Design and synthesis of new tricyclic indoles as potent modulators of the S1P1 receptor

Article abstract of DOI:10.1016/j.bmcl.2014.11.089

Modulators of S1P₁ have proven utility for the treatment of autoimmune disease and efforts to identify new agents with improved safety and pharmacokinetic parameters are ongoing. Several new S1P₁ chemotypes were designed and optimize

Full text of DOI:10.1016/j.bmcl.2014.11.089

Accepted Manuscript
Design and Synthesis of New Tricyclic Indoles as Potent Modulators of the
S1P1 Receptor
Daniel J. Buzard, Thomas O. Schrader, Xiuwen Zhu, Juerg Lehmann, Ben
Johnson, Michelle Kasem, Sun Hee Kim, Andrew Kawasaki, Luis Lopez,
Jeanne Moody, Sangdon Han, Yinghong Gao, Jeff Edwards, Jeremy Barden,
Jayant Thatte, Joel Gatlin, Robert M. Jones
PII:
S0960-894X(14)01296-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.11.089
BMCL 22244
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
7 November 2014
26 November 2014
28 November 2014
Please cite this article as: Buzard, D.J., Schrader, T.O., Zhu, X., Lehmann, J., Johnson, B., Kasem, M., Kim, S.H.,
Kawasaki, A., Lopez, L., Moody, J., Han, S., Gao, Y., Edwards, J., Barden, J., Thatte, J., Gatlin, J., Jones, R.M.,
Design and Synthesis of New Tricyclic Indoles as Potent Modulators of the S1P1 Receptor, Bioorganic & Medicinal
Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.11.089
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Design and Synthesis of New Tricyclic
Indoles as Potent Modulators of the S1P1
Receptor
Daniel J. Buzard, Thomas O. Schrader, Xiuwen Zhu, Juerg Lehmann, Ben Johnson, Michelle Kasem, Sun Hee
Kim, Andrew Kawasaki, Luis Lopez, Jeanne Moody, Sangdon Han, Yinghong Gao, Jeff Edwards, Jeremy
Barden, Jayant Thatte, Joel Gatlin and Robert M. Jones

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Design and Synthesis of New Tricyclic Indoles as Potent Modulators of the S1P1
Receptor
Daniel J. Buzard,a Thomas O. Schrader,a Xiuwen Zhu,a Juerg Lehmann,a Ben Johnson,a Michelle Kasem,a Sun Hee
Kim,a Andrew Kawasaki,a Luis Lopez,a Jeanne Moody,a Sangdon Han,a Yinghong Gao,a Jeff Edwards,a Jeremy
Barden,a Jayant Thatte,a Joel Gatlin,a and Robert M. Jonesa
a Arena Pharmaceuticals Inc. 6154 Nancy Ridge Drive, San Diego, CA 92121.
ARTICLE INFO
ABSTRACT
Article history:
Received
Modulators of S1P1 have proven utility for the treatment of autoimmune disease and efforts to
identify new agents with improved safety and pharmacokinetic parameters are ongoing. Several
new S1P1 chemotypes were designed and optimized for potency and oral bioavailability. These
new agents are characterized by a “tricyclic fused indole array” and are highly potent agonists of
the S1P1 receptor.
Revised
Accepted
Available online
Keywords:
S1P1
2014 Elsevier Ltd. All rights reserved
.
Sphingosine-1-phosphate
Gilenya®
HO
Gilenya® (1, Figure 1) is a sphingosine 1-phosphate (S1P)
receptor modulator indicated for the treatment of relapsing
remitting multiple sclerosis (RRMS). In vivo 1 is phosphorylated
to the (S)-monophosphate 2 which is a potent agonist of S1P1,
NH2
O
1
(Gilenya, R = H)
2 (Gilenya-P, R = PO(OH)2)
R
1
S1P3, S1P4 and S1P5. Sustained activation of lymphocyte
expressed S1P1 results in the internalization and degradation of
the receptor.2 Loss of S1P1 from the cell surface restricts T- and
B-cells to the lymphoid tissues and out of the periphery, thus
limiting the auto reactive potential of these cell types. This
mechanism is believed to be broadly applicable for the treatment
of various autoimmune diseases, and has encouraged efforts to
identify alternative S1P1 chemotypes.3
O
3
F3C
CO2H
cyclopenta[b]indoles
N
H
R1
We recently disclosed the medicinal chemistry effort leading
to the discovery of a new series of cyclopenta[b]indoles (e.g. 3,
Figure 1) as potent and selective S1P1 agonists.4 To expand upon
this motif, we sought to identify alternatives to the indole-fused
cyclopentane ring. We conceived of two generalized structures
that are defined by a six atom carbocycle or heterocycle, wherein
X = CH2, O, or NH (4 and 5).
R2
4
X
O
[X = CH2, O, NH)
R3
R4
CO2H
N
H
R1
R2
R3
Compound 11 was synthesized utilizing a Fischer Indole
strategy to prepare the 2,3,4,9-tetrahydro-1H-carbazole ring
system (Scheme 1). Intermediate 9 was synthesized in three steps
from 4-nitrophenol (6) utilizing sodium nitrite and tin chloride to
introduce the hydrazine. The Fischer Indole transformation was
carried out in acetic acid and was complete within one hour at 70
°C. Saponification of the resultant ester 10 with lithium
hydroxide afforded the target molecule 11.
R4
5
O
CO2H
[X = CH2, O, NH]
N
X
Figure 1. 1 (Gilenya®), 2 (monophosphate), 3
(cyclopenta[b]indoles), 4 and 5

