**5.3. Three-atom linkers**

Three-atom linkers have also been reported in the literature (**Figure 4**, **21**, **22**). It was suggested that the presence of a three-atom linker may increase RAR selectivity [45], and the ability for linkers to form hydrogen bonds may enhance RARγ selectivity [46]. As such, a series of urea/ thiourea derivatives were synthesized and evaluated (**Figure 1**, **14**–**20**) [6, 7]. Unlike conventional retinoids, these Flex-Hets were able to induce selective and potent apoptotic activity in cancer cells independent of RAR/RXR activation [44]. The only retinoid activity retained by Flex-Hets is the ability to induce differentiation and reverse the cancerous phenotype.

The X-ray crystal structure of the Flex-Hets showed that a unique lattice network formed through extensive intermolecular H-bonding between the NH and the oxygen of the highly polarized C=O urea linker in another molecule, whereas this was not observed in the C=S thiourea derivatives. It suggested that the urea derivatives may be more active than its thiourea counterpart. This observation is also supported by their *in vitro* growth inhibition activities where the urea derivative, SHetC2 (**17**), demonstrated to be slightly more potent (EC50= 1.02 μM) than its thiourea counterpart, SHetA2 (**16**) (EC50= 1.72 μM) [41].

Regardless, the fact remains that millions of dollars have been invested by the NCI RAID and RAPID programs in the preclinical development of SHetA2, and many studies involving animal models have shown that SHetA2 is a potent and selective inducer of apoptosis with no significant toxicities. While SHetC2 lacks these extensive studies, it does show potential as the next chemopreventive drug candidate following SHetA2. These results indicate that the inclusion of three-atom urea/thiourea linker is critical to induce potent anticancer activities independent of RAR/RXR activation as observed in these Flex-Hets. **Tables 2** and **3** summarize the effects of the structural modifications on the growth inhibition by these derivatives against various cancer cell lines reported to-date.


the thiochroman ring in enhancing the activity of Flex-Hets. Therefore, the thiochroman ring

To further increase the selectivity for each RAR and RXR receptors, various linkers were placed between the two aryl groups of the Hets by modifying their structure rigidity. Two-atom linker compounds such as amide (**Figure 1**, **8**) and ester (**Figure 1**, **9**) were reported [42, 43]. Compound (**8**) was found to be a receptor panagonist, while compound (**9**) was RXR selective. Both showed significant growth inhibitory activities against head and neck cancer using a tumor xenograft mouse model [42]; however, only compound (**8**) induced apoptosis in ovarian cancer cells. This indicated that RXR activation is sufficient to inhibit tumor growth, while activation of both RAR and RXR are required for the maximum activity, at the expense of toxicity. Other two ester-linked compounds (**Figure 1**, **10**, **11**) were found to activate both RARs and RXRs [44]. On the other hand, these ester-linked Hets appeared to only induce growth inhibition but not

Three-atom linkers have also been reported in the literature (**Figure 4**, **21**, **22**). It was suggested that the presence of a three-atom linker may increase RAR selectivity [45], and the ability for linkers to form hydrogen bonds may enhance RARγ selectivity [46]. As such, a series of urea/ thiourea derivatives were synthesized and evaluated (**Figure 1**, **14**–**20**) [6, 7]. Unlike conventional retinoids, these Flex-Hets were able to induce selective and potent apoptotic activity in cancer cells independent of RAR/RXR activation [44]. The only retinoid activity retained by Flex-Hets is the ability to induce differentiation and reverse the cancerous phenotype.

The X-ray crystal structure of the Flex-Hets showed that a unique lattice network formed through extensive intermolecular H-bonding between the NH and the oxygen of the highly polarized C=O urea linker in another molecule, whereas this was not observed in the C=S thiourea derivatives. It suggested that the urea derivatives may be more active than its thiourea counterpart. This observation is also supported by their *in vitro* growth inhibition activities where the urea derivative, SHetC2 (**17**), demonstrated to be slightly more potent (EC50= 1.02

Regardless, the fact remains that millions of dollars have been invested by the NCI RAID and RAPID programs in the preclinical development of SHetA2, and many studies involving animal models have shown that SHetA2 is a potent and selective inducer of apoptosis with no significant toxicities. While SHetC2 lacks these extensive studies, it does show potential as the next chemopreventive drug candidate following SHetA2. These results indicate that the inclusion of three-atom urea/thiourea linker is critical to induce potent anticancer activities independent of RAR/RXR activation as observed in these Flex-Hets. **Tables 2** and **3** summarize the effects of the structural modifications on the growth inhibition by these derivatives against

μM) than its thiourea counterpart, SHetA2 (**16**) (EC50= 1.72 μM) [41].

various cancer cell lines reported to-date.

forms one of the fundamental moieties of SHetA2 and its analogs (14–20).

**5.2. Two-atom linkers**

76 Anti-cancer Drugs - Nature, Synthesis and Cell

apoptosis.

**5.3. Three-atom linkers**


Growth inhibition (%) for renal cancer cell lines (1) Caki-1 and (2) 786-0; Normal renal cells (1) HK-2 and (2) RTC91696 [23]. Growth Inhibition (%) for ovarian cancer cell lines (1) CAOV-3, (2) OVCAR-3, and (3) SKOV-3 [6]. EC50 values for 50% growth inhibition for ovarian cancer cell line (4) A2780, and Normal endometrial cells (NE) [6, 41]. Growth inhibition (%) for cervical cancer cell lines (1) SiHa, (2) CC-1, (3) C33a, and (4) HT-3 [5]. Growth inhibition (%) for head and neck squamous cell cancer cell lines (HN) (1) SCC-2 [43], and (2) SCC-38 [32, 42]. Growth inhibition (%) for vulvar cancer cell lines (1) SW954 and (2) SW962 from Ref. [43]. "–" indicates no data available.

**Table 2.** Structural modifications of Hets, and their effects on cancer cell growth.


Growth inhibition (%) for renal cancer cell lines (1) Caki-1 and (2) 786-0; Normal renal cells (1) HK-2 and (2) RTC91696 [23]. Growth Inhibition (%) for ovarian cancer cell lines (1) CAOV-3, (2) OVCAR-3, and (3) SKOV-3 [6]. EC50 values for 50% growth inhibition for ovarian cancer cell line (4) A2780, and Normal endometrial cells (NE) [6, 41]. Growth inhibition (%) for cervical cancer cell lines (1) SiHa, (2) CC-1, (3) C33a, and (4) HT-3 [5]. Growth inhibition (%) for head and neck squamous cell cancer cell lines (HN) (1) SCC-2 [43], and (2) SCC-38 [32, 42]. Growth inhibition (%) for vulvar cancer cell lines (1) SW954 and (2) SW962 from Ref. [43].

"–" indicates no data available.

**Table 3.** Structural modifications of Flex-Hets, and their effects on cancer cell growth.

Substitutions on the phenyl group have also been evaluated [7]. The nitro (NO2) substitution (**Table 3**, **16**, **17**) consistently exhibited greater growth inhibitory and apoptotic activity than their methyl (**Table 3**, **18**–**20**) or ethyl ester counterparts (**Table 3**, **14**, **15**). This suggests that the nitro substitution may have enhanced the overall activity of the compound. Collectively, for the Flex-Hets, the thiochroman ring and nitro substitution are important for enhancing the anticancer activity, while the thiourea linker is crucial for RAR/RXR independent and selective anticancer activities against cancer cells.
