Syntheses and Therapeutic Potential of Hydroxamic Acid Based Siderophores and Analogues

Cf”, Rev. 1989, 89. 1563-1579 1563

MARVIN J. MILLER Department of Chemistry, Univ6rslfy of Notre Dam, Notre Dame. Indiana 46556

Received March 9, 1989 (Revised Manuscript Received June 15. 1989)

Contenfs I. Introduction 1563 11. Synthetic Design and Synthesis of SMerophore 1564 Components A. Simple Hydroxamates 1565 B. Siderophores Containing 1566 1-Amino-w-(hydroxyamino)alkanes C. More Complex Amino Acid Containing 1567 HydroxamatbBased Siderophores and Analogues D. Total Syntheses of Hydroxamate Amino 1569 Acid Based Sierophores and Analogues 111. Siderophores and Siderophore Derivatives as 1575 Antibiotics Marvin J. Miller was born on Jan 29, 1949, in Dickinson. ND. His interest in science was greatly stimulated at Assumption Abbey Prep, a Benedictine monastic high school in Richardton, ND. Un- I. Infroducflon dergraduate research with Professor S. Peter Pappas at North Dakota State University in Fargo. increased his fascination with Iron has played an essential role in the evolution of science, especially organic chemistry. Further studies during his nearly every form of life on earth. Although iron is one Ph.D. work with Professor G. Marc Loudon at Cornell and an NIH postdoctoral position with Professor Henry Rapoport at Ee&eley of the most abundant elements on the planet, its pivotal solidified an interest in bioorganic chemistry. Since joining the role depended on the development of effective methods faculty at #m University of Notre Dame in 1977, Dr. Miller has been for its assimilation. Ionic forms of iron, especially an A. P. Sloan Fellow, an NIH research career development iron(III), its most common state, are very insoluble awardee, and a Visiting Fellow at #m Australian National University. under physiological conditions. To circumvent the Dr. Miller’s current research interests involve the chemical and enzymatic synthesis and study of biologically important, heteroat- solubility problem, many microbial, plant, and even omcontaining systems including amino acids, peptides. antibiotics. higher organisms (i.e., octopi’) synthesize and utilize siderophores. and others. He and his wife. Paty. are also the very specific low molecular weight iron chelators called proud parents of four beautirul children. Chris. Katie. Joey, and Carl. siderophores.z4 When grown under iron-deficient conditions, many microbes will synthesize and excrete = 52!P that special reductive, proteolytic, and hydro- siderophores in excess of their own dry cell weight to lytic processes are apparently utilized by microbes to sequester and solubilize iron.5 This extreme focus on release the iron once it is in the ceIl?J4 On the basis the need for iron is reflected by its requirement for the of these structures and those of other siderophores, the proper function of the enzymes that facilitate electron types of ligands utilized by microbes have been divided transport, oxygen transport, and other life-sustaining into three general classes: catechols, hydroxamates, and processes. In fact, competition for iron between a host miscellaneous (including oxazoline, a-hydroxy carbox- and bacteria is one of the most important factors de- ylic acids, amides, and others) (Charts 1 and 2). Sev- termining the course of a bacterial infection? Because eral other subclasses have also been defined.15 Many of their ubiquitous nature, microbial siderophores have volumes and reviews have been written about the in- been extensively studied, yet much still needs to be organic and biological aspects of sideropbore~.~~~’”~’~ learned about their chemistry and biochemistry, as well The organic and inorganic chemical as well as biological as other important aspects of iron metabolism. aspects of multi-catechol-containing siderophores have The chemical properties of ionic forms of iron have also been recently described in detail.’6J7 This paper dictated the evolutionary design of siderophores. Thus, will focus on the chemical synthesis and therapeutic high-spin, octahedral ferric ion is most effectively potential of hydroxamate-based siderophores, their chelated by three bidentate oxygen-containing ligands. analogues, and derivatives. In many cases, the required three bidentate ligands are Hydroxamic acids, their derivatives, and often their incorporated into the same molecule. The result has iron complexes have long been implicated in a wide been the evolution of compounds such as enterobadin,7 spectrum of biological activities.’520 A number are agrobactin: ferrichrome? desferrioxamine,1° mycobac- antibacterialz1,z2and antifungal anticancer tin,” pseudobactin,’2 and others that are capable of agents,5sZ3and specific enzyme inhibitors?‘ Certain binding ferric ion extremely well. For example, the hydroxamic acids have been used to reactivate chy- formation constant for enterobactin is so high (log Kf motrypsin and acetylcholinesterase after inactivation

0009-2665/89/0789-1563$06.50/0 0 1989 American Chemical Society 1564 Chemical Reviews, 1989, Vol. 89, No. 7 Miller

CHART 1. CatechoI-Containing Siderophores chain of hemoglobin and therefore require frequent blood transfusions. Extensive iron overload induced by the multiple transfusions causes deposition of the metal in the heart, liver, endocrine glands, and other organs. k The ultimate result is organic malfunction and early fi calecholale death. Since desferrioxamine must be administered by injection and large amounts are needed to mobilize iron faster than it is accumulated, the search for more effective drug candidates is extremely important until a practical genetic solution is found. Siderophores and their analogues are logical models from which effective iron chelating drugs might be developed. The same or similarly tailored compounds are potentially useful for removal of other toxic metals, including plutonium,lksn from biological systems. Siderophore-producing mi- enterobactin (enterochelin), agrobactin, X=OH a trimer of 2,3-dihydroxybenzoyI- parabactin. X=H crobes have also been found to promote crop growth by L-serine. shown without iron (111) (without iron (Ill)) either inhibiting root colonization of pathogenic organisms by an iron starvation mechanism or providing CHART 2. Hydroxamate-Containing Siderophores the plant with a solubilized form of iron.28 Several approaches have been suggested for the development of antibiotics based on siderophores and their analogues.22 Very effective chelators may deprive pathogenic microbes of iron essential for growth. This could be accomplished by competitive chelation of iron or by blocking the iron-siderophore receptor site with a nonfunctional siderophore analogue or the natural siderophore substrate bound to another metabolically (")3 (")3 less useful Alternatively, direct attachment of (amino acid constituents and abbrenated formi lethal compounds to siderophores might lead to new forms of drug delivery that utilize the pathogenic organisms own iron transport system. As another mode of action, iron-mediated generation of hydroxyl radicals may induce microbial DNA damage.22a Exploitation of the tremendous therapeutic potential of siderophores, their analogues, and derivatives requires access to sufficient quantities of materials for

mycobadins deslernoxamines preliminary and long-term study. As indicated earlier, shown wnhcut Iron [ill) HO"KA1 anemaling NHy(CH& NHOH in iron-deficient media microbes can often be encour- 0 aged to produce significant quantities of their natural siderophores. Molecular cloning may eventually allow production of nearly unlimited supplies of sidero- phoresa3 In fact, the outer membrane receptor (the tonA protein) for ferrichromes,30the enterobactin gene complex of E. and other systems have already been cloned. Still, full exploration of desired struc-

rhodotoNllC acid ture-activity relationships and the development of (shown without iron) novel analogues with favorable therapeutic properties that do not also promote growth of other pathogens HU pseudobaclln require chemical modification (semisynthesis) of the natural siderophores or rational total synthesis. by the potent nerve poison sarin.25 While much needs to be learned about the application of hydroxamic acid II. Synthetic Design and Synthesis of derivatives to these and related biological and bio- Siderophore Components chemical problems, current interest has emphasized the study of hydroxamic acids, and especially siderophores, At first glance, the impressively complex appearing for their metal ion transport properties and possible use structures of most siderophores of interest would sug- for the development of either broad-spectrum or spec- gest that total synthesis might not be a viable approach ies-selective antibiotics.22 for exploring their therapeutic potential. Closer in- The potential use of siderophores and analogues for spection reveals that, in many instances, nature has the treatment of iron metabolism disorders was recog- been quite conservative in the design of siderophores. nized early on and has received considerable attention. As indicated earlier, most rely on the use of catechols One natural siderophore, desferrioxamine, is still the and hydroxamic acids as the iron chelating ligands. drug of choice for the treatment of iron overload asso- Even within these classes minimal structural variation ciated with the transfusional treatment of &thalassemia is observed. Catechol-containing siderophores are often or Cooley's anemia.26 Patients with this common ge- constructed from simple dihydroxybenzoyl derivatives netic disease produce inadequate amounts of the P- (i.e., enterobactin, agrobactin, and parabactin) or Hydroxamic Acid Based Siderophores and Analogues Chemical Reviews, 1989, Vol. 89, No. 7 1565

