Total synthesis of a direct derivative of intact halichondrins

Specifically, we recognized that our recent studies in the total syntheses of halichondrins could solve the problem of limited material supply via chemical synthesis. This approach has led us to E7130, as a promising anticancer drug candidate (Fig. 1a). The first batch of E7130 was prepared on a milligram scale via a modified version of the original synthesis13. This synthesis required 109 steps from commercially available materials, and HPLC purification of E7130 was essential to remove a number of unidentified impurities. Meanwhile, significant progress has been made in the total synthesis of halichondrins, including the discovery and development of catalytic, asymmetric Ni/Cr-mediated cross-coupling reactions and Zr/Ni-mediated one-pot ketone synthesis. These developments have been incorporated to this project. Thus, C52-halichondrin-B alcohol (E7130 precursor) was synthesized from the left and right halves via a C37-C38 bond formation in the new synthesis14,15,16, whereas the previous synthesis used a C38-C39 bond formation as a key step13 (Fig. 1b, and the relevant parts of E7130 synthesis are paragraph numbers [00868]-[00909] in WO2019/010363 and paragraph numbers [00387]-[00417], [00428]-[00432] and [00544]-[00547] in WO2016/176560). This modification resulted in remarkable improvements on the overall efficiency of synthesis in several respects. First, the longest linear sequence of the right half was reduced from 42 steps to 25 steps from a commercially available material. Second, in the new synthesis, the [6,6]-spiroketal in the left half was built before the final coupling, whereas it was constructed via an oxy-Michael reaction after the final coupling in the previous synthesis. Although both routes gave the desired product as the major product, the isolated yield of C52-halichondrin-B alcohol in the new route was dramatically higher than that in the original route. Third, a practical and scalable synthesis was developed by selecting the appropriate coupling conditions for four catalytic, asymmetric Ni/Cr-mediated coupling reactions as well as for other transformations17. Overall, we successfully synthesized 19.5 g of C52-halichondrin-B alcohol with 99.84% purity via a total synthesis14. From 15.0 g of this material, we obtained 11.5 g of E7130 with 99.81% purity, and the material was isolated by reverse-phase medium-pressure chromatography (Fig. 1b).

Figure 1 Chemical structures referenced in this work and the scale-up strategy. (a) Chemical structures of halichondrin B, norhalichondrin B, C52-halichondrin-B alcohol, and C52-halichondrin-B amine/E7130. (b) The strategy for scale-up. The previous synthesis relied on a C38-C39 bond formation strategy. The new synthesis relies on a C37-C38 bond formation strategy. The latter provided 19.5 g of C52-halichondrin-B alcohol with 99.84% purity. Fifteen grams of this compound was used to synthesize 11.5 g of E7130 with 99.81% purity as the first GMP batch (92 overall steps). Full size image

E7130 increases intratumoural microvessel density

With totally synthetic E7130 in hand, we began to study in vivo antitumour activity in mice reported for the halichondrin class of marine natural products. E7130 showed inhibitory activity towards tubulin polymerization in the cell-free system and caused the disappearance of the EB3 comet-structures, indicating that E7130 has the ability to suppress microtubule dynamics (Supplementary Fig. S1a,b, Supplementary Video 1 and 2). E7130 exhibited potent antiproliferative activities against several cancer cell lines in vitro with subnanomolar IC 50 values (Fig. 2a), which shows a more than 10-fold different in potency of E7130 across the cell lines. Although the reason of this has not been clarified, sensitivity to microtubule-targeted drugs is known to be affected by the expression of the drug efflux pump such as P-glycoprotein and mutations in and/or alteration of tubulin isotype levels18,19. E7130 also exerted significant and dose-dependent antitumour activities with tumour regression in KPL-4 HER2-positive breast cancer and OSC-19 squamous cell carcinoma of the head and neck (SCCHN) s.c. xenograft models (Supplementary Fig. S1c,d). In addition to its direct antitumour activities, we noted that E7130 increased the intratumoural microvessel density (MVD) in xenografts (Fig. 2b). To investigate whether the increased intratumoural microvessels are functional, we assessed the accumulation of cetuximab (CTX) in the presence of E7130 in the HSC-2 SCCHN orthotopic transplantation mouse model. Indeed, we found that E7130 significantly enhanced delivery of CTX into tumours (Fig. 2c). Moreover, notable tumour regression was observed when E7130 was used in combination with CTX. On the other hand, cisplatin (CDDP), which did not increase intratumoural MVD nor delivery of CTX, did not show tumour regression activity, even when used in combination with E7130 (Supplementary Fig. S2). The synergistic effect was also demonstrated by the apparent prolongation of survival in the model, and the prominent antitumour activity of the compounds together was also confirmed in the HSC-2 s.c. xenograft model (Fig. 2d,e). These data unequivocally supported that the increased intratumoural MVD caused by E7130 enhanced the delivery of CTX into tumours and led to a clear tumour regression and survival advantage. This mechanism of action is further supported by the data from two other cell line s.c. inoculation models. The first is the KPL-4 model, in which E7130 increased intratumoural MVD (Supplementary Fig. S3d). E7130 in combination with trastuzumab showed a stronger antitumour activity than each monotherapy in this model (Supplementary Fig. S3b,c). The second is the CT26 murine colon carcinoma model, in which E7130 in combination with an anti-mouse PD-1 antibody increased intratumoural accumulation of the antibody. The combination of these compounds delayed tumour growth relative to each monotherapy (Supplementary Fig. S4).

