Development of 2-thioxoquinazoline-4-one derivatives as dual and selective inhibitors of dynamin-related protein 1 (Drp1) and puromycin-sensitive aminopeptidase (PSA)

Article abstract of DOI:10.1248/cpb.c14-00333

An established inhibitor ot 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 Drpl, which is a potential target for treatment of Huntington's disease. Among the synthesized compounds, 3-(4-chloro3methoxyphenyl)-2-thioxoquinazoline-4-one 10g) exhibited more potent Drpl-inhibitory activity than mdivi-1 with high selectivity for Drpl over PSA.

Full text of DOI:10.1248/cpb.c14-00333

October 2014
Regular Article
Chem. Pharm. Bull. 62(10) 979–988 (2014)
979
Development of 2-Thioxoquinazoline-4-one Derivatives as Dual
and Selective Inhibitors of Dynamin-Related Protein 1 (Drp1) and
Puromycin-Sensitive Aminopeptidase (PSA)
,a
Akiyoshi Numadate,* Yusuke Mita,a Yotaro Matsumoto,b Shinya Fujii,a and Yuichi Hashimotoa
a Institute of Molecular and Cellular Biosciences, The University of Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032,
b
Japan: and Faculty of Pharma Sciences, Teikyo University; 2–11–1 Kaga, Itabashi-ku, Tokyo 173–8605, Japan.
Received April 28, 2014; accepted July 10, 2014
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 amino-
peptidase (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 identify-
ing 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.
Key words dynamin-related protein 1; Drp1; puromycin-sensitive aminopeptidase; 2-thioxoquinazoline-4-one
Dynamin related protein 1 (Drp1) is a member of the gua-
Since mdivi-1 is structurally closely related to PAQ-22,
nosine triphosphate hydrolase (GTPase) family. It is located we anticipated that mdivi-1 might also inhibit PSA. PSA is
mainly in cytosol, but upon activation it is recruited to the a single-chain protein of 99kDa that hydrolyzes N-terminal
mitochondrial surface, where it oligomerizes1–3) and bind to amino acids with a preference for basic and hydrophobic resi-
mitochondrial adaptors such as Fis1 and Mff to assemble a dues.14–19) This enzyme is an exopeptidase containing Zn2+ ion
fission site, at which mitochondrial membrane fission occurs at its catalytic site.15–17) It was reported that PSA hydrolyzes
in a GTP hydrolysis-dependent manner.4–7) Recently, Drp1 has polyQ chain and that PSA overexpression reduces polyQ ag-
been identified as a potential target for treatment of Hunting- gregate formation as well as the toxicity of mutant huntingtin
ton’s disease (HD).8) HD is an inherited neurodegenerative exon 1.20,21) These facts suggested that the PSA-inhibitory
disorder caused by abnormal polyglutamine (polyQ) expan- activity of mdivi-1 would be inappropriate in the treatment of
sion in huntingtin exon 1. PolyQ length determines disease HD. Indeed, we found that mdivi-1 strongly inhibits not only
onset and severity, with a longer expansion causing earlier Drp1, but also PSA.22)
onset.9,10) Treatment of HD remains a challenge. It is reported
On the basis of these considerations, we set out to inves-
that mutant huntingtin interacts with Drp1 and stimulates its tigate the structure–activity relationship (SAR) of mdivi-1
enzymatic activity, leading to an increase of mitochondrial derivatives and to conduct structural development studies
fragmentation and enhanced cell death.8,11) These defects targeting Drp1-selective inhibitors. In this paper, we describe
have been reported to be rescued by reducing Drp1 GTPase the design, synthesis, and biological evaluation of a series of
activity with dominant-negative mutant Drp1K38A 8,11)
findings suggest that Drp1 inhibitors are promising agents for hibitors.
.
These 2-thioxoquinazoline-4-one derivatives as Drp1-selective in-
treatment of HD to ameliorate neuronal cell death caused by
mutant huntingtin.
Structural Development and SAR Studies Mdivi-1 and
its oxo-analog (4) were synthesized as shown in Chart 1.
To develop Drp1 inhibitors, we selected the 3-phenyl- 2,4-Dichloro-5-aminoanisole (1) was amidated with 2-nitro-
2-thioxoquinazoline-4-one derivative mdivi-112) (Fig. 1, left), benzoyl chloride to afford 2. The nitro group of 2 was reduced
a known Drp1 inhibitor, as a lead compound. On the other to an amino group by using H2 in the presence of Pd/C. An-
hand, we previously developed PAQ-22 (Fig. 1, right), which nulation of 3 using CS2 in the presence of 1,8-diazabicyclo-
has 3-phenylquinazoline-2,4-dione structure,13) as an inhibitor [5.4.0]undec-7-ene (DBU) gave the 2-thioxoquinazoline-4-one
of puromycin-sensitive aminopeptidase (PSA).
derivative mdivi-1. Annulation of 3 using triphosgene gave
compounds bearing a quinazoline-2,4-dione ring.
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-
Fig. 1. Structures of Mdivi-1 (Left) and PAQ-22 (Right)
The authors declare no conflict of interest.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: 2466997510@mail.ecc.u-tokyo.ac.jp

980
Vol. 62, No. 10
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%.
Chart 1. Synthesis of Mdivi-1 and 4
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.
Table 1. Drp1-Inhibitory Activity of Mdivi-1 and Compounds 2–4 and
PAQ-22
Compound
Drp1 IC50 (µM)
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). Com-
pounds containing chlorine, i.e., 10c–h, were prepared by an-
nulation reaction of the corresponding anilines 9c–h with CS2
(path d in Chart 2).
Table 2 summarized the activities of the synthesized com-
pounds. Among them, 10g and 10f showed Drp1-inhibitory
activity, and 10g was about 3 times more potent than mdivi-1.
Mdivi-1
13
2
N.A.a)
N.A.a)
N.A.a)
N.A.a)
3
4
PAQ-22
Drp1-inhibitory activity was measured in a cell-free system by using recombinant
Drp1. The IC50 value is the molar concentration of test compound that affords 50%
of the maximal Drp1 GTPase activity. a) N.A. means no activity at 100µM.
Other compounds (10a–e, 10h) had no Drp1-inhibitory ac- ity of 19a–g was weaker than that of mdivi-1 (Table 3).
tivity. Next, we investigated the PSA-inhibitory activity of
Next, we optimized the alkoxy group of 10g. Compounds
these compounds, using the reported method.22) Briefly, PSA- bearing C2~C4 alkyl groups (24a–d) were synthesized by a
inhibitory activities were assessed by measuring 7-amino- similar method to that used for preparation of 10e, f, h, as
4-methylcoumarin (AMC) liberated from L-alanine 4-meth- shown in Chart 5.
ylcoumaryl-7-amide (Ala-MCA) using intact human acute
The Drp1- and PSA-inhibitory activities of 24a–d are sum-
lymphoblastic leukemia MOLT-4 cells. As we have reported, marized in Table 4. Ethyl derivative 24a and n-propyl deriva-
mdivi-1 exhibited strong PSA-inhibitory activity.22) On the tive 24b showed Drp1-inhibitory activity comparable to that
other hand, 10f showed only moderate PSA-inhibitory activity, of mdivi-1, but lower than that of methoxy derivative 10g.
and 10g exhibited little activity (inhibitory potency of only On the other hand, 24c and 24d bearing longer alkyl groups
46% at 100µM). Compounds without an R1 substituent, i.e., lacked Drp1-inhibitory activity. These results suggest that the
10a, 10b, 10e, 10g and 10h, did not exhibit PSA-inhibitory methoxy group is the most effective for potent Drp1-inhibitory
activity, and this result is consistent with our previous find- activity with high selectivity over PSA.
ing that ortho-substitution of the N-phenyl ring is important
for PSA-inhibitory activity.13) Among the compounds listed in
Conclusion
Table 2, 10g exhibited the most potent Drp1-inhibitory activity
Structural development of the known Drp1 inhibitor
and showed high selectivity for Drp1 over PSA.
mdivi-1 afforded 10g, which has a more potent Drp1-inhibi-
To optimize the para-substituent of the 3-phenyl ring tory activity than mdivi-1, together with high selectivity for
of compound 10g, various substituents, including halogen, Drp1 over PSA. Since our previous work showed that a high
methyl, methoxy, nitrile, and amide were introduced at the rotational barrier of the 3-phenyl ring was necessary for PSA-
para-position; these compounds (19a–d) were synthesized by inhibitory activity,13) the weak PSA-inhibitory activity of 10g
a similar method to that used for preparation of 10a, b (Chart may be due to the low rotational barrier of the 3-phenyl group,
3). Compounds 19e–g bearing a halogen atom were synthe- as evaluated by density functional theory (DFT) calculation.
sized in the same manner as 10e, f, h (Chart 4).
