Biochemical and pharmacological characterization of isatin ... · Review Biochemical and pharmacological characterization of isatin and its derivatives: from structure to activity

Review

Biochemical and pharmacologicalcharacterization of isatin and its derivatives:from structure to activity

Parvaneh Pakravan1, Soheila Kashanian2, Mohammad M. Khodaei2,

Frances J. Harding3

1Department of Chemistry, Zanjan Branch, Islamic Azad University, Zanjan, I.R. Iran

2Department of Chemistry, Sensor and Biosensor Research Center (SBRC) & Nanoscience and Nanotechnology

Research Center (NNRC), Faculty of Science, Razi University, Kermanshah, I.R. Iran

3Mawson Institute, University of South Australia, Adelaide, South Australia 5001, Australia

Correspondence: Soheila Kashanian, e-mail: [email protected]; Mohammad M. Khodaei,

e-mail: [email protected]

Abstract:

Isatin, 1H-indole-2,3-dione, is a heterocyclic compound of significant importance in medicinal chemistry. It is a synthetically versa-tile molecule, a precursor for a large number of pharmacologically active compounds. Isatin and its derivatives have aroused great at-tention in recent years due to their wide variety of biological activities, relevant to application as insecticides and fungicides and ina broad range of drug therapies, including anticancer drugs, antibiotics and antidepressants. The purpose of this review is to providean overview of the pharmacological activities of isatin and its synthetic and natural derivatives. Molecular modifications to tailor theproperties 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 againstmalaria, tuberculosis, AIDS, and trypanosomiasis isurgently needed in order to satisfy the overwhelmingdemand for disease treatment in Third World coun-tries [9]. Synthesis of new medicinal compoundsbased on candidate molecules with known biologicalactivity offers an opportunity to design effective safedrugs with minimal toxicity [60].

Isatin, 1H-indole-2,3-dione 1 (Fig. 1), is a versatilechemical building block, able to form a large number

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

Pharmacological Reports, 2013, 65, 313�335 313

Pharmacological Reports

2013, 65, 313�335ISSN 1734-1140

Copyright © 2013by Institute of PharmacologyPolish Academy of Sciences

Fig. 1. Structure of isatin 1 and indole 2

participate in a broad range of synthetic reactions,leading to its extensive use as a precursor molecule inmedicinal chemistry [10, 56, 60]. Here we discuss thepotential of isatin and its derivatives to create novelbioactive compounds. The basic chemistry and syn-thesis of isatin derivates are first reviewed. The ex-panse of biological, and particularly pharmacological,activities of isatin compounds is explored. During thisdiscussion, we propose molecular modifications totune and refine isatin compounds for use in specifictherapies.

The chemistry of isatin and its

derivatives

Fundamental reactivity of isatin and its

derivatives

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

a large number of reactions. The keto group at posi-tion 2 and particularly at position 3 can enter intoaddition reactions at the C-O bond and into condensa-tion reactions. Through the primary amine group,compounds of the isatin series are capable of enteringinto N-alkylation and N-acylation and into Mannichand Michael reactions [60]. These reactions are de-scribed in detail below.

Carbonyl reactions

Schiff bases of isatin 4 can be synthesized by condensa-tion of the keto group of isatin with different aromaticprimary amines 3 (Scheme 1) [63]. Bis-Schiff bases ofisatin can be prepared by reactions with aromatic dia-mines in the presence of catalytic amounts of glacialacetic acid in EtOH under reflux conditions [2, 22].

The straightforward synthesis of isatin-b-thiosemi-carbazones 6 by the condensation of isatin and a thio-semicarbazide 5 has been known for more than 50years (Scheme 2) [21, 25].

314 Pharmacological Reports, 2013, 65, 313�335

NH

O

O + RNH2NH

NHR

OEtOH, CH3COOH

reflux

3 4

Scheme 1. Schiff reaction

NH

O

O H2N

HNNH2

S+

NH

O

NHN

H2NS

5 6

Scheme 2. Preparation of isatin thio-semicarbazone

NH

O

O+ NH2OH

pH7.

0

pH7.6

NH

NH

NOH

O

O

NOH

7

8

9

Scheme 3. Formation of isatin 3-oxime8, isatin 2-oxime 9

Selectivity of isatinoxime formation reactions canbe controlled by pH (Scheme 3) [1]. The reaction ofisatin-2-oxime 8 with isocyanate gives bioactive car-bamoyl derivatives 10 as the main products (Scheme4) [1, 45].

N-Alkylation

N-substituted isatins have been frequently used as in-termediates and synthetic precursors for the prepara-tion of a wide variety of heterocyclic compounds. N-substituted isatin derivatives 12 can be synthesizedvia substitution reaction. The reaction between isatinand halohydrocarbons (11) can be carried out inNaOEt using EtOH as solvent or in the presence ofNaH using DMF as solvent (Scheme 5) [8]. A simple

and efficient technique to assisted synthesis of N-alkylisatins by N-alkylation of isatin using a house-hold microwave oven has been reported. The use ofmicrowave irradiation offers many advantages overconventional heating: it decreases reaction times, in-creases yields and requires less solvent, thus facilitat-ing reaction scale up (Scheme 6) [71].

N-Mannich bases of the Schiff base 15 can be syn-thesized by condensing the acidic amino group ofisatin 14 with formaldehyde and various secondaryamines (Scheme 7) [79].

Electrophilic aromatic substitution of isatin

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


Chemical and biological aspects of isatin and its derivativesParvaneh Pakravan et al.

