Chapter V

143

6.0 CHAPTER V SPIROOXINDOLES 6.1 INTRODUCTION The indole template is generally recognized as a privileged structure in medicinal

chemistry (Houlihan et al., 1992). Indole itself has been found to possess fungicidal, bactericidal,

antidepressant, anticonvulsant and analgesic activities (Kant et al., 2005).

In particular, oxindoles are important constituents of natural indole alkaloids as well as

drugs under development and also in the clinical trial (Jossang et al., 1991; Zhang and Zhang,

2002; Akai, et al., 2004). For example, the oxindole motif is present in the anti-Parkinson’s drug

ropinirole (Nagata et al., 2001), in non-opioid nociceptin receptor ligands and in the growth

hormone secretagogues. In addition, the oxindole moiety constitutes a key structural element in

several natural products including the antiobiotic speradin (Tsuda et al., 2003) and the cytostatin

welwistatin (Zhang and Smith, 1996). 3,3-Diaryloxindoles have shown to possess mechanism-

specific anti-proliferative, antibacterial, anti-protozoal and anti-inflammatory activities. These

compounds have also been used as laxatives and lead compounds for Ca2+-depletion-mediated

inhibition of translation initiation.

6.1.1 Biological Importance

NH

O

N

NO

H

H

O

O

OH

NH

OO

N

Cl

HH

S

ropinirole speradine A welwistatin

Chapter V

144

6.1.1.1 Indoles

Bajji et al. (1994) reported the biological activity of substituted 3-(4’-oxothiazolidine-2’-

aryl/ alkyl imino) indoles. All the thioureas were screened for antibacterial activity against S.

aureus, B. cirroflagelosus, S. typhi and P. fluorescence at 10 mg/ml and 13 mg/ml concentration.

Antifungal activity against A. niger and C. albicans were also assessed. Only some of the

compounds were active against organisms tested at both the doses. Some of the compounds were

screened for anti-inflammatory and anticonvulsant activities. None of the compounds showed

noteworthy anti-inflammatory and anticonvulsant activity.

Sarangapani et al. (2001) reported the CNS activity of 2 substituted-[1,3,4]oxadiazilo-

[5,6-b]indoles. The compounds exhibited reduction in the locomotor activity and potentiation of

pentobarbital sodium induced sleeping time in experimental animals at 100 mg/kg dose. Ashok

kumar et al. (2004) reported the anti-inflammatory, analgesic and cox II inhibitory activities of

indolyl pyrazolines. All the compounds were found to possess better anti-inflammatory,

analgesic and cox II inhibitory activities. Saundane et al. (1998) reported the analgesic, anti-

inflammatory, oxytocic, anthelmintic and antimicrobial activities of indoloisoquinoline

derivatives. Some of the compounds exhibited analgesic, anti-inflammatory, anthelmintic,

antibacterial and antifungal activity against S. citrus, P. aeruginosa, P. vulgaris, E. coli, C.

albicans and A. niger.

6.1.1.2 Spirooxindoles

The indole moiety is probably the most well known heterocycle, a common and

important feature of a variety of natural products and medicinal agents. Compounds carrying the

indole residue exhibiting antibacterial and antifungal activities have been extensively reported.

Chapter V

145

Spiro[indole-thiazolidinones] are one of the most studied classes of 3-spiroindole derivatives due

Chapter V

146

to a wide variety of bioactivity associated with them and are used in pharmaceuticals because of

Chapter V

147

their anti-inflammatory (Rovnyak et al., 1978), fungistatic (Dandia et al., 2004) and

Chapter V

148

bacteriostatic activities ( Ali and Alam, 1994). Dandia et al. (2006) reported the series of

Chapter V

149

benzindozolyl/triazolyl spiro[indole-thiazolidinone as potent antifungal agent against

Chapter V

150

Rhizoctonia solani, Fusarium oxysporum and Collectotrichum capsici by poison plate technique

in 1000 and 500 ppm concentrations and pot trial method. All compounds have shown good

activity against these pathogen. Incorporation of triazole ring enhances the activity of

compounds as compared parent skeleton. Oxindoles that incorporate a quaternary stereogenic

centre at C3 are attractive targets in organic synthesis because of their significant biological

activities as well as wide-ranging utility as synthetic intermediates for alkaloids, drug candidates

and clinical pharmaceuticals (Marti and Carreira, 2003; Dounay et al., 2003). Spirocyclic

compounds are systems containing one carbon atom common to two rings and are structurally

quite interesting (Heathcock et al., 1983; Sannigrahi, 1999). They represent an important class of

naturally occurring substances characterized by highly pronounced biological properties. The

spirooxindole framework represents yet another important structural organization present in a

number of bioactive natural products such as coerulescine, horsfiline, welwitindolinone A,

spirotryprostatin A, elacomine, alstonisine, etc (Chang et al., 2005; Baran and Richter, 2005).

Spirotryprostatin A, a natural alkaloid isolated from the fermentation broth of Aspergillus

fumigatus, has been identified as a novel inhibitor of microtubule assembly, and pteropodine and

N

NH

Me

OMeO

NH

NH

OOH

NH

O

N

N

O

O HH

MeO

NH

NO

O

HH

H

H CO2Me

Me

N

NH

Me

O

NH

O

H

Cl

CN

horsfiline elacomine

spirotryprostatin A alstonisine

coerulescine

welwitindolinone A

Chapter V

151

isopteropodine have shown to modulate the function of muscarinic serotonin receptors (Cui et

al., 1996).

