Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Communication to the Editor
The Design, Synthesis and Preliminary Pharmacokinetic Evaluation of d3-Poziotinib Hydrochloride
Shuchao MaLinan WangBen OuyangXinfa BaiQinglin JiLei Yao
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2019 年 42 巻 6 号 p. 873-876

詳細
Abstract

To establish a synthetic route to d3-poziotinib hydrochloride. Treatment of 4-chloro-7-hydroxyquinazolin-6-yl pivalate (1) with d3-methyliodide afforded the etherization product, which reacted with 3,4-dichloro-2-fluoroaniline to generate the key intermediate d3-4-(3,4-dichloro-2-fluorophenylamino)-7-methoxyquinazolin-6-yl pivalate (3). Followed the de-protection reaction, the nucleophilic substitution (SN2) reaction with tert-butyl 4-(tosyloxy)piperidine-1-carboxylate (TSP), and the de-protection reaction of t-butoxycarbonyl (Boc) group, and the amide formation reaction with acrylyl chloride, d3-poziotinib was obtained, which was converted to hydrochloride salt by treatment with concentrated hydrochloric acid (HCl). Starting from a known compound 4-chloro-7-hydroxyquinazolin-6-yl pivalate (1), after 7 steps transformation, d3-poziotinib hydrochloride was obtained with a total yield of 9.02%. The structure of d3-poziotinib hydrochloride was confirmed by 1H-NMR, 13C-NMR, and high resolution (HR)-MS. Meanwhile, the in vitro microsomal stability experiment showed that d3-poziotinib had a longer half time (t1/2 = 4.6 h) than poziotinib (t1/2 = 3.5 h).

INTRODUCTION

Poziotinib (Fig. 1, HM781-36B) is a pan-human epidermal growth factor receptor (HER) inhibitor. It was orally administrated alone or in combination with cytotoxic chemotherapeutic agents in treatment for gastric cancer,1) non-small cell lung cancer (NSCLC)2) and breast cancer.3) In the preclinical studies, it was found that poziotinib in vivo could be converted to an active metabolite M2 (Fig. 1) through O-demethylation metabolic pathway,4) and the M2 was considered to be the main cause of the drug’s major side effects such as severe diarrhea, vomiting, etc.

Fig. 1. The Structures of Poziotinib and Active Metabolite M2

Metabolic closure is a common strategy to improve the stability of drug metabolism. By blocking the drugs’ metabolic sites, the clearance rate of drugs could be delayed, and the production of active metabolites was decreased, so the effect time of drugs could be prolonged.

Deuterate is a stable non-radiation isotope of hydrogen. Due to the similarities of hydrogen and deuterate in space and shape, deuterated drugs should always keep the original biological activities and selectivity. Meanwhile, due to the bond strength of C–D bond is stronger than C–H bond, deuterated drugs are always bearing following advantages: 1. decrease the clearance, prolong the half time, and decrease side-effects; 2. decrease the metabolism of drugs in guts and liver, increase the therapeutic effects; 3. decrease the production of harmful metabolites, enhance the tolerance of drug.5,6)

The first U.S. Food and Drug Administration (FDA) approved deuterated drug was Austedo, a deuterated tetranaqine, which was used for treatment of Huntington’s disease-related dance symptoms.7) CTP-656, deuterated Ivacaftor, was an orphan drug developed by Concert for the treatment of cystic fibrosis.8) In order to decrease the production of undesirable metabolites, and enhance the patients’ tolerance of drugs, deuterated poziotinib was designed and synthesized in this article.

MATERIALS AND METHODS

Routine monitoring of reaction was performed by TLC using pre-coated GF254 TLC plate. 1H-NMR was recorded on a Bruker AVANCE 400 spectrometer at 400 MHz with tetramethylsilane used as an internal reference. MS were performed with electron spray ionization (ESI) mode. High-resolution (HR)-MS were recorded on an Agilent 6210 ESI/time-of-flight (TOF) mass spectrometer. Melting points (mp) were recorded on a Büchi B-540 melting point apparatus and are uncorrected. Flash column chromatographic separation was achieved using a silica gel from Qingdao Ocean Chemical (200 to 300 mesh) with a particle size from 54 to 74 µm using ethyl acetate and hexane (or petroleum ether) as the eluent.

