Selecting against S1P3 enhances the acute cardiovascular tolerability of 3-(N-benzyl)aminopropylphosphonic acid S1P receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2004.04.070

Structurally modified 3-(N-benzylamino)propylphosphonic acid S1P receptor agonists that maintain affinity for S1P₁, and have decreased affinity for S1P₃ are efficacious, but exhibit decreased acute cardiovascular toxicity in rodents than do nonselective agonists.

Full text of DOI:10.1016/j.bmcl.2004.04.070

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3501–3505
Selecting against S1P3 enhances the acute cardiovascular
tolerability of 3-(N-benzyl)aminopropylphosphonic acid S1P
receptor agonists
Jeffrey J. Hale,a,* George Doherty,a,ꢀ Leslie Toth,a Sander G. Mills,a Richard Hajdu,b
Carol Ann Keohane,b Mark Rosenbach,b James Milligan,b Gan-Ju Shei,b Gary Chrebet,b
James Bergstrom,b Deborah Card,b Michael Forrest,c Shu-Yu Sun,c Sarah West,c
Huijuan Xie,c Naomi Nomura,c Hugh Rosenb,ꢁ and Suzanne Mandalab
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Immunology and Rheumatology Research, Merck Research Laboratories, Rahway, NJ 07065, USA
cDepartment of Pharmacology, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 12 March 2004; accepted 19 April 2004
Available online
Abstract—Structurally modified 3-(N-benzylamino)propylphosphonic acid S1P receptor agonists that maintain affinity for S1P1,
and have decreased affinity for S1P3 are efficacious, but exhibit decreased acute cardiovascular toxicity in rodents than do non-
selective agonists.
Ó 2004 Elsevier Ltd. All rights reserved.
The immunosuppressive efficacy of FTY720 (1) has been
proposed to result from its conversion in vivo to the
corresponding phosphate ester (2), which is a potent
agonist of four of the five known sphingosine-1-phos-
phate (S1P) receptors.1;2 The systemic administration of
1 induces a dose–responsive lowering of circulating
lymphocytes and the efficacy of 1 has been attributed to
arise from this pharmacodynamic phenomenon.3 The
similarities observed for thymocyte emigration and
lymphocyte circulation in mice with S1P1 deleted from
their hematopoietic cells and normal mice treated with 1
strongly indicates that an agonist-driven functional
antagonism of S1P1 is a required component in the
mechanism of action of 1;4;5 several other recent reports
have also connected the S1P1 subtype to the efficacy of
S1P receptor agonists.6;7 The effectiveness of 1 in the
clinic has been demonstrated in allogenic renal trans-
plant patients8 and this compound has progressed into
Phase III trials.9
H3C
R
1, R = -OH (FTY720)
2, R = -OPO3H2
NH2
HO
H3C
H3C
PO3H2
NH2 OH
3
4
H
N
PO3H2
While 1 was reported to be well tolerated in Phase I
clinical trials, a transient, asymptomatic bradycardia
was an adverse event reported in 42% of the subjects.10
During the work to identify the nonselective S1P
receptor agonists 3 and 4,11;12 it was observed that these
compounds and related analogs (as well as 2) could
cause an acute toxicity in mice that appeared to be
* Corresponding author. Tel.: +1-732-594-2916; fax: +1-732-594-5966;
e-mail: jeffrey_hale@merck.com
ꢀ
Current address: Array BioPharma, 2620 Trade Center Avenue,
Longmont, CO 80503-755, USA.
ꢁ
Current address: The Scripps Research Institute, ICND-118, 10550
N. Torrey Pines Road, La Jolla, CA 92037, USA.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.070

