Discovery of anti-inflammatory clinical candidate E6201, inspired from resorcylic lactone LL-Z1640-2, III

Article abstract of DOI:10.1016/j.bmcl.2010.03.087

Inspired by natural product, LL-Z1640-2, clinical candidate, E6201 (22) was discovered in a medicinal chemistry effort through total synthesis. The modification on C14-position to N-alkyl substitution showed to be potent in vitro and orally active in vivo in anti-inflammatory assays.

Full text of DOI:10.1016/j.bmcl.2010.03.087

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3155–3157
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of anti-inflammatory clinical candidate E6201, inspired from
resorcylic lactone LL-Z1640-2, III
Yongchun Shen a, Roch Boivin a, Naoki Yoneda b, Hong Du a, Shawn Schiller a, Tomohiro Matsushima b,
Masaki Goto b, Hiroshi Shirota a, Fabian Gusovsky a, Charles Lemelin a, Yimin Jiang a, Zhiyi Zhang a,
Robert Pelletier a, Megumi Ikemori-Kawada b, Yoshiyuki Kawakami b, Atsushi Inoue b,
Matthew Schnaderbeck a, Yuan Wang a,
*
a Eisai Inc., 4 Corporate Drive, Andover, MA 01810, USA
b Eisai Tsukuba Research Laboratories, 1-3, Tokodai 5-chome, Tsukuba-shi, Ibaraki 300-2635, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Inspired by natural product, LL-Z1640-2, clinical candidate, E6201 (22) was discovered in a medicinal
chemistry effort through total synthesis. The modification on C14-position to N-alkyl substitution
showed to be potent in vitro and orally active in vivo in anti-inflammatory assays.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 18 January 2010
Revised 20 March 2010
Accepted 26 March 2010
Available online 30 March 2010
Keywords:
LL-Z1640-2
f152A1
Anti-inflammatory
Resorcylic lactone
In our effort of developing novel anti-inflammatory agent, in-
spired by natural product, f152A1 (1), we reported that the chem-
ical modifications at C4 and C14 positions of resorcylic lactone lead
to an in vitro and iv in vivo potent analog 2 (IC50: 15 nM in vitro;
ED50: 6.5 mg/kg iv) (Fig. 1).1–3 In the last letter we highlighted
the structure activity relationship in C14–oxygen series.3 However,
the oral bioavailability of 2 in mouse was very low (4.1%), and was
not suitable for exploring broader application outside of iv route. In
this Letter, we report the successful discovery of benzimidazole 7
and C14–N substitution analogs including the clinical candidate
E6201 (22).
Our previous investigations suggested that C14 region can tol-
erate substitutions. An imidazole group in 2 regained full potency
of the natural product.3 In addition, we found C13–halogen substi-
tution resulted in modest reduction of potency. Based on this
knowledge, we designed and synthesized analog 7, N-methylimid-
azole-fusion at C13, C14 positions to explore changes of electronic
characteristics on the aromatic ring.
benzimidazole intermediate 4 in 43% overall yield. Compound 4
was deprotected with TFA and re-protected with TBDPS-Cl in
89% yield for two steps. The properly protected intermediate was
brominated, displaced with thiophenol and followed by TBAF desi-
lylation to afford phenol 5 in 78% yield over three steps. Phenol 5
went through the sequential MOM-Cl protection (87% yield),
OH
O
1
OH
O
O
4
O
4
N
O
N
O
O
O
14
14
OH
OH
10
OH
OH
2
1: f152A1(LL-Z1640-2)
OH
O
OH
O
O
4
O
4
R
O
The synthetic route for analog 7 is illustrated in Scheme 1. Start-
ing with readily available aromatic compound 3 (synthesized from
commercially available Methyl Orsellinate in two steps as de-
scribed in Ref.3), a six-step sequence was employed to produce
14
N
N
H
14
O
OH
N
OH
OH
OH
21,
R=Me
7
22(E6201), R=Et
* Corresponding author. Tel.: +1 978 837 4859; fax: +1 978 837 4863.
E-mail address: yuan_wang@eisai.com (Y. Wang).
