Quantitative structure-activity relationships of 1,3,4-thiadiazol-2(3H)- ones and 1,3,4-oxadiazol-2(3H)-ones as human protoporphyrinogen oxidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.10.079

Protoporphyrinogen oxidase (Protox, EC 1.3.3.4) has attracted great interest during the last decades due to its unique biochemical characteristics and biomedical significance. As a continuation of our research work on the development of new PPO inhibitors

Full text of DOI:10.1016/j.bmc.2011.10.079

Bioorganic & Medicinal Chemistry 20 (2012) 296–304
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Bioorganic & Medicinal Chemistry
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Quantitative structure–activity relationships of 1,3,4-thiadiazol-2(3H)-ones and
1,3,4-oxadiazol-2(3H)-ones as human protoporphyrinogen oxidase inhibitors
Yang Zuo a, , Sheng-Gang Yang a, , Li-Li Jiang a, Ge-Fei Hao a, Zhi-Fang Wang a,b, Qiong-You Wu a,
a,
Zhen Xi b, , Guang-Fu Yang
⇑
⇑
a Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, PR China
b State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Protoporphyrinogen oxidase (Protox, EC 1.3.3.4) has attracted great interest during the last decades due
to its unique biochemical characteristics and biomedical significance. As a continuation of our research
work on the development of new PPO inhibitors, 23 new 1,3,4-thiadiazol-2(3H)-ones bearing benzothi-
azole substructure were designed and synthesized. The in vitro assay indicated that the newly synthe-
sized compounds 1a–w displayed good inhibition activity against human PPO (hPPO) with Ki values
Received 3 October 2011
Revised 29 October 2011
Accepted 29 October 2011
Available online 10 November 2011
ranging from 0.04 lM to 245 lM. To the knowledge, compound 1a, O-ethyl S-(5-(5-(tert-butyl)-2-oxo-
Keywords:
Human protoporphyrinogen oxidase
inhibitors
1,3,4-Thiadiazol-2(3H)-ones
1,3,4-Oxadiazol-2(3H)-ones
QSAR
1,3,4-thiadiazol-3(2H)-yl)-6-fluorobenzothiazol-2-yl)carbonothioate, with the Ki value of 40 nM, is so
far known as the most potent inhibitor against hPPO. Based on the molecular docking and modified
molecular mechanics/Poisson–Boltzmann surface area (MM-PBSA) calculations, the quantitative struc-
ture–activity relationships of 1,3,4-thiadiazol-2(3H)-ones and 1,3,4-oxadiazol-2(3H)-one derivatives
were established with excellent correlation relationships (r2 = 0.81) between the calculated and experi-
mental binding free energies. Some important insights were also concluded for guiding the future
rational design of new hPPO inhibitors with improved potency.
Molecular docking
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
in the research field of agricultural chemistry, PPO in plants has
been identified as one of the most important targets for herbicide
Protoporphyrinogen-IX oxidase (PPO; EC 1.3.3.4) is the last
common enzyme in the biosynthetic pathway leading to heme
and chlorophyll synthesis. Its function is to catalyze the transfor-
mation from protoporphyrinogen-IX to protoporphyrin-IX.1–3 In
mammals, there is only one isoform of protoporphyrinogen-IX
oxidase located on the cytosolic side of the inner mitochondrial
membrane.4–6 However, high plants have two isoforms, plastidic
PPO (PPO1) and the mitochondrial PPO (PPO2).7–11 During the last
decades, research interest in PPO has been increasing due to its un-
ique biochemical characteristics and biomedical significance. For
example, partial PPO deficiency in humans will cause an inherited
disease known as variegated porphyria (VP), whose symptoms in-
clude acute abdominal pain, neurological manifestations, and/or
cutaneous photosensitivity.12–16 This disease is relatively common
in the white African population of South Africa and be more com-
mon in women than in men,17,18 and may suddenly occur at any
age including the adolescent and elderly.17,19 It is now known that
the PPO deficiency is the primary genetic defect in VP,20 and the
activity of PPO in VP patients is reduced by at least 50%.21 Besides,
discovery since the introduction of herbicidal diphenyl ethers in
the 1970s.22 A great family of structurally diverse PPO inhibitors
has been developed as commercial herbicides for weed control in
agriculture.23–30
In addition, the most potentially useful application of PPO
inhibitors is to be used as a treatment for destroying cancer cells
via photodynamic therapy (PDT),31,32 which has been used as a fea-
sible medical technology in the detection and treatment of cancer
and other diseases.33 PDT always involves three key components: a
photosensitizer, light, and tissue oxygen.34 When the PPO is inhib-
ited, the substrate protoporphyrinogen-IX will accumulate and be
exported to the cytoplasm where it is slowly oxidized by O2 in
the tumor cells, producing protoporphyrin-IX. In the presence of
light, the photosensitive protoporphyrin-IX generates singlet
oxygen that causes lipid peroxidation and cell death. Therefore,
protoporphyrinogen-IX is an extremely effective photosensitizer
after it is activated by PPO inhibitors. Halling et al.35 showed that
PPO inhibitors could activate the photosensitizer protoporphyrino-
gen-IX and cause its accumulation within tumor cells.
Recently, we reported the high-resolution (1.9 Å) crystal
structure of human PPO (hPPO) bound to acifluorfen,36 which
was superimposed onto the PPOs from Nicotiana tabacum
⇑
Corresponding authors. Tel.: +86 27 67867800; fax: +86 27 67867141 (G.-F.Y.).
E-mail address: gfyang@mail.ccnu.edu.cn (G.-F. Yang).
Co-first authors.
(mtPPO)30 and gave an RMSD value of 1.8 Å for 411 C
a atoms.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.079

Y. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 296–304
297
O
O
X
X
O
Cl
O
S
N
N
S
S
O
Cl
N
N
N
N
N
N
RS
RS
RO
1a~w
1,3,4-Thiadiazol-2(3H)-ones
Oxadiazon [R = CH(CH3)2]
Oxadiargyl [R = CH2CCH]
1,3,4-Oxadiazol-2(3H)-ones
Scheme 1. Molecular design of the title compound 1a–w.
Although the conformation is highly similar to the FAD-binding
and substrate-binding domains, acifluorfen showed much weaker
2.1.1. Data for 4a
Overall yield: 25%; oil; 1H NMR (400 MHz, CDCl3): d 1.36 (s, 9H,
–C(CH3)3), 7.35–7.44 (m, 2H, Ar-H), 7.54–7.55 (m, 1H, Ar-H); ESI-
MS: 302.3 (M)+.
inhibition against hPPO (Ki = 1.71
lM) than against mtPPO
(Ki = 0.070 M). In addition, the Ki values against hPPO of the exist-
l
ing inhibitors are always on the order of micromolar potency, there
is so far no nanomolar hPPO inhibitor. Therefore, discovery of new
hPPO inhibitor with nanomolar potency is of great interest not only
for the elucidating of structural and functional implications of VP-
inducing mutations, but for the drug discovery associated with
PDT. Previously, we designed and synthesized a series of 1,3,4-oxa-
diazol-2-(3H)-one derivatives as hPPO inhibitors.37 Very interest-
2.1.2. Data for 4b
Overall yield: 36%; oil; 1H NMR (400 MHz, CDCl3): d 1.35 (s, 9H,
–C(CH3)3), 7.22–7.28 (m, 2H, Ar-H), 7.42–7.46 (m, 1H, Ar-H); ESI-
MS: 286.03 (M)+.
2.1.3. Data for 4c
ingly, some compounds with Ki values against hPPO under 1
lM
Overall yield: 39%; oil; 1H NMR (400 MHz, CDCl3): d 1.36 (s, 9H,
–C(CH3)3), 7.12–7.17 (m, 1H, Ar-H), 7.44–7.48 (m, 2H, Ar-H); ESI-
MS: 352.6 (M+Na)+.
were successfully discovered. In order to discover new hPPO inhib-
itors with higher potency, we further designed a series of new
1,3,4-thiadiazol-2(3H)-ones by the strategy of oxygen–sulfur bio-
isosterism substitution (Scheme 1). Very interestingly, a compound
with Ki value of 40.00 nM was successfully discovered, to the
knowledge, which is so far known as the most potent hPPO
inhibitor.
2.2. Preparation of 5-tert-butyl-3-(2,4-disubstituted-5-nitro-
phenyl)-1,3,4-thiadiazol-2(3H)-ones (5a–c)
A solution of HNO3 (1.07 g, 11.3 mmol) in concentrated H2SO4
(2.26 g, 22.6 mmol) was added dropwise to a stirred mixture of
4a–c (11.3 mmol) and concentrated H2SO4 (226 mL) in an ice bath.
