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Charge Transfer in the Mechanism of Drug Action Involving Quinoxaline Di-N-oxides
Charge Transfer in the Mechanism of Drug Action Involving Quinoxaline Di-N-oxides MICHAEL D.
RICHARDG. SCAMEHORN',
RYAN*,
AND PETER
KOVACIC*"
Received June 21,1984, from the Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53207 and the * Department of Accepted for publication November 26, 1984. Chemistry, Marquette University, Milwaukee, Wl 53233. Abstact 0 Cyclic voltammetry data were obtained for various 2,3disubstituted quinoxaline di-N-oxides: dimethyl, bishydroxymethyl, bisacetoxymethyl, bis-N-anilinomethyl, and dicarboxaldehyde hydrate. The dimethyl derivative exhibited the most negative EV,value, and along with the diol, showed reversible reduction for the first wave. Rationalizations of the El,. values are provided. Reasonable correlations exist for the electrochemical data and drug activity. The results support the diiminium theory of drug action.
While significant advances in our knowledge of the mechanism of drug action have been made during the past decades, suggested hypotheses generally lack detail at the molecular level. Recently, it was proposed',' that the conjugated iminium ion plays a vital role in physiological activity involving various important areas. Some of the more obvious examples include:
drugs3"(cyanines) ( l ) herbicides4 , (diquat) (2), redox enzymes3b [pyridoxal phosphate (3) and chlorophyll iminium"* (4)], vitamins3' (retinal iminium) ( 5 ) ,alkaloids (protonated myosmine (6),and carcinogen^"^^^'^ (0-alkylated guanine in DNA) (7). Generally, the iminium species is believed to be generated metabolically in vivo. The principal function is participation in charge transfer (CT) processes: beneficial transformations, interference with normal electron transfer, or generation of toxic oxy radicals. The positive charge enhances electron abstraction from cellular material. In some cases, the ions may exert their effect by electrophilic alkylation, e.g., of DNA." Diiminium ( B ) ,which is believed to possess a high free energy content and a positive reduction potential,'~2~12 is expected to undergo reversible one-electron reduction to the radical cation 9:
+
+
+*
2
1
Since the requisite structure is already present in certain heterocyclic di-N-oxides, e.g., the quinoxaline category, we have chosen this class of antibacterial agents for electrochemical investigation. There has been little discussion concerning the mode of action of these types of corn pound^.'^^'^ In contrast with 8, the di-N-oxide 10 is neutral overall and generates radical anion 11 on one-electron reduction:
I
0
0
I
--NZC--C=N-
I 3
e
Z=H,M"+
4
I
0
1
-
0
'
1
--N=C:C=N-
I
I
(2)
t 11
10
Experimental Section
5
492
Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
The known quinoxaline 1,4-dioxides 12 were prepared by standard literature procedures (mp corrected): 12b, mp 188189°C (lit.16mp 189-190°C); 12c, mp 171-172°C (lit.17mp 170172°C); 12d, mp 174-176°C (lit? mp 176-177°C); W e , mp 146-148°C (dec.) (lit.I7 mp 152-154°C); 13, mp 203-204°C (dec.) [lit.'* mp 202.5-203.0"C (dec.)]. NMR for the dialdehyde hydrate 13: (Me2SO-d6) (60 MHz, Varian EM360L): 6 6.77 and 7.10 (2 br s, 2), 8.20 (br, 2), 8.50 (m, 2), and 9.05 (m, 2), consistent with a mixture of cis and trans isomers; the MS (CI) contained a base peak at m/z 218 (M - H,O) and no parent peak at m/z 236. Dilution of an MezSO solution of 13 with water resulted in recovery, indicating stability in aprotic polar solvents. 0022-3549/85/0400-0492$0 1.00/0
0 1985,American PharmaceuticalAssociation
The cyclic voltammetric measurements were performed on an ECO model 550 potentiostat with a PARC model 175 waveform generator. The data were acquired with a California computer system 2210. All solutions were deaerated with prepurified nitrogen which was passed through an oxygen-scrubbing system. The working electrode was a platinum flag, while the reference electrode was a n IBM aqueous Ag/AgCl electrode in saturated KCl. The supporting electrolyte was tetrabutylammonium perchlorate (G. F. Smith Chemical Co.), and the solvent was dimethylformamide (DMF) of the highest available purity (Aldrich Chemical Co.).
