Synthesis of compounds 40–42 - 設計與合成人類胸苷酸激酶之抑制劑

2.4.3. SAR of dimeric compounds 35, 38, 39, and 42

Figure 29. Chemical structures of dimeric inhibitors 35, 38, 39, and 42 as well as suicide inhibitors 40 and 41.

Table 8. Normalized TMPK inhibition of dimeric compounds 35 and 38–42 at 2 μM.

(n = 6)^{a, 44}

Compound Inhibition (%)^b

35 80.7 ± 2.1

38 81.5 ± 3.8

39 79.1 ± 5.0

42 84.0 ± 1.2

40 51.3 ± 5.5

41 47.8 ± 3.3

a.Each of the data were obtained from 2 independent experiments and each assay was performed in triplicate.

b.Normalized TMPK inhibition based on YMU1 (70% inhibition) at 2 μM.

The luciferase-coupled TMPK assays (Table 8) indicated that all of the dimeric compounds at 2 μM revealed potent inhibitory activity, whereas, the iodo-substituted analogs 40 and 41 presented moderate inhibition against TMPK. The dimeric compounds 35, 38, 39, and 42 with decent inhibitory activity were chosen for the dosage-dependent inhibition experiments (Table 9).

Table 9. Dosage-dependent assays of YMU1 and dimeric compounds 35, 38, 39 and 42 against TMPK. (n = 9)^{a, 44}

Figure 30. Dosage-dependent inhibition of YMU1 and dimeric compounds 35, 38, 39 and 42 against TMPK.

All of the dimeric derivatives showed dosage-dependent inhibition with superior activity than YMU1 (blue line) at the indicated concentrations. In particular, the asymmetric dimer 42 (red line) demonstrated a great inhibitory activity at low concentrations (0.2–1 μM). The asymmetric dimer 42 also improved the water solubility, an Achilles’ heel of symmetric dimers.

2.5. Molecular docking of YMU1 derivatives

To further have an insight into the SAR, we chose compound 22 with a

hydroxyl-substituted heterocyclic core and the asymmetric dimer 42 for molecular docking experiments as well as inhibition constant (Ki) calculations (Figure 31).The docking experiments were performed by Sheu and coworkers at National Yang Ming University.

Figure 31. Selected compounds for molecular docking.

By using the NADH-coupled TMPK assay (Table 10), different concentrations of compounds 22 and 42 (0.1–2 μM) were preincubated with TMPK to measure the initial velocity. The K_m and V_max were obtained from nonlinear regression analysis, and the K_i values of compounds 22 and 42 were calculated by the equation of

K_i = [I] / (V_max/V_max’ – 1)

wherein V_max is the maximal velocity without treatment of compound, [I] and V_max’ are the concentration of compound and maximal velocity in the presence of compound,³⁹ to give 0.12 ± 0.03 and 0.18 ± 0.15 μM, respectively. Compared with YMU1 (K_i = 0.22 ± 0.03 μM), the hydroxyl-substituted core 22 and asymmetric dimer 42 indeed showed

59 (200 μM). Data obtained from nonlinear regression analysis were used to calculate the V_max and K_m. The K_i values represent mean values derived from five different concentrations of compounds 22 and 42.

a.Each of the data were obtained from 3 independent experiments.

As shown in Figure 32, three small molecules of 22 will be prone to insert into TMPK, with two bound at ATP pocket and one situated in YMU1 binding site according to the binding affinity of docking results. This may explain the superior inhibition of analog 22 at high concentration since the higher level of small molecules present, the more potential of these inhibitors to enter into the binding pocket of TMPK. On the

other hand, two molecules of dimeric compounds 42 may bind with TMPK, with one located at YMU1 binding site and the other covers the LID domain. Noteworthily, the dimeric analog contains two heterocyclic cores similar to the structure of adenine, one of the core fragments may bind to ATP pocket and the other one may situate at the original TMP binding site. Therefore, a dimeric compound will bind with TMPK more tightly to reveal higher inhibitory activity than YMU1 at either low or high concentrations.

Figure 32. Docking of compounds 22 (left) and 42 (right) with hTMPK. Inhibitors (green), ATP, TMP (light blue), LID domain (pink), and P-loop (blue) are shown.⁵²

2.6. Conclusion

Figure 33. Chemical structures of efficient hTMPK inhibitors.

In summary, we have synthesized 30 YMU1 derivatives and tested for their

inhibitory activity against hTMPK. Among these compounds, 7 analogs were proved to exhibit comparable or even better inhibitory activity than YMU1 (Figure 33). Based on the structure–activity relationships, we can establish a general structural feature as shown in Figure 34. First of all, the sulfur atom in bicyclic core plays an important role to maintain the inhibition potency. Second, additional hydroxyl functional group in the heterocyclic core can exert hydrogen bondings with the residues in hTMPK active site to promote the binding affinity. Third, more than one hydrophilic substituted bicyclic cores may be incorporated into the hTMPK binding site to attain good inhibitory activity.

Finally, dimeric analogs with diverse heterocyclic cores show superior inhibitory activity, whereas changing the end group R² to a carboxylic acid decreases the inhibition.

Figure 34. Structural features of YMU1 derivatives against hTMPK.

For further research, symmetric and asymmetric dimeric derivatives with hydrophilic functional groups at the R¹ position will be promising targets to enhance the inhibitory activity against hTMPK. In addition to co-crystallization of hTMPK–inhibitor for X-ray analysis, photoaffinity probe such as changing the R¹ into an azido group and introducing biotin at the R² position may be a prospective way to study the binding site.

Chapter 3. Experimental Section

3.1. General part

All reagents and solvents were reagent grade and used without further purification unless indicated otherwise. All solvents were anhydrous grade unless otherwise specified. Dichloromethane (CH2Cl2) was distilled from CaH2. All air or moisture sensitive experiments were performed under the atmosphere of argon. Reactions were tracked by analytical thin-layer chromatography (TLC) on 0.25 mm E. Merck silica gel 60 F254 glass plates. Compounds were visualized by UV, or using ninhydrine, p-anisaldehyde, KMnO₄, and phosphomolybdic acid (PMA) as visualizing agent. Flash chromatography was carried out by using E. Merck silica gel 60 (0.040–0.063 mm (triplet), dd (double of doublets), ddd (double of double of doublets), m (multiplet), and

br (broad). Coupling constants (J) are given in Hz. The ESI-MS experiments were conducted on a Bruker Daltonics BioTOF III high-resolution mass spectrometer.

