Removal of sphingosine 1-phosphate receptor-3 (S1P3) agonism is essential, but inadequate to obtain immunomodulating 2-aminopropane-1,3-diol S1P1 agonists with reduced effect on heart rate

Article abstract of DOI:10.1021/jm901776q

A series of 2-substituted 2-aminopropane-1,3-diols having a biphenyl moiety and their phosphate esters were synthesized to obtain sphingosine 1-phosphate receptor-1 (S1P₁) receptor agonists with potent immunomodulatory activity accompanied by little or no effect on heart rate. Many of the synthesized compounds sufficiently decreased the number of peripheral blood lymphocytes. Some of the phosphates had potent agonism at S1P₁ but no agonism at S1P₃, which had been reported to be a receptor responsible for heart rate reduction. Although high S1P₁/S1P ₃ selectivity was considered to be favorable to reduce the effect on heart rate, almost all the phosphates showed a remarkable heart rate lowering effect in vivo. The results suggest that other factors in addition to S1P ₃ agonism should be responsible for the heart rate reduction caused by S1P₁ agonists. Only 2-amino-2-[2-[2′-fluoro-4′-(4- methylphenylthio)biphenyl-4-yl]ethyl]propane-1,3-diol (6d) was identified as a desired S1P₁ receptor agonist having both the immunomodulatory activity and an attenuated effect on heart rate by a unique screening flow using in vivo evaluating systems primarily.

Full text of DOI:10.1021/jm901776q

3154 J. Med. Chem. 2010, 53, 3154–3168
DOI: 10.1021/jm901776q
Removal of Sphingosine 1-Phosphate Receptor-3 (S1P3) Agonism is Essential, But Inadequate to Obtain
Immunomodulating 2-Aminopropane-1,3-diol S1P1 Agonists with Reduced Effect on Heart Rate
Maiko Hamada,† Mitsuharu Nakamura,† Masatoshi Kiuchi,† Kaoru Marukawa,† Ayumi Tomatsu,† Kyoko Shimano,†
Noriko Sato,† Kunio Sugahara,† Mahoko Asayama,‡ Kan Takagi,‡ and Kunitomo Adachi*,†
†Research Division, Mitsubishi Tanabe Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan, and
‡Research Division, Mitsubishi Tanabe Pharma Corporation, 1-1, Kazusakamatari 1-chome, Kisarazu, Chiba 292-0818, Japan
Received December 1, 2009
A series of 2-substituted 2-aminopropane-1,3-diols having a biphenyl moiety and their phosphate esters
were synthesized to obtain sphingosine 1-phosphate receptor-1 (S1P1) receptor agonists with potent
immunomodulatory activity accompanied by little or no effect on heart rate. Many of the synthesized
compounds sufficiently decreased the number of peripheral blood lymphocytes. Some of the phosphates
had potent agonism at S1P1 but no agonism at S1P3, which had been reported to be a receptor
responsible for heart rate reduction. Although high S1P1/S1P3 selectivity was considered to be favorable
to reduce the effect on heart rate, almost all the phosphates showed a remarkable heart rate lowering
effect in vivo. The results suggest that other factors in addition to S1P3 agonism should be responsible
for the heart rate reduction caused by S1P1 agonists. Only 2-amino-2-[2-[20-fluoro-40-(4-methylphe-
nylthio)biphenyl-4-yl]ethyl]propane-1,3-diol (6d) was identified as a desired S1P1 receptor agonist
having both the immunomodulatory activity and an attenuated effect on heart rate by a unique
screening flow using in vivo evaluating systems primarily.
Introduction
phase 3 clinical trials. Additionally, (1R,2S,3R)-2-acetyl-
4-(1,2,3,4-tetrahydroxybutyl)imidazole (THI), which inhibits
S1P lyase in vivo, caused lymphopenia-like 112 and exerted an
immunomodulatory effect.13 These facts suggest that modu-
lators of lymphocyte circulation are highly promising as an
immnunomodulator.
On the other hand, the administration of 1 was typically
associated with a mild and transient reduction in heart rate as
an adverse event in the clinical trials.14 It was reported that
intravenous administration of 1-P at a dose of 0.005 mg/kg to
normal mice evoked a severe reduction of heart rate, whereas
in S1P3-deficient mice the administration of 1-P produced no
change in heart rate.15 This genetic evidence clearly indicates
that S1P3 is mainly responsible for the heart rate decreasing
effect of 1 in mice. Consequently, S1P1-selective agonists
against S1P3 would be expected as a next generation of the
S1P receptor modulator with little or no effect on heart rate.
On the basis of this presumption, research to find selective
S1P1 agonists have been actively performed.16,17
Our attempt to obtain a desired S1P1 receptor agonist
having both the immunomodulatory activity and a reduced
effect on heart rate was initiated by reassessment of 1-related
compounds in our library. Among the phosphates of 1-related
compounds, the phosphate of biphenyl compound 2 was
found to be inactive at the S1P3 receptor, although 2 showed
much weaker potency in lymphocyte-decreasing activity than
1 (Table 1). We modified the lipophilic side chain of 2 and
succeeded in obtaining compound 6d, which had a reduced
heart rate decreasing effect as well as a sufficient immunomo-
dulatory activity (Table 4). Interestingly, it was confirmed in
this research that most of the compounds with no S1P3
agonism still reduce heart rate remarkably. This fact strongly
suggests that some complicated mechanisms independent of
Sphingosine 1-phosphate (S1Pa) is a ubiquitous lysophos-
pholipid and influences multiple physiological systems.1
Genetic deletion of S1P receptor-1 (S1P1) in mice showed that
S1P has important roles in vascular maturation and resulted in
embryonic lethality.2,3 Our group previously reported that a
unique immunomodulator 1 (FTY720, Chart 1) was discovered
by the exploration of chemically modified analogues of a natural
compound myriocin using in vivo screens as the main evaluating
systems4,5 and that compound 1showed the activity by decreasing
the number of lymphocytes in peripheral blood and lymph
drastically.6 During the course of research to clarify the mechan-
ism of action of 1, it was revealed that 1 is converted to its
phosphate (1-P) by sphingosine kinase 27 and 1-P acts as an
agonist for four S1P receptors (S1P1,3,4,5) among five known S1P
receptors (S1P1-5).8,9 Currently, a large body of evidence shows
that S1P1 plays the main role in the lymphocyte-decreasing effect
of 1.10 Thus the physiological roles of S1P and its receptors have
become clear by complementary approaches of genetics and
medicinal chemistry.
Compound 1 was shown to be effective in the treatment of
multiple sclerosis in phase 2 clinical trials11 and is currently in
*To whom correspondence should be addressed. Phone: þ81-45-963-4293.
Fax: þ81-45-963-4211. E-mail: Adachi.Kunitomo@mc.mt-pharma.co.jp.
a Abbreviations: S1P, sphingosine 1-phosphate ; S1P1, sphingosine 1-
phosphate receptor-1; THI, (1R,2S,3R)-2-acetyl-4-(1,2,3,4-tetrahydroxybu-
tyl)imidazole; DMF, N,N-dimethylformamide; DME, 1, 2-dimethoxyethane;
S-Phos, 2-dicyclohexylphosphino-20,60-dimethoxybiphenyl; X-Phos, 2-dicy-
chlohexylphosphino-20,40,60-triisopropylbiphenyl; Xantphos, 4,5-bis(di-
phenylphosphino)-9,9-dimethylxanthene; Pd2(dba)3 CHCl3, tris(dibenzyl-
3
ideneacetone)dipalladium(0)-chloroform adduct; m-CPBA, 3-chloroperoxy-
benzoic acid; DIPEA, diisopropylethylamine; DMSO, dimethyl sulfoxide;
HPLC, high performance liquid chromatography; UPLC, ultra performance
liquid chromatography; TMS, tetramethylsilane; TFA, trifluoroacetic acid.
r
pubs.acs.org/jmc
Published on Web 03/25/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3155
Chart 1. Structures of FTY720 (1) and the Active Form, FTY720-P (1-P)
Table 1. Lymphocyte-Decreasing Effect, Agonistic Activity at S1P1 and S1P3, and Heart Rate Decreasing Effect of 1 and Lead Compounds
structure
compd
A
B
ED50a (mg/kg)
phosphate
S1P1 EC50b,c (nM)
S1P3 EC50b (nM)
ID50d (mg/kg)
1
0.02
0.40
0.11
0.10
1-P
2-P
3a-P
4-P
0.35 (0.14-0.91)
1.0 (0.33-3.5)
0.64 (0.20-2.1)
1.1 (0.40-3.0)
12 (11-13)
>1000
>1000
0.012
0.091
NTe
NT
2
3a
4
3-Cl
20-F
>1000
a Dose (mg/kg) required to decrease the number of lymphocytes by 50% of vehicle-treated control in mice (5 animals for each compound). Alcohol form of
compounds was administered intraperitoneally. b EC50 (nM) of phosphate form was determined by Ca mobilization assays. 95% confidence limits are given in
parentheses. c Concentration required to show 50% of agonistic activity at S1P (300 nM). d Dose (mg/kg) required to decrease heart rate by 50% of vehicle-
treated control in rats. Phosphate form of compounds was administered intravenously (3-6 animals for each compound). e Not tested.
Table 2. Lymphocyte-Decreasing Effect, Agonistic Activity at S1P1 and S1P3, and Heart Rate Decreasing Effect of Butoxy Derivatives
structure
compd
A
B
ED50a (mg/kg)
phosphate
S1P1 EC50b,c (nM)
S1P3 EC50b (nM)
ID50d (mg/kg)
3b
3c
3d
3e
3f
0.22
0.08
0.06
3.4
3b-P
3c-P
3d-P
0.81 (0.31-2.4)
0.62 (0.22-1.8)
0.53 (0.18-1.6)
>1000
>1000
0.008
0.013
0.009
3-Cl
20-F
51 (17-150)
20-Me
20-F, 50-F
30-F
0.15
0.21
1.3
3f-P
3g-P
3h-P
1.2 (0.40-3.3)
1.3 (0.40-4.3)
3.1 (0.42-23)
>1000
>1000
>1000
NTe
NT
NT
3g
3h
30-Cl
a Dose (mg/kg) required to decrease the number of lymphocytes by 50% of vehicle-treated control in mice (5 animals for each compound). Alcohol form of
compounds was administered intraperitoneally. b EC50 (nM) of phosphate form was determined by Ca mobilization assays. 95% confidence limits are given in
parentheses. c Concentration required to show 50% of agonistic activity at S1P (300 nM). d Dose (mg/kg) required to decrease heart rate by 50% of vehicle-
treated control in rats. Phosphate form of compounds was administered intravenously (3-6 animals for each compound). e Not tested.
S1P3 or species difference would contribute to heart rate
reduction caused by 1. In this paper, we describe the details
of the discovery of 6d and the relationships between S1P3
agonism and heart rate decreasing effect.
substituents on the biphenyl moiety of 2, were approximately
3-fold more potent than 2 and their phosphates retained the
inactivity at S1P3. These results prompted us to select 2 as a
lead compound and start our research to obtain S1P1
agonists with little or no effect on heart rate.
The first step of our research procedure was the synthesis
and evaluation of the alcohol form of designed compounds.
The immunomodulating activity of the alcohol form was
estimated by counting the number of lymphocytes in peri-
pheral blood. Since promoting the sequestration of peri-
pheral blood lymphocytes in secondary lymphoid organs
dependent on the S1P1 agonism has been considered to cause
immunomodulating activity of 1, the lymphocyte-decreasing
effect in peripheral blood can be used as a marker for the
Lead Compound and Pharmacological Evaluation. To ob-
tain a lead compound that had selective activity at S1P1,
we tried to check our 1-related compound library. We
synthesized the phosphate form of compounds in this library
(e.g., 1-P) and evaluated their S1P1 and S1P3 agonistic
activities. The results revealed that 2-P having a biphenyl
moiety in the hydrophobic part had no agonistic activity
at S1P3 (Table 1). Though the lymphocyte-decreasing
effect of 2 (ED50 = 0.40 mg/kg) was weaker than that of 1
(ED50 = 0.02 mg/kg), compounds 3a and 4, which have

3156 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Hamada et al.
Table 3. Lymphocyte-Decreasing Effect, Agonistic Activity at S1P1 and S1P3, and Heart Rate Decreasing Effect of Benzyloxy and Phenoxy Derivatives
structure
compd
A
B
R
ED50a (mg/kg)
phosphate
S1P1 EC50b,c (nM)
S1P3 EC50b (nM)
ID50d (mg/kg)
3i
3j
3k
3l
5
phenyl
phenyl
0.25
0.16
0.13
0.01
0.04
3i-P
3j-P
3k-P
3l-P
5-P
21e
>1000
>1000
0.14
3-Cl
3-Cl
4.7 (0.24-92)
1.6 (0.27-8.9)
1.1 (0.25-4.0)
1.8 (0.58-5.9)
0.087
0.017
0.014
0.016
benzyl
benzyl
4-Me-phenyl
13 (3.9-44)
4.1 (2.9-5.6)
83 (35-199)
20-F
20-F
a Dose (mg/kg) required to decrease the number of lymphocytes by 50% of vehicle-treated control in mice (5 animals for each compound). Alcohol
form of compounds was administered intraperitoneally. b EC50 (nM) of phosphate form was determined by Ca mobilization assays. 95% confidence
limits are given in parentheses. c Concentration required to show 50% of agonistic activity at S1P (300 nM). d Dose (mg/kg) required to decrease heart
rate by 50% of vehicle-treated control in rats. Phosphate form of compounds was administered intravenously (3-6 animals for each compound). e This
value is reported as mean for n = 2 determinations.
