An oral sphingosine 1-phosphate receptor 1 (S1P1) antagonist prodrug with efficacy in vivo: Discovery, synthesis, and evaluation

Article abstract of DOI:10.1021/jm3009508

A prodrug approach to optimize the oral exposure of a series of sphingosine 1-phosphate receptor 1 (S1P₁) antagonists for chronic efficacy studies led to the discovery of (S)-2-{[3′-(4-chloro-2,5- dimethylphenylsulfonylamino)-3,5-dimethylbiphen

Full text of DOI:10.1021/jm3009508

Article
pubs.acs.org/jmc
An Oral Sphingosine 1‑Phosphate Receptor 1 (S1P1) Antagonist
Prodrug with Efficacy in Vivo: Discovery, Synthesis, and Evaluation
Daniela Angst,* Philipp Janser, Jean Quancard, Peter Buehlmayer, Frederic Berst, Lukas Oberer,
Christian Beerli, Markus Streiff, Charles Pally, Rene Hersperger, Christian Bruns, Frederic Bassilana,
and Birgit Bollbuck
Novartis Pharma AG, Novartis Institutes for BioMedical Research, 4002 Basel, Switzerland
S
* Supporting Information
ABSTRACT: A prodrug approach to optimize the oral exposure of a
series of sphingosine 1-phosphate receptor 1 (S1P1) antagonists for
chronic efficacy studies led to the discovery of (S)-2-{[3′-(4-chloro-2,5-
dimethylphenylsulfonylamino)-3,5-dimethylbiphenyl-4-carbonyl]-
methylamino}-4-dimethylaminobutyric acid methyl ester 14. Methyl
ester prodrug 14 is hydrolyzed in vivo to the corresponding carboxylic
acid 15, a potent and selective S1P1 antagonist. Oral administration of
the prodrug 14 induces sustained peripheral blood lymphocyte reduction
in rats. In a rat cardiac transplantation model coadministration of a
nonefficacious dose of prodrug 14 with a nonefficacious dose of
sotrastaurin (19), a protein kinase C inhibitor, or everolimus (20), an
mTOR inhibitor, effectively prolonged the survival time of rat cardiac
allografts. This demonstrates that clinically useful immunomodulation
mediated by the S1P1 receptor can be achieved with an S1P1 antagonist
generated in vivo after oral administration of its prodrug.
INTRODUCTION
■
Sphingosine 1-phosphate (S1P), a metabolite of sphingomyelin,
is a bioactive sphingolipid and interacts with five S1P receptors
(S1P1−S1P5), a family of cell-surface G-protein-coupled
receptors.1 The S1P receptors are expressed in many tissues,
and S1P and the S1P receptors are involved in important
regulatory functions both in normal physiology and disease
processes such as apoptosis, cell migration, endothelial barrier
function, vascular tone, and neural cell communication.2
The S1P1 receptor is expressed in most immune cells,
including B and T cells, and plays a key role in controlling the
Figure 1. Structures of S1P1 antagonists with oral in vivo activity.
trafficking of lymphocytes from the lymphoid organs and the
thymus into blood.3 Modulation of the S1P1 receptor with an
b.i.d.15 At this dose 1 also effectively inhibited tumor
agonist and with an antagonist causes inhibition of lymphocyte
angiogenesis in mice.16 In a MDA-MB-231T mouse xenograft
egress from lymph nodes and thymus, leading to immunomo-
dulation.4−7 A broad range of chemical probes have been
model 2 (XL541) significantly inhibited tumor growth when
given orally for 14 days at 100 mg/kg po, q.d.17 Therapeutic
identified demonstrating that S1P1 agonists are efficacious in
various animal models for transplantation and autoimmune
diseases including experimental autoimmune encephalomyelitis,
treatment (26 days, 30 and 60 mg/kg po, b.i.d.) of 3 (NIBR-
0213) in a mouse experimental autoimmune encephalomyelitis
model resulted in gradual reduction in disease scores.18
There is a high need for S1P1 antagonists preferably with
high subtype selectivity to better understand their therapeutic
potential and potential mechanism-based liabilities. In partic-
ular, we aimed for a selective S1P1 antagonist with long lasting,
reversible lymphocyte sequestration from circulation at a
adjuvant- or collagen-induced arthritis, and lupus nephritis.8−13
Furthermore, the S1P1 agonist 2-amino-2-(4-octylphenethyl)-
propane-1,3-diol (fingolimod, FTY720) is approved for the
treatment of relapsing forms of multiple sclerosis.14 To the best
of our knowledge, only recently potent S1P1 antagonists with
demonstrated oral in vivo activity in animal models were
described in the literature (Figure 1). In a mouse collagen-
induced arthritis model 1 (TASP0277308) significantly sup-
pressed the development of arthritis at a dose of 100 mg/kg po,
Received: July 4, 2012
Published: October 15, 2012
© 2012 American Chemical Society
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Table 1. Activity of Carboxylic Acids in the GTPγ35S Binding
Assay
reasonable oral dose to explore efficacy in a rat heart
transplantation model.
RESULTS AND DISCUSSION
■
At the onset of this work, 4 was available as chemical starting
point, which resulted from our efforts to find a potent and
selective S1P1 antagonist (Figure 2).7 Compound 4 is highly
Figure 2. Structure of 4 and strategies for optimization.
potent in the GTPγ35S binding assay (Table 1) and efficiently
reduces peripheral lymphocyte counts in Lewis rats, both after
subcutaneous and after oral administration. However, the
duration of action was limited. A high oral dose (100 mg/kg)
was necessary to significantly reduce at 8 h the number of
lymphocytes in blood (20% of the control values), and control
lymphocyte counts were reestablished within 24 h (Table 2). It
was likely that the poor permeability (log Pe (permeability
coefficient defined by PAMPA) of −8.3 at pH 6.8) of 4 was
leading to absorption limited blood levels, thus contributing to
the short duration of action in the peripheral blood lymphocyte
(PBL) reduction assay. Our goal was to achieve sustained
systemic exposure to maintain full PBL reduction over 24 h
after a single dose of 30 mg/kg po, achieving the same effect as
with a selective S1P1 agonist.8
We envisioned two optimization strategies to modify the oral
exposure of the scaffold. This could be achieved by reducing the
polar surface area (PSA) of 4, either by alkylating the amide or
by masking the carboxylic acid as an ester prodrug and by
modulating the solubility by introducing basic moieties (Figure
2).19−22 To broadly explore the potential of such a prodrug
approach, we selected the corresponding carboxylic acids based
on potency in the GTPγ35S assay and evaluated all compounds
in the PBL reduction assay. For all carboxylic acids the
GTPγ35S values are reported in Table 1, and the in vivo efficacy
results of the corresponding prodrug esters in the PBL
reduction assay are reported in Table 2.
