α-Functionalized phosphonylphosphinates: Synthesis and evaluation as transcarbamoylase inhibitors

Article abstract of DOI:10.1021/jm991008q

Diverse α-methyl-substituted phosphonylphosphinates (P-C-P-C-X) are accessible from a protected, pentafluorophenylsulfonated phosphonylphosphinate via nucleophilic displacement. The utility of this route is demonstrated with several nitrogen nucleophiles. The resulting amine and amino acid phosphonylphosphinate derivatives were evaluated as inhibitors of Streptococcus faecalis ornithine transcarbamoylase (OTC). Compared with the structurally related phosphonoacetyl-L-ornithine (L-PALO), a known inhibitor of OTCs from various sources, the phosphonylphosphinates are surprisingly poor inhibitors, binding several orders of magnitude less tightly to the enzyme. These results suggest that the tetrahedral intermediate formed in the normal transcarbamoylase reaction is poorly mimicked by a tetrahedral and anionic phosphonate, either because of directly unfavorable interactions with a hydrogen-bond acceptor within the active site or because transition-state analogues are unable to induce the protein conformation changes that normally accompany reaction.

Full text of DOI:10.1021/jm991008q

J . Med. Chem. 1999, 42, 2633-2640
2633
r-F u n ction a lized P h osp h on ylp h osp h in a tes: Syn th esis a n d Eva lu a tion a s
Tr a n sca r ba m oyla se In h ibitor s
Alexander Flohr,‡,† Andreas Aemissegger,‡ and Donald Hilvert*,‡,†
Laboratorium fu¨r Organische Chemie, Swiss Federal Institute of Technology (ETH), Universita¨tstrasse 16,
CH-8092 Zu¨rich, Switzerland, and Departments of Chemistry and Molecular Biology, The Skaggs Institute of
Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La J olla, California 92037
Received J anuary 28, 1999
Diverse R-methyl-substituted phosphonylphosphinates (P-C-P-C-X) are accessible from a
protected, pentafluorophenylsulfonated phosphonylphosphinate via nucleophilic displacement.
The utility of this route is demonstrated with several nitrogen nucleophiles. The resulting amine
and amino acid phosphonylphosphinate derivatives were evaluated as inhibitors of Streptococcus
faecalis ornithine transcarbamoylase (OTC). Compared with the structurally related phospho-
noacetyl-L-ornithine (L-PALO), a known inhibitor of OTCs from various sources, the phospho-
nylphosphinates are surprisingly poor inhibitors, binding several orders of magnitude less
tightly to the enzyme. These results suggest that the tetrahedral intermediate formed in the
normal transcarbamoylase reaction is poorly mimicked by a tetrahedral and anionic phospho-
nate, either because of directly unfavorable interactions with a hydrogen-bond acceptor within
the active site or because transition-state analogues are unable to induce the protein
conformation changes that normally accompany reaction.
Sch em e 1
In tr od u ction
Phosphonates and phosphinates are useful moieties
for probing biological function and for creating high-
affinity enzyme inhibitors. Methylenebis(phosphonate),
for example, is a nonhydrolyzable surrogate for pyro-
phosphate that has been successfully incorporated into
inhibitors of several enzymes that process pyrophos-
phorylated substrates, including squalene synthase,1
prenyl transferase,2 glutamine synthetase,3 and a nu-
cleoside phosphorylase.4 Phosphonates have been simi-
larly employed as potent kinase inhibitors5 and, because
of their anionic character and tetrahedral geometry, as
transition-state analogues for acyl-transfer reactions6
and as haptens for eliciting catalytic antibodies.7,8
diate 2 which is formed when the δ-amino group of
ornithine attacks the carbonyl center of carbamoyl
phosphate. Compound 1 mimics the transition state
leading to this high-energy species: Its terminal phos-
phonate corresponds to the phosphate leaving group, the
phosphinate moiety mimics the tetrahedral, anionic
center undergoing attack, and the additional methylene
group inserted between the phosphinate phosphorus
and the nitrogen simulates the extended developing
bond between the incoming nucleophile and the carbo-
nyl center. Requirements with respect to the nucleophile
in this reaction can be explored by variation in the
amino acid side chain of 1.
Compounds such as 1 are not directly accessible by
existing methodologies for preparing substituted phos-
phonylphosphinates.11 Procedures involving alkylation
of phosphonylphosphonites with alkyl halides at el-
evated temperatures2 or of phosphonylphosphinyl di-
anions at low temperatures12 have been described, but
R-functionalized phosphonylphosphinates (P-C-P-
By analogy, R-functionalized phosphonylphosphi-
nates, which combine both motifs in a single molecule,
are potentially interesting as inhibitors of enzymatic
reactions involving phosphoanhydrides. Compounds
such as 1, for instance, could be considered as “expanded
transition-state analogues”9 for the reaction of L-orni-
thine with carbamoyl phosphate to give L-citrulline and
inorganic phosphate (Scheme 1). This reaction, which
is catalyzed by the enzyme ornithine transcarbamoylase
(OTC), is a key step in the biosynthesis of arginine and
also important in the detoxification of ammonia in
terrestrial vertebrates.10 It occurs via reactive interme-
* To whom correspondence should be addressed. Tel: (int +1) 632
3176. Fax: (int +1) 632 1486. E-mail: hilvert@org.chem.ethz.ch.
‡ ETH.
† The Scripps Research Institute.
10.1021/jm991008q CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/18/1999

2634 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Flohr et al.
Sch em e 2
°C no reaction occurs within 48 h, and 7a is quantita-
tively recovered. Pentafluorophenylsulfonates are known
to solvolyze at rates intermediate between the triflate
and the mesylate,21-23 but they have not been previously
exploited in synthesis, presumably because low yields
in their preparation have been claimed as a major
drawback.24 We have found, however, that the pen-
tafluorophenylsulfonate 7b can be prepared in even
higher yield than the corresponding mesylate. Moreover,
compound 7b is crystalline, is stable in air, and can be
stored at -78 °C for over a year without decomposition.
Even at 0 °C, only slow decomposition is observed.
Nevertheless, its reactivity is sufficient to allow intro-
duction of a variety of amine nucleophiles under mild
conditions (Scheme 3).
Pentafluorophenylsulfonate 7b was coupled in good
yield (72-98%) with an Nδ-unprotected ornithine de-
rivative (8), the corresponding R,γ-aminobutyric acid 14,
and azide (Scheme 3). Subsequent deprotection yielded
compounds 11 and 17. The product of the azide reaction
(18) was reduced by catalytic hydrogenation and depro-
tected using neat TMSBr to give amine 20 in good yield.