Table 4. Lymphocyte-Decreasing Effect, Agonistic Activity at S1P1 and S1P3, and Heart Rate Decreasing Effect of Benzylthio and Phenylthio
Derivatives
structure
compd
A
B
R
ED50a (mg/kg)
phosphate
S1P1 EC50b,c (nM)
S1P3 EC50b (nM)
ID50d (mg/kg)
6a
6b
6c
6d
6e
6f
3-Cl
benzyl
benzyl
phenyl
0.08
0.04
0.42
0.05
0.13
0.04
0.04
0.15
0.07
0.16
0.18
0.34
0.28
0.11
6a-P
6b-P
6c-P
6d-P
6e-P
6f-P
2.1 (0.77-5.8)
0.84 (0.24-3.0)
3.6f
>1000
0.018
0.008
NTe
20-F
20-F
20-F
20-F
20-F
20-F
20-F
20-F
20-F
20-F
20-F
20-F
20-F
4.9 (3.3-7.4)
>1000
>1000
4-Me-phenyl
3-Me-phenyl
4-Me-phenyl
4-Et-phenyl
4-i-Pr-phenyl
4-F-phenyl
3.0 (0.56-16)
2.0 (0.29-14)
4.2 (0.46-38)
0.10
0.063
0.016
>1000
>1000
3-Cl
6g
6h
6i
6j
3-F-phenyl
6k
6l
4-Cl-phenyl
4-CF3-phenyl
4-MeO-phenyl
3-MeO-phenyl
6m
6n
a Dose (mg/kg) required to decrease the number of lymphocytes by 50% of vehicle-treated control in mice (five animals for each compound). Alcohol
form of compounds was administered intraperitoneally. b EC50 (nM) of phosphate form was determined by Ca mobilization assays. 95% confidence
limits are given in parentheses. c Concentration required to show 50% of agonistic activity at S1P (300 nM). d Dose (mg/kg) required to decrease heart
rate by 50% of vehicle-treated control in rats. Phosphate form of compounds was administered intravenously (3-6 animals for each compound). e Not
tested. f This value is reported as mean for n = 2 determinations.
immunomodulating efficacy of the compounds. This method
has been adopted as a simple in vivo screening since the early
stage of 1-related research when its mechanism of action was
uncertain5 and has been widely used for the evaluation of
immnomodulatory effects of S1P receptor modulators.17,18
The number of lymphocytes was counted 24 h after intraper-
itoneal administration to mice, and the results are shown as
ED50 values. Next, the phosphate esters of alcohols having a
sufficient lymphocyte-decreasing effect (ED50 < 0.20 mg/
kg) were synthesized, and their S1P1/S1P3 selectivity was
evaluated by calcium mobilization assays using transfectants
stably expressing the hS1P1 or hS1P3 receptor. The EC50
values of 1-P for S1P1 and S1P3 were 0.35 and 12 nM,
respectively. Then, the influence of the phosphates on heart
rate was determined by intravenous administration to an-
esthetized rats. Compound 1-P lowered heart rate inten-
sively, and the ID50 value was 0.012 mg/kg. Pharmacokinetic
profiles usually reflect to in vivo results; however, the max-
imal heart rate reduction in this experiment was observed
promptly (in 1 min) after the intravenous administration of
1-P. Therefore, we assumed that the potency of phosphates
on heart rate decreasing effect could be evaluated with little
influence of pharmacokinetic difference among the com-
pounds by this method.
At the beginning of this study, we planned to evaluate
mainly the in vitro S1P receptor selectivity of the phosphates
because searching for S1P1 selective compounds seemed to
be a reasonable approach to obtain compounds with a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3157
Scheme 1. Synthesis of Intermediate Bromides 9a
a Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, rt; (b) NaI, 2-butanone, reflux; (c) N-protected diethyl aminomalonate, NaH, DMF, 60 °C; (d) NaBH4,
CaCl2, EtOH/H2O, rt; (e) Ac2O, pyridine, rt; (f ) 2,2-dimethoxypropane, p-TsOH H2O, acetone, rt; (g) (i) 4 M HCl/EtOAc, (ii) Ac2O, pyridine, rt.
3
Scheme 2. Synthesis of Biphenyl Compounds 3a
a Reagents and conditions: (method A) boronic acid, Pd(PPh3)4, NaHCO3, DME/H2O, 65 °C; (method B) (i) bis(pinacolate)diboron, PdCl2(Cy3P)2, AcOK,
dioxane, 100 °C, (ii) phenyl bromide, Pd(OAc)2, S-Phos, K3PO4, toluene, 100 °C; (a) 35% HCl, EtOH, reflux; (b) LiOH, THF/MeOH/H2O, reflux.
reduced effect on heart rate. On the basis of the result in S1P3
knock out mice, we expected that 2-P had no heart rate
deceasing effect. However 2-P clearly decreased heart rate.
Furthermore, the heart rate decreasing effect of 2-P was not
reduced at all despite the loss of S1P3 agonism. Although the
ID50 value of 2-P (0.091 mg/kg) was higher than that of 1-P
(0.012 mg/kg), the lymphocyte-decreasing effect of 2 was
much weaker than that of 1 (0.40 mg/kg/0.020 mg/kg). This
observation made us presume that in vitro agonistic activity
at S1P3 does not always correlate well with the in vivo
potential of heart rate reduction. Therefore, we decided to
adopt an in vivo evaluation of the effect of test compounds
on heart rate in addition to in vitro assay.
On the basis of the above preliminary results, the research
flow in this study was established as follows: (i) designing
compounds and syntheses of the alcohol form, (ii) determi-
nation of the lymphocyte-decreasing effect of the alcohols,
(iii) syntheses of the phosphate of sufficiently effective
compounds (ED50 < 0.20 mg/kg) and key compounds,
and (iv) evaluations of the heart rate decreasing effect in
anesthetized rats and in vitro S1P1/S1P3 selectivity of the
phosphates.
Synthesis. Scheme 1 describes the synthesis of inter-
mediate bromides 9. After conversion of phenethyl alco-
hols into iodides 7, condensation of 7 and N-protected
diethyl aminomalonate gave diesters 8. The ester groups
of 8 were reduced to hydroxymethyl groups, which
were subsequently protected to give the corresponding
bromides 9.
Biphenyl compounds 3a-l were synthesized from bro-
mides 9 through Suzuki coupling, followed by removal of the
protecting groups (Scheme 2).
The syntheses of compounds 4 and 5 were carried out as
shown in Scheme 3. Hydrogenolysis of biphenyl compound 10l
gave phenol 11. After activation of the phenolic hydroxy group
of 11 by conversion into a triflate, Sonogashira reaction of 12
and 1-pentyne, hydrogenation, and removal of the protecting
groups yielded the desired compound 4. The diaryl ether 5 was
obtained by coupling of phenol 11 and p-tolylboronic acid
using copper acetate, followed by deprotection.
Diaryl sulfides 6a-n were prepared from phenyl bromides
9 through biphenyl bromides 13 (Scheme 4). The bromides
13 transformed from 9 by a similar palladium-mediated
coupling to that described in Scheme 2 were converted to
the diaryl sulfides 6a-n by a coupling reaction using 4,5-
bis(diphenylphosphino)-9,9-dimethylxanthene as the phos-
phine ligand followed by deprotection.
The phosphate esters of 3, 4 and 5 (3-P, 4-P, and 5-P,
respectively) were prepared as outlined in Scheme 5. First,
the 2-aminopropane-1,3-diols were converted into oxazo-
lines to protect the amino group and one of the hydroxyl
groups. The remaining hydroxyl group was phosphorylated
by a phosphoramidite with 1H-tetrazole, followed by oxi-
dation to give the protected phosphate esters 14. Desired
phosphate esters were obtained by removing all protective
groups under acidic conditions.
The phosphate esters of compounds 6, which have a
sulfur atom, were synthesized through another route shown
in Scheme 6. Bromides 13 were phosphorylated to give
intermediates 15 before coupling with thiols because the
process of phosphorylation included an oxidative step.
Compounds 6-P were prepared by the palladium coupling
reaction of intermediates 15 with thiols, followed by acidic
deprotection.

3158 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Scheme 3. Synthesis of Compounds 4 and 5a
Hamada et al.
a Reagents and conditions: (a) 10% Pd/C, NH4CO2H, MeOH, rt; (b) Tf2O, pyridine, CH2Cl2, rt; (c) 1-pentyne, PdCl2(CH3CN)2, X-Phos, Cs2CO3,
CH3CN, 60 °C; (d) LiOH, MeOH/H2O, reflux; (e) 10% Pd/C, MeOH, rt; (f) 4 M HCl/EtOAc, MeOH/EtOAc, rt; (g) p-tolylboronic acid, Cu(OAc)2,
˚
pyridine, 4 A molecular sieves, CH2Cl2, rt.
Scheme 4. Synthesis of Diaryl Sulfides 6a
a Reagents and conditions: (a) bis(neopentyl glycolate)diboron, PdCl2(Cy3P)2, AcOK, dioxane, 100 °C; (b) iodobenzene, Pd(PPh3)4, NaHCO3,
DME/H2O, 100 °C; (c) thiol, Xantphos, Pd2(dba)3 CHCl3, DIPEA, dioxane, reflux; (d) 35% HCl, EtOH, 70 °C.
3
Scheme 5. Phosphorylation of 3, 4, and 5a
a Reagents and conditions: (a) CH3C(OEt)3, DIPEA, DMF, 120 °C; (b) (i) (t-BuO)2PN(i-Pr)2, 1H-tetrazole, CH2Cl2, rt; (ii) m-CPBA, rt; (c) 35% HCl,
EtOH, 50 °C.
Results
First, we searched for a strategy to improve the potency of
activity at S1P3. Replacing the fluorine with methyl (3e)
resulted in notably reduced activity. In attempts to increase
the number of fluorine (3f) and to change the substitution
position of halogens (3g, 3h), no improvement in activity was
observed compared to 3b although these phosphates had no
S1P3 agonism. The phosphates of the compounds having high
activity (3b-P, 3c-P, and 3d-P) were evaluated for the heart
rate decreasing effect. Compound 3d-P, which had agonistic
activity at S1P3, reduced heart rate intensively (ID50 = 0.009
mg/kg), supporting the hypothesis that the heart rate reduc-
tion caused by 1 is associated with S1P3 agonism. On the other
hand, ID50 values of 3b-P and 3c-P were also low (0.008 and
0.013 mg/kg, respectively) in spite of their inactivity at S1P3.
This result contradicts the reported observation that 1-P
had no effect on heart rate in S1P3-deficient mice. Although
the reason why our results of compound-based approach
disagreed with the genetic observation was not clear, we
lymphocyte-decreasing effect exhibited by 2 (Table 2). Repla-
cement of the pentyl group of 2 with butoxy (3b) resulted in a
slight increase in in vivo activity, and the agonistic activity of
the phosphate (3b-P) at S1P1 was slightly potentiated. This
result suggested that the heteroatom was tolerated as a linkage
between the biphenyl and the side chain binding to the 40
position. In addition, the heteroatom linkage was useful for
access to various types of side chains. To improve the potency
of 3b, introduction of chlorine or fluorine into the biphenyl
moietywas carried out. The introductions of chlorineintoring
A (3c) and fluorine into ring B (3d) were both useful to
improve the in vivo activity of 3b like the case of 2. The
fluorine on ring B was more effective than the chlorine on ring
A to improve in vivo activity; however, 3d-P showed agonistic

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3159
Scheme 6. Synthesis of Phosphate Esters of 6a
a Reagents and conditions: (a) 35% HCl, EtOH, 80 °C; (b) CH3C(OEt)3, DIPEA, DMF, 120 °C; (c) (i) (t-BuO)2PN(i-Pr)2, 1H-tetrazole, CH2Cl2, rt;
(ii) t-Butyl hydroperoxide, rt; (d) thiol, Xantphos, Pd2(dba)3 CHCl3, DIPEA, dioxane, reflux; (e) 35% HCl, EtOH, 50 °C.
3
continued our research mainly using in vivo evaluating system
rather than in vitro S1P1/ S1P3 selectivity.
phenyl ring to retain the total length of the side chain. Both the
para- and meta-substituted derivatives, 6d and 6e, respectively,
showed improved activity in terms of lymphocyte-decreasing
effect. Especially, 6d was equipotent to 6b. The results of the
phosphates (6d-P and 6e-P) in the heart rate measuring study
were interesting. The heart rate decreasing effect of 6d-P was
attenuated. More precisely, the ID50 values of 6d-Pand 1-Pwere
0.10 and 0.012 mg/kg, respectively whereas their ED50 values on
lymphopenia (0.05 and 0.02 mg/kg) were closer than their ID50
values. On the other hand, 6e-P produced more intense reduc-
tion of heart rate than 6d-P although 6e-P had a similar in vitro
profile to 6d-P. Chlorine was introduced into ring A of 6d to
explore more active compounds. The compound 6f was equi-
potent to 6d, but the phosphate 6f-P showed a strong effect on
heart rate like 1-P. Next, the substituents on the terminal phenyl
were examined. Although replacement of the methyl group of 6d
with an ethyl group (6g) was tolerated, replacement with a more
bulky group (6h) yielded less lymphocyte-decreasing activity.
Adoption of halogens (6i, 6j, and 6k) also led to weaker activity.
Among the halogens, fluorine (6i) was preferable to chlorine
(6k), and para-substituted derivative (6i) was better than meta-
substituted derivative (6j) like the case of the methyl substituent
(6d and 6e). Introduction of a trifluoromethyl group into
unsubstituted compound 6c did not influence activity (6l). The
activity of meta-methoxy compound 6n was stronger than that
of para-substituted compound 6m. The modification of the
terminal phenyl of 6d resulted in no significant improvement.
Consequently, 6d was the only compound having both the
significant lymphocyte-decreasing activity and the reduced effect
on heart rate in this study.
The results of replacement of the butoxy group of 3b with
phenoxy or benzyloxy groups were summarized in Table 3.
Compound 3i exhibited comparable lymphocyte-decreasing
activity to the butoxy analogue 3b regardless of weak S1P1
agonistic activity of the phosphate 3i-P. The heart rate decreas-
ing effect of 3i-P (ID50 = 0.14 mg/kg) was remarkably weaker
than that of 3b-P (0.008 mg/kg), suggesting that the phenyl ring
in the side chain would be a favorable substituent in order to
reduce the effect on heart rate without losing lymphocyte-
decreasing activity. The introduction of chlorine on ring A (3j
and 3k) led to a slight improvement in lymphocyte-decreasing
activity, and the introduction of fluorine on ring B (3l) drasti-
cally improved the activity equal to that of 1. The phosphate of
3l (3l-P) still had S1P3 agonistic activity; however, 5-P which
had a tolyl group instead of the benzyl group showed a weaker
agonistic activity at S1P3 than 3l-P. On the other hand, 5
maintained a comparable lymphocyte-decreasing effect to 3l.
These findings verified that the conversion of the alkoxy group
into the phenoxy or benzyloxy group was effective in increasing
the lymphocyte-decreasing effect and that the p-tolyl group was
a substituent to attenuate the S1P3 agonism. The phosphates of
compounds having the improved activity (3k-P, 3l-P, and 5-P)
strongly decreased heart rate like 1-P irrespective of their
different potencies of S1P3 agonism. Consequently, no desired
compounds having both potent lymphocyte-decreasing activity
and little or no effect on heart rate were found in Table 3.
Our next attempt was replacement of the side chain of 3b
with a phenylthio or benzylthio group (Table 4).
The most advantageous compound 6d was subjected to
detailed pharmacological evaluations. Compounds 1 and 6d
had comparable oral lymphocyte-decreasing activity in rats
with ED50 values of 0.040 mg/kg (0.029-0.056; 95% confi-
dence limit) and 0.084 mg/kg (0.064-0.11; 95% confidence
limit), respectively. Next, the effect of orally administered 6d on
heart rate was measured in conscious rats using a telemetry
system. Compound 1 significantly reduced the heart rate at
doses of 3 mg/kg and higher in this test. On the other hand, 6d
had no clear influence on heart rate at a dose of 10 mg/kg
(Figure 1). These data indicated that 6d have both the sufficient
immunomodulatory activity and a low adverse action on heart.
