Antineoplastic indole-containing compounds with potential VEGFR inhibitory properties

Cancer is one of the most significant health challenges worldwide. Various techniques, tools and therapeutics/materials have been developed in the last few decades for the treatment of cancer, together with great interest, funding and efforts from the scientific society. However, all the reported studies and efforts seem insufficient to combat the various types of cancer, especially the advanced ones. The overexpression of tyrosine kinases is associated with cancer proliferation and/or metastasis. VEGF, an important category of tyrosine kinases, and its receptors (VEGFR) are hyper-activated in different cancers. Accordingly, they are known as important factors in the angiogenesis of different tumors and are considered in the development of effective therapeutic approaches for controlling many types of cancer. In this case, targeted therapeutic approaches are preferable to the traditional non-selective approaches to minimize the side effects and drawbacks associated with treatment. Several indole-containing compounds have been identified as effective agents against VEGFR. Herein, we present a summary of the recent indolyl analogs reported within the last decade (2012–2023) with potential antineoplastic and VEGFR inhibitory properties. The most important drugs, natural products, synthesized potent compounds and promising hits/leads are highlighted. Indoles functionalized and conjugated with various heterocycles beside spiroindoles are also considered.


Introduction
Cancer is one of the most signicant health challenges worldwide.It is the second most fatal disease globally aer cardiovascular disorders. 1About 19-20 million people are diagnosed with different cancer types every year and many of them lose their life. 2Although many techniques, tools and therapeutics/ materials have been developed in the last few decades, none of them are effective in controlling the various types of cancer at different stages, especially the advanced ones. 3 To date, this challenge is still a signicant task despite the great interest, funding and efforts from the entire scientic society (including research institutes and pharmaceutical companies). 4Recently, progress has been achieved in cancer chemotherapy due to the efforts devoted to developing selective molecular therapeutics.Although traditional non-selective therapeutics are still employed clinically, their drawbacks/side effects and poor survival rates are major problems, limiting their applications.Additionally, the early detection of this disease is challenging, although it is the most appropriate opportunity for curing it. 5,6o date, 90 protein tyrosine kinases have been identied among the known 518 kinases. 7Tyrosine kinases are capable of many diverse cellular functions including growth, proliferation, differentiation and death. 8Tyrosine kinases can catalyze the phosphorylation of tyrosine utilizing the adenosine triphosphate (ATP) molecule, which can be classied into receptor and non-receptor types.The receptor category include transmembrane, extracellular and intracellular, whereas the non-receptors are intracellular. 9The overexpression of tyrosine kinases is associated with cancer proliferation and/or metastasis, indicating their importance as cancer chemotheraputics. 10,11ngiogenesis is an essential biological process for the formation/extension of new blood capillaries from the vessels present in the vascular system.Thus, it is an essential process for growth, menstruation, embryonic development, wound healing, functional repair and many pathological disorders including cancer.Furthermore, it is one of the main functions for delivering vital supplies including nutrients and oxygen to cells and removal of waste.Angiogenesis is also a critical process for tumor progression and metastasis.3][14][15] VEGF can be categorized into different classes (VEGF-A, B, C, and D), which can bind to diverse tyrosine kinase receptors (VEGFR-1, 2, and 3).Notably, VEGF and its receptors are hyper-activated in different cancers, and thus considered an important target for combating this disease.VEGFR-2 is the most well-known factor for angiogenesis of various solid tumors (colon, breast, ovary, lung, skin, renal, head, neck, lymphoma, etc.).Several drugs with inhibitory VEGFR-2 activity have been recognized and clinically approved as anticancer drugs. 12,13,16Anti-angiogenic active agents reduce the vascular permeability and enhance the extravasation of the therapeutic small molecules. 17However, some adverse effects have been reported to be associated with anti-VEGFR therapeutics including dermatologic disorders (skin rash, depigmentation and mucositis), painful hand-foot skin reactions and pruritus. 18ccordingly, multi-targeted inhibitory drugs/candidates have become a recent trend in cancer chemotherapy, attracting signicant attention and research interest.Interest in this strategy is attributed to the multifactorial nature of many cancer  (2005-2009).Subsequently, was granted two Postdoctoral Fellowships in Dundee (Scotland) and UT Health (Houston, Texas).His special interest is in the evaluation of compounds in various cell-based models such as cancer prevention, inammation inhibition and reversal of cancer multi-drug resistance.He also has experience in cancer cell biology and regulation of several members of the cytoprotective proteome using in vitro cell culture approaches, with experience in investigation the levels of expression of different mammalian proteins using western blotting.types. 7Additionally, carcinoma cell initiation and proliferation involve various receptors and signaling pathways.Moreover, multi-target inhibitors can overcome cancer cell resistance, which is an advantage for multi-targeted treatment compared to single-targeted treatment or cocktail of multi-component drugs. 7,19he indolyl scaffold occupies a unique position among the diverse alkaloids due to the wide range of bio-properties of its natural and synthesized analogs.Many natural alkaloids are well known, among which melatonin 1 is a natural hormone biosynthesized in the dark by the pineal gland 20 (Fig. 1).2][23][24] Serotonin 2 is a neurotransmitter that controls many human functions such as mood, appetite, sleep and social behavior. 25any indole-containing drugs have been approved and in clinical use for a long time, among which indomethacin 3 is a famous non-steroidal anti-inammatory drug (approved by the FDA "Food and Drug Administration" since 1965) 26 with inhibitory properties against cyclooxygenase (COX), an enzyme controlling the formation of prostaglandin from arachidonic acid. 27Delavirdine 4 (FDA approval in 1997) is an anti-HIV (human immunodeciency virus) drug. 28,29Umifenovir 5 (approved in Russia and China) is an anti-inuenza drug repurposed for the treatment of SARS-CoV-2. 30-33Pindolol 6 (antihypertensive drug, non-cardioselective b-blocker, FDA approval in 1982) 34 and Maxalt 7 (antimigraine agent, FDA approval in 1998) 35 are also indole analogs.Cipargamin 8 exhibits high efficacy against protein synthesis in Plasmodium falciparum.In addition, it has entered pre-clinical trial investigations as a potential antimalarial drug. 36he current study aims to describe and highlight the indolecontaining compounds with potential anti-VEGFR properties.Specically, the relevant keywords were input in different search engines such as Scopus, ScienceDirect and PubMed.The recent advances in this subject (last decade, 2023-2012) with the greatest diversication revealing promising bio-properties will be discussed.