The complete addition took about 15 min. After stirring for 3 h, the
solution was slowly poured into a mixture of ice and water
(500 mL). The resulted solid was collected by filtration and washed
with water (2000 mL), then dried to give to the desired solid
products.
2. Materials and methods
All chemical reagents were commercially available and treated
with standard methods before use. Solvents were dried in a routine
way and redistilled. 1H NMR spectra was recorded on a Mercury-
Plus 400 spectrometer in CDCl3 or DMSO-d6 with TMS as the inter-
nal reference. Mass spectral data were obtained on a MocroMass
platform by electrospray ionization (ESI-MS). Elementary analyses
were performed on a Vario EL III elementary analysis instrument.
Melting points were taken on a Buchi B-545 melting point appara-
tus and uncorrected. The starting materials of N0-(2,4-disubstitut-
edphenyl)pivalohydrazides (2a–c) were prepared according to
the existing method.37
2.2.1. Data for 5a
Yield: 65%; oil; 1H NMR (400 MHz, CDCl3): d 1.37 (s, 9H,
–C(CH3)3), 7.77 (s, 1H, Ar-H), 8.13 (s, 1H, Ar-H). ESI-MS: 369.1
(M+Na)+.
2.2.2. Data for 5b
Yield: 64%; mp: 89–90 °C; 1H NMR (600 MHz, CDCl3): d 1.37
(s, 9H, –C(CH3)3), 7.46 (d, J = 9.0 Hz, 1H, Ar-H), 8.21 (d, J = 6.6 Hz,
1H, Ar-H). ESI-MS: 331.4 (M)+.
2.1. Preparation of 5-(tert-butyl)-3-(2,4-disubstitutedphenyl)-
1,3,4-thiadiazol-2(3H)-ones (4a–c)
2.2.3. Data for 5c
To a refluxing solution of compound 2 in dimethylether (30 mL)
was added P4S10 (7.5 mmol). The solvent was evaporated after the
reaction accomplished according to the TLC detection. The residue
was dissolved in CH2Cl2, and washed successively with water
(200 mL) and saturated solution of NaCl (200 mL), and then dried
over anhydrous magnesium sulfate. After evaporating the solvent,
the oil products of 3 was obtained and used directly for the subse-
quent reaction. The oil intermediate of 3 was dissolved in the solu-
tion of CH2Cl2, (C2H5)3N (45 mmol) was added. The mixture was
cooled to 0 °C, and then a solution of bis(trichloromethyl) carbon-
ate (8.82 g, 30 mmol) in CH2Cl2 (50 mL) was added dropwise under
a nitrogen atmosphere. The resulted mixture was stirred overnight
at room temperature, and then was refluxed for two hours. After
cooling, the solution was washed with water (200 mL) and brine
(200 mL), then dried, filtered, and concentrated to give the desired
oil products of 4a–c.
Yield: 80%; mp: 96–97 °C; 1H NMR (400 MHz, CDCl3): d 1.37
(s, 9H, –C(CH3)3), 7.70 (d, J = 9.6 Hz, 1H, Ar-H), 8.24 (d, J = 6.8 Hz,
1H, Ar-H). ESI-MS: 399.1 (M+Na)+.
2.3. Preparation of 3-(5-amino-2,4-disubstitutedphenyl)-5-tert-
butyl-1,3,4-thiadiazol-2(3H)-ones (6a–c):38
The powder of Fe was added portionwise to a stirred solution of
NH4Cl (0.83 g), 5a–c (10.4 mmol) in a mixture of EtOH/H2O (v/v:
10:1, 220 mL) at reflux temperature. After TLC detection showing
that reaction finished, the reaction mixture was filtered, concen-
trated to dryness. The residue was dissolved in water (200 mL)
and extracted with EtOAc (200 mL), the organic phase was washed
with brine (150 mL), dried, filtered, then concentrated to give the
desired oil or solid products.

298
Y. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 296–304
2.3.1. Data for 6a
(d, J = 8.4 Hz, 1H, Ar-H), 8.00 (d, J = 6.6 Hz, 1H, Ar-H). ESI-MS:
415.3 (M+2H)+. Anal. Calcd for C16H16FN3O3S3: C, 46.47; H, 3.90;
N, 10.16. Found: C, 46.74; H, 3.70; N, 10.43.
Yield: 67%; mp: 102–103 °C; 1H NMR (400 MHz, CDCl3): d 1.35
(s, 9H, –C(CH3)3), 4.14 (broad, 2H, –NH2), 6.86 (s, 1H, Ar-H), 7.40 (s,
1H, Ar-H). ESI-MS: 317.7 (M+H)+.
2.5.3. Data for 1c
2.3.2. Data for 6b
Yield: 73%; mp: 124–126 °C. 1H NMR (400 MHz, CDCl3): d 1.29
(t, J = 7.2 Hz, 3H, –CH3), 1.40 (s, 9H, –C(CH3)3), 4.17 (s, 2H, –
SCH2), 4.23 (q, J = 7.2 Hz, 2H, –OCH2), 7.64 (d, J = 8.4 Hz, 1H, Ar-
H), 8.08 (d, J = 6.0 Hz, 1H, Ar-H). ESI-MS: 427.2 (M)+. Anal. Calcd
for C17H18FN3O3S3: C, 47.76; H, 4.24; N, 9.83. Found: C, 48.02; H,
4.13; N, 9.62.
Yield: 67%; mp: 115–116 °C; 1H NMR (400 MHz, CDCl3): d 1.35
(s, 9H, –C(CH3)3), 4.25 (broad, 2H, –NH2), 6.90 (d, J = 6.0 Hz, 1H, Ar-
H), 7.18 (d, J = 10.0 Hz, 1H, Ar-H). ESI-MS: 301.6 (M)+.
2.3.3. Data for 6c
Yield: 91%; mp: 96–97 °C; 1H NMR (400 MHz, CDCl3): d 1.36 (s,
9H, –C(CH3)3), 3.95 (broad, 2H, –NH2), 6.87 (d, J = 8.0 Hz, 1H, Ar-H),
7.28 (d, J = 10.4 Hz, 1H, Ar-H). ESI-MS: 369.3 (M+Na)+.
2.5.4. Data for 1d
Yield: 70%; mp: 86–88 °C. 1H NMR (400 MHz, CDCl3): d 1.26 (t,
J = 7.2 Hz, 3H, –CH3), 1.38 (s, 9H, –C(CH3)3), 1.71 (d, J = 7.2 Hz, 3H, –
CH(CH3)), 4.22 (q, J = 7.2 Hz, 2H, –OCH2), 4.69 (q, J = 7.2 Hz, 1H, –
CH), 7.65 (d, J = 8.4 Hz, 1H, Ar-H), 8.00 (d, J = 6.0 Hz, 1H, Ar-H).
ESI-MS: 441.4 (M)+. Anal. Calcd for C36H40BrFN6O6S6: C, 45.80; H,
4.27; N, 8.90. Found: C, 45.51; H, 4.06; N, 9.20.
2.4. Preparation of 5-tert-butyl-3-(6-substituted-2-
mercaptobenzo[d]thiazol-5-yl)-1,3,4-thiadiazol-2(3H)-
ones (7a–c):39
A solution of 6a–c (9.45 mmol), potassium O-ethyl dithiocar-
bonate (3.03 g, 18.9 mmol) in 142 mL anhydrous DMF was heated
at 120 °C for 3 h. After TLC detection showing that reaction fin-
ished, the reaction mixture was evaporated under reduced pres-
sure to remove most solvent and diluted with water (300 ml),
and acidified with concentrated HCl solution to induce precipita-
tion (pH 3). Stirring was continued for 10 min. The solid precipitate
was collected by filtration, and rinsed with water. The wet filter
cake was dried and then purified by flash column chromatography
to give the pure products 7a–c.
2.5.5. Data for 1e
Yield: 78%; mp: 121–123 °C. 1H NMR (400 MHz, CDCl3): d 1.22
(t, J = 7.2 Hz, 3H, –CH3), 1.40 (s, 9H, –C(CH3)3), 1.76 (s, 6H, –
SC(CH3)2), 4.20 (t, J = 7.2 Hz, 2H, –OCH2), 7.63 (d, J = 8.4 Hz, 1H,
Ar-H), 8.03 (d, J = 6.4 Hz, 1H, Ar-H). ESI-MS: 455.3 (M)+. Anal. Calcd
for C19H22FN3O3S3: C, 50.09; H, 4.87; N, 9.22. Found: C, 49.86; H,
5.07; N, 9.22.
