8414
Studies on Models for Tetrahydrofolic Acid. IV. Reactions of Amines with Formamidinium Tetrahydroquinoxaline Analogs S. J. Benkovic,*l” T. H. Barrows, and P. R. Farinalb
Contribution from the Department of Chemistry, The Pennsyluania State University, University Park, Pennsylvania 16802. Received June 2, 1973 Abstract: The condensation of amines with formamidinium model compounds for 5,lO-methenyltetrahydrofolic acid leads to N-formimidoyl products. The reaction involves decomposition of a presumed orthoamide intermediate to yield the formamidine product of kinetic control; the latter then isomerizes to the thermodynamically more stable N-formimidoyl derivative. In the absence of intramolecular effects, efficient transfer of the one-carbon unit to yield the N-formyl amine generally requires the pK, of the condensing amine to be less than the pK, of the formamidine nitrogens owing to the need for amine protonation prior to expulsion. The implications of kinetic us. thermodynamic control for the mechanisms of folate catalyzed transfer reactions are discussed.
T
he biosynthesis of inosinic acid, a precursor for adenine and guanine nucleotides, requires two transformylation reactions involving 5,6,7,8-tetrahydrofolic acid (FH4).2 Our previous work on tetrahydroquinoxaline models for F H 4 (Ia,b and IIa,b) has shown
I
OCH I. I
IIIa, X = C 0 , E t b , X = C1
IV
Elemental analysis confirms that the adduct is in the free base form, as expected from the method of synthesis. that they encompass many salient characteristics of the Assignment of the formimidoyl substituent to the natural coenzyme but possess fewer dissociable acidN-1 and/or N-4 position rather than N-10 is based base groups and greater stability toward redox proupon the following data. The nmr spectrum attributed cesses. These latter features coupled with systematic t o the aromatic protons of the tetrahydroquinoxaline variation of the para substitution should yield a degree moiety resembles the N-1 rather than N-10 formyl of insight into the formyl transfer process. We report derivative of Ia.3b In the N-10 formyl case the nmr herein experiments employing model compounds (IIa,b) spectrum reveals a near singlet corresponding to the in a study related t o the transfer of the formamidine four aromatic protons of the tetrahydroquinoxaline carbon of 5.10-methenyltetrahydrofolic acid (5,lOmoiety and, in the N-1 formyl case, a wider multiplet methenyl FH,) t o glycinamide ribonucleotide. arising from the same protons. The spectra of the three compounds are displayed in Figure 1. It is unResults likely, however, that this spectral region can be utilized Structural Assignments. Condensation of IIa with to distinguish between N-1 or N-4 substitution. A excess 2-methoxyethylamine yields an adduct which comparison of the aromatic proton chemical shifts was isolated as a white crystalline solid. Four possible of the benzocaine moiety for the methoxyethylamine structures were considered for this adduct; formimidoyl adduct with a series of structurally similar compounds substitution at N-4, N-1 (IIIa), or N-10 and orthoamide (Table I) likewise is informative. Compounds V and (IV).4,5 The orthoamide structure was ruled out by an VI were synthesized according to a modification of the ir absorption at 1640 cm-l (CH=N)B and an nmr signal procedure of Mandel and H i L 9 The assigned strucat 6 8.1 (s, 1 H), assigned to the formamidine h y d r ~ g e n . ~ tures of V and VI are consistent with spectral and eiemental data (cf, Experimental Section). It is apparent (1) (a) National Institutes of Health Career Development Awardee; (b) National Institutes of Health Postdoctoral Fellow, 1972-1973. that formimidoyl substitution at the N-10 position (2) For an extensive review see R. L. Blakley, “The Biochemistry of would shift the A protons of l a approximately 0.5 ppm Folic Acid and Related Pteridines,” North-Holland Publishing Co., Amsterdam, 1969. downfield. Since the A protons of the adduct and (3) (a) S . J. Benkovic, P. A. Benkovic, and D. R. Comfort, J. Amer. Ia have nearly identical chemical shift values, it can Chem. SOC., 91, 5270 (1969); (b) S . J. Benkovic, W. P. Bullard, and be concluded that the compound is not substituted P. A. Benkovic, ibid., 94, 7542 (1972). (4) J. Hocker and R. Merten, Chem. Ber., 105, 1651 (1972). at the N-10 position. Ia, X = C 0 , E t b , X = C1
IIa, X = C 0 , E t b , X = C1
( 5 ) P. A. S. Smith, “Open-chain Nitrogen Compounds,” Vol. 1, W. A . Benjamin, New York, N. Y . , 1965, Chapter 4. (6) I5 Evidently the presence of an amide function (XII) capable
z).
