222
Chem. Res. Toxicol. 1 9 8 8 , 1 , 222-227
N-Glycolylhydroxamic Acids: An Improved Synthetic Method and the in Situ Generation and Intramolecular Rearrangement of N-Acetoxy-N-glycolyl-2-aminofluorene Michael D. Corbett*ft and B e r n a d e t t e R. Corbettt Food Science & Human Nutrition Department and Department of Pharmacology & Therapeutics, University of Florida, Gainesville, Florida 3261 1 Received April 11, 1988
A new and improved method for the synthesis of glycolylhydroxamic acids is described. The two-step method involves acylation of arylhydroxylamines with acetoxyacetyl chloride, followed by saponification of the ester bond to give the desired products. The conversion of hydroxamic acids to their thallous salts followed by subsequent acetylation with acetyl chloride to give N-acyloxy esters is described. During the course of this investigation, it was observed that the N-acetoxy ester of an N-glycolylhydroxamic acid was highly unstable and underwent a novel 0-0 acyl migration. This facile rearrangement reaction was studied for the case of N-acetoxy-N-glycolyl-2-aminofluorene(N-OAc-GAF), which gave N-hydroxy-N-(acetoxyacetyl)-2aminofluorene (N-OH-AcAAF) as the sole product of this rearrangement. HPLC was used to investigate this reaction and included the assignment of an HPLC peak to be due to N-OAc-GAF. A competition study employed the amide N-glycolyl-2-aminofluorene (GAF) and demonstrated the absence of intermolecular transfer of the N,O-acyl (acetyl) group of N-OAc-GAF. The mechanism for the probable intramolecular rearrangement reaction is presented, along with a consideration of the possible significance it might have for the toxicity of glycolylhydroxamic acids.
Introduction We have previously demonstrated that aromatic Cnitroso compounds are converted to hydroxamic acids through the action of certain thiamine-dependent enzymes (1). Of particular interest is the ability of transketolase to effect the reduction/acylation sequence of aromatic nitroso compounds to give hydroxamic acids, which possess glycolic acid as the N-acyl moiety ( 2 ) (Scheme I). This adventitious reaction thus produces a unique type of hydroxamic acid, and the extent to which this biotransformation occurs under certain physiological conditions is of toxicological interest. Recently we have reported that some glycolylhydroxamicacids undergo certain metaboIic activation reactions in a manner quite different from the more common acetyl hydroxamic acids ( 3 , 4 ) . Most notable was the finding that N-hydroxy-N-glycolyl-2-aminofluorene (N-OH-GAF)' displayed a 10-fold greater rate of DNA binding than did N-OH-AAF when subjected to peroxidative bioactivation conditions ( 3 ) . Furthermore, N OH-GAF was found to be a strong and irreversible inhibitor of N,O-acyltransferase (5),which is an important enzyme for the bioactivation of acetyl hydroxamic acids such as N-OH-AAF (6,7). Both N-OH-GAF and N-OH-AAF were found to possess a similar degree of mutagenicity toward Salmonella typhimurium strains TA98 and TAlOO (8). Since nitroso aromatics are known to be important bioactivated metabolites of arylamines ( S l l ) ,it is important that the metabolic chemistry of glycolyl hydrox*Address reprint requests to: M. D. Corbett, University of Florida, IFAS - 0163 Gainesville, FL 32611-0163. 'Food Science and Human Nutrition Department and the Department of Pharmacology and Therapeutics. *Food Science and Human Nutrition Department.
0893-228x/88/2701-0222$01.50/0
Scheme I. Mechanism for the Transketolase-Catalyzed Conversion of C-Nitroso Aromatics to Glycolylhydroxamic Acids
Active Glycolaldehyde
amic acids be elucidated so that we can achieve a comprehensive understanding of the mechanisms of arylamine toxicity. The investigation of glycolylhydroxamicacids requires the development of reliable methods for their synthesis, and also for the synthesis of certain derivatives. Among our immediate needs was a method for the synthesis of N-OAc-GAF,which might serve as a model ultimate carcinogen for N-OH-GAF (3). We now report on a convenient method which should be generally applicable for the
' Abbreviations: N-OH-AAF, N-hydroxy-N-acetyl-2-aminofluorene; N-OAc-AAF,N-acetoxy-N-acetyl-2-aminofluorene; AcAAF, N-(acetoxyacetyl)-2-aminofluorene;N-OH-AcAAF,N-hydroxy-N-(acetoxyacetyl)-2aminofluorene; N-OAc-AcAAF, N-acetoxy-N-(acetoxyacetyl)-2-aminofluorene; GAF, N-glycolyl-2-aminofluorene; N-OH-GAF, N-hydroxy-Nglycolyl-2-aminofluorene; N-OAc-GAF, N-acetoxy-N-glycolyl-2-aminofluorene; N-OGly-GAF, N-(glycolyloxy)-N-glycolyl-2-aminofluorene; N-OMe-GAF, N-methoxy-N-glycolyl-2-aminofluorene. 0 1988 American Chemical Society
N-Glycolylhydroxamic Acid Reactions
preparation of glycolyl hydroxamic acids. In addition, we report on the use of thallous (TP)salts of hydroxamic acids as intermediates for the synthesis of 0-acylated hydroxamic acid derivatives. During the course of these studies, we discovered a previously unknown intramolecular migration of an acyl group from the oxygen of the N,Oacetylated glycolylhydroxamic acid (N-OAc-GAF)to the hydroxyl of the glycolyl group. This reaction causes N OAc-GAF to be too unstable to allow for its isolation and subsequent use as a synthetic intermediate.
