Thesynthesisof N-benzoylindolesasinhibitorsofrat … · 2018. 12. 20. · Received:23February2018...

Received: 23 February 2018 Revised: 12May 2018 Accepted: 25 June 2018

DOI: 10.1002/jbt.22193

The synthesis ofN-benzoylindoles as inhibitors of raterythrocyte glucose-6-phosphate dehydrogenase and6-phosphogluconate dehydrogenase

Sinan Bayindir1 Yusuf Temel2 Adnan Ayna1 Mehmet Ciftci1

1Department of Chemistry, Faculty of Sciences

andArts, Bingol University, 12000, Bingol,

Turkey

2Department ofHealth Services, Vocational

Schools, Bingol University, 12000, Bingol,

Turkey

Correspondence

SinanBayindir

E-mail: [email protected]

AbstractGlucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD)

play an important function in various biochemical processes as they generate reducing power of

the cell. Thus,metabolic reprogramming of reduced nicotinamide adenine dinucleotide phosphate

(NADPH) homeostasis is reported to be a vital step in cancer progression as well as in combina-

tional therapeutic approaches. In this study,N-benzoylindoles 9a--9d, which form themain frame-

work of many natural indole derivatives such as indomethacin and N-benzoylindoylbarbituric

acid, were synthesized through three easy and effective steps as an in vitro inhibitor effect of

G6PD and 6PGD. The N-benzoylindoles inhibited the enzymatic activity with IC50 in the range of

3.391505 𝜇M for G6PD and 2.19–990 𝜇M for 6PGD.

K EYWORDS

enzyme, glucose-6-phosphate dehydrogenase, indole, indomethacin, N-benzoylindoles, N-

substituted indole, 6-phosphogluconate dehydrogenase

1 INTRODUCTION

N-Benzoylindoles are known to play important roles at numer-

ous biological and pharmacological processes.[1–8] In recent years,

indoles have attracted considerable attention among synthetic organic

chemists due to its interesting reactivity. Therefore, a great deal of

attention has been given to develop effective, facile, and innovative

synthetic strategies for the development of the indole chemistry.[9–15]

While a number of methods have already been published for the syn-

thesis of 3-substituted indole derivatives, there are only few methods

for the synthesis of 2- or N-substituted indole derivatives through the

indole (1) ring (Figure 1). An alternative method that utilizes indoline

(4) for an easy and effective synthesis of N-substituted indoles was

developed by Saracoglu and group.[16–20] The indole exhibits reactiv-

ity at the C-3 position against the electrophiles and generally forms

the 3-substituent indole derivatives. Therefore, the synthesis of N-

substituted indole derivatives, which form the main framework of

many natural indole derivatives via reactions of indole, is quite diffi-

cult, and the reaction product is obtained with very low yields. Some-

times a two-step reaction is better than a one-step reaction for effec-

tive synthesis. The advantage of the strategy developed by Saracoglu

and co-workers is that the indoline is a pyrrolidine derivative. Thus,

the indoline is electrophilically reacted at the aza-position to give N-

substituted indole derivatives.

The pentose phosphate pathway (PPP) is one of the key compo-

nents of cellular metabolism. It is strongly connected to glycolysis as

a major consumer of glucose.[21,22] Glucose-6-phosphate dehydroge-

nase (G6PD) is a critical enzyme in mammalian erythrocytes that cat-

alyzes the first reaction and a rate-limiting enzyme in the PPP.[23] The

reduced nicotinamide adenine dinucleotide phosphate (NADPH) has

been shown to be essential for the protection of cells against oxida-

tive damage. 6-Phosphogluconate dehydrogenase (6PGD) is the third

enzyme in the PPP and converts 6-phosphogluconate into ribulose-

5-phosphate.[24] In the absence of these enzymes, the erythrocyte is

susceptible to oxidative damage. The association of G6PD with can-

cer, metabolic disorders, and cardiovascular diseases is also studied in

detail, demonstrating G6PD can be a cancer target. The suppression

of 6PGD decreased lipogenesis and RNA biosynthesis and increased

reactive oxygen levels in cancer cells, suggesting that 6PGD could also

be an anticancer target.[25–28]

In our previous studies, we developed an efficient, facile, and

atom economical protocol for the preparation of N-substituted indole

derivatives through the addition of indoline (4) using 𝛼, 𝛽-unsaturated

ketones as an electrophile followed by an oxidation step.[16,17] The

aim of this work was to synthesize N-substituted indole deriva-

tives from the main framework of important drugs such as N-

benzoylindoylbarbituric acid (2; see Figure 1) and indomethacin (3; see

Figure 1) and to investigate in vitro effects of these indole derivatives

J BiochemMol Toxicol. 2018;e22193. c© 2018Wiley Periodicals, Inc. 1 of 9wileyonlinelibrary.com/journal/jbthttps://doi.org/10.1002/jbt.22193

http://orcid.org/0000-0002-7845-4497

http://orcid.org/0000-0001-8148-3718

http://orcid.org/0000-0001-6801-6242

http://orcid.org/0000-0003-4971-4242

2 of 9 BAYINDIR ET AL.

