Design, Synthesis, and Evaluation of the Highly Selective and Potent G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitor for the Potential Treatment of Heart Failure

Article abstract of DOI:10.1021/acs.jmedchem.7b00443

A novel class of therapeutic drug candidates for heart failure, highly potent and selective GRK2 inhibitors, exhibit potentiation of β-adrenergic signaling in vitro studies. Hydrazone derivative 5 and 1,2,4-triazole derivative 24a were identified as hit compounds by HTS. New scaffold generation and SAR studies of all parts resulted in a 4-methyl-1,2,4-triazole derivative with an N-benzylcarboxamide moiety with highly potent activity toward GRK2 and selectivity over other kinases. In terms of subtype selectivity, these compounds showed enough selectivity against GRK1, 5, 6, and 7 with almost equipotent inhibition to GRK3. Our medicinal chemistry efforts led to the discovery of 115h (GRK2 IC₅₀ = 18 nM), which was obtained the cocrystal structure with human GRK2 and an inhibitor of GRK2 that potentiates β-adrenergic receptor (βAR)-mediated cAMP accumulation and prevents internalization of βARs in β2AR-expressing HEK293 cells treated with isoproterenol. Therefore, 115h appears to be a novel class of therapeutic for heart failure treatment.

Full text of DOI:10.1021/acs.jmedchem.7b00443

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Article
Design, Synthesis, and Evaluation of the Highly Selective
and Potent G-protein-Coupled Receptor Kinase 2 (GRK2)
Inhibitor for the Potential Treatment of Heart Failure
Tomohiro Okawa, Yoshio Aramaki, Mitsuo Yamamoto, Toshitake Kobayashi,
Shoji Fukumoto, Yukio Toyoda, Tsutomu Henta, Akito Hata, Shota Ikeda, Manami
Kaneko, Isaac D Hoffman, Bi-Ching Sang, Hua Zou, and Tetsuji Kawamoto
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00443 • Publication Date (Web): 12 Jul 2017
Downloaded from http://pubs.acs.org on July 12, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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of their duties.

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Design, Synthesis, and Evaluation of the Highly Selective and Potent GꢀproteinꢀCoupled
Receptor Kinase 2 (GRK2) Inhibitor for the Potential Treatment of Heart Failure
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*
,†
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Tomohiro Okawa, Yoshio Aramaki, Mitsuo Yamamoto, Toshitake Kobayashi, Shoji Fukumoto,
†
†
†
†
†
‡
Yukio Toyoda, Tsutomu Henta, Akito Hata, Shota Ikeda, Manami Kaneko, Isaac D. Hoffman,
‡
‡
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BiꢀChing Sang, Hua Zou and Tetsuji Kawamoto
†
Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd., Shonan Research Center,
2
6ꢀ1, MuraokaꢀHigashi 2ꢀChome, Fujisawa, Kanagawa 251ꢀ8555, Japan
‡
Takeda California, 10410 Science Center Drive, San Diego, CA 92121, USA
Keywords
heart failure, GRK2 inhibitors, (4ꢀPyridyl)ꢀ1,2,4ꢀtriazole derivatives, βꢀadrenergic signaling
Abstract
A novel class of therapeutic drug candidates for heart failure, highly potent and selective GRK2
inhibitors, exhibit potentiation of βꢀadrenergic signaling in vitro studies. Hydrazone derivative 5 and
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1,2,4ꢀtriazole derivative 24a were identified as hit compounds by HTS. New scaffold generation and
SAR studies of all parts resulted in
a
4ꢀmethylꢀ1,2,4ꢀtriazole derivative with an
Nꢀbenzylcarboxamide moiety with highly potent activity towards GRK2, and selectivity over other
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kinases. In terms of subtype selectivity, these compounds showed enough selectivity against GRK1,
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, 6, 7 with almost equipotent inhibition to GRK3. Our medicinal chemistry efforts led to the
discovery of 115h (GRK2 IC50 = 18 nM) which was obtained the coꢀcrystal structure with human
GRK2 and an inhibitor of GRK2 that potentiates βꢀadrenergic receptor (βAR)ꢀmediated cAMP
accumulation and prevents internalization of βARs in β2ARꢀexpressing HEK293 cells treated with
isoproterenol. Therefore, 115h appears to be a novel class of therapeutic for heart failure treatment.
Introduction
Heart failure (HF) is the most common disease for hospitalization in the elderly, with
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approximately 10 % of men and 8 % of women over the age of 60 affected. The prevalence of HF is
growing with the rise of an aging population in developed countries. There remains an intense need
for novel beneficial HF therapies with more than 3 million people in the United States diagnosed per
year, and HF related mortality and rehospitalization rates remaining high despite the modest
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improvement in survival rates seen from advances in device therapy and pharmacological therapy
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angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, and βꢀblockers ). A
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plethora of research into HF has revealed it to be a complex disease associated with various
pathogenetic mechanisms, including ventricular remodeling, excessive neurohormonal stimulation,
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abnormal Ca handling, and proliferation of the extracellular matrix. Although an overstimulation
of the sympathetic nervous system (SNS) initially compensates for cardiac dysfunction, the
subsequent release of catecholamine ultimately promotes disease progression via longꢀterm
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exposure. Activation of the SNS is mediated by adrenergic receptors (AR), and chronic βꢀAR
activation induces βꢀAR desensitization and downregulation, subsequently leading to the reduction
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of βꢀAR signaling. Gꢀprotein receptor kinase (GRK) 2 phosphorylates agonistꢀoccupied βꢀAR,
promotes the binding of βꢀAR arrestin to the Gβγ subunit of the Gꢀprotein, facilitates the Gꢀprotein
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uncoupling from βꢀAR, and results in βꢀAR desensitization and downregulation. In the hearts of HF
patients, GRK2 expression levels and activity were elevated, accompanied by lowered βꢀAR density
12ꢀ14
and signaling.
