Pharmacological Reports

2013, 65, 313335 ISSN 1734-1140

Copyright 2013 by Institute of Pharmacology Polish Academy of Sciences

Review

Biochemical and pharmacological characterization of isatin and its derivatives: from structure to activity
Parvaneh Pakravan1, Soheila Kashanian2, Mohammad M. Khodaei2, Frances J. Harding3
1

Department of Chemistry, Zanjan Branch, Islamic Azad University, Zanjan, I.R. Iran

Department of Chemistry, Sensor and Biosensor Research Center (SBRC) & Nanoscience and Nanotechnology Research Center (NNRC), Faculty of Science, Razi University, Kermanshah, I.R. Iran
3

Mawson Institute, University of South Australia, Adelaide, South Australia 5001, Australia

Correspondence: Soheila Kashanian, e-mail: kashanian_s@[Link]; Mohammad M. Khodaei, e-mail: mm_khodaei@[Link]

Abstract: Isatin, 1H-indole-2,3-dione, is a heterocyclic compound of significant importance in medicinal chemistry. It is a synthetically versatile molecule, a precursor for a large number of pharmacologically active compounds. Isatin and its derivatives have aroused great attention in recent years due to their wide variety of biological activities, relevant to application as insecticides and fungicides and in a broad range of drug therapies, including anticancer drugs, antibiotics and antidepressants. The purpose of this review is to provide an overview of the pharmacological activities of isatin and its synthetic and natural derivatives. Molecular modifications to tailor the properties of isatin and its derivatives are also discussed. Key words: isatin, derivatives, DNA and HSA binding, antimicrobial, anticancer, anticonvulsant, antioxidant

Introduction
The development of new, cost-effective drugs against malaria, tuberculosis, AIDS, and trypanosomiasis is urgently needed in order to satisfy the overwhelming demand for disease treatment in Third World countries [9]. Synthesis of new medicinal compounds based on candidate molecules with known biological activity offers an opportunity to design effective safe drugs with minimal toxicity [60]. Isatin, 1H-indole-2,3-dione 1 (Fig. 1), is a versatile chemical building block, able to form a large number

of heterocyclic molecules. The compound possesses an indole ring structure 2 (Fig. 1), common to many pharmaceuticals. Isatin itself possesses an extensive range of biological activities [4, 42]. Isatin is able to

Fig. 1. Structure of isatin 1 and indole 2

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participate in a broad range of synthetic reactions, leading to its extensive use as a precursor molecule in medicinal chemistry [10, 56, 60]. Here we discuss the potential of isatin and its derivatives to create novel bioactive compounds. The basic chemistry and synthesis of isatin derivates are first reviewed. The expanse of biological, and particularly pharmacological, activities of isatin compounds is explored. During this discussion, we propose molecular modifications to tune and refine isatin compounds for use in specific therapies.

a large number of reactions. The keto group at position 2 and particularly at position 3 can enter into addition reactions at the C-O bond and into condensation reactions. Through the primary amine group, compounds of the isatin series are capable of entering into N-alkylation and N-acylation and into Mannich and Michael reactions [60]. These reactions are described in detail below.
Carbonyl reactions

The chemistry of isatin and its derivatives

Fundamental reactivity of isatin and its derivatives

The presence of several reaction centers in isatin and its derivatives render them capable of participating in

Schiff bases of isatin 4 can be synthesized by condensation of the keto group of isatin with different aromatic primary amines 3 (Scheme 1) [63]. Bis-Schiff bases of isatin can be prepared by reactions with aromatic diamines in the presence of catalytic amounts of glacial acetic acid in EtOH under reflux conditions [2, 22]. The straightforward synthesis of isatin-b-thiosemicarbazones 6 by the condensation of isatin and a thiosemicarbazide 5 has been known for more than 50 years (Scheme 2) [21, 25].

O O N H
+

NHR RNH2 3 EtOH, CH3COOH reflux N H 4 O

Scheme 1. Schiff reaction

H2N O O N H H2N
+

S HN NH2 5 6

HN N N H

S
Scheme 2. Preparation of isatin thiosemicarbazone

NOH O N H 8
Scheme 3. Formation of isatin 3-oxime 8, isatin 2-oxime 9

O O N H + NH2OH 7

pH

0 7.

pH

O
7.6

NOH N H 9

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Selectivity of isatinoxime formation reactions can be controlled by pH (Scheme 3) [1]. The reaction of isatin-2-oxime 8 with isocyanate gives bioactive carbamoyl derivatives 10 as the main products (Scheme 4) [1, 45].
N-Alkylation

N-substituted isatins have been frequently used as intermediates and synthetic precursors for the preparation of a wide variety of heterocyclic compounds. Nsubstituted isatin derivatives 12 can be synthesized via substitution reaction. The reaction between isatin and halohydrocarbons (11) can be carried out in NaOEt using EtOH as solvent or in the presence of NaH using DMF as solvent (Scheme 5) [8]. A simple

and efficient technique to assisted synthesis of Nalkylisatins by N-alkylation of isatin using a household microwave oven has been reported. The use of microwave irradiation offers many advantages over conventional heating: it decreases reaction times, increases yields and requires less solvent, thus facilitating reaction scale up (Scheme 6) [71]. N-Mannich bases of the Schiff base 15 can be synthesized by condensing the acidic amino group of isatin 14 with formaldehyde and various secondary amines (Scheme 7) [79].
Electrophilic aromatic substitution of isatin

In 1925, Calvery, Noller and Adams reported the nitration of isatin at the C-5 position using fuming nitric

