1701. 7.1
I\TOTES
[ ) , 2 5inl. ( 4 millimoles) of base methyl iodide Cor 1 hours. The product was precipitated with water, centrifuged and washed. Hydrolysis was carried out as previously described. The yield was 0 . p g. or 90% of theoretical. 'The melting point was 68-70 . One recrystallization woulcl bring the melting point up to the desired 73-i5". The specific activity as determined with a n external counter (2.1 mg./cm.2) on a Raychronometer was 2.5-1 microcuries/mg.; this was 99.4% of theoretical. The overall radioactive yield based on the number of milligrams of conipound obtaiiied was 89.5%.
activityd up to 100 times greater than the activity of the protein preparations, has been prepared from
the cxtrncts of the pituitary gland.2fbg,h In addition, Dixon, et obtained a separation of the adrenal weight-increasing activity from the adrenal ascorbic acid-depleting activity in a pig ACTH protein preparation, using ion exchange chromatogr'iphv. These observations may be explained in p r t by the assumption that the protein preparations were not homogeneous. CANCERRESEARCH LABORATORY 1-SIVERSITYOF FLORIDA In an attempt to answer the question of homo~~AIh'ESVli,I,E,h O R I D h geneity, we have subjected the protein preparations to other separation techniques. In a recent conirnunication from this Laboratory5 it was reported Countercurrent Distribution of Sheep Adrenocor- that the ACTH protein preparation could be ticotropic Protein Preparations separated into two fractions by electrodialysis. D\r G E O R ~P. E HLSS, FREDERICK 11. CARPENTER A U D CIIOH One fraction, the cathode material, possessed nearly 7-11 the biological activity of the original protein EIAO LI preparation as measured by the adrenal ascorbic RECEIVED MARCH 3, 1952 acid depleting method.$ Further evidence for the Although the adrenocorticotropic hormone inhomogeneity of the ACTH protein preparations (ACTH) preparations isolated from -sheep and pig has now been obtained from investigations which pituitary glands by the method of Li, et a ] . , and have made use of the countercurrent distribution Sayers, et aI.,l have been found to behave as hoiiio- method of Craig.'j geneous proteins in electrophoretic, ultracentrifugal The ACTH protein preparations were submitted and solubility studies, considerable evidenceL has to countercurrent distribution between 2,4,cIaccumulated which indicates that the *ACTH collidine arid water, a solvent system which has activity is not restricted to a protein with the been described previously' for the investigations of properties described by Li, et al., and Sayers, et a1.l partial peptic hydrolysates of the ACTH protein. a'hen submitted to partial pepsin and acid hy- The results of a typical experiment in which 47 mg. drolysis, the protein preparations retained bio- of ACTH protein preparation was subjected to a logical These results could be intrr- 23-transfer distribution are shown in Table I and preted to inem that only a portion of the protein Fig. 1. At least three components were revealed. molecule was necessary for biological activity. The 11i:iin component (Tubes 0-14) contained However, material with an ascorbic acid depleting ?'I
TABLE I
SOLIDS AND BIOLOGIC ACTIVITYIN YARI(JUS F R A C T I U X S OBTAINED BY A 25-TRANSFER DISTRIBUTIOS oi: -4 SIIEEPACTH PROTEIN PREPARATION BETWEES CorI I I m m w I o N OF
?'
-
LIDINE A X D IVATER
\
+
ACTH potency, Ammint, Dose, Fraction mg. pg.
