(7-Benzyloxy-2,3-dihydro- 1H -pyrrolo[1,2-a]indol-1-yl)acetic acids as S1P1 functional antagonists

Article abstract of DOI:10.1021/ml500422m

S1P₁ is a validated target for treatment of autoimmune disease, and functional antagonists with superior safety and pharmacokinetic properties are being sought as second generation therapeutics. We describe the discovery and optimization of (7-benzyloxy-2,3-dihydro-1H-pyrrolo[1,2-a]indol-1-yl)acetic acids as potent, centrally available, direct acting S1P₁ functional antagonists, with favorable pharmacokinetic and safety properties.

Full text of DOI:10.1021/ml500422m

Letter
pubs.acs.org/acsmedchemlett
(7‑Benzyloxy-2,3-dihydro-1H-pyrrolo[1,2‑a]indol-1-yl)acetic Acids as
S1P1 Functional Antagonists
Daniel J. Buzard,* Luis Lopez, Jeanne Moody, Andrew Kawasaki, Thomas O. Schrader, Michelle Kasem,
Ben Johnson, Xiuwen Zhu, Lars Thoresen, Sun Hee Kim, Tawfik Gharbaoui, Dipanjan Sengupta,
Lorene Calvano, Ashwin Krishnan, Yinghong Gao, Graeme Semple, Jeff Edwards, Jeremy Barden,
Michael Morgan, Khawja Usmani, Chuan Chen, Abu Sadeque, Weichao Chen, Ronald J. Christopher,
Jayant Thatte, Lixia Fu, Michelle Solomon, Kevin Whelan, Hussien Al-Shamma, Joel Gatlin,
Ibragim Gaidarov, Todd Anthony, Minh Le, David J. Unett, Scott Stirn, Anthony Blackburn,
Dominic P. Behan, and Robert M. Jones
Arena Pharmaceuticals, Inc., 6154 Nancy Ridge Drive, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: S1P1 is a validated target for treatment of autoimmune disease, and
functional antagonists with superior safety and pharmacokinetic properties are
being sought as second generation therapeutics. We describe the discovery and
optimization of (7-benzyloxy-2,3-dihydro-1H-pyrrolo[1,2-a]indol-1-yl)acetic acids
as potent, centrally available, direct acting S1P1 functional antagonists, with
favorable pharmacokinetic and safety properties.
KEYWORDS: Sphingosine-1-phosphate, S1P1 agonist, FTY720, fingolimod, Gilenya
phingosine-1 phosphate (1, Figure 1) is a bioactive
sphingolipid that binds to the S1P1−5 receptors and
This cell type is associated with MS development, and
populations of TH17 cells in the cerebrospinal fluid of MS
patients are generally increased.12 The majority of medicinal
chemistry efforts has focused on identifying S1P3-sparing
molecules due to S1P3-dependent bradycardia observed in
rodents treated with 2.13 Bradycardia is also observed in
humans, but it is now thought to arise through activation of
S1P1 rather than S1P3,10,14 which is consistent with the finding
that S1P3-sparing agonists have been shown to cause a
reduction in heart rate in human trials.10,15 More recently,
S1P3 has been found to be associated with pro-fibrotic
properties.16,17 This may justify continued efforts to avoid
interaction with S1P3 in the absence of compelling evidence
suggesting a prominent role in disease amelioration.