3502
J. J. Hale et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3501–3505
cardiovascular in nature. Since this toxicity was poten-
tially mechanism based and seen with nonselective
compounds, an obvious first step in attempting to cir-
cumvent it would be to determine whether the toxicity
could be separated from the desired alterations in lym-
phocyte trafficking based on the agonism of different
S1P receptors. We wish to report herein that specific
structural alterations to 3-(N-alkyl)aminopropylphos-
phonic acid S1P receptor agonists12 afford new analogs
with high affinity for S1P1, which correlates with their
ability to lower circulating lymphocytes in rodents, but
attenuated affinity for S1P3, which correlates with an
enhanced acute tolerability in the same. A subset of
these new compounds has been employed as a tool in a
definitive demonstration that the agonism of S1P3
receptors can result in undesirable cardiovascular
effects.5
mercially available 4-(hydroxy)benzaldehyde. The ben-
zaldehydes needed for alcohol 18 and ketone 17 were
acquired by first alkylating terephthaldicarboxaldehyde
(7) with n-octylmagnesium bromide followed by oxida-
tion with Dess–Martin reagent.13 The coupling of 4-
(octyl)phenylbromide (10) and 4-(formyl)phenylboronic
acid (required for analog 20) was carried out using the
conditions described by Buchwald.14 Demethylation of
12 gave 13, which was used to prepare 26. The aldehydes
needed to prepare tetrazole 21 and 1,2,4-oxadiazole 22
were obtained by modifying the respective literature
procedures.15;16
Ligand competition studies between [33P]-S1P and all
the new compounds were carried out for each of the five
human S1P receptors stably expressed in Chinese
Hamster Ovary (CHO) cell membranes.1 S1P receptor
agonism by the test compounds was also determined by
measurement of ligand-induced [35S]-50-O-3-thiotri-
phosphate (GTPcS) binding; all of the compounds tes-
ted were found to be agonists of S1P receptors (data not
shown).17 No significant differences were observed for
compounds tested in assays conducted with mouse or
rat S1P receptors. The 3-(N-benzyl)aminopropylphos-
phonic acids in Table 1 were prepared to examine the
effect of the group linking the phenyl ring to the pendant
alkyl chain in analogs like 4 and 14. It was quickly
discovered that 4-alkoxy analogs of a proper chain
length could have S1P receptor profiles similar to 4-alkyl
analogs (compare 4 and 16). Changing the linker to a
ketone 17 decreased affinity for S1P1 by 3-fold and for
S1P3 by almost 100-fold as compared to 4 indicating
that this region of molecular space could be potentially
exploited to enhance S1P1/S1P3 selectivity. Neither
alcohol 18 nor ester 19 improved on the profile of 17.
Biphenyl analog 20 represents a different variation of the
side chain linker; a subnanomolar S1P1IC50 value was
maintained and its 200-fold selectivity for S1P1 over
S1P3 is on par with that seen for ketone 17. Heterocyclic
analogs 21 and 22 were prepared as potential hybrids of
17 and 20; while both compounds have increased affinity
for S1P3, they both maintain 100-fold selectivity for
S1P1 over S1P3. Attempts to transpose some of the side
chains of these analogs to the 3-position of the phenyl
ring or to extend the scope of linker structures to include
amides or sulfonamides gave analogs with greatly de-
creased affinity for all S1P receptors (data not shown).
The majority of the new S1P receptor agonist analogs
are 3-(N-benzyl)aminopropylphosphonic acids and were
synthesized using the previously described reductive
amination procedure (Scheme 1).12 Many of the benz-
aldehyde intermediates required for these reactions were
obtained by simply O-alkylating the appropriate com-
a
HO
CHO
O
CHO
R2
R1
R1
5
6
R1
b
N
H
PO3H2
R2
O
15, 16, 23-25, 27-42
c
HO
OHC
CHO
CHO
n-C8H17
7
8
O
d
CHO
n-C8H17
9
e
The ready availability of intermediates for the prepara-
tion of analogs of 15 allowed for their in depth investi-
gation (Table 2). For such analogs, the addition of small
substituents (–Cl, –OCH3) to the 2-position of the phen-
yl ring did not result in appreciable changes in overall
S1P receptor affinity or selectivity (data not shown). For
substituents added to the 3-position of the phenyl ring
(23–29), electron-withdrawing groups appeared to be
somewhat preferred for overall S1P receptor affinity
while increasing the size of the group was found to
influence S1P1/S1P3 selectivity as desired (compare
methoxy analog 24 to 15 or the trend 27 to 28 to 29).
Doubly flanking the 4-octyloxy group with the same
substituent could serve to enhance S1P1/S1P3 selectivity
(compare 30 to 23), but the compounds with the greatest
n-C6H13
CHO
n-C6H13
Br
11
10
12
CH3O
n-C8H17
HO
f
O
CHO
n-C8H17
O
CHO
13
Scheme 1. Reagents and conditions: (a) R2–X, K2CO3, CH3CN, reflux
(55–90%); (b) H2N(CH2)3PO3H2, 1 equiv Bu4NþOHꢀ, Na(CN)BH3,
MeOH, 50 °C (20–40%); (c) n-C8H17MgBr, THF, 0 °C (5%); (d) Dess–
Martin reagent CH2Cl2 (88%); (e) 4-(formyl)phenylboronic acid, KF,
cat. Pd(OAc)2, cat. 2-(dicyclohexylphosphino)-20-methylbiphenyl, 1,4-
dioxane, 80 °C (68%); (f) BBr3Æ(CH3)2S, CH2Cl2 (65%).

