Journal of Medicinal Chemistry, 197'0, Vol. 1,3, "Il.'o.3 415
A4NTILEUKE:YICS-TRITYLCPSTEISXS
cleaved in refluxing trifluoroacetic acid but less readily by HBr, and is riot affected by heavy metal salts (heavy metal halts can cleave the S-S bond on Ssubstituted cystine). Based on the results of Konig, et a1.,20 we postulate that the difference in sensitivity of I1 and I11 toward trifluoroacetic acid may be due to the presence of the benzylic H of I11 (and possibly of 117). It is probable that the rate and conditions of cleavage might be related to the inhibitory activity. Since practically no cleavage was observed for S-9fluorenylcysteirie (VIIIa) and S-9-(9-methyl)fluorenylcysteine (VIIIb) , preparation and activity correlation study of various derivatives with aryl substitution (IX) were carried out.
qp
(CsHj)3CNH(CHz)rCHCOOH
I
h-Hz
1 "
xVI I
SYIII
(C6H5)3CSCHzCHCOOH
I
SIX
(D
(CBH~)~CS(CH~)ZCHCOOH
I
NHz
NHz form)
XX
I n addition, for a comparison of biological activity, N-acetyl-S-9-(9-phenyl)fluorenylcysteine (XXI) and N-tritylserine Me est'er (XXII) were also prepared. HOCHICHCOOCHj
I
NHC(CGHj)] NHCOCH,;
I
2"
R IXa, R = H b, R = p-F c, R = p-C1 d, R = p C H e, R = p-CHJO f. R = 2,3-C4H,(a-naphthyl)
VIIIa, R = H b,R=CH;
Modification of the Side Chain of X-Trityl-L-cysteine. -This includes preparation of possible catabolic ( e . g . , X and XI) and anabolic products of S-trityl-L-cysteinesome of which might act as the actual inhibitor(s) in vivo-including certain polypeptides (detailed 1% ork of dipeptides will be treated in a separate part of our work) such as the glutathione derivativesz1 (XII). Other structural changes of the alanine moiety (XIIIXVI) of the parent compound, preparation of trityl derivatives of serine and lysine (XVII and XVIII), and preparation of X-trityl-D-cysteine (XIX) and X-tritylDL-homocysteine (xx)were also carried out. Compound XX might interfere with the transmethylation reaction.22 (C,H,),CSCHzCHiCOOH
(CsHj)3CSCHiCHzX;Hz.HCl XI
x
,(&W,H,MCH,CHCONHCH,COOH I R
NH
I
OCCH-CH-CHCOOH
I
NH2 XIIa, R = H
b,R=p-F (Ct,H,)jCSCHiCHCOOEt
I
NHz .HC1 SI11 (C,H,)aCSCHiCHCOOH "Et2
I
I
?;XI
SCH2CHCOOH
SH
(CaHj)3COCHzCHCOOH
( CsH0)3CSCHzCHCOOH
I
XHCOCHs XIV HJC
I
(C6HJ)jCS-CCHCOOH
I1
HaCNHz XVI (21) I n general, higher glutathione content is found in tumor t h a n in normal tissue. See, f o r example, V. I. Gorodyskii, I. V. Veselaya, and K. I. Uvarova, V o p . E k a p . Onkol, 147 (196.5); Ckem. Abstr., 66, 271728 (1967). ( 2 2 ) R . T. Taylor and H. \I-eissbacli, Arch. Biochem. Biophys.. 129, 728, 745 (1969).
