1) Novel compounds called O,O-diethyl 1-benzamido-2,2-biscarbamoylethanephosphonates were synthesized as substrates for HIV-1 protease (PR) to exploit activation of the phosphonate group. 2) One compound, O,O-diethyl 1-benzamido-2,2-bis[(1S)-N-(1-benzyl-2-hydroxyethyl)carbamoyl]ethanephosphonate, showed moderate anti-HIV activity in vitro. Its depsipeptide analogue inhibited HIV-1 PR with an IC50 of 31 μM. 3) The phosphonate group of these compounds was designed

1. Letters in Drug Design & Discovery, 2009, 6, 139-145 139
1570-1808/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.
Presentation of the -Carboxamidophosphonate Arrangement in
Substrate Structures Targeting HIV-1 PR
N.J. Wardle*,a
, H.R. Hudsonb
, R.W. Matthewsb
, C. Nunnb
, C. Vellab
and S.W.A. Bligha
a
Institute for Health Research and Policy; b
Department of Health and Human Sciences, London Metropolitan Univer-
sity, 166-220 Holloway Rd., London, N7 8DB, UK
Received October 27, 2008: Revised December 05, 2008: Accepted December 15, 2008
Abstract: Novel O,O-diethyl 1-benzamido-2,2-biscarbamoylethanephosphonates were synthesised as putative substrates
to HIV-1 PR, to exploit the state of activation of the phosphonate electrophilic function in -carboxamidophosphonate ar-
rangements. O,O-Diethyl 1-benzamido-2,2-bis[(1S)-N-(1-benzyl-2-hydroxyethyl)carbamoyl]ethanephosphonate exhibited
moderate anti-HIV activity in vitro (EC50 = 53 μ ), while its depsipeptide analogue; O,O-diethyl 1-benzamido-2,2-
bis[(1S)-N-(1-benzyl-2-{(2’S)-leucinyloxy}ethyl)carbamoyl]ethanephosphonate inhibited HIV-1 PR (IC50 = 31 μ ).
Keywords: -carboxamidophosphonates, HIV-1 PR inhibitors, Anti-HIV agents.
INTRODUCTION
Human immunodeficiency virus type-1 protease (HIV-1
PR) is critical to successful completion of the viral life-cycle,
initiating post-transcriptional cleavage of the viral poly-
protein precursor of gag (p55) and gag-pol (p160) viral pro-
teins, yielding structural proteins and enzymes essential to
virion-maturation. The protease constitutes a homodimer of
two 99-amino-acid-polypeptide monomers, each contribut-
ing an Asp25-Thr26-Gly27 triad to the active-site, located at
a cleft between the two domains as part of a four-stranded
beta turn. Its pivotal role in the viral life cycle has identified
HIV-1 PR as a target for therapeutic intervention, and prote-
ase inhibitors (PIs) have achieved potent inhibition of viral
replication in infected individuals [1-5]. PI candidates have
up to this point employed non-scissile P1-P1 [6] transition-
state isosteres in structures resembling natural substrates to
varying degrees. These have included C2-symmetric struc-
tures, exploiting the protease topology [7-15] (e.g. 1, Moz-
enavir, Fig. 1) both to enhance specificity for HIV-1 PR over
mammalian aspartic proteases and to optimise the P2-P2 / S2-
S2 (and peripheral) interactions critical to binding potency.
Structure-based approaches have achieved potent reversible,
competitive inhibitory profiles in clinical drug combination
(HAART) regimens [16]. Meanwhile, mechanism-based
approaches employing affinity labels have yielded irreversi-
ble, non-competitive inhibitors (e.g. 2, Fig. 1). While corre-
sponding PI candidates have yet to find clinical application
[17-22], the latter approach has the potential to provide po-
tent inhibition at lower concentrations than are required with
non-competitive inhibitors (thereby reducing toxicity).
Various hydrolytic studies have characterised an acute
activation of the -carboxyl/carboxamidophosphonate motif,
and its phosphinate analogue, to nucleophilic displacement at
phosphorus under acidic conditions, either via hydrogen-
bonded [23] or mixed cyclic carboxyl/carboxamidyl-
*Address correspondence to this author at the Institute for Health Research
and Policy, Tower Building, London Metropolitan University, 166-220
Holloway Rd., London, N7 8DB, UK; Tel: +44 207 133 2140; Fax: +44 207
133 2096; E-mail: n.wardle@londonmet.ac.uk
phosphonyl anhydride [24-28] intermediates (Scheme 1).
