synthesis and pp60c-src tyrosine kinase inhibitory activities of novel indole-3-imine and amine...

Arch. Pharm. Chem. Life Sci. 2009, 342, 333 – 343 Z. Kılıc̨ et al. 333

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Synthesis and pp60c-Src Tyrosine Kinase Inhibitory Activitiesof Novel Indole-3-Imine and Amine Derivatives Substituted atN1 and C5

Zuhal Kili�1, Yasemin G. Isg�r1, 2, and S�reyya �lgen1

1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ankara, Tandogan-Ankara,Turkey

2 Nanomedicine Research Center, Gazi University, G�lbasi, Ankara, Turkey

A series of novel 1,3,5-trisubstituted indole derivatives, namely, N-benzyl 5-phenyl indole-3-imine, N-benzyl-5-(p-fluorophenyl)indole-3-imine and their corresponding amine congeners,were designed and synthesized as pp60c-Src tyrosine kinase inhibitors, and their inhibitory activ-ities toward pp60c-Src tyrosine kinase were evaluated by in-vitro kinase assay. Pre-screening at twodoses of compounds against kinase target revealed that, except for the N-benzyl-5-phenyl indoleimine derivatives 7a–7d, all indole derivatives show the target inhibition at varying levels. Con-sequently, the compounds, 8c, 8f, 8g, and 8h, were selected for prescreening tests. The dose-response curves for up to six concentrations (250 to 7.8 lM) of the active compounds wereobtained by tyrosine kinase assay and the four-parameter logistic analysis of these data resultedin the IC50s of 4.69, 74.79, 75.06, and 84.23 lM for compounds 8c, 8f, 8g, and 8h, respectively.Therefore, compound 8c, 1-(1-benzyl-5-phenyl-1H-indole-3-yl)-N-(4-fluorobenzyl)methanami-ne N HCl, was the promising inhibitor for pp60c-Src, followed by compounds 8g and 8h. Under thesame conditions, compound 8f did not provide any reasonable inhibition pattern to be consid-ered as active compound. Therefore, among all four active compounds, compound 8f was notfound suitable for further analysis.

Keywords: Amine derivatives / Enzyme inhibition / Indole-3-imine / Tyrosine kinase pp60c-Src /

Received: November 26, 2008; accepted: January 9, 2009

DOI 10.1002/ardp.200800216

Introduction

The c-Src (pp60c-Src) protein tyrosine kinase (PTK) is a mem-ber of the largest family of nonreceptor PTKs, namely theSrc family kinases (SFKs), which is composed of enzymeshomologous to c-Src PTK [1]. Src tyrosine kinase was dis-covered as the first proto-oncogene product that pos-sesses intrinsic protein kinase activity [2]. The membersof SFK, and especially c-Src, have been implicated in the

regulation and promotion of diverse cellular processes,including cell growth and differentiation, motility, pro-liferation, adhesion, migration, and immune cell func-tion [3, 4]. Although Src itself is weakly oncogenic, recentadvances have implicated its role in the pathophysiologyof cancer, showing that the constitutive oncogenic acti-vation in cancer cells can be blocked by selective tyrosinekinase inhibitors [5]. Src tyrosine kinase activity has beenshown to be an important component in the epithelial tomesenchymal transition that occurs in the early stages ofinvasion of carcinoma cells [6], and elevated protein lev-els and catalytic activity of Src have also been detected inmany cancers including breast, colon, lung, pancreatic,skin, and head and neck [7, 8]. Src activity is also knownto be essential in the turnover of focal adhesion, a criticalcell motility component [9, 10]. Evidence from the clini-

Correspondence: Prof. S�reyya �lgen, Department of PharmaceuticalChemistry, Faculty of Pharmacy, Ankara University, 06100, Tandogan/Ankara, Turkey.E-mail: [email protected]: +90 312 213 1081

Abbreviations: Protein tyrosine kinase (PTK); Src family kinases (SFKs)

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334 Z. Kılıc̨ et al. Arch. Pharm. Chem. Life Sci. 2009, 342, 333 –343

cal study also supports a link between deregulated Srcactivity with increased invasive potential of tumor cells[11]. Therefore, with an emphasis on combination thera-pies with standard chemotherapeutic agents, Src kinaseinhibitors may enhance their therapeutic value byincreasing the sensitivity of tumors to the chemothera-peutic agents, and also by preventing metastasis toimprove the disease prognosis [12]. Consequently, consid-ering their ability to participate and control the manyphases of tumor progress and metastasis, targeting Srcand many members of SFKs is a promising way of discov-ering new anticancer therapeutics for metastatic dis-eases. Inhibition of Src by small molecule compoundscan be achieved at several sites, including protein-pro-tein interactions between the Src SH2 and SH3 domainsand their substrates, interaction between protein sub-strates and the substrate-binding site, and ATP-bindingsite within the tyrosine kinase domain of Src [13, 14]. Interms of selectivity, cellular potency, and possible thera-peutic applications, Src kinase inhibitors appear to bethe highly promising therapeutic agents. However, theonly dual src/abl (Abelson-Murine Leukemia) inhibitorthat has been approved as cancer therapeutics by theFDA is dasatinib, a derivative of aminothiazole to be usedfor chronic myelogenous leukemia (CML) [15, 16]. Severalclasses of small molecules which are structurally classi-fied as pyrazolo[3,4-d]pyrimidines, pyrrolo[2,3-d]pyrimi-dines, pyrido[2,3-d] pyrimidines, quinolines, and indoli-nones have been reported as ATP-competitive inhibitors

