Synthesis and biological evaluation of γ-aminophosphonates as potent, subtype-selective sphingosine 1-phosphate receptor agonists and antagonists

Article abstract of DOI:10.1016/j.bmc.2006.10.060

The synthesis of N-arylamide phosphonates and related arylether and arylamine analogues provided potent, subtype-selective agonists and antagonists of the five known sphingosine 1-phosphate (S1P) receptors (S1P_1-5). To this end, the syntheses of phosphoserine mimetics-selectively protected and optically active phosphonoserines-are described. In vitro binding assays showed that the implementation of phosphonates as phosphate mimetics provided compounds with similar receptor binding affinities as compared to their phosphate precursors. meta-substituted arylamide phosphonates were discovered to be antagonists of the S1P₁ and S1P₃ receptors. When administered to mice, an antagonist blocked the lymphopenia evoked by a S1P receptor agonist and caused capillary leakage in both lung and kidney.

Full text of DOI:10.1016/j.bmc.2006.10.060

Bioorganic & Medicinal Chemistry 15 (2007) 663–677
Synthesis and biological evaluation of c-aminophosphonates
as potent, subtype-selective sphingosine 1-phosphate receptor
agonists and antagonistsq
Frank W. Foss, Jr.,a,ꢀ Ashley H. Snyder,b Michael D. Davis,b Michael Rouse,c
Mark D. Okusa,c Kevin R. Lynchb,d and Timothy L. Macdonalda,*
aDepartment of Chemistry, University of Virginia, McCormick Road, PO Box 400319, Charlottesville, VA 22904, USA
bDepartment of Biochemistry and Molecular Genetics, University of Virginia, McCormick Road, PO Box 400319,
Charlottesville, VA 22904, USA
cDepartment of Medicine, University of Virginia, McCormick Road, PO Box 400319, Charlottesville, VA 22904, USA
dDepartment of Pharmacology, University of Virginia, McCormick Road, PO Box 400319, Charlottesville, VA 22904, USA
Received 28 September 2006; revised 25 October 2006; accepted 28 October 2006
Available online 1 November 2006
Abstract—The synthesis of N-arylamide phosphonates and related arylether and arylamine analogues provided potent, subtype-
selective agonists and antagonists of the five known sphingosine 1-phosphate (S1P) receptors (S1P1–5). To this end, the syntheses
of phosphoserine mimetics—selectively protected and optically active phosphonoserines—are described. In vitro binding assays
showed that the implementation of phosphonates as phosphate mimetics provided compounds with similar receptor binding affin-
ities as compared to their phosphate precursors. meta-substituted arylamide phosphonates were discovered to be antagonists of the
S1P1 and S1P3 receptors. When administered to mice, an antagonist blocked the lymphopenia evoked by a S1P receptor agonist and
caused capillary leakage in both lung and kidney.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
egress of T-lymphocytes from secondary lymphoid tis-
sues, and thus are thought to direct effector T-cells away
from sites of inflammation.4
Sphingosine 1-phosphate (S1P, Fig. 1) receptors
(S1P1–5) are integral membrane G protein-coupled
receptors that were initially referred to as endothelial
differentiation gene (EDG) receptors, EDG-1, -5, -3,
-6, and -8, respectively.1 These receptors provide control
over numerous aspects of cellular physiology when acti-
vated by endogenous S1P.2,3 Particular attention has
been accorded to the role of the S1P1 receptor in modu-
lating the immune system since the discovery of
FTY720, an agonist at the S1P1,3,4,5 receptors (Fig. 1).
S1P1 receptor agonists have been shown to inhibit the
FTY720 was developed from synthetic analogue studies
of myriocin (ISP-1).5 Eventually discovered to be a pro-
drug, FTY720 is activated in vivo when phosphorylated
by sphingosine kinase type 2 to form FTY720-P.6 The
biological activity of FTY720 indicated that S1P recep-
tors are valid targets for the treatment of autoimmune
disorders and allograft rejection.7 It is hoped that S1P
receptor agonists can modulate immune system function
without the toxic liabilities attendant to existing immu-
no-therapeutics such as calcineurin inhibitors and corti-
costeroids.7–9
Keywords: Sphingosine 1-phosphate; VPC23019; VPC44116; FTY720;
Immune-modulation.
q
Initial synthesis and binding affinities of compounds 12a, b, and d
The success of FTY720 in human investigation has
prompted synthetic efforts to provide both pharmaco-
logical tools to study S1P signaling and therapeu-
tics.10–15 We reported previously the synthesis of
aryl-amide containing phosphates (Fig. 1, VPC22173
and VPC23019) and profiled their binding affinities at
were presented: Foss, F. W. Jr.; Clemens, J. J.; Davis, M. D.; Lynch,
K. R.; Macdonald, T. L. Abstracts of Papers, 228th National
Meeting of the American Chemical Society, Philadelphia, PA, 2004.
*
ꢀ
Corresponding author. E-mail: ff2185@columbia.edu
Present address: Columbia University, Department of Chemistry,
Havemeyer MC-3145, 3000 Broadway, New York, NY 10027, USA.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.060

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F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
Figure 1. Structures of endogenous sphingosine 1-phosphate (S1P) and the S1P receptor ligands, FTY720, VPC22173, and VPC23019.
S1P receptors in vitro.14,16 Among these compounds
were the first S1P1 receptor antagonists, for example,
VPC23019.16 In an effort to discover compounds with
increased resistance to phosphatase-catalyzed hydrolysis
(the deactivation pathway of S1P analogues), the syn-
thesis of the corresponding phosphonates is reported
herein. Further, the synthesis of related aryl-amine and
aryl-ether containing phosphonates is discussed.
sponding phosphonate 12a. Phosphonate analogues
12c, 12d (VPC44116), and 12f, of our previously de-
scribed S1P1,3 antagonists,16 proved to retain their activ-
ity as antagonists and provided pharmacological tools
for in vivo studies. While a new class of arylether phos-
phonates 18a, 18b, and 19 were relatively weak partial
agonists or inactive, the aryl-amine 26 retained similar
activity to its amide precursor.
To initiate this work, strategies were pursued for the effi-
cient synthesis of chiral phosphonoserines 4a and 4b,
which are non-natural amino acids used to study protein
phosphorylation.17 Previous syntheses of note include
Barton and Vonder-Embse’s synthesis of the fully
unprotected phosphonoserine from N-Cbz-glutamic
acid in four steps and 58% yield, involving the use of
white phosphorus (P4).18 Perich and Johns published
two syntheses, the most recent in 42% yield and seven
steps, from properly protected glutamic acid and using
a Barton–McCombie deoxygenation.19 Finally, other
methods employed include enzymatic chiral resolutions
of racemic materials,20–22 and the induction of chirality
by chiral auxiliaries.23–25 Methods reported in this paper
lead to chiral phosphonoserines with protecting groups
amenable to our synthetic approach, as well as peptide
synthesis, in good yield from commercially available
L- or D-serine and R- or S-glycidol.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of aryl-amide-phosphonates 12a–f and 13.
The production of phosphonate analogues containing
an amide linker region was envisaged through the con-
densation of chiral phosphonoserines (L-or D-2-(N-
tert-butoxycarbonyl amine)-4-phosphonyl butyric acids)
4a with various substituted anilines. The initial efforts
(Scheme 1) towards this protected unnatural amino
acids began with the synthesis of Garner’s Aldehyde,
1, from commercially available L-serine over five steps.26
A Horner–Wadsworth–Emmons olefination performed
from one of two bisphosphonates installed the a-methy-
lene or a-fluorophosphonates in 2a or 2b, respectively.27
The fluorinated bisphosphonate used to synthesize 2b
was derived, as previously described by Prestwich,28
from the commercially available tetraethyl meth-
ylenebisphosphonate used to arrive at 2a.
Derived from protected R- and S-phosphonoserines 4a
and 4b, N-aryl-amide phosphonates 12a–f provided sim-
ilar binding affinities at S1P receptors as their phosphate
precursors. Synthesis of the a-fluorophosphonate 13
showed similarly potent binding compared to its corre-
The resulting olefins 2a and 2b were reduced by hydro-
genation over Pd/C to 3a and 3b. Selective acetonide
Scheme 1. Synthesis of (3R)-4a,b—Method A. Reagents and conditions: (a) i—SOCl2, MeOH, rt, 16 h; ii—Boc2O, Et3N, CH2Cl2, rt, 12 h; iii—2,2-
dimethoxypropane, p-TsOH, CH2Cl2, 0 ꢁC to rt, 2 h 62% (3 steps); iv—NaBH4, LiCl, 3:2 EtOH/THF, 0 ꢁC to rt, 4 h 89%; v—DMSO, (COCl)2,
CH2Cl2, ꢀ78 ꢁC, then Et3N ꢀ78 ꢁC to rt, 2–4 h, 97%; (b) tetraethyl methylenebisphosphonate, n-BuLi, THF, ꢀ78 ꢁC, rt, overnight, 75% (2a)
or tetraethyl 2-fluoromethynebisphosphonate, n-BuLi, THF, ꢀ78 ꢁC, rt, overnight, 31% (2b); (c) H2, Pd/C, rt, 12 h, EtOH, 99% (3a) and 88% (3b);
(d) Jones reagent, acetone, 0 ꢁC to rt, 12 h then, isopropyl alcohol, Celite, rt, 15 min, 59% (4a) and 48% (4b).

F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
665
Scheme 2. Synthesis of (3R)-[or (3S)-]4—Method B. Reagents and conditions: (a) BnBr, DMF, 60% NaH, 0 ꢁC to rt, 3 h, 78%; (b) CH3PO3Et2,
n-BuLi, BF3ÆOEt2, THF, ꢀ78 ꢁC to rt, 3 h then, NH4Cl, 1 h, 96%; (c) DPPA, DIAD, 3%-polymer-bound PhPPh2, CH2Cl2, 0 ꢁC to rt, 20 h, 96%; (d)
Boc2O, H2 (balloon), 20 w/w% Lindlar’s catalyst, MeOH, rt, 24 h 77%; (e) H2 (balloon), Pd/C, EtOH, rt, 24 h, 94%; (f) TEMPO,
bis(acetoxy)iodosobenzene, NaHCO3, 1:1 CH3CN/H2O, rt, 3 h, 38% or RuCl3Æhydrate, NaIO4, 3:2:2 H2O/CH3CN/CCl4, rt, 3 h, 76%.
deprotection proved to be low yielding in our initial
efforts;29 however, this led to the use of a convenient,
simultaneous deprotection and oxidation with the Jones
reagent in acetone.30 This un-optimized method led to
the protected amino-acids (R)-4a,b in 40% and 18%
yields, respectively, in three steps from Garner’s
Aldehyde.
plex reaction mixture (polar phosphonate 7 was nearly
inseparable from both triphenyl- and tributylphosphane
oxides). The use of commercially available polymer-
bound triphenylphosphine yielded equivalent conver-
sions to azide 7. With the use of three equivalents of
phosphine, the desired transformation was completed
in fewer than 20 h. The inclusion of a polymer-bound
phosphine reagent allowed for standard purification of
the crude reaction mixture, after filtration of the resul-
tant phosphane oxide.
A second avenue (Scheme 2) led to a shorter and more
efficient synthesis of both (S)- and (R)-4a. For example,
(R)-(+)-glycidol was benzyl protected,31 and the epoxide
was opened by the resultant carbanion of diethyl meth-
ylphosphonate and n-BuLi, in the presence of BF3ÆOEt2,
to yield alcohol 6.32 Installation of an azide at the 3-hy-
droxyl position proved to be the major impediment to
this approach, as has been noted by others for similar
substrates.33 Mesylate formation followed by azide sub-
stitution suffered from a tendency for elimination over a
range of temperatures. Therefore, a milder method of
azide formation was pursued.
A two-step reduction of 7, first in the presence of H2,
Boc2O, and Lindlar’s catalyst,35 followed by H2, Pd/C,
yielded the protected amino alcohol 9 in 72% over two
steps. Oxidation of the primary alcohol was performed
by means of RuCl3(cat)/NaIO4 conditions36 (76%) to
yield compound (R)-4a in 40% yield over six steps from
optically active glycidol. (R)- and (S)-4a were further de-
rived to the corresponding diastereomers, (R)- and (S)-
4a-Phe-OMe, through a PyBOP mediated condensation
with L-phenylalanine methyl ester. This was done to en-
sure high enantiomeric excess of the desired carboxylic
acids. These condensation reactions were high yielding
and arrived at individual stereoisomers as determined
by NMR.
Diphenylphosphoryl azide (DPPA) under Mitsunobu
conditions was successful in assembling the desired
azides, 7, despite reported difficulties arising from con-
gested secondary alcohols.34 After extensive isolation ef-
forts, near quantitative yields were found via this
procedure. Purification issues led to the investigation
of more convenient Mitsunobu reagents to preclude
the difficult separation of polar products from the com-
Compounds 12a–f and 13 (Scheme 3) were synthesized
by PyBOP initiated condensations37 of (R)- or (S)-4a
or (R)-4b with various aniline compounds 10a–d.
Scheme 3. Synthesis of arylamide phosphonates 12a–f and 13. Reagents and conditions: (a) PyBOP, 10a–d, di-iso-propyl ethylamine (DIEA),
CH2Cl2, 24–70%; (b) TMSBr, CH2Cl2, rt, 4–6 h then, 95:5 MeOH/H2O, rt, 1–4 h, 45–100%.

