[CONTRIBUTION BBOY
THE
LEDERLELABORATORIES DIVISION, . ~
R I C A NCYANAMIDCo.]
BIOTIN. VII. SYNTHESIS OF dl-epiBIOTIN B. R. BAKER, W. L. McEWEN,
AND
W. N. KINLEY
Received October 28, 19.46
In:? previous communication (1j were described the syntheses of dl-biotin and dl-epiallobiotin. The key intermediate for their preparation was 3-phenyl6-(b-carboxybutylj-5,6,8,9-tetrahydrothieno[3,4 ,e ,cis]uracil (111)prepared by inversion of the configuration of the 3-carboxyl group of 2-(b-carboxybutyl)4-uranilinothiophane-trans-3-carboxylicacid (11)to cis followed by ring closure. NH-C 0-N CaH6
NHCONHCsHj
I
COOH (CHJdCOOH
(CHhd COOH
trans I1
trans
I
Cis
I11 CsHs
I
CO-N-CO
COOH i-JNHCONHCd& \
__$
I
T [NH
(CHJrCOOH
(CHJ4COOH
trans
cis
IV
V
It should theoretically be possible to obtain the configuration of dl-epabiotin, which is epimeric to dl-biotin a t the C2 position, by inversion of the 4-carboxyl of a molecule such as 2-(b-carboxybutyl)-3-uranilinothiophane-trans-4-car-
cz + ct - + c4 - +
CHART I
Va c 2 c 3
c 4
+ -+ - +
+-+ c4 + Ia cz - + -+ c4 + c2
invert +--Ch
-
invert CC
t -
c 3
c 3
vb
+++IIIa -+ cs + +cz
invert
cs
c 3
c 4
c 2
invert -+
ca
c 4
Ib IIIb boxylic acid (IVj (synthesized from the triacid, I, by Curtius degradation of the 3-carboxyl group) to cis followed by ring closure to the thienouracil, V, no matter what the absolute configuration of the C2 side chain is in I, I1 and IV. This is readily demonstrated as in Chart I. 322
BIOTIN.
323
VI1
The trans-triacid racemate, I, may have either the total configuration Ia or Ib. The uranilinodiacids, I1 and IV, have the same configuration as I. If the configuration of the triacid is Ia, then inversion a t C3 (in the transformation I1 111) will give configuration IIIa. In contrast Ia, when inverted a t Cd (in the transformation IV =+ V) will give configuration Va. Both Va and IIIa
-
CHART I1 CONHCsHb
CONHCaHs
L
trans "I
trans
VI1
VI11
I -1
-1
I
CsHs CONHCsHs
I
CO-N-CO I
NH ~Z::CONHNHZ
'ZHZ),COOH
cis IX
v
are related cis a t C3 and C4, but are epimeric a t CI. The same argument may be applied if the configuration of the trans-triacid is Ib. Thus, both cis isomers of the biotin structure may be synthesized by utilization of this principle, which was modified for simplification in syntheses of dl-epbiotin as shown in Chart
11. 2-(6-Carbomethoxybutyl)-3-carbomethoxy-truns-4-carbanilidothiophane(VI) (l), when treated with one equivalent of alkali, was predominately selectively hydrolyzed a t the 3-carbomethoxy group to give VII. The monoester was characterized as the hydrazide, IX, soluble in both acid and alkali. The free carboxyl group was converted to the azide via the acid chloride with sodium azide. When the azide was heated in acetic anhydride containing sodium acetate, it rearranged t o the intermediate isocyanate, VIII. The reagents then caused inversion of the 4-carbanilido group from trans to cis followed by ring closure t o a cis-uracil.' The 1-acetyl and ester side chain groups were removed by acid hydrolysis. The resultant product was 3-phenyl-8-(6-care,cis]uracil(V). When the hydrazide, boxybuty1)d ,6,8,9-tetrahydrothieno[3,4, IX, was converted t o the azide and the latter treated under the same conditions, no crystalline product could be isolated. The ring closure of the iso1 The feasibility of this reaction was demonstrated on a model compound without the side chain, namely 4-carbanilidothiophane-3-isocyanate.