S
regulates a variety of cellular functions by signaling through
these GPCRs.1 S1P1 in particular plays an important role in
lymphocyte trafficking, and its immunomodulatory function led
to the development of Gilenya (2, fingolimod) (Figure 1), a
nonselective S1P1 agonist currently available in the US for
treatment of relapsing forms of multiple sclerosis (MS).2 In
vivo, fingolimod is phosphorylated by sphingosine kinase
(SPHK2) to (S)-monophosphate 3, which functionally
antagonizes S1P1 expressed on T-cells and B-cells through
activation (agonism), followed by internalization and protea-
somal degradation of the receptor.3,4 Loss of S1P1 prevents
lymphocytes from migrating out of the lymphatic system by
interrupting the S1P−S1P1 signaling axis responsible for
egress.5 In MS patients, this results in the sequestration of
autoreactive cells in the lymphoid tissues and occluding them
from the CNS. Additionally, fingolimod readily crosses the
blood−brain barrier and, upon phosphorylation with SPHK2,
acts directly on astrocytes via S1P1 to reduce neuro-
inflammation.6,7
Compound 3 is also an agonist of S1P3, S1P4, and S1P5. The
S1P5 receptor is present on oligodendrocytes and its activation
has been shown to improve barrier integrity and reduce trans
endothelial monocyte migration.8 Notably, several dual S1P1,5
agonists are currently being developed as new therapies for MS
(e.g., 4, Figure 1).9,10 S1P4 is expressed on lymphocytes,
hematopoietic cells, and dendritic cells (DCs), and the absence
of this receptor decreases TH17 differentiation of TH cells.11
Fingolimod is associated with a number of other events; such
as atrioventricular block, increased blood pressure, macular
edema, decreased pulmonary function, respiratory infection,
and elevated liver enzymes.18 Additionally, fingolimod is
teratogenic in rodents, and contraception in humans is required
during use and for two months following discontinuation of
treatment.18 Presumably the two month period is required due
to the relatively long half-life observed in humans (89−157 h).
Correspondingly, normalization of lymphocyte levels requires
>5 weeks following cessation of fingolimod treatment.19
A
Received: October 16, 2014
Accepted: November 23, 2014
Published: November 25, 2014
© 2014 American Chemical Society
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Table 1. Racemic 7-Benzyloxy SAR: hS1P1 EC50 [cAMP],
E
max, cLogP, and Lipophilic Efficiency (LiPE)
EC
E
maxb
50a
compd
R1
R2
(nM)
(%)
cLogP LiPE
12
13
14
15
16
17
18
19
20
CF3
CN
CF3
CF3
CN
CF3
CF3
CN
CF3
cyclopentyl
cyclopentyl
Cl
0.095
0.49
150
83
108
92
7.13
5.82
5.59
4.51
3.87
5.41
6.07
4.70
2.9
3.5
1.2
2.6
3.6
2.8
3.8
5.1
2.8
116
104
122
119
112
105
100
CN
OCH3
38
OCH2F
Oi-Pr
6.7
0.15
0.15
1.1
Oi-Pr
OCH2-cyclopropyl
6.21
a
b
EC50 values are the mean of three or more replicates. Percent
agonism relative to S1P.
our earlier series of pyrrolo[1,2a]indoles (e.g., 5, Figure 1).21 As
an extension of this earlier structure−activity relationship
(SAR), we now describe a related series of (7-benzyloxy-2,3-
dihydro-1-pyrrolo[1,2-a]indol-1-yl)acetic acids (e.g., 6) as
centrally available S1P1 functional antagonists.
Investigation of the (7-benzyloxy-2,3-dihydro-1H-pyrrolo-
[1,2-a]indol-1-yl)acetic acids began with the synthesis of 12.
This analogue was prepared by the synthetic route illustrated in
Scheme 1, wherein the key cyclopenta[a]indole ring system is
constructed in the first step using commercially available indole
7 and butyl acrylate. Subsequent decarboxylation under acidic
conditions afforded ketone 8, which provided a means to
introduce the vinyligous tert-butyl ester via a Wittig reaction.
The hydroxyl group was introduced in two steps by a palladium
catalyzed boronic ester synthesis, followed by treatment with
hydrogen peroxide. The unsaturated ester was hydrogenated
utilizing heterogeneous catalysis to afford intermediate 10.
Etherification with 4-(chloromethyl)-1-cyclopentyl-2-
(trifluoromethyl)benzene and removal of the tert-butyl ester
afforded the desired racemate 12.
