Thiocarbohydrazides: Synthesis and Reactions

Thiocarbohydrazides are an important class of compounds which possess applications in many fields. The chemistry of thiocarbohydrazides has gained increased interest in both synthetic organic chemistry and biological fields and has considerable value in many useful applications such as the assessment process of the three-dimensional ultrastructure examination techniques of interphase nuclei and tissues, besides their therapeutic importance. They are also described for use as fogging agents and are considered as safe, storable, and cool-burning pyrotechnic compounds for dissemination of smoke, chemical warfare agents. On the other hand, thiocarbohydrazides are used in performing a highly selective heavy metal ion adsorbent and as complexing agents for the solvent extraction separation methods. The present review covers the literature up to date for the synthesis, reactions and applications of such compounds.


Introduction
Study of thiocarbohydrazide and its derivatives is of great interest due to the wide use of these compounds and their valuable reactions. J. P. Thiery (1967) invented a technique for staining of cells involved in the assessment process of the ultrastructural cytochemical reactivity by scanning electron microscopy. This technique depends on periodic acid-thiocarbohydrazide-silver proteinate compound (TCH), where coating biological specimens of soft tissues with thiocarbohydrazide technique, the individual cells are clearly defined, surface contours are uniformly coated, intercellular borders are visible, cell-surface shape is outlined, and area contours are suitable for planimetry at higher magnification 1 , the carbohydrate content of atrial-specific granules of muscle cells 2 , intracellular glycoproteins 3 , ultrathin sections of Araldite-embedded atriums and ventricles were also stained in the assessment process of the ultra-structural cytochemical reactivity, renin activity, and cathepsin D activity of atriums and ventricles of the bullfrog 4,5 . The above technique was especially useful in the study of the roundish bodies and in the compositional and structural comparison of the siphon wall with the wound wall6. Also this method was applied for vicinal glycol group detection to the study of gastric mucosa endocrine cells e.g; cat, rabbit and mouse gastric mucosa endocrine cells 7 and the surface membrane of mouse myocardial cells 8 .
Polysaccharides associated with the cellular surface of a Gregarine (parasitic protozoan) were studied by periodic acid-thiocarbohydrazide-Ag proteinate cytochemical characterization and biochemical analysis of an isolated ghost fraction 9 . Conjugation of thiocarbohydrazide with a 40,000 Dalton dextran gave thiocarboxydrazyl-dextran, which is stable for a long time and suitable for electron microscopic demonstration of lectin receptors 10 . An ultrastructural analysis of Malus pumila was performed by examination of the adaxial leaf cuticle by electron microscopy to determine the transcuticular movement of foliarly applied chemicals, where an aqueous solution of uranyl acetate, AgNO 3 , and PhHgOAc was applied to the cuticle surface of leaf segments floated on solutions of phosphate salts or thiocarbohydrazide (TCH) which reacted within the cuticle to form insoluble electron-opaque deposits indicative of their pathways of transcuticular movement 11 . Also, ultrastructural cytochemistry of complex carbohydrates in lysosomal granules of human neutrophils, rabbit heterophils, rabbit and human eosinophils and basophils, and human monocytes was performed 12 . The carbohydrate content of human auricular specific granules was assessed by means of the periodic acid-thiocarbohydrazide-silver proteinate technique 13 . Methods for visualization of complex carbohydrates ultra-structurally were employed to study specific organelles of the rat bone marrow and peripheral blood monocyte and alveolar and peritoneal macrophages [14][15][16] . A thiocarbohydrazide-Ag-proteinate (TCH-SP) sequence was applied to thin sections of specimens that had been reacted with the high Fe diamine (HID) method for ultra-structural localization of sulfated complex carbohydrates 17 . Ultra-structural antibody localization of -macroglobulin in mem-brane-limited vesicles in cultured cells was performed by using post-sectioning staining with the osmium/ thiocarbohydrazide/osmium and uranyl acetate/lead citrate procedures 18 . Staining the outer hair cells of the guinea pig sections with the HIO 4 -thiocarbohydrazide-OsO 4 (PATCO) to study the effects of fixation and processing upon the morphololgical appearance of glycogen within these cells resulted in the appearance of distinct, coarse granules in the cytoplasm of the outer hair cells 19 . Ultra-structure of the cell wall of Candida albicans blastospores was studied, where thin sections of C. albicans blastospores were treated by the periodic acid-thiocarbohydrazide-silver proteinate method and the electron microscopic observations were correlated with literature data on the polysaccharide composition of C. albicans cell walls to give a model of the wall 20 . The HIO 4 (PA) /thiocarbohydrazide (TCH) /Ag proteinate (SP) staining method for electron microscopy of plant materials produced an intensified, finely-granular product over starch grains, walls, dictyosomes, and their secretion products 21 . Occurrence and biosynthesis of very long chain fatty acids and alkanes in plasmalemma enriched fractions from Saccharomyces cerevisiae was studied, where electron microscopic identification of plasmalemma is best achieved with a periodic acid/thiocarbohydrazide/Ag proteinate stain 22 . The ligand Os binding technique with thiocarbohydrazide is a successful alternative to evaporation or sputter coating of botanical specimens 23 . Studies of the chemistry of the positive contrast reaction of glycogen in Epon-thin sections with tungstophosphoric acid (TPA) treated with thicarbohydrazide were performed 24 . The glycogen of megakaryocytes and blood platelets was investigated in glutaraldehyde-and OsO 4 -fixed tissues by the HIO 4 -thiocarbohydrazide-Ag proteinate method (PA-TCH-SP) 25 . Enzymic activities and galactomannan mobilization in germinating seeds of fenugreek (Trigonella foenum-graecum L. Leguminosae) was studied by electron microscopy using periodate-thiocarbohydrazide-silver proteinate technique 26 . Mature and differentiating sieve tubes were examined cytochemically in Robinia pseudoacacia, Acer pseudoplatanus, and Dianthus caryophyllus by the thiocarbohydrazide-silver proteinate method 27 . The organization of the growing cell wall of Parenchyma, sieve cells, and collenchyma 28 and the filamentous structures of sieve elements of Mymphoides peltata petioles contained no polysaccharide components were demonstrated by this technique 29 . Demonstration of cholinesterases by the formation of osmium blacks at the sites of Hatchett's brown (Cu 2 Fe(CN) 6 .7H 2 O), which deposited at the sites of cholinesterase activity in tissues by the procedure of M. J. Karnovsky and L. Roots (1964), could be enhanced by bridging to Os through thiocarbohydrazide (TCH) 30 .
Reaction of thiocarbohydrazide with aromatic isothiocyanates afforded thiocarbobis(thiosemicarbazides). These compounds and their Cu and Ni chelates were found to have active biological effect upon plant protection against fungi 31 .
Thiocarbohydrazide as aldehyde blocking agent, block transformation of human peripheral lymphocytes by galactose oxidase 32,33 . NaBH 4 was shown to be an effective blocking reagent of aldehyde produced by HIO 4 oxidation of polysaccharides in thin sections of glutaraldehyde-Os fixed epoxy-embedded tissue prepared for thiocarbohydrazide-Ag proteinate staining in electron microscopy studies 34 . Aldehyde reagents in the presence of heat caused rapid degradation of apurinic acid to soluble fragments. The histochemical demonstration was carried out in case of stabilizing of apurinic by reaction with thiocarbohydrazide, where thiocarbohydrazone derivatives of apurinic acid were resistant to depolymerization during incubation in both Gomori's methenamine Ag solution and Protargol and were visualized with Ag solution 35 .