324
BAKER, MCEWEN, AND KINLEY
cyanate, VIII, to the uracil, V, proves that the monoester, VII, has the structure indicated and not that of the isomeric 2-(d-carboxybutyl)-3-carbomethoxytrans-4-carbanilidothiophane. The uracil, V, mas converted to dl-epzbiotin (XIV) in the same manner that the uracil, 111, of epimeric configuration was transformed into dl-biotin (1). The carboxyl group was blocked as the anilide, X. The uracil ring was easily ruptured with hydrazine forming the cis-hydrazide, XI.2 The latter was degraded to 2-(d-carbaniliclobutyl)-4-carbanilido-5-ketoimidazolido[4 ,5,c ,cislthiophane (XII) by butyl nitrite and hydrogen chloride in butanol. The blocking groups were removed by hydrolysis with barium hydroxide solution a t 170" and the resdtant diamine sulfate, XIII, treated with phosgene. The final product was dl-epzbiotin, XIV, r11.p. 190-191'. CsH6
I
0
co--N-c
v -
I
lr",
\ S /(CHI
-
ONHC6H5
F \
N
H
/(CHz)aCOOH
XIV
NH,.HzSOr c--
1"'
,!
XI11
~
I lo II / c\
II / c\ \
H
XI
0
I_/
E
(CHJrCONHCeH6
X
NH NH
C
+-
(CHdrCOOH
NH NCONHCeHj
b)
(CH2)4 C ONHCsH6
XI1
dl-epiBiotin was biologically inactive when assayed with S. cerewisiea. That it was epimeric to dl-biotin a t the Czposition was demonstrated by desulfurization with Raney nickel to the biologically active dl-desthiobiotin (2). Dittmer, Melville, and duVigneaud (3) have shown that yeast can utilize desthiobiotin by converting it to biotin. The fact that epzbiotin is inactive indicates that this microorganism cannot transform epzbiotin to biotin via desthiobiotin by removal and reintroduction of the sulfur atom t o give the biotin configuration. Thus, starting with only a single configuration, that of 2-(d-carboxybutyl)thiophane-3,4-trans-dicarboxylicacid (I), any one of three configurations related to biotin can be prepared a t will, namely, dl-biotin, dl-epiallobiotin, and dlepzbiotin. No attempt has been made to prepare the last of the four possible configurations, that of the known allobiotin (4),starting with I.
* It is interesting to note that the aniline residue used for a blocking group on the C4 functional group has now been transformed into the blocking group for the Cs functional group.
BIOTM.
VI1
325
Acknowledgment: The authors are indebted to Dr. Y. SubbaRow for his helpful suggestions, t o Mr. A. Dornbush for the microbiological assays, and to Messrs. Phillip Weiss and William Fulmor for the microanalyses. EXPERIMENTAL
8-(6-Carbomethoxybutyl) -4-carbanilidothiophane-trans-S-earboxylic acid (VZZ). T o a warm solution of 7 g. of 2-(6-carbomethoxybutyl) -3-carbomethoxy-trans-4-carbanilidothiophane (VI) (1) in 70 cc. of methanol was added 7 cc. of 10% sodium hydroxide solution. After seventeen hours at room temperature, the solution was concentrated on the steam-bath with an air stream to remove most of the methanol, then diluted with water containing sodium bicarbonate. The unchanged starting material (1.7 g.) was removed and the filtrate acidified; yield, 4.7 g. (69%) of product, m.p. 180-195", suitable for the next step. For analysis a sample was leached with hot ethyl acetate and the insoluble portion recrystallized from hot Methyl Cellosolve by dilution with water, white crystals, m.p. 207209'. Anal. Calc'd for ClsH,NO&4: C, 59.2; H, 6.3; N, 3.8. Found: C, 59.1; H, 6.1; N , 3.5. The 1.7 g. of unchanged diester was retreated as described above. An additional 1.1 g. (16%) of product, m.p. 185-205" was obtained. 