Scientific paper
Design and Synthesis of Novel UDP-Mur-.^Ac, UDP-Mur-^Ac-L-Ala and UDP-Mur-^Ac-L-Ala-D-Glu
mimetics
Rok Frlan,1 Franc Perdih,2 Nina Cirkven~i~,1 Slavko Pe~ar1'3 and Ale{ Obreza1*
1 University of Ljubljana, Faculty of Pharmacy, A{ker~eva 7, 1000 Ljubljana, Slovenia 2 University of Ljubljana, Faculty of Chemistry and Chemical Technology, A{ker~eva 5, 1000 Ljubljana, Slovenia. 3 "Jožef Stefan" Institute, Jamova 39, 1000Ljubljana, Slovenia * Corresponding author: E-mail: ales.obreza@ffa.uni-lj.si
Received: 26-09-2008 Dedicated to Professor Blanko Stanovnik on the occasion of his 70"' birthday
Abstract
A series of novel phenylsulfonylcarbamates were designed and synthesized as mimetics of UDP-Mur-^Ac, UDP-Mur-WAc-L-Ala and UDP-Mur-^Ac-L-Ala-D-Glu with the potential to inhibit ATP-dependent amide-forming ligases Mur-B, C, D and E, involved in the biosynthesis of bacterial cell wall. A detailed synthesis of these compounds is presented.
Keywords: MurB, MurC, MurD, MurE, inhibitors, antibacterial
1. Introduction
The discovery and use of antibiotics was one of the most significant factors in controlling infectious diseases caused by bacteria in the 20'h century. However, the subsequent emergence and spread of antibiotic resistance in pathogenic microorganisms has rendered several available antibiotics ineffective, and consequently several infectious diseases, such as tuberculosis, are re-emerging and posing a serious clinical threat.1 This situation has created an urgent need for the development of new antibacterial agents directed towards novel targets.2
One of the best known and most validated targets for antibacterial therapy is the peptidoglycan biosynthetic pathway. Peptidoglycan is an essential component of the bacterial cell wall that confers a defined cell shape and preserves cell integrity by compensating internal osmotic pressure. Any perturbation of the multi-step peptidogly-can biosynthesis may lead to cell lysis.3 Peptidoglycan is formed as a linear chain of repeating ^-acetylglucosamine (Glc^Ac) and ^-acetylmuramic acid (Mur^Ac) units, connected by short peptide chains. Four ATP-dependent
ligases (MurC, MurD, MurE and MurF) catalyze the assembly of the peptide moiety by the successive addition of L-alanine, D-glutamate, meso-diaminopimelate (or L-lysine) and D-alanyl-D-alanine to UDP-MurWAc which is synthesized from UDP-WAc enolpyruvate by a reductase MurB (Fig. 1). A divalent cation Mg2+ or Mn2+, is essential to the formation of an amide bond.4 Through their eamino group, meso-A2pm and L-Lys are involved in cross-linking the peptide units linked to the glycan chains, and they thus play a key role in the stability of the macro-molecule and the integrity of the bacterial cell.5
These essential cytoplasmic enzymes are highly specific and are present only in eubacteria, thus making them attractive targets for the development of new therapeutic antibacterial agents. However, research on these targets has been limited, due perhaps to the very few natural antibiotics that are known to inhibit them (bacilysin, fosfomycin, D-cycloserine).4
The reaction mechanism6 of Mur ligases Mur C through F is believed to be similar to that of other ATP-de-pendent amide-forming enzymes such as glutamine synthetase,7-10 glutathione synthetase11-12 and D-Ala-D-
OH
^ / ACNHÌ
HN
O
A
H3C
/ AcNH j
HN
COOH
O o
1.	MurB, NADPH
2.	MurC, D, E and F, ATP
HO OH
Figure 1: Reactions catalyzed by MurB, C, D, E and F.4
-p- -p
,0 0 0

ho"" "'oh
NH	^NH3
H3C—/	R
\=0	R=HorC02" .....CH, 2
OOC
Ala ligase.13,14 These enzymes all catalyze the formation of an acyl phosphate that is attacked by the incoming amino acid to form a tetrahedral intermediate that collapses to the amide product.
Several peptidomimetic compounds have been prepared that are structurally related to substrates, intermediates and products of the reactions catalyzed by MurC-F enzymes. Phosphinate transition state analogues are the most potent inhibitors, with IC50 in the low nanomolar range. However, none of these peptide-like compounds exhibit significant antibacterial activity, because of their high polarity and the resultant low cell permeability.4 Furthermore, Babi~ and colleagues also prepared O- and ^-sulfamoyl amides as diphosphate analogues of UDP-^-Ac-glucosamine and UDP-^-Ac-muramic acid. However, no inhibitory activity at Mur ligases have been reported so far for these compounds.15
In a search for new compounds with the potential to inhibit MurB, MurC, MurD and MurE, we focused on initially on an analysis of the active site within the X-ray crystal structures of MurB-F complexed with the reaction substrates or products.16-19 Initial docking of small peptide fragments using E-Hits20 showed that it was possible to omit the sugar residue in substrates/products while still retaining binding affinity, due to the interactions of other moieties with the active sites of these enzymes. In addition, a new diphosphate mimetic, ethyl (2-oxoethylamino) phenylsulfonylcarbamate, was introduced using molecular modelling. A general structure for good enzyme inhibition was proposed, which consisted of peptide mimetic, diphosphate mimetic and phenyl substitute for sugar residue, which orients both mimetics towards the proper conformation and, to some level restricts the molecule's degrees of freedom. (Fig. 2).
The hydroxamic acid functionality, a well-known metal complex forming group,21 was also introduced into some of the mimetics, in order to form strong interactions with the Mg2+ in the enzymes' active sites. This functional group was used previously in the inhibition of a variety of enzymes, including ureases, peroxidases, and matrix me-talloproteinases using the mechanism of metal complexa-tion. This functional group was also used in the design of therapeutics targeting cancer, cardiovascular disease, HIV, Alzheimer's, malaria, allergic diseases, tuberculosis, metal poisoning, and iron overload disease.22 However, to our knowledge, it has not used for inhibiting ligases.
Figure 2: General structure of substituted phenylsulfonylcarbama-
tes.
2. Results and Discussion
The overall synthetic approach is outlined in Figs. (3), (4) and (5). The synthesis of UDP-Mur-^Ac mimetics (Fig. (3)) was started from 3-nitrobenzenesulfonyl chlori-
de (4),23 which was coupled with GlyOMe hydrochloride24 to give sulfonamide 5, followed by the transformation of the methyl ester into the corresponding amide 6 via aminolysis.25 Compound 9 was synthesized from 6 in three steps. Compound 6 readily reacted with ethylchloro-formate in the presence of NaH as a base to give 7, followed by catalytic hydrogenation of the nitro functional group26 to give the aromatic amine 8 in 58% yield upon crystallization. The white crystals turned brown upon longer exposure to light, possibly due to the oxidation of the amino functional group. Compound 8 was then treated with glyoxyllic acid monohydrate under the conditions of reductive amination using NaCNBHg.27 The reaction was monitored with TLC until the starting material had disappeared and the product obtained was used for further reactions with amino acid derivatives (Fig. (3)).
The synthesis of compounds 13a and 13b is outlined in Fig. (4). These compounds were synthesized starting from Gly (12a) and L-Ala (12b) which were first protected with Boc-anhydride28 followed by coupling with commercially available O-benzylhydroxylamine hydrochlori-
de in the presence of EDC and HOBt.29 The Boc-protecti-ve group was removed by deprotection using CF3COOH30 or HCl31 to give 11a and 11b, respectively.