NH

O

O + X-RN

O

O

R

K2CO3

DMF

1112

Scheme 5. Synthesis of N-substitutedisatin derivatives

NH

O

NHN

H2NS

K2CO3, R1I

DMF, 100°C, 20 min

Mw IrradN

O

NHN

H2NS

R1

R1 = CH3, Et, Ph, Benzyl

6 13

Scheme 6. General synthetic route toN1-substituted thiosemicarbazones

N

NOH

R

ON

NOCONHR'

O

R

R'NCO

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

8 10

Scheme 4. Formation of the carba-moyl derivatives

NH

N

O

RHN R1

R2

N

N

O

NR1

R2

, HCHO

Mannich reaction

R

14 15

Scheme 7. Synthesis of N-Mannichbases

acid in concentrated sulfuric acid [5]. However,a more convenient method to synthesize 5-nitroisatininvolves the dropwise addition of a solution of isatinin sulfuric acid to a solution of potassium nitrate dis-solved in concentrated sulfuric acid at 0–4°C [24]. 5-Nitroindoline-2,3-dione has been prepared by reflux-ing of indoline-2,3-dione with a mixture of 95–100%sulfuric acid and 70% nitric acid in water bath at 60°Cfor 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 tempera-ture to specifically produce the 5-halo derivatives 17

as reported by De Souza et al. (Scheme 8) [13]. Thismethod is an alternative to the use of highly toxic andcorrosive Cl2 and Br2, which can lead to other prod-ucts such as 5,7-dibromo-3,3-dialkoxyoxindole whenthe bromination of isatin is attempted in alcoholic me-dia [40]. Moreover, others have reported using therelatively stable reagent trichloroisocyanuric acid(TICA), as an efficient new source of electrophilicchlorine and this provides a relatively inexpensiveroute to the chlorination of isatins [43, 74].

Complexes with bioactive Schiff bases

of isatin

Simple isatin based Schiff base compounds, contain-ing acyl, aroyl and heteroaroyl Schiff bases, possessadditional electron donor sites, for instance at C=Oand C=N-. These donor sites make such compoundsgood chelating agents, able to form a variety of com-plexes with various transition and inner transition

metals [37]. For example, isatin-b-thiosemicarbazonecan 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,Xligands to form stable coordination compounds withdifferent transition metal ions. Rodríguez-Argûelles etal. [64] reported the interesting biological propertiesof cobalt(II), nickel(II), copper(II) and zinc(II)complexes of 3-isatin- and N-methylisatin-3-pico-linoylhydra- zone as well as of 2-thiophenecarbonyland isonicotinoyl hydrazones of 3-(N-methyl)isatin.

Finally, complexes of divalent copper, cobalt,nickel and zinc ions with 3-salicylidenehydrazono-2-indolinone 18 can be isolated in the keto-form of theligand, as shown in Figure 2 [6].

Synthesis of new isatin derivatives

A number of novel reactions have been reported thatmay assist in the generation of pharmacologically ac-tive isatin compounds:

1. The synthesis of 3-(indol-3-yl)-3-hydroxy-indolin-2-ones 19 from isatins and indoles utilizingFe(III) as a recyclable homogeneous catalyst underultrasound irradiation has been described (Scheme 9).It was found that the conditions employed producedyields of 85–95% [33].

2. Electrocatalytic transformation of isatins andbarbituric acids in ethanol in an undivided cell in thepresence of sodium bromide as an electrolyte resultsin the formation of substituted 5,5’-(2-oxo-2,3-di-hydro-1H-indole-3,3-diyl)bis(pyrimidine-2,4,6(-1H, 3H,


NH

N

O

N

OMCl

[M(L)Cl]Cl

L = 3-Salicylidenehydrazono-2-indolinone

M = Co, Ni, Cu, Zn18

Fig. 2. 3-Salicylidenehydrazono-2-indolinone complexes

NH

O

O +

SN

O

X

O O

X = Cl, Br, I

NH

O

OXSiO2

CH2Cl2, rt

1617

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

5H)-triones) 22 with 89–95% substance yields and89–95% current yields (Scheme 10). This novel andefficient catalytic process is important due to itsdiversity-oriented large-scale processes and is anexample of easy environmentally benign synthesis viaelectrocatalytic tandem reaction [14].

5H)-triones) 22 with 89–95% substance yields and89–95% current yields (Scheme 10). This novel andefficient catalytic process is important due to itsdiversity-oriented large-scale processes and is anexample of easy environmentally benign synthesis viaelectrocatalytic tandem reaction [14].

3. An efficient and general method has been de-scribed for the synthesis of 3-hydroxy-3-(nitromethyl)-indolin-2-one 25 by the reaction of isatins with ni-tromethane/nitroethane in the presence of 1,4-diaza-bicyclo[2,2,2]octane (DABCO) (Scheme 11) [44]. The

reaction is catalytic and very rapid; yields are veryhigh and the reaction avoids the use of solvents.

4. A concise and efficient route for the synthesis ofhighly substituted imidazopyrroloquinoline deriva-tives 28 by simply refluxing a reaction mixture of dif-ferent types of isatins and heterocyclic ketene aminals(HKAs) by acetic acid has been developed (Scheme12). This method is suitable for combinatorial andparallel syntheses in drug discovery [85].

5. Activation of the pyridine nucleus has beenachieved via 1,5-electrocyclization of vinyl pyridinium



N

O

O

R1

R2

R3

R4R

+R5

NO2N

O

HOR5

NO2

RR4

R3

R2

R1

DABCO(5 mol%)

Solvent free, 1-5 min

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

R5 = H, Me

23 24 25

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

NO

R1

NH

O

NR2

N

R1

O

N NH

O

NR2

98 99

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

HN

+

NH

O

O

Fe(III), H2O:EtOH(60:40)

2-25 min, 50oC NH

O

OH

NH

19

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

ylides generated from bromo isomerized Morita-Baylis-Hillman adducts of isatin and pyridine underbasic conditions. The method has been successfullyapplied to achieve efficient synthesis of a number of3-spirodihydroindolizine-2-oxindoles 30, 31, whichmake up the core structure of secoyohimbane and het-eroyohimbane alkaloid natural products (Scheme 13)[84].