NOH
Scheme 4. Formation of the carbamoyl derivatives

NOCONHR' R'NCO N R 10 O

O N R 8

R = H, Me, Et; R' = Me, Ph

O
Scheme 5. Synthesis of N-substituted isatin derivatives

O O + X-R K2CO3 DMF N R 12

H 2N S K2CO3, R I O DMF, 100C, 20 min Mw Irrad 13 R1 = CH3, Et, Ph, Benzyl
HN R1 , HCHO R2 Mannich reaction N N R
1

N H

11

H2 N
Scheme 6. General synthetic route to N1-substituted thiosemicarbazones

HN N N H 6

HN N N R1

N R
Scheme 7. Synthesis of N-Mannich bases

O N H 14

O R1 N R2

15
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315

O O + N H

O SiO2 N X S O O 16 X = Cl, Br, I CH2Cl2, rt 17 X

O O N H
Scheme 8. Mono-halogenation (-Cl, -I, -Br) of isatin

N N N H

M O O Cl

[M(L)Cl]Cl L = 3-Salicylidenehydrazono-2-indolinone M = Co, Ni, Cu, Zn

Fig. 2. 3-Salicylidenehydrazono-2indolinone complexes

18

acid in concentrated sulfuric acid [5]. However, a more convenient method to synthesize 5-nitroisatin involves the dropwise addition of a solution of isatin in sulfuric acid to a solution of potassium nitrate dissolved in concentrated sulfuric acid at 04C [24]. 5Nitroindoline-2,3-dione has been prepared by refluxing of indoline-2,3-dione with a mixture of 95100% sulfuric acid and 70% nitric acid in water bath at 60C for 1 h [72]. Mono-halogenation (-Cl, -I, -Br) of isatin (17) can be achieved via reacting N-halosaccharins 16 with isatin in the presence of SiO2 at room temperature to specifically produce the 5-halo derivatives 17 as reported by De Souza et al. (Scheme 8) [13]. This method is an alternative to the use of highly toxic and corrosive Cl2 and Br2, which can lead to other products such as 5,7-dibromo-3,3-dialkoxyoxindole when the bromination of isatin is attempted in alcoholic media [40]. Moreover, others have reported using the relatively stable reagent trichloroisocyanuric acid (TICA), as an efficient new source of electrophilic chlorine and this provides a relatively inexpensive route to the chlorination of isatins [43, 74].

metals [37]. For example, isatin-b-thiosemicarbazone can complex manganese(II), iron(II), cobalt(II), nickel(II), copper(II), and zinc(II) ions [6]. Acylhydrazones can behave as k2-O,N or k3-O,N,X ligands to form stable coordination compounds with different transition metal ions. Rodrguez-Argelles et al. [64] reported the interesting biological properties of cobalt(II), nickel(II), copper(II) and zinc(II) complexes of 3-isatin- and N-methylisatin-3-picolinoylhydra- zone as well as of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-(N-methyl)isatin. Finally, complexes of divalent copper, cobalt, nickel and zinc ions with 3-salicylidenehydrazono-2indolinone 18 can be isolated in the keto-form of the ligand, as shown in Figure 2 [6].

Synthesis of new isatin derivatives

A number of novel reactions have been reported that may assist in the generation of pharmacologically active isatin compounds: 1. The synthesis of 3-(indol-3-yl)-3-hydroxyindolin-2-ones 19 from isatins and indoles utilizing Fe(III) as a recyclable homogeneous catalyst under ultrasound irradiation has been described (Scheme 9). It was found that the conditions employed produced yields of 8595% [33]. 2. Electrocatalytic transformation of isatins and barbituric acids in ethanol in an undivided cell in the presence of sodium bromide as an electrolyte results in the formation of substituted 5,5-(2-oxo-2,3-dihydro-1H-indole-3,3-diyl)bis(pyrimidine-2,4,6(-1H, 3H,

Complexes with bioactive Schiff bases of isatin

Simple isatin based Schiff base compounds, containing acyl, aroyl and heteroaroyl Schiff bases, possess additional electron donor sites, for instance at C=O and C=N-. These donor sites make such compounds good chelating agents, able to form a variety of complexes with various transition and inner transition

316

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H N
+

O O N H

Fe(III), H2O:EtOH(60:40) 2-25 min, 50oC N H

OH NH O 19

Scheme 9. Ultrasound-promoted, Fe(III) catalyzed 3-indolylation of isatins

O N H O N R1 98 NR
2

O N N H O N R1 99 NR2

Scheme 10. Efficient synthesis of functionalized (2-oxo-2,3-dihydro-1H-indole-3,3-diyl)bis (pyrimidine) system from isatin and barbituric acids

R1 R2 R3 R4 23

O O N R + R5 24 NO2 DABCO(5 mol%) Solvent free, 1-5 min

R2 R3

R1 HO R5 NO2 O N R4 R

R5 = H, Me R = H, CH3, Bn R1 = R2 = R3 = R4 = H, 5-CH3, 5-F, 5-Cl, 5-Br, 5-I , 5-NO2, 5-OCF3, 4-Br, 7-Cl, 7-Br, 4,6-dibromo, 5,7-dibromo.

25 1,4-diazabicyclo[2,2,2]octane (DABCO)

Scheme 11. 3-Hydroxy-3-(nitromethyl)indolin-2-one derivatives formation by using DABCO as catalyst

5H)-triones) 22 with 8995% substance yields and 8995% current yields (Scheme 10). This novel and efficient catalytic process is important due to its diversity-oriented large-scale processes and is an example of easy environmentally benign synthesis via electrocatalytic tandem reaction [14]. 3. An efficient and general method has been described for the synthesis of 3-hydroxy-3-(nitromethyl)indolin-2-one 25 by the reaction of isatins with nitromethane/nitroethane in the presence of 1,4-diazabicyclo[2,2,2]octane (DABCO) (Scheme 11) [44]. The

reaction is catalytic and very rapid; yields are very high and the reaction avoids the use of solvents. 4. A concise and efficient route for the synthesis of highly substituted imidazopyrroloquinoline derivatives 28 by simply refluxing a reaction mixture of different types of isatins and heterocyclic ketene aminals (HKAs) by acetic acid has been developed (Scheme 12). This method is suitable for combinatorial and parallel syntheses in drug discovery [85]. 5. Activation of the pyridine nucleus has been achieved via 1,5-electrocyclization of vinyl pyridinium
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R1

