Discovery of dual death-associated protein related apoptosis inducing protein kinase 1 and 2 inhibitors by a scaffold hopping approach

Article abstract of DOI:10.1021/jm5007929

DRAK2 emerged as a promising drug target for the treatment of autoimmune diseases and to prevent graft rejection after organ transplantation. Screening of a compound library in a DRAK2 binding assay led to the identification of an isothiazolo[5,4-b]pyridine derivative as a novel ligand for DRAK2, displaying a K_dvalue of 1.6 μM. Subsequent medicinal chemistry work led to the discovery of a thieno[2,3-b]pyridine derivative with strong DRAK2 binding affinity (K_d= 9 nM). Moreover, this compound also behaves as a functional inhibitor of DRAK2 enzymatic activity, displaying an IC₅₀value of 0.82 μM, although lacking selectivity, when tested against DRAK1. This paper describes for the first time functionally active dual DRAK1 and DRAK2 inhibitors that can be used as starting point for the synthesis of chemical tool compounds to study DRAK1 and DRAK2 biology, or they can be considered as hit compounds for hit-to-lead optimization campaigns in drug discovery programs.

Full text of DOI:10.1021/jm5007929

Article
pubs.acs.org/jmc
Discovery of Dual Death-Associated Protein Related Apoptosis
Inducing Protein Kinase 1 and 2 Inhibitors by a Scaffold Hopping
Approach
†,‡
Ling-Jie Gao,†,‡ Sona Kovackova,†,‡ Michal Sala, Anna Teresa Ramadori,†,‡ Steven De Jonghe,†,‡
̌
́
and Piet Herdewijn*,†,‡
†Rega Institute for Medical Research, Laboratory of Medicinal Chemistry, KU Leuven, Minderbroedersstraat 10, 3000 Leuven,
Belgium
‡Interface Valorisation Platform, KU Leuven, Kapucijnenvoer 33, 3000 Leuven, Belgium
ABSTRACT: DRAK2 emerged as a promising drug target for
the treatment of autoimmune diseases and to prevent graft
rejection after organ transplantation. Screening of a compound
library in a DRAK2 binding assay led to the identification of an
isothiazolo[5,4-b]pyridine derivative as a novel ligand for
DRAK2, displaying a Kd value of 1.6 μM. Subsequent
medicinal chemistry work led to the discovery of a
thieno[2,3-b]pyridine derivative with strong DRAK2 binding affinity (Kd = 9 nM). Moreover, this compound also behaves as
a functional inhibitor of DRAK2 enzymatic activity, displaying an IC50 value of 0.82 μM, although lacking selectivity, when tested
against DRAK1. This paper describes for the first time functionally active dual DRAK1 and DRAK2 inhibitors that can be used as
starting point for the synthesis of chemical tool compounds to study DRAK1 and DRAK2 biology, or they can be considered as
hit compounds for hit-to-lead optimization campaigns in drug discovery programs.
(EFGR inhibitor licensed for the treatment of nonsmall cell
lung cancer).
INTRODUCTION
■
Although the human kinome comprises more than 500 protein
kinases,1 only a small fraction of the kinome is currently being
targeted by reasonably selective and potent inhibitors.
However, small-molecule cell-permeable inhibitors of protein
kinases are of high value. They are useful tool compounds to
investigate the physiological role of protein kinases, because
they can be used to block the endogenous kinase activity in
cells, and, in addition, these compounds can be used as starting
points in hit-to-lead optimization campaigns in drug discovery
programs if the kinase is sufficiently validated as drug target.
Protein kinases are the second most exploited group of drug
targets, after the G-protein-coupled receptors (GPCR’s).
Hence, much research has been devoted to the identification
of small molecule kinase inhibitors and it has been stated that
currently 20−30% of the drug discovery programs in the
pharmaceutical industry are focused on kinases as drug targets.2
The vast majority of the kinase targets are being investigated
for the treatment of cancer, which has led to the FDA approval
of a number of small molecules (Figure 1).3 Imatinib was the
first kinase inhibitor drug to be developed targeting Abelson
tyrosine kinase and is used in the treatment of chronic myeloid
leukemia (Figure 1). Since its approval, many more compounds
have undergone clinical trials in different oncology indications
and a number of novel kinase inhibitors were licensed for
anticancer use. Examples include ruxolitinib (JAK1/2-inhibitor
approved for the treatment of myelofibrosis), dasatinib
(targeting Scr, ABL1, KIT, PDGFR, and Eph) and gefitinib
Although other therapeutic areas are lagging behind, there is
increasing interest in the development of kinase target
therapeutics for nononcology indications. Kinases have been
proposed as therapeutic targets for heart failure,4 CNS
disorders,5 and inflammatory diseases.6
In this paper, DAPK related apoptosis inducing protein
kinase 2 (DRAK2) was selected as a kinase target. DRAK2, also
known as STK17B, is a serine/threonine protein kinase which
belongs to the DAPK (death-associated protein kinase) family.7
The remaining four members are DAPK1, DAPK2, DAPK3,
and DRAK1.8 All members of the DAPK family have been
shown to induce apoptosis upon ectopic expression in various
cell types. Although DRAK2 is broadly expressed at low levels,
DRAK2 expression is highly enriched in T and B cells, whereas
it is not expressed at significant levels in NK cells, macrophages,
or dendritic cells.9 Due to the lymphoid specific nature of
DRAK2, its potential as a therapeutic target in immunological
diseases has been investigated. Genetic deletion of DRAK2
afforded mice that are resistant to autoimmune diseases, such as
EAE (experimental autoimmune encephalomyelitis, which is an
animal model of multiple sclerosis) and type 1 diabetes.10,11
The absence of DRAK2 in T cells in these animal models leads
to an increased death of T-cells and did not affect the survival
of T cells expanding during infections. It suggests that DRAK2
Received: May 22, 2014
© XXXX American Chemical Society
A
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Figure 1. Clinically used kinase inhibitors.
is essential for the survival of T cells in chronic, autoimmune
stimulation. Recent studies demonstrated that DRAK2 signal-
ing is required for efficient allorejections.12 It supports the
notion that DRAK2 inhibition may lead to long-term allograft
maintenance and that DRAK2 inhibition is a therapeutic option
to prevent graft rejection after organ transplantation.
Figure 2. Hit compound.
All together, these data suggest that DRAK2 inhibitors hold
promise for the treatment of a variety of autoimmune diseases,
as well as to prevent graft rejection after organ transplantation.
To the best of our knowledge, only one example of a small-
molecule inhibitor targeting DRAK2 has been reported in the
literature.13 In this paper, a low micromolar inhibitor of
DRAK2 was identified, which at a concentration of 1 nM is able
to induce neuronal differention. In this study, there seems to be
a discrepancy between the DRAK2 target affinity (low μM) and
the cellular activity (low nM). In addition, the exact chemical
structure of this compound in not mentioned. In order to
evaluate the therapeutic potential of DRAK2 inhibition, we
have set up a program aimed at the identification of DRAK2
inhibitors. Conventional kinase screenings rely on assays that
measure directly enzymatic activities. These types of assays
need to be optimized with respect to the selection of the
substrate, the ATP concentration, and the buffer composition.
Furthermore, when selectivity data are required against a broad
panel of kinases, this classical screening is expensive and time-
consuming. Therefore, we opted for a binding, rather than a
functional assay, for the identification of novel DRAK2
inhibitors, using the KinomeScan kinase platform. This
platform has been extensively used to assess the selectivity of
known kinase inhibitors.14 In contrast, we used this screening
platform as a primary tool to discover ligands for the DRAK2
enzyme, and then followed up with the conventional in vitro
enzymatic assays. Screening of a representative selection of 150
structurally diverse compounds from our proprietary com-
pound library for potential ligands for DRAK2 in the binding
assays of DiscoverX, led to the identification of a hit compound
based on an isothiazolo[5,4-b]pyridine scaffold (Figure 2),
displaying a Kd value of 1.6 μM. Moreover, this compound has
no affinity for other kinases when evaluated in a panel of 54
kinases at a concentration of 10 μM. Other, in silico determined
parameters, such as the Polar Surface Area (PSA: 53.82 Å2),
partition coefficient (clogP = 4.4), and ligand efficiency (LE =
0.34 kcal/mol/non-H) confirm this compound to be an
excellent starting point for medicinal chemistry work.
This manuscript describes our efforts toward the discovery of
novel DRAK2 inhibitors, starting from an isothiazolo[5,4-
b]pyridine derivative, using a combination of classical medicinal
chemistry (changing the decoration pattern of the original
scaffold) and a scaffold hopping approach.
CHEMISTRY
■
Synthesis of Isothiazolo[5,4-b]pyridines 10. A general
synthetic scheme used for the preparation of the isothiazolo-
[5,4-b]pyridine library is depicted in Scheme 1. Starting from
commercially available 2-chloro-3-cyano-pyridine 1, the corre-
sponding pyridinone derivative 2 was obtained under acidic
conditions. The introduction of a bromine atom by treatment
with bromine yielded the 2-hydroxy-3-cyano-5-bromo-pyridine
derivative 3. The tautomeric hydroxyl group at position 2 of the
pyridine scaffold was converted to its corresponding chlorine,
by treatment with POCl3.15 Nucleophilic displacement of the
chlorine by reaction with methyl 3-mercaptopropionate
afforded intermediate 5. Deprotection with sodium hydride
yielded the 2-thio-pyridine analogue 6, which was then in situ
ring closed with sodium hypochlorite as oxidizing agent,
affording the isothiazole ring system 7.16,17 This reaction
proceeds via a sulfenamide intermediate by the reaction of the
2-thio-pyridine analogue with chloramine (formed in situ from
sodium hypochlorite and ammonia).18,19 3-Amino-5-bromo-
isothiazolo[5,4-b]pyridine 7 was a key intermediate for library
synthesis around the isothiazolo[5,4-b]pyridine core. The
sequence that was followed depends on the need in the SAR
study. Either a palladium-catalyzed Suzuki reaction was
B
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a
Scheme 1
a
Reagent and conditions: (a) CH3COOH, reflux; (b) Br2, CH3COOH, rt; (c) POCl3, PCl5, reflux; (d) methyl 3-mercaptopropionate, NaOCH3,
DMF, rt; (e) NaH, THF, reflux; (f) NH3, NaOH, NaOCl, H2O, 0 °C; (g) K2CO3, Pd(PPh3)4, arylboronic acid, dioxane/water, 90 °C; (h) RC(O)Cl,
CH2Cl2, rt or RCOOH, TBTU, DMF, rt.
performed first, followed by amide formation, or alternatively,
the amide moiety was constructed, followed by Suzuki
coupling.20 Suzuki couplings were performed in a mixture of
dioxane/water, using potassium carbonate as a base and
(PPh3)4Pd as catalyst. For the construction of the amide
moiety, the coupling with a series of acid chlorides was effected.
If the acid chloride was not commercially available, we switched
to the coupling of the carboxylic acid using O-(benzotriazol-1-
yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU)
as coupling reagent. Alternatively, in a number of cases, the
carboxylic acid was converted to its corresponding acid
chloride, by treatment with thionyl chloride. These synthetic
procedures allowed us to prepare a library around the
isothiazolo[5,4-b]pyridine scaffold (see Tables 1, 2, and 3 for
the exact structures that have been synthesized).
Synthesis of Isothiazolo[5,4-b]pyrazine 15 and
pyrazolo[3,4-b]pyrazine 17. The synthesis of both fused
pyrazine bicycles is shown in Scheme 2 and starts from 6-
bromo-3-chloro-pyrazine-2-carbonitrile 11, which has been
synthesized according to literature procedures.21,22 A Suzuki
type of coupling with 3,4-dimethoxyphenylboronic acid yielded
pyrazine analogue 12 as a key intermediate. Nucleophilic
displacement with methyl 3-mercaptopropionate, followed by
alkaline deprotection and oxidative ring closure afforded the 3-
amino-isothiazolo[5,4-b]pyrazine 14, which was acylated in a
final step affording derivative 15. Alternatively, by using
hydrazine as nucleophile, the 3-pyrazolo[3,4-b]pyrazine 16 is
accessible. Condensation with cyclohexane carbonyl chloride
furnished target compound 17.
Scheme 3. Nucleophilic aromatic substitution of chloronitriles
11 and 18a−b with ethyl mercaptoacetate upon cooling
provides the ethyl carboxylates 19a−c. Hydrolysis of the
ethyl carboxylate to the carboxylic acid is followed by acidic
decarboxylation23 affording the 3-amino-thiophene derivatives
20a−c. Subsequently, the cyclohexyl residue and the aryl
moiety are introduced by acylation and Suzuki reaction,
respectively, to afford the final compounds 22a−c.
Synthesis of Pyrrolo[2,3-b]pyridine 25. Starting from
commercially available 5-bromo-3-nitro-pyrrolo[2,3-b]pyridine
23, the 3,4-dimethoxyphenyl moiety was introduced via
standard Suzuki reaction. Catalytic reduction of the nitro
group using Raney Nickel as catalyst yielded the 3-amino
pyrrolo[2,3-b]pyridine derivative.24 Subsequent coupling with
the appropriate acid chloride (Scheme 4) furnished the desired
pyrrolo[2,3-b]pyridine analogue 25.
Synthesis of Benzo[d]isothiazole 30 and Benzo[d]-
isoxazole 33. The synthesis of both scaffolds starts from
ortho-fluoronitrile 26 (Scheme 5). Nucleophilic aromatic
displacement of the fluorine with Na2S followed by treatment
with Zn/HCl provides the corresponding mercaptobenzonitrile
27.25 Oxidative ring closure with sodium hypochlorite allows
buildup of the isothiazole moiety. Acylation of the amino
group, followed by Suzuki reaction, affords the desired
benzo[d]isothiazole 30. 5-Bromobenzo[d]isoxazol-3-amine 31
is generated via an one-pot reaction of ortho-fluoronitrile 26
and acetohydroxamic acid in basic condition, with concomitant
intramolecular cyclization, furnishing the benzo[d]isoxazole
scaffold 31.26 Subsequent amide formation and Suzuki coupling
yields the desired benzo[d]isoxazole derivative 33.
Synthesis of Benzo[b]thiophene 22a, thieno[2,3-b]-
pyridine 22b and thieno[2,3-b]pyrazine 22c. The syn-
thesis of a series of fused thiophene derivatives is shown in
Synthesis of Indazole 36. In the first step, the endocyclic
nitrogen of the indazole building block 34 is protected with a
C
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Table 1. SAR of the 5-Aryl Moiety of Isothiazolo[5,4-b]pyridines
a
Values are the average of two independent experiments. ND = not determined.
Boc protecting group (Scheme 6). Acylation of the exocyclic
amino group is then followed by the standard Suzuki reaction.
Finally, acidic deprotection of the Boc protecting group yields
the desired indazole 36.
Synthesis of Pyrido[2,3-d]pyrimidine 40a and pyrido-
[3,2-d]pyrimidine 40b. The synthesis starts from suitable
amino-nicotinonitrile derivatives 37a−b (Scheme 7). Con-
struction of the pyrimidine moiety was achieved by heating
with triethyl orthoformate as one-carbon fragment, yielding the
pyrido[2,3-d]pyrimidine and pyrido[3,2-d]pyrimidine core
structures 38a and 38b, respectively.27 The dimethoxyphenyl
moiety is introduced via a Suzuki coupling, followed by reaction
of the exocyclic amino group with cyclohexane carboxylic acid
chloride, affording the desired final compounds 40a−b.
Synthesis of Quinazoline 45a and Pteridine 45b.
Starting from an appropriately substituted benzoic acid 41a or
pyrazine carboxylate 41b analogue, formation of the pyrimidin-
4(3H)-one moiety was achieved by treatment with triethyl
D
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Table 2. SAR of the Cyclohexyl Moiety
Table 3. SAR of the Amide Moiety
a
Values are the average of two independent experiments. ND = not
determined.
have %Ctrl = 0, while weaker binders have higher % control
values. For the most promising compounds, dose−response
curves were generated to determine Kd values.
In a first round of optimization, the isothiazolo[5,4-
b]pyridine scaffold, as well as the amide moiety were fixed,
and structural variation was introduced by the synthesis of a
number of substituted aryl and heteroaryl groups (Table 1).
Replacement of the 3-thienyl group of the hit compound 10a
by a 2-thienyl afforded compound 10b with similar DRAK2
affinity. In contrast, the presence of a 3-furanyl moiety
furnished analogue 10c with greatly reduced DRAK2 affinity.
Similarly, changing the thienyl into a phenyl (which is
considered as a bioisoster of the thiophene) led to compound
10d, displaying a strongly reduced DRAK2 affinity. The
presence of a 3,4-dimethoxyphenyl moiety affords an analogue
with potent DRAK2 binding affinity, displaying a Kd value of
1.6 μM. A small series of analogues was prepared around this
3,4-dimethoxyphenyl moiety. Dimethyl derivative 10f (carrying
two electron-donating groups as in the dimethoxyphenyl
substructure) was synthesized, but has no activity as DRAK2
ligand. Different regio-isomeric dimethoxyphenyl derivatives
(compounds 10g−h) were also prepared and were found to be
completely devoid of any DRAK2 affinity, indicating the
importance of the exact positioning of both methoxy groups.
The dioxolane analogue 10i (to be considered as the ring
closed derivative of the 3,4-dimethoxyphenyl analogue) is
completely devoid of DRAK2 affinity. The insertion of an
additional methoxy group affords the 3,4,5-trimethoxy analogue
10j, which lacks DRAK2 affinity.
a
Values are the average of two independent experiments. ND = not
determined.
formate.28 Suzuki reaction allowed the introduction of the 3,4-
dimethoxyphenyl moiety. Chlorination of the lactam group
with POCl3 and introduction of the amino group at position 4
was followed by amide formation by reaction with cyclo-
hexanecarboxylic acid chloride, affording the quinazoline and
pteridine derivatives 45a and 45b, respectively.
