Design, synthesis, and biochemical evaluation of phosphonate and phosphonamidate analogs of glutathionylspermidine as inhibitors of glutathionylspermidine synthetase/amidase from Escherichia coli

Article abstract of DOI:10.1021/jm970414b

Three phosphapeptides designed to mimic two distinct tetrahedral intermediates formed during either the synthesis or hydrolysis of glutathionylspermidine (Gsp) were synthesized and evaluated as inhibitors of the bifunctional enzyme Gsp synthetase/amidase.

Full text of DOI:10.1021/jm970414b

3842
J . Med. Chem. 1997, 40, 3842-3850
Design , Syn th esis, a n d Bioch em ica l Eva lu a tion of P h osp h on a te a n d
P h osp h on a m id a te An a logs of Glu ta th ion ylsp er m id in e a s In h ibitor s of
Glu ta th ion ylsp er m id in e Syn th eta se/Am id a se fr om Esch er ich ia coli
Shoujun Chen,† Chun-Hung Lin,§ David S. Kwon,§ Christopher T. Walsh,§ and J ames K. Coward*,†
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, and Department of Chemistry,
The University of Michigan, Ann Arbor, Michigan 48109-1055, and Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
Received J uly 14, 1997X
Three phosphapeptides designed to mimic two distinct tetrahedral intermediates formed during
either the synthesis or hydrolysis of glutathionylspermidine (Gsp) were synthesized and
evaluated as inhibitors of the bifunctional enzyme Gsp synthetase/amidase. While the
polyamine-containing phosphapeptides were determined to be potent and selective inhibitors,
they selectively inhibit the synthetase activity over the amidase domain. A phosphonate-
containing tetrahedral mimic is a reversible mixed-type inhibitor of Gsp synthetase with an
inhibition constant of 6 µM for the inhibitor binding to the free enzyme (Ki) and 14 µM for the
inhibitor binding to the enzyme-substrate complex (Ki′). The corresponding phosphonamidate
is a slow-binding inhibitor with a Ki of 24 µM and a Ki* (isomerization inhibition constant) of
0.88 µM. A non-polyamine-containing phosphonamidate exhibits no significant inhibition of
the synthetase or amidase activity.
In tr od u ction
Thus, interfering with the trypanothione biosynthetic
pathway is also attractive for antiparasitic drug de-
sign.16,17
Glutathionylspermidine (Gsp) synthetase, isolated
from Crithidia fasciculata18,19 or Escherichia coli,20 is
a key enzyme that participates in the first of two similar
steps of trypanothione biosynthesis (eq 1).15 It is an
Parasitic diseases are the major killer of millions of
children each year in the world. They are also the most
common opportunistic infections that affect patients
with acquired immunedeficiency syndrome (AIDS).1,2
However, compared to bacterial diseases, the develop-
ment of chemotherapy for the treatment of parasitic
diseases1,3 has been hindered by the close similarities
between parasite and host metabolism. Most of the
existing drugs are either ineffective or toxic to the host;
some are carcinogenic.1,2,4 Much effort has been made
during the last decade to differentiate between parasite
and host metabolism. It was first discovered that a
major difference exists in the biochemistry of defense
mechanisms against oxidative damage.5 Unlike host
cells which use a glutathione/glutathione reductase
couple to maintain the intracellular thiol redox balance
and therefore defend against oxidative stress, protozoal
parasites of the genera Trypanosoma and Leishmania
depend on a conjugate of glutathione and spermidine,
namely, trypanothione (N1,N8-diglutathionylspermi-
dine), for redox balance and oxidant defense.5 Subse-
quent discovery of trypanothione reductase (TR),6
a
ATP-dependent enzyme that couples the hydrolysis of
ATP to the ligation of GSH (R ) SH, 1a ) and spermidine
(2) to form Gsp (R ) SH, 3a ) and ADP.20 The catalytic
mechanism has been investigated.19 Substrate specific-
ity studies revealed that both glutamate and glycine
residues of GSH are important for the recognition of
substrate by Gsp synthetase, while the thiol group of
the cysteine is not essential for recognition; e.g., the
alanine-containing analog (R ) H, 1b) and ophthalmic
acid (R ) CH3, 1c) are also good substrates for Gsp
synthetase.18,21,22 Spermidine-binding site studies showed
that Gsp synthetase from E. coli recognizes a (ω-
aminoalkyl)-1,3-diaminopropane with a deprotonated
N-1 amino group as the likely reacting species.20 Sur-
prisingly, the E. coli Gsp synthetase was discovered to
be a bifunctional enzyme containing a hydrolytic activ-
ity.20 This activity, either as part of the full-length
parasitic enzyme with considerable structural7-10 and
mechanistic4,11 similarity to the host glutathione reduc-
tase, provides a means by which trypanothione can
regulate the intracellular thiol redox balance in the
parasite. The substrate specificity of TR and the unique
presence of trypanothione in trypanosomatid parasites
present two ideal targets for antiparasitic drug design.4
Consequently, inhibition of TR has been actively inves-
tigated.3,12 In addition, studies have demonstrated that
trypanosomatid parasites lack the enzymes catalase and
glutathione peroxidase and therefore may utilize try-
panothione to detoxify H2O2 and other organic hydro-
peroxides through non-enzyme-catalyzed reactions.3,13-15
† The University of Michigan.
§ Harvard Medical School.
X Abstract published in Advance ACS Abstracts, October 15, 1997.
S0022-2623(97)00414-7 CCC: $14.00 © 1997 American Chemical Society

Phosphapeptide Inhibitors of Gsp Synthetase/ Amidase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3843
Sch em e 1
bifunctional protein or as a proteolytic fragment, is
contained in a domain referred to as Gsp amidase.21 Gsp
amidase catalyzes the hydrolysis of Gsp to GSH and
spermidine (eq 1). Substrate specificity studies for the
Gsp amidase domain showed that this enzyme recog-
nizes predominantly the glutathione portion of glu-
tathionylspermidine.21
Although direct evidence is currently lacking, we
postulate that the reaction catalyzed by Gsp synthetase
proceeds through a tetrahedral intermediate, formed via
an acyl phosphate, based on extensive literature pre-
cedent with other ATP-dependent peptide biosynthesis
reactions.23-25 Phosphonate, phosphonamidate, and
phosphinates have been widely used as transition state
analog inhibitors26 of protease27-30 or ATP-dependent
ligase31-33 enzymes by mimicking the proposed unstable
tetrahedral intermediate in each case. We hypothesized
that phosphonate (4) and phosphonamidate (5) peptides
would be potent and specific inhibitors of Gsp syn-
thetase/amidase and would interfere with the trypano-
thione biosynthetic pathway.16 In addition, phosphapep-
tides such as 4 and 5 could act as inhibitors of the
amidase activity by mimicking the tetrahedral inter-
mediate formed as a result of direct attack of H2O at
the scissile amide bond.
catalytic hydrogenolysis. Thus, in the approach re-
ported herein, all the amines in the precursors were
protected by carbobenzyloxy (Cbz, Z) groups while the
carboxylic acids and phosphonic acid were masked as
their benzyl esters.
The synthesis of the Cbz-containing, selectively pro-
tected derivatives of 1-hydroxy-1-desaminospermidine
(8) and spermidine (9b) is outlined in Scheme 1.
Starting with 3-amino-1-propanol, the synthesis of 8
was effected, via the orthogonally protected spermidine
precursor 1-O-TBDPS-8-azido-4-azaoctane, 7,35 in seven
steps with an overall yield of 20%. Selectively protected
spermidine (9b) was obtained from 8 in two steps in 80%
yield. Benzyl hydrogen (phthalimidomethyl)phospho-
nate (10) was prepared from (phthalimidomethyl)-
phosphonic acid36 and benzyl alcohol according to a
general literature procedure.37 As depicted in Scheme
2, coupling of 10 with 8 proceeded smoothly by either
the oxalyl chloride approach16 or the Mitsunobu
method16,38,39 to afford the mixed phosphonate 11 in 89%
and 61% yields, respectively. Removal of the phthaloyl
group in 11 by hydrazinolysis afforded 12 in 82% yield.
Crude 12 could be used directly in the coupling with 13
to yield 14. Initially, partial success was achieved by
employing either DCC/HOBt or the mixed anhydride
method by which 14 was obtained in only 43% and 24%
yields, respectively. The BOP reagent, introduced
initially for peptide synthesis,40 proved to be much more
efficient and reliable; a much higher yield (81%) of 14
was obtained by using this reagent in the coupling
reaction.41,42 Catalytic hydrogenolysis of 14 followed by
cation exchange chromatography on AG 50W-X2 resin
afforded the target phosphonate 4 in 83% yield.