I-A_-
I - 9 6 10 14 18 22 Tube number. ig 1 -Twenty-four transfer distribution of sheep ACTH protein between 2,4,6-colIidirie and water
__
---
J
__.I-4
Startiilg material
47 2
Tubes" 0-14 Tubes" 15-30
31 5 12
1
1
(1) (a) C. TI. 1.i. H. ?.I E v a n s and I f , E. Simpson, J . Riol. Chem., 149, 413 (1943); ( b i G. Sayers, A. White a n d C. N. H. Long, i b i d . , 149, 425 (1943). (2) (a) C. 11.L i , T ~ O Mlfacy S C o n f . on Metabolic A s g e c t s ojConvalesc r ? i c P , 17, 118 ( 1 0 4 8 ) ; 0,) N. G . Brink, &A. I.P. ?\leisinger and K. Folkers, THISJ O T J R N A I . , 7 9 , 1010 (19301; (c) J . B. L r s h , J . I). Fisher, I . hl. Runding, J . J. Kixsis, L . S Walaszek, W . F. White and E. E. Hays. Science, 112, 43 (1950); ( d ) C. H. Li, THISJOURNAL, 73, 4146 (1951); (e) N. G . Brink, F. A. Kuehl, Jr., hl. A. P . Meisinger, M . N. Bishop a n d K. Folkers, ibid., 74, 480 (1952); ( f ) R. W. Payne, M. S. Raben a n d E. B. Astrvood, J . Bioi. Chem.. 187, 719 (1950): (g) B. Cortis-Jonr\. .\. C Crookr, X. .I. Hcnly, 1'. Llorris a n d C. J . 0. R . hiirrris, B:oriren:. J . . 4 6 , 173 (1830); (11) II. B 1'. Dixon, S. Moore, SI. P. S t a c k - I l u n n e , and F. G. Y o u n g , S a l i ~ r c ,168, 1044 (1951).
Tubes" 21-23 a
8 0.2
Depletion of ascorbic acid, mg./100 g. adrenal
-140, -118, -143, -117. -778, -99, -112, -142, - 101 -29, -62, -61, -57, f28, +:32 -68. -7.5, -i(\,-49, -59, -52, -120, -91, -41, -76, -44, -48, -49, -44 -69, -78, -778, -107, -81, -142, -89, -71, -85, -63, -116
I.U.1 mg.
3
il ( 1 1
1
111
See Fig. 1.
(3) M. Sayers, G. Sayers a n d L. A. Woodbury, Endocrinology, 42, 379 (1918). (4) H . R. F. Dixon, M. P. Stack-Dunne, F. G. Yonng and D B. C a t e r , .VValirre, 168, 1084 (1951). (5) G. P. Hess, J. I. Harris, F. H. Carpenter a n d C. 11. Li, 'lriis JOURNAL,73. 5918 (1961). (6) (a) L. C. Craig and D. Craig i n A. Weissberger, "Technique; of Organic Chemistry," Interscience Publ., h'ew York, N. Y.,Val I I 1 1950, C h a p . 4 ; (b) E. J. Harfenist a n d L. C. Craig, TITISJ O U H I \L 73, 877 (19.ili. ( 7 ) 1'. H. Carpentel-, " S i i t h International C o c i q g j f PLirr : L ~ I .I , plied Cliem.," Abstracts of Papers, pp. 69 (19>1),
NOTES
Oct. 5 , 1952
4957
61% of the recovered dry weight and was prac- substituted thiazolidines as I by dehydrohalogenatically devoid of potency, while the center com- tion to bicyclic @-lactamsas for example, 11. ponent (Tubes 15-20) contained 23% of the re(CHa)zC-CHCOzCHs (CH3)2C-CHC02CHa covered dry weight, and possessed about 13% of I 1 I I s s the total activity. The material in Tubes 21-25 contained 16% of the dry weight, and most of the ’c‘ “io biological potency. Thus i t is apparent that the /\/ adrenal ascorbic acid depleting activity of the HjC:OsC CII? hormone protein can be separated from the main CHaCl components. Whether other adrenal-stimulating I I1 activities (adrenal weight-increasing, adrenal repair, eosinopenic, etc.) are associated with the main Recently SUs3 has reported some experiments of components remains to be investigated. At any a similar nature. The first compound we investievent, i t must be concluded that the protein with an gated was 2-phenyl-3-chloroacetyl-4-carbomethoxyapparent molecular weight of 20,000 which was thiazolidine (111), obtained by the reaction of chloisolated from sheep pituitariesza is a mixture. roacetic anhydride with 2-phenyl-4-carbomethoxySince electrophoretic, ultracentrifugal and solu- thia~olidine.