Synthesis and bioevaluation of pyrazole-benzimidazolone hybrids as novel human 4-Hydroxyphenylpyruvate dioxygenase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.01.018

4-Hydroxyphenylpyruvate dioxygenase (HPPD), an essential enzyme in tyrosine catabolism, is an important target for treating type I tyrosinemia. Inhibition of HPPD can effectively alleviate the symptoms of type I tyrosinemia. However, only one commercial H

Full text of DOI:10.1016/j.ejmech.2015.01.018

European Journal of Medicinal Chemistry 92 (2015) 427e438
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and bioevaluation of pyrazole-benzimidazolone hybrids as
novel human 4-Hydroxyphenylpyruvate dioxygenase inhibitors
,
,
Yu-Ling Xu a 1, Hong-Yan Lin a 1, Xu Ruan a, Sheng-Gang Yang a, Ge-Fei Hao a,
Wen-Chao Yang a *, Guang-Fu Yang a
,
, b, *
a Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, PR China
b Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Hydroxyphenylpyruvate dioxygenase (HPPD), an essential enzyme in tyrosine catabolism, is an
important target for treating type I tyrosinemia. Inhibition of HPPD can effectively alleviate the symp-
Received 30 November 2014
Received in revised form
9 January 2015
Accepted 9 January 2015
Available online 10 January 2015
toms of type
I tyrosinemia. However, only one commercial HPPD inhibitor, 2-(2-nitro-4-
trifluoromethylbenzoyl) cyclohexane-1,3-dione (NTBC), has been available for clinical use so far. In the
present study, a series of novel pyrazole-benzimidazolone hybrids were designed, synthesized and
evaluated as potent human HPPD inhibitors. Most of the new compounds displayed significant inhibitory
activity against the recombinant human HPPD. Moreover, compound 9l was identified as the most potent
candidate with IC50 value of 0.021 mM against recombinant human HPPD, about 3-fold more potent than
NTBC. Thus the pyrazole-benzimidazolone hybrid has great potential to be further developed for the
treatment of type I tyrosinemia.
Keywords:
4-Hydroxyphenylpyruvate dioxygenase
Inhibitors
Benzimidazolone
© 2015 Elsevier Masson SAS. All rights reserved.
Type I tyrosinemia
1. Introduction
In human, abnormal metabolism in the tyrosine catabolism
pathway gives rise to various diseases, including type I tyrosinemia.
4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD),
catalyzing the conversion of 4-hydroxyphenylpyruvate (HPPA) to
homogentisate (HGA), is an essential enzyme implicated in
pigment synthesis and tyrosine catabolism in most organisms, and
has great importance in drug discovery in both agricultural and
therapeutic areas (Scheme 1) [1e3]. Inhibition of HPPD in plants
leads to the deficiency in isoprenoid redox cofactors such as plas-
toquinone and tocopherol, finally causing growth inhibition and
plant death. In the past thirty years, a series of inhibitors targeting
HPPD have been discovered as potent herbicides, such as 2-(2-
nitro-4-trifluoromethylbenzoyl) cyclohexane-1,3-dione (NTBC),
sulcotrione and mesotrione et al. [4e8].
More specifically, the excessive HPPD indirectly causes the accu-
mulation of succinylacetone which in turn induces severe liver and
kidney dysfunction along with mutagenic changes and hepatocel-
lular carcinoma, and even death [9]. At first, liver transplantation
was the only effective treatment for type I tyrosinemia. Later it was
found that the born defects in mammalian tyrosine catabolism
could be treated effectively by HPPD inhibitors. Animal model and
clinical test further indicated that NTBC, with a market name of
orfadin, dramatically changed the disease course by improving liver
and kidney functions and reducing risk of liver cancer [10e12].
Thereafter, NTBC was approved by the US FDA to clinically treat
hereditary type I tyrosinemia [13,14]. So far, considerably less
research has been focused on the discovery of new drugs targeting
human HPPD. In addition, recent study by animal model indicated
that some adverse effect occurred after NTBC treatment. Adminis-
tration of NTBC was reported to produce corneal lesions in rats fed
tyrosine in a low-protein diet [15e18]. Thus, it is of great interest to
explore novel HPPD inhibitor as drug candidate for type I tyrosi-
nemia treatment [19,20].
Abbreviations: HPPD, 4-hydroxyphenylpyruvate dioxygenase; HGA, homoge-
ntisate; DKN, diketonitriles; CDI: 1, 1'-carbonyldiimidazole; IPTG, isopropyl
thiogalactopyranoside; DMSO, dimethyl sulfoxide.
b-D-1-
* Corresponding authors. Key Laboratory of Pesticide & Chemical Biology of
Ministry of Education, College of Chemistry, Central China Normal University,
Wuhan 430079, PR China.
As heterocyclic aromatic compounds, benzimidazolones are
important chemical scaffolds and widely applied in pesticide and
pharmaceutical synthesis due to their extensive biological activities
E-mail addresses: tomyang@mail.ccnu.edu.cn (W.-C. Yang), gfyang@mail.ccnu.
edu.cn (G.-F. Yang).
1
These two authors made equal contributions.
http://dx.doi.org/10.1016/j.ejmech.2015.01.018
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

428
Y.-L. Xu et al. / European Journal of Medicinal Chemistry 92 (2015) 427e438
Scheme 1. The simple diagram for the catabolic pathways from tyrosine in plants and type I tyrosinemia patients.
[21e23]. Thus, the heterocyclic compounds incorporating the
benzimidazole moiety have received considerable attention
[24,25]. We envisioned that by applying scaffold hopping, hybrid (1
in Fig. 1) of quinazolone with an identified pharmacophore of
herbicides targeting HPPD (like pyrazole) would lead to novel HPPD
inhibitors with improved potency. Therefore, we designed and
synthesized a novel series of pyrazole-benzimidazolone hybrid,
and the structure-activity relationship (SAR) was subsequently
investigated in current study. As anticipated, more than ten new
HPPD inhibitors (compounds 9 in Table 1) were identified with
good potency at the nanomolar level in this study. Moreover, their
activities were significantly improved compared to NTBC. Among
them, compound 9l, was identified as the most active human HPPD
inhibitor, and its IC50 value is about 3-fold higher than that of NTBC.
Fig. 1. Design strategy of the pyrazole-benzimidazolone hybrids as novel human HPPD
inhibitors.
2. Results and discussion
covering almost 40% of commercial HPPD inhibitors which were
applied as herbicides, may also be a potential candidate to be
developed for medication, since pyrazole can tightly bind HPPD
through chelation with the ferrous ion. For example, pyrasulfotole
and topramezone, as two typical herbicides, are well-known HPPD
inhibitors bearing pyrazole moiety. Another compound, DNS869
(Fig. 1) [26], has been reported as a potent inhibitor of both plant
and mammalian HPPDs. As a heterocyclic aromatic compound,
benzimidazolone has been widely used in pharmaceutical synthe-
sis, and displays extensive biological activities, such as antihyper-
tensive, antidiabetic, anti-inflammatory, anticonvulsive properties.
For example, droperidol as a typical bnzimidzolone was developed
and used as neuroleptic pharmaceutical (Fig. 1) [27]. Thus, we
envisioned that hybrid of pyrazole-benzimidazolone would afford
novel HPPD inhibitors with improved potency (Fig. 1).
2.1. Chemistry
The preparation of the target compounds (9 and 10) was illus-
trated in Scheme 2. The synthesis followed an eight-step synthetic
route starting from the commercially available 4-chloro-3-nitro
benzoic acid. The esterification of 4-chloro-3-nitro benzoic acid
afforded the methyl benzoate in a yield of 83%, which was treated
with diverse alkylamine or aromatic amine to generate the inter-
mediate 3 ranging in yields from 60% to 90 %. The subsequent
reduction of the nitro group in the molecule 3 was achieved by
using palladium hydroxide in methanol to produce phenylenedi-
amine 4, which was subjected to the ring closure reaction without
further purification by treating with 1,1'-carbonyldiimidazole (CDI)
to give benzimidazolone derivatives 5. Then, the alkylation re-
actions of intermediates 5 with alkyl iodide afforded the corre-
sponding 6 in excellent yields (80%e95%). The raw material 6 was
treated with lithium hydroxide to afford carboxylic acids 7. The
transformation from 7 to 8, was furnished by a two-step one-pot
strategy that the carboxylic acid 7 was first converted to the cor-
responding acyl chloride by treating with PCl5 and the acyl chloride
was treated with pyrazol-5-ol to afford compounds 8. With the key
intermediates 8 in hand, the desired products 9 and 10 were pre-
pared though the rearrangement of the intermediates 8 in the
presence of acetone cyanohydrin and triethylamine in anhydrous
CH3CN. The structures of all the target compounds were charac-
terized by 1H NMR, 13C NMR and high resolution mass spectrum.
