Research and development of N,N′-diarylureas as anti-tumor agents

Abstract

Tumor neovascularization provides abundant nutrients for the occurrence and development of tumors, and is also an important factor in tumor invasion and metastasis, which has attracted extensive attention in anti-tumor therapy. Sorafenib is a clinically approved multi-targeted anti-tumor drug that targets vascular endothelial growth factor receptor (VEGFR) and inhibits the formation of tumor angiogenesis, thereby achieving the purpose of suppressing tumor growth. Since the approval of sorafenib, N,N′-diarylureas have received extensive attention as the key pharmacophore in its chemical structure. And a series of N,N′-diarylureas were designed and synthesized to screen a new generation of anti-tumor drug candidates through chemical modification and structural optimization. Moreover, the rational design of targeted drugs is beneficial to reduce toxic side effects and drug resistance and improve the curative effect. Here, this article reviews the research progress in the design, classification, structure–activity relationship (SAR) and biological activity of N,N′-diarylureas, in order to provide some prospective routes for the development of clinically effective anti-tumor drugs.

Tumor neovascularization provides abundant nutrients for the occurrence and development of tumors, and is also an important factor in tumor invasion and metastasis, which has attracted extensive attention in anti-tumor therapy.

1. Introduction

Tumor angiogenesis refers to the process of regenerating a large number of new blood vessels from the existing capillary network, which is an important pathological feature of tumor occurrence and development.¹ Previous reports indicate that rapid tumor growth and metastasis depend on angiogenesis to meet its demands for oxygen and nutrients.^2,3 Therefore, tumor angiogenesis is closely related to the growth of solid tumors. Most solid tumors generally do not exceed 1–2 mm³ in volume in the absence of angiogenesis.⁴ Subsequently, new endothelial cells provide tumor tissue growth-promoting factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), which stimulate the division and reproduction of vascular endothelial cells and form new vascular networks around the tumor.^5,6 These factors lead to the development, invasion and metastasis of malignant tumors. Tumor angiogenesis is heterogeneous. In addition to relying on endothelial cells to form vascular lumen on the basis of existing blood vessels, angiogenesis also includes new blood vessels formed from tumor stem cells, which leads to resistance to drugs targeting vascular endothelial cells. Hence, inhibiting tumor angiogenesis is an important target for anti-tumor therapy.

Angiogenesis is regulated by endogenous activators and inhibitors, and four families of receptor protein tyrosine kinases play key roles in new blood vessels.⁷ VEGF, vascular endothelial growth factor receptor (VEGFR), and fibroblast growth factor receptor (FGFR) are major regulators of tumor angiogenesis, among which VEGF secreted by tumor cells and surrounding stroma induces a non-mitotic response of endothelial cells, stimulates endothelial cell proliferation and survival, and induces the formation of new blood vessels.^8–10 VEGFR, a key intermediate in tumor angiogenesis and new blood vessels, provides nutrients and oxygen for tumor growth.¹¹ Moreover, VEGF/VEGFR signaling also activates many signaling pathways and physiological effects. FGFRs are a group of transmembrane receptor tyrosine kinases (RTKs) that can be activated by extracellular stimuli.^12,13 FGFRs bind to FGF ligands and are involved in cell proliferation, differentiation, and growth, as well as migration and selective apoptosis during embryogenesis and angiogenesis.¹⁴

Sorafenib, an oral multikinase inhibitor, was approved by the U.S. Food and Drug Administration (FDA) in 2005 as the first targeted drug for the treatment of advanced renal cell carcinoma (RCC).^15–17 It inhibits tumor growth by acting on VEGFR to inhibit the formation of new blood vessels and cut off the nutrient supply of tumor cells, and can also directly inhibit the proliferation of tumor cells by blocking the RAF/MEK/ERK-mediated cell signaling pathway.¹⁸ Interestingly, the unique N,N′-diarylurea group in the chemical structure of sorafenib has become a hot spot in anti-tumor drug research (Fig. 1). In order to improve the problems of poor solubility, fast metabolism, and low bioavailability of sorafenib, a variety of derivatives based on N,N′-diarylurea groups were designed and synthesized, and compounds with excellent anti-tumor activity were screened out.¹⁹ For example, a novel and potent multi-targeted VEGF and PDGFR receptor family inhibitor, linifanib (Fig. 1), has demonstrated significant anti-tumor effects on liver cancer in preclinical studies and phase II clinical trials.^20,21 In addition, regorafenib (Fig. 1) inhibits tumor growth by inhibiting tumor angiogenesis, tumor cell regeneration, and factors that maintain the tumor microenvironment. It has been approved by FDA for the treatment of colorectal cancer, advanced gastrointestinal stromal tumor, and liver cancer that have stopped responding to sorafenib.²²

Fig. 1. Chemical structures of sorafenib, regorafenib, and linifanib.

Based on the excellent biological activity of N,N′-diarylureas, they serve as important pharmacophores for the development of more receptor tyrosine kinase inhibitors for screening targeted anti-tumor drugs.^23,24 We found that most of the N,N′-diarylureas are usually designed to be substituted with different aromatic or heterocyclic rings on one side, and connect different electron withdrawing or electron donating groups such as halogen, methyl, methoxy, etc. on the other side. In this review, the N,N′-diarylurea compounds with excellent anti-tumor activity were classified into pyrazole, pyrazine, pyrimidine and pyridine substitutions (Fig. 2) according to their structure, and the SARs and corresponding activities were discussed. Here, the purpose of this review is to provide the research progress of N,N′-diarylureas with excellent anti-tumor activities, in order to provide reference for the development of targeted anti-tumor drugs.

Fig. 2. Classification of N,N′-diarylureas and common substituents for the R group.

2. Pyrazoles

2.1. Pyrazole

Pyrazoles are a class of five-membered heterocyclic compounds that are particularly useful in organic synthesis. As a unique structural scaffold, the pyrazole nucleus has attracted much attention due to its unique pharmacological properties and templates for combinatorial and medicinal chemistry.²⁵ A series of derivatives with pyrazole groups have been reported to exhibit anti-tumor activity.²⁶ Oh et al. synthesized a series of pyrazole-containing diarylurea compounds from 3,5-dichlorobenzoic acid and substituted aryl isocyanates (Fig. 3A).²⁷ Among them, compound 1 in which R₂ was 3,5-ditrifluoromethyl and R₁ was a methyl group showed stronger anti-tumor activity than that of sorafenib against 58 tumor cells. And most IC₅₀ values were in the sub-micron molar level. The results of the evaluation showed that compound 1 selectively inhibited V600E-mutated B-RAF kinase and might kill tumor cells by inducing apoptosis. This compound is a promising candidate for further structural modification and optimization of this series of derivatives.