3.2. Expression and purification of enzymes

Human TMPK cloned in pGEX-2T was transformed in E coli JM 109 strain and treated with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37 ^oC for 4 h to produce GTS-hTMPK fusion protein. After affinity purification by glutathione 4B beads (Amersgam Pharmacia Biotech), hTMPK protein was cleaved from GST-fusion protein bound to glutathione beads by thrombin digestion at 4 ^oC overnight.

3.3. Luciferase-coupled TMPK assay

The TMPK reaction was started by adding 0.01–1 μg of purified hTMPK in 50 μL

of TMPK assay buffer (100 mM Tris–HCl, pH 7.5, 100 mM KCl, 10 mM MgCl₂, 50 μM ATP and 100 μM dTMP) in 96-well plate at 25 ^oC, and then terminated by adding 200 μL of DTNB (100 μM). Afterwards, 10 μL of reaction solution was transferred to a

white 96-well plate containing 90 μL of luciferase assay buffer (50 mM glycine, 0.5 mM EDTA and 5 mM MgCl₂, pH 7.0, 0.1 μg of luciferase, 50 μM luciferin and 0.1% BSA).

The luminescence was measured with a luminescence counter (Packard).

3.4. NADH-coupled TMPK assay

All reactions were performed in 96-well plates in an assay volume of 100 μl. The activity of hTMPK was measured at 25 °C. Different concentrations of inhibitors (0.1–2 μM) with 10 μl and the purified hTMPK with 10 μl were preincubated for 10 min, and then 70 μl of the buffer containing 100 mM Tris–HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2, 0.5 mM phosphoenol pyruvate, 0.25 mM NADH, 5 units of lactate dehydrogenase, 4 units of pyruvate kinase, and 200 μM dTMP were added, followed by 10 μl of ATP with different concentrations (7.8–1000 μM) to underwent the reaction.

The change in NADH concentration was measured by reading the absorbance at 340 nm.

3.5. Molecular docking

The initial structure of TMPK for YMU1 MD simulation was taken from PDB 1E2D and subjected to energy minimization. The crystal structure of hTMPK for

compounds 22 and 42 molecular docking was provided from Nei-Li Chan’s team. YMU1

was built and the charge was assigned using Gaussian03 package. AutoDock program was

used to identify the binding sites and the interactions of YMU1 with TMPK. This study

involved MD simulation performed by the NAMD program with the CHARMM27 force

field.

3.6. Synthetic procedures and characterization of compounds

2-Mercapto-4,6-dimethylnicotinonitrile (2)²²

A mixture of 2-chloro-4,6-dimethylnicotinonitrile 1 (400 mg, 2.42 mmol) and thiourea (600 mg, 7.89 mmol) in n-butanol (12 mL) was heated at reflux (118 ^oC) for 4 h.

After cooling to room temperature, the solution turned to a suspension containing light yellow solids. The solids were collected by filtration, rinsed with n-butanol, and dried under reduced pressure to give compound 2 (333 mg, 84% yield). C₈H₈N₂S; light yellow powder; ¹H NMR (400 MHz, CDCl₃); δ 6.40 (1 H, s), 2.45 (3 H, s), 2.43 (3 H, s);

ESI–HRMS calcd for C₈H₉N₂S: 165.0486, found: m/z 165.0484 [M + H]⁺.

4,6-Dimethylisothiazolo[5,4-b]pyridin-3(2H)-one (3)²²

A solution of compound 2 (310 mg, 1.89 mmol) in conc. H₂SO₄(2.1 mL) was

stirred at 100 ^oC for 4 h. The mixture was then cooled and adjusted to pH 5–6 by addition of saturated NaHCO3(aq), producing white solids in suspension. The solids were then collected by filtration, rinsed with H₂O, and dried in vacuo to give compound 3

(237 mg, 69% yield). C8H8N2OS; pale yellow powder; ¹H NMR (400 MHz, CDCl3);

δ 6.95 (1 H, s), 2.75 (3 H, s), 2.62 (3 H, s); ¹³C NMR (100 MHz, CDCl3) δ 168.3, 166.5,

163.3, 149.9, 122.5, 115.0, 25.0, 18.3; ESI–HRMS calcd for C8H9N2OS: 181.0436, found: m/z 181.0437 [M + H]⁺.

Benzyl-1H-indazol-3(2H)-one (5)⁴¹

A mixture of 1H-indazol-3(2H)-one 4 (400 mg, 2.98 mmol) and NaOH (120 mg, 2.98 mmol) in H₂O (3 mL) was heated at 35 ^oC until it became a clear solution. Benzyl chloride (0.34 mL, 2.98 mmol) was then added slowly to the solution at 70 ^oC. The mixture was stirred for 2 h to give a suspension containing beige solids. After cooling to room temperature, the solids were collected by filtration, and then recrystallized from EtOAc to give compound 5 (40 mg, 60% yield). C₁₄H₁₂N₂O; white powder; ¹H NMR

(400 MHz, CD3OD); δ 7.68 (1 H, dt, J = 8.4, 1.0 Hz), 7.36–7.39 (2 H, m), 7.21–7.27 (3 H, m), 7.16–7.7.18 (2 H, m), 7.05 (1 H, ddd, J = 7.8, 6.4, 1.4 Hz), 5.25 (2 H, s); ¹³C NMR (100 MHz, CD3OD) δ 158.8, 145.1, 138.3, 130.2, 129.7 (2 ×), 128.9, 128.7 (2 ×), 122.3, 121.1, 115.6, 111.2, 53.4; ESI–HRMS calcd for C14H13N2O: 225.1028, found: m/z 225.1026 [M + H]⁺.

Isoindolin-1-one (7)⁴²

Phthalimide 6 (2 g, 13.5 mmol) was added to a suspension of AlCl₃ (9 g, 67.50 mmol) in cyclohexane (50 mL) at 110 ^oC for 15 h. The solids were removed by filtration, and the residue was extracted with CHCl₃ and H₂O. The organic phase was dried over MgSO₄, filtered, and concentrated to give yellow solids which were recrystallized from EtOAc to give compound 7 (1 g, 57% yield). C₈H₇NO; white powder; ¹H NMR (400 MHz, CDCl₃); δ 8.83 (1 H, s), 7.76 (1 H, d, J = 8.4 Hz), 7.42–7.45 (1 H, m), 7.33–7.36 (2 H, m), 4.36 (2 H, s); ¹³C NMR (100 MHz, CDCl₃) δ 171.6, 143.1, 131.6, 130.9, 127.2, 122.8, 122.5, 45.9; ESI–HRMS calcd for C₈H₈NO: 134.0606, found: m/z 134.0602 [M +

H]⁺.

