Design, synthesis, and biological evaluation of phosphinopeptides against Trypanosoma cruzi targeting trypanothione biosynthesis

Article abstract of DOI:10.1021/jm050922i

As a part of our project aimed at the search for new safe chemotherapeutic and chemoprophylactic agents against American trypanosomiasis (Chagas's disease); a series of phosphinopeptides structurally related to glutathione was designed, synthesized, and evaluated as antiproliferative agents against the parasite responsible for this disease, the hemoflagellated protozoan Trypanosoma cruzi. The rationale for the synthesis of these compounds was supported on the basis that the presence of the phosphinic acid moiety would mimic the tetrahedral transition state of trypanothione synthase (TryS), a typical C:N ligase, and the molecular target of these drugs. Of the designed compounds, 53 and 54 were potent growth inhibitors against the clinically more relevant form of T. cruzi (amastigotes) growing in myoblasts. The efficacy for these drugs was comparable to that exhibited by the well-known antiparasitic agent WC-9. The simple phosphinopeptide structure found as a pharmacophore in the present study constitutes a starting point for the development of straightforward optimized drugs.

Full text of DOI:10.1021/jm050922i

426
J. Med. Chem. 2006, 49, 426-435
Design, Synthesis, and Biological Evaluation of Phosphinopeptides against Trypanosoma cruzi
Targeting Trypanothione Biosynthesis
Esteban L. Ravaschino,† Roberto Docampo,‡ and Juan B. Rodriguez†,*
Departamento de Qu´ımica Orga´nica, Facultad de Ciencias Exactas y Naturales, UniVersidad de Buenos Aires, Pabello´n 2, Ciudad
UniVersitaria, C1428EHA, Buenos Aires, Argentina, and Center for Global and Emerging Diseases and Department of Cellular Biology,
UniVersity of Georgia, Athens, Georgia, 30602
ReceiVed September 16, 2005
As a part of our project aimed at the search for new safe chemotherapeutic and chemoprophylactic agents
against American trypanosomiasis (Chagas’s disease), a series of phosphinopeptides structurally related to
glutathione was designed, synthesized, and evaluated as antiproliferative agents against the parasite responsible
for this disease, the hemoflagellated protozoan Trypanosoma cruzi. The rationale for the synthesis of these
compounds was supported on the basis that the presence of the phosphinic acid moiety would mimic the
tetrahedral transition state of trypanothione synthase (TryS), a typical C:N ligase, and the molecular target
of these drugs. Of the designed compounds, 53 and 54 were potent growth inhibitors against the clinically
more relevant form of T. cruzi (amastigotes) growing in myoblasts. The efficacy for these drugs was
comparable to that exhibited by the well-known antiparasitic agent WC-9. The simple phosphinopeptide
structure found as a pharmacophore in the present study constitutes a starting point for the development of
straightforward optimized drugs.
Scheme 1. Oxidized and Reduced Form of Trypanothione
(compounds 1a and 1b, respectively)
Introduction
Prospects in antileishmanial and antitrypanosomal drug
research have changed substantially since the discovery of
trypanothione 1.1 The uniqueness of this metabolite and its
biosynthetic pathway in parasites of the order Kinetoplastida
confer to the involved enzymes of its biosynthesis a great
usefulness as a molecular target. Current chemotherapy is based
on empirically discovered drugs that interact with parasitic
metabolic pathways that have their corresponding counterparts
in mammals; therefore, the efficacy of these drugs is sustained
by the ability of the host to balance metabolic deficiencies with
alternate pathways. In addition, trypanothione is essential for
parasite survival for two reasons: (a) its crucial role in regulating
the cellular redox equilibrium, (b) pathogenic trypanosomes do
not have an alternate mechanism to protect against the oxidative
stress.2 Trypanothione (N1,N8-bis(glutathionyl)spermidine) is the
bisconjugated product between the tripeptide glutathione (2,
GSH) and the polyamine spermidine (3, Spd). The cysteine
residues present in the trypanothione moiety are involved in
the interconversion between its oxidized form [1a, T(S)2] and
reduced form [1b, T(SH)2] by cleaving and restoring an
intramolecular disulfide bridge. Both structures have a +1 net
charge at physiologic pH (Scheme 1).3
The enzyme responsible for the reduction of trypanothione
was isolated and named trypanothione reductase.4-7 This
enzyme, which is highly specific for trypanothione, does not
recognize glutathione as a substrate.
Trypanosomatids, as other aerobic organisms, are exposed
to highly reactive oxygen species such as O2-, H2O2, and HO•.
HO•, and RO• radicals can cause lethal damage if irreversibly
react with DNA or cellular membrane lipids.8 To avoid this
problem, the defense system maintains low levels of H2O2 and
O2-. Superoxide dismutases (SOD) have been identified in T.
cruzi,9 Leishmania donoVani,10 and C. fasciculata. Glutathione
reductase activity is not found in trypanosomes, but a highly
efficient trypanothione reductase activity is present instead.
Glutathione S-transferases are found in more developed organ-
isms, while typanothione S-transferase activity is present in
several parasites such as C. fasciculata, L. major, L. infantum,
L. tarentolae, and T. brucei, associated with the 1B eukaryotic
elongation factor (eEF1B).11 The eEF1B has been expressed,
cloned, and purified in L. major12 catalyzing conjugation
between a variety of electrophiles with 1 and glutathionylsper-
midine (4) but not with 2. Once again, this finding suggests
that trypanothione plays the same role in parasites that glu-
tathione does in mammals.
The last two steps of trypanothione biosynthesis are the most
relevant ones in terms of molecular targets, because they involve
enzymes that have no counterpart in mammals. These flavoen-
zymes are ATP-dependent C:N ligases, which require Mg2+ as
cofactor, catalyze the conjugation of 3 with two molecules of 2
* To whom correspondence should be addressed. E-mail:
jbr@qo.fcen.uba.ar.
† Universidad de Buenos Aires.
‡ University of Georgia.
10.1021/jm050922i CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/01/2005

Phosphinopeptides Targeting Trypanothione Biosynthesis
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 427
Scheme 2. Two Possibilities for Trypanothione Biosynthesis
to yield 1. At the present time is not completely clear how this
conjugation undergoes in all parasites of the order Kinetoplas-
tida. Two possibilities are being investigated: one comprises
two distinct enzymes (GspS and TryS) that catalyze sequentially
the conjugation of 2 with 3 to form 4, and the reaction of this
resulting product with another molecule of 2; the other one
involves only one enzyme (TryS), which is able to catalyze
biosynthesis of 1 directly from 2 and 3 (Scheme 2).13-26
Prospects in searching molecular targets began to change
substantially when T. cruzi TryS was cloned and expressed in
E. coli.27 This protein was not only able to synthesize trypan-
othione from glutathionylspermidine and glutathione but also
from spermidine and glutathione. At this point, no gene encoding
a GspS could be identified in T. cruzi, so it was postulated that
in this parasite a single enzyme catalyzed formation of trypan-
othione.27 The dual catalytic function of T.cruzi TryS, also found
in T. brucei brucei,28 led to investigate C. fasciculata TryS
catalytic properties. The enzyme was cloned and expressed in
E. coli as an active protein that catalyzed trypanothione
formation starting either from glutathionylspermidine and GSH
or from spermidine and GSH.29 In addition, Leishmania major
TryS was cloned and expressed in E. coli.30 On the other hand,
a pseudogene that encodes GspS (GSPS) was identified in this
parasite. This last finding suggests that an evolutionary link
occurs to resolve the observed divergence between C. fasciculata
and Trypanosoma spp.30 It seems reasonable to hypothesize that
an ancestral GSPS with a narrow substrate specificity may have
undergone duplication and divergence into two independent
genes (GSPS and TRYS). This is the case of the substrate-specific
enzymes (GspS and TryS) found in C. fasciculata. Trypanosoma
genome had only retained TRYS; therefore, TryS evolved into
a broad-specificity enzyme superseding the need for two
enzymes in trypanothione biosynthesis.30,31
and TryS have not been resolved yet. The lack of information
of the three-dimensional structure of the target proteins have
circumscribed the search of inhibitors of the enzymatic activity
against these enzymes to the classical chemical structure/
biological activity relationship (SAR). Therefore, the design of
inhibitors of the enzymatic activity for the mentioned ligases
should be based on isosteric replacements on the substrate or
on analogues of the transition state. The inhibition of the
enzymatic activity of these enzymes has been studied and some
data dealing with inhibitors of GSS activity are available. For
example, it has been found that analogues of glutathione in
which isoleucine replaced the cysteine residue to form L-γ-Glu-
L-Ile-Gly (5) or L-γ-Glu-L-Ile-L-Ala (6) exhibited noncompetitive
inhibition with Ki in the low millimolar range.35 Phosphonates
and phosphinates are well-known inhibitors of C:N-ligases.36,37
These findings have led to the design of phosphorus-containing
drugs that behave as inhibitors of the enzymatic activity of GspS.
For example, 7 exhibits linear noncompetitive inhibition with
a Ki value of 60 µM against GspS from C. fasciculata.38
Moreover, when compounds structurally related to 7 are coupled
with spermidine, slow tight-binding inhibitors of GspS were
obtained.39,40 For instance, 8 exhibits a Ki value of 3.2 µM for
binding to free enzyme (GspS from Escherichia coli) and 7.8
nM for binding to enzyme-substrate complex E‚I*, while 9
was much less potent than 7 showing inhibition constants of 6
µM and 14 µM for binding free enzyme and E‚I*, respec-
tively.39,40 These compounds would act as inhibitors of the
transition state at the active site of GspS.
Rationale
As trypanosomatids possess a strict requirement for trypan-
othione for survival and growth that cannot be replaced by
glutathione present in the mammalian host, depletion of this
metabolite will lead either to cell death or to growth impairment.
Bearing in mind that a rather similar mechanism of cell
protection is present in several pathogenic trypanosomes such
as T. cruzi, T. brucei rhodesiense, and T. brucei gambiense,
and Leishmania spp., this approach can be useful for the
treatment of different trypanosomiases and leishmaniasis. Since
this metabolic pathway is absent in the host, it is possible to
envisage that a highly selective inhibition of the enzymatic
activity either of TryS or GspS would not have any toxic effect
against mammalian cells. The crystal structure of TryR is now
available,32-34 while the corresponding X-ray structure for GspS
We have described a new series of analogues of glutathione
(L-γ-Glu-L-Leu-Gly), where the glycine carboxylic acid group
has been substituted by other acidic groups such as tetrazole,
hydroxamic acid, acylsulfonamide, and boronic acid. Among
this new family of compounds, the boronic acid derivative 10
is an effective inhibitor of GspS with a Ki value of 81 µM and
an IC50 value of 17 µM.41 In addition, substitution of the glycine
part on the same tripeptide leads to good inhibitors, specifically
when the glycine moiety is replaced by diaminopropionic acid

428 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
RaVaschino et al.