Fig. 3. Design of N,N′-diarylureas containing pyrazole (A), benzopyrazole (B and C) or N-phenylindazolyl (D).

Subsequently, they further investigated the effect of other derivatives on the V600E-B-RAF kinase.²⁸ Compound 2 in which R₂ was substituted with 4-chloro-3-trifluoromethyl and R₁ was hydrogen showed a significant kinase inhibitory effect with an IC₅₀ value of 7 nM. The molecular docking results showed that its binding to V600E-B-RAF kinase was based on hydrogen bonds established with Cys-532 and Glu-501 residues (Fig. 4). These suggest that this compound is a promising candidate for the future design and development of selective kinase inhibitors as potential anticancer drugs.

Fig. 4. The 2D structure of compound 2 (4A) and the binding mode of compound 2 at the V600E-B-RAF kinase site (4B). Green dotted line represents hydrogen bond interaction.

2.2. Benzopyrazole

Benzopyrazoles, also known as indolazoles or isoindolones, are a class of nitrogen-containing heterocycles related to natural indoles.²⁹ Nitrogen-containing heterocycles, especially the structurally diverse indolazole groups, have prompted a great deal of research due to their diverse biological activities, aiming at their synthetic routes and mechanisms of activity.^30–33 Due to the outstanding biological activities of benzopyrazole and diarylurea structures in drug development, many benzopyrazole-containing diarylureas have been designed and synthesized in recent years for the screening of anti-tumor drug candidates. Inspired by the chemical structure of sorafenib, Li et al. designed and synthesized a series of diarylureas containing indolazole or azaindazole groups (Fig. 3B).³⁴ Most of the compounds had better or similar anti-cell proliferative activity relative to sorafenib against non-small cell lung cancer cells NCI-H460 and A549, colon cancer cells HT-29 and Lovo with IC₅₀ values of 0.8 to 32 μM. These results provide guidance for the design of indolazole based diarylureas with anti-tumor effects.

Based on the previously designed compound 3, Shan et al. synthesized 38 diarylureas combined with aromatic heterocyclic structures as VEGFR-2 inhibitors by further optimization.³⁵ Pyridine, pyrimidine, indole, pyrazole and aminoindazole were used to replace the salicylic acid oxime moiety in compound 3. Substituents such as tertiary amines, halogens, ethanolamine esters, alkyl groups or methylenedioxy groups introduced on the terminal anilines increase the structural diversity. Unexpectedly, most of the aminoindazole substituted diarylurea derivatives had significantly reduced VEGFR-2 inhibitory activity relative to sorafenib. However, they still exhibited strong anti-tumor cell proliferation activity against A549 and SMMC-7721 cells, with IC₅₀ values ranging from 0.28 to 15.81 μM. Among them, compound 4 and compound 5 (Fig. 3C) exhibited VEGFR-2 inhibitory activity comparable to sorafenib (0.48 nM), with IC₅₀ values of 0.50 nM and 0.79 nM, respectively. It is worth mentioning that of the above compounds with other groups introduced, two pyridine-substituted compounds showed superior VEGFR-2 inhibitory activity to sorafenib. These derivatives all have the potential for further optimization and research.

2.3. N-Phenylindazolyl

Mereddy et al. synthesized and evaluated N,N′-diarylureas from methyl 1-H-indazole-3-carboxylate as potential anti-tumor candidates (Fig. 3D).³⁶ Compounds substituted with amido-amines on R₁ showed better solubility and stability than compounds with methyl and hydroxyl groups on R_1. The results of anti-proliferative activity in vitro showed that these derivatives exhibited good activity against several tumor cells with IC₅₀ values ranging from 0.4 to 50 μM. Among them, compound 7 showed excellent anti-proliferative activity against murine metastatic breast cancer cell 4T1, murine glioblastoma cell GL261, human triple-negative breast cancer cell MDA-MB-231, human pancreatic cancer cell MIAPaCa-2 and human colorectal cancer cell WiDr with IC₅₀ values of 6.8, 1.4, 1.7, 9.2 and 7.9 μM, respectively. In addition, compound 7 inhibited the tube length of human umbilical vein endothelial cells (HUVECs) and also showed strong tumor growth inhibitory activity in two invasive solid tumor models of WiDr cells and 4T1 cells.

3. Pyridines

3.1. Pyridine

In order to increase the diversity of aryl urea derivatives, Su et al. introduced pyridine, methylamine carbonyl pyridine and pivaloyl amide pyridine into the diaryl urea structure to obtain a series of novel diarylureas as VEGFR-2 inhibitors (Fig. 5A).³⁷ Most of the derivatives showed potent inhibitory effects on VEGFR-2, among which compounds 6 and 7 showed stronger activity with IC₅₀ values of 0.15 nM and 0.14 nM, which were higher than those of sorafenib (0.48 nM). In addition, SAR studies showed that the binding of 3-trifluoromethyl and methylamine carbonyl groups to terminal pyridine was beneficial to enhance the inhibitory effect of VEGFR-2. Molecular docking revealed two key hydrogen bonds between ureas and DFG residues of VEGFR-2, further suggesting the importance of ureas in inhibitor design.

Fig. 5. Design of N,N′-diarylureas containing pyridine (A and B), benzopyridine (C and D), thieno[3,2-b]pyridine (E), imidazo[1,2-a]pyridine (F) or isoxazol[3,4-b]pyridine (G). Chemical structure and anti-cell proliferation activity of compound 13 (H).

Subsequently, Wang et al. designed and synthesized a series of diarylurea derivatives containing N-pyridin-2-ylcyclopropane carboxamide based on a diarylurea antiangiogenic agent with salicylaldehyde keoxime structure (BPS-7) that they had previously discovered (Fig. 5B).³⁸ Among them, some of the derivatives showed inhibitory effects on all three pro-angiogenic RTKs, including VEGFR-2, TIE-2 and EphB4, while some of them also showed anti-proliferation activity on human vascular endothelial cells (EA.hy926). Compounds 8 and 9 showed the strongest activity against RTKs, and their IC₅₀ values against EA.hy926 cells were 16.11 μM and 14.54 μM, respectively. Furthermore, based on the results of the biological evaluation and molecular docking, N-pyridin-2-ylcyclopropane carboxamide is suitable for the development of multi-target antiangiogenic agents as a hinge-binding group.

