Fart Ii Some Experiments on the Chemistry of Osazones

FART II

SOME EXPERIMENTS ON THE CHEMISTRY OF OSAZONES. INTRODUCTION 103

SuKar Osazones.

Sugar osazones were first prepared by Emil Fischer (Ber., 579) in the year 1884; since then, these compounds have found widespread application both as derivatives for the identification of reducing sugars and as intermediates in the preparation of a variety of sugar compounds. The fact that the free sugars are brought to crystallisation only with great difficulty, together with the lack of a reagent that forms easily crystallisable derivatives with all reducing sugars, renders their identification a tedious process, especially when dealing with sugar mixtures. The osazones are unique in the ease with which they crystallise yet suffer from the disadvantage that every particular osazone is formed from a number of sugars, so that the complete identification of a sugar requires the use of further derivatives. Osazones can be prepared from compounds having in the O.—position to a carbonyl group another carbonyl ' group as in the case of osones, a hydroxyl group as in aldoses and ketoses, an amino group as in amino sugars, or a methoxyl or thio—alkyl group; in the latter two cases osazone formation proceeds with difficulty and 104 yields are poor.

Constitution of sugar osazones.

Fischer (Ber., 1887, 20, 821) ascribed to glucose phenyl-osazone structure (I); the other sugar osazones were likewise given open chain structures differing only in the configuration of the hydroxyl groups in the sugar molecules and in the substitution of the hydrazine residue. Fischer's formula has been criticised by several authors and at the present time, after more than sixty years, the structure of sugar osazones is not definitely established. Investigations have been mainly concerned with glucose phenyl-osazone where two structures deserve serious consideration, viz. Fischer's open chain structure (I) and the cyclic structure (II) proposed by Percival (J. Chem. Soc., 1935, 1398):-

CH =N-NHC6H5 CH = N-NHC6H5 C = N-NHC6 H 5 C-NHNHC6H5 HO C H HO - C H H - C - OH 0 H - C - OH H - C - OH H C - OH

CH2OH CH2 105

Evidence in support of structure (II) was given by Percival (loc.cit.) who obtained, by treating glucose phenyl-osazone (It) with dimethyl sulphate in alkali, a tetra-methyl derivative (III) which on treatment with 1-nitro benzaldehyde gave a trimethylglucosone (IV); this was finally reduced to the known 34:5-trimethyl- D-fructopyranose (V) denoting that only three of the methoxyl groups in (III) were attached to oxygen, the fourth being attached to a nitrogen atom in the hydrazone residue.

CH=N -NHC6H5 CH=N-NHC H I 6 5 ---- -C-NH-NHC H 0 -NH -N(CH )C H 6 5 3 6 5 1 1 HO-C-H CH 0 -C -H 1 (aH )SO 3 1 H -C -OH 3 4, 0 H -0 -CH alkali -6 3 H -C -OH H -C -0 -CH 3 ------CH2 ------CH2 (II) (III)

CHO CH/ 2 OH C OH ? -OH 1 nitro- CH30 -C -H Zn CH7 0 -C -H benzaldehydet 0 -I H -C -0 -CH acetic acid) 0 H -C -0 -CH I 3 i 3 H-! -0 -CH H -C -0 -CH 3 1 3 ! I CH2 CH2 (Iv) ( V) 106

Percival argues that the fact that the primary hydroxyl group in C6 was unattached while the secondary ones in positions 3-, 4- and 5—were, shows that position 6- was involved in ring formation. This was confirmed by Diels (Ber., 1938, 71, 1189) who showed that glucose phenylosazone failed to react with trityl chloride, which is known to react with primary hydroxyl groups. On the other hand, Wolfrom, Kbnigsberg and Saltzberg (J. Amer. Chem. Soc., 1936, 58, 490) obtained by acetylation a tetra-acetyl-glucose phenyl-osazone which they hydrolysed using a method developed by Kunz and Hudson (J. Amer. Chem. Soc., 1926, 48, 1982) which is claimed to hydrolyse 0-acetyl groups but not N-acetyl ones. This method consists in dissolving the acetate in acetone and adding 0.1N-KOH dropwise to the ice cooled solution; after two hours standing at room temperature the solution is back-titrated. All four acetate groups in the osazone were hydrolysed by this treatment and the authors concluded that glucose phenyl-osazone must have structure (I). Percival (J. Chem. Soc., 1937, 1320) doubted the significance of these results, as it is known, that N-acetyl groups are sometimes hydrolysed by the method of Kunz and Hudson, for example in D(;/3-diacetyl-phenyl-hydrazine, where an amount corresponding to about one acetyl group was removed under similar conditions. 107

The strongest argument for the open chain structure (I) of glucose phenyl-osazone was given by Chargaff and Magasnnik (J. Amer. Chem. Soc., 1947, 69, 1459); they showed that on oxidation with periodic acid one mot. of glucose phenyl-osazone yields 2 mols of formic acid, one mol. of formaldehyde and mesoxaldehyde phenyl osazone (VI) in 85% yield. They formulated their results as follows:-

CHZ-NHC H 6 5 C H=N-NHO6H5 I LN-NHC6H5 C=N-NHC6H5 HO- NCHO 311104 ,. H-C-OH ..- + I H-C-OH. 2H-000H 1 CH2OH + H-CHO (I) (VI) Engel (J. Amer. Chem. Soc., 1935, 2a, 2419) studied the ultra violet absorption spectra of sugar phenyl- osazones and found them similar to glyceraldehyde phenyl- osazone; this was taken to favour the open chain structure of sugar osazones since it is most unlikely that glyceraldehyde-phenyl-osazone has a ring structure. From the above review it is clear that there is strong evidence for both structures (I) and (II). This

108

contradiction could be explained if we assume an equilibrium between the two structures and that under methylation or tritylation conditions the cyclic structure (II) prevails while during periodic acid oxidation the equilibrium is shifted towards the open chain structure (I). An example of such a shift in structure is known in the case of free sugars which behave as open chain compounds towards periodic acid and as cyclic ones towards methylating agents.

Reactions of sugar-osazones.

1. Reduction.

Fischer (Ber., 1899, 22, 88) has shown that sugar- osazones when reduced with zinc in acetic acid yield 1-amino-ketoses (VII); later Maurer and Schiedt (Ber., 1935, 68, 2187) obtained the same products by catalytic hydrogenation of the osazones.

