Improved Synthesis of the Thiophenol Precursor N-(4-Chloro-3-mercaptophenyl)picolinamide for Making the mGluR4 PET Ligand

Abstract

Recently [¹¹C]mG4P012 (previously [¹¹C]KALB012 and presently named as [¹¹C]PXT012253 by Prexton Therapeutics) had been used as a biomarker during the preclinical development of a potential therapeutic drug, PXT0002331 (an mGluR4 PAM), for PD and L-dopa-induced dyskinesia. [¹¹C]mG4P012 was shown to be a promising PET radioligand for mGluR4 in the monkey brain and for further development in human subjects. However, the previously reported multi-step synthesis of the thiophenol precursor suffered from low yields and difficult workup procedures. To support the translational research of [¹¹C]mG4P012 and the other potential applications, we have developed a new route for synthesis of the thiophenol precursor and optimized the reaction conditions. The synthesis of N-(4-chloro-3-mercaptophenyl)picolinamide from 1-chloro-4-nitrobenzene has been greatly improved from 8% to 52% total yield with easy handling and in gram scales.

Graphical Abstract

1. Introduction

Metabotropic glutamate receptors (mGluRs) are involved in glutamate signaling in many excitatory synapses in CNS, which consist of three subgroups including eight known receptor subtypes (mGluR1–8).¹ mGluR4 is presynaptically expressed at multiple synapses throughout the basal ganglia and has been received much attention and research efforts due to the potential benefits of mGluR4 activation in treating numerous neuronal diseases.^1–8 Most efforts have focused on development of the mGluR4 positive allosteric modulators (PAMs) as new therapeutics for CNS disorders such as PD and L-dopa-induced dyskinesia.^9–11 An efficient PET radioligand for mGluR4 could be a useful tool for understanding the role of mGluR4 in healthy and disease conditions, and also for the development of new drugs targeting this receptor. The receptor occupancy is measured using a proper PET ligand to answer many vital questions such as the drug targeting and the therapeutic dose.^12,13 Therefore, extensive research efforts have been directed towards the development of PET ligands suitable for in vivo probing mGluR4, in which we and others had reported several mGluR4 PET radiotracers (Figure 1).^14–17 All these PET radiotracers have been designed or/and developed based on the mGluR4 PAMs. Some of these mGluR4 PAMs such as ML-128¹⁸ and ADX88178^19,20 had demonstrated the efficacy in a preclinical rodent model of motor impairments associated with PD.

Figure 1.

The PET tracers for mGluR4.

Among these PET tracers, the in vitro data revealed that mG4P012 has good mGluR4 binding affinity and selectivity over other mGluRs including mGluR1, mGluR5, mGluR6 and mGluR8, as well as an enhanced metabolic stability.^16,21 The PET studies in rats displayed that [¹¹C]mG4P012 (previously [¹¹C]KALB012) accumulated fast into the brain and had higher uptake, slower washout and 25% better contrast than [¹¹C]ML-128, indicating improved imaging characteristics as a PET radiotracer for mGluR4.¹⁶ The improved pharmacological properties and the enhanced imaging characteristics make [¹¹C]mG4P012 as a useful PET radiotracer for mGluR4 in biological research and drug development.

Recently [¹¹C]mG4P012 (renamed as [¹¹C]PXT012253 by Prexton Therapeutics) has been used as a biomarker during the preclinical development of a potential therapeutic drug, PXT0002331 (an mGluR4 PAM), for PD and L-dopa-induced dyskinesia.^22–24 [¹¹C]mG4P012 was reported to display binding in mGluR4-expressing regions in the brain of cynomolgus monkeys.²³ Competition of [¹¹C]mG4P012 with PXT002331 showed high specific binding in the total distribution volume, which was useful for the target occupancy or longitudinal studies. [¹¹C]mG4P012 was shown to be a promising PET radioligand for mGluR4 in the monkey brain and for further development in human subjects.²³

As Scheme 1 shows, [¹¹C]mG4P012 was radiolabeled by methylation of the thiophenol precursor 1 by using [¹¹C]CH₃I and K₂CO₃ in acetone at 50 °C for 3 min or by using [¹¹C]CH₃OTf and K₂CO₃ in acetone at rt.^16,23 On the other hand, mG4P012 is a mGluR4 PAM that was synthesized by methylation of 1 by using CH₃I and K₂CO₃/Cs₂CO₃ in DMF at rt for 1 h.¹⁶

Scheme 1.