Results and Discussion Cyclic Voltammetry-The compounds selected for electrochemical study were 2,3-disubstituted quinoxaline 1,4-dioxides [dimethyl ( 12b),15bishydroxymethyl ( 12c),17bisacetoxymethyl ( l2d),I7 bis-N-anilinomethyl ( 12e)I7] and the dicarboxaldehyde hydrate (13)."Fortunately, data on drug activity are available for most of the substances, which permits comparison with the electrochemical results. 0
I
0 I
I
bH
0
C,
QH
a,R= H ; b, R= CH 3 R = C H 20H ; d. R = CH2 0 A c
nitrogen, thus increasing electron attraction, and also stabilizes the oxyanions formed during charge transfer. It is interesting to compare 12c with iodinin 14a. O
R
R
O 14
Both compounds contain hydroxyl groups situated for hydrogen bonding via six-membered rings. The greater ease of reduction20,22 for 14a is expected due to the additional aromatic ring, the more acidic phenolic groups, and the greater degree of rigidity in the H-bonded system. An increase of 0.3 V is observed in going from 1 2 b to 1 2 c , and a similar increase (0.27 V) is seen for 14a versus phenazine 5,lO-dioxide 14b ( E , = -0.78 V, corre~ted).'~ Diacetate 12d had an E, which was 0.3 V more positive than for dimethyl 12b and gave an irreversible wave. The greater ease of reduction follows from the presence of the electronegative acetate group. The close correspondence in the potentials for diacetate and diol is notable. The effect of the ester moiety might be further enhanced by interaction with N-oxide as in 15. In a previous polarographic study of 1 2 d , E, = -1.60 V (Hg/AgClO, DMF), it was suggested that ease of reduction was associated with lability of methylene protons.'' The values for the difference between the E, figures for 12b and 1 2 d from the present work and the earlier investigation" correspond quite closely (0.31 versus 0.34).
e, R=CH2NHC6H5 12
13
The half-wave reduction potentials determined by cyclic voltammetry in DMF are summarized in Table I. In the cyclic voltammogram (AgJAgC1) of 1 2 b , the first wave entails reversible reduction with E, = -1.36 V, producing a stable radical anion. The effect of the methyl groups is to make the E , more negative by about 0.16 V (compare 12a and 12b). The literature value of -1.20 for 12a was determined with a standard calomel electrode (SCE)." Although the two electrodes are different, the E, figures derived from the two in prior studies are generally quite similar, with values -0.05 V more positive often being found with SCE." Kazakova et aLZ1 reported Eu = -1.94 (Hg/AgClO, DMF) for 12b. Diol 12c undergoes a reversible one-electron reduction with EIh= -1.06 V. The radical anion, stable only a t high scan rates, is less stable than in the case of the parent dimethyl 12b. The greater ease of reduction for 12c versus 12b can be rationalized by the inductive effect of hydroxyl and hydrogen bonding with N-oxide. Hydrogen bonding enhances the positive nature of
I
0
I
OAc
15
Diamine 12e also generated an irreversible wave with E,, = -0.86 V. As with 12c and 1 4 a , favorable geometry exists for H-bonding. Since there is not enforced coplanarity for 12c and 1 2 e , as compared to 14a,there should be a lesser effect on E,. While H-bonding from oxygen is normally stronger than from
Table I-Half-Wave Reduction Potentials of Quinoxaline Di-Noxides ~~
Compound 12a 12b 12c 12d 12e 13 14a 14a 14b
El,, versus AQIAgCI,V" -1.20b,= -1.36 -1.06 -1 .05d -0.86d -1 .05d -0.51" -0.35' -0.826
In DMF. Taken from ref. 19; versus SCE. Sweep rate (V.s-'), <1 Irreversible wave; 100 MV.s-'. Taken from ref. 22. 'Taken from ref. 20. a
x
0 I
NHCgHg I 16
nitrogen, the aromatic ring may help facilitate strong interaction and delocalization as in 16 and, thus, make reduction more facile. The instability of the radical anion product is not unexpected, since 12e itself decomposes in ~ o l u t i o n . ' ~ The dicarboxaldehyde hydrate 13gave a series of irreversible waves: E,, = -1.05 V, Ep2= -1.20 V, and Ep3= -1.38 V. The first one exhibited the same potential as for the diol and diacetate. Compared with the diol, a larger inductive effect would be expected based on the greater number of oxygens, and the more acidic, hemiacetal hydrogens should H-bond more Journal of Pharmaceutical Sciences / 493 Vol. 74, No. 4, April 1985
Table Il-Antibacterial ~-
Activity of Quinoxaline Derivatives”
~
Quinoxaline Derivative
Activity In Vitrob
In Vivo”
