Hydrazinolysis study of phthalimido-and phthalisoimido-penicillin amide derivatives

New phthalimido-penicillin amide (6-PhtPABn) and phthalisoimido-penicillin amide (6-isoPhtPABn) were prepared and the susceptibility of their different carbonyl functions toward hydrazine derivatives investigated in order to establish the feasibility of dephthaloylation within penicillin class compounds. Hydrazinolysis of phthalimido-penicillin amide (6-PhtPABn) resulted in dephthaloylation and β -lactam ring opening into thiazolidine derivatives, while in the case of phthalisoimido-penicillin amide (6-isoPhtPABn), the desired amino-penicillin amide (6-APABn) was isolated in low yield.


Introduction
β-Lactam antibiotics have been successfully used in the treatment of infectious diseases for many years. 1 Despite the large number of β-lactams that have already been synthesized and tested, there is still a need for new compounds of this kind, 2 due to the increasing resistance of bacterial strains to certain types of anti-infectives. 3t has been recognized that phthalimido-containing β-lactams are more stable to a wide variety of reaction conditions than the corresponding amido derivatives, making feasible many chemical reactions that were otherwise impracticable. 4 However, the obvious advantages of the phthaloyl protective group have been overshadowed by the lack of a selective removal method in the case of cephalosporins and penicillins.On these substrates, the general Ing-Manske method 5 for dephthaloylation with hydrazine derivatives can lead to possible β-lactam ring opening.
Several examples of successful dephthaloylation with hydrazine derivatives have been reported on monocyclic β-lactams, 6 but only a few examples on bicyclic derivatives like cephalosporins, 7 carbacephems 8 and isocephems 9 as well as tricyclic β-lactams. 10It has been found that in the case of penicillin-esters, 11 phthalimido group had to be converted to phthalisoimido group prior to hydrazinolysis in order to avoid β-lactam ring opening.
As a part of our work 12 on preparation of new β-lactam derivatives with potential biological activity, new phthalimido-penicillin amide (6-PhtPABn) and phthalisoimido-penicillin amide (6-isoPhtPABn) were prepared and feasibility of their dephthaloylation with hydrazines studied.

Results and Discussion
Synthesis of phthalimido-penicillin amide 1 (6-PhtPABn) was carried out by amidation of previously prepared 6-phthalimidopenicillanic acid (6-PhtPA) 13 with benzylamine (91% yield, Scheme 1).Amidation was carried out by a method commonly applied in organic synthesis and used in penicillin chemistry 14 .6-PhtPABn (1) was transformed to phthalisoimido-penicillin amide 3 (6-isoPhtPABn) via phthalic acid 2 by a modified procedure described on related phthalimido-penicillin esters. 11The first step in the synthesis is the hydrolysis of compound 1.The reaction is carried out by slow addition of aqueous sodium sulfide to THF solution of compound 1, followed by neutralization and evaporation of THF.It is important to maintain low reaction temperature and to adjust the pH of the solution prior to evaporation in order to minimize the β-lactam ring opening.Pure compound 2 can be obtained as a precipitate (46%) by acidifying water/acetonitrile mixture under the pH control.

Scheme 1
One of the by-products that remained in mother liquor was isolated (3%) and its structure assigned to thiazolidine compound 4 (Scheme 2), confirming β-lactam ring opening.