BIOLOGICAL EVALUATION AND
STRUCTURE−ACTIVITY RELATIONSHIP STUDIES
■
Electron-donating substituents, such as a hydroxyl function
(compound 10k) and an amino group (compound 10l)
furnishes compounds with affinity for the DRAK2 enzyme,
displaying Kd values in the low μM range. It seems although
that the exact position of the amino group is important, as the
4-amino analogue 10m does not show any affinity for the
DRAK2 enzyme.
As mentioned in the Introduction, we opted for a binding assay
to detect DRAK2 ligands. In a first round of screening,
compounds were tested at a concentration of 10 μM.
Compounds that bind to the kinase ATP site displace the
immobilized ligand from the kinase/phage, which is then
detected using quantitative PCR. The results are reported as
the percentage of kinase/phage remaining bound to the
ligands/beads, relative to a control. High affinity compounds
Finally, structural features endowed with promising DRAK2
affinity (i.e., the dimethoxyphenyl moiety and the amino/
E
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a
Scheme 2
a
Reagents and conditions: (a) K2CO3, Pd(PPh3)4, 3,4-dimethoxyphenylboronic acid, dioxane/water, 90 °C; (b) methyl 3-mercaptopropionate,
NaOCH3, DMF, rt; (c) (i) NaH, THF, reflux ; (ii) NH3, NaOH, NaOCl, H2O, 0 °C; (d) cyclohexanecarboxylic acid chloride, DCM; (e) N2H4,
EtOH, reflux.
a
Scheme 3
a
Reagents and conditions: (a) ethyl 2-mercaptoacetate, t-BuOK, DMF, 0 °C; (b) NaOH, EtOH, reflux; (c) H3PO4; (d) cyclohexanecarboxylic acid
chloride, DCM; (e) K2CO3, Pd(PPh3)4, 3,4-dimethoxyphenylboronic acid, DME, 90 °C.
a
Scheme 4
a
Reagents and conditions: (a) K2CO3, Pd(PPh3)4, 3,4-dimethoxyphenylboronic acid, dioxane/water, 90 °C; (b) Raney Ni/H2, methanol, rt; (c)
cyclohexanecarboxylic acid chloride, DIPEA, dichloromethane, rt.
hydroxyl substituent) were combined into one molecule. It
afforded the 4-hydroxy-3-methoxyphenyl derivative (compound
10o) and the 4-amino-3-methoxyphenyl congener 10n,
displaying Kd values of 2 and 0.66 μM, respectively.
As structural modifications of the aryl part did not lead to the
desired drastic improvement in DRAK2 affinity, a series of
analogues was prepared in which the 3,4-dimethoxyphenyl
group and the isothiazolo[5,4-b]pyridine core were kept intact
and the exocyclic amino group was used to introduce structural
variety by the synthesis of a series of amides. The study started
with the synthesis of a series of cyclo-aliphatic amides, ranging
from 3- to 7-membered rings (Table 2). A cycloheptyl ring
seems to optimal for activity, displaying a Kd value of 0.53 μM
(compound 10s). A cyclopentyl (compound 10r) and a
cyclohexyl derivative (compound 10e) only lose 3- to 5-fold
activity when compared to analogue 10s. The introduction of
F
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a
Scheme 5
a
Reagents and conditions: (a) Na2S, DMF; (b) Zn powder, 10% HCl; (c) NaClO, 3% NaOH, NH4OH; (d) cyclohexanecarboxylic acid chloride,
DIPEA, DCM; (e) K2CO3, Pd(PPh3)4, 3,4-dimethoxyphenylboronic acid, DME, 90 °C; (f) acetohydroxamic acid, t-BuOK, DMF.
a
Scheme 6
a
Reagents and conditions: (a) Boc2O, DMAP, DCM; (b) cyclohexanecarboxylic acid chloride, DIPEA, DCM; (c) K2CO3, Pd(PPh3)4, 3,4-
dimethoxyphenylboronic acid, DME, 90 °C; (d) 20% TFA/DCM.
a
Scheme 7
a
Reagents and conditions: (a) CH(OEt)3, NH4OAc, reflux; (b) K2CO3, Pd(PPh3)4, 3,4-dimethoxyphenylboronic acid, dioxane/water, 90 °C; (c)
cyclohexanecarboxylic acid chloride, DIPEA, DCM, rt.
unsaturation (compound 10t) and the insertion of a methylene
linker between the carbonyl and the cyclohexyl moiety
(compound 10u) give rise to less potent compounds. Similarly,
when the cyclohexyl ring is part of a bicyclic adamantyl ring
system (compounds 10v−w), the compounds have a much
lower affinity for the DRAK2 enzyme. Substitution at position 4
of the cyclohexyl moiety with a methyl group gives rise to a
strongly diminished DRAK2 binding. Replacement of the
cyclohexane ring by a piperidine ring afforded compound 10z
that retains reasonable DRAK2 affinity (Kd = 7.2 μM). This
compound offers the advantage of reduced lipophilicity and
increased aqueous solubility.
Besides cyclo-aliphatic amides, a number of aliphatic and
aromatic amides were prepared (Table 3). All these analogues
are completely devoid of DRAK2 affinity, pointing toward an
essential role of a cyclo-alkyl moiety in DRAK2 binding.
At this stage of the project, it was decided to evaluate the
most potent DRAK2 binders for their ability to functionally
inhibit the enzyme in a traditional, enzymatic assay, based on a
radioactive read-out. As can be derived from the data in Table
4, compounds 10s and 10n are completely devoid of functional
inhibitory activity.
Therefore, we felt it was necessary to further improve the
binding affinity, to identify compounds that could also
G
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a
Scheme 8
a
Reagents and conditions: (a) CH(OEt)3, NH4OAc, reflux; (b) K2CO3, Pd(PPh3)4, 3,4-dimethoxyphenylboronic acid, dioxane/water, 90 °C; (c) (i)
POCl3 (ii) NH3/Methanol; (d) cyclohexanecarboxylic acid chloride, DIPEA, DCM, rt.
avoid unwanted side effects, caused by inhibition of off-targets.
As DRAK2 belongs to the superfamily of DAPK, consisting of
DAPK1, DAPK2, DAPK3, DRAK1, and DRAK2, the most
promising compounds were profiled against this kinase panel in
the binding assay at a concentration of 10 μM (Table 6). The
data clearly indicate that the synthesized DRAK2 inhibitors
completely lack affinity for the different members of the DAPK
family (% Ctrl between 70 and 100), whereas they bind to the
DRAK1 enzyme (% Ctrl of 0.55 and 0.65, respectively). As
representative examples, the Kd values of compounds 22b and
22c for DRAK1 were experimentally determined to be 4.9 nM
and 13 nM, respectively. It can be concluded that these
compounds behave as dual DRAK1/DRAK2 ligands.
With compounds with strong DRAK1 and DRAK2 binding
affinity in hand, they were tested in a classical, biochemical
assay, to determine if they are functionally active DRAK1 and
DRAK2 inhibitors (Table 7). The two most potent DRAK1
and DRAK2 binders 22b and 22c do behave as inhibitors in
functional DRAK1 and DRAK2 assays. However, there seem to
be a discrepancy between the binding data (low nM affinity)
and the IC50 values (low μM values). This contradictory results
are quite surprising, given the fact that binding affinity data
usually correlate very well with the data obtained in functional
enzyme assays.29 Compound 25, although having strong affinity
for DRAK2, lacks completely functional inhibitory activity.
A possible reason for the greatly diminished activity could be
the fact that the compounds do not target the ATP binding site.
Therefore, enzyme kinetic experiments were performed, using
different concentrations of compound 22b (as representative
example) and ATP (Table 8). Increasing the concentration of
ATP from 1 μM to 500 μM leads to a 10-fold drop in activity of
compound 22b against DRAK1 and DRAK2, suggesting that
this class of compounds acts as ATP competitive inhibitors.
Table 4. Functional Inhibition of DRAK2 Activity
a
a
Cmpd
Kd (μM)
% inh @ 10 μM
10s
0.53 μM
0.66 μM
10%
0%
10n
a
Values are the average of two independent experiments.
functionally inhibit the DRAK2 enzymatic activity. It turns out
that by changing the decoration pattern of the isothiazolopyr-
idine scaffold, we were not able to improve DRAK2 affinity
substantially and only compounds were obtained with mediocre
binding affinity, lacking any functional, inhibitory activity.
Therefore, we switched the strategy toward a scaffold hopping
approach. Scaffold hopping is a useful approach to discover
structurally novel compounds starting from known active
molecules by changing the molecular core of the hit, yielding a
number of skeletons, keeping the substitution pattern intact.
Two types of heteroaromatic bicyclic scaffolds, i.e., the 6−5
and the 6−6 bicycles, were prepared (Table 5). Most of the 6−
5 bicycles do show some affinity for the DRAK2 enzyme, with
the exception of the isothiazolo[5,4-b]pyrazine derivative 15
and the indazole 36. The benzo[b]thiophene 22a, the
benzo[d]isothiazole 30, and the benzo[d]isoxazole 33 are
endowed with very similar DRAK2 affinity, with Kd values
ranging from 0.51 to 2.7 μM, which does not represent any
major step forward when compared to the DRAK2 affinity data
of the original hit compound. However, the thieno[2,3-
b]pyridine 22b, thieno[2,3-b]pyrazine 22c, and the pyrrolo-
[2,3-b]pyridine 25 display strong DRAK2 afffinity with Kd
values of 9 nM, 27 nM, and 33 nM, respectively. Among the 6−
6 type of bicycles, the pyrido[2,3-d]pyrimidine scaffold 40a as
well as the quinazoline scaffold 45a are completely devoid of
DRAK2 affinity. The pyrido[3,2-d]pyrimidine scaffold 40b
shows marginal activity, whereas the pteridine scaffold 45b is a
potent ligand for the DRAK2 enzyme, with a Kd value of 0.18
μM.
CONCLUSION
■
Starting from an isothiazolo[5,4-b]pyridine based hit com-
pound with weak affinity for the DRAK2 enzyme, classical
medicinal chemistry with systematic variation of the sub-
stituents afforded novel compounds with binding affinities of
approximately 0.5 μM, but lacking any functional inhibitory
activity. In a second round of optimization, a scaffold hopping
Selectivity is an important issue in kinase research. Selective
small-molecule kinase inhibitors are important when useful
conclusions regarding the physiological role of a certain kinase
need to be made. Moreover, when compounds are considered
as leads in drug discovery programs, specificity is important to
H
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Table 5. SAR of the Isothiazolopyridine Scaffold
Table 7. Functional DRAK1 and DRAK2 Inhibition
a
a
a
a
DRAK1 Kd
DRAK1 IC50
DRAK2 Kd
DRAK2 IC50
Cmpd
(nM)
(μM)
(nM)
(μM)
22b
22c
25
4.9
13
2.25
4.94
ND
9
27
33
0.86
1.78
ND
38% inh @
10 μM
a
Values are the average of two independent experiments. ND = not
determined.
Table 8. IC50 Determination of Compound 22b against
DRAK1 and DRAK2 at Different ATP Concentrations
a
a
ATP conc (μM)
DRAK1 IC50 (μM)
DRAK2 IC50 (μM)
1
0.56
0.63
0.71
0.94
1.33
2.11
2.85
5.91
0.37
0.51
0.53
0.65
0.72
0.99
1.47
2.22
5
10
25
50
100
250
500
a
Values are the average of two independent experiments.
between DRAK1 and DRAK2 and therefore need to be
considered as dual DRAK1 and DRAK2 inhibitors.
EXPERIMENTAL SECTION
General. For all reactions, analytical grade solvents were used. All
■
moisture-sensitive reactions were carried out in oven-dried glassware
1
(135 °C). H and 13C NMR spectra: Bruker Avance 300 MHz (1H
NMR: 300 MHz, 13C NMR: 75 MHz), 500 MHz (1H NMR: 500
MHz, 13C NMR: 125 MHz) and 600 MHz (1H NMR: 600 MHz, 13
C
1
NMR: 150 MHz) using tetramethylsilane as internal standard for H
NMR spectra and (D6)-DMSO (39.5 ppm) or CDCl3 (77.2 ppm) and
CD3OD (49.0 ppm) for 13C NMR spectra. Abbreviations used are s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad.
Coupling constants are expressed in Hz. Mass spectra are obtained
with a Finnigan LCQ Advantage Max (ion trap) mass spectropho-
tometer from Thermo Finnigan, San Jose, CA, USA. High resolution
mass spectrometry spectra were acquired on a quadrupole orthogonal
acceleration time-of-flight mass spectrometer (Synapt G2 HDMS,
Waters, Milford, MA). Samples were infused at 3 μL/min and spectra
were obtained in positive (or negative) ionization mode with a
resolution of 15000 (fwhm) using leucine enkephalin as lock mass.
Precoated aluminum sheets (Fluka Silica gel/TLC-cards, 254 nm)
were used for TLC. Column chromatography (CC) was performed on
ICN silica gel 63−200, 60 Å. All final compounds possess a purity of at
least 95% (with the exception of compound 10c which shows a purity
of 93%), as determined by analytical RP-HPLC analysis on an XBridge
column (C-18, 5 μm, 4.6 × 150 mm) in combination with a Waters
600 HPLC system, a Waters 717 plus Autosampler, and a Waters 2996
Photodiode Array Detector. The mobile phase consisted of a mixture
of solvent A (water with 0.1% trifluoroacetic acid) and solvent B
(acetonitrile with 0.1% trifluoroacetic acid). A gradient was used
starting with 0% solvent B, gradually increasing to 100% of solvent B,
over 30 min.
a
Values are the average of two independent experiments. ND = not
determined.
Table 6. Selectivity Profile of the Lead Compounds
a
a
a
a
a
Cmpd DAPK1 DAPK2 DAPK3
DRAK1
DRAK2
22b % Ctrl = % Ctrl = % Ctrl = % Ctrl = 0,55 Kd % Ctrl = 7.2 Kd
83 100 72 = 4.9 nM = 9 nM
22c
% Ctrl = % Ctrl = % Ctrl = % Ctrl = 0,65 Kd % Ctrl = 5.4 Kd
77 98 84 = 13 nM = 27 nM
a
Values are the average of two independent experiments.
2-Hydroxynicotinonitrile (2). A solution of 2-chloronicotinoni-
trile (1, 10 g, 72.5 mmol) in acetic acid (50 mL) was heated under
reflux for 60 h. After cooling to room temperature, the crystals was
collected by filtration and washed with acetic acid (10 mL), yielding
strategy was applied. It led to the discovery of a thieno[2,3-
b]pyridine and a thieno[2,3-b]pyrazine derivative as potent
ligands for the DRAK2 enzyme. In addition, these compounds
show promising activity as DRAK2 inhibitors. A preliminary
selectivity profile demonstrates that the compounds do not
bind to DAPK1, 2, and 3, but, however, do not discriminate
1
the title compound as white crystals (6.1 g, 70%). H NMR (300
MHz, DMSO-d6): δ = 6.35 (t, J = 6.8 Hz, 1H, arom H), 7.78 (dd, J =
6.8 Hz and J = 2.2 Hz, 1H, arom H), 8.13 (dd, J = 6.9 and 2.2 Hz, 1H,
arom H), 12.56 (br s, 1H, OH) ppm.
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1
5-Bromo-2-hydroxynicotinonitrile (3). To a stirring suspension
of 2-hydroxynicotinonitrile (2, 4.8 g, 40 mmol) in acetic acid (40 mL),
bromine was added dropwise (3.1 mL, 60 mmol). The resulting
mixture was stirred at room temperature for 2 h. Then, water (100
mL) was added and the mixture was extracted with dichloromethane
(3 × 50 mL). The combined organic phases were washed with brine
and water. After the solvents were evaporated under reduced pressure,
the title compound was obtained as white solid (6.0 g, 75%). 1H NMR
(300 MHz, DMSO-d6): δ = 8.09 (d, J = 2.8 Hz, 1H, arom H), 8.38 (d,
J = 2.8 Hz, 1H, arom H), 12.87 (br s, 1H, OH) ppm.
(119 mg, 85%). H NMR (300 MHz, DMSO-d6): δ = 3.82 (s, 3H,
OCH3), 3.89 (s, 3H, OCH3), 7.04 (br s, 2H, NH2), 7.13 (s, 1H, arom
H), 7.36 (m, 2H, arom H), 8.78 (d, J = 2.1 Hz, 1H, arom H), 9.06 (d, J
= 2.01 Hz, 1H, arom H) ppm.
N-(5-Bromo-isothiazolo)[5, 4-b ]pyrid ine- 3-yl)-
cyclohexanecarboxamide (9). To a solution of 3-amino-5-bromo-
isothiazolo[5,4-b]pyridine (7, 1.28 g, 5.56 mmol) in dry pyridine (17
mL) cyclohexanecarboxylic acid chloride (6.12 mmol, 823 μL) was
added dropwise at 0 °C and the reaction mixture was stirred at room
temperature for 2 h. The solvent was removed under vacuum and the
residue was partitioned between EtOAc and H2O. The organic layer
was washed with brine, dried over MgSO4, and evaporated in vacuo.
The crude product was purified by flash chromatography on silicagel
eluting with a mixture of cyclohexane and dichloromethane (in a ratio
gradually ranging from 6:4 to 1:9) as mobile phase. After
crystallization from MeOH, the title compound was obtained as a
white solid (1.39 g, 74%). 1H NMR (300 MHz, CDCl3): δ = 1.32 (m,
3H, cyclohexyl), 1.61 (m, 2H, cyclohexyl), 1.75 (m, 1H, cyclohexyl),
1.88 (m, 2H, cyclohexyl), 2.05 (m, 2H, cyclohexyl), 2.43 (m, 1H,
cyclohexyl H), 8.30 (br s, 1H, NH), 8.76 (d, 1H, J = 2.2 Hz, arom),
8.81 (d, 1H, J = 2.2 Hz, arom) ppm.