Materials

All reagents were purchased from commercial sources and were used as received. 3,4-dichloro-2-fluoroaniline (Maya, China), d3-methyliodide (CD3I) (Aldrich, U.S.A.), N-tert-butoxycarbonyl (Boc)-4-hydroxypiperidine (J&K, China), 7 N ammonia/MeOH (Aldrich), acrylyl chloride (Energy Chemical, China), reduced nicotinamide adenine dinucleotide phosphate (NADPH)+ (Acros, U.S.A.), tert-butyl 4-(tosyloxy)piperidine-1-carboxylate (TSP, 5): prepared by literature method.9) white solid, mp 97.9–98.7°C; 4-chloro-7-hydroxyquinazolin-6-yl pivalate (1): prepared by literature methods.10) white wax.

Chemistry

Staring from a known compound 4-chloro-7-hydroxyquinazolin-6-yl pivalate (1, Chart 1), the hydroxyl group was converted to corresponding methyl ether 2 by treatment with d3-methyl iodide under basic condition. Compound 4 was obtained after 2 steps transformation: 1: replacement of chloride group in compound 2 by 2-fluoro-3,4-dichloroaniline; 2: de-protection of Piv group by ammonia/methanol. Under basic condition, treatment of TSP (5) with compound 4, the key intermediate 5 was afforded with moderate yield. Followed the removal of Boc group by concentrated hydrochloric acid (HCl), amide formation reaction by treatment with acrylyl chloride, and salt formation with concentrated HCl, d3-poziotinib hydrochloride (7) was obtain as yellowish powder.

Chart 1. The Synthetic Route to d3-Poziotinib Hydrochloride

In Vitro Microsomal Stability Test

To a solution of rat liver microsomes solution 2.0 mL (1.0 mg/mL), was added the solution of test compounds 2.0 µL (5 µM). After mixing, the solution was divided into 3 parts, and incubated for 3 min at 37°C. When a solution of 10 µL of 400 mM NADPH regeneration solution was added to above mixture, aliquots sample were taken and terminated with 100 µL of cold methanol at 0, 5, 15, 30, 60, 120 min. Following centrifugation, the supernatant is analyzed on HPLC. (conditions: Column: Capcell pak C18 UG120-4.6 × 150 mm 5 micron; mobile phase A: 12.5 mM KH2PO4 + 50 mM NaClO4·H2O pH 2.5; mobile phase B: 100% acetonitrile; A : B = 41 : 59 (v/v)).

In the determination of the in vitro T1/2, the analytic peak areas were converted to percentage drug remaining, using the T = 0 peak area values as 100%. The slope of the linear regression from log percentage remaining versus incubation time relationships (−k) was used in the conversion to in vitro T1/2, values by in vitro T1/2 = −0.693/k. The percent remaining of test compound is calculated compared to the initial quantity at 0 time.

RESULTS

d3-4-Chloro-7-methoxyquinazolin-6-yl Pivalate (2)

To a solution of compound 1 (4.5 g, 16.1 mmol), potassium carbonate (K2CO3) (6.7 g, 48.3 mmol) in N,N-dimethylformamide (DMF) (50.0 mL), was added CD3I (1.5 mL, 24.1 mmol) slowly at 0°C. After addition, the reaction mixture was allowed to stir at room temperature for 16 h till TLC showed that all starting material consumed. The reaction mixture was partitioned between water and ethyl acetate, the organic layers were combined, dried, filtered, and concentrated. The residue was purified by chromatography to afford the tile compound as white powder (60.67%). 1H-NMR (CDCl3, 400 MHz) δppm: 8.95 (s, 1H, ArH), 7.86 (s, 1H, ArH), 7.40 (s, 1H, ArH), 1.42 (s, 9H, (CH3)3CO).

