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Article

Rational Design, Synthesis, Separation, and Characterization of New Spiroxindoles Combined with Benzimidazole Scaffold as an MDM2 Inhibitor

by
Saeed Alshahrani
1,
Abdullah Mohammed Al-Majid
1,
M. Ali
1,
Abdullah Saleh Alamary
1,
Marwa M. Abu-Serie
2,
Alexander Dömling
3,
Muhammad Shafiq
4,
Zaheer Ul-Haq
4 and
Assem Barakat
1,*
1
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
2
Medical Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications (SRTA-City), Alexandria 21934, Egypt
3
Department of Drug Design, Groningen Research Institute of Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands
4
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
*
Author to whom correspondence should be addressed.
Separations 2023, 10(4), 225; https://doi.org/10.3390/separations10040225
Submission received: 13 February 2023 / Revised: 9 March 2023 / Accepted: 16 March 2023 / Published: 24 March 2023
(This article belongs to the Special Issue Application of Separation and/or Purification Technology in Natural Products, Newly Synthetic Candidates, and Pharmaceuticals)
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Abstract

:
Rational design for a new spiroxindoles, combined with a benzimidazole scaffold to identify a new murine double minute two (MDM2) inhibitor was synthesized and characterized. The desired spiroxindoles were achieved via a [3+2] cycloaddition reaction approach which afforded the cycloadducts with four asymmetric centers separated in an excellent regioselective and diastereoselective compound. The separated spiroxindoles were subjected to a set of biochemical assays including an NCI cell panel assay, MTT assay, and MDM2 binding analysis by a microscale thermophoresis assay. The anticancer reactivity for the tested compounds showed IC50 (µM) in the range between 3.797–6.879 µM, and compound 7d with IC50 = 3.797 ± 0.205 µM was the most active candidate between the series. The results showed promising results that identified that compound 7a could be inhibited the MDM2 with KD = 2.38 μm. Compound 7a developed a network of interactions with the MDM2 receptor studied in silico by molecular docking.

1. Introduction

The MDM2–p53 protein–protein interaction inhibitor is a hot research topic and has been gaining a lot of attention recently [1,2,3,4]. The inhibition of the interaction between the two proteins, p53, and MDM2, leads to reactivation of the p53 which has many functionalities, including DNA repairing, apoptosis, cell cycle arrest, senescence, metabolic alteration, and tumor suppresser [5,6]. The mutant p53 protein has been found in approximately 50% of human cancer cells [7,8]. The dislocation between the MDM2 protein and p53 protein is a challenge and is important to the development of a new chemotherapeutic agent.
Based on the literature survey, it has been reported so far that more than 20 chemotypes of molecules have been identified as MDM2–p53 inhibitors such as spirooxindoles [9], nutlins [10], isoquinoline-1-one [11], chalcone [12], pyrrolin-2-one [13], piperidine [14], morpholinone [15], imidazolyl indole [16], benzodiazpinedione [17], diketopiperazines [18], chromenotriazolopyrimidines [19], and other pharmacophores. For this, protein–protein interaction (PPI) inhibitors have progressed into clinical trials including spirooxindoles such as APG-115 [20], SAR405838 [21], and other pharmacophores such as RG7388 [22], HDM201 [23], RG7112 [24], and AMG-232 [25]. Inhibiting the p53–MDM2 interaction is a promising strategy for cancer treatment, as it can help to restore normal cell growth and death.
Protein–protein interaction inhibitors (PPIs) are typically small molecules that are designed to bind to the p53 and MDM2 proteins and prevent them from interacting. Several PPIs have been developed and are currently in clinical trials for a variety of cancers. Common side effects including fatigue, nausea, and anemia were observed.
In between the small molecules reported as promising lead compounds for cancer research are the spirooxindoles. This scaffold is able to activate the p53 and bind with the MDM2 domain [26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47]. Spirotryprostatin B is an inspired natural product that exhibits anticancer reactivity [33] (Figure 1). Gollner A. et al. reported a novel chemically stable spiro [3H-indole-3, 2′-pyrrolidin]-2 (1H)-one lead compound and orally active inhibitors of the MDM2–p53 interaction [34]. Benzimidazole scaffold was introduced to many compounds which showed high efficacy against MDM2, MDMX, and NF-kB inhibitors [40,41,42,43,44,45,46]. Our research group has engaged in this research program for a couple of years and has been successful in designing and developing several molecules towards PPI [35,36,37,38,39]. Among the discovered molecules, a new spiroxindole [48], as a rigid structure with a combination of benzimidazole scaffold, has been discovered as a novel MDM2 protein inhibitor with dual effects of antimetastatic efficacy. Based on these findings, we have rationally designed and synthesized a new spirooxindoles-based benzimidazole unit as an MDM2 inhibitor.

2. Materials and Methods

2.1. General

“All chemicals were purchased from Aldrich, Sigma-Aldrich and Fluka, which were used without further purification unless otherwise stated. All melting points were measured using a Gallenkamp melting point apparatus in open glass capillaries and were uncorrected. Crude products were purified by column chromatography on silica gel of 100–200 mesh. IR spectra were measured as KBr pellets using a Nicolet 6700 FT-IR spectrophotometer. The NMR spectra were recorded using a Varian Mercury Jeol-400 NMR spectrometer. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopy were performed in either deuterated dimethylsulfoxide (DMSO-d6) or deuterated chloroform (CDCl3). Chemical shifts (δ) are reported in terms of ppm and coupling constants J are given in Hz. Elemental analysis was carried out using an Elmer 2400 Elemental Analyzer in CHN mode”.

