Carbazole scaffolds in cancer therapy: a review from 2012 to 2018

Abstract For over half a century, the carbazole skeleton has been the key structural motif of many biologically active compounds including natural and synthetic products. Carbazoles have taken an important part in all the existing anti-cancer drugs because of their discovery from a large variety of organisms, including bacteria, fungi, plants, and animals. In this article, we specifically explored the literature from 2012 to 2018 on the anti-tumour activities reported to carbazole derivatives and we have critically collected the most significant data. The most described carbazole anti-tumour agents were classified according to their structure, starting from the tricyclic–carbazole motif to fused tetra-, penta-, hexa- and heptacyclic carbazoles. To date, three derivatives are available on the market and approved in cancer therapy.


Introduction
Cancer is characterized by an uncontrolled growth of cells, which can spread to distant sites of the body with severe health consequences and is the second leading cause of death worldwide 1 . Around 14.1 million new cancer cases and 8.2 million cancerrelated deaths occurred in 2012, and 29.4 million new cases are estimated for 2035 with 18.8 million cancer-related deaths (GLOBOCAN 2012) 2 . The most commonly diagnosed cancers worldwide are those of the lung (1.8 million, 12.6% of the total), breast (1.7 million, 11.9%), colorectal (1.4 million, 9.8%) and prostate (1.1 million, 7.7%) cancers. Keeping in mind that both cancer cases are increasing and resistance to anti-cancer drug regimens are emerging, research and development of new powerful cancer treatments became extremely crucial for the next decades. Among the existing anti-cancer drugs, the carbazole scaffolds have been, for over half a century, the key structural motif of many biologically active compounds including natural and synthetic products 3 . Carbazole alkaloids originate in most cases from higher plants of the genera Murraya, Glycosmis, Clausena and Micromelum, all from the family of Rutaceae 4 . Other sources are bacteria (e.g. Streptomyces), algae (e.g. Hyella caespitosa) and fungi (e.g. Aspergillus species). The parent compound 9H-carbazole was isolated from coal tar in 1872 by Graebe and Glazer 5 . The first naturally occurring carbazole, the alkaloid murrayanine, was isolated from Murraya koenigii Spreng in 1962 6 . Later, many carbazole derivatives have been synthesized and are well known for their pharmacological activities such as anti-oxidant, anti-inflammatory, anti-bacterial, anti-tumour, anti-convulsant, anti-psychotic and anti-diabetic 7 . Many carbazole derivatives and related compounds have been studied. More interestingly, three derivatives have obtained marketing authorization with anti-cancer drug status in different countries. Ellipticine, which was discovered in 1959 ( Figure 1) and extracted from the leaves of Ochrosia elliptica (Apocynacae) before being entirely synthesized, could be considered as the first initial lead compound of carbazole analogues. Thereafter, an ellipticine analogue named N-methyl-9-hydroxyellipticinium acetate (Celiptium V R ) has been developed. Since 1982, Celiptium V R is still currently used in the treatment of metastatic breast cancer 8,9 . As reported by the National Cancer Institute (NCI) drug dictionary 10 , N-methyl-9-hydroxyellipticinium acetate acted as a topoisomerase II inhibitor and an intercalating agent, stabilizing the cleavable complex of topoisomerase II and inducing DNA breakages, thereby inhibiting DNA replication and RNA and protein synthesis. New N-thioalkylcarbazole derivatives were synthesized and evaluated in comparison to ellipticine. Among the bioactive carbazole-type derivatives, 7-(6-bromo-1,4-dimethyl-9Hcarbazol-9-yl)-heptane-1-thiol ( Figure 1) needs to be also mentioned 9 .
The second derivative to obtain marketing authorization was alectinib bearing a 5H-benzo[b]carbazol-11(6H)-one scaffold (AF802, CH 5424802, RG7853, RO5424802, Alecensa V R ) ( Figure 2). Alectinib, an orally available drug, was first approved in 2015 by the US Food and Drug Administration (FDA) 11 for Genentech and then by the European Medicines Agency (EMA) for Roche Pharmaceuticals 12 , with an indication as monotherapy for the treatment of adult patients with anaplastic lymphoma kinase (ALK)-positive advanced non-small-cell lung cancer (NSCLC) 13 .
Compared to the previously recent published reviews 18 , we focused this article on the carbazole derivatives exerting antitumour activity reported from 2012 to 2018, and we critically collected the most significant data. The term "carbazole" includes both the tricyclic molecular skeleton and diverse fused carbazoles including tetracyclic (with 5-, 6-and 7-membered rings), pentacyclic, hexacyclic and finally heptacyclic fused carbazoles ( Figure 4). Several databases, bibliographic information (articles) from namely ScienceDirect, Scifinder, Pubmed and Web of Science as well as technological (patents) information from INPI Patents Database, European Patent Office (EPO), as well as the World Intellectual Property Organization (WIPO), were used as literature sources.
The increased interest in the use of carbazole derivatives for the cancer therapy can also be expressed in the research of patents. In Table 1, the publication of 10 patents 19-28 is summarized including one European patent, three US patents and seven international patents for carbazole derivatives currently described for their anti-cancer activity.

