[CONTRIBUTION FROM
BIOTIN.
THE
LEDERLE LABORATORIES DIVISION, AMERICANCYANAMID COMPANY]
IX.' A SYNTHESIS OF dl-DEHYDROBIOTIX
S. R. SAFIR, SEYMOUR BERNSTEIN, B. R. BAKER, W. L. McEWEN, Y. SUBBAROW
AND
Received January 14, 1947
One of the methods contemplated for the synthesis of biotin called for the ,3,4,6-tetrahydro-l-thieno[3,4]imipreparation of 2-ket0-4-(8-carboxybutyl)-Z dazole (XV), which we have called dehydrobiotin. The transformation of dehydrobiotin into biotin by the addition of a molecule of hydrogen was a problem which would have to be faced when the synthesis of dehydrobiotin had been achieved. The main purpose in seeking dehydrobiotin as the key intermediate lay in the fact that separation of isomers would be deferred until the final step. Moreover, the fact that the urea bridge was already present in the dehydrobiotin molecule held out the hope that, if reduction could be made t o take place, it might, yield cis isomers exclusively. Since the completion of this work Harris and others (1) have argued, on the basis of resistance to hydrolysis, that biotin probably has the cis configuration with respect to the nitrogen atoms. In addition to the above reasons, it was of interest to find out whether or not dehydrobiotin itself possessed any biotin-like activity. The present paper reports the total synthesis of dl-dehydrobiotin. In the following paper in this series the synthesis of a model compound, 2-keto-4-methyl-2,3,4,6-tetrahydro-l-thieno[3 ,4]imidazole, will be presented. The starting material, 7-carbethoxy-2-heptenoic acid (I) (2), was treated with mercuric acetate in methanol, utilizing the excellent procedure of Carter and others (3) for the preparation of serine and threonine. The crystalline addition product [probably 2-acetoxymercuri-3-methoxy-7-carbethoxy-heptanoic acid (II)] was brominated with bromine dissolved in aqueous potassium bromide solution to give 2-bromo-3-methoxy-7-carbethoxyheptanoicacid (111) as an oily mixture of the two racemates. The latter was hydrolyzed in the cold with alkali to give 2-bromo-3-methoxysuberic acid (IV) in the form of an oil which was probably a mixture of the two possible racemates. The yield of IV from I was 72%. Treatment of the bromo acid IV with ammonia a t 100" gave 2-amino-3methoxysuberic acid (V) of which 36% was isolated as a pure crystalline isomer. In addition, there was present in the mother liquor a t least an equal amount of V which could be used in subsequent transformations. The amino acid V was benzoylated by the Schotten-Baumann method t o give a 97% yield of 2-benzamido-3-methoxysuberic acid (VI) which upon treatment with benzoyl chloride 1 Paper
VI11 in this series is to be found in J . Org. Chem., 12, 328 (1947). 475
476
SAFIR, BERNSTEIN, BAKER, MCEWEN, SUBBAROW
(CH2)4 CH=CHCOOH
Hg(OCOCHJ2 CHsOH
I
COOC ~ H ~
(CH2)4 CH-CHCOOH
1coo I
I
OCH3 HgOCOCH3 C2H5 I1
I
I
Brz
lKBr (CH2)4 CH-
(CH2)4 CH-CHCOOH
I
I
I
1
t---------
OCH3 Br COOH IV
CH CO OH
I
I
OCH3 Br COO C2Hs I11
NaoH
~NH’
(CH2)4 CH-CHCOOH
(CH2)d CH-CHCOOH
I
OCH3 NHz I
1
co CSI-IS
COOH
COOH
v
VI
(CH2)4CH=C-CO
I
I
1
N 0 \C/
COOH
I
CeHri
VI1
11
1
( CHO c 0)2 0 NaO C 0 CHI
HC1
(CH2)4 CH=C-CO
(CH2)4CH=CCOOH
I NH I
I
COOH
H CI
c -
I
I
COOCzHrj N
co C B H ~
IX
\C/
I
I
0
CsHa
VI11
SYNTRESIS OF
(CH2)6 CH=C-CO
I
I
COOH
477
CEZ-DEHYDROBIOTIN
I
0
N
I
CeHs
VI1
I '
NHCO CsH6
