Accepted Manuscript Review Natural hydrazine-containing compounds: biosynthesis, isolation, biological activities and synthesis Géraldine Le Goff, Jamal Ouazzani PII: DOI: Reference:

S0968-0896(14)00724-X http://dx.doi.org/10.1016/j.bmc.2014.10.011 BMC 11857

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Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

7 August 2014 7 October 2014 9 October 2014

Please cite this article as: Le Goff, G., Ouazzani, J., Natural hydrazine-containing compounds: biosynthesis, isolation, biological activities and synthesis, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/ 10.1016/j.bmc.2014.10.011

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Natural hydrazine-containing compounds: biosynthesis, isolation, biological activities & synthesis

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Géraldine Le Goff and Jamal Ouazzani Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles ICSN, Centre National de la Recherche Scientifique, CNRS, Avenue de la Terrasse 91198, Gif-sur-Yvette, cedex, France.

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Bioorganic & Medicinal Chemistry j o ur n al h om e p a g e : w w w . e l s e v i er . c o m

Review

Natural hydrazine-containing compounds: biosynthesis, isolation, biological activities and synthesis Géraldine Le Goff and Jamal Ouazzani* Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles ICSN, Centre National de la Recherche Scientifique, CNRS, Avenue de la Terrasse 91198, Gif-sur-Yvette, cedex, France.

A R T IC LE IN F O

A B S TR A C T

Article history: Received Received in revised form Accepted Available online

Hydrazine, hydrazone and hydrazide derivatives are nitrogen-nitrogen bond containing compounds. Such molecules are relatively scarce in nature and have been isolated from plants, marine organisms and microorganisms. These compounds exhibit remarkable structural diversity and relevant biological activities. The enzymes involved in the formation of the N-N bond are still unknown, but many lines of evidence support the involvement of N-nitrosation and N-hydroxylation activating steps. Beside the challenging N-N bond, N-acylases catalyzing the C-N bond formation contribute to the chemical diversity of N-N-containing natural products (N2NP). This review examines the state of knowledge regarding the biosynthesis of N2NP, for which only two biosynthetic gene clusters have been investigated. Biological properties and chemical synthesis of hydrazines, hydrazones and hydrazides are also reported.

Keywords: N-N bond synthesis and biosynthesis Hydrazine, hydrazone and hydrazide N-nitrosation/N-hydroxylation C-N bond biosynthesis Geralcins

2015 Elsevier Ltd. All rights reserved.

1. 2.

Introduction N-N bond in natural compounds: biochemical and biosynthetic considerations 2.1. Enzyme catalyzed N-nitrosation followed by N-nitroso-reduction to N-amine intermediate 2.2. Enzyme catalyzed N-hydroxylation flowed by inter- or intramolecular N-N bond formation 2.3. Enzyme catalyzed N-acylation 3. Sources, properties and biological relevance of naturally occurring hydrazines, hydrazones and hydrazides 3.1. Natural hydrazine derivatives 3.2. Natural hydrazone derivatives 3.2.1. Hydrazones from bacteria and fungi 3.2.2. Hydrazone from plants 3.2.3. Hydrazones from marine organisms 3.3. Natural hydrazide derivatives 3.3.1. Hydrazides from actinomycetes especially from Streptomyces genus 3.3.2. Hydrazides from filamentous fungi and mushrooms 3.3.3. Hydrazides from plants 3.3.4. Hydrazides from marine organisms 4. Synthesis of naturally occurring hydrazines, hydrazones and hydrazides 4.1. General procedures 4.1.1. Substitution of hydrazine and C-N bond formation 4.1.2. Condensation of hydrazines with activated carbonyl compounds 4.1.3. Diazotation/diazonium reduction or nitrosation/nitrosamine reduction 4.1.4. N-amination 4.1.5 Reduction of hydrazones and hydrazides 4.2. Application to the synthesis of natural hydrazines, hydrazones and hydrazides 4.2.1. Syntheses of natural hydrazones 4.2.2. Syntheses of natural hydrazides 5. Conclusions 6. References _________________ *Corresponding author. Tel.: +33 169823001; fax: +33 169077247. E-mail address: [email protected] (J. OUAZZANI).

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1. Introduction Despite considerable advances in the elucidation of biosynthetic routes to natural compounds, the limited occurrence of nitrogennitrogen bond remains enigmatic and their biosynthesis still unknown. To date, only four reviews have been dedicated to natural products containing an N-N bond (N2NP). The first review was published in 1977 as an inventory of the known compounds.1 Unfortunately, at that time, only a few compounds have been discovered. An updated inventory was recently published in 2013 and found that over 200 N2NP had been reported to date (less than 0.1% of the known natural products).2 Specific reviews on the naturally occurring diazo3 and piperazic acid4 derivatives were published in 2011. Although rare, N2NP exhibit exceptional structural diversity, including hydrazones, hydrazines, hydrazides, nitramines, nitrosamines, azoxy, diazo compounds and a range of heterocyclic scaffolds (pyrazole, pyridazine, azapteridine, triazolopyrimidine and piperazic acid derivatives). This review summarizes the enzymatic reactions that may lead to N-N bond formation and contribute to N2NP biosynthesis. It also gives an overview of the wide range of biological activities and synthetic efforts towards natural hydrazines, hydrazones and hydrazides. 2. N-N bond in natural compounds: biochemical and biosynthetic considerations This section summarizes the demonstrated and expected implication of enzymes in the biosynthesis of N-N bondcontaining natural compounds. For organic chemists, N-N bond is readily accessible from hydrazine and its derivatives.5 In nature, hydrazine production, catalyzed by hydrazine-synthase, is restricted to anaerobic ammonium-oxidising bacteria (anammox),6,7 while reported N2NP were mostly produced by aerobic organisms. This aroused the interest of the scientific community in the biosynthetic route to N-N bond containing compounds. In nature, the N-N bond is involved in the nitrogen cycle and contributes to the formation of molecular nitrogen N2 and nitrous oxide N2O gases. The N-N connection occurs via spontaneous chemical (abiotic) or enzyme-catalyzed (biotic) nitrosation (Scheme 1).8 During abiotic nitrosation, the nitrosating agent is the nitrosonium cation NO+, produced from nitrites under acidic conditions. In enzyme-catalyzed nitrosation, nitrite reductase uses NO2- as the nitrosating agent. Nitrite reductases are heme- or copper-containing enzymes that form a metal-nitrosyl complex in the presence of nitrites. This complex reacts with another nitrite molecule, forming the N-N containing nitrous oxide N2O. All of these pathways occur in the bacterial genus Streptomyces, the principal producer of N2NP.9 Furthermore, the NO-synthase enzyme that produces nitric oxide from arginine in mammals was also found in Streptomyces.10 NO-synthase provides nitric oxide NO•, which reacts in vivo with the superoxide ion O2•- to generate peroxynitrite OONO-, a highly nitrosating agent that catalyzes the nitration of tyrosine residues in proteins.11 In Streptomyces turgidiscabies, NO-synthase is involved in the nitration of tryptophan during the biosynthesis of thaxtomin, a toxin produced by Streptomyces.12,13

Scheme 1. The simplest N-N bond is found in N2 and N2 O, formed through abiotic and biotic nitrosations.

2.1. Enzyme catalyzed N-nitrosation followed by N-nitrosoreduction to N-amine intermediates. The biosynthesis of natural nitrosamines streptozotocin,14 analosine,15 and nitroglycine,16 has not yet been elucidated. Streptozotocin biosynthesis was partially investigated using 14Cand 3H-labelled precursors.17 Glucosamine, methionine and citrulline were indisputably involved in streptozotocin biosynthesis, and the nitroso-nitrogen atom derives from nitrous acid as suggested by the authors (Scheme 2). H 3C

S

COOH NH 2 Methionine

OH O HO

OH NH 2 OH

OH Glucosamine

O HO

CH 3

H N

N

O N

O

HNO 2 Nitrous acid

OH OH Streptozotocin O HOOC NH 2

N H

NH 2

Citrulline

Scheme 2. Labelled streptozotocin formed after feeding the producing microorganism with labelled precursors.

