Preparation and Stability of Some Esters of Vitamin A*

Preparation and Stability of Some Esters of Vitamin A* By ALBERT J. FORLANOt and LOYD E. HARRIS Vitamin A esters of fatty acids having electronegative groups in the number 2 osiAcids, with chlorine or a double bond, were used a n i the esters were tested for stability against acid and solvolytic action. The results indicate that (a)stability against proton attack, in an anhydrous solvent, varies directly with the K. of the acid portion, (6) the addition of water to an anhydrous solvent, containing HCI, decreases the rate of formation of anhydrovitamin A, and (c) stability in alcoholic solvents, without an acid catalyst, varies inversely with the K.. A combined mechanism of vitamin A ester degradation in the presence of mineral acids and hydrolytic solvents is presented. tion were prepared.

of lower alcohols and-strong acids vitamin A and its esters to produce anhydrovitamin A is a well established phenomenon. Shantz, el al. (l), refluxed vitamin A esters in ethanol and found that a mixture of anhydrovitamin A, fatty acid, vitamin A ethyl ether, and other products were formed and that the addition of HCl to the ethanol catalyzed this reaction. Vitamin A alcohol is more sensitive to elimination in acid solution than the esters; however, in the absence of the catalyst, vitamin A alcohol is more stable (2). Prolonged contact of anhydrovitamin A with ethanolic HC1 produces a substance called isoanhydrovitamin A (l),which is formed by the addition of a molecule of solvent to the double bond in the cyclohexene ring. JIeunier (3) suggested a reasonable mechanism for (H+) catalysis of anhydrovitamin A formation from vitamin A alcohol. No information has been found regarding the mechanism of proton catalyzed decomposition of the ester; however, the mechanism shown below appears to be satisfactory. HE ACTION

Tupon

TABLE I.-K.

X 101 OF ACIDS

Acetic acid Chloroacetic acid Dichhroacrtic acid Trichloroacetic- acid Propionic acid a-Chlorc)propic,iiic acid Acrylic acid But yric acid Crotonic acid Sorbic acid

1.75 15.5.00

5loo.00

120, m.00 1.34 14;.00 5.56 1.48 3.03 1.73

Shantz, et al. (l), further substantiated the mechanism of ester decomposition when they found that anhydrovitamin A and fatty acid were formed when the ester was refluxed in ethanol. The greater stability of the ester in the presence of acid could be related to the electronegative effect of the carhonyl group on the alkyl oxygen. Reduction of the electron density around the alkyl oxygen should result in decreased attraction ior protons, thus depressing the initial step of the reaction. Based on this assumption, two series of esters were synthesized having electronegative groups in the 2 and 4 position of the fatty acid: (a) those containing chlorine in the a position as vitamin .I chlomacetate and a-clilc iroprpionate, and ( b ) those containing unsaturation in the 2 position as vitamin A acrylate, crntonate, and sorbate, the latter also having unsaturntion in the 4 position. 1::;iJd i l ) : c p , ~ t c c ! t!:c K,’s of the acids pertinent to this sttidy. These are listed in Table I.

