Scientific paper
Design and Synthesis of New Peptidomimetics as Potential Inhibitors of MurE
v	1	1	2	1
Matej @ivec,1 Samo Turk,1 Didier Blanot2 and Stanislav Gobec1*
1 Faculty of Pharmacy, University of Ljubljana, A{ker~eva 7, SI-1000 Ljubljana, Slovenia.
2 Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS, Univ Paris-Sud,
F-91405 Orsay, France
* Corresponding author: E-mail: stanislav.gobec@ffa.uni-lj.si Tel: +386-1-4769500; Fax: +386-1-4258031
Received: 13-09-2010
Abstract
With the continuing emergence and spread of multidrug-resistant bacteria, there is an urgent need for the development of new antimicrobial agents. One possible source of new antibacterial targets is the biosynthesis of the bacterial cellwall peptidoglycan. The assembly of the peptide stem is carried out by four essential enzymes, known as the Mur ligases (MurC, D, E and F). We have designed and synthesised a focused library of compounds as potential inhibitors of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase (MurE) from Staphylococcus aureus. This was achieved using two approaches: (i) synthesis of transition-state analogues based on the methyleneamino core; and (ii) synthesis of MurE reaction product analogues. Two methyleneamino-based compounds are identified as initial hits for inhibitors of MurE.
Keywords: MurE, peptidoglycan, methyleneamino, peptidomimetics
1. Introduction
The emergence and spread of multidrug resistant bacteria represents a serious threat for community health. Although more rational use of antibiotics might alleviate the problem, there is an undisputable need for new antimicrobial agents.1-4 One possible source of new antibacterial targets is the biosynthesis of the bacterial cell-wall pepti-doglycan.5-7 Peptidoglycan is a heteropolymer that is composed of glycan chains that are cross-linked by short peptides. Its main functions are to preserve cell integrity, by withstanding the internal osmotic pressure, and to maintain a defined cell shape. Peptidoglycan is also involved in cell growth and division. Since peptidoglycan is found exclusively in eubacteria and has no known human counterpart, it represents an ideal target for selective toxicity.8,9 The biosynthesis of peptidoglycan is a complex process that involves several reactions that take place in the cytoplasm and on the inner and outer leaflets of the membrane.10-12 While most of the drugs that affect the bacterial cell wall target the enzymes involved in the late stages of this process, only a few agents target the enzy-
mes involved in the cytoplasmic steps, making them unde-rexploited as antibacterial targets.5-7
The assembly of the peptide stem of peptidoglycan is carried out by four essential enzymes, known as the Mur ligases (MurC, D, E and F). These are responsible for the sequential addition of L-Ala (MurC), D-Glu (MurD), a diamino acid (MurE) and dipeptide D-Ala-D-Ala onto the lactoyl group of UDP-MurNAc. The third amino acid is usually either meso-diaminopimelic acid (in most Gram-negative bacteria) or L-lysine (in most Gram-positive bacteria), although in some cases other amino acids are found at this position. The Mur ligases are ATP-dependent enzymes that catalyse the formation of an amide bond, with the concomitant formation of ADP and inorganic phosphate (Pi). Although they have only small overall sequence identities (between 15% and 22%), the Mur ligases share several common features. They are composed of three domains: the N-terminal domain that is responsible for the binding of the UDP precursor; a central ATP-bin-ding domain; and a C-terminal domain that is involved in the binding of the amino acid or dipeptide. They also have the same reaction mechanisms, which consist of the acti-
vation of the carboxyl group of the UDP-precursor with ATP, which leads to the formation of an acyl phosphate intermediate and ADP; this is followed by nucleophilic attack of the a-amino group of the amino acid (or dipepti-de), which results in the formation of a highly energetic tetrahedral intermediate that breaks down to form an amide and Pj.10
To assess the potential of the MurE enzymes (UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diamino-pimelate ligases) as antibacterial targets, several efforts have been made to define their structure and function. Studies of their substrate specificity have shown that the MurE enzymes are normally highly specific for their respective substrates. Although attempts have been made to crystallize various MurE enzymes, only the crystal structure of MurE from E. coli with its product, UDP-MurNAc-L-Ala-y-D-Glu-meso-A^m, has been defined to date, and the crystal structure of a lysine-adding enzyme has yet to be obtained.13 The early search for inhibitors identified some A^m analogues and N-acyl-dipeptide derivatives with poor to moderate inhibitory activities against MurE from E. coli.14-17 In 1998, a series of phosphinate-based transition-state analogues were synthesised the best inhibitor found was 1 (Figure 1) with an IC50 of 1.1 pM, which represents the best inhibitor of MurE to date. Interestingly, a derivative 2 (Figure 1), which is devoid of the UMP moiety, has an IC50 of only 700 pM.18
Later, some phosphinates and P-sulfonamides that were designed as transition-state analogues of the MurD enzyme were reported to have inhibitory activities against
both MurD and MurE.19,20 Similarly, a virtual screening investigation of a MurD crystal structure produced two benzene 1,3-dicarboxylic acid derivatives as inhibitors of MurD and MurE.21 Recently, some compounds based on a phosphorylated hydroxyethylamine scaffold have been reported to be micromolar inhibitors of Mur ligases, including MurE.22
Our aim was to obtain new inhibitors of MurE that could be used as hit molecules in antibacterial drug discovery and as molecular tools for the exploration of L-Lys addition by MurE from Gram-positive bacteria. With no three-dimensional X-ray structural data available, we decided to design and synthesise a small focused library of peptidomimetics as potential inhibitors of MurE from Staphylococcus aureus (Figure 2). As can be seen from the co-crystal structure of MurE from E. coli, the peptide moiety of the product (UDP-Mur-NAc-L-Ala-y-D-Glu-meso-A2pm) forms a number of H-bonds with the enzyme, making the peptide part of the MurE product a good starting point for the design of peptidomimetic inhibitors. The library was designed based on two approaches: (i) synthesis of transition-state analogues that were based on the methyleneamino core, and (ii) synthesis of product analogues (Figure 2). In both approaches, we wanted to further increase the binding affinities of these inhibitors by the incorporation of rigid fragments: 4-piperidinecarboxylic acid was used as a replacement for D-Glu, while (S)-2,6-diamino-4,5,6,7-tetrahydrobenzothiazole was used as a rigid mimetic of L-Lys.
Figure 1: Phosphinate inhibitors of MurE (from E. coli).
Figure 2: Reaction mechanism of MurE, showing the putative tetrahedral transition state intermediate, the product of the reaction, and the designed transition-state analogues and product analogues.
2. Results and Discussion
Compounds 8 and 10 were prepared using standard peptide chemistry approaches (Scheme 1). Here, 5-(benzyloxy)-4-[(Boc)amino]-5-oxopentanoic acid 3 (Boc representing tert-butoxycarbonyl) was first converted to benzyl 2-[(Boc)amino]-5-[methoxy(methyl)amino]-5-oxopentanoate 4. A second Boc protective group was introduced to the a-amino group of 4, to obtain the di-Boc-protected Weinreb amide 5. This second protecting group was needed to avoid the reaction of the monoprotected a-amino group with the y-aldehyde that is formed in the next reaction step, which would potentially lead to unwanted side products and a lower reaction yield.23 Weinreb amide 5 was then converted to benzyl N,N-di-Boc-glutamate y-semialdehyde 6 by reduction with tris-tert-butoxy lithium aluminium hydride (LiAl('BuO)3H) in diethyl ether at room temperature. The transition-state analogue, methyle-neamino pseudodipeptide D-Glu-y[CH2NH]-L-Lys 8, was prepared from aldehyde 6 in two steps. Reductive amination of 6 with H-L-Lys(Z)-OBzl using sodium tria-cetoxyborohydride in 1,2-dichloroethane (DCE) gave di-Boc-D-Glu-y[CH2NH]-L-Lys(Z)-OBzl 7, which was de-protected to give the target reduced amide 8. Alternatively, compound 7 was Boc deprotected with CF3CO2H in CH2Cl2, followed by coupling with Boc-L-Ala using the benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium
hexafluorophosphate (BOP) reagent to obtain the protected pseudotripeptide Boc-L-Ala-D-Glu-y[CH2NH]-L-Lys(Z)-OBzl 9. This was finally deprotected using standard procedures, to give the desired reduced methylenea-mino pseudotripeptide (L-Ala-D-Glu-y[CH2NH]-L-Lys) 10 (Scheme 1).
