Design, synthesis and pharmacological activity of novel enantiomerically pure phosphonic acid-based NAALADase inhibitors

Article abstract of DOI:10.1039/b615603g

Inhibitors of NAALADase have shown promise for a variety of diseases associated with glutamate excitotoxicity, and could be useful for the diagnosis and therapy of prostate cancer. A series of novel enantiomerically pure 2-(phosphonomethyl)pentanedioic ac

Full text of DOI:10.1039/b615603g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Design, synthesis and pharmacological activity of novel enantiomerically pure
phosphonic acid-based NAALADase inhibitors
Pingyu Ding, Paul Helquist and Marvin J. Miller*
Received 27th October 2006, Accepted 13th December 2006
First published as an Advance Article on the web 19th January 2007
DOI: 10.1039/b615603g
Inhibitors of NAALADase have shown promise for a variety of diseases associated with glutamate
excitotoxicity, and could be useful for the diagnosis and therapy of prostate cancer. A series of novel
enantiomerically pure 2-(phosphonomethyl)pentanedioic acid (2-PMPA) based NAALADase
inhibitors were synthesized. These compounds were prepared from previously reported
(S)-2-(hydroxyphosphinoylmethyl)pentanedioic acid benzyl ester 4. Biological test results showed that
the new compounds are good to outstanding NAALADase inhibitors. Compounds 8b and 10b showed
activity similar to the known potent inhibitor (S)-2-PMPA. Fluorescently labeled inhibitor 19b may
potentially be used to study binding to prostate cancer cells by fluorescence microscopy, and
siderophore-containing inhibitor 21b may be useful for detection of prostate-derived cancer cells by
magnetic resonance imaging (MRI).
potent and selective inhibitor was 2-(phosphonomethyl)-
Introduction
pentanedioic acid (2-PMPA) reported by Jackson et al. in 1996.6
The metalloprotease glutamate carboxypeptidase II (GCP II,
Since then, extensive structure–activity relationship studies have
also known as N-acetylated alpha-linked acidic dipeptidase,
been carried out using 2-PMPA as a template, and a few
potent inhibitors of GCP II, such as urea-based compounds7
and GPI52328 were identified. Berkman’s group reported the
synthesis of phenylalkylphosphonamidates as prostate-specific
membrane antigen (PSMA) inhibitors.9 Further studies on 2-
PMPA have demonstrated that potent inhibition of glutamate
carboxypeptidase II (GCP II) is specific to (S)-2-PMPA, which has
an absolute configuration corresponding to L-glutamate.10 Recent
studies on GPI5232 also showed that the (S)-enantiomer is 40-
fold more potent than the (R)-enantiomer in a GCP II inhibitory
assay.11 Therefore, it is essential to synthesize the enantiomerically
pure inhibitors prior to further preclinical characterization in
order to eliminate the potential for undesired pharmacological
effects or other interferences by the inactive enantiomers.
NAALADase) releases N-acetyl aspartate and glutamate from
both the neuronal peptide N-acetylaspartylglutamate (NAAG)
and folate polyglutamate (Scheme 1).1 Inhibitors of NAALADase
have shown efficacy in a variety of animal models of neurological
disease associated with glutamate excitotoxicity.2
Scheme 1
NAALADase is also expressed in the human prostate
parenchyma, from where it was first cloned and named prostate
specific membrane antigen (PSMA).3 It has been shown that
inhibitors of NAALADase also strongly bind to PSMA. As a
representative member of the phosphinate-based series of binders,
VA-033 was found to serve as a potent inhibitor of NAALADase
activity associated with PSMA that is expressed on LNCaP human
prostate cancer cells and by tumor cells in vivo.4a These inhibitors
themselves do not affect the viability of the LNCaP cells, nor do
they significantly alter cell cycle kinetics. They therefore provide
a biologically innocuous scaffold that can be used to target
diagnostic markers or chemotherapeutic drugs to the surface
membrane of prostate tumor cells. Recent studies have shown that
the NAALADase inhibitors may also have more direct therapeutic
potential.4b
Fig. 1 Inhibitors of NAALADase.
Based on these known results, we became interested in the syn-
thesis of enantiomerically pure phosphonates having the general
structure 1 and possessing the (S)-configuration. First, the phos-
phonate group of 1 was intended to both mimic the tetrahedral
intermediate formed during hydrolysis of NAAG and inhibit
catalytic activity of the enzyme by binding the essential zinc in the
active site. Second, the ability to derivatize the inhibitor at position
Over the past decade, tremendous efforts have been made in the
development of potent inhibitors of GCP II (Fig. 1).5 The first
Walther Cancer Research Center and Department of Chemistry & Biochem-
istry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame,
Indiana 46556, USA. E-mail: mmiller1@nd.edu; Fax: +1 574 631 6652;
Tel: +1 574 631 7571
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R by linking a suitably labeled head group was anticipated to allow
detection of prostate derived cells in circulation by fluorescence or
by magnetic resonance imaging (MRI) whilst not impairing the
ability of the inhibitor to access the active site.12 In this paper, we
describe the synthesis of a series of novel enantiomerically pure
2-PMPA based derivatives 1 and their inhibitory potencies in a
GCP II assay.