of Src-mediated tyrosine phosphorylation [17, 18].Toward the development of more effective SFK inhibi-tors, it is important to draw attention to the pyrazolopyr-imidines PP1[1-tert-Butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyri-midine-4-yl-amine] and PP2[1-tert-Butyl-3-p-chloro-1H-pyr-azolo[3,4-d] pyrimidine-4-yl-amine], which were found asselective inhibitors of Src-family member pp60c-Src withIC50 in the nanomolar range (Fig. 1) [19, 20]. Morerecently, several pyrrolopyrimidine compounds (7-pyrro-lidinyl- and 7-piperidinyl-5-pyrrolo[2,3-d]-pyrimidinesand 7-alkyl- and 7-cycloalkyl-5-aryl-pyrrolo[2,3-d]pyrimi-dines) were reported as potent tyrosine kinase pp60c-Src

inhibitors, which are useful in the treatment of osteopo-rosis [21, 22]. 2-Amino-4-oxo-pyrrolo[2,3-d] pyrimidine(LY231514, Pemetrexed) (Fig. 1) is also a pyrrolopyrimi-dine compound, which was reported as a strong anti-cancer agent [23].

Studies on indole-derived tyrosine kinase inhibitorsrevealed that several active indole derivatives have theability to inhibit several PTKs. On the other hand, com-pounds utilizing an indole scaffold were reported withmoderate activity for Src kinase as in the studies withhydroxyindole derivatives [24]. Moreover, recentlyreported substituted 3-[3-(aminopropyl)-4,5,6,7-tetrahy-dro-1H-indole-2-yl-methylene]-1,3-dihydro-indole-2-onederivatives were shown as potent and selective inhibitorsof the PTKs, namely Src and Yes. Among these derivatives,in particular, 3-[3-(dimethylaminopropyl)-4,5,6,7-tetrahy-dro-1H-indole-2-yl-methylene]-2-oxo-2,3-dihydro-1H-in-

i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.archpharm.com

Figure 1. Potent inhibitors of pp60c-Src tyrosine kinase.

Arch. Pharm. Chem. Life Sci. 2009, 342, 333 – 343 Novel Indole-3-Imine and Amine Derivatives 335

dole-5-sulfonic acid methylamide was reported as prom-ising inhibitor of Src kinase with IC50 of 0.1 lM (Fig. 1)[25].

Our previous studies on the indole scaffold bearinginhibitor research revealed that 1-benzyl-indole-2-piperi-dinoethyl carboxylate has the ability to inhibit Src withIC50 of 1.34 lM [26]. Our continuing interest in indolederivatives expanded with a study on a series of 3-(substi-tuted-benzylidene)-1,3-dihydro-indolin-2-thione deriv-atives and their corresponding indolin-2-one congenersas Src PTK inhibitors. Of the derivatives analyzed in thisstudy, we reported that (Z)-3-(49-dimethylamino-benzyli-dene)-1,3-dihydro-indolin-2-thione and (Z)-3-(29,69-di-chloro-benzylidene)-1,3-dihydro-indolin-2-thione aremoderately active Src PTK inhibitors, with IC50 of 21.91and 21.20 lM, respectively [27]. With regard to previousstudies, we focused on the design, synthesis, and activitytesting of 20 novel 1,3,5-trisubstituted indole derivativesas pp60c-Src tyrosine kinase inhibitors. With the guidanceof results from the literature, here, we particularlyfocused on substituents at 1,3,5-positions on indole ringto improve the inhibitory potency of molecules againstthe Src kinase target. Therefore, in this study, we reportthe synthesis and biological activity of 1,3,5-trisubsti-

tuted indole derivatives, their inhibitory activity on Srckinase, and the impact of the substituents on the indolering in terms of their activity on the kinase target. Thenew inhibitors were identified and their structure-activ-ity relationship with respect to the imine and amine sub-stitution at third position of indole was discussed asdescribed in the following section.

Results and discussion

The synthetic routes of N-benzyl-5-phenyl-indole-3-imine,N-benzyl-5-(p-fluorophenyl) indole-3-imine and theiramine congeners are described in Scheme 1. Palladium-catalyzed cross-coupling reaction between bromoindoleand arylboronic acids provided 5-phenyl indole 1 and 5-(p-fluorophenyl)indole 2 [28, 29]. Organoboron reagentsexhibit greater functional group compatibility thanorganozinc or Grignard reagents and they are nontoxicand thermally, air- and moisture-stable, and inexpensive.Therefore, they were chosen for the biaryl synthesis [30].For employing the arylboronic acids, Pd(Ph3)4 was used asthe catalyst, Na2CO3 as the base, and a mixture of toluene/ EtOH / H2O as solvent. The benzylation of 5-phenyl


Reagents and conditions: (i) Pd(PPh3)4, NaCO3, arylboronic acids, anhydrous toluene; (ii) NaH, BnBr, DMF, 08C to room temper-ature; (iii) DMF, POCl3, – 158C to room temperature; (iv) substituted benzylamines, MgSO4, CH2Cl2, reflux; (v) NaBH4, 508C.