Methyleneamino pseudopeptides are usually prepared by reductive amination reactions between an N-pro-tected a-amino aldehyde and an amino acid.24 Although several options are usually available for the preparation of ^-protected a-amino aldehydes,25 dicarboxylic amino acids are a special case, due to the presence of a second carboxylic group.26 Several options for the preparation of N,N-di-Boc-glutamate y-semialdehydes were presented in a review by Constantinou-Kokotou and Magrioti.26 Accordingly, N,N-di-Boc-glutamate y-semialdehydes were prepared mainly by two methods: (i) selective reduction of a y-methyl ester to the corresponding N,N-di-Boc-glutamate y-semialdehyde, using DIBAL at -78 °C; or (ii) reduction of a Weinreb amide with diisobutylaluminium hydride (DIBAL) at -78 °C.2327 All of the described methods were applied to the synthesis of either a-methyl or a-tert-butyl N,N-di-Boc-glutamate y-semialdehydes with good yields. Recently, the successful application of a method using DIBAL for the selective reduction of a y-methyl ester for the synthesis of benzyl N,N-di-Boc-glutamate y-semialdehyde 6 was reported.28 All of these methods require
Scheme 1: Synthesis of methyleneamino-containig pseudopeptides. Reagents and conditions: a) HCl X HN(OCH3)CH3, BOP, Et3N, CH2Cl2, 0 °C; b) Boc2O, DMAP, AcCN; c) LiAl(tBu)3H, diethyl ether, r.t.; d) H-L-Lys(Z)-OBzl, DCE, NaBH(OAc)3, r.t.; e) first H2, Pd/C, glacial acetic acid, then HCl(g), glacial acetic acid; f) first CH2Cl2/CF3CO2H = 9:1 for 1h, then Et3N, BOP, Boc-L-Ala-OH, CH2Cl2, 0 °C.
the synthesis to be performed at -78 °C. In a study where different reagents were assessed for their potential as reducing agents for the selective reduction of Weinreb amides to aldehydes, Paris et al. proposed the use of Li-Al(tBuO)3H as a mild reducing agent that allows for the reduction of Weinreb amides at room temperature.29 A comparison of the stabilities of the different ester functionalities (but not the benzyl ester) to various reducing agents at room temperature showed that ester functionalities were not affected by LiAl(tBuO)3H, while the appearance of polar compounds was detected for both LiAlH4 and DIBAL.29 We explored the potential of LiAl('BuO)3H for the reduction of Weinreb amide 5 to the desired benzyl N,N-di-Boc-glutamate y-semialdehyde 6. Our data show that LiAl(tBuO)3H can be used for the reduction of the Weinreb amide 5 to the semialdehyde 6 with good yield. Moreover, the reaction can be carried out at room temperature, as compared to other methods that need to be performed at -78 °C.
Transition-state analogues containing 4-piperidine-carboxylic acid (i.e. 15 and 17) (Scheme 2) were prepared in a similar fashion. 1-(Boc)piperidine-4-carboxylic acid 11 was converted to the Weinreb amide 12 by coupling with N,0-dimethylhydroxylamine. Reduction of the latter with LiAlH4 gave aldehyde 13, which was used in the subsequent reductive amination reaction with L-Lys(Z)-OB-
zl, to give the reduced dipeptide analogue 14. Deprotec-tion of the latter gave the target compound 15 as a constrained mimetic of the methyleneamino pseudodipeptide (D-Glu-y[CH2NH]-L-Lys) 8. The reduced tripeptide analogue 17 was prepared by Boc deprotection of the reduced dipeptide analogue 14, followed by coupling with Boc-L-Ala to give the protected tripeptide analogue 16. Deprotection of 16 by standard procedures gave compound 17 as a constrained mimetic of the methyleneamino pseudo-tripeptide (L-Ala-D-Glu-y[CH2NH]-L-Lys) 10.
The MurE product analogues of compounds 19 and 21 were prepared from 1-(Boc)piperidine-4-carboxylic acid 11 in two reaction steps (Scheme 3). Dipeptide analogue 18 was prepared by coupling of 11 with H-L-Lys(Z)-OBzl, using the diphenylphosphoryl azide (DP-PA) reagent. Compound 18 was then deprotected to give the target dipeptide analogue 19. To further increase the rigidity of compound 19, we replaced the Lys moiety with (S)-2,6-diamino-4,5,6,7-tetrahydrobenzothiazole, to give the target dipeptide analogue 21. This was prepared first by coupling of carboxylic acid 11 with (S)-2,6-dia-mino-4,5,6,7-tetrahydrobenzothiazole to give dipeptide analogue 20, followed by its deprotection to give the desired compound 21.
Product analogues containing an L-Ala moiety (27 and 29) were synthesized in five reaction steps from 4-pi-
Scheme 2: Synthesis of methyleneamino-containing pseudopeptides containing 4-piperidinecarboxylic acid. Reagents and conditions: a) HCl X HN(OCH3)CH3, BOP, Et3N, CH2Cl2, 0 °C; b) LiAlH4, ether, 0 °C; c) H-L-Lys(Z)-OBzl, DCE, NaBH(OAc)3, r.t.; d) first H2, Pd/C, glacial acetic acid, then HCl^, glacial acetic acid; e) first CH2Cl2/CF3CO2H = 9:1 for 1 h, then Et3N, BOP, Boc-L-Ala-OH, CH2Cl2, 0 °C.
Scheme 3: Syntheses of product-like pseudodipeptides. Reagents and conditions: a) H-L-Lys(Z)-OBzl, Et3N, DPPA, DMF, 0 °C; b) first H2, Pd/C, glacial acetic acid, then HCl(g), glacial acetic acid; c) (S)-2,6-diamino-4,5,6,7-tetrahydrobenzothiazole, DPPA, Et3N, DMF, 0 °C; d) HCl(g), EtOH.
Scheme 4: Synthesis of product analogues based on the 4-piperidinecarboxylic acid scaffold. Reagents and conditions: a) first p-TolSO3H, benzene, benzyl alcohol, then NaHCO3; b) Boc-L-Ala-OH, Et3N, DPPA, DMF, 0 °C; c) H2, Pd/C, MeOH, r.t.; d) H-L-Lys(Z)-OBzl, Et3N, DPPA, DMF, 0 °C; e) first CH2Cl2/CF3CO2H = 9:1, then H2, Pd/C, glacial acetic acid; f) (S)-2,6-diamino-4,5,6,7-tetrahydrobenzothiazole, BOP, CH2Cl2, 0 °C; g)
HCV THF.
peridinecarboxylic acid 22 (Scheme 4). Compound 22 was first transformed to its benzyl ester 23, which was next coupled with Boc-L-Ala to give L-Ala-D-Glu analogue 24. Catalytic hydrogenation of the latter yielded a car-boxylic acid 25, which was coupled to either H-L-Lys(Z)-OBzl or (S)-2,6-diamino-4,5,6,7-tetrahydrobenzothiazole to give compounds 26 and 28, respectively. These were finally deprotected to give the target L-Ala-D-Glu-L-Lys tripeptide analogues 27 and 29.
In compounds 15, 17, 19, 21, 27 and 29, D-glutamic acid was replaced by 4-piperidinecarboxylic acid, which confers a higher rigidity to these molecules and maintains the correct distance between the Na-atoms of the D-Glu and L-Lys fragments; however, this loses the a-Glu car-boxylic functionality. To assess the possibility of compensating for this loss by adding a carboxylic functionality through a methylene spacer on the amino group of the pi-peridine moiety, we synthesised MurE product analogue 33 (Scheme 5). Here, benzyl 4-piperidinecarboxylate 23 was alkylated with tert-butyl bromoacetate, to give benzyl
1-(2-tert-butoxy-2-oxoethyl)piperidine-4-carboxylate 30. After deprotection of the benzyl ester by catalytic hydrogenation, the free N-substituted 4-piperidinecarboxylic carboxylic acid 31 was coupled with L-Lys(Z)OBzl to give pseudodipeptide 32, which was deprotected to give the desired product analogue 33.
Specific rotations as well as NMR spectra, where no multiplications of signals could be observed, suggest that the chirality of the starting reagents was preserved during all reactions described in this paper.
All of these compounds were tested for inhibition of MurE from S. aureus. Among these, only compounds 8 (63% inhibition at 2 mM) and 15 (73% inhibition at 2 mM; IC50, 1.12 mM) showed MurE inhibitory activity. Both of these compounds are pseudodipeptides that are based on the methyleneamino core, and in both cases the inhibitory activity was lost upon extension of the molecule by an L-Ala moiety (i.e., compounds 10 and 17 showed no MurE inhibition). The similar inhibitory activities of compounds 8 and 15 indicated that 4-piperidine
Scheme 5: Synthesis of product analogue 33. Reagents and conditions: a) BrCH2CO2'Bu, Et3N, CH2Cl2, 0 °C; b) H2, Pd/C, MeOH, r.t.; c) H-L-Lys(Z)-OBzl, Et3N, DPPA, DMF, 0 °C; d) first H2, Pd/C, glacial acetic acid, then HCl^.
carboxylic acid is a plausible replacement for D-Glu. Also, the lack of activity of compound 19 indicated that the methyleneamino linker is needed between the piperidine and L-Lys moieties for the inhibitory activity of compound 15.
However, none of these active compounds had potent enough MurE inhibitory activity to be selected as potential lead compounds. Nevertheless, these results still offered insight into the future design of MurE inhibitors. Thus, if we compare the activities of compounds 8 and 15 with the phosphinate inhibitors of MurE from E. coli presented by Tanner et al. (their compounds 1 and 2),18 we can see that although our compounds have a 1,000-fold lower inhibitory activity than their compound 1, they were approximately only 2-fold less active than the truncated compound 2. As most of the reported inhibitors of MurE are most likely substrate analogues, we can conclude that design and synthesis of substrate analogues appears to be the best method to obtain inhibitors of this MurE.
3. Conclusions
We have designed and synthesised a focused library of peptidomimetic compounds as potential inhibitors of MurE from S. aureus. The compounds were designed as transition-state analogues based on the methyleneamino core, or as product analogues. Biological evaluation of these potential inhibitors identified two methyleneamino-based pseudodipeptides 8 and 15 that inhibited MurE in the mM range, and thus they represent initial hit compounds for further development.