N - ( 5 - A r y l - i s o t h i a z o l o ) [ 5 , 4 - b ] p y r i d i n e - 3 - y l ) -
cyclohexanecarboxamide analogues (10b−d and 10f−o).
General Procedure B. To a solution of N-(5-bromo-isothiazolo)[5,4-
b]pyridine-3-yl)cyclohexanecarboxamide (9, 100 mg, 0.3 mmol) in
dioxane or DME (1.5 mL) were added (PPh3)4Pd (5 mol %, 17 mg), a
2 M aqueous K2CO3 solution (2.0 equiv, 0.3 mL, 0.6 mmol), and the
appropriate boronic acid (2.0 equiv, 0.6 mmol). The resulting mixture
was heated in microwave at 150 °C and the reaction monitored by
TLC. Upon completion, the mixture was diluted with EtOAc, washed
with H2O and brine, and dried over MgSO4. After concentration in
vacuo, the crude residue was purified by flash chromatography on
silicagel to afford the title compounds.
General procedure C. To a solution of N-(5-bromobenzo[d]-
isothiazol-3-yl)cycloalkanecarboxamide (9) in DME (2 mL) was
added an appropriate boronic acid (1.5 equiv), potassium carbonate (2
equiv, 2 M solution in H2O), and Pd(PPh3)4 (10 mol %) was added.
The reaction was heated at 80 °C. After completion of reaction,
solvents were evaporated. The crude residue was purified by silica gel
flash chromatography, yielding the pure title compounds.
Synthesis of N-(5-thiophen-2-yl-isothiazolo)[5,4-b]pyridine-
3-yl)cyclohexane-carboxamide (10b). To a solution of N-(5-
bromo-isothiazolo)[5,4-b]pyridine-3-yl)cyclohexanecarboxamide (9,
100 mg, 0.3 mmol) in dioxane (1.5 mL) were added (PPh3)4Pd (5
mol %, 17 mg), a 2 M aqueous Na2CO3 solution (0.3 mL, 0.6 mmol),
and 2-thiopheneboronic acid (77 mg, 0.6 mmol). The resulting
mixture was heated at reflux overnight. One more equivalent of 2-
thiopheneboronic acid (38 mg, 0.3 mmol) was added and the reaction
was stirred at reflux for another 8 h. The mixture was diluted with
EtOAc, washed with H2O and brine, and dried over MgSO4. After
concentration in vacuo, the crude residue was purified by flash
chromatography eluting with a mixture of cyclohexane−EtOAc, in a
ratio of 9:1. The obtained compound was further purified by
preparative TLC using a mixture of heptane−EtOAc (in a ratio of
7:3) as mobile phase, yielding the pure title compound (35 mg, 34%).
1H NMR (300 MHz, CDCl3): δ = 1.17−1.48 (m, 3H, cyclohexyl),
5-Bromo-2-chloronicotinonitrile (4). A mixture of 5-bromo-2-
hydroxynicotinonitrile (3, 5.0 g, 25 mmol) and phosphorus
pentachloride (5.2 g, 25 mmol) in phosphorusoxychloride (50 mL)
was heated under reflux for 48 h. After concentration under reduced
pressure, ice water was added to the residue. The resulting reaction
mixture was stirred at room temperature for 3 h. The precipitate was
filtered off, washed with water, and dried to yield the title compound
as a gray solid (4.3 g, 79%). 1H NMR (300 MHz, CDCl3): δ = 8.14 (d,
J = 2.3 Hz, 1H, arom H), 8.68 (d, J = 2.3 Hz, 1H, arom H) ppm.
Methyl 3-(5-Bromo-3-cyanopyridin-2-ylthio)propanoate (5).
To a mixture of 5-bromo-2-chloronicotinonitrile (4, 4.3 g, 19.8 mmol)
and K2CO3 (4.15 g, 30 mmol) in DMF (50 mL) was added methyl 3-
mercaptopropionate (3.3 mL, 30 mmol). The mixture was stirred at
room temperature for 2 h. After addition of water (150 mL), the
precipitate was filtered off, washed with water, and dried, yielding the
1
title compound as a brown solid (4.5 g, 72%). H NMR (300 MHz,
CDCl3): δ = 2.73 (t, J = 7.2 Hz, 2H, CH2), 3.31 (t, J = 7.2 Hz, 2H,
CH2), 3.73 (s, 3H, OCH3), 7.93 (d, J = 1.9 Hz, 1H, Ar−H), 8.54 (d, J
= 1.9 Hz, 1H, Ar−H) ppm. MS m/z (%): 316.15 ([M + H]+, 100).
3-Amino-5-bromoisothiazolo[5,4-b]pyridine (7). To a solution
of 3-(5-bromo-3-cyanopyridin-2-ylthio)propanoate (5, 3.8 g, 12
mmol) in THF (60 mL) was added NaH (60%, 1.42 g, 36 mmol).
The mixture was heated under reflux for 2 h. After concentration
under reduced pressure, the residue was triturated with water (10 mL).
The crude product was collected by filtration and was used in the
following reaction without further purification.
To a solution of this crude product in a 25% ammonia in water (40
mL) were added NaOH (0.96 g, 24 mmol) and NaOCl (13% active
chlorine, 8.0 mL) at 0 °C. The mixture was stirred at 0 °C for 3 h. The
precipitate was collected by filtration, washed with water, and dried,
yielding the title compound as brown solid in 87% yield (2.4 g, 10.4
mmol). 1H NMR (300 MHz, DMSO-d6): δ = 7.09 (s, 2H, NH2), 8.80
(s, 2H, arom H) ppm.
3-Amino-5-aryl-isothiazolo[5,4-b]pyridines (8a−b). General
Procedure A. To a solution of 3-amino-5-bromo-isothiazolo[5,4-
b]pyridine (7, 230 mg, 1.0 mmol) in a mixture of dioxane (8 mL) and
water (2 mL) were added potasium carbonate (2 equiv, 296 mg, 2.0
mmol), (PPh3)4Pd (0.05 eq, 55 mg, 0.05 mmol) and an appropriate
arylboronic acid (1.2 equiv, 1.2 mmol). The reaction mixture was
stirred at 95 °C until the starting material was disappeared on TLC
(2−6 h). The solvents were evaporated and the crude residue was
purified by silicagel flash chromatography, yielding the pure title
compounds. The following compounds were made according to this
procedur.
3-Amino-5-(3-thienyl)-isothiazolo[5,4-b]pyridine (8a). This
compound was prepared from compound 7 according to the general
procedure A using 3-thiopheneboronic acid (124 mg, 0.98 mmol) and
the crude residue was purified on silicagel, using a mixture of
cyclohexane and ethyl acetate (in a ratio of 2:1) as mobile phase,
1.52−1.80 (m, 3H, cyclohexyl H), 1.82−1.96 (m, 2H, cyclohexyl),
2.00−2.14 (m, 2H, cyclohexyl), 2.39−2.57 (m, 1H, cyclohexyl), 7.15
(dd, 1H, J = 3.7, J = 5.2 Hz, arom H), 7.40 (dd, 1H, J = 1.2, 5.2 Hz,
arom), 7.44 (dd, 1H, J = 1.2, J = 4.7 Hz, arom), 8.32 (br s, 1H, NH),
8.76 (d, 1H, J = 2.0 Hz, arom H), 8.99 (d, 1H, J = 2.1 Hz, arom H)
ppm. 13C NMR (75 MHz, DMSO-d6): δ = 25.28 (CH2), 25.51 (CH2),
29.13 (CH2), 43.96 (CH), 122.09 (Cq), 125.83 (CH), 126.44 (Cq),
127.50 (CH), 129.02 (CH), 129.48 (CH), 139.17 (Cq), 148.85 (CH),
152.40 (Cq), 169.44 (Cq), 174.89 (CO) ppm. HRMS: calcd for
C17H18N3OS2 344.0885, found 344.0881.
1
yielding the title compound as a white powder (69 mg, 60%). H
NMR (300 MHz, DMSO-d6): δ = 7.03 (br s, 2H, NH2), 7.65 (dd, J =
0.96 Hz and J = 5.04 Hz, 1H, arom H), 7.77 (m, 1H, arom H), 8.04
(m, 1H, arom H), 8.84 (d, J = 1.98 Hz, 1H, arom H), 9.14 (d, J = 1.92
Hz, 1H, arom H) ppm.
3-Amino-5-(3,4-dimethoxy-phenyl)-isothiazolo[5,4-b]-
pyridine (8b). This compound was prepared from compound 7
according to the general procedure A using 3,4-dimethoxyphenylbor-
onic acid (178 mg, 0.98 mmol) and the crude residue was purified on
silica gel, using a mixture of cyclohexane and ethyl acetate (in a ratio of
2:1) as mobile phase, yielding the title compound as a white powder
N-(5-(Furan-3-yl)isothiazolo[5,4-b]pyridin-3-yl)-
cyclohexanecarboxamide (10c). This compound was prepared
according to general procedure C from compound 9 (40 mg, 0.117
mmol) using 3-furanboronic acid (0.175 mmol, 20 mg), 2 M K2CO3
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(0.117 mL), and Pd(PPh3)4 (0.0117 mmol, 13 mg). The crude
acetate in a ratio of 4:1, affording the title compound in 18% yield (8.4
mg, 0.021 mmol). H NMR (300 MHz, CDCl3): δ = 1.60−2.19 (m,
1
product was purified using a mixture of cyclohexane/ethyl acetate in a
1
ratio of 5:1, affording the pure title compound (22 mg, 56%). H
10H, 5 × CH2), 2.50 (m, 1H, CH), 3.96 (s, 6H, 2 × CH3), 6.55 (m,
1H, arom H), 6.76 (d, J = 2.22 Hz, 2H, arom H), 8.53 (br s, 1H, NH),
8.71 (m, 1H, arom H), 8.95 (d, J = 2.10 Hz, 1H, arom H) ppm. 13C
NMR (75 MHz, DMSO-d6): δ = 25.29 (CH2), 25.50 (CH2), 29.10
(CH2) 43.98 (CH), 55.61 (OCH3), 100.07 (CH), 105.78 (CH),
123.13 (Cq), 131.47 (CH), 132.16 (Cq), 138.71 (Cq), 150.34 (CH),
152.54 (Cq), 161.31 (Cq), 169.96 (Cq), 174.88 (CO) ppm. HRMS:
calcd for C21H24N3O3S 398.1532, found 398.1537.
NMR (300 MHz, CDCl3): δ = 1.60−2.06 (m, 10H, 5 × CH2), 2.50
(m, 1H, CH), 6.81 (m, 1H, arom H), 7.57 (m, 1H, arom H), 7.88 (s,
1H, arom H), 8.37 (bs, 1H, NH), 8.67 (d, J = 1.95 Hz, 1H, arom H),
8.91 (d, J = 2.07 Hz, 1H, arom H) ppm. 13C NMR (75 MHz, DMSO-
d6): δ = 25.32 (CH2), 25.52 (CH2), 29.09 (CH2), 44.00 (CH), 108.82
(CH), 122.30 (Cq), 122.44 (Cq), 124.57 (Cq), 129.40 (CH), 140.52
(CH), 145.08 (CH), 149.44 (CH), 152.44 (Cq), 169.14 (Cq), 174.93
(CO) ppm. HRMS: calcd for C17H18N3O2S 328.1114, found
328.1116. HPLC purity 93.2%.
N-(5-Phenyl-isothiazolo)[5, 4-b]pyridine-3-yl)-
cyclohexanecarboxamide (10d). This compound was prepared
according to general procedure B from compound 9 using
benzeneboronic acid (73 mg, 0.6 mmol) in dioxane. The reaction
was stirred for 45 min and the crude residue was purified by flash
chromatography eluting with a mixture of heptane−EtOAc (in a ratio
of 94:6) as mobile phase, affording the title compound in 50% yield
(50 mg, 0.15 mmol). 1H NMR (300 MHz, CDCl3): δ = 1.26−1.45 (m,
3H, cyclohexyl), 1.55−1.76 (m, 3H, cyclohexyl), 1.86−1.90 (m, 2H,
cyclohexyl), 2.04−2.09 (m, 2H, cyclohexyl), 2.39−2.55 (m, 1H,
cyclohexyl), 7.41−7.56 (m, 3H, arom H), 7.62−7.69 (m, 2H, arom
H), 8.32 (bs, 1H, NH), 8.76 (d, 1H, J = 2.1 Hz, arom H), 8.98 (d, 1H,
J = 2.1 Hz, arom H) ppm. 13C NMR (75 MHz, DMSO-d6): δ = 25.30
(CH2), 25.50 (CH2), 29.12 (CH2), 44.03 (CH), 122.17 (Cq), 127.46
(CH), 128.54 (CH), 129.46 (CH), 131.39 (CH), 132.28 (Cq), 136.67
(Cq), 150.22 (CH), 152.58 (Cq), 169.81 (Cq), 174.86 (CO) ppm.
HRMS: calcd for C19H20N3O1S1 338.13215, found 338.1316.
N-(5-(3,4-Dimethyl)phenyl-isothiazolo)[5,4-b]pyridine-3-yl)-
cyclohexanecarboxamide (10f). This compound was made
according to the general procedure B from compound 9 using 3,4-
dimethylphenylboronic acid (90 mg, 0.6 mmol) in dioxane. The
reaction was stirred for 30 min and the crude residue was purified by
flash chromatography eluting with a mixture of heptane−EtOAc (in a
ratio of 94:6) as mobile phase, affording the title compound in 38%
yield (42 mg, 0.114 mmol). 1H NMR (300 MHz, CDCl3): δ = 1.17−
1.41 (m, 3H, cyclohexyl), 1.55−1.77 (m, 3H, cyclohexyl), 1.86−1.90
(m, 2H, cyclohexyl), 2.04−2.08 (m, 2H, cyclohexyl), 2.36 (d, 6H, J =
9.5 Hz, 2CH3), 7.28 (s, 1H, arom H), 7.37−7.41 (m, 2H, arom H),
8.32 (br s, 1H, NH), 8.76 (d, 1H, J = 2.1 Hz, arom H), 8.98 (d, 1H, J =
2.01 Hz, arom H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 19.22
(CH3), 19.65 (CH3), 25.30 (CH2), 25.50 (CH2), 29.12 (CH2), 44.00
(CH), 122.17 (Cq), 124.74 (CH), 128.34 (CH), 130.53 (CH), 130.74
(CH), 132.35 (Cq), 134.07 (Cq), 136.83 (Cq), 137.38 (Cq), 150.13
(Cq), 152.47 (Cq), 169.44 (Cq), 174.82 (CO) ppm. HRMS: calcd for
C21H24N3O1S1 366.16345, found 366.1629.
Synthesis of N-(5-(Benzo[d][1,3]dioxol-5-yl)isothiazolo[5,4-
b]pyridin-3-yl)cyclohexanecarboxamide (10i). This compound
was prepared according to the general procedure B from compound 9
using 3,4-methylenedioxyphenylboronic acid (99 mg. 0.6 mmol) in
DME. The crude residue was purified by flash chromatography eluting
with DCM−MeOH (in a ratio of 99.5:0.5), followed by crystallization
from MeOH, affording the title compound as a white powder (11 mg,
1
10%). H NMR (300 MHz, CDCl3): δ = 1.33 (m, 3H, cyclohexyl),
1.61 (m, 2H, cyclohexyl H), 1.75 (m, 1H, cyclohexyl), 1.88 (m, 2H,
cyclohexyl), 2.06 (m, 2H, cyclohexyl), 2.47 (m, 1H, cyclohexyl), 6.04
(s, 2H, CH2), 6.94 (d, J = 8.5 Hz, 1H, arom H), 7.10 (m, 2H, arom
H), 8.37 (br s, 1H, NH), 8.66 (d, J = 2.0 Hz, 1H, arom), 8.90 (d, J =
2.1 Hz, 1H, arom H) ppm. 13C NMR (75 MHz, DMSO-d6): δ = 25.31
(CH2), 25.50 (CH2), 29.12 (CH2), 44.03 (CH), 101.60 (CH2), 107.74
(CH), 109.21 (CH), 121.33 (CH), 122.13 (Cq), 130.69 (Cq), 130.88
(CH), 132.07 (Cq), 147.81 (Cq), 148.42 (Cq), 150.14 (CH), 152.49
(Cq), 169.32 (Cq), 174.82 (CO) ppm. HRMS: calcd for
C20H20N3O3S1 382.12198, found 382.1223.
N-(5-(3,4,5-Trimethoxyphenyl)benzo[d]isothiazol-3-yl)-
cyclohexanecarboxamide (10j). This compound was prepared
according to the general procedure C from compound 9 (68 mg, 0.2
mmol) using 3,4,5-trimethoxyphenylboronic acid (0.3 mmol, 63.6
mg), 2 M K2CO3 (0.2 mL), and Pd(PPh3)4 (0.02 mmol, 23 mg). The
crude product was purified using a mixture of cyclohexane/ethyl
acetate in a ratio of 4:1, affording the title compound as a white
1
powder (44 mg, 51%). H NMR (300 MHz, CDCl3): δ = 1.60−2.03
(m, 10H, 5 × CH2), 2.56 (m, 1H, CH), 3.94 (s, 3H, CH3), 3.96 (s,
6H, 2 × CH3), 6.81 (s, 2H, arom H), 8.70 (d, J = 2.07 Hz, 1H, arom
H), 8.86 (br s, 1H, NH), 8.95 (d, J = 2.13 Hz, 1H, arom H) ppm. 13C
NMR (75 MHz, DMSO-d6): δ = 25.26 (CH2), 25.52 (CH2), 29.07
(CH2), 43.88 (CH), 56.35 (OCH3), 60.28 (OCH3), 105.28 (CH),
122.13 (Cq), 131.34 (CH), 132.39 (Cq), 132.53 (Cq), 150.53 (CH),
152.64 (Cq), 153.18 (Cq), 153.66 (Cq), 169.61 (Cq), 175.05 (CO)
ppm. HRMS: calcd for C22H26N3O4S 428.1638, found 428.1638.
