Discovery of CS-0777: A potent, selective, and orally active S1P 1 agonist

Article abstract of DOI:10.1021/ml100301k

CS-0777 (3) is phosphorylated in vivo, and the phosphate of CS-0777 (CS-0777-P) (4) acts as a selective S1P receptor-1 (S1P₁) modulator. We report herein the synthesis of CS-0777 and CS-0777-P, pharmacological effects such as S1P₁ an

Full text of DOI:10.1021/ml100301k

LETTER
pubs.acs.org/acsmedchemlett
Discovery of CS-0777: A Potent, Selective, and Orally
Active S1P1 Agonist
Takahide Nishi,*,† Shojiro Miyazaki,† Toshiyasu Takemoto,† Keisuke Suzuki,† Yukiko Iio,†
Katsuyoshi Nakajima,† Takashi Ohnuki,† Yumi Kawase,‡ Futoshi Nara,‡ Shinichi Inaba,§ Takashi Izumi,§
Hiroshi Yuita,|| Keiko Oshima,|| Hiromi Doi,^ Ryotaku Inoue,^ Wataru Tomisato,^ Takashi Kagari,^ and
Takaichi Shimozato*,^
†Lead Discovery & Optimization Research Laboratories I, ‡Cardiovascular-Metabolics Research Laboratories, and §Drug Metabolism &
Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo, 140-8710, Japan
Oncology Research Laboratories and ^Frontier Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13 Kitakasai, Edogawa-ku, Tokyo,
134-8630, Japan
ABSTRACT: CS-0777 (3) is phosphorylated in vivo, and the
phosphate of CS-0777 (CS-0777-P) (4) acts as a selective S1P
receptor-1 (S1P1) modulator. We report herein the synthesis of
CS-0777 and CS-0777-P, pharmacological effects such as S1P1
and S1P3 agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effect on experimental
autoimmune encephalomyelitis (EAE), and also the pharmacokinetics in rats. CS-0777-P had ∼320-fold greater agonist activity for
human S1P1 (EC50; 1.1 nM) relative to S1P3 (EC50; 350 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of
CS-0777 in rats, lymphocyte counts decreased significantly, with a nadir at 12 h postdose and recovery to vehicle control levels by 5
days postdose. In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were
observed for the 0.1 and 1 mg/kg CS-0777 groups in rats. CS-0777 is currently in clinical trials for the treatment of multiple sclerosis
(MS).
KEYWORDS: S1P1, agonist, CS-0777, lymphocyte, EAE, MS
phingosine 1-phosphate (S1P) receptor-1 (S1P1) is one of
five G-protein-coupled receptors which bind S1P, a bioactive
reported to be mediated via S1P3 in rodents.14,15 As a result,
selective S1P1 modulators have been proposed as a means to
avoid potential side effects, such as bradycardia, that have been
identified in clinical trials of fingolimod.16,17 We therefore
focused our attention on synthesizing compounds with greater
S1P1 selectivity. Comprehensive evaluation of various properties
of these synthesized compounds, such as the affinity for each S1P
receptor subtype, immunosuppressive efficacy in vivo, and toxi-
city and pharmacokinetic (PK) aspects, led to the identification
of a promising clinical candidate, CS-0777 (3), conclusively. We
report herein the synthesis of CS-0777 (3) and the phosphate of
CS-0777 (CS-0777-P) (4). In addition, we evaluated pharma-
cological effects such as S1P1 and S1P3 agonist activity in vitro,
peripheral blood lymphocyte lowering effects, and the suppres-
sive effect on the experimental autoimmune encephalomyelitis
(EAE) model and also the PK profile in rats.
The synthetic route of CS-0777 (3) is shown in Scheme 1.
