Design, synthesis and SAR study of Fluorine-containing 3^rd-generation taxoids

Abstract

It has been shown that the incorporation of fluorine or organofluorine groups into pharmaceutical and agricultural drugs often induces desirable pharmacological properties through unique protein-drug interactions involving fluorine. We have reported separately remarkable effects of the 2,2-difluorovinyl (DFV) group at the C3’ position, as well as those of the CF₃O and CHF₂O groups at the 3-position of the C2-benzoyl moiety of the 2^nd- and 3^rd-generation taxoids on their potency and pharmacological properties. Thus, it was very natural for us to investigate the combination of these two modifications in the 3^rd-generation taxoids and to find out whether these two modifications are cooperative at the binding site in the β-tubulin or not, as well as to see how these effects are reflected in the biological activities of the new 3^rd-generation DFV-taxoids. Accordingly, we designed, synthesized and fully characterized 14 new 3^rd-generation DFV-taxoids. These new DFV-taxoids exhibited remarkable cytotoxicity against human breast, lung, colon, pancreatic and prostate cancer cell lines. All of these new DFV-taxoids exhibited subnanomolar IC₅₀ values against drug-sensitive cell lines, A549, HT29, Vcap and PC3, as well as CFPAC-1. All of the novel DFV-taxoids exhibited 2–4 orders of magnitude greater potency against extremely drug-resistant cancer cell lines, LCC6-MDR and DLD-1, as compared to paclitaxel, indicating that these new DFV-taxoids can overcome MDR caused by the overexpression of Pgp and other ABC cassette transporters. Dose-response (kill) curve analysis of the new DFV-taxoids in LCC6-MDR and DLD-1 cell lines revealed highly impressive profiles of several new DFV-taxoids. The cooperative effects of the combination of the 3’-DFV group and 3-CF₃O/CHF₂O-benzoyl moiety at the C2 position were investigated in detail by molecular docking analysis. We found that both the 3’-DFV moiety and the 3-CF₃O/3-CHF₂O group of the C2-benzoate moiety are nicely accommodated to the deep hydrophobic pocket of the paclitaxel/taxoid binding site in the β-tubulin, enabling an enhanced binding mode through unique attractive interactions between fluorine/CF₃O/CHF₂O and the protein beyond those of paclitaxel and new-generation taxoids without bearing organofluorine groups, which are reflected in the remarkable potency of the new 3^rd-generation DFV-taxoids.

1. Introduction:

Paclitaxel [1–3] and docetaxel [4] are two most broadly used 1^st-generation taxane anticancer drugs and have been successfully applied to treat a variety of cancer types, e.g., ovarian, breast, lung, and non-small cell lung cancers, as well as Kaposi’s sarcoma and head/neck tumor, etc. for more than 25 years (Fig. 1) [2, 3, 5]. More recently, cabazitaxel [6] was also approved by the Food and Drug Administration, USA (FDA) for the treatment of metastatic castration-resistant prostate cancer (Fig. 1) [7]. The further exploration of new clinical applications of taxane anticancer agents with new formulations and drug combinations are being actively investigated [2], e.g., abraxane (nanoparticle albumin–bound paclitaxel) for breast and non-small cell lung cancer with mitigated undesirable side effects [8], and for the treatment of advanced pancreatic cancer in combination with gemcitabine [8–10]. However, the natural and acquired resistance to these taxane anticancer drugs have been limiting the scope of their therapeutic use. These limitations are, at least in part, due to multi-drug resistance (MDR) caused by overexpression of ABC cassette efflux pumps, overexpression of β-III tubulin isoform, point mutations in the microtubule binding site and cancer stem cells (CSCs) [4, 11, 12]. A number of new taxoids [13–19], new formulations [20–24] and new combination therapies [25–30] are currently in different stages of preclinical and clinical development [2, 19] to address these issues. New taxoids with high potency are also critical as payloads for tumor-targeting drug conjugates [3, 23, 31, 32]. Therefore, it is highly promising and significant to develop the next-generation taxoid anticancer agents with superior pharmacological properties and efficacy against drug-resistant and metastatic cancers, as well as CSCs, which are responsible for tumor recurrence and metastasis [2, 3].

Fig. 1.

Paclitaxel, docetaxel, cabazitaxel, and examples of 2^nd- and 3^rd-generation taxoids

We have been developing new generations of taxane anticancer agents, i.e., 2^nd-generation taxoids (modifications at C10, C3’ and C3’N) [16, 18, 33–36] and 3^rd-generation taxoids (modifications at C2, C10, C3’ and C3’N) [33, 34, 37]. These new-generation taxoids exhibit 2–3 orders of magnitude higher potency than the 1^st-generation taxane anticancer drugs, i.e., paclitaxel and docetaxel, against various drug-resistant cancer cell lines expressing the multidrug-resistance (MDR) phenotype (i.e., P-glycoprotein and other ABC efflux pumps), as well as point mutations at the taxane-binding site in β-tubulin [15].

Fluorine has been playing a significant role in medicinal chemistry and chemical biology, as demonstrated by the statistical fact that a large number of fluorine-containing compounds have been approved by the FDA for medical, pharmaceutical and agricultural use [38–42]. In fact, in the current drug discovery and development, fluorine is ranked second after nitrogen as “favorite heteroatom” [43]. The replacement of a C-H or C-O bond with a C-F bond in medicinally active compounds has been shown to induce or improve desirable pharmacological properties such as higher metabolic stability, increased affinity to target molecules, and enhanced membrane permeability [44, 45]. The unique properties that make fluorine and fluorine-containing organic groups exceptionally useful in drug discovery and development include fluorine’s small atomic radius and high electronegativity, as well as the low polarizability of the C-F bond. Therefore, we have been exploiting beneficial effects of fluorine and fluorine-containing organic groups on the potency and pharmacological properties of the 2^nd- and 3^rd-generation taxoid anticancer agents through their strategic incorporations [15, 34, 35, 46–48].

For instance, we strategically introduced a 2,2-difluorovinyl group in place of an isobutenyl group at the C3’ position of taxoid, which successfully blocked the hydroxylation of allylic methyl groups, as well as further oxidations of the taxoid molecule by a major human metabolic enzyme, CYP 3A4, of the cytochrome P450 family [34, 49].

Our extensive structure-activity relationship (SAR) studies of the 1^st-generation taxane anticancer agents led to the development of 2^nd-generation taxoids bearing non-aromatic substituents (isobutenyl or isobutyl) at the C3’ position and various acyl groups at the C10 position [33, 50], which exhibited excellent activities in vitro and in vivo, especially against taxane-resistant cancer cell lines and tumor xenografts in mice [33]. As a typical example of such 2^nd-generation taxoids, SB-T-1214 bearing a 3’-isobutenyl group (Fig. 1), is worth to mention. SB-T-1214 and its conjugate with docosahexaenoic acid, DHA-SB-T-1214, exhibited impressive efficacy in vivo against highly taxane-resistant DLD-1 tumor xenografts in mice [51]. Also, a nanoemulsion formulation of DHA-SB-T-1214 demonstrated clear efficacy against PPT2 patient-derived prostate CSC-initiated tumor xenografts in a mouse model, which is currently in late stage preclinical development [52]. SB-T-1214 was also found to possess high potency against CSCs from colon and prostate cancers by suppressing stemness gene expressions and restoring P21 and P53 expressions [53]. However, our study on the metabolic stability of 3’-isobutyl- and 3’-isobutenyl-taxoids revealed that there was a marked difference in metabolism between the 2^nd-generation taxoids and that of docetaxel and paclitaxel [49]. The metabolism study showed that CYP 3A4 metabolized the 2^nd-generation taxoids, such as SB-T-1214 and SB-T-1216, through hydroxylation primarily at the two allylic methyl groups of the 3’-isobutenyl group (Fig. 2) [49].

Fig. 2.

Primary sites of hydroxylation on the 2^nd-generation taxoids by the P450 family of enzymes (adapted from Reference [49]).

Thus, in order to prevent this enzymatic allylic hydroxylation, we decided to replace the 3’-isobutenyl group with a 2,2-difluorovinyl group, which we considered as its mimic [34]. Our computational analysis of 3’-(2,2-difluorovinyl)taxoids (DFV-taxoids) in the taxane binding site in β-tubulin indicated that 2,2-difluorovinyl group would nicely mimic an isobutenyl group in spite of a difference in size and electronic nature between the two groups [15]. The size of the 2,2-difluorovinyl group was predicted to be between that of vinyl and isobutenyl groups and two fluorine atoms would mimic electronically two hydroxyl groups rather than two methyl groups [15]. The examination of the metabolic stability of DFV-taxoids against P-450 family enzymes revealed that no appreciable metabolites were formed [49]. The results indicated that not only the metabolism at the C3’ position was effectively blocked, but also oxidative metabolism was shielded on other parts of the taxoid molecule, including the C3’N-t-Boc, C6-methylene, 3’-phenyl and 2-benzoyl moieties [49], which were identified as the major metabolism sites for docetaxel and paclitaxel [54–56]. Consequently, our strategic incorporation of a 2,2-difluorovinyl group at the C3’ position has proven successful in blocking the major metabolism in the 2^nd-generation taxoids.

DFV-taxoids, a variant of the 2^nd-generation taxoids, exhibited excellent potencies against human breast, ovarian, colon and pancreatic cancer cell lines, with 2–3 orders of magnitude higher potency than paclitaxel against taxane-resistant NCI/ADR ovarian cancer cell line [34]. A representative 2^nd-generation DFV-taxoid, SB-T-12854 (Fig. 1), was found to be highly potent against CSC-enriched HCT-116 human colon cancer cells [57]. It is impressive that SB-T-12854 was found to exhibit 230–33,000 times higher potency than conventional anticancer drugs (i.e., cisplatin, doxorubicin, methotrexate, topotecan and paclitaxel) against CSC-enriched HCT-116 cell lines [57]. Since CSCs are responsible for tumor metastasis and recurrence [58], this finding is highly significant. As mentioned above, new-generation taxoids that exhibit high potency against CSCs, were found to suppress the expression of “stemness genes”, promoting differentiation of the treated CSCs [53]. This finding adds a new mechanism of action (MOA) for taxoid anticancer agents whose major MOA is the blocking of the cell mitosis at the G2/M stage, leading to apoptosis [59–62].

Our extensive SAR studies disclosed that modifications of the C2-benzoate moiety at the meta-position with halogens (F, Cl), azide, methyl, vinyl and methoxy groups enhanced the potency of 2^nd-generation taxoids against MDR-cancer cell lines, which led to the development of 3^rd-generation taxoids [33]. These 3^rd-generation taxoids exhibited remarkable activities against taxane-resistant human cancer cell lines, NCI/ADR (ovarian) and LCC6-MDR (breast) [33]. Among the meta-substituents examined, the methoxy group was found to be most effective [33], and thus SB-T-121303 is shown as a representative 3^rd-generation taxoid in Fig. 1.

We also recently reported a new DFV-taxoid, DFV-ortataxel (Fig. 1) [48], which is a DFV analog of ortataxel (SB-T-11031) (Fig. 1) that advanced to the phase II clinical trials in humans [63]. DFV-ortataxel is a variant of the 2^nd-generation taxoids derived from 14β-baccatin III [50], and has exhibited promising efficacy against taxane-resistant MDA-MB-231R cell line, overexpressing ABCB1/ABCG2 efflux pumps, and MCF-7R cell line and its tumor xenografts in mice.

However, we did not examine the effects of fluoromethoxy groups, i.e., CF₃O and CHF₂O at that time. It has been shown that introduction of the CF₃O or CHF₂O group to drug candidates often improve their absorption, distribution, metabolism and excretion (ADME) properties, i.e., metabolic stability, membrane permeability and pharmacokinetics (PK) profile. Furthermore, the unique non-spherical structure of the CHF₂O group may provide beneficial characteristics. Accordingly, we synthesized a series of 3^rd-generation taxoids bearing CF₃O and CHF₂O groups as the meta-substituents of the C2-benzoyl moiety, investigated their anticancer activities, and reported recently [47, 64]. These new taxoids exhibited up to 7 times higher potency than paclitaxel against drug-sensitive cancer cell lines (MCF7 and LCC6-WT) and 2–3 orders of magnitude higher potency than paclitaxel against drug-resistant ovarian, breast and colon cancer cell lines with MDR-phenotype, as well as pancreatic cancer cell line. Since it has been shown that a bulky group at this position reduces potency [33], it is noteworthy that rather bulky CF₃O and CHF₂O groups are well tolerated and can virtually overcome MDR. It should also be noted that new taxoids, bearing a CHF₂O group at the C2 benzoate position (06 series), showed the highest potencies against MDR-cancer cell lines and CSC-enriched cancer cell lines. Among the new taxoids, bearing a CHF₂O group at the C2 benzoate position (05 series), SB-T-121205 (Fig. 1) exhibited the strongest activity against highly paclitaxel-resistant human breast cancer cell line, MCF-7/PTX, i.e., SB-T-121205 possesses 121 times higher potency than that of paclitaxel. Also, the MOA study on SB-T-121205 revealed that the PI3k/Akt pathway and epithelial-mesenchymal transition (EMT) were effectively suppressed by this new taxoid through upregulation of PTEN tumor suppressor [64]. This is another new and significant MOA associated with new-generation taxoids against highly drug-resistant cancer cells.