Benzopyrans and their derivatives occupy an important place in the realm of natural and

synthetic organic chemistry because of their biological and pharmacological properties such as

anti-sterility and anticancer agents. In addition, polyfunctionalized benzopyrans constitute a

structural unit of a number of natural products and because of the inherent reactivity of the

inbuilt pyran ring are versatile synthons. Moreover, they can

also be employed as cosmetics and pigments and utilized as potential biodegradable

agrochemicals. These findings stimulated the interest in the synthesis of heterocyclic derivatives

of these ring systems for their great importance.

Fused chromenes have been found to have a wide spectrum of activities such as

antimicrobial (Smith et al, 1998) antiviral (Hiramoto et al, 1997), mutagenicity (Dell et al,

1993), anti-proliferative (Bianchi and Tava, 1987), sex pheromone (Mohr et al, 1975), anti-

tumor (Elagamay and El-Taweel, 1990) and central nervous system activities

( Ballini et al, 2001).

6.1.2 Synthetic approaches

Padwa et al. (England et al., 2007) reported the synthesis of substituted spirooxindoles

from 3-hydroxysubstituted 1,3-dihydroindol-2-one by addition of various π-nucleophiles

followed by intramolecular cyclization.

Chapter V

152

NH

O

ORBF3.OEt2

NH

O

A simple and one-pot protocol for the synthesis of indene-spirooxindole derivatives via

TiCl4 mediated reaction between 1,1-diarylethylenes and isatin derivatives involving

construction of two carbon-carbon bonds through tandem Prins and intramolecular Friedel-Crafts

reaction has been described (Basaviah and Reddy, 2007).

NO

OR'

R R'' R''

TiCl4CH2Cl2, rt N

R'

R

R''R''

O+

Reaction of indole amides with tributylstannane gave spiroindolenines which were

readily converted into spiropyrrolidinyloxindoles (Hilton et al., 2000).

N

N

O

R

CH3

CN

Ph

N

N

R

O

CH3

CN

PhN

N

R

O

CH3

O

Ph

Bu3SnH KOtBu, O2, THF

200C, 1h

Miyamoto et al. (2006) reported a highly diastereoselective one-pot synthesis of

spirocyclic oxindoles through intramolecular Ullman coupling and Claisen

rearrangement.

Chapter V

153

N

OH

R

CH3

CuCl

NO

CH3

NO

CH3

2-amino pyridine

NaOMe, DME, MeOH1000C, 10 min

1500C, 8h

DME

R = H, I, OMe Diastereomeric three-, four-, five- and six-membered spirocycloalkyloxindoles were

successfully synthesized in a rapid and convenient manner from readily accessible starting

materials in moderate to high yields via a one-pot base-mediated double alkylation strategy

(Morales-Rios et al., 2007).

NO

CH3

CN

NCH3

O

BrCN

n

NaH, DMF

NO

H

CH3

CN

n

NO

CH3

HNC

n (CH2)nBr2

n=1-4 +

The spirooxindole ring system of citrinadin A was synthesized with excellent control

over the absolute stereochemistry at the spirocenter involving a novel diastereo selective

DMDO-mediated oxidative rearrangement employing an 8-phenylmenthol chiral auxiliary on the

indole ring (Pettersson et al., 2007).

N

OXc

DMDO

acetoneO

O

N

OXc

O silica

CH2Cl2, rt

OO

NO

OXc

00C

Xc = (-)-8-phenylmenthol

An efficient method was developed for the asymmetric synthesis of 2’-alkyl-4’-aryl-1H-

spiro[indole-3,3’-pyrrolidin-2-ones] which are potential inhibitors of the p53-MDM2 interaction

(Ding et al., 2005).

Chapter V

154

NH

O

ArRCHO

NH

OO Ph

Ph

NH

NH

ArR

O

O

NMe2

RR

+

An electrochemically induced catalytic multicomponent transformation of cyclic 1,3-

diketones, isatin and malononitrile in an undivided cell in the presence of NaBr as an electrolyte

results in the formation of spirooxindoles with fused functionalized 5,6,7,8-tetrahydro-4H-

chromene system (Elinson et al., 2007).

Zhu et al. (2007) carried out a simple and efficient one-pot method for the synthesis of

biologically important spirooxindoles by the reaction of isatin, activated methylene reagent and

1,3-dicarbonyl compounds in aqueous medium.

N

O

OR'

X

CN

O

O

R

O

N O XNH2

O

R'

R+ +H2O, TEBA

60oC

Shanmugam et al. (2006) reported a facile, high yield stereoselective synthesis of

functionalized diastereomeric 3-spirocyclopropane-2-indolones from the isomerized bromo

derivative of Baylis-Hillman adducts of isatin by reductive cyclization with NaBH4.

N

O

OR

R' CN

CN O

O

R''R''

O

N ONH2

O

R

CN

R'

R''R''

+ +electrolysis,0.1 F/mol

R'''OH, NaBr

Chapter V

155

N OR'

OH

RHBr, silica gel

N OR'

Br

RN OR'

BrR

RN OR'

HR

N OR'

H

MW, 750W+

+

NaBH4

THF, 0.5h

Z Z

Z

Z

Z

Nair et al. (2005) reported the synthesis of spirooxadiazolines from the reaction of N-

substituted isatins with the zwitterionic intermediate generated from dialkyl azodicarboxylate

and triphenylphosphine.