3.2. Benzopyridine

As an important pharmacophore, quinoline has been widely explored in the study of anti-cell proliferation. Oh et al. previously reported a class of diaramide derivatives with anti-proliferation activity at single-digit micromolar IC₅₀ values on antiproliferation and also showing inhibitory activity against C-RAF kinase (Fig. 5C).³⁹ Based on the remarkable clinical therapeutic effect of drugs with diaryl urea structure, they synthesized a series of new diaryl urea compounds with a quinoline group by replacing diaryl amide with diaryl urea structure. The SAR study showed that the compounds with a 6,7-dimethoxy modified quinoline ring exhibited better activity than those with a 6,7-dihydroxy modified quinoline structure. In addition, compounds with O linked between quinoline and diarylurea are better than those with other atoms as linkers. The end aryl group substituted by one or more electron withdrawing groups (such as –CF₃, –Cl) was conducive to the improvement of the biological activity of the compounds. For example, compound 10 with a 4-chloro-3-trifluoromethylphenyl terminal (Fig. 5C) has the highest activity for C-RAF kinase, with an IC₅₀ value of 0.1 μM. And it showed excellent anti proliferation activity on leukemia cell line RPMI-8226, non-small cell lung cancer cell line HOP-92 and colon cancer cell line COLO 205, with IC₅₀ values of 0.64 μM, 0.68 μM and 0.66 μM, respectively. Molecular docking studies showed that compound 10 formed three hydrogen bonds with Lys470, Asp486 and LYS431 residues of C-RAF kinase (Fig. 6), which was stronger than the binding ability of diarylamide derivatives.

Fig. 6. Best docked pose of compound 10 within the active site of C-RAF kinase. Hydrogen bond interactions are shown as green dashed lines (left) and its corresponding detailed interactions map (right).

Comprehensive analysis of protein–ligand interactions is also a potential strategy to find important binding sites. Liu et al. found that residues such as Asp1046, Ile1025, HIS1026, Cys919 and Lys868 are important sites for forming H bonds, while residues such as HIS1026, Asp1046, Glu885, Ile1025 and Leu840 also play a key role in non-bonding interactions. Then they designed and synthesized 10 new VEGFR-2 inhibitors with an N-methyl-4-oxo-N-propyl-1,4-dihydroquinoline-2-carboxamide scaffold using fragment-based drug design and structure-based virtual screening (Fig. 5D).⁴⁰ Although these compounds were tested to have lower inhibitory activity on VEGFR-2 than sorafenib, some of them showed moderate inhibitory activity on HepG-2 cells (33.65 μM) and HUVECs (19.54–57.98 μM).

3.3. Thieno[3,2-b]pyridine

Consistent with the above report, VEGFR-2 inhibitors mainly occupy adenine pockets with rings or ring systems, forming 1–3 hydrogen bonds in the hinge region of the kinase (Glu917–Cys919). It can also occupy hydrophobic pockets through the DFG-out conformation of Asp1046–Phe1047–Gly1048 in VEGFR-2. A pair of groups on the urea group that provide and receive hydrogen bonds can often form hydrogen bonds with Asp1046 and Glu885. Vera et al., based on their previous research on the thieno[3,2-d]pyrimidine-based VEGFR-2 inhibitor, found that the thioether linker (S-linker) can provide stronger inhibition than the oxyether linker (O-linker). Then they synthesized 1-aryl-3-[2-, 3- or 4-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas as VEGFR-2 inhibitors and evaluated their biological activity (Fig. 5E).⁴¹ Among them, the compounds with aryl urea in the middle of the thioether and hydrophobic groups (Me, F, CF₃ and Cl) on the end benzene ring showed significant inhibition on VEGFR-2 kinase, and the IC₅₀ value in the enzymatic test was 10–28 nM. Among them, compounds 11 and 12 were superior to sorafenib in inhibiting migration, cell proliferation and apoptosis of HUVECs in the concentration range of 0.1–5.0 μ M. This S-linker constitutes a new mode of substitution among known type II VEGFR-2 inhibitors, providing a new way to develop more active VEGFR-2 inhibitors. In addition, compound 11 showed strong anti-tumor activity against hormone-dependent (MCF-7) and non-hormone-dependent (MDA-MB-231) breast cancer cell lines with IC₅₀ values of 1.2 μM and 5.0 μM, respectively. They synthesized iron oxide nanoparticles with a particle size of about 12 nm by the co-precipitation method, and encapsulated them in liposomes with compound 11 for the development of magnetocaloric/chemotherapy combined therapy for breast cancer.⁴²

3.4. Imidazo[1,2-a]pyridine

Although sorafenib is considered to be the first non-selective B-Raf inhibitor for melanoma in vitro in clinical trials, its low activity in patients with advanced melanoma led to its failure to be approved. Diarylamides and diarylureas showed good anti-proliferative activity against melanoma. To further develop this class of derivatives, Garamvölgyi et al. synthesized diarylamides and diarylureas containing imidazole[1,2-a]pyridine scaffolds.⁴³ Compared with the derivatives of diarylamides, diarylureas (Fig. 5F) showed better anti-proliferation activity against human melanoma cell A375P, with IC₅₀ values lower than 5 μM in most cases. The introduction of a benzoyl group on the imidazole[1,2-a]pyridine substituent significantly improved the anti-proliferative activity, resulting in derivatives with lower IC₅₀ values.

3.5. Isoxazol[3,4-b]pyridine

The drug molecule with diaryl urea structure showed good activity against cell proliferation. In addition to clinical application of sorafenib, linifanib with this structure has also attracted considerable attention. In order to expand the structural diversity of linifanib and obtain new multi-target RTK inhibitors, Shi et al. designed and synthesized a series of diarylureas with 3-amino-isoxazol[3,4-b]pyridine structure (forming interaction with the ATP binding site) based on the chemical structure of linifanib, as multi-target inhibitors against receptor tyrosine kinase (RTK).⁴⁴ In addition, in the design of these compounds, methyl or aryl groups were substituted on isoxazol[3,4-b]pyridine, and different substituents were attached to the aryl urea termini, such as methyl, methoxyl, and trifluoromethyl. By SAR analysis, methyl-substituted isoxazol[3,4-b]pyridine compounds were generally more effective than phenyl-substituted compounds (Fig. 5G). Among the target compounds obtained, compound 13 was screened as the most potent inhibitor of several RTKs, including platelet-derived growth factor receptor (PDGFR-β), FMS-like tyrosine kinase 3 (FLT-3), and kinase insertion domain containing receptor (KDR) with IC₅₀ values of 4 nM, 3 nM and 8 nM, respectively. Compound 13 showed strong inhibitory activity against HUVECs, MCF-7 cells and MV4-11 cells with IC₅₀ values of 11.67 μM, 10.49 μM and 0.12 μM, respectively, which were lower than the positive control linifanib (Fig. 5H). Molecular docking results showed that compound 13 binds to the hydrophobic capsule and ATP-binding site of FLT3 by forming five hydrogen bonds with residues Cys 694, Glu 692, Asp 829 and Glu661. It is also combined with KDR by forming five hydrogen bonds, including two hydrogen bonds formed by nitrogen and oxygen atoms of the isoxazole ring with Cys 917, one hydrogen bond formed by the carbonyl group of the urea group, and two hydrogen bonds formed by two NH of the urea group with Glu 241. The two NH on the urea group of compound 13 formed two hydrogen bonds with Glu 241 on PDGFR-β. These results further indicate that isoxazole is a good pharmacophore, which plays an important role in binding to the ATP binding sites of FLT3 and KDR. In addition, the ureas play an important role in forming hydrogen bonds with various RTKs.

