PART 2, SECTION 2 Biological and Chemical Reduction of ...shodhganga.inflibnet.ac.in/bitstream/10603/29814/15/16_section 2.pdf · In the course of conducting a structure-activity

PART 2, SECTION 2

Biological and Chemical Reduction of Hydantoin Derivatives

2.2.1 Introduction

Hydantoins (imidazolidine-2,4-diones) serve as important building blocks for

enantioselective amino acid synthesis because enantiomerically pure amino acids can

be prepared from these by dynamic kinetic racemic resolution.102-104 Hydantoins and

their bi- and tricyclic derivatives represent an important class of biologically active

molecules that have broad medicinal (anticancer, anticovulsant, antimuscarinic,

antiulcer, and antiarrythmic) and agrochemical (herbicidal and fungicidal)

applications.105-110 Hydantoin and some of their derivatives are structural units found

in the naturally occurring substances of marine organisms, and in bacteria. Figure 5

shows some of the natural products containing hydantoin moiety.

H~N~ NH

H B

3'- Deimino-3'-oxoaplysinopsin (27) (+)- Hydantocidin (28) (E)Axinohydantoin (29)

Naamidinene A (30) Phenytoin (31) (-) - Deltoin (32)

Mukanadin B (33) Midpacamide (34)

Figure 5: Natural products containing hydantoin moiety

128

Part 2, Scetion 2, Introduction

Examples for many alkaloids extracted from sponges or corals which contain a

hydantoin moiety (shown below) are the well-known aplysinopsins (27) with

cytotoxic properties, axinohydantoins (29) from Axinella, Hymeniacidon and

Stylotella species inhibiting protein kinase C, naamidinene A (30), a dehydro

hydantoin derivative from the genus Leucetta, and mukanadin B (33) from Agelas

species.111-117 Hydantocidin (28) is a spiro nucleoside from Streptomyces

hygroscopicus, which possesses herbicidal and plant growth regulatory activity due to

the inhibition of adenylsuccinate synthetase.118-

122

2.2.2 Solution phase synthesis of hydantoins and their derivatives

Numerous hydantoin syntheses, both in the solution phase and on solid

supports, have been reported in the literature. There are several approaches to

hydantoins starting from different building blocks. The most important principles of

hydantoin construction are shown in Figure 6.

Hydantoins can be formed from (a) ureas and carbonyl compounds, one of the

methods being Biltz synthesis, (b) According to the Bucherer-Bergs method, N-1 and

N-3 unsubstituted hydantoins can be generated by the reaction of a carbonyl

compound with inorganic cyanide and introducing a second nitrogen and a carbonyl

unit by ammonium carbonate, (c) the Read-type reaction of amino acids (esters) with

inorganic iso(thio)cyanates gives hydantoins with an unsubstituted N-3 position, (d)

the use of alkyl or aryl iso(thio )cyanates results in substitution at nitrogen N-3, (e)

amino amides already contain four ring atoms, and an introduced C-1 unit can

complete the hydantoin ring and (f) unsubstituted hydantoins are generated when a

halogen amides are reacted with inorganic iso(thio)cyanates.

A few number of solution phase synthesis for the preparations of hydantoin

libraries have appeared in the literature. The synthesis of small-molecule libraries in

solution is less popular than the solid-phase methodology, although the former offers

advantages e.g. ease of scaleup and does not require a potentially redundant handle

c: . l'nk 123 124 Al . . f . c: . h b 1or resm 1 age. ' so, vast maJonty o orgamc trans1ormatwns ave yet to e

optimized for the solid phase. The main advantage of solid-phase synthesis lies in

ease of product purification. However, this can often be conveniently accomplished

129


for reactions in solution by liquid-liquid partitioning or the use of scavengers to

remove undesired material. 125-127

~ ~ I

/

X H

(a) (b) (c)

~ m I

: 12 H R

(d) (e) (f)

Figure 6: Synthetic strategies and building blocks in hydantoin formation

From monocarbonyl compounds or carbon dioxide and ureas

In an attempt to examine to what extent substituted hydantoins can be made

directly from simple, inexpensive starting materials, a one-pot synthesis of 5-, 3,5-,

and 1 ,3,5-substituted hydantoins that is based on the carbonylation of aldehydes in the

presence of urea derivatives has been described. 128 Scheme 9 shows the palladium

based carbonylation. To what extent sulfonamide, urethanes and urea derivatives can

be used as amide components in the carbonylation of aldehydes with amides

(amidocarbonylation) in the presence of a palladium catalyst has been studied.129•130

The conversion of cyclohexanecarbaldehyde with the respective amide component

served as a model reaction. In order to demonstrate the utility of this reaction, the

amidocarbonylation of cyclohexanecarbaldehyde was examined with a variety of

substituted urea derivatives (Scheme 9). Indeed, free urea gave very good selectivities

(90%) for the hydantoin 35 when water-absorbing agents such as triethyl

orthoformate or acetic anhydride were used. Free N-carbamoyl amino acid (36) can

be prepared in good yields by simply combining one equivalent of water with the

corresponding hydantoin.131-135 Hydantoin 37 was obtained in very good yield (86%).

130


The reaction with symmetrical dimethylurea led to the 5-cyclohexyl-1 ,3-

dimethylhydantoin (38) in comparably high yield (80%).

Scheme 9

~ ")-N.cn, n,c),Ncn~ HN~-cH3 z H H H H3?~-cH3

0 :JL 0 37 38

86% :··0<:·------·------------------ --~-------··: 80%

0 i c i 0 ~ : : ~

H2N NH 2 i ~ + i H2N NHz CH(OEt)j'··----------!P.d),-Br-.,.H------------· H

20

~H 0

35 90%

35 + 36 25% 45%

~H HN~NH2

0

36

55%

In the course of conducting a structure-activity relationship (SAR) study,

hydantoin 40 has been prepared by cyclizing (R)-amino amide 39 in the presence of

1, 1-carbonyldiimidazole (CDI) (Scheme 1 0). But the cyclization proceeded with

complete racemization.

Scheme 10

39 40

Initially, the base i.e. triethylamine was thought to be responsible for

racemization, so the reaction was tried with CDI in the absence of base, some

racemization still occurred, reducing the enantiomeric excess to 70%. In addition, the

reaction was sluggish and only gave the hydantoin product in 50% yield. Triphosgene,

131


was then selected as a coupling reagent. 136 This resulted in cyclization of 39 to give

enantiomerically pure hydantoin 40.