Scheme 1. Synthesis of 1,3,5-trisubstituted indole derivatives.


indole 1 and 5-(p-fluorophenyl)indole 2 was performed,following standard procedures using NaH 95% in dryDMF giving N-benzyl-5-phenyl indole 3 and N-benzyl-5-(p-fluorophenyl)indole 4 [31]. N-Benzyl-5-phenyl-indole-3-carbaldehyde 5 and N-benzyl-5-(p-fluorophenyl)-3-carbal-dehyde 6 were synthesized by using Vilsmaier formyla-tion [32]. Indole-3-carbaldehydes were then subjected to acondensation with substituted benzylamines in CH2Cl2

resulting in the Schiff' bases as Z / E (syn / anti) isomer mix-tures 7a-7j [33]. The reaction of imine compounds withNaBH4 afforded their oily amine congeners [34, 35] whichwere converted into a solid form as HCl salts 8a–8j.Attempts to separate the Z / E (syn / anti) isomer mixturesby flash chromatography were not successful, because ofthe decomposition of compounds during the procedure.All the synthesized compounds were characterized by 1H-NMR, IR, MS spectral methods, and the purity of finalcompounds was determined by elemental analysis. Alldata were consistent with the proposed structures. Thedata are shown in experimental part; for the Physico-chemical properties and spectral data of the compoundssee Table 1.

N-Benzyl protons of indole were observed as a sharpsinglet at 5.14–5.34 ppm for imine and 5.46–5.50 ppmfor amine derivatives. The chemical shifts of all aro-matics protons were detected at the 6.75–8.62 ppm forimine and 7.16–8.13 ppm for amine compounds. Thecharacteristic CH=N protons of imine compounds weredetected at 8.42–8.62 ppm as a mixture of Z / E (syn / anti)isomers. The CH2 peaks of CH=N-CH2-Ph (or substitutedPh) were found at 4.64–4.83 ppm. The NH peaks ofCH2NH-CH2-Ph (or substituted Ph) were obtained as broad

singlet at 9.42–9.77 ppm. The CH2 protons of CH2NH-CH2-Ph (or substituted Ph) were measured as triplet at 4.35–4.48 ppm and 4.17–4.28 ppm, respectively. The massspectra of compounds were taken with ESI methods andthe mass values of compounds were monitored as [M + 1]and [M+]. The expected isotop peaks of Cl and Br atomswere detected as a ratio of 3 : 1 and 1 : 1, respectively,and numbers of peaks were also detected depending onthe number of atoms. Elemental analysis of the com-pounds indicated purity in the range l 0.4%.

The biological activity of the compounds was eval-uated by an in-vitro tyrosine-kinase assay that measuresthe changes in the enzymatic activity of pp60c-Src tyrosinekinase by virtue of following the alterations in the phos-phorylation level of the immobilized substrate withrespect to DMSO (vehicle) control [36]. The activity of thesynthesized compounds was initially analyzed via pre-liminary screening (pre-screening) at two concentrationsof the compounds, reflecting the high (250 lM) and low(50 lM) doses, to determine those capable of altering thesubstrate-phosphorylation level with reasonable inhibi-tion profile. The pre-screening results were reported as%-inhibition of the Src activity at 50 lM and 250 lM of thecompounds, with respect to DMSO control. The pre-screening of each compound was performed as two inde-pendent experiments, each in duplicates, and the data ispresented as bar graphs, as given in Figs. 2 and 3. Tyro-sine-kinase assay was performed at 350610 – 7 unit/lLpp60c-Src for both preliminary and dose-response screen-ing of the compounds. Those compounds that yieldeda minimum 50% inhibition at 50 lM and more than 70%at 250 lM, were reported as “active” from pre-screening


Data shown here is the prescreening result of compounds at two different concentrations, each in duplicate (high dose,250 lM and low dose 50 lM).

Figure 2. Activity comparison of N-benzyl-5-phenyl indole derivatives 7a–7e and 8a–8eas%-inhibition exerted on pp60c-Src (650610 – 7 units/lL) kinase target activity.



Table 1. Physicochemical properties and spectral data of the compounds.

Comp. Isomers M.p.8C

% Yield MolecularFormula

MassESI-MS: m/z

Elemental Analysis(Calcd. / Found)

1H-NMR

C H N

7a E/Z 95 79.84 C29H24N2 401.26[M + 1]

86.9787.21

6.045.78

6.997.05

(CDCl3) d: 8.62 – 8.60 (m, 1H, CH=N, syn / anti mix-ture), 8.62 (m, 1H, H-b), 7.68 – 7.15 (m, 18H, aromaticprotons), 5.33 (s, 2H, CH2Ph), 4.83 (s, 2H, =N-CH2-Ph)

7b E/Z 89 78.80 C29H23ClN2 435.38[M + 1]

79.4279.27

5.375.60

N (0.2 H2O)

6.386.46

(CDCl3) d: 8.52 (m, J = 1.6 Hz, 1H, H-b), 8.42 (s, 1H,CH=N, syn / anti mixture), 7.56 – 6.99 (m, 17H, aro-matic protons), 5.14 (s, 2H, CH2Ph), 4.64 (s, 2H, =N-CH2-Ph)

7c E/Z 75 67.33 C29H23FN2 419.28[M + 1]

81.8181.57

5.635.80

N (0.4 H2O)

6.586.74

(CDCl3) d: 8.58 (m, 2H, CH=N, syn / anti mixture andH-b), 7.67-6.99 (m, 17H, aromatic protons), 5.34 (s,2H, CH2Ph), 4.78 (s, 2H, =N-CH2-Ph)