4. Pyrazine

Imidazo[1,2-a]pyrazine is a special heterocyclic ring, which is the core skeleton of many bioactive molecules and drug intermediates and exhibits a wide range of pharmacological properties.^45,46 Rita et al. designed derivatives containing imidazo[1,2-a]pyrazine in the process of exploring more effective anti-melanoma diarylamide and diarylurea compounds as mentioned above (Fig. 7).⁴³ The results showed that the derivatives containing imidazo[1,2-a]pyrazine exhibited superior anti-tumor activity in vitro to those containing imidazo[1,2-a]pyridine. Most of the derivatives showed IC₅₀ values lower than 5 μM against A375P melanoma cells. The IC₅₀ values of compounds 14, 15 and 16 for A375P were 0.06, 0.03 and 0.01 μM, respectively. In addition, these compounds showed excellent anti-proliferation activity against other cell lines, including A549, H358, PC9, PC9ER, HCT116 and HKE3. Therefore, SAR results showed that scaffolds containing imidazole[1,2-a]pyrazine substituents had better activity than scaffolds containing imidazole[1,2-a]pyridine, and there was no significant difference between amide and urea linkers. The introduction of benzoyl groups at site 8 of imidazole[1,2-a]pyrazine scaffolds was not required to enhance the anti-proliferative activity.

Fig. 7. Design of N,N′-diarylureas containing imidazo[1,2-a]pyridine or imidazo[1,2-a] pyrazine. Chemical structure and anti-cell proliferation activity of compounds 14, 15 and 16.

5. Pyrimidines

5.1. Benzopyrimidine

Benzopyrimidine, also known as quinazoline, is one of the most widely used structural scaffolds among natural or synthetic bioactive compounds.⁴⁷ These quinoxoline derivatives are important nitrogen-containing heterocycles with diverse biological activities, including anti-microbial and anti-tumor activities.^48–51

The epidermal growth factor receptor (EGFR), one of the protein tyrosine kinase receptors, plays a key role in the growth, differentiation and survival of a variety of cancer cells, including colon cancer, breast cancer, ovarian cancer, and head and neck cancer. Therefore, targeting EGFR and human epidermal growth factor receptor (HER2) has been widely studied and clinically validated in anti-tumor therapy. Among a large number of inhibitors developed, gefitinib, erlotinib and lapatinib have been approved for clinical treatment of cancer, and 4-aniline quinazoline as their common chemical group has attracted important attention. In order to explore novel EGFR inhibitors and anti-tumor drugs, Zuo et al. combined the important group aryl urea with 4-aniline quinazoline, and introduced a variety of tertiary amine groups to synthesize a series of derivatives (Fig. 8A).⁵² Tertiary amine groups, as water-soluble groups in drug development, play an important role in improving drug solubility and pharmacokinetic properties. These derivatives showed significant anti-proliferation activity against A431 and A549 cells. Compound 17 showed potent inhibitory activity against EGFR^wt and EGFR^L858R with IC₅₀ values of 4.1 nM and 11.5 nM. The molecular docking results showed that the urea and benzopyrimidine groups of compound 17 formed hydrogen bonds with residues Met769, Gln767, Thr830, Asp831 and Lys721 on EGFR^wt, and formed three hydrogen bonds with Met793 and Asp800 on EGFR^L858R. In a tumor-bearing mouse model established in A549 cells, compound 17 exhibited an anti-tumor effect in vivo comparable to that of the positive control gefitinib. The SAR showed that the activity of the compound decreased significantly when the R₂ portion was replaced by the amide bond form, while the tertiary amino portion attached to the benzyl group favored the antiproliferative activity. The trifluoromethyl of the R₁ group played an important role in enhancing the anti-proliferative activity of cancer cells. In addition, substitution of 2-(4-methylpiperazin-1-yl)ethylurea with phenylurea resulted in a complete loss of anti-proliferative activity, suggesting the importance of the activity of N,N′-diarylurea structure.

Fig. 8. Design of N,N′-diarylureas containing benzopyrimidines. Chemical structure of compounds 17 (A), 18 (B) and 19 (C).

Fibroblast growth factor receptors (FGFRs) are the major class of RTKs that constitute fibroblast growth factors (FGFs), including an extracellular ligand-binding domain and an intracellular tyrosine kinase domain. Overexpression of FGFR is closely related to the occurrence and development of tumor. For example, FGFR3, as an important member (FGFR1–4), participates in cell proliferation, differentiation and survival. In order to further explore effective FGFR3 inhibitors, Kim et al. designed and synthesized a series of novel diarylurea derivatives with an amino-quinoxaline skeleton (Fig. 8B).⁵³ By SAR analysis, compounds with electron-donating groups are generally found to be more efficient than those with electron-withdrawing groups (except for nitro groups). Among them, 13 compounds showed good efficacy against the RT112 bladder cancer cell line, with GI₅₀ values in the range of 1 micromole (GI₅₀ = 1.08–6.23 μM). Compounds of the aryl urea series were more active than compounds containing aryl amides. Compared with AZD4547 (Astrazeneca, phase II/III), a novel selective FGFR1, FGFR2, and FGFR3 tyrosine kinase inhibitor, compound 18 exhibited superior anti-proliferation activity with a GI₅₀ value of 0.0088 μM. However, these derivatives were less potent against FGFR3 than PD173074 (Pfizer), an important anti-tumor inhibitor that selectively inhibits FGFR1 and FGFR3. Nevertheless, diarylurea compounds containing quinazoline scaffolds have potential for further research and development in the treatment of bladder cancer.