2.2. Synthesis of Spirooxindole Analogues (7a-o) General Procedure)

Chalcone derivative 4a-o (0.5 mmol), octahydroindole-2-carboxylic acid 6 (84.62 mg, 0.5 mmol), and 5-chlorisatin 5 (90.79 mg, 0.5 mmol) were mixed in 20 mL MeOH then, heated up at 60–65 °C for 2–3 h. After the reaction was completed, as monitored by TLC, the crude material was subjected to column chromatography using ethylacetate/n-hexane (2: 6), yielding spiro compounds in pure form.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(4-chlorophenyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo [1,2-a]indol]-2-one (7a).
Pale yellow solid; yield (80%); m.p.:176–178 °C; IR (KBr, cm−1): 3434 (NH), 3277 (NH), 3093 (CH), 2929 (CH),1729 (CO), 1682 (CO); 1H-NMR (DMSO-d6, 400 MHz): δ 12.98 (1H, s, NH), 10.17 (1H, s, NH), 7.73 (1H, s, Ph-H) 7.45 (2H, d, J = 8.0 Hz, Ph-H), 7.42–7.26 (6H, m, Ph-H), 7.08 (1H, d, J = 8.0 Hz, Ph-H), 6.44 (1H, d, J = 8.0 Hz, Ph-H), 5.29 (1H, d, J = 11.9 Hz, CHCO), 4.00 (2H, m, CHN, CHPh), 3.23 (1H, d, J = 3.7 Hz), 2.16–2.08 (1H, m), 2.06–1.98 (1H, m), 1.55–0.70 (10H, m, aliphatic CH); Anal. for C32H28Cl2N4O2; calcd: C, 67.25; H, 4.94; N, 9.80 Exper.: C, 66.89; H, 5.03; N, 10.04.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(4-(trifluoromethyl)phenyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2-a]indol]-2-one (7b).
Pale yellow solid; yield (32%); m.p.:125–127 °C; IR (KBr, cm−1): 3429 (NH), 3282 (NH), 3099 (CH), 2927 (CH),1724 (CO), 1686 (CO); 1H-NMR (DMSO-d6, 400 MHz): δ 12.98 (1H, s, NH), 10.20 (1H, s, NH), 7.75 (1H, d, J = 8 Hz, Ph-H), 7.68 (4H, m, Ph-H), 7.48–7.20 (4H, m, Ph-H), 7.09 (1H, d, J = 8.8 Hz, Ph-H), 6.43 (1H, d, J = 8.3 Hz, Ph-H), 5.34 (1H, d, J = 11.7 Hz, CHCO), 4.11 (1H, m, CHN), 3.46 (1H, m, CHPh), 2.10 (2 H, d, J = 12.9Hz), 1.58–0.67 (10 H, m, aliphatic C-H); Anal. for C33H28ClF3N4O2; calcd: C, 65.51; H, 4.66; N, 9.26 Exper.: C, 66.09; H, 4.77; N, 9.14.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(p-tolyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2-a]indol]-2-one (7c).
Yellow solid; yield (41%); m.p.:163–165 °C; IR (KBr, cm−1): 3649 (NH), 3277 (NH), 3087(CH), 2926 (CH),1727 (CO), 1690 (CO); 1H-NMR (DMSO-d6, 400 MHz): δ 12.98 (1H, s, NH), 10.18 (1H, s, NH), 7.76 (1H, d, J = 8.0 Hz, Ph-H), 7.40 (1H, d, J = 2.2 Hz, Ph-H), 7.35–7.26 (5H, m, Ph-H), 7.10–7.06 (3H, d, J = 8.0 Hz, Ph-H), 6.44 (1H, d, J = 8 Hz, Ph-H), 5.34 (1H, d, J = 12.4 Hz, CHCO), 4.11–4.00 (1H, m, CHN), 3.96–3.86 (1H, m, CHPh), 2.20 (3H, s, CH3), 2.11 (2 H, d, J = 8.0Hz), 1.56–0.72 (10H, m, aliphatic C-H); 13C-NMR (DMSO-d6, 100 MHz): δ = 189.91, 180.05, 148.02, 143.07, 141.62, 136.60, 136.47, 135.18, 129.69, 127.85, 126.33, 125.17, 123.63, 111.02, 71.74, 63.94, 57.24, 52.79, 36.80, 28.19, 25.07; Anal. for C33H31ClN4O2; calcd: C, 71.92; H, 5.67; N, 10.17 Exper.: C, 71.59; H, 5.71; N, 10.44.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(thiophen-2-yl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2-a]indol]-2-one (7d).
Pale yellow solid; yield (48%); m.p.:130–132 °C; IR (KBr, cm−1): 3624 (NH), 3258 (NH), 3091(CH), 2927 (CH),1728 (CO), 1689 (CO); 1H-NMR (CDCl3, 400 MHz): δ 10.18 (1H, s, NH), 8.64 (1H, s, NH), 7.84 (1H, d, J = 8.0 Hz, Ph-H), 7.34 (1H, d, J = 8.0 Hz, thio-H), 7.31–7.20 (4H, m, Ph-H), 6.99 (1H, d, J = 3.5 Hz, thio-H), 6.95 (1H, dd, J = 8.2, 1.7 Hz, thio-H), 6.90–6.85 (1H, m, Ph-H), 6.39 (1H, d, J = 8.6 Hz, Ph-H), 5.30 (1H, d, J = 12.0 Hz, CHCO), 4.53–4.34(1H, m, CHN), 4.12 (1H, t, J = 11.0 Hz, CHPh), 3.20 (1H, d, J = 4.0 Hz), 2.16 (1H, d, J = 5.0Hz), 1.83–0.82 (10 H, m, aliphatic C-H); 13C-NMR (CDCl3, 100 MHz): δ = 189.65, 181.65, 146.83, 143.16, 141.99, 139.94, 133.80, 129.28, 126.77, 125.88, 123.80, 111.02, 72.12, 71.33, 65.76, 57.93, 48.77, 37.52, 28.38, 27.86, 19.70; Anal. for C30H27ClN4O2S; calcd: C, 66.35; H, 5.01; N, 10.32 Exper.: C, 66.49; H, 5.20; N, 10.14.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(4-fluorophenyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2-a]indol]-2-one (7e).