Indole-containing drugs and potent agents
2.1.8][39] It is clinically approved against advanced renal and imatinibresistant gastrointestinal (FDA approval in 2006) and pancreatic cancers (FDA approval in 2011).It has also been given FDA approval (2017) for adult adjuvant treatment at high risk of Fig. 1 Clinically approved indole-containing drugs (1-7) and cipargamin (8).
renal cancer. 40,41However, due to the side effects/drawbacks (diarrhea, fatigue, hypertension, hematologic toxicities, cardiotoxic effects and hand-foot syndrome) 39,[42][43][44] observed during clinical administration, many studies proposed the combination of sunitinib with another chemotherapeutic agent or radiation.This approach can reduce the unintended side effects and enhance the efficacy of the drug. 42lso, sunitinib can be useful in the treatment of melanoma, which was supported by pre-clinical studies revealing the initiation of tumor hypoxia in melanoma xenogras. 16An obvious objective response by solid tumors to sunitinib including metastatic breast, colon, neuroendocrine and nonsmall cell lung cancers (NSCLC) was reported. 39,42,45synergistic effect was observed upon the combination of sunitinib with erlotinib 10 (EGFR: epidermal growth factor receptor, inhibitor) for the treatment of NSCLC A549 xenogra mice 46 (Fig. 2).Pre-clinical model studies supported that the combination of PRX177561 (CXC4, chemokine receptor type 4 antagonist) with sunitinib enhanced the therapeutic efficacy (reducing the tumor proliferation and extending the diseasefree survival) against glioblastoma (brain cancer).47,48 In vivo studies supported that a COX-2 (cyclooxygenase-2) inhibitor (celecoxib 11) can enhance the activity of sunitinib in mice bearing human renal cancer xenogras via the observation of delay in tumor progression.FGFR-1, -2, -3, -4: broblast growth-factor receptor) [50][51][52][53][54] (Fig. 3  and 4).The FDA approved its use for the treatment of idiopathic lung brosis (2014), systemic sclerosis-associated interstitial lung disease (2019) and NSCLC (in combination with docetaxel 13).50,[52][53][54][55][56] However, the most notable side effects are diarrhea and increase in alanine and aspartate aminotransferase associated with the clinical administration of nintedanib.54 Many studies explained the promising efficacy of nintedanib against different types of cancer.58 Its clinical trial (phase III) also support its promising properties against ovarian cancer.59,60 A compensatory role was reported for nintedanib towards metastatic colon cancer.61 Meanwhile, clinical trial observation (20 patients, 200 mg twice per day) revealed that there was no considerable effect on salivary gland cancer except in controlling the rate of the disease.62 A phase I clinical study (13 elderly patients) also showed its efficacy towards myeloid leukemic cells, especially when used with cytarabine 14.

