Fluorinated aminoglycosides

Aminoglycosides (AGs) are natural or semisynthetic antibiotics that are inherently toxic, and their use is limited by the widespread presence of AG-resistant bacteria. However, AGs are still valued members of the antibiotic arsenal, particularly against Gram-negative bacteria. The efficiency and clinical value of this class of antibiotics have motivated the exploration of strategies to overcome or evade both the antimicrobial resistance and toxicity. The incorporation of a fluorine atom into AGs has attracted attention as a potential strategy to solve these problematics. This review provides an overview of the research on fluorinates aminoglycosides, their synthesis and, when available, their antimicrobial activity, relative toxicity toward mice or human cells and physicochemical properties

7][18][19] The correlation between the acute toxicity and basicity of AGs has been established, 20 and the © ARKAT USA, Inc introduction of an electronegative fluorine atom allows the modulation of the pK a of a neighbouring nitrogen. 217][28] To date, the primary synthetic tool for preparing fluorinated derivatives of AGs has been deoxyfluorination using fluorinating agents, particularly DAST, or by nucleophilic displacement using a fluoride salt (organic or inorganic) 29 .The principal obstacle to fluorination of aminoglycosides is obtaining an appropriately protected molecule in which only the target group is free to transform; this process may require several well-planned protection-deprotection sequences. 29Sometimes, the outcome of deoxyfluorination is difficult to predict a priori, 30 and several steps are required to achieve the desired substitution pattern, obtaining a single fluorinated AG derivative can be considered a scientific feat in and of itself.
2] Since then, the topic has been of particular interest, and several studies were published between 1980 and 1998.4][35][36] Our aim is to briefly review the synthetic procedures used to obtain fluorinated AGs described in the literature.When available, we also comment on the biological data and physicochemical parameters of the fluorinated AG.
Compounds 16, 17 and 18 were tested together with sporaricin A (9) against a panel of clinically important bacteria.The order of their antibacterial activity was 17 > sporaricin A (9) > 16 >> 18.The fluorinated analogues (16, 17 and 18) were less toxic than the parent compound, sporaricin A (9), with difluorinated analogue (18) being approximately two-fold less toxic than sporaricin A (9) in acute toxicity studies on mice.
Compound (23) proved to be a good reporter for monitoring the binding of A-site rRNA ligands.Taking advantage of the chemical-shift variations in their 19 F NMR spectra, the authors were able to determine the dissociation constants of paromomycin (7), neomycin (8), and neamine (19), and the obtained values were in good agreement with previous determinations. 42

Synthesis of 4'deoxy-4'-fluoro analogues of neamine (19)
. 43 Two syntheses were reported by Hanessian's group as part of a systematic study to obtain fluorinated analogues of neomycin B (8). 25 In their first approach (Scheme 3), paromamine derivative (24) was used as the starting material, and their second approach (Scheme 4) used neamine derivative (32).The deoxyfluorination reaction was first studied using 6azido-3-O-benzyl-6-deoxy--D-glucosamine pyranoside, a model compound that mimics ring I of neamine (19)  (not shown).The reaction with DAST and the S N 2 displacement of 4-sulfonate esters with fluoride anion were modelled.Treatment with DAST gave a complex mixture of products, making the approach from the 4sulfonate esters a more successful model.Both approaches were used in the synthesis of fluorinated neamine analogues. 