Based on the demonstrated highly beneficial effects of the DFV group at the C3’ position, as well as the CF₃O and CHF₂O groups as the meta-substituents of the C2-benzoyl moiety of the 2^nd- and 3^rd-generation taxoids, described above, it is very logical to assume that the combination of these two modifications in the 3^rd-generation taxoids might lead to extraordinary results. Although the 3’-DFV group was originally introduced to increase the metabolic stability of the 2^nd-generation taxoids, a molecular docking study on DFV-ortataxel disclosed that the two fluorine atoms have unique van der Waals interactions with β-tubulin, which do not exist in paclitaxel, docetaxel and 3’-isobutenyl/isobutyl-taxoids [48]. Also, a similar molecular docking study on the 3^rd-generation taxoids bearing a 3-CF₃O- and 3-CHF₂O-benzoate moiety at the C2 position indicated the conformational preference of the 2-(3-CF₃O/3-CHF₂O)benzoate moiety to be buried into the proximal site of the binding pocket, making hydrophobic contact with Leu230 and Leu275 [47]. Therefore, it is very important to find out whether these two modifications are cooperative at the binding site in the β-tubulin or not, and how these effects are reflected in the biological activities of the novel 3^rd-generation DFV-taxoids.

In the present work, we have found that some of these novel DFV-taxoids exhibit remarkable activities against drug-sensitive and drug-resistant human cancer cell lines, which are superior to the 2^nd-generation DFV-taxoids, as well as the the 3^rd-generation taxoids bearing a 3- CF₃O/CHF₂O-benzoyl moiety at the C2 position. The cooperative effects of the combination of the 3’-DFV group and 3- CF₃O/CHF₂O-benzoyl moiety at the C2 position have also been investigated in detail by molecular docking analysis.

2. Results and Discussion

2.1. Synthesis of 3rd-generation DFV-taxoids bearing 3-CF₃O or 3-OCF₂H group at the C2-benzoate moiety and difluorovinyl group at the C3’ position

New 3^rd-generation DFV-taxoids were synthesized using the procedures that we had developed by means of the β-lactam synthon method [65], through the Ojima-Holton coupling [66] of 2,10-modified baccatins 7, derived from 10-deacetylbaccatin III (10-DAB), with (3S,4R)-1-Boc-3-TIPSO-4-DFV-β-lactam 10 [34] as illustrated in Schemes 1–4.

Scheme 1.

Synthesis of key intermediates, baccatins 7(a-e)-05/06, 7f-05/06

Reagents and conditions: (i) 3-(trifluromethoxy)benzoic acid (2a) or 3-(difluromethoxy)benzoic acid (2b) (4–8 equiv.), DIC (8 equiv.), DMAP (8 equiv.), CH₂Cl₂, r.t., 2–5 days, 90–94%; (ii) HF/pyridine, pyridine/MeCN, 0 °C-r.t., overnight, 95–98%; (iii) acetic or propanoic anhydride (10 equiv.), CeCl₃·7H₂O (0.1 equiv.), THF, r.t., 4 h, 88–95%; (iv) TES-Cl (3 equiv.), imidazole (4 equiv.), DMF, 0 °C, 45 min, 85–88%; (v) TES-Cl (3 equiv.), imidazole (4 equiv.), DMF, 0 °C, 45 min, 85–90%; (vi) LiHMDS, THF, cyclopropanecarbonyl chloride, N,N-dimethylcarbamoyl chloride or methyl chloroformate, −40 °C, r.t., 2–4 h, 73–91%; 6e-05/6e-06: NaH (1.8 equiv.), CH₃I (10 equiv.), THF, 0 °C, 2–4 h, 48–49 %.

Scheme 4.

Synthesis of 3rd-generation DFV-taxoids

Reagents and conditions: (i) LiHMDS (1.5 equiv.), THF, −40 °C, 2 h; (ii) HF/pyridine, acetonitrile: pyridine (v:v = 1:1), 0 °C - r.t., overnight, 90% for two steps.

As Scheme 1 shows, 7-triethylsilylbaccatins 7a-d,g-05/06, bearing a 3-CF₃O- or 3-CHF₂O-benzoate moiety at C2 and various acyl groups at C10 were prepared by using the procedures described in a recent publication from our laboratory [47]. New baccatins 7e-05/06, bearing a methoxy group at C10, were also synthesized through the reaction of baccatins 6–05/06 with methyl iodide, following the procedure used for the acylation of the C10-OH with acid chlorides [47].

For the synthesis of new 7,10-dimethylbaccatins 7f-05/06, a procedure through the simultaneous methylation of the C7 and C10 hydroxyl groups of 10-DAB with methyl iodide, followed by the C2 modification was employed (Scheme 2). We also examined a procedure with a reversed order, i.e., the C2 modification first, followed by the 7,10-dimethylation, but found that the first procedure is more cost effective.

Scheme 2.

Synthesis of key intermediates, baccatins 7f-05,06

Reagents and conditions: (i) LiHMDS (2.6 equiv.), TfOMe (2.6 equiv.), THF, −78 °C, 4–8 h, 67%; (ii) 1) TESCl (2.1 equiv.), imidazole (2.8 equiv.), DMF, 0 °C, 5 days, 70%; (iii) Red-Al (3.5 equiv.), THF, −30 °C, 95%; (iv) 3-(trifluromethoxy)benzoic acid or 3-(difluromethoxy)benzoic acid (4 equiv.), DIC (8 equiv.), DMAP (8 equiv.), CH₂Cl₂, r.t., 3–5 days, 85–90%; (v) HF/pyridine, acetonitrile: pyridine (v:v = 1:1), 0 °C, overnight, 73–90%.

(3S,4R)-1-Boc-3-TIPSO-4-DFV-β-lactam 10b was synthesized through decarboxylative Wittig reaction [67, 68] of 1-PMP-3-TIPSO-4-formyl-β-lactam 9 with sodium chlorodifluoroacetate and triphenylphosphine to give 1-PMP-3-TIPSO-4-DFV-β-lactam 10a, followed by the deprotection of PMP with cerium ammonium nitrate (CAN) and introduction of a t-Boc group to the β-lactam nitrogen (Scheme 3). Previously we synthesized β-lactam 10a from 4-formyl-β-lactam 9 through a Wittig-type reaction using CF₂Br₂, hexamethylphosphorous triamide (HMPT), and Zinc [34], but the use CF₂Br₂ became prohibited and the decarboxylative Wittig reaction is found to be efficient.

Scheme 3.

Synthesis of key intermediate, DFV-β-lactam 10b

Reagents and conditions: (i) ClF₂CCO₂Na (3 equiv.), Ph₃P (3 equiv.), DMF, 90 °C, 3 hrs, 65% (ii) CAN (4 equiv.), MeCN-H₂O (1/1), −10 ᶱC, 2 h, 92 %; (iii) Boc₂O (1.2 equiv.), NEt₃, DMAP, CH₂Cl₂, r.t., 24 h, 96%.

The 3^rd-generation DFV-taxoids 11(a-g)-05/06 were synthesized in high yields through the Ojima-Holton coupling of the modified baccatins 7(a-g)-05/06, with 4-DFV-β-lactam 10b, followed by removal of TES and TIPSO groups with HF/pyridine (Scheme 4). Table 1 summarizes all 3^rd-generation DFV-taxoids 11(a-g)-05/06 synthesized and studied in this work.

Table 1.

3^rd-Generation DFV-taxoids

Taxoid	R1	R2	X
11a-05	Me-CO	H	OCF3
11b-05	c-Pr-CO	H	OCF3
11c-05	Et-CO	H	OCF3
11d-05	Me2N-CO	H	OCF3
11e-05	Me	H	OCF3
11f-05	Me	Me	OCF3
11g-05	MeO-CO	H	OCF3
11a-06	Me-CO	H	OCF2H
11b-06	c-Pr-CO	H	OCF2H
11c-06	Et-CO	H	OCF2H
11d-06	Me2N-CO	H	OCF2H
11e-06	Me	H	OCF2H
11f-06	Me	Me	OCF2H
11g-06	MeO-CO	H	OCF2H

2.2. Biological evaluations of 3^rd-generation DFV-taxoids

2.2.1. Cytotoxicity of 3^rd-generation DFV-taxoids against human cancer cell lines

The cytotoxicity (IC₅₀, nM) of the 3^rd-generation DFV-taxoids was evaluated by a standard MTT assay against various drug-sensitive and drug-resistant human cancer cell lines, i.e., A549 (lung), HT29 (colon), Vcap (prostate), PC3 (prostate), MCF7 (breast), PANC-1 (pancreas), DLD-1 (colon) and LCC6-MDR (breast). Results are summarized in Table 1. IC₅₀ values for paclitaxel, docetaxel and a representative 2^nd-generation taxoid SB-T-1214 are also shown for comparison. SB-T-1214 was used as the positive control.

As Table 2 shows, 3^rd-generation DFV-taxoids exhibit remarkable cytotoxicity against human breast, lung, colon, pancreactic and prostate cancer cell lines. All of these new DFV-taxoids exhibit subnanomolar IC₅₀ values against drug-sensitive cell lines, A549, HT29, Vcap and PC3, as well as PANC-1 which is moderately resistant to paclitaxel. Furthermore, all of the 3^rd-generation DFV-taxoids possess 2–4 orders of magnitude greater potency against extremely drug-resistant cancer cell lines, LCC6-MDR and DLD-1, as compared to paclitaxel, indicating that these new DFV-taxoids can overcome MDR caused by the overexpression of Pgp and other ABC cassette transporters. It appears that the new DFV-taxoids bearing a 3-CHF₂O group at the C2-benozate moiety (06 series, entries 11–17) possess slightly higher cytotoxicity than those bearing a 3-CF₃O-benzoate group at C2 (05 series, entries 4–10). Also, it should be noted that DFV-taxoid 11f-06, a DFV-mimic of cabazitaxel, exhibits the highest potency against Vcap, MCF7, PANC-1 and PC3 cancer cell lines, as well as the second highest potency (IC₅₀ 89 pM) against LCC6-MDR (entry 16).

Table 2.

IC₅₀ values (nM) of 3^rd-generation DFV-taxoids against drug-sensitive and drug-resistant human cancer cell lines

Entry	Taxoid	A549a	HT29b	Vcapc	PC3d	MCF7e	PANC-1f	DLD-1g	LCC6-MDRh
1	Paclitaxel	1.94	2.59	4.52	3.05	2.94	2.89	428*	503*
2	Docetaxel	0.42	0.65	1.05	0.94	0.50	0.55	-	118
3	SB-T-1214	0.28	0.40	0.40	0.48	0.20	0.33	4.00	2.59
4	11a-05	0.14	0.22	0.39	0.45	0.23	0.41	0.168	0.163
5	11b-05	0.22	0.38	0.41	0.44	0.14	0.27	0.079	0.951
6	11c-05	0.17	0.28	0.37	0.33	0.12	0.34	0.564	0.565
7	11d-05	0.26	0.32	0.42	0.29	0.18	0.62	0.211	0.490
8	11e-05	0.17	0.34	0.26	0.22	0.07	0.08	0.073	0.644
9	11f-05	0.30	0.22	0.22	0.23	0.15	0.33	0.089	0.132
10	11g-05	0.42	0.34	0.50	0.37	0.20	0.63	0.110	0.771
11	11a-06	0.14	0.15	0.23	0.22	0.08	0.22	0.177	0.904
12	11b-06	0.10	0.18	0.27	0.39	0.10	0.43	0.34	0.392
13	11c-06	0.12	0.21	0.20	0.22	0.08	0.29	0.093	0.248
14	11d-06	0.14	0.24	0.32	0.26	0.15	0.18	0.242	0.816
15	11e-06	0.19	0.24	0.36	0.25	0.13	0.19	0.073	1.280
16	11f-06	0.25	0.21	0.21	0.05	0.06	0.03	0.159	0.089
17	11g-06	0.10	0.24	0.34	0.26	0.13	0.43	0.024	0.034

^ahuman lung (NSCL)cancer cell line;

^bhuman colon cancer cell line;

^chuman prostate cancer cell line;

^dhuman metastatic prostate cancer cell line;

^ehuman breast cancer cell line;

^fhuman pancreatic cancer cell line;

^gmultidrug-resistant (Pgp+) human colon cancer cell line;

^hmultidrug-resistant (Pgp+) human breast cancer cell line.