Scheme 2
The target compound 3 was prepared by treatment of compound 2 in dry dioxan with the excess of trifluoroacetic acid anhydride (TFAA) and isolated by precipitation with water in very good yield (95%).The compound 3 has a tendency to convert back into compound 2 and therefore it should be prepared right before the next step of hydrazinolysis.
Structures of new compounds 1-4 were determined by spectroscopic data. 1 H NMR of compound 1 showed the retained Z-configuration of β-lactam ring: C 5 -H and C 6 -H gave corresponding doublets at 5.57 and 5.73 ppm, respectively, with coupling constant of 4.0 Hz.The CONH and its neighboring CH 2 group can be found as doublets at 8.78 and 4.33 ppm, respectively, with coupling constant of 5.9 Hz.Signals of phenyl and phthalimido aromatic hydrogens appeared as multiplets at 7.26-7.36and 7.89-7.94ppm, respectively.The ring opening of phthalimido moiety in structure 2 was evident from the splitting pattern of C 6 -H β-lactam proton.It is additionally coupled with the new CONH proton and appears as a doublet of doublets at 5.55 ppm.New signals of CONH at 9.27 and COOH at 12.94 ppm disappear after a D 2 O shake. 1 H NMR of compound 3 had no signals that could be attributed to COOH or CONH groups of phthalamic acid 2, in accordance with the closed structure of phthalisoimide ring.The β-lactam protons appeared as two doublets with coupling constant corresponding to Zconfiguration.The thiazolidine compound 4, formed as a byproduct during the preparation of compound 2, showed no signals characteristic for β-lactam hydrogens in 1 H NMR. Protonated thiazolidine NH was found at 3.35 ppm as a broad singlet.Geminal protons of new methylene group appeared as two doublet of doublets at 3.47 and 3.75 ppm with the geminal coupling constant of 13.9 Hz, each split with the neighboring chiral center with different coupling constants of 5.3 and 9.8 Hz.
The obtained phthalisoimido-penicillin amide 3 (6-isoPhtPABn) was further subjected to hydrazinolysis with methyl hydrazine (Scheme 3), according to the procedure described for penicillin esters. 11The hydrazinolysis requires very low temperature and even when it was carried out at 0 °C no penicillin compound 5 (6-APABn) could be isolated due to β-lactam ring opening.When the reaction was carried out at -75 °C in DCM, instead of THF as described in the literature, the hydrazide compound 6 precipitated after the reaction mixture has reached the RT, thus avoiding the evaporation step and chloroform work up.From the mother liquor the free amino-penicillin amide 5 (6-APABn), presumably formed via 7a and/or 7b intermediate, was isolated by acid-base extractions in 12% yield. 1 H and 13 C NMR of compound 5 showed no signals that could be attributed to phthaloyl moiety, confirming successful dephthaloylation and formation of new NH 2 group, found as a broad singlet at 2.16 ppm.When the hydrazinolysis was carried out under the same conditions but using hydrazine instead of methyl hydrazine, no product 5 was isolated.Monitoring the reaction by HPLC/MS corresponding intermediate hydrazino-adduct 8 was found (Scheme 3).We assume that hydrazine and methylhydrazine nitrogens are all basic enough for the first nucleophilic attack on ester-carbonyl group and formation of intermediates 7a, 7b and 8.However, the second intramolecular nucleophilic attack on amido-carbonyl group and the separation of phthalhydrazide in the case of intermediate 8 did not proceed under the same conditions.Based on this, it might be that in the reaction with methylhydrazine only intermediate 7a closes to hydrazide 6 giving the free amino-compound 5 that could contribute to the low yield of compound 5.The other side-reaction, adding further to the low yield of compound 5 is the competitive nucleophilic attack of hydrazine on β-lactam-carbonyl resulting in β-lactam ring opening and formation of thiazolidine derivatives.
As the overall yield on compound 5 obtained by dephthaloylation of 6-PhtPABn (1) via 6-isoPhtPABn (3) was very low (5%), the possibility to find the reaction conditions for direct dephthaloylation of 1 was explored.In the reaction of 6-PhtPABn (1), regardless of the reaction conditions and hydrazine derivative used, no compound 5 was found.Monitoring the reaction by HPLC/MS one-hydrazino-adducts 8 and 9 and two-hydrazino-adduct 10 were detected immediately after the first addition of hydrazine (Scheme 4).Intermediate 8, presumably also formed from 3 (Scheme 3), could be explained by initial nucleophilic attack on phthalimido moiety of compound 1.Compound 9 could be formed if the attack is on β-lactam ring.Both, 8 and 9, could undergo an additional hydrazine attack to give compound 10.The pure compound 10 was isolated (24%) from the reaction of 1 in toluene with the excess of hydrazine.On heating in alcoholic solution, the obtained compound 10 was found to transform by dephthaloylation (a) into compound 11 (36%) and by phthalimido ring closure (b) into compound 9 (45%) (Scheme 4).
The structures of obtained new thiazolidine derivatives 9, 10 and 11 were determined by spectroscopic data.The structure of one-hydrazino-adduct 9 was based on new NH signal pattern in 1 H NMR that could only be attributed to thiazolidine amine, as opposed to one-hydrazinoadduct 8. Namely, thiazolidine NH of compound 9 appears as a characteristic doublet of doublets at 3.84 ppm split by a neighboring thiazolidine C 2 -H and C 4 -H groups with coupling constants of J=8.1 and 13.3 Hz, respectively. 1H and 13 C NMR of compound 10 showed that both phthalimido and β-lactam rings were opened: new thiazolidine NH appeared as a corresponding doublet of doublets at 3.91 ppm while CONH from phthalimido ring opening appeared as a doublet at 8.38 ppm.Hydrazide NH groups were found as singlets at 9.21 and 9.59 ppm.All of the described NH signals disappear after a D 2 O shake. 13C NMR of compound 10 also showed four different carbonyl functions. 1H and 13 C NMR spectra of compound 11 were in accordance with open β-lactam ring and showed no signals corresponding to phthaloyl moiety, suggesting successful dephthaloylation and formation of free NH 2 group, found at 2.00 ppm as a broad singlet.

Conclusions
The dephthaloylation of phthalimido-penicillin amide 1 resulted in β-lactam ring opening and formation of new thiazolidine compounds.The nucleophilic attack of hydrazine on phthalimido moiety and β-lactam ring in penicillin-amide system is a competitive process.The aptitude to βlactam ring opening is driven by the formation of stable thiazolidine compounds.
By transforming phthalimido into more reactive phthalisoimido group it was possible to partially preserve β-lactam ring in compound 3 from hydrazinolysis and to isolate the desired amino-penicillin amide 5 in low yield.