3100
COMMUNICATIONS TO THE EDITOR
I'd. 7(i
dithi~hydantoin~ and 5-hexyl-5-methyl-2,4-dithioTABLE I hydantoin (m.p. 98.5-99.6'. Anal. Calcd.. N, COMPOSITION O F T H E RADIOACTIVE HYDROCARBONS FORXIBI) 12.16; S, 2'7.83. Found: N, 12.20; S, 27.83.) BY THE IRRADIATION OF A 5 MOLEPERCENT. S m u n t i y oii Doses of the order of 200-300 mg. /kg./day of 5ASILIKE IN NORMAL PEN~ANE n-heptyl-Zthiohydantoin were required to protect 5% of total C I 4 was extractable into 12 S FICl all mice infected intravenously with ill. tuberculosis Per cent. o f t!ic Chemical form totirl r:i well tolerated by these species. In view of these 2 45-2 90' 3 results it is felt that 5-n-heptyl-2-thiohydantoin is Residue 9 worthy of clinical trial as an antitubercular drug. I t seems more than a coincidence that the groupIt is clear from these preliminary results, which S were duplicated by a second run in which ethyl'1 ing, -NH-C-SHor a tautomeric form thereof amine was substituted for aniline, that a high velooccurs so frequently in in vivo tuberculostatically city carbon-14 on colliding with the liquid aliphatic active drugs. The thiosemicarbazones, the thio- hydrocarbon has a very good chance of entering the ureas reported by H ~ e b n e rthe , ~ mercaptotriazin- chain. The reasons for this may be debatable, but ones of Hagenbach5 and now the thiohydantoins the facts seem to be clear. I t should be realized that the hexane and heavier hydrocarbons produced all have in common the thioureido function. in the above-described bombardment were essen( 3 ) H C Carrington, J Chem Soc , 681 (1947) tially carrier free except for any which may have (4) C F Huebner, e l ~l , T H I SJOURNAL, 76, 2274 11953) been produced by gamma and fast neutron radia(5) R F Hagenbach, E Hodel and H Gysm, Expev:enlra, 10, 620 tion. It would seem therefore that the process (1954) E FROELICHdescribed can produce radioactive hydrocarbons of ALICE FRUEHANhigh specific activity. It is further clear that as STERLIUGWINTHROP MARYJACKMAN far as neutron economy is concerned, this process RESEARCH INSTITUTE FREDK RIRCHNER could well compete with any organic synthesis, for RENYSELAER, V Y E J ALEXANDER S ARCHER the sole labor involved is the purification of the original chemical, the preparation of the samples for RECEIVED MAY1, 1954 irradiation, and the subsequent distillation and separation. There is reason to believe that the DIRECT PRODUCTION OF RADIOACTIVE ALIPHATIC procedure outlined would also serve to introduce radiocarbon into heavy lubricating oils. HYDROCARBONS BY PILE IRRADIATION' )
Sir: Study of the hot atom chemistry of carbon-14 by the irradiation of nitrogenous organic materials in the heavy water pile a t the Argonne National Laboratory (CP-3') has led us to observe a method of producing saturated aliphatic hydrocarbons in radioactive form in high yield and high specific activity. -2 5 mole per cent. solution of aniline in normal pentane, 20 cc. of which was enclosed in a quartz tube and irradiated for one week in the CP-3' pile at a flux of 1011 neutrons per cm.2 per second, proved to yield about 25y0 of the radiocarbon in the form of radioactive normal pentane, with less than 1% as iso- or neo-pentane; about 15% in the form of radioactive hexane, which apparently is about two-thirds normal hexane; and the remainder in heavier hydrocarbons. The distribution is given in Table I.
DEPARTMENT OF CHEMISTRY AND INSTITUTE FOR S U C L E A R STUDIES UNIVERSITY OF CHICAGO CHICAGO, ILLINOIS
ARIEL G . SCHRODT F. T,rnnv
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RECEIVED MAY10, 1954 ____.~_._
-
ENZYMATIC REDUCTION OF CORTISONE'
Sir: Previous in viBo and in vitro studies reveal that the major pathway of cortisone metabolism is the reduction of the A4-3 ketone group to the saturated 3-alcohol by the l i ~ e r . ~ We , ~ .should ~ like to report the presence of an enzyme system in rat liver
(1) Thi? research was supported h y t h e United S t a t e s Air Force through t h e Office of Scientific Research of t h e Air Research and De-
(1) Abbreviations a s used in this communication are: cortisone (A4-pregnene-l7u,21 diol-3,11,20-trione), dihydrocortisone (pregnane17a.21-diol-3,11,20-trione), tetrahydrorortisone (pregnane-3a.17n.21triol-1 1,20-dione), T P N H a n d T P N (reduced and oxidized triphoqphopyridine nucleotide, respectively), D P N H and D E " (reduced a n d oxidized diphosphopyridine nucleotide, respectively). ( 2 ) J. J. Schneider, J . Biol. Chem., 194, 337 (1952). (3) J. J. Schneider a n d P. hl. Horstmann, i b i d . , 196, 629 (1952). , Levy a n d 0. M. Hechter. Arch. Biochcm., 46, I69 (4) E . Y C ~ s p iH.
\ c l o p m r n t Command.