First, we explored the effect of amide alkylation. Methyl-
amide of 4, 5 was highly potent in the GTPγ35S (6.1 1.02
nM) assay, and oral administration of 5 (100 mg/kg) induced
reduction of lymphocytes to 19% versus control for 12 h. The
increased duration of action in the PBL reduction assay was
likely due to better absorption (log Pe = −7.7 at pH 6.8). Next,
we investigated the effect of masking the carboxylic acid as an
ester, which could act as a prodrug and liberate the carboxylic
acid by in vivo hydrolysis. Methyl ester of 4, 6 is only weakly
active in the GTPγ35S assay (6733 1105 nM). However, oral
application of 6 (100 mg/kg) reduced the lymphocyte counts
to 24% versus control up to 14 h. This in vivo effect can only be
rationalized by the presence of the highly potent 4 in blood
which resulted from the hydrolysis of methyl ester 6. This was
confirmed by analysis of the blood samples.
a
IC50 values are reported as the mean of at least three separate
determinations SEM.
Finally, amide methylation and methyl ester were combined
to provide 7. We expected higher absorption for 7 versus 6 and
a longer duration of action in the PBL reduction assay.
Disappointingly, the duration of action was in the same range as
for 6 after oral administration of the same dose. We reasoned
that the low solubility of compound 7 (<0.004 mM at pH 6.8)
became the absorption limiting factor. As a result, we
concentrated our efforts on improving the solubility by
introducing solubilizing groups in the pro-moiety as well as
in the parent drug. First, introduction of a dimethylamino
group into the pro-moiety provided dimethylaminoethyl ester
8. This led to a significant longer duration of action. After an
oral dose of only 30 mg/kg lymphocytes were reduced to 25%
(14 h) and 49% (24 h) compared to control values, which was
likely because of better solubility (0.015 mM at pH 6.8).
Compounds with a basic group in the parent drug were then
evaluated. Administration of compound 9, with a dimethyla-
minomethyl side chain, induced only a reduction to 37% of
circulating lymphocytes at 14 h versus control despite a high
dose of 100 mg/kg po. Since 9 has high permeability (log Pe =
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Table 2. Administered Dose in PBL Reduction Assay and Percent Remaining Lymphocytes versus Control at Given Time
Points
a
b
Dose: mg/kg po. Remaining percent of circulating lymphocytes in blood versus control.
−4.9 at pH 6.8), the absorption is likely limited by the low
solubility (<0.004 mM at pH 6.8). Further side chain variations
were probed. Analogue 10, with an aminoethyl side chain,
displayed only marginal PBL reduction 14 h after a 100 mg/kg
po dose, although the corresponding carboxylic acid 11 was
promoted a persistent reduction of lymphocyte counts for 24 h.
The enantiomer of 14, 16 was also investigated. Only a modest
reduction of lymphocytes versus control values (67% at 14 h)
was observed after oral dosing of 30 mg/kg. This is not
unexpected given the reduced potency of the corresponding
carboxylic acid 17. Last, the ethyl ester of 15, 18, was explored.
The in vivo effect of ethyl ester 18 was somewhat reduced
compared to methyl ester 14 showing only 28% (14 h) and
48% (24 h) lymphocyte reduction in the PBL reduction assay
after a dose of 30 mg/kg po. Various factors can lead to this
reduced in vivo effect, such as permeability (log Pe = −5.0 at
pH 6.8), solubility (<0.004 mM at pH 6.8), and in vivo
hydrolysis rate.
In summary, we achieved our goal to significantly improve
the duration of action in the PBL reduction assay after oral
administration. Starting from 4, we identified 14, a prodrug
with sustained oral efficacy by amide methylation, masking the
carboxylic acid as a methyl ester and introducing a basic moiety
in the side chain.
highly potent in the GTPγ35S assay (2.1
0.71 nM). We
attributed the lack of in vivo activity of 10 to the low
permeability (log Pe = −6.8 at pH 6.8) of the primary amine
derivative. In addition, we had observed that the corresponding
lactam 12 was formed readily from 10 if the pH was not acidic.
The lactam 12 was only weakly active in the GTPγ35S assay
(163 42 nM) and did not lead to a reduction of lymphocyte
counts after an oral dose of 100 mg/kg. To improve
permeability and prevent potential in vivo lactam formation,
the amino group of 10 was dimethylated. Compound 13
displayed improved permeability (log Pe = −5.1 at pH 6.8), and
a dose of 100 mg/kg po induced full PBL reduction for 24 h
(24% lymphocytes remaining versus control values). Knowing
that amide methylation had a beneficial effect on the duration
of action, we investigated the combination with a basic moiety
in the side chain. The resulting analogue 14 had high
permeability (log Pe = −4.6 at pH 6.8), and the corresponding
carboxylic acid 15 was highly potent in the GTPγ35S assay (3.4
0.35 nM). Administration of compound 14 (30 mg/kg po)
Characterization of the Prodrug 14 and the Active
Moiety 15. Before starting further in vivo studies with 14, we
characterized the 14/15 prodrug/drug pair in more detail.
Schild plot analysis indicated that 15 is a competitive S1P1
antagonist with a calculated KD of 0.67 0.2 nM (Figure S1,
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Table 3. S1P Receptor Subtype Selectivity of Carboxylic Acid 15 in the GTPγ35S Binding Assays
hS1P1
hS1P2
hS1P3
hS1P4
>10000
1195 158
hS1P5
a
IC50, (nM)
3.4 0.35
>10000
>10000
>10000
802 76
>10000
978 59
>10000
a
EC50, (nM)
a
Antagonistic (IC50) and agonistic (EC50) activities for the subtype receptors are reported. IC50 and EC50 values are reported as the mean of at least
three separate determinations SEM.
Supporting Information). Compound 15 is a highly selective
S1P1 antagonist. No activity was measured on the S1P2 receptor
subtype (Table 3). The selectivity of 15 against the S1P3 and
S1P5 receptor subtypes is more than 200-fold, where a weak
antagonistic activity was measured in the GTPγ35S binding
assays. No antagonistic activity was measured on the S1P4
receptor subtype; however, 15 is a weak human S1P4 agonist.
Compound 15 was also tested for selectivity against a diverse
panel of receptors, and up to 10 μM no appreciable binding
affinity was observed.
Although S1P1 agonists like fingolimod and the S1P1
antagonist 15 have similar effects on PBL counts, they act
through different mechanisms. Agonists lead to S1P1-depend-
ent intracellular signaling followed by receptor internalization
and degradation.14,23,24 The S1P1 signaling is responsible for
the activation of the GIRK channel, which has been associated
with heart rate reduction in humans.25,26 In rodents S1P3 is
responsible for heart rate changes.27 It has been shown that the
selective S1P1 antagonist 3 does not lead to S1P1-dependent
signaling and does not induce S1P1 receptor internalization.18
In addition, 3 lacks GIRK activation and blocks agonist induced
receptor internalization. S1P1 antagonists, devoid of GIRK
channel activity, are not expected to cause bradycardia in
humans. S1P1 antagonist 15 does not induce S1P1-dependent
signaling and does not lead to GIRK activation.
at pH 1 not shown). However, at pH 7.4 hydrolysis to the
carboxylic acid was observed. Already after 2 h 9% of 15 was
present and the amount of hydrolyzed species continuously
increased to 48% (18 h) and 85% (49 h). After 82 h no more
methyl ester could be detected by UPLC. At pH 10 hydrolysis
of methyl ester 14 was significantly faster than at pH 7.4 with
only 52% ester remaining after 2 h. After 17 h no more methyl
ester 14 was detected. These data pointed out the importance
of carefully adjusting the pH of the formulations used for in
vivo experiments. All samples with methyl ester 14 were freshly
prepared before dosing by dissolving the compound in a
mixture of PEG200 and water and adjusting the pH to 5 with
aqueous HCl (0.1 M). For the determination of blood levels of
both the prodrug and the active moiety at different time points
during in vivo experiments it was necessary to prevent further
hydrolysis of the prodrug after sampling. This was ensured by
stabilizing the blood by sampling immediately in EDTA coated
Eppendorf vials containing aqueous citric acid (2 M). By use of
this procedure, the pH of the blood samples was adjusted to 4.4
and further chemical hydrolysis was inhibited. Seemingly, this
procedure also stopped further enzymatic hydrolysis, since the
same results were obtained when esterase inhibitors were
added. Furthermore, storage of the samples did not change the
blood levels.