Experimental Section Chemicals. N-Hydroxy-2-aminofluorenewas prepared by reduction of 2-nitrofluorene with Pd/C and hydrazine hydrate (12)and acetylated by a standard procedure to give N-OH-AAF (13). N-OAc-AAF was prepared by acetylation of the T1+ salt of N-OH-AAF and found to be identical with N-OAc-AAF previously reported in the literature (14). GAF was prepared by the DCC-driven condensation of 2-aminofluorene with glycolic acid in a manner similar to the DCC method for the preparation of glycolylhydroxamic acids (15);the mp for GAF was 187-189 "C [lit. (16) mp 187-188 "C], and the elemental analysis was acceptable. NMR and MS analyses were performed by the Department of Chemistry, Florida State University. Elemental analyses were conducted by Galbraith Laboratories. N-Hydroxy-N-(acetoxyacetyl)-2-aminofluorene(N-OHAcAAF). Freshly prepared N-hydroxy-2-aminofluorene (7.9 g, 0.04 mol) was placed in a 500-mL round-bottom flask equipped with a magnetic stirrer along with 300 mL of diethyl ether, NaHC03 (4.0 g, 0.048 mol), and 25 mL of H20. The contents of the flask were cooled to 0 "C in an ice bath and stirred vigorously while acetoxyacetyl chloride (6.0 g, 0.044 mol, Aldrich Chemical Co.) in 100 mL of anhydrous diethyl ether was added with a dropping funnel over the course of 90 min. The ice bath was removed, and the mixture was stirred for an additional 30 min. The reaction mixture was combined with 400 mL of ethyl acetate and 100 mL of H 2 0 in a separatory funnel, and then the aqueous layer was removed. The organic layer was washed twice with 50 mL of HzO, dried (Na2SO4),and evaporated in vacuo. The solid was chromatographed on silica gel with CHzC12by varying the methanol content from 1% to 4%. Appropriate fractions as inferred by TLC analysis on silica gel with 5% methanol/CH2C12 were combined and evaporated, followed by recrystallization from acetone/hexane to give 4.8 g (40% yield) of N-OH-AcAAF as pale yellow crystals: mp 186-190 "C dec. Anal. Calcd (C17HlSN04): C, 68.66; H, 5.09; N, 4.71. Found: C, 68.70; H, 5.16; N, 4.72. 'H NMR (CD,OD) b 2.15 (s,3 H), 3.9 (s, 2 H), 5.05 (s, 2 H) 7.6 (mult, 7 HI. MS, m / z [(MI+] 297, [(M - 161'1 281. N-Hydroxy-N-glycolyl-2-aminofluorene (N-OH-GAF). N-OH-AcAAF (1.1g, 3.7 mmol) was dissolved in 50 mL of 95% ethanol in a 500-mL separatory funnel and treated with 7.8 mL After the mixture was allowed of 1.0 N aqueous NaOH (7.8 "01). to stand for 5 min a t ambient temperature, 150 mL of HzO was added, and then the reaction was extracted twice with 200 mL of diethyl ether. The pH of the aqueous solution was adjusted to 5-6 with 2 N HCl, then extracted with 200 mL of diethyl ether, which was then dried (Na2SO4),evaporated and the solid recrystallized from acetone/hexane to give 0.74 g (78% yield) of N-OH-GAF as pale yellow crystals: mp 168-170 "C dec. Anal. Calcd (C15H13N09):C, 70.56; H, 5.14; N, 5.49; Found: C, 70.79; H, 5.32; N, 5.56. 'H NMR (CD3OD) 6 3.85 (9, 2 H), 4.45 (s,2 H), 7.6 (mult, 7 H). MS, m / z [(M)+] 255, [(M - IS)+] 239. Thallous Salt of N-OH-GAF. T o a finely dispersed suspension of N-OH-GAF (0.74 g, 2.9 mmol) in 20 mL of benzene and 1 mL of N,N-dimethylformamide (DMF) in a 50-mL round-bottom flask equipped with a magnetic stirrer was added T1(OC2H5)(0.75 g, 3 mmol) in 5 mL of benzene in the course of 5 min. The reaction was conducted under a N2 atmosphere, and all solvents had been purged with Nz. After being stirred for 10 min, the reaction mixture was diluted with 50 mL of diethyl ether and then fiitered and the solid washed with diethyl ether and dried in vacuo to give 1.3 g (98% yield) of Tl+[N-OH-GAF] as a pale tan solid. The salt was stored under vacuum in the dark until used. The physicochemical properties of the salt were not
