T H E J O U R N A L OF
Organic Chemistry 0 Copyright 1987 by the American Chemical Society
VOLUME52, NUMBER16
AUGUST7, 1987
Synthesis of Novel Imidazole-Containing DNA Minor Groove Binding Oligopeptides Related to the Antiviral Antibiotic Netropsin Krzysztof Krowicki and J. William Lown* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 Received February 18, 1987
Analysis of the structural and stereochemical requirements for the strict DNA base sequence recognition of (AT), and (AT), respectively for the oligopeptide minor groove binding agents netropsin and distamycin leads to proposals for the rational structure modification for altered base and sequence recognition. The syntheses of new analogues of the natural oligopeptide antiviral antibiotic netropsin bearing one or more imidazole moieties in place of pyrrole are described in order to test this hypothesis. In this regard nitration of ethyl l-methylimidazole-2-carboxylatewas investigated. During the reduction of nitroimidazole derivatives with stannous chloride, a novel chlorination of the imidazole nucleus was encountered.
Introduction Several alternative approaches may be considered in attempting to design sequence-specific DNA binding ligands for possible application as gene control agents. One approach, which is being developed by several groups, is to use the inherent specificity of oligonucleotide^.'-^ The latter have certain disadvantages associated with difficulties of transport across membranes,'P2 susceptibility,to rapid intracellular nuclease degradation,r5 and in requiring single stranded DNA as a cellular target.' We are currently addressing the problem of nuclease sensitivity of such probes by exploring the properties of unnatural a-oligoAlternatively groove selective agents offer advantages as probes for molecular recognition in that they target for duplex DNA and their binding does not incur helix distortion as in the use of intercalator~.~J~ We have chosen as our starting point netropsin1'J2 and distamycin,13
which are members of a modest family of natural oligopeptides including anthelvencin,', kikumycin B,15amidinomycin,16 and noformycin," which exhibit antibiotic, antiviral, and antitumor activity. Evidence from a study of their biochemical pharmacology indicates that they act to block the template function of DNA by binding to specific nucleotide sequences in the minor groove of double helical DNA.'? These sequences are (AT), and (AT), respectively for netropsin and distamycin.'? Examination of the structural requirements for the molecular recognition, deduced, in part, from recent X-ray studies on a complex of netropsin with a dodecamer,18 suggested that the replacement of one or more pyrrole rings by imidazole, or other appropriate heterocycle, should result in a rational alteration of base recognition from AT to GC.19 The latter prediction follows from the implied formation of new hydrogen bonds between G(2)-NH2in the minor groove and
(1)Galen, P. J.; Pines, 0.;Inouye, M. Annu. Rev. Biochem. 1986,55, 569. (2)Ts'O, P. 0.P.; Miller, P. S.; Gollne, J. J. In Development of Target-Oriented Anticancer Drugs; Cheng, Y.-C., Goz, B., Minkoff, M., Eds.; Raven Press: New York, 1983;p 189. (3) Toulene, J.-J.; Krisch, H. M.; Loreau, N.; Thuong, N. T.; Helene, C. Proc. Natl. Acad. Sci. U S A . 1986,83,1227. (4)Wickstrom, E. J. Biochem. Biophys. Methods 1986,13,97. (5)Zannechik, P. C.; Godchild, J.; Taguchi, Y.; Sarin, P. S. Proc. Natl. Acad. Sci. U.S.A. 1986,83,4143. (6)Morvan,F.;Rayner, F.; Imbach, J.-L.; Chang, D.-K.; Lown, J. W. Nucl. Acids Res. 1986, 14, 5019. (7)Morvan,F.; Rayner, B.; Imbach, J.-L.; Chang, D. K.; Lown, J. W. Nucleic Acids Res. 1987,15, 4241. (8)Morvan,F.;Rayner, B.; Imbach, J.-L.; Chang, D. K.; Lown, J. W. Nucleosides Nucleotides, in press. (9)Neidle, S., Waring, M. J., Eds. Molecular Aspects of Anticancer Drug Action; Topics in Molecular and Structural Biology: Vol. 3,Macmill&: London;1983. (10)Gale, E. F.; Cundliffe, E.;Reynolds, P. E.; Richmond, M. H.; Waring, M. J. The Molecular Basis of Antibiotic Action, 2nd ed.; Wiley: New York, 1981.