SCHEME 1 SCHEME 2

NHOH

modified versions of dihydroxyphenylalanine (DOPA, as in pseudobactin). The hydroxamate-based siderophores usually derive the iron chelating ligands 2 and 4 (Scheme 1) from l-amino-w-(hydroxyamin0)alkanes arpergtllic acid, 9 (1; also see desferrioxamine) or o-N-hydroxy amino acids (3; also see ferrichrome). Most often, the ligands SCHEME 3 are connected through ester or amide linkages that, by HiICs current synthetic methodology, should be routinely "'%~~NO€HZPh constructed. Thus, synthetic approaches to the syn- 0 HOzC%YH 00 N-OCHZPh 0 OCHzPh...*I1'b , +""%b thesis of the apparently complex hydroxamate-based 10 11 12 OCHZPh siderophores was limited only by the need for efficient syntheses of the corresponding hydroxamic acid ligands proiimivaline themselves. I Many hydroxamic acids can be easily prepared by reaction of esters or active esters with hydroxylamine

i- . itself or various N- or O-alkylated derivatives. Unfor- 'i 14 tunately, most of the natural hydroxamate components OH actinonin, 15 OH of siderophores are secondary (probably to avoid nu- cleophilic reactions of primary hydroxamates or oxi- A. Simple Hydroxamates dation to toxic acyl nitroso compounds under physiological conditions). Moreover, the secondary hydroxa- Not all hydroxamate-based siderophores contain mates are designed such that the N-alkyl substituent three hydroxamic acids within the same molecule. In is involved in the linkage of the hydroxamate groups an early re vie^,'^ Maehr subclassified the hydroxamates to the core of the siderophore. This arrangement has into mono-, di-, and trihydroxamic acids. Because of impeded the synthesis of many siderophores by re- their less complex appearance, early synthetic efforts quiring first the synthesis of unusual or sometimes focused on the synthesis of some of the known mono- complex hydroxylamine derivatives, including the w-N- hydroxamic acids. The syntheses of aspergillic acid and hydroxy a-amino acids. actinonin are representative. Designing chemical syntheses of natural products and Aspergillic acid (9), an N-hydroxylated pyrazine iso- their components is often greatly facilitated by infor- lated from Aspergillus fla~us,3~was one of the first mation obtained about the corresponding biosynthetic naturally occurring hydroxamic acids isolated. Its pathways. The key question concerning the biosyn- synthesis, first reported in 1966,40relied on the direct thesis of the hydroxamate-based siderophores has been preparation of the required hydroxamic acid from leu- the mechanism and timing involved for introduction of cine (Scheme 2). N-Leucyl-O-benzylhydroxylamine(6) the hydroxyl group of the hydroxamic acid. Detailed was alkylated with l-chloro-2-(hydroxyimino)-3- studies have shown that a,w-diamino acids are first methylpentane (5) to give the extended oxime 7. Hy- oxidized to the w-hydroxylamine (forms of 1 or 3) by drolysis of the oxime to the ketone 8 followed by re- a process requiring molecular oxygen and then acylated ductive debenzylation and treatment with ammonia by an acyl coenzyme A to give the hydroxamic acid.32 induced an intramolecular condensation which, followed This impressive direct molecular oxygen mediated by air oxidation, provided racemic aspergillic acid (9). amine oxidation has no equal in chemical synthesis. Several natural derivatives of aspergillic acid have Most direct oxidations of primary amines to hydrox- subsequently been isolated and synthesized. ylamines have utilized peroxide derivatives, including Actinonin (15), one of the few natural N-unsubsti- dibenzoyl peroxide,33tert-butyl pero~ybenzoate,3~pi- tuted hydroxamic acids, is a rather broad-spectrum valoyl peroxide,35arylsulfonyl peroxides,36and bis(di- antibiotic isolated from Streptomyces roseopallidus. phenylphosphinyl) peroxide.37 Many of these oxida- Like aspergillic acid, the synthesis of actinonin utilized tions are low yielding, primarily because of competitive an O-benzylhydroxylaminederivative to introduce the acylation of the amines before oxidation, or are not hydroxamic acid (Scheme 3).*l Thus, reaction of 0- compatible with other sensitive substituents. Amide benzylhydroxylamine with anhydride 10 gave hydrox- oxidation has also been reported.% Most other attempts amate 11, which upon treatment with dicyclohexyl- to chemically oxidize primary amines to hydroxylamines carbodiimide (DCC) gave a mixture of the N- and 0- result in overoxidation. Consequently, syntheses of acylated products 12 and 13. Treatment of this mixture hydroxamate siderophore components have usually with prolinolvaline followed by hydrogenation produced required introduction of preoxidized nitrogen in the actinonin. form of other hydroxylamine derivatives, nitrones, or Propioxatin A (19),42 a potent enkephalinase B in- nitro compounds. Several of these methods will be hibitor produced by Kitasatosporia setae SANK 60684, discussed followed by a summary emphasizing the is another N-unsubstituted hydroxamic acid structur- utility of direct incorporation of preformed hydroxyl- ally related to actinonin. The synthesis of propioxatin amine derivatives. A also utilized O-benzylhydroxylamine and closely re- 1588 Chemical Reviews, 1989, Vol. 89, No. 7 Miller

SCHEME 4 SCHEME 5 Zn cbzHN-NoZ -CbzHN-NHOH NHnCl 22 23

16 L i 17 1. AczO 1 succininc anhydride 2. Hp I Pd-C 2 AczO

sembled the synthesis of actinonin (Scheme 4). The simplest of the more common secondary hydroxamic acid derived siderophores is hadacidin (N- hydroxy-N-formylglycine (21), eq 1). This simple com- 3 HpIPd-C pound has antitumor activity and retards plant Yd.C growth.15 It has been formed by formylation of N- I: hydr~xyglycine~~and from the benzaldehyde-derived peo nitrone 20.44 $15 HO-N FeClj 0 o+ & - femoxamineB HCOOH 0 (y=-pH/ -AClO H+CO,H (1) OH 27. desfernoxamine B 20 Yadactdin 21 c:T

