An established inhibitor of dynamin-related protein 1 (Drp1), 3-(2,4-dichloro-5-methoxyphenyl)-2-thioxoquinazoline-4-one (mdivi-1), was recently reported also to show potent puromycin-sensitive aminopeptidase (PSA)-inhibitory activity. Herein, we report structural development of mdivi-1 derivatives and structure–activity relationship (SAR) analysis of the synthesized compounds, as well as the structurally related PSA-specific inhibitor 3-(2,6-diethylphenyl)quinazoline-2,4-dione (PAQ-22), with the aim of identifying key structural features for inhibitory activity in order to develop selective inhibitors of Drp1, which is a potential target for treatment of Huntington’s disease. Among the synthesized compounds, 3-(4-chloro-3-methoxyphenyl)-2-thioxoquinazoline-4-one (10g) exhibited more potent Drp1-inhibitory activity than mdivi-1 with high selectivity for Drp1 over PSA.

Dynamin related protein 1 (Drp1) is a member of the guanosine triphosphate hydrolase (GTPase) family. It is located mainly in cytosol, but upon activation it is recruited to the mitochondrial surface, where it oligomerizes^1–3) and bind to mitochondrial adaptors such as Fis1 and Mff to assemble a fission site, at which mitochondrial membrane fission occurs in a GTP hydrolysis-dependent manner.^4–7) Recently, Drp1 has been identified as a potential target for treatment of Huntington’s disease (HD).⁸⁾ HD is an inherited neurodegenerative disorder caused by abnormal polyglutamine (polyQ) expansion in huntingtin exon 1. PolyQ length determines disease onset and severity, with a longer expansion causing earlier onset.^9,10) Treatment of HD remains a challenge. It is reported that mutant huntingtin interacts with Drp1 and stimulates its enzymatic activity, leading to an increase of mitochondrial fragmentation and enhanced cell death.^8,11) These defects have been reported to be rescued by reducing Drp1 GTPase activity with dominant-negative mutant Drp1^K38A.^8,11) These findings suggest that Drp1 inhibitors are promising agents for treatment of HD to ameliorate neuronal cell death caused by mutant huntingtin.

To develop Drp1 inhibitors, we selected the 3-phenyl-2-thioxoquinazoline-4-one derivative mdivi-1¹²⁾ (Fig. 1, left), a known Drp1 inhibitor, as a lead compound. On the other hand, we previously developed PAQ-22 (Fig. 1, right), which has 3-phenylquinazoline-2,4-dione structure,¹³⁾ as an inhibitor of puromycin-sensitive aminopeptidase (PSA).

Fig. 1. Structures of Mdivi-1 (Left) and PAQ-22 (Right)

Since mdivi-1 is structurally closely related to PAQ-22, we anticipated that mdivi-1 might also inhibit PSA. PSA is a single-chain protein of 99 kDa that hydrolyzes N-terminal amino acids with a preference for basic and hydrophobic residues.^14–19) This enzyme is an exopeptidase containing Zn²⁺ ion at its catalytic site.^15–17) It was reported that PSA hydrolyzes polyQ chain and that PSA overexpression reduces polyQ aggregate formation as well as the toxicity of mutant huntingtin exon 1.^20,21) These facts suggested that the PSA-inhibitory activity of mdivi-1 would be inappropriate in the treatment of HD. Indeed, we found that mdivi-1 strongly inhibits not only Drp1, but also PSA.²²⁾

On the basis of these considerations, we set out to investigate the structure–activity relationship (SAR) of mdivi-1 derivatives and to conduct structural development studies targeting Drp1-selective inhibitors. In this paper, we describe the design, synthesis, and biological evaluation of a series of 2-thioxoquinazoline-4-one derivatives as Drp1-selective inhibitors.

Mdivi-1 and its oxo-analog (4) were synthesized as shown in Chart 1. 2,4-Dichloro-5-aminoanisole (1) was amidated with 2-nitrobenzoyl chloride to afford 2. The nitro group of 2 was reduced to an amino group by using H₂ in the presence of Pd/C. Annulation of 3 using CS₂ in the presence of 1,8-diazabicyclo- [5.4.0]undec-7-ene (DBU) gave the 2-thioxoquinazoline-4-one derivative mdivi-1. Annulation of 3 using triphosgene gave compounds bearing a quinazoline-2,4-dione ring.

Chart 1. Synthesis of Mdivi-1 and 4

Reagents and conditions: (a) 2-Nitrobenzoyl chloride, pyridine, DCM, r.t., 87%; (b) H₂, Pd/C, DMF, r.t., 87%; (c) CS₂, DBU, DMF, reflux, 96%; (d) Triphosgene, Et₃N, 1,2-dichloroethane, reflux, 83%.

Drp1-inhibitory activity of the synthesized compounds was evaluated by colorimetric assay using recombinant His-tagged Drp1. The assay is based on measurement of the change in light absorbance of the dye malachite green due to release of free phosphate upon hydrolysis of GTP by Drp1. As reported, mdivi-1 showed Drp1-inhibitory activity with an IC₅₀ value of 13 µM. On the other hand, its ring-opened derivatives (2 and 3) and oxo-analog (4) all lacked Drp1-inhibitory activity (Table 1). A structurally related PSA inhibitor, PAQ-22, also did not inhibit Drp1. This indicates that the 2-thioxo-quinazoline-4-one ring is essential for Drp1-inhibitory activity of mdivi-1. Therefore, we next examined the effects of substituents on the 3-phenyl ring of 2-thioxoquinazoline-4-one.

3-Substituted 2-thioxoquinazoline-4-ones were prepared as shown in Chart 2. Briefly, substituted aniline and methyl 2-isothiocyanatobenzoate were condensed in 1,4-dioxane to afford compounds 10a, and 10b (path a in Chart 2). Compounds containing chlorine, i.e., 10c–h, were prepared by annulation reaction of the corresponding anilines 9c–h with CS₂ (path d in Chart 2).

Chart 2. Synthesis of Compounds 10a–h

Reagents and conditions: (a) Methyl 2-isothiocyanatobenzoate, Et₃N, 1,4-dioxane, r.t., 76–91%; (b) 2-Nitrobenzoyl chloride, K₂CO₃, TBAHS, DCM, H₂O, r.t., 27%-q.y.; (c) 1. SOCl₂, THF, r.t., 2. Substituted aniline, pyridine, THF, r.t., 77%; (d) CS₂, DBU, DMF, 40°C, 34–60%, from 6 to 10d (34% in 2 steps) (e) SnCl₂·2H₂O, solvent, 36–65%; (f) 2-t-Butoxycarbonylaminobenzoic acid, HOBt, EDCI, DIEA, DMF, r.t.; (g) TFA, DCM, r.t., 44% from 5g in 2 steps.

Table 2 summarized the activities of the synthesized compounds. Among them, 10g and 10f showed Drp1-inhibitory activity, and 10g was about 3 times more potent than mdivi-1. Other compounds (10a–e, 10h) had no Drp1-inhibitory activity. Next, we investigated the PSA-inhibitory activity of these compounds, using the reported method.²²⁾ Briefly, PSA-inhibitory activities were assessed by measuring 7-amino-4-methylcoumarin (AMC) liberated from L-alanine 4-methylcoumaryl-7-amide (Ala-MCA) using intact human acute lymphoblastic leukemia MOLT-4 cells. As we have reported, mdivi-1 exhibited strong PSA-inhibitory activity.²²⁾ On the other hand, 10f showed only moderate PSA-inhibitory activity, and 10g exhibited little activity (inhibitory potency of only 46% at 100 µM). Compounds without an R¹ substituent, i.e., 10a, 10b, 10e, 10g and 10h, did not exhibit PSA-inhibitory activity, and this result is consistent with our previous finding that ortho-substitution of the N-phenyl ring is important for PSA-inhibitory activity.¹³⁾ Among the compounds listed in Table 2, 10g exhibited the most potent Drp1-inhibitory activity and showed high selectivity for Drp1 over PSA.

Table 2. Drp1-Inhibitory Activity of Mdivi-1 and Compounds 10a–h

Compound	R1	R2	R3	Drp1 IC50 ( µM)	PSA IC50 ( µM)
Mdivi-1	Cl	Cl	OMe	13	0.71
10a	H	H	H	N.A.a)	>100
10b	H	H	OMe	N.A.a)	30
10c	Cl	H	H	N.A.a)	44
10d	Cl	Cl	H	N.A.a)	45
10e	H	Cl	H	N.A.a)	>30
10f	Cl	H	OMe	9.6	31
10g	H	Cl	OMe	4.7	>100
10h	H	OMe	Cl	N.A.a)	>100

a) N.A. means no activity at 100 µM.

