3252
D, U. COSULICII, 2.K o ~ r rJ, . R1. SMITH, JK., M. E. IJIJL~QUIST ANU 1 1 . 9 0 Parallel
1.90
1.57
1.77
1.'17*
CHEMISTRY OF LEUCOVORIN
July 5, 1952 TABLE I1 ISOLEUCOVORIN CHLORIDE 6.88% C1 A.
I/Ima
12.3 9.5 9.0 7.5 6.6 6.4 6.1 5.83
1 .o 0.7 .4 .2 .2 .1 .1 .6
5.64 5.29 5.13 4.91 4.73 4.61
.4 .6
4.45 4.31 4.11
V.W.)
0.3 .2 .2 .2 .1 .4
-
9.8%
A. 12.4 9.4 9.0 7.2 6.6 6.2 6.2 5.79
5.32 5.11d' 4.91 4.72 4.60 4.48d 4.44 4.29 4.15 4.16
c1
-13.26% Cl
I/Im
0.8 1 .o 0.2 .3 .2 .3 .1 .6 .2 .2 .I .4
A. 12.5 9.5
7.3 6.7 6.3
IlIm
0.8 1 .o 0.2 V.W.
0.3
5.87 5.72d
.2
5.28 5.10 4.90 4.75
.1 .2 .2 .4
4.48d 4.35
.2
4.11
.3
.2
V.W.
0.2 0.1
.1
V.W.
0.3 .1
V.W. 3 . 99d0 3.88 V.W. .1 3.86 3.87 0.1 3.75 .1 V.W. 0.1 3.75 3.79 .1 3.62 3.65 0.1 .1 3.58 3.56 0.2 V.W. 3.47 V.W. 3.47 V.W. 3.48 3.41 3.41 1 .o 1.o 3.41 1.o 3.33 0.3 0.2 3.32d 3.33 0.7 3.27 .2 .2 3.22 3.23 .3 3.16 .3 3.16 .3 .1 3.19 3.09 .2 .3 3.11 .5 3.10 .3 3.06 3.03 .1 2.95d .1 .1 2.96 2.87 .1 .2 2.86 .2 2.88d .1 2.77 .1 2.77 .2 2.64 .2 2.63d V.W. 2.65 V.W. 2.61 2.50d .I 2.52 0.1 V.W. 2.41 2.42 .1 V.W. 2.34 .I 2.35 2.26 V.W. 2.27 .I 2.21 0.1 .I 2.20 .I 2.13 2.13 .2 2.05 .1 .2 2.0G 2.02 .1 0 The data are the interplanar spacings, in gngstrdms and the estimated relative intensities, a maximum intensity of 1.0 being arbitrarily assigned to the strongest line of each pattern. The radiation used was Ni-filtered CuKa. * v.w., very weak line. d, diffuse line.
and 0.01 N hydrochloric acid and then acetone, the chlorine analysis fell to 9.8%. Drying of this product a t 100' (2-3 mm.) over potassium hydroxide for two hours gave material which contained 8.76% chlorine. Drying under these conditions for 8 hours gave the pure monochloride. Anal. Calcd. for CmHnN,O&l: C, 48.8; H, 4.51; N, 19.9; C1, 7.21; -CHO, 5.9. Found (corrected for 6% water, by Karl Fischer titration): C, 49.4; H, 4.67; N, 20.2; C1, 7.32; -CHO, 5.74. These various products were examined crystallographically and by X-ray powder diagrams (Tables I and 11) and were found t o be almost identical. The differences between columns two and three may be due to the poorer pattern obtained for the latter. Measured as a growth factor for Le. citrovorum XI1 had
3261
an activity of only 0.2%. It was three times as active as PGA in reversing the aminopterin toxicity for S. faecalis R. The ultraviolet absorption spectra are shown in Fig. 3. Polarographic analysis0 a t PH 9 under strictly anaerobic conditions revealed a major tetrahydropteridine wave. A solution of 0.2890 g. of XI1 in concentrated hydrochloric acid was diluted slightly with water and cooled to 0". A considerable amount of crystallization occurred. Nevertheless, titration with 0.1 N sodium nitrite was undertaken; however, none was absorbed. Leucovorin from Isoleucovorin Chloride.-A mixture of 4.3 ml. of 1 N a n d 45.7 ml. of 0.1 N sodium hydroxide was boiled and cooled under a strong nitrogen stream. Then 0.5 g. of XI1 was added quickly. The solution was heated under a steady stream of nitrogen for 1 holir at 95-100". After cooling, filtering and adjusting to PH 8, a small sample was assayed with Le. citrovorlrm and was found to contain 10.1 mg./ml.; this indicated that conversion was 100%. The remainder of the filtrate was adjusted slowly t o PH 3.7 with dilute formic acid. After cooling. the precipitate, which appeared in the form of spherulites typical of lkucovorin, was filtered, washed and dried to yield 0.192 g. of material which contained 0.846 mg./mg. of leucovorin activity by bioassay. Cooling the filtrate from this fist fraction gave an additional 0.039 g. By ultraviolet absorption spectra analysis, it was 90% pure I. Anhpdroleucovorin-A10 (XIII) .