C-X compounds with variable X) cannot be prepared
by these methods. Addition of the carbanion derived
from diethyl methylphosphonate to an appropriately
derivatized and activated phosphonate ester to form the
central P-CH2-P moiety would be an alternative
approach,1,13,14 but this strategy cannot be used for
compounds such as 1 containing sensitive functionality
in the side chain. Here we report an efficient and
general route to R-functionalized phosphonylphosphi-
nates via nucleophilic substitution on an appropriately
activated derivative of a phosphonomethyl(hydroxy-
methyl)phosphinate. The scope of this approach is il-
lustrated with several nitrogen nucleophiles. The re-
sulting compounds were tested as inhibitors of OTC.
Compound 8 was synthesized by protection of the
R-amino group of commercially available H-L-Orn(Z)-
OtBu with Boc2O and subsequent catalytic hydrogena-
tion of the Z group in EtOH/HOAc.25,26 Compound 14
was synthesized starting from Boc-L-Asp(OH)-OtBu
(Scheme 4). Selective reduction of the side-chain car-
boxylic group with borane and subsequent treatment
with MesCl yielded the mesylate 12 as a stable com-
pound. Substitution of the mesylate with azide and final
hydrogenation gave 14.
Resu lts
Surprisingly, the protected phosphonylphosphinate
amino acid adducts 9 and 15 are less stable than the
pentafluorophenylsulfonate 7b and could only be stored
for a few weeks at -78 °C. These derivatives were
deprotected in a two-step procedure, involving initial
treatment with bromotrimethylsilane (TMSBr) in the
presence of the acid scavenger hexamethyldisilazane
(HMDS) to remove all the protecting groups except the
Boc moiety, followed by removal of the Boc group in
aqueous 3.2 M HCl (Scheme 3). HMDS could not be
replaced with allyltrimethylsilane or collidine, and
attempts to remove all protecting groups in a single step
using TMSBr or TMSI in the absence of HMDS resulted
in the cleavage of the amino acid side chain from the
methyl(phosphonomethyl)phosphinate unit. Decomposi-
tion was not observed when the terminal amine was
protected with an isobutyloxycarbonyl group, which is
stable against TMSBr even in the absence of an acid
scavenger, suggesting that the free amine facilitates the
cleavage reaction. Phosphonate/phosphinate esters are
deprotected more slowly than the primary amine with
TMS halides in the absence of an acid scavenger, and
it is known that functionalized phosphinate esters with
an electrophilic group in the R-position are more sensi-
tive to fragmentation than their corresponding acids.16
The final amino acid adducts 11 and 17 are stable in
aqueous solution between pH 1 and 12. Amine 20 also
proved stable for months.
Syn th esis of P h osp h on ylp h osp h in a tes. The syn-
thesis of R-functionalized phosphonylphosphinates is
complicated by the base sensitivity of methylphospho-
nates bearing electron-withdrawing R-substituents15,16
and by the very low reactivity at the phosphinyl methyl
carbon.17 We anticipated, however, that R-functionalized
phosphonylphosphinates might be readily derived from
6 (Scheme 2). Activation of the alcohol as a suitably
tuned sulfonate ester, followed by nucleophilic displace-
ment, would yield the desired compounds. Such an
approach would have the significant advantage that
multiple derivatives bearing different side chains could
be prepared from a common intermediate.
Alcohol 6 was synthesized in gram quantities starting
from diethyl phosphite in a six-step sequence (Scheme
2). Addition of formaldehyde to diethyl phosphite,
followed by the protection of the hydroxymethyl group
with benzyl bromide/silver(I) oxide, yielded 4. In our
hands, this sequence of reactions was found to be
superior to previous routes to 4.18,19 Partial saponifica-
tion with potassium hydroxide in ethanol/water, fol-
lowed by treatment with oxalyl chloride and catalytic
amounts of DMF, yielded acid chloride 5. The latter was
easily coupled with lithiated diethyl methylphospho-
nate. Deprotection of the resulting adduct by catalytic
hydrogenation gave alcohol 6 in an overall yield of 30%.
Although diethyl hydroxymethylphosphonate can be
triflated in good yield by various methods (unpublished
results),20 all attempts to triflate 6 failed. Mesylate 7a ,
in contrast, is readily formed. Unfortunately, its reactiv-
ity is so low that even with sodium azide in DMF at 65
In h ibition of Or n ith in e Tr an scar bam oylase. Phos-
phonylphosphinates 11, 17, and 20 were evaluated as
inhibitors of OTC from Streptococcus faecalis27-29 using
a standard colorimetric assay.30 For comparison, inhibi-

R-Functionalized Phosphonylphosphinates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2635
Sch em e 3
Sch em e 4
tion constants for phosphate, pyrophosphate, methyl-
enebis(phosphonate), and the known OTC inhibitor
L-PALO31,32 (21) were also determined. All reactions
were performed in either maleate (pH 6) or Tris (pH
7-8) buffers, which are known to be compatible with
the assay.27,33 The concentration of ornithine was 4.8
mM; at this concentration the enzyme should be nearly
saturated (Km ) ca. 740 µM27,34) but not subject to
substrate inhibition (which occurs at concentrations of
ornithine greater than 6 mM28,35). The concentration of
carbamoyl phosphate was varied between 70 and 500
µM to bracket its Km value (ca. 200 µM in the absence
of inhibitor27,28).
F igu r e 1. Lineweaver-Burk plots for the inhibition of OTC
from S. faecalis with various concentrations of L-PALO (21):
4.8 mM ornithine, 10 ng/mL OTC, 50 mM Tris‚HCl, 0.5 mM
EDTA, pH 8.0, 37 °C.
ety as a leaving group of intermediate reactivity. In
contrast to previously described methodologies, which
do not permit ready introduction of side chains after the
formation of the phosphonylphosphinate unit, a wide
variety of R-functionalized phosphonylphosphinates can
be prepared via displacement of the pentafluorophenyl-
sulfonate in 7b by an appropriate nucleophile. A major
advantage of this approach is its compatibility with side
chains containing acid- or base-sensitive groups. More-
over, the current approach is convergent inasmuch as
7b serves as a common intermediate for the introduction
of different side chains (Scheme 3). Orthogonality
problems between the side-chain protecting groups and
the phosphonylphosphinate moiety are also avoided
since the side chain is introduced at the end of the
synthesis via a very mild coupling step. By using acid-
labile protecting groups in the side chain, as in the
examples presented here, the final deprotection can be
accomplished in a single pot using mild TMS-Br/HMDS
or neat TMS-Br.
Compounds 11 and 20, like L-PALO (21) and pyro-
phosphate, were found to be competitive inhibitors with
respect to carbamoyl phosphate (Figure 1), whereas 17
displays noncompetitive behavior. Inhibition constants
were determined from Dixon plots (1/v vs [I]) at several
pH values and are summarized in Table 1. Compared
to L-PALO, the phosphonylphosphinates prepared in
this study are relatively weak inhibitors of OTC. The
tightest binder, amine 20, is recognized ca. 103-fold less
well than L-PALO. The ornithine derivative 11 is a
somewhat weaker inhibitor still, comparable to pyrophos-
phate36 but substantially better than the corresponding
NR-Boc-protected derivative 10, phosphonylphosphinate
17, phosphate,36 and methylenebis(phosphonate).