Figure 2 Biochemical, cellular, and in vivo mechanistic activity of E7130. (a) The effect of E7130 on the viability of 4 cell lines after 3 days represented as the concentration of E7130 required to decrease cell viability to 50% (IC 50 ) and 95% confidence interval (CI). (b) HSC-2 squamous cell carcinoma of the head and neck orthotopically transplanted tumours were collected 4 days after the administration. Data show the mean tumour vessel ratios of treated to non-treated ± s.e.m. (n = 3). *P = 0.0228, **P = 0.0030 versus non-treated (Dunnett’s multiple comparison test). (c) The accumulation of fluorescent-labelled cetuximab (FPI-CTX) was analysed using an In Vivo Imaging System (IVIS) 5 days after the indicated administration. Representative in vivo bioluminescence images and ex vivo fluorescence labelling in resected tongues are shown. The values of total flux (photon/second) were normalized with each bioluminescent value (photon/second). The graph shows the mean FPI-CTX accumulation ratios to the accumulation in the FPI-CTX mono-administration group ± s.e.m. (n = 4). *P = 0.0440 versus FPI-CTX mono-administration (Two-tailed unpaired t test). (d) Effect of the indicated administration on day 1, day 8, and day 15 on survival in the HSC-2 orthotopic transplantation mouse model (n = 16). (e) Effect of the indicated administration on day 1, day 8, and day 15 on the relative tumour volume of the subcutaneous HSC-2 subcutaneous xenograft model. In this study, nine days after the cell inoculation subcutaneously in the right flank of Balb/C-nu mice, 36 mice were selected based on their tumour volumes and shapes of tumours, and were randomly allocated into 6 groups (day 1). The mean tumour volume of mice assigned to the groups on day 1 were 321.6 mm3. The mean relative tumour volume to the tumour volume on day 1 ± s.e.m. is shown (n = 6). Full size image

E7130 ameliorates the tumour microenvironment to improve cancer treatment

We further analysed the combined effect of E7130 with CTX in the FaDu SCCHN s.c. xenograft model. As in the case of the HSC-2 model, a remarkable combined effect was observed above the dose of 90 μg/kg, one-half of the maximum tolerated dose in mice, even after a single administration. Interestingly, tumour regression in the E7130 combination groups (90 μg/kg and 180 μg/kg) was sustained for a longer period than was seen in the paclitaxel combination group (Fig. 3a). These observations led us to the hypothesis that an additional function of E7130 inhibits tumour regrowth in the combinational groups. To test this hypothesis, we analysed the changes in the intratumoural cancer-associated fibroblasts (CAFs) in the xenografts because intratumoural CAFs are key characteristics of SCCHN and are negative prognostic factors contributing to worse clinical outcomes for those with the disease20,21. The effect on CAFs was analysed by quantifying the area of α-SMA-positive cells, which is a well-known activated CAF marker in SCCHN22, and the immunohistochemical analyses were performed with tumour tissues collected 10 days after the administration, which was just before the tumour regrowth in the paclitaxel combination group. Interestingly, E7130 in the combination treatment significantly reduced the α-SMA-positive CAFs at 90 μg/kg and 180 μg/kg in the xenografts, whereas paclitaxel did not show this effect (Fig. 3b,c). In contrast, ER-TR7 (pan-fibroblast marker) staining clearly indicated that the E7130 combination did not reduce the overall stroma area (Supplementary Fig. S5a), which suggests that E7130 specifically reduced the α-SMA-positive CAFs, and the E7130 combination modulated the phenotypes of the fibroblasts. Furthermore, Ki67-positive cancer cells were observed adjacent to the α-SMA-positive CAFs in tumours treated with the paclitaxel combination, whereas such staining patterns were much less frequently observed in the E7130 combination (Fig. 3d). As the candidate for the downstream effector in α-SMA-positive CAFs, which can account for its tumour-promoting function, we examined the expression of tenascin-C and EDA-fibronectin, extracellular matrix (ECM) proteins23,24. Immunohistochemical analysis demonstrated that tenascin-C and EDA-fibronectin were reduced by E7130 in combination with CTX but not by paclitaxel in combination with CTX (Fig. 3e, Supplementary Fig. S5b–d). These results strongly suggested that α-SMA-positive CAFs provide some growth-promoting signals to neighbouring cancer cells, which led to tumour regrowth, and the difference in the potencies of the treatments for reducing such tumour-promoting α-SMA-positive CAFs can account for their different antitumour activities in terms of the suppression of regrowth between treatment with E7130 and CTX and treatment with paclitaxel and CTX. In addition, we confirmed the potency of E7130 monotherapy for reducing α-SMA-positive CAFs in this model and the other types of SCCHN xenografts (Supplementary Fig. S5e–h).