To optimize the para-substituent of the 3-phenyl ring of compound 10g, various substituents, including halogen, methyl, methoxy, nitrile, and amide were introduced at the para-position; these compounds (19a–d) were synthesized by a similar method to that used for preparation of 10a, b (Chart 3). Compounds 19e–g bearing a halogen atom were synthesized in the same manner as 10e, f, h (Chart 4).

Chart 3. Synthesis of Compounds 19a–d

Reagents and conditions: (a) MeNH₂, DMT–MM, DMF, r.t., q.y.; (b) MeI, K₂CO₃, DMF, r.t. 74%; (c) H₂, Pd/C, MeOH, r.t., 95%-q.y.; (d) Methyl 2-isothiocyanatobenzoate, Et₃N, 1,4-dioxane, r.t., 16–64%.

Chart 4. Synthesis of Compounds 19e–g

Reagents and conditions: (a) 16f; 1. NaNO₂, H₂SO₄, H₂O, 2. CuBr, HBr, 0°C to r.t., 80%, 16g; 1. NaNO₂, HCl, H₂O, 2. KI aq. 0°C to r.t., 71%; (b) 14f; Zn, AcOH, MeOH, r.t. 63%, 14g; Fe, NH₄Cl, THF/MeOH/H₂O, reflux, q.y.; (c) 2-Nitrobenzoyl chloride, K₂CO₃, TBAHS, DCM/H₂O, 0°C to r.t., from 14e to 17e (95%); (d) SnCl₂·2H₂O, AcOEt, 60°C, from 17e to 18e (80%), Zn, AcOH, MeOH, r.t. from 14f to 18f (81%), Fe, NH₄Cl, THF/MeOH/H₂O, reflux, from 14g to 18g (51%); (e) CS₂, DBU, DMF, 40°C, 42–93%.

The Drp1- and PSA-inhibitory activities of compounds 19a–g are summarized in Table 3. The Drp1-inhibitory activity of 19f was comparable to that of 10g. All compounds other than 19e and 19f lacked Drp1-inhibitory activity (Table 3). These results indicate that Cl or Br at the para-position in the phenyl group is important for potent Drp1-inhibitory activity. The lack of Drp1-inhibitory activity of 19g might be attributed to the large size of the iodine atom. The PSA-inhibitory activity of 19a–g was weaker than that of mdivi-1 (Table 3).

Table 3. Drp1-Inhibitory Activity of Compounds 10g and 19a–g

Compound	R	Drp1 IC50 ( µM)	PSA IC50 ( µM)
10g	Cl	4.7	>100
19a	CONHMe	N.A.a)	>100
19b	CN	N.A.a)	>100
19c	Me	N.A.a)	>100
19d	OMe	N.A.a)	>100
19e	F	44	>100
19f	Br	2.2	51
19g	I	N.A.a)	>100

a) N.A. means no activity at 100 µM.

Next, we optimized the alkoxy group of 10g. Compounds bearing C₂~C₄ alkyl groups (24a–d) were synthesized by a similar method to that used for preparation of 10e, f, h, as shown in Chart 5.

Chart 5. Synthesis of Compounds 24a–d

Reagents and conditions: (a) Alkyl bromide or Alkyl iodide, K₂CO₃, DMF, 60°C, 50–68%; (b) 2-Nitrobenzoyl chloride, K₂CO₃, TBAHS, DCM/H₂O, 0°C to r.t., 95%-q.y.; (c) SnCl₂·2H₂O, AcOEt, 60°C, 74–87%; (d) CS₂, DBU, DMF, 40°C, 53–98%.

The Drp1- and PSA-inhibitory activities of 24a–d are summarized in Table 4. Ethyl derivative 24a and n-propyl derivative 24b showed Drp1-inhibitory activity comparable to that of mdivi-1, but lower than that of methoxy derivative 10g. On the other hand, 24c and 24d bearing longer alkyl groups lacked Drp1-inhibitory activity. These results suggest that the methoxy group is the most effective for potent Drp1-inhibitory activity with high selectivity over PSA.

Table 4. Drp1-Inhibitory Activity of Compounds 10g and 24a–d

Compound	R	IC50 ( µM)
		Drp1	PSA
10g	Me	4.7	>100
24a	Et	14	>30b)
24b	n-Pr	10	>30b)
24c	i-Pr	N.A.a)	>100c)
24d	n-Bu	>100	>30b)

a) N.A. means no activity at 100 µM. b) Precipitation at 100 µM final DMSO concentration amounted to 11%. c) Final DMSO concentration was 11%.

Structural development of the known Drp1 inhibitor mdivi-1 afforded 10g, which has a more potent Drp1-inhibitory activity than mdivi-1, together with high selectivity for Drp1 over PSA. Since our previous work showed that a high rotational barrier of the 3-phenyl ring was necessary for PSA-inhibitory activity,¹³⁾ the weak PSA-inhibitory activity of 10g may be due to the low rotational barrier of the 3-phenyl group, as evaluated by density functional theory (DFT) calculation. A Cl or Br atom at the p-position of the 3-phenyl group is essential for Drp1-inhibitory activity, and a moderately long alkyl chain in the substituent on the O atom is also important. The structure–activity relationship of mdivi-1 derivatives is summarized in Fig. 2.

Fig. 2. Key Features in the Structure–Activity Relationship of Drp1 Inhibitors

Compound 10g should be a promising lead compound for development of highly selective Drp1 inhibitors to treat Huntington’s disease.

¹H-NMR spectra were recorded on a JEOL JNM-GX500 or JNMECA-500 (500 MHz) spectrometer. The following abbreviations are used for spin multiplicity: s=singlet, d=doublet, t=triplet, q=quartet, sep=septet, m=multiplet, br=broad. ¹³C-NMR spectra were recorded on a JEOL JNM-GX500 or JNMECA-500 spectrometer at 125 MHz. Mass spectra were recorded on a JEOL JMS-HX110 spectrometer. Melting points were determined on a MP-J3 melting point apparatus (Yanaco, Japan). Elemental analyses were carried out in the Microanalytical Laboratory, Faculty of Pharmaceutical Sciences, the University of Tokyo, and results were within ±0.3% of the theoretical values.

To a solution of 5-amino-2,4-dichloroanisole (386 mg, 2.01 mmol) and pyridine (0.1 mL) in DCM (1.0 mL) was added 2-nitrobenzoyl chloride (264 µL, 2.01 mmol) at 0°C, and the whole was stirred at r.t. After 1 h, a pale yellow solid precipitated. The precipitate was collected by filtration and washed with AcOEt to afford the title compound (595 mg, 1.74 mol, 87%) as a pale yellow solid (recrystallized from CHCl₃/n-hexane). mp 176–177°C. ¹H-NMR (500 MHz, CDCl₃) δ: 8.28 (1H, s), 8.16 (1H, d, J=8.5 Hz), 7.88 (1H, br s), 7.78 (1H, m), 7.69 (2H, m), 7.42 (1H, s), 3.98 (3H, s). Anal. Calcd for C₁₄H₁₀Cl₂N₂O₄: C, 49.29; H, 2.95; N, 8.21. Found: C, 49.28; H, 3.08; N, 8.04.

Compound 2 (341 mg, 1.00 mmol) was dissolved in DMF (5.0 mL) and hydrogenated (1 bar H₂) over 10% palladium on charcoal. The mixture was filtered through a pad of Celite. The filtrate was diluted with AcOEt and washed with water and brine. The organic layer was dried over MgSO₄ and concentrated to afford the title compound (272 mg, 0.873 mmol, 87%) as a pale yellow solid (recrystallized from CHCl₃/n-hexane). mp 127–130°C. ¹H-NMR (500 MHz, CDCl₃) δ: 8.34 (2H, m), 7.52 (1H, d, J=8.0 Hz), 7.40 (1H, s), 7.29 (1H, m), 6.75 (2H, m), 5.59 (2H, br s), 3.96 (3H, s). Anal. Calcd for C₁₄H₁₂Cl₂N₂O₂: C, 54.04; H, 3.89; N, 9.00. Found: C, 53.98; H, 3.93; N, 9.03.

To a solution of 3 (80.6 mg, 260 µmol) and CS₂ (300 µL, 5.00 mmol) in DMF (2.0 mL) was added DBU (38.7 µL, 258 µmol). The reaction mixture was stirred for 15.5 h at r.t., and then diluted with AcOEt. The organic layer was washed with 2 N HCl aq, water and brine, then dried and concentrated to afford the title compound (87.7 mg, 0.248 mmol, 96%) as a pale yellow solid (recrystallized from CHCl₃/MeOH). mp 293–294°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.98 (1H, d, J=7.5 Hz), 7.83–7.80 (1H, m), 7.78 (1H, s), 7.47–7.45 (2H, m), 7.39–7.36 (1H, m), 3.80 (3H, s). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 174.86, 158.90, 154.19, 139.64, 136.29, 136.15, 129.76, 127.48, 124.73, 123.41, 121.93, 115.88, 115.56, 115.22, 56.77. FAB-MS m/z 353 (MH⁺), 355, 357. High resolution (HR)-MS(FAB) Calcd for C₁₅H₁₀Cl₂N₂O₂S 351.9840, Found 351.9850.