CH=N-NHC6 H5 1 red. Cr.g-NHC6H5 (I)

The importance of this reaction lies in the fact that it

109

offers a means of converting aldoses into ketoses; thus, by starting with glucose and reducing its osazone 1-amino-fructose is obtained which, on deamination with nitrous acid, yields fructose (Fischer and Tafel,. Bert, 1887, 20, 2566).

2. Oxidation with molecular oxygen in alkali.

Diels (Ber., 1938, 71, 1189) oxidised glucose phenyl- osazone with oxygen in alkaline solution, and obtained a dehydro-osazone to which he ascribed structure (VIII):-

CHZ.4THC H 6 5 CH =N`NO H C-NHNHC H CI-N ,/ 6 5 6 5 .---NHC6H5 HO-C-H (0) HO-6-H > 0 H-C-OH H-C-OH H-C-OH H-C-OH

CH2 CH2

(II) (VIII)

The structure of the dehydro-osazone was based on the following experiments:- Acetylation of the dehydro-osazone gave a triacetate denoting the presence of three hydroxyl- groups; trityl chloride failed to react with the dehydro-osazone showing that the primary hydroxyl group

).10

in C6 was engaged in ring-formation; lastly it was found that substituted osazones such as methyl-phenyl-osazones failed to give on oxidation a dehydro-osazone, denoting that the hydrogen atoms in the hydrazone residues were involved in the oxidation. In the light of this evidence Diels (loc.cit.) suggested structure (VIII) for glucose dehydro-phenyl-osazone.

3. Action of Alkali.

Diels (Ann., 1936, 525, 94) has shown that sugar phenyl-osazones, when heated with 1% alcoholic alkalies, break up to form glyoxal phenyl-osasone (IX) thus

CH=N-NH H C6 5 CH=N7NHC6H5

CZ--NHC H 6 5 CHZ-NHC6 5 (1)

4. Conversion of osazones into osones.

Fischer (Ber., 1888, 21, 2631; 1889, 22, 88) found that on heating sugar osazones with concentrated hydrochloric acid the hydrazone residues are removed yielding dicarbonyl compounds, the osones. These. 111

can be reduced with zinc and acetic acid yielding ketoses, thus offering another route for the conversion of aldoses to ketoses (Fischer, Ber., 1889, 22, 188). Sugar osones have recently acquired great interest as intermediates in the preparation of ascorbic acid and its homologues (Haworth, Hirst, Jones and Smith, J. Chem. Soc., 1931+, 1192; Reichstein, GrUssner and Oppenauer, Hely. 1933, 16, 561, 1019; 1934, 17, 510). The yield of ascorbic acid was found to depend on the purity of the osone. This. led to a search for a method for the preparation of osones in a high state of purity; the old methods using aldehydes such as benzaldehyde and o-nitro benzaldehyde (Morell and Ballard, J. Chem. Soc., 1905, 87, 280) were found to produce the osones in poor yields; BrUll (Ann. Chim. applicata, 1936, 26, 415) obtained glucosone in 40% yield and in a high state of purity by treating glucose phenyl-osazone with pyruvic acid. During the course of the present study on sugar osazones, a method has been developed which yields the osone from a substituted osazone in a high state of purity and in a yield of 80%. This method will be . discussed in greater detail in the theoretical section.

5. Conversion of Osazones to Osotriazoles.

The use of sugar osazones for the identification of 112

sugars has the disadvantage, among other things, that the osazones melt with decomposition and that their melting points depend to a great extent on the rate of heating; furthermore their optical rotations are not constant owing to mutarotation. Hudson and Hann (J. Amer. Chem. Soc., 1944; 66, 735) have converted sugar osazones into osotriazoles by boiling them with copper sulphate. These osotriazoles are colourless compounds which melt without decomposition, their solutions do not mutarotate and they can easily be purified by sublimation. The present writer has shown (Disc., Zurich, 1950, p.28) that the conversion of osazones to triazoles is a general reaction and succeeded in preparing triazoles from different substituted osazones, such as the 2-toly1-, 1-chlorophenyl- and /3 -naphthyl-derivatives. The structure of sugar osotriazoles was determined by Hudson (loc.cit.) who showed that glucose phenyl- osotriazole (X) formed a tetracetate and a tetrabenzoate which, on hydrolysis, gave back the starting osotriazole, thus denoting the presence of four hydroxyl groups in the molecule. Periodic acid oxidation showed that 3 mols, of acid were consumed per mol.of triazole and that 1 mol. of formaldehyde, and 2 cools. of formic acid were produced together with the crystalline 2-phenyl-4- formy1-1:2:3- osotriazole (XI). Hudson formulated these results as follows: 113

OH N-0 H c =N,_ / 6 5 194 3 ' "6"5 C=N C t;=N I CHO 3 HIO4 H-C-OH I H-C-OH 2HCOOH

CH2OH HCHO

The sent writer (Diss., Zurich, 1950, p.32) succeeded in oxidising glucose phenyl-osotriazole (X) with 20% nitric acid to 2-pheny1-1:2:3-osotriazole-4-carboxylic acid (XII), thus confirming the presence of the osotriazole ring system.

= N,, 1 2N - C6H5 N3' COON

(XII)

6. Conversion of Osazones to anhydro-osazones.

Percival (J. Chem. Soc., 1938, 1384) has shown that, on deacetylating osazone-acetates with alkali, the elimination of the acetyl groups is accompanied by a loss of 2 molecules of water with the formation of dianhydro- osazones. Percival found that the dianhydro-osazones prepared from glucose, galactose and gulose were identical and, as these sugars differ in the configuration at C-atoms 3 and 4, he concluded that during the dehydration of the osazones a Walden inversion must have occurred in C and C 3 The dianhydro-osazone failed to react with trityl chloride denoting the absence of a primary hydroxyl group in the compound. In the light of these results Percival ascribed structure (XIII) to the dianhydro-osazone obtained from glucose, galactose and gulose.

CH = C - NH I I .1 6H5 I I H C —N CH5 (XIII) H C - OH

CH2

Mono-anhvdro-osazones were prepared by Diels (Ann., 1935, 519, 157) by heating sugar phenyl-osazones with methanol containing a few drops of Sulphuric acid- and the glucose compound was ascribed the structure of a 3:6-anhydro-glucose7phenyl-osazone (XIV). In a later

11-5

publication Diels (Ann., 1936, 525, 94) abandoned structure (XIV) in favour of a pyrazolone structure (XV) because he found that mono-anhydro-osazones failed to give osones on treatment with hydrochloric acid, as they should have done on the basis of structure (XIV). Diels also failed to obtain anhydro-osazones from methyl-phenyl- osazones and concluded that the hydrazone residue must be involved in the anhydro-ring formation.

