Synthesis and Cytotoxicity of 7,9-O-Linked Macrocyclic C-Seco Taxoids

A series of novel 7,9-O-linked macrocyclic taxoids together with modification at the C2 position were synthesized, and their cytotoxicities against drug-sensitive and P-glycoprotein and βIII-tubulin overexpressed drug-resistant cancer cell lines were evaluated. It is demonstrated that C-seco taxoids conformationally constrained via carbonate containing-linked macrocyclization display increased cytotoxicity on drug-resistant tumors overexpressing both βIII and P-gp, among which compound 22b, bearing a 2-m-methoxybenzoyl group together with a five-atom linker, was identified as the most potent. Molecular modeling suggested the improved cytotoxicity of 22b results from enhanced favorable interactions with the T7 loop region of βIII.


Introduction
Antitubulin agent paclitaxel (1a, Figure 1) and its semi-synthetic derivative docetaxel (1b, Figure 1) are successfully used in the clinic for the treatment of ovarian, breast, and non-small cell lung and prostate cancers and Kaposi's sarcoma.

Introduction
Antitubulin agent paclitaxel (1a, Figure 1) and its semi-synthetic derivative docetaxel (1b, Figure  1) are successfully used in the clinic for the treatment of ovarian, breast, and non-small cell lung and prostate cancers and Kaposi's sarcoma. However, their clinical uses are severely restricted by drug resistance. Various resistance mechanisms toward taxane-based anticancer drugs have been revealed, among which only overexpression of P-glycoprotein (P-gp) and βIII-tubulin have been confirmed clinically [1][2][3][4].
A great deal of effort has been made to overcome multi-drug resistance (MDR) mediated by overexpression of P-gp. Nevertheless, many taxane-based drug candidates targeting P-gp overexpression did not achieve the expected efficacy in clinical trials [5,6]. However, their clinical uses are severely restricted by drug resistance. Various resistance mechanisms toward taxane-based anticancer drugs have been revealed, among which only overexpression of P-glycoprotein (P-gp) and βIII-tubulin have been confirmed clinically [1][2][3][4].
A great deal of effort has been made to overcome multi-drug resistance (MDR) mediated by overexpression of P-gp. Nevertheless, many taxane-based drug candidates targeting P-gp overexpression did not achieve the expected efficacy in clinical trials [5,6].
A correlation between βIII-tubulin overexpression and poor prognosis has been reported in several human tumors, including various advanced malignancies upon treatment of taxanes, such as breast, non-small cell lung, and ovarian cancers [7][8][9][10]. It was reported that a C-seco taxane IDN5390 (2, Figure 1) is more active than paclitaxel in several paclitaxel-resistant human ovarian adenocarcinoma cell lines (e.g., A2780TC1, A2780TC3, and OVCAR-3), expressing high βIII-tubulin and P-gp levels. However, IDN5390 was nearly 10-fold less active than paclitaxel in paclitaxel-sensitive cells [11].
To find a modified taxane effective against both paclitaxel-sensitive and -resistant tumors, the 7,9-O-linked C-seco taxoid 3 ( Figure 2) was synthesized in our lab and shown to exhibit significant activity enhancement against drug-sensitive Hela and βIII-tubulin overexpressing HeLa-βIII tumor cells compared to the C-seco paclitaxel derivative 4 (an analog of IDN5390, Figure 2) [12]. Those macrocyclic taxoids provided a successful example for the compromise between structural pre-organization and sufficient flexibility in the C ring of C-seco taxoids to improve the cytotoxicity against tumor cells that overexpress βIII tubulin [12].
To find a modified taxane effective against both paclitaxel-sensitive and -resistant tumors, the 7,9-O-linked C-seco taxoid 3 ( Figure 2) was synthesized in our lab and shown to exhibit significant activity enhancement against drug-sensitive Hela and βIII-tubulin overexpressing HeLa-βIII tumor cells compared to the C-seco paclitaxel derivative 4 (an analog of IDN5390, Figure 2) [12]. Those macrocyclic taxoids provided a successful example for the compromise between structural preorganization and sufficient flexibility in the C ring of C-seco taxoids to improve the cytotoxicity against tumor cells that overexpress βIII tubulin [12]. It has long been observed that modification of taxane at the C2 position could play a critical role in its interaction with tubulin. Consistent with this observation, the 7,9-O-linked C-seco taxoid with C2 modification 5 ( Figure 3) was much more cytotoxic than the C-seco taxoid 6 ( Figure 3) in drugresistant A2780AD and KB-V1 cells (P-gp overexpression) and Hela-βIII (βIII overexpression) [12]. It was demonstrated that the meta-substitution (i.e., -OMe, -F, -Cl) of the C2-benzoate moiety of C-seco taxoids derivatives 7a-c ( Figure 3) could increase the interaction of C-seco-taxoids with βIII-tubulin to overcome paclitaxel-resistance [13]. In order to explore the effect of macrocyclization in a different way from that employed in our previous study (i.e., via the carbonate formation to restrict the conformation), the synthesis and It has long been observed that modification of taxane at the C2 position could play a critical role in its interaction with tubulin. Consistent with this observation, the 7,9-O-linked C-seco taxoid with C2 modification 5 ( Figure 3) was much more cytotoxic than the C-seco taxoid 6 ( Figure 3) in drug-resistant A2780AD and KB-V1 cells (P-gp overexpression) and Hela-βIII (βIII overexpression) [12]. It was demonstrated that the meta-substitution (i.e., -OMe, -F, -Cl) of the C2-benzoate moiety of C-seco taxoids derivatives 7a-c ( Figure 3) could increase the interaction of C-seco-taxoids with βIII-tubulin to overcome paclitaxel-resistance [13].
To find a modified taxane effective against both paclitaxel-sensitive and -resistant tumors, the 7,9-O-linked C-seco taxoid 3 ( Figure 2) was synthesized in our lab and shown to exhibit significant activity enhancement against drug-sensitive Hela and βIII-tubulin overexpressing HeLa-βIII tumor cells compared to the C-seco paclitaxel derivative 4 (an analog of IDN5390, Figure 2) [12]. Those macrocyclic taxoids provided a successful example for the compromise between structural preorganization and sufficient flexibility in the C ring of C-seco taxoids to improve the cytotoxicity against tumor cells that overexpress βIII tubulin [12]. It has long been observed that modification of taxane at the C2 position could play a critical role in its interaction with tubulin. Consistent with this observation, the 7,9-O-linked C-seco taxoid with C2 modification 5 ( Figure 3) was much more cytotoxic than the C-seco taxoid 6 ( Figure 3) in drugresistant A2780AD and KB-V1 cells (P-gp overexpression) and Hela-βIII (βIII overexpression) [12]. It was demonstrated that the meta-substitution (i.e., -OMe, -F, -Cl) of the C2-benzoate moiety of C-seco taxoids derivatives 7a-c ( Figure 3) could increase the interaction of C-seco-taxoids with βIII-tubulin to overcome paclitaxel-resistance [13]. In order to explore the effect of macrocyclization in a different way from that employed in our previous study (i.e., via the carbonate formation to restrict the conformation), the synthesis and In order to explore the effect of macrocyclization in a different way from that employed in our previous study (i.e., via the carbonate formation to restrict the conformation), the synthesis and biological activity assessment of a series of 7,9-O-linked macrocyclic taxoids together with modification at the C2 position were performed and are reported here.