By modification of the above three-step approach for the
preparation of benzyl ester 7, the four benzyl esters 9a–12a
(Fig. 2) were obtained in the indicated overall yields from the
corresponding commercially available phenalkanols.
Results and discussion
Our first synthesis of a form of 1 focused on preparation of
8a (Scheme 2). The synthesis of TBS protected alcohol 3 has
been previously accomplished by selective deprotection of the
corresponding bis(TBS) derivative with I2 in methanol.13 We used
the route shown in Scheme 2 to obtain this product in excellent
yield. The starting material 2 was readily prepared from ethyl 4-
hydroxyphenylacetate.14 The coupling reaction between 3 and op-
tically pure (S)-2-(hydroxyphosphinoylmethyl)pentanedioic acid
dibenzyl ester 45 afforded phosphinate 5 smoothly. Product 5 was
then subjected to oxidation by NaIO4 to provide phosphonic acid
6. Treatment of 6 with BnBr–K2CO3 in DMF gave benzyl ester
7 in 85% yield, while the esterification reaction employing ben-
zylisourea or using BOP as an activating agent did not give good
yields.15 Desilylation using HF in CH3CN16 gave phenol 8a cleanly.
Fig. 2 Benzyl esters 9a–12a.
Several additional analogs were obtained by elaboration of
10a (Scheme 3). Thus, Boc deprotection of 10a gave aniline 13
as a crude oil that was used directly in the next step without
purification. Acetylation of 13 provided 14a. Boc-Gly-OH, Boc-
Gly-Pro-OH and Boc-(Gly)3-OH were also coupled to aniline 13
to give compounds 15a–17a in excellent yields.
Scheme 3
Removal of the benzyl groups from each of the protected
products gave the corresponding potential inhibitors 8b, 9b, 10b,
11b, 12b, 14b, 15b, 16b, and 17b (Table 1). 31P NMR analysis
of each final acid revealed one sharp singlet, supporting the
formation of a single diastereomer.
Scheme 2
Table 1 Inhibition of GCP II by the proposed inhibitors generated by debenzylation
Compound
R
IC50/nM
SEM
na
8b
CH2CH2(4-OH-C6H4)
CH2CH2(4-F-C6H4)
CH2CH2(4-BocNH-C6H4)
CH2CH2C6H5
CH2CH2CH2C6H5
CH2CH2(4-AcNH-C6H4)
CH2CH2(4-Boc-Gly-NH-C6H4)
CH2CH2(4-Boc-Gly-Pro-NH-C6H4)
CH2CH2[4-Boc-(Gly)3-NH-C6H4]
0.2
300
0.1
0.8
4
4
0.7
7
8
2
5
0.4
0.05
0
0.01
0.05
2
4
2
4
2
4
1
2
1
1
4
4
4
9b
10b
11b
12b
14b
15b
0
16b
17b
19b
0.8
2
0.09
21b
(S)-2-PMPA
a n = the number of times the assay was performed.
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displayed activity with IC50 values of 2 nM and 5 nM, respectively,
indicating that potentially diagnostically useful labeling groups
are compatible with inhibitor design.
Conclusions
Based on the known potent NAALADase inhibitor (S)-2-
(phosphonomethyl)pentanedioic acid (2-PMPA), we synthesized
a series of eleven enantiomerically pure forms of phosphonate
1. Most of the final products showed good to outstanding
NAALADase inhibitory activity. Our studies clearly demonstrate
that the bulky, phenyl-derived linker group is tolerated in the newly
prepared NAALADase inhibitors. These results should facilitate
future development of other NAALADase and PSMA inhibitors.
Additional future studies will determine the feasibility of using
fluorescently labeled inhibitor 19b to study prostate cancer cells
by fluorescence microscopy. Siderophore-containing inhibitor 21b
will be studied as iron and gadolinium complexes for the detection
of prostate cancer cells by MRI. These and other chemical agents
are being developed for the early detection and treatment of
prostate cancer.
