Design and synthesis of piperazine-indole p38α MAP kinase inhibitors with improved pharmacokinetic profiles

Article abstract of DOI:10.1016/j.bmcl.2009.12.091

Derivatives of the 4-fluorobenzyl dimethylpiperazine-indole class of p38α MAP kinase inhibitors are described. Biological evaluation of these compounds focused on maintaining activity while improving pharmacokinetic (PK) properties. Improved properties were observed for structures bearing substitutions on the benzylic methylene.

Full text of DOI:10.1016/j.bmcl.2009.12.091

Bioorganic & Medicinal Chemistry Letters 20 (2010) 828–831
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of piperazine-indole p38a MAP kinase inhibitors with
improved pharmacokinetic profiles
*
Xuefei Tan , Richland W. Tester, Gregory R. Luedtke, Sarvajit Chakravarty, Babu J. Mavunkel,
John J. Perumattam, Qing Lu, Imad Nashashibi, Joon Jung, Jie Hu, Albert Liclican, Ramona Almirez,
*
Jocelyn Tabora, Vinh Tran, Maureen Laney, Daniel E. Levy , Sundeep Dugar
Department of Medicinal Chemistry and Biology, Scios inc., 6500 Paseo Padre Parkway, Fremont, CA 94555, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Derivatives of the 4-fluorobenzyl dimethylpiperazine-indole class of p38
a MAP kinase inhibitors are
Received 26 October 2009
Revised 23 December 2009
Accepted 24 December 2009
Available online 4 January 2010
described. Biological evaluation of these compounds focused on maintaining activity while improving
pharmacokinetic (PK) properties. Improved properties were observed for structures bearing substitutions
on the benzylic methylene.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
p38
MAP
Kinase
Inhibitor
Inflammatory
The effectiveness of modulating pro-inflammatory cytokines in
the treatment of diseases including rheumatoid arthritis and psori-
SCIO-469 is a potent inhibitor of p38a (IC50 = 9 nM), with mod-
est isoform selectivity over p38b (IC50 = 98 nM). Furthermore, it is
over 1000-fold selective against a number of additional kinases
and receptors. The compound inhibits LPS-induced cytokine re-
lease in human peripheral blood mononuclear cells (PBMCs)
asis is demonstrated by the beneficial effects of anti-TNF
a thera-
peutics such as Enbrel and Remicade.1–4 However, due to
disadvantages including high costs and inconvenient dosing regi-
mens, new generations of anti-inflammatory therapies call for
the development of safe and effective orally active small mole-
cules. Of particular interest is the development of selective inhibi-
tors of the p38 MAP kinase pathway.5–7
(EC50 = 20–50 nM for TNFa, IL-1b, IL-6, IL-8) and blocks the LPS-in-
duced release of TNF
a
in 10Â diluted human whole blood
(EC50 = 300 nM). It has oral bioavailability in rat (F% 15), dog (F%
69), and monkey (F% 12) with a dose-dependent linear PK profile
and is active in a rat collagen induced arthritis model at 40 mg/
kg, QD. Unfortunately, the half life (T1/2) of SCIO-469 is relatively
short as revealed in various animal PK evaluations. In fact, from
both in vitro and in vivo metabolism studies, a major debenzylated
metabolite (1) was observed and was thought to be responsible for
the shortened half life (Fig. 2). Due to these problems, considerable
effort was placed on the development of next-generation backups
with improved pharmacokinetic profiles compared to SCIO-469.
The
a isoform of p38 MAP kinase has been shown to be a con-
trol point which, when activated, leads to the downstream release
of a pro-inflammatory cassette of cytokines including interleukin-
1b (IL-1b), interleukin-6 (IL-6) and tumor necrosis factor
a (TNFa).
As p38 activation is not involved in normal physiology, increases
a
in the levels of these cytokines are thought to play a pathophysio-
logical role in inflammatory processes associated with rheumatoid
arthritis,8–10 inflammatory bowel disease,11 congestive heart fail-
ure12 and psoriasis.13 Since p38
a
activation leads to cytokine
expression, it is thought that inhibition of p38 with compounds
a
such as SCIO-469 (Fig. 1) can normalize this aberrant physiology
and play a key role in the treatment of inflammatory disorders.
In fact, SCIO-469 was evaluated in human phase I and phase II clin-
ical trials.14,15
* Corresponding authors. Tel.: +11 86 22 66282937 (X.T.); +1 650 704 3051
(D.E.L.).
E-mail addresses: Xuefei827@gmail.com (X. Tan), del345@gmail.com (D.E. Levy).
Figure 1. Structure and p38a enzymatic potency of SCIO-469.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.091