Moreover, GRK2 inhibition by overexpression of the βARKct, the peptide
inhibitor of GRK2, or cardiac specific GRK2 gene ablation, improved cardiac function and survival
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5ꢀ17
with the increases in βꢀAR density and βꢀAR responses in several HF models.
These results
suggest that GRK2 has a strong relationship with HF, and inhibition of GRK2 is a promising
mechanism for the treatment of HF. Although there are no small molecular GRK2 inhibitors
18ꢀ26
currently in clinical trials, there are several small molecules
reported in preclinical studies.
Furthermore, several papers to discover the other chemotypes of highly selective GRK2 inhibitors
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8ꢀ20
were reported by use of the information of 115h and 139d.
Moreover, 115h and the related
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compounds were introduced by reveiws.
Here we describe the design, synthesis, structureꢀactivity relationships (SAR), and discovery of
(
4ꢀpyridyl)ꢀ1,2,4ꢀtriazole derivatives and the novel, selective, GRK2 inhibitor 115h. Furthermore, we
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describe the acquisition and utilization of crystal structures of human GRK2 with the inhibitors as
well as relevant biological studies.
Design and Synthetic Plans
A high throughput screen testing inhibition of GRK2 mediated phosphorylation of Ulight TopoIIα
as an artificial substrate yielded a 4ꢀpyridylhydrazone derivative (hit compound 5) and a
4ꢀpyridylꢀ1,2,4ꢀtriazole derivative (hit compound 24a) as hit compounds. These compounds act by
competitive inhibition of ATP, binding to the kinase active site. Chemical modifications of
compounds 5 (IC50 = 1800 nM) and 24a (IC50 = 1200 nM) were carried out to develop novel GRK2
inhibitors with potent activity, as well as good selectivity against Rhoꢀassociated protein kinase 2
(
ROCK2) and Protein kinase C alpha (PKCα), with minimal effects on blood pressure in vivo. First,
a series of compounds related to hit compounds 5 (acyclic hydrazone derivative) is described as
follows (Figure 1). i) Introduction of substituent(s) on the benzene ring at the right hand side (RHS),
ii) Cyclization to produce patentable ring systems, iii) Optimization of the substituent at the
3
ꢀposition of the benzene ring at the RHS. Other strategies from acyclic hydrazone derivatives to
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discover
isoquinoline
derivatives
(for
examples,
Nꢀisoquinolinꢀ6ꢀylꢀNꢀ2ꢀ(3ꢀmethoxyphenyl)glycinamide, IC50 (nM): GRK2 / ROCK2 = 12 / 1.3) and
ꢀ(4ꢀpyridyl)pyrazoline derivatives (for examples,
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Nꢀ(2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀyl)ꢀ3ꢀ((2ꢀoxoꢀ2ꢀ(3ꢀ(pyridinꢀ4ꢀyl)ꢀ4,5ꢀdihydroꢀ1Hꢀpyrazolꢀ1ꢀyl)ethyl)ami
no)benzamide, IC50 (nM): GRK2 / ROCK2 = 4.9 / 250) are detailed as well.
[
Figure 1]
Second, a series of compounds related to hit compound 24a (1,2,4ꢀtriazole derivative) is described
as follows (Figure 2). i) Modification at the linker moiety, ii) Modification at the benzene ring at the
RHS, iii) Transformation into the compounds bearing a 5ꢀmembered heterocyclic ring other than
1
Hꢀ1,2,4ꢀtriazole ring, and iv) Modification of the pyridine ring. These two plans were designed, and
the compounds were synthesized, to investigate their biological activities.
[
Figure 2]
Chemistry
Acyclic Hydrazone Derivatives
Acyclic hydrazone derivatives 5a–f were prepared as described in Scheme 1. Reaction of anilines
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1a–f with TFAA activated the reactivity of the nitrogen atom and protected, then the alkylation with
ethyl bromoacetate gave 3a–f in near quantitative yield. Formation of acid hydrazides 4a–f with
hydrazine hydrate was achieved with simultaneous removal of trifluoroacetyl. The 4ꢀPyridyl part, as
a hinge, was introduced by use of 4ꢀpyridinecarboxaldehyde via dehydration reaction resulting in the
final compounds 5a–f.
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[
Scheme 1]
Cyclic Hydrazone Derivatives
Cyclic hydrazone derivatives 12a–e were prepared as described in Scheme 2. Acid hydrazides
11a–e were prepared in a similar way from anilines 6d–e shown in scheme 1. Cyclic hydrazones
1
2a–e were produced by use of 7,8ꢀdihydroisoquinolinꢀ5(6H)ꢀone, instead of
4
ꢀpyridinecarboaldehyde, in good yields.