Treatment of 518 with Ac2O and NEt3 resulted in providing
diacetate 6 in quantitative yield. Then, acylation at the 5-posi-
tion on the pyrrole ring was carried out in the presence of an
excess amount of 4-(4-methylphenyl)butanoyl chloride (7) and
4-(dimethylamino)pyridine (DMAP) in toluene at 110 ꢀC to
afford enol ester 8. The two acetyl groups and the enol ester
S
lipid mediator involved in cell differentiation, morphogenesis,
angiogenesis, motility, and multiple other processes.1,2 Modula-
tion of S1P receptors may have important clinical applications. In
particular, targeting S1P1 with synthetic modulators appears
to have clinical utility in the suppression of autoimmunity
by affecting lymphocyte trafficking.1-4 Knowledge of the phy-
siology and pharmacology of S1P1 has evolved rapidly through
studies involving fingolimod (FTY720) (1), a S1P-like derivative
of the fungal metabolite myriocin that modulates S1P1 signaling
on lymphocytes and other cells (see Figure 1).5-7 The systemic
administration of fingolimod induces a dose-responsive decrease
in circulating lymphocytes, which contributes to its efficacy in
animal models of autoimmune and inflammatory diseases.
According to recent information, the active phosphorylated form
of fingolimod (FTY720-P) (2),8,9 which is generated in vivo via a
sphingosine kinase, acts as an S1P1 agonist or functional antago-
nist. Binding of FTY720-P to S1P1 induces receptor internaliza-
tion and inhibits receptor recycling, depriving T and B cells of
their ability to sense an S1P gradient and, thus, preventing their
exit from lymphoid organs.3-7 Recently, the clinical efficacy of
fingolimod has been demonstrated in phase 3 studies in patients
with relapsing remitting multiple sclerosis (MS).10,11 However,
FTY720-P lacks specificity due to its affinity for S1P3, S1P4, and
S1P5.12,13 While the beneficial effects of fingolimod are thought
to be mediated via S1P1, these other S1P receptor subtypes may
contribute to off-target effects such as bradycardia, which is
Received: December 20, 2010
Accepted: February 13, 2011
Published: March 02, 2011
r
2011 American Chemical Society
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Figure 1
Scheme 1a
a Reagents and conditions: (a) Ac2O, NEt3, DMAP, CH2Cl2 (quant). (b) 4-(4-Methylphenyl)butanoyl chloride (7), DMAP, PhMe (47%). (c) LiOH,
THF, MeOH, H2O (83%).
Scheme 2a
a Reagents and conditions: (a) AllocCl, KHCO3, AcOEt, H2O. (b) (AllylO)2PN(i-Pr)2, tetrazole. (c) mCPBA, CH2Cl2 (72%, 3 steps). (d) Pd(PPh3)4,
pyrrolidine, CH3CN (73%).
moiety were removed by saponification with aqueous LiOH in
THF-MeOH to afford CS-0777 (3) in good yield.
of ML-236B,20 were found to convert CS-0777 (3) to CS-0777-P
(4) in 73% yield, as shown in Scheme 3. Thus, the biotransfor-
mation method is more practical and efficient than the previous
route shown in Scheme 2.
Next, agonist-specificity CS-0777-P (4) was determined by
measuring agonist-evoked [35S]GTPγ-S binding activity to rat
and human S1P1 and S1P3 expressed in transfected CHO-K1
cells as shown in Table 1.21 The active FTY720-P (2) was
included as a comparator. CS-0777-P (4) demonstrated potent
agonist activity for rat and human S1P1 (EC50 = 1.8 and 1.1 nM,
respectively) and at least 100-fold greater activity for S1P3
(EC50 = 200 and 350 nM, respectively). In comparison, the
The chemical synthesis of CS-0777-P (4) was carried out as
shown in Scheme 2. After protection of the amino group of 3 with
allyl chloroformate (AllocCl) and KHCO3 in aqueous AcOEt,
phosphorylation using (AllylO)2PN(i-Pr)2 was performed in the
presence of tetrazole, and subsequent treatment with 3-chlor-
operoxybenzoic acid (mCPBA) in CH2Cl2 cleanly provided 9 in
moderate yield. Both the allyl and Alloc groups of 9 were
deprotected with Pd(PPh3)4 and pyrrolidine in CH3CN to give
the desired CS-0777-P (4).