-Isoleucovorin chloride ( X I I ) (3.1 g.), when added to 378 ml. of boiling water, dissolved immediately to yield a solution (PH 2) which was treated with charcoal, filtered, and allowed t o cool slowly. The mats of hair-like bright yellow needles which crystallized were filtered when the temperature reached 35', washed with 0.01 N hydrochloric acid, and dried to yield 1.3 g. Further cooling of the filtrate gave 0.72 g. of gelatinous material containing undifferentiated crystals. XI11 was purified by dissolving 1.3 g. in 0.01 N hydrochloric acid (180 ml.) at 96' and after charcoal decolorization, it precipitated as mats of hair-like needles. After a second recrystallization 0.5282 g. of material was obtained. Since the material dried i n vacuo over calcium chloride was hygroscopic, no attempt t o dry it t o an anhydrous form for analysis was made. The water content was determined by drying a sample at 100' over phosphorus pentoxide and weighing rapidly. The Karl Fischer titration for water content was unsuccessful because the matted character of the material prevented dispersion in the Karl Fischer reagent, in which XI11 was largely insoluble. Anal. Calcd. for C~OH~LN,OV~H,O: C, 45.5; H, 5.42; N, 18.6; CHO, 5.5; H20, 13.7. Found: C, 45.4; H, 5.44; N, 18.6; CHO,5.68; HzO, 13.4. The melting point of the dried sample was, with preliminary shrinking a t 245', 250-257' (dec.). The crystallographic data of the acicular crystals are shown in Table I . The material after drying had the same crystallographic indices and essentially the same X-ray powder diagram (Table 111)as the hydrated forrn.lg In the polarographg at PH 9 under anaerobic conditions XI11 was found to exhibit a major tetrahydropteridine wave. The ultraviolet absorption spectrum in 0.1 N hydrochloric acid was the same as that of isoleucovorin chloride, as would be expected, since these are the conditions for the transformation to the latter. In 0.01 N hydrochloric acid the spectrum was quite similar to that of XII. In aging in PH 4 buffer, the spectrum was the same as
qv.
Biologically, X I I I had 0.0024 mg./mg. of leucovorin activity, but was three times as active as PGA in reversing the aminopterin toxicity for S.faecalis R. To a mixture of 4.1 ml. of 98-100% formic acid and 0.41 ml. of acetic anhydride was added 0.1375 g. of XIII. After allowing the solution to stand overnight a t room temperature, it was poured into 50 ml. of ether. The bright yellow precipitate was filtered, washed with ether and dried in vacuo over calcium chloride to yield 0.104 g. of solid which was similar t o the starting material, by both ultraviolet absorption spectrum analysis and polarographic behavior. The formyl analysis (4.79%) indicated that a second formyl group had not been introduced (theory for one formyl, 6.37%; theory for two, 12%). For the conversion t o leucovorin, XI11 (6.1 mg.) was heated under nitrogen with 6 ml. of air-free 0.1 N sodium hydroxide a t 95' for one hour. After adjustment to PH
x9m TABLE 111 ASHYDROLECCOVORISS
A. 12.2 8.0 i .a 7.0 6.6 0.2 j.87 j.57 5.28 4.60 4.48 4.27tlC 4.w 3.86 3.72 3.62 3.49 3.34 3.12 3.05 2.93 2.83 2.78 "
C
A i,Im,'
1.0 . ,I
v. w , v,\v. '1 .a. 0. 1
1, ( I I). 1 .4
.,>
3
.1
11.2 9.7 7.6 t i . 2d 5.64 5.33 5 .os 4.81i
4.62 1,40 4.21 3 97 3 7ti 8 6ii
3 , 55 3.33 3 18 2 93 2 76 2.66 2.59 2 ,
July 5 , 1952
CHEMISTRY OF LETJCOVORIN
3263
Product from Leucovorin and Sulfurous Acid (XVIII).acid. The identification was made bv means of the crvsA 1.00-g. sample of leucovorin was mixed with 125 ml. of tallographic properties. Anhydroleucovorin-B (0.021 g.) was obtained by boiling water a t room temperature, and sulfur dioxide was bubbled 0.1 E. of XV with 12.6 ml. of bH 4 buffer for five minutes. in until a saturated solution (ca. 2 N ) was obtained. The Identification was made by mefting point (> 330") and crys- leucovorin dissolved, and a yellow solution was obtained, which stood a t room temperature overnight. The excess tallographic data. Leucovorin Nitrate (XVI).