CH=N-NHC6H5 pH=N-NHe-C8H5 I C=N-NHC,H CH Z• ----ccz;-17-c05 1 .5 1 \N-CH 1 r----C-H C6H5NH.-NH-C= r 6 ' Ho-w-11 I I H-C-OH H-C-OH 0 H-C-OH 0 I 1 H-C-OH H-C-OH 1 H-C I I I CH OH I ---- CH 2 ------' CH2 2

(XIV) (XV) (XVI)

Perci,Tal (J. Chem. Soc., 1937, 1320; 1945, 783) found that Diels, anhydro-osazone failed to react with trityl chloride, as it should do on the, basis of structure (XV); on acetylation it gave a diacetate instead of the expected triacetate, and could not be made to react with acetone to give an isopropylidene compound. In the light of these experiments Percival suggested structure (XVI) for glucose mono-anhydro-phenyl-osazone. The present author (Dins., Zurich, 1950, p.38) obtained an anhydro-osotriazole by treating Dielse anhydro-glucose-phenyl-osazone with copper sulphate. The investigation of the structure of this anhydro- osotriazole which is described in the theoretical part revealed it to be a 3:6-anhydro-osotriazole, thus favouring Diels' original structure (XIV) for the parent osazone; a full discussion of the structure of the osazone is given in the theoretical part. THEORETIC AL 112 The work described in this section of the thesis is a continuation of the author's study of_the reaction of sugar osazones and hydrazones with copper sulphate (piss. Zurich, 1950) and is concerned with .three problems. The first was to establish the structure of the anhydro- osotriazole which the author had previously prepared (Disc., Zurich, 1950, p.82) from glucose mono-anhydro- phenyl-osazone; in the light of these experiments, the much disputed structure of the parent osazone could be satisfactorily_settled._ The action of capper sulphate on disubstituted osazones was then studied taking glucose methyl-phenyl-osazone as representative of this class of compounds; in the course of this work a new method for the preparation of sugar osones in high yield was developed. Lastly, attempts were made to prepare sugar osotriazoles using heavy metal salts other than_ copper sulphate; this study was intended to shed some light on the mechanism of osotriazole formation.

Structure of Glucose mono -anhydro -phenyl -osotriazole.

In his previous study of sugar-osotriazoles, the writer (loc.cit.) found that Diels' glucose monoanhydr - phenyl-osazone reacted with copper sulphate to yield a compound, 012H1303N3, which he suspected to be an anhydro-osotriazole. The investigation of the structure 118

of this compound was then carried .out independently in London by the writer and in Zurich by his former supervisor Dr.- E. Hardegger. The_. crude anhydro-osotriazole was found to melt over the range of 84-89°; repeated sublimation and recrystallisation raised the melting point to 90-93°. It was suspected that it might be contaminated with some other material but no separation could be achieved by recrystallisation. To show the presence of an osotriazole ring in the compound, it was heated. with 30% nitric acid and 2- pheny1-1:2:3-osotriazole-4-carboxylic acid (XII) m.p. 190° was obtained; identical with that obtained from glucose phenyl-osotriazole (X) by oxidation with nitric acid. The anhydro-osotriazole__. formed a diacetate and a dibenzoate which could not be crystallised but_were purified by distillation_ under reduced pressure._ On treatment with alkali these compounds yielded the starting anhydro-osotriazole. _It was thus clear that the anhydro osotriazole contained two hydroxyl_ groups_ in the molecule. In order to determine whether these were adjacent and possessed the same steric configuration, the anhydro-osotriazole was treated with acetone containing 1% of hydrogen chloride; an isopropylidene derivative, m.p. 71-72°, was isolated,

119

showing that the two hydroxyl groups were adjacent and had the same configuration. Three structures (XVII), 0 le vn (XVIII) and (XIX) are therefore possible; the latter two possibilities arise because of the_possfbility of free rotation of the carbon atoms Aring the hydroxyl riazole.

CH.-N‘ CH-NN dli=4 NC H t NCe:11 „441 Os 6 5 =N C=N' u 5 I

Ip 0

(xvIII) (XIX)

It is clear, then, that the anhydro-ring may be either five membered (as in XVII)_or three membered as in (XVIII)_or in (XIX). It is well known that_the former type is stable to_acid and alkali treatment . whereas the three-membered ethylene oxide type of ring is easily opened by_treatment with acids or alkalies (see Peat, Adv. Carbohydrate Chem., 1946,_2, 37). The anhydro- osotriazole was therefore heated at 100° for two hours

with 2N-H2SO4 and 2N-NaOH_respectively; it was recovered from both reaction mixtures unaltered, thus favouring the five membered ring structure (XVII). 120

Proof that the carbon atom in position 6 was engaged in ring formation was obtained from the fact that the anhydro-osotriazole failed to react with trityl_chloride and that its ditosylate4 m.p. 138°, could not be made to react with sodium iodide. In_ the light of the above experiments it_was concluded that the_anhydro-osotriazole was the 3:6- anhydro-glucose-phenyl-osotriazole. To confirm this, glucose phenyl-osotriazole_was tosylated and_the 6-tosyl derivative m.p.140°, treated with alkali, yielding an anhydro-osotriazole9 m.p. 96°, which, on admixture with anhydro-osotriazole under investigation, melted at 930.. Final proof of the structure of the anhydro-osotriazole

,..s obtained when authentic 3:6-anhydro-glucose (kindly made available by Dr. L.N. Owen) was converted into the 3:6-anbydro-glucose phenyl-osotriazole by treating its osazone with copper sulphate. _Authentic 3:6-anhydro triazole prepared in this way melted at 96-97°, slightly higher than the osotriazole under investigation 90-93°); the mixed melting point had 'an intermediate value of 94°the difference in melting point being clearly due to some contaminant. The nature of this contaminant was investigated by the Zurich group of workers under _ Hardegger (private communication)_who isolated from the crude anhydro-osotriazole, obtained from Dielst aahydro

