n 144
[COSTRIBUTIOS FROM THE RESEARCH
BY
ITOl.77
~V.~I,TOX, \I*AGNBR, U.ICIIEI,OR, PETERSON, HOLLYAXD FOLKERS
li;DLV.4RD
LABORATORIES O F THE
CHEMICAL
DIVISION, hfERCK & CO.,
Synthesis of (+)-a-Lipoic Acid and its Optical Antipodel n'.ILTOX, . i R T H U R F. TTAGNER, FRAKK Iv.BACHELOR, LOUIS13. P E T E R S O h - ,
INC.]
FREDERICK
It'.
HOLLY AND KARLFOLICERS RECEIVED MARCH 15, 1956 (.-f )-oc-Lipoicacid has been synthesized. DL-3-Acetylthio-7-carbethoxyheptanoic acid lyas converted into its acid chloride whlch was reduced with sodium borohydride to yield a mixture of reduction products. Alkaline hydrolysis of this mixture produced 8-hydroxy-6-thioloctanoic acid. Replacement of the hydroxyl with sulfhydryl followed by oxidation gave DL-alipoic acid. Resolution of ~~-3-acetylthio-7-carbethoxyheptai~oic acid supplied the starting materials for the synthesis of the (+)- and ( - )-a-lipoic acids.
a-Lipoic acid has been isolated from natural ~ o u r c e and s ~ ~has ~ been shown to be active as a coenzyme in the oxidative decarboxylation of pyruvate. On the basis of degradative?~~ syntheticb-8 and spectroscopicg evidence, a-lipoic acid is the cy5- [3-(1,2-dithiolanyl)1-pentanoic clic disulfide, acid (1'111' The rxemic form of this compound, 0
VI11
tiesignatedI0 either ~r,-a-lipoicacid5P7,8or 6-thioctic (1) .4 preliminary account of this work appeared in a Cornmunical i o r i t o t h e Editor, TAISJOURNAL, 76, 4748 (1964).
( 2 ) L. J. Reed, I. C. Gunsalus, G. H. F. Schnakenherg, Q. F. Soper, H. E. Boaz, S. F. Kern and T. 1 '. Parke. ibid.,76, 1267 (1953). ( 3 ) E . I,. Patterson, J. V. Pierce, E. L. R. Stokstad. C. E. Hoffmann. J. A Brockman, J r , F. E' Day, M E:. Rfacchi and T. H . Jukes, ibid., 76, 1823 (1954). (4) J. A . Brockman, Jr., E. L. R.Stokstad, E. L. Patterson, J V. Pierce and 31. E. Maccbi, ibid.. 76, 1827 (1954) ( 5 ) C. S.Hornberger, Jr., R. F. Heitmiller, I. C. Gunsalus, G. H. F. Schnakenberg a n d L. J. Reed, ibid.,76, 1273 (1953). ( 6 ) M. W. Bullock, J. A. Brockman, Jr., E. L. Patterson, J. V. Pierce, >,I. H. von Saltza, F. Sanders and E. L. R. Stokstad, ibid.,7 6 , 1828 (1954). 17) Q . F. Soper, W. E . Ruting, J. E. Cochran, Jr., and A . Pohland. ibici., 76, 410:) (19543. (81 L. J . Reed and Ching-I Xiu, ibid., 77, 416 (1955). (9) bf. Calvin and J. A . Barltrop, ibid., 74, 61.53 (19.52). (10) For a discussion concerning nomenclature scr: G , \V. Kiildrr, F e d u a t i o n Proc., 13, 695 (19.54).