Pale yellow solid; yield (35%); m.p.:148–150 °C; IR (KBr, cm−1): 3431 (NH), 3268 (NH), 3096 (CH), 2960 (CH),1732 (CO), 1684 (CO); 1H-NMR (CDCl3, 400 MHz): δ 10.02 (1H, s, NH), 8.55 (1H, s, NH), 7.82 (1H, d, J = 8.3 Hz, Ph-H), 7.40 (2 H, dd, J = 8.5, 5.4 Hz), 7.35–7.12 (5H, m, Ph-H), 6.94 (3H, m, Ph-H), 6.36 (1H, d, J = 8 Hz, Ph-H), 5.32 (1H, d, J = 12.4 Hz, CHCO), 4.31 (1H, q, J = 7.4 Hz, CHN), 3.79 (1H, t, J = 12.4 Hz, CHPh), 3.19–0.87 (12 H, m, aliphatic C-H); 13C-NMR (CDCl3, 100 MHz): δ = 189.71, 181.92, 163.19, 160.76, 146.85, 143.08, 139.91, 134.65, 134.62, 133.69, 129.56, 129.49, 129.30, 127.35, 126.82, 126.64, 126.09, 123.68, 122.61, 115.63, 115.42, 112.08, 110.91, 72.01, 71.26, 65.28, 57.78, 53.18, 41.88, 37.45, 29.79, 28.43, 27.82, 24.79, 19.75; Anal. for C32H28ClFN4O2; calcd: C, 69.25; H, 5.08; N, 10.09 Exper.: C, 69.49; H, 5.18; N, 10.44.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(2,4-dichlorophenyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2-a]indol]-2-one (7f).
Pale yellow solid; yield (48%); m.p.:138–140 °C; IR (KBr, cm−1): 3436 (NH), 3281 (NH), 3090 (CH), 2925 (CH),1730 (CO), 1685 (CO); 1H-NMR (CDCl3, 400 MHz): δ 10.14 (1H, s, NH), 8.40 (1H, s, NH), 7.84 (1H, d, J = 8.0 Hz, Ph-H), 7.55 (1H, d, J = 8.7 Hz, Ph-H), 7.37 (1H, s, Ph-H), 7.35–7.14 (5H, m, Ph-H), 6.95 (1H, d, J = 8.0 Hz, Ph-H), 6.37 (1H, d, J = 8.0 Hz, Ph-H), 5.39 (1H, d, J = 12.3 Hz, CHCO), 4.47 (1H, t, J = 11.0 Hz, CHPh), 4.20 (1H, q, J = 8.5 Hz, CHN), 3.20 (1 H, d, J = 4.4 Hz), 2.14–0.89 (12 H, m, aliphatic C-H); 13C-NMR (CDCl3, 100 MHz): δ = 189.44, 181.66, 146.72, 143.03, 139.94, 135.42, 135.35, 133.76, 133.00, 129.56, 126.98, 125.98, 110.98, 71.87, 48.25, 27.66, 19.79; Anal. for C32H27Cl3N4O2; calcd: C, 63.43; H, 4.49; N, 9.25 Exper.: C, 63.59; H, 5.04; N, 9.04.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(3-hydroxyphenyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2-a]indol]-2-one (7g).
Pale yellow solid; yield (62%); m.p.:186–188 °C; IR (KBr, cm−1): 3626 (NH), 3257 (OH), 2931 (CH), 1726 (CO), 1688 (CO); 1H-NMR (CDCl3, 400 MHz): δ 9.00 (1H, s, NH), 8.25 (1H, s, NH), 7.54 (1H, d, J = 4.0 Hz, Ph-H), 7.46 (1H, s, OH), 7.22 (1H, d, J = 8.0 Hz, Ph-H), 7.08–7.04 (3H, m, Ph-H), 6.89–6.72 (5H, m, Ph-H), 6.23 (1H, d, J = 8.0 Hz, Ph-H), 5.31 (1H, d, J = 12.5 Hz, CHCO), 4.49–4.38 (1H, m, CHN), 3.75–3.65 (1H, m, CHPh), 3.14 (1H, d, J = 4 Hz), 2.16–0.81 (12 H, m, aliphatic C-H); Anal. for C32H29ClN4O3; calcd: C, 69.49; H, 5.29; N, 10.13 Exper.: C, 69.69; H, 5.14; N, 9.94.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(3,4,5-trimethoxyphenyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2-a]indol]-2-one (7h).
Pale yellow solid; yield (88%); m.p.:160–162 °C; IR (KBr, cm−1): 3430 (NH), 3269 (NH), 3093 (CH), 2996 (CH),1722 (CO), 1683 (CO); 1H-NMR (CDCl3, 400 MHz): δ 10.15 (1H, s, NH), 8.500 (1H, s, NH), 7.81 (1H, d, J = 8.0 Hz, Ph-H), 7.37–7.19 (4H, m, Ph-H), 6.97 (1H, d, J = 8.4 Hz, Ph-H), 6.66 (2H, s, Ph-H), 6.38 (1H, d, J = 8.5 Hz, Ph-H), 5.38 (1H, d, J = 12.3 Hz, CHCO), 4.33 (1H, m, CHPh), 3.78 (6H, s, OCH3), 3.73 (3H, s, OCH3), 3.19 (1H, m, CHN), 2.02–0.78 (12 H, m, aliphatic C-H); 13C-NMR (CDCl3, 100 MHz): δ = 189.75, 181.68, 153.30, 146.99, 143.14, 139.85, 136.95, 134.53, 133.80, 133.31, 126.78, 126.18, 123.39, 122.48, 110.86, 104.89, 72.25, 71.07, 60.86, 56.33, 56.18, 54.56, 41.84, 37.58, 28.44, 27.83, 24.76.; Anal. for C35H35ClN4O5; calcd: C, 67.03; H, 5.63; N, 8.93 Exper.: C, 67.59; H, 5.34; N, 9.05.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(2-hydroxyphenyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2-a]indol]-2-one (7i).