Because we established and reported an efficient synthetic
method for the preparation of diphenyl sulfide in the research of
S1P receptor modulators,19 the method was applied to the
biphenyl compounds in this study. First we replaced the side
chain with the benzylthio group because the benzyloxy deriva-
tives 3k and 3l showed good activity. Compound 6a, whichhasa
chlorine atom on ring A, exhibited comparable activity to the
phenoxy analogue 3k. Changing chlorine into fluorine (6b) led
to better in vivo activity. However, the phosphate of 6b (6b-P)
showed miserable results in S1P1/S1P3 selectivity and the heart
rate decreasing effect. The potency of 6b-P on heart rate
decreasing (ID50 = 0.008 mg/kg) was even higher than that of
1-P(0.012 mg/kg). On the basis of results of Table 3, we expected
that replacement of the benzylthio group with the phenylthio
group could be effective to reduce agonistic activity at the S1P3
receptor. Fortunately, our expectation was supported by the
result that 6c-P was not an agonist at S1P3 although the
lymphocyte-decreasing effect of 6c was approximately 10-fold
weaker than that of 6b. Next, we added substituents on the
terminal phenyl ring of 6c to improve the activity. Because the
phenylthio group is smaller than the benzylthio group by one
methylene, a methyl group was introduced into the terminal
Discussion
The genetic studies using fetal liver chimeric mice with
specific deletion of S1P1 from hematopoietic cells and S1P3-
deficient mice revealed that S1P1 and S1P3 regulate lympho-
cyte circulation and reduction of heart rate, respectively.10,15
It was reported that KRP-203 (not shown), which has the
same 2-aminopropane-1,3-diol skeleton as 1, had a compar-
able lymphocyte-decreasing effect to 1 and a reduced heart
rate decreasing effect.20 It appears that KRP-203 reduced the

3160 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Hamada et al.
Figure 1. Effects of compounds 1 (A) and 6d (B) on heart rate in conscious rats (10 mg/kg, po). ΔHeart rate means the change (beat/min) of
post-treatment value (day 0) compared to the mean of pretreatment values (from day -3 to day -1) at the corresponding time of each day in the
same animals. *: p < 0.05, ** : p < 0.01, significantly different from the corresponding value in the vehicle group (n = 3 or 4, paired t-test).
Vehicle background date represent the mean ( SD of each time point data (n = 38).
effect on heart rate to almost the same level as compound 6d
according to the report. The phosphate (KRP-203-P) was an
agonistatS1P1, butinactive atS1P3. TheS1P1/S1P3 selectivity
was presumed to be important for reduction of the effect on
heart rate.
reduced effectonheart rateamong them. Thestructureof6dis
similar to that of KRP-203, which has two additional phenyl
rings compared to 1 and a sulfur atom in the hydrophobic
part. These structural and physicochemical properties may
relate to the reduced effect on heart rate of both compounds.
Furthermore, the pathogenic mechanism of S1P receptor
modulators on the heart rate reduction may be revealed by
using compounds having a similar structure and a different
potency on heart rate reduction (e.g., 6d and 6f).
Because S1P3 agonism showed no consistent relationship to
the heart rate decreasing effect at the early stage of this
research, the in vivo evaluation in rats was promptly adopted
to estimate the potency of the phosphates for heart rate
reduction. Thereby we constructed the original screening
strategy based on not only in vitro S1P1/S1P3 selectivity but
also simple in vivo systems and found compound 6d whose
effect on heart rate was attenuated. The detailed pharmaco-
logical evaluation revealed that oral administration of 6d in
rats provided the excellent lymphocyte-decreasing effect and
the attenuated effect on heart rate. To the best of our knowl-
edge, this is the first successful example on finding S1P1
agonists with sufficient immunomodulatory activity and little
or no effect on heart rate by a medicinal chemical approach.
In the beginning of our research, we identified 2 as a lead
compound that was inactive against S1P3 on this hypothesis
by the screening of our 1-related compound library. Then the
pentyl group of 2 was converted into alkoxy, phenoxy, and
phenylthio groups to improve the activity. As a result, lots of
compounds showed a remarkable lymphocyte-decreasing
effect. All the phosphates of the favorably active compounds
having S1P3 agonism (e.g., 3k-P, 3l-P, 5-P, and 6b-P) pro-
voked an intensive decrease of heart rate. This result sup-
ported that S1P3 was responsible for the reduction of heart
rate caused by 1. Additionally, the fact that 6d-P was inactive
at S1P3 receptor-like KRP-203-P indicates that elimination of
S1P3 receptor agonism shouldbenecessary toreduce the effect
on heart rate. However, most of the phosphates having no
agonism at S1P3 also significantly influenced heart rate. For
instance, the phosphates of both meta-methyl (6e-P) and
chloro-substituted (6f-P) derivatives remarkably lowered
heart rate, although neither of them showed any agonistic
activity at S1P3. This fact contradicted the result of the genetic
study that 1 had no effect on heart rate in S1P3-deficient mice
and exploded the previously reported assumption that the
research of compounds having no agonism at S1P3 would
directly lead to find the desirable S1P1 agonist with little or no
effect on heart rate. Although the cause of the disagreement
between the genetic and our compound-based results is
unclear, there are some possibilities: (i) species difference
of the heart rate reduction mechanism between the S1P3-
deficient mice15 and the rats used here, (ii) difference of
intravital environments between S1P3-deficient mice and
wild-type animals exposed to no exogenous S1P3 agonism
but to endogenous S1P3 agonism by natural S1P, and
(iii) existence of other signaling pathways undetectable by
using our calcium mobilization assay.21 It is likely that
other factors in addition to S1P3 participate in the heart
rate reduction caused by S1P receptor modulators.
Conclusion
2-Substituted 2-aminopropane-1,3-diols having a biphenyl
moiety were synthesized to obtain immunomodulating S1P1
receptor agonists with little or no effect on heart rate. The
phosphate of 2 was identified as a lead compound with S1P1
selectivity against S1P3 by a screening of 1 analogue library.
The structural evolution of 2 led to lots of biphenyl compounds
showing considerable lymphocyte-decreasing effect. Several
phosphate esters of them were potent S1P1 receptor agonists
having high selectivity against S1P3; however, significant ef-
fects on heart rate remained in almost all of the phosphates.
The results suggested that other factors in addition to S1P3
agonism should be considered to clarify the cause of heart
rate reduction by S1P1 receptor agonists. The syntheses of
analogues and our original screening strategy using in vivo
evaluating systems primarily revealed that 6d had potent
lymphocyte-decreasing effect by intraperitoneal administra-
tion to mice and that the effect on heart rate of the phosphate
Although it is relatively easy to obtain compounds without
S1P3 agonism, it is rather difficult to find compounds with a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3161
6d-P was attenuated by intravenous administration to anesthe-
tized rats. It was then confirmed that the oral administration of
6d to rats brought a similar immnomodulatory efficacy to that
of 1 and no influence on heart rate at a dose of 10 mg/kg.
room temperature overnight, 1 M aqueous HCl was added to
the reaction mixture and the mixture was extracted with EtOAc.
The organic layer was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was dissolved in pyr-
idine (300 mL). Acetic anhydride (25.0 mL, 340 mmol) was
added to the solution at 0 °C. After stirring at room temperature
overnight, ice and water (300 mL) were added to the reaction
mixture. The mixture was extracted with EtOAc, and the
organic layer was washed successively with water, 0.5 M aqu-
eous HCl, saturated NaHCO3, and brine. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue was
precipitated from hexane/EtOAc (1/1) to give the title com-
pound (11.4 g) as a white solid. The mother liquid was concen-
trated in vacuo. Purification of the residue by silica gel column
chromatography (hexane/EtOAc) gave the second crop of the
title compound (4.8 g). The total yield was 16.2 g (46%).
1H NMR (CDCl3) δ 1.99 (3H, s), 2.10 (6H, s), 2.16-2.22 (2H,
m), 2.53-2.59 (2H, m), 4.32 (4H, s), 5.68 (1H, brs), 7.06 (2H, d,
J = 8.4 Hz), 7.40 (2H, d, J = 8.4 Hz).
Experimental Section
Chemistry. Silica gel column chromatography was performed
on a Moritex Purif-R2 system using Purif-Pack 30 or 60 μm silica
gel columns and the described solvents as eluent under gradient
condition. Preparative HPLC was performed on a Shimadzu
HPLC system using Capcell Pak C18 UG80 (5 μm 20 mm ꢀ 250
mm) under gradient condition (CH3CN/H2O þ 0.05% TFA).
1H NMR spectra were recorded on a Bruker AVANCE 400 (400
MHz) spectrometer. Chemical shifts are expressed in ppm
relative to tetramethylsilane (TMS) as an internal standard.
Mass spectra were measured in a combination with a Waters
Acquity UPLC system (CH3CN þ 0.05% TFA/H2O þ 0.05%
TFA) and a Micromass ZQ (ESI) spectrometer. Melting points
N-[5-[2-(4-Bromophenyl)ethyl]-2,2-dimethyl-1,3-dioxane-5-yl]-
acetamide (9b). NaBH4 (21.8 g, 576 mmol) was added portion-
wise to a solution of 8a (57.8 g, 144 mmol) and CaCl2 (32.0 g, 288
mmol) in water (100 mL) and EtOH (500 mL). After stirring at
room temperature overnight, 1 M aqueous HCl was added to
the reaction mixture, and the mixture was extracted with
EtOAc. The organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was dissolved
in acetone (200 mL). 2,2-Dimethoxypropane (41.9 g, 402 mmol)
and a catalytic amount of p-toluenesulfonic acid monohydrate
was added to the solution. After stirring at room temperature
overnight, the mixture was concentrated in vacuo. The residue
was washed with saturated NaHCO3 to give the title compound
as a white solid (41.5 g, 81%). 1H NMR (CDCl3) δ 1.43 (6H, s),
2.03 (3H, s), 2.01-2.06 (2H, m), 2.46-2.52 (2H, m), 3.67 (2H, d,
J = 11.7 Hz), 3.95 (2H, d, J = 12.0 Hz), 5.76 (1H, brs), 7.05 (2H,
d, J = 8.4 Hz), 7.38 (2H, d, J = 8.4 Hz).
2-Acetamido-2-[2-(4-bromo-2-chlorophenyl)ethyl]propane-1,3-
diol Diacetate (9c). NaBH4 (66.6 g, 1760 mmol) was added
portionwise to a solution of 8b (217 g, 440 mmol) and CaCl2
(97.7 g, 880 mmol) in THF (543 mL), water (1085 mL), and
EtOH (2170 mL) at 0 °C. After stirring at room temperature for
14 h, 1 M HCl (1085 mL) was added to the reaction mixture and
the mixture was extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, and concentrated in
vacuo. The residue was dissolved in 4 M HCl solution in EtOAc
(326 mL). After stirring at 30 °C for 1 h, the mixture was
concentrated in vacuo. Aqueous NaOH (0.1 M, 2713 mL) was
added to the reaction mixture. The mixture was extracted with
EtOAc, and the organic layer was washed with brine. The
organic layer was dried over MgSO4 and concentrated in vacuo.
The residue was dissolved in pyridine (271 mL). Acetic anhy-
dride (270 mL) was added to the solution at 0 °C. After stirring
at room temperature for 11 h, EtOAc (2710 mL), 1 M NaOH
(1084 mL), and 9% NaHCO3 (5420 mL) were added to the
reaction mixture. The mixture was extracted with EtOAc, and
the organic layer was washed successively with 1 M HCl, 9%
NaHCO3, and brine. The organic layer was dried over Na2SO4
and concentrated in vacuo. The residue was precipitated from
IPE/n-heptane (1/1) to give the title compound (135 g, 62%) as a
white solid. 1H NMR (CDCl3) δ 2.00 (3H, s), 2.10 (6H, s),
2.12-2.16 (2H, m), 2.66-2.72 (2H, m), 4.36 (4H, s), 5.75 (1H,
brs), 7.10 (1H, d, J = 8.2 Hz), 7.32 (1H, dd, J = 1.9, 7.5 Hz), 7.70
(1H, d, J = 1.9 Hz).
€
were obtained on a Buchi 535 melting point apparatus and are
uncorrected. Elemental analyses were performed on a Perkin-
Elmer 2400 II CHN analyzer. All compounds showed >95%
purity, according to UPLC or the elemental analyses.
2-(4-Bromophenyl)ethyl Iodide (7a). Methanesulfonyl chlor-
ide (11.5 mL, 148 mmol) was added to a solution of 2-(4-
bromophenyl)ethyl alcohol (25.0 g, 124 mmol) and triethylamine
(22.6 mL, 163 mmol) in CH2Cl2 (250 mL) at 0 °C. The suspension
was stirred at room temperature for 2 h. The reaction mixture was
washed with brine, dried over Na2SO4, and concentrated in vacuo
to yield a red oil (39.1 g). Sodium iodide (18.6 g, 124 mmol) was
added to a solution of the oil in 2-butanone (400 mL), and the
suspension was heated under reflux for 4.5 h. The reaction mixture
was diluted with EtOAc and washed with water, 10% aqueous
sodium thiosulfate, and brine. The organic layer was dried over
Na2SO4 and concentrated in vacuo. Purification of the residue by
silica gel column chromatography (hexane/EtOAc) gave the title
compound (34.2 g, 91%) as a colorless oil. 1H NMR (CDCl3) δ
3.13 (2H, t, J = 7.5 Hz), 3.32 (2H, t, J = 7.5 Hz), 7.07 (2H, d, J =
8.4 Hz), 7.44 (2H, d, J = 8.4 Hz).
2-(4-Bromo-2-chlorophenyl)ethyl Iodide (7b). The title com-
pound was synthesized from 2-(4-bromo-2-chlorophenyl)ethyl al-
cohol using a similar procedure to that described for 7a. 1H NMR
(CDCl3) δ 3.22-3.28 (2H, m), 3.32-3.38 (2H, m), 7.12 (1H, d, J =
8.1 Hz), 7.36 (1H, dd, J = 1.8, 8.4 Hz), 7.53 (1H, d, J = 2.1 Hz).
Diethyl 2-Acetamido-2-[2-(4-bromophenyl)ethyl]malonate (8a).
A solution of diethyl acetamidomalonate (28.7 g, 132 mmol) in
DMF (55 mL) was added dropwise to a suspension of NaH (60%
dispersion in mineral oil, 5.72 g, 143 mmol) in DMF (110 mL) at
0 °C. After stirring at room temperature for 45 min, 7a (34.2 g,
110 mmol) was added dropwise to the reaction mixture at 0 °C.
After stirring at room temperature for 1 h and an additional 2 h at
60 °C, the reaction mixture was poured into iced water and
extracted with EtOAc. The organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo. Purification of the
residue by silica gel column chromatography (hexane/EtOAc)
gave the title compound (35.4 g, 78%) as a white solid. 1H NMR
(CDCl3) δ 1.23 (6H, t, J = 7.2 Hz), 2.00 (3H, s), 2.44 (2H, dd, J =
9.0, 10.8 Hz), 3.32 (2H, dd, J = 9.0, 10.8 Hz), 4.16-4.25 (4H, m),
6.76 (1H, brs), 7.02 (2H, d, J = 8.4 Hz), 7.38 (2H, d, J = 8.4 Hz).