2.5.6. Data for 1f
Yield: 60%; mp: 86–87 °C. 1H NMR (600 MHz, CDCl3): d 1.27 (d,
J = 6.6 Hz, 6H, –CH(CH3)2), 1.40 (s, 9H, –C(CH3)3), 4.13 (s, 2H, –
SCH2), 5.08–5.10 (m, 1H, –OCH), 7.64 (d, J = 8.4 Hz, 1H, Ar-H),
7.97 (d, J = 6.6 Hz, 1H, Ar-H). ESI-MS: m/z 441.4 (M)+. Anal. Calcd
for C18H20FN3O3S3: C, 48.96; H, 4.57; N, 9.52. Found: C, 48.70; H,
4.37; N, 9.35.
2.4.1. Data for 7a
Yield: 60%; mp: 170–171 °C; 1H NMR (400 MHz, CDCl3): d 1.38
(s, 9H, –C(CH3)3), 7.23 (s, 1H, Ar-H), 7.56 (s, 1H, Ar-H), 10.52
(broad, 1H, –SH). ESI-MS: 357.5 (M)+.
2.4.2. Data for 7b
Yield: 76%; mp: 180–182 °C; 1H NMR (400 MHz, CDCl3): d 1.40
(s, 9H, –C(CH3)3), 7.38 (d, J = 8.4 Hz, 1H, Ar-H), 7.45 (d, J = 6.0 Hz,
1H, Ar-H), 10.92 (br, 1H, –SH). ESI-MS: 341.01 (M)+.
2.5.7. Data for 1g
Yield: 49%; mp: 88–89 °C. 1H NMR (600 MHz, CDCl3): d 0.93 (t,
J = 7.2 Hz, 3H, –CH3), 1.40 (s, 9H, –C(CH3)3), 1.67–1.70 (m, 2H, –
CH2–), 4.14 (t, J = 6.6 Hz, 2H, –OCH2), 4.17 (s,2H, –SCH2), 7.64 (d,
J = 8.4 Hz, 1H, Ar-H), 7.98 (d, J = 5.4 Hz, 1H, Ar-H). ESI-MS: 442.0
(M)+. Anal. Calcd for C18H20FN3O3S3: C, 48.96; H, 4.57; N, 9.52.
Found: C, 48.89; H, 4.69; N, 9.36.
2.4.3. Data for 7c
Yield: 68%; mp: 179–180 °C; 1H NMR (400 MHz, CDCl3): d 1.39
(s, 9H, –C(CH3)3), 7.21 (s, 1H, Ar-H), 7.72 (s, 1H, Ar-H), 10.45
(broad, 1H, –SH). ESI-MS: 400.2 (MꢀH)ꢀ.
2.5.8. Data for 1h
2.5. General synthetic procedure for the target compounds
1a–w
Yield: 63%; mp: 83–84 °C. 1H NMR (600 MHz, CDCl3): d 1.28 (t,
J = 7.2 Hz, 3H, –CH3), 1.40 (s, 9H, –C(CH3)3), 2.92 (t, J = 7.2 Hz, 2H, –
CH2CO), 3.60 (t, J = 7.2 Hz, 2H, –SCH2), 4.19 (q, J = 7.2 Hz, 2H, –
OCH2), 7.63 (d, J = 8.4 Hz, 1H, Ar-H), 7.99 (d, J = 6.0 Hz, 1H, Ar-H).
ESI-MS: 441.6 (M)+. Anal. Calcd for C18H20FN3O3S3: C, 48.96; H,
4.57; N, 9.52. Found: C, 48.73; H, 4.77; N, 9.31.
K2CO3 powder (0.33 g, 2.4 mmol) was added to a solution of
7a–c (1.2 mmol) in acetone (20 mL). After stirring for 10 min at
room temperature, RX (1.8 mmol) in acetone solution was added
dropwise to the mixture. The resulted mixture reacted for about
30 min, then filtered, concentrated. The residue was purified via
flash chromatography to give the pure products 1a–w in yields of
42–78%.
2.5.9. Data for 1i
Yield: 50%; oil. 1H NMR (400 MHz, CDCl3): d 1.24 (t, J = 7.2 Hz,
3H, –CH3), 1.40 (s, 9H, –C(CH3)3), 2.14–2.21 (m, 2H, –CH2–), 2.49
(t, J = 7.2 Hz, 2H, –CH2CO), 3.40 (t, J = 7.2 Hz, 2H, –SCH2), 4.12 (q,
J = 7.2 Hz, 2H, –OCH2), 7.63 (d, J = 8.8 Hz, 1H, Ar-H), 7.98 (d,
J = 6.4 Hz, 1H, Ar-H). ESI-MS: 455.5 (M)+. Anal. Calcd for
2.5.1. Data for 1a
Yield: 69%; mp: 146–148 °C. 1H NMR (600 MHz, CDCl3): d 1.37–
1.43 (m, 12H, –C(CH3)3, –CH3), 4.47 (q, J = 7.2 Hz, 2H, –OCH2), 7.77
(d, J = 8.4 Hz, 1H, Ar-H), 8.15 (d, J = 6.6 Hz, 1H, Ar-H). ESI-MS: 415.4
(M+2H)+. Anal. Calcd for C16H16FN3O3S3: 46.47; H, 3.90; N, 10.16.
Found: C, 46.73; H, 3.65; N, 10.43.
C19H22FN3O3S3: C, 50.09; H, 4.87; N, 9.22. Found: C, 49.70; H,
4.96; N, 8.93.
2.5.10. Data for 1j
Yield: 60%; mp: 154–157 °C. 1H NMR (600 MHz, CDCl3): d 1.40
(s, 9H, –C(CH3)3), 2.31 (s, 1H, –CH), 4.13 (s, 2H, –SCH2), 7.66 (d,
J = 9.0 Hz, 1H, Ar-H), 8.05 (d, J = 6.0 Hz, 1H, Ar-H). ESI-MS: 380.2
2.5.2. Data for 1b
Yield: 74%; mp: 111–112 °C. 1H NMR (600 MHz, CDCl3): d 1.40
(s, 9H, –C(CH3)3), 3.79 (s, 3H, –OCH3), 4.18 (s, 2H, –SCH2), 7.64

Y. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 296–304
299
(M+H)+.Anal. Calcd for C16H14FN3OS3: C, 50.64; H, 3.72; N, 11.07.
Found: C, 50.45; H, 3.50; N, 10.89.
3H, –CH(CH3)), 4.22 (q, J = 7.2 Hz, 2H, –OCH2), 4.68 (q, J = 7.2 Hz,
1H, –SCH), 7.94 (s, 1H, Ar-H), 8.11 (s, 1H, Ar-H). ESI-MS: 501.9
(M+H)+. Anal. Calcd for C18H20BrN3O3S3: C, 43.03; H, 4.01; N,
8.36. Found: C, 43.04; H, 3.85; N, 8.21.
2.5.11. Data for 1k
Yield: 65%; mp: 137–138 °C. 1H NMR (600 MHz, CDCl3): d 1.37
(s, 9H, –C(CH3)3), 1.40 (t, J = 7.2 Hz, 3H, –CH3), 4.45 (q, J = 7.2 Hz,
2H, –OCH2), 8.04 (s, 1H, Ar-H), 8.08 (s, 1H, Ar-H). ESI-MS: 429.9
(M+H)+. Anal. Calcd for C16H16ClN3O3S3: C, 44.69; H, 3.75; N,
9.77. Found: C, 44.80; H, 4.00; N, 9.60.
2.5.20. Data for 1t
Yield: 75%; mp: 94–95 °C. 1H NMR (400 MHz, CDCl3): d 1.21 (t,
J = 7.2 Hz, 3H, –CH3), 1.38 (s, 9H, –C(CH3)3), 1.76 (s, 6H, –SC(CH3)2),
4.17 (q, J = 7.2 Hz, 2H, –OCH2), 7.98 (s, 1H, Ar-H), 8.10 (s, 1H, Ar-H).
ESI-MS: 515.9 (M+H)+. Anal. Calcd for C19H22BrN3O3S3: C, 44.18; H,
4.29; N, 8.14. Found: C, 44.11; H, 4.50; N, 8.15.