[
Scheme 2]
Incorporation of the amide at the 3ꢀposition of the benzene ring at the RHS was performed as
shown in Scheme 3. When alkylation of 3ꢀtrifluoroacetoamideꢀbenzoate 14 was finished, the crude
product was treated with TFAA again because a part of the trifluoroacetyl group was removed during
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the alkylation reaction. Hydrolysis of the corresponding ester was differentiated via hydrogenation
by use of palladium – on activated carbon and gave 16. Condensation with Bocꢀhydrazine followed
by hydrolysis of the ester group gave the key intermediate benzoic acid 18 having NꢀBoc acid
hydrazide. Finally, cyclic hydrazone derivatives 12f and 12 g with amide were produced by WSC
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and
HOBt
amidation,
removal
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trifluoroacetyl,
and
dehydration
with
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,8ꢀdihydroisoquinolinꢀ5(6H)ꢀone.
[
Scheme 3]
Modification of the Linker Moiety of 1,2,4ꢀtriazole derivatives
Synthesis of 24c, 24g and 24h
The typical methods of preparation of 1Hꢀ1,2,4ꢀtriazole derivatives are shown in Scheme 4.
Reaction of 4ꢀcyanopyridine 21 with hydrazine and the following acylation of 22 with chloroacetyl
chloride in the presence of DBU and heating in acetic acid gave the useful 1Hꢀ1,2,4ꢀtriazole
intermediate 23a in moderate yield. 1Hꢀ1,2,4ꢀTriazole intermediate having 2ꢀchloroethyl group at
C(5) position 23b was synthesized in a similar method in low yield. Compounds 23a–b were treated
with appropriate anilines or benzylamine to give 24c, 24g and 24h, respectively.
[
Scheme 4]
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Synthesis of 24d, 24f and 24e
Reaction of isonicotinic hydrazide 25 with ammonium thiocyanate, and subsequent treatment with
aqueous sodium hydroxide afforded 3ꢀmercaptoꢀ5ꢀ(4ꢀpyridyl)ꢀ1Hꢀ1,2,4ꢀtriazole 27a in 79% yield.
Alkylation of the mercapto group with benzyl halide in the presence of potassium hydroxide gave
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4d in 66% yield in Scheme 5. Treatment of isonicotinic acid 26 with aminoguanidine hydrochloride
yielded 3ꢀaminoꢀ5ꢀ(4ꢀpyridyl)ꢀ1Hꢀ1,2,4ꢀtriazole 27b in 92 % yield. Acylation of the nitrogen atom
on the 1,2,4ꢀtriazole of 27b, and the subsequent rearrangement of the acyl group to the amino group
by heating, gave the amide derivative 24f in 91% yield. Reduction of the amide with BH
3
afforded
24e in 69 % yield, as shown in Scheme 5.
[
Scheme 5]
Introduction of Substituents into Phenyl Moiety
The synthetic routes to prepare 4ꢀpyridylꢀ1Hꢀ1,2,4ꢀtriazole derivatives are shown in Schemes 6ꢀ10.
Several anilines were purchased and the other anilines were prepared as follows. Treatment of 23a
with various anilines gave desired products exhibited in Table 4, 28a–i in 22 – 74% yield,
respectively, as shown in Scheme 6.
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[Scheme 6]
Furthermore, treatment of 23a with the other various anilines 29 gave desired products 30a–q in
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9 – 63% yield, respectively, shown in Scheme 7.
[
Scheme 7]
Because 30i demonstrated potent GRK2 inhibitory activity (vide infra) and was required for
several further studies, an alternative synthetic route to 4ꢀpyridylꢀ1Hꢀ1,2,4ꢀtriazole derivatives,
having an amide group on the RHS, was investigated. Carboxylic acid bearing benzoate 16, which
was utilized for synthesis of the other chemotype, was condensated with benzamidrazone 22 by use
of WSC and HOBt to produce 31. After removal of the trifluoroacetyl group, formation of the
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Hꢀ1,2,4ꢀtriazole ring, under acidic conditions, followed by hydrolysis of the ester group, gave the
key intermediate 34, having a carboxylic acid. Amide analogues 30h and 30i were prepared by
condensation with WSC and HOBt in good yields.
[
Scheme 8]
Several anilines which were not purchased were prepared as follows in Scheme 9.
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3ꢀNitrobenzamides 39i, 39j and 39l were prepared by the condensation of 3ꢀnitrobenzoic acid 36
with the appropriate amines in 57% – quantitative yield. Alkylation of 3ꢀnitrophenol 37 using
phenylethylbromide and potassium carbonate gave 3ꢀnitrobenzene bearing ether 39c in 83% yield,
and the treatment of 3ꢀnitrophenylisocyanate 38 with benzylamine obtained the corresponding
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ꢀnitrobenzene having a urea bond 39p in 97% yield. Hydrogenation of the nitro group of these
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compounds 39 under mild conditions, by use of reduced iron in the presence of CaCl in ethanol,
afforded anilines 29 in 89% – quantitative yield. Alternatively, 29i, 29k and 29q were directly
obtained by use of WSC and HOBt from 3ꢀaminobenzoic acid 35a–b and various amines in 42 –
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4% yield.