Synthesis of [¹¹C]mG4P012 and mG4P012 from the thiophenol precursor 1.

However, the previously reported multi-step synthesis of the thiophenol precursor 1 suffered from low yields and difficult workup procedures.¹⁶ To support the translational research of [¹¹C]mG4P012 and the other potential applications, we have developed a new route for synthesis of the thiophenol precursor 1 as well as synthesis of mG4P012.

2. Results and discussion

To scale up the synthesis of 1, we re-examined the original synthetic route (Scheme 2) and tried to optimize the reaction conditions. The previous synthesis of 1 was carried out in 5 steps from the starting compound 2 in total yield only 8%.¹⁶

Scheme 2.

The original synthesis of the precursor 1.

In the synthesis of 3, the chlorosulfonation of 2 took place slowly because the benzene ring was significantly deactivated by the strong electron-withdrawing effect of 4-nitro substitution. Therefore, a harsh condition was applied to push the reaction to complete, in which the reaction was carried out at high temperature (120 °C) for 2 days. After reaction, the mixture was extracted with EtOAc and DCM, dried over anhydrous Na₂SO₄, filtered, and evaporated. The residue was purified by flash chromatography to give 3 in 52% yield. Since the reaction mixture was dirty and the workup procedure was difficult, we checked the different reaction conditions and found when the reaction was carried out at a lower temperature (100 °C) under a nitrogen atmosphere for 7 days, the chlorosulfonation of 2 led to a higher yield (72%) of 3 and less side products at a 20-gram scale. Under this procedure the product 3 was obtained by precipitating out in cold water, leading to product 3 ready for use in the next step without the column purification.

The next reaction was to reduce both the nitro group and the sulfonyl chloride group of 3 by tin(II) chloride to get 4. It was found that the reaction mixture was a thick suspension that made isolation of the product 4 by extraction or filtration very difficult. The tedious extraction workup procedure required copious amounts of organic solvents, which not only led to a lower yield but also made it difficult to scale up. To solve this problem, we checked an alternative approach, in which compound 3 was reduced to 4 in two steps of reactions (Scheme 3).

Scheme 3.

The two steps reduction of 3 to 4.

First step, the reduction of sulfonyl chloride group of 3 was accomplished by refluxing 3 with the reducing reagent PPh₃ in toluene, affording the thiophenol 7 in up to 92% yield without observing disulfide formation. This reaction was completed within 5 min at a 2-gram scale. The by-product triphenyl phosphine oxide was more polar than product 7, making the purification simple. The reaction mixture was filtered through a short pad of silica gel followed by elution with hexanes and EtOAc to give compound 7 as a light-yellow crystal, which was very stable in air and heat. It was suggested that the nitro group greatly reduces the electron density of the benzene ring, which leads to the decreased reactivity of the thiophenol 7 to prevent the formation of the disulfide.

Second step, the Pd/C catalyzed hydrogenation reaction was carried out to reduce the nitro group of 7. Interestingly, under the neutral condition, the disulfide 5 was formed as the dominant product with only trace amount of 4 detected (Scheme 4). It is indicated that the electron-rich thiophenol 4 is easily oxidized to the diphenyl disulfide 5. It was also found that if the reaction was carried out under the acidic conditions, the thiophenol 4 can be obtained as a major product (using TFA, 52%) or a predominant product (using HCl, 99%). Compared to the previous one-step reduction of 3 by using tin(II) chloride, this two-step procedure was much easier to run, which provided the thiophenol 4 or/and the diphenyl disulfide 5 in high yield by using different conditions. It was noticed that as a more advanced intermediate in the original synthesis route, the diphenyl disulfide 5 could be obtained under the neutral condition.

Scheme 4.

The hydrogenation of 7.

However, it was found in the following synthesis (Scheme 2) that the solubility of disulfides 5 and 6 was very poor in both polar and non-polar organic solvents such as MeOH and DCM. The acylation of 5 gave only <50% yield of 6. The poor solubility of the disulfides limited the reaction scale and might also be the reason for the low yield of the amide formation.