The thiocarbohydrazones RR 1 C:NNHC(S)NHNC:R 2 R 3 (R, R 1 , R 2 and R 3 = H, alkyl or aryl) are fungicides for textile fabrics, where prevented the growth of Aspergillus niger and Chaetomium globsum on cotton 36 . Thiocarbohydrazide (TCH) besides the other sulfur-containing hydrazides such as methyldithiocarbazinate (MDTC), and thiosemicarbazide (TSC) have convulsant action, where the 3 drugs inhibited pyridoxal phosphokinase in vitro. Long-latency convulsions after MDTC are comparable to those seen after TSC 37 .
In the endothelial cells lining the rat splenic blood vessels, neutral carbohydrates were studied by means of combined periodic acid-thiocarbohydrazide-silver protein (PA-TCH-SP) and -amylase digestion methods 38 . A variety of sialic acids contained in the rat epididymis were histochemical examined, where hydrated sections were subjected either to the lectin methods using biotinylated Limax flavus, Sambucus nigra, Sambucus sieboldiana or Maackia amurensis lectins or to the selective periodate oxidn.-phenylhydrazine-thiocarbohydrazide-silver protein-physical development technique with or without sapon 39,40 . Epididymides from male Sprague-Dawley rats on post-natal days 14, 21, 30, 39, 49, 56 and 70 were fixed in Bouin's fluid and embedded routinely in paraffin wax and the hydrated sections were subjected to the selective periodate oxidn.-phenylhydrazine-thiocarbohydrazide-silver protein-physical deve lopment technique with or without sapon 41 .
Thiocarbohydrazide showed structural reinforcement effect in the preparation of rabbit knee articular cartilage for the scanning electron microscope by enhancing the binding of osmium tetroxide to it, possibly along with that of other soluble tissue constituents 42 .The ontogeny of pepsinogen C-producing cells in rat fundic glands was studied, where the maturation stages of the chief cells could be traced by electron microscopy along the longitudinal axis of the rat fundic gland by double-staining with anti-pepsinogen C antibody and HIO 4 -thiocarbohydrazide-Ag proteinate 43 .The ontogeny of sulfated glycoconjugate-producing cells in the rat fundic gland has been also studied using HID-thiocarbohydrazide (TCH)-silver proteinate (SP)-physical development (PD) technique under electron microscopy 44 . Sequential secretory changes in granular tubule cells caused by the secretagogue cyclocytidine were studied at the ultrastructural level using the periodic acid-thiocarbohydrazide-silver proteinate technique (PA-TCH-SP) 45 . An electron microscopic analysis with specific histochemical stainings for acidic glycocon-jugates was carried out to examine the endothelium lining blood vessels of the rat spleen using high or low iron diamine-thiocarbohydrazide-silver protein-physical development (HID or LID-TCH-SP-PD) method, with or without prior digestion with acidic glycoconjugate-degrading enzymes, such as heparitinase, testicular hyaluronidase, chondroitinase B and neuraminidase 46 . Micro-heterogeneity of pectins and calcium distribution in the epidermal and cortical parenchyma cell walls of flax hypocotyls was determined using a combination of subtractive treatment and PATAg (periodic acid-thiocarbohydrazide-silver proteinate) staining method 47 . Detection of acidic glycoconjugates in a variety of mammalian connective tissues by means of a physical development procedure into high or low iron diamine-thiocarbohydrazide-silver protein (HID or LID-TCH-SP) methods 48 . The cellulose synthesized in the mannan medium by Acetobacter xylinum in the presence of acetyl glucomannan was stained heavily by the periodic acid-thiocarbohydrazide-silver proteinate (PATAg) method for X-ray diffractometry and FT-IR spectroscopy determination of its crystal structure 49 . The presence and localization of storage polysaccharides and of polysaccharides as cell structure constituents of Paracoccidioides brasiliensis yeast-like cells and protoplasts were studied using the same technique 50 . Identification of two glycosylated components of Mycoplasma penetrans using transmission electron microscopy was performed by staining with periodic acid-thiocarbohydrazide silver proteinate 51 . Lysozyme-expressing cells in human labial salivary glands were electron-lucent and exhibited reactivity for mucus staining by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH -SP) technique 52 .
