X
July 196s
6
-
,
l
~
~
~
~
~
~
~
n
-
~
~
~ 717 ~
pounds (25, 45, 51, 54, 57). As the group in the 5
TABLE VI11
~ I O L . ICONCENTRATIONS R FOR 507, INHIBITION OF DIHYDROPOLIC position increases above methyl, activity is sharply decreased. The rat liver erizyme is not as sensitive to ACID REDUCTION BY THREE ISOLATED ENZYILIES -5O%
CoIniid
1
7 1% 14 15 18 20 21 2% 23 24 23 31 3 ;I 35 37 88 39 40 41 42 43 44 45
53 54 58
R a t liver
20,000 4,400 2880 49
inhibitory concn X108, 7 E . coli P s . aeruginosa
14,000 140 14
46
1.0 50
25
40
$2
2,200 1880 7.0 26 6.0
3.0 3.5 4.0 25 6.0 1.5
270 23 1.5 2.0 0.20 0.38
0.92 0.78 1.0
100 14 70 30 37 40 600 700 46 160 320 85 3.0 2.0 0.80 1.2 0.70 0.40 1.4 1.8 0.23
120
derivatives (14, 18, 21, 22, 23) activity reaches a peak in 18 (R, = C4H9). For antibacterial activity, the size of the 5 substituent is quite limited since only 3-methyl and kmsubstituted compounds show appreciable activity. This is illustrated in the series of G-benzyl-7-unsubstituted com-
changes in the 5 substituent as are the bacterial enzymes. As is showri in Table VIII, 54 is nearly as inhibitory as 45 to the rat liver enzyme, whereas 54 is only l/loo as inhibitory as 45 toward the E. coli enzyme. There is a notable change in the spectra of activity between the 5-unsubstituted and &methyl series of derivatives which has been documented prev i o ~ s l y and , ~ which can be seen in Tables I and IV. Generally the 5-unsubstituted derivatives are more active against S. aureus than against E. coli or P . odgaris and the 5-methyl derivatives are more active against E. coli arid P.vulgaris than against S.aureus (compare 18 with 38, and 25 with 45). An over-all picture has been developed of a 2,4diaminopyrido [',3-cl]1)yriniidiiie derivative having uo substituent in the 7 position, no substituent or a methyl group in the 3 position, arid a substituent of medium bulk, consisting of an alkyl group of four or five carbon atoms or a benzyl group, in the G position. Such derivatives are potent inhibitors of bacterial dihydrofolate reductases and thus have general antibacterial activity. The spectrum of this activity depends primarily on the substituent in the 3 position. The action of the derivative is potentiated by sulfonamides and combinations of the two drugs have useful chemotherapeutic indices for the treatment of bacterial infectioris in mammalian species. Acknowledgments.-The authors \\ish to thank Uessrs. X. Iannotti, N.Rigopoulos, and G. Tharrington for technical assistance. Our thxnlis are also due It. A. Seal, 11. L. Clarke, arid W. H. G. Richards for the tests against protozoal infections.
Synthesis and Biological Activity of Some N6-Alkyladenosines 11. H. FLEYSHER, :\I. T. HAKALA, A. BLOCH, AND
R.H. HALL
Depurtiiient of Experimental Therapeutics, Roswell Park Meniorzal Inslilute, Buffalo,
\L
ew l'o&
Recei'uecl January 19, 1968 The synthesis of N6-isoamyl-, K%-propyl-, N6-isopropy1-, and N6-allyladeno~iiiemid S6-isoainyladenine was carried out by a general method iiivolvirig the coiidensation of 6-chloropurine riboside or its base with an excess of the appropriate amine in an alcoholic medium in the presence of calcium carbonate. By this method essentially pure products were readily obtained. The biological effects of these compo~undswere examined with cell cultures of Sarcoma 180 and its subline A H / S and with cultnres of Streptococcus faecalis and Escherichia coli. All the compounds synthesized showed growth inhibitory activity in oiie or the other of these test systems.
S6-(A2-Isopentenyl)adenosine1~2 (IPA) is a very potent cytokinin with activity equal to or better than kinetin and zeatin in the bud tests of Wickson and T h i m a ~ i n . ~This compound mas also shown to inhibit the growth of human and mouse tumor cells in vitro.* Because of this biological activity of IPA and because several S6-alkylpurine bases have shown kinetin activ(1) R. H . Hall. &I. J. Robins, L. Stasiuk, a n d R. Thedford, J. A m . Chem' Soc., 88, 2614 (1966). (2) XI. J. Robins, R. H . Hall, and R. Thedford, Bzochemzstry, 6, 1837 (1967). (3) JI. Wickson a n d K. V. Thimann, P h y s i o l . Plantarum, 11, 62 (1968). (4) J. T. Grace, hI. T. Hakala. R . H. Hall, and J. Blakeslee, Proc. A m . dssoc. Cancer R e s . . 8 , 23 (1967).