Yellow solid; yield (82%); m.p.:128–130 °C; IR (KBr, cm−1): 3311 (OH), 3063 (CH), 2925 (CH),1716 (CO), 1667 (CO); 1H-NMR (CDCl3, 400 MHz): δ 10.57 (1H, s, NH), 8.93 (1H, s, NH), 7.80 (1H, d, J = 8.0 Hz, Ph-H), 7.49 (1H, d, J = 8.0 Hz, Ph-H), 7.29 (2H, d, J = 7.3 Hz, Ph-H), 7.23 (1H, s, OH), 7.08 (2H, t, J = 7.7 Hz, Ph-H), 6.97 (2H, d, J = 8 Hz, Ph-H), 6.87 (2H, d, J = 8 Hz, Ph-H), 6.45 (1H, d, J = 8 Hz, Ph-H), 5.07 (1H, dd, J = 11.7, 6.6 Hz, CHCO), 4.54 (1H, q, J = 7.3 Hz, CHN), 4.43 (1H, t, J = 11.0 Hz, CHPh), 3.22 (1H, d, J = 4.4 Hz), 2.16–0.83 (12 H, m, aliphatic C-H); 13C-NMR (CDCl3, 100 MHz): δ = 191.67, 181.97, 154.84, 146.69, 142.54, 139.78, 133.34, 129.39, 128.16, 127.44, 126.93, 125.55, 125.42, 124.12, 122.07, 121.41, 118.44, 112.22, 111.31, 85.63, 85.25, 83.06, 73.14, 57.83, 46.34, 41.88, 40.99, 37.30, 28.55, 28.44, 27.76, 24.73, 23.97; Anal. for C32H29ClN4O3; calcd: C, 69.49; H, 5.29; N, 10.13 Exper.: C, 69.55; H, 5.16; N, 10.34.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(4-(dimethylamino)phenyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2-a]indol]-2-one (7j).
Orange solid; yield (45%); m.p.:140–142 °C; IR (KBr, cm−1): 3434 (NH), 3275 (NH), 3094 (CH), 2929 (CH),1729 (CO), 1682 (CO); 1H-NMR (CDCl3, 400 MHz): δ 9.81 (1H, s, NH), 8.01 (1H, s, NH), 7.78 (1H, d, J = 8.0 Hz, Ph-H), 7.31 (5H, m, Ph-H), 7.17 (1H, d, J = 2.2 Hz, Ph-H), 6.94 (1H, dd, J = 8.3, 2.3 Hz, Ph-H), 6.65 (2H, d, J = 8.8 Hz, Ph-H), 6.31 (1H, d, J = 8.6 Hz, Ph-H), 5.32 (1H, d, J = 12.4 Hz, CHCO), 4.38–4.27 (1H, m, CHN), 4.10 (1H, t, J = 7.3 Hz, CHPh), 3.19 (1H, d, J = 3.8 Hz), 2.87 (6H, s, NCH3), 2.07–0.83 (12 H, m, aliphatic C-H); 13C-NMR (CDCl3, 100 MHz): δ = 189.78, 181.66, 149.77, 147.05, 139.62, 133.54, 129.10, 128.85, 128.72, 127.56, 126.77, 126.36, 112.88, 110.59, 72.04, 71.13, 65.09, 57.80, 53.18, 40.70, 31.67, 28.50, 27.81, 24.84, 22.74, 19.86; Anal. for C34H34ClN5O2; calcd: C, 70.39; H, 5.91; N, 12.07 Exper.: C, 70.65; H, 6.06; N, 11.94.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-1’-(4-bromophenyl)-5-chloro-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2-a]indol]-2-one (7k).
Yellow solid; yield (72%); m.p.:159–161 °C; IR (KBr, cm−1): 3625 (NH), 3422 (NH), 3088 (CH), 2913 (CH),1725 (CO), 1682 (CO); 1H-NMR (CDCl3, 400 MHz): δ 10.08 (1H, s, NH), 8.46 (1H, s, NH), 7.80 (1H, d, J = 8.2 Hz, Ph-H), 7.36 (2H, d, J = 8.6 Hz, Ph-H), 7.31–7.16 (7H, m, Ph-H), 6.99–6.93 (1H, m, Ph-H), 6.34 (1H, d, J = 8.2 Hz, Ph-H), 5.30 (1H, d, J = 12.3 Hz, CHCO), 4.36–4.24 (1H, m, CHN), 3.75 (1H, t, J = 12.4 Hz, CHPh), 3.19 (1H, d, J = 3.9 Hz), 2.22–0.82 (12 H, m, aliphatic C-H); 13C-NMR (CDCl3, 100 MHz): δ = 189.58, 181.78, 146.77, 143.06, 139.92, 138.08, 133.72, 131.76, 129.87, 129.35, 127.32, 126.82, 126.65, 126.03, 122.61, 120.92, 112.11, 110.94, 71.97, 71.19, 65.20, 57.78, 53.43, 41.86, 37.41, 28.43, 27.81, 24.78, 19.75; Anal. for C32H28BrClN4O2; calcd: C, 62.40; H, 4.58; N, 9.10 Exper.: C, 62.60; H, 4.34; N, 9.03.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(3-fluorophenyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2- a]indol]-2-one (7l).