Chem. Res. Toxicol., Vol. 1, No. 4, 1988 223 characterized directly, since subsequent reactions sufficed to confirm the structure of the product. Methylation of Tl+[N-OH-GAF]. The T1+ salt of N-OHGAF (0.92 g, 2 mmol) was stirred as a fine suspension in 15 mL of DMF with methyl iodide (0.85 g, 6 "01) for 2 h. The reaction mixture was combined with 50 mL of benzene and filtered and the filtrate washed twice with 10 mL of H20. The organic solution was evaporated to give a residue that was chromatographed on silica gel with 0.5-1.0% methanol/CHzC12 Appropriate fractions as identified by TLC were combined and evaporated in vacuo. The solid was recrystallized from acetone/hexane to give 0.46 g (85% yield) of N-OMe-GAF as cream-colored plates: mp 128-129 "C. Anal. Calcd (C16H15N03):C, 71.34; H, 5.62; N, 5.20. Found: C, 71.68; H, 5.50; N, 5.01. 'H NMR (CDCld 6 3.75 (s, 3 H), 3.92 (s, 2 H), 4.45 (s, 2 H), 7.6 (mult, 7 H). MS, m/z [(M)'] 269, [(M - 30)+] 239. N-Acetoxy-N-(acetoxyacetyl)-2-aminofluorene (N-OAcAcAAF). A solution of N-OH-AcAAF (1.2 g, 4 mmol) in 4 mL of DMF and 25 mL of benzene was treated with T1(OC2H,) (1.0 g, 4 mmol) in 10 mL of benzene and then, after 2 min, was diluted with 150 mL of diethyl ether and cooled in an ice bath to facilitate precipitation of the T1+salt. The salt was collected by filtration and washed with diethyl ether to give 1.9 g (97% yield) of the salt (3.8 mmol), which was then suspended in 30 mL of benzene in a 100-mL round-bottom flask equipped with a magnetic stirrer. Acetyl chloride (0.3 g, 3.8 mmol) in 20 mL of anhydrous diethyl ether was added dropwise with stirring over the course of 20 min, and the mixture was stirred for an additional 20 min. The mixture was fiitered to remove TlCl, and then the fitrate was washed twice with 20 mL of H 2 0 ,dried (Na2SO4),and evaporated in vacuo to give a yellow oil, which was crystallized from diethyl ether/hexane to give 1.0 g (78% yield) of N-OAc-AcAAF as a cream-colored solid: mp 103-105 "C dec. Anal. Calcd (C19H17N05):C, 67.23; H, 5.05; N, 4.13. Found: C, 67.47; H, 5.18; N, 4.14. 'H NMR (CDC13) 6 2.15 (9, 3 H), 2.2 (9, 3 H), 3.9 (9, 2 H), 4.65 (9, 2 H), 7.6 (mult, 7 H). MS, m / z [(M)+] 339. N-(Acetoxyacetyl)-2-aminofluorene(AcAAF). To a stirred suspension of 2-aminofluorene (2.5 g, 14 mmol) in 200 mL of benzene containing pyridine (1.1g, 14 mmol) was added acetoxyacetyl chloride (1.9 g, 14 mmol) in 100 mL of diethyl ether in the course of 5 min. The reaction was stirred for 30 min, then washed with 80 mL of H20, dried (Na2S04),and evaporated in vacuo. The residue was chromatographed on silica gel with 1-2% methanol/CH2C12 and appropriate fractions were combined on the basis of TLC analysis. After evaporation, the solid was recrystallized from acetone/hexane to give 1.6 g (41% yield) of white needles: mp 157-159 "C. Anal. Calcd (C17H15N03):C, 72.57; H, 5.38; N, 4.98. Found: C, 72.70, H, 5.43; N, 5.01. 'H NMR (CDCl,) 6 2.25 (s, 3 H), 3.9 (s, 2 H), 4.7 (s, 2 H), 7.6 (mult, 7 H), 7.9 (br s, 1 H). In Situ Production and Investigation of the Decomposition of N-OAc-GAF. To 23 mg (50 pmol) of Tl+(N-OH-GAF), which was finely suspended in 5 mL of DMF (certified ACS reagent grade) through rapid stirring by means of a magnetic stir bar and contained in a 10-mL round-bottom flask, was rapidly added 3.6 pL (3.9 mg, 50 pmol) of acetyl chloride by means of a syringe. The reaction was conducted a t ambient temperature under a N2 atmosphere and the DMF had been purged with N2 gas and dried over molecular sieves (Davison Type 4A). Aliquots of 0.10 mL were taken a t various times from 30 s to 3 min and rapidly combined with 0.90 mL of DMF, which had been precooled to -70 "C in a dry ice/isopropyl alcohol bath. The quenched aliquots were then filtered through an Acrodisc filter (Gelman Co., 5.0-pm pore size) and kept a t -70 "C until HPLC analyses were performed or until an aliquot was rapidly warmed to 20 "C, followed by HPLC analysis. In competition studies, 48 pg (0.2 pmol) of GAF as a solution in 30 WLof DMF was added to a 1-mL volume of a quenched and filtered aliquot, which had been obtained a t T = 30 s and kept a t -70 "C. This gave a final concentration of 0.2 mM GAF in a reaction mixture that contained about 0.1-0.2 mM N-OH-GAF (hydrolyzed starting material), which, together with products, gave a total concentration of 1mM. After warming the reaction mixture to 20 "C, its composition was determined by HPLC a t various times over a period of 1h. The HPLC system employed to study the normal production and fate of N-OAc-GAF (Figure 1)consisted of the solvent 55% aqueous
Corbett and Corbett
224 C h e m . Res. Toxicol., Vol. I , No. 4, 1988
Scheme 111. Use of Tl(OC2H,)for the Synthesis of an
0 16-
N,O-Acylated Hydroxamic Acid
a -
TI
P F1
H? FL-N-CCH20CCH3
2
012-
TI(OCzH5)
?-?