Scheme 4
As shown in Scheme 4, fluorescent-labeled compound 19b
was prepared in two steps. Refluxing the mixture of free
amine 13 and commercially available 7-(diethylamino)coumarin-
3-carbonyl-azide 18 in benzene followed by column chromato-
graphic purification in the presence of Et3N provided the fully
protected intermediate 19a in good yield.17 To avoid overreduction,
debenzylation was performed by using 1,4-cyclohexadiene in the
presence of 10% Pd–C catalyst in MeOH–EtOH (v/v 3 : 1) to give
the desired product (19b). As will be subsequently reported, this
fluorescently labeled compound will be used to study binding to
prostate cancer cells by fluorescence microscopy.
Siderophore-containing moiety 21b was readily obtained in two
steps as shown in Scheme 5. The coupling reaction between aniline
13 and tripeptide acid 2018 gave the fully protected precursor 21a.
Upon removal of the benzyl protecting groups under standard
catalytic hydrogenolysis conditions, conjugate 21b was obtained
in quantitative yield. The iron and gadolinium complexes of this
compound will be studied for the detection of prostate cancer cells
by MRI.
Experimental
All reactions were carried out under argon by using standard
techniques. Solvents were dried under nitrogen by standard proce-
dures, distilled before use and stored under argon. NMR spectra
were recorded on a Varian Unityplus 300 MHz spectrometer
or Varian Inova 500 MHz spectrometer. Optical rotations were
recorded on a Perkin Elmer model 343 polarimeter. Mass spectra
were recorded on a Jeol JMS-AX505 HA Double Sector mass
spectrometer.
2-(4-tert-Butyldimethylsilyloxyphenyl)ethanol (3)
To a suspension of LiAlH4 (1.08 g, 28.46 mmol) in THF (70 mL)
was added a solution of 214 (4.00 g, 13.58 mmol) in THF (15 mL)
portionwise at 0 ◦C, and the resulting mixture was stirred at that
temperature for 1.5 h. Then H2O (1.1 mL) was added slowly,
followed by 1.1 mL of 15% NaOH solution and 1.65 mL of
◦
Scheme 5
H2O. After stirring for another 30 min at 0 C, the mixture was
filtered through a Celite pad to remove the resulting white solid.
The solid was washed with THF. The combined organic layers were
concentrated to provide the crude product which was purified by
flash column chromatography on silica gel (hexanes–EtOAc 3 : 1
→ 2 : 1) to afford 3.18 g of the product as a colorless oil (93%). 1H
NMR (500 MHz, CDCl3) d: 7.08 (d, J = 8.5 Hz, 2H), 6.79 (d, J =
8.5 Hz, 2H), 3.82 (t, J = 6.5 Hz, 2H), 2.81 (t, J = 6.5 Hz, 2H), 1.27
(brs, 1H), 0.99 (s, 9H), 0.19 (s, 6H); 13C NMR (125 MHz, CDCl3)
d: 154.46, 131.11, 130.13, 120.36, 64.03, 38.57, 25.89, 18.39, −4.22.
The in vitro GCP II inhibitory potencies of these obtained
PSMA inhibitors were measured at Guilford Pharmaceuticals
Inc. using N-acetyl-L-aspartyl-[3H]-L-glutamate as a substrate
and human recombinant GCP II19 as previously reported.20 The
assay results are shown in Table 1 along with the assay reference
compound (S)-2-PMPA. Eight concentrations of each inhibitor in
the 0.1 pM−1 lM range were evaluated for each IC50 determination.
Xcelfit software was used to calculate the IC50 values. We plotted
log of concentration vs. % inhibition. The fluoro compound 9b
showed low activity, whereas compounds 8b and 10b demonstrated
the best activity with IC50 values of 0.2 nM and 0.1 nM,
respectively. Compared to compound 11b, compound 12b with
the increased number of methylene units was five-fold less active.
Acetyl derivative 14b possessed good activity with an IC50 value of
4 nM. Again, compared to compound 15b, compounds 16b and
17b, with the increased side chain length of the amide attached to
the phenyl group, had ten-fold decreased activity. Fluorescently
labeled inhibitor 19b and siderophore-containing inhibitor 21b
General procedure for the synthesis of benzyl esters (7, 9a, 10a,
11a, and 12a) from the corresponding phenalkanols
(S)-2-[(4-tert-Butyldimethylsilyloxyphenyl)ethoxy]-phosphinoyl-
methyl-pentanedioic acid dibenzyl ester (5). To a solution of 3
(200 mg, 0.792 mmol) and (S)-(−)-2-[hydroxyphosphinoylmethyl]-
pentanedioic acid dibenzyl ester 45 (309 mg, 0.792 mmol) in THF
(9 mL) were added DCC (172 mg, 0.833 mmol) and DMAP
(12 mg, 0.095 mmol). The reaction mixture was stirred overnight,
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and the resulting white solid (DCU) was filtered and washed with
THF (7 mL). The filtrate was concentrated and purified by flash
column chromatography on silica gel (hexanes–EtOAc 3 : 1 →
General procedure for the synthesis of final acids (8b, 9b, 10b, 11b,
12b, 14b, 15b, 16b, 17b and 21b)
(S)-2-[[(4-Hydroxyphenyl)ethoxy]phosphonoyl]methyl-pentane-
dioic acid (8b). To a solution of 8a (25 mg, 0.031 mmol) in MeOH
(5 mL) was added 10% Pd–C (30 mg). The reaction mixture was
stirred under an atmosphere of H2 for 3 h. The resulting mixture
was filtered through a pad of Celite and washed with methanol.
