Recent advances of tryptanthrin and its derivatives as potential anticancer agents

Abstract

Tryptanthrin is one of the well-known natural alkaloids with a broad spectrum of biological activities and can act as anti-inflammatory, anticancer, antibacterial, antifungal, antiviral, antitubercular, and other agents. Owing to its potent anticancer activity, tryptanthrin has been widely explored for the therapy of various cancers besides being effective against other diseases. Tryptanthrin with a pharmacological indoloquinazoline moiety can not only be modified by different functional groups to achieve various tryptanthrin derivatives, which may realize the improvement of anticancer activity, but also bind with different metal ions to obtain varied tryptanthrin metal complexes as potential anticancer agents, due to their higher anticancer activities in comparison with tryptanthrin (or its derivatives) and cisplatin. This review outlines the recent advances in the syntheses, structures, and anticancer activities of tryptanthrin derivatives and their metal complexes, trying to reveal their structure–activity relationships and to provide a helpful way for medicinal chemists in the development of new and effective tryptanthrin-based anticancer agents.

Tryptanthrin with a broad spectrum of biological activities can be not only decorated by various functional groups, but also coordinated by bio-metal ions, generating varied tryptanthrin derivatives as potential anticancer agents with low toxicity.

1. Introduction

Cancer has become one of the main causes of human suffering and death worldwide, and its incidence will increase in the next decades based on the predictability of global demographic characteristics, with about 420 million new cancer cases likely occurring annually by 2025.^1–5 Consequently, the development of new and highly efficient anticancer agents is very necessary to protect human health. To date, a large variety of natural and synthetic anticancer agents have acquired remarkable success,^6–10 and the typical case is plant-derived natural alkaloids, such as paclitaxel,⁶ camptothecin,^7,8 and vinblastine,^9,10 with diverse pharmacological activities, which have been successfully applied as chemotherapy agents that are highly effective in treating various cancers, remarkably decreasing the mortality of cancer patients. However, natural alkaloids have also some drawbacks including resistance against drug molecules, toxicity, and low selectivity that cannot discriminate between cancer and normal cells, reducing the efficacy of clinically used anticancer agents.^6a,7a,10b,c Therefore, there is always an urgent need to develop new and highly efficient anticancer agents with minimum toxic side effects.

The natural tryptanthrin chemically named as indolo[2,1-b]quinazoline-6,12-dione is a weakly basic indoloquinazoline alkaloid derived from medicinal plants and contains a quinazoline ring (A and B rings) fused with an indole (C and D rings) moiety with two ketone (C O) groups in the 6- and 12-positions.^11,12 A review article described the preparation of tryptanthrin and some related compounds in 2012.¹¹ Subsequently, the study on tryptanthrin alkaloid was discussed in another review article in 2013, where various synthetic methods were applied for the preparation of tryptanthrin.^12a Owing to its pharmacologically aromatic indoloquinazoline moiety with a wide range of biological and pharmacological activities, tryptanthrin has been extensively applied as a parental backbone via the modification of different functional groups for the development of new tryptanthrin derivatives with significant medicinal benefits as antitrypanosomal,¹³ antifungal,^14,15 anti-phytopathogenic bacterial,¹⁶ antibacterial,^17–21 anti-MRSA,²² antiviral,^23–26 anti-angiogenic,²⁷ anti-inflammatory,^28–39 anti-leishmanial,^40,41 antimalarial,⁴² antiplasmodial,⁴³ antiparasitic,⁴⁴ anti-tubercular,^45–47 and anti-cancer^48–64 agents for the treatment of different diseases (Fig. 1). The most important biological activities of tryptanthrin derivatives are their anti-cancer properties. The typical examples are benzo[b]tryptanthrin,^63,64 halogen-substituted tryptanthrin,^56–61 and N-substituted tryptanthrin (D ring) derivatives^49–54 achieved by the reaction of halogen-substituted tryptanthrins and secondary amines, which exhibit anti-cancer activities against different cancer cell lines (Fig. 1), such as chronic myeloid leukemia (K562), liver (HepG2), lung (A549), prostate (PC3), hepatocellular carcinoma (HCC) and breast (MCF7) cancer with IC₅₀ mean values at low micro-molar levels.^{49,51–53,56,60} The ketone group in the sixth position of the indolo[2,1-b]quinazoline-6,12-dione core can be also replaced with different functional groups to produce other spiro-tryptanthrin,^65,66 tryptanthrin aminothiazole,⁶⁷ tryptanthrin-6-oxime⁵² and 8-halogen-substituted-6-hydrazono tryptanthrin⁶⁸ derivatives with various in vitro antitumor activities. In addition, tryptanthrin derivatives can also emit near-infrared fluorescence suitably applied as highly sensitive and selective fluorescent probes for biological metal ions,^69,70 indicating environmental significance. Kaur et al. in 2017 reviewed the syntheses and biological activities of tryptanthrin and its related derivatives in last two decades,^12b but there has been no consideration of tryptanthrin metal complexes with anticancer activities in this review. Soon afterwards, great efforts were made in the syntheses of tryptanthrin derivatives^{49–51,53–55,58,60,61,65–67} and their metal complexes^71–75 with excellent anticancer activities in the past six years.

Fig. 1. Biological activities of tryptanthrin and its derivatives.

Furthermore, some indispensable bio-metal cations are commonly used for the treatment and diagnosis of cancer, because these bio-metal cations have a significant role in different biological pathways.^76–82 The imbalance of bio-metal ions (such as Cu²⁺, Zn²⁺, and other metal cations) in the human body may cause some pathological response, for instance, their imbalances may induce certain cancers and other diseases,^79–81,83 while their complexes are found to interact with genetic DNA inside the cancer cell nucleus and interfere with the transcription and replication of DNA, finally resulting in cancer cell death.^84–91 Considering the bioactive tryptanthrin and its derivatives with O and N donors, the combination of bioactive metal ions and tryptanthrin (or its derivatives) may generate a new class of tryptanthrin derivatives denoted as tryptanthrin metal complexes with improved cytotoxic activities. Consequently, a large number of tryptanthrin metal complexes incorporating d-block metal ions like Cu²⁺, Zn²⁺, Ru²⁺ and Pt²⁺ (ref. 71–75) with various spatial configurations have been achieved, and showed a broader spectrum of anticancer activity.

Generally, synthetic modification via various functional groups or bio-metal coordination can improve the anticancer activity of the original tryptanthrin. However, owing to differences in types of functional groups, substituents at different sites, water solubility, and spatial configuration of tryptanthrin metal complexes, the anticancer activities of tryptanthrin derivatives are different. As a result, the study of the function mechanism and structure–activity relationship of synthetically modified tryptanthrins is still in the preliminary exploration stage. This review will offer readers information about the recent advances in the syntheses, structures, and anticancer activities of tryptanthrin derivatives, which will not only reveal the important structure–activity relationship of tryptanthrin derivatives, but also provide some references for the future development of new tryptanthrin-based anticancer agents.