N-Acylcarbazoles
N-Acylated carbazoles were synthesized and evaluated for their anti-proliferative activities against CAL 27 (squamous cell carcinoma) cell lines. The IC 50 values of the most active compounds 2a and 2b ( Figure 6) were 0.028 and 0.45 mM, respectively 30 .

N-f3-[3-(9-Methyl-9H-carbazol-3-yl)-acryloyl]-phenylgbenzamide
These derivatives ( Figure 8) were synthesized and evaluated for their in vitro xanthine oxidase (XO), tyrosinase and melanin production inhibitory activity. Most of the target compounds (4a, 4c, 4d, 4e, 4g, 4i and 4j) inhibited XO with IC 50 values comprised between 4.3 and 5.6 mM. Furthermore, these derivatives showed a better activity than the standard drug allopurinol (IC 50 value of 8.5 mM). Interestingly, compound 4a bearing a cyclopropyl ring was found to be the most potent inhibitor of XO with an IC 50 of 4.3 mM. Compounds 4b, 4d, 4f, 4h and 4j were found to be potent inhibitors of tyrosinase (IC 50 values ranging from 14.01 to17.52 mM). These results suggest the possible use of these compounds for the design and development of novel XO and tyrosinase inhibitors 32 .  BMVC or compound 5 ( Figure 9) is known for its ability (i) to suppress the telomerase activity, (ii) to induce senescence of cancer cells and (iii) to destroy the intra-tumour vasculature. BMVC was studied for tumour targeting as well as for its photo-induced antitumour effect. The properties of this fluorescent molecule provided a design of photosensitizer (PS) for photodynamic therapy (PDT) treatment. PDT results showed that BMVC inhibited the growth of tumour cells both in vitro and in vivo 33 . BMVC is the most studied to this date as "G-quadruplex" ligand, which interacted with different forms of nucleic acids and stabilizes G-quadruplex structures. BMVC suppressed the tumour-related properties of cancer cells, including cell migration, colony-forming ability and anchorage-independent growth 34 . In clinical tests (overall, 114 outpatients), the use of fluorescent BVMC was investigated for the cancer diagnosis (needle aspirates of neck masses) 35 . Many analogue derivatives of BVMC have been analysed and used as probes due to the fluorescent electron donating optical chromophore properties of this carbazole derivative. Recently a new derivative has been used for the detection of bcl-2 2345 quadruplex structures 36 .

Benzopsoralen and 3-hydroxy-N-alkylcarbazole
Benzopsoralen derivatives and its carbazole analogues were synthesized, tested against MDA MB231 (breast carcinoma) and TCC-SUP (urinary bladder cell carcinoma) cell lines, and their mechanism of action was investigated by means of molecular docking studies. Every benzosporalen and carbazole derivative showed interesting anti-proliferative activities, with GI 50 values in the nanomolar range against both cell lines. Among carbazole derivatives, compound 6 ( Figure 10) had very strong activity with GI 50 values of 0.198 and 0.025 mM against MDA MB231 and TCC-SUP cell lines, respectively 37 .