Reduction of the disulfide intermediate 6 by sodium borohydride gave the thiophenol 1 quantitatively, which was monitored by LC-MS under acidic condition (0.1% TFA/CH₃CN/H₂O). Since sodium borohydride would decompose in neutral or acidic aq solution, the reaction was carried out in the basic condition. Therefore, the workup process was very crucial. It was observed that the thiophenol 1 was prone to the disulfide formation under neutral or slight basic conditions. The extraction by water and DCM without acidification resulted in exclusively the disulfide 6 in 2 min. The resulting mixture should be acidified quickly by adding 10% HCl to avoid disulfide bond formation. However, the good solubility of the resultant hydrogen chloride salt of 6 in water made the extraction with organic solvents not efficient. It was time-consuming to remove the salts involved in this step by semi-preparative HPLC.

Although a couple of reactions in the original synthesis route were optimized including the chlorosulfonation of 2 to 3, the reduction of 3 to 4 (or 5) and the reduction of 6 to 1, this approach still suffered from the low solubility of the disulfides 5 and 6 and the tedious workup procedure for the reduction of the disulfide intermediate 6 by sodium borohydride. These issues greatly limited the scale and total yield for the synthesis.

In the original synthesis route, the disulfide 5 was introduced for protection of the thiol group of 4, since both its thiol and amino groups could react with picolinic acid. In order to avoid the problems caused by the diphenyl disulfides and to improve the synthesis, a new synthetic route was designed and studied for synthesis of the thiophenol precursor 1 (Scheme 5).

Scheme 5.

The synthesis by using PMB as the thiol protection group.

The protection of the thiol group of 7 with p-methoxybenzyl chloride (PMB-Cl) was first carried out with K₂CO₃ or NaOH, in which the reactions were sluggish and starting material 7 was remained. It was discovered later that compound 8 could be easily synthesized in 97% yield by using K₂CO₃ and 1 eq of KI. The reduction of the nitro group of 8 was then carried out by sodium borohydride and NiCl₂.6H₂O to give the aniline 9 in 92% yield within 5 min. The acylation of 9 with picolinic acid and N,N’-diisopropylcarbodiimide (DIC) gave the compound 10 in 90% yield.

The PMB protecting group of 10 could be removed by using TFA to give the final product 1 in almost quantitative yield in 2 h, which was easily precipitated out by adding MeOH into the reaction mixture solution. However, we noticed that the TFA salt of 1 was not very stable at rt and approx. 40% of the disulfide formed after 3 weeks. Therefore, the HCl salt of 1 was prepared for long time storage.

As shown in Scheme 6, the major modifications in the new synthesis route were: 1) the selective reduction of sulfonyl chloride group of 3 to give the thiophenol 7, in which 7 did not favor the formation of the disulfide because of its strong electron-withdrawing nitro group; 2) the thiol group of 7 was protected by PMB, followed by sodium borohydride enabled selective reduction of nitro group to form 9 and acylation to give 10; 3) the removal of PMB group under acidic condition to give the thiophenol precursor 1. Starting from compound 2, the thiophenol precursor 1 was synthesized in 52% total yield via 6-steps of reactions in this new route. At the same time, the involved workup and purification procedures were much simpler and more efficient than those used in the previous synthesis route.

Scheme 6.

The new synthesis of the precursor 1.

On the other hand, mG4P012 is a potent mGluR4 PAM, which has been used to study the pharmacology of mGluR4 and as a blocking agent to determine specificity of the developmental mGluR4 PET tracers.²¹ In order to support new studies based on mG4P012, we have also scaled up its synthesis. Although mG4P012 was previously prepared by methylation of 1 (Scheme 1), it could be efficiently synthesized via a shorter route as shown in Scheme 7.

Scheme 7.

The synthesis of mG4P012.

The methylation of the thiophenol 7 by using CH₃I, K₂CO₃ and KI was done smoothly. However, it required a large amount of CH₃I (5.0 eq) and a long reaction time depending on the reaction scale (2–7 h). Compound 11 could also be prepared in high yield (97%) with 1.5 eq of CH₃I and NaH as the base in 1 h. Hydrogenation of 11 gave almost quantitative yield of 12, however, it took 5–7 h to completion depending on the reaction scale. In an alternative approach, reduction of 11 by using NaBH₄ and NiCl₂.6H₂O was accomplished by reflux for 10 min with gram scales. Compound 12 coupled with picolinic acid to give the final product, mG4P012, in up to 92% yield. The synthesis of mG4P012 was highly improved with shorter reaction time, good yield, easy workup procedures, and possibility to scale up all involved reactions.

