ISS.\ 11367.8377

Synthesis of a potent antagonist of substance P by replacing the CHzSCH3 and the a-carboxamide groups of the methionine at

[ Orn6]-SP~1 1 by benzyl ester groups

KOSTAS KARAGIANNIS I. GEORGE STAVROPOULOS I, CONSTANTINE POULOS I . CHRISTOPHER C. JORDAN and RUSSELL M. HAGAN'

' Depcirtriierit of Cheriiistri.. Uriiversitj. of Patras, Patras. Greece. Department of Gastroirirestirial Pharmacologj~ arid -? Neurophnrr?inco(~ Department, Glum Group Research Lid., Ware, Herfordshire. L'K

Received 11 September 1992, accepted for publication 30 May 1993

Analogues of [Orn"]-SP6-ll have been synthesized in which the CHzSCH3 group of Met" is replaced by a COOCHI or a COOBzl group. These analogues, which were tested for agonist and antagonist activity in three iri vitro preparations representative of NK-1, NK-2 and NK-3 receptor types, were full agonists at NK-I receptors, showed very weak agonist activity at NK-2 receptors and were weak antagonists at NK-3 recep- tors. The above analogues were modified by substituting the a-carboxamide of residue 11 by a COOCH3 and a COOBzl group, respectively. The resulting analogues were found to be devoid of agonist activity in each of the functional assays. However, they showed weak antagonist activity at each receptor subtype, with the exception of the dibenzyl analogue, which was a potent and selective NK-1 receptor antagonist. It is con- cluded that appropriate modification of the side chain of Met" and its r-carboxamide leads to a potent and selective at NK-1 receptor antagonist. 0 Munksgaard 1993.

Kej. wrd.v; analogues; antagonists; Q-dialkyl aspartates; guinea pig ileum; rat colon: rat portal vein: substance P

The mammalian tachykinins substance P (SP), neuro- kinin A (NKA) and neurokinin B (NKB) are members of a family of peptides which share the common C-terminal sequence Phe-X-Gly-Leu-Met-NH2 (X = Phe, Val) and have a wide spectrum of biological properties. Receptors for SP, NKA and NKB have been classified into three sub-groups named NK- 1, NK-2 and NK-3, respectively (1). The above classifi- cation has been based upon the relative order of po- tency, in various bioassays, of natural peptides, their fragments and synthetic agonists selective toward one of the receptor subtypes (2, 3). Very recently, several novel agonists and antagonists have been synthesized which are either peptides derived from appropriate modifications in the natural tachykinins or their C-terminal fragments (4-8), or non-peptides (9), and

Abbreviations used are in accordancc with rules of IUPAC-IUB Commission on Biochemical Nonienclature (1984) Eur. J . Biocheni. 138, 9-37; (1989) J . B i d Chew. 264, 663-673. Other abbreviations are: AcOEt. ethyl acetate; DCC, dicyclohexylcarbodiimide; DCU, dicyclohexylurea; HOBt, I-hydroxybenrotriazol; DMF, N.N- dimcthylformamide; NMM. N-mcthylmorpholine; TLC, thin-layer chromatography.

are characterized by high potency and selectivity. The C-terminal hexapeptide of substance P, H-Gln-Phe- Phe-Gly-Leu-Met-NH? (SPs-I I), is the minimal pep- tide fragment of substance P (SP) that retains substan- tial SP-like activity in most biological preparations (10-13). In view of the importance of the C-terminal hexapeptide of SP for biological activity, this sequence should provide a basis for examining some aspects of the structure-activity relationship for tachykinin ago- nists.

In our recent work (14) with glutamic pester ana- logues at position l l , in the model hexapeptide [Om6]- SP6-11 (1) (15, 16) we have shown the importance of the Met" side chain for the activity of SP, and that the nature of the group attached at the :-position of the side chain in the amino acid at position 11 is a deter- mining factor for activity. The above results prompted us to investigate the effect of a COOCH3 or a COOBzl group in the side chain of residue 11, which is closer to the peptide backbone by a methylene group. The fact that replacement of the r-carboxamide group of Met" in S P by a methyl ester group leads to a selective ag- onist at the NK-1 receptor (17) prompted us to inves- tigate if the latter applies to other analogues with modi-

565

K. Karagiannis et al.

fications of the SCH3 group of Met". In addition, we wanted to obtain information on the effect of a double modification at the Met" residue consisting of the re- placement of CHzSCH3 and the r-carboxamide groups by the same group. Thus we synthesized analogues where the methionyl amide residue in the peptide 1 was replaced by the Asp(OCH3)-NHz, Asp(0Bzl)-NHz, Asp(OCH3)-OCH3 and Asp(OBz1)-OBzl residues. The resulting analogues were tested for agonist and antago- nist activity in the three different neurokinin receptor types.