H
H
/
,N\ HN=CH +
0 XI1
of intercepting the intermediate formimidoyl species (N-1 or N-10) still is insufficient to redirect the reaction toward net formyl transfer. *@
Experimental Section Materials. Synthesis of ethyl p4N-2'4 1,2,3,4)tetrahydroquinoxalinylmethylene]aminobenzoate and the related p-C1 derivative (Ia and Ib) has been previously described.8a,21 The corresponding formamidinium fluoroborate salts (IIa and IIb) were prepared (15) S . C. Hartman, B. Levenberg, and J. M. Buchanan, J . Bid. Chem., 221, 1057 (1956). (16) B. D. Perrin, "Dissociation Constants of Organic Bases in Aqueous Solution," Butterworths, London, 1965. (17) E. R. Bissel and M. Finger, J . Org. Chem., 24, 1256 (1959). (18) S . J. Benkovic and W. P. Bullard, "Progress in Bioorganic Chemistry," Vol. 2, E. T.Kaiser and F. J. Kizdy, Ed., Wiley-Interscience, New York, N. Y . , 1973. (19) J. Hocker and R. Merten, Angew. Chem., Int. Ed. Engl., 11, 964 (1972), and references cited therein. (20) T. H. Barrows, Ph.D. Thesis, The Pennsylvania State University. (21) S . J. Benkovic, P. A. Benkovic, and R. L. Chrzanowski, J . Amer. Chem. Soc., 92, 523 (1970).
according to the published procedure by condensation of Ia and Ib with triethyl orthoformate in ethanol and acidification with aqueous fluoroboric acidS3b The N-1 and N-10 formyl derivatives of Ia and the N-1 formyl derivative of Ib are known compounds.3b The preparation of the unknown N-10 formyl deriyative of Ib is described below. The purity of tetrahydroquinoxaline derivatives was analyzed on silica gel tlc (Brinkmann PF-254) by migration with ethyl acetate: cyclohexane (80% :20%, v/v) and visualization with uv light or by spraying with 5 % FeC13 in 0.1 N HCI. The ferric chloride spray produced characteristic colors which were stable for about 0.5 hr. Melting points were taken with a Fisher-Johns melting point apparatus and are uncorrected. Infrared spectra were taken on a Perkin-Elmer Model 257 and were calibrated with polystyrene film. Nuclear magnetic resonance spectra were taken on a Varian Associates A-60 instrument, and chemical shifts are reported in 6 units relative to tetramethylsilane. Ultraviolet spectra were taken on a Cary 14 instrument. Elemental analyses were performed by Midwest Microlab, Ltd., Indianapolis, Ind. Solvents, unless otherwise stated, were reagent grade and used as received. Ethyl p-[[[l,2,3,4-Tetrahydro-l-[N-(2-methoxyethyl)formimidoyI]2-quinoxalinyl]methyI]amino]benzoate(IIIa). 2-Methoxyethylamine (Aldrich) (0.10 ml, 1.4 mmol) was added to a solution of p-carbethoxyfcrmamidine fluoroborate (Ira) (200 mg, 0.49 mmol) in acetonitrile (5 ml). White thread-like crystals formed upon cooling to -45" (Dry Ice-acetonitrile) and were filtered and washed with Recrystallization from absolute ethanol cold ether (172 mg, 91 and drying in uucuo gave a white powder: mp 124-126"; ir (KBr) 1685, 1640, and 1610 cm-1; uv A":," 308 nm ( E 30,500) and 275 (18,000); nmr (CDCh) 6 8.10(s, 1 H) [7.78 and 6.57 (qAB, J = 9 Hz), 7.2 t o 6.4 (m)] (8 H), 4.3 to 3.3 (m, 14 H), and 1.33 (t, 3 H. J = 7 Hz); m / e 396(M+). Anal. Calcd for C22H28N103:C, 66.67; H , 7.07; N , 14.14. Found: C,66.79; H,7.44; N, 14.05. 