Figure 1. Compounds 1 [Sphingosine-1-phosphate (S1P)], 2
[Gilenya], 3 [Gilenya monophosphate], 4 [siponimod], 5 [pyrrolo-
[1,2-a]indoles], 6 [7-benzyloxy analogues.
shorter acting agent may be desirable in the event of an
opportunistic infection where a rapid return to normal immune
functioning would be advantageous.
We, and others, have sought to avoid S1P1 modulators
requiring bioactivation (e.g., phosphorylation), instead focusing
on carboxylates as phosphate mimetics. Identification of
centrally available, nonprodrug modulators has been challeng-
ing,20 and was one factor precluding further development of
Engagement of S1P1 was evaluated in a homogeneous time-
resolved fluorescence (HTRF) cyclase assay, and 12 was found
a
Scheme 1. (7-Benzyloxy-2,3-dihydro-1-pyrrolo[1,2-a]indol-1-yl)acetic Acid Synthesis
a
Reagents and conditions: (a) butyl acrylate, NaH, toluene, 110 °C; (b) HCl, AcOH, reflux, 55% (2 steps); (c) (tert-butoxycarbonylmethylene)
triphenylphosphorane, THF, 65 °C, 72%; (d) pinacol diborane, PdCl2(dppf), dioxane, 90 °C, 100%; (e) hydrogen peroxide, NaOH, THF, rt, 78%;
(f) 10% Pd/C, hydrogen (95 psi), EtOAc, rt, 57%; (g) ArCH2Cl, Cs2CO3, DMF, 84%; (h) TFA, CH2Cl2, room temperature or LiOH, dioxane,
70 °C, 70%.
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ACS Medicinal Chemistry Letters
Letter
Table 2. Human S1P1‑5 EC50 Data
β-arrestin EC50 (nM) (Emax %)
racemate
enantiomers
S1P1 cAMP EC50, (nM) (Emax %)
S1P1
S1P2
S1P3
S1P4
S1P5
12
12a
12b
18a
18b
19a
19b
0.041 (97)
0.32 (100)
0.067 (107)
0.64 (116)
0.13 (98)
2.5 (105)
20 (100)
8.6 (108)
40 (104)
11 (128)
160 (122)
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10,000
>10000
570 (47)
420 (49)
>10000
>10000
>10000
>10000
107 (83)
81 (59)
18
19
>10000
>10000
410 (54)
9800 (133)
1.5 (104)
Table 3. Sprague-Dawley Rat Pharmacokinetics, CNS Distribution, and Lymphocyte Lowering (LL) IC50
enantiomer
Clsys (L/h/kg)
Vss(L/kg)
Cmax (μg/mL)
tmax (h)
AUC0‑inf (μg·h/mL)
t1/2 po (h)
%F
B/P
LL IC50 (ng/mL)
12a
19a
0.0721
0.131
0.986
0.732
0.526
0.569
2.67
2.00
5.43
3.99
9.16
3.99
39.0
49.8
0.53
24.1
0.016
210.0
a
racemates, as well as compound 12, were resolved into their
respective enantiomers using chiral chromatography and were
evaluated in human S1P1 HTRF, and S1P1−5 β-arrestin agonist
assays (Table 2). The absolute stereochemistry of each
enantiomer tested was not determined at this time. All test
compounds were active at S1P1 with one enantiomer in each
pair having increased potency relative to the other. A potency
shift between HTRF and β-arrestin was observed and is
consistent with what we have seen with previous chemical
series. Every enantiomer tested was selective against S1P2 and
S1P3 with 12a and 12b showing appreciable activity at S1P4 and
S1P5.
Scheme 2. Stereoselective Synthesis of 24 and 25
Given the favorable LiPE value of 5.1, 19a was selected for
rat pharmacokinetic and CNS distribution analysis (Table 3).