B. Siderophores Containing +Amino-@-( hydroxyamin0)alkanes Synthesis of the dozens of more common and apparently complex siderophores required the development of efficient methods for their constituent 1- amino-w-(hydroxyamino)alkanes or o-N-hydroxyamino a-amino acids since simple acylations would provide the hydroxamate iron ligands (1 to 2 and 3 to 4, Scheme 1). Early approaches to the synthesis of these hydroxylamines relied on generation and reduction of the corresponding nitro compounds, generation and hydrolysis of aldehyde-derived nitrones, generation and hydrolysis of oxaziridines, and alkylation of N-alkoxysulfonamides. Several applications of these processes are summarized in the following schemes and discussions. The 1-amino-5-(hydroxyamino) pentane component (23) of ferrioxamine B (the iron complex of desferrioxamine B) was first prepared45 by reduction of the R ’OH 338 R =H corresponding nitro derivative 22 with zinc (Scheme 5). 33b R = COzH Acylation followed by hydrogenolytic removal of the Cbz protecting group produced the parent hydroxamic anhydride gave the acid amide 30. DCC-mediated acid 24. Reaction of the same hydroxylamine 23 with condensation of succinate 30 and amine 31 produced succinic anhydride provided 25, which served the dual dihydroxamate 32 in 88% yield. Hydrogenation of the role of N-acylating the hydroxylamine nitrogen with the nitrile of 32 to the amino group was followed by a required succinate and activating the other succinate second condensation with succinate 30 to give the carbonyl group. Thus, direct reaction of amine 24 and corresponding trihydroxamate. Finally, reduction of the active ester 25 resulted in formation of the di- terminal nitrile and reductive removal of all of the hydroxamate 26. Deprotection of 26 followed by a benzyl protecting groups to provide desferrioxamine B second reaction with 25 and subsequent removal of the were accomplished with hydrogen and 10% Pd-C in 0.1 Cbz group and addition of ferric chloride gave ferriox- N HCl in methanol. This promises to be a versatile amine B. route for the synthesis of a number of important ana- A more efficient synthesis of desferrioxamine B has logues of desferrioxamineB. An indication of the types been recently reported by Bergeron (Scheme 6).46 In of analogues that may be of interest was provided by this synthesis, the amine backbone was constructed by Bergeron in the same paper in which he alluded to the condensing 4-cyanobutanal with O-benzylhydroxyl- extension of the ferrioxamine chelation system by amine and reduction of the resulting oxime 28 to the continuing the sequential synthetic process to include protected hydroxylamine 29 with sodium cyanoboro- additional chelation sites on a single molecule. Related hydride. Acetylation of 29 with acetic anhydride fol- concepts were also described by Raymond.47 He found lowed by hydrogenation of the nitrile provided the re- that acylation of the N-terminus of ferrioxamine B with quired l-amino-5-(N-acetyl-N-(benzyloxy)amino)pen- dihydroxybenzoic acid derivatives, followed by depro- tane (31). Alternatively, reaction of 29 with succinic tection, gave the catechoyl-modified siderophores 33. Hydroxamic Acid Based Siderophores and Analogues Chemical Reviews, 1989, Vol. 89, No. 7 1567 Unlike desferrioxamine B itself, 33b was able to ki- netically remove transferrin-bound iron. The hydroxylamine and succinoyl components of ferrioxamine are present in a number of other natural siderophores. One of these, the recently discovered bishydroxamate bisucaberine (34), isolated from the

HO\N-A

04 u l4 lYo k’-N\OH 0 34 marine bacteria Alteromonas haloplanktis SB-1123, effectively sensitizes tumor cells to macrophage-medi- 0 ated cytolysis.48 Although no synthesis of bisucaberine has been rep0rted,4~the methods used for the synthesis of ferrioxamine should be directly applicable. A number of analogues of the w-(hydroxyamin0)-1- aminoalkane-based siderophores have also been synthesized. Interestingly, the crystal structure of aliphatic SCHEME 8 dimeric N-isopropylhydroxamic acids 35 revealed that Ar

51 47 40 49 50

with lithium borohydride gave alcohol 39, which upon in the absence of metal ion coordination, the hydrox- treatment with HBr gave the corresponding bromide amic acid groups existed in the trans rather than the 40. The required nitrogen was introduced by reaction usual cis conformation.s0 Another series of synthetic with sodium nitrite to give 41. Zinc reduction provided bishydroxamates called hexamates have been report- the hydroxylamine 42, which was hydrolyzed with 6 N ed.51 These molecules consist of a 1,6-hexanediamine HC1 to give the synthetic sample of 6-N-hydroxy-L- backbone acylated at both ends with malonyl- (n = l), ornithine (43)in about 7% overall yield. Prior to that, succinoyl- (n = 2), or glutaroyl-N-methylhydroxamates. the only source of this unusual amino acid was hy- None of the hexamates were able to stimulate the drolysis of siderophores, such as cis-fusarinine (44),73 growth of E. coli or Vibrio cholerae and several mu- which contained it. Keller-Schierlein subsequently tants, suggesting a preference for hexadentate or amino reported an alternate route to the 6-nitro-L-norvaline acid based ligands for more effective iron transport. precursor of 6-N-hydroxy-~-ornithine.~~In his review, Maehr15i75reported an improved synthesis of 6-N- C. More Complex Amino Acid Containing hydroxyornithine from 5-hydroxy-2-aminovalericacid Hydroxamate-Based Siderophores and (45),which, in turn, can be prepared by reduction of Analogues the y-carboxyl group of glutamic acid. The nitrone 46 was the key intermediate. Hydrolysis of 46 provided The most common hydroxamic acid components of the desired amino acid 43 directly. siderophores are derivatives of 6-N-hydroxyornithine. The first syntheses of eN-hydroxy-L-lysinealso relied This amino acid is the principal component of fusari- on the use of a nitro groupS5and nitroneT6as the hy- nine,52 fusarinine B and C,53 rhodotorulic acid,M droxlyamine precursors. This amino acid, a homologue dimerumic c~progen,~~ferriba~tin,~~ ferri- of 6-N-hydroxy-L-ornithine,is the functional constituent chr~me,~~~~ferrichrome A,60*61ferrichrome C:2 neu- of aeroba~tin~~and the mycobactins.lla rosp~rin,~~albomy~ins,~~*~~ ferrichrochin,66 ferri- Ornithine is also a logical choice of starting material chr~sin,~’~~ferrirubin,B8 ferrirhodin,68 pseud~bactin,~~ for the synthesis of 6-N-hydroxyornithine. As indicated, pseudobactin A,70 the pyoverdines,7l and other sidero- direct chemical oxidation of the 6-amino group is pro- phores. Because of its prevalence, synthesis of 6-N- blematic. However, Keller-Schierlein’s group was able hydroxyornithine has received considerable attention. to perform the oxidation indirectly (Scheme 8).78 As in the synthesis of desferrioxamine, a nitro group Reaction of a-amino- and carboxyl-protected ornithine was used as the hydroxylamine precursor in the first or ornithinyl peptides with p-methoxybenzaldehyde synthesis of 6-N-hydroxy-L-ornithine reported by formed the imine derivatives 49. Epoxidation with Neilands (Scheme 7).72 This synthesis, though low m-chloroperbenzoic acid gave the oxaziridine 50, which yielding, served as a model for many related syntheses. upon hydrolysis produced the desired N-hydroxy- Neilands recognized that L-glutamic acid (36)contained ornithine derivatives 51. This became a very attractive the appropriate carbon framework for the synthesis of route to hydroxamate constituents of siderophores. 6-N-hydroxy-L-ornithine.He first protected the a-am- However, the yields are often variable and the final ino and carboxyl groups as the hydantoin 37 and then hydrolysis often gives mixtures of products, including esterified the y-carboxyl to produce ester 38. Reduction regeneration of the starting ornithine and aldehyde. 1588 Chemical Reviews, 1989, Vol. 89, No. 7 Miller

SCHEME 9 SCHEME 11

,CH=NOCH2Ph /CHo

52 53 62 63 1 NaCNBHq t TS, ,OCHzPh TS, ,OCHzPh TS, ,OCHzPh Ts, ,OCH2Ph

HBr / HOAc NaOH (,sH (,sH + ? H - LH ACHN CONHPh ACHNXCOzH ACHN COzH AcHN(,COzEl COzEI L.66 0-65 65 64