*
P
FL-N-CCH20CCH3
/?
N - OH- Ac A A F
/
? . .
E
CH3CCI
0
a N
w
+
f
oos-
FL=
I
0
z
8
I:
CH3E0 0
4
m
i l l
FL-N-CCH20CCH3
Lz
0
m
N-OAc-AcAAF
m
a
004-
Scheme IV. The Synthesis and Reactions of the T1' Salt of N-OH-GAF
3
P
A
IO
0
H? FL-N-CCH20H 20
0
20
IO
TIME
0
IO
N-OH-GAF
20
(min)
TI+
?-
FL-N-CCH20H
Scheme 11. Alternative Methods for the Synthesis of Glycolylhydroxamic Acids FL-N-CCH20H Ar-NHOH
+
P F I CH3COCHzCCI
4
H?FI P Ar-N-CCHz0CCH3
I
Ie
TI(OCzH5)
Figure 1. HPLC chromatograms of the aliquot quenched at T = 30 s from the reaction of the T1+ salt of N-OH-GAF with acetyl chloride: chromatograma, aliquot taken at T = 30-9 reaction time and kept at -70 "C until analysis was made; chromatogramb, same aliquot analyzed 2 min after rapidly warming to 20 "C; chromatogram c, same aliquot analyzed 20 min after rapidly warming to 20 "C. Peak assignments: 1, N-OH-GAF; 2, N-OAc-GAF (putative);3, N-OH-AcAAF;4, N-OAc-AcAAF.
N-OAc-GAF
1
y 3
PF1
FL- N-CCH2OH N-OMe-GAF
I
\ ,HOCCH20H t
0 )!/,
\
OHH+
J
\OCC
fl
P
H? FL-N-CCH20CCH3 N - OH- A C A A F
Ar-N-CCHZOH H?!
methanol buffered to pH 3.5 with 0.01 M KHzPOl buffer and containing 0.01% desferrioxaminemesylate (Desferal,Ciba Geigy Co.) at a flow rate of 1.5 mL/min through a WBondapak C18 column (30 cm X 3.9 mm, Waters Assoc.). In the competition studies with GAF, the HPLC system was identical except that 50% rather than 55% aqueous methanol was used. In thk solvent system, the retention times (min) were found to be as follows: N-OH-GAF, 16; GAF, 18; putative N-OAc-GAF, 23; N-OHA ~ A A F ,28; N-OAC-ACAAF, 38.