ity in tobacco callus growth tests,j it was of interest to determine whether other K6-alliyl derivatives of adenosine would also show biological activity. For this reason the following adenosine derivatives were prepared and their biological activity was exaniined: N6-isoamyl- (I), S6-n-propyl- (11), S6-isopropyl- (III), N6-allyl- (IV), K6-isoamyl- (V) , and Y-methyladenosine. Compounds I-IV have not been reported thus far, while V has been studied by Strongsaand has been shown to possess kinin activity; however, the method of ( 5 ) (a) F. AI. Strong, "Topics in Rficrohial Chemistry." John K i l e y a n d Sons, Inc., Nen. Tork. N. Y., 1958. pp 125-131; (I,) H. Q. Hamzi and F. Skoog, Proc. S a l l . Acud. Sei. C. S., 51, iti (1961).
~
~
71s
l'(11.
11
N
July 1968
6
are inhibitory a t 1 X and 7 X 10-751, respectively, whereas X.faecalis is not affected by these nucleosides even a t 1 X M. To gain some information concerning the mode of action of these compounds, the reversal of growth inhibition by S6-isopentenyladenine and IPA was investigated in both the parent 5180 and in the AH/S subline systems. Inhibition of growth was not prevented by adenine, adenosine, hypoxanthine, inosine, guanosine, deoxyguanosine, cytidine, deoxycytidine, or deoxyuridine. This would indicate that in these cells the de novo synthesis of purines or pyrimidines was not affected by the presence of the inhibitor. The fact that these two compounds were as inhibitory when AH/S cells were grown in an amethopterin medium (containing thymidine, hypoxanthine, and g l y ~ i n e ) ~ as they were in Eagle's medium would also support this assumption. When in the amethopterin mediumg hypoxanthine was replaced by isopentenyladenosine, and no growth of S180 cells was observed, indicating that the analog cannot support cellular multiplication. Whereas the IPA inhibition of mammalian cells could not be reversed by pyrimidines, the inhibition of E . coli by isopentenyladenosine was preventable by cytosine and its nucleoside, and by uridine, deoxyuridine, and thymidine."J The purines, on the other hand, enhanced the inhibitio11.l~ This indicates that in this microbial system, interference with the pyrimidine metabolism mag' be responsible for interference with cell growth. This differential response of various cell types in vitro to the action of the N6-alkyladenosine derivatives also has its parallel in man and the selectivity of action of these compounds appears to be of value in chemotherapy."
Experimental Section The melting points were determined on a Mel-Temp melting point apparatus and are not corrected. Uv spectra were obtained on a Cary Model 14 recording spectrophotometer. The optical rotation was measured on a Jasco hlodel ORD-UV5 optical rotatory dispersion recorder. The solvent systems used for chromatography are given in Table I below. Where analyses are indicated only by symbols of the elements, analytical results obtained for those elements were within *0.47, of the theoretical values. N6-Isoamyladenosine (I).-6-Chloropurine 9p-n-ribofuranoside (2.00 g, 7 mmoles), isoamylamine (1.83 g, 21 mmoles), CaC03 (1.4 g, 14 mmoles), and 100 ml of EtOH were refluxed for 18 hr with stirring. Chromatographic examination of the reaction mixture showed one absorbing spot with an R f different from that of 6-chloropurine or its ribonucleoside. The mixture was filtered, while still hot, to remove calcium salts. The product ciptallized from the filtrate on cooling. It was filtered off, wazhed with chilled EtOH, and dried zn vacuo; yield 2.02 g (865)),mp 138". iln additional yield of 0.34 g (14OG), mp 156O, [ a I z 6-86.5' ~ (c 0.104, EtOH), was obtained from the filtrate. . I d . (C16H23N604) C, H, N. Chromatographic data for this and other preparations and the uv-absorption spectra data are presented in Table I . N'-n-Propyladenosine (11).-6-Chloropurine 9p-n-riboside (2 g, 7 mmoles), 1.239 g (21 mmoles) of n-propylamine, and 1.40 g (14 mmoles) of CaC03were refluxed in 100 ml of EtOH as in I for 20 hr. Chromatographic examination of the reaction mixture revealed one uv spot of product uncontaminated by 6-chloro(9) M . T.Hakala, Science, 126, 255 (1957). (10) .4. Bloch and C. A . Nichol, Proc. A m . Assoc. Cancer Res., 9, 6 (1968). (11) R.Jones, Jr., J. T. Grace, Jr., A. hlittelman, a n d R . E. Gerner, ibid., 9, 36 (1868).