A Cl or Br atom at the p-position of the 3-phenyl group is
The Drp1- and PSA-inhibitory activities of compounds essential for Drp1-inhibitory activity, and a moderately long
19a–g are summarized in Table 3. The Drp1-inhibitory activ- alkyl chain in the substituent on the O atom is also important.
ity of 19f was comparable to that of 10g. All compounds other The structure–activity relationship of mdivi-1 derivatives is
than 19e and 19f lacked Drp1-inhibitory activity (Table 3). summarized in Fig. 2.
These results indicate that Cl or Br at the para-position in the
Compound 10g should be a promising lead compound for
phenyl group is important for potent Drp1-inhibitory activity. development of highly selective Drp1 inhibitors to treat Hun-
The lack of Drp1-inhibitory activity of 19g might be attributed tington’s disease.
to the large size of the iodine atom. The PSA-inhibitory activ-

October 2014
981
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.
Chart 2. Synthesis of Compounds 10a–h
2-nitrobenzoyl chloride (264µL, 2.01mmol) at 0°C, and the
Table 2. Drp1-Inhibitory Activity of Mdivi-1 and Compounds 10a–h
whole was stirred at r.t. After 1h, a pale yellow solid precipi-
tated. The precipitate was collected by filtration and washed
with AcOEt to afford the title compound (595mg, 1.74mol,
87%) as a pale yellow solid (recrystallized from CHCl3/n-
1
hexane). mp 176–177°C. H-NMR (500MHz, CDCl3) δ: 8.28
(1H, s), 8.16 (1H, d, J=8.5Hz), 7.88 (1H, brs), 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
R1
R2
R3
Drp1 IC50 (µM)
PSA IC50 (µM)
Mdivi-1
10a
Cl
H
Cl OMe
13
0.71
>100
30
H
H
H
OMe
H
N.A.a)
N.A.a)
N.A.a)
N.A.a)
N.A.a)
9.6
10b
10c
H
2-Amino-N-(2,4-dichloro-5-methoxyphenyl)benzamide (3)
Compound 2 (341mg, 1.00mmol) was dissolved in DMF
(5.0mL) 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 con-
centrated to afford the title compound (272mg, 0.873mmol,
87%) as a pale yellow solid (recrystallized from CHCl3/n-
Cl
Cl
H
H
44
10d
10e
Cl
Cl
H
H
45
H
>30
31
10f
Cl
H
OMe
10g
Cl OMe
OMe Cl
4.7
N.A.a)
>100
>100
10h
H
a) N.A. means no activity at 100µM.
1
hexane). mp 127–130°C. H-NMR (500MHz, CDCl3) δ: 8.34
(2H, m), 7.52 (1H, d, J=8.0Hz), 7.40 (1H, s), 7.29 (1H, m),
Experimental
Chemistry General Comments 1H-NMR spectra were 6.75 (2H, m), 5.59 (2H, brs), 3.96 (3H, s). Anal. Calcd for
recorded on a JEOL JNM-GX500 or JNMECA-500 (500MHz) C14H12Cl2N2O2: C, 54.04; H, 3.89; N, 9.00. Found: C, 53.98; H,
spectrometer. The following abbreviations are used for spin 3.93; N, 9.03.
multiplicity: s=singlet, d=doublet, t=triplet, q=quartet,
3-(2,4-Dichloro-5-methoxyphenyl)-2-thioxoquinazolin-
sep=septet, m=multiplet, br=broad. 13C-NMR spectra were 4-one (Mdivi-1) To a solution of 3 (80.6mg, 260µmol)
recorded on a JEOL JNM-GX500 or JNMECA-500 spec- and CS2 (300 µL, 5.00mmol) in DMF (2.0mL) was added
trometer at 125MHz. Mass spectra were recorded on a JEOL DBU (38.7µL, 258µmol). The reaction mixture was stirred
JMS-HX110 spectrometer. Melting points were determined on for 15.5h at r.t., and then diluted with AcOEt. The organic
a MP-J3 melting point apparatus (Yanaco, Japan). Elemental layer was washed with 2N HCl aq, water and brine, then
analyses were carried out in the Microanalytical Laboratory, dried and concentrated to afford the title compound (87.7mg,
Faculty of Pharmaceutical Sciences, the University of Tokyo, 0.248mmol, 96%) as a pale yellow solid (recrystallized from
1
and results were within 0.3% of the theoretical values.
CHCl3/MeOH). mp 293–294°C. H-NMR (500MHz, DMSO-
N-(2,4-Dichloro-5-methoxyphenyl)-2-nitrobenzamide
(2) d6) δ: 7.98 (1H, d, J=7.5Hz), 7.83–7.80 (1H, m), 7.78 (1H, s),
To
a
solution of 5-amino-2,4-dichloroanisole (386mg, 7.47–7.45 (2H, m), 7.39–7.36 (1H, m), 3.80 (3H, s). 13C-NMR
2.01mmol) and pyridine (0.1mL) in DCM (1.0mL) was added (125MHz, DMSO-d6) δ: 174.86, 158.90, 154.19, 139.64,

982
Vol. 62, No. 10
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-isothiocyanatobenzo-
ate, Et3N, 1,4-dioxane, r.t., 16–64%.
Chart 3. Synthesis of Compounds 19a–d
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%.
Chart 4. Synthesis of Compounds 19e–g
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 (73.9mg, 0.219mmol,
Table 3. Drp1-Inhibitory Activity of Compounds 10g and 19a–g
1
83%) as a white solid. mp 277–278°C. H-NMR (500MHz,
CDCl3) δ: 8.84 (1H, s), 8.18 (1H, d, J=7.3Hz), 7.68–7.65 (1H,
m), 7.59 (1H, s), 7.31–7.28 (1H, m), 7.05 (1H, d, J=8.0Hz),
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.
3-Phenyl-2-thioxoquinazoline-4-one (10a) To a solution
of methyl 2-isothiocyanatobenzoate (46.7mg, 0.242mmol)
and aniline (22.1µL, 0.242mmol) in 1,4-dioxane (2.0mL) was
added Et3N (37.4µL, 0.242mmol). The mixture was stirred
at r.t. for 2h, 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 (47.0mg,
Compound
R
Drp1 IC50 (µM)
PSA IC50 (µM)
10g
19a
19b
19c
19d
19e
19f
Cl
4.7
>100
>100
>100
>100
>100
>100
51
CONHMe
N.A.a)
N.A.a)
N.A.a)
N.A.a)
44
CN
Me
OMe
F
Br
2.2
N.A.a)
19g
I
>100
a) N.A. means no activity at 100µM.
136.29, 136.15, 129.76, 127.48, 124.73, 123.41, 121.93, 115.88, 0.185mmol, 76%) as a white solid. mp>300°C. 1H-NMR
115.56, 115.22, 56.77. FAB-MS m/z 353 (MH+), 355, 357. High (500MHz, DMSO-d6) δ: 7.94 (1H, dd, J=1.4, 7.7Hz), 7.79–7.76
resolution (HR)-MS(FAB) Calcd for C15H10Cl2N2O2S 351.9840, (1H, m), 7.48–7.43 (3H, m), 7.41–7.38 (1H, m), 7.36–7.33 (1H,
Found 351.9850.
m), 7.28–7.25 (2H, m). 13C-NMR (125MHz, DMSO-d6) δ:
3-(2,4-Dichloro-5-methoxyphenyl)quinazoline-2,4-dione (4) 176.05, 159.81, 139.40, 139.30, 135.61, 128.99, 128.89, 128.10,
Triphosgene (30.0mg, 0.101mmol) in 1,2-dichloroethane 127.41, 124.35, 116.20, 115.69, FAB-MS m/z 255 (MH+). Anal.
(10mL) was dropped into a solution of 3 (82.6mg, 0.265mmol) Calcd for C14H10N2OS·0.6H2O: C, 63.42; H, 4.26; N, 10.57.
and Et3N (50.0µL, 0.324mmol) in 1,2-dichloroethane (5.0mL) Found: C, 63.34; H, 4.38; N, 10.36.

October 2014
983
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%.