43he per-O-benzylation of 24 using sodium hydride, benzyl bromide and tetrabutylammonium iodide (TBAI) as the catalyst, followed by cleavage of the benzylidene protecting group under acidic conditions, gave compound (25) (Scheme 3).The regioselective tosylation of the primary 6'-OH followed by azide displacement afforded 26.Unlike the monosaccharide model, the reaction of compound 26 with DAST in dichloromethane afforded a separable mixture of 27 and 28 in 32% and 33% yields, respectively.Derivative (28), with retention of the configuration at C-4', was generated via neighbouring group participation by the C-3' benzyl ether through a 3',4'-oxiranium ring intermediate.Although the yields were reasonable, scale-up proved challenging, and the use of 4-sulfonate esters was explored.Formation of the triflate and displacement with fluoride using tetrabutylammonium fluoride (TBAF) provided 27.A Staudinger reduction of the azide groups followed by hydrogenolysis of the benzyl ethers gave 29.The treatment of 26 with Tf 2 O in pyridine/CH 2 Cl 2 followed by S N 2 displacement with acetate and Zemplén deacetylation afforded a 4'-epi derivative (30).Compound 30 was treated with Tf 2 O and TBAF to give a 4'-deoxy-4'-fluorinated derivative (28).Compound 31 was obtained after removal of the protecting groups under standard conditions. 43he second approach started from perazido neamine derivative 32 (Scheme 4).Treatment of 32 with triisopropylsilyl trifluoromethanesulfonate (TIPSOTf) followed by reaction with mesyl chloride (MsCl) and cleavage of the silyl protecting group with concomitant formation of the 3'-4'-oxirane moiety afforded epoxide (33) in 35% yield.Treatment of 33 with TBAF gave 4'-deoxy-4'-fluoroglucosamine derivative (34) and vinyl azide 35.The vinyl azide is formed by syn elimination promoted by the basicity of the fluoride ion by a method that was previously reported in the literature. 44The deprotection of compound 34 gave 31 in 68% yield.The reaction of 32 with DAST in dichloromethane afforded two compounds: a 4'-deoxy-4'-epi-4'-fluoro neamine derivative (36) and compound 37, which was formed by ring contraction.

O
Both compounds (29 and 31) were crystallized as complexes with the rRNA A-site, [45][46] showing that these compounds effectively interact with the macromolecule.
The reaction of 80 with Ac 2 O in DMSO followed by treatment with DAST afforded difluorinated analogue 81.5-Deoxy-5,5-difluoronetilmicin (82) was obtained from 81 after cleavage of the protecting groups under standard conditions (Scheme 11).
6´-Deamino-6´-fluorosisomicin (87) shows selective binding to the eukaryotic ribosome over the prokaryotic ribosome, supporting the presumption that substitution with fluoride at the C-6' position is detrimental to the antibacterial activity. 33Compound (87) does not show antibacterial activity against Escherichia coli or Staphylococcus aureus strains but shows antiparasitic activity against Tripanosoma brucei brucei, T. brucei rhodesiense, T. cruzi, Leishmania major, and Plasmodium falciparum.Outstandingly, sisomicin (65) possesses both antibacterial and antiparasitic activity, being approximately three-fold more potent against parasitic cells than 87, except for against P. falciparum, which showed some susceptibility only to the fluorinated derivative.These differences between the activities of sisomicin (65) and 6'-deamino-6'fluorosisomicin (87) are attributed to their interactions within the ribosomal A-site of bacteria and protozoa.A single nucleobase change from an adenine in position 1408 in the bacterial ribosome to guanine in the protozoal ribosome A-sites seems to be responsible for the selectivity shown by 87.Fluorine can only function as a H-bond acceptor and is unable to form a stable interaction with adenine, while ring I of the AG can interact with guanine through the C-6' fluorine, and the oxygen at C-5' that forms part of the pyranoside ring and the OH at C-1' can form a characteristic stable pseudo base-pair.Additionally, their cytotoxic activities toward HeLa cells were determined, and both sisomicin (65) and 6´-Deamino-6´-fluorosisomicin (87) are toxic in the hundreds of micromolar range, with 87 being at least 20-fold more toxic in vitro toward protozoan cells than toward human cells. 33 59 um et al. claimed 59 that the addition of 2,5-dideoxy-5-fluorostreptamine (70) into a fermentation culture of Micromonospora purpurea strain ATCC 31,119 yielded 5-deoxy-5-fluorogentamicins C 1 (88), C 2 (89) and C 1a (90) according to the reported procedure, and the compounds were most likely obtained as their 5-deoxy-5fluoro-5-epi fluoro derivatives (Scheme 13).The compounds were chemically modified by the introduction of an N-(S)-4-amino--hydroxybutyryl (HABA) moiety in positions 1-N, 3-N and 2´-N; unfortunately, neither the yields nor the characterization data nor the results of antibacterial tests were reported for these compounds.