2-[(p-Chloroanilino)methyl]-l,2,3,4-tetrahydro-l-[N-(2-methoxyethyl)formimidoyl]quinoxaline (IIIb). 2-Methoxyethylamine (0.12 ml, 1.7 mmol) was added to a solution of p-chloroformamidinium fluoroborate (IIb) (300 mg, 0.8 mmol) in acetonitrile (15 ml) and the resulting solution stored in the cold for several hours. Crystallization was induced by scratching and cooling to -45'. After recrystallization from acetonitrile the product was dried in cnciio at 56" to yield a white powder (75 mg, 2 6 z ) : mp 105-107"; ir (KBr) 1630 and 1590 cm-1; uv ": :A: 318 nm (e 8700) and 250 (25,600); nmr (acetone-&) 6 8.26 (s, 1 H) [7.08 and 6.72 (qAB, J = 9 Hz), 7.2 to 6.5 (m)] (8 H), 3.58 (s, 5 H), 3.35 (s, 6 H), and 2.69 (s, 3 H); m / e 358 (M+). Anal. Calcd for C19H2,NaOCl: C, 63.60; H, 6.42; N, 15.62. Found: C, 63.40; H , 6.27; N , 15.74. Ethyl p4Methylamino)benzoate (I). Sulfuric acid (1 1 ml) was added slowly to a suspension of p-(methy1amino)benzoic acid (Eastman) (15.1 g, 0.1 mol) in ethanol (100 ml). The reaction was refluxed with stirring for 2.5 hr to give a clear solution. The solution was concentrated under reduced pressure to approximately half its volume and neutralized with saturated aqueous sodium bicarbonate. Extraction with methylene chloride and drying (MgSOJ gave a white crystalline product (13.4 g, 75 %). Recrystallization from heptane gave ethyl p-(methy1amino)lbenzoate: mp 66-67" [lit.22 65-67"], Ethyl p-[(N-Formyl)methylamino]benzoate. Ethyl p-(methylamino)benzoate (21 g, 0.120 mol) was added to a flask containing 75 ml of benzene and 75 ml of formic acid ( 9 0 z ) fitted with a DeanStark trap. The reaction mixture was refluxed with constant stirring for 3 hr and the water which collected in the separator was removed periodically. Evaporation of the solution gave a syrup which was dissolved in ether, washed several times with aqueous saturated sodium bicarbonate, washed with water, and dried (MgSOJ. The solvent was evaporated to dryness to yield ethyl p-[(N-formyl)methylamino]benzoate (22.1 g, 89%), mp 56-57". Reciystallizations from 1 :1 diethyl ether :petroleum ether (low boiling) gave an analytical sample: my 57-58.5"; ir (Nujol) 1718, 1680, 1610, 780, and 715 cm-1; nmr (CDC1,) 6 8.63 (s, 1 H), 8.05 and 7.24 (qAB, 4 H, JAB = 8.5 Hz), 4.36 (9, 2 H, J = 7 Hz), 3.3 3 (s, 3 H), and 1.38 (t , 3 H , J = 7 Hz); uv, , ,A (pH 7.5) 265 nm (e 17,300); ni/e 207 (MA). Aid. Calcd for CllHI3NO3: C, 63.76; H, 6.32; N , 6.76. Found: C,63.96; H , 6.39; N,6.76. Ethyl p-[[N-(2-MethoxyethyI)formimidoyl]methylamino]benzoate Hydrochloride (V). A flask containing ethyl p-[(N-formy1)methyl-
z).