(19.53)
capable of catalyzing the reduction of cortisone t o tetrahydrocortisone. This activity is found solely in the particle free supernatant of a sucrose or phosphate homogenate and i s precipitated between 55 and 70% saturation with ammonium sulfate. This+ reaction, which results in loss of the cr,p unsaturation of cortisone, can be followed by the disappearance of the characteristic absorption of the steroid a t 240 mp. The hydrogen donor is T P N H ; D P N H is completely inactive. Reversal cannot be observed upon the addition of T P N and either dihydrocortisone or tetrahydrocortisone. The reaction can be coupled to the oxidation of d-isocitrate by T P N and isocitric dehydrogenase or to the oxidation of glucose-6-phosphate by T P N with glucose-6-phosphate dehydrogenase (Table I). The reaction proceeds faster under nitrogen than air. I n this system, with cortisone as the s u t s h t s , tetrahydrocortisone appears to be the sole product. The allopregnane isomer is not detected. The metabolite has been identified by (a) paper chromatography of the free alcohol and of the acetate in two solvent systems; (b) spectrophotometric comparison of the sulfuric acid chromogens of isolated and authentic tetrahydroc~rtisone~; and (c) comparison of infrared spectra with known compounds. TABLE I COFACTOR REQUIREMENT FOR CORTISONE REDUCTION Reaction components: 5pM. MgClg, 5 pM. nicotinamide, 50 pM. phosphate buffer PH 7.4, 0.28 p M cortisone, 7 mg. protein of ammonium sulfate fraction; final vol. 1.5 ml ; incubated 1 hour a t 38'. under nitrogen. pM Cortisone metabolized
Additions
D P N (0.5pM.) D P N (0.5pM.) lactate (20pM.) lactic dehydrogenase (0.2 mg.) T P N (0.lpM.) T P N (0.1pM.) d-isocitrate (1pM.) isocitric dehydrogenase ( 5 mg. protein) 'TlVT(lT.lpiW) glucose-6-phosphate (1pM.) 4- glucose-6-phosphate dehydrogenase (0.2 mg.)
+
+
+
+
+
0 0
0 0 0 0 0 22 0 18
Since the reduction of cortisone to tetrahydrocortisone involves the addition of four hydrogens, it is likely that the reaction proceeds in two steps with dihydrocortisone as the intermediate. In support of this, the same liver fraction also has been found to catalyze the conversion of dihydrocortisone to tetrahydrocortisone. This latter reaction requires either DPNH or TPNH. It can be followed spectrophotometrically by the absorption of the reduced pyridine nucleotides a t 340 mp and is readily reversible with the equilibrium toward the reduced product (Fig. 1). The reaction is completely inhibited by p-chloromercuribenzoate ( 5 X M ) and the inhibition can be reversed by glutathione or cysteine (5 x 10-3
M). It is probable, therefore, that the formation of tetrahydrocortisone from cortisone proceeds thus
TPNH
Cortisone
!o.lor
COMMUNICATIONS TQ THE EDITOR
June 5, 195.2
T P N H or D P N H
*
Dihydrocortisone
T P N or D P N Tetrahydrocortisone
( 6 ) A. Zsffaroni, THISJOURNAL, T I , 3828 (1950).
-.
3LQL
o'20
-0 0.05
t0
I
I
I
1
2
4
6
I
t
I
e
10
12
MINUTES
Fig. 1.-Ascending curve represents formation of T P N H as a result of the oxidation of tetrahydrocortisone on adding 1 pM. TPN, 1 pM. tetrahydrocortisone arid 15 mg. protein of ammonium sulfate fraction a t PH 7.4. Reversibility is demonstrated a t (1) when 0.15 pM dihydrocortisone is added, causing decrease in optical density as dihydrocortisone is reduced.
That dihydrocortisone does not accumulate in detectable amounts when cortisone is the substrate may be explained by the observation that the reduction of dihydrocortisone to tetrahydrocortisone is more rapid than the over-all reaction. Definitive proof of this mechanism awaits the separation of the enzymes involved. The ammonium sulfate fraction is also capable of catalyzing the disappearance of other A4,3ketosteroids in the presence of TPNH. These include hydrocortisone, desoxycorticosterone, progesterone, testosterone, adrenosterone, and cholestenone. It is not yet known whether the enzymes mediating these reactions a r e t h e same as those concerned with the reduction of cortisone. NATIONAL HEARTINSTITUTE, AND NATIONAL INSTITUTE OF ARTHRITIS G. TOMKINS AND METABOLIC DISEASES K. J. ISSELBACHER INSTITUTES OF HEALTH NATIONAL 14, MARYLAND BETHESDA RECEIVED MAY10, 1954
A NEW TYPE OF METALLIC BONDING IN MOLECU-
Sir:
LAR COMPLEXES1
Evidence for weak metallic bonding in planar Ni(II), Pd(I1) and Pt(I1) complexes has been presented.2 It has been suggested that metallic chains may result when normal dsp2square bonding leaves a vacant p-orbital, allowing mixing with an octahedral state involving d2sp3orbitals and metal bonds2 Au(II1) complexes should, then, be capable of forming weak metal bonds. Examining this possibility led to the preparation of Au(DMG)2+AuC12- (HDMG = dimethylglyoxime) which has linear gold chains with Au-Au (1) Work was performed in p a r t in t h e Ames L a b o r a t o r y of t h e Atomic E n e r g y Commission. (2) (a) S. Yamada. Bull. Chem. SOC.J a p a n , I C , 125 (1915),a n d references therein. (b) L. E. Godycki a n d R . E. R u n d l e , Acto Cryrt., 6, 487 (1953).
