M a y 20, 193s
COMMUNICATIO~VS TO THE EDITOR
the chloro sugar XX, prepared as described above (cf. preparation of X X I ) from 5.29 g. (10 mmoles) of 1-0-acetyl-2,3di-O-benzoyl-5-deoxy-5-phthalimido-D-ribofuranose, in 50 cc. of xylene was added to an azeotropically dried suspension of 1.75 g. ( 5 mmoles) of acetylcytosinemercury (II1)l1in 180 cc. of xylene. T h e stirred mixture was allowed t o reflux for three hours and was then evaporated i n uacuo. The residual brown gum was dissolved in chloroform (200 cc.) and the solution was Tvashed with t\To 20-cc. portions of 3070 aqueous potassium iodide solution and with 40 cc. of water. The organic phase was dried and partially decolorized over magnesium sulfate and Norit. The filtered solution was evaporated i n nacuo and the residual gum (6.05 g.) was dissolved in 10 cc. of chloroform and 10 cc. of benzene. T h e solution was chromatographed on a column prepared from 120 g. of silica gel in benzene-chloroform (1: 1). The column (3.5 X 29.5 cm.) mas eluted with 400 cc. of this solvent mixture and the 0.22 g. of gum obtained from the pooled fractions v a s discarded. Elution 'ims continued with 200 cc. of chloroform, 200 cc. of chloroform-acetone (98:2) and 150 cc. of chloroforxn-acetone (97:3). Solid material was isolated from the following fractions which were eluted with 100 cc. of chloroform-acetone (97:3) and 100 cc. of chloroform-acetone (95: 5 ) . By crystallization and one recrystallization from ethanol-ethl-l acetate there was obtained 1.03 g. (29Fc based on 10 mmoles of sugar derivative X X ) , m.p. 145-146', undepressed by admixture of the C20HlsSOa compound isolated in the preparation of XXI. Continued elution of the column x i t h the last mentioned solvent mixture (150 cc.), with 350 cc. of chloroformacetone (9: 1) and with 100 cc. of chloroform-acetone (85: 15) afforded gummy material (1.06 g.) n-hich \vas discarded. IVashing the column with 450 cc. of chloroform-acetone (65:35) yielded a solid which was crystallized and recrystallized from acetone t o afford 1.69 g. (277, based on 10 mmoles of XX; S l y o based on 111), m.p. 147-150'. For
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analysis the substance was recrystallized twice more from acetone; m.p. 146-148". Material dried a t 74" in vacuo still contained acetone of crystallization. T h e compound showed [ c Y ] ~ ~+37.8' D (G 0.98 in CHC18); "::A : 283 m P (E 10,880 in acid), 298 mk (E 7,480 in methanol) and 272 mp (E 11,700 in base).31 Anal. Calcd. for C B ~ H Z ~ N ~ O Q . C C,~63.52; H ~ O : H , 4.74; Ii,8.23; C-CH,, 4.42. Foupd: C, 63.24; H, 4.83; hT, 8.01; C-CH3, 3.91. 5'-Amino-5'-deoxycytidine [ 1-(5-Amino-5-deoxy-/3-~ribofuranosy1)-cytosine (XXIV) ] .--4 mixture of 1.86 g. (3 mmoles) of the 4acetamidopyrimidinone derivative X X I I I , 40 cc. of absolute methanol and 7 cc. of butylamine was heated under reflux for 16 hours. T h e reaction mixture was worked up as described in the preparation of VI1 and the residue (0.74 9.) obtained by evaporation of the combined water layers was dissolved in 30 cc. of hot methanol and The filtrate was evaporated t o a filtered through S o r i t . small volume and decanted from a small amount of yellow gum. Ethanol was added a t the b.p. until the solution became cloudy. The solid which precipitated on standing was collected, washed with ethanol and ether. T h e vacuumdried material (0.37 g.) was amorphous and melted around 145'; ultraviolet analysis indicated a purity of 8 4 s 4 Q ; the 0.37 g. of product, therefore, represented a 74y0 yield. T h e compound as such could not be purified further and was characterized through the picrate prepared in methanol and recrystallized from methanol-ethanol; m.p. 218-220' dec., [ c Y ] ~ ~+21.5' D (c 1.02 in methyl Cellosolve). Anal. Calcd. for CloHliSi011.H20: C, 36.81; H , 3.91; h-,20.04. Found: C,36.51; H , 3 . 2 3 ; S ,19.37. PEARLRIVER,YEW TORK (49) T h e extinction of cytidine a t 269 mii was used for comparison.