CF3
CF3
HO
a
b
O
O
NO2
NO2
NH2
6
7
8
c
CF3
CF3
d
O
O
CO2R
N
N
NH2
H
H
10
11
9
(R = Et)
(R = H)
e
Scheme 1. (a) 4-(chloromethyl)-1-cyclopentyl-2-(trifluoromethyl)benzene, K2CO3, IPA, 80 °C, 72% (b) Zn, 3.0M aqueous NH4Cl,
MeCN, rt, 75% (c) NaNO2, 12M aqueous HCl, SnCl2, water, 0 °C to rt, 95% (d) ethyl 2-(2-oxocyclohexyl)acetate, AcOH, 70 °C, 44%
(e) 1.0 M LiOH, dioxane, rt, 66%
Scheme 2. (a) 4-(chloromethyl)-1-cyclopentyl-2-(trifluoromethyl)benzene, Cs2CO3, DMF, rt, 94% (b) methyl 3-methoxyacrylate, BF3
etherate, DCM or methyl 3-oxobutanoate, TsOH, toluene, 80 °C, 56% (c) 1.0 M LiOH, dioxane, rt, 95%
NH2
a
b, c
NH
NBoc
CO2Me
BnO
BnO
HO
CO2Me
N
N
H
N
H
H
18
19
20
d
CF3
CF3
e, f
NH
NBoc
CO2Me
O
O
CO2H
N
N
H
H
22
21
Scheme 3. (a) methyl 3,3-dimethoxypropanoate, TFA, MeCN, H2O, 80 °C, 79% (b) Pd/C, H2, MeOH/THF, 95% (c) Boc2O, THF, rt,
67% (d) 4-(chloromethyl)-1-cyclopentyl-2-(trifluoromethyl)benzene, Cs2CO3, DMF, rt (e) 4.0M HCl in dioxane, rt 62% (2 steps) (f) 1.0
M LiOH, dioxane, rt, 74%