Barley chromosomes were prepared for high-resolution scanning electron microscopy SEM using a combination of enzyme maceration, treatment in acetic acid and osmium impregnation using thiocarbohydrazide. Using this technique, the three-dimensional ultrastructure of interphase nuclei and mitotic chromosomes was examined 53 . Pollen grains made conductive for SEM by coating in a series of osmium and thiocarbohydrazide solutions (OTOTO) show charging artifacts when nonconductive detritus (sand grains, plant fragments, etc.) is present in the immediate sample area of the SEM scanning probe 54 .Transmission light microscopy of structurally colored semithin cartilage sections using silver proteinate by thiocarbohydrazones staining method 55 and cytochemical investigations on tunic morphogenesis in the sea peach Halocynthia papillosa (Tunicata, Ascidiacea) demonstrated by the same method 56 . Keratan sulfate glycosaminoglycans in murine eosinophil-specific granules were examined using high iron diamine-thiocarbohydrazide-silver proteinate physical development method (HID-TCH-SP-PD) for staining 57 . On the other hand, thiocarbohydrazide derivatives; isatin -thiocarbohydrazone (IsTCH) and Nethylisatin--thiocarbohydrazone (N-Et-IsTCH) showed antiproliferative action towards healthy human peripheral blood mononuclear cells (PBMC) and on two neoplastic cell lines 58 .
A highly selective heavy metal ion adsorbent could be obtained when coconut shell activated carbon is soaked in acidic aqueous thiocarbohydrazide and contacted with an aqueous heavy metal ion, e.g. Hg (II), which is effective at a lower pH without leakage of thiocarbohydrazide 59 . Also, the S-containing triazine resin used to adsorb metals from wastewaters is prepared by reacting cyanuric chloride with thiocarbohydrazide and polyether diamine or an aminosulfonic acid or its salt in a polar nonprotic solvent which is inert to the polar solvent [60][61][62][63][64] . Thiocarbohydrazide was used as a complexing agent for the solvent extraction separation of some bivalent metalssuch as Cd from Co, Cu, and Pb and of Pb from Ni and Pd and Cu from Zn and Hg and of Pb from Zn and Cd using various masking agents 65,66 . Bis(p-sulfophenyl)thiocarbohydrazide (disodium salt, Na 2 H 2 L) was used as metallochromic indicator for determination of Zn by complexmetric titration 67 .
Thiocarbohydrazides, thiosemicarbazides and their hydrazones are described for use as fogging agents for the fogging development of unfogged direct-positive Ag halide emulsions 68 . The formation process of photographic images is described by processing the photographic material in the presence of thiocarbohydrazide compounds of the type R 1 R 2 NNHCSNR 5 NR 3 R 4 (I; R 1 ,R 4 = H, saturated or olefinic aliphatic, aryl, heterocyclic, or acyl group; R 1 with R 2 and R 3 with R 4 may form a methylene group or an alkyl, aryl, or heterocyclic substituted methylene group and the substituents on the methylene group may together form a carbocyclic or heterocyclic ring; R 5 = H or R 1 with R 4 or R 5 may comprise the necessary atoms to form a 5-or 6-membered heterocyclic ring) in the emulsion layer as stabilizers. Thus, Thiocarbohydrazides with aliphatic, aromatic or heterocyclic substituents have an antifoggant effect during the 1 st , and exert a fogging effect during the 2 nd color development of reversal materials so that an overall exposure or chemical fogging is unnecessary [69][70][71] .
Weatherability improvers for jet-printing ink compositions and inks contain water-soluble dyes, wetting agents, water, and discoloration inhibitors selected from thioureas, thiosemicarbazides, and thiocarbohydrazides 72 . The sheet especially useful in ink-jet recording with a coating of a porous ink-receptive layer containing selected derivatives from thiourea, thiosemicarbazide, and thiocarbohydrazide shows good ink absorption and dye fixability and provides high-quality images showing no discoloration upon storage 73 .
Receptor sheets with good ink absorptivity and colorant-fixing ability for the nonimpact printing comprise a porous ink-receiving layer containing iodine or iodides as quenching agents and of thiourea, thiosemicarbazide, and thiocarbohydrazide derivatives as antifading agents 74 .