Chart 5. Synthesis of Compounds 24a–d
Table 4. Drp1-Inhibitory Activity of Compounds 10g and 24a–d
IC50 (µM)
Compound
R
Drp1
PSA
>100
Fig. 2. Key Features in the Structure–Activity Relationship of Drp1
Inhibitors
10g
24a
24b
24c
24d
Me
Et
4.7
14
>30b)
>30b)
>100c)
>30b)
n-Pr
i-Pr
n-Bu
10
N.A.a)
hexane:AcOEt=3:1) to afford the title compound (52.3mg,
0.212mmol, 36%) as a yellow solid. 1H-NMR (500MHz,
CDCl3) δ: 7.71 (1H, brs), 7.51 (2H, d, J=8.5Hz), 7.43 (1H,
dd, J=8.3, 1.8Hz), 7.31 (2H, d, J=8.5Hz), 7.26–7.23 (2H, m),
6.72–6.69 (2H, m), 5.48 (2H, brs).
>100
a) N.A. means no activity at 100µM. b) Precipitation at 100µM final DMSO con-
centration amounted to 11%. c) Final DMSO concentration was 11%.
3-(3-Methoxyphenyl)-2-thioxoquinazoline-4-one
(10b)
3-(4-Chlorophenyl)-2-thioxoquinazoline-4-one (10e) This
This compound was prepared from 3-methoxyaniline by a compound was prepared from 9e by a method similar to
similar method to that described for preparation of 10a. Yel- that used for preparation of mdivi-1. Yellow solid (24.9mg,
low solid (76.7mg, 0.270mmol, 91%) (recrystallized from 0.0862mmol, 34%) (recrystallized from CHCl3/MeOH).
1
CHCl3). H-NMR (500MHz, CDCl3) δ: 9.72 (1H, s), 8.17 (1H, mp>300°C. 1H-NMR (500MHz, CDCl3) δ: 8.06 (1H, dd,
d, J=8.0Hz), 7.72–7.68 (1H, m), 7.47–7.44 (1H, m), 7.36–7.33 J=8.0, 1.0Hz), 7.66–7.62 (1H, m), 7.44–7.42 (2H, m), 7.28–7.25
(1H, m), 7.11 (1H, d, J=7.9Hz), 7.04–7.02 (1H, m), 6.88–6.87 (1H, m), 7.18 (1H, d, J=8.0Hz), 7.15–7.12 (2H, m). FAB-MS
(1H, m), 6.81 (1H, s), 3.83 (3H, s). FAB-MS m/z 285 (MH+). m/z 289 (MH+). HR-MS(FAB, MH+) Calcd for C14H10ClN2OS
Anal. Calcd for C15H12N2O2S·H2O: C, 59.59; H, 4.67; N, 9.27. 289.0202, Found 289.0230.
Found: C, 59.86; H, 4.82; N, 9.07.
N-(3-Chloro-4-methoxyphenyl)-2-nitrobenzamide
(7h)
N-(4-Chlorophenyl)-2-nitrobenzamide (7e) To
a
mix- This compound was prepared from 3-chloro-4-methoxyaniline
ture of 4-chloroaniline (259mg, 2.03mmol), K2CO3 by a method similar to that used for preparation of 7e. Yellow
1
(299mg, 2.17mmol), and TBAHS (29.1mg, 0.0857mmol) in solid (952mg, 3.03mmol, quant.). H-NMR (500MHz, CDCl3)
DCM:H2O=2:1 was added 2-nitrobenzoyl chloride (290µL, δ: 8.08 (1H, d, J=8.0Hz), 7.70–7.67 (1H, m), 7.59–7.57 (3H,
2.20mmol) at 0°C. The reaction mixture was stirred for 2h at m), 7.50 (1H, dd, J=2.5, 8.5Hz), 6.88 (1H, d, J=9.5Hz), 3.85
r.t., then CHCl3 and MeOH were added. The organic layer was (3H, s).
washed with water and brine, dried and concentrated to give
2-Amino-N-(3-chloro-4-methoxyphenyl)benzamide
(9h)
the title compound (165mg, 0.596mmol, 27%) as a pale yellow This compound was prepared from 7h by a method similar
1
solid. H-NMR (500MHz, CDCl3) δ: 8.12 (1H, d, J=8.5Hz), to that used for preparation of 9e. Pale yellow solid (61.5mg,
1
7.74–7.71 (1H, m), 7.64–7.62 (2H, m), 7.53 (2H, d, J=8.5Hz), 0.222mmol, 65%). H-NMR (500MHz, CDCl3) δ: 7.63 (1H,
7.45 (1H, brs), 7.33 (1H, d, J=8.5 Hz).
d, J=2.0Hz), 7.62 (1H, brs), 7.42 (1H, d, J=7.5Hz), 7.40 (1H,
2-Amino-N-(4-chlorophenyl)benzamide (9e) To a solu- dd, J=2.5, 9.0Hz), 7.26–7.22 (1H, m), 6.91 (1H, d, J=8.5Hz),
tion of 7e (165mg, 0.596mmol) in DMF (4.0mL) was added 6.71–6.68 (1H, m), 5.49 (2H, brs), 3.89 (3H, s).
SnCl2·2H2O (417mg, 1.99mmol). The mixture was stirred
3-(3-Chloro-4-methoxyphenyl)-2-thioxoquinazoline-4-one
at 90°C for 4h, then NaHCO3 aq. was added and the whole (10h) This compound was prepared from 9h by a method
was filtered through a pad of Celite. The filtrate was di- similar to that used for preparation of mdivi-1. Yellow solid
luted with AcOEt and washed with water and brine. The (74.8mg, 0.235mmol, 60%) (recrystallized from CHCl3/
1
organic layer was dried over MgSO4 and concentrated. The MeOH). mp>300°C. H-NMR (500MHz, DMSO-d6) δ: 7.94
residue was purified by silica gel column chromatography (n- (1H, dd, J=1.2, 8.0Hz), 7.79–7.76 (1H, m), 7.44–7.43 (2H, m),

984
Vol. 62, No. 10
7.35–7.32 (1H, m), 7.24 (1H, dd, J=2.0, 8.9Hz), 7.22 (1H, d, rification.
J=8.6Hz), 3.91 (3H, s). 13C-NMR (125MHz, DMSO-d6) δ:
3-(2,4-Dichlorophenyl)-2-thioxoquinazoline-4-one
(10d)
176.23, 159.91, 154.20, 139.54, 135.60, 132.22, 130.40, 129.06, This compound was prepared from 9d by a method similar
127.42, 124.36, 120.62, 116.23, 115.68, 112.55, 56.27. FAB-MS to that used for preparation of mdivi-1. Yellow solid (118mg,
m/z 319 (MH+), 321. HR-MS(FAB) Calcd for C15H11ClN2O2S 0.365mmol, 34% from anthracitic acid). mp 256–260°C,
318.0261, Found 318.0261.
1H-NMR (500MHz, CDCl3) δ: 9.96 (1H, brs), 8.17 (1H, d,
N-(2-Chloro-5-methoxyphenyl)-2-nitrobenzamide
(7f) J=8.0Hz), 7.73–7.70 (1H, m), 7.60 (1H, d, J=1.9Hz), 7.43 (1H,
This compound was prepared from 2-chloro-5-methoxyaniline dd, J=2.5, 8.6Hz), 7.38–7.35 (1H, m), 7.28 (1H, d, J=8.6Hz),
hydrochloride by a method similar to that used for prepara- 7.14 (1H, d, J=8.0Hz). 13C-NMR (125MHz, CDCl3) δ: 175.65,
1
tion of 7e. Yellow solid (288mg, 0.940mmol, 88%). H-NMR 159.13, 138.83, 136.13, 135.83, 134.73, 133.47, 131.18, 130.47,
(500MHz, CDCl3) δ: 8.15–8.13 (2H, m), 7.91 (1H, brs), 128.96, 128.46, 125.37, 115.98, 114.95. FAB-MS: not detected.
7.76–7.75 (1H, m), 7.69–7.67 (2H, m), 7.28 (1H, d, J=8.6Hz), Anal. Calcd for C14H8Cl2N2OS·0.2H2O: C, 51.45; H, 2.59; N,
6.69 (1H, dd, J=3.1, 8.6Hz), 3.85 (3H, s).