Scheme 3a
a Reagents and conditions: (a) (i) H2PO2-NH4+, HMDS, 110 °C, 2 h, (ii) RI, CH2Cl2, rt, 16 h; (b) BnOH, DCC, DMAP, THF, rt, 16 h; (c) TrNdCH2,
BF3‚Et2O, MePh, rt, 3 d; (d) TFA (2.5%), CH2Cl2, rt, 30 min; (e) (i) N-hydroxysuccinimide, EDC‚HCl, DMF, rt, 16 h, (ii) Et3N, DMF, rt, 16 h; (f) H2, Pd/C,
MeOH, 1 atm, rt, 1 h.
to form 11. This compound was a potent inhibitor of GspS
activity with Ki ) 7.2 µM.42
Results and Discussion
Compound 8 and 9 represent interesting examples of slow,
tight-binding inhibitors of the enzymatic activity of GspS. These
compounds act by mimicking the transition state for amide bond
In addition, D’Silva et al.43,44 have reported the biological
activity of glutathione derivatives targeting trypanothione
metabolism, where the protected tripeptide 12 arises as main
member of this class of gluthation analogues. This compound
is moderately potent against T. brucei (ED50 ) 1.9 µM) but
exhibits very weak biological activity against T. cruzi.43,44 The
above results encouraged us to prepare analogues assuming that
the optimum glutathione tripeptide derivative should be L-γ-
Glu-L-Leu-Gly. Unexpectedly, N-alkyl amide derivatives of
gluthatione of general formula 13 are devoid of activity against
T. cruzi growth.45 The lack of biological action of 13 might be
attributable to the amidase activity of the target enzyme. This
enzyme, despite being unique for trypanosomatids with a
specific function of catalyzing the first of the two ultimate steps
of trypanothione biosynthesis, would act as a bifunctional
synthetase/amidase.46,47 The loss of the N-alkylamine moiety
would be responsible for the marginal activity observed. A
similar approach for glutathione biosynthesis inhibition was
depicted employing buthionine sulfoxime as substrate ana-
logue.48
formation of this C:N ligase. However, any attempt to optimize
the biological action of these compounds would be impractical
from the synthetic point of view. In fact, the chemical synthesis
of these compounds, or structurally related analogues, requires
many synthetic steps due to the complexity of their structures.
In the search for a simpler pharmacophore than 8, keeping this
structure as lead drug, a series of phosphinopeptide analogues
50-56 was prepared. The peptidic moiety was modified by
replacing the L-cysteine residue by L-leucine on the basis that
this replacement converts an enzyme substrate into an inhibitor
of the enzymatic activity of the same enzyme.38 The phosphinate
unit was retained, while the polyamine residue was changed to
an n-alkyl group. The preparation of the designed compounds
was carried out starting from ammonium hypophosphite, which
on reaction with hexamethyldisilazane at 120 °C for 2 h,49
followed by alkylation with the appropriate alkyl iodide, and
further esterification with benzylic alcohol in the presence of
dicyclohexylurea and 4-(dimethylamino)pyridine was converted
into the corresponding benzyl alkylphosphinate (compounds
22-28) (Scheme 3). The P-alkylation reaction occurred smoothly
if the alkylating agent had up to four carbon atoms. The

Phosphinopeptides Targeting Trypanothione Biosynthesis
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 429
Table 1. Effect of Phosphinopeptides against Trypanosoma cruzi
(amastigotes and epimastigotes) for Compounds 50-56
metabolically stable analogues with similar antiparatic activities.
Efforts in these aspects are currently being pursued in our
laboratory.
amastigotes:
epimastigotes:
compd
IC50 (µM)a
IC50 (µM)a
Experimental Section
50
51
52
53
54
55
56
57
>50
>50
>50
>50
>50
>50
>50
>50
>50
The glassware used in air and/or moisture sensitive reactions
was flame-dried and carried out under a dry argon atmosphere.
Unless otherwise noted, chemicals were commercially available and
used without further purification. Solvents were distilled before use.
Tetrahydrofuran and toluene were distilled from sodium/benzophe-
none ketyl. Anhydrous N,N-dimethylformamide was used as
supplied from Aldrich.
Nuclear magnetic resonance spectra were recorded using a Bruker
AC-200 MHz or a Bruker AM-500 MHz spectrometers. Chemical
shifts are reported in parts per million (δ) relative to tetramethyl-
silane. Coupling constants are reported in Hertz. 13C NMR spectra
were fully decoupled. Splitting patterns are designated as s, singlet;
d, doublet; t, triplet; q, quartet.
High-resolution mass spectra was conducted using a Micromass
Q-Tof Ultima apparatus, which is a hybrid quadrupole time-of-
flight mass spectrometer with MS/MS capability.
Melting points were determined using a Fisher-Johns apparatus
and are uncorrected. IR spectra were recorded using a Nicolet
Magna 550 spectrometer.
>50
9.8
14.2
>50 (45%)b
>50 (31%)b
2.0
a Values are means of three experiments. b Maximum inhibition values
obtained at the indicated concentrations are given in parentheses.
preparation of alkyl derivatives of more than four carbon atoms
required more reactive electrophiles such as 2-alkenyl iodides,
which were reduced to the corresponding saturated n-alkyl
derivatives, together with protecting groups, removed by
catalytic hydrogenation during the last synthetic step. The
resulting O-benzyl alkylphosphinates treated with methylene
tritylamine and boron trifluoride etherate in toluene afforded
the respective O-benzyl alkyl [(N-tritylamine) methyl]phosphi-
nate (compounds 29-35). Removal of the trityl protecting group
yielded the corresponding O-benzyl alkyl(aminomethylmethyl)-
phosphinates (compounds 36-42) that was conjugated with
dipeptide 14 to give the immediate precursor of the title
compounds (43-49). Protecting group cleavage by catalytic
hydrogenation afforded the desired compounds (50-56) that
were evaluated against the clinically more relevant form of the
parasite (the intracellular amastigotes) and the dividing nonin-
fective epimastigote forms of the parasite. Of the designed
compounds, 53 and 54 were potent inhibitors against intracel-
lular T. cruzi proliferation possessing IC50 values of 9.8 µM
and 14.2 µM, respectively. The inhibitory efficacy of these
compounds was comparable to the potency exhibited by
4-phenoxyphenoxyethyl thiocyanate 57 (WC-9),50-53 a well-
known antiparasitic agent, which was used as a positive control.
Derivatives with short aliphatic chains were not active against
amastigotes, while 55 and 56 were moderately potent against
this form of the parasite. All of these compounds were devoid
of activity against the noninfective epimastigote forms of the
parasite at concentrations up to 50 µM. The inhibition data are
illustrated in Table 1. This particular behavior has also been
observed in other antiparasitic agents with a marked different
mode of action such as bisphosphonates, which are very potent
growth inhibitors of the amastigote forms of T. cruzi but have
very weak action toward epimastigotes.54,55
Column chromatography was performed with E. Merck silica
gel (Kieselgel 60, 230-400 mesh). Analytical thin-layer chroma-
tography was performed employing 0.2 mm coated commercial
silica gel plates (E. Merck, DC-Aluminum sheets, Kieselgel 60
F254).
General Procedure for the One-Pot Preparation of Phos-
phinic Acid Benzyl Ester. A mixture of ammonium phosphinate
(1.00 g, 12.04 mmol) and hexamethyldisilazane (2.53 mL, 12.04
mmol) under an argon atmosphere was stirred at 110 °C for 2 h.
The reaction mixture was cooled at 0 °C and anhydrous methylene
chloride (12 mL) added, followed by addition of the corresponding
alkyl halide (12.04 mmol). The mixture was allowed to reach room
temperature and stirred overnight. A yellow precipitate appeared,
and the mixture was filtered through Celite under nitrogen.
Anhydrous benzyl alcohol (2.46 mL, 24.08 mmol) was added to
the filtrate and the solvent removed at room temperature under
reduced pressure. Pressure was restored with argon. Anhydrous
tetrahydrofuran (20 mL) was added followed by addition of an
additional equivalent of benzyl alcohol (1.23 mL, 12.04 mmol),
4-(dimethylamino)pyridine (100 mg), and dicyclohexylcarbodiimide
(3.23 g, 15.65 mmol) at 0 °C. The reaction mixture was stirred at
room-temperature overnight. White dicyclohexylurea precipitated,
and the suspension was filtered through a glass frit filter. The solvent
was evaporated and the residue purified by column chromatography
(silica gel) employing mixtures of CH2Cl2-methanol ranging from
100:0 to 97:3. The oil obtained was redissolved in a minimum of
a mixture of hexanes-EtAcO (1:1) and filtered through Celite to
eliminate the remaining dicyclohexylurea. The solvent was evapo-
rated, and the purified compound was immediately used in next
step reaction before hydrolysis could take place. Reaction yields
were based on ammonium phosphinate.
These compounds, at concentrations up to 50 µM, exhibited
no toxicity to the host cells, as assessed by phase contrast
microscopy observation of detachment, vacuolation, and round-
ing of the cells. In conclusion, we found a simple pharmacoph-
ore structure that can be straightfowardly prepared from
inexpensive starting materials involving few synthetic steps. This
simple pharmacophore prompts new approaches in the field of
tropical diseases chemotherapy. Compound 53 is an encouraging
model of an effective antiparasitic agent, which presents
potential prospects as a pharmacophore to be used for further
structure optimization. It has two well-defined parts: (a) a
peptidic moiety, and (b) an aliphatic unit. Both of these parts
of the molecule can be easily modified. Metabolic stability, cell
penetration, bioavailavity, etc., are common problems associated
with peptide analogues. Therefore, the design of peptidomi-
metics that retain the pharmacophoric unit would provide
Methylphosphinic Acid Benzyl Ester (22). Colorless oil, 46%
1
yield; H NMR (500.13 MHz, CD3OD) δ 1.47 (dd, JP-H ) 15.3
Hz, JH-H ) 2.1 Hz, 3H, PCH3), 4.59 (2H, OCH2Ph), 7.13 (d, JP-H
) 546.8 Hz, PH), 7.21-7.25 (m, 1H, Ph); 7.28-7.36 (m, 4H,
Ph).13C NMR (125.77 MHz, CD3OD) δ 17.0 (d, J ) 93.3 Hz,
PCH3), 65.2 (OCH2Ph), 128.0 (Ph), 128.2 (Ph), 129.3 (Ph), 142.7
(Ph); 31P NMR (202.45 MHz, CD3OD) δ 30.1.
Ethylphosphinic Acid Benzyl Ester (23). Colorless oil, 32%
1
yield; H NMR (200 MHz, CDCl3) δ 1.16 (d, JP-H ) 21.4 Hz,
JH-H ) 7.7 Hz, 3H, CH3), 1.7-1.95 (m, 2H, PCH2CH3), 5.08 (dd,
JP-H ) 24.7 Hz, JH-H ) 11.7 Hz, 1H, PCHaPh), 5.12 (dd, JP-H
)
23.0 Hz, JH-H ) 11.7 Hz, 1H, PCHaPh), 7.09 (dt, JP-H ) 530.0
Hz, JH-H ) 1.8 Hz, 1H, HP), 7.27-7.50 (m, 5H, Ph).
Allylphosphinic Acid Benzyl Ester (24). Colorless oil, 43%
1
yield; H NMR (200 MHz, CDCl3) δ 2.63-2.67 (m, 2H, PCHa-

430 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
RaVaschino et al.