With the exception of compound 20, which lacks the
amino acid side chain, the R-functionalized phospho-
nylphosphinates are surprisingly poor inhibitors of S.
faecalis OTC (Table 1). Even 20 is orders of magnitude
weaker than L-PALO, a previously described inhibitor
of rat liver OTC31,32,37 that also binds to the Streptococ-
cus enzyme with submicromolar affinity (Table 1). Given
the structural similarities between L-PALO, 11, and 17,
Discu ssion
This study provides a general route to diverse phos-
phonylphosphinates (P-C-P-C-X) and establishes the
synthetic utility of the pentafluorophenylsulfonate moi-

2636 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Ta ble 1. Inhibition of S. faecalis OTC at 37 °C
Flohr et al.
a
b
50 mM Tris‚HCl, 0.5 mM EDTA, pH 8.0. 50 mM Tris‚HCl, 0.5 mM EDTA, pH 7.0. c 50 mM maleate, 0.5 mM EDTA, pH 6.0. nc,
noncompetitive inhibition.
such weak inhibition was unexpected and suggests that
the amino acid side chains in the phosphonylphosphi-
nates make few productive interactions with the OTC
active site. This conclusion is supported by the similarity
in Ki values determined for 11 and pyrophosphate,
another known competitive inhibitor of OTCs,29 and by
the fact that protection of the primary amine of 11 with
a Boc group has a relatively minor effect on binding,
except at pH 6 where the differences between the
inhibitors are more pronounced but the enzyme is
substantially less active.27,38
Compound 11 is longer than L-PALO by a methylene
group, and this could conceivably account for the
observed differences in affinity, but 17, which lacks this
group, is an even poorer inhibitor. In fact, L-PALO and
17 differ only in replacement of the neutral, planar
amide with a zwitterionic, R-amino-substituted, tetra-
hedral phosphinate. It is possible that OTC, as sug-
gested for the related enzyme aspartate transcarbam-
oylase,39,40 functions via a “closed transition state”,41
accessible only by a protein conformational change
induced by ground-state analogues containing a trigonal
carbonyl group.42 Alternatively, and perhaps more
likely, the additional oxygen at the tetrahedral phos-
phinate center may make unfavorable contacts within
the active site. A crystal structure of the S. faecalis
enzyme is not available, but the complex between
L-PALO and an E. coli OTC was recently solved at 2.8-Å
resolution.43 This structure suggests that Gln136 might
stabilize tetrahedral intermediate 2 by hydrogen bond-
ing to the amine derived from carbamoyl phosphate via
the carbonyl group in its side-chain amide. An analogous
interaction (with Gln171) has been postulated for the
human enzyme,44 and if a similar situation prevails in
S. faecalis OTC, replacement of the amine in the inhib-
itor with a phosphinate oxygen would be unfavorable.
Covalent inactivation of OTC by phaseolotoxin (22)45,46
(Harg ≡ homoarginine) shows that potent inhibition can
be achieved with molecules containing tetrahedral
phosphorus. Future attempts to develop more potent
transition-state analogues for OTC might therefore

R-Functionalized Phosphonylphosphinates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2637
removed in vacuo and the residue codistilled twice with
anhydrous benzene. The yellow oil (3.7 g) was >90% pure by
NMR and used without further purification. 1H NMR (CDCl3,
400 MHz): δ 1.42 (t, J ) 14 Hz, 3H, OEt), 4.01 (d, J ) 2.5 Hz,
2H, CH2OBn), 4.27-4.38 (m, 2H, OEt), 4.73 (s, 2H, CH2Ph),
7.32-7.40 (m, 5H, Ph). 13C NMR (CDCl3, 125.7 MHz): δ 16.3
(s, OCH2CH3), 64.1 (d, J ) 10 Hz, OCH2CH3), 68.5 (d, J ) 143
Hz, CH2Bn), 74.9 (d, J ) 10 Hz, CH2Ph), 128.1 (s, C-2′), 128.3
(s, C-4′), 128.5 (s, C-3′), 136.8 (s, C-1′). 31P NMR (CDCl3, 161.9
MHz): δ 35.23 (s, P).
profitably focus on replacing the phosphinate moiety in
11 and 17 with a phosphinic amide or sulfonamide. Such
compounds would be valuable for studies of the enzyme
and as haptens for the generation of catalytic antibodies
with OTC activity.
Exp er im en ta l Section
Gen er a l Exp er im en ta l Con d ition s. Carbamoyl phos-
phate and ornithine were obtained from Sigma, H-L-Orn(Z)-
OtBu and Boc-L-Asp(OH)-OtBu from Bachem, and all other
chemicals from Aldrich. Nδ-(Phosphonoacetyl)-L-ornithine was
synthesized by a modified literature procedure (see Supporting
Information).47,48 Ornithine transcarbamoylase from S. faecalis
was purchased from Sigma and showed a specific activity of
156 nmol of citrulline formed/mg of enzyme/min at 37 °C in
50 mM Tris‚HCl at pH 8.5. THF was freshly distilled from
potassium, benzene was distilled from sodium, and pyridine
was distilled from calcium hydride. Dichloromethane was
distilled from phosphorus pentoxide and checked for the
absence of chloride before use. All reactions were performed
under a dry argon atmosphere with the exception of those that
used water as solvent. Reaction progress was monitored by
thin-layer chromatography (TLC) or HPLC. TLC analyses were
performed with 0.25-mm silica gel plates on glass support
containing F-254 indicator or with octadecyl-modified silica on
glass support and visualized using UV light (254 nm), alkaline
potassium permanganate, or ninhydrin/nBuOH/HOAc. Flash
chromatography was performed on silica 60 from Merck,
Darmstadt. HPLC analyses were performed on LiChrospher
100 CH18/2 (5 µm) and LiChrosorb RP 18 (10 µm) with water/
MeCN (containing 0.005% TFA) and monitored at 220 nm.
Melting points are uncorrected.
Dieth yl Hyd r oxym eth ylp h osp h on a te. Diethyl phosphite
(15 mL, 120 mmol), triethylamine (16 mL, 120 mmol), and
paraformaldehyde (4.0 g, 130 mmol) were mixed in a reaction
vessel. The vessel was tightly sealed under vacuum and then
heated to 90 °C for 12 h. All volatile components were
subsequently removed under reduced pressure, and the re-
maining semisolid was dissolved in 40 mL of dichloromethane.