2-Amino-N-(2-chloro-5-methoxyphenyl)benzamide
8.57. Found: C, 51.39; H, 2.80; N, 8.51.
t-Butyl-2-(4-chloro-3-methoxyphenylcarbamoyl)phenylcar-
(9f)
This compound was prepared from 7f by a method simi- bamate (8) To a solution of 2-t-butoxycarbonylaminobenzoic
lar to that used for preparation of 9e. Yellow solid (130mg, acid (115mg, 0.485mmol) in DMF (2.0mL) was added a
1
0.470mmol, 52%). H-NMR (500MHz, CDCl3) δ: 8.35 (1H, s), solution of 4-chloro-3-methoxyaniline (82.2mg, 0.522mmol),
8.18 (1H, d, J=2.7Hz), 7.53 (1H, d, J=8.0Hz), 7.29–7.27 (1H, HOBt (70.0mg, 0.518mmol), EDCI·HCl (104mg, 0.542mmol)
m), 6.75–6.73 (2H, m), 7.16 (1H, dd, J=2.7, 9.0Hz), 5.59 (brs, and DIEA (90.0µL, 0.522mmol) in DMF (1.0mL). The result-
2H), 3.84 (s, 3H).
ing mixture was stirred at r.t. overnight. After completion of
3-(2-Chloro-5-methoxyphenyl)-2-thioxoquinazoline-4-one the reaction, AcOEt was added. The organic layer was washed
(10f) This compound was prepared from 9f by a method with water and brine, dried, and concentrated to give the title
similar to that used for preparation of mdivi-1. Yellow solid compound as a pale yellow paste, which was used for the next
(111mg, 0.349mmol, 95%) (recrystallized from 1,4-dioxane/ step without further purification.
1
DMF/H2O). mp 232–234°C. H-NMR (500MHz, CDCl3) δ:
2-Amino-N-(4-chloro-3-methoxyphenyl)benzamide
(9g)
9.88 (1H, brs), 8.18 (1H, d, J=7.9Hz), 7.72–7.69 (1H, m), 7.47 To a solution of 8 (96.0mg, crude) in DCM (2.0mL) was
(1H, d, J=9.2Hz), 7.37–7.34 (1H, m), 7.13 (1H, d, J=7.9Hz), slowly added TFA (250µL) at r.t. The mixture was stirred for
6.99 (1H, dd, J=3.1, 9.2Hz), 6.88 (1H, d, J=2.5Hz), 3.82 (3H, 4.5h, and then NaHCO3 aq. and AcOEt were added to it. The
s). FAB-MS m/z 319 (MH+). Anal. Calcd for C15H11ClN2O2S· organic layer was washed with water and brine, dried and
0.25H2O: C, 55.73; H, 3.59; N, 8.67. Found: C, 55.84; H, 3.48; concentrated to afford the title compound as a yellow paste
N, 8.64.
(61.5mg, 0.222mmol, 87%). This compound was used for the
2-Amino-N-(2-chlorophenyl)benzamide (9c) To a solu- next step without further purification. 1H-NMR (500MHz,
tion of anthranilic acid (147mg, 1.07mmol) in THF (10mL) CDCl3) δ: 7.75 (1H, brs), 7.57 (1H, d, J=1.9Hz), 7.46 (1H,
was added SOCl2 (2.0mL). The mixture was stirred at r.t. for d, J=7.9Hz), 7.32–7.31 (2H, m), 6.90 (1H, dd, J=1.9, 8.6Hz),
1h, and then 2-chloroaniline (255mg, 2.00mmol) in pyridine 6.74–6.71 (1H, m), 5.49 (2H, brs), 3.95 (3H, s).
(1.0mL) was added. The whole was stirred at r.t. overnight,
3-(4-Chloro-3-methoxyphenyl)-2-thioxoquinazoline-4-one
then diluted with AcOEt and the organic layer was washed (10g) This compound was prepared from 9g by a method
with H2O and brine, dried and concentrated. Column chroma- similar to that used for preparation of mdivi-1. Yellow solid
tography (n-hexane:AcOEt=5:1 to 3:1) of the residue gave (41.4mg, 0.130mmol, 59%) (recrystallized from DMF/H2O).
1
the title compound (203mg, 0.821mmol, 76%) as a yellow mp>300°C. H-NMR (500MHz, DMSO-d6) δ: 7.95 (1H, dd,
1
solid. H-NMR (500MHz, CDCl3) δ: 8.45 (1H, d, J=7.9Hz), J=1.4, 7.8Hz), 7.80–7.76 (1H, m), 7.51 (1H, d, J=8.0Hz), 7.44
8.34 (1H, brs), 7.54 (1H, d, J=8.0Hz), 7.41 (1H, d, J=7.9Hz), (1H, d, J=8.1Hz), 7.36–7.33 (1H, m), 7.19 (1H, d, J=1.7Hz),
7.34–7.27 (2H, m), 7.09–7.06 (1H, m), 6.76–6.73 (1H, m), 5.60 6.91 (1H, dd, J=2.3, 8.6Hz), 3.79 (3H, s). 13C-NMR (125MHz,
(2H, brs). FAB-MS m/z 246 (M+), 247 (MH+).
DMSO-d6) δ: 175.85, 159.72, 154.93, 139.64, 139.32, 135.67,
3-(2-Chlorophenyl)-2-thioxoquinazoline-4-one (10c) This 129.81, 127.41, 124.38, 122.27, 120.69, 116.22, 115.73, 113.98,
compound was prepared from 9c by a method similar to 56.31. FAB-MS m/z 319 (MH+). HR-MS(FAB) Calcd for
that used for preparation of mdivi-1. Yellow solid (38.7mg, C15H11ClN2O2S 318.0230, Found 318.0274
0.134mmol, 50%). mp 247–251°C. 1H-NMR (500MHz,
2-Methoxy-N-methyl-4-nitrobenzamide (13a) To a solution
CDCl3) δ: 10.19 (1H, brs), 8.18 (1H, d, J=7.9 Hz), 7.72–7.69 of 2-methoxy-4-nitrobenzoic acid (202mg, 1.03mmol) and
(1H, m), 7.61–7.59 (1H, m), 7.48–7.44 (2H, m), 7.37–7.35 (2H, aqueous MeNH2 (125 µL, 1.50mmol) in DMF (1.0mL) was
m), 7.16 (1H, d, J=8.0Hz). FAB-MS m/z 289 (MH+). Anal. added DMT–MM (430mg, 1.55mmol). The whole was stirred
Calcd for C14H9ClN2OS: C, 58.23; H, 3.14; N, 9.70. Found: C, at r.t. overnight and then diluted with AcOEt. The organic
58.06; H, 3.14; N, 9.70.
layer was washed with water and brine, dried and concen-
2-Amino-N-(2,4-dichlorophenyl)benzamide (9d) To a so- trated to give the title compound (225mg, 1.03mmol, quant.)
1
lution of anthranilic acid (147mg, 1.07mmol) in Et2O (5.0mL) as a white solid. H-NMR (500MHz, CDCl3) δ: 8.35 (1H, d,
was added SOCl2 (2.0mL). The mixture was stirred at r.t. for J=8.6Hz), 7.89 (1H, dd, J=2.3, 8.8Hz), 7.81 (1H, d, J=1.8Hz),
1h, then 2,4-dichloroaniline (337mg, 2.08mmol) in pyridine 7.70 (1H, brs), 4.05 (3H, s), 3.01 (3H, d, J=4.6 Hz).
(1.0mL) was added. The whole was stirred at r.t. overnight
2-Methoxy-1-methyl-4-nitrobenzene (13c) To a solution
and then diluted with AcOEt. The organic layer was washed of 2-methyl-5-nitrophenol (309mg, 2.02mmol) and K2CO3
with H2O and brine, dried and concentrated to afford the title (400mg, 2.90mmol) in DMF (4.0mL) was added MeI (855mg,
compound, which was used for next step without further pu- 6.02mmol). The reaction mixture was stirred for 9h at r.t.

October 2014
985
and then diluted with AcOEt. The organic layer was washed similar to that used for preparation of 10a. White solid
with water and brine, dried over MgSO4 and concentrated to (71.0mg, 0.238mmol, 43%) (recrystallized from DMF/H2O).
1
give compound 13c as a dark yellow solid (251mg, 1.50mmol, mp>300°C. H-NMR (500MHz, DMSO-d6) δ: 7.94 (1H, dd,
74%), which was used for the next step without further puri- J=1.2, 8.1Hz), 7.78–7.75 (1H, m), 7.43 (1H, d, J=8.0Hz),
1
fication. H-NMR (500MHz, CDCl3) δ: 7.74 (1H, dd, J=2.0, 7.35–7.32 (1H, m), 7.20 (1H, d, J=8.0Hz), 6.89 (1H, d,
8.0Hz), 7.64 (1H, d, J=2.0Hz), 7.23 (1H, d, J=8.0Hz), 3.90 J=1.7Hz), 6.74 (1H, dd, J=1.7, 8.0Hz), 3.72 (3H, s), 2.19
(3H, s), 2.28 (3H, s).