2. Syntheses of tryptanthrin and its derivatives

2.1. Syntheses of tryptanthrin

Tryptanthrin was first achieved by decompressed sublimation of indigo,⁹² after that it was also obtained from other natural plant species, as exemplified by Chinese woad,^93,94 isatis,^95,96 isatidis,⁹⁷ Calanthe,⁹⁸ Wrightia,⁹⁹ Couroupita,¹⁰⁰ Strobilanthes,¹⁴ and others.^101,102 The exact structure of tryptanthrin was confirmed by X-ray crystallography,^103,104 and its structural feature is a practically planar indolo[2,1-b]quinazoline-6,12-dione core. Moreover, there might be different approaches for the preparation of tryptanthrin (1, Scheme 1). Clearly, the utilization of one or more reaction materials and the change of reaction conditions may result in the formation of tryptanthrin. For instance, Friedlander et al.¹⁰⁵ have successfully prepared tryptanthrin by treating an aqueous KMnO₄ solution of isatin as the source material, but with a very low yield. Indigo is oxidized by KMnO₄ to also obtain yellow tryptanthrin.¹⁰⁶ Isatin was reacted with isatoic anhydride for 1 h to give tryptanthrin with a high yield of 90% in toluene solvent in the presence of triethylamine (Et₃N).^62,107 The reaction of isatin chloride (or α-isatinanilide) and anthranilic acid can also produce tryptanthrin, which offers a new vista for the chemically synthetic approach of tryptanthrin.¹⁰⁵ In addition, the copper-catalyzed tandem reaction of 2-bromobenzoic acid with aqueous ammonia and 2-(2-bromophenyl)-2-oxoacetaldehyde was also used in the preparation of tryptanthrin.¹⁰¹

Scheme 1. Different synthetic approaches for tryptanthrin.

2.2. Syntheses of tryptanthrin derivatives

The tryptanthrin derivatives are obtained from natural sources and chemical syntheses. Phaitanthrins A (2), B (3), C (4), D (5), and E (6), methylisatoid (7) and candidine (8) can be achieved from Phaius mishmensis,¹⁰¹ while ophiuroidine (9) is isolated from the brittle star Ophiocoma riisei,¹⁰⁸ and the structures of 2–9 are shown in Scheme S1.† Although tryptanthrin has a wide range of biological activity, its poor aqueous solubility and moderate anticancer activities are considered problematic. To solve the solubility issues and improve the anticancer activities, its derivatives are designed to (i) incorporate solubilizing groups (such as imines), where the D ring substitutions are beneficial for improving biological activity and (ii) combine with other pharmacophores (such as aminothiazole and pyrano[2,3-c]pyrazole groups) in one core, synergistically enhancing the anticancer activities. Most substituted tryptanthrin derivatives are prepared by the reaction of substituted isatins and isatoic anhydrides,^49–52 while different functional groups are applied for decorating different positions of the tryptanthrin skeleton to also achieve tryptanthrin derivatives.^53,55 The rational syntheses of tryptanthrin derivatives are presented in Schemes 2–11.

Scheme 2. Syntheses of nitrotryptanthrin derivatives.

Scheme 3. Syntheses of halogenated, alkylated and methoxyl tryptanthrins.

Scheme 4. Synthetic route of N-aryl substituted tryptanthrins 20–24 (I) and 25–26 (II) [(a) Et₃N, toluene, reflux; (b) Pd(OAc)₂, BINAP, amine, Cs₂CO₃, toluene, reflux; (c) ethyl acrylate, Pd(OAc)₂, K₃PO₄, DMA, N₂, 8 h, 140 °C; (d) PdCl₂(dppf), KOAc, DMF, bis(pinacolato)diboron, N₂, 18 h, 80 °C; (e) NaOH, EtOH, H₂O, 5 h, room temperature; (f) NaIO₄, THF, H₂O, 15 min, room temperature].

Scheme 5. Synthetic route of N-aryl/benzyl substituted tryptanthrins 27–28 (I), 29a–f (II) and 30–31 (III) [(a) DMSO, Cs₂CO₃, 110 °C, organic amine; (b) HNO₃/H₂SO₄, 0 °C, 0.5 h; (c) SnCl₂·2H₂O, ethanol/HCl, 80 °C, 2 h; (d) RCOCl or RSO₂Cl, Et₃N, DCM, 0 °C, 0.5–1 h; (e) N-bromosuccinimide, azobisisobutyronitrile, CH₂Cl₂, reflux; (f) Et₃N, KI, DMF, organic amine].

Scheme 6. Syntheses of benzo-annulated tryptanthrins.

Scheme 7. Syntheses of oxime and dicyanomethylene substituted tryptanthrins 33–35 (I) and 36a–c (II) [(a) (H₂NOH)₂·H₂SO₄, pyridine, MeOH, reflux, 4 h; (b) RX, K₂CO₃, D MF, 90 °C, 12–20 h].

Scheme 8. Syntheses of 6-imine substituted tryptanthrins 37a–r (I) and 38–39 (II) [(a) NH₂–NH₂·H₂O, 12 h, THF, reflux; (b) Na₂CO₃ (20 mL), PdCl₂(PPh₃)₂, DMF, 9 h, 110 °C, 4-pyridineboronic acid (or 4-carboxyphenyl boronic acid); (c) o-vanillin, CH₃OH, reflux 12 h (or acetic acid, benzoyl acetone, reflux)].

Scheme 9. Syntheses of tryptanthrin derivatives.

Scheme 10. Syntheses of 6-spiro tryptanthrin derivatives 42a–i (I) and 43a–d (II).

Scheme 11. Syntheses of TA and its zinc complexes [(a) K₂CO₃, NaI, DMF, 45 °C, 12 h; (b) ZnCl₂, EtOH, 65 °C, 24 h; (c) curcumin (H-Cur), CH₃OH, NaOH, 80 °C, 12 h].

Nitro tryptanthrins

Tryptanthrin has a strong electron-directing influence from nitro on the substituent position, so that the 8-position of tryptanthrin is more reactive than the 2-position. The reaction of one equivalent of nitric acid and tryptanthrin at 0 °C can produce a new 8-nitrotryptanthrin (10a, Scheme 2).⁵⁵ An excess of HNO₃/H₂SO₄ is employed in the preparation of the substituted tryptanthrin at 40 °C, forming a new 2,8-dinitrotryptanthrin (10b).¹¹

Halogenated, alkylated and methoxyl tryptanthrins

The substitution of halogen, alkyl and alkoxy groups onto the A and D aromatic rings has been investigated by Liu, Sharma and other groups.^49–62 A series of substituted tryptanthrins (11–19) are prepared by the optimized Bergman condensation reaction of substituted isatins with substituted isatoic anhydrides (Scheme 3). The usage of substituted anilines as reaction materials to prepare substituted isatin derivatives needs two steps. Firstly, substituted anilines are reacted with Cl₃CCHO and HONH₂·HCl to generate anilide derivatives. Secondly, a Beckmann rearrangement was implemented in H₂SO₄ (95%) to achieve the substituted isatins.¹⁰⁹ The anilines with adjacent or opposite substituents usually yield only one product after Beckman rearrangement,¹⁰⁹ while the aromatic ring of anilines with o-dihalogeno groups can result in the mixture of two isomers upon electrophilic substitution of a benzene ring,^51,56 for instance, when 3-chloro-4-fluoaniline was utilized as the starting material, isomers (6-chloro-5-fluoro-isatin and 4-chloro-5-fluoro-isatin) with a ratio of 1 : 1 are produced,⁵⁶ and they are very difficult to separate owing to their very similar polarities.