CHCH2), 2.67-2.70 (m, 2H, PCHbCHCH2), 5.06 (dd, JH-H ) 11.8
Hz, JP-H ) 8.7 Hz), 5.15 (dd, JH-H ) 11.8 Hz, JP-H ) 10.1 Hz),
5.23 (dddt, 1H, 3JH-H ) 17.1 Hz, JP-H ) 6.1 Hz, 2JH-H ) 1.3 Hz,
3JH-H ) 1.4 Hz, PCH2CHCHa), 5.27 (dddt, 1H, 3JH-H ) 10.2 Hz,
5.9 Hz, OCH2Ph), 72.7 (d, J ) 17.0 Hz, CPh3), 127.7 (Ph), 128.9
(Ph), 129.0 (Ph), 129.6 (Ph), 129.7 (Ph), 129.8 (Ph), 137.9 (d, J )
5.1 Hz, Ph), 146.2 (Ph); 31P NMR (202.45 MHz, CD3OD) δ 56.6.
O-Benzyl Ethyl[(N-tritylamino)methyl]phosphinate (30). Col-
2
3
1
JP-H ) 5.4 Hz, JH-H 1.3 Hz, JH-H ) 1.1 Hz, PCH2CHCHb),
orless oil, 52% yield; H NMR (500.13 MHz, CDCl3) δ 1.18 (dt,
)
5.73 (dddt, 3JH-H, trans ) 17.1 Hz, 3JH-H, cis ) 10.1 Hz, JP-H ) 6.9
JP-H ) 18.1 Hz, JH-H ) 7.7 Hz, 3H, CH3), 1.65-2.13 (bs, 1H,
NH), 1.94 (dq, 2H, JH-P ) 13.9 Hz, JH-H ) 7.7 Hz, PCH2CH3),
2.43 (dd, JH-H ) 14.1 Hz, JH-P ) 10.0 Hz, 1H, PCHHaNH), 2.53
(dd, JH-H ) 14.1 Hz, JH-P ) 12.1 Hz, 1H, PCHHbNH), 4.98 (dd,
3
Hz, JH-H ) 7.5 Hz, 1H, PCH2CHCH2), 7.04 (ddd, JP-H ) 546.8
Hz, JH-H ) 2.2 Hz, JH-H ) 1.8 Hz, 1H, HP), 7.24-7.31 (m, 1H,
Ph), 7.31-7.41 (m, 4H, Ph); 13C NMR (125.77 MHz, CDCl3) δ
35.3 (d, J ) 90.7 Hz, PCH2CHCH2), 65.1 (OCH2Ph), 122.3 (d, J
) 14.2 Hz, PCH2CHCH2), 125.6 (d, J ) 9.3 Hz, PCH2CHCH2),
127.0 (Ph), 127.5 (Ph), 128.5 (Ph); 31P NMR (202.45 MHz, CDCl3)
δ 34.5.
JH-H ) 12.1 Hz, JP-H ) 7.7 Hz, 1H, OCHHaPh), 5.04 (dd, JH-H
)
12.1 Hz, JP-H ) 8.1 Hz, 1H, OCHHbPh), 7.15-7.43 (m, 20H, Ph);
13C NMR (125.77 MHz, CDCl3) δ 5.7 (d, J ) 5.1 Hz, PCH2CH3),
20.4 (d, J ) 93.3 Hz, PCH2CH3), 41.0 (d, J ) 101.7 Hz, PCH2-
NH), 66.0 (d, J ) 5.9 Hz, OCH2Ph), 71.4 (d, J ) 15.3 Hz, CPh3),
126.6 (Ph), 127.7 (Ph), 128.0 (Ph), 128.5 (Ph), 128.6 (Ph), 136.8
(d, J ) 5.9 Hz, Ph), 144.7 (Ph); 31P NMR (202.45 MHz, CDCl3)
δ 57.0.
1-Butylphosphinic Acid Benzyl Ester (25). Colorless oil, 15%
1
yield; H NMR (500.13 MHz, CDCl3) δ 0.91 (t, 1H, J ) 7.2 Hz,
CH3), 1.41 (dist. sext., 2H, J ) 7.4 Hz, P(CH2)2CH2CH3), 1.53-
1.62 (m, 2H, PCH2CH2CH2CH3), 1.75-1.83 (m, 2H, PCH2(CH2)2-
CH3), 5.03 (dd, 1H, JH-H ) 11.9 Hz, JP-H ) 8.4 Hz, PCHHaPh),
5.14 (dd, 1H, JH-H ) 11.8 Hz, JP-H ) 10.1 Hz, PCHHbPh), 7.32-
7.41 (m, 5H, Ph), 7.65 (dt, 1H, JP-H ) 530.0 Hz, JH-H ) 2.0 Hz,
HP); 13C NMR (125.77 MHz, CDCl3) δ 13.5 (PCH2(CH2)2CH3),
22.7 (d, J ) 2.5 Hz, PCH2CH2CH2CH3), 23.5 (d, J ) 16.1 Hz,
PCH2CH2CH2CH3), 28.5 (d, J ) 92.4 Hz, PCH2(CH2)2CH3), 67.5
(d, J ) 6.8 Hz, OCH2Ph), 128.1 (Ph), 128.6 (Ph), 128.7 (Ph), 135.8
(d, J ) 5.9 Hz); 31P NMR (202.45 MHz, CDCl3) δ 39.2.
2-Pentenylphosphinic Acid Benzyl Ester (26). Colorless oil,
45% yield; 1H NMR (200 MHz, CDCl3) δ 0.97 (t, J ) 7.5 Hz, 3H,
PCH2CHCHCH2CH3), 2.01-2.11 (m, 2H, PCH2CHCHCH2CH3),
2.50-2.63 (m, 2H, PCH2CHCHCH2CH3), 5.04 (dd, JH-H ) 11.9
Hz, JP-H ) 8.7 Hz, 1H, OCHHaPh), 5.14 (dd, 1H, JH-H ) 11.9
Hz, JP-H ) 10.1 Hz, OCHHbPh), 5.27-5.39 (m, 1H, PCH2CH-
CHCH2CH3), 5.62-5.72 (m, 1H, PCH2CHCHCH2CH3), 6.99 (ddd,
JP-H ) 544.5 Hz, JH-H ) 2.4 Hz, JH-H ) 1.8 Hz, 1H, HP), 7.31-
7.40 (m, 5H, Ph).
O-Benzyl 2-Propenyl[(N-tritylamino)methyl]phosphinate (31).
1
Colorless oil, 70% yield; H NMR (500.13 MHz, CDCl3) δ 2.05
(dt, 1H, JH-P ) 8.0 Hz, JH-H ) 3.3 Hz, NH), 2.49 (ddd, JH-H
)
14.4 Hz, JP-H ) 10.0 Hz, JH-H ) 7.5, 1H, PCHHaNH), 2.56 (ddd,
JH-H ) 14.2 Hz, JP-H ) 12.0 Hz, JH-H ) 8.7, 1H, PCHHbNH),
2.82 (dd, JP-H ) 17.2 Hz, JH-H ) 7.4, 2H, PCH2CHCH2), 5.01
(dd, JH-H ) 12.0, JP-H ) 8.1 Hz, 1H, OCHaPh), 5.05 (dd, JH-H
)
12.0, JP-H ) 8.2 Hz, 1H, OCHbPh), 5.18-5.26 (m, 3H, PCH2-
CHCH2), 5.83 (dddt, 3JH-H,trans ) 17.2 Hz, 3JH-H,cis ) 9.9 Hz, JP-H
) 5.9 Hz, 3JH-H ) 7.3 Hz, 1H, PCH2CHCH2), 7.16-7.42 (m, 20H,
Ph); 13C NMR (125.77 MHz, CDCl3) δ 33.6 (d, J ) 88.2 Hz, PCH2-
CHCH2), 41.2 (d, J ) 104.3 Hz, PCH2NH), 66.3 (d, J ) 6.8 Hz,
OCH2Ph), 71.5 (d, J ) 16.1 Hz, CPh3), 120.4 (d, J ) 12.7 Hz,
PCH2CHCH2), 126.6 (Ph), 127.2 (d, J ) 8.5 Hz, PCH2CHCH2),
127.7 (Ph), 128.0 (Ph), 128.3 (Ph), 128.5 (Ph), 128.6 (Ph), 136.5
(d, J ) 5.1 Hz, Ph); 31P NMR (202.45 MHz, CDCl3) δ 51.5.
O-Benzyl 1-Butyl[(N-tritylamino)methyl]phosphinate (32).
Colorless oil, 51% yield; 1H NMR (500.13 MHz, CDClD3) δ 0.92
(t, J ) 7.3 Hz, 3H, CH3), 1.37-1.48 (dist. sext, J ) 7.3 Hz, 2H,
P(CH2)2CH2CH3), 1.51-1.64 (m, 2H, PCH2CH2CH2CH3), 1.85-
2-Hexenylphosphinic Acid Benzyl Ester (27). Colorless oil,
1
45% yield; H NMR (200 MHz, CDCl3) δ 0.96 (t, J ) 7.7, 3H,
PCH2CHCH(CH2)2CH3), 1.37 (dist. sext, J ) 7.3 Hz, 2H, PCH2-
CHCHCH2CH2CH3), 1.92-2.09 (m, 2H, PCH2CHCHCH2CH2CH3),
2.51-2.58 (m, 1H, PCHHaCHCH(CH2)2CH3), 2.60-2.67 (m, 1H,
2.09 (m, 3H, PCH2(CH2)2CH3), 2.42 (dd, JH-H ) 14.2 Hz, JH-P
)
10.0 Hz, 1H, PCHHaNH), 2.53 (dd, JH-H ) 14.2 Hz, JH-P ) 12.1
Hz 1H, PCHHbNH), 4.98 (dd, JH-H ) 12.0 Hz, JH-P ) 7.9 Hz,
1H, OCHaPh), 5.04 (dd, JH-H ) 12.0 Hz, JH-P ) 8.1 Hz, 1H, OCHb-
Ph), 7.12-7.47 (m, 20H, Ph); 13C NMR (125.77 MHz, CDCl3) δ
13.6 (P(CH2)2CH2CH3), 23.6 (d, J ) 4.2 Hz, P(CH2)2CH2CH3),
23.9 (d, J ) 16.1 Hz, PCH2CH2CH2CH3), 27.1 (d, J ) 91.6 Hz,
PCH2(CH2)2CH3), 41.4 (d, J ) 100.1 Hz, PCH2NH), 65.9 (d, J )
5.9 Hz, OCH2Ph), 71.4 (d, J ) 15.3 Hz, CPh3), 126.7 (Ph), 127.8
(Ph), 128.1 (Ph), 128.4 (Ph), 128.6 (Ph) 128.7 (Ph), 136.8 (d, J )
5.1, Ph), 144.8 (Ph); 31P NMR (202.45 MHz, CDCl3) δ 56.1.
O-Benzyl 2-Pentenyl[(N-tritylamino)methyl]phosphinate (33).
Colorless oil, 47% yield; 1H NMR (500.13 MHz, CDClD3) δ 0.94
(t, J ) 7.4 Hz, 3H, PCH2CHCHCH2CH3), 1.96-2.08 (m, 3H, PCH2-
CHCHCH2CH3, NH), 2.46 (dist. t, J ) 11.9 Hz, 1H, PCHaNH),
2.54 (dist. t, J ) 13.6 Hz, 1H, PCHbNH), 2.74 (dd, JP-H ) 16.8
Hz, J H-H ) 7.3, 2H, PCH2CHCHCH2CH3), 5.00 (dd, JH-H ) 12.0
Hz, JH-P ) 7.9 Hz, 1H, OCHHaPh), 5.06 (dd, JH-H ) 12.0 Hz,
JH-P ) 7.9 Hz, 1H, OCHHbPh), 5.36-5.46 (m, 1H, PCH2CHCHCH2-
CH3), 5.62-5.71 (m, 1H, PCH2CHCHCH2CH3), 7.14-7.51 (m,
20H, Ph); 13C NMR (125.77 MHz, CDCl3) δ 13.7 (PCH2-
CHCHCH2CH3), 25.7 (d, J ) 2.5 Hz, PCH2CHCHCH2CH3), 32.3
(d, J ) 89.0 Hz, PCH2CHCHCH2CH3), 41.0 (d, J ) 103.4 Hz,
PCH2NH), 66.1 (d, J ) 6.8 Hz, OCH2Ph), 71.4 (d, J ) 16.1 Hz,
CPh3), 117.2 (d, J ) 9.3 Hz, PCH2CHCHCH2CH3), 126.6 (Ph),
127.7 (Ph), 128.0 (Ph), 128.3 (Ph), 128.5 (Ph), 128.6 (Ph), 136.6
(d, J ) 5.9 Hz, Ph), 138.2 (d, J ) 11.9 Hz, PCH2CHCHCH2CH3),
144.7 (Ph); 31P NMR (202.45 MHz, CDCl3) δ 52.5.