Benzo[d]isoxazol-3(2H)-one (9)⁴³

A mixture of salicylhydroxamic acid 8 (100 mg, 0.65 mmol) and carbonyldiimidazole (127 mg, 0.78 mmol) in anhydrous THF (5 mL) was stirred for 15 h at 70 ^oC. The solution was concentrated under reduced pressure, and washed successively with 1 M HCl_(aq) and saturated NaHCO_3(aq). The organic phase was dried over MgSO₄, filtered, and concentrated to give pink solids which were recrystallized from EtOAc to give compound 9 (70 mg, 80% yield). C₇H₅NO₂; white powder; ¹H NMR (400 MHz, CD₃OD); δ 7.63 (1 H, dd, J = 8.8, 1.2 Hz), 7.34–7.36 (1 H, m), 6.85–6.91 (2 H, m); ¹³C NMR (100 MHz, CD₃OD) δ 168.5, 160.3, 134.6, 128.4, 120.2, 118.3, 115.5; ESI–HRMS calcd for C₇H₄NO₂: 134.0242, found: m/z 134.0240 [M – H]^–.

Tert-butyl 4-(2-chloroacetyl)piperazine-1-carboxylate (11)²²

A mixture of piperazine (1.10 g, 12.8 mmol) and DIEA (2.24 mL, 12.8 mmol) in anhydrous CH2Cl2 (60 mL) was stirred at room temperature, and a solution of di-tert-butyl dicarbonate (1.46 mL, 6.4 mmol) in anhydrous CH2Cl2 (80 mL) was added slowly via a separatory funnel over a period of 3 h. The mixture was extracted with CH₂Cl₂ and H₂O. The organic phase was dried over MgSO₄, filtered, and concentrated to give compound 10 (1.39 g, 58% yield).

Chloroacetyl chloride (0.63 mL, 7.32 mmol) was added slowly to a solution of compound 10 (1.24 g, 6.64 mmol) and DIEA (3.5 mL, 19.94 mmol) in anhydrous CH₂Cl₂at 0 ^oC. The mixture was then stirred at room temperature for 4 h and extracted with CH₂Cl₂ and H₂O. The organic phase was dried over MgSO₄, filtered, and purified by flash chromatography on a silica gel column with elution of EtOAc/hexane (1:2) to give compound 11 (1.14 g, 71% yield). C₁₁H₁₉ClN₂O₃; white solid; ¹H NMR (400 MHz, CDCl₃) δ 4.05 (2 H, s), 3.56–3.58 (2 H, m), 3.47 (4 H, s), 3.42–3.43 (2 H, m), 1.45 (9 H, s); ¹³C NMR (100 MHz, CDCl₃) δ 164.8, 154.0, 80.1, 45.9, 43.1 (2 ×), 41.8, 40.7, 28.2

(3×); ESI–HRMS calcd for C11H20ClN2O3: 263.1162, found: m/z 263.1165 [M + H]⁺.

Tert-butyl 4-(2-iodoacetyl)piperazine-1-carboxylate (12)²²

A mixture of compound 11 (297 mg, 1.13 mmol) and sodium iodide (514 mg, 3.43 mmol) in acetone (12 mL) was stirred at room temperature for 16 h. The solids were removed by filtration, and the solution was concentrated under reduced pressure. The residue was extracted with CH₂Cl₂ and H₂O. The organic phase was dried over MgSO₄, filtered, and concentrated to give compound 12 (353 mg, 88% yield). C₁₁H₁₉IN₂O₃; brown solid; ¹H NMR (400 MHz, CDCl₃) δ 3.74 (2 H, s), 3.56–3.59 (2 H, m), 3.51–3.53 (2 H, m), 3.40–3.42 (4 H, m), 1.46 (9 H, s); ¹³C NMR (100 MHz, CDCl₃) δ 166.2, 153.9, 80.1, 46.8, 42.6 (2 ×), 41.7, 28.2 (3 ×), –4.0; ESI–HRMS calcd for C₁₁H₂₀IN₂O₃: 355.0519, found: m/z 355.0515 [M + H]⁺.

Tert-butyl 4-[2-(1-benzyl-3-oxo-1H-indazol-2(3H)-yl)acetyl]piperazine-1-

carboxylate (13)

A mixture of compound 5 (100 mg, 0.45 mmol), compound 12 (189 mg, 0.54 mmol) and DIEA (0.39 mL, 2.23 mmol) in anhydrous CH2Cl2 (2 mL) was stirred for 8 h at room temperature. The solution was concentrated under reduced pressure, and then purified by flash chromatography on a silica gel column with elution of EtOAc/hexane (1:1) to give compound 13 (82 mg, 41% yield). C₂₅H₃₀N₄O₄; transparent liquid; TLC (EtOAc/hexane = 1:1) R_f = 0.2; IR ν_max(neat) 2976, 2929, 2864, 1704, 1462, 1417, 1366, 1235, 1168, 1030, 996, 919, 862, 754, 730, 700, 645, 542 cm^–1; ¹H NMR (400 MHz, CDCl₃) δ 7.84 (1 H, d, J = 8.0 Hz), 7.51–7.55 (1 H, m), 7.15–7.25 (6 H, m), 7.10 (1 H, br, s), 4.94 (2 H, br, s), 4.57 (2 H, s), 3.43–3.46 (2 H, m), 3.22–3.31 (6 H, m), 1.43 (9 H, s); ¹³C NMR (100 MHz, CDCl₃) δ 165.1, 154.4, 150.2, 135.2, 132.7, 128.7 (2 ×), 128.2 (2 ×), 127.7 (2 ×), 124.4, 122.3, 117.8, 111.8, 80.3, 54.3, 45.9, 44.9, 43.4 (2 ×), 41.9, 28.3 (3 ×); ESI–HRMS calcd for C₂₅H₃₁N₄O₄: 451.2345, found: m/z 451.2341 [M + H]⁺.