Triphosgene (30.0 mg, 0.101 mmol) in 1,2-dichloroethane (10 mL) was dropped into a solution of 3 (82.6 mg, 0.265 mmol) and Et₃N (50.0 µL, 0.324 mmol) in 1,2-dichloroethane (5.0 mL) and the mixture was stirred at r.t. overnight. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (n-hexane : AcOEt=3 : 1 to 1 : 1) to afford the title compound (73.9 mg, 0.219 mmol, 83%) as a white solid. mp 277–278°C. ¹H-NMR (500 MHz, CDCl₃) δ: 8.84 (1H, s), 8.18 (1H, d, J=7.3 Hz), 7.68–7.65 (1H, m), 7.59 (1H, s), 7.31–7.28 (1H, m), 7.05 (1H, d, J=8.0 Hz), 6.92 (1H, s), 3.90 (3H, s). FAB-MS m/z 337 (MH⁺), 339. Anal. Calcd for C₁₅H₁₀Cl₂N₂O₃·1/9H₂O: C, 53.12; H, 3.04; N, 8.26. Found: C, 53.41; H, 3.34; N, 8.14.

To a solution of methyl 2-isothiocyanatobenzoate (46.7 mg, 0.242 mmol) and aniline (22.1 µL, 0.242 mmol) in 1,4-dioxane (2.0 mL) was added Et₃N (37.4 µL, 0.242 mmol). The mixture was stirred at r.t. for 2 h, and the precipitated white solid was collected by filtration, washed with n-hexane, and recrystallized from CHCl₃ and n-hexane to afford the title compound (47.0 mg, 0.185 mmol, 76%) as a white solid. mp>300°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.94 (1H, dd, J=1.4, 7.7 Hz), 7.79–7.76 (1H, m), 7.48–7.43 (3H, m), 7.41–7.38 (1H, m), 7.36–7.33 (1H, m), 7.28–7.25 (2H, m). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 176.05, 159.81, 139.40, 139.30, 135.61, 128.99, 128.89, 128.10, 127.41, 124.35, 116.20, 115.69, FAB-MS m/z 255 (MH⁺). Anal. Calcd for C₁₄H₁₀N₂OS·0.6H₂O: C, 63.42; H, 4.26; N, 10.57. Found: C, 63.34; H, 4.38; N, 10.36.

This compound was prepared from 3-methoxyaniline by a similar method to that described for preparation of 10a. Yellow solid (76.7 mg, 0.270 mmol, 91%) (recrystallized from CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 9.72 (1H, s), 8.17 (1H, d, J=8.0 Hz), 7.72–7.68 (1H, m), 7.47–7.44 (1H, m), 7.36–7.33 (1H, m), 7.11 (1H, d, J=7.9 Hz), 7.04–7.02 (1H, m), 6.88–6.87 (1H, m), 6.81 (1H, s), 3.83 (3H, s). FAB-MS m/z 285 (MH⁺). Anal. Calcd for C₁₅H₁₂N₂O₂S·H₂O: C, 59.59; H, 4.67; N, 9.27. Found: C, 59.86; H, 4.82; N, 9.07.

To a mixture of 4-chloroaniline (259 mg, 2.03 mmol), K₂CO₃ (299 mg, 2.17 mmol), and TBAHS (29.1 mg, 0.0857 mmol) in DCM : H₂O=2 : 1 was added 2-nitrobenzoyl chloride (290 µL, 2.20 mmol) at 0°C. The reaction mixture was stirred for 2 h at r.t., then CHCl₃ and MeOH were added. The organic layer was washed with water and brine, dried and concentrated to give the title compound (165 mg, 0.596 mmol, 27%) as a pale yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.12 (1H, d, J=8.5 Hz), 7.74–7.71 (1H, m), 7.64–7.62 (2H, m), 7.53 (2H, d, J=8.5 Hz), 7.45 (1H, br s), 7.33 (1H, d, J=8.5 Hz).

To a solution of 7e (165 mg, 0.596 mmol) in DMF (4.0 mL) was added SnCl₂·2H₂O (417 mg, 1.99 mmol). The mixture was stirred at 90°C for 4 h, then NaHCO₃ aq. was added and the whole was filtered through a pad of Celite. The filtrate was diluted with AcOEt and washed with water and brine. The organic layer was dried over MgSO₄ and concentrated. The residue was purified by silica gel column chromatography (n-hexane : AcOEt=3 : 1) to afford the title compound (52.3 mg, 0.212 mmol, 36%) as a yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ: 7.71 (1H, br s), 7.51 (2H, d, J=8.5 Hz), 7.43 (1H, dd, J=8.3, 1.8 Hz), 7.31 (2H, d, J=8.5 Hz), 7.26–7.23 (2H, m), 6.72–6.69 (2H, m), 5.48 (2H, br s).

This compound was prepared from 9e by a method similar to that used for preparation of mdivi-1. Yellow solid (24.9 mg, 0.0862 mmol, 34%) (recrystallized from CHCl₃/MeOH). mp>300°C. ¹H-NMR (500 MHz, CDCl₃) δ: 8.06 (1H, dd, J=8.0, 1.0 Hz), 7.66–7.62 (1H, m), 7.44–7.42 (2H, m), 7.28–7.25 (1H, m), 7.18 (1H, d, J=8.0 Hz), 7.15–7.12 (2H, m). FAB-MS m/z 289 (MH⁺). HR-MS(FAB, MH⁺) Calcd for C₁₄H₁₀ClN₂OS 289.0202, Found 289.0230.

This compound was prepared from 3-chloro-4-methoxyaniline by a method similar to that used for preparation of 7e. Yellow solid (952 mg, 3.03 mmol, quant.). ¹H-NMR (500 MHz, CDCl₃) δ: 8.08 (1H, d, J=8.0 Hz), 7.70–7.67 (1H, m), 7.59–7.57 (3H, m), 7.50 (1H, dd, J=2.5, 8.5 Hz), 6.88 (1H, d, J=9.5 Hz), 3.85 (3H, s).

This compound was prepared from 7h by a method similar to that used for preparation of 9e. Pale yellow solid (61.5 mg, 0.222 mmol, 65%). ¹H-NMR (500 MHz, CDCl₃) δ: 7.63 (1H, d, J=2.0 Hz), 7.62 (1H, br s), 7.42 (1H, d, J=7.5 Hz), 7.40 (1H, dd, J=2.5, 9.0 Hz), 7.26–7.22 (1H, m), 6.91 (1H, d, J=8.5 Hz), 6.71–6.68 (1H, m), 5.49 (2H, br s), 3.89 (3H, s).

This compound was prepared from 9h by a method similar to that used for preparation of mdivi-1. Yellow solid (74.8 mg, 0.235 mmol, 60%) (recrystallized from CHCl₃/MeOH). mp>300°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.94 (1H, dd, J=1.2, 8.0 Hz), 7.79–7.76 (1H, m), 7.44–7.43 (2H, m), 7.35–7.32 (1H, m), 7.24 (1H, dd, J=2.0, 8.9 Hz), 7.22 (1H, d, J=8.6 Hz), 3.91 (3H, s). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 176.23, 159.91, 154.20, 139.54, 135.60, 132.22, 130.40, 129.06, 127.42, 124.36, 120.62, 116.23, 115.68, 112.55, 56.27. FAB-MS m/z 319 (MH⁺), 321. HR-MS(FAB) Calcd for C₁₅H₁₁ClN₂O₂S 318.0261, Found 318.0261.

This compound was prepared from 2-chloro-5-methoxyaniline hydrochloride by a method similar to that used for preparation of 7e. Yellow solid (288 mg, 0.940 mmol, 88%). ¹H-NMR (500 MHz, CDCl₃) δ: 8.15–8.13 (2H, m), 7.91 (1H, br s), 7.76–7.75 (1H, m), 7.69–7.67 (2H, m), 7.28 (1H, d, J=8.6 Hz), 6.69 (1H, dd, J=3.1, 8.6 Hz), 3.85 (3H, s).

This compound was prepared from 7f by a method similar to that used for preparation of 9e. Yellow solid (130 mg, 0.470 mmol, 52%). ¹H-NMR (500 MHz, CDCl₃) δ: 8.35 (1H, s), 8.18 (1H, d, J=2.7 Hz), 7.53 (1H, d, J=8.0 Hz), 7.29–7.27 (1H, m), 6.75–6.73 (2H, m), 7.16 (1H, dd, J=2.7, 9.0 Hz), 5.59 (br s, 2H), 3.84 (s, 3H).