Analogous activation within an enzyme-inhibitor (EI) com-
plex of HIV-1 PR could offer scope for interaction with
various enzymatic residues, subject to the positioning of the
inhibitor’s “warhead” [29] function. Potential interactions
would include covalent adduct formation (e.g. with Thr
26/26’ or Thr31/31’ residues), or alternatively sequestration
of enzymatic water (e.g. the water molecule hydrogen-
bonded to Ile50/50’ binding-flap residues) in hydrolysis. The
water molecule specified in parenthesis is critical to ES bind-
ing interactions [3,30], and its functional replacement has
previously featured as a target for inhibitor design [11-16] in
order to exploit the entropic gain arising from liberation of
the protein-bound water to bulk solvent [31]. In the context
of the titled structures, hydrolytic sequestration of this water
molecule could release the alcohol by-product of displace-
ment to bulk solvent, while retaining hydrogen-bonding in-
teractions with Ile50/50’ residues through the modified
phosphonate function. Therefore, the titled compounds were
developed to present the above motif in pseudo-C2-
symmetric, peptidomimetic structures commensurate with
the binding criteria of HIV-1 PR.
1 Mozenavir
N N
O
HO OH
N
H
N
N
H
O
O
S
O
N
S
O
O
O O
2
H2N NH2
Fig. (1). Examples of reversible (1) and irreversible (2) inhibitors of
HIV-1 PR.

2. 140 Letters in Drug Design & Discovery, 2009, Vol. 6, No. 2 Wardle et al.
RESULTS AND DISCUSSION
Modelling
“Molegro” virtual docking experiments indicated flexi-
ble docking of ligand 3 (Scheme 2) within the negatively
charged active-site binding pocket of the protease dimer, and
hydrogen-bonding interactions were exhibited in top-ranked
poses, between components of the N-(1-phenyl-2-
hydroxyethyl)carbamoyl functions of 3 and the Gly27 and
Gly48 enzymatic residues respectively (of 1.7Å and 2.3Å
distance in the lowest energy conformation, Fig. 2). The
lowest energy conformation also exhibits one -
biscarbamoyl oxo-function of 3 proximal (2.1Å) to the ac-
tive-site Asp25 carboxyl, and suggests scope for interaction
between the ligand’s phosphonate function and the Ile50/50 -
bound structural water molecule described above. With re-
spect to enzymatic Ile50/50 N-atoms, the ligand P=O phos-
phorus atom is placed at 6.3Å and 6.0Å distances respec-
tively and the corresponding oxygen atom at 5.1Å and 4.8Å
distances.
Chemistry
The synthetic route followed for the titled O,O-diethyl 1-
benzamido-2,2-biscarbamoyl-ethanephosphonates is illus-
trated in Scheme 2. AdN-E reaction between diethyl
ethoxymethylenemalonate (4) and benzamide at 180-200o
C
over 6 h gave diethyl benzamidomethylenemalonate (5,
40%). Thereafter, a base-catalysed Michael addition of di-
ethyl phosphite across the olefinic function therein [32,33]
generated O,O-diethyl benzamido[diethoxyphosphinyl]
methyl malonate (6, 82%) from which the biscarbamoyl
structure would be constructed by aminolysis. The state of
activation towards nucleophilic substitution recorded in eno-
lizable -dicarbonyl ester systems [34-36] led to an anticipa-
tion of desired chemoselectivity of substitution, despite the
presence in malonate 6 of two functions (i.e. carboxylate and
phosphonate) potentially reactive to the same reagents and
catalysts. Accordingly, desired bis[N-(1-phenyl-2-hydroxy-
ethyl)carbamoyl] derivative 3 was generated in a methanolic
medium in 18% yield from 6 using a minor excess of (2S)-
phenylalaninol at maximum practicable concentration and
mildly elevated temperature (45-55o
C, 14 days). Use of simi-
lar conditions in preliminary procedures involving benzy-
lamine and straight-chain alkanolamine reagents abnegated
P
O
O
RO
X
intramolecular
H-bonded
intermediates
mixed cyclic carboxyl/
carboxamidyl-phosphonyl
anhydride
X = O, N R''R'''
P
O
O
O
RO
O
H or
P
O
O
O
RO
H
OR
R
P
R'
O
RO
RO
O
R = alkyl
R' = OH, NR''R'''
reactive
intermediate
P
R'
O
HO
HO
O
Scheme 1. Accelerated hydrolysis of phosphonate functions via active intermediates.