Streptozotocin, alanosine and nitroglycine were isolated from Streptomyces species. Streptomyces produces a variety of nitrate reductases, which transform the nitrate into nitrites.18 Under acidic conditions, nitrites (NO2-) lead to the nitrosating agent nitrosonium ion (NO+) through the intermediate formation of nitrous acid HNO2. In the example of alanosine, 2,3diaminopropionic acid produced by Streptomyces,19,20 may undergo a regioselective nitrosation of the β-amine to the nitrosoamine intermediate which could be further hydroxylated to analosine by cytochrome P-450 type enzymes, often encountered in Streptomyces species (Scheme 3).21,22

NH 2 OH NO +

H 2N

O 2,3-diaminopropionic acid

H N

N

NH 2

OH OH Ox N

O

O

O

R1

NH 2

N

OH

O Alanosine

R2

O HN

O H 2N

NO + OH

N

C

H N

NO +

OH

N ON

O

H N

Ox OH

O2N

O

Glycine

N H

O

O OH

R1 R1 R1 R1

N-nitroglycine

C

Red

OH

N H 2N

O

Proline

C

OH

A

O

O HN

OH

N H

NH 2

= CH2OH, R2 = H, Agaritine = CHO, R2 = H, Agaritinal = OH, R2 = H, Xanthodermine = H, R2 = OH, Pygmeine COOH

O

Spinamycin

COOH

COOH

COOH

PABA

O

?

Synthase

N-aminoproline

Scheme 3. Nitrosation followed by nitrosamine oxidation or reduction may explain the formation of N-nitroso, N-nitro and N-amino derivatives.

HO

OH

O

OH Shikimate

Such a situation is commonly encountered in mammals. When proline and nitrites are co-administrated to rats, nitrosoproline was detected in the urine and feces.23 Under standard diet, traces of nitrosoproline were also detected in human urine.24,25 Nnitrosoproline may undergo reduction to N-aminoproline (Scheme 3),26 which interacts with glutamic acid to give linatine isolated together with N-aminoproline from Linum usitatissimum (Scheme 4).27

COOH

OH Chorismate

NH 2 PABA

HN

NH 2

Folic acid (Vit B 9) O H 3C

CH3

O2N

CH 3

CH 3 O

CH 3

O

CH 3

Spectinabilin

NH 2 HO

OH

B O

OH

N H

O

Glutamic acid + O N OH NH 2 N-aminoproline

HO

NH 2

H N

HO O

R

NH 2 H 2N

O

COOH O

+ HN

NH 2

N

O Linatine

GGT

R

NH 2 N H

Scheme 4. Linatine formation through the N-acylation of Naminoproline by glutamic acid.

H N

GGT. Gamma-glutamyl tranferase

COOH O

Scheme 5. Potential biosynthetic route to agaritine-related compounds.

N-nitrosation followed by nitroso-reduction is also suspected in the case of agaritine and agaritinal, hydrazide derivatives produced by the mushroom Agaricus campestris (Scheme 5).28 These compounds were isolated together with their suspected precursor p-hydrazinobenzoic acid. This latter may derive from p-aminobenzoic acid (PABA), involved in the biosynthesis of folic acid, and considered as a starter unit in spectinabilin by Streptomyces spectabilis.29 PABA could be N-nitrosated, and the nitroso-amine derivative reduced to p-hydrazinobenzoic acid (Scheme 5-A). A potential biosynthetic sequence involving the enzyme gamma-glutamyl transferase was recently proposed (Scheme 5-B).30 This hypothetical biosynthetic route also applies to xanthodermin, isolated from the basidiomycete mushroom Agaricus xanthoderma,31 pygmeine isolated from the marine lichen Lichina pygmaea,32 and spinamycin, isolated from Streptomyces albospinus.33

2.2. Enzyme catalyzed N-hydroxylation followed by inter or intramolecular N-N bond formation. Lyophillin is an azoxycarboxamide N2NP produced by the mushroom Lyophyllum connatum.34 Feeding young fruit-bodies with labelled suspected precursors provided evidence for the potential involvement of hydroxylamines (Scheme 6). O

H3C N H3C

O NH OH

13C-hydroxyurea

H3C +

NH Lyophyllum connatum OH

N-13CH3 -hydroxylamine

H3 C

O-

N+ N N CH3 CH3 Lyophyllin

Scheme 6. Lyophillin biosynthesis using labelled precursors.

Valanimycin isolated from Streptomyces viridifaciens,35,36 and kutznerides37 isolated from the actinomycete Kutzneria sp., were the only N2NP to be investigated at the biosynthetic genetic level. In both cases, the N-N bond requires the conversion of a primary amine to the corresponding hydroxylamine. The enzymes responsible for this oxidation are NADP(H) flavin-dependent Nhydroxylating monooxygenases. In valanimycin biosynthesis,

4

VlmH and VlmR catalyzes the oxidation of isobutylamine to isobutylhydroxylamine (Scheme 7).38 The latter reacts with Lseryl-tRNA, leading to O-(L-seryl)isobutylhydroxylamine.39 The intramolecular rearrangement of O-(L-seryl)isobutyl hydroxylamine leading to the N-N bond remains speculative, and the enzymes involved in this step are not yet identified.

derivative was demonstrated for pyrinadine A, an N2NP isolated from the marine sponge Cribochalina sp. (Scheme 9).42 Here again, the aliphatic primary hydroxylamine did not dimerize spontaneously and required a second oxidation step to give the nitroso derivative. 9 N

COOH

HO

NH2 Valine

O tRNA P O O-

COOH NH2 Serine

VlmD CO2

9

OH OH

N O tRNA P O O-

N+ O-

O

9

OH

N

NH

N

OH

O

OH

9 N

OH VlmA

9

NH2

OH

+ -O N N COOH Valanimycin Hydrate

Pyrinadine A

NH2

NH-OH Isobutylhydroxylamine

VlmJ, VlmK

N 9

O

FAD - NADPH

+ -O N N COOH Valanimycin

N

Ade

O

VlmH, VlmR NH2 Isobutylamine

O2 CH 2Cl2, 48h

Ade

O

VlmL, ATP

NH OH

N H

O

OH

N

N

N

N 9

OH

H2O

O

O-(L-seryl)isobutylhydroxylamine

Pyrinadine A

Scheme 9. Biomimetic synthesis of pyrinadine A.

Scheme 7. Valanimycin biosynthetic genes cluster and corresponding enzymes.

In addition to NADP(H) flavin-dependent monooxygenases, cytochrome P-450 oxidases have been reported to catalyze the Nhydroxylation of tyrosine and tryptophan during the biosynthesis of the defense compounds dhurrin and indole glucosinolates (Scheme 10).43 In these cases, oxidation leads to the corresponding oxime intermediates.

Kutznerides are piperazic acid derivatives. The flavindependent N-monooxygenase KtlZ catalyzes the N-hydroxylation of ornithine.40,41 N-hydroxyornithine is not able to spontaneously cyclize into piperazic acid, suggesting an activation step, probably through a second oxidation to the nitroso derivative, which reacts spontaneously or enzymatically with the remaining primary amine (Scheme 8).

COOH

COOH NH 2

HO

NH 2 N H Tryptophane

Tyrosine CYP79A1

CYP79B2 COOH

CYP79B3

HN OH CYP79A1

HO

N

HO

HO

N N OH H Indole-3-acetaldoxime

OH

CYP83B1

CYP79A1

N HO OH p-hydroxphenylacetaldoxime

N H

N OH

Glu O

S Glu

Scheme 8. Biosynthesis of the piperazic acid moiety in kutznerides. N

In a biomimetic synthesis approach, the spontaneous dimerisation of N-hydroxylamine to the corresponding azoxy

HO Dhurrin

5

N

N OSO 3H Glucobrassicin

enzymes involved in N-nitrosation, N-hydroxylation and Nacylation could help the genetic targeting of potential biosynthetic clusters. The organism of choice for these investigations is indisputably the microbial Streptomyces genus, from which 65% of the known N2NP were isolated.