d3-4-(3,4-Dichloro-2-fluorophenylamino)-7-methoxyquinazolin-6-yl Pivalate (3)

A solution of compound 2 (2.9 g, 9.76 mmol), 3,4-dichloro-2-fluoroaniline (2.1 g, 11.7 mmol) in acetonitrile (20 mL) was stirred at 90 ± 5°C for 8 h till TLC showed that all starting material consumed. The reaction mixture was cooled to room temperature, and the white precipitate was isolated and washed with acetonitrile (2 × 20 mL) and dried to afford the title compound as white solid (yield: 67.44%). The filtrate was concentrated, and the residue was purified by a chromatography to afford the second crop of product. 1H-NMR (CDCl3, 400 MHz) δppm: 11.16 (br s, 1H, ArNH), 8.78 (s, 1H, ArH), 8.43 (s, 1H, ArH), 7.67 (s, 1H, ArH), 7.50–7.54 (t, 1H, J = 8.2 Hz, ArH), 7.20–7.23 (dd, 1H, J = 8.7, 1.5 Hz, ArH), 1.52 (s, 9H, (CH3)3CO).

d3-tert-Butyl-4-(4-(3,4-dichloro-2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)piperidine-1-carboxylate (6)

To a solution of compound 3 (2.9 g, 6.6 mmol) in methanol, was added ammonia solution 30 mL (7 N in methanol). After addition, the reaction mixture was allowed to stir at room temperature for 10 h. The yellow precipitate was collected and dried to afford the de-protection product quantitatively, which used in next step directly.

To a solution of TSP (5) (1.21 g, 3.4 mmol), and K2CO3 (0.7 g, 5.1 mmol) in DMF (4 mL), was added above yellow compound (0.6 g, 1.7 mmol). The reaction mixture was allowed to stir for 24 h at 67–70°C till TLC showed that all starting material consumed. After cooling to room temperature, water (4.8 mL) was added to the reaction mixture slowly. The resulting mixture was stirred for 3 h at room temperature, and the solid was collected by filtration and washed with water (2 × 10 mL). After drying, compound 5 was obtained as off-white to pale yellow solid (43.57%). 1H-NMR (DMSO-d6, 400 MHz) δppm: 9.6 (br s, 1H, ArNH), 8.39 (s, 1H, ArH), 7.86 (s, 1H, ArH), 7.58 (m, 2H, ArH), 7.23 (s, 1H, ArH), 4.68–4.73 (m, 1H, OCH), 3.67–3.71 (m, 2H, NCH2), 3.25 (m, 2H, NCH2), 1.98–2.01 (m, 2H, CH2), 1.57–1.67 (m, 2H, CH2), 1.42 (s, 9H, OC(CH3)3).

d3-Poziotinib Hydrochloride (7)

To a solution of compound 6 (8.0 g, 14.9 mmol) in acetone (80 mL), was added conc. HCl (14.4 mL) slowly. After addition, the reaction mixture was allowed to stir at room temperature for 5 h, till TLC showed that all starting material consumed. The precipitate was isolated, washed with acetone (2 × 10 mL), and dried to afford the de-protection intermediate (7.4 g, 97.6%) as yellow solid, which used in following step directly.

To a solution of above solid (8.7 g, 17.0 mmol) in tetrahydrofuran (THF) (61 mL), was added a solution of sodium bicarbonate (NaHCO3) (5.71 g, 68 mmol) in water (87 mL) under violent stirring. The resultant solution was cooled to 0–3°C, and a solution of acryly chloride (1.6 mL, 19.9 mmol) in THF (61 mL) was added through an addition funnel within 30 min. After addition, the reaction mixture was allowed to stir for 30 min at such temperature till TLC showed the completion of the reaction. Water (140 mL) was added to the reaction mixture, the aqueous solution was extracted with ethyl acetate (3 × 100 mL). The organic layers were combined, dried, filtered, and concentrated. The residue was purified by a chromatography to afford the d3-poziotinib free base as pale yellow foam (5.93 g, 70.73%).