After isolation and purification of 14 several minor signals
were observed in the NMR as well as an additional peak (peak
A) in the UPLC (Figure 4). According to UPLC the ratio of
peak A:B was 7:93. We anticipated that because of the amide
methylation, the rotation around the amide bond might be
hindered and therefore the minor peak A could arise from the
(Z) amide conformer of 14. This hypothesis was supported by
the fact that the same mass was observed for peak A and peak
B. If peak A and peak B were indeed amide conformers, a
sample enriched with peak A should equilibrate to provide
again a peak ratio A:B of 7:93. It is possible to separate peak A
and peak B on silica, and column chromatography of 14
provided a fraction with a peak ratio A:B of 55:45. This sample
was monitored by UPLC over time. The amount of peak A
decreased over time while the amount of peak B increased
(Figure 5). After 24 h a ratio of 14:86 was observed, and after 6
days the equilibrium ratio of 7:93 was reached, matching the
initially determined ratio for 14 after isolation. For additional
confirmation and for unambiguous assignment of the amide
Ideally, the prodrug 14 should be chemically stable under the
formulation conditions and during oral administration.
Compound 14 should undergo enzymatic cleavage in vivo
during the absorption and distribution processes to release the
active species 15 into the circulation. For the determination of
the blood levels of both species, further ester cleavage after
blood sampling has to be stopped. To investigate potential
contributions of nonenzymatic cleavage through chemical
hydrolysis, we probed the chemical stability of 14 at different
pH by UPLC. At pH 1 and 5 methyl ester 14 was stable for >53
h and none of the active moiety 15 was detected (Figure 3, data
1
conformers we also investigated the equilibration by H NMR.
We were able to separate peak A and peak B by preparative
HPLC. Removal of the solvent at low temperature and storage
of the isolated sample on dry ice afforded a sample with a ratio
1
A:B of 65:35 as determined by H NMR (Figure 6). The
proton in the position α to the ester (H-32) and the methyl
group of the ester (H-40) were monitored (Figure 7). The
signals of conformer A decreased over time, and the signals of
conformer B increased. After 14.4 h the ratio of A:B was 14:86,
and after 6 days equilibrium was reached with A:B of 7:93.
Furthermore, the conformers A and B were characterized by 1H
and 13C NMR. The 13C shift differences between the two
Figure 3. Hydrolysis of methyl ester 14 to carboxylic acid 15 at
different pH.
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Figure 4. UPLC of 14 at equilibrium.
1
expected H shift difference of N-methyl groups between (Z)
and (E) isomers is 0.12 ppm. In conformer B the N-methyl
group H-31 is high field shifted. In contrast, in conformer A the
α-proton H-32 and H-35, H-36, H-38, and H-41 are high field
shifted. These high field shifts can be explained by the shielding
effect by the inner ring current of the aromatic system attached
to the amide. The data suggest that in conformer A the
complete amino acid side chain including H-32 is placed above
the aromatic ring and the methyl ester group points to the
other direction, leading to the conclusion that conformer A
corresponds to the (Z) amide. In conformer B only the methyl
group H-31 is affected by the high field shift and the amino acid
side chain has normal shifts, which is in agreement with the (E)
amide. The chemical shift differences of protons and carbons of
the amino acid residue in both conformers allow the
unambiguous assignment of the amide conformers, conformer
A corresponding to the (Z) amide and conformer B to the (E)
Figure 5. Equilibration of methyl ester 14 amide conformers A and B
monitored by UPLC.
1
amide of 14. In summary, both UPLC and H NMR analyses
confirmed the presence of two amide conformers in 14 with the
(E) conformer being the major at equilibrium.
In Vivo Evaluation of 14. Oral administration of 14 (30
mg/kg po) induced a rapid decrease in the number of
conformers are 4.7 ppm for C-31 and 4.0 ppm for C-32 (Figure
7). These differences are comparable with those observed in
(Z) and (E) amide conformers.28,29 The 1H NMR shifts of the
N-methyl groups H-31 show a difference of 0.32 ppm, while the
1
Figure 6. Equilibration of methyl ester 14 amide conformers A and B monitored by H NMR.
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1
Figure 7. Assignment of amide conformers by H and 13C NMR.
peripheral lymphocytes in Lewis rats (Figure 8). Reduction of
circulating lymphocytes was maintained for 24 h, and peripheral
a single dose, suggesting that the methyl ester 14 acted as a
reservoir, continuously generating active species 15.
Having shown that a single oral dose of 14 leads to
lymphocyte sequestration over 24 h, repeated dosing was
investigated to probe the potential of the compound in a
chronic setting. Prodrug 14 was dosed daily (30 mg/kg po)
over 5 days, and lymphocytes were measured once a day before
administration of the next dose. Circulating lymphocytes were
reduced to around 20% of control values over the whole
experiment, confirming the immunomodulation after repeated
application (data not shown). It has been shown that S1P1
antagonists induce capillary leakage in rodent lungs.15,30 We
investigated the acute and chronic effects of 14 in an Evans blue
dye (EBD) lung leakage model.30 Compound 14 was applied
b.i.d. (30 mg/kg po) to Lewis rats, and the EBD increase versus
control was measured at 6 h (after one dose), 24 h (two doses),
and 96 h (eight doses). At 6 h a (3.0 0.4)-fold EBD increase
was measured, at 24 h a (2.4 0.4)-fold increase, and at 96 h a
(2.2
0.3)-fold increase (Figure 9). Fluid was found in the
Figure 8. Lymphocyte counts and blood levels of 14 and 15 after
administration of methyl ester 14 (30 mg/kg po). The baseline value
of PBL (100%) corresponds to 7745 lymphocytes per microliter.
thorax at 6 h (1.00 0.22 mL) and 24 h (2.00 0.57 mL).
However, after 96 h no fluid could be detected in the thorax.
These data suggest that the pulmonary leakage is a transient
effect that decreases after multiple dosing. The physiological
consequences of such an effect are not yet known. Further
safety investigations are necessary to understand the long-term
effect of S1P1 antagonists on lung permeability.
These data encouraged us to test the prodrug 14 in a rat
heart transplantation model to investigate if the observed
immunomodulation translates into prolonged allograft survival.