Ch em istr y
In a previous paper, we described efforts made toward
the synthesis of intermediates for the target molecules
4 and 5.16 Initially, the azido group was chosen as a
precursor of the N-8 primary amino group, whereas the
tosyl group was chosen to protect the N-4 secondary
amino group in spermidine derivatives. Thus, suitably
protected precursors of 1-hydroxy-1-desaminospermi-
dine and spermidine were coupled in satisfactory yield
with methyl hydrogen (phthalimidomethyl)phosphonate
(activation with either oxalyl chloride or via the Mit-
sunobu method) to provide, respectively, blocked phos-
phonate and phosphonamidate precursors of 4 and 5.16
However, it soon became evident that the deprotection
of such polyfunctionalized molecules was very difficult,
especially in the presence of the obstinate N-tosyl group.
As a result, all attempts to isolate the target molecule
4 using such a protective strategy were unsuccessful.34
One possible reason for these failures is the harsh
conditions required for removal of the tosyl group that
may affect the P-O bond (phosphonate, 4) and/or the
P-N bond (phosphonamidate, 5) in the target mol-
ecules. In order to solve this problem, we decided to
choose protective groups that can be removed simulta-
neously at the final stage under neutral conditions, e.g.,
In contrast to the facile coupling of 10 with 8 to form

3844 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Chen et al.
Sch em e 2
the complex phosphonate 11, the coupling of 10 with 9
to form the corresponding phosphonamidate 15 proved
to be very idiosyncratic. Although the phosphonic
monomethyl ester corresponding to benzyl ester 10
coupled smoothly with simple amines, including a
protected form of spermidine similar to 9b, following
generation of the corresponding phosphonochloridate,16
the coupling of 10 with N-butylamine (9a ) or 9b using
the oxalyl chloride method was not satisfactory. How-
ever, other amines such as 3-chloropropylamine and
diethyl glutamate were coupled with 10 to provide the
corresponding phosphonamidates in moderate to good
yield under the same conditions (S. Chen, J . K. Coward,
unpublished results). Fortunately, using the BOP
method, 10 coupled smoothly with both 9a ,b to give
15a ,b, respectively, in satisfactory yields.
The lability of P-N bonds in phosphonamidates
compared to those in phosphoramidates is well docu-
mented. The P-N bond in a phosphonamidate is
hydrolyzed faster than that in a phosphoramidate.43
Nonetheless, N-terminal protected phosphonamidate
peptides, stabilized as the corresponding lithium salt,
were successfully prepared as inhibitors of some pro-
tease enzymes.28,44 However, in the case of completely
deprotected phosphonamidate peptides, even the lithium
salts could not be isolated due to their extreme lability.45
In order to determine whether this is also the case for
nonpeptidic P-N bonds, 17 was prepared from 15a in
a similar way as described for the synthesis of 14
(Scheme 2). Subsequent hydrogenolysis of this material
in EtOH under mild basic conditions failed to give any
trace of the desired phosphonamidate 5a . Instead, a
product resulting from ethanolysis of the P-N bond was
isolated and characterized by NMR and MS analyses.
Instability of the P-N bond is of concern for enzyme
inhibitor and drug design. For instance, some phos-
phoramidates are known to be potent inhibitors of
several proteases such as collagenase.46 However, these
phosphoramidates could not be developed as useful
drugs.47 Considering the instability of 5 and literature
precedent for the use of methyl or phenyl phosphon-
amidates as inhibitors of serine proteases such as
chymotrypsin,48 we attempted to stabilize the target
molecule 5 by blocking the free hydroxyl group on phos-
phorus as a methyl ester. Therefore, esters 21a ,b were
synthesized (Scheme 3) as surrogates of 5 for inhibition
studies. Intermediates 19a ,b were obtained in reason-
able yields from 1816 and 9a ,b, respectively, using either
the BOP or oxalyl chloride method. The desired methyl
phosphonamidates, 21a ,b, were obtained from 19a ,b by
chemistry similar to that described for the synthesis of
4.
Bioch em ica l Eva lu a tion
Compounds 4 and 21a ,b were assayed for inhibition
of the E. coli Gsp synthetase using a pyruvate kinase/

Phosphapeptide Inhibitors of Gsp Synthetase/ Amidase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3845
Ta ble 1. Inhibition of Gsp Synthetase by 4 and 21a ,b
Sch em e 3
a
For definition of inhibition constants, see Schemes 4 and 5.
Percent inhibition at 1 mM inhibitor. c See ref 21.
b
Sch em e 4
Sch em e 5
The phosphonate and phosphonamidates all exhibit
ATP-independent inhibition; i.e., phosphorylation of
these inhibitors does not occur, in contrast to ATP-
dependent inhibition by a closely related phosphinate
inhibitor, which has been interpreted as reflecting
formation of an enzyme-bound phosphinophosphate.49
The phosphonate 4 was determined to exhibit noncom-
petitive or mixed-type inhibition (Scheme 4),21 whereas
the phosphonamidate 21b was shown to be a time-
dependent (slow-binding) inhibitor. The observed 30-
fold tightening (Ki f Ki*) is typically interpreted as an
isomerization of a collisional E‚I complex to a rear-
ranged E‚I* complex (Scheme 5).52 It is possible,
however, that some portion of the slow-binding inhibi-
tion is due to a slow decomposition of 21b (vide supra)
and more potent inhibition by the product(s).
For inhibition of the Gsp amidase domain, we hy-
pothesized21 that the synthesized phosphapeptides would
mimic the tetrahedral intermediate that would result
if the hydrolysis involved direct attack of H2O (e.g., zinc
or aspartyl proteases). Poor inhibition, orders of mag-
nitude less than observed for the synthetase domain
(Table 1), argues against such a reaction mechanism
and suggests an amidase mechanism of covalent cataly-
sis by a serine or cysteine nucleophile forming an acyl-
enzyme intermediate.21 Recent studies using rapid
kinetic methods and mutagenesis indicate that such a
covalent acyl-enzyme mechanism does apply; cysteine
appears to be a likely candidate for the nucleophile (C.
T. Walsh, et al. Biochemistry, in press).
lactate dehydrogenase coupled assay, as previously
described.20,21 As noted above, the E. coli enzyme is a
bifunctional protein; the second activity, Gsp amidase,
catalyzes the hydrolysis of Gsp back to glutathione and
spermidine. Amidase and synthetase activities are
contained in separate N-terminal and C-terminal do-
mains, respectively, of the 70 kDa protein.21 Therefore,
the inhibition of Gsp amidase was also investigated
using an alcohol dehydrogenase/aldehyde dehydroge-
nase coupled assay, as previously described.21 To avoid
the potential artifacts due to negative regulation of the
amidase activity by the synthetase domain, the 225-
amino acid amidase fragment (25 kDa)21 was used in
all amidase inhibition studies.
Inhibition results of Gsp synthetase are summarized
in Table 1, and several initial observations are note-
worthy. First, the spermidine portion is essential for
inhibition. Compound 21b, containing the spermidine
moiety, is much more potent than 21a , which has an
N-butyl group substituted for spermidine. Presumably,
these results reflect a binding preference which has been
also observed for spermidine analogs20 and Gsp-phos-
phinate analogs.49 Second, the phosphonamidate 21b,
the more stable surrogate of 5, was observed by HPLC
to decompose gradually during inhibition assays (30
min, 37 °C). The observed instability is consistent with
that observed for phosphonamidate inhibitors of D-Ala-
D-Ala ligase.50,51
In summary, the phosphapeptides 4 and 21b selec-
tively inhibit Gsp synthetase with inhibition constants

3846 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Chen et al.
(80 mL) at 0 °C. To this solution was added dropwise a
solution of CbzCl (3.01 mL, 21.2 mmol) in CH2Cl2 (10 mL)
during a period of 30 min. The resultant yellow solution was
stirred at room temperature for 24 h before being diluted with
150 mL of CH2Cl2, then washed with water (100 mL), 5% citric
acid (100 mL), 5% NaHCO3 solution (100 mL), and brine (100
mL), and dried over Na2SO4. After removal of the organic
solvent under reduced pressure, the crude product was purified
by column chromatography on silica gel using hexane/EtOAc
in the micromolar range. Taken together with data on
the corresponding phosphinate,49 where X in 4 is CH2
rather than O, these studies define the structural
determinants for effective design of potent inhibitors of
Gsp synthetase. Unfortunately, neither 4 nor 21b is
an effective inhibitor of the growth of Tryanoma brucei
(C. Bacchi, personal communication) or Leishmania
donovani and Trypanoma cruzi (S. Croft, personal
communication) in cell culture assays. A variety of
structural modifications can be envisioned that may
allow for these phosphapeptides to penetrate the cell
membranes. Synthetic studies to access such modified
tetrahedral mimics are currently underway in our
laboratory.
