Design and synthesis of a novel ZB716-d6 as a stable isotopically labeled internal standard

GRAPHICAL ABSTRACT

ZB716 is a synthetic, steroidal, orally active anti-estrogen agent that is under clinical development for the treatment of estrogen receptor (ER)-positive metastatic breast cancer. The stable isotope-labeled ZB716 was required for use as an internal standard in LC-MS/MS assays. Therefore, a novel deuterated ZB716 (ZB716-d6) as an isotopically labeled internal standard was designed and synthesized through a newly developed route, which prepared ZB716-d6 in eight steps from the commercially available deuterium-labeled starting material [²H₆]pentafluoropentanol. This procedure is very practicable and gives the final compound in good yield (19% total yield) and high purity (D, >99%, chemical purity 98%). At present, ZB716-d6 has been successfully used as an internal standard in clinical bioanalysis.

Introduction

ZB716, also known as fulvestrant-3-boronic acid, is a synthetic, steroidal, orally active anti-estrogen agent that is under development for the treatment of estrogen receptor (ER) positive, metastatic or advanced breast cancer.^[1] The drug is an analog of fulvestrant, a selective estrogen receptor degrader (SERD), in which the C3 hydroxy group has been partially replaced by a boronic acid moiety. It competitively binds to ERα (IC₅₀ = 4.1 nM) and effectively down-regulates ERα in both tamoxifen-sensitive and tamoxifen-resistant breast cancer cells.^[2,3] Moreover, it has excellent oral bioavailability (AUC = 2547.1 ng · h/mL) in vivo.^[2,4] As such, whereas fulvestrant is not orally active and must be administered via intramuscular injection,^[5–7] ZB716 could potentially be used as an oral agent and achieve greater systemic exposure and therapeutic benefit.^[2,3] Currently, it is in a phase 1/2 clinical trial for ER+/HER2- metastatic/advanced breast cancer.^[8]

The stable-isotope labeled internal standards (SIL-IS) yield better assay performance results for quantitative bioanalysis and can more accurately correct the matrix effects in mass spectrometry-based quantitation.^[9–11] For this reason, the stable isotope-labeled ZB716 was needed to support ongoing clinical studies. The SIL-IS of ZB716 is not commercially available and requires custom design and synthesis. When designing SIL-IS, to prevent the naturally occurring isotope of the analyte from interfering with the labeled internal standard, the mass difference between the analyte and the corresponding SIL-IS should be at least 3 Da. ^[12] The isotopic labels have to be incorporated at positions that are chemically stable and are not subject to biological attack. In addition, material availability, synthetic feasibility, and economic cost must also be considered. Introducing the isotopes with a commercially available isotope building block is the desired approach to constructing a SIL-IS. In this case, the side chain of ZB716 can be derived from pentafluoropentanol, which has a commercially available deuterated counterpart [²H₆]pentafluoropentanol, providing a feasible location for isotope labeling of ZB716. So, our efforts were directed at the synthesis of [²H₆]pentafluoropentanol derived deuterium-labeled ZB716 (ZB716-d6) (Fig. 1).

Figure 1.

Structures of ZB716-d6 and ZB716.

In addition, recently, the incorporation of deuterium into active drugs has gone beyond the only potentially beneficial improvement in the pharmacokinetic parameters. Deuterated drugs might provide broader opportunities to reduce toxicity, increase bioactivation, stabilize stereoisomers, reduce the dose of coadministered boosters, and elucidate the mechanism of action. ^[13–14] Moreover, the unique bulky side chain of SERDs such as fulvestrant and ICI 164384 obstructs the folding of the H 12 helix of estrogen receptors and determines their novel mode of action. ^[15] ZB716-d6 has a similar C-7 side chain with six deuterium-labeled modifications. Therefore, ZB716-d6 may have pharmacological differences from ZB716 with potential applications as a SERD molecule.

Results and discussion

Previously, we reported the synthesis of ZB716 through a four-step synthetic route from 17-O-acetyl S-deoxo fulvestrant.^[2] For introducing labeled isotope, starting from commercially available deuterium-labeled 4,4,5,5,5-pentafluoropentanol, we first obtained deuterium-labeled 17-O-acetyl S-deoxo fulvestrant (4) through three-step reactions ^[16] (Scheme 1). Next, according to the reference method ^[2], the deprotection of pinacol borate to free boric acid is carried out by oxidation of m-chloroperbenzoic acid (mCPBA), but this method is usually not easy to control (oxidative deboronization) and sometimes the resulting product is difficult to be purified. Considering these difficulties, we chose an effective approach to convert 6 into ZB716-d6 through potassium 17-acetyl S-deoxyfulvestrant 3-trifluoroborate-d6 (7).^[17–18] (Scheme 1) Finally, we developed a synthetic route for the synthesis of ZB716-d6. After further optimizing the reaction conditions and post-treatments of most steps, ZB716-d6 has been prepared with high efficiency and high quality through eight-step reactions from [²H₆]pentafluoropentanol.