Figure 3 E7130 showed an anti-CAF effect leading to combinational effect against FaDu xenografts. (a) Effect of the indicated administration on day 1 on the relative tumour volume of FaDu subcutaneous xenografts. The mean relative tumour volume to the volume on day 1 ± s.e.m. is shown (n = 6). (b–e) FaDu xenografts were collected 10 days after the indicated administration. The areas of α-SMA (b–d), Ki67 (d), and tenascin-C (e) were analysed by immunohistochemistry. The data shown are the mean area of α-SMA-positive CAFs (b) or tenascin-C (e) to those of the non-treated group ± s.e.m. (n = 5). (b) **P = 0.0021, ***P = 0.0002, (e) *P = 0.0360, **P = 0.0031 versus the non-treated group (Dunnett’s multiple comparison test). Representative images are shown in (c,d). Full size image

E7130 impedes the TGF-β-induced myofibroblast transdifferentiation process

Next, we analysed the molecular mechanisms of the α-SMA-positive CAF reduction using an in vitro culture system. We first confirmed that the expression of α-SMA was induced in BJ normal human fibroblasts upon co-cultivation with FaDu cells, and the expression was attenuated by treatment with A83-01, a potent selective inhibitor of the TGF-β-receptor (Fig. 4a). These results suggested that TGF-β plays a major role as a mediator of α-SMA induction in this system. In addition, immunofluorescence analysis revealed that treatment with E7130 interfered with α-SMA induction by TGF-β in BJ cells without growth inhibitory activity (Fig. 4b, Supplementary Fig. S6a). We further found that E7130 did not significantly change the TGF-β-induced phosphorylation and nuclear localization of Smad2/3 (Supplementary Fig. S6d,e), but it reduced the activation of the PI3K/AKT/mTOR pathway, which plays essential roles in TGF-β-induced α-SMA expression (Fig. 4c,f and Supplementary Fig. S6b,c). Moreover, TGF-β treatment enhanced β-tubulin expression and microtubule network formation, which were diminished by co-treatment with E7130 in BJ cells (Fig. 4d). Under the same experimental settings, the enhanced formation of focal adhesions, which were detected as punctate structures with the antibody against phosphorylated FAK at tyrosine-397, after stimulation with TGF-β was decreased in BJ cells by treatment with E7130 (Fig. 4e). Many signalling complexes are assembled in focal adhesion sites, and those complexes dispatch several downstream signals, including those involved in the PI3K/AKT/mTOR pathway25. In fact, treatment with defactinib, an FAK inhibitor, clearly decreased the level of α-SMA expression as well as S6 ribosomal protein phosphorylation induced by TGF-β in BJ cells (Fig. 4g,h). We confirmed that all of the abovementioned phenomena were also observed in other normal human fibroblasts (TIG3 cells) (Supplementary Fig. S7). Considering that several reports have also found that plus ends of microtubules interact with focal adhesion sites and modulate their functions in interphase cells26, our results strongly suggest that E7130 impedes the TGF-β-induced myofibroblast transdifferentiation process by disrupting microtubule network formation, which is important for focal adhesion assembly and thereby the downstream activation of the PI3K/AKT/mTOR pathway.