2.5.12. Data for 1l
Yield: 58%; mp: 103–105 °C. 1H NMR (400 MHz, CDCl3): d 1.25
(t, J = 7.2 Hz, 3H, –CH3), 1.37 (s, 9H, –C(CH3)3), 4.17 (s, 2H, –
SCH2), 4.22 (q, J = 7.2 Hz, 2H, –OCH2), 7.91 (s, 1H, Ar-H), 7.95 (s,
1H, Ar-H). ESI-MS: 443.0 (M)+. Anal. Calcd for C17H18ClN3O3S3: C,
45.99; H, 4.09; N, 9.46. Found: C, 46.11; H, 4.42; N, 9.54.
2.5.21. Data for 1u
Yield: 63%; oil. 1H NMR (400 MHz, CDCl3): d 1.26 (t, J = 7.2 Hz,
3H, –CH3), 1.38 (s, 9H, –C(CH3)3), 2.90 (t, J = 6.8 Hz, 2H, –CH2CO),
3.59 (t, J = 6.8 Hz, 2H, –SCH2), 4.16 (q, J = 7.2 Hz, 2H, –OCH2), 7.96
(s, 1H, Ar-H), 8.07 (s, 1H, Ar-H). ESI-MS: 501.8 (M+H)+. Anal. Calcd
for C18H20BrN3O3S3: C, 43.03; H, 4.01; N, 8.36. Found: C, 42.80; H,
4.30; N, 8.23.
2.5.13. Data for 1m
Yield: 65%; oil. 1H NMR (400 MHz, CDCl3): d 1.26 (t, J = 7.2 Hz,
3H, –CH3), 1.38 (s, 9H, –C(CH3)3), 1.71 (d, J = 7.2 Hz, 3H, –CH(CH3)),
4.22 (q, J = 7.2 Hz, 2H, –OCH2), 4.67 (q, J = 7.2 Hz, 1H, –SCH), 7.91 (s,
1H, Ar-H), 7.95 (s, 1H, Ar-H). ESI-MS: 458.0 (M + H)+. Anal. Calcd for
C18H20ClN3O3S3: C, 47.20; H, 4.40; N, 9.17. Found: C, 47.42; H, 4.69;
N, 9.03.
2.5.22. Data for 1v
Yield: 62%; oil. 1H NMR (400 MHz, CDCl3): d 1.25 (t, J = 7.2 Hz,
3H, –CH3), 1.38 (s, 9H, –C(CH3)3), 2.13–2.20 (m, 2H, –CH2–), 2.49
(t, J = 7.2 Hz, 2H, –CH2CO), 3.41 (t, J = 7.2 Hz, 2H, –SCH2), 4.12 (q,
J = 7.2 Hz, 2H, –OCH2), 7.94 (s, 1H, Ar-H), 8.07 (s, 1H, Ar-H). ESI-
MS: 537.8 (M+Na)+. Anal. Calcd for C19H22BrN3O3S3: C, 44.18; H,
4.29; N, 8.14. Found: C, 44.44; H, 4.50; N, 8.19.
2.5.14. Data for 1n
Yield: 54%; mp: 98–99 °C. 1H NMR (400 MHz, CDCl3): d 1.21 (t,
J = 7.2 Hz, 3H, –CH3), 1.38 (s, 9H, –C(CH3)3), 1.76 (s, 6H, –SC(CH3)2),
4.17 (q, J = 7.2 Hz, 2H, –OCH2), 7.92 (s, 1H, Ar-H), 7.98 (s, 1H, Ar-H).
ESI-MS: 472.0 (M+H)+. Anal. Calcd for C19H22ClN3O3S3: C, 48.34; H,
4.70; N, 8.90. Found: C, 48.50; H, 4.90; N, 8.61.
2.5.23. Data for 1w
Yield: 70%; mp: 131–133 °C. 1H NMR (600 MHz, CDCl3): d 1.38
(s, 9H, –C(CH3)3), 2.30 (t, J = 2.4 Hz, 1H, –CH), 4.13 (d, J = 2.4 Hz,
2H, –SCH2), 8.00 (s, 1H, Ar-H), 8.10 (s, 1H, Ar-H). ESI-MS: 440.4
(M)+. Anal. Calcd for C16H14BrN3OS3: C, 43.64; H, 3.20; N, 9.54.
Found: C, 43.83; H, 3.53; N, 9.74.
2.5.15. Data for 1o
Yield: 42%; oil. 1H NMR (400 MHz, CDCl3): d 1.26 (t, J = 7.2 Hz,
3H, –CH3), 1.38 (s, 9H, –C(CH3)3), 2.90 (t, J = 7.2 Hz, 2H, –CH2CO),
3.59 (t, J = 7.2 Hz, 2H, –SCH2), 4.16 (q, J = 7.2 Hz, 2H, –OCH2), 7.90
(s, 1H, Ar-H), 7.96 (s, 1H, Ar-H). ESI-MS: 457.4 (M)+. Anal. Calcd
for C18H20ClN3O3S3: C, 47.20; H, 4.40; N, 9.17. Found: C, 47.43; H,
4.51; N, 9.22.
2.6. X-ray diffraction
A colorless block shaped crystal of 1n (0.16 ꢁ 0.12 ꢁ 0.10 mm)
was selected and mounted on a Bruker SMART APEX CCD Single
Crystal Diffraction System with graphite-monochromated MoK
a
2.5.16. Data for 1p
radiation (k = 0.71073 Å) and x-ray provided by a fine-focus sealed
tube operated at 50 kV and 30 mA.40 The crystal determined at
298(2) K was crystallized in the monoclinic, C2/c space group with
a = 21.842(2) Å, b = 9.3205(8) Å, c = 24.884(2) Å, b = 115.026(3)°,
Yield: 52%; oil. 1H NMR (400 MHz, CDCl3): d 1.25 (t, J = 7.2 Hz,
3H, –CH3), 1.37 (s, 9H, –C(CH3)3), 2.15–2.19 (m, 2H, –CH2–), 2.49
(t, J = 6.8 Hz, 2H, –CH2CO), 3.41 (t, J = 6.8 Hz, 2H, –SCH2), 4.12 (q,
J = 7.2 Hz, 2H, –OCH2), 7.90 (s, 1H, Ar-H), 7.95 (s, 1H, Ar-H). ESI-
MS: 471.6 (M+H)+. Anal. Calcd for C19H22ClN3O3S3: C, 48.34; H,
4.70; N, 8.90. Found: C, 48.63; H, 4.91; N, 8.93.
V = 4590.1(8) Å3, Z = 4, dcalcd = 1.366 g/cm3 and la(MoK ) =
a
0.464 mmꢀ1. Lattice constants were determined with the Bruker
SAINT software package using peak centers for 6195 reflections.41
Then a full hemisphere of diffracted intensities with an
x scan
2.5.17. Data for 1q
width of 0.30° was measured. A total of 14627 integrated reflection
intensities having 2.06° < h < 26.0° were produced using the Bruker
program SAINT and 4517 of these reflections were unique. The
Bruker software package SHELXTL42 was used to solve the structure
using ‘direct methods’ techniques. All stages of weighted full-
matrix least-squares refinement were conducted using F2O data
with the SHELXTL Version 6.10 software package. All the non-
hydrogen atoms were found from the difference maps and refined
anisotropically. All the hydrogen atoms were located their ideal
positions with C–H@0.98 Å (methyl) and 0.93 Å (aromatic) and
their isotropic thermal parameters of were fixed at values 1.5
(methyl) or 1.2 (aromatic) times of the equivalent isotropic ther-
mal parameter of their parent carbon atoms. A total of 268 param-
eters were refined in the refinement. Final agreement factors at
convergence are: R1 = 0.0535, wR2 = 0.1410 and Gof = 1.068 for
Yield: 79%; mp: 111–112 °C. 1H NMR (400 MHz, CDCl3): d 1.38
(s, 9H, –C(CH3)3), 2.30 (t, J = 2.4 Hz, 1H, –CH), 4.13 (d, J = 2.4 Hz,
2H, –SCH2), 7.93 (s, 1H, Ar-H), 8.00 (s, 1H, Ar-H). ESI-MS: 395.5
(M)+. Anal. Calcd for C16H14ClN3OS3: C, 48.53; H, 3.56; N, 10.61.
Found: C, 48.60; H, 3.80; N, 10.65.
2.5.18. Data for 1r
Yield: 44%; mp: 104–105 °C. 1H NMR (400 MHz, CDCl3): d 1.27
(t, J = 7.2 Hz, 3H. –CH3), 1.37 (s, 9H, –C(CH3)3), 4.16 (s, 2H, –
SCH2), 4.22 (q, J = 7.2 Hz, 2H, –OCH2), 7.94 (s, 1H, Ar-H), 8.08 (s,
1H, Ar-H). ESI-MS: 487 (MꢀH)+, 489.0 (M+H)+. Anal. Calcd for
C17H18BrN3O3S3: C, 41.80; H, 3.71; N, 8.60. Found: C, 41.62; H,
3.90; N, 8.52.