PCHHbCHCH(CH2)2CH3), 5.03 (dd, 1H, JP-H ) 8.9 Hz, JH-H
)
12.0 Hz, OCHHaPh), 5.14 (dd, 1H, JP-H ) 10.2 Hz, JH-H ) 12.0
Hz, OCHHbPh), 5.21-5.41 (m, 1H, PCH2CHCH(CH2)2CH3), 5.51-
5.75 (m, 1H, PCH2CHCH(CH2)2CH3), 6.98 (dt, 1H, JP-H ) 543.7
Hz, JH-H ) 2.1 Hz, HP), 7.29-7.44 (m, 5H, Ph).
2-Heptenylphosphinic Acid Benzyl Ester (28). Colorless oil,
55% yield; 1H NMR (200 MHz, CDCl3) δ 0.88 (t, J ) 6.9 Hz, 3H,
PCH2CHCH(CH2)3CH3), 1.17-1.38 (m, 4H, PCH2CHCHCH2-
(CH2)2CH3), 1.94-2.08 (m, 2H, PCH2CHCHCH2CH2CH2CH3),
2.47-2.58 (m, 1H, PCHHaCHCH(CH2)3CH3), 2.60-2.68 (m, 1H,
PCHHbCHCH(CH2)3CH3), 5.04 (dd, JH-H ) 12.0 Hz, JP-H ) 10.0
Hz, 1H, OCHHaPh), 5.15 (dd, JH-H ) 12.0 Hz, JP-H ) 10.0 Hz,
1H, OCHHbPh), 5.19-5.43 (m, 1H, PCH2CHCH(CH2)3CH3), 5.57-
5.78 (m, 1H, PCH2CHCH(CH2)3CH3), 6.99 (dt, JP-H ) 543.5 Hz,
JH-H ) 2.1 Hz, 1H, HP), 7.28-7.40 (m, 5H, Ph).
General Procedure for the Preparation of O-Benzyl Alkyl
[(N-Tritylamine)methyl]phosphinates. To a solution of methylene
tritylamine (726 mg, 2.68 mmol) in anhydrous toluene (20 mL)
cooled at 0 °C was added phosphinic acid benzyl ester (2.30 mmol)
under argon atmosphere. A 5% v/v solution of boron trifluoride
etherate in toluene (5 mL) was added dropwise, and the reaction
mixture was allowed to reach room temperature. The mixture was
stirred for 72 h. The solvent was evaporated, and the residue was
purified by column chromatography (silica gel) employing a mixture
of hexane-AcOEt (4:1) as eluant to afford the pure product.
O-Benzyl Methyl[(N-tritylamino)methyl]phosphinate (29).
Colorless oil, 46% yield; 1H NMR (500.13 MHz, CD3OD) δ 1.63
(d, J ) 14.0 Hz, 3H, CH3), 2.30 (dd, JH-H ) 14.6, JP-H ) 10.2
Hz, 1H, PCHaNH), 2.38 (dd, JH-H ) 14.6 Hz, JP-H ) 11.6 Hz,
1H, PCHbNH), 5.01 (d, J P-H ) 9.1 Hz, 2H, OCH2Ph), 7.06-7.52
(m, 20H, Ph); 13C NMR (125.77 MHz, CD3OD) δ 12.9 (d, J )
93.2 Hz, PCH3), 43.9 (d, J ) 107.7 Hz, PCH2NH), 67.6 (d, J )
O-Benzyl 2-Hexenyl[(N-tritylamino)methyl]phosphinate (34).
Colorless oil, 74% yield; 1H NMR (500.13 MHz, CDClD3) δ 0.85
(t, J ) 7.4 Hz, PCH2CHCH(CH2)2CH3), 1.32 (dist. sext, J ) 7.3
Hz, 2H, PCH2CHCHCH2CH2CH3), 1.93-2.00 (m, 2H, PCH2-
CHCHCH2CH2CH3), 2.00-2.08 (bs, 1H, NH), 2.41-2.60 (m, 2H,
PCH2NH), 2.74 (dd, JH-H ) 7.3, JP-H ) 17.1 Hz, 2H, PCH2CHCH-

Phosphinopeptides Targeting Trypanothione Biosynthesis
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 431
(CH2)2CH3), 5.01 (dd, JH-H ) 12.1 Hz, JP-P ) 7.8 Hz, 1H, OCHHa-
Ph), 5.06 (dd, JH-H ) 12.2 Hz, JP-P ) 7.8 Hz, 1H, OCHHbPh),
5.37-5.46 (m, 1H, PCH2CHCH(CH2)2CH3), 5.57-5.65 (m, 1H,
PCH2CHCH(CH2)2CH3), 7.14-7.44 (m, 20H); 13C NMR (125.77
MHz, CDCl3) δ 13.5 (PCH2CHCH(CH2)2CH3), 22.1 (d, J ) 3.4
Hz, PCH2CHCH(CH2)CH2CH3), 32.2 (d, J ) 89.0 Hz, PCH2-
CHCH(CH2)2CH3), 35.9 (d, J ) 1.7 Hz, PCH2CHCHCH2CH2CH3),
40.9 (d, J ) 103.4 Hz, PCH2NH), 66.0 (d, J ) 5.9 Hz, OCH2Ph),
71.3 (d, J ) 16.1 Hz, CPh3), 118.1 (d, J ) 9.3 Hz, PCH2CHCH-
(CH2)2CH3), 126.4 (Ph), 127.6 (Ph), 127.8 (Ph), 128.1 (Ph), 128.38
(Ph), 128.43 (Ph), 136.4 (d, J ) 12.7 Hz, PCH2CHCH(CH2)2CH3),
136.5 (d, J ) 5.1 Hz, Ph), 144.5 (Ph); 31P NMR (202.45 MHz,
CDCl3) δ 52.4.
2H, P(CH2)2CH2CH3) 1.36-1.50 (m, 2H, PCH2CH2CH2CH3), 1.85
(dt, JH-P ) 15.4 Hz, JH-H ) 8.2 Hz, 2H, PCH2(CH2)2CH3), 3.21
(d, J ) 8.0 Hz, 2H, NH2), 5.01 (dd, JH-H ) 11.8 Hz, JH-P ) 8.7
Hz, 1H, OCHaPh), 5.09 (dd, JH-H ) 11.8 Hz, JH-P ) 8.4 Hz, 1H,
OCHbPh); 13C NMR (125.77 MHz, CDCl3) δ 13.2 (PCH2-
(CH2)2CH3), 23.1 (d, J ) 5.1 Hz, PCH2CH2CH2CH3), 23.5 (d, J )
17.0 Hz, P(CH2)2CH2CH3), 27.6 (d, J ) 95.8, PCH2(CH2)2CH3),
35.7 (d, J ) 91.6 Hz, NHCH2P), 67.0 (d, J ) 6.8 Hz, OCH2Ph),
127.9 (Ph), 128.7 (Ph), 135.7 (d, J ) 5.9 Hz, Ph); 31P NMR (202.45
MHz, CDCl3) δ 50.9.
O-Benzyl 2-Penten(aminomethylmethyl)phosphinate (40). Pale
1
yellow oil. 76% yield; H NMR (500.13 MHz, CDCl3-CD3OD,
4:1) δ 0.95 (t, J ) 7.5 Hz, 3H, PCH2CHCHCH2CH3), 2.02 (dist.
sext, J ) 6.8 Hz, 2H, PCH2CHCHCH2CH3), 2.60-2.75 (m, 2H,
PCH2NH2), 3.04-3.25 (m, 2H, PCH2CHCHCH2CH3), 5.07 (dd,
O-Benzyl 2-Heptenyl[(N-tritylamino)methyl]phosphinate (35).
Colorless oil, 74% yield; 1H NMR (500.13 MHz, CDClD3) δ 0.86
(t, J ) 7.1 Hz, PCH2CHCH(CH2)3CH3), 1.21-1.34 (m, 4H, PCH2-
CHCHCH2(CH2)2CH3), 1.95-2.05 (m, 3H, PCH2CHCHCH2(CH2)2-
CH3, NH), 2.45 (dd, JH-H ) 13.4 Hz, JH-P ) 10.5 Hz, 1H,
PCHaNH), 2.54 (dist. t, JH-H ) 13.1 Hz, 1H, PCHbNH), 2.73 (dd,
JH-H ) 11.8 Hz, JH-P ) 9.0 Hz, 2H, OCHaPh), 5.11 (dd, JH-H
)
11.8 Hz, JH-P ) 8.3 Hz, 2H, OCHbPh), 5.21-5.30 (m, 2H,
PCH2CHCHCH2CH3), 5.62-5.72 (m, 2H, PCH2CHCHCH2CH3),
7.31-7.44 (m, 5H, Ph).
JP-H ) 16.9 Hz, J
) 7.3, 2H, PCH2CHCH(CH2)3CH3), 5.01
O-Benzyl 2-Hexen(aminomethylmethyl)phosphinate (41). Pale
H-H
1
(dd, JH-H ) 12.1 Hz, JP-P ) 7.7 Hz, 1H, OCHaPh), 5.06 (dd, JH-H
) 12.1 Hz, JP-P ) 7.7 Hz, 1H, OCHbPh), 5.35-5.45 (m, 1H,
PCH2CHCH(CH2)3CH3), 5.56-5.65 (m, 1H, PCH2CHCH(CH2)3-
CH3), 7.16-7.43 (m, 20H); 13C NMR (125.77 MHz, CDCl3) δ 13.8
(PCH2CHCH(CH2)2CH2CH3), 22.1 (PCH2CHCH(CH2)2CH2CH3),
31.1 (d, J ) 2.5 Hz, PCH2CHCHCH2CH2CH2CH3), 32.26 (d, J )
88.2 Hz, PCH2CHCH(CH2)3CH3), 32.27 (d, J ) 1.7 Hz, PCH2-
CHCHCH2CH2CH2CH3), 41.0 (d, J ) 103.4 Hz, PCH2NH), 66.1
(d, J ) 6.8 Hz, OCH2Ph), 71.4 (d, J ) 15.3 Hz, CPh3), 118.0 (d,
J ) 9.3 Hz, PCH2CHCH(CH2)2CH2CH3), 126.5 (Ph), 127.6 (Ph),
127.9 (Ph), 128.2 (Ph), 128.47 (Ph), 128.51 (Ph), 136.6 (d, J )
5.9 Hz, Ph), 136.8 (d, J ) 11.9 Hz, PCH2CHCH(CH2)2CH2CH3),
144.6 (Ph); 31P NMR (202.45 MHz, CDCl3) δ 52.5.
yellow oil. 94% yield; H NMR (500.13 MHz, CDCl3-CD3OD,
4:1) δ 0.89 (t, J ) 7.4 Hz, 3H, CH3), 1.37 (dist. sext, J ) 7.3 Hz,
2H, PCH2CHCHCH2CH2CH3), 1.95-2.05 (m, 2H, PCH2CHCHCH2-
CH2CH3), 2.61-2.69 (m, 2H, PCH2NH), 2.92-3.06 (m, 2H, PCH2-
CHCH(CH2)2CH3), 5.08 (d, J ) 8.4 Hz, 2H, OCH2Ph), 5.28-5.42
(m, 2H, PCH2CHCH(CH2)2CH3), 5.57-5.71 (m, 2H, PCH2-
CHCH(CH2)2CH3), 7.31-7.44 (m, 5H, Ph).
O-Benzyl 2-Hepten(aminomethylmethyl)phosphinate (42).
Pale yellow oil. 85% yield.
1H NMR (500.13 MHz, CDCl3-CD3OD, 4:1) δ 0.89 (t, J ) 7.2
Hz, 3H, CH3), 1.25-1.37 (m, 4H, PCH2CHCHCH2(CH2)2CH3),
1.98-2.07 (m, 2H, PCH2CHCHCH2(CH2)2CH3), 2.61-2.68 (m,
2H, PCH2NH2), 2.96-3.01 (m, 2H, PCH2CHCH(CH2)3CH3), 5.07
(d, J ) 8.7 Hz, 2H, OCH2Ph), 5.28-5.39 (m, 2H, PCH2CHCH-
(CH2)3CH3), 5.59-5.69 (m, 2H, PCH2CHCH(CH2)3CH3), 7.32-
7.43 (m, 5H, Ph).