Scheme 10. Implication of cytochrome P-450 enzymes in hydroxylamines formation.

2.3. Enzyme catalyzed N-acylation Enzymatic N-acylation of hydrazines also plays a key role in N2NP biosynthesis and account for the chemodiversity of this class of compounds. Lipases have been investigated for their stereo- and chemo-selectivity, resulting in several applications in the field of biocatalysis.44,45 The acylation of hydrazine precursors by lipases may explain the formation of hydrazides, as illustrated by ibuprofen (Scheme 11).46

3. Sources, properties and biological relevance of naturally occurring hydrazines, hydrazones and hydrazides. The rising awareness and risk assessment around hydrazines, hydrazones and hydrazides, bring regulatory authorities to classify these compounds as potential genotoxic molecules. Specific guidance was issued from the US Food and Drug Administration (FDA) and the EU Committee for Human Medicinal Products (CHMP), especially for the analysis of these compounds in active ingredients and drugs.48 Since the seventies, almost all the N2NP were investigated according to their antibiotic profiles or multi-target toxicity. Thereafter, broad screenings revealed a diversity of relevant biological activities, especially for hydrazines, hydrazones and hydrazides. This section compiled available data on N2NP producing organisms and associated bioactivities (Table 1).

OH O Ibuprofen 1. H + /CH3OH 2. H 2N-NH 2 H N

NH 2

O

3.1. Natural hydrazines

HOOC-C 7H 15 Lipase

Naturally occurring hydrazine derivatives are very scarce, and only four non-acylated compounds were known to date. Three were isolated from plants and the fourth, from a marine mollusk. N-amino-D-proline (1) was isolated from the flax seed Linum usitatissimum.27 In chicks, acute doses of 1 caused convulsions and characteristic symptoms of vitamin B6 deficiency. Moreover, 1 exhibited weak antibacterial activity against Gram-negative bacteria.49 Munroniamide (2), an α,β-seco-tetranortriterpenoid lactam, was isolated from the methanol extract of the whole bodies of the plant Munronia henryi in 2003.50 Compound 2 exhibited moderate antifeedant activity against the larvae of Pieris brassicae L. In 2006, the ethanolic stem bark extract of Aspidosperma nitidum afforded a novel indole alkaloid braznitidumine (3), with an unusual bridged scaffold (1,2,9triazabicyclo-[7.2.1] system).51 The Aspidosperma tree also named carapanaúba, is widely distributed in Amazonas State in Brazil, and the stem bark is traditionally employed in folk medicine as a contraceptive, antimalarial, anti-inflammatory, anticarcinogenic, antidiabetic and antileprosy. In 2006, ostrerine A (4) was isolated from the Quanzhou marine mollusk Ostrea rivularis,52 without highlighting any biological activity.

O

H N

N H

O

C7H15

Scheme 11. Lipase-catalyzed acylation of ibuprofen hydrazine to the corresponding hydrazide.

Recently, a multicomponent synthesis of dihydropyrano[2,3c]pyrazoles was achieved by a lipase from Aspergillus niger (Scheme 12).47 H 2N-NH 2 O O

+ NC

O

CN

CN

Lipase 30 °C, 50 h

O R1

R1 R 2

R2

HN N

O

NH 2

dihydropyrano[2,3-c]pyrazoles

Scheme 12. Lipase-catalyzed one pot synthesis of pyrazole derivatives.

In conclusion, about 200 N2NP were discovered since the sixties, while the formation of the N-N bond in natural compounds remains poorly understood. Identification of the

6

Table 1. Sources, properties and biological relevance of naturally occurring hydrazines, hydrazones and hydrazides. Compounds

Producing organisms

Biological activities or properties

References

Antimicrobial Antifeedant Nr* Nr*

27, 49 50 51 52

Hydrazines

N-amino-D-proline Munroniamide Braznitidumine Osterine A Katorazone Yoropyrazone NG-061 Gyromitrins Leucoagaricone Schaefferals Farylhydrazones Rubroflavin an derivatives Veratrylidenehydrazide Psammaplin G Phosphorus-containing hydrazine Negamycin and derivatives XK-90 FR-900137 Fosfazinomycins Spinamycin Desferrimaduraferrin Elaiomycins and Hydrazidomycins Geralcins Anthglutin Stephanosporin Agaritine and derivatives Xanthodermine and derivatives

Leucorubroflavin Linatine 1-carboxy-2-carbamoylhydrazine N,N’-dicrotonoylhydrazine Montamine Butyrolactam hydrazide Caribbazoins 5-fluorouracilhydrazine Dinohydrazides 11-membered macrocyclic hydrazide * Not reported

Linum usitatissimun Munronia henryi Aspidosperma nitidum Ostrea rivularis Hydrazones Streptomyces sp. IFM 11299 Streptomyces sp. IFM 11307 Penicillium minioluteum Gyromitra esculenta Agaricus xanthoderma Agaricus silvicola and A. arvensis Cordyceps sinensis Calvatia rubroflava, C. craniformis Wedelia biflora Pseudoceratina purpurea Gymnodinium breve Hydrazides Streptomyces purpeofuscus Streoptomyces sp. MK-90 Streptomyces unzenensis Streptomyces lavendofoliae Streptomyces albospinus Actinomadura madurae Streptomyces sp. BK 190, Streptomyces atratus Streptomyces sp. LMA-545 Penicillium oxalicum SANK 10477 Stephanospora caroticolor Agaricus bisporus, A. campestris Agaricus xanthoderma

Anticancer Anticancer Enhancer of NGF (Nerve Growth Effect) Toxic and carcinogenic Chromogen Chromogen Nr* Pigments Nr* DNA methyltransferase inhibitor Ichthyotoxic

58 59 60 61-66 31 67 68 69-71 75, 76 77 78-80

Antimicrobial, proteins biosynthesis inhibitor Antimicrobial Antimicrobial Antifungal Antifungal and anticancer Siderophore Antimicrobial, anticancer, Acetylcholinesterase inhibitor Anticancer, DnaG primase inhibitor Specific γ-glutamyltransferase inhibitor Pigment Carcinogenic Antimicrobial, anticancer, antioxidant

81-89 91, 92 93 94 33, 95 96 97-99

Calvatia rubroflava Linum usitatissimum Butea monosperma Crinum defixum Centaurea montana Schizonepeta multifida Cliona caribboea Phakellia fusca Dinoflagellate Sargassum vachellianum

Nr* Vitamin B6 antagonist, antimicrobial Nr* Antigenotoxic, antioxidant Antioxidant, anticancer Anticancer Hypotensive Nr* Anticancer Nr*

100, 101 107, 108 109 28, 110, 111 31, 32, 115117 69-71 27, 118, 119 120, 121 122 123 124 125 126 127 128

O

O

O

N

N OH

NH 2

O

O O

N NH 2 H

O

1

2 O

O O

O N N

O

O OH

NH

N

N

HO

NH 2

N O NH

N

O N N

N O

O

3 O

7

4 N HO

NH 2

N

OH

N

O

3.2. Natural hydrazones R

Natural and synthetic hydrazones possess a wide spectrum of biological activities including antimicrobial, anticonvulsant, analgesic, anti-inflammatory, antiplatelet, antitubercular and antitumour activities.53,54 In particular, acyl hydrazones with an azomethine proton -NHN=CH-, represent relevant candidates for drug discovery.55 One of the most representative example is the anti-tubercular drug isoniazid found to be less toxic and more efficient than its corresponding hydrazide.56 Hydrazones are also good candidates for metal-based drugs. For illustration, Lonibala et al. synthesized salicylidine-(N-benzoyl)glycylhydrazone complexes and investigated their coordination behavior towards some bivalent metal ions. Generally, the ligands act synergistically with metals to improve biological activity.57

O H N O

HN

N

(CH 2) 3-CH3, (CH 2) 4-CH3, CH=CH-(CH 2) 3-CH3.