Compound 12a was also included in this study. A maximal
plasma concentration of approximately 500 ng/mL was
achieved for both compounds following a 1.0 mg/kg oral
dose, and each possessed adequate oral bioavailability (39−
49.8%). Compound 12a exhibited a reduced clearance rate
relative to 19a and had an elimination half-life of 9.16 h,
approximately double that of 19a. Compounds 12a and 19a
were examined in an abbreviated CNS distribution study
wherein brain and plasma drug levels were measured over a 6 h
period following a single 1.0 mg/kg oral dose. Interestingly, 19a
showed reduced central exposure relative to 12a as evidenced
by the brain-to-plasma (B/P) ratio (0.016 vs 0.53).
A rat pharmacokinetic/pharmacodynamic (PD/PK) model
was utilized to determine the plasma concentration required to
reduce lymphocyte counts by 50%. A reduction in circulating
lymphocytes is the hallmark property of an S1P1 agonist and is
predictive of its immunomodulatory potential. Lymphocyte
lowering (LL) IC50 determination showed approximately a 10-
fold difference in in vivo potency between the two analogues
(12a IC50 = 24.1 ng/mL; 19a IC50 = 210 ng/mL). This
disparity was also observed in vitro, where rat S1P1 EC50 values
in the β-arrestin platform were 2.5 (12a) and 11 nM (19a),
respectively. In our experience, β-arrestin data has been
historically a more reliable predictor of in vivo potency than
a
Reagents and conditions: (a) Cs2CO3, DMF, rt, 82% (25); (b) NCS,
CH2Cl2, rt, 96% (25); (c) NaOH, dioxane, methanol, rt, 94% (25).
to be a picomolar agonist (Table 1). Efficacy was measured
relative to S1P, and 12 was determined to have an Emax value of
108%.
We subsequently examined other 7-benzyloxy groups and
screened them in the HTRF assay (Table 1). Lipophilic
efficiency (LiPE) values were derived from the functional data
point to the 1-isopropoxy-2-(trifluoromethyl)benzene (18) and
2-isopropoxy benzonitrile (19) as preferable motifs. These
Table 4. Preclinical Species S1P1 and Human S1P1‑5 EC50 Data
preclinical species S1P1 EC50 cAMP (nM) (Emax %)
human S1P1−5 EC50 β-arrestin (nM) (Emax %)
compd
human
mouse
rat
dog
monkey
S1P1
S1P2
S1P3
S1P4
S1P5
24
25
0.025 (94)
0.021 (99)
0.050 (104)
0.064 (95)
0.072 (93)
0.050 (81)
0.049 (66)
0.049 (59)
0.038 (96)
0.058 (84)
0.63 (112)
0.62 (112)
>10000
>10000
>10000
>10000
8600 (85)
85 (29)
47 (84)
19 (59)
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Table 5. Sprague-Dawley Rat Pharmacokinetics and Lymphocyte Lowering (LL)
compd dose iv/po (mg/kg) Clsys (L/h/kg) Vss (L/kg) Cmax (μg/mL) tmax (h) AUC0‑inf (μg·h/mL) t1/2 po (h)
%F
rat LL IC50 (ng/mL)
24
25
1/1
1/1
0.192
0.900
0 831
0.618
0 584
4.00
4 67
4.21
6 16
3.44
5 09
76.4
56.6
10.4
25.4
0 0926
correspond to a B/P ratio of 1.61. A subsequent rat dose
escalation study was performed, and 25 was dose proportional
up to 1000 mg/kg po where it reached a maximal concentration
(Cmax) of 72.2 μg/mL.
