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'. N. J. RAHWAY,
[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. T o 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 s t u d y was supported in part by U. S. Public Health Service Research g r a n t N o . G-3585 ( C 2 ) ( 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. B r a t t o n , J r . , of Parke, Davis S. C o . , Detroit. hlichigan. ( . 5 ) D . I.. Davis and F. F. Foldes. Federation P Y O C .13, , 346 (1064). ( G ) F. F . Foldes a n d D . H . Rhodes, A m s f h . & Anaig., 32, 305 (1963). (7) F. F. Foldes. D . L. Davis a n d 0. J. Plekss, "Anesthesiology." in press.
Cholase S u b s t r a t e 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 S u b s t r a t e 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 ) F r o m here on, f o r t h e sake of brevity, t h e various compounds will b e 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).
F.F.FOLDES, D. L. DAVIS,S . SHANOR AND G.VAN HEES
5150
aities, observed at a wave length of 313 m r , were recorded a t 30 or 60 second intervals until completion of the hydrolysis, in a model DU Beckman spectrophotometer. The absorption chamber \\as kept at a temperature of 37 + 0.2" by circulating n a t e r n i t h an electric pump, from a thermostatically controlled source, through two thermospacers mounted on the sides of the sample compartment. Readings were made against a blank in which the substrate solution was replaced by an equal amount of distilled water. This blank, which does not correct for alkaline hydrolysis, was selected because preliminary observations indicated t h a t the alkaline hydrolysis of the substrates, with the experimental conditions used, was negligible. The K,,, values were determined, as suggested by Lineu eaver and Burk,Io by plotting S / V against S (where S is the substrate concentration and V the reaction rate with the corresponding
SI.
All determinations were done in duplicate. represent the averages of several experiments.
Vol. i'i
The introduction of a fluoride or bromide radical in the 2-position into I also increased the enzymatic hydrolysis rate both in human plasma and in Cholase (see Table I17). T.4BLE
THEISFLUESCE OF THE SUBSTITUTIOS OF VARIOUSHaroGEN RADICALS IT THE PROCAINE MOLECULE O N EXZYMATIC HI'DROLYSIS RATE Halogen substituted
Fluroide Chloride Bromide
The data
K substituted K non-substituted Cholase Plasma
8 6 .i(i
3 1 3 0
2 8
2 6
The influence of the various halogens was chlorine > fluorine > bromine; and the effect was more marked when the enzyme source was Cholase than when it was plasma. The Km values of four of the eight compounds investigated (see Table 11) were of the order of TABLE I1 both with plasma and Cholase. The order ~ I I C H A E(LKInS, ) ASD REACTIOSRATE ( K ) CONSTANTS of magnitude of the K , of I1 and VII, however, K m ( mole/l ) K ( moleil. min ) Compd Cholase Plasma Cholase Plasma was 10-j. The difference of the K , values of 2 . 2 X 1 0 ~ 6 V and VI with plasma and Cholase cannot be 3 . 0 X 10-6 ii X 1 0 - 3 I 2 . 6 X 10 e 1 . 2 x 10-5 I , ; < x 10-2 6 . 8 X 10-6 I1 1 . 3 X 10-5 readily explained. It is possible that because of 111 3 1 X 10-6 1 . 8 X 10-6 2 0 X 10-2 1 . 0 X IO-' considerable self inhibition observed with the 1 . 0 x 10-2 5 . 8 X 10-6 IV 2 . 7 X 10-5 1 . 2 x 10-6 enzymatic hydrolysis of V and VI the K and the 9 6 X 10-4 7 9 X 10-6 V 2 . 8 X 10-6 J 0 2: 10-7 VI s.fi x 10-7 I I x 10-6 4 2 x in-.. :{.I x io Km values could not be determined as accurately TI1 1 . 3 X 10-c 1 . 3 X 10-5 1 . 5 X 10-3 i . 3 X 10-8 as those of the other compounds. VIII 3.0 x 10-6 2 . 3 x 10-5 8 . 