The solution was extracted with saturated NH4Cl, K2CO3, and
again with NH4Cl and dried with MgSO4 and the solvent
removed under reduced pressure. Compound diethyl hy-
droxymethylphosphonate was obtained as a pale-yellow oil
(11.7 g, 7.0 mmol, 60%). All spectroscopic data agreed with
previously published data.20,49
Eth yl Ben zyloxym eth yl[(d ieth oxyp h osp h or yl)m eth yl]-
p h osp h in a te. n-Butyllithium (2.0 M in cyclohexane, 7.5 mL,
15 mmol) was slowly added to a solution of diethyl meth-
ylphosphonate (2.2 mL, 15 mmol) in 60 mL of absolute THF
at -78 °C. After 15 min a white suspension formed, and 3.7 g
(∼15 mmol) of 5 in 30 mL of absolute THF was added over a
period of 30 min, after which the suspension clarified. The
reaction was stirred 1 h at -78 °C and 1 h at 0 °C and then
quenched by the addition of 10% HCl and diethyl ether. The
pH was adjusted to ca. 1, and after separation the aqueous
phase was extracted with dichloromethane. The combined
organic phases were extracted with saturated NaHCO3 and
dried with MgSO4, and the solvent was removed in vacuo.
Flash chromatography on silica gel with EtOAc/5% MeOH
yielded 4.0 g of ethyl benzyloxymethyl[(diethoxyphosphoryl)-
methyl]phosphinate (11 mmol, 73% based on 5) as a pale-
1
yellow oil. H NMR (CDCl3, 250 MHz): δ 1.20-1.40 (m, 9H,
OEt), 2.40-2.60 (m, 2H, P-CH2-P), 3.80-3.95 (m, 2H, CH2-
OBn), 4.0-4.25 (m, 6H, OEt), 4.55-4.70 (m, 2H, CH2Ph),
7.25-7.40 (m, 5H, Ph). 13C NMR (CDCl3, 100.6 MHz): δ 16.3
(d, J ) 6 Hz, P2OCH2CH3), 16.4 (d, J ) 6 Hz, P1OCH2CH3),
25.2 (dd, J ) 135 Hz, 83 Hz, P-CH2-P), 64.6 (d, J ) 6.5 Hz,
P1OCH2CH3), 62.5 (d, J ) 6.2 Hz, P2OCH2CH3), 62.7 (d, J )
6.2 Hz, P2OCH2CH3), 65.6 (d, J ) 119 Hz, CH2OBn), 75.2 (d,
J ) 13.5 Hz, CH2Ph), 128.1 (s, C-2′), 128.1 (s, C-4′), 128.4 (s,
C-3′), 136.8 (s, C-1′). 31P NMR (CDCl3, 161.9 MHz): δ 20.38
(d, J ) 4.5 Hz, P-1), 41.82 (d, J ) 4.5 Hz, 1P, P-2). HRMS
(FAB, NBA):
365.1275.
C
15H27O6P2 (M + H+) calcd 365.1283, found
Eth yl [(Dieth oxyp h osp h or yl)m eth yl]h yd r oxym eth -
ylp h osp h in a te (6). A solution of ethyl benzyloxymethyl-
[(diethoxyphosphoryl)methyl]phosphinate (4.0 g, 11 mmol) in
300 mL of ethanol was treated with 0.4 g of palladium on
carbon (5%, contained 50% H2O) and then hydrogenated at
ambient pressure for 12 h. After separation from the catalyst
and evaporation under reduced pressure, 3.0 g (11 mmol,
1
>98%) of 6 was obtained as a colorless, clear resin. H NMR
Dieth yl Ben zyloxym eth ylp h osp h on a te (4). A solution
of diethyl hydroxymethylphosphonate (14 g, 82 mmol) in 50
mL of absolute dichloromethane was treated successively with
silver(I) oxide (10 g, 50 mmol) and benzyl bromide (14 mL,
115 mmol). The reaction was stirred in the dark at room
temperature for 48 h. After filtration and removal of solvent
in vacuo, benzyl bromide was distilled off at 50 °C and 0.1
mbar. Flash chromatography on silica with ethyl acetate as
eluent afforded 14 g (54 mmol, 66%) of 4 as a pale-yellow oil.
All data agreed with the previously published data.18,50
Eth yl Hyd r ogen Ben zyloxym eth ylp h osp h on a te. A so-
lution of 4 (4.0 g, 15 mmol) in ethanol (125 mL) and 1 M
aqueous potassium hydroxide (85 mL) was heated to 75 °C
for 2.5 h. The solution was cooled to 0 °C and then brought to
pH 7.0 with 3.2 M HCl. The ethanol was removed under
reduced pressure. The remaining solution was diluted with
400 mL of CH2Cl2 and acidified with 3.2 M HCl to pH 1.0.
The aqueous phase was separated and extracted again with
CH2Cl2. The combined organic extracts were washed with 50
mL of saturated NaCl and dried over MgSO4. Removal of the
solvent under reduced pressure yielded ethyl hydrogen ben-
zyloxymethylphosphonate as a pale-yellow oil (3.5 g, 15 mmol,
>98%). All data agreed with previously published data.50
Eth yl Ben zyloxym eth yl(ch lor o)p h osp h on a te (5). Ox-
alyl chloride (3.8 mL, 43 mmol) was added slowly to a solution
of ethyl hydrogen benzyloxymethylphosphonate (4.0 g, 17.3
mmol) and DMF (10 µL) in 50 mL of anhydrous benzene. After
stirring for 2.5 h at room temperature, the solvent was
(CDCl3, 200 MHz): δ 1.33-1.40 (m, 9H, OEt), 2.47-2.65 (m,
2H, P-CH2-P), 4.03-4.07 (m, 2H, CH2OH), 4.09-4.24 (m, 6H,
OEt). 13C NMR (CDCl3, 100.6 MHz): δ 16.1 (d, J ) 5.5 Hz,
P2OCH2CH3), 16.2 (d, J ) 7 Hz, P2OCH2CH3), 16.3 (s, J ) 6
Hz, P1OCH2CH3), 25.3 (dd, J ) 135 Hz, 78 Hz, P-CH2-P), 59.0
(d, J ) 112 Hz, CH2OH), 61.5 (d, J ) 6.5 Hz, P1OCH2CH3),
62.7 (d, J ) 6.5 Hz, P2OCH2CH3), 62.8 (d, J ) 6 Hz, P2OCH2-
CH3). 31P NMR (CDCl3, 161.9 MHz): δ 21.68 (d, J ) 36.8 Hz,
P-1), 44.23 (d, J ) 44.4 Hz, 1P, P-2). HRMS (FAB, NBA/NaI):
C8H20O6P2Na (M + Na+) calcd 297.0633, found 297.0638.
[(Diet h oxyp h osp h or yl)m et h yl(et h oxy)p h osp h or yl]-
m eth yl Meth a n esu lfon a te (7a ). A solution of 6 (260 mg, 1
mmol) in 20 mL of absolute pyridine was treated with
methanesulfonyl chloride (180 µL, 2.3 mmol, dissolved in 7
mL of pyridine) at 0 °C over a period of 30 min. The solution
was kept at this temperature for 24 h, then poured into ice
water, extracted three times with CH2Cl2, and dried with
Na2SO4 and the solvent removed in vacuo. Flash chromatog-
raphy on silica with CH2Cl2/MeOH/acetone (30:2:2) afforded
179 mg (53%) of 7a as a colorless oil. 1H NMR (CDCl3, 300
MHz): δ 1.35 (t, J ) 6 Hz, 9H, OEt), 2.50 (td, J ) 19 Hz, 6
Hz, 2H, P-CH2-P), 3.12 (s, 3H, SO2Me), 4.15 (m, 6H, OEt),
4.59 (m, 2H, CH2OMes).