3. Conclusion

The original synthetic route of the thiophenol precursor 1 was carried out from 2 via 5 steps of the reactions to give the precursor 1 in only 8% total yield and with some tedious workup and purification procedures. The new route was accomplished from 2 in 6 steps of the reactions including the selective reduction of the sulfonyl chloride group of 3, PMB protection of the thiophenol of 7, the reduction of the nitro group of 8, the amide coupling and removing PMB protection to give 1 in 52% total yield and more efficient workup procedures. The gram-scale synthesis of the thiophenol precursor 1 can now be efficiently achieved to warrant the translational research of [¹¹C]mG4P012 as well as other mGluR4 PET tracers. On the other hand, the new and efficient synthesis of mG4P012, a potent mGluR4 PAM, was also developed.

4. Experimental Section

General Methods.

All reagents and starting materials were obtained from the commercial sources including Sigma-Aldrich (St. Louis, MO), Thermo Fisher Scientific, Oakwood Products, Inc and used as received. The reactions were monitored by TLC using a UV lamp monitored at 254 nm. If necessary, the reactions were also checked by LC−MS. The LC-MS was performed using the Agilent 1200 series HPLC system coupled with a multiwavelength UV detector and a model 6310 ion trap mass spectrometer (Santa Clara, CA) equipped with a Luna C18 column (Phenomenex, 100 × 2 mm, 5 μm, 100 Å). The RP-HPLC was carried out by using a 7-min gradient method (LC-Method 1): eluent A: 0.1% formic acid/ H₂O; eluent B: 0.1% formic acid/CH₃CN; gradient: 5% B to 95% B from 0 to 3 min, 95% B from 3 to 4.5 min, 95% to 5% B from 4.5 to 5 min, 5% B from 5 to 7 min; flow rate at 0.7 mL/min. HRMS was acquired using a DART-SVP ion source (IonSense, Saugus, MA) attached to a JEOL AccuTOF 4G LC-plus mass spectrometer (JEOLUSA, Peabody, MA) in positive-ion mode. The silica gel used in flash column chromatography was from Aldrich (Cat. 60737, pore size 60 Å, 230–400 mesh). The products were identified by ¹H NMR and ¹³C NMR using a Varian 500 MHz spectrometer. NMR samples were dissolved in chloroform-d (CDCl₃), methanol-d₄ (CD₃OD) or DMSO-d₆ containing tetramethylsilane as a reference standard. Chemical shifts were expressed as ppm and calculated downfield from the NMR signal of reference standard. J was expressed as Hz, and its splitting patterns were reported as s, d, t, q, or m. Unless otherwise specified, the purities of all new compounds were over 95% determined by HPLC.

2-Chloro-5-nitrobenzenesulfonyl chloride (3)

1-Chloro-4-nitrobenzene 2 (25.2 g, 161 mmol) was carefully added to chlorosulfuric acid (70 mL) in a large (500 mL) high neck round flask. The reaction mixture was stirred under nitrogen at 100 °C for 7 days until 2 was consumed, which was monitored by TLC. The reaction mixture was cooled down in a freezer (−20 °C), and the cold reaction mixture solution was slowly added to a beaker (1 L) containing a large amount of icy water (700 mL) with vigorous stirring. The precipitation was collected to give a brown solid (29.5 g, 115 mmol, 72%). The raw product was a dark brown solid and could be used without purification. The raw product could also be chromatographed on silica gel eluting with EtOAc and hexane (1:7) to afford 3 as large colorless needle crystals. ¹H NMR (500 MHz, CDCl₃) δ 9.00 (s, 1H), 8.52 (d, J = 7.9 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 146.1, 142.1, 139.7, 134.3, 130.1, 125.8. LCMS (LC-MS Method 1, ESI): t_R = 4.64 min, m/z: [M + Na]⁺ Calcd for C₆H₃Cl₂NO₄SNa 277.9; Found 277.2.

