Identification of novel pyrrolopyrazoles as protein kinase C β II inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.10.032

A novel series of pyrrolopyrazole-based protein kinase C β II inhibitors has been identified from high-throughput screening. Herein, we report our initial structure-activity relationship studies with a focus on optimizing compound ligand efficiency and physicochemical properties, which has led to potent inhibitors with good cell permeability.

Full text of DOI:10.1016/j.bmcl.2010.10.032

Bioorganic & Medicinal Chemistry Letters 21 (2011) 584–587
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of novel pyrrolopyrazoles as protein kinase C b II inhibitors
⇑
Hui Li , Yufeng Hong, Seiji Nukui, Jihong Lou, Sarah Johnson, Stephanie Scales, Iriny Botrous,
Eileen Tompkins, Chunfeng Yin, Ru Zhou, Mingying He, Jordan Jensen, Djamal Bouzida,
⇑
Gordon Alton, Jennifer Lafontaine , Stephan Grant
Pfizer Global Research & Development, La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of pyrrolopyrazole-based protein kinase C b II inhibitors has been identified from high-
throughput screening. Herein, we report our initial structure–activity relationship studies with a focus
on optimizing compound ligand efficiency and physicochemical properties, which has led to potent
inhibitors with good cell permeability.
Received 17 August 2010
Revised 5 October 2010
Accepted 6 October 2010
Available online 13 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Protein kinase C
Kinase inhibitor
Pyrrolopyrazole
Diabetic complications
Protein kinase C is a super family of serine/threonine kinases
that includes over 10 different isozymes.1 Based on structural
homology in their regulatory domains and different requirements
for activating co-factors, the PKC isozymes are classified into three
Several accounts of PKC b II inhibitor designs have been re-
ported in the literature,9–11 almost all of which were based on
the bisindomaleimide/carbazole class of inhibitors, which have tra-
ditionally suffered from low kinase selectivity and poor physico-
chemical properties. Here, we report our own effort in the
identification of a novel series of pyrrolopyrazole-based PKC b II
inhibitors.
The pyrrolopyrazole (PP), an extension of the more classical
3-aminopyrazole, has been utilized as a hinge binder for a number
of protein kinase targets (Fig. 2).12–16 Recently, PHA-739358, an
Aurora kinase inhibitor, achieved good pharmacokinetic properties
and general safety profiles in a phase I clinical study.17 Compared
with many other hinge binding templates, the PP core offers
categories:2 conventional PKC (PKC
(PKCd, , and h), and atypical PKC (PKCf and i
a
, bI, bII, and
c), novel PKC
e,
g
/k). The PKC signal
transduction cascade coordinates complex physiological events
including normal tissue function and repair. Disruption of cellular
environment can induce pathological PKC activation, which has
been implicated in a wide variety of disease states, including can-
cer, diabetes, and cardiovascular diseases.3–5
Although PKCs were originally identified over 30 years ago,6 the
detailed understanding of the unique function of each individual
isozyme only became possible recently. Among all the PKC iso-
zymes, PKC b has been one of the most well studied, particularly
with regards to its association with diabetic complications. PKC b
I and PKC b II are splice variants from a single gene and it is the
PKC b II isozyme that is more directly associated with microvascu-
lar leakage.4,7 It has been established that in the diabetic state, the
increased level of PKC b activation initiated by hyperglycemia is
closely associated with many microvascular abnormalities, includ-
ing diabetic retinopathy, nephropathy, and neuropathy. This has
H
N
H
N
O
O
N
O
O
N
N
N
N
been supported by clinical findings from
a selective PKC b
O
inhibitor, ruboxistaurin from multiple phase II/III clinical trials,
N
both for retinopathy and nephropathy.8
N
1 (Ruboxistaurin)
2 (Enzastaurin)
⇑
Corresponding authors. Tel.: +1 858 210 9472.
E-mail address: hui.li@ppdi.com (H. Li).
Figure 1. Molecular structures of selected PKC b inhibitors in clinical evaluation.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.032

H. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 584–587
585
From our internal high-throughput screening, we identified a
series of PKC b II inhibitors, such as compound 5, that shared a
3-amino-6,6-dimethyl-pyrrolopyrazole core structure. Although
no X-ray cocrystal structure of this class of kinase inhibitor in com-
plex with PKC b II has been obtained,18 extensive biochemical char-
acterization has validated that they are competitive with regards
to ATP and interact with the protein in the hinge region.19 Similar
to previous reports,16 a common synthetic intermediate 11 can be
prepared in an efficient manner (Scheme 1) and offered two
orthogonal vectors for further synthetic modifications. Treatment
of 2-amino-2-methylpropanoic acid (6) with acrylonitrile under
basic conditions afforded the Michael addition product 7 in excel-
lent yield. The resulting amine was then protected with a Boc
group, followed by conversion of the carboxylic acid to the corre-
sponding methyl ester to give intermediate 9. The acidic proton
adjacent to the nitrile group was removed by the action of potas-
sium t-butoxide and the resulting anion subsequently attacked
the carbonyl group of the ester to provide cyclic product 10 in
89% overall yield. Treatment of cyanoketone 10 with hydrazine
in the presence of acetic acid afforded the pyrroloypyrazole tem-
plate 11 in 91% yields. Protection of the pyrazole NH group as
the ethyl carbamate gave two regio-isomers in about 3:1 ratio
(13:12). The two isomers were easily separated by column chro-
matography and both worked well in the following synthetic steps.
From the protected PP template, 12, the 1st diversity point at the
3-amino group was introduced by reaction with various acid chlo-
rides to provide the corresponding amides in generally good yields.
Subsequently the Boc protection can be selectively removed under
acidic conditions without affecting the ethyl carbamate. Function-
alization at the 2nd diversity point at the 5-amino group of
intermediate 15 was then accomplished by the formation of the
urea. It should be noted, as a result of the steric hinderance
introduced by the gem-dimethyl group at the 6-position, formation
N
NH
H
N
N
HN
N
O
N
HN
4
O
N
N
N
O
O
O
3
(PHA-739358)
(PHA-793887)
Figure 2. Molecular structures of pyrrrolopyrazole-based kinase inhibitors.
1
H
6
N
2
N
O
N
5
O
3
HN
Head Region
4
Tail region
N
Ph
2'
O
5'
H3C
N
5
: PKC β Ki = 541 nM
Figure 3. Molecular structure and properties of the HTS hit. The biochemical assay
for PKC b was carried out as previously described.18
efficient hydrogen bond interactions without the requirement of
multiple co-planar aromatic systems, which has provided greatly
improved physicochemical properties, such as aqueous solubility.
However, most previous PP kinase inhibitors have been designed
for intravenous administration. Achievement of good cell
permeability and oral bio-availability still remain a challenge for
this chemical series (Fig. 3).
NC
NC
NaOH
95%
Me NOH
NC
Boc2O
74%
4
N
Boc
CO2H
N
CO2H
H2N
COOH
H
6
7
8
KHCO3, MeI
96%
O
N
H2NNH2
KOtBu
89%
Boc
N
NH
Boc
N
NC
N
CO2Me
AcOH, EtOH
91%
CN
Boc
NH2
11
12
10
9
ClCO2Et
CO2Et
H
N
O
N
N
N
N
N
R1
Boc
N
N
CO2Et
Boc
N
N
+
R2
NH
O
NH2
NH2
Ar1
13
17
Ar1COCl, Et3N
Et3N, MeOH
O
CO2Et
N
N
CO2Et
COOEt
N
N
N
Boc
N
HN
N
N
1R2RN
triphosgine,
HCl
NH
1
2
NH
NH
Ar1
then R R NH
O
O
O
Ar1
Ar1
15
14
16
Scheme 1. Synthesis of pyrrolopyrazoles.