OH

O

O

O

O

HO

O

O

N H

H N

OH

O

OH

N OH

O

12

O

13

Gyromitrin (8) was the first natural hydrazine isolated from the false morel Gyromitra esculenta in 1967.61,62 This compound and six higher homologues were identified as toxins in the wild edible mushroom.63 Compound 8 and analogues caused gastrointestinal and neurological disorders, through the reduction of pyridoxine contents in the CNS, the inhibition of GABA synthesis and glutathione depletion in red blood cells.64 At 37°C and under acidic conditions compatible with human gastric pH, gyromitrin hydrolysis leads to aldehyde and Nmethyl-N-formylhydrazine, which further hydrolyzes to the carcinogen N-methylhydrazine derivative.65,66 The fungus genus Agaricus provided several natural hydrazones and hydrazides. Leucoagaricone (9), was isolated in 1985 from Agaricus xanthoderma.31 The compound is easily oxidised to its azine form agaricone, accounting for the intense yellow coloration observed when the fruiting body is damaged. Schaefferal A (10) and B (11) were isolated in 2010 from the methanol extract of the edible mushrooms Agaricus silvicola and Agaricus arvensis.67 These chromogens are responsible for the development of an orange to red color after treating the caps of certain Agaricus species by Schaeffer’s cross-reaction. Discovered in 2011, farylhydrazones B (12) and A (13), were isolated from the cultivation broth of the fungus Isaria farinose, which colonize the caterpillar fungus Cordyceps sinensis.68

O

OH N H

N

R

10 R = H 11 R = OH

O

H N

N

N

O H

OH

5 H2N

N H

N 9

H N

7

OH O

H N

H N

OAc N

H

8 R = CH 3

N

O

N

Gyromitrin natural homologs R = CH 2CH 3, (CH 2) 2-CH3, CH 2-CH-(CH 3) 2,

3.2.1. Hydrazones from bacteria and fungi. Two anticancer hydrazones were recently isolated from Streptomyces species. The first compound was the alkaloid katorazone (5) with a 2-azaquinone-phenylhydrazone structure, isolated from the ethyl acetate extract of Streptomyces sp. IFM 11299, found in a Japanese soil.58 The same group also discovered a naphthopyridazone compound yoropyrazone (6) produced by Streptomyces sp. IFM 11307.59 Compounds 5 and 6 exhibit a synergistic effect in combination with TRAIL (TNF-related apoptosis-inducing ligand) and decreased the viability of human gastric adenocarcinoma cells.

O

N

OH

6

A neural growth stimulator, named NG-061 (7), was isolated in 1999 from the fermentation broth of the fungus Penicillium minioluteum F-4627.60 Compound 7 enhanced the neurotrophic effect of the nerve growth factor (NGF) on neurite outgrowth in the rat pheochromocytoma cell line P12 at doses of 1-10 µg/ml.

H2N O

N

S

O NH

N S

O 14

H2N

S

O NH

O S

O 15

H2N

O

N

NH O

S

S

O 16

The orange natural pigment rubroflavin (14) was isolated in 1987 from the North American puffball Calvatia rubroflava and from Calvatia craniformis in 1997.69,70 Compound 14 was also detected in its colourless form named leucorubroflavin. In 2001, 14 was further isolated along with two novel pigments, deoxyrubroflavin (15) and oxyrubroflavin (16).71

8

3.2.2. Hydrazone from plants. The Thai plant Wedelia biflora is commonly used in folk medicine for headache and fever.72-74 In 1993, veratrylidenehydrazide (17) was isolated from the methylene chloride extract of the dried leaves of W. biflora, and screened as antifungal and antifeedant. Unfortunately, the compound did not exhibit any bioactivity.75,76

O

OH

O

O N H

20

O 17

3.2.3. Hydrazones from marine organisms. Psammaplin G (18), a potent DNA methyltransferase inhibitor, was isolated in 2003 from the sponge Pseudoceratina purpurea, collected in Papua New Guinea.77 The unique characteristics of 18 are the original bisulphide bromotyrosine scaffold with a dense N-functionalization and the presence of sulfur and bromide heteroatoms. The toxic phosphorus-containing hydrazone 19, was isolated in 1982 from the chloroform extract of a culture of the dinoflagellate Gymnodinium breve.78 This organism is responsible for the toxic red tides along the gulf coast of Florida.79 Compound 19 was classified as a highly ichthyotoxic against fish at 0.9 ppm in the case of Lebistes reticulates (skin disorder). The toxicity of the compound to rodents has also been demonstrated.80

NH 2

OH

H N

NH 2

NH 2

O N H

21

O

O

H 2N

N

OH

O N H

N

OH

22

O NH

O

O N H

OH

N

OH

23

Br O O

N

OH

O

NH 2

HO

N

In 1971, leucylnegamycin (21) was isolated from Streptomyces sp. M890-C2 during the early growth phase and was proposed as a biosynthetic precursor of 20.85 Compound 21 was 16 times less active than 20.86 In 1977, 3-epi-deoxynegamycin (22) and leucyl-3-epideoxynegamycin (23) were isolated from Streptomyces goshikiensis MD967-A2. These compounds were previously synthesized as negamycin analogs.87 Compound 22 exhibited a significant antibiotic activity with a very narrow antimicrobial spectrum against Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris and Pseudomonas fluorescens in comparison to (+)-negamycin. Compound 23 showed no inhibition of Proteus vulgaris and Pseudomonas fluorescens.88, 89 In 2014, compounds 22 and 23 were claimed to have more potent readthrough activity against nonsense mutation in eukaryote than (+)-negamycin (20)90.

O

N H

O

H2N

O N

NH2

N H

S 18

S

O

O

H N

NH 2

P

NH

S

N

N

NH 2

The strain Streptomyces sp. MK-90 produced the antibiotic hydrazide XK-90 (24), revealing a broad antibiotic activity against Gram-positive and Gram-negative bacteria.91,92 The original phosphorus-containing hydrazide antibiotic FR-900137 (25) was produced by Streptomyces unzenensis. The antibiotic was detected in the fermentation broth using a 7aminocephalosporanic acid supersensitive mutant of Pseudomonas aeruginosa. Compound 25 was also found to be active against Bacillus subtilis and Escherichia coli.93 Related phosphorus-containing compounds fosfazinomycins A (26) and B (27), were isolated in 1983 from Streptomyces lavendofoliae No.630 as antifungal agents.94 The phenylhydrazide derivative spinamycin (28) was isolated from Streptomyces albospinus in 1966,33 as antifungal and cytotoxic against rat tumor cells.95

N

OH

OH 19

3.3. Natural hydrazide derivatives 3.3.1. Hydrazides from actinomycetes and especially from the Streptmyces genus. Various antibacterial and antifungal natural hydrazides were isolated from Streptomyces species. In 1970, negamycin (20) was isolated from the culture filtrate of three Streptomyces purpeofuscus strains (M890-C2, MA91-M1 and MA104-M1). Compound 20 exhibited a strong inhibition against resistant Gram-negative bacteria, including the genus Pseudomonas.81 In 1972, the structure of 20 was confirmed by total synthesis from D-galacturonic acid.82 Compound 20 was also reported to inhibit protein biosynthesis with miscoding activity.83 In 2012, compound 20 was claimed to be active against Duchenne muscular dystrophy and diseases associated with nonsense mutations.84

9

HO

NH2 N H

O

H N

O

H N

O O P N OH

O

O

24

O

N

N H

25

NH

H2N HN

O

NH2

N H

H N

O N

O

OH O P

O

OH

N H

O

31

O

32

O

33

N

N H

O

28

O

30

O

O HN

O

OH

26

O HO

O P

O

O

NH 2

H N

H N

O

NH 2

N OH

O

N

N H

NH 27

Desferrimaduraferrin (29) was first identified as a peptide iron complex, constitutive of the siderophore maduraferrin. Compound 29 was isolated from the microorganism Actinomadura madurae in 1988.96 OH

O

O N H

N H

H N

O

O N

O

N

OH

O

N

N H

During a multidisciplinary approach to discover novel antibiotics from microorganisms, a new family of natural alkylhydrazides, named geralcins A to E (34-38), was discovered in our laboratory. These original hydrazide derivatives were produced by the bacterial strain Streptomyces sp. LMA-545, isolated from a sample soil collected in the Mare Longue forest (La Réunion Island, France).100,101 Geralcins were isolated from liquid-state and agar-supported fermentation using in-situ Amberlite XAD-16 solid-phase extraction.102 Structure assignment involved extensive 1H, 13C, and 15N NMR spectroscopy, high-resolution mass spectrometry and molecular modelling calculations.100,101 The molecular scaffolds of geralcins C (36) and D (37) are unique. Geralcin C (36) contains two different N-N bonds consisting in an azoxy and a hydrazide groups, and the seven-membered heterocycle of geralcin D (37) has never been described so far, in any natural or synthetic compounds.