1
(7:3) as eluant: yield 1.7 g (50%) of an oil; H NMR (DMSO-
d6, 334 K) δ 0.99 (s, 9 H), 1.28-1.37 (m, 2 H), 1.39-1.51 (m,
2 H), 1.65-1.74 (m, 2 H), 2.95-3.01 (m, 2 H), 3.17 (t, 2 H, J )
7.6), 3.30 (t, 2 H, J ) 8.0), 3.64 (t, 2 H, J ) 8.0), 5.00 (s, 2 H),
5.03 (s, 2 H), 6.89-7.01 (br, 1 H), 7.25-7.36 (m, 10 H), 7.37-
7.44 (m, 6 H), 7.52-7.61 (m, 4 H); 13C NMR (CDCl3) δ 156.5,
156.1, 137.1, 135.6, 133.9, 129.8, 129.7, 128.6, 128.2, 128.0,
127.9, 127.8, 127.3, 67.1, 66.8, 61.7, 46.9, 44.1, 40.8, 32.4, 27.4,
27.1, 25.5, 19.5; MS (CI with methane/NH3) m/ z (rel intensity)
653 (MH+, 100), 545 (45), 91 (58); HRMS (CI) calcd for
C39H48N2O5SiH (MH+) 653.3411, found 653.3443.
A solution of this fully protected amino alcohol (0.4 g, 0.6
mmol) and TBAF (1 M solution in THF, 2.41 mL, 2.41 mmol)
in THF (20 mL) containing acetic acid (1 mL) was stirred at
room temperature for 6 h. The reaction mixture was then
concentrated, and the residue was partitioned between CH2-
Cl2 (30 mL) and water (20 mL). The organic layer was
separated, and the aqueous layer was extracted with CH2Cl2
(2 × 20 mL). Organic extracts were combined and dried over
Na2SO4. After being concentrated and purified by column
chromatography on silica gel using hexane/EtOAc (1:1) as
eluant, 0.20 g (81%) of the desired product 8 was obtained as
a colorless oil: Rf 0.24 (hexane/EtOAc (1:1)); 1H NMR (DMSO-
d6, 332 K) δ 1.34-1.42 (m, 2 H), 1.42-1.53 (m, 2 H), 1.57-
1.66 (m, 2 H), 2.95-3.03 (m, 2 H), 3.15-3.29 (m, 4 H), 3.34-
3.43 (m, 2 H), 4,18-4.28 (br, 1 H), 5.01 (s, 2 H), 5.06 (s, 2 H),
6.95-7.08 (br, 1 H), 7.15-7.44 (m, 10 H); 13C NMR (CDCl3) δ
157.7, 156.6, 136.8, 129.9, 128.7, 128.3, 128.1, 127.3, 67.5, 66.9,
58.7, 46.8, 43.7, 40.8, 30.9, 27.4, 25.8, 21.6; MS (CI with CH4/
NH3) m/ z (rel intensity) 415 (MH+, 87), 307 (100), 173 (37),
108 (26); HRMS (CI) calcd for C23H30N2O5H (MH+) 415.2233,
found 415.2256.
N-(Ben zyloxyca r bon yl)-N′-(ben zyloxyca r bon yl)-N′-(3-
a m in op r op yl)-1,4-d ia m in obu ta n e (9b). To a stirred solu-
tion of 8 (1.3g, 3.12 mmol), phthalimide (0.46g, 3.12 mmol),
and Ph3P (0.86g, 3.28 mmol) in dry THF (16 mL) under N2
was added DEAD (0.53g, 3.12 mmol) gradually at room
temperature. The resultant yellow solution was stirred at
room temperature overnight and then concentrated under
reduced pressure. After usual workup, the desired product
was purified by column chromatography (98-33% hexanes in
EtOAc) to give 1.45 g (85%) of the phthaloyl derivative as a
clear oil: Rf 0.35 (1:1 Hex/EtOAc); 1H NMR (DMSO-d6, 331
K) δ 1.31-1.44 (m, 2 H), 1.45-1.53 (m, 2 H), 1.71-1.89 (m, 2
H), 2.95-3.04 (m, 2 H), 3.19-3.27 (m, 4 H), 3.57 (t, 2 H, J )
7), 5.01 (s, 2 H), 5.02 (s, 2 H), 6.98-7.07 (br, 1 H), 7.25-7.36
(m, 10 H), 7.78-7.85 (m, 4 H); 13C NMR δ 168.4, 156.6, 156.2,
136.8, 134.1, 132.1, 128.6, 128.1, 128.0, 127.9, 123.3, 67.1, 66.6,
47.4 (46.8), 45.4 (45.0), 40.7, 36.6, 35.8, 31.5, 28.0 (27.6), 27.2,
26.0 (25.6).
Exp er im en ta l Section
Gen er a l Meth od s. 1H NMR spectra were recorded at 300
or 360 MHz on Bruker spectrometers. Chemical shifts in ppm
were measured relative to TMS. All coupling constants are
reported in hertz (Hz). 13C NMR spectra were recorded on a
Bruker 360 spectrometer at 90.6 MHz or a Bruker 300
spectrometer at 75.5 MHz, and chemical shift data are
reported in reference to TMS. When appropriate, carbon-
phosphorus coupling is reported with the chemical shift data
(multiplicity, coupling constant in Hz). 31P NMR spectra were
recorded on a Bruker 360 spectrometer at 145 MHz with 85%
H3PO4 (δ ) 0 ppm) as an external reference and with broad-
1
band H decoupling. All NMR spectra were obtained at room
temperature (ca 298 K) unless otherwise indicated. Mass
spectra and high-resolution mass spectra were recorded on a
Finnigan 4500 GC/MS-EICI system or on a VG Analytical
system, Model 70-250S. Elemental analyses were obtained
from Atlantic Microlab Inc., at Norcross, GA, or at the
Elemental Analysis Labs, Department of Chemistry, The
University of Michigan. Melting points were determined using
a Thomas-Hoover capillary apparatus and are uncorrected.
Acetonitrile, DMF, ethylene glycol dimethyl ether (DME), and
benzyl alcohol were distilled and stored over molecular sieves
(4 Å). N-Methylmorpholine (NMM) and triethylamine (TEA)
were stored over KOH pellets after distillation. Tetrahydro-
furan (THF) was distilled under N2 from violet sodium
benzophenone ketyl before use. Dichloromethane (CH2Cl2)
was distilled from calcium hydride. Ethyl acetate (EtOAc),
hexane, and other solvents were Baker HPLC grade and used
without further treatment except as otherwise indicated.
Commercially available amino acid and dipeptide precursors
were purchased from Bachem-Bioscience Inc. or Fisher Sci-
entific. Other reagents were obtained from Aldrich or as
otherwise indicated. Compound 6 was prepared according to
the procedures of Lakanen.35 Synthesis of 7 was carried out
by a slight modification of the original procedure35 as detailed
in the Supporting Information. The synthesis of Z-Glu-γ-Ala-
OH (13) was carried out by standard methods and is described
in the Supporting Information. Methyl hydrogen (phthalimi-
domethyl)phosphonate (18) was synthesized as previously
described.16
N-(Ben zyloxyca r bon yl)-N′-(ben zyloxyca r bon yl)-N′-(3-
h yd r oxyp r op yl)-1,4-d ia m in obu ta n e (8). To a stirred solu-
tion of 7 (3.0 g, 5.3 mmol) in dry DME (30 mL) in an oven-
dried round bottom flask under dry nitrogen was added 1 M
sodium naphthalenide-DME53 solution dropwise at -78 °C (dry
ice-2-propanol) until a green color persisted for at least 5 min.
The resultant green solution was stirred at -78 °C for 1 h,
the reaction was then quenched with water (2 mL), and solvent
was removed by concentration under reduced pressure; the
residue was partitioned between ethyl ether (50 mL) and water
(50 mL). The organic layer was separated and washed with
water (30 mL), 5% NaHCO3 solution (40 mL), and brine (40
mL), dried over sodium sulfate, and then evaporated to
dryness. The crude secondary amine, obtained as a semisolid
material with naphthalene contamination, was thoroughly
dried over P2O5 in vacuo and then dissolved, with triethyl-
amine (3.7 mL, 26.5 mmol) and DMAP (0.3 g), in dry CH2Cl2
A mixture of the phthaloyl derivative (1.55 g, 2.8 mmol) and
NH2NH2‚H2O (1.4g, 28 mmol) in MeOH was stirred at room
temperature for 48 h. The resultant precipitate was removed
by filtration, and the filtrate was concentrated under vacuum.
The residue was then partitioned between CH2Cl2 and NH4-
OH. The two layers were separated, the aqueous layers were
extracted by CH2Cl2, and the combined organic extracts were
dried over anhydrous Na2SO4. Removal of the solvent under
reduced pressure afforded 1.1 g (94%) of the desired product
9b as an oil: 1H NMR (DMSO-d6, 331 K) δ 1.43-1.54 (m, 2
H), 1.55-1.70 (m, 4 H), 2.63 (t, 2 H, J ) 6.6), 3.07-3.15 (m, 2
H), 3.28-3.39 (m, 4 H), 5.13 (s, 2 H), 5.18 (s, 2 H), 7.08-7.19
(br, 1 H), 7.32-7.49 (m, 10 H); 13C NMR (CDCl3) δ 156.6, 156.4,
136.9, 136.8, 128.6, 128.2, 128.1, 128.0, 67.1, 66.7, 46.8 (46.4),
44.7, 40.8, 39.3, 33.4 (33.0), 27.3, 26.0 (25.6); MS (EI, 70 eV)
m/ z (rel intensity) 413 (M+, 0.7), 149 (14), 91 (100); HRMS
(EI, 70 eV) calcd for C23H31N3O4 413.2315, found 413.2294.