N-Aryl/benzyl substituted tryptanthrins

The syntheses of N-aryl substituted tryptanthrins are shown in Schemes 4 and 5I and II. The 7 (or 9)-halogeno-substituted tryptanthrins are obtained by the reaction of 7 (or 9)-halogeno-isatin and isatoic anhydride. The nucleophilic substitution between different organic amines and 7 (or 9)-halogeno-substituted tryptanthrins can generate N-benzyl substituted tryptanthrins 20a–j and 23a–c.^49,51 But 20k–l cannot be obtained by a similar method. Notably, the position of the halogen atom on the D ring of tryptanthrin affects the selectivity and activity of the nucleophilic reaction. Owing to the C-8 halogen atom and the C-6 carbonyl group enhancing nucleophilicity, the C-7 position shows better reactivity than the C-9 position. The C-8 position is the most stable, so their activities follow the order of C-7 position > C-9 position > C-8 position.⁵¹ When the molar ratio of di-halogeno-substituted tryptanthrin and different organic amines was 1 : 1, the C-7-substituted tryptanthrins 21a–h, 22a–j and 24a–g are preferentially generated.⁵¹ The C-2-substituted tryptanthrins 25a–f (Scheme 4II) are also achieved with Pd(OAc)₂ as a catalyst.⁵⁰ In addition, the C-2-substituted tryptanthrins can be obtained by other reactions, for instance, 26a is acquired by the Heck reaction of 2-bromo-8-fluoro-tryptanthrin with ethyl acrylate in the presence of K₃PO₄ with Pd(OAc)₂ as a catalyst.⁵⁴26b is achieved via the hydrolysis of 26a in the CH₃CH₂OH solution of NaOH.⁵⁴26c is prepared by the Miyaura reaction of 2-bromo-8-fluoro-tryptanthrin with bis(pinacolato)diboron in K(CH₃COO) alkaline solution with [PdCl₂(dppf)] (dppf = 1,1′-bis(diphenylphosphino)ferrocene) as a catalyst.⁵⁴26d is isolated by the similar hydrolysis of 26c in the aqueous solution of tetrahydrofuran.⁵⁴

To consider the C-6 carbonyl group enhancing the electrophilicity of the C-7 and C-9 positions, the coupling reaction of tryptanthrin, 8-fluorotryptanthrin or 8-chlorotryptanthrin with secondary amines can also generate the N-aryl C-7-substituted tryptanthrins 20a, 20c, 22a, 22c, 22e, 22i, 22f, 23a, 23c and 27, and N-aryl C-9-substituted tryptanthrins 28a–b (Scheme 5I).⁵³ These reactions are regioselective, and the halogen at the 8-position cannot undergo substitution. In addition, the C-8-substituted tryptanthrins 29a–f can be obtained by the reaction of 8-amino-tryptanthrin with alkanoylhalides or alkylsulfonylhalides (Scheme 5II).⁵² Due to benzyl bromide having a better reactivity than aryl bromide, the nucleophilic reaction of benzyl bromide of 2-bromomethyl-tryptanthrin or 2-bromomethyl-8-fluorotryptanthrin (BMFT) with different organic amines in the presence of KI as a catalyst easily gives N-benzyl substituted tryptanthrins 30a–f (Scheme 5III).⁵⁰ BMFT is also used as a substrate to obtain other N-benzyl substituted tryptanthrins 31a–h,⁵⁴ for instance, BMFT is oxidized by N-methylmorpholine-N-oxide to transform into 31a. The reaction of BMFT with NaN₃ introduces an azide group, which is further reacted with propiolic acid in the presence of cuprous iodide and sodium ascorbate as a catalyst, generating 31b. The reaction of BMFT with methyl 4-piperidinecarboxylate, methyl 3-piperidinecarboxylate and proline methyl ester hydrochloride, respectively, in the presence of NEt₃ and KI gives new N-benzyl substituted tryptanthrins 31c, 31e and 31g. The reaction of BMFT with 4-piperidinecarboxylic acid and 3-piperidinecarboxylic acid, respectively, under the catalysis of KI generates 31d and 31f. 31g is hydrolyzed in the alcohol solution of NaOH to afford 31h.

Benzo-annulated tryptanthrins

The Bergman condensation reaction of isatin and its benzo-analogues with anthranilic acid and 3-amino-2-naphthoic acid in the presence of SOCl₂, respectively, generates tryptanthrin 1 and benzo-annulated tryptanthrins 32a–g in 51–84% yields (Scheme 6).⁶⁴ In addition, the similar condensation reaction of isatin and its benzo analogues with isatoic anhydride and its benzo-analogues, respectively, also gives tryptanthrin 1 and benzo-annulated tryptanthrins 32a–g in lower yields.⁶⁴

6-Oxime and 6-imine substituted tryptanthrins

The 6-carbonyl (C O) group has polarity, and the electronegativity of its O atom is larger than that of the C atom, so the O atom is negative and the C atom is positive. Nitrogen/carbon nucleophiles easily attack the positively charged C atom, and undergo nucleophilic reaction. The typical example is tryptanthrin and its substituted derivatives reacting with (H₂NOH)₂·H₂SO₄ to produce various oximes (Scheme 7I), such as indolo[2,1-b]quinazoline-6,12-dione 6-oxime 32.⁵² These oximes can further react with acid halides and alkyl halides to generate an oxime ester (33d) and ethers (33a–c, 33e–h, 34a–c and 35a–b), respectively.⁵² Malononitrile is a carbon nucleophile, so the reaction of tryptanthrin and its substituted derivatives with malononitrile in DMSO or NaH/THF can obtain dicyanomethylene derivatives 36a–c (Scheme 7II).⁶²

The condensation reaction of primary amines with ketones or aldehydes can usually generate various imines. Hydrazine has two –NH₂ groups, and one –NH₂ as a nitrogen nucleophile reacts with tryptanthrin analogues in the presence of acid to produce 6-imine substituted tryptanthrins 37a–d (Scheme 8I).⁶⁸ The Suzuki coupling reaction of 2-bromo-12-hydrazono-12H-indolo[2,1-b]quinazolin-6-one and 4-pyridineboronic acid or 4-carboxyphenyl boronic acid can produce other 6-imine substituted tryptanthrins 37e–f.⁶⁸ Due to the existence of a free –NH₂ group in 37a–f they can be further converted into 37g–r after the reaction of 37a–f with benzoyl acetone and o-vanillin, respectively.⁶⁸ The condensation of 1 and thiosemicarbazide or 4-phenylthiosemicarbazide generates tryptanthrin thiosemicarbazones, which further react with α-bromo-4-substituted acetophenones to give aminothiazole substituted tryptanthrins 38a–h and 39a–h (Scheme 8II).⁶⁷ Moreover, the aldol reaction between tryptanthrin and various CH-acidic ketones in the presence of diethylamine produces tryptanthrin derivatives 2 and 40a–d (Scheme 9),¹⁰¹ which are racemic diastereomers. The similar tryptanthrin derivatives were also prepared by the Grignard reaction. The typical example is that isatin reacts with different alkylmagnesiumbromides in THF solution at 0 °C to achieve various 3-hydroxy-3-alkyl-2-indanones, which are further condensed with isatoic anhydrides in the presence of diethylamine (Scheme 9), generating the target products 41a–d.⁵²

6-Spiro tryptanthrin derivatives

Bayat and Stepakov's groups have investigated the preparation of 6-spiro tryptanthrin derivatives.^65,66 The Bayat group chooses the domino reaction of tryptanthrin and its derivatives, malononitrile as a CH-acid, Et₃N as a basic catalyst and 3-methyl-pyrazolone in CH₂Cl₂ at room temperature.⁶⁵ The conjugate base of malononitrile is formed in the Et₃N basic medium, which further participates in the Knoevenagel condensation reaction with tryptanthrin and its derivatives, respectively, generating the dicyanomethylene tryptanthrin derivatives. 3-Methyl-pyrazolone attacks the dicyanomethylene tryptanthrin derivatives by its nucleophilic C₄ center, resulting in the formation of 6-spiro tryptanthrin derivatives 42a–42i (Scheme 10I).⁶⁵ Moreover, tryptanthrin with a conjugated O C–C N system can interact with α-amino acids to form azomethine ylides, which further react with non-electrophilic 1,2-diphenylcyclopropenes to form complex 6-spiro tryptanthrin derivatives 43a–d (Scheme 10II),⁶⁶ which possess new spirocyclic skeletons, namely spiro[cyclopropa[a]pyrrolizine-2,6′-indolo[2,1-b]quinazolin]-12′-one and 3-azaspiro[bicyclo[3.1.0]hexane-2,6′-indolo[2,1-b]quinazolin]-12′-one.