586
H. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 584–587
Table 1
is a much more efficient inhibitor with a ligand efficiency of 0.30.
Similar analog, 20, with a fused bicyclic octahydropyrrolopyrazine
ring system in the head region, further improved the inhibitory po-
tency (Ki = 131 nM) while maintaining the improved ligand effi-
ciency (LE = 0.31). Although compound 19 was similar in potency
to the initial hit 5, it dramatically reduced its molecular weight
(from 564 to 382) and lipophilicity (c log P from 6.90 to 2.72)
and was deemed a much more attractive starting point for subse-
quent inhibitor design.
Intramolecular hydrogen bonds have been utilized extensively in
the literature to pre-organize molecular conformations.20 At the
same time, it also reduces the number of effective hydrogen bond
donors, a key factor that hinders compound permeability. By intro-
ducing a hydrogen bond acceptor atom at the ortho position of the
benzene ring in the tail region, we have observed marked improve-
ment in their permeability. As shown in Figure 4, the picolinamide
analog 21 had achieved a fivefold improvement in Caco-2 A to B per-
meability as compared to the simple benzamide analog 20, presum-
ably through the added interaction between the pyridine nitrogen
and the proton from the amide. Similarly, introduction of a fluorine
atom at the ortho position (compound 22) also demonstrated a ca.
fourfold increase in Caco-2 permeability.
Identification of more ligand efficient lead through truncation strategy
H
N
O
N
R3
N
H
N
O
R4
Compd
R3
R4
Ki (nM)
MW
564
LE
c log P
N
N
5
OPh
541
0.21
6.90
Bn
N
N
18
H
189
472
0.26
4.82
Bn
N
19
20
H
H
541
131
382
408
0.30
0.31
2.73
2.96
N
N
N
Encouraged by these results, we turned our attention to the fur-
ther optimization in the head region (Fig. 1). In the octahydropyrrol-
opyrazine sub-series, the stereochemistry at the 30 and 80 centers
(Table 2) had major influences on the inhibitory potency. We found
that the S-methyl group at the 30-position was critical for potent PKC
b II inhibition, and that the 80-S analogs were ca. threefold more po-
tent than the corresponding 80-R analogs (compound 23 compared
to 24). In contrast, the 30-R-methyl analogs were not active regard-
less of their stereochemistry at the 80-position (13% and 24% inhibi-
of urea analogs at the 5-amino group of the PP template turned out
to be more challenging. Treatment of triphosgine was required to
provide the corresponding acyl chloride, which in many cases
can be purified before reacting with a diverse set of amines. Carba-
mate protection of the resulting urea 16 can be effectively removed
by the action of Et3N in MeOH.
Despite its enzymatic potency against PKC b II, compound 5 was
not a very attractive lead for further structural–activity relation-
ship (SAR) development. As a result of its high molecular weight
(MW = 564) and lipophilicity (c log P = 6.9), compound 5 had a
low ligand efficiency (LE = 0.21) and high clearance in human liver
microsome stability assay (2.1% remaining in HLM after 60 min
incubation). In addition, its low permeability and potential for ef-
flux as measured in Caco-2 assay (AB = 0.4 ꢀ 10ꢁ6 cm/s, BA/
AB = 12.5) remained concerns for the development of an oral agent.
In order to identify a more attractive lead molecule, a truncation
strategy was employed that systematically removed portions of
the molecule to establish the minimum pharmacophore for PKC
b II inhibition. As summarized in Table 1, removal of the phenyoxyl
group at the meta position in the tail region of compound 5 (R3)
provided compound 18, which increased potency by ca. threefold,
and more importantly, improved ligand efficiency from 0.21 to
0.26. Further elimination of the benzyl group from the piperidine
ring in the head region afforded less potent analog 19 with a Ki
of 541 nM. However, because of the reduced molecular weight, it
tion at 1 l
M, respectively). Further extension at the 30-position from
methyl to ethyl group provided compound 27 with similar potency,
while introduction of the gem-dimethyl group at the same position
resulted in a fourfold drop in potency (compound 28, Ki = 125 nM).
The gem-dimethyl group at the 6-position of the PP core appeared
to be optimal: larger substitutions, such as a di-ethyl group, gave
similar potency (compound 29, Ki = 62 nM) and a cyclopropyl analog
at the 6-position resulted in a nearly 10-fold loss of potency (com-
pound 30, Ki = 338 nM). The PKC b II inhibitory activity was also di-
rectly correlated to the basicity of the amino group in the head
region. Replacing one hydrogen at the 70-position of the octahydro-
pyrrolopyrazine head group with a fluorine atom lowered the pKa of
the amino group from 9.2 (ACD calculation, compound 31) to 6.9,
and reduced its inhibitory potency from 29 to 109 nM. A difluoro
substitution at the 70-position further reduced the pKa to 4.5 and
completely abolished inhibitory potency against PKC. These obser-
vations were consistent with the hypothesis that an acidic residue
is present in the binding site near the inhibitor’s head region.
O
O
O
N
NH
N
N
N
N
N
NH
N
N
NH
N
N
N
N
N
O
H
F
N
HN
O
H
O
N
F
20
0.57/32.4
21
2.8/32.4
22
Caco-2 AB/BA
(x10 cm/s)
2.2/15.8
-6
Figure 4. Improvement of compound permeability through intramolecular hydrogen bonds.

H. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 584–587
587
Acknowledgments
Table 2
Further optimization of pp series in the head region
The authors would like to thank Drs. Simon Bailey, Michael
Jirousek, Michael Niesman, Achim Krauss for their helpful discus-
sions while carrying out this work.
H
N
R5
O
N
R6
O
N
H
N
N
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8'
R
N
S
S
7'
23
Me
Me
29
3'
N
N
24
25
26
27
28
Me
Me
Me
Me
Me
Me
Me
Me
92
N
S
N
R
13%a
24%a
46
N
R
N
S
N
R
N
N
N
Et
N
Me
Me
Me
Et
125
N
N
29
30
31
32
62
N
N
R5, R6 = cyclopropyl
338
N
F
F
Me
Me
Me
Me
109
N
N
>10,000
N
F
a
Percentage inhibition at 1 lM.
In conclusion, we have identified a novel series of pyrrolopyraz-
oles as highly active protein kinase C b II inhibitors. With the prop-
er control of stereochemistry and physicochemical properties,
including pKa and intramolecular hydrogen bonds, potent and per-
meable inhibitors, such as compound 23, have been achieved. Fur-
ther optimization in cell-based assays and their pharmacokinetic
profiles of this series of PKC b II inhibitors will be reported in the
near future.
20. Kuhn, B.; Mohr, P.; Stahl, M. J. Med. Chem. 2010, 53, 2601.