General Procedure for Trityl Cleavage. To a solution of
O-benzylmethyl[(N-tritylamine)alkyl]phosphinate (0.68 mmol) in
methylene chloride (5 mL) was added dropwise a 5% v/v solution
of trifluoroacetic acid in methylene chloride (5 mL) at room
temperature. The reaction was stirred for 30 min. The solvent was
evaporated, and the residue was redissolved in methanol (10 mL).
Then, strong cationic resin Amberlite CG-400 (1.00 g) was added,
and the mixture was stirred for 1 h. The mixture was filtered off
and the filtrate washed with methanol (20 mL). The solvent was
evaporated, and the residue was purified by column chromatography
(silica gel) eluting with AcOEt f AcOEt-methanol (4:1) to yield
the corresponding free amine as an oil that had to be rapidly used
in next step to prevent N-oxide formation.
General Procedure for Amide Bond Formation. To solution
of N-benzyloxycarbonyl-L-glutamyl(R-benzylester)-L-leucine (300
mg, 0.619 mmol) and N-hydroxysuccinimide (106.9 mg, 0.923
mmol) in anhydrous N,N-dimethylformamide (5 mL) cooled at 0
°C was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride (EDC; 237.3 mg, 2.24 mmol). The reaction mixture
was stirred at room-temperature overnight. Then, a solution of
corresponding amine (0.495 mmol) and triethylamine (15 µL, 2.1
mmol) in N,N-dimethylformamide (3 mL) was added, and the
mixture was stirred at room temperature for 16 h. Ethyl acetate
(20 mL) was added, and the solution washed with brine (2 × 20
mL), 5% aqueous hydrochloric acid (2 × 10 mL), saturated sodium
bicarbonate (2 × 10 mL), and water (2 × 10 mL). The organic
layer was dried (Na2SO4), and the solvent was evaporated. The
product was purified by chromatography column (silica gel) using
a mixture of hexanes-EtAcO (4:1) as eluant to yield the corre-
sponding protected phosphinopeptide.
O-Benzyl Methyl-[[(N-benzyloxy-L-γ-glutamyl(r-benzyl ester)-
L-leucyl)amino]methyl]phosphinate (43). White solid, hygro-
scopic, 42% yield; 1H NMR (500.13 MHz, CD3OD) δ 0.83-0.96
(m, 6H, H3C(δ)Leu), 1.43-1.69 (m, 6H, PCH3, HC(â)Leu, HC-
(γ)Leu), 1.83-1.97 (m, 1H, HaC(γ)Glu), 2.09-2.22 (m, 1H, HbC-
(γ)Glu), 3.58-3.76 (m, 2H, NHCH2P), 4.18-4.26 (m, 1H, HC-
(R)Leu), 4.30-4.38 (m, 1H, HC(R)Glu), 4.99-5.18 (m, 6H,
OCH2Ph), 7.22-7.41 (m, 15H, Ph); 13C NMR (125.77 MHz, CD3-
OD) δ 12.8 (d, J ) 93.3 Hz, PCH3, major diast.), 12.9 (d, J )
93.3 Hz, PCH3, minor diast.), 21.9 (H3Ca(δ)Leu, minor diast.), 22.0
(H3Ca(δ)Leu, major diast.), 23.3 (H3Cb(δ)Leu, minor diast.), 23.3
(H3Cb(δ)Leu, minor diast.), 25.9 (C(γ)Leu, minor diast), 25.9 (C(γ)-
Leu, major diast.), 28.3 (C(γ)Glu), 32.7 (C(â) Glu), 38.8 (d, J )
102.6 Hz, NHCH2P, minor diast.), 38.9 (d, J ) 102.6 Hz, NHCH2P,
major diast.), 41.7 (C(â)Leu), 53.3 (C(R)Leu), 55.1 (C(R)Glu), 67.6
(d, J ) 5.9 Hz, POCH2Ph), 67.7 (OCH2Ph), 68.0 (OCH2Ph), 128.8
(Ph), 128.8 (Ph), 129.0 (Ph), 129.1 (Ph), 129.2 (Ph), 129.5 (Ph),
129.5 (Ph), 129.6 (Ph), 129.7 (Ph), 129.7 (Ph), 172.6 (CO), 175.2
O-Benzyl Methyl(aminomethylmethyl)phosphinate (36). Pale
yellow oil. 64% yield; 1H NMR (500.13 MHz, CD3OD) δ 1.68 (d,
J ) 14.8 Hz, 3H, PCH3), 3.36 (d, J ) 9.8 Hz, 2H, NH2CH2P),
5.12 (dd, JH-H ) 11.7 Hz, JH-P ) 9.5 Hz, 1H, OCHHaPh), 5.17
(dd, JH-H ) 11.7 Hz, JH-P ) 8.8 Hz, 1H, OCHHbPh), 7.28-7.49
(m, 5H, Ph). 13C NMR (125.77 MHz, CD3OD) δ 13.8 (d, J ) 96.6,
CH3), 37.7 (d, J ) 97.5 Hz, NHCH2P), 68.4 (d, J ) 6.8 Hz, OCH2-
Ph), 129.4 (Ph), 129.8 (Ph), 129.9 (Ph), 137.2 (d, J ) 5.9 Hz, Ph).
31P NMR (202.45 MHz, CD3OD) δ 47.6.
O-Benzyl Ethyl(aminomethyl)phosphinate (37). Pale yellow
1
oil; H NMR (500.13 MHz, CD3OD) δ 1.13 (dt, JH-P ) 19.1 Hz,
JH-H ) 7.7 Hz, 3H, PCH2CH3), 1.94 (dq, JH-P ) 15.2 Hz, JH-H
)
7.7 Hz, 2H, PCH2CH3), 3.27 (d, J ) 8.8 Hz, 2H, NHCH2P), 5.03
(dd, J
) 11.8 Hz, JH-P ) 8.5 Hz, 1H, OCHHaPh), 5.15 (dd,
H-H
JH-H ) 11.8 Hz, JH-P ) 10.1 Hz, 1H, OCHHbPh), 7.36-7.50 (m,
5H, Ph).
O-Benzyl 2-Propen(aminomethylmethyl)phosphinate (38).
1
Pale yellow oil, 89% yield; H NMR (200 MHz, CDCl3) δ 2.67
(dd, JH-P ) 17.9 Hz, JH-H ) 7.4 Hz, 3H, PCH2NH2), 2.96-3.14
(m, 2H, PCH2CHCH2), 3.14-3.44 (broad s, 2H, NH2), 5.05 (d, J
) 8.5 Hz, 2H, OCH2Ph), 5.09-5.25 (m, 2H, PCH2CHCH2), 5.54-
5.87 (m, 1H, PCH2CHCH2), 7.26-7.37 (m, 5H, Ph).
O-Benzyl 1-Butyl(aminomethylmethyl)phosphinate (39). Pale
1
yellow oil. 87% yield; H NMR (500.13 MHz, CDCl3) δ 0.81 (t,
J ) 7.3 Hz, 3H, PCH2(CH2)2CH3), 1.29 (dist. sept., J ) 7.1 Hz,

432 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
RaVaschino et al.
(CO), 175.6 (CO), 137.2 (Ph), 137.7 (d, J ) 6.8 Hz, Ph), 138.1
(Ph), 158.6 (CO), 173.5 (CO), 174.7 (CO), 175.0 (CO); 31P NMR
(202.45 MHz, CDCl3) δ 52.9 (minor diast.), 53.1 (major diast.).
O-Benzyl Ethyl-[[(N-benzyloxy-L-γ-glutamyl(r-benzyl ester)-
L-leucyl)amino]methyl]phosphinate (44). White solid, hygro-
scopic. 40% yield. White solid, hygroscopic. 40% yield.; 1H NMR
(500.13 MHz, CD3OD) δ 0.83-0.93 (m, 9H, H3C(δ)Leu, PCH2CH3),
1.01 (dt, JH-P ) 18.8 Hz, JH-H ) 7.6 Hz, 2H, PCH2CH3, minor
diast.), 1.11 (dt, JH-P ) 18.8 Hz, JH-H ) 7.6 Hz, 2H, PCH2CH3,
major diast.), 1.44-1.69 (m, 3H, HC(â)Leu, HC(γ)Leu), 1.73-
1.83 (m, 2H, PCH2CH3), 1.83-1.95 (m, 1H, HaC(γ)Glu), 2.07-
2.21 (m, 1H, HbC(γ)Glu), 2.27-2.39 (m, 2H, HC(â)Glu), 3.59-
3.77 (m, 2H, NHCH2P), 4.17-4.27 (m, 1H, HC(R)Leu), 4.27-
4.40 (m, 1H, HC(R)Glu), 4.98-5.19 (m, 6H, OCH2Ph), 7.23-7.42
(m, 15H, Ph); 13C NMR (125.77 MHz, CD3OD) δ 5.52 (d, J ) 4.8
Hz, PCH2CH3, major diast.), 5.54 (d, J ) 4.8 Hz, PCH2CH3, minor
diast.), 20.4 (d, J ) 93.5 Hz, PCH2CH3, major diast.), 20.6 (d, J )
92.5 Hz, PCH2CH3, minor diast.), 21.87 (H3Ca(δ)Leu, minor diast.),
21.93 (H3Ca(δ)Leu, major diast.), 23.35 (H3Cb(δ)Leu, minor diast.),
23.40 (H3Cb(δ)Leu, minor diast.), 25.86 (C(γ)Leu, major diast),
25.89 (C(γ)Leu, minor diast.), 28.2 (C(γ)Glu), 32.7 (C(â) Glu),
37.0 (d, J ) 98.2 Hz, NHCH2P, major diast.), 37.1 (d, J ) 98.2
Hz, NHCH2P, minor diast.), 41.7 (C(â)Leu), 53.2 (C(R)Leu), 55.1
(C(R)Glu), 67.6 (d, J ) 6.7 Hz, POCH2Ph), 67.7 (OCH2Ph), 68.0
(OCH2Ph), 128.8 (Ph), 129.0 (Ph), 129.2 (Ph), 129.3 (Ph), 129.5
(Ph), 129.6 (Ph), 129.64 (Ph), 129.66 (Ph), 129.7 (Ph), 137.2 (Ph),
137.9 (d, J ) 5.7 Hz, Ph), 138.1 (Ph), 158.7 (CO), 173.5 (CO),
174.7 (CO), 175.0 (CO); 31P NMR (202.45 MHz, CD3OD) δ 55.4
(minor diast.), 55.6 (major diast.).
NHCH2P, major diast.), 41.7 (C(â)Leu), 53.2 (C(R)Leu), 55.1
(C(R)Glu), 67.57 (d, J ) 5.9 Hz, POCH2Ph, major diast.), 67.59
(d, J ) 5.9 Hz, POCH2Ph, minor diast.), 67.7 (OCH2Ph), 68.0
(OCH2Ph), 128.8 (Ph), 129.0 (Ph), 129.1 (Ph), 129.2 (Ph), 129.3-
(Ph), 129.48 (Ph), 129.51(Ph), 129.57 (Ph), 129.64 (Ph), 129.66
(Ph), 137.88 (d, J ) 5.9 Hz, Ph, minor diast.), 137.89 (d, J ) 5.9
Hz, Ph, major diast.), 138.1 (Ph), 158.7 (CO), 173.5 (CO), 174.7
(CO), 174.9 (d, J ) 11.0 Hz, CO); 31P NMR (202.45 MHz, CDCl3)
δ 54.4 (minor diast.), 54.5 (major diast.).
