α- and β-Substituted phosphonate analogs of LPA as autotaxin inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.11.078

Autotaxin (ATX) is an attractive pharmacological target due to its lysophospholipase D activity which leads to the production of lysophosphatidic acid (LPA). Blockage of ATX produced LPA by small molecules could be a potential anticancer chemotherapy. In our previous study, we have identified the two β-hydroxy phosphonate analogs of LPA (compounds f17 and f18) as ATX inhibitors. With this work, we investigated α- and β-substituted phosphonate analogs of LPA and evaluated them for ATX inhibitory activity. The stereochemistry of β-hydroxy phosphonates was also studied.

Full text of DOI:10.1016/j.bmc.2007.11.078

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2212–2225
a- and b-Substituted phosphonate analogs
of LPA as autotaxin inhibitors
Peng Cui,a,* William F. McCalmont,a Jose L. Tomsig,b
Kevin R. Lynchb and Timothy L. Macdonalda
aDepartment of Chemistry, University of Virginia, McCormick Road, PO Box 400319, Charlottesville, VA 22904, USA
bDepartment of Pharmacology, University of Virginia, 1300 Jefferson Park Avenue, PO Box 800735, Charlottesville, VA 22908, USA
Received 4 October 2007; revised 28 November 2007; accepted 29 November 2007
Available online 4 December 2007
Abstract—Autotaxin (ATX) is an attractive pharmacological target due to its lysophospholipase D activity which leads to the pro-
duction of lysophosphatidic acid (LPA). Blockage of ATX produced LPA by small molecules could be a potential anticancer che-
motherapy. In our previous study, we have identified the two b-hydroxy phosphonate analogs of LPA (compounds f17 and f18) as
ATX inhibitors. With this work, we investigated a- and b-substituted phosphonate analogs of LPA and evaluated them for ATX
inhibitory activity. The stereochemistry of b-hydroxy phosphonates was also studied.
Published by Elsevier Ltd.
1. Introduction
There are a limited number of ATX inhibitors which
have been reported.28–32 In our laboratory, we have
developed a series of b-keto and b-hydroxy phosphonate
derivatives and tested the ATX inhibitory activity in a
choline detection assay.33 Among them, two b-hydroxy
phosphonates originated from L-tyrosine were identified
as the lead compounds and they inhibited approximately
70% of ATX activity at 1 lM. The pKa of the second
phosphonate proton (ꢀ7.6) can be influenced by the
substitutions on the a- and b-positions of the phospho-
nate.34 This electronic property is believed to affect
enzyme–substrate interaction.35 In this report, we syn-
thesized both a- and b-substituted phosphonate analogs
based on the lead compounds and investigated their
activities on ATX inhibition.
Autotaxin (ATX, NPP2) is a member of the nucleotide
pyrophosphatase and phosphodiesterase (NPP) family
of enzymes which are able to hydrolyze phosphoester
bonds in nucleotides.1,2 It can be biosynthesized by a
variety of cells and tissues and highly expressed in vari-
ous malignancies.3–8 ATX is a multifunctional enzyme
which potently stimulates cancer cell proliferation and
tumor cell motility, augments the tumorigenicity, and
induces angiogenic response.3,9–14 In 2002, two indepen-
dent laboratories discovered that ATX is identical to
plasma lysophospholipase D (lysoPLD) and acts by
hydrolyzing lysophosphatidylcholine (LPC) into lyso-
phosphatidic acid (LPA).15,16 LPA is a lipid mediator
that signals cell proliferation, migration, and survival
by activating its specific extracellular and intracellular
receptors.17–27 The generation of LPA by ATX can fully
account for the biological effects of ATX in cell culture.
Considering the fact that ATX is the major enzyme in
biosynthesis of LPA in plasma, the development
of ATX inhibitors could lead to novel anticancer
chemotherapies.
2. Results and discussion
2.1. Chemistry
Synthesis of a-fluoro phosphonates (Scheme 1) began
with esterification of the free acid of N-tert-butyl carba-
mate, O-benzyl protected L-tyrosine 5, with trimethyl-
silyl diazomethane. The methyl ester 6 was reduced to
the corresponding alcohol with sodium borohydride
in the presence of calcium chloride and then oxidized
to the desired aldehyde 8 under the Swern oxidative con-
dition. The tetraethyl monofluoromethylene diphospho-
nate 2 was formed by the fluorination of the tetraethyl
Keywords: Autotaxin; ATX; Phosphonates; LPA.
*
Corresponding author at present address: Department of Chemistry
and Biochemistry, University of California, Los Angeles, 1224
Young Hall/3210 MSB CHEM, 607 Charles E Young Dr. Los
Angeles, CA 90095, USA. Tel.: +1 434 227 1270;
e-mail: pengcui@chem.ucla.edu
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2007.11.078

P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
2213
O
O
P
O
P
OEt
O
O
P
O
P
OEt
a
b
EtO
OEt
EtO
EtO
OEt
EtO
OMs
F
OH
N
N
3
4
1
2
O
O
NHBoc
NHBoc
HO
NHBoc
MeO
6
HO
c
d
e
BnO
BnO
BnO
7
5
O
EtO
P
O
EtO
P
F
F
NHBoc
EtO
EtO
O
NHBoc
NH2TFA
f
g
BnO
BnO
BnO
8
9
i
10
O
P
O
P
EtO
EtO
EtO
EtO
F
F
H
H
N
h
N
C15H31
C15H31
j
O
O
BnO
HO
11
12
O
P
EtO
EtO
O
P
F
HO
HO
F
H
N
H
N
C15H31
k
C15H31
O
N
O
O
R =
RO
RO
13
14
Scheme 1. Synthesis of 14. Reagents and conditions: (a) NaH, THF, 0 ꢀC; Selectfluor, rt, overnight, 56%; (b) NEt3, CH2Cl2, 0 ꢀC, then MsCl, 5 h,
90%; (c) TMS–CHN2, 0 ꢀC, 10 min, anhydrous MeOH, 100%; (d) NaBH4, CaCl2, THF, EtOH, 3 h, 0 ꢀC—rt, 94%; (e) oxalyl chloride, DMSO,
CH2Cl2, À78 ꢀC, NEt3, 2 h, 83%; (f) 2, n-BuLi, THF, À78 ꢀC, 2 h, 70%; (g) TFA, CH2Cl2, rt, 5 h; (h) palmitoyl chloride, TEA, 0 ꢀC, 3 h; (i) Pd(OH)2,
H2, EtOH, rt, overnight, 85%, three steps; (j) 4, K2CO3, acetone, 18-crown-6, reflux, overnight, 90%; (k) TMS–Br, then MeOH and H2O, rt,
overnight, 82%.
diphosphonate anion with Selectfluor.36 Next, treatment
of the monofluorodiphosphonate 2 with n-butyl lithium
at À78 ꢀC generated the lithiated carbanion, which con-
densed with the aldehyde to afford a-fluorovinylphosph-
onate 9. The protecting N-Boc group was removed by
treatment of trifluoroacetic acid and the remaining
ammonium trifluoroacetic acid salt was acylated with
palmitoyl chloride to generate amide 11. After dual
hydrogenation/hydrogenolysis of the olefin and benzyl
group, respectively, the resulting phenol 12, which was
a mixture of two diastereomers, was coupled with the
mesylate 4 under basic conditions to give the ether mix-
ture 13. The a-fluoro phosphonate 14 was obtained by
trimethylsilyl bromide mediated deprotection of diethyl
esters.37
Synthesis of a-hydroxy phosphonates (Scheme 2) com-
menced with the Arndt–Eistert reaction. Treatment of
N-tert-butyl carbamate, O-benzyl ether protected L-
tyrosine 5 with isobutyl chloroformate gave the mixed
anhydride. The diazoketone 16 was prepared by adding
the pre-cooled freshly distilled diazomethane to the
mixed anhydride. Next, a carbene mediated Wolff rear-
rangement furnished the transformation from a diazok-
etone to a ketene, which underwent a nucleophilic
addition by methanol to afford the desired homologated

2214
P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
O
OMe
NHBoc
O
O
N2
NHBoc
NHBoc
HO
a, b
d
c
BnO
O
BnO
OH
BnO
O
P
O
P
MeO
MeO
MeO
MeO
OH
NHBoc
H
HO
NHBoc
N
C15H31
NHBoc
g
e
f
O
BnO
BnO
BnO
BnO
O
MeO
P
O
P
O
P
OH
MeO
MeO
HO
HO
MeO
OH
H
OH
h
i
H
H
j
N
C15H31
N
C15H31
N
C15H31
O
O
O
R
R
HO
O
O
O
N
R =
Scheme 2. Synthesis of 24a and b. Reagents and conditions: (a) KOH, 65 ꢀC, isobutyl chloroformate, THF, NEt3, 0 ꢀC; (b) CH2N2, overnight, 51%;
(c) silver benzoate, NEt3, MeOH, THF, À78–0 ꢀC, overnight, 73%; (d) NaBH4, CaCl2, THF, EtOH, 0 ꢀC—rt, overnight, 97%; (e) oxalyl chloride,
DMSO, CH2Cl2, À78 ꢀC, NEt3, 2 h, 94%; (f) dimethyl phosphite NaH, THF, 0 ꢀC, 1 h, 60%; (g) TFA, CH2Cl2, rt, 5 h, then palmitoyl chloride, NEt3,
CH2Cl2, 0 ꢀC, 3 h, 82%; (h) Pd(OH)2, H2, EtOH, rt, overnight, 58–73%; (i) 4-methoxy-3,5-dimethyl-2-pyridine-methanol, DIAD, PPh3, NEt3,
CH2Cl2, 0 ꢀC—rt, overnight, 80%; (j) TMS–Br, then MeOH and H2O rt, overnight, 90%.
methyl ester 17. The aldehyde 19 was obtained by a two-
step reduction/oxidation process. Then the sodium an-
ion of dimethyl phosphite was generated and reacted
with the aldehyde to give a-hydroxy phosphonates 20.
Next, the N-Boc protecting group was removed and
the resulting ammonium trifluoroacetate salt was acyl-
ated with palmitoyl chloride to generate amides 21a
and b. The two diastereomers in terms of a-hydroxy
group were separated by column chromatography at
this step. The exact stereochemistry for each diastereo-
mer was determined (unpublished results). The benzyl
ether was cleaved by treatment with palladium hydrox-
ide on carbon and the resulting phenols 22a and b were
reacted with 4-methoxy-3,5-dimethyl-2-pyridine-metha-
nol under Mitsunobu condition to give 23a and b. Final-
ly, the fully functionalized a-hydroxy phosphonates
were obtained by the same method mentioned above.
2.2. Biological evaluation
The ATX activity was measured in the presence of the
compounds under different concentrations (100 lM,
10 lM, and 1 lM). The ATX activity without com-
pounds was used as the standard (100% activity). The
data are shown in Table 1. a-Hydroxy (24a and b) and
a-fluoro (14a and b) phosphonates did not show any
substantial ATX inhibition. The b-fluoro phosphonate
demonstrated the same levels of activity as the lead com-
pounds (f17 and f18)33 at higher concentrations (10 lM
and 100 lM); however it lost some potency at the lowest
concentration (1 lM). Further optimization was at-
tempted on b-hydroxy and b-keto phosphonates. We
investigated a variety of natural amino acid backbones
and the compounds and data are shown in Table 2.
Most of the compounds could inhibit 50–80% ATX
activity at the highest concentration and lost the potency
when lower concentrations were applied. The b-keto
phosphonate derived from L-proline 38 has proved to
be the most potent b-keto phosphonate which could in-
hibit 80% of ATX activity at medium concentration
(10 lM). Tryptophan was chosen to compare with the
linker region in tyrosine. The compounds were not as
The transformation of the b-hydroxy phosphonates (49a
and b)33 to b-fluoro phosphonates (25a and b) was
accomplished by using diethylaminosulfur trifluoride
(DAST) (Scheme 3). The product was then formed with
the above-mentioned deprotection of the phosphonate
group using TMS–Br.