^*Data from Ref. [47], normalized by the IC₅₀ value of SB-T-1214 as the positive control.

In order to examine the effects of 3’-DFV and 2-(3-CF₃O/CHF₂O-benzoyl) groups on the potency of new-generation taxoids, the cytotoxicities of a representative 2^nd-generation taxoid, SB-T-1214, and its 3’-DFV-analog, SB-T-12852, it’s 2-(3-CF₃O/CHF₂O-benzoyl) analogs, SB-T-121405/SB-T-121406, and 3’-DFV/2-(3-CF₃O/CHF₂O-benzoyl) analogs, SB-T-12852–05 (11c-05)/SB-T-12852–06 (11c-06), were compared.

As Table 3 shows, the introduction of these two kinds of fluoro groups, individually or together exhibits incremental increase in potency against drug-sensitive human breast cancer cell line, MCF7. However, the introduction of 3’-DFV group alone, i.e., SB-T-12852, does not show any improvement in the potency against drug-sensitive human pancreatic cancer cell line, PANC-1 and drug-resistant human ovarian cancer cell line, NCI/ADR, as compared to SB-T-1214 (entry 2). Thus, the introduction of 3’-DFV group is only beneficial to block the metabolism by CYP 3A4 enzyme (Fig. 2). The introduction of 2-(3-CF₃O/CHF₂O-benzoyl) groups to SB-T-1214 exhibits incremental increase in potency against NCI/ADR cell line, as well as two other drug-resistant human colon (DLD-1) and breast (LCC6-MDR) cancer cell lines (entries 3 and 4). In contrast, the introduction of both 3’-DFV and 2-(3-CF₃O/CHF₂O-benzoyl) groups to SB-T-1214 demonstrates one order of magnitude increase in potency against drug-resistant cancer cell lines, DLD-1 and LCC6-MDR, which clearly indicate synergy between these two fluoro groups (entries 5 and 6).

Table 3.

Cytotoxicity (IC₅₀ nM) of 2^nd-and 3^rd-generation taxoids, bearing a cyclopropanecarbonyl group at C10, against drug-sensitive and drug-resistant human cancer cell lines

Entry	Taxoid	MCF7	PANC-1	NCI/ADRc	DLD-1	LCC6-MDR
1	SB-T-1214	0.20	0.33	1.2	4.00	2.59
2a	SB-T-12852	0.14	0.52	3.45	--	--
3b	SB-T-121405	0.15	--	0.75	2.98	1.71
4b	SB-T-121406	0.10	--	0.97	3.65	1.46
5	11c-05	0.12	0.34	--	0.56	0.565
6	11c-06	0.08	0.43	--	0.09	0.248

^aTaken from Ref. 37 and normalized based on the IC₅₀ values of SB-T-1214.

^bTaken from Ref. 47 and normalized based on the IC₅₀ values of SB-T-1214.

^cPaclitaxel IC₅₀ 177 nM; Taken from Ref. 47 and normalized based on the IC₅₀ values of SB-T-1214.

2.2.2. Dose-response (kill) curve analysis of 3^rd-generation DFV-taxoids in drug-resistant cancer cell lines

As Fig. 3 shows, paclitaxel is essentially ineffective (IC₅₀ 503 nM, Table 2) against the LCC6-MDR drug-resistant breast cancer cell line. While the treatment of this cell line with 20 nM dose of paclitaxel only killed 20% of the cancer cells, less than 10% cells were viable after the treatment of the same cancer cells with the same dose (20 nM) of any of 3^rd-generation DFV-taxoids. It is worthy of note that all of the 3^rd-generation DFV-taxoids exhibited better dose-repsonse (kill) curves as compared to those of paclitaxel and SB-T-1214. In the 05 series of DFV-taxoids, 11f-05 showed the best dose-dependent potency. Among all 3^rd-generation DFV-taxoids, however, two of the 06 series DFV-taxoids, 11f-06 and 11g-06, exhibited the most impressive dose-response (kill) curves.

Fig. 3.

Dose-response (kill) curves of 3^rd-generation DFV-taxoids in LCC6-MDR breast cancer cell line: (A) 05 series with CF₃O and (B) 06 series with CHF₂O

In the DLD-1 drug-resistant colon cancer cell line (Fig. 4), paclitaxel is practically ineffective (IC₅₀ 428 nM, Table 2). With 4 nM dose of paclitaxel and SB-T-1214, 90% and 50% of DLD-1 cells were viable, respectively. In sharp contrast, less than 10% of the same cells were viable with the same dose (4 nM) of 3^rd-generation DFV-taxoids, except for 11d-05 (ca. 18% viability). Among all the 3^rd-generation DFV-taxoids, 11e-05 and 11g-06 exhibited the most impressive dose-response (kill) curves. It is noteworthy that 11g-06 was able to kill ca. 90% of the cancer cells even at 0.16 nM dose and completely eradicated the cancer cells at 20 nM dose.

Fig. 4.

Dose-response (kill) curves of new 3^rd-generation taxoids in DLD-1 colon cancer cell line: (A) 05 series with CF₃O and (B) 06 series with CHF₂O

2.2.3. Molecular docking analysis of 3^rd-generation DFV-taxoids

To evaluate the effects of the 2,2-difluorovinyl modification on the binding mode of 3^rd-generation taxoids, we produced the structures of all 3^rd-generation DFV-taxoids in the β-tubulin binding site for molecular docking analysis based on the paclitaxel-bound β-tubulin cocrystal structure (PDB ID: 1JFF). The binding mode of the resulting structures of the 3^rd-generation DFV-taxoids bound to β-tubulin conserves the position of the baccatin framework, as well as the H-bond between the Gly362 backbone NH and the C2’-OH in 1JFF. An overlay of DFV-taxoid 11b-05 with paclitaxel, 2^nd-generation taxoid SB-T-1214, and 3^rd-generation taxoid SB-T-121405 exemplifies the consistency in the docking pose of new-generation taxoids with paclitaxel (Fig. S1 A,B).

Stereoelectronic effects of the DFV group at the C3’ position of the 2^nd-generation DFV-taxoids, as well as the 3-CF₃O and 3-CHF₂O groups at the C2-benzoate position of the 3^rd-generation taxoids, on the binding mode and affinity of those taxoids to β-tubulin have been studied by means of molecular docking analyses and recently reported by us [47, 48]. These computational analyses have indicated that the 3’-DFV moiety and the 3-CF₃O/3-CHF₂O group of the C2-benzoate moiety are, separately, accommodated in the proximal area of the paclitaxel binding site, consisting of the His229, Phe272, Leu275, and Leu230 residues in β-tubulin. This deep hydrophobic pocket, where the 3’-DFV moiety, as well as the 3-CF₃O/3-CHF₂O group of the C2-benzoate moiety, extends the binding mode through unique attractive interactions beyond those of paclitaxel and new-generation taxoids bearing no organofluorine groups.

The most potent compounds, 11e-05 and 11f-06 were analyzed in detail to investigate possible effects of the introduction of the 3’-DFV group to the 3^rd-generation taxoids bearing the 3-CF₃O/CHF₂O group at the C2-benzoate moiety. First, the molecular docking analysis confirmed that the basic paclitaxel binding pose, involving the H-bonding interactions of C2’-OH with Gly362 and the oxetane oxygen with Thr276, is conserved (see Fig. S1 A,B). It was also found that the simultaneous introduction of the 3’-DFV moiety and the 3-CF₃O/CHF₂O group at the C2-benzoate moiety brought in only positive effects on the binding mode of these new taxoids as compared to that of either the 2^nd-generation DFV-taxoids or the 3^rd-generation taxoids with the 3-CF₃O/CHF₂O-benzoate moiety at C2.

One of the most distinct features of the 3^rd-generation DFV-taxoids is the attractive interactions of fluorine substituents with the aromatic π-system of Phe272 (Fig. 5 A,B) [69, 70], as well as the H-bonding of the exo-fluorine of the 2,2-difluorovinyl group with the hydroxyl group of Ser236 [71]. In addition, favorable van der Waals contacts between the CF₃O/CHF₂O moiety and hydrophobic residues, Leu217, Leu230 and Leu275, in the proximal binding site were recognized although not highlighted (Fig. 5 C,D). It is worthy of note that the 3-CHF₂O group at the C2 benzoate moiety of 11f-06 is oriented to form a H-bond to the (π)N3 of His229, which indicates that the CHF₂O group can make a unique interaction (OF₂C-H---N) [72], leading to higher affinity in β-tubulin (Fig. 5 F). On the other hand, the 3-CF₃O group at the C2 benzoate moiety of 11f-05 is shown to form a H-bond with the (π)N3-H of His229 (OF₂C-F---HN) [71, 73] by taking advantage of facile tautomerization in the imidazole moiety of His229 (Fig. 5 E).

Fig. 5.

Key attractive interactions of the fluorine substituents with specific amino acid residues in the taxoid binding site. (A,B): Fluorine interactions of 11e-05 (A) and 11f-06 (B) with the π-system of Phe272 and H-bonding with Ser236. (C,D): Predicted binding mode of 11e-05 (C) and 11f-06 (D) in a hydrophobicity surface representation of the proximal binding site, showing hydrophobic residues nearby. (E,F): H-bonding of the CF₃O group of 11e-05 (E) and the CHF₂O group of 11f-06 (F) with His229. (G): Overlay of 11e-05 (cyan) and SB-T-121405 (light brown), showing a consistent docking mode of the CF₃O group in contrast to the deviated DFV group position. (H): The overlay of 11f-06 (pink) and SB-T-121706 (blue), exhibiting considerable difference in the positions of the 3-CHF₂O-benzoate group at C2 between these two taxoids and the loss of the OF₂C-H---N interaction, in addition to the anticipated difference between the DFV and isobutenyl groups.

As anticipated, the DFV group of 3^rd-generation DFV-taxoids mimics the binding mode of the isobutenyl group of 2^nd- and 3^rd-generation taxoids, e.g. SB-T-1214 or SB-T-121405, in spite of the fact that a fluorine is substantially smaller than a methyl group in size. Partly due to this difference in size, the DFV group is positioned in closer proximity to Phe272 and Ser236 than the isobutenyl group, which enables the attractive interaction of fluorine with the aromatic π-system of this key amino acid residue. This appears to be a critical merit for the replacement of isobutyl group with DFV group to enhance the affinity (Fig. 5 G,H). It should be noted that the 3-CF₃O-benzoate moieties at C2 of 11e-05 and SB-T-121405 are perfectly overlaid (Fig. 5 G). On the other hand, the overlay of 11f-06 and SB-T-121706 revealed a very interesting difference in the positioning and orientation of the 3-CHF₂O-benzoate moiety at C2 between these two taxoids. As described above for 11f-06, a H-bonding (OF₂C-H---N) was indicated between the 3-CHF₂O group and the (π)N3 nitrogen of His229 (Fig. 5 F,H). In sharp contrast, no possible H-bonding was observed between the 3-CHF₂O group of SB-T-121706 and His229 (Fig 5 H). This is attributed to the rotation of the CHF₂O group, which makes the orientation of the F₂C-H bond impossible to form a H-bond with the the (π)N3 nitrogen of His229. The C2-benzoate moiety is deviated away from His229 in SB-T-121706, which appears to favor this rotation of the CHF₂O group.

