3,5-Diarylazoles as novel and selective inhibitors of protein kinase D

Article abstract of DOI:10.1016/j.bmcl.2011.01.014

The synthesis and preliminary studies of the SAR of novel 3,5-diarylazole inhibitors of Protein Kinase D (PKD) are reported. Notably, optimized compounds in this class have been found to be active in cellular assays of phosphorylation-dependant HDAC5 nuclear export, orally bioavailable, and highly selective versus a panel of additional putative histone deacetylase (HDAC) kinases. Therefore these compounds could provide attractive tools for the further study of PKD / HDAC5 signaling.

Full text of DOI:10.1016/j.bmcl.2011.01.014

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1447–1451
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3,5-Diarylazoles as novel and selective inhibitors of protein kinase D
Gabriel G. Gamber a, , Erik Meredith a, Qingming Zhu a, Wanlin Yan a, Chang Rao a, Michael Capparelli a,
⇑
Robin Burgis a, Istvan Enyedy a, Ji-Hu Zhang a, Nicolas Soldermann a, Kimberley Beattie a, Olga Rozhitskaya a,
Keith A. Koch b, Nikos Pagratis b, Vinayak Hosagrahara a, Richard B. Vega a, Timothy A. McKinsey b,
Lauren Monovich a
a Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA
b Gilead Colorado, Inc., Boulder, CO 80301, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and preliminary studies of the SAR of novel 3,5-diarylazole inhibitors of Protein Kinase D
(PKD) are reported. Notably, optimized compounds in this class have been found to be active in cellular
assays of phosphorylation-dependant HDAC5 nuclear export, orally bioavailable, and highly selective ver-
sus a panel of additional putative histone deacetylase (HDAC) kinases. Therefore these compounds could
provide attractive tools for the further study of PKD / HDAC5 signaling.
Received 20 May 2010
Revised 5 January 2011
Accepted 6 January 2011
Available online 11 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Kinase inhibitor
PKD
Protein kinase D
Histone deacetylase
Heart failure
Cardiac hypertrophy
Cardiac hypertrophy is a common response to stress signals in
the heart that arise from a variety of cardiovascular disorders,
including myocardial infarction and chronic hypertension.1 Pro-
longed hypertrophy has been recognized as a major determinant
of heart failure,2 a disease of increasing prevalence and high mor-
bidity and mortality.
moderate activity (Figure 1). The synthesis and preliminary SAR
studies of benzamide analogs of this chemotype, which resulted
in the development of potent, selective, and orally available small
molecule inhibitors of PKD, are reported herein.7
The synthesis of 3,5-diarylazole PKD inhibitors is shown in
Schemes 1–5. The 3,5-diarylpyrazole core8 of compounds 4a
(Scheme 1), 4b (Scheme 4), and 4c (Scheme 5) was constructed via
Recently, it has been reported that histone deacetylase 5
(HDAC5), acts as a signal responsive repressor of cardiac hypertro-
phy when it is co-localized with transcription factors, such as
MEF2, in the nucleus.3 In response to stress signals, phosphoryla-
tion of HDAC5 by one or more HDAC kinases triggers its export
from the nucleus into the cytoplasm and releases the repression
of MEF2 responsive genes.4 This is accompanied by an increase in
cardiomyocyte cell size, assembly of sarcomeres, and the activation
of a ‘fetal’ program of gene expression, leading to pathological car-
diac hypertrophy. Protein kinase D 1 (PKD1) has been implicated as
an HDAC kinase mediating the subcellular localization and signal-
ing of HDAC5 in cardiac tissue.5,6 Therefore, inhibition of PKD1
would be predicted to block the nuclear export of HDAC5 and blunt
the hypertrophic response to stress in cardiac tissue, potentially
providing a novel therapy for heart failure.
Claisen condensation of
a suitably substituted acetophenone
(2a–c) and dimethyl isophthalate3, followed by further condensation
of theresultant diketone with hydrazine. Analogously, 3,5-diarylisox-
azoles9 could be formed by reaction of the diketone products of the
Claisen condensation with hydroxylamine (not shown). However,
this approach generally resulted in mixtures of 3,5-isoxazole
regioisomers. Therefore, the 3,5-diarylisoxazole core of compounds
10a (Scheme 3) and 10b (Scheme 5) was constructed through the
[3+2] cycloaddition of nitrile N-oxides (formed in situ from hydroxi-
moyl chlorides 8a–b) with substituted phenylacetylenes 9a–b, which
provided isoxazoles as single, predictable, isomers.