Salmonella Pseudomonas Salmonella Pseudomonas typhi aeruginosa typhi aeruginosa Inactive 500 Inactive 2,3-Dimethyl >500-2500 125 200 200 12a 15.6-62.5 12b >500-2500 >500 50 400 50 200 125-250 12c 3.9-31.2 12d 31 -2-500 500 50 400 a Taken from ref 23. Mininum suppressive concentration, pg/rnL. Optimum therapeutic dose, rng/kg; degree of activity. strongly, but H-bonding would not involve desired coplanarity due to the position of hydroxyl on the fused aliphatic ring. Instability of the radical anion is not surprising in light of the polar, labile hemiacetal structure. Correlation of Electrochemical Properties w i t h D r u g Activity-Almost all of the quinoxalines investigated have been shown to exhibit antibacterial and other pharmacological action,’5.1x.2:i.24 Details for comparative antibacterial activity of some members are provided in Table 11. Dioxidine (12c), which has been studied rather extensively, is one of the more potent agents. Quinoxidine (12d) is rapidly hydrolyzed to 1 2 c in mice.” Dimethyl 12b possesses higher activity in vivo than in vitro and is converted to 1 2 c in mice.I5 Furthermore, iodinin 14a, a natural antibiotic, exhibits more powerful drug action than any of the quinoxalines or their metabolites.I5 Thus, a reasonably consistent picture emerges relating structure, activity, electrochemical characteristics, and metabolism, in keeping with the proposed theoretical framework. The main criteria for high activity in the ultimate agent generally appear to be ( a ) existence of the di-N-oxide system 1 0 , ( b ) facilitation of CT by hydrogen bonding, and ( c ) adequate radical anion stability to permit electron transfer in a catalytic manner. The importance of t,he first feature is demonstrated by comparison of E , for 1 2 b and 2,3-dimethylquinoxaline. The dioxide is found to be 0.4 V more positive than the deoxygenated counterpart which is essentially Phenazine 5,lO-dioxide ( E , = -0.8 V) and phenazine (E,,2 = -1.13 V) comprise a similar situation.’” The most active quinoxalines (e.g., 12c) and phenazines (e.g., 14a) usually exhibit hydrogen bonding, and the more powerful 14a also is characterized by a more positive E,. Compounds which show irreversible reduction waves are found to be less active, e.g., 13,’* or else there is metabolic conversion to an active form which possesses a reversible wave, e.g., 12d to 12c. For 13, only weak activity has been observed, and there is apparently no report concerning the activity of 12e. Although 1 2 d , 12e, and 13 have favorable reduction potentials, they give products too unstable for reversible electron transfer. Rapid metabolic breakdown could also take place giving rise to impotent products. Concerning the more specific aspects of drug action based on the working premise, the diiminium type moiety may serve as a conduit for electron transfer which might disrupt normal CT processes. Alternat,ively, electron transfer from microbial constituents to oxygen could result in the formation of toxic oxy radicals via superoxide. Formation of oxy radicals is known to occur in the natural leukocyte (phagocyte) response to invading organi~rns.‘~~’~ The dual characteristic of antibacterial action and mutagenicity is observed for an appreciable number of the di-N-oxide5..2:1.29 This lends further credence to the hypothesis that both processes may involve oxy radicals.’,’ There are previous reports which indicate a correlation between biological activity and half-wave reduction potential. For example, mitomycins possessing less negative E,,2values exhib494
/ Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 7985
ited more powerful antibiotic activity.:’”Also, recent data reveal a relationship between carcinogenic activity and E,,g values of purines and pyrimidines in ionic form resulting from alkylation or protonation.” The range for the E , figures (-0.98 to -1.2 V) obtained from the purine salts is similar to that of the heterocyclic di-N-oxides (Table I). Kaye and Stonehill pointed out that an objection to the theory may be raised because of the rather negative reduction potentials.”’ However, they provided a number of answers to the possible criticism, including beneficial influences to be expected in vivo. In addition, favorable solvation, stereochemical, and counterion effects might well be operative in living systems during reduction. Appreciable alteration in half-wave redzction potentials with change in the medium has been observed in other studies.3’ Of course, an absolute correlation between drug activity and electrochemical behavior is unreasonable. Many other factors pertain, such as solubility, diffusion, absorption, metabolic behavior (either destructive or beneficial), stereochemistry, and active site binding. The literature contains only meager treatment of the mechanistic aspects.’:’ I d McIlwain proposed inhibition of vitamin K by competition for common receptor sites due to similarity in structure.’” Alt,ernatively, a molecular model based on NMR data and energy calculations indicates that quinoxaline antibiotics form a bis-intercalated complex with DNA in which two base pairs are sandwiched between the chromophore~.’~ If intercalation occurs followed by oxy radical generation via CT, then this would represent another instance of “site specific, free radical” formation.:” In summary, the overall results support the participation of electron transfer processes in the mechanism of drug action involving quinoxaline di-N-oxides.