F IGURE 1 The indole (1) and N-benzoylindole-based naturalcompounds.

on the enzymatic activity of G6PD and 6PGD. As these enzymes are

anticancer target, the derivatives synthesizing and investigating their

potential inhibitory actions are important. In this work, we synthe-

sized new N-benzoylindoles via Bi(NO3)3.5H2O catalyzed reactions

of indoline (4) with nitro olefins followed by two oxidation steps. In

the continuation of the work, we purified two important enzymes of

the PPP, G6PD, and 6PGD from rat blood erythrocytes and studied

the effect ofN-benzoylindoles derivatives on the enzymatic activity of

both enzymes.

2 EXPERIMENTAL AND METHODS

2.1 General information

All chemicals, reagents and solvents were commercially available from

Sigma-Aldrich (St. Louis, MO) or Fluka (Munich, Germany) and used

as received. 2′,5′-ADP Sepharose 4B was purchased from Pharmacia.

Melting pointswere determinedon aBuchi 539 capillarymelting appa-

ratus and uncorrected. Infrared spectra were recorded on a Mattson

1000 FT-IR spectrophotometer. 1H NMR and 13C NMR spectra were

recorded on 400 (100) MHz Varian and Bruker spectrometers and are

reported in terms of chemical shift (𝛿, ppm) with SiMe4 as an inter-

nal standard. Data for 1H NMR are recorded as follows: chemical shift

(𝛿, ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quar-

ted, p = pentet, m = multiplet, bs = broad singlet, bd = broad dou-

blet, qd = quasi doublet) and coupling constant (s) in Hz, integration.

Elemental analyses were carried out on a Leco CHNS-932 instrument.

Column chromatography was carried out on silica gel 60 (230–400

mesh ASTM). The reaction progress was monitored by thin layer chro-

matography (TLC) (0.25 mm thick precoated silica plates: Merck Fer-

tigplatten Kieselgel (60 F254). UV–vis spectra were recorded on a Shi-

madzu UV-3101PL UV-vis-NIR spectrometer.

2.2 General procedure for synthesis of nitro olefins

Nitro olefins 6a–6e were prepared according to the literature

method.[29] Amixture of aldehyde (1mmol), nitro alkane (6mmol), and

piperidine (0.1mmol) were added sequentially to oven-dried flask con-

taining toluene as solvent (10 mL), then anhydrous FeCl3 (0.1 mmol)

was added into the reactionmedium. Themixturewas heated to reflux

slowly for 3–10 h in air; the reactionwasmonitored by TLC. After com-

pletion, the mixture was cooled to room temperature. The excess sol-

vent was removed under reduced pressure, and the compounds were

purified by silica gel column chromatography (EtOAc/hexane; v/v: 1/9)

to afford the nitro olefins.

2.3 General procedure for synthesis of

N-substituted indoline derivatives

N-Substituted indoline derivatives 7a–7e were prepared according to

the literature method.[16,17] To a solution of indoline (1.0 mmol) and

nitro olefine (1.0 mmol) in CH2Cl2 (10mL), Bi(NO3)3.5H2O (0.1 mmol)

was added. The mixture was stirred until the starting material disap-

peared (TLC, hexane/ethyl acetate). After evaporation of the solvent,

the crude product was dissolved with EtOAc (30 mL), and the organic

phase was washed with water (3×30 mL). The EtOAc extract was

dried over Na2SO4, filtered, evaporated in vacuo, and the compound

was purified by silica gel column chromatography (EtOAc/hexane; v/v:

1/9).


N-substituted indole derivatives

N-Substituted indole derivatives 8a–8e were prepared according

to the literature method.[16,17] N-substituted indoline derivative

(1.0 mmol) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ;

1.0 mmol) were dissolved in CH2Cl2 (10 mL). The mixture was stirred

at room temperature for overnight. After completion of the reac-

tion, it was quenched with a saturated aqueous solution of NaHCO3

(5%, 20 mL). A CH2Cl2 layer was separated and washed with water

and brine solution and dried over anhydrous MgSO4 and filtered.

The solvent was removed in vacuo, and then the compound was

purified by silica gel column chromatography (EtOAc/hexane; v/v:

2/8).


N-benzoylindole derivatives

A solution of N-substituted indole derivative (8a–8d, 1 mmol), sodium

nitrite (3 mmol), and acetic acid (10 mmol) were dissolved in dimethyl-

sulfoxide (DMSO) (15 mL). The mixture was stirred at room tempera-

ture for 1–5 h; the reactionwasmonitored by TLC. After completion of

the reaction, 1 MHCl (15 mL) was added to the solution; after 15 min,

the aqueous phase was extracted with CH2Cl2 (3×50 mL). The com-

bined organic phases were then dried over MgSO4 and filtered. The

solvent was removed in vacuo, and then the compoundwas purified by

silica gel column chromatography (methylene chloride/ methanol; v/v:

3/2).