4. Experimental
4. 1. Chemistry
Chemicals were from Sigma-Aldrich, Acros Orga-nics and Bachem, and were used without further purification. Solvents were used without purification or drying,
unless otherwise stated. Analytical TLC was performed on Merck silica gel (60F254) plates (0.25 mm), and the compounds were visualised under ultraviolet light. Column chromatography was carried out on silica gel 60 (particle size, 240-400 mesh). Melting points were determined on a Reichert hot-stage microscope and are uncorrected. 1H NMR spectra were recorded on a Bruker AVANCE DPX spectrometer at 300 MHz in CDCl3, DM-SO-d6, MeOH-d4 and D2O solution, with TMS as the internal standard. IR spectra were obtained on a Perkin-El-mer 1600 FT-IR spectrometer. Microanalyses were performed on a 240 C Perkin-Elmer C, H, N analyser. Mass spectra were obtained using a VG-Analytical Autospec Q mass spectrometer.
(R)-Benzyl 2-[(feri-Butoxycarbonyl)amino]-5-[met-hoxy(methyl)amino]-5-oxopentanoate (4)29
Boc-D-Glu-OBzl (1.67 g, 5 mmol) was dissolved in 30 mL CH2Cl2 and cooled in an ice-bath. Then BOP (2.30 g, 5.20 mmol) and Et3N (0.55 g, 5.50 mmol) were added to the solution and left to react for 10 min, followed by addition of Et3N (0.55 g, 5.50 mmol) and N,O-dimethylhy-droxylamine (0.54 g, 5.50 mmol). The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with 100 mL CH2Cl2. The organic layer was washed successively with 1 M aqueous HCl (3 x 30 mL), a saturated aqueous solution of NaHCO3 (3 x 30 mL), and brine (40 mL), and dried over Na2SO4. The solvent was evaporated under reduced pressure to give an oily residue. The product was purified by column chroma-tography using an ethyl acetate/hexane elution system. This gave title compound 4 as a colourless oil (1.62 g, 85%). [a]20D = 6.95 (c = 0.285, CHCl3). IR (nujol) v/cm-1: 3337, 2976, 1743, 1711, 1658, 1512, 1366, 1250, 1170, 999. 1H NMR (300 MHz, CDCl3) 5 1.37-1.50 (m, 9H, 3 x CH3), 1.91-2.30 (m, 2H, CH2CO), 2.42-2.56 (m, 2H, CHCH2CH2), 3.15 (s, 3H, NCH3), 3.61 (s, 3H, OCH3), 4.36 (d, J = 4.7 Hz, 1H, CH), 5.10-5.23 (m, 2H, OCH2), 5.32 (d, J = 6.8 Hz, 1H, NHCO), 7.29-7.39 (m, 5H, Ar-H). MS (ESI) m/z: 381 (M+H, 10), 281 (100). HRMS-ESI (m/z): [M+H]+ calcd for C19H29N2O6, 381.2026; found:
381.2024. Anal. Calcd for C19H28N2O6: C 59.98, H 7.42, N 7.36. Found: C 59.94, H 7.(55, IN 77.44.
(^)-Benzyl 2-[Bis(fert-butoxycarbonyl)ammo]-5-[met-hoxy(methyl)amino]-5-oxopentanoate (5)
Di-íerí-butyl dicarbonate (2.18 g, 10.0 mmol) and 4-dimethylaminopyridine (DMAP; 0.31 g, 2.50 mmol) were added to a solution of Weinreb amide 4 (1.90 g, 5.00 mmol) in acetonitrile (30 mL) and left to react at room temperature for 24 h. Di-íerí-butyl dicarbonate (1.09 g, 5.00 mmol) and DMAP (0.15 g, 1.25 mmol) were again added, and the solution was left stirring for 18 h. After completion of the reaction, the solvent was evaporated under reduced pressure and the residue dissolved in diethyl ether (150 mL). The organic phase was washed with 1 M aqueous HCl (3 x 30 mL) and brine (40 mL), and dried over Na2SO4. The solvent was evaporated and the product purified by column chromatography using an ethyl aceta-te/hexane elution system. This gave title compound 5 as a colourless oil that solidified upon standing (1.89 g, 79%), mp 55-57 °C. [a]20D = 26.7 (c = 0.220, CHCl3). IR (nujol) v/cm-1: 2979, 2361, 1735, 1697, 1667, 1455, 1367, 1245, 1144, 995. 1H NMR (300 MHz, CDCl3) 5 1.45 (s, 18H, 6 x CH3), 2.14-2.19 (m, 1H, 1H of CH2), 2.45-2.60 (m, 3H, 1H of CH2, CH2), 3.16 (s, 3H, NCH3), 3.65 (s, 3H, OCH3), 4.98-5.03 (m, 1H, CH), 5.16 (s, 2H, OCH2), 7.30-7.40 (m, 5H, Ar-H). HRMS-ESI (m/z): [M+H]+ calcd for C24H37N2O8, 481.2550; found: 481.2548. Anal. Calcd for C24H36N2O8: C 59.98, H 7.55, N 5.83. Found: C 59.99, H 7.778, IN 52.56.
(R)-2-[Bis(íerí-butoxycarbonyl)amino]-5-oxopenta-noate (6)
Compound 5 (1.44 g, 3.00 mmol) was dissolved in diethyl ether (30 mL) and LiAl(O'Bu)3H (1.52 g, 6.00 mmol) was added at room temperature. After 2 h, the solution was hydrolysed with a 5% aqueous solution of KHSO4 (20 mL).The layers were separated, and the aqueous layer was extracted with diethyl ether (4 x 20 mL). The combined organic layers were washed successively with a saturated solution of NaHCO3 (3 x 20 mL) and brine (30 mL), and dried over Na2SO4. The solution was concentrated in vacuo and the oily residue was purified by column chromatography using an ethyl acetate/hexane elu-tion system. This gave title compound 6 as a pale yellow
(S)-Benzyl 2-({(fl)-5-(Benzyloxy)-4-[bis(teri-butoxy-carbonyl)amino]-5-oxopentyl}amino)-6-benzyloxycar-bonylaminohexanoate (7)
Compound 6 (1.49 g, 3.5 mmol) was dissolved in DCE (30 mL), and HCl x H-L-Lys(Z)-OBzl (1.57 g, 3.80 mmol) and Et3N (0.38 g, 3.80 mmol) were added. Then, NaBH(OAc)3 (1.06 g, 5.00 mmol) was added, and the mixture was left to react overnight at room temperature under an Ar atmosphere. The reaction mixture was quenched by adding 10% aqueous solution of NaHCO3 (40 mL), and the product was extracted with diethyl ether (4 x 30 mL). The solution was dried over Na2SO4 and then concentrated in vacuo. The residue was purified by column chromatography using an ethyl acetate/hexa-ne elution system. This gave title compound 7 as a co-
lourless oil (1.49 g, 55%). [a]2
10.47 (c = 0.235,
CHCl3). IR (nujol) v/cm-1: 3386, 2978, 2361, 1731, 1524, 1368, 1247, 1130, 1028, 854. 1H NMR (300 MHz, CDCl3) 5 1.22-1.73 (m, 27H, 6 x CH3, 4 x CH2, NH), 1.84-2.01 (m, 1H, 1H of CH2p-Glu), 2.10-2.26 (m, 1H, 1H of CH2p-Glu), 2.35-2.52 (m, 1H, 1H of CH25-Glu), 2.55-2.70 (m, 1H, 1H of CH25-Glu), 3.13 (dd, Jj = 12.8 Hz, J2 = 6.4 Hz, 2H, CH2e-Lys), 3.24 (t, J = 6.6 Hz, 1H, CHa Lys), 4.78 (bs, 1H, NHCO2), 4.89 (dd, J1 = 9.6 Hz, J2 = 5.2 Hz, 1H, CHa-Glu), 5.06-5.25 (m, 6H, 3 x OCH2), 7.29-7.44 (m, 15H, Ar-H). 13C NMR (75.5 MHz, CDCl3) 5 22.95, 26.93, 27.13, 27.92, 33.07, 47.62, 58.07, 61.35, 66.39, 66.70, 83.00, 126.95, 127.93, 128.02, 128.06, 128.35, 128.40, 128.47, 128.57, 135.72, 135.81, 136.67, 152.26, 156.32, 170.69, 175.27. MS (ESI) m/z: 776 (M+, 57), 676 (100). Anal. Calcd for C43H57N3O10: C 66.56, H 7.40, N 5.42. Found: C 66.61, H 7.677, IN 5.38.
(S)-6-Ammo-2-((^)-4-ammo-4-carboxybutylamino)he-xanoic Acid Dihydrochloride (8)
Compound 7 (1.00 g, 1.29 mmol) was dissolved in acetic acid (20 mL) and Ar was passed through the solution. Pd/C (0.10 g) was added, and the reaction mixture was stirred under H2 until no starting material was detected by TLC. After the reaction, the Pd/C was filtered off and the solution was treated with gaseous HC-l for 0.5 h. Acetic acid was removed in vacuo and the residue was freeze-dried. This gave title compound 8 as a very hygroscopic brown foam (0.38 g, 88%). [a]20D =
oil (0.74 g, 59%). [a] D = 41.7 (c = 0.225, CHCl3). IR 0.91 (c = 0.286, H2O). IR (nujol) v/cm1: 3394, 2962,
(nujol) v/cm1: 3483, 2980, 2367, 1747, 1456, 1368, 1250, 1143, 994, 853. 1H NMR (300 MHz, CDCl3) 5 1.45 (s, 18H, 6 x CH3), 2.14-2.27 (m, 1H, 1H of CH2), 2.46-2.64 (m, 3H, 1H of CH2, CH2), 4.89-4.94 (m, 1H, CH), 5.16 (dd, J1 = 12.5 Hz, J2 = 1.7 Hz, 2H, OCH2), 7.30-7.40 (m, 5H, Ar-H), 9.77 (s, 1H, CHO). 13C NMR (75.5 MHz, CDCl3) x 5 22.16, 27.88, 40.46, 57.46, 66.87, 83.38, 127.97, 128.14, 128.43, 135.50, 152.07, 170.08, 200.83. HRMS-ESI (m/z): [M-H]- calcd for C22H31NO7, 420.2022; found: 420.2026.