MHY407
The carbazole derivative MHY407 ( Figure 11) is active against breast cancer cell lines by inhibiting cellular proliferation with IC 50 around 5 mM. This compound increased DNA damage and triggered cell cycle arrest in S phase. In combination with various chemotherapeutic treatments, such as doxorubicin, etoposide or radiation, MHY407 improved the efficiency of the treatments by reducing cell viability and increasing apoptosis 38 .

Amide-containing carbazole derivatives
Amide-containing carbazole derivatives 7a-d and 8 ( Figure 12) have been synthesized and their in vitro anti-proliferative activities against NPC-TW01 (nasopharyngeal carcinoma), NCI-H661 (lung carcinoma) and Jurkat (leukaemia) cell lines were evaluated. All carbazole derivatives were inactive or weakly active, with IC 50 values ranging from 11.09 to 42.77 mM 39 .

Clauszoline-I
Extracted from Clausena vestita Tao, clauszoline-I ( Figure 14) showed effective ability to induce the cell cycle arrest in the S and G2/M phases. The mechanism is linked with the inhibition of the phosphorylation of the Ser-643 of the protein kinase C delta (PKCd). PKCd is a prototypical class of serine/threonine kinases, and implicated in nearly all stages of cancer 41 , and the induction of the cell cycle arrest. Clauszoline-I displayed a growth inhibitory activity against four cancer cell lines (cervical carcinoma, glioblastoma, nasopharyngeal carcinoma, hormone-independent breast cancer), with IC 50 values in the micromolar range (13.3-71.6 mM) 42 .

Excavatine A
A carbazole alkaloid, excavatine A ( Figure 16), was isolated from the stems and leaves of Clausena excavata BURM. f. (Rutaceae) and its cytotoxic activities against A549 lung carcinoma and HeLa cervix adenocarcinoma cell lines were assessed, showing IC 50 values of 17.77 and 6.47 mM, respectively 44 .

Clausenawalline F
Twenty-two compounds were isolated from the roots of Clausena wallichii (Rutaceae) and tested for both anti-bacterial and cytotoxicity activities. After evaluations, clausenawalline F ( Figure 17

EHop-016
EHop-016 was serendipitously discovered to be the most potent Rac1 inhibitor. The small GTPase Rac1 is a member of the Ras superfamily of GTPases and has been implicated in the regulation of cellular migration and invasion in breast cancer cells. EHop-016 reduced metastatic cancer cell viability at a concentration inferior to 5 mM. Additionally, its anti-cancer activity (tumour growth and metastasis) was demonstrated in vivo by using a mouse model of breast cancer. The carbazole group contributed to Rac1 inhibitory activity and then new compounds 13a and 13b ( Figure 19) were designed and synthesized. The most potent Rac1 inhibitor was 13b, which inhibited by 55% at a concentration of 250 nM and was four times more potent inhibitor of Rac1 than EHop-016 with reduced cellular toxicity 47 .

Carbazole-3,6-diamine derivatives
Carbazole derivatives bearing diamine groups presented a new potential for telomerase inhibition. Using three different docking programs (CDOCKER, Ligandfit, Autodock) and interaction analysis demonstrated that compounds 14a and 14b ( Figure 20) had the best telomerase inhibition activity more interestingly with the introduction of a pyrazole ring 48 .

Carbazole sulfonamide derivatives
The carbazole sulfonamide IG-105 49 ( Figure 21) was described as a potent anti-mitotic agent that inhibited microtubule assembly through specific interactions within the tubulin structure. The introduction of a hydroxyl group (7-OH) on the carbazole-ring increased the solubility and provided a new derivative named SL-3-19 ( Figure 21). This compound was active against HepG2 liver cancer (IC 50