O-Benzyl 2-Penten-[[(N-benzyloxy-L-γ-glutamyl(r-benzyl ester)-
L-leucyl)amino]methyl]phosphinate (47). White solid, hygro-
1
scopic. 36% yield. H NMR (500.13 MHz, CD3OD) δ 0.82-1.00
(m, 9H, H3C(δ)Leu, PCH2CHCHCH2CH3), 1.47-1.57 (m, 2H,
H2C(â)Leu),1.57-1.69 m, 1H, HC(γ)Leu), 1.83-1.95 (m, 1H, HaC-
(â)Glu), 1.97-2.07 (2H, 2.09-2.21, PCH2CHCHCH2CH3), 2.10-
2.20 (m, 1H, HbC(â)Glu), 2.28-2.39 (m, 2H, HbC(γ)Glu), 2.58-
2.68 (m, 2H, PCH2CHCHCH2CH3), 3.59-3.76 (m, 2H, NHCH2P),
4.19-4.28 (m, 1H, HC(R)Leu), 4.33-4.51 (m, 1H, HC(R)Glu),
5.99-5.19 (m, 6H, OCH2Ph), 5.31-5.41 (m, 1H, PCH2CHCHCH2-
CH3), 5.58-5.68 (m, 1H, PCH2CHCHCH2CH3), 7.22-7.40 (m,
15H, Ph); 13C NMR (125.77 MHz, CD3OD) δ 13.79 (PCH2-
CHCHCH2CH3, minor diast.), 13.82 (PCH2CHCHCH2CH3, major
diast.), 21.90 (H3Ca(δ)Leu, minor diast), 21.93 (H3Ca(δ)Leu, major
diast), 23.38 (H3Cb(δ)Leu, minor diast.), 23.41 (H3Cb(δ)Leu, major
diast.), 25.8 (C(γ)Leu, minor diast.), 25.9 (C(γ)Leu, major diast.),
26.7 (PCH2CHCHCH2CH3, minor diast.), 26.8 (PCH2CHCHCH2-
CH3,major diast.), 28.2 (C(γ)Glu), 32.1 (d, J ) 89.0 Hz, PCH2-
CHCHCH2CH3, minor diast.), 32.3 (d, J ) 88.3 Hz, PCH2CHCHCH2-
CH3, major diast.), 32.7 (C(â) Glu), 37.2 (d, J ) 100.0 Hz,
NHCH2P, minor diast.), 37.3 (d, J ) 100.0 Hz, NHCH2P, major
diast.), 41.7 (C(â)Leu), 53.2 (C(R)Leu), 55.1 (C(R)Glu), 67.7 (O-
CH2-Ph), 67.8 (d, J ) 6.8 Hz, OCH2Ph), 68.0 (OCH2Ph), 117.4
(d, J ) 10.2 Hz, PCH2CHCHCH2CH3, major diast.), 117.5 (d, J )
10.2 Hz, PCH2CHCHCH2CH3, major diast.), 128.96 (CO), 129.0
(CO), 129.2 (CO), 129.3 (CO), 129.5 (CO), 129.6 (CO), 129.61
(CO), 129.62 (CO), 137.2 (Ph), 137.87 (d, J ) 5.9 Hz, Ph, major
diast.), 137.89 (d, J ) 5.9 Hz, Ph, major diast.), 138.1 (Ph), 140.1
(d, J ) 12.7 Hz, PCH2CHCHCH2CH3), 158.65 (CO, minor diast.),
158.67 (CO, major diast.), 173,5 (CO), 174.40 (CO, major diast.),
174.73 (CO, minor diast.), 174.85 (CO, minor diast), 174.88 (CO,
major diast.), 174.5 (CO); 31P NMR (202.45 MHz, CD3OD) δ 50.1
(major diast.), 50.2 (minor diast.).
O-Benzyl 2-Propen-[[(N-benzyloxy-l-γ-glutamyl(r-benzyl ester)-
L-leucyl)amino]methyl]phosphinate (45). White solid, hygro-
1
scopic, 38% yield. H NMR (500.13 MHz, CD3OD) δ 0.80-0.96
(m, 6H, H3C(δ)Leu), 1.45-1.57 (m, 2H, H2C(â)Leu),1.57-1.69
(m, 1H, HC(γ)Leu), 1.83-1.94 (m, 1H, HaC(â)Glu), 2.08-2.20
(m, 1H, HbC(â)Glu), 2.25-2.37 (m, 2H, HC(γ)Glu), 2.61-2.78
(m, 2H, PCH2CHCH2), 3.57-3.78 (m, 2H, NHCH2P), 4.13-4.27
(m, 1H, HC(R)Leu), 4.29-4.41 (m, 1H, HC(R)Glu), 4.95-5.31
(m, 8H, OCH2Ph, PCH2CHCH2), 5.68-5.83 (m, 1H, PCH2CHCH2),
7.17-7.45 (m, 15H, Ph). 13C NMR (125.77 MHz, CD3OD) δ 21.86
(H3Ca(δ)Leu, major diast), 21.90 (H3Ca(δ)Leu, minor diast), 23.36
(H3Cb(δ)Leu), 25.85 (C(γ)Leu, minor diast.), 25.89 (C(γ)Leu, major
diast.), 26.3 (PCH2CHCH2), 28.2 (C(γ)Glu), 32.7 (C(â) Glu), 33.6
(d, J ) 88.0 Hz, PCH2CHCH2, minor diast.), 33.7 (d, J ) 88.0
Hz, PCH2CHCH2, major diast.), 37.3 (d, J ) 100.5 Hz, NHCH2P),
41.7 (C(â)Leu), 53.2 (C(R)Leu), 55.1 (C(R)Glu), 67.7 (OCH2Ph),
67.9 (d, J ) 7.2 Hz, OCH2Ph), 67.9 (OCH2Ph), 121.6 (d, J ) 12.6
Hz, PCH2CHCH2), 127.6 (d, J ) 9.9 Hz, PCH2CHCH2, minor
diast.), 127.7 (d, J ) 9.9 Hz, PCH2CHCH2, major diast.), 128.3
(Ph), 129.0 (Ph), 129.2 (Ph), 129.3 (Ph), 129.5 (Ph), 129.56 (Ph),
129.62 (Ph), 137.2 (Ph), 137.8 (d, J ) 7.2 Hz, Ph), 138.1 (Ph),
158.7 (CO), 173.5 (CO), 174.7 (CO), 174.9 (d, J ) 12.6 Hz, CO);
31P NMR (202.45 MHz, CD3OD) δ 49.3 (major diast.), 49.4 (minor
diast.).
O-Benzyl 2-Hexen-[[(N-benzyloxy-L-γ-glutamyl(r-benzyl ester)-
L-leucyl)amino]methyl]phosphinate (48). White solid, hygro-
scopic, 63% yield; 1H NMR (500.13 MHz, CD3OD) δ 0.83-0.93
(m, 9H, H3C(δ)Leu, PCH2CHCH(CH2)2CH3), 1.30-1.41 (m, 2H,
PCH2CHCHCH2CH2CH3), 1.48-1.57 (m, 2H, H2C(â)Leu), 1.57-
1.68 (m, 1H, HC(γ)Leu), 1.85-2.02 (m, 3H, HaC(â)Glu, PCH2-
CHCHCH2CH2CH3), 2.09-2.21 (m, 1H, HbC(â)Glu), 2.29-2.37
(m, 2H, HbC(γ)Glu), 2.57-2.68 (m, 2H, PCH2CHCH(CH2)2CH3),
3.58-3.74 (m, 2H, NHCH2P), 4.19-4.27 (m, 1H, HC(R)Leu),
4.33-4.40 (m, 1H, HC(R)Glu), 5.99-5.17 (m, 6H, OCH2Ph),
5.31-5.41 (m, 1H, PCH2CHCH (CH2)2CH3), 5.58-5.69 (m, 1H,
PCH2CHCH(CH2)2CH3), 7.21-7.40 (m, 15H, Ph); 13C NMR
(125.77 MHz, CD3OD) δ 13.79 (PCH2CHCH(CH2)2CH3), 21.93
(H3Ca(δ)Leu, minor diast), 21.97 (H3Ca(δ)Leu, major diast), 23.29
(H3Cb(δ)Leu, minor diast.), 23.31 (H3Cb(δ)Leu, major diast.), 23.4
(d, J ) 2.5 Hz, PCH2CHCHCH2CH2CH3), 25.88 (C(γ)Leu, major
diast.), 25.91 (C(γ)Leu, minor diast.), 28.2 (C(γ)Glu), 31.9 (d, J
) 89.0 Hz, PCH2CHCH(CH2)2CH3, major diast.), 32.3 (d, J ) 89.0
Hz, PCH2CHCH(CH2)2CH3, minor diast.), 32.7 (C(â) Glu), 35.8
(d, J ) 2.5 Hz, PCH2CHCHCH2CH2CH3), 37.31 (d, J ) 100.0
Hz, NHCH2P, major diast.), 37.33 (d, J ) 100.0 Hz, NHCH2P,
major diast.), 41.7 (C(â)Leu), 53.2 (C(R)Leu), 55.1 (C(R)Glu), 67.7
(OCH2Ph), 67.8 (d, J ) 6.8 Hz, OCH2Ph), 68.0 (OCH2Ph), 118.5
(d, J ) 9.3 Hz, PCH2CHCH(CH2)2CH3, major diast.), 118.6 (d, J
) 10.2 Hz, PCH2CHCH(CH2)2CH3, major diast.), 128.8 (Ph),
128.98 (Ph), 129.03 (Ph), 129.14 (Ph), 129.3 (Ph), 129.45 (Ph),
129.48 (Ph), 129.57 (Ph), 129.61 (Ph), 129.63 (Ph) 137.2 (Ph), 137.9
(d, J ) 5.9 Hz, Ph), 138.1 (Ph), 138.5 (d, J ) 12.7 Hz, PCH2-
CHCH(CH2)2CH3), 158.7 (CO), 173,5 (CO), 173.5 (CO), 174.7
O-Benzyl 2-Butyl-[[(N-benzyloxy-L-γ-glutamyl(r-benzyl ester)-
L-leucyl)amino]methyl]phosphinate (46). White solid, hygro-
scopic. 39% yield; 1H NMR (500.13 MHz, CD3OD) δ 0.81-0.94
(m, 9H, H3C(δ)Leu, P(CH2)3CH3), 1.30-1.42 (m, 2H, P(CH2)2CH2-
CH3), 1.44-0.1.68 (m, 5H, PCH2CH2CH2CH3, H2C(â)Leu, HC-
(γ)Leu), 1.71-1.84 (m, 2H, PCH2(CH2)2CH3), 1.84-1.95 (m, 1H,
HaC(γ)Glu), 2.08-2.22 (m, 1H, HbC(γ)Glu), 2.27-2.39 (m, 2H,
H2C(γ)Glu), 3.58-3.76 (m, 2H, NHCH2P), 4.15-4.26 (m, 1H, HC-
(R)Leu), 4.30-4.7 (m, 1H, HC(R)Glu), 4.99-5.12 (m, 4H, OCH2-
Ph), 5.14 (s, 2H, OCH2Ph), 7.23-7.42 (m, 15H, Ph); 13C NMR
(125.77 MHz, CD3OD) δ 13.9 (P(CH2)3CH3), 21.88 (H3Ca(δ)Leu,
minor diast.), 21.94 (H3Ca(δ)Leu, major diast.), 23.32 (H3Cb(δ)-
Leu, major diast.), 23.37 (H3Cb(δ)Leu, minor diast.), 24.3 (d, J )
4.2 Hz, P(CH2)2CH2CH3), 24.8 (d, J ) 14.4 Hz, PCH2CH2CH2-
CH3), 25.84 (C(γ)Leu, major diast), 25.89 (C(γ)Leu, minor diast.),
27.0 (d, J ) 91.6 Hz, PCH2NH, minor diast.), 27.2 (d, J ) 91.6
Hz, PCH2NH, major diast.), 28.2 (C(γ)Glu), 32.7 (C(â) Glu), 37.5
(d, J ) 98.3 Hz, NHCH2P, minor diast.), 37.6 (d, J ) 98.3 Hz,

Phosphinopeptides Targeting Trypanothione Biosynthesis
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 433
(CO), 174.9 (d, J ) 11.9 Hz, CO); 31P NMR (202.45 MHz, CD3-
OD) δ 49.9 (major diast.), 50.1 (minor diast.).