In a similar manner, the molecular docking analysis was performed for six other 05-series and six other 06-series DFV-taxoids. The energy minimized docked structures of all (14) of the 3^rd-generation DFV-taxoids in β-tubulin, together with paclitaxel and SB-T-1214 for comparison, are collectively shown in Fig. S2. In all of the 05-series DFV-taxoids (Fig. S2), one of the fluorine of the CF₃O group forms a H-bond with the (π)N3-H of His229 (OF₂C-F---HN) in exactly the same manner as that found in 11e-05 (Fig. 5 E). Also, in all of the 06-series DFV-taxoids (Fig. S2), the methine of the CHF₂O group forms a H-bond with (π)N3 of His229 (OF₂C-H---N) in exactly the same manner as that found in 11f-06 (Fig. 5 F). As mentioned above, gem-fluorines of all 3^rd-generation DFV-taxoids hold attractive interactions with Phe272 and H-bonding with Ser236 regardless of the 05-series or 06-series.

In order to assess the positioning of the 3’-DFV group and the attractive interaction of its gem-fluorines with the aromatic π-system (i.e., phenyl group) of Phe272, as well as the H-bonding with the hydroxyl group of Ser236, the energy-minimized docked structures of SB-T-12852, SB-T-12857, 11e-05 and 11f-06 were overlaid (Fig. S1 C), which clearly shows an excellent overlap regardless of the substituent at C10 and the CF₃O/CHF₂O group of the benzoate moiety at C2. The overlay of SB-T-12852 (no substituent on the C2-benzoate) and its 3-CF₃O-benzoate counterpart, 11e-05, shows a very good overlap of their 3’-DFV moieties, regardless of the presence or absence of a substitution on the C2-benzoate moiety (Fig. S1 D). In a similar manner, the overlay of SB-T-12857 (no substituent on the C2-benzoate) and its 3-CHF₂O-benzoate counterpart, 11f-06, exhibits an excellent overlap of their 3’-DFV moieties, regardless of the presence or absence of a 3-CHF₂O group on the C2-benzoate moiety (Fig. S1 E). As mentioned above, 3rd-generation DFV-taxoids bearing a methoxy group at C10 or two methoxy groups at C7 and C10, i.e., 11e-05, 11e-06, 11f-05 and 11f-06, are some of the most potent compounds (Table 1). However, it appears that the observed high cytotoxicity is not attributed to their stronger affinity to β-tubulin based on particular attractive interaction with amino acid residues in the binding site, since the methoxy group at C10 (11e) and those at C7 and C10 (11f) are in the solvent-exposed region (Fig. S1 F).

3. Conclusions

A series of 3^rd-generation taxoids bearing a CF₂=CH- (DFV) group at C3’ and a CF₃O/CHF₂O group at C2 benzoate moiety were synthesized and fully characterized. The cytotoxicity of these 3^rd-generation DFV-taxoids was evaluated in A549, HT29, Vcap, PANC-1, DLD-1 and LCC6-MDR human cancer cell lines. All of these new DFV-taxoids exhibited 2–4 orders of magnitude higher potency against extremely drug-resistant cancer cell lines, LCC6-MDR and DLD-1, as compared to paclitaxel, which is practically ineffective against these two cell lines, and also better dose-response (kill) curves than paclitaxel and SB-T-1214 (representative 2^nd-generation taxoid). The results clearly indicate that these new DFV-taxoids can effectively overcome MDR caused by the overexpression of Pgp and other ABC cassette transporters. Furthermore, several highly potent new DFV-taxoids exhibited impressive dose response (kill curve) profiles. In general, the new DFV-taxoids bearing a 3-OCF₂H group at the C2-benozate moiety (06 series) appear to possess slightly higher potency than those bearing a 3-CF₃O-benzoate group at C2 (05 series). A DFV-mimic of cabazitaxel, 11f-06, exhibited remarkably high potency (IC₅₀ 30–89 pM) against MCF7, PANC-1, PC3 and LCC6-MDR cancer cell lines.

In order to assess the multiple “fluorine effects” on the binding mode of the 3^rd-generation DFV-taxoids, which combined the 2^nd-generation DFV-taxoids with the 3^rd-generation taxoids bearing a 3-CF₃O/CHF₂O-benzoate at C2, we performed an extensive molecular docking analysis. This computational study has revealed that these new DFV-taxoids occupy the paclitaxel binding site in β-tubulin in a manner similar to paclitaxel (PDB: 1JFF) and SB-T-1214 (2^nd-generation taxoid), keeping critical H-bonding interactions between C2’-OH and Gly362, as well as oxetane oxygen and Thr276. Also, the 3-CF₃O/CHF₂O-benzoate at C2 moiety of the new DFV-taxoids overlays very well with that of SB-T-121405, as well as other 3^rd-generation taxoid with a 3-CF₃O-benzoate at C2. However, the 3’-DFV moiety induces a little more compact folded structure in the new DFV-taxoids, which is attributed to a smaller size of the DFV group than the isobutenyl group. This compact structure allows unique attractive interactions between the gem-difluoro group and Phe272, as well as a H-boding with Ser236. In addition, the two fluorine atoms of the CF₃O/CHF₂O group have the same attractive interactions with Phe272. Furthermore, the CF₃O group has a H-bonding (OF₂C-F---HN) interaction with His229 and the CHF₂O group has another type H-bonding (OF₂C-H---N) interaction with His229.

These unique attractive interactions, involving fluorine, enable an enhanced binding mode of the new DFV-taxoids with β-tubulin beyond those of paclitaxel and new-generation taxoids without bearing organofluorine groups, and we believe that these unique features are reflected in the remarkable potency of the new DFV-taxoids.

It should also be noted that these new 3^rd-generation DFV-taxoids will serve as promising candidates for highly potent chemotherapeutic agents with proper formulations, e.g., nanoformulations, and also as payloads for tumor-targeted drug delivery systems, e.g., (a) antibody drug conjugates (ADCs) wherein the 1^st-generation ADCs require a 10–100 pM level IC₅₀ potency, whereas 2^nd-generation ADCs require only 1 nM level IC₅₀ potency [74], and (b) small molecule drug conjugates (SMDCs) [75].

4. Experimental Section

4.1. General Methods.

¹H NMR and ¹³C NMR spectra were measured on a Varian 300 or 500 MHz spectrometer or a Bruker 300, 400, 500, or 700 MHz NMR spectrometer. Melting points were measured on a Thomas-Hoover capillary melting point apparatus and are uncorrected. Thin-layer chromatography (TLC) analyses were performed on aluminum-backed Silica G TLC plates (Sorbent Technologies, 200 μm, 20 cm × 20 cm), visualized with UV light, and stained with 10% sulfuric acid- EtOH, 10% PMA-EtOH or 10% vanillin-EtOH with 1% sulfuric acid. Column chromatography was performed on silica gel 60 (Merck, 230−400 mesh ASTM). High-resolution mass spectrometry analysis was carried out on an Agilent LC-UV- TOF mass spectrometer using Jupiter C18 analytical column with 2.6 μm, 100 Å, 2.1 mm × 100 mm, with 0.1% TFA in water (optima grade) as solvent A and 0.1% TFA in CH₃CN (optima grade) as solvent B, running temperature 25 °C, and flow rate 0.5 mL/min, at the Institute of Chemical Biology and Drug Discovery (ICB&DD), Stony Brook, NY.

4.2. Materials

The chemicals were purchased from Sigma-Aldrich, Fisher Scientific, or VWR International. Dichloromethane and methanol were dried before use by distillation over calcium hydride under nitrogen. Ether and tetrahydrofuran were dried before use by distillation over sodium benzophenone kept under nitrogen. 10-Deacetylbaccatin III was obtained from Indena SpA, Italy. Reaction flasks were dried in a 100 °C oven and allowed to cool to room temperature in a desiccator over calcium sulfate and assembled under an inert nitrogen gas atmosphere. (3R,4S)-4-(2,2-Difluoroethenyl)-1-(4-methoxyphenyl)-3-(triisopropylsiloxy)azetidin-2-one[34, 76], 7,10,13-tris(triethylsilyl)-2-debenzoyl-10-deacetylbaccatin III (1) [33], 2-debenzoyl-2-(3-trifluoromethoxy)-10-deacetylbaccatin III (3–05) [47], 2-debenzoyl-2-(3-difluoromethoxy)-10-deacetylbaccatin III (3–06) [47], 2-debenzoyl-2-(3-trifluromethoxybenzoyl)-10-deacetylbaccatin III (4–05) [47], 10-deacetyl-2-debenzoyl-2-(3-difluromethoxybenzoyl)baccatin III (4–06) [47], 2-debenzoyl-2-(3-trifluromethoxybenzoyl)baccatin III (5a-05) [47], 2-debenzoyl-2-(3-difluromethoxybenzoyl)baccatin III (5a-06) [47], 2-debenzoyl-2-(3-trifluromethoxybenzoyl)-10-propanoyl-10-deacetylbaccatin III (5c-05) [47], 2-debenzoyl-2-(3-difluromethoxybenzoyl)-10-propanoyl-10-deacetylbaccatin III (5c-06) [47], 2-debenzoyl-2-(3-trifluromethoxybenzoyl)-7-triethylsilyl-10-deacetylbaccatin III (6–05) [47], 2-debenzoyl-2-(3-difluromethoxybenzoyl)-7-triethylsilyl-10-deacetylbaccatin III (6–06) [47], 2-debenzoyl-2-(3-trifluromethoxybenzoyl)-7-triethylsilylbaccatin III (7a-05) [47], 2-debenzoyl-2-(3-difluromethoxybenzoyl)-7-triethylsilylbaccatin III (7a-06) [47], 2-debenzoyl-2-(3-trifluromethoxybenzoyl)-7-triethylsilyl-10-cyclopropanecarbonyl-10-deacetylbaccatin III (7b-05) [47], 2-debenzoyl-2-(3-difluromethoxybenzoyl)-7-triethylsilyl-10-deacetyl-10-cyclopropanecarbonylbaccatin III (7b-06) [47], 2-debenzoyl-7-triethylsilyl-2-(3-trifluromethoxybenzoyl)-10-propanoyl-10-deacetylbaccatin III (7c-05) [47], 2-debenzoyl-7-triethylsilyl-2-(3-difluromethoxybenzoyl)-10-propanoyl-10-deacetylbaccatin III (7c-06) [47], 2-debenzoyl-2-(3-trifluromethoxybenzoyl)-7-triethylsilyl-10-(N,N-dimethylcarbamoyl)-10-deacetylbaccatin III (7d-05) [47], 2-debenzoyl-2-(3-difluromethoxybenzoyl)-7-triethylsilyl-10-deacetyl-10-N,N-demthylcarbamoylbaccatin III (7d-06) [47], 10-deacetyl-2-debenzoyl-10-methoxycarbonyl-7-TES-2-(3-trifluoromethoxybenzoyl)-baccatin III (7g-05) [47], 2-debenzoyl-2-(3-difluromethoxybenzoyl)-10-deacetyl-10-methoxycarbonyl-7-triethylsilylbaccatin III (7g-06) [47], 7,10-dimethyl-10-deacetylbaccatin III (8) [77], 13-triethylsilyl-7,10-dimethyl-10-deacetylbaccatin III (8–1) [77], and 2-debenzoyl-7,10-dimethyl-13-triethylsilyl-10-deacetylbaccatin III (8–2) [77] were prepared by the literature methods. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), was purchased from Aldrich Co. and Sigma. Biochemical materials, including RPMI-1640 cell culture media, DPBS buffer, fetal bovine serum (FBS), PenStrep, and TrypLE, were obtained from Gibco and VWR International and used as received for cell-based assays. Cancer cell lines A549, HT29, Vcap, MCF7, PANC-1 and PC3 were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. The DLD-1 cell line was purchased from ATCC. The LCC6-MDR was a gift from Dr. Robert Clarke (Georgetown University/University of Minnesota).