2364, 1993, 1735, 1604, 1500, 1219. 1H NMR (300 MHz, MeOH-d4) 5 1.39-1.78 (m, 4H, 2 x CH2), 1.80-2.12 (m, 6H, 3 x CH2), 2.84-3.01 (m, 2H, CH2e-Lys), 3.02-3.21 (m, 2H, CH2g-Glu), 3.88-4.07 (m, 2H, 2 x CH). 13C NMR (75.5 MHz, MeOH-d4) 5 23.08, 23.45, 28.04, 28.70, 30.50, 40.32, 47.38, 53.50, 61.27, 171.24, 171.55. HRMS-ESI (m/z): [M-H]- calcd for C11H22N3O4, 260.1610; found: 260.1603. Anal. Calcd for CnH23N3O4 x 2 HCl x 3.5 H2O: C 33.25, H 8.12, N 10.58. Found: C 33.49, H 8.18, N 10.42.
(6S,9Ä,14S)-Benzyl 9-Benzyloxycarbonyl-14-(4-benzy-loxycarbonylaminobutyl)-2,2,6-trimethyl-4,7-dioxo-3-oxa-5,8,13-triazapentadecan-15-oate (9)
Compound 7 (1.49 g, 1.92 mmol) was dissolved in a mixture of CH2Cl2/CF3CO2H (9:1; 20 mL) and stirred at room temperature until disappearance of the starting material, as determined by TLC. The solvent was evaporated in vacuo. The residue was dissolved in CH2Cl2 (10 mL) and Et3N was added to pH 8. Boc-L-Ala (0.38 g, 2.00 mmol) was dissolved in CH2Cl2 (10 mL), and BOP (0.93 g, 2.10 mmol) and Et3N (0.22 g, 2.10 mmol) were added at 0 °C. The two solutions were mixed together and the reaction mixture was left to react for 2 h. After completion of the reaction, CH2Cl2 (80 mL) was added. The organic layer was washed successively with a saturated aqueous solution of NaHCO3 (3 x 20 mL) and brine (20 mL), and dried over Na2SO4. The solvent was removed in vacuo to give an oily residue. The residue was purified by column chromatography using an ethyl acetate/hexane elution system. This gave title compound 9 as a colourless oil (1.00 g, 70%). [a]20D = -16.58 (c = 0.205, CHCl3). IR (nu-jol) v/cm-1: 3347, 2937, 1718, 1522, 1456, 1366, 1250, 1167, 1026, 751. 1H NMR (300 MHz, DMSO-d6) 5
I.10-1.80	(m, 23H, 4 x CH3, 5 x CH2, NH), 2.27-2.50 (m, 2H, CH2), 2.93 (m, 2H, CH2), 3.10-3.14 (m, 1H, CH), 3.98-4.02 (m, 1H, CH), 4.22-4.29 (m, 1H, CH), 5.00 (s, 2H, OCH2), 5.12 (s, 4H, 2 x OCH2), 6.79-6.87 (m, 1H, CONH), 77.18 (t, J = 7.5 Hz, 1H, NHCO), 7.30-7.40 (m, 15H, Ar-H), 8.11 (d, J = 7.5 Hz, 1H, CONH). MS (ESI) m/z: 747 (M+H, 100). HRMS-ESI (m/z): [M+H]+ calcd for C41H55N4O9, 747.3969; found: 747.3974. Anal. Calcd for C41H54N4O9 x H2O: C 64.38, H 7.38, N 7.32. Found: C 64.02, H 7.56, N 7.(51.
(S)-6-Amino-2-{[(R)-4-((S)-2-ammopropanamido)-4-carboxybutyl]amino}hexanoic Acid Dihydrochloride (10)
Compound 9 (0.75 g, 1.00 mmol) was dissolved in acetic acid (15 mL), and Ar was passed through the solution. Pd/C (0.07 g) was added and the reaction mixture was stirred under H2 until no starting material was detected by TLC. After the reaction, the Pd/C was filtered off and the solution was treated with gaseous HCl for 0.5 h. Acetic acid was removed in vacuo, and the residue was freeze-dried. This gave title compound 10 as a very hygroscopic colourless foam (0.34 g, 84%). [a]20D =
II.23	(c = 0.285, H2O). IR (nujol) v/cm1: 3430, 2968, 1993, 1735, 1684, 1560, 1498, 1397, 1210, 1149, 1004, 854. 1H NMR (300 MHz, MeOH-d4) 5 1.42-2.15 (m, 13H, 5 x CH2, CH3), 2.98 (t, J = 7.3 Hz, 2H, CH2e-Lys), 3.07-3.20 (m, 2H, CH25-Glu), 3.96-4.17 (m, 2H, 2 x CH), 4.38-4.50 (m, 1H, CH). 13C NMR (75.5 MHz, MeOH-d4) 5 18.05, 23.03, 24.05, 27.99, 29.53, 29.95, 40.30, 47.64, 50.38, 53.33, 60.99, 170.97, 171.35, 174.27. HRMS-ESI (m/z): [M+H]+ calcd for C14H29N4O5, 333.2138; found: 333.2130. Anal. Calcd for C14H28N4O5 x 2 HCl x 2 H2O x
0.5 CH3CO2H: C 38.22, H 7.70, N 11.89. Found: C 38.25, H 8.07, N 11.79.
tert-Butyl 4-[Methoxy(methyl)carbamoyl]piperidine-1-carboxylate (12)31
1-(Boc)piperidine-4-carboxylic acid (2.29 g, 10 mmol) was dissolved in CH2Cl2 (50 mL) and cooled in an ice-bath. Then BOP (4.86 g, 11 mmol) and Et3N (1.11 g, 11 mmol) were added to the solution, and left to react for 10 min, followed by addition of Et3N (1.11 g, 11 mmol) and N,O-dimethylhydroxylamine (1.07 g, 11 mmol). The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with CH2Cl2 (150 mL). The organic phase was washed successively with 1 M aqueous HCl (3 x 40 mL), a saturated aqueous solution of NaHCO3 (3 x 40 mL) and brine (50 mL), and dried over Na2SO4. The solvent was evaporated under reduced pressure to give an oily residue. The product was purified by column chromatography using an ethyl aceta-te/hexane elution system. This gave title compound 12 as a colourless oil that solidifies on air (2.30 g, 85%), mp 51-54 °c (lit. 54 °C). IR (nujol) v/cm1: 3568, 2972, 1692, 1420, 1365, 1234, 1170, 984, 870. 1H NMR (300 MHz, CDCl3) 5 1.46 (s, 9H, 3 x CH3), 1.59-1.79 (m, 4H, 2 x CH2), 23.73-2.87 (m, 3H, CH2, CH), 3.19 (s, 3H, NCH3), 3.71 (s, 3H, OCH3), 4.29-4.00 (m, 2H, CH2). MS (ESI) m/z: 273 (M+H, 50), 217 (100). HRMS-ESI (m/z):
[M+H]+ calcd for C13H25N2O4, 273.1814; found: 273.1818. 13 25 2 4
tert-Butyl 4-Formylpiperidine-1-carboxylate (13)32
Compound 12 (1.36 g, 5.00 mmol) was dissolved in diethyl ether (30 mL) and LiAlH4 (0.23 g, 6 mmol) was added at -10 °C. After 1 h, the solution was hydrolysed with a 5% aqueous solution of KHSO4 (20 mL) and the product was extracted with diethyl ether (4 x 30 mL). The organic phase was washed successively with a saturated solution of NaHCO3 (3 x 40 mL) and brine (40 mL), and dried over Na2SO4. The solution was concentrated in vacuo and the oily residue was purified by column chroma-tography using an ethyl acetate/hexane elution system. This gave title compound 13 as a colourless oil (0.92, 86%). IR (nujol) v/cm-1: 3422, 2974, 2361, 1691, 1424, 1366, 1237, 1167, 865. 1H NMR (300 MHz, CDCl3) 5 1.45 (s, 9H, 3 x CH3), 1.47-1.62 (m, 2H, CH2), 1.80-1.97 (m, 2H, CH2), 2.35-2.43 (m, 1H, CH), 2.92 (m, 2H, CH2), 3.97 (m, 2H, CH2), 9.65 (s, 1H, CHO).