[Scheme 9]
The synthesis of 30n and 30o is shown in Scheme 10. The 1Hꢀ1,2,4ꢀtriazole derivative 40 was
prepared in a similar fashion to that shown in Scheme 6 and 7. The nitro group of 40 was converted
into an amino group under the aboveꢀmentioned mild reductive conditions, to maintain the chlorine
atom, in 85% yield. Finally, the condensation of 41 with the corresponding carboxylic acids, and
subsequent hydrolysis to remove the acyl group attached to the 1,2,4ꢀtriazole ring, gave 30n and 30o
in 66 and 63% yield, respectively.
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[Scheme 10]
Introduction of Substituent into 4ꢀPyridine Ring
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Synthesis of 47a and 47b
The synthesis of 47a and 47b, containing 2ꢀmethylpyridinꢀ4ꢀyl and 3ꢀmethylpyridinꢀ4ꢀyl moieties,
respectively, is shown in Scheme 11. The methyl group at the 4ꢀposition of 2,4ꢀdimethylpyridine 42a
and 3,4ꢀdimethylpiridine 42b were lithiated selectively, and the lithiated products were subsequently
treated with dimethylamine and DMF to give enamines 43a–b. The treatment of the enamines 43a–b
4
with aqueous NaIO solution afforded 2ꢀmethyl or 3ꢀmethylpyridineꢀ4ꢀcarbaldehyde 44a–b in 85 –
92% yield. The aldehyde intermediates 44a–b were converted into 2ꢀmethyl or
3ꢀmethylpyridineꢀ4ꢀcarbonitorile 45a–b via oximes in two steps. 47a and 47b were prepared by
similar methods to those shown in Scheme 6 and 7.
[
Scheme 11]
Synthesis of 47c and 47d
The synthesis of 47c having 3ꢀhydroxymethylpyridinꢀ4ꢀyl, and 47d having
3
ꢀhydroxyethylpyridinꢀ4ꢀyl moieties, is shown in Scheme 12. Esterification of
4 2
pyridineꢀ3,4ꢀdicaroxylic acid 48 in 65% yield, and subsequent reduction using NaBH – CaCl gave
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pyridineꢀ3,4ꢀdimethanol 50 in 85% yield. Each monoꢀprotection of a hydroxyl group of 22 with
TBSCl in the presence of imidazole afforded 51 (15% yield) and 52 (25% yield), respectively. Swern
oxidation of 51, condensation with hydroxylamine, and subsequent dehydration, gave the desired
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ꢀsubstituted 4ꢀpyridinecarbonitrile 53 in 81% yield. Finally, treatment of 53 with hydrazide 20g in
ethanol in the presence of sodium ethoxide, generated by sodium hydride, resulted in the formation
of the 1Hꢀ1,2,4ꢀtriazole ring and removal of the TBS group, and affording 47c in 81% yield. Using
the Mitsunobu reaction, 52 was transformed into nitrile 54 in 85% yield. The 4ꢀpyridinecarbonitorile
5
5 was prepared from 54 in 40% yield in 5 steps, and 47d was prepared in a similar fashion to that of
7c in 78% yield.
4
[Scheme 12]
Synthesis of 47e
Selective protection of the nitrogen atom in the linker moiety of 47c in two steps gave 56 in 87%
yield. Subsequent carbamoylation of the hydroxyl function, and removal of the trifluoroacetyl group,
afforded 47e in 40% yield, as shown in Scheme 13.
[
Scheme 13]
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Synthesis of 68a
The synthesis of 68a, having 2ꢀacetamidepyridinꢀ4ꢀyl, is shown in Scheme 14. After acetylation of
7 at the initial step, oxidation of the methyl group by KMnO yielded the corresponding carboxylic
5
4
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60
acid 59. Thereafter, acid hydrazide 61 was prepared by condensation with monoꢀBocꢀprotected
hydrazine, followed by deprotection of the Boc group. On another front, the precursor of
4
ꢀmethylꢀ1,2,4ꢀtriazole, thioamide 64, was prepared from 62 in two steps. After treatment of 64 with
Meerwein reagent, the alkylated intermediate was reacted with acid hydrazide 61 to construct the
ꢀmethylꢀ1,2,4ꢀtriazole ring 65a. Chloromethylꢀ4ꢀmethylꢀ1,2,4ꢀtriazole intermediate 67a was
prepared by hydrolysis of the acetate group and chlorination of the hydroxyl group. Finally, synthesis
4
3
of target product 68a was completed by reaction with the aniline containing 2ꢀCF ꢀbenzyl amide 69a,
which was prepared from 3ꢀaminobenzoic acid 35a, and the corresponding substituted benzylamine,
in good yield.
[
Scheme 14]
Changing the 2ꢀsubstitution of the pyridine ring was performed as follows in Scheme 15. At first,
the acid hydrazide bearing 2ꢀchloropyridine 71 was prepared from methyl 2ꢀchloroisonicotinate 70.
The thioamide containing ethyl ester 74 was prepared from ethyl chloroglyoxylate 72 in two steps.