This compound was prepared from 9f by a method similar to that used for preparation of mdivi-1. Yellow solid (111 mg, 0.349 mmol, 95%) (recrystallized from 1,4-dioxane/DMF/H₂O). mp 232–234°C. ¹H-NMR (500 MHz, CDCl₃) δ: 9.88 (1H, br s), 8.18 (1H, d, J=7.9 Hz), 7.72–7.69 (1H, m), 7.47 (1H, d, J=9.2 Hz), 7.37–7.34 (1H, m), 7.13 (1H, d, J=7.9 Hz), 6.99 (1H, dd, J=3.1, 9.2 Hz), 6.88 (1H, d, J=2.5 Hz), 3.82 (3H, s). FAB-MS m/z 319 (MH⁺). Anal. Calcd for C₁₅H₁₁ClN₂O₂S·0.25H₂O: C, 55.73; H, 3.59; N, 8.67. Found: C, 55.84; H, 3.48; N, 8.64.

To a solution of anthranilic acid (147 mg, 1.07 mmol) in THF (10 mL) was added SOCl₂ (2.0 mL). The mixture was stirred at r.t. for 1 h, and then 2-chloroaniline (255 mg, 2.00 mmol) in pyridine (1.0 mL) was added. The whole was stirred at r.t. overnight, then diluted with AcOEt and the organic layer was washed with H₂O and brine, dried and concentrated. Column chromatography (n-hexane : AcOEt=5 : 1 to 3 : 1) of the residue gave the title compound (203 mg, 0.821 mmol, 76%) as a yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.45 (1H, d, J=7.9 Hz), 8.34 (1H, br s), 7.54 (1H, d, J=8.0 Hz), 7.41 (1H, d, J=7.9 Hz), 7.34–7.27 (2H, m), 7.09–7.06 (1H, m), 6.76–6.73 (1H, m), 5.60 (2H, br s). FAB-MS m/z 246 (M⁺), 247 (MH⁺).

This compound was prepared from 9c by a method similar to that used for preparation of mdivi-1. Yellow solid (38.7 mg, 0.134 mmol, 50%). mp 247–251°C. ¹H-NMR (500 MHz, CDCl₃) δ: 10.19 (1H, br s), 8.18 (1H, d, J=7.9 Hz), 7.72–7.69 (1H, m), 7.61–7.59 (1H, m), 7.48–7.44 (2H, m), 7.37–7.35 (2H, m), 7.16 (1H, d, J=8.0 Hz). FAB-MS m/z 289 (MH⁺). Anal. Calcd for C₁₄H₉ClN₂OS: C, 58.23; H, 3.14; N, 9.70. Found: C, 58.06; H, 3.14; N, 9.70.

To a solution of anthranilic acid (147 mg, 1.07 mmol) in Et₂O (5.0 mL) was added SOCl₂ (2.0 mL). The mixture was stirred at r.t. for 1 h, then 2,4-dichloroaniline (337 mg, 2.08 mmol) in pyridine (1.0 mL) was added. The whole was stirred at r.t. overnight and then diluted with AcOEt. The organic layer was washed with H₂O and brine, dried and concentrated to afford the title compound, which was used for next step without further purification.

This compound was prepared from 9d by a method similar to that used for preparation of mdivi-1. Yellow solid (118 mg, 0.365 mmol, 34% from anthracitic acid). mp 256–260°C, ¹H-NMR (500 MHz, CDCl₃) δ: 9.96 (1H, br s), 8.17 (1H, d, J=8.0 Hz), 7.73–7.70 (1H, m), 7.60 (1H, d, J=1.9 Hz), 7.43 (1H, dd, J=2.5, 8.6 Hz), 7.38–7.35 (1H, m), 7.28 (1H, d, J=8.6 Hz), 7.14 (1H, d, J=8.0 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ: 175.65, 159.13, 138.83, 136.13, 135.83, 134.73, 133.47, 131.18, 130.47, 128.96, 128.46, 125.37, 115.98, 114.95. FAB-MS: not detected. Anal. Calcd for C₁₄H₈Cl₂N₂OS·0.2H₂O: C, 51.45; H, 2.59; N, 8.57. Found: C, 51.39; H, 2.80; N, 8.51.

To a solution of 2-t-butoxycarbonylaminobenzoic acid (115 mg, 0.485 mmol) in DMF (2.0 mL) was added a solution of 4-chloro-3-methoxyaniline (82.2 mg, 0.522 mmol), HOBt (70.0 mg, 0.518 mmol), EDCI·HCl (104 mg, 0.542 mmol) and DIEA (90.0 µL, 0.522 mmol) in DMF (1.0 mL). The resulting mixture was stirred at r.t. overnight. After completion of the reaction, AcOEt was added. The organic layer was washed with water and brine, dried, and concentrated to give the title compound as a pale yellow paste, which was used for the next step without further purification.

To a solution of 8 (96.0 mg, crude) in DCM (2.0 mL) was slowly added TFA (250 µL) at r.t. The mixture was stirred for 4.5 h, and then NaHCO₃ aq. and AcOEt were added to it. The organic layer was washed with water and brine, dried and concentrated to afford the title compound as a yellow paste (61.5 mg, 0.222 mmol, 87%). This compound was used for the next step without further purification. ¹H-NMR (500 MHz, CDCl₃) δ: 7.75 (1H, br s), 7.57 (1H, d, J=1.9 Hz), 7.46 (1H, d, J=7.9 Hz), 7.32–7.31 (2H, m), 6.90 (1H, dd, J=1.9, 8.6 Hz), 6.74–6.71 (1H, m), 5.49 (2H, br s), 3.95 (3H, s).

This compound was prepared from 9g by a method similar to that used for preparation of mdivi-1. Yellow solid (41.4 mg, 0.130 mmol, 59%) (recrystallized from DMF/H₂O). mp>300°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.95 (1H, dd, J=1.4, 7.8 Hz), 7.80–7.76 (1H, m), 7.51 (1H, d, J=8.0 Hz), 7.44 (1H, d, J=8.1 Hz), 7.36–7.33 (1H, m), 7.19 (1H, d, J=1.7 Hz), 6.91 (1H, dd, J=2.3, 8.6 Hz), 3.79 (3H, s). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 175.85, 159.72, 154.93, 139.64, 139.32, 135.67, 129.81, 127.41, 124.38, 122.27, 120.69, 116.22, 115.73, 113.98, 56.31. FAB-MS m/z 319 (MH⁺). HR-MS(FAB) Calcd for C₁₅H₁₁ClN₂O₂S 318.0230, Found 318.0274

To a solution of 2-methoxy-4-nitrobenzoic acid (202 mg, 1.03 mmol) and aqueous MeNH₂ (125 µL, 1.50 mmol) in DMF (1.0 mL) was added DMT–MM (430 mg, 1.55 mmol). The whole was stirred at r.t. overnight and then diluted with AcOEt. The organic layer was washed with water and brine, dried and concentrated to give the title compound (225 mg, 1.03 mmol, quant.) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.35 (1H, d, J=8.6 Hz), 7.89 (1H, dd, J=2.3, 8.8 Hz), 7.81 (1H, d, J=1.8 Hz), 7.70 (1H, br s), 4.05 (3H, s), 3.01 (3H, d, J=4.6 Hz).

To a solution of 2-methyl-5-nitrophenol (309 mg, 2.02 mmol) and K₂CO₃ (400 mg, 2.90 mmol) in DMF (4.0 mL) was added MeI (855 mg, 6.02 mmol). The reaction mixture was stirred for 9 h at r.t. and then diluted with AcOEt. The organic layer was washed with water and brine, dried over MgSO₄ and concentrated to give compound 13c as a dark yellow solid (251 mg, 1.50 mmol, 74%), which was used for the next step without further purification. ¹H-NMR (500 MHz, CDCl₃) δ: 7.74 (1H, dd, J=2.0, 8.0 Hz), 7.64 (1H, d, J=2.0 Hz), 7.23 (1H, d, J=8.0 Hz), 3.90 (3H, s), 2.28 (3H, s).

Compound 13a (221 mg, 1.05 mmol) was dissolved in MeOH (8.0 mL) and hydrogenated (1 bar H₂) over 10% palladium on charcoal for 5.5 h at r.t. The mixture was filtered through a pad of Celite and the filtrate was concentrated. The residue was recrystallized from DCM and n-hexane to afford the title compound (193 mg, 1.05 mmol, quant.) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.02 (1H, d, J=8.6 Hz), 7.63 (1H, br s), 6.32 (1H, dd, J=2.3, 8.6 Hz), 6.18 (1H, d, J=2.3 Hz), 4.03 (2H, d, J=4.6 Hz), 3.88 (3H, s), 2.95 (3H, d, J=4.6 Hz).