(S)-tert-Butyl 4-{[(1-Benzyloxy-6-benzyloxycarbonyla-mino-1-oxohexan-2-yl)amino]methyl}piperidine-1-carboxylate (14)
Compound 13 (0.75 g, 3.50 mmol) was dissolved in DCE (30 mL), and HCl x H-L-Lys(Z)-OBzl (1.51 g, 3.70 mmol) and Et3N (0.37 g, 3.70 mmol) were added. Then, NaBH(OAc)3 (1.00 g, 4.7 mmol) was added, and the mixture was left to react overnight at room temperature under
an Ar atmosphere. The reaction mixture was quenched by adding 1 M NaOH (30 mL) and the product was extracted with diethyl ether (3 X 40 mL). The organic layer was dried over Na^O4 and concentrated in vacuo. The residue was purified by column chromatography using an ethyl acetate/hexane elution system. This gave title compound 14 as a colourless oil (1.40 g, 71%). [a]20D = -7.36 (c = 0.315, CHCl3). IR (nujol) v/cm-1: 3337, 2931, 2365, 1726, 1691, 1533, 1425, 1365, 1247, 1170, 725. 1H NMR (300 MHz, CDCl3) 5 1.04 (m, 2H, CH2), 1.28-1.75 (m, 19H, 3 x CH3, 4 x CH2, CH, NH), 2.28 (dd, J1 = 11.4 Hz, J2 = 6.4 Hz, 1H, 1H of CH2), 2.44 (dd, J1 = 11.4 Hz, J2 = 6.9 Hz, 1H, 1H of CH2), 2.65 (m, 2H, CH2), 3.23-3.07 (m, 3H, CH2, CHa), 4.09-3.99 (m, 2H, CH2), 4.74 (s, 1H, NHCO), 5.08 (s, 2H, OCH2), 5.15 (dd, J1 = 12.3 Hz, J2 = 6.1 Hz, 2H, OCH2), 7.40-7.27 (m, 10H, Ar-H). MS (ESI) m/z: 568 (M+H, 100). Anal. Calcd for C32H45N3O6: C 67.70, H 7.99, N 7.40. Found: C 67.82, H 8.228, IN 73.55.
(S)-6-Amino-2-[(piperidin-4-ylmethyl)ammo]hexanoic Acid Dihydrochloride (15)
Compound 14 (0.62 g, 1.1 mmol) was dissolved in acetic acid, and Ar was passed through the solution. Pd/C (0.06 g) was added, and reaction mixture was stirred under H2 until no starting material was detected by TLC. After the reaction the Pd/C was filtered off and the solution was treated with gaseous HCl for 0.5 h. Acetic acid was removed in vacuo, and the residue was freeze-dried. This gave title compound 15 as a very hygroscopic foam (0.28 g, 89%). [a]20D = 6.74 (c = 0.270, H2O). IR (nujol) v/cm-1: 3400, 2963, 2043, 1735, 1618, 1458, 1397, 1211, 1005, 958. 1H NMR (300 MHz, MeOH-d4) 5 1.85-1.48 (m, 6H, 3 x CH2), 2.31-2.01 (m, 5H, 2 x CH2, CH), 3.19-2.93 (m, 6H, 3 x CH2), 3.45 (m, 2H, CH2), 4.06 (t, J = 6.12 Hz, 1H, CHa). 13C NMR (75.5 MHz, MeOH-d4) 5 23.29, 27.54, 27.58, 27.99, 30.00, 32.69, 40.29, 44.47, 44.49, 52.65, 61.61, 170.96. MS (ESI) m/z: 244 (M+H, 100). Anal. Calcd for C12H25N3O2 x 2 HCl x 3.3 H2O x 0.3 CH3CO2H: C 38.44, H 8.91, N 10.67. Found: C 38.64, H 9.143, N 10.42.
(S)-Benzyl 6-Benzyloxycarbonylammo-2-[({1-[(S)-2-(ieri-butoxycarbonylammo)propanoyl]piperidin-4-yl}methyl)amino]hexanoate (16)
Compound 14 (1.00 g, 1.76 mmol) was dissolved in a mixture of CH2Cl2/CF3CO2H (9:1; 10 mL) and stirred at room temperature until disappearance of the starting material, as determined by TLC. The solvent was evaporated in vacuo and the residue dissolved in CH2Cl2 (10 mL). Et3N was added to pH 8. Boc-L-Ala (0.34 g, 1.80 mmol) was dissolved in CH2Cl2 (10 mL) and BOC (0.84 g, 1.90 mmol) and Et3N (0.20 g, 2.00 mmol) were added at 0 °C. The two solutions were mixed together and the reaction mixture was left to react for 2 h at room temperature. After completion of the reaction, CH2Cl2 (100 mL) was added. The organic layer was washed successively with a sa-
turated aqueous solution of NaHCO3 (3 x 20 mL) and brine (40 mL), and dried over Na2SO4. The solvent was removed in vacuo, to give an oily residue that was purified by column chromatography using an ethyl acetate/hexane elution system. This gave title compound 16 as a colourless oil (0.88 g, 78%). [a]20D = -10.57 (c = 0.210, CHCl3). IR (nujol) v/cm1: 3328, 2933, 2364, 1712, 1637, 1528, 1455, 1367, 1249, 1168, 1054, 1026. 1H NMR (300 MHz, DMSO-d6) 5 0.78-1.06 (m, 2H, CH2), 1.11 (d, J = 6.2 Hz, 3H, CH3), 1.22-1.77 (m, 18H, 3 x CH3, 4 x CH2, CH), 2.16-2.41 (m, 2H, CH2NH), 2.43-2.50 (m, 1H, 1H of CH2), 2.94 (dd, J1 = 12.52 Hz, J2 = 6.4 Hz, 3H, CH2e, 1H of CH2), 3.08-3.22 (m, 1H, CH), 3.74-3.94 (m, 1H, 1H of CH2), 4.21-4.53 (m, 2H, CHa-Ala, 1H of CH2), 5.00 (s, 2H, CH2O), 5.13 (s, 2H, CH2O), 6.76-6.90 (m, 1H, CONH), 7.19 (t, J = 5.4 Hz, 1H, CONH), 7.26-7.46 (m, 10H, Ar-H). MS (ESI) m/z: 639 (M+H, 48), 180 (100). Anal. Calcd for C35H50N4O7 x 0.3 H2O: C 65.20, H 7.92, N 8.70. Found: C 65.26, H 8.31, N 8^0.
(S)-6-Amino-2-({[1-((S)-2-aminopropanoyl)piperidm-4-yl]methyl}amino)hexanoic Acid Dihydrochloride (17)
Compound 16 (0.70 g, 1.10 mmol) was dissolved in acetic acid, and Ar was passed through the solution. Pd/C (0.07 g) was added, and reaction mixture was stirred under H2 until no starting material was detected by TLC. After the reaction, the Pd/C was filtered off and the solution was treated with gaseous HCl for 0.5 h. Acetic acid was removed in vacuo, and the residue was freeze-dried. This gave title compound 17 as a very hygroscopic colourless foam (0.38 g, 89%). [a]20D = 11.23 (c = 0.285, H2O). IR (nujol) v/cm1: 3429, 2946, 1993, 1735, 1640, 1490, 1388, 1270, 1107, 1002, 729. 1H NMR (300 MHz, MeOH-d4) 5 1.14-1.83 (m, 9H, 3 x CH2, CH3), 1.86-2.24 (m, 5H, 34 x CH2, CHCH2NH ), 2.70-23.27 3(m, 6H, 2 x CH2, 2 x 1H of CH2), 3.86-3.96 (m, 1H, 1H of CH2), 3.982-4.05 (m, 1H, CHa-Lys), 4.35-4.60 (m, 2H, 1H of CH2, CHa-Ala). 13C NMR (300 MHz, MeOH-d4) 5 17.36, 23.35, 28.03, 30.06, 30.27, 31.03, 34.82, 40.31, 43.08, 45.80, 48.23, 53.19, 61.65, 169.15, 171.03. HRMS-ESI (m/z): [M+H]+ calcd for C15H31N4O3, 315.2396; found: 315.2403. Anal. Calcd for C15H30N4O3 x 2 HCl x 3 H2O: C 40.82, H 8.68, N 12.69. Found: C 40.90, H 8^ N 12.43.
(S)-feri-Butyl 4-[(1-Benzyloxy-6-benzyloxycarbonyla-mino-1-oxohexan-2-yl)carbamoyl]piperidine-1-car-boxylate (18)
1-(Boc)piperidine-4-carboxylic acid (0.92 g, 4.00 mmol) and HCl x H-L-Lys(Z)-OBzl (1.67 g, 4.1 mmol) were dissolved in dimethylformamide (DMF). Et3N (0.81 g, 8 mmol) and DPPA (1.13 g, 4.1 mmol) were added at 0 °C, and the reaction was left to react for 18 h. The solution was concentrated in vacuo, and the residue dissolved in CH2Cl2 (150 mL). The organic layer was washed successively with 10% citric acid (3 x 30 mL), a saturated solu-
tion of NaHCO3 (3 x 30 mL) and brine (40 mL), and dried over Na2SO4. The product was precipitated from ethyl acetate. This gave title compound 18 as a white solid (1.25
2368, 1677, 1537, 1418, 1365, 1168, 1121, 948. 1H NMR (300 MHz, DMSO-d6) 5 1.32-1.47 (m, 11H, 3 x CH3, CH2), 1.54-1.90 (m, 4H, 2 x CH2), 2.21-2.48 (m, 4H, 2 x
g, 54%), mp 91-94 °C. [a]20D = -3.52 (c = 0.210, CHCl3). CH2), 2.60-2.80 (m, 3H, CHO, CH2), 3.94 (d, J = 10.8
IR (nujol) v/cm-1: 3350, 2948, 1748, 1689, 1643, 1541, 1425, 1263, 1175, 952, 752. 1H NMR (300 MHz, DMSO-d6) 5 1.21-1.49 (m, 15H, 3 x CH2 3 x CH3), 1.49-1.80 (m, 4H, 2 x CH2), 2.37 (ddd, J1 = 14.3 Hz, J2 = 6.9 Hz, J3 = 2.9 Hz, 1H, CHCO), 2.72 (t, J = 9.5 Hz, 2H, CH2e), 2.96 (m, 2H, CH2), 3.90 (d, J = 11.3 Hz, 2H, CH2), 4.23 (dd, J1 = 13.4 Hz, J2 = 8.2 Hz, 1H, CHa), 5.00 (s, 2H, OCH2), 5.10 (dd, J1 = 12.6 Hz, J2 = 3.6 Hz, 2H, OCH2), 7.21 (t, J = 5.0 Hz, 1H, NHCO), 7.26-7.46 (m, 10H, Ar-H), 8.17 (d, J = 7.4 Hz, 1H, NHCO). HRMS-ESI (m/z): [M+H]+ calcd for C32H44N5O7, 582.3179; found 582.3196. Anal. Calcd for C32H43N3O7: C 66.07, H 7.45, N 7.22. Found: C 66.32, H 7.45, IN 73.22.