From a-dicarbonyl compounds and ureas

Here, Biltz synthesis is employed to generate hydantoin moiety containing

pharmaceutically important products. Phenytoin and phenytoin derivatives have been

synthesized by irradiating an alkaline mixture of (thio)ureas and benzils in DMSO

with 750 W microwave pulses (Scheme 11).137'138 A solvent-free microwave-assisted

synthesis of disubstituted hydantoins and thiohydantoins is also reported in

literature. 139

Scheme 11

41

H R'GrNlNH2 X=O,S

42

PPE _. MW R~~ I ~ )::::/

~X H

43

Arylglyoxals 41 when reacted with phenylurea or phenylthiourea 42 and

polyphosphoric ester as reaction mediator produced the corresponding disubstituted

hydantoin/thiohydantoin, 43. Biltz synthesis occurs by pinacol-pinacolone

rearrangement. There are other reactions between a-dicarbonyl compounds and ureas

building hydantoin derivatives which deviate from the mechanism of the Biltz

synthesis. Ishii et.al. have illustrated the condensation of oxalyl chloride with

monosubstituted ureas to form 2,4,5-trioxoimidazolidines, which represent substituted

parabanic acids. 140 Ring opening of a carbamoylizatin derivative by urea gave the

oxalylurea analogue, which could be cyclized in two different mechanisms: (i) first

generating the quinazolin-2-one unit and followed by fonnation of the hydantoin ring

under acidic conditions or (ii) first forming the hydantoin moiety and followed by

generation of the quinazolin-2-one ring using primary amines.

132


Methods based on the Bucherer-Bergs synthesis

The Bucherer-Bergs synthesis is a practical and suitable route to synthesize

hydantoins. The synthesis involves the reaction of carbonyl compounds with

potassium cyanide and ammonium carbonate. The aldose reductase inhibitor, sorbinil

(46), has been prepared from benzopyranone (44) as shown below (Scheme 12). 141

Scheme 12

KCN, (NH4hC~ C2H 50H, H 20

~NH ~NH F~ F'CQN ~ 1. Brucin

~ --------~~ ~ ,~ 2. HCI

44 45 46

The synthesis of PET ligands for tumor detection via hydantoins has been

reported in literature.142 Ultrasonication accelerates hydantoin formation using the

Bucherer-Bergs reaction. 143 a-Amino nitrites have been reacted with carbon dioxide to

give the disubstituted ureas which underwent cyclization in water at room temperature

followed by hydrolyzation of the imino compounds to the corresponding

h d . 144-145 y antoms.

Methods based on the Read synthesis

A frequently applied method for preparation of (thio) hydantoins is the Read

synthesis. 146-147 During their efforts to obtain silicon-containing hydantoins 49, Smith

eta!. treated silylated amino acids 47 with potassium cyanate in pyridine followed by

acid cyclization as shown (Scheme 13). 148

Scheme 13

49

Access to the 5-methylenehydantoin has been achieved by conversion of

cystine via a double Read synthesis and cleavage of the dimer under standard

alkylation conditions. 149

133


From amino acids or esters and isocyanates

Hydantoins can be prepared by treatment of a-amino acids with aryl or alkyl

isocyanates via the intermediate ureido acids. Esters or amides of a-amino acids and

even peptides can also act as starting materials. The Edman degradation has been

varied that heterocyclic modification of the N-terminus of a peptide takes place.150

Thus, the thiourea formed from the amino acid and the aryl isocyanate was subjected

to dehydrothiolation reaction and subsequent trapping of the intermediate

carbodiimide by the adjacent amide nitrogen resulting in a small library of 2-

iminohydantoins. The intermediate ureido acids are reported to undergo both acid

catalyzed and base catalyzed cyclizations. 151-156

From amino acid amides and carbonic acid derivatives

Hydantoins can also be obtained from amino acid amide. 157 Coupling Boc

protected amino acids to primary amines and subsequent deprotection affords the

desired amino acid amides, which can be cyclized with carbonyldiimidazole (CDI).

This cyclization strategy has been used in solid-phase synthesis.

Miscellaneous conversions of carboxamides

Cyclopropane dicarboxylic acid derivatives undergo Hofmann rearrangement

to form 1 ,3-unsubstituted hydantoins (Scheme 14). 158

Scheme 14

50

~ONHBr

~ONHBr

51

Conversions of other heterocyclic compounds to hydantoins

Conversion reactions from three-membered rings

~ H

52

1,5-Disubstituted hydantoins 55 could be prepared from reacting aziridinones

53 with NH2CN and treatment of the formed iminohydantoins 54 with HN02

(Scheme15).159

134


Scheme 15

55

Conversion reactions from other five-membered rings

Transformations of other five-membered rings to hydantoins are important for

the synthesis of naturally occurring compounds with a hydantoin moiety.

Pyrroloazepinones containing a 2-imidazolone substituent have been synthesized and

oxidized by three equivalents of bromine to afford the axinohydantoin derivatives 57

and 58 as shown in (Scheme 16). 115

Scheme 16

56

Acolr B NaOAc

45%

57

+

Ring contraction reactions from six-membered rings

B

35%

58

Ring contraction reactions from six-membered nngs to hydantoins started

from pyrimidine derivatives, such as barbiturates. A photochemical conversion of 5-

allyl( ethyl)-1-methyl-5-phenylbarbituric acid to 5-allyl( ethyl)-3-methyl-5-

phenylhydantoin has been described, the reactions involved the loss of carbon

monoxide. Another approach is based on the new aminobarbituric acid-hydantoin

rearrangement.160-161 First, diethyl acetamidomalonates were treated with ureas and

the intermediate 5-acetaminobarbituric acids 59 undergoes rearrangement to yield 5,

5-disubstituted hydantoins 60 in a one-pot synthesis as shown (Scheme 17).

135

Scheme 17

59


OR

R~ H

60

t

An efficient one-pot procedure for the preparation of hydantoins and

thiohydantoins is depicted in Scheme 18 (X =0, S).162 The synthesis begins with

imine formation between an (R)-amino acid ester 61 and an aldehyde 62, followed by

in situ reduction by sodium triacetoxyborohydride to secondary amine 63. An

isothiocyanate 64 is then added to give a thiourea intermediate 65, which cyclizes to

the thiohydantoin 66. This sequence occurs in nearly quantitative yields with

stoichiometric reagent quantities.

