262 Journal of Medicinal Chemistry, 1971, Vol. 14, N o . 3
soln, IT20, NaHC08, and HzO, then dried ( NazSOp), and concd in uacuo to give the product as an oil. The product was chromatographed on a column of SilicAR CC7 using CHC13 as eluant. The fully blocked tripeptide failed to crystallize from EtOAchexane and was used as the oil; yield 17.3 g (74.49;). A suspension of 0.83 g of 10% Pd:C in 5 ml of AcOH and 40 ml of MeOH containing 0.68 g (18.6 mmoles) of dry HC1 was hydrogenated for 10 min. A soln of 8.65 g (18.6 mmoler) of %-?-totBu-Glu-Gly-G1y >le was added i n 100 ml of XleOIl mid hydrogenation was continued until there was iio further evoliition of CO?. The reaction mixt was filtered and the filtrate was evapd under reduced pressure. The resulting oil was F;t& to yield 5.8 g (84.8%,): mp 76' dec; [a]'% 38.X' i c 1.16, IIiLlF). Anal. (Ci4H26ClN306) C, H, 3. Z-0-tert-Bu-Tyr-y-tert-Bu-Glu-Gly-Gly M e (3).--To a niixtiire of 11.6 g (31.6 mmoles) of y-2ert-biit,yl-Glii-Gly-C;ly Me eater.ITC1 in 200 ml of CI12C12 was added 3.2 g (31.6 mmoles) of E t & and 13.5 g (29.9 mmoles) of Z-0-tert-buiyltyrosine penlachloropheiiyl ester. The solii was stirred a t room temp for 4S hr, t,heii coiicd in t m u o . The residiie was dissolved in EtOAc and washed with lo!; cit,ric acid s o h , 1120, NaHC03, and HtO, t,hen dried (NarS04)mid coiicd in uucuo. The oily product was chromatographed oil a wliiinii of Silicar CC7, eluted with le (1). The first, 2 weeks they were inject,ed iiit,erdermally using complet,e Freinids adjuvant as siispeiisioii medium, atid the 3rd week they were injected sc. The injection on the 4th week was done iv using bliffered saline. Bleedings were coiiducted o n the following week aiid the serrini from each aniriinl was not found to give a precipitin reaction with up to 10,000 pg of the polymer 1 . To 1-ml aliquots of rabbit, ant isera to poly(Try-C:Iri-Ala-(;Iy)C:ly-f-"C Et3 were added inrremeiital amts of u p to 500 ~g of polypeptide 1. To earh tiibe was added the equiv point amt of the antigen ( 3 0 p g ) and the mixt was theii iiicubated at 37". Each tuhe gave a precipitiii reaction. The precipitates were kept at 4 " for 48 hi, washed twice with buffered saline, and collected by cenlrigugation. The I~otalanit of proteiii pptd was estimated by the Kjeldahl method. No iiihibitioii of the precipit iri reaction way observed using Ihe polypeptide 1.
Acknowledgment.--This \vorlt \vas supported by a gr:iiit froin tlic Sntional Science I ~ o u i i d ii ~ o iti .
Antifungal Activity of the Thiosemicarbazones of Some Heterocyclic Aldehydes' 1). fir.
WILhS* \ N D T. SL'PRGNCHUh
l h i ?ion qf ('hewinsiry, iYataonal Research Counczl of Canada,
Ottawa, Canada lieceived August 18, 1970 r.