6-Amino-2-(piperidine-4-carboxamido)hexanoic Acid Hydrochloride (19)
Compound 18 (1.16 g, 2.00 mmol) was dissolved in acetic acid (20 mL), and Ar was passed through the solution. Pd/C (0.1 g) was added, and the reaction mixture was stirred under H2 until no starting material was detected by TLC. After the reaction, the Pd/C was filtered off and the solution was treated with gaseous HCl for 0.5 h. Acetic acid was removed in vacuo, and the residue was freeze-dried. This gave title compound 19 as a very hygroscopic colourless foam (0.53 g, 89%). [a]20D = -13.46 (c = 0.260, D2O). IR (nujol) v/cm1: 3403, 2962, 2044, 1725, 1649, 15246, 1455, 1396, 1221, 959. 1H NMR (300 MHz, D2O) 5 1.35-1.50 (m, 2H, CH2), 1.58-1.95 (m, 6H, 3 x CH2), 1.97-2.10 (m, 2H, CH2), 2.69 (tt, J1 = 11.4 Hz, J2 = 3.8 Hz, 1H, CHCO), 2.93-3.00 (m, 2H, CH2), 3.06 (dt, J1 = 12.8 Hz, J2 = 3.0 Hz, 2H, CH2), 3.46 (td, J1 = 13.0 Hz, J2 = 3.3 Hz, 2H, CH2), 4.32 (m, 1H, CHa). HRMS-ESI (m/z): [M-H]- calcd for C^H^N^ 256.16(51; found 256.1663. Anal. Calcd for C12H23N3O3 x HCl x 3 H2O: C 41.44, H 8.69, N 12.08. Found: C 41,57, H 8,78, N 22,04.
tert-Butyl 4- [(2-Amino-4,5,6,7-tetrahydrobenzo[d] thiazol-6-yl)carbamoyl]piperidine-1-carboxylate (20)
1-(Boc)piperidine-4-carboxylic acid (1.15 g, 5.00 mmol) and (S)-2,6-diamino-4,5,6,7-tetrahydrobenzothia-zole (0.93 g, 5.50 mmol)33 were dissolved in DMF (15 mL). Et3N (1.01 g, 10.00 mmol) and DPPA (1.51 g, 5.50 mmol) were added at 0 °C, and the reaction was left to react for 18 h. The solution was concentrated in vacuo, and the residue dissolved in CH2Cl2 (150 mL). The organic layer was washed successively with 10% citric acid (3 x 30 mL), a saturated solution of NaHCO3 (3 30 mL) and brine (30 mL), and dried over Na2SO4. The solvent was removed in vacuo to give an oily residue. Title compound 20 was crystallized from ethyl acetate to give a white solid (1.18 g, 62%), mp 153-155 °C. [a]20D = -27.82 (c = 0.220, MeOH). IR (nujol) v/cm-1: 3355, 3143, 2930,
Hz, 3H, CHNH, CH2), 6.63 (s, 2H, NH2), 7.86 (d, J = 7.6 Hz, 1H, NHCO). MS (ESI) m/z: 403 (M+Na), 381 (M+H, 79), 325 (100). Anal. Calcd for C18H28N4O3S x 0.5 H2O: C 55.50, H 7.50, N 14.38. Found: C 55.50, H 7^ N 14.29.
(S)-N-(2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)piperidine-4-carboxamide Dihydrochloride (21)
Compound 20 (0.51, 1.33 mmol) was dissolved in absolute EtOH (20 mL) and treated with gaseous HCl. The solvent was evaporated in vacuo, and the product triturated with diethyl ether. This gave title compound 21 as a hygroscopic white solid (0.45 g, 95%), mp 235-240 °C. [a]20D = -19.61 (c = 0.205, H2O). IR (nujol) v/cm1: 3420, 3246, 3093, 2948, 2802, 2495, 1618, 1438, 1311, 1244, 953. 1H NMR (300 MHz, D2O) 5 1.73-2.07 (m, 6H, 3 x CH2), 2.39-2.70 (m, 4H, CH2CS, CH2CN), 2.77-2.89 (m, 1H, CHCO), 2.94-3.09 (m, 2H, CH2NH), 3.40-3.50 (m, 2H, CH2NH), 4.07-4.22 (m, 1H, CHNH). HRMS-ESI (m/z): [M+H]+ calcd for C13H21N4OS, 281.1436; found: 281.1429. Anal. Calcd for C^H^OS x 2 HCl x 3.3 H2O: C 37.83, H 6.98, N 13.57. Found: C 38.00, H 6.89, N 13.21.
4-Benzyloxycarbonylpiperidin-1-ium 4-methylbenze-nesulfonate (23)
Piperidine-4-carboxylic acid (6.52 g, 50 mmol), p-TolSO3H (9.70 g, 0.051 mmol), benzyl alcohol (25 mL) and benzene (70 mL) were refluxed in a Dean-Stark apparatus overnight. The solvent was removed in vacuo, and the residue was triturated with diethyl ether. This gave title compound 23 as a white solid (15.18 g, 78%), mp 68-72 °C, which was used in the next step of the reaction without further purification. IR (nujol) v/cm-1: 3448, 3032, 2980, 1724, 1453, 1191, 1034, 1010, 817. 1H NMR (300 MHz, DMSO-d6) 5 1.64-1.81 (m, 2H, CH2CH2CH), 2.00 (dd, J1 = 14.3 Hz, J2 = 3.2 Hz, 2H, CH^^CH), 2.29 (s, 3H Ar-CH3), 2.75 (tt, J1 = 11.0 Hz, J2 = 3.8 Hz, 1H, CHCO), 2.94 (dt, J1 = 12.5 Hz, J2 = 2.9 Hz, 2H, CH2NH), 3.25 (td, J1 = 12.6 Hz, J2 = 3.5 Hz, 2H, CH2NH), 5.12 (s, 2H, ArCH2O), 7.12 (d, J = 7.9 Hz, 2H, Ar-H), 7.51 (d, J = 8.1 Hz, 2H, Ar-H), 7.43-7.29 (m, 5H, Ar-H), 8.42 (bs, 2H, NH2). HRMS-ESI (m/z): [M+H]+ calcd for C13H18NO2, 220.1338; found: 220.1333.
(S)-Benzyl 1-(2-tert-Butoxycarbonylaminopropanoyl) piperidine-4-carboxylate (24)
4-[(Benzyloxy)carbonyl]piperidin-1-ium 4-methyl-benzenesulfonate (1.96 g, 5 mmol) was partitioned between ethyl acetate and aqueous NaHCO3. The organic layer was separated, and evaporated to dryness. The residue was dissolved in DMF, and Boc-L-Ala (0.94 g, 5 mmol),
Et3N (1.01 g, 10 mmol) and DPPA (1.51 g, 5.5 mmol) were added at 0 °C. The reaction was left to react for 18 h. The solution was concentrated in vacuo, and the residue dissolved in CH2Cl2 (150 mL). The organic layer was washed successively with 10% citric acid (3 x 30 mL), a saturated solution of NaHCO3 (3 x 30 mL) and brine (40 mL), and dried over Na2SO4. The solvent was removed in vacuo, to give an oily residue. The residue was purified by column chromatography using an ethyl acetate/hexane elution system. This gave title compound 24 as a colourless oil (1.17 g, 59%). [a]20D = 10.75 (c = 0.225, CHCl3). IR (nujol) v/cm-1: 3422, 3310, 2977, 2362, 1731, 1644, 1454, 1366, 1250, 1168, 1025, 752. 1H NMR (300 MHz, DMSO-d6) 5 1.11 (d, J = 6.0 Hz, 3H, CH3), 1.36 (s, 9H, 3 x CH3), 1.38-1.60 (m, 2H, CH2), 1.86 (d, J = 12.1 Hz, 2H, CH2), 2.60-2.88 (m, 2H, CHCO, 1H of CH2), 3.00-3.20 (m, 1H, 1H of CH2), 3.74-3.96 (m, 1H, 1H of CH2), 4.09-4.33 (m, 1H, 1H of CH2), 4.36-4.48 (m, 1H, CHa), 5.11 (s, 2H, OCH2), 6.79-7.04 (m, 1H, NHCO), 7.45-7.26 (m, 5H, Ar-H). HRMS-ESI (m/z): [M+Na]+ calcd for C21H30N2O5Na, 413.2052; found 413.2051. Anal. Calcd for C21H30N2O5: C 64.59, H 7.74, N 7.17. Found: C 64.48, H 8.08, N 7.177.