121

osazone, besides 3:6-anhydro-glucose phenyl-osotriazole he_ (XXI8) a considerable amount of 3:6-anhydro-allose pehnyl- osotriazole (XX11) m.p. 88°. The separation of the two osotriazoles was achieved by chromatography on alumina. The formation of an allose derivative from a glucose compound involves a change in the configuration of carbon atom 3. A Walden inversion probably takes place during the treatment of glucose, phenyl-osazone with methanol containing sulphuric acid to form the anhydro-osotriazole:- CH=N-NHC6H5 CH=N, -NC H 0=N-NHC6H5 CuS0 C=N' 6 5 =N-NHC6H5 7-H r I C=N-NHC6 H5,H-C-OH I 0 H-C-OH 0 (XI V) (XXI) HO-C-H H-C-OH H-C-OH

H-C-OH --CH ----- 2 CH2 H-C-OH

CH2OH CH=N-NHC H CH=N, 1 6 5 ro H C=N-NEC6H5 C-_-4N" 6 5 H-C ----- 11-? -I H-C-OHH . . H-C-OH ' H-C-O 0 I 0 H -C -OH H -C -OH (XXII XX) I CH r-- CH2

Structure of Diels' Anhydro-osazone

The fact that on heating Diels' glucose monoanhydro- phenyl-osazone with copper sulphate a mixture of glucose 122

and allose 3:6-anhydro-phenyl-osotriazoles is obtained4 sheds a new light on the structure of the anhydrwazone. The evidence against the 3:6-anhydro structure given by Diels (Ann., 1936, 5254 94) was the fact that the osazone failed to give an osone on treatment_with concentrated hydrochloric acid and that glucose methyl-phenyl osazone failed to give an anhydro-compound on_treatment with methanol and sulphuric acid. These results were taken as denoting that the hydrazone residues in the anhydro compound must be involved in ring formation4 otherwise both these reactions should have been possible. This, conclusion can, however, only be accepted with sober reserve in view _.of the fact that Diels' evidence is based on negative results and is contradicted by the fact that it was possible to obtain a triazole from the anhydro-osazone, clearly indicating that the.hydrazone residues were not involved in any stable ring structure. _Percival!S finding that the anhydro-osazone formed a diacetate is in agreement with the 3:6-anhydro_ structure, as is the fact that the_anhydro-osazone failed to react with trityl chloride. Percival, _however, failed to obtain from the_osazone the isopropylidene derivative which might be expected from_a 3:6-anhydro-compound. The author,_however, succeeded in condensing_the osotriazole with acetone and, rather than assume a change in the anhydro-ring during osotriazole formation is inclined 123 to disregard Percival's negative evidence and to conclude - that Diels' glucose mono-anhydro-phenylosazone is a mixture of 3:6-anhydrp-glucose phenyl osazone (RIB') and 3:6- anhydro-allose phenyl-osazOne (XX).

H=N-NHC H ,CH=N-NHC H 6 5 1 6 5 C=U-NHC6H5 C=N-NHC H 1 6 5 -H H-C - I ICH-C-OH H-C-OHI 0 1 i () H-C-OH H-C-OH I i -CH 2 CH2

(XIV) (XX)

Reaction of Glucose methyl-phenyl-osazone with copper sulphate.

The present writer (Dins., Zurich, 1950) found that sugar hydrazones react with copper sulphate yielding_ the_parent sugar in nearly quantitative yields. The liberation of the free sugar was always accompanied by the, decomposition of the hydrazone residue to the aromatic amine and nitrogen, thus:- 124 CuSO4 2 ?H=N-NHR > 2CHO + 2RNH2 + N2

On the other hand sugar osazones, such as the phenyl- -tolyl-, 2-chloro-phenyl- and p-naphthylr derivatives, react with_ copper sulphate, forming the very stable osotriazole ring system with removal of the aromatic amine, thus:-

CH= NN H R CH-N, CuSO4 I + RNH C= N- NHR C= N' 2 I - It was, therefore, of interest to determine whether copper sulphate would react with an_osazone in which the hydrogen atoms attached to the nitrogen of the hydrazone residues are substituted by methyl groups, i.e. where no aromatic amine can be split off, and if so whether the hydrazone residue would be_completely removed, as_in the case of the sugar hydrazones. Glucose methyl-phenyl-osazone (XXIII) was refluxed for ten minutes with a copper sulphate solution and from the reaction mixture a faintly yellow crystalline compound was isolated which had m.p. 163°[ c. 1f)0 = - 22.6 -16.5° (c = 1.15 in ethanol). This compound was found to be identifal with glucosone methyl-phenyl-hydrazone (XXIV), m.p. 162°, first prepared by Fischer (Ber.,_1889, 22, 88) by treating glucosone (XXVI) with methylrphenyl- hydrazine. With acetic anhydride the hydrazone (XXIV) _ formed a tetra-acetate, m.p. 112°, and with benzoyl chloride 125

a tetrabenzoate, m.p. 187°. On treatment withphenyl- hydrazine an osazone (XXV) m.p'. 203° was obtained which had [0c] 32)0 = -55.5 - 14° (c = 1.09 in pyridine). Votocek and Vondracek (Ber., 1904, 37, 3848) by reacting_ glucose phenyl-hydrazone with methyl-phenylhydrazine, obtained two isomeric osazones; "A", m.p. 192° had [Dg 1,7= - 53 -6° and "B", m.p. 205°, [c/,] 1D7= -60 —t -15° (both in pyridine-alcohol)., Percival (J. Chem.Soc., 1941, 750) obtained osazones SI.A.1? and "B" by reacting phenyl-hydrazine_with the two isomeric fructose methyl-phenyl-hydrazones; m.p. 170° and 118° respectively. Isomers "A" and "B are an and anti forms of_glucose 1-phenyl-2-methyl-phenyl-osazone. It is clear that osazone (XXV) was very similar to osazone "B"_of Votocek; to prove their identity osazone "B" was prepared by the method of Percival (loc.cit.) i.e. by treating fructose methyl-phenyl-hydrazone (XXVII), m.p. 118°, with phenyl-hydrazine; the product melted at 204° and gave no depression on mixing with osazone (XXV); it was therefore concluded that_osazone (XXV) was glucose 1-phenyl-2-methyl-phenyl osazone_and that hydrazone (XXIV) was glucosone 2-methyl-phenyl-hydrazone. -126