P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
2215
O
P
OH
O
F
H
N
H
N
MeO
MeO
HO
HO
C15H31
P
C15H31
O
O
a,b
N
N
O
O
OMe
OMe
49a,b
25a,b
Scheme 3. Synthesis of 25a and b. Reagents and conditions: (a) DAST, CH2Cl2, À78 ꢀC, 1 h, 50%; (b) TMS–Br, CH2Cl2, rt, 4 h, then H2O and
MeOH, overnight, 62%.
Table 1. ATX inhibitory evaluation of compounds 14, 24, and 25
Compounds
X
a
Y
b
ATX activity (%)
1 lM
10 lM
100 lM
24a
24b
14
OH
OH
Fa
H
S
H
71
73
74
63
27
59
70
68
68
21
13
22
53
87
25
8
R
H
RS
H
f18
f17
25
OH
OH
Fa
bb
a
H
6
11
H
RS
a Racemic products were tested.
b a refers to the diastereomer that elutes first, b refers to the diastereomer that elutes second from normal phase silica gel based column
chromatography.
potent as the lead compounds and the b-hydroxy phos-
phonate with 4-methoxy-3,5-dimethyl-pyridyl moiety 44
did show higher potency than the one with benzyl group
45.
Karplus relationship.38 Due to the single bond rotation
the coupling constants are revealed as an average value
contributed from relatively stable rotational isomers. It
3
is expected that the J difference between syn and anti
isomers could be enlarged if the hydroxyl group and
amide in the b-hydroxy phosphonate substrate are fixed
in a ring form which prevents a free rotation of carbon
bond. Oxazolidines 47 and 48 were prepared (Scheme 4)
from b-hydroxy phosphonates 49a and 49b (49a was the
less polar isomer and 49b was the more polar isomer).
The results of the decoupling study show that the J val-
ues between geminal benzylic protons H3 and H4 are
approximately 14 Hz in both oxazolidines (Fig. 1).
These two protons couple with H2 to give J values cor-
responding to 6 Hz and 9 Hz, respectively. The 3J values
between H1 and H2 are close to 0 Hz in 47 and 5 Hz in
48. According to the Karplus relationship, 47 has the
anti configuration and the less polar isomer 49a corre-
sponds to the R alcohol; 48 has the syn configuration
and the more polar isomer 49b corresponds to the S
alcohol. This result is consistent with the reported 3J val-
ues of oxazolidone derivatives of a-amino-b-hydroxy
acids.39,40 Taken into account the outcome of diastere-
2.3. Structure determination of lead compounds
The lead compounds f17 and f18 are diastereomers orig-
inating from reduction of b-keto phosphonate. To fur-
ther study this reaction and determine the absolute
stereochemistry of b-hydroxy phosphonates, a variety
of reductive agents and reaction conditions were applied
(Table 3). Sodium borohydride gave diastereoselectivity
in 1:2.5 ratio favoring the more polar isomer. Lewis acid
mediated reduction gave higher reaction yields but lost
the diastereoselectivity. Application of bulky hydride
reducing reagents such as lithium triethylborohydride
(Super-Hydride) and lithium tris[(3-ethyl-3-pen-
tyl)oxy]aluminohydride resulted in lower reaction yields
but significantly improved the selectivity.
The relationship between the dihedral angle and the vic-
inal coupling constant J was given theoretically by the
3

2216
P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
Table 2. ATX inhibitory evaluation of compounds 26–46
O
P
X
H
N
HO
HO
R
O
Compounds
X
R
ATX activity (%)
1 lM
10 lM
100 lM
26
27
28
29
30
31
32
33
34
35
36
37
38
39
@O
–H
–H
79
88
60
50
109
115
72
89
68
93
88
92
49
83
95
88
67
14
77
57
78
48
53
64
70
99
18
43
76
56
72
25
24
13
23
25
23
52
70
67
9
–OHa
@O
–CH3
–CH3
–OHa
@O
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH2CH(CH3)2
–CH2CH(CH3)2
–CH2CH(CH3)2
–CH2Ph
–OH (a)b
–OH (b)b
@O
–OH (a)
–OH (b)
–OH (a)
–OH (b)
@O
–CH2Ph
–CH2CH2CH2–c
–CH2CH2CH2–c
–OHa
16
40
41
42
@O
91
88
93
70
51
75
28
25
26
N
H
–OH (a)
–OH (b)
N
H
N
H
43
44
–OHa
80
64
35
28
28
13
N
–OHa
N
N
OMe
O
O
45
46
–OH (a)
–OH (b)
83
78
41
46
19
61
a Racemic products were tested.
b a refers to the diastereomer that elutes first, b refers to the diastereomer that elutes second from normal phase silica gel based column
chromatography.
c The compounds were synthesized from protected L-proline.
oselectivity, the reaction is likely governed by Felkin–
Ahn model (Fig. 2).
inhibitory activity. The b-substituted analogs showed
higher potency than the a-substituted analogs. Further
structural optimization was attempted on b-keto and
b-hydroxy phosphonates. We investigated a variety of
amino acid backbones. Some analogs showed compara-
ble potency with the lead compounds (f17 and f18) at
high concentrations (10 lM and 100 lM). However, at
the lowest concentration (1 lM), these newer analogs
3. Conclusion
We have synthesized a series of a-/b-substituted phos-
phonate analogs of LPA and evaluated them for ATX

P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
2217
Table 3. Reduction of b-keto phosphonate
O
P
OH
*
O
P
O
H
N
H
N
MeO
MeO
MeO
MeO
C15H31
C15H31
O
O
[H]
N
N
O
O
OMe
OMe
Entry
Hydride
Conditions
Yield (%)
Ratio (a:b)a
1
2
3
4
5
6
7
NaBH4
EtOH, 0 ꢀC
EtOH, À78 ꢀC
EtOH, 0 ꢀC
EtOH, 0 ꢀC
THF, 0 ꢀC
THF, 0 ꢀC
THF, 0 ꢀC
75
82
1:2.5
1:1
NaBH4, CeCl3
NaBH4, CeCl3
NaBH4, MgCl2
Li(C2H5)3BH
LiAl[OC(C2H5)3]3H
Catecholborane
85
60
1:1
1:1
33
40
NRb
1:9
1:11
NR
a a refers to the diastereomer that elutes first, b refers to the diastereomer that elutes second.
b NR, no reaction.
O
P
O
O
P
OH
H
N
MeO
MeO
N
C15H31
MeO
MeO
C15H31
O
O
a
N
N
O
O
OMe
OMe
49a,b
47 and 48
Scheme 4. Synthesis of 47 and 48. Reagents and conditions: (a) 2-methoxypropene, CSA, CH2Cl2, 0 ꢀC, 30–35%.
showed reduced potency compared to the lead com-
pounds. The stereochemistry of the b-hydroxy phospho-
nates was also determined by 1H homonuclear
decoupling study. The most potent compound (f17)
was proven to be a b-hydroxy phosphonate with R-hy-
droxy moiety. Additional synthetic efforts are needed
to fully elucidate the SAR of the phosphonate analogs
and toward the goal of developing potent ATX inhibi-
tors as the potential anticancer chemotherapy.
THF was filtered through alumina and subsequently dis-
tilled over a mixture of sodium and benzophenone
immediately prior to use. Merck silica gel F-254 pre-
coated, aluminum backed plates and Merck Silica Gel
60 (230–400 mesh) or Silicycle Ultra pure silica gel
(230–400 mesh) were used in thin layer chromatography
and flash column chromatography, respectively. Anal-
tech (Newark, NJ) alumina GF Preparative TLC plates
(500 lm, 1000 lm, and 2000 lm 20 · 20 cm) were used
for preparative TLC separation. All nuclear magnetic
resonance (NMR) spectra were collected using a Varian
UnityInova 300 NMR spectrometer at 300 MHz and
chemical shifts are reported in ppm.
4. Experimental
4.1. Materials
4.2. General procedures
Chemicals for syntheses were purchased from Aldrich
Chemical Company (Milwaukee, WI), Acros Chemical
Company (Morris Plains, NJ), Novabiochem Chemical
Company (Laufelfingen, Switzerland), and Fluka Chem-
ical Company (Milwaukee, WI), and were used without
further purification. All reactions were carried out under
an inert atmosphere of nitrogen unless otherwise stated.
All solvents were filtered through alumina (activity 1)
immediately prior to use in reactions. In certain case,
4.2.1. General procedure A: acylation of amines. To a
solution of the amine (1 equiv) to be acylated and
TEA (3 equiv) in CH2Cl2 at 0 ꢀC was added the appro-
priate acyl chloride dropwise by syringe. The reaction
mixture was then allowed to stir for several hours. After
completion of the reaction as determined by TLC, it was
quenched with 10% aqueous HCl and further extracted
with 10% aqueous HCl. The aqueous layer was then ex-