SCHEME 10

?r HzNhH COzH HzNk.H COzH 67 688, R = CHzPh 69 M)b,R=H

Several attempts have been made to synthesiz w-N- hydroxy amino acids by incorporating intact h hox- ylamines and hydroxamic acids into preformed or ex- isting amino acid frameworks. The keys to these processes have been to circumvent the ambident nucleo- philcity of hydroxylamine and to incorporate appropriate functionality at the w-terminus of the amino acids. In one approach (Scheme 9),79 the hydroxyl- amino group was initially introduced by formation of an oxime 53 from 0-benzylhydroxylamine and the glutamic acid derived aldehyde 52. Subsequent reduction with sodium cyanoborohydride in the presence of acetic anhydride produced the 0-protected hydroxamic acid 54 directly. Further deprotection of the am- anilide with papain produced the L-amino acid L-66. ino acid proceeded normally to ultimately give 8-N- Subsequent reactions produced b-N-acetyl-8-N- acetyl-8-N-hydroxy-L-ornithine(56). Complete pro- hydroxy-L-ornithine derivatives suitable for elaboration tection of the a-amino group of the amino acid was to the parent amino acid 69 and its dimer, the diketo- necessary to avoid intramolecular cyclization of the piperazine rhodotorulic acid. amino group onto the aldehyde as it was formed.s0 A Direct N-alkylation of an intact hydroxamic acid very similar route, in which the a-amino group was conceptually provides the most efficient method for protected as the diallyl derivative, was used by Bedl preparing a number of hydroxamate constituents of in his studies related to the synthesis and structure siderophores. The acidity and reactivity of the NH determination of albomycin. bond of 0-protected hydroxamic acids are well docu- Fujii also utilized 0-benzylhydroxylamine as the mented, and N-alkylation has proven to be synthetically source of the hydroxamic acid (Scheme Alkyl- useful.21 However, intermolecular alkylation of 0-alkylation of 0-benzylhydroxylamine with bromide 57, itself or 0-acylhydroxamicacids [70, R1= alkyl (pK - 9-12), derived from diethyl acetamidomalonate by a standard R1= acyl (pK - 6-8)] often provides mixtures of N- alkylation, followed by saponification, decarboxylation, alkylation and carbonyl O-alkylation products [hy- and acetylation produced the racemic hydroxamic acid droxamates (73) and hydroximates (74), Scheme 58. Enzymatic resolution (Taka-diastase) gave the op- 121.81984785 Slight modification of the hydroxamate al- tically active amino acid 59. Problems with poly-N- lows direct N-alkylation. Thus, replacement of the acyl alkylation of the 0-benzylhydroxylamine might be ex- group of the hydroxamate with an &oxycarbonyl group pected with this approach. However, enough material (70, R = OR) promoted clean N-alkylation with a was prepared to enable a total synthesis of rhodotorulic number of alkyl halides.&*%This approach has been acid to be accomplished. used to prepare a number of N-alkylhydroxylamines Isowa recognized the need for further protection of and hydroxamic acids, including 8-N-hydroxy-L- the hydroxylamine precursors (Scheme ll).83 His ornithine and E-N-hydroxy-L-lysine. This route and group found that 0-benzyl-N-(ptolylsulfony1)- oxime formation followed by reduction and acylation hydroxylamine (60) could be cleanly N dkylated. Thus, (75 - 76 - 73) are now the most frequently used reaction of 60 with 1,3-dibromopropt.ne (61) gave the methods for the preparation of the hydroxamate con- substituted hydroxylamine 62, which upon reaction with stituents of siderophores. Applications of these meth- diethyl acetamidomalonate anion (63) followed by hy- ods to the total synthesis of a number of amino acid drolysis gave the racemic 6-N-hydroxyornithine deriv- based siderophores and siderophore analogues will be ative 65. Enzymatic resolution of the corresponding described in the next section. Hydroxamic Acid Based Slderophores and Analogues Chemical Reviews, 1989, Vol. 89, No. 7 1589

SCHEME 13

T%°CH2Ph

H2N 'C02H H 80 67

D. Total Syntheses of Hydroxamate Amino Acid Based Slderophores and Analogues Many of the methods described for the syntheses of hydroxamic acids were developed with the intent of incorporating them into total syntheses of siderophores and therapeutically useful analogues. Several of these 59 syntheses will be summarized in this section. Since SCHEME 16 syntheses of ferrioxamine B have already been de- P scribed, emphasis will be on, but not limited to, the siderophores containing the amino acid based hydroxamic acids. P-CIjXH202CP-PhCHfiC (Cbz) [tm) t .P The 6-N-hydroxyornithine-containingsiderophores 12 AcQ ferrichrome and rhodotorulic acid have received the most synthetic attention. Rhodotorulic acid (77a) is a

R H NO' AH o~N~~-"yOOH 50Hy-HyJ: 7 OH OH H R 01 77. rhodotmlic acid R = CH3 77b dlmeNmC aad R = HGC(CH3)CHzOH Ho8N 40""-F H H 03 HNr OH 78 coprogen

All structures shown without iron

0 79, foroxymithine 6-nitro-L-norvaline(81), reducing the two nitro groups diketopiperazine derived from dimerization of two 6- with zinc and acetylating the bis(hydroxy1amine) N-hydroxy-L-ornithine residues.55 Several natural (Scheme 14).90 As expected, acetylation occurred at analogues of rhodotorulic acid have also been found. both the nitrogen and oxygen of the hydroxylamines. Most of these, including dimerumic acid (77b),% differ The 0-acetyl groups were readily removed by solvolysis. only in the type of their 6-N-acyl substituents. Others, Fujii first prepared the monomeric O-benzyl-6-N- such as coprogen (78)57and foroxymithine (79),% con- hydroxy-6-N-acetyl-L-ornithine(59) as previously de- tain more extensive 6-N-acyl groups that also incorpo- scribed and then converted it to the diketopiperazine rate additional iron chelating ligands. As a di- by stepwise peptide chemistry. Catalytic hydrogenation hydroxamate, rhodotorulic acid is quadridentate and removed the benzyl groups without overreduction of the cannot effectively serve as a hexadentate ligand for hydroxamate N-0 bonds (Scheme 15).82 ferric ion. Consequently, the rhodotorulate-iron com- In a similar approach, Lee, Gerfen, and Millerg1first plex must have a minimum of a 3:2 st~ichiometry.~~ prepared the a-N- and 0-protected forms (87 and 89) Despite this, early studies indicated that rhodotorulic respectively of 6-N-acetyl-6-N-benzyloxy-L-ornithine, acid was more effective than desferrioxamine in pro- using the hydroxamate N-alkylation approach (Scheme moting iron excretion from iron-overloaded animal 12). Thus, as shown in Scheme 16, protected glutamic models. Unfortunately, the painful local reaction to the acid 83 was converted to the mixed anhydride and re- molecule when administered by injection diminished duced with sodium borohydride to give 6-hydroxy-L- its use.88 norvaline derivative 84, which upon treatment with Isowa's group first synthesized rhodotorulic acid by protected 0-benzylhydroxylamines under the Mitsu- converting the previously described b-N-tosyl-6-N- nobu conditions produced the fully protected 6-N- benzyloxy-L-ornithine (67) to diketopiperazine 80 using hydroxy-L-ornithines 85. Deprotections and 6-N- conventional peptide synthetic techniques. Detosyla- acetylation gave the free amino acid 86. Protection of tion with HBr, followed by 6-N-acetylation and catalytic 86 with the Boc group gave 87, which was also esterified hydrogenation to remove the benzyl protecting groups with an 0-methyldiisopropylisourea (R = i-Pr). Re- gave rhodotorulic acid (Scheme 13).89 moval of the Boc group from the resulting ester 88 gave Keller-Schierlein's group synthesized rhodotorulic the free amine 89. Conversion to the diketopiperazines acid by first assembling the diketopiperazine 82 from was accomplished by both conventional peptide tech- 1570 Chemical Reviews, 1989, Vol. 89, No. 7 Miller

SCHEME 17 SCHEME 19 H NO2 PhPN)