N-(5-(4-Hydroxy)phenyl-isothiazolo)[5,4-b]pyridine-3-yl)-
cyclohexanecarboxamide (10k). This compound was prepared
according to the general procedure B from compound 9 using 4-
hydroxyphenylboronic acid (83 mg, 0.6 mmol) in DME. The crude
residue was purified by flash chromatography eluting with DCM−
MeOH 99:1, affording the title compound as a white powder (21 mg,
N-(5-(2,5-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
cyclohexanecarboxamide (10g). This compound was prepared
according to the general procedure C from compound 9 (40 mg, 0.117
mmol) using 2,5-dimethoxyphenylboronic acid (0.175 mmol, 32 mg),
2 M K2CO3 (0.117 mL), and Pd(PPh3)4 (0.0117 mmol, 13 mg).
Product was purified using a mixture of cyclohexane/ethyl acetate in a
ratio of 4:1, affording the title compound in 64% yield (30 mg, 0.075
1
20%). H NMR (300 MHz, MeOD): δ = 1.36 (m, 3H, cyclohexyl),
1.59 (m, 2H, cyclohexyl), 1.75 (m, 1H, cyclohexyl), 1.87 (m, 2H,
cyclohexyl), 2.47 (m, 2H, cyclohexyl), 2.59 (m, 1H, cyclohexyl), 6.94
(d, 2H, J = 8.6 Hz, arom H), 7.56 (d, 2H, J = 8.6 Hz, arom), 8.59 (d,
1H, J = 2.0 Hz, arom H), 8.96 (d, 1H, J = 2.0 Hz, arom H) ppm. 13C
NMR (125 MHz, DMSO-d6): δ = 25.31 (CH2), 25.50 (CH2), 29.13
(CH2), 44.02 (CH), 116.30 (CH), 117.30 (Cq), 127.18 (CH), 128.62
(CH), 130.14 (CH), 132.21 (Cq), 149.94 (CH), 152.61 (Cq), 158.13
(Cq), 168.75 (Cq), 174.89 (CO) ppm. HRMS: calcd for
C19H20N3O2S1 354.12706, found 354.1264.
1
mmol). H NMR (300 MHz, CDCl3): δ = 1.58−2.06 (m, 10H, 5 ×
CH2), 2.52 (m, 1H, CH), 3.94 (s, 3H, CH3), 3.80 (s, 3H, CH3), 3.85
(s, 3H, CH3), 6.96 (m, 3H, arom H), 8.67 (d, J = 1.53 Hz, 1H, arom
H), 8.72 (br s, 1H, NH), 8.94 (d, J = 1.59 Hz, 1H, arom H) ppm. 13C
NMR (75 MHz, DMSO-d6): δ = 25.26 (CH2), 25.49 (CH2), 29.11
(CH2) 43.88 (CH), 55.73 (OCH3), 56.33 (OCH3), 113.40 (CH),
114.59 (CH), 116.72 (CH), 121.91 (Cq), 126.55 (Cq), 129.92 (Cq),
133.81 (CH), 150.51 (Cq), 151.97 (CH), 152.67 (Cq), 153.69 (Cq),
169.39 (Cq), 174.95 (CO) ppm. HRMS: calcd for C21H24N3O3S
398.1532, found 398.1528.
N-(5-(3-Aminophenyl)isothiazolo[5,4-b]pyridin-3-yl)-
cyclohexanecarboxamide (10l). This compound was prepared
according to the general procedure C from compound 9 (40 mg, 0.117
mmol) using 3-aminophenylboronic acid (0.175 mmol, 32 mg), 2 M
K2CO3 (0.117 mL) and Pd(PPh3)4 (0.0117 mmol, 13 mg). The crude
product was purified using a mixture of cyclohexane/ethyl acetate in a
ratio of 4:1, affording the title compound as a white powder (25 mg,
N-(5-(3,5-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
cyclohexanecarboxamide (10h). This compound was prepared
according to the general procedure C from compound 9 (40 mg, 0.117
mmol) using 3,5-dimethoxyphenylboronic acid (0.175 mmol, 32 mg),
2 M K2CO3 (0.117 mL) and Pd(PPh3)4 (0.0117 mmol, 13 mg). The
crude product was purified using a mixture of cyclohexane/ethyl
1
60%). H NMR (300 MHz, DMSO-d6): δ = 1.45−1.89 (m, 10H, 5 ×
CH2), 2.59 (m, 1H, CH), 3.94 (s, 3H, CH3), 5.31 (s, 2H, NH2), 6.65
(d, J = 7.59 Hz, 1H, arom H), 6.88 (m, 2H, arom H), 7.21 (t, J = 7.71
K
dx.doi.org/10.1021/jm5007929 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Hz, J = 7.74 Hz, 1H, arom H), 8.62 (d, J = 1.95 Hz, 1H, arom H), 9.02
(d, J = 2.01 Hz, 1H, arom H), 10.91 (br s, 1H, NH) ppm. 13C NMR
(75 MHz, CDCl3): δ = 25.31 (CH2), 25.59 (CH2), 29.14 (CH2),
44.01 (CH), 112.44 (CH), 114.12 (CH), 114.90 (CH), 122.16 (Cq),
129.98 (CH), 130.95 (Cq), 133.21 (Cq), 137.29 (Cq), 149.63 (Cq),
150.06 (CH), 152.55 (Cq), 169.71 (Cq), 174.85 (CO) ppm. HRMS:
calcd for C19H21N4OS 353.1430, found 353.1432.
under reduced pressure, the crude residue was purified by flash
chromatography on silica, yielding the title compounds.
General Procedure F. A solution of the carboxylic acid analogue
(0.3 mmol) in thionyl chloride (2 mL) was heated under reflux for 30
min. The excess of thionyl chloride was removed under reduced
pressure. The residue was dissolved in dichloromethane (2 mL) and
added to a solution of 3-amino-5-(3,4-dimethoxyphenyl)isothiazolo-
[5,4-b]pyridine (57 mg, 0.2 mmol) in pyridine (2 mL). The mixture
was stirred at room temperature for 30 min. After concentration under
reduced pressure, the residue was purified by flash chromatography on
silica, yielding the pure title compounds.
N-(5-(Thiophen-3-yl)isothiazolo[5,4-b]pyridin-3-yl)-
cyclohexanecarboxamide (10a). This compound was prepared
according to the general procedure D from 3-amino-5-(3-thienyl)-
isothiazolo[5,4-b]pyridine (8a, 0.41 mmol, 95 mg) and cyclo-
hexanecarbonyl chloride (0.147 mL, 1.09 mmol). The crude residue
was purified by flash chromatography on silicagel using a mixture of
cyclohexane and ethyl acetate (in a ratio of 5:1) as mobile phase,
affording the pure title compound as a white powder (62 mg, 44%).
1H NMR (300 MHz, DMSO-d6): δ = 1.25 (m, 2H, cyclohexyl H),
1.29 (br s, 1H, cyclohexyl H), 1.33 (br s, 1H, cyclohexyl H), 1.48 (m,
2H, cyclohexyl H), 1.68 (m, 1H, cyclohexyl H), 1.79 (m, 2H,
cyclohexyl H), 1.93 (m, 2H, cyclohexyl H), 7.67 (dd, J = 1.35 and 5.04
Hz, 1H, arom H), 7.78 (dd, J = 2.91 and 5.01 Hz, 1H, arom H), 8.11
(dd, J = 1.35 and 2.88 Hz, 1H, arom H), 8.70 (d, J = 2.04 Hz, arom
H), 9.23 (d, J = 2.13 Hz, arom H), 10.85 (s, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6): δ = 25.32 (CH2), 25.52 (CH2), 29.11 (CH2),
44.02 (CH), 122.25 (Cq), 123.01 (CH), 126.34 (CH), 127.51 (Cq),
128.21 (CH), 130.11 (CH), 137.59 (Cq), 149.86 (CH), 152.54 (Cq),
169.24 (Cq), 174.90 (CO) ppm. HRMS: calcd for C17H18N3OS2
344.0885, found 344.0887.
N-(5-(4-Aminophenyl)isothiazolo[5,4-b]pyridin-3-yl)-
cyclohexanecarboxamide (10m). This compound was prepared
according to the general procedure C from compound 9 (40 mg, 0.117
mmol) using 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
(0.175 mmol, 38 mg), 2 M K2CO3 (0.117 mL) and Pd(PPh3)4
(0.0117 mmol, 13 mg). Product was purified using a mixture of
cyclohexane/ethyl acetate in a ratio of 3:2, affording the title
1
compound as a white powder (15 mg, 36%). H NMR (300 MHz,
CDCl3): δ = 1.64−2.05 (m, 10H, 5 × CH2), 2.50 (m, 1H, CH), 6.82
(d, J = 8.43 Hz, 2H, arom H), 7.62 (d, J = 8.43 Hz, 2H, arom H), 8.42
(br s, 1H, NH), 8.65 (s, 1H, arom H), 8.94 (d, J = 2.04 Hz, 1H, arom
H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 25.31 (CH2), 25.51
(CH2), 29.14 (CH2), 44.01 (CH), 114.53 (CH), 122.32 (Cq), 123.29
(Cq), 127.97 (CH), 128.97 (CH), 132.94 (Cq), 149.47 (CH), 149.61
(Cq), 152.39 (Cq), 168.54 (Cq), 174.99 (CO) ppm. HRMS: calcd for
C19H21N4OS 353.1430, found 353.1428.
N-(5-(4-Amino-3-methoxyphenyl)benzo[d]isothiazol-3-yl)-
cyclohexanecarboxamide (10n). This compound was prepared
according to the general procedure C from compound 9 (68 mg, 0.2
mmol) using 4-amino-3-methoxyphenylboronic acid pinacol ester (0.3
mmol, 74.7 mg), 2 M K2CO3 (0.2 mL) and Pd(PPh3)4 (0.02 mmol, 23
mg). Product was purified using a mixture of cyclohexane/ethyl acetate
in a ratio of 4:1, affording the title compound (25 mg, 33%). 1H NMR
(300 MHz, CDCl3): δ = 1.65−2.08 (m, 10H, 5 × CH2), 2.56 (m, 1H,
CH), 3.96 (s, 3H, CH3), 6.85 (d, J = 7.95 Hz, 1H, arom H), 7.06 (d, J
= 1.89 Hz, 1H, arom H), 7.11 (dd, J = 1.92 Hz, J = 7.95 Hz, 1H, arom
H), 8.64 (d, J = 2.01 Hz, 1H, arom H), 8.74 (br s, 1H, NH), 8.95 (d, J
= 2.13 Hz, 1H, arom H) ppm. 13C NMR (75 MHz, CDCl3): δ = 25.29
(CH2), 25.52 (CH2), 29.12 (CH2), 43.94 (CH), 55.67 (OCH3),
109.58 (CH), 114.18 (CH), 120.21 (CH), 122.31 (Cq), 124.04 (Cq),
129.36 (CH), 133.13 (Cq), 138.62 (Cq), 146.93 (Cq), 149.95 (CH),
152.42 (Cq), 168.28 (Cq), 174.90 (CO) ppm. HRMS: calcd for
C20H23N4O2S 381.1390, found 381.1387.
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
cyclohexanecarboxamide (10e). This compound was prepared
according to the general procedure D from 3-amino-5-(3,4-
dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.33 mmol, 95
mg) and cyclohexanecarbonyl chloride (0.102 mL, 0.76 mmol). The
crude residue was purified by flash chromatography on silicagel using a
mixture of cyclohexane and ethyl acetate (in a ratio of 5:1) as mobile
phase, affording the pure title compound as a white powder (113 mg,
1
86%). H NMR (300 MHz, DMSO-d6): δ = 1.24 (m, 3H, cyclohexyl
N-(5-(4-Hydroxy-3-methoxyphenyl)isothiazolo[5,4-b]-
pyridin-3-yl)cyclohexanecarboxamide (10o). This compound
was prepared according to the general procedure C from compound
9 (68 mg, 0.2 mmol) using 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenol (75 mg, 0.3 mmol), 2 M K2CO3 (0.2 mL)
and Pd(PPh3)4 (0.02 mmol, 23 mg). Product was purified using a
mixture of cyclohexane/ethyl acetate in a ratio of 4:1, affording the
title compound as a white powder (27 mg, 32%). 1H NMR (300 MHz,
DMSO-d6): δ = 1.29−1.94 (m, 10H, 5 × CH2), 2.59 (m, 1H, CH),
3.84 (s, 3H, CH3), 7.07−7.21 (m, 3H, arom H), 8.61 (d, J = 2.07 Hz,
1H, arom H), 9.04 (d, J = 2.10 Hz, 1H, arom H), 9.28 (bs, 1H, NH),
10.88 (s, 1H, OH) ppm. 13C NMR (75 MHz, CDCl3): δ = 25.62
(CH2), 25.82 (CH2), 29.46 (CH2), 44.35 (CH), 56.18 (OCH3),
113.28 (CH), 114.68 (CH), 118.68 (CH), 122.50 (Cq), 129.60 (Cq),
130.74 (CH), 132.57 (Cq), 147.64 (Cq), 148.68 (Cq), 150.21 (CH),
152.80 (Cq), 169.45 (Cq), 174.18 (CO) ppm. HRMS: calcd for
C20H22N3O3S 384.1376, found 384.1377.
Synthesis of 3-(Amino-acyl)-5-aryl-isothiazolo[5,4-b]-
pyridines (10a, 10e, 10p-10af). General Procedure D. To a
solution of an appropriate 3-amino-5-aryl-isothiazolo[5,4-b]pyridine
(0.32 mmol) in pyridine (10 mL) was added a suitable acid chloride
(1.5 equiv). The reaction mixture was stirred at room temperature
overnight. The solvents were evaporated in vacuo and the crude
residue was purified by flash chromatography on silicagel yielding the
pure title compounds.
H), 1.33 (br s, 1H, cyclohexyl H), 1.46 (m, 2H, cyclohexyl H), 1.67
(m, 1H, cyclohexyl H), 1.78 (m, 2H, cyclohexyl H), 1.92 (m, 2H,
cyclohexyl H), 3.82 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 7.14 (d, J =
8.37 Hz, 1H, arom H), 7.30 (dd, J = 2.01 and 8.31 Hz, 1H, arom H),
7.37 (d, J = 8.37 Hz, 1H, arom H), 8.63 (d, J = 2.01 Hz, arom H), 9.15
(d, J = 2.13 Hz, arom H), 10.87 (s, 1H, NH) ppm. 13C NMR (75
MHz, DMSO-d6): δ = 25.29 (CH2), 25.51 (CH2), 29.96 (CH2) 43.95
(CH), 55.79 (OCH3), 55.92 (OCH3), 111.18 (CH), 112.66 (CH),
119.88 (CH), 122.18 (Cq), 129.21 (Cq), 130.68 (CH), 132.30 (Cq),
149.44 (Cq), 149.49 (Cq), 150.23 (CH), 152.54 (Cq), 169.20 (Cq),
174.93 (CO) ppm. HRMS: calcd for C21H24N3O3S 398.1532, found
398.1535.
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
cyclopropanecarboxamide (10p). This compound was prepared
according to the general procedure E from 3-amino-5-(3,4-
dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.25 mmol, 72
mg) and cyclopropanecarbonyl chloride (1.2 equiv, 0.3 mmol). The
crude residue was purified by flash chromatography on silica gel using
a mixture of cyclohexane and ethyl acetate (in a ratio of 5:1) as mobile
phase, affording the pure title compound as a white powder (50 mg,
1
56%). H NMR (300 MHz, CDCl3): δ = 1.00−1.70 (m, 4H, CH2),
1.91 (m, 1H, CH), 3.97 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 7.01 (d, J
= 8.3 Hz), 7.14 (d, J = 2.1 Hz, 1H, arom H), 7.21 (dd, J = 8.3 Hz and J
= 2.1 Hz, 1H, arom H), 8.69 (d, J = 2.3 Hz, 1H, arom H), 8.90 (br s,
1H, NH), 8.97 (d, J = 2.3 Hz, 1H, arom H) ppm. 13C NMR (150
MHz, DMSO-d6): δ = 175.50, 169.20, 152.29, 150.22, 149.41, 149.33,
132.26, 130.88, 129.14, 122.04, 119.82, 112.54, 111.05, 55.84, 55.72,
14.05, 8.31 ppm. HRMS: calcd for C18H17N3O3S1 356.1063, found
356.1069.
General Procedure E. To a solution of an appropriate 3-amino-5-
(3,4-dimethoxyphenyl)isothiazolo[5,4-b]pyridine (72 mg, 0.25 mmol)
in dichloromethane (10 mL) was added diisopropylethlyamine (0.5
mmol) and an appropriate acid chloride (0.3 mmol). The resulting
mixture was stirred at room temperature for 1.5 h. After concentration
L
dx.doi.org/10.1021/jm5007929 | J. Med. Chem. XXXX, XXX, XXX−XXX

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Article
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
cyclobutanecarboxamide (10q). This compound was prepared
according to the general procedure D from 3-amino-5-(3,4-
dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.20 mmol, 57
mg) and cyclobutanecarbonyl chloride (0.2 mmol). The crude residue
was purified by flash chromatography on silicagel using a mixture of
cyclohexane and ethyl acetate (in a ratio of 5:1) as mobile phase,
affording the pure title compound as a yellowish solid (60 mg, 81%).