Synthesis of 4''-deoxy-4''-epi-4''-fluorokanamycin A (100)
. 60 The regioselective protection of the primary OH of 94 as a trityl ether followed by per-O-acetylation afforded compound 95 in 77% yield (Scheme 14).Cleavage of the trityl protecting group with BF 3 •2MeOH at room temperature afforded compound (96) in quantitative yield, 61 and this compound was treated with a 1:1 mixture of pyridine in water to promote the acetyl migration from position 4" to position 6", providing 97.Alternatively, the slow addition of 0.1 N NaOH in ethanol also promotes the migration of the acetyl group, but in a lower yield.The reaction of 97 with trifluoromethanesulfonic anhydride followed by treatment with tetrabutylammonium fluoride (TBAF) afforded 4''-deoxy-4''-fluoro-4''-epi derivative (98) in 73% yield.Oxazolidinone (99) was formed as a by-product (12% yield) in this reaction.Both reactions proceeded with inversion of the configuration at C-4''.The formation of 99 can be explained by nucleophilic attack of the N-Boc carbonyl on the C-4'' triflate promoted by fluoride. 62 The potential use of the p-bromobenzenesulfonyl (brosyl) ester as the leaving group at the C-4'' oxygen of 97 was also examined (not shown).Unfortunately, the reaction of the 4''-brosyl derivative with TBAF resulted in a complex mixture of products that was not analysed.Reactions with other nucleophiles, however, resulted either in S N 2 substitution products or elimination products, generating the C3''-C4''eno derivatives.Final cleavage of the protecting groups afforded 4''-deoxy-4''-epi-4''-fluorokanamycin A (100).
Compounds 100, 103, 104, 106, and 107 were tested against several bacterial strains, and with the exception of compound 104, all showed potencies comparable to kanamycin A (1).The pK b values of the 3''amino groups of 100 and kanamycin A (1) were calculated from the pH dependency of the chemical shift of C-2'' in the 13 C NMR spectrum.The obtained values were pK b 7.4 and pK b 8.3 for 100 and kanamycin A (1), respectively.The introduction of a fluorine atom in this series is compatible with their antibacterial activity and has a profound impact on the basicity of the amino groups near the introduced halogen. 63age 196 © ARKAT USA, Inc An alternative synthesis of 106 by the direct fluorination of tetra N-benzyloxycarbonyl kanamycin A and 1-N acyl derivatives directly with DAST was reported in the literature, but only some NMR data of this compounds are discussed. 64 Scheme 16.Synthesis of 6''-deoxy-6''-fluorokanamycin A (106) and 5,6''-dideoxy-5-epi-5,6''-difluorokanamycin A (107).Compounds 100 (Scheme 14), 103 (Scheme 15), 110, 113, and 116 were tested against S. aureus, Streptococcus faecalis, P. aeruginosa, E. coli and Proteus mirabilis, and in general, their potencies were higher or similar to that of kanamycin A (1).It is known that kanamycin A (1) does not possess antibacterial activity against P. aeruginosa, but compounds with a fluorine atom at position C-6'' presented a slight potency against this bacterial strain. 65

Synthesis of 4''-deoxy-4''-fluorokanamycin A (120)
. 67 The reaction of 97 with triflic anhydride in pyridine followed by treatment with NaNO 2 in DMF [68][69] afforded the 4''-epi derivative (117) in good yield (Scheme 18).The attack of the ambident nitrite nucleophile produces the nitrite ester, which is then hydrolysed during routine work-up.The reaction of 117 with triflic anhydride followed by treatment with TBAF gave the desired compound (118) and derivative (119) as a by-product.The inseparable mixture was deprotected using standard conditions and afforded 120 after purification.