2-Chloro-5-nitrobenzenethiol (7)

Compound 3 (2.55 g, 9.96 mmol) was dissolved in 50 mL of toluene, which was heated to reflux. To the mixture was added PPh₃ (7.86 g, 30.0 mmol) slowly over 10 min. After 3 was consumed, the reaction mixture was cooled to rt and then concentrated under vacuum. The residue was chromatographed on silica gel eluting with EtOAc and hexane (1:5) to afford 7 as yellow crystals (1.74 g, 9.16 mmol, 92%). ¹H NMR (500 MHz, CDCl₃) δ 8.46 (d, J = 2.6 Hz, 1H), 8.07 (dd, J = 8.7, 2.6 Hz, 1H), 7.60 (d, J = 8.7 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 147.4, 138.7, 136.5, 130.9, 123.1, 122.3. LC-MS (LC-MS Method 1, ESI): t_R = 3.98 min. No molecular ion peak was detected.

(2-Chloro-5-nitrophenyl)(4-methoxybenzyl)sulfane (8)

To the solution of 7 (1.33 g, 7.01 mmol) in acetonitrile (20 mL) were added K₂CO₃ (2.76 g, 20.0 mmol), KI (1.20 g, 7.23 mmol) and 4-methoxybenzyl chloride (1.50 g, 9.58 mmol). The mixture was refluxed for 2 h. The reaction mixture was cooled to rt and then concentrated under vacuum. The residue was dissolved in DCM (20 mL) and washed with 20 mL of water. The aq phase was further extracted with 20 mL of DCM twice. The combined organic extract was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The crude product was chromatographed on silica gel eluting with EtOAc and hexane (1:7) to afford 8 as light-yellow needle crystals (2.11 g, 6.81 mmol, 97%). ¹H NMR (500 MHz, CDCl₃) δ 8.12 (d, J = 2.6 Hz, 1H), 7.92 (dd, J = 8.7, 2.6 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.35 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 4.22 (s, 2H), 3.80 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 159.3, 146.7, 139.5, 138.8, 130.2, 130.0, 126.5, 121.8, 120.6, 114.3, 55.3, 36.6. LC-MS (LC-MS Method 1, ESI): t_R = 4.50 min, m/z: [M + NH₄]⁺ Calcd for C₁₄H₁₆ClN₂O₃S 327.0; Found 327.0; m/z: [M + Na]⁺ Calcd for C₁₄H₁₂ClNNaO₃S 332.0; Found 331.9.

4-Chloro-3-((4-methoxybenzyl)thio)aniline (9)

To the solution of 8 (1.55 g 5.00 mmol) and NiCl₂·6H₂O (1.19 g, 5.00 mmol in 2 mL of MeOH) in 20 mL of THF was added NaBH₄ (567 mg, 15.1 mmol). The reaction was slowly heated to reflux until 8 was consumed (10 min). The reaction mixture was cooled to rt and then concentrated under vacuum. To the residue were added 20 mL of distilled water and 30 mL of EtOAc. The aq phase was extracted with EtOAc (2 × 30 mL). The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated under vacuum. The residue was chromatographed on silica gel eluting with EtOAc and hexane (1:2) to afford 9 as a white solid (1.29 g, 4.61 mmol, 92%). ¹H NMR (500 MHz, CDCl₃) δ 7.27 (d, J = 6.95 Hz, 2H), 7.12 (d, J = 8.5 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 6.57 (dd, J = 8.5, 2.7 Hz, 1H), 6.44 (dd, J = 8.5, 2.7 Hz, 1H), 4.07 (s, 2H), 3.79 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 158.9, 145.3, 136.3, 130.1, 130.0, 129.9, 128.3, 122.7, 115.6, 114.0, 113.8, 55.3, 37.0. LC-MS (LC-MS Method 1, ESI): t_R = 4.00 min, m/z: [M + H]⁺ Calcd for C₁₄H₁₅ClNOS 280.1; Found 280.0. HRMS (DART-SVP-AccuTOF) m/z: [M + H]⁺ Calcd for C₁₄H₁₅ClNOS 280.0563; Found 280.0561.