O O N H 26 +
n(
)

H O N N H

O R3 AcOH toluene reflux R1 N

n ( )

R2

R2

27

R3

R1 = H, Br, Cl, F

n = 1, 2, 3; R2 = H, CH3 R3 = CH3O, CH3, H, Cl

28: 7894%

Scheme 12. Synthesis of highly substituted imidazopyrroloquinoline derivatives

R4 Z R1 N O R2 30 N N K2CO3, CH3CN reflux R1

Br O N

R3 R1

Z N

N O R2

R3

N R2 29: E/Z isomers

K2CO3, CH3CN reflux

1, 5-electro cyclization R1 = H, F, Br, Me, CHO; R2 = Me, allyl, benzyl, propargyl, ethyl; R3 = Me, OH; Z = CO2Me; R4 = Br R1 N R2 31
Scheme 13. Synthesis of 3-spirodihydroindolizine oxindole

N O

R3

ylides generated from bromo isomerized MoritaBaylis-Hillman adducts of isatin and pyridine under basic conditions. The method has been successfully applied to achieve efficient synthesis of a number of 3-spirodihydroindolizine-2-oxindoles 30, 31, which make up the core structure of secoyohimbane and heteroyohimbane alkaloid natural products (Scheme 13) [84]. 6. Organocatalytic asymmetric Henry reaction of isatins with nitromethane has been achieved with the use of C6-OH cinchona alkaloid catalyst, and a variety of chiral 3-substituted 3-hydroxyoxindoles 34 have been successfully synthesized giving in 95% yields with high enantioselectivities (Scheme 14) [86].
318
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Most of the currently available methods for the construction of 3-substituted 3-hydroxyindolin-2-ones via nucleophilic addition to isatins generate only one stereocenter and involve stepwise construction of structures with adjacent stereocenters. There is clearly a demand for novel strategies to efficiently construct this important class of polyfunctional substance with multistereocenters [19].

Biological activity
The indole ring is found in many naturally occurring compounds, notably alkaloids, fungal metabolites and marine natural products. Indole derivatives have been

Chemical and biological aspects of isatin and its derivatives

Parvaneh Pakravan et al.

S R2 O O N R1 32
+

CH3NO2

Q-1e (10 mol%) THF, 5C OH OR N H N R = 3, 5-(CF3)2Bz Q-1e

R2 HO N 33 R1

NO2 O

HO

N H O

SCH3

N H 34: (R)-(+)-dioxibrassinin

Scheme 14. Highly enantioselective synthesis of 3-hydroxy-2-oxindoles

found to possess several biological properties, including antimicrobial, antibiotic, anti-inflammatory, analgesic, anticonvulsant, antimalarial, anticancer, antiulcer, antileishmanial, contraceptive and antioxidant activities [80]. Isatin itself is known to possess CNS-MAO inhibition, anticonvulsant and anxiogenic activities. Isatin derivatives show such diverse activity, including fibrinolytic, muscle relaxant, antiallergic, immunosuppressant, and antithrombotic activity

[51]. Isatin derivatives also have agonistic effects on several receptors [80]. In particular, 3-substituted 3hydroxyoxindole has been found to be a structural motif in alkaloid based natural and synthetic pharmaceuticals (Fig. 3) [84, 86]. In this section, we concentrate on the antimicrobial, antiviral, anticonvulsant and anti-tumorigenic properties. DNA binding is discussed as a mechanism to explain the variable anticancer activity of isatin derived compounds.

O RH N HC O

H N H O

SCH Br

Br HO COMe O N H HN 36: Convolutamydine A

Cl F C HO O N

N H CH

A: R= 35: Maremyclin B: R=

OH OH

O NEt HCl 37: SM-130686 H S N O N H 40: Dioxibrassinin H CS S N

HO O MeO NMe N H Me 38: CPC-1 HO N H

OH O NH NH O CONH HO

SMe

NH O O N H O 39: TMC-95A

O N H 41: Spirobrassinin

Fig. 3. Selected examples of natural products and bioactive compounds containing 3-substituted 3-hydroxyoxindole motifs

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Antimicrobial/antiviral activity of isatin and its derivatives

Isatin compounds have well documented potent antimicrobial and antiviral properties. Schiff and Mannich bases of isatin derivatives 44 (Scheme 15) have shown significant antibacterial and moderate antifungal activities [60]. Various isatin-N-Mannich bases of isatin-3thiosemicarbazones and substituted indolinones have shown tuberculostatic activity [51]. Moreover, several compounds in a series of isatin derivatives linked to 1, 4-benzothiazine moiety 48 (Scheme 16) have demonstrated promising antifungal activity [55]. Recently, it has been reported that a bis-imine of isatin has antimicrobial properties. Antiviral properties of isatin compounds have also been reported, including thiosemicarbazone and their Mannich bases [23]. Methisazone is an effective compound against variola and vaccinia viruses [21]. Carbamoyl derivatives 10 are noted as antivirals [1, 45]. The N-dimethyl and morpholino derivative of 5-methylisatin and trimethoprim exhibited antiviral activity against HIV-1 with half maximal effective concentration (EC50) of approximately 4.3 and 17.7 mg/ml, respectively [51]. The introduction of electron withdrawing groups at positions 5, 6, and 7 of the indole ring greatly increases the antimicrobial activity of isatin, with substitution at the 5th position being most favorable [57]. This is not surprising, as C-5 substitution has previously been associated with increased biological activity for a range of indole-based compounds. The substitution of an aromatic ring at the 3rd position has been reported to be associated with antimicrobial properties [52, 57]. The