The combined solution was evaporated to afford 10.1 mg of the
product as a colorless oil (95%. [a]2D0 = +4.4◦ (c = 0.75, MeOH);
1H NMR (500 MHz, CD3OD) d: 6.98 (2H, d, J = 8.5 Hz), 6.62
(2H, d, J = 8.5 Hz), 3.98 (2H, q, J = 7.0 Hz), 2.76 (2H, q, J =
7.0 Hz), 2.63–2.58 (1H, m), 2.30–2.18 (2H, m), 2.08–2.00 (1H,
m), 1.90–1.65 (3H, m); 13C NMR (125 MHz, CD3OD) d: 177.93,
176.65, 157.26, 131.24, 129.89, 116.37, 67.41, 40.78, 37.33, 32.37,
29.76, 28.62; 31P NMR (121 MHz, CDCl3) d: 26.05; HRFABMS
calcd. for C14H20O8P (M + H)+ 347.0896, found 347.0913.
1
1 : 1) to afford 346 mg of 5 as a colorless oil (70%). H NMR
(500 MHz, CDCl3) d: 7.38–7.35 (m, 10H), 7.08 (dd, J = 553,
10 Hz, 1H), 7.07–7.04 (m, 2H), 6.80–6.77 (m, 2H), 5.14 (s, 2H),
5.12 (s, 2H), 4.30–4.05 (m, 2H), 3.01–2.80 (m, 3H), 2.43–1.80 (m,
6H), 0.99 (s, 9H), 0.20 (s, 6H); HRFABMS calcd. for C34H46O7PSi
(M + H)+ 625.2750, found 625.2731.
(S)-2-[[(4-tert-Butyldimethylsilyloxyphenyl)ethoxy]-phospho-
noyl]methyl-pentanedioic acid dibenzyl ester (6). Compound 5
(327 mg, 0.534 mmol) was dissolved in dioxane–water (5 : 1, 6 mL),
and NaIO4 (134 mg, 0.628 mmol) was added. The reaction mixture
was stirred at room temperature for 20 h, and then diluted with
EtOAc (150 mL) and water (20 mL). The layers were separated,
and the organic layer was washed with saturated aqueous KHSO4
(25 mL), 0.3 M Na2S2O3 (25 mL), H2O (20 mL) and brine (25 mL).
After being dried, filtered, and concentrated, 335 mg of the product
was obtained as a semi-solid (100%). 1H NMR (500 MHz, CDCl3)
d: 7.36–7.22 (m, 10H), 7.02 (d, J = 8.0 Hz, 1H), 6.67 (d, J =
8.0 Hz), 5.09–4.98 (m, 4H), 3.95–3.85 (m, 2H), 2.80–2.70 (m, 3H),
2.32–2.22 (m, 2H), 2.03–1.80 (m, 4H), 1.65–1.55 (m, 1H), 0.93 (s,
9H), 0.11 (s, 6H); 13C NMR (125 MHz, CDCl3) d: 176.96 (d, J =
6.2 Hz), 174.65, 155.56, 137.72, 137.66, 132.95, 131.18, 129.65,
129.42, 129.31, 129.22, 121.06, 67.74, 67.46, 66.16, 41.90, 37.76,
32.75, 29.71, 29.58, 26.34, 19.15, −4.08; HRFABMS calcd. for
C34H45KO8PSi (M + K)+ 679.2253, found 679.2258.
(S) - 2 - [[(4 - Fluorophenyl)ethoxy]phosphonoyl]methyl - pentane -
dioic acid tribenzyl ester (9a). Yield: 60%. 1H NMR (500 MHz,
CDCl3) d: 7.42–7.31 (15H, m), 7.13–7.09 (2H, m), 6.98–6.95 (2H,
m), 5.17–4.95 (6H, m), 4.20–4.02 (2H, m), 2.85 (2H, q, J = 7.0 Hz),
2.83–2.78 (1H, m), 2.40–2.24 (3H, m), 2.04–1.96 (2H, m), 1.93–
1.77 (1H, m); 31P NMR (121 MHz, CDCl3) d: 29.78; HRFABMS
calcd. for C35H37FO7P (M + H)+ 619.2261, found 619.2272.