Syntheses and crystal structures of tryptanthrin metal complexes

Tryptanthrin and its derivatives have O- and N-donors, which coordinate to metal ions, generating various metal complexes. The Qin group has investigated the zinc complexes of the new tryptanthrin derivative, namely 5-(bis-pyridin-2-ylmethyl-amino)-pentanoic acid (6,12-dioxo-6,12-dihydro-indolo[2,1-b]quinazo-lin-8-yl)-amide (TA, 44) firstly obtained by the synthetic method in Scheme 11.⁷¹ The reaction of TA with ZnCl₂ in the presence of EtOH at 65.0 °C gives [Zn(TA)Cl₂] (45), which reacts with H-Cur at a 1 : 1 molar ratio for 12 h, achieving the product [Zn(TA)(Cur)]Cl (46). Tryptanthrin (Try, 1) and 8-bromo-tryptanthrin (BrTry, 12b) react with an excess of CuCl₂·2H₂O in the mixed solution of methanol and CHCl₃ (v/v = 1 : 4) at 80 °C for 3 days to achieve the copper complexes [Cu(Try)₂Cl₂] (47) and [Cu(BrTry)₂Cl₂] (48).⁷³ The reaction of the dimer [Ru(p-cym)Cl₂]₂ (p-cym = p-cymene), [Ru(C₆Me₆)Cl₂]₂ or [Ru(C₆H₆)Cl₂]₂ with the appropriate indolo[2,1-b]quinazoline-6,12-dione 6-oxime (IQO, 32) at a 1 : 1 (or 1 : 2) stoichiometric ratio in methanol or methanol/DMF mixture (1 : 1) at room temperature achieves arene–ruthenium(ii) complexes [Ru(p-cym)(IQO)Cl] (49),⁷⁵ [Ru(C₆Me₆)(IQO)Cl]·0.5MeOH (50),⁷² and [Ru(C₆H₆)(IQO)Cl] (51).⁷² In addition, the reaction of 1, 8-iodo-tryptanthrin (ITry, 15a) and 12b with cis-Pt(DMSO)₂Cl₂ in the mixed solution of CH₃OH and CHCl₃ (v : v = 1 : 1) at 55 °C produces platinum(ii) complexes [Pt(Try)(DMSO)Cl₂] (52), [Pt(ITry)(DMSO)Cl₂] (53) and [Pt(BrTry)(DMSO)Cl₂] (54).⁷⁴

Tryptanthrin and its derivatives can usually act as bidentate chelating or mono-dentate ligands. 47 confirms that the bidentate chelating Try ligand via the N and O atoms in combination with two Cl⁻ anions coordinates to the Cu²⁺ ion, creating a distorted octahedral geometry (Fig. 2a).⁷³ The crystal structure of 48 is similar to that of 47.⁷³49 crystallizes in a monoclinic space group of P2₁/n,⁷⁵ while 51 crystallizes in a triclinic space group of P1̄.⁷² In 49 and 51, three coordination sites of the quasi-octahedral ruthenium center are occupied by the η⁶ arene ligand and the other three coordination sites are occupied by one Cl⁻ anion and two N atoms of the bidentate chelating IQO molecule (Fig. 2b). The Pt²⁺ ion in 52–54 is four-fold coordinated by two Cl⁻ anions, one S atom of the DMSO ligand and one N atom from tryptanthrin and its derivatives, generating a square-planar geometry (Fig. 2c and d).⁷⁴ Notably, 52 (Fig. 2c) and 54 show the trans-configuration, where two Cl⁻ anions occupy two trans-positions, whereas 53 (Fig. 2d) exhibits the cis-configuration, in which two Cl⁻ anions occupy two cis-positions.

Fig. 2. Crystal structures of 47 (a), 49 (b), 52 (c) and 53 (d) [symmetry operation: (i) 1 − x, 1 − y, −z]. Hydrogen atoms are omitted for clarity.

3. Anticancer activities of tryptanthrin and its derivatives

Tryptanthrin and its derivatives are potential cytotoxic agents used for cancer treatment, because they can suppress tumor growth by modulating various targets. Firstly, dysregulation of STAT3 and ERK signaling is often found in human leukemia cells, resulting in an increase in their proliferation, growth and uncontrolled division. Hence, the STAT3 and ERK signaling pathways are primarily targeted to induce the apoptosis in human leukemia cell lines, and the typical case is 8-bromo-tryptanthrin (12b) that can early suppress p-STAT3 signaling with concomitant up-regulation of p-ERK, causing the activation of intrinsic and extrinsic pathways of apoptosis in HL-60 cells.¹¹⁰ Secondly, human topoisomerases, grouped into topoisomerase I and topoisomerase II, are ubiquitous enzymes for solving DNA topological problems encountered during DNA transcription, replication, recombination, and chromosome segregation in mitosis following replication, where topoisomerase IIα overexpression reflects a high mitotic rate, showing that it can be a marker of proliferation in cancer cells, and 7-(2-(dimethylamino)ethyl)amino-tryptanthrin (20k)⁴⁹ and benzo-annulated tryptanthrin (32b)⁶⁴ represent promising leads for the development of new topoisomerase II inhibitors as anti-cancer therapeutic agents. Thirdly, mitogen-activated protein kinases (MAPKs) are essential components of the intracellular signal transduction pathways that regulate cell proliferation and apoptosis, and the phosphorylation of p38 MAPK, which belongs to the MAPK superfamily in mammalian systems, may play a key role in apoptosis in human cancer cells,¹¹¹ so p38 MAPK should be investigated as a new molecular target for human cancer therapies.^112,113 Fourthly, multidrug resistance (MDR), which can be caused by the membrane proteins actively transporting drugs out of cells that result in a decrease of intracellular drug concentration, is a major cause of chemotherapy failure in cancer. The first identified ATP-binding cassette transporter is P-glycoprotein (named as MDR1) and its expression correlates with the degree of resistance. Tryptanthrin can suppress the activity of the MDR1 gene promoter in the breast cancer cell line MCF-7/adr and may be a new adjuvant agent for chemotherapy.¹¹⁴ Moreover, tryptanthrin derivatives are also applied for potential anticancer therapy via inhibiting indoleamine 2,3-dioxygenase (IDO),^55,57,59,68 vascular endothelial growth factor receptor 2 (VEGFR2)¹¹⁵ and telomerase targeting the c-myc promoter.^73,74