~A benzene solution of I11 was heated bility methods, as they were applied, did not reveal under reflux with triethylamine but it failed to afthis inhomogeneity, the results reported here are a ford a @-lactam. I n the expectation that the sulfurther demonstration6b of the utility of the fone of I11 might be more reactive, I11 was submitcountercurrent method in homogeneity studies on ted to oxidation with potassium permanganate or low molecular weight proteins. hydrogen peroxide. The only oxidation product isolated was the sulfoxide, which was unchanged Experimental after heating in dioxane solution with triethylamBecause of the formation of emulsions, the distributions were performed in glass centrifuge tubes. Volumes of 5 ml. ine. It seemed that a more favorable case for closing for the upper and 5 ml. for the lower layer were used. The solvent was obtained by equilibrating 7 parts of 2,4,6-colli- the fused @-lactam-thiazolidinering would be that dine (Reilly Coal Tar and Chemical Company, b.p. 170’) of a derivative of 2-carboxythiazolidine since the with 11 parts of water. The experiment was performed with the lower layers as the moving phase, but the data are methenyl group would be activated by the carboxy recorded as though the upper layers were the moving phase. function and perhaps also by the sulfur atom. The solid in each tube was determined by transferring the Therefore, 2,4-dicarbethoxythiazolidine (IV) and contents of the tube to a tared Florence flask weighing about 2 - carbethoxy- 4 - carbomethoxy-5,5-dimethylthiazo2.2 g.,* and removing the solvents by evaporation from the frozen state. The residues were finally kept for several lidine (V) were prepared by condensing ethyl glyhours over phosphoric anhydride in vacuo before being oxalate alcoholate with L-cysteine ethyl ester weighed. A total of 51 mg. bf material was present in the hydrochloride and D-penicillamine methyl ester hytubes. This increase in weight over the starting material drochloride, respectively. An evaporative distillawas probably due to the formation of a small amount ( C U . 5 mg.) of non-volatile collidine hydrochloride. The resi- tion of a crude sample of V afforded a crystalline dues were dissolved in water and the solutions were pooled (Va) and a liquid (Vb) distillate. It was found as indicated in Table I. These pooled solutions were as- that Va and Vb had identical elemental analysis sayed by the adrenal ascorbic acid depleting method3s9in and infrared spectra but different optical rotations hydrophysectomized male rats. A preparation of the In([ci]”D -20.7’ and +28.2”, respectively). Eviternational Standard was employed for comparison to estidently Va and Vb are diastereoisomers formed when mate the ACTH potency of the various fractions obtained. Acknowledgment.-This work was aided in part the new center of asymmetry was created. The N-chloroacetylated thiazolidines (VI, I) by grants from the National Institutes of Health, (corresponding to IV and V, respectively) were unthe United States Public Health Service, the Armour Laboratories, Merck and Company, Inc., affected by treatment with triethylamine. Efforts to oxidize VI and I to sulfoxides or sulfones failed. and the Eli Lilly Laboratories. R’?C-
(8)L. C. Craig, G. H. Hogeboom, F. H. Carpenter and V. d u Vigneaud, J . Biol. Chem.. 168, 665 (1947). (9) I. I. Geschwind, B. S Williams and C. H. Li,Acto Endocrin., 8, 247 (1951).
DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF CALIFORNIA BERKELEY 4, CALIFORNIA
Attempted Cyclization of N-Chloroacetylthiazolidines BY JOHN C. SHEEHAN AND AJAYKUMAR BOSEI RECEIVED APRIL18, 1952
I n an effort to extend our new p-lactam synthesis,2 we have conducted a preliminary study of the possibility of cyclizing such N-chloroacetylated (1) Oversea8 scholar of the Government of India. (2) J. C. Sheehan and A. K. Bose, THISJOURNAL,
‘la, 5158 (1950).
CHR
$\ / 4\ CH
R“‘
H , R” = CBH5, R”’ = ClCHzCO 111, R = C02CH3, R’ IV, R = C02CzH5, R’ = H, R’ = COzCgH,, R”’ = H V. R = C02CHs. R’ = CH1. R ” = CO~CIHE.R”’ H Experimental5
2-Phenyl-3-chloroacetyi-4-carbomethoxythiazolidine (111). -Benzaldehyde and L-cysteine hydrochloride were condensed‘ and the crude thiazolidine ( 5 9.) suspended in ether was treated with diazomethane to give the methyl ester (3) 0.Sus, A n n . , 668, 129 (1950). M.P. Schubert, J . Biof. Chcm., 114, 347 (1936). ( 5 ) All melting points are corrected. We are indebted to Dr. S. M. Nagy and his associates for the microanalyses. (4)