7d E/Z 105 – 107 77.52 C29H22Cl2N2 469.28[M+]

73.0872.89

4.825.02

N (0.4 H2O)

5.875.99

(CDCl3) d: 8.66 (d, J = 1.6 Hz, 1H, H-b), 8.53 (s, 1H,CH=N, syn / anti mixture), 7.67 – 7.11 (m, 16H, aro-matic protons), 5.27 (s, 2H, CH2Ph), 4.80 (s, 2H, =N-CH2-Ph)

7e E/Z 88 – 90 76.51 C29H22F2N2 437.33[M + 1]

79.4679.14

5.104.82

(0.1 H2O) N

6.396.48


7f E/Z 90 – 91 64.50 C29H23FN2 419.35[M + 1]

82.8782.70

5.565.55

N (0.1 H2O)

6.666.71


7g E/Z 99 – 101 55.00 C29H22ClFN2 453.36[M + 1]

76.9077.07

4.904.67

6.186.21

(CDCl3) d: 8.55 (s, 1H, CH=N, syn / anti mixture), 8.53(s, 1H, H-b), 7.45 (s, 1H, H-a), 7.60 – 7.07 (m, 15H, aro-matic protons), 5.31 (s, 2H, CH2Ph), 4.76 (s, 2H, =N-CH2-Ph)

7h E/Z 100 – 102 87.48 C29H22F2N2 437.37[M + 1]

79.8079.55

5.084.87

6.426.50

(CDCl3) d: 8.55 (s, 1H, CH=N, syn / anti mixture), 8.55(s, 1H, H-b), 7.44 (s, 1H, H-a), 7.59 – 6.98 (m, 15H, aro-matic protons), 5.29 (s, 2H, CH2Ph), 4.76 (s, 2H, =N-CH2-Ph)

7i E/Z 101 – 103 83.03 C29H21Cl2FN2 487.31[M+]

71.2070.97

4.364.08

N (0.1 H2O)

5.725.75

(CDCl3) d: 8.59 (s, 1H, H-b), 8.57 (s, 1H, CH=N, syn / antimixture), 7.61 – 7.08 (m, 15H, aromatic protons), 5.33(s, 2H, CH2Ph), 4.82 (s, 2H, =N-CH2-Ph)

7j E/Z 81 – 82 66.61 C29H21F3N2 455.43[M + 1]

76.6476.54

4.664.70

6.166.30

(CDCl3) d: 8.57 (s, 1H, CH=N, syn / anti mixture), 8.53(d, J = 1.6 Hz, 1H, H-b), 7.48 (s, 1H, H-a), 7.60 – 6.77 (m,14H, aromatic protons), 5.33 (s, 2H, CH2Ph), 4.78 (s,2H, =N-CH2-Ph)

8a – 186 78.12 C29H27ClN2 403.00[M + 1]

78.6978.79

6.236.16

N (0.2 H2O)

6.326.56

(DMSO-d6) d: 9.51 (bs, 2H, +NH2), 8.02 (d, J = 1.6 Hz, 1H,H-b), 7.74 (s, 1H, H-a), 7.70 – 7.22 (m, 17H, aromaticprotons), 5.46 (s, 2H, CH2Ph), 4.35 (t, 2H, CH2NH), 4.17(t, 2H, NH-CH2-Ph)

8b – 214 – 216 91.80 C29H26Cl2N2 437.29[M + 1]

73.5773.51

5.545.63

5.925.92


8c – 204 – 205 85.91 C29H26ClFN2 421.27[M + 1]

75.9275.90

5.756.01

N (0.1 H2O)

6.106.12


8d – 196 – 198 93.50 C29H25Cl3N2 473.00[M + 2]

68.0967.92

5.004.70

N (0.2 H2O)

5.475.54

(DMSO-d6) d: 9.77 (bs, 2H, +NH2), 8.13 (d, J = 1.6 Hz, 1H,H-b), 7.81 (s, 1H, H-a), 7.83 – 7.24 (m, 15H, aromaticprotons), 5.50 (s, 2H, CH2Ph), 4.48 (s, 2H, CH2NH), 4.28(s, 2H, NH-CH2-Ph)

8e – 195 – 197 87.95 C29H25ClF2N2 439.39[M + 1]

73.3373.54

5.315.59

5.905.98


8f – 198 86.49 C29H26ClFN2 421.41[M + 1]

76.2276.16

5.735.67

6.136.30



where the compounds were dissolved in the maximum-tolerated DMSO-containing assay buffer. The active com-pounds were further analyzed to obtain dose-responsecurves with varying concentrations of compounds(DMSO a 0.6% of the assay). Their potential to inhibitenzyme activity was reported as IC50 and determined byvirtue of nonlinear regression analysis (four parameterlogistic equation, sigmoidal dose-response) performedusing GraphPad Prism version 4.0 for Windows (Graph-Pad Software, San Diego Ca, USA; www.graphpad.com).The pre-screening of N-benzyl-5-phenyl 7a–8e and N-ben-zyl-5-(p-fluoro)phenyl 7f–8j indole derivatives revealedthat, except for 7a–7d, all compounds showed some

activity against the kinase target (Figs. 2 and 3) at bothhigh and low doses of the compounds. Among N-benzyl-5-phenyl indole imine derivatives 7a–7e, reduced solubil-ity was observed only for compounds 7a–7d and thesewere eventually inactive. Of these, slightly active 7e wasonly partially soluble at the tolerated levels of DMSO,whereas solubility of 7a–7d could not be improvedunder the same conditions (Fig. 2). At 250 lM, of all com-pounds analyzed, more than 70% inhibition wasobserved for the amine salts of the N-benzyl-5-phenyl 8a–8e and N-benzyl-5-(p-fluoro)phenyl indole 8f–8j deriv-atives. At 50 lM, however, the 50% activity criterion wasonly fullfilled by five compounds, and these were N-ben-


Table 1. Continued.