Guanidinocarbazoles
Several alkylguanidines derived from carbazole were prepared and tested for their anti-cancer activity. Three compounds 17a-c ( Figure 23) were tested at 10 À5 M against KB and HeLa cell lines, and the best IC 50 values (3.1, 3.5 and 4 mM, respectively) against HL60 acute promyelocytic leukaemia cell line. Compound 17a, which was found to be the most active, also demonstrated a high inhibition at 10 À5 M against MCF-7, HCT116, PC3 (prostate cancer) and MRC5 (human fatal lung fibroblast) cell lines. Furthermore, fluorescence measurements were carried out and showed that 17a had some DNA binding properties 52 , which was described as a cycle-dependent cytotoxic activity. In summary, various pharmacomodulations in the series of tricyclic carbazoles were performed to obtain new anti-tumour compounds and to investigate the structure-activity relationships. Several substituents were essentially introduced on the nitrogen atom of the indole moiety or on the benzene moiety. Figure 24 shows the substituents that have contributed to the improvement of the anti-tumour activity of the compounds described in this section. and RCC45 (renal cell carcinoma) cell lines. The highest activity (EC 50 value of 0.08 mM) was observed with compound 18 ( Figure  25) having an acetyl group at C6 and N9 substituted with (1methylpyrrolidin-2-yl)ethyl 53 .

Furanocarbazoles
Fourteen compounds, including mafaicheenamines D and E, were isolated from the roots of Clausena lansium (Rutaceae) and evaluated against KB, MCF-7 and NCI-H187 cell lines. All compounds were non-cytotoxic against the tested cell lines, except mafaicheenamine E with a methoxy group at C 1 and bearing a substituted furanone ring on C 2 -C 3 ( Figure 26) which exhibited
Other pyrrolo[2,3-a]carbazole derivatives with substituent on position 4 were synthesized and their biological activities were evaluated as Pim kinase inhibitors and in vitro anti-proliferative agents. Compound 21 (Figure 29), bearing a methoxycarbonyl group at the 4-position, was found to be active, especially on Pim-3 kinase with IC 50 around 0.5 mM. The latter also showed antiproliferative activities on fibroblasts (IC 50 ¼8 mM) and on PC3 cells (IC 50 around 6 mM) 60 .
Natural C-glycosyl pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione derivatives were tested as Checkpoint kinase 1 (Chk1) inhibitors. Compounds 22 and 23 ( Figure 30) substituted at the C 1 with a glycosyl group and at C 6 with a hydroxyl group were the most active compounds among this series and exhibited IC 50 values from 0.5 to 9.5 mM 61 .
Briefly, when comparing the mentioned tetracyclic carbazoles containing a five-membered ring, SAR study can be correlated with substitutions at the carbazole ring ( Figure 33).

Benzocarbazoles
Alectinib/CH5424802 (compound 27a, Figures 2 and 34) is the second generation ALK inhibitor bearing a 5H-benzo[b]carbazol-11(6H)-one structural scaffold which presented a high selective ALK inhibition at a nanomolar scale (IC 50 value of 1.9 nM) 64 . Additionally, it is a potent anti-proliferative compound against KARPAS-299 cell line (human T cell lymphoma) carrying the nucleophosmin (NPM)-ALK fusion gene with an IC 50 value of 3.0 nM. In vivo studies in mice, using ALK fusion gene-positive NSCLC xenograft model, showed that orally administrated compound 27a significantly regressed tumours. Currently, this compound is being evaluated in phase I/II clinical trials for the treatment of ALK-positive NSCLC 65 .
Compared to the first generation non-carbazole derivative crizotinib (Figure 35), in vitro and in vivo studies showed that alectinib was more potent and selective against wild and mutant ALK. Kodama et al. 66 showed that the inoculation of alectinib reduced the tumour size and avoided its regrowth. As previously mentioned, alectinib received in 2015 approvals by FDA and EMA for anti-cancer use.  A series of 2-(4-aminobenzosulfonyl)-5H-benzo[b]carbazole-6,11-dione derivatives has been synthesized. In vitro anti-proliferative activity was performed against SiHa (cervical carcinoma) cell lines. Compounds 28, 29a,b ( Figure 36) exhibited a good cytotoxicity with IC 50 values of 52.2, 53.8 and 33.5 mM, respectively. The interaction between all compounds and HDAC8 was also carried out by performing molecular docking studies with the use of the GLIDE program 67 .

