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Synthesis of seven semi-synthetic analogs of NDGA is described. An approach to NDGA derivitization is described in which the ortho-phenolic groups are tethered together by one atom, forming a 5-membered heterocyclic ring. The analogs were evaluated for cytotoxicity in four cancer cell lines and compared to NDGA and tetra-O-methyl-NDGA (M4N) (1a). NDGA bis-cyclic sulfate (2a), NDGA bis-cyclic carbonate (2b), and methylenedioxyphenyl-NDGA (2d) and NDGA tetra acetate (1b) showed anti-cancer activity in vitro. Two compounds, (1b) and (2b), were evaluated for anticancer activity in a mouse xenograft model of human melanoma and showed dose-dependent activity.
Nordihydroguaiaretic acid (NDGA) is a lignan that is found at up to 10–15% by dry weight in the leaves and twigs of the creosote bush (Larrea tridentata (Sesse & Moc.) Coville, Zygophyllaceae).1 NDGA is a 5-lipoxygenase inhibitor with a well-defined mechanism of action requiring “free” phenolic groups for activity.2 Tetra-substituted, “masked” phenolic group NDGA analogs were found to be devoid of inhibitory activity against soybean, human 12 and human 15- lipoxygenase.3 NDGA has shown many biological activities including anticancer activity. In vitro, NDGA inhibited DNA synthesis in K562 chronic myelogenous leukemia blast cells with an IC50 of 230 µM.4 NDGA inhibited the proliferation of human small cell lung5, non-small cell (NSCLC)6 lung, human pancreatic and cervical cancer cells in vitro.7 In vivo, NDGA inhibited tumor growth in esophageal adenocarcinoma8 and NSCLC xenografts in mice.6
Natural 9,10 and semi-synthetic, O-methylated11,12 analogs of NDGA have been studied for their antiviral activities. The mechanism of antiviral activity was reported to be inhibition of host Sp-1 transcription factor binding with subsequent inhibition of Sp-1-dependent viral gene expression.12 The degree of inhibition was directly proportional to the degree of phenolic group methylation with the tetra-O-methylated NDGA (M4N) showing the strongest inhibition.11 Tetra-acetyl13 and tetra-glycinated14,15 NDGA analogs were shown to have a similar antiviral mechanism. Recently, NDGA analogs have shown anti-HIV activity in an in vitro Tat-regulated secreted alkaline phosphatase assay16.
Heller and colleagues have reported that M4N arrested C3 cells, a HPV-16/ras-transformed, tumorigenic mouse embryo cell line, in the G2 phase of the cell cycle. The growth inhibitory activity of M4N was associated with a decrease in the expression of cyclin-dependent kinase (Cdc2), an Sp-1-promoter dependent gene which progresses the cell through G2/M. In vivo, intratumoral injections of M4N caused a decrease in C3 xenograft tumor size in mice which was correlated with an observed decrease in protein levels of CDC2.17 M4N inhibition of Sp-1 binding was further investigated as a mechanism of anticancer activity. Sp-1 promoter binding is responsible for Cdc2 and survivin gene expression. Survivin, an inhibitor of apoptosis, is overexpressed in most cancer cells. Its expression is G2/M specific and Sp-1 dependent. M4N-induced apoptosis in the C3 cell line was correlated to inhibition of survivin, suggesting that inhibition of survivin expression may be an underlying mechanism of action of M4N. M4N-treated C3 cells showed a decrease in CDC2 and survivin at the mRNA and protein level. A non-Sp-1 dependent promoter showed resistance to M4N-induced cytotoxic activity.18 M4N suppressed tumor growth in Hep3B hepatocellular carcinoma, LnCaP prostate carcinoma, HT-29 colorectal carcinoma, MCF-7 breast carcinoma and K-562 erythroleukemia in a nude mouse xenograft model. A decrease in Cdc2 and survivin gene expression was correlated to tumor growth inhibition.19
M4N inhibited growth in MCF-7 human breast, A549 human lung, SW480 human colon cancer cell lines and was especially potent against A375 and ACC375 human melanoma cells with IC50 values of 2.5 and 5.0 µM, respectively. In the ACC375 cell line, M4N was non-schedule dependent and induced apoptosis. At 50 µM, M4N inhibited DNA synthesis by 66% after 1 hr and by 95% of control after 24 hr demonstrating an early event in growth inhibition. M4N arrested melanoma cells in G1/G0 and G2/M phases of the cell cycle, suggesting it may affect cell cycle checkpoint proteins. In vivo, M4N inhibited tumor growth in the B16 murine melanoma model, as well as in SW480 human colon cancer and A375 human melanoma xenografts in SCID mice.20
Tetra-O-substituted NDGA analogs show decreased in vivo toxicity, for example, the lethal dose 50% (LD50) of NDGA was found to be 75 mg/kg i.p.21 M4N was well-tolerated at 1,000 mg/kg i.p.18, approximately 11 times the LD50 of NDGA.