2.6 Characterization of organic compounds

1-(2-Nitro-1-phenylethyl)indoline (7a)[17]: Orange crystal, mp. 125–

126◦C, yield 94%. 1H NMR (400 MHz, CDCl3): 𝛿 7.38–7.32 (m, =CH,3H), 7.30–7.26 (m, =CH, 2H), 7.11–7.04 (m, =CH, 2H), 6.70–6.66(m, =CH, 2H), 5.62 (t, J = 7.6 Hz, CH, 1H), 5.01 (dd, J = 12.4, 7.6 Hz,

BAYINDIR ET AL. 3 of 9

CH2, A part of AB system, 1H), 4.92 (dd, J = 12.4, 7.6 Hz, CH2, A part

of AB system, 1H), 3.46–3.41 (m, CH2, B part of AB system, 1H), 3.17–

3.13 (m, CH2, B part of AB system, 1H), 2.96–2.90 (m, CH2, 2H).13C

NMR (100 MHz, CDCl3): 𝛿150.1, 135.1, 129.8, 129.2 (=CH), 128.8(=CH), 127.8 (=CH), 127.7 (=CH), 125.0 (=CH), 118.6 (=CH), 107.3(=CH), 75.6 (CH), 57.7 (CH2), 47.5 (CH2), 28.4 (CH2). Anal. Calcd. for

C16H16N2O2: C, 71.62; H, 6.01; N, 10.44. Found: C, 71.46; H, 6.00; N,

10.47. IR (KBr, cm–1) 3025, 2913, 2846, 2325, 1608, 1555 1457, 1373,

1309, 1231, 1194, 1083, 742.

1-(2-Nitro-1-(2-nitrophenyl)ethyl)indoline (7e): Pale yellow crystals,

m.p. 155–156◦C, yield 81%. 1H NMR (400 MHz, CDCl3): 𝛿 7.91 (d,

J = 8.6 Hz, = CH, 1H), 7.52–7.45 (m, =CH, 3H), 7.10–7.00 (m, =CH,2H), 7.11–7.04 (m, =CH, 2H), 6.73 (t, J = 7.6 Hz, =CH, 1H), 6.49 (d,

J =7.6 Hz, =CH, 1H), 6.16 (t, J = 7.0 Hz, CH, 1H), 5.03 (dd, J = 13.3,

7.0 Hz, CH2, A part of AB system, 1H), 4.93 (dd, J = 13.3, 7.1 Hz, CH2,

B part of AB system, 1H), 3.66–3.63 (m, CH2, A part of AB system, 1H),

3.37–3.34 (m, CH2, B part of AB system, 1H), 2.97–2.94 (m, CH2, 2H).13CNMR(100MHz,CDCl3): 𝛿 149.5, 141.3, 137.4, 133.6, 132.0, 130.6,

127.8, 125.2, 124.2, 121.2, 116.5, 106.6, 68.3, 60.3, 51.7, 30.0. Anal.

Calcd. for C16H15N3O4: C, 61.34; H, 4.83; N, 13.41. Found: C, 61.36;

H, 4.29; N, 13.45. IR (KBr, cm–1) 3035, 2923, 2746, 1603, 1505 1427,

1273, 1241, 1184, 1084, 742.

1-(2-Nitro-1-phenylethyl)-1H-indole (8a)[17]: White crystal, m.p. 97–

98◦C, yield 95%. 1H NMR (400 MHz, CDCl3): 𝛿 7.63 (dd, J = 6.9,

0.9 Hz, =CH, 1H), 7.38–7.34 (m, =CH, 4H), 7.23–7.15 (m, =CH, 4H),7.13 (dd, J = 6.9, 0.9 Hz, =CH, 1H), 6.61 (d, J = 3.3 Hz, =CH, 1H), 6.44(dd, J = 9.0, 6.1 Hz, =CH, 1H), 5.23 (dd, J = 13.3, 9.0 Hz, CH2, A part of

AB system, 1H), 5.15 (dd, J = 13.3, 6.1 Hz, CH2, B part of AB system,

1H). 13C NMR (100 MHz, CDCl3): 𝛿136.2. 135.9, 129.6 (=CH), 129.3(=CH), 129.1, 126.7 (=CH), 124.6 (=CH), 122.6 (=CH), 121.5 (=CH),120.6 (=CH), 109.7 (=CH), 103.9 (=CH), 77.4 (CH), 57.3 (CH2). Anal.


H, 5.30; N, 10.40. IR (KBr, cm–1) 3025, 2913, 2846, 2325, 1608, 1555

1457, 1373, 1309, 1231, 1194, 1083, 742.

1-(1-(4-Chlorophenyl)-2-nitroethyl)-1H-indole (8b): Red crystals, m.p.