1 1 mc~ize

3 AC2O l2 z;i ferrichrome

SCHEME 18

Ts,N,OCHzPh

s, 98 enamiofer+chrome

peptide 1 cyclize 1 2 HBr I HOAc SCHEME 20

\" 0/3 " 0 4 HZjPd-C 93 5 FeClJ niques (87 + 89 90 rhodotorulic acid) and direct - - 1018, R =CHI dimerization of the Leuch anhydride (91) derived from 101b. R I H 86 and phosgene. Subsequent removal of the 0-benzyl protecting groups gave rhodotorulic acid in good overall the N- and 0-peracetylated peptide 97. Simple treat- yields. ment with methanolic ammonia removed the 0-acetyl The first total synthesis of ferrichrome, a cyclic groups, and reaction with ferric chloride in methanol- hexapeptide containing three sequential residues of / water produced the enantioferrichrome. The optical 6-N-hydroxy-L-ornithineand three sequential residues rotation and CD curves were the opposite of natural of glycine, was described by Keller-Schierlein's group ferrichrome. Quite interestingly, the ability of the en- (Scheme 17).92 On the basis of Neilands' and his own antioferrichrome to support microbial growth was com- precedent, Keller-Schierlein used 6-nitro-L-norvalineas parable to that of ferrichrome itself. the precursor to the 6-N-hydroxy-L-ornithineresidues. These syntheses have prompted the preparation of Using conventional peptide synthetic methods of the a number of analogues of ferrichrome containing 6-N- time, he constructed hexapeptide 92. Glycine residues hydroxyornithine or its "retro" analogues 101. Akiyama were judiciously placed at both the N- and C-termini described the synthesis and properties of a number of of the peptide to avoid racemization during the p-di- sequence isomers of linear and cyclic hexapeptides nitrophenyl ester mediated activation required for containing three 6-N-hydroxy-L-ornithine and three forming the corresponding cyclic hexapeptide. Subse- glycine residues.% Unfortunately, no microbial trans- quent reduction of all three of the nitro groups to the port studies or other biological tests were described in hydroxylamines with zinc followed by acetylation with the original report. Olsen described the synthesis of two acetic anhydride and extensive purification provided ferrichrome analogues [ lOla (R = CH3) and lOlb (R = a 10.5% yield of ferrichrome from the trinitro cyclo- H)] in which the side-chain N-hydroxyl and acyl hexapeptide. functions were transposed relative to natural ferri- Using the previously described b-N-tosyl-6-N- chrome; hence the term "retro ferrichrome" (Scheme benzyloxy-L-ornithine (67), Isowa's group prepared two 20)?5 The peptides were constructed from glycine and different sequence hexapeptides 92 and 93 by conven- hydroxamic acids 100 derived from a-L-aminoadipic tional means. Both of these precursors were cyclized acid (99). The N-methyl compound lOla was as ef- with DCC in dilute solution to the desired cyclo-tri- fective as natural ferrichrome as a microbial transport glycyl-tri-(6-N-hydroxy-6-N-benzyloxy-L-ornithinyl) agent, while the demethyl analogue lOlb was com- peptide. Subsequent removal of the tosyl groups fol- pletely inactive.% lowed by N-acetylation and hydrogenolytic removal of Many other non amino acid based "rhodotorulic acid the benzyl protecting groups and addition of ferric ion and ferrichrome analogues" (di- and trihydroxamates) gave ferrichrome (Scheme 18Lg3 have also been prepared. The general hydroxamate Keller-Schierlein's group later published a synthesis alkylation and 0-protected oxime reduction processes of enantioferrichrome (Scheme 19).78 In this case, the (Scheme 12) were especially useful for the syntheses of cyclohexapeptide (D-or~~)~-Gly~(94) was first prepared most of these. In many the constituent hy- by conventional methods. Reaction of the &amino droxylaminea and hydroxamic acids were prepared from groups with benzaldehyde followed by oxidation with a-amino w-alcohols by first N-protection and then ela- m-chloroperbenzoic acid gave the trioxaziridine 95. boration either by reaction with N- and 0-diprotected Subsequent hydrolysis with aqueous trifluoroacetic acid hydroxylamines under Mitsunobuge conditions or by produced the tri-(6-N-hydroxy-Dornithinyl)peptide 96, oxidation to the corresponding aldehydes, conversion which upon acetylation with acetic anhydride produced to the oximes, and sodium cyanoborohydride reduction Hydroxamic Acid Based Siderophores and Analogues Chemical Reviews, 1989, Vol. 89, No. 7 1571

SCHEME 21

R - (CH&N(OH)Ac FHSONHR YH$ONHR FHCONHR Y-CHzCONHR CHzCONHR CHzCONHR 1138, n I3 1148. n - 3 107 108 109 113b, n = 5 114b, n - 5

SCHEME 22

FHzCONHR’ ~H~CONHR’ CH2CONHR’

115 hi HkR R’ 116 R’ I (CH2)&ON(Me)OH for the treatment of Cooley’s anemia. Chiral ferrichrome analogues 125 and 126 have also been recently synthesized by Shanzer’s group.1oo Both of these compounds are tripodal structures assembled from L-amino acid hydroxamates linked by a-N- acylation to a tricarboxylate core. They are “retro” hydroxamates and like “retro ferrichrome“, they function effectively as microbial growth promoters.lol P-

Several trihydroxamate (128a-c, spermexatins) and OANAO mixed hydroxamate-catechol ( 133a-c, spermexatols) I H tie analogues of the catechol-containing spermidine-based 124.-C ~O~D~ON~ICPad siderophores agrobactin and parabactin have also been 0 OH (quadradenlate) synthesized and screened for microbial transport ability E mli mutatl pmwvl SupPorl S!ructures shown wflhout iron n-3 + (abp”eimnexcremnin (eq 2 and Scheme 23).61 Although no known natural n = 4 ++ inn fats upon wcbon) n-5 + H to the required 0-benzylhydroxylamine. Specific ex- 8210 amples are shown for the preparation of diamino- pentane (104) and diaminopropane (110) derivatives (Scheme 21). Reactions of these a-amino-w-hydroxyaminoderiva- n-1.2.3 tives (104 and 110) with a variety of multiacyl-con- hydroxamate-containing siderophores are spermidine taining substrates and minor elaboration provided a or norspermidine based, these synthetic compounds number of siderophore analogues (11 1-1 16).97 Essen- were found to substitute for the natural catechol-con- tially all of these analogues, which were water soluble, taining siderophore vibriobactin in iron transport in behaved nutritionally like ferrichrome when tested for mutants of Vibrio cholerae and E. coli. Compound their ability to support microbial growth. 128c stimulated growth of mutant organisms lacking Extensions of the analogue studies led to the syn- a functional catechol receptor for transport, implying thesis of several tris[ (acetylhydroxyamino)alkyl] iso- that this spermidine-based iron chelator was recognized cyanurates (124a-c, Scheme 22).% The biological ac- by other receptors, such as the hydroxamate ferri- tivity of these compounds was quite dependent on the chrome receptor. length of the alkyl chains. E. coli strains not mutated Albomycin (134a) is another natural siderophore that in any of the functions required for iron hydroxamate can be considered an analogue of ferrichrome. This uptake were able to use all of these trihydroxamates for very interesting compound has been “discovered” sev- iron transport. Interestingly, tris[(acetylhydroxy- eral times.22 It was first isolated from Streptomyces amino)butyl] isocyanurate 124b (n = 4) and to some griseus in 1947 and given the name grisein.Io2 Four extent ita pentyl analogue 124c (n = 5) were able to years later, the same compound was isolated from support the growth of fhuB mutants, which are un- Streptomyces subtropicus and given the name albo- responsive to other analogues and all natural sidero- mycin.lo3 Several years later, careful comparisons phores tested. Compound 124b was also found to demonstrated that the two compounds were identical.’O4 stimulate excretion of iron from hypertransfused mice Extensive structural studies indicated that, like ferri- in a model study for the development of iron chelators chrome, the iron chelating component was based on 1572 Chemical Reviews, 1989, Vol. 89, No. 7 Miller

SCHEME 24

- iH* iH* -*;O2H H2tkOzC[CH3), HN 140 141 142

9:: \ OH 36 143 144 CHI% 0$cH3T0H HN-N-H 0

vtbriobaain (shown without iron) tri- ( &N-acetyl-6-N-hydroxy-~-ornithine).lo5 In fact, TFA zn 1 AQO i HOA~ until recently, albomycin was also thought to be a cyclic CH CH (c CH CbzHN 147C02H CbzHN 146C02C(CHi), CbZHNc 145C02C(CH3), hexapeptide as is ferrichrome. However, in 1982, Benz provided conclusive evidence that the structure is com- prised of a pentapeptide with a thionucleoside attached to the C-terminal amino acid residue (134a).lM The extensive studies of albomycin have been well justified 1 1471EEDa since it is a rather broad-spectrum antibiotic. A de- 2 [PhlPjrPd scription of the implications of its biological activity will 3. 140 I EEDO be deferred until the last section of this paper.