Chen et al. synthesized a series of quinazolinyl-arylurea derivatives on the basis of structural modifications of the targeted anti-tumor drug sorafenib (Fig. 8C).⁵⁴ These compounds were found to be more sensitive to human bladder cancer cell T24 in the evaluation of anti-proliferative activity of six human cancer cells. Compound 19 displayed the most potent proliferative inhibitory activity against T24 cells with an IC₅₀ value of 3.97 ± 0.23 μM and favorable selectivity, which was obviously superior to that of sorafenib. After treatment of T27 cells with compound 19, it was found that the lower concentration of compound 19 within 8 h could lead to apoptosis-dependent cell death. At higher concentration and longer incubation time, ferroptosis and autophagy occurred through effective regulation of Sxc⁻/GPx4/ROS and PI3K/Akt/mTOR/ULK1 pathways. These forms of death were closely associated with compound 19-induced intracellular ROS production and decreased mitochondrial membrane potential. The molecular docking results showed that compound 19 could bind well to the active site of the corresponding receptor glutathione peroxidase 4 (GPx4). The compound also provides a promising drug candidate for the treatment of bladder cancer. Analysis of the SAR showed that ureyl was a key pharmacophore. One or more electron-absorbing groups on the terminal benzene ring, such as Cl-, CF₃-, were conducive to the improvement of activity. Moreover, the volume of the molecule played a crucial role in binding to the receptor GPx4 and influenced the anti-proliferative activity. For example, aryl urea derivatives with 3,4-dichlorobenzene rings showed suitable molecular size and good proliferation inhibition activity.

5.2. Oxazolo[5,4-d]pyrimidines

The oxazolidine ring is considered to be a 9-oxapurine analog of purine, where 2-phenyl oxazolo[5,4-d]pyrimidine has been reported to have adenosine kinase inhibitory activity.⁵⁵ Sun et al. combined oxazolidine–pyrimidine rings with diarylamides or diarylureas in the hope of developing effective vascular inhibitors (Fig. 9A).⁵⁶ These compounds showed inhibitory activity against HUVECs in vitro. The IC₅₀ value of compound 20 on HUVECs was 12.43 ± 0.52 μM, and that of sorafenib was 18.24 ± 1.27 μM. Compound 20 could effectively inhibit the migration of human umbilical vein endothelial cells and the formation of capillary-like tubes, and also showed a concentration dependent inhibition of capillary sprouting from the rat aorta rings. The inhibition rate of angiogenesis was 51.2% at 5 μM concentration. It inhibited protein kinase activation by decreasing phosphorylation of PI3K and ERK 1/2. Based on the theory that tumor growth depends on angiogenesis, these results suggested that these compounds could be developed as anti-tumor drugs targeting tumor vessels. SAR analysis showed that the diarylurea bond was more favorable to anti-proliferative activity than the amide bond. In the derivatives containing the amide bond, when the Cl atom of the R₁ group was replaced by the MeO group, the inhibitory activity of VEGF-HUVEC was almost completely lost. The introduction of hydrophilic side chains, such as methylpiperazine, was beneficial to enhance the inhibition activity of VEGF-HUVEC. Methyl substitution of the R₂ group reduced the antiproliferative activity of the compound, and the compound was even less effective after replacing the benzene ring with a thiazole ring, suggesting that the benzene ring may be necessary for receptor binding.

Fig. 9. Design of N,N′-diarylureas containing oxazolo[5,4-d]pyrimidine (A) or thiazolo[5,4-d]pyrimidine (B). Chemical structure of compounds 20 and 21.

5.3. Thiazolo[5,4-d]pyrimidines

Based on the excellent activity of oxazolo[5,4-d]pyrimidines against angiogenesis, Sun et al. envisioned replacing oxazolopyrimidine with thiazolopyrimidine in the hope of developing a more potent inhibitor of VEGFR-2 (Fig. 9B).⁵⁷ Among the 13 synthesized diarylurea derivatives containing thiazolo[5,4-d]pyrimidine, 12 derivatives showed stronger anti-HUVEC cell proliferation activity (IC₅₀ = 5.3–66.5 μM) than the positive control sorafenib (IC₅₀ = 73.16 μM). However, we also investigated the IC₅₀ values of two reported positive controls sorafenib against HUVEC cells, which were 18.24 ± 1.27 μM and 73.16 μM, respectively, and the large difference between them brought uncertainty to the determination of activity. SAR analysis showed that the R₂ group had a better inhibition effect as an electron-withdrawing substituent than as an electron-donating substituent. Unfortunately, they found that the substitution of thiazolopyrimidine for oxazolopyrimidine did not increase the inhibitory activity of the new compounds against VEGFR-2. Even so, diarylurea compound 21 with oxazolo[5,4-d]pyrimidine showed the best anti-proliferation activity against HUVECs with an IC₅₀ value of 5.3 μM. It has certain inhibitory activity on VEGF-stimulated cell migration, but it is not obvious enough. These results suggested that these compounds have the potential to inhibit tumor angiogenesis, but the specific mechanism and compound optimization still need to be explored.

6. Quinoxalinedione

Diarylurea derivatives showed a wide range of biological activities and clinical applications. Hassanzadeh et al. studied the SAR of sorafenib and its analogues by retaining the diarylureas' function as key pharmacophores and focusing their major modifications on the partial replacement of the pyridyl carboxamide group of sorafenib with a quinoxalindione moiety (Fig. 10).⁵⁸ The exploration could investigate the effect of the increase of the end rigidity of the skeleton on the bioactivity of the synthesized compounds. Most of the synthesized compounds showed comparable or better anti-proliferation activity than sorafenib against human breast cancer cell line MCF-7 and liver cancer cell line HepG-2. Six of the compounds had IC₅₀ values in the range of 10–18 μM for both tumor cells. SAR analysis showed that the anti-proliferation activity of the compound was lost when the R₁ group was OMe.

Fig. 10. Design of N,N′-diarylureas containing quinoxalinedione and the chemical structure of compound 22.

Furthermore, these novel diarylurea compounds were shown to bind to B-RAF kinase binding sites, and the binding energy range was −11.22–−12.69 kcal mol⁻¹, while the binding energy of sorafenib as the lead compound was −11.74 kcal mol⁻¹. Molecular dynamics simulation also showed that the binding of compound 22 at the B-RAF active site was stable. The IC₅₀ values of compound 22 on MCF-7 and HepG-2 cells were 14 ± 2.89 and 17 ± 3.63 μM, respectively. These results suggest that they may be potential B-RAF kinase inhibitors.

