, calcium-calmodulin protein kinase kinase 2;
AMPK, AMP-activated protein kinase; AKT, protein kinase
B (PKB); siRNA, small interfering RNA; HCC, hepatocellular
carcinoma; NAFLD, non-alcoholic fatty liver disease; GRK3, G
protein-coupled receptor kinase 3; CK2, casein kinase 2;
CDKL2, cyclin-dependent kinase-like 3; AhR, aryl hydro-
carbon receptor; ATP, adenosine triphosphate; SEM,
trimethylsilylethoxymethyl; CDI, 1,1-carbodiimidazole; DME,
dimethoxyethane; DSF, differential scanning fluorimetry; PoC,
percentage of control; ADMET, absorption, distribution,
metabolism, excretion, and toxicology; LE, ligand efficiency;
LLE, lipophilic ligand efficiency; DMSO, dimethylsulfoxide;
BRET, bioluminescence resonance energy transfer
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02274
Author Contributions
⊥⊥B.J.E., S.N.O., and L.T. contributed equally to this work.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This research was funded in part by the National Cancer
Institute of the National Institutes of Health (grant numbers
R01CA218442 and R01CA184208) and was supported by the
NIH Illuminating the Druggable Genome program (grant
number 1U24DK116204-01), FAPESP (Fundaca
̧
o
̃
de Amparo
Paulo) (2013/50724-5 and 2014/
50897-0), and CNPq (Conselho Nacional de Desenvolvimen-
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beamline is funded by an NIH-ORIP HEI grant (S10
RR029205). This research used resources of the Advanced
Photon Source, a U.S. Department of Energy (DOE) Office of
Science User Facility, operated for the DOE Office of Science
by Argonne National Laboratory under contract no. DE-AC02-
06CH11357. We thank Diamond Light Source for access to
beam line I03 (proposal number MX10619-74).
Notes
The authors declare no competing financial interest.
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