Tert-butyl 4-[2-(3-oxo-1H-indazol-2(3H)-yl)acetyl]piperazine-1-carboxylate (14)

Compound 13 (28 mg, 0.06 mmol) and Pd/C (10%, 3 mg, 0.02 mmol) in MeOH (1 mL) was stirred at room temperature for 24 h under an atmosphere of hydrogen. After filtrated through a pad of Celite, the filtrate was concentrated, and purified by flash chromatography on a silica gel column with elution of EtOAc/hexane (1:1) to give compound compound 14 (12 mg, 53% yield). C₁₈H₂₄N₄O₄; yellow liquid; TLC (EtOAc/hexane = 1:1) R_f= 0.1; IR ν_max(neat) 3453, 2974, 2929, 1638, 1634, 1469,

1462, 1422, 1365, 1238, 1170, 1031, 996, 757, 679 cm^–1; ¹H NMR (400 MHz, CDCl₃) δ 7.77 (1 H, d, J = 8.0 Hz), 7.56 (1 H, ddd, J = 8.4, 7.2, 1.2 Hz), 7.29 (1 H, d, J = 8.4

Hz), 7.18 (1 H, td, J = 8.4, 0.8 Hz), 4.89 (2 H, s), 3.58–3.61 (4 H, m), 3.53 (2 H, s), 3.46 (2 H, s), 1.48 (9 H, s);¹³C NMR (100 MHz, CDCl₃) δ 167.3, 164.4, 156.4, 148.7, 133.4, 124.3, 123.0, 118.2, 113.6, 81.9, 46.8, 46.0, 43.2, 28.7 (3 ×); ESI–HRMS calcd for C₁₈H₂₃N₄O₄: 359.1719, found: m/z 359.1719 [M – H]^–.

Tert-butyl 4-[2-(1,3-dioxoisoindolin-2-yl)acetyl]piperazine-1-carboxylate (15)

A mixture of phthalimide 6 (33 mg, 0.23 mmol), compound 12 (81 mg, 0.32 mmol), TBAI (84 mg, 2.30 mmol) and DIEA (0.2 mL, 1.14 mmol) in anhydrous CH2Cl2 (10 mL) was stirred at 40 ^oC for 24 h. The solution was concentrated under reduced pressure, and the residue was extracted with CH2Cl2 and H2O. The organic phase was dried over MgSO₄, filtered, concentrated, and purified by flash chromatography on a silica gel column with elution of EtOAc/hexane (1:1) to give compound 15 (57 mg, 67% yield).

C₁₉H₂₃N₃O₅; white solid; mp 243–244 ^oC; TLC (EtOAc/hexane = 1:1) R_f= 0.2; IR ν_max

(neat) 2922, 2852, 1774, 1726, 1458, 1420, 1395, 1236, 1169, 1115, 957, 716, 666 cm^–1;

1H NMR (400 MHz, CDCl₃) δ 7.85 (2 H, dd, J = 5.6, 3.2 Hz), 7.70 (2 H, dd, J = 5.6, 3.2

Hz), 4.48(2 H, s), 3.51–3.57 (6 H, m), 3.43–3.45 (2 H, m), 1.47 (9 H, s); ¹³C NMR (100 MHz, CDCl₃) δ 167.9 (2 ×), 164.1, 154.3, 134.0 (2 ×), 132.1 (2 ×), 123.4 (2 ×), 80.3, 44.5, 43.1 (2 ×), 41.9, 38.9, 28.2 (3 ×); ESI–HRMS calcd for C₁₉H₂₄N₃O₅: 374.1716, found: m/z 374.1712 [M + H]⁺.

Tert-butyl 4-[2-(1-oxoisoindolin-2-yl)acetyl]piperazine-1-carboxylate (16)

A mixture of compound 7 (30 mg, 0.23 mmol), compound 12 (96 mg, 0.27 mmol) and Cs2CO3 (220 mg, 0.68 mmol) in anhydrous CH3CN(1 mL) was stirred at 50 ^oC for 36 h. The solution turned to a suspension containing white solids. After concentrated under reduced pressure, the residue was extracted with CH2Cl2 and H2O. The organic phase was dried over MgSO4, filtered, concentrated, and purified by flash chromatography on a silica gel column with elution of EtOAc/hexane (1:1) to give compound 16 (40 mg, 49% yield). C₁₉H₂₅N₃O₄; yellow solid; mp 212–214 ^oC; TLC (EtOAc/hexane = 1:1) R_f= 0.1; IR ν_max(neat) 2974, 2924, 2856, 1684, 1663, 1458, 1413, 1365, 1238, 1166, 1031, 996, 736 cm^–1; ¹H NMR (400 MHz, CDCl₃); δ 7.84 (1 H, d, J = 7.6 Hz), 7.52–7.56 (1 H, m), 7.44 (2 H, t, J = 7.6 Hz), 4.54 (2 H, s), 4.44 (2 H, s),

3.54–3.59 (4 H, m), 3.40–3.45 (4 H, m), 1.45 (9 H, s); ¹³C NMR (100 MHz, CDCl₃) δ 168.5, 166.3, 154.2, 141.5, 131.6, 131.5, 127.9, 123.8, 122.7, 80.4, 50.8, 45.5, 45.1,

44.1 (2 ×), 41.9, 28.4 (3 ×); ESI–HRMS calcd for C₁₉H₂₆N₃O₄: 360.1923, found: m/z 360.1919 [M + H]⁺.

Tert-butyl 4-[2-(3-oxobenzo[d]isoxazol-2(3H)-yl)acetyl]piperazine-1-carboxylate

(17)

A mixture of compound 9 (120 mg, 0.89 mmol), compound 12 (376 mg, 1.06 mmol) and DIEA (0.92 mL, 5.30 mmol) in anhydrous CH2Cl2 (5 mL) was stirred for 72 h at room temperature. The solution was concentrated under reduced pressure, and then purified by flash chromatography on a silica gel column with elution of EtOAc/hexane (1:2) to give compound 17 (20 mg, 6% yield). C₁₈H₂₃N₃O₅; white solid; mp 221–223 ^oC;

TLC (EtOAc/hexane = 1:1) R_f= 0.5; IR ν_max(neat) 2976, 2929, 2865, 1791, 1695, 1668, 1489, 1461, 1419, 1366, 1241, 1168, 1022, 919, 754, 690 cm^–1; ¹H NMR (400 MHz, CDCl₃) δ 7.21 (1 H, d, J = 7.6 Hz), 7.14 (2 H, td, J = 13.4, 7.6 Hz), 6.99 (1 H, d, J = 7.6 Hz); 4.62 (2 H, s), 3.58–3.60 (2 H, m), 3.53–3.55 (2 H, m) 3.50 (2 H, s), 3.41–3.44 (2 H, m), 1.45 (9 H, s); ¹³C NMR (100 MHz, CDCl₃) δ 163.7, 154.2 (2 ×), 142.5, 130.9, 124.0, 122.8, 110.1, 109.1, 80.6, 45.2, 43.7 (2 ×), 43.3, 42.1, 28.5 (3 ×); ESI–HRMS calcd for C₁₈H₂₄N₃O₅: 362.1716, found: m/z 362.1716 [M + H]⁺.