Methyl ester 14 was tested in the stringent DA (Dark Agouti)
to Lewis rat heterotopic vascular heart allotransplantation
model in combination with a nonefficacious dose of 3-(1H-
indol-3yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]-
pyrrole-2,5-dione (sotrastaurin, 19),31 a protein kinase C
inhibitor, or everolimus (20),32 an mTOR inhibitor. In vehicle
treated recipient rats, DA grafts were acutely rejected on day 6
(Table 4). In recipient rats treated with 14 alone at 30 mg/kg
po, b.i.d., grafts were rejected within 10 days with a median
graft survival time (MST) of 8 days, and severe acute rejection
lymphocyte counts returned to pretreatment levels 48 h after
compound administration. Analysis of the blood levels revealed
that methyl ester 14 and the corresponding carboxylic acid 15
were present over 24 h. The methyl ester 14 itself is not an
S1P1 antagonist. Compound 14 is hydrolyzed in vivo to the
potent S1P1 antagonist 15 which is responsible for the in vivo
effect. For the prodrug 14 a two-phase absorption was observed
leading to an early peak after 0.25 h (146 32 ng/mL) and a
maximum concentration after 8 h (364 65 ng/mL). After 24
h low levels of 14 could still be detected in the blood (50
ng/mL). For the active moiety 15 the levels were low over 24
h. The maximum concentration was detected at 8 h (29
4
2
ng/mL), and after 24 h only very low levels of 15 were
measured (5 0 ng/mL). Despite the low levels of the active
species, lymphocyte sequestration was maintained for 24 h after
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revealed no drug−drug interactions. Combination therapy did
not affect the blood levels of both immunomodulating agents.
These results are comparable to treatment with S1P1 agonists
in the same model. Fingolimod (0.1 mg/kg po, q.d.) co-
treatment with 19 (10 mg/kg po, b.i.d.) or 20 (0.3 mg/kg po,
q.d.) led to a MST of >68.5 or 38 days, respectively.33,34 With
the selective S1P1 agonist (3-(((2-(2-(trifluoromethyl)-[1,1′-
biphenyl]-4-yl)benzo[b]thiophen-5-yl)methyl)amino)-
propanoic acid) (10 mg/kg po, q.d.) in combination with 20
(0.3 mg/kg po, q.d.) all grafts reached the termination point of
26 days, showing moderate acute cellular rejection.8 Thus, oral
combination therapy of the S1P1 antagonist prodrug 14 with 19
or with 20 significantly prolonged cardiac allograft survival. It
was shown that the selective S1P1 antagonist 15, generated in
vivo after oral administration of its methyl ester prodrug 14,
mediates potent immunomodulation leading to increased graft
protection in a transplantation model.
Figure 9. EBD accumulation in rat lungs after single and repeated
dosing of 14 (30 mg/kg po b.i.d.).
CHEMISTRY
■
Intermediate 21 was obtained by Suzuki coupling of 4-bromo-
2,6-dimethylbenzoic acid (22) with 3-aminophenylboronic acid
to afford 23 followed by reaction with 4-chloro-2,5-
dimethylphenylsulfonyl chloride (Scheme 1). The amide
coupling reaction to introduce the different amino acid head
groups was carried out by formation of the acid chloride of 21
followed by addition of the corresponding amines. For the
synthesis of 6, (S)-2-aminopropionic acid methyl ester
hydrochloride (24) was used in the amide coupling reaction
(Scheme 2). Compound 7 was obtained by using (S)-2-
methylaminopropionic acid methyl ester hydrochloride (25)
for the coupling. Ester hydrolysis of 7 provided 5. Subsequent
esterification with 2-dimethylaminoethanol led to 8. Com-
pound 9 was obtained from intermediate 21 and (S)-2-
methylamino-3-hydroxypropanoic acid methyl ester hydro-
chloride (26) and a subsequent conversion of the alcohol 27
t o t h e t e r t i a r y a m i n e w i t h ( c y a n o m e t h y l ) -
trimethylphosphonium iodide and dimethylamine hydrochlor-
ide.35 Ester hydrolysis generated 28. Amide coupling of (S)-2-
amino-4-((tert-butoxycarbonyl)amino)butanoic acid methyl
ester hydrochloride (29) with compound 21 provided the
Boc protected intermediate 30 which was deprotected to give
10 (Scheme 3). To obtain the carboxylic acid 11, intermediate
30 was first treated with aqueous base to provide the carboxylic
acid 31 followed by Boc cleavage with HCl in dioxane. The
lactam derivative 12 was obtained by deprotection of 30 with
TFA followed by cyclization in refluxing toluene. The amino
group of 10 was dialkylated by reductive amination with
formaldehyde to yield 13. Hydrolysis of the ester provided 32.
Reductive amination of 29 with benzaldehyde followed by an in
was confirmed by histology. At this dose 14 did not prevent
graft rejection, but it was capable of strongly mediating
sustained lymphocyte counts. In recipients treated with 19 (10
mg/kg po, b.i.d.) or with 20 (0.3 mg/kg po, q.d.) all grafts were
rejected at day 7, with severe acute rejection confirmed by
histology. In contrast, combination therapy of 14 (30 mg/kg
po, b.i.d.) with either 19 (10 mg/kg po, b.i.d.) or 20 (0.3 mg/
kg po, q.d.) showed a synergistic effect leading to pronounced
prolongation of heart allograft survival. For both combination
treatments all grafts remained functional until the predeter-
mined termination point of 50 days. Histological analysis of the
grafts confirmed that acute rejection was only mild to moderate
in the group co-treated with 19, whereas it was absent to mild
in the group co-treated with 20. Cardiac allograft vasculopathy
developed in all grafts co-treated with 19 (mild to severe) but
in none of the grafts co-treated with 20. Multifocal interstitial
fibrosis was also observed in three grafts co-treated with 19 and
in only one graft co-treated with 20. Despite the acute vascular
leakage in the EBD model, the treatment was well tolerated and
a normal increase of body weight was observed. Besides mild
perivascular edema, there were no histopathological findings in
the lung in both co-treatment groups. The number of
peripheral lymphocytes was measured once a week, always
before treatment. Lymphocytes were reduced to 12−29%
versus control in the group co-treated with 19 and to 9−22%
versus control in the co-treatment group with 20 over the
whole treatment period of 50 days. PK analysis confirmed in
vivo hydrolysis of the ester 14 to the active moiety 15 and
Table 4. Effect of Treatment of Oral 14 Alone and When Combined with Oral 19 or 20 on Survival of Rat Heart Allografts
a
treatment dose (mg/kg po, b.i.d.) dose (mg/kg po, q.d.)
graft survival (days)
median survival time (MST) (days)
acute cellular rejection scores
control
14
0
6, 6, 6, 6
6
3R%, 3R%, 3R%, 3R%
30
10
8, 8, 10, 8
8
3R, 3R, 3R, 3R
19
7, 7, 7, 7
7
3R, 3R, 3R, 3R
20
0.3
0.3
7, 7, 7, 7, 7
7
3R, 3R, 3R, 3R, 3R
2R1#−3#$, 1R1#,$, 2R1#,$, 1R1#−3#
0, 0, 1R, 1R$
14 + 19
14 + 20
30 + 10
30
>50, >50, >50, >50
>50, >50, >50, >50
>50
>50
a
Acute cellular rejection score: 0 = no rejection, 1R = mild rejection, 2R = moderate rejection, 3R = severe rejection, 1# = mild cardiac allograft
vasculopathy, 2# = moderate cardiac allograft vasculopathy, 3# = severe cardiac allograft vasculopathy, $ = multifocal interstitial fibrosis, % = diffuse
necrosis.