Phosphapeptide Inhibitors of Gsp Synthetase/ Amidase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3847
This material was used without further purification in the
synthesis of 15b and 19b.
123.6, 68.4 (d, J ) 6), 67.1, 66.6, 64.3, 47.5, 43.8, 40.8, 34.8,
32.7, 29.8, 27.2, 25.6; 31P NMR (CDCl3) δ 20.7; MS (CI with
NH3) m/ z (rel intensity) 728 (MH+, 6), 620 (11), 512 (23), 422
(58), 307 (100), 91 (37); HRMS (CI) calcd for C39H43N3O9P
(MH+) 728.2737, found 728.2803. Anal. (C39H42N3O9P‚0.5H2O)
C, H, N.
Ben zyl Hyd r ogen (P h th a lim id om eth yl)p h osp h on a te
(10). To a stirred solution of diethyl (phthalimidomethyl)-
phosphonate (4.0 g, 13.4 mmol) in dry CH2Cl2 (40 mL) was
added TMSBr (8.0 g, 52 mmol) at room temperature, under
N2. After 12 h stirring at room temperature the reaction
mixture was concentrated under reduced pressure. The
residue was then triturated with MeOH (45 mL) and AcOH
(1.25 mL). Precipitated product was collected and washed
thoroughly with MeOH to give 2.82 g (100%) of (phthalimi-
domethyl)phosphonic acid: mp 302-304 °C dec (lit.36 mp 274-
277 °C). This material was used directly in the next step.
A stirred suspension of (phthalimidomethyl)phosphonic acid
(1.0 g, 0.47 mmol) in dry DMF (10 mL) was chilled in a dry
ice-acetone bath (ca. -35 °C). Thionyl chloride (0.51 mL, 0.7
mmol) was then added through a syringe, and the reaction
mixture solidified over a period of 30 min. The bath temper-
ature was allowed to rise to -25 °C when benzyl alcohol (2.5
mL) was added slowly through a dropping funnel as the solid
mass dissolved. The resultant clear solution was stirred at 0
°C for 1 h and then at room temperature for 12 h. The volatile
components were removed on a rotary evaporator, and the
residue was dissolved in 5% aqueous NaHCO3 (15 mL) which
was extracted with EtOAc (2 × 10 mL). The aqueous solution
was acidified to pH 2-3 with concentrated hydrochloric acid
at 0 °C and extracted with EtOAc (4 × 20 mL). The EtOAc
extracts were washed with brine (30 mL) and dried over
sodium sulfate. The solid product obtained after removal of
the solvent was crystallized from hexane/CHCl3 to give 1.37 g
(88%) of a white powder: mp 176.5-178 °C; Rf 0.25 (CHCl3/
MeOH (2:1)); 1H NMR (DMSO-d6) δ 4.0 (d, 2 H, J ) 10.9), 5.01
O-Ben zyl-O-[3-[N-[4-[N-(b en zyloxyca r b on yl)a m in o]-
b u t yl]-N -(b e n zyloxyca r b on yl)a m in o]p r op yl](a m in o-
m eth yl)p h osp h on a te (12). A solution of 11 (0.12 g, 0.16
mmol) and hydrazine monohydrate (0.12 mL, 1.6 mmol) in
MeOH (8 mL) was stirred at room temperature for 24 h. The
precipitated byproducts were removed by filtration, and the
filtrate was concentrated to afford a solid material which was
then partitioned between CH2Cl2 and water (20 mL each); the
organic layer was separated while the aqueous layer was
extracted with CH2Cl2 (3 × 10 mL). The combined organic
extracts were dried over sodium sulfate and evaporated to
dryness. The desired product, 12 (81 mg, 85%), was obtained
1
as a colorless oil: Rf 0.2 (EtOAc); H NMR (CDCl3) δ 1.35-
1.60 (m, 6 H), 1.80-1.91 (br, 2 H), 2.85-2.96 (m, 2 H), 3.15-
3.35 (br, 6 H), 3.92-4.13 (m, 2 H), 5.05-5.11 (m, 6 H), 7.15-
7.34 (m, 16 H); 13C NMR (CDCl3) δ 156.6, 156.2, 136.8, 136.4,
129.9, 128.8, 128.6, 128.4, 128.2, 128.0, 127.3, 67.9, 67.2, 66.7,
63.6, 53.6, 47.5, 44.4, 40.7, 38.6 (d, 148.5), 30.6, 27.2, 25.4; 31
P
NMR (CDCl3) δ 20.5; MS (FAB) m/ z (rel intensity) 598 (MH+,
8), 688 (9), 508 (5), 397 (5), 91 (100); HRMS (FAB) calcd for
C31H40N3O7PH (MH+) 598.2682, found 598.2628. This mate-
rial was used in the next step without further purification.
O-Ben zyl-O-[3-[N-[4-[N-(b en zyloxyca r b on yl)a m in o]-
b u t yl]-N -(b e n zy lox yc a r b o n y l)a m in o ]p r o p y l][[[(r-O-
b en zyl-Z-glu t a m yl)a la n yl]a m in o]m et h yl]p h osp h on a t e
(14). To a stirred solution of 13 (61 mg, 0.14 mmol), 12 (82
mg, 0.14 mmol), and BOP reagent (67 mg, 0.15 mmol) in dry
CH2Cl2 (8 mL) was added DIEA (54 mg, 0.41 mmol) slowly
through a syringe at 0 °C. The reaction mixture was stirred
at 0 °C for 15 min and then at room temperature for 1.5 h.
After removal of the volatile components, the residue was
partitioned between EtOAc and H2O (40/20 mL), the layers
were separated, and the aqueous layer was extracted with
EtOAc (2 × 20 mL). The combined EtOAc extracts were
washed successively with 5% citric acid (2 × 30 mL), H2O (30
mL), 5% NaHCO3 solution (2 × 30 mL), H2O (30 mL), and
brine (40 mL) and then dried over Na2SO4. After removal of
the solvent, the product was purified by column chromatog-
raphy on silica gel eluting with a gradient of EtOAc to MeOH/
EtOAc (1:9) to give 0.11 g (81%) of 14 as a colorless oil: 1H
NMR (DMSO-d6) δ 1.14 (d, 3 H, J ) 7), 1.25-1.38 (m, 2 H),
1.40-1.50 (m, 2 H), 1.70-1.86 (m, 3 H), 1.94-2.03 (br, 1 H),
2.22 (t, 2 H, J ) 1.7), 2.94-3.02 (m, 2 H), 3.11-3.23 (m, 4 H),
3.54-3.67 (m, 2 H), 3.89-3.97 (m, 2 H), 4.06-4.14 (m, 1 H),
4.28-4.36 (m, 1 H), 4.95-5.14 (m, 10 H), 7.36 (m, 27 H), 7.80
(d, 1 H, J ) 6), 8.04 (br, 1 H), 8.32 (br, 1 H); 13C NMR (CDCl3)
δ 173.0, 172.6, 172.5, 156.5, 136.8, 136.4, 136.1, 136.7, 135.4,
128.8, 128.7, 128.4, 128.2, 127.9, 68.3, 68.2, 68.1, 67.4, 67.2,
66.8, 64.2, 63.7, 53.8, 49.0, 46.7, 43.9, 40.7, 35.8-34.1 (d, 1J P-C
) 158.7), 32.1, 29.1, 28.0, 27.2, 25.8, 25.4; 31P NMR (CDCl3) δ
23.8; MS (FAB) m/ z (rel intensity) 1022 (MH+, 1.3), 91 (100).
Anal. (C54H64N5O13P‚0.5H2O) C, H, N.
(d, 2 H, J ) 7.2), 7.20-7.40 (m, 5 H), 7.75-7.95 (m, 4 H); 13
C
NMR (DMSO-d6) δ 166.2, 136.0, 134.4, 131.3, 128.1, 127.7,
127.3, 123.0, 66.2 (d, J ) 5.51), 34.5 (d, J ) 150.1); 31P NMR
(D2O/NaOD) δ 14.3. Anal. (C16H14NO5P‚0.5H2O) C, H, N.
O-Ben zyl-O-[3-[N-[4-[N-(b en zyloxyca r b on yl)a m in o]-
b u t yl]-N -(b e n zyloxyca r b on yl)a m in o]p r op yl](p h t h a l-
im id om et h yl)p h osp h on a t e (11). A. Oxa lyl Ch lor id e
Meth od . To a suspension of the phosphonic monoester 10
(0.14 g, 0.42 mmol) in dry CH2Cl2 (15 mL) was added oxalyl
chloride (0.2 g, 1.65 mmol) slowly at room temperature. After
2 h stirring, the resultant clear solution was concentrated
under reduced pressure to afford a white solid which was
redissolved in dry toluene, reconcentrated, and dried in vacuo.