The bioactivity of these newly synthesized compounds against
human HPPD were evaluated (Table 1), using NTBC as the positive
control. As expected, all compounds showed significant inhibitory
activity against human HPPD. More than ten compounds have IC50
values in the nanomolar range. As indicated in Table 1, compound
9l, displayed the highest activity with IC50 of about 0.021
3-fold higher than of the commercial control NTBC
M).
mM, about
(IC50 ¼ 0.07
m
For the sake of clarity, all the compounds were divided into two
groups according to the substitution on the pyrazole ring. As seen in
Table 1, the inhibitory activities of the series of compounds 9 (R1 is
H) are generally about 10-fold more potent than that of the series of
compounds 10 (R1 is CH3). More specifically, compound 9a, 9f, 9l
and 9q exhibited excellent inhibition potency with respective IC50
2.2. Lead compound design and SAR study
values of 0.059 mM, 0.022 mM, 0.021 mM and 0.040 mM, which are
about 17-fold, 44-fold, 45-fold and 18-fold higher than the corre-
sponding compounds with methyl substitution at 3-position of
pyrazole (10a, 10f, 10k and 10n), respectively. This interesting
Although plenty of potent HPPD inhibitors have been
commercialized, only NTBC, a triketone HPPD inhibitor, was
approved for type I tyrosinemia therapy. Pyrazoles derivatives,

Y.-L. Xu et al. / European Journal of Medicinal Chemistry 92 (2015) 427e438
429
Table 1
benzimidazolones moiety, various substitutions were introduced to
the N atoms (R2 position and R3 position) for both series of the title
compounds. In general, the SAR of the title compounds (9 and 10)
on R2 position is not in good order, but the potency order of R3
substitution is quite clear. The presence of steric bulky group for R3
substitution, particularly the cyclopropyl and phenyl groups, would
significantly reduce the potency of new compounds. Three repre-
sentative compounds (9f, 9e and 9h) were selected to analyze the
binding modes by molecular modeling. As depicted in Fig. 2BeD, all
the three compounds showed similar binding modes in the HPPD
active site, and the strength for the Fe(II)-chelation didn't exhibit big
difference. However, the steric clash between the compounds and
the active site consistently increased with the enhancement of the
bulky substitution on R3 position. The detailed volume of the three
compounds and the distances for the steric clash were listed in
Table 2. The introduction of larger group resulted in the bigger
volume of the compounds, which definitely lead to the closer
proximity to the key residues (Leu289 and Phe387). Thus, com-
pound 9l tightly binding in the HPPD active site with less steric
hindrance, was identified as the most active compound (with IC50
Chemical structures of new compounds 9aer, 10aeo, and their biological activity
against recombinant human HPPD.
Compound
R1
R2
R3
IC50 (mM)
NTBC
NO
0.068 0.0047
O
O
O
F C
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3CH2CH2e
CH3CH2CH2CH2e
CH3CH(CH3)2e
eCH(CH2)3CH2e
CH(CH3)2e
0.059 0.0052
0.17 0.023
0.14 0.010
0.13 0.012
0.061 0.0076
0.022 0.0052
0.10 0.011
0.25 0.0090
0.13 0.0041
0.10 0.0012
0.022 0.0022
0.021 0.0020
0.064 0.0047
0.15 0.0087
0.22 0.0093
0.039 0.0063
0.040 0.0081
0.077 0.0031
1.03 0.015
0.64 0.042
2.71 0.043
0.93 0.051
0.94 0.088
0.97 0.074
0.99 0.13
CH3CH2e
C (CH3)3e
of 0.021 mM), displaying about 3-fold higher activity than NTBC.
4-CH3eC6H4e
C6H4CH2e
eCHCH2CH2e
CH3CH2CH2e
CH3CH2e
CH(CH3)2e
C(CH2)3e
eCHCH2CH2e
CH3CH2e
CH3CH2CH2e
CH(CH3)2e
CH3CH2CH2e
CH3CH2CH2CH2e
CH3CH(CH3)2e
eCH(CH2)3CH2e
CH(CH3)2e
CH3CH2e
C(CH3)3e
4-CH3eC6H4e
C6H4CH2e
CH3CH2CH2e
CH3CH2e
3. Conclusion
In summary, a series of hybrids of pyrazole-benzimidazolone
were rationally designed through scaffold hopping, and synthe-
sized and evaluated as human HPPD inhibitors, leading to the
discovery of several novel human HPPD inhibitors with excellent
inhibitory potency. The bioevaluation assay indicated that most of
the newly synthesized compounds displayed significant human
HPPD inhibition activity. Some of them showed superior activity
compared to NTBC. In particular, compounds 9l, characterized by a
pyrazole moiety linked to a substituted benzimidazolone, was
identified as the most potent human HPPD inhibitor with IC50 value
of ~21 nM, over 3-fold more potent than NTBC. Based the afore-
mentioned results, hybrids of pyrazolone-quinazolone could be
regarded as a novel lead scaffold for discovering new human HPPD
inhibitors to treat type I tyrosinemia.
H
H
9r
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
0.99 0.066
0.67 0.087
0.88 0.084
0.94 0.014
0.97 0.018
0.74 0.13
CH3CH2
CH3CH2
CH3CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH(CH3)2e
CH3CH2e
CH3CH2CH2e
CH(CH3)2e
4. Experimental section
0.72 0.046
1.19 0.025
4.1. Methods and synthesis
All chemical reagents were commercially available and treated
with standard methods before use. Solvents were dried and
redistilled before use. 1H NMR were recorded on a VARIAN
Mercury-Plus 600 or 400 spectrometer in CDCl3 or DMSO-d6 with
TMS as the internal reference, The following abbreviations are used
to designate mutiplicities: s ¼ singlet, d ¼ doublet, t ¼ triplet,
m ¼ multiplet, br ¼ broad. High resolution mass spectra (HRMS)
were obtained on an Agilent 6224 TOF LC/MS (USA). Melting points
were taken on a Buchi B-545 melting point apparatus and are
uncorrected.
observation indicates that the introduction of methyl group at the
3-position of pyrazole ring would result in a significant decrement
of the biological activity. To illustrate the structural basis of the
above observation, molecular modeling was applied to understand
the binding modes of representative compounds 9f and 10f. As
showed in Fig. 2A and B, the binding of compound 10f in the human
HPPD active site was very similar to that of compound 9f, but the
strength of the chelation reduced and remarkable steric clash arose
due to the introduction of methyl group at R1 position. The specific
distances for the chelation and steric clash were listed in Table 2.
Notably, the distances for the Fe(II)-chelation with compound 10f
are 3.4 Å and 1.8 Å, respectively, whereas the distances for the Fe(II)
-
4.1.1. General procedure for intermediate 2 [28]
4-Chloro-3-nitro benzoic acid (40.0 g, 0.02 mol) was suspended
in methanol (200 mL) and cooled down to 0 ꢀC. Concentrated
sulfuric acid (22 mL) was slowly added with stirring, and then the
mixture was heated at reflux for 17 h. Upon cooling to room tem-
perature, a precipitate formed, which was collected by filtration
and washed with cold methanol (2 ꢁ 30 mL) and hexane
(2 ꢁ 30 mL) to afford the methyl ester as a white solid (35.5 g, 83%).
chelation with compound 9f are 2.4 Å and 1.9 Å, respectively. As to
the steric clash, stronger hindrance could be interpreted due to the
closer distances for compound 10f to the residues Asn241 (3.4 Å
and 3.9 Å), Leu289 (4.2 Å) and Phe347 (3.3 Å and 3.7 Å), comparing
to the distances for compound 9f to the residues Asn241 (longer
than 5 Å), Leu289 (4.9 Å) and Phe347 (3.3 Å and 4.0 Å). Thus, it
revealed that the decreased Fe(II)-chelation and the steric clash
were attributed to the less potency of the compounds 10.
4.1.2. General procedure for intermediate 3b1 [29]
Next, in order to understand the substituent effect on the
Methyl 4-chloro-3-nitrobenzoate (8.0 g, 0.037 mol) was

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Y.-L. Xu et al. / European Journal of Medicinal Chemistry 92 (2015) 427e438
Reagents and conditions: a) MeOH, Con. H2SO4, refluxed for 20 h; b1) CH3CN/H2O, AcONa, alkylamine,
refluxed for 17-24 h; b2) MeOH/DMF , N,N-diisopropylethylamine, cycloalkylamine, refluxed for 18 h; b3)
DMSO (or DMF), 70 ºC, 8 h; c) MeOH, Pd(OH)2, r.t., 5 h; d) anhyd. dioxane, CDI, r.t., 24h; e) DMSO,
NaH, R3I, 22 ºC; f) THF, H2O, LiOH, 50 ºC 2 h; g) anhyd. CH2Cl2, PCl5, Et3N, rt. 24-36 h; h) anhyd.
CH3CN, Et3N, (CH3)2C(OH)CN, rt. 4-10 h.
Scheme 2. Synthetic route of the title compounds.
dissolved in recently distilled acetonitrile (60 mL) under stirring.
