**6. Cytotoxic activity of colchicine and its derivatives**

Cytotoxic activity of colchicine has been known for many decades. In 1968, it was known that colchicine can efficiently bind to tubulin. Its antitumor activity derives from its tubulin binding activity [39]. Nowadays, it is known that colchicine can act with α and β tubulin in microtubules and disrupt the formation of microtubules. In past decades, many attempts have been made to design and synthesize new colchicine derivatives which could be less toxic and more effective compounds than colchicine as cytotoxic agents. On the basis of years of screening colchicine derivatives, their activity against human cancer cell lines structure: activity relationship has been established. It was found out that derivatives with alkylthio substituents at C-10 position and modified at C-7 usually are more active and less toxic than colchicine. One of the most known active semi-synthetic colchicine derivatives is thiocolchicine (10-methylthiocolchicine) **31**. Some of the obtained derivatives seem to be effective and promising agents against selected human cancer cell lines and possibly in the future could be used as anticancer drugs. Cytotoxic activity of colchicine derivatives has been tested in *in vitro* experiments on mice (KLN205, A2C12, yB8, yD12, βD10, yA7, yA3, B3, βD5, A2B1, yD1) [70] or hamster (CHO-K1) [45] cancer cell lines and human cancer cell lines such as: MFC-7 human breast adenocarcinoma [40, 45, 47, 54, 71, 72] and MDA-MB-231 [47, 72] human Caucasian breast adenocarcinoma, SK-Br-3 human breast cancer cell line [46], DLD-1 [47] and LOVO [47] human colon adenocarcinoma, HCT-5 colon cancer, HCT-15 colon carcinoma [44, 45], A549 human lung adenocarcinoma [44, 52–55, 57, 58, 70], DMS-114 small lung cell cancer [44], SKOV-3 ovarian cancer [46], OVCAR-3 ovarian carcinoma [44], A2780 human ovarian carcinoma cell line [73], 1A9 human ovarian carcinoma [53], KB human epidermoid carcinoma [46, 53, 57], PC-3 prostate cancer [40], H460 human large cell lung carcinoma [71], SF268 human astrocytoma [71], HTC-8 human ileocecal carcinoma cell [46, 57], DU-145 human prostate carcinoma [46], SKMEL-2 human skin malignant melanoma [46, 54], SKMEL-5 human skin malignant melanoma [44], RXF-631 renal carcinoma [44], SNB-19 CNS carcinoma [44], RPMI-7951 malignant melanoma [56], TE671 human medulloblastoma [56], HepG2 human hepatocyte carcinoma [70], CaCo-2 human colon carcinoma [70], and CAKI-1 kidney carcinoma [54]. As a positive control in cytotoxic tests were used: colchicine, doxorobucin or camptothecin and MTT tests [39, 57] or SRB tests MTS assay [70]. Values of IC50 for compounds **1**, **4**, **7**, **8**, **31**, **32**, **33**, **34**, and **35** are given in **Table 1**. Naturally occurring colchicine and other colchicine-like alkaloids were tested against human cancer cell lines and usually showed much better activity than parent compound.

tumor cell lines: A549, 1A9, and KB with values of IC50 0.02–0.06 μg/mL [53]. Hybrids of vindoline, anhydrovinblastine, and vinorelbine with thiocolchicine have been tested in arresting

**Table 1.** The IC50 values (nM) of compounds tested against cancer cell lines: MCF-7 [47], MDA-MB-231 [47], DLD-1 [47], LoVo [47], H460 [71] and SF268 [71]. Data were obtained from triplicate experiments. Doxorubicin was used as positive

Camptothecin — — 0.309 [71] — 0.024 [71] 0.043 [71]

**Cell line compound DLD-1 LoVo MCF-7 MDA MB-231 H460 SF268** 43.0 [47] 118.8 [47] 41.3 [47] 25.3 [47] 32 [71] 25 [71] — — 52 [71] — 44 [71] 39 [71] — — 151 [71] — 165 [71] 354 [71] — — 2440 [71] — 3200 [71] 981 [71] 4.2 [47] 13.6 [47] 55.5 [47] 81.2 [47] — — 51.2 [47] 19.5 [47] 56.1 [47] 148.3 [47] — — 71.8 [47] 56.1 [47] 764.4 [47] 704.2 [47] — — 177.3 [47] 149.6 [47] 564.2 [47] 1103.8 [47] — — 316.7 [47] 438.0 [47] 873.6 [47] 1773.3 [47] — — Doxorubicin 510.6 [47] 520.2 [47] 1210.1 [47] 935.5 [47] — —