H N

N

OH 29

O O

H

In 2011, two alkylhydrazides, elaiomycins B (30) and C (31),97 were isolated from the culture broth of Streptomyces sp. BK 190 together with the azoxy antibiotic elaiomycin.98 The compounds showed moderate antibiotic activity against Staphylococcus lentus DSM 6672 and moderate inhibition of the acetylcholinesterase and phosphodiesterase enzymes. Few month later hydrazidomycin A (32), B (33) and C (31) were isolated from Streptomyces atratus.99 The structure of hydrazidomycin C is identical to 31, and hydrazidomycin B 33 is similar to 30 even if the assignment of the double bond position was debated for this last compound. Hydrazidomycins exhibited moderate to strong cytotoxic activities against a large panel of 42 cancer cell lines, with hydrazidomycin A (32) being the most active compound displaying a mean IC50 of 0.37 µM, followed by hydrazidomycin B (33) with an IC50 of 10.7 µM.

O N

O

O

H N

OH

N

O

H N

O

O

O

O 34

35

O

O N+

N

N

H N

OHO

N O

O

O

O

O

36

OH HO

37

O O N

38

10

N

NH

While geralcins did not exhibit significant antibiotic activity, geralcin B (35) was found to be cytotoxic against MDA231 breast cancer cells with an IC50 of 5 µM, and geralcin C (36) exhibited an IC50 of 0.8 µM against KB and HCT116 cancer cell lines. Furthermore, this compound inhibited the E. coli DnaG primase, a Gram-negative antimicrobial target, with an IC50 of 0.7 mM.100,101 MH-031, an hepatoprotective α,β-unsaturated γ-lactone,103 is part of the structure of geralcins A (34), B (35) and D (37). This compound was also isolated from Streptomyces sp. LMA545 and suspected to be a potential biosynthetic precursor.100 We recently synthetized MH-031 and achieved a bio-inspired total synthesis of geralcin A.104 In order to investigate the biosynthesis of geralcins, the genome of Streptomyces sp. LMA-545 was totally sequenced and assembled. According to the structural similarity between MH-031 and the signaling butyrolactones in Streptomyces,105,106 and the potential involvement of N-oxidases in N2NP compounds, a putative biosynthetic cluster was identified and our current efforts are dedicated to interrupt the butyrolactone synthase AfsA and Nhydroxylase putative genes.

O HO

O

N H

O

O

NH2

H N

OH

CO2H O

39

HO

N H

NH2 41

40

O

O

HO

O

H N

N H

H N

NH2

N H 42

N H

NH 2

H N

OH

43

O O

O

N H

NH2

H N

OH O

O

HO

N H

NH 2

NH2 O

H N

OH

45

O

44

HN

H N

S

S

O

OH 46

Xanthodermine (43) was isolated first in 1985 from the fruitbodies of Agaricus xanthoderma, together with the azine agaricone, and the hydrazides agaritine (41) and leucoagaricone (9) previously described.31 The structure was confirmed by total synthesis.114 In 2010, 43 was isolated from the methanolic extract of the marine lichen Lichina pygmea32 together with two aryl-hydrazide L-glutamic acid derivatives: L-glutamic acid-5-[(2,4-dimethoxyphenyl)hydrazide] (44), and pygmeine (45).32,115 Compound 43 inhibited human (A375) and murine (B16) melanoma cell growth with an IC50 of 1.6 µM. Compound 45 was found 10 times less cytotoxic than 43, suggesting that the p-hydroxyl position is required for the biological activity. The same year, another team published the structure of ramalin, produced by the Antarctic lichen Ramalina terebrata.116 The absolute configuration of the glutamic acid moiety was not assigned, but ramalin and 45 appeared to be identical. Ramalin shows antioxidant properties in vivo and antimicrobial activity against Bacillus subtilis. Ramalin was patented in 2011.117 The hydrazide derivative leucorubroflavin (46) was isolated in 2001 from the North American puffball Calvatia rubroflava together with the previously described hydrazones 14, 15 and 16.69-71 The easy re-oxidation of 46 to 14 explained the color changes from orange to white, shown by the fruiting bodies when bruising.

3.3.2. Hydrazides from filamentous fungi and mushrooms. Anthglutin (39) was isolated from the culture broth of Penicillium oxalicum SANK 10477. The structure of 39 was established as an aryl-hydrazide L-glutamic acid derivative on the basis of classical spectroscopic analysis and chemical degradations.107 Anthglutin inhibited γ-glutamyl transpeptidase; kinetic analysis revealed that the compound did not affect other enzymes related to the metabolism of glutamic acid, such as glutamate dehydrogenase or γ-aminobutyrate transaminase.108 In 2001, the chloronitroarene stephanosporin (40) was isolated from the carrot truffle Stephanospora caroticolor.109 Upon injury of the fruiting body, 40 is transformed into the toxic 2-chloro-4-nitrophenolate, a chemical deterrent against predation.

CO2H

O

OH Cl

NO 2

3.3.3. Hydrazides from plants. Linatine (47) was isolated in 1967 from linseed meal and flax (Linum usitatissimum)27 and exhibited significant antibiotic activity against Azotobacter vinelandii. Compound 47 inhibited the growth of chicks when seedflax is present in the poultry diet. The symptoms of a vitamin B6 deficiency were observed during the growth phase of chicks while no toxic effects were reported in mature poultry.118,119 Compound 48 was isolated from the ethanolic extract of the seed coats of Butea monosperma (Lam.) Kuntze,120 commonly used in Indian ethnopharmacology.121

H N O

OH

In 1961, the toxic compound agaritine (41) was the first naturally occurring phenylhydrazide. 41 was isolated from the commercial mushroom Agaricus bisporus.28,110,111 The compound accounts for the potential health risks after ingestion.112 The aldehyde derivative agaritinal (42) was isolated from Agaricus campestri.28 The concomitant production of 41 and 42 in Agaricus species was confirmed later and their postulated biosynthesis from shikimatechorismate pathway investigated.28,113

11

O NH2

N

HO

NH O

OH

H 3C H N

O 47

O

H N

O

O

growth of human endothelial and cancer cells (HL60 leukaemia and B16 melanoma).127 The original 11-membered macrocyclic hydrazide (57) was isolated from the algae Sargassum vachellianum collected in the South China Sea in 1999.128

NH2 N H

O

O

48

N H

O

HN

49

HO

O

O HN

O

HN

O

O

O

OMe

O NH N HO

H3C

O

O

N

OH

NH

N

H2N

NH

HN OMe

O

O

H N

N

H N NH

O

51

53

O

52

O

O N H

55

O 54

OH

N

N

O

F

50

HN H N

A bioguided fractionation of the wild garlic bulbs extract (Crinum defixum) resulted in 2009 in the isolation of an aminohydrazide derivative N,N’-dicrotonoyl-hydrazine (49). Compound 49 was assayed for anti-genotoxic and chromosomal alteration (Allium test). Compound 49 showed an anti-oxidant protective effect against H2O2 genotoxicity.122 In 2006, the methanol extract of the seeds of Centaurea montana produced a unique dimeric indole alkaloid with an N,N’-diacylhydrazide moiety. The structure of montamine (50) was established unequivocally by UV, IR, MS and a series of 1D- and 2D NMR analyses.123 The antioxidant properties of 50 were investigated by the DPPH assay (free radical scavenging activity). Its toxicity towards brine shrimp and cytotoxicity against Caco2 colon cancer cells were evaluated by brine shrimp lethality and the MTT cytotoxicity assays, respectively. Compound 50 revealed significant in vitro anti-colon cancer activity (IC50 43.9 µM). In comparison, the monomeric form moschamine, also isolated from Centaurea montana, showed an IC50 of 81.0 µM when assayed in the same conditions. The hydrazine containing butyrolactam (51) was isolated from the Chinese medicinal plant Schizonepeta multifida (L.) Briq. The structure was unambiguously assigned by X-ray single-crystal diffractometry. Compound 51 was identified as 3-imino-N-(α-imidoethylamino)butyrolactam.124 Compound 51 possessed in vitro antitumour activity against liver tumour cells (SMMC-7721).