2218
P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
O
O
O
P
N
C15H31
O
MeO
MeO
N
C15H31
O
H1
O
H2
H2
H4
P
H
1H3
MeO
MeO
H3
H4
N
N
O
O
OMe
OMe
47
48
O
OH
O
P
OH
MeO
MeO
MeO
P
NH
C15H31
NH
C15H31
MeO
O
O
N
N
O
O
OMe
OMe
Figure 1. 1H homonuclear decoupling study.
O
P
OH
S N
H
MeO
MeO
C15H31
S
OH
O
R1HN
O
1
2
R HN
CH R
2
CH2R2
N
O
H-
H
H
R
R
H
OMe
Figure 2. Modified Felkin–Ahn model of reductive reaction.
tracted once with CH2Cl2 followed by a brine extraction
of the organic layer. The organic layer was then dried
using magnesium sulfate, filtered, and concentrated un-
der reduced pressure. Then the product was purified
using the appropriate purification techniques.
any purification that was deemed necessary was
undertaken.
4.2.3. General procedure C: sodium borohydride reduction
of esters. NaBH4 (2 equiv) and CaCl2 (2 equiv) are dis-
solved in ethanol so that a solution of milky appearance
is formed. This solution is cooled to 0 ꢀC and the ester to
be reduced is dissolved in THF, so that a 2:1 ethanol/
THF mixture will be the resulting solution ratio. Then
the ester in THF is added dropwise to the reaction mix-
ture via canula. The reaction mixture is allowed to
slowly warm to rt and then it is stirred overnight. The
following day the reaction is quenched with a saturated
solution of aqueous ammonium chloride, then extracted
with EtOAc, brine, dried with sodium sulfate, and con-
centrated under reduced pressure. The product is then
4.2.2. General procedure B: palladium hydroxide on
carbon mediated hydrogenolysis of benzyl ethers. To a
solution of the benzyl ether to be cleaved in ethanol
was added a catalytic amount of 20% palladium hydrox-
ide on carbon; the reaction was then subjected to a
hydrogen atmosphere (typically 1ATM) and was al-
lowed to stir overnight. The following day the reaction
mixture was filtered through a pad of Celite to remove
all palladium using methanol as the eluent followed by
removal of all solvent under reduced pressure. Then

P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
2219
either purified via flash column chromatography or used
unpurified.
is added dropwise to the reaction mixture via canula.
The reaction mixture is allowed to slowly warm to rt
and then it is stirred overnight. The following day the
reaction is quenched with a saturated solution of aque-
ous ammonium chloride, then extracted with EtOAc,
brine, dried with sodium sulfate, and concentrated un-
der reduced pressure. The product was then purified
using flash column chromatography.
4.2.4. General procedure D: Swern oxidation of alcohols.
To dry dichloromethane in a flame-dried RBF was
added oxalyl chloride (1.5 equiv, 2 M in CH2Cl2), the
mixture was then cooled to À78 ꢀC before a solution
of dimethylsulfoxide (3 equiv) in CH2Cl2 was added
dropwise via syringe. The resulting mixture was stirred
at this temperature for 30 min before a solution of the
alcohol (1 equiv) to be oxidized in CH2Cl2 was added
dropwise via canula. The mixture was stirred for
30 min before triethyl amine was added via syringe;
the resulting mixture was stirred for an additional
15 min before it was warmed to rt. Then a 10% HCl
solution in water was added to the reaction mixture until
it turned acidic as indicated by pH paper. Then the or-
ganic layer was extracted with another small portion
of 10% HCl, then the aqueous layer was twice back ex-
tracted with CH2Cl2. The organic layer was further ex-
tracted with brine, dried using magnesium sulfate,
filtered, and then concentrated under reduced pressure.
The resulting aldehyde was either purified using flash
column chromatography or used unpurified.
4.2.8. General procedure H: bromotrimethylsilane medi-
ated deprotection of phosphate and phosphonate esters.
To a stirring solution of the ester to be deprotected is
added bromotrimethylsilane (10 equiv). The reaction
mixture is then allowed to stir for about 4 h and pro-
gress is monitored by TLC; after completion of the reac-
tion the solvent is removed under reduced pressure and
then the resulting oil is solvated with 95% methanol 5%
water and the reaction mixture is allowed to stir over-
night. The following day the solvent is removed under
reduced pressure and the appropriate purification tech-
nique is employed.
4.2.9. General procedure I: mitsunobu coupling. To a
solution of the alcohol (2 equiv), the coupling phenol
or acid (1 equiv), diisopropylethyl amine (2 equiv), and
polymer bound triphenylphine (2 equiv) in CH2Cl2 at
0 ꢀC was added diisopropylazodicarboxylate (2 equiv).
The reaction mixture was then allowed to slowly warm
to room temperature and to stir overnight; the following
day the reaction mixture is diluted with methanol and
filtered through a pad of Celite using methanol as the
eluent. The organic layer is then concentrated under re-
duced pressure and the product is purified using flash
column chromatography.
4.2.5. General procedure E: mesylate coupling. To a stir-
ring solution of phenol (1 equiv), potassium carbonate
(4 equiv), and appropriate mesylate (or alkyl halide)
(1.2 equiv) in acetone was added a catalytic amount of
18-crown-6 and the reaction mixture was heated to re-
flux. The reaction was maintained at this temperature
overnight and the next morning allowed too cool to
room temperature. After sufficient cooling the reaction
mixture was concentrated under reduced pressure and
then the resulting mixture was solvated with ethyl ace-
tate and extracted with ammonium chloride, then the
aqueous layer was further extracted with ethyl acetate,
the organic layer was washed with brine, dried with so-
dium sulfate, filtered, and then concentrated under re-
duced pressure. The product was then purified using
flash column chromatography.
4.3. Synthesis of compounds 2–25, 47, and 48
4.3.1. [(Diethoxy-phosphoryl)-fluoro-methyl]-phosphonic
acid diethyl ester (2). To a solution of sodium hydride
(700 mg, 60%, 17.5 mmol) in THF at 0 ꢀC was added
a solution of (diethoxy-phosphorylmethyl)-phosphonic
acid diethyl ester (5 g, 17.3 mmol) in THF. The reaction
mixture was allowed to stir at this temperature for 1 h
before solid Selectfluor (6.146 g, 17.3 mmol) was added
to the mixture. The reaction mixture was stirred at room
temperature overnight and then the solid was filtered off
through a pad of Celite using methanol as the eluent,
concentrated under reduced pressure, and purified using
flash column chromatography (4% MeOH:EtOAc,
stained with Henessian stain) to yield the monofluori-
nated product in 3.0 g (56%). 1H NMR (300 MHz,
CDCl3) d 1.37 (t, 12 H, J = 7.31 Hz), 4.28 (p, 8H,
J = 8.05 Hz), 5.50 (m, 1H).
4.2.6. General procedure F: synthesis of b-keto phospho-
nates. To a solution of diethylmethylphosphonate
(1 equiv) in THF at À78 ꢀC was added n-BuLi
(1.1 equiv, 2.5 M) dropwise via syringe. The reaction
mixture was allowed to stir at this temperature for 1 h
before a solution of the ester (1.05 equiv) in THF was
added. The reaction mixture was stirred for an addi-
tional hour before a saturated aqueous solution of
ammonium chloride was added and the reaction mixture
was allowed to slowly warm to room temperature. Then
the reaction mixture was extracted with ethyl acetate,
followed by brine, dried over sodium sulfate and con-
centrated under reduced pressure. The product was then
purified using flash column chromatography.
4.3.2. Methanesulfonic acid 4-methoxy-3,5-dimethyl-pyri-
din-2-ylmethyl ester (4). To a stirring solution of (4-
methoxy-3,5-dimethyl-pyridin-2-yl)-methanol (500 mg,
3.0 mmol) and triethylamine (0.63 ml, 4.52 mmol) in
CH2Cl2 at 0 ꢀC was slowly added methane sulfonylchlo-
ride (0.28 ml, 3.62 mmol) via syringe. The reaction mix-
ture was slowly warmed to room temperature and
stirred for an additional 4 h at which time the reaction
was stopped. It was stopped prematurely and some
4.2.7. General procedure G: sodium borohydride reduction
of ketones. NaBH4 (2 equiv) is dissolved in ethanol so
that a solution of milky appearance is formed. This solu-
tion is cooled to 0 ꢀC and the ketone to be reduced is dis-
solved in THF, so that a 2:1 ethanol/THF mixture will
be the resulting solution ratio. Then the ester in THF