H
H
N
O
N N
High throughput screening against recombinant PKD1 identi-
fied 3,5-diarylpyrazole 1 as a novel kinase inhibitor scaffold with
N
MeN
1
PKD1 IC50 = 2.3 uM
⇑
Corresponding author.
E-mail address: gabriel.gamber@novartis.com (G.G. Gamber).
Figure 1. Initial 3,5-diarylazole hit.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.014

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G. G. Gamber et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1447–1451
HATU mediated amide bond formation (Scheme 1).10 Compounds
7a–b were synthesized by a further Suzuki–Miyaura coupling of
the bromide of 6i with the respective aryl boronic acid (Scheme
2).11
Synthesis of the piperazine-containing isoxazole compounds
14a–c (Scheme 3) required protection of bromo-acid 10a as a
methyl ester (11) before installation of the piperazine ring using
Buchwald–Hartwig amination12 conditions to provide intermedi-
ate 12. By analogy to the route illustrated in Scheme 1, isoxazole
ester 12 was saponified to provide carboxylic acid 13, which could
be coupled with amines to provide amides 14a–c. Chiral, non-race-
mic compounds 15a–b were resolved from the racemate 14b by
standard HPLC techniques on a chiral stationary phase. Alterna-
O
R1
N
O
H
R1
N
N N
(a), (b)
MeN
MeO
R2
2a
+
O
O
MeN
4a, R2 = OMe or OEt
5, R2 = OH
OMe
(c)
(d)
6a-j , R2 = NHR
3
Scheme 1. R and R1 are defined in Table 1. Reagents: (a) NaH, THF or NaOEt, EtOH;
(b) hydrazine, EtOH; (c) LiOH, THF:H2O; (d) HATU, R-NH2, DiPEA, DMF.
tively, dehydration of chiral, non-racemic D-alanine amide 14c pro-
ceeded with complete retention of enantiopurity to provide 15a.13
In addition to providing convenient access to enantiomerically
pure (>98% ee) materials from a chiral pool source, this orthogonal
strategy also allowed the assignment of the absolute stereochem-
istry of the aminonitrile enantiomers.
O
H
R1
N N
N
H
N
MeN
6i, R1 = Br
(a)
7a, R1 = Ph; 7b, R1 = 4-Py
Pyrazole compounds 18a–d, in which the piperazine ring has
been replaced with a benzylic amine, were synthesized from bro-
mo ester 4b (Scheme 4). Amidation of the methyl ester was med-
iated by trimethylaluminum14 to afford bromo-amide 16.
Lithium–halogen exchange followed by quenching with DMF pro-
vided aldehyde 17, which was converted to benzylic amines 18a–d
by reductive amination. The benzylic amine of pyrazole 20
(Scheme 5, X = NH) was installed through an alternate sequence
starting from dimethyl acetal 4c. Hydrolysis of the acetal with
aqueous acid provided aldehyde 19, which was converted to a ben-
zylic amine by reductive amination. Benzylamine-substituted isox-
azole 20 (Scheme 5, X = O), was synthesized from compound 10b
by radical bromination of the tolyl group with NBS, followed by
trapping of the benzyl bromide intermediate with amines.
Saponification of the methyl ester of 20 (Scheme 5, X = N or O)
to carboxylate 21, followed by amide bond formation provided
Scheme 2. Reagents and conditions: (a) ArylB(OH)2, Pd(dppf)Cl2, K2CO3 (aq), 1:10
EtOH in PhMe, 110 °C.
HO
N
O
O
Cl
OH
O N
R2
(a)
8a
Br
+
10a , R2 = -OH
11 ,R2 = -OMe
(b)
Br
9a
(c)
14b , R2 =
O
CN
N
N
H
O N
compounds 22a–d. Further transformation of the D-alanine amide
R2
+
15b
15a
(f)
of 22d by dehydration with trifluoroacetic anhydride provided
aminonitrile 23, in which the benzylic amine was simultaneously
protected by trifluoroacetic anhydride. The resulting trifluoroace-
tamide could be removed cleanly and chemoselectively by treat-
ment with sodium borohydride in methanol15 to provide
compounds 24a–c in excellent enantiopurity (>98% ee). Compound
24d, the enantiomer of compound 24c, was synthesized by the
N
12 , R2 = -OMe
13 , R2 = -OH
(d)
(e)
14c R2 =
14a-c , R2 = -NHR
NH2
N
H
O
(g)
same methods and in similar enantiopurity by substituting L-ala-
nine amide in the amide bond formation step from acid 21.