acid,6 has been synthesized. This paper describes the details of a synthesis of (+)-, ( - ) - and DL-a lipoid acid.I Addition of thiolacetic acid to 7-carbethoxy-2heptenoic acid (I)11 yielded ~~-3-acetylthio-'i-carbethoxyheptanoic acid (11) which was converted into the acid chloride 111. Reduction of the acid chloride I11 led t o a mixture of esters (IVa, b and c). By alkaline hydrolysis, the mixture was converted to ~~-8-hydroxy-Gthioloctanoic acid (Ir). Replacement of the S-hydroxyl by sulfhydryl yielded m-dihydroa-lipoic acid (V-I]. The dithiol TI was oxidized to DL-a-lipoic acid. \Then the (+I and (-1 isomers of 3-acetylthio-7-carbethoxyheptanoic acid (11) were used in the above sequence, (+)- and ( - I-a-lipoic acids, respectively, were produced. Thiolacetic acid was added to the a,?-unsaturated acid I to gire ~~-3-acetylthio-7-carbethoxyheptanoic acid (11). Thionyl chloride converted D1.-3acetylthio - 7 - carbethoxyheptanoic acid 111)into DL-3-acetylthio - 7 - carbethoxyheptanoyl chloride (111). This acid chloride was used immediately in the next step, since it decomposed on standing. DL -3 - .I cetylthio - 7 - carbethouvheDtmoy1 chloride (111)was reduce'd bi- sodium borohydride'? suspended in dioxane. The reduction product was a mixture which contained varying amounts of ethyl ~~-8-hydroxy-6-thioloctanoate ( It'b) and ethyl DL-8-acetoxy-6-thioloctanoate (Ia'cj as well as the major product, ethyl DL-G-acetvlthio-r;-h!--droxyoctanoate (IVa). The presence of the 0acetyl thiol IVc can best be explained bv the migration of acetyl from sulfur to oxygen during the later part of the reaction. ri six-Inelnbereci cyclic orthoacetate has been proposed as the intermediate for this type of migration.13 The hydroxy thiol IVb is produced by alkaline hydrolysis of the acetal group. (11) G. B. Brown, hf. D. Armstrong, A. W.Ifoper, 1%'. P. Anslow, Jr., €3. R. Baker, M. V. Querry, S. Bernstein and S. R . Safir, .l O r : Chcnz., 12, 1GO (1947). (12) 5. rT. Chaiken and IT. G . Brown, THISJ r i r . n x 4 1 , 71, 122 (1949).
(I:'.
A 95-mg. sample of ( +)-a-lipoic acid was sublimed at 8590" (25 p ) to yield 82 mg. of sublimate. This was recrystallized from 1.5 ml. of cyclohexane to give 41 mg. of purified product, m.p. 46-48' (micro-block), [ 0 1 ] * 3 ~ +104" ( G 0.88, benzene), 333 m,u ( E 150). Anal. Calcd. for CUHI4O&: C, 46.60; H , 6.84; S, 31.05; neut. equiv., 206; mol. wt., 206. Found: C , 46.95; H, 6.8.5; S, 31.00; neut. equiv., 208 (pK, 5.4); mol. Ivt. (ebul.), 194 i 2. B. From ( +)-8-Hydroxy-6-thioloctanoic Acid.--A mixture of 7.6 g. (0.04 mole) of (+)-8-hydroxy-6-thioloctanoic acid, 27 g . (0.36 mole) of thiourea and 70 ml. of 40% h y drobromic acid was refluxed for 16 hours. The reactioll mixture was neutralized with about 60 ml. of 30% aqueous sodium hydroxide and made 0.5 S with an additional 1:j ml. of 30% aqueous sodium hydroxide. The alkaline misture was refluxed 30 minutes. I t was acidified and extracted with 3 portions of chloroform. The combined chloroform extracts were mashed with water, dried over anhydrous magnesium sulfate and filtered. The filtrri te was concentrated a t reduced pressure to yield 6.8 g. of crude ( - )-6,8-dithioloctanoic acid, n 2 6 ~ 1.5238, [CY] -8.8" (c 6.92, methanol), neut. equiv. 203 (calcd. 208). A solution of 5.8 g. of the (-))-dithiol in 75 ml. of w t c r and 3.8 g. of potassium carbonate was adjusted to PH 7 by adding 2.5 11' hydrochloric acid. The solution was treated with 1.5 ml. of ferric chloride (1%) which produced a dark coloration. Air was bubbled through the solution until the color changed to yellow. The reaction solution was acidified with hydrochloric acid and the liberated product was extracted into 3 portions of chloroform. The combined chloroform extracts were washed with \vater, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated at reduced pressure to leave a. 3.5%. residue. The residue was leached with t:vo 25-ml. portions of boiling cyclohexane. The first cyclohexane extract vielded 1.47 g. (20% from ( +)-5-hydroxyA-thioloctanoic acid) of (+)-a-lipoic acid, m.p. 45-47", [CY]~:D+99" ( c 1.07, benzene). The filtrate and second cyclohexane extract were combined and concentrated to a 10-ml. volume. On cooling the concentrate, a second crop (0.65 9.) of product im.p. 43-47') mas obtained. ( - )-a-Lipoic Acid. A . From ( +)-6,6'-Dithiobis-(8-hydroxyoctanoic Acid).