/ C6H5 CH=N-N CH=0 CH=NNHC H CH 6 5 3 I / 005 ,C6H5 C,,H C-Z-N ..\ C=W41's C=IT.N/ u 2 CH CH \ I 3 1 3 CH7 HO-CH HO-CH HO -C-H 1 uS0 I H-C-OH ----C 4--7 H-C-OH henyl H-C -OH I 4 . 1 gydrazine H-C-OH H-C-Oli H-c-ai i CH OH CH2O 2 H CH2OH (xxIII) (XXIV) (XXV)

CH 2OH CH2OH 1 I Call C=0 C=1I-N/ `' 2 I I ‘. HO-C-H HO-C-H CH H-C-OH methylphenyl > HC-OH- hydrazine i H-C-OH EIC•-OH 1 I CH2OH CH2OH

(XXVI) (XXVII)

Glucose 1-phenyl-2-methyl-phenyl osazone (XXV) has one hydrazone residue containing a hydrogen atom while in the other, attached to C-atom 2, the hydrogen atom is 127

substituted by a methyl group. It was now of interest to determine whether such an osazone on treatment with copper sulphate would form an osotriazole or an osone. Treatment of osazone (XXV) with_copper sulphate was, in facts found to yield glucose phenyl-osotriazole in 67% yield. It has already been mentioned that sugar hydrazones react with copper sulphate yielding the parent sugar_ by removal of the hydrazone residue. Glucosone. 2-methyl- phenyl-hydrazone (XXIV) would, therefore, be expected to react in a similar manner yielding the parent sugar, glucosone. This was folind to be the case, prolonged treatment of the hydrazone with copper sulphate giving glucosone in a yield of nearly 80% which is by far the highest recorded in the literature. It was also found that, when glucose-methyl-phenyl osazone (XXIII) was heated for 45 minutes with copper sulphate the osone was formed_directly there_being no need to isolate the intermediate hydrazone; the yield of osone by this method was 75% of the theory._ The osone obtained by both methods was completely colourless and on paper chromatograms behaved as a homogenous substance, giving only one spot with-out any tailing. TO sum up these results on the_action of copper sulphate on sugar hydrazones and osazones, we find that an osazone 128'

can be converted into an osotriazole with copper sulphate if at least one of the hydrazone residp.es in the osazone is mono substituted, i.e. has a hydrogen atom attached to_ nitrogen. The osotriazole formed will then be substituted, with the_, same group that was attached on the mono substituted hydrazone, thus:-

.CH=N- N H R CH =N\ NR }IV C = N -N/R C \R

If bothl-hydrazone residues are disubstituted,_i.e. there is no available hydrogen attached to_the nitrogen- of the hydrazone residues, then the osazone reacts in_ the ;Jame manner.as a hydrazone and loses the hydrazone residues leaving the free carbonyl compound:-

_ N _ r /R C \Ri CHO CHO C = N N'R C = 0 C = - N'R \R1

Reaction of Glucose phenyl-osazone with heavy metal salts.

Hudson (J. Amer. Chem. Soc., 1944, 66, 735) suggested

129

that on reacting Bugar osazones with_copper sulphate to form a triazole, the osazone formed a_complex with copper which was then decomposed_with the elimination of aniline and the liberation of the osotriazolev the copper was then precipitated as such. • The present. author found that cuprous salts such as cuprous chloride failed to react_with glucose phenyl-osazone and that the sulphate ions were not responsible for the osotriazole formation since_ nickel sulphate, for instance, failed to yield a triazole. On the other hand salts such as ferric_chloride and silver nitrate, readily form_osotriazoles when heated with glucose phenyl-osazone. These salts are known to be oxidising agents and it is_therefore suggested that in the course of osotriazole formation oxidation is involved in a manner similar to the mode of formation of simple oSotriazoles (viz. Pechmann, Ann., 1891, 262, 294) where the first stage of the reaction is an oxidation of the osazone to the osotetrazine, which is then converted to the osotriazole by the action of acid:-

R R R \ \ \ C = N-NH-C H CNN--C H C=N 6 5 6 1 \\N-C H oxidation s -8----ei-7-C1- 6 5 1 I C= N-NH-C6H5 C=N N-0 H5 CAN / / 6 / R R R 1 1 1 EXPERIMENTAL 130

Structure of Diels' Anhydro-Osazone

Preparation of D-glucose anhydro-phenyl-osazone.

The method of Diels and Meyer (Ann., 1935, 519, 157) was adopted; D--glucose phenyl-osazone (20 g.) was suspended in methanol (750 ml.) to which 20% sulphuric acid was added (3 ml.). The mixture was refluxed for five hours on the steam bath, during which time all the osazone went into solution. Hot water was now added to the hot methanolic solution until the solution became slightly turbid; the mi ture was then left to crystallise in the cold room. The crystalline anhydro-osazone (12 g.' was recrystallised from warm acetone (100 ml.) by adding hot water to turbidity; the product melted at 174-176° (Found: C, 63.42; H, 5.9. Cale. for C 18H20°3N4:

C, 63.51; H, 5.910). [,e;11)20 °= -150° ( c = 0.5 in methanol).

Preparation of anhydro-osotriazole.

The anhydro-osazone (25 g.) was dissolved in bo'linp7 methanol (150 ml.) and a hot solution of copper sulphate (10 g.) in water (150 ml.) added. The mixture was 131

refluxed for 15 mins. and filtered before cooling. On cooling, the solution was shaken four times with ether and the combined ether extracts dried over anhydrous sodium sulphate and evaporated to dryness. The residue crystallised on addition of a drop of methanol and was recrystallised from hot water, after treatment with charcoal, forming colourless elongated needles m.p. 84-89°; the yield was 15 g. Repeated sublimation at 130° (0.001 mm.) and recrystallisation from ether-pet.ether (bp. 40-60°) and from water, brought the melting point to 90-93°, (Found: C, 58.60; H, 5.4; N, 16.56. Cale. A0O C1211,303N3: C, 58.29; H, 5.3; N, 17.00%). [qDp0° -44.0° (c = 1 in ethanol).

Preparation of the anhydro-osotriazole diacetate.