Comp. Isomers M.p.8C

% Yield MolecularFormula

MassESI-MS: m/z

Elemental Analysis(Calcd. / Found)

1H-NMR

C H N

8g – 209 88.77 C29H25Cl2FN2 455.44[M + 1]

70.8871.05

5.135.35

5.705.85

(DMSO-d6) d: 9.42 (bs, 2H, +NH2), 8.03 (d, J = 1.2 Hz, 1H,H-b), 7.75 – 7.24 (m, 16H, aromatic protons), 5.48 (s,2H, CH2Ph), 4.38 (s, 2H, CH2NH), 4.21 (s, 2H, NH-CH2-Ph)

8h – 200 88.48 C29H25ClF2N2 439.51[M + 1]

73.3373.02

5.315.12

5.905.94


8i – 193 82.82 C29H24Cl3FN2 489.24[M+]

66.0065.73

4.624.28

N (0.1 H2O)

5.305.42

(DMSO-d6) d: 9.70 (bs, 2H, +NH2), 8.11 (d, J = 1.2 Hz, 1H,H-b), 7.81 – 7.24 (m, 15H, aromatic protons), 5.50 (s,2H, CH2Ph), 4.48 (s, 2H, CH2NH), 4.28 (s, 2H, NH-CH2-Ph)

8j – 187 – 188 89.17 C29H24ClF3N2 457.36[M + 1]

70.6670.50

4.914.52

5.685.77


Data shown here is the prescreening result of compounds at two different concentrations, each in duplicate (high dose,250 lM and low dose 50 lM).

Figure 3. Activity comparison of N-benzyl-5-(p-fluoro)phenyl indole derivatives 7f–7j and8f–8j as%-inhibition exerted on pp60c-Src (650610-7 units/lL) kinase target activity.


zyl-5-(p-fluoro)phenyl containing indole imine 7j, aminesalts of N-benzyl-5-(p-fluoro)phenyl indole compounds8f–8h, and N-benzyl-5-phenyl containing indole amine8c. Therefore, based on these prescreening results, onlyfour compounds, 8c, 8f, 8g, and 8h, were selected asbeing active. The dose-response curves of these com-pounds were obtained from two to three independenttyrosine-kinase assays (each in duplicates, n = 3–6) forfive concentrations (250–15.62 lM) for compounds 8f–8g and six concentrations (250–7.8 lM) for compound 8c(Fig. 4, 5, 6, and 7). The dose-response curves of these com-pounds were obtained using tyrosine-kinase assay for sixconcentrations (250–7.8 lM) of compound 8c and fiveconcentrations (250–15.62 lM) of compounds 8f, 8g, and8h. In the dose-response curves, 10–25% decrease in per-cent inhibition was observed, as expected, compared

with the prescreening results. This was due to various lev-els of DMSO content used in these two assays. To get allpossible active compounds throughout the prescreeningat two doses of the compounds, the solubility wasincreased with DMSO using its maximum-tolerated lev-els. Knowing that DMSO interferes with enzyme activityto some level of the vehicle used in compound dilutions(data not shown), compounds with up to six doses wereprepared in the lowest levels of DMSO and so interferencethat could possibly arise from the vehicle was eliminatedwhile constructing the dose-response curves. Theobserved reductions at%-inhibition of the maximumdoses were therefore observed with the dose-responsecurves of all four active compounds. The analysis of dose-response data using the four-parameter logistic analysismethod (GraphPad Prism 4.0) revealed that the com-pound 8c, 1-(1-benzyl-5-phenyl-1H-indole-3-yl)-N-(4-fluoro-benzyl)methanamine … HCl, is the most potent inhibitorof pp60c-Src tyrosine kinase with IC50 of 4.69 l 1.23 lM(Fig. 4), followed by compounds 8g and 8h with IC50s of75.06 l 1.24 (Fig. 6) and 84.2 3l 1.19 lM (Fig. 7), respec-tively. Under the same assay conditions, compound 8fwith IC50 of 74.79 l 1.43 could not provide a reasonablyhigh maximum inhibition, and therefore a narrow assaywindow, to be considered as active compound (Fig. 5).Hence, among all four actives, compound 8f was not suit-able to be further developed as drug lead or biologicaltarget probe.

To understand the role of indole-ring substitutionsand the biological activity of derivatives, we evaluatedsubstituents on the indole scaffold and their activity


Tyrosine kinase assay was performed at 350610 – 7 unit/lL pp60c-Src at six doses ofcompound 8c. Data represents the results of three independent experiments each induplicate (n = 3 – 6) and the IC50 values obtained by non-linear regression analysis.

Figure 4. The dose-response curve for the compound 8c withIC50 4.69 l 1.23 lM.

Tyrosine kinase assay was performed at 350610 – 7 unit/lL pp60c-Src at five doses ofcompound 8f. Data represents the results of two independent experiments each induplicate (n = 3 – 6) and the IC50 values obtained by non-linear regression analysis.