Fig. (2). Lowest docked energy conformation of ligand 3 showing
hydrogen bonding interactions.

3. Presentation of the -Carboxamidophosphonate Arrangement Letters in Drug Design & Discovery, 2009, Vol. 6, No. 2 141
chemoselectivity, necessitating procedures conducted at
room temperature to generate the corresponding bis-N-
carbamoyl derivatives. Significant excess of amine reagent
invariably resulted in concomitant mono-dealkylation of the
phosphonate function, while use of organonitrogen nucleo-
philic/base catalysts (i.e. aniline, pyridine, imidazole etc.)
also diminished chemoselectivity unacceptably.
In addition to their function as putative binding determi-
nants, the hydroxyl functions of 3 permit modification of
lipophilicity/water-solubility characteristics via derivatiza-
tion to depsipeptides - an established method of enhancing
drug bioavailability. DCC/DMAP-mediated coupling [37]
furnished enantiopure Cbz-leucinyl derivative 7 (66%),
which was deprotected by catalytic hydrogenolysis at atmos-
pheric pressure over Pd/C (5% w/w, CAUTION) and iso-
lated as dihydrochloride salt 8 of the enantiopure depsipep-
tide (72%).
4 R = EtO
5 R = BzNH
i
5, ii
3 R' = OH
7 R' = O-(2S)-Leu-Cbz
8 R' = O-(2S)-Leu-NH2.HCl
iv
v
iii
6
EtO OEt
O O
R
OEtEtO
OO
NH(EtO)2P
O
O
R'
H
N
H
N
R'
O O
(EtO)2P NH
O
O
Scheme 2. (i) BzNH2, ; (ii) HP(O)(OEt)2, NaOEt (cat), ; (iii) (S)-
phenylalaninol (2.1 eqv.), 45-55o
C; (iv) Cbz-leu-OH, DCC, DMAP
(cat.); (v) Pd/C (5%), H2, then dry HCl/Et2O.
Biological Activity
Compounds were tested for in vitro anti-HIV activity at
the National Institutes of Health, USA (NIH) against the T-
cell line adapted isolate HIV-1LAI/3B (3B) in CEM-SS cells
using a standard XTT assay [38]. Compound 3 was found to
be moderately active (EC50 = 53 μM), while attempts to en-
hance cellular uptake through development of depsipeptide
structures resulted in a loss of in vitro activity in leucinyl
derivatives 7 and 8 (EC50 > 200μM). However, in HIV-1 PR
binding studies, the binding of 8 proved superior to that of 3
(IC50 = 31 μM and >50 μM respectively) [39].
CONCLUSIONS
In summary, a putative ligand to HIV-1 PR, i.e. 3, consti-
tuting a -carboxamidophosphonate arrangement, has been
developed; exhibiting moderate anti-HIV activity in vitro.
While the enhanced HIV-1 PR binding of depsipeptide 8
compared with that of 3 is explicable in terms of alternative
binding modes and participation of the leucinyl function in
EI interactions, the lack of anti-HIV activity of 8 (and 7) in
vitro compared with 3 has yet to be explained, although a
correlation with the relative efficacies of intracellular trans-
port of these compounds may be possible.
EXPERIMENTAL SECTION
General
NMR spectra were recorded at ambient temperature (un-
less otherwise stated), on a Bruker AM-250 (1
H, 1
H-1
H
COSY, 250.13 MHz; 13
C, 135-DEPT, 62.90 MHz; 31
P,
101.26 MHz) or Bruker Avance-500 (1
H, 1
H-1
H COSY,
500.13 MHz; 13
C, 135-DEPT, 125.77 MHz; 31
P, 202.45
MHz) spectrometer in pulse Fourier transform (pFt) mode.