SCHz COO C2H5
XI
COC6H5
0 NH2.HC1
I
0 NH
I l l
( S / L C HI 2 ) . COOH XIV
I
co
/ \
lKCNO
NH NH
/=I
COOCnHs XI11
478
RAFIR, BERNSTEIN, BARER, MCEWEh', SUBBAROW
and pyridine yielded the azlactone VIP mixed with benzoic acid. The crude mixture was esterified with ethanol and sulfuric acid and gave, after the removal of ethyl benzoate, 70% of ethyl 2-benzamido-7-carbethoxy-2-heptenoate(X). The free .acid IX corresponding to X was obtained in a pure, crystalline form from the interaction of ethyl &formylvalerate with hippuric acid followed by acid hydrolysis. I X was also obtained from an acid hydrolysis of the crude azlactone VII. The addition of ethyl thioglycolate to X took place in the presence of piperidine to give ethyl 2-benzamido-3-(carbethoxymethylthio)suberate (XI) in the form of an oil. The latter was found to undergo the Dieckmann reaction in the presence of sodium ethoxide and dry ether to give a crystalline sodium salt which on acidification yielded 2-(b-carbethoxybutyl)-3-benzamido-4-keto-5carbethoxythiophane (XII). The latter was obtained as an oil in 17% yield from X and was characterized as the 2,4-dinitrophenylhydrazone. The keto ester XI1 and its sodium salt give a dark green color with alcoholic ferric chloride. Hydrolysis of XI1 with a mixture of alcohol and 12 N hydrochloric acid led to stripping of the 5-carbethoxy group and gave 2-(6-carbethoxybutyl)-3-benzamido-4-ketothiophane (XIII) which was characterized as the phenylhydrazone and as the 2,4-dinitrophenylhydrazone. More drastic hydrolysis of XII, with a mixture of acetic and hydrochloric acids, stripped the benzoyl group and the ester group of the side-chain as well as the 5-carbethoxy group, to give 2-(6-carboxybutyl)-3-amino-4-ketothiophane hydrochloride (XIV). The latter substance upon treatment with potassium cyanate in water yielded dl-dehydrobiotin (XV). The yield of pure XIr from XI1 was 28%. Dehydrobiotin was found to be biologically inactive with L. casei. Acknowledgment. The authors are grateful to Messrs. Louis Brancone, Louis Dorfman, Philip Weiss, and Miss Barbara Eames for the microanalyses and to Mr. A. C. Dornbush for the biological assays. EXPERIMENTAL
8-Bromo-3-methosysubericacid ( I V ) . A solution prepared from 20 g. of 7-carbethoxy-2heptenoic acid (2)) 31.9 g. of mercuric acetate, and 190 cc. of methanol was shaken at room temperature for two days. The white, crystalline addition product (11) was filtered, wwhed with methanol, and airdried; yield 37.6 g. To a cold (5"), gently stirred solution of 37.6 g. of I1 and 17.9 g. of potassium bromide in 100 cc. of water was added, dropwise, during the course of one hour, a solution composed of 16 g. of bromine, 17.9 g. of potassium bromide, and 35 cc. of water. The reaction was carried out in an open beaker illuminated with a 200-watt photoflood lamp. The mixture 2 The lability of a &hydroxy group under azlactonizing conditions was discovered by Erlenmeyer, Jr. and Friihstuck (4) in the conversion of p-phenylserine into the azlactone of a-acetamidocinnamic acid. Carter e t al. (5) showed that methoxy and acetoxy groups were also labile. Thus X-benzoyl-dl-allothreonine, PI'-benzoyl-0-methyl-dl-threonine, and Nbenzoyl-O-acetyl-dl-allothreonineall were found by Carter's group to give the same aalactone upon treatment with benzoyl chloride and pyridine.