July 1960

SC~ENTIPIC EDITION

4 3

SYNTHESIS

The saponification equivalents of these e5trr5 were determined pcitentinrnetricallv by the Schriner Materials Used in This Study.-Vitamin A alco- and Fuson metliod ( T ) . The results are tabulatd hol, Rwhe; chloroacetyl chloride, Eastman Organic in Table 11. (redistilled) b. p. 105-106' ; trichloroacetyl chloride; Infrared and U. V. Spectra.-The infrared spectra Eastman Organic (redistilled) b. p. 114-116'; of these compounds mere determined on a Baird ina-chloropropionyl chloride, Eastman Organic (redis- frared spectrophotometer, using sodium chloride tilled) b. p. 109-111"; pyridine dried over CaC12 plates. The spectra showed definite ester carbonyl (redistilled) b. p. 114-115' ; ethylene dichloride, peaks and the absence of hydroxyl peaks. The p i Eastman Organic b. p. 82-84'; n'oelm neutral ac- tions of the carbonyl peaks are tabulated in Table tivated alumina, Grade I for chromatography; 111. The E(176,lcm.) values of the esters were petroleum ether b. p. 30-60", Mallinckrodt (-1R); determined in petroleum ether (3&60') on a Beckquinoline, Eastman Organic b. p. 11C-l1l0/14 man DU spectrophotometer a t the U. Y.niaxirnum. mm. ; N.N-dimethylaniline. Matheson, Coleman, and The extinction coefficients and wavelength of the Bell b. p. 192-1935'; sorbic acid, cr6tonyl chlo- maxima are given in Table 111. ride, and acrylylchloride, Delta Chemical Co. General Method of Preparation of Esters.-This method, a modification of the Baiter and Robeson 111.-I. R. ASD U. V. PHYSICAL CONSTASTS method ( 5 ) . was used for all the esters, except when TABLE OF THESE ESTERS modifications are specified. Four and one-half grams (0.016 11) of vitamin -4 Position Of alcohol was dissolved in 25 ml. of ethylene dichloride I. R. & u. v. containing 5 ml. of pyridine and cooled t o 10' (I). (l%,lcm.) Maximum Peak Vitamin A Esters (P) (U.V.) (mu) In a separate flask, 0.018 M of the acid chloride was 5 .i l 1300 328 dissolved in 25 ml. ethylene dichloride (11). Solu- a-Chloropropionate 5.64 1046" 325 tion I1 was added slowly t o solution I with stirring Chloroacetate 5.80 1318 32; and set in a dark place for two hours. The solvent Sorbate Acrylate 5.74 1387 32i was removed in vacuo and the residue was dissolved Crotonate 5.76 1318 32; in 10 ml. of petroleum ether (30-60"). The insta- Palmitate 5.53 963 32; bility of some of these esters necessitated their purification on alumina columns. The alumina was a This low extinction coefficient is an indication of its lack deactivated by the addition of 870 water in a glass- of stability. stoppered flask. After two ,hours of hydration, enough petroleum ether (3C-60') was added to the The complete U. V. spectra were determined on alumina t o make a slurry which was poured into a glass column. Fifty grams of deactivated alumina a Cary recording spectrophotometer in petroleum were used for each Gm. of ester. The entire re- ether (3Ck60'). All the esters, except the chloroaction mixture was poured on the column followed acetate, had typical vitamin A spectra (maxima by petroleum ether (30-60"). Anhydrovitamin A 328328 m p ) . The chloroacetate showed impurity was eluted first, followed by the ester. The former peaks a t 310 mp. 341 mp, and 360 mp. Ktamin was distinguished from the latter by its orange color .i sorbate had two peaks, one due to the chromophore of the vitamin A molecule. niaximurn a t 327 in U. V.light; the latter had a yellpm-green color. The ester fraction was evaporated in I'UCUO with mp, and the second due t o the sorbate part a t '755 the aid of a nitrogen bleeder, redissolved in petro- ma. Determination of Rf Values and Purity.--Using leum ether, and rechromatographed as directed above. The residue was subjected to a high vacuum the system described in the previocs paper (6). [6C, (0.5 mm.) for six hours a t 3.5' to remove the pyridine liquid petrolatum i n petroleum ether (3crSO')or other bases. Quinoline and S,S-dimethylaniline ethanol, U. S. P.], the esters were chromatographed were used in place of pyridine for the crotonate on paper as a means of identification and determinaand acrylate esters, respectively. -ittempts a t tion of purity. See Table I\'. The new esters with the exception of vitamin 1 crystallization from a variety of polar, nonpolar, chloroacetatr, showed only one spot on the paper. and combination of both solvents were unsuccessful. The per cent yields are listed in Table 11. Sorbic indicating that they were pure. The chloroncet3te acid chloride was prepared by treating sorbic acid appeared t o be a mistue of anhydrovitamiri -1and with thionyl chloride, s. a., and collecting the frac- ester, indicatiiig the sensitivity of the ester to ethanol. tion that distilled at 79.-80' and 13 nun.

TABLE lI.-PEK

CEST

YIELD ASD SAPOSlFlCATIOS

EQL'IVALESTS O F THESEESTEKS

~-

Vitamin A Esters

9.:

;ic-.-'

1-. ;.