Figure 5. The dose-response curve for the compound 8f withIC50 74.79 l 1.43 lM.

Tyrosine kinase assay was performed at 350610 – 7 unit/lL pp60c-Src at five doses ofcompound 8g. Data represents the results of two independent experiments each induplicate (n = 3 – 6) and the IC50 values obtained by non-linear regression analysis.

Figure 6. The dose-response curve for the compound 8g withIC50 75.06 l 1.24 lM.


behavior on prescreening of compounds. Analyzing the5-substitution on the indole ring, it was apparent that a5-(4-fluoro)-phenyl substitution, regardless of any sub-stituent at other positions of the indole ring, improvedthe activity of the compounds compared with their corre-sponding 5-phenyl-substituted compounds. This ten-dency was also true for both imine and amine derivativesof these compounds, and the remarkable difference wasseen from the screening at low dose (50 lM). In moredetailed analysis of screening results, compounds 8a–8efrom 5-phenyl-substituted indole amine series, were com-pared with their corresponding members 8f–8j from 5-(4-fluoro)-phenyl substituted indole amine series. At thethird position of the indole, substitutions were made onthe phenyl ring (Scheme 1, substitutions R2 and R3). Here,the comparison of 3-substitution on the indole scaffoldrevealed that except compounds 8c, 8d, and 8e, none ofthe compounds from this series showed neither closernor better activities than their corresponding com-pounds in the 5-(4-fluoro)-phenyl-substituted indoleamine series, when screening was performed at the lowdose (clear bars, Figs. 2 and 3). Compounds 8c and 8ewere the indole derivatives bearing 3-substitution withmono- and di-fluoro-substituted phenlyamines, respec-tively. Among the 5-(4-fluoro)-phenyl-indole amine deriv-atives, the compounds' activities were highest whenunsubstituted or mono-halogen-substituted (8f, 8g, and8h) compared to their corresponding derivatives with di-halogen substituents (8i and 8j). Here, interestingly, theactive mono-halogen-bearing derivatives were those hav-ing p-fluoro or p-chloro substituents in the benzyl ring atthird position of the indole scaffold. Of the compounds

analyzed, the four passing the prescreening criteria wereindoles having substitution on the third position witheither unsubstituted 8f or mono-halogen-substituted 8c,8g, and 8h phenlyamines. As explained before, com-pound 8f with a IC50 value of 74.79 l 1.43 was not consid-ered as active compound. These different behaviors ofsubstitutions on activity might be due to their favorablecontribution to lipophilic and electronic factors of thecompounds. While no significant correlation wasobserved, all active compounds were identified as thosepossessing electron-withdrawing groups on the benzylring at the third position of indole. This general consider-ation may help to understand the concept of the SAR ofthe derivatives analyzed here. The activity differences ofboth imine and amine derivatives seem to be particularlyinteresting, considering that the imine derivatives wereeither inactive or less active compared with their corre-sponding amine derivatives which were identified withremarkable activity. One possible reason might be thatthe amine compounds are more soluble in assay mediumand more efficiently reacted with enzyme.

In the light of these results, more advanced structuraloptimizations with further biological analyses may facili-tate efforts to investigate the role of substituents at the1,3,5-positions of indoles to design and synthesize morepotent compounds as potential Src kinase inhibitors tobe develop them as drug leads or chemical probes for Srckinase target in the future.

This work was partially supported by a grant from the TurkishScientific and Technical Research Institute (106S127 SBAG-HD-141).

The authors have declared no conflict of interest.

Experimental

ChemistryAnhydrous magnesium sulphate, sodium sulphate, hexane,ethyl acetate, anhydrous dimethylformamide, sodium dihydro-gen phosphate, potassium hydrogen phosphate, sodium chlor-ide, dimethylsulfoxide, sulfuric acid, mercaptoethanol, phos-phorous oxychloride, tetrakistriphenylphosphine palladium,sodium carbonate, ethanol (from Merck, Darmstadt, Germany);deutero dimethylsulfoxide, deutoro chloroform, benzylamine,4-chlorobenzylamine, 4-fluorobenzylamine, 2,4-dichlorobenzyl-amine, 2,4-difluorobenzylamine, indole, 5-bromoindole, phenyl-boronic acid, 4-fluorophenylboronic acid (from Acros Organics,Geel, Belgium); methanol, hydrochloric acid, dichloromethane,anhydrous ethanol, sodium hydride, benzyl bromide, anhy-drous toluene (from Sigma-Aldrich, St. Louis, MO, USA). TakaraUniversal Tyrosine Assay Kit (from Takara-Bio Inc., Shiga, Japan)was used to analyze the biological activity of the compounds syn-


Tyrosine kinase assay was performed at 350610 – 7 unit/lL pp60c-Src at five doses ofcompound 8h. Data represents the results of two independent experiments each induplicate (n = 3 – 6) and the IC50 values obtained by non-linear regression analysis.