NO

O

R

R' NN

CO2R''

R''2OCPPh3

DME, Arrt

O

NN

NO

R

R'R''2OC

OR''

+ +

A novel regioselective synthesis of a number of functionalized 3-spiropyrrolizidine and

3-spiropyrroline oxindoles from Baylis Hillman adducts of isatin via [3+2] cycloaddition of

azomethine ylides in excellent yields has been reported (Shanmugam et al., 2007).

N OR'

OH

NO

O

R''

NH

CO2H

N OR''

N N

O

R'OH

+ +montmorillonite K 10

MeOH, reflux, 0.5h

Z

Z

A microwave-assisted three-component regioselective one-pot cyclocondensation method

has been developed for the synthesis of novel spiro[indole-thiazolidinones] using an

environmentally benign procedure at atmospheric pressure in open vessel (Dandia et al., 2006).

Chapter V

156

NH

O

OX HetNH2 SH

R

CO2HX

SN

NH

OHet

OR

+ +MW

One-pot synthesis of spiro[cyclohexane-1,3’-indoline]-2’,4-diones starting from

Danishefsky’s diene and 3-chloromethylene-2-indolones is described (Beccalli et al., 2003)

N O

H

Cl

CO2Et

OSiMe3

OMeN OCO2Et

OSiMe3Cl

OMe

p-TSAreflux

N OCO2Et

O

H2, Pd/C

AcOEtN OCO2Et

O

+toluene

reflux

Spiro dihydrofuran oxindole derivatives were prepared via (3+2) oxidative cycloaddition

of 1,3-dicarbonyl compounds to 3-(phenyl-2-oxoethylidene)-1-methyloxindole and 3-

benzylidene-1-methyloxindole derivatives mediated by CAN (Savitha et al., 2007).

NO

CH3

O

R' R

O

OR''

R''O

N

O

OR''

R''

O

CH3

R

R'CAN/NaHCO3+CH3CN, OoC

Chapter V

157

6.2 OBJECTIVES

Compounds carrying the indole residue, exhibiting antibacterial and antifungal activities,

have been extensively reported. Further more, it has been reported that sharing of the indole3–

carbon atom in the formation of spiroindoline derivatives highly enhances biological activity.

Spiroindolines are used in pharmaceuticals because of their anti-inflammatory, fungistatic,

bacteriostatic and anticonvulsant activities. The extensive literature survey revealed that the

presence of two or more different heterocyclic moieties in a single molecule often enhances the

biocidal profile. Hence the present study was designed with the following objectives.

To synthesize and characterize a few substituted spirooxindole derivatives of

biocidal interest

To study the possible antibacterial, antifungal and antiviral activities of the

synthesized spirooxindole derivatives

Chapter V

158

6.3 EXPERIMENTAL

6.3.1 Materials and methods

Zinc (II) chloride, bismuth (III) chloride, ferric chloride hexahydrate, stannous chloride,

isatin, α-naphthol, β-naphthol, malononitrile and sodium hydrogen sulphate monohydrate were

obtained from S.D. Fine Chemicals. THF was distilled over sodium-benzophenone before use.

All melting points are uncorrected. IR spectra were recorded on a Perkin Elmer FT-IR

spectrophotometer. 1H and 13C NMR spectra were recorded in DMSO-d6 using TMS as an

internal standard on a JEOL spectrometer at 500 MHz and 125 MHz respectively. Mass spectra

were recorded on a JEOL DX 303 HF spectrometer. Elemental analyses were recorded using a

Thermo Finnigan FLASH EA 1112 CHN analyzer. Column chromatography was performed on

silica gel (100-200 mesh, SRL, India). Analytical TLC was performed on precoated plastic

sheets of silica gel G/UV-254 of 0.2 mm thickness (Macherey-Nagel, Germany).

6.3.2 General procedure for the synthesis of spirooxindoles (4a-i)

6.3.2.1 Method A (Microwave method)

Isatin (0.147g, 1 mmol), malononitrile (0.066g, 1 mmol) and α-naphthol (0.144g, 1

mmol)/β-naphthol/cyclohexanone/dimedone/1-phenyl-3-methyl-pyrazole-5-one/4-

hydroxycoumarin (1 mmol) were added to silica gel impregnated with sodium hydrogen sulphate

monohydrate (20 mol %), prepared by adding a solution of NaHSO4.H2O in a minimum amount

of THF to silica gel (2g, 100-200 mesh activated by heating for 4h at 150oC before use),

Chapter V

159

followed by complete evaporation of solvent under vacuum. The whole mixture was stirred for 5

min for uniform mixing and then irradiated in a microwave oven (BPL SANYO) at 450 W for

about 10 min. On completion, the reaction mixture was directly charged on a small silica gel

column and eluted with a mixture of ethyl acetate-hexane (4:6) to afford the pure product in 70

% yield as a white solid. This procedure was followed for the synthesis of all the spirooxindoles

(4a-i). The structures of compounds (4a-i) were confirmed by IR, 1H and 13C NMR spectroscopy

and elemental analysis.

6.3.2.2 Method B (Conventional method)

To the reaction mixture containing isatin (1 mmol), malononitrile (0.066g, 1 mmol) and

α-naphthol (1 mmol) / β-naphthol/cyclohexanone/dimedone/1-phenyl-3-methyl-pyrazole-5-

one/4-hydroxycoumarin (1 mmol). Sodium hydrogen sulphate monohydrate, (20 mmol %) was

added and stirred at reflux for about 1.5 h. On completion, the reaction mixture was poured into

crushed ice and the precipitate formed was filtered, dried and purified by column

chromatography to afford the pure product in 70 % yield. This procedure was followed for the

synthesis of all the spirooxindoles (4a-i).