2-(6-Carboxhydrazidobutyl) -~-cu~banilidothiophane-trans-d-carboxylic acid ( I X ) , A solution of 280 mg. of pure 2-(6-carbomethoxybutyl)-4-carbanilidothiophane-trans-3-carboxylic acid (VII) in 0.6 cc. of 100% hydrazine hydrate was heated on the steam-bath for twenty minutes, then diluted with about 20 cc. of water, acidified with 2 cc. of acetic acid, and cooled in an ice-bath. The product consisted of white crystals, m.p. 243-245'dec.; v e l d , 230 mg. (82%). This compound readily dissolves in dilute acid or alkali. Anal. Calc'd for C1,HzsNp04S:C, 55.8; H, 6.3. Found: C, 55.4; H, 6.0. trans-3-Carbomethoxy-4-carbanilidothiophune.A mixture of 8 g. of trans-3-carbomethoxythiophane-4-carboxylic acid (5), 8 cc. of thionyl chloride and 40 cc. of benzene was refluxed for twenty minutes, then evaporated t o dryness in vacuo. The evaporation was repeated after the addition of 50 cc. of benzene. The residual acid chloride, dissolved in 50 cc. of benzene, was treated with 16 cc. of aniline. After two minutes, the mixture was diluted with 50 cc. of ethyl acetate and washed with dilute hydrochloric acid. The turbid organic layer was evaporated to dryness i n vacuo and the residue extracted with 150 C C . of hot benzene. The filtered solution was diluted with 300 cc. of petroleum ether and the product collected; yield, 7.2 g. (6573, m.p. 147-149'. Recrystallization from benzene gave voluminous white needles, m.p. 15C-151". Anal. Calc'd for Cl~HlsKOsS:N , 5.3. Found: N , 5.5. Hydrolysis with hot dilute alcoholic alkali gave trans-4-carbanilidothiophane-3-carboxylic acid in 93% yield, m.p. 223-224". Anal. Calc'd for C12HllN03S: C, 57.3; H, 5.2. Found: C, 57.4; H, 4.9. trans-4-Carbanilidothiophane-3-carboxhydrazide. A mixture of 3.8 g. of trans-3-carbomethoxy-4-carbanilidothiophane and 16 cc. of 100% hydrazine hydrate was heated t o boiling. The resultant solution was heated on the steam-bath for forty-five minutes, then diluted to about 75 cc. with water. The solid was washed with water; yield, 3.7 g. (97%), m.p. 198-200". For analysis a sample was recrystallized from 50% alcohol, white crystals, m.p. 200-201". Anal. Calc'd for C1SH15N302S: N, 15.8. Found: N , 15.7. l-Aeetyl-S-phenyl-6,6,8,9-tetrahydrothieno[S,4,e,cis]uracil. T o a solution of 320 mg. of trans-4-earbanilidothiophane-3-carboxhydrazide in 4 cc. of 0.5 N hydrochloric acid cooled in an ice-bath was added with stirring over a period of five minutes a solution of 100 mg. of sodium nitrite in 1 cc. of water. The gummyazide was extracted with reagent ethyl acetate
326
BAKER, McEWEN, AND KINLEY
and the extracts dried with magnesium sulfate a t 0". The solution was refluxed for twenty minutes, then evaporated to dryness in vacuo. The residual isocyanate was refluxed with 150 mg. of anhydrous sodium acetate and 3 cc. of acetic anhydride for twenty minutes. Dilution with about 20 cc. of water gave white crystals, m.p. 236-239";yield, 230 mg. (66%). h mixture with this same compound prepared by a different method (5)gave no depression in m p. trans-3-Carbetlioxyamino-4-carbanilidothiophane. trans-4-Carbanilidothiophane-3-carboxhydrazide (3.2 9.) was converted to the azide in ethyl acetate solution as described above. The solution was diluted with 35 cc. of absolute ethanol, refluxed fifty minutes, then evaporated. The solid residue was heated to boiling with 50 cc. of absolute ethanol, then cooled in an ice-bath; yield. 