Oh

Esters, amides ketones and alcohols ACTIVE

Hemiasterlin (30): Ar=A/-Methylindol-2-yl HTI-286 (31): Ar=Ph

Figure 10 Hemiasterlin SAR.

Cryptophycin 52, the C-6 gem dimethyl analog of cryptophycin 1 29, was initially the most clinically promising member of this family.101 Preclinical studies indicated that cryptophycin 52 was 40-400 times more potent than paclitaxel and broad-spectrum in vivo activity was observed in several xenograft models, including multidrug resistant (MDR)-expressing tumors.102 A phase I dose-escalating study (0.1-1.92 mgm_2) monitored toxicity in patients who received 2-h infusions once every 3 weeks. The most common toxicities observed included acute peripheral neuropathy and myalgia.103 This totally synthetic cryptophycin analog was withdrawn from clinical trials in 2002 due to limited efficacy; however, a number of other cryptophycin analogs are presently in preclinical studies.104

7.04.2.1.2.3 Hemiasterlin tripeptides

Hemiasterlin 30, a potent cytotoxic peptide that was originally isolated from the sea sponge, Hemiasterella minor, noncompetitively inhibits vincristine-tubulin binding through interaction at the vinca peptide site ( ). Its inhibition of tubulin polymerization resulted in IC50 values as low as 0.0014 mgmL~1 in human cancer cells.106'107 Consequently, hemiasterlin has generated substantial interest as a lead compound in the search for new cancer chemotherapies.108-110 SAR data reveal that structural diversification of either the aryl substituent or the carboxylic acid may provide analogs that maintain antiproliferative activity.111,112

HTI-286 31 (Figure 10) is a synthetic derivative of hemiasterlin that has the advantage of maintaining potency in MDR cell lines that overexpress P-glycoprotein (P-gp). Although in vitro resistance of the KB-3-1 epidermoid carcinoma cell line has been observed, this phenomenon results from a point mutation in alpha-tubulin and from a novel efflux pump, not P-gp.113 Preclinical mouse xenograft studies, dosing of HTI-286 at the maximum tolerated dose (1.6 mgkg _ 1 intravenously) resulted in up to 98% growth inhibition of both paclitaxel-sensitive and paclitaxel-resistant tumor cells.114 Phase I human clinical trials involved administering this molecule intravenously over 30min every 21 days at doses ranging from 0.06 to 2.0 mgm_ 2 and generated toxicities including neutropenia, nausea, alopecia, and pain. The resulting recommended phase II dose of HIT-286 was near 1.5 mgm_2.115

7.04.2.2 Inhibitors Targeting the Colchicine Alkaloid Binding Domain

Binders to the colchicine domain of tubulin are inhibitors of mitosis, although precise details of the mechanism are unclear. For example, binding of the drug molecule to microtubule ends, which destabilizes the microtubules leading to depolymerization, only occurs at drug concentrations well above those required to show activity. At lower concentrations these compounds bind to the alpha, beta-tubulin dimer, which is subsequently incorporated into the growing polymer. Although this binding does not stop the ability of the microtubule to grow, it does appear to disrupt the lattice, impairing the delicately balanced microtubule dynamics required for progression of mitosis.116 Early labeling experiments had localized the binding of colchicines to alpha-tubulin.117,118 More recently, these results were confirmed through a crystal structure of colchicine itself bound to a tubulin heterodimer,119 which shows that the heterodimer undergoes a conformational change to a more bent form in the presence of the drug. This conformational change results in a loss of lateral contacts between the heterodimers, which it is believed results in the observed lower microtubule dynamic stability. It is inferred from these results that compounds that bind to this site achieve the antimitotic effect via a similar mechanism.