4.3. Chemical synthesis

4.3.1. 2-Debenzoyl-2-(3-trifluoromethoxybenzoyl)-7-triethylsilyl-10-methyl-10-deacetylbaccatins (7e-05)

Compound 6–05 (410 mg, 0.55 mmol) and NaH (~ 60% wt. in oil; 40 mg, 0.994 mmol) were dissolved in tetrahydrofuran (THF) (8.2 mL) and cooled to 0 °C for 10 minutes. To this mixture was added excess of CH₃I (15.95 mmol; 0.33 mL, 5.52 mmol). The mixture was allowed being stirred for 2–4 hours at this temperature and the whole process was monitored via thin-layer chromatography (TLC) (hexanes:ethyl acetate = 70:30). Upon completion, the reaction was quenched with saturated NH₄Cl solution (4 mL), extracted with ethyl acetate (3 × 100 mL) and washed the collected organic layers with brine (3 × 100 mL), dried over anhydrous MgSO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (hexanes: ethyl acetate = 67:33) to afford 7e-05 (201 mg, 0.27 mmol, 48%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 8.04 (d, J = 8.00 Hz, 1H), 8.00 (s, 1H), 7.53 (t, J = 8.00 Hz, 1H), 7.45 (t, J = 8.00 Hz, 1H), 5.58 (d, J = 7.04 Hz, 1H), 4.89 – 4.98 (m, 3H), 4.43 (dd, J₁ = 10.44 Hz, J₂ = 6.76 Hz, 1H), 4.27 (AB, J_AB = 8.16 Hz, 1H), 4.13 (AB, J_AB = 8.16 Hz, 1H), 3.89 (d, J = 6.96 Hz, 1H), 3.42 (s, 3H), 2.46 – 2.54 (m, 1H), 2.24 – 2.29 (m, 5H), 2.11 (s, 3H), 1.86 – 1.93 (m, 1H), 1.67 (s, 3H), 1.17 (s, 3H), 1.07 (s, 3H), 0.92 – 0.98 (m, 9H), 0.51 – 0.62 (m, 6H). This synthetic intermediate was used in the next step without further characterization.

4.3.2. 2-Debenzoyl-2-(3-difluoromethoxybenzoyl)-7-triethylsilyl-10-methyl-10-deacetylbaccatins (7e-06)

Compound 6–06 (295 mg, 0.407 mmol) and NaH (~ 60% wt. in oil; 28 mg, 0.692 mmol) were dissolved in THF (5.8 mL) and cooled to 0 °C for 10 minutes. To this mixture was added excess of CH₃I (1.14 mL, 19.14 mmol). The mixture was allowed being stirred for 2–4 hours at this temperature and the whole process was monitored via TLC (hexanes:ethyl acetate = 70:30). Upon completion, the reaction was quenched with saturated NH₄Cl solution (2 mL), extracted with ethyl acetate (3 × 80 mL) and washed the collected organic layers with brine (3 × 60 mL), dried over anhydrous MgSO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (hexanes: ethyl acetate = 67:33) to afford 7e-06 (148 mg, 0.20 mmol, 49%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, J = 8.00 Hz, 1H)), 7.90 (s, 1H), 7.48 (t, J = 8.00 Hz, 1H), 7.35 – 7.37 (m, 1H), 6.57 (t, J = 73.20 Hz, 1H), 5.58 (d, J = 7.04 Hz, 1H), 4.89 – 4.98 (m, 3H), 4.44 (dd, J₁ = 10.44 Hz, J₂ = 6.76 Hz, 1H), 4.29 (AB, J_AB = 8.16 Hz, 1H), 4.14 (AB, J_AB = 8.16 Hz, 1H), 3.89 (d, J = 6.96 Hz, 1H), 3.41 (s, 3H), 2.46 – 2.53 (m, 1H), 2.24 – 2.27 (m, 5H), 2.11 (s, 3H), 1.86 – 1.92 (m, 1H), 1.67 (s, 3H), 1.17 (s, 3H), 1.07 (s, 3H), 0.94 – 0.98 (m, 9H), 0.53 – 0.63 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 204.69, 171.07, 166.11, 151.21, 143.41, 133.86, 131.56, 130.31, 127.42, 125.20, 121.11, 118.42, 115.82, 113.22, 84.54, 82.88, 81.13, 79.07, 77.55, 77.43, 77.23, 76.91, 76.69, 75.53, 73.14, 68.12, 58.43, 55.99, 47.52, 43.01, 38.51, 37.50, 29.90, 27.04, 22.75, 19.74, 15.40, 10.05, 6.99, 5.62; ¹⁹F NMR (376 MHz, CDCl₃) δ −81.03 (ABq, J = 167 Hz, 2F). This synthetic intermediate was used in the next step without further characterization.

4.3.3. 2-Debenzoyl-7,10-dimethyl-13-triethylsilyl-2-(3-trifluromethoxybenzoyl)-10-deacetylbaccatin III (8-3-05)

Compound 8–2 (479 mg, 0.822 mmol), 4-N,N-dimethylamionopyridine (DMAP) (782 mg, 6.40 mmol) and 3-trifluromethoxybenzoic acid (659 mg, 3.20 mmol) were dissolved in CH₂Cl₂ (7 mL), and purged with nitrogen. To the mixture N,N’-diisopropylcarbodiimide (DIC) (1.0 mL, 6.40 mmol) was added dropwise under inert conditions. The mixture was allowed at room temperature and the reaction progress was monitored by TLC (hexanes:ethyl acetate = 80:20). Upon completion, the reaction was quenched with saturated aqueous NH₄Cl solution (7 mL), diluted with water (70 mL) and extracted with ethyl acetate (3 × 70 mL). The organic layers were collected, washed with brine (3 × 70 mL), dried over anhydrous MgSO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (hexanes:ethyl acetate = 90:10) to afford 8-3-05 (558 mg, 0.72 mmol, 88%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 8.04 (d, J = 8.05 Hz, 1H)), 7.99 (s, 1H), 7.53 (t, J = 8.05 Hz, 1H), 7.45 (d, J = 8.05 Hz, 1H), 5.58 (d, J = 7.16 Hz, 1H), 4.96 – 5.04 (m, 2H), 4.83 (s, 1H), 4.28 (AB, J_AB = 8.20 Hz, 1H), 4.15 (AB, J_AB = 8.20 Hz, 1H), 3.94 (dd, J₁ = 10.72 Hz, J₂ = 6.40 Hz, 1H), 3.86 (d, J = 7.08 Hz, 1H), 3.47 (s, 3H), 3.32(s, 3H), 2.68 – 2.76 (m, 1H), 2.29 (s, 3H), 2.11 – 2.23 (m, 2H), 2.07 (d, J = 0.92 Hz, 3H), 1.75 – 1.82 (m, 1H), 1.71 (s, 3H), 1.17 (s, 3H), 1.15 (s, 3H), 1.03 (t, J = 8.00 Hz, 1H), 0.65 – 0.72 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 205.8, 170.36, 165.76, 149.47, 144.64, 133.52, 131.75, 130.44, 128.79, 126.34, 122.22, 84.37, 83.35, 81.43, 80.99, 79.87, 77.50, 76.49, 75.90, 68.68, 57.65, 57.31, 56.80, 47.72, 43.21, 40.00, 32.15, 26.99, 23.72, 22.41, 21.25, 15.47, 10.55, 7.10, 5.04. This synthetic intermediate was used in the next step without further characterization.

4.3.4. 2-Debenzoyl-7,10-dimethyl-13-triethylsilyl-2-(3-difluromethoxybenzoyl)-10-deacetylbaccatin III (8-3-06)

Compound 8–2 (602 mg, 0.88 mmol), DMAP (860 mg, 7.04 mmol) and 3-trifluromethoxybenzoic acid (827 mg, 4.40 mmol) were dissolved in CH₂Cl₂ (8 mL) and purged with nitrogen. To the mixture DIC (1.1 mL, 6.40 mmol) was added dropwise under inert conditions. The mixture was allowed at room temperature and the reaction progress was monitored by TLC (hexanes:ethyl acetate = 80:20). Upon completion, the reaction was quenched with saturated NH₄Cl (5 mL), diluted with water (60 mL) and extracted with ethyl acetate (3 × 60 mL). The organic layers were collected, washed with brine (3 × 50 mL), dried over anhydrous MgSO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel with increasing amounts of ethyl acetate in hexanes (hexanes:ethyl acetate = 90:10) to afford 8-3-06 (545 mg, 0.73 mmol, 83% yield) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 7.94 (d, J = 8.00 Hz, 1H)), 7.88 (s, 1H), 7.48 (t, J = 8.00 Hz, 1H), 7.35 – 7.37 (m, 1H), 6.56 (d, J = 73.2 Hz, 1H), 5.56 (d, J = 7.12 Hz, 1H), 4.95 – 5.02 (m, 2H), 4.82 (s, 1H), 4.27 (AB, J_AB = 8.20 Hz, 1H), 4.13 (AB, J_AB = 8.20 Hz, 1H), 3.90 – 3.95 (m, 1H), 3.84 (d, J = 7.08 Hz, 1H), 3.45 (s, 3H), 3.30 (s, 3H), 2.66 – 2.74 (m, 1H), 2.28 (s, 3H), 2.14 – 2.18 (m, 2H), 2.07 (d, J = 0.96 Hz, 3H), 1.73 – 1.80 (m, 1H), 1.69 (s, 3H), 1.15 (s, 3H), 1.13 (s, 3H), 1.00 – 1.04 (m, 9H), 0.64 – 0.71 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 205.8, 170.39, 166.04, 151.22, 151.19, 151.17, 144.59, 133.50, 131.53, 130.32, 127.41, 125.28, 120.98, (118.41, 115.81, 113.20), 84.34, 83.31, 81.37, 80.96, 79.75, 77.43, 76.49, 75.73, 68.66, 57.57, 57.26, 56.77, 47.67, 43.18, 40.00, 32.11, 26.95, 22.43, 21.21, 15.42, 10.52, 7.09, 5.01. This synthetic intermediate was used in the next step without further characterization.

4.3.5. 2-Debenzoyl-7,10-dimethyl-2-(3-trifluromethoxybenzoyl)-10-deactylbaccatin III (7f-05)

Compound 8-3-05 (558 mg, 0.72 mmol) was dissolved in a 1:1 mixture of acetonitrile:pyridine (24 mL total) and cooled to 0 °C under inert conditions. To the mixture excess HF, 70% in pyridine (7 mL), was added dropwise. The reaction was stirred at room temperature and monitored by TLC (hexanes : ethyl acetate = 70:30). Upon completion the reaction was quenched with 10% aqueous citric acid (15 mL), neutralized with saturated NaHCO₃ (70 mL) and extracted with ethyl acetate (3 × 70 mL). The organic layer was collected, washed with saturated CuSO₄ solution (3 × 60 mL), water (60 mL) and brine (3 × 70 mL). The extract was then dried over anhydrous MgSO₄, and concentrated in vacuo. Purification was done by column chromatography on silica gel with increasing amounts of methanol in DCM (methanol : DCM = 2:98) to afford 7f-05 (349 mg, 0.531 mmol, 73%) as a crystalline white solid: ¹H NMR (400 MHz, CDCl₃, ppm) δ 8.04 (d, J = 8.15 Hz, 1H)), 8.00 (s, 1H), 7.53 (t, 1H), 7.45 (d, J = 8.15 Hz, 1H), 5.57 (d, J = 7.08 Hz, 1H), 5.03 (d, J = 8.16 Hz, 1H), 4.90 (t, 1H), 4.85 (s, 1H), 4.28 (AB, J_AB = 8.16 Hz, 1H), 4.14 (AB, J_AB = 8.16 Hz, 1H), 3.92 – 3.95 (m, 2H), 3.47 (s, 3H), 3.32 (s, 3H), 2.69 – 2.77 (m, 1H), 2.20 – 2.30 (m, 5H), 2.12 (d, J = 1.02 Hz, 3H), 1.75 – 1.82 (m, 1H), 1.71 (s, 3H), 1.53 (bs, OH), 1.17 (s, 3H), 1.08 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 205.63, 171.09, 165.72, 149.47, 149.46, 143.03, 134.63, 131.69, 130.44, 126.30, 122.35, 121.92, 119.35, 84.37, 83.34, 81.51, 81.06, 79.11, 77.43, 76.53, 75.36, 68.17, 57.51, 57.25, 57.11, 48.03, 42.96, 38.50, 32.27, 27.25, 22.71, 20.07, 15.54, 10.42; ¹⁹F NMR (376 MHz, CDCl₃, 25 °C) δ −57.86 (3F). HRMS (TOF) m/z: Calcd. For C₃₂H₃₉F₃O₁₁Na⁺, 679.2337. Found, 679.2334.