Anlotinib
5][66][67][68][69] Hypertension and gastrointestinal problems are the most signicant side effects associated with its administration. 65inger print loss for about two months was observed during a case study on its treatment of lung cancer. 65It has also been reported that anlotinib is capable of inhibiting lymphangiogenesis and lymphatic metastasis, which is probably due to the suppression of VEGFR-3 phosphorylation. 70Antiproliferation properties were also observed against colon cancer cells (HCT-116 and LOVO). 71Moreover, anlotinib showed promising properties against thyroid and metastatic renal cell cancers. 68,72A phase II clinical study supported the possibility for the therapeutic utilization of anlotinib in combination with oxaliplatin 16 and capecitabine 17 to treat patients with metastatic colon cancer. 734.Surufatinib (HMPL-012, sulfatinib) Surufatinib (Sulanda) 18 (Fig. 6) is an oral multi-kinase inhibitor with dual function against angiogenesis (VEGFR-1, -2, -3 and FGFR) and tumor immune evasion (CSF-1R: colony stimulating factor-1 receptor).It is approved (in China, 2020) for the treatment of extrapancreatic neuroendocrine tumor (NET).74 However, it has been led with the FDA for approval for the treatment of advanced NET (2021).75 Hypertension and proteinuria are the most severe adverse effects reported with the administration of Sulanda.76 A phase II clinical study adopting 39 patients (300 mg, 28 day cycles, once daily) supported its possibility for moderate biliary tract cancer.77 2.5.SU5416 (semaxanib) SU5416 (semaxanib) 19 (Fig. 6) is a human solid tumor antiangiogenic VEGFR inhibitor under investigation.78-80 Its antiproliferation of murine cardiac endothelial cells was reported.80 Also, pathophysiological effects in respiratory disorders were mentioned, which was supported by the lipopolysaccharideinduced acute lung injury in mice via restrain/modulate vascular permeability.79

Vorolanib
Vorolanib (CM082) 20 (Fig. 6) is an oral VEGFR and PDGFR inhibitor. 81A phase I study (22 patients) exhibited promising results for the combination of vorolanib with everolimus 21 towards renal and neuroendocrine cancers. 82It has also mentioned that vorolanib enhances the antiproliferation and apoptosis properties of getinib (Iressa) 22 (EGFR: epidermal growth factor receptor, inhibitor) towards NSCLC cell lines (HCC827 and H3255).This was explained by the strong inhibitory properties of the combined therapeutics on STAT3 phosphorylation compared to that of the mono-therapeutic. 83

Toceranib
Toceranib (Palladia, Zoetis) 25 (Fig. 6) is a multi-targeted tyrosine kinase inhibitor (VEGFR-2, PDGFRs and c-Kit) used as a phosphate salt for the treatment of bone cancer (canine osteosarcoma) in veterinary medicine (dogs).Cell growth inhibition of toceranib phosphate on canine osteosarcoma (Penny and Wall) in vitro was reported. 86S49076 26 (Fig. 6) is a VEGF and HIF-1-a (hypoxia-inducible factor 1-alpha) inhibitor. 87The inhibition of VEGF and HIF-1 expression can explain the mode of action of S49076 in ovarian cancer cells. 87The administration of S49076 as a monotherapeutic resulted in the arrest of colon bevacizumab-resistant tumor growth.Moreover, the combination of S49076 and bevacizumab (Avastin, anti-vascular endothelial growth factor antibody) showed total growth inhibition of colon cancer xenogra models. 88A phase I study (103 patients) also revealed its effect on solid tumors (colon, lung, mesothelioma and uveal melanoma) upon oral administration. 89heme 13 Synthesis of indolyl hydrazones 113.