Scheme 18

H Rz-CHO (62) RtryR1

NHz Na(OAc))BH

61

66

136

RJtyR, NH......,...R2

63

I RrNCX(64) fEt3N

65


2.2.3 Solid-phase syntheses

Solid-phase synthesis of structurally diverse, non-peptidic heterocycles

bearing one or more nitrogen atoms, in particular, the synthesis of small organic

molecules which have improved pharmacological properties over peptides is a major

focal point in search of leads utilizing automated high-throughput screening (HIS).

The hydantoin scaffold is quiet often selected as it provides a chemically tractable

molecular framework. Cyclization and cleavage (involved in the solid phase

synthesis) from the resin typically occurrs in two ways: (i) by cyclo-elimination, i.e.

cyclization of the acyclic resin-bound compound and spontaneous autocleavage and

(ii) by performing cyclization and cleavage in separate steps. The reactions are

divided into two groups.

Cyclo-elimination release strategies

(i) Acid-catalyzed cyclizations

DeWitt et.al. reported synthesis of hydantoins on a solid support in 1993

using both a combinatorial and automated approach.163 A resin-bound amino acid

(linked to the resin through C-terminal ester functionality) was linked with a variety

of substituted isocyanates to generate the urea precursor. The desired products were

cyclized and spontaneously cleaved with strong acid at elevated temperatures.

Apart from polystyrene Wang resin used by Dewitt et.al., other polymers for

the acid-catalyzed cyclo-elimination release of hydantoins have also been used eg 2-

polystyrylsulfonyl ethanol support and high-loading radiation grafted polymers. 164-165

(ii) Base-catalyzed cyclizations

Analogous to the synthetic route employed by DeWitt et al. for the acidic

cyclo-elimination, Kim et al. applied milder, basic cleavage conditions using neat

diisopropylamine at room temperature.166 This method is fast (less than 1 h),

convenient, mild, and affords high yield and purity.

Separate cyclization and cleavage steps

(i) Cyclizations induced by carbonyldiimidazole or phosgene derivatives

Primary or secondary amine functionalities of amino acids have been treated

with carbonyldiimidazole or triphosgene to fonn intermediate isocyanates which then

underwent a ring closure reaction to yield the corresponding hydantoins. 167 Cleavage

137


of the obtained di- or trisubstituted hydantoins resulted from treatment of the resin

with HF /anisole in a separate step as shown (Scheme 19). Instead of triphosgene, use

of diphosgene in solid-phase hydantoin synthesis has also been reported.168

Scheme 19

~R, H R, W'

' N)fNH2 Triphoge"i, '}N NH H 0 orCDI H lr

-~O N NH Boc removal and acylati~ ~ )( HF/ anisole 0

BocNH BocNH

HN

~Rz 67 68 69

(ii) Other separate cyclization and cleavage steps

Attaching aldehydes to solid support, e.g. a 5-hydroxymethylfurfural template

and reacting them stepwise with an amino acid, NaBH3CN and an isocyanate leading

to the formation of the hydantoin ring after treatment with a base has been reported.169

Release from the resin was performed with TF A. Heine et al. introduced a spot

hydantoin synthesis on cellulose membranes.170 Acid treatment led to the cyclization

of ureas to hydantoins. Depending on the linker type chosen, simultaneous cleavage

occurred or release from a photo-linker was achieved by irradiation.

2.2.4 Reactivity of hydantoins and their derivatives

Hydrolysis of hydantoins

Hydrolysis of hydantoins can be performed either in an acidic or basic

medium. C-5 substituted hydantoin derivatives are of synthetic utility as precursors to

a-amino acids. The hydrolytic degradation proceeds through the intermediacy of

ureido acids. This can be performed by biocatalytic conversion, e.g. using microbial

or plant hydantoinases to produce ureido acids. The further transformation to amino

acids can then be catalyzed by other enzymes or acids. 171-172 The formation of amino

acids from hydantoins can be achieved non-enzymatically also. Rare unnatural amino

acids can be prepared from hydantoins both under acidic or basic conditions.

Aminobicyclo[2.2.1 ]heptane dicarboxylic acids have been prepared from

spirohydantoins by acidic hydrolysis. 173

138


N-Alkylations with electrophilic reagents

N-unsubstituted hydantoins can easily be monoalkylated at the imide nitrogen

in position 3, whereas substitution of both nitrogens in one step requires much harder

conditions. Alkylation at amide N-1 could be done after first protecting the N-3.174-175

N-3 alkylation of hydantoins is a commonly applied reaction to modify the core

scaffold.

N-Alkylations by Mitsunobu coupling

Hydaritoins have been shown to react with 4-nitrophenyl alcohol or 5-ethyl

alcohol trytamine derivative under Mitsunobu conditions (Scheme 20). Similarly,

Mitsunobu couplings have also been shown in the synthesis of novel P2X receptor 0 176 antagomsts.

Scheme 20

HN-f ~H

70 71

Aldol-type reactions

Hydantoins having a free methylene group in the C-5 position can be

condensed with aldehydes resulting in C-5-unsaturated compounds. Synthesis of the

aplysinopsin derivative is shown (Scheme 21 ).

Scheme 21

or+ H

CH3 _/fJ N\ Pyridine,Reflux

)rN'cH, 73 74 75

139


Cycloaddition reactions of hydantoins

Diels-Alder reaction of a 5-methylene hydantoin (R=(S)-1-phenylethyl) acting

as dienophile with cyclopentadiene acting as diene has been reported (Scheme 22). 177

Scheme 22

76 77

Other reactions of hydantoins

l.Lewis acid, -70 °C, 1 h

2. 0°,3 h, DCM

78

5,5-Disubstituted hydantoins have been shown to react with 3-

(dimethylamino)-2,2-dimethyl-2H-azirine to give 4H-imidazoles in a very complex

ring transformation reaction (Scheme 23).

Scheme 23

79 80 81

Complexation of hydantoins with metal ions

Interactions ofhydantoins with metal ions, such as copper(II) (Zwikker test) or

cobalt(Il) (Parri test) are widely used in colour reactions for identification.