This compound was prepared from 2-methoxy-4-nitrobenzonitrile by a method similar to that used for preparation of 14a. White solid (290 mg, 1.95 mmol, 96%). ¹H-NMR (500 MHz, CDCl₃) δ: 7.26 (1H, d, J=8.6 Hz), 7.23 (1H, s), 6.19 (1H, dd, J=2.3, 8.7 Hz), 6.12 (1H, d, J=2.3 Hz), 4.11 (2H, br s), 3.82 (3H, s).

This compound was prepared from 13c by a method similar to that used for preparation of 14a. Brown solid (195 mg, 1.42 mmol, 95%). ¹H-NMR (500 MHz, CDCl₃) δ: 6.87 (1H, d, J=7.5), 6.21–6.19 (2H, m), 3.90 (3H, s), 3.53 (2H, br s), 2.08 (3H, s), FAB-MS m/z 138 (MH)⁺.

To a solution of methyl 2-isothiocyanatobenzoate (65.4 mg, 0.338 mmol) and 14a (62.4 mg, 0.346 mmol) in dioxane (2.0 mL) was added Et₃N (80 µL, mmol). The mixture was stirred at r.t. for 10 h, and the precipitated white solid was collected by filtration and washed with n-hexane to give the title compound (18.9 mg, 0.0541 mmol, 16%) as a white solid (recrystallized from CHCl₃/MeOH/n-hexane). mp>300°C. ¹H-NMR (500 MHz, CDCl₃) δ: 8.24 (1H, d, J=8.6 Hz), 8.05 (1H, dd, J=1.2, 8.0 Hz), 7.86 (1H, d, J=4.6 Hz), 7.65–7.61 (1H, m), 7.27–7.24 (1H, m), 7.20–7.17 (1H, m), 6.91 (1H, dd, J=1.7, 8.0 Hz), 6.82 (1H, d, J=2.3 Hz), 3.89 (3H, s), 2.94 (3H, d, J=4.6 Hz). FAB-MS m/z 342 (MH⁺). Anal. Calcd for C₁₇H₁₅N₃O₃S·0.3H₂O: C, 58.88; H, 4.53; N, 12.12. Found: C, 58.61; H, 4.64; N, 11.96.

To a solution of methyl 2-isothiocyanatobenzoate (98.8 mg, 0.511 mmol) and 14b (74.0 mg, 0.499 mmol) in dioxane (2.0 mL) was added Et₃N (80 µL, mmol). The mixture was stirred at r.t. for 10 h, and the precipitated white solid was collected by filtration and washed with n-hexane to give the title compound (18.9 mg, 0.0818 mmol, 16%) as a white solid. mp>300°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.98 (1H, dd, J=1.2, 8.0 Hz), 7.88 (1H, d, J=8.1 Hz), 7.83–7.80 (1H, m), 7.47 (1H, d, J=8.0 Hz), 7.39–7.36 (2H, m), 7.12 (1H, dd, J=1.7, 8.0 Hz), 3.89 (3H, s). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 175.30, 161.55, 159.60, 145.42, 139.87, 135.73, 133.91, 127.33, 124.40, 122.48, 116.24, 116.09, 115.94, 113.78, 99.97, 56.63. FAB-MS m/z 310 (MH⁺). Anal. Calcd for C₁₆H₁₁N₃O₂S: C, 62.12; H, 3.58; N, 13.58. Found: C, 61.98; H, 3.66; N, 13.47.

This compound was prepared from 14c by a method similar to that used for preparation of 10a. White solid (71.0 mg, 0.238 mmol, 43%) (recrystallized from DMF/H₂O). mp>300°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.94 (1H, dd, J=1.2, 8.1 Hz), 7.78–7.75 (1H, m), 7.43 (1H, d, J=8.0 Hz), 7.35–7.32 (1H, m), 7.20 (1H, d, J=8.0 Hz), 6.89 (1H, d, J=1.7 Hz), 6.74 (1H, dd, J=1.7, 8.0 Hz), 3.72 (3H, s), 2.19 (3H, s). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 176.14, 159.77, 157.62, 139.57, 138.10, 135.57, 130.21, 127.41, 125.40, 124.31, 120.60, 116.19, 115.63, 111.17, 55.40, 15.87, FAB-MS m/z 315 (MH)⁺. HR-MS(FAB) Calcd for C₁₆H₁₄N₂O₂S 298.0776, Found 298.0771.

This compound was prepared from 3,4-dimethoxyaniline by a method similar to that used for preparation of 10a. Brown solid (114 mg, 0.363 mmol, 64%). mp 292–293°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.96 (1H, dd, J=1.2, 8.0 Hz), 7.80–7.77 (1H, m), 7.45 (1H, d, J=8.1 Hz), 7.37–7.33 (1H, m), 7.03 (1H, d, J=8.6 Hz), 6.93 (1H, d, J=2.3 Hz), 6.80 (1H, dd, J=2.3, 8.0 Hz), 3.82 (3H, s), 3.71 (3H, s). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 176.38, 159.92, 148.90, 148.38, 139.63, 135.50, 132.04, 127.41, 124.25, 121.05, 116.21, 115.69, 112.76, 111.39, 55.62, 55.55. FAB-MS m/z 315 (MH)⁺. HR-MS(FAB) Calcd for C₁₆H₁₄N₂O₃S 314.0725, Found 314.0715.

H₂SO₄ (1.5 mL) was added slowly to a cold (0°C) stirred mixture of 2-methoxy-4-nitrobenzenamine (340 mg. 2.02 mmol) and NaNO₂ (186 mg, 2.69 mmol) in H₂O (4.0 mL). After the addition was completed, stirring was continued for an additional 30 min at 0°C. Then a solution of CuBr (345 mg, 2.40 mmol) in HBr (1.5 mL) was added slowly at 0°C. The whole was stirred at r.t. overnight and diluted with CHCl₃. The organic layer was washed with water and brine, dried and concentrated. Column chromatography (n-hexane : AcOEt=3 : 1) gave the title compound (358 mg, 1.62 mmol, 80%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ: 7.72–7.67 (3H, m), 3.97 (3H, s).

To a mixture of 16f (358 mg, 1.61 mmol) and Zn powder (697 mg, 10.66 mmol) in MeOH (16 mL) was added AcOH (4.8 mL). The whole was stirred at r.t. for 4.5 h and then extracted with AcOEt three times. The organic layer was combined, washed with water and brine, dried and concentrated. Column chromatography (n-hexane : AcOEt=3 : 1 to 2 : 1) of the residue gave the title compound (205 mg, 1.01 mmol, 63%) as a yellow paste.

HCl (2.0 mL) was added slowly to a cold (0°C) stirred solution of 2-methoxy-4-nitrobenzenamine (343 mg. 2.04 mmol) and NaNO₂ (170 mg, 2.46 mmol) in H₂O (5.0 mL). After the addition was completed, the solution was stirred for a further 30 min at 0°C. Then a solution of KI (524 mg, 3.16 mmol) in H₂O (2.0 mL) was added slowly at 0°C. The whole was stirred at r.t. overnight and quenched with CHCl₃. The organic layer was washed with Na₂S₂O₃ aq., water and brine, dried and concentrated. Column chromatography (n-hexane : AcOEt=5 : 1 to 3 : 1) gave the title compound (404 mg, 1.45 mol, 71%) as a pale yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ: 7.96 (1H, d, J=8.2 Hz), 7.62 (1H, d, J=2.8 Hz), 7.59 (1H, dd, J=2.8, 8.2 Hz), 3.99 (3H, s).

To a mixture of 16g (404 mg, 1.45 mmol) and NH₄Cl (130 mg, 2.43 mmol) in MeOH (7.0 mL), THF (7.0 mL) and H₂O (3.5 mL) was added Fe powder (443 mg, 7.93 mmol). The whole was stirred at 80°C for 4.5 h and then the reaction was quenched with AcOEt and H₂O. The resulting mixture was filtered through a pad of Celite and the filtrate was extracted with AcOEt three times. The organic layers were combined, washed with water and brine, dried and concentrated. Column chromatography (n-hexane : AcOEt=3 : 1 to 2 : 1) gave the title compound (372 mg, 1.45 mmol, quant.) as a brown paste. ¹H-NMR (500 MHz, CDCl₃) δ: 7.44 (1H, d, J=8.5 Hz), 6.21 (1H, d, J=2.8 Hz), 6.11 (1H, dd, J=2.8, 8.5 Hz), 3.81 (3H, s), 3.72 (2H, br s). FAB-MS m/z 249 (MH⁺).