(S)-1-(2-tert-Butoxycarbonylaminopropanoyl)piperi-dine-4-carboxylic acid (25)
Compound 24 (1.80 g, 4.60 mmol) was dissolved in MeOH, and Ar was passed through the solution. Pd/C (0.18 g) was added, and the reaction mixture was stirred under H2 until no starting material was detected by TLC. After the reaction, the Pd/C was filtered off and the solution was concentrated in vacuo. This gave title compound 25 as a white solid (1.27 g, 92%), mp 85-90 °C. [a]20D = -27.41 (c = 0.205, MeOH). IR (nujol) v/cm1: 3452, 17D0, 1542, 1250, 1168, 1115, 1025, 924, 861. 1H NMR (300 MHz, DMSO-d6) 5 1.10 (d, J = 6.0 Hz, 3H, CH3), 1.36 (s, 9H, 3 x CH3), 1.39-1.58 (m, 2H, CH2), 1.71-1.90 (m, 2H, CH2), 2.41-2.55 (m, 1H, CHCO), 2.73 (m, 1H, 1H of CH2), 3.08 (m, 1H, 1H of CH2), 3.82 (s, 1H, 1H of CH2), 4.00-4.31 (m, 1H, 1H of CH2), 4.33-4.48 (m, 1H, CHa), 6.74-7.07 (m, 1H, NHCO), 12.32 (bs, 1H, COOH). HRMS-ESI (m/z): [M-H]- calcd for C14H23N2O5, 299.1607; found: 299.1605. Anal. Calcd for C14H24N2O5: C 55.98, H 8.05, N 9.33. Found: C 55.83, H 8.09, N 9.32.
(S)-Benzyl 6-Benzyloxycarbonylamino-2-[1-((S)-2-tert-butoxycarbonylaminopropanoyl)piperidine-4-carbo-xamido]hexanoate (26)
Compound 25 (1.20 g, 4.00 mmol) and HCl x L-Lys(Z)OBzl (1.62 g, 4 mmol) were dissolved in DMF. Et3N (0.81 g, 8 mmol) and DPPA (1.21 g, 4.4 mmol) were added at 0 °C, and the reaction was left to react for 18 h. The solution was concentrated in vacuo, and the residue dissolved in CH2Cl2 (150 mL). The organic layer was washed successively with 10% citric acid (3 x 30 mL), a saturated solution of NaHCO3 (3 x 30 mL) and brine (30
mL), and dried over Na2SO4. The solvent was removed in vacuo, to give an oily residue, which was purified by column chromatography using a CH2Cl2/MeOH elution system. This gave title compound 26 as a colourless oil (2.08 g, 80%). [a]20D = -10.30 (c = 0.20, CHCl3). IR (nujol) v/cm1: 3315, 2933, 2362, 1702, 1638, 1534, 1456, 1366, 1251, 1169, 1024, 954. 1H NMR (300 MHz, CDCl3) 5 1.21-1.32 (m, 5H, CH3, CH2), 1.42 (s, 9H, 3 x CH3), 1.44-1.51 (m, 2H, CH2), 1.57-1.94 (m, 6H, 3 x CH2), 2.36 (tt, Jj = 10.8 Hz, J2 = 3.9 Hz, 1H, CH2C#CH2), 2.61-2.81 (m, 1H, CHCO), 2.94-3.18 (m, 3H, 1H of CH2, CH2e), 3.77-3.91 (m, 1H, 1H of CH2), 4.38-4.63 (m, 3H, 1H of CH2, 2 x CHa), 4.74-4.85 (m, 1H, NHCO), 5.07 (s, 2H, OCH2), 5.15 (m, 2H, OCH2), 5.48-5.58 (m, 1H, NHCO), 6.10-6.24 (m, 1H, NHCO),7.30-7.40 (m, 10H, Ar-H). HRMS-ESI (m/z): [M+H]+ calcd for C35H49N4O8, 653.3564; found: 653.3569. Anal. Calcd for C35H48N4O8: C 64.40, H 7.41, N 8.58. Found: C 64.16, H 7.64, NN 8.42.
(S)-6-Amino-2-[1-((S)-2-aminopropanoyl)piperidi-ne-4-carboxamido]hexanoic Acid 2,2,2-Trifluoroace-tate (27)
Compound 26 (1.49 g, 2.28 mmol) was dissolved in a mixture of CH2Cl2/CF3CO2H (9:1; 10 mL) and stirred at room temperature until disappearance of the starting material, as determined by TLC. The solvent was evaporated in vacuo, and the residue dissolved in glacial acetic acid. Ar was passed through the solution. Pd/C was added, and reaction mixture was stirred under H2 over night. After the reaction, the Pd/C was filtered off and the acetic acid was evaporated in vacuo. The wet product was then freeze-dried. This gave title compound 27 as a very hygroscopic colourless foam (0.89 g, 88%). [a]20D = 2.48 (c = 0.355, H2O). IR (nujol) v/cm1: 3430, 2948, 1668, 1538, 1452, 13292, 1201, 1134, 1024, 957. 1H NMR (300 MHz, MeOH-d4) 5 1.95-1.26 (m, 13H, 5 x CH2, CH3), 2.48-2.61 (m, 1H, CHCO), 2.66-2.81 (m, 1H, 1H of CH2), 2.85 (t, J = 7.5 Hz, 2H, CH2e), 3.13 (m, 1H, 1H of CH2), 3.82 (m, 1H, 1H of CH2), 4.47-4.20 (m, 3H, 1H of CH2, 2 x CHa). HRMS-ESI (m/z): [M+H]+ calcd for C15H29N4O4, 329.2189; found: 329.2190. Anal. Calcd for C^H^NO x 1.5 CF3CO2H x 1.5 H2O: C 41.06, H 6.22, N 10^4. Found: C 41.31, H 6.13, N 10.28.
tert-Butyl {(S)-1-[4-((S)-2-Amino-4,5,6,7-tetrahydro-benzo[d]thiazol-6-ylcarbamoyl)piperidin-1-yl]-1-oxo-propan-2-yl}carbamate (28)
Compound 25 (0.90 g, 3.00 mmol) was dissolved in CH2Cl2 (20 mL) and cooled in an ice-bath. Then BOP (1.39 g, 3.1 mmol) and Et3N (0.31 g, 3.10 mmol) were added to the solution and left to react for 10 min, followed by addition of Et3N (0.31 g, 3.10 mmol) and (S)-2,6-diamino-4,5,6,7-tetrahydrobenzothiazole (0.59 g, 3.5 mmol). The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with CH2Cl2 (100 mL). The organic phase was washed successively with 1 M
aqueous HCl (3 x 30 mL), a saturated aqueous solution of NaHCO3 (3 x 30 mL) and brine (30 mL), and dried over Na^O4. The solvent was evaporated under reduced pressure to give an oily residue. The product was purified by column chromatography using a CH2Cl2/MeOH elution system. This gave title compound 28 as a pale yellow solid (0.78 g, 58%), mp 140-143 °C. [a]20D = -31.74 (c = 0.235, MeOH). IR (nujol) v/cm-1: 3315, 2977, 2932, 1698, 1637, 1526, 1453, 1367, 1165, 1062, 948. 1H NMR (300 MHz, MeOH-d4) 5 1.28 (dd, J1 = 12.7 Hz, J2 = 7.0 Hz, 3H, CH3), 1.46 (s, 9H, 3 x CH3), 1.54-2.05 (m, 6H, 3 x CH2), 2.36-2.98 (m, 6H, 2 x CH2, CHCO, 1H of CH2), 3.26-3.06 (m, 1H, 1H of CH2), 3.97-4.26 (m, 2H, CHNH, 1H of CH2), 4.41-4.73 (m, 2H, CHa, 1H of CH2). HRMS-ESI (m/z): [M+H]+ calcd for C21H34N5O4S, 452.2332; found 452.2321. Anal. Calcd for C21H33N5O4S x 1.3 H2O: C 53.10, H 7.55, N 14.74. Found: C 53.18, H 7.78, N 14.67.
N-[(S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl]-1-[(S)-2-ammopropanoyl]piperidine-4-carboxami-de Hydrochloride (29)
Compound 28 (0.55 g, 1.22 mmol) was dissolved in anhydrous THF (20 mL) and treated with gaseous HCl. The solvent was evaporated in vacuo, and the product triturated with diethyl ether. The wet solid was freez-dried to give title compound 29 as a very hygroscopic colourless solid (0.46 g, 90%). [a]20D = -28.49 (c = 0.308, H2O). IR (nujol) v/cm-1: 3409, 2949, 1640, 1543, 1448, 1370, 1267, 1209, 1095, 948. 1H NMR (300 MHz, MeOH-d4) 5 1.49 (dd, J1 = 10.1 Hz, J2 = 7.0 Hz, 3H, CH3), 1.55-2.13 (m, 6H, 3 x CH2), 2.45-2.98 (m, 6H, 2 x CH2, CHCO, 1H of CH2), 3.17-3.30 (m, 1H, 1H of CH2), 3.86-4.03 (m, 1H, 1H of CH2), 4.15-4.26 (m, 1H, CHNH), 4.38-4.60 (m, 2H, CHa, 1H of CH2). HRMS-ESI (m/z): [M+H]+ C16H26N5O2S, 352.1807; found: 352.1823. Anal. Calcd for C16H25Ni5O2S x 1.5 HCl x 3.3 H2O: C 41.27, H 7.17, N 15.04. Found: C 41.57, H 7.49, N 14.69.
