Synthesis and structure determination of diastereomeric carbapenems in the Ad N E-reaction of (±)-4,4-dimethyl-3-mercaptodihydrofuran-2(3 H )-one with chiral carbapenem enol phosphate

Incorporation of a γ -lactone ring into the structure of carbapenems is considered as a potential “trap” for metallo-β -lactamases that destructively act on carbapenems. New carbapenems containing a (±)-4,4-dimethyl-3-mercaptodihydrofuran-2(3 H )-one fragment at C 3 have been synthesized. On chromatographing a mixture of the diastereomeric carbapenems on a column with silica gel deactivated with NEt 3 , a change of the ratio of isomers occurs. The remaining enantiomerically enriched (3 S )-4,4-dimethyl-3-mercaptodihydrofuran-2(3 H )-one with 20. ] [ D  -34 o was also isolated. The determining factor in the structural assignment of the diastereomeric carbapenems was the presence, in both diastereomers, of NOE interactions between the C 3ʹ -H proton of the lactone with the C 4 -H and C 4 -Me protons of the bicyclic carbapenems


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-34 o was also isolated.The determining factor in the structural assignment of the diastereomeric carbapenems was the presence, in both diastereomers, of NOE interactions between the C 3ʹ -H proton of the lactone with the C 4 -H and C 4 -Me protons of the bicyclic carbapenems.

Introduction
5][6] Though carbapenems are resistant to the majority of lactamases (i.e.][9] For this reason, the synthesis and introduction of new types of carbapenems with more stable structures are relevant in medicine in the fight against infectious diseases.In this work, we report the synthesis of a new carbapenem 1 by coupling of the well-known carbapenem 2 10 with thiol 3 which was obtained from pantolactone for the first time.During the reaction, a noticeable kinetic optical resolution of thiol 3 is also observed.