X. Tan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 828–831
829
Figure 3. Proposed binding of SCIO-469 (yellow) and compound 9 (magenta) to
p38 using a published p38 X-ray crystal structure (PDB code: 1ove). Carbonyl
Figure 2. Major metabolites of SCIO-469 identified from liver microsomal
incubation.
a
a
oxygen of SCIO-469 and 9 make bi-dentate hydrogen bonds to backbone nitrogen of
Met-109 and Gly-100. 4-Fluoro benzyl group of SCIO-469 and one of the phenyl
rings of the diphenylmethane analog of 9 occupy a hydrophobic pocket commonly
known as the gatekeeper pocket. In addition, the substituted indole moieties
occupy a second hydrophobic pocket near the solvent front. The second phenyl ring
of the bis-phenyl moiety of 9 occupies an opening near the conserved residues Lys-
53 and Asp-168.
Docking studies of SCIO-469 into a p38a MAP kinase crystal
structure revealed additional space at the benzylic position for fur-
ther substitution (Fig. 3). In an effort to improve the metabolic sta-
bility of SCIO-469, introduction of steric bulk at and around the site
of metabolism was pursued. Thus, the introduction of methyl, cya-
no, and phenyl groups at the SCIO-469 benzylic position, as well as
the preparation of cyclic compounds (7–12 and 19) were proposed.
The common intermediate 6, used in the preparation of com-
9 were completedby initialtreatment of compound 6 with appropri-
ate substituted benzyl bromides followed by reacting with oxalyl
chloride and dimethylamine. Compound 10 was prepared similarly
but employed a reductive amination instead of a direct displace-
ment. Compounds 11 and 12 were prepared by initial reaction with
4-fluorobenzaldehyde and ethyl aluminum cyanide.17
pounds 7–12, was prepared from the D-tartaric acid salt of chiral
2,5-dimethyl-4-fluorobenzyl piperazine, compound 4.16 As shown
in Scheme 1, Boc-protection of compound 4 followed by debenzyla-
tion afforded Boc-protected piperazine 5. Subsequent coupling with
6-chloro-indole carboxylic acid yielded compound 6. Compounds7–
As illustrated in Scheme 2, the synthesis of cyclized indazole
analog 19 originated from commercially available ortho-chloro
Scheme 1. Reagents and conditions: (a) 10% NaOH, EtOAc 100%; (b) (Boc)2O, Na2CO3, dioxane, 46%; (c) Pd(OH)2/C, MeOH, AcOH, H2, 30 psi, 100%; (d) EDCI, 6-chloro-1-
methyl-1H-indole-5-carboxylic acid,16 DMF, Et3N, 86%; (e) HCl, dioxane, 98%; (f) bromide/ethanol, Et3N, 66–90%; (g) oxalyl chloride, DCM then dimethyl amine, 74–93%; (h)
indan-1-one, Ti(O-ipr)4, NaBH3CN, 48%; (i) 4-fluoro-benzaldehyde, Ti(O-ipr)4, EtAlCN,17 53%; (j) KHMDS, THF then CH3I, 65%.