-A mixture of 11.9 g. (0.031 mole) of ( +)-6,6'-dithiobis-(8-hpdroxyoctanoic acid), 19.2 g. (0.25 mole) of thiourea and 63 ml. of 40% hydrobromic acid \vas refluxed for 16 hours. The products of the reaction 7w-e worked up in a manner similar t o that described for the corresponding (+)-isomer above. The chloroform solution after iodine oxidation yielded 2.2 g. of product. This material was leached with 25 ml. of hot cyclohexane from which was obtained 800 mg. (6yc)of (-))-a-lipoic acid, m.p. 45-47'. A 300-mg. sample was recrystallized from 11 ml. of cyclohexane to give 200 mg. of purified (-)-a-lipoic acid, m.p. 45-47.5' (micro-block), 1 ~ 1 2 -11.3" 3 ~ ( c 1.88 benzene), AE::~~ 330 mp ( E 140). Anal. Calcd. fpr CsHla02S2:C, 46.60; H, 6.84; s, 31.05; neut. equiv., 206, mol. w t . , 206. Found: C , 46.65; H, 6.66; S, 31.32; neut. equiv., 208 (pK, 5.4); mol. wt. (ebul.), 212 z!c 2. mixB. From ( - )-8-Hydroxy-6-thioloctanoic Acid.-A ture of 12.5 g. (0.065 mole) of ( -)-8-hydroxy-6-thioloctanoic acid, 38 g. (0.50 mole) of thiourea and 100 ml. of 40% hydrobromic acid was refluxed for 16 hours. The reaction mixture was worked u p in the manner described for the corresponding (+)-isomer above to give 11.6 g. of crude (+)-6,8-dithioloctanoic acid, ? z * ~ D1.5267, [ u ] " ~4-11" ( ( 5.51, methanol), neut. equiv. 228 (calcd. 208). A solution of 11.4 g. of (+)-6,8-dithioloctanoic acid in 150 ml. of water and 7.4 g. of potassium carbonate was adjusted to pH 7 and oxidized with air and ferric chloride (1 ml. of 1%). The reaction product was worked up as before to yield a chloroform-soluble fraction. This material was leached with one 100-ml. and two 50-ml. portions of hot cyclohexane. The 100-ml. extract yielded 3.65 g. (27%) of (-)-u-lipoic acid, m.p. 45-47', [ a I z 7-105' ~ (c 0.9, benzene). The filtrate and two 50-ml. extracts were combined, concentrated and cooled to yield a second crop of 0.7 g. (:% of protluc!, in.1). 45 48". 1
Oct. 5 , 1955
5149
HYDROLYSIS OF ESTERLOCAL ANESTHETICS
DL-a-Lipoic Acid from (+)- and (-)-a-Lipoic Acid.Samples of 12.9 mg. of both (+)-a-lipoic acid and ( -)-w lipoic acid were mixed and ground together in a mortar.
The mixture (m.p. 55-57') was recrystallized from cyclohexane to give DL-cu-lipOiC acid, m.p. 60-61'. RAHWAY, N. J.
[CONTRIBGTION FROM THE DEPARTMENT OF ANESTHESIA, MERCYHOSPITAL AXD SECTIOS o s ANESTHESIOLOGY, DEPARTMENT O F SURGERY, USIVERSITY O F PITTSBERGH SCHOOL O F MEDICIXE]
Hydrolysis of Ester-type Local Anesthetics and their Halogenated Analogs by Purified Plasma Cholinesterase1 BY FRANCIS F. FOLDES, DAVIDL. DAVIS,SYDNEY
SHANOR A N D
GERTRUDE V A N HEES
RECEIVED APRIL2, 1955 The reaction rate constants ( K ) and the Michaelis constants (K,) of three +aminobenzoic acid esters and their 2-chloro substituted derivatives2 were determined in fresh human plasma and a human plasma cholinesterase concentrate ( Cholase3). The K and K,,, values of the 2-fluor04 and 2-bromo2 substituted procaine HCl were also determined. Halogen substitution in the 2-position caused a 4- t o &fold increase in the K values of the compounds investigated. The influence of halogen substitution on the K , values was less marked.
The influence of halogen substitution on the enzymatic hydrolysis rate of p-aminobenzoic acid esters in human plasma5 and on the local anesthetic activity and systemic toxicity of these compounds6~' have been previously reported. In these studies the hydrolysis rates of the various compounds were observed in plasma samples obtained on different days from different individuals and were expressed as the time necessary for 50% hydrolysis. Despite precautions taken to obtain valid comparative values for the hydrolysis rates i t was felt that more characteristic data could be obtained by using a uniform source of enzyme (Cholase), and by determining, instead of the 50% hydrolysis time, the K and Km values of the different compounds.