Scheme 3. Unless otherwise indicated, R is defined in Table 1. Reagents and
conditions: (a) DiPEA, CH2Cl2; (b) TMSCl, MeOH; (c) Pd(OAc)2, BINAP, Cs(CO3)2,
PhMe, reflux; (d) LiOH, THF:H2O; (e) HATU, R-NH2, DiPEA, DMF; (f) Chiral HPLC,
Chiralpak IA column, 0.2% TEA:1% EtOH; ACN; (g) TFAA, Et3N.
Preliminary structure–activity relationship (SAR) data is pro-
vided in Tables 1 and 2. In general, equivalently substituted pyra-
zole- and isoxazole-derived compounds show similar potency as
PKD1 inhibitors (cf. Table 1, 6c, 14a; Table 2, 24a, 24b).
In order to improve the moderate PKD1 inhibitory potency ob-
served with 1 (Figure 1), a series of varied benzamide derivatives
were synthesized (Table 1, 6a–i). Alkyl amides (6a–b) show po-
O
H
O
N N
R2
(a), (b)
O
3
+
H
Br
Br
tency against PKD1 similar to 1. However, an a-aminonitrile amide
4b, R2 = -OMe
16 , R2 = -NHi Pr
2b
(c)
(6c) provides a significant (ꢀ18-fold) improvement in potency.
While the nature of the interaction of the nitrile with the PKD1
binding site is unknown, the effect of this substituent appears to
be quite specific, as amide substituents bearing other polar and/
or hydrogen bond-accepting functionality (6e–h), or even an addi-
tional methylene spacer (b-aminonitrile 6d), uniformly fail to pro-
N N
N
H
R4
(d)
H
17 , R4 = (O=)
18a -d, R4 = H-, R3NH-
(e)
vide increases in potency similar to the
some cases, even detrimental to PKD1 inhibitory activity compared
to a simple alkyl amide (6a–b). The beneficial effect of the -ami-
a-aminonitrile, and are, in
Scheme 4. Substituent R3 is defined in Table 2. Reagents and conditions: (a) NaH,
THF; (b) hydrazine, DiPEA; (c) AlMe2, NH2(iPr); (d) i) BuLi, ꢁ78 °C; (ii) DMF, ꢁ78 °C;
(e) NH2-R3, Na(OAc)3BH.
a
nonitrile is also observed with the 3,5-diarylisoxazole core (14a,
also cf. 22a, 22b).
Further elaboration of pyrazole ester 4a to compounds 6a–j was
achieved through saponification to carboxylate 5, followed by
Notably, a modest but consistent increase in potency is ob-
served with addition of a-branching on the aminonitrile substitu-

G. G. Gamber et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1447–1451
1449
O
H
O
HO
Cl
N N
N
O
OMe
(a), (b)
+
3
+
OMe
R1
(MeO)2CH
4c, R1 = (MeO)2CH-
19 , R1 = (O)CH-
2c
(c)
9b
8b
(j)
(d)
O
O
O
R3
X N
X N
R3
O N
R3
CN
(g)
(k)
OMe
N
H
HN
N
R5
22d , R2 =
20 , R2 = -OMe
21 , R2 = -OH
23 , R5 = TFA-
24a-c , R5 = H
NH2
10b
(e)
(f)
(h)
N
H
22a -d, R2 = -NHR
O
Scheme 5. Unless otherwise indicated, substituents R, R3, and X are defined in Table 2. Reagents and conditions: (a) NaH, THF; (b) hydrazine, DiPEA; (c) TFA, H2O:DCM; (d)
NH2-R3, Na(OAc)3BH, DCM; (e) LiOH, H2O:THF; (f) NH2-R, HATU, DiPEA, DMF; (g) TFAA, TEA, THF; (h) NaBH4, MeOH; (j) DiPEA, DCM; (k) (i) NBS, AIBN, CCl4, reflux; (ii) NH2-R3.