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a
Scheme 1. Synthesis of Intermediate 21
a
Reagents: (a) 3-aminophenylboronic acid, aqueous NaHCO3, Pd(PPh3)4, DME; (b) 4-chloro-2,5-dimethylphenylsulfonyl chloride, pyridine,
dichloromethane.
a
Scheme 2. Synthesis of Prodrugs 6−9 and Carboxylic Acid Derivatives 5 and 28
a
Reagents: (a) (i) SOCl2, dichloromethane, (ii) DIPEA, THF; (b) aqueous LiOH, THF; (c) HOCH2CH2NMe2, Boc2O, DMAP, THF; (d)
Me2N·HCl, DIPEA, (Me3PCH2CN)I, propionitrile.
a
Scheme 3. Synthesis of Prodrugs 10 and 13, Lactam 12, and
Carboxylic Acid Derivatives 11 and 32
Scheme 4. Synthesis of Building Blocks 34 and 38
a
a
Reagents: (a) (i) benzaldehyde, DIPEA, AcOH, NaCNBH3,
methanol, (ii) aqueous formaldehyde; (b) H2, Pd(OH)2, methanol.
a
Reagents: (a) (i) SOCl2, dichloromethane, (ii) DIPEA, THF; (b)
HCl, dioxane; (c) aqueous LiOH, THF; (d) (i) TFA, dichloro-
methane, (ii) toluene; (e) aqueous formaldehyde, NaCNBH3,
methanol.
ide (37) (Scheme 4 and Scheme 5). Basic hydrolysis of 16
yielded the carboxylic acid 17. Esterification of 15 with ethanol
provided ethyl ester 18.
situ second reductive amination with formaldehyde afforded
intermediate 33 (Scheme 4). Debenzylation gave amine 34,
which was used as crude in the amide coupling with
intermediate 21 to provide 35 (Scheme 5). Deprotection of
the Boc group generated the amine 36, which was converted to
14 by reductive amination. Hydrolysis of 14 generated
carboxylic acid 15. The enantiomer of 14, 16 was obtained
following the same sequence starting with (R)-2-amino-4-((tert-
butoxycarbonyl)amino)butanoic acid methyl ester hydrochlor-
CONCLUSIONS
■
A prodrug approach was successfully applied to modulate the
duration of the pharmacodynamic effect of the selected scaffold
for chronic efficacy studies in a rat heart transplantation model.
A broad scaffold exploration by amide methylation, masking the
carboxylic acid as an ester and introducing basic moieties, led to
the identification of 14.
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a
Scheme 5. Synthesis of Prodrugs 14, 16, and 18 and Carboxylic Acid Derivatives 15 and 17
a
Reagents: (a) (i) SOCl2, dichloromethane, (ii) DIPEA, THF; (b) HCl, dichloromethane; (c) aqueous formaldehyde, AcOH, NaBH(OAc)3,
acetonitrile; (d) aqueous LiOH, THF; (e) EtOH, Et3N, DMAP, propylphosphonic anhydride, THF.
after drug application or 14, 18, 24, and 48 h after drug application).
Whole blood was sampled in EDTA-coated Eppendorf tubes and
subjected to hematology analysis. Lymphocyte counts were measured
by an automated hematology analyzer (ADVIA 120, Bayer
Diagnostics, Zurich, Switzerland). Individual changes in peripheral
lymphocyte counts after vehicle or compound application were
compared with the respective baseline value determined before start of
treatment. Changes were then calculated as percent change compared
to baseline values.
EBD Lung Leakage Model. Male Lewis rats (Harlan CPB) were
treated orally (gavage), twice per day, with either vehicle (control) or
14 at 30 mg/kg (freshly prepared in a 30% PEG200/buffer solution).
Leakage of plasma proteins in the lung was assessed using the standard
Evans blue dye (EBD) leakage technique.30 Briefly, EBD (Fluka) was
administered intravenously (20 mg/2 mL NaCl/kg) under anesthesia
at 5, 23, or 95 h after treatment. In each case, 1 h after EBD injection,
the rats were bled under deep anesthesia by incision of the vena cava
and the pulmonary vessels perfused via the right-ventricle with 10 mL
of saline to remove blood and EBD from the vascular spaces. The
lungs were collected en bloc and dried at 60 °C for 24 h. Dried lungs
were weighed and immersed into 2 mL of formamide (Sigma) for 24 h
at 37 °C in order to extract EBD from tissues. Each extract was then
assessed by spectrophotometry at 620 and 740 nm to correct for
contaminating heme pigments with the following formula: OD620
(EBD) = OD620 − [(1.426)(OD740) + 0.030]. The EBD
concentrations in lung extracts were finally estimated with the help
of parallel EBD-standard curve.
Heterotopic Vascular Heart Transplantation DA-to-Lewis
Rat Model. The heterotopic cardiac transplantation was performed as
described previously.36 The cooled and heparinized donor heart was
removed following ligation of all vessels except the ascending aorta and
the right pulmonary artery. These vessels were then anastomosed end-
to-side to the recipient’s abdominal aorta and inferior vena cava. After
release of the clamps, the heart started to beat within less than 2 min.
Graft function was assessed daily by palpation for ventricular
contraction. Hearts in which ventricular motion had ceased were
considered rejected, which was confirmed subsequently by histology.
Body weight was monitored regularly.
Recipient rats received the following treatments: vehicle (po, b.i.d.),
14 (30 mg/kg po, b.i.d.), 19 (10 mg/kg po, b.i.d.), or 20 (0.3 mg/kg
po, q.d.), all as monotherapy, or 14 (30 mg/kg po, b.i.d.) in
combination with 19 (10 mg/kg po, b.i.d.) or 14 (30 mg/kg po, b.i.d.)
in combination with 20 (0.3 mg/kg po, q.d.). Compound 14 was
dissolved in PEG200, distilled water and acidified to pH 5 by addition
of aqueous HCl (0.1 M). Compound 19 was mixed in D-glucose and
dissolved in PEG400, distilled water, and aqueous HCl (0.1 M).
Upon oral administration 14 was hydrolyzed in vivo to the
corresponding carboxylic acid 15, a highly potent and selective
S1P1 antagonist, which induced a long lasting, reversible
reduction of circulating lymphocytes in the rat (full PBL
reduction over 24 h after a single dose of 30 mg/kg po). In
addition, oral administration of 14 in combination with a
subtherapeutic dose of 19 or 20 in a stringent rat heart
transplantation model effectively prolonged survival of cardiac
allografts with excellent histology. These results suggest a
synergy between complementary immunosuppressive modes of
action. It is of note that 14 was very well tolerated during the
entire observation period of up to 50 days after transplant.