From Hydrazine and Carbon Disulphide
The scale-up technology for synthesis of thiocarbohydrazide (TCH) 1 was studied, using 2-chloroethanol as catalyst, CS 2 and hydrazine hydrate as starting materials. The results showed that the optimized reaction conditions were CS 2 /hydrazine hydrate 1:3.1-3.2 mol, time 10-11 h, temperature 80 o C, and the consumption of catalyst 0.15 mol/CS 2 mol. It was found the turbine agitator was preferable, and the total yield of TCH was >90% 76

Cyclocondensation with S-Methylated Compounds
Cyclocondensation of thiocarbohydrazide 1 with S-methylated compounds in PhMe in pyridine gave 13 88

Reaction with Acetyl Chloride Derivatives
Thiadiazines [14, R = 3,4-(HO) 2 C 6 H 3 , 2,5-(HO) 2 C 6 H 3 , 2,3,4-(HO) 3 C 6 H 2 ] were obtained by treatment of RCOCH 2 Cl with thiocarbohydrazide 1 89,90 . ; n = 2 or 0) were prepared and characterized on the basis of analytical, spectral and magnetic moment data. The complex Ni(bstcz).nH 2 O was isolated in 2 forms, a 4-coordinate diamagnetic and a 6-coordinate paramagnetic form. Complexes of Ni(II) (diamagnetic) and Pd(II) were assigned square-planar structures, Zn(II) tetrahedral, Cu(II) distorted octahedral, and the acetato complexes of Pb(II) and Cd(II) were assigned dimeric structures 96 . The Distorted octahedral complex of nickel(II) with o-hydroxy-4-benzamidothiosemicarbazide (1-salicyloyl-3-t hiocarbohydrazide) showed a magnetic moment of 2.59 B, and 4 absorption bands at 9,600, 13,000, 16,000, and 31,000 cm -1 , respectively 97 . The IR and Raman spectra (30-4000 cm -1 ) of thiocarbohydrazide and deuterated thiocarbohydrazide were measured. Vibrational assignments were discussed by referring to characteristic group frequencies of related molecules and force constant calculations 98 . Also, Raman and IR spectra (30-4000 cm -1 ) of dichlorobis(thiocarbohydrazide)cadmium(II) 16 was measured where, the vibrational spectra of the Cd complex of thiocarbohydrazide were discussed in relation to that of the free ligand 99  thiosemicarbazide] gave 4 bands at 13600, 17420, 27400, and 34500 cm -1 from which Racah interelectronic repulsion parameters B35 and B55 were calculated, which yielded a covalency parameter 0.89 102 . Several mono-and bis-carbono-and thiocarbonohydrazone ligands and metal complexes of Cu(II), Fe(II), and Zn(II) salts were tested in vitro against Gram positive and Gram negative bacteria, yeasts and moulds. In general, the bisthiocarbonohydrazones possess the best antimicrobial properties and Gram positive bacteria are the most sensitive microorganisms 103 . Cobalt(II) chloride forms a 1:1 complex with Schiff base derived from salicylaldehyde and thiocarbohydrazide. Kinetic parameters like apparent activation energy, frequency factor, activation entropy and apparent order of reaction of complex have been determined employing Freeman-Carroll, Fuoss et al., Coats-Redfern, Karkhanawala-Dharwadker and Doyle methods as modified by Zsako using non-isothermal TG curves. The complex decomposes regularly without any distinguishable rest with random nucleation mechanism 104 . The complexes of tin(IV) chloride with tetradentate Schiff bases H 2 L derived from the 1:2 condensation of thiocarbohydrazide with benzoin, were prepared in alcoholic medium 105 . Sn(II) and Sn(IV) com-plexes of dibenzaldehyde thiocarbohydrazone, diacetophenone thiocarbohydrazone, dicyclohexanone thiocarbohydrazone, disalicylaldehyde thiocarbohydrazone, dicinnamaldehyde thiocarbohydrazone and di-o-hydroxyacetophenone thiocarbohydrazone were prepared as chloro and iodo heteroleptic complexes 106 . The Cu(II), Ni(II) and Zn(II) metal complexes of Schiff bases derived from thiocarbohydrazide and salicylaldehyde, o-vanillin, 2,4-dihydroxybenzaldehyde or 2-hydroxy-1-naphthaldehyde were showed fluorescence, antibacterial and pigmentation properties 107 . The ligand 1,5-bis(p-aminoacetophenone) thiocarbohydrazone was synthesized by condensation reaction of thiocarbohydrazide with 4'-aminoacetophenone in ethanol in the presence of acetic acid by refluxing for 4 hours. Its complexes with Cu(I), Zn(II), Fe(II), Sn(II), and Pb(II) were synthesized by electrochemical oxidation of anodic metal method with Cu, Zn, Fe, Sn, or Pb as anodes in the ligand/nonaqueous solution 108 .