666
F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
Aqueous soluble carbodiimide (EDC methiodide) was
also investigated as a condensation reagent; however,
this method generally produced lower yields. The alkane
hydrocarbons of unavailable alkylanilines were installed
through Sonogashira couplings with 3-iodo-nitroben-
zene,38 followed by concomitant reduction of the nitro
group and resulting triple bond. Pd couplings were also
successfully performed following the amide formation
with m- or p-iodoaniline to complete the desired phos-
phonates in a linear fashion. The compounds 11a–f were
deprotected with bromotrimethylsilane followed by
hydrolysis of the ensuing phosphonate silyl-oxy-esters.
These conditions conveniently deprotected the N-Boc
group, as well, to yield compounds 12a–f. a-Fluoro-
phosphonate 13 was synthesized by the same protocol
from 4b and 10a.
10% Pd/C (10 w/w%), added after one day of vigorous
stirring, converted the remaining material to the reduced
alcohol 9a in less than 72 h.39 This selective acetonide
deprotection proved to be effective with greater than five
grams of material and yielded more efficient conversions
(>95%) than 1.5 equivalents of p-toluenesulfonic acid in
ethanol (65–75%). The alcohol was then converted to 4a
with RuCl3/NaIO4 conditions, as previously described.
2.1.2. Synthesis of aryl ether phosphonates 18a, 18b and
19. Using glycidol as an alternative starting material to
serine led conveniently to phenolic ether compounds
(Scheme 5). The synthesis began with a Mitsunobu con-
densation between p-octylphenol and glycidol. Chiral
epoxides 14 were then opened, as previously described,
to alcohols 15. For these substrates, DPPA/Mitsunobu
conditions yielded products with sufficiently disparate
polarities from tributylphosphane oxide. The azides,
16, were reduced and deprotected as described above
to give enantiomers 18a and 18b. Compound 15a was
deprotected to form 19 to confirm the overall effects of
an amine at the 3-position of 18b.
While the synthesis described from glycidol retained the
reported efficiency up to half-gram scale, increasing
material to greater than one gram proved detrimental
to the formation of azide 7. Due to the demand for
greater quantities of compounds 13b and 13d, further
optimization of our synthesis from serine was undertak-
en (Scheme 4). Acetonide protected vinyl phosphonate 2
was converted to phosphonoserine 4a in two convenient
steps. Compound 2, as displayed in Scheme 1, undergoes
reduction within 4–6 h under an atmosphere of H2 and
in the presence of 10% Pd/C (20 w/w%). It was observed
that a trace amount of the alcohol 9a was present at this
time. The reaction was allowed to stir for 1 day at room
temperature, and it was estimated by TLC that nearly
half of the acetonide was hydrolyzed. Following further
investigation, the use of an additional half equivalent of
2.1.3. Synthesis of aryl amine phosphonate 26. Secondary
amine 26 was synthesized to view the effects of reducing
the amide bond in 12d (Scheme 6) while retaining a func-
tional group capable of donating a hydrogen bond.
After activation of p-octylaniline by mono-tosyl protec-
tion, the amine was condensed under Mitsunobu condi-
tions to form epoxide 21. Consecutive nucleophilic ring
opening, azide formation with DPPA/Mitsunobu condi-
tions, and reduction yielded amine 24. Deprotection of
the N-tosyl group proved difficult. Sluggish reaction
Scheme 4. Efficient synthesis of 4a from 2a. Reagents and conditions: (a) H2, 10% Pd/C (20 w/w% followed by 10 w/w%), EtOH, rt, 3d, >95%; (b)
RuCl3ÆH2Ox, NaIO4, 2:2:3 CCl4/CH3CN/H2O, rt, 1–3 h, 79%.
Scheme 5. Synthesis of arylether phosphonate analogues 18a–b and 19. Reagents and conditions: (a) DIAD, PPh3, THF, 0 ꢁC to rt, overnight, 80–
84%; (b) CH3PO3Et2, n-BuLi, BF3ÆOEt2, THF, ꢀ78 ꢁC to rt, 3 h then, NH4Cl, 1 h, 72–87%; (c) DPPA, DIAD, PPh3, CH2Cl2, 0 ꢁC to rt, 20 h, 67–
98%; (d) H2 (balloon), Pd/C, EtOH, formic acid cat, rt, 3.5 h, quantitative; (e) TMSBr, CH2Cl2, rt, 4–6 h then, 95:5 MeOH/H2O, rt, 2–4 h, 79–100%.

F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
667
Scheme 6. Synthesis of arylamine phosphonate analogue 26. Reagents and conditions: (a) DIAD, PPh3, THF, 0 ꢁC to rt, overnight, 69%; (b)
CH3PO3Et2, n-BuLi, BF3ÆOEt2, THF, ꢀ78 ꢁC to rt, 2 h then, NH4Cl, 2 h, 97%; (c) DPPA, DIAD, PPh3, CH2Cl2, 0 ꢁC to rt, 20 h, 85% (containing 5%
OPPh3); (d) H2 (balloon), 20 w/w% Pd(OH)2, 20:1 MeOH/concd HCl, 1 h, 100%; (e)Na(s), NH3(l), ꢀ78 ꢁC, 5 min then EtOH, 25% (recovered 28%
starting material); (f) TMSBr, CH2Cl2, rt, 4–6 h then, 95:5 MeOH/H2O, 4 h, rt, 95%.
times and low yields were discovered with the planned
Mg/MeOH deprotection,41 and dissolving metal condi-
tions were used. Solid sodium in NH3(l) unmasked the
desired secondary amine in an un-optimized 26% yield.
Compound 25 was deprotected with TMSBr to yield
the ammonium bromide salt 26.
ity to antagonize S1P’s endogenous activity in the [c35S]-
GTP assays. The effects on S1P’s endogenous binding
constant were determined as previously discussed.16
Para-substituted phosphonates 12a, 12b (VPC44152),
12e, 18a, 18b and 19 showed various activities as ago-
nists. Phosphonate 12b (VPC44152) was twice as potent
as corresponding phosphate VPC22173 at S1P1 and
S1P3, while less potent at S1P4 and S1P5. Phosphonate
12a gained activity across all receptors with comparison
to VPC22173 and displayed similar potency to FTY720-
P and S1P at S1P1. The replacement of the amide linkage
with an ether resulted in the loss of activity, at S1P1 and
S1P3, for 18a compared to 12b, implicating the impor-
tance of available hydrogen-bond donation alpha to
the phenyl ring. Interestingly, epimer 18b was consider-
ably less potent than 18a at S1P1 but displayed modest
activity at all five S1P receptors. c-Hydroxyphosphonate
19 was less potent than 18a at S1P1 and functionally
inactive at S1P2–5, which is consistent with the two-point
2.2. Biological evaluation
2.2.1. [c35S]-GTP binding assay. [c35S]-GTP-dependent
receptor binding activity (Table 1) was determined in vi-
tro for S1P, FTY720-P, and all final compounds, as
previously communicated.16 Briefly, the expression of
individual human S1P receptors and individual G pro-
tein subunits was forced in HEK293T cells. The mem-
brane-bound G protein a subunits yielded data by
binding the labeled, non-hydrolyzable [c35S]-GTP when
activated by an extracellular ligand. Following our
discovery of the S1P1,3 antagonist VPC23019 (B), the
meta-substituted analogues were analyzed for their abil-
Table 1. [c-35S]-GTP binding assay in HEK293T cells over-expressed with subtype specific S1P receptorsa
Compound Linker
Head Group
Receptors
S1P3
S1P1
S1P2
Emax EC50 ECmax EC50
S1P4
S1P5
ECmax EC50 ECmax
a
EC50
ECmax EC50
FTY720-P
VPC22173
VPC23019
H2CCH2 Phosphate
1.3
1.00
1.10
0.00
0.96
0.93
0.00
0.00
NA
NA
NA
0.00
0.00
0.00
0.1
450.0
NA
0.50
0.35
0.00
0.88
0.38
0.00
0.00
4.0
0.90
1.10
1.13
0.67
0.80
n/a
4.0
52.0
0.56
0.79
Amide
Amide
Amide
Amide
Amide
Amide
Amide
Amide
Amide
Ether
Phosphate
Phosphate
58.0
NA
3.6
500.0
120.0
230.0
2300.0
n/a
480.0 0.57
12a
b
Phosphonate
Phosphonate
Phosphonate
Phosphonate
Phosphonate
Phosphonate
270.0 0.50
43.0
270.0
NA
30.0
76.0
n/a
0.77
0.64
n/a
27.0
NA
NA
NA
n/a
0.00
n/a
c
d
NA
NA
NA
0.00
0.00
0.00
NA
6100.0
n/a
n/a
1.42
n/a
33.0
n/a
0.73
n/a
e
1200.00 NA
15,000 0.30
f
NA
0.00
0.99
0.70
0.64
0.43
0.56
NA
23.0
NA
100.0
NA
NA
0.00
0.96
0.00
0.53
0.00
0.00
n/a
n/a
n/a
13
18a
b
Fluorophosphonate 2.1
490.0 0.47
NA 0.00
530.0 0.50
170.0
147.0
150.0
0.68
0.71
0.90
19.0
31.0
47.0
NA
41.0
0.77
0.41
0.50
0.00
0.61
Phosphonate
Phosphonate
Phosphonate
Phosphonate
68.0
Ether
Ether
Amine
140.0
33.0
30.0
19
26
NA
NA
0.00
0.00
440000.0 0.45
340.0 1.13
Emax values are normalized to the maximal activation of endogenous S1P, at each receptor.
NA, no activation.
n/a, not available.
a EC50s are nM and determined by the mean of at least three experiments.