However, this synthetic route was still unsatisfactory because
of the low yield and multistep reactions. In order to circumvent
these issues, we already reported one-step biotransformation
of R,R-disubstituted R-amino alcohol derivatives.19 Among
our microbial library, strains belonging to Circinella muscae,
which was reported to have microbial phosphorylation activity
agonist activity of FTY720-P (2) for rat and human S1P1 (EC50
=
0.29 and 0.37 nM, respectively) was approximately 5- to 10-fold
greater than that for rat and human S1P3 (EC50 = 1.3 and 3.3 nM,
respectively). Relative to S1P3 agonist activity, CS-0777-P
(4) appears to have greater selectivity for S1P1 compared to
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LETTER
Scheme 3a
a Reagents and conditions: (a) Circinella muscae (NBRC 4457) (73%).
Table 1. In Vitro Agonist-Evoked GTPγ-S Binding to Rat and
a
Human S1P1 and S1P3
EC50(nM)
rat
human
selectivity
compd
S1P1 S1P3 S1P1 S1P3 (human S1P1 vs S1P3)
4 (CS-0777-P) 1.8
2 (FTY720-P) 0.29
200
1.3
1.1
350
3.3
320
9
0.37
a Note: EC50 is defined as the midpoint between the binding ratio of the
vehicle and the maximum response of the test compound.
Figure 3. Suppressive effect of CS-0777 on rat EAE.
dose levels, respectively, 12 h after CS-0777 administration. By
48 h postdose, lymphocyte counts had recovered to 71.5%
and 30.1% of control for 0.1 and 1 mg/kg doses, respectively,
and by 5 days postdose, lymphocyte counts of both CS-0777
administered groups were similar to those of vehicle-treated rats
(Figure 2).21
Figure 2. Effects of a single oral dose of CS-0777 on lymphocyte counts
in rats. Vehicle (1% MC solution, ]), 0.1 mg/kg of CS-0777 (b), or 1
mg/kg of CS-0777 (9) was orally administered to rats. The rats were
anesthetized and abdominally dissected 3 h, 8 h, 12 h, 1 day, 2 days, 3
days, 5 days, and 7 days after oral administration. The blood was drawn
from the inferior vena cava of administered rats, and lymphocyte number
was counted by an automatic hemacytometer. The blood of untreated
rats was also drawn, and lymphocyte number was counted ([). Five rats
were used for each group. Lymphocyte numbers are shown as mean
( SE.
Next, we evaluated the suppressive effect of CS-0777 on EAE
in rats. EAE has been used as a preclinical animal model for proof
of concept studies for multiple sclerosis therapy. EAE was
induced to Lewis rats (6-week old females) by injection of
myelin basic protein guinea pig fragment 68-82 with adjuvant
into both hind footpads of rats on day 0. Vehicle or 0.01, 0.1, or
1 mg/kg of CS-0777 was orally administered every day from day
0 to day 20. The EAE score was evaluated daily from day 8 to day
21 in accordance with the following criteria: 0, no abnormality; 1,
flaccid tail; 2, mild paralysis of limb and/or ataxia; 3, unilateral
hind limb paralysis; 4, bilateral hind limb paralysis; 5, tetraplegia;
6, dead.21 Cumulative EAE scores were calculated by summing
up the daily scores. Vehicle-administered rats began to show
clinical signs of EAE from day 12, and thereafter, an acute
increase of the EAE score was observed, reaching a maximal
level on day 15. Mean cumulative EAE scores, calculated by
summing the daily scores, were 14.0, 9.6, 0, and 0 for the vehicle
and 0.01, 0.1, and 1 mg/kg of CS-0777 groups, respectively.
FTY720-P (2). As a result, CS-0777 is expected to avoid potential
side effects corresponding to S1P3. In addition, the agonist activity of
CS-0777-P (4) for human S1P5 (EC50 = 21 nM) was approximately
60-fold weaker than that of FTY720-P (EC50 = 0.34 nM), and both
compounds had no ability to bind human S1P2 (data not shown).