Figure 1. Structures of f152A1 and synthetic analogs 2, 7, 21 and 22 (E6201).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.087

3156
Y. Shen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3155–3157
Table 1
OMOM
CO2Me
OMOM
CO2Me
OH
Structures, activity and bioavailability data for selected analogs
CO2Me
SPh
b
a
Analog
R on C14
TNF PLAP IC50 (nM)
Mouse F% po
O
MeN
OH
MeN
13
14
15
16
17
18
19
20
21
22
Et2N–
Benzyl-NH–
nPrNH–
Me2N–
H2N–
Piperazine–
Morpholine–
HO–(CH2)2–NH–
MeNH–
2066
1251
26
437
360
351
129
71
—
TMS
N
N
—
4
O
5
—
3
21
49
—
c
O
48
—
29
12
O
Me
N
TBSO
I
85
56
N
OH
EtNH–
OBz
O
OH
O
7
— Not tested.
6
Scheme 1. Synthesis of analog 7. Reagents and conditions: (a) (i) TBAF, 95%; (ii)
Among the C14-substituted analogs, both the di-N-substituted
compounds (16 and 19) and the unsubstituted amino analog (17)
showed excellent oral bioavailability in mice but their potencies
were lower than compound 7 (Table 1). Mono-substituted analogs
with large R group (14, R = Benzyl) showed no improvement either.
However, methyl substituted analog 21 demonstrated excellent
bioavailability in mice (29%) and good in vitro potency
(IC50 = 85 nM). The ethyl analog, 22, showed similar potency as
21 and its oral bioavailability in mice was acceptable.
With this success, we decided to evaluate 21 and 22 in various
inflammatory animal models. The synthetic route described above
was flexible for diverse analog synthesis in small scale. However,
the employment of selenium chemistry and long synthetic se-
quence (17 steps) presented significant challenge for larger scale
synthesis, which was necessary for in vivo studies. In order to sup-
þ
NO2 BF4ꢀ, 80%; (iii) Tf2O, 89%; (iv) BnNHMe, 75%; (v) H2, 20% Pd(OH)2/C; (iv)
HC(OEt)3, KSF clay (85% two steps); (b) (i) TFA; (ii) TBDPSCl (89% two steps); (iii)
NBS; AlBN; (iv) PhSH, Cs2CO3; (v) TBAF (78% three steps); (c) (i) MOMCl, 87%; (ii)
KOH, 74%; (iii) TMS(CH2)2OH, Ph3P, DEAD, 100%; (iv) LiHMDS, 40%; (v) mCPBA; (vi)
Et3N; (vii) TBAF; (vii) 2-chloro-1-methylpyridinium iodode; (ix) NaOH, 55% five
steps; (x) TFA, 50% two steps.
OH
N
O
OH
O
OH O
O
O
Me
N
Me
N
N
Me
N
N
OH
OH
Me
7
8
9
TNFα-PLAP(nM)
108
23
43
ACT-PLAP(nM)
>10000
49%
>10000
>10000
O
O
O
O
Oral BA (%)
P(t-Bu) , Pd(dba)
3
O
O
BOC
BOC-NH
N
H
OTf
2
Figure 2. C13–14 Benzylimidazole series.
TfO
OTf
100% C.S., 62%
24
23
saponification (74% yield), Mitsunobu esterification (100% yield),
coupling with acyclic iodide (40% yield), MCPBA oxidation, E3N
promoted elimination, TBAF deprotection, lactone cyclization un-
der the influence of Mukaiyama’s reagent,4 basic debenzoylation
(55% yield over five steps), PCC oxidation and acidic deprotection
to furnish analog 7 in 50% yield over the final two steps.
Analog 7 was potent (IC50: 23 nM in vitro) and had impressive
oral bioavailability in mouse (49%). With this encouraging result,
we further expanded to a limited set of substitution on benzimid-
azole. Analogs 8 and 9 showed slight loss of potency (IC50: 108 and
43 nM, respectively, Fig. 2). Encouraged by the SAR of C14–O series,
represented by 2, we turned our focus to analogs with simple N-
substituted side chains at C14 position. From advanced intermedi-
ate 10, Buchwald chemistry was employed to transform O-substi-
tution at C14 into N-substituent (Scheme 2).3 From intermediates
11 and 12, dozens of analogs bearing N-substitution at C14 were
prepared.5
O
O
O
BOC
N
H
OTf
OTBS
O
O
24
+
a
O
b
Boc
N
MPMO
21 or 22
TBS
O
H
PMBO
O
O
O
O
25
Scheme 3. Amination reaction and simplified synthetic route. Reagents: (a)
Pd2(DBA)3, Cy2NMe, 65%; (b) (i) LiHMDS, R–I (R = methyl of ethyl), 82%; (ii) TBAF,
imidazole-HCL; (iii) KHMDS, 76% for two steps; (iv) TBDMSCl, imidazole, 99%; (v)
DDQ, 98%; (vi) PCC, 81%; (vii) TFA, 96%.