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F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
binding model for S1P receptor interaction.42 Compared
with our previously described phosphate agonist
VPC22173, phosphonates 12a, 12b (VPC44152) and
12e retained similar potency and efficacy. meta-substitut-
ed compounds 12c, 12d (VPC44116) and 12f showed no
agonist activity at S1P1 and S1P3 receptors; rather, meta-
substituted compounds displayed antagonist activity
against S1P binding to the S1P1 and S1P3 receptors.
To characterize these compounds, Schild regressions
were performed as described in earlier work.16 These
experiments revealed arylamides 12d and 12f as potent
antagonists at the S1P1 and S1P3 receptors. Arylamine
26 displayed antagonist activity at both receptors with
a preference for S1P3. The most promising antagonist,
12d (VPC44116), was compared with its phosphate pre-
cursor VPC23019. VPC23019 and VPC44116 were near-
ly indistinguishable in their affinity for the S1P1 and
S1P3 receptors (Ki values of about 30 and 300 nM,
respectively). This was described in more detail by
radioligand displacement experiments, described
previously,16 revealing IC50s for the phosphate and
phosphonate to be 31 and 72 nM, respectively (not
shown).
Figure 3. Effect of VPC44116 on vascular permeability. C57BL/6 mice
were treated with vehicle (2% hydroxypropyl b-cyclodextrin) or
VPC44116 (25 mg/kg) two hours prior to injection 2% Evans blue
dye (EBD) (20 mg/kg) into the jugular vein 30 min before harvesting
tissues. EBD was extracted into formamide, measured in a spectrom-
eter, and the amount of extravasated EBD in tissues was calculated
from a standard curve. Values are means SE; n = 4 for each group.
**P < 0.05, **P < 0.01 compared with vehicle treatment.
We have demonstrated previously that VPC44116
opposes the protective effect of FTY720 in a mouse
model of acute renal injury.43 To characterize this com-
pound further, we injected mice with doses up to 45 mg/
kg body weight and measured blood lymphocytes. Lym-
phopenia (a decrease in circulating lymphocytes below
the normal range) is a convenient index of S1P1 receptor
agonist action. The quintessential S1P agonist, FTY720,
has been proposed to operate as a functional antagonist
through receptor desensitization mechanisms44—a
hypothesis that suggests a direct receptor antagonist
would behave likewise. Nevertheless, no significant
change in circulating lymphocyte numbers was observed
at any dose of VPC44116 tested (not shown). However,
12d (VPC44116 (meta)) blocked the lymphopenia
evoked by its isomer, the agonist 12b (VPC44152 (para))
(Fig. 2). A S1P receptor antagonist similar to VPC44116
but containing a hexyl (vs octyl in VPC44116) group
caused vascular leakage in lung when administered to
mice.45 To learn whether VPC44116 behaved similarly,
we injected VPC44116 into mice followed by Evans blue
dye as described previously.43 After sacrifice, we found
extravasation of the dye into lung and kidney, but not
into heart, liver, testes or intestine (Fig. 3).
3. Conclusion
The synthetic methods described provide entry to
multiple oxidation states and/or orthogonal protecting
groups of phosphonoserines, including an isoelectric
a-fluorophosphonate. This is accomplished from two
convenient materials of the current chiral pool, L- or
D-serine (eight steps and 39% yield) and R- or S-glycidol
(six steps and 40% yield). Optically active agonists and
antagonists of the S1P receptors are described. Replac-
ing the phosphorus–oxygen bond with a phosphorus–
carbon bond provides bioactive agents with putative
resistance to degradative phosphatase activity. The
phosphonate-containing antagonist, 12d (VPC44116),
opposed the lymphopenia evoked by its agonist isomer,
12b (VPC44152), but did not affect numbers of circulat-
ing lymphocytes when injected alone. However, injec-
tion of VPC44116 alone caused capillary leakage in
lung and kidney.
4. Experimental
Figure 2. The lymphopenia evoked by the S1P agonist, 12b
(VPC44152), is blocked by co-administration of the S1P receptor
antagonist 12d (VPC44116). Groups of 3 C57BL/6 · sv129/J mice
injected with vehicle (2% hydroxypropyl b-cyclodextrin), VPC44116
(22 mg/kg) and/or VPC44152 (18 mg/kg). After 16 h, blood was drawn
from the orbital sinuses and lymphocytes were measures with a
Hemavet blood analyzer. Data are presented as means SE.
4.1. General experimental
All reactions were performed under an inert atmosphere
using flame-dried glassware. Reaction solvents methy-
lene chloride, diethyl ether, tetrahydrofuran and toluene

F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
669
were obtained from OptiDry canisters (<50 ppm H2O,
Fisher Scientific) and passed through an activated alu-
mina (activity I) column directly into the reaction flask
when possible. Dimethylformamide was obtained from
an OptiDry canister without further drying prior to
use. All other solvents were used as obtained. All com-
mercially available reagents were purchased from either
Aldrich (Milwaukee, WI), Sigma (St. Louis, MO), Acros
(Pittsburg, PA), or Advanced Chem Tech (Louisville,
KY) and used as obtained unless otherwise stated. The
reactions were monitored by analytical thin-layered
chromatography using Merck silica gel F-254 pre-coated
aluminum-backed plates. Rf values refer to column
chromatography eluent, unless otherwise noted. When
not reported, Rf value ꢁ0.00. Silicycle Ultra Pure Silica
Gel (230–400 mesh) or Fisher Scientific Silica Gel 60
Sorbent (230–400 mesh) was used for all normal phase
chromatography. All yields refer to chromatographical-
ly and spectroscopically pure compounds, unless other-
wise indicated.
(3· 25 mL). The organic layer was dried over Na2SO4(s)
,
1
filtered, and concentrated. H NMR (300 MHz, CDCl3,
23ꢁ, d): 5.54 (br d, 1H), 4.35 (m, 1H), 3.89 (ddd,
J = 13.8, 11.2, and 3.7 Hz, 2H), 3.76 (s, 3H), 2.76 (br
s, 1H), 1.43 (s, 9H) ppm.
The crude oil was dissolved in acetone (120 mL) and 2,2-
dimethoxypropane (87 mL, 15 equiv). The solution was
stirred at room temperature and BF3ÆOEt2 (1.2 mL,
9.52 mmol) was added. The reaction mixture turned a
yellow-orange hue and was stirred for 2.5 h. When the
reaction was complete, by TLC analysis, the solution
was treated with 99% Et3N (1.2 mL) and the solvent
was removed. The brown oil was then partitioned be-
tween diethyl ether and saturated NaHCO3(aq). The
aqueous layer was extracted with diethyl ether
(4· 25 mL) and the organic layers were combined, dried
(Na2SO4), and concentrated to a yellow oil (>90% pure
by 1H NMR). Rf
EtOAc/hexanes) = 0.73. 1H NMR
(1:1
(300 MHz, CD3OD, 23 ꢁC, d): Major rotamer = 4.37
(dd, J = 7.0, 3.1 Hz, 1H), 4.13 (dt, J = 9.23, 7.0 Hz,
2H), 3.74 (S, 3H), 1.52 (s, 3H), 1.48 (s, 3H), 1.40 (s,
9H) ppm; Minor rotamer = 4.48 (dd, J = 6.6, 2.6 Hz,
1H), 4.04 (dt, J = 7.3, 2.9 Hz, 2H), 3.74 (s, 3H), 1.66
(s, 3H), 1.62 (s, 3H) 1.48 (s, 9H) ppm. 13C NMR
(300 MHz, CD3OD, 23 ꢁC, d): Major rotamer = 151.38,
80.51, 66.45, 59.46, 52.49, 28.46, 27.60, 25.15,
24.57 ppm; Minor rotamer = 151.38, 80.51, 66.20,
59.38, 52.61, 28.54, 27.60, 26.21, 25.35 ppm.
Optical rotations were measured on a Perkin-Elmer
a sodium lamp at
model 343 polarimeter with
23 2 ꢁC in the stated solvent; [a]D values are given in
10ꢀ1 degcm2 gꢀ1. 1H and 13C NMR spectra were record-
ed on UnityInova 300 (75) and 500 (125) MHz spec-
trometers (Varian). Chemical shifts are reported in d
1
(ppm) units using H (residual) and 13C signals from
CDCl3 as an internal standard (7.26 and 77.23 ppm,
respectively) unless otherwise specified. Elemental anal-
ysis was performed by Atlantic Microlab, Inc. (Nor-
cross, GA) for C, H, and N. Elemental analyses were
run in duplicate after thorough drying, before submis-
sion and when obtained by the vendor. Low-Resolution
Electrospray Ionization (ESI) was performed at the
University of Virginia Mass Spectometry Laboratory.
High-Resolution Mass Spectrometry (HRMS) was
performed at the Mass Spectrometry Laboratory at
University of Illinois Urbana-Champaign (Micromass
Q-T of Ultima).
A mixture of NaBH4 (2.247 g, 59.08 mmol) and LiCl
(2.505 g, 59.08 mmol) was prepared in EtOH (42 mL),
at 0 ꢁC. A solution of the purified acetonide (7.659 g,
29.54 mmol) dissolved in THF (30 mL) was then added
dropwise to the reaction mixture. The mixture was
warmed to room temperature and stirred for four hours.
After 4 h, the precipitate was filtered over Celite and
washed with EtOH. The filtrate was then concentrated
and reconstituted in H2O (50 mL) and EtOAc (50 mL).
The layers were separated and the aqueous layer was
extracted with EtOAc (3· 25 mL). The organic layers
were combined and washed with brine (2· 25 mL), dried
(Na2SO4(s)), concentrated, and the crude oil was purified
by column chromatography (25–50% EtOAc/hexanes)
to give 6.101 g (89%) of the alcohol as a white solid.
4.1.1.
4-Formyl-2,2-dimethyloxazolidine-3-carboxylic
acid tert-butyl ester (1). L-Serine (5.00 g, 0.048 mol)
was dissolved in 100 mL of methanol and cooled to
<0 ꢁC (brine/ice). Thionyl chloride (20.8 mL,
0.286 mol) was added slowly by syringe. The mixture
was stirred overnight and then concentrated and co-
evaporated with ether multiple times to eliminate excess
thionyl chloride and provide the desired methyl ester
that was shown to be >95% pure by 1H NMR. 1H
NMR (300 MHz, CD3OD, 23 ꢁC, d): 4.07 (t,
J = 4.0 Hz, 1H), 3.98 (d, J = 4.0 Hz, 2H), 3.83 (s, 3H)
ppm.
1
Rf (1:1EtOAc/hexanes) = 0.86. H NMR (300 MHz, CDCl3,
35 ꢁC, d) = 4.07 (m, 2H), 3.75 (s, 1H), 3.68 (m, 2H),
1.52 (s, 6H), 1.49 (s, 9H) ppm.
To a stirring solution of 2.0 M oxalyl chloride, (COCl)2,
(11.78 mL, 23.56 mmol) at ꢀ78 ꢁC was added dropwise
a solution of DMSO (2.1 mL, 23.56 mmol) in CH2Cl2
(33 mL). This solution was allowed to stir at ꢀ78 ꢁC
for 15 min before a solution of the primary alcohol in
CH2Cl2 was added dropwise. The solution was stirred
for 35 min before Et3N was added in a dropwise manner
at ꢀ78 ꢁC. Following the addition of base, the reaction
mixture was allowed to warm to 0 ꢁC and the reaction
was quenched with saturated NH4Cl(aq). The mixture
was separated and the organic layer was washed with
sat. NaHCO3(aq) (2· 25 mL) followed by brine (2· 25
mL). The organic layer was dried (MgSO4) and concen-
trated to a clear oil which was used immediately in the
The amino ester was reconstituted in CH2Cl2 (100 mL)
and triethylamine (16.6 mL, 0.119 mol) was added drop-
wise at 0 ꢁC. To this stirring solution was added di-tert-
butyl dicarbonate (11.420 g, 0.052 mol) in one portion.
The reaction mixture was stirred until the starting mate-
rial was consumed, as determined by TLC (1:1 EtOAc/
hexanes). The reaction mixture was concentrated and
dissolved in EtOAc (50 mL) and then washed with satu-
rated NaHCO3(aq) (3· 25 mL) followed by brine