In a second series of studies, the effects of CS-0777 (3) on
peripheral blood lymphocyte counts and the suppression of the
development of EAE were investigated in rats. In Lewis rats
administered a single oral dose of 0.1 or 1 mg/kg of CS-0777,
peripheral blood lymphocyte counts decreased to 27.4% and
18.4% of vehicle-treated control values for the 0.1 and 1 mg/kg
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LETTER
Table 2. Pharmacokinetic Parameters and Bioavailability of CS-0777 and CS-0777-P to Ratsa
compd
dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUC0-inf (ng h/mL)
Fb (%)
CLc (mL/(min kg))
Vssc (L/kg)
3
3
CS-0777
0.1
1
1.89 ( 0.20
18.2 ( 2.0
26.4 ( 3.4
220 ( 19
4.75 ( 3.50
3.25 ( 2.06
9.50 ( 1.00
9.00 ( 2.00
5.40 (n = 1)
10.4 ( 1.2
10.9 ( 1.0
11.6 ( 0.6
31.7 (n = 1)
346 ( 35
72.2 (n = 1)
69.5 ( 6.9
80.6 ( 15.9
90.8 ( 7.7
38.0 ( 2.3
33.7 ( 2.7
nad
17.1 ( 0.7
18.1 ( 1.0
nad
CS-0777-P
0.1
1
686 ( 136
6280 ( 540
nad
nad
a Note: Each value is the mean ( standard deviation (SD) of four animals in the rat study. b The F value (%) of CS-0777-P was calculated by dividing the
CS-0777-P area under the curve (AUC) after oral administration of CS-0777 by that after intravenous administration of CS-0777 at the same dosage.
c The total body clearance (CL) and distribution volume at steady state (Vss) were calculated based on the data after intravenous administration of CS-
0777. d Not applicable.
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: þ81-3-3492-3131. Fax: þ81-3-5436-8563. E-mail:
nishi.takahide.xw@daiichisankyo.co.jp.
Author Contributions
T. Nishi, S. Miyazaki, T. Takemoto, K. Suzuki, Y. Iio contributed
to the Design, Synthesis of CS-0777; K. Nakajima and T. Ohnuki
contributed to the Synthesis of CS-0777-P and FTY720-P; Y.
Kawase and F. Nara contributed to the In vitro study of CS-0777-P
and FTY-720-P; S. Inaba and T. Izumi contributed to the PK
study of CS-0777 and CS-0777-P; and H. Yuita, K. Oshima, H.
Doi, R. Inoue, W. Tomisato, T. Kagari, and T. Shimozato
contributed to the In vivo study of CS-0777.
Figure 4. Blood concentration versus time profiles of CS-0777 and CS-
0777-P to rats.
’ ACKNOWLEDGMENT
We thank Dr. Tsuyoshi Nakamura, Dr. Takuya Ikeda, Dr.
Takaaki Jojima, Dr. Takahiro Yamane, Taiji Goto, Takeshi
Fukuda, Takashi Tsuji, and Yumiko Mizuno for preparing the
Compared to the vehicle-treated group, statistically significant
decreases in the cumulative EAE scores were observed for the 0.1
key intermediates and their helpful discussions; Dr. Masakazu
and 1 mg/kg of CS-0777 groups (nonparametric Dunnett test
[joint ranking], p < 0.01) (Figure 3).21 Thus, CS-0777 has a
potent suppressive effect on EAE in rats, and from the above data,
CS-0777 is expected to be a promising agent for the treatment of
Tamura, Rie Ohya, and Kiyoaki Yonesu for performing the S1P1-
3 agonist selectivity assays and their helpful discussions; and Taku
Moriguchi, Yasunori Ono, and Emi Kurosawa for performing
screenings to provide the biotransformation.
MS.
The pharmacokinetics of CS-0777 and CS-0777-P were
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’ NOTE ADDED AFTER ASAP PUBLICATION
The author contribution for T. Shimozato was erroneously
reported in the version of this paper published on March 2,
2011. The correct version was reposted on March 8, 2011.
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