RESULTS AND DISCUSSION

The analogues of the C-terminal hexapeptide of SP were synthesized in solution by coupling the protected N-terminal tetrapeptide acid Boc-Orn(Boc)-Phe-Phe- Gly-OH to the C-terminal dipeptides H-Leu-X

OCH3, Asp(OBz1)-OBzl] using the DCC/HOBt method. Final products were obtained by deprotection with HCI in acetic acid.

Structural modifications in the model hexapeptide IOrn6]-SP6-~ I (1) involved replacement of the methio- nyl residue by P-methyl or P-benzyl esters and by r,P- dimethyl or r,P-dibenzyl esters of aspartic acid. The resulting analogues were tested in guinea pig ileum long- itudinal smooth muscle (GPI), rat colon muscularis mucosae (RC) and rat portal vein (RPV). Agonist ac- tivity is expressed as equipotent molar ratios [ EPMR; ratio ECjo (test compound)/ECso (standard)]. The standards used were substance P methyl ester (SP- OCH3) in GPI, NKA in RC and NKB in RPV. For antagonist activity, dose ratios (DR) were estimated

[ X = A s ~ ( O C H ~ ) - N H ~ , Asp(OBzl)-NHz, Asp(OCH3)-

from the rightward shift of the agonist concentration- response curve obtained in the presence of the antago- nist, and mean ~ K B values were calculated from the equation PKB = log[(DR) - 11 - IogfB], where [B] is the molar concentration of antagonist. The results are summarized in Table 1.

At NK-1 receptors in GPI, the hexapeptide amides 14 and 15 are full agonists, although a dramatic de- crease in activity is observed compared to the parent hexapeptide (1). Their activities compared to those of the corresponding glutamic acid analogues (18) and (19) (Table 1) are much lower (14). This is an indica- tion that not only the nature of the group attached at the y-position in the side chain of the residue 11 in SP is important, but also its position in the side chain seems to be a determining factor. This is in agreement with our previous results (14, 16, 18). Thus, the pres- ence of a COOCH3 group at the P-position of the side chain of residue 11 in analogue 14 reduces the activity at the NK-1 receptor compared to analogue 18. The same effect is also observed with a COOBzl group in 15 compared to 19. In the other two assay preparations (NK-2, NK-3) the analogues 14 and 15 show different behaviour from the corresponding analogues 18 and 19 in the glutamic acid series, as they are very weak ago- nists at NK-2 and weak antagonists at NK-3 receptors. Considering that the Met'l side chain is associated with the affinity and/or efficacy of SP, we would expect our modifications to affect it.

The low affinity analogues 14 and 15 are candidates for further modifications in vital groups in order to obtain antagonist activity. Such a group is the r-carboxamide of methionine. Analogues 16 and 17 resulted from replacement of the r-carboxamide group

TABLE 1

Agoiiist uiid antagoiiist uctivity of peptides H-Om-Phe-Phe- Gly-Leu-X

No. X Equipotent molar ratio (EPMR) PKn

NK-1 NK-2 NK-3 NK-1 NK-2 NK-3 SP-OCH, = 1 NKA= 1 N K B = 1

1 M CL-N H 2 2.6 148.1 2000 - - - 14 Asp(OCH ?)-NH: 6426 > 3000.' > 3000b <4.5 15 AsP(OBZI)-NH: 838.6 > 3000' > 3000b < 4.5 16 As~(OCH~)-OCH? > lOOOOd > 3000e > IOOOb < 5.0 1 4 . 5 1 4.5 17 Asp(OBz1)-OBzl > 7 I4Ob > 3000h > 3000h 6.92 f 0.08' 1 4 . 5 <4.5

- - - -

18 Glu(OCHq)-NH:S 44 1688 > 3000h C - -

19 GIu(OBZI)-NH~B 7.1 79.2 - - - -

" 1 1 u o NKA E,,,,s, at 30 p ~ . No significant effect at 30 p ~ . 47", NKA E,,, , , at 30 p ~ . 9", SPOCH, E,,,,, at 25 p M .