(S) - 2 - [[(4 - Fluorophenyl)ethoxy]phosphonoyl]methyl - pentane-
dioic acid (9b). Yield: 93%. [a]2D0 = +20.4◦ (c = 0.5, MeOH); 1H
NMR (500 MHz, CD3OD) d: 7.10–7.04 (2H, m), 6.83–6.77 (2H,
m), 3.96 (2H, q, J = 6.5 Hz), 2.75 (2H, t, J = 6.5 Hz), 2.52–2.43
(1H, m), 2.23–2.05 (2H, m), 2.02–1.93 (1H, m), 1.80–1.58 (3H,
m); 31P NMR (121 MHz, CDCl3) d: 27.23; HRFABMS calcd. for
C14H19FO7P (M + H)+ 349.0852, found 349.0834.
(S)-2-[[(4-tert-Butyldimethylsilyloxyphenyl)ethoxy]-phospho-
noyl]methyl-pentanedioic acid tribenzyl ester (7). Compound 6
(70 mg, 0.109 mmol) was dissolved in DMF (4 mL), and K2CO3
(90 mg, 0.655 mmol) and benzyl bromide (78 lL, 0.665 mmol) were
added. The resulting mixture was stirred at room temperature for
10 h, diluted with CH2Cl2, and washed with cold water and brine.
After being dried, filtered, and concentrated, the residual DMF
was evaporated under high vacuum. The residue was purified by
flash column chromatography eluting with hexanes–ethyl acetate
(2 : 1 → 1 : 1 → 1 : 2) to afford 64 mg of product as a colorless oil
(85%). 1H NMR (500 MHz, CDCl3) d: 7.42–7.25 (15H, m), 7.06–
6.97 (2H, m), 6.80–6.72 (2H, m), 5.18–4.90 (6H, m), 4.17–4.06
(2H, m), 2.84–2.80 (3H, m), 2.35–2.24 (3H, m), 2.07–1.80 (3H,
m), 1.00 (9H, s), 0.20 (6H, s); HRFABMS calcd. for C41H52O8PSi
(M + H)+ 731.3169, found 731.3138.
(S)-2-[[(4-N-tert-Butoxylcarbonylphenyl)ethoxy]-phosphonoyl]-
1
methyl-pentanedioic acid tribenzyl ester (10a). Yield: 71%. H
NMR (500 MHz, CDCl3) d: 7.36–7.24 (17H, m), 7.12–7.02 (2H,
m), 6.52 (1H, NH, brs), 5.10–4.92 (6H, m), 4.20–4.01 (2H, m),
2.90–2.73 (3H, m), 2.37–2.19 (3H, m), 2.02–1.72 (3H, m), 1.54
(9H, s); 31P NMR (121 MHz, CDCl3) d: 29.03, 28.96; HRFABMS
calcd. for C40H47NO9P (M + H)+ 716.2988, found 716.2963.
(S)-2-[[(4-N-tert-Butoxylcarbonylphenyl)ethoxy]-phosphonoyl]-
methyl-pentanedioic acid (10b). Yield: 97%. [a]2D0 = −32.0◦ (c =
1
0.5, MeOH); H NMR (500 MHz, CD3OD) d: 7.32–7.26 (2H,
m), 7.18–7.12 (2H, m), 4.15–3.97 (2H, m), 2.95–2.85 (3H, m),
2.42–1.63 (6H, m), 1.49 (9H, s); 31P NMR (121 MHz, CDCl3) d:
24.33; MS calcd. for C19H28NO9P (M + H + Na)+ 469.1478, found
469.1490.
(S)-2-[[(4-Hydroxyphenyl)ethoxy]phosphonoyl]methyl-pentane-
dioic acid tribenzyl ester (8a). To a flask containing 7 (280 mg,
0.383 mmol) was added 40% HF–CH3CN (5 : 95) (21 mL), and the
reaction mixture was stirred at room temperature for 6 h and then
diluted with EtOAc (100 mL). The layers were separated, and the
organic layer was washed with saturated NaHCO3 solution and
brine. After being dried, filtered, and concentrated, the residue was
purified by flash column chromatography eluting with hexanes–
ethyl acetate (2 : 1 → 1 : 1 → 1 : 2) to afford 200 mg of product
(S)-2-[(2-Phenylethoxy)phosphonoyl]methyl-pentanedioic acid
1
tribenzyl ester (11a). Yield: 80%. H NMR (500 MHz, CDCl3)
d: 7.41–7.17 (20H, m), 5.12–4.92 (6H, m), 4.25–4.11 (2H, m),
2.91 (2H, q, J = 6.5 Hz), 2.88–2.78 (1H, m), 2.38–2.22 (3H,
m), 2.03–1.96 (2H, m), 1.88–1.77 (1H, m); 31P NMR (121 MHz,
CDCl3) d: 29.58, 29.51; HRFABMS calcd. for C35H38O7P (M +
H)+ 601.2355, found 601.2349.