Coupling of 9 with various amines 13a-13d yielded compounds 14a-14d followed by hydrogenolysis of the benzyl ester moiety under conditions of heterogeneous catalytic transfer hydrogenation using Pd/C as catalyst and ammonium formate as hydrogen donor32 to give UDP-Mur-^Ac-L-Ala mimetics 15a-15d (Fig. 5). At this stage H2/Pd/C was also used for hydrogenolysis of benzyl protective group. However, with some compounds (15a and 15b), the sample decomposed and therefore ammonium formate was preferentially used.
15d was further used for the synthesis of UDP-Mur-^Ac-L-Ala-D-Glu mimetic, which was obtained by coupling 14d with Glu(OBzl)Obzl ■ pTolSO3H to give 16, followed by hydrogenolysis with H2/Pd/C to give 17.
Our modelling procedure for 15d revealed that the mode of binding for one of the conformations of the molecule is similar to that of UMA(UDP-Mur^Ac-L-Ala), which is also presented in Fig. 7. Comparison of
OoN
O
„
0=S-NH O

44%
OoN
91%
0=S-NH O
OoN
/V
58%
HoN
59%
HOOC

Figure 3: Synthesis of UDP-Mur-WAc mimetic compounds 9 and 11. i. H2NCH2COOMe,24 Et3N, CH2a2, ii. NH3(g), CH3OH, iii. aCOOCH2CH3, NaH, DMF, iv. H2/Pd/C, MeOH:THF=1:1, v. OHC-COOH ■ H2O, MeOH, N[aCNBH3, vi. H2NOBz ■ HCl, Ei:)C, HOBt, NMM, CH2a2, vii. NHHCO, Pd/C, MeOH.
Figure 4: Synthesis of peptidomimetic building blocks 13a and 13b. i. (Boc)2O, dioxan, water, ii. H2NOBz ■ HCl, EDC, HOBt, NMM, DMF, iii. CF3C00H/ CH2Cl2 (1:9) for 13a, HCl(g), diethyl ether for 13b, iv. NaHCO3, H20.
ver, inhibitory activity has to be measured, and will be reported in due course before any firm conclusions can be made.
In conclusion, 7 new compounds with the potential to inhibit MurB, C, D and E have been synthesized. The peptide fragments, which strongly resemble the peptide chains of UDP-Mur-Nac, UDP-Mur-NAc-L-Ala and UDP-Mur-NAc-L-Ala were introduced, and linked to a phenyl fragment attached to a sulfonocarbamoyl mimetic of diphosphate. The biological activity of the synthesized compounds is under investigation.
Figure 5: Synthesis of UDP-Mur-NAc-L-Ala mimetics 15a-e. i. Suitable amino acid derivative, EDC, HOBt, NMM, DMF, 0 °C ^ r.t., ii. NH4HC02, Pd/C, MeOH.
0=S-H NH2
O-^OH

15d
0=S-N NH2
Figure 6: Synthesis of UDP-Mur-NAc-LAla-D-Glu mimetic 17. i. Glu(OBzl)Obz ■ pTolSO3H, EDC, HOBt, NMM, DMF, 0 °C ^ r.t., ii. H2/Pd/C, MeOH
UMA and 15d reveals that the sulfonocarbamate moiety forms 6 H-bonds with the active site, according to our model, and could mimic diphosphate. Furthermore, an additional interaction with Asp 175, which is not present in the binding mode of UMA, is also seen. Howe-
3. Acknowledgements
This work was supported by the European Union FP6 Integrated Project EUR-INTAFAR (Project No. LSHM-CT-2004-512138) under the thematic priority of Life Sciences, Genomics and Biotechnology for Health.
Figure 7: Proposed binding mode of 15d (green) into the active site of MurC. The binding mode of UMA (grey) is also presented for comparison.
The support from the Ministry of Higher Education, Science and Technology of the Republic of Slovenia and the Slovenian Research Agency is also acknowledged. The authors are thankful to dr. Du{an @igon and dr. Bogdan Kralj of Jožef Stefan Institute for the mass spectra and dr. Roger H. Pain for critical reading of the manuscript.
4. Experimental 4. 1. General Experimental Methods
Chemicals from Fluka and Sigma-Aldrich Chemical Co. were used without further purification. Anhydrous tetrahydrofuran, dichloromethane and Et3N were dried and purified by distillation over Na, K2CO3 and KOH, respectively. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel (6OF254) plates (0.25 mm). Column chromatography was performed on silica gel 60 (Merck, particle size 240^00 mesh). Melting points were determined on a Reichert hot stage microscope and are uncorrected. ^H-, COSY-, HMQC- and 13C-NMR spectra were recorded on a Bru-ker AVANCE DPX300 spectrometer in CDCl3 or DMSO-d6 solution with TMS as internal standard. Chemical shifts were reported in ppm (6) downfield from TMS. All the coupling constants (J) are in hertz. IR spectra were recorded on a Perkin-Elmer FTIR 1600 spectrometer. Mass spectra were obtained with a VG-Analytical Au-
tospec Q mass spectrometer with EI or FAB ionization (MS Centre, Jožef Stefan Institute, Ljubljana). Elemental analyses were performed by the Department of Organic Chemistry, Faculty of Chemistry and Chemical Technology, Ljubljana, on a Perkin Elmer elemental analyzer 240 C. All reported yields are yields of purified products.
4. 2. Molecular Modelling
The three dimensional structure of the synthesized 15d was generated with Pymol v0.99 (DeLano Scientific LLC). The geometry of compound 15d was minimized using MM+ force field, where a Polak-Ribiere (conjugate gradient) algorithm was applied until the gradient value was smaller than 0.001 kcal/Àmol. The initial crude optimization was followed by further minimization by a semi-empirical AMI method where the same algorithm was applied again using the same parameters as for the initial crude optimization. The X-ray structures of UMA bound into the MurC active site was obtained from the Protein Data Bank (PDB, 1p3d). E-Hits molecular docking tool20 was used to determine possible binding modes. UMA was taken as a reference molecule and MurC was clipped by 6 À around UMA. All docking solutions were inspected by using Pymol v0.99 and compared to the experimentally determined structure of the ligand-enzyme complex. Fig. (7), which represents binding mode with the closest resemblance to the binding mode of UMA (pKi = -2.7), was then generated.