L_ l o , N K A E ,,,,,, at 25 {IM.

8 EPMR values obtained from ref. 14. Schild slope 1.08 (95", C.L. 0.72-1.44)

28", NKB E,,,, at 100 p ~ .

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Antagonists of substance P

chrosorb RP-18 column 250 x 4 mm 5 p with the fol- lowing solvent systems: (A) O.l:o TFA in water, (B) 0.1% TFA in CH3CN, 809,-20~, (A:B) isocratic elution for 5 min, and then linear gradient 80~o-20",b (A:B) to 20%-80% (A:B) for 25 min, UV detection at 257 nm, flow rate 1 mL/min. The elemental analysis of amino acid derivatives and dipeptides were within f 0.40% of the calculated values. Methodology for amino acid analyses of the final products and for FAB mass spectal analysis have been previously reported (15, 16). D M F was distilled immediately before use over CaH2.

in 14 and 15 by a methyl and a benzyl ester group, respectively. Analogues 16 and 17 were devoid of ag- onist activity in all receptor types. However, 17 is a moderately potent antagonist (pKs = 6.92) at NK-1 re- ceptor in GPI. In terms of selectivity, the antagonist 17 is approximately 1000-fold selective for NK- 1 receptor type and shows no appreciable antagonist activity at NK-2 and NK-3 receptors.

Compared to the currently available peptide antago- nists (19,20), the potent and selective NK-1 antagonist [ O m i i,Asp(OBzl)i I I -SPS-II-OB~I (17) has the follow- ing characteristics: (a) It does not contain D-amino acids. (b) It has modifications only to one amino acid. (c) It has higher activity and selectivity compared to previously reported hexapeptide antagonists of SP, while it is equipotent and with higher selectivity to the octa- and undecapeptide antagonists. (d) It is less po- tent than the non-peptide antagonist (k )CP 96345, while the only common feature that we may note, al- though it is very speculative, is the presence, in both, of two phenyl rings arranged at a certain angle (21).

The above results show that the antagonist activity is strongly associated with structural changes at the amino acid at position 11 of SP. These changes may not necessarily create substantial changes in the mo- lecular conformation, although these can not be ruled out. It is also interesting to note that simultaneous modification of the side chain and the x-carboxamide of methionine as well as the presence of two benzene rings in this series seems to be necessary for high an- tagonist activity. This is also in agreement with our recent results (22) in the glutamic acid series, where the corresponding z, y-dibenzyl analogue is an antagonist, but less potent and less selective than the analogue 17.

In conclusion, the Meti1 residue of SP has a multi- functional role in the process of recognition and stimu- lation of the tachykinin receptors and depends on the nature of the side chain and its z-carboxamide. Appro- priate structural modifications at the Met" residue, such as the presence of bulky and lipophilic groups, may result in potent and selective antagonists at NK-1 receptor.

EXPERIMENTAL PROCEDURES

Capillary melting points were determined on a Buchi SMP-20 apparatus and are reported uncorrected. Op- tical rotations were measured with a Carl Zeiss preci- sion polarimeter ( f 0.005 "). Analysis by TLC was on precoated plates of silica gel F254 (Merck) with the following solvent systems: Rfl chloroform-methanol (6:1), Rfi 1-butanol-acetic acid-water (4:l:l) and Rf3 1 -butanol-acetic acid-water-pyridine (30:6:24:20), Rf4 I-butanol-acetic acid-water (4: 1:5 upper phase). The products on TLC plates were detected by UV light and either chlorination followed by a solution of 1% starch- 1 KI (1: 1 v/v) or ninhydrin. Retention times (tR) of peptides were measured by RP-HPLC with Li-

Preparatiott of x-amides of atmtzo acid deriwtives. To a solution of the Nx-tert-butyloxycarbonyl amino acid de- rivative (3 mmol) in T H F (10 mL) cooled to -15 'C was added NMM (3 mmol) followed by isobutylchlo- roformate (3 mmol). After 2 min a solution of 58:, NH40H (0.9 mL, 4.5 mmol) precooled to - 15 C was added and the reaction mixture left to stand at the above temperature for 2 h and then warmed up to room temperature. At the end saturated NaHCO3 was added and the mixture was extracted with AcOEt, which was then washed with 5% NaHCO3, water and dried (Na2S04). The solvent was evaporated it? ~ c u o and the residue was crystallized with the addition of petro- leum ether 60-80 " C to yield the corresponding amides.