Results A two-step procedure for the conversion of arylhydroxylamines to the corresponding glycolylhydroxamic acids is summarized in Scheme 11. This procedure was found to be a much better method than the single-step procedure which uses dicyclohexylcarbodiimide (DCC) to effect the condensation of arylhydroxylamines with glycolic acid (15). The acylation of the arylhydroxylamines with acetoxyacetyl chloride was conducted by a method which had been previously developed to minimize acid-catalyzed decomposition and polymerization of reactive arylhydroxylamines (13). The intermediate acetoxyacetyl hydroxamic acids were readily obtained in good yield and high purity. The saponification of the ester bond was found to proceed selectively in the presence of 2 equiv of NaOH to give the desired glycolylhydroxamic acids in high yields. There was no hydrolysis of the hydroxamic acid acyl group, which would have been detected through the
formation of the hydroxylamine and its decomposition products. The detailed methods for the preparation of N-OH-AcAAF and N-OH-GAF are presented in the Experimental Section. Similar methods were also used for preparation of the analogous compounds derived from other arylhydroxylamines (data not shown). We have previously reported that thallous ethoxide converts hydroxamic acids to their T1+ salts (17), which are readily acylated by acyl halides. In the present study, both N-OH-AcAAF and N-OH-GAF were converted to readily isolable and stable T1+ salts in nearly quantitative yields (Schemes I11 and IV). The acylation of Tl+[NOH-AcAAF] with acetyl chloride gave N-OAc-AcAAF in 78% yield and of analytical purity by recrystallization alone (Scheme 111). On the other hand, the synthetic-scale reaction of Tl+[N-OH-GAF]with acetyl chloride resulted only in the production of N-OH-AcAAF, as demonstrated by TLC analysis (Scheme IV). We had expected to obtain N-OAc-GAF via this reaction, since the hydroxamic acid OH is much more acidic than the glycolyl OH group. In support of our postulate that the anionic charge of T1'[N-OH-GAF] resides on the hydroxamate group, it was found that the reaction of this salt with methyl iodide gave N-OMe-GAF in 85% yield (Scheme IV). That the methyl group addition occurred at the hydroxamate oxygen was unambiguously demonstrated by the fact that the product was negative in the Fe3+test for free hydroxamate OH (18). The isomer in which the methyl group is attached to the glycolyl oxygen would be positive in this test; but no such
Chem. Res. Toxicol., Vol. 1, No. 4, 1988 225
N-GlycolylhydroxamicAcid Reactions
0.8.
060)
c n
049 VI
a
-
0.2
O‘
‘
2;O ’ 2;O‘
2;O’
-. .270’ 260’ 40 ‘ 3
Wavelength
... 0
(nm)
Figure 2. UV spectra of HPLC peaks obtained by a rapid-
scanning spectrophotometric detector. Assignment of spectra: 1, N-OH-GAF; 2, N-OAc-GAF (putative);3, N-OH-AcAAF; 4, N-OAc-AcAAF;5, N-OAc-AAF. Spectra 1-4 were obtained on the products of the reaction described in Figure 1. Spectra 5 was obtained on a sample of synthetic N-OAc-AAF. The spectra represented by 1, 3, and 4 were identical with those generated by synthetic N-OH-GAF, N-OH-AcAAF,and N-OAc-AcAAF, respectively. product was detected by TLC analysis of the reaction mixture. The reaction of Tl+[N-OH-GAF] with acetyl chloride was investigated more thoroughly by the use of HPLC in order to determine if N-OAc-GAF might be the initial reaction product. The evidence obtained strongly supports this possibility. The reaction conditions included the use of more dilute concentrations of reactants (Le., 10 mM) than are normally used for preparative-scale runs (5Ck100 mM) and also the use of a much more polar aprotic solvent (DMF) than is normally employed (e.g., benzene or diethyl ether) for preparative purposes. Aliquots were removed at short intervals and immediately chilled by 10-fold dilution into DMF that had been cooled to -72 “C. HPLC analyses were conducted within a few minutes after sampling the reaction. The HPLC solvent contained desferrioxamine mesylate (19) and was designed to effect the separation of N-OH-GAF, N-OH-AcAAF, and N-OAcAcAAF. Our expectation that such an HPLC solvent would also allow for the chromatographic resolution of N-OAc-GAF appears to be correct (Figure 1). The sequence of chromatograms a to c in Figure 1shows that an intermediate product (evidenced by peak 2) was formed immediately following the start of the reaction and that as this intermediate disappeared rapidly with time, the major final product, N-OH-AcAAF, was produced simultaneously. The identities of the chemicals that gave rise to the HPLC peaks were confirmed by comparison of their UV spectra with those of the three authentic standards (Figure 2). These spectra were obtained by use of a rapid-scanning spectrophotometric detector with the HPLC system. The UV spectrum of the unstable intermediate (peak 2), which is thought to be N-OAc-GAF is presented in Figure 2, along with those for the hydrolyzed starting material (N-OH-GAF)and final product (N-OHAcAAF). The spectrum for N-OAc-AAF obtained under identical conditions is also presented. The general appearances of the UV spectra for all these derivatives of 2-AF are very similar; however, the 0-acylated derivatives (N-OAc-AcAAF,N-OAc-AAF, and the putative intermediate, N-OAc-GAF) all show a hypsochromic shift of the absorption maximum to 273 nm from the 280-nm maxi-
mum observed for the free hydroxamic acids (N-OH-GAF and N-OH-AcAAF). This spectral evidence strongly supports our proposal that the unstable intermediate, which gives rise to peak 2 in the HPLC chromatogram, is NOAC-GAF. A quantitative material balance was obtained upon completion of the reaction (about 30 min), as evidenced by the near absence of HPLC peak 2 (Figure 1). Under the conditions described in the experimental section, the composition of the final reaction mixture was 18% NOH-GAF, 79% N-OH-AcAAF, and 3% N-OAc-AcAAF. The presence of N-OH-GAF in the reaction is most likely due to simple hydrolysis of the T1+ salt. This would be expected to the extent that HC1 is present in the acetyl chloride or produced in the reaction mixture due to hydrolysis of acetyl chloride. The amounts of both N-OHGAF and N-OAc-AcAAF were constant in the reaction mixture throughout the course of the reaction, which was sampled from 30 s through 60 min. This suggests that all the possible acylation and hydrolysis reactions of acetyl chloride occurred very rapidly following the addition of acetyl chloride to the suspension of the T1+ salt in DMF. The relative amount of N-OH-GAF that was present in the reaction was found to increase as more dilute concentrations of reactants in DMF were employed (data not shown). This is likely due to the fact that more total H20, which is present as a trace contaminant of DMF, is available when the reaction is carried out in more dilute solutions.