1
as a colorless oil (85%). H NMR (500 MHz, CDCl3) d: 7.99–
7.93 (1H, m), 7.37–7.32 (15H, m), 6.94–6.91 (2H, m), 6.82–6.80
(2H, m), 5.11–4.94 (6H, m), 4.20–4.01 (2H, m), 2.90–2.78 (3H,
m), 2.40–2.23 (3H, m), 2.02–1.77 (3H, m); 31P NMR (121 MHz,
CDCl3) d: 29.30, 29.19; HRFABMS calcd. for C35H38O8P (M +
H)+ 617.2304, found 617.2324.
(S)-2-[(2-Phenylethoxy)phosphonoyl]methyl-pentanedioic acid
(11b). Yield: 91%. [a]2D0 = +5.8◦ (c = 1.2, MeOH); H NMR
1
(500 MHz, CD3OD) d: 7.08–6.95 (5H, m), 3.93 (2H, q, J =
7.0 Hz), 2.76 (2H, t, J = 7.0 Hz), 2.58–2.40 (1H, m), 2.19–2.01
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(2H, m), 1.99–1.51 (4H, m); 31P NMR (121 MHz, CDCl3) d:
26.75; HRFABMS calcd. for C14H20O7P (M + H)+ 331.0947,
found 331.0959.
column chromatography eluting with hexanes–ethyl acetate (1 : 4)
to provide the product as a colorless oil in 89% yield (234 mg). 1H
NMR (500 MHz, CDCl3) d: 8.47 (1H, NH, brs), 7.46–7.24 (17H,
m), 7.10–7.05 (2H, m), 5.46 (1H, NH, brs), 5.09–4.94 (6H, m),
4.16–4.02 (2H, m), 3.93 (2H, brs), 2.84–2.72 (3H, m), 2.38–2.20
(3H, m), 1.99–1.71 (3H, m), 1.49 (9H, s); 31P NMR (121 MHz,
CDCl3) d: 29.17, 29.07; HRFABMS calcd. for C42H50N2O10P (M
+ H)+ 773.3203, found 773.3229.
(S) - 2 - [(3 - Phenylpropanoxy)phosphonoyl]methyl - pentanedioic
acid tribenzyl ester (12a). Yield: 76%. 1H NMR (500 MHz,
CDCl3) d: 7.40–7.17 (20H, m), 5.12–5.02 (6H, m), 4.03–3.88 (2H,
m), 2.97–2.83 (1H, m), 2.70–2.62 (2H, m), 2.44–2.28 (3H, m),
2.12–1.82 (5H, m); 31P NMR (121 MHz, CDCl3) d: 29.59, 29.54;
HRFABMS calcd. for C36H40O7P (M + H)+ 615.2512, found
615.2487.
Compound 15b. Yield: 97%. [a]2D0 = −5.4◦ (c = 1.0, MeOH);
1H NMR (500 MHz, CD3OD) d: 7.28–7.24 (2H, m), 7.14–6.92
(2H, m), 3.95 (2H, q, J = 6.5 Hz), 3.66–3.55 (2H, m), 2.75 (2H,
t, J = 6.5 Hz), 2.63–2.58 (1H, m), 2.26–1.50 (6H, m); 31P NMR
(121 MHz, CDCl3) d: 26.04; EI-MS calcd. for C21H32N2NaO10P
(M + H + Na)+ 526, found 526.
(S) - 2 - [(3 - Phenylpropanoxy)phos◦phonoyl]methyl - pentanedioic
acid (12b). Yield: 93%. [a]2D0 = +5.3 (c = 1.1, MeOH); 1H NMR
(500 MHz, D2O) d: 7.40–7.21 (5H, m), 3.90–3.38 (2H, m), 2.80–
2.33 (5H, m), 2.20–1.74 (6H, m); 31P NMR (121 MHz, CDCl3) d:
26.04; FABMS calcd. for C15H22O7P (M + H)+ 345, found 345.