(3H, s). 13C-NMR (125MHz, DMSO-d6) δ: 176.14, 159.77,
4-Amino-2-methoxy-N-methylbenzamide (14a) Compound 157.62, 139.57, 138.10, 135.57, 130.21, 127.41, 125.40, 124.31,
13a (221mg, 1.05mmol) was dissolved in MeOH (8.0mL) 120.60, 116.19, 115.63, 111.17, 55.40, 15.87, FAB-MS m/z 315
and hydrogenated (1 bar H2) over 10% palladium on char- (MH)+. HR-MS(FAB) Calcd for C16H14N2O2S 298.0776, Found
coal for 5.5h at r.t. The mixture was filtered through a pad 298.0771.
of Celite and the filtrate was concentrated. The residue was
3-(3,4-Dimethoxyphenyl)-2-thioxoquinazoline-4-one (19d)
recrystallized from DCM and n-hexane to afford the title com- This compound was prepared from 3,4-dimethoxyaniline by
1
pound (193mg, 1.05mmol, quant.) as a white solid. H-NMR a method similar to that used for preparation of 10a. Brown
(500MHz, CDCl3) δ: 8.02 (1H, d, J=8.6Hz), 7.63 (1H, brs), solid (114mg, 0.363mmol, 64%). mp 292–293°C. 1H-NMR
6.32 (1H, dd, J=2.3, 8.6Hz), 6.18 (1H, d, J=2.3Hz), 4.03 (2H, (500MHz, DMSO-d6) δ: 7.96 (1H, dd, J=1.2, 8.0Hz),
d, J=4.6Hz), 3.88 (3H, s), 2.95 (3H, d, J=4.6 Hz).
7.80–7.77 (1H, m), 7.45 (1H, d, J=8.1Hz), 7.37–7.33 (1H, m),
4-Amino-2-methoxybenzonitrile (14b) This compound 7.03 (1H, d, J=8.6Hz), 6.93 (1H, d, J=2.3Hz), 6.80 (1H, dd,
was prepared from 2-methoxy-4-nitrobenzonitrile by a meth- J=2.3, 8.0Hz), 3.82 (3H, s), 3.71 (3H, s). 13C-NMR (125MHz,
od similar to that used for preparation of 14a. White solid DMSO-d6) δ: 176.38, 159.92, 148.90, 148.38, 139.63, 135.50,
1
(290mg, 1.95mmol, 96%). H-NMR (500MHz, CDCl3) δ: 7.26 132.04, 127.41, 124.25, 121.05, 116.21, 115.69, 112.76, 111.39,
(1H, d, J=8.6Hz), 7.23 (1H, s), 6.19 (1H, dd, J=2.3, 8.7Hz), 55.62, 55.55. FAB-MS m/z 315 (MH)+. HR-MS(FAB) Calcd for
6.12 (1H, d, J=2.3Hz), 4.11 (2H, brs), 3.82 (3H, s).
3-Methoxy-4-methylaniline (14c) This compound was pre-
C16H14N2O3S 314.0725, Found 314.0715.
1-Bromo-2-methoxy-4-nitrobenzene (16f) H2SO4 (1.5mL)
pared from 13c by a method similar to that used for prepara- was added slowly to a cold (0°C) stirred mixture of 2-meth-
1
tion of 14a. Brown solid (195mg, 1.42mmol, 95%). H-NMR oxy-4-nitrobenzenamine (340mg. 2.02mmol) and NaNO2
(500MHz, CDCl3) δ: 6.87 (1H, d, J=7.5), 6.21–6.19 (2H, m), (186mg, 2.69mmol) in H2O (4.0mL). After the addition was
3.90 (3H, s), 3.53 (2H, brs), 2.08 (3H, s), FAB-MS m/z 138 completed, stirring was continued for an additional 30min
(MH)+.
at 0°C. Then a solution of CuBr (345mg, 2.40mmol) in HBr
3-(3-Methoxy-4-methylaminocarbonylphenyl)-2-thioxoquin- (1.5mL) was added slowly at 0°C. The whole was stirred at
azoline-4-one (19a) To a solution of methyl 2-isothio- r.t. overnight and diluted with CHCl3. The organic layer was
cyanatobenzoate (65.4mg, 0.338mmol) and 14a (62.4mg, washed with water and brine, dried and concentrated. Column
0.346mmol) in dioxane (2.0mL) was added Et3N (80µL, chromatography (n-hexane:AcOEt=3:1) gave the title com-
1
mmol). The mixture was stirred at r.t. for 10h, and the precipi- pound (358mg, 1.62mmol, 80%) as a white solid. H-NMR
tated white solid was collected by filtration and washed with (500MHz, CDCl3) δ: 7.72–7.67 (3H, m), 3.97 (3H, s).
n-hexane to give the title compound (18.9mg, 0.0541mmol,
4-Bromo-3-methoxyaniline (14f) To a mixture of 16f
16%) as a white solid (recrystallized from CHCl3/MeOH/n- (358mg, 1.61mmol) and Zn powder (697mg, 10.66mmol) in
hexane). mp>300°C. 1H-NMR (500MHz, CDCl3) δ: 8.24 MeOH (16mL) was added AcOH (4.8mL). The whole was
(1H, d, J=8.6Hz), 8.05 (1H, dd, J=1.2, 8.0Hz), 7.86 (1H, d, stirred at r.t. for 4.5h and then extracted with AcOEt three
J=4.6Hz), 7.65–7.61 (1H, m), 7.27–7.24 (1H, m), 7.20–7.17 times. The organic layer was combined, washed with water
(1H, m), 6.91 (1H, dd, J=1.7, 8.0Hz), 6.82 (1H, d, J=2.3Hz), and brine, dried and concentrated. Column chromatography
3.89 (3H, s), 2.94 (3H, d, J=4.6Hz). FAB-MS m/z 342 (MH+). (n-hexane:AcOEt=3:1 to 2:1) of the residue gave the title
Anal. Calcd for C17H15N3O3S·0.3H2O: C, 58.88; H, 4.53; N, compound (205mg, 1.01mmol, 63%) as a yellow paste.
12.12. Found: C, 58.61; H, 4.64; N, 11.96.
1-Iodo-2-methoxy-4-nitrobenzene (16g) HCl (2.0mL) was
3-(4-Cyano-3-methoxyphenyl)-2-thioxoquinazoline-4-one added slowly to a cold (0°C) stirred solution of 2-methoxy-
(19b) To a solution of methyl 2-isothiocyanatobenzoate 4-nitrobenzenamine (343mg. 2.04mmol) and NaNO2 (170mg,
(98.8mg, 0.511mmol) and 14b (74.0mg, 0.499mmol) in di- 2.46mmol) in H2O (5.0mL). After the addition was completed,
oxane (2.0mL) was added Et3N (80µL, mmol). The mixture the solution was stirred for a further 30min at 0°C. Then a
was stirred at r.t. for 10h, and the precipitated white solid solution of KI (524mg, 3.16mmol) in H2O (2.0mL) was added
was collected by filtration and washed with n-hexane to give slowly at 0°C. The whole was stirred at r.t. overnight and
the title compound (18.9mg, 0.0818mmol, 16%) as a white quenched with CHCl3. The organic layer was washed with
solid. mp>300°C. 1H-NMR (500MHz, DMSO-d6) δ: 7.98 Na2S2O3 aq., water and brine, dried and concentrated. Column
(1H, dd, J=1.2, 8.0Hz), 7.88 (1H, d, J=8.1Hz), 7.83–7.80 (1H, chromatography (n-hexane:AcOEt=5:1 to 3:1) gave the title
m), 7.47 (1H, d, J=8.0Hz), 7.39–7.36 (2H, m), 7.12 (1H, dd, compound (404mg, 1.45mol, 71%) as a pale yellow solid.
J=1.7, 8.0Hz), 3.89 (3H, s). 13C-NMR (125MHz, DMSO-d6) 1H-NMR (500MHz, CDCl3) δ: 7.96 (1H, d, J=8.2Hz), 7.62
δ: 175.30, 161.55, 159.60, 145.42, 139.87, 135.73, 133.91, 127.33, (1H, d, J=2.8Hz), 7.59 (1H, dd, J=2.8, 8.2Hz), 3.99 (3H, s).