Chemistry
Our synthesis began with the preparation of 2 -tert-butyldimethylsilyl-paclitaxel 12a, which was then converted to 10-deacetylpaclitaxel 13a upon treatment with hydrazine hydrate in methanol. Synthesis of the analogues 12b-e of 2 -tert-butyldimethylsilyl-paclitaxel with modifications at the C2 positions was then realized by selective C2 debenzoylation and then elaboration with various m-substituted benzoyl group. C2-modified 10-deacetylpaclitaxel 13b-e were afforded in the same manner as compound 13a. Then the compounds 13a-e were oxidized by copper (II) diacetate to furnish 14a-e as a mixture of two epimers. Reductive trapping of the ring C-seco tautomers 15a-e were obtained by treatment with l-selectride. Subsequent deprotection with pyridine hydrofluoride (HF-pyridine) afforded 16a-e. The 9-OH in 15a-e was hydroxylalkylated by 2-bromoethanol or 3-bromo-1-propanol in the presence of potassium carbonate and potassium iodide in N,N-dimethylformamide (DMF). Cyclization of compounds 17a-e could be accomplished by treatment with a slight excess of triphosgene, affording the cyclic carbonate analogues 19a-e. Desilylation of 19a-e with HF-pyridine afforded macrocyclic analogues 21a-e. Accordingly, macrocyclic taxoids 22a-e bearing a linkage with one more carbon atom were synthesized from 18a-e in a similar manner (Scheme 1).
Molecules 2019, 24, x 3 of 23 biological activity assessment of a series of 7,9-O-linked macrocyclic taxoids together with modification at the C2 position were performed and are reported here.