1.5 g. (420/,),m.p. 221-223". Additional material was obtained from the filtrate. For analysis a sample was recrystallized from aqueous Methyl Cellosolve, white crystals, m.p. 225-226". Anal. Calc'd for Cl~Hl~N208S: C, 57.2;H, 6.2;N,9.5. Found: C,57.9;H, 6.4; N,9.5. Attempts to convert this urethan to l-acetyl-3-phenyl-5,6,8,9-tetrahydrothieno[3,4,e,cisluracil by boiling with acetic anhydride containing sodium acetate for two hours via inversion of the configuration and elimination of the elements of ethanol resulted in unchanged starting material. J-Phenyl-8-(6-earboxybutyl) -6,6,8,9-tetrahydrothieno[J, 4,e,eis]uracil (V). A mixture of 7.0 g. of 2-(6-carbomethoxybutyl)-J-carbanilidothiophane-trans-3-carboxylicacid (VII), 25 cc. of dry ether containing 0.5% pyridine, and 18 cc. of thionyl chloride was shaken occasionally for thirty minutes. The mixture was evaporated t o dryness in vacuo (bath 50") and the evaporation repeated s.fter addition of 25 cc. of chloroform. The residual acid chloride, dissolved in 50 cc. of acetone at O', was added to a stirred and ice-cooled solution of 5 g. of sodium azide in 50 cc. of water over a period of five minutes, 10 cc. more of acetone being used as a rinse. After being stirred for thirty minutes a t O", the mixture was diluted to 250 cc. with ice-water containing 15 cc. of saturated sodium bicarbonate solution. The slightly gummy azide was washed with ice-water and dried at room temperature at 1 mm. T o the dry azide was added 105 cc. of acetic anhydride and 3.5 g. of anhydrous sodium acetate. On being heated, the mixture rapidly evolved nitrogen. After being refluxed for one hour, the hot mixture r z t s cautiously diluted with 52 cc. of water. When the acetic anhydride had decomposed, the solution was diluted with 52 cc. of concentrated hydrochloric acid and refluxed thirty minutes more. The solution was concentrated in vacuo until the product started t o separate, then i t was diluted to about 250 cc. with ice-water. The product was washed with water and slurried with ethyl acetate, then washed with ethyl acetate (about 100 cc.) until no more color was removed; yield, 2.7 g. (41%), m.p. 203-208". Recrystallization from aqueous methanol did not change the m.p. Anal. Calc'd for C1~H2~TLT2O4S: C, 58.7;H,5.8;K , 8 0. Found: C, 59.0;H,5.4;N,8.3,7.9. 2-(6-Carbanilidobut yl)-3-uranilinothiophane-cis-.&carboxhydrazide ( X I ) . A mixture of 0.90 g. of 3-phenyl-8-(6-carboxpbutyl)-5,6,8,9-tetrahydrothieno[3,4,e,c~s]uracil (V), 18 cc. of chloroform, 9 cc. of thionyl chloride, and 0.05cc. of pyridine was refluxed for five minutes, then the solution was evaporated to dryness in vacuo. Suspended in 20 cc. of acetone, the acid chloride was treated with 3 cc. of aniline and heated on the steam-bath for about five minutes until all the acid chloride had reacted. The mixture was poured into 160 cc. of water containing 10 cc. of concentrated hydrochloric acid. The 3-phenyl-8-(6-carbanilidobutyl)-5,6,8,9-tetrahydrothieno[3,4,e,cis]uracil(X) was rashedwithwater; yield, 1.05g. (90%). The anilide could not be satisfactorily purified for analysis. A mixture of 1.05g. of the anilide and 10 cc. of 100% hydrazine hydrate was heated on the steam-bath for five minutes. A nearly clear solution rapidly formed, then the product began to separate. The mixture was diluted with water and the product removed; yield, T20 mg. (65%), m.p. 193-195'. A similar preparation was obtained as a white solid, m.p. 195-197", and was analyzed. Anal. Calc'd for C B H ~ ~ N ~ ON, S S15.4. : Found: K,15.4.
BIOTIN.