Discussion In previous studies, we prepared glycolylhydroxamic acids via a DCC-driven condensation of the arylhydroxylamine with glycolic acid (2,15). In the cases of several substrates (e.g., (2,6-dimethylphenyl)hydroxylamine), this process was found not to work or to give only low yields of a highly impure product, respectively. An enzymatic method that we developed for the preparation of glycolylhydroxamicacids on a microscale involves the use of transketolase to convert the nitroso analogue to the glycolylhydroxamicacid (3). Although this method gave excellent results in terms of percent conversion to the desired product, it is practical only for microscale radiosyntheses. The use of acetoxyacetyl chloride to convert arylhydroxylaminesto their N-(acetoxyacety1)hydroxamic acids has been found to work very well for those substrates employed to date, including those which gave poor results when the DCC method was employed. The method should be generally applicable and even useful at the microscale. The selective hydrolysis of the ester group in the presence of the hydroxamic acid group is most likely due to the fact that the hydroxamic acid functional group exists in its ionized form in aqueous base, and the anionic charge should decrease the ability of hydroxyl ion to attack the hydroxamate carbonyl carbon. We had previously discovered that hydroxamic acids are converted almost quantitatively to their T1+ salts upon treatment with T1(OC2H,) in aprotic solvents (17). These salts, as suspensions in benzene or diethyl ether, react rapidly with acetyl chloride to produce N-acetoxy esters and TlC1. We routinely use this method to prepare NOAc-AAF rather than the standard method for the preparation of this “model” ultimate carcinogen, which employs reaction of acetic anhydride with the hydroxamic acid in aqueous base (14). These T1+salts also react readily with other acid chlorides, including methanesulfonyl chloride to yield N-mesyloxy esters of hydroxamic acids (Corbett et al., unpublished results).
226 Chem. Res. Toxicol., Vol. 1, No. 4 , 1988
Corbett and Corbett
Scheme V. Mechanism for the Intramolecular Rearrangement of N-OAc-GAF H
-
~?\cICHS 0’ L T H
I
Ar-N,C,CH2 I1
0
A?
,CH3
0’
“0
K-c I
I
-
H?f ?
Ar-N-CCH20&CH3
Ar-NYCH2 0
Scheme VI. Design of Competition Experiment
FI
7:
CH3C0 0 I
I1
+
FL-N-CCH20H
FL-N-CCH20H
N - O A c - GAF
GAF
HO 0 I
II
H O I l l
B
F L - N - C CHzOH
F L - N- CCH2OC CH3
N-OH-GAF
AcAAF
With the ultimate goal of preparing the arylamidation adducts of DNA, which would result from certain bioactivation reactions of N-OH-GAF ( 3 , 4 ) ,we attempted to synthesize the necessary reactive species, N-OAc-GAF, via the T1+ salt method (Scheme IV). Our discovery that this attempt actually gave N-OH-AcAAF was not too surprising since the acyl bond of N-acyloxy esters of hydroxamic acids is known to be somewhat reactive, even though N-OAcAAF is routinely recrystallized from aqueous acetone (14). For example, it is known that certain N-acetoxy-N-arylacetamides, including N-OAc-AAF, can acetylate protein amino groups (20) and nucleoside hydroxyl groups (21). The parent hydroxamic acid is released during the course of such acetylation reactions. In a similar manner, the solvolysis of the 0-acyl group of 0-acetylated hydroxamic acids in aqueous solvents appears to be a rather general reaction, which is highly dependent upon pH and, most significantly, upon the nature of the aryl group attached to the hydroxamate nitrogen (22). A competing reaction of certain 0-acetylated hydroxamic acids is heterolysis of the N-0 bond to produce a putative nitrenium ion. It is this nitrenium ion producing reaction that resulted in the proposal that 0-acetylated forms of carcinogenic hydroxamic acids might be ultimate bioactivated species (reviewed in ref 3). It is particularly interesting that NOAc-AAF has been found to undergo N-O bond heterolysis almost exclusively a t or below pH 7 in aqueous acetone (23). In contrast, our results with N-OAc-GAF,which was generated in situ in DMF, showed no evidence of products that might have arisen from heterolysis of the N-0 bond. We propose that the instability of N-OAc-GAF is due to an intramolecular reaction, which is analogous to the solvolysis reaction of 0-acetylated hydroxamic acids. The mechanism for this reaction is presented in Scheme V, although the details on probable prototropic