Pyranocarbazoles
Girinimbine (Figure 37), a carbazole alkaloid isolated from the stem bark of M. koenigii, had a strong anti-tumour promoting activity. The expression of the Epstein-Barr Virus Early Antigen (EA-EBV) in Raji cells was inhibited by more than 90% when tested at 16 mg/mL (50% inhibition at 22.8 mM). The compound did not alter Raji cell's viability due to very low cytotoxicity. Girinimbine showed strong anti-oxidant properties comparable to a-tocopherol    Das et al. 71 reported that the combination of mahanine with 5fluorouracilenhanced the reactive oxygen species (ROS) production, increased the activation of tumour suppressor proteins and suppressed chemo-migration. In another study 72 , the same authors reported that when associated to cisplatin, mahanine  could overcome cisplatin-toxicity and drug resistance. Mahanine synergically improved the apoptosis induced by cisplatin in cervical cancer cells and inhibited migration property. The combination at molar ratio 1:4 of cisplatin: mahanine showed a growth inhibitory effect on HeLa and SiHA cell lines (IC 50 of 1.6-1.8 mM, respectively). This effect on cell inhibition was 10 times higher to the inhibition induced only by mahanine.

Pyridocarbazoles
The commercial analogue of ellipticine, Celiptium V R (Figure 1), is active against metastatic breast cancer and acts as an inhibitor of type II topoisomerase. Several studies have also reported apoptosis induction by ellipticine involving the p53 tumour suppressor protein. Prudent et al. 73 have recently reported a novel mechanism of action of ellipticine as a new inhibitor of the casein kinase CK2 and new analogues are currently developed.
Mori et al. 74 reported the synthesis of new ellipticine and pyridocarbazole derivatives and their evaluation against HeLa S-3 cell lines. Most of the compounds showed anti-tumour activity with IC 50 values between 2.50 mM and 60 mM. It appeared that compound 31 (Figure 40), an ellipticinium-analogue linked to a methylnitrosourea group, showed the best anti-tumour activity with an IC 50 value of 1.3 mM which was two times more potent than ellipticine (2.1 mM).
Quantitative structure activity analysis (QSAA) was carried out on olivacine and compounds 32a-i ( Figure 41) using the software TSAR to determine the structural features responsible for their activity 75 .
Hetero annulated carbazoles were designed, synthesized and their in vitro cytotoxicity was evaluated against HeLa and MCF-7 cell lines by MTT assay and compared to the standard drug ellipticine. Compound 33 ( Figure 42) demonstrated 1.2-fold stronger activity than ellipticine's cytotoxic activity against HeLa. Then, molecular docking studies were carried out using CK2 as a target, in which compound 33 showed the lowest binding energy and best ligand efficiency 76 . SAR studies revealed that, the compound bearing the pyrimido moiety and the electron-withdrawing chlorine in the carbazole displayed excellent cytotoxic activity (IC 50 value of 8.11 mM) against HeLa cells.
The in vitro cytotoxicity of the pyrido[2,3-a]carbazoles was evaluated by SRB (sulfo-rhodamine B) assay against MCF-7, HeLa and A549 cell lines. Among these derivatives, compound 34 ( Figure  43) showed the best activity with an IC 50 of 13.42 mM against HeLa cells (cisplatin, 13.20 mM). All the designed compounds demonstrated a higher potency against HeLa than against the other tested cell lines 77 .
Pyrido[3,2-a]carbazole derivatives and their analogues were tested against A549 and HT29 cell lines with IC 50 values ranging from 0.07 mM to 4.45 mM. For example, compound 35 ( Figure 44) was active against A549 cells with an IC 50 value of 0.07 mM and with 0.11 mM against HT29 cells 78 .
Ditercalinium is a dimer of two 7H-pyrido [4,3-c]carbazole units ( Figure 45). It is a bis-interacting agent in the major groove of DNA. NMR studies and X-ray crystal structure revealed that both rings of the dimer allowed intercalation with base pairs and caused structural changes in the DNA 79 .