References and Notes 1. Kovacic, P . “Fundamental Chemistry of Life and Death”; UWM Bookstore: University of Wisconsin-Milwaukee, 1983; pp 1-151. 2. Kovacic, P. Kem. Ind. 1984, 33, 473; Chem. Abstr. 1985, 102, 41873. 3. Kovacic, P. “Abstracts, Joint Great Lakes and Central Regional Meeting, Kalamazoo, MI,” (a) Medicinal Chemistry Division, No. 230, (b) Biological Chemistry Division, No. 50, (c) Medicinal Chemistry Division, No. 231; Am. Chem. SOC.:Washington, DC, 1984. 4. Kovacic, P. “Abstracts, 186th National Meeting, American Chemical Society, Agricultural and Food Division”; Am. Chem. SOC.: Washington, DC, 1983; p 162. 1984, 106, 5. Ward, R.; Chang, C . K.; Young, R. J. Ant. Chem. SOC. 3943. 6. Hanson, L. K.; Chang, C. K.; Ward, B.; Callahan, P. M.; Babcock, G. T.; Head, J . D. J. Am. Chem. SOC.1984, 106, 3950. 7. Petke, J. D.; Maggiora, G. M. J . Am. Chem. Soc. 1984, 106, 3129. 8. Maggiora, L. L.; Maggiora, G. M. Photochem. Photobiol. 1984,39, 847
9. Kovacic, P. Age 1983,6, 144. 10. Kovacic. P.: Ryan. - . M. D.: Nelson, V. C.; Crawford, P. W., unpublished results. 11. Sariaslani, F. S.; Eckenrode, F. E.; Beale, J. M., Jr.; Rosazza, J . P. J . Med. Chem. 1984, 27, 749. 12. Eberlein, G.; Bruice, T. C. J . Am. Chem. Soc. 1983, 105, 6679. 13. McIlwain, H. J . Chem. SOC.1943, 322. 14. Waring, M. J . Annu. Reu. Biochem. 1981,50, 159. 15. Bambury, R. E. in “Burger’s Medicinal Chemistry,” 4th ed., part 2; Wolff, M. E., Ed.: Wiley: New York, 1979; pp 69-71. 16. Landquist, J . K.; Stacey, G. J . J. Chem. SOC.1953, 2822. 17. Landquist, J. K.; Silk, J. A. J. Chem. SOC.1956, 2052. 18. Musatova, I. S.; Elina, A. S.; Anisimova, 0. S.; Padeiskaya, E. N.; Novitskaya, N. A. Khim. Farm. Zh. 1979, 13, 42; Chem. Abstr. 1979,91, 157687. 19. Miyazaki, H.; Matsuhisa, Y.; Kubota, T. Bull. Chem. Soc. Jpn. 1981,54, 3850. 20. Mann, S. Arch. Mikrobiol. 1970, 71, 304. 21. Kazakova, V. M.; Sokol, 0. G.; Dvoryantseva, G. G . ; Musatova, I. S.; Elina, A. S. Chem. Heterocycl. Comp. 1980, 16, 284. 22. Ryan, M. D.; Hollstein, U.; Scamehorn, R. G.; Kovacic, P., unpublished results.