Sodium acetate (6.1 g, 0.074 mol) and then alkylamine (0.055 mol)
and distilled water (20 mL) were added to this solution under
vigorous stirring, and the obtained mixture was refluxed for
17e24 h, during which time the course of the reaction was moni-
tored by TLC. The yellow reaction mixture was concentrated under
reduced pressure to remove MeCN (30 mL). The residue was cooled
and diluted with cold water (1.2 L). The obtained yellow precipitate
was triturated in aqueous suspension, separated by filtration,
washed with cold water (2 ꢁ 500 mL) and dried to give the title
compound as bright yellow powder (96%).
for 8 h. The reaction was cooled to 22 ꢀC and poured into water
(1000 mL). a precipitate formed, which was collected by filtration
and washed with cold water to afford the product (9.4 g, 82%) as an
orange solid. 3-nitro-4-phenylaminobenzoic acid methyl ester.
4.1.5. General procedure for intermediate 4
Intermediate of 3 (5 g), 160 mL of ethanol, and Pd hydroxid
(1.6 g) was stirred under atmosphere of hydrogen gas for 5 h. The
mixture was filtered through nylon and concentrated to give 3.9 g
of 4 as a brown liquid.
4.1.6. General procedure for intermediate 5
4.1.3. General procedure for intermediate 3b2 [28]
Intermediate 4（0.027 mol) was dissolved in anhydrous dioxane
(60 mL). 1,1'-carbonyldiimidazole (CDI; 5 g, 0.0308 mol) was added
to this solution in several small portions with stirring. The reaction
mixture was stirred at room temperature for 24 h. The solvent was
removed under reduced pressure, and the residue was washed with
cold ether (3 ꢁ 50 mL), and dried to give the intermediate 5 as off-
white crystals in yield 81%.
Methyl 4-chloro-3-nitrobenzoate (3.84 g, 0.0178 mol) was dis-
solved in a mixture of methanol (25 mL) and N,N-dimethylforma-
mide (3 mL) and treated with cycloalkylamine (21.4 mmol) and
N,N-diisopropylethylamine (3.72 mL, 21.4 mmol). This mixture was
heated at reflux for 18 h. Upon cooling to ambient temperature, a
precipitate formed, which was collected by filtration and washed
with cold methanol (2 ꢁ 10 mL) and hexane (2 ꢁ 10 mL) to afford
the ester 3b as a yellow powder (60%e85%).
4.1.7. General procedure for intermediate 6 [33]
To a stirred solution of intermediate 5 (12 mmol) in 30 mL of
dimethyl sulfoxide (DMSO) was added sodium hydride (60%,
0.576 g, 24 mmol). The resulting mixture was stirred for 1 h at 22 ꢀC
before the addition of alkyl iodide (24 mmol). The mixture was
stirred for 48 h at 25 ꢀC; the reaction was quenched with water, and
4.1.4. General procedure for intermediate 3b3 [30e32]
To
a solution of methyl-4-chloro-3-nitrobenzoate (8.6 g,
0.04 mol) in DMSO (60 mL) was added 0.08 mol 4-methylaniline (or
benzylamine in DMF). The reaction was heated to 70 ꢀC and stirred

Y.-L. Xu et al. / European Journal of Medicinal Chemistry 92 (2015) 427e438
431
Fig. 2. Simulated binding modes of the representative compounds (10f, 9f, 9e and 9h) in human HPPD. The red dash line represents the distance for the Fe(II)-chelation, and the
black dash line refers to the distance for the steric clash. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the mixture was extracted with methylene chloride (3X). The
combined organic layers were orderly washed with water and
brine, dried Na2SO4, and filtered, and the filtrate was concentrated
to give of intermediate 6, yield 80%e95%.
(dd, J ¼ 8.4, 1.2 Hz, 1H), 7.77 (d, J ¼ 1.2 Hz, 1H), 7.48 (d, J ¼ 1.8 Hz,
1H), 7.10 (d, J ¼ 8.4 Hz, 1H), 6.20 (d, J ¼ 1.8 Hz,1H), 3.92 (t, J ¼ 7.2 Hz,
2H), 3.80 (s, 3H), 3.51 (s, 3H), 1.91e1.71 (m, 2H), 1.00 (t, J ¼ 7.8 Hz,
3H). EI-MS (m/z): 314.18 (M)þ.
4.1.11. 1,3-dimethyl-1H-pyrazol-5-yl 3-methyl-2-oxo-1-propyl-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
4.1.8. General procedure for intermediate 7 [34]
To a solution of the intermediate 6 (10 mmol) in THF (50 mL)/
H2O (50 mL) was added 0.84 g (20 mmol) LiOH, and the mixture
was stirred at 50 ꢀC for 2 h, followed by evaporation of THF under
reduced pressure. To the residue solution, 30 mL H2O was added,
and then 6 N hydrochloric acid was utilized to adjust the pH to
2.0e3.0. The resulting precipitate was collected by filtration,
washed with water and dried to afford the intermediates 7 as white
powder (85%e95%).
Yield: 51%. m.p. 135e137 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.99
(dd, J ¼ 8.4, 1.2 Hz, 1H), 7.76 (d, J ¼ 1.2 Hz, 1H), 7.08 (d, J ¼ 8.4 Hz,
1H), 5.99 (s, 1H), 3.91 (t, J ¼ 7.2 Hz, 2H), 3.72 (s, 3H), 3.50 (s, 3H),
2.28 (s, 3H), 1.82 (dd, J ¼ 14.4, 7.2 Hz, 2H), 0.99 (t, J ¼ 7.8 Hz, 3H). EI-
MS (m/z): 328.77(M)þ.
4.1.12. 1-methyl-1H-pyrazol-5-yl 1-butyl-3-methyl-2-oxo-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 41%. m.p. 83e85 ꢀC. 1H NMR (600 MHz, CDCl3)
d 8.01 (d,
4.1.9. General procedure for intermediate 8
J ¼ 8.4 Hz, 1H), 7.76 (s, 1H), 7.48 (s, 1H), 7.09 (d, J ¼ 7.8 Hz, 1H), 6.20
(s, 1H), 3.94 (t, J ¼ 7.2 Hz, 2H), 3.80 (s, 3H), 3.50 (s, 3H), 1.86e1.65
(m, 2H), 1.41 (dd, J ¼ 15, 7.2 Hz, 2H), 0.97 (t, J ¼ 7.2 Hz, 3H). EI-MS
(m/z): 328.25(M)þ.
Intermediate 7 (4 mmol), dissolved in dry dichloromethane
(60 mL), and PCl5 (4.2 mmol) were added and stirred at room
temperature for 1 h. The solvent was moved out under reduced
pressure to afford acid chloride, the acid chloride was then dis-
solved in dry CH2Cl2 (60 mL), Then 1-methyl-1H-pyrazol-5-ol
(4 mmol) and Et3N (4.8 mmol) were added to the above solution,
and stirred for a further 18e24 h at room temperature. After adding
dichloromethane (30 mL), the resultant mixture was washed
sequentially with 2 N hydrochloric acid (100 mL), saturated
NaHCO3 and saturated NaCl and dried over Na2SO4. The rest of the
solution was evaporated in vacuum and purified by silica gel col-
umn chromatography using gradient elution of ethyl acetate/hex-
ane (V:V ¼ 1:2) to obtain the desired intermediate 8 as a write
powder (30%e70%).
4.1.13. 1,3-dimethyl-1H-pyrazol-5-yl 1-butyl-3-methyl-2-oxo-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 40%. m.p. 123e125 ꢀC. 1H NMR (600 MHz, CDCl3)
d 8.00 (d,
J ¼ 7.8 Hz, 1H), 7.76 (s, 1H), 7.09 (d, J ¼ 7.8 Hz, 1H), 6.02 (s, 1H), 3.95
(t, J ¼ 6.6 Hz, 2H), 3.75 (s, 3H), 3.50 (s, 3H), 2.30 (s, 3H), 1.87e1.70
(m, 2H), 1.51e1.28 (m, 2H), 0.97 (t, J ¼ 6.0 Hz, 3H). EI-MS (m/z):
342.26(M)þ.
4.1.14. 1,3-dimethyl-1H-pyrazol-5-yl 1-isobutyl-3-methyl-2-oxo-
2,3-dihydro-1H-benzo[d] imidazole-5-carboxylate
4.1.10. 1-methyl-1H-pyrazol-5-yl3-methyl-2-oxo-1-propyl-2,3-
dihydro-1H-benzo[d] imidazole- 5-carboxylate
Yield: 41%. m.p. 142e144 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.99 (d,
J ¼ 7.8 Hz, 1H), 7.77 (s, 1H), 7.09 (d, J ¼ 7.8 Hz, 1H), 6.01 (s, 1H),
Yield: 45%. m.p. 107e108 ꢀC. 1H NMR (600 MHz, CDCl3)
d 8.01
3.79e3.67 (m, 5H), 3.51 (s, 3H), 2.29 (s, 3H), 2.26e2.19 (m, 1H), 0.99

432
Y.-L. Xu et al. / European Journal of Medicinal Chemistry 92 (2015) 427e438
Table 2
4.1.22. 1,3-dimethyl-1H-pyrazol-5-yl 1-tert-butyl-3-methyl-2-oxo-
2,3-dihydro-1H-benzo[d] imidazole-5-carboxylate
Volume of the representative compounds (10f, 9f, 9e and 9h), distances for Fe(II)
-
chelation, and the steric clash between the two key residues (Leu289 and Phe347) of
human HPPD and the representative compounds.