(m, 2H, H2C(â)Glu), 2.43-2.56 (m, 2H, H2C(γ)Glu), 3.49 (dd, JH-H
) 15.6 Hz, JP-H ) 9.5 Hz, 2H, NHCHaP, minor diasteromer), 3.50
(dd, JH-H ) 15.5 Hz, JP-H ) 9.5 Hz, 2H, NHCHaP, major
diasteromer ), 3.57 (dd, JH-H ) 15.5 Hz, JP-H ) 9.5 Hz, 1H,
O-Benzyl 2-Hepten-[[(N-benzyloxy-L-γ-glutamyl(r-benzyl es-
ter)-L-leucyl)amino]methyl]phosphinate (49). White solid, hy-
1
NHCHbP, major diasteromer ), 3.58 (dd, JH-H ) 15.6 Hz, JP-H
)
groscopic, 56% yield; H NMR (500.13 MHz, CD3OD) δ 0.75-
9.3 Hz, 1H, NH-CHb-P, minor diasteromer ), 3.99 (t, J ) 6.5 Hz,
1H, HC(R)Leu, minor diasteromer), 4.00 (t, J ) 6.1 Hz, 1H, HC-
(R)Leu), 4.26 (dd, J ) 9.2, 4.7 Hz, 1H, HC(R)Glu, minor
diasteromer), 4.27 (dd, J ) 9.4, 5.1 Hz, 1H, HC(R)Glu, major
diasteromer); 13C NMR (125.77 MHz, D2O) δ 7.4 (d, J ) 5.1 Hz,
PCH2CH3), 22.7 (d, J ) 93.2 Hz, PCH2CH3), 23.2 (H3Ca(δ)Leu),
24.7 (H3Cb(δ)Leu), 26.9 (C(γ)Leu), 28.1 (C(γ)Glu), 33.3 (C(â)
Glu), 40.1 (d, J ) 98.3 Hz, NH-CH2-P), 42.4 (C(â)Leu), 55.0
(C(R)Leu), 55.3 (C(R)Glu), 174.4 (CO), 176.9 (CO), 177.2 (CO);
31P NMR (202.45 MHz, D2O) δ 50.1 (major diasteromer), 49.9
(minor diasteromer). HRMS (ESI), Calcd. for C14H29N3O6P
(MH+): 366.1794, found: 366.1790.
1-Propyl-[[(L-γ-glutamyl-L-leucyl)amino]methyl]phosphi-
nate (52). HPLC purification: eluent solvent H2O-MeCN-TFA (85:
15:0.2), tR ) 10.73 min: white solid, 99% yield; 1H NMR (500.13
MHz, D2O) δ 0.97 (d, J ) 6.1 Hz, 3H, H3Ca(δ)Leu), 1.01 (d, J )
6.2 Hz, 3H, H3Cb(δ)Leu), 1.06 (t, J ) 7.6 Hz, 2H, PCH2 CH2CH3),
1.56-1.79 (m, 5H, H2C(â)Leu, HC(γ)Leu, P(CH2)2CH3), 2.23-
2.36 (m, 2H, H2C(â)Glu), 2.56-2.69 (m, 2H, H2C(γ)Glu), 3.58
(dd, JH-H ) 15.6, JP-H ) 9.2 Hz, 2H, NHCHaP, minor diasteromer),
3.59 (dd, JH-H ) 15.7, JP-H ) 9.5 Hz, 2H, NHCHaP, major
diasteromer ), 3.66 (dd, JH-H ) 15.7, JP-H ) 9.6 Hz, NHCHbP,
major diasteromer), 3.67 (dd, JH-H ) 15.6, JP-H ) 9.6 Hz,
NHCHbP, minor diasteromer), 4.10 (t, J ) 6.5 Hz, 1H, HC(R)-
Leu), 4.26 (dd, J ) 9.6, 5.2 Hz, 1H, HC(R)Glu); 13C NMR (125.77
MHz, D2O) δ 15.8 (d, J ) 4.2 Hz, P(CH2)2CH3), 15.8 (d, J ) 16.1
Hz, PCH2CH2CH3), 21.5 (H3Ca(δ)Leu), 22.9 (H3Cb(δ)Leu), 25.2
(C(γ)Leu), 26.4 (C(γ)Glu), 30.1 (d, J ) 93.2 Hz, PCH2CH2CH3),
31.6 (C(â) Glu), 38.9 (d, J ) 97.4 Hz, NH-CH2-P), 40.6 (C(â)-
Leu), 53.5 (C(R)Leu), 53.6 (C(R)Glu), 172.9 (CO), 175.2 (CO),
175.4 (CO); 31P NMR (202.45 MHz, D2O) δ 49.7 (major diaster-
omer), 49.5 (minor diasteromer). HRMS (ESI), Calcd. for
C15H31N3O6P (MH+): 380.1950, found: 380.1945.
1-Butyl-[[(L-γ-glutamyl-L-leucyl)amino]methyl]phosphinate
(53). HPLC purification: eluent solvent H2O-MeCN-TFA (80:20:
0.2), tR ) 9.14 min: white solid; 1H NMR (500.13 MHz, CD3OD)
δ 0.91 (d, J ) 6.5 Hz, 3H, H3Ca(δ)Leu), 0.93 (t, J ) 6.6 Hz, 3H,
P(CH2)3CH3), 0.96 (d, J ) 6.5 Hz, 3H, H3Cb(δ)Leu), 1.36-1.44
(m, 4H, P(CH2)2CH2CH3), 1.50-1.72 (m, 7H, P(CH2)2CH2CH3,
H2C(â)Leu, HC(γ)Leu), 2.14-2.21 (m, 2H, H2C(â)Glu), 2.48-
2.59 (m, 2H, H2C(γ)Glu), 3.36 (dd, JH-H ) 14.8 Hz, JP-H ) 10.1
Hz, 2H, NH-CHa-P ), 3.46 (dd, JH-H ) 14.8 Hz, JP-H ) 7.9 Hz,
NHCHbP), 3.89 (t, J ) 6.0 Hz, 1H, HC(R)Leu), 4.27 (t, J ) 7.3
Hz, 1H, HC(R)Glu); 13C NMR (125.77 MHz, CD3OD) δ 14.0
(P(CH2)3CH3), 21.8 (H3Ca(δ)Leu), 23.4 (H3Cb(δ)Leu), 24.9
(P(CH2)2CH2CH3), 25.1 (d, J ) 16.7 Hz, P(CH2CH2CH2CH3), 25.9
(C(γ)Leu), 27.0 (C(γ)Glu), 32.3 (C(â) Glu), 41.6 (C(â)Leu), 53.6
(C(R)Leu), 174.5 (CO), 174.7 (CO); 31P NMR (202.45 MHz, CD3-
OD) δ 45.0.; HRMS (ESI), Calcd. for C16H33N3O6P (MH+):
394.2107, found: 394.2124.
1-Pentyl-[[(L-γ-glutamyl-L-leucyl)amino]methyl]phosphi-
nate (54). HPLC purification: eluent solvent H2O-MeCN-TFA (75:
25:0.2), tR ) 10.39 min: white solid; 100% yield; 1H NMR (500.13
MHz, CD3OD) δ 0.89 (t, J ) 6.6 Hz, 3H, P(CH2)4CH3), 0.90 (d,
J ) 6.6 Hz, 3H, H3Ca(δ)Leu), 0.94 (d, J ) 6.6 Hz, 3H, H3Cb(δ)-
Leu), 1.30-1.41 (m, 4H, P(CH2)2(CH2)2CH3) 1.54-1.63 (m, 4H,
P(CH2)2(CH2)2CH3) 1.63-1.73 (m, 3H, H2C(â)Leu, HC(γ)Leu),
2.07-2.28 (m, 2H, H2C(â)Glu), 2.48-2.59 (m, 2H, H2C(γ)Glu),
3.39 (dd, JH-H ) 15.5 Hz, JP-H ) 9.8 Hz, 2H, NH-CHa-P), 3.53
(dd, JH-H ) 15.5 Hz, JP-H ) 8.9 Hz, NHCHbP), 4.00 (t, J ) 6.4
Hz, 1H, HC(R)Leu), 4.30 (t, J ) 7.5 Hz, 1H, HC(R)Glu); 13C NMR
(125.77 MHz, CD3OD) δ 14.2 (P(CH2)4CH3), 21.8 (H3Ca(δ)Leu),
22.2 (d, J ) 4.2 Hz, PCH2CH2(CH2)2CH3), 23.2 (P(CH2)3CH2CH3),
23.4 (H3Cb(δ)Leu), 25.9 (C(γ)Leu), 27.0 (C(γ)Glu), 28.5 (d, J )
91.6 Hz, PCH2(CH2)3CH3), 32.2 (C(â) Glu), 34.2 (d, J ) 16.1 Hz,
P(CH2)2CH2CH2CH3), 40.0 (d, J ) 100.9 Hz, NHCH2P), 41.7
0.96 (m, 9H, H3C(δ)Leu, PCH2CHCHCH2(CH2)2CH3) 1.18-1.36
(m,4H, PCH2CHCHCH2(CH2)2CH3), 1.43-1.56 (m, 2H, H2C(â)-
Leu), 1.56-1.68 (m, 1H, HC(γ)Leu), 1.81-2.05 (m, 3H, HaC(â)Glu,
PCH2CHCHCH2(CH2)2CH3), 2.07-2.21 (m, 1H, HbC(â)Glu),
2.24-2.38 (m, 2H, HbC(γ)Glu), 2.53-2.69 (m, 2H, PCH2-
CHCHCH2(CH2)2CH3), 3.55-3.75 (m, 2H, NHCH2P), 4.15-4.27
(m, 1H, HC(R)Leu), 4.29-4.40 (m, 1H, HC(R)Glu), 5.91-5.17
(m, 6H, OCH2Ph), 5.27-5.40 (m, 1H, PCH2CHCH(CH2)3CH3),
5.56-5.69 (m, 1H, PCH2CHCH(CH2)3CH3), 7.18-7.41 (m, 15H,
Ph); 13C NMR (125.77 MHz, CD3OD) δ 14.2 (PCH2CHCH-
(CH2)3CH3), 21.96 (H3Ca(δ)Leu, major diast), 22.0 (H3Ca(δ)Leu,
minor diast), 23.2 (PCH2CHCH(CH2)2CH2CH3), 23.38 (H3Cb(δ)-
Leu, major diast), 23.41 (H3Cb(δ)Leu, minor diast), 25.88 (C(γ)-
Leu, major diast.), 25.91 (C(γ)Leu, minor diast.), 28.3 (C(γ)Glu),
32.2 (d, J ) 88.1 Hz, PCH2CHCH(CH2)3CH3, major diast.), 32.4
(d, J ) 88.1 Hz, PCH2CHCH(CH2)3CH3, minor diast.), 32.4 (d, J
) 3.4 Hz, PCH2CHCHCH2CH2CH2CH3), 32.7 (C(â) Glu), 33.4
(d, J ) 2.5 Hz, PCH2CHCHCH2(CH2)2CH3), 37.3 (d, J ) 99.2
Hz, NHCH2P), 41.7 (C(â)Leu), 53.2 (C(R)Leu), 55.1 (C(R)Glu),
67.7 (OCH2Ph), 67.8 (d, J ) 6.8 Hz, OCH2Ph), 68.0 (OCH2Ph),
118.36 (d, J ) 9.3 Hz, PCH2CHCH(CH2)3CH3, major diast.), 118.41
(d, J ) 10.2 Hz, PCH2CHCH(CH2)3CH3, major diast.), 128.8 (Ph),
128.96 (Ph), 129.03 (Ph), 129.14 (Ph), 129.3 (Ph), 129.45 (Ph),
129.49 (Ph), 129.57 (Ph), 129.62 (Ph), 129.64 (Ph), 137.2 (Ph),
137.9 (d, J ) 5.9 Hz, Ph), 138.1 (Ph), 138.7 (d, J ) 12.7 Hz, PCH2-
CHCH(CH2)3CH3), 158.6 (CO), 173.5 (CO), 173.5 (CO), 174.7
(CO), 174.9 (d, J ) 9.3 Hz, CO); 31P NMR (202.45 MHz, CD3-
OD) δ 49.9 (minor diast.), 50.0 (major diast.).