[(Diet h oxyp h osp h or yl)m et h yl(et h oxy)p h osp h or yl]-
m eth yl 2,3,4,5,6-P en ta flu or oben zen esu lfon a te (7b). A
solution of 6 (1.0 g, 3.6 mmol) and N-ethyldiisopropylamine
(1.0 mL, 7.8 mmol) in 30 mL of absolute CH2Cl2 was cooled to

2638 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Flohr et al.
-78 °C and slowly treated with a solution of pentafluoroben-
zenesulfonyl chloride (800 µL, 5.4 mmol) in 10 mL of absolute
CH2Cl2. After 3 h at -78 °C the solution was slowly warmed
to -15 °C and then kept for 12 h. The solvent was evaporated
at reduced pressure and the yellow oil loaded immediately on
a silica column, preequilibrated with CH2Cl2/MeOH/acetone
(30:2:2). The column was rapidly eluted with the same solvent,
and the fractions containing product (Rf 0.6) were combined.
Removal of the solvent in vacuo yielded 7b (1.5 g, 3.0 mmol,
84%) as pale yellow-crystals. Mp: 65 °C. 1H NMR (CDCl3, 500
MHz): δ 1.34 (t, J ) 7 Hz, 3H, P2OCH2CH3), 1.35 (t, J ) 7
Hz, 3H, P2OCH2CH3), 1.39 (t, J ) 7 Hz, 3H, P1OCH2CH3), 2.51
(m, 2H, P-CH2-P), 4.17 (m, 6H, OCH2CH3), 4.66 (m, 2H,
CH2OPf). 13C NMR (CDCl3, 125.7 MHz): δ 16.0 (d, J ) 6 Hz,
P2OCH2CH3), 16.1 (d, J ) 6 Hz, P2OCH2CH3), 16.2 (d, J ) 5
Hz, P1OCH2CH3), 25.4 (dd, J ) 90 Hz, 45 Hz, P-CH2-P), 62.3
(d, J ) 6 Hz, P2OCH2CH3), 62.5 (d, J ) 7.5 Hz, P2OCH2CH3),
63.2 (d, J ) 6 Hz, P1OCH2CH3), 64.7 (d, J ) 113 Hz,
CH2OPf), 111.0 (m, C1′-SO3), 137.9 (dm, J ) 239 Hz, C3′-F),
145.3 (dm, J ) 264 Hz, C2′-F, C4′-F). 31P NMR (CDCl3, 161.9
MHz): δ 18.07 (s, P-1), 35.29 (s, P-2). 19F NMR (CDCl3, 376.3
MHz): δ -154.5 (m, 2F, 2-F), -138.9 (m, 1F, 4-F), -130.2 (m,
3-F). HRMS (FAB, NBA/CsI): C14H19O8F5P2SCs (M + Cs+)
calcd 636.9250, found 636.9263.
n-CO2tBu), 171.96 (s, CO2-). 31P NMR (121.4 MHz, D2O, pH
5.0): δ 16.9 (s, P-1), 23.0 (s, P-2).
N δ-{[D ih y d r o x y p h o s p h o r y lm e t h y l(h y d r o x y )p h o s -
p h or yl]m eth yl}-L-or n ith in e Sod iu m Sa lt (11). Compound
10 (34 mg, 75 µmol) was dissolved in 2 mL of 3.2 M HCl at 0
°C and lyophilized after 30 min. The white powder was
dissolved in water, and the pH was adjusted to 8.0 with 2 M
NaOH. Chromatography on RP-18 silica with water/methanol
(1:1) and subsequent lyophilization yielded 30 mg (75 µmol,
>98%) of 11 as a white, hygroscopic powder. 1H NMR (500
MHz, D2O, pH 6.0): δ 1.70-2.05 (m, 4H, γ-CH2, â-CH2), 2.13
(t, J ) 37 Hz, 2H, P-CH2-P), 3.09 (m, 2H, δ-CH2), 3.11 (t, J
) 11 Hz, 2H, P-CH2-N), 3.78 (J ) 6 Hz, t, R-CH). 13C NMR
(125.7 MHz, D2O, pH 8.0) δ 24.51 (s, â-CH2), 30.19 (s, γ-CH2),
36.4 (dd, J ) 116 Hz, 85 Hz, P-CH2-P), 41.63 (s, δ-CH2), 49.9
(d, J ) 91 Hz, P-CH2N), 51.02 (s, R-CH), 176.9 (s, CO2). 31P
NMR (161.9 MHz, D2O, pH 8.0): δ 16.6 (s, P-1), 23.0 (s, P-2).
HRMS: (ESI) [C7H15N2O7P2]3-‚2H+ (M3-+ 2H+) calcd 303.0511,
found 303.0519; [C7H15N2O7P2]3-‚2Na+ (M3-+ 2Na+) calcd
347.0150, found 347.0159.
Nr-ter t-Bu toxyca r bon yl-L-or n ith in e ter t-Bu tyl Ester
(8). Compound 8 was prepared in >98% yield from H-L-Orn-
(Z)-OtBu by protection with Boc2O in NaOH/THF, subsequent
catalytic hydrogenation in EtOH/HOAc with Pd/C as catalyst,
1
Nr-ter t-Bu t oxyca r b on yl-Nδ-{[(d iet h oxyp h osp h or yl)-
m eth yl(eth oxy)p h osp h or yl]m eth yl}-L-or n ith in e ter t-Bu -
tyl Ester (9). Pentaflate 7b (500 mg, 1.0 mmol) was dissolved
in 35 mL of absolute CH2Cl2 and cooled to 0 °C. Then 8 (721
mg, 2.5 mmol), in 15 mL of absolute CH2Cl2, was added in
one batch and the solution immediately heated to 40 °C for 3
h. After cooling to ambient temperature, the solution was
diluted with 200 mL of Et2O and extracted with 20 mL of
saturated K2CO3. After back-extraction of the aqueous layer,
the combined organic phases were dried over MgSO4 and
evaporated under reduced pressure and the yellow oil was
chromatographed on silica with CH2Cl2/MeOH/acetone (30:2:
2) as eluent. 9 (392 mg, 0.72 mmol, 72%) was obtained as a
and final extraction with saturated aqueous K2CO3/Et2O. H
NMR, 13C NMR, and melting point were consistent with
previously published data.26,51 HRMS (FAB, NBA): C14H29N2O4
25
(M + H+) calcd 289.2127, found 289.2122. [R]D +1.85 (c ) 1,
25
CH2Cl2); [R]436 ) +5.45 (c ) 1, CH2Cl2).
ter t-Bu t yl 2(S)-ter t-Bu t oxyca r b on yla m in o-4-m et h yl-
su lfon yloxybu tyr a te (Boc-L-Hse(Mes)-OtBu ) (12). To a
solution of 2(S)-2-tert-butoxycarbonylamino-4-hydroxybutyric
acid tert-butyl ester52 (250 mg, 0.9 mmol, prepared from Boc-
L-Asp(OH)-OtBu by borane reduction in THF) in 5 mL of
CH2Cl2 was added 360 µL (2.7 mmol) of N-ethyldiisopro-
pylamine and 78 µL (1.0 mmol) of methanesulfonyl chloride
at 0 °C. The resulting solution was stirred for 20 min and then
washed with saturated aqueous NH4Cl. The aqueous layer was
back-extracted three times with CH2Cl2 and dried over MgSO4,
and the combined organic phases were evaporated in vacuo.