Determination of Metal ions
Online separation and sequential determination of trace amounts of heavy metals in biological materials by flow injection inductively coupled plasma atomic emission spectrometry were conducted based on the complexation of the metal ions with 1,5-bis(di-2-pyridyl)methylene thiocarbohydrazide 109 .

Zinc Determination
A simple and rapid method is described for the preconcentration and determination of zinc in water using column packed with silica gel functionalized with 1,5-bis (di-2-pyridyl)methylene thiocarbohydrazide (DPTH-gel) 110 .

Copper Determination
Cu (1.8-15.0 g) was determined spectrophotometrically at 455 nm (molar absorptivity was 2.06  104) after complexation at pH 2.5 with 1,5-diphenylthiocarbohydrazide and extraction of the complex into BuOAc. Beer's law was obeyed for 0.18-1.5 ppm Cu. Ag present in amts. equivalent to Cu interfered. The method was used to determine 0.735 ppm Cu in a 100-g mushroom sample 111 .

Bismuth Determination
Reaction of di(p-sulfophenyl)thiocarbohydrazide with bismuth ions at pH 1.15, where Bi 3+ replaces 1 proton from each of 3 molecules of the dianion of di(p -sulfo phenyl)thiocarbohydrazide to form a red complex, with maximum absorbance at 510 nm, and a formation const. of (3.4  1.2)  10 -22 . This reagent is a satisfactory metal indicator for use in the EDTA titration of Bi 3+ 112 .

Cadmium Determination
An FI-ICP-AES method for the determination of trace levels of cadmium in biological samples is described based on the complexation of the metal ion with 1,5 -bis(di -2-pyridyl)methylene thiocarbohydrazide (DPTH) and its subsequent online extraction into iso-Bu Me ketone (IBMK) 113 .

Palladium Determination
The use of thiocarbohydrazide as a masking agent provides rapid masking at room temperature, enabling an EDTA titration to give an indirect determination of palladium 115 .

Mercury Determination
Automated online separation-preconcentration system for inductively coupled plasma atomic emission spectrometry and its application to mercury determination, where the preconcentration step is performed on a chelating resin microcolumn [silica gel functionalized with 1,5 -bis(di -2-pyridyl) methylene thiocarbohydrazide] placed in the injection valve of a simple flow manifold 116 .

Reaction with Organic Acids
Cyclocondensation of thiocarbohydrazide 1 with organic acids RCOOH and condensation of the resulting aminotriazolethiones with ketones R 1 COR 2 , the (methyleneamino)

Reaction with Glycine Derivatives
Condensation of thiocarbohydrazide 1 and glycine afforded N-(dihydrazinomethylene)glycine 28 which was assayed for reduction of hyperglycemia and obesity in mice 132 .

Reaction with Hydroxybenzaldehyde
Reaction of thiocarbohydrazide 1 with 2-hydroxybenzaldehyde gave 1,5 -bis(2-hydroxybenzaldehyde) dithiocarbohydrazone 40 143  Reaction of thiocarbohydrazide 1 with representative unsaturated ketones led to the exclusive formation of monothiocarbohydrazones. The cytotoxicity of these compounds against murine P388 and L1210 cells, human T-lymphocytes, and human tumor cell lines was undertaken to ascertain their bioactivity, whereby the carbohydrazones would be predicted to be more than twice as cytotoxic as the ketones and Mannich bases from which they were derived. In general, conversion of the arylpentenones 44 (R = H) into corresponding biscarbohydrazones 45 was accompanied by a decrease of cytotoxicity. On the other hand, the compounds in series 45 (R = N-piperidylmethyl) were more potent cytotoxic agents than their corresponding precursors 44. Some compounds displayed activity comparable with or exceeding that of melphalan 146 .

Conclusions
The review covers recent literature regarding the synthesis and chemical reactions of thiocarbohydrazides. Thiocarbohydrazides are easily synthesized and are useful in the synthesis of highly functionalized heterocyclic compounds and used in the design of novel highly effective pharmaceuticals with a broad spectrum, besides their applications in several methods for metal ions determination and structural determination of cells and tissues.