The reaction of unprotected triol 121 with DAST was fairly selective, giving the 6''-deoxyfluorinated analogue (122) in 71% yield (Scheme 19).Compound 122 was then deprotected under standard conditions affording 123.Treatment of 123 with acetic acid-free zinc acetate in DMSO followed by Cbz-OSu gave 124. 71Compound 124 was reacted with ethyl trifluoroacetate in DMF, resulting in the selective introduction of a trifluoroacetamide moiety at 3''-N.First, a weak trifluoroacetyl ester is formed at O-2'', and subsequent migration of the © ARKAT USA, Inc trifluoroacetyl group to the neighbouring 3''-N resulted in the more stable trifluoroacetamide.The HABA side chain was then installed using Cbz-protected HABA-OSu in p-dioxane, giving 125, which afforded the 1-N-[(S)-4-amino-2-hydroxybutanoyl] (HABA) derivative (126) after a standard deprotection.131). 70The 6'-oxo kanamycin C derivative (127) was first alkylated with nitromethane and the nitro moiety was reduced © ARKAT USA, Inc with Adam's catalyst in a 25:5:1 mixture of MeOH/water/acetic acid.Final N-tosylation afforded a separable diastereomeric mixture of 128a and 128b (Scheme 20).The isomers were separated and characterized, but the absolute stereochemistry at C-6' was not determined, and their syntheses were carried out using two parallel pathways.The reaction with 1,1-dimethoxycyclohexane and p-TsOH followed by phenylmethanesulfonyl chloride in pyridine and treatment with KHF 2 in DMF gave 129a and 129b.Final cleavage of the protecting groups of 129a and 129b afforded epimeric 6'-C-(fluoromethyl)kanamycin C 130a and 130b.The more active of the two isomers (130a) was 1-N acylated with a HABA under standard conditions, affording 6'-C-(fluoromethyl) arbekacin (131).
The antibacterial potencies of compounds 130a, 130b and 131 were generally lower than those of dibekacin (3) and arbekacin (93).Compound 131 was at least two-fold more active than 130a and 130b.It is theorized that the lack of activity of 123 and 126 is due to the absence of a hydrogen donor at C-6'; such a group is present in kanamycin C (92) and is vital for its interaction with the AG receptor.This explanation also satisfies the results obtained with compounds 130a, 130b and 131, which are active but to a lesser extent than their C-6' hydroxylated counterparts.Other AGs, such as gentamicin C 1 and C 2 (5 and 6, Figure 1) that possess good antibacterial potency also have a methyl group at the C-6' position, indicating the fluoromethyl group would not have an adverse steric effect.Instead, the electron-withdrawing character of the fluorine atom lowers the basicity of the C-6'' amine group, decreasing its ability to form hydrogen bonds, and limiting the capacity of the molecule to bind its receptor. 70  cyanide as a promotor gave 139 in a 44% yield.The α anomer was the major product, and only traces of the β anomer were observed.Compound (140) was obtained after deprotection and purification. 14,24 eme 21.Synthesis of 3'-deoxy-3'-fluorokanamycin A (140).