1H NMR (300 MHz, DMSO-d6): δ = 1.96 (m, 2H, CH2), 2.7 (m, 4H,
CH2), 3.50 (m, 1H, CH), 3.83 (s, 3H, OCH3), 3.88 (s, 3H, OCH3),
7.14 (d, J = 8.3 Hz, 1H, arom H), 7.32 (d, J = 8.3 Hz, 1H, arom H),
7.39 (s, 1H, arom H), 8.72 (d, J = 1.9 Hz, 1H, arom H), 9.16 (d, J =
1.9 Hz, 1H, arom H), 10.85 (s, 1H, NH) ppm. 13C NMR (150 MHz,
DMSO-d6): δ = 173.75, 169.14, 152.37, 150.22, 149.41, 149.36,
132.27, 130.72, 129.13, 122.01, 119.83, 112.55, 111.08, 55.85, 55.72,
38.94, 24.67, 17.79 ppm. HRMS: calcd for C19H19N3O3S1 370.1220,
found 370.1219.
mg) and 2-cyclohexylacetyl chloride (0.2 mmol). The crude residue
was purified by flash chromatography on silica gel using a mixture of
dichloromethane and ethyl acetate (in a ratio of 8:1) as mobile phase,
affording the pure title compound as a yellowish solid (60 mg, 73%).
1H NMR (300 MHz, DMSO-d6): δ = 1.20 (m, 5H, CH2) 1.7 (m, 6H,
CH2), 2.42 (d, J = 6.5 Hz, 2H, CH2), 3.83 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 7.14 (d, J = 8.3 Hz, 1H, arom H), 7.32 (d, J = 8.3 Hz, 1H,
arom H), 7.36 (s, 1H, arom H), 10.96 (s, 1H, NH), 8.67 (d, J = 1.9
Hz, 1H, arom H), 9.16 (d, J = 1.9 Hz, 1H, arom H) ppm. 13C NMR
(150 MHz, DMSO-d6): δ = 171.27, 169.17, 152.46, 150.19, 149.43,
149.37, 132.21, 130.73, 129.08, 122.12, 119.75, 112.54, 110.96, 55.78,
55.73, 43.29, 34.66, 32.61, 25.90, 25.70 ppm. HRMS: calcd for
C22H25N3O3S1 412.1689, found 412.1681.
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
3-noradamantanecarboxamide (10v). This compound was
prepared according to the general procedure D from 3-amino-5-
(3,4-dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.20 mmol, 57
mg) and 3-noradamantanecarbonyl chloride (0.2 mmol). The crude
residue was purified by flash chromatography on silica gel using a
mixture of dichloromethane and ethyl acetate (in a ratio of 6:1) as
mobile phase, affording the pure title compound as a white solid (70
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
cyclopentanecarboxamide (10r). This compound was prepared
according to the general procedure D from 3-amino-5-(3,4-
dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.30 mmol, 86
mg) and cyclopentanecarbonyl chloride (0.30 mmol). The crude
residue was purified by flash chromatography on silica gel using a
mixture of cyclohexane and ethyl acetate (in a ratio of 10:1) as mobile
phase, affording the pure title compound as a white solid (90 mg,
1
mg, 80%). H NMR (300 MHz, DMSO-d6): δ = 1.60−2.30 (m, 13H,
CH and CH2), 3.83 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 7.14 (d, J =
8.3 Hz, 1H, arom H), 7.32 (d, J = 8.3 Hz, 1H, arom H), 7.37 (s, 1H,
arom H), 8.38 (d, J = 1.9 Hz, 1H, arom H), 9.16 (d, J = 1.9 Hz, 1H,
arom H), 10.41 (s, 1H, NH) ppm. 13C NMR (150 MHz, DMSO-d6):
δ = 176.20, 169.22, 153.32, 150.32, 149.40, 149.35, 132.36, 131.01,
129.14, 122.95, 119.87, 112.60, 111.14, 55.78, 55.72, 46.65, 43.25,
42.53, 37.15, 34.29 ppm. HRMS: calcd for C24H25N3O3S1 436.1689,
found 436.1686.
1
84%). H NMR (300 MHz, CDCl3): δ = 1.69−2.10 (m, 8H, CH2),
2.99 (m, 1H, CH), 3.97 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 7.02 (d, J
= 8.3 Hz), 7.15 (d, J = 2.1 Hz, 1H, arom H), 7.23 (dd, J = 8.3 Hz and J
= 2.1 Hz, 1H, arom H), 8.64 (br s, 1H, NH), 8.71 (d, J = 2.3 Hz, 1H,
arom H), 8.97 (d, J = 2.3 Hz, 1H, arom H) ppm. 13C NMR (150
MHz, DMSO-d6): δ = 175.14, 169.16, 152.50, 150.20, 149.41, 149.35,
132.24, 130.74, 129.13, 122.11, 119.79, 112.56, 111.05, 55.82, 55.72,
44.43, 30.12, 25.79 ppm. HRMS: calcd for C20H21N3O2S1 384.1376,
found 384.1374.
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
1-adamantanecarboxamide (10w). This compound was prepared
according to the general procedure D from 3-amino-5-(3,4-
dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.20 mmol, 57
mg) and adamantanecarbonyl chloride (0.2 mmol). The crude residue
was purified by flash chromatography on silica gel using a mixture of
dichloromethane and ethyl acetate (in a ratio of 6:1) as mobile phase,
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
cycloheptanecarboxamide (10s). This compound was prepared
according to the general procedure F from 3-amino-5-(3,4-
dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.20 mmol, 57
mg) and cycloheptanecarbonic acid (0.30 mmol). The crude residue
was purified by flash chromatography on silica gel using a mixture of
cyclohexane and ethyl acetate (in a ratio of 10:1) as mobile phase,
1
affording the pure title compound as a white solid (70 mg, 78%). H
NMR (300 MHz, DMSO-d6): δ = 2.02−1.73 (m, 15H, CH and CH2),
3.83 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 7.14 (d, J = 8.3 Hz, 1H,
arom H), 7.29 (d, J = 8.3 Hz, 1H, arom H), 7.36 (s, 1H, arom H), 8.32
(d, J = 2.0 Hz, 1H, arom H), 9.16 (d, J = 2.0 Hz, 1H, arom H), 10.42
(s, 1H, NH) ppm. 13C NMR (150 MHz, DMSO-d6): δ = 177.13,
169.20, 153.40, 150.32, 149.40, 149.35, 132.37, 130.87, 129.14, 122.91,
119.88, 112.63, 111.16, 55.80, 55.73, 40.97, 38.23, 36.03, 27.69 ppm.
HRMS: calcd for C25H27N3O3S1 450.1846, found 450.1841.
1
affording the pure title compound as a white solid (70 mg, 85%). H
NMR (300 MHz, DMSO-d6): δ = 1.40−2 (m, 12H, CH and CH2),
2.79 (m, 1H, CH), 10.88 (s, 1H, NH), 3.83 (s, 3H, OCH3), 3.88 (s,
3H, OCH3), 7.14 (d, J = 8.3 Hz, 1H, arom H), 7.29 (d, J = 8.3 Hz, 1H,
arom H), 7.38 (s, 1H, arom H), 8.63 (d, J = 2.0 Hz, 1H, arom H), 9.16
(d, J = 2.0 Hz, 1H, arom H) ppm. HRMS: calcd for C22H25N3O3S1
412.1689, found 412.1683.
trans-N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-
3-yl)-4-methylcyclohexanecarboxamide (10x). This compound
was prepared according to the general procedure F from 3-amino-5-
(3,4-dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.20 mmol, 57
mg) and trans-4-methylcyclohexanecarboxylic acid (0.3 mmol). The
crude residue was purified by flash chromatography on silica gel using
a mixture of dichloromethane and ethyl acetate (in a ratio of 7:1) as
mobile phase, affording the pure title compound as a white solid (70
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
cyclohex-3-enecarboxamide (10t). This compound was prepared
according to the general procedure D from 3-amino-5-(3,4-
dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.20 mmol, 57
mg) and cyclohex-3-enecarbonyl chloride (0.2 mmol). The crude
residue was purified by flash chromatography on silicagel using a
mixture of dichloromethane and ethyl acetate (in a ratio of 8:1) as
mobile phase, affording the pure title compound as a yellowish solid
1
mg, 85%). H NMR (300 MHz, DMSO-d6): δ = 0.90 (d, J = 6.5 Hz,
3H, CH3), 1.44−2.00 (m, 10H, CH and CH2), 3.83 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 7.14 (d, J = 8.3 Hz, 1H, arom H), 7.30 (d, J = 8.3
Hz, 1H, arom H), 7.38 (s, 1H, arom H), 8.64 (d, J = 2.0 Hz, 1H, arom
H), 9.16 (d, J = 2.0 Hz, 1H, arom H), 10.90 (s, 1H, NH) ppm. 13C
NMR (150 MHz, DMSO-d6): δ = 174.97, 169.13, 152.47, 150.21,
149.40, 149.36, 132.26, 130.65, 129.13, 122.12, 119.83, 112.57, 111.09,
55.85, 55.72, 43.77, 34.00, 31.64, 29.03, 22.60 ppm. HRMS: calcd for
C22H25N3O3S1 412.1689, found 412.1687.
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
pivalamide (10aa). This compound was prepared according to the
general procedure E from 3-amino-5-(3,4-dimethoxyphenyl)-
isothiazolo[5,4-b]pyridine (8b, 72 mg, 0.25 mmol) and pivaloyl
chloride (0.3 mmol). The residue was purified by flash chromatog-
raphy on silica, the mobile phase being a mixture of ethyl acetate and
dichloromethane (in a ratio of 1/8), yielding the title compound (60
1
(70 mg, 89%). H NMR (300 MHz, DMSO-d6): δ = 1.60−2.50 (m,
6H, CH2), 2.83 (m, 1H, CH), 3.83 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 5.67 (d, J = 11.8 Hz, 1H, CCH), 5.73 (s, 1H, CCH),
7.14 (d, J = 8.3 Hz, 1H, arom H), 7.32 (d, J = 8.3 Hz, 1H, arom H),
7.38 (s, 1H, arom H), 8.81 (d, J = 1.9 Hz, 1H, arom H), 9.16 (d, J =
1.9 Hz, 1H, arom H), 10.99 (s, 1H, NH) ppm. 13C NMR (150 MHz,
DMSO-d6): δ = 176.58, 169.15, 152.41, 150.23, 149.37, 132.27,
130.67, 129.12, 126.60, 125.61, 122.12, 119.83, 112.57, 111.08, 55.85,
55.73, 38.49, 27.52, 25.39, 24.32 ppm. HRMS: calcd for C21H21N3O3S1
396.1376, found 396.1371.
2-Cyclohexyl-N-(5-(3,4-dimethoxyphenyl)isothiazolo[5,4-b]-
pyridin-3-yl)acetamide (10u). This compound was prepared
according to the general procedure D from 3-amino-5-(3,4-
dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.20 mmol, 57
M
dx.doi.org/10.1021/jm5007929 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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1
mg, 65%) as white solid. H NMR (300 MHz, CDCl3): δ = 1.42 (s,
mg) and 2-(4-chlorophenoxy)acetyl chloride (0.2 mmol). The residue
was purified by flash chromatography on silica, the mobile phase being
a mixture of ethyl acetate and dichloromethane (in a ratio of 1/10),
9H, CH3), 3.97 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 7.01 (d, J = 8.3
Hz), 7.15 (d, J = 2.1 Hz, 1H, arom H), 7.22 (dd, J = 8.3 Hz, 2.1 Hz,
1H, arom H), 8.40 (br s, 1H, NH), 8.63 (d, J = 2.1 Hz, 1H, arom H),
8.95 (d, J = 2.1 Hz, 1H, arom H) ppm. 13C NMR (150 MHz, DMSO-
d6): δ = 177.70, 169.21, 153.36, 150.32, 149.41, 149.36, 132.37,
130.90, 129.12, 122.89, 119.86, 112.60, 111.14, 55.79, 55.73, 33.43,
27.11 ppm. HRMS: calcd for C19H21N3O3S1 372.1376, found
372.1377.
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
propionamide (10ab). This compound was prepared according to
general procedure E from 3-amino-5-(3,4-dimethoxyphenyl)-
isothiazolo[5,4-b]pyridine and propionyl chloride. The crude residue
was purified by flash chromatography on silica gel using a mixture of
cyclohexane and ethyl acetate (in a ratio of 2:1) as mobile phase,
1
yielding the title compound (90 mg, 79%) as a white solid. H NMR
(300 MHz, CDCl3): δ = 3.97 (s, 3H, OCH3), 4.00 (s, 3H, OCH3),
4.76 (s, 2H, OCH2), 6.85−7.40 (m, 7H, arom H), 8.75 (d, J = 2.1 Hz,
1H, arom H), 9.00 (d, J = 2.1 Hz, 1H, arom H), 9.27 (br s, 1H, NH)
ppm. HRMS: calcd for C22H18N3O4S1 456.0779, found 456.0775.
1-Acetyl-N-(5-(3,4-dimethoxyphenyl)isothiazolo[5,4-b]-
pyridin-3-yl)piperidine-4-carboxamide (10y). To a suspension of
1-acetylpiperidine-4-carboxylic acid (0.3 mmol) in dichloromethane
(10 mL) were added O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluro-
nium tetrafluoroborate (TBTU, 0.3 mmol) and DIPEA (0.4 mmol),
respectively. After stirring for 20 min, 3-amino-5-(3,4-
dimethoxyphenyl)isothiazolo[5,4-b]pyridine (57 mg, 0.2 mmol) was
added. The resulting reaction mixture was stirred at room temperature
for 12 h. After concentration under reduced pressure, the crude
residue was purified by flash chromatography on silica, the mobile
phase being a mixture of methanol and dichloromethane (in a ratio of
1/40), yielding the pure title as a white solid (60 mg, 68).
1
affording the pure title compound in 80% yield. H NMR (300 MHz,
DMSO-d6): δ = 1.13 (t, J = 7.47 Hz, 3H, CH3), 2.57 (q, J = 7.41 Hz,
2H, CH2), 3.82 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 7.13 (d, J = 8.31
Hz, 1H, arom H), 7.32 (dd, J = 2.01 and 8.22 Hz, 1H, arom H), 7.38
(d, J = 2.01 Hz, 1H, arom H), 8.73 (d, J = 1.89 Hz, arom H), 9.15 (d, J
= 2.07 Hz, 1H, arom H), 10.94 (s, 1H, NH) ppm.
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
benzamide (10ac). This compound was prepared according to the
general procedure E from 3-amino-5-(3,4-dimethoxyphenyl)-
isothiazolo[5,4-b]pyridine (8b, 0.20 mmol, 57 mg) and benzoyl
chloride (0.3 mmol). The crude residue was purified by flash
chromatography on silica gel using a mixture of dichloromethane
and ethyl acetate (in a ratio of 6:1) as mobile phase, affording the pure
title compound as a white solid in (50 mg, 51%).
1H NMR (300 MHz, DMSO-d6): δ = 2.02 (s, 3H, CH3), 2.00−1.45
(m, 4H), 3.01, 2.65 (m, 4H, CH2), 3.45 (m, 1H, CH), 3.83 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 4.40 (m, 1H, CH), 7.14 (d, J = 8.3 Hz,
1H, arom H), 7.31 (d, J = 8.3 Hz, 1H, arom H), 7.38 (s, 1H, arom H),
8.65 (d, J = 2.0 Hz, 1H, arom H), 9.16 (d, J = 2.0 Hz, 1H, arom H),
11.02 (s, 1H, NH) ppm. 13C NMR (150 MHz, DMSO-d6): δ =
173.65, 169.15, 168.13, 152.31, 150.27, 149.41, 149.37, 132.29, 130.66,
129.09, 122.10, 119.83, 112.56, 111.09, 55.85, 55.73, 45.24(41.78),
40.33, 28.63(28.07), 21.38 ppm. HRMS: calcd for C22H24N4O4S1
441.1591, found 441.1592.
tert-Butyl-4-(5-(3,4-dimethoxyphenyl)isothiazolo[5,4-b]-
pyridin-3-ylcarbamoyl)piperidine-1-carboxylate. This com-
pound was prepared according to the procedure, described for the
synthesis of compound 10y from 3-amino-5-(3,4-dimethoxyphenyl)-
isothiazolo[5,4-b]pyridine (8b, 1.0 mmol, 287 mg) and 1-(tert-
butoxycarbonyl)piperidine-4-carboxylic acid (1.0 mmol). The crude
residue was purified by flash chromatography on silica gel using a
mixture of dichloromethane and ethyl acetate (in a ratio of 7:1) as
mobile phase, affording the pure intermediate as a yellowish solid (375
mg, 85%). 1H NMR (300 MHz, CDCl3): δ = 1.46 (s, 9H, CH3), 1.40−
1.80 (m, 4H, CH2), 3.90−2.06 (m, 5H, CH and CH2), 3.97 (s, 3H,
OCH3), 4.00 (s, 3H, OCH3), 7.02 (d, J = 8.3 Hz, 1H, arom H), 7.16
(s, 1H, arom H), 7.22 (d, J = 8.3 Hz, 1H, arom H), 8.67 (d, J = 2.0 Hz,
1H, arom H), 8.97 (d, J = 2.0 Hz, 1H, arom H), 9.17 (br s, 1H, NH)
ppm. MS m/z (%): 499.2 ([M + H]+, 100).
1H NMR (300 MHz, CDCl3): δ = 3.74 (s, OCH3), 3.76 (s, OCH3),
3.95 (s, OCH3), 3.99 (s, OCH3), 6.70−7.70 (m, 8H, Ar−H), 8.02 (d, J
= 2.3 Hz, 1H, arom H), 8.76 (d, J = 2.3 Hz, 1H, arom H), 8.90 (br.,
1H, NH) ppm. 13C NMR (150 MHz, DMSO-d6): δ = 169.25, 166.13,
152.89, 150.38, 149.38, 149.34, 132.48, 131.08, 129.33, 129.13, 129.04,
128.59, 128.40, 122.03, 119.55, 112.50, 111.26, 55.89, 55.71 ppm.
HRMS: calcd for C21H17N3O3S1 392.1063, found 392.1060.