.-..It'.

b

\-iAd

a-Cliloropropii,riatc Chloroace ta t e Trichloroacetate Sorbate Crotonate A,

0

.______

c-

8;

sn 53

YC Deviation

XIolecular \\;eight

Saponification Equivaleut

376 362 ... 381 3.54

3S5 359 ... 38.5

+L'.t;!

3R9 3 17

+2.51 J-2 15

nin

The low sa~wnificalioovalue is an inr?icarion of its poor stability. This ester could not be made under these conditions.

from Thwretical

-9.13"

...

+1.z

JOURNAL OF

460

TABLE IV.-&

THE

AMERICAX PHARMACEUTICAL ASSOCIATION

L’ol. 49, No. 7

VALUESOF VITAMIN A ESTERSAND RELATED COMPOUNDS

Compound

R f Value

Compound

R f Value

Seovitamin A alcohol Vitamin A alcohol Vitamin A methyl ether Vitamin A acetate Vitamin A acrylate

0.960 0.920 0.725 0.651 0.620

Vitamin A sorbate Vitamin A crotonate Vitamin A chloroacetate Vitamin A a-chloropropionate Vitamin A palmitate

0.570 0.W 0.525 0.515 0.100

Stability of These Esters.-The relative stability of these esters and related compounds toward eliminative degradation was determined in: (a) 0.01 N HCI in anhydrous ethanol [water content 0.148% determined by Karl Fischer method ( S ) ] ; and ( b ) 0.01 N HCI in ethanol U.S. P. A sufficient quantity of material, such that the U. V. extinction a t 326 mp would be 0.500, was added t o the acid solutions. These solutions were then transferred to silica cells and placed into a Beckman DU spectrophotometer and remained there through the entire determination. The temperature of the water flowing through the hydrogen lamp housing was maintained a t 15 f 1’. Anhydrovitainin A has a n extinction a t 326 mp equal to 0.2 EW m,,. The latter quantity was subtracted from the E E observed ~ to give a valid measurement of the vitamin A content a t any time @bserr& -0.2 Eaol)us. t was 1. X plot of log linear. Since all the ingredients except vitamin .I were in a large excess the rate was calculated from the slope of the line. The rates are given in Table V.

in the’previous paper ( 6 ) under sections dealing with vitamin -1esters in isopropanol and “95%’’ isopropanol. They were assayed by the method of Cama. el a l . (9). The starting concentrations of these solutions ranged from 6000-8000 u./ml. The initial 45’ rates of decomposition of the various esters are given in Table VI according to the method previously described (6)by plotting log concentration us. time. That the fatty acid did not appear to stabilize vitamin A chloroacetate in “95%” isopropanol is unexplainable a t this time. The rates of the chloroacetate and a-chloropropionate degradation a t 45’ and the other temperatures indicate their extreme sensitivity to solvolysis. The 35’ and 45’ sample of the chloro esters reached equilibrium in five hours, whereas the other esters required twenty to thirty hours to equilibrate. Chromatography of the Reaction Mixtures.-The reaction mixtures from the isopropanolic solutions were paper chromatographed, using the method described previously (6) under the section dealing with vitamin A acetate in “95%” isopropanol. The TABLE V.-RATES OF ELIVISATION OF VITAMIN A identification of the zones was based on their R, SUBSTANCES I N 0.01 .V HCI I N ANHYDROUS ETH- values previously described and U. \‘. spectra A N O L ~A N D ETHANOL U. S. P. in ethanol U. S. P.; vitamin A esters maxima 325-328 mp; anhydrovitamin .I 351, 371, and 391 k k mp; and oxidation products 275, 290, and 310 2,303 X 104 min.-l 2.303 X lo‘ min.-’ mp. Vitamin A _Substance (Anhydrous Ethanol) (Ethanol U. S. P.) All the samples contained the ester originally introduced, vitamin A alcohol, and oxidation Vitamin A chloroacetate b b products. The pcrylate, crotonate, and sorbate Vitamin A samples with fatty acids also contained small alcohol 150.0 6.60 quantities of vitamin A decanoate, and larger Seovitamirr amounts of vitamin .I alcohol than the controls. A alcohol 65.0 .. The samples of the chloro esters with the fatty acids Vitamiti A did not appear to contain vitamin X decanoate. 1.15 methyl ether . 23.0 Vitamin A sorbste 15.0 0.50 DISCUSSION Vitamin A acetate 12.5 1.10 Mechanism of Decomposition of Vitamin A AlcoVitamin A hol and Esters.-The series of esters described in this palrnitatc 9.65 0.30 \?tamin A paper was synthesized, based on the assumption that crotonate 9.35 0.60 decreased electronegativity around the alkyl oxygen Vitaniiri A or carbonyl oxygen wmld decrease proton catalyzed acryl at e 4.77 0.20 attack. Vitaruin A aWheii.the stability of these esters was determined cIil~~qm)in “99:;” and . 95C,:” isopropanol, with and with2.33 6 82 pinnate out a fatty acid present, their stability varied in Water content 0.148%. b Too last to measure. this order:

Tiit. rbters i\trc tcstccl fur stability against elitninative dcgradation in isnprnpanol and dccanoic acid in isopropuml. The si)lvents were ”99‘;” isopropancil and “9.5‘’; ” isiJpropnid (containing O . O S ~ c arid 4.6t;‘-;s water. rcspectiv~~ly, :IS dctcrinined by the Karl Fiwher n i e t h d ( S ) ] . m d a second scries c i ~ ~ ~ t a i i ~O.IX33 i r t g -Y ~ L . C L L I I I Bacid ~C iii both solvaits. The solutions wcre prcparcd as dcscribcd

3

<1 :. I ;5 5

to

5 1

~

,<

-

x Vitamin A trichloroacctate Y .Vitamin A chloroacetate Vitamin A a-cti~oropropionate Vitamin A arrylate Vitaniin A c r i i t ~ i n n t e n Vitamiti A sorbate a L l-itmnin A acetate ; Vitamin h palrnitatc

4 $z .-c (

SCIENTIFIC EDITION

July 1960

461

T ~ LVI.-INITIAL E DECOVPOSITIOS RATESOF VITAMIN A ESTERSIS ISOPROPASOLIC SOLUTIONS AT 45’ (k/2.303X 10’ hr.-*) ‘‘0970*’ Isopropanol

Ester

Isopropanol

7.50 85.00 4.16 56.10 5.75 1.37

Acrylate Chloroacetate Sorbate a-Chloropropionate Crotonate Palmitate“ Acetate” 8

“05%”

22.50 43.20 5.00

5.00 59.0 1.66

83.50

50.0

6.66

...

...

0.033 S Decanoic Acid in “99%” lsopropanol

4.17

0.033 S Decanoic Acid in “9572” Isopropanol

3.13 1.25

...

10.00 83.00 3.22 80.00 4.55

...

2.45

Values derived from ref. (6).

Obviously there was a correlation between the Kn of the acid proton and decreased stability against elimination in isopropanol. The addition of 570 water to the anhydrous media catalyzed the elimination reaction which was to be expected. It appeared t h a t the electronegative group in the 2 position increased the K . of the acid by reduction of electron density around the alkyl oxygen; this factor also resulted in a weakening of the covalent alkyl oxygen bond (X). This weakening rendered i t more susceptible to solvolysis in hydroalcoholic

1

Vitamin A chloroacetatel Vitamin A alcohol Neovitamin A alcohol Vitamin A methyl ether Vitamin A sorbate Vitamin A acetate Vitamin A palmitate Vitamin A crotonate Vitamin A acrylate Vitamin A a-chloropropionate

.-C

-.-.-x Y

2

G M .-

$ t;

2

The reason for the position of the chloroacetate can only be explained by the fact that the -C-& bond is very sensitive to solvolysis in this compound, consequently, ethanol had sufficient solvolytic properties to destroy it rapidly. The stability of the other esters varied directly with the K O of the acid radicals. The degradation rates of these compounds in and alcoholic media as shown by the stability tests. This was further substantiated by the fact HC1-ethanol U. S. P. did not follow any definite that little or no anhydrovitamin -1was formed in pattern. Their rates of decomposition appeared to hydrocarbon solvents. The reaction could be be a composite of proton attack and solvolysis; the acrylate ester was the most stable. This represented as follows: addition of 5% water to the anhydrous media reduced the rate of elimination about 10 times. This appears to be related to a competition of a a t e r ROH molecules and the proton on the cyclohexene ring I ROH ROH for the oxygen of the ethanol, thus reducing the basicity of the media and rate of elimination of the proton.