3.1. Anticancer activity of tryptanthrin

The cytotoxicity of tryptanthrin has been evaluated in various cancer cells, revealing that it shows moderate to good cytotoxicity towards these cancer cell lines.^112–126 A typical case is that tryptanthrin has a significant effect on inhibiting the proliferation of breast cancer MCF-7 cells in the concentration range of 12.5–100 μM and the cell cycle distribution of MCF-7 cells, arresting cells in the G1 phase, inhibiting the synthesis of their DNA, and ultimately inducing the apoptosis of MCF-7 cells.¹¹⁶ Considering the poor solubility of tryptanthrin in water, limiting its access into cancer cells, it is necessary to improve delivery systems for increasing the inhibition of multidrug resistance (MDR). The Fang group has employed nanoparticles encapsulating tryptanthrin to improve the delivery into cultured MCF7 cells, promoting the sustained release of this drug.¹¹⁷ Tryptanthrin can not only inhibit MDR and reverse doxorubicin resistance in the breast cancer cell line MCF-7 via down-regulation of MDR1 gene expression,¹¹⁴ but also downregulate GSTπ expression, contributing to the sensitization of doxorubicin-resistant MCF-7 cells by the c-jun NH₂-terminal kinase-mediated apoptosis pathway,¹¹⁹ indicating that it may serve as a new chemoadjuvant agent. Tryptanthrin has effective cytocidal effects on different leukemia cells in vitro,^119–122 for instance, tryptanthrin has an obvious inhibiting effect on the proliferation of human chronic myeloid leukemia K562 cells in vitro and inducing the apoptosis of K562.^119,120 The underlying mechanism may be related to an increase in cytosol cyt-c, Bax and activated caspase-3 expression, with a decrease in mitochondria membrane potential, Bcl-2, mito cyt-c and pro-caspase-3 contents.¹¹⁹ Tryptanthrin may exert its anti-proliferative effect on murine myelomonocytic leukemia WEHI-3B JCS cells (IC₅₀ value = 1.5 μM at 48 h) by causing cell cycle arrest at the G0/G1 phase and by inducing cell differentiation.¹²¹ In addition, tryptanthrin may also be an effective suppressor of non-melanoma skin cancer,¹¹² neuroblastoma,¹²³ esophageal cancer¹²⁴ and other cancers.^{113,115,125,126}

3.2. Anticancer activities of tryptanthrin derivatives

Anticancer activities of nitro, halogenated, alkylated and methoxyl tryptanthrins

The in vitro anticancer activity of A and D aromatic ring-substituted tryptanthrins is presented along with the corresponding cytotoxicity in Table 1. Because the poor water solubility of tryptanthrin becomes an important factor hindering its further development, the introduction of polar groups, such as nitro, amino, and halogeno, may improve its activity. The typical example is 8-nitro tryptanthrin (10a) with a solubilizing –NO₂ group, which improves its solubility, realizing better permeability and retention effects in tumor.⁵⁵10a shows relatively high cytotoxicity, and its cytotoxicity in HeLa (IC₅₀ value = 0.34 μM) and B16-F10 (IC₅₀ value = 0.49 μM) tumor cells is about two times higher than normal LX-2 (IC₅₀ value = 0.83 μM) cells. The nanoparticles of 10a can kill cancer cells by mitochondrion-mediated cytotoxicity, inhibit indoleamine 2,3-dioxygenase (IDO) activity in tumor and induce immunogenic cell death (ICD). The in vitro cytotoxic activities of 1 and 11a–11p against A549 and MC3T3-E1 cancer cells are evaluated by the MTT assay.⁵⁶ Compounds 1b–c, 1i–j, and 1p show stronger inhibitory activity of A549 cancer cells with IC₅₀ values of 3.58, 0.99, 1.03, 2.10, and 0.51 μM, respectively, stronger than parent 1 (74.71 μM) and positive control gefitinib (5.44 μM), but weaker than camptothecin (0.39 μM). The IC₅₀ values of compounds 11d–e against A549 cancer cells are 10.88 and 22.64 μM, respectively, and weaker than gefitinib, but stronger than the parent 1. The remaining compounds 11a, 11f–h, and 11k–o with IC₅₀ values greater than 100 μM do not exhibit significant inhibitory activity. According to the inhibition result of the MC3T3-E1 cell line (mouse osteoblasts), compounds 11b–d, 11i, and 11o–p have some cytotoxicity. Compounds 10a and 12b containing bromo and amino groups on the C-10 position exhibit better anticancer activity, especially, the IC₅₀ value of 10a towards MCF7/ADR, H522, M14 and DU145 cell lines is less than 1 μM, indicating strong cytotoxic activity.⁵²

Cytotoxicity of 1–43 toward cancer cell lines after an incubation time of 24 h (or 48 h)^{1–19,49–54,56,59–68,101,126}.

Considering that tryptophan 2,3-dioxygenase (TDO) along with indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase-2 (IDO2) is becoming a promising therapeutic target for the treatment of various cancer diseases, the cellular TDO, IDO1 and IDO2 inhibitory activities of compounds 1, 10a, 11c, 12a–f, and 19b are evaluated by the Yang group.^57–59 Compounds 10a, 11c and 12b show stronger IDO-1 inhibitory activity {IC₅₀ value = 0.103 (10a), 0.534 (11c) and 0.574 (12b) μM} than 1 (IC₅₀ value = 7.15 μM), and all these tryptanthrin derivatives have an electron-withdrawing group (such as F, Br, or –NO₂) at the 8-position, which may be related to their IDO-1 inhibitory activity,⁵⁹ but 12c with a F atom at the 2-position exhibits poorer activity (IC₅₀ value = 864.4 μM). 19b with 4-phenyl-1,2,3-triazol at the 2-position and a F atom at the 8-position shows stronger IDO-1 inhibitory activity than 4-aryl-1H-1,2,3-triazole with 4-phenyl-1H-1,2,3-triazol (IC₅₀ value = 143 μM),¹²⁷ but its analogue 19a with 4-phenyl-1,2,3-triazol at the 8-position and a F atom at the 2-position has no inhibitory activity. In addition, 11c can suppress the growth of Lewis lung cancer (LLC) tumor-bearing mice and decrease the numbers of Foxp³⁺ regulatory T cells, which is a potent immunomodulatory IDO-1 inhibitor applied as an immunotherapeutic agent. The IC₅₀ values of compounds 1, 10a, 11c, 12b and 12d are 5.0 ± 1.5, 0.45 ± 0.2, 1.2 ± 0.5, 3.2 ± 0.8 and 8.2 ± 3.4 μM, respectively, and they show higher cellular hIDO2 inhibitory activity than that of the frequently-used inhibitor d-methyl-tryptophan (IC₅₀ value = 148.9 ± 20 μM),⁵⁷ suggesting that these tryptanthrin derivatives may act as promising small molecule drugs to restrain tumor immune escape. For compounds 1, 10a, 11c and 12a–f, the IC₅₀ values of the U87 MG assay are very close to those of the HEK293-hTDO assay.⁵⁸ Compounds 10a, 11c, 12b, and 12d show 3–4 times higher cellular inhibition activities than 1, indicating that the electron-withdrawing F, Br or –NO₂ substituent at the 8-position increases the TDO inhibitory activity. The IC₅₀ value of 12a is similar to that of 1, implying that the electron donor –CH₃ substituent at the 8-position may have no meaningful effect on the TDO inhibitory activity. 12b can significantly inhibit the proliferation of human leukemia cell lines HL-60, MOLT-4 and K-562 with IC₅₀ values of 2, 8 and 5 mM for 48 h, inducing the cytotoxicity in HL-60 cells via the activation of Bax by the ERK pathway, and down regulation of STAT signaling.⁵¹