6.3.3 Antibacterial activity

Anti-bacterial study was carried out for the synthesized spirooxindoles (4a-i) by disc

diffusion method against ATCC gram positive and gram negative bacterial strains at 1000 µg,

500 µg and 100 µg concentrations.

6.3.3.1 Materials requirement

• The gram positive organism used for this study was Staphylococcus aureus and the gram

negative organism was and Klebsiella pneumoniae (The strains were received from

Department of Veterinary Microbiology, Madras Veterinary College, Chennai-600 007).

Chapter V

160

• The medium Tryptose Soy Agar (TSA) powder (HiMedia, Mumbai) was used at 4 g /100

ml to prepare solid agar plates and was used for both Gram positive and Gram negative

bacteria.

6.3.3.2 Method The same procedure was followed as given in chapter -1.Students‘t’ test was used for

statistical analysis. P values < 0.001 and <0.01 were considered to be statistically significant.

6.3.4 Antifungal activity

The in vitro anti-fungal activity of the spirooxindoles(4a-i) were studied

against Candida albicans using disc diffusion method at 1000 µg, 500 µg and 100 µg

concentrations.

6.3.4.1 Materials requirement

• The ATCC strain of Candida albicans was used for the anti-fungal study. (The strain was

received from Department of Veterinary Microbiology, Madras Veterinary College,

Chennai-600 007)

• The medium of Sauboraud’s Dextrose Agar (HiMedia, Mumbai) was used at 6.5 g/100

ml concentration for preparing solid agar plates.

6.3.4.2 Method

The same procedure was followed as given in chapter -3.

6.3.5 Cytotoxic assay The same procedure was followed as given in chapter -4. 6.3.6 Antiviral assay The same procedure was followed as given in chapter -4. 6.4 SPECTRAL DATA

Chapter V

161

2-Amino spiro[(4H)-benzo(h)chromen-4,3’-(3’H)-indol]-(1’H)-2’-one-3-carbonitrile 4a

White solid mp: 222-224°C. υmax (KBr): 3479, 3300, 3173, 2193, 1670, 1655, 1370, 751 cm-1.

1H NMR (DMSO-d6, 500 MHz): δ 6.52 (d, 1H, J = 9.2 Hz), 6.96 (m, 2H), 7.05 (d, 1H, J = 7.5

Hz), 7.25 (t, 1H, J = 7.5 Hz), 7.43 (br s, 2H, NH2, D2O exchangeable), 7.53 (d, 1H, J = 9.2 Hz),

7.58 (t, 1H, J = 8.0 Hz), 7.63 (t, 1H, J = 8.1 Hz), 7.85 (d, 1H, J = 8.0 Hz), 8.24 (d, 1H, J = 8.1

Hz), 10.67 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6, 125 MHz): δ 52.42, 54.8,

110.6, 115.3, 119.1, 121.3, 123.2, 123.3, 123.7, 125.1, 125.6, 127.6, 127.9, 128.2, 129.8, 133.6,

135.2, 142.4, 144.1, 161.6, 179.3. MS (m/z): 339 (M+). Anal. Calcd. for C21H13N3O2: C, 74.33;

H, 3.86; N, 12.38 . Found: C, 74.29; H, 3.81; N, 12.32 .

2-Amino spiro[(4H)-benzo(f)chromen-4,3’-(3’H)-indol]-(1’H)-2’-one-3-carbonitrile 4b

White solid. mp: 236-237°C. υmax (KBr): 3446, 3321, 1704, 1653, 1170 cm-1. 1H NMR (DMSO-

d6, 500 MHz): δ 6.86 (m, 2H), 7.00 (d, 1H, J = 8.0 Hz), 7.04 (d, 1H, J = 8.6 Hz) 7.16 (br s, 2H,

NH2, D2O exchangeable), 7.23 (t, 2H, J = 7.5 Hz), 7.29 (d, 1H, J = 9.2 Hz), 7.32 (t, 1H, J = 7.5

Hz), 7.88 (d, 1H, J = 8.0 Hz), 7.96 (d, 1H, J = 8.6 Hz), 10.98 (s, 1H, NH, D2O exchangeable).

13C NMR (DMSO-d6, 125 MHz): δ 50.80, 57.8, 110.80, 111.7, 117.7, 118.4, 123.2, 123.4, 124.8,

125.4, 127.9. 129.5, 129.7, 130.7, 131.5, 131.6, 135.9, 141.3, 148.5, 159.9. 179.1. MS (m/z): 339

(M+). Anal. Calcd. for C21H13N3O2: C, 74.33; H, 3.86; N, 12.38. Found: C, 74.26; H, 3.80; N,

12.34.

2-Amino spiro[(4H)-5,6,7,8-tetrahydrochromene-4,3’-(3’H)-indol]-(1’H)-2’one-3-

carbonitrile 4c

Brown solid. mp: 236-238°C. υmax (KBr): 3443, 3290, 2194, 1710, 1631,1470, 1218, 1148 cm-1.

