Organometallic complexes with biological molecules. XI. Solid state and in vivo investigations of some diorganotin(IV)-chloramphenicol and cycloserine derivatives

Journal of Inorganic Biochemistry 72 (1998) 115±125

Organometallic complexes with biological molecules. XI. Solid state and in vivo investigations of some diorganotin(IV)-chloramphenicol and cycloserine derivatives A. Pellerito a, T. Fiore a, C. Pellerito a, A. Fontana a, R. Di Stefano a, L. Pellerito M.T. Cambria b, C. Mansueto c

a,*

,

a

b

Dipartimento di Chimica Inorganica, Universit a di Palermo, 26 Via Archira®, 90123 Palermo, Italy Istituto di Scienze Biochimiche e Farmacologiche, Universita di Catania, 6 Viale A. Doria, 95125 Catania, Italy c Dipartimento di Biologia Animale, Universit a di Palermo, 18 Via Archira®, 90123 Palermo, Italy Received 26 May 1998; received in revised form 17 August 1998; accepted 24 August 1998

Abstract Diorganotin(IV) derivatives of chloramphenicol, { ˆ D-(-)threo-2,2-dichloro-N-[b-hydroxy-a-(hydroxymethyl)-b-(4-nitrophenyl)ethyl]acetamide ( ˆ Hchloramph)}, and D-cycloserine, { ˆ (R)-4-amino-3-isoxazolidone [ ˆ Hcyclos]} have been prepared. The stoichiometries of the obtained compounds were R2 SnClantib and R2 Snantib2 (antibÿ1 ˆ chloramphÿ1 , R ˆ methyl and phenyl; antibÿ1 ˆ cyclosÿ1 , R ˆ methyl). The solid state con®guration of the complexes was investigated by I.R. and M ossbauer spectroscopy, from which structural hypotheses were inferred. In particular, the experimental data suggested monomer structures both for R2 Sn(IV)Clchloramph and R2 Sn(IV)chloramph2 , in which chloramphenicolate anion behaved as monoanionic monodentate ligand through the oxygen atom of the deprotonated secondary alcoholic group, with formation of tetrahedral R2 SnOCl and R2 SnO2 environments. In R2 Sn(IV)Clcyclos and R2 Sn(IV)cyclos2 derivatives, M ossbauer spectroscopy, and in particular the narrowness of the full width at half height of the resonant peaks, C1 and C2 , suggested the occurrence of two di€erent absorbing tin sites with di€erent environments around the tin(IV) atoms. According to calculations performed by applying the point charge model formalism, one site was constituted by a tin(IV) tetrahedrically coordinated by monoanionic monodentate cycloserinate groups, through the oxygen atom of the resonance stabilised hydroxamate anion, originating R2 SnClO and R2 SnO2 polyhedrons both in R2 Sn(IV)Clcyclos and R2 Sn(IV)cyclos2 , respectively. The second site would correspond to a tin(IV) in a polymeric octahedral con®guration with Me2 SnCl2 ON and Me2 SnO2 N2 environments, in Me2 Sn(IV)Clcyclos and Me2 Sn(IV)cyclos2 derivatives, respectively, in which the second donor atoms was the amino nitrogen atom. 1 H and 13 C NMR spectra, of both chloramphenicol and its diorganotin(IV) derivatives were carried in DMSO-d6 solution, in which R2 Sn(IV)Clchloramph and R2 Sn(IV)chloramph2 underwent total, (R ˆ Me), or partial, (R ˆ Ph), dissociation. As far as the organotin(IV)-D-cycloserine derivatives were concerned, 1 H and 13 C NMR spectra, also carried out for the free D-cycloserine, showed that, owing to the coordinating properties of the solvent, octahedral and trigonal bipyramidal isomers were present in DMSO solution of Me2 Sn(IV)Clcyclos and Me2 Sn(IV)cyclos2 . Finally, the cytotoxic activity of the free chloramphenicol, D-cycloserine and of their dimethyltin(IV) derivatives has been investigated towards Ciona intestinalis and Ascidia malaca fertilised eggs, at di€erent developing stages. Ó 1998 Elsevier Science Inc. All rights reserved. Keywords: Organotin; Antibiotics; Chloramphenicol; D-cycloserine; Infrared; M ossbauer; NMR; Ascidian eggs; Development

1. Introduction Diorgano and triorganotin(IV) antibiotic derivatives (antibiotic ˆ amoxicillin, ampicillin, methicillin and penicillin G; R ˆ Me, n Bu, Ph), because of their consid*

Corresponding author. E-mail: [email protected].

erable biological activity in vivo, were the aim of previously reported research [1±5]. On the basis of I.R. and M ossbauer spectroscopy, carried out on the obtained complexes, trigonal bipyramidal con®guration was proposed for R2 SnClantibiotic, cis-R2 Sn, and R3 SnClantibioticNa, eq-R3 Sn, derivatives, while distorted R2 Sn skew trapezoidal environments have been hypothesised for R2 Snantibiotic2 compounds