Preparation of the 1,3,4,9-tetrahydropyrano[3,4-b]indole
analogs (16, 17) was somewhat more difficult depending on the
nature of the R4 substituent (Scheme 2). Both compounds were
derived from 13 utilizing either methyl 3-methoxyacrylate or
methyl 3-oxobutanoate to introduce the pyran ring. 16 proved the
most challenging, with the ring forming step proceeding in very
low yield (i.e. <5%). Compound 17 on the other hand, was
synthesized in moderate yield (i.e. 56%) from methyl 3-
oxobutanoate.
2,3,4,9-Tetrahydro-1H-pyrido[3,4-b]indole 22 was made by the
synthetic route outlined in Scheme 3. The synthesis was
relatively facile despite the need for several protecting group
manipulations.
A Pictet-Spengler reaction was utilized to
construct the 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole ring
system and proceeded in good yield (79%). Etherification of this
product afforded 21 which provided the target piperidinyl
carboxylic acid 22 after treatment with acid and base.
(33) affording racemic mixtures of comparable potency to 22a
and 22b.
22a was selected for further profiling and was evaluated against
mouse, rat, dog, and monkey S1P1 (Table 3). 22a was observed
to retain good potency, being in the low picomolar range across
all preclinical species. Additionally, human S1P1 selectivity was
evaluated in the β-arrestin platform, and 22a was found to
possess no agonist activity for S1P2 or S1P3. Discovery of S1P3-
sparing agonists have been a major focus in this area due to the
association of this receptor with bradycardia,6 though this effect
appears to be species dependent. 22a is an agonist of S1P4 and
S1P5, though it did not produce as large a response as S1P in this
assay. S1P4 and S1P5 may play a role in disease amelioration in
MS patients,7,8 but they are not involved in the primary
mechanism of lymphocyte trafficking. 22a was stable in mouse,
rat, and human liver microsomes (Table 4), and no significant
interaction with any of the major CYP p450 enzymes was
observed. Sprague-Dawley rat pharmacokinetics were
subsequently assessed and 22a was observed to have very low
oral bioavailability (i.e. <2%). This is perhaps due to the
zwitterionic properties of this molecule. Intravenous
administration revealed a sufficiently long half-life (9.49 h) and
Compounds 11, 16, 17, and 22 were all obtained as racemic
mixtures, thus requiring separation into their respective
enantiomers. This was accomplished chromatographically for 11,
17, and 22, but due to the low yield in preparing 16, this
compound was characterized as the racemic mixture. The
stereochemistry of each enantiomer was not determined at this
time. Human S1P1 EC50 values for these compounds were
determined in a homogeneous time resolved fluorescence
(HTRF) cyclase assay (Table 1). All of these analogs exhibited
good activity against S1P1, with the 2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indole enantiomers 22a and 22b being the most
potent in this assay.
In light of the favorable potency of the 2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indoles, a brief SAR effort focused on benzyl
substitution was performed (Table 2). Various R1, R2, and R3
substituents were explored and were consistent with the SAR
observed for the cyclopenta[b]indoles. Larger lipophilic R2
substituents improved potency, with isobutyl (32) and isopropoxy

prompted
a
lymphocyte lowering pharmacokinetic
/
deprotection gave the fully elaborated compound 44. Lastly,
chlorine could be optionally introduced in the 10-position by
treatment with NCS (e.g. 45).
pharmacodynamic (PK/PD) analysis. A reduction in circulating
lymphocytes (lymphocyte lowering) is the primary mechanism
by which S1P1 agonists exert their immunomodulating effect. An
indirect PK/PD model was created to estimate the blood
concentration of the drug needed to suppress lymphocyte levels
by 50% (IC50). 22a was determined to have an in vivo
lymphocyte lowering IC50 value of 13.0 ng/mL.
Piperiazine 47 was prepared from mesylate 46 via ammonia
promoted intramolecular cyclization. Debenzylation, alkylation,
and cleavage of the Boc and tert-butyl ester under acidic
conditions gave 50. Chlorination to afford 51 was possible via
treatment with N-chlorosuccinimide. The analogous N-methyl
adducts 67 and 68 (Table 5) were prepared in a similar matter
utilizing methyl amine instead of ammonia.
Several tricyclic motifs wherein the indole ring fusion is present
on the opposite side were also prepared (e.g. 39, 45, 51)
Preparation of indoles 39, 45 and 51 was carried out utilizing the
chemistry shown in Schemes 4 and 5. The carbocyclic indole 39
was synthesized in a similar method employed previously.5
Alkylation of commercial 34 with ethyl 4-bromobutyrate
followed by Dieckmann Condensation afforded 36. Heating this
intermediate in acetic acid and water provided 37 in good yield.
Horner-Emmons olefination followed by hydrogenation provided
phenol 38. Etherification and subsequent saponification of the
ester under basic conditions afforded the final product 39.
Morpholine 45 and piperizine 51 were constructed from common
intermediate 42, which was synthesized in three steps from
commercially available 40. Intramolecular cyclization to generate
the morpholine ring was carried out in moderate yield upon
treatment of 42 with CsF in THF. Hydrogenation, alkylation, and
The chemistry described in Schemes 4 and 5 was utilized to
synthesize a variety of analogs and human S1P1 EC50 values were
obtained (Table 5). Disubstitution was exclusively examined and
larger R2 groups provided compounds of improved potency. Of
the compounds prepared, morpholines 62-64 were the most
potent in the cyclase assay, and appear to benefit from the
chlorine in the 10-position. Racemate 63 was resolved into its
respective enantiomers using chiral chromatography, and both
enantiomers were very potent in the S1P1 agonist assay (i.e. 63a
IC50 = 0.23 nM, Emax = 93%; and 63b IC50 = 0.01 nM, Emax
=
98%). 63b was also evaluated in β-arrestin and had an EC50 value
of 0.83 nM (Emax = 112%) in this platform. Most importantly, this
enantiomer was observed to be orally bioavailable in rat when
dosed as a suspension in 0.5% methylcellulose in sterile water
BnO
BnO
BnO
OH
CO2Et
a
b
CO2Et
CO2Et
N
N
H
N
CO2Et
34
35
36
c
ArCH2O
HO
BnO
O
f, g
d, e
CO2H
CO2Et
N
N
N
39
38
37
Scheme 4.
(a) ethyl 4-bromobutyrate, tetrabutylammonium iodide, NaH, DMF, rt, 97% (b) KOt-Bu, THF, rt, 96% (c) acetic acid /
water, 220 °C, 80% (d) ethyl 2-(diethoxyphosphoryl)acetate, NaH, DMF, 65 °C, 45% (e) Pd(OH)2/C, NH4CO2H, MeOH, THF, 60
°C, 86% (f) ArCH2Cl, Cs2CO3, DMF, 75 °C, 62-84% (g) 1.0 M LiOH, dioxane, rt, 91%