670
F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
following reaction without further purification. Rf (1:3
EtOAc/hexanes) = 0.35. 1H NMR (300 MHz, CDCl3,
23 ꢁC, d) = Major rotamer: 9.35 (d, 1H, J = 2.4 Hz),
4.02 (m, 1H), 3.88 (m, 2H), 1.43 (s, 3H), 1.34 (s, 3H),
1.22 (9H) ppm; and Minor rotamer: 9.38 (m, 1H), 4.13
(m, 1H), 3.88 (m, 2H), 1.39 (s, 3H), 1.33–1.27 (m,
12H) ppm. 13C NMR (300 MHz, CDCl3, 23 ꢁC,
d) = Major rotamer: 198.84, 151.07, 94.72, 80.55,
64.53, 63.58, 27.99, 25.51, 23.54 ppm; and Minor rot-
amer: 199.02, 152.29, 94.05, 80.92, 64.60, 63.16, 28.03,
26.45, 24.47 ppm.
Following three to five repetitions, the reaction mixture
was allowed to stir under an H2 atmosphere for 4 h.
The mixture was filtered over Celite and washed with
EtOH. The filtrate, which required no further purifica-
tion, was concentrated to 844 mg (99%) of clear oil.
Rf(EtOAc) = 0.29. 1H NMR (300 MHz, CDCl3, 23 ꢁC,
d): 4.09 (dq, J = 3.3, 7.0 Hz, 4H), 3.94 (m, 1H), 3.68
(m, 2H), 1.80 (m, 4H), 1.59 (s, 3H) 1.54 (s, 3H),
and 1.32 (t, J = 7.0 Hz, 6H) ppm. 13C NMR
(300 MHz, CDCl3, 23 ꢁC, d): 156.4, 92.5, 89.8, 65.0,
61.9 (d, J = 6.6 Hz), 31.2, 28.6, 23.4 (d, J = 20.1 Hz),
21.6 (d, J = 20.1 Hz), 16.7 (d, J = 6.0 Hz) ppm.
4.1.1.1. 4-[2-(Diethoxyphosphoryl)vinyl]-2,2-dimethyl-
oxazolidine-3-carboxylic acid tert-butyl ester ((3R)-2a).
To tetraethyl methylenebisphosphonate (4.28 mL,
17.278 mmol) in THF (40 mL) at ꢀ78 ꢁC was added
2.5 M n-BuLi in hexanes (6.28 mL, 15.707 mmol). After
15 min of stirring at low temperature, aldehyde 1
(3.601 g, 15.707 mmol) was added in THF (40 mL) and
the reaction was allowed to warm to room temperature
with continued stirring overnight. The reaction mixture
was concentrated to 1–2 mLs and purified by column
chromatography (500 mL SiO2, 5% MeOH in CHCl3)
to yield 5.440 g (95%, two steps) of clear oil. Rf = 0.65.
1H NMR (300 MHz, CDCl3, 23 ꢁC, d) = 6.63 (dt, 1H,
J = 5.9, 17.8 Hz), 5.72 (dt, 1H, J = 15.8, 17.8 Hz), 4.40
(dt, 1H), 4.04 (m, 5H), 3.77 (m, 1H), 1.59 (m, 3H), 1.42
(m, 12H), 1.25 (dt, 6H, J = 1.5, 7.0 Hz) ppm. 13C
NMR (300 MHz, CDCl3, 23 ꢁC, d): 157.4, 150.3, 127.3,
119.7, 117.2, 92.6, 67.5, 62.1, 59.6 (d, J = 22.7 Hz),
4.1.4. 4-[2-(Diethoxyphosphoryl)-2-fluoroethyl]-2,2-dim-
ethyloxazolidine-3-carboxylic acid tert-butyl ester (3b).
General procedure I was performed on 2b (141 mg,
0.37 mmol) to give 125 mg (88%) of 3b as a clear oil.
1H NMR (300 MHz, CDCl3, 23 ꢁC, d): 5.24 (d,
J = 14.3 Hz, 1H), 4.76 (m, 1H), 4.14 (m, 4H), 4.03 (m,
1H), 3.84 (dd, J = 8.1 Hz, 2H), 3.60 (m, 1H), 2.17 (m,
2H), 1.50 (m, 3H), 1.40 (m, 12H), 1.28 (m, 6H) ppm.
13C NMR (300 MHz, CDCl3, 23 ꢁC, d): 1.52.96, 68.24,
66.42, 63.34, 55.68, 28.44, 23.20, 16.55 ppm.
4.1.5. 2-tert-Butoxycarbonylamino-4-(diethoxyphospho-
ryl)butyric acid ((2R)-4a). In a stirring solution of 3a
(844 mg, 2.31 mmol) and 5 mL of acetone, at 0 ꢁC was
added Jones’ reagent (1.73 mL, 4.62 mmol). The reac-
tion mixture was allowed to warm to room temperature
and the stirring was continued for 12 h. After this time
the mixture was transferred to a larger flask and then
Celite and isopropyl alcohol were added. The mixture
was stirred for 15 min and the precipitate was filtered,
washed with acetone, and made alkaline by the addition
of sat. NaHCO3(aq). The solution was concentrated to
remove organic solvents and washed with EtOAc
(3· 25 mL). The aqueous layer was acidified to a
pH ꢄ 3 by the addition of solid Citric acid and extracted
with CH2Cl2 (5· 25 mL). The combined extracts were
washed with brine (3· 15 mL) and dried over solid
MgSO4. The solvent was concentrated to 460 mg
(59%) of a white solid, which could be recrystallized
from Et2O/hexanes. 1H NMR (300 MHz, CDCl3,
23 ꢁC, d): 10.41 (br s, 1H), 5.38 (d, J = 7.5 Hz, 1H),
4.32 (m, 1H), 4.10 (t, J = 7.3 Hz, 4H), 1.95 (m, 4H),
1.43 (s, 9H), 1.31 (dt, J = 7.0, 1.5 Hz, 6H) ppm. 13C
NMR (300 MHz, CDCl3, 23 ꢁC, d): 190.58, 157.35,
62.47, 53.57, 28.49, 25.81, 22.44, 16.50 ppm.
28.5, 26.7, 25.7, and 16.6 (d, J = 8.1 Hz) ppm.
23
D
lated m/z = 364.1889, experimental m/z = 364.1893.
½aꢂ ¼ ꢀ64:1ꢃ (c = 1.00, MeOH). HRMS (ES+) calcu-
4.1.1.2. 4-[2-(Diethoxyphosphoryl)vinyl]-2,2-dimethyl-
oxazolidine-3-carboxylic acid tert-butyl ester ((3S)-2a).
The above procedure for the synthesis of (3R)-2a was
employed using the epimer of 1 (1.146 g, 5.00 mmol) in
THF (15 mL), 2.5 M n-BuLi in hexanes (2.00 mL,
5.00 mmol), and tetraethyl methylenebisphosphonate
(1.36 mL, 5.5 mmol) yielding 1.728 g (>95%, two steps)
1
of clear oil. Rf, H and 13C NMR were consistent with
23
D
data reported for (3R)-2a. ½aꢂ ¼ þ65:9ꢃ (c = 1.03,
MeOH). HRMS (ES+) calculated m/z = 364.1889,
experimental m/z = 364.1893.
4.1.2.
4-[2-(Diethoxyphosphoryl)-2-fluorovinyl]-2,2-di-
methyl-oxazolidine-3-carboxylic acid tert-butyl ester
(2b). The above procedure for synthesis of 2a was em-
ployed using 1 (229 mg, 1.00 mmol) in THF (2.5 mL),
tetraethyl 2-fluoromethylene-bisphosphonate (337 mg,
1.10 mmol) in 2.5 mL THF, and 2.5 M n-BuLi in hex-
anes (0.4 mL, 1.00 mmol). The reaction yielded 119 mg
(31%) of 2b as a clear liquid. Rf(EtOAc) = 0.36. 1H
NMR (300 MHz, CDCl3, 23 ꢁC, d): 5.92 (dt, J = 37.6,
7.9 Hz, 1H), 3.71 (m, 1H), 4.10 (m, 5H), 3.72 (m, 1H),
1.41 (m, 6H), 1.39 (m, 9H), 1.29 (m, 6H) ppm.
4.1.6. 2-tert-Butoxycarbonylamino-4-(diethoxyphospho-
ryl)-4-fluorobutyric acid ((2R)-4b). Similar procedures
for the synthesis of (2R)-4a were followed using 3b
(125 mg, 0.326 mmol), 1 mL of acetone, and Jones’ re-
agent (0.25 mL, 0.625 mmol) to yield 40 mg (48%) of
1
(2R)-4b as a white solid. H NMR (300 MHz, CDCl3,
23 ꢁC, d): 5.90 (m, 1H), 5.50 (m, 2H), 5.00 (dt,
J = 46.4, 8.8 Hz, 1H), 4.31 (m, 1H), 4.17 (m, 4H), 2.38
(m, 2H), 1.41 (s, 9H), 1.32 (dt, J = 7.0, 2.2 Hz, 6H) ppm.
4.1.3. General procedure I: Hydrogenation/or hydrogen-
olysis (3a). To a solution of phosphonate 2a (845 mg,
2.33 mmol) dissolved in 25 mL of anhydrous EtOH
was added 10% Pd/C (20% w/w). The reaction flask
was repeatedly filled with H2 (balloon) and evacuated.
4.1.6.1. 2-Benzyloxymethyloxirane ((2S)-5). To a stir-
ring mixture of BnBr (0.72 mL, 6 mmol), 60% NaH
(193 mg, 4.8 mmol) suspended in mineral oil, and
5 mL of DMF was added R-glycidol (0.26 mL, 4 mmol)