0162-0134/98/$ ± see front matter Ó 1998 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 2 - 0 1 3 4 ( 9 8 ) 1 0 0 6 5 - X

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A. Pellerito et al. / J. Inorg. Biochem. 72 (1998) 115±125

(antibiotic ˆ amoxicillin and ampicillin; R ˆ Me, n Bu, Ph) [1,3,4]. Furthermore, equilibrium study on the mixed-ligand complex formation of M2‡ ions (M2‡ ˆ Co2‡ , Ni2‡ , Cu2‡ and Zn2‡ ) with a-d(-)aminobenzylpenicillin ( ˆ ampicillin) and nucleic bases, in aqueous solution at 37°C and ®xed ionic strength, was recently reported [6]. Following our interest in the organotin(IV) antibiotic interactions, in this paper we report the interaction of diorganotin(IV) moieties with chloramphenicol and D-cycloserine. The interest in the D-cycloserine-organotin(IV) derivatives is correlated to the well-known observation that metallic complexes of D-cycloserine are more active than the free ligand [7]. Furthermore, owing to the existence, in polar solvents, of dipolar ions according to previously reported equilibria [8], the cyclic and the exocyclic nitrogen and oxygen atoms of the D-cycloserine may compete with each other on co-ordinating Lewis acids [8±12]. Finally, to the best of our knowledge, the diorganotin(IV) chloramphenicol compounds, here reported, represent the ®rst example of interaction of chloramphenicol with metallic centres.

On cooling, the complexes precipitated and the solids, recovered by ®ltration, were recrystallized from methanol or methanol/ether solutions and analysed for C, H, N, Sn and Cl content (Table 1). C, H and N analyses were performed at Laboratorio di Chimica Organica (University of Padova). Sn and Cl contents were determined in our laboratory according to standard procedures [13,14]. I.R. spectra were registered, as Nujol and Hexachlorobutadiene mulls, on a Perkin±Elmer grating spectrometer mod.983G, between CsI windows. The spectra were analysed through a Perkin±Elmer 3600 data station with Perkin±Elmer PE983 software (Table 2 (a) and (b)). The M ossbauer spectra were recorded with the apparatus elsewhere described [1,3,4]. The calculated M ossbauer parameters are reported in Table 3. 1 H and 13 C NMR spectra of all the organotin(IV) complexes were registered with an AC 250E Bruker instrument, operating, at 250 and 63 MHz, respectively, using tetramethylsilane (TMS) as an internal standard, DMSO-d6 as solvents (Table 4(a)±(d)).

2. Experimental

2.2. Biological materials and methods

2.1. Chemical materials and methods

Eggs of Ciona intestinalis and Ascidia malaca, which belong to the phylum of Chordata living along the coast of Palermo, were used for the in vivo cytotoxicity studies. Female and male gametes were removed from their gonaducts. Four batches of eggs were fertilised, in antibiotics and their derivative solutions, or incubated 30 min after fertilisation or at the 2-cell stage up to controls reared to the larval stage. For each experiment, eggs of the same treated batch were fertilised in Millipore ®ltered sea water and allowed to develop up to the larva stage as controls. The used concentrations of chloramphenicol and cycloserine, and of their organotin(IV) derivatives were 10ÿ4 and 10ÿ5 mol dmÿ3 in Millipore ®ltered sea water solutions (Fig. 1).

R2 SnClantib and R2 Snantib2 (antibÿ ˆ chloramphÿ , R ˆ Me, Ph; antibÿ ˆ cyclosÿ , R ˆ Me) were prepared according to the following procedures. 1. R2 SnClantib complexes were obtained as white solids by re¯uxing methanolic solutions of R2 SnCl2 (gift from Witco GMBH, Bergkamen) with methanolic suspensions of the sodium salt of the antibiotics, chloramphenicol and D-cycloserine (Fluka, Bucks, Switzerland), in the molar ratio 1:1; 2. R2 Snantib2 were synthesised by re¯uxing R2 SnO, freshly prepared by hydrolysing the parent diorganotin(IV)dichloride, and methanolic suspension of the antibiotics, (molar ratio 1 : 2), until clear solutions were obtained. Table 1 Analytical data (Found (calcd) (%)) for R2 SnCln antib…2-n† derivatives

a

Compound

C

H

N

Sn

Cl

Me2 SnClchloramph

30.15 (30.84) 43.69 (43.82) 36.04 (36.35) 44.95 (44.53) 20.72 (21.05) 26.81 (27.38)

3.18 (3.38) 3.25 (3.36) 3.67 (3.56) 3.64 (3.52) 3.69 (3.89) 4.50 (4.60)

5.16 (5.53) 4.69 (4.44) 6.89 (7.06) 6.38 (6.11) 9.95 (9.82) 15.56 (15.97)

23.97 (23.44) 18.44 (18.82) 14.42 (14.97) 12.67 (12.94) 42.1 (41.6) 33.65 (33.82)