Boc-Asp(OCH3)-NH2 (2), yield 53:,, m.p. 115-

Boc-Asp(OBz1)-NHz (3), yield 62",, m.p. 147- 117 "C, [ r ] E -28.48

149 "C, [ c Y ] ~ -21.69

(C 1 DMF), Rfi 0.54, Rfi 0.70.

( C 1 DMF), Rfi 0.61, Rf2 0.77.

Deprotection of the tert-but?,/os?.o,u?'carboti?,l group. A sample (2 mmol) of the N'-Boc protected amino acid derivative or peptide was dissolved in 1 N HCI in acetic acid (10 mL). After 1 h at room temperature the solvent was removed in vucuo at 25 'C and the residue was solidified by the addition of dry ether. The resultant hydrochloride salt was filtered, washed with dry ether, dried in vucuo over KOH pellets and was then used in the coupling without further purification.

Preparation of dipeptides. To a solution of the N'- protected leucine (4.8 mmol) in T H F (8 mL) cooled to -15 " C was added NMM (4.8 mmol) followed by isobutylchloroformate (4.8 mmol). After 2 min a solu- tion of the hydrochloride Salt of the amino component (3 mmol) in D M F ( 5 mL) precooled to - 15 'C was added, and the reaction mixture was left to stand at the above temperature for 3 h and then allowed to warm up to room temperature. At the end the solvent was evapo- rated in vucuo, and the residue was dissolved in AcOEt, which was then washed with 59; NaHCO3, water, 10% citric acid, water and dried (Na2S04). The sol- vent was removed iii i'acuo and the residue was so- lidified with the addition of petroleum ether 60-80 ' C to yield the desired product. The compounds HCI.H-Asp(OCH3)-OCHi (4) and p-TSA.H-Asp-

567

K. Karagiannis et al.

(0Bzl)-OBzl (5), used in the above couplings, were prepared according to literature procedures (20). All the dipeptides were recrystallized from AcOEt/ petroleum ether 60-80 "C. Boc-Leu-Asp(OCH3)-NHz(6),yield81%, m.p. 113-

Boc-Leu-Glu(OBzl)-NH2 (7), yield 66%, m.p. 124-

Boc-Leu-Asp(OCH3)-OCH3 (S), yield 79%, m.p. 99-100 "C, [ x]g -33.44 O (c 1 DMF), RfI 0.73,

Boc-Leu-Asp(0Bzl)-OBzl (9), yield 867;, m.p. 78-

115 "C, [ ~ ] g -6.32 O (C 1 DMF), RfI 0.57, Rf2 0.74.

125 "C, [ r ] E -40.25 " (C 1 DMF), Rfi 0.58, Rfi 0.87.

Rfi 0.82.

80 "C, [z]E -26.27 ' (C 1 DMF), Rfl 0.67, Rfi 0.79.

Preparatioii qf protected hexapeptide analogues. A por- tion (1 mmo1)ofHCl.H-Leu-X [X = Asp(OCH3)-NH2,

OBzl] was dissolved in D M F (5 mL), neutralized with NMM and allowed to react with a sample of Boc- Orn(Boc)-Phe-Phe-Gly-OH (1 mmol) dissolved in DMF (10 mL) and preactivated at 0 "C for 0.5 h with HOBt (1.6 mmol) and DCC (1 mmol). The reaction mixture was left to stand for 2 h at 0 " C and then for 24 h at room temperature, while the pH of the reaction mixture was adjusted to 7.5-8 with NMM. The pre- cipitated DCU was filtered, and the solvent was evapo- rated in ~'acuo. The remaining residue was solidified by trituration with saturated NaHCO3, filtered, washed on the filter with water, 10% citric acid, water and then dried iiz vucuo over P205. Recrystallization from the appropriate solvent gave the desired product. Boc-Orn(Boc)-Phe-Phe-Gly-Leu-Asp(OCH3)-NH2