2.5.19. Data for 1s
3892 independent reflections with I >2r(I); R1 = 0.0606, wR2 =
0.1463 and Gof = 1.068 for all the 4517 independent reflections.
The largest shift/s.u. was 0.000 in the final refinement cycle. The
Yield: 60%; mp: 88–90 °C. 1H NMR (600 MHz, CDCl3): d 1.26 (t,
J = 7.2 Hz, 3H, –CH3), 1.40 (s, 9H, –C(CH3)3), 1.71 (d, J = 7.2 Hz,

300
Y. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 296–304
final difference map had maxima and minima of 0.561 and
suffered from sulfuration with P4S10 to afford N0-(2,4-disubsti-
tuted-phenyl)-2,2-dimethylpropanethiohydrazide (3a–c), which
reacted directly without purification with bis(trichloromethyl)
carbonate under a nitrogen atmosphere to afford 5-(tert-butyl)-
ꢀ0.289 eꢀ/Å3, respectively.
2.7. Determination of the Ki values of the title compounds
against human PPO
3-(2,4-disubstitutedphenyl)-1,3,4-thiadiazol-2(3H)-ones
(4a–c).
The intermediates of 4a–c reacted with a solution of HNO3 in con-
centrated H2SO4 to give 5-tert-butyl-3-(2,4-disubstituted-5-nitro-
phenyl)-1,3,4-thiadiazol-2(3H)-ones (5a–c) in yields of 64–80%,
which were reduced by Fe powder to afford 3-(5-amino-2,4-disub-
stitutedphenyl)-5-tert-butyl-1,3,4-thiadiazol-2(3H)-ones (6a–c).
The intermediates of 6a–c underwent ring-closing reaction with
potassium O-ethyl carbonodithioate in the DMF solution to pro-
duce the key intermediate of 5-tert-butyl-3-(6-substituted-2-mer-
captobenzo[d]thiazol-5-yl)-1,3,4-thiadiazol-2(3H)-ones (7a–c) in
acceptable yields (60–76%). Finally, intermediate 7a–c reacted
with diverse alkylation reagent to give the target compounds 1a–
w in yields of 42–78%. The structures of all intermediates and title
compounds were confirmed by elemental analyses, 1H NMR and
ESI-MS spectral data. In addition, the crystal structure of 1n was
determined by X-ray diffraction analyses. As can be observed from
the structure of 1n shown in Figure 1, the thiadiazole ring is almost
perpendicular to the benzothiazol ring with a dihedral angle of
89.9° between them. In the crystal packing, the adjacent molecules
are linked into a one-dimensional chain running parallel to the
[010] direction by the C9–H9ꢂ ꢂ ꢂO3 (x, 1+y, z, dC9ꢂ ꢂ ꢂO3 = 3.278(3) Å)
hydrogen bond. Further analysis shows that these neighboring
chains are joined together into the final three-dimensional
The detailed method for enzyme expression, purification and
inhibition kinetic analysis has been described previously.37,43 The
kinetic parameters were evaluated by Sigma Plot software 10.0
(SPSS, Chicago, USA). IC50 was determined by measuring PPOX
activity over a range of inhibitor concentrations at a single sub-
strate concentration. IC50 values were calculated by fitting
[I] data to a single binding site model described by Eq. 1
v
versus
max ꢀ min
y ¼ min þ
50ꢀx
ð1Þ
1 þ 10log IC
where y is the percentage of maximal rate, max and min are the y
values at which the curve levels off, x is the logarithm of inhibitor
concentration, and IC50 is the inhibitor concentration that elicits
50% of the total inhibition. Calculated Ki value is obtained by
applying the following relationship, which exists for competitive
inhibition among Ki, Km, and IC50 at any saturating substrate con-
centration (S).
IC50
S=Km þ 1
Ki ¼
ð2Þ
network by two
p p interactions for one between the
ꢂ ꢂ ꢂ
2.8. Computational simulations and binding free energies cal-
culations
C7–C12 benzene ring and the S2-involved thiazole ring
(dcentroid-to-centroid = 3.758(2) Å), and the other one between two
symmetry-related thiadiazole rings (dcentroid-to-centroid = 3.950(2) Å).
The chemical structures of compounds were constructed by
using SYBYL 7.0. The optimized geometries were used as the start-
ing structures for docking study. Docking calculations were per-
formed by using AutoDock4.0.44 The structures of protein and
ligand were prepared with AutoDock Tools.45 A total of 256 runs
were launched for each molecule. Most of the parameters for the
docking calculation were set to the default values recommended
by the program. For each docked structure, a modified molecular
mechanics/Poisson–Boltzmann surface area (MM/PBSA) described
before46 was performed to compare binding energies by analyzing
binding strength between the protein and the ligand determined
by electrostatic, hydrophilic, and hydrophobic interactions. Before
the MM/PBSA calculation, the complex structure was further re-
fined with the steepest descent algorithm first and then the conju-
gated gradient algorithm by using Amber9 package.47 The partial
atomic charges of ligands were determined using the AM1-BCC
method implemented in the Antechamber module of the Amber9
program.47 During the energy minimization process, the receptor
was first fixed and only the ligand was kept free, then the ligand
and residue sidechains were kept free. Finally, all atoms of the sys-
tem were kept free and refined to a convergence of 0.01 kcal/
(mol Å). The refined structure was used in the final binding energy
calculation. The energy minimization calculations of the fluorine,
chlorine and bromine derivatives for the comparison of the dihe-
dral angle between the oxadiazole or thiadiazole and the benzothi-
azole ring were performed using the GAUSSIAN 03 program.48
3.2. Inhibition activity of 1,3,4-thiadiazol-2(3H)-ones against
hPPO
The ki values against hPPO of the newly synthesized 1,3,4-thia-
diazol-2(3H)-ones 1a–w were listed in Table 1. Oxadiazon, a com-
mercial herbicide acting as PPO inhibitor, were used as a control.
For the sake of clarity, compounds 1a–j, 1k–q and 1r–w will be
named as fluorine, chlorine, and bromine derivatives, respectively
through the text. Results as shown in Table 1 indicated that com-
pound 1a (R = COOC2H5, Ki = 0.04
hPPO-inhibition activity, about 73-fold higher than oxadiazon
(Ki = 2.93 M). In addition, compound 1k (R = COOC2H5, Ki =
0.20 M) also displayed the highest hPPO inhibition activity within
lM) displayed the highest
l
l
the chlorine derivatives. These results showed that COOC2H5 might
be the best substituent among the investigated R groups. As far as
the same R group be concerned, fluorine derivatives (compounds
1a–1j) always displayed much higher hPPO-inhibition activity
than chlorine derivatives (compounds 1k–1q) and bromine
derivatives (compounds 1r–1w). However, the chlorine derivative
1o (Ki = 1.28
exception and became the stronger hPPO inhibitor than the
corresponding fluorine derivative 1h (Ki = 9.26 M) and bromine
derivative 1v (Ki = 27.5 M).
lM), whose R group is CH2CH2COOCH2CH3, is an
l
l
In order to understand the structure–activity relationships, we
compared the hPPO-inhibition activity of compounds 1a–w with
their corresponding 1,3,4-oxadiazol-2(3H)-one derivatives which
were previously synthesized.37 As shown in Table 1, apart from
compounds 1a– c and 1k, other compound 1 displayed lower
hPPO-inhibition activity than their corresponding 1,3,4-oxadiazol-
2(3H)-one derivatives. Very interestingly, for the 1,3,4-oxadiazol-
2(3H)-one derivatives, the fluorine derivative sometimes displayed
higher activity than the corresponding chlorine derivatives
containing the same R group, while sometimes lower activity than
the corresponding chlorine derivatives. However, in most cases,
3. Results and discussion
3.1. Synthetic chemistry of the title compounds
As shown in Scheme 2, the target compounds 1a–w were pre-
pared by a six-step synthetic route. The starting materials of
N0-(2,4-disubstitutedphenyl)pivalohydrazides (2a–c) were pre-
pared according to the existing method. Compounds 2a–c were

Y. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 296–304
301
X
X
O
X
a
b
S
Y
NH
HN
S
Y
NH
HN
O
Y
N
N
2a, X = Cl, Y = Cl
4a, X = Cl, Y = Cl
4b, X = F, Y = Cl
3
a, X = Cl, Y = Cl
2
2
b, X = F, Y = Cl
c, X = Br, Y = F
3b, X = F, Y = Cl
3c, X = Br, Y = F
4
c, X = Br, Y = F
c
O
X
O
O
X
X
S
S
S
d
e
Y
N
Y
N
N
S
N
N
N
N
H2N
O2N
HS
5a, X = Cl, Y = Cl
5b, X = F, Y = Cl
7
a, X = Cl
6
a, X = Cl, Y = Cl
7b, X = F
6b, X = F, Y = Cl
6c, X = Br, Y = F
5
c, X = Br, Y = F
7c, X = Br
f
X
O
S
N
S
N
R
N
S
1 ~
a w
Reagents and conditions: a, P4S10, DME, reflux; b, (COCl2)3, NEt3, toluene, N2; c, HNO3/H2SO4, 00C; d, iron powder,
NH4Cl, EtOH/H2O, ref lux; e, EtOC(S)SK, DMF, relux, con. HCl; f , acetone, K2CO3, RX, rt.