7. 1,3,4-Oxadiazole

In recent years, many studies have shown that compounds with 1,3,4-oxadiazole scaffold chemical groups show potential anti-proliferation activity, in addition to anti-inflammatory, anti-bacterial, anti-diabetes and other aspects of the activity.^59–63 Oh et al. designed and synthesized a series of diaramides and diarourea analogues containing 1,3,4-oxadiazole (Fig. 11).⁶⁴ The anti-proliferative activities of the compounds on 58 cell lines from 9 different cancer types were investigated in vitro, using sorafenib as a control. The results showed that the anti-proliferative activity of the diarylurea derivatives was superior to that of the amide derivatives, possibly due to the longer spacing of the diarylurea structure and the ability of the terminal NH group to form additional hydrogen bonds at the receptor site. Among them, compound 23 of diarylurea with a 4-chloro-3-(trifluoromethyl)phenyl terminal moiety showed about 100% inhibition rate on 58 cell lines at a concentration of 10 μM. Compounds 24, 25 and 26 also showed an average inhibition rate of more than 90% at 10 μM. The IC₅₀ values of compound 24 against the PC-3 prostate cancer cell line, HCT-116 colon cancer cell line and ACHN renal cancer cell line were 0.67, 0.80 and 0.87 μM, respectively, which were comparable to sorafenib. These results suggest that diarylurea compounds with a 1,3,4-oxadiazole scaffold are promising candidates for the development of anti-tumor drugs.

Fig. 11. Design of N,N′-diarylureas containing 1,3,4-oxadiazole and the chemical structure of compounds 22, 24, 25 and 26.

8. Biphenyl

The natural alkaloid taspine has been reported to inhibit angiogenesis by inhibiting VEGFR-2, and the biphenyl scaffold in its structure has attracted extensive attention. Further studies of biphenyl derivatives obtained through structural optimization revealed that these biphenyl derivatives significantly inhibited VEGFR-2, thereby reducing angiogenesis. SAR analysis also indicated that the biphenyl scaffolds played an important role in maintaining the bioactivity of the compounds. Ureyl is an important pharmacophore of type II VEGFR-2 inhibitors, which can form hydrogen bonds with the DFG domain of VEGFR-2. To further optimize the compound structure to find new potent VEGFR-2 inhibitors, Zhang et al. added urea units to biphenyl scaffolds to obtain a series of derivatives.⁶⁵ In addition, biphenyl has poor water solubility, and the introduction of tertiary amine in the terminal aryl group can improve the hydrophilicity of the compounds. The potential of these compounds to inhibit VEGFR-2 was investigated. Among them, compounds 27, 28 and 29 showed significant enzyme inhibition activity with IC₅₀ values of 4.06, 4.55 and 5.26 nM, respectively. Compound 27 exhibited significant anti-proliferation activity against human neuroblastoma cell SH-SY5Y, human colon cancer cell LOVO and human immortalised myelogenous leukemia line K562, with IC50 values of 10.4, 11.1 and 2.23 μM, respectively. SAR analysis showed that the introduction of –CH₃ in the ortho-position of the biphenyl urea and tertiary amine moiety could improve the inhibitory activity against VEGFR-2 and the anti-proliferation effect. As previously reported, the urea group in the synthesized compound was observed to form four hydrogen bonds with DFG residues by molecular docking methods. These results indicated that biphenylurea could be used as a lead compound with anti-tumor potential.

Based on the above activity, they further extended the structural diversity of biphenylurea to optimize the structure of biphenylurea to obtain better VEGFR-2 inhibitors and anti-tumor drug candidates.⁶⁶ The methoxy group of the A-ring was demethylated to a hydroxyl group, and the acetyl group was optimized to an oxime by reaction (Fig. 12A). These salicylaldoximes form a pseudo six-membered ring via hydrogen bonds, which can mimic quinazoline, a known competitive inhibitor of ATP. Moreover, intermolecular hydrogen bonds help to stabilize the planar conformation of molecules, so that molecules can obtain certain rigidity. The conformation of salicylaldoxime oxime and quinazoline is similar, and they are both presumed to act on the hinge region of VEGFR-2. Among these derivatives, compound 27 (Fig. 12B) showed excellent anti-cell proliferation activity against a variety of cells, and the inhibitory effect on A549, MDA-MB-435S and MDA-MB-231 cells was stronger than that of sunitinib. It also showed a significant inhibitory effect on VEGFR-2, with an IC₅₀ value of 5.3 nM. The molecular docking (Fig. 12C) results showed that the hydroxyl group of the oxime of compound 27 formed two hydrogen bonds with Cys917 with a distance of 2.05 Å and 1.97 Å, respectively, and the carbonyl group of urea formed hydrogen bonds with the skeleton NH of Asp 1044 with a distance of 2.21 Å. In addition, as speculated, the salicylaldoxime group was shown to bind to the hinge region of VEGFR-2 (Cys917), suggesting that this structural optimization strategy holds promise for the development of novel potent inhibitors of VEGFR-2.

Fig. 12. Design of N,N′-diarylureas containing biphenyl (A). The chemical structure (B) and molecular docking (C) of compound 27.

9. Benzoxazole or benzimidazole

Benzoxazole and benzimidazole are a class of molecules with a wide range of biological activities and are commonly found in the structures of pesticides, drugs and active natural products. Their derivatives have been studied for anti-tumor, anti-bacterial, anti-inflammatory, analgesic and other activities.^67–73

Previously, anti-tumor strategies to inhibit tumor neovascularization mainly focused on targeting VEGF and three receptors VEGFR-1, VEGFR-2 and VEGFR-3. Of these, VEGFR-2 has attracted the most attention, and its inhibitors are in various stages of clinical development. In addition, angiopoietin 1 (Ang1) is considered as a novel angiogenic factor. It is a ligand of the endothelial-specific receptor tyrosine kinase TIE-2 (tyrosine kinase containing the Ig and EGF homologous domains). TIE was found to be expressed only in vascular endothelial cells and early hematopoietic cells, and was used to identify a class of RTKs, suggesting that the development of its inhibitors may contribute to the inhibition of angiogenesis. Hasegawa et al. synthesized a series of benzimidazole-containing urea derivatives as inhibitors of VEGFR-2 and TIE-2 kinase receptors in order to inhibit tumor neovascularization.⁷⁴ Most of the derivatives showed significant inhibitory activity against VEGFR-2 and TIE-2, with the IC₅₀ values of compound 28 (Fig. 13A) for VEGFR-2 and TIE-2, respectively, being 3.5 and 6.9 nM. SAR studies have shown that the N1 and urea components of benzimidazole play a key role in the activity, and X-ray crystallography confirms their key hydrogen bond interactions with enzymes.

Fig. 13. Chemical structure of compound 28 (A). Design of N,N′-diarylureas containing benzoxazole or benzimidazole (B) and the chemical structure of compound 29 (C).