Platinum(II) complexes with 5-methyl-5-phenylhydantoin (83) have been synthesized

and found to be effective in cytotoxicity tests (Scheme 24).178

Scheme 24

r-=\. ~NH AgN03 ~N~o -c-is---[P-t(_N_H.;_3_h_C-112~] H

82 83

140

Part 2, Scetion 2, Results and discussion

Among other transition metal complexes with hydantoin ligands iron (II),

Nickel (II), copper(ll) and gold (I) complexes have been synthesized and

characterized.179-182 Moreover, the complexations of 5,5-diphenylhydantoin or

hydantoin itself with silver (1), zinc (II), and cadmium (II) ions or with antimony (V)

and mercuric (II) ions have also been reported.

2.2.5 Present work: Results and Discussion

The objective of the present work was to study the biological and chemical

reduction of the carbonyl group of hydantoin derivatives. Hydantoin derivatives are

important industrial intermediates especially for the preparation of natural and non

natural amino acids. Synthesis of hydantoin derivatives is well established (Section

2.2.2). Conversion of hydantoins to corresponding carbamyl derivative or a-amino

acids by enzymatic and chemo-enzymatic methods has been well documented

(Section 2.2.4). But, till date, no report has appeared in literature that documents the

study of enzymatic reduction of carbonyl function of hydantoin derivatives. We

envisaged that the reduction of carbonyl group of a suitably substituted hydantoin

could lead to a quick entry into optically active cyclic, heterocyclic and alicyclic 1 ,2-

diamines as described in Scheme 25.

Scheme 25

84

H2N~NH2 ----~-~

X H 0'Y COOH

Jl =~tN NR~ Xn, H

Y OOH

85 86

R~o R2 =protecting groups X= CH2, n = I or 2 Y= CH2 or heteroatom

87

Moreover, oxidation-reduction cycles could lead to optically active 1 ,2·

aminoalcohols 92 and a-amino aldehydes 90 (Scheme 26). In addition, an alternate

route for the preparation of a-amino acids 94 is also feasible.

141

Scheme 26

0

HN)lNH

RKO 88

Reduction

Part 2, Scetion 2, Results and discussion

0 Jl HN)lNH :::;;:=~::: HN NH2 Reductiolj. ~ ~CHO

If "oH If 89

94

90

! Oxid•tion

0

HN)lNH2

r_cooH 93

0

HN)lNH2

rCH20H

91

!

We initiated our work with the study of chemical and enzymatic reduction of

the carbonyl group of 1,3-dibenzyl-5-methylhydantoin (96) and 3-benzyl-5-

methylhydantoin (97). Compounds 96 and 97 were prepared by benzylation of 5-

methylhydantoin (95) (Scheme 27). 5-methylhydantoin (95) was prepared by a two

step literature method starting from racemic alanine. 183 Hydantoin 95 was purified by

crystallization and characterized by 1HNMR and IR spectral data. In NMR, the 5-

methyl group of 95 resonated as a doublet at 8 1.39 (J=6.6 Hz), while the quartet for

the methine proton appeared at 8 4.15 (J=6.6 Hz). IR spectra showed bands for

carbonyl stretching at 1720 and 1740 cm-1•

Benzylation of 95 with 2.5 equivalent of benzyl bromide in the presence of

potassium carbonate in DMF produced dibenzyl derivative, 96 and monobenzyl

derivative, 97 in 9:1 ratio (Scheme 27a).

Scheme 27

HNJl.NH

0 0

DMF,K2C03 Jl. Jl. (a) K Benzyl bromide (2.5 eq) ~N N:'O + HN N:'O

H3C 0 overnight stirring at rt H3CHO H3CHO h

95 96 97 96/97 = 9:1

HNJl.NH

0 0

DMF,K2C03 Jl. Jl.

(b) K Benzyl bromide (1.1 eq) ~N N:'O + HN N:'O

H3C 0 overnight stirring at rt H3CHO H3CHO h

95 96 97 96/97 = 2:8

142

Part 2, Section 2, Results and discussion

To obtain monobenzyl derivative as maJor compound, the reaction was

repeated under same conditions using 1.1 equivalent of benzyl bromide. Under these

conditions 96 and 97 were obtained in the ratio of2:8 (Scheme 27b).

The compounds 96 and 97 were separated by flash chromatography (ethyl

acetate/hexane, 1 :3). The faster moving fraction in the flash chromatography was

identified as N,N-dibenzyl-5-methylimidazolidine-2,4-dione (1,3-dibenzyl-5-

methylhydantoin, 96) based on 1 HNMR spectral data. Doublet for the -CH3 protons at

C-5 appeared at 8 1.27 (1=7.1 Hz) while the -CH proton appeared as quartet at 8 3.68

(1=7.1 Hz). An interesting observation made in the 1HNMR spectrum of this product

was that the methylene protons of the benzyl group not only at N-1, but also at N-3

showed geminal coupling and appeared as separate doublets because of the presence

of an asymmetric carbon centre at C-5. The protons of the methylene group present on

the N-1 appeared as two doublets (1=15.2 Hz) at 8 4.07 and 8 4.88 while the protons

of the methylene group present on N-3 appeared as doublets at (1=18 Hz) 8 4.62 and 8

4.56. The aromatic protons appeared as multiplets at 8 7.2-7.4.

The slow moving fraction obtained from the flash chromatography was

identified as N-benzyl-5-methylimidazolidine-2,4-dione (3-benzyl-5-

methylhydantoin, 97) on the basis of 1 HNMR spectral data. Doublet for the -CH3

protons at C-5 appeared at 8 1.01 (1=6.6 Hz) while the methine proton appeared as

quartet at 8 3.75 (1=6.6 Hz). The methylene protons ofthe benzyl group appeared as a

singlet at 8 4.63. The ureido proton appeared at 8 6.3 as a singlet while the aromatic

protons appeared at 8 7.18-7.28.

2.2.5.1 Sodium borohydride reduction of 1,3-dibenzyl-5-methylhydantoin (96)

Initially sodium borohydride reduction of dibenzyl derivative, 96 was studied

(i) to understand the behaviour of carbonyl group at position 4 and (ii) to obtain

standard samples of alcohols, if reduction occurs. Moreover, the behaviour of 4-

carbonyl group in borohydride reduction would have implications for the enzymatic

reduction since borohydride and NAD(P)H reductions exhibit several similarities.

