Other Novel Antiproliferative Agents

In the discovery phase, compounds continue to be identified and optimized by assay in a tumor cell line, although an MOA or biochemical target is not known. The compounds are identified from a variety of sources, including natural products, or from modification of structures that are known to be bioactive, or by de novo chemical synthesis. Authors often describe the compounds as ''novel'' because they have a previously unreported structural modification or they possess unique activity profiles in tumor cells. The data in the initial reports of these compounds are focused on SAR in an in vitro tumor cell assay, and may contain evidence of in vivo activity in a human tumor xenograft model. Information on ADME and PK properties is generally not available for the examples in Table 3.

An initial hypothesis that 2-phenylbenzothiazoles could mimic ATP and thus inhibit tyrosine kinases led to a series of compounds in which a 2-(dimethoxyphe-nyl)benzothiazole, GW610 (7), was found to be the most active member (Fig. 3) [20]. The initial in vitro assay was in four tumor cell lines (MCF-7, MDA 468, KM 12, and HCC2998), with follow up of the most active members in the NCI's full panel of 60 human tumor cell lines [21]. In the smaller set of tumor cells, 7 was most active in the breast cell line MDA 468. In the larger NCI panel, the compound exhibited its greatest activity in the colon and NSCLC subpanels. Interestingly, 7 appears to complement a more extensively studied 2-(aminophenyl)benzothiazole, in that breast tumor cell lines which acquire resistance to the (aminophenyl)benzothiazole retain sensitivity to the dimethoxyphenyl analog. A COMPARE analysis was not reported, a molecular target was not identified from this study, and in vivo studies were not undertaken, but the authors indicate such work will be reported at a later time.

A series of anthranilic acid analogs were prepared and assayed in the NCI's full panel of 60 tumor cell lines, and the most active compound was found to be entry 8, a 2-trifluoromethylpyridyl derivative [22]. The compound was especially active in

Table 3.

Novel antiproliferatives with new structures or

biological characterization

Cmpd

no.

Name

Source of structure

Assay for SAR

In vivo model

Mechanism

7

GW 610

Bioactive skeleton

MCF-7 (breast)

None reported

None reported

(benzothiazole)

MDA 468 (breast)

KM 12 (colon)

HCC 2998 (colon)

8

N-(2-(trifluoro-methyl)-

Bioactive skeleton

NCI's full panel of

U251 (CNS)

COMPARE performed;

pyridin-4-yl)

(anthranilic acid)

60 human

possibly antimetabolite

anthranilic ester

tumor cell lines

or topoisomerase II

inhibitor

9

NSC-686288; TK-2339

Bioactive skeleton

MCF7 (breast)

MCF7 (breast)

Induces DNA strand

(flavone)

breaks

10

Cyclopenta-[e]azepine-

Bioactive skeleton

In vitro cell line

None reported

COMPARE performed;

4,10(1 H,5 H)-diones

(benzazepine-

screen at NCI

possibly tyrosine

diones)

kinases

11

Pyrrolo [3,2-f]quinolin-

Bioactive skeleton

Included:

BNL 1ME A.7R.1

Antimitotic via tubulin

9-one

(flavone)

MCF7 (breast)

murine liver

depolymerization

H295R

carcinoma

HT-29 (colon)

12

EPC-2407; MX-116407

Synthetic library

T47D (breast),

Calu-6 (lung)

Antimitotic via tubulin

H1299

inhibition

(NSCLC)

DLD-1 (colon)

13

Decursin

Natural product

DU145 (prostate)

Murine sarcoma

Cell cycle arrest,

PC-3 (prostate)

180

apoptosis via caspase-

LNCaP (prostate)

dependent and

independent pathways

CD CD D

leukemia and colon tumor cell lines in vitro, but subsequent assay in hollow fibers and xenografts in vivo showed its greatest activity in a central nervous system tumor, U251, when dosed intraperitoneally (ip) at 100mg/kg daily for 4 days. The growth inhibition was stated as "moderate" by the authors (67% of control under the assay conditions), but the PK properties of 8 were not described; potentially dosing on a different schedule or route could result in greater tumor growth inhibition. With respect to mechanism of action, the COMPARE PCC for 8 was greatest with the antimetabolite, morpholino-ADR, and the topoisomerase II inhibitor, vincristine. The conclusion drawn at present is that these anthranilates act by inhibiting cell cycle progression, but additional studies will be necessary to determine a specific target.