The 4ꢀMethylꢀ1,2,4ꢀtriazole intermediate bearing ethyl ester 75 was obtained as described previously
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in Scheme 14, but with improved yield (63%). After reduction of the ester by use of NaBH and
2
CaCl gave hydroxy methyl, introduction of various alkyl groups at the 2ꢀposition of the pyridine
ring furnished the 2ꢀalkylaminopyridine derivatives 66b–e in moderate yield. The alternative
synthesis of the target compounds is shown as follows. Reaction of the aldehyde 77b–e, derived
from the corresponding alcohol 66b–e, and the aniline containing 2ꢀFꢀ6ꢀFꢀbenzyl amide 69b, formed
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the imine intermediate, and subsequent reduction by NaBH
improved yield (68 – 94%).
4
achieved the target products 68b–e, with
[
Scheme 15]
Replacement of 5ꢀMembered Heterocyclic Ring System
Compounds bearing 5ꢀmembered heterocyclic aromatic rings other than the 1Hꢀ1,2,4ꢀtriazole ring
were designed in order to investigate the effects of the 1Hꢀ1,2,4ꢀtriazole ring system on biological
functions. Thus, compounds bearing thiazoles, 1,2,4ꢀ and 1,3,4ꢀoxadiazoles, pyrazoles, isoxazoles,
imidazoles, pyrroles and 4ꢀmethylꢀ1,2,4ꢀtriazoles were synthesized. These compounds were prepared
from the corresponding chloromethyl or aldehyde intermediates and the appropriate aniline
derivatives as follows.
Synthesis of 81a and 81b
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Treatment of 78a–b with LHMDS, followed by reaction with diethyl oxalate, gave
ꢀpyridylꢀ2,4ꢀdioxopropionates, which were treated with hydrazine to afford pyrazole derivatives
9a–b in 64 and 16% yield. Carboxylic acid 79a was converted into alcohol 80a, via the Weinreb
4
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amide, followed by reduction by two steps in 77% yield. Also, ester 79b was converted into alcohol
0b in low yield. These alcohols 80a–b were converted into chloromethyl intermediates, and were
treated with aniline 29i to provide 81a and 81b, respectively, in 42 – 63% yield, as shown in Scheme
6.
8
1
[
Scheme 16]
Synthesis of 81c
The Suzuki coupling reaction between the borate 82 and 4ꢀiodoꢀNꢀtritylimidazole (83) gave 84 in
8
3% yield. The 4ꢀ(4ꢀpyridyl)ꢀNꢀtritylimidazole 84 was lithiated, and subsequent reaction with DMF
4
gave 85 in 49% yield. Reduction of the aldehyde 85 using NaBH gave alcohol that was chlorinated
to afford the chloromethyl intermediate. Finally, the chloromethyl intermediate was treated with
aniline 29i to provide 81c in 54% yield, as shown in Scheme 17.
[
Scheme 17]
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Synthesis of 81d and 81e
ꢀPyridylꢀ2,4ꢀdioxopropionate prepared in Scheme 16 was treated with hydroxylamine to afford
4
4
the mixture of two isoxazole isomers. Reduction of the ester function using LiAlH , and subsequent
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chlorination of resulting alcohols afforded the chloromethyl intermediates 86 and 87, which were
separated by column chromatography on silica gel. Finally, both chloromethyl intermediates 86 and
8
7 were treated with aniline 29i to provide 81d (42% yield) and 81e (39% yield), respectively, as
shown in Scheme 18.
[
Scheme 18]
Synthesis of 81f
The imidazole ring, containing trifluoromethyl moiety 89, was constructed by using
3
,3ꢀdibromoꢀ1,1,1ꢀtrifluoroacetone (88) and 4ꢀpyridinecarboaldehyde in 31% yield. Conversion of
the trifluoromethyl moiety of 89 into a cyano group was carried out by treatment with 5% aqueous
4
ammonia solution in 84% yield. Reduction of the nitrile 90, using DIBAL and then NaBH to the
corresponding alcohol and subsequent chlorination afforded chloromethyl intermediate. Finally, the
chloromethyl intermediate was treated with aniline 29i to provide 81f in 60% yield, as shown in
Scheme 19.
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[Scheme 19]
Synthesis of 81g
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60
3
ꢀ(4ꢀPyridyl)pyrrole (92) was prepared from 4ꢀvinylpyridine (91) and tosylmethyl isocyanide
(
TOSMIC) using a [2+3] cycloaddition reaction in 49% yield. Introduction of the formyl function
into 57 was achieved by using the Vilsmeier reagent in low yield. Finally, reductive amination of 93
with 29i produced the desired product 81g in 21% yield, as shown in Scheme 20.
[
Scheme 20]
Synthesis of 81h
Treatment of 4ꢀaminopyridine 94 with 95 in the presence of acetic acid afforded formylated
1
ꢀ(4ꢀpyridyl)pyrrole (96) in 22% yield. Reductive amination of 96 with 29i yielded the desired
product 81h in 63% yield, as shown in Scheme 21.
[
Scheme 21]
Synthesis of 81i
3
The reaction of 4ꢀcyanopyridine 21 with NaN gave the corresponding tetrazole 97 in 34% yield,
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which was converted into 1,3,4ꢀoxadiazole 98 by treatment with ethyl oxalyl chloride in the presence
4 2
of 2,4,6ꢀcollidine in 27% yield. Reduction of the ester, using NaBH and CaCl , into alcohol,
followed by chlorination, afforded chloromethyl intermediate. Finally, the chloromethyl intermediate
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was treated with aniline 29i to provide 81i in 57% yield, as shown in Scheme 22.