SYNTHESIS OF
dZ-DEHYDROBIOTIN
479
waa stirred an additional fifteen minutes, the slight excess of bromine was removed with sodium bisulfite, the solution was shaken once with ether, and the aqueous layer was acidified to pH 1-2. The mixture was extracted with ether, the ether solution was washed with saturated salt solution, dried with magnesium sulfate, and evaporated to dryness. A nearly colorless oil composed of I11 was obtained; yield 23.3 g. A stirred mixture of 6.2 g. of I11 and 8.4 cc. of 0.5 N alkali was cooled to 15-20' and treated during thirty-five minutes with 8.4 cc. of 5 N alkali. The mixture waa stirred an additional fifty minutes at 25", washed once with ether, and acidified. The solution was saturated with salt and extracted with ether. The ether solution was washed with saturated salt solution, dried, and the solvent was evaporated. The resulting 2-bromo-3methoxyeuberic acid (IV) was obtained as a colorless oil; yield 5.4 g. Anal. Calc'd for CsHl6BrOa: C, 38.2; H, 5.3; neutr. equiv., 142. Found: C, 38.6; H, 5.6; neutr. equiv., 146. It is very probable that IV is a mixture of the two theoretically possible racemates. 8-Amino-3-methoxysuberic acid (V). A solution of 56 g. IV in 600 cc. of 28% ammonia was heated in a glass-lined autoclave for six hours at 100". The solution was treated with Norit, filtered, and the filtrate was evaporated to dryness i n vacuo. The gummy residue was dissolved in water and the solution was again evaporated to dryness i n vacuo. This process was repeated twice more. Finally, the residue was slurried in water and filtered. The yield of crystalline, nearly white amino acid V was 15.4 g. (36%), m.p. 235-237" (dec.). After one recrystallization from water the m.p. was unchanged. Anal. Calc'd for CsHlrNOs: C, 49.3; H, 7.8; N, 6.4. Found: C, 49.2; H, 6.9; N, 6.4. The mother liquor remaining after filtration of the crystalline amino acid was found to contain at least an equal amount of V (probably a mixture of racemates). This could be utilized by acidifying the solution to pH 3, evaporating to dryness, and benzoylating the resulting crude residue. R-Benzamido-3-methoxysubericacid ( V I ) . To an ice-cold, stirred solution of 2.8 g. of V in 25.6 cc. of N alkali was added, alternately, during one-half hour, 3.6 g. of benzoyl chloride and 38.4 CC. of N alkali. After being stirred for an additional fifteen minutes, the mixture was acidified and extracted with ether. The ether solution was dried with magnesium sulfate and evaporated to dryness. The gummy residue was leached three times with hot petroleum ether (b.p. 90-100"). The residual colorless gum consisted of 4.0 g. of nearly pure VI. Anal. Calc'd for ClaHsrNOs: C, 59.5; H, 6.5; N, 4.3. Found: C, 58.8; H, 6.5; N, 4.8. Ethyl %-benzamido-7-carbethoxy-8-heptenoate ( X ). To an ice-cold solution of 10.1 g. of VI in 75 cc. of dry pyridine was added, with swirling, 8.8 g. of benzoyl chloride. After iifteen minutes at 0" the mixture was poured into cold, dilute hydrochloric acid containing 100 cc. of the 12 N acid. The mixture was saturated with salt and extracted with ether. The ether layer was washed with dilute hydrochloric acid, then with saturated salt solution, dried with magnesium sulfate, and evaporated to dryness. The residue, 15.6 g., consisted of a mixture of the azlactone of 8-benzamid0-7-carboxyd-heptenoic acid ( V I I ) and benzoic acid. The crude product was dissolved in 100 cc. of dry ethanol containing 5 cc. of sulfuric acid, 100 cc. of benzene was added, and the whole was refluxed sixteen hours in a Soxhlet apparatus containing magnesium sulfate in the thimble. The resulting solution was cooled, diluted with an equal volume of water, the mixture was saturated with salt and extracted with ether. The ether solution was washed i n succession with water, bicarbonate solution, and finally with water. After drying, the solution was evaporated in vacuo. In order to remove the ethyl benzoate, the residual oil was distilled from a water-bath at 80"/0.5 mm. The product (X) was obtained as a light yellow oil remaining in the distillation flask; yield 7.7 g. (70% from VI).