21.83 (21.01) 16.81 (16.87) 17.71 (17.88) 15.27 (15.46) 12.86 (12.43)

Ph2 SnClchloramph Me2 Snchloramph2 Ph2 Snchloramph2 Me2 SnClcyclos Me2 Sncyclos2 a

Antibÿ ˆ chloramphÿ ˆ chloramphenicolateÿ ; n ˆ 0, 1, R ˆ Me, Ph; antibÿ ˆ cyclosÿ ˆ D-cycloserinateÿ , n ˆ 0, 1, R ˆ Me.

b

a

3305 s 3118 s 3273 m,bd

m(NH2 )

3469 s 3450 s

3475 s 3462 s

m(OH)

s s s s s

3300-2100 s,bd

m(NH‡ 3)

3342 3347 3372 3345 3340

mas (NH)

s s s s s

1667 s

1665 s

1630s

mas (NH)

3258 3257 3257 3257 3257

ms (NH)

s s s s s

1600-1500 s,bd

m(C ˆ O)

1688 1687 1687 1687 1687

m(C@O)

s s s s s

1589 s

1587 s

m(C ˆ N)

1563 1564 1563 1563 1562

m(C±NO2 )

s s s s s

582 s

580 s

mas (SnC2 )

1518 1519 1524 1518 1518

d(NH)

s s s s s

525 m

540 w

ms (SnC2 )

1349 1348 1347 1349 1348

m(C±NO2 )

420 w

454 m

m(SnO)

1065 vs

d(OH)

579 m

578 m

mas (SnC2 )

510 w

511 w

ms (SnC2 )

464 m,bd

464 m,bd

m(SnO)

450 m

450 m

Whi€en mode

Hchloramph ˆ chloramphenicol; chloramphÿ ˆ chloramphenicolateÿ ; i.r. spectra taken as Nujol and Hexachlorobutadiene mulls; s ˆ strong, m ˆ medium, w ˆ weak, bd ˆ broad. Hcyclos ˆ D-cycloserine; cyclosÿ ˆ D-cycloserinateÿ ; i.r. spectra taken as Nujol and hexachlorobutadiene mulls; s ˆ strong, m ˆ medium, w ˆ weak, bd ˆ broad.

Me2 Sncyclos2

Me2 SnClcyclos

Hcyclos

Part b

Hchloramph Me2 SnClchloramph Ph2 SnClchloramph Me2 Snchloramph2 Ph2 Snchloramph2

Part a

Compounds

Table 2 Part a: Assignment of more relevant absorption bands of diorganotin(IV)-chloramphenicol derivatives in the 4000±250 cmÿ1 region a ; part b: Assignment of more relevant absorption bands of dimethyltin(IV)-D-cycloserine derivatives in the 4000±250 cmÿ1 region b

A. Pellerito et al. / J. Inorg. Biochem. 72 (1998) 115±125 117

3.27

3.38

2.51 2.15 2.10 1.67

jDexpj (mm sÿ1 )

c

1.67

1.38

0.89 0.83 0.91 0.81

C1

(mm sÿ1 )

c

1.69

1.51

0.92 0.85 0.91 0.85

C2

(mm sÿ1 )

d

2.44 3.65

jDj

1.06 1.16

(mm sÿ1 )

2.61 3.93

d

1.15 1.23

d

d

0.75 0.76

0.76 0.74

(mm sÿ1 ) C1

d

0.75 0.75

0.76 0.74

C2

112 148

113 108 97 101 117 160

C±Sn±C angle ‹13°

ÿ2.78 ÿ2.53 ÿ2.24 ÿ1.99 ÿ2.78 4.00 4.14 ÿ2.24 3.86 3.90

Dcalcd

e

3(a) 3(a) 3(b) 3(b) 3(a) 3(c) 3(d) 3(b) 3(e) 3(f)

Figure

Experimental M ossbauer parameters measured at liquid nitrogen temperature; isomer shift, d ‹ 0.02 mm sÿ1 , with respect to R.T. Ca119 SnO3 ; nuclear quadrupole splitting, jDexp j ‹ 0.03 mm sÿ1 ; b Chloramphÿ ˆ chloramphenicolateÿ ; cyclosÿ ˆ D-cycloserinateÿ . c C1 and C2 values are the full width at half height of the resonant peaks, respectively at greater and lower velocity with respect to the centroid of the M ossbauer spectra; d Experimental spectra ®tted as two doublets. e Partial quadrupole splittings, p.q.s., mm sÿ1 , used for D calculations, according to the point charge model formalism applied to the idealized structures of Fig. 3(a)±(f), were Refs. [18,21,22]: 1. tetrahedral structures: ([Me]±[Cl]) ˆ ÿ1.37; ([Ph]±[Cl]) ˆ ÿ1.26; ([C±O]±[Cl])chloramph ˆ ([C±O]±[Cl])cyclos ˆ ([OH]±[Cl]) ˆ ÿ0.40; 2. octahedral structures: ([Me]±[Cl]) ˆ ÿ1.03; ([C±O]±[Cl])cyclos ˆ ([OH]±[Cl]) ˆ ÿ0.14; ([NH2 ]±[Cl])cyclos ˆ 0.011.