[
Scheme 22]
Synthesis of 81j
Compound 99 was prepared in 15% yield by the reacting 4ꢀcyanopyridine 21 with hydroxylamine
(98% yield), followed by treatment with chloroacetyl anhydride. Compound 99 was treated with
aniline 29i to provide 81j in 71% yield, as shown in Scheme 23.
[
Scheme 23]
Synthesis of 81k
Compound 100 was prepared by condensation of 4ꢀ(bromoacetyl)pyridine with ethyl thiooxamate
in 59% yield. Reduction of the ester function using DIBAL, and subsequent chlorination of alcohol
2
using SOCl , afforded the chloromethyl intermediate 101, containing a thiazole ring. Finally,
compound 101 was treated with aniline 29i to provide 81k, as shown in Scheme 24.
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[
Scheme 24]
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Synthesis of 81l
Intermediate 103, containing a thiazole ring, was prepared by condensation of thioamide 102 with
,3ꢀdichloroacetone in 35% yield. Compound 103 was treated with trifluoroacetoanilide 104 in the
1
presence of potassium carbonate, and the trifluoroacetyl group was removed to provide 81l in 48%
yield, as shown in Scheme 25. Compound 104 was prepared from aniline 29i with trifluoroacetic
anhydride in 93% yield.
[Scheme 25]
Synthesis of 81m
The treatment of Weinreb amide 105 (Intermediate 80a in Scheme 16), with iodine in the presence
of ceric ammonium nitrate (CAN), afforded the corresponding 4ꢀiodopyrazole 106 in 82% yield.
Suzuki coupling of 106 with phenylboronic acid gave 107 in 21% yield. Reduction of Weinreb amide
1
07 with LiAlH
4
, and subsequent reductive amination with 29i, afforded 81m in 7% yield, as shown
in Scheme 26.
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[Scheme 26]
Introduction of Substituent into Phenyl Moiety of NꢀBenzylcarboxamide
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The substituted aroyl thiosemicarbazide was prepared by the reaction of isonicotinic hydrazide 25
with methyl isothiocyanate, followed by heating the obtained thiosemicarbazide in the presence of
sodium bicarbonate, giving 1,2,4ꢀtriazoleꢀ3ꢀthiol 108 in quantitative yield. Compound 108 was
treated with HNO
3
to take off the mercapto group, and afford 109 in 90% yield.
4
ꢀMethylꢀ1,2,4ꢀtriazole 109 was lithiated, and subsequent reaction with DMF gave 110 in 80% yield.
Reductive amination with 13 yielded the desired intermediate, 113, in 68% yield, as shown in
Scheme 27. Compound 110 can also be prepared as follows. Treatment of ethyl oxalyl chloride (72)
with methylamine and subsequent reaction of the resulting amide with Lawesson's reagent gave the
thioamide. Alkylation of the thioamide with Meerwein reagent, followed by condensation with
isonicotinic hydrazide (25), led to 4ꢀmethylꢀ1,2,4ꢀtriazole (111) in 60% yield. Reduction of the ester
4 2 2
functionality using NaBH and CaCl in 80% yield and subsequent oxidation using MnO afforded
the above aldehyde 110 in 96% yield, as shown in Scheme 27. Hydrolysis of the ester of 113 in 99%
yield and subsequent condensations with appropriate amines or anilines gave the desired compounds
115a–j in 60 – 98% yield, except for 115e, which was prepared from the aldehyde 110 by reductive
amination in 2 steps.
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[Scheme 27]
Because it was difficult to change the substitution of the 4ꢀposition of the 1,2,4ꢀtriazole after
formation of this ring system, appropriate amines had to be used at the initial step in Scheme 28. The
desired products 119a–f were prepared via the same procedure as described in Scheme 27.
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[
Scheme 28]
A novel 3ꢀring system (133) was constructed as follows. 3ꢀHydroxyisonicotinate ethyl ester 123
was prepared from 3,4ꢀpyridinecarboximide 120 in three steps. Homologation of 123 by the
Mitsunobu reaction with Zꢀprotected aminoethanol, and deꢀprotection of the Zꢀgroup, gave the
pyridineꢀfused 1,4ꢀoxazepine derivative 125. After thiocarbonylation by Lawesson's reagent,
derivatives 133a–b were produced by use of carboxamide containing acid hydrazides 132a–b, as
shown in Scheme 29.
[
Scheme 29]
Synthesis of pyrimidine derivatives is shown in Scheme 30. Pyrimidineꢀ4ꢀcarbohydrazide 136 was
prepared from 4ꢀmethylpyrimidine 134 in 2 steps. Treatment of the corresponding thioimidates,
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derived from ethyl oxalyl chloride 72 and appropriate amines, in 2 steps, as described in Schemes 26
and 27, gave 137a–b in moderate yield. The desired products 139a–d were synthesized by the same
methods (vide supra).