6. Organocatalytic asymmetric Henry reaction ofisatins with nitromethane has been achieved with theuse of C6’-OH cinchona alkaloid catalyst, and a vari-ety of chiral 3-substituted 3-hydroxyoxindoles 34

have been successfully synthesized giving in 95%yields with high enantioselectivities (Scheme 14)[86].

Most of the currently available methods for the con-struction of 3-substituted 3-hydroxyindolin-2-ones via nu-cleophilic addition to isatins generate only one stereocenterand involve stepwise construction of structures with adja-cent stereocenters. There is clearly a demand for novelstrategies to efficiently construct this important class ofpolyfunctional substance with multistereocenters [19].

Biological activity

The indole ring is found in many naturally occurringcompounds, notably alkaloids, fungal metabolites andmarine natural products. Indole derivatives have been


R1

NR2

N

O

Z

K2CO3, CH3CN

reflux

N

R4

NO

Z Br

R2

R1

29: E/Z isomers

N

R3

K2CO3, CH3CN

reflux

NO

N R3Z

R2

R1

1, 5-electro

cyclization

NO

N R3Z

R1

R2

R1 = H, F, Br, Me, CHO; R2 = Me, allyl, benzyl, propargyl, ethyl;

R3 = Me, OH; Z = CO2Me; R4 = Br

30

31

Scheme 13. Synthesis of 3-spirodihydroindolizine oxindole

NH

O

O

R1

+NH

HN

R2

OR3

R1 = H, Br, Cl, F

n()

n = 1, 2, 3; R2 = H, CH3

R3 = CH3O, CH3, H, Cl

AcOH

toluene

reflux N

NON

R2

R3

R1

( )n

28: 78–94%

26 27

Scheme 12. Synthesis of highly substituted imidazopyrroloquinoline derivatives

found to possess several biological properties, includ-ing antimicrobial, antibiotic, anti-inflammatory, anal-gesic, anticonvulsant, antimalarial, anticancer, antiul-cer, antileishmanial, contraceptive and antioxidantactivities [80]. Isatin itself is known to possessCNS-MAO inhibition, anticonvulsant and anxiogenicactivities. Isatin derivatives show such diverse activ-ity, including fibrinolytic, muscle relaxant, antialler-gic, immunosuppressant, and antithrombotic activity

[51]. Isatin derivatives also have agonistic effects onseveral receptors [80]. In particular, 3-substituted 3-hydroxyoxindole has been found to be a structuralmotif in alkaloid based natural and synthetic pharma-ceuticals (Fig. 3) [84, 86]. In this section, we concen-trate on the antimicrobial, antiviral, anticonvulsantand anti-tumorigenic properties. DNA binding is dis-cussed as a mechanism to explain the variable anti-cancer activity of isatin derived compounds.



N

O

O

R2+ CH3NO2

Q-1e (10 mol%)

THF, 5°C N

R1

O

HONO2

R1

R2

NH

O

HO NH

SCH3

S

OH

NN

OR

H

R = 3, 5-(CF3)2Bz

Q-1e

34: (R)-(+)-dioxibrassinin32

33

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

N OH C

RNH

HNO

OCH

H

HSCH

A: R= OHB: R= OH

NH

O

HOCOMe

Br

Br

36: Convolutamydine A

N

NMe

Me

MeO

H

38: CPC-1

NO

HO

ClF C

H N

O

37: SM-130686

NH

O

HO HN

SMe

S

40: Dioxibrassinin

NH

O

S N

H CS

41: Spirobrassinin

NH

O

HO

NH

O

NH

OH

NH

O

O

HOO

NHO

CONH

39: TMC-95A

NEt HCl35: Maremyclin

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

Antimicrobial/antiviral activity of isatin

and its derivatives

Isatin compounds have well documented potent anti-microbial and antiviral properties. Schiff and Mannichbases of isatin derivatives 44 (Scheme 15) have shownsignificant antibacterial and moderate antifungal activi-ties [60]. Various isatin-N-Mannich bases of isatin-3-thiosemicarbazones and substituted indolinones haveshown tuberculostatic activity [51]. Moreover, severalcompounds in a series of isatin derivatives linked to 1,4-benzothiazine moiety 48 (Scheme 16) have demon-strated promising antifungal activity [55]. Recently, ithas been reported that a bis-imine of isatin has antimi-crobial properties. Antiviral properties of isatin com-pounds have also been reported, including thiosemicar-bazone and their Mannich bases [23]. Methisazone isan effective compound against variola and vaccinia vi-ruses [21]. Carbamoyl derivatives 10 are noted as an-tivirals [1, 45]. The N-dimethyl and morpholino de-rivative of 5-methylisatin and trimethoprim exhibitedantiviral activity against HIV-1 with half maximal ef-fective concentration (EC50) of approximately 4.3 and17.7 mg/ml, respectively [51].

