Synthesis of 1,4-bis(indolin-1-ylmethyl)benzene derivatives and their structure-activity relationships for the interaction of human carbonic anhydrase isoforms I and II

Synthesis of 1,4-bis(indolin-1-ylmethyl)benzene derivatives and their

structure–activity relationships for the interaction of human carbonic

anhydrase isoforms I and II

Oktay Talaz

a,⇑

, Hüseyin Çavdar

b

, Serdar Durdagi

c

, Hacer Azak

a

, Deniz Ekinci

d a_{Karamanog˘lu Mehmetbey University, Kamil Özdag˘ Science Faculty, Chemistry Department, 70100 Karaman, Turkey}

b_{Dumlupınar University, Education Faculty, 43100 Kütahya, Turkey} c

Institute for Biocomplexity and Informatics, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada

d

Ondokuz Mayıs University, Faculty of Agriculture, Department of Agricultural Biotechnology, 55139 Samsun, Turkey

a r t i c l e

i n f o

Article history:

Available online 10 October 2012 Keywords: Carbonic anhydrase Docking Bisindole Glaucoma

a b s t r a c t

Several 1,4-bis(indolin-1-ylmethyl)benzene-based compounds containing substituents such as five, six and seven cyclic derivatives on indeno part (9a–c) were prepared and tested against two members of the pH regulatory enzyme family, carbonic anhydrase (CA). The inhibitory potencies of the compounds at the human isoforms hCA I and hCA II targets were analyzed and KIvalues were calculated. KIvalues

of compounds for hCA I and hCA II human isozymes were measured in the range of 39.3–42.6lM and 0.17–0.29lM, respectively. The structurally related compound indole was also tested in order to under-stand the structure–activity relationship. Most of the compounds showed good CA inhibitory efficacy. In silico docking studies of these derivatives within hCA I and II were also carried out and results are sup-ported the kinetic assays.

Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Indole ring containing derivatives continue to receive substan-tial interest because of their broad biological activity range.1,2

In-dole moieties occur in many synthetic and natural products, some of them are physiologically relevant and therapeutically used.3–5_{For example bisindole alkaloids are prevalent subclass of}

compounds containing indole ring, such as nortopsentins A–C (1), arcyriaflavin A (2), hyrtiosin B and psychotrimine (4) have at-tracted interest of researchers due to their biologicalactivities in recent years (Fig. 1).6,7

Most of the methods used to form bisindoles have been in-spired over the years by various synthetic strategies developed for bisindole ring formation. Two indole ring of 3-subtituted, 2-substitued, fused benzene ring and N-substituted bisindoles can be possibly found in fused or opens systems. N-substituted and C2 substituted bisindoles are not common due to less reactive N and C2 position compared to C3 position.8_{However bisFischer}

indolizations have remained elusive to date. In this study we re-port synthesis of bisphenylhydrazine using Fischer indolization using several ketones.

Carbonic anhydrases (CAs, EC 4.2.1.1) form a family of metallo-enzymes that play an essential role in several physiological and pathological processes. There are sixteen CA isoforms identified in mammals (humans have only 15 isoforms) that differ in their subcellular localization and catalytic activity.9_{Inhibitors or}

activa-tors of this metalloenzyme family have several medical applica-tions. For example treatment of glaucoma, in the management of several neurological disorders including epilepsy, possibly in the treatment of Alzheimer’s disease, and several agents show clinical evaluations as antiobesity or antitumor drugs/diagnostic tools.9–12

So far inhibitory effects of different sulfonamide derivatives, an-ions, metal an-ions, phenols and drugs have been investigated against many CAs.11–15

In a recent study, the antioxidant activities of 5,10-dihydroinde-no[1,2-b]indole derivatives were reported by one of our groups.16

For this reason, we extend the previous study through investigat-ing the effects of these molecules on the CA activity, with the goal to discover new CA inhibitors (CAIs).

2. Results and discussion 2.1. Chemistry

Based on our structure–activity relationship analysis, we syn-thesized a series of new N-connected bisindole alkoloids as shown

0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.bmc.2012.09.027

⇑ Corresponding author. Tel.: +90 338 2262431. E-mail address:otalaz@kmu.edu.tr(O. Talaz).

Contents lists available atSciVerse ScienceDirect

Bioorganic & Medicinal Chemistry

inScheme 1. NaH base was added to the dissolved phenlhydrazine mixture contained in THF. Phenylhydrazine base developed anion by replacing

a

-NH proton. New developed anion attacked on 1,2-bis(bromomethyl)benzene as nucleophyllic attack and resulted in the synthesis of 1,4-bis((1-phenylhydrazinyl)methyl)benzene.