Diethyl 2-[2-(4-Bromo-2-chlorophenyl)ethyl]-2-(tert-butoxy-
carbonylamino)malonate (8b). The title compound was synthe-
sized from 7b and diethyl tert-butoxycarbonylaminomalonate
1
using a similar procedure to that described for 8a. H NMR
(CDCl3) δ 1.26 (6H, t, J = 7.1 Hz), 1.44 (9H, s), 2.57 (4H, brs),
4.16-4.30 (4H, m), 6.01 (1H, brs), 7.05 (1H, d, J = 8.1 Hz), 7.30
(1H, dd, J = 1.8, 8.4 Hz), 7.49 (1H, d, J = 1.8 Hz).
2-Acetamido-2-[2-(4-bromophenyl)ethyl]propane-1,3-diol Dia-
cetate (9a). NaBH4 (13.4 g, 354 mmol) was added portionwise to
a solution of 8a (35.4 g, 88.4 mmol) and CaCl2 (20.6 g, 185
mmol) in water (55 mL) and EtOH (310 mL). After stirring at
2-[2-(4-Bromo-2-chlorophenyl)ethyl]-2-(tert-butoxycarbonyla-
mino)propane-1,3-diol Diacetate (9d). The title compound was
synthesized from 8b using a similar procedure to that described
for 9a. 1H NMR (CDCl3) δ 1.46 (9H, s), 2.03-2.07 (2H, m), 2.09
(6H, s), 2.67-2.73 (2H, m), 4.29 (4H, s), 4.75 (1H, brs), 7.09 (1H,
d, J = 8.2 Hz), 7.32 (1H, dd, J = 2.0, 8.4 Hz), 7.50 (1H, d, J =
2.0 Hz).

3162 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Hamada et al.
2-(tert-Butoxycarbonylamino)-2-[2-(3-chloro-40-pentylbiphe-
nyl-4-yl)ethyl]propane-1,3-diol Diacetate (10a). A mixture of 9d
(493 mg, 1.00 mmol), 4-pentylphenylboronic acid (230 mg, 1.19
mmol), tetrakis(triphenylphosphine)palladium(0) (12 mg, 0.010
mmol), and NaHCO3 (504 mg, 5.99 mmol) in 1,2-dimethox-
yethane (6.0 mL) and water (2.0 mL) was heated at 65 °C for 4 h.
Water was added to the reaction mixture, and the resulting mix-
ture was extracted with EtOAc. The organic layer was washed
with brine, dried over Na2SO4, and concentrated in vacuo.
Purification of the residue by silica gel chromatography
(hexane/EtOAc) gave the title compound (450 mg, 80%) as a
2-(tert-Butoxycarbonylamino)-2-[2-(3-chloro-40-phenoxybi-
phenyl-4-yl)ethyl]propane-1,3-diol Diacetate (10j). 1H NMR
(CDCl3) δ 1.47 (9H, s), 2.10 (6H, s), 2.08-2.15 (2H, m), 2.75-
2.81 (2H, m), 4.32 (4H, s), 4.78 (1H, brs), 7.04-7.13 (4H, m),
7.25-7.28 (2H, m), 7.34-7.39 (3H, m), 7.49-7.54 (3H, m).
2-[2-(40-Benzyloxy-3-chlorobiphenyl-4-yl)ethyl]-2-(tert-butoxy-
carbonylamino)propane-1,3-diol Diacetate (10k). 1H NMR
(CDCl3) δ 1.47 (9H, s), 2.10 (6H, s), 2.04-2.14 (2H, m), 2.74-
2.80 (2H, m), 4.32 (4H, s), 4.77 (1H, brs), 5.11 (2H, s), 7.04 (2H,
d, J = 8.4 Hz), 7.23-7.26 (1H, m), 7.35-7.52 (9H, m).
2-Acetamido-2-[2-(40-benzyloxy-20-fluorobiphenyl-4-yl)ethyl]pro-
pane-1,3-diol Diacetate (10l). 1H NMR (CDCl3) δ 1.98 (3H, s),
2.10 (6H, s), 2.19-2.28 (2H, m), 2.62-2.68 (2H, m), 4.37 (4H, s),
5.09 (2H, s), 5.68 (1H, brs), 6.76-6.84 (2H, m), 7.23-7.44
(10H, m).
1
colorless oil. H NMR (CDCl3) δ 0.90 (3H, t, J = 6.6 Hz),
1.32-1.35 (4H, m), 1.47 (9H, s), 1.64-1.66 (2H, m), 2.04-2.20
(2H, m), 2.09 (6H, s), 2.64 (2H, t, J = 7.5 Hz), 2.75-2.80 (2H,
m), 4.32 (4H, s), 4.79 (1H, brs), 7.23-7.27 (3H, m), 7.39-7.47
(3H, m), 7.56 (1H, d, J = 2.0 Hz).
2-Amino-2-[2-(3-chloro-40-pentylbiphenyl-4-yl)ethyl]propane-
1,3-diol Hydrochloride (3a). A solution of 10a (450 mg, 0.803
mmol) in EtOH (9.0 mL) was added to 35% HCl (3.0 mL), and
the mixture was refluxed for 3 h. After the organic solvent was
distilled off in vacuo, the yielded solid was collected to afford the
title compound (320 mg, 97%) as white crystals; mp 185-
The compounds 10b-l were synthesized using a similar
procedure to that described for 10a. Synthetic route for each
compound is summarized in the Supporting Information.
2-Acetamido-2-[2-(40-butoxybiphenyl-4-yl)ethyl]propane-1,3-
1
diol Diacetate (10b). H NMR (CDCl3) δ 0.99 (3H, t, J = 7.2
1
187 °C; MS (ESIþ) m/z 376 [M þ H]. H NMR (DMSO-d6) δ
Hz), 1.48-1.54 (2H, m), 1.75-1.82 (2H, m), 1.96 (3H, s), 2.10
(6H, s), 2.22-2.26 (2H, m), 2.62-2.66 (2H, m), 4.00 (2H, t, J =
6.4 Hz), 4.37 (4H, s), 5.65 (1H, brs), 6.94-6.96 (2H, m), 7.23
(2H, d, J = 8.0 Hz), 7.46-7.50 (4H, m).
0.87 (3H, t, J = 6.6 Hz), 1.19-1.41 (4H, m), 1.49-1.66 (2H, m),
1.73-1.89 (2H, m), 2.52-2.63 (2H, m), 2.68-2.82 (2H, m), 3.57
(4H, d, J = 4.5 Hz), 5.40-5.41 (2H, m), 7.28 (2H, d, J = 8.1 Hz),
7.42 (1H, d, J = 8.1 Hz), 7.58-7.60 (3H, m), 7.68 (1H, d, J = 1.8
2-[2-(40-Butoxy-3-chlorobiphenyl-4-yl)ethyl]-2-(tert-butoxycar-
bonylamino)propane-1,3-diol Diacetate (10c). 1H NMR (CDCl3)
δ 0.99 (3H, t, J = 7.5 Hz), 1.46 (9H, s), 1.47-1.58 (2H, m), 1.70-
1.88 (2H, m), 2.05 (6H, s), 2.04-2.10 (2H, m), 2.73-2.79 (2H,
m), 4.00 (2H, t, J = 6.3 Hz), 4.32 (4H, s), 4.78 (1H, brs), 6.95
(2H, d, J = 8.4 Hz), 7.23-7.26 (1H, m), 7.35-7.38 (1H, m),
7.45-7.52 (3H, m).
Hz), 7.84 (3H, brs). Anal. (C22H30ClNO2 HCl) C, H, N.
3
2-Amino-2-[2-(40-butoxybiphenyl-4-yl)ethyl]propane-1,3-diol
Hydrochloride (3b). A solution of 10b (570 mg, 1.21 mmol) in
MeOH (5.0 mL) and THF (4 mL) was added to a solution of
lithium hydroxide monohydrate (255 mg, 6.07 mmol) in water
(5.0 mL), and the mixture was refluxed for 4 h. After cooling to
room temperature, the reaction mixture was poured into water
and extracted with EtOAc. The organic layer was dried over
Na2SO4 and concentrated in vacuo. The residue was dissolved in
4 M HCl solution in EtOAc (6.0 mL) and MeOH (6.0 mL), and
the solvent was distilled off in vacuo. The yielded white crystal-
line solid was collected and washed with EtOAc to give the title
compound (384 mg, 83%); mp 201-203 °C; MS (ESIþ) m/z 344
[M þ H]. 1H NMR (DMSO-d6) δ 0.94 (3H, t, J = 7.2 Hz), 1.40-
1.50 (2H, m), 1.68-1.75 (2H, m), 1.80-1.85 (2H, m), 2.61-2.66
(2H, m), 3.54 (4H, d, J = 5.2 Hz), 4.00 (2H, t, J = 6.4 Hz), 5.39
(2H, t, J = 5.2 Hz), 6.98-7.01 (2H, m), 7.26 (2H, d, J = 8.4 Hz),
2-Acetamido-2-[2-(40-butoxy-20-fluorobiphenyl-4-yl)ethyl]pro-
pane-1,3-diol Diacetate (10d). 1H NMR (CDCl3) δ 0.99 (3H, t,
J = 7.6 Hz), 1.48-1.53 (2H, m), 1.75-1.82 (2H, m), 1.96 (3H, s),
2.09 (6H, s), 2.23-2.27 (2H, m), 2.63-2.67 (2H, m), 3.98 (2H, t,
J = 6.4 Hz), 4.37 (4H, s), 5.64 (1H, brs), 6.67-6.76 (2H, m), 7.24
(2H, d, J = 8.0 Hz), 7.30 (1H, t, J = 8.8 Hz), 7.42-7.44 (2H, m).
2-Acetamido-2-[2-(40-butoxy-20-methylbiphenyl-4-yl)ethyl]pro-
1
pane-1,3-diol Diacetate (10e). H NMR (CDCl3) δ 0.99 (3H,
t, J = 7.4 Hz), 1.48-1.54 (2H, m), 1.74-1.80 (2H, m), 1.97
(3H, s), 2.10 (6H, s), 2.24 (3H, s), 2.26-2.29 (2H, m), 2.63-
2.68 (2H, m), 3.98 (2H, t, J = 6.4 Hz), 4.37 (4H, s), 5.66
(1H, brs), 6.75-6.81 (2H, m), 7.11 (1H, d, J = 8.4 Hz), 7.21
(4H, brs).
7.53-7.57 (4H, m), 7.87 (3H, brs). Anal. (C21H29NO3 HCl)
3
C, H, N.
2-Acetamido-2-[2-(40-butoxy-20,50-difluorobiphenyl-4-yl)ethyl]-
propane-1,3-diol Diacetate (10f). 1H NMR (CDCl3) δ 0.99 (3H,
t, J = 7.6 Hz), 1.49-1.57 (2H, m), 1.79-1.86 (2H, m), 1.97 (3H,
s), 2.10 (6H, s), 2.23-2.27 (2H, m), 2.63-2.67 (2H, m), 4.04 (2H,
t, J = 6.4 Hz), 4.36 (4H, s), 5.65 (1H, brs), 6.76 (1H, dd, J = 7.1,
11.7 Hz), 7.15 (1H, dd, J = 7.3, 11.8 Hz), 7.24-7.26 (2H, m),
7.41 (2H, d, J = 8.3 Hz).
The compounds 3c-l were synthesized using a similar pro-
cedure to that described for 3a or 3b. The synthetic route for
each compound is summarized in the Supporting Information.
Some compounds were isolated as free base before converting
into hydrochloride salt.
2-Amino-2-[2-(40-butoxy-3-chlorobiphenyl-4-yl)ethyl]propane-
1,3-diol Hydrochloride (3c). mp 188-190 °C; MS (ESIþ) m/z 378
[M þ H]. 1H NMR (DMSO-d6) δ 0.94 (3H, t, J = 7.2 Hz), 1.41-
1.49 (2H, m), 1.69-1.84 (4H, m), 2.73-2.76 (2H, m), 3.57 (4H, d,
J = 4.8 Hz), 4.01 (2H, t, J = 6.3 Hz), 5.44 (2H, t, J = 4.8 Hz), 7.01
(2H, d, J = 8.7 Hz), 7.40 (1H, d, J = 8.1 Hz), 7.55-7.70 (4H, m),
7.87 (3H, brs). Anal. (C21H28ClNO3 HCl 0.25H2O) C, H, N.
2-Acetamido-2-[2-(40-butoxy-30-fluorobiphenyl-4-yl)ethyl]pro-
pane-1,3-diol Diacetate (10g). 1H NMR (CDCl3) δ 0.95 (3H, t,
J = 7.6 Hz), 1.50-1.55 (2H, m), 1.79-1.86 (2H, m), 1.97 (3H, s),
2.10 (6H, s), 2.22-2.64 (2H, m), 2.62-2.67 (2H, m), 4.07 (2H, t,
J = 6.4 Hz), 4.36 (4H, s), 5.67 (1H, brs), 7.00 (1H, t, J = 8.6 Hz),
7.22-7.31 (4H, m), 7.44 (2H, d, J = 8.0 Hz).
3
3
2-Amino-2-[2-(40-butoxy-20-fluorobiphenyl-4-yl)ethyl]propane-
1,3-diol Hydrochloride (3d). mp 163-165 °C; MS (ESIþ) m/z 362
[M þ H]. 1H NMR (DMSO-d6) δ 0.94 (3H, t, J = 7.6 Hz), 1.42-
1.47 (2H, m), 1.68-1.75 (2H, m), 1.81-1.85 (2H, m), 2.63-2.67
(2H, m), 3.55 (4H, d, J= 4.8 Hz), 4.02 (2H, t, J= 6.8 Hz), 5.39 (2H,
t, J = 4.8 Hz), 6.85-6.92 (2H, m), 7.29 (2H, d, J = 8.4 Hz), 7.38-
2-Acetamido-2-[2-(40-butoxy-30-chlorobiphenyl-4-yl)ethyl]pro-
pane-1,3-diol Diacetate (10h). 1H NMR (CDCl3) δ 1.01 (3H, t,
J = 7.6 Hz), 1.52-1.58 (2H, m), 1.82-1.86 (2H, m), 1.97 (3H, s),
2.01 (6H, s), 2.22-2.26 (2H, m), 2.62-2.67 (2H, m), 4.07 (2H, t,
J = 6.4 Hz), 4.36 (4H, s), 5.67 (1H, brs), 6.97 (1H, d, J = 8.4 Hz),
7.24 (2H, d, J = 8.0 Hz), 7.38-7.40 (1H, m), 7.44 (2H, d, J = 8.0
Hz), 7.57 (1H, d, J = 2.4 Hz).