Yield: 41%. m.p. 187e189 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.89 (d,
J ¼ 9.0 Hz, 1H), 7.70 (s, 1H), 7.50 (d, J ¼ 8.4 Hz, 1H), 6.00 (s, 1H), 3.72
(s, 3H), 3.44 (s, 3H), 2.28 (s, 3H), 1.82 (s, 9H). EI-MS (m/z):
342.19(M)þ.
Comp. Volume (Å3) Distance for chelation (Å) Distance for the steric clash
(Å)
Leu289 Phe347 Asn241
10f
9f
9e
9h
208.20
195.51
205.79
233.92
3.4, 1.8
2.4, 1.9
2.3, 1.9
2.6, 2.1
4.2
4.9
3.4
3.5
3.3, 3.7 3.3, 3.9
4.1.23. 1,3-dimethyl-1H-pyrazol-5-yl 3-methyl-2-oxo-1-p-tolyl-
2,3-dihydro-1H-benzo[d] imidazole-5-carboxylate
3.3, 4.0
3.3, 3.4
2.9, 3.2
e
e
e
Yield: 39%. m.p. 184e186 ꢀC. 1H NMR (400 MHz, CDCl3)
d 7.96 (d,
J ¼ 12 Hz, 1H), 7.82 (s, 1H), 7.54e7.32 (m, 4H), 7.12 (d, J ¼ 12.2 Hz,
1H), 6.03 (s, 1H), 3.75 (s, 3H), 3.57 (s, 3H), 2.45 (s, 3H), 2.30 (s, 3H).
EI-MS (m/z): 376.01(M)þ.
(d, J ¼ 4.8 Hz, 6H). EI-MS (m/z): 342.26(M)þ.
4.1.24. 1-methyl-1H-pyrazol-5-yl 1-tert-butyl-3-methyl-2-oxo-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
4.1.15. 1-methyl-1H-pyrazol-5-yl 1-ethyl-3-methyl-2-oxo-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 41%. m.p. 171e173 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.90 (d,
Yield: 41%. m.p. 100e102 ꢀC. 1H NMR (600 MHz, CDCl3)
J ¼ 8.4 Hz, 1H), 7.70 (s, 1H), 7.51 (d, J ¼ 8.4 Hz, 1H), 7.48 (s, 1H), 6.21
(s, 1H), 3.80 (s, 3H), 3.44 (s, 3H), 1.83 (s, 9H). EI-MS (m/z):
328.28(M)þ.
d
8.09e7.96 (m, 1H), 7.78 (s, 1H), 7.48 (d, J ¼ 1.8 Hz, 1H), 7.11 (d,
J ¼ 8.4 Hz, 1H), 6.20 (d, J ¼ 1.2 Hz, 1H), 4.02 (q, J ¼ 7.2 Hz, 2H), 3.81
(s, 3H), 3.50 (s, 3H), 1.38 (t, J ¼ 7.2 Hz, 3H). EI-MS (m/z): 300.28(M)þ.
4.1.16. 1,3-dimethyl-1H-pyrazol-5-yl 1-ethyl-3-methyl-2-oxo-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
4.1.25. 1-methyl-1H-pyrazol-5-yl 1-cyclopropyl-3-methyl-2-oxo-
2,3-dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 48%. m.p. 186e188 ꢀC. 1H NMR (600 MHz, CDCl3)
d
8.01 (d,
Yield: 38%. m.p. 157e158 ꢀC. 1H NMR (600 MHz, CDCl3)
d 8.01 (d,
J ¼ 7.8 Hz, 1H), 7.76 (s, 1H), 7.10 (d, J ¼ 7.8 Hz, 1H), 6.00 (s, 1H), 4.01
(q, J ¼ 7.2 Hz, 2H), 3.73 (s, 3H), 3.50 (s, 3H), 2.28 (s, 3H), 1.38 (t,
J ¼ 7.2 Hz, 3H). EI-MS (m/z): 314.31(M)þ.
J ¼ 7.8 Hz, 1H), 7.73 (s, 1H), 7.50 (s, 1H), 7.31 (d, J ¼ 8.4 Hz, 1H), 6.22
(s, 1H), 3.82 (s, 3H), 3.46 (s, 3H), 2.99e2.90 (m, 1H), 1.24e1.13 (m,
2H), 1.08e0.99 (m, 2H). EI-MS (m/z): 312.30(M)þ.
4.1.17. 1-methyl-1H-pyrazol-5-yl 1-isopropyl-3-methyl-2-oxo-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
4.1.26. 1,3-dimethyl-1H-pyrazol-5-yl 1-benzyl-3-methyl-2-oxo-
2,3-dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 65%. m.p. 160e162 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.98 (d,
Yield: 35%. m.p. 163e165 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.91 (d,
J ¼ 7.2 Hz, 1H), 7.76 (s, 1H), 7.48 (s, 1H), 7.23 (d, J ¼ 8.4 Hz, 1H), 6.20
(s, 1H), 4.92e4.66 (m, 1H), 3.80 (s, 3H), 3.48 (s, 3H), 1.58 (s, 3H), 1.57
(s, 3H). EI-MS (m/z): 314.40(M)þ.
J ¼ 7.8 Hz, 1H), 7.76 (s, 1H), 7.42e7.28 (m, 5H), 6.97 (d, J ¼ 8.4 Hz,
1H), 5.99 (s, 1H), 5.13 (s, 2H), 3.71 (s, 3H), 3.54 (s, 3H), 2.27 (s, 3H).
EI-MS (m/z): 376.29(M)þ.
4.1.18. 1,3-dimethyl-1H-pyrazol-5-yl 1-isopropyl-3-methyl-2-oxo-
2,3-dihydro-1H-benzo[d] imidazole-5-carboxylate
4.1.27. 1-methyl-1H-pyrazol-5-yl 1-benzyl-3-methyl-2-oxo-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 58%. m.p. 183e185 ꢀC. 1H NMR (600 MHz, cdcl3)
d 7.97 (d,
Yield: 34%. m.p. 137e138 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.92 (d,
J ¼ 8.4 Hz, 1H), 7.76 (s, 1H), 7.23 (d, J ¼ 7.8 Hz, 1H), 6.00 (s, 1H),
4.89e4.68 (m, 1H), 3.72 (s, 3H), 3.49 (s, 3H), 2.28 (s, 3H),1.58 (s, 3H),
1.57 (s, 3H). EI-MS (m/z): 328.24(M)þ.
J ¼ 7.8 Hz, 1H), 7.77 (s, 1H), 7.53 (s, 1H), 7.39e7.28 (m, 5H), 6.98 (d,
J ¼ 7.8 Hz, 1H), 6.25 (s, 1H), 5.14 (s, 2H), 3.84 (s, 3H), 3.55 (s, 3H). EI-
MS (m/z): 362.43(M)þ.
4.1.19. 1-methyl-1H-pyrazol-5-yl 1-cyclopentyl-3-methyl-2-oxo-
2,3-dihydro-1H-benzo[d] imidazole-5-carboxylate
4.1.28. 1-methyl-1H-pyrazol-5-yl 3-methyl-2-oxo-1-p-tolyl-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 45%. m.p. 123e125 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.98 (d,
Yield: 46%. m.p. 173e175 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.96 (d,
J ¼ 8.4 Hz, 1H), 7.76 (s, 1H), 7.48 (s, 1H), 7.16 (d, J ¼ 8.4 Hz, 1H), 6.21
(s,1H), 4.90 (s,1H), 3.80 (s, 3H), 3.49 (s, 3H), 2.23e2.05 (m, 4H), 2.00
(s, 2H), 1.77 (s, 2H). EI-MS (m/z): 340.72(M)þ.
J ¼ 8.4 Hz, 1H), 7.82 (s, 1H), 7.57 (s, 1H), 7.43e7.34 (m, 4H), 7.13 (d,
J ¼ 8.4 Hz, 1H), 6.31 (s, 1H), 3.89 (s, 3H), 3.58 (s, 3H), 2.45 (s, 3H),
2.18 (s, 3H). EI-MS (m/z): 362.38(M)þ.