The reduction of 1 ,3-dibenzyl-5-methylhydantoin (96) was done in the

presence of 0.5 eq. of sodium borohydride in ethanol at 0 °C (Scheme 28). Progress of

the reaction was monitored by TLC and the the products were identified by 1 HNMR

spectroscopy. The chemical reduction resulted in the formation of two diastereomeric

alcohols, 98 and 99, in approximately 1:1 ratio, structures of which were confirmed

143


by 1HNMR of the crude product. Methyl protons at C-5 for the two diastereomers

appeared as doublet at 8 1.26 (J=6.6 Hz) and 8 1.02 (J=6.6 Hz). Doublet of quartet for

C-5 proton for the two diastereomers resonated at 8 3.25 (J=6.6 and 7.1 Hz) and 8

3.18 (J=6.6 and 8.0 Hz). The doublet for the single proton at C-4 was observed at 8

4.28 (J=6.9 Hz) and 8 4.27 (J=7.8 Hz). The -OH protons appeared as broad singlet at

8 3.27 and 8 1.72. The doublets for the methylene protons at N-1 for the diastereomers

appeared at 8 4.13 (J=6.9 Hz) and 8 4.62 (J=6.9 Hz) and 8 4.10 (J=7.1 Hz) and 8 4.60

(J=7.1 Hz) and the doublets for the methylene protons at N-3 were observed at 8 4.7

(J=6.9 Hz) and 8 4.87 (J=6.9 Hz) and 8 4.4 (J=7.2 Hz) and 8 4.54 (J=7.2 Hz). The

aromatic protons were observed as multiplet at 8 7.0-7.35. The crude product also

showed the presence of trace amount of starting material 96.

Scheme 28

Jl:tCJ N\. __ _l 1 NaBHiEthanol ~ h ooc

H3C 0

0 0

~~'U) + 0~)~-u) H3C OH H3C OH

96 98 99

Crude product was subjected to flash chromatography over silica-gel (ethyl

acetate/hexane, 3 :7). The following fractions were isolated.

Fraction 1: The fastest moving fraction corresponded with the unreacted starting

material, 1,3-dibenzyl-5-methyl-imidazolidine-2,4-dione (96), confirmed by 1HNMR

spectral data.

Fraction 2: The second fraction was assigned the structure, 1,3-dibenzyl-5-methyl-

4,5-dihydroimidazol-2-one (100) based on 1HNMR spectral data, which showed the

methyl protons as a singlet at 8 1.86. The olefinic proton appeared as a singlet at 8

5.77. The methylene protons of the benzyl groups at N-1 and N-3 resonated at 8 4.67

and 8 4.74 each as a singlet while the aromatic protons were observed as multiplet at 8

7.0-7.35. This product was not present in the crude mixture, suggesting that a silica

gel mediated dehydration of alcohol has occurred on the column.

Fraction 3: The slowest moving fraction was a mixture of diastereomeric alcohols,

the 1HNMR of which has been described above.

144


The diastereomeric alcohols were very labile and prone to undergo

dehydration over silica-gel. Doping the silica-gel with triethylamine did not prevent

dehydration. The compound was unstable even at 4 oc and dehydrated within 48 h of

storage, presumably due to the presence of trace amounts of acetic acid left after ethyl

acetate evaporation (Scheme 29).

An interesting observation made during the course of this work was that when

a solution of sample in CDCh was scanned for 1 HNMR after standing for about two

h, one of the diastereomers (slow moving compound on TLC) almost completely

disappeared in favour of the olefin 100, leaving behind the second diastereomer

(faster moving compound on TLC)

Scheme 29

Jl C(:N N:)) H3C)---lz,"OH

98/99

Silica gel chromatography

or Storage at 4°C for 48 h

100

Based on this observation, the faster movmg compound on TLC was

tentatively assigned cis-structure, 99. Its 1HNMR spectral data (abstracted from the

crude mixture, which contained starting material, olefin and the alcohol) 1s giVen

below:

Doublet for the -CH3 appeared at 8 1.26 (1=6.6 Hz) and the doublet of quartet

for C-5 proton was resonated at 8 3.25 (1=6.6 Hz and 7.1 Hz). The doublet for the

proton at C-4 was observed at 8 4.28 (1=6.9 Hz). The -OH group at C-4 appeared at 8

3.27 as a broad singlet. The doublets for the methylene protons at N-1 appeared at 8

4.13 (1=6.9 Hz) and 8 4.62 (1=6.9 Hz) and the doublets for the methylene protons at

N-3 were observed at 8 4.7 (1=6.9 Hz) and 8 4.87 (1=6.9 Hz). The aromatic protons

appeared as multiplet at 8 7.21-7.35.

Manual subtraction of the peaks of tentatively assigned cis-alcohol from the

mixture of diastereomeric alcohols produced the spectral data for the trans-alcohol,

98. Its 1HNMR spectral data is given below:

145


Doublet for the C-5 proton appeared at () 1.02 (1=6.6 Hz) and the doublet of

quartet for the C-5 proton was seen at() 3.18 (1=6.6 Hz and 8.0 Hz). The doublet for

the single proton at C-4 was observed at () 4.27 (1=7.8 Hz). The -OH group at C-4

appeared at () 1. 72 as a broad singlet. In this case also, the methylene protons of the

benzyl groups at N-1 and N-3 showed geminal coupling and appeared as separate

doublets. The doublets for the methylene protons at N-1 appeared at & 4.10 (1=7 .1 Hz)

and () 4.60 (1=7.1 Hz) and the doublets for the methylene protons at N-3 were

observed at() 4.4 (1=7.2 Hz) and() 4.54 (1=7.2 Hz). The aromatic protons appeared as

multiplet at () 7.17-7 .26.

2.2.5.2 Enzymatic reduction of 1,3-dibenzyl-5-methylhydantoin (96)

(i) Strain selection

Purified cultures of more than 300 bacterial and 200 fungal strains isolated

from different soil samples were screened for their ability to reduce the carbonyl

functionality of hydantoins and their benzyl derivatives. Microbial cells (0.5 g) were

suspended in 20 mM phosphate buffer, pH 7.5 (5 mL) and 5 mg of 1,3-dibenzyl-5-

methylhydantoin was added from a stock solution in DMSO to the cell suspension

and the contents were incubated at 30 oc (fungal) or 37 oc (bacterial) at 200 rpm.