7.04.2.2.1 Colchicine and analogs

Colchicine 32 is a naturally occurring alkaloid from the plant meadow saffron (Colchium autumnnale L.). It was used as a poison in Roman times but was not isolated in pure form until 1820. The structure caused much debate and was finally confirmed by x-ray crystallography in the early 1950s (Figure 11).121 While the molecule was not successful as an anticancer agent, due primarily to its toxicity, it has been used clinically for the treatment of gout,122 familial Mediterranean fever,123 and liver cirrhosis.124

Colchicine was first produced synthetically in 1959 and, while there are now many syntheses employing a range of strategies in the literature,125 it still remains a synthetic challenge to integrate all the complex structural features into this molecule. Using total synthesis and semisynthetic approaches, many derivatives of colchicine have been synthesized. From these compounds it has been shown that the B ring is not involved in binding, but does hold the molecule in the active conformation with respect to the A and C rings. Indeed the unnatural 7R-colchicine 33, which exists as the other atropisomer, does not bind tubulin. Furthermore, the 7-acetamide is not required for tubulin binding. The naturally occurring 7-ketone, colchicone,126 is similar to colchicine, while introducing a double bond into the B ring 34 improves binding to tubulin.127

In the tropolone C ring, swapping the keto and methoxy groups leads to inactive compounds. The 10-methoxy group can be varied without affecting tubulin binding (Figures 12 and 13). Halides, alkyl, alkoxy, and amino groups have all been synthesized without affecting activity.128 However, activity is decreased as the steric bulk increases at this position. Moving from a seven-membered tropolone to the aromatic phenol compound, androbiphenylene 35, a natural product, does not impact tubulin binding. In a study of aromatic biphenyls,129 tubulin binding could be maintained upon replacement of the C ring by a 4-methylketone, but was lost with a 4-methoxy group. This study also investigated the effect of methoxy groups on the A ring. It was found that the 4-methoxy was required for strong binding. By contrast, the 2- and 3-methoxy groups are not critical for strong binding, although the 2-methoxy group might be important in setting the correct conformation of the molecule.

MeO Colchicine (32)

MeO Colchicine (32)

Figure 11 Structure of colchicine.

Figure 12 Structures of colchicine analogs.

Figure 12 Structures of colchicine analogs.

Figure 13 Colchicine SAR.

7.04.2.2.2 Podophyllotoxin and steganacin

Podophyllotoxin 36 is found in a number of plants together with a number of related compounds (Figure 14). The chemistry and biology of this compound and related plant natural products have been reviewed.130 The biology and SAR for this class of compounds have been complicated by the presence of a second potential mechanism for cytotoxicity, inhibition of topoisomerase II (topo II). The SAR described in this section is, unless otherwise noted, for tubulin inhibition.

Podophyllotoxin itself is a potent antimitotic agent, which binds in the colchicine-binding site of tubulin at submicromolar concentrations. In cell culture experiments, the subnanomolar cytotoxicity of this compound led to its investigation as an anticancer agent. This compound failed clinically due in part to its toxicity; however, podophyllotoxin has been used topically for the treatment of venereal warts for several decades.

From the many related compounds, a good understanding of the SAR has been obtained.131 The stereochemistry of the C-1 position is important for positioning the E ring, for the enantiomer is inactive. Replacing the methyl of the 4'-methoxy with bulky groups lowers the ability of the molecule to bind tubulin; however, its removal has little effect. Removal of the 3'-methoxy in combination with the methyl of the 4'-methoxy lowers the activity of the compound. The configuration of the trans fused D-ring lactone is crucial to activity, with the C-2 epimer, obtained upon treatment with mild base, being inactive. The lactone moiety itself is not necessary. Indeed, opening of the lactone with hydrazines yields the podophyllic acid hydrazides, which are weaker, but the ethyl hydrizide 37 has been used clinically as an anticancer agent. The cyclic ether 38 is only twofold less active than podophyllotoxin, but increasing the size of the heteroatom lowers the activity, suggesting a steric constraint in this portion of the molecule.

Aromatization of the C ring or ring opening leads to compounds with low or no activity; presumably this is due to the E ring no longer being positioned correctly. However, dehydration to the apopicropodophyllotoxin 39 has improved activity. Inversion of the 4-hydroxyl leads to a set of naturally occurring and semisynthetic compounds that are usually glycosylated. These are characterized by etoposide 40, which is used clinically for the treatment of a range of cancers. However, etoposide does not bind to tubulin; it is a topoisomerase II inhibitor. All analogs appear to exert their activity by this mechanism.132

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