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
furan-2-carboxamide (10ad). This compound was prepared
according to the general procedure E from 3-amino-5-(3,4-
dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.20 mmol, 57
mg) and furan-2-carbonyl chloride (0.2 mmol). The residue was
purified by flash chromatography on silica, the mobile phase being a
mixture of ethyl acetate and dichloromethane (in a ratio of 1/20),
1
yielding the title compound 50 mg (53%) as a white solid. H NMR
(300 MHz, CDCl3): δ = 3.96 (s, 3H, OCH3), 3.99 (s, 3H, OCH3),
6.50−7.70 (m, 6H, arom H), 8.84 (d, J = 2.3 Hz, 1H, arom H), 8.98
(d, J = 2.3 Hz, 1H, arom H), 9.12 (br s, 1H, NH) ppm. HRMS: calcd
for C19H15N3O4S1 382.0856, found 382.0863.
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
piperidine-4-carboxamide (10z). To a solution of tert-butyl-4-(5-
(3,4-dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-ylcarbamoyl)-
piperidine-1-carboxylate (220 mg, 0.5 mmol) in dichloromethane (5
mL) was added trifluoroacetic acid (2 mL). The mixture was stirred at
room temperature for 30 min. After concentration under reduced
pressure, the residue was purified by flash chromatography on silica,
the mobile phase being a mixture of methanol and dichloromethane
(in a ratio of 1/10 to 1/5), yielding the pure title compound as a
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyridin-3-yl)-
nicotinamide (10ae). This compound was prepared according to the
general procedure E from 3-amino-5-(3,4-dimethoxyphenyl)-
isothiazolo[5,4-b]pyridine (8b, 0.20 mmol, 57 mg) and nicotinoyl
chloride (0.2 mmol). The residue was purified by flash chromatog-
raphy on silica, the mobile phase being a mixture of ethyl acetate and
dichloromethane (in a ratio of 1/20), yielding the title compound (50
mg, 51%) as white solid. 1H NMR (300 MHz, DMSO-d6): δ = 3.81 (s,
3H, OCH3), 3.87 (s, 3H, OCH3), 7.11 (d, J = 8.3 Hz), 7.34 (dd, J =
8.3 and 2.1 Hz, 1H, Ar−H), 7.40 (d, J = 2.1 Hz, 1H, arom H), 7.62
(dd, J = 8.0 and 5.0 Hz, 1H, arom H), 8.41 (dt, J = 8.0 and 1.9 Hz, 1H,
arom H), 8.73 (d, J = 2.1 Hz, 1H, arom H), 8.83 (dd, J = 5.0 and 1.9
Hz, 1H, arom H), 9.19 (d, J = 2.1 Hz, 1H, arom H), 9.23 (d, J = 1.9
Hz, 1H, arom H), 11.63 (br s, 1H, NH) ppm. 13C NMR (150 MHz,
DMSO-d6): δ = 169.26, 164.83, 152.83, 152.47, 150.44, 149.38,
136.15, 132.51, 131.09, 129.20, 129.06, 123.65, 122.66, 120.05, 112.48,
111.27, 55.90, 55.71 ppm. HRMS: calcd for C20H16N4O3S1 393.1016,
found 393.1014.
1
yellowish solid (190 mg, 95%). H NMR (300 MHz, CDCl3): δ =
1.40−1.80 (m, 4H, CH2), 2−17−3.90 (m, 5H, CH and CH2), 3.86 (s,
3H, OCH3), 3.89 (s, 3H, OCH3), 6.83 (d, J = 8.3 Hz, 1H, Ar−H), 7.01
(s, 1H, arom H), 7.02 (d, J = 8.3 Hz, 1H, arom H), 8.72 (d, J = 2.0 Hz,
1H, arom H), 8.77 (d, J = 2.0 Hz, 1H, arom H), 9.30 (br s, 1H, NH)
ppm. 13C NMR (150 MHz, DMSO-d6): δ = 171.54, 169.11, 151.84,
150.30, 149.44, 149.39, 132.31, 130.66, 129.02, 122.07, 119.83, 112.52,
111.10, 55.87, 55.75, 43.95, 43.03, 26.14 ppm. HRMS: calcd for
C20H22N4O3S1 399.1485, found 399.1476.
3-Chloro-6-(3,4-dimethoxyphenyl)pyrazine-2-carbonitrile
(12). This compound was prepared starting from 6-bromo-3-
chloropyrazine-2-carbonitrile (218 mg, 1.0 mmol) and 3,4-dimethox-
yphenylboronic acid (1.2 equiv) according to general procedure A.
The crude residue was purified by flash chromatography on silica, the
mobile phase being a mixture of methanol and dichloromethane (in a
ratio of 1/15), yielding the title compound as a yellow solid (170 mg,
2-(4-Chlorophenoxy)-N-(5-(3,4-dimethoxyphenyl)-
isothiazolo[5,4-b]pyridin-3-yl)acetamide (10af). This compound
was prepared according to the general procedure E from 3-amino-5-
(3,4-dimethoxyphenyl)-isothiazolo[5,4-b]pyridine (8b, 0.20 mmol, 57
N
dx.doi.org/10.1021/jm5007929 | J. Med. Chem. XXXX, XXX, XXX−XXX

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62%). 1H NMR (300 MHz, DMSO-d6): δ = 3.86 (s, 3H, OCH3), 3.88
(s, 3H, OCH3), 7.15 (d, J = 8.5 Hz, 1H, arom H), 7.68 (s, 1H, arom
H), 7.79 (d, J = 8.5 Hz, 1H, arom H), 9.42 (s, 1H, arom H) ppm.
5-(3,4-Dimethoxyphenyl)isothiazolo[5,4-b]pyrazin-3-amine
(14). This compound was prepared starting from 3-chloro-6-(3,4-
dimethoxyphenyl)pyrazine-2-carbonitrile (12, 83 mg, 0.3 mmol),
following the same reaction pathway as described for the synthesis
of compound 7. The crude residue was purified by flash
chromatography on silica, the mobile phase being a mixture of ethyl
acetate and dichloromethane (in a ratio of 1/20), yielding the title
and ethyl 2-mercaptoacetate (344 μL, 3.12 mmol) were dissolved in
dry DMF (20 mL) and the mixture was cooled to 0 °C. To this
mixture tBuOK (699 mg, 6.25 mmol) was added portionwise over a
period of 45 min. The reaction mixture was stirred an additional 30
min at 0 °C, and then 1 h at room temperature. After the reaction was
completed, ice (20 mL) was added to the mixture. The yellow
precipitate was filtered off and dried affording the title compound as a
yellow powder (692 mg, 80%). HRMS: calcd for C10H10BrN2O2S
300.9641, found 300.9638.
Ethyl 7-Amino-2-bromothieno[2,3-b]pyrazine-6-carboxylate
(19c). 5-Bromo-2-chloro-3-cyanopyrazine (11, 1.06 g, 5.0 mmol) and
ethyl 2-mercaptoacetate (610 μL, 11.0 mmol) were dissolved in dry
DMF (30 mL) and the mixture was cooled to 0 °C. To this mixture
tBuOK (1.24 mg, 11.0 mmol) was added portionwise over a period of
60 min. Mixture was stirred additional 60 min at 0 °C. After the
reaction was complete, the solvent was evaporated and the residue was
purified on silicagel using a mixture of cyclohexane/ethyl acetate (in a
ratio of 3:1) as mobile phase, yielding the pure title compound (303
mg, 20%). 1H NMR (300 MHz, CDCl3): δ = 1.42 (t, J = 7.14 Hz, 3H,
CH3), 4.39 (q, J = 7.14 Hz, 2H, CH2), 6.14 (br s, 2H, NH2), 8.58 (s,
1H, arom H) ppm. HRMS: calcd for C9H9BrN3O2S 301.9593, found
301.9596.
1
compound as a yellow solid (57 mg, 86%). H NMR (300 MHz,
DMSO-d6): δ = 3.86 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 7.14 (d, J =
8.5 Hz, 1H, arom H), 7.22 (s, 2H, NH2), 7.94 (d, J = 8.5 Hz, 1H, arom
H), 7.98 (s, 1H, arom H), 9.44 (s, 1H, arom H) ppm.
N-(5-(3,4-Dimethoxyphenyl)isothiazolo[4,5-b]pyridin-3-yl)-
cyclohexanecarboxamide (15). This compound was synthesized
starting from compound 14 (50 mg, 0.17 mmol) and cyclo-
hexanecarbonyl chloride (0.17 mmol), according to general procedure
E. The crude residue was purified by flash chromatography on silica,
the mobile phase being a mixture of ethyl acetate and dichloromethane
(in a ratio of 1/15), yielding the title compound as a yellow solid (55
1
mg, 81%). H NMR (300 MHz, DMSO-d6): δ = 1.23−1.98 (m, 10H,
CH2), 2.71 (m, 1H, CH), 3.87 (s, 3H, OCH3), 3.91 (s, 3H, OCH3),
7.16 (d, J = 8.5 Hz, 1H, arom H), 7.91 (s, 1H, arom H), 7.97 (d, J =
8.5 Hz, 1H, arom H), 9.54 (s, 1H, arom H), 10.73 (s, 1H, NH) ppm.
13C NMR (150 MHz, DMSO-d6): δ = 174.73, 161.26, 152.58, 151.20,
149.60, 149.28, 142.94, 136.50, 127.73, 120.72, 111.99, 110.46, 55.75,
55.71, 43.80, 29.19, 25.50, 25.19 ppm. HRMS: calcd for C20H22N4O3S1
399.1485, found 399.1479.
5-Bromobenzo[b]thiophen-3-amine (20a). Ethyl 3-amino-5-
bromobenzo[b]thiophene-2-carboxylate (19a, 450 mg, 1.5 mmol) was
treated with a 30% aqueous NaOH solution (3 mL) in refluxing
ethanol (10 mL). After completion of the reaction (2 h), solvents were
evaporated. The residue was dissolved in water (45 mL) and the
mixture was carefully neutralized with a 1 M HCl solution. The
intermediate was then extracted into ethyl acetate. A light yellow
powder was obtained upon evaporation. This residue was suspended
in H3PO4 (3 mL). The mixture was stirred at room temperature for 2
h. The reaction mixture was diluted with water/ice and the pH was
adjusted to 7−8 with 5 M NaOH. The product was then extracted into
ethyl acetate. The organic phase was dried over MgSO4 and finally
filtered. Upon evaporation the crude product 20a was obtained as a
dark yellow powder (50 mg, 15%) and was used in the next step
5-(3,4-Dimethoxyphenyl)-1H-pyrazolo[3,4-b]pyrazin-3-
amine (16). To a solution of 3-chloro-6-(3,4-dimethoxyphenyl)-
pyrazine-2-carbonitrile (12, 138 mg, 0.5 mmol) in EtOH (5 mL) was
added a 60% solution of hydrazine in water (2.5 mmol). The mixture
was refluxed for 2 h. After concentration under reduced pressure, the
crude residue was purified by flash chromatography on silicagel, the
mobile phase being a mixture of methanol and dichloromethane (in a
gradient, gradually ranging from 1:40 to 1:10), yielding the pure title
compound as yellow solid (40 mg, 29%). 1H NMR (300 MHz,
DMSO-d6): δ = 3.83 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 7.09 (d, J =
8.5 Hz, 1H, arom H), 7.70 (d, J = 8.5 Hz, 1H, arom H), 7.75 (s, 1H,
arom H), 9.02 (s, 1H, arom H), 12.35 (s, 1H, NH) ppm.
N-(5-(3,4-Dimethoxyphenyl)-1H-pyrazolo[3,4-b]pyrazin-3-
yl)cyclohexanecarboxamide (17). This compound was prepared
from 5-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-b]pyrazin-3-amine
(16, 40 mg, 0.15 mmol) and cyclohexanecarbonyl chloride (0.2
mmol) according to general procedure E. The crude residue was
purified by flash chromatography on silica gel using a mixture of
dichloromethane and ethyl acetate (in a ratio of 15:1) as mobile phase,
affording the pure title compound as a yellowish solid (31 mg, 55%).
1H NMR (300 MHz, DMSO-d6): δ = 1.2−2.0 (m, 11H, CH and
1
without further purification. H NMR (300 MHz, CDCl3): δ = 3.78
(bs, 1H, NH2), 6.37 (s, 1H, arom H), 7.43 (dd, J = 1.83 Hz, J = 8.55
Hz, 1H, arom H), 7.63 (d, J = 8.55 Hz, 1H, arom H), 7.76 (d, J = 1.80
Hz, 1H, arom H) ppm.
5-Bromothieno[2,3-b]pyridin-3-amine (20b). This compound
was obtained from ethyl 3-amino-5-bromothieno[2,3-b]pyridine-2-
carboxylate (19b, 117 mg, 0.32 mmol) according to the procedure
described for the synthesis of 20a. The crude product 20b was
obtained as yellow powder (75 mg, 100%) and was used in the next
step without further purification.
2-Bromothieno[2,3-b]pyrazin-7-amine (20c). This compound
was obtained from ethyl 7-amino-2-bromothieno[2,3-b]pyrazine-6-
carboxylate (19c, 303 mg, 1.0 mmol) according to the procedure
described for the synthesis of 20a. The crude product 20c was
obtained as yellow powder (40 mg, 17%) and was used in the next step
CH2), 3.83 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 7.08 (d, J = 8.5 Hz,
1H, arom H), 7.73 (d, J = 8.5 Hz, 1H, arom H), 7.77 (s, 1H, arom H),
8.18 (s, 1H, NH) 9.25 (s, 1H, arom H) ppm. HRMS: calcd for
C20H23N5O3 382.1874, found 382.1870.
1
without further purification. H NMR (300 MHz, CDCl3): δ = 3.85
(bs, 1H, NH2), 6.67 (s, 1H, arom H), 8.58 (s, 1H, arom H) ppm.
N - ( 5 - B r o m o b e n z o [ b ] t h i o p h e n - 3 - y l ) -
cyclohexanecarboxamide (21a). Cyclohexanecarboxylic acid chlor-
ide (33 μL, 0.24 mmol) was added in one portion to a stirred mixture
of 5-bromobenzo[b]thiophen-3-amine (20a, 50 mg, 0.21 mmol) in
dichloromethane (4 mL). After completion of reaction (24 h) solvents
were evaporated. The crude residue was purified on silicagel using a
mixture of cyclohexane/ethyl acetate in a ratio of 2:1, affording the
pure title compound (46 mg, 62%). 1H NMR (300 MHz, CDCl3): δ =
1.59−2.06 (m, 10H, 5 × CH2), 2.38 (m, 1H, CH), 7.46−7.52 (m, 2H,
arom H, NH), 7.70−7.73 (m, 2H, arom H), 8.04 (s, 1H, arom H)
ppm.
Ethyl 3-Amino-5-bromobenzo[b]thiophene-2-carboxylate
(19a). 5-Bromo-2-chlorobenzonitrile (18a, 864 mg, 4.0 mmol) and
ethyl 2-mercaptoacetate (393 μL, 4.4 mmol) were dissolved in dry
DMF (20 mL) and the mixture was cooled to 0 °C. To this mixture,
tBuOK (985 mg, 8.8 mmol) was added portionwise over a period of
45 min. The reaction mixture was stirred an additional hour at 0 °C.
The solvent was evaporated and the crude residue was purified by
silicagel flash chromatography, the mobile phase being a mixture of
cyclohexane/ethyl acetate in a ratio 3:2, yielding the pure title
1
compound as a white powder (450 mg, 38%). H NMR (300 MHz,
CDCl3): δ = 1.40 (t, J = 7.11 Hz, 3H, CH3), 4.36 (q, J = 7.11 Hz, 2H,
CH2), 5.86 (br s, 1H, NH), 7.54 (m, 2H, arom H), 7.76 (d, J = 1.38
Hz, 1H, arom H) ppm. HRMS: calcd for C11H11BrNO2S 299.9688,
found 299.9692.
Ethyl 3-Amino-5-bromothieno[2,3-b]pyridine-2-carboxylate
(19b). 5-Bromo-2-chloro-3-cyanopyridine (18b, 617 mg, 2.84 mmol)
N - ( 5 - B r o m o t h i e n o [ 2 , 3 - b ] p y r i d i n - 3 - y l ) -
cyclohexanecarboxamide (21b). This compound was prepared
according to the procedure for the synthesis of compound 21a,
starting from 5-bromothieno[2,3-b]pyridin-3-amine (20b, 75 mg,
0.329 mmol). The crude product was purified on silicagel using a
mixture of cyclohexane/ethyl acetate as mobile phase (in a ratio of
O
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9:1), affording the title compound as white powder (74 mg, 66%). 1H
NMR (300 MHz, CDCl3): δ = 1.63−2.05 (m, 10H, 5 × CH2), 2.37
(m, 1H, CH), 7.55 (br s, 1H, NH), 7.97 (s, 1H, arom H), 8.03 (d, J =
2.07 Hz, 1H, arom H), 8.62 (d, J = 1.98 Hz, 1H, arom H) ppm.
N - ( 2 - B r o m o t h i e n o [ 2 , 3 - b ] p y r a z i n - 7 - y l ) -
cyclohexanecarboxamide (21c). This compound was prepared
according to the procedure for the synthesis of compound 21a,
starting from crude 2-bromothieno[2,3-b]pyrazin-7-amine (20c, 40
mg, 0.17 mmol) The crude product was purified on silicagel using a
mixture of cyclohexane/ethyl acetate (in a ratio of 9:1) as mobile
(CO) ppm. HRMS: calcd for C21H24N3O3S 398.1532, found
398.1529.
5-(3,4-Dimethoxyphenyl)-3-nitro-1H-pyrrolo[2,3-b]pyridine
(24). A mixture of 5-bromo-3-nitro-1H-pyrrolo[2,3-b]pyridine 23
(242 mg, 1.0 mmol), 3,4-dimethoxyphenylboronic acid (1.2 mmol),
K2CO3 (2.0 mmol), and tetrakis(triphenylphosphine)palladium(0)
(0.03 mmol) in 1,4-dioxane (4 mL) and water (1 mL) was refluxed for
12 h. After cooling to room temperature, the precipitate was filtered
off, and the crude product 24 was used as such in the following step.