The introduction of electron withdrawing groups atpositions 5, 6, and 7 of the indole ring greatly increasesthe antimicrobial activity of isatin, with substitution atthe 5th position being most favorable [57]. This is notsurprising, as C-5 substitution has previously been as-sociated with increased biological activity for a rangeof indole-based compounds. The substitution of an aro-matic ring at the 3rd position has been reported to be as-sociated with antimicrobial properties [52, 57]. The

ways in which Schiff base compounds react with bac-teria and fungi varies with molecular structure. Gener-ally, the antimicrobial activity of compounds in-creases with the introduction of halogens (Tab. 1) [20,49]. A comparison of antimicrobial action in a seriesof halogenated compounds revealed that substitutionat the 5th position of isatin with chlorine, bromine orfluorine produced more active compounds [54]. Elec-tron withdrawing substituents and the presence of nitrogroups may modulate efficacy as an antimicrobialagent. Comparing compounds 53 and 54 (Fig. 4), thepresence of electron withdrawing substituents in thephenyl ring in 3rd position and also the presence of ni-tro group at 5th position, in compound 54 would be ex-pected to increase the lipophilic character of the mole-cule, facilitating transport across the microorganismcell membrane and increasing antimicrobial activity[57].

Complexes are noted to be more effective at inhib-iting microbial growth than ligands alone (Tab. 1). Al-though it is difficult to make out an exact structure-activity relationship between the microbial activityand the structure of these complexes, it is possiblethat chelation enhances the activity of the complexes.Chelation reduces the polarity of the central ion,mainly because of the partial sharing of its positivecharge with the donor groups and possible p-electrondelocalization within the whole chelate ring. Thischelation increases the lipophilic nature of the centralatom, which favors the permeation through the lipidlayer of the membrane. As mentioned above, this in-creases the toxicity of the compound in question [34,36, 64, 70].


NH

O

NR1

+

HN

NN

F COOH

O

CiprofloxacinSchiff bases of isatin

N

O

NR1

NN

N

F

COOH

OMannich reactions

HCHO

42 43 44

Scheme 15. Schiff’s and Mannich bases of isatin

Investigation of new antimicrobial agents with re-duced toxicity and lower side effects is an ongoingcontinuous process. Based on the work cited here, forexample, it would be worthwhile to design and syn-thesize compounds containing both 1,4-benzothiazineand isatin derivatives of Mannich bases to generatea series of new 1,4-benzothiazine derivatives toscreen for antimicrobial activity.

Anticonvulsant activity of hydrazones,

Schiff and Mannich bases of isatin

derivatives

Isatin is endogenously produced in the central nerv-ous system. Its effect as a selective monoaminooxi-dase (MAO) inhibitor is its most potent in vitro action



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

Compounds S. aureus B. subtilis E. coli P. aeruginosa K. pneumoniae A. niger C. albicans Ref.

49 (6.25) (25) (50) – – – (6.25) [20, 35]

50 (6.25) (6.25) (6.25) – – – (6.25) [20]

51 6.29 6.25 50 – – – 6.25 [20]

52 (25) (50) (50) – – – (50) [20]

53 22(13.4) – 19 (19.2) 19 (19.9) 19 (19.8) 20 (21.5) – [57]

54 24(9.40) – 25 (12.3) 23 (12.0) 27 (10.8) 26 (12.3) – [57]

55 (16.3) – (16.6) (14.8) (14.5) (12.8) (15.7) [61]

56 (1.4) – (1.2) (1.6) (1.9) (2) (4.9) [61]

57 12 13 12 – – 12 12 [54]

58 12 16 7 – – 8 10 [7]

NH2

SH

O OHN

S COOH

NR

O

O

HN

S

+

N

O

O

H

N

O

O

R

HCOH/HNR2

DMF/gl. acetic acid

45 46 47

48

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

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

activity [8]. 3-Hydroxy-3-substituted oxindoles de-rived from isatin, 3-(4-thiazolidone-2-hydrazono)-isatin,1-morpholinomethyl-3-(aryloxy-arylthio-acetyl hydra-zone)-isatin and isatin based spiroazetidinones areknown to possess anticonvulsant activity. Therefore,it would be expected that hydrazones, Schiff andMannich bases of isatin would also exhibit significantanticonvulsant activity [78]. Compound 59 (Fig. 5) isan example of a potent anticonvulsant, with 87% pro-


HN

O

N

55

HN O

NN

N

N

NCu

56

NH

BrN

O

NH

O

NN

O

57

N

N

N

Br

O

58

NH

N

O

NH

49

NH

N

O

NH

Br

50

NH

N

O

NHBr

51

NH

N

O

NH

ON

52

NH

N

O

N

OCH

OCH

OCH

O N

53

NH

N

O

N

NO

O N

54

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

tection at 100 mg/kg and an EC50 of 53.61 mg/kg (me-trazol-induced convulsions, MET).

The effect of 5-substitution (of CH3, NO, Cl and Br)60 (Fig. 5) into the isatin ring significantly affects theanticonvulsant activity [78]. The presence of electronwithdrawing groups such as Cl or Br at the 5-position

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



NH

O

N

Cl

H3CR

D

HBD

59

NO

NR1

R3

R2

60

NH

N

O

NR

NH

ONH

O2N

O

NNH

ONHR

R2

R1

R = H, Cl, NO2

R = 2-Cl, 3-Cl, 4-Cl, 4-Br, 4-NO2,

4-SO2, NH2

R1 = CH3, COCH3,

R2 = H, NO2

61

62

NO

N

Cl

Br

CH3

68

N

O

O

H2C

R

HN

O

NH

HN

S

R1

A

R

HBD HBD

D

64

NH

O

HO

O

R

A: R = H

B: R = Me

NH

O

HOO

65

66

NH

O

HO

O

67

NR'

N

O

NH

OHN

X

R

a) R = H, R' = -COCH3, X = 4-Cl

b) R = H, R' = -COCH3, X = 4-NH2

c) R = 5-Br, R' = -COCH3, X = 4-SO2NH2

63

Fig. 5. Isatin derivatives possessing anticonvulsant activity

experimental dose levels, revealing the hydrogenbonding domain as essential for anticonvulsant activ-ity [78].