1liiosernic:trbazuiics,*~~ including tliosc i i d e from heterocyclic : ~ l d e h y d e s ~sho~v - ~ tuberculostthc, :tnt'ibacteri:~I,~ :md carcitiostatic10 activity. The qucstion lias been raised as to the relative act'ivit? of 4-alkyl substit'uted t1iiosemic:irbazones. Thiosemic:irb:tzoties"-'" atid dithiosemicnrbazo~ies'~ n u d e from diphatic and aromatic :Lldehj.des and ketones 1i:ir.e already been shown to be futigist:itic. It is report,ed licre that some thiosemicarbazories (I) and 4substituted thiosemicarbazorles (11) made from liet'eroc! clic :ildchydes :we also effective against cellulolytic fungi. The compounds prepared in this work :we listed in Table I ; ir : L T elemeutal ~ data confirm t h e assigned structures. Satisfactory :Lgreemcnt of melting (1) Issued a s K R C C K O ,l l . i 4 3 (2) 11. C . Calrlwell and TV, L . l";ol,les, J . Amer. Phnrm. .4ss., Sci. E d . , 46, 279 (1956). (3) D . F. llodgen and \Y,I,. Nobles, ibid., 16, 437 (1957). ( 4 ) R . E . Hagenhach and H . Gysin, E z p e r i e n t i n , 8 , 181 (1952). (5) N g . Pli. I3uu-Hoi, Ng. D . Xuong, R . Royer, and D. Lavit, J . Chem. S o c . , 547 (1953). (6) M . Tisler, E z p e r i e n l i a , 19, 261 (1956). (7) J. Bernstein, H . L. Yale, K . Losee, h l . Holsing, J. Martins, and JV,A . Lott, J . Amer. Chem. S o c . , 7 8 , 906 (1951). (8) F. E. Anderson, C . .J. Duca, and J . V. Scudi, i b i d . , 1 8 , 4967 (1951). (9) D . Nardi, $2. Massarani, .i. Tajana, L . Degsn, and 31,J . Magistretti, J . M e d . Chem., 10, 530 (1967). (10) F . A . French and E. J . I3lanz J r . , ibid., 8, 585 (1966). (11) D . M .Wiles and T. Suprunchuk, ibid., 13, 526 (1969). (12) B . G . 13enns. B. A . Ginzras, and C . H . Bayley, A p p l . Microbiol.. 8 , 353 (1960). (13) B . .%.Gingras, G Colin, and C I I . Bayley. J . Pharm. S c i . , 64, 1674 (1965). (14) D. M . \Viles, 13. .I,Gingras, and T . Suprunchuk, Can. J . Chem.. 46, 1375 (1967). (15) JVhere analyses are indicated only b>- symbols of the elements, analytical results ohtained for those elements were within 4 ~ 0 . 4 % of t h e theoretical values.
Journal of Medicinal Chemistry, 1971, Vol. i4,N o . 3 253
NOTES
TABLE I HETEROCYCLIC ALDEHYDETHIOSEMICARBAZONES
Yield,
No..
X"
R
%
MP, O C
180 8lC Ph N 1 170 71d Allyl N 2 186 756 Me N 3 199 61d Et N 4 227 2-Naphthyl 55e N 5 192 45c 3-FCe" N 6 219 95C m-o~Nc& N 7 218 9oe N p-OzNCeH4 8 121 701 N 9 Ph(CHz)z 203 730 H N 10 187 74d Ph 0 11 139 591 Allyl 12 0 163 731 Me 0 13 143 491 Et 0 14 199 73d 1-N aph thy1 0 15 186 70" 2-N aphthy1 0 16 192 8gh 0 17 p-OzNC& 122 71, 0 18 Ph(CHz)z 160 710 H 0 19 200 79* Ph 20 S 160 641 Allyl 21 S 159 881 Me 22 S 188 611 Et 23 S 222 806 1-Naphthyl 24 S 200 49e 2-N aph t h y 1 25 S 208 94e 26 S m-OzNCsHc 192 84" 27 S p-OzNCeHc 169 7 11 28 S Ph(CHz)z 193 74h 29 H S 140 7gd 30 Ph N 95 31 451 Allyl N 172 808 32 Me N 61d 144 Et 33 N 45d 183 34 2-Naphthyl N 411 154 35 3-FCeH4 N 181 36 916 N p-OzNCeHd 116 651 37 N Ph(CHz)z 166 N 77d H 38 157 Ph 39 79, S 163 40 941 Allyl S 202 41 971 S Me 178 42 521 S Et 192 43 1-Naphthyl 881 S 44 2-Naphthyl 195 811 S 45 186 S 971 m-OzNCeH4 46 205 911 S p-OzNCsH4 47 175 S 791 Ph(CHz)z Y = H for 1-38; Y = CHa for 39-47; Z = H for 1-10; Z = CH3 for 30-38. b Anal. C, H, N, S (also F for 6 and 35). Where analyses are indicated only by symbols of the elements, analytical results for those elements were within *0.4% of the theoretical values. CeH6; a MezCO-H20; CeHghexane; 8 EtOH-H20; EtOH. Previous report of this Solvents used for recrystn: MeOH-Ht0; compd i n ' F. Fujikawa, F. Hirao, T. Shiota, M. Natio, and S. Tsukuma, Yakugaku Zasshi, 87, 1493 (1967); Chem. Abstr., 69, 43751 (1967). R. E . Hagenbach and H. Gysin, Esperatia, 8, 184 (1952). k F. E . Anderson, C. J. Duca, and J. V. Scudi, J . Amer. Chem. F. FujiSOC.,73, 4967 (1951). J. Bernstein, H. L. Yale, K. Losee, M. Hoking, J. Martins, and W. A . Lott, ibid., 73, 906 (1931). kawa, Japanese Patent 14,685, July 25 1964); Chem. Abstr., 62, 527b (1963). % L. W. Clemence and H. J. Eichel, U. S. Patent 2,746,972, May 22, 1956; Chem. Abstr., 51, 2871a (1957). (I