Progress of the reaction was monitored by TLC using samples of, 98, 99 and olefin

100 obtained by the borohydride reduction of96 as standards. An aliquot of 1 mL was

withdrawn from the reaction mixture and extracted with ethyl acetate (1 mL). The

organic layer was used for TLC. Two strains, one bacterium and one fungal, were

able to reduce the carbonyl of the substrate 96.

(ii) Strain identification

The fungal strain was a known strain, previously isolated in our laboratory for

the enantioselective reduction of ethyl 4-chloro-3-oxobutanoate. The strain has been

previously identified as Penicillium funiculosum and has been assigned accession

number MTCC 5246. 184

The bacterial strain, designated as B4W is a new isolate from the soil samples

collected from Lothal, Gujrat. The strain has been identified as Bacillus pumilis based

on morphological, biochemical characterization (Table 1) and full 16s rDNA

sequence. Strain has been assigned accession number MTCC B6033.

146


Table 1: Summary of the results of the morphological, biochemical and physiological tests conducted on strain B4W

Morphological Characteristics Rods, Gram's positive, Motile, Opaque colonies, Motile, Endospore forming.

Biochemical and Physiological Tests

1. Growth on MacConkey agar 16. Acid production from a) lactose fermentor (-) carbohydrates b) non lactose fermentor (+) Adonitol (-)

2. Indole test (-) Arabinose (+) 3. Methyl red test (+) Cellobiose (+) 4. Voges Proskauer test (+) Dextrose (+) 5. Citrate utilization (+) Fructose (+) 6. Casein hydrolysis (+) Galactose (-) 7. Starch hydrolysis (+/-) Inositol (-) 8. Urea hydrolysis (-) Lactose (-) 9. Nitrate reduction (+/-) Maltose (-) I 0. H2S production (-) Malibiose (-) 11. Oxidase test (+) Raffinose (-) 12. Catalase test (+) Sialcin (+) 13. Oxidation/fermentation (-) Sorbitol (-) 14. Gelatin liquefaction (+) Sucrose (+) 15. Arginine dihydroxylase (+) Trehalose (-)

17. Growth temp. 15-42 °C 18. Growth pH 5.7-11.0 19. NaCl tolerance 2.5-10%

16 s rDNA Analysis

The chromosomal DNA of strain B4W was isolated according to the procedure

described by Rainey et a/. 185 The 16S rRNA gene was amplified with primers 8-27f (5'

AGAGTTTGATCCTGGCTCAG-3') and 1500r (5'AGAAAGGAGGTGATCCAGGC-

3'). The amplified DNA fragment was separated on 1 % agarose gel, eluted from the

gel and purified using QIAquick gel extraction kit (Qiagen, Germany). The purified

PCR product was sequenced with four forward and three reverse primers namely 8-27f

(5'AGAGTTTGATCCTGGCTCAG-3'), 357f (5'-CTCCTACGGGAGGCAGCAG-'),

704f5'-TAGCGGTGAAATGCGTAGA-3'), 1114f( 5'- GCAACGAGCGCAACC-3' ),

685r (5'-TCTACGCATTTCACCGCTAC-3'),1110r (5'-GGGTTGCGCTCGTTG-3')

and 1500r (5'-GAAAGGAGGTGATCCAGGC-3'), respectively (Escherichia coli

numbering system). The rDNA sequence was determined by the dideoxy chain

termination method using the Big-Dye terminator kit using ABI 310 Genetic Analyzer

(Applied Biosystems, USA). The 16S rDNA sequence of the strain B4W generated in

this work (1352 bases; Figure 7) was aligned with the 16S rDNA sequence of other

closely related members of the genus Bacillus. A sequence similarity search was done

147


using GenBank BLASTN .186 Sequences of closely related taxa were retrieved; aligned

using Clustal X programme187 and the alignment was manually corrected. For the

neighbour-joining analysis, 188 the distances between the sequences were calculated

using Kimura's two-parameter model.189 Bootstrap analysis was performed to assess the

confidence limits ofthe branching.190 The results are summarized in Figure 8.

TGCANTCGAGCGGANAGAAGGGAGCTTGCTCCCGGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGT AACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCAT GGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTG AGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAG ACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGC AACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCAAGAG TAACTGCTTGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAA TACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGA TGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAG TGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCT GGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACG CCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCAC TCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGA GCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAG ATAGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATG TTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTNAGTTGGGCACTCTAAGG TGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGC TACACACGTGCTACAATGGACAGAACAAAGGGCTGCGAGACCGCAAGGTTTAGCCAATCCCACAAATCT GTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAG CATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC

Figure 7: 1352 bases generated by the 16S rDNA sequence of the strain B4W

0.01 Bacillus licheniformis NCOO 1772 T (X60623)

acillus atrophaeus NCIMB 12899T (X60607)

99 Bacillus amylo/iquefaciens A TCC 23350T (X60605)

51 Bacillus pumilus NCOO 1766T (X60637)

100 Strain B4W

50 '----------Bacillus cereus lAM I2605T (016273)

.-----------Bacillus coagulans JCM 2257T (078313)

'--------Bacillus badius ATCC 14574T (X77790)

,------Bacilus circulans lAM 12462 T (078312)

'-------Bacillus flrmus JCM 2512 T (078314)

'-----------Bacillus inso/itus OSM5T (X60642)

Figure 8: Neighbour-joining tree based on 16S rDNA (1352 bases) sequences showing the phylogenetic relationship between strain B4W and other closely related species of the genus Bacillus. Bootstrap values (expressed as percentage of 1000 replications) greater than 50% are given at the nodes. Bar, 1 %sequence variation.

148


In a parallel programme on screemng of microorganisms for vanous

biocatalytic activities in our laboratory, the strain B4W has also shown excellent

sulphide oxidation activity. Characterization of enzyme and its application in organic

synthesis is the subject matter of another thesis submitted to JNU, New Delhi (Shefali

Sangar, 2007).

2.2.5.3 Bacillus pumilis catalyzed reduction of 1,3-dibenzyl-5-methylhydantoin

(96)

Cells of Bacillus pumilis (5.0 g) were washed with phosphate buffer, pH 7.5

and resuspended in 20 mM phosphate buffer (pH 7.5, 50 mL). 50 mg of 1,3-dibenzyl-

5-methylhydantoin (96) was added from a DMSO stock solution to the cell

suspension and the contents were incubated at 3 7 °C at 200 rpm. Progress of the

reaction was monitored by TLC for which an aliquot of 5 mL was withdrawn from the

reaction mixture and extracted with ethyl acetate (5 mL). The organic layer was used

for TLC. Final work up of the reaction mixture involved extraction of the reaction

mixture with ethyl acetate (2x40 mL). The organic layer was separated, washed twice

with brine solution, dried over sodium sulphate and evaporated under reduced

pressure. Structures of the products were confirmed by 1HNMR spectroscopy.