COMMUNICATIONS T O T H E EDITOR ~~v~-DIPHENYLPYROPHOSPHORODIAMIDIC ACID : A
NEW
SUBSTRATE FOR THE COLORIMETRIC ESTIMATION OF ENZYMES
Sir : S-Phosphorylated nitrogen mustards which have been shown to be non-toxic are potentially cytotoxic agents with a high selectivity for cells with high phosphamidase activity. la-g The distribution of phosphamidase enzymes in malignant tissues is, therefore, a matter of interest in chemotherapy. Studies have shown that some tumors do have high conphosphamidase activity. 2a-d However, firmation, extension and exploitation of these results have been hampered by the fact t h a t the substrates used are unstable below P H 5.0 where the enzyme(s) is a ~ t i v e . ~ On ~ - the ~ basis of the struc(1) ( a ) 0. 31. Friedman a n d A . R I . Seligman, THISJ O G R K A L , 70' :3082 (1948); (h) 7 6 , I j > 3 (1951); ic) 1 6 , 0 3 8 ( 1 0 3 1 ) ; ( d ) A. hf. Selig' m a n , R I . R I . Nachias, L. H. I l a n h e i m e r , 0 . A I . Friedman and G. Wi>lf.A n n . S i r v ~ . ,130, 333 (1949); ( e ) A . If. Seligman, R I . XIilden and 0. 11, Friedman, C u n c e i , 2 , 701 ( 1 9 4 9 ) ; ( f ) A. M. R u t e n b u r g , I.. P e h k y , 0. PI[. Friedman and A . 11. Selignian, J . Pharin. E x p . Tilerap., 111, &82 (195L); (6) 0. ;\I Friedman a n d E. Boger, THIS J O U R N A L , 1 8 , 4659 (19513). (2) (a) G. Gomori, Pvoc. SOL.Ex15il. B i d . M e d . , 69, 407 (1948): ( b ) J . hieyer a n d J. P. Weinmann, J . Dint. R e s , 32, GO9 (1963); (c) J . l l c y c r and J. P. n'einmann, J . Ilisiocheii;. o i i t l Cy/cvh?i1i., 1, :SO:-:{l4 (I%,]), ( d ) 5 , ;4!),< ( 1 9 5 i ) . (3) (a) J I). Chanley and I?. Feageson. Al>,t of Paper, 130th A C S RIeeting. Sept , 19.X. 4 9 - 0 : (11) H. Holter and Si Oh I.i, Coinpi. Rend.
tural features apparently necessary for enzymatic activity4 and from theoretical considerations with regard to stability, pyrophosphoroamidic acids, although previously unreported, appeared attractive as substrates for enzymes of the phosphamidase type. In addition, chromogenic pyrophosphoroamidic acids would be satisfactory prototypes for toxagenic analogs potentially useful in the treatment of cancer. \\'e report here the synthesis of sym-diphenylpyrophosphorodiamidic acid (11). O-Benzylphenylphosphoroamidic acid,j m.p. 118-119', on treatment with dicyclohexyl carbodiimide in dimethylformamide gave sym-0,O-dibenzyldiphenylpyrophosphorodiamidate (I) (1n.p. 142-143 O ; Anal. Calcd. for C26H26NYP205: C , 61.44; H, 5.16; N, 5.52. Found: C, '61.44: H, 5.53; iX, 5.49). Debenzylation of I m ith hydrogen over palladium afforded the dibasic pyrophosphoroamidic acid I1 which was isolated both as the dicyclohexylarnmonium salt (m.p. 223-284"; -1nc~I.Calcd. for C24H40N40jP2; C, 54.75; H, i . 6 2 ; K , 10.64. Trau. Lab. Cavlsbevg, Ser. Chim., 27, 393 (1951): (c) K. h l . AColler, Biochem. et B i o p h y s . A c l u , 1 6 , 162 (1955). (4) Active substrates have in common structures with a t least one ionizable phosphoric acid group, ref. 2a. ( 5 ) V. 31. Clark a n d A . R . ' r u d d . J . C i i f i x Sot., 20:M (1950); 1499 (195;).