ways in which Schiff base compounds react with bacteria and fungi varies with molecular structure. Generally, the antimicrobial activity of compounds increases with the introduction of halogens (Tab. 1) [20, 49]. A comparison of antimicrobial action in a series of halogenated compounds revealed that substitution at the 5th position of isatin with chlorine, bromine or fluorine produced more active compounds [54]. Electron withdrawing substituents and the presence of nitro groups may modulate efficacy as an antimicrobial agent. Comparing compounds 53 and 54 (Fig. 4), the presence of electron withdrawing substituents in the phenyl ring in 3rd position and also the presence of nitro group at 5th position, in compound 54 would be expected to increase the lipophilic character of the molecule, facilitating transport across the microorganism cell membrane and increasing antimicrobial activity [57]. Complexes are noted to be more effective at inhibiting microbial growth than ligands alone (Tab. 1). Although it is difficult to make out an exact structureactivity relationship between the microbial activity and the structure of these complexes, it is possible that chelation enhances the activity of the complexes. Chelation reduces the polarity of the central ion, mainly because of the partial sharing of its positive charge with the donor groups and possible p-electron delocalization within the whole chelate ring. This chelation increases the lipophilic nature of the central atom, which favors the permeation through the lipid layer of the membrane. As mentioned above, this increases the toxicity of the compound in question [34, 36, 64, 70].

N O N R1 O N H
+

R1

O F N N COOH HCHO Mannich reactions N N N N 44 F O

HN Ciprofloxacin 43 COOH

Schiff bases of isatin

42

Scheme 15. Schiffs and Mannich bases of isatin

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NH2
+

O 46

H N S 47 COOH

SH 45

O O N H HCOH/HNR2 O

O DMF/gl. acetic acid N R H N S O N R O 48

Scheme 16. Schiff and Mannich bases of isatin (antibacterial and antifungal)

Tab. 1. In vitro antimicrobial activity of some new isatin derivatives zone of inhibition in mm (MIC in g/ml)

Compounds 49 50 51 52 53 54 55 56 57 58

S. aureus (6.25) (6.25) 6.29 (25) 22(13.4) 24(9.40) (16.3) (1.4) 12 12

B. subtilis (25) (6.25) 6.25 (50) 13 16

E. coli (50) (6.25) 50 (50) 19 (19.2) 25 (12.3) (16.6) (1.2) 12 7

P. aeruginosa 19 (19.9) 23 (12.0) (14.8) (1.6)

K. pneumoniae 19 (19.8) 27 (10.8) (14.5) (1.9)

A. niger 20 (21.5) 26 (12.3) (12.8) (2) 12 8

C. albicans (6.25) (6.25) 6.25 (50) (15.7) (4.9) 12 10

Ref. [20, 35] [20] [20] [20] [57] [57] [61] [61] [54] [7]

Investigation of new antimicrobial agents with reduced toxicity and lower side effects is an ongoing continuous process. Based on the work cited here, for example, it would be worthwhile to design and synthesize compounds containing both 1,4-benzothiazine and isatin derivatives of Mannich bases to generate a series of new 1,4-benzothiazine derivatives to screen for antimicrobial activity.

Anticonvulsant activity of hydrazones, Schiff and Mannich bases of isatin derivatives

Isatin is endogenously produced in the central nervous system. Its effect as a selective monoaminooxidase (MAO) inhibitor is its most potent in vitro action
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Br N N H O N H 49 N H 50 N N H O Br N H 51 OCH OCH N ON N O N H 53 N OCH ON N O N H 54 H N O O N 55 O Br N H 57 N N H O N N N N 58 O 56 Br N O N N N Cu N N N N H O N H 52

O N N H O N

NO

H N

Fig. 4. The structure of a newer class of anti-bacterial and anti-fungal agents

recorded to date with an inhibitory concentration (IC50) of about 3 g/ml [51]. Isatin has been reported to possess anti-MES (maximal electroshock) activity and it appears to have a range of actions in the brain [75]. Derivatives have also been proposed as antiepileptic drugs [75]. Many isatin derivatives, such as isatin hydrazone, isatin Mannich bases, isatin based spiroazetidinones and 3-(methylene)indolin-2-ones, have also been reported to possess neuroprotective
322

activity [8]. 3-Hydroxy-3-substituted oxindoles derived from isatin, 3-(4-thiazolidone-2-hydrazono)-isatin, 1-morpholinomethyl-3-(aryloxy-arylthio-acetyl hydrazone)-isatin and isatin based spiroazetidinones are known to possess anticonvulsant activity. Therefore, it would be expected that hydrazones, Schiff and Mannich bases of isatin would also exhibit significant anticonvulsant activity [78]. Compound 59 (Fig. 5) is an example of a potent anticonvulsant, with 87% pro-

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Chemical and biological aspects of isatin and its derivatives

Parvaneh Pakravan et al.

tection at 100 mg/kg and an EC50 of 53.61 mg/kg (metrazol-induced convulsions, MET). The effect of 5-substitution (of CH3, NO, Cl and Br) 60 (Fig. 5) into the isatin ring significantly affects the anticonvulsant activity [78]. The presence of electron withdrawing groups such as Cl or Br at the 5-position

produces compounds with no anticonvulsant activity, probably due to the disruption of the hydrophobic unit of the molecule. A 5-methyl substituent resulted in the least inductive effect on the aromatic ring, and was found to yield the most active compounds. All the Mannich bases tested were found to be inactive at the