1H NMR (DMSO-d6, 500 MHz): δ 1.24 (m, 8H), 6.79 (d, 1H, J = 7.6 Hz), 6.82 (br s, 2H, NH2,

Chapter V

162

D2O exchangeable), 6.96 (t, 1H, J = 7.7 Hz), 7.05 (d, 1H, J = 7.7 Hz), 7.16 (t, 1H, J = 7.7 Hz),

10.44 (br s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6, 125 MHz): δ 22.1, 22.4, 23.0,

26.2, 52.3, 54.8, 106.8, 110.1, 119.4, 122.9, 124.8, 129.2, 133.5, 142.3, 145.0, 161.2, 178.9. MS

(m/z): 293 (M+). Anal. Calcd. for C17H15N3O2: C, 69.56; H, 5.06; N, 14.22 . Found: C, 69.61; H,

5.15; N, 14.33.

2-Amino-5-oxo-7,7-dimethyl spiro[(4H)-5,6,7,8-tetrahydrochromene-4,3’-(3’H)-indol]-

(1’H)-2’one-carbonitrile 4d

Colorless solid. mp: 268-270°C. υmax (KBr): 3410, 3306, 3136, 2959, 2191, 1719, 1679, 1653,

1601, 1218, 1163 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ 0.97 (s, 3H), 1.00 (s, 3H), 2.05 (d, 1H,

J = 16.1 Hz), 2.12 (d, 1H, J = 16.1 Hz), 2.52 (d, 2H, J = 6.1 Hz), 6.75 (d, 1H, J = 7.7 Hz), 6.84

(t, 1H, J = 6.9 Hz), 6.94 (d, 1H, J = 6.9 Hz), 7.09 (t, 1H, J = 7.7 Hz), 7.19 (br s, 2H, NH2, D2O

exchangeable), 10.36 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6, 125 MHz): δ 19.1,

27.6, 28.1, 47.3, 50.5, 56.6, 58.0, 109.8, 111.3, 117.9, 122.2, 123.6, 128.7, 134.9, 142.6, 159.3,

164.7, 178.6, 195.4. MS (m/z): 335 (M+). Anal. Calcd. for C19H17N3O3: C, 67.98; H, 5.03; N,

12.42 . Found: C, 68.05; H, 5.11; N, 12.53.

6-Amino-3-methyl-1-phenyl spiro[(3’H)-indol-3’,4-4(H)-pyrano(3,2-d)pyrazol]-(1’H)-2’-

one-5-carbonitrile 4e

Colorless solid. mp: 248-250°C. υmax (KBr): 3460, 3284, 2195, 1701, 1652, 1518, 1393, 753 cm-

1. 1H NMR (DMSO-d6, 500 MHz): δ 1.55 (s, 3H), 6.95 (d, 1H, J = 7.4 Hz), 7.02 (t, 1H, J = 7.5

Hz), 7.17 (d, 1H, J = 6.9 Hz), 7.28 (t, 1H, J = 7.5 Hz), 7.35 (t, 1H, J = 7.5 Hz), 7.51 (t, 2H, J =

Chapter V

163

7.5 Hz), 7.58 (br s, 2H, NH2, D2O exchangeable), 7.79 (d, 2H, J = 7.5 Hz), 10.75 (s, 1H, NH,

D2O exchangeable). 13C NMR (DMSO-d6, 125 MHz): δ 12.2, 48.3, 56.7, 96.9, 110.4, 118.5,

120.7, 123.2, 125.4, 127.1, 129.8, 130.0, 132.6, 137.8, 142.1, 144.5, 145.5, 161.6, 178.0. MS

(m/z): 369 (M+). Anal. Calcd. for C21H15N5O2: C, 68.28; H, 4.09; N, 18.96 . Found: C, 68.16; H,

4.03; N, 18.90.

Ethyl 6-amino-3-methyl-1-phenyl spiro[(3’H)-indol-3’,4-4(H)-pyrano(3,2-d) pyrazol]-(1’H)-

2’-one-5-carboxylate 4f

White solid. mp: 208-209°C. υmax (KBr): 3441, 3301, 1710, 1652, 1601, 1574, 1156 cm-1. 1H

NMR (DMSO-d6, 500 MHz): δ 0.69 (t, 3H, J = 6.9 Hz), 1.56 (s, 3H), 3.71 (m, 2H), 6.82 (m,

2H), 6.93 (d, 1H, J = 7.5 Hz), 7.12 (t, 1H, J =7.5 Hz), 7.29 (t, 1H, J = 7.5 Hz), 7.46 (t, 2H, J =

8.0 Hz), 7.77 (d, 2H, J = 8.0 Hz), 8.19 (br s, 2H, NH2, D2O exchangeable), 10.49 (s, 1H, NH,

D2O exchangeable). 13C NMR (DMSO-d6, 125 MHz): δ 12.3, 13.6, 48.0, 59.5, 75.1, 98.7, 109.4,

120.5, 122.3, 123.7, 126.9, 128.3, 129.9, 136.3, 137.9, 142.7, 144.5, 144.8, 161.9, 168.4, 179.8.

MS (m/z): 416 (M+). Anal. Calcd. for C23H20N4O4: C, 66.34; H, 4.84; N, 13.45. Found: C, 66.28;

H, 4.80; N, 13.39.