A hot solution of d3-poziotinib free base (5.8 g, 11.7 mmol) in methanol (50 mL) was acidified to pH = 2–3 by conc. HCl. The reaction mixture was allowed to stir at room temperature for 24 h. The precipitate was isolated, washed with acetone (3 × 10 mL), and dried to afford d3-poziotinib hydrochloride as pale yellow powder (4.6 g, 74.2%). mp 230–232°C (decomposed). 1H-NMR (DMSO-d6, 400 MHz) δppm: 12.0 (br s, 1H, ArNH), 8.83 (s, 1H, ArH), 8.57 (m, 1H, ArH), 7.59–7.67 (m, 2H, ArH), 7.36 (s, 1H, ArH), 6.81–6.87 (m, 1H, ArH), 6.81–6.13 (dd, J = 16.68, 2.32 Hz, 1H, ArH), 5.67–5.70 (dd, J = 10.44, 2.32 Hz, 1H, ArH), 5.02 (m, 1H, OCH), 3.87–3.99 (m, 2H, NCH2), 3.55–3.67 (m, 2H, NCH2), 2.08–2.11 (m, 2H, CH2), 1.57–1.67 (m, 2H, CH2). 13C-NMR (DMSO-d6, 100 MHz) 164.3, 158.9, 157.3, 154.8, 152.3, 149.0, 147.8, 135.8, 131.2, 128.4, 127.8, 127.2, 125.7(d), 125.2(d), 120.2(d), 107.0, 106.1, 100.2, 73.7, 42.5, 40.1, 30.9, 30.0. HR-MS Calcd for C23H19D3Cl2FN4O3 [M + H]+ 494.1236. Found 494.1263.

Microsomal Stability Test

The results of percent remaining from the in vitro study were presented in Fig. 2. The percent remaining value of poziotinib after 60 and 120 min incubation with rat microsomes were 84 ± 1.2 and 72 ± 1.3%, respectively. On the other hand, the percent remaining value of d3-poziotinib were 89 ± 1.7 and 79 ± 1.6%, respectively. And then, the half-life (t1/2) values of poziotinib and d3-poziotinib were 3.5 h and 4.6 h in the rat respectively. Compared with poziotinib, d3-poziotinib was more metabolic stable, and had a longer half time.

Fig. 2. The Results of in Vitro Microsomal Stability Test

DISCUSSION

The only difference between poziotinib and d3-poziotinib is the methyl group on the quinazoline ring. So on the NMR spectrum, poziotinib and d3-poziotinib should share same signals except the methyl group. Figure 3 compares the 1H-NMR and 13C-NMR of poziotinib hydrochloride (a) with d3-poziotinib (b). In the 1H-NMR spectra, the poziotinib has a single methyl-peak signal near 4 ppm, and the d3-poziotinib has no signal at this location. Meanwhile, in the 13C-NMR spectra, poziotinib has a methyl signal peak at 56.59 ppm, however, d3-poziotinib has no signal in this position.

Fig. 3. The NMR Spectra Comparison of Poziotinib (a) and d3-Poziotinib (b)

CONCLUSION

Starting from a known compound 4-chloro-7-hydroxyquinazolin-6-yl pivalate (1), after 7 steps transformation, d3-poziotinib hydrochloride was obtained with a total yield of 9.02% (conditions not optimized). The reaction conditions of this synthetic route were mild. Meanwhile, the in vitro microsomal stability assay showed that the half time of d3-poziotinib was a little longer than Poziotinib, which means that d3-poziotinib was more metabolic stable than Poziotinib. The further pharmacological and toxicological investigation of d3-poziotinib was still undergoing, and the results will report soon.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES
 
© 2019 The Pharmaceutical Society of Japan
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