830
X. Tan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 828–831
Table 3
In vivo PK profiles of diphenylmethane analog 9
Species
Rat
Dog
Monkey
CL (L/h/kg)
V (L/kg)
1.2
2.7
0.2
6.5
—
—
Cmax (ng/mL)
367
1.2
810
1272
31
899
1.3
3430
5609
33
360
2
—
1829
—
Tmax (h)
*
*
AUCIV
AUCPO
%F
(
(
l
g
h/L)
h/L)
lg
As indicated in Table 1, the p38
analogs was below 50 nM, indicating that the predictive model was
an effective tool in the design of potent p38 inhibitors. However,
a activity for all, but one of the
a
the cellular activities (dWBA IC50) of these analogs were clearly
dependent upon a compound’s cell permeability and not predictive
based on modeling. Interestingly, both a more hydrophobic analog
8 and a more polar analog 11 gave the worst cellular activities. The
microsomal incubation study shows that only the diphenylme-
thane analog 9 and indazole analog 19 gave significantly lower lev-
els of the debenzylated metabolite while maintaining potency.
Analogs 7 and 10 yielded higher percentages of the debenzylated
metabolite whereas 11 and 12 were much less stable under the
microsomal incubation conditions. Finally, while microsomal
digestion of analog 19, produced less than 5% of the corresponding
debenzylated metabolite, its oral availability (determined in rat PK
studies) was found to be much lower than expected. As this com-
pound is structurally quite different than SCIO-469, its poor PK
may be due to other factors.
Scheme 2. Reagents and conditions: (a) TsNHNH2, toluene, 97%; (b) SOCl2, 100%;
(c) (2R,5S)-dimethyl-piperazine-1-carboxylic acid tert-butyl ester,18 THF, rt, 65%;
(d) K2CO3, NMP, 57%; (e) TFA, DCM, 96%; (f) 6-chloro-1-methyl-1H-indole-5-
carboxylic acid,16 HATU, Et3N, DMF, 87%; (g) oxalyl chloride, DCM then dimethyl
amine, 83%; (h) KOH, MeOH–H2O, 90%.
Table 1
Potency and metabolism comparison of SCIO-469 and new analogs
Further microsomal studies of the diphenylmethane analog 9
demonstrate that it is, in fact, more metabolically stable that
SCIO-469. Additionally, this trend was observed in multiple spe-
cies. While it was observed that metabolism of 9 also occurs at
the dimethyl amide region of the molecule, a higher percentage
of demethylated metabolite, compared to SCIO-469, was formed
in every species (Table 2).
Evaluation of compound 9 in in vivo PK studies indicated it has
a bioavailability of 31% in rat and 33% in dog. Furthermore, PK
parameters such as oral exposure, clearance and volume of distri-
bution, measured in multiple species (Table 3), warranted consid-
eration of compound 9 as a backup to SCIO-469. While lower
exposure was observed in monkey, this was consistent with the in-
creased amount of demethylated metabolite noted in the in vitro
studies mentioned above.
19
20
Compds
p38
a
IC50
dWBA IC50
M)
Debenzylated
(l
M)
(l
metabolitea (%)
SCIO-469
7
8
9
10
11
12
19
0.009
0.006
0.024
0.009
0.029
0.183
0.047
0.048
0.1–0.2
ND
24.8
29.6
ND
5.1
24%@0.5
0.5
0.5
32%@0.3
0.3
l
l
Mb
Mb
40.8
Not stablec
Not stablec
<5%
0.47
a
b
c
Values are based on MS signal intensity.
Percentage of TNF inhibition at certain inhibitor concentration.
Significant loss of cyano group was observed in microsomal incubation.
a
benzoyl chloride. Reaction with tosyl hydrazide followed by treat-
ment with thionyl chloride yielded the chloroimine 14. Displace-
ment of 14 with (2R,5S)-dimethyl-piperazine-1-carboxylic acid
tert-butyl ester18 provided intermediate 15, which upon treatment
with K2CO3, cyclized into indazole 16. Following the removal of the
Boc protecting group, the amine was coupled with 6-chloro-1-
methyl-1H-indole-5-carboxylic acid16 to afford compound 18. Sub-
sequent treatment of 18 with oxalyl chloride and dimethyl amine
followed by cleavage of the tosyl protecting group gave the desired
indazole analog 19.
In summary, through efforts to discover backup molecules with
improved pharmacokinetic profiles over SCIO-469, we identified
the diphenylmethane group as a superior alternative to the 4-fluo-
robenzyl moiety in the indole-piperazine class of p38
a MAP kinase
inhibitors. This modification resulted in improved in vitro and
in vivo pharmacokinetic profiles compared to SCIO-469, presum-
ably due to the sterically hindered environment around the site
of metabolism. Furthermore, this steric hindrance suppressed the
formation of a potential major metabolite while maintaining over-
all biological properties and potency against p38a MAP kinase.
Table 2
Microsomal metabolism of diphenylmethane analog 9 and SCIO-469 in various species
Microsomal metabolism
Compds
Parenta(%)
SCIO-469
Debenzylated metabolite (%)
Demethylated metabolite (%)
SCIO-469
9
SCIO-469
9
9
Rat
Dog
Monkey
Human
65.8
94.5
51.2
65.1
83.9
91.7
71.4
90.8
24.8
2.6
25.2
25.8
5.1
2.5
2.9
1.1
3.2
2.3
11.9
4.9
9.4
4.8
19.5
6.3
a
Values are based on MS signal intensity.

X. Tan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 828–831
831
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