23. Tolcheev, Y. D.; Padeiskaya, E. N.; Smolyanskaya, A. Z.; Pershin, G. N.; Elina, A. S.; Musatova, I. S. Khim. Farm. Zh. 1982, IS, 1482; Chem. Abstr. 1983,98, 137204. 24. Rudzit, E. A,; Lisitsa, L. J.; Tagirov, R. F. Zh. Mikrobiol. Epidemiol. Immunobiol. 1973, 112; Chem. Abstr. 1973, 79, 87603. 25. Padeiskaya, E. N.; Polukhina, L. M.; Budanova, L. I.; Kuzovkin, V. A.; Pershin, G. N.; Sokolov, S. D. “Curr. Chemother. Proc. Int. Congr. Chemother., loth, 1977,” vol. 1, 1978; p 579; Chem. Abstr. 1978,89, 99565. 26. Sawyer, D. T.; Komai, R. V. Anal. Chem. 1972, 44, 715. 27. Baehner, R. L.; Boxer, L. A,; Ingraham, L. M. in “Free Radicals in Biology,” vol. V; Pryor, W. A,, Ed.; Academic Press: New York, 1982; chap. 4, pp 91-93. 28. Weitberg, A. B.; Weitzman, S. A.; Destrempes, M.; Litt, S. A.; Stossel, T. B. N . Engl. J. Med. 1983,308, 26.
29. Hashimoto, T.; Negishi, T.; Namba, T.; Hayakawa, S.; Hayatsu, H. Chem. Pharm. Bull. 1979, 27, 1954; Chem. Abstr. 1979, 91, 187227. 30. Kinoshita, S.; Uzu, K.; Nakano, K.; Shimuzu, M.; Takahashi, T.; Matsui, M. J. Med. Chem. 1971, 14, 103,109. 31. Kaye, R. C.; Stonehill, H. I. J . Chem. SOC.1952,3244. 32. Kolthoff, I. M.; Lingane, J. J. “Polarography,” vol. 11; Interscience: New York, 1952, p 625. 33. Bachur, N. R.; Gee, M. W.; Freedman, R. D. Cancer Res. 1982, 42, 1078.
Acknowledgments This work was presented in part a t the 188th National Meeting, American Chemical Society, Philadelphia, PA, Abstr. MED 173 (1984).
Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
/ 495
RICHARDG. SCAMEHORN',
RYAN*,
AND PETER
KOVACIC*"
Received June 21,1984, from the Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53207 and the * Department of Accepted for publication November 26, 1984. Chemistry, Marquette University, Milwaukee, Wl 53233. Abstact 0 Cyclic voltammetry data were obtained for various 2,3disubstituted quinoxaline di-N-oxides: dimethyl, bishydroxymethyl, bisacetoxymethyl, bis-N-anilinomethyl, and dicarboxaldehyde hydrate. The dimethyl derivative exhibited the most negative EV,value, and along with the diol, showed reversible reduction for the first wave. Rationalizations of the El,. values are provided. Reasonable correlations exist for the electrochemical data and drug activity. The results support the diiminium theory of drug action.
While significant advances in our knowledge of the mechanism of drug action have been made during the past decades, suggested hypotheses generally lack detail at the molecular level. Recently, it was proposed',' that the conjugated iminium ion plays a vital role in physiological activity involving various important areas. Some of the more obvious examples include:
drugs3"(cyanines) ( l ) herbicides4 , (diquat) (2), redox enzymes3b [pyridoxal phosphate (3) and chlorophyll iminium"* (4)], vitamins3' (retinal iminium) ( 5 ) ,alkaloids (protonated myosmine (6),and carcinogen^"^^^'^ (0-alkylated guanine in DNA) (7). Generally, the iminium species is believed to be generated metabolically in vivo. The principal function is participation in charge transfer (CT) processes: beneficial transformations, interference with normal electron transfer, or generation of toxic oxy radicals. The positive charge enhances electron abstraction from cellular material. In some cases, the ions may exert their effect by electrophilic alkylation, e.g., of DNA." Diiminium ( B ) ,which is believed to possess a high free energy content and a positive reduction potential,'~2~12 is expected to undergo reversible one-electron reduction to the radical cation 9:
+
+
+*
2
1