J. J. Hale et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3501–3505
Table 1. Inhibition (IC50, nM) of [33P]-S1P binding to S1P receptorsa
3503
4
H3C
X
H
N
1
3
PO3H2
2
Compd
X
S1P1
S1P2
S1P3
S1P4
S1P5
4
–CH2CH2CH2–
–CH2CH2–
–CH2CH2O–
0.2
0.6
1.5
0.3
0.9
32
750
2.7
21
40
26
0.7
5.5
14
15
16
17
18
19
>10,000
4600
840
8.5
1.1
530
74
6.3
1.7
–CH2CH2CH2O–
–CH2CH2(C@O)–
–CH2CH2CH(OH)–
–CH2O(C@O)–
8600
5400
260
110
280
250
2100
700
4.0
110
680
2.3
>10,000
20
21
22
0.9
0.5
0.5
3400
2300
1500
180
120
270
120
2.6
1.4
1.8
N
N
N
N
55
N
N
O
46
a Displacement of [33P]-labeled sphingosine-1-phosphate (S1P) by test compounds from human S1P receptors expressed on CHO cell membranes.
Data are reported as mean for n ¼ 3 determinations. SD were generally 20% of the average. See Ref. 1 for assay protocol.
Table 2. Inhibition (IC50, nM) of [33P]-S1P binding to S1P receptorsa
R2
4
H3C
O
H
N
1
PO3H2
S1P3
R1
2
Compd
R1
R2
S1P1
S1P2
4600
S1P4
S1P5
15
23
24
25
26
27
28
29
30
31
32
33
34
35
–H
–H
–H
–H
–H
–H
–H
–H
–H
1.5
0.5
1.9
2.5
14
8.5
6.0
530
32
6.3
1.8
24
–CH3
–OCH3
–OEt
–OH
–F
>10,000
>10,000
>10,000
>10,000
4700
99
74
450
420
1200
59
12
41
110
0.7
0.3
0.4
0.4
0.6
1.3
2.3
2.4
4.1
1.2
4.9
26
2.0
1.7
2.9
0.8
1.0
3.0
19
–Cl
–Br
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
55
36
–CH3
–Cl
–Br
–CH3
–Cl
–Br
44
13
15
6.6
94
23
89
57
80
–CH3
–Cl
–Br
–OCH3
–OCH3
–OCH3
1000
610
2100
8.0
10
a Displacement of [33P]-labeled sphingosine-1-phosphate (S1P) by test compounds from human S1P receptors expressed on CHO cell membranes.
Data are reported as mean for n ¼ 3 determinations. SD were generally 20% of the average. See Ref. 1 for assay protocol.
window were obtained on combining 3-substituents
previously found in analogs with enhanced S1P1 affinity
as compared to 15 with the selectivity-enhancing meth-
oxy group of 24. Compounds 33–35 all exhibit 250–500-
fold selectivity for S1P1 over S1P3 with the S1P3IC50
values for 33 and 35 being 1–2 lM.
cost of losing 3-fold in selectivity for S1P1 over S1P3 on
going from hexyl ether 36 to undecyl ether 40. Regard-
less, analogs such as 35, 38, and 39 maintain good
affinity for S1P1 while being the most selective against
S1P3 prepared as part of this work.
New compounds were screened for their ability to lower
circulating lymphocyte levels in mice as a surrogate
marker for immunosuppressive efficacy.11 Simple ether
analogs 15 and 16 and substituted octyl ethers 23, 24,
and 27–31 were all found to be lethal 1–2 min after the
administration of 4 mpk iv doses as was previously ob-
served with 4.12 Lower doses given via the peritoneal
cavity were better tolerated and several of these
A factor that influenced S1P receptor affinity in analogs
of 4 was the overall length of the lipophilic region.12 The
compounds in Table 3 are straight chain ether analogs
of 35 in which the alkyl group ranges from 6 to 11
carbon atoms. Overall affinity for all S1P receptors
(except for S1P2) does correlate with increased alkyl
chain length for these compounds, but this occurs at the

