Recrystallization frorii n-&)rlJpylal~~oliol tiid not (Gliaiige the dvc~iinpositionrange. Anal. Calcd. for Cj,&tlSO:: ( ' > 77.0!1: H, 4 . 4 4 : S , .-1,!i2, Fourid: C, 7 7 . 3 6 ; H, 4.31 : 2-Nitrosofluoranthen-3-0 ra~itlieli-l;-iiI9 ( 2 . 0 g . ) i t i ; ~ I J solute ethanol (50 ml.) a t 0' w:is treated w i t h r o i i ( drcichloric acid ( L mi.) and n-butyl nitrite ( 0 . 5 g ( 35 nil.). The iiiixture WLS stirred at !I" for 1 lir. and heated :it IOUo for 1.5 hr. The resulting bro\vri prwipitate after frac~tioii:tI (.rystallization from absolute etharicil anti sncc,essive crystallizations from absolute ethanol arid froni beiizerie give y?llo\vorange needles rrieltirig at 216" der. : yield 0,133 g. AInaZ. Calcd. for CIGHYSC)::: (', 7 7 . 2 : H, 3.ii7: S , .i.(i7. l o u r i d : C, 78.29; H,3.83; K,5.11, 2-Amino-1,2,3,10b-tetrahydrofiuoranthen-3-one Hydrochloride (III).--%-Osirnino-1,2,3,1Ilt)-tetrah~(~rofliuor:inthen-:l-oiie (:('ifcoiideiisatioii of the tetraIiniitiopyriiiiidiiir.. Th ib did iiot occur wlivii ttic reactioii was carried out with ail o\;idatioii i l l pyt~diii(~ aiid i i i t l i v pi ( w i i w of dicyclolic\ylcai,t)odiitiiide, hiit I I I thi\ ( Y M ~ tltc> yicltl of tho I,rodLlct \va* \ Ply IO\\ . 011the otlict halid. just as i l l tlie (YM' ( i t tht. c l a , ~ ~ i c u l folic acid sy~ithesis,~ coiideiisatioii with coiiroiiiitaiit oxidation to t h r pteridine occurred rcatlily if the rc'actaiits weie siiiiply coiiibirird i i i a11 aquc'oiis (alcoholics) wlutioii aiid allowed t o react a t roo111tciiipei,atur(. \z.itli qtii~iiigfot ail extcliidcd pwiod of ttiiic swicas 0 1 r~.periiiieiit.i \\as carried oiit to cstablidi the optiliial p H ; it irac f o i u i t i that coiideiisatioii at pH 7.5 8.0
S UTE:$ TABLE I RAT.k?;TIANDROGEN C'ompound
Daily dose, a y
Levator ani, ing.
...
Controls Testosterone Testosterone I11 I11
1 9.c. 10 s.c. 100 S.C.
I11
100 S.C.
l b 9.c.
1,
1 S.C. \ I11 l b s.c.1 Testosterone 1 s.c.) 111 100 p.0. TI1 l h p.0. 111 100 p.0. Testosterone 1 s.c. \ I11 l h p.0.). Testosterone 1 s.c. \ P.c.. = subcutaneous injection; p.0. = per os.
Testosterone
a
Seminal yes, cl e ,
prostat?,
Ventral
70
ing.
rng.
inhibition
1.8 3.2
8.8 9 2 11.3 10.8 9.8
2.8 2.5
9.0 14.5 24.3 9.5 '3.3
10.0
3.5
14.5
10.5
3.3
13.7
9.8 8.3
2.5 2.0
9.3
7.5
2.0
10.0
82
.3
2.s
Il.ri
55
9
i.0
i.5
I n mg.