1
(S) - 2 - [[(4 - Aminophenyl)ethoxy]phosphonoyl]methyl - pentane -
dioic acid tribenzyl ester (13). To a solution of 10a (440 mg,
0.714 mmol) in CH2Cl2 (10 mL) was added 1 mL of TFA. The
resulting reaction mixture was stirred at room temperature for 1 h
and then diluted with CH2Cl2 (100 mL). The mixture was washed
with saturated aqueous Na2CO3 to remove the excess TFA. The
separated aqueous layer was extracted with CH2Cl2 twice, and
the combined extracts were washed with 30 mL of brine, dried
over MgSO4, filtered and concentrated. The crude product was
obtained as a colorless oil, which was not stable and used directly
for the next steps without further purification. HRFABMS calcd.
for C35H39NO7P (M + H)+ 616.2464, found 616.2469.
Compound 16a. Yield: 90%. H NMR (500 MHz, CDCl3) d:
9.32 (bs, 1H), 7.50–7.01 (m, 19H), 5.51 (bs, 1H), 5.12–4.91 (m, 6H),
4.71 (m, 1H), 4.18–3.90 (m, 4H), 3.59–3.38 (m, 2H), 2.85–2.75 (m,
3H), 2.50–1.79 (m, 10H), 1.45 (s, 9H); 31P NMR (121 MHz, CDCl3)
d: 29.59, 29.53; 31P NMR (121 MHz, CDCl3) d: 26.41; HRFABMS
calcd. for C47H57N3O11P (M + H)+ 870.3731, found 870.3768.
Compound 16b. Yield: 99%. [a]2D0 = −6.7◦ (c = 0.6, MeOH);
1H NMR (500 MHz, CD3OD) d: 7.60–7.13 (m, 4H), 4.64–
4.52 (m, 1H), 4.20–3.80 (m, 3H), 3.39–3.28 (m, 1H), 2.99–1.64
(m, 15H), 1.45 (s, 9H); 31P NMR (121 MHz, CDCl3) d: 26.41;
HRFABMS calcd. for C26H39KN3O11P (M + H + K)+ 639.1959,
found 639.1942.
(S)-2-[[(4-Acetaminophenyl)ethoxy]phosphonoyl]methyl-pentane-
dioic acid tribenzyl ester (14a). To a solution of crude 13 (80 mg,
0.13 mmol) in CH2Cl2 were added Ac2O (37 lL, 0.39 mmol) and
pyridine (32 lL, 0.39 mmol) at 0 ◦C. The reaction mixture was
stirred and warmed to room temperature overnight. The volatiles
were evaporated and the residue was purified by flash column
chromatography eluting with hexanes–ethyl acetate (1 : 2 → 1 :
1
Compound 17a. Yield: 92%. H NMR (500 MHz, CDCl3) d:
9.05 (bs, 1H), 7.95–7.05 (m, 19H), 6.03 (bs, 1H), 5.12–4.90 (m,
6H), 4.20–3.77 (m, 8H), 3.01–2.93 (m, 4H), 2.40–2.21 (m, 3H),
2.05–1.78 (m, 3H), 1.39 (s, 9H); 31P NMR (121 MHz, CDCl3) d:
29.40, 29.35; HRFABMS calcd. for C46H55N4NaO12P (M + Na)+
909.3452, found 909.3440.
1
3) to afford 65 mg (76%) of product as a colorless oil. H NMR
(500 MHz, CDCl3) d: 7.20–7.15 (16H, m), 7.25–7.22 (2H, m),
7.05–7.03 (2H, m), 5.09–4.92 (6H, m), 4.24–4.06 (2H, m), 2.92
(2H, q, J = 6.5 Hz), 2.88–2.80 (1H, m), 2.40–2.78 (3H, m), 2.27
(3H,s), 2.05–1.80 (3H, m); 31P NMR (121 MHz, CDCl3) d: 29.29;
HRFABMS calcd. for C37H41NO8P (M + H)+ 658.2570, found
658.2568.
Compound 17b. Yield: 96% yield. [a]2D0 = −7.2◦ (c = 0.7,
1
MeOH); H NMR (500 MHz, CD3OD) d: 7.56–7.38 (m, 2H),
7.20–7.02 (m, 2H), 4.05–3.93 (m, 3H), 3.80–3.60 (m, 3H), 2.88–
1.15 (m, 11H), 1.36 (s, 9H); 31P NMR (121 MHz, CDCl3) d: 26.04;
HRFABMS calcd. for C25H37N4NaO12P (M + Na)+ 639.2043,
found 639.2011.