Pale yellow solid; yield (42%); m.p.:143–145 °C; IR (KBr, cm−1): 3625 (NH), 3422 (NH), 3088 (CH), 2913 (CH),1725 (CO), 1682 (CO); 1H-NMR (CDCl3, 400 MHz): δ 9.51 (1H, s, NH), 7.86 (1H, d, J = 8.0 Hz, Ph-H), 7.42 (1H, s, NH), 7.32 (2H, d, J = 4 Hz, Ph-H), 7.29–7.27 (2H, m, Ph-H), 7.23–7.13 (3H, m, Ph-H), 6.96 (1H, dd, J = 8.5, 2.2 Hz, Ph-H), 6.91–6.85 (1H, m, Ph-H), 6.32 (1H, d, J = 8.4 Hz, Ph-H), 5.31 (1H, d, J = 12.0 Hz, CHCO), 4.41–4.30 (1H, m, CHN), 3.79 (1H, t, J = 12.4 Hz, CHPh), 3.22 (1H, d, J = 4.3 Hz, CHPh), 2.17–0.88 (12 H, m, aliphatic C-H); Anal. for C32H28ClFN4O2; calcd: C, 69.25; H, 5.08; N, 10.09 Exper.: C, 69.40; H, 4.94; N, 10.13.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(furan-2-yl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2- a]indol]-2-one (7m).
Pale yellow solid; yield (62%); m.p.:165–167 °C; IR (KBr, cm−1): 3435 (NH), 3256 (NH), 3090 (CH), 2928 (CH),1729 (CO), 1687 (CO); 1H-NMR (CDCl3, 400 MHz): δ 10.31 (1H, s, NH), 8.77 (1H, s, NH), 7.83 (1H, d, J = 8 Hz, Ph-H), 7.36 (1H, d, J = 8 Hz, Ph-H), 7.28 (1H, t, J = 7.5 Ph-H), 7.22 (2H, d, J = 7.9 Hz, Ar), 7.11 (1H, s, Ph-H), 6.93 (1H, d, J = 8.7 Hz, Ph-H), 6.39 (1H, d, J = 8.7 Hz, Ph-H), 6.20 (1H, t, J = 1.6 Hz, fur-H), 6.12 (1H, d, J = 3.6 Hz, fur-H), 5.37 (1H, d, J = 12.3 Hz, CHCO), 4.39 (1H, q, J = 8.3 Hz, CHN), 3.97 (1H, t, J = 11.4 Hz, CHPh), 3.17 (1H, d, J = 4.4 Hz), 2.16–0.85 (12 H, m, aliphatic C-H); 13C-NMR (CDCl3, 100 MHz): δ = 189.68, 181.80, 153.04, 146.83, 143.19, 141.75, 141.71, 141.67, 139.98, 133.89, 129.32, 127.40, 126.74, 126.63, 125.97, 123.69, 122.69, 112.20, 111.05, 110.27, 106.01, 71.94, 68.30, 62.85, 57.68, 46.89, 41.94, 37.74, 28.39, 27.79, 24.80, 19.69; Anal. for C30H27ClN4O3; calcd: C, 68.37; H, 5.16; N, 10.63 Exper.: C, 68.60; H, 4.94; N, 10.33.
  • 2’-(1H-Benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(4-nitrophenyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2- a]indol]-2-one (7n).
Pale yellow solid; yield (46%); m.p.:165–167 °C; IR (KBr, cm−1): 3437 (NH), 3255 (NH), 3094 (CH), 2929 (CH),1728 (CO), 1686 (CO); 1H-NMR (CDCl3, 400 MHz): δ 9.81 (1H, s, NH), 8.12 (1H, s, NH), 8.12 (2H, d, J = 8.6 Hz, Ph-H), 7.80 (1H, d, J = 8.0 Hz, Ph-H), 7.63 (2H, d, J = 8.6 Hz, Ph-H), 7.33–7.23 (3H, m, Ph-H), 7.14 (1H, s, Ph-H), 6.97 (1H, dd, J = 8.3, 1.8 Hz, Ph-H), 6.36 (1H, d, J = 8.6 Hz, Ph-H), 5.35 (1H, d, J = 11.9 Hz, CHCO), 4.41–4.35 (1H, m, CHN), 3.90 (1H, t, J = 12 Hz, CHPh), 3.23 (1 H, d, J = 3.9 Hz), 2.03- 0.84 (12 H, m, aliphatic C-H); 13C-NMR (CDCl3, 100 MHz): δ = 189.08, 181.27, 147.16, 146.89, 146.55, 143.00, 139.84, 133.60, 129.53, 129.10, 129.04, 126.98, 126.86, 125.72, 123.92, 122.54, 112.06, 110.85, 71.90, 71.25, 65.46, 57.83, 53.73, 41.86, 37.38, 31.67, 28.38, 27.80, 24.73, 23.95; Anal. for C32H28ClN5O4; calcd: C, 66.03; H, 4.85; N, 12.03 Exper.: C, 66.10; H, 4.74; N, 12.11.
  • 2’-(1H-benzo[d]imidazole-2-carbonyl)-5-chloro-1’-(3-nitrophenyl)-1’,2’,4a’,5’,6’,7’,8’,8a’,9’,9a’-decahydrospiro[indoline-3,3’-pyrrolo[1,2- a]indol]-2-one (7o).
Pale yellow solid; yield (52%); m.p.:135–137 °C; IR (KBr, cm−1): 3438 (NH), 3257 (NH), 3092 (CH), 2930 (CH),1729 (CO), 1688 (CO); 1H-NMR (CDCl3, 400 MHz): δ 9.55 (1H, s, NH), 8.36 (1H, s, NH), 8.06 (1H, dd, J = 8.1, 2.3 Hz, Ph-H), 7.86 (2H, d, J = 8.2 Hz, Ph-H), 7.57 (1H, s, Ph-H), 7.47 (1H, t, J = 8 Hz, Ph-H), 7.31 (3H, d, J = 4.0 Hz, Ph-H), 7.14 (1H, d, J = 2.1 Hz, Ph-H), 6.98 (1H, dd, J = 8.3, 2.4 Hz, Ph-H), 6.33 (1H, d, J = 8.0 Hz, Ph-H), 5.31 (1H, d, J = 12.0 Hz, CHCO), 4.46–4.36 (1H, m, CHN), 3.92 (1H, t, J = 12.0 Hz, CHPh), 3.24 (1H, d, J = 4.3 Hz), 2.19–0.81 (12 H, m, aliphatic C-H); Anal. for C32H28ClN5O4; calcd: C, 66.03; H, 4.85; N, 12.03 Exper.: C, 66.09; H, 4.73; N, 12.10.