Scheme 1.

Synthesis of ZB716-d6

As shown in Scheme 1, the mesylation of [²H₆]pentafluoropentanol by methanesulfonyl chloride in dichloromethane (DCM) afforded 1 quantitatively, which was condensed with thiourea under microwave irradiation at 80 °C to quickly give 2 without solvent or in a small volume of i-propanol. In the reaction of 2 and (7a,17b)-7-(9-bromononyl)-estra-1,3,5(10)-triene-3,17-diol 17-acetate (3) to form 4, using the freshly ground solid sodium hydroxide instead of NaOH aqueous solution^[19] greatly reduced the formation of deacetylation by-product of 4. Following the trifluoromethanesulfonylation of 4 to 5, the preparation of 6 from 5 through palladium-catalyzed Miyaura borylation reaction is a key reaction. The microwave technique was successfully applied to facilitate this reaction.^[20] Compared with the synthesis of 6 by traditional heating, microwave-assisted borylation of 5 at 100 °C not only shortened the reaction time from more than 10 h to 2 h but also reduced the formation of unknown byproducts (including a possible debromination product) and increased the yield of about 20%, leading to more facile workup after the reaction. The reaction of 6 with KHF₂ gave 7 as a salt, which was easily purified by recrystallization. 7 was efficiently oxidized by NaIO₄ to 8, which was directly used in the next step without further purification after a simple workup. Compared with the previous procedure,^[2] the improved process of 7 and 8 is very economical, especially suitable for large-scale manufacturing. 8 was smoothly deacetylated and hydrolyzed to ZB716-d6 at room temperature in a one-pot reaction by LiOH in acetonitrile-water with 90% isolated yield. Although the NaIO₄ synthesis route from 6 to ZB716-d6 has one more step than the mCPBA route, the 66% yield using NaIO₄ is significantly higher than the ~55% yield of the two-step synthesis using mCPBA.^[2] The total yield of ZB716-d6 is 19% from starting [²H₆]pentafluoropentanol through eight reactions.

Higher-energy collisional dissociation (HCD) MS-MS spectrum of ZB716-d6 at m/z 641.3721 shows a strong signal at m/z 623.3616. It corresponds to [M + H-H₂O]⁺, which supports the supposed structure of ZB716-d6 (Fig. S13). The same loss of H₂O is observed with a strong signal at m/z 617.3278 in the HCD MS-MS spectrum of the unlabeled compound, ZB716 (Fig. S14). That further supports the assignment of the structure of ZB716-d6. Finally, the internal standard ZB716-d6 was confirmed to have an isotope purity of >99% and a chemical purity of 98% by HPLC/MS and HRMS analysis (Fig. S11, S12, S13). The biological activity of the novel deuterium-labeled ZB716-d6 is being investigated.

Conclusions

A novel stable isotopically labeled internal standard of ZB716 (ZB716-d6) was synthesized with a feasible route in eight steps from the commercially available deuterium-labeled starting material [²H₆]pentafluoropentanol and (7a,17b)-7-(9-bromononyl)-estra-1,3,5(10)-triene-3,17-diol 17-acetate (3). ZB716-d6 was of acceptable purity and most importantly did not contain any unlabeled compound. It is currently being successfully used as an internal standard in the bioanalysis of ZB716 in clinical samples. The biological evaluation of the novel deuterium-labeled ZB716-d6 is underway in our laboratory.

Experimental section

General

[²H₆]-Pentafluoropentanol was purchased from Sigma-Aldrich (St. Louis, MO), (7a,17b)-7-(9-bromononyl)-estra-1,3,5(10)-triene-3,17-diol 17-acetate (3) was obtained from Sinopep-Allsino (Lianyungang, Jiangsu, China), and others from Fisher Scientific, CombiPhos Catalysts (Princeton, NJ) and Greenfield (Shelbyville, KY). Microwave irradiated reactions were performed on CEM Microwave Synthesizer Discover (Matthews, NC). ¹H-NMR and ¹³C-NMR spectra were recorded on a Bruker Fourier 300 and 400 MHz FT-NMR spectrometer (Billerica, MA). Chemical shifts are reported as parts per million (ppm) relative to TMS. HRMS spectra data were collected on a Thermo LTQ Orbitrap-XL mass spectrometer (Waltham, MA) in positive ion mode. The melting point was measured by Haake Buchler Melting Point Apparatus (UK). The purity of internal standard ZB716-d6 was confirmed by the Thermo HRMS Q-Exactive HPLC system (Waltham, MA).