H 3C

D N R N H O HBD

Cl

R1 N

N R2 O R3

O2N NH N N H O NH O

60 59

61 R = H, Cl, NO2 H N X

NH N

O R N NH O N R'
63

O NH O

R2 N R1 62

R = 2-Cl, 3-Cl, 4-Cl, 4-Br, 4-NO2, 4-SO2, NH2 R1 = CH3, COCH3, R2 = H, NO2

a) R = H, R' = -COCH3, b) R = H, R' = -COCH3, c) R = 5-Br, R' = -COCH3,

X = 4-Cl X = 4-NH2 X = 4-SO2NH2

R
HBD A HBD

O HO O O N D H2 C R R N H 65 A: R = H B: R = Me 66 O N H O HO O

R1 HN

O H N N H

64

O HO O N H 67
Fig. 5. Isatin derivatives possessing anticonvulsant activity

Cl N Br O N CH3 68

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experimental dose levels, revealing the hydrogen bonding domain as essential for anticonvulsant activity [78]. A series of p-nitrophenyl substituted semicarbazones 61 (Fig. 5) compounds were found to be active in pentylenetetrazol-induced convulsions (scPTZ) and maximal electric shock (MES) tests. A series of N-methyl/acetyl-5-(un)substituted-isatin-3-semicarbazones 62 (Fig. 5) revealed compounds with 4-bromo and 2-chloro substitutions possessing promising activity and were also active in MES, scPTZ and subcutaneous strychnine (scSTY) induced tests [51]. Smith et al. [71] synthesized a series of N-methyl/acetyl 5-(un)- substituted isatin-3-semicarbazoles 63 (Fig. 5) and screened the newly synthetized compounds for

their anticonvulsant activity. Products a, b and c emerged as broad-spectrum compounds as indicated by their protection in MES, scPTZ and scSTY screens [72]. Other promising anticonvulsants derived from isatin include amino-N-arylmethanethio)-3-(1-substituted benzyl-2,3-dioxoindolin-5-yl)urea 64 (Fig. 5) [73], Nmethyl-5-bromo-3-(p-chlorophenylimino)isatin and 3-cycloalkanone-3-hydroxy-2-oxindoles. For example, compounds A and B 65 (Fig. 5) are effective in anti-MES test and 66 (Fig. 5) antagonizes PTZ convulsions in mice. Raj et al. performed highly enantioselective catalytic synthesis of 3-cycloalkanone-3hydroxy-2-oxindoles 80 to produce potential anticonvulsants [58]. N-methyl-5-bromo-3-(p-chlorophen-

Fig. 6. New isatin derivatives with antioxidant activity

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Parvaneh Pakravan et al.

ylimino)isatin 68 (Fig. 5) exhibited anticonvulsant activity in MES and ScMet tests with an LD50 600 mg/ kg, showing better activity than the standard drugs phenytoin, carbamazepine and valproic acid [82].

Antioxidant activity of isatin derivatives

Recently, new isatin derivatives 6972 with potential antioxidant activity were investigated [3, 46, 47, 62]. The ketolactam ring is responsible for initiating free radical scavenging activity due to its N-H and C=O moieties [48]. George et al. studied antioxidant activity of synthesized indole derivatives of oxadiazolylpyrimidinones [17]. They revealed that compound 74 with isopropyl substitution showed the best free radical scavenging activity, comparable to that of ascorbic acid. Systems incorporating the halogens chlorine and bromine sharply enhanced the antioxidant potency (76, 78) (Fig. 6) [66]. Naik et al. [48] synthesized a series of novel isatin conjugated with aniline and substituted anilines, then examined the newly synthetized products for their antioxidant activity. Initially, the model compound (isatin) showed negligible activity. Coupling of aniline and substituted anilines significantly increased activity (Tab. 2) (8083) (Fig. 6) [48]. Among the synthesized compounds, compound 83, bearing an electron donating methoxy substituent addition to the phenolic moiety, showed dominant 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity com-

pared to butylated hydroxyanisole (BHA). The presence of an electron donating methoxy substituent in the phenolic compounds is known to increase the stability of the radical, and hence antioxidant activity. In comparison, compound 82, bearing a nitro group (electron withdrawing group) exhibited slightly lower activity. Bromine substitution also resulted in lower activity.

Anticancer activity of isatin derivatives

It is well known that isatin heterocycles are potent chemotherapeutic agents. The 2-oxoindoles derivatives (in Fig. 7) of SU-5416 (semaxanib, 84) and SU-11248 (sunitinib, 85) (Fig. 7) possess tyrosine kinase inhibitory and antiangiogenic properties. Sunitinib inhibits at least eight receptor protein-tyrosine kinases including vascular endothelial growth factor receptors 13 (VEGFR1VEGFR3), platelet-derived growth factor receptors (PDGFRa and PDGFRb), stem cell factor receptor (Kit), Flt-3, and colonystimulating factor-1 receptor (CSF-1R). VEGFR1 and VEGFR2 play key roles in vasculogenesis and angiogenesis. PDGFRb, which is found in pericytes that surround capillary endothelial cells, plays a pivotal role in stabilizing the vascular endothelium. Sunitinib inhibits angiogenesis by diminishing signaling through VEGFR1, VEGFR2, and PDGFRb. Renal cell cancers that have metastasized, or spread from the primary tumor, exhibit extensive vascularity, and sunitinib is approved for the treatment of these neoplasms. Activating Kit mutations occur in about 85% of gastrointestinal stromal tumors and activating PDGFRa mutations occur in about 5% of these tumors. Sunitinib binds reversibly to the ATP binding site of their target kinases and thereby inhibits their catalytic activity [65]. Sunitinib possesses very good oral bioavailability, has been shown highly efficacious in a number of preclinical tumor models, and is well tolerated at efficacious doses. It is currently in clinical phase I trials for the treatment of cancers [81]. Another structurally similar molecule, SU9516 86 (Fig. 7), is a potential inhibitor of cyclin-dependent kinases (CDKs) that can induce apoptosis in colon carcinoma cells. CDK inhibitory properties in isatin derived phenylhydrazones have also been reported (87) (Fig. 7). Based on this information, Abadi et al.
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Tab. 2. Inhibition (50%) of DPPH radical by isatin and its analogues