Natural and synthetic flavonoid derivatives exhibit a variety of biological activities, including antitumor activity, although the antiproliferative mechanisms are not fully understood. Researchers at Kyowa Hakko made two key observations: hydroxy-substituted flavonoids exhibit activity associated with breast cancer or estrogenic action, and that few analogs existed in which the hydroxyl group had been replaced by an amine. A series of modifications were analyzed, first confirming that an amino flavone inhibited in vitro antiproliferative activity in estradiol-stimu-lated MCF-7 breast cancer cells with an IC50 value of 7.2 nM [23]. Subsequently, fluorine was introduced to both the fused and pendant aromatic rings to enhance the metabolic stability of the scaffold, and this analog potently inhibited the growth of both estrogen receptor (ER) positive breast cancer cell lines and cells from ER-negative cancers of the breast, ovaries, endometrium, and liver [24]. The final iteration of the series explored the 7-position of the flavone ring, providing NSC-686288 (9), and it was found that methyl substitution was preferred at this position in vitro and in vivo in MCF7 cell and xenograft assays, respectively [25]. Mechanistic studies at the NCI have shown that 9 is metabolized by CYP1A1, and that it induces the expression of CYP1A1 in MCF-7 cells [26]. It additionally has been demonstrated that 9 involves the aryl hydrocarbon receptor (AhR) in the regulation of the CYP1A1 expression [27], and more recently 9 has been shown to induce DNA single strand breaks and DNA-protein cross-links [28].

The fused tricycle cyclopenta-[e]azepine-4,10(1 H,5 H)-dione 10 was discovered via modification of the synthesis which had provided cytotoxic antitumor benzaze-pines [29]. A tandem Michael-Aldol reaction in the presence of base led to a new compound class of 6/7/5 fused tricyclics as mixtures of diastereomers [30]. Separation and analysis of the diastereomers in the NCI's in vitro cell line screening project (IVCLSP) found that alteration of the substitution pattern on the saturated 5-membered ring (1,3-disubstitution vs. 2,3-disubstitution as shown in 10) indicated distinctly different mechanisms of action. The highest correlations of 10 with known agents were with bleomycin and 4-nitro-estrone-4-methyl ester, which do not have a common molecular target. To further explore activity suggested by the NCI's database of molecular targets, the most active diastereomer of 10 was assayed at 10 mM concentration in 16 cancer-related kinases, but only moderate inhibitory activity was not found. Thus, it is proposed that 10 could interact with molecules of the signaling cascades in which kinases are involved, although it may not be a kinase inhibitor itself. Additional investigations will be necessary to establish the mechanism of action of this novel ring system.

The finding that some natural flavonoids have antimitotic cytotoxic activity by interfering with tubulin polymerization spurred the synthesis of another analog, 2-phenylpyrroloquinolin-4-one. Two structural variants have been reported, varying the fusion position of a pyrrole ring to the quinolone [31,32]. The SAR was developed in human and mouse tumor cell lines including those derived from liver, pancreas, colon, thyroid, ovary, and breast solid tumors. The daily ip injection of 11 (40mg/kg) caused a significant (83%) inhibition of tumor growth relative to control groups in the hormone-sensitive syngeneic murine hepatocarcinoma BNL 1ME A.7R.1 [32]. Based on evidence of G2/M arrest in two cell lines by flow-activated cell sorting analysis and dose-dependent inhibition of tubulin polymerization similar to vincristin, the mechanism of cytotoxicity is hypothesized to be antimitotic via tubulin depolymerization, although a specific molecular target has not been identified.

A commercially obtained compound library yielded a novel series of 2-amino-4-(3-bromo-4,5-dimethoxy-phenyl)-3-cyano-4H chromenes as potent apoptosis inhibitors in high-throughput assay utilizing HL60 B-cell leukemia cancer cells [33]. Selected compounds were profiled further and found to have activity only toward proliferating cells and to interact with colchicines at the tubulin binding site, thereby inhibiting tubulin polymerization and leading to cell cycle arrest and apoptosis [34]. The most potent member of the family, MX-116407 (12), was found to be highly active in the human lung tumor xenograft, Calu-6, and is claimed as the lead candidate, although its structure has not been disclosed [35]. The SAR and structural variation within the family has been explicitly described in the literature, and the 7-dimethylamino analog, MX-58151, demonstrated potency similar to col-chicines and vinblastine with an EC50 value of 19 nM in a caspase activation assay [33]. The compounds also are active in cells resistant to other antimitotic agents such as the taxanes and vinca alkaloids, and therefore might offer an advantage for the treatment of drug-resistant cancers.

Screening natural products for anticancer activity remains productive, and novel compounds continue to be identified in this manner. Decursin 13 is a coumarin compound that was isolated from Korean angelica gigas root and prepared via total synthesis in 2003 [36]. Like the flavonoids, it exhibits a variety of biological activities, including antibacterial, neuroprotective, and antioxidant. However, only recently has 13 been demonstrated to possess in vivo activity in murine sarcoma 180 [37] and to inhibit growth and induce cell death in human prostate cancer cell lines DU145, PC-3, and LNCaP [38]. Its inhibitory mechanism appears to be associated with G1 arrest in DU145 and LNCaP cells and its apoptotic effect is linked to both caspase-dependent and -independent pathways. Notably, it has no effect on normal PWR-1E prostate epithelial cells.

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