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[
Scheme 30]
Results and Discussion
SAR for Benzene Ring Substitutions of Acyclic Hydrazone Derivatives
[Table 1]
Benzene ring substitution SAR for hit compound 5 (acyclic hydrazone derivative) is summarized
in Table 1. These compounds were tested for inhibitory effects on kinase activity of GRK2,
Rhoꢀassociated protein kinase 2 (ROCK2), and Protein kinase C alpha (PKCα). ROCK2 is one of the
most significant kinases involved in vascular smooth muscle cell contraction. We found that our
compounds with potent ROCK2 inhibition induced vasorelaxing effects in rat aorta rings (data not
shown). PKCα is a member of the AGC family of kinases (PKA, PKG and PKC), regulating
modulation of membrane structure and skeletal reorganization, receptor desensitization,
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transcriptional control, cell growth and differentiation, and mediation of immune response, among
others.
Although selectivity against ROCK2 was not achieved, introduction of a chlorine atom (5a) and a
methoxy group (5e) on the metaꢀposition of the benzene ring led to ~2.5ꢀfold and ~35ꢀfold increases
in GRK2 inhibitory activity, respectively. However, introduction of substituents on the ortho and
paraꢀposition of the benzene ring significantly decreased GRK2 inhibitory activity. Therefore,
further optimization at the metaꢀposition of the benzene ring to optimize the activity and selectivity
was performed next.
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60
SAR for Benzene Ring Substitutions of Cyclic Hydrazone Derivatives
[
Table 2]
Effects of metaꢀposition modifications on the RHS of benzene ring 12a (cyclic hydrazone
derivative) are summarized in Table 2. Novel cyclic hydrazone derivatives (Figure 1) displayed both
GRK2 inhibitory activity and a desired trend in selectivity against ROCK2. Therefore, a variety of
substituents on the metaꢀposition of the benzene ring were introduced to increase GRK2 inhibitory
activity utilizing the SAR information in Table 1. The resulting compounds, except for
morpholinoꢀamide derivative 12f, show a dramatic increase in GRK2 inhibitory activity. In particular,
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12e shows not only potent GRK2 inhibitory activity, but also excellent selectivity against ROCK2.
Moreover, these cyclic hydrazone derivatives do not show affinity for PKCα. However, 12g exhibits
potent inhibitory activity towards GRK2, but does not show high selectivity over ROCK2.
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Linker Moiety Modification SAR for 4ꢀPyridylꢀ1,2,4ꢀTriazole Derivatives
[
Table 3]
Linker moiety SAR for hit compound 24a (4ꢀpyridylꢀ1,2,4ꢀtriazole derivative) is summarized in
Table 3. Displacement of the linker sulfur atom by an oxygen (hit compound 24b) reduced GRK2
inhibitory activity, while changing it to nitrogen (24c) led to ~30ꢀfold increase. Replacing carbon
with sulfur (24a to 24d) and nitrogen (24c to 24e) resulted in drastic decreases in GRK2 inhibition.
Amide derivative (24f) and methylene derivative (24g and 24h) showed low activity. Next, based on
this SAR information, further modification of the benzene ring of 24c was implemented.
Benzene Ring Substitution SAR for 4ꢀPyridylꢀ1,2,4ꢀTriazole Derivatives
[
Table 4]
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Benzene ring substitution SAR for 28a is summarized in Table 4. Introduction of a methoxy group
on the metaꢀposition of benzene ring (28c) showed almost no change in GRK2 inhibitory activity,
while a methoxy group in the ortho and paraꢀposition drastically decreased activity. Among
chloroꢀderivatives, the paraꢀposition derivative 28g showed the most potent activity. Furthermore,
the 3ꢀsulfonamide derivative 28h showed stronger inhibitory activity than the corresponding 4ꢀ
sulfonamide derivative 28i. While all compounds showed high levels of selectivity against PKCα, we
observed no improvement in ROCK2 selectivity. Next, further modifications of the 3ꢀposition of the
benzene ring were implemented based on this SAR information.
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[Table 5]
Initial SAR for benzene ring substitutions at the 3ꢀposition of 28a are summarized in Table 5.
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the RHS was preferable. Therefore, optimization of R was implemented as follows. 28a, 30a, 30b
and 30c introducing various alkoxy groups did not increase the inhibitory activity. Conversion of the
benzyloxy of 30b into benzylthio (30d), benzyl sulfonamide (30e) and benzylsulfone (30f) also did
not increase the inhibitory activity. However, several secondary amide derivatives (30g, 30h, 30i, 30j,
and 30k) undoubtedly gave rise to increases of inhibitory activity. In particular, the benzyl amide
derivative (30i) demonstrated the most potent inhibitory activity in Table 5. The crystal structure of
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human GRK2 in complex with 30i obtained in Takeda California (TCAL) (Figure 3) revealed the
compound formed the obligatory hinge interaction between the pyridine head group and the
backbone amide of Met274. It also made significant interactions with the side chain of the
DLGꢀmotif residue Asp335, and the backbone amide of Phe202, in the Pꢀloop. Additionally, the
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,2,4ꢀtriazole engaged in an interesting network of waterꢀmediated interactions. These interactions
contributed to increases in GRK2 inhibitory activity. This similar information has been already
reported by use of coꢀcrystallization between bovine GRK2 and CMPD101 (vide infra, 115h) by
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Thal D. M. et. al. in University of Michigan. On the other hand, a tertiary amide (30l) evidently
decreased the activity, compared with 30i. These results indicate that the direction of the carbonyl
group of the amide plays an important role and/or the orientation of the benzyl part is important in
terms of pushing up the Pꢀloop to enhance inhibition of GRK2. Revised amide derivatives (30m, 30n
and 30o) and a urea derivative (30p) apparently maintained the potent activity. Judging from the
SAR information between 30m and 30n, the chlorine atom at R2 does not affect GRK2 inhibitory
activity. Furthermore, 30o having reversed amide of 30i which showed most potent activity in Table
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maintained the potent activity. Introduction of a methylene (30q) between the amide and the central
benzene ring markedly decreased the activity.