2220
P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
starting material was retained. The solvent was removed
under reduced pressure and then the resulting deep red
oil was placed directly onto a flash column and purified
via flash column chromatography (1:1 EtOAc/hexanes)
to give 660 mg product (90%). 1H NMR (300 MHz,
CDCl3) 2.15 (s, 3H), 2.24 (s, 3H), 3.67 (s, 3H), 4.57 (s,
2H), 8.10 (s, 1H); 13C NMR d 11.11, 13.54, 46.02,
60.12, 126.04, 126.79, 149.60, 154.73, 164.53.
hexanes) to afford the product in 600 mg (70%) yield.
1H NMR (300 MHz, CDCl3) d 1.33 (m, 6H); 1.46 (m,
9H); 2.04 (s, 1H), 2.78 (m, 2H), 4.07 (m, 4H), 4.88 (m,
1H), 5.02 (s, 2H), 5.15 (m, 1H), 5.95 (m, 1H), 6.92 (d,
2H, J = 8.45 Hz), 7.14 (d, 2H, J = 8.46 Hz), 7.44 (m,
5H).
4.3.7. [4-(4-Benzyloxy-phenyl)-1-fluoro-3-hexadecanoyla-
mino-but-1-enyl]-phosphonic acid diethyl ester (11). A
solution of 9 (600 mg, 1.18 mmol) in dichloromethane
and trifluoroacetic acid (2:1) was stirred for several
hours and progress was monitored by TLC. After
the reaction had proceeded to completion the solvent
was removed under reduced pressure and the remain-
ing TFA was removed after repeated azeotropic distil-
lations with both diethyl ether and methanol. The
product was prepared from palmitoyl chloride accord-
ing to the general procedure A. The crude products
were collected without purification in a yield of
860 mg.
4.3.3. 3-(4-Benzyloxy-phenyl)-2-tert-butoxycarbonylami-
no-propionic acid methyl ester (6). To a solution of N-
Boc O-Bnz L-tyrosine (2.5 g, 6.74 mmol) in anhydrous
methanol was added trimethylsilyl diazomethane (2 M
in ether, 5.1 ml, 10.1 mmol) dropwise to the stirring
mixture at 0 ꢀC until no more nitrogen gas was
evolved and a bright yellow color persisted (about
20 min). The mixture was then concentrated under re-
duced pressure and purified by flash column chroma-
tography (20% EtOAc:hexanes) to yield the crude
product 2.8 g. 1H NMR (300 MHz, CDCl3) d 1.43
(s, 9H), 3.03 (m, 2H), 3.69 (s, 3H), 4.57 (m, 1H),
5.02 (s, 2H), 5.08 (m, 1H), 6.91 (d, 2H, J = 8.42 Hz),
7.05 (d, 2H, J = 8.42 Hz), 7.31–7.44 (m, 5H). 13C
NMR d 28.58, 37.72, 52.41, 54.86, 70.22, 115.20,
127.75, 128.23, 128.63, 128.85, 130.61, 137.31,
158.17, 172.70.
4.3.8. [1-Fluoro-3-hexadecanoylamino-4-(4-hydroxy-phe-
nyl)-butyl]-phosphonic acid diethyl ester (12). This com-
pound was synthesized according to the general
procedure B from 11 (860 mg) in a yield of 570 mg
1
(85%, three steps). H NMR (300 MHz, CDCl3) d 0.84
(t, 3H, J = 5.12 Hz), 1.21 (s, 24H), 1.30 (m, 6H), 1.52
(m, 2H), 2.09 (t, 3H, J = 7.31 Hz), 2.72 (m, 2H),
3.61(s, 1H), 4.14 (m, 4H), 4.39 (b, 1H), 4.82 (m, 1H),
6.11 (t, 1H, J = 8.79 Hz), 6.72 (d, 2H, J = 8.05 Hz),
6.95 (d, 2H, J = 7.32 Hz). 13C NMR d 14.32, 16.52,
16.60, 16.65, 22.89, 25.90, 25.99, 29.47, 29.49, 29.57,
29.73, 29.86, 29.89, 29.91, 32.12, 36.99, 37.01, 63.54,
64.02, 70.71, 115.73, 127.25, 128.00, 128.09, 130.48,
130.50, 156.02, 173.87, 174.13.
4.3.4. [1-(4-Benzyloxy-benzyl)-2-hydroxy-ethyl]-carbamic
acid tert-butyl ester (7). This compound was synthesized
according to general procedure C from 6 (2.8 g,
7.27 mmol) to yield the resulting alcohol in 2.26 g
(94%). 1H NMR (300 MHz, CDCl3) d 1.42 (s, 9H),
2.78 (d, 2H, J = 7.32 Hz), 3.60 (m, 2H), 3.82 (b, 1H),
5.04 (s, 2H), 6.92 (d, 2H, J = 8.78 Hz), 7.13 (d, 2H,
J = 8.42 Hz), 7.31–7.45 (m, 5H).
4.3.5. [1-(4-Benzyloxy-benzyl)-2-oxo-ethyl]-carbamic acid
tert-butyl ester (8). This compound was synthesized
according to general procedure D from 7 (2.26 g,
8.79 mmol) to yield the resulting aldehyde in 1.87 g
(83%).1H NMR (300 MHz, CDCl3) d 1.45 (s, 9H),
3.06 (d, 2H, J = 6.58 Hz), 4.38 (d, 1H, J = 6.59 Hz),
5.04 (s, 2H), 5.11 (m, 1H), 6.92 (d, 2H, J = 8.42 Hz),
7.09 (d, 2H, J = 8.42 Hz), 7.31–7.44 (m, 5H), 9.62 (s,
1H). 13C NMR d 28.54, 34.78, 61.14, 70.26, 80.37,
115.34, 127.72, 128.25, 128.85, 128.91, 130.63, 137.18,
158.14, 199.91.
4.3.9. {1-Fluoro-3-hexadecanoylamino-4-[4-(4-methoxy-
3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-phos-
phonic acid dimethyl ester (13). This compound was
synthesized according to the general procedure E from
12 (1.098 g, 1.97 mmol) and 4 (531 mg, 2.17 mmol) in
a yield of 1.151 g (83%). The product was then purified
by using flash column chromatography (5%
1
MeOH:CH2Cl2). H NMR (300 MHz, CDCl3) d 0.84
(t, 3H, J = 6.5 Hz), 1.21 (m, 26H), 1.29 (m, 8H), 1.52
(p, 2H, J = 6.5 Hz), 2.08 (m, 3H), 2.23 (s, 3H), 2.29 (s,
3H), 2.77 (m, 2H), 3.76 (s, 3H), 4.135 (q, 4H,
J = 8.1 Hz), 4.36 (m, 1H), 5.09 (s, 2H), 5.69–5.76 (m,
1H), 6.92 (d, 2H, J = 8,8 Hz), 7.06 (d, 2H, J = 8.5 Hz),
8.20 (s, 1H).
4.3.6. [4-(4-Benzyloxy-phenyl)-3-tert-butoxycarbonylami-
no-1-fluoro-but-1-enyl]-phosphonic acid diethyl ester (9).
To a solution of 2 (500 mg, 1.63 mol) in THF at À78 ꢀC
was added a solution of n-butyl lithium (0.67 ml, 2.5 M
in THF). The reaction mixture was allowed to stir for
1 h and then a solution of 8 (775 mg, 2.18 mmol) in
THF was added and the reaction mixture was allowed
to slowly warm to room temperature. The reaction mix-
ture was allowed to stir overnight and the next day the
reaction was quenched with ammonium chloride and
then the aqueous layer was extracted with EtOAc, then
the organic layer was extracted with brine, the organic
was then dried with sodium sulfate, filtered, and then
concentrated under reduced pressure. The product was
purified via flash column chromatography (1:1 EtOAc/
4.3.10. {1-Fluoro-3-hexadecanoylamino-4-[4-(4-methoxy-
3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-phos-
phonic acid (14). This product was formed by using gen-
eral procedure H from 13 (70 mg, 0.099 mmol) to afford
41 mg yield (63%). 1H NMR (300 MHz, CD3OD) d 0.90
(t, J = 6.8 Hz, 3H); 1.27 (m, 26H); 1.52 (m, 2H); 1.53 (m,
2H); 2.15 (m, 4H); 2.39 (s, 3H); 2.46 (s, 3H); 2.76 (m,
2H); 4.10 (s, 3H); 4.30 (m, 1H); 5.35 (s, 2h); 7.05 (d,
J = 8.4 Hz, 2H); 7.23 (d, J = 7.9 Hz, 2H); 8.42 (s, 1H);
elemental analysis CHN calcd
%
C = 64.59%,
H = 8.67%, N = 4.30%; found C = 62.51%, H = 8.61%,
N = 4.22%.