135–136◦C, yield 86%. 1H NMR (400 MHz, CDCl3): 𝛿 7.72 (d,

J= 7.7 Hz,=CH, 1H), 7.53–7.48 (m,=CH, 2H), 7.43 (t, J= 6.5 Hz,=CH,1H), 7.37–7.32 (m, =CH, 3H), 7.25–7.23 (m, =CH, 2H), 6.67 (d,

J = 3.2 Hz, =CH, 1H), 6.53 (t, J = 7.1 Hz, CH, 1H), 5.27 (dd, J = 13.3,

7.1Hz, CH2, A part of AB system, 1H), 5.19 (dd, J= 13.3, 7.1Hz, CH2, B

part of AB system, 1H). 13C NMR (100 MHz, CDCl3): 𝛿 131.6, 131.1,

127.3, 126.6, 124.6, 123.7, 122.9, 121.6, 121.0, 120.3, 110.1, 102.7,

72.5, 60.3. Anal. Calcd. for C16H13ClN2O2: C, 63.90; H, 4.36; N, 9.31.

Found: C, 63.98; H, 4.54; N, 9.37. IR (KBr, cm–1) 3126, 2923, 2836,

1628, 1505 1427, 1319, 1218, 1114, 1063, 742.

1-(1-(Furan-2-yl)-2-nitroethyl)-1H-indole (8c)[17]: Pale yellowcrystals,

m.p. 124–125◦C, yield 93%. 1H NMR (400 MHz, CDCl3): 𝛿 7.63 (d,

J = 8 Hz, =CH, 1H), 7.43 (m, =CH, 2H), 7.26 (m, =CH, 1H), 7.17–7.13(m, =CH, 2H), 6.58 (d, J = 3.28 Hz =CH, 1H), 6.44 (t, J = 7.7 Hz, CH,

1H), 6.39–6.33 (m,=CH, 2H), 5.18 (dd, J= 13.36, 6.77 Hz, A part of AB

system, CH2, 1H), 5.10 (dd, J = 13.36, 7.87 Hz, B part of AB system,

CH2, 1H).13C NMR (400 MHz, CDCl3): 𝛿 148.3, 143.7, 135.8, 129.1,

125.3, 122.7, 121.6, 120.7, 111.1, 109.6, 109.3, 104.1, 76.0, 51.8. Anal.


H, 4.66; N, 10.72. IR (KBr, cm–1)3053, 2913, 1646, 1556, 1458, 1373,

1307, 1236, 1194, 1146, 1014.

1-(2-Nitro-1-(thiophen-2-yl)ethyl)-1H-indole (8d)[17]: Pale red crys-

tals, m.p. 131–132◦C, yield 92%. 1H NMR (400 MHz, CDCl3): 𝛿 7.63

(d, J = 7.0 Hz, =CH, 1H), 7.41 (d, J = 8.4 Hz, =CH, 1H), 7.31 (m, =CH,1H), 7.25–7.00 (m, =CH, 5H), 6.64 (t, J = 7.5 Hz, CH, 1H), 6.60 (d,

J = 3.3 Hz, =CH, 1H), 5.25–5.14 (m, CH2, 2H).13C NMR (400 MHz,

CDCl3): 𝛿 138.6, 135.9, 129.2, 127.5, 126.8, 126.2, 124.7, 122.8, 121.7,

120.8, 109.5, 104.3, 77.8, 53.5. Anal. Calcd. for C14H12N2O2S: C,

61.75; H, 4.44; N, 10.29. Found: C, 62.01; H, 4.40; N, 10.44. IR (KBr,

cm–1) 3025, 2919, 2846, 1604, 1550, 1485, 1375, 1247, 744, 698.

N-Benzoylindole (9a)[6,17,30]: Pale yellow crystals, m.p. 58–59◦C,

yield 95%. 1HNMR (400MHz, CDCl3): 𝛿 8.41 (bd, J=7.6Hz,=CH, 1H),7.75–7.73 (m, =CH, 2H), 7.63–7.59 (m, =CH, 2H), 7.55–7.51 (m, =CH,2H), 7.39 (t, J = 7.6 Hz, =CH, 1H), 7.34–7.25 (m, =CH, 2H), 6.62 (d,

J = 3.7 Hz, =CH, 1H); 13C NMR (100 MHz, CDCl3): 𝛿 168.9, 136.3,

134.9, 132.1, 131.0, 129.4, 128.8, 127.8, 125.1, 124.1, 121.1, 116.6,

108.8. Anal. Calcd. for C15H11NO: C, 81.43; H, 5.01; N, 6.33. Found: C,

81.30; H, 4.92; N, 6.44. IR (KBr, cm–1): 2917, 2849, 1731, 1684, 1450,

1376, 1337, 1178, 1055, 937, 884, 749.

N-(4-Chlorobenzoyl) indole (9b)[31]: Yellow crystals, m.p.

