CO2Me O2N C OMe O O2N C OH O O2N C N O OBn H O2N C N O OBn R H2N C N O O2N-Me + OH R O2N C N O i 1 ii 3 3 3'a OTr H 3 2 O2N C N O 3 3a 3b–d 3 3 4'a OH H 3 4a–d iii iii' iv v' v a R = H b R = Me c R = Bun d R = n-C8H17 218 J. CHEM. RESEARCH (S), 1997 J. Chem. Research (S), 1997, 218–219† Phase-transfer-catalysed Preparation of N-Alkylated Trihydroxamic Acids† Pascal Hoffmann, Jean-Baptiste Doucet, Wenhao Li, Laurent Vergnes and Serge Labidalle* Laboratoire de Synth`ese, Physico-Chimie et Radiobiologie, JE 175, Universit�e Paul Sabatier, Facult�e des Sciences Pharmaceutiques, 35, chemin des Mara�ýchers, 31062 Toulouse cedex 04, France We describe a synthetic route involving phase transfer catalysis leading to a series of tripodal N-alkylated hydroxamic acids as models of desferrioxamine; iron(III) exchange reactions between their iron complexes and EDTA were investigated.Naturally occurring hydroxamic acids act variously as growth factors, antibiotics, tumour inhibitors, cell division factors or lipoxygenase inhibitors, and play a major role as iron transfer agents.1 Most of these biological activities are due to their complexing properties towards transition metal ions, particularly with iron(III).Desferrioxamine, a natural trihydroxamic acid, is used therapeutically for the treatment of iron-overloaded patients,2 particularly in patients with AIDS, but the lack of oral activity and its short biological half-life limits its use.Otherwise, iron chelation by desferrioxamine, and other chelators, protects against the cytotoxic and reactivating effects of hydrogen peroxide,3 and thus decreases NF-kB activation due to oxidative stress, and subsequent activation of HIV-1 transcription. The natural products with an iron(III)trihydroxamate centre form a class comprised of many known members. When three hydroxamate functions are present in the same molecule with an appropriate spacing between the hydroxamic acid units, the complex tends to retain the 1:1 structure, even at low pH.In this work, we report the synthesis of a series of N-H or N-alkylated tripodal hydroxamic acids 4 (Scheme 1) as simple models of desferrioxamine. We use phase transfer catalysis4 (PTC) on the one hand to access the triester 1, and on the other hand to perform the N-alkylation reactions. The relative stabilities of the iron(III) complexes were investigated. PTC was used for the first step to synthesize the triester 1, a building block that is often used as a starting material for dendrimeric macromolecule synthesis.5 Michael-type addition of nitromethane to methyl acrylate, without added organic solvent but in the presence of potassium carbonate and with benzyltriethylammonium chloride as phase-transfer reagent, followed by saponification of the triester 1 afforded the triacid 2.Condensation of O-protected hydroxylamines with the triacid chloride gave the O-benzylated trihydroxamic acid 3a, and the O-tritylated trihydroxamic acid 3pa.As the deprotection conditions of the benzyl group are often incompatible with the presence of other functional groups, we used two different protective groups for the synthesis of compounds 3. Although synthetic methods for hydroxamic acids are well documented,6 direct acylation of O-protected hydroxylamine derivatives with acid chlorides remains the most commonly used method. A direct condensation with unprotected hydroxylamine gave a mixture of N- and Oacylated products.Hydroxamic acids have been alkylated with a large variety of electrophiles. Phase-transfer catalysis in