O

NH

O N H O

56

N H

N H

O

57

4. Synthesis of naturally occurring hydrazines, hydrazones and hydrazides. 4.1. General procedures This section gives a global view of the strategies applied in the synthesis of natural hydrazines, hydrazones and hydrazides (Fig. 1). These strategies can be summarized in four options: substitution of hydrazines, diazotation followed by diazonium reduction, N-amination and reduction of hydrazones and hydrazides. R1 N

N

R3

O

R2 R1

N

N

R1 N R4

N R4 R2 R3 Hydrazines Hydrazides Hydrazones R1, R 2, R 3, R 4 = H, alkyl, aryl R3

R4

R2

Figure 1. General structural motifs of hydrazines, hydrazides and hydrazones.

Hydrazine H2N-NH2 is a highly reactive base with reducing properties used for more than a century in organic synthesis.129,130 Hydrazine has been used for the production of several drugs such as nifuroxazide, carbidopa, hydralazine, dihydralazine, isoniazid and iproniazid. Hydrazine itself, as its sulphate salt, has been used in the treatment of tuberculosis, sickle anaemia and various chronic diseases.131 Moreover, the widespread use of hydrazine derivatives as precursors for heterocycles and peptidomimetics has led to the development of various specific methodologies. However, despite all efforts in this area, only few general methods have been described so far.5

3.3.4. Hydrazides from marine organisms. In 1990, two novel hydrazides caribbazoins A (52) and B (53) were isolated from the marine sponge Cliona caribboea collected in the Caribbean Sea.125 Because of the limited quantities isolated from the marine sponge, excluding intensive investigations, the authors admitted doubt about the natural occurrence of caribbazoins, which might have originated from pollutants. In the first biological investigations, these compounds were found to possess mild hypotensive activity in rats. In 2003, 5-fluorouracil derivatives were isolated from the marine sponge Phakellia fusca collected around Yongxing Island of the Xisha Islands in the South Sea of China. Among these compounds, the authors isolated a 5fluorouracilhydrazine (54).126 In 2010, dinohydrazides A (55) and B (56) were isolated from a symbiotic marine dinoflagellate of the marine sponge Xeospongia sp. Compounds 55 and 56 moderately inhibited the

4.1.1. Substitution of hydrazine and C-N bond formation. The direct substitution of free hydrazine with halides RX is difficult to control and produces mixtures, containing overalkylated products. An excess of hydrazine is generally applied to reduce the formation of over-substituted derivatives, but yields did not exceed 40%. Steric hindrance of R can also

12

promote the mono-subtitution. The reaction of free hydrazine with electron-deficient alkenes such as acrylamide, various acrylonitriles and styrene, is easier to control.132 A series of reagents were evaluated for hydrazine alkylation. Among them dialkyl sulfates, sulfonates, alcohols in the presence of phosphoric acid and hydrogen halides, oxiranes and aziridines. In most cases, yields were modest including fastidious purification procedures. To avoid the over-alkylation of free hydrazine, protected derivatives can be employed as starting material. This method generally involved more synthetic steps, but as nowadays various convenient protecting groups for amino functions are available, the total yields are higher and the purification steps easier to perform.5 Recently, efforts have been dedicated to develop selective alkylation reactions of hydrazine through polyanions formation. This method offers fast and convenient access to multi-alkylated derivatives (Scheme 13),133,134 even if the method turns inefficient when the substituent R is a long aliphatic chain.

aromatic hydrazines. 1,1-disubstituted hydrazines, with both aliphatic and aromatic substituents, were obtained by nitrosation/reduction of the corresponding secondary amines (Scheme 15). 137 Diazotation

ArNH2

R1 NH

Nitrosation

Reduction

ArN2

R1 N

Boc

3 equiv BuLi Boc

HN

NH 2

Li N

N

Li Boc HN NH 2

2 equiv BuLi

Li

Boc

H N

Li

N Li

iv R qu 4e

2 eq uiv

X

Scheme 15

4.1.4. N-amination of primary amines. In analogy with the industrial Raschig synthesis of hydrazine, chloramine and primary amines are allowed to react to give alkylhydrazine derivatives (Scheme 16). Hydroxylamine-Osulfonic acid could replace chloramine to unsure mild reaction condition.138 RNHNH2

RNH2 + NH2O-SO 3H

N N R

Boc

R

X vR qui e 2 rt 1 eq uiv R -50° X C

N

4.1.5. Reduction of hydrazones and hydrazides. The reduction of the C=N bond of hydrazones by catalytic hydrogenation leads to the corresponding hydrazines (Scheme 17). The distal nitrogen is generally protected to stabilize the N-N bond against cleavage. Other mild reducing agents like NaBH4 and NaBH3CN can be used. Alternatively, the reduction of hydrazides with LiAlH4 provides the corresponding alkylhydrazine derivatives.5

N

H

R

Boc

R N

H

H

Scheme 13. Alkylation of hydrazine derivatives using polyanions

R1

4.1.2. Condensation of hydrazines with activated carbonyl compounds. Hydrazides can be obtained from the reaction between hydrazines and active carbonyl compounds. Direct condensation of hydrazines with carboxylic acids remains inefficient and requires activation of the carboxyl group. The standard method to prepare carboxylic acid hydrazides is hydrazinolysis of esters in alcoholic solutions. Carboxyl group can also be activated by using halogenating or peptide coupling reagents. A stronger activation, employing the acid halide technique, is frequently recommended when the starting material is particularly hindered.135,136 Aldehyde or ketone are allowed to react with hydrazine derivatives to afford the corresponding hydrazones.

R1

R2 OH

H2N N R3

O R1

R3 R2

H2N N R4

N R2

Acid activation R1

NH2

HN

H2 / cat

R2

NH HN

O

R1 H2 / cat R2

R3

NH NH 2

O R

N H

NH2

LiAlH 4/AlCl3 R

N H

NH 2

Scheme 17

4.2. Application to the synthesis of natural hydrazines, hydrazones and hydrazides.

NH N R2

Only a few natural hydrazines, hydrazones and hydrazides were synthetized. Among the four natural hydrazines, Naminoproline 1 was easily synthetized27 and used to investigate peptide conformation.139 To pursue synthetic and biological investigation, 8 natural hydrazones and 8 hydrazides were synthetized together with their analogs.