A series of p-nitrophenyl substituted semicarba-zones 61 (Fig. 5) compounds were found to be activein pentylenetetrazol-induced convulsions (scPTZ) andmaximal electric shock (MES) tests. A series ofN-methyl/acetyl-5-(un)substituted-isatin-3-semicarba-zones 62 (Fig. 5) revealed compounds with 4-bromoand 2-chloro substitutions possessing promising ac-tivity and were also active in MES, scPTZ and subcu-taneous strychnine (scSTY) induced tests [51]. Smithet al. [71] synthesized a series of N-methyl/acetyl5-(un)- substituted isatin-3-semicarbazoles 63 (Fig. 5)and screened the newly synthetized compounds for

their anticonvulsant activity. Products a, b and cemerged as broad-spectrum compounds as indicatedby their protection in MES, scPTZ and scSTY screens[72].

Other promising anticonvulsants derived from isatininclude amino-N-arylmethanethio)-3-(1-substituted ben-zyl-2,3-dioxoindolin-5-yl)urea 64 (Fig. 5) [73], N-methyl-5-bromo-3-(p-chlorophenylimino)isatin and3-cycloalkanone-3-hydroxy-2-oxindoles. For exam-ple, compounds A and B 65 (Fig. 5) are effective inanti-MES test and 66 (Fig. 5) antagonizes PTZ con-vulsions in mice. Raj et al. performed highly enantio-selective catalytic synthesis of 3-cycloalkanone-3-hydroxy-2-oxindoles 80 to produce potential anticon-vulsants [58]. N-methyl-5-bromo-3-(p-chlorophen-


Fig. 6. New isatin derivatives with antioxidant activity

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

Antioxidant activity of isatin derivatives

Recently, new isatin derivatives 69–72 with potentialantioxidant activity were investigated [3, 46, 47, 62].The ketolactam ring is responsible for initiating freeradical scavenging activity due to its N-H and C=Omoieties [48]. George et al. studied antioxidant activ-ity of synthesized indole derivatives of oxadiazolyl-pyrimidinones [17]. They revealed that compound 74

with isopropyl substitution showed the best free radi-cal scavenging activity, comparable to that of ascorbicacid. Systems incorporating the halogens chlorine andbromine sharply enhanced the antioxidant potency(76, 78) (Fig. 6) [66]. Naik et al. [48] synthesizeda series of novel isatin conjugated with aniline andsubstituted anilines, then examined the newly synthe-tized products for their antioxidant activity. Initially,the model compound (isatin) showed negligible activ-ity. Coupling of aniline and substituted anilines sig-nificantly increased activity (Tab. 2) (80–83) (Fig. 6)[48]. Among the synthesized compounds, compound83, bearing an electron donating methoxy substituentaddition to the phenolic moiety, showed dominant2,2-diphenyl-1-picrylhydrazyl (DPPH) activity com-

pared to butylated hydroxyanisole (BHA). The pres-ence of an electron donating methoxy substituent inthe phenolic compounds is known to increase the sta-bility of the radical, and hence antioxidant activity. Incomparison, compound 82, bearing a nitro group(electron withdrawing group) exhibited slightly loweractivity. Bromine substitution also resulted in loweractivity.

Anticancer activity of isatin derivatives

It is well known that isatin heterocycles are potentchemotherapeutic agents. The 2-oxoindoles deriva-tives (in Fig. 7) of SU-5416 (semaxanib, 84) andSU-11248 (sunitinib, 85) (Fig. 7) possess tyrosine ki-nase inhibitory and antiangiogenic properties. Suniti-nib inhibits at least eight receptor protein-tyrosine ki-nases including vascular endothelial growth factor re-ceptors 1–3 (VEGFR1–VEGFR3), platelet-derivedgrowth factor receptors (PDGFRa and PDGFRb),stem cell factor receptor (Kit), Flt-3, and colony-stimulating factor-1 receptor (CSF-1R). VEGFR1 andVEGFR2 play key roles in vasculogenesis and angio-genesis. PDGFRb, which is found in pericytes thatsurround capillary endothelial cells, plays a pivotalrole in stabilizing the vascular endothelium. Sunitinibinhibits angiogenesis by diminishing signalingthrough VEGFR1, VEGFR2, and PDGFRb. Renalcell cancers that have metastasized, or spread fromthe primary tumor, exhibit extensive vascularity, andsunitinib is approved for the treatment of these neo-plasms. Activating Kit mutations occur in about 85%of gastrointestinal stromal tumors and activatingPDGFRa mutations occur in about 5% of these tu-mors. Sunitinib binds reversibly to the ATP bindingsite of their target kinases and thereby inhibits theircatalytic activity [65]. Sunitinib possesses very goodoral bioavailability, has been shown highly effica-cious in a number of preclinical tumor models, and iswell tolerated at efficacious doses. It is currently inclinical phase I trials for the treatment of cancers [81].

Another structurally similar molecule, SU9516 86

(Fig. 7), is a potential inhibitor of cyclin-dependentkinases (CDKs) that can induce apoptosis in coloncarcinoma cells. CDK inhibitory properties in isatinderived phenylhydrazones have also been reported(87) (Fig. 7). Based on this information, Abadi et al.



Tab. 2. Inhibition (50%) of DPPH radical by isatin and its analogues

Compounds IC50 (µg/ml) Remarks Ref.