The Bacillus pumilis catalyzed reduction of 1,3-dibenzyl-5-methylhydantoin

(96) resulted in the exclusive formation of one diastereomer of 1,3-dibenzyl-4-

hydroxy-5-methylhydantoin. On TLC the alcohol corresponded with the slow moving

diastereomer obtained in sodium borohydride reduction and was therefore assigned

trans-structure 98 (Scheme 30).

Scheme 30

96

Bacillus pumilis Phosphate buffer, pH 7.t;

16 h

149

98

100


The 1HNMR spectral data of the biocatalyzed product corresponded with the 1HNMR spectral data assigned to the trans diastereomer in borohydride reduction. No

olefin was produced under the reaction conditions. But storage of the pure sample

after evaporation of ethyl acetate resulted in dehydration to alkene 100, the 1HNMR

spectra of which corresponded with the spectra of alkene obtained in borohydride

reduction.

2.2.5.4 Sodium borohydride reduction of 3-benzyl-5-methylhydantoin (97)

Next we investigated the reduction of 3-benzyl-5-methylhydantoin (97) with

sodium borohydride. The reaction was done in absolute ethanol at 0 °C. But to our

surprise, desired alcohols could not be obtained by the chemical reduction of 3-

benzyl-5-methylhydantoin (97) even after many attempts. Instead, complete reduction

of the carbonyl functionality at C-4 occurred tb form 3-benzyl-5-methyl

imidazolidine-2-one (101; Scheme 31).

Scheme 31

N aBH4/Ethanol

0°C

101

Structure of the product 101 was confirmed by 1HNMR spectroscopy in which

doublet for the -CH3 appeared at 8 1.01 (1=6.6 Hz) and the methine proton was seen

as a multiplet at 8 3.75. The two methylene protons at C-4 showed germinal coupling

because of the presence of an asymmetric centre and thus each proton appeared as

doublet of a doublet at 8 3.45 (1=10.8 Hz, 3.9 Hz) and 8 3.33 (1=10.8 Hz, 6.9 Hz).

The methylene protons of the benzyl group were observed as a singlet at 8 4.63. The

aromatic protons appeared as multiplet at 8 7.18-7 .28. The -NH proton appeared as a

broad singlet at 8 5.3.

The results may be explained as shown in Scheme 32. Sodium borohydride

reduction of 97 may initially produce 102, which undergoes rapid dehydration to

produce olefin 103a, which inturn exists in ene-amine tautomeric forms. The imine

form 103b is further reduced with sodium borohydride to give the product 101.

150


Overall, it is an interesting result as carbonyl group has been reduced to methylene

group with sodium borohydride under very mild conditions.

Scheme 32

97

NaBHiEthanol

0°C

102 103a

2.2.5.5 Bacillus pumilis catalyzed reduction of 3-benzyl-5-methylhydantoin (97)

The bacterium was grown in rich medium comprising of peptone (0.5%), meat

extract (0.2%), yeast extract (0.1 %) and NaCl (0.5%) for 22 hr. The cells were

separated by centrifugation and washed with phosphate buffer (20 mM, pH 7 .5). Cells

(5.0 g) were suspended in 20 mM phosphate buffer (pH 7.5, 50 mL) and 50 mg of 3-

benzyl-5-methylhydantoin was added from a stock solution in DMSO to the cell

suspension and the contents were incubated at 3 7 oc at 200 rpm. Progress of the

reaction was monitored by TLC (ethyl acetate/hexane, 2:3) for which an aliquot of 5

mL was withdrawn from the reaction mixture and extracted with ethyl acetate (5 mL).

The organic layer was used for TLC. Final work up of the reaction mixture involved

extraction of the reaction mixture with ethyl acetate (2 x 40 mL). The organic layer

was separated, washed twice with brine solution, dried over sodium sulphate and

evaporated under reduced pressure. To our surprise, in this case also complete

reduction of the substrate took place and the product that we could isolate from the

reaction was only 3-benzyl-5-methylimidazolidine-2-one (101; Scheme 33). 1HNMR

of the product is in agreement with the assigned structure and corresponded with the

product obtained by sodium borohydride reduction.

151

Scheme 33

Bacillus pumilis ., Phosphate buffer, pH 7.5;

18h


97 101

2.2.5.6 Sodium borohydride reduction of methyl 2-{(1,3-dibenzyl-2,5-

dioxoimidazolidin-4-yl)methylthio }acetate (1 04)

Substrate, 104 was selected as the next example because its reduction followed

by cyclization would lead to the synthesis of an advanced intermediate of biotin

(Scheme 34).

Scheme 34

reduction

104

Steps

106

~Steps Biotin

First, the reduction of 104 with sodium borohydride was attempted. The

reaction was done in absolute ethanol at 0 oc. Progress of the reaction was monitored

by TLC. Complete consumption of the starting material occurred with the formation

of a mixture of three compounds, which were separated by flash chromatography over

silica-gel (Scheme 35).

Scheme 35

100

+ --.... _OH Hs- ......., 107

152


Fraction 1: The fastest moving compound showed the absence of -SCH2COOCH3

residue in the 1HNMR. Instead a methyl group was observed as a singlet at() 1.86. In

addition, a singlet appeared in the olefinic region at () 5.76. The benzylic protons

appeared as two singlets at () 4.67 and () 4.74. The aromatic protons resonated as a

multiplet at() 6.9-7.4. The 1HNMR spectral data was indistinguishable from the olefin

100, obtained during the reduction of 1 ,3-dibenzyl-5-methyl hydantoin with sodium

borohydride.