changes have been left out for simplicity. The possibility that the apparent 0-0 acyl migration might actually be due to an intermolecular reaction between N-OH-GAF and N OAc-GAF seemed less likely. This possibility was virtually eliminated through competition studies (Scheme VI) in which the amide, GAF, was added to the reaction solution immediately after N-OAc-GAF had been generated. The absence of production of any AcAAF during the disappearance of N-OAc-GAF suggests that the intermolecular transfer of the hydroxamate 0-acetyl group of N-OAc-GAF to the glycolyl hydroxyl group of another molecule does not occur to any significant degree. This study assumed
that the nucleophilicity of the glycolyl OH is about the same in both N-OH-GAF and GAF. In contrast to the putative N-OAc-GAF, N-OAc-AAF was found to be quite stable as a solution in DMF, even in the presence of HzO. No hydrolysis of N-OAc-AAFwas detected for a 1mM solution of this compound in 5% H 2 0 in DMF (2.7 M concentration of H20 in DMF) for at least 1 h (data not shown). This is further indirect evidence against an intermolecular mechanism for the decomposition of N-OAc-GAF in DMF. The pronounced instability of N-OAc-GAF relative to N-OAc-AAF is the result of this internal “solvolysis” reaction and could be a very important determinant of the genotoxic and necrotic actions of N-OH-GAF. On an a priori basis, such an acyl migration gives a less reactive product with respect to the possible generation of a nitrenium ion species and thus might be considered as a “self-detoxifying” reaction. To assess this possibility, it will be necessary to elucidate the reaction(s) of N-OAcGAF following its in situ generation under physiological conditions, particularly in aqueous buffers. At present, there is no evidence that N-OAc-GAF is produced as an actual metabolite under normal physiological conditions. In a previous study, we reported on the intermediary production of the N-glycolyloxy ester of N-OH-GAF (NOGly-GAF) via the action of horseradish peroxidase on N-OH-GAF ( 3 ) . N-OGly-GAF was proposed to be responsible for the much greater covalent binding to DNA than was observed with N-OAc-AAF when both were produced via peroxidative bioactivation. This observation suggested that more rapid cleavage of the N,O-bond to produce nitrenium ions occurred with glycolate rather than acetate as the leaving group (3). No evidence was obtained in that study to suggest any 0-0 acyl transfer of the glycolyl group during the reaction. It is obvious that the nature of the acyl groups attached to the hydroxamate nitrogen and oxygen atoms has a profound effect upon the subsequent reactions of these proximate and ultimate carcinogenic species. Further studies are in progress in an attempt to elucidate these structural effects. Acknowledgment. This work was supported by Grant No. ES 03631 from the National Institute of Environmental Health Sciences, DHHS. Registry No. GAF, 51480-57-0; N-OH-AAF, 53-95-2; N-OHAAF (thallium salt), 115244-69-4; N-OAc-AAF, 6098-44-8; NOH-AcAAF, 115227-92-4; N-OH-GAF, 111959-98-9; N-OH-GAF (thallium salt), 115244-70-7;N-OMe-GAF, 115227-93-5; N-OHAcAAF (thallium salt), 115244-71-8;N-OAc-AcAAF, 115227-94-6; AcAAF, 75871-36-2; N-OAc-GAF, 115227-95-7; CH31, 74-88-4; 53-94-1; 2H3CCOCl, 75-36-5; N-hydroxy-2-aminofluorene, nitrofluorene, 607-57-8; 2-aminofluorene, 153-78-6; glycolic acid, 79-14-1; acetoxyacetyl chloride, 13831-31-7.
References (1) Corbett, M. D., and Corbett, B. R. (1985) ”The reactions of C-nitroso aromatics with cy-oxo acids”. In Biological Oxidation of Nitrogen in Organic Molecules (Gorrod, J. W., and Damani, L. A., Eds.) pp 400-408, Ellis Horwood Ltd., Chichester, England. (2) Corbett, M.D. and Corbett, B. R. (1986)“Effect of ring substituents on the transketolase-catalyzed conversion of nitroso aromatics to hydroxamic acids”. Biochem. Pharmacol. 35, 3613-3621. (3) Corbett, M. D.,and Corbett, B. R. (1987) “HRP-catalyzed bioactivation of carcinogenic hydroxamic acids. The greater reactivity of glycolyl versus acetyl derived hydroxamic acids”. Chem.-Biol. Interact. 63,249-264. (4) Corbett, M. D., Lim, L. O., Corbett, B. R., Johnston, J. J., and Wiebkin, P. (1988) “Covalent binding of N-hydroxy-N-acetyl-2to rat aminofluorene and N-hydroxy-N-glycolyl-2-aminofluorene hepatocyte DNA: in vitro and cell suspension studies”. Chem. Res. Toxicol. 1, 41-46.