(11)Julia, M.;PrBau-Joseph, N. Bull. SOC.Chim. Fr. 1967,4348;C. R. Hebd. Seances Acad. Sci. 1963,257,1155. (12) Hahn, F. E. In Antibiotics III. Mechanism of Action of Antimicrobial and Antitumor Agents; Corcoran, J. W., Hahn, F. E.,Eds. Springer-Verlag: New York, 1975;p 79. (13)Arcamone, F.;Orezzi, P. G.; Barbieri, W.; Nicolella, V.; Penco, S. Gazz. Chim. Ital. 1967,97, 1097. (14)Probst, G.W.; Hoehn, M. M.; Woods, B. L. Antimicrob. Agents Chemother. 1965,789. (15)Takaishi, T.;Sugaware, Y.; Suzuki, M. Tetrahedron Lett. 1972, 19,1873. (16)Nakamura, S.;Karasawa, K.; Yonehara, H.; Tanaka, N.; Umezawa, H. J . Antibiot., Ser. 1961,14, 103. (17)Diana, G. D. J.Med. Chem. 1973,16, 857. (18)Kopka, M.L.; Yvon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E. Proc. Natl. Acad. Sci. U.S.A. 1985,82,1376. (19)R. E. Dickerson, Molecular Biology Institute, UCLA has independently suggested the substitution of imidazole for pyrrole in netropsin on the basis of X-ray diffraction analysis of the antibiotic cocrystallized with a dodecamer:18 Dr. Dickerson and his co-workers are currently attmpting similar cocrystallization and X-ray analysis with out compounds 26 and 36 bound to a duplex oligodeoxyribonucleotide.
I
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0 1987 American Chemical Society
Krowicki and Lown
3494 J . Org. Chem., Vol. 52, No. 16, 1987 the N3 of the imidazole moiety. We have recently confirmed this prediction20 and now report details of the syntheses of these novel oligopeptide analogues. This report includes certain synthetic aspects of imidazole nitration, reduction of nitroimidazoleswith stannous chloride and the concomitant chlorination of the imidazole ring, as well as efforts to improve the efficiency of introduction of the guanidineacetyl moiety in this class of oligopeptide agents. Synthetic Strategy. The structure of the antibiotic netropsin was established in 1963 by Julia and Pr6au-Joseph.ll Since that time syntheses of both the parent antibiotic'l and that of certain analogues have been reported. The latter are those bearing additional pyrrole units or in which the pyrrole rings were replaced by benzene, pyridine. trimethylpyrrole, or thiophene,21or by changing the mode of pyrrole ring substitution from 2,4 to 2,5.22 The syntheses of these latter compounds have been based largely on the original method of Julia and PrBau-Joseph." The anticipated need to examine a number of new structures in order to establish the molecular recognition characteristics for DNA binding required development of an efficient and flexible general synthesis. Therefore we recently reported new efficient total syntheses of netropsin and distamycin, which are sufficiently adaptable for the present requirement^.^^ The strategy adopted involves significant changes in the methods and order of introduction of the amidine and guanidineacetyl moieties from those reported hitherto.'l,*l This resulted in better yields of the final products (Schemes 1-111). An additional important advantage was the avoidance of chromatography, which is not suitable for such polar compounds as netropsin, because of contamination of the final products with inorganic salts eluted from the absorbents. The syntheses of the imidazole analogues are essentially based on our method of synthesis of n e t r o p ~ i nand ~ ~distamycinZ3but with modifications necessitated by the presence of imidazole moieties. Nitration of 1-Methylimidazole-2-carboxylic Acid. Reaction of 1-methylimidazolewith ethyl chloroformate in the presence of triethylamine afforded the ester 2.24 Nitration of 2 afforded three products, 3, 4, and 5 , which were readily separable and of which only the 5-nitro derivative 4 has been described previously25(Scheme I). Development of this reaction showed that the highest yield of the desired compound 5 is obtained by using a 1:l mixture of 100% nitric acid and sulfuric acid at a temperature of 95 "C for 50 min. Longer reaction times tend to give more of the undesired 4,5-dinitro derivative 3. Reaction of 2 with a mixture of nitric acid and acetic anhydride is very slow whereas a similar reaction using trifluoroacetic anhydride is very rapid and affords largely the dinitro compound 3. Compound 5 was isolated from the preferred procedure in crystalline form in 46.5% yield. Alkaline hydrolysis of 5 afforded the imidazole-2-carboxylic acid 6 after acidification in 95% yield. Compound 6 proved to be sensitive to decarboxylation upon heating under acidic conditions. (20) Lown, J. W.; Krowicki, K.; Bhat, N. G.; Skorobagaty, A.; Ward, B.; Dabrowiak, J. C. Biochemistry 1986, 25, 7408. (21) (a) Julia, M.; Gombert, R. Bull. SOC.Chim. Fr. 1968.36. (h) Julia, M.; Gombert, R. Bull. SOC.Chim. Fr. 1968, 376. (c) Jones, D. H.; Wooldridge, K. R. M. J. Chem. SOC.C 1968, 550. (d) Gendler. P. I-.: Rapoport, H. J . Med. Chem. 1981, 12, 33. (22) Bialer, M.; Yagen, B.; Mechoulam, R.; Becker. Y. J . Pharm. Sci. 1980,69, 1334. (23) Lown, J. W.; Krowicki, K. J. Org. Chem. 1985, 50: 3774. (24) Regel, E.; Buchel, K. M. Liebigs Ann. Chem. 1977, 145. (25) Merck and Co. Neth. Appl. 6409117: Chem. Ahstr. 1965. 63. P606.