1.16

Me2 Sncyclos2

a

1.17

Me2 SnClcyclos

d (mm sÿ1 )

1.03 0.98 0.93 0.62

b

Me2 SnClchloramph Ph2 SnClchloramph M2 Snchloramph2 Ph2 Snchloramph2

Compound

Table 3 Experimental M ossbauer parameters a , Isomer shift, d,mm sÿ1 , and Nuclear Quadrupole Splittings, jDexpj, mm sÿ1 , measured at liquid N2 temperature and calculated Nuclear Quadrupole Splittings, Dcalcd , according to the point charge formalism applied to the idealized structures of Fig. 3(a)±(f)

118 A. Pellerito et al. / J. Inorg. Biochem. 72 (1998) 115±125

A. Pellerito et al. / J. Inorg. Biochem. 72 (1998) 115±125

119

Table 4 Part a: 1 H NMR of Hchloramph, R2 Sn(IV)Clchloramph and R2 Sn(IV)chloramph2 , at 298 K (B0 ˆ 5.87 or 9.40 T). Chemical shifts are in ppm from TMS; multiplicity in parentheses (m ˆ multiplet; d ˆ doublet; bd ˆ broad); solvent: DMSO-d6 ; part b: 13 C NMR of Hchloramph, R2 Sn(IV)Clchloramph and R2 Sn(IV)chloramph2 , at 298 K (B0 ˆ 5.87 or 9.40 T). Chemical shifts are in ppm from TMS; solvent: DMSO-d6 ; part c: 1 H NMR of Hcyclos, Me2 Sn(IV)Clcyclos and Me2 Sn(IV)Clcyclos, at 298 K (B0 ˆ 5.87 or 9.40 T). Chemical shifts are in ppm from TMS; multiplicity in parentheses (m ˆ multiplet; bd ˆ broad); solvent: DMSO-d6 ; part d: 13 C NMR of Hcyclos, Me2 Sn(IV)Clcyclos and Me2 Sn(IV)cyclos2 at 298 K (B0 ˆ 5.87 or 9.40 T). Chemical shifts are in ppm from TMS; solvent: DMSO-d6 a Assignment

Hchloramph

Me2 SnClchloramph

Ph2 SnClchloramph

Me2 Snchloramph2

Ph2 Snchloramph2

Part a NH C…30 † H, C…50 † H C(20 † H, C…60 † H C…5† H C…1† OH C…3† OH C…1† H C…2† H C…3† H2 SnR

8.40(d) 8.20(d), 7.68(d) 6.53 6.12(d) 5.14(bd) 5.07 4.00(m) 3.65(m), 3.44(m)

8.50(d) 8.20(d), 7.68(d) 6.56 6.15(d) 5.15(bd) 5.11 3.98(m) 3.65(m), 3.39(m)

8.52(d) 8.20(d), 7.68(d) 6.56 6.15(d) 5.14(bd) 5.14 3.98(m) 3.64(m), 3.45(m) 8.08±7.91 7.41±7.35

8.41(d) 8.20(d), 7.67(d) 6.53 6.12(d) 5.13(bd) 5.05 3.98m 3.65(m), 3.42(m)

8.40(d) 8.21(d), 7.68(d) 6.54 6.14(d) 5.14(bd) 5.08 4.00(m) 3.67(m), 3.45(m) 7.89 7.51±7.42

Part b C…4† @O C…40 † C…10 † C…20 ;60 † C…30 ;50 † C…5† C…1† C…2† C…3† SnR

163.74 151.55 146.72 127.61 123.19 69.31 66.75 60.60 57.12

163.58 151.50 146.58 127.55 123.07 69.16 66.66 60.38 57.02

Assignment

Hcyclos

Me2 SnClcyclos

Me2 Sncyclos2

4.49(m) 3.80(m) 4.71(bd)

4.58(m), 4.08(m) 3.80(m), 3.30(m)

4.66(m), 4.02(m) 3.77(m), 3.30(m)

8.18, 6.55 0.64, 0.49

8.18, 6.18 0.66, 0.48

Part c C…4† H C…5† H2 NH‡ 3 NH2 SnR 2 119 J( Sn1 H) CSnC angle Assignment

Hcyclos

Me2 SnClcyclos

Part d C…3† @O C…5† C…4† SnR

174.68 75.29 55.63

170.79 75.13 53.77 11.29

a

163.58 151.50 146.57 127.54 123.06 69.16 66.65 60.38 57.01 137.07±136.29 129.50±127.73

98.57 159.7 Me2 SnClcyclos 170.79 72.79 52.58 11.15

163.62 151.48 146.65 127.54 123.12 69.21 66.66 60.49 57.03

Me2 Sncyclos2 170.69 76.56 57.30 9.43

163.59 151.46 146.61 127.51 123.08 69.19 66.64 60.46 57.01 136.96±135.44 129.48±127.98

83.00 134.7 Me2 Sncyclos2 165.54 72.62 53.94 9.43

Hchloramph ˆ chloramphenicol; chloramphÿ ˆ chloramphenicolateÿ ; R ˆ Me, Ph, Hcyclos ˆ D-cycloserine; cyclos ˆ D-cycloserinate.