were also determined.8 The K and K , of all compounds were also measured in freshly obtained, heparinized, pooled human plasma samples and occasionally also in individual plasmas. To avoid extreme variations in the times necessary for the completion of the experiments and in order t o obtain more accurate K , values the quantity of the enzymes used was varied according t o the hydrolysis rate of the compound investigated. Since too high plasma concentrations interfered with spectrophotometric readings, this principle could be followed more closely with Cholase than with plasma. Cholase dilutions of 1 t o 125, 1 t o 375, 1 t o 750 and 1 to 1500 were made with a phosphate buffer (containing 6.07 g. of NalHPOd and 2.00 g. of NaH2POa.H20in 1 liter of distilled water). 10-3 M solutions of all substrates were prepared in distilled water. The systems used for the hydrolysis studies were prepared by adding a t zero time, 0.2 nil. of the substrate solution, to a mixture of 1 .O ml. of huffer,
TABLE 1 CHOLASE A N D PLASMA DILUTIOXS ASD RELATED SUBSTRATE CONCENTRATIONS Substrate
Dilution used
Procaine.HC1 ( I ) 2-Fluoroprocaine.HC1 (11) 2-Chloroprocaine.HC1 (111) 2-Bromoprocaine.HC1 (IT) Tetracaine.HC1 ( V ) 2-Chlorotetracaine.HC1 ( V I ) 2-sec-Butylarninoethyl-3-arninobenzoate~HC1 (IrII) 2-sec-Butylaminoethyl-2-chloro-4-aminobenzoate~HCl (VIII)
1-375 1-1500 1-1500 1-750 1-125 1-750 1-126 1-780
Material and Methods The K and K , of procaine.HC1 ( I ) , tetracaineHC1 ( V ) and 2-sec-butylaminoethyl-4-aminobenzoate~HC1( V I I ) as well as their 2-chloro substituted analogs (111, V I and VIII, respectively) were measured using a purified human plasma cholinesterase concentrate (Cholase). The K and K , of the 2-bromo ( I V ) and 2-fluor0 (11) analogs of procaineHC1 ( 1 ) This study was supported in part by U. S. Public Health Service Research grant N o . G-3585 (C2) ( 2 ) These compounds were kindly supplied b y Dr Robert H Hall of Wallace & Tiernan. Inc., Belleville. N e w Jersey. 13) Cholase was kindly supplied b y Dr E d n i n R . AlcLean, C u t t r r Laboratories, Berkeley. California. (4) T h e 2-Fluoroprocaine HCI was placed a t niir disposal by D r . A . C. Bratton, J r . , of Parke, Davis S. Co., Detroit. hlichigan. ( . 5 ) D. I.. Davis and F. F. Foldes. Federation P Y O C .13, , 346 (1064). ( G ) F. F . Foldes and D . H . Rhodes, A m s f h . & Anaig., 32, 305 (1963). (7) F. F. Foldes. D . L. Davis and 0. J. Plekss, "Anesthesiology." in press.
Cholase Substrate concn. in cholase, moles/ml.
3.75 x 1.30 x 1.50 x 7.50 X 1.25 X 7.50 x 1.23 x 7.50 X
10-5 10-4
10-4 10-5 10-5
10-5
10"
Dilution used
1-8.0 1-10.0 1-10.0 1-10.0 1-2.5 1-5.0 1-2,3 1-5.0
Plasma Substrate concn. in plasma, moles/ml.
5.0 X 1.0 x 1.0 x 1.0 x 2.5 x 3.0 x 2.5 x
5.0 x
lo-' 10-6 10-6 10-6 10-7 10-7 10-7 10-7
0.8 ml. of distilled water and 2.0 ml. of the appropriate dilution of Cholase or plasma used. All solutions were warmed to 37" before mixing. The 0.8 ml. of distilled water was included in the systems to allow for the use of cholinesterase inhibitors in subsequent studies. The p H (7.4) and the K a + concentration (0.025 M ) were identical with those present in the systems generally used for the measurement of the activities of the various enzyme source5 with acetylcholine substrate in ITarburg experiments. The volume of all systems was 4 ml. and their final substrate concentration was 5 X 10-5 M . The only variables in the composition of the systems were the dilution of enzymes used and, consequently, the enzymes substrate ratio. These variables are summarized in Table I. The hydrolysis rates of the various substrates were determined with a modification of the ultraviolet spectrophotometric method of Ka10w.~ The changes of the optical den( 8 ) From here on, for t h e sake of brevity, t h e various compounds will be referred t o by their serial number in Table 11. (9) W. Kalow. J . Pharmacol 6' E r p e r . Thcrap., 104, 122 (1952).