R2 N R1

O R3

R3 Condensation

NH R2

R1 N

R2

R1

H2 / cat NH 2

R1

O Coupling reaction

RNHNH2

Scheme 16 RX

N

O

N NH 2 R2

R2

R2

R

R

R1

Reduction

NO

RNH2 + NH2Cl Boc

ArNHNH2

N R3 R4

Scheme 14. General methods for condensation of hydrazines with carboxylic acids

4.2.1. Syntheses of natural hydrazones. The natural hydrazone NG-061 (7) and its derivatives were synthesized to evaluate their biological activity as NGF enhancers. The synthesis of 7 was straightforward and achieved in a single step from 2-methoxy-1,4-benzoquinone and phenylacetylhydrazine. After recrystallization, 7 was obtained in 12% yield (Scheme 18).140

4.1.3. Diazotation/diazonium reduction or nitrosation /nitrosamine reduction. The first synthetized hydrazine was phenylhydrazine.137 The synthesis was based on aniline diazotation and direct reduction of the corresponding diazonium salt by sulphite salts or other reducing agents. As aromatic amines are easily available, this simple method remains a convenient way to mono-substituted

13

eq), NH4Cl (10.0 eq), acetone/water (4:1), 40 °C, 12 h, 74%; (c) NaNO2 (0.90 eq), SnCl2.2H2O (1.8 eq), HCl, H2O, 0 °C, 1 h; (d) Pyruvic acid (1.7 eq) HCl, H2O, rt, 45 min, 46% (two steps), (e) NaOH (0.5 N), H2O/MeOH (1:1), reflux, 1 h, 99%; (f) Gly-OMe.HCl (1.0 eq), DMAP (2.0 eq), EDC.HCl (1.0 eq), CH2Cl2, 0 °C to rt, 16 h, 52%; (g) NaOH (0.5 eq), H2O/MeOH (1:1), reflux, 1 h, 70%.

O

O H N

O

H N

O

NH2

O

O

N

O

A similar approach was applied for the synthesis of deoxyrubroflavin 15, except for the final step in which the semicarbazone moiety derives from the diazo cyanide group (Scheme 21).142 Deoxyrubroflavin 15 in hand, rubroflavin 14 was synthetized through an enantioselective sulfoxidation based on Kagan’s reaction.142

7

Scheme 18. One-step synthesis of NG-061 (7). Reagents and conditions. CHCl3, rt, 48 h, 12%.

Schaefferals A 10 and B 11 were synthetized according to the general procedure involving the alkylation of a hydrazine precursor (Scheme 19).67 H N

OCH 2C6H5

a

N

O

Cl

R

10 R = H 11 R = OH

H

OCH2C6H5

OH

b Cl

Cl

Cl

H3CS NO2 OCH 2C6H5

OCH2C6H5

CH2NH2

CH2NHCOCF 3

CH 2NHCOCF 3

b

e

d

c

a

c

H3CS

H 3CS

SCH3

NH2

N

NH

e

N

H

CN

CH 2NH 2

d

Scheme 21. Synthesis of deoxyrubroflavin (15). Reagents and conditions. (a) C6H5CH2Cl, aq. NaOH, EtOH, 86%; (b) CH3SNa, acetone, 84%; (c) Sn, aq. HCl, MeOH, 90%; (d) NaNO2, HCl, H2O, 0 °C, then Na2CO3, KCN, 95%; (e) TiCl4, AcOH, H2O, 73%.

10 or 11

NH

4.2.2. Synthesis of natural hydrazides. Abundant literature concerning the synthesis of the hydrazide negamycin 20 is available for racemic and optically active forms.143,144 Recent strategies for the synthesis of 20, employed the conjugate addition of an enantiopure lithium amide to an α,β-unsaturated ester (Scheme 22).145, 146

H

R

R

Scheme 19 Synthesis of schaefferals A (10) and B (11). Reagents and conditions. (a) F3CCO2Me, MeOH, rt, 1 h, 96%; (b) NaNO2, HCl, -5 °C, 30 min, then SnCl.2H2O, 0 °C, 54%; (c) 4RC6H4CHO, acetate buffer pH 5, overnight, 70%; (d) Ba(OH)2, aq MeOH, rt, (e) isonicotinaldehyde, DBU, DMF, rt, overnight, then aq KHSO4, 55% (10), 33% (11), 36% (nitro analogue).

Ph CO2CHEt 2 Ph

O Cl

NO 2

a-c

OH O H N

N O

H N

e

Ph

N

O

NH2

O

H2N 20

OH

CO 2Bn

O

OH O

N

Boc

e,f

OH

N H

N

13

H N

O

c or d

Boc

f-g H N

N

O

O

O

O

Ph CO 2H

N

OH

OH

Ph

N

OH

N

Boc

Ph

b

O

O N

Boc

O

O

CO2CHEt2

O N

d

NH 2

N

a

CO2Et

A short, scalable and convergent total synthesis of farylhydrazones A (12) and B (13) was reported in 2012 (Scheme 20).141 Compound 13 was obtained in five steps from o-nitrobenzoic acid with a 30% overall yield, and 12 was synthetized from 13 methyl ester in six steps with 11% yield. H N

N

N

HNNH 2.HCl

CH 2NHCOCF3

15

SCH3

NH3+Cl-

NH2

SCH3

NO2

NO2

O N H

N

OH

Scheme 22 An efficient synthesis of negamycin (20). Reagents and conditions. (a) lithium (R)-N-benzyl-N-(α-methylbenzyl) amide, THF, 78 °C 1 h, then NH4Cl (sat, aq), 85%; (b) LiOH, MeOH/THF/H2 O (3:1:1), reflux, 24 h; (c) benzyl [N(1)-methylhydrazino]acetate, DCC, HOBt, Et3N, THF, 0 °C to rt, 5 h, 79%; (d) Et3N, ClCO2Et, CH2Cl2, -15

O 12

Scheme 20. Total synthesis of farylhydrazones 12 and 13. Reagents and conditions. (a) H2SO4, MeOH, 65 °C, 5 days, 90%; (b) Zn dust (5.0

14

°C, 5 min, then 0 °C, 30 min, 57%; (e) TFA, THF, H2 O (1:1), rt, 16 h; (f) H2 (5 atm), Pd(OH)2/C, AcOH (five drops), MeOH, rt, 22 h, 71%.

O N H

Stephanosporin 40 (Scheme 23)109 agaritine 41 (Scheme 24)28 and linatine 47 (Scheme 25)27 were also synthetized by condensing an activated carbonyl group with a hydrazine precursor. O N2

Cl

N OH

OBz

NH 2

Proline

N3 BzO

OH

d O O Glutamic acyl azide

OH

H N

+H N 3

a

O

a,b,c

Cl

Cl-

O NH 2

NO2

NO2

N

HO

OH

NH

b O O

N H

OH

40

H N

O

OH Cl

O COOH O

Ph

O

47

Upon the discovering of xanthodermine 43 and other glutamic acid hydrazines 44 and 45, Roullier et al. developed in 2010 an efficient synthesis by coupling L-glutamic acid with phenylhydrazine moieties.32 The peptide coupling reagent TBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3tetramethyluroniumtetrafluoroborate) was used in the presence of iPr2NEt for the carboxyl activation. Arylhydrazide analogues were obtained with an overall yield of 48-66% (Scheme 26).

NO 2

Scheme 23. Synthesis of stephanosporin (40). (a) SnCl2, HCl, HOAc, 0 °C, 77%; (b) succinic anhydride, Et3N, MeOH, 25 °C, 70%.

Ph

O

Scheme 25. Biomimetic synthesis of linatine (47). (a) NaNO2/H2SO4, (b) Zn/CH3COOH, (c) BzCl/Pyridine, (d) CHCl3, -5 °C.

NH O a, b

BnO

O Ph

O

Ph

O

O

BnO

a

NH 2

N 2+

H N N H

NH

b

NH 2

BnO

NH

COOH

c

.HCl

O O Ph

O

Ph

O

O

H N

HN

c

N H

NH

HO 2C

d O

O

HO

N H

NH2

CO 2Bn BnO

OH

O

CO 2Bn

HN

H N

N H

H N

CO 2Bn CO 2Bn

O d

41

HO

OH

Scheme 24. Synthesis of agaritine (41). Reagents and conditions. (a) EtOCOCl, Et3N, THF, -10 °C then -5 °C, 30 min; (b) 4carboxyphenylhydrazine, THF/Et3N/H2O, -10 °C to rt, overnight, 60%; (c) BH3/THF, -10 °C, 3 h, 48%; (d) H2, Pd/C, THF, rt, 24 h, 88%.