The pH of the antibiotics and of their organotin(IV) derivative solutions ranged within 7.76±7.78 (as for sea water).

In vivo observations were made with a Leitz microscope and photographs were taken with a Leitz orthoplan microscope, using an Ilford FP4 Plus ®lm.

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A. Pellerito et al. / J. Inorg. Biochem. 72 (1998) 115±125

Me2 SnClchloramph and Me2 Snchloramph2 derivatives, respectively. The m(SnO) stretchings, for the above mentioned diorganotin(IV) derivatives, were present at 464 cmÿ1 , as medium absorptions. Finally, in the spectra of the Ph2 Sn derivatives, a strong band at 450 cmÿ1 , characteristic of the so-called y Sn±Ph mode in the Whi€en notation, was present [16]. From the above reported considerations, it might be concluded that, in the investigated diorganotin(IV)chloramphenicol derivatives, chloramphenicolate behaved as monoanionic oxygen donor ligand through the deprotonated oxygen donor atom of the secondary alcoholic group.

Fig. 1. View of the ligands (a) Chloramphenicol, (b) D-cycloserine with the numbering scheme referred to NMR assignments.

3. Results and discussion 3.1. Solid state investigations 3.1.1. I.R. spectra 3.1.1.1. Diorganotin(IV)-chloramphenicol derivatives. The I.R. spectrum of chloramphenicol (Table 2(a)) showed, in the range 4000 ÿ 2000 cmÿ1 , bands attributable to m(OH) (3475 cmÿ1 ), mas (NH) and ms (NH) of the acetoamide group (3342 and 3258 cmÿ1 ), respectively [15]. Furthermore, at 1688 and 1518 cmÿ1 , bands due to the amide I m(C@O) and amide II m(NH) absorptions were present [15]; the bands at 1563 and 1349 cmÿ1 were characteristic of the nitro group. Finally, a band at 1065 cmÿ1 was attributed to the m(OH) of the secondary alcoholic group [15]. Coordination of the chloramphenicolate (chloramphÿ ) to both R2 SnCl‡ and R2 Sn2‡ moieties, in R2 SnClchloramph and R2 Snchloramph2 derivatives (R ˆ Me, Ph), respectively, was inferred on the basis of the comparison of the above reported bands, referred to the free ligand, with those of the diorganotin(IV) chloramphenicol derivatives. The most relevant di€erences, in the diorganotin(IV) derivatives, were: (i) the disappearance of the band at 1065 cmÿ1 , in the free chloramphenicol, involving deprotonation of the secondary alcoholic OH group and (ii) the appearance, in the 600±200 cmÿ1 range, of bands attributable to the organometallic moieties and, in Me2 SnClchloramph and R2 Snchloramph2 , to the formed Sn±O bonds as a consequence of the coordination of the ligand to the organotin(IV) moieties. In particular, the bands due to mas (SnC2 ) and ms (SnC2 ) of the Me2 Sn moiety, were found at 578 and 511 cmÿ1 , 579 and 510 cmÿ1 , in

3.1.1.2. Dimethyltin(IV)-cycloserine derivatives. The assignment of more relevant I.R. peaks for the D-cycloserine (Hcyclos) is reported in Table 2(b), together with those of the Me2 SnClcyclos and Me2 Sncyclos2 derivatives. In the free D-cycloserine, the formation of a zwitterion and of a resonance stabilised hydroxamate anion was evident from the broad NH stretch absorption, due to the NH‡ 3 , extended by combination bands, ranging from 3200 up to 2100 cmÿ1 . Furthermore, a strong band at 1630 cmÿ1 , attributable to mas (NH) was present, and ®nally the 1600±1500 broad band was due to the absorption of the resonance stabilised anion [8±12,17]. The I.R. spectra of diorganotin(IV) cycloserine derivatives, as expected, were similar in the occurrence of stretchings attributable to: (i) NH2 (3305, 3118 and 1665 cmÿ1 in Me2 SnClcyclos; 3273 and 1667 cmÿ1 in Me2 Sncyclos2 ); (ii) C@N (1587cmÿ1 in Me2 SnClcyclos and at 1589 cmÿ1 in Me2 Sncyclos2 ); (iii) mas (SnC2 ) and ms (SnC2 ) (580 and 540 cmÿ1 in Me2 SnClcyclos; 582 and 525 cmÿ1 in Me2 Sncyclos2 ) ; (iv) SnO (454 cmÿ1 and 420 cmÿ1 , in Me2 SnClcyclos and Me2 Sncyclos2 , respectively) and in the disappearance of the NH‡ 3 characteristic absorptions of the free D-cycloserine. These ®ndings, all together, would point towards a coordination of the D-cycloserine as a monoanionic bidentate ligand through the oxygen atom of the resonance stabilised hydroxamate anion and the amino nitrogen atom. Coordination of the amino group, in a ring closed con®guration [10], could not be excluded, a priori, mainly on the basis of the I.R. spectra of the previous reports on copper(II)-D-cycloserine complexes [10], according to which coordination of the amino group was suggested by the presence of a mas (NH) at ~1610 cmÿ1 . In fact, both in Me2 SnClcyclos and Me2 Sncyclos2 , mas (NH) was present, even if at higher wavenumbers (1665 and 1667 cmÿ1 , respectively), than in the free cycloserine and in the copper(II)-D-cycloserine complexes [10]. The above mentioned I.R. ®ndings, together with the stoichiometries of the synthesised derivatives would suggest that in the diorganotin(IV) chloramphenicol