Since the requisite structure is already present in certain heterocyclic di-N-oxides, e.g., the quinoxaline category, we have chosen this class of antibacterial agents for electrochemical investigation. There has been little discussion concerning the mode of action of these types of corn pound^.'^^'^ In contrast with 8, the di-N-oxide 10 is neutral overall and generates radical anion 11 on one-electron reduction:
I
0
0
I
--NZC--C=N-
I 3
e
Z=H,M"+
4
I
0
1
-
0
'
1
--N=C:C=N-
I
I
(2)
t 11
10
Experimental Section
5
492
Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
The known quinoxaline 1,4-dioxides 12 were prepared by standard literature procedures (mp corrected): 12b, mp 188189°C (lit.16mp 189-190°C); 12c, mp 171-172°C (lit.17mp 170172°C); 12d, mp 174-176°C (lit? mp 176-177°C); W e , mp 146-148°C (dec.) (lit.I7 mp 152-154°C); 13, mp 203-204°C (dec.) [lit.'* mp 202.5-203.0"C (dec.)]. NMR for the dialdehyde hydrate 13: (Me2SO-d6) (60 MHz, Varian EM360L): 6 6.77 and 7.10 (2 br s, 2), 8.20 (br, 2), 8.50 (m, 2), and 9.05 (m, 2), consistent with a mixture of cis and trans isomers; the MS (CI) contained a base peak at m/z 218 (M - H,O) and no parent peak at m/z 236. Dilution of an MezSO solution of 13 with water resulted in recovery, indicating stability in aprotic polar solvents. 0022-3549/85/0400-0492$0 1.00/0
0 1985,American PharmaceuticalAssociation
The cyclic voltammetric measurements were performed on an ECO model 550 potentiostat with a PARC model 175 waveform generator. The data were acquired with a California computer system 2210. All solutions were deaerated with prepurified nitrogen which was passed through an oxygen-scrubbing system. The working electrode was a platinum flag, while the reference electrode was a n IBM aqueous Ag/AgCl electrode in saturated KCl. The supporting electrolyte was tetrabutylammonium perchlorate (G. F. Smith Chemical Co.), and the solvent was dimethylformamide (DMF) of the highest available purity (Aldrich Chemical Co.).
Results and Discussion Cyclic Voltammetry-The compounds selected for electrochemical study were 2,3-disubstituted quinoxaline 1,4-dioxides [dimethyl ( 12b),15bishydroxymethyl ( 12c),17bisacetoxymethyl ( l2d),I7 bis-N-anilinomethyl ( 12e)I7] and the dicarboxaldehyde hydrate (13)."Fortunately, data on drug activity are available for most of the substances, which permits comparison with the electrochemical results. 0
I
0 I
I
bH
0
C,
QH
a,R= H ; b, R= CH 3 R = C H 20H ; d. R = CH2 0 A c
nitrogen, thus increasing electron attraction, and also stabilizes the oxyanions formed during charge transfer. It is interesting to compare 12c with iodinin 14a. O
R
R
O 14
Both compounds contain hydroxyl groups situated for hydrogen bonding via six-membered rings. The greater ease of reduction20,22 for 14a is expected due to the additional aromatic ring, the more acidic phenolic groups, and the greater degree of rigidity in the H-bonded system. An increase of 0.3 V is observed in going from 1 2 b to 1 2 c , and a similar increase (0.27 V) is seen for 14a versus phenazine 5,lO-dioxide 14b ( E , = -0.78 V, corre~ted).'~ Diacetate 12d had an E, which was 0.3 V more positive than for dimethyl 12b and gave an irreversible wave. The greater ease of reduction follows from the presence of the electronegative acetate group. The close correspondence in the potentials for diacetate and diol is notable. The effect of the ester moiety might be further enhanced by interaction with N-oxide as in 15. In a previous polarographic study of 1 2 d , E, = -1.60 V (Hg/AgClO, DMF), it was suggested that ease of reduction was associated with lability of methylene protons.'' The values for the difference between the E, figures for 12b and 1 2 d from the present work and the earlier investigation" correspond quite closely (0.31 versus 0.34).