'+
J
I
c7i$ s
CONu,CC212cN
"Reaction conditions: (a) C1CO2Et, Et,N; (b) H z S 0 4 + HN03; ( c ) aqueous NaOH, then HCl; (d) (COCl)z or pivaloyl chloride and
NEt3, add NH2(CH2)2CN;(e) SnC12.2H20 + HCl, acetyl chloride, chloroacetyl chloride or acyl chloride of 6; (f) SnC12.2H20 + HCl; (g) EtN(i-Pr),, acyl chloride of 6; (h) Pd/H,.
The initial attempt to condense the acid 6 with aminopropionitrile employed oxalyl chloride, which, however, afforded, in addition to the main product 8 in 80% yield, a small amount of the 5-formyl derivative 7 (Scheme I). The latter plausibly arises from initial oxalylation at position 5 followed by hydrolysis of the acyl chloride and decarboxylation. For this reason an alternative procedure was adopted employing the mixed anhydride of 6 and pivalic acid to give 8 in 91% yield. Reduction of Nitroimidazole Derivatives with Stannous Chloride. Since catalytic reduction of nitroheterocycles was inconvenient on a larger scale, an alternative procedure was investigated. Reduction of 8 with stannous chloride afforded a mixture of two compounds 9a and loa, which were not separated but were identified by means of their N-acetyl and N-chloroacetyl derivatives 9b, lob, and 9c, lOc, respectively (Scheme I). The compositions of the derivatives 9(b,c),10(b,c)were established unequivocally by MS exact mass measurement. In the case of chloroacetyl derivatives 9c and 1Oc the ratio of the 5-chloro derivative to the nonchlorinated one was 56:44. However, this ratio depends on the order of addition of the reactants, being higher if SnC1, is added slowly to the nitro compound dissolved in aqueous HC1. and lower if the nitro compound is added to the solution of SnC1, in aqueous HCI. Coupling of the compounds 9a
J. Org. Chem., Vol. 52, No. 16, 1987 3495
DNA Minor Groove Binding Oligopeptides
Scheme XIID
Scheme XI. Possible Mechanism for the 5-Chlorination of Imidazole Which Accompanies SnC1, Reduction of 8
+ ck
H20NH
0 -
H o N \ ~ coNHcH2cH2cN
H+ -H +
coNHcH,cH,cN
CH3
18
17
I
CH3
NH
4
b
C
H
3
e NHCOCH,Br
C H 3 ~ N H C O C H 2 N H - C \NH-CN
19
cH3-@NHCOCH2NH%NH2
-
20
-&
21
LCH3-@NHCOCH2NHCN
1
d,
22
HN , NU
CIQ - c o N H C H 2 c H 2 c N I CH3 1Oa
and 10a (as a mixture) with the acid chloride of 6 gave the corresponding peptides 11 and 12 (Scheme I), which were readily isolable in 50% and 12% yields, respectively, and were identified individually. Stannous chloride reduction of compound 12 also led to two products, 13a and 14,which were readily separated by chromatographyto give 30% and 57% yields, respectively. No such chlorination of the second imidazole ring of 12 occurs, so this reaction is evidently connected with the process of reduction of the nitro group. A large excess of stannous chloride caused a decrease in the proportion of chlorination products from about 56% to about 2 % . However this cannot be due to reduction of the chloro substituent by stannous chloride, similar to that described by Rinkes,26because the chloro derivatives 10 and 13b survive such prolonged treatment. We also found that neither compound 9a nor 14 reacts with stannous chloride. We suggest that this 5-imidazole chlorination may follow the mechanism suggested in Scheme 11, which is analogous to that of the reaction of N-phenylhydroxylamine with HF affording p-aminofluorobenzenen and that of the reduction of nitrobenzene with stannous chloride and hydrochloric acid producing a mixture of 53% of chloroaniline and 47% of aniline.28 Small amounts of chloro derivatives formed in the presence of a large excess of SnC1, means that in the case the reduction of the intermediate hydroxylamine group prevails over the rearrangement. Although the reduction of the nitro compounds with stannous chloride affords some of the side product due to imidazole chlorination, it was faster and more convenient on a larger scale for intermediate stages than catalytic reduction in the presence of palladium. However, in order to avoid chlorination in the step leading to the final three ring compound 15, the nitro compound 12 was reduced catalytically with hydrogen in the presence of palladium on charcoal to give 14 in an excellent yield of 80% (Scheme 1). Coupling of the dipeptide 14 with the acid chloride of 6 afforded the tripeptide 15 in 68% yield. Similar catalytic reduction of 15 gave the amino tripeptide 16 in 65% yield. Introduction of the Guanidineacetyl Moiety. With the bis(imidazo1e) dipeptide 14 and the corresponding tris(imidazo1e)tripeptide 16 in hand, we now turned to the attachment of the guanidineacetylend group. The existing (26) Rinkes, I. J. Organic Syntheses; Wiley: New York, 1943; Collect. VOl. 2, p 393. (27) Titov, A. I.; Baryshnikova,A. N. Zh. Obshch. Khim. 1953,23,346. (28) Blanksma, J. J. Recl. Trau. Chim. Pays-Bas 1906, 25, 365.