116–117◦C, yield 82%. 1H NMR (400 MHz, CDCl3): 𝛿 8.43 (bd,

J= 8.0Hz,=CH, 1H), 7.78–7.76 (m,=CH, 2H), 7.65–7.63 (m,=CH, 1H),7.57 (d, J = 7.3 Hz, =CH, 2H), 7.41 (d, J = 7.3 Hz, =CH, 2H), 7.34–7.32(m, =CH, 2H), 6.65 (d, J = 3.6 Hz, =CH, 1H); 13C NMR (100 MHz,

CDCl3): 𝛿 165.9, 141.4, 140.4, 137.6, 137.0, 134.8, 131.8, 131.0, 129.2,

128.2, 125.2, 120.5, 113.0; Anal. Calcd. for C15H10ClNO: C, 70.46; H,

3.94; N, 5.48. Found: C, 71.04; H, 3.82; N, 5.41. IR (KBr, cm–1): 3125,

2817, 2809, 1735, 1682, 1420, 1366, 1324, 1128, 10,565, 888, 741.

1H-Indol-1-il)(furan-2-il)methanon (9c): Pale yellow viscous liquid,

yield 91%. 1H NMR (400 MHz, CDCl3): 𝛿 8.42 (bd, J = 8.2 Hz, =CH,1H), 7.92–7.85 (m, =CH, 3H), 7.57 (d, J = 7.6 Hz, =CH, 1H), 7.39(t, J = 7.6 Hz, =CH, 1H), 7.33 (t, J = 7.6 Hz, =CH, 1H), 7.21 (t,

J=3.5Hz,=CH, 1H), 6.70 (d, J=3.6Hz,=CH, 1H); 13CNMR (100MHz,

CDCl3): 𝛿 163.8, 143.2, 138.3, 135.5, 134.9, 134.7, 129.8, 129.2, 127.1,

126.1, 123.1, 118.1, 111.0; Anal. Calcd. for C13H9NO2: C, 73.92; H,

4.30; N, 6.63. Found: C, 73.84; H, 4.22; N, 6.68. IR (KBr, cm–1): 3475,

3225, 2985, 2856, 1734, 1617, 1578, 1498, 1252, 1237, 1145, 1117,

938.

1H-Indol-1-il)(tiyofen-2-il)methanon (9d)[6]: Pale red viscous liquid,

yield 93%. 1H NMR (400 MHz, CDCl3): 𝛿 8.40 (bd, J = 8.1 Hz, =CH,1H), 7.71–7.67 (m, =CH, 3H), 7.61 (d, J = 7.7 Hz, =CH, 1H), 7.38(t, J = 7.7 Hz, =CH, 1H), 7.31 (t, J = 7.7 Hz, =CH, 1H), 7.19 (t,

J=4.4Hz,=CH, 1H), 6.68 (d, J=3.6Hz,=CH, 1H); 13CNMR (100MHz,

CDCl3): 𝛿 161.9, 137.4, 136.4, 133.6, 132.9, 130.8, 127.8, 127.3, 125.2,

124.2, 121.2, 116.5, 109.0; Anal. Calcd. for C13H9NOS: C, 68.70; H,

3.99; N, 6.16; S, 14.11. Found: C, 68.84; H, 4.02; N, 6.24. IR (KBr, cm–1):

3082, 3028, 2972, 2839, 1731, 1605, 1479, 1452, 1384, 1332, 1256,

1232, 1184, 1023.

2.7 Preparation of hemolysate

Fresh blood samples were obtained from rats and placed in ethylene-

diaminetetraacetic acid (EDTA)-containing tubes. To distinguish the


erythrocyte from plasma, blood samples were filtered to remove any

impurities then centrifuged for 15 min at 2500×g to remove plasma.

Following that, the precipitated erythrocyteswerewashed three times

with 0.16 M KCl and hemolyzed with 5 volume of cold water. Then, to

remove the cell membranes and intact cells 30 min centrifugation at

10.000×gwas performed.[32]

2.8 5′-ADP sepharose 4B affinity chromatography

Following preparation of the hemolysates, G6PD was passed through

the 2′,5′-ADP Sepharose 4B affinity column, which was equilibrated

with 50 mM KH2PO4, 1 mM EDTA, and 1 mM dithiothreitol (DTT) at

pH 7.3. The protein was eluted with 80 mM KH2PO4, 10 mM EDTA,

80 mM KCI, and 5 mMNADP+ at pH 7.3. All procedures were carried

out at 4˚C.[32]

6PGD was purified with the 2′,5′-ADP Sepharose 4B affinity col-

umn, which was equilibrated with 50 mM KH2PO4, 1 mM EDTA ,and

1 mM DTT at pH 7.3. The protein was eluted with 80 mM KH2PO4,

10mMEDTA, 80mMKCI, and50mMNADP+ at pH7.3. All procedures

were carried out at 4˚C.[32]

2.9 Measurement of enzyme activity

G6PD and 6PGD activity assays were run at 25oC according to

Beutler's method, which depends on the reduction of NADP+ by

both enzymes, in the presence of glucose-6-phosphate (G6P) or 6-

phosphogluconate (6PGA). For this spectrophotometric measurement

of G6PD, the reaction medium contained 0.1 mMTris–HCl (pH 8) with

0.2 mM NADP+, 0.5 mM EDTA, 0.01 mM MgCl2 and 0.6 mM G6P in

a total volume of 1 mL. The reaction started with the addition of the

enzyme. One unit of enzyme (EU) activity was defined as the amount

of enzyme that reduced 1 mmol NADP+ per minute at pH 8.[32] The

activity of 6PGAwasmeasured according to the Beutler method using

1mMTris–HCl (pH 8) with 2mMNADP+, 5 mMEDTA, 0.1mMMgCl2,

and 6mM6PGA in a total volume of 1mL.