P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
2221
4.3.11.
[1-(4-Benzyloxy-benzyl)-3-diazo-2-oxo-propyl]-
115.14, 127.78, 128.26, 128.90, 130.53, 130.63, 137.39,
157.10, 157.77.
carbamic acid tert-butyl ester (16). To N-Boc, O-benzyl
L-tyrosine acid (4 g, 10.8 mmol) in THF at 0 ꢀC in a
erlenmeyer flask free of internal scratches is added iso-
butyl chloroformate (2.206 g, 16.2 mmol). The reaction
mixture is allowed to stir for 30 min before a solution
of ethereal diazomethane (approximately 2.3 g,
54 mmol) is added via canula. Reaction mixture is al-
lowed to slowly warm to room temperature and then al-
lowed to stir overnight. The following day the reaction
mixture was then stirred open to air in order to allow
for unreacted diazomethane to dissipate. After a few
hours the reaction mixture is extracted (sodium bicar-
bonate, then brine), dried (sodium sulfate), and then
concentrated under reduced pressure. The reaction mix-
ture is purified via flash chromatography (50% EtOAc in
4.3.14. [1-(4-Benzyloxy-benzyl)-3-oxo-propyl]-carbamic
acid tert-butyl ester (19). This compound was prepared
from 18 (1.41 g, 3.81 mmol) by using general proce-
dure D. The product was purified by flash column
chromatography (20% EtOAc in hexanes) in a yield
of 1.32 g (94%). 1H NMR (300 MHz, CDCl3) d
1.42 (s, 9H), 2.55 (m, 2H), 2.77 (m, 2H), 4.22 (b,
1H), 4.85 (d, 1H, J = 8.06 Hz), 5.03 (s, 2H), 5.06
(b, 1H), 6.94 (d, 2H, J = 8.60 Hz), 7.11 (d, 2H,
J = 8.43 Hz), 7.32–7.46 (m, 5H), 9.68 (s, 1H); 13C
NMR d 28.63, 40.15, 47.78, 48.05, 70.27, 79.86,
115.28, 127.78, 128.26, 128.87, 130.02, 130.63,
137.31, 157.98, 201.44.
1
hexanes) to yield the product (2.16 g, 51%). H NMR
(300 MHz, CDCl3) d 1.42 (s, 9H), 2.96 (d, 2H,
J = 6.77 Hz), 4.36 (b, 1H), 5.05 (s, 2H), 5.16 (b, 1H),
5.23 (b, 1H), 6.91 (d, 2H, J = 8.71 Hz), 7.10 (d, 2H,
J = 8.71 Hz), 7.30–7.44 (m, 5H); 13C NMR d 28.58,
37.98, 54.73, 58.90, 70.27, 115.28, 127.78, 128.82,
128.87, 130.69, 137.23, 158.09.
4.3.15. [4-(4-Benzyloxy-phenyl)-3-tert-butoxycarbonyla-
mino-1-hydroxy-butyl]-phosphonic acid dimethyl ester
(20). To a solution of 60% NaH in mineral oil
(215 mg, 5.38 mmol) in THF at 0 ꢀC was added di-
methyl phosphite (0.5 ml, 5.38 mmol) dropwise via syr-
inge. The resulting mixture was allowed to stir at this
temperature for 1 h before a solution of aldehyde 19
(1.32 g, 3.58 mmol) was added. The reaction mixture is
then allowed to stir for 15 min before it was quenched
with saturated ammonium chloride in water. The organ-
ic layer is then extracted with EtOAc, followed by brine,
dried with sodium sulfate, and then concentrated under
reduced pressure. The product was purified by flash col-
umn chromatography (EtOAc) in a yield of 1.02 g
(60%). 1H NMR (300 MHz, CDCl3) d 1.37 (s, 9H),
1.66 (m, 1H), 1.89 (m, 1H), 2.75 (d, 2H, J = 6.41 Hz),
3.75 (m, 6H), 4.05 (m, 2H), 4.80 (d, 1H, J = 8.97 Hz),
4.99 (s, 2H), 6.88 (d, 2H, J = 7.33 Hz), 7.07 (d, 2H,
J = 7.33 Hz), 7.25–7.40 (m, 5H); 13C NMR d 28.55,
37.02, 40.47, 48.08, 48.29, 53.61, 63.28, 65.52, 70.22,
80.26, 115.01, 115.20, 127.70, 128.18, 128.82, 130.07,
130.50, 130.80, 157.40, 157.82.
4.3.12. 4-(4-Benzyloxy-phenyl)-3-tert-butoxycarbonyla-
mino-butyric acid methyl ester (17). To a solution of
the a-diazoketone 16 (2.16 g, 5.47 mmol), TEA (2.3 ml,
16.4 mmol) in 25 ml anhydrous methanol, and a small
amount of THF (added to help solubilize the diazoke-
tone) in a flask wrapped with tin foil, to protect reaction
from light, and cooled to À78 ꢀC was added a catalytic
amount of silver benzoate. Silver benzoate was dissolved
in a small amount of TEA before it is added to reaction
mixture. Reaction mixture was allowed to stir at À78 ꢀC
for several hours before a second portion of silver ben-
zoate (catalytic amount, dissolved in TEA) was added.
Then reaction mixture was allowed to slowly warm to
room temperature and then allowed to stir overnight.
The next day the reaction mixture is filtered through sil-
ica gel by using EtOAc as eluent to remove any silver
salts that are left over from the reaction. The mixture
is then concentrated under reduced pressure and purified
via flash column chromatography (20% EtOAc in hex-
anes) to yield the product (1.63 g, 73%). 1H NMR
(300 MHz, CDCl3) d 1.42 (s, 9H), 2.47 (m, 2H), 2.75
(m, 2H), 3.66 (s, 3H), 4.12 (b, 1H), 5.02 (s, 2H), 5.11
(b, 1H), 6.90 (d, 2H, J = 8.61 Hz), 7.10 (d, 2H,
J = 8.42 Hz), 7.30–7.44 (m, 5H); 13C NMR d 28.66,
37.88, 39.80, 49.25, 51.92, 70.22, 79.59, 115.14, 127.75,
128.20, 128.85, 130.34, 130.66, 137.39, 155.45, 157.85,
172.41.
4.3.16. [4-(4-Benzyloxy-phenyl)-3-hexadecanoylamino-1-
hydroxy-butyl]-phosphonic acid dimethyl ester (21a and
b). This compound was prepared from 20. The N-Boc
protecting group was removed with the same method
in preparation of 11 and then the TFA salt was reacted
with palmitoyl chloride according to the general proce-
dure A. The products were purified by column chroma-
tography (2% MeOH in CH2Cl2) in a yield of 1.1 g (82%
two steps). Two diastereomers were separated in the ra-
tio of 1:4.
1
Compound 21a: H NMR (300 MHz, CDCl3) d 0.87 (t,
4.3.13. [1-(4-Benzyloxy-benzyl)-3-hydroxy-propyl]-car-
bamic acid tert-butyl ester (18). This product was synthe-
sized according to general procedure C using 17 as the
starting material (1.63 g, 3.95 mmol). The product was
purified by flash column chromatography (EtOAc) and
the yield was 1.41 g (97%). 1H NMR (300 MHz, CDCl3)
d 1.44 (s, 9H), 1.81 (b, 2H), 2.75 (d, 2H, J = 4.95 Hz),
3.65 (b, 2H), 4.02 (b, 1H), 4.78 (d, 1H, J = 8.98 Hz),
5.03 (s, 2H), 5.06 (b, 1H), 6.92 (d, 2H, J = 8.72 Hz),
7.11 (d, 2H, J = 8.73 Hz), 7.30–7.46 (m, 5H); 13C
NMR d 28.69, 37.85, 40.81, 48.69, 59.19, 70.30, 79.94,
3H, J = 5.85 Hz), 1.25 (s, 24H), 1.48 (p, 2H,
J = 6.96 Hz), 1.69 (m, 1H), 2.00 (m, 1H), 2.12 (dt, 2H,
J = 2.56, 7.30 Hz), 2.79 (dq, 2H, J = 5.49, 15.37 Hz),
3.81 (dd, 6H, J = 2.92, 10.61 Hz), 3.92 (m, 1H), 4.33
(m, 1H), 5.01 (s, 2H), 6.18 (d, 1H, J = 8.79 Hz), 6.89
(d, 2H, J = 8.05 Hz), 7.08 (d, 2H, J = 8.42 Hz), 7.25–
7.41 (m, 5H); 13C NMR d 14.40, 22.95, 26.07, 29.41,
29.62, 29.76, 29.97, 32.19, 36.75, 37.50, 40.23, 47.09,
47.28, 53.71, 53.79, 63.20, 65.47, 70.25, 115.25, 127.67,
128.20, 128.82, 129.81, 129.94, 130.31, 137.23, 157.93,
165.76, 175.72.

2222
P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
1
Compound 21b: H NMR (300 MHz, CDCl3) d 0.87 (t,
3H, J = 6.22 Hz), 1.23 (s, 24H), 1.51 (p, 2H,
J = 6.95 Hz), 1.85 (m, 1H), 1.99 (m, 1H), 2.12 (t, 2H,
J = 8.42 Hz), 2.80 (d, 2H, J = 6.59 Hz), 3.74 (t, 6H,
J = 9.88 Hz), 4.04 (m, 1H), 4.25 (m, 1H), 5.00 (s, 2H),
6.18 (d, 1H, J = 7.69 Hz), 6.88 (d, 2H, J = 8.42 Hz),
7.09 (d, 2H, J = 8.79 Hz), 7.25–7.42 (m, 5H); 13C
NMR d 14.40, 22.95, 25.94, 29.57, 29.62, 29.68, 29.81,
29.92, 29.97, 32.19, 35.90, 37.13, 40.17, 49.49, 49.71,
53.45, 53.53, 53.82, 65.47, 67.66, 70.25, 115.06, 127.70,
128.20, 128.82, 130.25, 130.66, 157.82, 173.96.
1H), 5.11 (s, 2H), 5.99 (d, 1H, J = 7.7 Hz), 6.93 (d,
2H, J = 8.8 Hz), 7.07 (d, 2H, J = 8.8 Hz), 8.23 (s, 1H).
4.3.19. {3-Hexadecanoylamino-1-hydroxy-4-[4-(4-meth-
oxy-3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-phos-
phonic acid (24a and b). The two diastereomers were
synthesized according to general procedure H from
23a (56 mg, 0.0825 mmol) and 23b (86 mg, 0.127 mmol)
in the yields of 43 mg (80%) and 70 mg (86%).
Compound 24a: 1H NMR (300 MHz, CD3OD) d 0.89 (t,
3H, J = 6.8 Hz, 3H), 1.26 (m, 26H), 1.51 (m, 2H), 1.83
(m, 2H), 2.13 (t, 2H, J = 6.6 Hz), 2.35 (s, 3H), 2.39 (s,
3H), 2.75 (d, 2H, J = 6.6 Hz), 3.38 (m, 1H), 3.99 (s,
2H), 4.32 (m, 1H), 4.94 (s, 2H), 5.25 (m, 1H), 6.99 (d,
2H, J = 9.2 Hz), 7.19 (d, 2H, J = 8.2 Hz), 8.35 (s, 1H);
13C NMR (300 MHz, CD3OD) d 14.03, 14.46, 23.75,
27.20, 30.32, 30.48, 30.61, 30.83, 33.10, 37.29, 41.67,
93.43, 98.51, 115.92, 131.68, 133.47, 157.96, 170.14; ele-
mental analysis CHN calcd % C = 64.79%, H = 8.86%,
N = 4.32%; found C = 64.06%, H = 9.14%, N = 4.56%.
4.3.17. [3-Hexadecanoylamino-1-hydroxy-4-(4-hydroxy-
phenyl)-butyl]-phosphonic acid dimethyl ester (22a and
b). The two diastereomers were synthesized according to
general procedure B from 21a (290 mg, 0.46 mmol) and
21 b (190 mg, 3.01 mmol) in the yields of 95 mg (58%)
and 180 mg (73%).
1
Compound 22a: H NMR (300 MHz, CDCl3) d 0.86 (t,
3H, J = 6.59 Hz), 1.23 (s, 24H), 1.50 (p, 2H,
J = 5.85 Hz), 1.68 (m, 1H), 1.95 (m, 1H), 2.13 (t, 2H,
J = 8.05 Hz), 2.74 (m, 2H), 3.79 (dd, 6H, J = 4.76,
10.24 Hz), 3.89 (m, 1H), 4.34 (m, 1H), 6.14 (d, 1H,
J = 8.78 Hz), 6.73 (d, 2H, J = 8.42 Hz), 6.96 (d, 2H,
J = 8.42 Hz); 13C NMR d 14.34, 22.90, 26.02, 29.44,
29.54, 29.58, 29.72, 29.89, 29.94, 32.14, 36.74, 36.87,
40.14, 53.67, 53.74, 53.96, 54.06, 115.88, 128.08,
130.31, 155.94, 175.84.
Compound 24b: 1H NMR (300 MHz, CD3OD) d 0.89 (t,
3H, J = 6.5 Hz), 1.27 (m, 26H), 1.46 (p, 2H, J = 7.3 Hz),
1.84 (m, 1H), 1.99 (m, 1H), 2.06 (t, 2H, J = 6.9 Hz), 2.36
(s, 3H), 2.41 (s, 3H), 2.60 (m, 1H), 2.90 (m, 1H), 3.82 (m,
1H), 4.01 (s, 2H), 4.30 (m, 1H), 5.27 (s, 2H), 6.68 (d, 1H,
J = 8.1 Hz), 7.01 (m, 2H), 7.20 (d, 2H, J = 7.0 Hz), 8.37
(s, 1H); 13C NMR (300 MHz, CD3OD) d 9.95, 12.88,
13.27, 22.57, 25.89, 29.04, 29.07, 29.31, 29.34, 29.41,
29.45, 29.64, 31.90, 36.15, 36.67, 38.41, 60.38, 66.73,
95.54, 114.65, 114.79, 130.22, 130.54, 132.42, 174.37;
1
Compound 22b: H NMR (300 MHz, CDCl3) d 0.86 (t,
3H, J = 6.95 Hz), 1.23 (s, 24H), 1.51 (p, 2H,
J = 6.90 Hz), 1.82 (m, 1H), 1.99 (m, 1H), 2.12 (t, 2H,
J = 7.68 Hz), 2.72 (d, 2H, J = 5.49 Hz), 3.73 (t, 6H,
J = 9.88 Hz), 4.00 (t, 1H, J = 7.69 Hz), 4.24 (m, 1H),
6.38 (m, 1H), 6.74 (d, 2H, J = 8.41 Hz), 6.95 (d, 2H,
J = 8.42 Hz); 13C NMR d 14.34, 22.92, 25.92, 29.54,
29.59, 29.79, 29.95, 32.14, 37.04, 53.49, 53.58, 53.76,
54.06, 115.78, 128.43, 130.58, 155.80, 174.36.
elemental analysis CHN calcd
H = 8.86%, N = 4.32%; found C = 62.91%, H = 8.94%,
N = 4.03%.
%
C = 64.79%,
4.3.20. {2-Fluoro-3-hexadecanoylamino-4-[4-(4-methoxy-
3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-phos-
phonic acid (25a). To a solution of 49a (30 mg,
0.04 mmol) in CH2Cl2 at À78 ꢀC was added via cannula
a pre-cooled solution of DAST (0.015 ml, 0.11 mmol)
CH2Cl2. The reaction mixture was allowed to warm to
room temperature and after 1 h, a saturated solution
of sodium bicarbonate was added and the mixture was
diluted with EtOAc. The mixture was extracted with
EtOAc and combined organic layers were washed with
brine and dried over magnesium sulfate and concen-
trated under vacuum. The residue was purified by col-
umn chromatography (4% MeOH:CH2Cl2) to give the
dimethyl ester product in 14 mg (47%). 1H NMR
(300 MHz, CDCl3) d 0.87 (t, 3H, J = 7.57 Hz), 1.25 (s,
24H), 1.61 (p, 2H, J = 7.10 Hz), 2.05 (m, 2H), 2.26 (s,
3H), 2.28 (t, 2H, J = 7.48 Hz), 2.32 (s, 3H), 2.75 (dq,
2H, J = 8.32, 15.05 Hz), 3.75 (m, 6H), 3.77 (s, 3H),
4.38 (q, 1H, J = 7.48 Hz), 4.87 (dq, 1H, J = 3.49,
10.48 Hz), 5.12 (s, 2H), 6.95 (d, 2H, J = 8.73 Hz), 7.12
(d, 2H, J = 8.73 Hz), 8.23 (s, 1H). MS (ESI) m/z 679.6
[M+H]+. Compound 25a was prepared according to
4.3.18. {3-Hexadecanoylamino-1-hydroxy-4-[4-(4-meth-
oxy-3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-
phosphonic acid dimethyl ester (23a and b). The two
diastereomers were synthesized according to general
procedure I from 22a (250 mg, 0.474 mmol) and 22b
(106 mg, 0.201 mmol) in the yields of 216 mg (67%)
and 86 mg (63%).
1
Compound 23a: H NMR (300 MHz, CDCl3) d 0.87 (t,
3H, J = 7.0 Hz), 1.24 (m, 26H); 1.49 (p, 2H, J = 7.0 Hz),
1.68 (m, 1H), 2.02 (m, 1H), 2.12 (m, 2H), 2.27 (s, 3H),
2.32 (s, 3H), 2.79 (dq, 2H, J = 7.9, 14.9 Hz), 3.78 (s,
3H), 3.81 (dd, 6H, J = 4.9, 10.3 Hz), 3.87 (m, 1H),
4.36 (m, 1H), 5.12 (s, 2H), 5.72 (d, 1H, J = 8.6 Hz),
6.95 (d, 2H, J = 8.8 Hz), 7.07 (d, 2H, J = 8.8 Hz), 8.23
(s, 1H).
1
1
Compound 23b: H NMR (300 MHz, CDCl3) d 0.86 (t,
the general procedure H to give 8 mg (62%). H NMR
3H, J = 6.9 Hz), 1.23 (m, 26H), 1.53 (m, 2H), 1.83 (m,
1H), 2.03 (m, 1H), 2.09 (t, 2H, J = 8.5 Hz), 2.26 (s,
3H), 2.31 (s, 3H), 2.79 (d, 2H, J = 6.2 Hz), 3.75 (m,
9H), 4.01 (m, 1H), 4.12 (q, 1H, J = 6.5 Hz), 4.21 (m,
(300 MHz, CD OD) d 0.89 (t, 3H, J = 6.35 Hz), 1.28
3
(s, 24H), 1.64 (p, 2H, J = 6.80 Hz), 2.19 (dd, 2H,
J = 7.26, 19.05 Hz), 2.40 (s, 3H), 2.45 (t, 2H,
J = 7.71 Hz), 2.50 (s, 3H), 2.63 (dd, 1H, J = 9.53,