Table 1
PKD1 inhibitory activity, microsomal stability, and solubility of 3,5-diarylazolesa
O
R1
N
R
X N
N
H
MeN
Compd
-NHR
R1-
-H
-X-
PKD1 IC50
2.5
(
lM)
RLM t1/2 (min)
Sol (
lM)
N
H
6a
–NH–
4
330
6b
6i
-H
–NH–
–NH–
–NH–
–NH–
1.0
3
270
40
N
H
-Br
0.28
0.98
0.20
5
N
H
7a
7b
-Ph
-4-Py
22
51
<10
<10
N
H
N
H
N
H
CN
CN
6c
6j
-H-
-Br
–NH–
–NH–
0.14
3
9
50
N
H
0.087
<10
N
H
6d
6e
6f
-H
-H
-H
–NH–
–NH–
–NH–
0.75
6.9
4
650
760
170
CN
N
H
4
OMe
N
H
1.6
11
OH
N
H
6g
-H
–NH–
2.0
11
>960
NMe2
N
H
CF
3
6h
-H
-H
–NH–
–O–
1.1
2
170
<10
N
H
CN
CN
CN
14a
0.20
<2
15a
15b
-H
-H
–O–
–O–
0.060
0.43
<2
<2
<10
<10
N
H
N
H
a
All in vitro assay results reported as mean of n P 2 experiments.

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G. G. Gamber et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1447–1451
Table 2
PKD1 inhibitory activity, microsomal stability, and solubility of 3,5-diarylazolesa
O
R
X N
N
H
H
N
R3
Compd
-NHR
(R3)NH-
-X-
PKD1 IC50
0.10
(l
M)
RLM t1/2 (min)
Sol (lM)
H
N
18a
–NH–
107
>970
910
N
H
H
N
18b
18c
–NH–
–NH–
0.43
0.39
67
16
N
H
MeO
H
N
220
N
H
H
N
18d
24a
24b
–NH–
–NH–
–O–
6.0
21
<10
N
H
F3C
H
N
0.0037
0.009
>198
54
>990
>870
N
H
CN
CN
H
N
N
H
H
N
O
22a
22b
22c
–O–
–O–
–O–
0.22
41
140
170
70
N
H
H
N
N
H
CN
O
O
0.019
0.049
>176
40
H
N
N
H
CN
CN
H
N
O
O
24c
24d
–O–
–O–
0.0055
0.095
144
58
240
110
N
H
H
N
N
H
CN
a
All in vitro assay results reported as mean of n P 2 experiments.
Table 3
ent (cf. Table 1, 14a, 15a; Table 2, 22b, 24c). This effect is stereo-
specific, with the R-nitrileamide inhibiting PKD1 more potently
than the S-enantiomer (cf. Table 1, 15a, 15b; Table 2, 24c, 24d).
Consistent with this observation,
served potency intermediate to the two mono-methyl enantiomers
PKD inhibitor 24c is highly selective against a panel of additional putative hHDAC
kinases
Kinase
% inhibition at 1
l
Ma
IC50
(l
M)
b
a,
a0-dimethylation provides ob-
hPKD1
hPKD2
hPKD3
hCaMKId
94
85
0.0055
0.048
0.017
(cf. Table 2, 22c, 24c and 24d).
With compounds bearing a methylpiperazine moiety (Table 1)
poor metabolic stability in liver microsomes was generally
observed,16 presumably due to oxidation of the piperazine ring.
This metabolism can be moderated by the introduction of sterically
bulky R1 groups on the aromatic core ortho- to the piperazine ring,
and, in some cases, these modifications also provide an increase in
PKD1 inhibitory potency (cf. 6b, 6i, 7a–b; 6c, 6j). However, this
solution is less than ideal, as increasing the size of R1 also results
in reduced solubility,17 to undetectable levels in some cases.
Modification of the linker between the basic amine moiety and
the aromatic core proved to be a more effective solution to balanc-
ing potency, metabolic stability, and solubility. Notably, when the
piperazine (Table 1) is replaced with a benzylic amine (Table 2) a
significant improvement in all three of these parameters is ob-
served (cf. 6b, 18a; 14a, 22b; 15a, 24b and 24c). These effects
might be partially due to the increased basicity of the benzylic
amine versus the aryl piperazine, as, consistent with this interpre-
tation, substituents that would reduce the pKa of the benzylic
amine, such as cyclopropyl (18c), methoxyethyl (18b), or trifluoro-
ethyl (18d), generally result in attenuated inhibition of PKD1, re-
duced metabolic stability, and reduced solubility.