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, (404mg, 1.45mmol) and NH4Cl (130mg, 2.43mmol) in MeOH
62.12; H, 3.58; N, 13.58. Found: C, 61.98; H, 3.66; N, 13.47. (7.0mL), THF (7.0mL) and H2O (3.5mL) was added Fe pow-
4-Iodo-3-methoxyaniline (14g) To a mixture of 16g
3-(3-Methoxy-4-methylphenyl)-2-thioxoquinazoline-4-one der (443mg, 7.93mmol). The whole was stirred at 80°C for
(19c) This compound was prepared from 14c by a method 4.5h and then the reaction was quenched with AcOEt and

986
Vol. 62, No. 10
H2O. The resulting mixture was filtered through a pad of for 5h and then diluted with AcOEt and H2O. The resulting
Celite and the filtrate was extracted with AcOEt three times. mixture was filtered through a pad of Celite. The combined
The organic layers were combined, washed with water and organic layer was washed with water and brine, dried and
brine, dried and concentrated. Column chromatography (n- concentrated. Column chromatography (n-hexane:AcOEt=5:1
hexane:AcOEt=3:1 to 2:1) gave the title compound (372mg, to 3:1) gave the title compound (276mg, 0.750mmol, 51% for
1
1.45mmol, quant.) as a brown paste. 1H-NMR (500MHz, 2 steps) as a yellow paste. H-NMR (500MHz, CDCl3) δ: 7.78
CDCl3) δ: 7.44 (1H, d, J=8.5Hz), 6.21 (1H, d, J=2.8Hz), 6.11 (1H, brs), 7.68 (1H, d, J=8.6Hz), 7.47–7.44 (2H, m), 7.28–7.23
(1H, dd, J=2.8, 8.5Hz), 3.81 (3H, s), 3.72 (2H, brs). FAB-MS (2H, m), 6.76 (1H, dd, J=2.3, 8.0Hz), 6.72–6.69 (2H, m), 5.48
m/z 249 (MH+).
(2H, brs), 3.91 (3H, s).
N-(4-Fluoro-3-methoxyphenyl)-2-nitrobenzamide
(17e)
3-(4-Fluoro-3-methoxylphenyl)-2-thioxoquinazoline-4-one
To
a
mixture of 4-fluoro-3-methoxyaniline (142mg, (19e) This compound was prepared from 18e by a method
1.01mmol), K2CO3 (157mg, 1.14mmol) and TBAHS (19.2mg, similar to that used for preparation of mdivi-1. White solid
0.0565mmol) in DCM:H2O=2:1 was added 2-nitrobenzoyl (18.9mg, 61.1µmol, 16%) (recrystallized from DMF/H2O).
chloride (162µL, 1.10mmol) at 0°C. The reaction mixture was mp>300°C. 1H-NMR (500MHz, DMSO-d6) δ: 7.95 (1H,
stirred at r.t. overnight, and then CHCl3 was added to it. The dd, J=1.4, 7.7Hz), 7.79–7.76 (1H, m), 7.44 (1H, d, J=8.1Hz),
organic layer was washed with water and brine, dried and 7.36–7.28 (1H, m), 7.19 (1H, dd, J=2.6, 7.7Hz), 6.89–6.86 (1H,
concentrated to give the title compound (277mg, 0.960mmol, m), 3.78 (3H, s). 13C-NMR (125MHz, DMSO-d6) δ: 176.08,
95%) as a pale yellow solid.1H-NMR (500MHz, CDCl3) δ: 159.77, 151.97, 150.03, 147.45, 147.36, 139.55, 135.73, 135.62,
8.11 (1H, d, J=8.0Hz), 7.73–7.70 (1H, m), 7.62–7.61 (2H, m), 127.39, 124.35, 121.66, 121.61, 116.19, 115.87, 115.71, 115.65,
7.53–7.52 (1H, m), 7.43 (1H, s), 7.02 (1H, dd, J=8.6, 10.9Hz), 115.02, 56.16. FAB-MS m/z 303 (MH+). Anal. Calcd for
6.86–6.83 (1H, m), 3.90 (3H, s).
C15H11FN2O2S·0.5H2O: C, 57.87; H, 3.89; N, 9.00. Found: C,
2-Amino-N-(4-fluoro-3-methoxyphenyl)benzamide
(18e) 58.11; H, 4.03; N, 9.20.
To a solution of 17e (277mg, 0.955mmol) in AcOEt (5.0mL)
3-(4-Bromo-3-methoxylphenyl)-2-thioxoquinazoline-4-one
was added SnCl2·2H2O (690mg, 2.92mmol). The mixture (19f) This compound was prepared from 18f by a method
was stirred at 60°C for 3h, and then NaHCO3 aq. was added. similar to that used for preparation of mdivi-1. Yellow solid
The whole was filtered through a pad of Celite, and the fil- (61.6mg, 0.169mmol, 42%) (recrystallized from DMF/H2O).
1
trate was diluted with AcOEt and then washed with water mp>300°C. H-NMR (500MHz, DMSO-d6) δ: 7.95 (1H, d,
and brine. The organic layer was dried and concentrated. J=8.1Hz), 7.80–7.77 (1H, m), 7.67 (1H, d, J=8.6Hz), 7.44
The residue was purified by column chromatography (n- (1H, d, J=8.1Hz), 7.37–7.33 (1H, m), 7.16 (1H, d, J=1.9Hz),
hexane:AcOEt=3:1) to afford the title compound (198mg, 6.86 (1H, dd, J=1.9, 8.3Hz), 3.79 (3H, s). 13C-NMR (125MHz,
1
0.764mmol, 80%) as a pale yellow solid. H-NMR (500MHz, DMSO-d6) δ: 175.78, 159.67, 155.90, 140.01, 139.60, 135.67,
CDCl3) δ: 7.71 (1H, s), 7.47 (1H, dd, J=2.6, 7.7Hz), 7.25–7.21 132.84, 127.41, 124.40, 122.80, 116.21, 115.69, 113.89, 110.27,
(1H, m), 7.01 (1H, dd, J=8.9, 11.2Hz), 6.85–6.82 (1H, m), 56.43. Anal. Calcd for C15H11BrN2O2S·0.8H2O: C, 47.71; H,
6.70–6.67 (2H, m), 5.45 (2H, brs), 3.88 (3H, s).
3.36; N, 7.42. Found: C, 47.62; H, 3.11; N, 7.31.
2-Amino-N-(4-bromo-3-methoxyphenyl)benzamide
(18f)
3-(4-Iodo-3-methoxylphenyl)-2-thioxoquinazoline-4-one
To
a
mixture of 14f (203mg, 1.00mmol), K2CO3 (19g) This compound was prepared from 18g by a method
(281mg, 1.18mmol) and TBAHS (21.8mg, 0.0642mmol) in similar to that used for preparation of mdivi-1. Yellow solid
DCM:H2O=2:1 was added 2-nitrobenzoyl chloride (191µL, (121mg, 0.295mmol, 93%) (recrystallized from n-hexane/
1.30mmol) at 0°C. The mixture was stirred at r.t. overnight, AcOEt/MeOH). mp>300°C. 1H-NMR (500MHz, DMSO-
and then the reaction was quenched with CHCl3. The organic d6) δ: 7.95 (1H, dd, J=1.2, 8.0Hz), 7.85 (1H, d, J=8.1Hz),
layer was washed with water and brine, dried, and concen- 7.79–7.76 (1H, m), 7.44 (1H, d, J=8.0Hz), 7.37–7.33 (1H, m),
trated (17f). To a suspension of 17f and Zn powder (421mg, 7.04 (1H, d, J=2.3Hz), 6.71 (1H, dd, J=2.0, 8.3Hz), 3.76 (3H,
6.44mmol) in MeOH (10mL) was added AcOH (3.0mL). The s). 13C-NMR (125MHz, DMSO-d6) δ: 175.74, 159.64, 158.36,
whole was stirred at r.t. for 3h and then diluted with AcOEt. 140.90, 139.59, 138.82, 135.65, 127.39, 124.37, 123.36, 116.18,
The organic layer was washed with water and brine, dried and 115.68, 112.79, 85.81, 56.55, FAB-MS m/z 411 (MH+). HR-
concentrated. Column chromatography (n-hexane:AcOEt=3:1 MS(FAB) Calcd for C15H11IN2O2S 409.9586, Found 409.9594.
to 2:1) gave the title compound (262mg, 0.816mmol, 81% for
4-Chloro-3-ethoxyaniline (21a) To a mixture of 5-amino-
1
2 steps) as a yellow solid. H-NMR (500MHz, CDCl3) δ: 7.76 2-chlorophenol (148mg, 1.03mmol) and K2CO3 (296mg,
(1H, brs), 7.54 (1H, d, J=2.3Hz), 7.48–7.44 (1H, m), 7.27 (1H, 2.14mmol) in DMF (2.0mL) was added EtI (100µL,
dd, J=1.2, 7.5Hz), 6.73–6.70 (2H, m), 5.49 (2H, brs), 3.93 (3H, 1.25mmol). The whole was stirred at 60°C for 2.5h and then
s).
diluted with AcOEt. The organic layer was washed with
2-Amino-N-(4-iodo-3-methoxyphenyl)benzamide
(18g) water and brine, dried and concentrated. Column chroma-
To
a
mixture of 14g (366mg, 1.47mmol), K2CO3 tography (n-hexane:AcOEt=3:1) of the residue gave the title
(218mg, 1.58mmol) and TBAHS (29.1mg, 0.0857mmol) in compound (88.5mg, 0.515mmol, 50%) as a pale yellow paste.