Oxazinocarbazoles
A set of various oxazinocarbazoles was synthesized 81 and their activities were studied using a CE-based assay for CK2 activity measurement, a cytotoxicity assay using IPC-81 cells. Three oxazinocarbazoles 37a-c ( Figure 47         Briefly, when comparing the mentioned tetracyclic carbazoles containing a six-membered ring SAR can be correlated with substitutions at the carbazole ring ( Figure 49).    PARP-1 inhibitors. They were also tested in a cell-based assay that evaluated their ability to attenuate the depletion of NAD þ levels following hydrogen peroxide insult in PC12 (rat pheochromocytoma) cells. Results showed that two analogues, compounds 42a and 42b (Figure 51), displayed potent enzyme activity with IC 50 values of 18 and 25 nM, respectively, as well as high cell permeability (100% NAD þ recovery at 30 mM) 84 .

4.2.
Pentacyclic fused carbazoles containing a five-membered ring and a six-membered ring

Tetrahydroindolo[2,3-b]carbazoles
Tetrahydroindolo [2,3-b]carbazoles were synthesized to undergo a one dose screening at 10 À5 M, followed by a five dose screening for the best compounds using the NCI 60 cell lines list. Compound    43 ( Figure 52) exhibited the highest anti-cancer activity with growth inhibition at lowest mean value of 21.63%, and GI 50 values ranging from 1.07 to 9.56 mM against the tested cell lines 85 .
Several inert metal complexes such as pyridocarbazole-rhodium (III) were synthesized and characterized by X-ray crystallography. Stability studies were carried out including evaluation of Pim-1 kinase inhibitory activity. Compound 44 (Figure 53) was found to be a stable rhodium (III) complex and extremely potent inhibitor of Pim-1 kinase (IC 50 around 160 pM) 86 .
The iridium-pyridocarbazole complexes 45a and 45 b ( Figure  54) are highly photocytotoxic compounds. Their anti-angiogenic properties were investigated in a 3D angiogenesis assay. It resulted that 45a and 45b are light-independent potent antiangiogenic agents, very active on the vascular endothelial growth factor 87 .

Carbazole-amonafide structural hybrids
Preliminary anti-proliferative assays revealed that structural hybrids [4,5-bc]carbazole-amonafide derivatives possessed a good cytotoxic activity with IC 50 values in the sub-micromolar to micromolar range against HTC116 cell line, and were also selective for cancer

Indenopyrrolocarbazoles
A strong lead candidate, compound 47 ( Figure 58), was synthesized from staurosporine aglycone (K252c) 90 . The structure-activity relationship showed that compound 47 is a powerful tropomyosine kinase TrkA inhibitor; therefore, it was selected as a proof of concept for in vitro and in vivo studies 53 .   FGFR1), which are potent targets for tumour angiogenesis and vascular maintenance. Furthermore, CEP-11981 exhibits excellent permeability, metabolic stability and pharmacokinetic properties. It was advanced into full development and was in clinical phase I study 91 .

Indolopyrrolocarbazoles
Among the carbazole derivatives, four synthetic indolopyrrolocarbazoles ( Figure 60) are currently in clinical trials for cancer therapy. CEP-2563 is active against MTC (medullary thyroid carcinoma) and blocked tyrosine kinase receptors such as Trk family and the platelet-derived growth factor (PDGF) receptor tyrosine kinase 92 . Edotecarin (J-107088) and becatecarin (XL119) which both could intercalate into DNA and edoteacrin could additionally stabilize the DNA-topoisomerase I complex. Edotecarin (J-107088) is currently in phase III trials (Pfizer) and becatecarin (XL119) (NCI) is in clinical trials (phase II) and represent promising approaches for the cancer therapy. UCN-01 is a protein kinase C (PKC) inhibitor and is currently in Phase II trials (NCI) for its activity against pancreatic, lymphoma and breast cancers 92 .

Staurosporine and analogues
A series of indolocarbazoles and staurosporine analogues ( Figure  61) were synthesized and tested as anti-proliferative agents against HUVEC (Human Umbilical Vein Endothelial Cells), LoVo (colorectal adenocarcinoma), DLD-1 (colorectal adenocarcinoma) and ST-486 (Burkitt's lymphoma) cell lines. Their anti-angiogenesis activity was also investigated by capillary tube formation in 3-D matrigel matrix. Acero et al. 93 observed on all cell lines that the dimethylaminoalkyl chain in R 1 (Figure 61) enhances both activity and selectivity. Analog 48 (Figure 61), with an IC 50 of 0.1 mM against HUVEC, was one of the most active compounds and the most selective one. The in vivo anti-angiogenic assay using the Lewis lung mice carcinoma model revealed that no tumour reduction was observed, although a slight reduction in metastasis number was noticed 93,94 .