Scheme 2. Synthetic route for the title compound 1a–w.
Figure 1. Crystal structure of compound 1n.
the fluorine derivatives of 1,3,4-thiadiazol-2(3H)-ones displayed
higher activity than the corresponding chlorine derivatives. One
similarity is that the bromine derivatives of 1,3,4-oxadiazol-
2(3H)-one and 1,3,4-thiadiazol-2(3H)-one always displayed the
lowest hPPO-inhibition activity.
the experimental binding free energies range from ꢀ4.93 to
ꢀ10.10 kcal/mol. Obviously, the MM-PBSA calculations overesti-
mated the absolute binding affinities of the ligands with the en-
zyme. However, the calculated values are expected to reflect the
relative change of the binding affinities of the compounds. Qualita-
tively in comparison with the experimental data, there is a good
3.3. Binding free energies calculation and quantitative
structure–activity relationships
linear correlation (r2 = 0.81) between
DGcal and DGexp derived from
the experimental data (Fig. 2), suggesting that the computational
models constructed and tested in this study should be reliable.
Because the oxygen atom in oxadiazole is more electronegative
but less bulky than sulfur atom in thiadiazole, compared with the
oxadiazole ring, the thiadiazole ring might have different
Summarized in Table 1 are the energetic results obtained from
the binding free energy calculations. The binding free energies cal-
culated for all ligands range from ꢀ9.28 to ꢀ14.01 kcal/mol, while

302
Y. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 296–304
Table 1
The Ki values (lM) of compounds 1 and 8 against hPPO, and their calculated binding free energies (DGcal, kcal/mol)
X
O
X
O
S
O
N
N
S
S
N
N
N
N
RS
RS
1a~w
8a~w
b
No.
X
R
D
EELE
D
EVDW
D
EGAS
D
GSOL
D
GPB
ꢀTD
S
D
Gcal
Kia
DGexp
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
F
F
F
F
F
F
F
F
COOCH2CH3
CH2COOCH3
ꢀ17.84
ꢀ24.12
ꢀ20.03
ꢀ25.46
ꢀ24.35
ꢀ24.74
ꢀ25.24
ꢀ27.44
ꢀ25.88
ꢀ6.40
ꢀ56.43
ꢀ54.44
ꢀ58.75
ꢀ56.51
ꢀ57.67
ꢀ51.85
ꢀ57.47
ꢀ55.68
ꢀ58.94
ꢀ52.52
ꢀ58.27
ꢀ56.39
ꢀ58.09
ꢀ60.79
ꢀ62.40
ꢀ61.14
ꢀ54.24
ꢀ55.90
ꢀ56.45
ꢀ57.82
ꢀ61.90
ꢀ53.69
ꢀ57.85
ꢀ53.67
ꢀ54.17
ꢀ55.53
ꢀ56.22
ꢀ57.25
ꢀ52.65
ꢀ58.22
ꢀ57.37
ꢀ57.14
ꢀ54.92
ꢀ57.29
ꢀ56.41
ꢀ56.39
ꢀ58.33
ꢀ57.63
ꢀ60.50
ꢀ52.92
ꢀ53.23
ꢀ55.15
ꢀ57.86
ꢀ56.71
ꢀ57.92
ꢀ52.16
ꢀ74.27
ꢀ78.58
ꢀ78.78
ꢀ81.97
ꢀ82.02
ꢀ76.58
ꢀ82.71
ꢀ83.12
ꢀ84.82
ꢀ58.92
ꢀ73.43
ꢀ77.83
ꢀ82.99
ꢀ76.81
ꢀ85.25
ꢀ89.62
ꢀ61.34
ꢀ79.19
ꢀ75.96
ꢀ79.55
ꢀ84.54
ꢀ74.11
ꢀ69.01
ꢀ69.24
ꢀ78.16
ꢀ79.79
ꢀ81.28
ꢀ79.55
ꢀ76.23
ꢀ82.15
ꢀ82.04
ꢀ83.54
ꢀ58.95
ꢀ73.26
ꢀ81.03
ꢀ78.09
ꢀ83.93
ꢀ85.57
ꢀ89.27
ꢀ59.33
ꢀ77.34
ꢀ76.01
ꢀ79.07
ꢀ81.54
ꢀ84.31
ꢀ58.73
35.81
36.00
37.61
37.72
34.01
34.97
40.34
39.09
37.57
20.12
33.04
32.88
36.46
32.35
40.02
44.71
21.46
35.44
32.61
35.24
40.46
35.53
29.19
31.57
35.66
35.85
36.89
30.81
35.11
37.23
36.20
37.85
16.24
31.95
37.98
32.15
37.21
37.55
44.76
19.22
31.65
31.62
31.16
36.15
38.72
20.17
ꢀ38.45
ꢀ42.56
ꢀ41.17
ꢀ44.25
ꢀ48.01
ꢀ41.61
ꢀ42.38
ꢀ44.04
ꢀ47.25
ꢀ38.79
ꢀ40.39
ꢀ44.95
ꢀ46.54
ꢀ44.45
ꢀ45.23
ꢀ44.90
ꢀ39.88
ꢀ43.75
ꢀ43.36
ꢀ44.31
ꢀ44.08
ꢀ38.58
ꢀ39.82
ꢀ37.68
ꢀ42.50
ꢀ43.95
ꢀ44.39
ꢀ48.73
ꢀ41.12
ꢀ44.91
ꢀ45.84
ꢀ45.69
ꢀ42.71
ꢀ41.31
ꢀ43.05
ꢀ45.95
ꢀ46.73
ꢀ48.02
ꢀ44.50
ꢀ40.11
ꢀ45.69
ꢀ44.39
ꢀ47.91
ꢀ45.39
ꢀ45.60
ꢀ38.56
24.44
30.57
27.60
32.76
35.44
27.96
30.11
31.92
35.45
27.44
26.57
32.75
34.72
33.49
32.63
34.25
28.14
32.83
34.08
32.79
33.26
28.60
28.00
24.09
30.47
30.95
31.59
36.09
27.71
31.92
31.74
31.87
30.10
27.56
29.46
32.39
33.71
34.20
31.95
28.48
33.12
32.30
35.24
32.36
35.57
27.51
ꢀ14.01
ꢀ11.99
ꢀ13.57
ꢀ11.49
ꢀ12.57
ꢀ13.65
ꢀ12.27
ꢀ12.12
ꢀ11.80
ꢀ11.35
ꢀ13.82
ꢀ12.20
ꢀ11.83
ꢀ10.96
ꢀ12.60
ꢀ10.65
ꢀ11.74
ꢀ10.92
ꢀ9.28
0.04
3.05
0.34
5.89
3.46
1.26
1.31
9.26
8.67
9.73
0.20
5.82
12.00
34.4
1.28
17.70
21.40
15.60
173.0
43.50
27.50
2450
20.70
0.72
3.99
1.58
1.42
1.54
1.17
0.70
0.32
0.36
1.92
0.33
1.07
1.69
1.35
0.25
2.02
8.61
6.96
8.13
1.58
1.69
5.83
11.12
2.93
ꢀ10.10
ꢀ7.53
ꢀ8.84
ꢀ7.14
ꢀ7.46
ꢀ8.06
ꢀ8.04
ꢀ6.88
ꢀ6.91
ꢀ6.85
ꢀ9.15
ꢀ7.15
ꢀ6.72
ꢀ6.10
ꢀ8.05
ꢀ6.49
ꢀ6.38
ꢀ6.57
ꢀ5.14
ꢀ5.96
ꢀ6.23
ꢀ4.93
ꢀ6.40
ꢀ8.39
ꢀ7.38
ꢀ7.92
ꢀ7.99
ꢀ7.94
ꢀ8.10
ꢀ8.41
ꢀ8.87
ꢀ8.80
ꢀ7.81
ꢀ8.85
ꢀ8.16
ꢀ7.88
ꢀ8.02
ꢀ9.02
ꢀ7.78
ꢀ6.92
ꢀ7.05
ꢀ6.95
ꢀ7.92
ꢀ7.88
ꢀ7.15
ꢀ6.77
CH2COOCH2CH3
CH(CH)3COOC2H5
C(CH3)2COOC2H5
CH2COOCH(CH3)2
CH2COOC3H7
CH2CH2COOC2H5
CH2(CH2)2COOC2H5
CH2CCH
F
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
F
F
F
F
F
F
F
F
F
COOC2H5
CH2COOC2H5
ꢀ15.16
ꢀ21.44
ꢀ24.90
ꢀ16.02
ꢀ22.85
ꢀ28.48
ꢀ7.10
CH(CH3)COOC2H5
C(CH3)2COOC2H5
CH2CH2COOC2H5
CH2(CH2)2COOC2H5
CH2CCH
CH2COOC2H5
ꢀ23.29
ꢀ19.51
ꢀ21.73
ꢀ22.64
ꢀ20.42
ꢀ11.16
ꢀ15.57
ꢀ23.99
ꢀ24.27
ꢀ25.07
ꢀ22.29
ꢀ23.58
ꢀ23.93
ꢀ24.67
ꢀ26.4
1s
1t
CH(CH3)COOC2H5
C(CH3)2COOC2H5
CH2CH2COOC2H5
CH2(CH2)2COOC2H5
CH2CCH
ꢀ11.52
ꢀ10.82
ꢀ9.98
1u
1v
1w
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
ꢀ11.82
ꢀ13.59
ꢀ12.03
ꢀ13.00
ꢀ12.80
ꢀ12.64
ꢀ13.41
ꢀ12.99
ꢀ14.10
ꢀ13.82
ꢀ12.61
ꢀ13.78
ꢀ13.59
ꢀ13.57
ꢀ13.02
ꢀ13.82
ꢀ12.56
ꢀ11.63
ꢀ12.57
ꢀ12.09
ꢀ12.67
ꢀ13.03
ꢀ10.03
ꢀ11.05
COOCH2CH3
CH2COOCH3
CH2COOCH2CH3
CH(CH)3COOC2H5
C(CH3)2COOC2H5
CH2COOCH(CH3)2
CH2COOC3H7
CH2CH2COOC2H5
CH2(CH2)2COOC2H5
CH2CCH
F
ꢀ4.03
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
COOC2H5
CH2COOC2H5
ꢀ15.97
ꢀ24.62
ꢀ21.7
8m
8n
8o
8p
8q
8r
CH(CH3)COOC2H5
C(CH3)2COOC2H5
CH2CH2COOC2H5
CH2(CH2)2COOC2H5
CH2CCH
ꢀ25.6
ꢀ27.94
ꢀ28.76
ꢀ6.41
CH2COOC2H5
ꢀ24.11
ꢀ20.86
ꢀ21.21
ꢀ24.82
ꢀ26.4
8s