Isatin 111 Less active [48]

73 7.03 Highly active [17]




77 25.38 Moderate activity [66]



80 19 Highly active [48]

81 96 Less active [48]




NH

O

NCl

O

NN

SH

90

NH

O

O

Br

Br

Br

91

NH

N

O

NH

O

H NBr

NH

N

O

NH

O

H NCl

92 93

Fig. 8. The chemical structure of new anticancer therapeutic agents

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

Compound HeLa(cervical)

IMR-32(neuroblastoma)

MCF-7(breast)

U937 jurkat MDA-MB-231 PC-3 HCT-116 Ref.

Isatin 521.9 352.74 410.95 565 – – – – [18, 83]

90 12.84 15.84 16.68 – – – – – [18]

91 – – 13.3 6.76 5.8 21.8 25.9 15.9 [83]

92 175 194.00 – – – – – – [66]

93 200 285.50 – – – – – – [66]

U937: human monocyte-like, histiocytic lymphoma.

NH

O

NH

H CCH

84: SU-5416, Semaxanib

NH

O

NH

H C

CH

O

NH N

85: Su-11248, Sunitinib

NH

O

NH

N

H CO

86: Su-9516

NH

R'

X

O

NH

R

X = N, CH

NH

N

O

HOOC

NH

N

Br

8987

NH

O

OR

88

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

synthesized several 2-indolone imino derivatives (88)(Fig. 7) to examine their antitumor and antiangiogenicproperties. Vine et al. also synthesized several substi-tuted isatin analogs (89) (Fig. 7), and screened againsta panel of five human cancer cell lines. These authorsconcluded that substituted isatins could be effective asanticancer drugs [4, 6, 76].

Bis-diisatin derivatives, bis-isatin thiocarbohydra-zone metal complexes, 3-o-nitrophenyl hydrazones ofisatin possess cytotoxicity activity [50]. The potency(IC50 values) of anticancer activity of compounds 90

(Fig. 8) was comparable with that of known antican-cer agent, cisplatin. Among the synthesized 2-indo-linones, compounds 90 with a halogen atom (electronwithdrawing group) at C5 position showed potent ac-tivity (Tab. 3) [18]. These results indicate that C5 sub-stituted derivatives may be useful leads for anticancerdrug development in the future. Structure-activity re-lationship studies identified C5, C6, and C7 substitu-tion as greatly enhancing activity with some di- andtri-halogenated isatins giving IC50 values of 10 µM,indicating activity exceeding that of cisplatin (IC50

value between 13.54 and 14.08 µM) [18]. Introduc-tion 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 favor-able (94, 95) (Fig. 9) [83]. Halogenation yielded themost active compounds, with di- and tri-substitution91 (Fig. 8) increasing activity up to > 100-fold fromthe parent molecule, isatin (Tab. 3). These results in-dicate that structural modification of di- and tri-substituted isatins may lead to new derivatives withenhanced and selective anticancer activity [83]. Previ-ous studies have shown that strong electronegativeatom substitution, such as Cl and Br, at the C-5 posi-tion of the aromatic ring increases the lipophilicity of

molecules and is responsible for enhanced cytotoxic-ity [18]. Elsewhere, halogenated hydrazine deriva-tives have also been reported to exhibit anticanceractivity. For example, 5-bromo-3-o-nitrophenylisatinhydrazone was found to be active intramuscularlyagainst Walker carcinoma-256 and a series of 5-bro-mo-(2-oxo-3-indolinyl)-thiazolidine-2,4-diones substi-tuted by various Mannich bases were found to exhibitanti-leukemic activity against P388 lymphocytic leu-kemia in mice [83].

Compounds 92 and 93 (Fig. 8) have been screenedfor their cytotoxic activity against HeLa cell lines andIMR-32 cell lines (Tab. 3) [17]. However, neither ofthese compounds is comparable in cytotoxicity withthat of cisplatin. Two recent synthesized isatin Schiffbase copper(II) complexes Cu(isaepy)2 96 and Cu(is-apn) 97 (Fig. 9) are able to induce apoptosis via themitochondrial pathway in neuroblastoma SH-SY5Ycells and in other tumor histotypes, mainly bycopper-dependent oxidative stress and nuclear/mito-chondrial site-directed damage [16]. In contrast, N-alkyl isatins possess potent and selective caspase inhi-bition [71].

On the basis of above discussion, isatin and its de-rivative should be regarded as strong candidates forfuture anticancer treatment investigations.

Interaction of isatin and its derivatives

with DNA and HSA

Investigation of the binding of isatin and its deriva-tives with DNA are important in order to delineatefurther their pharmacological properties. Many com-



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

pounds exert antitumor effects through DNA binding,manipulating the replication of DNA and inhibitingthe growth of the tumor. Thus DNA binding may formthe basis of antitumor drug design. In this case, the ef-fectiveness of the therapy would be expected to be in-fluenced by the binding mode and affinity [39]. Themechanism of action exerted by some isatin com-pounds has been proposed to occur via distortion anddamage to the DNA structure [16, 61]. However, fewstudies explicitly examining binding with doublestranded (ds-) DNA have been carried out.

One example of an investigation linking the DNAbinding capacity with biological activity is providedby Karpenko et al. [26]. This work compared theDNA-binding properties of isatin and benzoisatin hy-drazones. The interferon induction capability and cy-totoxicity of these compounds varied in phase withtheir DNA affinity. The DNA affinity of benzoisatinderivatives 98 was determined to be two orders ofmagnitude 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

bond (H-bond) allows the formation of the third(pseudo) cycle in these structures, making them po-tential DNA intercalators. Intramolecular hydrogenbonding extends the aromatic rings in isatin com-pounds and enhances DNA binding affinity [12].