XXII
Chemistry.-Among the lmon-n met'hods for the preparation of S-substituted cysteines7~20,23-26 and St'ritylation r e a c t i ~ n s , ~ 'condensation -~~ of cysteine with the appropriate aryl-substituted carbinols with BF,,*rz0 which involves initial carbonium ion formation of the carbinol followed by nucleophilic attack of the S atom of cysteine or related compounds, was adapted for the present preparation. The carbinols and fluorenols were obtained through conventional Grignard synthesis. The BF3-AcOH catalyzed condensation reactions between various carbinols with cysteines and related compounds can be generalized as follows. (1) Compounds containing primary SH groups (such as cysteine, p-mercaptopropionic acid, P-mercaptoethylamine, etc.) readily condense with tritanol or tri(substituted aryl)carbinols in good (70-90%) yield and the product's are usually of high purity; ( 2 ) electron-donating groups substitut'ed on the aromatic rings favor the condensation, whereas electron-wit'hdrawing groups retard the condensation; no reaction was noted between cysteine and p-nitrotritanol or diphenyl-4-pyridinylmethanol (the relative ease of carbonium ion formation of various triphenylmethanols in another medium has been reported33and is in accord with the present observation) ; (3) condensat'ion of tertiary SH derivatives, such as penicillamine, and tritanols give fair (50-70%) yields of condensation products; (4) condensation of cyst'eine and secondary aralkyl alcohols, such as 9-fluorenol, gives poor (10-20%) yields, perhaps due to the relative instability of the carbonium ion; ( 5 ) in the case of 0 analogs of cysteine, condensation of tritanols with primary alcohols ( e . g . , serine) give moderate (20-40y0) yields; no reaction was observed between tritanols and secondary alcohols (e.g., threonine) or between tritanols and phenolic derivatives ( e . g . , tyrosine) ; (6) tritanols do not condense with the N analogs of cysteine (e.g., lysine) because of prot'onation of the amino group by AcOH. Nf-Trityllysine (XVIII) was prepared by the (23) J . L. \Toad and V. du Vigneaud, J. Biol. Chem., 130, 109 (1939). (21) H. Zaiin and K . Traumann, Justus Liebigs A n n . Chem., 681, 168 (1953). (25) d u Vigneaud, J. L. Wood, a n d F. Binkley, J . B i d . Chem., 138, 369 (1941). (26) H. D . West and G. R. Mathura, ibid.. 208, 315 (1954). (27) C. Finzi and V. Bellavita, Gam. Chirn. Ifal., 62, 699 (1932). ( 2 8 ) H. Burton a n d G. W.H. Cheeseman, J . Chem. S O L , 832 (1953). (29) C. A . XacKenzie and G . Chuchani, J . O r g . Chem.. 2 0 , 336 (1955). (30) G . Cliucliani, H. Diaz, and J. Zahicky, ibid.. 31, 15i3 (1966). (31) N. Darroeta. G. Chuchani, and J. Zahicky. ibid., 31, 2330 (1966). (32) G. Chuchani and K. S. Heckmann, J . Chem. Soc. C, 1136 (1969). (33) E. M. Arnett and R. D. Bushick. J . Amer. Chem. SOC.,86, 1564 (1964).
V.
I:! 12 (i (i (i (i ti ti (i li I:! 12
I 2 I:! 10 I:!
I:!
I"
I:!
I2
I!:
I:!
IS
Is
hSTILEUKEMIC
Journal 4f Jlcrlicr'nui
S-TKI'I.~LCYSTI:I~~,~S
C h i t i i s t r y , l Y ? U , 1'01. l.j8.Yo.
.;
417
TABLE I (Continucd) 7 -
.\I olecular Compd
Yieldb
hly,
formula"
O C
(70)
d e r ( e = 1, 0.1 N NaOH)
[cr]*4~,
Dose, mg/kg
dntileukemic activity against L-1210"--Wt. diff, Survivors T-C
T,'C (7c)
12.5 6/6 -0.9 134 50-200 Nontoxic, inact xx Cz3H23NOPS 215-217 75 Nontoxic, inact 80 -17.9 28-200 XXI CzIH21NO3Y 120-122 5Ck400 Sontoxic, inact 72 f44.8'1 XXII Ce3H23K03T1 142-144 Vehicle: saliiie (ip). Hydrate. e 2 cures (see ref 8 3 ) . All vonipouiids analyzed correctly for C, H, N. * Purified compounds. J Formation of a gel was noted in 0.1 : Y XaOH during optical rotation determination. e Quarterhydrate. 6 '6 survived, T 'C below 124. Prepared by the method described in ref 7. i Hemihydrate. k Lit. [E. Billmari, and N. 1.. Due, H u l l . SOC.Chim. Fr., The hydrochloride salt of this compound was found to be identical with t.hat prepared b>- a different 35, 384 (1924)] mp 208-209". Refererice 10. ,,Immediate cloud formatioil method [F.I. Carroll, H. 11.Dickson, and 31. E. Wall, J . Org. Chrm. 30, 33 (1963)l. was noted ill 0.1 S NaOH. Lit,.34mp 230". p Prepared by the method described in ref 9. q Measured in MeOH (c = 1). 1
0