(22) M . Sekiya and K. Ito, Chem. Pharm. Bull., 14, 1007 (19661,
Benkovic, Barrows, Farina
1 Studies on Models for
Tetrahydrofolic Acid
8420 N-Ethyl[N-(2-methoxyethyl)formimidoyl]-p~chloroaniline (IX). NEthyl-p-chloroformaiiilide was prepared from p-chloroaniline, triethyl orthoformate, and sulfuric acid according to the procedure of Roberts andVogt.23 N-Ethyl-p-chloroformanilide (9.5 g, 0.052 mol), 2-methoxyethylamine (4.0 g, 0.054 mol), and phosphorus pentachloride (1 6.0 g, 0.076 mol) were dissolved in 200 ml of chloroform and the solution was refluxed for 3 hr. The solvent was removed under vacuum, and 250 ml of 1 N NaOH was added to the resulting oil. The mixture was extracted with ether (3 X 100 ml), dried (MgSOd, and evaporated under vacuum. The product was obtained as a colorless liquid by fractional distillation (8.0 g, 64%): bp 114" (0.02 mm); ir (neat) 1640 and 1590 cm-l; uv": ;A: 267 nm ( e 28,200); nmr(DMSO-d6)67.97(s,1 H),7.27(qAB, 4 H , J = 9 H z ) , 3.87(q,2 H, J = 7 Hz), 3.49 (s, 4 H). 3.28 ( s , 3 H), 1.10 (t, 3 H , J = 7 Hz); m / e 240(M+). Anal. Calcd for Cl1H1,N20CI: C, 59.87; H, 7.07; N , 11.64. Found: C, 59.86; H,6.94; N , 11.53. The N-10 Formyl Derivative of Ib. Attempted recrystallization of crude IIIb (200 mg, 0.56 mmol) from hot 2-propanol (20 ml, Fisher purified grade) gave the N-10 formyl product as colorless flakes(74my,42%): mp 115-117"; ir(KBr) 1655 and 1590cm-I; Ethyl 2-[(N-Methoxyformimidoyl)methylamino]henzoate (VI). uv A?,"," 310 nm ( e 4400); (CDCI,) 6 8.36 (s, 1 H), 7.33 and 7.07 This compound was synthesized in a similar manner to V. Methoxy(qAB, 4 H , J = 9 Hz), 6.48 (broad s , 4 H), and 4.0 to 3.0 (m, 7 H ) ; amine was prepared, from methoxyamine hydrochloride (Eastman), m/e 301 (M+). by dissolving the salt in a small amount of water and making it A/?aI. Calcd for C16HieN,OC1: C , 63.68; H , 5.31 ; N. 13.93. alkaline with sodium hydroxide pellets. I t was distilled (bp 50"), Found: C,63.82; H , 5.47; N, 13.99. weighed, diluted with chloroform, and dried (MgSOJ. The addiAn authentic sample of the N-1 formyl derivative of' Ib (mp 167tion of phosphorus pentachloride to the solution of methoxyamine 170")3bwas found to have an Rfof0.52(light yellow to FeCI3)whereas and ethyl p-[(N-formyl)methylamino]benzoate produced vigorous the above compound exhibited an Rf of 0.48 (bright blue to FeCh). bubbling which subsided in several minutes, after which the solution The presence of N-1 formyl as well as some additional N-10 formyl was raised to reflux temperature. The fine, white solid collected product in the mother liquor was detected by tlc. after ether precipitation was briefly suspended in aqueous sodium Kinetics. The instruments employed have been previously bicarbonate with stirring and filtered ( 3 7 z yield), mp 66-68". described.z4 All reagents and solvents used were reagent grade. Recrystallization from petroleum ether (low boiling) gave white Kinetic runs (25") were initiated by addition of 0.02 ml of a stock crystals which were dried i~ cucuo: mp 68-69"; ir (Nujol) 1699, solution of the desired compound to a cuvette containing 2 ml of the (pH 7.5) 305 nm ( e 26,000); nmr 1605, and 1570 cm-l; uv ,,,A, aqueous buffer. The final reactant concentration was 5 X loT5M , (CDCI,) 6 8.36 (s, 1 H), 7.98 and 7.00 (qAB. 4 H , JAB= 9 Hz), 4.35 ( q , 2 H , J = 7Hz),3.79(~,3H),3.30(~,3H),andl.40(t,3H,J=71.1 = 0.2, KCI. The stock solutions were freshly prepared with either tetrahydrofuran (tetrahydroquinoxaline derivatives) or acetonitrile Hz); rn/e 236(M+). (benzocaine derivatives). The course of the reactions was monitored A t i d . Calcd for C12H1603N2:C , 61.00; H, 6.83; N, 11.86. by observing the decrease in O D at 305 (IIIa), 308 (IIIb), 300 (V), or Found: C,60.77; H,6.58; N, 11.63. 