P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
2223
14.06 Hz), 3.19 (dd, 1H, J = 5.44, 14.97 Hz), 3.68 (m,
4.4. Synthesis of compounds 26–46
6H), 3.91 (m, 1H), 4.16 (s, 3H), 5.45 (s, 2H), 7.23 (d,
2H, J = 8.62 Hz), 7.38 (d, 2H, J = 8.62 Hz), 8.50 (s,
1H). MS (ESI) m/z 651.2 [M+H]+.
These compounds were synthesized from appropriate
amino acids by using General procedures A, E, F, G,
and H.24
4.3.21. {2-Fluoro-3-hexadecanoylamino-4-[4-(4-methoxy-
3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-phos-
phonic acid (25b). It was prepared from 49b with the
same method as in the preparation of 25a with same
4.4.1. (3-Hexadecanoylamino-2-oxo-propyl)-phosphonic acid
(26). 1H NMR (300 MHz, CD3OD) d 0.90 (t, 3H,
J = 7.03 Hz), 1.28 (s, 24H), 1.62 (q, 2H, J = 7.03 Hz),
2.25 (t, 2H, J = 7.03 Hz), 3.15 (m, 2H), 4.20 (s, 2H).
MS (ESI) m/z 392.5 [M+H]+.
1
reaction yield. H NMR (300 MHz, CD3OD) d 0.89 (t,
3H, J = 6.54 Hz), 1.28 (s, 24H), 1.67 (p, 2H,
J = 6.94 Hz), 2.15 (m, 2H), 2.40 (s, 3H), 2.47 (t, 2H,
J = 6.54 Hz), 2.50 (s, 3H), 2.95 (m, 1H), 3.94 (m, 1H),
4.16 (s, 3H), 5.39 (m, 1H), 5.45 (s, 2H), 7.10 (m, 1H),
7.29 (m, 4H), 8.51 (s, 1H). MS (ESI) m/z 651.8 [M+H]+.
4.4.2. (3-Hexadecanoylamino-2-hydroxy-propyl)-phosphonic
1
acid (27). H NMR (300 MHz, CD3OD) d 0.90 (t, 3H,
J = 6.93 Hz), 1.28 (s, 24H), 1.62 (q, 2H, J = 6.93 Hz),
1.90 (m, 2H), 2.22 (t, 2H, J = 6.94 Hz), 3.22 (dd, 1H,
J = 6.63, 14.47 Hz), 3.38 (dd, 1H, J = 3.38, 14.32 Hz),
4.03 (m, 1H). MS (ESI) m/z 394.3 [M+H]+.
4.3.22. {3-Hexadecanoyl-4-[4-(4-methoxy-3,5-dimethyl-
pyridin-2-ylmethoxy)-benzyl]-2,2-dimethyl-oxazolidin-5-
ylmethyl}-phosphonic acid dimethyl ester (47). 2-Meth-
oxypropene (0.06 ml, 0.63 mmol) was added to a
solution of 49a (80 mg, 0.12 mmol) in CH2Cl2 at 0 ꢀC,
then CSA (5.5 mg, 0.02 mmol) was added and the
mixture was allowed to warm to room temperature
overnight. The next day the reaction mixture was diluted
with CH2Cl2 and washed with saturated sodium bicar-
bonate solution then dried over magnesium sulfate and
concentrated under vacuum. The residue was purified
by preparative TLC (4% MeOH:CH2Cl2) to give
4.4.3. (3-Hexadecanoylamino-2-oxo-butyl)-phosphonic acid
(28). 1H NMR (300 MHz, CD3OD) d 0.90 (t, 3H,
J = 6.93 Hz), 1.28 (s, 24H), 1.33(d, 3H, J = 7.42 Hz),
1.61 (q, 2H, J = 7.28 Hz), 2.23 (t, 2H, J = 7.43
Hz), 3.20 (m,2H), 4.50 (m, 1H). MS (ESI) m/z 406.5
[M+H]+.
4.4.4. (3-Hexadecanoylamino-2-hydroxy-butyl)-phosphonic
1
acid (29). H NMR (300 MHz, CD3OD) d 0.90 (t, 3H,
1
27 mg product (32%). H NMR (300 MHz, CDCl3) d
J = 6.96 Hz), 1.14 (d, 3H, J = 6.50 Hz), 1.29 (s, 24H),
1.61 (q, 2H, J = 6.86 Hz), 1.91 (m, 2H), 2.19 (t, 2H,
J = 6.97 Hz), 3.70 (m, 1H), 3.91(m, 2H). MS (ESI) m/z
408.4 [M+H]+.
0.86 (t, 3H, J = 6.06 Hz), 1.24 (bs, 24H), 1.60 (m, 2H),
1.63 (s, 3H), 1.66 (s, 3H), 2.00 (m, 2H), 2.25 (s, 3H),
2.30 (s, 3H), 2.85 (m, 2H), 3.50 (m, 6H), 3.77 (s, 3H),
4.13 (dd, 1H, J = 5.30, 9.60 Hz), 4.33 (m, 1H), 5.12 (s,
2H), 6.98 (d, 2H, J = 8.61 Hz), 7.11 (d, 2H,
J = 8.61 Hz), 8.24 (s, 1H). 13C NMR d 11.14, 13.62,
14.37, 22.95, 25.43, 28.13, 28.58, 29.62, 29.70, 29.81,
29.94, 31.25, 32.16, 33.07, 35.85, 40.68, 52.46, 52.54,
60.15, 64.61, 64.66, 70.99, 74.89, 115.41, 126.76,
129.57, 130.58, 149.33, 154.33, 154.38, 158.01, 170.59.
4.4.5.
(3-Hexadecanoylamino-4-methyl-2-oxo-pentyl)-
1
phosphonic acid (30). H NMR (300 MHz, CD3OD) d
0.90 (m, 9H), 1.28 (s, 24H), 1.61 (m, 2H), 2.13 (m,
1H), 2.26 (t, 2H, J = 7.52 Hz), 4.31 (m, 1H), 8.21 (b,
1H). MS (ESI) m/z 434.6 [M+H]+.
4.4.6. (3-Hexadecanoylamino-2-hydroxy-4-methyl-pentyl)-
1
4.3.23. {3-Hexadecanoyl-4-[4-(4-methoxy-3,5-dimethyl-
pyridin-2-ylmethoxy)-benzyl]-2,2-dimethyl-oxazolidin-5-
ylmethyl}-phosphonic acid dimethyl ester (48). 2-Meth-
oxypropene (0.06 ml, 0.63 mmol) was added to a
solution of 49b (80 mg, 0.12 mmol) in CH2Cl2 at 0 ꢀC,
then CSA (5.5 mg, 0.02 mmol) was added and the
mixture was allowed to warm to room temperature
overnight. The next day the reaction mixture was diluted
with CH2Cl2 and washed with saturated sodium bicar-
bonate solution then dried over magnesium sulfate and
concentrated under vacuum. The residue was purified
by preparative TLC (4% MeOH:CH2Cl2) to give
phosphonic acid (31). H NMR (300 MHz, CD3OD) d
0.93 (m, 9H), 1.29 (s, 24H), 1.63 (p, 2H, J = 7.09 Hz),
1.92 (m, 2H), 2.28 (m, 1H), 2.40 (t, 2H, J = 7.10 Hz),
3.58 (m, 2H). MS (ESI) m/z 436.1 [M+H]+.
4.4.7. (3-Hexadecanoylamino-2-hydroxy-4-methyl-pentyl)-
1
phosphonic acid (32). H NMR (300 MHz, CD3OD) d
0.89 (m, 9H), 1.28 (s, 24H), 1.62 (p, 2H, J = 6.62 Hz),
1.95 (m, 2H), 2.28 (m, 1H), 2.25 (t, 2H, J = 7.66 Hz),
3.67 (m, 2H), 3.78 (m, 1H). MS (ESI) m/z 436.1
[M+H]+.
1
25 mg product (30%). H NMR (300 MHz, CDCl3) d
4.4.8. (3-Hexadecanoylamino-5-methyl-2-oxo-hexyl)-phos-
phonic acid (33). H NMR (300 MHz, CD3OD) d 0.94
(m, 9H), 1.28 (s, 24H), 1.64 (m, 5H), 2.25 (t, 2H,
J = 6.73 Hz), 3.16 (m, 2H), 4.59 (m, 1H). MS (ESI)
m/z 448.4 [M+H]+.
1
0.87 (t, 3H, J = 6.72 Hz), 1.24 (br s, 24H), 1.60 (s,
3H), 1.65 (m, 2H), 1.77 (s, 3H), 2.10 (m, 2H), 2.26 (s,
3H), 2.30 (s, 3H), 2.65 (m, 1H), 2.95 (dd, 1H, J = 5.60,
4.18 Hz), 3.77 (s, 3H), 3.78 (m, 6H), 4.06 (m, 1H), 4.60
(m, 1H), 5.11 (s, 2H), 6.95 (d, 2H, J = 8.21 Hz), 7.07
(d, 2H, J = 8.21 Hz), 8.25 (s, 1H). 13C NMR d 11.17,
13.65, 14.40, 22.95, 24.25, 25.16, 27.22, 27.65, 29.49,
29.62, 29.73, 29.78, 29.97, 32.19, 35.34, 35.63, 52.19,
52.75, 53.21, 61.38, 61.49, 70.97, 72.17, 101.76, 115.54,
129.89, 130.74, 149.38, 154.35, 158.04, 171.31.
4.4.9. (3-Hexadecanoylamino-2-hydroxy-5-methyl-hexyl)-
1
phosphonic acid (34). H NMR (300 MHz, CD3OD) d
0.92 (m, 9H), 1.28 (s, 24H), 1.62 (m, 5H), 1.90 (m,
2H), 2.25 (t, 2H, J = 7.27 Hz), 3.72 (m, 1H), 3.99 (m,
1H). MS (ESI) m/z 450.4 [M+H]+.