Synthesis

(7R,8R,9S,13S,14S,17S)-13-Methyl-7-(9-((4,4,5,5,5-pentafluoropentyl-1,1,2,2,3,3-d6)thio)nonyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclo-penta[a]phenanthren-17-yl acetate (6):

To a solution of 5 (2.30 g, 2.94 mmol) in dioxane (3.0 mL), was added bis(pinacolato)-diboron (1.49 g, 5.88 mmol), Pd(dppf)Cl₂ (0.21 g, 10%), and potassium acetate (0.49 g, 4.99 mmol). The reaction mixture was irradiated at 100 °C for 2 h under microwave heating. The reaction mixture was filtered and washed the residue with ethyl acetate. The filtrate was concentrated and purified by flash column with hexane-ethyl acetate eluent to afford 6 (1.42 g, 65% yield). Pale yellow viscous oil.¹H-NMR (300 MHz, CDCl₃): δ 0.83 (s, 3H), 1.02 (m, 1H), 1.12–1.88 (m, 36H), 2.05 (s, 3H), 2.23 (m, 1H), 2.38 (m, 2H), 2.49 (t, J=7.2 Hz, 2H), 2.83 (d, J=16.2 Hz, 1H), 2.91 (dd, J=17.1, 4.5 Hz, 1H), 4.70 (t, J=8.1 Hz, 1H), 7.30 (d, J=7.2 Hz, 1H), 7.54 (s, 1H), 7.58 (d, J=7.8 Hz, 1H). ¹³C-NMR (75 MHz, CDCl₃): 171.17, 143.00, 136.70, 134.79, 132.01, 125.39, 119.17 (qt, J_CF¹ = 283.4 Hz and J_CF² = 36.1 Hz), 115.75 (tq, J_CF¹ = 250.0 Hz and J_CF² = 37.3 Hz), 83.62, 82.78, 46.39, 42.96, 41.45, 38.80, 37.15, 34.30, 33.22, 31.83, 29.94, 29.67, 29.59, 29.51, 29.20, 28.85, 28.24, 27.53, 26.83, 25.63, 24.88, 24.79, 22.78, 21.15, 19.34 (m), 12.04. HRMS (+ESI): calcd for C₄₀H₅₅D₆BF₅O₄S [M+H]⁺ 749.4680, found 749.4687

((7 R,8R,9S,13S,14S,17S)-17-Hydroxy-13-methyl-7-(9-((4,4,5,5,5-pentafluoropentyl-1,1,2,2,3, 3-d6)sulfinyl)nonyl)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl)b oronic acid (ZB716-d6):

To a solution of 8 (0.37 g, 0.49 mmol) in acetonitrile (5.0 mL), was added a solution of LiOH (0.24 g, 10 mmol) in H₂O (5 mL). The reaction mixture was stirred at rt for 48 h under nitrogen. The reaction mixture was diluted with ethyl acetate and NH₄Cl aqueous solution, neutralized with concentrated HCl (0.8 mL) to pH < 7, separated, and extracted with ethyl acetate. The combined organic layer was washed with brine, dried over MgSO₄, filtered, concentrated, and purified by flash chromatography with DCM-MeOH as eluent to afford ZB716-d6 (0.28 g, 90% yield). Colorless crystal, Mp. 288–290 °C (dec.). ¹H-NMR (400 MHz, DMSO-d₆): δ 0.66 (s, 3H), 0.87 (m, 1H), 1.14–1.63 (m, 22 H), 1.71 (d, J=9.6 Hz, 1H), 1.81 (d, J=12.0 Hz, 1H), 1.89 (m, 1H), 2.30 (m, 2H), 2.61–2.72 (m, 3H), 2.82 (dd, J=16.0 and 4.1 Hz, 1H), 3.54 (m, 1H), 4.52 (bs, 1H), 7.22 (d, J=8.0 Hz, 1H), 7.44 (s, 1H), 7.50 (d, J=7.6 Hz, 1H), 7.95 (bs, 2H). ¹³C-NMR (100 MHz, DMSO-d₆): 141.41, 136.05, 133.80, 131.53, 124.95, 118.97 (qt, J_CF¹ = 283.9 Hz and J_CF² = 36.5 Hz) , 115.96 (tq, J_CF¹ = 249.7 Hz and J_CF² = 36.9 Hz) , 80.23, 51.00, 46.21, 43.05, 41.67 (m), 41.58, 40.43–38.66 (1C), 36.93, 34.16, 32.83, 29.93, 29.43, 29.11, 28.89, 28.73, 28.20, 27.65, 26.85, 25.17, 22.38, 22.14, 13.18 (m), 11.38. HPLC purity: 98%, isotopic purity: >99% by HPLC/MS analysis (Fig. S11 and S12). HRMS (+ESI): calcd for C₃₂H₄₃D₆BF₅O₄S [M + H]⁺ 641.3741, found 641.3721 (Fig. S13).