Synthesis of methyl 2-(3-nitrophenylsulfonamido)ace-
tate (5) To a solution of triethylamine (21.3 mL, 153.5 mmol) in dichloromethane (200 mL) glycine methyl ester hydrochloride24 was added and the mixture stirred for 15 minutes at 0 °C. A solution of 3-nitrobenzensulfonyl chloride (22.7 g, 102.3 mmol) (4) in dichloromethane (50 mL) was added dropwise and then stirred for 24 hours at room temperature. The solution was extracted with 1M HC-l (2 X 50 mL), saturated NaHCO3 solution (30 mL) and brine (30 mL), and the organic phase was dried over anhydrous Na2SO4. After evaporation of solvent in vacuo, the crude product was crystallized from diethyl ether and filtered to give white crystals. Yield = 44%. mp 125-127 °C. 1H NMR (300 MHz, CDCl3) 6 3.67 (s, 3H, CH3OCO), 3.90 (d, J 5.5 Hz, 2H, CH2COO), 5.23 (t, J = 5.5 Hz, 1H, NHCH2), 7.73-7.76 (m, 1H, ArH), 8.18-8.22 (m, 1H, Ar-H), 8,42-8,46 (m, 1H, ArH), 8.69-8,74 (m, 1H, ArH) ppm. 13C NMR (75 MHz, DMSO-d6) 6 43.6 (CH2C(O)NH2), 51.8 (OCH3), 121.3 (ArC), 126.9 (ArC), 131.0 (ArC), 132.4 (ArC), 142.4 (ArC), 147.7 (ArC), 169.2 ppm. MS (m/z): 275 (M+H, 29), 297 (MNa+, 100). IR (KBr): v 3458, 3209, 2361, 1735, 1609, 1529, 1437, 1310, 1355, 1233, 1171, 1124, 974, 881, 839, 734, 670, 581 cm-1. Anal. Calcd for C9H10N2O6S: C 39.42, H 3.68, N 10.21. Found: C 39.63, H 3.588, ^ ^0.20
Synthesis of 2-(3-nitrophenylsulfonamido)acetamide (6). NHj^g) was bubbled into a solution of 5 in a mixed solvent of methanol and THF (1:2, 150 mL) for 45 minutes and the solution then stirred for 24 hours. After evaporation of the solvent in vacuo, the crude product was crystallized from diethyl ether to give a white solid. Yield = 91%. mp 178-180 °C. 1H NMR (300 MHz, DMSO-d6) 5 3.50 (s, 2H, CH2CONH2), 7.05 (s, 1H, CONH2), 7.36 (s, 1H, CONH2), 7.85-7.91 (m, 1H, ArH), 8.20-8.23 (d, 1H, Ar-H, J 8.0 Hz), 8.27 (s, 1H, SO2NH), 8.45-8.48 (m, 1H, Ar-H), 8.55-8.59 (m, 1H, ArH) ppm. 13C NMR (75 MHz, DMSO-d6) 5 47.8 (CH2C(O)NH2), 121.7, 127.0, 131.0, 132.7, 142.2, 147.8, 169.4 ppm. MS (m/z): 260 (M+H, 92), 282 (M+Na, 100). IR (KBr): v 3449, 3364, 3098, 3363, 1929, 1670, 1596, 1525, 1421, 1357, 1179, 1131, 1087, 912, 881, 839, 811, 764, 736, 697, 580 cm-1. Anal. Calcd for C8H9N3O5S: C 37.06, H 3.50, N 16.21. Found: C 37.29, H 3.50, N 16.19
Synthesis of ethyl 2-amino-2-oxoethyl(3-nitrophenyl-sulfonyl)carbamate (7). Sodium hydride (60% in mineral oil) (1.11 g, 27.76 mmol) was suspended in DMF under argon atmosphere and cooled to -10 °C. 6 (6.485 g, 25.0 mmol) was then added slowly in portions over 30 minutes and stirring of the suspension was continued for an additional hour at room temperature. The reaction mixture was cooled to 0 °C and ethyl chloroformate (2.51 ml, 26.3 mmol) was added dropwise. After stirring the reaction mixture for 24 hours, dichloromethane was added (100 mL) and then extracted with 10% citric acid (3 x 25 mL), water (15 mL) and brine (20 mL). The organic layer was dried over anhydrous Na2SO4, evaporated in vacuo and the crude product crystallized from diethyl ether to give white crystals. Yield = 68%. mp 180-182 °C. 1H NMR (300 MHz, DMSO-dg) 5 1.06 (t, J 7.1 Hz, 3H, CH3CH2), 4.06 (q, 2H, CH3CH2), 4.44 (s, 2H, CH2CONH2), 7.27 (s, 1H, CH2CONH2), 7.63 (s, 1H, CH2CONH2), 7.90-7.98 (m, 1H, ArH), 8.40-8.46 (m, 1H, ArH), 8.54^8.59 (m, 1H, ArH), 8.78-8.84 (m, 1H, ArH) ppm. DEPT-135 NMR (300 MHz, DMSO-dg): 5 13.3, 47.6, 63.3, 123.4, 128.1, 130.4, 134.4 ppm. MS (m/z): 332 (M+H, 100). IR (KBr): v 3445, 3192, 3005, 2362, 1727, 1686, 1617, 1532, 1475, 1368, 1332, 1251, 1173, 1125, 1016, 944, 881, 849, 803, 766, 733, 671, 588, 569 cm-1. Anal. Calcd for C11H13N3O7S: C 39.88, H 3.69, N 12.68. Found: C 40.02, H 4.15, N 12.39.
yield crude product which was crystallized from mixed solution of diethyl ether and petroleum ether (5:1, 60 mL) to yield pure white crystals. Yield 58%. mp 193-195 °C. 1H NMR (300 MHz, DMSO-dg) 5 1.06 (t, J 7.1 Hz, 3H, CH3CH2), 4.02 (q, 2H, CH2CH2), 4.31 (s, 2H, CH2CO1S[H2), 5.61 (s, 2H, Ar-NH), 6.^0-6.85 (m, 1H, Ar-H), 7.05-7.10 (m, 2H, ArH + CH2CONH2), 7.12-7.16 (m, 1H, ArH), 7.17-7.25 (m, 1H, Ar-H), 7.48 (s, 1H, CH2CONH2) ppm. DEPT-135 NMR (300 MHz, DMSO-dg): 5 13.3, 47.4, 62.6, 112.2, 114.2, 118.0, 128.9 ppm. MS (m/z): 302 (M+H, 100). IR (KBr): v 3479, 3425, 3348, 3006, 2360, 1714, 1687, 1644, 1596, 1487, 1355, 1283, 1180, 1079, 1023, 937, 805, 770, 681, 626, 566 cm1. Anal. Calcd for C11H15N3O5S: C 43.85, H 5.02, N 13.95. Found: C 44.04, H 4.78, N 13.97.
Synthesis of 2-(3-[N-(2-amino-2-oxoethyl)-N-(ethoxy-carbonyl)sulfamoyl]phenylamino)acetic acid (9). To a
solution of 8 (2.838 g, 9.42 mmol) and of glyoxylic acid monohydrate (1.04 g, 11.3 mmol) in methanol (70 mL), NaCNBH3 was added in portions over 20 minutea at 0 °C. After the mixture was stirred at room temperature for 24 hours, the solvent was evaporated in vacuo. The crude reaction product was dissolved in dichloromethane (100 mL) and extracted with 0.5M HCl (50 mL). When the aqueous phase was extracted with dichloromethane (4 x 20mL), yellow crystals appeared, which were filtered off and the filtrate was washed with dichloromethane (3 x 20 mL). To the combined organic extracts petrolether was added and they were left at 0 °C overnight. Pure yellow crystals were filtered off and found to be the same as those that crystallized from water phase. Yield = 59%. mp 160-164 °C. 1H NMR (300 MHz, DMSO-dg) 5 1.04 (t, J 7.1 Hz, 3H, CH3CH2), 3.71 (s, 2H, NHCH2COOH), 4.02 (q, 2H, CH3CH2), 4.33 (s, 2H, CH2CONH2), 6.45 (br, 1H, Ar-NH), 6.83-7.87 (m, 1H, ArH), 7.11-7.17 (m, 3H, ArH + CONH2), 7.25-7.29 (m, 1H, ArH), 7.53 (s, 1H, CH2CONI12 ), 12.62 (br, 1H, NHCH2COOH) ppm. 13C NMR (75 MHz, DMSO-dg) 5 13.7 (^3CH2), 44.4 (NHCH2COOH), 47.7 (CH2C(O)NH2), 63.1 (CH3CH2), 110.8 (ArC), 115.2 (ArC), 116.9 (ArC), 129.2, 139.9, 148.5, 151.6, 168.6, 172.1 ppm. MS (m/z): 360 (M+H, 100). IR (KBr): v 3407, 3193, 3360, 1742, 1674, 1602, 1526, 1488, 1350, 1269, 1178, 1136, 1084, 1004, 931, 771, 676, 568 cm1. Anal. Calcd for C13H17N3O7S: C 43.45, H 4.77, N 11.69. Found: C 43.46, H 4.68, N 11.64.