Chemistry
Our synthesis began with the preparation of 2′-tert-butyldimethylsilyl-paclitaxel 12a, which was then converted to 10-deacetylpaclitaxel 13a upon treatment with hydrazine hydrate in methanol. Synthesis of the analogues 12b-e of 2′-tert-butyldimethylsilyl-paclitaxel with modifications at the C2 positions was then realized by selective C2 debenzoylation and then elaboration with various msubstituted benzoyl group. C2-modified 10-deacetylpaclitaxel 13b-e were afforded in the same manner as compound 13a. Then the compounds 13a-e were oxidized by copper (II) diacetate to furnish 14a-e as a mixture of two epimers. Reductive trapping of the ring C-seco tautomers 15a-e were obtained by treatment with L-selectride. Subsequent deprotection with pyridine hydrofluoride (HF-pyridine) afforded 16a-e. The 9-OH in 15a-e was hydroxylalkylated by 2-bromoethanol or 3bromo-1-propanol in the presence of potassium carbonate and potassium iodide in N,Ndimethylformamide (DMF). Cyclization of compounds 17a-e could be accomplished by treatment with a slight excess of triphosgene, affording the cyclic carbonate analogues 19a-e. Desilylation of 19a-e with HF-pyridine afforded macrocyclic analogues 21a-e. Accordingly, macrocyclic taxoids 22a-e bearing a linkage with one more carbon atom were synthesized from 18a-e in a similar manner (Scheme 1).

Bioactivity Evaluation of Taxoids 21a-e and 22a-e
Taxoids 21a-e, 22a-e, and 16a-e were evaluated for their in vitro cytotoxicities against cervical carcinoma HeLa cells and their corresponding drug-resistant cell lines (HeLa-βIII).
As shown in Table 1, all the 7,9-O-linked macrocyclic analogues (21a-e, 22a-e) possessed a remarkably higher potency against HeLa and HeLa-βIII cells than their corresponding C-seco taxoids analogues 16a-e. The R/S values (IC50 in drug resistant cells/IC50 in drug sensitive cells) of all

Bioactivity Evaluation of Taxoids 21a-e and 22a-e
Taxoids 21a-e, 22a-e, and 16a-e were evaluated for their in vitro cytotoxicities against cervical carcinoma HeLa cells and their corresponding drug-resistant cell lines (HeLa-βIII).
As shown in Table 1, all the 7,9-O-linked macrocyclic analogues (21a-e, 22a-e) possessed a remarkably higher potency against HeLa and HeLa-βIII cells than their corresponding C-seco taxoids analogues 16a-e. The R/S values (IC 50 in drug resistant cells/IC 50 in drug sensitive cells) of all macrocyclic analogues were lower than those of their corresponding C-seco taxoids analogues 16a-e. These findings demonstrate that conformational restraint via carbonate-containing linked macrocyclization can improve the cytotoxicity against human carcinoma cell lines overexpressing βIII tubulin.
Furthermore, all of the 7,9-O-linked macrocyclic analogues bearing the four-atom linker (21a-e) turned out to be generally more potent against sensitive HeLa cells and HeLa-βIII cells than the corresponding macrocyclic analogues bearing the five-atom linker (22a-e). Since the cytotoxicity of 21a-d was approximately equal to that of paclitaxel, these results suggest that a four-atom tether is optimal in these macrocyclic C-seco taxoids.
We investigated this effect in the 7,9-O-linked C-seco taxoids 21b-e and 22b-e reported here. Thus, introduction of a meta substituent (-OMe, -F, -Cl) on derivatives bearing the four-atom linker showed similar cytotoxicity against HeLa sensitive cell lines and Hela-βIII cells lines as their corresponding C-seco taxoid 21a bearing a 2-benzyloxy group. In contrast, the meta-substituted (-OMe, -F, -Cl) derivatives bearing five-atom linker possessed higher potency against HeLa-βIII cells lines than its corresponding C-seco taxoid 22a (by a factor of 3.1-8.9). Nonetheless, the 2-m-CF 3 analogues 21e and 22e were considerably less cytotoxic against both drug sensitive and resistant cells by one to two orders of magnitude.
The growth inhibition effects of the 7,9-O-linked macrocyclic analogues were also measured on human breast cancer MCF-7 cells and their corresponding P-gp-overexpressing drug-resistant MCF-7/R cells. As shown in Table 1, the 7,9-O-linked macrocyclic analogues possessed remarkable potency against MCF-7/R cells compared to their C-seco counterparts, except for the 2-m-CF 3 derivatives. Strikingly, macrocyclic taxoids bearing the five-atom linker were almost inactive. The cytotoxicity against MCF-7/R cells of the 7,9-O-linked macrocyclic derivatives (-OMe, -F, -Cl) bearing the five-atom linker were slightly higher than that of the compouds bearing the four-atom linker. Compounds 22b-d (-OMe, -F, -Cl) bearing the five-atom linker and 21a, 21d (-H, -Cl) bearing the four-atom linker displayed lower R/S values than that of paclitaxel. All in all, 22b turned out to be the best compound in the whole series.