6-Amino-1’,3-dimethyl-1-phenyl spiro[(3’H)-indol-3’,4-4(H)-pyrano(3,2-d) pyrazol]-2’-one-

5-carbonitrile 4g

White solid. mp: 200-201°C. υmax (KBr): 3450, 3310, 2198, 1686, 1603, 1572, 1179 cm-1. 1H

NMR (DMSO-d6, 500 MHz): δ 1.43 (s, 3H), 3.22 (s, 3H), 7.07 (t, 1H, J = 7.4 Hz), 7.12 (d, 1H, J

= 8.0 Hz), 7.21 (d, 1H, J = 6.9 Hz), 7.31 (m, 2H), 7.47 (t, 2H, J = 8.6 Hz), 7.62 (br s, 2H, NH2,

D2O exchangeable), 7.75 (d, 2H, J = 8.0 Hz). 13C NMR (DMSO-d6, 125 MHz): δ 12.2, 27.0,

Chapter V

164

47.9, 56.2, 96.7, 109.4, 118.4, 120.7, 123.9, 125.1, 127.1, 129.9, 131.9, 137.7, 143.6, 144.4,

145.5, 161.7, 176.3. MS (m/z): 383 (M+). Anal. Calcd. for C22H17N5O2: C, 68.92; H, 5.15; N,

13.02 . Found: C, 68.79; H, 5.10; N, 12.97.

2-Amino-5-oxaspiro[(3’H)-indol-3’,4-4(H)-pyrano(3,2-c)chromen]-(1’H)-2’-one-3-

carbonitrile 4h

Off white solid. mp: 249-250°C. υmax (KBr): 3419, 3301, 2203, 1714, 1671, 1169 cm-1. 1H NMR

(DMSO-d6, 500 MHz): δ 6.82 (d, 1H, J = 7.5 Hz), 6.89 (t, 1H, J = 7.5 Hz), 7.17 (m, 2H), 7.45 (d,

1H, J = 8.6 Hz), 7.49 (t, 1H, J = 8.1 Hz), 7.66 (br s, 2H, NH2, D2O exchangeable), 7.72 (t, 1H, J

= 7.5 Hz), 7.91(d, 1H, J = 8.0 Hz), 10.67 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6,

125 MHz): δ 48.1, 57.5, 101.9, 110.1, 112.9, 117.5, 122.6, 123.2, 124.7, 125.6, 129.5, 133.6,

134.2, 142.7, 152.6, 155.6, 158.8, 158.9, 177.7. MS (m/z): 357 (M+). Anal. Calcd. for

C20H11N3O4: C, 67.23; H, 3.10; N, 11.76 . Found: C, 67.19; H, 3.04; N, 11.71.

2-Amino-1’-methyl-5-oxaspiro[(3’H)-indol-3’,4-4(H)-pyrano(3,2-c)chromen]-2’-one-3-

carbonitrile 4i

Off white solid. mp: 242-244°C. υmax (KBr): 3452, 2195, 1712, 1672, 1601, 1168 cm-1. 1H NMR

(DMSO-d6, 500 MHz): δ 3.17 (s, 3H,Nme) 6.98 (t, 1H, J=7.5 Hz), 7.04 (d, 1H, J=8.1 Hz), 7.25

(d, 1H, J=7.4 Hz), 7.28 (t, 1H, J=7.4 Hz), 7.45 (d, 1H, J=8.6 Hz), 7.50(t, 1H, J=7.5 Hz), 7.71 (br

s, 2H, NH2, D2O exchangeable), 7.73 (m, 1H), 7.91 (d, 1H, J=7.5 Hz). 13C NMR (DMSO-d6, 125

MHz): δ 27.1, 47.8, 57.1, 101.8, 109.1, 112.9, 117.2, 117.4, 123.3, 123.4, 124.4, 125.6, 129.7,

Chapter V

165

132.8, 134.3. 144.2, 152.6, 155.7, 158.8, 159.1, 176.2. MS (m/z): 371 (M+). Anal. Calcd. for

C21H13N3O4: C, 67.92; H, 3.53; N, 11.32. Found: C, 67.87; H, 3.47; N, 11.26.

6.5 RESULTS AND DISCUSSION

Spirocyclic compounds, which are systems containing one carbon atom common to two

rings, are structurally quite interesting. Among them, the heterocyclic spirooxindole framework

is an important structural motif in biologically relevant compounds as natural products and

pharmaceuticals. Azaspiro derivatives are well-known, but the synthesis of the corresponding

oxa analogues have evolved at a relatively slow pace.

As a part of our endeavour to discover spirooxindoles of biocidal interest, and guided by

the observation that the presence of two or more different heterocyclic moieties in a single

Chapter V

166

molecule often enhances the biocidal profile remarkably, we investigated a three-component

reaction involving isatin, malononitrile and α-naphthol, in order to synthesize spirooxindoles

with fused chromenes. Fused chromenes have been found to have a wide spectrum of activities

such as antimicrobial, antiviral, mutagenicial, antiproliferative, sex pheromones, antitumor and

central nervous system activity.

Initially, evaluation of various catalysts and solvent systems was carried out for the

synthesis of spirooxindole with fused chromenes. After systematic screening, NaHSO4.H2O was

found to be the best. The microwave assisted NaHSO4.H2O/SiO2 catalyzed method was superior

to the conventional one, as the reaction was high yielding, fast and clean without any side

products. The amount of NaHSO4.H2O required for this transformation was also evaluated. As

little as 10 mol% of NaHSO4.H2O catalyzed the reaction but required a longer reaction time (10–

15 min). With silica gel alone, the reaction was extremely slow with very poor yield (10%)

(Table 5.1).