Results and Discussion
Thiol 3 was prepared from (-)-pantolactone 4 (Scheme 2) by sequential mesylation, SN2-substitution of mesylate 5 with potassium thioacetate and hydrolysis of thioacetate 6 by treatment with LiOH in THF-H2O.Unfortunately, the resulting thiol 3 was racemic.2][13] We also observed the formation of disulfide 7 in a number of experiments.Next, the racemic thiol 3 was used in the substitution reaction with enol phosphate 2 under the reported conditions. 10As expected, the formation of two main diastereomers 1a and 1b was observed, where diastereomer 1a, which is more polar on SiO2, was predominant (~2:1, by 1 H NMR). Rapid purification of the reaction mixture by flash chromatography allows one to isolate this mixture in a total yield of 90%.Surprisingly, the remaining thiol 3 was enantiomerically enriched ).The product of kinetic control 1a predominates in the 1a+1b mixture.This means that 3(R)-3 reacted almost completely, while 3(S)-3 reacted partially, so the residual thiol 3 has an S-configuration.Individual diastereomers 1a and 1b were isolated by repeated column chromatography of the 1a+1b mixture (SiO2, eluent: CHCl3:MeOH, 150:1).However, during the separation of the diastereomers by column chromatography on silica gel deactivated with NEt3, we observed a significant change in the ratio of diastereomers with predomination of the less polar diastereomer 1b.The yield of 1a+1b is 78%.The ratio of diastereomeric carbapenems changes from 1a:1b = 2:1 to 1a:1b ~ 1:3.3 (by 1 H NMR). Assessment of the material balance in the chromatography indicates that 1a is consumed in two directions.The base-catalyzed isomerization 1a→1b and adverse destruction of thermodynamically less stable 1a (see below) occur in parallel.Similar chromatography of individual 1a gives a mixture of 1a + 1b with a total yield of 45% and a ratio of 1:5.The chemical shifts of all protons and carbon atoms for the individual diastereomers 1a and 1b were assigned based on the 13 C NMR spectra recorded with proton decoupling and in the DEPT-90 and DEPT-135 modes, as well as two-dimensional correlation spectra { 1 H, 13 C} HSQC, { 1 H, 13 C} HMBC, { 1 H, 1 H} COSY and { 1 H, 1 H} NOESY.The structure of the bicyclic part known to be 4R, 5S, 6S is confirmed by the constants of spin-spin coupling between the protons at the C 4 , C 5 and C 6 carbon atoms.The connection of the five-membered lactone through the S atom to the bicycle gives two diastereomers differing in configuration at C 3ʹ (R or S).Thus, the proton at C 3ʹ for the less polar isomer 1b resonates at δH 3.56 ppm as a singlet, and according to the HSQC spectrum, its carbon atom corresponds to the signal at δC 51.20 ppm.The chemical shifts of the remaining carbon atoms in the lactone part were determined from the cross-peaks in the HMBC spectrum: δC(C 2ʹ ) = 174.01,δC(C 4ʹ ) = 40.44,δC(C 5ʹ ) = 77.74ppm.
For the more polar isomer 1a, the signals of the proton and carbon at C 3ʹ -H position are observed at δH 3.65 ppm and δC 54.26 ppm, correspondingly.
Furthermore, the structures of diastereomers 1a and 1b were accurately assigned by NOESY spectra, where the cross peaks of the C 3ʹ -H coupling at δH 3.65 and δC 3.56 ppm with the protons at C 4 and with the protons of the methyl group at C 4 were observed (Figure 1 Thus, the diastereomer with an R configuration at the C 3ʹ center is the more polar isomer 1a and the less polar diastereomer 1b has the S configuration at the C 3ʹ center with an anti C 4ʹ Me2 position relative to the methyl group at the C 4 bicycle.The steric hindrance that arises in this case results in downfield shift of the signals of one of the methyl groups at C 4ʹ (δC 26.13 ppm).Considering that in the reaction of (±)-3 with 2, the predominant formation of 1a is observed at first, the S-configuration of the chiral center should be assigned to the residual thiol (Scheme 2).
In the next stage, in order to improve the yields and optical purity of thiol 3, the sequence 4 → 8 → 6 was used to synthesize thiol 3 from pantolactone 4 under milder conditions via trifluoromethanesulfonate derivative 8 14 followed by hydrolysis (LiOH-THF-H2O).The thiol 3 obtained in this case had a The assignment of 1a and 1b was confirmed by quantum chemical calculations.For the stationary points corresponding to the NOESY interactions found, we performed optimization of the geometric parameters, vibrational frequency calculation, calculation of the total energy and geometric corrections in the B3LYP / 6-311 + G (d,p) approximation for standard conditions (298.15 K, 1 atm) in the Gaussian program. 15Estimation of the relative thermodynamic stability of the isomers, ΔG 298 , showed that diastereomer 1b is the most favorable with ΔG 298 rel = 2.7 kJ/mol.The structure of 1b was confirmed by X-ray diffraction data (Figure 6).
Due to the significant degradation during the isolation and purification, unstable acids 10a,b after hydrogenolysis of 1a,b were converted to methyl esters 11a, 11b by treatment with MeI in MeCN-DIPEA.Unlike the acids 10a,b, the latter were stable under the conditions of chromatographic purification on SiO2 and were characterized by spectral methods.
We believe that methyl esters 11a,b are more lipophilic prodrugs than 10a,b, since the methyl esters are rapidly hydrolyzed enzymatically in vivo to the corresponding acids.The enantiomeric purity of the thiol 3 obtained was determined by 1 H NMR with a chiral shift reagent (-)-Eu(hfc)3 (Figures 7, 8).Accordingly, sequential addition of (-)-Eu(hfc)3 to a solution of (±)-3 causes a shift and doubling of the SH signal in the 1 H NMR spectra.In the case of S-3, doublet signals of both enantiomers are observed in the 1 H NMR spectra upon addition of the europium complex (Figure 8), which indicates the high enantiomeric purity of the thiol (-)-3 obtained (ee=89%).
The fact that a mixture of 1a and 1b with predominance of 1a (1a:1b = 2:1) along with enantiomerically enriched residual thiol (-)-3 are isolated quickly by flash chromatography in the reaction of (±)-3 with chiral 2 indicates that the substitution occurs under conditions of considerable kinetic control.
However, during the chromatography of mixture 1a, 1b on NEt3-deactivated silica gel, the equilibrium in the 1a+1b mixture shifts towards the thermodynamically more stable 1b.The reason for this lies in the steric hindrance in structures 1a,b.As a result, predominantly the thermodynamically favorable product is formed (product development control).There is no need to start from (-)-pantolactone in the synthesis of 1a,b.The same result can be obtained using the cheap (±)-pantolactone.