All 13
C and 31
P NMR spectra are 1
H-broad-band decoupled.
1
H and 13
C NMR spectra were referenced internally to
Me4Si. Aqueous phosphoric acid (85% H3PO4) was used as
the external reference for 31
P NMR spectra. Infrared spectra
were recorded on a Bruker Vector 22 FT-IR or Nicolet IR-
100 FT-IR spectrophotometer, with samples prepared as
16mm diameter KBr discs. Mass spectrometry was per-
formed either using a Kratos Profile HV3 mass spectrometer
in “Liquid Secondary Ionization Mass Spectrometry” mode
(employing glycerol as a matrix), or a Finnigan 710C spec-
trometer with an electrospray source in positive ion mode.
Melting points were obtained on an Electrothermal Eng. Ltd.
digital melting-point apparatus, and are uncorrected. A Carlo
Erba 1108 Elemental Analyser was used for C, H and N mi-
croanalyses. TLC was performed on precoated silica plates
(Whatman Al Sil G/UV, 250 μm layer) using
CH2Cl2/MeOH, 50:3 (v/v) as solvent eluent; denoted “sys-
tem A” in the experimental section.
Reagents were used as purchased. (2S)-Cbz-Leucine was
prepared according to the methodology of Zervas et al. [40].
Drying (and storage) of solvents when necessary were car-
ried out according to established methods [41].
Synthesis
Diethyl Benzamidomethylenemalonate (5). A suspen-
sion of benzamide (78.74 g, 650.0 mmol) in diethyl
ethoxymethylenemalonate 4 (140.55 g, 650.0 mmol) was
heated at 180-200o
C in a distillation apparatus fitted with a
Dean-Stark trap, until one equivalent of ethanol had been
collected (6 h). When cool, the oily mixture was washed
with petroleum ether (3x150 cm3
), triturated in petroleum
ether (300 cm3
) and left to crystallize in the ether over sev-
eral days. The solid formed was isolated, and recrystalliza-
tion from 99IMS/petroleum ether (1:1) solution yielded 5 as
a waxy, yellow solid (76.23 g, 40%). Rf 0.87 (system A, u.v.
abs.), mp 42-45o
C; IR: 3446, 1724, 1706, 1666, 1604, 1453,
1377, 1267, 1246, 1216, 1077, 1068, 1059; H(CDCl3): 1.35,
1.40 [6H, 2x t, J 7.1, 2x CH3]; 4.28, 4.36 [4H, 2x q, J 7.1, 2x
CH2]; 7.40-7.68 [3H, m’s, m,p-Ar-H]; 7.975, 7.98 [2H, 2x d,

4. 142 Letters in Drug Design & Discovery, 2009, Vol. 6, No. 2 Wardle et al.
J 7.3, 7.0 resp., o-Ar-H]; 8.78 [1H, d, J 11.8, C=CH]; 12.02
[1H, br.d., J 11.8, NH]; C(CDCl3): 14.17, 14.27 [2x CH3];
60.88, 61.41 [2x CH2]; 102.74 [HC=C]; 127.54, 128.00,
128.60, 129.12, 131.46 (ipso), 133.61 [Ar-C]; 146.94
[HC=C]; 164.26, 164.47, 167.98 [C=O’s]; MS (LSIMS): 292
(81%, [MH]+
). Anal. Calcd for C15H17NO5: C, 61.85; H,
5.88; N, 4.81%. Found: C, 62.07; H, 6.00; N, 4.81%.