The resulting crude phosphonochloridate (31P NMR (CDCl3) δ
32) was used in the next step without further purification; it
was dissolved in dry CH2Cl2 (10 mL) followed by the addition
of 7 (0.12 g, mmol) and DMAP (10 mg) at 0 °C under N2. The
resultant solution was allowed to stir at room temperature
overnight. The reaction mixture was diluted with CH2Cl2 (30
mL) and washed subsequently with H2O (1 × 30 mL), 5% citric
acid (2 × 30 mL), H2O (30 mL), 5% NaHCO3 solution (2 × 30
mL), H2O (30 mL), and brine (30 mL). After being dried over
sodium sulfate, the solvent was removed to afford 0.22 g of a
colorless oil which was purified by column chromatography
eluting with EtOAc; 0.19 g (89%) of 11 was obtained as an oil.
Spectral properties are identical with those described for 11
prepared via method B.
Hyd r ogen O-[3-[N-(4-Am in obu tyl)a m in o]p r op yl][[(γ-
glu ta m yla la n yl)a m in o]m eth yl]p h osp h on a te (4). A mix-
ture of 13 (56 mg, 0.077 mmol) and 10% Pd/C (49 mg) in MeOH
(15 mL) and AcOH (1 mL) was shaken under H2 at 44 psi for
28 h. The catalyst was removed by filtration and the oily
residue, obtained after the removal of MeOH, was dissolved
in H2O and purified by ion exchange chromatography (AG
B. Mitsu n obu Meth od . To a stirred solution of 10 (0.11
g, 0.34 mmol) were added 8 (0.14 g, 0.34 mmol) and tri-
phenylphosphine (88 mg, 0.34 mmol) in dry THF (10 mL)
followed by dropwise addition of DEAD (63.3 mg, 0.37 mmol)
in dry THF (2 mL) at room temperature. The resultant yellow
solution was stirred at room temperature for 8 h and then
concentrated under reduced pressure. The residue was dis-
solved in EtOAc (40 mL) and washed successively with 5%
citric acid (2 × 20 mL), H2O (20 mL), and brine (30 mL).
Removal of the solvent after being dried over Na2SO4 afforded
crude 11 as a dense oil which was purified by column
chromatography eluting with 100% EtOAc; 0.15 g (61%) of 11
was obtained as an oil: 1H NMR (CDCl3) δ 1.32-1.60 (br, 4
H), 1.77-1.89 (br, 2 H), 3.11-3.28 (m, 6 H), 3.94-4.22 (m, 4
H), 5.04-5.28 (m, 7 H), 7.30 (s, 15 H), 7.64-7.73 (m, 2 H),
7.75-7.86 (m, 2 H); 13C NMR (CDCl3) δ 166.8, 156.5, 156.1,
136.9, 134.2, 131.9, 129.7, 128.6, 128.5, 128.2, 128.0, 127.9,
+
50W-X8 cation exchange resin, NH4 form) eluting with 0.08
M NH4HCO3 to afford 19.5 mg (83%) of 4 as a hygroscopic
solid: Rf 0.32 (MeOH/AcOH (2:1), cellulose); [R]25 -22.8° (c
D
1
0.24, MeOH); H NMR (D2O) δ 1.36 (d, 3 H, J ) 7.4), 1.70-
1.81 (m, 4 H), 1.90-2.08 (m, 4 H), 2.40 (t, 2 H, J ) 8.8), 2.95-
3.12 (m, 6 H), 3.46 (d, 2 H, J ) 11.8), 3.45-3.54 (m, 1 H), 3.9-
3.98 (q, 2 H), 4.24-4.32 (q, 1 H); 13C NMR (MeOH-d4) δ 178.6,
176.1, 175.6, 63.6, 56.0, 51.0, 48.3, 46.4, 40.1, 36.8 (d, J )
148.1), 32.8, 29.5, 28.3, 25.8, 24.3, 17.8; 31P NMR (D2O) δ 18.5;
MS (FAB) m/ z (rel intensity) 440 (MH+, 100), 309 (52); HRMS

3848 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Chen et al.
(FAB) calcd for C16H34N5O7PH (MH+) 440.2274, found 440.2297.
Anal. (C16H34N5O7P‚H2CO3‚0.5H2O) C, H, N.
The organic extracts were combined and dried over Na2SO4.
Removal of the solvent in vacuo afforded the corresponding
(aminomethyl)phosphonamidate which was dissolved in dry
CH2Cl2 (20 mL/mmol 15 or 19). Dipeptide 13 (1 mol equiv)
and BOP reagent (1.2 mol equiv) were then added followed by
the slow addition of DIEA (2.0 mol equiv) at 0 °C. The reaction
was continued at 0 °C for 10 min and then at room tempera-
ture for 1.5 h. After a standard extractive workup, the product
was purified by recrystallization or column chromatography
on silica gel. Crystalline products (17, 20a ) melt over a broad
range due to the presence of diastereomeric mixtures.
Gen er a l P r oced u r e for t h e Syn t h esis of P h osp h on -
a m id a tes. Meth od A.16 Phosphonochloridate (1.0-1.4 mol
equiv), prepared from the corresponding phosphonate mo-
noester (10 or 18) and oxalyl chloride, was dissolved in dry
CH2Cl2 (10-30 mL/mmol); TEA (1.2-2.4 mol equiv) and a
catalytic amount of DMAP were added followed by the slow
addition of the corresponding amine (1 mol equiv) at 0 °C. The
reaction was continued at 0 °C for 30 min and at room
temperature overnight. The reaction mixture was then worked
up as described under method B.
Meth od B. To a stirred solution of the phosphonate
monoester (10 or 18, 1 mol equiv), the amine 9 (1 mol equiv),
and BOP reagent (1.2 mol equiv) in dry CH2Cl2 was added
DIEA (2 mol equiv) dropwise at 0 °C. The reaction mixture
was then allowed to stir at 0 °C for 10 min and at room
temperature for 1.5 h. The volatile components were removed
under reduced pressure, the residue was partitioned between
EtOAc/H2O (1:1, 60 mL), the EtOAc layer was separated, and
the aqueous layer was extracted with EtOAc (2 × 30 mL). The
combined EtOAc extracts were washed successively with 5%
citric acid (2×), H2O (1×), 5% NaHCO3 solution (2×), H2O (1×),
and brine (1×), and dried over sodium sulfate. After removal
of the solvent, the products were purified by recrystallization
or column chromatography.
15a : mp 124-126 °C; Rf 0.62 (EtOAc); method A, 19%;
method B, 100%; 1H NMR (CDCl3) δ 0.86 (t, 3 H, J ) 7), 1.22-
1.51 (m, 4 H), 2.86-3.10 (m, 3 H), 4.05-4.20 (m, 2 H), 5.10 (d,
2 H, J ) 7), 7.16-7.43 (m, 5 H), 7.71-7.78 (m, 2 H), 7.80-
7.89 (m, 2 H); 13C NMR δ 166.5, 134.2, 132.1, 128.6, 128.3,
127.9, 124.9, 123.6, 120.5, 60.4, 40.6, 35.2 (d, J ) 127), 34.3,
20.3, 14.2; 31P NMR δ 24.4; MS (FAB) m/ z (rel intensity) 387
(MH+, 25), 259 (26), 219 (66), 145 (45), 136 (41), 91 (100);
HRMS (FAB) calcd for C20H23N2O4PH (MH+) 387.1474, found
387.1468.
17: mp 150-158 °C; 89% from 15a ; 1H NMR (CDCl3) δ
0.81-0.90 (q, 3 H), 1.25-1.40 (m, 7 H), 1.90-2.25 (m, 4 H),
2.75-2.95 (m, 2 H), 3.25-3.50 (m, 2 H), 3.75-3.86 (m, 1 H),
4.36-4.54 (m, 2 H), 4.90-5.00 (m, 1 H), 5.05-5.12 (m, 6 H),
6.30 (br d, 1 H), 6.65 (br d, 1 H), 7.15-7.35 (m, 15 H); 13C
NMR δ 173.0, 172.2, 172.1, 155.6, 136.4, 135.4, 128.8, 128.7,
128.5, 128.4, 127.9, 67.4, 67.2, 66.0, 53.9 (53.8), 49.4 (49.2),
36.8 (d, J ) 143), 35.8 (d, J ) 143), 34.1, 32.2, 28.2 (28.0),
19.9, 18.5 (18.2), 13.9; 31P NMR δ 28.6, 28.5; MS (FAB) m/ z
(rel intensity) 681 (MH+, 3), 608 (11), 154 (21), 91 (100); HRMS
(FAB) calcd for C35H45N4O8PH (MH) 681.3052, found 681.3064.