Figure 7. The dose-response curve for the compound 8h withIC50 84.23 l 1.19 lM.


thesized in this study. The contents of a kit: PTK substrate immo-bilized microplate (8 wells612), kinase reacting solution(11.0 mL), 40 mM ATP-2 Na (0.55 mL), extraction buffer(11.0 mL), PTK control (0.50 mL), anti-phoshotyrosine (PY20-HRP,for 5.5 mL/H2O), blocking solution (11.0 mL), HRP coloring solu-tion (TMBZ = Tetra Methyl Benzidine, 12.0 mL). Melting pointswere measured with a capillary melting point apparatus (Elec-trothermal, Essex, UK) and are uncorrected. The Nuclear Mag-netic Resonance (1H-NMR) spectra were recorded on Varian Mer-cury 400 NMR spectrometer 400 MHz (Varian Inc., Palo Alto, CA,USA). The chemicals shift values were expressed in parts per mil-lion (ppm) relative to tetramethylsilane as an internal standardand signals were reported as s (singlet), d (doublet), t (triplet), q(quartet), m (multiplet). Mass spectra were recorded on a WatersZQ Micromass LC-MS spectrometer (Waters Corporation, Mil-ford, MA, USA) Electrosprey Ionization (ESI) method. Infrared(IR) spectra were measured on Jasco FT/IR-420 (Jasco, Tokyo,Japan). Elemental analysis was taken on a Leco-932 CHNS-O ana-lyzer (Leco, St. Joseph, MI, USA). molecular Devices Spectra MAX190 (from molecular Devices Corporation, Sunnyvale, CA, USA)was used to measure of absorbance of phoshorylation reaction.Analytical TLC was carried out on Merck 0.2 mm pre-coatedsilica gel (60 F-254) aluminium sheets (Merck), visualization byirradiation with an UV lamp. The flash column chromatographywas accomplished on silica gel 60 (230–400 mesh; Merck). Thedose-response curves of the compounds and non-linear regres-sion analysis were performed using GraphPad Prism 4.0 (Graph-Pad Software for Windows, San Diego, California, USA).

5-Phenyl indole 15-Bromoindole (2.88 g, 0.014 mol) was dissolved in 20 mL anhy-drous toluene and Pd(PPh3)4 (0.849 g, 0.735 mol were added andthen stirred at room temperature for 30 min. Na2CO3 (21 mL,2 M) and phenylboronic acid (2.69 g, 0.022 mol) in 15 mL abso-lute ethanol were added respectively. The mixture was boiled at908C for 4 h and the layers were separated. The water layer wasextracted by ethylacetate (36100 mL) and dried over anhydrousNa2SO4. The Crude product was purified by column chromatog-raphy (hexane / ethylacetate = 9.5 : 0.5) and crystallized withethanol resulted in 1.59 g of pure compound with 56.0% yield.M.p.: 7408C (lit. m.: 69 –708C) [37].

5-(4-Fluorophenyl) indole 25-Bromoindole (5.02 g, 0.025 mol) was dissolved in 50 mL anhy-drous toluene and Pd(PPh3)4 (1.48 g, 1.28 mmol) was added andthen stirred at room temperature for 30 min. Na2CO3 (37 mL,2M) and 4-fluoro phenylboronic acid (5.37 g, 0.038 mol) in10 mL absolute ethanol were added respectively. The mixturewas boiled at 908C for 4 h and the layers were separated. Thewater layer was extracted by ethylacetate (36100 mL) and driedover anhydrous Na2SO4. The Crude product was purified by col-umn chromatography (hexane / ethylacetate = 9.5 : 0.5) and crys-tallized with ethanol resulted in 2.88 g of pure compound with53.4% yield. M.p.: 89 –908C [38].

N-Benzyl-5-phenyl indole 35-Phenyl indole (2.17 g, 0.01 mol) was dissolved in DMF (6 mL)and the solution was cooled to 08C. NaH (0.54 g, 0.02 g) wasadded and the mixture stirred for 15 min at 08C and then45 min at room temperature. Benzyl bromide (2.7 mL, 0.02 mol)was added dropwise to the reaction mixture and stirred for 72 h

at room temperature. It was diluted with water and extractedwith EtOAc (3650 mL). The combined organic phase dried overanhydrous Na2SO4. Evaporation of the solvent gave crude com-pounds, which were purified by column chromatography (hex-ane / ethylacetate = 9.5 : 0.5) and following EtOH crystallization.2.81 g pure compound was obtained with 88.3% yield. M.p.: 99 –1008C [39].

N-Benzyl-5-(4-fluorophenyl) indole 45-(4-Fluoro)phenyl indole (2.88 g, 0.013 mol) was dissolved inDMF (7 mL) and the solution was cooled to 08C. NaH (0.72 g,0.03 g) was added and the mixture stirred for 15 min at 08C andthen 45 min at room temperature. Benzyl bromide (3.3 mL,0.027 mol) was added dropwise to the reaction mixture andstirred for 72 h at room temperature. It was diluted with waterand extracted with EtOAc (3650 mL). The combined organicphase was dried over anhydrous Na2SO4. Evaporation of the sol-vent gave crude the compounds, which were purified by columnchromatography (hexane / ethylacetate = 9.5 : 0.5) and followingEtOH crystallization, 3.15 g pure compound was obtained with76.0% yield. M.p.: 7508C. 1H-NMR (CDCl3) d: 7.95 (s, 1H, H-c), 7.71 –7.21 (m, 12H, aromatic protons), 6.73 (d, 1H, H-b), 5.37 (s, 2H,CH2Ph); ESI-MS: m/z 302.21 [M + 1].