2.3. NCI Screening

The compounds have been processed according to the standard method NCI-60 Human Tumor Cell Lines Screen for the organic compound at the development therapeutic program (DTP) (see Supplementary Materials, Table S2; Figures S1 and S2).

2.4. Anticancer Activity Protocol

The anticancer activity protocol was carried out according to the method reported in [48]. “The cytotoxicity of tested compounds was investigated on a human normal lung fibroblast (Wi-38) cell line, triple-negative breast (MDA-MB 231) cells, and prostate cancer (PC3) cells. These cells were cultured in DMEM containing 10% fetal bovine serum. After cell seeding (10,000, 4000, and 5000 cells, respectively, per well) in 96-well cell culture plates and incubating for 24 h in a 5% CO2 incubator, serial dilutions (2,4,6,8, and 10 μM) of the tested compounds were added. Following 48 h in a 5% CO2 incubator, 20 µL of MTT (5 mg/mL) was added and incubated for 4 h, then this solution was removed and 100 µL of DMSO was added. The absorbance was measured at 590 nm (BMG LabTech, Ortenberg, Germany). The half-inhibitory growth concentration (IC50) was calculated by GraphPad Prism software” [48].

2.5. MDM2 Binding Analysis by Microscale Thermophoresis (MST) Assay

The full protocol has been provided in SI and the binding curve is shown in the Supplementary Material (Figures S3–S6).

2.6. Methodology for Molecular Docking

Two-dimensional structures of compounds 7a, 7g, 7h, and 7k were drawn via the builder and subjected to preliminary structure preparation, namely energy minimization with the force field MMFF94x and the subsequent application of partial charges. The X-Ray crystal structure of MDM2 with PDB ID: 5LAZ, having a cocrystallized ligand with structural similarity to the studied compounds, was retrieved from the RCSB Protein Data Bank for the docking studies. Structure preparation of the protein was brought about by energy minimization with force field Amber10:EHT and partial charges were then applied to the protein. The cocrystallized ligand (6ST), after the necessary structure preparations, was used as a reference compound for validation of the results in docking studies of the aforementioned compounds [34]. Benchmarking of the docking protocols was performed well before the docking studies. Redocking of the 6ST ligand was brought about to observe the deviation of the ligand conformation from the original one (SI Figure S7). All the operations were performed in the molecular operating environment (MOE 2019.01) [49], which was chosen based on the RMSD value (0.14 Å) between the coordinates of the cognate ligand and the simulated pose. Induced fit docking was directed to the ligand atoms, brought about by placement of the ligand into the binding site of MDM2 utilizing the triangle matcher algorithm followed by determining the scores of the generated fifteen conformations through the London dG scoring function. Finally, five top-scored conformations were retained and evaluated by the GBVI/WSA dG scoring function. Thereafter, the same protocols were followed for the docking simulation of the studied compounds. The interaction patterns of the ligands with binding site residues were analyzed by the Protein-Ligand Interaction Profiler [50].

2.7. Statistical Analysis

The data are expressed as mean ± standard error of the mean (SEM) and values were considered significantly different at p < 0.05, using one-way analysis of variance (ANOVA) and Tukey’s test (SPSS software version 16).

3. Results and Discussion

3.1. Chemistry

Based on the recently published idea of spiroxindole having the benzimidazole nucleus and showing promising results against cancer cell lines and an antimetastatic effect. To study the cytotoxicity and structure reactivity relationship, a new library 7a-o has been synthesized and characterized (Scheme 1). Different electronic effects on the aromatic ring, including electron-donating and electron-withdrawing effects, also achieved a heterocycle aromatic ring and were explored. The synthetic methodology was carried out based on a multicomponent one-pot reaction via the [3+2] cycloaddition reaction approach [48]. The desired dipolarphiles were synthesized from orthophenylene diamine in a consequential step. Mixing the chalcones 4a-o with the 5-chloroisatin, 5 and the key amino acid 6 in methanol under reflux for 2–3 h, afforded the final compounds in a high chemical yield and a regio- and diasetero-selective manner. The stereochemistry for the final cycloadduct is matched with the previous lead compound published by our research group [48].

3.2. In Vitro Anti-Cancer Activity Assays

3.2.1. NCI Screening (Development Therapeutic Program, DTP)

The successfully synthesized spirooxindoles (7a-o) were submitted for NCI for screening against 60 various cancer cell lines, classified into nine subpanels: breast, kidney, melanoma colon, prostate, CNS, ovary, melanoma, leukemia, and lung cancers. The initial single dose assay for the assessed spirooxindoles were tested at a 10 µM and the results were then expressed as a percentage of growth inhibition (GI%) (Table S2). As observed, the synthesized compounds inhibited the growth of the NCI cell-line panel according to the following order: breast > renal > leukemia cancer cell lines > other tested cancer cell lines (Table S2). The initial results afforded the most active compound, 7g, which entered the five dose assays, and the results are shown in Supporting Materials (Figures S1 and S2).

3.2.2. MTT Assay

In order to determine the IC50 (µM) of the synthesized spirooxindoles, 7a-o were subjected to an MTT assay in vitro against the two-cancer cell line MDA-MB 231 and PC3 cells, and the data reported in Table 1. For the breast cancer line (MDA-MB 231) the compounds are shown in IC50 (µM) in the range between 3.797–6.879 µM; the most active candidate between the series was compound 7d with IC50 = 3.797 ± 0.205 µM, the chemical structure compromises a thiophene ring. On the other hand, the least reactivity was compound 7n with IC50 = 6.879 ± 0.308 µM. All other compounds are shown in the range of 4 µM reactivity. In the case of prostate cancer (PC3), the reactivity of the synthesized compounds exhibited the range of IC50 = 4.252 to 7.567 µM. In the case of compound 7a with the Cl-atom in the fourth position showed IC50 = 4.763 ± 0.069 and 4.574 ± 0.011 µM, for both tested two-cancer cells MDA-MB 231, and PC3, respectively. The reactivity slightly improved compared with compound 7a when the CF3-group was introduced into the aromatic ring, as indicated in compound 7b which showed IC50 = 4.284 ± 0.007 and 4.404 ± 0.008 µM; 7e contain the p-fluoro atom on the aromatic ring compared with 7l having the m-fluoro atom on the aromatic, where no differences were observed in the cytotoxicity. In the isosteric analog of the compound 7d, which replaced the thiophene with furan heterocycle, as shown in compound 7m, the cytotoxicity dropped to IC50 = 6.039 ± 0.111 and 5.098 ± 0.119 µM with less than 1.6 and 1.18 times, compared to the compound 7d. Introducing the electron-withdrawing effect of the NO2 group either in the para- or meta-position, we observed that NO2 in the meta (compound 7o) was more active than the para-position (compound 7n) with 1.66 and 1.77 folds. The existence of electron donating groups such as methyl group (compound 7c); hydroxyl group (compound 7g or compound 7c); trimethoxy groups (compound 7h); and dimethyl amine (compound 7j) did not alter the reactivity.