Binding Mode of 22b and Rationale for the Increased Affinity for the βIII Isotype
Inspection of the paclitaxel-β-tubulin complex in high-resolution cryo-electron microscopy (cryo-EM) structures of mammalian microtubules reveals a very tight fit between the bulky drug and the taxane-binding site, in which the M-loop is structured as an α-helix [14]. The oxetane ring oxygen of paclitaxel (1a) accepts a hydrogen bond from the backbone NH of Thr276, whereas the C-3 benzamide carbonyl oxygen acts as a hydrogen bond acceptor for N ε of His229. These two hydrogen bonds are likely to be present in the 22b:β-tubulin complex as well (Figure 4), and definitely contribute to the binding affinity, but these two residues are common to both βIIB and βIII isotypes. In the search for sequence differences that can account for the resistance to taxanes, a lot of emphasis has been placed on the Ser→Ala replacement at position 277, but we believe that the most important replacement in relation to drug resistance possibly involves Cys241→Ser. The reason is that, as a consequence of this substitution, disulfide bond formation with Cys356 is precluded in the βIII isotype and the T7 loop region, which closes over the bound taxane, is likely to be more flexible. By directing the macrocyclic region of 22b towards this loop, favorable interactions within the taxane-binding site are enhanced and the affinity towards the βIII isotype is improved. We hypothesize that the improved cytotoxicity of 22b relative to 1a, and the other taxanes described here, results from enhanced favorable interactions with the T7 loop region of βIII, an isotype in which Cys356 cannot engage in a disulfide bond with the amino acid present at position 241 (Ser). As shown in this modelling study, the binding of these taxoids to microtubules are similar to that of paclitaxel. Although cyclization does increase the cytotoxicity either in drug-sensitive andresistant cells in most cases, the 7,9-O-linked macrocyclic taxoids only showed comparable cytotoxicity to that of paclitaxel. The enhanced activity is not only arisen from the known C-2 modifications [15,16], but also from the cyclization of the cleaved C-ring of taxane.  . 22b docked into homology models of human tubulins IIb and III bound to guanosine diphosphate (GDP)-Mg 2+ . Colored in magenta and labeled are those residues close to the taxane-binding site that differ between these two isotypes.
As shown in this modelling study, the binding of these taxoids to microtubules are similar to that of paclitaxel. Although cyclization does increase the cytotoxicity either in drug-sensitive and -resistant cells in most cases, the 7,9-O-linked macrocyclic taxoids only showed comparable cytotoxicity to that of paclitaxel. The enhanced activity is not only arisen from the known C-2 modifications [15,16], but also from the cyclization of the cleaved C-ring of taxane.