Flash chromatography on silica with EtOAc/hexane (1:1)
afforded 229 mg (640 mmol, 71%) of 12 as white needles. Mp:
1
clear, colorless resin. H NMR (500 MHz, CDCl3): δ 1.32 (m,
9H, OEt), 1.41 (s, 9.5H, NH-CO2CMe3, γ-CH2), 1.43 (s, 9.5H,
CH-CO2CMe3, γ-CH2), 1.52 (m, 1H, γ-CH2), 1.63 (m, 1H,
â-CH2), 1.75 (m, 1H, â-CH2), 2.51 (m, 2H, P-CH2-P), 2.68 (m,
2H, δ-CH2), 3.6 (m, 2H, P-CH2-N), 4.15 (m, 7H, OEt, R-CH),
5.23 (m, 1H, NH). 13C NMR (CDCl3, 125.7 MHz): δ 16.2 (m,
P2CH2CH3), 16.4 (m, P1CH2CH3), 25.3 (s, â-CH2), 25.9 (dd, J
) 252 Hz, 180 Hz, P-CH2-P), 27.9 (s, CH-CO2CMe3), 28.2
(s, NH-CO2CMe3), 30.3 (s, γ-CH2), 47.6 (d, J ) 114 Hz,
P-CH2N), 50.6 (d, J ) 109 Hz, R-CH), 53.7 (s, δ-CH2), 61.2 (m,
P1OCH2CH3), 62.4 (m, P2OCH2CH3), 79.3 (NH-CO2CMe3),
81.5 (s, CH-CO2CMe3), 155.9 (s, NH-CO2tBu), 172.5 (s, CH-
CO2tBu). 31P NMR (161.9 MHz, CDCl3): δ 20.84 (d, J ) 2.5
Hz, P-1), 44.84 (dd, J ) 12 Hz, 3 Hz, P-2). HRMS (FAB, NBA/
CsI): C22H46N2O9P2 + Cs+ (M + Cs+) calcd 677.1730, found
1
85-87 °C. H NMR (400 MHz, CDCl3): δ 1.45 (s, 9H, OtBu),
1.48 (s, 9H, Boc), 2.03-2.33 (m, 2H, â-CH2), 3.03 (s, 3H, OMes),
4.25-4.33 (m, 3H, R-CH, γ-CH2), 5.17 (s, 1H, NH). 13C
NMR (100.6 MHz, CDCl3): δ 27.97 (s, OtBu), 28.30 (s, Boc),
32.22 (s, â-CH2), 37.30 (s, OMes), 50.95 (s, R-CH), 66.18 (s,
γ-CH2), 80.13 (s, NHCO2tBu), 82.78 (s, CHCO2tBu), 155.39 (s,
NHCO2tBu), 170.80 (s, CHCO2tBu). HRMS (FAB, NBA +
PEGMEE):
354.1584. [R]D +11.6 (c ) 1.03, CHCl3).
C
14H28NO7S (M + H+) calcd 354.1586, found
25
ter t-Bu tyl 4-Azid o-2(S)-ter t-bu toxyca r bon yla m in obu -
tyr a te (13). A solution of 229 mg (0.65 mmol) of mesylate 12
and 211 mg (3.2 mmol) of sodium azide in 10 mL of dimeth-
ylformamide was stirred at 40 °C for 12 h. The solvent was
removed in vacuo, the residue dissolved in water/CH2Cl2, and
after separation of the layers the aqueous layer extracted three
times with CH2Cl2. The organic layers were combined and
dried over MgSO4, and evaporation of the solvent afforded 138
mg (0.46 mmol, 71%) of 13 as a clear, colorless oil. 1H NMR
(400 MHz, CDCl3): δ 1.45 (s, 9H, tBu), 1.48 (s, 9H, tBu), 1.8-
2.1 (m, 2H, â-CH2), 3.39 (t, J ) 6.5 Hz, 2H, γ-CH2), 4.25 (d, J
) 4 Hz, 1H, R-CH), 5.15 (s, 1H, NH). 13C NMR (100.6 MHz,
CDCl3): δ 27.97 (s, tBu), 28.32 (s, tBu), 32.10 (s, â-CH2), 47.85
(s, γ-CH2), 51.91 (s, R-CH), 79.96 (s, Boc), 82.49 (s, CO2tBu),
25
25
677.1746. [R]D ) +1.64 (c ) 1.1, CH2Cl2), [R]436 ) +4.73 (c
) 1.1, CH2Cl2).
Nr-ter t-Bu t oxyca r b on yl-Nδ-{[d ih yd r oxyp h osp h or yl-
m eth yl(h ydr oxy)ph osph or yl]m eth yl}-L-or n ith in e Sodiu m
Sa lt (10). Hexamethyldisilazane (210 µL, 1.0 mmol) was added
to a solution of 9 (50 mg, 97 µmol) in 5 mL of absolute
dichloromethane, and the clear solution was cooled to 0 °C.
After addition of bromotrimethylsilane (130 µL, 1.0 mmol) the
solution was slowly warmed to room temperature and then
stirred for 12 h. After a second addition of both reagents and
stirring for an additional 24 h, all volatile components were
removed under reduced pressure. Water (4 mL) was added and
the pH adjusted to 8.0 with 2 M NaOH at 0 °C. Chromato-
graphy on RP-18 silica with water as eluent and subsequent
lyophilization yielded 10 as a white powder (34 mg, 75 µmol,
155.34 (s, Boc), 171.04 (s, CO2tBu). HRMS (FAB, NBA):
25
C
13H25N4O4 [M + H+] calcd 301.1876, found 301.1879. [R]D
1
+20.1 (c ) 1.17, CHCl3).