Treatment of 141 with Raney nickel reduced the 6'-azide, which was then protected as a tosylate.Peracetylation followed hydrogenolysis of the benzyl groups afforded 142 (Scheme 22).The treatment of 142 with sulfuryl chloride chemoselectively halogenation C-4' and afforded 143 in 92% yield.The reduction of 143 under Birch conditions resulted in loss of both the chlorine and fluorine atoms and the formation of a 3',4´eno derivative (not shown).Tosyl deprotection was achieved by first reducing the 4'-Cl moiety with tributyltin hydride and AIBN followed by reaction under Birch conditions to remove the tosyl groups.Compound 144 was obtained after cleavage of the remaining protecting groups under standard reaction conditions. 14s expected, compound (144) was active against resistant bacteria producing both APH(3') and ANT(4'') AMEs. 14 724][75][76] Using this approach, the treatment of © ARKAT USA, Inc kanamycin A (1) with zinc acetate in DMSO followed by the addition of Cbz-OSu allowed the selective introduction of a Cbz protecting group at 6'-N to afford 145 (Scheme 23).Subsequent treatment with Amberlite CG-50(H + ) ion exchange resin to remove the chelate, tosylation of the remaining amino moieties, 4'',6''-cyclohexylidene formation, followed by treatment with NaH in DMF to protect 4'-O as a cyclic carbamate afforded 146 in 67% overall yield.The selective O-acetylation of 146 with 1-acetylimidazole (AcIm) in a 1:10 mixture of pyridine-DMSO followed by treatment with benzylsulfonyl chloride in pyridine gave compound 147 (Scheme 24).The acetates were cleaved with a catalytic amount of sodium methoxide in methanol, and the 2',3'-oxirane was formed upon treatment of the deacetylated product with a hot solution of 2% sodium methoxide in methanol, affording 148.Model experiments and molecular mechanics calculations were necessary to establish suitable conditions for maximizing the formation of the 3'-deoxy-3'fluoroglucose derivative (150) over the 2-deoxy-2fluoroaltrose derivative (149).Based on these optimization studies, a reaction with potassium hydrogen difluoride in ethylene glycol at 150 °C for 3.5 h was used to prepare 150.Global deprotection afforded 151. 72  Pseudomonas.In general, the fluorinated derivatives exhibit greater antibacterial potency than tobramycin (2) against sensitive and resistant bacteria containing AMEs. 24heme 24.Synthesis of 3'-deoxy-3'-fluorokanamycin A (151).
Both epimers of 2',3'-Dideoxy-2'-fluorokanamycin A (158 and 159) were tested for their antibacterial activities; 159 was practically inactive, and 158 was only slightly less potent than tobramycin (2).Both compounds (163 and 165) showed antibacterial activity, and the potencies of 163 were similar to those of tobramycin (2), 24 while 165 was active against bacteria producing 3' and 4' AMEs. 84he stereochemical outcome of the attack of 2,3-(N-tosylepimino)--D-allopyranosides (166) by a fluoride nucleophile depends on the initial conformation (Figure 10).The attack can occur at position C-2, affording an altroside derivative (167), or at position C-3, affording a glucoside derivative (168).Model studies 84 with 2,3-(N-tosylepimino)-α-D-allopyranosides (166) revealed that while the attack to form 168 is irreversible, the altroside (167) is in equilibrium with the 2,3-(N-tosylepimino)--D-allopyranoside (166) despite the high C-F bond energy.When the derivative is rigid, for example, when an acetal is formed at C-4 and C-6 OH, compounds 167 and 168 can be isolated, but at longer reaction times, the only fluorinated product detected is 168.When derivative (166) is flexible, only 168 is isolated regardless of the reaction times.It is postulated that the attack at position C-3 occurs when 166 adopts a 5 H O conformation, which is stabilized by the acidic KHF 2 and the solvent, instead of the O H 5 (half-chair with the oxygen up and C-5 down) conformation that favours the attack at C-2 following the Fürst-Plattner rule; this creates a torsionally stable transition state despite the stereoelectronic effects of the lone pair on the pyranoside oxygen. 86If a nucleophile is present at C-6 (e.g., OH), it could open the N-tosylepimino ring at C-3 to form the 3,6-anhydroglucose derivative (169). 84

Synthesis of 4'-deoxy-4'-fluorokanamycin A (176)