2.10 In vitro enzyme inhibition studies

The N-benzoylindole derivatives synthesized in this study were used

as inhibitors/activators of the enzymes. In the reactionmediumwith or

without inhibitor, the substrate (G6P and 6PGA) concentrations were

6.25, 15.30, 62.5, and 90 𝜇M. The N-benzoylindole derivatives were

added to the reaction medium, resulting in three different fixed con-

centrations of inhibitors in 1mL of total reaction volume. To determine

IC50 values, activities were calculated with a 0.60 mM constant sub-

strate (G6P/6PGA) and different inhibitor concentrations. The enzyme

activities in the absence of compounds were acknowledged as 100%.

Activity %versus compound concentration graphs were drawn and

used to calculate the drug concentrations (IC50 values) causing a 50%

decrease in enzymeactivity. Inhibition andKi constants forG6PDwere

determined via Lineweaver–Burk graphs. The study was carried out in

compliance with the protocol confirmed by Local Ethic Committee of

Animal Experiments of Bingol University (85680299/020).

2.11 Homologymodeling

The three-dimensional models G6PD was generated by Phyre2

(Protein Homology/AnalogY Recognition Engine)[33] as described by

Bayindir and co-workers.[13] For the modeling of structure of rat

erythrocyte G6PD, the sequences were retrieved from Uniprot

(http://www.uniprot.org/uniprot/P05370) as the FASTA format. The

sequences were then submitted to Phyre2 for protein structure deter-

mination. The X-ray crystal structures of humanG6PD (PDB-ID: 1QKI)

was used as the starting structure for molecular modeling.

3 RESULTS

3.1 Synthesis of organic compounds

Initially, nitro olefin derivatives 6a–6e were synthesized via reac-

tion of aldehydes 5a–5e with nitro methane (Scheme 1A). The sec-

ond synthetic step was the conjugate addition of indoline with nitro

olefins and followed by an oxidation reaction by DDQ in CH2Cl2(Scheme 1B and 1C). After synthesis of N-substituted indoles 8a–

8e, the N-benzoylindole compounds 9a–9e were obtained through

the reaction of N-substituted indole derivatives with NaNO2/AcOH in

DMSO (Scheme 1D). Similarly, synthesis of N-benzoylindoline deriva-

tives was attempted by reacting N-substituted indoline derivatives

7a–7e with NaNO2/AcOH in DMSO. Unfortunately, the oxidation of

the N-substituted indoline derivatives produced unexpected starting

molecules, indoline (4) and nitromethane derivatives, instead of the

expected products N-benzoylindoline (Scheme 1E). Looking at the 1H

NMR spectrum of the N-benzylindole 9a, it appears that 10 aromatic

CH protons are resonant at a range of 8.41–6.62 ppm. In the 13C

NMR spectrum, the presence of four quaternary and nine aromatic

CH carbon signals, one of which is the carbonyl group resonating at

168.9 ppm, also support the structure of 9a.[17]

3.2 Purification and in vitro enzyme kinetic studies

In the present study,G6PDand6PGD from rat erythrocyteswere puri-

fied in a single chromatographic step with high purity. Purification of

rat erythrocyte G6PD was carried out with a specific activity of 13.7

EU/mg, a yield of 67.7 and 155.6 purification fold.[32] 6PGD was puri-

fied with 61.5% purification yield from rat erythrocytes with a specific

activity of 1.37 EU/mg.We investigated the inhibition and activation of

G6PD and 6PGDwith a panel ofN-benzoylindole derivatives. The inhi-

bition/activation observed with these derivatives against G6PD and

6PGDare shown inTable1. StudieswithG6PDsuggested that the least

effective derivatives were 7a and 7e, which did not affect the enzy-

matic activity up to a concentration of 10 mM derivatives. Moderately

weak inhibitory action, in the micromolar range, was observed for the

following derivatives 8d, 9a, and 9c, with a Ki of 483.31, 92.98, and

230.01 𝜇M, respectively. The most effective inhibitors of G6PD were

the compounds 9b and 9d, inhibiting the enzyme activity with an IC50

of 3.39 and 3.85 𝜇M and Ki of 2.56 and 2.60 𝜇M, respectively. While

the aforementioned compounds inhibited the activity of the enzyme

http://www.uniprot.org/uniprot/P05370


TABLE 1 Chemical characteristics of indole derivatives and determination of IC50, Ki values, and inhibition types of the indole derivatives