(lo), yield 670/,, m.p. 213-215 "C from ethanol, [ r ] g

Boc-Orn(Boc)-Phe-Phe-Gly-Leu-Asp(OBzl)-NH2 (ll), yield 73:,, m.p. 195-197 "C from ethanol/ether,

Boc - Orn(Boc) - Phe - Phe - Gly - Leu - Asp(OCH3)- OCH3 (12), yield 64'4, m.p. 190-192 "C from DMF/ ether, [ r ] E -36.65 ' (c 1 DMF), Rfl 0.67, Rfi 0.83, Rf3 0.77. Boc-Orn(Boc)-Phe-Phe-Gly-Leu-Asp(OBz1)-OBzl

(13), yield 69%, m.p. 202-204 O C from ethanol/ether,

Asp(OBzl)-NHz, A s ~ ( O C H ~ ) - O C H ~ , Asp(OBz1)-

-48.82 ' (C 1 DMF), Rfi 0.57, Rf2 0.76, Rf3 0.81.

[ r ] E -43.38 ( C 1 DMF), Rfi 0.60, Rfi 0.73, Rf3 0.77.

[ ~ ] s -28.48 ( C 1 DMF), Rfi 0.76, Rf2 0.81, Rf3 0.85.

Preparation of H-Orii-Phe-Phe-G!y-Leu-X. A sample of Boc-Orn(Boc)-Phe-Phe-Gly-Leu-X (200-300 mg) was deprotected according to the general procedure de- scribed above. The deprotected hexapeptides were dis- solved in water, filtered through a Millipore filter and lyophilized. The peptides were purified by gel filtration on Sephadex (3-15 (2 x 85 cm) using as eluent 1 M ace- tic acid followed by partition chromatography on Sephadex G25F (2 x 85 cm) with 1-butanol-acetic acid-water (4:1:5 vjv, upper phase). H-Orn-Phe-Phe-Gly-Leu-Asp(OCH3)-NHi (14):

m.p. 117-118"C, [ r ] E -42.7' (c 1, DMF), Rfi 0.34,

568

Rf3 0.56, Rf4 0.18, tR 23.0 min, FAB-MS mjz: 725 (M t H)+ , amino acid anal. Phe1.98 Glyi.oo Leul.01 ASpo.96.

H-Orn-Phe-Phe-Gly-Leu-Asp(OBzl)-NH2 (15): m.p. 110-112"C, [cx]i3 -39.7" (c 1, DMF), Rf2 0.48, Rf3 0.58, Rf4 0.42, tR 24.5 min, FAB-MS mjz: 801 ( M t H ) + , amino acid anal. Phe1.97 G1yl.00 Leu1.02 Aspo.98.

H-Orn-Phe-Phe-Gly-Leu-Asp(OCH3)-OCH3 (16): m.p. 62-64 "C, [a]:: -37.9" (c 1, DMF), Rf2 0.45, Rf3 0.65, Rf4 0.33, t R 22.5 min, FAB-MS nijz: 740 ( M + H ) + , amino acid anal. Phe1.99 Gly1.01 Leui.oo ASpo.98.

H-Om-Phe-Phe-Gly-Leu-Asp(0BzI)-OBzl (17): m.p. 156-158 "C, [ z ] E -26.0 (c 1, DMF), Rf2 0.42,

( M + H ) + , amino acid anal. Phe1.96 Gly1.02 Leul.oo A spo. 97.

Rf3 0.63, Rf4 0.31, t R 27.5 min, FAB-MS P ? I / Z : 892

Bioassays Agonist activity at NK-1 receptors was determined from contractile responses of guinea pig ileum longitu- dinal smooth muscle (GPI) recorded under isotonic conditions, at 37 "C, in the presence of atropine (1 p ~ ) , mepyramine (1 p ~ ) , methysergide (1 VM) and in- domethacin (1 p ~ ) . Agonist activity at NK-2 receptors was determined from contractile responses of the rat colon muscularis rnucosae preparation (RC) in the presence of antagonists as described for the GPI. Tis- sues were mounted in 2 mL organ baths and doses of agonist were added in volumes of <220 p L (exposure time 20 s for GPI; 45 s for RC). Preparations were washed thoroughly between doses and the inter-dose interval was 7 min (GPI) or 15 min (RC).

