SYNTHESIS AND BIOLOGICAL ACTIVITY OF PANTETHEINE ANALOGS AND DERIVATIVES by Charles Jack Stewart A THES IS submitted to OhEGON STATE COLlEGE in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY June 195'5 APPROVED: Professor of Chemistry In Charge of Major Associate Professor of Chemistry In Charge of Major Chairman of Department of Chemistry Chairman of School Graduate Committee Dean of Graduate School Date thesis is presented August 17, 1954 Typed by Gladys McGinnis s ACKNOWLEDGMENT The author wishes to convey his sincere gratitixe to Dr. Vernon H. Cheldelin for his kindness, patience, encouragement, perception of the problem, advice, and ability to make the difficult task easy. To Dr. Tsoo E. King he wishes to state his deep appreciation for many fruitful hours of discussion, heartening advice, and the keen solution to many a difficult problem. Gratitude is also expressed to the Office of Naval Research, whose financial support facilitated the work, and to Mr. H. J. Sharkey for assistance in the preparation of intermediates. He also wishes to thank his fellow workers for their frequent advice and assistance. TABLE OF CONTENTS g e INT1SODTJCTION i EXPERIMENTAL 7 Synthetic Methods 7 Compound II -N-pentoy1-2-aminoethantho1 7 Compound III - N-Pantoy1ethanoi9nine 7 Cornpoux.d Iv - oiurr N-pantothenyi twrine 7 Compound \'III - bis(N-( 7_hydroxvbutyry1_f - alanyl)-2-arninoethyl) disulfide 9 Compound V - N-pantotheny1ethnc1eniine 9 Compound VI - Ì-(pantoy1g1ycy1)-2- aminoethanthiol 13 Compound VII - N-(Dantoylalanyl)-2- amioethnthio1 16 Cornpound IX -bi(-(ntey1norva1y1) aminoethyl) disulfide 21 S-acetyl pantetheine 25 S-acetyl Coenzyine A 26 Biological Methods 27 RESULTS AND DISCUSSION 28 SvMARY 42 BIBLIOGRAPUY 44 LIST OF TABIS Page Table I - Pantetheine and Analos 4 Table II - Growth resonse of Lactobacillus huiggricus to pantetheine and 5-acetylpantetheine 29 Table III - Growth response of tobacillus elveticua 80 to pantetheine, N-pantoyl-2--amlnoethanthiol, and N-pantoylethanolamine 30 Table IV - Growth stimulation of j. helvetipus by N-(pantoylglycyl)-2.-arninoethanthiol, N-(pantoylalanyl )-2-aminoethanthiol, and bis(N-( 'Y-.hydroxybutyryl- (alany1.) -2-.arninoethyl) clisulfide 32 Table V - Effect of pantoyl lactone on the growth of . helyeticu in the presence of pantetheine 33 Table VI - Growth stimulation of L. belvet.icus by pantothenyl taurine 36 Table VII - Growth stimulation of j. feimenti by pantothenyl taurine 36 Table VIII - Inhibition of J. helveticus by "Oxy-pantetheine" (V) 38 Table IX - Competitive inhibition of L. helveticus by '1oxy-pantetheine't (V) 39 - Table X Stimulation of growth of . fermenti by "oxy-pantetheine" (V) 40 SYNTUESIS AND BIOLOGICAL ACTIVITY OF PANTETHEINE ANALOGS AND DERIVATIVES INTRODUCTION The enzymatic studies (9,25) of an unidentified growth factor for Lactobci1ius bulgaricus (L. bulgaricu,s factor; LBF (43) ), revealed the presence of pantothenic acid as an integral part of the molecule. The chemical structure was fully elucidated by Snell , (33) as N-antotherìylmercaptoethylam1ne, CH OH C(CH ) CHOHCONHCH CH CONHCH CH SII 2 32 2 2 2 2 They proposed the name pantetheine for the free thiol and pantethine for the dimeric disulfide. The demonstration by King and Strong (20) of the enzymatic conversion of pantetheine into coenzyme A and the microbial growth studies of Craig and Snell (13) ar:used further interest in pantetheine. The inadequacies of the original synthetic method (33) soon became apparent and sev'ral laboratories developed more reliable prepara- tive methods (2,18,31,38,39,40,45). As a result of the foregoing syntheses, the ready availability of pantetheine facilitated the investigation of its relationship to coenzyme A. The synthetically produced 4'-pantetheine phosphate was shown to be 2 identical to a degradation product of OcA1 (3) and could be enzymatically converted into the coenzyne (y). Pante- theme was then demonstrated by Novelli (28) to be an essential intermediate in the biosynthesis of CoA. The recent observations of King and Cheldelin (17) and of Brown and Snell (10,11) that N-pantothenylcystelne had high growth stimulatory effects for Acetohact suboxrdpns led the latter workers to conclude that the immediate precursor of pantetheine . is panto- thenylcyste±ne. Hoagland and Novelli (1) have con- firmed this In their enzymatic biosynthesis of CoA and have carefully investigated the requirements of their system. The enzymatic formation of pantetheino from pantothenic acid fails to occur unloss cysteine is adeed. In the complete system the formation of CoA involves five individual reactions starting with pantothenic acid: 1) pantothenic acid + cysteine -i- \TP pantothenylcysteine -1- Pi -4- ADP 2) pantothenylcysteine pantetheine4CO2 3) pantetheine4ATP _ pantetheinephosphateADP 4) pantetheinephosnhate-l-ATP -desphostho CoA-4-PP 5) desphospho CÖA-fATP 'CoA-+ADP 1The following abbreviations are used throughout this Thesis. CoA - coenzyrne A, desphospho CoA - desohospho coenzyme A, ATP - adenosine triphosphate, ADP - adenosine diphosphate, AMP - adenosine monophosphate, Pi - inorganic phosphate, PP - pyrophosphate. 