The anhydro-osotriazole (0.4 g.) was dissolved in anhydrous pyridine (1 ml.) and cooled to 0°; acetic anhydride (0.5 ml.) was added to the solution and the mixture kept 24 hrs. at room temperature. Ice water was now added and the emulsion shaken with ether several times. The combined ether extracts was washed with 0.5 N.HC1, to eliminate the pyridine, then with 0.5 N Na2CO3 and finally with water. The dried ether extract was 132 evaporated and the residue distilled at 1000 (0.01 mm. pressure). The product failed to crystallise (Found: C, 58.06; H, 5.2; N, 12.59. Cale. for C16H1705N3: C, 58.00; H, 5.2; N, 19.70%).

Hydrolysis of the anhydro-osotriazole diacetate.

The acetate (0.1 g.) was dissolved in ethanol (5 ml.) and heated for 15 mins. with N-NaOH (4 ml.). The excess alkali was neutralised with 0.1 N-HC1 and the solution evaporated to dryness. The residue was extracted with ether; evaporation of the extract yielded the crystalline anhydro-osotriazole, m.p. 90-920, not depressed on admixture with a sample of the anhydro-osotriazole which had not been acetylated.

Preparation of the anhydro-osotriazole dibenzoate.

The anhydro-osotriazole (0.2 g.) was dissolved in pyridine (0.5 ml.) and treated with 0.1 ml. benzoyl chloride; the mixture was left to react for 24 hrs. Absolute alcohol (0.5 ml.) was added to decompose the excess benzoyl chloride; after 20 mins. water was added and the mixture extracted with ether. The ether extract 133 was washed with 0.5 NHC1, 0.5 N Na2CO3 and water successively and then dried and evaporated to dryness. The residue, which contained the osotriazole benzoate and ethyl benzoate was distilled under reduced pressure (0.001 ram.); the first fraction distilling at 50°, was ethyl benzoate; at 120° the anhydro-osotriazole dibenzoate distilled as an amorphous glass which could not be crystallised (Found: C, 68.70; H, 4.6; N, 9.49. Calc. for C26H2105N3: C, 68.50; H, 4.6; N, 9.25%).

Hydrolysis of the anhydro-osotriazole dibenzoate..

The dibenzoate (50 mg.) was dissolved in ethanol (2 ml.) and refluxed for 15 mins. with 2 ml. of N-Na0H. The excess alkali was neutralised with 0.1 N-HC1 and the solution evaporated to dryness. The residue was extracted with ether; evaporation yielded the crystalline anhydro-osotriazole, m.p. 900. The recrystallised anhydro-osotriazole melted at 90-92°, not depressed when mixed with a sample of unbenzoylated anhydro-osotriazole.

Preparation of the isopropylidene derivative of the anhydro-osotriazole. The osotriazole (0.2 g.) was dissolved in 3 ml. of dry acetone containing 1% HC1; to the solution 1 g. of 134

anhydrous sodium sulphate (previously heated to redness) was added and the mixture shaken for 24 hrs. at room temperature and then filtered. The filtrate was neutralised with silver carbonate and evaporated to dryness: the residue was distilled under reduced pressure (0.01 mm.) at 15e; the distillate crystallised on standing, m.p. 71-72° (Found: C, 62.30; H, 6.0;

N, 14.66. Cale. for Ci5H1703N3: C, 62.71; H, 5.9; N, 14.62); [DK]D0 = —24° (c = 1 in chloroform).

Preparation of the anhydro—osotriazole ditosylate.

The anhydro—osotriazole (0.2 g.) was dissolved in pyridine (2 ml.) and the solution cooled at 0°; tosyl chloride (0.2 g.) was added to the solution and left to react for 24 hrs. at room temperature. The pyridine was now evaporated under reduced pressure and the residue crystallised from hot methanol. The twice recrystallized material melted at 138° (Founth C, 55.84; H, 4.5; N, 7.36. Calc. for C26H2507N3S2: C, 56.20; H, 4.5; N, 7.56). [I,X1E° = (c = 1 in chloroform).

Reaction of the tosyl derivative with sodium iodide..

To determine whether the'primary hydroxyl group on C6 was tosylated, 150 mg. of the ditosylate were dissolved in acetone (5 ml.) and the solution refluxed with sodium iodide (150 mg.) for a period fo two hours. Water was added to the hot solution until it was turbid; on cooling, crystals of the anhydro-osotriazole ditosylate separated (120 mg.), m.p. 138°, denoting that no reaction had occurred.

Reaction of the anhydro-osotriazole with trityl chloride.

The anhydro-osotriazole (0.2 g.) was dissolved in pyridine and 0.2 g. of trityl chloride added; the mixture was kept for 24 hrs. at room temperature. No pyridine hydrochloride was formed in the reaction mixture, denoting that no reaction had taken place; the mixture was then heated on the steam bath for 2 hrs. and on addition of water crystals of triphenyl carbinol, m.p. 1660, and of the .unchanged anhydro-osotriazole, m.p. 94°, separated. It was therefore concluded that no reaction had taken place.

Resistance of the anhydro-osotriazole towards acid.

The anhydro-osotriazole was heated at 100° for 2 hrs. with 2N-HC1 and 2N H2SO4 respectively. After neutralisation 136 with 0.5 N-Na0H, the solutions were evaporated to dryness under reduced pressure and the residues extracted with ' ether. On evaporating the ether extracts only unchanged anhydro-osotriazole was obtained, m.p. 89°, not depressed on mixing with the original anhydro-osotriazole.

Resistance of the anhydro-osotriazole towards alkali.

The anhydro-osotriazole (0.2 g.) was heated at 100 for 2 hrs. with 2N NaOH (5 ml.), neutralised with 0.5 N-HC1 and then evaporated to dryness under reduced pressure.. The residue was extracted with ether: evaporation yielded the unchanged anhydro-osotriazole m.p. 89° not depressed on mixing with the original anhydro-osotriazole.

Oxidation of the anhydro-osotriazole with nitric acid.

The anhydro-osotriazole (1 g.) was dissolved in hot 30% nitric acid (10 ml.) and the mixture heated for 4 hrs. on the steam bath. On cooling the reaction mixture was diluted with 20 ml. of water and shaken several times with ether. The ether extract was dried over sodium sulphate and evaporated to dryness yielding crystals of 2-phenyl-1:2:3-osotriazole-4-carboxylic acid, m.p. 190°, identical with a specimen prepared in the same way 137

from D-glucose phenyl-osotriazole and melting at 190°.

Preparation of D-glucose phenyl-osotriazole-6-tosylate.