O+f

Ho-N\ I

HoQ;+cHO 151a.R-H 151b. R-OH R 134a, X - S (albomyan 61) 134b,X = 0 (BEN s anakg) q0 to the synthesis of the triornithinyl peptide 142 and In an elegant sequence of papers, Benz also described extended tetra- and pentapeptides (149,150, and 151, chemical and enzymatic methods for the cleavage of the Scheme 25).'11 In limited assays, several of these hydroxamate components from the seryl nucleosidelo7 peptides stimulated microbial growth, but neither 6-N- which also allowed him to determine that the absolute acetyl-6-N-hydroxy-L-ornithineitself nor the corre- configuration of the constituent amino acids were of the sponding dipeptide displayed any growth-promoting natural L form.lo8 He followed this with syntheses of activity. The failure of the diornithinyl peptide to serve 6-N-acetyl-6-N-benzyloxy-L-ornithinederivatives as a microbial growth promoter is especially interesting 139-141 from glutamic acid:' the tripeptide 142 (tri- since the related diketopiperazine (rhodotorulic acid) (6-N-acetyl-6-N-hydroxy-L-ornithine)),lO9 and the oxy- is a very effective siderophore. As described later, gen analogue (134b) of the deferriform of 61-albomycin several of these peptides have also been attached to (Scheme 24).'1° Quite interestingly, the simple sub- antibiotics for antimicrobial studies. stitution of the oxygen for the sulfur resulted in loss of Other natural siderophores containing three residues antibiotic activity. of 6-N-hydroxy-L-ornithineinclude the coprogens (78) An alternate synthesis of 6-N-acetyl-d-N-hydroxy-L- and foroxymithine (79). These interesting compounds ornithine from glutamic acid has also been elaborated resemble rhodotorulic acid by incorporating two of the Hydroxamic AcM Based Siderophores and Analogues Chemical Reviews, 1989, Vol. 89, No. 7 1573

SCHEME 27

163 161 162 68b 152,cylo-6-N-Hydroxy. L-ornithine

-N ACNHCCH2Ph

PHN N HCI 2. 1 166 164 53 165 P=AC 153b 154 SCHEME 28 DIAD/ PhzP 0

), ~ OC H2P h 1 AczO HO CO2H 2 PCIS

NHOCHzPh 167 168 144 155 0 1 H2iPd-C 12 Phlh=NCOpEI/ DMAP CH-NOCH2Ph 1 TFA f 2 HONSulDCC hH 2. HzNOCHzPh LH 153 aerobaclin 3. N~BH~CNIHOA~(PHN = Phlh-Nl 169 Phth-N GQC(CH3)j Ph1h.N CQC(CH3)3 156 157 which is the protected fluorescent portion of azoto- bactin.'15 hydroxamates in a diketopiperazine, but, as described earlier, they also contain a third peripheral hydroxa- CCH2Ph mate. Although foroxymithine is also an effective ACE (angiotensin converting enzyme) inhibitor, no efforts pH" N OCHzPh related to its synthesis have been reported. The pseudobactins are the most complex 6-N- hydroxyornithine-containingsiderophores. The parent As indicated earlier, fewer of the known siderophores compound, pseudobactin,'2 itself contains the 6-N- contain eN-acetyl-t-N-hydroxylysine the homo- hydroxyornithine in its cyclic form (152). A hydroxyl (160), group and a carboxyl group from a @-hydroxyaspartic logue of 6-N-acetyl-6-N-hydroxyornithine.Both aero- acid component and a catechol, apparently derived from DOPA, provide the additional iron chelation sites. Hz=@OH Pseudobactin AI2 contains both cyclo-6-N-hydroxy-L- is0 A. ornithine and the more common linear form as well as the same catechol derivative as the iron ligands. Several bactin and mycobactin, which contain e-N-hydroxy- syntheses of cyclo-6-N-hydroxyornithinehave been re- lysine, have been ~ynthesized.l'~J~'As with most ported (Scheme 26). Simple treatment of 6-N- siderophore syntheses, the key was the preparation of hydroxyornithine (68b) itself with methanolic HC1 hydroxamate-containing amino acid constituents. In produces the cyclic form 152.112 Sodium cyanoboro- these cases, incorporation of the hydroxamate group hydride reduction of the oxime precursor (53) in the was accomplished by directly alkylating 0-benzyl ace- absence of acetic anhydride also produced the doubly tohydroxamate (Scheme 27) rather than utilizing the protected form of cyclo-6-N-hydroxyornithine153a?9 previous nitro or oxime approaches. The amino acid Fujii noted that saponification of the malonate 154 backbone was ultimately derived from dihydropyran followed by acidification gave racemic cyclo-6-N- (161). Thus, hydrolysis of dihydropyran to the open- hydroxyornithine 153b. Alternate syntheses from glu- chain hydroxy aldehyde followed by a Strecker reaction tamic acid derivative 144 have also been effective. gave the racemic amino acid 162. The amino acid was Conversion of 144 to the a-hydroxamate 155 followed resolved by chloroacetylation and subsequent hydrolysis by a Mitsunobu cyclization produced 153a. Protecting with acylase. a-Amino and carboxyl protection followed group exchange of 144 gave the phthalimide 156, which by conversion of the w-hydroxyl group to the bromide upon oxidation and reaction with O-benzylhydroxyl- 164 provided the substrate for the hydroxamate al- amine gave oxime 157. a-Carboxyl deprotection and kylation. Treatment of 164 with 0-benzyl aceto- activation as the N-hydroxysuccinimide ester with hydroxamate in the presence of potassium carbonate subsequent oxime reduction allowed direct cyclization and potassium iodide produced the N-alkylated hy- to the desired cyclo-6-N-hydroxy-~-ornithine.~~~ droxamate 165 surprisingly well. Removal of the amino Synthesis of the remaining peptide component of protecting group (Boc) produced free amine 166 suitable pseudobactin has also been completed.'14 Protected for elaboration to aerobactin. forms of the characteristically fluorescent dihydroxy- The two e-N-hydroxylysineresidues of aerobactin are quinoline portion (158) have been prepared from attached to the terminal carboxyl groups of citric acid DOPA.'14 Slight modification of the reaction se- (167). To avoid reaction of 166 with citric acid's internal quence114also provided the analogous tetracycle 159, carboxyl, it and the hydroxyl group were protected by 1574 Chemical Reviews, 1989, Vol. 89, No. 7 Miller

SCHEME 29 SCHEME 30 OH DEAD OH 1 (BOC120

HiNHrr CO2H 2 HZNOCHZPh 'OCHZPh OCHiPl 163 RN.C=NR' 177 0 178 mycobactins

mycobam S2 R, R4 = Me

R2 iRJ I Rg= H HO*NKR' 0

NADH I H* a NAD* A

172, mycobactic acid 173, cobactin

HOCNKMe 173. (-).cobaclin a 0 a

WiGOH HOMuoH H2NHQ,oH OH 0 0 174, 0-hydroxybenZOylSerine (R).p-hydroxy butyrate 176, cyclo-HYLYS