Compound G6PD 6PGDNo. IC50 (𝝁M) Ki (𝝁M) Inhibition type IC50 (𝝁M)

1

NNO2

7a Noneffect – – Noneffect

2

N

NO2

NO2

7e Noneffect – – Noneffect

3

NNO2

8a Activator – – Activator

4

N

CI

NO2

8b Activator – – Activator

5

o

NNO2

8c Activator – – Activator

6

N

S

NO2

8d 1505 483.31 Competitive 990

7

o

N

9a 86.6 92.98 Noncompetitive 2.75

8

o

N

CI

9b 3.39 2.56 Competitive 2.19

9

oo

N

9c 173.2 230.01 Noncompetitive 346

10

oS

N

9d 3.85 2.60 Competitive 3.36


SCHEME 1 Synthesis ofN-benzoylindole derivatives 9a–9e

derivatives, the derivatives 8a, 8b, and 8cwere the weak activators of

the enzyme (Table 1; Figures S21–S26 in the Supporting Information).

The inhibition type of the each inhibitor was also studied, revealing

compounds 9a and 9c inhibited the enzyme activity noncompetitively

whereas compounds 8d, 9b, and 9dwere the competitive inhibitors of

the enzyme. The effect of the samederivatives on6PGDdemonstrated

that moderate inhibition was observed with compounds 8d, 9a, 9b, 9c,

and 9dwith an IC50 of 990, 2.75, 2.19, 346, and 3.36 𝜇M, respectively.

The least effective derivativeswere7a and7e as they did not show any

inhibitory and activator properties with up to 10 𝜇M concentration.

The N-substituted indole derivatives 8a–8c were the activator of the

enzyme, whereas derivatives 8d and 9a–9d were potent inhibitors of

the enzyme (Table 1; Figures S27–S29 in the Supporting Information).

3.3 Structural analysis

Following in vitro enzyme activity studies, a homology model of

the enzyme was generated to explore NADP-binding site of the rat

erythrocyte G6PD and compared it to a ligand-binding site in human

G6PD. The analysis of homology model demonstrated that overall

fold of the both enzymes is similar (Figures 2a and 2c). After careful

examination of the sequence alignment, the ligand-binding sites of the

enzymeswere compared structurally, further revealing NADP-binding

site interactions. In human G6PD, the aromatic rings of NADP+ are

sandwiched between delocalized 𝜋-electron clouds; the adenine lies

between Tyr503 and Arg487 and the nicotinamide between Trp509

and Tyr401. The 2′-phosphate makes hydrogen bonds to Arg487,

Arg357, Lys238, and Lys366; the diphosphate interacts with another

arginine (Arg370), whereas Arg393 and Asp421 interact with the

amide function of the nicotinamide (Figure 2b).[34] The comparison

of these structures revealed that the NADP-binding site is very well

conserved as NADP is interacted with the same residues in both

structures. Glucose-6-phospahate interacted with Lys360, Asp258,

Glu239, His201, His202, His263, Lys171, and Gln395 in both human

and rat G6PD (Figure 2d).[35] The histidine acts as the general base

that abstracts the proton from the C1-hydroxyl group of glucose 6-

phosphate, and the carboxylate group of Asp stabilizes the positive

charge that forms on His in the transition state. In our work, we pos-

tulated that the indole derivatives could make interactions with Asp

and His at active sites of the enzyme and hence block the enzymatic

activity.[36]

4 DISCUSSION

The objective of this study was to synthesize N-benzoylindoles, which

constitute the main framework of many natural products and pharma-

ceutical analysts, and investigate their effects on the activities of rat

blooderythrocyteG6PDand6PGD. For this purpose, initially, the reac-

tion conditions were optimized by changing parameters such as tem-

perature, time, catalysts, and solvents to identify favorable reaction

conditions. It was determined that the most favorable reaction condi-

tions for the reactions were NaNO2-catalyzed reaction conditions in

DMSO/AcOH (Table2, entry6). The synthesis of otherN-benzylindoles

was then carried out under these conditions.

The previous study by our group demonstrated that the overall

structure and residues involved in NADP and G6P binding are very

well conserved, suggesting they may behave similarly in vitro.[13,21–24]

To date, only a few G6PD inhibitors have been available with high

IC50 values, which negatively affect their use as therapeutics and basic

research probes. The NADPH, generated during reaction catalyzed by

G6PD, is essential for scavenging the reactive oxygen species (ROS)

mainly derived from oxidative phosphorylation.[37] Therefore, more

NADPH might be required to fight against harmful effects of ROS,

thus the enzyme might need to be more active and produce more

NADPH. In this context, following synthesis the indole derivatives,

containing the skeletons of N-substituted indole, N-substituted indo-

line, andN-benzoylindoles, investigation of effects of these derivatives


F IGURE 2 Comparison of putative G6P and NADP binding sites in human G6PD (a) and rat erythrocyte G6PD (shown in magenta) (c) and theirequivalent in human (shown in green) (b) and rat erythrocyte (shown in cyan) G6PDs (d).