8 x 10-3 4.3 x in-5 I t was previously reported by Kalowg that the Independent of the source of the enzyme the Km affinity of I to human plasma cholinesterase is 220 values of most of the compounds ranged from 1.2 times greater than the affinity of XCh to this ento 3.1 X the K, of I1 and VII, however, were zyme. Since the Kmof ,4Ch in Cholase and fresh 1.2 to 1.3 X 10-j. Whereas the K , of Vin Cholase human plasma determined by LVarburg's manoand that of its 2-chloro analog in plasma were in metric technique was found to be 1.8 X lo-? AI line with the other K , values, the K , of V in plasma and 2.0 X 10-3 JI,respectively,ll our data indicate was 5 X lo-' and the K , of VI in Cholase was that the affinity of I to plasma cholinesterase, xs calculated from their K , values, is about 700 times 8.6 x 10-7. It is evident from Table I1 that halogen sub- greater than that of ACh. The affinities of 111, stitution markedly increased the hydrolysis rate IV, T', VI and VI11 are of the same order of rnagboth in Cholase and in plasma, of all compounds nitude, whereas these I1 and T'II are about one-tenth as great. investigated. Measuring the hydrolysis rate of ,ICh with Discussion llichel's electrometric methodl2 the activity of 1 The data presented indicate that the introduc- ml. of Cholase was found to be equivalent to the tion of a chlorine atom in the %position markedly activity of 300 ml. of fresh pooled human plasma " increased the enzymatic hydrolysis rate of p- n'ith Warburg's manometric technique, also usin: aminobenzoic acid esters. The factor by which XCh substrate, this ratio was found to be 220 iri the K of the three compounds investigated was in- our laboratory.ll In contrast to this the relative creased by the chloro substitution was the great- ability of Cholase to hydrolyze various p-aminoest for VII, less for I, and the least for V. The rate TABLE T of increase in the K values was about the same ABILITYOF CIIOLASEA Y D with heparinized plasma and with Cholase (see VARIATIONIN THE COMPARATIVE FRESHHUMAN PLASMA TO HYDROLYZE ACh A b D p - ~ a r I v o Table 111).
Results The K , and K values for the eight compounds investigated are presented in Table 11.
TABLE I11 THEINFLUESCE OF CHLORINE SUBSTITUTIOS ON THE KEACTIOS RATE CONSTAXTS ( K ) OF p-AMIXOBENZOIC ACID ESTERS K chlorine substituted Compd. compared
I and 111 V and 17 TI1 and T'III
K non-substituted Cholase Plasma
5 6 4 4 :9
5 0 4 0 G O
(10) H . Linemeaver and D . Burk. l f n s J O U R ~ Y A I . . 5 6 , 658 (1934,.
BEUZOIC
Compd
Cholase Placma activity ratio
ACh I I1 I11 11-
220 161 192 200 173
ACID ESTERS
-~
Compd
Chola3e Plasma activity r a t i o
v
1-30
VI
1% 203 202
1'11 1.111
(I I i F. F. Foldes, G. R. Van Hees and S P Shanor, t o be published ( 1 2 ) H 0. Michel, J . Lab. Clin. . l t e d . , 3 4 , 1564 (1949). 13 I E B . I I c L e a n , personal communication
Oct. 5 , 1953
PREPARATION OF
FOUR 3,IiP-DIHYDROXYESTRENES
benzoic acid derivatives varied from a 120- to 200fold as compared to plasma (see Table V). The discrepancy in the relative potency of plasma and Cholase to hydrolyze ACh could be explained partly by loss of potency in storage and partly by the different methods used. It is more difficult, however, to explain the relatively lower activity of Cholase against p-aminobenzoic acid esters in general, and the considerable variation of its activity against various members of this group of compounds (see Table V). The possibilities to be considered include: (1) That more than one enzyme capable of hydrolyzing XCh is present in human plasma and that the activity of these different enzymes toward other esters hydrolyzed by human plasma is variable. It is furthermore conceivable that with the method of purification used in the preparation of Cholase relatively less of the fraction primarily responsible for the hydrolysis of p-aminobenzoic acid esters is extracted. (2) That there is only one plasma cholinesterase but the activity of this one enzyme toward various substrates is affected to a variable degree by the extraction process and/or storage. An answer to these questions would have considerable theoretical and practical importance.
[CONTRIBUTIOS FROM THE
ROLLISH.