Diethyl Benzamido[diethoxyphosphinyl]methyl Malo-
nate (6). A vigorously stirred reaction mixture of 5 (58.50 g,
200.0 mmol) in freshly distilled diethyl phosphite (30.38g,
220.0 mmol) was heated to 65o
C, at which time heating was
stopped as ethanolic sodium ethoxide solution was added
dropwise (~ 1 M, 1 cm3
). An exothermic reaction was ob-
served immediately, the reaction temperature rising to
100o
C, and the temperature was maintained at 90-95o
C for
2.5 h after the exothermic reaction had subsided. Diethyl
ether (20 cm3
) was added to the cooled mixture and the re-
sulting solution was immediately filtered. Material crystal-
lized from the filtrate over several days was isolated in two
crops and washed with diethyl ether (20 cm3
, dropwise) to
leave 6 as a crystalline white solid (70.38 g, 82%). Rf 0.52
(system A, I2 vis.), mp 86-90o
C; IR: 3274, 1700, 1671, 1545,
1539, 1321, 1311, 1245, 1227, 1047, 1015, 983, 966;
H(CDCl3): 1.21, 1.29, 1.34 [12H, 3x t, J 7.1, 7.1, 7.2 resp.,
4x CH3]; 4.02 [1H, dd, J1 5.3, J2 3.1, C H]; 4.08-4.39 [8H,
m’s, 4x CH2]; 5.39 [1H, ddd, J1 17.9, J2 9.8, J3 3.1, C H];
7.40-7.53 [3H, m’s, m,p-Ar-H]; 7.81, 7.82 [2H, 2x d, J 6.8,
6.7 resp., o-Ar-H]; 7.89 [1H, br.d, J ~9.8, NH]; C(CDCl3):
13.93, 13.97 [2x C(O)OCH2CH3]; 16.33, 16.39 [2x d, J 5.7,
5.5 resp., P(OCH2CH3)2]; 45.76 [d, J 161.8, C ]; 50.59 [d, J
5.1, C ]; 62.12, 62.45 [2x C(O)OCH2]; 63.16, 63.33 [2x d, J
6.6, 6.9 resp., P(OCH2CH3)2]; 127.14, 127.81, 128.59,
128.68, 131.86 [o,m,p-Ar-C], 133.73 [ipso-Ar-C]; 166.45,
166.69, 168.62 [3x d, J 3.8, 18.1, 2.8 resp., C=O’s];
P(CDCl3): 21.06; MS (LSIMS): 430 (100%, [MH]+
). Anal.
Calcd for C19H28NO8P: C, 53.15; H, 6.57; N, 3.26%. Found:
C, 53.46; H, 6.67; N, 3.17%.
O,O-Diethyl 1-benzamido-2,2-bis[(1S)-N-(1-benzyl-2-hyd-
roxyethyl)carbamoyl]ethanephosphonate (3). A methanolic
solution (10 cm3
) of malonate 6 (6.87 g, 16.0 mmol) and
(2S)-2-amino-3-phenyl-1-propanol (5.41 g, 35.8 mmol, ~ 2.1
equiv.) was stirred at 45-55o
C for 14 days (monitored by
TLC; solvent system A) under anhydrous conditions. The
reaction mixture was evaporated in vacuo, redissolved in
ethyl acetate (200 cm3
), and the solution was washed with
deionised water (2x100 cm3
), 0.2N HCl solution (2x100
cm3
), sat’d NaHCO3 solution (2x100 cm3
) and sat’d brine
(2x100 cm3
) respectively. The organic phase was dried over
MgSO4 and evaporated in vacuo. Column chromatography
over silica (CH2Cl2/MeOH; 89:11 v/v) yielded a clear oil
from which a white solid was crystallized in methanol (5
cm3
). Washing of the isolated solid with acetone and diethyl
ether yielded 3 as a white powder (1.87 g, 18%). Rf 0.33
(system A, u.v. abs.), mp 186-187o
C; IR: 3419, 3305, 1700,
1692, 1672, 1652, 1539, 1329, 1252, 1232, 1052, 1032, 971;
H(DMSO-d6): 1.15, 1.21 [6H, 2x t, J 6.9, 7.0 resp., 2x
CH3)]; 2.58-2.82 [4H, m’s, 2x CH2Ph]; 3.08-3.38 [4H, 2x m,
2x CH2OH]; 3.78 [1H, dd, J1J2 7.7, C H]; 3.72-4.07 [6H,
m’s, P(OCH2CH3)2 and 2x NCHBn]; 4.78, 4.81 [2H, 2x t, J
5.3, 5.6 resp., 2x OH]; 5.08 [1H, ddd, J1 14.7, J2 8.2, J3 7.7,
C H]; 7.13-7.29 [10H, m’s, Ar-H {Bn}]; 7.43-7.58 [3H, m’s,
m,p-Ar-H {Bz}]; 7.73 [1H, d, J 8.2, NHCH]; 7.75 [2H, d, J
8.1, o-Ar-H {Bz}]; 8.00 [1H, d, J 8.2, NHCH]; 8.68 [1H, d, J
8.2, BzNH]; C(DMSO-d6): 16.14 [2x d, J 5.9, 2x CH3];
35.89, 36.12 [2x CH2Ph]; 46.39 [d, J 158.5, C ]; 51.46 [d, J
5.6, C ]; 52.51, 52.64 [2x CHBn]; 61.03, 61.43 [2x HOCH2];
62.04, 62.31 [2x d, J 6.6, 6.9 resp., P(OCH2CH3)2]; 125.91,
127.00, 128.05, 128.10, 128.31, 128.96, 129.10, 131.30
[o,m,p-Ar-C], 134.08 [ipso-Ar-C {Bz}]; 138.59, 138.63 [2x
ipso-Ar-C {Bn}]; 165.59, 165.68, 167.12 [3x d, J 5.5, 11.3,
10.2 resp., C=O’s]; P(DMSO-d6): 23.59; MS (LSIMS): 641
(100%, [MH]+
). Anal. Calcd for C33H42N3O8P: C, 61.96; H,
6.62; N, 6.57%. Found: C, 61.96; H, 6.73; N, 6.46%.