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~ORIAIUNICilTIONSTO THE
EDITOR
Vol. 80
METHYLATION OF ALCOHOLS WITH Found: C, 54.85; H, 7.90; N, 10.35) and as the DIAZOMETHANE dipotassium salt (A nul. Calcd. for Cl2HI2O5P2K2 : C, 33.64; H,2.99; N. 6.93. Found: C,35,.40; H, Siu: Methylation of alcohols b y the IVillianison sytl3.06; Lu, 7.14) in yields of about 60%. This substrate I1 underwent no spontaneous thesis requires strongly basic conditions and there hydrolysis a t pH 4.0 to pH 11.5 after two hours a t is no satisfactory general methylation procedure 37". It slowly liberated aniline a t pH 3.5 and did so effective under neutral or mildly acidic conditions. more rapidly at pH 2.5. Aniline was measured I n principle, diazomethane with a n acidic catalyst colorimetrically.6 The evidence, moreover, clearly should methylate alcohols, b u t the usual protonic indicates t h a t no hydrolysis of the pyrophosphate, acid, such as hydrochloric acid, is unsatisfactory P-0-P, bond had occurred under any of these because i t is itself methylated with diazomethane. conditions and t h a t liberation of aniline in acid re- Fluoroboric acid, however, promised to serve as a sulted solely from phosphamide, P-N, bond cleav- useful catalyst since it would be consumed in reacage. If hydrolysis of the pyrophosphate bond tion with diazoinethane only by some proces: inabove pH 4.0 had occurred phenylphosphoroamidic volving rupture of a B-F bond.' This expectation acid (111) would be formed. This product 111' is was indeed realized and a novel method has been essentially completely hydrolyzed after 30 minutes developed for methylation of alcohols in high yields at pH 4.0, conditions a t which the substrate I1 under mild conditions. Methylations of alcohols were carried out in diethyl ether or methylene chloride a t 0-23" in thc presence of 0.6-8 mole per cent. of fluoroboric acid. Methyl ethers of simple primary or unhindered secondary alcohols thus were formed rapidly in 84OSyoyields. Moderately hindered secondary alcoI, R = C6HjCHa hols and tertiary alcohols reacted more slowly, 11, R = H yields were lower and methylation was accompanied gives no hydrolysis. Thus I11 can be assayed for. by some polymethylene formation, minimized a t Cleavage of the pyrophosphate bond in I1 below lower temperatures. Typical cases were n-octanol p H 4.0 is measurable simply b y assay for inorganic ( S i % ) , cyclohexanol (927,), chol orthophosphate. These assays showed that in- cholestanol (98yG), 3-cholestanol significant amounts of either phenylphosphoro- phenylcarbinol (307;) and t-amyl Competition experiments s h o w amidic acid I11 or inorganic phosphate were prothe rates of primary, secondary and tertiary butyl duced above or below p H 4.0, respectively. As regards stability to spontaneous hydrolysis alcohols = 2,.2:1.3:1.0 and ,3 (equatorial) and cy this substrate I1 is, therefore, eminently satisfac- (axial) cholestanol = 1.3: l . Clearly, these acidtory for enzymatic work. Extracts of intestine catalyzed methylations lack high steric selectivity. with high alkaline phosphatase activity8 active Triphenylcarbinol and isoborneol could not be against various phosphamidesg were significantly methylated by this method. The new reagent provides a unique tool for the active against this substrate 11. Preliminary results, however, suggest t h a t the methylation of certain alcohols containing other active enzyme is not alkaline phosphatase since sensitive groups. For example, testosterone and maximum hydrolysis occurred a t a new p H between desoxycorticosterone have been converted directly 7.6-8.0, and of the two intestinal extracts studied to their inethyl ether:---a dificult, if not impossible, the partially purified one was significantly more ac- transformation by any previously axrailable direct tive against @-glycerol phosphate and P-napthyl methods. Testosterone methyl ether, which docs phosphate than i t was against the substrate 11. not appear to have been described before, melts a t T h e relationship of this "pyrophospharnidase" to 127-1%i.,j0, [ a ] , > C H C ' a +106.3" (found: C,7!).%1 ; phosphamidase and its distribution in tissues are H, 0.71; OCH::, 10.3). Ascorbic acid gave a hitherto unknown trimethyl ether, 1n.p. 09.3-101 ', under study t h a t will be reported later. Acknowledgments.-This work was s u j p r t e c i ( Q ) ? ~ D 33" (HYO) ; C, 49.48; H , 6.49;OCH3, 4%.:3; by a research grant ( N o . C-2130) from the Sational which we consider to be 2,3,6-triiiiethylascorbic Institutes of Health, U.S. Public Health Service. acid on the basis 01 its oxidation with weakly The authors also gratefully acknowledge assistance alkaline periodate, and b y its infrared and ultrafrom A h . Phyllis R. Kaplan and Mr. Harold violet spectra and the changes in the latter in the presence of dilute alkali. Sominer. Fluoroboric acid also has been s1~ow.n to catDEPARTMEKT OF CHEMISTRY E L r a r x BOGER alyze the othern-ise sluggish reaction of weakly BRANDEIS UNIVERSITY ORRIE52. FRIEDMAN acidic phenols with diazomethane. Estradiol was lV.4LTHAM 54, >IASSACHUSETTS thus converted t o the dimethyl ether i n Slyc yield RECEIVEE JANWART 22, tR68 urider Conditions which, although forcillg, gave 110 reaction a t all in the absence of thc catalyst. ( t i ) F. hIichael and J . Schierhcrlt, Rev., 89, 1089 (1952). Other meakly acidic phenols of PI