6-Amino-2-mercapto-4-methylnicotinonitrile (19)⁴⁸

A mixture of 2-cyanothioacetamide (3.0 g, 30 mmol), 3-aminocrotononitrile (2.46 g, 30 mmol) in 1,4-dioxane (30 mL) was stirred at 100^oC for 2 h. After cooling to room temperature, the solids were collected and recrystallized from EtOH to give compound

19 (1.18 g, 24% yield). C₇H₇N₃S; light yellow solid; ¹H NMR (400 MHz, DMSO-d₆) δ 5.89 (1 H, s), 2.18 (3 H, s); ¹³C NMR (100 MHz, DMSO-d6) δ 175.4, 155.2, 153.5,

118.1, 100.2, 99.0, 20.8; ESI–HRMS calcd for C₇H₈N₃S: 166.0439, found: m/z 166.0437 [M + H]⁺.

6-Amino-4-methylisothiazolo[5,4-b]pyridin-3(2H)-one (20)

A mixture of compound 19 (145 mg, 0.88 mmol) and conc.H₂SO₄(1.5 mL) was stirred at 100 ^oC for 5 h. The mixture was cooled and adjusted to pH 5–6 by addition of

NaHCO3(s), producing white solids in suspension. The solids were collected by filtration, rinsed with H₂O, and dried in vacuo to give compound 20 (156 mg, 98% yield).

C7H7N3OS; pale yellow solid; mp 204–206 ^oC; TLC (CH2Cl2/MeOH = 9:1) Rf = 0.2;

IR νmax (neat) 3421, 3344, 3069, 1632, 1600, 1550, 1453, 1375, 1209, 1170, 1028, 983, 838, 619, 550, 500 cm^–1; ¹H NMR (400 MHz, DMSO-d6) δ 6.82 (2 H, s), 6.19 (1 H, s), 2.45 (3 H, s); ¹³C NMR (100 MHz, DMSO-d6) δ 168.0, 166.0, 161.8, 147.5, 107.2, 106.9, 17.4; ESI–HRMS calcd for C7H8N3OS: 182.0388, found: m/z 182.0395 [M + H]⁺.

6-Hydroxy-4-methylisothiazolo[5,4-b]pyridin-3(2H)-one (22)⁴⁹

A mixture of 2-cyanothioacetamide (200 mg, 2.0 mmol) and morpholine (0.17 mL, 2.0 mmol) in EtOH (2 mL) was stirred in seal tube for 5 min. Once dissolved, ethyl acetoacetate (0.25 mL, 2.0 mmol) was added. The mixture was heated at 160^oC by microwave irridation for 10 min. After cooling to room temperature, the solution was concentrated under reduced pressure to give dark red oil. The oil mixture was treated with CH₂Cl₂ at reflux for 10 min. Once cooling, the yellow solids of compound 21 (141 mg) were collected by filtration and dried in vacuo.

The above-prepared compound 21 (141 mg) was added to conc.H₂SO₄ (1 mL) and stirred under 80^oC for 5 h. After cooling, the solution was modulated to pH 5–6 by NaHCO3(s), producing yellow solids in suspension. The solids were collected by filtration, rinsed with H₂O, and dried in vacuo to give compound 22 (78 mg, 22% yield for 2 steps).

C7H6N2O2S; pale yellow solid; TLC (CH2Cl2/MeOH = 9:1) Rf = 0.2; ¹H NMR (400 MHz, DMSO-d6) δ 6.13(1 H, s), 2.41 (3 H, s); ¹³C NMR (100 MHz, DMSO-d6) δ 164.1, 163.0, 160.8, 148.6, 114.3, 106.5, 18.2; ESI–HRMS calcd for C7H7N2O2S: 183.0228, found: m/z 183.0231 [M + H]⁺.

Tert-butyl 4-(2-azidoacetyl)piperazine-1-carboxylate (23)

A mixture of compound 12 (67 mg, 0.14 mmol) and sodium azide (20 mg, 0.40 mmol) in DMF (0.5 mL) was sitrred at 80 ^oC for 12 h. The solution was concentrated under reduced pressure and then extracted with CH₂Cl₂ and H₂O. The organic phase was dried over MgSO_4, filtrated, and concentrated to give compound 23 (36 mg, 95% yield).

C₁₁H₁₉N₅O₃; yellow solid; mp 226–228 ^oC; TLC (EtOAc/hexane = 2:1) R_f= 0.2; IR

νmax (neat) 3518, 2977, 2929, 2865, 2098, 1684, 1650, 1459, 1421, 1366, 1285, 1236,

1169, 1127, 995, 863, 770, 553 cm^–1;¹H NMR (400 MHz, CDCl3) δ 3.93 (2 H, s), 3.60 (2 H, s, br), 3.44 (4 H, s), 3.34-3.345 (2 H, m), 1.46 (9 H, s); ¹³C NMR (100 MHz, CDCl3) δ 165.4, 154.0, 80.3, 50.5, 44.8, 43.4 (2 ×), 41.7, 28.3 (3 ×) ; ESI–HRMS calcd for C11H20N5O3: 270.1566, found: m/z 270.1564 [M + H]⁺.

Tert-butyl 4-(2-aminoacetyl)piperazine-1-carboxylate (24)

Compound 23 (33 mg, 0.12 mmol) and Pd/C (10%, 7 mg, 0.05 mmol) in MeOH (1.2 mL) was stirred at room temperature for 12 h under an atmosphere of hydrogen.

The mixture was filtrated through a pad of Celite, and the filtrate was concentrated under reduced pressure to give compound 24 (28 mg, 96% yield). C₁₁H₂₁N₃O₃; pale yellow solid; mp 226–228 ^oC; TLC (EtOAc/hexane = 1:2) R_f= 0.1; IR ν_max(neat) 3014, 2980, 2687, 2600, 1681, 1654, 1504, 1434, 1364, 1244, 1179, 1009, 763, 545 cm^–1; ¹H NMR (400 MHz, CD₃OD) δ 3.96 (2 H, s), 3.59–3.62 (2 H, m), 3.42–3.50 (6 H, m), 1.48 (9 H, s); ¹³C NMR (100 MHz, CD₃OD) δ 166.1, 156.1, 81.8, 45.8, 43.1, 41.3, 28,8 (3×);

ESI–HRMS calcd for C11H22N3O3: 244.1661, found: m/z 244.1660 [M + H]⁺.