STEVENS MEMORIAL
5151
It might clarify whether the variation in activity toward ACh and I observed in certain mammalian plasmas as compared to human plasmal4 is due to marked species variation in the properties of a single enzyme or the uneven distribution of several enzymes in different mammalian plasmas. Of the halogen substituted compounds included in this study, 111, I V and VI11 have proved to be excellent as local anesthetic agents under clinical circumstances. IIIlj was found to be about twice and I V and VIII16 about four times as potent as I. Their activity was characterized by rapid onset, great intensity, and excellent penetrating capacity. Because of their relatively fast enzymatic hydrolysis rate in human plasma, the possibility fur the accumulation of toxic concentrations in the organism is limited. Indeed, no systemic absorption reactions have been encountered with the clinical use of these halogen substituted local anesthetic agents. (14) hI. H. Aven, A. Light a n d F. F . Foldes, Feiieiaiiun P Y O C .12, , ?99 (1953). (15) F. F. Foldes a n d P. G . McKall, Aizcstheriology, 13,287 (1952 (10) F. F . Foldes, J. IV. Covaliucenzo a n d J. H . Birch, Anastizeri a i d Analgesia, in press.
PITTSBURGH, PA.
LABORATORY O F T H E DETROIT ISSTITUTE
O F CANCER
RESEARCH
The Preparation of Four 3,17P-Dihydroxyestrenes1 BY JOHN A. HARTMAN RECEIVED APRIL26, 1955 Preparations of the unsaturated alcohols derived from Ab(1o1-estrene-a-one, 19-nortestoster-one and the enoldiacetate of the latter are described.
The catalytic hydrogenation of estrone, estradiol and estriol has yielded mixtures of mono-, di- and triols, along with some incompletely saturated material.*-j The stereochemistry of the newly formed asymmetric centers is unknown with the exception that both estrone and estradiol give the same dihydroxyestrane, reported to be identical with a naturally occurring steroid found in human pregnancy urine.6 The partial reduction of estradiol by the Birch’ method provides a convenient synthesis of two liP-hydroxyestrene-3-ones, i.e., the A b ( l o j - ( I ) and A4-(II). The latter has been assigned the “normal” or anti configuration a t Clo by Wilds on the basis of (1) T h i s work was supported b y institutional grants t o t h e Detroit I n s t i t u t e of Cancer Research from t h e American Cancer Society, Inc., T h e American Cancer Society, Southeastern Michigan Division, a n d T h e Kresge Foundation. W e also wish to t h a n k t h e Schering Corporation for a gift of estradiol used t o prepare t h e starting materials. (2) A. B u t e n a n d t a n d U. Westphal, Z. phyriol. Chem., 223, 147 (1934). (3) W . Dirscherl, ibid., 239, 49 (1936). (4) J. F. Danielli, G. M. Rlarrian and G. A . D . Haslewood, Biochein. J . , 27, 311 (1933). ( 3 ) R. E. M a r k e r and E. R o h r m a n n , THIS J O U R N A L60, , 2927 (1938). ( G ) R . E. Marker, E. R o h r m a n n , E. L. Wittle a n d E. J. Lawson, ibid., 60, 1901 (1938). (7) A. J. Birch, J . Cheni. SOL.,2531 (1949); A. L. Wilds a n d N. A. SelSOn, THISJ O U R N I L , 75, 5366 (1953); A superior preparation is described using t h e 3-methyl ether a n d lithium in liquid ammonia.
molecular rotation.’ Since it may be converted into a A3.j-enolacetate(111)8the formation of three estrene diol epimer pairs (3a- and 3P-hydroxy) is possible with unsaturation a t carbon five common to all, and the stereochemistry a t C,; and Clo known. Since we were primarily interested in obtaining the 3p-epimers for biological testing, conditions were selected on the assumption that any hydrogen attached to Clo would have qualitatively, and perhaps quantitatively, the same effect as an angular methyl group on reduction with complex hydrides. Thus the ketones I and I1 were reduced with lithium aluminum hydrideg and the enol diacetate I11 was reduced with sodium borohydride.1° None of the crude reduction mixtures formed an insoluble fraction when treated with a warm saturated 95% ethanol solution of digitonin. Negative results with this test are not indicative of the absence of the 3P-alcohols and previous work has indicated the lack of digiton in precipitates in the completely saturated compounds.3 , 6 However, Dirscherl reported that two different “hexahydro” derivatives of estrone formed insoluble digitonides. (8) A . S . Dreiding a n d J. A. H a r t m a n , to be published. (9) W. G. Dauhen, R. A . Lficheli a n d J. F. E a s t h a m , THISJ O U R K A I . , 74, 3852 (1952). (10) B. Belleau and T. F. Gallagher, ibid , 73, 4458 (1931): R’ ti Dauben a n d J, F. E a s t h a m , ibid., 73, 4463 (1931)