The in vitro anticancer activity of tryptanthrin derivatives 10a, 11c–d, 12e, 13a, 14a and 14c towards A549, K562, PC3, and HepG2 tumor cell lines is superior to that of 1,⁶⁰ indicating that the integration of a halogen or –NO₂ group into the D ring of 1 can improve the anticancer activity. 10a and 12b show multi-fold higher cytotoxicity in Hep3B hepatocellular carcinoma cells (IC₅₀ value = 1.4 ± 0.3 μM for 10a and 2.0 ± 0.3 μM for 12b) than the parent 1.⁶¹ Flow cytometric analysis reveals that 10a and 12b induce cytotoxicity in hepatocellular carcinoma cells partly by ROS generation, caspase activation and modulating Akt and MAPK signaling. 15a and 37a show cytotoxicity towards MCF-7, HeLa, SKOV3, and A498 cancer cells with IC₅₀ values of 0.65–8.51 μM.⁶²

Anticancer activities of N-aryl/benzyl substituted and benzo-annulated tryptanthrins

Human topoisomerases I/II are essential enzymes for the regulation of DNA topology, where topoisomerase I can implement its regulatory function including DNA replication, transcription and chromosome segregation by means of cleaving only one DNA strand, and topoisomerase II can cleave both DNA strands.^128,129 Overexpression of topoisomerase IIα may be related to a high mitotic rate, so it is generally regarded as a marker of proliferation in cancer cells. Topoisomerase I/II inhibition has become an important target for cancer treatment. The inhibitory activities of compounds 1, 20e and 32a–g towards topoisomerases I and/or II are evaluated.^49,63,64 The parent 1 exhibits strong selectivity on topoisomerase I inhibition in comparison with that of CPT at the 100 μM level, but the benzo-annulated tryptanthrins 32a–g do not exhibit inhibitory activities on topoisomerase I, and the activities on topoisomerase II are evidently enhanced.⁶⁴ The parent 1 shows high cytotoxicities against T47D, HCT15, DU145 and HEK293 cell lines, while benzo-annulated 32a–g do not result in any significant improvement of anticancer activity except for 32d and 32e towards HCT15 and HEK293, respectively. 32a also acts as a DNA non-intercalative topoisomerase I and II dual catalytic inhibitor, and especially inhibits topoisomerase II by blocking adenosine triphosphate (ATP) binding and ATP hydrolysis of topoisomerase II.⁶³20e can effectively inhibit topoisomerase II with an IC₅₀ value of 26.6 ± 4.7 μM, and its activity is about 2.5 times better than that of positive control etoposide (IC₅₀ value = 68.3 ± 5.4 μM) under the same experimental conditions,⁴⁹ which exhibits promising anticancer potential on acute leukemia, colon and breast cancer cell lines with IC₅₀ values of 7.13–30.48 μM. Compound 29d with terminal chloro functionality towards MCF7/ADR, U251, SW620, H522, M14, SKOV3, DU145 and A498 tumor cells has more potent cytotoxicity with an average IC₅₀ value of 1 μM.⁵²

The inhibitory efficacy of 25a–f, 26a–d, 30a–f, and 31a–h on IDO1, TDO, and IDO2 is evaluated by the Kuang group.^50,54 The N-benzyl substituted tryptanthrins 30a–f exhibit higher IDO1, IDO2, and TDO inhibitory activities than their N-aryl substituted analogues 25a–f.⁵⁰30a–f and 25a–f show stronger inhibitory activities than the frequently-used inhibitor l-methyl-tryptophan on IDO1 and IDO2, and their inhibitory activities on IDO1 and TDO are significantly superior to that of IDO2 with an order of magnitude difference, indicating that 30a–f and 25a–f can act as IDO1/TDO dual inhibitors. Carr–Purcell–Meiboom–Gill (CPMG) and saturation transfer difference (STD) experiments reveal that 25a can directly interact with IDO1, TDO, and IDO2, which promotes T cell proliferation, blocks the kynurenine pathway and effectively inhibits tumor growth of LLC and H22 tumor-bearing mice. 26a–d and 31a–h also show IDO1/TDO dual inhibitory activity, especially, the IDO1 inhibitory activity of 26a and 26b is similar to that of INCB024360, and the TDO inhibitory activity of 26b and 31a is superior to that of the well-known TDO inhibitor LM10.⁵⁴ The cellular IDO1 inhibitory activity of 26a–d and 31a–h is determined using HeLa cells. 26a, 26b, and 31b comprising cinnamic acid ester, cinnamic acid and triazole groups show relatively high cytotoxicity, and their cytotoxicity in HeLa {IC₅₀ value = 0.02, 0.06 and 0.08 μM, respectively} is very close to that of INCB024360 (IC₅₀ value = 0.02 μM), implying that the cinnamic acid ester, cinnamic acid and triazole groups might contribute to the IDO1 inhibitory activity of these tryptanthrin derivatives.

The in vitro anticancer activities of D ring-substituted tryptanthrins 1, 11a, 17a–c, 20a–d, 21a–h, 22a–i, 23a–c, 24a–g, 27 and 28a–b are measured on A549, HCT116 and MDA-MB-231 cell lines.^51,5311a and 17a show better activity than 1,⁵¹ because the halogen group at the D ring might enhance their anticancer activities. Compounds 20a, 21b, 21e, 21g, 21h, 22h and 28b show better anticancer activity than 1, particularly, the IC₅₀ value of 21h, 22h and 28b towards the three cell lines is less than 2 μM and ten times more potent than 1 (IC₅₀ value = 74.71 μM for A549, 17.49 μM for HCT116, and >100 μM for MDA-MB-231).^51,53 Other tryptanthrin derivatives 20b, 20d, 21c, 22a, 22e, 22g, 23b, 24b–d and 24g show weak anticancer activity on one or two cell lines. The cell apoptosis assay result reveals that 28b induces G2/S cell cycle arrest and apoptosis in A549 cells.⁵³

Anticancer activities of 6-oxime, 6-imine and 6-spiro tryptanthrins

Considering the importance of the 6-carbonyl modified functionality in the biological activities of tryptanthrins, their cytotoxic activities towards various cancer cell lines are evaluated using the MTT assay. 6-Oxime ether tryptanthrins 33a, 33e–h, 34a–c and 35a–b exhibit high cytotoxicity towards MCF7/ADR, U251, SW620, H522, M14, and SKOV3 cancer cell lines with IC₅₀ values from 0.01 to 30 μM.⁵²33a with a terminal –OH group in the alkyl chain is less active than 33e–h comprising terminal N,N-dialkyl-amino groups in the side chains. 33f containing two C atoms in the terminal alkylamino side chain is slightly more active in comparison with three C atoms in 33e. 33e–f with n-alkylamino side chains are also more active than 33g–h with cyclicamino side chains. The in vitro anti-cancer activities of 6-imine tryptanthrins 37a–r, 38a–h and 39a–h against the MCF-7, A549 and HeLa cancer cell lines are investigated.^67,6837h–i and 37k–l exhibit better anti-cancer activity towards MCF-7, A549 and HeLa cell lines than others 37a–g, 37j and 37m–r.⁶⁸ 6-Aminothiazole tryptanthrins 38b–c show potent cytotoxic activity against the growth of the MCF-7, A549 and HeLa cell lines with IC₅₀ values in the range of 6.05–10.15 μM, owing to the existence of cyano and methoxy groups on the thiazole phenyl ring, but 5a–h show lower activity, possibly due to the existence of the phenyl ring on the N atom of the thiazole ring.⁶⁷