Cytotoxic Colchicine Alkaloids: From Plants to Drugs http://dx.doi.org/10.5772/intechopen.72622 53

Many of tested colchicine derivatives and thiocolchicine derivatives obtained by partial synthesis were assayed measuring mitotic arrest in L1210 murine leukemia cell cultures [70], their binding to tubulin *in vitro*, their antitumor activity against the P388 lymphocytic leukemia screen in mice, and their inhibition of swelling produced in rat paws by injection with uric acid. To measure inhibition in binding different colchicine derivatives to tubulin, many tests have been used *in vitro* and *in vivo*: CD spectra [74], radiolabeled compounds, and cancer cell lines. The effect on tubulin can be assessed *in vitro* by measuring inhibition of tubulin polymerization [53, 66, 70] and binding of radiolabeled colchicine to tubulin [75]. Significant inhibition in binding to tubulin greater than colchicine **1** was observed with 3-demethylcolchicine (**3**), 10-methylthiocolchicine **31**, and 3-demethyl-10-methylthiocolchicine **48** [76]. Significant inhibition of binding radiolabeled colchicine to purified tubulin was observed with thiocolchicine

Colchicine showed to be too much toxic to be used as a drug candidate for cancer diseases. Colchicine is much more less toxic than colchicine [77]. Through past decades many derivatives were tested against cancer cell lines to checked their cytotoxic activity and activity in vitro to disrupt microtubule network and spindle formation. Binding of colchicine analogs to tubulin measured by competition for labeled colchicine is for **1** 5 × 10−6, **31** 4 × 10−6, and **41** 2–3 × 10−5 [76]. Inhibition of tubulin assembly by thiocolchicine derivatives **69** and **70** is IC50 8.7 μM and IC50 3.8 μM, respectively [53]. The compounds **72**, **76**, and **77** showed potent inhi-

cell cycle and against A549 cell lines [57].

control (MTT test) [47].

and 3-demethylthiocolchicine (**Table 2**).

Thiocolchicine **31** showed good activity against A2780 human ovarian carcinoma cell line with value of IC50 1.6 nM [73]. The water-soluble compound **69** (salt of succinic acid of *N*,*N*-dimethyl-*N*-deacetylthiocolchicine) showed selective activity against HTC-8 0.022 μg/mL and SK-BR-3 0.012 0.022 μg/mL cancer cells [46]. The second salt of succinic acid of *N*-deacetylthiocolchicine **72** showed activity against five of tested cancer cell lines 0.001–0.005 μg/mL (HTC-8, SK-BR-3, A549, DU145, KB) [46].

Thiocolchicine derivative **83** modified at C-7 position showed good cytotoxic activity against A549, RPMI-7951, and TE671 cancer cell lines 0.001 nM/mL [56]. Derivatives **66**, **67**, and **68** showed good cytotoxic activity against A549, SKOV-3, SKMEL-2, HCT-15, and MCF-7 cancer cell lines with IC50 values 5.2–29.8 nM [52]. **69** and **70** showed significant activity against


**6. Cytotoxic activity of colchicine and its derivatives**

52 Cytotoxicity

lines and usually showed much better activity than parent compound.

A549, DU145, KB) [46].

Thiocolchicine **31** showed good activity against A2780 human ovarian carcinoma cell line with value of IC50 1.6 nM [73]. The water-soluble compound **69** (salt of succinic acid of *N*,*N*-dimethyl-*N*-deacetylthiocolchicine) showed selective activity against HTC-8 0.022 μg/mL and SK-BR-3 0.012 0.022 μg/mL cancer cells [46]. The second salt of succinic acid of *N*-deacetylthiocolchicine **72** showed activity against five of tested cancer cell lines 0.001–0.005 μg/mL (HTC-8, SK-BR-3,

Thiocolchicine derivative **83** modified at C-7 position showed good cytotoxic activity against A549, RPMI-7951, and TE671 cancer cell lines 0.001 nM/mL [56]. Derivatives **66**, **67**, and **68** showed good cytotoxic activity against A549, SKOV-3, SKMEL-2, HCT-15, and MCF-7 cancer cell lines with IC50 values 5.2–29.8 nM [52]. **69** and **70** showed significant activity against