20a : mp 98-103 °C; 82% from 19a ; 1H NMR (CDCl3) δ
0.80-0.92 (m, 3 H), 1.15-1.45 (m, 7 H), 1.95-2.15 (dm, 2 H),
2.26-2.38 (m, 2 H), 2.65-2.88 (m, 2 H), 3.40-3.70 (m, 5 H),
4.05-4.35 (m, 1 H), 4.31-4.49 (m, 2 H), 4.99-5.12 (m, 5 H),
6.25-6.45 (m, 1 H), 7.05-7.45 (m, 10 H), 7.62-7.76 (br, 1 H);
13C NMR δ 173.5, 172.6, 172.2, 156.6, 136.3, 135.4, 128.8,
128.6, 128.4, 128.3, 67.4, 67.2, 53.7, 50.9, 49.5, 40.6, 36.7 (d, J
) 143) (35.5 (d, J ) 145)), 34.0 (d, J ) 5), 31.9, 27.9, 19.9,
18.1, 13.9; 31P NMR δ 29.8; MS (FAB) m/ z (rel intensity) 605
(MH+, 4), 532 (19), 134 (9), 91 (100); HRMS (FAB) calcd for
C29H41N4O8PH (MH) 605.2740, found 605.2724.
20b: oil; Rf 0.5 (EtOAc/MeOH/TEA (16:4:1)); 91% from 19b;
1H NMR (CDCl3) δ 1.31 (d, J ) 7), 1.41-1.74 (overlap, 6 H),
2.05 (dm, 2 H), 2.20-2.29 (overlap, 2 H), 2.81-2.95 (overlap,
2 H), 3.05-3.31 (m, 6 H), 3.41-3.72 (overlap, 5 H), 3.80-4.02
(br, 1 H), 4.27-4.62 (overlap, 2 H), 4.95-5.10 (m, 8 H), 5.21-
5.32 (br, 1 H), 6.50-6.63 (br, 1 H), 6.95-6.62 (br, 1 H), 7.15-
7.45 (m, 20 H), 7.70-7.82 (br, 1 H); 13C NMR δ 173.0, 172.1
(×2), 156.5, 156.0, 136.7, 136.3, 135.3, 128.5, 128.1, 127.7, 67.1,
66.8, 66.5, 53.8, 50.8, 48.9, 46.5, 44.4, 41.0 (40.6), 38.3 (37.7),
35.8 (d, J ) 142), 32.0, 30.1, 27.6, 27.1, 25.7 (25.3), 18.4; 31P
NMR δ 29.2, 29.1, 28.9; MS (FAB) m/ z (rel intensity) 945
(MH+, 1.3), 793 (1.6), 523 (8.2), 414 (47), 91 (100); HRMS (FAB)
calcd for C48H61N6O12PH (MH+) 945.4163, found 945.4199.
Anal. (C48H61N6O12P) H, N; C: calcd, 60.98; found, 60.54.
O-Meth yl-N-bu tyl[[(γ-glu ta m yla la n yl)a m in o]m eth yl]-
p h osp h on a m id a te (21a ). O-Methyl-N-butyl[[[(Z-R-O-benzyl-
γ-glutamyl)alanyl]amino]methyl]phosphonamidate (20a ; 50
mg, 0.08 mmol) and 10% Pd/C (20 mg) in EtOH (15 mL) were
shaken under H2 (40 psi) on a Parr hydrogenator for 12 h, and
the catalyst was then removed by filtration. The filtrate was
concentrated to afford the product as a syrup which was
triturated with ether to afford a white powder; 30 mg (95%)
of pure 21a was obtained by crystallization from EtOH/Et2O:
mp 178-189 °C dec (softens at 118 °C); 1H NMR (MeOH-d4) δ
0.87 (t, 3 H, J ) 7), 1.21-1.48 (m, 7 H), 2.08-2.21 (m, 2 H),
2.47 (t, 2 H, J ) 6.5), 2.81-2.94 (m, 2 H), 3.45-3.72 (m, 5 H),
3.86 (t, 1 H, J ) 6), 4.20-4.39 (q, 1 H); 13C NMR δ 175.3, 174.5,
172.6, 54.3, 51.8, 50.9, 41.5, 37.0 (d, J ) 144), 35.6, 32.5, 27.4,
21.0, 18.0, 14.3; 31P NMR δ 31.0; MS (FAB) m/z (rel intensity)
381 (MH+, 100), 308 (94), 201 (12), 180 (21); HRMS (FAB) calcd
for C14H29N4O6PH (MH+) 381.1903, found 381.1888.
15b: oil; Rf 0.45 (EtOAc); method A, trace; method B, 73%;
1H NMR (CDCl3 + MeOH-d4) δ 1.40-1.49 (overlap, 4 H), 1.51-
1.73 (m, 2 H), 2.89-3.10 (br, 2 H), 3.10-3.78 (m, 6 H), 4.05-
4.13 (m, 2 H), 5.06 (m, 6 H), 7.15-7.76 (m, 15 H), 7.68-7.75
(m, 2 H), 7.78-7.89 (m, 2 H); 13C NMR δ 167.3, 157.0, 155.8,
136.4, 134.3, 131.7, 129.7, 128.4, 128.3, 128.0, 127.7, 127.6,
126.9, 123.4, 67.1, 66.4, 46.4 (46.1), 4.2, 37.8 (37.3), 40.3 (d, J
) 12), 37.8 (37.3), 34.9 (d, J ) 143), 30.7 (29.8), 26.8 (d, J )
10), 25.6 (25.0); 31P NMR δ 24.7; MS (FAB) m/ z (rel intensity)
727 (MH+, 9), 511 (5), 307 (8), 153 (48), 91 (100); HRMS (DCI/
CH4) calcd for C39H43N4O4PH 727.2897, found 727.2903.
19a : mp 113-115 °C; Rf 0.44 (MeOH/EtOAc (8:92)); method
1
A, 70%; H NMR (CDCl3) δ 0.89 (t, 3 H, J ) 7.2), 1.25-1.39
(m, 2 H), 1.40-1.54 (m, 2 H), 2.75-2.89 (m, 1 H), 2.90-3.06
(m, 2 H), 3.73 (d, 3 H, J ) 11), 3.95-4.14 (m, 2 H), 7.70-7.80
(m, 2 H), 7.85-7.95 (m, 2 H); 13C NMR δ 167.4, 134.3, 132.0,
123.6, 51.8, 40.6, 34.8 (d, J ) 141), 34.4, 20.0, 13.9; 31P NMR
δ 25.3. Anal. (C14H19N2O4P) C, H, N.
19b: mp 57-59 °C; Rf 0.45 (EtOAc/TEA (4:1)); method A,
1
48%; method B, 61%; H NMR (CDCl3) δ 1.35-1.65 (overlap,
4 H), 1.65-1.75 (t, 2 H), 2.90-3.05 (overlap, 2 H), 3.10-3.42
(m, 7 H), 3.55-3.70 (m, 3 H), 3.75-4.05 (m, 2 H), 5.04 (s, 4
H), 7.15-7.40 (m, 10 H), 7.60-7.70 (m, 2 H), 7.75-7.85 (m, 2
H); 13C NMR δ 167.0, 156.4, 136.5, 134.0, 131.6, 128.3, 128.2,
127.8, 127.5, 123.2, 66.8, 66.2, 51.2 (d, J ) 5.9), 46.9, 46.2,
46.2, 43.9, 40.3, 37.8, 37.2, 34.5 (d, J ) 141), 34.3 (d, J ) 141),
30.9, 30.0, 26.9, 25.5, 25.0; 31P NMR δ 25.5; MS (DCI/CH4)
m/ z (rel intensity) 651 (MH+, 11), 442 (31), 414 (93), 273 (100);
HRMS (DCI/CH4) calcd for C33H39N4O8PH 651.2584, found
651.2581. Anal. (C33H39N4O8P) C, H, N.
O-Met h yl-N-[3-[N-(4-a m in ob u t yl)a m in o]p r op yl][[(γ-
glu ta m yla la n yl)a m in o]m eth yl]p h osp h on a m id a te (21b).
To a solution of 20b (0.1 g, 0.1 mmol) in absolute EtOH (14
mL) was added a suspension of 10% Pd/C in EtOH (4 mL),
and the mixture was shaken under H2 (40 psi) on a Parr
hydrogenator for 12 h. The catalyst was then removed by
filtration, and the filtrate was concentrated under reduced
pressure. The resultant residue was triturated with Et2O/
EtOH (20:1, v/v), and 38 mg (78%) of 21b was obtained as a
hygroscopic powder: Rf 0.64 (MeOH/AcOH/H2O (4:1:1), cel-
lulose); 1H NMR (D2O + MeOH-d4) δ 1.34 (d, 3 H, J ) 7), 1.62-
1.75 (br, 4 H), 1.76-1.87 (overlap, 2 H), 2.04-2.10 (m, 2 H),
Gen er a l P r oced u r e for th e Syn th esis of O-Alk yl-N-
a lk yl-[[[(Z-r-O-ben zyl-γ-glu ta m yl)a la n yl]a m in o]m eth yl]-
p h osp h on a m id a tes. A mixture of phosphonamidate 15 or
19 (1 mol equiv) and H2NNH2‚H2O (10 mol equiv) in MeOH
(ca. 10 mL/mmol) was stirred at room temperature for 48 h.