-To 0.50 g. of leucovorin in sulfur dioxide was then removed by applying a slight vacuum 3 ml. of water was added sufficient ammonium hydroxide to to the solution, over a five-hour period. A light yellow dissolve the compound, followed by 13 rnl. of 5 N nitric crystalline product separated slowly, which consisted of acid. A white precipitate formed, which was filtered off, long hair-like needles. After the five hours, the mixture washed with 5 N nitric acid, alcohol, and acetone, and dried was cooled, the product isolated, and dried in vacuo over calcium chloride; weight, 0.393 g. An additional 0.198 g. in vacuo over calcium chloride to yield 0.35 g. Anal. Calcd. for CzoH23N1O1.2HNOa.Hz0: C, 38.9; was obtained from the mother liquor on standing. XVIII H, 4.41; S , 20.4; H30, 2.92. Found: C, 38.8, 39.9; H, decomposed about 248', and was inactive in the bioassay 4.49, 4.88; N, 20.0, 18.5; H20, 4.0, 4.2 (by Karl Fischer (0.019 mg./mg.) with Le. citrovorum and by polarographic assay.9 The ultraviolet absorption spectrum was similar titration). to other acid transformation products of leucovorin and the The analyses given are for two different preparations. infrared absorption did not indicate the presence of sulfite. The ultraviolet absorption spectrum in 0.1 N sodium Crystallographically, hydroxide was identical with that of leucovorin. The bio- covorin-A (Table I ) . it was identical with anhydroleuassay was the same as that obtained for leucovorin, when Anal. Calcd. for CzoH~~r\i&~/zSOa:C, 48.4; H, 4.47; corrected for molecular weight differences. The polarograph9 showed no oxidation or reduction waves a t pH 9, in- N. 19.8: S. 3.23. Found (corrected for 7.0. 16.3. 11.5% dicating that the substance retained the leucovorin struc- HiO, respectively); C, 48.8,' 50.4,53.8; H, 3:97, 3:5, 3 . ~ : ture. This compound could not be purified by recrystalli- S , 20.0, 20.9, 21.9; S, 3.24, 2.12, 1.72. The first set of analyses represents one preparation, the zation from water without substantial loss of the biological water content being determined by Karl Fischer. The secactivity. ond series represent another preparation, the water being Hydrolysis Product of Leucovorin Nitrate (XVII).-Five grams of calcium leucovorin was converted to the nitrate determined by weight loss (the sulfur analysis did not change (XVI) by dissolving in water and adding 5 N nitric acid as on drying). The third set of analyses resulted after drying the second preparation over potassium hydroxide for 8 hours above. The product was isolated and placed in 100 rnl. of water, which caused it to dissolve in part, and form some a t 100' in vacuo (the water content was by Karl Fischer tigummy material. This mixture was allowed to stand a t tration). The X-ray powder diagram of XVIII is shown in room temperature for 24 hours, a t the end of which time the Table V. The pattern resembled that given by XVII (hygummy material had changed to a heavy yellow crystalline drolysis product of leucovorin nitrate), the principle differprecipitate, which appeared as needles under the micro- ence being that the lines of XVII appeared a t consistently smaller spacing than equivalent lines of XVIII. This scope. The pH of the solution was 1.75. The product would seem to indicate that the structures of XVII and was isolated and dried in alacuo; weight 2.2 g. C,B46.3; O ~ :H, 4.14; iV,.21.6. XVIII are essentially the same, but that the unit of XVII Anal. Calcd. for C ~ ~ H ~ ~ N Found (corrected for 3.3. 12.5 and 7.0% H20. resDectivelv. is smaller. by Kad Fischer titration): C, 46.2, 47.6,. 46.4;' H, 4 . i 7 TABLE V 3.87, 4.00; N, 21.4, 21.5, 21.7. XVIII The analyses shown represent two preparations; the last I/Im" A. I/Irn4 series of figures represent the material obtained by drying 0 . 4 4.82 0 .2 15.5 the second preparation over potassium hydroxide for eight .2 4.46 .2 12.2 hours a t 100" (2-3 mm.). No leucovorin activity was present in XVII either by .2 10.2 .9 4.17 polarographic or biological assay (0.0098 mg./mg.). Crys.1 8.3 V.W.* 4.00 tallographically, XVII was identical with anhydroleuco0.1 3.76 .5 7.9 vorin-A (Table I ) . The ultraviolet absorption spectrum .1 3.56 .8 7.5 was similar to the other acid-transformation products. The X-ray powder diagram (Table IV) was quite different 6.8 V.W. 3.41 1 .o from isoleucovorin nitrate a1.d somewhat different V.W. 2.97 6.5 0.1 from anhydroleucovorin-A (Table 111). 0.5 2.85 .1 5.95 When 0.8 g. of XVII was recrystallized twice from 805.37 .6 2.69 .1 100 mi. of boiling water 0.293 g. of material which did not melt below 330" was obtained. The crystallographic .1 2.62 V.W. 5.07 properties were identical with those of anhydroleucovorin-B BOUNDBROOK,NEWJERSEY (XIV).