Tert-butyl 4-[(6-amino-4-methyl-3-oxoisothiazolo[5,4-b]pyridin-2(3H)-2-yl)acetyl]

piperazine-1-carboxylate (25)

A mixture of compound 20 (100 mg, 0.55 mmol), compound 12 (98 mg, 0.28 mmol) and DIEA (0.38 mL, 2.27 mmol) in anhydrous DMF(3.8 mL) was stirred for 3 h at room temperature. The solution was concentrated under reduced pressure, and then purified by flash chromatography on a silica gel column with elution of EtOAc/hexane (1:1 to 3:1) to give compound 25 (78 mg, 68% yield). C₁₈H₂₅N₅O₄S; white solid; mp 226–228 ^oC;

TLC (EtOAc/hexane = 1:1) R_f = 0.1; IR ν_max(neat) 3260, 3109, 2944, 2887, 1708, 1603, 1568, 1495, 1379, 1298, 1194, 1142, 1080, 928, 795, 667, 432 cm^–1; ¹H NMR (400 MHz, CD₃OD) δ 6.29 (1 H, s), 4.70 (2 H, s), 3.56–3.57 (4 H, m), 3.53 (2 H, br, s), 3.45 (2 H, s), 2.54 (3 H, s), 1.48 (9 H, s); ¹³C NMR (100 MHz, CDCl₃) δ 167.8, 167.7, 166.5, 163.8, 156.4, 150.9, 109.1, 108.1, 81.9, 45.9, 45.3, 43.2, 28.8 (3 ×), 18.1; ESI–HRMS calcd for C₁₈H₂₆N₅O₄S: 408.1706, found: m/z 408.1706 [M + H]⁺.

Tert-butyl 4-(2-[(6-amino-4-methylisothiazolo[5,4-b]pyridin-3-yl)oxy)acetyl]

piperazin-1-carboxylate (26)

A mixture of compound 20 (52 mg, 0.29 mmol), compound 12 (51 mg, 0.14 mmol) and DIEA (0.2 mL, 1.19 mmol) in anhydrous DMF(2 mL) was stirred for 2 h at room temperature. The solution was concentrated under reduced pressure, and then purified by flash chromatography on a silica gel column with elution of EtOAc/hexane (1:1) to give compound 26 (9 mg, 16% yield). C₁₈H₂₅N₅O₄S; white solid; mp 206–208 ^oC; TLC (EtOAc/hexane = 1:1) R_f = 0.2; IR ν_max(neat) 3530, 3366, 3190, 2975, 2931, 1691, 1643, 1593, 1422, 1383, 1255, 1206, 1163, 1133, 1017, 840, 684, 590 cm^–1; H NMR (400 MHz, CD₃OD) δ 6.36 (1 H, s), 5.23 (2 H, s), 3.57–3.58 (4 H, m), 3.54 (2 H, s, br), 3.46 (2 H, s, br), 2.58 (3 H, s), 1.48 (9 H, s); ¹³C NMR (100 MHz, CD₃OD) δ 168.5 (2

×), 162.6, 162.2, 156.1, 147.7, 111.1, 109.1, 81.8, 65.9, 45.8, 43.0, 28.8 (3 ×), 19.7;

ESI–HRMS calcd for C₁₈H₂₆N₅O₄S: 408.1706, found: m/z 408.1705 [M + H]⁺.

6-Amino-4-methyl-2-[2-oxo-2-(piperazin-1-yl)ethyl]isothiazolo[5,4-b]pyridin-3(2H)-one (27)

Compoound 25 (48 mg, 0.12 mmol) and TFA (1.9 mL, 24.40 mmol) in anhydrous CH2Cl2 (14 mL) was stirred for 2 h at room temperature. The solution was concentrated under reduced pressure and washed successively with Et2O and MeOH to give compound 27 as the TFA salt (55 mg, 86% yield). C₁₃H₁₇N₅O₂S; white powder; mp 214–216 ^oC; TLC (CH₂Cl₂/MeOH = 9:1) R_f = 0.1; IR ν_max(neat) 3414, 3335, 3228, 2924, 1680, 1648, 1598, 1543, 1444, 1375, 1340, 1249, 1203, 1145, 1026, 799, 723, 545, 465 cm^–1; ¹H NMR (400 MHz, CD₃OD) δ 6.31(1 H, s), 4.72 (2 H, s), 3.84 (4 H, s, br),

3.37–3.84 (2 H, m), 3.25–3.26 (2 H, m), 2.55 (3 H, s); ¹³C NMR (100 MHz, CD₃OD) δ 167.9 (2 ×), 166.4, 163.9, 151.0, 109.2, 108.0, 45.1, 44.5 (2 ×), 43.1, 40.2, 18.0;

ESI–HRMS calcd for C₁₃H₁₈N₅O₂S: 308.1181, found: m/z 308.1179 [M + H]⁺.

4-Nitrophenyl-4-[2-(6-amino-4-methyl-3-oxoisothiazolo[5,4-b]pyridin-2(3H)-yl)

acetyl]piperazine-1-carboxylate (28)

A mixture of compound 27 (55 mg, 0.10 mmol), 4-nitrophenyl chloroformate (22 mg, 0.11 mmol) and DMAP (37 mg, 0.30 mmol) in anhydrous DMF(0.5 mL) was stirred for 72 h at room temperature. The solution was concentrated under reduced pressure, and washed with MeOH to give brown solids. The solids were removed and the supernatant was purified by flash chromatography on a silica gel column with elution of CH₂Cl₂/MeOH (30:1 to 20:1) to give compound 28 (10 mg, 21% yield).

C₂₀H₂₀N₆O₆S; white solid; mp 232–234 ^oC; TLC (EtOAc) R_f = 0.3; IR ν_max(neat) 3321, 3211, 2923, 2852, 2486, 1731, 1638, 1593, 1523, 1442, 1422, 1344, 1028, 1158, 1109, 1018, 858, 746, 542 cm^–1; ¹H NMR (400 MHz, DMSO-d₆) δ 8.29(2 H, d, J = 8.8 Hz), 7.47 (2 H, d, J = 8.8 Hz), 6.93 (2 H, br, s), 6.22 (2 H, s), 4.67 (2 H, s), 2.46 (3 H, s) ; ¹³C NMR (100 MHz, DMSO-d₆) δ 165.3, 164.9, 164.3, 161.7, 155.9, 151.6, 147.9, 144.3, 125.0 (2 ×), 122.8 (2 ×), 107.1, 105.6, 43.8, 17.2; ESI–HRMS calcd for C₂₀H₂₁N₆O₆S:

473.1243, found: m/z 473.1237 [M + H]⁺.

Tert-butyl 4-[(6-hydroxy-4-methyl-3-oxoisothiazolo[5,4-b]pyridin-2(3H)-2-yl)

acetyl]piperazine-1-carboxylate (29)

Compound 25 (133 mg, 0.33 mmol) was added to a solution of conc. H2SO₄ (0.04 mL, 0.7 mmol) and H2O (0.5 mL) below 0^oC. The resultant solution was treated with a solution of NaNO2 (24.1 mg, 0.35 mmol) in H2O (0.5 mL) below 5 ^oC and stirred for 45 min. After then, the mixture was heated to 95^oC for 1 h and turned into transparent solution. Once cooling, the solution was neutralized by NaHCO_3(aq)to pH 6–7, and dried under reduced pressure to give pale solids.