0
rCaMKII
hCaMKIIb
hCaMKIId
hCaMKIV
hMARK1
hSIK1
a
ꢁ2
22
37
26
29
15
ꢁ4
ꢁ9
ꢁ2
>7.9c
hGRK5
hPKCd
hPKC
e
a
Mean of n = 2 measurements, inhibition assays conducted by Milllipore (http://
www.millipore.com/drugdiscovery/dd3/KinaseProfiler).
Mean of n P 2 experiments.
b
c
Values of 5.7 lM, >10 lM were obtained.
Combination of the R-a-aminopropionitrile amide substituent
with benzylic basic amine substituents identified through our
SAR studies resulted in compounds with low nanomolar potencies
against PKD1, good in vitro metabolic stability, and moderate to
excellent solubility (24a–c).
Isoxazole compound 24c was further profiled against a panel of
kinases that have been implicated in the regulation of HDAC sub-

G. G. Gamber et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1447–1451
1451
Table 4
6a–j, 7a–b, 14a–b, 15a–b, 18a–d, 22a–c, and 24a–d) associated
with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2011.01.014.
PKD1 inhibitors block HDAC5 nuclear export (EC50
>1
lM observed with other compounds in Tables 1
and 2)a
Compd
EC50 (lM)
6j
0.68
0.57
0.53
0.46
0.24
References and notes
15a
24a
24b
24c
1. (a) McKinsey, T. A.; Kass, D. A. Nat. Rev. Drug Disc. 2007, 6, 617; (b) Mann, D. L.;
Bristow, M. R. Circulation 2005, 111, 2837; (c) MacLellan, W. R.; Schneider, M. D.
Ann. Rev. Physiol. 2000, 62, 289.
2. (a) Gosse, P. J. Hypertens. 2005, 23, S27; (b) Frey, N.; Katus, H. A.; Olson, E. N.;
Hill, J. A. Circulation 2004, 109, 1580; (c) Gardin, J. M.; Lauer, M. S. JAMA 2004,
292, 2343; (d) Frey, N.; Olson, E. N. Ann. Rev. Physiol. 2003, 65, 45.
3. (a) Zhang, C. L.; McKinsey, T. A.; Chang, S.; Antos, C. L.; Hill, J. A.; Olson, E. N. Cell
2002, 110, 479; (b) Chang, S.; McKinsey, T. A.; Zhang, C. L.; Richardson, J. A.;
Hill, J. A.; Olson, E. N. Mol. Cell. Biol. 2004, 24, 8467.
4. Grozinger, C. M.; Scheiber, S. L. Proc. Nat. Acad. Sci. 2000, 97, 7835.
5. (a) Monovich, L.; Vega, R. B.; Meredith, E.; Miranda, K.; Rao, C.; Capparelli, M.;
Lemon, D. D.; Phan, D.; Koch, K. A.; Chapo, J. A.; Hood, D. B.; McKinsey, T. A. FEBS
Lett. 2010, 584, 631; (b) Vega, R. B.; Harrison, B. C.; Meadows, E.; Roberts, C. R.;
Papst, P. J.; Olson, E. N.; McKinsey, T. A. Mol. Cell. Biol. 2004, 24, 8374; (c) Rykx,
A.; De Kempe, L.; Mikhalap, S.; Vantus, T.; Seufferlein, T.; Vandenheede, J. R.;
Van Lint, J. FEBS Lett. 2003, 546, 81.
a
Mean of n P 2 experiments.