Benzyl 1-(2-feri-Butoxy-2-oxoethyl)piperidine-4-car-boxylate (30)
4-[(Benzyloxy)carbonyl]piperidin-1-ium 4-methyl-benzenesulfonate (1.96 g, 5 mmol) was partitioned between a 10% aqueous solution of NaHCO3 (50 mL) and ethyl acetate (3 x 30 mL). The combined organic layers were evaporated to dryness to give an oily residue. This was dissolved in THF and cooled in an ice bath, and Et3N (1.21 g, 12 mmol) was added. Then BrCH2CO2'Bu (1.95,
10	mmol) was added dropwise over 5 min at 0 °C. The reaction was left to proceed at room temperature overnight. Triethylammonium bromide was filtered off by suction filtration, and the filtrate was concentrated in vacuo to give an orange oily residue. The residue was purified by column chromatography using an ethyl acetate/hexane elution system. This gave title compound 30 as an orange
011	(1.23 g, 74%). IR (nujol) v/cm1: 3448, 2944, 2362, 1735, 1454, 1368, 1260, 1158, 1046, 1015, 751. 1H NMR
(300 MHz, CDCl3) 5 1.46 (s, 9H, 3 x CH3), 1.77-1.99 (m, 4H, 2 x CH2CH), 2.18-2.41 (m, 3H, CH2N, CHCO), 2.86-2.94 (m, 2H, CH2N), 3.10 (s, 2H, NCH2CO), 5.12 (s, 2H, OCH2), 7.30-7.38 (m, 5H, Ar-H). MS (ESI) m/z: 334, 278 (100). Anal. Calcd for C19H27NO4 x 0.5 H2O: C 66.64, H 8.24, N 4.09. Found: C 66.55, H 8.42, N 3.89.
1-(2-feri-Butoxy-2-oxoethyl)piperidine-4-carboxylic Acid (31)
Compound 30 (1.00 g, 3.00 mmol) was dissolved in MeOH, and Ar was passed through the solution. Pd/C (0.18 g) was added, and reaction mixture was stirred under H2 until no starting material was detected by TLC. After the reaction, the Pd/C was filtered off and the solution was concentrated in vacuo. This gave title compound 31 as a yellow solid (0.672 g, 92%), mp 148-152 °C. IR (nujol) v/cm-1: 3447, 2976, 1718, 1458, 1373, 1282, 1157, 942, 847. 1H NMR (300 MHz, CDCl3) 5 1.45 (s, 9H, 3 x CH3), 1.74-2.05 (m, 4H, 2 x CH2CH), 2.30 (tt, J1 = 10.8 Hz, J2 = 4.1 Hz, 1H, CHCO), 2.36-2.48 (m, 2H, CH2N), 2.98-3.10 (m, 2H, CH2N), 3.23 (s, 2H, NCH2CO), 10.73 (bs, 1H, CO2H). MS (ESI) m/z: 266 (M+Na, 4), 244 (M+H, 21), 188 (100). Anal. Calcd for C12H21NO4: C 59.24, H 8.70, N 5.76. Found: C 59.34, H 8.95, 1ST 5.74.
(S)-Benzyl 6-Benzyloxycarbonylamino-2-[1-(2-ieri-butoxy-2-oxoethyl)piperidine-4-carboxamido]hexa-noate (32)
Compound 31 (0.66 g, 2.72 mmol) and HCl x L-Lys(Z)-OBzl (1.11 g, 2.72 mmol) were dissolved in DMF. Et3N (0.61 g, 6 mmol) and DPPA (0.82 g, 3.00 mmol) were added at 0 °C, and the reaction was left to react for 18 h. The solution was concentrated in vacuo, and the residue dissolved in CH2Cl2 (150 mL). The organic layer was washed successively with 10% citric acid (3 x 30 mL), a saturated solution of NaHCO3 (3 x 30 mL) and brine (40 mL), and dried over Na2SO4. The solvent was removed in vacuo, and the product was purified by column chromatography using a CH2Cl2/MeOH elution system. This gave title compound 32 as a white solid (1.19 g, 74%), mp 70-74 °C. [a]20D = -6.87 (c = 0.230, CHCl3). IR (nujol) v/cm1: 3344, 2941, 1747, 1691, 1642, 1542, 1264, 1151, 956. 1H NMR (300 MHz, CDCl3) 5 1.12-1.92 (m, 19H, 3 x CH3, 5 x CH2), 2.06-2.30 (m, 3H, CH2N, CHCO), 2.89-2.99 (m, 2H, CH2N), 3.07-3.16 (m, 4H, NCH2CO, CH2e), 4.63 (dt, J1 = 7.7 Hz, J2 = 5.1 Hz, 1H, CHa), 4.80 (s, 1H, NHCO), 5.03-5.25 (m, 4H, 2 x OCH2), 6.10 (d, J = 7.6 Hz, 1H, NHCO), 7.27-7.44 (m, 10H, Ar-H). HRMS-ESI (m/z): [M+H]+ C33H46N3O7, 596.3336; found: 596.3333. Anal. Calcd for C33H45N3O7: C 66.53, H 7.61, N 7.05. Found: C 66.31, H 7.83, N 7.05.
(S)-6-Amino-2-[1-(carboxymethyl)piperidine-4-carbo-xamido]hexanoic Acid Hydrochloride (33)
Compound 32 (1.00 g, 1.68 mmol) was dissolved in acetic acid, and Ar was passed through the solution. Pd/C
was added, and the reaction mixture was stirred under H2 until no starting material was detected by TLC. After the reaction, the Pd/C was filtered off and the solution was treated with gaseous HCl for 0.5 h. Acetic acid was removed in vacuo, and the residue was freeze-dried. This gave the title compound 33 as a very hygroscopic colourless foam (0.55 g, 95%). [a]20D = -8.16 (c = 0.245, H2O). IR (nujol) v/cm1: 3422, 2954, 1994, 1736, 1654, 1543, 1401, 1232, 1160, 953. 1H NMR (300 MHz, MeOH-d4) 5 0.79-0.96 (m, 2H, CH2y), 1.01-1.33 (m, 4H, CH2R, CH25),
2ß>
1.36-1.62 (m, 4H, CH2CH), 2.07-2.22 (m, 1H, CHCO), 2.27-2.39 (m, 2H, CH2e), 2.57-2.79 (m, 2H, CH2NH), 2.97-3.17 (m, 2H, CH2NH), 3.48 (s, 2H, CH2CO2H), 3.63-3.72 (m, 1H, CHa). 13C NMR (75.5 MHz, MeOH-d4) 5 23.80, 27.75, 27.78, 31.45, 40.35, 40.43, 53.49, 168.02, 175.03, 175.79. HRMS-ESI (m/z): [M+H]+ C14H26N3O5, 316.1872; found: 316.1868. Anal. Calcd for CmH25N3O5 x HCl x 2.9 H2O: C 41.61, H 7.93, N 10.40. Found: C 41.88, H 7.93, N 10.40.
4. 2. Biochemical Evaluation of the Compounds
The compounds were tested for inhibition of addition of L-[14C]Lys to UMAG in a mixture (final volume, 50 pl) containing 0.1 M Tris-HCl, pH 8.6, 15 mM MgCl2, 5 mM ATP, 0.1 mM UMA, 198.4 pM L-Lys, 1.6 pM L-[14C]Lys (50,000 cpm), 30 pM Tween-20, 5% (v/v) DMSO, purified MurE from S. aureus (diluted in 20 mM potassium phosphate (pH 7.2), 1 mM DTT), and the test compound (compounds were soluble in the enzyme assay mixture containing 5% DMSO at all of the concentrations used). The samples were incubated for 30 min at 37 °C, and the reaction was stopped by addition of 10 pL glacial acetic acid. The mixture was lyophilised and dissolved in water. This solution was then analysed by TLC. The plates were run in a mobile phase of «-propanol:NH3:water (6:3:1) for 8 h. The radioactive substrate and product were detected and quantified with a radioactivity monitor (Berthold France, Thoiry, France). The residual activity for each inhibitor concentration was calculated with respect to a similar assay without inhibitor, and the IC50 values were calculated from the fitted regression equation using the logit-log plot.
5. 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. Support from the Ministry of Higher Education, Science and Technology of the Republic of Slovenia and the Slovenian Research Agency are also acknowledged.
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Povzetek
Zaradi pojavljanja in širjenja bakterijskih sevov, ki so hkrati odporni na več vrst protimikrobnih učinkovin, obstaja velika potreba po razvoju novih zdravilnih učinkovin. Eden izmed pomembnih virov novih tarč za razvoj protimikrobnih učinkovin je biosinteza bakterijskega peptidoglikana. Biosintezo peptidne verige v peptidoglikanu katalizirajo encimi ligaze Mur (C, D, E in F), ki so nujni za bakterijsko preživetje. Načrtovali in sintetizirali smo serijo spojin kot potencialnih inhibitorjev UDP-N-acetilmuramoil-L-alanil-D-glutamat:L-lizin ligaze (MurE) iz Staphylococcus aureus. Pri tem smo uporabili dva pristopa: (i) sintezo metilenaminskih derivatov kot mimetikov prehodnega stanja in (ii) sintezo pro-duktnih ananlogov reakcije MurE. Dve spojini z metilenaminskim ogrodjem sta primerni za nadaljni razvoj inhibitorjev MurE.