F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
671
dissolved in 3 mL of DMF, dropwise and at 0 ꢁC. The
solution was added via syringe over 30 min at which
time the reaction was allowed to stir for an additional
3 h while warming to room temperature. The crude
material was diluted with 50 mL of EtOAc and washed
with NH4Cl (3· 50 mL), NaHCO3 (3· 50 mL), LiBr
(3· 25 mL), and brine (3· 50 mL). The organic layer
was dried over Na2SO4, filtered, and concentrated to
clear oil. The crude oil was purified by flash chromatog-
raphy (ꢄ200 mL SiO2, 1:4 EtOAc/hexanes) to yield
515 mg (78%) of clear oil. Rf(1:4 EtOAc/hexanes) = 0.31.
1H NMR (300 MHz, CDCl3, 23 ꢁC, d): 7.31 (m, 5H),
4.56 (dd, J = 18.7, 12.1 Hz, 2H), 3.74 (dd, J = 11.4,
2.9 Hz, 1H), 3.39 (dd, J = 11.4, 5.9 Hz, 1H), 3.14 (dq,
J = 2.9, 0.9 Hz, 1H), 2.73 (t, J = 4.6 Hz, 1H), 2.57 (dd,
J = 5.1, 2.6 Hz, 1H) ppm. 13C NMR (300 MHz, CDCl3,
23 ꢁC, d): 137.83, 128.21, 128.19, 127.49, 72.96, 70.65,
50.58, 43.85 ppm.
(0.74 mL, 3.450 mmol) and then DIAD (0.68 mL,
3.450 mmol) via syringe. The reaction mixture was al-
lowed to warm to room temperature and was stirred
overnight. By morning no starting material was present
by TLC analysis. The reaction mixture was filtered
through Celite, which was rinsed with methanol,
and the filtrate was concentrated to about 2 g of
crude yellow oil. Further purification was achieved by
column chromatography (ꢄ250 mL of SiO2, 1:9
acetone/chloroform) to yield 952 mg (96%) of clear
1
oil. Rf(1:9 acetone/chloroform) = 0.31. H NMR (300 MHz,
CDCl3, 23 ꢁC, d): 7.34 (m, 5H), 4.56 (s, 2H), 4.09 (m,
4H), 3.56 (m, 3H), 1.80 (m, 4H), 1.32 (dt, J = 7.0,
1.1 Hz, 6H) ppm. 13C NMR (300 MHz, CDCl3,
23 ꢁC, d): 137.78, 128.62, 128.51, 127.98, 127.76,
73.54, 72.58, 61.87, 24.37, 23.33, 21.44, 16.61 (d,
J = 6.1 Hz) ppm.
4.1.6.6. (3-Azido-4-benzyloxybutyl)phosphonic acid
diethyl ester ((3S)-7). Compound (3R)-6 (741 mg,
2.342 mmol) was converted to (3S)-7 as the above reac-
tion to yield 585 mg (73% isolated yield) of the title com-
pound. Rf(1:9 acetone/chloroform) = 0.31. See (3S)-7 for
4.1.6.2. 2-Benzyloxymethyloxirane ((2R)-5). The
above procedures for the formation of (2S)-5 were used
to form 1.232 g (75%) of the title compound from S-
glycidol (0.66 mL, 10.0 mmol). Rf(1:4
=
EtOAc/hexanes)
0.31. 1H and 13C NMR data were consistent with
(2S)-5.
1
corresponding H and 13C NMR data.
4.1.6.7. (4-Benzyloxy-3-tert-butoxycarbonylaminobu-
tyl)phosphonic acid diethyl ester ((3R)-8). Compound
(3R)-7 (1.412 g, 4.137 mmol) was stirred in 32 mL of
methanol and Boc2O (0.993 g, 4.551 mmol) followed
by Lindlar’s catalyst (285 mg, 20% by weight) were add-
ed. The reaction mixture was stirred vigorously and a
balloon of H2(g) was affixed. The apparatus was purged
numerous times by cycling between vacuum (5· 5 min)
and H2(g). Finally H2 atmosphere was applied and
maintained for 24 h at room temperature. At this time
no starting material was visible by TLC analysis
and the reaction mixture was filtered through Celite
545 with the aid of methanol. The solvent was
condensed and the resultant clear oil was purified by
column chromatography (ꢄ150 mL of SiO2, 1:9
4.1.6.3. General procedure II: Nucleophilic epoxide
opening ((3S)-6). To a solution of diethyl methylphosph-
onate (2.04 mL, 14.1 mmol) dissolved in THF (14 mL),
stirring at ꢀ78 ꢁC, was added 2.5 M n-BuLi (5.65 mL,
14.1 mmol) dropwise. The mixture was stirred for
15 min at ꢀ78 ꢁC and (3S)-5 (773 mg, 4.71 mmol) dis-
solved in THF (2.4 mL) followed by BF3ÆOEt2
(2.32 mL, 18.8 mmol) were added dropwise at ꢀ78 ꢁC.
Stirring was continued at ꢀ78 ꢁC for two additional
hours before the reaction was quenched by the dropwise
addition of NH4Cl(aq) (ꢄ2 mL). The mixture was al-
lowed to warm to room temperature overnight, concen-
trated to a yellow oil, and purified by column
chromatography (ꢄ300 mL of SiO2, 1:1 acetone/chloro-
form) to give 1.424 g (96%) of a faintly yellow liquid.
Rf(1:1 acetone/chloroform) = 0.39. 1H NMR (300 MHz,
CDCl3, 23 ꢁC, d): 7.34 (m, 5H), 4.55 (s, 2H), 4.10 (m,
4H), 3.85 (m, 1H), 3.49 (dd, J = 9.5, 3.3 Hz, 1H), 3.38
(dd, J = 9.7, 7.3 Hz, 1H), 1.85 (m, 4H), 1.31 (t,
J = 7.0 Hz, 6H) ppm. 13C NMR (300 MHz, CDCl3,
23 ꢁC, d): 137.74, 127.81, 127.12, 127.07, 127.00, 73.63,
72.69, 69.20 (d, J = 16.6 Hz), 60.99 (d, J = 6.6 Hz),
acetone/chloroform) to yield 1.324 g (77%) of clear oil.
1
Rfð5% MeOH in CHCl Þ ¼ 0:35. H NMR (300 MHz, CDCl3,
3
23 ꢁC, d): 7.30 (m, 5H), 4.94 (d, J = 8.5 Hz, 1H), 4.48 (d,
J = 4.2 Hz, 2H), 4.06 (m, 4H), 3.74 (m, 1H), 3.45 (d,
J = 3.5 Hz, 2H), 1.81 (m, 4H), 1.42 (s, 9H), 1.29 (t,
J = 6.9 Hz, 6H) ppm. 13C NMR (300 MHz, CDCl3,
23 ꢁC, d): 155.63, 137.95, 128.38, 128.22, 127.70,
127.59, 79.25, 73.15, 71.73, 61.54 (d, J = 6.8 Hz), 50.65
(d, J = 19.3 Hz), 28.35, 25.33, 23.34, 21.46, 16.45 (d,
J = 6.8 Hz) ppm.
26.12 (d, J = 4.5 Hz), 22.03, 20.15, 15.93 (d,
23
D
J = 6.0 Hz) ppm. ½aꢂ ¼ ꢀ11:9ꢃ (c = 1.13, MeOH).
4.1.6.4. (4-Benzyloxy-3-hydroxybutyl)phosphonic acid
diethyl ester ((3R)-6). Compound (2R)-5 (1.232 g,
7.50 mmol) was converted to (3R)-6 as in the above
reaction to yield 1.311 g (55%) of the title compound
as a faintly yellow liquid. Rf(1:1 acetone/chloroform) = 0.39.
4.1.6.8. (4-Benzyloxy-3-tert-butoxycarbonylaminobu-
tyl)phosphonic acid diethyl ester ((3S)-8). Compound
(3S)-7 was converted to (3S)-8 by the above method to
yield 605 mg of a clear oil, which was >95% pure by
NMR and was carried on to reaction 4.1.12.2 without
1
See (3S)-6 for corresponding H and 13C NMR data.
further purification. Rfð5% MeOH in CHCl Þ ¼ 0:35. See
3
23
D
1
½aꢂ ¼ þ10:4ꢃ (c = 1.06, MeOH).
(3S)-8 for corresponding H and 13C NMR data.
4.1.6.5. (3-Azido-4-benzyloxybutyl)phosphonic acid
diethyl ester ((3R)-7). To (3S)-6 (0.992 g, 3.136 mmol)
dissolved in CH2Cl2 (4 mL) at 0 ꢁC was added poly-
mer-bound-Ph3P (3.1 g, ꢄ9.3 mmol), followed by DPPA
4.1.6.9.
(3-tert-Butoxycarbonylamino-4-hydroxybu-
tyl)phosphonic acid diethyl ester ((3R)-9). General proce-
dure I was utilized for the hydrogenolysis of compound
(3R)-8 (1.201 g, 2.891 mmol) to give 2.714 g (94%) of

672
F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
(3R)-9 as a clear oil. No further purification was neces-
layers were combined, dried over Na2SO4(s), filtered, and
evaporated to an amorphous solid. The desired material
was used without further purification in the next
series of reactions. RfðAcOH=MeOH=CH Cl Þ ¼ 0:40. 1H
sary. Rfð5% MeOH in CHCl Þ ¼ 0:09. 1H NMR (300 MHz,
3
CDCl3, 23 ꢁC, d): 5.33 (d, J = 6.9 Hz), 4.00 (m, 5H),
3.52 (m, 2H), 1.69 (br m, 5H), 1.34 (s, 9H), 1.23 (t,
J = 6.9 Hz, 6H) ppm. 13C NMR (300 MHz, CDCl3,
23 ꢁC, d): 156.20, 79.25, 64.31, 61.76, 57.30, 52.74 (d,
2
2
NMR (300 MHz, CDCl3, 23 ꢁC, d): 9.91 (br s, 1H),
5.41 (m, 1H), 4.31 (m, 1H), 4.08 (m, 4H), 2.02 (m,
4H), 1.41 (s, 9H), 1.29 (m, 6H) ppm. 13C NMR
(300 MHz, CDCl3, 23 ꢁC, d): 173.79, 135.57, 80.15,
62.53 (d, JP = 6.0 Hz), 57.58, 53.52, 28.41, 25.66,
22.40, 20.51, 16.46 (d, JP = 6.0 Hz) ppm. HRMS
(ES+) calculated m/z = 340.1525, experimental m/z =
340.1514.
JP = 19.3 Hz), 33.31 (d, JP = 15.5 Hz), 28.44, 24.32,
23
D
23.17, 16.46 (d, JP = 5.8 Hz) ppm. ½aꢂ ¼ ꢀ10:8ꢃ
(c ꢁ 1.00, MeOH).
4.1.6.10. (3-tert-Butoxycarbonylamino-4-hydroxybu-
tyl)phosphonic acid diethyl ester ((3S)-9). The crude
material from (3S)-8 (605 mg, 1.4 mmol) was converted
to (3S)-9 by General procedure I. The resultant oil was
purified by flash chromatography (100 mL SiO2, 5%
methanol in chloroform) to yield 347 mg (72% over
4.1.6.14. RuCl3/NaIO4 oxidation: ((2S)-4a). To (3S)-9
(231 mg, 0.710 mmol) in 1.4 mL of CCl4, 1.4 mL MeCN,
and 2.2 mL H2O was added NaIO4 (456 mg,
2.130 mmol) followed by RuCl3 hydrate (3 mg,
0.016 mmol). By TLC analysis all the alcohol was con-
sumed in 5 min (presumably to the aldehyde;
two steps) as clear oil. Rfð5% MeOH in CHCl Þ ¼ 0:09. See
3
(3R)-9 for corresponding 1H and 13C NMR data.
23
D
½aꢂ ¼ þ10:4ꢃ (c = 1.00, MeOH).
Rfð1:9MeOH=CHCl Þ ꢁ 1). After 1.5 h, only a baseline spot
3
4.1.6.11. (3-tert-Butoxycarbonylamino-4-hydroxybu-
tyl)-phosphonic acid diethyl ester ((3R)-9) from (3R)-2a.
To a solution of compound 2a (5.211 g, 14.340 mmol) in
EtOH (145 mL) was added 10% Pd/C (2.8 g, 20% w/w)
[caution: Pd/C may spark on contact with ground glass
joint; funnel and joint were rinsed with EtOH prior to
attaching ground glass apparatus]. The round-bottomed
flask was affixed with a three-way adapter w/stopcock.
The adapter was fitted with a balloon of H2 and a vacu-
um hose. The system was purged of air and filled with H2
repeatedly in 5 min increments. After three cycles, the
vacuum was removed and mixture was opened to H2
atmosphere. After 1.5 days, the reaction had preceded
ꢄ60% by TLC and a second portion of Pd/C (1.4 g,
10% w/w) was added as well as more EtOH (50 mL). Fol-
lowing another 1.5 days, the reaction had progressed to
one spot by TLC analysis. The mixture was filtered over
Celite and washed with MeOH (4· 50 mL). The solvent
was evaporated and NMR analysis showed the resultant
was visible by TLC and the reaction mixture was diluted
with H2O and extracted with CHCl3 (5· 15 mL). The
organic layers were combined and dried over MgSO4,
then concentrated and purified by flash chromatography
(ꢄ75 mL of SiO2, 10–25% MeOH in CH2Cl2) to yield
184 mg (76%) of an amorphous white solid.
RfðAcOH=MeOH=CH Cl Þ ¼ 0:40. 1H NMR (300 MHz,
2
2
CDCl3, 23 ꢁC, d): 11.32 (s, 1H), 5.39 (d, J = 7.2 Hz),
4.23 (m, 1H), 4.03 (dq, J = 7.2, 2.4 Hz, 4H), 1.93 (m,
4H), 1.35 (s, 9H), 1.23 (t, J = 7.0 Hz, 6H) ppm. 13C
NMR (300 MHz, CDCl3, 23 ꢁC, d): 173.81, 155.59,
79.94, 62.30, 53.32 (d, J = 19.8 Hz), 28.29, 25.55,
22.30, 20.40, 16.33 (d, J = 6.1 Hz) ppm. HRMS (ES+)
calculated m/z = 340.1525, experimental m/z = 340.1525.
4.1.6.15. General procedure III: PyBOP condensation
(11a). To a solution of the protected amino acid (2R)-4a
(50 mg,0.147 mmol) in dry CH2Cl2 (4 mL) were added
PyBOP (77 mg, 0.147 mmol) and DIEA (0.03 mL,
0.147 mmol), followed by p-decylaniline (34 mg,
0.147 mmol). The reaction was stirred at room tempera-
ture for 12 h and then concentrated and purified by col-
umn chromatography (0–20% acetone in chloroform) to
give 35 mg of clear oil. Rf(1:1 EtOAc/hexanes)=0.15. 1H
NMR (300 MHz, CDCl3, 23 ꢁC, d): 9.16 (s, 1H), 7.46
(d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 5.69 (d,
J = 6.5 Hz, 1H), 4.44 (m, J = 7.3, 1.2 Hz, 1H), 4.12 (m,
4H), 2.54 (t, J = 7.7 Hz, 2H), 2.07 (m, J = 7.7 Hz, 2H),
1.88 (m, 2H), 1.56 (p, J = 6.9 Hz, 2H), 1.44 (s, 9H),
1.32 (dt, J = 11.9, 7.3 Hz, 6H), 0.87 (t, J = 6.9 Hz, 3H)
ppm.
1
clear oil (4.72 g, 14.34 mmol) to be >95% pure. H and
13C NMR are consistent with data reported for 9a from
23
D
culated m/z = 326.1733, experimental m/z = 326.1737.
8a. ½aꢂ ¼ ꢀ10:2ꢃ (c = 1.00, MeOH). HRMS (ES+) cal-
4.1.6.12. 3-tert-Butoxycarbonylamino-4-hydroxybutyl-
phosphonic acid diethyl ester ((3S)-9) from (3S)-2a. The
above procedure was repeated with (3S)-2a (1 g,
2.75 mmol), 10% Pd/C (1.5 g then 0.75 g) in EtOH
(75 mL then 25 mL) to yield 0.905 g (>95%) of clear
oil. The physical data were consistent with those of com-
pound (3S)-9 derived from (3S)-8. HRMS (ES+) calcu-
lated m/z = 326.1733, experimental m/z = 326.1729.
4.1.6.16. [3-tert-Butoxycarbonylamino-3-(4-octylphe-
nylcarbamoyl)propyl]-phosphonic acid diethyl ester
(11b). General procedure III was used to convert (2R)-
4a (50 mg, 0.147 mmol) and p-octylaniline (0.05 mL,
0.147 mmol) to the title compound. The crude material
was purified through flash chromatography (ꢄ50 mL
of SiO2, 1:9 acetone/CHCl31) to give 54 mg (70%) of clear
4.1.6.13. TEMPO oxidation: ((2R)-4a). To a mixture
of (3R)-9 (181 mg, 0.72 mmol) and iodosobenzenediace-
tate (430 mg, 1.335 mmol) and NaHCO3 (1.44 mmol)
stirring in 22 mL of 1:1 MeCN/H2O was added 2,2,
6,6-tetramethyl-1-piperidinyloxy (20 mg, 0.128 mmol).
The mixture was stirred vigorously at room temperature
for 3 h and then diluted with 10 mL of chloroform and
extracted with Na2CO3. Ethyl acetate was added to the
aqueous layer and the solution was acidified with 1 N
HCl and extracted (5· 15 mL) with EtOAc. The organic
oil. Rfð1:9acetone=CHCl Þ ¼ 0:2. H NMR (300 MHz, CDCl3,
3
23 ꢁC, d): 9.22 (s, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.08 (d,
J = 8.4 Hz, 2H), 5.77 (d, J = 8.1 Hz, 1H), 4.44 (m, 1H),
4.10 (m, 4H), 2.53 (t, J = 7.9 Hz, 2H), 2.09 (m, 2H), 1.87