A. Pellerito et al. / J. Inorg. Biochem. 72 (1998) 115±125

compounds, the tin(IV) atom would attain, at least, tetra coordination, while in the dimethyltin(IV)-D-cycloserine complexes, the tin(IV) atom could be coordinated in several di€erent ways. 3.1.2. M ossbauer spectra The M ossbauer parameters, isomer shifts, d (mm sÿ1 ), and nuclear quadrupole resonance, D (mm sÿ1 ), both for R2 SnClchloramph and R2 Snchloramph2 derivatives (R ˆ Me, Ph) and Me2 SnClcyclos and Me2 Sncyclos2 were characteristic of diorganotin(IV) derivatives [18,19]. The isomer shift, d, of all the derivatives increased with the total 5s electronic density at the nucleus of the absorbing tin atoms, according to the electronegativity of organic groups and donor atoms of the ligands bonded to the tin(IV). The narrowness of the experimental C1 and C2 values, the full width at half height of the resonant peaks, respectively at greater and lower velocity with respect to the centroid of the M ossbauer spectra, reported in Table 3, implied for R2 SnClchloramph and R2 Snchloramph2 the occurrence of a single tin site in each compound, while multiple sites, with di€erent environments, might be hypothesised in Me2 SnClcyclos and Me2 Sncyclos2 . The M ossbauer spectra of Me2 SnClcyclos and Me2 Sncyclos2 were re®ned by imposing a two doublets spectrum. Fig. 2 reports the experimental M ossbauer spectrum of Me2 SnClcyclos (white circles), together with the 2 subspectra (a) and (b), (dashed lines) corresponding to the two tin sites with octahedral or

121

tetrahedral environment, respectively. The solid line (c) is a least squares computer ®t to the experimental spectrum. The results of such re®nements are reported in Table 3, together with calculated nuclear quadrupole splittings, Dcalcd (mm sÿ1 ), obtained from the rationalisation of the experimental nuclear quadrupole splittings, |Dexp |, according to the point charge model formalism applied to the idealised structures of Fig. 3(a)±(f) and the C±Sn±C angles calculated according to Sham [20]. The partial quadrupole splittings, (p.q.s., mm sÿ1 ; Table 3), employed in the calculations were literature or calculated from literature reported values [18,21,22]. Calculated D for R2 SnClchloramph and R2 Snchloramph2 , according to the tetrahedral structures of Fig. 3(a) and (b), did not exceed ‹0.4 mm sÿ1 with respect to experimental nuclear quadrupole splittings, |Dexp |, strongly supporting the I.R. ®ndings, according to which both in R2 SnClchloramph and R2 Snchloramph2 , tin atoms were four coordinated, in tetrahedral environments [23]. On the other hand, as far as Me2 SnClcyclos and Me2 Sncyclos2 complexes were concerned, the two D values for each complex, calculated from the re®nement of the experimental spectra, allowed us to distinguish two di€erent environments around the tin atoms. Once again the calculated D, according to the point charge formalism applied to the idealised structures of Fig. 3(a)±(f), i.e. tetrahedral and octahedral arrangements around the two di€erent absorbing tin(IV) sites, did not di€er more than ‹0.4 mm sÿ1 , the higher dif-

Fig. 2. M ossbauer spectrum of Me2 SnClcyclos at liquid N2 temperature. The experimental points are reported as white circles. The dashed (a) and (b) subspectra correspond to the two tin sites with octahedral or tetrahedral environment, respectively. The parameters of the subspectra are reported in Table 3. The solid line (c) is a least squares computer ®t to the experimental spectrum.