-

H

R

‘ 0 ’

I

v\ (9’

\\’hen the esters, vitamin A alcohol. rieovitaniin A a1culiu1, arid methyl ether were treated w i t h HC1 in anhydrous ethanol, the stability was changed iri

thisorder:

The differences in stability of these compounds in alcoholic solutions, with and without HCI suggested two mechanisms of decomposition. (a) Profon Catalyzed -4ttacR.-The esters contnining fatty acids with largc Ka’s were attacked the least and this appeared to be related to the reduced electmn dencity nmtcnd t h e nlkyl nr!.,orr!. S i ~ r r the electroriegative effect of hydrogen is less thari that of a c a r h n y l group it is riot surprising that vitamin .I alcnhnl is ninre sensitive to the prutnn catalyzed attack t k u i vitamin .I esters. (i, j Suivoiyric . . i ~ k ~ i . - i i i aicuiiuiic ur iiyiirvaicc1

This compound is an exception to the rule.

462

JOURNAL OF THE :\MERICAN

PHARMACEUTICAL .~SSOCIATIOS

holic media the stability against elimination is dependent on the strength of the carbon-oxygen bond ( S ) . I t has been observed that vitamin A alcohol is more stable than vitamin A esters in uncatalyzed solvent systems (2). It is reasonable to assume that the elcctronegative e5ect of the carbony1 group and the groups in the a position weakened the bond through an inductive effect on the electrons of the covalent bond. The electronegative effect of hydrogen (in vitamin -4 alcohol) is small compared to the strong electronegative groups mentioned. Solvolysis was further substantiated by Higuchi and Reinstein (2) who showed that pyridine in ethanolic vitamin X acetate solutions had no effect on the rate of elimination. Since pyridine would remove protons it appeared that the mechanism was solvolysisand not acid attack. Stabilization by the one mechanism caused a corresponding decrease in stability by the other mechanism, therefore, it would appear most desirable to synthesize a molecule which is stable against solvolytic attack. This could possibly be done by the use of fatty acids with very low K,,values. The combined mechanism of decomposition is as shown above.

SUhfhIARY AND CONCLUSIONS 1. A method of synthesis for esters of vitamin X sensitive to water was developed.

r.01. 49,No.

7

2. T h e rate of eliminative degradation of vitamin A esters in isopropanol. aqueous-isopropanol, and 0.01 N HCI in ethanol was measured. 3. The physical constants for these new esters were determined. 4. T h e previous information on vitamin A alcohol, its esters, and the information in this study was correlated into t h e possible mechanisms of vitamin A decomposition. 5. Paper chromatography was used to determine the reaction products.

REFERENCES (1) Sbantz, E. S l.. Cawley. J. D.. and Embree. N. D.. J . A m . Chon. SOL..65, WI(1943). (2) Higuchi, T.. and Reinstein. J.. THISJ O U U ~ A L 48, ,

I55(1959).

(3) hfeunier. P . . Bull. sac.chim. biol.. 25.371(1943). (4) Ingold. C. K.,“Structure and Mechanisms in Organic Chemistry.” Cornell University Press, Ithaca N.Y. 1953. (5) Baxter, J. G.. and Robeson, C., J . Am.’Chrm.’Soc.. 64.

I.”.\.”.-,.

9dn71i (113)

( 6 ) Forlano. A. J.. and Harris, L. E., THISJOIJRXAL. 49, 451 (1960): (?)~Schr,~ner, R. L.. and Fuson, C. R.. “The Systematic Identrficatron of Organic Compounds,“ John Wiley & Sons. New York. N. Y., 1956. p. 235. (8) ”United States Pharmacopeia,” 15th rev., Slack Publishing Co.. Easton, Pa., 1955. p. 941. (9) Cama. R.,Collins, F. D., and Xtorton, R. A.. Biochrm. J . . 50.48(1932).