Other isatin derivatives also possess the ability tointercalate with DNA. Isatin-3-isonicotinylhydrazone(INH, 100, Fig. 11) has been also found to be poten-tially capable of intercalation with DNA [31]. The ab-sorption spectrum of the INH shows that as the con-centration of DNA increases, a large degree of hypo-chromism develops in the spectrum. Hypochromismusually arises from the strong stacking interaction be-tween the aromatic chromophore and the base pairs.The intrinsic binding constant observed (1.0 × 105

M–1) was roughly comparable to other intercalators.The viscosity increase of DNA can be ascribed to theintercalative binding mode of the INH, due to effec-tive DNA length increase (Fig. 12) [29]. In Figure 13,the changes in the circular dichroism (CD) spectrumof CT-DNA in the presence of increasing concentra-tions of INH are depicted. INH molecules stack in be-tween the base pairs of DNA, thus leading to an en-


NH

O

N N

O

H

N

100

N

N

H

NH

SH2N

O

101

Fig. 11. Structures of isatin-3-isoni-cotinylhydrazone (INH) 100 andisatin-b-thiosemicarbazone (IBT) 101

NO

R1

NH

O

NR2

N

R1

O

N NH

O

NR2

98 99

Fig. 10. Structure of isatin and benzoi-satin aminoacetylhydrazones

hancement in the positive band. An increase in thefluorescence of methylene blue (MB)-DNA solutionsin the presence of increasing amounts of INH indi-cates that INH is able to competitively bind DNA atsimilar sites, releasing the intercalated MB com-pletely [31]. Formation constants of INH-DNA com-plex at different temperatures, calculated by Benesi-Hildebrand equation [27], are shown in Table 4. Fromthermodynamic parameters of INH-DNA formationdetermined via Van’t Hoff equation [15], the reactionis exothermic and enthalpy favored. Negative entropyconfirms the intercalative binding mode of INH toDNA [31].

Binding mode of isatin-b-thiosemicarbazone (IBT,101, Fig. 11) to calf thymus DNA (CT-DNA) has alsobeen characterized to be intercalative. Considerablehypochromism in the absorption and a small shift inthe absorption maximum (from 356 to 357 nm) wereobserved during the interaction of IBT with DNA.The Kb of IBT with DNA was calculated at 1.03 × 105

M–1. By comparison of the Kb of IBT with the Kb ofother DNA intercalative drugs [69], we can deducethat the IBT binds CT-DNA via an intercalationmechanism. Several other pieces of experimental evi-dence also support an intercalative binding mecha-nism:

1. The binding of IBT molecules to DNA led toa marked increase in emission intensity, as observedfor other intercalators.

2. A competitive reaction monitored between neu-tral red (NR) dye, DNA and IBT showed that the in-

tercalated NR was displaced from the DNA-NR sys-tem by IBT.

3. Fluorescence titration data also was used to de-termine the binding constant (Kf) (Tab. 4) [77].Evaluation of the formation constants for the IBT-DNA complex at four different temperatures allowthermodynamic parameters of IBT-DNA formationvia Van’t Hoff equation to be determined (Tab. 4).The results indicated that the van der Waals interac-tions or hydrogen bonds are the main forces in the



Fig. 13. Circular dichroism spectra of DNA (8.0 ´ 10�5 M) in 10 mMTris-HCl buffer, in the presence of increasing amounts of IBT (A),isatin (B), INH (C)

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) onthe viscosity of CT-DNA (5 ´ 10-5 M) in 0.01 M Tris-HCl buffer (pH 7.4)

binding of the investigated drug to CT-DNA, and themode of binding is intercalation [30].

4. The viscosity increase of DNA is ascribed to theintercalative binding mode of the IBT, due to effectiveDNA length increase (Fig. 12) [28].

5. Changes in the CD spectra of CT-DNA in thepresence of increasing concentrations of IBT are de-picted in Figure 13. The IBT molecules stack in be-tween the base pairs of DNA thus leading to the en-hancement in the positive band [59].

Recently, we reported complexation of isatin withDNA which occurred by the mechanism of groovebinding [32]. Isatin binding to DNA was characterized

by hypochromism in the absorption spectrum. The in-trinsic binding constant of isatin with DNA observedwas comparable to other groove binders [41]. Evalua-tion of the formation constant for the isatin-DNAcomplex at four different temperatures allowed ther-modynamic parameters of isatin-DNA formation to bedetermined via Van’t Hoff equation [67] (Tab. 4).These results indicated that the isatin-DNA binding isentropically favored but enthalpically disfavored.Groove binding is predominantly entropically driven,whereas intercalation is enthalpically driven. Experi-mentation involving DNA viscosity (Fig. 12), fluores-cence quenching and DNA spectral band analysis


Fig. 14. Chemical structures of indole derivatives

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

Intercalatorand groove binder

T(K) Kf DG(kJ/mol)

DH(kJ/mol)

DS(J/ mol K)

Ref.

Isatin

(groove binder)

277

288

298

310

1.9 ´ 105

3.1 ´ 105

5.0 ´ 105

6.3 ´ 105

–28.35

–30.56

–32.57

–34.99

+27.42 +201.35 [32]

100

(intercalator)

277

288

298

310

4.7 ´ 104

2.2 ´ 104

1.7 ´ 104

1.1 ´ 104

–24.519

–24.294

–24/.89

–23.844

–30.187 –20.46 [31]

101

(intercalator)

290

298

303

310

1.04 ´ 105

7.52 ´ 104

4.77 ´ 104

2.82 ´ 104

–71.664

–72.265

–72.641

–73.167

–49.87 –75.152

(Fig. 13) in the presence of isatin also support the hy-pothesis that isatin can be categorized as a DNAgroove binder. Pandya et al. report the binding modeof several biologically important indole derivatives tobe DNA groove binders (Fig. 14) [53]. All of the in-dole derivatives mentioned possess only one indolering, just as for isatin. The objective of this work wasto avoid increasing the overall stacking forces duringmolecular synthesis, thereby specifically targeting theminor groove of DNA [53].