The precipitate that formed was removed by filtration, and
the filtrate was concentrated under reduced pressure. The
resultant residue (white semisolid in most cases) was parti-
tioned between CH2Cl2 and NH4OH. CH2Cl2 layer was
separated, and the aqueous layer was extracted with CH2Cl2.

Phosphapeptide Inhibitors of Gsp Synthetase/ Amidase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3849
(6) Shames, S. L.; Fairlamb, A. H.; Cerami, A.; Walsh, C. T.
Purification and characterization of trypanothione reductase
from Crithidia Fasciculata, a newly discovered member of the
family of disulfide-containing flavoprotein reductases. Biochem-
istry 1986, 25, 3519-3526.
2.35-2.46 (t, 2 H, J ) 6), 2.89-3.09 (overlap, 8 H), 3.51-3.70
(m, 6 H), 4.29-4.28 (q, 1 H); 13C NMR δ 176.0, 175.6, 175.1,
55.3, 52.8, 51.0, 48.0, 46.2, 39.9, 38.3, 36.8 (d, J ) 143), 32.2,
29.1, 27.8, 25.1, 23.9, 17.6; 31P NMR δ 31.5, 31.4; MS (FAB)
m/ z (rel intensity) 453 (MH+, 9), 202 (38), 180 (100); HRMS
(FAB) calcd for C17H37N6O6PH (MH) 453.2590, found 453.2575.
All attempts to obtain an analytical sample of 21b by use of
ion exchange chromatography failed due to product instability
during chromatography.
En zym e In h ibition Assa ys. Inhibition of Gsp synthetase
by the phosphonate 421 and phosphonamidates 21a ,b was
observed spectrophotometrically by coupling the hydrolysis of
ATP to oxidation of NADH via pyruvate kinase/lactate dehy-
drogenase reactions.20 The assay was initiated by adding
purified Gsp synthetase (12.8 nM) to an assay mixture which
contained the following components (final concentration): 1.56
mM glutathione, 10 mM spermidine, 2 mM ATP, 2.7 mM
MgCl2, 1 mM phospho(enol)pyruvate, 0.2 mM NADH, 50 µg/
mL lactate dehydrogenase, 100 µg/mL pyruvate kinase, and
various concentrations of inhibitor in 50 mM NaPIPES (pH
6.8) at 37 °C.
(7) Kuriyan, J .; Kong, X. P.; Krishna, T. S.; Sweet, R. M.; Murgolo,
N. J .; Field, H.; Cerami, A.; Henderson, G. B. X-ray structure of
trypanothione reductase from Crithidia fasciculata at 2.4-A
resolution. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8764-8768.
(8) Stoll, V. S.; Simpson, S. J .; Krauth-Siegel, R. L.; Walsh, C. T.;
Pai, E. F. Glutathione reductase turned into trypanothione
reductase: Structural analysis of an engineered change in
substrate specificity. Biochemistry 1997, 36, 6437-6447.
(9) Henderson, G. B.; Murgolo, N. J .; Kuriyan, J .; Osapay, K.;
Kominos, D.; Berry, A.; Scrutton, N. S.; Hinchliffe, N. W.;
Perham, R. N.; Cerami, A. Engineering the substrate specificity
of glutathione reductase toward that of trypanothione reduction.
Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8769-8773.
(10) Faerman, C. H.; Savvides, S. N.; Strickland, C.; Breidenbach,
M. A.; Ponasik, J . A.; Ganem, B.; Ripoll, D.; Krauth-Siegel, R.
L.; Karplus, P. A. Charge is the major discriminating factor for
glutathione reductase versus trypanothione reductase inhibitors.
Bioorg. Med. Chem. 1996, 4, 1247-1253.
(11) Cenas, N. K.; Arscott, D.; Williams, J . C. H.; Blanchard, J . S.
Mechanism of reduction of quinones by Trypanosoma congolense
trypanothione reductase. Biochemistry 1994, 33, 2509-2515.
(12) J acoby, E. M.; Schlichting, I.; Lantwin, C. B.; Kabsch, W.;
Krauth-Siegel, R. L. Crystal structure of the Trypanosoma cruzi
trypanothione reductase‚mepacrine complex. Proteins: Struct.,
Funct., Genet. 1996, 24, 73-80.
(13) Krauth-Siegel, R. L.; Scho¨neck, R. Trypanothione reductase and
lipoamide dehydrogenase as targets for a structure-based drug
design. FASEB J . 1995, 9, 1138-1146.
(14) Yin, H.; Chan, C.; Garforth, J .; Douglas, K. T.; Bolgar, M. S.;
Gaskell, S. J .; Fairlamb, A. H. Fluphenazine photoaffinity
labelling of binding sites for phenothiazine inhibitors of
trypanothione reductase. J . Chem. Soc., Chem. Commun. 1996,
973-974.
(15) Fairlamb, A. H. Metabolism and functions of trypanothione in
the kinetoplastida. Annu. Rev. Microbiol. 1992, 46, 695-729.
(16) Malachowski, W. P.; Coward, J . K. The chemistry of phos-
phapeptides: Formation of functionalized phosphonochloridates
under mild conditions and their reaction with alcohols and
amines. J . Org. Chem. 1994, 59, 7616-7624.
(17) Verbruggen, C.; De Craecker, S.; Rajan, P.; J iao, X.-Y.; Borloo,
M.; Smith, K.; Fairlamb, A. H.; Haemers, A. Phosphonic acid
and phosphinic acid tripeptides as inhibitors of glutathionyl-
spermidine synthetase. Bioorg. Med. Chem. Lett. 1996, 6, 253-
258.
(18) Smith, K.; Nadeau, K.; Bradley, M.; Walsh, C.; Fairlamb, A. H.
Purification of glutathionylspermidine and trypanothione syn-
thetases from Crithidia fasciculata. Protein Sci. 1992, 1, 874-
883.
(19) Koenig, K.; Menge, U.; Kiess, M.; Wray, V.; Flohe´, L. Convenient
isolation and kinetic mechanism of glutathionylspermidine
synthetase from Crithidia fasciculata. J . Biol. Chem. 1997, 272,
11908-11915.
(20) Bollinger, J . M., J r.; Kwon, D. S.; Huisman, G. W.; Kolter, R.;
Walsh, C. T. Glutathionylspermidine metabolism in Escherichia
coli. J . Biol. Chem. 1995, 270, 14031-14041.
(21) Kwon, D. S.; Lin, C.-H.; Chen, S.; Coward, J . K.; Walsh, C. T.;
Bollinger, J . M., J r. Dissection of glutathionylspermidine syn-
thetase/amidase from E. coli into autonomously folding and
functional synthetase and amidase domains. J . Biol. Chem.
1997, 272, 2429-2436.
(22) Craecker, S. D.; Verbruggen, C.; Rajan, P. K.; Smith, K.;
Haemers, A.; Fairlamb, A. H. Characterization of the peptide
substrate specificity of glutathionylspermidine synthetase from
Crithidia fasciculata. Mol. Biochem. Parasitol. 1997, 84, 25-
32.
(23) Banerjee, R. V.; Shane, B.; McGuire, J . J .; Coward, J . K.
Dihydrofolate synthetase and folylpolyglutamate synthetase:
Direct evidence for intervention of acyl phosphate intermediates.
Biochemistry 1988, 27, 9062-9070.
(24) Mullins, L. S.; Zawadzke, L. E.; Walsh, C. T.; Raushel, F. M.
Kinetic evidence for the formation of D-alanyl phosphate in the
mechanism of D-alanyl-D-alanine ligase. J . Biol. Chem. 1990,
265, 8993-8998.
(25) Fan, C.; Moews, C.; Walsh, C. T.; Knox, J . R. Vancomycin
resistance: Structure of D-alanine:D-alanine ligase at 2.3 Å
resolution. Science 1994, 266, 439-443.
Noncompetitive or mixed-type inhibition was analyzed
according to Scheme 4. Scheme 5 was used as the basis for
the analysis of slow-binding inhibition. Ki and Ki* are defined
as shown in Scheme 5.52
The inhibition of Gsp amidase activity was assayed by
coupling the production of EtOH (due to the hydrolysis of a
substrate analog, glutathione ethyl ester) to the reduction of
NAD through the activities of alcohol dehydrogenase and
aldehyde dehydrogenase.21 The assay mixture contained the
following components: 1 mM NAD, 0.5 mg (170 units) of
alcohol dehydrogenase, 0.07 mg (4 units) of aldehyde dehy-
drogenase, 1.28 nM purified Gsp amidase fragment, 2.5 mM
GSH ethyl ester, and various concentrations of inhibitor in
50 mM NaPIPES (pH 6.8) at 37 °C.
Ack n ow led gm en t. This investigation received fi-
nancial support from the UNDP/WORLD BANK/WHO
Special Programme for Research and Training in Tropi-
cal Diseases (J .K.C.). Additional support was received
from NIH Grant GM20011 (C.T.W.). Much of the
groundbreaking research on phosphapeptide synthesis
in our laboratory was done by Dr. William Malachowski,
and we are indebted to him for his contributions to this
research. The syntheses of 6 and 7 were originally
carried out by Dr. J ohn Lakanen, and we gratefully
acknowledge his contribution to this research. We would
like to thank Ms. Carol Capelle for careful preparation
of this manuscript and Prof. J . Martin Bollinger (Penn-
sylvania State University) for his interest and helpful
discussions.