Fraction 2: This fraction also showed the absence of -SCH2COOCH3 moiety, but

showed the presence methyl protons at C-5 as doublet at () 1.26 (1=6.6 Hz) and() 1.02

(1=6.6 Hz). Doublet of quartet for C-5 proton resonated at() 3.25 (1=6.6 and 7.1 Hz)

and() 3.18 (1=6.6 and 8.0 Hz). The doublet for the single proton at C-4 was observed

at () 4.28 (1=6.9 Hz) and () 4.27 (1=7.8 Hz). The -OH protons appeared as broad

singlet at() 3.27 and() 1.72. The doublets for the methylene protons at N-1 appeared

at() 4.13 (1=6.9 Hz) and() 4.62 (1=6.9 Hz) and() 4.10 (1=7.1 Hz) and() 4.60 (1=7.1

Hz) and the doublets for the methylene protons at N-3 were observed at() 4.7 (1=6.9

Hz) and() 4.87 (1=6.9 Hz) and() 4.4 (1=7.2 Hz) and() 4.54 (1=7.2 Hz). The aromatic

protons were observed as multiplet at () 7.0-7.35. The 1 HNMR spectral data of the

compound was indistinguishable from the diastereomeric alcohols 98 and 99 obtained

from the reduction of 1 ,3-dibenzyl-5-methyl hydantoin with sodium borohydride.

Fraction 3: This was identified as 2-hydroxyethanethiol, 107.

The desired alcohol, 105 was however, not obtained. The formation of

products may be explained as shown in Scheme 36. In the first step, elimination of the

thiol moiety, -SCH2COOCH3 may occur to produce 1,3-dibenzyl-5-

methylenehydantoin (108). Michael type reduction of 108 would produce 1,3-

dibenzyl-5-methylhydantoin (96), which is then further reduced to diastereomeric

alcohols 98/99. The dehydration of 98 over silica-gel would produce olefin 100 as

described earlier (Section 2.2.5 .1 ).

153

Scheme 36

100

~)lN~ ~ Co V -HSCH2COOCH\.

s-n0.....CH3

104 0

dehydration

(Silica-gel)

Jl ~NN~ ~ K~--~ OH .

98/99


108

96

2.2.5.7 Bacillus pumilis catalyzed reduction of methyl 2-{(1,3-dibenzyl-2,5-

dioxoimidazolidin-4-yl)methylthio }acetate (1 04)

Microbial reduction of 104 with Bacillus pumilis was studied next. The

reaction conditions for the biocatalyzed reduction were same as described before. The

biocatalyzed reduction proceeded in a manner similar to the chemical reduction,

except that only one diastereomer of alcohol, trans-1 ,3-dibenzyl-4-hydroxy-5-

methylhydantoin (98) was obtained along with the 4,5-dihydro product (100) (Scheme

37). The structure of the products was confirmed by 1HNMR spectral data. The

products were same as those obtained during Bacillus pumilis catalyzed reduction of

1 ,3-dibenzyl-5-methylhydantoin (96).

Scheme 37

Jl v.N N:'O~ 0 Bacillus pumilis

0 h Phosphate bufftr, ~ pH7.0;

s II CH3 14 h

0 104 98 100

The reduction of substrate 104 was also studied with Penicillium funiculosum.

The fungus was grown in rich medium comprising of peptone (0.2%), KH2P04

(0.2%), yeast extract (0.2%) and 50% glucose (0.2%). The cells were harvested by

centrifugation after 45 hand washed with phosphate buffer (20 mM, pH 7.0). Cells

(10.0 g) were resuspended in 20 mM phosphate buffer (pH 7.5, 50 mL) and 50 mg of

104 was added. The contents were incubated at 30 °C at 200 rpm in an orbital shaker.

154


TLC of the crude product showed the presence of two compounds. The slower

moving compound was identified as trans-1 ,3-dibenzyl-4-hydroxy-5-methylhydantoin

(98) based on 1 HNMR and TLC of the crude product. The faster moving compound

was isolated by preparative TLC. Its 1HNMR spectral data showed the presence of an

exo-methylene group which appeared as two singlets at 8 5.1 and 8 5.35. 1HNMR

spectral data was in agreement with the structure of 1,3-dibenzyl-5-methylene

hydantoin (108; Scheme 38).

Scheme 38

108

Thus, sodium borohydride reduction and the biocatalyzed reduction of

substrate 104 also followed similar pathway. The isolation of 5-methylene hydantoin

derivative 108 supports the proposed mechanism as shown in Scheme 36.

2.2.5.8 Sodium borohydride and Bacillus pumilis catalyzed reduction of 1,3-

dibenzyl-5-phenylhydantoin (109)

1,3-dibenzyl-5-phenylhydantoin (109) was synthesized according to literature

method as described in experimental section.183 First, the reduction of 1,3-dibenzyl-5-

phenylhydantoin (109) was performed with sodium borohydride in absolute ethanol

at 0 oc. However, no reduction occurred even after prolonged reaction time, refluxing

the reaction mixture and using excess of sodium borohydride also failed to reduce

109. Only the starting material was recovered unchanged.

Biocatalyzed reduction was studied next. Bacillus pumilis was suspended in

phosphate buffer (pH 7.0, 20 mM, 50 mL). 109 (50 mg) was added to the cell

suspension and the contents incubated at 37 oc for 24 h. No reaction was observed

and only the starting material was recovered unchanged. A close analysis of the 1HNMR of 1,3-dibenzyl-5-phenylhydantoin (109) revealed that this compound

primarily exists in the enol form (110). The methylene protons of the benzyl group

appeared as two singlets at 8 3.06 and 8 3.44. The singlet for C-5 proton could not be

observed at its predicted place; instead a singlet corresponding to enol appeared at 8

155


8.24. The aromatic protons were observed between 8 7.17-7.59 as multiplet. The

resistance of 1,3-dibenzyl-5-phenylhydantoin (109) to undergo reduction with sodium

borohydride or biocatalyst may thus be attributed to the existence of the compound in

the enol form 110.

109 110

2.2.6 Conclusion

In conclusion, we have shown that borohydride and biocatalyzed reduction of

hydantoin derivatives follows exactly the same path, only difference being that

whereas biocatalyzed reduction is highly stereoselective and results in the production

of only one diastereomer, the borohydride reduction is non stereoselective and

produces a diastereomeric mixture of alcohols. Interesting results were obtained in

borohydride or biocatalyzed reduction of 3-benzyl-5-methylhydantoin (97). Under

both the conditions, carbonyl was completely reduced to methylene. 1 ,3-dibenzyl-5-

phenylhydantoin (109) failed to undergo reduction with borohydride or biocatalyst,

presumably due to the occurance of this compound in predominantly enol form, (110).

156

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