Compounds Isatin 73 74 75 76 77 78 79 80 81 82 83

IC50 (g/ml) 111 7.03 6.2 7.21 15.36 25.38 15.89 14.34 19 96 14 6

Remarks Less active Highly active Highly active Highly active Highly active Moderate activity Highly active Highly active Highly active Less active Highly active Highly active

Ref. [48] [17] [17] [17] [66] [66] [66] [66] [48] [48] [48] [48]

325

HC CH N H O N H 84: SU-5416, Semaxanib

HC N H

O NH N CH H CO N H 86: Su-9516 HOOC N O N H 89 N N H N N OH

O N H 85: Su-11248, Sunitinib R O

R' N H 87

X N H O X = N, CH

Br R N H 88 O

Fig. 7. Isatin analogs with anticancer activity [76]

Tab. 3. Anticancer activity (IC50 (M)) of isatin and its derivatives

Compound Isatin 90 91 92 93

HeLa (cervical) 521.9 12.84 175 200

IMR-32 (neuroblastoma) 352.74 15.84 194.00 285.50

MCF-7 (breast) 410.95 16.68 13.3

U937 565 6.76

jurkat 5.8

MDA-MB-231 21.8

PC-3 25.9

HCT-116 15.9

Ref. [18, 83] [18] [83] [66] [66]

U937: human monocyte-like, histiocytic lymphoma.

N N N Cl N H 90 O Br N H 92
Fig. 8. The chemical structure of new anticancer therapeutic agents

O O

SH

Br Br Br 91 N H

O O

O Cl N H 93 N N H O HN

N N H O HN

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synthesized several 2-indolone imino derivatives (88) (Fig. 7) to examine their antitumor and antiangiogenic properties. Vine et al. also synthesized several substituted isatin analogs (89) (Fig. 7), and screened against a panel of five human cancer cell lines. These authors concluded that substituted isatins could be effective as anticancer drugs [4, 6, 76]. Bis-diisatin derivatives, bis-isatin thiocarbohydrazone metal complexes, 3-o-nitrophenyl hydrazones of isatin possess cytotoxicity activity [50]. The potency (IC50 values) of anticancer activity of compounds 90 (Fig. 8) was comparable with that of known anticancer agent, cisplatin. Among the synthesized 2-indolinones, compounds 90 with a halogen atom (electron withdrawing group) at C5 position showed potent activity (Tab. 3) [18]. These results indicate that C5 substituted derivatives may be useful leads for anticancer drug development in the future. Structure-activity relationship studies identified C5, C6, and C7 substitution as greatly enhancing activity with some di- and tri-halogenated isatins giving IC50 values of 10 M, indicating activity exceeding that of cisplatin (IC50 value between 13.54 and 14.08 M) [18]. Introduction of electron withdrawing groups at positions 5, 6, and 7 greatly increased activity from that of isatin, with substitution at the 5-position being most favorable (94, 95) (Fig. 9) [83]. Halogenation yielded the most active compounds, with di- and tri-substitution 91 (Fig. 8) increasing activity up to > 100-fold from the parent molecule, isatin (Tab. 3). These results indicate that structural modification of di- and trisubstituted isatins may lead to new derivatives with enhanced and selective anticancer activity [83]. Previous studies have shown that strong electronegative atom substitution, such as Cl and Br, at the C-5 position of the aromatic ring increases the lipophilicity of

molecules and is responsible for enhanced cytotoxicity [18]. Elsewhere, halogenated hydrazine derivatives have also been reported to exhibit anticancer activity. For example, 5-bromo-3-o-nitrophenylisatin hydrazone was found to be active intramuscularly against Walker carcinoma-256 and a series of 5-bromo-(2-oxo-3-indolinyl)-thiazolidine-2,4-diones substituted by various Mannich bases were found to exhibit anti-leukemic activity against P388 lymphocytic leukemia in mice [83]. Compounds 92 and 93 (Fig. 8) have been screened for their cytotoxic activity against HeLa cell lines and IMR-32 cell lines (Tab. 3) [17]. However, neither of these compounds is comparable in cytotoxicity with that of cisplatin. Two recent synthesized isatin Schiff base copper(II) complexes Cu(isaepy)2 96 and Cu(isapn) 97 (Fig. 9) are able to induce apoptosis via the mitochondrial pathway in neuroblastoma SH-SY5Y cells and in other tumor histotypes, mainly by copper-dependent oxidative stress and nuclear/mitochondrial site-directed damage [16]. In contrast, Nalkyl isatins possess potent and selective caspase inhibition [71]. On the basis of above discussion, isatin and its derivative should be regarded as strong candidates for future anticancer treatment investigations.