Table 5
Pharmacokinetic parameters for compound 24ca
AUC0-8h (nM h)
CL (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
840 80
93
6.0 1.4
1.2 0.1
38
9
F (%)
6. McKinsey, T. A. Cardiovasc. Res. 2007, 667.
a
Sprague–Dawley rat PK. Animals were dosed
7. For other small molecule inhibitors of PKD, see: (a) Meredith, E. L.; Ardayfio, O.;
Beattie, K.; Dobler, M.; Enyedy, I.; Gaul, C.; Hosagrahara, V.; Jewell, C.; Koch, K.;
Lee, W.; Lehmann, H.; McKinsey, T. A.; Miranda, K.; Pagratis, N.; Pancost, M.;
Patnaik, A.; Phan, D.; Plato, C.; Qian, M.; Rajaramna, V.; Rao, C.; Rozhitskaya, O.;
Ruppen, T.; Shi, J.; Siska, S. J.; Springer, C.; van Eis, M.; Vega, R. B.; von Matt, A.;
Yang, L.; Yoon, T.; Zhang, J.-H.; Zhu, N.; Monovich, L. G. J. Med. Chem. 2010, 53,
5422; (b) Meredith, E. L.; Beattie, K.; Burgis, R.; Capparelli, M.; Chapo, J.;
DiPietro, L.; Gamber, G.; Enyedy, I.; Hood, D. B.; Hosagrahara, V.; Jewell, C.;
Koch, K. A.; Lee, W.; Lemon, D. D.; McKinsey, T. A.; Miranda, K.; Pagratis, N.;
Phan, D.; Plato, C.; Rao, C.; Rozhitskaya, O.; Soldermann, N.; Springer, C.; van
Eis, M.; Vega, R. B.; Yan, W.; Zhu, Q.; Monovich, L. G. J. Med. Chem. 2010, 53,
5400; (c) Sharlow, E. R.; Giridhar, K. V.; LaValle, C. R.; Chen, J.; Leimgruber, S.;
Barrett, R.; Bravo-Altamirano, K.; Wipf, P.; Lazo, J. S.; Wang, Q. J. J. Biol. Chem.
2008, 283, 33516; (d) Gschwendt, M.; Kittstein, W.; Johannes, F.-J. FEBS Lett.
1998, 421, 165; (e) Gschwendt, M.; Dietrich, S.; Rennecke, J.; Kittstein, W.;
Mueller, H.-J.; Johannes, F.-J. FEBS Lett. 1996, 392, 77; (f) Singh, R.; Li, H.; Zhao,
H.; Payan, D. G.; Kolluri, R.; Tso, K.; Ramphal, J.; Gu, S. WO 2009012421.; (g)
Yokoyama, S. WO 2008088006.; (h) Raynham, T. M.; Hammonds, T. R.; Charles,
M. D.; Pave, G. A.; Foxton, C. H.; Blackaby, W. P.; Stevens, A. P.; Ekwuru, C. T.
WO 2008074997.; (i) Raynham, T. M.; Hammonds, T. R.; Gilliat, J. H.; Charles,
M. D.; Pave, G. A.; Foxton, C. H.; Carr, J. L.; Mistry, N. S. WO 2007125331.
8. For reviews of pyrazoles, see: (a) Yet, L. In Comprehensive Heterocyclic Chemistry
III; Joule, J. A., Ed.; Elsevier: Oxford, 2008; Vol. 4, p 1; (b) Makino, K.; Kim, H. S.;
Yoshihisa, K. J. Heterocycl. Chem. 1999, 36, 321; (c) Elguero, J. In Comprehensive
Heterocyclic Chemistry II; Shinkai, I., Ed.; Elsevier: Oxford, 1996; Vol. 3, p 1; (d)
Kost, A. N.; Grandberg, I. I. Adv. Heterocycl. Chem. 1966, 6, 347.
9. For reviews of isoxazoles, see: (a) Giomi, D.; Cordero, F. M.; Machetti, F. In
Comprehensive Heterocyclic Chemistry III; Joule, J. A., Ed.; Elsevier: Oxford, 2008;
Vol. 4, p 365; (b) Pinho e Mehlo, T. M. V. D. Curr. Org. Chem. 2005, 9, 925; (c)
Sutharchandevi, M.; Murugan, R. In Comprehensive Heterocyclic Chemistry II;
Shinkai, I., Ed.; Elsevier: Oxford, 1996; Vol. 3, p 221; (d) Wakefield, B. J.; Wright,
D. J. Adv. Heterocycl. Chem. 1979, 25, 147; (e) Kochetkov, N. K.; Sokolov, S. D.
Adv. Heterocycl. Chem. 1963, 2, 365.