7.44 (3H, m), 7.84 (3H, brs). Anal. (C21H28FNO3 HCl) C, H, N.
3
2-Amino-2-[2-(40-butoxy-20-methylbiphenyl-4-yl)ethyl]propane-
1,3-diol Hydrochloride (3e). mp 136-138 °C; MS (ESIþ) m/z
358 [M þ H]. 1H NMR (DMSO-d6) δ 0.94 (3H, t, J = 7.6 Hz),
1.40-1.49 (2H, m), 1.67-1.74 (2H, m), 1.82-1.87 (2H, m),
2.20 (3H, s), 2.63-2.67 (2H, m), 3.55 (4H, d, J = 5.2 Hz),
3.98 (2H, t, J = 6.4 Hz), 5.38 (2H, t, J = 5.2 Hz), 6.80 (1H, dd,
2-Acetamido-2-[2-(40-phenoxybiphenyl-4-yl)ethyl]propane-1,3-
diol Diacetate (10i). 1H NMR (CDCl3) δ 1.97 (3H, s), 2.10 (6H,
s), 2.22-2.28 (2H, m), 2.63-2.68 (2H, m), 4.37 (4H, s), 5.68 (1H,
brs), 7.04-7.11 (5H, m), 7.23-7.26 (2H, m), 7.32-7.38 (2H, m),
7.47-7.54 (4H, m).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3163
J = 2.4, 8.4 Hz), 6.85 (1H, d, J = 2.4 Hz), 7.08 (1H, d, J = 8.4
Hz), 7.21-7.26 (4H, m), 7.85 (3H, brs). Anal. (C22H31NO3
CH2Cl2 (1.0 mL) was added dropwise to a solution of 11 (507 mg,
1.17 mmol) and pyridine (0.58 mL, 11 mmol) in CH2Cl2 (3.0 mL)
at 0 °C. After stirring at room temperature for 2.5 h, the reaction
was quenched with saturated NaHCO3 and extracted with CHCl3.
The organic layer was washed with brine, dried over Na2SO4,
and concentrated in vacuo. Purification of the residue by silica
gel chromatography (hexane/EtOAc) gave the title compound
3
HCl H2O) C, H, N.
2-Amino-2-[2-(40-butoxy-20,50-difluorobiphenyl-4-yl)ethyl]pro-
pane-1,3-diol Hydrochloride (3f). mp 182-184 °C; MS (ESIþ)
3
1
m/z 380 [M þ H]. H NMR (DMSO-d6) δ 0.95 (3H, t, J =
7.2 Hz), 1.42-1.48 (2H, m), 1.70-1.78 (2H, m), 1.80-1.85 (2H,
m), 2.50-2.67 (2H, m), 3.54 (4H, d, J = 5.1 Hz), 4.10 (2H, t, J =
6.6 Hz), 5.39 (2H, t, J = 4.9 Hz), 7.21 (1H, dd, J = 7.4, 12.3 Hz),
7.30 (2H, d, J = 8.3 Hz), 7.40 (1H, dd, J = 7.0, 12.2 Hz), 7.46 (2H,
1
(460 mg, 70%) as a pale-yellow solid. H NMR (CDCl3) δ 1.98
(3H, s), 2.01 (6H, s), 2.25-2.29 (2H, m), 2.65-2.69 (2H, m), 4.36
(4H, s), 5.68 (1H, brs), 7.11-7.18 (2H, m), 7.29 (2H, d, J =
8.0 Hz), 7.43 (2H, dd, J = 1.3, 8.3 Hz), 7.49 (1H, t, J = 8.2 Hz).
2-Amino-2-[2-(20-fluoro-40-pentylbiphenyl-4-yl)ethyl]propane-
1,3-diol Hydrochloride (4). To a mixture of 12 (150 mg, 0.266
mmol), bis(acetonitrile)dichloropalladium(II) (2.8 mg, 0.010
mmol), 2-dicyclohexylphosphino-20,40,60-triisopropylbiphenyl (X-
Phos, 15.2 mg, 0.0318 mmol), and cesium carbonate (225 mg,
0.690 mmol) in CH3CN (1.0 mL) was added 1-pentyne (0.13 mL,
1.3 mmol). The mixture was stirred at 60 °C for 5 h, poured into
water, and extracted with EtOAc. The organic layer was washed
with brine, dried over Na2SO4, and concentrated in vacuo. The resi-
due was dissolved in MeOH (2.0 mL) and added to a solution of
lithium hydroxide monohydrate (56 mg, 1.3 mmol) in water (2.0
mL). The mixture was refluxed for 3 h. After cooling to room tem-
perature, the reaction mixture was poured into water and extracted
with EtOAc. The organic layer was dried over Na2SO4 and con-
centrated in vacuo. Purification of the residue by silica gel chroma-
tography (CHCl3/MeOH) gave 2-acetamido-2-[2-[20-fluoro-40-(1-
pentynyl)biphenyl-4-yl]ethyl]propane-1,3-diol diacetate (67 mg,
70%) as a pale-yellow solid. A solution of the solid in MeOH (10
mL) was stirred under hydrogen atmosphere in the presence of 10%
Pd/C (20 mg) for 4 h at room temperature. After removal of the
catalyst by filtration, the filtrate was concentrated in vacuo. To a
solution of the residue in EtOAc (2.0 mL) and MeOH (2.0 mL) was
added 4 M HCl solution in EtOAc (1.0 mL). After the solution was
concentrated in vacuo, the residue was collected by filtration to give
the title compound (44 mg, 87%) as a white solid. mp 154-156 °C;
MS (ESIþ) m/z 360 [M þ H]. 1H NMR (DMSO-d6) δ 0.88 (3H, t,
J = 6.8 Hz), 1.27-1.35 (4H, m), 1.56-1.64 (2H, m), 1.81-1.85
(2H, m), 2.51-2.67 (4H, m), 3.54 (4H, d, J = 5.0 Hz), 5.41 (2H, t,
J = 4.9 Hz), 7.11-7.15 (2H, m), 7.30 (2H, d, J = 8.0 Hz), 7.40 (1H,
t, J = 8.5 Hz), 7.46 (2H, d, J = 6.7 Hz), 7.84 (3H, brs). Anal.
(C22H30FNO2 HCl 0.5H2O) C, H, N.
d, J=6.7Hz), 7.85(3H, brs). Anal. (C21H27F2NO3 HCl) C, H, N.
3
2-Amino-2-[2-(40-butoxy-30-fluorobiphenyl-4-yl)ethyl]propane-
1,3-diol Hydrochloride (3g). mp 164-166 °C; MS (ESIþ) m/z 362
1
[M þ H]. H NMR (DMSO-d6) δ 0.95 (3H, t, J = 7.6 Hz),
1.43-1.48 (2H, m), 1.70-1.77 (2H, m), 1.80-1.84 (2H, m),
2.61-2.66 (2H, m), 3.54 (4H, d, J = 4.8 Hz), 4.08 (2H, t, J = 6.4
Hz), 5.38 (2H, t, J = 4.8 Hz), 7.22 (1H, t, J = 8.8 Hz), 7.27 (2H,
d, J = 8.0 Hz), 7.40-7.43 (1H, m), 7.50-7.53 (1H, m), 7.58
(2H, d, J = 8.0 Hz), 7.81 (3H, brs). Anal. (C21H28FNO3 HCl
3
3
0.25H2O) C, H, N.
2-Amino-2-[2-(40-butoxy-30-chlorobiphenyl-4-yl)ethyl]propane-
1,3-diol Hydrochloride (3h). mp 154-156 °C; MS (ESIþ) m/z 378
1
[M þ H]. H NMR (DMSO-d6) δ 0.95 (3H, t, J = 7.6 Hz),
1.45-1.51 (2H, m), 1.71-1.78 (2H, m), 1.80-1.85 (2H, m),
2.62-2.66 (2H, m), 3.54 (4H, d, J = 5.2 Hz), 4.10 (2H, t, J = 6.4
Hz), 5.39 (2H, t, J = 5.2 Hz), 7.21 (1H, d, J = 8.8 Hz), 7.28 (2H,
d, J = 8.4 Hz), 7.56-7.59 (3H, m), 7.69 (1H, d, J = 2.4 Hz), 7.85
(3H, brs). Anal. (C21H28ClNO3 HCl 0.5H2O) C, H, N.
3
3
2-Amino-2-[2-(40-phenoxybiphenyl-4-yl)ethyl]propane-1,3-diol
(3i). mp 149-150 °C; MS (ESIþ) m/z 364 [M þ H]. H NMR
1
(CDCl3) δ 1.73-1.78 (2H, m), 2.67-2.73 (2H, m), 3.53 (2H, d,
J = 10.8 Hz), 3.63 (2H, d, J = 10.8 Hz), 7.04-7.12 (4H, m),
7.25-7.27 (4H, m), 7.32-7.38 (1H, m), 7.47-7.54 (4H, m).
Anal. (C23H25NO3 0.3H2O) C, H, N.
3
2-Amino-2-[2-(3-chloro-40-phenoxybiphenyl-4-yl)ethyl]propane-
1,3-diol Hydrochloride (3j). mp 211-212 °C; MS (ESIþ) m/z 398
[M þ H]. 1H NMR (DMSO-d6) δ 1.80-1.86 (2H, m), 2.75-2.81
(2H, m), 3.58 (4H, d, J = 4.8 Hz), 5.41 (2H, t, J = 4.8 Hz), 7.08
(4H, d, J = 8.4 Hz), 7.18 (1H, t, J = 7.5 Hz), 7.40-7.45 (3H, m),
7.59-7.62 (1H, m), 7.69-7.72 (3H, m), 7.88 (3H, brs). Anal.
(C23H24ClNO3 HCl) C, H, N.
3
2-Amino-2-[2-(40-benzyloxy-3-chlorobiphenyl-4-yl)ethyl]propane-
1,3-diol Hydrochloride (3k). mp 186-187 °C; MS (ESIþ) m/z 412
[M þ H]. 1H NMR (DMSO-d6) δ 1.79-1.85 (2H, m), 2.73-2.79
(2H, m), 3.57 (4H, d, J = 4.8 Hz), 5.16 (2H, s), 5.40 (2H, t, J =
5.1 Hz), 7.10 (2H, d, J = 8.7 Hz), 7.38-7.48 (6H, m), 7.55-7.66
3
3
2-Amino-2-[2-[20-fluoro-40-(4-methylphenoxy)biphenyl-4-yl]-
ethyl]propane-1,3-diol (5). A mixture of 11 (278 mg, 0.644
mmol), 4-methylphenylboronic acid (175 mg, 1.28 mmol), Cu-
(OAc)2 (117 mg, 0.644 mmol), pyridine (255 mg, 2.84 mmol),
˚
(4H, m), 7.85 (3H, brs). Anal. (C24H26ClNO3 HCl) C, H, N.
and powdered molecular sieves 4 A (500mg) inCH2Cl2 (5.0mL) was
3
2-Amino-2-[2-(40-benzyloxy-20-fluorobiphenyl-4-yl)ethyl]pro-
pane-1,3-diol (3l). mp 166-167 °C; MS (ESIþ) m/z 396 [M þ H].
1H NMR (DMSO-d6) δ 1.30 (2H, s), 1.47-1.55 (2H, m),
2.59-2.65 (2H, m), 3.23-3.26 (4H, m), 4.48 (2H, brs), 5.17
(2H, s), 6.93-7.03 (2H, m), 7.26 (1H, d, J = 8.4 Hz), 7.36 (2H, d,
stirred at room temperature for 9 h. The reaction mixture
was poured into 2% aqueous solution of citric acid and ex-
tracted with CHCl3. The organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo. Purification of
the residue by silica gel chromatography (hexane/EtOAc) gave
the protected title compound (260 mg, 77%) as a white solid. To
a solution of the solid in MeOH (10 mL) was added 1 M aqueous
LiOH (10 mL), and the mixture was refluxed for 2 h. The
reaction mixture was concentrated in vacuo, and the resultant
solid was collected by filtration. Purification of the solid by
preparative HPLC gave the title compound (59 mg, 21%) as a
white solid. mp 154-156 °C; MS (ESIþ) m/z 396 [M þ H]. 1H
NMR (DMSO-d6) δ 1.72-1.78 (2H, m), 2.36 (3H, s), 2.67-2.72
(2H, m), 3.54 (2H, d, J = 10.5 Hz), 3.63 (2H, d, J = 10.8 Hz),
6.72-6.83 (2H, m), 6.98 (2H, d, J = 8.7 Hz), 7.18 (2H, d, J = 8.4
Hz), 7.25-7.28 (2H, m), 7.34 (1H, t, J = 8.6 Hz), 7.44 (2H, dd,
J = 1.5, 8.1 Hz). Anal. (C24H26FNO3) C, H, N.
J = 8.4 Hz), 7.39-7.49 (7H, m). Anal. (C24H26FNO3 1.25H2O)
3
C, H, N.
2-Acetamido-2-[2-(20-fluoro-40-hydroxybiphenyl-4-yl)ethyl]-
propane-1,3-diol Diacetate (11). A mixture of 10l (0.86 g, 1.6
mmol) and ammonium formate (517 mg, 8.19 mmol) in MeOH
(30 mL) was stirred under hydrogen atmosphere in the presence
of 10% Pd/C (0.30 g) for 6 h. After removal of the catalyst by
filtration, the filtrate was concentrated in vacuo, diluted with
water, and extracted with EtOAc. The organic layer was dried
over Na2SO4 and concentrated in vacuo. Purification of the
residue by silica gel chromatography (hexane/EtOAc) gave the
title compound (0.56 g, 87%) as a white solid. 1H NMR (CDCl3)
δ 2.04 (3H, s), 2.11 (6H, s), 2.29-2.34 (2H, m), 2.61-2.67 (2H,
m), 4.38 (4H, s), 6.06 (1H, brs), 6.64-6.71 (2H, m), 7.10-7.27
(3H, m), 7.38 (2H, dd, J = 1.2, 8.1 Hz), 8.42 (1H, brs).
2-Acetamido-2-[2-[20-fluoro-40-(trifluoromethanesulfonyloxy)-
biphenyl-4-yl]ethyl]propane-1,3-diol Diacetate (12). A solution
of trifluoromethanesulfonic anhydride (0.30 mL, 1.7 mmol) in
N-[5-[2-(40-Bromo-20-fluorobiphenyl-4-yl)ethyl]-2,2-dimethyl-
1,3-dioxane-5-yl]acetamide (13a). A mixture of 9b (7.13 g, 20.0
mmol), bis(neopentyl glycolato)diboron (4.97 g, 22.0 mmol),
potassium acetate (5.89 g, 60.0 mmol), and dichlorobis(tricyclo-
hexylphosphine)palladium(II) (738 mg, 1.00 mmol) in 1,4-diox-
ane (80 mL) was heated at 100 °C. After 6 h, the mixture was

3164 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Hamada et al.
allowed to cool to room temperature and poured into water. The
mixture was extracted with EtOAc and washed with brine. The
organic layer was dried over Na2SO4 and concentrated in vacuo.