Interaction of isatin and its derivatives with DNA and HSA

Investigation of the binding of isatin and its derivatives with DNA are important in order to delineate further their pharmacological properties. Many com-

Fig. 9. Structures of the studied copper(II) complexes with oxindole-Schiff base ligands

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O N H O N R1 98 NR2

O N N H O N R1 99 NR2
Fig. 10. Structure of isatin and benzoisatin aminoacetylhydrazones

N H2 N O N N N H 100
pounds exert antitumor effects through DNA binding, manipulating the replication of DNA and inhibiting the growth of the tumor. Thus DNA binding may form the basis of antitumor drug design. In this case, the effectiveness of the therapy would be expected to be influenced by the binding mode and affinity [39]. The mechanism of action exerted by some isatin compounds has been proposed to occur via distortion and damage to the DNA structure [16, 61]. However, few studies explicitly examining binding with double stranded (ds-) DNA have been carried out. One example of an investigation linking the DNA binding capacity with biological activity is provided by Karpenko et al. [26]. This work compared the DNA-binding properties of isatin and benzoisatin hydrazones. The interferon induction capability and cytotoxicity of these compounds varied in phase with their DNA affinity. The DNA affinity of benzoisatin derivatives 98 was determined to be two orders of magnitude greater than that of isatin derivatives 99 (Fig. 10). Isatin hydrazones are bicyclic compounds, which do not intercalate into the ds-DNA structure. However, the presence of an intramolecular hydrogen
328

S H O

Fig. 11. Structures of isatin-3-isonicotinylhydrazone (INH) 100 and isatin-b-thiosemicarbazone (IBT) 101

N N H N H 101

bond (H-bond) allows the formation of the third (pseudo) cycle in these structures, making them potential DNA intercalators. Intramolecular hydrogen bonding extends the aromatic rings in isatin compounds and enhances DNA binding affinity [12]. Other isatin derivatives also possess the ability to intercalate with DNA. Isatin-3-isonicotinylhydrazone (INH, 100, Fig. 11) has been also found to be potentially capable of intercalation with DNA [31]. The absorption spectrum of the INH shows that as the concentration of DNA increases, a large degree of hypochromism develops in the spectrum. Hypochromism usually arises from the strong stacking interaction between the aromatic chromophore and the base pairs. The intrinsic binding constant observed (1.0 105 M1) was roughly comparable to other intercalators. The viscosity increase of DNA can be ascribed to the intercalative binding mode of the INH, due to effective DNA length increase (Fig. 12) [29]. In Figure 13, the changes in the circular dichroism (CD) spectrum of CT-DNA in the presence of increasing concentrations of INH are depicted. INH molecules stack in between the base pairs of DNA, thus leading to an en-

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Ri

Fig. 12. Effect of increasing amounts of INH (ri = 0.0, 0.5, 0.7, 0.9, 1, 1.2, 1.5, 1.8, and 2), IBT, isatin (ri = 0.0, 0.5, 1, 1.2, 1.5, 1.8, and 2) on the viscosity of CT-DNA (5 10-5 M) in 0.01 M Tris-HCl buffer (pH 7.4)

hancement in the positive band. An increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of INH indicates that INH is able to competitively bind DNA at similar sites, releasing the intercalated MB completely [31]. Formation constants of INH-DNA complex at different temperatures, calculated by BenesiHildebrand equation [27], are shown in Table 4. From thermodynamic parameters of INH-DNA formation determined via Vant Hoff equation [15], the reaction is exothermic and enthalpy favored. Negative entropy confirms the intercalative binding mode of INH to DNA [31]. Binding mode of isatin-b-thiosemicarbazone (IBT, 101, Fig. 11) to calf thymus DNA (CT-DNA) has also been characterized to be intercalative. Considerable hypochromism in the absorption and a small shift in the absorption maximum (from 356 to 357 nm) were observed during the interaction of IBT with DNA. The Kb of IBT with DNA was calculated at 1.03 105 M1. By comparison of the Kb of IBT with the Kb of other DNA intercalative drugs [69], we can deduce that the IBT binds CT-DNA via an intercalation mechanism. Several other pieces of experimental evidence also support an intercalative binding mechanism: 1. The binding of IBT molecules to DNA led to a marked increase in emission intensity, as observed for other intercalators. 2. A competitive reaction monitored between neutral red (NR) dye, DNA and IBT showed that the in-

Fig. 13. Circular dichroism spectra of DNA (8.0 105 M) in 10 mM Tris-HCl buffer, in the presence of increasing amounts of IBT (A), isatin (B), INH (C)

tercalated NR was displaced from the DNA-NR system by IBT. 3. Fluorescence titration data also was used to determine the binding constant (Kf) (Tab. 4) [77]. Evaluation of the formation constants for the IBTDNA complex at four different temperatures allow thermodynamic parameters of IBT-DNA formation via Vant Hoff equation to be determined (Tab. 4). The results indicated that the van der Waals interactions or hydrogen bonds are the main forces in the

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Tab. 4. Thermodynamic parameters and binding constants for binding of isatin and its hydrazones to CT-DNA

Intercalator and groove binder Isatin (groove binder)

T(K) 277 288 298 310 277 288 298 310 290 298 303 310

Kf 1.9 105 3.1 105 5.0 105 6.3 105 4.7 104 2.2 104 1.7 104 1.1 104 1.04 105 7.52 104 4.77 104 2.82 104

DG (kJ/mol) 28.35 30.56 32.57 34.99 24.519 24.294 24/.89 23.844 71.664 72.265 72.641 73.167

DH (kJ/mol) +27.42

DS (J/ mol K) +201.35

Ref. [32]

100 (intercalator)

30.187

20.46

[31]

101 (intercalator)

49.87

75.152

binding of the investigated drug to CT-DNA, and the mode of binding is intercalation [30]. 4. The viscosity increase of DNA is ascribed to the intercalative binding mode of the IBT, due to effective DNA length increase (Fig. 12) [28]. 5. Changes in the CD spectra of CT-DNA in the presence of increasing concentrations of IBT are depicted in Figure 13. The IBT molecules stack in between the base pairs of DNA thus leading to the enhancement in the positive band [59]. Recently, we reported complexation of isatin with DNA which occurred by the mechanism of groove binding [32]. Isatin binding to DNA was characterized

by hypochromism in the absorption spectrum. The intrinsic binding constant of isatin with DNA observed was comparable to other groove binders [41]. Evaluation of the formation constant for the isatin-DNA complex at four different temperatures allowed thermodynamic parameters of isatin-DNA formation to be determined via Vant Hoff equation [67] (Tab. 4). These results indicated that the isatin-DNA binding is entropically favored but enthalpically disfavored. Groove binding is predominantly entropically driven, whereas intercalation is enthalpically driven. Experimentation involving DNA viscosity (Fig. 12), fluorescence quenching and DNA spectral band analysis