10. Speicher, A.; Klaus, T.; Eicher, T. J. Prakt. Chem. 1998, 340, 581.
11. (a) Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457; (b) Bellina, F.; Carpita, A.;
Rossi, R. Synthesis 2004, 2419; (c) Martin, R.; Buchwald, S. L. Acc. Chem. Res.
2008, 41, 1461.
either iv (2 mg/kg, n = 2) or po (5 mg/kg, n = 3) with
compound in 10% NMP, 10% Cremophor, 80% pH 4.63
buffer vehicle. PK parameters derived from iv dosing,
and are reported as mean SD.
cellular localization and signaling (Table 3).6,18 This compound was
found to inhibit all PKD family members with nanomolar IC50s,
although with 9-fold and 3-fold selectivity for PKD1 versus PKD2
and PKD3, respectively. Notably, compound 24c appears to be
>1000-fold selective versus other members of the panel. Further,
in a wider panel of 230 kinases, compound 24c inhibited only 4
with >50% efficacy at 1 lM. Therefore, the observed sub-micromo-
lar inhibition of the phosphorylation-dependant nuclear export of
GFP-HDAC5 over-expressed in neonatal rat ventricular myocytes
(NRVMs)5a,7a,b is consistent with inhibition of PKD family members
in the cellular environment by this compound, and, by extension,
other compounds of this series (Table 4).
Pharmacokinetic studies in rats (Table 5) indicate that com-
pound 24c is orally available (38% F) with a relatively short plasma
t1/2 (Table 5). The large volume of distribution observed with this
compound suggests distribution to peripheral tissues, potentially
driven by the basicity of the benzylic amine (pKa = 7.7).
In summary, a novel 3,5-diarylazole PKD inhibitor chemotype
has been identified. SAR studies have allowed the development
of compounds with low nanomolar potency against the isolated
enzyme, sub-micromolar activity in a cellular assay of PKD signal-
ing, and impressive selectivity against a panel of additional kinases
(including putative HDAC kinases). As such, these compounds
could provide excellent tools for the further study of PKD signaling.
12. (a) Tasler, S.; Mies, J.; Lang, M. Adv. Synth. Catal. 2007, 349, 2286; (b) Hartwig, J.
F. Synlett 2006, 1283; (c) Muci, A. R.; Buchwald, S. L. Top. Curr. Chem. 2002, 219,
131.
13. (a) Campagna, F.; Carotti, A.; Casini, G. Tetrahedron Lett. 1977, 21, 1813; (b)
Altmann, E.; Betschart, C.; Buhl, T.; Green, J.; Lattmann, R.; Missbach, M. Bioorg.
Med. Chem. Lett. 2006, 2549.
14. Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett. 1977, 18, 4171.
15. Weygland, F.; Frauendorfer, E. Chem. Ber. 1970, 103, 2437.
Acknowledgments
We thank Patrick Cronan for high resolution mass spectrometry
data, the NIBR Analytical Sciences group for LC–MS and NMR sup-
port, and the NIBR Metabolism and Pharmacokinetics group for
in vitro solubility and liver microsome stability data, as well as
in vivo and bioanalytical support for pharmacokinetic studies.
16. Lau, Y. Y.; Krishna, G.; Yumibe, N. P.; Grotz, D. E.; Sapidou, E.; Norton, L.; Chu, I.;
Chen, C.; Soares, A. D.; Lin, C. C. Pharm. Res. 2002, 19, 1606. Compounds were
incubated at 1 lM in 96-well assay plates with rat liver microsomes and
selected cofactors. Half-life is determined by the measurement of test
compound concentration at four time points.
17. Zhou, L.; Yang, L.; Tilton, S.; Wang, J. J. Pharm. Sci. 2007, 96, 3052.
18. (a) Berdeaux, X.; Goebel, N.; Banaszynski, L.; Takemori, H.; Wandless, T.;
Shelton, G. D.; Montminy, M. Nat. Med. 2007, 13, 597; (b) Martini, J. S.; Raake,
P.; Vinge, L. E.; DeGeorge, B. R., Jr.; Chuprun, J. K.; Harris, D. M.; Gao, E.; Eckhart,
A. D.; Pitcher, J. A.; Koch, W. J. Proc. Natl. Acad. Sci. 2008, 105, 12457.
Supplementary data
Supplementary data (experimental details for the PKD1 and
HDAC export assays and characterization data for compounds