. 87 The C-5 configuration of 170 88 was inverted at C-4' by the S N 2 displacement of the previously formed triflate with sodium nitrite in DMF, affording 171 in a low yield (Scheme 27).The treatment of 171 with DAST resulted in the formation of an unexpected product (172).The formation of this compound was postulated to occur via a 1,2-hydride shift in which the hydrogen at C-3' displaced the anti −O−SF 2 −NEt 2 leaving group at C-4'. 87 However, a 3',4'-elimination reaction promoted by the © ARKAT USA, Inc basic fluoride ion with concomitant cleavage of the 2',3'-O-cyclohexylidene during work-up cannot be excluded.Due to this discouraging result, a different approach was adopted.Compound 170 was treated sequentially with phenylmethanesulfonyl chloride, 80% aqueous acetic acid and 0.1 M sodium methoxide solution in methanol to afford epoxide (173).The treatment of 173 with KHF 2 in ethylene glycol afforded fluorinated derivatives 174 and 175 as a 1:5 mixture of inseparable compounds.This reaction also afforded non-fluorinated by-products as a result of nucleophilic attacks by ethylene glycol and water (not shown).A mixture 174 and 175 was deprotected under Birch conditions, and then the compounds were separated using ionic exchange chromatography, allowing the isolation of 176.

Synthesis of 4'-deoxy-4'-fluorokanamycin B (182)
. 87 Compound 177 was first reacted with phenylmethanesulfonyl chloride in pyridine and then treated with sodium methoxide to afford 178 (Scheme 28).The reaction of 178 with KHF 2 in ethylene glycol resulted in a complex mixture of products, of which only three were identified.The major product was bicyclic compound 180 (52% yield), followed by the desired 4'deoxy-4'-fluoro derivative (179), which was obtained in 24%.Traces of 3'-deoxy-3'-fluoro-4'-epi kanamycin B derivative (181) were also isolated.Several attempts to improve the yield of 179 by running the reaction in various protic and polar aprotic high-boiling solvents resulted only in the formation of compound 180.Compound 178 preferentially adopts an O H 1 conformation that exists in rapid equilibrium with a higher energy 1 H O conformation.Following the Fürst-Plattner rule, attack of the O H 1 conformer is not favoured because of the stereoelectronic effects of the lone pair orbital on the oxygen of the glycosidic bond hindering the approach of the nucleophile from the bottom face.Fluoride attack at C-4' occurs on the highest energy 1  Compounds 176 and 182 were slightly less potent than kanamycins A (1) and B, but they showed broader activity.Bacteria expressing AAD(4') enzyme were susceptible to 176 and 182, but bacteria expressing APH(3´) were resistant to the fluorinated AGs, indicating that the presence of an equatorial fluorine at C-4' does not influence enzymes modifying the C-3' position. 87

lividomycin 229 lividomycin B
7 paromomycin  95 The selective tritylation of the C-5'' OH of 230 with phenyl boronate and trityl chloride followed by peracetylation afforded 231 (Scheme 37).The removal of the C-5'' trityl group under acidic conditions followed by reaction with DAST produced 232 (59% yield) as the major component and desired 5''-deoxy-5''fluoro derivative 233 in 27% yield.Standard protecting group removal afforded 234.Hydrogenolysis of the benzylidene protecting group of compound 235 afforded 236 (Scheme 38).A selective deoxyfluorination with DAST in dichloromethane followed by cleavage of the protecting groups using standard procedures afforded 238.Compared with paromomycin (7), 6'-deoxy-6'-fluoro analogue (238) showed a lower potency against both wild-type 1408A and 1408G mutant ribosomes 96 and against methicillin-resistant strains of S. aureus, E. coli and P. aeruginosa. 97The synthesis started with a suitably protected neomycin B analogue (239, Scheme 39).Treatment of 239 with DAST gave a complex mixture of products (not shown).Compound 239 was then reacted with triflic anhydride in dichloromethane and then treated with TBAF in THF to give 240.Cleavage of the protecting groups under standard conditions afforded 241.To synthesize 243, the alcohol at 4'-C was epimerized by sequential oxidation with Dess-Martin periodinane in dichloromethane followed by stereoselective reduction using L-Selectride®, affording 4-epi derivative 242.The reaction of 242 with triflic anhydride followed by treatment with fluoride using TBAF in THF and deprotection gave compound (243).The selective reduction of the C-1 and C-3 azides of 240 under Staudinger conditions [98][99] followed by acylation with N-Cbz-HABA-OSu afforded compounds 244 and 245 (Scheme 40).The reaction gave a 1:0.8 ratio of 244:245.The regioselectivity of the Staudinger reduction correlates with the electron density at the corresponding -nitrogen atoms as determined by natural abundance 15 N NMR; the azide bearing the most electron-deficient -nitrogen was preferentially reduced. 100Compound 244 was deprotected under standard conditions, affording 246.