In addition, the 2-fluoro-5-(trifluoromethyl) phenyl portion of compound 28 was buried in the hydrophobic orifice pocket of VEGFR-2, and the benzimidazole ring occupied the adenine pocket and interacted with the Cys919 residue of the enzyme. The aryl urea binding group bent toward the hinge region of the enzyme. Zi et al. speculated that shortening the length of the urea linker might better match the active site.⁷⁵ 6-Substituted benzoxazole or benzimidazole derivatives have been developed as inhibitors of a variety of kinases, including tyrosine kinases and serine/threonine kinases. In order to develop better anti-angiogenesis inhibitors, they synthesized a series of derivatives with 6-arylurea-2-arylbenzoxazole and 6-arylurea-2-arylbenzimidazole (Fig. 13B). SAR analysis showed that the electron-absorbing groups in aryl urea increased the kinase inhibitory activity, especially the introduction of larger electron-absorbing groups in the opposition was beneficial to improve the kinase inhibitory activity. Compound 29 (Fig. 13C) showed the strongest antiangiogenic effect by a chicken chorionic allantoic method. The IC₅₀ values of HUVEC, H1975, A549 and Hela cells were 8.46, 1.40, 7.61 and 0.28 μM, respectively. Moreover, compound 29 also showed an inhibitory effect on VEGFR-2 kinase with an IC₅₀ value of 0.25 μM. Overall, these derivatives have potential anti-angiogenic properties.

10. N,N′-Diarylureas

A series of fluorinated N,N′-diarylureas were synthesized as adenosine monophospho-activated kinase (AMPK) activators, which play a central role in regulating energy homeostasis and cell maintenance and cell cycle progression.⁷⁶ The results showed that they could activate AMPK at 1–3 μM, which is much lower than other known AMPK activators, such as metformin. And these new derivatives could strongly inhibit the proliferation of colon cancer cells. Subsequently, they selected eight fluorinated N,N′-diarylureas and investigated their effects on colorectal cancer metastasis and stem cell lines.⁷⁷ All fluorinated N,N-diarylureas at micromolar levels significantly induced cell-cycle inhibition and apoptosis in metastatic colorectal cancer cells and cancer stem cells. Notably, compound 30 (Fig. 14) significantly inhibited the viability of metastatic colorectal cancer cells in a dose-dependent manner. They then continued to investigate to determine the effects of compound 30 alone or in combination with PI-103 (PI3K/mTOR inhibitor) or SN-38 (active metabolite of irinotecan) on cell cycle arrest and apoptosis in colorectal cancer cell lines.⁷⁸ The results showed that phosphorylated AMPK expression was significantly increased in colorectal cancer cells treated with compound 30, while cell proliferation markers such as cyclin D1 were decreased. In addition, the apoptosis induction and proliferation inhibition effects of compound 30 were also demonstrated. Binding of compound 30 to PI-103 resulted in increased cell death in all cell lines, while binding to SN-38 resulted in increased cell death in some cell lines.

Fig. 14. Chemical structure of compound 30 and compound 31.

Based on the isotope effect, a deuterium-substituted drug structure can improve the pharmacokinetic characteristics of drugs. Zhong et al. reported the synthesis and bioactivity evaluation of deuterium-rich sorafenib derivatives.⁷⁹ The in vitro anti-tumor activity of these derivatives against HepG-2 and HeLa cells was comparable to that of sorafenib. The high lipid water distribution coefficient (log P) (1.66) and low clearance rate (CL) (0.58 L h⁻¹ kg⁻¹) of compound 31 (Fig. 14) allowed it to be more easily absorbed into the bloodstream and metabolized slowly in the body than other derivatives. This suggests that it has better druggability and is conducive to the development of new anti-tumor drugs.

11. Chemistry

The synthesis of N,N′-diarylureas often involves the acquisition of amino groups, except for the aminobenzene derivatives, through nitro-reduction is a common way (Table 1). In the process of nitro reduction, Pd/C/H₂ catalysis is the most common method, which can be used at room temperature for a short time. In addition, SnCl₂·2H₂O or iron power/NH₄Cl is also used to react under reflux conditions, and their catalytic efficiency is roughly equivalent to that of Pd/C/H₂.

Chemical reaction conditions for reduction of nitrobenzene to aminobenzene.

Entry	Cat.	Solvent	T (°C)/time	Yield (%)	Ref.
1	Pd/C, H2	EtOH	rt/3 h	87	53
2	Pd/C, H2	MeOH	rt/4 h	90	39
3	10% Pd/C, H2	THF	rt/2 h	87	27
4	5% Pd/C, H2	MeOH	rt/0.5 h	97.4	40
5	SnCl2·2H2O	EtOH	Reflux	89	44
6	Iron power, NH4Cl	MeOH/H2O	Reflux/1 h	93	52

Finally, the synthesis of N,N′-diarylureas with aminobenzene derivatives usually uses three compound reactions, including phenylisocyanate, aminobenzene derivatives and acyl azide (Table 2). Among them, the target product can be obtained by the reaction of aminobenzene derivatives with phenyl isocyanate under mild conditions and without the presence of catalysis. The highest average reaction efficiency was achieved in the presence of trimethylamine (Et₃N). The synthesis of the N,N′-diarylureas from two aminobenzene derivatives was conducted in the presence of Et₃N/triphosgene (BTC) at low temperature (0 °C) and in the low volatile solvent dichloromethane (DCM). And acylazide as a raw material can also undergo a reflux reaction with aminobenzene derivatives in toluene solvent without the presence of a catalyst to obtain the target compound. In general, the different substituents will affect the reaction efficiency to some extent, but there is no significant difference in the reaction efficiency of these three synthesis methods.

Chemical conditions for the reaction of aminobenzene derivatives with phenylisocyanate (a), aminobenzene derivatives (b) and acyl azide (c) respectively to produce N,N′-diarylureas.