4.1.20. 1,3-dimethyl-1H-pyrazol-5-yl 1-cyclopentyl-3-methyl-2-
oxo-2,3-dihydro-1H-benzo[d] imidazole-5-carboxylate
4.1.29. 1-methyl-1H-pyrazol-5-yl 1,3-diethyl-2-oxo-2,3-dihydro-
1H-benzo[d] imidazole-5-carboxylate
Yield: 66%. m.p. 116e118 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.97 (d,
Yield: 42%. m.p. 135e137 ꢀC. 1H NMR (600 MHz, CDCl3)
d 8.01 (d,
J ¼ 7.8 Hz, 1H), 7.76 (s, 1H), 7.16 (d, J ¼ 8.4 Hz, 1H), 6.01 (s, 1H),
5.00e4.75 (m, 1H), 3.73 (s, 3H), 3.49 (s, 3H), 2.29 (s, 3H), 2.16e2.04
(m, 4H), 2.03e1.91 (m, 2H), 1.86e1.69 (m, 2H). EI-MS (m/z):
354.63(M)þ.
J ¼ 8.4 Hz, 1H), 7.78 (s, 1H), 7.53 (s, 1H), 7.11 (d, J ¼ 8.4 Hz, 1H), 6.26
(s,1H), 4.07e3.99 (m, 4H), 3.86 (s, 3H),1.39 (dd, J ¼ 13.2, 6.6 Hz, 6H).
EI-MS (m/z): 314.20(M)þ.
4.1.21. 1-methyl-1H-pyrazol-5-yl 1-isobutyl-3-methyl-2-oxo-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
4.1.30. 1-methyl-1H-pyrazol-5-yl 1-ethyl-2-oxo-3-propyl-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 40%. m.p. 141e143 ꢀC. 1H NMR (600 MHz, CDCl3)
d
8.00 (d,
Yield: 36%. m.p. 95e97 ꢀC. 1H NMR (600 MHz, CDCl3)
d 8.00 (d,
J ¼ 7.8 Hz, 1H), 7.77 (s, 1H), 7.50 (s, 1H), 7.09 (d, J ¼ 8.4 Hz, 1H), 6.22
(s, 1H), 3.82 (s, 3H), 3.75 (d, J ¼ 7.2 Hz, 2H), 3.51 (s, 3H), 2.35e2.12
(m, 1H), 1.00 (s, 3H), 0.99 (s, 3H), 0.00 (s, 3H). EI-MS (m/z):
328.39(M)þ.
J ¼ 8.4 Hz, 1H), 7.77 (s, 1H), 7.51 (s, 1H), 7.11 (d, J ¼ 8.4 Hz, 1H), 6.23
(s, 1H), 4.01 (q, J ¼ 7.2 Hz, 2H), 3.92 (t, J ¼ 7.2 Hz, 2H), 3.83 (s, 3H),
1.83 (dd, J ¼ 15, 7.2 Hz, 2H), 1.38 (t, J ¼ 7.2 Hz, 3H), 1.00 (t, J ¼ 7.2 Hz,
3H). EI-MS (m/z): 328.07(M)þ.

Y.-L. Xu et al. / European Journal of Medicinal Chemistry 92 (2015) 427e438
433
4.1.31. 1-methyl-1H-pyrazol-5-yl 3-ethyl-2-oxo-1-propyl-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
3.96e3.87 (m, 4H), 3.72 (s, 3H), 2.28 (s, 3H), 1.90e1.75 (m, 4H), 0.99
(dd, J ¼ 12.0, 7.2 Hz, 6H). EI-MS (m/z): 356.27(M)þ.
Yield: 39%. m.p. 89e91 ꢀC. 1H NMR (600 MHz, CDCl3)
d 8.00 (d,
J ¼ 8.4 Hz, 1H), 7.79 (s, 1H), 7.49 (s, 1H), 7.10 (d, J ¼ 8.4 Hz, 1H), 6.21
(s, 1H), 4.02 (q, J ¼ 7.2 Hz, 2H), 3.91 (t, J ¼ 7.2 Hz, 2H), 3.81 (s, 3H),
1.82 (dd, J ¼ 14.4, 7.2 Hz, 2H), 1.39 (t, J ¼ 7.2 Hz, 3H), 1.00 (t,
J ¼ 7.2 Hz, 3H). EI-MS (m/z): 328.25(M)þ.
4.1.40. 1,3-dimethyl-1H-pyrazol-5-yl 3-ethyl-1-isopropyl-2-oxo-
2,3-dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 70%. m.p. 129e131 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.96 (d,
J ¼ 8.4 Hz, 1H), 7.77 (s, 1H), 7.23 (d, J ¼ 7.8 Hz, 1H), 6.00 (s, 1H),
4.84e4.70 (m, 1H), 4.00 (q, J ¼ 7.8 Hz, 2H), 3.72 (s, 3H), 2.28 (s, 3H),
1.57 (d, J ¼ 6.6 Hz, 6H), 1.38 (t, J ¼ 7.2 Hz, 3H). EI-MS (m/z):
342.27(M)þ.
4.1.32. 1-methyl-1H-pyrazol-5-yl 1-tert-butyl-3-ethyl-2-oxo-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 41%. m.p. 126e128 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.89 (d,
J ¼ 9.0 Hz, 1H), 7.72 (s, 1H), 7.51 (d, J ¼ 8.4 Hz, 1H), 7.48 (s, 1H), 6.21
(s, 1H), 3.96 (q, J ¼ 7.2 Hz, 2H), 3.81 (s, 3H), 1.83 (s, 9H), 1.36 (t,
J ¼ 7.2 Hz, 3H). EI-MS (m/z): 342.21(M)þ.
4.1.41. 1,3-dimethyl-1H-pyrazol-5-yl 1-isopropyl-2-oxo-3-propyl-
2,3-dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 51%. m.p. 110e112 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.96 (d,
J ¼ 8.4 Hz, 1H), 7.75 (s, 1H), 7.23 (d, J ¼ 8.4 Hz, 1H), 6.00 (s, 1H),
4.85e4.73 (m, 1H), 3.90 (t, J ¼ 7.2 Hz, 2H), 3.72 (s, 3H), 2.28 (s, 3H),
1.88e1.74 (m, 2H), 1.57 (d, J ¼ 7.2 Hz, 6H), 1.00 (t, J ¼ 7.2 Hz, 3H). EI-
MS (m/z): 356.18(M)þ.
4.1.33. 1-methyl-1H-pyrazol-5-yl 2-oxo-1,3-dipropyl-2,3-dihydro-
1H-benzo[d] imidazole-5-carboxylate
Yield: 40%. m.p. 97e99 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.99 (d,
J ¼ 8.4 Hz,1H), 7.77 (s, 1H), 7.48 (d, J ¼ 1.8 Hz,1H), 7.09 (d, J ¼ 8.4 Hz,
1H), 6.20 (d, J ¼ 1.8 Hz, 1H), 3.96e3.87 (m, 4H), 3.81 (s, 3H),
1.88e1.74 (m, 4H), 1.03e0.96 (m, 6H). EI-MS (m/z): 342.39(M)þ.
4.1.42. 1-methyl-1H-pyrazol-5-yl 1-cyclopropyl-3-ethyl-2-oxo-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
4.1.34. 1-methyl-1H-pyrazol-5-yl 3-ethyl-1-isopropyl-2-oxo-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 40%; m.p. 117e118 ꢀC. 1H NMR (600 MHz, CDCl3)
d 8.00 (d,
J ¼ 7.2 Hz, 1H), 7.75 (s, 1H), 7.49 (s, 1H), 7.31 (d, J ¼ 8.4 Hz, 1H), 6.21
(d, J ¼ 1.8 Hz,1H), 3.98 (q, J ¼ 7.2 Hz, 2H), 3.81 (s, 3H), 2.99e2.88 (m,
1H), 1.37 (t, J ¼ 7.2 Hz, 3H), 1.18 (q, J ¼ 7.2 Hz, 2H), 1.09e1.02 (m, 2H).
EI-MS (m/z): 326.43(M)þ.
Yield: 41%. m.p. 107e108 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.97
(dd, J ¼ 7.8,1.8 Hz,1H), 7.78 (d, J ¼ 1.2 Hz,1H), 7.49 (d, J ¼ 1.8 Hz,1H),
7.24 (d, J ¼ 8.4 Hz, 1H), 6.21 (d, J ¼ 1.2 Hz, 1H), 4.85e4.71 (m, 1H),
4.00 (q, J ¼ 7.2 Hz, 2H), 3.81 (s, 3H), 1.58 (d, J ¼ 7.2 Hz, 6H), 1.39 (t,
J ¼ 7.2 Hz, 3H). EI-MS (m/z): 328.18(M)þ.
4.1.43. General procedure for target compounds 9 and 10
The mixture of corresponding intermediate 8 (1 mmol) dis-
solved in acetonitrile (15 mL), triethylamine (0.32 mL), and acetone
cyanohydrin (0.01 mL) was stirred at room temperature for 5e12 h.