Synthesis of ethyl 2-amino-2-oxoethyl(3-aminophenyl-sulfonyl)carbamate (8). Argon was bubbled into a solution of 7 (5.811 g, 17.6 mmol) in a mixed solvent of THF and methanol (1:1, 200 mL) for 30 minutes. 10% Pd/C, unreduced, was then added and H2 was bubbled into the resulting mixture which was stirred for 30 minutes. Hydrogenation and stirring of the resulting mixture were continued for 24 hours under hydrogen atmosphere. Pd/C was filtered off and the solution concentrated in vacuo to
Synthesis of ethyl 2-amino-2-oxoethyl{3-[2-(benzylox-yamino)-2-oxoethylamino]phenylsulfonyl}carbamate
(10). To a solution of 9 (975 mg, 2.72 mmol) and O-benzylhydroxylamine hydrochloride (434 mg, 2.72 mmol) in dichloromethane (40 mL) was added N-methyl-morpholine (0.90 mL, 8.16 mmol) at 0 °C. The mixture was stirred for 10 minutes followed by the addition of HOBt (367 mg, 2.72 mmol) and EDC (521 mg, 2.72 mmol). The reaction mixture was allowed to warm to
room temperature and stirred for 24 hours under argon atmosphere. The solvent was removed under reduced pressure and the residue dissolved in dichloromethane (120 mL), extracted with 10% citric acid (3 x 10 mL), saturated NaHCO3 (2 x 10 mL) and brine (15 mL), and dried over anhydrous Na2SO4. After filtration and evaporation of the solvent in vacuo the crude product was crystallized from diethyl ether to yield white crystals. Yield 54%. mp 140-145 °C. 1H NMR (300 MHz, DMSO-d6) 5 1.03 (t, J
7.1	Hz, 3H, CH3CH2), 3.67 (d, J 5.1 Hz, 2H, NHCH2CO),
4.02	(q, 2H, C^3CH2), 4.33 (s, 2H, CH2CONH2), 4.79 (s, 2H, CH2Ph), 6.49 (s, 1H, ArH ), 6,82 (d, J 7.2 Hz, 1H, Ar-H), 7.13-7.37 (m, 8H, ArH + CONH2), 7.52 (s, 1H, CH2C0NH2), 12.25 (s, 1H, CONHOCH2Ph) ppm. DEPT-135 NMR (300 MHz, DMSO-d6): 5 13.4, 43.7, 47.4, 76.7, 110.8, 116.5, 128.0, 128.5, 128.9 ppm. MS (m/z): 465 (M+H, 100). IR (KBr): v 3414, 3197, 2976, 2361, 1742, 1678, 1603, 1516, 1425, 1366, 1235, 1166, 1011, 939, 867, 801, 764, 701, 681, 612, 579, 530 cm-1. Anal. Calcd for C20H24N407S: C 51.72, H 5.21, N 12.06. Found: C 51.55, H 5.37, IN 12.07.
Synthesis of 2-amino-^-(benzyloxy)acetamide (13a).
To a solution of Boc-Gly35 (552 mg, 3.15 mmol) (12a) and 0-benzylhydroxylamine hydrochloride (503 mg, 3.15 mmol) in DMF (40 mL), N-methylmorpholine (1.0 mL, 9.42 mmol) was added at 0 °C. The mixture was stirred for 10 minutes followed by the addition of HOBt (425 mg, 3.15 mmol) and EDC (0.604 mg, 3.15 mmol). The reaction mixture was warmed to room temperature and stirred for 24 hours under argon atmosphere. The solvent was removed under reduced pressure and the residue dissolved in ethyl acetate (80 mL), washed with 10% citric acid (3 x 20 mL), saturated NaHCO3 (2 x 20 mL) and brine (20 mL), and dried over anhydrous Na2SO4. After filtration and evaporation of the solvent in vacuo the crude product was purified by flash chromatography on a silica gel column using hexane/ethyl acetate (1/1) as eluent (Rf = 0.17) to give pure white crystals which were used for the next reaction. Yield = 33%. mp 68-70 °C. 1H NMR (300 MHz, CDCl3) 5 1.42(s, 9H, t-Bu) 3.68 (s, 2H, NHCH2C0N^), 4.87 (s, 2H, CH2Ph), 5.41 (s, 1H, OCONH), 7.36 (s, 5H, ArH), 9.50 (s, 1H, NHOCH2Ph) ppm. DEPT-135 NMR (300 MHz, DMSO-dg): 5 27.8, 40.7, 76.6, 127.9, 128.4 ppm. MS (m/z): 281 (M+H, 49), 225 (100). IR (KBr): v 3412, 3234, 2976, 2360, 1721, 1683, 1530, 1417, 1364, 1291, 1165, 1033, 949, 919, 900, 860, 786, 742, 698, 608, 503 cm-1. Anal. Calcd for C14H20N204: C 59.99, H 7.19, N 9.99. Found: C 60.24, H 7.06, ^ ^0.15.
The product of the previous step (2.260 g, 8.07 mmol) was dissolved in a mixed solvent of dichloromet-hane and trifluoroacetic acid (9:1, 78 mL) and stirred for 1 hour. After the removal of solvent in vacuo the residue was dissolved in water and treated with dichloromethane (2 x 20 mL). Water phase was alkalized to pH 8 using Na-
HCO3. The white crystals obtained were filtered, washed with water and dried to afford compound 13a. Yield = 58%. mp 171-173 °C. 1H NMR (300 MHz, DMSO-dd) 5 3.02 (s, 2H, CH2NH2), 4.79 (s, 2H, CH2Ar), 7.35-7.39 (m, 5H, ArH ) ppm. DEPT-135 NMR (300 MHz, D20 + D2S04): 5 38.6, 78.8, 129.1, 129.6, 123.0 ppm. MS (m/z): 1^1 (IVI+H, 100). IR (KBr): v 2989, 2365, 1991, 1686, 1617, 1492, 1428, 1400, 1373, 1319, 1252, 1215, 1110, 1064, 1016, 952, 896, 837, 742, 696, 606, 545, 468 cm-1. Anal. Calcd for C5H12N2O4: C 59.99, H 6.71, N 15.55. Found: C 59.72, H 6.^7, N 15.17.