SIM010603
SIM010603 27 (Fig. 6) is an oral multi-targeted tyrosine kinase inhibitor (VEGFR-2, -3; PDGFR-b and stem cell factor receptor "ckit"). 90Antiproliferation properties were reported against NCI-H460 (human lung), LLC-SW44 (Lewis lung) and MDA-MB-435 (breast) cancer cells in addition to the inhibition of xenogra tumor growth models and angiogenesis in mice. 90Adverse effects including gastrointestinal, pancreatic and skeletal toxicities, bronchopneumonia and cardiovascular dysfunction were mentioned in the toxicological studies of SIM010603 in rats and dogs (0-20 and 0-10 mg kg −1 per day oral administration, respectively, for 28 followed by 14 recovery days).However, no mortality rates were recorded for dogs receiving 10 mg kg −1 . 91
A set of indolyl Schiff bases incorporated in urea 73 was synthesized through the condensation reaction of the appropriate indoles 54 with the corresponding 1-(4-aminophenyl)-3substituted urea 72.The latter was obtained through reduction (H 2 , Pd/C, and MeOH) of the corresponding nitro analogs, which were prepared through reaction of 4-nirophenylisocyanate 69 with the appropriate anilines 70 in reuxing acetonitrile 104 (Scheme 4).Some of the synthesized Schiff bases revealed promising antiproliferation properties (SRB "sulforhodamine B" technique) against the HepG2 (liver) cancer cell line relative to that of doxorubicin and sorafenib.The VEGFR-2 properties were determined for the discovered promising agents, which showed comparable observations to that of the antiproliferation efficacies.The efficacy observed for most of the synthesized agents followed order of phenyl substitution of 4-Cl > 3-Cl > 3-CF 3 .The most promising agent discovered was 73x (R = SO 2 NH 2 , R 0 = Cl; IC 50 = 3.15 ± 0.36 and 0.31 ± 0.04 mM for HepG2 cell line and VEGFR-2, respectively), which exhibited comparable activity to that of sorafenib (IC 50 = 3.40 ± 0.25, 0.10 ± 0.02 mM for the HepG2 cell line and VEGFR-2, respectively) 104 (ESI Fig. S4 †).
The condensation reaction of isatins 54 with L-phenylalanine secondary amine conjugates (obtained from the reaction of Boc amino acid with secondary amines in THF (tetrahydrofuran) in the presence of IBCF (iso-butyl chloroformate) and NMM (Nmethyl morpholine) at room temperature and inert atmosphere followed by removal of the Boc group (HCl gas in dioxane)) in EtOH containing triethylamine (TEA) at room temperature gave the corresponding Schiff bases 76 (Scheme 5).Additionally, the reaction of isatins 79/80 with 4-amino antipyrine 81 in ethanolic solution at room temperature afforded the corresponding Schiff bases 83 and 82, respectively.Isatin derivatives 80 were obtained through alkylation with excess dibromoalkane (DMF/ K 2 CO 3 ), which gave monoalkylated derivatives 78 (major products) and bis-isatin derivatives 79 (minor products).The monoalkylated isatins were coupled with secondary amines (DMF/ K 2 CO 3 at room temperature) affording the corresponding isatin derivatives 80 (Schemes 6-8).
Similarly, Schiff bases 88 were obtained through the condensation of 4-amino antipyrine 81 with isatin triazol conjugates 87 (EtOH, room temperature).Isatin triazol conjugates 87 were synthesized through the click reaction of the appropriate aryl azides 86 with N-propargylated isatins 85 (tbutanol/H 2 O, CuSO 5 $5H 2 O, sodium D-isoascorbate, microwave, 100 °C) 15 (Scheme 9).Some of the synthesized Schiff bases exhibited promising antiproliferation properties against MCF7 (breast), HCT116 (colon) and PaCa2 (pancreatic) cancer cell lines (MTT assay) compared to the reference standards (sunitinib and 5-uorouracil) (ESI Fig. S5 †).Compound 88f (R = Me, R 1 = H, and R 2 = OMe) was the highest potent analog observed against MCF7 (2.1times potency relative to the standard reference sunitinib).Additionally, some of the compounds prepared (88b, 88d and 88f) exhibited higher efficacies than that of the standard drug 5-urouracil (approved drug for colon cancer 105 ).The safe prole of all the tested analogs (IC 50 = >50.00mM) against the non-cancer RPE1 cell line is good support, especially for the high potent analogs towards more detailed studies for assigning promising hits.The CAM assay (chick chorioallantoic membrane) of fertilized chicken eggs in addition to VEGFR-2 inhibitory properties (% inhibition ± SD utilizing the IC 50 values observed against MCF7 cell line of the tested agents relative to that of sunitinib) (ESI Fig. S5 †) support their capability towards antiangiogenesis. 15