In addition to rat, a PK/PD analysis was also performed in
mouse, and 25 was determined to have an in vivo lymphocyte
lowering IC50 value of 18.3 ng/mL. Given the good lymphocyte
lowering achieved in mouse, 25 was subsequently evaluated in a
prophylactic experimental autoimmune encephalomyelitis
(EAE) model that simulates MS by inducing a demyelinating
autoimmune response in female C57Bl/6 mice.23 Mice were
dosed with test compound for 24 days and scoring began on
day 10. The following scoring system was utilized: 0 = healthy;
1 = limp tail or hind limb weakness; 2 = limp tail and limb
weakness or weakness of 2 or more limbs; 3 = severe limb
weakness or single limb paralysis; 4 = paralysis of 2 or more
limbs; 5 = death. At a 1.0 mg/kg oral dose, groups treated with
either 25 or 2 remained disease free throughout the dosing
period (i.e., mean disease score = 0), whereas the vehicle
treated group reached a maximal mean disease score of 3.0 at
day 20. By day 24, 90% of the animals in the vehicle group had
developed disease. At 0.1 and 0.3 mg/kg, 25 exhibited a dose
dependent effect on prevention of disease with the 0.3 mg/kg
group maintaining a mean disease score of zero at day 24.
Minor signs of disease, however, were evident for the 0.1 mg/
kg treatment arm (i.e., day 24 mean disease score = 0.7).
The ability of 25 to activate inward-rectifying potassium
channels (IKAch) expressed on isolated human atrial myocytes
was examined in vitro, as this mechanism is believed to be in
part responsible for the transient bradycardia observed in
humans treated with S1P1 agonists. Both 25 and S1P were
tested against this G protein-gated potassium channel and were
found to have EC50 values of 8.9 and 2.1 nM, respectively. This
outcome suggests that 25 may produce a similar reduction in
heart rate as has been observed in humans treated with other
S1P1 agonists.
Further development of 25 required identification of a
suitable solid form for additional preclinical and potential
clinical studies. In the course of developing the stereoselective
synthesis of 25, this molecule was observed to form a
crystalline, high melting solid (179 °C) upon precipitation in
the final step of the synthetic sequence. Larger crystals of the
same crystal phase were grown by slow evaporation from
acetone and methanol and were suitable for X-ray structure
determination. X-ray diffraction revealed a pseudocentrosym-
metric hydrogen bonded dimer and confirmed the tentative
stereochemical assignment (Figure 2). This crystal phase was
found to be poorly soluble in water (<0.001 mg/mL) and
prompted a salt screen focused on the conjugate base of the
carboxylic acid. Various inorganic and organic counterions were
examined before identifying an L-lysine salt of 25 as the
preferred development form. The L-lysine adduct is highly
crystalline and, unlike the free acid, possesses excellent aqueous
solubility (>100 mg/mL). Subsequent physical characterization
revealed that the L-lysine salt is anhydrous, nonhygroscopic, and
has a high melting onset of 215 °C. Lastly, no thermodynami-
Figure 2. X-ray crystal structure: 25 is triclinic, space group P1, with
two independent molecules in the asymmetric unit related by a pseudo
center of symmetry forming an R22(8) hydrogen bonded dimer.
HTRF, wherein low single digit nanomolar compounds
identified in β-arrestin often are quite potent in vivo.
Suboptimal in vivo potency and limited central exposure
prompted further optimization of 19a. We found the
pyrrolo[1,2-a]indole ring system amenable to electrophilic
substitution in the 9-position, which ultimately led to the
identification of 24 and 25 as lead molecules in this chemical
series (Scheme 2). A stereoselective synthesis of the (R)-ethyl
2-(7-hydroxy-2,3-dihydro-1H-pyrrolo[1,2-a]indol-1-yl)acetate
intermediate 22 was developed22 and was utilized for
preparation of multigram quantities of enantiopure 24 and 25
(Scheme 2). The corresponding S-isomers (not depicted) were
also evaluated but were significantly less potent.