After synthesis of bisphenylhydrazine, we first used Fischer ind-olization for one-pot synthesis of bisindole derivatives. Bisindole alkaloids 9a–c were synthesized using cyclopentanone, cyclohexa-none and cycloheptacyclohexa-none Fischer indolization with 1,4-bis((1-phenylhydrazinyl)methyl)benzene by AcOH/H2SO4 acidic media.

The structures were confirmed by 1_{H NMR characteristic peaks.}

(i.e., CH2protons observed between 5.19 and 5.27 ppm). We tried

also to synthesize eight-membered ring derivatives using cyclooc-tanone, however synthesis was not successful. Although we tried different acidic media and temperature, synthesis of this com-pound could not be achieved (Scheme 2).

2.2. CA inhibition studies

The 1,4-bis(indolin-1-ylmethyl)benzene derivatives 9a–c and indole have been tested for the inhibition of two cytosolic ubiqui-tous isozymes of human origin, that is, hCA I and hCA II (Table 1).

Table 1

Ki Values obtained from regression analysis graphs for hCA I and hCA II in the

presence of different inhibitors concentrations (lM)

Compound hCA I hCA II

9a 42.6 0.17 9b 39.3 0.25 9c 41.7 0.29 Indolea 27.4 32.5 a Ref.13a. NH NH₂ NaH THF CH₂Br BrH2C N NH2 : : N2 N N H₂N NH2 THF 5 4 7

Scheme 1. Synthesis of 1,4-bis((1-phenylhydrazinyl)methylbenzene (7).

N N H2N NH2 + O N N G. AcOH TFA n n n 7 _8a-c 9a-c

Scheme 2. Synthesis of compounds 9a–c.

N H N HN NH R _R

1

NH NH H N O O 2 N H O O H N OH HO 3 N H N MeHN N H NHMe 4 Figure 1.

The following should be noted regarding CA inhibitory data of Table 1:

(i) The indole derivatives investigated here against the slow cytosolic izoform hCA I, showed weak to moderate inhibitory properties. Thus, derivatives 9a and 9c exhibited moderate inhibition of this isoform, with KI-s in the range of 41.7 and

42.6

l

M (Table 1). The compounds 9b and indole were more effective inhibitors against hCA I, with KI-s in the range of

27.4–39.3

l

M (Fig. 2). These results demonstrate the contri-bution of the hydrophobic groups to the inhibition efficacy. Interestingly, hydrophobic indole group containing com-pounds 9a–c were as effective as indole. This trend also shows the efficacy of indole moiety. The most potent deriva-tives were the compounds 9a–c against hCA II and this data is supported by the in silico docking data (Table 2). As these compounds do not possess any of the zinc-anchoring groups present in known CAIs, presumably such compounds may bind in the coumarin/activator binding site.16

(ii) Indole acted as weak inhibitor against the ubiquitous and dominant rapid cytosolic isozyme hCA II (Table 1). However, 9a–c derivatives acted as strong hCA II inhibitors

(KI-s: 0.17–0.29

l

M) (Table 1). The most potent inhibitor

was the bulky 1,4-bis(indolin-1-ylmethyl)benzene 9a (KI

0.17

l

M). ChemScore docking scores of compounds 9a–c show similar results. However GOLD Fitness score of com-pound 9a is better than other derivatives and supports the obtained experimental results regarding this compound (Table 2). Also, hydrophobic group containing compounds 9a–c were much more effective compared to indole. Obtained docking scores of 9a–c and indole against hCAII supports in vitro data.

2.3. In silico studies

In this study, a flexible docking methodology was employed using the GOLD docking algorithm.13,14_{The tested derivatives were}

docked at the binding site of the hCA I and hCA II targets. Average GOLD Fitness and ChemScore docking scores of docked inhibitors at hCA I and hCA II targets and corresponding binding interactions for the most potent inhibitors were tabulated atTables 2 and 3, respectively. (Chemscores results for compounds are also detailed at theSupplementary data Fig. 1.)