SCHEME 2 Proposedmechanism for 6PGD -catalyst oxidation of 8awith NADP

on rat blood erythrocyte G6PD and 6PGD was aimed. In our work,

the results demonstrated that thederivatives containingN-substituted

indolines had no effect on the activity of G6PD and 6PGD whereas

the compounds with N-benzoylindoles inhibited the enzymatic activ-

ity and compoundswithN-substituted indoline skeletons increased the

activity of the enzyme, suggesting the derivatives containing the same

skeletons show similar effect on the activity in vitro. This is important

as it gives important clues about what might be the effects of natural

products and drugs bearing this skeleton on G6PD and 6PGD, the two

important enzymes of the PPP.

In the current research, it has been determined that organic

molecules containing sulfur, nitrogen, oxygen, and halide groups in

the structure of molecules are strong inhibitors of enzymes.[13,38,39]

It is of interest that these groups have free electrons and suitable

geometries that can easily interact with the active site amino acids in

the structure of the enzymes. The N-benzoylindoles 9b and 9d con-

taining chloride and sulfur groups were found to inhibit the enzyme

at a moderate level, whereas the other derivatives, which are dif-

ficult to interact noncovalently, showed no effect or increased the

enzymatic activity. G6PD oxidizes G6P to 6-phospho-D-glucono-1,5-

lactone, whereas 6PGD oxidizes 6PGA to ribulose-5-phosphate. The

indolines are oxidized to the corresponding indole derivative by oxida-

tion with DDQ in a manner analogous to the oxidation mechanism of

NADP, and the corresponding indole derivative is similarly oxidized to

over oxidizedN-benzoylindoles by oxidation with DDQ.[16,17] As a rea-

son for theN-substituted indoles to be an activator of the enzyme, the


TABLE 2 Optimization of reaction conditionsa

Entry Catalyst SolventTemperature(oC) Time

Yields% (9a)b

1 – DMSO/AcOH 50 1 day 0

2 NaNO2 – 50 1 day 0

3 NaNO2 DMSO 50 1 day 0

4 NaOH DMSO 50 1 day 24c

5 NaNO2 DMSO/AcOH 0 2 h 26

6 NaNO2 DMSO/AcOH 25 2 h 96

7 NaNO2 DMSO/AcOH 50 2 h 78c

8 NaOH H2SO4/H2O 25 2 h 21c

9 NaNO2 DMSO/HCl 25 30min 15c

10 KOH DMSO/AcOH 25 2 h 56

11 NaOH H2O 25 2 h 76

aConditions: 1-(2-nitro-1-phenylethyl)-1H-indole (8a, 1 equiv.), catalyst(3 equiv.) and solvent (10mL).bIsolated yields of 5.cComplex reactionmixture.

possibility that NADPH can be transformed into the N-substituted

indoles after oxidizing the substrate and this can be considered as an

activation (Scheme 2; Figure S30 in the Supporting Information).[13]

Another remarkable subject is the question ofwhyN-substituted indo-

lines do not affect the enzymatic activity. In the literature, it is consid-

ered that theN-substituted indolines are unstable in theworking envi-

ronment andmight turn into degradation products.[17]

5 CONCLUSION

In this study, the inhibitory effects of N-benzoylindole derivatives on

two important enzymes of PPP, G6PD, and 6PGD were investigated

in vitro. Initially,N-substituted indole andN-benzoylindole derivatives,

which are the skeleton of natural and important medicinal chemi-

cals, were synthesized via an effective and facile method. Following

that the enzymes were purified in a single chromatographic step with

high purity. Subsequently, these derivativeswere investigated for their

G6PD and 6PGD inhibition properties. The results suggested that

some of these derivatives were moderate inhibitors of the enzymes,

whereas some of them increased the activity of both enzymes. The

derivatives bearing the same groups showed similar inhibitor or acti-

vator properties.

ACKNOWLEDGMENTS

Authors are indebted toDepartment ofChemistry at BingolUniversity

(BAP-209-324-2015), and the author also would like to thank Prof. Dr.

Nurullah Saraçoglu for practical support and helpful discussions.

ORCID

Sinan Bayindir http://orcid.org/0000-0002-7845-4497

Yusuf Temel http://orcid.org/0000-0001-8148-3718

Adnan Ayna http://orcid.org/0000-0001-6801-6242

Mehmet Ciftci http://orcid.org/0000-0003-4971-4242

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SUPPORTING INFORMATION

Additional supporting informationmay be found online in the Support-

ing Information section at the end of the article.

How to cite this article: Bayindir S, Temel Y, Ayna A, Ciftci

M. The synthesis of N-benzoylindoles as inhibitors of rat

erythrocyte Glucose-6-phosphate dehydrogenase and 6PGD.

J Biochem Mol Toxicol. 2018;e22193. https://doi.org/10.1002/

jbt.22193

https://doi.org/10.1002/jbt.22193

https://doi.org/10.1002/jbt.22193

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