e, R=CH2NHC6H5 12
13
The half-wave reduction potentials determined by cyclic voltammetry in DMF are summarized in Table I. In the cyclic voltammogram (AgJAgC1) of 1 2 b , the first wave entails reversible reduction with E, = -1.36 V, producing a stable radical anion. The effect of the methyl groups is to make the E , more negative by about 0.16 V (compare 12a and 12b). The literature value of -1.20 for 12a was determined with a standard calomel electrode (SCE)." Although the two electrodes are different, the E, figures derived from the two in prior studies are generally quite similar, with values -0.05 V more positive often being found with SCE." Kazakova et aLZ1 reported Eu = -1.94 (Hg/AgClO, DMF) for 12b. Diol 12c undergoes a reversible one-electron reduction with EIh= -1.06 V. The radical anion, stable only a t high scan rates, is less stable than in the case of the parent dimethyl 12b. The greater ease of reduction for 12c versus 12b can be rationalized by the inductive effect of hydroxyl and hydrogen bonding with N-oxide. Hydrogen bonding enhances the positive nature of
I
0
I
OAc
15
Diamine 12e also generated an irreversible wave with E,, = -0.86 V. As with 12c and 1 4 a , favorable geometry exists for H-bonding. Since there is not enforced coplanarity for 12c and 1 2 e , as compared to 14a,there should be a lesser effect on E,. While H-bonding from oxygen is normally stronger than from
Table I-Half-Wave Reduction Potentials of Quinoxaline Di-Noxides ~~
Compound 12a 12b 12c 12d 12e 13 14a 14a 14b
El,, versus AQIAgCI,V" -1.20b,= -1.36 -1.06 -1 .05d -0.86d -1 .05d -0.51" -0.35' -0.826
In DMF. Taken from ref. 19; versus SCE. Sweep rate (V.s-'), <1 Irreversible wave; 100 MV.s-'. Taken from ref. 22. 'Taken from ref. 20. a
x
0 I
NHCgHg I 16
nitrogen, the aromatic ring may help facilitate strong interaction and delocalization as in 16 and, thus, make reduction more facile. The instability of the radical anion product is not unexpected, since 12e itself decomposes in ~ o l u t i o n . ' ~ The dicarboxaldehyde hydrate 13gave a series of irreversible waves: E,, = -1.05 V, Ep2= -1.20 V, and Ep3= -1.38 V. The first one exhibited the same potential as for the diol and diacetate. Compared with the diol, a larger inductive effect would be expected based on the greater number of oxygens, and the more acidic, hemiacetal hydrogens should H-bond more Journal of Pharmaceutical Sciences / 493 Vol. 74, No. 4, April 1985
Table Il-Antibacterial ~-
Activity of Quinoxaline Derivatives”
~
Quinoxaline Derivative
Activity In Vitrob
In Vivo”
Salmonella Pseudomonas Salmonella Pseudomonas typhi aeruginosa typhi aeruginosa Inactive 500 Inactive 2,3-Dimethyl >500-2500 125 200 200 12a 15.6-62.5 12b >500-2500 >500 50 400 50 200 125-250 12c 3.9-31.2 12d 31 -2-500 500 50 400 a Taken from ref 23. Mininum suppressive concentration, pg/rnL. Optimum therapeutic dose, rng/kg; degree of activity. strongly, but H-bonding would not involve desired coplanarity due to the position of hydroxyl on the fused aliphatic ring. Instability of the radical anion is not surprising in light of the polar, labile hemiacetal structure. Correlation of Electrochemical Properties w i t h D r u g Activity-Almost all of the quinoxalines investigated have been shown to exhibit antibacterial and other pharmacological action,’5.1x.2:i.24 Details for comparative antibacterial activity of some members are provided in Table 11. Dioxidine (12c), which has been studied rather extensively, is one of the more potent agents. Quinoxidine (12d) is rapidly hydrolyzed to 1 2 c in mice.” Dimethyl 12b possesses higher activity in vivo than in vitro and is converted to 1 2 c in mice.I5 Furthermore, iodinin 14a, a natural antibiotic, exhibits more powerful drug action than any of the quinoxalines or their metabolites.I5 Thus, a reasonably consistent picture emerges relating structure, activity, electrochemical characteristics, and metabolism, in keeping with the proposed theoretical framework. The main criteria for high activity in the ultimate agent generally appear to be ( a ) existence of the di-N-oxide system 1 0 , ( b ) facilitation of CT by hydrogen bonding, and ( c ) adequate radical anion stability to permit electron transfer in a catalytic manner. The importance of t,he first feature is demonstrated by comparison of E , for 1 2 b and 2,3-dimethylquinoxaline. The dioxide is found to be 0.4 V more positive than the deoxygenated counterpart which is essentially Phenazine 5,lO-dioxide ( E , = -0.8 V) and phenazine (E,,2 = -1.13 V) comprise a similar situation.’” The most active quinoxalines (e.g., 