The crude solids were then treated with di-tert-butyl dicarbonate (0.1 mL, 0.5 mmol) and DIEA (0.3 mL, 1.63 mmol) in anhydrous DMF (1 mL), and stirred at room temperature for 2 h. The solution was concentrated under reduced pressure and purified by flash chromatography on a silica gel column with elution of CH₂Cl₂/MeOH (40:1 to 9:1) to give compound 29 (10 mg, 8% yield). C₁₈H₂₄N₄O₅S; white solid; mp 195–197 ^oC;

TLC (EtOAc/hexane = 4:1) R_f = 0.1; IR ν_max(neat) 3455, 3241, 2975, 2928, 2855, 1703, 1644, 1546, 1464, 1418, 1384, 1287, 1230, 1170, 1127, 1030, 860, 765, 556 cm^–1; ¹H NMR (400 MHz, CD₃OD) δ 6.25 (1 H, s), 4.73 (2 H, s), 3.54–3.60 (6 H, m), 3.46 (2 H,

s), 2.56 (3 H, s), 1.48 (9 H, s); ¹³C NMR (100 MHz, CD3OD) δ 167.3 (2 ×), 166.4, 156.1, 153.1, 115.3, 81.8, 45.9, 45.3, 44.1 (2 ×), 43.2, 28.8 (3 ×), 18.6; ESI–HRMS calcd for C18H25N4O5S: 409.1546, found: m/z 409.1547 [M + H]⁺.

Methyl 1-(2-chloroacetyl)piperidine-4-carboxylate (31)

Chloroacetyl chloride (0.31 mL, 3.90 mmol) was added slowly to a solution of

methyl piperidine-4-carboxylate 30 (0.48 mL, 3.54 mmol) and DIEA (1.9 mL, 10.63 mmol) in anhydrous CH₂Cl₂at 0 ^oC. The mixture was stirred at room temperature for 17 h, and extracted with CH₂Cl₂ and H₂O. The organic phase was dried over MgSO₄, filtered, and purified by flash chromatography on a silica gel column with elution of EtOAc/hexane (1:2) to give compound 31 (482 mg, 62% yield). C₉H₁₄ClNO₃; yellow oil;

TLC (EtOAc/hexane = 1:2) R_f= 0.1; IR ν_max(neat) 2954, 2863, 1731, 1650, 1452, 1320, 1204, 1178, 1040, 956, 788, 654 cm^–1; ¹H NMR (400 MHz, CDCl₃) δ 4.31 (1 H, dt, J = 13.2, 3.6 Hz), 4.05 (2 H, s), 3.81 (1 H, dt, J = 7.0, 3.4 Hz), 3.69 (3 H, s), 3.20 (1 H, ddd, J = 13.6, 10.8, 2.8 Hz), 2.90 (1 H, ddd, J = 13.6, 10.8, 2.8 Hz), 2.56 (1 H, tt, J = 10.4,

4.0 Hz), 1.93–2.00 (2 H, m), 1.66–1.82 (2 H, m); ¹³C NMR (100 MHz, CDCl3) δ 174.0, 164.7, 51.8, 45.5, 41.5, 41.0, 40.4, 28.2, 27.6; ESI–HRMS calcd for C9H15ClNO3: 220.0740, found: m/z 220.0739 [M + H]⁺.

Methyl 1-(2-iodoacetyl)piperidine-4-carboxylate (32)

A solution of compound 31 (482 mg, 2.19 mmol) and sodium iodide (985 mg, 6.57 mmol) in acetone (23 mL) was stirred at room temperature for 13 h. The solids were removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was extracted with CH₂Cl₂ and H₂O. The organic phase was dried over MgSO₄, filtered, concentrated to give compound 32 (642 mg, 94% yield). C₉H₁₄INO₃; brown oil;

IR ν_max(neat) 2998, 2951, 2860, 1730, 1644, 1454, 1374, 1323, 1173, 1040, 899, 606 cm^–1; ¹H NMR (400 MHz, CDCl₃) δ 4.29–4.34 (1 H, m), 3.73 (2 H, t, J = 4.0 Hz), 3.69 (3 H, s), 3.13 (1 H, ddd, J = 13.6, 10.8, 2.8 Hz), 2.89 (1 H, ddd, J = 13.6, 10.8, 2.8 Hz), 2.6 (1 H, tt, J = 10.4, 4.0 Hz), 1.9–2.0 (2 H, m), 1.77–1.87 (1 H, m), 1.60–1.70 (2 H, m);

13C NMR (100 MHz, CDCl₃) δ 173.6, 165.6, 52.0, 46.7, 41.7, 40.7, 28.0, 27.9, –3.5;

ESI–HRMS calcd for C9H15INO3: 312.0093, found: m/z 312.0097 [M + H]⁺.

Methyl 1-[2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetyl]piperidine-4-carboxylate (33)

A mixture of BT (188 mg, 1.24 mmol), compound 32 (425 mg, 1.37 mmol) and DIEA (1.1 mL, 6.21 mmol) in anhydrous CH2Cl2 (5.6 mL) was stirred for 2 h at room temperature. The solution was concentrated under reduced pressure, and then purified by flash chromatography on a silica gel column with elution of EtOAc/hexane (1:1) to give compound 33 (360 mg, 87% yield). C₁₆H₁₈N₂O₄S; white solid; mp 148–150 ^oC; TLC (EtOAc/hexane = 1:1) R_f = 0.1; IR ν_max(neat) 2951, 2859, 1730, 1650, 1448, 1340, 1314, 1174, 1039, 783, 743, 673 cm^–1; ¹H NMR (400 MHz, CDCl₃) δ 8.02 (1 H, dd, J = 8.0, 1.0 Hz), 7.54 (1 H, dt, J = 7.2, 0.8 Hz), 7.60 (1 H, ddd, J = 8.0, 7.0, 1.0 Hz), 7.37 (1 H, ddd, J = 8.0, 7.0, 1.0 Hz), 4.69 (2 H, d, J = 8.0 Hz), 4.32–4.35 (1 H, m), 3.90 (1 H, d, J = 13.2 Hz), 3.67 (3 H, s), 3.22 (1 H, ddd, J = 13.6, 10.8, 2.8 Hz), 2.89–2.96 (1 H, m),

2.56 (1 H, tt, J = 10.4, 4.0 Hz), 1.94–1.97 (2 H, m), 1.63–1.75 (2 H, m); ¹³C NMR (100 MHz, CDCl₃) δ 173.5, 165.0, 164.1, 140.1, 131.6, 126.3, 124.9, 123.0, 119.9, 52.0, 45.0, 44.6, 41.7, 40.7, 28.5, 27.9; ESI–HRMS calcd for C₁₆H₁₉N₂O₄S: 335.1066, found: m/z

335.1067 [M + H]⁺.