3504
J. J. Hale et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3501–3505
Table 3. Inhibition (IC50, nM) of [33P]-S1P binding to S1P receptorsa
Br
H3C
O
n
H
N
PO3H2
CH3O
Compd
n
S1P1
S1P2
6500
S1P3
S1P4
S1P5
36
37
35
38
39
40
5
6
7
8
9
7.3
13
8200
>10,000
2100
260
33
80
33
18
42
6.2
6.1
10
>10,000
>10,000
>10,000
>10,000
>10,000
4.1
1.4
0.4
1.2
830
200
3.9
1.5
1.9
10
430
a Displacement of [33P]-labeled sphingosine-1-phosphate (S1P) by test compounds from human S1P receptors expressed on CHO cell membranes.
Data are reported as mean for n ¼ 3 determinations. SD were generally 20% of the average. See Ref. 1 for assay protocol.
compounds (15, 23, 31) were found to induce a maximal
lymphocyte lowering response 3 h after an 0.1–0.25 ip
dose. The severe toxicity was found to be attenuated
when ketone 17, biphenyl analog 20, and dibromide 32
were screened while no visible signs of distress were seen
when ethers 33–40 were given similarly. Dose–titration
data for circulating lymphocyte reduction for selected
compounds is shown in Table 4; the ED50 values are
consistent with the trend observed for the S1P1 IC50s.
These new compounds did not exhibit appreciable oral
bioavailability in rodents, however pharmacokinetic
data for 20, 32, and 35 in the rat indicate that many
of these compounds would be expected to produce
comparable plasma coverage after iv administration.
against S1P3.5 First, iv bolus administration of com-
pounds with different degrees of S1P1/S1P3 selectivity to
anesthetized Sprague–Dawley rats resulted in a brady-
cardia that could be readily monitored. The extent of the
bradycardia, measured as the peak decrease in heart rate
as a percentage change from average baseline rate, was
found to be dose–responsive for compounds 2, 15, 35,
and 36 with decreased S1P3 affinity correlating with the
higher doses required to elicit similar absolute heart rate
lowering responses for the various compounds. Second,
administration of a 1.0 mpk iv bolus of the nonselective
agonist 15 to male S1P3 null mice was found to elicit a
maximal lymphocyte response at a 3 h time point and
was well tolerated. A similar experiment in wild-type
mice was found to result in death within 90 s. Taken as a
whole, these data indicate that S1P3 receptor agonism is
not required for efficacy, but does play a significant role
in the observed acute toxicity of nonselective S1P
receptor agonists.19
The severity of the acute toxicity for various test com-
pounds in mice ranged from mild to severe independent
of their selectivity for S1P1 against either S1P4 and S1P5
(e.g., compare severely toxic 15 to mildly toxic 20 or
mildly toxic 23 to well-tolerated 35), while compounds
that were at least 100-fold selective for S1P1 against
S1P3 and had absolute S1P3 IC50 values greater than
100 nM were all better tolerated. Two sets of experi-
ments were conducted to support the supposition that
these subjective observations were based on selection
In conclusion, several reports have appeared that
strongly implicate S1P1 receptors in the immunosup-
pressive efficacy of 1,4–7 while a more complete under-
standing of the undesirability of S1P3 receptor agonism
is just emerging.6;7;20 It has been shown that appropri-
ately substituted 3-(N-benzyl)aminopropylphosphonic
acid S1P receptor agonists can maintain high affinity for
S1P1, but have decreased affinity for S1P3 with the steric
constraints imposed by the added substituents appearing
to be the basis for the observed selectivity. Such changes
resulted in efficacious analogs that were less acutely
toxic in rodents. This work helps to establish that the
exploitation of structural factors that lead to the sepa-
ration of affinities for S1P1 and S1P3 represents a
practical approach for enhancing the cardiovascular
tolerability of S1P receptor agonists. The identification
and characterization of S1P receptor agonists with en-
hanced selectivity and pharmacokinetic profiles will be
the subject of future reports.
Table 4. Mouse peripheral lymphocyte loweringa (PLL) and rat
pharmacokineticb data for selected S1P receptor agonists
Compd
Murine PLL
ED50 (mpk iv)
Rat PK (1.0 mpk iv)
17
20
0.4
0.3
NDc
Clp ¼ 4:6 mL/min/kg
Vdss ¼ 1:8 L/kg, t1=2 ¼ 1:9 h
32
35
0.6
2.1
Clp ¼ 8:3 mL/min/kg
Vdss ¼ 1:0 L/kg, t1=2 ¼ 2:6 h
Clp ¼ 9:2 mL/min/kg
Vdss ¼ 1:4 L/kg, t1=2 ¼ 2:1 h
a Individual data points for dose–titrations were the average percent-
age decrease of peripheral blood lymphocyte counts in n ¼ 3 animals
versus control (n ¼ 3) 3 h after iv administration of the test com-
pound. SD were generally 20% of the average. See Ref. 11 for assay
protocol.
References and notes
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b See Ref. 18.
c Not determined.

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