When the reaction monitored by TLC completed, the solvent was
removed under reduced pressure. After adding 20 mL dichloro-
methane, the solution was washed sequentially with 1 N hydro-
chloric acid (20 mL), saturated brine (40 mL ꢁ 2), and dried Na2SO4.
Then the solvent was removed under reduced pressure. Finally, the
desired products were recrystallized from ethyl acetate/petroleum
ether (V:V ¼ 1:10) after solvent evaporation under reduced
pressure.
4.1.35. 1-methyl-1H-pyrazol-5-yl 1-isopropyl-2-oxo-3-propyl-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
Yield: 50%. Yellow oil. 1H NMR (600 MHz, CDCl3)
d 7.97 (d,
J ¼ 7.8 Hz,1H), 7.77 (s, 1H), 7.49 (d, J ¼ 1.2 Hz, 1H), 7.24 (d, J ¼ 8.4 Hz,
1H), 6.21 (d, J ¼ 1.2 Hz, 1H), 4.84e4.73 (m, 1H), 3.90 (t, J ¼ 7.2 Hz,
2H), 3.81 (s, 3H), 1.89e1.75 (m, 2H), 1.58 (d, J ¼ 6.6 Hz, 6H), 1.00 (t,
J ¼ 7.2 Hz, 3H). EI-MS (m/z): 342.34(M)þ.
4.1.36. 1,3-dimethyl-1H-pyrazol-5-yl 1,3-diethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazole-5-carboxylate
Yield: 32%. m.p. 157e158 ꢀC. 1H NMR (600 MHz, CDCl3)
d 8.00 (d,
J ¼ 7.8 Hz, 1H), 7.78 (s, 1H), 7.10 (d, J ¼ 8.4 Hz, 1H), 6.00 (s, 1H),
4.11e3.89 (m, 4H), 3.73 (s, 3H), 2.28 (s, 3H), 1.39 (q, J ¼ 7.2 Hz, 6H).
EI-MS (m/z): 328.22(M)þ.
4.1.44. 5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-3-methyl-
1-propyl-1H-benzo[d] imidazol-2(3H)-one 9a
Yellow solid, Yield: 56%. m.p. 144e146 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.82 (s, 1H), 7.77 (d, J ¼ 7.8 Hz, 1H), 7.58 (s, 1H), 7.09 (d,
4.1.37. 1,3-dimethyl-1H-pyrazol-5-yl 1-ethyl-2-oxo-3-propyl-2,3-
dihydro-1H-benzo[d] -imidazole-5-carboxylate
J ¼ 7.8 Hz, 1H), 3.90 (t, J ¼ 7.2 Hz, 2H), 3.72 (s, 3H), 3.49 (s, 3H),
1.94e1.69 (m, 2H), 0.99 (t, J ¼ 7.8 Hz, 3H). 13C NMR (100 MHz, CDCl3)
Yield: 39%. m.p. 146e148 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.99 (d,
d
188.83, 159.85, 154.45, 138.36, 133.34, 130.33, 130.25, 122.99,
J ¼ 7.8 Hz, 1H), 7.76 (s, 1H), 7.10 (d, J ¼ 8.4 Hz, 1H), 6.00 (s, 1H), 4.01
(q, J ¼ 7.2 Hz, 2H), 3.91 (t, J ¼ 7.2 Hz, 2H), 3.73 (s, 3H), 2.29 (s, 3H),
1.89e1.75 (m, 2H), 1.38 (t, J ¼ 7.2 Hz, 3H), 1.00 (t, J ¼ 7.2 Hz, 3H). EI-
MS (m/z): 342.25(M)þ.
107.23, 106.91, 101.83, 42.91, 32.90, 27.21, 21.66, 11.21. HRMS (ESIþ)
cal. for [MþHþ]: cal. 315.1457, found. 315.1463. Anal. Calcd. For
C
16H18N4O3: C, 61.13; H, 5.77; N, 17.82. Found: C, 61.10; H, 5.46; N,
17.92.
4.1.38. 1,3-dimethyl-1H-pyrazol-5-yl 3-ethyl-2-oxo-1-propyl-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
4.1.45. 1-butyl-5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-
3-methyl-1H-benzo[d] imidazol-2(3H)-one 9b
Yield: 35%. m.p. 154e156 ꢀC. 1H NMR (600 MHz, CDCl3)
d 7.99 (d,
J ¼ 7.8 Hz, 1H), 7.78 (s, 1H), 7.09 (d, J ¼ 8.4 Hz, 1H), 6.00 (s, 1H), 4.02
(q, J ¼ 7.2 Hz, 2H), 3.91 (t, J ¼ 7.2 Hz, 2H), 3.73 (s, 3H), 2.29 (s, 3H),
1.82 (dd, J ¼ 14.4, 7.2 Hz, 2H), 1.39 (t, J ¼ 7.2 Hz, 3H), 0.99 (t,
J ¼ 7.2 Hz, 3H). EI-MS (m/z): 342.34(M)þ.
Yellow solid, Yield: 48%. m.p. 125e126 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.83 (s, 1H), 7.77 (d, J ¼ 7.8 Hz, 1H), 7.58 (s, 1H), 7.09 (d,
J ¼ 7.8 Hz, 1H), 3.93 (t, J ¼ 7.2 Hz, 2H), 3.73 (s, 3H), 3.49 (s, 3H),
1.88e1.67 (m, 2H), 1.51e1.31 (m, 2H), 0.97 (t, J ¼ 7.8 Hz, 3H). 13C
NMR (100 MHz, CDCl3)
d 188.89, 159.90, 154.46, 138.38, 133.33,
4.1.39. 1,3-dimethyl-1H-pyrazol-5-yl 2-oxo-1,3-dipropyl-2,3-
dihydro-1H-benzo[d] imidazole-5-carboxylate
130.36, 130.31, 123.03, 107.26, 106.93, 101.88, 41.16, 32.95, 30.41,
27.25, 19.98, 13.64. HRMS (ESIþ) cal. for [MþHþ]: cal. 329.1614,
found. 329.1616. Anal. Calcd. For C17H20N4O3: C, 62.18; H, 6.14; N,
17.06. Found: C, 62.30; H, 6.04; N, 16.91.
Yield: 72%. m.p. 137e138 ꢀC. 1H NMR (600 MHz, CDCl3)
d
7.98 (d,
J ¼ 8.4 Hz, 1H), 7.76 (s, 1H), 7.09 (d, J ¼ 8.4 Hz, 1H), 5.99 (s, 1H),

434
Y.-L. Xu et al. / European Journal of Medicinal Chemistry 92 (2015) 427e438
4.1.46. 5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-1-
isobutyl-3-methyl-1H-benzo[d] imidazol-2(3H)-one 9c
Yellow solid, Yield: 74%. m.p. 168e170 ꢀC. 1H NMR (600 MHz,
(ESIþ) cal. for [MþHþ]: cal. 363.1457, found. 363.1451. Anal. Calcd.
For C20H18N4O3: C, 66.29; H, 5.01; N, 15.46. Found: C, 66.13; H, 5.28;
N, 15.57.
CDCl3)
d
8.83 (s, 1H), 8.76 (dd, J ¼ 7.8, 1.2 Hz, 1H), 8.58 (s, 1H), 8.09
(d, J ¼ 8.4 Hz, 1H), 4.74 (s, 1H), 4.73 (s, 4H), 4.50 (s, 3H), 3.35e3.17
4.1.52. 1-benzyl-5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-
3-methyl-1H-benzo[d] imidazol-2(3H)-one 9i
(m, 1H), 2.00 (s, 3H), 1.98 (s, 3H). 13C NMR (100 MHz, CDCl3)
d
188.80, 159.82, 154.66, 138.36, 138.33, 133.63, 130.30, 130.1,
Yellow solid, Yield: 59%. m.p. 237e238 ꢀC. 1H NMR (600 MHz,
122.94, 107.17, 101.81, 48.74, 32.88, 27.90, 27.23, 20.00. HRMS (ESIþ)
CDCl3)
J ¼ 1.8 Hz, 4H), 7.31e7.27 (m,1H), 6.97 (d, J ¼ 7.8 Hz,1H), 5.13 (s, 2H),
3.74 (s, 3H), 3.53 (s, 3H). 13C NMR (100 MHz, DMSO)
186.88, 56.30,
d
7.80 (s, 1H), 7.67 (d, J ¼ 7.8 Hz, 1H), 7.58 (s, 1H), 7.34 (d,
cal. for [MþHþ]: cal. 329.1614, found. 329.1620. Anal. Calcd. For
C
17H20N4O3: C, 62.18; H, 6.14; N, 17.06. Found: C, 61.93; H, 5.99; N,
d
17.01.
