View
1
Download
0
Category
Preview:
Citation preview
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: sbayindir@bingol.edu.tr
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
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).
2.4 General procedure for synthesis of
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).
2.5 General procedure for synthesis of
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.
Calcd. for C16H14N2O2: C, 72.16; H, 5.30; N, 10.52. Found: C, 72.08;
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.
Calcd. for C14H12N2O3: C, 65.62; H, 4.72; N, 10.93. Found: C, 65.85;
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
4 of 9 BAYINDIR ET AL.
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
BAYINDIR ET AL. 5 of 9
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
6 of 9 BAYINDIR ET AL.
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
BAYINDIR ET AL. 7 of 9
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
8 of 9 BAYINDIR ET AL.
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
REFERENCES
[1] L. Kun, M. B. Regina, J. A. John, M. Ralph D. Sheryl, A. Ramon, Y. Meng,
T. G. Richard, C. Lawrence, L. Cherrie et al., Bioorg. Med. Chem. Lett.2005, 15, 2437.
[2] R. P. Narsimha, R. P. Purushothama, K. Vinod, A. C. Peter, Bioorg. Med.Chem. Lett. 2013, 23, 1442.
[3] W.M. Dai, D. S. Guo, l. P. Sun, Tetrahedron Lett. 2001, 42, 5275.
[4] P. Franck, B. Eckhard, R.Wolfgang,W.H. Rolf,Bioorg.Med. Chem.2000,8, 1479.
[5] W. Jingsong, L. Nhut, H. Alonso, S. Haijing, R. Robert, L. X. Wang, Org.Biomol. Chem. 2005, 3, 1781.
[6] X. F. Wu, S. Oschatz, M. Sharif, P. Langer, Synthesis 2015, 47,2641.
[7] D. S. Dhanoa, S. W. Bagley, R. S. L. Chang, V. J. Lotti, T. B. Chen, S. D.
Kivlighn, G. J. Zingaro, P. K. S. Siegl, A. A. Patchett, W. J. Greenleet, J.Med. Chem. 1993, 36, 4230.
[8] J. Kaur, A. Bhardwaj, Z. Huang, E. E. Knaus, Bioorg. Med. Chem. Lett.2012, 22, 2154.
[9] M. Kim, N. K.Mishra, J. Park, S. Han, Y. Shin, S. Sharma, Y. Lee, E. K. Lee,
J. H. Kwak, I. S. Kim, Chem. Commun. 2014, 50, 14249.
[10] B. E. Maki, K. A. Scheldt,Org. Lett. 2009, 11, 1651.
[11] Z. Song, R. Samanta, A. P. Antonchick,Org. Lett. 2013, 15, 5662.
[12] H. Kilic, S. Bayindir, E. Erdogan, C. S. Agopcan, F. A. S. Konuklar, S. K.
Bali, N. Saracoglu, V. Aviyente,New J. Chem. 2017, 41, 9674.
[13] S. Bayindir, A. Ayna, Y. Temel, M. Ciftci, Turk J. Chem. 2018, 42, 332.https://doi.org/10.3906/kim-1706-51.
[14] H. Kilic, S. Bayindir, N. Saracoglu, Curr. Org. Chem. 2014, 11, 167.
[15] S. Bayindir, N. Saracoglu, RSC Adv. 2016, 6, 72959.
[16] S. Bayindir, E. Erdogan, H. Kilic, N. Saracoglu, Synlett 2010, 10, 1455.
[17] H. Kilic, S. Bayindir, E. Erdogan, N. Saracoglu, Tetrahedron 2012, 68,5619.
[18] S. Bayindir, E. Erdogan, H. Kilic, O. Aydin, N. Saracoglu, J. HeterocyclicChem. 2015, 52, 1589.
[19] O. Aydin, H. Kilic, S. Bayindir, E. Erdogan, N. Saracoglu, J. HeterocyclicChem. 2016, 52, 1540.
[20] H. Kilic, O. Aydin, S. Bayindir, N. Saracoglu, J. Heterocyclic Chem. 2016,53 6, 2096.
[21] R.C. Stanton, IUBMB Life. 2012, 64, 362.
[22] H.A. Krebs, L.V. Eggleston, Advances in Enzyme Regulation. Oxford:
Pergamon Press Ltd. 1978, 12, 421.
[23] M.D. Cappellini, G. Fiorelli, Lancet. 2008, 371, 64.
[24] M.D.E. Beutler, Academic press. 1971, 68.
[25] C. Zhang, Z. Zhang, Y. Zhu, S. Qin, Anticancer Agents Med Chem. 2014,14, 280.
[26] J. Park, H.K. Rho, K.H. Kim, S.S. Choe, Y.S. Lee, J.B. Kim, Mol. Cell Biol.2004, 25, 5146.
[27] J. Park, S.S. Choe, A.H. Choi, K.H. Kim, M.J. Yoon, T. Suganami, Y.
Ogawa, J.B. Kim,Diabetes 2006, 55, 2939.
[28] S.A. Gupte, Curr. Opin. Investig. Drugs. 2008, 9, 993.
[29] S. Jalal, S. Sarkar, K. Bera, S. Maiti, U. Jana, Eur. J. Org. Chem. 2013, 22,4823.
[30] Q. Han, S. Fu, X. Zhang, S. Lin, Q. Huang, Tetrahedron Lett. 2016, 57,4165.
[31] M. Terashima,M. Fujioka,Heterocycles 1982, 19, 91.
[32] Y. Temel, U.M. Kocyigit, J. BiochemMol Toxicol. 2017, 31, 21927.
BAYINDIR ET AL. 9 of 9
[33] L. A. Kelley, S. Mezulis, C.M. Yates, M.N. Wass, M.J. E. Sternberg, Nat.Protoc. 2015, 10, 845.
[34] W.N.S. Au, G. Sheila, M.S. Veronica, M. Lam, J. Adams, Structures 2000,8, 293.
[35] A.T. Ranzani, A.T. Cordeiro, FEBS Lett. 2017, 591, 1278.
[36] M.S. Cosgrove, C. Naylor, S. Paludan, M.J. Adams, H.R Levy, Biochem-istry 1998, 37, 2759.
[37] E.S. Cho, Y.H. Cha, H.S. Kim, N.H. Kim, J.I Yook, Biomol Ther. 2018, 26,29.
[38] A. Akıncıoglu, H. Akıncıoglu, I. Gülçin, S. Durdagı, C. T. Supuran, S.
Göksu, Bioorg. Med. Chem. 2015, 23, 3592.
[39] K. Aksu, M. Nar, M. Tanç, D. Vullo, I. Gülçin, S. Göksu, F. Tümer, C. T.
Supuran, Bioorg. Med. Chem. 2013, 21, 2925.
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
Recommended