N-(4-Chloro-3-((4-methoxybenzyl)thio)phenyl)picolinamide (10)

To the solution of picolinic acid (152 mg, 1.24 mmol) and 9 (279 mg, 0.997 mmol) in DCM (12 mL) were added N,N’-diisopropylcarbodiimide (253 mg, 2.00 mmol) and 4-dimethylaminopyridine (245 mg, 2.00 mmol). The mixture was stirred at rt overnight. The reaction mixture was then filtered, and the filtrate was dried under vacuum. The residue was chromatographed on silica gel eluting with EtOAc and hexane (1:2) to afford 10 as a white needle crystal (345 mg, 0.897 mmol, 90%). ¹H NMR (500 MHz, CDCl₃) δ 10.03 (s, 1H), 8.63 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H), 8.30 (dd, J = 7.8, 1.0 Hz, 1H), 7.97 (d, J = 2.3 Hz, 1H), 7.94 (tdd, J = 7.8, 1.6, 1.0 Hz, 1H), 7.56 – 7.48 (m, 1H), 7.44 (ddd, J = 8.6, 2.4, 0.7 Hz, 1H), 7.36 (dd, J = 6.7, 1.8 Hz, 2H), 7.33 (s, 1H), 6.86 (d, J = 8.1 Hz, 2H), 4.20 (s, 2H), 3.79 (d, J = 0.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 162.0, 159.0, 149.4, 148.0, 137.8, 137.3, 136.9, 130.3, 129.8, 127.8, 127.8, 126.7, 122.4, 119.1, 117.6, 114.0, 55.3, 36.9. LC-MS (LC-MS Method 1, ESI): t_R = 4.53 min, m/z: [M + H]⁺ Calcd for C₂₀H₁₈ClN₂O₂S 385.1; Found 385.0. HRMS (DART-SVP-AccuTOF) m/z: [M + H]⁺ Calcd for C₂₀H₁₈ClN₂O₂S 385.0778; Found 385.0774.

N-(4-Chloro-3-mercaptophenyl)picolinamide (1)

Compound 10 (269 mg, 0.699 mmol) was dissolved in TFA (5.0 mL). The mixture was refluxed for 2 h. The solvent was removed under vacuum and the residue was recrystallized from 5 mL of MeOH containing hydrogen chloride (37%, 0.12 mL) to give 1 as a white solid (204 mg, 0.677 mmol, 97%). ¹H NMR (500 MHz, DMSO-d₆) δ 10.74 (s, 1H), 8.74 (s, 1H), 8.21 (d, J = 1.9 Hz, 1H), 8.15 (d, J = 7.3 Hz, 1H), 8.08 (t, J = 7.5 Hz, 1H), 7.69 (s, 1H), 7.61 (dd, J = 8.7, 2.0 Hz, 1H), 7.41 (d, J = 8.7 Hz, 1H), 5.80 (s, 1H). ¹H NMR (500 MHz, CD₃OD) δ 8.41 (br, 1H), 7.90 (br, 1H), 7.73 (br, 1H), 7.69 (s, 1H), 7.32 (br, 1H), 7.26 (d, J = 7.3 Hz, 1H), 7.06 (d, J = 8.6 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 163.1, 150.0, 148.9, 138.7, 138.0, 133.3, 129.9, 127.6, 125.6, 123.0, 121.6, 119.3. LC-MS (LC-MS Method 1, ESI): t_R = 4.09 min, m/z: [M + H]⁺ Calcd for C₁₂H₁₀ClN₂OS 265.0; Found 265.0.

(2-Chloro-5-nitrophenyl)(methyl)sulfane (11):

Method 1. To the solution of 7 (945 mg, 4.98 mmol) in dry acetonitrile (20 mL) were added KI (995 mg, 5.99 mmol), K₂CO₃ (2.27 g, 16.4 mmol) and CH₃I (3.55 g, 25.0 mmol). The mixture was refluxed overnight. The reaction mixture was cooled to rt and then concentrated under vacuum. To the residue were added 50 mL of distilled water and 30 mL of DCM, and the aq phase was further washed with DCM (2 × 30 mL). The combined organic extract was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The residue was chromatographed on silica gel eluting with EtOAc and hexane (1:12) to afford 11 as large colorless needle crystals (965 mg, 4.74 mmol, 95%).