310 nm (VIII), corresponding to the disappearance of reactant. Ethyl p-(3,4,4a,5-Tetrahydroimidazo[l,5-a]quinoxaIin-2(lH)-yI)k o b s d (i 5 %) was taken as slope X 2.303 from a plot of log [(OD0 benzoate (VII). Compound IIIa (4.4 mg, 0.011 mmol) was disOD,)/(OD, - OD,)] cs. time. Product distributions for IlIa were solved in DMSO (1 ml) and NaBHI added (0.25 ml of a 0.05 M determined from ultraviolet spectra after 8-10 half-lives by the solution in DMSO, 0.012 mmol). The solution exhibited a single previously described m e t h ~ d . ~ bThe pH of reaction solutions taken FeCl, positlve spot on tlc (Rr 0.59). The product was isolated from before and after runs agreed within 0.02 unit. Values of UOH- were preparative tlc and recrystallized from ethanol, mp 168-169". The taken as 10-14/aH -. mp, ir, uv, mass spectrum, and tlc are identical with an authentic Product distributions for the hydrolysis of IIb and Illb were sample of VII.3= Ethyl p-[[[1,2,3,4-Tetrahydro-l-[N-(2,2,2-trifluoroethyI)formimi- determined as follows, After 10 half-lives, 2-ml buffered solutions containing 5 X 10-6 M products were concentrated to a small doyl]-2-quinoxalinyI]methyl]amino]henzoate(VIII). 2,2,2-Trifluorovolume, extracted with absolute ethanol, and separated by preparaethylamine (Columbia Organic Chemical Co.) (3 ml) was added to tive tlc. The concentration of product in each band was obtained p-carbethoxyformamidinium fluoroborate (Ha) (200 mg, 0.49 mmol) by extraction with absolute ethanol (3 X 2 ml) and summation of the and the mixture allowed to stand for several hours, whereupon the uv spectrum for each extract. Comparison of the spectra of the two solid dissolved and white thread-like crystals slowly formed. The products with spectra of original mixtures showed recovery of product was collected and washed with ether (180 mg, 8873, mp >90%. 161-1 63 '. Recrystallization several times from acetonitrile and drying in cucuo at 56" yielded a white powder: mp 162-163", ir Acknowledgment. Support from the National 306 nm ( e 30,900) and (KBr) 1690, 1635, and 1590 cm-1; uv": :A: Science Foundation is greatly appreciated. 274 (17,300); nmr (DMSO-&), 6 8.46 (s, 1 H) [7.64 and 6.75 (qAB, J = 9Hz),7.2to6.4(m)]@H),4.13(q,2H,J= 7Hz),3.5to2.9 (23) R. M. Roberts and P. J. Vogt, J . Amer. Chem. SOC., 7 8 , 4778 ( m , 7 H ) , 1 . 2 5 ( t , 3 H , J = 7Hz); m/e419(M+). (1956). Anal. Calcd for CllH2*N4F3: C , 60.14; H , 5.25; N , 13.36. (24) S. J. Benkovic and P. A . Benkovic, J . Amer. Chem. SOC., 88, 5504 (1966). Found: C, 59.90; H, 5.46; N, 13.43.
aminoJbenzoate (5.5 g, 0.026 mol), 2methoxyethylamine (2.0 g, 0.027 mol), and phosphorus pentachloride (8.0 g, 0.038 mol) in 100 ml of chloroform was fitted with a condenser and drying tube and the solution refluxed for 3 hr. The solution was transferred to a I I. flask and cooled in Dry Ice-acetone, followed by the addition of 500 ml of anhydrous ether to give a white semisolid. The precipitate was allowed to warm to room temperature, filtered, and dried in cacuo (4.4 g, 57%). Recrystallization from ethyl acetate: acetonitrile gave white needles: mp 151-152"; ir (Nujol) 1719, (pH 7.5) 261 nm ( e 18,800) and, , ,A 1698, and 1610 cm-l; uv, , ,A (pH 10.5) 303 nm ( e 28,500); nmr (CDCl,) 6 8.67 (broad d, 1 H), 8 . 2 3 a n d 7 . 6 7 ( q A B , 4 H , J ~=~8.5Hz),5.17(broads,lH),4.46(q, 2 H, J = 7 Hz), 3.87 (broad s, 7 H), 3.45 (s, 3 H), and 1.42 (t, 3 H, J = 7 Hz); m/e 263 (M+ - HCI). Anal. Calcd for ClaH.,103N2CI: C, 55.90; H, 7.04; N, 9.43. Found: C, 55.98; H, 6.92; N,9.24. The free base form of V was prepared by rapid neutralization with aqueous sodium bicarbonate, extraction with chloroform, and evaporation to give a colorless oil: nmr (CDCIJ [6 8.11 ( s ) , 7.98 and 7.07 (qAB, J = 9 Hz)] ( 5 H); 4.33 (q, 2 H, J = 7 Hz) [3.58 (s) and 3.37(m)](lOH); and 1.35(t, 3 H , J = 7Hz).
Journal of the American Chemical Society
/
95.25
/ December 12, 1973