We have performed docking studies of compounds 9a–c within the hCA I and II active sites, using as templates hCA I and II adducts for which the X-ray crystal structures have been reported in com-plexes with activators (e.g., hCA I–L-His, PDB code 2FW4)15 or inhibitors (the hCA II–phenol adduct PDB code 2HNC).12,13 Top docking poses derived for the best inhibitor 9a (against hCA II). It may be observed that as for phenol5_{or hydroxycinnamic acids/}

lacosamide20_{—for which the X-ray crystal struyctures in adduct}

with hCA II have been reported—the indoles investigated here do not coordinate to the metal ion but are bound more towards the exit of the cavity, interacting eventually with the zinc-coordinated water molecule. This is a new binding mode within the CA active site, which was less investigated to date.

Figure 2. Top docking pose of compound 9a at the hCA-II.

Table 3

Binding interactions for top docking poses of 9a–c within hCAI and II

Compound hCA-I hCA-II

9a Ala121, Leu131, Ala135, Leu198, Trp5, Pro201, His200, Thr199, His94

Phe70, Ile91, Glu69, His119, Val135, Trp123, Gln92, Leu141, Val135, Val121, His122, Leu198, Pro202, Trp123

9b Tyr20, Phe91, Leu141, Pro201, His200, Phe95, His96, His94 Glu69, Ile91, Val121, Pro202, Val135, Phe131, Leu198, Gln92

9c His94, Phe91, Leu141, Pro201, His64 Ln92, Phe131, Ile91, Trp123, Val121, Val135, Pro202, Ile91, Glu69, Phe70, Leu141, Leu57 Table 2

Molecular docking binding scores of the compounds 9a–c, EZA and ZNS within hCA I and II Compound Average ChemScore for hCA-I (kJ/mol) Average GOLDFitness Score for hCA-I

Average ChemScorefor hCA-II(kJ/mol) Average GOLDFitness Scorefor hCA-II 9a 33.00 48.56 48.63 79.10 9b 33.65 49.11 48.66 65.18 9c 33.57 48.01 48.95 64.32 Indolea 34.98 58.15 41.50 43.59 a Ref.13a

3. Conclusions

Here we provide an effective synthesis which is expected to be of great value for improved synthesis of new bisindole derivatives as well as for new development in the field of Fischer indole chem-istry. Several 1,4-bis(indolin-1-ylmethyl)benzene-based com-pounds have been assayed for the inhibition of the physiologically relevant human CA isozymes hCA I and II. These compounds showed inhibition constants in the range of 39.3– 42.6

l

M and 0.17–0.29

l

M, against hCA I and hCA II, respectively. Molecular docking studies of derivatives 9a–c within hCA I and hCA II were also carried out and supported the kinetic assays. 4. Experimental

4.1. Chemicals

Sepharose 4B, protein assay reagents, 4-nitrophenylacetate and chemicals for electrophoresis were purchased from Sigma–Aldrich Co. All other chemicals were of analytical grade and obtained from Merck.

4.2. Synthesis

4.2.1. Synthesis of 1,4((1-phenylhydrazine)methyl)benzene A solution of 5 (1 g, 9.24 mmol) in dry THF cooled 20 °C after added NaH (225 mg, 9.35 mmol) mixture was stirred at 20 °C for 2 h under N2. Then (1.2 g, 4.56 mmol)

1,4-bis(bromomethyl)ben-zene was added and mixture was stirred at room temperature for 12 h. After removal of the solvent, the residue was dissolved EtOAc (50 mL) and washed with water (2  50 mL), dried over MgSO4, and solvent was evaporated. The product 7 was obtained

quantitative (yield yellow oil,1.3 g, 90%).

1_{H NMR (400 MHz, CDCl} 3) d (ppm): d 7.32–7.22 (m, ArH, 8H), 7.09 (d, J = 0.4 Hz, ArH, 4H), 6.83 (t, J = 0.4 Hz, ArH, 2H), 4.61 (s, 4H, CH2), 3.70 (s, 4H, NH2). 13C NMR (50 MHz, CDCl3) d (ppm):153.82, 138.88, 131.09, 130.26, 120.65, 115.67, 62.12. IR (KBr, cm1_{): 3327.7, 3182.1, 3020.6, 2918.8, 2829.1, 1595.4,}

1495.8, 1350.0, 1220.5, 1152,4, 989.1. Anal. Calcd for C20H22N4

(318.18): C, 55.68; H, 6.96; N, 17.60. Found: C, 55.69; H, 6.95; N, 17.58.