77%). H NMR (500 MHz, D2O, pH 5.0): δ 1.49 (s, 9H, tBu),
1.75-2.02 (m, 4H, â-CH2, γ-CH2), 2.13 (t, J ) 19 Hz, 2H,
P-CH2-P), 3.11 (m, 4H, P-CH2-N, δ-CH2), 4.00 (t, J ) 6.5
Hz, R-CH2). 13C NMR (125.7 MHz, D2O, pH 5.0) δ 22.51 (s,
â-CH2), 29.77 (s, tBu), 29.85 (s, â-CH2), 36.26 (dd, J ) 85 Hz,
32 Hz, P-CH2-P), 49.80 (d, J ) 92 Hz, P-CH2N), 50.65 (d, J )
7.5 Hz, δ-CH2), 55.39 (s, R-CH2), 88.39 (s, tBu), 131.22 (s,
ter t-Bu tyl 4-Am in o-2(S)-ter t-bu toxyca r bon yla m in obu -
tyr a te (14). A solution of 13 (90 mg, 0.30 mmol) in 10 mL of
ethanol and 17 µL (0.30 mmol) of acetic acid was treated with
21 mg of palladium on carbon (5%) and hydrogenated at
ambient pressure for 1 h. Separation from the catalyst and
evaporation of the solvent at reduced pressure afforded 103

R-Functionalized Phosphonylphosphinates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2639
mg (0.30 mmol, >98%) of 14 as a clear, colorless resin.
P-CH2-P), 3.21 (d, J ) 8 Hz, 2H, P-CH2-NH2), 4.22-4.15
(m, 6H, OEt). 13C NMR (CDCl3, 125.7 MHz): δ 16.2 (d, J ) 5
Hz, P(OEt)2), 16.4 (d, J ) 5 Hz, P(CH2NH2)OEt), 25.3 (dd, J
) 77 Hz, 59 Hz, P-CH2-P), 40.9 (d, J ) 106 Hz, CH2NH2),
61.1 (d, J ) 5 Hz, P(CH2NH2)OEt), 62.4 (d, J ) 5 Hz, OEt)
62.7 (d, J ) 5 Hz, OEt). 31P NMR (CDCl3, 161.9 MHz): δ 20.9
(s, P-1), 45.6 (s, P-2). HRMS (NBA/NaI): C8H22NO5P2 (M +
H+) calcd 274.0973, found 274.0977.
Sod iu m Am in om eth yl(d ih yd r oxyp h osp h or ylm eth yl)-
p h osp h in a te (20). Compound 19 (50 mg, 183 µmol) was
treated with 240 µL (1.8 mmol) of bromotrimethylsilane at -40
°C. After 1 h, the solution was slowly warmed to 0 °C and
stirred for 60 h. Workup and purification in the same way as
described for 10 yielded 27 mg (69%) of 20 as a white powder.
1H NMR (D2O, pH 12.0, 400 MHz): δ 1.80 (t, J ) 18 Hz, 2H,
P-CH2-P), 3.47 (m, 2H, P-CH2NH2). 13C NMR (D2O, pH 6.0,
75.4 MHz): δ 35.0 (dd, J ) 83 Hz, 39 Hz, P-CH2-P), 41.7 (d,
J ) 95 Hz, P-CH2N). 31P NMR (D2O, pH 12.0, 161.9 MHz): δ
12.6 (d, J ) 4 Hz, P-1), 39.2 (d, J ) 4 Hz, P-2); (D2O, pH 6,
121.4 MHz): 16.1 (s, P-1), 25.0 (s, P-2); (D2O, pH 1, 121.4
MHz): 19.5 (s, P-1), 26.2 (s, broad, P-2). MS (ESI): C2H8NO5P2
(M3-‚2H+): 188.
In h ibition Stu d ies w ith Or n ith in e Tr a n sca r ba m oy-
la se. Ornithine (96 mM, adjusted to pH 8.5 with 2 M KOH),
and carbamoyl phosphate (96 mM), and OTC (2 µg/mL) were
frozen in aliquots and thawed immediately prior to use. A
chromogenic assay described by Pastra-Landis30 was used. For
the color reagent, antipyrin (10 mL, 250 mg in 50 mL of 50%
H2SO4) and 2,3-butanedione monoxime (5 mL, 200 mg in 25
mL of 5% HOAc, freshly prepared before use) were mixed just
before use and stored on ice. The assays were initiated by the
addition of carbamoyl phosphate, quenched by the addition of
color reagent, and then stored in the dark for 12 h at room
temperature. The samples were then heated 24 min to 45 °C
under illumination by a 60-W bulb at a distance of 70 cm. Then
the absorption was measured at 466 nm. Blank samples
contained all reagents with the exception of OTC, and controls
contained all reagents but no inhibitor.
The concentration of the different inhibitors was varied
stepwise between 0.5 µM and 2.5 mM. Typically, the appropri-
ate inhibitor, 4.8 mM ornithine, 10 ng/mL OTC, and 71-500
µM carbamoyl phosphate in a total volume of 0.1 mL were
incubated at 37.0 °C for 8 min and then quenched by the
addition of 0.1 mL of color reagent; 50 mM Tris‚HCl, 0.5 mM
EDTA (pH 8.0 and 7.0), and 50 mM maleate, 0.5 mM EDTA
(pH 6.0) were used as buffers. Lineweaver-Burk plots (1/v vs
1/[carbamoyl phosphate]) were used to assess competitive
behavior, and Dixon plots of 1/v vs [I] were used to determine
the Ki values.53 Km values for carbamoyl phosphate at the
different pH values in the buffers used were taken from Kurtin
et al.:27 180 µM (pH 8.0), 111 µM (pH 7.0), and 30 µM (pH
6.0).
1
Compound 14 was characterized as the acetate salt. H NMR
(200 MHz, CDCl3): δ 1.42 (s, 9H, tBu), 1.45 (s, 9H, tBu), 1.8-
2.1 (m, 2H, â-CH2), 1.94 (s, 3H, OAc), 2.9-3.1 (m, 2H, γ-CH2),
4.15 (d, J ) 4 Hz, 1H, R-CH2), 5.15 (s, 1H, NH), 6.5 (s, 3H,
NH3). 13C NMR (100.6 MHz, CDCl3): δ 22.8 (s, OAc), 28.9 (s,
2 tBu), 33.03 (s, â-CH2), 36.9 (s, γ-CH2), 51.7 (s, R-CH), 80.3
(s, tBu), 82.6 (s, tBu), 156.1 (s, OAc), 171.2 (s, N-CO2tBu), 177.1
(s, CH-CO2tBu). HRMS (FAB, NBA): C13H27N2O4 (M + H+)
25
calcd 275.1971, found 275.1971.; [R]D -2.6 (c ) 4.2, CHCl3).