DCM:H2O=2:1 was added 2-nitrobenzoyl chloride (297µL, 1H-NMR (500MHz, CDCl3) δ: 7.07 (1H, d, J=8.6Hz), 6.25
2.03mmol) at 0°C. The reaction mixture was stirred at r.t. (1H, d, J=2.9Hz), 6.19 (1H, dd, J=2.6, 8.3Hz), 4.02 (2H, q,
overnight, and then quenched with CHCl3. The organic layer J=7.0Hz), 3.63 (2H, brs), 1.43 (3H, t, J=7.0 Hz).
was washed with water and brine, dried, and concentrated
4-Chloro-3-propoxyaniline (21b) This compound was
(17g). To a suspension of 17g and NH4Cl (85.6mg, 1.60mmol) prepared from 5-amino-2-chlorophenol and n-PrI by a method
in MeOH (7.0mL), THF (5.0mL), H2O (3.0mL) was added Fe similar to that used for preparation of 21a. Pale yellow paste
powder (363mg, 6.50mmol). The whole was stirred at 80°C (107mg, 0.572mmol, 54%). 1H-NMR (500MHz, CDCl3) δ:

October 2014
987
7.07 (1H, d, J=8.0Hz), 6.25 (1H, d, J=2.3Hz), 6.19 (1H, dd, This compound was prepared from 22b by a method similar
J=2.6, 8.3Hz), 3.90 (1H, d, J=6.6Hz), 3.63 (2H, brs), 1.83 to that used for preparation of 23a. Pale yellow paste (146mg,
1
(1H, qt, J=7.5, 6.6Hz), 1.04, (3H, t, J=7.5 Hz).
0.479mmol, 87%). H-NMR (500MHz, CDCl3) δ: 7.71 (1H,
4-Chloro-3-isopropoxyaniline (21c) This compound was brs), 7.48 (1H, d, J=2.3Hz), 7.42–7.41 (1H, m), 7.27 (1H, d,
prepared from 5-amino-2-chlorophenol and i-PrBr by a meth- J=8.6Hz), 7.25–7.21 (1H, m), 6.85 (1H, dd, J=2.6, 8.3Hz),
od similar to that used for preparation of 21a. Yellow paste 6.70–6.67 (2H, m), 5.45 (2H, brs), 4.00 (2H, t, J=6.8Hz), 1.85
(110mg, 0.592mmol, 58%). 1H-NMR (500MHz, CDCl3) δ: (2H, qt, J=6.8, 7.4Hz), 1.04 (3H, t, J=7.4 Hz).
7.06 (1H, d, J=8.6Hz), 6.27 (1H, d, J=2.3Hz), 6.19 (1H, dd,
J=2.3, 8.6Hz), 4.44 (1H, sep, J=6.3Hz), 3.60 (2H, brs), 1.33 This compound was prepared from 22c by a method similar
(6H, d, J=6.3 Hz). to that used for preparation of 23a. Yellow solid (144mg,
2-Amino-N-(4-chloro-3-isopropoxyphenyl)benzamide (23c)
1
3-Butoxy-4-chloroaniline (21d) This compound was pre- 0.474mmol, 80%). H-NMR (500MHz, CDCl3) δ: 7.70 (1H,
pared from 5-amino-2-chlorophenol and n-BuI by a method brs), 7.49 (1H, d, J=2.3Hz), 7.43–7.41 (1H, m), 7.27 (1H, d,
similar to that used for preparation of 21a. Yellow paste J=8.6Hz), 7.25–7.21 (1H, m), 6.86 (1H, dd, J=2.3, 8.6Hz),
(141mg, 0.700mmol, 68%). 1H-NMR (500MHz, CDCl3) δ: 6.70–6.67 (2H, m), 5.45 (2H, brs), 4.57 (1H, sep, J=5.9Hz),
7.05 (1H, d, J=8.6Hz), 6.23 (1H, d, J=2.3Hz), 6.17 (1H, 1.37 (6H, d, J=5.9 Hz).
dd, J=2.6, 8.3Hz), 3.93 (2H, t, J=6.6Hz), 3.61 (2H, brs),
1.80–1.74 (2H, m), 1.52–1.45 (2H, m), 0.94 (3H, t, J=7.5 Hz).
2-Amino-N-(3-butoxy-4-chlorophenyl)benzamide
This compound was prepared from 22d by a method similar
(23d)
N-(4-Chloro-3-ethoxyphenyl)-2-nitrobenzamide (22a) This to that used for preparation of 23a. Yellow solid (158mg,
1
compound was prepared from 21a by a method similar to 0.497mmol, 74%). H-NMR (500MHz, CDCl3) δ: 7.71 (1H,
that used for preparation of 7e. Pale yellow solid (153mg, brs), 7.47 (1H, d, J=2.3Hz), 7.42–7.41 (1H, m), 7.26 (1H, d,
0.474mmol, 95%). 1H-NMR (500MHz, CDCl3/CD3OD) δ: J=8.6Hz), 7.25–7.21 (1H, m), 6.85 (1H, dd, J=2.3, 8.6Hz),
8.02–7.99 (1H, m), 7.63–7.59 (1H, m), 7.51–7.49 (2H, m), 6.70–6.67 (2H, m), 5.45 (2H, brs), 4.03 (2H, t, J=6.3Hz),
7.43–7.42 (1H, m), 7.16–7.13 (1H, m), 6.87 (1H, dd, J=2.3, 1.83–1.78 (2H, m), 1.54–1.47 (2H, m), 0.96 (3H, t, J=7.4 Hz).
8.6Hz), 3.90 (1H, d, J=6.6Hz), 4.01 (2H, q, J=6.8Hz), 1.33,
(3H, t, J=6.8Hz).
3-(4-Chloro-3-ethoxyphenyl)-2-thioxoquinazoline-4-one
(24a) This compound was prepared from 23a by a method
(22b) similar to that used for preparation of mdivi-1. White solid
N-(4-Chloro-3-propoxyphenyl)-2-nitrobenzamide
This compound was prepared from 21b by a method similar (47.8mg, 0.143mmol, 61%) (recrystallized from DMF/H2O).
1
to that used for preparation of 7e. Pale yellow solid (187mg, mp>300°C. H-NMR (500MHz, DMSO-d6) δ: 7.95 (1H, dd,
0.560mmol, 97%). 1H-NMR (500MHz, CDCl3/CD3OD) δ: J=1.2, 8.0Hz), 7.78–7.76 (1H, m), 7.50 (1H, d, J=8.1Hz), 7.44
8.00 (1H, d, J=8.1Hz), 7.63–7.60 (1H, m), 7.51–7.49 (1H, m), (1H, d, J=8.0Hz), 7.36–7.33 (1H, m), 7.18 (1H, d, J=2.3Hz),
7.44 (1H, d, J=2.3Hz), 7.14 (1H, d, J=8.6Hz), 6.86 (1H, dd, 6.89 (1H, dd, J=2.3, 8.6Hz), 4.05 (2H, q, J=6.9Hz), 0.96
J=2.3, 8.6Hz), 3.90–3.87 (2H, m), 1.75–1.71 (2H, m), 0.93 (3H, (3H, t, J=6.9Hz). 13C-NMR (125MHz, DMSO-d6) δ: 175.88,
t, J=7.4 Hz).
159.67, 154.09, 139.58, 139.16, 135.65, 129.81, 127.40, 124.38,
(22c) 122.10, 120.99, 116.20, 115.67, 115.02, 64.47, 14.43. FAB-
N-(4-Chloro-3-isopropoxyphenyl)-2-nitrobenzamide
This compound was prepared from 21c by a method similar MS m/z 333 (MH+). HR-MS(FAB) Calcd for C16H13ClN2O2S
to that used for preparation of 7e. Pale yellow solid (199mg, 332.0386, Found 332.0372.