4.3.6. 2-Debenzoyl-7,10-dimethyl-2-(3-difluromethoxybenzoyl)-10-deacetylbaccatin III (7f-06)

In the same manner as that for the synthesis of 7f-05, 7f-06 (349 mg, 73% yield) was synthesized from 8-3-06 (450 mg, 0.481 mmol), as a crystalline white solid: ¹H NMR (400 MHz, CDCl₃, ppm) δ 7.96 (d, J = 8.00 Hz, 1H)), 7.90 (s, 1H), 7.49 (t, J = 8.00 Hz, 1H), 7.35 – 7.38 (m, 1H), 6.57 (d, J = 73.2 Hz, 1H), 5.57 (d, J = 7.08 Hz, 1H), 5.02 (d, J = 8.16 Hz, 1H), 4.87 – 4.92 (m, 1H), 4.85 (s, 1H), 4.29 (AB, J_AB = 8.20 Hz, 1H), 4.14 (AB, J_AB = 8.20 Hz, 1H), 3.89 – 3.95 (m, 2H), 3.46 (s, 3H), 3.32(s, 3H), 2.69 – 2.76 (m, 1H), 2.22 – 2.28 (m, 5H), 2.19 (d, J = 5.24 Hz, 1H), 2.11 (d, J = 0.92 Hz, 3H), 1.74 – 1.81 (m, 1H), 1.70 (s, 3H), 1.54 (s, 1H), 1.16 (s, 3H), 1.07 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 205.68, 171.14, 166.06, 151.20, 143.07, 134.60, 131.50, 130.34, 127.41, 125.24, 121.11, 115.81, 84.38, 83.34, 81.50, 81.06, 79.06, 77.43, 76.58, 75.24, 68.15, 57.48, 57.25, 57.11, 48.01, 42.97, 38.55, 32.27, 27.23, 22.75, 20.08, 15.52, 10.43. HRMS (TOF) m/z: Calcd. For C₄₄H₅₇F₂NO₁₇H⁺, 661.2431. Found, 661.2429.

4.3.7. (3R,4S)-4-(2,2-Difluoroethenyl)-1-(4-methoxyphenyl)-3-(triisopropylsiloxy)azetidin-2-one ((+)-10a) [34]

A solution of (+)-9 (163 mg, 0.43 mmol), 3 eq. of sodium chlorodifluoroacetate (197 mg, 1.29 mmol) and 3 eq. of trophenylphosphine (339 mg, 1.29 mmol) in DMF (2 mL) was heated to 90 °C for 5 hours. The reaction mixture was cooled to room temperature and quenched with saturated aqueous NH₄Cl and extracted with ethyl acetate and washed with brine. The organic layer was dried over MgSO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel with hexanes/ethyl acetate (3:1) as eluent to give (+)-10a (160 mg, 0.87 mmol, 68%) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 7.33 (d, J = 8.8 Hz, 2H)), 6.87 (d, J = 8.8 Hz, 2H), 5.14 (d, J = 4.9 Hz, 1H), 4.83 (m, 1H), 4.54 (dd, J₁ = 24.7 Hz, J₂ = 10.1 Hz, 1H), 3.79 (s, 3H), 1.07–1.26 (m, 21H); ¹³C NMR (100 MHz, CDCl₃) δ 164.81, 160.90, 157.99, 156.63, 155.09, 130.79, 118.50, 114.67, 77.43, 77.36, 75.92, 75.77, 75.68, 75.53, 55.66, 54.01, 53.94, 17.87, 17.82, 12.08; ¹⁹F NMR (376 MHz, CDCl₃, 25 °C) δ −81.20 (d, J = 31.0 Hz, 1 F), −85.83 (dd, J = 5.6 Hz, 31.0 Hz, 1F). All data are in agreement with literature values [34].

4.3.8. 2-Debenzoyl-2-(3-trifluoromethoxybenzoyl)-10-acetyl-3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11a-05)

Compound 7a-05 and (+)-10 were dissolved in THF and cooled to −30 °C under inert conditions. To the mixture was added LiHMDS, 1M in tert-butyl methyl ether, dropwise. The reaction was monitored at low temperature by TLC and, upon completion, was quenched with saturated aqueous NH₄Cl solution. The mixture was then allowed to warm to room temperature, diluted with water and extracted with ethyl acetate. The organic layer was then washed with brine, dried over anhydrous MgSO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel to afford the coupling product as a white solid, which was subjected to the subsequent deprotection without characterization. An aliquot of the coupling product was dissolved in a 1:1 mixture of acetonitrile:pyridine and cooled to 0 °C under inert conditions. To the mixture excess HF, 70% in pyridine, was added dropwise. The reaction mixture was stirred at room temperature and monitored by TLC. Upon completion the reaction was quenched with 10% aqueous citric acid, neutralized with saturated NaHCO₃ and extracted with ethyl acetate. The organic layer was collected, washed with saturated CuSO₄ solution, water and brine. The extract was then dried over anhydrous MgSO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel with increasing amounts of ethyl acetate in hexanes (hexanes:ethyl acetate = 100:0–50:50) to afford 11a-05 (90% for 2 steps) as a crystalline white solid: mp 134.7–135.6 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.06 (d, J = 8.0 Hz, 1H), 7.99 (s, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 6.30 (s, 1H), 6.20 (t, J = 8.0 Hz, 1H), 5.65 (d, J = 7.2 Hz, 1H), 4.97 (d, J= 8.4 Hz, 1H), 4.92 – 4.80 (m, 2H), 4.58 (dd, J = 25.2, 9.2 Hz, 1H), 4.47–4.39 (m, 1H), 4.30–4.23 (m, 2H), 4.17 (d, J = 8.4 Hz, 1H), 3.82 (d, J = 7.2 Hz, 1H), 3.47 (d, J = 5.6 Hz, 1H), 2.61 – 2.47 (m, 2H), 2.35 (d, J = 12.0 Hz, 5H), 2.24 (s, 3H), 1.92–1.85 (m, 4H), 1.70 (s, 1H), 1.68 (s, 3H), 1.63 (s, 4H), 1.31(s, 9H), 1.25 (s, 5H), 1.15 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 203.70, 171.35, 170.39, 165.77, 149.51, 142.46, 131.41, 130.52, 128.72, 126.31, 122.46, 84.58, 81.23, 79.34, 76.47, 75.82, 75.71, 73.29, 72.84, 72.30, 58.74, 45.83, 43.36, 35.73, 35.54, 29.86, 28.25, 26.83, 22.24, 20.96, 15.04, 9.67; ¹⁹F NMR (470 MHz, CDCl₃) δ −57.96 (s), −83.91 (d, J = 36.2 Hz), −85.79 (d, J = 36.2 Hz). HRMS (TOF) m/z: Calcd. For C₄₂H₅₀F₅NO₁₆Na⁺, 942.2942. Found, 942.2970.

In a similar manner, the other 13 novel taxoids were synthesized.

4.3.9. 2-Debenzoyl-2-(3-trifluoromethoxybenzoyl)-10-cyclopropancarbonyl-3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11b-05)

White solid; 94% yield; mp 138.2–140.3 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.05 (d, J = 8.0 Hz, 1H), 7.99 (s, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 6.30 (s, 1H), 6.19 (t, J = 8.8 Hz, 1H), 5.64 (d, J = 6.8 Hz, 1H), 5.01 – 4.90 (m, 2H), 4.89–4.80 (m, 1H), 4.57 (dd, J = 24.5, 9.6 Hz, 1H), 4.45–4.35 (m, 1H), 4.30–4.20 (m, 2H), 4.15 (d, J = 8.0 Hz, 1H), 3.82 (d, J = 6.4 Hz, 1H), 3.51 (d, J = 3.2 Hz, 1H), 2.65 – 2.47 (m, 2H), 2.36 (s, 3H), 2.33 (s, 2H), 1.88 (s, 4H), 1.81–1.74 (m, 1H), 1.73–1.68 (m, 2H), 1.67 (s, 3H), 1.30 (s, 9H), 1.26 (s, 3H), 1.15 (s, 5H), 1.06 – 0.93 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ203.6, 175.07, 170.18, 165.58, 156.53, 154.82, 149.30, 142.33, 133.04, 131.20, 130.34, 128.55, 126.14, 122.28, 121.42, 119.37, 84.44, 80.36,81.02, 79.17, 76.28, 75.65, 75.32, 73.10, 72.65, 72.17, 58.53, 47.99, 45.65, 43.16, 35.48, 35.35, 28.06, 26.69, 22.08, 21.91, 12.99,14.89, 9.46, 9.41, 9.17; ¹⁹F NMR (471 MHz, CDCl₃) δ −57.96 (s), −83.93 (d, J = 35.2 Hz), −85.80 (d, J = 35.2 Hz). HRMS (TOF) m/z: Calcd. For C₄₃H₅₃F₅NO₁₆H⁺, 946.3279. Found, 942.3294.

4.3.10. 2-Debenzoyl-2-(3-trifluoromethoxybenzoyl)-10-propanoyl-3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11c-05)

White solid; 89% yield; mp 136.4–138.7 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.05 (d, J = 8.0 Hz, 1H), 7.98 (s, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 6.31 (s, 1H), 6.19 (t, J = 8.0 Hz, 1H), 5.64 (d, J = 7.2 Hz, 1H), 5.01 – 4.90 (m, 2H), 4.89–4.80 (s, 1H), 4.57 (dd, J = 24.5, 9.3 Hz, 1H), 4.46–4.38 (m, 1H), 4.30–4.22 (m, 2H), 4.16 (d, J = 8.0 Hz, 1H), 3.82 (d, J = 6.8 Hz, 1H), 3.54 (d, J = 5.2 Hz, 1H), 2.62 – 2.42 (m, 4H), 2.36 (s, 5H), 1.95 – 1.81 (m, 4H), 1.76 (d, J = 3.2 Hz, 2H), 1.67 (s, 3H), 1.30 (s, 9H), 1.24 (s, 3H), 1.23 (t, J= 7.2 Hz, 3H), 1.14 (s, 3H); ¹³C NMR (125 MHz, CDCl₃, ppm) δ203.60, 174.57, 172.45, 170.21, 165.57, 156.53, 154.84, 149.30, 142.14, 133.06, 131.20, 130.34, 128.54, 126.14, 122.27, 121.42, 119.37, 84.41, 81.02, 80.36, 79.12, 76.28, 75.63, 75.34, 73.10, 72.63, 72.13, 58.51, 48.05, 45.67, 43.16, 35.55, 35.35, 28.06, 27.54, 26.62, 22.06, 21.81, 14.85, 9.49, 8.99; ¹⁹F NMR (471 MHz, CDCl₃) δ −57.96 (s), −83.94 (d, J = 37.7 Hz), −85.81 (d, J = 37.7 Hz). HRMS (TOF) m/z: Calcd. For C₄₂H₅₃F₅NO₁₆H⁺, 934.3279. Found, 934.3279.

4.3.11. 2-Debenzoyl-2-(3-trifluoromethoxybenzoyl)-10-(N,N’-dimethylcarbamoyl)-3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11d-05)

White solid; 92% yield; mp 144.4–145.5 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.05 (d, J = 8.0 Hz, 1H), 7.98 (s, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 6.25 (s, 1H), 6.20 (t, J = 8.4 Hz, 1H), 5.63 (d, J = 7.2 Hz, 1H), 4.98 (d, J = 9.2 Hz, 2H), 4.84 (t, J = 8.8 Hz, 1H), 4.57 (dd, J = 24.4, 9.6 Hz, 1H), 4.48 – 4.40 (m, 1H), 4.27 (d, J = 8.0 Hz, 2H), 4.16 (d, J = 8.4 Hz, 1H), 3.81 (d, J = 7.2 Hz, 1H), 3.61 (d, J= 4.4 Hz, 1H), 3.30–3.21 (m, 1H), 3.04 (s, 3H), 2.95 (s, 3H), 2.60 – 2.49 (m, 1H), 2.36 (s, 3H), 2.35–2.29 (m, 2H), 1.90 (s, 3H), 1.89–1.78 (m, 4H), 1.66 (s, 3H), 1.30 (s, 9H), 1.24 (s, 3H), 1.15 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 205.61, 170.30, 165.74, 156.72, 156.26, 155.01, 149.47, 142.72, 133.54, 131.44, 130.48, 128.70, 126.25, 122.45, 121.60, 119.55, 84.79, 81.27, 80.50, 79.40, 76.44, 76.32, 75.96, 73.27, 72.82, 72.53, 58.64, 48.19, 45.80, 43.34, 36.79, 36.17, 35.60, 35.55, 29.82, 28.50, 28.23, 26.99, 22.34, 22.23, 15.07, 9.49; ¹⁹F NMR (470 MHz, CDCl₃) δ −57.97 (s), −84.01 (d, J = 36.2 Hz), −85.87 (d, J = 36.2 Hz). HRMS (TOF) m/z: Calcd. For C₄₃H₅₃F₅N₂O₁₆H⁺, 949.3388. Found, 949.3388.