F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
673
(m, 2H), 1.55 (p, J = 6.8 Hz, 2H), 1.43 (s, 9H), 1.31 (dt,
J = 10.6, 7.0 Hz, 6H), 0.86 (t, J = 7.0, 3H) ppm. 13C
NMR (300 MHz, CDCl3, 23 ꢁC, d): 169.75, 155.96,
139.21, 135.76, 128.99, 120.12, 80.30, 62.29 (d,
J = 22.2 Hz), 35.63, 32.13, 31.78, 29.72, 29.51, 28.59,
26.52 (d, J = 5.0 Hz), 23.08, 22.91, 21.20, 16.67 (d,
J = 7.6 Hz), 14.35 ppm.
(34 mg, 0.100 mmol) and m-octylaniline39 (0.02 mL,
0.100 mmol) to the title compound. The crude material
was purified through flash chromatography (ꢄ50 mL
of SiO2, 1:1 EtOAc/hexanes) to give 23 mg (45%) of an
off-white solid. Rf(1:1 EtOAc/hexanes)=0.10. See 11d for cor-
1
responding H and 13C NMR data.
4.1.6.21. General procedure IV: Phosphonate diester
deprotection (12a). The phosphonate diester 11a (35 mg,
0.063 mmol) was dissolved in 0.65 mL CH2Cl2 to which
bromotrimethylsilane (0.08 mL, 0.630 mmol) was added
via syringe. The solution was allowed to stir for four to
6 h. The off-white solution was then concentrated and
reconstituted in >0.65 mL of 95% methanol in water.
The new solution was allowed to stir for 4 h to ensure
hydrolysis and was concentrated and co-evaporated
with methanol and diethyl ether until a pasty solid was
attained. The compound was triturated to a fine white
solid on addition of water. The solid was filtered
through a fine fritted funnel and washed with water
(3·) followed by cold pentane (3·) to yield 31 mg of
the desired compound as a white solid. 1H NMR
(300 MHz, CD3OD, 23 ꢁC, d): 7.51 (d, J = 8.4 Hz,
2H), 7.16 (d, J = 8.4 Hz, 2H), 4.12 (t, J = 6.4 Hz, 1H),
2.58 (t, J = 7.7 Hz, 2H), 2.24 (m, 2H), 1.86 (m, 2H),
1.60 (p, J = 6.7 Hz, 2H), 1.28 (m, 12H), 0.90 (t,
J = 7.0 Hz, 3H) ppm. 13C NMR (300 MHz, CD3OD,
23 ꢁC, d): 141.04, 130.04, 121.40, 114.14, 56.36, 36.47,
33.21, 32.88, 30.89, 30.86, 30.74, 30.60, 30.41, 23.88,
14.59 ppm. MS (ESI+) m/z 399 [M+H]+.
4.1.6.17. [3-tert-Butoxycarbonylamino-3-(3-heptylphe-
nylcarbamoyl)propyl]-phosphonic acid diethyl ester (11c).
General procedure III was used to convert (2R)-4a
(50 mg, 0.147 mmol) and m-heptylaniline39 (0.28 mg,
0.147 mmol) to the title compound. The crude material
was purified through flash chromatography (ꢄ50 mL
of SiO2, 1:3 acetone/CHCl3) to give 35 mg (46%) of clear
oil. Rfð1:3acetone=CHCl Þ ¼ 0:55. 1H NMR (300 MHz,
3
CDCl3, 23 ꢁC, d): 9.22 (s, 1H), 7.40 (s, 1H), 7.39 (d,
J = 9.2 Hz, 1H), 7.19 (t, J = 7.7 Hz, 1H), 6.91 (d,
J = 7.3 Hz, 1H), 5.68 (d, J = 7.3 Hz, 1H), 4.46 (m,
1H), 4.11 (m, 4H), 2.55 (t, J = 7.7 Hz, 2H), 2.07 (m,
2H), 1.89 (m, 2H), 1.58 (p, J = 7.3 Hz, 2H), 1.44 (s,
9H), 1.32 (dt, J = 11.9, 6.9 Hz, 6H), 0.86 (t,
J = 6.9 Hz, 3H) ppm. 13C NMR (300 MHz, CDCl3,
23 ꢁC, d): 169.72, 144.14, 138.00, 128.90, 124.61,
123.12, 119.93, 117.28, 78.50, 36.21, 62.15, 36.21,
31.99, 31.67, 29.53, 29.38, 28.52, 26.44, 22.58, 21.18,
16.64, 14.29 ppm.
4.1.6.18. [3-tert-Butoxycarbonylamino-3-(4-octylphe-
nylcarbamoyl)propyl]-phosphonic acid diethyl ester
(11d). General procedure III was used to convert (2R)-
4a (1.122 g, 3.307 mmol) and m-octylaniline (0.815 g,
3.968 mmol) to the title compound. The crude material
was purified through flash chromatography (ꢄ300 mL
of SiO2, 1:9 acetone/CHCl3) to give 1.134 g (65%) of
4.1.6.22. [3-Amino-3-(4-octylphenylcarbamoyl)propyl]-
phosphonic acid (12b–VPC44152). General procedure IV
was used to globally deprotect compound 11b in forma-
tion of the title compound as 47 mg (100%) of an off-
white solid. 1H NMR (300 MHz, CD3OD, 23 ꢁC, d):
7.51 (d, J = 8.3 Hz, 2H), 7.17 (d, J = 8.3 Hz, 2H), 4.13
(t, J = 6.5 Hz, 1H), 2.59 (t, J = 7.9 Hz, 1H), 2.24 (m,
2H), 1.88 (m, 2H), 1.60 (p, J = 6.2 Hz, 2H), 1.31 (m,
10H), 0.89 (t, J = 7.0 Hz, 3H) ppm. 13C NMR
(300 MHz, CD3OD, 23 ꢁC, d): 140.90, 136.48, 129.69,
121.25, 66.69, 57.13, 36.32, 33.02, 32.72, 30.56, 30.41,
30.27, 23.71, 22.89, 22.46, 14.43 ppm. MS (APCI) m/
z = 371.9 [M+1]+, 370.80 [M, 100%]+. Elemental CHN:
calculated C, 47.90; H, 7.15; N, 6.21; found C, 48.21;
H, 7.25; N, 6.02%.
translucent oil. Rfð3:7acetone=CHCl Þ ¼ 0:58. 1H NMR
3
(300 MHz, CDCl3, 23 ꢁC, d): 9.28 (s, 1H), 7.41 (s, 1H),
7.37 (d, J = 8.1 Hz, 1H), 7.18 (t, J = 8.1 Hz, 1H), 6.90
(d, J = 7.7 Hz, 1H), 5.74 (d, J = 8.1 Hz, 1H), 4.46 (m,
1 H), 4.12 (ddt, J = 24.6, 7.3, 3.5 Hz, 4H), 2.54 (t,
J = 7.3 Hz, 2H), 2.07 (sep., J = 6.5 Hz, 2H), 1.90 (dq,
J = 18.4, 13.5, 6.5 Hz, 2H), 1.57 (p, J = 7.7 Hz, 2H),
1.43 (m, 9H), 1.31 (dt, J = 10.0, 7.3 Hz, 6H), 1.28 (m,
12H), 0.86 (t, J = 6.5 Hz, 3H) ppm. 13C NMR
(300 MHz, CDCl3, 23 ꢁC, d): 169.77, 144.07, 138.04,
128.85, 124.53, 120.33, 119.94, 118.11, 117.29, 80.20,
62.36, 62.04, 54.79, 36.20, 32.06, 31.65, 30.07, 29.66,
29.56, 29.43, 29.13, 28.51, 26.46, 23.03, 22.83, 22.78,
21.16, 16.64, 16.55, 14.28 ppm.
4.1.6.23. [3-Amino-3-(3-heptylphenylcarbamoyl)pro-
pyl]-phosphonic acid (12c). General procedure IV was
used to globally deprotect compound 11c in formation
of the title compound as 25 mg (84%) of a white solid.
1H NMR (300 MHz, (CD3)2SO, 23 ꢁC, d): 8.26 (m,
1H), 7.42 (m, 1H), 7.26 (t, J = 6.8 Hz, 1H), 6.97 (d,
J = 7.5 Hz, 1H), 3.98 (m, 1H), 2.55 (m, 2H), 2..03 (m,
2H), 1.60 (m, 4H), 1.26 (m, 8H), 0.85 (m, 3H) ppm.
13C NMR (300 MHz, (CD3)SO, 23 ꢁC, d): 166.79,
143.28, 137.83, 128.94, 124.37, 119.27, 116.95, 53.25,
35.17, 31.27, 30.87, 28.60, 28.54, 25.36, 24.32, 22.51,
22.11, 13.99 ppm. MS (ESI+) m/z 357 [M+H]+.
4.1.6.19. [3-tert-Butoxycarbonylamino-3-(4-octylphe-
nylcarbamoyl)propyl]-phosphonic acid diethyl ester
(11e). General procedure III was used to convert (2S)-
4a (50 mg, 0.147 mmol) and p-octylaniline (0.05 mL,
0.147 mmol) to the title compound. The crude material
was purified through flash chromatography (ꢄ50 mL
of SiO2, 1:9 acetone/CHCl3) to give 54 mg (70%) of clear
oil. Rfð1:9acetone=CHCl Þ ¼ 0:2. See 11b for corresponding 1H
3
and 13C NMR data.
4.1.6.20. [3-tert-Butoxycarbonylamino-3-(3-octylphe-
nylcarbamoyl)propyl]-phosphonic acid diethyl ester (11f).
General procedure III was used to convert (2S)-4a
4.1.6.24. [3-Amino-3-(3-octylphenylcarbamoyl)propyl]-
phosphonic acid (12d–VPC44116). General procedure IV
was used to globally deprotect compound 12d in