122

A. Pellerito et al. / J. Inorg. Biochem. 72 (1998) 115±125

ference between experimental and calculated D allowed to accept the proposed structures [23]. It must be pointed out that, at this stage of the calculations, it is not possible to distinguish between the structures reported in Fig. 3(c) and (d) for the octahedral site of Me2 SnClcyclos, and between those of Fig. 3(e) and (f) for the octahedral sites of Me2 Sncyclos2 , both structures being within the above allowed di€erence value of ‹0.4 mm sÿ1 . Finally, from the ratios of the area under the resonant peaks of the subspectra (a) and (b), as those

reported in Fig. 2 for Me2 SnClcyclos, it has been calculated that both Me2 SnClcyclos and Me2 Sncyclos2 derivatives, octahedral and tetrahedral isomers are present in equal percentage (50%). 3.2. Solution studies of organotin(IV)-derivatives 3.2.1. NMR spectra 1 H and 13 C NMR spectra for the diorganotin(IV)chloramphenicol, the D-cycloserine derivatives and for the free ligands are reported in Table 4(a)±(d), in DMSO-d6 and at 298 K, while, owing to the solubility of the compounds (lower than 10ÿ3 mol dmÿ3 ), it was not possible to perform 1 H and 13 C NMR spectra in D2 O. 3.2.2. 1 H and 13 C NMR spectra of chloramphenicol and its R2 Sn(IV), R ˆ Me, Ph, complexes The 1 H-NMR spectral parameters of the chloramphenicol and its dimethyl and diphenyltin(IV) derivatives are reported in Table 4(a). The comparison of the integrated intensities of the spectra of the free chloramphenicol with those of its complexes, clearly indicates that, at least in DMSO solution, both the dimethyl and diphenyltin(IV) chloramphenicol derivatives are, completely or partially, dissociated into free chloramphenicol and diorganotin(IV) moieties, respectively. In fact, 1 H-NMR signals attributable to the hydrogen atoms of organic radicals were not present in both the dimethyltin(IV)-chloramphenicol derivatives, while they were present in both the diphenyltin(IV)-chloramphenicol derivatives, in their characteristic ppm ranges, with integrated intensities lower than those expected for 1:1 and 1:2 stoichiometries in Ph2 SnClchloramph and Ph2 Snchloramph2 , respectively (Table 4(a)). The above reported ®ndings should be due to complete dissociation both in the case of Me2 SnClchloramph and Me2 Snchloramph2 and to a partial dissociation in the case of Ph2 SnClchloramph and Ph2 Snchloramph2 (~65% in Ph2 SnClchloramph and ~50% in Ph2 Snchloramph2 derivatives). The 13 C NMR parameters are shown in Table 4(b). The lack of signals attributable to the CH3 carbons of the dimethyltin moiety, and the presence, in the 13 C NMR spectra of the diphenyltin(IV) chloramphenicol derivatives, also of signals attributable to the carbon atoms of the organotin(IV) moieties, besides those of the chloramphenicol, would con®rm the previously reported considerations.

Fig. 3. Regular structures of tin assumed to estimate the nuclear quadrupole splittings according to the point charge model formalism (Table 3) for R2 SnClantib and R2 Snantib2 derivatives. The partial quadrupole splittings, p.q.s., mm sÿ1 , used in the calculations are reported in Table 3.

3.2.3. 1 H and 13 C NMR spectra of D-cycloserine and its Me2 Sn(IV) derivatives The 1 H and 13 C NMR spectra of D-cycloserine and of its dimethyltin(IV) derivatives were rather simple, and their spectral parameters are reported in Table 4(c) and (d), respectively. In the 1 H NMR spectrum of the Dcycloserine only three signals were present, and their attribution is summarised in Table 4(c). Table 4 (c) and (d) clearly show that, upon coordination, the D-cycloserine signals in 1 H and 13 C NMR

A. Pellerito et al. / J. Inorg. Biochem. 72 (1998) 115±125

spectra were doubled as a consequence of the contemporary presence of two structural isomers, as already evidenced in the solid state by M ossbauer spectroscopy. In the 1 H NMR spectra, two coupling constant [2 J(119 Sn,1 H)] values, in the range for ®ve coordinated and six coordinated diorganotin(IV) complexes, were found, which, by applying Lockhart equation [24] 2

119

h ˆ 0:0161j J…

2

2

Sn;1 H†j ÿ 1:32j J…

119

Sn;1 H†j ‡ 133:4;

123

allowed to calculate C±Sn±C angles, Table 5(c). The obtained angle value, C±Sn±C ˆ 135° for the ®ve coordinated species, would suggest the presence of a distorted cis-Me2 Sn trigonal bipyramidal moiety which originated from the coordination of the solvent to the tetrahedral species present in the solid state, while the CSnC angle obtained as 160° for the six coordinated species would suggest a rather distorted trans-Me2 Sn octahedral structure.