153.99, 139.22, 136.63, 132.00, 131.54, 129.68, 128.59, 127.49, 127.32,
122.68, 107.90, 107.59, 102.88, 43.90, 32.85, 27.12. HRMS (ESIþ) cal.
for [MþHþ]: cal. 363.1457, found. 363.1435. Anal. Calcd. For
4.1.47. 1-cyclopentyl-5-(5-hydroxy-1-methyl-1H-pyrazole-4-
carbonyl)-3-methyl-1H-benzo[d] imidazol-2(3H)-one 9d
Yellow solid, Yield: 50%. m.p. 140e141 ꢀC. 1H NMR (600 MHz,
C
20H18N4O3: C, 66.29; H, 5.01; N, 15.46. Found: C, 66.30; H, 5.34; N,
15.71.
CDCl3)
d
7.83 (s, 1H), 7.74 (d, J ¼ 8.4 Hz, 1H), 7.58 (s, 1H), 7.16 (d,
J ¼ 8.4 Hz, 1H), 4.98e4.81 (m, 1H), 3.73 (s, 3H), 3.48 (s, 3H),
2.19e2.10 (m, 1H), 2.09e2.03 (m, 1H), 1.99 (t, J ¼ 7.8 Hz, 2H), 1.76 (t,
4.1.53. Cyclopropyl-5-(5-hydroxy-1-methyl-1H-pyrazole-4-
carbonyl)-3-methyl-1H-benzo[d] imidazol-2(3H)-one 9j
Yellow solid, Yield: 72%. m.p. 219e221 ꢀC. 1H NMR (600 MHz,
J ¼ 5.4 Hz, 2H). 13C NMR (100 MHz, CDCl3)
d 188.62, 159.73, 154.10,
138.24, 131.92, 130.37, 129.93, 122.57, 107.90, 107.17, 101.76, 53.57,
32.78, 28.75, 27.05, 24.87. HRMS (ESIþ) cal. for [MþHþ]: cal.
341.1614, found. 341.1618. Anal. Calcd. For C18H20N4O3: C, 63.52; H,
5.92; N, 16.46. Found: C, 63.08; H, 6.21; N, 16.38.
DMSO)
d
7.76 (s, 1H), 7.66 (d, J ¼ 7.8 Hz, 1H), 7.55 (s, 1H), 7.32 (d,
J ¼ 7.8 Hz, 1H), 3.59 (s, 3H), 3.36 (s, 3H), 3.07e2.82 (m, 1H), 1.07 (q,
J ¼ 6.6 Hz, 2H), 0.90 (t, J ¼ 7.2 Hz, 2H). 13C NMR (100 MHz, CDCl3)
d
188.83, 159.82, 154.63, 138.24, 134.13, 130.55, 129.96, 122.91,
107.79, 107.01, 101.84, 32.87, 27.05, 22.65, 5.97. HRMS (ESIþ) cal. for
4.1.48. 5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-1-
isopropyl-3-methyl-1H-benzo[d] imidazol-2(3H)-one 9e
Yellow solid, Yield: 65%. m.p. 182e184 ꢀC. 1H NMR (600 MHz,
[MþKþ]: cal.351.0859, found. 351.0797. Anal. Calcd. For
C
16H16N4O3: C, 61.53; H, 5.16; N, 17.94. Found: C, 61.41; H, 5.52; N,
18.05.
CDCl3)
J ¼ 8.4 Hz, 1H), 4.90e4.65 (m, 1H), 3.73 (s, 3H), 3.47 (s, 3H), 1.58 (s,
3H), 1.56 (s, 3H). 13C NMR (100 MHz, CDCl3)
188.58, 159.66, 153.77,
138.21, 131.98, 130.30, 129.84, 122.59, 107.95, 107.13, 101.74, 45.24,
32.74, 26.97, 20.04. HRMS (ESIþ) cal. for [MþHþ]: cal. 315.1457,
found. 315.1462. Anal. Calcd. For C16H18N4O3: C, 61.13; H, 5.77; N,
17.82. Found: C, 61.44; H, 6.16; N, 18.09.
d
7.83 (s, 1H), 7.75 (d, J ¼ 8.4 Hz, 1H), 7.58 (s, 1H), 7.23 (d,
4.1.54. 3-ethyl-5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-
1-propyl-1H-benzo[d] imidazol-2(3H)-one 9k
d
Yellow solid, Yield: 52%. m.p. 130e132 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.83 (s, 1H), 7.76 (d, J ¼ 8.4 Hz, 1H), 7.60 (s, 1H), 7.10 (d,
J ¼ 7.8 Hz, 1H), 4.01 (q, J ¼ 7.2 Hz, 2H), 3.90 (t, J ¼ 7.8 Hz, 2H), 3.74 (s,
3H), 1.88e1.73 (m, 2H), 1.38 (t, J ¼ 7.2 Hz, 3H), 1.00 (t, J ¼ 7.8 Hz, 3H).
13C NMR (100 MHz, DMSO)
d 187.07, 156.19, 153.46, 139.26, 132.59,
4.1.49. 1-ethyl-5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-
3-methyl-1H-benzo[d] imidazol-2(3H)-one 9f
131.21, 128.50, 122.70, 107.73, 107.50, 102.96, 42.12, 35.38, 32.97,
21.29, 13.48, 11.04. HRMS (ESIþ) cal. for [MþKþ]: cal. 367.1172,
found. 367.1132.
Yellow solid, Yield: 86%. m.p. 238e240 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.82 (s, 1H), 7.78 (d, J ¼ 8.4 Hz, 1H), 7.58 (s, 1H), 7.10 (d,
J ¼ 8.4 Hz, 1H), 4.00 (q, J ¼ 7.2 Hz, 2H), 3.73 (s, 3H), 3.49 (s, 3H), 1.38
4.1.55. 1,3-diethyl-5-(5-hydroxy-1-methyl-1H-pyrazole-4-
carbonyl)-1H-benzo[d] imidazol-2(3H)-one 9l
(t, J ¼ 7.8 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d 188.93, 159.94,
154.21, 138.36, 138.34, 132.97, 130.44, 123.08, 107.33, 106.80, 101.92,
36.20, 32.96, 27.24, 13.60. HRMS (ESIþ) cal. for [MþHþ]: cal.
301.1301, found. 301.1312. Anal. Calcd. For C15H16N4O3: C, 59.99; H,
5.37; N, 18.66. Found: C, 59.83; H, 5.19; N, 18.33.
Yellow solid, Yield: 56%. m.p. 177e179 ꢀC. 1H NMR (600 MHz,
CDCl3)
J ¼ 8.1 Hz, 1H), 4.06e3.94 (m, 4H), 3.75 (s, 3H), 1.38 (t, J ¼ 7.2 Hz,
6H). 13C NMR (100 MHz, DMSO-d6)
187.09, 156.18, 153.11, 139.27,
d
7.84 (s, 1H), 7.77 (d, J ¼ 8.0 Hz, 1H), 7.60 (s, 1H), 7.11 (d,
d
132.13, 131.23, 128.59, 122.72, 107.75, 107.35, 102.97, 35.44, 32.97,
13.51. HRMS (ESIþ) cal. for [MþKþ]: cal. 353.1016, found. 353.0948.
Anal. Calcd. For C16H18N4O3: C, 61.13; H, 5.77; N, 17.82. Found: C,
61.06; H, 6.02; N, 18.03.
4.1.50. tert-butyl-5-(5-hydroxy-1-methyl-1H-pyrazole-4-
carbonyl)-3-methyl-1H-benzo[d] imidazol-2(3H)-one 9g
Yellow solid, Yield: 57%. m.p. 165e166 ꢀC. 1H NMR (600 MHz,
DMSO)
7.52 (s,1H), 3.59 (s, 3H), 3.35 (s, 3H),1.74 (s, 9H). 13C NMR (100 MHz,
CDCl3) 188.62, 159.91, 154.34, 138.33, 133.23, 130.76, 129.56,
122.18, 110.79, 106.76, 101.86, 58.85, 32.87, 29.29, 26.82. HRMS
(ESIþ) cal. for [MþKþ]: cal. 367.1172, found. 367.1143. Anal. Calcd.
For C17H20N4O3: C, 62.18; H, 6.14; N, 17.06. Found: C, 62.07; H, 5.95;
N, 16.83.
d
7.78 (s, 1H), 7.61 (d, J ¼ 8.4 Hz, 1H), 7.55 (d, J ¼ 9.0 Hz, 1H),
4.1.56. 3-ethyl-5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-
1-isopropyl-1H-benzo[d] imidazol-2(3H)-one 9m
d
Yellow solid, Yield: 40%. m.p. 131e132 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.83 (s, 1H), 7.74 (d, J ¼ 7.8 Hz, 1H), 7.59 (s, 1H), 7.24 (d,
J ¼ 8.4 Hz, 1H), 4.87e4.68 (m, 1H), 3.99 (q, J ¼ 7.2 Hz, 2H), 3.74 (s,
3H), 1.57 (d, J ¼ 6.6 Hz, 6H), 1.38 (t, J ¼ 7.2 Hz, 3H). 13C NMR
(100 MHz, DMSO)
d 187.03, 156.17, 152.77, 139.25, 131.32, 130.94,
4.1.51. 5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-3-methyl-
1-p-tolyl-1H-benzo[d] imidazol-2(3H)-one 9h
128.66, 122.45, 108.36, 107.69, 102.99, 44.71, 35.28, 32.95, 19.84,
13.44. HRMS (ESIþ) cal. for [MþNaþ]: cal. 351.1433, found. 351.1439.
Anal. Calcd. For C17H20N4O3: C, 62.18; H, 6.14; N, 17.06. Found: C,
62.50; H, 6.29; N, 16.95.