Synthesis of (S)-2-amino-N-(benzyloxy)propionamide (13b). To a solution of Boc-L-Ala36 (1.112 g, 5.88 mmol) (12b) and 0-benzylhydroxylamine hydrochloride (935 mg, 5.88 mmol) in DMF (50 mL) N-methylmorpholine (1.95 mL, 17.75 mmol) was added at 0 °C. The mixture was stirred for 10 minutes followed by the addition of HOBt (794 mg, 5.88 mmol) and EDC (1.128 g, 5.88 mmol). The reaction mixture was warmed to room temperature and stirred for 24 hours under argon atmosphere. The solvent was removed under reduced pressure and the residue dissolved in ethyl acetate (80 mL), washed with 10% citric acid (3 x 20 mL), saturated NaHCO3 (2 x 20 mL) and brine (20 mL), and dried over anhydrous Na2SO4. After filtration and evaporation of the solvent in vacuo the crude product was purified by crystallization from diethyl ether/petroleum ether to give white crystals which were used in the next reaction. Yield = 60%. mp 102-105 °C. [a] 20d -55,769 (c 0.260, MeOH). 1H NMR (300 MHz, DMSO-dd) 5 1.31 (d, J 7.2 Hz 3H, CHCH3), 1.41 (s, 9H, (CH3)3C), 3.98-4.03 (m, 1H, COCH), 4.87 (s, 2H, CH2Ph), 5.21 (d, J 7.5 Hz, 1H, COCHNH), 7.35-7.40 (m, 5H, Ar-H), 9.49 (s, 1H, CONHO) ppm. DEPT-135 NMR (300 MHz, DMSO-dg): 5 17.7, 27.8, 47.2, 76.4, 127.9, 128.5 ppm. MS (m/z): 295 (M+H, 7), 317 (M+Na, 100). IR (KBr): v 3337, 3256, 2988, 2875, 2363, 1672, 1520, 1450, 1367, 1320, 1255, 1163, 1072, 942, 905, 853, 787, 749, 699, 640, 570, 523, 498 cm-1. Anal. Calcd for C15H22N204: C 61.21, H 7.53, N 9.52. Found: C 61.39, H 7.43, N 9.55.
The product of the previous reaction (2.78 g, 9.56 mmol) was dissolved in diethyl ether (50 mL) and treated with HCl(g) at 0 °C for 30 minutes. Solvent was evaporated in vacuo, the residue dissolved in methanol/dichloro-methane = 1/4 (50 mL) and extracted with saturated Na-HCO3 solution (3 x 10 mL). The aqueous phase was acidified to pH 8 with 1M HCl and extracted with dichloromet-hane (3 x 10 mL). The combined extracts were quenched with NaCl, dried over anhydrous Na2SO4 and evaporated to yield a pure brown powder. Yield = 66%. mp 90-94 °C. [a]20D= -15.405 (c 0.185, MeOH). 1H NMR (300 MHz, DMSO-dd) 5 1.08 (d, J 6.8 Hz, 3H, CHCH3), 3.17 (q, 1H, CHCH3), 4.79 (s, 2H, CH2Ph), 7.35-7.40 (m, 5H, ArH) ppm. MAS (m/z): 195 (M+H, 100). IR (KBr): v 3670, 3069, 2853, 2102, 1606, 1508, 1452, 1361, 1281, 1205, 1181,
1085, 1052, 921, 857, 830, 753, 733, 702, 672, 613, 494 cm-1. Anal. Calcd for C10H14N2O2: C 61.84, H 7.27, N 14.42. Found: C 61.65, H 7.20, N 14.34.
4. 3. General Procedure for the Synthesis of 14a-d
To a solution of 9 (1 mmol) and the corresponding amino acid derivative (14a-14d) (1.1 mmol) in DMF (10 mL) N-methylmorpholine (3 mmol) was added. The solution was stirred at 0 °C for 10 minutes followed by addition of EDC (1 mmol) and HOBt (1 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 24 hours under argon atmosphere. Solvent was removed under reduced pressure and the residue dissolved in dichloromethane (50 mL), extracted with 10% citric acid (3 X 5mL), saturated NaHCO3 (2 x 5 mL) and brine (5 mL), and dried over anhydrous Na2SO4. After filtration and evaporation of the solvent in vacuo the crude product was purified by flash column chromatography or by crystallization.
Synthesis of ethyl 2-amino-2-oxoethyl{3-[2-(2-(benzy-loxyamino)-2-oxoethylamino]-2-oxoethylamino}phen-ylsulfonyl)carbamate (14a). Compound 14a was prepared by the reaction of compound 9 with (S)-2-amino-N-2-amino-N-(benzyloxy)acetamide (13a) following the procedure described above and crystallized from diethyl ether to give white crystals. Yield = 51%. mp 138-140 °C. 1H NMR (300 MHz, DMSO-d6) 5 1.04 (t, J 7.1 Hz, 3H, CH3CH2), 3.67 (d, J 5.5 Hz, 2H, NHCH2CO), 3.76 (s, 2H, NHCH2CONH), 4.02 (q, 2H, CH3^2), 4.32 (s, 2H, CH2CONH2), 4.78 (s, 2H, CH2Ph), ^.48 (t, J 5.5 Hz, 1H, NHCH2CO), 6.80-6.89 (d, J 6.3 Hz, 1H, Ar-H), 7.13-7.28 (m, 4H, Ar-H + CONH2), 7.32-7.40 (m, 5H, Ar-H), 7.51 (s, 1H, CH2CONH2), 8.18-8.22 (m, 1H, CONHCH2), 11.15 (s, 1H, CONHO) ppm. 13C NMR (75 MHz, DMSO-dg) 5 13.6 (CH3), 46.2 (NHCH2CO), 47.7 (CH2C(O)NH2), 63.0 (^2CH3), 76.2 (CH2Ar), 111.1 (ArC), 115.2 (ArC), 116.8 (ArC), 128.2 (ArC), 128.1 (ArC), 128.7 (ArC), 129.2 (ArC), 135.8, 139.8, 148.5, 151.5, 165.9, 168.4, 169.9 ppm. MS (m/z): 522 (M+H, 100), 558 (M+Na, 100). IR (KBr): v 3373, 1658, 1600, 1504, 1371, 1272, 1233, 1173, 686, 572 cm-1. Anal. Calcd for C22H27N5O8S: C 50.66, H 5.22, N 13.43. Found: C 50.53, H 5.13, N 13.54.
Synthesis of (S)-ethyl 2-amino-2-oxoethyl(3-{2-[1-
(benzyloxyamino)-1-oxopropan-2-ylamino]-2-oxoeth-ylamino}phenylsulfonyl)carbamate (14b). Compound 14b was prepared by the reaction of compound 9 with (S)-2-amino-N-(benzyloxy)propanamide (13b) following the procedure described above. The crude product was purified by flash chromatography using dichloromethane:met-hanol = 9:1 (Rf = 0.32) as eluent to give a product which was purified again with preparative chromatography using
dichloromethane:methanol:acetone = 10:1:1 (Rf = 0.47) as eluent to give a pure, yellow solid. Yield = 57%. mp 132-135 °C. [a] 20D -18.358 (c 0.335, 100 ml, MeOH). 1H NMR (300 MHz, DMSO-dg) 5 1.03 (t, J 7.1 Hz, 3H, CH3CH2), 1.17 (d, J 5.4 Hz, 3H, CH3CH), 3.76 (d, J 5.8 Hz, 2H, NHCH2CO), 4.01 (q, 2H, CH3CH2), 4.2 (m, 1H, CH3CHNH), 4.33 (s, 2H, CH2CONH2), 4.77 (s, 2H, CH2Ph ), 6.44 (t, J 5.8 Hz, 1H, NHCH2CO), 6.83-6.87 (m,1H, ArH), 7.14-7.31 (m, 4H, Al.H + CONH2), 7.36-7.40 (m, 5H, ArH), 7.52 (s, 1H, CH2CONH2), 8.14 (d, J 7.6 Hz, 1H, CHNHC(O)), 11.24 (s, 1H, CON-HOCH2Ph) ppm. DEPT-135 NMR (300 MHz, DMSO-dg): 5 14.6, 19.2, 46.9, 47.0, 48.6, 63.9, 77.7, 112.0, 116.1, 117.8, 129.2, 129.8, 130.1 ppm. MS (m/z): 536 (M+H, 25), 558 (M+Na, 100). IR (KBr): v 3436, 3209, 2982, 2360, 1742, 1678, 1605, 1511, 1239, 1164, 1081, 1023, 939, 866, 765, 703, 677, 575, 532 cm-1. Anal. Calcd for C23H29N5O8SxH2O: C 49.90, H 5.64, N 12.65. Found: C 452.87, H5.18, N13.05.