1-[2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)acetyl]piperidine-4-carboxylic acid (34)

A mixture of compound 33 (100 mg, 0.30 mmol) and LiOH (18 mg, 0.44 mmol) in a mixed solvent of EtOH (0.34 mL), THF (0.34 mL) and H2O (0.17 mL) was stirred for 1 h at room temperature. The solution was neutralized with 1M HCl(aq), and then concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column with elution of CH₂Cl₂/MeOH (4:1) to give compound 34 (10 mg, 11% yield). C₁₅H₁₆N₂O₄S; white solid; mp 259–261 ^oC; TLC (CH₂Cl₂/MeOH = 4:1) R_f

= 0.3; IR ν_max(neat) 3194, 2983, 1778, 1681, 1668, 1566, 1471, 1395, 1312, 1182, 1033, 942, 575, 455 cm^–1; ¹H NMR (400 MHz, CD₃OD) δ 8.02 (1 H, d, J = 7.6 Hz), 7.40–7.43 (1 H, m), 7.26–7.34 (2 H, m), 4.25 (1 H, t, J = 15.0 Hz), 3.90 (1 H, t, J = 13.4 Hz), 3.14–3.22 (1 H, m), 2.79 (1 H, t, J = 12.0), 2.34–2.36 (1 H, m), 1.87–1.97 (2 H, m), 1.49–1.67 (2 H, m); ¹³C NMR (100 MHz, CD₃OD) δ 168.0, 167.2, 135.0, 133.8, 130.6, 130.1, 128.5, 127.7, 127.0, 53.3, 46.9, 46.7, 43.8, 43.2, 29.9; ESI–HRMS calcd for C₁₅H₁₇N₂O₄S: 321.0909, found: m/z 321.0910 [M + H]⁺.

1,4-Bis[(3-oxo-benzo[d]isothiazol-2-yl)acetyl]piperazine (35)²²

A mixture of BT (63 mg, 0.46 mmol), compound 37 (80 mg, 0.19 mmol) and DIEA (0.16 mL, 0.95 mmol) in anhydrous CH2Cl2 (2.2 mL) was stirred at room temperature for 12 h to give a suspension containing white solids. The suspension was concentrated under reduced pressure and washed with MeOH. The residual solids were collected by centrifugation, rinsed successively with CH2Cl2 and EtOAc, and dried in vacuo to give compound 35 (72 mg, 82% yield). C₂₂H₂₀N₄O₄S₂; white solid; TLC (CH₂Cl₂/MeOH = 30:1) R_f= 0.1; ¹H NMR (400 MHz, DMSO-d₆) δ 7.99 (2 H, d, J = 8.0 Hz), 7.88 (2 H, d, J = 8.0 Hz), 7.70 (2 H, t, J = 7,6 Hz), 7,43 (2 H, t, J = 7.6 Hz), 4.80–4.82 (4 H, m), 3.63

(2 H, br s), 3.57 (4 H, br s), 3.48 (2 H, br s); ¹³C NMR (100 MHz, CDCl₃) δ 165.2 (2 ×), 165.0 (2 ×), 141.4 (2 ×), 132.0 (2 ×), 125.7 (2 ×), 125.4 (2 ×), 123.5 (2 ×), 121.7 (2 ×), 44.5 (2 ×), 44.0 (2 ×); ESI–HRMS calcd for C₂₂H₂₁N₄O₄S₂: 469.1004, found: m/z 469.1003 [M + H]⁺.

1,4-Bis(chloroacetyl)piperazine (36)²²

Chloroacetyl chloride (0.63 ml, 7.31 mmol) was added slowly to a solution of piperazine (300 mg, 3.48 mmol), Et3N (1.9 ml, 13.93 mmol) in anhydrous CH2Cl2 at 0 ^oC. The mixture was then stirred at room temperature for 3 h. After washed with 1 M HCl(aq) and extracted with CH2Cl2 and H2O, the organic phase was dried over MgSO₄, filtered, and concentrated under reduced pressure to give compound 36 (419 mg, 56% yield). C₈H₁₂Cl₂N₂O₂; brown solid; ¹H NMR (400 MHz, CDCl₃) δ 4.07 (4 H, s), 3.69–3.70 (3 H, m), 3.61–3.64 (3 H, m), 3.54–3.55 (2 H, m); ¹³C NMR (100 MHz, CDCl₃) δ 165.2 (2 ×), 46.2, 45.8, 42.0, 41.7, 40.7 (2 ×); ESI–HRMS calcd for C₈H₁₃Cl₂N₂O₂: 239.0354, found: m/z 239.0356 [M + H]⁺.

1,4-Bis(iodoacetyl)piperazine (37)²²

A mixture of compound 36 (968 mg, 4.06 mmol) and sodium iodide (2.44 g, 16.25 mmol) in acetone (12 mL) was stirred at room temperature for 16 h. The solids were removed by filtration, and the solution was concentrated under reduced pressure. The residue was extracted with CH2Cl2 and H2O. The organic phase was dried over MgSO4, filtered, and concentrated to give compound 37 (1.21 g, 70% yield). C₈H₁₂I₂N₂O₂; white solid; ¹H NMR (400 MHz, CDCl₃) δ 3.76 (4 H, s), 3.72–3.74 (2 H, m), 3.62 (2 H, s), 3.57 (2 H, s) 3.44–3.47 (2 H, m); ¹³C NMR (100 MHz, CDCl₃) δ 166.2, 165.9, 46.6, 46.5, 41.7, 41.5, –3.9, –4.0; ESI–HRMS calcd for C₈H₁₃I₂N₂O₂: 422.9067, found: m/z 422.9076 [M + H]⁺.

1,4-Bis[(3-oxo-4,6-dimethylisothiazolo[5,4-b]pyridin-2-yl)acetyl]piperazine (38)

A mixture of compound 3 (83 mg, 0.46 mmol), compound 37 (89 mg, 0.21 mmol) and DIEA (0.22 mL, 1.3 mmol) in anhydrous CH2Cl2 (2.2 mL) was stirred at room temperature for 1.5 h to give a suspension containing brown solids. The suspension was

在文檔中設計與合成人類胸苷酸激酶之抑制劑 (頁 76-197)