Su p p or tin g In for m a tion Ava ila ble: Detailed procedures
for the synthesis of 7 and 13, including peptide precursors of
13 (2 pages). Ordering information is given on any current
masthead page.
Refer en ces
(1) Despommier, D. D.; Gwadz, R. W.; Hotez, P. J . Parasitic
Diseases, 3rd ed.; Springer-Verlag New York, Inc.: New York,
1995.
(2) Hightower, R. C.; Santi, D. V. Drug action and drug resistance:
Antifolate resistance in parasitic protozoa. In Parasitic Infec-
tions; Churchill Livingstone Inc.: New York, 1988; Vol. 7, pp
81-95.
(3) Schirmer, R. H.; Mu¨ller, J . G.; Krauth-Siegel, R. L. Disulfide-
reductase inhibitors as chemotherapeutic agents: The design
of drugs for trypanosomiasis and malaria. Angew. Chem., Int.
Ed. Engl. 1995, 34, 141-154.
(4) Walsh, C.; Bradley, M.; Nadeau, K. Molecular studies on
trypanothione reductase, a target for antiparasitic drugs. TIBS
1991, 16, 305-309.
(26) Radzicka, A.; Wolfenden, R. Transition state and multisubstrate
analog inhibitors. Methods Enzymol. 1995, 249, 284-312.
(27) Rahil, J .; Pratt, R. F. Mechanism of inhibition of the Class C
Y-lactamase of Enterobacter cloacae P99 by phosphonate mo-
noesters. Biochemistry 1992, 31, 5869-5878.
(28) Morgan, B. P.; Holland, D. R.; Matthews, B. W.; Bartlett, P. A.
Structure-based design of an inhibitor of the zinc peptidase
thermolysin. J . Am. Chem. Soc. 1994, 116, 3251-3260.
(5) Fairlamb, A. H.; Blackburn, P.; Ulrich, P.; Chait, B. T.; Cerami,
A. Trypanothione: A novel bis(glutathionyl)spermidine cofactor
for glutathione reductase in trypanosomatids. Science 1985, 227,
1485-1487.

3850 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Chen et al.
(29) J ira´cek, J .; Yiotakis, A.; Vincent, B.; Lecoq, A.; Nicolaou, A.;
Checler, F.; Dive, V. Development of highly potent and selective
phosphinic peptide inhibitors of zinc endopeptidase 24-15 using
combinatorial chemistry. J . Biol. Chem. 1995, 270, 21701-
21706.
(30) Bartlett, P. A.; Giangiordano, M. A. Transition state analogy of
phosphonic acid peptide inhibitors of pepsin. J . Org. Chem. 1996,
61, 3433-3438.
(31) Parsons, W. H.; Patchett, A. A.; Bull, H. G.; Schoen, W. R.; Taub,
D.; Davidson, J .; Combs, P. L.; Springer, J . P.; Gadebusch, H.;
Weissberger, B.; Valiant, M. E.; Mellin, T. N.; Busch, R. D.
Phosphinic acid inhibitors of D-alanyl-D-alanine ligase. J . Med.
Chem. 1988, 31, 1772-1778.
(32) Logusch, E. W.; Walker, D. M.; McDonald, J . F.; Franz, J . E.
Substrate variability as a factor in enzyme inhibitor design:
Inhibition of ovine brain glutamine synthetase by R- and
γ-substituted phosphinothricins. Biochemistry 1989, 28, 3043-
3051.
(33) Hiratake, J .; Kato, H.; Oda, J . Mechanism-based inactivation
of glutathione synthetase by phosphinic acid transition-state
analogue. J . Am. Chem. Soc. 1994, 116, 12059-12060.
(34) Malachowski, W. P. Ph.D. Thesis, The University of Michigan,
Ann Arbor, MI, May 1993.
(35) Lakanen, J . R. Ph.D. Thesis, The University of Michigan, Ann
Arbor, MI, J anuary 1994.
(36) O¨ sapay, G.; Szila´gyi, I.; Seres, J . Conversion of amino acids and
dipeptides into their phosphonic analogs. Tetrahedron 1987, 43,
2977-2983.
(37) Hoffmann, M. A convenient synthesis of alkyl hydrogen 1-(benz-
yloxycarbonylamino)-alkanephosphonates. Synthesis 1986, 557-
558.
phate-mediated activation of monophosphonate esters: Synthesis
of mixed phosphonate diesters, the reactivity of the benzotria-
zolyl phosphonic esters vs the reactivity of the benzotriazolyl
carboxylic esters. J . Org. Chem. 1995, 60, 5214-5223.
(43) Rahil, J .; Haake, P. Reactivity and mechanism of hydrolysis of
phosphonamides. J . Am. Chem. Soc. 1981, 103, 1723-1734.
(44) J acobsen, N. E.; Bartlett, P. A. A phosphonamidate dipeptide
analogue as an inhibitor of carboxypeptidase A. J . Am. Chem.
Soc. 1981, 103, 654-657.
(45) Yamauchi, K.; Ohtsuki, S.; Kinoshita, M. Synthesis of peptide
analogues containing (2-aminoethyl)phosphonic acid (ciliatine).
J . Org. Chem. 1984, 49, 1158-1163.
(46) Kortylewicz, Z. P.; Galardy, R. E. Phosphoramidate peptide
inhibitors of human skin fibroblast collagenase. J . Med. Chem.
1990, 33, 263-273.
(47) Bird, J .; De Mello, R. C.; Harper, G. P.; Hunter, D. J .; Karran,
E. H.; Markwell, R. E.; Miles-Williams, A. J .; Rahman, S. S.;
Ward, R. W. Synthesis of novel N-phosphonoalkyl dipeptide
inhibitors of human collagenase. J . Med. Chem. 1994, 37, 158-
169.
(48) Bartlett, P. A.; Lamden, L. A. Inhibition of chymotrypsin by
phosphonate and phosphonamidate peptide analogs. Bioorg.
Chem. 1986, 14, 356-377.
(49) Chen, S.; Lin, C.-H.; Walsh, C. T.; Coward, J . K. Novel inhibitors
of trypanothione biosynthesis: synthesis and evaluation of a
phosphinate analog of glutathionyl spermidine (Gsp), a potent,
slow-binding inhibitor of Gsp synthetase. Bioorg. Med. Chem.
Lett. 1997, 7, 505-510.
(50) Lacoste, A. M.; Chollet-Gravey, A. M.; Vo-Quang, V.; Vo-Quang,
Y.; Goffic, F. L. Time-dependent inhibition of Streptococcus
faecalis D-alanine:D-alanine ligase by R-aminophosphonamidic
acids. Eur. J . Med. Chem. 1991, 26, 255-260.
(38) Mitsunobu, O. The use of diethyl azodicarboxylate and tri-
phenylphosphine in synthesis and transformation of natural
products. Synthesis 1981, 1-28.
(39) Campbell, D. A.; Bermak, J . C. Phosphonate ester synthesis
using a modified Mitsunobu condensation. J . Org. Chem. 1994,
59, 658-660.
(51) Ellsworth, B. A.; Tom, N. J .; Bartlett, P. A. Synthesis and
evaluation of inhibitors of bacterial D-alanine:D-alanine ligases.
Chem. Biol. 1996, 3, 37-44.
(40) Castro, B.; Dormoy, J . R.; Evin, G.; Selve, C. Reactifs de couplage
peptidique IV (1) - L’hexafluorophosphate de benzotriazolyl
N-oxytrisdimethylamino phosphonium (B.O.P.). (Reagents for
peptide coupling IV (1) - The hexafluorophosphate of benzotria-
zolyl-N-trisdimethylaminooxyphosphonium (B.O.P.).) Tetra-
hedron Lett. 1975, 1219-1222.
(52) Morrison, J .; Walsh, C. T. The behavior and significance of slow-
binding enzyme inhibitors. In Advances in Enzymology and
Related Areas of Molecular Biology; Meister, A., Ed.; J ohn Wiley
& Sons: New York, 1988; Vol. 61, pp 201-301.
(53) Lakanen, J . R.; Pegg, A. E.; Coward, J . K. Synthesis and
biochemical evaluation of adenosylspermidine, A nucleoside-
polyamine adduct inhibitor of spermidine synthase. J . Med.
Chem. 1995, 38, 2714-2727.
(41) Campagne, J .-M.; Coste, J .; Guillou, L.; Heitz, A.; J ouin, P. Solid
phase synthesis of phosphinic peptides. Tetrahedron Lett. 1993,
34, 4181-4184.
(42) Campagne, J .-M.; Coste, J .; J ouin, P. (1H-Benzotriazol-1-yloxy)-
tris(dimethylamino)phosphonium hexafluorophosphate- and (1H-
benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophos-
J M970414B