Synthesis of benzyl 2-(2-{3-[^-(2-amino-2-oxoethyl)-^-(ethoxycarbonyl)sulfamoyl]phenylamino}acetami-do)acetate (14c). Compound 14c was prepared by the reaction of compound 9 with glycine benzyl ester p-tolue-nesulfonate,33 following the procedure described above, and crystallized from diethyl ether to give white crystals. Yield = 55%. mp 90-92 °C. 1H NMR (300 MHz, DMSO-dg) 5 1.04 (t, J 7.1 Hz, 3H, CH3CH2), 3.76 (d, J 5.7 Hz, 2H, NHCH2CO), 3.92 (d, J 6.0 Hz, 2H, NHCH2COO), 4.02 (q, 2H, CH3CH2), 4.32 (s, 2H, CH2CONH2), 5.13 (s, 2H, CH2Ph), 6.56 (t, J = 5.9 Hz, 1H, NHCH2CO), 6.86 (d, J 7.8 Hz, 1H, ArH), 7.12-7.45 (m, 9H, ArH + CH2CONH2), 7.50 (s, 1H, CH2CONH2), 8.40 (t, J 6.0 Hz, 1H, NHCH2COO) ppm. 13C NMR (75 MHz, DMSO-dg) 5 13.7 (CH3), 46.4 (NHCH2CO), 47.9 (CH2C(O)NH2), 63.1 (CH2CH3), 65.9 (CH2Ar), 111.5 (ArC), 115.6 (ArC), 116.8 (A.rC), 127.9 (ArC), 128.1 (ArC), 128.4 (ArC), 129.3 (ArC), 135.9, 139.8, 148.6, 151.6, 168.7, 169.6, 170.5 ppm. MS (m/z): 507 (M+H, 73), 529 (M+Na, 100). IR (KBr): v 3394, 3198, 2975, 1740, 1674, 1603, 1526, 1344, 1364, 1316, 1233, 1165, 1011, 939, 869, 765, 683, 577, 535 cm1. Anal. Calcd for C22H2gN4O8S: C 52.17, H 5.17, N 11.06. Found: C 52.20, H 5.0(5, NN 11.05.
Synthesis of benzyl 2-(2-{3-[^-(2-amino-2-oxoethyl)-^-(ethoxycarbonyl)sulfamoyl]phenylamino}acetami-do)acetate (14d). Compound 14d was prepared by the reaction of compound 9 with benzyl ß-alanine benzyl ester p-toluenesulfonate34 following the procedure described above. The product was recrystallized from diethyl ether to give a yellow powder (62%), mp 81-83 °C. 1H NMR (300 MHz, DMSO-dg) 5 1.03 (t, J 7.1 Hz, 3H, CH3CH2), 2.49-2.55 (m, 2H, CH2COOCH2Ph), 3.37 (m, 2H, N^CH2CH2), 3.67 (d, J 5.7 Hz, 2H, NHCH2CO), 4.02 (q, 2H, CH3CH2), 4.32 (s, 2H, CH2CONH2), 5.07 (s, 2H, CH2Ar), 6.48 (t, J 5.7 Hz, 1H, NHCH2CO), 6,77-6.83
(m, 1H, ArH), 7.12-7.26 (m, 4H, ArH + CONH2), 7.33-7.37 (m, 5H, ArH), 7.52 (s, 1H, CH2CONH2), 8.05 (t, J 5.6 Hz, 1H, CONHCH2CH2) ppm. 13C NMR (75 MHz, DMSO-d6) 6 13.6 (CH3), 33.6 (CH2COO), 34.5 (NHCH2CH2), 46.3 (NHCH2CO), 47.6 (CH2C(O)NH2), 62.9 (CH2CH3), 65.4 (CH2Ar), 111.1 (ArC), 115.2 (ArC]), 116.7 (ArC), 127.8 (ArC), 127.9 (ArC), 128.3 (ArC), 129.1 (ArC), 135.9, 139.7, 148.4, 151.4, 168.4, 169.4, 171.0 ppm. MS (m/z): 521 (M+H, 100). IR (KBr): v 3422, 3326, 3184, 1734, 1685, 1601, 1515, 1486, 1349, 1315, 1268, 1177, 1082, 1010, 937, 856, 750, 729, 701, 682, 626, 566 cm-1. Anal. Calcd for C23H28N4O8S: C 53.07, H 5.42, N 10.76. Found: C 52.82, H 5.1^, NN 10.38.
4. 3. General Procedure for the Synthesis of 11 and 15a-d
Into a solution of 10 or 14a-d (1 mmol) in methanol (10 mL), argon was bubbled for 10 minutes. 10% Pd/C, unreduced (10% wt/wt), was added followed by ammonium formate (3 mmol) and the mixture stirred for 24 hours. The mixture was filtered, solvent was evaporated in vacuo and crude product was purified by flash column chromatography followed by crystallization from diethyl ether.
Synthesis of ethyl 2-amino-2-oxoethyl{3-[2-(hydroxya-mino)-2-oxoethylamino]phenylsulfonyl}carbamate (11). Compound 11 was prepared from compound 10 following the procedure described above. The product was purified by flash chromatography using dichlorometha-ne:methanol = 9:1 as eluent (Rf = 0.17) and recrystallized from diethyl ether to give white crystals. Yield = 26%. mp 85-87 °C. 1H NMR (300 MHz, DMSO-dg) 6 1.07 (t, J 7.1 Hz, 3H, CH3CH2), 3.64 (d, J 5.5 Hz, 2H, NHCH2CO), 4.03 (q, 2H, CH3CH2), 4.32 (s, 2H, CH2CONH2), 6.43 (t, J 5.5 Hz, 1H, NHCH2CO), 6.85 (d, J 77.2 Hz, 1H, ArH), 7.15-7.28 (m, 4H, ArH + CONH2), 7.52 (s, 1H, CH2CONH2 ), 8.84 (s, 1H, CONHOH), 10.61 (s, 1H, CONHOH) ppm. DEPT-135 NMR (300 MHz, DMSO-dg): 6 13.2, 47.4, 62.6, 73.5 110.9, 115.0, 116.8, 128.8 ppm. MS (m/z): 375 (M+H, 60), 324 (100). IR (KBr): v 3364, 1693, 1601, 1335, 1158, 1009, 936, 766, 682 cm-1. Anal. Calcd for C13H18N4O7S ■ 7/6 H2O: C 37.79, H 4.85, N 14.69. Found: C 38.02, ìì 5.24, N 14.25.