1, natural tryptanthrin derivative 2 and synthetic tryptanthrin derivatives 40c–d exhibit moderate cytotoxicity towards MCF-7, NCI-H460, and SF-268 cell lines with IC₅₀ values of 9.0–43.9 μM, while other natural tryptanthrin derivatives 3–8 show no corresponding activity.¹⁰¹ 6-Hydroxy-6-alkyl/aryl disubstituted derivatives 41a and 41c–d are observed to be inactive, while 41b shows moderate cytotoxicity towards MCF7/ADR, U251, SW620, H522, M14, SKOV3, DU145 and A498 cell lines with IC₅₀ values of 6.0–20.0 μM, potentially due to the existence of an ethylene group.⁵² 6-Spiro tryptanthrin 42a shows better activity against pancreatic Panc1 cancer cells (IC₅₀ value = 21.41 ± 0.005 μM) than the well-known etoposide (IC₅₀ value = 24.35 ± 0.001 μM), because its two H groups in R1 and R2 may reduce tumor growth in Panc1 cells.⁶⁵ 6-Spiro tryptanthrin 43a with a bioactive isopropyl-carbamoyl group at a cyclopropane ring shows significant activity (IC₅₀ value = 4.8 μM) against the K562 cell line than other 6-spiro tryptanthrins 43b–d.⁶⁶

3.3. Anticancer activities of tryptanthrin metal complexes

The in vitro anticancer activities of metal complexes 48–54 (Table 2) are evaluated using the MTT assay towards various human tumor cell lines. The IC₅₀ values of 45 and 46 are 3.04 ± 0.69 and 0.14 ± 0.03 μM, respectively, for A549/DDP cancer cells, indicating superior cytotoxicity, but their cytotoxicity against A549 cells (IC₅₀ value = 15.65 ± 1.22 for 45 and 10.29 ± 0.49 μM for 46) is lower.⁷¹ The anticancer activity of 46 is clearly superior to that of the corresponding ligands {PA, Try, TA (44) and H-Cur}, ZnCl₂ and metal complexes {45, cisplatin and [Zn(PA)] (PA = di-(2-picolyl)amine)} against the A549/DDP and A549 cancer cell lines, which may be related to the synergistic effect of the bioactive Zn²⁺ ion, 44 and Cur. Compared with cisplatin against normal human liver HL-7702 cells (IC₅₀ value = 14.01 ± 1.33 μM), the cytotoxicity of 45 (IC₅₀ value = 86.30 ± 1.02 μM) and 46 (IC₅₀ value = 45.21 ± 0.80 μM) is lower, suggesting that 46 might be a promising therapeutic anticancer drug, due to its selectivity and low cytotoxicity. 45 and 46 are found to accumulate in the cell nuclei, inducing cell cycle arrest, DNA damage, mitochondrial dysfunction, and apoptosis both in vivo and in vitro. 46 shows a higher anti-proliferative effect on the A549/DDP tumor xenograft {tumor growth inhibition (TGI) = 57.4%} than that of either 45 (TGI = 50.0%) or cisplatin (TGI = 33.1%). 47 exhibits a higher activity towards the cancer cell lines BEL-7402, T-24, MGC80-3, and HepG2 with IC₅₀ values of 4.02–23.07 μM than 48 (IC₅₀ value = 30.88–108.23 μM) and cisplatin (IC₅₀ value = 15.12–19.61 μM), while 47 displays a high IC₅₀ value against normal HL-7702 cells and a low IC₅₀ value against the BEL-7402 cancer cell line, indicating its selective cytotoxicity.⁷³47 can cause cell cycle arrest in the S phase, DNA damage, and cell apoptosis of the BEL-7402 cells, and inhibit telomerase by its interaction with the c-myc promoter. It is found that 47 can significantly increase the levels of cytochrome c, apaf-1, caspase-3/9 ratio, Ca²⁺, and ROS, decrease bcl-2 protein expression and lose Δψ in the BEL-7402 tumor cells, demonstrating that 47 triggers apoptosis in the BEL-7402 cells through a mitochondrial dysfunction pathway.

Cytotoxicity of 44–54 toward cancer cell lines after an incubation time of 24 h (or 48 h).

Compd.	IC50 (μM)			Ref.	Compd.	IC50 (μM)			Ref.
	A549	A549/DDP	HL-7702			MCF10A	MCF7	MCF7CR
1	>100	>100	>100	71	49	26.2 ± 4.0	66.6 ± 11.7	53.3 ± 14.5	75
44	63.71 ± 0.55	52.38 ± 1.92	>100	71	50	5.8 ± 2.3	9.0 ± 4.4	8.9 ± 1.5	72
45	15.65 ± 1.22	3.04 ± 0.69	86.30 ± 1.02	71	51	>75	>75	>75	8
H-Cur	35.82 ± 1.08	67.24 ± 0.57	>100	71	Cisplatin	18.4 ± 1.3	36.4 ± 3.1	>75	—
46	10.29 ± 0.49	0.14 ± 0.03	>100	71	Compound	A549	A549/DDP	HL-7702	Ref.
PA	>100	>100	>100	71	Zn(PA)	38.11 ± 0.46	34.25 ± 0.11	45.21 ± 0.80	71

Compd.	T-24 [48 h]	BEL-7402 [48 h]	—	MGC80-3 [48 h]	—	—	HepG2 [48 h]	HL-7702 [48 h]	Ref.
1	>150	41.29 ± 0.31	—	70.19 ± 1.39	—	—	90.14 ± 0.47	90.12 ± 1.12	73
47	6.00 ± 0.45	4.02 ± 0.61	—	9.03 ± 1.12	—	—	23.07 ± 1.06	39.79 ± 2.13	73
BrTry	15.07 ± 0.88	27.16 ± 0.75	—	59.06 ± 0.39	—	—	99.56 ± 0.69	50.11 ± 0.82	73
48	108.23 ± 2.05	30.88 ± 1.72	—	56.57 ± 1.04	—	—	98.22 ± 1.19	41.32 ± 0.56	73
Cisplatin	15.19 ± 1.23	19.61 ± 1.06	—	15.12 ± 0.43	—	—	18.02 ± 0.55	17.69 ± 1.26

Compd.	T-24	A549	—	SK-OV-3	—	HeLa	Hep-G2	HL-7702	Ref.
1	>150	>150	—	85.09 ± 0.74	—	>150	90.14 ± 0.47	90.12 ± 1.12	75
52	67.77 ± 1.57	>150	—	51.79 ± 0.33	—	>150	93.33 ± 1.86	100.20 ± 0.55	75
15a	85.23 ± 1.08	>150	—	79.06 ± 1.05	—	>150	88.56 ± 0.72	94.06 ± 1.19	75
53	18.51 ± 1.07	112.10 ± 1.22	—	19.94 ± 0.29	—	135.98 ± 0.44	52.40 ± 0.36	90.52 ± 0.53	75
12b	15.07 ± 0.88	89.64 ± 1.20	—	75.0 ± 1.98	—	>150	99.56 ± 0.69	50.11 ± 0.82	75
54	0.21 ± 0.25	31.41 ± 0.74	—	1.80 ± 0.45	—	100.70 ± 1.85	27.54 ± 0.85	92.06 ± 1.17	75
Cisplatin	15.19 ± 1.23	14.85 ± 1.97	—	14.25 ± 1.57	—	13.02 ± 1.03	18.02 ± 0.55	17.69 ± 1.26	75