3.2.3. Microscale Thermophoresis Assay (MST) for MDM2 Binding Detection

Compounds 7a, 7g, 7h, and 7k shown very good anti-cancer potential as well as high safety profiles as obtained by the MTT assay. Accordingly, the compound 7a, 7g, 7h, and 7k were then tested for MDM2 binding analysis. The tested compound was incubated with a fluorescently labeled MDM2 at increasing concentrations (0.763 nM to 25 μM; Solubility play a crucial role to reach maximum concentration). MST binding curves showed that spirooxindole-based benzimidazole 7a, 7g, and 7h showed moderate binding affinity in the range of (KD; 2.38–38 μm). Compound 7a showed better binding reactivity compared to our previous spirooxindole-based benzimidazole, which has been shown (KD; 7.94 μM) [48]. The 7k did not show any binding detection (Table 2).

3.3. Molecular Docking of the Studied Compound

Results of the docking simulations of the spirooxindoles molecules were validated relative to the reference compound (6ST), which was firmly held into the binding site by a diverse network of interactions. The nitrogen of the imidazole ring of His96 and oxygen in the carboxylic acid moiety of Leu54 accepted hydrogens from the N-H of indolinone and the N-H of pyrrolidine rings, resulting in hydrogen bonds with 1.99 Å and 2.16 Å lengths, respectively. Additionally, His96 had π-stacking and halogen bonding with the chloroindolinone moiety. There were formed salt bridges by the carboxylate group of this compound with His73 and Lys94. This binding was further enhanced by hydrophobic interactions with seven amino acid residues.
Compound 7a developed a network of interactions similar to that of the reference compound. A hydrogen bond was established with the ligand at a distance of 2.08 Å, where hydrogen was donated by the N-H of benzimidazole to Leu54. Furthermore, His96 formed π-stacking and halogen bonding with the chlorobenzene ring the same way it was created with the reference compound. Similarly, hydrophobic interactions formed by seven residues well accommodated this compound into the binding site. In contrast to 7a, compound 7g formed two hydrogen bonds. One of the hydrogen bonds between benzimidazole moiety and Val93 was of 3.02 Å length, and the other bond resulted between the amino group of Lys96, being the donor, and phenolic group of the ligand was accepting at 2.59 Å distance. His96 provided π-stacking with the aromatic rings of benzimidazole moiety. However, fewer hydrophobic interactions were observed for this compound as compared to 7a. In the case of 7h, only one hydrogen bond was formed, which was between the amino group of Lys94 and the central methoxy group of the trimethoxybenzene substituent in the compound. The benzimidazole of this compound was anchored by π-stacking, twice with His96 and once with Tyr100. Three hydrophobic interactions were also developed. For compound 7k, the observed interactions included a halogen bond between its bromobenzene substituent and His96, and hydrophobic interactions with six residues of the MDM2 binding site.
The docked poses of the studied compounds are presented in Figure 2, and Table 3 enlists all the observed interactions, the interacting groups, and docking scores.

4. Conclusions

New spiroxindoles, combined with a benzimidazole scaffold, were synthesized, characterized, and identified as an MDM2 inhibitor. The requisite spiroxindoles were successfully achieved via the [3+2] cycloaddition reaction approach, which separated in an excellent regioselective and diastereoselective manner. The separated spirooxindoles showed promising results against cancer cells including MDA-MB 231 and PC3 in microscale reactivity. The anticancer reactivity for compound 7d showed potential activity with IC50 = 3.797±0.205 µM and was recognized as the most active candidate in the series. MDM2 binding analysis showed that compound 7a could be inhibited by the MDM2 with KD = 2.38 μm. This finding could be of possible use for cancer research development in the future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/separations10040225/s1, Figure S1: one dose for compound 7g; Figure S2: Five doses for the compound 7g; Figure S3–S6: Binding curve for MST assay; Figure S7: validation of the docking protocol. Figure S8–S38: Selected NMR and IR spectrum; Table S1: Characterization of the chalcones 4a-o; Table S2: GI % at 10 μM concentration for compounds 7a-o; Table S3: The percentage of Wi-38 viability and the toxicity percentage of MDA-MB231, and PC3 cells after incubation with 5 µM of different tested compounds (7a-o).

Author Contributions

Conceptualization, A.B. and A.D.; methodology, S.A., M.A. and A.S.A.; software, M.S. and Z.U.-H.; validation, S.A., M.A. and A.S.A.; formal analysis, S.A., M.A. and A.S.A.; investigation, S.A., M.A. and A.S.A.; resources, A.B.; data curation, S.A. and Z.U.-H.; Biological activity assays: A.D. and M.M.A.-S.; writing—original draft preparation, A.B.; writing—review and editing, A.B.; visualization, A.M.A.-M.; supervision, A.M.A.-M.; project administration, M.A.; funding acquisition, A.B. All authors have read and agreed to the published version of the manuscript.

Funding

The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number (IFKSURG–2-361).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number (IFKSURG–2-361).