Table 5.1 The effect of catalysts and a comparison between conventional and microwave assisted methods for the synthesis of spirooxindoles

Entry Catalyst Refluxa MW (solvent-free)a Time (h) Yield (%)b Time (min) Yield (%)b

1 ZnCl2 3 45 4.5 55 2 FeCl3.6H2O 6 20 5 25 3 BiCl3 5 42 3.5 48 4 SnCl22H2O 4 45 3.5 50 5 NaHSO4.H2O 2.5 60 8 75

aReactions carried out on a 1mmol scale. bIsolated yield.

The scope and limitations of the one-pot reactions involving isatin, malononitrile with 1-

naphthol/2-naphthol were explored (Scheme 5.1). Results of the investigation involving

Chapter V

167

microwave irradiation (method A) as well as thermal heating (method B) are presented in Table

5.2.

Scheme 5.1

NH

O

O

NC CN

Y

z

NH

O

ONH2

NC

O

NH

O

NH2

NC

OH H

H

+ +

(or)

1 2 3

4

Y =O H, Z = HY = H, Z = OH

NaHSO4.H2O/ SiO2/ MW

Y Z

4a

4b

(20 mol %)

NaHSO4.H2O/CH3CN reflux

H OH

4a=4b=

Chapter V

168

Table 5.2 Synthesis of spirooxindoles from 1-naphthol/2-naphthol

aIsolated yield

The structure 4b was assigned to the product on the basis of spectral data. The IR

spectrum of 4b showed absorptions at 3453, 3321, 2198, 1704 and 1655 cm-1 indicating the

presence of –NH2, cyano, carbonyl and olefinic functionalities respectively (Spectrum 5.3). In

the 1H NMR spectrum (Spectrum 5.1), aromatic signals were seen at δ 6.86 – 7.96, a broad

singlet at δ 7.16 and 10.98 showed the presence of -NH2 and -NH groups (D2O exchangeable).

The carbonyl resonated at δ 179.1 in the 13C NMR spectrum (Spectrum 5.2). Mass spectral

analysis also supported the structural assignment.

NH

O

ONH2

NC

O

NH

O

NH2

NC

4a

4b

1

2

Entry Product Yielda MW (min)

Yielda (h)

58

61

2.0

2.4

(%) (%)

8

8

75

72

Chapter V

169

Encouraged by these results, this protocol could be extended to a three-component

coupling reaction involving an equimolar quantity of isatin, malononitrile and

cyclohexanone/dimedone (Scheme 5.2). This gave spirooxindoles 4c and 4d in excellent yields

without the formation of any side products.

Scheme 5.2

To further explore the potential of this protocol for various heterocyclic synthesis, we

investigated one-pot reactions involving 1-phenyl-3-methyl pyrazolon-5-one and 4-hydroxy

coumarin and obtained spirooxindoles in excellent yields (Scheme 5.3 and Table 5.3).

NH

O

O

NC CN

NO

ONH2

H

NC

NO

H

ONH2

O

NC

O O

O+ +

1 2

NaHSO4.H2O/SiO2

MW

(or)

4d4c

(or)

5

NaHSO4.H2O/CH3CN

Reflux(20 mol %)

Chapter V

170

Scheme 5.3

NO

O

R

XNC NNOPh

OO

OH

NO

ONH2 NNPh

X

RN

O

R

ONH2

O

OX

+ +

1 2R = HR = CH3

X = CNX = CO2Et

NaHSO4.H2O/SiO2

MW

(or)

(or)

4h-i4e-g

7

NaHSO4.H2O/CH3CN

Reflux(20 mol %)

Chapter V

171

Table 5.3 Synthesis of spirooxindoles from enones

aIsolated yield

NH

O

ONH2 NNPh

NC

NH

O

ONH2 NNPh

EtO2C

NO

ONH2 NNPh

NC

CH3

NH

O

ONH2

NC

NH

O

ONH2

O

NC

O

O

O

NNPh

O

NNPh

O

NNPh

O

NH

O

ONH2

O

ONC

NO

ONH2

O

ONC

CH3

O

O

O

O

O

O

4e

4f

4g

3

4

5

67

73

75

Entry Product YieldaYielda

8.5

8.5

9

2.2

2.0

2.2

57

60

60

(%) (%)

4d

2 705 2.5 57

4c

1 68 2.5 58

(h)MW(min)

Reactant

4h

4i

6

7

75

72

9

9

2.5

2.5

62

61

8.5

8.5

Chapter V

172

6.5.1 Antibacterial and Antifungal activity (Disc Diffusion Method)

Certain possible modification on the chemical structure by the addition of diverse

substituents may lead to products with better biological profiles.

Bajji et al. (1994) reported the biological activity of substituted 3-(4’-oxothiazolidine-2’-

aryl/alkyl imino) indoles. All the thio ureas were screened antibacterial activity against S. aureus,

B. cirroflagelosus, S. typhi and P. fluorescence at 10 mg/ml and 13 mg/ml concentration.

Antifungal activity against A. niger and C. albicans were also assessed. Only some of the

compounds were active against organisms tested at both the doses.

Saundane et al. (1998) reported the antimicrobial activities of indoloisoquinoline

derivatives. Some of the compounds exhibited antibacterial and antifungal activity against S.

citrus, P. aeruginosa, P. vulgaris, E. coli, C. albicans and A. niger at 50 and 75 μg/ml

concentrations .Only compounds carrying chloro or methyl group in C-8 exhibited activity.