The residue was precipitated from water to give a brown solid. To
a mixture of the solid, 1-bromo-3-fluoro-4-iodobenzene (5.95 g,
19.7 mmol) and NaHCO3 (9.48 g, 112 mmol) in 1,2-dimethox-
yethane (120 mL) and water (40 mL) was added tetrakis-
(triphenylphosphine)palladium(0) (434 mg, 0.375 mmol). After
stirring at 100 °C for 8 h, 1-bromo-3-fluoro-4-iodobenzene (2.83
g, 9.4 mmol) and tetrakis(triphenylphosphine)palladium(0) (217
mg, 0.188 mmol) was added to the mixture. The mixture was
stirred at 100 °C for another 8 h and extracted with EtOAc. The
organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. Purification of the residue by silica gel
chromatography (hexane/EtOAc) gave the title compound (5.28
g, 58%) as a white solid. 1H NMR(DMSO-d6) δ 1.35 (6H, s), 1.87
(3H, s), 1.98-2.04 (2H, m), 2.47-2.53 (2H, m), 3.71 (2H, d, J =
11.8 Hz), 3.97 (2H, d, J = 11.6 Hz), 7.27 (2H, d, J = 8.1 Hz),
7.44-7.52 (4H, m), 7.61-7.66 (2H, m).
2-Amino-2-[2-[20-fluoro-40-(3-methylphenylthio)biphenyl-4-yl]-
ethyl]propane-1,3-diol Hydrochloride (6e). mp 148-150 °C; MS
(ESIþ) m/z 412 [M þ H]. 1H NMR (DMSO-d6) δ 1.79-1.85 (2H,
m), 2.35 (3H, s), 2.63-2.68 (2H, m), 3.53-3.54 (4H, m), 5.39
(2H, t, J = 4.7 Hz), 7.10-7.14 (2H, m), 7.23-7.49 (9H, m), 7.81
(3H, brs). Anal. (C24H26FNO2S HCl 0.25H2O) C, H, N.
3
3
2-Amino-2-[2-[3-chloro-20-fluoro-40-(4-methylphenylthio)bi-
phenyl-4-yl]ethyl]propane-1,3-diol (6f). mp 98-100 °C; MS
1
(ESIþ) m/z 446 [M þ H]. H NMR (DMSO-d6) δ 1.29 (2H,
brs), 1.47-1.53 (2H, m), 2.35 (3H, s), 2.72-2.77 (2H, m), 3.21-
3.30 (4H, m), 4.49 (2H, t, J = 4.7 Hz), 7.01-7.06 (2H, m), 7.30
(2H, d, J = 8.0 Hz), 7.38-7.52 (6H, m). Anal. (C24H25ClFNO2-
S 0.25H2O) C, H, N.
3
2-Amino-2-[2-[40-(4-ethylphenylthio)-20-fluorobiphenyl-4-yl]-
ethyl]propane-1,3-diol (6g). mp 115-117 °C; MS (ESIþ) m/z 426
[M þ H]. 1H NMR (DMSO-d6) δ 1.20 (3H, t, J = 7.5 Hz), 1.29
(2H, brs), 1.49-1.54 (2H, m), 2.60-2.68 (4H, m), 3.21-3.29
(4H, m), 4.46 (2H, t, J = 5.1 Hz), 7.05-7.08 (2H, m), 7.09-7.34
(4H, m), 7.40-7.49 (5H, m). Anal. (C25H28FNO2S 0.5H2O) C,
3
2-Acetamido-2-[2-(40-bromo-3-chloro-20-fluorobiphenyl-4-yl)-
ethyl]propane-1,3-diol Diacetate (13b). The title compound was
synthesized from 9c using a similar procedure to that described
for 13a. 1H NMR (CDCl3) δ 2.01 (3H, s), 2.11 (6H, s), 2.18-2.24
(2H, m), 2.75-2.81 (2H, m), 4.39 (4H, s), 5.74 (1H, brs),
7.24-7.38 (5H, m), 7.50 (1H, s).
H, N.
2-Amino-2-[2-[20-fluoro-40-(4-isopropylphenylthio)biphenyl-4-
yl]ethyl]propane-1,3-diol Hydrochloride (6h). mp 245-246 °C;
MS (ESIþ) m/z 440 [M þ H]. 1H NMR (DMSO-d6) δ 1.20 (6H,
d, J = 6.9 Hz), 1.77-1.83 (2H, m), 2.61-2.66 (2H, m), 2.91 (1H,
sept, J = 6.9 Hz), 3.52 (4H, d, J = 4.6 Hz), 5.39 (2H, t, J = 4.6
Hz), 7.05-7.09 (2H, m), 7.28-7.35 (4H, m), 7.41-7.46 (5H, m),
2-Amino-2-[2-(40-benzylthio-20-fluorobiphenyl-4-yl)ethyl]pro-
pane-1,3-diol Hydrochloride (6b). A mixture of 13a (450 mg,
0.999 mmol), benzyl mercaptan (137 mg, 1.13 mmol), tris(di-
benzylideneacetone)dipalladium(0)-chloroform adduct (25.9 mg,
0.0250 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxan-
thene (Xantphos, 29.8 mg, 0.0515 mmol), and N,N-diisopropy-
lethylamine (258 mg, 1.99 mmol) in 1,4-dioxane (5.0 mL) was
refluxed for 7 h. After being poured into water, the mixture was
extracted with EtOAc. The organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo. Purification of
the residue by silica gel chromatography (hexane/EtOAc) gave
the protected title compound. The compound was dissolved in
MeOH (6.0 mL), and 35% HCl (3.0 mL) was added to the
solution. The mixture was stirred at 70 °C for 3.5 h, concentrated
in vacuo, and diluted with diisopropyl ether. The yielded solid
was collected by filtration to give the title compound (362 mg,
94%) as a pale-yellow solid. mp 218-219 °C; MS (ESIþ) m/z 412
[M þ H]. 1H NMR (DMSO-d6) δ 1.80-1.85 (2H, m), 2.63-2.68
(2H, m), 3.54 (4H, d, J = 4.5 Hz), 4.34 (2H, s), 5.40 (2H, t, J =
4.9 Hz), 7.22-7.35 (7H, m), 7.41-7.47 (5H, m), 7.83 (3H, brs).
7.83 (3H, brs). Anal. (C26H30FNO2S HCl) C, H, N.
3
2-Amino-2-[2-[20-fluoro-40-(4-fluorophenylthio)biphenyl-4-yl]-
ethyl]propane-1,3-diol (6i). mp 137-139 °C; MS (ESIþ) m/z 416
[M þ H]. 1H NMR (DMSO-d6) δ 1.29 (2H, brs), 1.49-1.55 (2H,
m), 2.60-2.66 (2H, m), 3.19-3.29 (4H, m), 4.46 (2H, t, J = 5.2
Hz), 7.07-7.26 (2H, m), 7.29-7.60 (9H, m). Anal. (C23H23-
F2NO2S) C, H, N.
2-Amino-2-[2-[20-fluoro-40-(3-fluorophenylthio)biphenyl-4-yl]-
ethyl]propane-1,3-diol (6j). mp 137-139 °C; MS (ESIþ) m/z 416
[M þ H]. 1H NMR (DMSO-d6) δ 1.30 (2H, brs), 1.50-1.56 (2H,
m), 2.61-2.67 (2H, m), 3.19-3.30 (4H, m), 4.47 (2H, t, J = 5.4
Hz), 7.23-7.33 (7H, m), 7.44-7.55 (4H, m). Anal. (C23H23-
F2NO2S 0.5H2O) C, H, N.
3
2-Amino-2-[2-[40-(4-chlorophenylthio)-20-fluorobiphenyl-4-yl]-
ethyl]propane-1,3-diol (6k). mp 135-136 °C; MS (ESIþ) m/z 432
[M þ H]. 1H NMR (DMSO-d6) δ 1.31 (2H, brs), 1.50-1.55 (2H,
m), 2.61-2.66 (2H, m), 3.20-3.28 (4H, m), 4.46 (2H, brs),
7.16-7.30 (4H, m), 7.43-7.55 (7H, m). Anal. (C23H23ClF-
NO2S 0.5H2O) C, H, N.
3
2-Amino-2-[2-[20-fluoro-40-[4-(trifluoromethyl)phenylthio]bi-
phenyl-4-yl]ethyl]propane-1,3-diol (6l). mp 128-130 °C; MS
Anal. (C24H26FNO2S HCl) C, H, N.
3
The compounds 6a,c-n were synthesized using a similar
procedure to that described for 6b. Synthetic route for each
compound is summarized in the Supporting Information. Some
compounds were isolated as free base before converting into
hydrochloride salt.
1
(ESIþ) m/z 466 [M þ H]. H NMR (DMSO-d6) δ 1.32 (2H,
brs), 1.51-1.56 (2H, m), 2.62-2.68 (2H, m), 3.22-3.29 (4H, m),
4.48 (2H, t, J = 5.2 Hz), 7.30-7.38 (3H, m), 7.44-7.52 (5H, m),
7.61 (1H, t, J = 8.2 Hz), 7.74 (2H, d, J = 8.4 Hz). Anal. (C24H23-
2-Amino-2-[2-(40-benzylthio-3-chlorobiphenyl-4-yl)ethyl]pro-
pane-1,3-diol Hydrochloride (6a). mp 167-168 °C; MS (ESIþ)
m/z 428 [M þ H]. 1H NMR (DMSO-d6) δ 1.78-1.84 (2H, m),
2.74-2.79 (2H, m), 3.57 (4H, d, J = 4.8 Hz), 4.30 (2H, s), 5.42
(2H, t, J = 4.8 Hz), 7.24-7.43 (8H, m), 7.60-7.70 (4H, m), 7.87
(3H, brs). Anal. (C24H26ClNO2S HCl 0.25H2O) C, H, N.
F4NO2S 0.5H2O) C, H, N.
3
2-Amino-2-[2-[20-fluoro-40-(4-methoxyphenylthio)biphenyl-4-
yl]ethyl]propane-1,3-diol Hydrochloride (6m). mp 246-247 °C;
MS (ESIþ) m/z 428 [M þ H]. 1H NMR (DMSO-d6) δ 1.75-1.81
(2H, m), 2.59-2.65 (2H, m), 3.50 (4H, d, J = 4.3 Hz), 3.80 (3H, s),
5.36 (2H, brs), 6.92-6.96 (2H, m), 7.05 (2H, d, J = 6.6 Hz), 7.28
(2H, d, J = 8.2 Hz), 7.41-7.43 (3H, m), 7.50 (2H, d, J = 8.8 Hz),
7.69 (3H, brs). Anal. (C24H26FNO3S HCl 0.75H2O) C, H, N.
3
3
2-Amino-2-[2-(20-fluoro-40-phenylthiobiphenyl-4-yl)ethyl]pro-
pane-1,3-diol Hydrochloride (6c). mp 154-155 °C; MS (ESIþ)
m/z 398 [M þ H]. 1H NMR (CDCl3) δ 1.79-1.85 (2H, m),
2.63-2.69 (2H, m), 3.54 (4H, d, J = 4.5 Hz), 5.41 (2H, t, J = 5.0
Hz), 7.13-7.17 (2H, m), 7.32 (2H, d, J = 8.4 Hz), 7.44-7.53
3
3
2-Amino-2-[2-[20-fluoro-40-(3-methoxyphenylthio)biphenyl-4-
yl]ethyl]propane-1,3-diol (6n). mp 108-110 °C; MS (ESIþ) m/z
428 [M þ H]. 1H NMR (DMSO-d6) δ 1.55-1.61 (2H, m),
2.61-2.66 (2H, m), 3.26-3.33 (4H, m), 3.76 (3H, s), 4.64 (2H,
brs), 6.92-7.08 (3H, m), 7.15-7.58 (8H, m). Anal. (C24H26F-
(8H, m), 7.83 (3H, brs). Anal. (C23H24FNO2S HCl) C, H, N.
3
2-Amino-2-[2-[20-fluoro-40-(4-methylphenylthio)biphenyl-4-yl]-
ethyl]propane-1,3-diol (6d). The title compound was obtained by
purification using preparative HPLC. mp 135-136 °C; MS
NO3S 1.5H2O) C, N. H: calcd, 6.43; found, 6.00.
3
4-(Di-tert-butylphosphoryloxymethyl)-4-[2-(3-chloro-40-pen-
tylbiphenyl-4-yl)ethyl]-2-methyl-2-oxazoline (14a). A solution of
3a (180 mg, 0.436 mmol), N,N-diisopropylethylamine (0.16 mL,
0.89 mmol), and triethyl orthoacetate (0.095 mL, 0.52 mmol) in
DMF (2.0 mL) was heated at 120 °C for 5 h. The reaction mixture
1
(ESIþ) m/z 412 [M þ H]. H NMR (DMSO-d6) δ 1.30 (2H,
brs), 1.49-1.54 (2H, m), 2.35 (3H, s), 2.60-2.65 (2H, m), 3.19-
3.29 (4H, m), 4.47 (2H, brs), 7.04 (2H, d, J = 9.9 Hz), 7.29 (4H,
m), 7.39-7.48 (5H, m). Anal. (C24H26FNO2S) C, H, N.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3165
was poured into water and extracted with EtOAc. The organic
layer was washed with brine, dried over Na2SO4, and concentrated
in vacuo. To a solution of the residue in CH2Cl2 (4.4 mL) were
added 1H-tetrazole (61 mg, 0.87 mmol) and di-tert-butyl N,N-
diisopropylphosphoramidite (0.28 mL, 0.88 mmol) at 0 °C. After
stirring at room temperature for 2 h, m-CPBA (65%, 201 mg, 0.75
mmol) was added to the mixture at 0 °C. The mixture was stirred at
room temperature for 1 h, poured into saturated NaHCO3, and
extracted with CHCl3. The organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo. Purification of the
residue by silica gel chromatography (hexane/EtOAc) gave the title
compound (159 mg, 62%) as a yellow oil. 1H NMR (CDCl3) δ0.90
(3H, t, J = 6.6 Hz), 1.23-1.45 (4H, m), 1.48 (18H, s), 1.56-1.72
(2H, m), 1.78-1.98 (2H, m), 2.03 (3H, s), 2.63 (2H, t, J = 7.5 Hz),
2.66-2.88 (2H, m), 3.85-4.02 (2H, m), 4.12 (1H, d, J = 9.0 Hz),
4.38 (1H, d, J = 8.7 Hz), 7.23-7.28 (3H, m), 7.38-7.47 (3H, m),
7.55 (1H, d, J = 1.8 Hz).
The compounds 14b-n were synthesized using a similar
procedure to that described for 14a. Synthetic route for each
compound is summarized in the Supporting Information.