To a mixture of 4-fluoro-3-methoxyaniline (142 mg, 1.01 mmol), K₂CO₃ (157 mg, 1.14 mmol) and TBAHS (19.2 mg, 0.0565 mmol) in DCM : H₂O=2 : 1 was added 2-nitrobenzoyl chloride (162 µL, 1.10 mmol) at 0°C. The reaction mixture was stirred at r.t. overnight, and then CHCl₃ was added to it. The organic layer was washed with water and brine, dried and concentrated to give the title compound (277 mg, 0.960 mmol, 95%) as a pale yellow solid.¹H-NMR (500 MHz, CDCl₃) δ: 8.11 (1H, d, J=8.0 Hz), 7.73–7.70 (1H, m), 7.62–7.61 (2H, m), 7.53–7.52 (1H, m), 7.43 (1H, s), 7.02 (1H, dd, J=8.6, 10.9 Hz), 6.86–6.83 (1H, m), 3.90 (3H, s).

To a solution of 17e (277 mg, 0.955 mmol) in AcOEt (5.0 mL) was added SnCl₂·2H₂O (690 mg, 2.92 mmol). The mixture was stirred at 60°C for 3 h, and then NaHCO₃ aq. was added. The whole was filtered through a pad of Celite, and the filtrate was diluted with AcOEt and then washed with water and brine. The organic layer was dried and concentrated. The residue was purified by column chromatography (n-hexane : AcOEt=3 : 1) to afford the title compound (198 mg, 0.764 mmol, 80%) as a pale yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ: 7.71 (1H, s), 7.47 (1H, dd, J=2.6, 7.7 Hz), 7.25–7.21 (1H, m), 7.01 (1H, dd, J=8.9, 11.2 Hz), 6.85–6.82 (1H, m), 6.70–6.67 (2H, m), 5.45 (2H, br s), 3.88 (3H, s).

To a mixture of 14f (203 mg, 1.00 mmol), K₂CO₃ (281 mg, 1.18 mmol) and TBAHS (21.8 mg, 0.0642 mmol) in DCM : H₂O=2 : 1 was added 2-nitrobenzoyl chloride (191 µL, 1.30 mmol) at 0°C. The mixture was stirred at r.t. overnight, and then the reaction was quenched with CHCl₃. The organic layer was washed with water and brine, dried, and concentrated (17f). To a suspension of 17f and Zn powder (421 mg, 6.44 mmol) in MeOH (10 mL) was added AcOH (3.0 mL). The whole was stirred at r.t. for 3 h and then diluted with AcOEt. The organic layer was washed with water and brine, dried and concentrated. Column chromatography (n-hexane : AcOEt=3 : 1 to 2 : 1) gave the title compound (262 mg, 0.816 mmol, 81% for 2 steps) as a yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ: 7.76 (1H, br s), 7.54 (1H, d, J=2.3 Hz), 7.48–7.44 (1H, m), 7.27 (1H, dd, J=1.2, 7.5 Hz), 6.73–6.70 (2H, m), 5.49 (2H, br s), 3.93 (3H, s).

To a mixture of 14g (366 mg, 1.47 mmol), K₂CO₃ (218 mg, 1.58 mmol) and TBAHS (29.1 mg, 0.0857 mmol) in DCM : H₂O=2 : 1 was added 2-nitrobenzoyl chloride (297 µL, 2.03 mmol) at 0°C. The reaction mixture was stirred at r.t. overnight, and then quenched with CHCl₃. The organic layer was washed with water and brine, dried, and concentrated (17g). To a suspension of 17g and NH₄Cl (85.6 mg, 1.60 mmol) in MeOH (7.0 mL), THF (5.0 mL), H₂O (3.0 mL) was added Fe powder (363 mg, 6.50 mmol). The whole was stirred at 80°C for 5 h and then diluted with AcOEt and H₂O. The resulting mixture was filtered through a pad of Celite. The combined organic layer was washed with water and brine, dried and concentrated. Column chromatography (n-hexane : AcOEt=5 : 1 to 3 : 1) gave the title compound (276 mg, 0.750 mmol, 51% for 2 steps) as a yellow paste. ¹H-NMR (500 MHz, CDCl₃) δ: 7.78 (1H, br s), 7.68 (1H, d, J=8.6 Hz), 7.47–7.44 (2H, m), 7.28–7.23 (2H, m), 6.76 (1H, dd, J=2.3, 8.0 Hz), 6.72–6.69 (2H, m), 5.48 (2H, br s), 3.91 (3H, s).

This compound was prepared from 18e by a method similar to that used for preparation of mdivi-1. White solid (18.9 mg, 61.1 µmol, 16%) (recrystallized from DMF/H₂O). mp>300°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.95 (1H, dd, J=1.4, 7.7 Hz), 7.79–7.76 (1H, m), 7.44 (1H, d, J=8.1 Hz), 7.36–7.28 (1H, m), 7.19 (1H, dd, J=2.6, 7.7 Hz), 6.89–6.86 (1H, m), 3.78 (3H, s). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 176.08, 159.77, 151.97, 150.03, 147.45, 147.36, 139.55, 135.73, 135.62, 127.39, 124.35, 121.66, 121.61, 116.19, 115.87, 115.71, 115.65, 115.02, 56.16. FAB-MS m/z 303 (MH⁺). Anal. Calcd for C₁₅H₁₁FN₂O₂S·0.5H₂O: C, 57.87; H, 3.89; N, 9.00. Found: C, 58.11; H, 4.03; N, 9.20.

This compound was prepared from 18f by a method similar to that used for preparation of mdivi-1. Yellow solid (61.6 mg, 0.169 mmol, 42%) (recrystallized from DMF/H₂O). mp>300°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.95 (1H, d, J=8.1 Hz), 7.80–7.77 (1H, m), 7.67 (1H, d, J=8.6 Hz), 7.44 (1H, d, J=8.1 Hz), 7.37–7.33 (1H, m), 7.16 (1H, d, J=1.9 Hz), 6.86 (1H, dd, J=1.9, 8.3 Hz), 3.79 (3H, s). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 175.78, 159.67, 155.90, 140.01, 139.60, 135.67, 132.84, 127.41, 124.40, 122.80, 116.21, 115.69, 113.89, 110.27, 56.43. Anal. Calcd for C₁₅H₁₁BrN₂O₂S·0.8H₂O: C, 47.71; H, 3.36; N, 7.42. Found: C, 47.62; H, 3.11; N, 7.31.

This compound was prepared from 18g by a method similar to that used for preparation of mdivi-1. Yellow solid (121 mg, 0.295 mmol, 93%) (recrystallized from n-hexane/AcOEt/MeOH). mp>300°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.95 (1H, dd, J=1.2, 8.0 Hz), 7.85 (1H, d, J=8.1 Hz), 7.79–7.76 (1H, m), 7.44 (1H, d, J=8.0 Hz), 7.37–7.33 (1H, m), 7.04 (1H, d, J=2.3 Hz), 6.71 (1H, dd, J=2.0, 8.3 Hz), 3.76 (3H, s). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 175.74, 159.64, 158.36, 140.90, 139.59, 138.82, 135.65, 127.39, 124.37, 123.36, 116.18, 115.68, 112.79, 85.81, 56.55, FAB-MS m/z 411 (MH⁺). HR-MS(FAB) Calcd for C₁₅H₁₁IN₂O₂S 409.9586, Found 409.9594.

To a mixture of 5-amino-2-chlorophenol (148 mg, 1.03 mmol) and K₂CO₃ (296 mg, 2.14 mmol) in DMF (2.0 mL) was added EtI (100 µL, 1.25 mmol). The whole was stirred at 60°C for 2.5 h and then diluted with AcOEt. The organic layer was washed with water and brine, dried and concentrated. Column chromatography (n-hexane : AcOEt=3 : 1) of the residue gave the title compound (88.5 mg, 0.515 mmol, 50%) as a pale yellow paste. ¹H-NMR (500 MHz, CDCl₃) δ: 7.07 (1H, d, J=8.6 Hz), 6.25 (1H, d, J=2.9 Hz), 6.19 (1H, dd, J=2.6, 8.3 Hz), 4.02 (2H, q, J=7.0 Hz), 3.63 (2H, br s), 1.43 (3H, t, J=7.0 Hz).

This compound was prepared from 5-amino-2-chlorophenol and n-PrI by a method similar to that used for preparation of 21a. Pale yellow paste (107 mg, 0.572 mmol, 54%). ¹H-NMR (500 MHz, CDCl₃) δ: 7.07 (1H, d, J=8.0 Hz), 6.25 (1H, d, J=2.3 Hz), 6.19 (1H, dd, J=2.6, 8.3 Hz), 3.90 (1H, d, J=6.6 Hz), 3.63 (2H, br s), 1.83 (1H, qt, J=7.5, 6.6 Hz), 1.04, (3H, t, J=7.5 Hz).