Cytotoxic activity of colchicine has been known for many decades. In 1968, it was known that colchicine can efficiently bind to tubulin. Its antitumor activity derives from its tubulin binding activity [39]. Nowadays, it is known that colchicine can act with α and β tubulin in microtubules and disrupt the formation of microtubules. In past decades, many attempts have been made to design and synthesize new colchicine derivatives which could be less toxic and more effective compounds than colchicine as cytotoxic agents. On the basis of years of screening colchicine derivatives, their activity against human cancer cell lines structure: activity relationship has been established. It was found out that derivatives with alkylthio substituents at C-10 position and modified at C-7 usually are more active and less toxic than colchicine. One of the most known active semi-synthetic colchicine derivatives is thiocolchicine (10-methylthiocolchicine) **31**. Some of the obtained derivatives seem to be effective and promising agents against selected human cancer cell lines and possibly in the future could be used as anticancer drugs. Cytotoxic activity of colchicine derivatives has been tested in *in vitro* experiments on mice (KLN205, A2C12, yB8, yD12, βD10, yA7, yA3, B3, βD5, A2B1, yD1) [70] or hamster (CHO-K1) [45] cancer cell lines and human cancer cell lines such as: MFC-7 human breast adenocarcinoma [40, 45, 47, 54, 71, 72] and MDA-MB-231 [47, 72] human Caucasian breast adenocarcinoma, SK-Br-3 human breast cancer cell line [46], DLD-1 [47] and LOVO [47] human colon adenocarcinoma, HCT-5 colon cancer, HCT-15 colon carcinoma [44, 45], A549 human lung adenocarcinoma [44, 52–55, 57, 58, 70], DMS-114 small lung cell cancer [44], SKOV-3 ovarian cancer [46], OVCAR-3 ovarian carcinoma [44], A2780 human ovarian carcinoma cell line [73], 1A9 human ovarian carcinoma [53], KB human epidermoid carcinoma [46, 53, 57], PC-3 prostate cancer [40], H460 human large cell lung carcinoma [71], SF268 human astrocytoma [71], HTC-8 human ileocecal carcinoma cell [46, 57], DU-145 human prostate carcinoma [46], SKMEL-2 human skin malignant melanoma [46, 54], SKMEL-5 human skin malignant melanoma [44], RXF-631 renal carcinoma [44], SNB-19 CNS carcinoma [44], RPMI-7951 malignant melanoma [56], TE671 human medulloblastoma [56], HepG2 human hepatocyte carcinoma [70], CaCo-2 human colon carcinoma [70], and CAKI-1 kidney carcinoma [54]. As a positive control in cytotoxic tests were used: colchicine, doxorobucin or camptothecin and MTT tests [39, 57] or SRB tests MTS assay [70]. Values of IC50 for compounds **1**, **4**, **7**, **8**, **31**, **32**, **33**, **34**, and **35** are given in **Table 1**. Naturally occurring colchicine and other colchicine-like alkaloids were tested against human cancer cell

**Table 1.** The IC50 values (nM) of compounds tested against cancer cell lines: MCF-7 [47], MDA-MB-231 [47], DLD-1 [47], LoVo [47], H460 [71] and SF268 [71]. Data were obtained from triplicate experiments. Doxorubicin was used as positive control (MTT test) [47].

tumor cell lines: A549, 1A9, and KB with values of IC50 0.02–0.06 μg/mL [53]. Hybrids of vindoline, anhydrovinblastine, and vinorelbine with thiocolchicine have been tested in arresting cell cycle and against A549 cell lines [57].

Many of tested colchicine derivatives and thiocolchicine derivatives obtained by partial synthesis were assayed measuring mitotic arrest in L1210 murine leukemia cell cultures [70], their binding to tubulin *in vitro*, their antitumor activity against the P388 lymphocytic leukemia screen in mice, and their inhibition of swelling produced in rat paws by injection with uric acid. To measure inhibition in binding different colchicine derivatives to tubulin, many tests have been used *in vitro* and *in vivo*: CD spectra [74], radiolabeled compounds, and cancer cell lines.

The effect on tubulin can be assessed *in vitro* by measuring inhibition of tubulin polymerization [53, 66, 70] and binding of radiolabeled colchicine to tubulin [75]. Significant inhibition in binding to tubulin greater than colchicine **1** was observed with 3-demethylcolchicine (**3**), 10-methylthiocolchicine **31**, and 3-demethyl-10-methylthiocolchicine **48** [76]. Significant inhibition of binding radiolabeled colchicine to purified tubulin was observed with thiocolchicine and 3-demethylthiocolchicine (**Table 2**).