the N-alkylation of organic molecules seems to be effective especially when the nitrogen atom has a low basicity which renders it less reactive towards alkylating agents. Here, we used PTC in the absence of solvent for the N-alkylation4c of 3a with methyl iodide, n-butyl bromide, and n-octyl bromide in the presence of potassium tert-butoxide and Aliquat 336 (tricaprylylmethylammonium chloride) to yield respectively the N-alkylated-O-protected hydroxamic acids 3b–d.Finally, deprotection of the benzylated derivatives 3a–d by catalytic hydrogenation with ammonium formate and 10% palladium –carbon gave the amino hydroxamic acids 4a–d in moderate yields (20–35%), while removal of the trityl group of compounds 3pa in acidic diethyl ether gave the nitro derivative 4pa. The N-n-octylated product (4d) was isolated by chromatography on silica gel, whereas 4a–c and 4pa were purified by reversed-phase preparative TLC with a mixture of methanol and water as eluent.All final hydroxamic acids gave a deep red colour in the presence of Fe3+ with a wide characteristic absorption band in the visible range with a maximum located between 420 and 440 nm. In order to investigate the relative stabilities of the iron complexes, the pseudo-first-order constants for iron(III) *To receive any correspondence. †This is a Short Paper as defined in the Instructions for Authors, Section 5.0 [see J.Chem. Research (S), 1997, Issue 1]; there is therefore no corresponding material in J. Chem. Research (M). Scheme 1 Reagents and conditions: i, PTC: benzyltriethylammonium chloride, K2CO3, RT; ii, NaOH–methanol, reflux; iii, SOCl2, reflux; O-benzylhydroxylamine, Et3N–THF, RT; iiip, SOCl2, reflux; O-tritylhydroxylamine, Et3N–THF, RT; iv, PTC: methyl iodide, n-butyl bromide or n-octyl bromide, Aliquat 336, potassium tert-butoxide, 50 °C (reflux for methyl iodide, 42 °C); v, ammonium formate, 10% Pd/C– methanol, reflux; vp, HCl–diethyl ether, RT (RT=room temperature)J.CHEM. RESEARCH (S), 1997 219 exchange reactions between the FeIII–4 complexes and EDTA were determined by following the decrease in absorbance at 425 nm in the presence of a large excess of EDTA. The relative stability constants of the iron(III) complexes of 4pa, 4a, 4b, 4c and 4d were respectively 0.8Å10µ2, 1.5Å10µ2, 1.0Å10µ2, 0.2Å10µ2 and 0.4Å10µ2 sµ1, suggesting that the lipophilic compounds 4c and 4d hold iron a little more tightly than do the other ligands.However, the order of magnitude of the observed constants indicates that the tripodal ligands 4 form much less stable complexes than does desferrioxamine, a linear trihydroxamic acid that has an optimal nine-atom spacing between hydroxamate moieties, and whose exchange constant towards EDTA is 6.5Å1µ6 sµ1 under the same experimental conditions.The shape of the molecule (linear or tripodal) and the spacing between the hydroxamic acid units seem to play a major role in creating stable iron(III) complexes. The final hydroxamic acids will be tested as potent antiviral agents in future work; moreover, as part of a program to develop novel chelators for the radioimaging of organs and tumours, the affinity of the trihydroxamic acids 4 towards gallium(III) and indium(III) will be measured.Experimental NMR spectra were recorded on a Bruker AC 250 instrument at 250 MHz and UV spectra with a UVIKON 931 spectrometer (Kontron instruments). Compound 1. Yield: 97%, oil; dH (CDCl3) 2.26–2.28 (12 H, m, CH2CH2), 3.66 (9 H, s, CH3). Compound 2. Yield: 65%, solid, mp 175 °C; dH [2H6]DMSO) 2.16–2.17 (12 H, m, CH2CH2), 12.31 (3 H, s, COOH). Compound 3a. Yield: 77%, solid, mp 107 °C; dH ([2H6]DMSO) 1.93–2.13 (12 H, m, CH2CH2), 4.78 (6 H, s, PhCH2), 7.34–7.40 (15 H, m, C6H5), 11.09 (3 H, s, NH).Compound 3pa. Yield: 58%, oil; dH ([2H6]DMSO) 1.88–2.08 (12 H, m, CH2CH2), 7.25–7.48 (15 H, m, C6H5), 10.89 (3 H, s, NH). General Procedure for PTC N-alkylation.