1H NMR (300 MHz, DMSO-d6): δ = 3.81 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 7.09 (d, J = 8.3 Hz, 1H, arom H), 7.32 (s, 1H, arom H), 7.32
(d, J = 8.3 Hz, 1H, arom H), 13.4 (br s, 1H, NH), 8.84 (s, 1H, arom
H), 8.73 (d, J = 2.0 Hz, 1H, arom H), 8.55 (d, J = 2.0 Hz, 1H, arom
H) ppm.
1
phase, affording the pure title (17 mg, 27%). H NMR (300 MHz,
CDCl3): δ = 1.63−2.04 (m, 10H, 5 × CH2), 2.47 (m, 1H, CH), 8.28
(br s, 1H, NH), 8.45 (s, 1H, arom H), 8.64 (s, 1H, arom H) ppm.
N-(5-(3,4-Dimethoxyphenyl)benzo[b]thiophen-3-yl)-
cyclohexanecarboxamide (22a). To a solution of N-(5-
bromobenzo[b]thiophen-3-yl)cyclohexanecarboxamide (21a, 46 mg,
0.13 mmol) in DME (2 mL) was added 3,4-dimethoxyphenyl boronic
acid (47 mg, 0.26 mmol) and potassium carbonate (0.13 mL, 2 M
solution in H2O). The mixture was degassed and Pd(PPh3)4 (15 mg,
0.013 mmol) was added. The reaction was heated to 80 °C. After
completion of the reaction (24 h), solvents were evaporated. The
crude product was purified on silicagel using a mixture of cyclohexane/
ethyl acetate (in a ratio of 1:1) as mobile phase, affording the pure title
N-(5-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-
cyclohexanecarboxamide (25). To a suspension of 5-(3,4-
dimethoxyphenyl)-3-nitro-1H-pyrrolo[2,3-b]pyridine 24 (100 mg,
0.33 mmol) in methanol (10 mL) was added Raney Ni (100 mg).
The mixture was stirred at room temperature under an atmosphere of
hydrogen for 48 h. The reaction mixture was filtered through Celite.
After concentration under reduced pressure, the crude residue was
treated with cyclohexanecarbonyl chloride according to general
procedure E. The crude residue was purified by silicagel flash
chromatography, the mobile phase being a mixture of methanol and
dichloromethane (in a ratio gradually ranging from 1/50 to 1/25),
1
compound (12.2 mg, 23%). H NMR (300 MHz, CDCl3): δ = 1.61−
2.06 (m, 10H, 5 × CH2), 2.40 (m, 1H, CH), 3.95 (s, 3H, CH3), 3.99
(s, 3H, CH3), 6.98 (d, J = 8.28 Hz, 1H, arom H), 7.16 (d, J = 2.04 Hz,
1H, arom H), 7.22 (dd, J = 2.10 Hz, J = 8.19 Hz, 1H, arom H), 7.57
(d, J = 8.34 Hz, arom H), 7.67 (bs, 1H, NH), 7.88 (d, J = 8.37 Hz, 1H,
arom H), 8.03 (s, 1H, arom H) ppm. 13C NMR (125 MHz, CDCl3): δ
= 25.32 (CH2), 29.35 (CH2), 29.49 (CH2) 45.75 (CH), 55.71
(OCH3), 55.83 (OCH3), 110.84 (CH), 111.32 (CH), 112.77 (CH),
116.46 (CH), 119.57 (CH), 123.10 (CH), 124.18 (CH), 128.19 (Cq),
132.69 (Cq), 134.01 (Cq), 136.33 (Cq), 137.39 (Cq), 148.50 (Cq),
148.97 (Cq), 173.76 (CO) ppm. HRMS: calcd for C23H26NO3S
396.1627, found 396.1626.
1
yielding the pure title compound as a white solid (40 mg, 32%). H
NMR (300 MHz, DMSO-d6): δ = 1.98−1.23 (m, 10H, CH2), 2.48 (m,
1H, CH), 3.81 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 7.08 (d, J = 8.3
Hz, 1H, arom H), 7.22 (d, J = 8.3 Hz, 1H, arom H), 7.26 (s, 1H, arom
H), 7.82 (s, 1H, arom H), 11.31 (s, 1H, NH), 8.44 (d, J = 2.0 Hz, 1H,
arom H), 8.51 (d, J = 2.0 Hz, 1H, arom H), 9.88 (s, 1H, arom H)
ppm. 13C NMR (150 MHz, DMSO-d6): δ = 173.34, 149.26, 148.25,
144.94, 141.99, 131.98, 127.44, 124.13, 119.01, 115.54, 114.38, 112.67,
112.49, 110.76, 55.78, 55.69, 44.06, 29.45, 25.54, 25.41 ppm. HRMS:
calcd for C22H26N3O3 380.1969, found 380.1967.
5-Bromo-2-mercaptobenzonitrile (27). Into an oven-dried flask
was added 5-bromo-2-fluorobenzonitrile (26, 400 mg, 2 mmol), Na2S
(172 mg, 2.2 mmol), and DMF (2 mL) under nitrogen gas. The
reaction mixture was stirred at room temperature for 2.5 h. Then, a 1
M NaOH solution (25 mL) was added and the aqueous phase was
washed with dichloromethane (2 × 25 mL). The aqueous layer was
acidified to pH 1−2 with a 6 M HCl solution and extracted with
dichloromethane (2 × 25 mL). The combined organic layers were
washed with brine (50 mL), dried over MgSO4, filtered, and
concentrated under reduced pressure to provide a crude residue. To
the residue was added 10% HCl (20 mL) and cooled with an ice−
water bath. Then, zinc dust (1 g) was added and the mixture was
stirred for 1 h. Then, ethyl acetate (100 mL) was added and the
mixture was stirred for an additional 30 min. The organic layer was
separated and washed with water (20 mL) and brine (20 mL), dried
over MgSO4, filtered, and concentrated to provide the title compound
(209 mg, 98%). 1H NMR (300 MHz, CDCl3): δ= 4.11 (br s, 1H, SH),
7.28 (d, J = 8.55 Hz, 1H, arom H), 7.55 (dd, J = 8.49 Hz, J = 1.47 Hz,
1H, arom H), 7.70 (s, 1H, arom H) ppm.
5-Bromobenzo[d]isothiazol-3-amine (28). To a solution of 5-
bromo-2-mercaptobenzonitrile (27, 446 mg, 1.08 mL) in a 25%
aqueous ammonia solution (13 mL) were added 3% NaOH (5 mL)
and NaOCl (13% active chlorine, 1.3 mL) at 0 °C. The mixture was
stirred at 0 °C for 4 h. The precipitate was filtered off, washed with
water, and dried, yielding the pure title compound (244 mg, 51%). 1H
NMR (300 MHz, CDCl3): δ = 5.31 (br s, 2H, NH2), 7.53−7.60 (m,
2H, arom H), 7.63 (d, J = 1.35 Hz, 1H, arom H) ppm.
N-(5-(3,4-Dimethoxyphenyl)thieno[2,3-b]pyridin-3-yl)-
cyclohexanecarboxamide (22b). This compound was prepared
according to the procedure described for the preparation of 22a,
starting from N-(5-bromothieno[2,3-b]pyridin-3-yl)-
cyclohexanecarboxamide (21b, 74 mg, 0.21 mmol). The crude residue
was purified on silicagel using a mixture of cyclohexane/ethyl acetate
(in a ratio of 3:2) as mobile phase, affording the title compound as
white powder (34 mg, 39%). 1H NMR (300 MHz, CDCl3): δ = 1.64−
2.00 (m, 10H, 5 × CH2), 2.39 (m, 1H, CH), 3.94 (s, 3H, CH3), 3.96
(s, 3H, CH3), 6.97 (d, J = 8.28 Hz, 1H, arom H), 7.10 (d, J = 2.01 Hz,
1H, arom H), 7.16 (dd, J = 2.01 Hz, J = 8.22 Hz, 1H, arom H), 7.75
(br s, 1H, NH), 7.97 (d, J = 1.95 Hz, 1H, arom H), 8.03 (s, 1H, arom
H), 8.78 (d, J = 1.86 Hz, 1H, arom H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 25.62 (CH2), 29.69 (CH2), 29.78 (CH2), 45.96 (CH),
56.04 (OCH3), 56.16 (OCH3), 110.89 (CH), 111.87 (CH), 114.09
(CH), 119.06 (CH), 124.36 (CH), 126.30 (Cq), 126.70 (Cq), 130.83
(Cq), 132.68 (Cq), 146.32 (CH), 149.29 (Cq), 149.50 (Cq), 158.07
(Cq), 174.55 (CO) ppm. HRMS: calcd for C22H23N2O3S 395.1434,
found 395.1434.
N-(2-(3,4-Dimethoxyphenyl)thieno[2,3-b]pyrazin-7-yl)-
cyclohexanecarboxamide (22c). This compound was prepared
according to the procedure described for the preparation of 22a,
starting from N-(2-bromothieno[2,3-b]pyrazin-7-yl)-
cyclohexanecarboxamide (21c, 17 mg, 0.05 mmol). The crude residue
was purified on silica gel using a mixture of cyclohexane/ethyl acetate
(in a ratio of 3:2), affording the title compound as a white powder
1
(12.5 mg, 62%). H NMR (300 MHz, CDCl3): δ = 1.61−2.09 (m,
N - ( 5 - B r o m o b e n z o [ d ] i s o t h i a z o l - 3 - y l ) -
cyclohexanecarboxamide (29). This compound was synthesized
according to the procedure for the synthesis of compound 21a,
starting from 5-bromobenzo[d]isothiazol-3-amine (28, 229 mg, 1
mmol) The crude product was purified by silicagel flash chromatog-
raphy, using a mixture of cyclohexane/ethyl acetate (in a ratio of 1:1)
as mobile phase, affording the title compound as a white powder (116
10H, 5 × CH2), 2.45 (m, 1H, CH), 3.99 (s, 3H, CH3), 4.04 (s, 3H,
CH3), 6.05 (d, J = 8.22 Hz, 1H, arom H), 7.66 (m, 2H, arom H), 8.39
(s, 1H, arom H), 8.51 (br s, 1H, NH), 8.98 (s, 1H, arom H) ppm. 13C
NMR (75 MHz, CDCl3): δ = 25.28 (CH2), 25.34 (CH2) 29.31 (CH2),
45.45 (CH), 55.75 (OCH3), 55.83 (OCH3), 109.96 (CH), 111.24
(CH), 113.90 (Cq), 120.00 (CH), 126.82 (Cq), 128.88 (Cq), 139.00
(CH), 148.53 (Cq), 149.32 (CH), 150.61 (Cq), 151.08 (Cq), 174.90
1
mg, 34%). H NMR (300 MHz, DMSO-d6): δ = 1.60−1.91 (m, 10H,
P
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5 × CH2), 2.51 (m, 1H, CH), 7.73 (dd, J = 2.13 Hz, J = 8.28 Hz, 1H,
arom H), 7.74 (dd, J = 8.70 Hz, J = 1.80 Hz, 1H, arom H), 8.13 (d, J =
8.70 Hz, 1H, arom H), 8.26 (d, J = 1.26 Hz, 1H, arom H), 10.65 (s,
1H, NH) ppm.
were evaporated. The crude product was purified by silicagel flash
chromatography, using a mixture of cyclohexane/ethyl acetate as
mobile phase (in a ratio of 3:1), affording the pure title compound
1
(200 mg, 64%). H NMR (300 MHz, CDCl3): δ = 1.70 (s, 9H, 3 ×
N-(5-(3,4-Dimethoxyphenyl)benzo[d]isothiazol-3-yl)-
cyclohexanecarboxamide (30). This compound was synthesized
according to the procedure for the synthesis of compound 22a,
starting from N-(5-bromobenzo[d]isothiazol-3-yl)-
cyclohexanecarboxamide (29, 100 mg, 0.29 mmol). The crude product
was purified on silicagel using a mixture of cyclohexane/ethyl acetate
as mobile phase (in a ratio of 3:2), affording the title compound as a
white powder (40 mg, 34%). 1H NMR (300 MHz, CDCl3): δ = 1.60−
2.04 (m, 10H, 5 × CH2), 2.64 (m, 1H, CH), 3.95 (s, 3H, CH3), 3.98
(s, 3H, CH3), 6.97 (d, J = 8.28 Hz, 1H, arom H), 7.00 (d, J = 2.04 Hz,
1H, arom H), 7.22 (dd, J = 2.13 Hz, J = 8.28 Hz, 1H, arom H), 7.73
(dd, J = 8.49, J = 1.56 Hz, 1H, arom H), 7.87 (d, J = 8.46 Hz, 1H,
arom H), 8.17 (s, 1H, arom H), 8.52 (s, 1H, NH) ppm. 13C NMR (75
MHz, DMSO-d6): δ = 25.31 (CH2), 25.55 (CH2), 29.22 (CH2), 43.93
(CH), 55.78 (OCH3), 55.81 (OCH3), 111.00 (CH), 112.53 (CH),
119.42 (CH), 121.13 (CH), 121.48 (CH), 127.52 (CH), 129.73 (Cq),
132.40 (Cq), 137.20 (Cq), 148.95 (Cq), 149.32 (Cq), 149.95 (Cq),
153.31 (Cq), 174.98 (CO) ppm. HRMS: calcd for C22H25N2O3S
397.1580, found 397.1583.
CH3), 4.50 (s, 2H, NH2), 7.60 (dd, J = 1.86 Hz, J = 8.88 Hz, 1H, arom
H), 7.69 (d, J = 1.77 Hz, 1H, arom H), 7.96 (s, 1H, arom H) ppm.
tert-Butyl 5-bromo-3-(cyclohexanecarboxamido)-1H-inda-
zole-1-carboxylate (35). This compound was prepared according
to the procedure for the synthesis of compound 21a, starting from tert-
butyl 3-amino-5-bromo-1H-indazole-1-carboxylate (200 mg, 0.64
mmol). After reaction completion (3 h), solvents were evaporated.
The product was purified on silica gel using a mixture of cyclohexane/
ethyl acetate (in a ratio of 7:1), affording the title compound (178 mg,
1
65%). H NMR (300 MHz, CDCl3): δ = 1.60−2.01 (m, 19H, 3 ×
CH3, 5 × CH2), 2.39 (m, 1H, CH), 7.62 (dd, J = 1.92 Hz, J = 8.97 Hz,
1H, arom H), 7.98 (d, J = 9.03 Hz, 1H, arom H), 8.34 (s, 1H, arom
H), 8.41 (s, 1H, NH) ppm.
tert-Butyl 5-bromo-3-(cyclohexanecarboxamido)-1H-inda-
zole-1-carboxylate. This compound was prepared according to the
procedure described for the preparation of 22a, starting from tert-butyl
5-bromo-3-(cyclohexanecarboxamido)-1H-indazole-1-carboxylate (35,
178 mg, 0.42 mmol). The crude residue was purified on silica gel using
a mixture of cyclohexane/ethyl acetate (in a ratio of 3:2) as mobile
1
phase, affording the pure title compound (101 mg, 50%). H NMR
5-Bromobenzo[d]isoxazol-3-amine (31). Acetohydroxamic acid
(150 mg, 2 mmol) and tBuOK (224 mg, 2 mmol) were suspended in
dry DMF and the resulting reaction mixture was stirred at room
temperature for 40 min. Then 5-bromo-2-fluorobenzonitrile (26, 400
mg, 2 mmol) was added in one portion. After completion of the
reaction (24 h), the solvent was evaporated. The crude product was
purified on silica gel using a mixture of cyclohexane/ethyl acetate as
mobile phase (in a ratio of 5:1), affording the pure title compound
(300 MHz, CDCl3): δ = 1.61−2.07 (m, 19H, 3 × CH3, 5 × CH2), 2.42
(m, 1H, CH), 3.94 (s, 3H, CH3), 3.98 (s, 3H, CH3), 6.96 (d, J = 8.31
Hz, 1H, arom H), 7.16 (d, J = 2.04 Hz, 1H, arom H), 7.21 (dd, J =
2.10 Hz, J = 8.25 Hz, 1H, arom H), 7.74 (dd, J = 8.82, J = 1.77 Hz, 1H,
arom H), 8.11 (d, J = 8.76 Hz, 1H, arom H), 8.14 (s, 1H, arom H),
8.25 (s, 1H, NH) ppm.
N-(5-(3, 4-Dimethoxyphenyl)-1H-indazol-3-yl)-
cyclohexanecarboxamide (36). Tert-butyl 5-bromo-3-(cyclohexa-
necarboxamido)-1H-indazole-1-carboxylate (101 mg, 0.21 mmol) was
suspended in 20% TFA/DCM (10 mL). The reaction was stirred for
20 min at room temperature. Then, the mixture was carefully
neutralized with solid NaHCO3. The solids were filtered off and the
solvents were evaporated. The crude product was purified on silica gel
using a mixture of cyclohexane/ethyl acetate (in a ratio of 1:6) as
1
(214 mg, 50%). H NMR (300 MHz, CDCl3): δ = 4.47 (br s, 2H,
NH2), 7.32 (d, J = 8.62 Hz, 1H, arom H), 7.60 (dd, J = 8.82 Hz, J =
1.35 Hz, 1H, arom H), 7.69 (d, J = 1.33 Hz, 1H, arom H) ppm.
N-(5-Bromobenzo[d]isoxazol-3-yl)cyclohexanecarboxamide
(32). This compound was prepared according to the procedure for the
synthesis of compound 21a, starting from 5-bromobenzo[d]isoxazol-3-
amine (31, 200 mg, 0.93 mmol). The mixture was heated to 50 °C.