4.2.2. General procedure for bisindalizations

A solution of ketone (6.28 mmol), 1,4((1-phenylhydr-azine)methyl)benzene (3.14 mmol) and TFA (five drops) in acetic acid (20 mL) was refluxed for 2 h and cooled to room temperature. The reaction mixture was evaporated to remove glacial acetic acid under reduced pressure, crude product was dissolved EtOAc (100 mL) then washed with water (1  50 mL) and (2  50 mL), dried over MgSO4 and solvent evaporated The residues were

recrystallized from the below giving solvent mixture for every molecules, yield (89–92%).

4.2.3. 1,4-bis((2,3-Dihydrocyclopenta[b]indol-4(1H)-yl)methyl)benzene (9a)

Light green crystals (2.4 g, 92%) from EtOAc/Hexane; mp 147– 148 °C;1_{H NMR (400 MHz, CDCl}

3) d (ppm): d 7.49–7.44 (m, ArH,

2H), 7.21–7.15 (m, ArH, 2H), 7.12–7.06 (m, ArH, 4H), 7.02 (s, ArH, 4H), 5.19 (s, CH2, 4H), 2.92–2.86 (m, CH2, 4H), 2.80–2.73 (m, CH2,

4H), 2.60–2.49 (m, CH2, 4H).13C NMR (50 MHz, CDCl3) d (ppm):

148.09, 143.19, 139.28, 129.00, 126.89, 122.16, 121.19, 120.59, 120.41, 111.77, 49.89, 30.38, 27.12, 26.55. IR (KBr, cm1_{): 3025.2,}

2924.4, 2845.9, 1675.5, 1611.2, 1580.4, 1457.7, 1435.0, 1376.2, 1345.5, 1163.6, 1018.8. Anal. Calcd for C30H28N2(416.23): 86.50;

H, 6.78; N, 6.72. Found: C, 86.49; H, 6.79; N, 6.73.

4.2.4. 1,4-bis((2,3-dihydro-1H-Carbazol-9(2H)-yl)methyl)benzene (9b)

Light yellow crystals (2.4 g, 89%) from EtOAc/Hexane; mp 160– 161 °C;1_{H NMR (400 MHz, CDCl}

3) d (ppm): d 7.53–7.49 (m, ArH,

2H), 7.21–7.07 (m, ArH, 6H), 6.80 (s, ArH, 4H), 5.20 (s, ArH, 4H), 2.77–2.75 (m, CH2, 4H), 2.61–2.59 (m, CH2, 4H), 1.90–1.89 (m,

CH2, 8H). 13C NMR (50 MHz, CDCl3) d (ppm): 139.33, 139.56

137.42, 129.55, 122.74, 120.85, 118.78, 111.97, 110.88, 47.90, 25.24, 25.19, 24.15, 23.10. IR (KBr, cm1_{): 3053.2, 2924.4, 2851.4,}

1767.8, 1605.6, 1465.7, 1437.8, 1421.0, 1265.5. Anal. Calcd for C32H32N2(444.26): C, 86.44; H, 7.25; N, 6.30. Found: C, 86.43; H,

7.24; N, 6.29.

4.2.5. 1,4-bis((7,8,9,10-Tetrahydrocyclohepta[b]indol-5(6H)-yl)methyl)benzene (9c)

Light green crystals (2.7 g, 91%) from EtOAc/Hexane; mp 179– 180 °C;1_{H NMR (400 MHz, CDCl} 3) d (ppm): d 7.56–7.51, (m, ArH, 2H), 7.21–7.07 (m, ArH, 8H), 6.89 (s, ArH, 4H), 5.27 (s, CH2, 4H), 2.91–2.85 (m, CH2, 4H), 2.78–2.72 (m, CH2, 4H), 1.89–1.70 (m, CH2, 12H). 13C NMR (50 MHz, CDCl3) d (ppm): 140.74, 139.49, 137.78, 130.08, 128.31, 122.55, 120.93, 119.64, 116.30, 110.98, 47.91, 33.57, 30.38, 29.01, 28.46, 26.39. IR (KBr, cm1_{): 3019.6,} 2919.6, 2851.5, 1770.62, 1683.9, 1605.6, 1467.6, 1443.4, 1415.4, 1345.5, 1317.5. Anal. Calcd for C34H36N2 (472.29): C, 86.40; H,

7.68; N, 5.93. Found: C, 86.39; H, 7.67; N, 5.92.