O,O-Diethyl 1-benzamido-2,2-bis[(1S)-N-(1-benzyl-2-
{(2’S)-N-[benzyloxycarbonyl]leucinyloxy}ethyl) carbamoyl]-
ethanephosphonate (7). A dichloromethane solution (25
cm3
) containing DCC (0.70 g, 3.4 mmol), (2S)-Cbz-leucine
(0.83 g, 3.1 mmol), phosphonate 6 (0.83 g, 1.3 mmol) and
DMAP (0.06 g, 0.5 mmol) was stirred at 0o
C for 30 min, and
at rt for 72 h thereafter, with moisture excluded. The mixture
was filtered, the isolated solid was washed with dichloro-
methane (3x20 cm3
), and the combined filtrate and washings
were evaporated in vacuo (40o
C). An ethyl acetate solution
of the residue (250 cm3
, filtered after 3 days) was washed
with ice-cold 1N HCl solution (2x250 cm3
), sat’d NaHCO3
solution (2x250 cm3
) and sat’d brine (2x250 cm3
) respec-
tively, dried over MgSO4, and evaporated in vacuo to leave a
semi-solid residue. Column chromatography over silica
(CH2Cl2/MeOH; 91:9 v/v) yielded 7 as a clear glass (0.97 g,
66%). Rf 0.40 (system A, u.v. abs.), softening pt 56-59o
C;
IR: 3413, 3291, 2957, 1749, 1724, 1676, 1532, 1243, 1048,
1026, 978; H(DMSO-d6): 0.88, 0.90 [12H, 2x d, J 6.2, 6.3
resp., 4x CH3 {Leu}]; 1.15, 1.20 [6H, 2x t, J 7.0,
P(OCH2CH3)2]; 1.43-1.82 [6H, m’s, 2x CHCH2Me2]; 2.55-
2.74 [4H, m’s, 2x CH2Ph {Bn}]; 3.77-3.89 [4H, 2x m, 2x
CH2OLeu]; 3.80-3.90 [1H, m, C H]; 3.89-4.11 [4H, m,
P(OCH2CH3)2]; 4.08-4.29 [4H, m, 2x NCHBn and 2x NCH
{Leu}]; 5.03-5.14 [1H, m, C H]; 5.03, 5.04 [4H, 2x s, 2x
CH2 {Cbz}]; 7.15-7.35 [20H, m’s, Ar-H {Cbz, Bn}]; 7.41-
7.54 [3H, m, m,p-Ar-H {Bz}]; 7.74 [2H, d, J 7.3, o-Ar-H
{Bz}]; 7.79, 7.97, 8.12 [4H, 3x d, J 8.3, 7.8, 7.8 resp., 4x
NHCH]; 8.72 [1H, d, J 9.1, BzNH]; C(DMSO-d6): 16.10 [d,
J 5.2, P(OCH2CH3)2]; 20.97, 22.63 [4x CH3 {Leu}]; 24.15
[2x CHMe2]; 35.90, 36.00 [2x CH2 {Leu}]; 39.33 [2x CH2
{Bn}]; 46.44 [d, J 175.5, C ]; 49.04 [2x NCHLeu]; 51.37
[C ]; 52.18 [2x NCHBn]; 61.97, 62.24 [2x d, J 6.7,
P(OCH2CH3)2]; 64.05, 64.37 [2x CH2OLeu]; 65.43 [CH2
{Cbz}]; 126.17, 126.91, 127.56, 127.69, 128.19, 128.33,
128.83, 128.95 [o,m,p-Ar-C], 133.90 [ipso-Ar-C {Bz}];
136.72, 136.74, 137.46 [ipso-Ar-C {Cbz, Bn}]; 156.03,
156.06 [C=O’s {Cbz}]; 165.65, 165.71, 165.87, 166.98,
167.13 [C=O’s, {Cbm, Bz}]; 172.31, 172.41 [C=O’s, {es-
ter}]; P(DMSO-d6): 23.31; MS (LSIMS): 1133 (63%,
[MH]+
). Anal. Calcd for C61H74N5O14P: C, 64.71; H, 6.59; N,
6.19%. Found: C, 64.94; H, 6.65; N, 6.09%.