These data demonstrate that graft protection can be achieved
by targeting the S1P1 receptor with a selective antagonist,
generated in vivo by hydrolysis of a prodrug, leading to similar
efficacy as with S1P1 agonists.
EXPERIMENTAL SECTION
■
GTPγ35S. Membranes were prepared from CHO cell clones stably
expressing a human S1P1. The desired amount of membranes (1−5
μg/well) was diluted with assay buffer (50 mM HEPES, pH 7.4, 5 mM
MgCl2, 1 mM CaCl2, 1% fatty acid-free BSA) containing 10 μM GDP,
25 μg/mL saponin, and 5 mg/mL WGA-SPA beads (Perkin-Elmer).
Serial dilutions of compounds were incubated with 200 μL of
membrane-WGA-SPA bead slurry for 15 min followed by the addition
of the agonist S1P (4 nM final concentration). Then GTPγ35S (0.2 nM
final concentration) in assay buffer was added. After incubation at
room temperature for 120 min under constant shaking the plates were
centrifuged for 10 min at 1000g to pellet the membrane−SPA beads
slurry. Then the plates were measured in a TOPcount NXT
instrument (Perkin-Elmer). Eight different concentrations of com-
pound were used to generate a concentration response curve (using
two data points per concentration) and the corresponding IC50 using
the curve-fitting tool of GraphPad Prism.
S1P2, S1P3, S1P4, and S1P5 dependent GTPγ35S assays were carried
out in a manner comparable to that of the S1P1 GTPγ35S assay using
membranes from CHO cells stably expressing the S1P receptors.
Peripheral Blood Lymphocyte Reduction Assay. Male Lewis
rats (Harlan CPB) were subjected to a single dose of either vehicle
(control) or compound (all prepared fresh in a 30% PEG200/buffer
solution) via oral administration. Longitudinal blood sampling (<200
μL) was performed under anesthesia (isoflurane 5% v/v; Forene,
Abbott, Baar, Switzerland) by sublingual punctures at given time
points (before compound administration (baseline) and 4, 8, 12, 24 h
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Microemulsion preconcentrate of 20 was diluted with distilled water.
All solutions were freshly prepared daily. All treatments were
performed by oral gavage using rat-feeding needles. Treatments with
14 and 20 were performed using a volume of 2 mL/kg, and treatments
with 19 were performed using a volume of 3 mL/kg. In combination
treatments the compounds were given one after the other, with 14
always applied first. Treatments started 3 days before transplantation.
The experiments were terminated at rejection or at predefined
termination points.
For histological examination, cardiac allografts were fixed in 10%
buffered formalin, processed according to standard procedures, and
embedded in paraffin. Tissue blocks were cut to 3 μm sections that
underwent staining with hematoxylin and eosin (H&E) and trichrome.
The degree of acute cardiac rejection was scored according to
Stewart:37 0R, no rejection; 1R, mild; 2R, moderate; 3R, severe. In
addition, the grade of cardiac allograft vasculopathy (CAV) was
scored: 0, no CAV; 1, mild (<25% lumen occlusion); 2, moderate
(26−50% occlusion); 3, severe (>50% occlusion).
NMR Study. The minor conformer A was separated from peak B
by preparative HPLC, injecting a sample of the free base of 14. The
solvent was removed at low temperature, and the isolated sample was
stored on dry ice. The NMR measurements were performed on a
Bruker AV 600 spectrometer at 298 K, using a 1 mm TXI microliter
probe. 1H NMR spectra were measured with 32 scans, 4.233 s
repetition time, 64 k time domains, and 2.733 s acquisition time, using
a 30° pulse angle (zg30). The ROESY spectrum was obtained with
377 t1 values (TD = 2 k, 16 scans), using a 200 ms 180° spinlock.
The HSQC spectrum was run with 256 t1 values (TD = 2 k, 8 scans)
using adiabatic pulses for 13C decoupling. The integration of the two
conformers was done on H-32 (Figure 7), at 5.0−5.3 ppm for the
major conformer B and at 3.9−4.2 ppm for the minor conformer A,
after a manual baseline correction was applied between 3.8 and 5.4
ppm on each spectrum.
The NMR solution was prepared by dissolving 1 mg of compound
(free base) in 18 μL of DMSO-d6. An amount of ∼9 μL was
transferred into a capillary NMR tube. Nine measurements at different
time points were acquired, where the decay of conformer A was
observed. By use of the remaining solution, 1H NMR, 2D-HSQC, and
2D-ROESY experiments were performed. 13C shifts of the N-methyl
group H-31 and the α-CH group H-32 of both conformers were taken
from the HSQC spectrum for comparison. The protons of both
conformers were assigned on the basis of the ROESY and the HSQC
spectra.
accuracy below 1 ppm was obtained by using a lock mass. All final
compounds were purified to ≥95% purity as assayed by analytical
UPLC−MS (Waters Acquity UPLC instrument equipped with an
PDA detector and a SQ mass spectrometer using a Waters Acquity
UPLC BEH C18, 2.1 mm × 30 mm, 1.7 μm column) at a 1.0 mL/min
flow rate with a gradient of 5−98% acetronitrile (containing 0.04%
formic acid) in 0.05% aqueous formic acid (containing 3.75 mM
ammonium acetate) for 9.4 min and a total run time of 10 min.
3′-(4-Chloro-2,5-dimethylphenylsulfonylamino)-3,5-dime-
thylbiphenyl-4-carboxylic Acid (21). To a mixture of 4-bromo-2,6-
dimethylbenzoic acid (22) (1.66 g, 7.23 mmol) and tetrakis-
(triphenylphosphine)palladium (25 mg, 0.022 mmol) in dimethoxy-
ethane (200 mL) and aqueous sodium bicarbonate (10 wt %, 45 mL,
50.6 mmol) was added 3-aminophenylboronic acid (1.09 g, 7.95
mmol). The mixture was stirred at 100 °C for 1 h. When the mixture
cooled, a brownish oily layer was formed which was carefully decanted.
The solvents were evaporated. The residue was taken up in water and
washed with diethyl ether. The pH of the aqueous layer was adjusted
to about 4 with aqueous HCl (2 M), upon which a slightly sticky solid
precipitated. The solid was filtered off, redissolved in ethyl acetate, and
dried over sodium sulfate. Filtration and evaporation afforded 3′-
amino-3,5-dimethylbiphenyl-4-carboxylic acid (23) (1.42 g, 84%) as a
beige powder which was used without further purification for the next
step. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.25 (s, 2H), 7.09 (t, J
= 7.8 Hz, 1H), 6.83 (br t, J = 1.9 Hz, 1H), 6.7 (v br d, J = 7.8 Hz, 1H),
6.57 (br ddd, J = 7.8, 1.9, 1.1 Hz, 1H), 3.35 (v br s, 2H), 2.33 (s, 6H).
UPLC−MS m/z (ES−) (M − H)− = 240.3. HRMS m/z (EI) (M +
H)+ = 242.1173 (calcd for C15H16NO2, 242.1181).