4-[2-(40-Butoxybiphenyl-4-yl)ethyl]-4-(di-tert-butylphosphoryl-
oxymethyl)-2-methyl-2-oxazoline (14b). 1H NMR (CDCl3) δ
0.98 (3H, t, J = 7.2 Hz), 1.49 (18H, s), 1.50-1.56 (2H, m), 1.75-
2.04 (4H, m), 2.02 (3H, s), 2.65-2.70 (2H, m), 3.88-3.91 (1H,
m), 3.95-4.02 (4H, m), 4.35 (1H, d, J = 8.7 Hz), 6.95 (2H, d,
J = 8.7 Hz), 7.23 (2H, d, J = 8.2 Hz), 7.46 (2H, d, J = 8.1 Hz),
7.48 (2H, d, J = 8.7 Hz).
4-[2-(40-Butoxy-3-chlorobiphenyl-4-yl)ethyl]-4-(di-tert-butylphos-
phoryloxymethyl)-2-methyl-2-oxazoline (14c). 1H NMR (CD-
Cl3) δ 0.99 (3H, t, J = 7.2 Hz), 1.36-1.58 (2H, m), 1.50 (18H, s),
1.68-1.97 (4H, m), 2.03 (3H, s), 2.65-2.90 (2H, m), 3.86-4.02
(2H, m), 4.00 (2H, t, J = 6.6 Hz), 4.12 (1H, d, J = 8.7 Hz), 4.38
(1H, d, J = 8.7 Hz), 6.95 (2H, d, J = 8.7 Hz), 7.25 (1H, d, J =
6.9 Hz), 7.36 (1H, dd, J = 1.5, 7.8 Hz), 7.45-7.52 (3H, m).
4-[2-(40-Butoxy-20-fluorobiphenyl-4-yl)ethyl]-4-(di-tert-butylphos-
phoryloxymethyl)-2-methyl-2-oxazoline (14d). 1H NMR (CD-
Cl3) δ 0.99 (3H, t, J = 7.4 Hz), 1.48 (18H, d, J = 3.2 Hz),
1.51-1.55 (2H, m), 1.75-1.82 (2H, m), 1.85-1.89 (1H, m),
1.97-2.02 (1H, m), 2.02 (3H, s), 2.66-2.71 (2H, m), 3.88-3.92
(1H, m), 3.96-4.03 (4H, m), 4.36 (1H, d, J = 8.8 Hz), 6.67-6.71
(1H, m), 6.73-6.76 (1H, m), 7.24 (2H, d, J = 8.0 Hz), 7.31 (1H,
t, J = 8.8 Hz), 7.41-7.43 (2H, m).
2.50-2.56 (1H, m), 2.60-2.68 (1H, m), 3.77-3.84 (2H, m), 4.07
(1H, d, J = 8.8 Hz), 4.14 (1H, d, J = 8.8 Hz), 7.05-7.08 (4H, m),
7.16 (1H, t, J = 7.2 Hz), 7.29 (2H, d, J = 8.2 Hz), 7.39-7.44
(2H, m), 7.55 (2H, d, J = 8.1 Hz), 7.64-7.68 (2H, m).
4-(Di-tert-butylphosphoryloxymethyl)-4-[2-(3-chloro-40-phe-
noxybiphenyl-4-yl)ethyl]-2-methyl-2-oxazoline (14j). 1H NMR
(CDCl3) δ 1.49 (18H, s), 1.80-2.02 (2H, m), 2.04 (3H, s), 2.68-
2.92 (2H, m), 3.86-4.02 (2H, m), 4.13 (1H, d, J = 8.6 Hz), 4.39
(1H, d, J = 8.8 Hz), 7.04-7.13 (5H, m), 7.28 (1H, d, J = 8.0 Hz),
7.33-7.39 (3H, m), 7.49-7.54 (3H, m).
4-[2-(40-Benzyloxy-3-chlorobiphenyl-4-yl)ethyl]-4-(di-tert-butyl-
phosphoryloxymethyl)-2-methyl-2-oxazoline (14k). 1H NMR
(CDCl3) δ 1.49 (18H, s), 1.78-2.00 (2H, m), 2.03 (3H, s), 2.64-
2.90 (2H, m), 3.86-4.00 (2H, m), 4.12 (1H, d, J = 8.9 Hz), 4.38
(1H, d, J = 8.9 Hz), 5.11 (2H, s), 7.04 (2H, d, J = 8.5 Hz), 7.25-
7.52 (10H, m).
4-[2-(40-Benzyloxy-20-fluorobiphenyl-4-yl)ethyl]-4-(di-tert-butyl-
phosphoryloxymethyl)-2-methyl-2-oxazoline (14l). 1H NMR
(CDCl3) δ 1.49 (18H, s), 1.76-1.94 (2H, m), 2.02 (3H, s), 2.61-
2.72 (2H, m), 3.87-4.03 (3H, m), 4.33-4.37 (1H, m), 5.09 (2H,
s), 6.76-6.84 (2H, m), 7.23-7.46 (10H, m).
4-(Di-tert-butylphosphoryloxymethyl)-4-[2-(20-fluoro-40-pen-
tylbiphenyl-4-yl)ethyl]-2-methyl-2-oxazoline (14m). 1H NMR
(CDCl3) δ 0.91 (3H, t, J = 6.9 Hz), 1.33-1.38 (4H, m), 1.48
(18H, s), 1.61-1.68 (2H, m), 1.82-1.89 (1H, m), 1.97-2.05 (1H,
m), 2.02 (3H, s), 2.62 (2H, t, J = 7.9 Hz), 2.69 (2H, t, J = 8.6
Hz), 3.89 (1H, dd, J = 4.7, 10.0 Hz), 3.97 (1H, dd, J = 4.9, 10.1
Hz), 4.01 (1H, d, J = 8.8 Hz), 4.36 (1H, d, J = 8.8 Hz), 6.94-
7.01 (2H, m), 7.24-7.26 (2H, m), 7.31 (1H, t, J = 8.0 Hz), 7.45
(2H, dd, J = 1.3, 8.3 Hz).
4-(Di-tert-butylphosphoryloxymethyl)-4-[2-[20-fluoro-40-(4-
methylphenoxy)biphenyl-4-yl]ethyl]-2-methyl-2-oxazoline (14n).
1H NMR (CDCl3) δ 1.49 (18H, s), 1.80-2.10 (2H, m), 2.02
(3H, s), 2.36 (3H, s), 2.69 (2H, t, J = 8.6 Hz), 3.90 (1H, dd, J =
4.5, 9.9 Hz), 3.98 (1H, dd, J = 4.8, 9.9 Hz), 4.02 (1H, d, J = 8.4
Hz), 4.36 (1H, d, J = 8.7 Hz), 6.72-6.82 (2H, m), 6.96-6.99
(2H, m), 7.17-7.44 (7H, m).
2-Amino-4-(3-chloro-40-pentylbiphenyl-4-yl)-2-(phosphoryl-
oxymethyl)butanol (3a-P). A solution of 14a (159 mg, 0.268
mmol) in EtOH (2.5 mL) and 35% HCl (0.50 mL) was heated at
50 °C for 3 h. After addition of water (20 mL), the yielded solid
was collected and washed with water to afford the title com-
pound (79 mg, 64%) as a white solid. mp 229-238 °C (dec); MS
(ESIþ) m/z 456 [M þ H]. 1H NMR (CD3OD) δ 0.93 (3H, t, J =
6.6 Hz), 1.29-1.48 (4H, m), 1.60-1.74 (2H, m), 1.95-2.08 (2H,
m), 2.66 (2H, t, J = 7.5 Hz), 2.84-2.94 (2H, m), 3.72-3.85 (2H,
m), 3.98-4.14 (2H, m), 7.27 (2H, d, J = 8.1 Hz), 7.43 (1H, d,
J = 8.1 Hz), 7.50-7.53 (3H, m), 7.62 (1H, d, J = 1.5 Hz). Anal.
(C22H31ClNO5P) C, H, N.
4-[2-(40-Butoxy-20,50-difluorobiphenyl-4-yl)ethyl]-4-(di-tert-butyl-
phosphoryloxymethyl)-2-methyl-2-oxazoline (14f). 1H NMR
(CDCl3) δ 0.99 (3H, t, J = 7.4 Hz), 1.48 (18H, d, J = 3.3 Hz),
1.51-1.55 (2H, m), 1.79-1.88 (3H, m), 1.96-2.02 (1H, m), 2.01
(3H, s), 2.67-2.71 (2H, m), 3.89 (1H, dd, J = 4.6, 9.8 Hz), 3.98
(1H, dd, J = 5.2, 10.0 Hz), 4.02-4.13 (3H, m), 4.35 (1H, d, J =
8.9 Hz), 6.75 (1H, dd, J = 7.1, 11.7 Hz), 7.14 (1H, dd, J = 7.3,
11.7 Hz), 7.23-7.26 (2H, m), 7.41 (2H, d, J = 7.8 Hz).
4-[2-(40-Butoxy-30-fluorobiphenyl-4-yl)ethyl]-4-(di-tert-butylphos-
phoryloxymethyl)-2-methyl-2-oxazoline (14g). 1H NMR (CD-
Cl3) δ 1.00 (3H, t, J = 7.2 Hz), 1.48 (18H, d, J = 3.2 Hz), 1.51-
1.57 (2H, m), 1.79-1.88 (3H, m), 1.96-2.03 (1H, m), 2.02 (3H,
s), 2.66-2.70 (2H, m), 3.89 (1H, dd, J = 4.8, 10.0 Hz), 3.98 (1H,
dd, J = 5.2, 10.0 Hz), 4.01 (1H, d, J = 8.8 Hz), 4.07 (2H, t, J =
6.4 Hz), 4.35 (1H, d, J = 8.8 Hz), 7.00 (1H, t, J = 8.4 Hz), 7.22-
7.32 (4H, m), 7.41-7.43 (2H, d, J = 8.4 Hz).
The compounds 3(b-l)-P, 4-P, and 5-P were synthesized
using a similar procedure to that described for 3a-P. Synthetic
route for each compound is summarized in the Supporting
Information.
2-Amino-4-(40-butoxybiphenyl-4-yl)-2-(phosphoryloxymethyl)-
butanol (3b-P). mp 222-225 °C (dec); MS (ESIþ) m/z 424
[M þ H]. 1H NMR (CD3OD) δ 1.00 (3H, t, J = 7.2 Hz), 1.50-
1.55 (2H, m), 1.75-1.79 (2H, m), 1.97-2.02 (2H, m), 2.69-2.75
(2H, m), 3.72 (2H, brs), 4.00 (4H, t, J = 6.1 Hz), 6.96 (2H, d, J =
8.4Hz), 7.29(2H, d,J=7.6Hz), 7.48-7.51(4H, m). Anal. (C21H30-
NO6P) C, H, N.
4-[2-(40-Butoxy-30-chlorobiphenyl-4-yl)ethyl]-4-(di-tert-butyl-
phosphoryloxymethyl)-2-methyl-2-oxazoline (14h). 1H NMR
(CDCl3) δ 1.00 (3H, t, J = 7.2 Hz), 1.48 (18H, d, J = 3.6 Hz),
1.48-1.56 (2H, m), 1.81-1.88 (3H, m), 1.95-2.04 (1H, m), 2.03
(3H, s), 2.66-2.70 (2H, m), 3.89 (1H, dd, J = 4.8, 10.0 Hz), 3.97
(1H, dd, J = 5.2, 10.0 Hz), 4.01 (1H, d, J = 8.8 Hz), 4.07 (2H, t,
J = 6.4 Hz), 4.35 (1H, d, J = 8.8 Hz), 6.96 (1H, d, J = 8.8 Hz),
7.23 (2H, d, J = 8.0 Hz), 7.39 (1H, dd, J = 2.4, 8.4 Hz), 7.43 (2H,
d, J = 8.0 Hz), 7.58 (1H, d, J = 2.0 Hz).
2-Amino-4-(40-butoxy-3-chlorobiphenyl-4-yl)-2-(phosphoryl-
oxymethyl)butanol (3c-P). mp 219 °C (dec); MS (ESIþ) m/z 458
[M þ H]. 1H NMR (CD3OD) δ 1.02 (3H, t, J = 7.3 Hz),
1.46-1.60 (2H, m), 1.74-1.85 (2H, m), 1.99-2.05 (2H, m),
2.85-2.90 (2H, m), 3.72-3.85 (2H, m), 3.98-4.14 (4H, m), 7.00
(2H, d, J = 8.6 Hz), 7.40 (1H, d, J = 7.9 Hz), 7.46-7.54 (3H, m),
7.59 (1H, d, J = 1.6 Hz). Anal. (C21H29ClNO6P) C, H, N.
2-Amino-4-(40-butoxy-20-fluorobiphenyl-4-yl)-2-(phosphoryl-
oxymethy)butanol (3d-P). mp 220-223 °C (dec); MS (ESIþ) m/z
4-(Di-tert-butylphosphoryloxymethyl)-2-methyl-4-[2-(40-phe-
noxybiphenyl-4-yl)ethyl]-2-oxazoline (14i). 1H NMR (DMSO-
d6) δ 1.40 (18H, d, J = 2.2 Hz), 1.72-1.86 (2H, m), 1.92 (3H, s),
1
442 [M þ H]. H NMR (CD3OD) δ 1.00 (3H, t, J = 7.2 Hz),

3166 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Hamada et al.
1.49-1.55 (2H, m), 1.74-1.81 (2H, m), 1.99-2.04 (2H, m),
2.70-2.76 (2H, m), 3.69-3.76 (2H, m), 3.97-4.06 (4H, m),
6.72-6.75 (1H, m), 6.78-6.81 (1H, m), 7.29-7.36 (3H, m), 7.41
(2H, d, J = 8.4 Hz). Anal. (C21H29FNO6P) C, H, N.
(25 mL). After stirring at 80 °C for 4 h, the reaction was con-
centrated in vacuo and neutralized with 1 M aqueous NaOH.
The yielded precipitate was collected by filtration to give the
deprotected compound (1.36 g, 86%). A solution of the com-
pound, N,N-diisopropylethylamine (0.57 g, 4.4 mmol), and
triethyl orthoacetate (0.72 g, 4.4 mmol) in DMF (5.0 mL) was
heated at 120 °C for 3 h. The reaction mixture was poured into
water and extracted with EtOAc. The organic layer was washed
with brine, dried over Na2SO4, and concentrated in vacuo. To
the solution of the residue in CH2Cl2 (37 mL) was added 1H-
tetrazole (389 mg, 5.55 mmol) and di-tert-butyl N,N-diisopro-
pylphosphoramidite (1.54 g, 5.55 mmol) at 0 °C. After stirring at
room temperature for 2 h, a solution of tert-butyl hydroperoxide
in decane (5-6 M, 2.22 mL) was added to the solution at 0 °C.