General Methods
All chemicals and reagents were purchased from Beijing Innochem Science and Technology Co. Ltd. (Beijing, China), Sinopharm Chemical Reagent Co. Ltd. (Beijing, China), and thin-layer chromatography (TCI). The 200-300 mesh silica gel used for flash column chromatography was purchased from Rushanshi Shuangbang Xincailiao Co. Ltd. (Rushan, Shandong, China). Visualization on TLC (analytical thin layer chromatography) was achieved by the use of UV light (254 nm) and treatment with phosphomolybdic acid or KMnO 4 followed by heating. All solvents were purified and dried according to the standard procedures. The purification was performed on flash column chromatography. The high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry (MS) analysis was carried out in an Agilent 1260 Infinity HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a reversed phase 4.68 × 50 mm (1.8 um) XDB-C18 Column and consisted of a binary solvent delivery system, an auto sampler, a column temperature controller, and an UV detector. The mass spectra were acquired by a 6120 Quadrupole LC-MS mass spectrometer (Agilent Technologies, Waldbronn, Germany) connected to the HPLC system via an ESI interface. Proton and carbon magnetic resonance spectra ( 1 H-NMR and 13 C-NMR) were recorded on a Bruker BioSpin AG 300 or 400 MHz spectrometer or Varian 300, 400, or 600 MHz spectrometer. 1 H-NMR data were reported as follows: Chemical shifts, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m=multiplet), coupling constant(s) in Hz, integration. All tested compounds 16a-e, 21a-e, and 22a-e were ≥95% pure by HPLC (column XDB C18 4.6 × 50 mm 1.8 µm, mobile phase: acetonitrile-water (10:90-100:0 gradient in 4.5 min), flow rate 1.0 mL/min, detected at 220 nm).  1.41 mmol). The suspension was stirred at room temperature overnight (open air) and then diluted with ethyl acetate. The organic phase was washed sequentially with saturated aqueous NaCl (50 mL), 2 N NH 3 to remove copper salts and saturated aqueous NH 4 Cl. After drying (Na 2 SO 4 ) and evaporation under reduced pressure, the crude product was purified by silica gel chromatography (ethyl acetate: petroleum ether = 1:2) to give a 4:1 mixture of epimers 14a (14-β-OH and 14-α-OH, 223.8 mg, 85.8% total yield). 1 13

Cell Assays
Cell viability after taxoids treatment was evaluated using CCK-8 assay. Briefly, 3000 cells per well were seeded in 96-well plates and incubated under normal conditions for 24 h. Cells were treated with different concentrations of the test agent for 72 h, then the medium was removed, 100 µL of CCK-8 working solution was added to each well for 1 h at 37 • C. The absorbance was measured at 450 nm with a microplate reader (Tecan Trading, AG, Switzerland). Vehicle-only treated cells served as the indicator of 100% cell viability. The 50% inhibitory concentration (IC 50 ) was defined as the concentration that reduced the absorbance of the vehicle-only treated wells by 50% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Model Building and Molecular Dynamics Simulations
The X-ray crystal structure of paclitaxel provided a template for the construction of 18b and 22b using the molecular editing tools implemented in PyMOL [17]. Geometry optimization of 18b and 22b was achieved by means of the updated AM1 hamiltonian implemented in the sqm program, which also produced atomic charge distributions for both ligands that can reproduce the molecular electrostatic potential. The ff14SB AMBER force field was used to assign bonded and non-bonded parameters to protein and ligand atoms [18,19]. 18b was immersed in a cubic box containing TIP3P water molecules and simulated under periodic boundary conditions for 50 ns at 300 K. Subsequent gradual cooling, from 300 to 273 K over 1 ns, of snapshots taken regularly every 2.5 ns, followed by energy minimization until the root-mean-square of the Cartesian elements of the gradient was less than 0.1 kcal·mol −1 ·Å −1 , provided representative structures of this molecule; those displaying the shortest distance between the free hydroxyl groups were chosen to build 22b by means of a C=O linkage. The simulated macromolecular ensemble representing a short piece of a microtubule with bound 22b was constructed as previously reported for D-seco taxol derivatives [20].

Conclusions
In summary, this study demonstrated that C-seco taxoids conformationally constrained via carbonate-containing linked macrocyclization display increased cytotoxicity on drug-resistant tumors overexpressing both βIII and P-gp, compared to the non-cyclized C-seco taxoid counterparts Among them, compound 22b, bearing a 2-m-methoxybenzoyl group together with a five-atom linker, was identified as the most potent compound in the series.

Conflicts of Interest:
The authors declare no conflicts of interest regarding the publication of this paper.