A comparison shows the Kb of IBT-DNA (1.03× 105 M–1) and INH-DNA (1.0 × 105 M–1) to be greaterthan that of isatin-DNA (7.34 × 104 M–1). It seemsreasonable that, since INH and IBT possess greaterplanar area and a more extended p system than that ofisatin. INH and IBT penetrate more deeply into thestacked base pair of DNA structure than isatin, witha concomitant increase in DNA binding. The possibil-ity of intercalation of indole derivatives (isatin) be-tween the DNA base pairs is small, mainly due toless-effective stacking forces between the indole nu-cleus and the DNA bases [53]. These data emphasizethat the mode of binding has a significant effect on thestrength of DNA binding.

The strength of DNA binding of isatin compoundscan be increased by metal complexation. Mixedligand Cu(II)/Zn(II) complexes using 3-(phenyl-imino)-1,3-dihydro-2H-indol-2-one (obtained by thecondensation of isatin and aniline) as the primaryligand and 1,10-phenanthroline (phen)/2,2’-bipyri-dine (bpy) as an additional ligand (Fig. 15) showed an

increase in intrinsic binding constant values comparedto free ligands [61]. Both copper and zinc complexesexhibited hypochromicity and a red-shifted chargetransfer peak maxima in the absorption spectra. Theviscosity of the DNA solution increased with increas-ing ratio of both the copper and zinc complexes toDNA, further suggesting an intercalating bindingmode of the complexes with DNA [68]. Cyclic volt-ammograms performed using carbon electrode in so-lutions containing [CuL(phen)2]Cl2 during incre-mental addition of DNA to the complex resulted ina negative shift in the potential of the second cathodicpeak and a decrease in the current intensities duringDNA addition. Changes in the voltammetric currentsin the presence of CT-DNA can be attributed to theslow diffusion of the metal complex bound to thelarge, slowly diffusing DNA molecule. The changesof the peak currents observed for the complexes uponaddition of CT-DNA may indicate that the complexespossess a higher DNA binding affinity. The results ofcleavage studies using pUC19 DNA showed that thecomplexes had higher nuclease activities than theisatin-based ligand. Higher binding affinity of com-plexes to DNA may be attributed to the nature of thebinding of the mixed ligand complexes with DNA,which is significant due to p-stacking or hydrophobicinteractions of the aromatic phenyl rings. However,the metal ions play essential role in DNA binding bythese complexes. Chelating effect (metal ion to freeligand) can enhance the planar functionality of metalcomplex, so the complexes can insert and stack be-



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)

tween the base pairs of double helical DNA more eas-ily than the free ligands [38].

A limited amount of data examining isatin interac-tion with model proteins is available. Da Silveira et al.investigated interactions of oxindole-Schiff base cop-per(II) complexes (Fig. 9) with HSA [11]. Modifica-tions in the characteristic a-helix feature of the pro-tein induced by interactions with these complexeswere detected. Since copper ions inserted in the pro-tein can catalyze the formation of reactive oxygenspecies (ROS), especially in the presence of hydrogenperoxide or molecular oxygen, oxidative damage tothe protein was also verified and monitored. Only thecomplexes [Cu(isaepy)H2O]2+, [Cu(isaepy)2]

2+ and[Cu(isapn)]2+ were able to modify significantly thisprotein feature, as observed by the decreasing of theband at 208 nm, indicating ~25% reduction of itshelical content after addition of two copper ions perprotein. In the presence of hydrogen peroxide all thecompounds tested exhibited an increased level ofprotein oxidation, depending on the ligand, in theorder of: [Cu(isaepy)2]

2+ >> [Cu(isapn)]2+ >[Cu(isaepy)H2O]+ >> [Cu(isaenim)]2+ > [Cu(H2O)4]2+.The two complexes exhibiting the greatest proteinoxidation power, [Cu(isaepy)2]

2+ and [Cu(isapn)]2+,are indeed those which modified protein a-helixstructure most significantly. Only the complex[Cu(isaepy)2]

2+ was able to cause notable degradationof HSA [11].”

Conclusions

The isatin structural motif can be found in a broadrange of natural and synthetically derived pharmaco-logically active compounds. The creation of novelisatin derivatives as drug targets is an active area ofmedicinal chemistry. The review compiles publisheddata on the synthesis and biological action of newisatin derivatives. Our literature survey indicated thatC-5 substituted derivatives may be especially usefulto consider when designing anticancer drugs. Intro-duction of electron withdrawing groups at positions 5,6, and 7 of the indole ring greatly increased the activ-ity in comparison with isatin, however, substitution atthe C-5 position can be considered most favorable.This is not surprising, as C-5 substitution has previ-ously been associated with increased biological activ-

ity for a range of indole-based compounds. In general,complexes of isatin derivatives have greater biologi-cal activity than their ligands alone. Further in vivo

and in vitro studies are necessary to establish and con-firm the mentioned mechanisms. We hope that ourdiscussion will prove helpful in further developmentand progress of the pharmacological applications ofthese molecules.

Acknowledgment:

Financial support from Razi University Research Center is gratefully

acknowledged.

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Received: January 6, 2012; in the revised form: November 19, 2012;

accepted: November 26, 2012.



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Biochemical and pharmacological characterization of isatin ... · Review Biochemical and pharmacological characterization of isatin and its derivatives: from structure to activity