Conflicts of Interest

The authors declare no conflict of interest.

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Separations 10 00225 g001 550
Figure 1. Reported spirooxindoles and benzimidazoles with anticancer activity and our rationally designed compound 7a-o [34,36].
Figure 1. Reported spirooxindoles and benzimidazoles with anticancer activity and our rationally designed compound 7a-o [34,36].
Separations 10 00225 g001
Separations 10 00225 sch001 550
Scheme 1. Synthetic methodology for the desired spirooxindole derivative 7a-o.
Scheme 1. Synthetic methodology for the desired spirooxindole derivative 7a-o.
Separations 10 00225 sch001
Separations 10 00225 g002 550
Figure 2. Docked conformations of the ligands into the binding site of MDM2 (PDB ID: 5LAZ).
Figure 2. Docked conformations of the ligands into the binding site of MDM2 (PDB ID: 5LAZ).
Separations 10 00225 g002
Table
Table 1. The estimated IC50 (µM) of 7a-o on Wi-38 viability, the growth of MDA-MB 231, and PC3 cells.
Table 1. The estimated IC50 (µM) of 7a-o on Wi-38 viability, the growth of MDA-MB 231, and PC3 cells.
Chemical StructureWi-38MDA-MB 231PC3
IC50 (μM)
Separations 10 00225 i001
7a		5.178 ± 0.3334.763 ± 0.0694.574 ± 0.011
Separations 10 00225 i002
7b		5.157 ± 0.0394.284 ± 0.0074.404 ± 0.008
Separations 10 00225 i003
7c		4.466 ± 0.0884.221 ± 0.0704.504 ± 0.059
Separations 10 00225 i004
7d		4.325 ± 0.0623.797 ± 0.2054.314 ± 0.036
Separations 10 00225 i005
7e		4.024 ± 0.0114.143 ± 0.1644.252 ± 0.128
Separations 10 00225 i006
7f		5.084 ± 0.1524.274 ± 0.1674.388 ± 0.008
Separations 10 00225 i007
7g		5.120 ± 0.1154.329 ± 0.1634.294 ± 0.023
Separations 10 00225 i008
7h		4.938 ± 0.1614.261 ± 0.0954.241 ± 0.045
Separations 10 00225 i009
7i		4.854 ± 0.0094.371 ± 0.1744.495 ± 0.034
Separations 10 00225 i010
7j		4.267 ± 0.1534.261 ± 0.0794.420 ± 0.008
Separations 10 00225 i011
7k		4.316 ± 0.0074.479 ± 0.3134.539 ± 0.094
Separations 10 00225 i012
7l		4.401 ± 0.0924.156 ± 0.1354.360 ± 0.208
Separations 10 00225 i013
7m		4.698 ± 0.1296.039 ± 0.1115.098 ± 0.119
Separations 10 00225 i014
7n		3.959 ± 0.0466.879 ± 0.3087.567 ± 0.709
Separations 10 00225 i015
7o		5.187 ± 0.0894.138 ± 0.1864.275 ± 0.005
All values are presented as mean ± SEM.
Table
Table 2. MST binding assay results of MDM2.
Table 2. MST binding assay results of MDM2.
#CodeChemical StructuresKD (µM)
17aSeparations 10 00225 i0162.38
27gSeparations 10 00225 i01738
37hSeparations 10 00225 i01810.6
47kSeparations 10 00225 i019No binding detected
Table
Table 3. Docking scores and network of interactions formed between the ligands and residues of MDM2.
Table 3. Docking scores and network of interactions formed between the ligands and residues of MDM2.
Cpd.
ID		S (kcal/mol)Hydrogen BondingHydrophobic InteractionsOther Interactions
ResiduesLigand ResiduesDistance (Å)
7a−7.05Leu54N-H of benzmidazole2.08Leu54, Leu57, Ile61, Phe91, Val93, Ile99, Ile103π-stacking, Halogen bonding with His96
7g−7.09Val93N-H of benzmidazole3.02Leu54, Tyr67, Ile99, Tye100π-stacking with His96
Lys94O of phenol ring2.59
7h−7.43Lys94O of methoxy3.29Leu54, Ile61, Ile99π-stacking with His96, Tyr100
7k−7.29---Lys51, Leu54, Leu57, Ile61, Phe91, Ile99halogen bonding with His96
6ST−8.97Leu54N-H of indolinone1.99Leu54, Leu57, Ile61, Phe91, Val93, Lys94, Ile99π-stacking, Halogen bonding with His96; salt bridge with His73, Lys94
His96N-H of pyrrolidine2.16
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Alshahrani, S.; Al-Majid, A.M.; Ali, M.; Alamary, A.S.; Abu-Serie, M.M.; Dömling, A.; Shafiq, M.; Ul-Haq, Z.; Barakat, A. Rational Design, Synthesis, Separation, and Characterization of New Spiroxindoles Combined with Benzimidazole Scaffold as an MDM2 Inhibitor. Separations 2023, 10, 225. https://doi.org/10.3390/separations10040225

AMA Style

Alshahrani S, Al-Majid AM, Ali M, Alamary AS, Abu-Serie MM, Dömling A, Shafiq M, Ul-Haq Z, Barakat A. Rational Design, Synthesis, Separation, and Characterization of New Spiroxindoles Combined with Benzimidazole Scaffold as an MDM2 Inhibitor. Separations. 2023; 10(4):225. https://doi.org/10.3390/separations10040225

Chicago/Turabian Style

Alshahrani, Saeed, Abdullah Mohammed Al-Majid, M. Ali, Abdullah Saleh Alamary, Marwa M. Abu-Serie, Alexander Dömling, Muhammad Shafiq, Zaheer Ul-Haq, and Assem Barakat. 2023. "Rational Design, Synthesis, Separation, and Characterization of New Spiroxindoles Combined with Benzimidazole Scaffold as an MDM2 Inhibitor" Separations 10, no. 4: 225. https://doi.org/10.3390/separations10040225

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