674
F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
formation of the title compound as 28 mg (90%) of a white
solid. H NMR (300 MHz, CD3OD, 23 ꢁC, d): 7.45 (m,
2H), 1.29 (m, 12H), 0.90 (t, J = 6.6 Hz, 3H) ppm. 13C
NMR (300 MHz, CD3OD, 23 ꢁC, d): 166.71, 140.61,
135.83, 129.57, 120.97, 36.04, 32.64, 32.29, 30.32,
30.21, 30.05, 29.89, 23.36, 16.81, 14.37 ppm. MS
(ESI+) m/z 417 [M+H]+.
1
2H), 7.23 (t, J = 7.8 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1H),
4.15 (m, 1H), 2.59 (t, J = 7.3 Hz, 2H), 2.25 (m, 2H), 1.89
(m, 2H), 1.61 (m, 2H), 1.31 (m, 10H), 0.89 (t,
J = 6.8 Hz, 3H) ppm. 13C NMR (300 MHz, CD3OD,
23 ꢁC, d): 167.99, 139.32, 130.01, 126.19, 121.28, 119.44,
118.72, 55.39, 55.20, 37.04, 33.17, 32.77, 30.74, 30.47,
29.72, 27.11, 25.42, 23.87, 23.61, 14.58 ppm. MS (APCI)
m/z = 371.9 [M+1]+, 370.80 [M, 100%]+. HRMS (ES+)
calculated m/z = 371.2100, experimental m/z = 371.2108.
Elemental CHN: calculated C, 47.90; H, 7.15; N 6.21;
found C, 48.11, H, 7.14, N, 6.14%.
4.1.6.28. General procedure V: Standard Mitsunobu
conditions (14a). To a solution of p-octylphenol (1.000 g,
4.847 mmol) in 5 mL of anhydrous THF was added (R)-
(+)-glycidol (0.35 mL, 5.331 mmol) and Ph3P (1.398 g,
5.331 mmol). The reaction mixture was cooled to 0 ꢁC
and DIAD (1.05 mL, 5.331 mmol) was added dropwise.
The reaction mixture was allowed to warm to room tem-
perature and stirred for 24 h. A white precipitate formed
upon addition of Et2O and was filtered off through a fine
fritted funnel. The filtrate was concentrated and purified
by column chromatography (ꢄ200 mL SiO2, CHCl3) to
give 1.023 g (80%) of 14a as colorless oil.
4.1.6.25. [3-Amino-3-(4-octylphenylcarbamoyl)propyl]-
phosphonic acid (12e). General procedure IV was used to
globally deprotect compound 11e in formation of the ti-
tle compound as 25 mg (96%) of a white solid. 1H NMR
(300 MHz, CD3OD, 23 ꢁC, d): 7.47 (d, J = 8.4 Hz, 2H),
7.15 (d, J = 8.1 Hz, 2H), 4.25 (m, 1H), 3.95 (t,
J = 6.8 Hz, 1H), 2.57 (t, J = 7.5 Hz, 2H), 2.22 (m, 2H),
1.97 (m, 1H), 1.84 (m, 2H), 1.58 (m, 2H), 1.26 (m,
10H), 0.87 (t, J = 6.6 Hz, 3H) ppm. 13C NMR
(300 MHz, CD3OD, 23 ꢁC, d): 140.90, 136.48, 129.69,
121.25, 66.69, 57.13, 36.32, 33.02, 32.72, 30.56, 30.41,
30.27, 23.71, 22.89, 22.46, 14.43 ppm. MS (ESI+) m/z
371 [M+H]+.
RfðCHCl Þ ¼ 0:66. 1H NMR (300 MHz, CDCl3, 23 ꢁC,
3
d): 7.09 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H),
4.19 (dd, J = 11.2, 3.3 Hz, 1H), 3.95 (dd, J = 11.0,
5.5 Hz, 1H), 3.35 (p, J = 4.8 Hz, 1H), 2.90 (t,
J = 4.2 Hz, 1H), 2.75 (dd, J = 4.8, 2.6 Hz, 1H), 2.54 (t,
J = 7.5 Hz, 2H), 1.56 (m, 2 H), 1.28 (m, 10H), 0.88 (t,
J = 7.0 Hz, 3H) ppm. 13C NMR (300 MHz, CDCl3,
23 ꢁC, d): 156.70, 135.92, 129.51, 114.63, 69.02, 50.44,
45.01, 35.26, 32.10, 31.93, 29.70, 29.49, 22.89,
14.33 ppm.
4.1.6.26. [3-Amino-3-(3-octylphenylcarbamoyl)propyl]-
phosphonic acid (12f). General procedure IV was used to
globally deprotect compound 11f in formation of the ti-
tle compound as 20 mg (100%) of a white solid. 1H
NMR (300 MHz, CD3OD, 23 ꢁC, d): 7.44 (s, 1H), 7.42
(d, J = 10.0 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 6.98 (d,
J = 7.8 Hz, 1H), 4.08 (t, J = 6.6 Hz, 1H), 2.60 (t,
J = 7.3 Hz, 2H), 2.23 (m, 2H), 1.89 (m, 2H), 1.61 (q,
J = 6.8 Hz, 2H), 1.32 (m, 10H), 0.89 (t, J = 6.6 Hz,
3H) ppm. 13C NMR (300 MHz, CD3OD, 23 ꢁC, d):
167.99, 139.32, 130.01, 126.19, 121.28, 119.44, 118.72,
55.39, 55.20, 37.04, 33.17, 32.77, 30.74, 30.47, 29.72,
27.11, 25.42, 23.87, 23.61, 14.58 ppm. MS (ESI+) m/z
371 [M+H]+.
4.1.6.29. 2-(4-Octylphenoxymethyl)-oxirane (14b).
The title compound was formed, through General pro-
cedure V, starting with 1.000 g of (S)-(ꢀ)-glycidol, as
1.073 g (84%) of a clear oil following column chroma-
1
tography (ꢄ200 mL SiO2, CHCl3) RfðCHCl Þ ¼ 0:66. H
3
and 13C NMR were consistent with those of epimer 14a.
4.1.6.30. [3-Hydroxy-4-(4-octylphenoxy)-butyl]-phos-
phonic acid diethyl ester (15a). General procedure II
was used to transform 14a (1.023 g, 3.899 mmol) to
1.400 g (87%) of 15a as a clear oil after column chroma-
tography (ꢄ300 mL SiO2, 1:4 acetone/chloroform).
Rf(1:4 acetone/chloroform) = 0.33. 1H NMR (300 MHz,
CDCl3, 23 ꢁC, d): 7.09 (d, J = 8.8 Hz, 2H), 6.82 (d,
J = 8.5 Hz, 2H), 4.11 (m, 4H), 4.02 (m, 1H), 3.90 (m,
2H), 2.53 (t, J = 7.7 Hz, 2H), 1.92 (m, 4H), 1.56 (p,
J = 8.1 Hz, 2H), 1.30 (m, 16H), 0.87 (t, J = 6.9 Hz,
3H) ppm. 13C NMR (300 MHz, CDCl3, 23 ꢁC, d):
156.70, 135.89, 129.54, 114.54, 71.77, 70.08, 62.02,
35.26, 32.11, 31.95, 29.70, 29.49, 26.59, 23.12, 22.89,
21.23, 16.69, 14.33 ppm.
4.1.6.27. [3-Amino-3-(4-decylphenylcarbamoyl)-2-flu-
oropropyl]-phosphonic acid (13). Compounds (3R)-4b
and p-decylaniline were converted to the title compound
by application of first; General procedure III to yield
22 mg (24%) of a clear liquid, Rf(1:1 EtOAc/hexanes) = 0.21.
1
H NMR (300 MHz, CDCl3, 23 ꢁC, d): 8.78 (d, J = 38.9,
1H), 7.43 (d, J = 7.7 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H),
5.56 (d, J = 22.6 Hz, 1H), 4.99 (m, 1H), 4.56 (m, 1H),
4.22 (m, 4H), 2.55 (t, J = 7.9 Hz, 2H), 2.40 (m, 2H),
1.56 (p, J = 7.0 Hz, 2H), 1.45 (s, 9H), 1.36 (dt, J = 7.2,
7.0 Hz, 6H), 1.25 (m, 12H), 0.87 (t, J = 6.6 Hz, 3H)
ppm. 13C NMR (300 MHz, CDCl3, 23 ꢁC, d): 129.01,
120.11, 100.16, 63.98, 63.47, 51.81, 51.50, 25.59, 32.10,
31.74, 29.83, 29.81, 29.71, 29.53, 29.45, 28.50, 28.48,
22.89, 16.63, 14.33 ppm. General procedure IV was then
implemented to give 19 mg (100%) of the title compound
4.1.6.31. [3-Hydroxy-4-(4-octylphenoxy)-butyl]-phos-
phonic acid diethyl ester (15b). General procedure II
was used to transform 14b (1.073 g, 4.089 mmol) to
1.215 g (72%) of 15b as a clear oil after column chroma-
tography (ꢄ300 mL SiO2, 1:4 acetone/chloroform).
Rf(1:4 acetone/chloroform) = 0.33.
4.1.6.32. General procedure VI: Azide formation with
free phosphine (16a). To a stirring solution of 15a
(500 mg, 1.206 mmol) in 1.6 mL CH2Cl2 at 0 ꢁC was
added Ph3P (348 mg, 1.327 mmol). The solution was
stirred for 15 min and then DIAD (0.26 mL,
1
as a white solid. H NMR (300 MHz, CD3OD, 23 ꢁC,
d): 7.50 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H),
5.02 (m, 1H), 4.26 (m, 1H), 4.11 (m, 1H), 2.59 (t,
J = 7.6 Hz, 2H), 2.39 (m, 1H), 1.60 (p, J = 7.0 Hz,