Glucose phenyl-osotriazole (1 g.), prepared by treating glucose phenyl-osazone with copper sulphate, was dissolved in pyridine (3 ml.) and treated with tosyl chloride (1.1 g.) for 24 hrs. at room temperature. Water was then added (2 ml.) and the mixture extracted with ether and the extract washed with 0.5 N-HC1, 0.5 N-NaHCO and water successively. 3 The dried ether extract yielded on evaporation the crystalline tosylate (0.8 g.) which, on recrystallisation from ethanol, melted at 140o (Found: C, 54.43; H, 4.9. Cale. for C19H210,14'3S; C, 541440s. H, 5.05%). [ck]p = -50° (c . 1 in pyridine).

Preparation of 3:6-anhydro-glucose yhenyl-osotriazole from the tosylate.

The osotriazole 6-tosylate (0.2 g.) was dissolved in methanol (5 ml.) and refluxed with sodium hydroxide (0.1 g.) for a period of 30 mins; the mixture was then neutralised with 0.5 N-HC1 and evaporated to dryness. The residue was extracted with ether; evaporation gave the crystalline 3:6-anhydro-glucose phenyl osotriazole 138

m.p. 96°, not depressed on admixture pith authentic 3:6-anhydro glucose phenyl-osotriazole prepared from. 3:6-anhydro-glucose, on mixture with the osotriazole from Dieis, anhydro osazone melted at 93°.

Preparation of-3:6-anhydro-glucose phenyl-osotriazole from 3:6-anhydro-glucose phenyl-osazone.

The aahydro-osazone (1 g.), prepared from_3:6- anhydro-glucose and phenyl hydrazine, was suspended in water _(20 ml.) containing 0.8 g of copper sulphate_and refluxed for 30 mins. The cooled solution was then _ filtered and extracted with ether_and the extract dried over sodium sulphate and_evaporatedto dryness, The residue was recrystallised frnm ether-pet. ether (b.p. . 40-60°) and melted at 96-97°. (Found: C, 58.63; H, 5.0. Calc. for 012111303N3: C, 58.291 1_11 5.3%).

D = -40.0° (c = 1 in ethanol). The melting point of a mixture_ with the osotriazole obtained from Diels' anhydrosazone was 94°. 139

Reaction of Glucose methyl-phenyl-osazone with Copper sulphate.

Preparation of glucosone methyl -phenyl -hydrazone from glusoce methyl -phenyl -osazone.

Glucose methyl-phenyl-osazone (7.g.) was dissolved in dioxan (100 ml.) and_the solution added in portions to a hot solution of copper sulphate (5 g.) in water (10Q ml.).; after the initial vigorous reaction had subsided the_ mixture was refluxed for 10 minutes, and then filtered hot from the copper precipitated during the reaction. The cold solution was free from copper ions by passing in hydrogen sulphide until, on filtration, no mare copper sulphide was precipitated.. The solution, now free from copper ions, was treated_with barium carbonate, to eliminate sulphate ions, filtered and evaporated to dryness. The residue, on addition of few drops_of methanol, crystallised spontaneously and was purified by recrystallisation from hot water. _ The pure product, which was faintly yellow in colour, melted at 163° (decomp.) (Found: 0, 55.21; H, 6.6; N, 10.07. Calc. for 013H1805N2: 0, 55.31; H, 6.A4; N, 9.92%)

o< 20 = - 22.6 --* -16.5° (c = 1.15 in ethanol.) 140

Preparation of glucosone methyl-phenyl-hydrazone from - glucosone.

Glucosone (0.5 g.) was dissolved in water 5 ml.) and treated with methyl-phenyl-hydrazine (0.3 g.) in 5 ml. of 10% acetic acid. The mixture was kept at room,_, temperature for 15_mins. and then filtered from the resulting hydrazone. The latter was twice recrystallised from water, m.p. 162°; a_mixed melting point with_the hydra one described in the previous paragtaph showed no depression. (Found: 0, 55.71; H, 6.6. Cale. for 013 H18 05 N2 * • C, 55.31; H, 6.4%).

Preparation of glucosone methyl phenyl-hydrazone-tetra- acetate.

The hydrazone (0.1 g.) was dissolved in pyridine ml.) and treated with acetic anhydride (0.5 g.)_; the reaction mixture was kept for 24 hrs. at room temperature; water was then added (5_ml.) and the mixture_kept for another 20 mins. and then extracted with ether. The ether extract was washed with 0.5 N-E01, 0.5 N-NaHCO3 and finally with water, and then dried over sodium sulphite and evaporated. The residue crystallised on addition of a few drops of methanol_and was recrystallised from methanol The pure prOduct melted at 112° and. had 141

[rXj 20 = + 12.6 + 8.6° (c = 1.03 in ethanol) (Found: 0, 55.75; H, 5.9; N, 6.53. Calc.for 021 H26 09 N2'• C ' 56.00%; H, 5.8; N, 6.23%).

Preparation of glucosone methyl-phexyl-hydrazone tetrabenzoate.

The hydrazone_(0.1 was dissolved in pyrldine (l_ml. and treated with benzoyl chloride_(0.5 g.). The mixture was kept for 24 hrs. at room temperaturel_then treated with ethanol (2 ml,) and kept for a further 20 minx. The mixture was then extracted with other and the extract_ washed_with .0.5 N-11_01, 0.5 EN-NaH003 and water_cgnsecutively., and then dried and evaporated to dryness. The_ethylbenzoate was eliminated by distillation under reduced pressure (b.p. 45°, 0.0005 mm.); the_residue crystallised 2n addition of a few drops of methanol. The benzoate was recrystallised from ethanol, m.p. 187°, = 3.8° - 1.1° (c = 10.9 in chloroform). (Found: C, 70.63; H, 5.1; N, 4.33. Cale. for 041113909N2: C, 70.48; H, 4.9; N, 4.05).

`Preparation of glucose 1-phev1-2-methyl-phenyl-osazone from glucosone.2-methyl-phenyl-hydrazone.

Glucosone methyl-phenyl-hydrazone (1 g.) was dissolved 142

in water (5 ml.) and treated with a solution of phenylhydrazine (0.4 g.) in 5 ml. of 10% acetic acid. The mixture was left to react at 30° for 2 hrs. and the crystalline osazone which separated filtered off and twice recrystallised from alcohol-water; m.p. 203°, [c)(40 = —55.5 — 14° (c = 1.09 in pyridine). (Found: C, 61.56; H, 6.7; N, 15.22. Cale. for C19H2404N4: C, 61.26; H, 6.6; N, 15.05%).