3 The ubiquitous requirement for CoA in biological systems could be more easily stuóîed if some substance cou.ld be prepared which v:ould inhibit CoA dependent reactions. The inhibited reactions might accumulate the unreacted intermediates and thus indicate still unknown mechanisms of metabolism. CoA is a relatively large dinucleotide (28) whIch will be extremely difficult to synthesize by chemical means. The most facile method to prepare a CoA inhibitor would he to produce a compound that could block the synthesis of this coenzyme, or that might even be trans- formed vivo into a CoA analog of inhibitory action. By the proper alteration in the structure of pantetheine it should be possIble to prepare a compound which would have this action. Moreover, the biolobical activity of these "alteid pantetheines" would yield information regarding the steric specificity and points of enzymatic attachment of the intermediate compounds Involved in the biosynthesis of CoA arid CoA catalyzed reactions. For the purpose of this investigation the compounds shown in Table I, were prepared and their biological activities determined. The importance of CoA as a transacvlating agent (23,24,35) together with the acetylating ability of diacetyl mercaptoethylamlne (1) and S-acetyl glutathione TABLE I Pantetheine and Analogs I s -G- OH -C- -à- OH OH I I S OH i S i I I I S i 5 I S S S I S n I, s S -C-C-C-C-N-C-C-S-H Ç-C-C-C-N C -C-C-N-C-C-S-H - C-C-C-C-N-C-C-O-H s ' SS 0GOi i e H'H H'H H'H Il -G- OH OH -ò- 0H OH S i S I ill I S I I S I n I I I i S IS -C-G-C-C-N-C-C-C-N-C-C-S 0 N a iv y - C-C-C-C-N-C-C-C-N-C-C-O-H S I S I i S i 0GO i S S I H'H H'HO.C.O -d- OH OH -C- OH OH I S S II S S I i I I I N I SI I S VI VII i e -C-G- C-C-N-C-C-N-C-C-S-H -C-C-C-C-N-C-C-N-C-C-S-H I S S I I t i I i I OCO O.C.O -C- H'H H'H i OH OH -C- OH OH I S I is i i S II P I I i iii 1)< s , I il IN t ( -C-C-C-C-N-C-C-C-N-C-C-S-) ( -C-C-C-C-N-C-C-N-C-C-S-) I I S I i t i S i I i 2 O O.C.0 CH i i H H 'H Table I - Legend I Iantetheine1 II N-pant oyl-2-aminoethanthiol III N-pantoylethanolamine IV sodium N-pantothenyl taurine V N-pantothenylethanolamine VI N_(pantoy1rLcyl )-2-aminoethanthiol VII N-(pantoylalanyl)-2-aminoethanthiol VIII bis(N-(?'-hydroxybutyryl-/' -alanyl)-2-aminoethyl) disuJ.ficle IA bis (-(pantoylnorva1yl )aniinoethyl) di sulfide 1Each of these compounds has been prepared for use in the resnt tudy. All except Nos. I, II and III are new compounds. No. VIII has recently been prepared ndeDedetly in anothar laboratorr (P) (41) prompted the preoaration of S-acetylpantetheirie as a flAOdCl conipound of acetyl coenzyrne A The transacetyla- t.ion method of ie1and and Bakeirnarm (41) employing pheriylthioacetate was found to be satisfactory for the preparation of S-acetylpantetheine as well as acetyl CoA. 7 EXPER IME NTAL Synthetic Method s For use as intermediates or reagents in the fol- lowing syntheses, several imown compounds had to be pre- pared. This was done, a follows: i) erformic acid was produced by adding 2 ml. of 3c hydrogen peroxide to 18 ml. of 9 formic acid (37); 2) carbobenzoxy chloride according to the directions of Bergmann and Zervas (7); 3) 2-aminöethanthlol as des- cribed by Mills and ]3ogert (26); 4) (3-alethelne and 5) panthetheine, by the method of King, Stewart, and Cheldelin (18); 6) N-pantoylethanolamlne (III) by fusion of pantoyl lactone with ethanolamine (34); 7) N-pantoyl-2-aminoethanthiol (II) by fusion of pantoyl lactone and 2-aminoethantlìlol according to Barnett (6). Pantothenyl taurine-sodium salt (IV) The sodium salt of pantetheine was prepared by dissolving l.9 g. of crude pantetheine (obtained as a fusion mixture) In 4 ml. of l.11SN sodium methoxide. This mixture was added to 20 ml. of performic acid. After standing at room temperature for l minutes the reaction solution was poured into 200 ml. of acetone. To encourage orecipitation of sodium f ormate approximately 50 ini. of ether were added to the acetone solution. The turbid mixture was left at room temperature for three days.