Letters in Drug Design & Discovery, 2009, 6, 139-145 139
1570-1808/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.
140 Letters in Drug Design & Discovery, 2009, Vol. 6, No. 2 Wardle et al.
“Molegro” virtu...
Presentation of the -Carboxamidophosphonate Arrangement Letters in Drug Design & Discovery, 2009, Vol. 6, No. 2 141
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, ...
Presentation of the -Carboxamidophosphonate Arrangement Letters in Drug Design & Discovery, 2009, Vol. 6, No. 2 143
for 3 ...
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]...
Presentation of the -Carboxamidophosphonate Arrangement Letters in Drug Design & Discovery, 2009, Vol. 6, No. 2 145
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  1. 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. 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. 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. 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. 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
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