-
A
~
~
~
~
~
719 ~
purine riboside. The mixture was worked up as in I ; yield 2.10 g (977,), mp 161", [ ( Y ] ~ ~-90.5' D (c 0.116, EtOH). Anal. (C13H19NsO4'0.2HzO) C, H, N. Ne-Isopropyladenosine (111) was prepared exactly as was 11 except that 1.239 g of isopropylamine was employed. The reaction mixture showed the absence of 6-chloropurine riboside after 8 hr of refluxing. After filtration from calcium salts, the filtrate was evaporated to dryness and the residue was crystallized from 3 : l (vol) MeCN-EtOH and dried (P206)i n vacuo since the product was found to be very soluble in EtOH; yield 1.40 g ~ (c 0.164, EtOH). Anal. (C13H1e(65'30), mp l.57", [ a I z 5-82.5' N~O4.0.33HzO)C, H, N. No-Allyladenosine (IV).-A mixture of 6-chloropurine riboside (2.00 g, 7 mmoles), allylamine (1.197 g, 21 mmoles), CaC03 (1.40 g, 14 mmoles), and 100 ml of EtOH was refluxed for 18 hr so that no residual 6-chloropurine riboside remained. The filtrates from the Ca salts were evaporated to dryness by several additions of anhydrous EtOH during evaporation. The residue was recrystallized from H20 (charcoal) containing a trace of EtOH and in 2iacuo a t 78"; yield 1.35 g (637,), mp 166-167', dried (P206) [a] - 101' (c 0.094, EtOH). Anal. (Cl3H17N504) C, H, X. This compound was also made by a modification of the method employed by Jones and Robins' and Leonard, et al.12 Bdenosine (7.5 mmoles) and allyl bromide (22.5 mmoles) in D h l F (30 ml) were agitated a t 33" for 24 hr to form N1-allyladenosine (65%). After removal of the excess of allyl bromide and D h I F by evaporation, the residue was heated to 80-90" in xH4OH (pH 10-12) to convert the N1-allyladenosine to W-allyladenosine. The product was obtained by chromatography on a Celite column using solvent system A. The product, P-allyladenosine, came off the column first leaving the residual adenosine adsorbed on the Celite. Evaporation of the eluate to dryness, followed by crystallization of the product from H20 containing a trace of EtOH, gave a 55yc yield of IV. The physical properties were the same as given above. No-Isoamyladenine (V).-The biological propertie of this compound have been previously reported by however, its synthesis and physical properties have not been described. It was synthesized and tested in connection with this work. A mixture of 6-chloropurine (1.082 g, 7 mmoles), isoamylamine (1.827 g, 21 mmoles), CaC03 carbonate (1.40 g, 14 mmoles), and 2-methoxyethanol (60 ml) was stirred and refluxed for 5 hr, after which time chromatographic examination revealed the absence of 6-chloropurine. The hot reaction mixture was filtered and the Ca salts were washed with hot methoxyethanol. The combined filtrates were evaporated in vacuo and the tan residue azeotroped five times with EtOH. The material was crystallized from H 2 0 containing a trace of EtOH (charcoal) and after evaporation of most of the EtOH the crystals were filtered, washed (H20),an! dried (P&) in vacuo to yield 1.17 g (81.57,) of V, mp 201 Anal. ( C I ~ H ~ SC, X H, ~ ) K. Effect on the Growth of Sarcoma 180 Cells in Vitro.-The compounds were added in aqueous solution to the cultures of S180 cells and to a subline AH/S13 grown as monolayers in T-15 flasks in Eagle's14 medium containing 57, horse serum. The cells were exposed to the compounds for 7 days involving four changes of the medium. During this time the control cultures increased by 15-20-fold, as estimated by protein determinations.'& The results are given in Table 11. Microbial Assay Procedures.-The microbial assays were carried out according to previously published techniques.'e E. coli K-12 was grown in the synthetic medium described by Gray and Tatum." S. faecalis 8043 was grown in the medium of Flynn, et al.,'* from which uracil and the purines had been omitted and to which 1 mpg/ml of folic acid was added. The compounds were added to the medium at 10-3 to 10-6 Jf concentrations. Results are given in Table 11.
.
(12) N. J. Leonard, S. Achmatowicz, R. N. Loepky, K. L. Carraway. W. A. Grim, A. Saweykowska. H . Q. Hamai, a n d F. Skoog, Proc. Natl. Acad. Sci. U . S.,56, 709 (1966). (13) hl. T.Hekals a n d T. Ishihara, Cancer Res., 22, 987 (1962). (14) H. Eagle, Science, 130, 432 (1959). (15) 0. H . Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J . B i d . Chem., 192, 265 (1951). (16) A. Bloch a n d C. Coutsogeorgopoulos, Biochemistry, 6, 3345 (1966); A. Bloch, M.H. Fleysher, R . Thedford, R. J. hlaue, a n d R. H. Hall, J . Med. Chem., 9, 886 (1966). (17) C. H.Gray a n d E. L. T a t u m , Proc. Nati. Acad. Sci. U . S., SO, 404 (1944). (18) L. AI. Flynn, V. B. Williams, B. L. O'Dell, and -4. G. Hogan, Anal. Chem., 23, 180 (1951).