O,O-Diethyl 1-benzamido-2,2-bis[(1S)-N-(1-benzyl-2-
{(2’S)-leucinyloxy}ethyl)carbamoyl]-ethanephosphonate
Di-hydrochloride Salt (8). Pd/C (5% Pd w/w, 0.51 g, CAU-
TION; pyrophoric in air) was added to a suspension of
phosphonate ester 7 (0.29 g, 0.25 mmol) in absolute ethanol
(50 cm3
) at 0o
C under an atmosphere of N2. The mixture was
stirred under N2 for 10 min and under a stream of dry H2 gas

5. Presentation of the -Carboxamidophosphonate Arrangement Letters in Drug Design & Discovery, 2009, Vol. 6, No. 2 143
for 3 h thereafter (1.5 h at rt, and 1.5 h at 50o
C), then filtered
through a column of Celite 521 filter agent under N2. The
filtrate was evaporated in vacuo, redissolved in diethyl ether
(50 cm3
, seeded with methanol), and bubbled with dry HCl
gas for 30 min at 0o
C. Evaporation in vacuo, and trituration
of the residue in petroleum ether afforded 8 as a cream-white
powder (0.17 g, 72%), mp 134-136o
C; IR: 3431, 3300, 2960,
1752, 1686, 1533, 1221, 1048, 1026, 977; H(DMSO-d6):
0.88, 0.90 [12H, 2x d, J 5.9, 4x CH3 {Leu}]; 1.12, 1.18 [6H,
2x t, J 7.1, 7.2, P(OCH2CH3)2]; 1.45-1.93 [6H, m’s, 2x
CH2CHMe2]; 2.70-3.00 [4H, m’s, 2x CH2Ph]; 3.70-4.38
[13H, m’s, P(OCH2CH3)2, C H, 2x NCHBn, 2x NCH {Leu}
and 2x CH2OLeu]; 5.11 [1H, m, C H]; 7.18-7.40 [10H, m,
Ar-H {Bn}]; 7.43-7.54 [3H, m, m,p-Ar-H {Bz}]; 7.82 [2H,
d, J 7.3, o-Ar-H {Bz}]; 8.20-9.05 [~9H, m’s, 7x NH and 2x
HCl]; C(DMSO-d6): 16.17 [d, J 2.7, P(OCH2CH3)2]; 21.89,
21.97 [4x CH3 {Leu}]; 23.66 [2x CHMe2]; 35.83, 36.11 [2x
CH2 {Leu}]; 38.71 [2x CH2 {Bn}]; 45.96 [d, J 157.2, C ];
49.25, 49.88 [4x NCH]; 50.67 [C ]; 62.00, 62.24 [2x d, J 7.0,
6.6 resp., P(OCH2CH3)2]; 65.71, 65.88 [2x CH2OLeu];
126.22, 127.11, 128.22, 128.92, 128.99, 131.27 [o,m,p-Ar-
C], 133.95 [ipso-Ar-C {Bz}]; 137.85, 137.94 [ipso-Ar-C
{Bn}]; 163.21, 165.46, 165.96, 166.07, 167.02, 169.09
[C=O’s]; P(DMSO-d6): 23.29, 24.87 (95:5 resp., rt)
22.70 (100o
C); MS (Es-MS): 866 (100%, [MH]+
{free
amine}); MS (LSIMS): 866 (32%, [MH]+
{free amine}).