F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
675
1.327 mmol) and DPPA (0.29 mL, 1.327 mmol) were
added consecutively, dropwise. The reaction mixture
was allowed to warm to room temperature and stirred
overnight. Mixture was concentrated and purified by
chromatography (ꢄ150 mL SiO2, 1:9 acetone/CHCl3)
followed by (ꢄ100 mL SiO2, 50–80% Et2O in petroleum
ether) to yield 518 mg (98%) of non-viscous clear liquid.
25.28, 24.70, 23.87, 14.58 ppm. MS (ESI+) m/z 358
[M+H]+.
4.1.6.37.
[3-Amino-4-(4-octylphenoxy)-butyl]-phos-
phonic acid (18b). General procedure IV was used to
deprotect compound 17b in formation of the title com-
pound as 278 mg (79%) of an off-white solid. 1H
NMR, 13C NMR, and MS (ESI+) were consistent with
those obtained for compound 18a. Elemental CHN: cal-
culated C, 60.49; H, 9.02; N, 3.92; found C, 60.21; H,
9.00%; N, 3.68%.
Rfð1:9acetone=CHCl Þ ¼ 0:44. 1H NMR (300 MHz, CDCl3,
3
23 ꢁC, d): 7.04 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 8.8 Hz,
2H), 4.05 (m, 4H), 3.95 (m, 2H), 3.75 (m, 1H), 2.48 (t,
J = 7.9 Hz, 2H), 1.80 (m, 4H), 1.51 (p, J = 7.3 Hz,
2H), 1.27 (t, J = 7.0 Hz, 6H), 1.23 (m, 10H), 0.82 (t,
J = 6.6 Hz, 3H) ppm. 13C NMR (300 MHz, CDCl3,
23 ꢁC, d): 156.11, 135.80, 129.29, 114.30, 70.45, 61.67,
60.95, 35.01, 31.84, 31.68, 29.44, 29.22, 24.16, 23.10,
22.63, 21.21, 16.41, 14.06 ppm.
4.1.7. [3-Hydroxy-4-(4-octylphenoxy)-butyl]-phosphonic
acid (19). General procedure IV was used to deprotect
compound 15a (25 mg, 0.060 mmol) in formation of the
title compound. Further purification was performed by
recrystallization from EtOAc and hexanes to yield
22 mg (79%) of a white solid. 1H NMR (300 MHz,
CD3OD, 23 ꢁC, d): 7.07 (m, 2H), 6.85 (m, 2H), 3.90 (m,
3H), 2.53 (m, 2H), 1.95 (m, 2H), 1.78 (m, 2H), 1.29 (m,
10H), 0.89 (m, 3H) ppm. MS (ESIꢀ) m/z 357 [MꢀH]ꢀ.
4.1.6.33.
[3-Azido-4-(4-octylphenoxy)-butyl]-phos-
phonic acid diethyl ester (16b). General procedure VI
was used to form the title compound from alcohol
15b. Purification consisted of two chromatography
steps. First a column (ꢄ250 mL of SiO2, 1:9 acetone/
CHCl3) was run to yield the desired product with
OPPh3. The crude material was then run through a short
plug (ꢄ50 mL of SiO2) with CHCl3 as the eluent. The
phosphine oxide did not move over this system and
the plug yielded 354 mg (67%) of a clear liquid. Data
were consistent with that of 16a.
4.1.8.
4-Methyl-N-(3-octylphenyl)-benzenesulfonamide
(20). To a stirring solution of m-octylaniline40 (1.000 g,
4.87 mmol) in 5 mL of pyridine at 0 ꢁC was added tosyl
chloride (928 mg, 4.87 mmol). The reaction mixture was
warmed to room temperature. After one hour, the mix-
ture was diluted with EtOAc and washed with 1 N HCl
(3·), NaHCO3 (2·), and brine (2·). The organic layer
was dried over Na2SO4, filtered, and concentrated. The
crude material was purified by column chromatography
(ꢄ150 mL SiO2, 1:19 acetone/chloroform) to yield
4.1.6.34.
[3-Amino-4-(4-octylphenoxy)-butyl]-phos-
phonic acid diethyl ester (17a). General procedure I
was used to reduce the azide 16a to 488 mg of amine
17a as a yellow liquid. Further purification was not nec-
essary and the product was carried on to the final depro-
tection. 1H NMR (300 MHz, CD3OD, 23 ꢁC, d): 6.97 (d,
J = 8.4 Hz, 2H), 6.74 (d, J = 8.1 Hz, 2H), 5.43 (m, 2H),
3.99 (m, 4H), 3.90 (m, 2H), 2.43 (t, J = 7.9 Hz, 2H), 1.89
(m, 4H), 1.47 (p, J = 7.0 Hz, 2 H), 1.21 (m, 16H), 0.79 (t,
J = 6.7 Hz, 3H) ppm. 13C NMR (300 MHz, CD3OD,
23 ꢁC, d): 168.83, 156.23, 135.57, 129.18, 114.35, 69.57,
61.82, 51.05, 49.84, 34.96, 31.81, 31.66, 29.41, 29.20,
24.83, 22.73, 22.58, 20.85, 16.34, 14.03 ppm.
1.700 g (97%) of a white solid. Rf(1:19 acetone/chloroform)
=
1
0.70. H NMR (300 MHz, CDCl3, 23 ꢁC, d): 7.72 (d,
2H), 7.59 (bs, 1H), 7.19 (d, J = 7.8 Hz, 2H), 7.03 (q,
4H), 2.51 (t, J = 7.2 Hz, 2H), 2.34 (s, 3H), 1.54 (p,
J = 7.6 Hz, 2H), 1.27 (m, 10H), 0.88 (t, J = 6.8 Hz,
3H) ppm. 13C NMR (300 MHz, CDCl3, 23 ꢁC, d):
144.2, 140.5, 136.7, 134.7, 130.1, 129.7, 127.9, 122.4,
35.8, 32.4, 31.9, 30.0, 29.8, 23.2, 22.0, 14.6. MS (ESI)
m/z 360 [M+H]+.
4.1.9. 4-Methyl-N-(3-octylphenyl)-N-oxiranylmethylben-
zenesulfonamide (21). General procedure V was used to
couple 20 (0.809 g, 2.25 mmol) with (R)-(+)-glycidol
(0.23 mL, 3.375 mmol) to form 644 mg (69%) of the
title compound 21 after flash chromatography
(ꢄ150 mL SiO2, 1:4 EtOAc/hexanes), as a clear oil.
4.1.6.35.
[3-Amino-4-(4-octylphenoxy)-butyl]-phos-
phonic acid diethyl ester (17b). General procedure I
was used to reduce the azide 16b to 333 mg of amine
17b as a light yellow liquid. Further purification was
not necessary and the product was carried on to the final
deprotection. Data were similar to those obtained for
17a.
1
Rf(1:4 EtOAc/hexanes) = 0.36. H NMR (300 MHz, CDCl3,
23 ꢁC, d): 7.45 (d, J = 8.1 Hz, 2H), 7.20 (m, 2H), 7.07
(m, 1H), 7.02 (m, 1H), 6.89 (m, 1H), 6.82 (m, 1H),
3.65 (ddd, J = 35.2, 14.3, 5.3 Hz, 2H), 3.08 (m, 1H),
2.63 (m, 1H), 2.49 (t, J = 7.5 Hz, 2H), 2.37 (s, 3H),
2.16 (m, 1H), 1.48 (p, J = 6.8 Hz, 2H), 1.25 (m, 10H),
0.85 (t, J = 7.0 Hz, 3H). 13C NMR (300 MHz, CDCl3,
23 ꢁC, d): 144.01, 143.51, 139.60, 138.83, 129.39,
127.68, 126.10, 53.65, 50.24, 45.78, 35.56, 31.84, 31.24,
29.41, 29.15, 22.64, 21.47, 14.10 ppm.
4.1.6.36.
[3-Amino-4-(4-octylphenoxy)-butyl]-phos-
phonic acid (18a). General procedure IV was used to
deprotect compound 17a in formation of the title com-
pound as 500 mg (97%) of an off-white solid. 1H
NMR (300 MHz, CD3OD, 23 ꢁC, d): 7.12 (d,
J = 8.4 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 4.22 (dd,
J = 10.4, 3.5 Hz, 1H), 4.07 (dd, J = 10.7, 6.1 Hz, 1H),
3.68 (m, 1H), 2.55 (t, J = 7.3 Hz, 2H), 2.09 (m, 2H),
1.88 (m, 2H), 1.57 (p, J = 7.7 Hz, 2H), 1.30 (m, 10H),
0.89 (t, J = 6.8 Hz, 3H) ppm. 13C NMR (300 MHz,
CD3OD, 23 ꢁC, d): 13764, 130.64, 115.70, 101.52,
68.14, 36.14, 33.18, 33.07, 30.73, 30.58, 30.39,
4.1.10. {3-Hydroxy-4-[(3-octylphenyl)-(toluene-4-sulfo-
nyl)-amino]-butyl}-phosphonic acid diethyl ester (22). Gen-
eral procedure II completed the synthesis of 853 mg (97%)
of compound 22 from 21 as a clear oil following column

676
F. W. Foss, Jr., et al. / Bioorg. Med. Chem. 15 (2007) 663–677
chromatography (SiO2, 5–20% acetone in CHCl3). Rf(1:4
EtOAc/hexanes) = 0.05. 1H NMR (300 MHz, CDCl3, 23 ꢁC,
d): 7.41 (dd, J = 8.1, 6.5 Hz, 2H), 7.18 (d, J = 7.7 Hz,
2H), 7.13 (d, J = 8.1, 1H), 7.04 (d, J = 7.7 Hz, 1H), 6.85
(m, 1H), 6.73 (m, 1H), 4.00 (m, 4H), 3.61 (m, 2H), 3.47
(m, 2H), 2.45 (t, J = 8.1 Hz, 2H), 1.76 (m, 4H), 1.45 (m,
2H), 1.24 (m, 16H), 0.83 (t, J = 6.9 Hz, 3H) ppm. 13C
NMR (300 MHz, CDCl3, 23 ꢁC, d): 143.63, 139.60,
138.92, 134.77, 129.50, 129.44, 128.93, 128.31, 127.88,
126.04, 68.80 (d, J = 13.5 Hz), 61.74 (d, J = 5.8 Hz),
56.23 (d, J = 7.7 Hz), 35.66, 31.93, 31.35, 29.49, 29.30,
28.98, 27.10 (d, J = 4.8 Hz), 22.70, 21.59, 20.48, 16.47
(d, J = 5.8 Hz), 14.18 ppm.
2-necked flask fitted with a cold finger cooled to ꢀ78 ꢁC
and stir bar. Compound 24 (182 mg, 0.306 mmol) diluted
in a minimal amount of THF was quickly added to the
solution and the reaction mixture was allowed to proceed
for no longer than 5 min, at which time ethanol was added
under vigorous stirring and the reaction was allowed to
warm to room temperature. The reaction mixture was
concentrated to dryness, dissolved in EtOAc, and washed
with NaHCO3 (3·) and brine (1·). The organic layer was
dried and then columned (ꢄ75 mL of SiO2, 5% MeOH in
CHCl3). The desired product was found to bea clear oil, in
25 mg (25%) quantities. Starting material (28%) was also
recovered.
Rfð5%MeOH in CHCl Þ ¼ 0:17.
1H
NMR
3
(300 MHz, CDCl3, 23 ꢁC, d): 7.07 (t, J = 7.5 Hz, 1H),
6.53 (t, J = 7.0 Hz, 1H), 6.44 (m, 2H), 4.08 (m, 4H), 3.70
(m, 1H), 3.11 (m, 3H), 2.50 (t, J = 7.9 Hz, 2H), 1.83 (m,
4H), 1.57 (p, J = 7.3 Hz, 2H), 1.31 (m, 16H), 0.87 (t,
J = 7.0 Hz, 3H) ppm. 13C NMR (300 MHz, CDCl3, 23
ꢁC, d): 157.35, 148.46, 129.30, 118.14, 113.44, 110.41,
62.05, 61.72, 58.20, 47.13, 36.41, 34.55, 32.10, 31.72,
29.92, 29.73, 29.64, 29.49, 28.18 (d, J = 8.6 Hz), 22.88,
16.69 (d, J = 7.1 Hz), 14.32 ppm.
4.1.11. {3-Azido-4-[(3-octylphenyl)-(toluene-4-sulfonyl)-
amino]-butyl}-phosphonic acid diethyl ester (23). General
procedure VI transformed alcohol 22 (302 mg,
0.532 mmol) into azide 23. The crude material was puri-
fied twice by chromatography (ꢄ150 mL of SiO2, 10%
acetone/CHCl3) followed by (ꢄ100 mL of SiO2, 7:3
EtOAc/hexanes) to yield 267 mg (85%) as a clear oil.
(Another 5% of the desired product was isolated with
residual phosphine oxide.) Rfð1:9acetone=CHCl Þ ¼ 0:43. 1H
3
NMR (300 MHz, CDCl3, 23 ꢁC, d): 7.38 (d, J = 8.4 Hz,
2H), 7.19 (d, J = 7. Hz, 2H), 7.07 (d, J = 7.7 Hz, 1H),
6.86 (d, J = 13.2 Hz, 1H), 6.78 (m, 1H), 4.02 (m, 4H),
3.53 (m, 3H), 2.48 (t, J = 7.5 Hz, 2H), 2.37 (s, 3H), 1.75
(m, 4H), 1.46 (p, J = 7.0 Hz, 2H), 1.25 (m, 16H), 0.84 (t,
J = 7.0 Hz, 3H) ppm. 13C NMR (300 MHz, CDCl3,
23 ꢁC, d): 144.36, 143.80, 139.38, 134.55, 132.04, 129.48,
129.05, 128.45, 127.87, 125.73, 61.75 (d, J = 6.6 Hz),
61.32 (d, J = 16.1 Hz), 54.74, 35.68, 31.92, 31.33, 29.49,
29.28, 28.97, 28.56, 25.30 (d, J = 4.5 Hz), 22.70, 21.59,
16.49 (d, J = 6.0 Hz), 14.16 ppm.
4.1.14.
[3-Amino-4-(3-octylphenylamino)-butyl]-phos-
phonic acid (26). The deprotection of 25 (23 mg,
0.056 mmol) was carried out by General procedure IV
to give 20 mg (83%) of the title compound as a colorless
solid. 1H NMR (300 MHz, CD3OD, 23 ꢁC, d): 7.36 (dd,
J = 7.9, 3.7 Hz, 1H), 7.26 (t, J = 1.7 Hz, 1H), 7.21 (d,
J = 7.9 Hz, 1H), 7.13 (d, J = 7.7 Hz, 1H), 3.70 (m,
3H), 2.65 (t, J = 7.5 Hz, 2H), 2.14 (m, 2H), 1.95 (m,
2H), 1.64 (p, J = 7.5 Hz, 2H), 1.31 (m, 10H), 0.89 (t,
J = 7.0 Hz, 3H) ppm. MS (ESI+) m/z 357 [M+H]+.
4.1.12. {3-Amino-4-[(3-octylphenyl)-(toluene-4-sulfonyl)-
amino]-butyl}-phosphonic acid diethyl ester (24). Com-
pound 23 (267 mg, 0.450 mmol) was dissolved in
50 mL of a 20:1 MeOH/HCl solution. To this solution
was added ꢄ0.5 g of Pd(OH)2 and the apparatus was
assembled and experiment run analogous to that of
General procedure I. When no starting material re-
mained (ꢄ4 h by TLC), the mixture was filtered over
Celite and washed with 2% Et3N in MeOH. The filtrate
was concentrated to yield 268 mg (100%) of the title
compound as yellow oil. No further purification was
Acknowledgment
This research was funded by Grants from the NIH (R01
GM067958 and F31 GM064101).
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