ter t-Bu tyl
2(S)-ter t-Bu toxyca r bon yla m in o-4-({[(d i-
m eth oxyph osph or yl)m eth yl(eth oxy)ph osph or yl]m eth yl}-
a m in o)bu tyr a te (15). Compound 15 was synthesized from
14 (as the free base by extraction with K2CO3/Et2O) and 7b in
the manner described for 9. 15 was obtained as a clear,
1
colorless resin. H NMR (500 MHz, CDCl3): δ 1.35 (td, J ) 7
Hz, 2.5 Hz, 3H, OEt), 1.44 (s, 9H, OtBu), 1.47 (s, 9H, OtBu),
1.77-2.04 (m, 2H, â-CH2), 2.59 (m, 2H, P-CH2-P), 2.78 (t, J
) 7 Hz, 2H, γ-CH2), 3.11 (d, J ) 11 Hz, 2H, P-CH2N), 3.83
(m, 6H, OMe), 4.18 (m, 2H, OEt), 4.23 (m, 1H, R-CH), 5.20
(dd, J ) 11 Hz, 8 Hz, NHBoc). 13C NMR (125.7 MHz, CDCl3):
δ 16.6 (s, OCH2CH3), 24.6 (dd, J ) 78 Hz, 134 Hz, P-CH2-
P), 28.1 (s, OtBu), 28.4 (s, OtBu), 32.3 (d, J ) 27 Hz, â-CH2),
47.4 (s, γ-CH2), 47.9 (d, J ) 109 Hz, P-CH2N), 52.4 (d, J ) 8
Hz, OMe), 53.0 (d, J ) 7 Hz, OMe), 53.1 (dd, J ) 16 Hz, 7 Hz,
R-CH), 61.6 (d, J ) 7 Hz, OCH2CH3), 79.6 (s, NCO2CMe3), 81.8
(s, OCO2CMe3), 155.6 (s, NCO2), 171.9 (s, OCO2). 31P NMR
(121.4 MHz, CDCl3): δ 24.0 (s, P-1), 45.0 (d, J ) 50 Hz, P-2).
HRMS (FAB): C19H41N2O9P2 (M + H+) calcd 503.2287, found
25
503.2299. [R]D +5.75 (c ) 0.4, CHCl3).
Sod iu m
2(S)-ter t-Bu toxyca r bon yla m in o-4-({[d ih y-
d r oxyp h osp h or ylm eth yl(h yd r oxy)p h osp h or yl]m eth yl}-
a m in o)bu tyr a te (16). Compound 15 was deprotected to give
16 as described for 10. 1H NMR (300 MHz, D2O): δ 1.55 (s,
9H, tBu), 2.32 (m, 2H, P-CH2-P, â-CH2), 3.25 (d, J ) 11 Hz,
2H, P-CH2N), 3.37 (m, 2H, γ-CH2), 4.19 (m, 1H, R-CH). 31P
NMR (121.4 MHz, D2O): δ 16.31 (s, P-1), 22.65 (s, P-2).
Sod iu m 2(S)-Am in o-4-({[d ih yd r oxyp h osp h or ylm eth yl-
(h yd r oxy)p h osp h or yl]m eth yl}a m in o)bu tyr a te (17). Com-
pound 16 was deprotected to 17 using the same method as
1
that described for 11. H NMR (500 MHz, D2O): δ 2.20-2.30
(m, 4H, P-CH2-P, â-CH2), 3.21 (dd, J ) 11 Hz, 3.5 Hz, 2H,
P-CH2N), 3.24-3.33 (m, 2H, γ-CH2), 3.84 (m, 1H, R-CH2). 13
C
NMR (125.7 MHz, D2O): δ 29.76 (s, â-CH2), 35.22 (dd, J )
121 Hz, 85 Hz, P-CH2-P), 39.16 (s, γ-CH), 48.36 (d, J ) 8 Hz,
R-CH2), 49.76 (d, J ) 93 Hz, P-CH2N), 175.74 (s, CO2). 31P
NMR (121.4 MHz, CDCl3): δ 15.9 (s, P-1), 23.1 (s, P-2). HRMS
(ESI): [C6H13N2O7P2]3-‚3H+ (M3- + 2H+) calcd 289.0355, found
289.0366.
E t h yl
Azid om et h yl[(d iet h oxyp h osp h or yl)m et h yl]-
p h osp h in a te (18). A solution of 7b (300 mg, 0.60 mmol) in
12 mL of absolute ethanol was treated with sodium azide (200
mg, 3.0 mmol), and the suspension stirred at ambient tem-
perature for 24 h. After filtration, the solvent was removed in
vacuo and the residue suspended in dry Et2O and refiltered.
Removal of solvent under reduced pressure yielded 179 mg
Ack n ow led gm en t. This work was supported by the
National Institutes of Health (DK45232 to D.H.) and a
fellowship from the scientific board of the NATO by the
DAAD (to A.F.). We thank Dr. Arnd Ingendoh from the
Bruker-Franzen, Bremen, Germany, for the high-
resolution ESI mass spectra.
1
(0.60 mmol, >98%) of 18 as a clear, colorless resin. H NMR
(400 MHz, CDCl3): δ 1.31-1.40 (m, 9H, OEt), 3.72 (dd, J ) 5
Hz, 4.5 Hz, 2H, P-CH2-P), 4.20 (m, 2H, P-CH2N3), 4.25-
4.17 (m, 6H, OEt). 13C NMR (100.6 MHz, CDCl3): δ 16.3 (d, J
) 7 Hz, P(OEt)2), 16.5 (d, J ) 6 Hz, P(CH2N3)OEt), 26.0 (dd,
J ) 83 Hz, 52 Hz, P-CH2-P), 47.8 (d, J ) 107 Hz, P-CH2N3),
62.2 (d, J ) 7 Hz, OEt), 62.7 (d, J ) 6 Hz, OEt), 63.0 (d, J )
7 Hz, OEt). 31P NMR (161.9 MHz, CDCl3): δ 19.3 (s, P-1), 40.1
(s, P-2). HRMS (FAB, NBA/NaI): C8H19N3O5P2Na (M + Na+)
calcd 322.0698, found 322.0692.
Su p p or tin g In for m a tion Ava ila ble: Preparation and
characterization of ethyl chloromethyl[(diethoxyphosphoryl)-
methyl]phosphinate, NR-isobutoxycarbonyl-L-ornithine tert-
butyl ester, and NR-isobutoxycarbonyl-Nδ-{[(diethoxyphospho-
ryl)methyl(ethoxy)phosphoryl]methyl}-L-ornithine tert-butyl
ester; modified procedures for the preparation of dibenzyl
methylphosphonate and (dibenzyloxyphosphoryl)acetic acid.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Eth yl Am in om eth yl[(d ieth oxyp h osp h or yl)m eth yl]-
p h osp h in a te (19). A solution of 18 (179 mg, 0.60 mmol) in
60 mL of ethanol was treated with 50 mg of palladium on
carbon (5%) and hydrogenated at ambient pressure for 17 h.
After separation from the catalyst and removal of the solvent
under reduced pressure, 164 mg (0.60 mmol, >98%) of 19 was
obtained as a clear, colorless resin. 1H NMR (CDCl3, 500
MHz): δ 1.37 (m, 9H, OEt), 2.53 (dd, J ) 16.5 Hz, 4 Hz, 2H,
Refer en ces
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M.; Harrity, T.; Rich, L. C.; Ciosek, C. P. J . Am. Chem. Soc. 1991,
113, 8522-8524.

2640 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
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