Indole heterocycle conjugates
Bio-conjugation is one of the most powerful and attractive rational drug design strategies used for the development of new drug candidates by connecting two or more therapeutic     with 2-aminobenzothiazoles 200 (in CH 2 Cl 2 containing Et 3 N at room temperature) (Scheme 28).The synthesized conjugates exhibited considerable inhibitory properties (Fig. 13) compared to that of sunitinib (% inhibition of VEGFR-2 at 10 mM = 98.1). 140

Indole-pyrimidine conjugates
Pazopanib 203 (Fig. 14) is an FDA approved VEGFR-2 inhibitor for the treatment of advanced renal cell cancer (2009) and so tissue sarcoma. 141,142In this molecule, the pyrimidinyl pharmacophoric heterocycle is attached to an indazole heterocycle.This inspired the design, construction and VEGFR-2 inhibitory properties investigation of indole pyrimidine conjugates linked through an ether linkage. 143A set of indole pyrimidine conjugates 211 was synthesized through the coupling of 5-hydroxyindole derivative 209 with 2,4-dicholoropyrimidine or 4,6dicholoropyrimidine in Me 2 CO containing aq.NaOH, followed by reaction with various primary amines (HCl "36%", i-PrOH, sealed tube, 100 °C) (Scheme 29).Some of the synthesized  ene).The reaction was assumed to take place via the basecatalyzed Michael addition of indole to the Knoevenagel adduct formed from the condensation of the salicaldehyde with 1,3cyclohexanone assisted by the basic catalysis of DBU 145 (Scheme 31).The synthesized conjugates 225 exhibited mild antitumor properties (MTT assay) against the PC-3 (prostate) and SKOV-3 (ovarian) cancer cell lines compared to that of doxorubicin (Fig. 15).Their VEGFR-2 inhibitory properties were reported based on the molecular modeling technique (PDB ID: 4ASD, AutoDock Tools 4.2). 145However, theoretical (molecular modeling) studies are insufficient supporting evidence for assigning the mode of action, and thus experimental data is required.

Conclusion
Tyrosine kinases are capable of many diverse cellular functions including growth, proliferation, differentiation and death.VEGFR is one of the targeted therapeutic approaches that is preferable to the classical non-selective therapies to minimize the associated side effects or drawbacks.VEGF is an important category of tyrosine kinases, which can stimulate angiogenesis.VEGFR-2 is the most well-known factor in the angiogenesis of different solid tumors (colon, breast, ovary, lung, skin, renal, head, neck, lymphoma, etc.).Indolyl therapeutics have been approved against some serious types of cancer.Additionally, research efforts identied natural and synthesized antitumor indole-containing compounds with promising anti-VEGFR properties.Computational/theoretical studies can assist in designing and identifying novel hits/leads of anti-VEGFR agents; however, without experimental supporting their enzymatic properties, the predictions cannot be considered for further investigations or utilization.

Abbreviations
Mohamed S: Bekheit Mohamed S. Bekheit, Associate Professor in the National Research Center, Egypt since 2018.He received his PhD from Ain-Shams University, Egypt (2013).His research studies focus on design and synthesis of heterocyclic compounds of potential biological properties, together with interest in organophosphorus chemistry with special interest in computational chemistry techniques.He has participated in several national and international research projects.Ahmed R: Hamed Ahmed Ragab Hamed, Professor of Therapeutic Biochemistry at the National Research Centre, Egypt.He was awarded his PhD in Biomedical Sciences from the University of Nottingham, UK
alkanesulfonate 118 was synthesized in a two-step reaction in excellent yields.The reaction of isatins 54 in EtOH containing a quantitative amount of Et 2 NH gave the corresponding 3hydroxy-2-oxoindole derivatives 117.Acidic dehydration (EtOH and HCl) of 117 gave the corresponding 118 92 (Scheme 14). 92

Fig. 14
Fig. 14 Pazopanib VEGFR-2 inhibitor FDA approved for treatment of advanced renal cell cancer and soft tissue sarcoma.
cell lung cancer NT Not tested PDGFR Platelet-derived growth-factor receptor SAR Structure-activity relationship