method of Amiard and Gofinetd4using the principle of selective detritylation. Under the reaction conditions emplo?-ed, tritylation of the E t ester of cysteine resulted in partial hydrolysis (to the extent of 10-20%) of the ester group. The products, which consist of a mixture of tritylated ester and tritylated acid, were readily separated since the acid is insoluble in either water or ether. The amide linkage, on the other hand, was found to be quite stable during the tritylation reaction. Consequently, the S-trityl and S-9-fluorenyl-9-pheiiyl derivatives of mercapturic acid were obtained in good yield. From the preceding study, it was found possible to selectively tritylate S H or primary OH groups in the presence of amino, secondary hydroxy, and/or amido groups. Thus compounds such as the S-tritylglutathione derivatives X I I a and X I I b can be readily prepared from glutathione in a single step. Biological Activity and Discussion.-Preliminary test results3j of derivatives of S-trityl-L-cysteine and related compounds in leukemia L-1210 (see Table I) are summarized as folloms. (a) 3-Tritylthiopropionic acid (X) and the HC1 salt of S-tritylcysteamine (XI), two possible metabolites of S-trityl-L-cysteine, are inactive against leukemia L-1210. Compound XI was found to be rather toxic. (b) All fluorenyl derivatives (VIII, IX, and X X I ) are inactive. Toxicity was noted with S-9-(9-phenyl)fluorenylmercapturicacid ( X X I ) . (c) ilryl-substituted S-trityl-L-cysteines (V) containing moderate electron-donating substituents retained or somewhat lowered the original activity; with moderate electron-withdrawing substituents the derivatives showed similar or slightly improved antileukemic activity when compared with that of S-trityl-L-cysteine. (d) S-(1-Sapthyldiphenylmethy1)-L-cysteine (VII) possesses no antileukemic activity. The corresponding 2-naphthyl isomer VI, on the other hand, exhibits better activity than the original compound. (e) S-Tritylmercapturic acid (XIV) is totally inactive. S-Tritylglutathione (XIIa) possesses very low antileukemic activity, whereas the corresponding p-fluorotrityl analog (XIIb) shows slightly better activity. The E t ester of S-trityl-L-cysteine .HC1 (XIII), which is more soluble in HZ0 than the parent compound 11, has only a trace of activity. The Et&H salt of S,N-ditrityl-Lcysteine (XV) is not active. These results indicate that substitution on the amino group of the original compound invalidates the activity. (f) The biological (3.1) G. Amiard and 13. Goffinet, Bull. Soc. Chim. F r . , 1133 (1988). ( 3 6 ) Test results were provided by contract screeners of CCNSC. Detailed interpretations of test data are provided in Cancer Chemother. R e p . , 26, 1 (1962).
activity of S-trityl-u-cysteine (XIX) is somewhat reduced as compared to that of the corresponding L isomer. (g) 8-Tritylpenicillamine (XVI) possesses no antileukemic activity. (h) 0-Trityl-L-serine (XVII) shows low activity against leukemia L-1210; Ne-tritylL-lysine (XVIII) and the ester of N-tritylserine (XXII) are completely inactive. (i) S-Trityl-uL-homocysteine (XX) is nontoxic and inactive over a wide range of dose levels. (j) The S a salt of S-trityl-L-cysteine, which is readily soluble in HzO, is only 50yoas active as the original compound. This, together with the test results of amino-substituted derivatives (XII, XIY, XV, etc.), suggests that the internal zwitterion form might be important for biological activity. Although the mode of antileukemic action of Strityl-L-cysteine is not yet understood, two possible mechanisms for its action can perhaps be deduced based on the results of the present study. Blocking Mechanism.-This amino acid may participate in certain peptide synthesis and subsequently block the S-S cross-linking of the peptide chains. Carrier Mechanism.-The cysteine portion of this Compound may act as an active carrier for the transport of the trityl radical for certain biological alkylating action. The low but definite activity of S-trityl-u-cysteine as me11 as 0-trityl-L-serine suggests that the second postulation is more probable. Experimental Section All meltiiig points (corrected) were take11 011 a ThoniabHoover meltiiig poiiit apparatus. Since the decomposition points of amino acids are usually irregular and depend on the rate of heating, the data reported were determined as follows. An approximate decomposition poiiit of the amino acid was initially obtained by rapid heating. A second sample was heated rather rapidly to ea. 20" below the approximate decomposition point of the amino acid, at which point the temperature of the bath was then increased at the rate of 2O,'min. The optical rotations were determined with a Hilger standard polarimeter. Where analyses are indicated only by symbols of the elements, analytical results obtained for those elements were within 4~0.4%of the theoretical values. S-(Substituted trity1)-L-cysteines, S-[S-(I)-Substituted phenyl)fluorenyl] -L-cysteines and Related Compounds.-To a stirred suspension of 0.03 mol of anhydrous bcysteine .HC1 or related compounds iri 25 ml of AcOH was added 0.033 mol of the appropriate alcohol followed by 6.5 ml of purified BF3-etherate.36 The temperature of the reaction mixture throughout the addition was maintained a t ca. 25'. An intense coloration (color varies from yellow to dark blue, depending on the iiature of substituents on t,he ring system) iisiially developed immediately and the SIIS( 3 6 ) Cf. L. F. Fieser and AT, Fieser, "Reagents for Organic Syntliesis." John W l e y & Sons, Inc., New York, Pi. Y..1Y67, p 70.