Table 5 Part a: Results of development of fertilised eggs incubated in sea water solutions of chloramphenicol and D-cycloserine a ; part b: Results of development of eggs and embryos of Ciona intestinalis and Ascidia malaca or incubated 30 min after fertilisation or at the 2-cell stage in sea water solutions of Me2 Snchloramph2 and Me2 Sncyclos2 derivatives; part c: Results of development of eggs and embryos of Ciona intestinalis incubated for 1 hour in sea water solutions of Me2 Snchloramph2 and Me2 Sncyclos2 derivatives and subsequently in normal sea water Species

Compound

Concentration (mol dmÿ3 )

30' after fertilisation

Incubation at 2-cell stage

Part a Ciona intestinalis Ascidia malaca Ciona intestinalis

10ÿ4 chloramphenicol, pH ˆ 7.78

Ciona intestinalis

10ÿ4

Ascidia malaca

Compound

70(90) 80(80) 80(90) 80(80)

95(95)

75(90)

D-cycloserine, pH ˆ 7.73

80(80)

10ÿ5

Ascidia malaca b

90(95)

10ÿ5

Ascidia malaca

Ciona intestinalis

80(80)

ÿ3

Concentration (mol dm )

80(90)

Development stage after fertilisation

a;c

0'

30 min

2-cell stage

Unsegmented eggs 90(90) Anomalous larvae 90(90) Unsegmented eggs 100(90) anomalous larvae 90(90)

Unsegmentated eggs 90(100)

Blocked embryos with fused blastomeres 100(90)

Unsegmented eggs 100(90)

Blocked embryos with fused blastomeres 100(90)

30 min

2-cell stage

Late gastrula

Larvae 90(90)

Larvae 90(90) Larvae 90(90) Block with anomalous disposition of blastomeres

Part b Me2 Snchloramph2

10ÿ4 10ÿ5

Me2 Sncyclos2

10ÿ4 10ÿ5

Part c Me2 Snchloramph2 Me2 Sncyclos2 a b c

10ÿ4 10ÿ4

In parentheses are reported the percentage of control larvae. chloramphÿ ˆ chloramphenicolateÿ ; cyclosÿ ˆ D-cycloserinateÿ ; In parentheses are reported the percentage of developed control larvae.

124

A. Pellerito et al. / J. Inorg. Biochem. 72 (1998) 115±125

3.3. Biological results 3.3.1. Experiments with antibiotics The results reported in Table 5 (a) show that the fertilised eggs of Ciona intestinalis and Ascidia malaca, incubated in 10ÿ4 and 10ÿ5 mol dmÿ3 sea water solutions of both chloramphenicol and D-cycloserine, developed up to normal swimming larvae (Fig. 4(a) and (b)). They constituted a trunk containing a nervous system with two sensorial black spots, and a tail with chordal and muscle cells. 3.3.2. Experiments with organotin(IV) derivatives of chloramphenicol and D-cycloserine The eggs fertilised in the 10ÿ4 mol dmÿ3 solutions of organotin(IV) chloramphenicol and D-cycloserine derivatives, or incubated 30 min after fertilisation, did not

cleave into the blastomeres. If they were incubated at the 2-cell stage, in the 10ÿ4 mol dmÿ3 solutions of organotin(IV) chloramphenicol and D-cycloserine derivatives, development stopped and the cytoplasm of two blastomeres was fragmented into several parts that often could refuse (Fig. 4(c), Table 5(b)). The fertilised eggs incubated in 10ÿ5 mol dmÿ3 solutions of organotin(IV) chloramphenicol and D-cycloserine derivatives developed into anomalous larvae (Fig. 4(d), Table 5(b)). They were inside the membrane with an open neural plate lacking sensorial organs and a very short tail. Eggs and embryos incubated for 1 h in 10ÿ4 mol dmÿ3 Me2 Snchloramph2 and then transferred into normal sea water originated larvae as the controls, but slightly delayed; those incubated in Me2 Sncyclos2 blocked at the 2-cell stage with an anomalous disposition of blastomeres (Fig. 4(e), Table 5(c)).

Fig. 4. (a) Ciona intestinalis larvae of fertilised eggs treated with 10ÿ5 mol dmÿ3 chloramphenicol solutions (magni®cation ´ 56). (b) Ciona intestinalis 2-cell stage incubated in 10ÿ4 mol dmÿ3 Me2 Snchloramph2 solution.The blastomeres stop to divide and in some case they refuse (magni®cation ´ 56). (c) Ascidia malaca larvae of fertilised eggs incubated in 10ÿ5 mol dmÿ3 cycloserine solution (magni®cation ´ 56). (d) Ascidia malaca anomalous larvae obtained by eggs fertilised in 10ÿ5 mol dmÿ3 Me2 Sncyclos2 . The anomalous larvae are inside the ovular envelopes (magni®cation ´ 56). (e) Ciona instestinalis 2-cell stage incubated for 1 h in 10ÿ4 mol dmÿ3 Me2 Sncyclos2 and afterwards transferred into normal sea water. The eggs block to develop and the blastomeres are arranged following an anomalous spatial pattern (magni®cation ´ 56).

A. Pellerito et al. / J. Inorg. Biochem. 72 (1998) 115±125

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