HOJK Me 0 1 6 0 (HYLYS) g -N.hydroxy.6-N-acefyI-L-lyUne reaction with formaldehyde to form the anhydro- methylenecitrate, which was then converted to the di- acid chloride 168 (Scheme 28). Direct coupling of 168 PhCH20*EI)i'Me with 166 produced the fully protected aerobactin 169. 0 0 Saponification and hydrogenation produced the first 1 180 i DEAD i PhiP synthetic sample of aerobactin. I2 Hi/ W.C Although it has not yet been isolated from natural mvcobactin 52 sources, the citric acid based 6-N-hydroxy-L-ornithine- containing siderophore awaitin A (170a) has been syn- attention to be focused on the synthesis of the known thesized as a process directly related to that used for mycobactin hydrolysis products, mycobactic acid (172) aerobactin ( 170d).11* Similar approaches were used for and cobactin (173). Each of these fragments could be the synthe~es~~~J~~of other citrate-based siderophores, broken down to two additional components (Scheme schizokinen (170e),lz1schizokinen A (171), and arthro- 29). bactin (170f).lZ2Formation of imide during the syn- The synthesis of cobactin required the preparation thesis of schizokinen led to the assignment of the un- of cyclo-e-N-hydroxylysine (176) and D-(R)-P-hydroxy- known structure of schizokinen A as 171. butyric acid (175). As shown in Scheme 30, the cyclic hydroxamic acid was prepared from the alcohol 163 ~NHWkOHYo used previously for the synthesis of the linear form of e-N-hydroxy-L-lysine. Thus, the cy-amino group of 163 was first protected with a Boc group. The carboxyl group was then coupled with 0-benzylhydroxylamine OR to give hydroxamate 177. An intramolecular Mitsunobu reaction gave the seven-membered-ring cyclic hydrox- 1708, wamA R-COOH, n - 1 171, schizokinen A (n - 0) 1708,WB R-H,n- 1 amic acid plus considerable amounts of the corre- 170c,e!&t.nC R-CWH,n-O sponding carbonyl 0-alkylated (hydroximate) products. 1704, ae- R - COOH, n- 2 1700,achiroklnen R-H,n -0 The P-hydroxybutyrate was prepared by enzymatic 170f, arth-n R-H,n-2 reduction of lithio acetoacetate with P-hydroxybutyrate The mycobactins (Scheme 29, R groups are varia- dehydr0gena~e.l~~Since this enzyme requires NADH ble)"* contain two residues of t-N-hydroxy-L-lysine,one for the reduction, the reaction was coupled with the in the usual linear form and one cyclic. This group of galactose dehydrogenase mediated conversion of ga- siderophores was isolated from Mycobacterium tuber- lactose to galactonate. Thus, the NADH was recycled culoci, the organism that causes tuberculosis. To sim- and the ultimate reducing agent was galactose. Re- plify its synthesis, an analogue, mycobactin S2 (R, = moval of the Boc group from 178 followed by EEDQ- R4 = Me, R2 = R3 = R5 = H), was chosen as the first mediated coupling of the fl-hydroxybutyrate (175) and target."' Since e-N-hydroxy-L-lysine derivatives were the a-amino group of resulting 0-benzyl-protected cy- made available during the synthesis of aerobactin, clo-€-N-hydroxy-L-lysinegenerated 180, and subsequent preparations of the cyclo-eN-hydroxy-L-lysine(cobactin hydrogenation produced (-)-cobactin T (173). (173)) and 2-(o-hydroxyphenyl)oxaoline (the third iron The synthesis of the mycobactic acid fragment was chelating group) residues were of primary concern.112 initiated by coupling 2-benzyloxy benzoate active ester Dissection of the molecule at the ester linkage allowed 181 with L-serine to give 183 (Scheme 31). Reaction Hydroxamic Acid Based Siderophores and Analogues Chemical Reviews, 1989, Vol. 89, No. 7 1575 of 183 with 166, the eN-hydroxy-L-lysine derivative SCHEME 32 used in the aerobactin synthesis, in the presence of SOzNHCQA EEDQ produced peptide 184. Cyclization to the desired 1, fenioxamine B oxazoline was accomplished by reaction with thionyl chloride under carefully controlled conditions. Sapo- )y$yI NO2 187 OH nification generated dibenzylmycobactic acid S2 (185). HO 04 Interestingly, attempts to first prepare the oxazoline from 183 and then couple it to amino acid derivative 166 failed. A Mitsunobu-mediated coupling of pro- 6 6 tected mycobactic acid and (-)-cobactin fragments 185 and 180, respectively, resulted in formation of the final ester linkage with the desired and required inversion at the carbinol center of mycobactin S2. Hydrogenation removed all of the benzyl groups to complete the synthesis of mycobactin S2. In summary, it appears that the fundamentally sim- S02N-C-0 ple reactions of N-alkylation of hydroxamic acid de- SGNHCO-R 0 rivatives or oxime formation, followed by reduction and acylation, provide direct and versatile access to im- R- NOz portant iron-binding constituents. Commonly em- 187 191 NH2 ployed protecting groups and routine coupling reactions now allow these constituents to be combined to chemically prepare many of the known siderophores and 1. Soch analogues. These studies have evolved so well that 2. lenkrccinI pyridine p attention can now be shifted to specific therapeutic NH 3. Hz /PdG applications of synthetic siderophores and their ana- NH logues. lenkman 1. - H) I I I I. Siderophores and Siderophore Derivatives 192 NHz as Ant/6iotics the sulfonamides were attached by reaction with iso- An excellent review on iron-containing antibiotics has cyanate 187 or the N-hydroxysuccinimide derived from been published by Neilands and Valenta.% The review 189. Ferricrocin differs from ferrichrome only by the focuses on natural siderophores with antimicrobial ac- replacement of the central glycine residue with a serine. tivity. Thus, this rather brief section will review only The primary hydroxyl group of the serine residue pro- attempts to couple siderophores or analogues with an- vided a convenient point for attaching the sulfonamides. timicrobial agents to take advantage of iron transport Of the four compounds made, only the two sulfanil- mechanisms for antibiotic delivery. Albomycin and the amidonicotinic acid derivatives 190 and 192 displayed ferrioxamine B derivative ferrimycin AI ( 186)22a9124are any antimicrobial activity and it was limited to Sta- phylococuss aureus. Recently, many groups have attempted to attach catechol or hydroxamate groups to 0-lactamantibiotics HNnit) with the anticipation that the corresponding iron com-

bH H? 03 plexes may be carried into microbial cells by iron H "ykNH transport mechanisms. This approach has met with CHI 0 considerable success. Ureidopenicillins (193a,b)having n: a terminal catechol moiety were synthesized and found to have 30-60-fold increase in activity relative to pi- peracillin against various microbes, including Pseudo- natural examples of the use of an iron transport system monas aeruginosa strains, which are generally less to deliver a toxic substance to a microbe. Evidence now sensitive to &lactam antibi~tics.l~~J~Related catechol indicates that albomycin is actively carried into mi- derivatives of ureidocephalosporins and ureidocepha- crobial cells by normal iron transport processes and, mycins also displayed enhanced activity against Pseu- once in the cells, the toxic thioribosyl moiety is enzy- domonas ~eruginosa.~~~It was speculated that all of matically released, perhaps by a peptidase-mediated these compounds owed their enhanced antimicrobial ~1eavage.I~~Again, it is worth recalling that Benz's activity to their ability to bind iron and be actively oxygen analogue of the thioribosyl group was apparently carried into cells by the iron transport mechanisms. devoid of antimicrobial activity. Similarly, the antimicrobial activity of the catechol- Attempts to mimic albomycin's antimicrobial activity containing cephalosporin M14659 (194) was found to with other less labile siderophore-antimicrobial agent be enhanced in low-iron environments, suggesting that conjugates have been of considerable interest. The it may be assimilated by microbial iron transport sys- earliest tests of this possibility utilized a semisynthetic tems." E-0702 (195)is a new semisynthetic iron che- approach in which various sulfonamides were covalently lating cephalosporin derivative which has been shown bound to ferrioxamine B and ferricrocin to produce the to be incorporated into E. coli cells by the tonB-de- adducts 188, 190, 191, and 192 (Scheme 32).lZ6 Fer- pendent iron transport ~ystem.~~~J~~All of these rioxamine B contains a terminal amino group to which cephalosporin derivatives have the catechol moiety 1576 Chemical Reviews, 1989, Vol. 89, No. 7 Miller SCHEME 33 ""* 0 CI 200 C02pNB 1938, 4 =CHI 193b, R = ICHlllOH h 11

HoqCOzNaHO "WX'3&sYN;:Ho 195. €4702 cqNa N-N CkC0,Na

Co2Na VN 194 M14659 CM

acetyl-6-N-hydroxy-L-ornithine)(142) had been shown to be an effective microbial growth promoter, covalent