480
SAFIR, BERNSTEIN, BAKER, MCEWEN, SUBBAROW
Anal. Calc'd for CleHzsN05:C, 65.7;H, 7.2;N,4.0. Found: C, 64.4;H, 7.4; N,3.5. I-Benzumido-7-carboxy-I-heptenoic acid ( I X ) . A number of experiments were performed on the condensation of ethyl b-formylvalerate (2) with hippuric acid to form the uzEuctone of I-benzamido-7-carbethoxy-2-heptenoicacid ( V I I I ). The crude products were then subjected to acid hydrolysis and, in the following procedure, an 8% yield of IX was obtained. A mixture of 24.6 g. of freshly distilled ethyl b-formylvalerate, 21.4 g. of hippuric acid, 55 g. of acetic anhydride, and 9.8 g. of anhydrous sodium acetate was stirred vigorously at 55" for five and one-quarter hours. The resulting, nearly clear, orange solution w w poured into cold water. The mixture was extracted with ether and the ether solution was evaporated to dryness in vucuo,the bath temperature being finally raised to 85". The residue was a light brown oil consisting of 39.9 g. of impure VIII. A mixture of 5 g. of the latter, 100 cc. of 3-A alcohol, and 200 cc. of 2 N hydrochloric acid was refluxed one-quarter hour, then concentrated in vucuo to 50-60 cc. The clear solution was decanted from the gum, and on cooling and standing 348 mg. (8%) of I X separated in the form of colorless crystals; m.p. 187-188". The m.p. was unchanged after one recrystallization from water. A mixture of I X and hippuric acid (m.p. 187-188') melted a t 165475". Anal. Calc'd for CI5Hl7N05:C, 61.8;H, 5.8;N,4.8;neutr. equiv., 146. Found: C, 62.1;H, 5.6;N,4.6;neutr. equiv., 146. Similarly, acid hydrolysis of the crude mixture of azlactone VI1 and benzoic acid obtained from the reaction of VI with benzoyl chloride and pyridine gave an acid identical in m.p. and mixed m.p. with I X . The independent synthesis of IX by the two described methods confirms the presence of the azlactone structures assigned to VI1 and VIII. Ethyl B-benzamido-3-(curbethoxymethylthio)suberute( X I ) . A mixture of 101 g. of X, 42 g. of ethyl thioglycolate, and 250 cc. of dry ethanol was treated with 2 cc. of piperidine. The mixture was kept at 25" for seventeen hours, then refluxed seven hours. The solution was evaporated to dryness in vucuo,the resulting oil was dissolved in ether, the ether solution was cooled to 0" and washed in succession with ice-cold N alkali and saturated salt solution, dried with magnesium sulfate, and evaporated to dryness. The yield of X I was 90 g. Anal. Calc'd for CzaHIaNO,S:C, 59.1;H, 7.1;N,3.0. Found: C, 58.2;H, 6.9;X, 3.0. 2-(b-CarbethoxybutyE)-3-benzamido-~-keto-6-curbethoxythiophane (XIZ). The sodium ethoxide obtained by evaporation of a solution of 9 g. sodium in absolute ethanol followed by baking in vacuo at 125-130"was suspended in 300 cc. of dry ether and a solution of 90 g. of XI in 600 cc. of ether was admitted, with cooling, under a nitrogen atmosphere. The stoppered flask was shaken a t room temperature for one day and the contents were poured into iced water. The crystalline sodium salt was filtered, washed with water and ether, and then added with stirring to cold, dilute excess acetic acid. The mixture was extracted with ether, the ether layer was washed thoroughly with sodium bicarbonate solution followed by saturated salt solution, dried, and evaporated to dryness in vacuo. The residue consisted of 20.6 g. (17y0from X) of the keto ester XI1 in the form of B very light yellow oil. Anal. Calc'd for C21Hz,NOaS:C, 59.7;H, 6.4;N,3.3. Found: C, 59.4; H, 7.0; N,3.3. XI1 and the sodium salt of X I 1 give a dark green color on treatment with alcoholic ferric chloride. The 2,4-dinitrophenylhyd~azoneseparates in lemon-yellow needles from a solution of XI1 in 2,4dinitrophenylhydrazine reagent (prepared by dissolving 2 g. of the hydrazine in 300 cc. of 3-A alcohol and 25 cc. of 12 N hydrochloric acid). The m.p. after one recrystallization from benzene-alcohol was 191-192" (dec.) . AnaE. Calc'd for Cz,HalNsOsS:C, 53.9;H, 5.2;N , 11.7. Found: C, 53.8;H, 5.0;N,11.4.