N H

NH 2

H N

CO 2H O

Scheme 26. Synthesis of aryl-hydrazide L-glutamic acid derivatives. Reagents and conditions. (a) NaNO2, 6M HCl, H2 O, 0 °C; (b) SnCl2, 6M HCl, 0 °C, 69%; (c) TBTU, iPr2NEt, DMF, rt, 60-75%; (d) H2, Pd/C, MeOH, rt, 75-95%.

The synthesis of caribbazoins A (52) started from 2-acetyl1,3-indanedione. Reaction with ethyl carbazate through a Schiff base addition gives the enolic form of the corresponding hydrazone with a good yield. The generated intermediate was acylated with acetic anhydride to produce the desired product (Scheme 27).125

15

O

The same year, another strategy has been developed by Meyer et al. for compound 32 (Scheme 29).150 The authors used a versatile reaction sequence involving a metal-catalyzed Zselective N-alkenylation. Inspired from the synthesis of E- and Z-configured enamides, the key step of the synthesis was the ruthenium-catalyzed hydroamination of tetradec-1-yne and the hydrazide-protected phthalimide, predominantly yielding the Zconfigured enehydrazide. The final N-acylation of the deprotected compound with methoxyacetic acid was performed using the peptide coupling system DCC/DMAP to afford the naturally occurring enehydrazide derivative 32.

O O a HN

O

NH O

O

O

O

O b HN N O

O O

52

Scheme 27. Synthesis of caribbazoin A (52). Reagents and conditions. (a) ethyl carbazate (1.0 eq), EtOH, reflux, 30 min, 90%; (b) acetic anhydride (excess), H2SO4, toluene, 100 °C, 30 h, 64%.

O

a, b

O

TMS

HN

d C12H 25

TMS

d

N

Boc

OH

TMS

f

N

O O

N H

O

H2N

N

C 9H19

O e

O

C12H25 N H

N

C9H19 O

The last reported N2NP are geralcins isolated in our laboratory.100,101 The total synthesis of geralcin A 34 was recently achieved, consisting of the condensation of the α,βunsaturated γ-lactone moiety MH-031 with the N-substituted alkylhydrazine (Scheme 30).104 The strategy involves concomitant electronic and steric effects to allow a full reactivity differentiation between the two N-atoms of the hydrazide. Our current efforts are dedicated to the synthesis of the highly challenging geralcins 35, 36 and 37.

e

C12H 25

C12H 25 N

C 9H19

Scheme 29. Synthesis of hydrazidomycin A (32) (Meyer et al. approach). Reagents and conditions. (a) NH2NH2.H2 O, EtOH, 24 h, reflux, 83%; (b) phtalic anhydride, toluene, 3 h, reflux with Dean-Stark apparatus, 82% ; (c) (cod)Ru(met)2 (cod = 1,5-cyclooctadiene, met = 2methallyl), 1,4-bis(dicyclohexylphosphanyl)butane, Yb(OTf)3, DMF, 24 h, 60 °C, 11%; (d) MeNHNH2, THF, 24 h, 0 °C to rt, 68%; (e) MeOCH2COOH, 4-(dimethylamino)pyridine (DMAP), N,N'dicyclohexylcarbodiimide (DCC), CH2Cl2, 2 h, rt, 81%.

C12H 25 g, h

N

c

Hydrazidomycin A (32)

C 9H 19

HN

C 12H 25 N O

O

O C 9H19

b

O

C 12H25

C12H 25

O Boc HN

OH

C9H19

E/Z = 1/5.5

C12H 25 NH

H N

C9H19 O

O

Boc O

H2N

O

H N

N

c TMS

a

O

The most recently isolated compounds were the trisubstituted hydrazides, elaiomycins 30-31, hydrazidomycins 32-33, and geralcins 34-38. Synthesis of trisubtituted hydrazide derivatives is strongly challenging and requires controlled and selective reactions to avoid multiple alkylations or acylations. Another main synthetic challenge is the central cis-enehydrazide subunit. In comparison to the structurally similar and wellstudied enamides,147 there are very few examples concerning enehydrazide synthesis148 and only two publications were dedicated to stereo-controlled cis-enehydrazide syntheses of 32.149,150 In 2013, Ramsay et al. developed a Lewis acid catalyzed Boc-carbazate silyl epoxide ring-opening followed by a Peterson elimination. This innovative methodology involving a highly stereoselective formation of Z-enehydrazides was successfully applied for the first synthesis of 32 (16% overall yield in 8 steps, Scheme 28).149 Functionalization of the hydrazide core was performed by condensing the desired alkylacid chloride with the Boc-carbazate silyl epoxide before undergoing the Peterson elimination under mild conditions. This strategy allowed the authors to complete the syntheses of 30 and 33 with 6.7% and 7.3% overall yields respectively, in 11 steps. C12H 25

C9H 19

C 9H19 O

Hydrazidomycin A (32)

Scheme 28 Synthesis of hydrazidomycin A (32) (Beveridge et al. approach). Reagents and conditions. (a) n-Buli, Me3SiCl; (b) DIBAL-H, 53% > 15:1 Z:E (1H-NMR); (c) mCPBA, 85%; (d) Boccarbazate (4.0 equiv.), BF3.OEt2(10 mol %), THF, 45 °C, 16 h, 75%; (e) C9H19C(O)Cl, , Et3N, CH2Cl2, rt, 18 h, 88%; (f) t-BuOK, THF, 45 °C, 16 h; (g) KHMDS, MeOCH2C(O)Cl, THF, -78 °C to rt, 1 h; then (h) Mg(ClO4)2 (10 mol %), MeCN, 55 °C, 16 h, 53%.

16

SH

O

a

+

Ph

S

O

O

1. 2. 3. 4.

O b

O Ph

S

O

c

O

Ph

S

OH

5. 6.

O

d O

O

S

Ph

O

O

+ H 2N

7.

Boc NH

g

e

8.

O

O

N O

O

N H

9.

Boc

10.

h

f

H N

O

O

OH

O

N H

Boc

11. 12.

i

13. O

O N

O

H N

Boc

14. 15.

j O O

16.

O N

17.

H N

OH

18.

O

19. Geralcin A (34)

20. 21.

Scheme 30. Total synthesis of geralcin A (34). Reagents and conditions. (a) H2O, rt, 15 min, 90%; (b) LiAlH4, THF, rt, 30 min, 100%; (c) ClCO2Me, Pyridine, 1 h, 0 °C then NaIO4, MeOH, H2O, 24 h, 4 °C, 83%; (d) LDA, THF, 12 h, -78 to 0 °C, 65%; (e) NaH cat, THF,15 min, then methylacrylate, 50 °C, 3 h, 68%; (f) LiOH, THFH2 O, rt, 3 h, 100%; (g) Heptane reflux, 20 min, 92%; (h) BH3.THF, rt, 10 min, 45%; (i) (COCl)2, DMF, CH2Cl2, rt, 1 h, then pyridine, CH2Cl2, rt, 5 h, 72%; (j) HCl, CH2Cl2, rt, 1 h then DCC, HOAt, DMF, 30 min, then, HOCH2COOH, rt, 24 h, 64%.

22. 23. 24.

25.

5. Conclusion

26.

Besides the chemical diversity and the relevant biological activities of N2NP, the discovery of the enzymes involved in their biosynthesis, and especially those involved in N-N bond formation, represents the future challenge. N-acylases and Nhydroxylases catalyzing Nitrogen-Carbon and NitrogenOxygen bonds respectively are of extreme interest for fundamental and applied biochemistry and highly desirable in biocatalysis. Nitrogen-Nitrogen bond forming enzymes are puzzling and still keep their secret hidden after 63 years since the discovery of the first N2NP, macrozamin.151 Despite sustained efforts, molecules such as geralcins C and D isolated in our laboratory, still remain defiant to total synthesis and further synthetic and structural determination work remains necessary.

27. 28. 29. 30. 31. 32.

33. 34. 35.

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