Conclusions
New carbapenems 1, 11 containing a (±)-4,4-dimethyl-3-mercaptodihydrofuran-2(3H)-one fragment at C 3 have been synthesized.In the NOESY spectra, the characteristic NOE interactions between the C 3 ʹ-H proton of the lactone with C 4 -Me and C 4 -H protons in the bicyclic part of both diastereomers was the determining factor in the structural assignments of 1a and 1b.The main structural differences in the diastereomers are caused by the syn-or anti-orientation of the gem-dimethyl groups in the lactone part with respect to the C 4 -Me of the bicycle.This assignment of diastereomers was subsequently confirmed by X-ray single-crystal diffraction analysis of 1b.
Hydrogenolysis of the PNB-protecting groups of 1a, 1b gave the corresponding unstable acids, which were converted to methyl esters 11a, 11b without isolation.
A potentially important aspect of the application 1, 11 is as follows: as it is well known, pantolactone 4 undergoes ring opening under mild conditions under the action of N-nucleophiles and anionic O-nucleophiles.In addition, the compounds 1, 11 obtained are of interest as carbapenems sterically screened at C 3 , which increases their chemical stability.In the case of 1, 11 the lactone ring is a potential moiety that reacts with Nnucleophiles (serine β-lactamases) 16 and O-nucleophiles (metallo-β-lactamases). 17 Thus, incorporation of a lactone moiety into carbapenems simultaneously allows one to suppress the degrading action of lactamases.
Also, the tandem transformations with kinetic resolution of thiol 3 and the thermodynamically controlled selection in a mixture of 1a + 1b are of a significant synthetic interest.

Experimental Section
General.IR spectra were recorded on a Shimadzu IR Prestige-21 spectrophotometer in films or Nujol mulls.NMR spectra were recorded on a Bruker Avance-III 500MHz spectrometer [operating frequencies 500.13 ( 1 H) and 125.77 ( 13 C) MHz], in CDCl3, in Acetone-d6 for internal reference (δH 7.27, 2.05, δC 77.00, 29.84 ppm).The elemental compositions were determined with a Euro EA3000 CHNS analyzer.Atmospheric pressure chemical ionization (APCI) mass spectra were obtained on an HPLC LCMS-2010EV mass-spectrometer (Shimadzu) (direct syringe sample inlet; sample solution in acetonitrile; acetonitrile/water (95:5) mobile phase) in positive and negative ion mode at ionizing electrode potentials of 4.5 kV and −3.5 kV, respectively.The mobile phase flow rate was 0.1 mL min −1 .The temperature of the interface for APCI was 250 ºC, the heater and desolvation line temperatures were 200 and 230 ºC, respectively.The nebulizer gas (nitrogen) flow rate was 2.5 L min −1 .The progress of the reactions was monitored by TLC on Sorbfil plates; the spots were detected by treatment with a 10% solution of 4-methoxybenzaldehyde in ethanol acidified with sulfuric acid.Optical rotations were determined on a Perkin-Elmer 341 M polarimeter.To determinate enantiomeric purity of thiol 3, a 0.05% solution of (-)-Eu(hfc)3 in CDCl3 was sequentially added (in 0.02 mL portions) with a syringe to a solution of thiol 3 in CDCl3.An NMR spectrum was recorded after each addition.This work was performed using the equipment of the "Khimiya" Center for Collective Use of the Ufa Institute of Chemistry UFCR RAS.X-Ray diffraction data were obtained using the equipment in the "Agidel" Regional Center for Collective Use at the UFCR RAS.
On chromatographing a mixture of 1a+1b on a column with silica gel deactivated with NEt3, the ratio of 1a and 1b changes from ~2:1 to ~1:3.3, but the total yield decreases (78%) due to partial decomposition of the less stable 1a and its epimerization to 1b.

Figure 6 .Scheme 4 .
Figure 6.The molecular structure of 1b with atom labelling.The atoms are drawn as thermal ellipsoids at the 50% probability level.
D  AUTHOR(S)