h."
CH3-Q)--NH~~~~2~~-~\ NHCN 20 a Reaction conditions: (a) CNNH2, NaOEt, dicyclohexano-18crown-6; (b) CNNHLi; (c) Hg(OAc)Z, CNNHNa.
literature method of coupling the amine with guanidineacetic acid hydrochloride in the presence of dicyclohexylcarbodiimide (DCC) was unsatisfactory because of the low yield caused by a side reaction between DCC and aminopyrrole derivative^.^^ Therefore alternative procedures were examined. Attempted couplings of the chloroacetyl group in 9c with guanidine carbonate, acetate, or free base were unsuccessful. A report on cyanoaminoacetic acid formation from disodium cyanamide and chloroacetic acid30 suggested an alternative method.31 Therefore reactions with model compounds 17 and 19 and sodium cyanamide designed to afford 22 were investigated. Reaction of the chloro compound 17 with sodium cyanamide in ethanol afforded the cyclic product 18. In a similar reaction of the bromo compound 19 in protic solvents the cyanoguanidinyl derivative 20 was isolated (Scheme 1111, although the desired compound 22 is implicated as an intermediate. Presumably compound 22 is too reactive to survive in the presence of the cyanamide sodium salt. Therefore in order to avoid the basic conditions of the reaction the thiourea derivative 21 was allowed to react with mercuric acetate. Although the desired compound 22 was formed, it again proved to be unstable under the reaction conditions. Its formation was confirmed by the reaction with sodium cyanamide to afford 23. The above efforts being unsuccessful, we turned back to the condensation in the presence of DCC by optimizing the conditions. The best results were obtained by using 1 equiv of the nitro compound, e.g., 8, 12, or 15, to be reduced, 1.5 equiv of guanidineacetic acid hydrochloride, and 1.5 equiv of DCC. Introduction of the Amidine Moiety by Modified Pinner Reaction. Formation of the amidine moiety was effected by a modification of the Pinner reaction.32 Our observations agree with those of B a k ~ h e e vthat ~ ~ the first step of the reaction of the cyano group, i.e., formation of the imino ester with an alcohol in the presence of hydrogen chloride, is completed in 90 min and that longer reaction (29) Bialer, M.; Yagen, B.; Mechoulam, R. J.Heterocycl. Chem. 1980, 17(8), 1797. (30) Fromm, E.Liebigs Ann. Chem. 1925,442, 147. (31) All our attempts to repeat the original reactionmfailed. Since the cyanoaminoacetic acid has not been reported in the literature subsequently, it appears unlikely that it can be isolated in monomeric form. (32) Pinner, A.;Klein, F. Chem. Ber. 1877, 10, 1889. (33) Baksheev, A. N.; Gavrilov, N. I. Zh.Obshch. Khim. 1952,22,2021; Chem. Abstr. 1952, 47, 864/e.
3496 J. Org. Chem., Vol. 52, No. 16, 1987
Krowicki and Lown Scheme V4
Scheme IV"
CH,
NH,
CH-. 8
24
CH3 H,N
32 Y = C H Z = N 33 Y = N Z ' C H
30 Y = C H 31 Y = N
%-NHCH,CONH H~N' CLCONHCH2CH2CN CH3
cl-
+
CH3 25
H2N\\ C-NHCH2CONH
-
H,N'
14 16
c26 n = l 27 n=2
+
34 Y = C H . Z = N 35 Y = N . Z = C H
d
I
NHCHzCHzCN
+ H2N_
C-NHCH2CONH 31 Y = N . Z = C H
HZN
28 n = l 29 n.2
I CH?