N - Glycolylhydroxamic Acid Reactions ( 5 ) Lim, L. O., Corbett, B. R., and Corbett, M. D. (1987), “Irreversible inhibition of the cytosolic metabolism of Nhydroxy-2-acetylaminofluoreneby its glycolyl analog”. Cancer Lett. 37,205-211. (6) King, C. M. and Glowinski, I. B. (1983)“Acetylation, deacetylation and acyl-transfer”. EHP, Enuiron. Health Perspect. 49, 43-50. (7) Hanna, P. E., and Banks, R. B. (1985)“Arylhydroxylamines and arylhydroxamic acids: conjugation reactions”. In Bioactiuation of Foreign Compounds (Anders, M. W., Ed.) vp _ - 375-402, Academit, Orlando: (8) Corbett, M.D., Corbett, B. R., Wei, C. I., Owusu-Yaw, J. D., and and the relative mutaLim, L. 0. “Microsomal N-hydroxylation genicities of the glycolic and acetic amide derivatives of 2aminofluorene”. Carcinogenesis (submitted for publication). (9) Neumann, H.-G., Metzler, M., and Topner, W. (1977)“Metabolic activation of diethylstilbestrol and aminostilbene derivatives”. Arch. Toxicol. 39,21-30. (IO) Eyer, P. (1985)”Reactions of nitrosoarenes with sulfhydryl groups: reaction mechanism and biological significance”. In Biological Oxidation of Nitrogen in Organic Molecules (Gorrod, J. W., and Damani, L. A,, Eds.) pp 386-399, Ellis Horwood Ltd., Chichester, England. (11) Corbett, M. D., Wei, C. I., Johnston, J. J., Chang, P. F., and Corbett, B. R. (1987)“Mutagenicity of the C-nitroso analog of fenitrothion”. Toxicol. Lett. 35,201-207. (12) Westra, J. G. (1981)“A rapid and simple synthesis of reactive metabolites of carcinogenic aromatic amines in high yield”. Carcinogenesis 2, 355-357. (13) Smissman, E. E.,and Corbett, M. D. (1972)“A facile method for N-acylation of ring activated phenylhydroxylamines”. J. Org. Chem. 37, 1847-1849.
Chem. Res. Toxicol., Vol. 1, No. 4, 1988 227 (14) Lotlikar, P. D., Scribner, J. D., Miller, J. A., and Miller, E. C. (1966)“Reaction of esters of aromatic N-hydroxyamines and amides with methionine in vitro: a model for in uiuo binding of amine carcinogens to protein”. Life Sci. 5, 1263-1269. (15) Corbett, M. D., and Chipko, B. R. (1978)“Synthesis of Naryl-N-glycolyl hydroxamic acids: Nonmicrosomal metabolites of nitrosoaromatics”. Experientia 34, 1125. (16) Banks, R. B., Smith, T. J., and Hanna, P. E. (1983)“N-Arylhydroxamic acid N,O-acyltransferase. Positional requirements for the substrate hydroxyl group” J.Med. Chem. 26, 1780-1784. (17) Corbett, M. D., and Chipko, B. R. (1978)“Synthesis and antibiotic properties of chloramphenicol reduction products”. Antimicrob. Agents Chemother. 13, 193-198. (18) Corbett, M. D., and Corbett, B. R. (1980)“Reaction of nitroso aromatics with glyoxylic acid. A new path to hydroxamic acids”. J. Org. Chem. 45, 2834-2839. (19) Corbett, M. D., and Chipko, B. R. (1979)“Quantitative determination of N-arylaceto- and N-arylglycolhydroxamic acids in biochemical reaction mixtures”. Anal. Biochem. 98, 169-177. (20) Barry, E. J., and Gutmann, H. R. (1973)“Protein modifications by activated carcinogens”. J. Biol. Chem. 248,2730-2737. (21) Scribner, J. D. and Scribner, N. K. (1982)“Acetylation of nucleosides by N-acetoxy-N-arylacetamides:dependence on base, aryl group, and buffer composition”. J.Org. Chem. 47,3143-3145. (22) Underwood, G. R., and Callahan, R. J. (1987)“The solvolysis of N-acetoxyacetanilides: the elucidation of two major reaction pathways for model ultimate carcinogens under neutral conditions”. Tetrahedron Lett. 28,5427-5430. (23) Underwood, G. R., and Kirsch, R. B. (1985)“Thesolvolysis of N-acetoxy-2-acetylaminofluoreneand N-acetoxy-4-acetylaminobiphenyl: delicate balance between nitrenium ion formation and hydrolysis”. J. Chem. SOC.,Chem. Commun. 136-138.