f
points has been obtained for the 12 compounds reported previously. I n a standard tube-dilution test,12with pure fungal cultures, the effectiveness of the thiosemicarbazones against Chaetomiumglobosum (2-week incubation) was as follows: (a) 14 and 19 were effective a t 10 pg/ml,
(b) 1,6,7,9,17, and 31 were effective a t 100 &ml, and (c) 3, 10, 13, 20, 22, 24, 29, 35, and 38 were effective a t 1000 pg/ml. Against Aspergillus niger (48-hr incubation), 1, 6, 7, and 9 were effective a t 100 pg/ml; 13, 14, 19, 20, 22, 29, and 31 were effective a t 1000 pg/ml. The antifungal activity of 14 and 19 is equivalent to
254 Journal of Medicinal Chemistry, 1971, Vol. 14, No. S
that of good commercial fungicides'? and some dithios e m i c a r b a ~ o n e s . ~Nevertheless, ~ the thiosemicarbazones of none of the 3 types of heterocyclic aldehydes reported here are as effective as a class against C. globosum as are thiocarbohydrazones,'6 for example. Since moderate effectiveness against both organisms is important, however, some useful generalizations about chemical structure and activity can be made. The order of decreasing effectiveness (by aldehyde class) of the thiosemicarbazones is pyrrole carboxaldehyde furfural > N-methylpyrrolecarboxaldehyde Ei thiopheriecarboxaldehyde. Substitution a t the 4 position does not seem to be important for the first and last types but is for the second and third (cf. ref 3 ) . The order of decreasing effectiveness of substituents is aromatic > aliphatic in the case of the pyrrole carboxaldehyde thiosemicarbazories but is reversed for the N-methyl analogs. There is no obvious reason why the 5-methylthiophene derivatives (30-38) should be completely ineffective (cf. ref 4). Experimental Section Thiosemicarbazide, the substituted isothiocyanates, and the heterocyclic aldehydes were the purest grades obtainable from commerical sources, and were used as received. The preparation of the 4-substituted thiosemicarbazides has been described. 17 General Preparation for Thiosemicarbazones (Table I).-To a warm s o h of [substituted] thiosemicarbazide (0.01 mole) ill Ii2O[Et,OH] (50 nil) containing HAC (1 ml) was added dropwise a s o h of heterocyclic aldehyde (0.01 mole) in EtOH (50 ml). The mixt was heated gently o n a steam bath for 1 hr; Ii20 was added \inti1 the oiiset of ppt8ri. The ppt, which formed on sitbsequent, cooling was sepd by filtrration, washed with cold 507; EtOH-HzO, dried, and recrystd to constant mp. Characteristic ir absorptions: all compds, 3360-3140 ( N H ) ; 1630-159.5 (CN); 1560-1535 (CNH); 1160-1110 (NCN); 836-800 (CS); 2,12, 21, 31, 40, 1640 tC=C). I r spectra were measured with a Model 621 Perkin-Elmer spectrophotometer. Elemental analyses were carried out at the microanalytical lab of Drs. Weiler and Strauss in Oxford, England. Melting points were determined using a Fisher-Johns apparatus and have been corrected. The antimicrobial activity of the compds listed in Table I has been evaluated11 by the tube-dilution method,12 using pure cultures of C. globosum (Strain USIIA 1042.4) and Aspergillus niger (Strain USDA 215-5373.16).
Acknowledgments.-The authors thank Aliss G . Colin for the microbiological screening data, and R. Ironside and V. Boyko for recording the ir spectra. (16) I). A I . Wiles and T Suprunchuk, J . .Wed. Chem., 13, 323 (1970). (17) 1). hI. Wiles and T. Siiprunchuk, Cun. J . Chem.. 46, 1865 (1968). ..~-.
Potential Antitumor Agents. Selenoguanosine and Related Compounds' SHIH-" CHU Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island 02912 Received June 5, 1970
It has been shown previously that 6-selenog~anine~ exhibits antitumor activity on ascites cells of Sarcoma 1SO and against lymphomas L1210 and L-5178Y. (1) This work has been supported by Grant T-536 from the American Cancer Society and Grant 16538-01 from the United States Public Health Service. (2) H. G. Mautner, 8 . H. Chu, J. J. Jaffe, and A. C. Sartorelli, J. Med. Chem. 6, 36 (1963).