This compound was prepared from 5-amino-2-chlorophenol and i-PrBr by a method similar to that used for preparation of 21a. Yellow paste (110 mg, 0.592 mmol, 58%). ¹H-NMR (500 MHz, CDCl₃) δ: 7.06 (1H, d, J=8.6 Hz), 6.27 (1H, d, J=2.3 Hz), 6.19 (1H, dd, J=2.3, 8.6 Hz), 4.44 (1H, sep, J=6.3 Hz), 3.60 (2H, br s), 1.33 (6H, d, J=6.3 Hz).

This compound was prepared from 5-amino-2-chlorophenol and n-BuI by a method similar to that used for preparation of 21a. Yellow paste (141 mg, 0.700 mmol, 68%). ¹H-NMR (500 MHz, CDCl₃) δ: 7.05 (1H, d, J=8.6 Hz), 6.23 (1H, d, J=2.3 Hz), 6.17 (1H, dd, J=2.6, 8.3 Hz), 3.93 (2H, t, J=6.6 Hz), 3.61 (2H, br s), 1.80–1.74 (2H, m), 1.52–1.45 (2H, m), 0.94 (3H, t, J=7.5 Hz).

This compound was prepared from 21a by a method similar to that used for preparation of 7e. Pale yellow solid (153 mg, 0.474 mmol, 95%). ¹H-NMR (500 MHz, CDCl₃/CD₃OD) δ: 8.02–7.99 (1H, m), 7.63–7.59 (1H, m), 7.51–7.49 (2H, m), 7.43–7.42 (1H, m), 7.16–7.13 (1H, m), 6.87 (1H, dd, J=2.3, 8.6 Hz), 3.90 (1H, d, J=6.6 Hz), 4.01 (2H, q, J=6.8 Hz), 1.33, (3H, t, J=6.8 Hz).

This compound was prepared from 21b by a method similar to that used for preparation of 7e. Pale yellow solid (187 mg, 0.560 mmol, 97%). ¹H-NMR (500 MHz, CDCl₃/CD₃OD) δ: 8.00 (1H, d, J=8.1 Hz), 7.63–7.60 (1H, m), 7.51–7.49 (1H, m), 7.44 (1H, d, J=2.3 Hz), 7.14 (1H, d, J=8.6 Hz), 6.86 (1H, dd, J=2.3, 8.6 Hz), 3.90–3.87 (2H, m), 1.75–1.71 (2H, m), 0.93 (3H, t, J=7.4 Hz).

This compound was prepared from 21c by a method similar to that used for preparation of 7e. Pale yellow solid (199 mg, 0.568 mmol, quant.). ¹H-NMR (500 MHz, CDCl₃) δ: 8.11 (1H, d, J=8.0 Hz), 7.73–7.71 (1H, m), 7.64–7.61 (1H, m), 7.54 (1H, d, J=2.3 Hz), 7.44 (1H, br s), 7.29 (1H, d, J=8.6 Hz), 6.86 (1H, dd, J=2.3, 8.6 Hz), 3.90 (1H, d, J=6.5 Hz), 1.38 (6H, d, J=6.5 Hz).

This compound was prepared from 22d by a method similar to that used for preparation of 7e. Yellow solid (234 mg, 0.670 mmol, 98%). ¹H-NMR (500 MHz, CDCl₃) δ: 8.07 (1H, d, J=8.0 Hz), 7.70–7.67 (1H, m), 6.19 (1H, dd, J=2.6, 8.3 Hz), 3.90 (1H, d, J=6.6 Hz), 3.63 (1H, br s), 1.83 (1H, qt, J=7.5, 6.6 Hz), 1.04, (3H, t, J=7.5 Hz).

To a solution of 22a (149 mg, 0.464 mmol) in AcOEt (4.0 mL) was added SnCl₂·2H₂O (334 mg, 1.48 mmol). The reaction mixture was stirred at 60°C for 4.5 h, then quenched with NaHCO₃ aq. and filtered through a pad of Celite. The filtrate was diluted with AcOEt and washed with water and brine). The organic layer was dried and concentrated. Column chromatography (n-hexane : AcOEt=3 : 1) gave the title compound (108 mg, 0.371 mmol, 80%) as a pale yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ: 7.71 (1H, br s), 7.49 (1H, d, J=2.3 Hz), 7.43–7.41 (1H, m), 7.27 (1H, d, J=8.6 Hz), 7.25–7.22 (1H, m), 6.85 (1H, dd, J=2.3, 8.6 Hz), 6.70–6.67 (2H, m), 5.45 (2H, br s), 4.12 (2H, q, J=6.9 Hz), 1.45 (3H, t, J=6.9 Hz).

This compound was prepared from 22b by a method similar to that used for preparation of 23a. Pale yellow paste (146 mg, 0.479 mmol, 87%). ¹H-NMR (500 MHz, CDCl₃) δ: 7.71 (1H, br s), 7.48 (1H, d, J=2.3 Hz), 7.42–7.41 (1H, m), 7.27 (1H, d, J=8.6 Hz), 7.25–7.21 (1H, m), 6.85 (1H, dd, J=2.6, 8.3 Hz), 6.70–6.67 (2H, m), 5.45 (2H, br s), 4.00 (2H, t, J=6.8 Hz), 1.85 (2H, qt, J=6.8, 7.4 Hz), 1.04 (3H, t, J=7.4 Hz).

This compound was prepared from 22c by a method similar to that used for preparation of 23a. Yellow solid (144 mg, 0.474 mmol, 80%). ¹H-NMR (500 MHz, CDCl₃) δ: 7.70 (1H, br s), 7.49 (1H, d, J=2.3 Hz), 7.43–7.41 (1H, m), 7.27 (1H, d, J=8.6 Hz), 7.25–7.21 (1H, m), 6.86 (1H, dd, J=2.3, 8.6 Hz), 6.70–6.67 (2H, m), 5.45 (2H, br s), 4.57 (1H, sep, J=5.9 Hz), 1.37 (6H, d, J=5.9 Hz).

This compound was prepared from 22d by a method similar to that used for preparation of 23a. Yellow solid (158 mg, 0.497 mmol, 74%). ¹H-NMR (500 MHz, CDCl₃) δ: 7.71 (1H, br s), 7.47 (1H, d, J=2.3 Hz), 7.42–7.41 (1H, m), 7.26 (1H, d, J=8.6 Hz), 7.25–7.21 (1H, m), 6.85 (1H, dd, J=2.3, 8.6 Hz), 6.70–6.67 (2H, m), 5.45 (2H, br s), 4.03 (2H, t, J=6.3 Hz), 1.83–1.78 (2H, m), 1.54–1.47 (2H, m), 0.96 (3H, t, J=7.4 Hz).

This compound was prepared from 23a by a method similar to that used for preparation of mdivi-1. White solid (47.8 mg, 0.143 mmol, 61%) (recrystallized from DMF/H₂O). mp>300°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.95 (1H, dd, J=1.2, 8.0 Hz), 7.78–7.76 (1H, m), 7.50 (1H, d, J=8.1 Hz), 7.44 (1H, d, J=8.0 Hz), 7.36–7.33 (1H, m), 7.18 (1H, d, J=2.3 Hz), 6.89 (1H, dd, J=2.3, 8.6 Hz), 4.05 (2H, q, J=6.9 Hz), 0.96 (3H, t, J=6.9 Hz). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 175.88, 159.67, 154.09, 139.58, 139.16, 135.65, 129.81, 127.40, 124.38, 122.10, 120.99, 116.20, 115.67, 115.02, 64.47, 14.43. FAB-MS m/z 333 (MH⁺). HR-MS(FAB) Calcd for C₁₆H₁₃ClN₂O₂S 332.0386, Found 332.0372.

This compound was prepared from 23b by a method similar to that used for preparation of mdivi-1. White solid (51.9 mg, 0.150 mmol, 53%) (recrystallized from DMF/H₂O). mp>300°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.94 (1H, dd, J=1.7, 8.1 Hz), 7.78–7.75 (1H, m), 7.49 (1H, d, J=8.0 Hz), 7.43 (1H, d, J=8.0 Hz), 7.35–7.32 (1H, m), 7.17 (1H, d, J=2.3 Hz), 6.88 (1H, dd, J=2.0, 8.3 Hz), 3.94 (2H, t, J=6.3 Hz), 1.72 (2H, tt, J=6.3, 7.4 Hz), 0.97 (3H, t, J=7.4 Hz). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 175.86, 159.67, 154.23, 139.57, 139.16, 135.63, 129.77, 127.39, 124.36, 122.08, 121.02, 116.19, 115.66, 115.01, 70.18, 21.78, 10.25. FAB-MS m/z 347 (MH⁺). Anal. Calcd for C₁₇H₁₅ClN₂O₂S·0.5H₂O: C, 57.38; H, 4.53; N, 7.87. Found: C, 57.49; H, 4.57; N, 7.98.