The purpose of this project was to generate new tetra-O-substituted NDGA analogs for the continued investigation of in vivo, anti-melanoma therapeutic activity demonstrated in our prior studies.20 This work compares the in vitro cytotoxicity of NDGA and M4N to NDGA tetra-acetate and NDGA tetra-methanesulfonate (scheme 1). In addition, we investigate an approach to tetra-O-substitution of NDGA by the tethering together of the ortho phenolic groups by a single atom forming a 5-membered heterocyclic ring (scheme 1). Analogs from this group were evaluated for their cytotoxicity in A375, human malignant melanoma cells, HT-29, human colorectal adenocarcinoma cells, MCF-7, human mammary gland adenocarcinoma cells and HepG2 human hepatoma cancer cell lines. Cytotoxicity was evaluated by 5-day 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and is summarized in Table 1. One analog from each group was evaluated in vivo using the A375 xenograft model in SCID mice.
The analogs 2a, 2b and 2c were synthesized by a modification of the procedure of Tickner et al.22 Analog 2d was synthesized my a method described by Grazia23. Analogs 1a, 2a, 2d were all purified to >95% by semi-preparative HPLC and purity was assessed by 3-dimensional UV scan by this method. Analogs 1b, 1c, 2b, 2c were all purified to >95% (as assessed by 3-dimensional UV scan) using recrystallization from isopropanol.
The MTT assay was used to assess in vitro cytotoxicity of the test compounds. All IC50 determinations are for 5-day treatment times. Compounds 1a, 1b and NDGA showed the highest potency in the MTT assay against the panel of cell lines with IC50 values ranging from 8.5 – 79.4 µM (Table 1). Compounds 2a and 2b had moderate inhibitory activity against MCF-7 cells. Compounds 1c and 2d were 2.2 – 116-fold less potent than NDGA against the panel. Compound 2c showed no significant cytotoxicity against any of the cell lines tested.
The cytotoxicity of the analogs against A375 cells was further evaluated by determining the inhibitory effects of the analogs against DNA, RNA and protein synthesis by a method described by Mayr24 The results are shown in Table 1. There was relatively good agreement between cytotoxicity and inhibition of macromolecular synthesis inhibition with the exception of 1c. This compound was inactive below 1,000 µM in the MTT assay but showed strong inhibitory activity against RNA and DNA synthesis at 85 and 100 µM, respectively. Further studies of this discrepancy are needed.
Effects of the test compounds on tumor growth were determined in two studies. In study 1, 1b, 2a and 2b were suspended in 100% Tween 80 (Sigma-Aldrich, St. Louis, MO.). Mice were implanted s.c. with 10 × 106 A375 human melanoma cells in 100 µl phosphate buffer saline in the rear right flank. The mice were implanted on day 0 and were dosed with test compounds on days 1, 5 and 9. Controls (n=4) received 100% Tween 80. The method for study 2 was identical to study 1, except there were eight mice per group and the analogs were suspended in 100% PEG 300. Controls (n=4) received 100% PEG 300. Tumor volumes were measured 3 times/week and were determined based on the following formula: V(mm3)= length X (width)2/2. The in vivo tumor growth inhibition model data was analyzed by determining % T/C as described by Bissery et. al.25 and are summarized in Table 2. A percent T/C between 42% and 10% is considered moderate tumor growth inhibition. Both 1b and 2b showed moderate growth inhibitory effects against xenograft tumors in the first experiment, however in the second study, tumor growth inhibition was observed only for 1b at the 300mg/kg dose. This may be the result of lower solubility of 2b in a PEG 300 formulation compared to the Tween 80 formulation in study 1. In study 2, PEG 300 was used as the vehicle because Tween 80 displayed some tumor growth inhibition compared to untreated controls in study 1 (data not shown). Compounds 1b and 2b were well-tolerated at doses of 100 to 300 mg/kg and no sign of general toxicity was observed compared to control mice. Compound 2a displayed significant toxicity after a single injection at the doses tested and studies of this compound were discontinued.
In conclusion, among the tetra-substituted analogs, 1a and 1b were clearly more potent in vitro than 1c, although 1c was rather insoluble in vitro above 100 µM. The compounds in scheme 2 showed markedly reduced cytotoxicity compared to scheme 1, with the exception of analogs 2a and 2b which showed IC50 values similar to scheme 1 in the MCF-7 cell line. Moreover, compounds 1b and 2b demonstrated significant antitumor activity in vivo and were well-tolerated at doses 2.6 and 3.4 times the LD50 of NDGA. The fused-ring heterocyclic derivatives of NDGA, such as 2b, represent a new class of derivatives that should be further investigated for their potential antitumor activity and the possible underlying mechanisms.
Materials, Methods and results of synthesis, purification, characterization and bioassay.
The authors would like to acknowledge Bhashyam S. Iyengar and William A. Remers for their assistance in the naming and characterization of the compounds described in this article.
Sponsorship: The study was funded by grant CA017094 from the National Institutes of Health, Bethesda, Maryland, USA.
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