The crude residue was purified by flash chromatography on silica gel
using a mixture of cyclohexane/ethyl acetate (in a ratio of 9:1),
1
mobile phase, affording the pure title compound (27 mg, 34%). H
NMR (300 MHz, MeOD): δ = 1.60−2.03 (m, 10H, 5 × CH2), 2.56
(m, 1H, CH), 3.88 (s, 3H, CH3), 3.93 (s, 3H, CH3), 7.05 (d, J = 8.13
Hz, 1H, arom H), 7.20 (m, 2H, arom H), 7.49 (d, J = 8.73 Hz, 1H,
arom H), 7.64 (d, J = 8.40 Hz, 1H, arom H), 7.91 (s, 1H, arom H)
ppm. 13C NMR (75 MHz, DMSO-d6): δ = 25.39 (CH2), 25.59 (CH2),
29.37 (CH2), 43.99 (CH), 55.77 (OCH3), 110.50 (CH), 110.96
(CH), 112.56 (CH), 117.12 (Cq), 119.06 (CH), 119.43 (Cq), 126.17
(CH), 132.16 (Cq), 134.10 (CH), 140.38 (Cq), 140.85 (Cq), 148.20
(Cq), 149.17 (CH), 174.62 (CO) ppm. HRMS: calcd for C22H26N3O3
380.1968, found 380.1974.
N-(6-(3,4-Dimethoxyphenyl)pyrido[2,3-d]pyrimidin-4-yl)-
cyclohexanecarboxamide (40a). Step a. A mixture of 2-amino-5-
bromonicotinonitrile 37a (200 mg, 1.0 mmol) and NH4OAc (5
mmol) in triethyl orthoformate (5 mL) was refluxed for 2 h. After
cooling to room temperature, the precipitate was filtered off, washed
with water, and dried affording crude 6-bromopyrido[2,3-d]pyrimidin-
4-amine 38a as a gray solid (225 mg, 99%).
1
affording the title compound (110 mg, 36%). H NMR (300 MHz,
CDCl3): δ = 1.58−2.07 (m, 10H, 5 × CH2), 2.47 (m, 1H, CH), 7.39
(d, J = 8.91 Hz, 1H, arom H), 7.66 (dd, J = 8.88 Hz, J = 1.98 Hz, 1H,
arom H), 8.45 (br s, 1H, NH), 7.49 (d, J = 1.74 Hz, 1H, arom H)
ppm.
N-(5-(3,4-Dimethoxyphenyl)benzo[d]isoxazol-3-yl)-
cyclohexanecarboxamide (33). To a solution of N-(5-
bromobenzo[d]isoxazol-3-yl)cyclohexanecarboxamide (32, 110 mg,
0.34 mmol) in DME (2 mL) was added an 3,4-dimethoxyphenyl
boronic acid (92 mg, 0.51 mmol) and potassium carbonate (0.68 mL,
2 M solution in H2O). Mixture was degassed and Pd(PPh3)4 (36 mg,
0.034 mmol) was added. The reaction was heated to 80 °C. After the
completion of reaction (6 h), solvents were evaporated. Product was
purified on silica gel using a mixture of cyclohexane/ethyl acetate in a
ratio of 3:2, affording the title compound in 21% yield (27 mg, 0.07
1
mmol). H NMR (300 MHz, CDCl3): δ = 1.59−2.17 (m, 10H, 5 ×
Step b. The crude product 38a (225 mg, 1.0 mmol), 3,4-
dimethoxyphenylboronic acid (1.2 mmol), K2CO3 (2.0 mmol), and
tetrakis(triphenylphosphine)palladium(0) (0.03 mmol) in a mixture of
1,4-dioxane (4 mL) and water (1 mL) was heated at 90 °C for 2 h.
After cooling to room temperature, the precipitate was filtered off,
yielding crude 6-(3,4-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-4-
amine 39a, which was used for further reaction without any further
purification.
CH2), 3.09 (m, 1H, CH), 3.95 (s, 3H, CH3), 3.94 (s, 3H, CH3), 6.98
(d, J = 8.28 Hz, 1H, arom H), 7.11−7.18 (m, 3H, arom H), 7.62 (dd, J
= 2.37 Hz, J = 8.64 Hz, 1H, arom H), 8.21 (d, J = 2.34 Hz, 1H, arom
H), 8.52 (s, 1H, NH) ppm. 13C NMR (125 MHz, CDCl3): δ = 24.94
(CH2), 25.10 (CH2), 29.86 (CH2), 35.94 (CH), 55.68 (OCH3), 55.71
(OCH3), 109.87 (CH), 110.51 (Cq), 111.25 (CH), 117.67 (CH),
118.74 (CH), 125.53 (CH), 131.28 (CH), 132.79 (Cq), 132.88 (Cq),
148.13 (Cq), 148.90 (Cq), 155.96 (Cq), 166.51 (Cq), 181.98 (CO)
ppm. HRMS: calcd for C22H25N2O4 381.1808, found 381.1808.
tert-Butyl 3-amino-5-bromo-1H-indazole-1-carboxylate. To
a solution of 5-bromo-1H-indazol-3-amine (34, 212 mg, 1 mmol) in
dichloromethane (5 mL) was added dropwise a solution of di-tert-
butyl dicarbonate (218 mg, 1 mmol) in dichloromethane (5 mL) at
room temperature. After completion of the reaction (2 h), the solvents
Step c. Compound 39a was treated with cyclohexanecarbonyl
chloride according to the general procedure E. The crude residue was
purified by flash chromatography on silicagel using a mixture of
dichloromethane and methanol (in a ratio of 50:1) as mobile phase,
affording the pure title compound as a yellowish solid (290 mg, 74%
1
over 3 steps). H NMR (300 MHz, DMSO-d6): δ = 1.23−1.98 (m,
Q
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Journal of Medicinal Chemistry
Article
11H, CH and CH2), 3.85 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 7.16
(d, J = 8.2 Hz, 1H, arom H), 7.47 (d, J = 8.2 Hz, 1H, arom H), 7.48 (s,
1H, arom H), 8.76 (d, J = 2.5 Hz, 1H, arom H), 9.12 (s, 1H, arom H),
9.58 (d, J = 2.5 Hz, 1H, arom H), 10.94 (s, 1H, NH) ppm. 13C NMR
(150 MHz, DMSO-d6): δ = 171.27, 169.17, 152.46, 150.19, 149.43,
149.37, 132.21, 130.73, 129.08, 122.12, 119.75, 112.54, 110.96, 55.78,
55.73, 43.29, 32.61, 25.70, 25.20 ppm. HRMS: calcd for C22H24N4O3
393.1921, found 393.1919.
8.07 (s, 1H, arom H), 8.10 (d, J = 8.5 Hz, 1H, arom H), 9.06 (s, 1H,
arom H), 9.91 (s, 1H, arom H), 10.67 (s, 1H, NH) ppm.
13C NMR (150 MHz, DMSO-d6): δ = 174.61, 158.00, 157.00,
152.46, 151.83, 151.12, 150.70, 149.39, 127.28, 125.21, 121.66, 111.92,
110.99, 55.81, 55.76, 44.52, 28.99, 25.48, 25.21 ppm. HRMS: calcd for
C21H23N5O3 394.1874, found 394.1870.
Binding Assays for DRAK1, DRAK2, DAPK1, DAPK2, and
DAPK3. Compounds were screened at a single concentration of 10
μM, using binding assays previously described.14 Briefly, T7 kinase-
tagged phage strains were grown in parallel in 24- or 96-well blocks in
an E. coli host derived from the BL21 strain. E. coli were grown to log
phase and infected with T7 phage from a frozen stock (multiplicity of
infection ∼0.1) and incubated with shaking at 32 °C until lysis (∼90
min). The lysates were centrifuged (6000g) and filtered (0.2 μM) to
remove cell debris. Streptavidin-coated magnetic beads were treated
with biotinylated small molecule ligands for 30 min at 25 °C to
generate affinity resins for kinase assays. The liganded beads were
blocked with excess biotin and washed with blocking buffer (SeaBlock
(Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound
ligand and to reduce nonspecific phage binding. Binding reactions
were assembled by combining phage lysates, liganded affinity beads,
and test compounds in 1 × binding buffer (20% SeaBlock, 0.17 × PBS,
0.05% Tween 20, 6 mM DTT). Test compounds were prepared as
1000× stocks in DMSO and rapidly diluted into the aqueous
environment (0.1% DMSO final). DMSO (0.1%) was added to
control assays lacking a test compound. All reactions were carried out
in polystyrene 96-well plates that had been pretreated with blocking
buffer in a final volume of 0.1 mL. The assay plates were incubated at
25 °C with shaking for 1 h, long enough for binding reactions to reach
equilibrium, and the affinity beads were washed four times with wash
buffer (1 × PBS, 0.05% Tween 20, 1 mM DTT) to remove unbound
phage. After the final wash, the beads were resuspended in elution
buffer (1 × PBS, 0.05% Tween 20, 2 mM nonbiotinylated affinity
ligand) and incubated at 25 °C with shaking for 30 min. The phage
titer in the eluates was measured by standard plaque assays or by
quantitative PCR. The compounds were tested at a single
concentration of 10 μM. The results of this primary screening is
reported as ‘% Ctrl’, where lower numbers indicate stronger hits.
Binding Constant Measurements for DRAK1 and DRAK2. For
selected compounds where single concentration results at 10 μM had
shown % control <5, Kd values were determined. The equilibrium
binding equations yield the following expression for the binding
constant for the interaction between the free test compound and the
kinase (Kd(test)), assuming that the phage concentration is below
N-(6-(3,4-Dimethoxyphenyl)pyrido[3,2-d]pyrimidin-4-yl)-
cyclohexanecarboxamide (40b). This compound was prepared
using the 3-step procedure, as described for the preparation of
compound 40a, starting from 3-amino-6-chloropicolinonitrile 37b
(154 mg, 1.0 mmol). The final crude residue was purified by flash
chromatography on silica gel using a mixture of dichloromethane and
methanol (in a ratio of 50:1) as the mobile phase, yielding the title
1
compound as a yellowish solid (134 mg, 34% over 3 steps). H NMR
(300 MHz, DMSO-d6): δ = 1.98−1.23 (m, 10H, CH2), 3.05 (m, 1H,
CH), 3.87 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 7.14 (d, J = 8.5 Hz,
1H, arom H), 7.94 (d, J = 8.5 Hz, 1H, arom H), 8.05 (s, 1H, arom H),
8.34 (d, J = 8.9 Hz, 1H, arom H), 8.60 (d, J = 8.9 Hz, 1H, arom H),
8.93 (s, 1H, arom H), 10.45 (s, 1H, NH) ppm. 13C NMR (150 MHz,
DMSO-d6): δ = 174.17, 156.08, 154.45, 151.19, 149.20, 144.59,
136.96, 130.75, 129.58, 126.23, 120.88, 111.74, 110.77, 55.73, 55.52,
44.66, 28.97, 25.48, 25.24 ppm. HRMS: calcd for C22H24N4O3
393.1921, found 393.1914.
N - ( 6 - ( 3 , 4 - D i m e t h o x y p h e n y l ) q u i n a z o l i n - 4 - y l ) -
cyclohexanecarboxamide (45a). Step a. A mixture of ethyl 2-
amino-5-bromobenzoate 41a (230 mg, 1.0 mmol) and NH4OAc (5
mmol) in triethyl orthoformate (5 mL) was refluxed for 1 h. After
cooling to room temperature, the precipitate was filtered off, washed
with water, and dried, yielding crude 6-bromoquinazolin-4(3H)-one
42a as a gray solid (225 mg, 99%).
Step b. A mixture consisting of crude 42a (225 mg, 1.0 mmol), 3,4-
dimethoxyphenylboronic acid (1.2 mmol), K2CO3 (2.0 mmol), and
tetrakis(triphenylphosphine)palladium(0) (0.03 mmol) in 1,4-dioxane
(4 mL) and water (1 mL) was refluxed for 12 h. After cooling to room
temperature, the precipitate was filtered off, affording crude 43a, which
was used as such in the following step.
Step c. Crude 43a (1 mmol) in POCl3 (2 mL) was heated at 125
°C for 2 h. After concentration under reduced pressure, the residue
was treated with a 7 N ammonia solution in methanol at room
temperature for 30 min. The solvents were evaporated affording crude
44a as a gray residue, which was used as such for further reaction.
Step d. Crude 44a was treated with cyclohexanecarbonyl chloride
according to the general procedure D. The final crude residue was
purified by flash chromatography on silica gel using a mixture of
dichloromethane and methanol (in a ratio of 40:1) as mobile phase,
yielding the pure title compound as white solid (240 mg, 61% over 4
steps).
Kd(test): Kd(test) = (Kd(probe)/(Kd(probe) + [Probe])) × [test]1/2
.
Kd(probe) is the binding constant for the interaction between the kinase
and the immobilized ligand, [Probe] is the concentration of the
immobilized ligand, and [test]1/2 is the concentration of the free test
compound at the midpoint of the transition. If [Probe] is below
Kd(probe) the expression simplifies to Kd(test) = [test]1/2. Under these
conditions the binding constants measured for the interaction between
kinases and test compounds (Kd(test)) are therefore independent of the
affinity of the immobilized ligand for the kinase (Kd(probe)). T7 phage
grow to a titer of 108−1010 plaque forming units (PFU)/mL, and the
concentration of phage-tagged kinase in the binding reaction is
therefore in the low picomolar range. The concentration of the
immobilized ligand is kept in the low nanomolar range, below its
binding constant for the kinase. Binding data were fit to the equation
PFU = L + ((H − L) × (Kd(test)/(Kd(test) + [test]))), where L is the
lower baseline, H is the upper baseline, Kd(test) is the binding constant
for the interaction between the test compound and the kinase, and
[test] is the free test compound concentration. Binding constants
measured in duplicate on the same day as part of the same experiment
generally were within 2-fold. Duplicate measurements performed on
separate days generally varied by no more than 4-fold. Clustering and
visualization was performed with Cluster 3.0 (M. Eisen, Stanford
University) and Mapletree software (M. Eisen, Stanford University; L.
Simirenko, Lawrence Berkeley National Lab). For kinase/compound
combinations where no interaction was observed, the binding constant
was arbitrarily set to 1 M.
1H NMR (300 MHz, DMSO-d6): δ = 1.98−1.23 (m, 10H, CH2),
2.89 (m, 1H, CH), 3.83 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 7.13 (d, J
= 8.6 Hz, 1H, arom H), 7.38 (d, J = 8.6 Hz, 1H, arom H), 7.39 (s, 1H,
arom H), 8.00 (d, J = 8.6 Hz, 1H, arom H), 8.30 (d, J = 2.0 Hz, 1H,
arom H), 8.35 (dd, J = 8.6, J = 2.0 Hz, 1H, arom H), 8.97 (s, 1H, Ar−
H), 10.78 (s, 1H, NH) ppm. 13C NMR (150 MHz, DMSO-d6): δ =
176.05, 157.69, 155.23, 153.98, 150.02, 149.31, 138.66, 133.07, 131.59,
128.40, 121.58, 119.72, 117.90, 112.40, 110.90, 55.75, 55.72, 44.22,
29.10, 25.50, 25.23 ppm. HRMS: calcd for C23H25N3O3 392.1969,
found 392.1963.
N - ( 6 - ( 3 , 4 - D i m e t h o x y p h e n y l ) p t e r i d i n - 4 - y l ) -
cyclohexanecarboxamide (45b). This compound was prepared
using the 4-step procedure as described for the synthesis of 45a,
starting from ethyl 3-amino-6-bromopyrazine-2-carboxylate 41b (116
mg, 0.5 mmol). The final residue was purified by flash chromatography
on silica gel using a mixture of dichloromethane and methanol (in a
ratio of 60:1) as mobile phase, yielding the title compound as a white
1
solid (60 mg, 30% yield over 4 steps). H NMR (300 MHz, DMSO-
d6): δ = 1.98−1.23 (m, 10H, CH and CH2), 3.02 (m, 1H, CH), 3.88
(s, 3H, OCH3), 3.94 (s, 3H, OCH3), 7.16 (d, J = 8.5 Hz, 1H, arom H),
R
dx.doi.org/10.1021/jm5007929 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
DRAK1 and DRAK2 Functional, Biochemical Assays. The
DRAK1 and DRAK2 to be employed in the compound profiling
process were cloned, expressed, and purified in-house at SignalChem
using proprietary methods (www.signalchem.com). The enzymes are
generated from the full-length human genes and are mutation free.
Quality control testing is routinely performed on each of the targets to
ensure compliance to acceptable standards. 33P-ATP was purchased
from PerkinElmer. All other materials were of standard laboratory
grade. A stock solution of the compound in DMSO was prepared. The
stock solution was then diluted to form an assay stock solution and
this was used to profile against DRAK1 and DRAK2.
SignalChem uses a radioisotope assay format for profiling evaluation
of protein kinase target and all assays are performed in a designated
radioactive working area. Protein kinase assays (in duplicate) were
performed at ambient temperature for 30 min in a final volume of 25
μL according to the following assay reaction recipe:
maximal (50%) inhibitory concentration; Kd, dissociation
constant; ND, not determined; rt, room temperature; SAR,
structure−activity relationship; TBTU, N,N,N′,N′-tetramethyl-
O-(benzotriazol-1-yl)uronium tetrafluoroborate; TFA, tri-
fluoroacetic acid; THF, tetrahydrofuran
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AUTHOR INFORMATION
Corresponding Author
■
*Phone: +32 16 337387. Fax: +32 16 337340. E-mail: Piet.
Herdewijn@rega.kuleuven.be.
́
Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lelias, J.; Mehta, S.
Author Contributions
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by a grant from the IWT
(Agentschap voor Innovatie door Wetenschap en Technologie-
Vlaanderen (Grant IWT-SBO 100014; to P.H). Mass
spectrometry was made possible by the support of the Hercules
Foundation of the Flemish Government (grant 20100225−7).
ABBREVIATIONS
■
DAPK, death-associated protein kinase; DRAK, DAPk related
apoptosis inducing protein kinase; Boc, tert-butyloxycarbonyl;
BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate; DCM, dichloromethane; DIPEA,
diisopropylethylamine; DME, dimethoxyethane; DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; IC50, half
S
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