Method 2. To the solution of 7 (913 mg, 4.81 mmol) in dry THF (20 mL) were added NaH (192 mg, 8.01 mmol) and CH₃I (1.02 g, 7.19 mmol) at 0 °C. The mixture was warmed to rt for 1 h. The reaction was quenched by addition of water (2 mL), and the mixture was concentrated under vacuum. To the residue were added 50 mL of distilled water and 30 mL of DCM, and the aq phase was washed with DCM (2 × 30 mL). The combined organic extract was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The residue was chromatographed on silica gel eluting with EtOAc and hexane (1:12) to afford 11 as large colorless needle crystals (952 mg, 4.68 mmol, 97%). ¹H NMR (500 MHz, CDCl₃) δ 7.98 (d, J = 7.3 Hz, 1H), 7.92 (s, 1H), 7.50 (d, J = 8.5 Hz, 1H), 2.58 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 147.1, 141.2, 129.9, 120.0, 119.5, 15.2. LCMS (LC-MS Method 1, ESI): t_R = 4.72 min; No molecular ion peak was detected.

4-Chloro-3-(methylthio)aniline (12)

Method 1. To the solution of 11 (548 mg, 2.69 mmol) in DCM (20 mL) and EtOH (30 mL) was added Pd/C (100 mg). The reaction flask was filled with hydrogen by a balloon and stirred at rt overnight. The mixture was filtered to give 12 (458 mg, 2.64 mmol, 98%), which could be used without purification.

Method 2. To the solution of 11 (550 mg, 2.70 mmol) in 20 mL of THF were added NiCl₂·6H₂O (642 mg, 2.70 mmol in 2.0 mL of MeOH) and NaBH₄ (513 mg, 13.6 mmol). The reaction mixture was slowly heated to reflux until 11 was consumed (within 10 min). The reaction mixture was cooled to rt and then concentrated under vacuum. To the residue were added 20 mL of distilled water and 30 mL of DCM, and the aq phase was washed with DCM (2 × 30 mL). The combined organic extract was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The residue was chromatographed on silica gel eluting with EtOAc and hexane (1:3) to afford 12 as an oil (435 mg, 2.50 mmol, 93%), which was hard to be recrystallized and was sensitive to oxidation in air. After 1 mL of HCl (4.0 M in THF) was added into the solution of the purified product in EtOAc (5 mL), large amount of white solid was precipitated out from the solution, which was collected for long-time storage. ¹H NMR (500 MHz, CD₃OD) δ 7.41 (d, J = 8.3 Hz, 1H), 7.17 (s, 1H), 7.04 (d, J = 8.1 Hz, 1H), 2.43 (s, 3H). ¹³C NMR (126 MHz, CD₃OD) δ 141.4, 131.2, 130.3, 119.5, 119.4, 13.6. LC-MS (LC-MS Method 1, ESI): t_R = 3.37 min, m/z: [M + H]⁺ Calcd for C₇H₉ClNS 174.0; Found 174.0.

N-(4-Chloro-3-(methylthio)phenyl)picolinamide (mG4P012)

To the solution of picolinic acid (443 mg, 3.60 mmol) and 12 (520 mg, 3.00 mmol) in DCM (20 mL) were added by N,N’-diisopropylcarbodiimide (505 mg, 4.00 mmol) and 4-dimethylaminopyridine (490 mg, 4.01 mmol). The resulting mixture was stirred at rt overnight. The reaction mixture was then filtered, and the filtrate was dried under vacuum. The residue was chromatographed on silica gel eluting with EtOAc and hexane (1:2) to afford the title product as white needle crystals (767 mg, 2.75 mmol, 92%). ¹H NMR (500 MHz, CDCl₃) δ 10.1 (s, 1H), 8.6 (s, 1H), 8.28 (d, J = 7.2 Hz, 1H), 7.91 (d, J = 7.1 Hz, 1H), 7.85 (d, J = 6.1 Hz, 1H), 7.49 (s, 1H), 7.33 (t, J = 7.2 Hz, 2H), 2.55 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 162.2, 149.5, 148.1, 138.9, 137.9, 137.2, 129.7, 126.8, 126.6, 122.5, 116.6, 116.3, 15.3. LC-MS (LC-MS Method 1, ESI): t_R = 4.53 min, m/z: [M + H]⁺ Calcd for C₁₃H₁₂ClN₂OS 279.0; Found 279.0. HRMS (DART-SVP-AccuTOF) m/z: [M + H]⁺ Calcd for C₁₃H₉D₂ClFN₂OS 299.0390; Found 299.0388.