To a solution of 23 (1.34 g, 5.55 mmol) in pyridine (9 mL) was
added dropwise a solution of 4-chloro-2,5-dimethylphenylsulfonyl
chloride (1.33 g, 5.55 mmol) in dichloromethane (18 mL) over a
period of 5 min. The resulting mixture was stirred at room
temperature for 2 h. The mixture was diluted with dichloromethane
and washed with aqueous HCl (2 M, 3×), water, and brine. The
organic layer was dried over sodium sulfate, filtered, and concentrated
1
to afford 21 (2.07 g, 83%) as an off-white solid. H NMR (400 MHz,
DMSO-d6): δ (ppm) 13.22 (br s, 1H), 10.59 (s, 1H), 7.98 (s, 1H),
7.49 (s, 1H), 7.23−7.36 (m, 3H), 7.15 (s, 2H), 7.06 (br dt, J = 7.6, 1.8
Hz, 1H), 2.54 (s, 3H), 2.35 (s, 3H), 2.33 (s, 6H). UPLC−MS m/z
(ES−) (M − H)− = 442.3. HRMS m/z (EI) (M + H)+ = 444.1029
(calcd for C23H23ClNO4S, 444.1036).
(S)-4-tert-Butoxycarbonylamino-2-methylaminobutyric Acid
Methyl Ester (34). To a solution of (S)-2-amino-4-((tert-
butoxycarbonyl)amino)butanoic acid methyl ester hydrochloride
(29) (25.0 g, 93.0 mmol) in methanol (900 mL) was added DIPEA
(17.8 mL, 102.3 mmol). The pH of the solution was adjusted to pH
4−5 by addition of acetic acid. Benzaldehyde (10.7 g, 97.7 mmol) was
added. The solution was stirred at room temperature for 1 h, and the
formation of the imine was monitored by MS. After complete
formation of the imine, sodium cyanoborohydride (6.4 g, 102.3 mmol)
was added. The resulting mixture was stirred at room temperature for
1 h. Formaldehyde (37% in H2O, 24.9 mL, 334.4 mmol) was added,
and the reaction mixture was stirred at room temperature for 45 min.
The mixture was quenched with saturated aqueous sodium
bicarbonate, and the organic solvent was evaporated under reduced
pressure. The residue was extracted with ethyl acetate (3×). The
combined organic layers were washed with brine, dried over
magnesium sulfate, filtered, and concentrated. The crude was purified
by flash chromatography (cyclohexane/ethyl acetate, 9:1) to afford
(S)-2-(benzylmethylamino)-4-tert-butoxycarbonylaminobutyric acid
Chemistry. All reagents and solvents were purchased from
commercial suppliers and used without further purification or were
prepared according to published procedures. All reactions were
1
performed under inert conditions (argon) unless otherwise stated. H
NMR spectra were recorded on a Bruker 360 MHz or a Bruker 400
MHz NMR spectrometer. Chemical shifts are reported in parts per
million (ppm) relative to an internal solvent reference. Significant
peaks are tabulated in the order multiplicity (s, singlet; d, doublet; t,
triplet; q, quartet; quintet; m, multiplet; br, broad; v, very), coupling
constants, and number of protons. Analytical UPLC−MS was
performed on a Waters Acquity UPLC instrument equipped with a
PDA detector and a SQ mass spectrometer using a Waters Acquity
UPLC BEH C18 2.1 mm × 30 mm, 1.7 μm column. Electrospray
ionization (ESI) mass spectra were recorded on an Agilent 1100 series
mass spectrometer. Mass spectrometry results are reported as the ratio
of mass over charge. For all compounds containing amide conformers
the percentage of each conformer was determined by analytical UPLC
(Waters Acquity UPLC instrument equipped with a PDA detector and
a Waters Acquity UPLC BEH C18, 2.1 mm × 50 mm, 1.7 μm
column). Peak detection is reported as the maximum plot from 190 to
350 nm wavelength. The NMR data of both conformers are reported
when the percentage of the minor is >5%; otherwise only the data of
the major are given. The accurate mass analyses were performed by
using electrospray ionization in positive mode after separation by LC.
The elemental composition was derived from the averaged mass
spectra acquired at a high resolution of about 30 000 on an LTQ
Orbitrap XL mass spectrometer (Thermo Scientific). The high mass
1
methyl ester (33) (27.8 g, 82.6 mmol, 89%) as a colorless liquid. H
NMR (360 MHz, CDCl3): δ 7.29−7.13 (m, 5H), 4.81 (br s, 1H), 3.70
(d, J = 13.0 Hz, 1H), 3.66 (s, 3H), 3.57 (d, J = 13 Hz, 1H), 3.29 (dd, J
= 8.5, 6.7 Hz, 1H), 3.18−3.11 (m, 2H), 2.21 (s, 3H), 1.85−1.78 (m,
2H), 1.36 (s, 9 H). UPLC−MS m/z (ES+) (M + H)+ = 337.3. HRMS
m/z (EI) (M + H)+ = 337.2123 (calcd for C18H29N2O4, 337.2127).
To a solution of 33 (9.00 g, 26.75 mmol) in methanol (270 mL)
was added palladium hydroxide on carbon (20 wt % Pd, 0.50 g). The
flask was evacuated and purged with hydrogen three times. The
mixture was stirred under a hydrogen atmosphere (1 bar) overnight.
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The flask was purged with argon. The mixture was diluted with
dichloromethane and filtered over Celite. The filtrate was concentrated
to afford 34 (6.60 g, 25.58 mmol, 96%) as a light yellow oil, which was
used without further purification. 1H NMR (360 MHz, CDCl3): δ 5.25
(br s, 1H), 3.75 (s, 3H), 3.39−3.28 (m, 1H), 3.25−3.14 (m, 2H), 2.38
(s, 3H), 1.97−1.81 (m, 1H), 1.81−1.64 (m, 1H), 1.46 (s, 9 H). MS m/
z (ES+) (M + H)+ = 247.1.
3.31−3.19 (m, 1H), 3.16 (s, 0.21H), 3.12−2.97 (m, 1H), 2.84−2.78
(m, 5.79H), 2.75 (s, 3H), 2.56 (s, 3H), 2.53−2.43 (m, 1H), 2.37 (s,
3H), 2.34−2.23 (m, 1H), 2.28 (s, 5.79H), 2.10 (s, 0.21H). UPLC−MS
m/z (ES+) (M + H)+ = 600.3. HRMS m/z (EI) (M + H)+ = 600.2291
(calcd for C31H39ClN3O5S, 600.2299).