12c) and phenazines (e.g., 14a) usually exhibit hydrogen bonding, and the more powerful 14a also is characterized by a more positive E,. Compounds which show irreversible reduction waves are found to be less active, e.g., 13,’* or else there is metabolic conversion to an active form which possesses a reversible wave, e.g., 12d to 12c. For 13, only weak activity has been observed, and there is apparently no report concerning the activity of 12e. Although 1 2 d , 12e, and 13 have favorable reduction potentials, they give products too unstable for reversible electron transfer. Rapid metabolic breakdown could also take place giving rise to impotent products. Concerning the more specific aspects of drug action based on the working premise, the diiminium type moiety may serve as a conduit for electron transfer which might disrupt normal CT processes. Alternat,ively, electron transfer from microbial constituents to oxygen could result in the formation of toxic oxy radicals via superoxide. Formation of oxy radicals is known to occur in the natural leukocyte (phagocyte) response to invading organi~rns.‘~~’~ The dual characteristic of antibacterial action and mutagenicity is observed for an appreciable number of the di-N-oxide5..2:1.29 This lends further credence to the hypothesis that both processes may involve oxy radicals.’,’ There are previous reports which indicate a correlation between biological activity and half-wave reduction potential. For example, mitomycins possessing less negative E,,2values exhib494
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ited more powerful antibiotic activity.:’”Also, recent data reveal a relationship between carcinogenic activity and E,,g values of purines and pyrimidines in ionic form resulting from alkylation or protonation.” The range for the E , figures (-0.98 to -1.2 V) obtained from the purine salts is similar to that of the heterocyclic di-N-oxides (Table I). Kaye and Stonehill pointed out that an objection to the theory may be raised because of the rather negative reduction potentials.”’ However, they provided a number of answers to the possible criticism, including beneficial influences to be expected in vivo. In addition, favorable solvation, stereochemical, and counterion effects might well be operative in living systems during reduction. Appreciable alteration in half-wave redzction potentials with change in the medium has been observed in other studies.3’ Of course, an absolute correlation between drug activity and electrochemical behavior is unreasonable. Many other factors pertain, such as solubility, diffusion, absorption, metabolic behavior (either destructive or beneficial), stereochemistry, and active site binding. The literature contains only meager treatment of the mechanistic aspects.’:’ I d McIlwain proposed inhibition of vitamin K by competition for common receptor sites due to similarity in structure.’” Alt,ernatively, a molecular model based on NMR data and energy calculations indicates that quinoxaline antibiotics form a bis-intercalated complex with DNA in which two base pairs are sandwiched between the chromophore~.’~ If intercalation occurs followed by oxy radical generation via CT, then this would represent another instance of “site specific, free radical” formation.:” In summary, the overall results support the participation of electron transfer processes in the mechanism of drug action involving quinoxaline di-N-oxides.
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23. Tolcheev, Y. D.; Padeiskaya, E. N.; Smolyanskaya, A. Z.; Pershin, G. N.; Elina, A. S.; Musatova, I. S. Khim. Farm. Zh. 1982, IS, 1482; Chem. Abstr. 1983,98, 137204. 24. Rudzit, E. A,; Lisitsa, L. J.; Tagirov, R. F. Zh. Mikrobiol. Epidemiol. Immunobiol. 1973, 112; Chem. Abstr. 1973, 79, 87603. 25. Padeiskaya, E. N.; Polukhina, L. M.; Budanova, L. I.; Kuzovkin, V. A.; Pershin, G. N.; Sokolov, S. D. “Curr. Chemother. Proc. Int. Congr. Chemother., loth, 1977,” vol. 1, 1978; p 579; Chem. Abstr. 1978,89, 99565. 26. Sawyer, D. T.; Komai, R. V. Anal. Chem. 1972, 44, 715. 27. Baehner, R. L.; Boxer, L. A,; Ingraham, L. M. in “Free Radicals in Biology,” vol. V; Pryor, W. A,, Ed.; Academic Press: New York, 1982; chap. 4, pp 91-93. 28. Weitberg, A. B.; Weitzman, S. A.; Destrempes, M.; Litt, S. A.; Stossel, T. B. N . Engl. J. Med. 1983,308, 26.
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Acknowledgments This work was presented in part a t the 188th National Meeting, American Chemical Society, Philadelphia, PA, Abstr. MED 173 (1984).
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