Synthesis of ethyl 2-amino-2-oxoethyl(3-{2-[2-(hydrox-yamino)-2-oxoethylamino]-2-oxoethylamino}phenyl-sulfonyl)carbamate (15a). Compound 15a was prepared from compound 14a following the procedure described above. The product was purified by flash chromatography (dichloromethane:methanol =5:1 followed by dichloro-methane:methanol =1:1) and recrystallized from diethyl ether to give white crystals. Yield = 44%. Rf (dichloromet-hane:methanol=1:1) = 0.70. mp 158-160 °C. 1H NMR (300 MHz, DMSO-dg) 6 1.05 (t, J 7.1 Hz, 3H, CH3CH2),
3.65 (d, J 5.4 Hz, 2H, NHCH2CH2), 3.67 (d, J 5.5 Hz, 2H, NH^2CONH,), 4.03 (q, 2H, CH3^2), 4.32 (s, 2H, CH2CONH2), 6.49 (t, J 5.5 Hz, 1H, ^CH2CO), 6.85 (d, J 7.8 Hz, 1H, ArH,), 7.14-7.16 (m, 3H, ArH + CONH2), 7.26-7.34 (m, 1H, ArH), 7.52 (s, 1H, CH2CONH2), 8.19 (t, J 4.9 Hz, 1H, NHCH2CH2), 8.89 (s, 1H, CONfHOH), 10.56 (s, 1H, CONHOH) ppm. 13C NMR (75 MHz, DM-SO-dg) 6 13.6 (CH3), 46.1 (NHCH2CO), 47.7 (CH2C(O)NH2), 63.0 (CH2CH3), 111.0 (ArC), 115.2 (ArC), 116.8 (ArC), 119.1 (ArC), 139.8, 148.6, 151.5, 168.4, 169.8 ppm. MS (m/z): 454 (M+H, 100). IR (KBr): v 3466, 3391, 3345, 3282, 2996, 2918, 2343, 1732, 1681, 1635, 1605, 1528, 1510, 1479, 1431, 1410, 1374, 1359, 1334, 1301, 1247, 1185, 1161, 1090, 1036, 1011, 989, 937, 877, 844, 790, 766, 701, 674, 611, 571, 528 cm-1. Anal. Calcd for C15H21N4O8S: C 40.63, H 5.08, N 15.79. Found: C 40.42, H 41.68, ^ 15.47.
Synthesis of (S)-ethyl 2-amino-2-oxoethyl(3-{2-[1-(hy-droxyamino)-1-oxopropan-2-ylamino]-2-oxoethylami-no}phenylsulfonyl)carbamate (15b). Compound 15b was prepared from compound 14b following the procedure described above. The resulting crude yellow oil was purified by flash chromatography using dichlorometha-ne:methanol = 4:1 (Rf = 0.19) as eluent and recrystallized from diethyl ether to give white crystals. Yield = 52%. mp 119-111 °C. [a] 20D -11.725 (c 0.255, MeOH). 1H NMR (300 MHz, DMSO-dg) 6 1.04 (t, J 7.1 Hz, 3H, CH3CH2), 1.18 (d, J 5.4 Hz, 3H, CH3CHNH), 3.75 (d, J 5.8 Hz, 1H, NHCH2CO), 4.02 (q, ^H, CH3CH2), 4.25 (m, 1H, CH3CHNH), 4.33 (s, 1H, CH2CO]S[H2), 6.45 (t, J 5.8 Hz, 1H, NHCH2CO), 6.85 (d, J 7.2 Hz, 1H, ArH), 7.15-7.17 (m, 3H, Ar^H), 7.25-7.31 (m, 1H, CH2CONH2), 7.53 (s, 1H, CH2CONH2), 8.08 (d, J 7.2 Hz, 1H, CHH.CHNH), 8.87 (s, 1H, CONHOH) 10.65 (s, 1H, CONHOH) ppm. MS (m/z): 446 (M+H, 18), 468 (MNa+, 100). IR (KBr): v 3417, 2357, 1684, 1601, 1519, 1349, 1268, 1177, 1013, 937, 858, 765, 682, 567 cm-1. Anal. Calcd for C16H23N5O8S: C 43.14, H 5.20, N 15.72. Found: C 43.43, H 5.5^, NN 15.32.
Synthesis of 2-(2-{3-[^-(2-amino-2-oxoethyl)-^-(et-hoxycarbonyl)sulfamoyl]phenylamino}acetami-do)acetic acid (15c). Compound 15c was prepared from compound 14c following the procedure described above. The product was purified by flash chromatography (dich-loromethane:methanol =5:1 followed by dichlorometha-ne:methanol =1:1) and recrystallized from diethyl ether to give white crystals Yield = 53%. Rf (dichlorometha-ne:methanol) = 0.73. mp 162-165 °C. 1H NMR (300 MHz, DMSO-dg) 6 1.03 (t, J 7.0 Hz, 3H, CH3CH2), 3.57 (d, J 3.64 Hz, 1H, NHCH2COOH), 3.74 (s, 1H, NH^2CONH), 4.00 (q, 1H, CH3CH2), 4.35 (s, 1H, CH2CONH2), 6.21 (br, 1H, NHCH2CO), 6.85 (d, J 7.8 Hz, 1H, ArH), 7.11-7.20 (m, 3H, Ar-^ + CONH2), 7.17-7.31 (m, 1H, ArH), 7.62 (s, 1H, CONH2), 7.69 (s, 1H,
CH2CONH2 ), 7.81 (s, 1H, NHCH2COOH) ppm. 13C NMR (75 MHz, DMSO-dg) 5 13.7 (CH3), 42.7 (CH2COO), 46.5 (NHCH2CO), 47.8 (CH2C(O)NH2), 63.1 (CH2CH2), 111.1 (ArC), 115.4 (ArC), 117.0 (ArC), 129.3 (ArC), 139.9, 148.7, 151.6, 168.7, 169.7, 171.8 ppm. MS (m/z): 417 (M+H, 100). IR (KBr): v 3398, 2977, 2362, 1734, 1669, 1604, 1396, 1373, 1342, 1244, 1165, 1077, 1013, 986, 941, 867, 798, 767, 680, 608, 575, 528 cm-1. Anal. Calcd for C15H20N4O8S ■ 7/3H2O: C 39.30, H 5.42, N 12.22. Found: C 39.63, H 5.18, N 11.86.
Synthesis of 3-(2-{3-[iV-(2-amino-2-oxoethyl)-^-(etho-xycarbonyl)sulfamoyl]phenylamino}acetamido)pro-panoic acid (15d). Compound 15d was prepared from compound 14d following the procedure described above. The product was purified by flash chromatography (dich-loromethane:methanol =5:1 followed by dichlorometha-ne:methanol =1:1) and recrystallized from diethyl ether to give white crystals (Yield 53%). Rf (dichlorometha-ne:methanol=1:1) = 0.53. mp 122-124 °C. 1H NMR (300 MHz, DMSO-dg) 5 1.03 (t, J 7.1 Hz, 3H, CH3CH2), 2.35 (t, J 6.9 Hz, 3H, CH2^2COOH), 3.29 (q, 2H, NHCH2CH2), 3.68 (d, J4.0 Hz, 2H, NHCH2CONH), 4.02 (q, 2H, CH3CH2), 4.32 (s, 2H, CH2CONH2), 6.48 (t, J 4.0 Hz, 1H, S[HCH2CO), 6.82 (d, J 8.7 Hz, 1H, ArH), 7.15-7.18 (m, 3H, Ar-H + CONH2), 7.27-7.32 (m, 1H, ArH), 7.54 (s, 1H, CH2CONH2), 8.01 (t, J 5.5 Hz, 1H, NHCH2CH2) ppm. 13C SSMR (75 MHz, DMSO-dg) 5 13.7 (CH3), 34.3 (CH2COO), 34.9 (C(O)NHCH2), 46.3 (NHCH2CO), 47.7 (CH2C(O)NH2), 63.0 (CH2CH2), 111.0 (ArC), 115.3 (ArC), 116.9 (ArC), 129.2 (AArC), 139.8, 148.6, 151.5, 168.5, 169.4, 173.4 ppm. MS (m/z): 431 (M+H, 9), 453 (M+Na, 100). IR (KBr): v 3524, 3418, 3323, 3177, 2936, 2363, 1732, 1685, 1601, 1528, 1487, 1407, 1375, 1349, 1320, 1275, 1245, 1183, 1138, 1085, 1018, 939, 876, 812, 768, 712, 683, 629, 569, 535 cm-1. Anal. Calcd for C1gH22N4O8S x 2/3 H2O: C 34.43, H 5.32, N 12.66. Found: C 4^.34, H 5.27, N 12.32.