4.3.12. 2-Debenzoyl-2-(3-trifluoromethoxybenzoyl)-10-methyl-2’-triisopropylsilyl-3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11e-05)

White solid; 71% yield; mp 208.8–209.7 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.03 (d, J = 8.0 Hz, 1H), 7.97 (s, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 6.19 (t, J = 8.4 Hz, 1H), 5.63 (d, J = 7.2 Hz, 1H), 5.10 (d, J = 7.6 Hz, 1H), 5.0–4.92 (m, 2H), 4.84 (t, J = 8.8 Hz, 1H), 4.57 (dd, J= 24.4, 9.6 Hz, 1H), 4.27 (d, J = 8.0 Hz, 2H), 4.14 (d, J = 8.0 Hz, 1H), 3.90 – 3.77 (m, 2H), 3.41 (s, 3H), 2.60 – 2.49 (m, 1H), 2.35 (s, 3H), 2.34–2.28 (m, 2H), 2.15 (s, 1H), 1.92 (s, 3H), 1.87 – 1.77 (m, 2H), 1.67 (s, 3H), 1.31 (s, 9H), 1.19 (s, 3H), 1.17 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 207.87, 173.34, 171.34, 165.86, 157.49, 156.78, 150.18, 140.98, 135.67, 133.11, 131.31, 129.50, 126.77, 123.02, 122.54, 120.50, 118.45, 85.72, 83.55, 82.24, 80.62, 78.99, 78.78, 78.73, 78.47, 77.31, 76.75, 73.92, 72.78, 72.26, 60.27, 58.82, 56.43, 49.51, 48.49, 47.95, 44.30, 37.28, 36.37, 28.56, 26.80, 22.60, 21.54, 14.66, 10.19; ¹⁹F NMR (376 MHz, CDCl₃) δ −57.94 (s), −83.91 (d, J = 36.0 Hz), −85.79 (d, J = 36.0 Hz). HRMS (TOF) m/z: Calcd. For C₄₁H₅₀F₅NO₁₅Na⁺, 914.2993. Found, 914.3015.

4.3.13. 2-Debenzoyl-2-(3-trifluoromethoxybenzoyl)-3’-(2,2-difluoroethenyl)-3’-dephenylcabazitaxel (11f-05)

White solid; 83% yield; mp 130.5–131.7 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.04 (d, J = 8.0 Hz, 1H), 7.97 (s, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 6.20 (t, J = 8.4 Hz, 1H), 5.60 (d, J = 7.2 Hz, 1H), 5.00 (d, J = 9.2 Hz, 1H), 4.85 (m, 1H), 4.85 (t, J = 8.8 Hz, 1H), 4.80 (s, 1H), 4.57 (dd, J= 23.6, 9.6 Hz, 1H), 4.28 (d, J = 8.0 Hz, 1H), 4.15 (d, J = 8.4 Hz, 1H), 3.90 – 3.80 (m, 2H), 3.68–3.60 (m, 1H), 3.45 (s, 3H), 3.30 (s, 3H), 2.75–2.65 (m, 1H), 2.36 (s, 3H), 2.35–2.21 (m, 2H), 1.94 (s, 3H), 1.83 – 1.72 (m, 2H), 1.71 (s, 3H), 1.66 (s, 1H), 1.33 (s, 9H), 1.20 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 204.93, 172.36, 170.53, 165.55, 159.00, 156.69, 154.97, 154.38, 149.35, 138.82, 135.68, 131.41, 130.36, 128.62, 126.14, 123.57, 122.30, 121.52, 119.47, 117.41, 84.19, 82.70, 81.75, 80.77, 80.43, 78.86, 76.43, 75.33, 73.26, 72.74, 57.37, 57.06, 56.97, 48.18, 47.46, 43.36, 35.19, 32.14, 29.73, 28.18, 26.78, 22.24, 20.75, 14.72, 10.36; ¹⁹F NMR (470 MHz, CDCl₃) δ −57.95 (s), −84.03 (d, J = 35.5 Hz), −85.84 (d, J = 35.5 Hz). HRMS (TOF) m/z: Calcd. For C₄₁H₅₃F₅NO₁₅H⁺, 906.3330. Found, 906.3326.

4.3.14. 2-Debenzoyl-2-(3-trifluoromethoxybenzoyl)-10-methoxycarbonyl-3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11g-05)

White solid; 91% yield; mp 140.1–141.3 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.06 (d, J = 8.0 Hz, 1H), 7.97 (s, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 6.20 (t, J = 8.4 Hz, 1H), 6.12 (s, 1H), 5.65 (d, J = 7.2 Hz, 1H), 4.97 (d, J = 8.4 Hz, 1H), 4.92 (d, J = 9.2 Hz, 1H), 4.84 (t, J = 8.8 Hz, 1H), 4.57 (dd, J= 24.4, 9.6 Hz, 1H), 4.41–4.32 (m, 1H), 4.30–4.22 (m, 2H), 4.16 (d, J= 8.4 Hz, 1H), 3.87 (s, 3H), 3.80 (d, J= 7.2 Hz, 1H), 3.50 (d, J= 6.0 Hz, 1H), 2.60 – 2.51(m, 1H), 2.49 (d, J= 4.0 Hz, 1H), 2.37 (s, 3H), 2.36–2.30 (m, 2H), 1.92 (s, 3H), 1.91 – 1.77 (m, 1H), 1.69 (s, 3H), 1.67 (s, 3H), 1.30 (s, 9H), 1.24 (s, 3H), 1.15 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 203.75, 172.40, 170.26, 165.55, 158.83, 156.52, 155.70, 154.86, 154.21, 149.30, 149.29, 143.11, 132.65, 131.18, 130.34, 128.55, 126.14, 123.47, 122.26, 121.42, 119.37, 117.32, 84.36, 80.96, 80.39, 79.12, 78.22, 76.26, 75.58, 73.12, 72.54, 72.01, 58.51, 55.56, 48.08, 45.64, 43.11, 43.06, 35.59, 35.29, 28.33, 28.05, 26.54, 22.05, 22.04, 21.70, 14.93, 9.43; ¹⁹F NMR (470 MHz, CDCl₃) δ −57.96 (s), −84.03 (d, J = 37.6 Hz), −85.79 (d, J = 37.6 Hz). HRMS (TOF) m/z: Calcd. For C₄₁H₅₁F₅NO₁₇H⁺, 936.3072. Found, 936.3093.

4.3.15. 2-Debenzoyl-2-(3-difluoromethoxybenzoyl)-10-acetyl-3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11a-06)

White solid; 88% yield; mp 140.2–141.8 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.94 (d, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 6.77 (t, J = 80.0 Hz, 1H), 6.29 (s, 1H), 6.22 (t, J = 8.4 Hz, 1H), 5.64 (d, J = 6.8 Hz, 1H), 4.97 (d, J = 5.6 Hz, 2H), 4.88 (t, J = 8.0 Hz, 1H), 4.57 (dd, J = 24.4, 9.6 Hz, 1H), 4.38–4.46 (m, 1H), 4.32 – 4.27 (m, 2H), 4.16 (d, J = 8.4 Hz, 1H), 3.81 (d, J = 6.8 Hz, 1H), 3.51 (d, J = 4.0 Hz, 1H), 2.60–2.50 (m, 2H), 2.38 (s, 3H), 2.36 – 2.26 (m, 2H), 2.24 (s, 3H), 1.92 – 1.82 (m, 4H), 1.75 (d, J = 10.4 Hz, 2H), 1.67 (s, 3H), 1.26 (s, 9H), 1.25 (s, 3H), 1.14 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ203.55, 172.45, 171.22, 170.32, 165.95, 156.52, 154.88, 151.33, 142.18, 133.05, 130.93, 130.26, 127.09, 124.99, 119.71, 117.75, 115.68, 113.60, 84.40, 80.96, 80.38, 79.20, 76.28, 75.57, 75.51, 73.04, 72.61, 72.05, 58.47, 47.91, 45.62, 43.16, 35.50, 35.38, 28.04, 26.66, 22.13, 21.92, 20.80, 14.81, 9.54; ¹⁹F NMR (470 MHz, CDCl₃) δ −80.62 (d, J = 169.6 Hz), −83.12 (d, J = 169.6 Hz), −84.00 (d, J = 33.0 Hz), −85.76 (d, J = 33.0 Hz). HRMS (TOF) m/z: Calcd. For C₄₂H₅₁F₄NO₁₆H⁺, 902. 3217. Found, 902.3231.

4.3.16. 2-Debenzoyl-2-(3-difluoromethoxybenzoyl)-10-cyclopropancarbonyl-3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11b-06)

White solid; 90% yield; mp 133.9–134.5 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.95 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.36 (dd, J = 8.0, 1.7 Hz, 1H), 6.76 (t, J = 80.0 Hz, 1H), 6.29 (s, 1H), 6.23 (t, J = 8.4 Hz, 1H), 5.64 (d, J = 7.1 Hz, 1H), 4.99 – 4.83 (m, 3H), 4.57 (dd, J = 24.8, 8.5 Hz, 1H), 4.48 – 4.37 (m, 1H), 4.34–4.25 (m, 2H), 4.15 (d, J = 8.0 Hz, 1H), 3.81 (d, J = 8.0 Hz, 1H), 3.44 (d, J = 4.7 Hz, 1H), 2.62 – 2.49 (m, 2H), 2.41 – 2.22 (m, 5H), 1.93 – 1.82 (m, 4H), 1.81–1.73 (m, 1H), 1.68 (s, 6H), 1.26 (s, 9H), 1.25 (s, 3H), 1.19 – 1.08 (m, 5H), 1.05 – 0.95 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 205.07, 175.13, 173.62, 171.85, 166.55, 157.85, 157.20, 152.85, 142.32, 134.97, 133.21, 131.41, 127.95, 125.47, 121.21, 119.65, 117.59, 115.53, 85.87, 82.37, 80.76, 79.11, 77.45, 76.84, 76.70, 74.16, 72.79, 72.34, 59.33, 49.51, 48.49, 48.02, 44.56, 37.51, 36.96, 36.74, 31.67, 28.64, 26.97, 22.77, 22.31, 14.81, 13.73, 10.37, 9.13, 9.10; ¹⁹F NMR (470 MHz, CDCl₃) δ −80.06 (d, J = 166.9 Hz), −83.13 (d, J = 166.9 Hz ), −83.99 (d, J = 37.6 Hz), −85.75 (d, J = 37.6 Hz). HRMS (TOF) m/z: Calcd. For C₄₄H₅₃F₄NO₁₆H⁺, 928. 3373. Found, 928.3380.

4.3.17. 2-Debenzoyl-2-(3-difluoromethoxybenzoyl)-10-propionyl −3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11c-06)

White solid; 91% yield; mp 138.5–139.9 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.95 (d, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.36 (dd, J = 8.0, 1.6 Hz, 1H), 6.77 (t, J = 76.0 Hz, 1H), 6.30 (s, 1H), 6.23 (t, J = 8.4 Hz, 1H), 5.64 (d, J = 7.2 Hz, 1H), 5.00 – 4.93 (m, 2H), 4.88 (t, J = 8.0 Hz, 1H), 4.57 (dd, J = 24.5, 9.5 Hz, 1H), 4.48 – 4.40 (m, 1H), 4.34 – 4.25 (m, 2H), 4.16 (d, J = 8.4 Hz, 1H), 3.82 (d, J = 7.2 Hz, 1H), 3.47 (d, J = 5.2 Hz, 1H), 2.63 – 2.42 (m, 4H), 2.41 – 2.23 (m, 5H), 1.93 – 1.82 (m, 4H), 1.76 – 1.63 (m, 5H), 1.27 (s, 9H), 1.25 (s, 3H), 1.23 (t, J= 7.6 Hz, 3H), 1.14 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ203.62, 174.58, 172.47, 170.30, 165.96, 156.53, 154.86, 151.29, 142.05, 133.14, 130.93, 130.26, 127.09, 124.99, 119.71, 117.75, 115.67, 113.60, 84.42, 80.98, 80.39, 79.22, 76.29, 75.31,75.58, 73.04, 72.65, 72.10, 58.49, 47.93, 45.63, 43.16, 35.50, 35.38, 28.04, 27.54, 26.67, 22.16, 21.94, 14.80, 9.54, 8.99; ¹⁹F NMR (470 MHz, CDCl₃) δ −80.61 (dd, J = 164.5 Hz), −83.13 (dd, J = 164.5 Hz), −83.97 (d, J = 37.6 Hz), −85.73 (d, J = 37.6 Hz). HRMS (TOF) m/z: Calcd. For C₄₃H₅₃F₄NO₁₆H⁺, 916.3373. Found, 916.3424.