8t
CH(CH3)COOC2H5
C(CH3)2COOC2H5
CH2CH2COOC2H5
CH2(CH2)2COOC2H5
CH2CCH
8u
8v
8w
Oxadiazon
ꢀ6.58
a
The Ki values of compounds 8a–w against hPPO was taken from Ref. 37.
b
D
Gexp = ꢀRTlnKi.
interaction with the surrounding residues, which can be reflected
by the energy decomposition of 1a–w and 8a–w. As shown in Table
1, compound 1a is the most potent inhibitor, displaying about 18-
times higher PPO-inhibition activity than the corresponding 1,3,4-
oxadiazol-2(3H)-one 8a. In order to understand the molecular ba-
sis why compound 1a displayed higher activity than 8a, we com-
pared their binding modes. As shown in Figure 3, we found that
the simulated binding modes of compounds 1a and 8a were very
similar. The oxadiazole and thiadiazole rings were sandwiched be-
tween the residues of M368 and G169, and formed hydrophobic
interaction with these two residues. The benzothiazole moiety of
compounds 1a and 8a was located between the residues of V347
F331. In addition, the side chain of the benzothiazole cycle formed
hydrogen bond interactions with R97. As listed in Table 1, the bind-
ing free energies (DGcal) of compounds 1a and 8a are, respectively,
ꢀ14.01 and ꢀ13.59 kcal/mol, which are qualitatively in good
agreement with the experimentally-derived binding free energies
(D
Gexp) (ꢀ10.10 and ꢀ8.39 kcal/mol). The energy component anal-
ysis showed that the difference came from the electrostatic and
van der Waals interactions, and solvation effect. Compound 1a
had a stronger electrostatic (ꢀ17.84 kcal/mol) and van der Waals
interactions (ꢀ56.43 kcal/mol) than compound 8a (ꢀ15.57 and
ꢀ53.67 kcal/mol) of, but higher solvation (35.81 kcal/mol) penalty
than that (31.57 kcal/mol) of compound 8a. From Figure 3, we can
also conclude that replacement of oxadiazole by thiadiazole
and L334, and formed a T–
p interaction with the side chain of

Y. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 296–304
303
-2
-3
-4
-5
-6
-7
-8
-9
benzothiazole ring of the energy-minimized structures of the fluo-
rine, chlorine and bromine derivatives based on HF/6–31G⁄ level.
The results indicated that the dihedral angle of fluorine derivatives
is very similar to that of 4-bromo-3-(50-carboxy-40-chloro-20-fluo-
rophenyl)-1-methyl-5-trifluoro-methyl-pyrazol (INH) in the crys-
tal structure of tobacco (N. tabacum) PPO. However, the dihedral
angles of chlorine and bromine derivatives are significantly differ-
ent from that of INH. That means that there is a little energy differ-
ence between the energy-minimized structures and the bioactive
conformation of fluorine derivatives. However, the chlorine and
bromine derivatives need to overcome a larger energy barrier
when they bind to the enzyme.
∆Gexp= 0.8087∆Gcal+ 2.4741
R2 = 0.8072
-10
-11
-15
-14
-13
-12
-11
-10
-9
-8
-7
4. Conclusion
Calculated Binding Free Energy
(kcal/mol)
In summary, a series of 1,3,4-thiadiazol-2(3H)-ones containing
benzothiazole substructure were designed and synthesized as po-
tential hPPO inhibitors. The in vitro results indicated that com-
pound 1a, O-ethyl S-(5-(5-(tert-butyl)-2-oxo-1,3,4-thiadiazol-
Figure 2. Correlation between the calculated and experimental binding free
energies.
3(2H)-yl)-6-fluorobenzo-thiazol-2-yl)carbonothioate,
displayed
the highest PPO-inhibition activity (ki = 40 nM), about 73-fold
higher than oxadiazon. To our knowledge, it is so far the most po-
tent hPPO inhibitor. The results of computational simulations indi-
cated that compound 1a has a very similar binding mode as its
corresponding 1,3,4-oxadiazol-2(3H)-one derivative 8a. The bind-
ing free energy calculations showed that the higher activity of
compound 1a is mainly from the stronger electrostatic and van
der Waals interactions. According to the established computational
models, we can conclude that: (1) Improving the hydrogen bond
interaction between R97 and the side chain of inhibitors should
be favorable to the binding affinity. (2) Too long side chain will
led to higher conformation flexibility of inhibitors and decrease
the binding affinity. (3) The dihedral angle between the oxadiazole
or thiadiazole and the benzothiazole ring is an important factor
which should be taken into account when designing new inhibi-
tors. We believe that these insights will be helpful for the future ra-
tional design of novel hPPO inhibitors with improved potency.
resulted in a stronger hydrogen bond interaction between R97 and
the oxygen atom of the side-chain ester group.
Very interestingly, when the carbonyl oxygen atom rather than
the ester oxygen atom formed hydrogen bonds with R97, the bind-
ing affinity will not be improved. For example, compounds 1h and
8h displayed lower hPPO-inhibition activity than compounds 1a
and 8a, respectively. As shown in Table 1, after inserting two more
carbon atoms between the carbonyl and sulfur atom, the hydrogen
bond interactions between the carbonyl oxygen atom and R97
were improved significantly, and their electrostatic interaction
energies were also improved. However, due to their longer side
chain, the entropy penalties of 1h and 8h are 31.92 kcal/mol and
31.74 kcal/mol, which are greatly higher than that of compounds
1a (24.44 kcal/mol) and 8a (24.09 kcal/mol), respectively.