Preparation of glucose 1-phenyl-2-metIvi-Thenyl-omazone from fructose methyl-phenyl hydrazone.

Fructose methyl-phenyl-hydrazone, m.p. 116°, (1 g.) was added to a solution of phenylhydrazine (1 g.) in 10 ml. of 5% acetic acid and the mixture heated on the water bath for 30 mins. After 5 mins. all the hydrazone had gone into solution and after 15 mins. the osazone began to separate. The osazone so formed was recrystallised from ethanol-water; it had m.p. 204° and on mixture with the osazone prepared from glucosone methyl-phenyl- hydrazone melted at 205° (Found: C, 61.13; H, 6.6. Calc. for C19 H240 4 N4* C, 61.26; H, 6.60; the product [0( had 10 = - 56° 15° (c = 1 in pyridine). 143

Preparation of Rlucose ?henylosotriazole from glucose 1-phenyl-2-methyl-phenyl-csazone.

Glucose 1 phenyl-2-methyl-phenyl-osazone (0.5 g.) was suspended in water (10 ml.) and added in portions to a boiling solution of copper sulphate (0.5 g.) in 12 ml. of water. After completion of the addition of the osazone, the mixture was refluxed for one hour, and then filtered hot. The filtrate was freed from copper ions by passing hydrogen sulphide into the solution until the filtrate gave no further precipitates with hydrogen sulphide. The solution was now treated with barium carbonate to eliminate sulphate ions. The precipitate was removed by filtration and the solution evaporated to dryness. On addition of few drops of methanol the osotriazole crystallised and was purified by recrystallisation from ethanol; the product had m.p. 196°, not depressed on admixture with authentic glucose phenyl-osotriazole prepared from glucose phenylosazone; the yield was 0.25 g. (67%). The product had.[D(h) = -80.7° (c = 1 in pyridint); lit. = 81.8°.

Preparation of g1ucosone from glucosone 2-methyl-phenyl- hydrazone.

The hydrazone (1 g.) was suspended in a solution of 144

copper sulphate (0.5 g.) in 15 ml. of water and the mixture refluxed for 30 minutes and then filtered. Hydrogen sulphide was passed into the solution to eliminate the copper ions; after 15 minutes all the copper was precipiated and the filtrate was then treated with barium carbonate to eliminate sulphate ions. The filtrate was now decolourised with charcoal and evaporated to dryness giving a colourless glassy mass of glucosone. To eliminate all traces of inorganic matter the crude osone was redissolved in 20 ml. of water and passed through a column of cation-exchange resin and then through another filled with anion-exchange resin. On evaporation of the solution 0.5 g. of glucosone were 4.tot obtained (y4cd1 79% of the theoretical). [c(,]D20 = —3o (c = 1.5 in pyridine).

Preparation of glucosone from glucose methyl-phenyl-osazone.

The osazone (2 g.) was suspended in a solution of copper sulphate (1 g.) in 2.5 ml. of water. The mixture oc was refluxed for a period e 45 mins and then filtered. A current of hydrogen sulphide was passed into the solution until no more precipitate was formed. The solution was then treated with barium carbonate and the filtrate, after treatment with charcoal, evaporated to 1A5

dryness giving a colourless glass which was treated with ion exchange resins (as on piaiWto remove all traces of inorganic matter. The final product weighed 0.7 g. (75% of the theoretical) and had [ c4, 13) 20= (c = 1.1 in water). The glucosone readily reacted with phenyl hydrazine in the cold forming glucose 'phenyl-osazone, m.p. 207°. To test the homogenity of the glucosone a spot was applied to Whatmann No.1 filter paper and chromatographed with collidine; on spraying with ammoniacal silver nitrate only one spot developed which had RE = 0.21; the RF value in phenol-water was 0.133; No movement occurred with butanol saturated with water.

Reaction of Glucose phenyl-osazone with heavy- metal salts.

1. Reaction with chlorides.

a) Ferric chloride.

Glucose phenyl osazone (1 g.) was suspended in a solution of ferric chloride (0.5 g.) in 20 ml. of water and the mixture refluxed for a period of 30 mins. The osazone went into solution after 5 mins. heating. The solution was filtered hot; on cooling crystals of glucose phenyl- osotriazole separated. The first crop of crystals was 146 collected by filtration; the solution was then concentrated to 5 ml. and a further crop of osotriazole was thus obtained. The combined yield was 0.6 g. (81% of the theoretical). The osotriazole, on recrystallisation, had m.p. 1960, not depressed by glucose phenyl-osotriazole obtained by treating glucose phenyl-osazone with copper sulphate.

b) Cuprous chloride.

Glucose phenyl-osazone (1 g.) was suspended in a solution of cuprous chloride (0.5 g.) in 20 ml. of water and refluxed for 2 hrs. The osazone had not passed into solution, so the heating was continued for another 6 hrs. without any sign of reaction; the solution was then filtered and the starting osazone recovered (0.8 g.), m.p. 2070, not depressed on mixing with pure glucose phenyl-osaone.

2. Reaction with sulphates.

a) Nickel sulphate.

The (=zone (1 g.) was suspended in a solution of nickel sulphate (0.5 g.) in 20 ml. of water and refluxed for 6 hrs.; no reaction took place and the osazone (0.8 g.) 14.7

was recovered from the solution.

b) Manganous sulphate.

The osazone was treated with manganous sulphate but no reaction took place after 6 hrs. refluxing.

Reaction of glucose Phenyl-osazone with silver nitrate.

Glucose phenyl-osazone (1 g.) was suspended in a solution of silver nitrate (1 g.) in 20 ml. of water and the mixture refluxed. After 5 mins. all the osazone had gone into solution; the heating was continued for 15 mins. and the solution then filtered from the precipitated silver. On cooling glucose phenyl- osotriazole crystallised out. A further crop of crystals was obtained by concentrating the solution to 10 ml. The combined yield of osotriazole was 0.55 g. (74% of the theoretical). The product melted at 196° and was not depressed on admixture with authentic glucose phenyl- osotriazole (m.p. 1960).

Typewriting by - Mildred Furst, 267 Goldhurst Terrace, London N.W.6. MAI.7479.