Colchicine showed to be too much toxic to be used as a drug candidate for cancer diseases. Colchicine is much more less toxic than colchicine [77]. Through past decades many derivatives were tested against cancer cell lines to checked their cytotoxic activity and activity in vitro to disrupt microtubule network and spindle formation. Binding of colchicine analogs to tubulin measured by competition for labeled colchicine is for **1** 5 × 10−6, **31** 4 × 10−6, and **41** 2–3 × 10−5 [76]. Inhibition of tubulin assembly by thiocolchicine derivatives **69** and **70** is IC50 8.7 μM and IC50 3.8 μM, respectively [53]. The compounds **72**, **76**, and **77** showed potent inhi-


liver cirrhosis, disk problems, Behçet syndrome, prevention of post-pericardial syndrome, primary biliary cirrhosis, hepatic cirrhosis, dermatitis herpetiformis, Paget's disease of bone, pseu-

Cytotoxic Colchicine Alkaloids: From Plants to Drugs http://dx.doi.org/10.5772/intechopen.72622 55

Colchicine can be used to treat familial Mediterranean fever in children 4 years of age and older. Colchicine is available as a tablet, capsule, and a gel. In tablet form, it is available in a generic 0.6 mg tablet and as Colcrys 0.6 mg tablet. It is available as a capsule in a generic form of 0.6 mg and as Mitigare 0.6 mg capsule. There is a topical gel form of *Colchicum autumnale*, available as ColciGel. Colchicine is commonly administered orally, and use of the topical gel is rare. Due to toxicity of colchicine from 2009, the injectable form is not available. Dosing is

Usually, colchicine is a major component of tablets or capsules in which in a single tablet or capsule its amount is in range of 0.5 or 0.6 mg, sometimes is used as an injection (disk problems). Usually a man/woman of 60 kg takes a dose of 0.5–4.8 mg/day [82, 83]. Since 2008, only oral use of colchicine for patients is possible because of 50 cases of serious adverse events [84]. The known medicines with colchicine are: Colchicum Dispert®, Colcrys, Mitigare, and Colchimax. Col-Benemid or

One of the known colchicine derivatives that has been used for the treatment of Hodgkin's lymphoma and chronic granulocytic leukemia is *N*-deacetyl-*N*-methylcolchicine, brand name is Colcemid [72]. Moreover, its efficacy against melanoma and prostatic cancer has been established. Thiocolchicoside (=glucopyranosyl derivative of the semi-synthetic 3-*O-*demethylthiocolchicine **41**), is well-known as a muscle relaxing agent and as an anti-inflammatory drug substance [85]. This compound is registered in different countries under the trade names of Colcamyl, Coltramyl, Coltrax, Miorel, and Musco-Ril. Muscle spasm is one of the main factors responsible for chronic pain, and because this particular drug reduces muscle tone, it is used in therapy for the treatment of contractures and inflammatory conditions that affect the muscular system [48].

A new tool for searching new potent anticancer agents is docking studies. Some years ago it became possible to study new compounds of possible biological activity by new technical

The way to search new colchicine derivatives especially thiocolchicine derivatives seems to be worth trying because of its promising cytotoxicity. Many new derivatives have been obtained, have been tested for many different cancer cell lines, and many of them seem to be promising

methods like molecular modeling and docking studies [37, 86–90].

Proben-C is a drug where next to colchicine probenecid is added as uricosuric agent.

dogout, and idiopathic pulmonary fibrosis.

dependent on age of patient and kind of illness.

**7.3. Drugs with colchicine derivatives**

**8. Docking studies**

**9. Conclusion**

anticancer agents in the future.

**Table 2.** Inhibition [%] of binding radiolabeled colchicine to purified tubulin [76].

bition of tubulin assembly IC50 = 0.8–1.1 μM, for comparison for **1** is 1,5 μM [54]. Compound **83** showed good inhibition of tubulin polymerization and inhibition of colchicine binding (%), IC50 3.4 μM and 60% and **79** IC50 2.4 μM and 91%; **82** IC50 6.6 μM and 78% [56]. Hybrid thiocolchicine-vindoline causes cell cycle arrest in the G2/M phase [57]. Inhibition of tubulin polymerization has been studied with thiocolchicine-podophyllotoxin conjugates, where **31** was modified at C-7 substituent [63].

*In vivo* P388 mouse leukemia test data P388 for colchicine **1** is 0.5, **31** is 0.18, and **48** is 5 [mg/kg] [76].