4.3.18. 2-Debenzoyl-2-(3-difluoromethoxybenzoyl)-10-(N,N’-dimethylcarbamoyl) −3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11d-06)

White solid; 93% yield; mp 141.2–142.3 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, J = 8.0 Hz, 1H), 7.90 (s, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.37 (dd, J = 8.0, 1.6 Hz, 1H), 6.76 (t, J = 76.0 Hz, 1H), 6.28–6.22 (m, 2H), 5.64 (d, J = 7.2 Hz, 1H), 4.99 (d, J= 8.0 Hz, 1H), 4.91–4.85 (m, 2H), 4.64–4.51 (m, 1H), 4.49 – 4.41 (m, 1H), 4.32 (d, J= 8.4 Hz, 1H), 4.28 (d, J= 4.8 Hz, 1H), 4.17 (d, J = 8.4 Hz, 1H), 3.82 (d, J = 6.8 Hz, 1H), 3.39 (d, J = 5.2 Hz, 1H), 3.22–3.17 (m, 1H), 3.04 (s, 3H), 2.96 (s, 3H), 2.59–2.49 (m, 1H), 2.39 (s, 3H), 2.38–2.31 (m, 1H), 2.30–2.21 (m, 1H), 1.93 – 1.83 (m, 4H), 1.67 (s, 3H), 1.63 (s, 1H), 1.58 (s, 6H), 1.27 (s, 9H), 1.25 (s, 3H), 1.15 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 206.72, 170.51, 166.07, 156.69, 154.99, 151.39, 139.22, 135.26, 131.16, 130.34, 127.22, 125.09, 119.96, 117.87, 115.80, 113.71, 84.37, 82.72, 81.31, 80.51, 79.06, 76.56, 75.57, 73.19, 72.81, 71.98, 57.91, 56.98, 48.01, 46.86, 43.29, 37.08, 35.39, 28.19, 26.48, 22.28, 21.04, 14.56, 9.83; ¹⁹F NMR (470 MHz, CDCl₃) δ −80.64 (d, J = 167.1 Hz), −83.04 (d, J = 167.1 Hz), −83.95 (d, J = 36.5 Hz), −85.73 (d, J = 36.5 Hz); HRMS (TOF) m/z: Calcd. For C₄₃H₅₄F₄N₂O₁₆H⁺, 931.3482. Found, 931.3482.

4.3.19. 2-Debenzoyl-2-(3-difluoromethoxybenzoyl)-10-methyl-3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11e-06)

White solid; 71% yield; mp 139.3–141.6 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.95 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.36 (dd, J = 8.0, 1.6 Hz, 1H), 6.75 (t, J = 76.0 Hz, 1H), 6.24 (t, J= 8.8 Hz, 1H), 5.65 (d, J = 7.2 Hz, 1H), 5.00–4.93 (m, 2H), 4.92–4.85 (m, 2H), 4.58 (dd, J= 24.8, 9.6 Hz, 1H), 4.33 – 4.20 (m, 3H), 4.17 (d, J= 8.4 Hz, 1H), 3.88 (d, J = 7.2 Hz, 1H), 3.54 (d, J= 8.0 Hz, 1H), 3.44 (s, 3H), 2.62–2.53 (m, 1H), 2.38 (s, 3H), 2.34–2.22 (m, 2H), 1.92 (s, 3H), 1.86–1.74 (m, 1H), 1.69 (s, 6H), 1.58 (s, 6H), 1.29 (s, 9H), 1.21 (s, 3H), 1.19 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 205.49, 172.48, 170.22, 166.00, 156.11, 154.85, 151.32, 142.51, 133.47, 131.01, 130.25, 127.11, 124.99, 119.83, 117.78, 115.71, 113.63, 84.66, 81.10, 79.37, 76.31, 76.11, 75.73, 73.05, 72.76, 72.38, 58.49, 45.58, 43.20, 36.65, 36.01, 35.46, 35.34, 28.07, 26.89, 22.16, 14.89, 9.36; ¹⁹F NMR (376 MHz, CDCl₃); ¹⁹F NMR (376 MHz, CDCl₃) δ −80.58 (d, J = 168.0 Hz), −83.03 (d, J = 168.0 Hz), −83.95 (d, J = 36.1 Hz), −85.73 (d, J = 36.1 Hz). HRMS (TOF) m/z: Calcd. For C₄₁H₅₁F₄NO₁₅H⁺, 874.3268. Found, 874.3267.

4.3.20. 2-Debenzoyl-2-(3-difluoromethoxybenzoyl)-3’-(2,2-difluoroethenyl)-3’-dephenylcabazitaxel (11f-06)

White solid; 91% yield; mp 137.6–138.1 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.95 (d, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.36 (dd, J = 8.0, 1.6 Hz, 1H), 6.73 (t, J = 76.0 Hz, 1H), 6.23 (t, J = 8.8 Hz, 1H), 5.61(d, J = 7.2 Hz, 1H), 5.00 (d, J = 8.4 Hz, 1H), 4.96–4.85 (m, 2H), 4.80 (s, 1H), 4.58 (dd, J = 23.6, 9.2 Hz, 1H), 4.33–4.26 (m, 2H), 4.16 (d, J = 8.0 Hz, 1H), 3.90 – 3.81 (m, 2H), 3.52 (d, J= 8.0 Hz, 1H), 3.46 (s, 3H), 3.31 (s, 3H), 2.77–2.67 (m, 1H), 2.38 (s, 3H), 2.29 (d, J = 9.2 Hz, 2H), 1.94 (s, 3H), 1.83 – 1.75 (m, 1H), 1.71 (s, 3H), 1.61 (s, 1H), 1.56 (s, 1H), 1.30 (s, 9H), 1.20 (s, 3H), 1.19 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 204.81, 170.56, 165.90, 131.07, 130.22, 127.14, 124.98, 84.11, 82.60, 81.65, 80.68, 78.90, 75.16, 73.09, 72.81, 57.40, 57.02, 56.86, 47.36, 43.28, 35.08, 32.04, 28.10, 26.75, 22.29, 10.33; ¹⁹F NMR (470 MHz, CDCl₃) δ −80.57 (d, J = 164.9 Hz), −82.77 (d, J = 164.9 Hz), −83.88 (d, J = 37.7 Hz), −85.64 (d, J = 37.7 Hz). HRMS (TOF) m/z: Calcd. For C₄₂H₅₃F₄NO₁₅H⁺, 888. 3424. Found, 888. 3433.

4.3.21. 2-Debenzoyl-2-(3-difluoromethoxybenzoyl)-10-methoxycarbonyl-3’-(2,2-difluoroethenyl)-3’-dephenyldocetaxel (11g-06)

White solid; 87% yield; mp 130.9–131.8 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.37 (dd, J = 8.0, 1.6 Hz, 1H), 6.76 (t, J = 76.0 Hz, 1H), 6.23 (t, J = 8.8 Hz, 1H), 6.12 (s, 1H), 5.65 (d, J = 6.8 Hz, 1H), 4.97 (d, J = 8.4 Hz, 1H), 4.92–4.83 (m, 2H), 4.58 (dd, J = 24.5, 8.4 Hz, 1H), 4.43–4.38 (m, 1H), 4.33 (d, J = 8.4 Hz, 1H), 4.28 (d, J = 4.0 Hz, 1H), 4.17 (d, J = 8.0 Hz, 1H), 3.87 (s, 3H), 3.80 (d, J = 6.8 Hz, 1H), 3.44 (d, J =5.2 Hz, 1H), 2.61 – 2.51 (m, 1H), 2.50–2.43 (m, 1H), 2.39 (s, 3H), 2.38–2.21 (m, 2H), 1.94 – 1.75 (m, 4H), 1.69 (s, 3H), 1.65 (s, 3H), 1.27 (s, 9H), 1.24 (s, 5H), 1.15 (s, 3H), 0.90–0.81 (m, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 203.88, 170.46, 166.04, 155.83, 154.99, 151.43, 143.13, 132.86, 131.05, 130.37, 127.21, 125.11, 119.85, 117.86, 115.79, 113.72, 84.48, 81.07, 80.55, 79.32, 78.30, 76.39, 75.63, 73.16, 72.70, 72.08, 58.63, 55.67, 48.01, 45.71, 43.18, 35.67, 35.45, 28.15, 26.70, 22.25, 21.93, 14.98, 14.17, 9.59, 1.09; ¹⁹F NMR (470 MHz, CDCl₃) δ −80.63 (d, J = 167.1 Hz), −83.09 (d, J = 167.1 Hz), −83.96 (d, J = 36.2 Hz), −85.73 (d, J = 36.2 Hz). HRMS (TOF) m/z: Calcd. For C₄₂H₅₁F₄NO₁₇H⁺, 918.3166. Found, 918.3110.

4.4. Cytotoxicity Assay

Human cancer cells A549 (lung), HT29 (colon), MCF7 (breast), Vcap (prostate), PC3 (prostate), PANC-1 (pancreatic), DLD-1 (colon) and LCC6-MDR (breast) were cultured as monolayers on 100 mm tissue culture dishes in the RPMI-1640 cell culture medium (ATCC) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) as well as 1% (v/v) penicillin at 37 °C in a humidified atmosphere with 5% CO₂. The cells were harvested, collected, and resuspended in 100 μL of medium at a concentration of ~0.8 ×10⁵ cells per well over a 96-well plate. The cells were allowed to descend to the bottom of the plates overnight, and fresh medium was added to each well upon removal of the old medium. Taxoids were dissolved in DMSO to 5 mM and were further diluted in appropriate medium prior to addition to cells. After 72 h of treatment, the medium was aspirated and the cells were washed in warm PBS. MTT reagent (Sigma) was diluted in RPMI-1640 medium without phenol red (ATCC) and added to the cells at 0.5 mg/mL. After 3 h of incubation, the reagent was aspirated and the as-produced insoluble violet formazan crystals were dissolved in 50 μL of 0.1 N HCl in isopropanol to give a violet solution. The plate was allowed to shake for 8 min to fully dissolve the violet formazan crystals and then the spectrophotometric absorbance measurement of each well in the 96-well plate was run at 568 nm. The IC₅₀ values and their standard errors were calculated from the viability-concentration curve using the Four Parameter Logistic Model of Sigmaplot. The concentration of DMSO per well was ≤ 1% in all cases. Each experiment was run in triplicate.

4.5. Molecular docking analysis

Docking analyses were performed using AutoDock Vina [78] with default parameters and scoring function. The number of scored docking results was set to 25 and an exhaustiveness parameter of 120 was set for each calculation. The protein model was generated using the available cocrystal structure of paclitaxel-bound β-tubulin (PDB:1JFF) [79] wherein the coordinates of the baccatin core was used to construct the 3^rd-generation DFV-taxoids, as well as other new-generation taxoids, used for comparisons, in the Avogadro [80] molecular editor. Taxoid structures were then minimized using the MMFF94 forcefield [81–85] for 5,000 steps of steepest descent followed by 5,000 steps of conjugate gradient minimization, or extended until a relative tolerance gradient of 10⁻³ dE was reached. Atomic partial charges were added using the Gasteiger method [86, 87], rotatable bonds were assigned by AutoDockTools [88] and finally the structures were used as input for docking. Resulting structures, showing excellent overlay with the paclitaxel structure bound to β-tubulin (PDB: 1JFF), were identified as the lowest energy binding pose in most cases and were further visualized in the UCSF Chimera [89] for relevant interactions, distances, angles, and illustrations.

Supplementary Material

Appendix A. Supplementary data

Supplementary data related to this article can be found at https://doi.org/...........................................

Abbreviations

MDR

multi-drug resistance

CSCs

cancer stem cells

SAR

structure-activity relationship

MOA

mechanism of action

ADME

absorption, distribution, metabolism and excretion

MMPA

matched molecular pair analysis

ADCs

antibody-drug conjugates

DPBS

Dulbecco’s phosphate-buffered saline

DFV

2,2-difluorovinyl

H-bonding

hydrogen-bonding

PMP

para-methoxyphenyl

TES

triethylsilyl

TIPS

triisopropylsilyl

PI3k/Akt

phosphatidylinositol 3-kinase/ Protein kinase B

EMT

epithelial-mesenchymal transition

PTEN

phosphatase and tensin homolog