Synthesis of (Ä)-dibenzyl 2-[2-(2-{3-[V-(2-amino-2-ox-oethyl)-V-(ethoxycarbonyl)sulfamoyl]phenylami-no}acetamido)acetamido]pentanedioate (16). To a solution of 15d (505 mg, 1.214 mmol) and D-glutamic acid, dibenzyl ester p-toluenesulfonate (605 mg, 1.214 mmol) in DMF (5 mL) N-methylmorpholine (0.40 mL, 3.63 mmol) was added. A solution was stirred at 0 °C for 10 minutes followed by the addition of EDC (256 mg, 1.335 mmol) and HOBt (164 mg, 1.214 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 24 hours under argon atmosphere. Solvent was removed under reduced pressure and the residue dissolved in dichloromethane (50 mL), extracted with 10% citric acid (3 x 5 mL), saturated NaHCO3 (2 x 5 mL) and brine (5 mL), and dried over anhydrous Na2SO4. After filtration and evaporation of the solvent in vacuo the crude brown oil was purified by flash column chromatography using
dichloromethane:methanol = 9:1 (Rj = 0.42) as eluent to obtain 16, which was crystallized from diethyl ether to give a white solid. Yield = 21%. mp 85-87 °C. [a]20D +7.939 (c 0.330, MeOH). 1H NMR (300 MHz, DMSO-dg) 5 1.04 (t, J 7.1 Hz, 3H, CH3CH2), 1.85-1.83 (m, 1H, CHCH2CH2COO), 2.00-2.09 (m, IH, CHCH2CH2COO), 2.44 (t, J 7.6 Hz, 2H, CH2CH2COO), 3.76-3.81 (m, 4H, ArNH^2CO + NH^2C0NHCH2), 4.02 (q, 2H, CH3CH2), 4.35 (s, 2H, CH2CONH2), 4.37-4.43 (m, 1H, NH3:H(Č(0)), 5.06-5.15 (A^B, J 1^.5 Hz, v 25 Hz, 4H, 2xCH2Ph), 6.50 (t, J 5.8 Hz, 1H, NHCH2CO), 6.86 (d, J 7.7 Hz, 1H, ArH), 7.15 (d, J 8.2 Hz, 1H, ArH), 7.20 (s, 2H, ArH), 7.24-7.42 (m, 11H, ArH + CONH2), 7.52 (s, 1H, CH2C0NH2), 8.15 (t, J 5.6 Hz, 1H, CONHCH2), 8.33 (d, J 7.6 Hz, 1H, CONHCH) ppm. 13C NMR (75 MHz, DMSO-dg) 5 13.6 (CH3), 26.0 (CH2CH2CO), 29.7 (CH2CH2C0), 41.5 (CH2NHC0), 46.2 (NHCH2CO), 47.7 (CH2C(0)NH2), 51.1 (NHCHC(O)), 62.9 (CH2CH2), 65.4 (CH2Ar), 66.0 (CH2Ar), 111.2 (ArC), 115.3 (AArC), 116.7 (ArC), 127.7 (ArC), 127.8 (ArC), 127.9 (ArC), 128.3 (ArC), 129.2 (ArC), 135.8, 136.0, 139.8, 148.5, 151.5, 168.4, 168.9, 169.8, 171.3, 171.9 ppm. MS (m/z): 726 (M+H, 100). IR (KBr): v 3373, 1738, 1660, 1599, 1503, 1356, 1231, 1171, 685cm-1. Anal. Calcd for C34H39N5O11S: C 56.27, H 5.42, N 9.65. Found: C 56.18, H ^.27, SS 9.70.
Synthesis of (Ä)-2-[2-(2-{3-[V-(2-amino-2-oxoethyl)-V-(ethoxycarbonyl)sulfamoyl]phenylamino}acetami-do)acetamido]pentanedioic acid (17). Into a solution of 16 (0.5 mmol) in methanol (50 mL) argon was bubbled for 10 minutes. 10% Pd/C, unreduced (10% wt/wt), was added and the mixture then bubbled with hydrogen for 30 minutes followed by stirring under hydrogen atmosphere for 24 hours. The mixture was filtered, solvent was evaporated in vacuo and crude product was crystallized from di-ethyl ether to yield a pure white solid. Yield = 63%. mp 78-80 °C. [a] 20d -5.582 (c 0.335, MeOH). 1H NMR (300 MHz, DMSO-dg) 5 1.09 (t, J 7.1 Hz, 3H, ^3CH2), 1.70-1.83 (m, 1H, CH2CH2COO), 1.83-1.91 (m, 1H, ^2CH2C00), 2.25 (t, J 7.7 Hz, 2H, CH2^2COO), 3.73-3.78 (m, 4H, ArNH^2CO + NH^2CONHCH), 4.03 (q, 2H, CH3CH2), 4.15-4.19 (m, 1H, SSHCH), 4.33 (s, 2H, CH2C0NH2), 6.51 (t, J 5.8 Hz, 1H, NHCH2CO), 6.50 (t, J 5.3 Hz, 1H, NHCH2CO), 6.84-6.88 (m, 1H, Ar-H), 7.15 (d, J 8.2 Hz, 1H, Ar^), 7.13-7.33 (m, 3H, ArH + C0NH2), 7.35-7.40 (m, 1H, ArH), 7.52 (s, 1H, CH2C0NH2), 8.01 (t, J 5.6 Hz, 1H, CONHCH2), 8.33 (d, J 7.6 Hz, 1H, CONHCH), 8.15 (t, J 5.6 Hz, 1H, CONHCH), 8.33 (d, J 7.6 Hz 1H, CONHCH2) ppm. 13C NMR (75 MHz, DMSO-dg) 5 13.6 (CH3), 26.6 (CH2CH2CO), 30.2 (CH2CH2C0), 41.7 (CH2NHCO), 46.2 (NHCH2CO), 47.7 (CH2C(0)NH2), 51.3 (NHCHC(O)), 63.0 (CH2CH2), 111.2 (ArC), 115.3 (ArC), 116.7 (ArC), 129.3 (ArC), 139.8, 148.5, 151.5, 168.4, 168.5, 169.8, 173.0, 173.8 ppm. MS (m/z): 546 (M+H, 14), 568 (M+Na, 100). IR (KBr): v 3372, 2979, 2363, 1734, 1684, 1604, 1534, 1413,
1374, 1344, 1244, 1167, 1012, 986, 938, 877, 766, 681, 623, 570, 538 cm-1. Anal. Calcd for C2oH27N5O11Sx7/3 H20: C 40.88, H 5.43, N 11.92. Found: C 40.84, H 5.65, N11.63.
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Povzetek
Načrtovali in sintetizirali smo serijo novih fenilsulfonilkarbamatov, ki oponašajo strukturo UDP-Mur-NAc, UDP-Mur-NAc-L-Ala in UDP-Mur-NAc-L-Ala-D-Glu in so kot takšni potencialni inhibitorji od ATP odvisnih ligaz MurB, C, D in E, udeleženih v biosintezi bakterijske celične stene. Predstavljena je podrobna sinteza omenjenih spojin