These findings have led us to prepare the additional uiireported methylseleno 9-P-D-ribosyl derivatives of (iselenoguanine. We hope these niorc solublc :innlogs will offer further improvement in antitumor :nctivity. This coniniuiiicatiori describes thc iyritlicsis of (i-sclcnoguanosine3 (l),G-methq.lselenogu31iiiic, &met Ii~~lsolcnoguanosine, arid G-metli~lseleiioi1io5i!i~arid prdiminary studies of their biological propcrt ics.
Hoed HO
OH
Experimental Section'
%-Amino-6-seleno-9-p-~-ribofuranosylpur~ne (6-Selenoguanosine) (l).-Condensed ILSe (1.5 ml) was bubbled through a so111 of 0.3 g (0.013 mole) of N a in 30 ml of abs MeOH. 2-Amino-6chloro-9-~-~-ribofuranosylpurine5(3.0 g, 0.00906 mole) in 70 ml of abs MeOH was introduced into the well-stirred orange solli. The mixture was stirred under N2 at room temp for I hr. The greenish solid was collected by filtration and taken up iii 35 ml of 37, NaZCO, arid the colloidal Se was filtered off. The filtrate was acidified with glacial AcOR to pH 4 and cooled. The bright yellow solid was collected, washed with cold HzO, and dried. The yield was 1.75 g (54.4%) mp 197.5' dec. On t l P the Br value in Hz0 is 0.40. Anal. (CIOH1BN,04Se.0.T,HzO) C, H, N . 2-Amino-6-methylseleno-p-u-ribofuranosylpurine(6-Methylselenoguanosine).-A solii of 1.2 g (0.00338 mole) of 1 i n S.45 ml of 0.4 N NaOH (0.00338 mole) was stirred at room temp and 0.22 ml (0.00348 mole) of Me1 was added. The wln was stirred a t room temp for 1 hr. The resulting mixture was filtered and the filtrate extd continuously with E t 2 0 . After 24 hr, the solid was collected and dried in vacuo. The yield was 0.65 g (52.35;,). The product was recrystd from Et,OH-pet ether (30°-60"), mp 144-147". On tlq6 the Rr value in H2O is 0.49. Anal. (CIIHIP NjO4Se.O.5H20) C , H, II'. 2-Amino-6-methylselenopurine (6-Methylselenoguanine).-A soln of 2.23 g (0.01 mole) of selenoguanine in 25 ml (0.01 mole) of 0.4 N NaOH was stirred at room temp and 0.65 ml (0.01 mole) of Me1 added. The soln was kept a t room temp for 1 hr. The light yellow solid was collected, washed with H20, and dried. The yield was 1.85 g (81.170). It was recrystd from h/leOH, mp 218'. On tlce the Rr value in H 2 0 is 0.30. Anal. (C6H?N;Se) C, H, N. 6-Methylseleno-9-p-~-ribofuranosylpurine(6-Methylselenoinosine).-A soln of 0.192 g (0.00058 mole) of selenoinosine' in 1.45 ml (0.00058 mole) of 0.4 N NaOH was stirred a t room temp, and 0.073 ml(0.00058 mole) of Me1 was added. The soln was stirred at room temp for 1 hr. The resulting mixture was extd continuously with EtrO. After 24 hr the solid was collected by filtratioii and dried in vacuo. The Et20 s o h was evapd to dryness. The yield was 0.175 g (85.00/,). The product was recrystd from On tlca the Rr value in EtOH-pet ether (30-60°),mp 154-155'. HzO is 0.70. Anal. ( C ~ I H M N ~ O ~ S ~C, . HH, Z ON.) Dissociation Constants.-pK, values were determined by potentiometric tit,ration using a Radiometer pH meter 26. The selenoguanine, which is very insol in HzO, was dissolved in boiling (3) Very recently 6-aelenoguanosine was synthesized by L. B. Townsend and G . H. Milne, J . Heterocycl. Chem., 7, 753 (1970). (4) All melting points are uncorrected. Analyses were carried out at Midwest Microlab, Inc., Indianapolis, I n d . (5) J. F. Gerster, J. W. Jonea, and R . K . Robins, J. Org. Chem., 26, 945 (1963). (6) Polygram C E L 300 P E I from Brinkmann Instruments, Inc., Westbury, N . Y. (7) H. G. Mautner and J. J. Jaffe, Cancer Res., 10, 381 (1960)