. Abdel Rehman et al. (2004) reported the antibacterial and antifungal activities of some new

spiroindoline based heterocycles against Bacillus subtilis, Bacillus megatherium, Escherichia

coli, Aspergillus niger and Aspergillus oryzae. The results revealed that the prepared spiro 3 H-

indoles -3, 4’-pyrano(3’,2’-d) oxazole derivative showed comparable anti-bacterial activity, spiro

3H-indole-3,4’-pyrazolo(3’,4’-b) pyrano(3’,2’-d) oxazole derivatives revealed very high anti-

bacterial activity and this might be as a result of the presence of the extended fused pyrazole

moiety in their structure. On the other hand, all the compounds exhibited an interesting high

antifungal activity.

Compounds (4a-i) were tested against K. pneumoniae, S. aureus, and C. albicans at 100,

500, and 1000 μg concentration. Compounds (4b-i) produced significant antibacterial activity

against S. aureus. Compound 4a did not show any activity S. aureus. Compounds (4c-g, 4i) did

Chapter V

173

not have any activity against K. pneumoniae. Compounds (4a, 4b, 4h) showed significant

antibacterial activity against K. pneumoniae (Table 5.4).

Dandia et al. (2006) reported potent anti-fungal activity of triazolyl spiro(indole-

thiazolidinones) against Rhizoctonia sonani, Fusarium oxysporum and Collectotrichum

capsici.None of the compounds had any activity against C. albicans (Table 5.4).

6.5.2 Antiviral activity of spirooxindoles

Antiviral activity of spirooxindoles (4a-i) was evaluated against peste des petits ruminant

virus (PPRV) in Vero cell line by CPE inhibition assay. Peste des petits ruminant’s virus (PPRV)

is a RNA virus of the Morbilli virus genus and as the members are serologically related, the

antiviral effect of these compounds against PPRV could very well be applied to other viruses

also.

The synthesized compounds (4a-i) were tested for in vitro anti-viral activity against

PPRV by CPE inhibition assay. All the compounds were tested for cytotoxicity against normal

vero cell using MTT assay method. Concentrations that were non-toxic to the vero cells were

selected for anti-viral activity screening. The cytotoxic concentration (CC50) of the compounds

were between 12.5 and 500 µg/100 µl. Compound 4a, 4b, 4d and 4i had, 85 %,90 %, 80 % and

75 % CPE inhibition against PPRV in Vero cells while the rest of the compounds had less than

50 % CPE inhibition against PPRV in Vero cells. The results are presented in Table 5.5

6.6 SUMMARY

A quick, clean and simple method was developed in this study for the synthesis of

spirooxindoles catalyzed by sodium hydrogen sulphate monohydrate under solvent-free

conditions and also by thermal heating. Further merits of this method are its generality, shorter

reaction times and easy work-up. The compounds were screened for antimicrobial and antiviral

Chapter V

174

activity. Compounds (4b-i) produced significant antibacterial activity against S. aureus.

Compounds (4a, 4b, 4h) showed significant antibacterial activity against K. pneumoniae. The

compounds (4a-i) did not have any activity against C. albicans. The cytotoxic concentration

(CC50) of the compounds was between 12.5 and 500 µg/100 µl. Compound 4a, 4b, 4d and 4i

had, 85 %, 90 %, 80 % and 75 % CPE inhibition against PPRV in vero cells and found to be

comparable with the standard drug used. Further biological evaluation to delineate the mode of

action as well as study of animal models to assess the full potential of bis(pyrazolyl)methanes is

warranted.

Chapter V

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Chapter V

Spectrum 5.1 1H NMR spectrum of Compound 4b

Chapter V

Spectrum 5.2 13C NMR spectrum of Compound 4b

Spectrum 5.3 IR spectrum of Compound 4b

4000.0 3000 2000 1500 1000 400.00.0

10

20

30

40

50

60

70

80

90

100.0

cm-1

%T

3452.49

3188.04 2198.33

1703.731654.71

1512.91

1470.721410.95

1322.62

1222.95

1174.35

1107.30

1056.25

913.55

852.17

749.02

682.71614.93

579.09494.63

3321.03

Chapter V

Table 5.4 Antibacterial and anti fungal activities of Spirooxindoles

Compound

Name

Zone of inhibition of different conc. of compounds for different

organisms in mm

Klebsiella

pneumoniae

Staphylococcus

aureus

Candida albicans

1000µg 500µg 100µg 1000µg 500µg 100µg

6a 8** 7* 7* - - -

All the three concentrations

of different compounds did

not have any activity against

this yeast organism

6b 8** 7* 7* 8** 8** 7*

6c - - - 8** 8** 7.5*

6d - - - 8.5** 8.5** 8**

6e - - - 10** 9.5** 8**

6f - - - 9** 9** 8**

6g - - - 9** 9** 8**

6h 8** 8** 7* 9** 8** 8**

6i - - - 8** 8** 7.5**

Standard Gentamicin- 32**

(10 µg)

Ciprofloxacin- 35**

(5 µg)

Nystatin- 25**

(100 units)

* P<0.01, ** P<0.001 when compared with control (6 mm) – student’s‘t’ - test

Chapter V

Table 5.5 Antiviral activities of spirooxindoles

Compound

No

Cytotoxic

concentration

(μg/100 μL)

Effective concentration

(μg/100 μL)

CPE inhibition (%)

4a 12.5 6.25 85

4b 12.5 6.25 90

4c 12.5 - <50

4d 500 250 80

4e 100 - <50

4f 100 - <50

4g 25 - <50

4h 250 - <50

4i 500 250 75

Ribavirin 25 90