The mixture was stirred for 10 min, poured into saturated
NaHCO3, and extracted with CHCl3. The organic layer was
washed with brine, dried over Na2SO4, and concentrated in
vacuo. Purification of the residue by silica gel chromatography
(hexane/EtOAc) gave the title compound (0.98 g, 30%) as a
colorless oil. 1H NMR (CDCl3) δ 1.49 (18H, s), 1.87-2.02
(2H, m), 2.04 (3H, s), 2.70 (2H, t, J = 8.6 Hz), 3.86-4.01 (3H,
m), 4.36 (1H, d, J = 8.9 Hz), 7.26-7.44 (7H, m).
2-Amino-4-(40-butoxy-20,50-difluorobiphenyl-4-yl)-2-(phosphoryl-
oxymethyl)butanol (3f-P). mp 228-231 °C (dec); MS (ESIþ) m/z
1
460 [M þ H]. H NMR (CD3OD) δ 1.00 (3H, t, J = 7.5 Hz),
1.49-1.58 (2H, m), 1.77-1.84 (2H, m), 1.99-2.04 (2H, m),
2.70-2.77 (2H, m), 3.72 (2H, d, J = 2.2 Hz), 3.98-4.04 (2H, m),
4.08 (2H, t, J = 6.4 Hz), 6.96 (1H, dd, J = 7.2, 12.0 Hz), 7.19
(1H, dd, J = 7.6, 11.8 Hz), 7.32 (2H, d, J = 8.2 Hz), 7.43 (2H, d,
J = 8.3 Hz). Anal. (C21H28F2NO6P 0.25H2O) C, H, N.
3
2-Amino-4-(40-butoxy-30-fluorobiphenyl-4-yl)-2-(phosphoryl-
oxymethyl)butanol (3g-P). mp 224-227 °C (dec); MS (ESIþ)
m/z 442 [M þ H]. 1H NMR (CD3OD) δ 1.00 (3H, t, J = 7.2 Hz),
1.49-1.58 (2H, m), 1.76-1.83 (2H, m), 1.99-2.04 (2H,
m), 2.69-2.76 (2H, m), 3.69-3.76 (2H, m), 3.97-4.04 (2H,
m), 4.08 (2H, t, J = 6.4 Hz), 7.10-7.14 (1H, m), 7.30-7.35 (4H,
m), 7.50 (2H, d, J = 8.0 Hz). Anal. (C21H29FNO6P 0.1H2O)
3
C, H, N.
2-Amino-4-(40-butoxy-30-chlorobiphenyl-4-yl)-2-(phosphoryl-
oxymethylbutanol (3h-P). mp 221-224 °C (dec); MS (ESIþ) m/z
1
4-[2-(40-Bromo-3-chloro-20-fluorobiphenyl-4-yl)ethyl]-4-(di-
tert-butylphosphoryloxymethyl)-2-methyl-2-oxazoline (15b). The
title compound was synthesized from 13b using a similar procedure
to that described for 15a. 1H NMR (CDCl3) δ 1.49 (18H, s), 1.85-
1.95 (2H, m), 2.03 (3H, s), 2.73-2.85 (2H, m), 3.92 (1H, dd, J=4.8,
9.9 Hz), 3.99 (1H, dd, J = 4.8, 9.9 Hz), 4.11 (1H, d, J = 9.0 Hz),
4.38 (1H, d, J = 8.7 Hz), 7.24-7.37 (5H, m), 7.49 (1H, s).
458 [M þ H]. H NMR (CD3OD) δ 1.01 (3H, t, J = 7.5 Hz),
1.54-1.59 (2H, m), 1.78-1.83 (2H, m), 1.98-2.02 (2H, m),
2.69-2.73 (2H, m), 3.72 (2H, d, J = 2.3 Hz), 3.99-4.04 (2H, m),
4.09 (2H, t, J = 6.3 Hz), 7.10 (1H, d, J = 8.6 Hz), 7.31 (2H, d,
J = 8.0 Hz), 7.46-7.50 (3H, m), 7.59 (1H, d, J = 2.2 Hz). Anal.
(C21H29ClNO6P) C, H, N.
2-Amino-4-(40-phenoxybiphenyl-4-yl)-2-(phosphoryloxymethyl)-
butanol (3i-P). mp 207-209 °C (dec); MS (ESIþ) m/z 444 [M þ
2-Amino-4-(40-benzylthio-20-fluorobiphenyl-4-yl)-2-(phosphoryl-
oxymethyl)butanol (6b-P). A mixture of 15a (280 mg, 0.479
mmol), benzyl mercaptan (66 mg, 0.53 mmol), tris(dibenzyl-
ideneacetone)dipalladium(0)-chloroform adduct (12.4 mg, 0.0119
mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xant-
phos, 14.3 mg, 0.0247 mmol), and N,N-diisopropylethylamine
(124 mg, 0.959 mmol) in 1,4-dioxane (2.0 mL) was refluxed for 5
h. After being poured into water, the mixture was extracted with
EtOAc. The organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. Purification of the residue
by silica gel chromatography (hexane/EtOAc) gave the protected
title compound. To a solution of the compound in EtOH (5.0 mL)
was added 35% HCl (1.0 mL). The mixture was stirred at 50 °C for
4 h and diluted with water. The yielded solid was collected by
filtration to give the title compound (74 mg, 31%) as a white solid.
mMp 167-170 °C; MS (ESIþ) m/z 492 [M þ H]. 1H NMR
(CD3OD) δ 1.99-2.06 (2H, m), 2.70-2.80 (2H, m), 3.73 (2H, s),
4.09 (2H, d, J = 5.1 Hz), 4.22 (2H, s), 7.10-7.37 (10H, m), 7.43-
1
H]. H NMR (CD3OD) δ 1.98-2.04 (2H, m), 2.70-2.76 (2H,
m), 3.73 (2H, d, J = 1.1 Hz), 3.98-4.07 (2H, m), 7.01-7.05 (4H,
m), 7.12 (1H, t, J = 7.7 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.36 (2H, t,
J = 8.3 Hz), 7.53 (2H, d, J = 8.0 Hz), 7.58 (2H, d, J = 8.6 Hz).
Anal. (C23H26NO6P 0.25H2O) C, H, N.
3
2-Amino-4-(3-chloro-40-phenoxybiphenyl-4-yl)-2-(phosphoryl-
oxymethyl)butanol (3j-P). mp 218 °C (dec); MS (ESIþ) m/z 478
[M þ H]. 1H NMR (CD3OD) δ 2.00-2.05 (2H, m), 2.86-2.92
(2H, m), 3.72-3.85 (2H, m), 3.98-4.14 (2H, m), 7.03-7.08 (5H,
m), 7.36-7.51 (3H, m), 7.60-7.64 (4H, m). Anal. (C23H25ClN-
O6P) C, H, N.
2-Amino-4-(40-benzyloxy-3-chlorobiphenyl-4-yl)-2-(phosphoryl-
oxymethyl)butanol (3k-P). mp 241 °C (dec); MS (ESIþ) m/z 492
[M þ H]. 1H NMR (CD3OD) δ 2.00-2.04 (2H, m), 2.85-2.90
(2H, m), 3.72-3.85 (2H, m), 3.98-4.14 (2H, m), 5.15 (2H, s),
7.09 (2H, d, J = 8.4 Hz), 7.32-7.60 (10H, m). Anal. (C24H27-
ClNO6P H2O) C, H, N.
3
2-Amino-4-(40-benzyloxy-20-fluorobiphenyl-4-yl)-2-(phosphoryl-
oxymethyl)butanol (3l-P). mp 241-243 °C; MS (ESIþ) m/z 476
[M þ H]. 1H NMR (CD3OD) δ 2.02-2.05 (2H, m), 2.72-2.77
(2H, m), 3.71-3.73 (2H, m), 4.07-4.09 (2H, m), 5.12 (2H, s),
6.82-6.90 (2H, m), 7.29-7.46 (10H, m). Anal. (C24H27FNO6-
7.46 (2H, m). Anal. (C24H27FNO5PS 1.5H2O) C, H, N.
3
The compounds 6(a,c-f)-P were synthesized using a similar
procedure to that described for 6b-P. Synthetic route for each
compound is summarized in the Supporting Information.
2-Amino-4-(40-benzylthio-3-chlorobiphenyl-4-yl)-2-(phosphoryl-
oxymethyl)butanol (6a-P). mp 236-238 °C; MS (ESIþ) m/z 508
[M þ H]. 1H NMR (CD3OD) δ 1.98-2.02 (2H, m), 2.84-2.88
(2H, m), 3.72-3.80 (2H, m), 4.00-4.09 (2H, m), 4.19 (2H, s),
7.19-7.29 (3H, m), 7.33 (2H, d, J = 7.4 Hz), 7.38 (2H, d, J = 8.2
Hz), 7.42 (1H, d, J = 7.9 Hz), 7.48-7.52 (3H, m), 7.61 (1H, d,
P H2O) C, H, N.
3
2-Amino-4-(20-fluoro-40-pentylbiphenyl-4-yl)-2-(phosphoryloxy-
methyl)butanol (4-P). mp 225-228 °C (dec); MS (ESIþ) m/z 440
[M þ H]. 1H NMR (CD3OD) δ 0.92 (3H, t, J = 6.8 Hz),
1.33-1.37 (4H, m), 1.61-1.67 (2H, m), 1.98-2.04 (2H, m),
2.62-2.66 (2H, m), 2.70-2.76 (2H, m), 3.72 (2H, d, J = 2.4 Hz),
3.97-4.04 (2H, m), 6.98 (1H, d, J = 11.1 Hz), 7.05 (1H, d, J =
8.0 Hz), 7.31-7.36 (3H, m), 7.45 (2H, d, J = 7.9 Hz). Anal.
(C22H31FNO5P) C, H, N.
J = 1.5 Hz). Anal. (C24H27ClNO5PS 1.25H2O) C, H, N.
3
2-Amino-4-(20-fluoro-40-phenylthiobiphenyl-4-yl)-2-(phosphoryl-
oxymethyl)butanol (6c-P). mp 208-212 °C; MS (ESIþ) m/z 478
[M þ H]. 1H NMR (CD3OD) δ 1.94-2.05 (2H, m), 2.68-2.80
(2H, m), 3.73 (2H, s), 3.93-4.08 (2H, m), 7.03-7.06 (1H, m),
7.13 (1H, d, J = 8.4 Hz), 7.34-7.49 (10H, m). Anal. (C23H25-
2-Amino-4-[20-fluoro-40-(4-methylphenoxy)biphenyl-4-yl]-2-(phos-
phoryloxymethyl)butanol (5-P). mp 218-220 °C; MS (ESIþ) m/z
1
476 [M þ H]. H NMR (CD3OD) δ 1.96-2.08 (2H, m), 2.35
FNO5PS 0.25H2O) C, H, N.
3
2-Amino-4-[20-fluoro-40-(4-methylphenylthio)biphenyl-4-yl]-
2-(phosphoryloxymethyl)butanol (6d-P). mp 236-238 °C; MS
(ESIþ) m/z 492 [M þ H]. 1H NMR (CD3OD) δ 1.98-2.08 (2H,
m), 2.38 (3H, s), 2.68-2.82 (2H, m), 3.72 (2H, s), 3.94-4.10 (2H,
m), 6.91 (1H, d, J = 11.4 Hz), 7.05 (1H, d, J = 8.1 Hz), 7.24-
7.45 (9H, m). Anal. (C24H27FNO5PS) C, H, N.
(3H, s), 2.64-2.80 (2H, m), 3.72 (2H, s), 3.95-4.08 (2H, m),
6.71-6.83 (2H, m), 6.96 (2H, d, J = 8.4 Hz), 7.22 (2H, d, J = 8.4
Hz), 7.31-7.45 (5H, m). Anal. (C24H27FNO6P) C, H, N.
4-[2-(40-Bromo-20-fluorobiphenyl-4-yl)ethyl]-4-(di-tert-butyl-
phosphoryloxymethyl)-2-methyl-2-oxazoline (15a). To a solution
of 13a (2.50 g, 5.55 mmol) in EtOH (50 mL) was added 35% HCl

Article
2-Amino-4-[20-fluoro-40-(3-methylphenylthio)biphenyl-4-yl]-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3167
was inserted into the abdominal aorta. Test compounds were
homogeneously suspended with 0.5% HPMC (METOLOSE
60SH-50, Shin-Etsu Chemical Co., Ltd.) and administered orally.
Heart rate was monitored by data acquisition system in the
telemetry system (Dataquest, A.R.T., Science International).
Frequency of data sampling was for 15 s in every 5 min.
2-(phosphoryloxymethyl)butanol (6e-P). mp 212-213 °C; MS
(ESIþ) m/z 492 [M þ H]. 1H NMR (CD3OD) δ 1.97-2.05 (2H,
m), 2.34 (3H, s), 2.68-2.82 (2H, m), 3.72 (2H, s), 3.92-4.10 (2H,
m), 6.97 (1H, d, J = 11.4 Hz), 7.09 (1H, d, J = 9.9 Hz), 7.18-
7.47 (9H, m). Anal. (C24H27FNO5PS 0.75H2O) C, H, N.
3
2-Amino-4-[3-chloro-20-fluoro-40-(4-methylphenylthio)biphenyl-
4-yl]-2-(phosphoryloxymethyl)butanol (6f-P). mp 214-216 °C;
MS (ESIþ) m/z 526 [M þ H]. 1H NMR (CD3OD) δ 1.93-2.02
(2H, m), 2.39 (3H, s), 2.85-2.91 (2H, m), 3.68-3.82 (2H, m),
3.95-4.10 (2H, m), 6.91 (1H, d, J = 11.1 Hz), 7.04 (1H, d, J =
8.4 Hz), 7.27 (2H, d, J = 7.8 Hz), 7.35-7.42 (5H, m), 7.53 (1H,
s). Anal. (C24H26ClFNO5PS) C, H, N.
Acknowledgment. We thank Sachiko Mochizuki and Makio
Ohtsuki for performing the lymphocyte reduction assay.
Supporting Information Available: Tables giving the synthetic
routes of alcohol compounds and phosphate compounds, pre-
paration details of 6a and 6a-P, and elemental analysis data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Intracellular Ca2þ Mobilization Assay. Chem-1 or CHO cells
stably expressing human S1P receptors (hS1P)1 or hS1P3
respectively were loaded with 5 μM Fura-2 AM (Dojindo,
Kumamoto, Japan) for 90 min at 37 °C. After two washes, the
cells were suspended at a concentration of 3 ꢀ 105 cells/mL in
HBSS containing 0.1% BSA (fatty acid free) and 1.25 mM
probenecid (pH 7.4). The cells (3 ꢀ 104 cells/well) in HBSS
containing 0.1% BSA (fatty acid free) and 1.25 mM probenecid
(pH 7.4) were plated in black wall 96-well plates, and then the
changes in fluorescence were monitored at 37 °C at excitation
wavelengths of 340 and 380 nm and an emission wavelength of
540 nm using a functional drug screening system (FDSS3000,
Hamamatsu Photonics, Shizuoka, Japan). Ten seconds after the
start of monitoring, the serial dilutions of S1P or test com-
pounds were added and the maximal change in Ca2þ concentra-
tion after stimulator addition was quantitated.
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