1
0.568mmol, quant.). H-NMR (500MHz, CDCl3) δ: 8.11 (1H,
3-(4-Chloro-3-n-propoxyphenyl)-2-thioxoquinazoline-4-one
d, J=8.0Hz), 7.73–7.71 (1H, m), 7.64–7.61 (1H, m), 7.54 (1H, d, (24b) This compound was prepared from 23b by a method
J=2.3Hz), 7.44 (1H, brs), 7.29 (1H, d, J=8.6Hz), 6.86 (1H, dd, similar to that used for preparation of mdivi-1. White solid
J=2.3, 8.6Hz), 3.90 (1H, d, J=6.5Hz), 1.38 (6H, d, J=6.5 Hz). (51.9mg, 0.150mmol, 53%) (recrystallized from DMF/H2O).
1
N-(3-Butoxy-4-chlorophenyl)-2-nitrobenzamide (22d) This mp>300°C. H-NMR (500MHz, DMSO-d6) δ: 7.94 (1H, dd,
compound was prepared from 22d by a method similar to that J=1.7, 8.1Hz), 7.78–7.75 (1H, m), 7.49 (1H, d, J=8.0Hz), 7.43
used for preparation of 7e. Yellow solid (234mg, 0.670mmol, (1H, d, J=8.0Hz), 7.35–7.32 (1H, m), 7.17 (1H, d, J=2.3Hz),
1
98%). H-NMR (500MHz, CDCl3) δ: 8.07 (1H, d, J=8.0Hz), 6.88 (1H, dd, J=2.0, 8.3Hz), 3.94 (2H, t, J=6.3Hz), 1.72 (2H,
7.70–7.67 (1H, m), 6.19 (1H, dd, J=2.6, 8.3Hz), 3.90 (1H, d, tt, J=6.3, 7.4Hz), 0.97 (3H, t, J=7.4Hz). 13C-NMR (125MHz,
J=6.6Hz), 3.63 (1H, brs), 1.83 (1H, qt, J=7.5, 6.6Hz), 1.04, DMSO-d6) δ: 175.86, 159.67, 154.23, 139.57, 139.16, 135.63,
(3H, t, J=7.5 Hz).
129.77, 127.39, 124.36, 122.08, 121.02, 116.19, 115.66, 115.01,
2-Amino-N-(4-chloro-3-ethoxyphenyl)benzamide (23a) To 70.18, 21.78, 10.25. FAB-MS m/z 347 (MH+). Anal. Calcd for
a solution of 22a (149mg, 0.464mmol) in AcOEt (4.0mL) was C17H15ClN2O2S·0.5H2O: C, 57.38; H, 4.53; N, 7.87. Found: C,
added SnCl2·2H2O (334mg, 1.48mmol). The reaction mixture 57.49; H, 4.57; N, 7.98.
was stirred at 60°C for 4.5h, then quenched with NaHCO3 aq.
3-(4-Chloro-3-isopropoxyphenyl)-2-thioxoquinazoline-4-one
and filtered through a pad of Celite. The filtrate was diluted (24c) This compound was prepared from 23c by a method
with AcOEt and washed with water and brine). The organic similar to that used for preparation of mdivi-1. Pale yel-
layer was dried and concentrated. Column chromatography low solid (60.1mg, 0.172mmol, 74%) (recrystallized from
(n-hexane:AcOEt=3:1) gave the title compound (108mg, CHCl3/n-hexane/MeOH). mp>300°C. 1H-NMR (500MHz,
1
0.371mmol, 80%) as a pale yellow solid. H-NMR (500MHz, DMSO-d6) δ: 7.91 (1H, dd, J=1.2, 8.0Hz), 7.75–7.72 (1H, m),
CDCl3) δ: 7.71 (1H, brs), 7.49 (1H, d, J=2.3Hz), 7.43–7.41 (1H, 7.44 (1H, d, J=8.0Hz), 7.39 (1H, d, J=8.0Hz), 7.32–7.30 (1H,
m), 7.27 (1H, d, J=8.6Hz), 7.25–7.22 (1H, m), 6.85 (1H, dd, m), 7.16 (1H, d, J=2.3Hz), 6.83 (1H, dd, J=2.3, 8.6Hz), 4.05
J=2.3, 8.6Hz), 6.70–6.67 (2H, m), 5.45 (2H, brs), 4.12 (2H, q, (1H, sep, J=5.7Hz), 0.96 (6H, t, J=5.7Hz). FAB-MS m/z 347
J=6.9Hz), 1.45 (3H, t, J=6.9 Hz).
2-Amino-N-(4-chloro-3-propoxyphenyl)benzamide
(MH+). Anal. Calcd for C17H15ClN2O2S·0.4H2O: C, 57.67; H,
(23b) 4.50; N, 7.91. Found: C, 57.77; H, 4.43; N, 7.73.

988
Vol. 62, No. 10
3-(3-Butoxy-3-chlorophenyl)-2-thioxoquinazoline-4-one 50µL of Ala-AMC (10mM) was added at 37°C and incuba-
(24d) This compound was prepared from 23d by a method tion was continued for exactly 30min. At this point, 1.5mL of
similar to that used for preparation of mdivi-1. Yellow solid 1M AcONa–AcOH (pH 4.0) was added to stop the enzymatic
(122mg, 0.338mmol, 98%) (recrystallized from CHCl3/n- reaction. The amounts of liberated AMC were measured in
1
hexane/MeOH). mp 292–293°C. H-NMR (500MHz, CDCl3) terms of fluorescence intensity (excitation at 355nm, emis-
δ: 10.20 (1H, brs), 8.13 (1H, dd, J=1.2, 8.0Hz), 7.68–7.65 sion at 460nm) with a Wallac 1420 multilabel counter (Per-
(1H, m), 7.47 (1H, d, J=7.5Hz), 7.33–7.30 (1H, m), 7.10 (1H, kinElmer, Inc., Life Science) and a Wallac Envision 2104 mul-
d, J=8.0Hz), 6.78–6.76 (1H, m), 4.02–3.95 (2H, m), 1.81–1.76 tilabel reader (PerkinElmer, Inc., Life Science). The assay was
(2H, m), 1.52–1.44 (2H, m), 0.94 (3H, t, J=7.5Hz). FAB-MS performed at least in duplicate, and the mean value was taken.
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.
Acknowledgments This work was supported by Platform
for Drug Discovery, Informatics, and Structural Life Science
from the Ministry of Education, Culture, Sports, Science and
Biology
Cell Culture MOLT4 cells were cultured in RPMI me- Technology of Japan. We thank Dr. Kenji Ohgane for helpful
dium supplemented with 10% FBS and penicillin and strepto- discussion.
mycin at 37°C in a humidified incubator (5% CO2 in air).
Expression and Purification of Drp1 Protein Histi-
dine-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 0.1mM isopropyl-β-D-
thiogalactopyranoside (IPTG) at 25°C for 19h. Cell suspen-
sions were centrifuged (7500×g for 5min, 4°C). Bacterial
pellets were resuspended in 25mM Tris–HCl (pH 8.0) buffer
containing 300mM NaCl, 10% glycerol (v/v), 1mM phenyl-
methylsulfonyl fluoride (PMSF). Cell pellets were homog-
enized by sonication (for 4min×3 times) and further centri-
fuged (16000×g for 5min, 4°C). Supernatants were purified
over Ni-NTA resin. Soluble His-tagged proteins were loaded
on Ni-NTA resin, which was washed with 25mM Tris–HCl
buffer containing 300mM NaCl, 10% glycerol (v/v) for twice,
and 25mM Tris–HCl buffer containing 300mM NaCl, 20mM
imidazole, 10% glycerol (v/v) for 4 times and then eluted with
25mM Tris–HCl buffer containing 300mM NaCl, 200mM im-
idazole, 10% glycerol (v/v). Eluted proteins were quantified
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) with Oriole staining.
Assay of Drp1-Inhibitory Activity This assay was per-
formed using QuantiChrom™ ATPase/GTPase Assay Kit
(BioAssay Sytems, DATG-200) according to the protocol rec-
ommended by the supplier. Briefly, the reaction mixture con-
sisting of assay buffer (20µL), approximately 1µM Drp1 (5µL)
and test compound in DMSO (5µL) was preincubated at 37°C
for 15min. GTP solution (1mM, 10µL) was added, and the re-
action mixture was incubated at 37°C for 60min. The reagent
(200µL) was added and incubation was continued at r.t. for
30min. OD600 was measured with a Wallac 1420 multilabel
counter (PerkinElmer, Inc., Life Science) and a Wallac Envi-
sion 2104 multilabel reader (PerkinElmer, Inc., Life Science).
The assay was performed at least in duplicate, and the mean
value was taken.
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