4.3. Purification of carbonic anhydrase isoenzymes from human and bovine by affinity chromatography

Purification of hCA I and hCA II were previously described.13–15

4.4. CA inhibition

Carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25 °C using a spec-trophotometer according to the method described by Verpoorte et al.21a_{The enzymatic reaction, in a total volume of 3.0 mL,}

con-tained 1.4 mL 0.05 M Tris–SO4 buffer (pH 7.4), 1 mL 3 mM

4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution. A

ref-erence measurement was obtained by preparing the same cuvette without enzyme solution. The inhibitory effects of compounds 9a– c is compared with indole. All compounds were tested in triplicate at each concentration used. Different inhibitor concentrations were used. Different inhibitor concentrations were used. Control cuvette activity in the absence of inhibitor was taken as 100%. For each inhibitor an Activity %–[Inhibitor] graph was drawn. The curve-fit-ting algorithm allowed us to obtain the IC50values, working at the

lowest concentration of substrate of 0.15 mM, from which KI

val-ues were calculated by using the Chenge–Prusoff equation. 21b

The catalytic activity (in the absence of inhibitors) of these en-zymes was calculated from Lineweaver–Burk plots, as reported earlier, and represent the mean from at least three different deter-minations. The enzymes used here were purified from human blood as described earlier.21c

4.5. Protein determination

Protein during the purification steps was determined spectro-photometrically at 595 nm according to the Bradford method, using bovine serum albumin as the standard.21d

4.6. SDS polyacrylamide gel electrophoresis

SDS polyacrylamide gel electrophoresis was performed after purification of the enzymes. It was carried out in 10% and 3%

acrylamide for the running and the stacking gel, respectively, con-taining 0.1% SDS according to Laemmli method.21e

4.6.1. In silico docking studies

Crystal structures of CAs in complex with activators (e.g., hCA I– L-His, PDB code 2FW4)18bor inhibitors (the hCA II–phenol adduct PDB code 2HNC)13_{were used for the molecular docking calculations}

of compounds 9a–c within the active sites of the hCA I and II. Expli-cit water molecules from the X-ray structures were kept for all the calculations. Before the docking simulations, the target structure were submitted to the protein preparation module of the Schro-dinger molecular modeling package.19_{Compounds 9a–c were}

con-structed using the Schrodinger’s Maestro module and then geometry optimization was performed for these ligands using Po-lak-Ribiere conjugate gradient (PRCG) minimization (0.0001 kJ Å1_mol1_{, convergence criteria).}17

Protonation states of ligands and residues were tested using LigPrep and Protein Prepara-tion modules under Schrodinger package19–22_{at neutral pH}

(exper-imentally the compounds have been tested at pH of 7.4). Genetic Optimisation for Ligand Docking (GOLD) program is used.22_In

dock-ing the maximum number of generic algorithm runs was set to 100 for each compound. The default generic algorithm parameters were selected (100 population size, 5 number of islands, 100,000 number of generic operations and 2 for the niche size). Default cutoff values of 2.5 Å (dH-X) for hydrogen bonds and 4.0 Å for van der Waals dis-tance were employed. The two scoring functions (GoldScore Fitness and the ChemScore) were used.22_{The GoldScore Fitness function is a}

molecular mechanics-like function with four terms:

GoldScore Fitness ¼ SHB-extþ SVDW-extþ SHB-intþ SVDW-int

where SHB-extis the protein–ligand hydrogen-bond score and S VDW-extis the protein–ligand van der Waals score. SHB-intis the

contribu-tion to the Fitness due to intramolecular hydrogen bonds in the li-gand; SVDW-intis the contribution due to intramolecular strain in the

ligand.

On the other hand, the ChemScore function estimates the free energy of binding of the ligand to a protein as follows:

D

Gbinding¼

D

G0þ

D

GHBondSHBondþ

D

GmetalSmetalþ

D

GlipoSlipo

þ

D

GrotHrot

where SHbond, Smetal, and Slipo are scores for hydrogen-bonding,

acceptor-metal, and lipophilic interactions, respectively. Hrot is a

score representing the loss of conformational entropy of the ligand upon binding to the protein. The final ChemScore value is obtained by adding in a clash penalty and internal torsion terms, which mil-itate against close contacts in docking and poor internal conforma-tions. Covalent and constraint scores may also be included:

ChemScore ¼

D

Gbindingþ Eclashþ Eint

Acknowledgments

The authors are grateful to Karamanoglu Mehmetbey University Scientific Research Council, (BAP) (Project No.: BAP-24-M-11) for (O.T.).

Supplementary data

Supplementary data associated with this article can be found, in the online version, athttp://dx.doi.org/10.1016/j.bmc.2012.09.027. References and notes

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