(S)-2-[[(4-Acetaminophenyl)ethoxy]phosphonoyl]methyl-pentane-
dioic acid (14b). Yield: 83%. [a]2D0 = −12.1◦ (c = 0.3, MeOH);
1H NMR (500 MHz, CD3OD) d: 7.41 (2H, d, J = 8.0 Hz), 7.14
(2H, d, J = 8.0 Hz), 4.12–4.03 (2H, m), 3.66 (2H, t, J = 7.0 Hz),
2.73–2.68 (1H, m), 2.30–2.20 (3H, m), 2.04 (3H, s), 1.98–1.85
(3H, m); 31P NMR (121 MHz, CDCl3) d: 26.88; FABMS calcd.
for C16H23NO8P (M + H)+ 388.1, found 388.1.
Fluorescent-labeled compound precursor (19a). To a solution
of aniline 13 (100 mg, 0.162 mmol) in dry benzene (5 mL) were
added commercially available (Fluka) 7-(diethylamino)coumarin-
3-carbonyl azide 18 (25 mg, 0.087 mmol) and DMAP (16 mg,
0.131 mmol). The resulting mixture was heated to reflux for 12 h.
After cooling to room temperature, the solvent was evaporated.
The residue was subjected to column chromatographic purification
(EtOAc–hexanes–triethylamine 100 : 25 : 1 to 100 : 25 : 0.5) to
afford 35 mg of the product as a yellow solid (46%): mp 63–65 ◦C;
1H NMR (500 MHz, CDCl3) d: 8.90 (bs, 1H), 8.60–8.40 (m, 2H),
7.46–7.03 (m, 23H), 6.80–6.45 (m, 1H), 5.14–4.98 (m, 6H), 4.20–
4.04 (m, 2H), 3.58–3.41 (m, 3H), 3.01–2.80 (m, 3H), 2.50–2.31 (m,
3H), 2.09–1.74 (m, 4H), 1.42–1.12 (m, 7H); 31P NMR (121 MHz,
General procedure for the synthesis of 15a, 16a and 17a
Compound 15a. To a suspension of aniline 13 (210 mg,
0.341 mmol) and Boc-Gly-OH (72 mg, 0.409 mmol) in CH3CN
(5 mL) were added diisopropylethylamine (71 lL, 0.409 mmol),
HOAt (56 mg, 0.409 mmol) and EDCI (79 mg, 0.409 mmol).
The reaction mixture was stirred at room temperature for 18 h.
After removal of the solvent, the residue was purified by flash
830 | Org. Biomol. Chem., 2007, 5, 826–831
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CDCl3) d: 29.741; HRFABMS calcd. for C49H53N3O10P (M + H)+
874.3469, found 874.3489.
References
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1
+72.4 (c = 0.5, MeOH); H NMR (500 MHz, CDCl3) d: 7.40–
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2H), 2.79–2.60 (m, 1H), 2.40–1.60 (m, 5H), 1.40–0.81 (m, 8H);
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temperature for 24 h, and then diluted with EtOAc (60 mL).
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satd. aq. NaHCO3 (5 mL), brine (10 mL), and then dried.
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chromatography eluting with EtOAc–MeOH (10 : 1 to 8 : 1) to
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1
provide 91 mg of the product as a colorless oil (89%). H NMR
(500 MHz, CDCl3) d: 7.78–7.25 (m, 34 H), 7.03–6.80 (m, 3H),
5.10–4.78 (m, 12H), 4.72–4.40 (m, 3H), 4.18–3.40 (m, 7H), 2.90–
2.60 (m, 6H), 2.40–20 (m, 4H), 2.12–1.92 (m, 12H), 1.90–1.58 (m,
14H); 31P NMR (121 MHz, CDCl3) d: 29.522; EI-MS calcd. for
C79H94N7O17P (M+) 1444, found 1444.
◦
1
21b. Yield: 100%. [a]D20 = −5.1 (c = 2.4, MeOH); H NMR
(500 MHz, CD3OD) d: 7.54–7.38 (m, 2H), 7.20–7.11 (m, 2H),
4.43–4.02 (m, 6H), 3.78–3.40 (m, 8H), 3.84 (bs, 2H), 2.38–2.20 (m,
2H), 2.18–1.52 (m, 26H); 31P NMR (121 MHz, CDCl3) d: 28.382;
HRFABMS calcd. for C37H59N7O17P (M + H)+ 904.3705, found
904.3675.
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Acknowledgements
We thank Dr Vincent Kalish and Dr Camilo Rojas at Guilford
Pharmaceuticals Inc. for generously providing the biological
assays. We gratefully acknowledge financial support by the
Department of Defense and collaboration with the Walther
Cancer Institute as well as NIH grant AI054193 (MJM, PD).
We also appreciate the use of the NMR facilities provided by the
Lizzadro Magnetic Center and the mass spectrometry services (Dr
W. Boggess and Ms N. Sevova) provided by the University of Notre
Dame.
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