Anal. Calcd for C45H64N5O10P.2HCl: C, 57.56; H, 6.87; N,
7.46%. Found: C, 57.41; H, 6.88; N, 7.58%.
Computational Details
The X-ray crystal structure of HIV-1 protease (PDB code
1HSG) was used as a model for the HIV-1 protease. The 3-D
coordinates for the structure of ligand 3, including hydrogen
atoms, were generated using Chem3D Ultra version 10.0
(ChemOffice 2006, CambridgeSoft Corporation) and energy
minimised. Modelling of the ligand into the active site of
HIV-1 protease was carried out using the Molegro virtual
docker which utilises the MolDock algorithm [42]; a hybrid
search algorithm called “guided differential evolution”
which combines the differential evolution optimisation tech-
nique with a cavity prediction algorithm. The docking scor-
ing function of MolDock is based upon a piecewise linear
potential [43], and the docking procedure was randomized
with a minimum of 10 runs and 5000 iterations. The best five
poses (ligand orientation) generated were recalculated and
re-ranked by analyzing the energy scores, binding affinities,
and ligand-residue (H-bond) interactions, and the top-ranked
pose was visualized using the Molegro virtual docker visu-
alization software.
Anti-HIV Activity: XTT Assay
The XTT assay was performed at NIH as described [38].
Briefly, stock solutions of the compounds were prepared in
DMSO and serial half log10 dilutions (10-3
M to 10-7
M) added
to 3B-infected CEM-SS cells. After 6 days, the tetrazolium
salt XTT and phenazine methosulfate were added to the
cells, and the assay incubated to allow formazin colour de-
velopment by metabolically active viable cells. Colour de-
velopment was analysed spectrophotometrically to quantitate
formazin production in individual wells. Compound-treated,
virus-infected wells were compared both with untreated,
virus-infected wells and with controls. Uninfected, treated
cells served as a control for compound-induced cytotoxicity
while AZT-treated, infected cells served as a positive control
for protection from 3B-induced cell death. The concentration
protecting 50% of cells (EC50) from 3B-induced cell death
was calculated.
ACKNOWLEDGEMENTS
We thank Mr. J. Crowder for running NMR spectra, Mr.
W. Dissanayake for running mass spectra, and Mr. S. Boyer
for running C, H, N microanalysis. We also thank the
Thrombosis Research Institute for the mass spectrometry
performed on compound 8. Dr T. Lowinger of Bayer AG,
Wuppertal, Germany is thanked for bioevaluation against
HIV-1 PR; and NIH, Bethesda, USA, is also thanked for
evaluating anti-HIV activity.
ABBREVIATIONS
abs = absorption
AdN-E = addition-elimination
Asp = aspartic acid
Bn = benzyl
br = broad
Bz = benzoyl
Cbz = carbobenzyloxy
Cbm = carbamoyl
COSY = correlated spectroscopy
DCC = N,N-dicyclohexylcarbodiimide
DEPT = distortionless enhancement by polarisation
transfer
DMAP = 4-dimethylaminopyridine
DMSO = dimethyl sulfoxide
EI = enzyme-inhibitor complex or interaction
ES = enzyme-substrate complex or interaction
Es-MS = electrospray mass spectrometry
Gly = glycine
HAART = “highly active anti-retroviral therapy;”
imp = impurity
99IMS = industrial methylated spirits (denatured etha-
nol)
Leu = leucine
LSIMS = liquid secondary ionization mass spectrome-
try
resp = respectively
rt = room temperature
sat’d = saturated
Thr = threonine
vis = visualization

6. 144 Letters in Drug Design & Discovery, 2009, Vol. 6, No. 2 Wardle et al.
XTT- = 2,3-bis[2-methoxy-4-nitro-5-sulfo-phenyl]-5-
[(phenylamino)carbonyl]-2H-tetrazolium
hydroxide
o-, m-, p- = ipso- are used according to the standard
convention for aromatic substituents
C and C = denote carbons of the central ethane motif at
positions and respectively to phospho-
rus.
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