General Procedure for Benzyl Ether Cleavage and Alkene
Hydrogenation. A solution of the respective protected phosphi-
nopeptide (0.1 mmol) in methanol (10 mL) in the presence of
palladium on charcoal (catalyst) was treated with hydrogen at 1
atm. The reaction mixture was stirred at room temperature for 1 h.
The mixture was filtered through a glass frit filter, and the solvent
was evaporated. The residue was purified by high performance
liquid chromatography employing a Beckman Ultrasphere ODS-2
5 µM column (10 mm × 250 mm) at a rate of 3 mL/min. Eluent
solvents and retention times are indicated in each case. Peak elution
was monitored at 230 nm.
Methyl-[[(L-γ-glutamyl-L-leucyl)amino]methyl]phosphinate (50).
HPLC purification: eluent solvent H2O-MeCN-TFA (90:10:0.2),
tR ) 10.45 min: white solid; 100% yield; 1H NMR (500.13 MHz,
D2O) δ 0.97 (d, J ) 6.1 Hz, 3H, H3Ca(δ)Leu), 1.01 (d, J ) 6.4
Hz, 3H, H3Cb(δ)Leu), 1.49 (d, J ) 13.7 Hz, 3H, P-CH3), 1.62-
1.78 (m, 3H, H2C(â)Leu, HC(γ)Leu), 2.20-2.36 (m, 2H, H2C(â)-
Glu), 2.57-2.70 (m, 2H, H2C(γ)Glu), 3.48 (dd, JH-H ) 15.5, JP-H
) 9.6 Hz, 2H, NH-CHa-P , minor diasteromer), 3.49 (dd, JH-H
)
15.6, JP-H ) 9.6 Hz, 2H, NH-CHa-P, major diasteromer ), 3.55
(dd, JH-H ) 15.6 Hz, JP-H ) 10.0 Hz, NHCHbP, major diasteromer
), 3.56 (dd, JH-H ) 15.5 Hz, JP-H ) 9.8 Hz, NHCHbP, minor
diasteromer), 3.99 (t, J ) 6.6 Hz, 1H, HC(R)Leu, minor diaster-
omer), 4.00 (t, J ) 6.6 Hz, 1H, HC(R)Leu, major diasteromer),
4.27 (dd, J ) 9.5, 5.1 Hz, 1H, HC(R)Glu); 13C NMR (125.77 MHz,
D2O) δ 13.8 (d, J ) 92.4 Hz, PCH3), 21.5 (H3Ca(δ)Leu), 22.9
(H3Cb(δ)Leu), 25.2 (C(γ)Leu), 26.3 (C(γ)Glu), 31.6 (C(â) Glu),
40.6 (C(â)Leu), 40.1 (d, J ) 105.1 Hz, NHCH2P), 40.6 C(γ)Leu),
53.3 (C(R)Leu), 53.6 (C(R)Glu), 172.6 (CO), 175.2 (CO), 175.6
(CO); 31P NMR (202.45 MHz, D2O) δ 49.1 (major diasteromer),
48.9 (minor diasteromer). HRMS (ESI), Calcd. for C13H27N3O6P
(MH+): 352.1637, found: 352.1637.
Ethyl-[[(L-γ-glutamyl-L-leucyl)amino]methyl]phosphinate (51).
HPLC purification: eluent solvent H2O-MeCN-TFA (87:13:0.2),
1
tR ) 10.25 min: white solid; 99% yield; H NMR (500.13 MHz,
D2O) δ 0.96 (d, J ) 6.5 Hz, 3H, H3Ca(δ)Leu), 1.01 (d, J ) 6.4
Hz, 3H, H3Cb(δ)Leu), 1.03 (dt, J ) 13.7, 7.4 Hz, 3H, PCH2CH3),
1.47-1.71 (m, 5H, H2C(â)Leu, HC(γ)Leu, PCH2CH3), 2.08-2.23

434 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
RaVaschino et al.
(C(â)Leu), 53.5 (C(R)Leu), 55.6 (C(R)Glu), 171.5 (CO), 174.5
(CO), 174.8 (CO); 31P NMR (202.45 MHz, CD3OD) δ 47.3; HRMS
(ESI), Calcd. for C17H35N3O6P (MH+): 402.2263, found: 402.2278.
1-Hexyl-[[(L-γ-glutamyl-L-leucyl)amino]methyl]phosphi-
nate (55). HPLC purification: eluent solvent H2O-MeCN-TFA (75:
25:0.2), tR ) 17.38 min: white solid; 98% yield; 1H NMR (500.13
MHz, CD3OD) δ 0.90 (t, J ) 7.0 Hz, 3H, P(CH2)5CH3), 0.92 (d,
J ) 6.4 Hz, 3H, H3Ca(δ)Leu), 0.96 (d, J ) 6.4 Hz, 3H, H3Cb(δ)-
Leu), 1.26-1.41 (m, 6H, P(CH2)2(CH2)3CH3), 1.54-1.62 (m, 4H,
P(CH2)2(CH2)2CH3), 1.63-1.72 (m, 3H, H2C(â)Leu, HC(γ)Leu),
2.08-2.29 (m, 2H, H2C(â)Glu), 2.48.-2.59 (m, 2H, H2C(γ)Glu),
3.46 (dd, JH-H ) 15.4 Hz, JP-H ) 8.8 Hz, 2H, NH-CHa-P), 3.61
(dd, JH-H ) 15.4 Hz, JP-H ) 8.7 Hz, NHCHbP), 4.00 (t, J ) 6.3
Hz, 1H, HC(R)Leu), 4.36 (t, J ) 7.5 Hz, 1H, HC(R)Glu); δ 14.4
(P(CH2)5CH3), 21.8 (H3Ca(δ)Leu), 22.7 (PCH2CH2(CH2)3CH3), 23.4
(H3Cb(δ)Leu), 23.5 (P(CH2)4CH2CH3), 26.0(C(γ)Leu), 27.0 (C(γ)-
Glu), 31.3 (d, J ) 89.9 Hz, PCH2(CH2)4CH3), 34.3 (d, J ) 14.4
Hz, P(CH2)2CH2(CH2)2CH3), 32.2 (C(â) Glu), 32.5 (P(CH2)3CH2-
CH2CH3), 41.6 (d, J ) 100.9 Hz, NHCH2P), 41.7 (C(â)Leu), 53.5
(C(R)Leu), 55.6 (C(R)Glu), 171.6 (CO), 174.5 (CO), 174.8 (CO);
31P NMR (202.45 MHz, CD3OD) δ 46.4; HRMS (ESI), Calcd. for
C18H37N3O6P (MH+): 422.2420, found: 422.2421.
Drug Testing against Epimastigotes. Trypanosoma cruzi epi-
mastigotes (Y strain) were grown in LIT + 5% NCS + 1% P/S
medium. Five day old culture was centrifuged and resuspended in
fresh medium to get a 2-3 × 106 cell/mL suspension (as determined
by counting in a Neubauer chamber). Parasites were then placed
in sterile screw-cap tubes (2 mL/tube). Two tubes were filled with
medium for blank. Each drug was tested at four different concentra-
tions (1, 5, 15, and 25 µg/mL), each one in triplicate. Drugs were
dissolved in absolute ethanol. A control without drug was done
for each group tested. An equivalent amount of ethanol (20 µL)
was added to blank and control tubes. The concentration of cells
was determined by measuring the absorbance of the culture medium
containing parasites at 600 nm against a blank with culture medium
alone. To calculate percent inhibition, the following formula was
used: Percent inhibition ) 100 - (∆Ad × 100)/∆Ac, where ∆Ac
and ∆Ad are the differences in the absorbance of control cultures
and drug-treated cultures, respectively, at the beginning and at the
end of the experiment. The maximum amount of solvent used (1%
ethanol) did not have any significant effect on the epimastigotes
growth. 57 was used as a positive control.
Acknowledgment. This work was supported by grants from
the European Commission INCO-DC, Fundacio´n Antorchas, the
National Research Council of Argentina (PIP 635/98), and the
Universidad de Buenos Aires (X-252) to J.B.R., and the U.S.
National Institutes of Health (GM-65307) to R.D.
1-Heptyl-[[(L-γ-glutamyl-L-leucyl)amino]methyl]phosphi-
nate (56). HPLC purification: eluent solvent H2O-MeCN-TFA (70:
1
30:0.2), tR ) 13.96 min: white solid; 99% yield; H NMR (500
MHz, CD3OD) δ 0.89 (t, J ) 7.0 Hz, 3H, P(CH2)6CH3), 0.91 (d,
J ) 6.2 Hz, 3H, H3Ca(δ)Leu), 0.95 (d, J ) 6.2 Hz, 3H, H3Cb(δ)-
Leu), 1.23-1.42 (m, 4H, P(CH2)2(CH2)4CH3), 1.45-1.75 (m, 7H,
H2C(â)Leu, HC(γ)Leu, P(CH2)2(CH2)4CH3), 2.07-2.28 (m, 2H,
H2C(â)Glu), 2.44.-2.63 (m, 2H, H2C(γ)Glu), 3.26 (dd, JH-H ) 14.7
Hz, JP-H ) 9.8 Hz, 2H, NH-CHa-P ), 3.38 (dd, JH-H ) 114.7 Hz,
JP-H ) 8.8 Hz, NHCHbP), 3.89 (t, J ) 6.0 Hz, 1H, HC(R)Leu),
4.36 (dd, J ) 10.1, 4.4 Hz, 1H, HC(R)Glu); 13C NMR (125.77
MHz, CD3OD) δ 14.4 (P(CH2)6CH3), 21.8 (H3Ca(δ)Leu), 22.8
(PCH2CH2(CH2)4CH3), 23.4 (H3Cb(δ)Leu), 23.7 (P(CH2)5CH2CH3),
26.0 (C(γ)Leu), 27.0 (C(γ)Glu), 28.9 (d, J ) 87.3 Hz, PCH2(CH2)5-
CH3), 30.0 (P(CH2)3CH2(CH2)2CH3), 32.1 (d, J ) 15.3 Hz,
P(CH2)2CH2(CH2)3CH3), 32.3 (C(â) Glu), 32.8 (P(CH2)4CH2CH2-
CH3), 39.2 (d, J ) 101.7 Hz, NHCH2P), 41.7 (C(â)Leu), 53.7
(C(R)Leu), 54.4 (C(R)Glu), 172.5 (CO), 174.4 (CO), 174.8 (CO);
31P NMR (202.45 MHz, CD3OD) δ 45.0; HRMS (ESI), Calcd. for
C19H39N3O6P (MH+): 436.2576, found: 436.2584.
Supporting Information Available: 1H, 13C, and 31P NMR
spectra, copies of the HRMS data, and HPLC analyses for the title
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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