MOM
O
O
MOM
O
O
N
O
BzO
a
11
12
R = HN
R =
O
BzO
HO
R
b
O
H2N
O
O
10
O
Scheme 2. Introducing N-substitution at C14 position. Reagents: (a) (i) Tf2O, Et3N; (ii) piperazine, Pd(OAc)2, BINAP, 21% for two steps; (b) (i) Tf2O, Et3N; (ii) benzophenone
imine, Pd(OAc)2, BINAP; (iii) NH2OHꢁHCL, NaOAc, 93% for three steps.

Y. Shen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3155–3157
3157
Table 2
In conclusion, our medicinal chemistry efforts have led to the
discovery of E6201 (22), a fully synthetic analog of f152A1 (1) with
desirable in vitro and in vivo pharmacological properties.8–10
Development of practical synthetic routes afforded SAR investiga-
tion and detailed biological evaluation. E6201 entered clinical trial.
In vivo effects of selected analogs and reference drugs on short-term arthritis
Analogs
Anti-arthritis (ED50
mg/kg)
,
Tolerated dose (TD;
mg/kg)
Ratio (ED50
TD)
/
7
5
<5
1
8
9
16
21
10
10
15
5
5–10
1
<10
<10
ꢂ60
>30
>30
3
1
1
4
6
3–6
3
3
Acknowledgment
We thank Dr. Yoshito Kishi, chairman of Eisai Scientific Advi-
sory Board on helpful discussion, scientific insight and encourage-
ment over the years. We acknowledge contribution from
researchers who supported this effort, Drs. Kenichi Chiba, Makoto
Kotake, Yoshihito Eguchi, Hideki Sakurai, Hatsue Ito-Igarashi, Aki-
fumi Kimura, Yoshikazu Kuboi, Kenzo Muramoto, Yoshiharu Mizui,
Isao Tanaka, and Takatoshi Kawai. We thank Drs. Seiichi Kobayashi
(Eisai, Japan), Ieharu Hishinuma and Nancy Wong (Eisai Inc.) for
discussion and helpful advices.
22
Indomethacin
Prednisolone
3
10
References and notes
1. Ellestad, G. A.; Lovell, F. M.; Perkinson, N. A.; Hargreaves, R. T.; McGahren, W. J.
J. Org. Chem. 1978, 43, 2339.
2. Du, H.; Matsushima, T.; Spyvee, M.; Goto, M.; Shirota, H.; Gusovsky, F.; Chiba,
K.; Kotake, M.; Yoneda, N.; Eguchi, Y.; DiPietro, L.; Harmange, J-C.; Gilbert, S.; Li,
X-Y.; Davis, H.; Jiang, Y.; Zhang, Z.; Pelletier, R.; Wong, N.; Sakurai, H.; Yang, H.;
Ito-Igarashi, H.; Kimura, A.; Kuboi, Y.; Mizui, Y.; Tanaka, I.; Ikemori-Kawada,
M.; Kawakami, Y.; Inoue, A.; Kawai, T.; Kishi, Y.; Wang, Y. Bioorg. Med. Chem.
Lett. 2009, 19, 6196.
3. A manuscript describing the SAR study on C14-oxygen substitution series has
been submitted for publication in the same journal.
4. Mukaiyama’s reagent as activating agent for carboxylic acids: (a) Mukaiyama,
T.; Usui, M.; Saigo, K. Chem. Lett. 1976, 49; (b) Narasaka, K.; Maruyama, K.;
Mukaiyama, T. Chem. Lett. 1978, 885.