AGs with Fluorinated -Amino
Acid Lateral Chains at Position 1 5.1.Synthesis of amikacin analogues with a -amino-α-fluoroalkanoyl side chain 23 Kanamycin A (1) was 1-N-acylated with a series of -amino--fluoro amino acids to determine their impact on the antibacterial activity (Figure 14).The fluorinated amino acids required for the side chains were synthesized by deoxyfluorination of suitably protected -hydroxy or -oxo esters with DAST.The most promising chain in terms of biological activity, the (S)-4-amino-2-fluorobutyric acid moiety, was also introduced to other AGs, to get acylated derivatives of kanamycin B (252), tobramycin (253), dibekacin (254), and gentamycin B (not shown) (Figure 15).Of all the -amino-2-fluoro amino acids coupled with kanamycin A, 2'''-deoxy-2'''-fluoroamikacin (248) showed the best biological activity profile (similar to that of kanamycin).The other analogues showed © ARKAT USA, Inc dramatically lower antibacterial potencies.It was also noted that the antibacterial potency of the fluorinated analogues parallel the potencies of the corresponding 2-hydroxybutyryl analogues, leading to the conclusion that the 2-fluoro group may play the same role as the 2-hydroxy group when present in the side chain.The acute toxicity of 248 (LD 50 280 mg/kg, iv mice) was found to be the same as that reported for amikacin (
The pK a values of the C-4''' amino groups of amikacin (4) and arbekacin (93) were measured by 13 C NMR spectroscopy and were approximately two units higher than the pK a values determined for compounds 255 Page 218 © ARKAT USA, Inc and 257 (10.2 vs 8.7).The acute toxicity values determined for 255 (LD 50 250 mg/kg) and 257 (LD 50 80 mg/kg), on the other hand, were quite similar to those of the reference compounds (4, LD 50 220 mg/kg and 93, LD 50 80 mg/kg), indicating that the toxicity was not influenced by the presence of the fluorine atom.he homologous (2S,4S) and (2R,4R)-5-amino-4-fluoro-2-hydroxypentanoic acids (prepared from malic acid) were coupled to 1-N of the aminoglycoside dibekacin (3) to obtain compounds 259 and 260 (Figure 17).Compounds 259 and 260 had the same potency as arbekacin (93).The acute toxicities in mice determined for 259 (~130 mg/kg) and 260 (~125 mg/kg) were approximately 1.7-fold weaker than that of arbekacin (93) (~75 mg/kg).This decrease in toxicity was attributed to the length of the chain attached to the AG.To further test this hypothesis, compounds 93, 261, 262 and 263 were synthesized (Figure 17), and a direct relationship between the chain length and the toxicity was found.The acute toxicities of these compounds were in the order 261>93>262>263.The LD 50 for 262 (~120 mg/kg), which possesses a 5-carbon chain, is similar to that of 259 (~130 mg/kg), indicating that the chain length and not the presence of the fluorine atom at C-4''' controls the toxicity.With respect to antibacterial activity, 263 was less potent than arbekacin (93), while 261 and 260 are almost equipotent to the aforementioned AG, 102 indicating that a butanoic derivative presents the best balance between high antibacterial potency and low acute toxicity.to 271, and subsequent reduction of the azides and olefins by catalytic hydrogenation afforded 273 and 274.A similar procedure was used to prepare 276 and 277.Diols 275 and 278 were also synthesized.Compounds 272-275, which have the natural L-HABA configuration at C-2'''', and unnatural D-HABA analogues (276-278) were tested against a broad panel of susceptible and resistant strains of ESKAPE pathogens to examine the effects of γ-N pK a modulation on their antibacterial potencies.The L-HABA analogues (273-275) were as potent as the parent compound (272) against wild-type strains and retained the activity of the parent compound through a panel of AG-resistant strains.The D-HABA analogues (276-278) were generally less potent than the analogues in the L-series but much more potent than the reference compounds. 28A strong correlation between the γ-N pK a and the antibiotic potency was observed in the D-HABA series.