CH3
+
NH
0
NHCH2CH2C \ 1
SO4
NH2
"Reaction conditions: (a) CH,OH + HC1, NH3; (b) DCC; (c) (H2N)2CfNHCH2C02H, Cl- and DCC; (d) EtOH + HCl, NH,, Na2S04.
times promote side reactions resulting in lower yields. The imino ester reacts readily with ammonia in ethanolic solution within l h at ambient temperature. The conditions for both reactions, i.e., Pinner and condensation with guanidineacetic acid, were first optimized with the monoimidazole derivatives 8 and 9a to give 24 and 25, respectively. In the final step of the synthesis, involving compounds 14 and 16, the latter were first allowed to react with guanidineacetic acid since the products, 26 and 27, respectively, could be readily purified. Subsequent introduction of the amidine moiety to afford the final target molecules 28 and 29 proceeded almost quantitatively (Scheme IV). The latter products were isolated initially as the hydrochloride salts contaminated with a little ammonium chloride and were very soluble in protic solvents. Exchange of the counterion with sodium sulfate afforded the corresponding sulfate salts which were more readily purified by recrystallization. The composition of these polar compounds was established by FAB-MS.34 In the case of the chloride salts (M - C1)+or (M - HCl - C1)+ions were observed for one or two salt functional groups, respectively. For the corresponding sulfates both MH+ and (M - HS04)+ions were observed. Synthesis of Oligopeptides Containing Both Pyrrole and Imidazole Moieties. The isomeric imidazolepyrrole and pyrrole-imidazole dipeptides 32 and 33 were synthesized following similar procedures from 30 and 31 and l-methyl-4-nitroimidazole-2-carboxylic acid and 1methyl-4-nitropyrrole-2-carboxylicacid, respectively (Scheme V). After the reduction of the nitro groups, the resulting amino compounds 34 and 35 were condensed with guanidineacetic acid hydrochloride in the presence of DCC to give 36 and 37. The Pinner reaction conditions on the (34) Hogg, A. M. Int. J . Mass Spectrom. Ion Phys. 1983, 49, 25
I 38 Y = C H . Z = N 39 Y=N Z=CH
CH3
"Reaction conditions: (a) 5% Pd/C/H2 acyl chloride of 6 or acyl acid; (b) 10% chloride of l-methyl-4-nitropyrrole-2-carboxylic Pd/C/H,; (c) (H2N)2C+NHCH2C02H, Cl- and DCC; (d) EtOH + HCl, NH,.
latter compounds produced the target compounds 38 and 39. Initial findings on the altered DNA sequence specificity of these novel oligopeptides have been reported.20 These studies while confirming the predicted GC-recognizing character of the imidazole moiety in turn led to other predictions concerning the contribution of certain van der Waals contacts in their molecular recognition of DNA. Appropriate models are being synthesized to elucidate these factors and their syntheses and DNA specificity will be reported in due course.
Experimental Section Melting points were determined on a Fisher-Johns apparatus and are uncorrected. The IR spectra were recorded on a Nicolet 7199 F.T. spectrophotometer, and only the principal sharply defined peaks are reported. The 'H NMR spectra were recorded on Bruker WH-200and WH-400spectrometers. The spectra were recorded on approximately 525% (w/v) solutions, depending upon the spectrometers, in appropriate deuteriated solvents with tetramethylsilane as internal standard. Line positions are recorded in ppm from reference. Electron impact and FAB mass spectra were determined on an Associated Electrical Industries (AEI) MS-9 and MS-50 focussing high resolution mass spectrometers. Kieselgel 60 (230-400 mesh) of E. Merck was used for flash chromatography and precoated sheets of Kieselgel60 FZb4 of E. Merck were used for thin layer chromatography. TLC systems: (i) for covalent peptidic compounds, chloroform-methanol 9:l; (ii) for ionic compounds with one ionic pair, methanol with some acetic acid; (iii) for ionic compounds with two ionic pairs, methanol with some formic acid. Ethyl 1-Methylimidazole-2-carboxylate (2). A solution of 3.2 mL (40 mmol) of 1-methylimidazole (Aldrich Chem. Co.) in 20 mI, of anhydrous acetonitrile and 10 mL of triethylamine was
DNA Minor Groove Binding Oligopeptides cooled to -30 "C and a solution of 7 mL (66 mmol) of ethyl chloroformate in 10 mL of acetonitrile added rapidly while maintaining the temperature a t