COzNa N-N linkage of P-lactam antibiotics was expected to provide R, 198 an interesting test of the conceptual use of siderophores OANIRi, as drug delivery agents. To avoid problems that might OH be associated with the eventual hydrogenolytic removal attached to the a-acyl group of the P-lactam. of several benzyl protecting groups, non-sulfur-con- Several compounds have also been prepared in which taining &lactams were fmt considered. Two such types catechols are incorporated at other positions around the of @-lactamsare the oxamazins ( 199)13' and the carba- periphery of the cephalosporin nucleus. Most of these cephalosporons (200).138 Both of these types of anti- are C-3 methyl substituted derivatives. BO-1236 (196) biotics were acylated with protected D-phenylglycine or is a new anti Pseudomonal cephalosporin which ap- D-p-hydroxyphenylglycineto give derivatives 201 and parently owes its enhanced activity to the iron-binding 202, which were then covalently attached to the C- capability of the pendant catech01.l~~The activity of terminal end of the protected triornithinyl peptide (203, dihydroxybenzoyl cephalosporin derivatives 197 has also Scheme 33). The phenylglycyl residues were added been correlated with tonB-dependent illicit transport since they are common side chains for many @-lactam across the outer membranes of microbes." As ex- antibiotics (but not the oxamazins). The penultimate pected, the patent literature contains numerous exam- peptideantibiotic conjugates were completely protected ples of related catechol-containing cephalosporin de- with hydrogenolytically labile groups. Thus, in each rivative~.'~~One patent also disclosed a cephalosporin case, careful catalytic hydrogenation released the final derivative (198) that incorporated a hydroxamic acid, products. Neither of the oxamazin derivatives (204a presumably for promoting transport by iron shuttle nor 204b) displayed antibiotic activity against a variety processes.136 The compound exhibited significant ac- of organisms. This should not have been too disap- tivity against both Gram negative and Gram positive pointing since the activity of the oxamazins is signifi- bacteria. cantly affected by the side chain. Incorporation of the The apparent ability of many of these iron chelating ATMO (aminothiazolone methoxime) side chain in- P-lactam antibiotics to utilize iron transport processes duces considerable activity against Gram negative for cellular delivery is quite impressive since most of bacteria, whereas the phenylglycyl side chains are nearly the compounds would not be considered efficient hex- devoid of activity. Still it would have been quite in- adentate iron ligands. Presumably the 1igand:iron teresting if the oxamazin derivatives would have had stoichiometry is not crucial for transport. This was demonstrable activity because of more effective iron- previously demonstrated with rhodotorulic acid, which mediated transport into the microbial cells. forms a 3:2 complex with ferric ion, yet is a very ef- In sharp contrast, the carbacephalosporin-sidero- fective siderophore. On the other hand, and as indi- phore conjugates 205a and 205b displayed significant, cated earlier, 6-N-acetyl-6-N-hydroxy-L-ornithineand but very species-selective, antimicrobial activity. In- the corresponding linear dipeptide did not promote cubation with P-lactam hypersensitive E. coli X580 microbial growth in select studies and, if covalently (Lilly) resulted in delayed growth (Figure 1). The bound to antibiotics, they presumably would not be able bacteria that did eventually grow were isolated and to utilize iron transport mechanisms for drug delivery. separately incubated again in the presence of each of Thus, some attention has been given to the synthesis the carbacephalosporin derivatives. In both cases, no and study of antibiotics containing hexadentate iron delay of bacterial growth was observed. Among other ligands. possibilities, these results suggest that in the first in- Albomycin mimics were chosen as logical sidero- cubation, a mutant of the parent E. coli strain had been phore-antibiotic synthetic targets. Since tri(6-N- selected that was deficient in some form of iron trans- Hydroxamic Acid Based Slderophores and Analogues Chemical Reviews, 1989, Vol. 89, No. 7 1577

(4) Swinburne, T. R., Ed. Iron, Siderophores, and Plant Dis- eases; Nato AS1 Series; Series A Life Sciences Vol. 117; Plenum: New York, 1986 and references therein. (5) Neilands, J. B. Bacteriol. Rev. 1957, 21, 101. (6) Payne, S. M. CRC Crit. Rev. Microbiol. 1988, 16,81. (7) (a) Kroth, H. Arch. Mikrobiol. 1970,70,297. (b) O'Brien, I. G.; Cox, G. B.; Gibson, F. Biochim. Biophys. Acta 1970,215, 393. (8) Peterson, T.; Falk, K. E.; Leong, S. A.; Klein, M. P.; Neilands, J. B. J. Am. Chem. SOC.1980,102, 7715.

fl* (9) (a) Emery, T. F.; Neilands, J. B. J. Am. Chem. SOC.1961,83, 0 -,s* 1626. (b) Prelog, V.; Walser, A. Helu. Chim. Acta 1962,45, 0 10 20 31 3 631. ilma (hn) (10) (a) Keller-Schierlein, W.; Prelog, V. Helu. Chim. Acta 1961, 44, 1981. (b) Keller-Schierlein, W.; Mertens, P.; Prelog, V.; Figure 1. Effects of preformed Fe(II1) complexes of compounds Walser, A. Helu. Chim. Acta 1965,48, 710. (c) Prelog, V.; 205a and 205b (10 pM)in Mueller-Hinton broth on the growth Walser, A. Helo. Chim. Acta 1962, 45, 631. (d) Keller- rate of E. coli X580. Schierlein, W.; Prelog, V. Helu. Chim. Acta 1961,44,709. (e) Prelog, V.; Walser, A. Helu. Chim. Acta 1962, 45, 1732. (11) (a) Snow, G. A. Bacteriol. Reu. 1970,34,99. (b) Ratled e, C. port since its growth was unaffected by the presence of In Iron Transport in Microbes, Plants and Animals, *ink- either or Iron transport deficient mutants elmann, G., van der Helm, D., Neilands, J. B., Eds.; VCH: 205a 205b. Weinheim, New York, 1987; Chapter 12. might not be expected to be virulent pathogens. The (12) (a) Teintze, M.; Hossain, M. B.; Barnes, C. L.; Leong, J.; van same compounds (205a and 205b) displayed moderate der Helm, D. Biochemistry 1981,20,6446. (b) Demange, P.; Wendenbaum, S.; Bateman, A.; Dell, A.; Abdallah, M. A. In activity against a number of bacteria and significant Iron Transport in Microbes, Plants and Animals; Winkel- activity against Salmonella X514 (MIC = 1.0 and 0.06 mann, G., van der Helm, D., Neilands, J. B., Eds.; VCH. Fg/L, respectively). The normal broad-spectrum ac- Weinheim, New York, 1987; Chapter 10. (c) Meyer, J.-M.; Halle, F.; Hohnade, D.; Lemanceu, P.; Ratefiarivelo, H. In tivity of the carbacephalosporins was apparently di- Iron Transport in Microbes, Plants and Animals; Winkel- minished because either they were not carried into other mann, G., van der Helm, D., Neilands, J. B., Eds.; VCH: microbial cells by the iron transport system and also Weinheim, New York, 1987; Chapter 11. (13) Harris, W. R.; Carrano, C. J.; Cooper, S. R.; Sofen, S. R.; no longer recognized by the normal @-lactamtransport Avdeef, A. E.; McArdle, J. V.; Raymond, K. N. J. Am. Chem. processes or they were actively transported by an iron Soc. 1979,101,6097. shuttle system, but the antibiotic group was not intra- (14) For recent discussions on the mechanism for the release of iron from siderophores, see: (a) Cass, M. E.; Garrett, T. M.; cellularly released. These preliminary exciting results Raymond, K. N. J. Am. Chem. SOC.1989, 111, 1677. (b) indicate that albomycin analogues and perhaps many Ecker, D. J.; Loomis, L. D.; Cass, M. E.; Raymond, K. N. J. Am. Chem. SOC.1988,110,2457. (c) Raymond, K. N.; Cass, other siderophores and analogues may effectively carry M. E.; Evans, S. L. Pure Appl. Chem. 1987,59, 771. unnatural antibiotics into cells, perhaps selectively, and (15) Maehr, H. Pure Appl. Chem. 1971,28,603. inhibit microbial growth. (16) (a) Bergeron, R. J. Chem. Rev. 1984,84,587. (b) Bereeron, R. J. In Iron Transport in Microbes, Plants and Animals; While it may be easy to predict that the concept of Winkelmann, G., van der Helm, D., Neilands, J. B., Eds.; iron transport mediated illicit drug delivery has con- VCH Weinheim, New York, 1987; Chapter 16. siderable potential, much work needs to be done. (17) Wierenga, W. Total Synthesis of Natural Products; Wiley: New York, 1981; Vol. 4, Chapter 3, p 274. Further synthetic and biological studies are certain to (18) For recent accounta of the synthesis and biological impor- reveal extremely interesting aspects of iron metabolism tance of related N-hydroxy amino acids, see: (a) Ottenheijm, well new modes of chemotherapy. This review H. C. J.; Herscheid, J. D. M. Chem. Rev. 1986,86,697. (b) as as has Chimiak, A.; Mitewska, M. J. Prog. Chem. Org. Nat. 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