Entry	Cat.	Solvent	T (°C)/time	Yield (%)	Ref.
1 (a)	—	THF	rt/3 h	19–62	53
2 (a)	—	THF	rt/12 h	24–85	27
3 (a)	—	THF	rt/1–2 h		36
4 (a)	—	DCM : THF (1 : 1)	rt/12 h	50–72	41
5 (a)	—	Anhydrous pyridine	rt/2 h	56–90	43
6 (a)		DCM	rt		40
7 (a)	K2CO3	THF	rt/overnight	40–78	39
8 (a)	Et3N	EtOH	25 °C/2 h	73–88	44
9 (a)	—	THF	rt/24 h	27–46	64
10 (a)	—	Toluene	Reflux/1–4 h	85–91	75
11 (a)	—	DCM	0 °C	36–87	79
12 (b)	BTC, Et3N	DCM	Ice-bath	∼40	35
13 (b)	BTC, Et3N	DCM	rt	73	37
14 (b)	BTC, Et3N	DCM	0 °C to rt	∼13	38
15 (b)	DCI	MeCN	rt/8 h to reflux/4 h	20–90	52
16 (b)	i) BTC, Et3N, ii) Et3N	1,2-Dichloroethane	i) 0 °C/5 min to rt/0.5 h to 85 °C/8 h, ii) rt	17–82	54
17 (b)	BTC, Et3N	DCM	rt	30–46	65
18 (b)	BTC, Et3N	DCM	rt	35	66
19 (c)	—	Toluene	Reflux/4 h	20–68	52

In addition, the benzene ring substituted group of diarylurea derivatives is often synthesized on nitrobenzene or aminobenzene and then the final product is obtained by the reaction of the above two steps. For example, halogenated nitrobenzene reacted under the condition of basic catalysis to obtain nitrobenzene derivatives containing substituents (Fig. 15A), p-aminobenzoborate reacted with bromo-benzene derivatives to obtain aniline-containing derivatives (key intermediates of compounds 1 and 2), and compounds 6 and 7 were obtained by reacting with substituted anilines (Fig. 15B). In addition, it can also synthesize the structure of diarylurea after protecting the active group, and then react with the active group to get the final product. For example, the synthesis of compound 4 was conducted to obtain the structure of diarylurea on the basis of protecting the boric acid group of aminobenzoborate, and then it reacted with the protective boric acid group (Fig. 15C).

Fig. 15. (A) Synthesis of nitrobenzene derivatives as key intermediates of compounds 1 and 2. (B) The synthesis of aminobenzene derivatives and compounds 6 and 7. (C) Protection of the boric acid group of aminobenzoborate and synthesis of compound 4.

Some new cyclic urea compound synthesis methods have been reported which may provide additional approaches for the synthesis of structurally diverse diarylurea derivatives. For example, the [4 + 2] cyclic addition between α and β-unsaturated imines and isocyanates, similar to the reaction forms in Table 1, produces chiral diarylureas at high yields (Fig. 16A),⁸⁰ which facilitate the preparation of chiral diarylurea derivatives with diverse structures. Optically active cyclic ureas can also be obtained by palladium-catalyzed asymmetric hydrogenation of pyrimidines containing tautomeric hydroxyl groups (Fig. 16B).⁸¹ In addition, mild activation of carbamyl azide to the corresponding nitro group using a blue light/[Ir]-catalytic strategy resulted in obtaining chemically robust N-trifluoromethyl-containing cyclic urea compounds and further derivations of subsequent diverse compounds (Fig. 16C).⁸² The establishment of new chemical synthesis methodologies is expected to expand the fabrication of more active drug molecules with novel and interesting structures.

Fig. 16. Synthesis method of cyclic urea compounds. (A) Cycloaddition of α, β-unsaturated imines and isocyanates; (B) asymmetric hydrogenation of 2-hydroxypyrimidines. (C) Synthesis of cyclic N-trifluoromethyl ureas through photocatalytic activation of carbamoyl azides.

12. Discussion and future aspects

Tumor angiogenesis is one of the key factors for rapid tumor metastasis and invasion. At present, tyrosine kinase inhibitors are the main inhibitors of tumor angiogenesis. However, few anti-tumor agents that inhibit tumor angiogenesis and development have been approved for clinical use in the last decade (Table 3). Compared with traditional chemotherapy drugs, anti-tumor drugs targeting tumor vasculature have the advantages of high selectivity and low side effects. Over the past decade, several anti-tumor agents that inhibit tumor angiogenesis and development have been approved for clinical practice (Table 3). In addition, a number of drug candidates are in various stages of clinical development.

Multi-target kinase inhibitors already on the market.

Name		Target	Development status	Ref.
Vandetanib	Medullary thyroid cancer	VEGFR-1/2	Approved in 2011	83
		EGFR, RET
Axitinib	Renal cell cancer	VEGFR-1/2/3, PDGFR-β, c-kit	Approved in 2012	84
Bosutinib	Chronic myelogenous leukemia	Bcr-Abl	Approved in 2012	85
Regorafenib	Hepatic carcinoma	VEGFR-1/2/3, PDGFR-β, RAF, FGFR-1, RET, c-kit	Approved in 2012	86
Ponatinib	Chronic myeloid leukemia	VEGFR-2/3, PDGFR-α, RET, Flt-3, c-kit	Approved in 2012	87
Cabozantinib	Medullary thyroid cancer and renal-cell carcinoma	VEGFR-2, Flt-3, c-kit, MET, RET	Approved in 2012	88
Nintedanib	Non-small cell lung cancer, colorectal cancer and ovarian cancer	VEGFR-1/2/3, PDGFR-α/β, FGFR-1	Launched in 2014	89, 90
Lenvatinib	Thyroid cancer	VEGFR-1/2/3, FGFR-1, RET, Kit, PDGFR	Launched in 2015	91
Apatinib	Metastatic gastric cancer	VEGFR-1/2, RET, ATM, PLK-1	Launched in 2014	92
Anlotinib	Non-small cell lung cancer	VEGFR-2/3, c-kit, PDGFR, EGFR	Launched in 2018	93, 94
Fruquintinib	Metastatic colorectal cancer	VEGFR-1/2/3, EGFR	Launched in 2018	95

Since the approval of sorafenib, the structure of N,N′-diarylurea as a key pharmacophore has received extensive attention. In recent years, anti-tumor compounds that inhibit tumor angiogenesis have been synthesized by introducing heterocyclic rings, such as oxazole, quinoline, indole or other modifications to the structure of N,N′-diarylurea. Most of the reported compounds showed significant inhibitory effects on tumor cell proliferation and target protein expression. However, with the development of research and clinical efficacy, it is difficult to completely kill tumor cells only by inhibiting tumor angiogenesis. The unique energy acquisition and immune escape of tumor cells lead to the uncontrolled development of tumor. The search for a new generation of multi-pathway anti-tumor drugs may be a potentially effective strategy. For example, the introduction of the key pharmacodynamic groups that can promote apoptosis, inhibit the cell cycle and inhibit glycolysis into N,N′-diarylurea may be beneficial to the development of novel anti-tumor compounds with multiple effects. In addition, drug combination is also an effective way to reduce the toxic side effects and the development of resistance of these drugs. In future studies, the solubility, biodistribution, in vivo pharmacokinetic properties and in vivo pharmacodynamics of compounds remain to be further improved.

Conflicts of interest

No potential conflicts of interest were disclosed.