In addition, as shown in Figure 4, we also compared the dihedral
angle between the oxadiazole or thiadiazole ring and the
Figure 3. Computational simulated binding models of compounds 1a, 1h, 8a, and 8h.

304
Y. Zuo et al. / Bioorg. Med. Chem. 20 (2012) 296–304
Figure 4. Comparison of the dihedral angles between the oxadiazole or thiadiazole rings and the benzothiazole ring of the fluorine, chlorine and bromine derivatives. The
energy minimization was performed at HF/6–31G⁄ level. The conformation of INH was taken from its PPO complex crystal structure.
27. Theodoridis, G. U.S. Patent 5798,316, 1989.
28. Theodoridis, G.; Princetons, N. J. U.S. Patent 4818,275, 1989.
Acknowledgments
29. Corradi, H. R.; Corrigall, A. V.; Boix, E.; Mohan, C. G.; Sturrock, E. D.; Meissner, P.
We should thank the financial support from the National Basic
Research Program of China (2010CB126103), and the National Nat-
ural Science Foundation of China (Nos. 20925206, 21002038 and
20932005), and the PCSIRT (No. IRT0953).
N.; Acharya, K. R. J. Biol. Chem. 2006, 281, 38625.
30. Koch, M.; Breithaupt, C.; Kiefersauer, R.; Freigang, J.; Huber, R.; Messerschmidt,
A. J. EMBO 2004, 23, 1720.
31. Duke, S. O.; Rebeiz, C. A. In ACS Symposium Series; American Chemical Society:
Washington, DC, 1994; Vol. 559, p 71.
32. Fingar, V. H.; Wieman, T. J.; McMahon, K. S.; Haydon, P. S.; Halling, B. P.; Yuhas,
D. A.; Winkelman, J. W. Cancer Res. 1997, 57, 4551.
References and notes
33. Volker, A.; Burkhard, G. U.S. Patent 20050049228, 2005.
34. Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Nat. Rev. Cancer 2003, 3, 380.
35. Halling, B. P.; Yuhas, D. A.; Fingar, V. F.; Winkelmann, J. W. Protophyrinogen
Oxidase Inhibitors for Tumor Therapy, in Porphyric Pesticides: Chemistry,
Toxicology and Pharmaceutical Applications; American Chemical Society:
Washington, DC, 1994.
36. Qin, X. H.; Tan, Y.; Wang, L. L.; Wang, Z. F.; Wang, B. F.; Wen, X.; Yang, G. F.; Xi,
Z.; Shen, Y. Q. FASEB J. 2011, 25, 653.
37. Jiang, L. L.; Tan, Y.; Zhu, X. L.; Wang, Z. F.; Zuo, Y.; Chen, Q.; Xi, Z.; Yang, G. F. J.
Agric. Food Chem. 2010, 58, 2643.
38. Bosch, J.; Roca, T.; Catena, J. L.; Farrerons, C.; Miquel, I. Synthesis 2000, 721.
39. Zhu, L.; Zhang, M. B.; Dai, M. J. Heterocycl. Chem. 2005, 42, 727.
40. Bruker. SMART (Version 5.628), Bruker AXS, Inc., Madison, Wisconsin, USA,
2001.
41. Bruker. SAINT-Plus (Version 6.45), Bruker AXS, Inc., Madison, Wisconsin, USA,
2001.
42. Sheldrick, G. M. Acta Crystallogr., Sect. A 2008, 64, 112.
43. Tan, Y.; Sun, L.; Xi, Z.; Yang, G. F.; Jiang, D. Q.; Yan, X. P.; Yang, X.; Li, H. Y. Anal.
Biochem. 2008, 383, 200.
1. Gerald, S. M. Bot. Rev. 1966, 32, 56.
2. Poulson, R.; Polglase, W. J. J. Biol. Chem. 1975, 250, 1269.
3. Poulson, R. J. Biol. Chem. 1976, 251, 3730.
4. Deybach, J. C.; Da Silva, V.; Grandchamp, B.; Nordmann, Y. Eur. J. Biochem. 1985,
149, 431.
5. Ferreira, G. C.; Andrew, T. L.; Karr, S. W.; Dailey, H. A. J. Biol. Chem. 1988, 263,
3835.
6. Ferreira, G. C.; Dailey, H. A. Biochem. J. 1988, 250, 597.
7. Beale, S. I. Photosynth. Res. 1999, 60, 43.
8. Heinemann, I. U.; Diekmann, N.; Masoumi, A.; Koch, M.; Messerschmidt, A.;
Jahn, M.; Jahn, D. Biochem. J. 2007, 402, 575.
9. Jacobs, J. M.; Jacobs, N. J. Arch. Biochem. Biophys. 1984, 229, 312.
10. Jung, S.; Lee, Y.; Yang, K.; Lee, S. B.; Jang, S. M.; Ha, S. B.; Back, K. Plant Cell
Environ. 2004, 27, 1436.
11. Lermontova, I.; Kruse, E.; Mock, H. P.; Grimm, B. Proc. Natl. Acad. Sci. U.S.A. 1997,
94, 8895.
12. Brenner, D. A.; Bloomer, J. R. N. Engl. J. Med. 1980, 302, 765.
13. Deybach, J. C.; De Verneuil, H.; Nordmann, Y. Hum. Genet. 1981, 58, 425.
14. Meissner, P. N.; Day, R. S.; Moore, M. R.; Disler, P. B.; Harley, E. Eur. J. Clin. Invest.
1986, 16, 257.
15. Nordmann, Y.; Puy, H. Clin. Chim. Acta. 2002, 325, 17.
16. Whatley, S. D.; Puy, H.; Morgan, R. R.; Robreau, A. M.; Roberts, A. G.; Nordmann,
Y.; Elder, G. H.; Deybach, J. C. Am. J. Hum. Genet. 1999, 65, 984.
17. Dean, G. The porphyrias: A story of inheritance and environment, 2nd ed.; Pitman
Medical: London, 1971.
44. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
45. Sanner, M. F. J. Mol. Graphics Modell. 1999, 17, 57.
46. Xiong, Y.; Li, Y.; He, H.; Zhan, C. G. Bioorg. Med. Chem. Lett. 2007, 17, 5186.
47. Case, D. A.; Darden, T. A.; Cheatham; Iii, T. E.; Simmerling, C. L.; Wang, J.; Duke,
R. E.; Luo, R.; Merz, K. M.; Pearlman, D. A.; Crowley, M. AMBER 9, University of
California, San Francisco, 2006.
48. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
Cheeseman, J. R.; Montgomery, J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.;
Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.;
Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.;
Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,
O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken,
V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A.
J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G.
A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghava-chari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.;
Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe,
M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.
Gaussian 03, revision B-03, Gaussian, Inc.: Pittsburgh, PA, 2003.
18. Jenkins, T. Nat. Genet. 1996, 13, 7.
19. Day, R. S. Semin. Dermatol. 1986, 5, 138.
20. Deybach, J. C.; Puy, H.; Robreau, A. M.; Lamoril, J.; Da Silva, V.; Grandchamp, B.;
Nordmann, Y. Hum. Mol. Genet. 1996, 5, 407.
21. Meissner, P. N.; Dailey, T. A.; Hift, R. J.; Ziman, M.; Corrigall, A. V.; Roberts, A. G.;
Meissner, D. M.; Kirsch, R. E.; Dailey, H. A. Nat. Genet. 1996, 13, 95.
22. Duke, S. O.; Lydon, J.; Becerril, J. M.; Sherman, T. D.; Lehnen, L. P., Jr.;
Matsumoto, H. Weed Sci. 1991, 465.
23. Liu, Y. X.; Cui, Z. P.; Liu, B.; Cai, B. L.; Li, Y. H.; Wang, Q. M. J. Agric. Food Chem.
2009, 58, 2685.
24. Nagano, E.; Hashimoto, S.; Yoshida, R.; Matsumoto, H.; Oshio, H.; Kamoshita, K.
EP Patent 61,741, 1982.
25. Gitsopoulos, T. K.; Froud-Williams, R. J. Weed Res. 2004, 44, 329.
26. Hiraki, M.; Ohki, S.; Sato, Y.; Jablonkai, I.; Boger, P.; Wakabayashi, K. Pestic.
Biochem. Phys. 2001, 70, 159.