The cytotoxicity of 49–51 against the breast adenocarcinoma human cell line (MCF7), its cisplatin-resistant variant (MCF7CR) and non-tumorigenic epithelial cell line (MCF10A) depends the nature of the oxime and arene ligands,^72,75 and the cytotoxicity increases upon the variation of the arene from benzene (51), cymene (49) to hexamethylbenzene (50) in agreement with the increase of their lipophilicities (log P value = 2.14 for benzene, 4.07 for p-cymene and 4.98 for hexamethylbenzene) that allow better cell membrane transport.^130,13150 is considerably more cytotoxic than 49, 51 and cisplatin against human breast cancer cells MCF-7 (IC₅₀ value = 9.0 ± 4.4 μM) and MCF-7CR (IC₅₀ value = 8.9 ± 1.5 μM), but it is not cytotoxic towards carcinogenic and non-carcinogenic cell lines. 54 shows the highest inhibitory effect on T-24 (IC₅₀ value = 0.21 ± 0.25 μM) and SK-OV-3 (IC₅₀ value = 1.80 ± 0.45 μM) cell lines in comparison with 52, 53, and cisplatin.⁷⁴ Notably, 54 is more sensitive to the T-24 cell line, and about 386.0 times and 72.3 times higher than that of the free ligand BrTry (IC₅₀ value = 81.06 ± 0.84 μM) and cisplatin (IC₅₀ value = 15.19 ± 1.23 μM), respectively, indicating the synergistic effect of the Pt²⁺ ion and BrTry ligand. Moreover, 54 exhibits low toxicity against normal HL-7702 cells, suggesting that it has the potential clinical application for inhibitory activity against T-24 tumor cells. Cytotoxicity studies show that 54 can significantly up-regulate the levels of ROS, cytochrome c, bax, apaf-1 and caspase-3/9 ratio, down-regulate cyclin A and CDK2 proteins, and lose Δψ_m in T-24 cells, causing T-24 cell cycle arrest in the S phase. In addition, 54 acts as a telomerase inhibitor targeting the c-myc promoter, triggering apoptosis and mitochondrial dysfunction of T-24 cells.

4. Structure–activity relationships of tryptanthrin derivatives

To inquire into the relationship between the structure and in vitro anticancer activity of tryptanthrin derivatives, their cytotoxicity towards various cancer cell lines is shown in Tables 1 and 2. The SARs may be related to the substituent present on different aromatic rings, functional substituent groups, and second ligands. Firstly, the substituent present on the different aromatic rings has a certain effect on anticancer activity. The integration of a halogen group into the D ring of tryptanthrin can greatly improve the anticancer activity, and the double halogen substituent or the nitro group can further enhance its activity due to the strength of the electron-withdrawing group, while the same substituent in the aromatic A-ring diminishes the anticancer activity.^56–61 For instance, two chlorine substitutions on the aromatic D-ring significantly increase the anticancer activity (IC₅₀ value = 0.99 μM), but two chlorine substitutions on the aromatic A-ring are less active than the parent tryptanthrin (IC₅₀ value > 100 μM).⁵⁶ Tryptanthrin derivatives with substituents on aromatic A- and D-rings are less active than the parent tryptanthrin, resulting from the synergetic effect of A-ring substitution decreasing the activity and D-ring substitution enhancing the activity.⁵⁶ Secondly, different substituents have a significant effect on anticancer activity.^57–59,68 Tryptanthrin derivatives with an electron withdrawing group on the D-ring show higher cytotoxicity in comparison with the analogue with an electron-donating group on the D-ring, and the potency of tryptanthrin derivatives decreases along with the decrease of the electron-withdrawing ability of the substituents.^57–59 Thirdly, their cytotoxicity may be related to the lipophilicity of second ligands, and the typical case is that the cytotoxicity of tryptanthrin metal complexes increases upon the variation of the arene ligand from benzene to cymene to hexamethylbenzene in agreement with the increasing lipophilicity.⁸⁸ Fourthly, most of the tryptanthrin metal complexes can enhance the already strong anticancer activity of free tryptanthrin or its derivatives, due to the synergetic effect of coordination.^87,89–91 In addition, the preparation and structure of some tryptanthrin derivatives are reported, but their anticancer activities are not investigated,^132–143 in particular, some literature data on the anticancer tryptanthrin derivatives are not always homogeneous, such as different incubation times used for the test of cytotoxicity, different units of IC₅₀, no IC₅₀ values in many tryptanthrin derivatives, and different types of structural modes, so the cytotoxicity of most tryptanthrin derivatives might be not invariably comparable.

5. Conclusion and perspective

Owing to the therapeutic versatility and immense potency, tryptanthrin and its derivatives have become important pharmacological indoloquinazoline moieties for the development of anticancer agents. Synthetic modification using different functional groups and bio-metal coordination can not only address potentially the solubility issues and induce better absorption, but also improve the anticancer activity of the parent tryptanthrin. Generally, the inclusion of an electron withdrawing group into the D ring of tryptanthrin can greatly enhance the cytotoxicity towards various cancer cell lines, for instance, 11c with one electron withdrawing fluorine group at the 8-position shows IDO-1 inhibitory activity at a nano-molar level (IC₅₀ value = 2.30 × 10⁻² μM), dramatically augmenting the proliferation of T cells and efficiently suppressing LLC tumor growth.⁵⁹N-Aryl substituted halo-tryptanthrins 21h and 22h exhibit anticancer activities against the human tumor A549, HCT116 and MDA-MB-231 cell lines with mean IC₅₀ values at low micro-molar levels (less than 2 μM) and are ten times more potent than tryptanthrin.⁵¹ Complexation of tryptanthrin or its derivatives with bio-metal ions usually shows better anticancer activity than known cisplatin and parent tryptanthrin, and the typical example is that the cytotoxicity of zinc complexes 45 and 46 against A549/DDP cancer cells is superior with IC₅₀ values of 3.04 ± 0.69 and 0.14 ± 0.03 μM, respectively.⁸⁹ Therefore, the superior cytotoxicity of these tryptanthrin derivatives in comparison with cisplatin and other prevailing chemotherapeutic agents might make them a new type of anticancer agent.

The development of more potent bioactive tryptanthrin derivatives as anticancer agents in the future is prospected as follows: (a) so far, the in vitro anticancer activity of tryptanthrin and its derivatives has been extensively studied, while the evaluation of the in vivo anticancer efficacy is still rare. Therefore, a systematic study on the in vivo anticancer activity of tryptanthrin and its derivatives before clinical application is an urgent issue. (b) Their anticancer activities should be related to the structural and physicochemical properties, but the studies on the structure–activity relationships (SARs) are still very scarce, so SARs must be further disclosed. (c) The study on the anticancer activity of tryptanthrin and its derivatives is still on the surface level due to the complex pathway of cancer cell apoptosis, and the rational mechanism of action needs to be further investigated and clarified. (d) Different synthetic modifications and conditions will produce different tryptanthrin derivatives, but the synthetic modification methods have certain limitations, so it is very necessary to further improve the synthetic modification methods, achieving ideal anticancer tryptanthrin derivatives. (e) In brief, this review will offer large aid for the future development of novel anticancer tryptanthrin derivative agents with less toxic side effects.

Author contributions

Xiaofeng Zhou wrote, reviewed & edited the manuscript.

Conflicts of interest

There are no conflicts of interest to declare.