·To a dry mixture of 3a (9 mmol) and potassium tert-butoxide (45 mmol) was added Aliquat 336 (0.1 g). After 2 h under stirring at 50 °C, methyl iodide, n-butyl or n-octyl bromide (5–10 mol equiv.) were added and the mixture was stirred for 2 h at 50 °C. Chromatography on silica gel with a silica gel with a mixture of dichloromethane and methanol as eluent (ranging from 99:1 to 95:5) provided the desired compounds. 3b (yield: 67%), oil, dH (CDCl3 2.15–2.20 (12 H, m, ), 3.17 (9 H, s, NCH3), 4.77 (6 H, s, PhCH2), 7.36 (15 H, s, C6H5). 3c (yield: 55%), oil, dH (CDCl3) 0.87–0.93 (9 H, t, CH3), 1.25–1.35 (6 H, m, CH2), 1.54–1.63 (6 H, m, CH2), 2.08–2.27 (12 H, m, CH2CH2), 3.58–3.64 (6 H, t, NCH2), 4.75 (6 H, s, PhCH2), 7.36 (15 H, s, C6H5). 3d (yield: 48%) oil 0.87 (9 H, s, CH3), 1.25–1.30 (36 H, m, CH2), 2.08–2.23 (12 H, m, CH2CH2), 3.56–3.60 (6 H, t, NCH2), 4.75 (6 H, s, PhCH2), 7.36 (15 H, s, C6H5).General Procedure for Debenzylation.·To a solution of 3a–d (9 mmol) in methanol (50 ml) was added under argon 10% Pd–C (2.5 g) and a solution of ammonium formate (90 mmol) in methanol (100 ml). The mixture was heated at 65 °C for 12 h under argon. After cooling, the mixture was filtered through a Celite pad.The palladium was washed three times with methanol, and the filtrate was evaporated. 4a–c were chromatographed on reversed-phase TLC and 4d on silica gel. 4a (yield: 59%), solid (hygroscopic): dH ([2H6]DMSO) 1.75–2.18 (14 H, m, CH2CH2+NH2), 5.02 (3 H, br s, OH), 7.35 (3 H, s, NH). 4b (yield: 70%), oil dH ([2H6]DMSO) 2.02–2.27 (14 H, m, CH2CH2+NH2), 3.02 (9 H, s, NCH3), 7.87 (3 H, br s, OH). 4c (yield: 95%), oil: dH ([2H6]DMSO) 0.82–0.88 (9 H, t, CH3), 1.19–1.34 (6 H, m, CH2), 1.42–1.53 (6 H, m, CH2), 2.05–2.32 (14 H, m, CH2CH2+NH2), 3.43–3.48 (6 H, t, NCH2), 6.70 (3 H, br s, OH). 4d (yield: 79%), oil, dH ([2H6]DMSO) 0.85–0.87 (9 H, t, CH3), 1.26–1.33 (36 H, m, CH2), 2.10–2.30 (14 H, m, CH2CH2+NH2), 3.51–3.59 (6 H, t, NCH2), 8.43 (3 H, br s, OH). RF values on reversed-phase TLC (C18): 4a 0.75 (H2O–MeOH, 80:20); 4b 0.62 (H2O–CH3OH, 60:40); 4c: 0.17 (H2O–MeOH, 40:60). RF value on silica gel TLC: 4d: 0.20 (CH2Cl2–MeOH, 95:5). Compound 4pa. To a solution of 3pa (5.6 g, 5.34 mmol) in dichloromethane (190 ml) was added a solution of hydrochloric acid (1.1 M) in diethyl ether (30 ml).The solution was stirred for 2 h at room temperature. The precipitate was filtered off, washed with dichloromethane and dried under reduced pressure. The residue was dissolved in water, filtered and lyophilised to give 4pa (yield: 74%), solid (hygroscopic); dH ([2H6]DMSO) 1.92–2.10 (12 H, m, CH2CH2), 9.90 (3 H, br s, OH), 10.66 (3 H, s, NH). RF=0.70 [reversed-phase TLC (C18); H2O–MeOH, 80:20].Iron-exchange Reactions with EDTA.·The exchange reactions between the iron complexes and EDTA were carried out by UV spectroscopy, noting the decrease in the absorbance at 425 nm in the presence of an excess of EDTA as previously described.7 Received, 13th January 1997; Accepted, 28th February 1997 Paper E/7/00300E References 1 J. B. Neilands, Science, 1967, 156, 1443; R. J. Bergeron, Chem. Rev., 1984, 84, 587. 2 R. J. Bergeron, C. Z. 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