Compound 273 was co-crystallized with the A-site decoding rRNA region.The data showed that the fluorinated acyl groups adopt the same conformation inside the A-site as the HABA chain as was observed for amikacin (4), and the (3''''R) fluorine atom forms hydrogen bonds to two cytosine residues inside the A-site rRNA.
Dose-response caspase-3/7 activation on the human kidney cell line HK2 triggered by compounds 272-278 revealed that apoptosis induction depends on the pK a of the derivative.Compounds with higher γ-N pK a values elicited apoptosis with an average EC 50 = 26 mg/mL, which is equal to that of the parent compound (272) (EC 50 = 27 mg/mL) within assay error.Difluorinated analogues 274 and 277 were less toxic, requiring a 2-fold higher concentration to elicit apoptosis (EC 50 = 47 and 58 mg/mL, respectively).

Conclusions
The present review provides an overview of the methods used for the incorporation of fluorine into AGs.It can be seen that the major synthetic challenge continues to be the somewhat cumbersome protecting-group strategies employed herein.The development of milder and more selective fluorinating agents, e.g., PhenoFluor®, which allows the fluorination of unprotected complex molecules, will undoubtedly simplify this process in the future.The biological importance of the fluorination of AGs parallels the importance of the introduction of fluorine into biologically active molecules and remains an active field of research given the importance of AGs as antibiotics.
Additionally, current progress in the biosynthetic pathways of aminoglycosides [104][105] could furnish tools to access fluorinated aminoglycosides by designed mutasynthesis.

Figure 9 .
Figure 9.The kanamycin family of AGs.
5-fluorokanamycin derivative 101 and the 5-deoxy-5-epi-5-fluoro derivative (102) (Scheme 15).The reaction intermediate (4''-O-SF 2 -NEt 2 ) undergoes neighbouring group attack from the carbonyl group of the Boc moiety at C-3'' rather than attack by an external fluoride.The 101/102 ratio obtained depended on the reaction time with the formation of 102 being favoured at longer reaction times, which suggested that 101 was formed during work-up.Compounds 101 and 102 were deprotected using standard procedures to afford AGs (103 and 104).

Figure 11 .
Figure 11.Influence of fluorination on pK a .

Page 191 © ARKAT USA, Inc
and the reaction afforded 86 in good yield.Final deprotection under Staudinger conditions afforded 87. .
The reaction of 111 with TBAF in acetonitrile followed by treatment with tetrabutylammonium bromide (TBAB) afforded derivative 112.The 6''-bromo moiety was reduced with Raney nickel, and the protecting groups were removed under standard conditions, affording 113.The reaction of 111 with sodium acetate in dimethylformamide proceeded with inversion at C-4'', giving 114.Treatment of 114 with sodium methoxide to remove the acetates and with TBAF to introduce a fluorine atom at C-6'' gave 115, which was then deprotected using TFA to afford 116.