This compound was prepared from 23c by a method similar to that used for preparation of mdivi-1. Pale yellow solid (60.1 mg, 0.172 mmol, 74%) (recrystallized from CHCl₃/n-hexane/MeOH). mp>300°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 7.91 (1H, dd, J=1.2, 8.0 Hz), 7.75–7.72 (1H, m), 7.44 (1H, d, J=8.0 Hz), 7.39 (1H, d, J=8.0 Hz), 7.32–7.30 (1H, m), 7.16 (1H, d, J=2.3 Hz), 6.83 (1H, dd, J=2.3, 8.6 Hz), 4.05 (1H, sep, J=5.7 Hz), 0.96 (6H, t, J=5.7 Hz). FAB-MS m/z 347 (MH⁺). Anal. Calcd for C₁₇H₁₅ClN₂O₂S·0.4H₂O: C, 57.67; H, 4.50; N, 7.91. Found: C, 57.77; H, 4.43; N, 7.73.

This compound was prepared from 23d by a method similar to that used for preparation of mdivi-1. Yellow solid (122 mg, 0.338 mmol, 98%) (recrystallized from CHCl₃/n-hexane/MeOH). mp 292–293°C. ¹H-NMR (500 MHz, CDCl₃) δ: 10.20 (1H, br s), 8.13 (1H, dd, J=1.2, 8.0 Hz), 7.68–7.65 (1H, m), 7.47 (1H, d, J=7.5 Hz), 7.33–7.30 (1H, m), 7.10 (1H, d, J=8.0 Hz), 6.78–6.76 (1H, m), 4.02–3.95 (2H, m), 1.81–1.76 (2H, m), 1.52–1.44 (2H, m), 0.94 (3H, t, J=7.5 Hz). FAB-MS m/z 361 (MH⁺). Anal. Calcd for C₁₈H₁₇ClN₂O₂S: C, 59.91; H, 4.75; N, 7.76. Found: C, 59.79; H, 5.05; N, 7.69.

MOLT4 cells were cultured in RPMI medium supplemented with 10% FBS and penicillin and streptomycin at 37°C in a humidified incubator (5% CO₂ in air).

Histidine-tagged DRP1 protein (plasmid was purchased from GeneCopoeia™) was expressed in transformed Rosetta (DE3) competent cells (Novagen), derived from Escherichia coli BL21, grown in LB by induction with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 25°C for 19 h. Cell suspensions were centrifuged (7500×g for 5 min, 4°C). Bacterial pellets were resuspended in 25 mM Tris–HCl (pH 8.0) buffer containing 300 mM NaCl, 10% glycerol (v/v), 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell pellets were homogenized by sonication (for 4 min×3 times) and further centrifuged (16000×g for 5 min, 4°C). Supernatants were purified over Ni-NTA resin. Soluble His-tagged proteins were loaded on Ni-NTA resin, which was washed with 25 mM Tris–HCl buffer containing 300 mM NaCl, 10% glycerol (v/v) for twice, and 25 mM Tris–HCl buffer containing 300 mM NaCl, 20 mM imidazole, 10% glycerol (v/v) for 4 times and then eluted with 25 mM Tris–HCl buffer containing 300 mM NaCl, 200 mM imidazole, 10% glycerol (v/v). Eluted proteins were quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Oriole staining.

This assay was performed using QuantiChrom™ ATPase/GTPase Assay Kit (BioAssay Sytems, DATG-200) according to the protocol recommended by the supplier. Briefly, the reaction mixture consisting of assay buffer (20 µL), approximately 1 µM Drp1 (5 µL) and test compound in DMSO (5 µL) was preincubated at 37°C for 15 min. GTP solution (1 mM, 10 µL) was added, and the reaction mixture was incubated at 37°C for 60 min. The reagent (200 µL) was added and incubation was continued at r.t. for 30 min. OD600 was measured with a Wallac 1420 multilabel counter (PerkinElmer, Inc., Life Science) and a Wallac Envision 2104 multilabel reader (PerkinElmer, Inc., Life Science). The assay was performed at least in duplicate, and the mean value was taken.

PSA activity was evaluated by means of the usual assay, by measuring 7-amino-4-methylcoumarin (AMC) liberated from L-methylcoumarylamide (Ala-AMC). Briefly, to a solution of 395 µL of 50 mM Tris–HCl (pH 7.4) in the presence or absence of a test inhibitor (5 µL in DMSO, various concentrations) was added 50 µL of intact MOLT4 cell suspension (2.0×10⁶ cells/mL) at 37°C and the system was pre-incubated for exactly 10 min. Then 50 µL of Ala-AMC (10 mM) was added at 37°C and incubation was continued for exactly 30 min. At this point, 1.5 mL of 1 M AcONa–AcOH (pH 4.0) was added to stop the enzymatic reaction. The amounts of liberated AMC were measured in terms of fluorescence intensity (excitation at 355 nm, emission at 460 nm) with a Wallac 1420 multilabel counter (PerkinElmer, Inc., Life Science) and a Wallac Envision 2104 multilabel reader (PerkinElmer, Inc., Life Science). The assay was performed at least in duplicate, and the mean value was taken.

This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Dr. Kenji Ohgane for helpful discussion.

• 1) Chan D. C., Annu. Rev. Cell Dev. Biol., 22, 79–99 (2006).
• 2) Chang C. R., Blackstone C., Ann. N. Y. Acad. Sci., 1201, 34–39 (2010).
• 3) Fannjiang Y., Cheng W. C., Lee S. J., Qi B., Pevsner J., McCaffery J. M., Hill R. B., Basañez G., Hardwick J. M., Genes Dev., 18, 2785–2797 (2004).
• 4) James D. I., Parone P. A., Mattenberger Y., Martinou J. C., J. Biol. Chem., 278, 36373–36379 (2003).
• 5) Palmer C. S., Osellame L. D., Laine D., Koutsopoulos O. S., Frazier A. E., Ryan M. T., EMBO Rep., 12, 565–573 (2011).
• 6) Zhao J., Liu T., Jin S., Wang X., Qu M., Uhlén P., Tomilin N., Shupliakov O., Lendahl U., Nistér M., EMBO J., 30, 2762–2778 (2011).
• 7) Otera H., Wang C., Cleland M. M., Setoguchi K., Yokota S., Youle R. J., Mihara K., J. Cell Biol., 191, 1141–1158 (2010).
• 8) Bossy-Wetzel E., Petrilli A., Knott A. B., Trends Neurosci., 31, 609–616 (2008).
• 9) Lin M. T., Beal M. F., Nature (London), 443, 787–795 (2006).
• 10) Song W., Chen J., Petrilli A., Liot G., Klinglmayr E., Zhou Y., Poquiz P., Tjong J., Pouladi M. A., Hayden M. R., Masliah E., Ellisman M., Rouiller I., Schwarzenbacher R., Bossy B., Perkins G., Bossy-Wetzel E., Nat. Med., 17, 377–382 (2011).
• 11) Shirendeb U. P., Calkins M. J., Manczak M., Anekonda V., Dufour B., McBride J. L., Mao P., Reddy P. H., Hum. Mol. Genet., 21, 406–420 (2012).
• 12) Cassidy-Stone A., Chipuk J. E., Ingerman E., Song C., Yoo C., Kuwana T., Kurth M. J., Shaw J. T., Hinshaw J. E., Green D. R., Nunnari J., Dev. Cell, 14, 193–204 (2008).
• 13) Kakuta H., Tanatani A., Nagasawa K., Hashimoto Y., Chem. Pharm. Bull., 51, 1273–1282 (2003).
• 14) Look A. T., Ashmun R. A., Shapiro L. H., Peiper S. C., J. Clin. Invest., 83, 1299–1307 (1989).
• 15) Hersh L. B., McKelvy J. F., J. Neurochem., 36, 171–178 (1981).
• 16) Bauer W. O., Nanda I., Beck G., Schmid M., Jacob F., Cytogenet. Cell Genet., 92, 221–224 (2001).
• 17) Tobler A. R., Constam D. B., Schmitt-Gräff A., Malipiero U., Schlapbach R., Fontana A., J. Neurochem., 68, 889–897 (1997).
• 18) Johnson G. D., Hersh L. B., Arch. Biochem. Biophys., 276, 305–309 (1990).
• 19) Rawlings N. D., Barrett A. J., Biochem. J., 290, 205–218 (1993).
• 20) Bhutani N., Venkatraman P., Goldberg A. L., EMBO J., 26, 1385–1396 (2007).
• 21) Menzies F. M., Hourez R., Imarisio S., Raspe M., Sadiq O., Chandraratna D., OʼKane C., Rock K. L., Reits E., Goldberg A. L., Rubinsztein D. C., Hum. Mol. Genet., 19, 4573–4586 (2010).
• 22) Matsumoto Y., Noguchi-Yachide T., Nakamura M., Mita Y., Numadate A., Hashimoto Y., Heterocycles, 86, 1449–1463 (2012).