Methylenedioxy-and ethylenedioxy-fused indolopyrrolocarbazoles
The biological activity of indolo[2,3-a]carbazole derivatives ( Figure  63) was determined as potential anti-cancer agents. Among the analogues, compounds 49a-d were the most potent compounds against human topoisomerase I and exhibited inhibitory activities with IC 50 values in the micromolar range (from 3.2 to 5.4 mM) 96 .
In summary, the scaffold pyrrolocarbazole was extensively used as a part of hexacyclic fused carbazoles. Three sub-series of related compounds were developed, namely indeno-, indazo-and indolo-pyrrolocarbazoles ( Figure 64). For some the additional presence of a sugar moiety is also to notice (e.g. edotecarin, compounds 49a-d). It is an important point to modulate their physicochemical properties such as hydrosolubility and then to facilitate in vivo investigation.   was evaluated through topoisomerase II inhibition and in cellulo assay using the NCI-60 cell line. Although no topoisomerase II inhibition was observed, compound 52a ( Figure 66) was found to inhibit in vitro the growth of HCT-15 (colon carcinoma), SK-MEL-2 (melanoma), and ACHN, CAKI-1 and UO-31 (renal adenocarcinoma) cell lines with GI 50 values in the low micromolar range. The less toxic azaindolocarbazole 52b (Figure 66) also showed cytostatic activity against NCI-H522 (non-small-cell lung cancer) and UO-31 cell lines 98 .

Indolopyrazolocarbazoles
In the particular point of the replacement of the pyrrolo moiety of indolopyrrolocarbazole either by a pyrazolo or by a pyrimido ring systems (Figure 67), new active hexacyclic derivatives demonstrated cytotoxic activity on colon carcinoma cell lines (e.g. HCT116, HCT-15).
6. Heptacyclic fused carbazoles 6.1. Carbazole derivatives of ursolic acid A series of carbazole derivatives of ursolic acid was synthesized and assayed against two human liver cancer cell lines (SMMC-7721 and HepG2) using the MTT colorimetric method. From the results, compounds 53a-f ( Figure 68) displayed pronounced cytotoxic activities with IC 50 values below 10 mM. Compound 53e was found to be the most active compound with IC 50 values of 1.08 ± 0.22 and 1.26 ± 0.17 mM against SMMC-7721 and HepG2 cells, respectively, comparable to those of doxorubicin. In addition, 53e showed reduced cytotoxicity against noncancerous LO 2 cells with an IC 50 value of 5.75 ± 0.48 mM 99 .

Conclusion
Cancer is a very complex disease and the increase of the biological targets can be synergistically coordinated to relieve patients from cancer burden. Many new cancer therapies have been developed in the last years, but this research field still presents many challenges. Among the natural products, the carbazole alkaloids have shown several biological activities ( Figure 69). Since 2012, we presented the major anti-tumoural activities of natural and synthetic carbazole derivatives.
In a recent study, Iman et al. 100 showed that the combination of many mechanisms of action were observed in the case of girinimbine which resulted in an induction of G0/G1 phase arrest, an upregulation of two cyclin-dependent kinase proteins p21 and p27, an activation of caspase-3 and caspase-9, downregulation of Bcl-2 and upregulation of Bax in girinimbine-treated cells. Another activity was seen on the upregulation of p53. Induction of apoptosis by girinimbine was also investigated in vivo by using zebrafish embryos, with results demonstrating significant distribution of apoptotic cells in embryos after a 24-h treatment period 100 . Some compounds are currently following clinical trial phases and the optimal structure has not yet been found. Carbazole derivatives have been recently described by Diaz et al. for their anti-tumour activity with the microtubule targeting and could inhibit tubulin assembly 101 . Many potential compounds can be the future candidate for the cancer chemotherapy, with the purpose of a multitarget therapy.