Table 5.
Human S1P1 cAMP EC50 Values
R1
R2
R3
R4
10
O
CO2H
N
X
EC50, nMa
Emax (%)b
R1
R2
R3
R4
X
Compound
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
CF3
CN
CF3
CF3
OEt
CN
CF3
CF3
CN
Cl
CF3
CN
CF3
CF3
CF3
CN
H
H
CF3
OCF3
H
H
H
OCF3
H
H
H
H
H
H
H
H
H
OCF3
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
H
H
Cl
Cl
CH2
CH2
CH2
CH2
CH2
O
O
O
O
O
O
O
O
NH
NH
NMe
NMe
22
25
2.3
3.3
170
6.8
0.14
11
1.7
0.48
0.04
0.04
0.04
2.1
103
99
102
105
105
98
cyclopentyl
isopropoxy
OEt
H
cyclopentyl
OCH2F
99
101
104
94
94
104
96
101
99
101
95
isopropoxy
OCH(CH2F)2
isopropoxy
isopropoxy
cyclopentyl
isobutyl
isopropoxy
H
0.67
1.7
0.15
CF3
cyclopentyl
a EC50 values are the mean of three or more replicates. b % agonism relative to S1P (Emax).

Mediated via Distinct Receptor Subtypes J. Pharm. Exp. Ther.
2004, 309, 758-768
(%F = 39). At a 1.0 mg/kg oral dose, this molecule achieved
Cmax and AUClast levels of 0.319 µg/mL and 0.973 hr·µg/mL,
respectively. In addition, this molecule has an acceptable half-life
(t1/2) of 3.4h in rat.
7. Van Doorn, R.; Lopes Pinheiro, M. A.; Kooij, G.; Lakeman,
K.; von het hof, B.; van der Pol, S.; Geerts, D.; van Horssen, J.;
van der Valk, P.; van der Kam, E.; Ronken, E.; Reijerkerk, A.; de
Vries, H. E. Sphingosine 1-phosphate Receptor 5 Mediates the
Immune Quiescence of the Human Brain Endothelial Barrier J.
Neuroinflamm. 2012 10.1186/1742-2094-9-133.
8. Schulze, T.; Golfier, S.; Tabeling, C.; Rabel, K.; Graler, M.;
Witzenrath, M.; Lipp, M. Sphingosine-1-phosphate Receptor 4
(S1P4) Deficiency Profoundly Affects Dendritic Cell Function
and TH17-cell Differentiation in a Murine Model FASEB 2011,
25, 4024-4036.
In summary, we have described the synthesis of several new
S1P1 agonist series. One of these new S1P1 chemotypes, the
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indoles (e.g. 22a), was found
to be highly potent; however, development of this series was
limited by poor systemic exposure following oral dosing. Further
exploration revealed a morpholinyl chemical series, from which
several molecules were identified as low picomolar agonists of
S1P1. Moreover, a selected molecule from within this series was
shown to have appreciable systemic exposure in rat after oral
delivery as a suspension in 0.5% methylcellulose. Further
evaluation of this new scaffold is ongoing and results from these
studies will be reported in due course.
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