Figure 3. Section of the Xꢀray crystal structure of human GRK2 in complex with the small molecule
inhibitor 30i
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SAR for Substituent on 4ꢀPyridine Ring
[
Table 6]
SAR for substitutions on the 4ꢀpyridine ring of 24c or 30i are summarized in Table 6. As shown in
Tables 3, 4 and 5, a series of 4ꢀpyridylꢀ1Hꢀ1,2,4ꢀtriazole derivatives showed potent GRK2 inhibitory
activity, but also simultaneous inhibition of CYP3A4. As listed in Table 6, to reduce the inhibitory
potency towards CYP3A4, a methyl group was introduced at the C(2) position of the pyridine ring of
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4c (47a). As a result, although the inhibition of CYP3A4 was decreased, the GRK2 inhibitory
activity was also decreased. In further modifications, the introduction of methyl (47b),
hydroxymethyl (47c), 2ꢀhydroxyethyl (47d) and carbamoyloxymethyl (47e) groups at the C(3)
position of the pyridine ring of 24c or 30i also resulted in decreases in GRK2 inhibitory potency.
These compounds also still showed potent inhibition of CYP3A4.
[
Table 7]
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Further investigation to decrease the inhibition of CYP3A4 by introducing substitutions at the
C(2) position of the pyridine ring by use of 4ꢀmethylꢀ1,2,4ꢀtriazole derivatives (vide infra: the SAR
of 5 membered heterocyclic ring system including introduction of methyl group at the 4ꢀposition of
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,2,4ꢀtriazole) was performed as follows. These results were shown in Table 7. Introduction of
acetamide (68a), methylamine (68b) and benzylamine (68c) decreased the inhibition of CYP3A4
while keeping moderate inhibitory activity towards GRK2. However, extending the alkyl portion of
the benzylamine (68d and 68e) showed a trend of increased inhibition of CYP3A4.
SAR for 5ꢀMembered Heterocyclic Ring System Replacement
[Table 8]
SAR for 5ꢀmembered heterocyclic ring system replacement of 30i are summarized in Table 8.
Although replacement of the 1Hꢀ1,2,4ꢀtriazole ring with a pyrazole (81a), 4ꢀmethylpyrazole (81b),
imidazole (X = N, Y = N, Z = C, 81c) and two isoxazole isomers (81d and 81e) resulted in
equipotent inhibition of GRK2 as 30i, the derivatives also showed potent inhibition of ROCK2 and
CYP3A4. Interestingly, 81c, containing a 4ꢀ(4ꢀpyridyl)imidazole, showed more than 20ꢀfold greater
inhibition of GRK2 as 81f (X = N, Y = C, Z = N), containing a 2ꢀ(4ꢀpyridyl)imidazole. This result
suggests that the location of the nitrogen atom plays a crucial role for inhibition of GRK2. It is
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worthwhile to notice that introduction of a methyl group at the N(4) position of the 1,2,4ꢀtriazole ring
(
115e), which is described at a later point (Table 9), results in potent inhibition of GRK2 and lower
activity against ROCK2, PKCα and CYP3A4. In addition to this result, introduction of a methyl
group at the C(4) position of the pyrazole ring (81b) maintains potent inhibition of ROCK2 and
CYP3A4. Based on the SAR information for the 1,2,4ꢀtriazole, pyrazole and imidazole derivatives,
the nitrogen atom at the Y position of the 5ꢀmembered heterocyclic ring system might be important
to show the activity. However, the pyrrole derivative bearing only a nitrogen atom at the Y position
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(81g) exhibited about a 20ꢀfold loss in potency for GRK2. Moreover, Nꢀlinked 4ꢀpyridylꢀpyrrole
derivative (81h) gave a dramatic decrease in potency for GRK2. Further investigation of the
5ꢀmembered heterocyclic ring system to generate two kinds of oxadiazoles and thiazoles did not
maintain the same inhibitory activity (81i, 81j, 81k and 81l). Finally, incorporation of a bulky group
(
phenyl group, 81m) at the 4ꢀposition of the pyrazole ring of 81a decreased the activity. Next, further
modifications of the phenyl moiety of the Nꢀbenzylcarboxamide of the 1Hꢀ1,2,4ꢀtriazole derivative
0i and the 4ꢀmethylꢀ1,2,4ꢀtriazole derivative 115e were implemented based on this SAR
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information.
SAR for Phenyl Moiety Modification of NꢀBenzylcarboxamide
[
Table 9]
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