Figure 3. Analog 21 CAIA efficacy results by oral dosing.9
5. All routes for analog preparations were detailed in this publication: Boivin, R.;
Chiba, K.; Chow, J.; Du, H.; Eguchi, Y.; Fujita, M.; Goto, M.; Gusovsky, F.;
Harmange, J. C.; Inoue, A.; Jiang, Y.; Kawada, M.; Kawai, T.; Kawakami, Y.;
Kimura, A.; Kotake, M.; Kuboi, Y.; Lemelin, C.; Li, X. Y.; Matsushima, T.; Mizui,
Y.; Muramoto, K.; Sakurai, H.; Shen, Y.; Shirota, H.; Spyvee, M.; Tanaka, I.;
Wang, Y. J.; Yamamoto, S.; Yoneda, N.; (Eisai Co. Ltd, Japan; et al.) PCT Int. Appl.,
2003, PCT/US03/07377, WO 2003076424 A1 20030918.
6. Buchwald–Hartwig amination: (a) Wolfe, J. P.; Buchwald, S. L. Org. Synth. 2004,
Coll. Vol. 10, 423; (b) Louie, J.; Hartwig, J. F. Tetrahedron Lett. 1995, 36, 3609.
7. (a) Heck, R. F.; Nolley, J. P., Jr. J. Org. Chem. 1972, 37, 2320; (b) Heck, R. F. Org.
React. 1982, 27, 345.
ply large amount of 21 and 22, a simplified route featuring selec-
tive Buchwald–Hartwig amination reaction and Heck reaction
were developed.6,7 Careful survey of conditions for Buchwald–Har-
twig amination reactions revealed that ditriflate 23 could be selec-
tively aminated when P(t-Bu)3 was used as the Pd ligand (Scheme
3). We found that all the amination took place at C14 position and
no undesired regio-isomer was observed. Desired amide 24 was
produced in 62% isolated yield from ditriflate 23. The coupling of
triflate 24 and acyclic olefin 25 proceeded smoothly under modi-
fied Heck conditions (65% yield after purification), which success-
fully replaced the selenium chemistry and the synthetic sequence
was shortened to nine steps from ditriflate 23 with an overall yield
of 19% (83% average yield for each step).
Using the new synthetic route, sufficient quantities of analogs
21, 22 and other analogs were produced for detailed biological
evaluation. In a collagen antibody induced arthritis model (CAIA)
in mice, a selected group of analogs were evaluated (Table 2). A
representative dosing and responses in the CAIA model were
shown in Figure 3. In this case, 21 showed potent effect with the
ED50 of ꢂ5 mg/kg by oral dosing, QD ꢃ 2. Overall, 21 and 22
showed good anti-inflammatory effects and wider therapeutic
windows in in vivo model of arthritis with ED50 around 5–10 mg/
kg (Table 2). Additional in vivo models will be reported separately.8
Based on overall profile, 22 was chosen as our clinical candidate,
and named E6201.8,9
8. A manuscript describing the detailed in vivo anti-inflammatory studies of
synthetic analogs is being prepared.
9. E6201, [(3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-
3,4,9,19-tetrahydro-1H-2-benzoxzcyclotetradecine-1,7(8H)-dione],
a
Novel
Kinase Inhibitor of Mitogen-Activated Protein Kinase/Extracellular Signal-
Regulated Kinase (MEK)-1 and MEK Kinase-1: In vitro characterization of its
anti-inflammatory and anti-hyperproliferative activities: Goto, M.; Chow, J.;
Muramoto, K.; Chiba, K.-i.; Yamamoto, S.; Fujita, M.; Obaishi, H.; Tai, K.; Mizui,
Y.; Tanaka, I.; Young, D.; Yang, H.; Wang, Y. J.; Shirota, H.; Gusovsky, F. J. Pharm.
Exp. Ther. 2009, 331, 485.
10. Detailed description on anti-arthritic effects of 21 on collagen antibody
induced arthritis model in mice: The arthritis was induced in male BALB/c
mice according to the protocol from Chondrex Inc. (Redmond, WA). The mice
started to develop arthritis on day 2 after the injection of the antibody cocktail
for type II collagen, then the mice were randomized to six groups according to
the arthritis score (mean score; 1.5–1.7). Vehicle (0.5% methyl cellulose
solution) or serial doses of ER-805940 (3, 5, 10, 20, 30 mg/kg) were orally
administered therapeutically on day
2
and
3
(QD ꢃ 2). Further arthritis
development in each mouse was evaluated on day 4, and the increased
arthritis score from the score on day 2 was calculated. Each column represents
the mean with SEM (n = 6–7) of the increased arthritis score in each treatment.
*
**
P <0.05,
control group in unpaired t-test.
P <0.01; statistically significant as compared with the vehicle