gave the cleanest product. The condensation reaction was found to proceed for about 72 hr.; after this, no further increase of yield was observed. Details of the purification procedure are given in the Experimental part. The analytically pure pteridinosteroid is a yellow crystalline solid which is soluble in alcohol and chloroform. It has the characteristic ultraviolet absorption spectrum of pteridines (two maxima, a t 257 and 375 nip) and o p t i d activity due to the steroid portion of the molecule. Its infrared spectrum is almost identical with that of the [4,3]-fused pteridinosteroid? except for some minor differences in the fingerprint region. Based on analogy with established reactions's* the halogel? is expected to react preferentially with the niorc basic 3-amino group of the pyrimidine, while the carbonyl group condenses with the imino-type 4- or 6-substituents (which are symnietrical) ; consequently, of the two possible position isoniers, I11 and IT', the former represents the proposed structure of this conipound. I11 niicrobiological experinients using the Lactobacillzts leichinannii s y s t e ~ i i ,the ~ new, linear polycyclic compound (111) appears to be more active as a n antifolic agent than the [4,3-g]pteridinosteroid.2 Half-maximal inhibition is obtained a t 1-7/nil. concentration, while approxiniately 5-y/ml. concentration is required for the [4,3-g]pteridinosteroid. Figure 1 shows the reversal of this inhibitory effect by folinic acid. The inhibition appears within this range to be of the competitive type with a n inhibition index of approxiniately 3000. I n animal experiments, I11 showed no androgenic activity in the rat or chick, but it appears to be a somewhat more effective antiandrogen than the [4,3-g]pteridinosteroid.2 The results of the rat assay are shown in Table I. It is interesting to note that the compound is inactive on parenteral administration but shows substantial inhibition of testosterone if it is given orally. I n the chick assay (Table 11),the inhibition of testosterone stimulation of the chick comb averaged 307c. (8) D. R. Seeper. D. B. Cosulrrh, J. AI. Smith, Jr., a n d 11. E. Hultquist, J . A m . Chem. Soc., 71, 1753 (1949). (9) T. J. Rardos, G. hl. Leiine, R. R. Herr, and H. L. Gordon, $bid.. 7 7 , 4279 (1955).
ASSAY
TABLE I1 CHICKAXTIANDROGEN ASSAY NO.
Compound
Controls Testosterone Testosterone Testosterone enanthate 111
I11 I11
Daily dose,a y
of chich
...
15 14 14 15 15 13
1 10 0.9 50
.iooc
Comb wt., mg.
5% inhibition
18.4 50.1 81.7 74.5 16.9 20.5
1
Testosterone I11 'jooC 14 38 0 38 Testosterone 1 I11 'OoC 14 47.6 48 0 , 5 *( Testosterone enanthate c1 Applied to comb locally. Single intramuscular injection; in mg. c Suspension.
Due to insufficient material, antitumor testing of I11 was done only against Sarcoma 180 in mice, using intraperitoneal route of administration. The results were inconclusive. However, the above mentioned dependence of the antiandrogen activity of this compound on the route of administration indicates the need for additional testing in this tumor system. Experimental10 17p-Hydroxy-2a-bromo-50-androstan-3-one (Ia).-To a solution of 17p-hydroxyLt5a-androstan-3-one (50 g.) in glacial acetic acid (200 ml.) was added rapidly a solution of bromine in glacial acetic acid (200 ml., 5.0% Brpin acetic acid v./v.). When the bromine color disappeared, the solution was diluted with water, and the precipitate was collected; recrystallization from acetone-petroleum ether, followed by aqueous methanol, gave I a ; m.p. 182-183" dec.; lit,.4 m.p. 180-181' dec.; yield, 42 g. (667,). 17p-Acetoxy-2a-bromo-50-androstan-3-one (Ib).-One gram of Ia was dissolved in pyridine (2 ml.) and cooled in ice, while acetic anhydride (2 ml.) was added with stirring. After standing a t room temperature for 6 hr., the solution was poured in a thin stream and under vigorous stirring into ice-cold water. The solids were filtered with suction and repeatedly mashed with hot water, then dried in an oven at 110' for 4 hr. (900 mg.). I t was dissolved in 5 ml. of benzene-petroleum ether (b.p. 30-60") mixture (1 : 1 ) and chromatographed over a column of Woelm neutral (10) Melting points were taken i n a Kofler m i r r o hot stage and are corrected. Microanalyses b y Galbraith Laboratories. Inr., Iinoxville, Tenn.