2224
P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
4.4.10. (3-Hexadecanoylamino-2-hydroxy-5-methyl-hex-
yl)-phosphonic acid (35). H NMR (300 MHz, CD3OD)
d 0.92 (m, 9H), 1.28 (s, 24H), 1.43 (m, 2H), 1.61 (m,
3H), 1.91 (m, 2H), 2.22 (t, 2H, J = 7.41 Hz), 3.72 (m,
1H), 3.84 (m, 1H), 3.95 (b, 1H). MS (ESI) m/z 450.2
[M+H]+.
(br s, 26H), 2.03 (t, 2H, J = 6.76 Hz), 3.73 (d, 2H,
J = 9.85 Hz), 4.02 (m, 1H), 4.27 (m, 1H), 5.30 (s, 2H),
7.01–7.07 (m, 3H), 7.09 (s, 1H), 7.20–7.33 (m, 5H),
7.62 (d, 1H, J = 7.88 Hz). MS (ESI) m/z 613.3 [M+H]+.
1
4.4.19. {3-Hexadecanoylamino-2-hydroxy-4-[1-(4-meth-
oxy-3,5-dimethyl-pyridin-2-ylmethyl)-1H-indol-3-yl]-
butyl}-phosphonic acid (44). 1H NMR (300 MHz,
CD3OD) d 0.90 (t, 3H, J = 7.40 Hz), 1.28 (s, 26H),
1.65 (m, 2H), 2.01 (t, 2H, J = 7.93 Hz), 2.40 (s, 3H),
2.46 (s, 3H), 3.24 (m, 2H), 3.34 (s, 3H), 3.37 (m, 2H),
4.15 (s, 2H), 5.73 (d, 1H, J = 7.17 Hz), 7.10 (s, 1H),
7.08–7.18 (m, 2H), 7.24 (d, 1H, J = 6.86 Hz), 7.70 (d,
1H, J = 7.17 Hz), 8.36 (s, 1H). MS (ESI) m/z 672.3
[M+H]+.
4.4.11. (3-Hexadecanoylamino-2-hydroxy-4-phenyl-butyl)-
1
phosphonic acid (36). H NMR (300 MHz, CD3OD) d
0.90 (t, 3H, J = 7.05 Hz), 1.28 (s, 24H), 1.46 (p, 2H,
J = 7.05 Hz), 1.92 (m, 2H), 2.13 (t, 2H, J = 7.40 Hz),
2.80 (m, 1H), 2.93 (m, 1H), 4.07(m, 1H), 4.16 (m, 1H),
7.24 (m, 5H). MS (ESI) m/z 484.4 [M+H]+.
4.4.12. (3-Hexadecanoylamino-2-hydroxy-4-phenyl-butyl)-
1
phosphonic acid (37). H NMR (300 MHz, CD3OD) d
0.90 (t, 3H, J = 6.74 Hz), 1.29 (s, 24H), 1.37 (p, 2H,
J = 6.37 Hz), 1.90 (m, 2H), 2.04 (t, 2H, J = 7.11 Hz),
2.60 (m, 1H), 3.15 (m, 1H), 3.95 (m, 1H), 4.12 (m,
1H), 7.22 (m, 5H). MS (ESI) m/z 484.5 [M+H]+.
4.4.20. (4-Benzyloxy-3-hexadecanoylamino-2-hydroxy-
butyl)-phosphonic acid (45). 1H NMR (300 MHz,
CD3OD) d 0.89 (t, 3H, J = 7.22 Hz), 1.27 (s, 24H),
1.61 (q, 2H, J = 7.23 Hz), 1.88–1.96 (m, 2H), 2.26 (t,
2H, J = 7.22 Hz), 3.60 (m, 2H), 4.16 (m, 1H), 4.25 (m,
1H), 4.53 (s, 2H), 7.25–7.35 (m, 5H). MS (ESI) m/z
514.3 [M+H]+.
4.4.13. [2-(1-Hexadecanoyl-pyrrolidin-2-yl)-2-oxo-ethyl]-
1
phosphonic acid (38). H NMR (300 MHz, CD3OD) d
0.90 (t, 3H, J = 7.16 Hz), 1.29 (s, 24H), 1.59 (q, 2H,
J = 7.16 Hz), 2.00 (m, 2H), 2.21 (m, 2H), 2.36 (t, 2H,
J = 7.47 Hz), 3.12 (m, 1H), 3.21 (m, 1H), 3.62 (m, 2H),
4.66 (m, 1H). MS (ESI) m/z 432.4 [M+H]+.
4.4.21. (4-Benzyloxy-3-hexadecanoylamino-2-hydroxy-
butyl)-phosphonic acid (46). 1H NMR (300 MHz,
CD3OD) d 0.89 (t, 3H, J = 6.86 Hz), 1.27 (s, 24H),
1.59 (q, 2H, J = 6.86 Hz), 1.89–2.00 (m, 2H), 2.22 (t,
2H, J = 6.86 Hz), 3.86 (m, 2H), 4.07 (m, 1H), 4.52 (s,
2H), 7.27–7.35 (m, 5H). MS (ESI) m/z 514.2 [M+H]+.
4.4.14. [2-(1-Hexadecanoyl-pyrrolidin-2-yl)-2-hydroxy-
ethyl]-phosphonic acid (39). 1H NMR (300 MHz,
CD3OD) d 0.90 (t, 3H, J = 7.51 Hz), 1.24 (s, 24H),
1.61 (q, 2H, J = 7.00 Hz), 1.92 (m, 2H), 2.05 (m, 4H),
2.38 (t, 2H, J = 7.51 Hz), 3.59 (m, 2H), 4.10 (m, 1H),
4.25 (m,1H). MS (ESI) m/z 434.7 [M+H]+.
Acknowledgment
This work was supported by a Grant from the NIH
(R01 GM052722).
4.4.15. [3-Hexadecanoylamino-4-(1H-indol-3-yl)-2-oxo-
butyl]-phosphonic acid (40). 1H NMR (300 MHz,
CD3OD) d 0.90 (t, 3H, J = 6.98 Hz), 1.28 (s, 24H),
1.45 (q, 2H, J = 6.98 Hz), 2.13 (t, 2H, J = 7.29 Hz),
3.10 (m, 2H), 3.17 (m, 2H), 7.05 (m, 2H), 7.08 (s, 1H),
7.31 (d, 1H, J = 8.25 Hz), 7.58 (d, 1H, J = 8.25 Hz).
MS (ESI) m/z 521.6 [M+H]+.
References and notes
1. Bollen, M.; Gijsbers, R.; Ceulemans, H.; Stalmans, W.;
Stefan, C. Crit. Rev. Biochem. Mol. Biol. 2000, 35, 393–
432.
2. Goding, J. J. Leukoc. Biol. 2000, 67, 285–311.
3. Stracke, M.; Krutzsch, H.; Unsworth, E.; Arestad, A.;
Cioce, V.; Schiffmann, E.; Liotta, L. J. Biol. Chem. 1992,
267, 2524–2529.
4. Lee, H.; Murata, J.; Clair, T.; Polymeropoulos, M.;
Torres, R.; Manrow, R.; Liotta, L.; Stracke, M. Biochem.
Biophys. Res. Commun. 1996, 218, 714–719.
5. Narita, M.; Goji, J.; Nakamura, H.; Sano, K. J. Biol.
Chem. 1994, 269, 28235–28242.
4.4.16. [3-Hexadecanoylamino-2-hydroxy-4-(1H-indol-3-
yl)-butyl]-phosphonic acid (41). 1H NMR (300 MHz,
CD3OD) d 0.89 (t, 3H, J = 7.09 Hz), 1.27 (s, 24H),
1.46 (m, 2H), 1.93 (m, 2H), 2.16 (t, 2H, J = 7.00 Hz),
3.06 (m, 2H), 4.15 (m, 1H), 4.25 (m, 1H), 7.03 (m,
2H), 7.09 (s, 1H), 7.30 (d, 1H, J = 8.42 Hz), 7.62 (d,
1H, J = 8.42 Hz), 8.25 (s, 1H). MS (ESI) m/z 523.9
[M+H]+.
6. Kawagoe, H.; Soma, O.; Goji, J.; Nishimura, N.; Narita,
M.; Iinzawa, J.; Nakamura, H.; Sano, K. Genomics 1995,
30, 380–384.
7. Fuss, B.; Baba, H.; Phan, T.; Tuohy, V.; Macklin, W.
J. Neurosci. 1997, 17, 9095–9103.
8. Stefan, C.; Gijsbers, R.; Stalmans, W.; Bollen, M.
Biochim. Biophys. Acta 1999, 1450, 45–52.
9. Yang, S.; Lee, J.; Park, C.; Kim, S.; Hong, S.; Chung, H.;
Min, S.; Han, J.; Lee, H.; Lee, H. Clin. Exp. Metastasis
2002, 19, 603–608.
4.4.17. [3-Hexadecanoylamino-2-hydroxy-4-(1H-indol-3-
yl)-butyl]-phosphonic acid (42). 1H NMR (300 MHz,
CD3OD) d 0.89 (t, 3H, J = 7.38 Hz), 1.28 (br s, 26H),
2.11 (m, 2H), 2.14 (t, 2H, J = 7.98 Hz), 2.93 (m, 1H),
3.21 (m, 1H), 4.09 (m, 1H), 4.37 (m, 1H), 7.01 (m,
2H), 7.08 (s, 1H), 7.30 (d, 1H, J = 8.60 Hz), 7.61 (d,
1H, J = 8.60 Hz). MS (ESI) m/z 523.9 [M+H]+.
4.4.18. [4-(1-Benzyl-1H-indol-3-yl)-3-hexadecanoylami-
no-2-hydroxy-butyl]-phosphonic acid (43). 1H NMR
(300 MHz, CD3OD) d 0.89 (t, 3H, J = 6.51 Hz), 1.27
10. Kishi, Y.; Okudaira, S.; Tanaka, M.; Hama, K.; Dai, S.;
Kitayama, J.; Yamori, T.; Aoki, J.; Fujimaki, T.; Arai, H.
J. Biol. Chem. 2006, 281, 17492–17500.