(S)-2-{[3′-(4-Chloro-2,5-dimethylphenylsulfonylamino)-3,5-
dimethylbiphenyl-4-carbonyl]methylamino}-4-dimethylami-
nobutyric Acid Hydrochloride (15). To a solution of 14 (free base,
1.50 g, 2.50 mmol) in THF (70 mL) was added aqueous lithium
hydroxide (1 M, 12.5 mL, 12.50 mmol). The reaction mixture was
stirred at room temperature for 3 h. The mixture was acidified with
aqueous HCl (1 M) to pH 3 and concentrated. The residue was
purified by preparative HPLC (water/acetonitrile gradient) to give a
white solid, which was taken up in dichloromethane and treated with
an excess HCl (2 M in diethyl ether). The mixture was concentrated
to afford 15 (0.94 g, 1.50 mmol, 60%) as a white solid. UPLC: rotamer
(S)-2-{[3′-(4-Chloro-2,5-dimethylphenylsulfonylamino)-3,5-
dimethylbiphenyl-4-carbonyl]methylamino}-4-dimethylami-
nobutyric Acid Methyl Ester Hydrochloride (14). To a
suspension of 21 (7.50 g, 16.92 mmol) in dichloromethane (250
mL) was added a catalytic amount of DMF followed by the dropwise
addition of thionyl chloride (4.10 g, 2.50 mL, 34.46 mmol). The
reation mixture was refluxed for 30 min, and a yellow solution was
obtained. The mixture was concentrated under reduced pressure, and
the residue was dried in vacuo for 15 min. To a solution of the residue
in THF (250 mL) was added 34 (5.00 g, 20.30 mmol) followed by
DIPEA (10.90 g, 14.7 mL, 84.40 mmol). The reaction mixture was
stirred at room temperature overnight. The mixture was concentrated.
The residue was taken up in EtOAc and washed with aqueous HCl (1
M, 2×), water (2×), saturated aqueous sodium bicarbonate (2×), and
brine (2×). The organic layer was dried over magnesium sulfate,
filtered, and concentrated. The residue was purified by flash
chromatography (cyclohexane/ethyl acetate, 2:1) to afford (S)-2-
{[3′-(4-chloro-2,5-dimethylphenylsulfonylamino)-3,5-dimethylbiphen-
yl-4-carbonyl]methylamino}-4-tert-butoxycarbonylaminobutyric acid
methyl ester (35) (9.18 g, 13.62 mmol, 80%) as an off-white solid.
UPLC: rotamer A 6%, rotamer B 94%. 1H NMR (360 MHz, CDCl3):
δ (ppm) 7.91 (s, 1H), 7.70 (s, 1H), 7.31−7.24 (m, 3H), 7.14 (s, 1H),
7.12−7.06 (m, 2H), 7.03 (dt, J = 6.5, 2.3 Hz, 1H), 5.55 (dd, J = 10.9,
4.4 Hz, 0.94H), 5.25 (br s, 1H), 4.59−4.54 (m, 0.06H), 3.82 (s,
2.82H), 3.76 (s, 0.18H), 3.66−3.43 (m, 1H), 3.17 (s, 0.18H), 3.11−
2.98 (m, 1H), 2.79 (s, 2.82H), 2.62 (s, 3H), 2.37 (s, 6H), 2.36−2.29
(m, 3.94H), 2.25 (s, 0.06H), 2.07−2.01 (m, 1H), 1.47 (s, 9H).
UPLC−MS m/z (ES+) (M + H)+ = 672.5. HRMS m/z (EI) (M + H)+
= 672.2502 (calcd for C34H43ClN3O7S, 672.2510).
1
A 4%, rotamer B 96%. H NMR (360 MHz, DMSO-d6): δ (ppm)
13.23 (br s, 1H), 10.63 (s, 1H), 10.60 (br s, 1H), 8.00 (s, 1H), 7.51 (s,
1H), 7.37−7.24 (m, 3H), 7.22−7.15 (m, 2H), 7.08 (dt, J = 7.1, 2.0 Hz,
1 H), 5.12 (dd, J = 9.9, 5.3 Hz, 1H), 3.30−3.19 (m, 1H), 3.09−2.97
(m, 1H), 2.81 (br s, 6H), 2.73 (s, 3H), 2.55 (s, 3H), 2.50−2.41 (m,
1H), 2.37 (s, 3H), 2.30−2.24 (m, 1H), 2.28 (s, 6H). UPLC−MS m/z
(ES+) (M + H)+ = 586.3. HRMS m/z (EI) (M + H)+ = 586.2133
(calcd for C30H37ClN3O5S, 586.2142).
ASSOCIATED CONTENT
■
S
* Supporting Information
Determination of KD for 15 by Schild plot analysis and
synthesis procedures and spectral data for compounds 5−13,
16−18, 27, 28, and 30−32. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +41 61 3246858. E-mail: daniela.angst@novartis.com.
To a solution of 35 (6.00 g, 8.93 mmol) in dichloromethane (65
mL) was added HCl (2 M in diethyl ether, 44.6 mL, 89.20 mmol)
dropwise. The reaction mixture was stirred at room temperature
overnight, and a white precipitate was formed. The mixture was
concentrated and the residue was dried in vacuo to afford (S)-2-{[3′-
(4-chloro-2,5-dimethylphenylsulfonylamino)-3,5-dimethylbiphenyl-4-
carbonyl]methylamino}-4-aminobutyric acid methyl ester hydro-
chloride (36) (5.40 g, 8.93 mmol, quantitative) as an off-white solid.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Eric Francotte for the isolation of the enriched minor
amide conformer and Christian Guenat for the HRMS analyses.
1
UPLC: rotamer A 5%, rotamer B 95%. H NMR (360 MHz, DMSO-
d6): δ (ppm) 10.66 (s, 1H), 8.23 (br s, 3H), 8.00 (s, 1H), 7.50 (s, 1H),
7.36−7.28 (m, 3H), 7.19 (s, 2H), 7.10 (dt, J = 7.2, 1.9 Hz, 1H), 5.10
(dd, J = 9.7, 5.2 Hz, 1H), 3.73 (s, 3H), 3.07−2.91 (m, 1H), 2.91−2.78
(m, 1H), 2.75 (s, 3H), 2.55 (s, 3H), 2.47−2.38 (m, 1H), 2.36 (s, 3H),
2.27 (s, 3H), 2.26 (s, 3H), 2.25−2.16 (m, 1H). UPLC−MS m/z (ES+)
(M + H)+ = 572.3. HRMS m/z (EI) (M + H)+ = 572.1979 (calcd for
C29H35ClN3O5S, 572.1986).
ABBREVIATIONS USED
■
CAV, cardiac allograft vasculopathy; DA, Dark Agouti; EBD,
Evans blue dye; GTPγ35S, [35S]guanosine 5′-O-(3-thiotriphos-
phate); MST, median survival time; PBL, peripheral blood
lymphocyte; S1P, sphingosine 1-phosphate
To a suspension of 36 (4.40 g, 7.23 mmol) in acetonitrile (79 mL)
was added acetic acid (0.41 mL, 0.43 g, 7.23 mmol) followed by
formaldehyde (37% in water, 5.40 mL, 72.52 mmol). The mixture was
sonicated until a yellow solution was obtained. Sodium triacetoxybor-
ohydride (4.90 g, 23.14 mmol) was added. The reaction mixture was
stirred at room temperature for 40 min. The mixture was diluted with
dichloromethane (360 mL) and washed with saturated aqueous
sodium bicarbonate (2×) and water (2×). The organic layer was dried
over magnesium sulfate, filtered, and concentrated. The residue was
purified by flash chromatography (dichloromethane/methanol, 16:1)
to afford a white solid, which was dissolved in dichloromethane and
treated with an excess HCl (2 M in diethylether). The mixture was
concentrated and the residue was dried in vacuo to provide 14 (2.40 g,
3.77 mmol, 52%) as a white solid. UPLC: rotamer A 7%, rotamer B
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