~
~
Acknowledgments.-The authors uish to thalik 1)r. Charles h.Nichol for his interest arid encouragement of this nork. Thiq research T\ ab supported ill p:wt I)\
grant- from the S:ltlotlal C a n c c ~I ~ l ~ i i i u t[( .~h. , lbl)ll(~ H w l t h Scrvicv ( Ci.I-0.XY7 : i l l t i ('.I( iraiit T-43; f 1 0111 t h c .hiericaii ( ' : i i i w i
Antiviral Agents. I. Analogs and Derivatives of 2-Diethylaminoethyl 4-Methylpiperazine-1-carboxylate
'i Variety of alialogs ai id derivatives of L'-tiiethyinriliiitJet~i~l 4-1tlelhylpiperaziiit~-t - ( w h . hynthesized arid examined for aiitiviral activity 1's. iiifliieiiaa i n mire. Xiiieteei~c ~ r ~ ~ n p o ~ i i i active brit none was siiperior to 1. A str~ictriie-activity velatioilship ii dihviissed a i d iiietl described. A t elevated temperat iirea some ~-dialk~l:rri~iiroethylii~ethaiis \vc're f ~ l i l l dtlJ a c t ~ o ~ ~ t(I\-), h~l toward their corresponding 2-dkylamiiio ethoxide ions to f o r m syrnrnetrical ~ ) i ~ - L ' - d i a l k ~ ~ l a r i i i iethers
The diicovery2 that 2-diethylaminoethyl 4-niethj 1I)ii)erazine-l-carboxS.late (1) exhibit< significant antiviral activity against an Influenza (PR8) infection in riiice led US to synthesize variou- arialogs and derivat ivei. Thih report d e ~ ~ i b ethe s structure-xtivity ielationships of thebe compounds ti9 n ell :is ioinc of the. rhcmiitrj involved in their iyntheies. a
CH,
b
\
I
n
N I \ /
\ I
A'COO
c
! I
d
CH2CH2 N(C,H,). I
I
1
Chemistry.-For purposes of discussion 1 ma? he considered t o consist of four parts, a, b, c, arid cl. The synthesis of analogs arid derivatives of 1 will be considered under those four headings. Variations in a (Table I).-Various alkyl groups nerc attached by the reaction of 2-diethylaminoethyl piper:izine-l-carbox?l:te (2) with an alkyl halide, as i i i procedure '1. Other variations in a are listed in Table I. Their syntheses n ere classical arid uiiconiplicated and are described in the Experimental Section. Variations in c, d, and c
+ d (Tables I1 and 111).
1Lost of the compounds of this class uere prepared b! i)ne of the folloning three methods: procedure B, L: tr:iii.;csterific:Ltioii reaction using ethyl -hiethylpipernzine-l-carboxyl:ite (1) as the starting material; procedure C, from phosgeiie 1 iu B chloroformate ester or D , from ~-nieth;\-lpiper:tziiie-l-\-lc:irboii~-l chloride. The transesterificxtioii method described as pr(1cedure B is a normal base-catalyzed reaction. Howwer. we should emphasize the fact that in order to obtain high yieldh and avoid the production of byproduct the reaction must be c:irricd out at a temper:iture not to exceed ea. 1%". In the early l'hases of the iri\-c.stigtttion the coriditioiis uhed for this reaction were >imilar to those described by Turrier3a in which ethyl 4( 1 ) (a) Yurnriier employee, Career Traininp Propram. 1964; (1)) slimmer employee, Career Trainin4 Program, 1'363. ( 2 ) H. F,Lindlr a n d AI, Forbes, l'roc. S O C B . x p . Bioi. .lfed., 121, 65 (19661. ( 3 ) (a) I t . J. Turner, U. 8 . Patent 2,617,803 (1952). 0))h related reaction, a n N-alkylation of hindered amines by dialkylaminoethyl carbonate esters, was recently described b y L. \Veintraub a n d R . Terrel, .I. O r a . Chem.,
90, 2470 (19fi5).
li
I1 1
k Ill
L
-
J
IIlA
nun1ber oi :1na1ogi (\X) of 1 \\ere pX!p:Lrcd b) dlov-jrig an S-tertiary :imino alcohol T' to react first IT itli I)ho.gciir to give :I cliloroforniate intermcldiate \'I not I~o1utt.d).: i d then n ith 4-niethylpiper:izit1e o r other : i i i i i t w \ (i)rorrdure C). .llthough t l w J ields laiiged iron1 moderately good to I c ) ~ , thii a1)])roach was versatile and, i n contrast to ester iiitei-