P. Cui et al. / Bioorg. Med. Chem. 16 (2008) 2212–2225
2225
11. Nam, S.; Clair, T.; Kim, Y.; McMarlin, A.; Schiffmann,
E.; Liotta, L.; Stracke, M. Cancer Res. 2001, 61, 6938–
6944.
12. Kehlen, A.; Englert, N.; Seifert, A.; Klonisch, T.; Dralle,
H.; Langner, J.; Hoang-Vu, C. Int. J. Cancer 2004, 109,
833–838.
26. Lee, C.; Rivera, R.; Gardell, S.; Dubin, A.; Chun, J.
J. Biol. Chem. 2003, 281, 23589–23597.
27. McIntyre, T.; Pontsler, A.; Silva, A.; St. Hilaire, A.; Xu,
Y.; Hinshaw, J.; Zimmerman, G.; Hama, K.; Aoki, J.;
Arai, H.; Prestwich, G. Proc. Natl. Acad. Sci. U.S.A. 2003,
100, 131–136.
13. Ferry, G.; Tellier, E.; Try, A.; Gres, S.; Naime, I.; Simon,
M.; Rodriguez, M.; Boucher, J.; Tack, I.; Gesta, S.;
Chomarat, P.; Dieu, M.; Raes, M.; Galizzi, J.; Valet, P.;
Boutin, J.; Saulnier-Blache, J. J. Biol. Chem. 2003, 278,
18162–18169.
14. Song, J.; Clair, T.; Noh, J.; Eun, J.; Ryu, S.; Lee, S.; Ahn,
Y.; Kim, S.; Lee, S.; Park, W.; Yoo, N.; Lee, J.; Nam, S.
Biochem. Biophys. Res. Commun. 2005, 337, 967–975.
15. Umezu-Goto, M.; Kishi, Y.; Taira, A.; Hama, K.;
Dohmae, N.; Takio, K.; Yamori, T.; Mills, G.; Inoue,
K.; Aoki, J.; Arai, H. J. Cell Biol. 2002, 158, 227–233.
16. Tokumura, A.; Majima, E.; Kariya, Y.; Tominaga, K.;
Kogure, K.; Yasuda, K.; Fukuzawa, K. J. Biol. Chem.
2002, 277, 39436–39442.
17. Siess, W.; Zangl, K.; Essler, M.; Bauer, M.; Brandl, R.;
Corrinth, C.; Bittman, R.; Tigyi, G.; Aepfelbacher, M.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6931–6936.
18. Zhang, C.; Baker, D.; Yasuda, S.; Makarova, N.; Balazs,
L.; Johnson, L.; Marathe, G.; McIntyre, T.; Xu, Y.;
Prestwich, G.; Byun, H.; Bittman, R.; Tigyi, G. J. Exp.
Med. 2004, 199, 763–774.
28. Durgam, G.; Virag, T.; Walker, D.; Tsukahara, R.;
Yasuda, S.; Liliom, K.; van Meeteren, A.; Moolenaar,
H.; Wilke, N.; Siess, W.; Tigyi, G.; Miller, D. J. Med.
Chem. 2005, 48, 4919–4930.
29. Durgam, G.; Tsukahara, R.; Makarova, N.; Walker, D.;
Fujiwara, Y.; Pigg, R.; Baker, L.; Sardar, M.; Parrill, L.;
Tigyi, G.; Miller, D. Bioorg. Med. Chem. Lett. 2006, 16,
633–640.
30. Gududuru, V.; Zeng, K.; Tsukahara, R.; Makarova, N.;
Fujiwara, Y.; Pigg, K.; Baker, D.; Tigyi, G.; Miller, D.
Bioorg. Med. Chem. Lett. 2006, 16, 451–456.
31. Baker, L.; Fujiwara, Y.; Pigg, R.; Tsukahara, R.; Kobay-
ashi, S.; Murofushi, H.; Uchiyama, A.; Murakami-Mur-
ofushi, K.; Koh, E.; Bandle, W.; Byun, H.; Bittman, R.;
Fan, D.; Murph, M.; Mills, B.; Tigyi, G. J. Biol. Chem.
2006, 281, 22786–22793.
32. Uchiyama, A.; Mukai, M.; Fujiwara, Y.; Kobayashi, S.;
Kawai, N.; Murofushi, H.; Inoue, M.; Enoki, S.; Tanaka,
Y.; Niki, T.; Kobayashi, T.; Tigyi, G.; Murakami-Mur-
ofushi, K. Biochim. Biophys. Acta Mol. Cell Biol. Lipids
2007, 1771, 103–112.
19. Sugiura, T.; Nakane, S.; Kishimoto, S.; Waku, K.;
Yoshioka, Y.; Tokumura, A. J. Lipid Res. 2002, 43,
2049–2055.
33. Cui, P.; Tomsig, J.; McCalmont, W.; Lee, S.; Becker, C.;
Lynch, K.; Macdonald, T. Bioorg. Med. Chem. Lett. 2007,
17, 1634–1640.
20. Strum, A.; Sudermann, T.; Schulte, K.; Goebell, H.;
Dignass, A. Gastroenterology 1999, 117, 368–377.
21. Sturm, A.; Dignass, A. Biochim. Biophys. Acta 2002, 1582,
282–288.
22. Deng, W.; Balazs, L.; Wang, D.; Van Middlesworth, L.;
Tigyi, G.; Johnson, L. Gastroenterology 2002, 123, 206–216.
23. Contos, J.; Ishii, I.; Chun, J. Mol. Pharm. 2000, 58, 1188–
1196.
34. Nieschalk, J.; Batsanov, A.; O’Hagan, D.; Howard, J.
Tetrahedron 1992, 52, 165–176.
35. Adams, P.; Harrison, R. Biochem. J. 1974, 141, 729–732.
36. Xu, Y.; Qian, L.; Prestwich, G. Org. Lett. 2003, 5, 2267–
2270.
37. Chakravarty, P.; Combs, P.; Roth, A.; Greenlee, W.
Tetrahedron Lett. 1987, 28, 611–612.
38. Karplus, M. J. Am. Chem. Soc. 1963, 85, 2870–2871.
39. Futagawa, S.; Inui, T.; Shiba, T. Bull. Chem. Soc. Jpn.
1973, 46, 3308–3310.
24. Noguchi, K.; Ishii, S.; Shimizu, T. J. Biol. Chem. 2003,
278, 25600–25606.
25. Lee, C.; Rivera, R.; Dubin, A.; Chun, J. J. Biol. Chem.
2007, 282, 4310–4317.
40. Dufour, M.; Jouin, P.; Poncet, J.; Pantaloni, A.; Castro,
B. J. Chem. Soc., Perkin Trans. 1986, 1, 1895–1899.