327
VI1
The yield of hydrazide varied from W80% depending upon the purity of the uracil anilide, X. 2-(8-Carbanilidobutyl) -4-carbanilido-b-ketoirnidazolido[4,6, c ,cislthiophane (XZZ) To a hot solution of 720 mg. of 2-(6-carbanilidobutyl)-3-uranilinothiophane-n's-4-carboxhydrazide (XI) in 7.2cc. of dry butanol containing 85 mg. of dry hydrogen chloride was added 0.21 cc. of butyl nitrite. The solution, although gas evolution was complete in five minutes, was heated on the steam-bath for thirty minutes, then evaporated t o dryness in vacuo. The residue was heated to boiling with 10 cc. of methanol, then cooled in an ice-bath. After several hours, the product was collected and washed with cold methanol; yield, 310 mg. (45744,m.p. 169-173". An additional 110 mg. (16%), m.p. 170-173",separated from the filtrate after standing for one week a t -3'. Recrystallization from dilute alcohol gave white crystals, m.p. 173-174'. Ana2. Calc'd for CZSHZUNIO~S: C, 63.0;H, 6.0; N,12.8. Found: C, 62.8; H , 6.3; N, 12.9, 13.1. Attempted diazotization of the hydrazide, X I , in 0.5 N hydrochloric acid with sodium nitrite failed t o give any of the desired product. epi-%-(8-Carboxybutyl)-3,4-cis-diaminothiophane sulfate (XZZZ). A mixture of 380 mg. of 2-(8-carbanilidobutyl)4-carbanilido-5-ketoimidazolido[4,5,c,cis]thiophane(XII), 2 g. of barium hydroxide octahydrate, and 5 cc. of 50% methanol was shaken in a bomb a t 170-180" for twenty hours. The product was isolated in the same manner as described for the diamine derived from dl-biotin (1); yield, 210 mg. (77%), m.p. 240-250"dec. For analysis a sample was recrystallized from aqueous methanol, white crystals, m.p. W245"dec. Anal. Calc'd for CBHZONPOUSZ: C, 34.2; H, 6.4. Found: C, 34.2; H, 6.3. epi-2-(8-Carboxybutyl) -6-ketoimidazolido[4,6 ,c ,eis]thiophane (dl-epibiotin) (XZV). A solution of 180 mg. of epi-2-(6-carboxybutyl) -3,4-cis-diaminothiophanesulfate (XIII) in 16 cc. of 10% sodium carbonate was treated with phosgene with ice cooling until acidic. The product separated slowly in two hours at 0'; yield, 110 mg. (79%) of white crystals, m.p. 190-191". After several days the filtrate deposited an additional 15 mg. (ll%), m.p. 190-191". Recrystallization from water, in which it is much more soluble hot than dlbiotin, gave long white needles of the same m.p. Anal. Calc'd for CI~H,UN,O~S: C,49.2;H,6.6;N, 11.5. Found: C, 49.4;H, 7.2;N, 11.5. Desulfurization with Raney nickel gave dl-desthiobiotin, m.p. 160-161' (2). A mixture with an authentic sample of dl-desthiobiotin prepared by the desulfurization of dl-biotin gave no depression in m.p. The identity of the desulfurized product was also demonstrated by its biotin activity (6),being 50% as active as biotin when assayed by the yeast method.
.
SUMMARY
The synthesis of dl-epbiotin, a previously unknown isomer of dZ-biotin, has been described and the stereochemistry of its method of preparation discussed. PEARLRIVER,NEW YORK REFERENCES
(1) BAKER,QUERRY,MCEWEN,BERNSTEIN,SAFIR,DORFMAN, AND SWBAROW, J. Org. Chem.,12, 186 (1947). (2) HARRIS,MOZINGO, WOLF,WILSON,AND FOLKERS, J. Am. Chem.SOC.,67, 2102 (1945). (3) DITTMER,MELVILLE, AND DUVIGNEAUD, Science,99, 203 (1944). (4) HARRIS, WOLF,MOZINGO, ARTH, ANDERSON,EASTON, AND FOLKERS, J. Am. Chem.Soc., 67,2096 (1945). (5) BAKER,QUERRY,SAFIR,MCEWEN,AND BERNSTEIN, J. Org. Chem.,12, 174 (1947). (6) MELVILLE, DITTMER, BROWN,AND DUVIGNEAUD, Science,98,497 (1943).