Experiments were conducted as 3 + 3 (GPI) or 2 + 2 (RC) assays against SP-OCH3 or NKA, respectively, as standards using a randomized block design. Each concentration of agonist was tested four times. Data were analyzed by analysis of variance. Only those as- says demonstrating no significant deviation from par- allelism and no Significant deviation from linearity were used to calculate equipotent molar ratios (EPMR).

Agonist activity at NK-3 receptors was determined from contractile activity in the everted rat portal vein preparation (RPV) (24) at 25 "C. Isometric contrac- tions (resting tension 0.5 g) were recorded in response to serially applied doses of agonist administered at in- tervals of 15 min. Concentrafion-response curves were established for standard (neurokinin B) and test com- pound. Results were standardized by determining EPMR values for each compound, and ECso values were determined.

Guinea pig ileum and rat colon preparations were bathed in Tyrode solution of the following composition: (mM) N a + 149.1, K' 2.8, Ca2+ 1.8, Mg2+ 2.1, CI- 147.5, H2PO; 0.3, HCO; 11.9, glucose 5.6, bubbled with 95% oxygen/5% carbon dioxide. Rat portal vein preparations were bathed in Krebs-Henseleit solution

Antagonists of substance P

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C., Jordan, C.C. & Hagan. R.M. (1991) f r t r . J . Pepride Proteiri Res. 38, 350-356

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16. Poulos, C., Stavropoulos. G.. Brown. J.R. & Jordan, C.C. (1987) J . Med. Chewi. 30, 15 12- 15 15

17. Watson, S.P., Sandberg, B.E.B., Hanley, M.R. & Iversen. L.L. (1983) Eur. J . Pharrriucol. 87, 77-84

18. Theodoropoulos, D., Poulos, C.. Gatos, D., Cordopatis. P., Couture, R., Regoli, D. & Escher, E. (1982) in Peptides 1982 Proc. 17th Eur. Pept. Symp. (Blaha, K. & Malon. P.. eds.). pp. 521-526, Walter de Gruyter, Berlin

19. Regoli, D., Escher, E. & Mizrahi, J. (1984) Phoniiucohgi. 28,

20. Jukic, D., Rouissi, N., Laprise, R., Boussougou, M. & Regoli, D. (1991) Life Sci. 49, 1463-1469

21. Howson, W., Hodgson, J., Richardson, R.. Walton. L., Guard. S. & Waking, K. (1992) Bioorg. Med. Cheni. Leu. 2 (6), 559-564

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29, 1281-1284

301-320

of the following composition: (mM) Na’ 143, K + 5.9, Ca’+ 1.25, Mg2+ 0.6, CI- 125.2, H2PO; 1.2, HC 0; 25, SO: ~ 0.6, glucose 11.1 and bubbled with 95% oxygen/5 O 0 carbon dioxide.

For determination of antagonist activity, isolated tis- sue preparations were prepared as for the agonist stud- ies. Antagonists were pre-equilibrated with the tissue for 15 min. Antagonist-induced parallel displacements of agonist concentration-response curves were quan- tified as the ratio of equi-active molar concentrations and estimated graphically at the level of half-maximal response. The apparent affinity (pKe) of the antagonist was estimated as the mean ( k SE mean) of the indi- vidual values using the relationship:

pK, = log(concentration ratio - 1) - log(molar antagonist concentration).

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I . Henry. J.L. (1987) in Suhruiice P utid Netrrokiniris (Henry, J.L., Couture. R., Pelletier, G.. Quirion, R. & Regoli, D., eds.), pp. n i i . Springer-Verlag, New York

2 . Regoli. D.. Drapeau, G.. Dion, S. & DOrleans-Juste. P. (1989) Phurritcicologv 38, 1 - 15 and references cited therein

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9. Rouissi. N.. Gitter, B.D.. Waters, D.C.. Howbert, J.J., Nixon,

Address:

Dr. Coristuiitiiie Pottlor Department of Chemistry University of Patras Patras Greece

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