In vitro, 24 and 25 were evaluated for S1P1 activity in
multiple preclinical species and against the five human S1P
subtypes (Table 4). Activity was maintained in all species, and
neither molecule activated human S1P2 or S1P3. As anticipated,
improved β-arrestin potency (24 IC50 = 0.63 nM; 25 IC50
=
0.62 nM) translated into robust lymphocyte lowering at
relatively low plasma concentration (24 LL IC50 = 10.4 ng/
mL; 25 LL IC50 = 25.4 ng/mL) (Table 5). Rat PK was similar
for both molecules, with 25 possessing a longer elimination
half-life (5.09 h vs 3.44 h). Notably, these half-lives are
significantly lower than that observed for 12a. Compound 25
was determined to have a superior brain-to-plasma (B/P) ratio
relative to 24 (i.e., 0.27 versus 0.09) in an abbreviated rat CNS
distribution study. This may result from the increased
lipophilicity of the 1-isopropoxy-2-trifluoromethylbenzene
group. Early safety profiling of 24 and 25 revealed no affinity
for the hERG channel, and no cardiovascular effects were noted
in telemeterized rat. CYP studies showed a weak 2C9 inhibitory
potential for 24 (IC50 = 8.5 μM), while 25 did not inhibit any
of the five major isozymes (>50 μM). Selectivity profiling in a
broad panel of receptors and enzymes revealed that 24
possessed moderate affinity for rat P2Y in a [35S]dATPαS
competitive binding assay (IC50 = 390 nM), but no significant
interactions were observed for 25.
Compound 25 was selected for further preclinical studies
including a repeat dose rat CNS distribution study. Rats were
dosed with 1.0 mg/kg for six consecutive days and plasma,
brain, and cerebrospinal fluid (CSF) samples were taken over a
24 h time period following the last dose. Average plasma and
brain concentrations of 352 and 568 ng/mL were achieved and
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(9) Komiya, T.; Sato, K.; Shioya, H.; Inagaki, Y.; Hagiya, H.; Kozaki,
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Gardin, A.; Wang, N.; Crumb, W. J.; Saltzman, M.; Rosenberg, M.;
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Deficiency Profoundly Affects Dendritic Cell Function and TH17-cell
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cally more stable polymorphs were identified in physical
stability testing.
In summary, we have described the discovery and
optimization of (7-benzyloxy-2,3-dihydro-1-pyrrolo[1,2-a]-
indol-1-yl)acetic acids as a new series of selective S1P1 agonists.
These agonists were optimized for CNS penetration, in vivo
potency, and favorable pharmacokinetic and physicochemical
properties. Additional information regarding future develop-
ment of this chemical series will be reported elsewhere.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis and analytical data for compounds 12−20, 24, and
25. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: dbuzard@arenapharm.com.
■
(13) Forrest, M.; Sun, S.-Y.; Hajdu, R.; Bergstrom, J.; Card, D.;
Doherty, G.; Hale, J.; Keohane, C.; Meyers, C.; Milligan, J.; Mills, S.;
Nomura, N.; Rosen, H.; Rosenbach, M.; Shei, G.-J.; Singer, I. I.; Tian,
M.; West, S.; White, V.; Xie, J.; Proia, R. L.; Mandala, S. Immune Cell
Regulation and Cardiovascular Effects of Sphingosine 1-Phosphate
Receptor Agonists in Rodents Are Mediated via Distinct Receptor
Subtypes. J. Pharm. Exp. Ther. 2004, 309, 758−768.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
DMF, dimethylformamide; DIAD, diisopropyl azodicarbox-
ylate; THF, tetrahydrofuran; EtOAc, ethyl acetate; hERG,
■
(14) Koyrakh, L.; Roman, M. I.; Brinkmann, V.; Wickman, K. The
Heart Rate Decrease Caused by Acute FTY720 Administration is
Mediated by the G Protein-Gated Potassium Channel I IKACh. Am. J.
Transplant. 2005, 5, 527−536.
̀
human Ether-a-go-go-Related Gene; CYP, Cytochrome P450;
CSF, cerebrospinal fluid; AcOH, acetic acid; CNS, central
nervous system; NCS, N-chloro succinimide
(15) Goodman, A. News from the AAN Annual Meeting: Novel
Agent Reduces MS. Lesions in Early Trial. Neurol. Today 2012, 12,
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