N-Benzyl-5-phenyl indole-3-carbaldehyde 5DMF (3.1 mL, 0.039 mol) and POCl3 (1.02 mL, 0.01 mol) werestirred at –158C for 15 min and N-benzyl-5-phenyl indole (2.81 g,0.01 mol) in 5 mL DMF was added. The mixture was stirred atroom temperature for 45 min. It was poured into cold water andadjusted to basic pH by adding 10 mL of 2 M NaOH (1.91 g,0.047 mol) solution. The collected crude solid was purified bycolumn chromatography (hexane / ethylacetate = 8 : 2) and fol-lowed by crystallization with EtOH / H2O mixture. 2.08 g purecompound was obtained with 67.2% yield. M.p.: 1018C. 1H-NMR(CDCl3) d: 10.03 (s, 1H, CHO), 8.56 (s, 1H, H-b), 7.74 (s, 1H, H-a),7.56 –7.20 (m, 12H, aromatic protons), 5.39 (s, 2H, CH2Ph); IR(KBr, cm – 1): 1656 (C=O); ESI-MS: m/z 312.14 [M + 1].

N-Benzyl-5-(4-fluorophenyl)indole-3-carbaldehyde 6DMF (3.25 mL, 0.041 mol) and POCl3 (1.07 mL, 0.011 mol) werestirred at –158C for 15 min and N-benzyl-5-(4-fluoro)phenylindole (3.15 g, 0.01 mol) in 5 mL DMF were added. The mixturewas stirred at room temperature for 45 min. It was poured intocold water and adjusted to basic pH by adding 10 mL of 2 MNaOH (2.0 g, 0.05 mol) solution. The collected crude solid waspurified by column chromatography (hexane / ethylacetate =8 : 2) and followed by crystallization with EtOH / H2O mixture.2.03 g pure compound was obtained with 59.2% yield. M.p.:102 –1048C. 1H-NMR (CDCl3) d: 10.01 (s, 1H, CHO), 8.51 (s, 1H, H-b), 7.74 (s, 1H, H-a), 7.49-7.10 (m, 11H, aromatic protons), 5.38 (s,2H, CH2Ph); IR (KBr, cm – 1): 1651 (C=O); ESI-MS: m/z 330 [M + 1].

General procedure for N-[(1-benzyl-5-phenyl-1H-indole-3-yl)methylene]-1-phenyl / substitutedphenylmethanamine 7a–7e and N-[(1-benzyl-5-(4-fluorophenyl)-1H-indole-3-yl)methylene]-1-phenyl /substituted phenylmethanamine 7f–7jCompounds 5 or 6 (1 eq.) were dissolved in CH2Cl2 and MgSO4 (0.5eq.) and corresponding amine derivatives (1 eq.) were added. Themixture was refluxed for 40 h, MgSO4 was filtered off and the sol-



vent was evaporated. The crude compounds were purified byEtOH crystallization.

General procedure for 1-(1-benzyl-5-phenyl-1H-indole-3-yl)-N-(benzyl / substituted benzyl)methanamine N HCl8a–8e and 1-(1-benzyl-5-(4-fluorophenyl)-1H-indole-3-yl)-N-(benzyl / substituted benzyl)methanamine N HCl 8f–8jCompounds 7a –7j were dissolved in 5 mL MeOH and 5 eq.NaBH4 was added. The mixture was heated 1 h at 508C. After thesolvent removed under reduced pressure, the residue wasdiluted with water. The water layer was extracted with ethyl ace-tate, and the organic layer was separated and dried over Na2SO4.The evaporation of the solvent yielded oily products, which weresolidified by making their HCl salts.

BiologyIn-vitro tyrosine-kinase (pp60c-Src) assayThe inhibition of the synthesized compounds was verified by vir-tue of the ELISA-based in-vitro tyrosine-kinase assay where thepp60c-Src tyrosine-kinase activity was measured by monitoringthe phosphorylation level of immobilized substrate (poly (Glu-Tyr) peptide) using the classical ELISA-sandwich method (TakaraUniversal Protein Tyrosine Kinase Assay, Tokyo, Japan). The kin-ase assay was performed at 378C in a final assay volume of 50 lL.The concentrations of the pp60c-Src used to construct the calibra-tion curve were as follows: 1520, 760, 380, 190, 94.8, 47.4610 – 7

units/lL for pp60c-Src. The kinase reactions were performed with350610 – 7 unit/lL pp60c-Src at 40 nM ATP. Following severalwashing and incubation steps, the phosphorylation of the sub-strate was probed with HRP-conjugated anti-phosphotyrosine(PY20) antibody and the absorbance of the reaction mixture wasmeasured at 450 nm with a microplate reader. The inhibitoryactivities of compounds against tyrosine kinase were monitoredby the diminished activity of kinase at 450 nm. The pp60c-Src tyro-sine kinase activity (control) is measured as the differencebetween the total activity in the absence and presence of vehicle(DMSO), and the activity of enzyme in the presence of a com-pound (in DMSO) is determined with respect to control. IC50

value was determined as the concentration of a compoundrequired to achieve 50% inhibition of pp60c-Src tyrosine kinaseactivity with respect to control. Compounds to be tested wereprepared at final concentrations of 7 to 650 lM and all measure-ments and 50% inhibitory concentration (IC50) determinationsfrom the dose-response curves were made within this range. TheIC50 values were determined by non-linear regression analysis,the four-parameter logistic equation (Sigmoidal dose-response,GraphPad Prism version 4.0 for Windows, GraphPad Software,San Diego California USA).

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synthesis and pp60c-src tyrosine kinase inhibitory activities of novel indole-3-imine and amine...

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