SYNTHESIS OF
dl-DEHYDROBIOTIN
481
R-(&-Carbethoxybutyl)-S-benzamido-~-~etothiophane ( X I I I ) . L4 mixture of 1.4 g. of X I I , 17 cc. of 3-A alcohol, and 14 cc. of 12 N hydrochloric acid was refluxed forty minutes; 3 cc. of alcohol and 2 cc. of 12 N hydrochloric acid were added and the mixture was refluxed an additional five minutes. An equal volume of water was added, the mixture was saturated with salt and extracted with ether. The ether layer was washed with saturated salt solution until the washings were neutral, dried, and evaporated. The ketone XI11 was obtained as a light yellow oil; yield 0.85 g. Anal. Calc'd for C18H23NO&: C, 61.9; H , 6.6; K',4.0. Found: C, 62.0; H, 6.5; N, 3.2. I n contrast to XII, XI11 gives a negative color reaction with alcoholic ferric chloride. The 2,4-dinitrophenylhydrazoneseparates in yellow needles from an ice-cold solution of 2,4-dinitrophenylhydrazinein alcoholic-hydrochloric acid; m.p. 16&170°. The m.p. was unchanged after one recrystallization from 3-A alcohol. Anal. Calc'd for C2dH2,NaOlS: C, 54.4; H, 5.1; N, 13.2. Found: C, 54.0;H, 5.0; N, 13.3. The phenylhydrazone of XI11 separates as colorless crystals from 60% alcohol; m.p. 145147'. Anal. Calc'd for C Z ~ H Z ~ N ~C,O65.6; ~ S : H, 6.6; S , 9.6. Found: C, 65.7; H, 7.3; N, 9.8. 2 -Keto -4-(6-carboxybutyl) -2,3,4,6 -tetrahydro-1 - thieno[3,4]imidazole (dl-dehydrobiotin) (XV). A mixture of 3.1 g. of X I I , 20 cc. of acetic acid, and 20 cc. of 6 N hydrochloric acid was refluxed five and one-half hours. During the first three hours 20 cc. of 12 N hydrochloric acid was added in five portions. The mixture was cooled to 5" and filtered from benzoic acid. The filtrate was diluted with an equal volume of water and extracted thoroughly with ether. The aqueous solution was evaporated to dryness in vacuo, the light brown crystalline residue was dissolved in 30 cc. water, treated with Norit, filtered, and the filtrate was evaporated to dryness. The residue consisted of 1.1 g. of crude %(&-carboxybutyl)-S-amino-4-ketothiophanehydrochloride ( X I V ). The latter was dissolved in 10 cc. of water, the solution was treated mith Norit and filtered. To the filtrate was added a solution of 0.7 g. of potassium cyanate in 5 cc. of water. The nearly colorless solid which separated was filtered and washed with a small amount of cold water; yield 700 mg.; m.p. 168-170". One recrystallization from water gave 491 mg. (28y0 from XII) of dehydrobiotin as colorless crystals, m.p. 175-176". The m.p. was not raised upon further recrystallizations. Anal. Calc'd for C,oHl,N20aS: C, 49.6; H, 5.8; N, 11.6; S, 13.2. Found: C, 49.8; H, 5.5; N, 11.3; S, 13.4. Dehydrobiotin is relatively insoluble in cold water, quite soluble in hot water, and soluble in sodium bicarbonate solution. The methyl ester was obtained by refluxing a mixture of 0.5 g. of XV, 60 cc. of methanol, 60 cc. of chloroform, and 0.5 cc. of sulfuric acid seven hours in a Soxhlet apparatus containing anhydrous magnesium sulfate in the thimble. The mixture was diluted with water, the chloroform layer was separated and washed with bicarbonate solution followed by water, dried and evaporated to dryness in vacuo. The residue, upon recrystallization from water gave 200 mg. colorless crystals, m.p. 123-124'. Anal. Calc'd for C11H&'20$3: C, 51.6; H, 6.3; N,10.9. Found: C, 51.8; H, 6.2; N, 10.7. SUMMhRY
Addition of ethyl thioglycolate to ethyl 2-benzamido-7-carbethoxy-2-heptenoate and Dieckmann cycl zation of the resultant adduct gives 2-(6-carbethoxybutyl)-3-benzamido-4-keto-5-carbethoxythiophane.Drastic acid hydrolysis of the latter forms 2-(6-carboxybutyl)-3-amino-4-ketothiophanehydrochloride,
482
SAFLR, BERNSTEIN, BAKER, MCEWE", SUBBAROW
which, on treatment with potassium cyanate, is readily converted to dl-dehydrobiotin. PEARLRIVEB,N. Y. REFERENCES
(1) HARRIS,MOZINGO, WOLF,WILSON,AND FOLKERS, J . Am. Chem. Soc., 67, 2102 (1945). MOYER,ANSLOW,JR., BAKER,QUERRY,BERNSTEIN,AND SAFIR, (2) BROWN,ARMSTRONG, J. Org. Chem., 12, 160 (1947). (3) CARTERAND WEST,Organic Syntheses, 20, 81, 101 (1940). JR. AND FROHSTUCK, Ann., 284, 36 (1895). (4) ERLENMEYER, (5) CARTER, HANDLER, AND MELVILLE, J . Biol. Chem., 129, 359 (1939).