Fig. 14. Chemical structures of indole derivatives

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Fig. 15. The structural formulas of the complexes [ML(phen)2]Cl2 110 and [ML(bpy)2]Cl2 111; M = Cu(II) or Zn(II)

(Fig. 13) in the presence of isatin also support the hypothesis that isatin can be categorized as a DNA groove binder. Pandya et al. report the binding mode of several biologically important indole derivatives to be DNA groove binders (Fig. 14) [53]. All of the indole derivatives mentioned possess only one indole ring, just as for isatin. The objective of this work was to avoid increasing the overall stacking forces during molecular synthesis, thereby specifically targeting the minor groove of DNA [53]. A comparison shows the Kb of IBT-DNA (1.03 105 M1) and INH-DNA (1.0 105 M1) to be greater than that of isatin-DNA (7.34 104 M1). It seems reasonable that, since INH and IBT possess greater planar area and a more extended p system than that of isatin. INH and IBT penetrate more deeply into the stacked base pair of DNA structure than isatin, with a concomitant increase in DNA binding. The possibility of intercalation of indole derivatives (isatin) between the DNA base pairs is small, mainly due to less-effective stacking forces between the indole nucleus and the DNA bases [53]. These data emphasize that the mode of binding has a significant effect on the strength of DNA binding. The strength of DNA binding of isatin compounds can be increased by metal complexation. Mixed ligand Cu(II)/Zn(II) complexes using 3-(phenylimino)-1,3-dihydro-2H-indol-2-one (obtained by the condensation of isatin and aniline) as the primary ligand and 1,10-phenanthroline (phen)/2,2-bipyridine (bpy) as an additional ligand (Fig. 15) showed an

increase in intrinsic binding constant values compared to free ligands [61]. Both copper and zinc complexes exhibited hypochromicity and a red-shifted charge transfer peak maxima in the absorption spectra. The viscosity of the DNA solution increased with increasing ratio of both the copper and zinc complexes to DNA, further suggesting an intercalating binding mode of the complexes with DNA [68]. Cyclic voltammograms performed using carbon electrode in solutions containing [CuL(phen)2]Cl2 during incremental addition of DNA to the complex resulted in a negative shift in the potential of the second cathodic peak and a decrease in the current intensities during DNA addition. Changes in the voltammetric currents in the presence of CT-DNA can be attributed to the slow diffusion of the metal complex bound to the large, slowly diffusing DNA molecule. The changes of the peak currents observed for the complexes upon addition of CT-DNA may indicate that the complexes possess a higher DNA binding affinity. The results of cleavage studies using pUC19 DNA showed that the complexes had higher nuclease activities than the isatin-based ligand. Higher binding affinity of complexes to DNA may be attributed to the nature of the binding of the mixed ligand complexes with DNA, which is significant due to p-stacking or hydrophobic interactions of the aromatic phenyl rings. However, the metal ions play essential role in DNA binding by these complexes. Chelating effect (metal ion to free ligand) can enhance the planar functionality of metal complex, so the complexes can insert and stack be-

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tween the base pairs of double helical DNA more easily than the free ligands [38]. A limited amount of data examining isatin interaction with model proteins is available. Da Silveira et al. investigated interactions of oxindole-Schiff base copper(II) complexes (Fig. 9) with HSA [11]. Modifications in the characteristic a-helix feature of the protein induced by interactions with these complexes were detected. Since copper ions inserted in the protein can catalyze the formation of reactive oxygen species (ROS), especially in the presence of hydrogen peroxide or molecular oxygen, oxidative damage to the protein was also verified and monitored. Only the complexes [Cu(isaepy)H2O]2+, [Cu(isaepy)2]2+ and [Cu(isapn)]2+ were able to modify significantly this protein feature, as observed by the decreasing of the band at 208 nm, indicating ~25% reduction of its helical content after addition of two copper ions per protein. In the presence of hydrogen peroxide all the compounds tested exhibited an increased level of protein oxidation, depending on the ligand, in the order of: [Cu(isaepy)2]2+ >> [Cu(isapn)]2+ > [Cu(isaepy)H2O]+ >> [Cu(isaenim)]2+ > [Cu(H2O)4]2+. The two complexes exhibiting the greatest protein oxidation power, [Cu(isaepy)2]2+ and [Cu(isapn)]2+, are indeed those which modified protein a-helix structure most significantly. Only the complex [Cu(isaepy)2]2+ was able to cause notable degradation of HSA [11].

ity for a range of indole-based compounds. In general, complexes of isatin derivatives have greater biological activity than their ligands alone. Further in vivo and in vitro studies are necessary to establish and confirm the mentioned mechanisms. We hope that our discussion will prove helpful in further development and progress of the pharmacological applications of these molecules.

Acknowledgment: Financial support from Razi University Research Center is gratefully acknowledged.

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Conclusions
The isatin structural motif can be found in a broad range of natural and synthetically derived pharmacologically active compounds. The creation of novel isatin derivatives as drug targets is an active area of medicinal chemistry. The review compiles published data on the synthesis and biological action of new isatin derivatives. Our literature survey indicated that C-5 substituted derivatives may be especially useful to consider when designing anticancer drugs. Introduction of electron withdrawing groups at positions 5, 6, and 7 of the indole ring greatly increased the activity in comparison with isatin, however, substitution at the C-5 position can be considered most favorable. This is not surprising, as C-5 substitution has previously been associated with increased biological activ332
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Received: January 6, 2012; in the revised form: November 19, 2012; accepted: November 26, 2012.

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