Yellow solid, Yield: 78%. m.p. 254e256 ꢀC. 1H NMR (600 MHz,
CDCl3)
J ¼ 8.4 Hz, 2H), 7.36 (d, J ¼ 7.8 Hz, 2H), 7.13 (d, J ¼ 7.8 Hz, 1H), 3.74 (s,
3H), 3.56 (s, 3H), 2.44 (s, 3H). 13C NMR (100 MHz, CDCl3)
188.94,
d
7.82 (s, 1H), 7.73 (d, J ¼ 7.8 Hz, 1H), 7.64 (s, 1H), 7.40 (d,
d
4.1.57. 1-tert-butyl-3-ethyl-5-(5-hydroxy-1-methyl-1H-pyrazole-
4-carbonyl)-1H-benzo[d] imidazol-2(3H)-one 9n
160.03, 153.78, 138.46, 138.25, 133.46, 131.37, 131.28, 130.49, 130.26,
125.87, 123.24, 108.09, 107.52, 102.04, 33.01, 27.47, 21.19. HRMS
Write solid, Yield: 48%. m.p. 157e158 ꢀC. 1H NMR (600 MHz,

Y.-L. Xu et al. / European Journal of Medicinal Chemistry 92 (2015) 427e438
435
DMSO)
d
7.74 (s, 1H), 7.62 (d, J ¼ 9.0 Hz, 1H), 7.57e7.52 (m, 2H), 3.91
N, 17.17.
(q, J ¼ 7.2 Hz, 2H), 3.59 (s, 3H), 1.74 (s, 9H), 1.20 (t, J ¼ 7.2 Hz, 3H). 13
C
NMR (100 MHz, DMSO-d6)
d
186.83, 156.17, 153.14, 139.08, 132.17,
4.1.63. 1-butyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-
130.47, 128.90, 121.91, 111.07, 107.10, 102.90, 58.07, 34.92, 32.88,
28.84, 13.14. HRMS (ESIþ) cal. for [MþNaþ]: cal. 365.1590, found.
365.1599. Anal. Calcd. For C18H22N4O3: C, 63.14; H, 6.48; N, 16.36.
Found: C, 63.03; H, 6.10; N, 16.46.
carbonyl)-3-methyl-1H-benzo[d] imidazol-2(3H)-one 10b
Yellow solid, Yield: 47%. m.p. 131e133 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.43 (dd, J ¼ 8.4, 1.2 Hz, 1H), 7.32 (s, 1H), 7.05 (d, J ¼ 7.8 Hz,
1H), 3.93 (t, J ¼ 7.8 Hz, 2H), 3.66 (s, 3H), 3.47 (s, 3H), 2.12 (s, 3H),
1.81e1.67 (m, 2H), 1.47e1.33 (m, 2H), 0.97 (t, J ¼ 7.8 Hz, 3H). 13C
4.1.58. 1-cyclopropyl-3-ethyl-5-(5-hydroxy-1-methyl-1H-pyrazole-
4-carbonyl)-1H-benzo[d] imidazol-2(3H)-one 9o
NMR (100 MHz, CDCl3) d 191.44, 159.80, 154.21, 146.30, 132.36,
131.00, 129.66, 122.53, 107.10, 106.31, 101.95, 40.91, 32.21, 30.18,
26.98, 19.75, 15.62, 13.41. HRMS (ESIþ) cal. for [MþHþ]: cal.
343.1770, found. 343.1781. Anal. Calcd. For C18H22N4O3: C, 63.14; H,
6.48; N, 16.36. Found: C, 63.34; H, 6.92; N, 16.45.
Write solid, Yield: 60%. m.p. 144e145 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.84 (s, 1H), 7.76 (d, J ¼ 7.8 Hz, 1H), 7.56 (s, 1H), 7.31 (d,
J ¼ 7.8 Hz, 1H), 3.97 (q, J ¼ 7.2 Hz, 2H), 3.75 (s, 3H),2.97e2.88 (m,
1H),1.36 (t, J ¼ 7.2 Hz, 3H),1.16 (q, J ¼ 6.6 Hz, 2H),1.09e1.00 (m, 2H).
13C NMR (100 MHz, DMSO)
d
187.08, 156.14, 153.50, 139.24, 133.41,
4.1.64. 5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-1-
isobutyl-3-methyl-1H-benzo[d] imidazol-2(3H)-one 10c
131.41, 128.28, 122.63, 107.87, 107.48, 102.96, 35.24, 32.96, 22.48,
13.41, 5.69. HRMS (ESIþ) cal. for [MþNaþ]: cal. 349.1277, found.
349.1288. Anal. Calcd. For C17H18N4O3: C, 62.57; H, 5.56; N, 17.17.
Found: C, 62.18; H, 5.28; N, 17.10.
Write solid, Yield: 51%. m.p. 191e193 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.42 (d, J ¼ 7.8 Hz, 1H), 7.32 (s, 1H), 7.04 (d, J ¼ 7.8 Hz, 1H),
3.73 (d, J ¼ 7.2 Hz, 2H), 3.67 (s, 3H), 3.47 (s, 3H), 2.32e2.18 (m, 1H),
2.13 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H). 13C NMR (100 MHz, CDCl3)
4.1.59. 1-ethyl-5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-
3-propyl-1H-benzo[d] imidazol-2(3H)-one 9p
d 191.60, 160.00, 154.60, 146.38, 132.87, 131.08, 129.74, 122.59,
107.12, 106.71, 102.09, 48.69, 32.32, 27.83, 27.14, 19.95, 15.69. HRMS
(ESIþ) cal. for [MþHþ]: cal. 343.1770, found. 343.1788. Anal. Calcd.
For C18H22N4O3: C, 63.14; H, 6.48; N, 16.36. Found: C, 63.11; H, 6.70;
N, 16.54.
Yellow solid, Yield: 48%. m.p. 116e118 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.82 (s, 1H), 7.76 (d, J ¼ 8.4 Hz, 1H), 7.59 (s, 1H), 7.10 (d,
J ¼ 7.8 Hz,1H), 4.00 (q, J ¼ 7.2 Hz, 2H), 3.91 (t, J ¼ 7.2 Hz, 2H), 3.73 (s,
3H), 1.88e1.76 (m, 2H), 1.38 (t, J ¼ 7.2 Hz, 3H), 1.00 (t, J ¼ 7.2 Hz, 3H).
13C NMR (100 MHz, DMSO)
d
187.07, 156.12, 153.45, 139.20, 132.01,
4.1.65. 1-cyclopentyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-
carbonyl)-3-methyl-1H-benzo[d] imidazol-2(3H)-one 10d
Write solid, Yield: 64%. m.p. 185e186 ꢀC. 1H NMR (600 MHz,
131.24, 129.05, 122.68, 107.85, 107.33, 102.97, 41.97, 35.51, 32.97,
21.30, 13.48, 11.10. HRMS (ESIþ) cal. for [MþNaþ]: cal. 351.1433,
found. 351.1394. Anal. Calcd. For C17H20N4O3: C, 62.18; H, 6.14; N,
17.06. Found: C, 62.48; H, 6.41; N, 16.98.
CDCl3)
d
7.41 (d, J ¼ 8.4 Hz, 1H), 7.32 (s, 1H), 7.12 (d, J ¼ 7.8 Hz, 1H),
4.90 (p, J ¼ 9.0 Hz, 1H), 3.66 (s, 3H), 3.45 (s, 3H), 2.13 (s, 3H), 2.11 (d,
J ¼ 8.4 Hz, 2H), 2.10e2.03 (m, 2H), 2.02e1.93 (m, 2H), 1.82e1.69 (m,
4.1.60. 5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-1,3-
dipropyl-1H-benzo[d]imidazol- 2(3H)-one 9q
2H). 13C NMR (100 MHz, CDCl3)
d 191.66, 160.19, 154.11, 146.39,
131.26, 130.83, 130.03, 122.26, 107.51, 107.21, 102.06, 53.64, 32.37,
28.76, 27.06, 24.87, 15.85. HRMS (ESIþ) cal. for [MþHþ]: cal.
355.1770, found. 355.1774. Anal. Calcd. For C19H22N4O3: C, 64.39; H,
6.26; N, 15.81. Found: C, 64.22; H, 5.98; N, 15.69.
Yellow solid, Yield: 47%. m.p. 121e124 ꢀC. 1H NMR (600 MHz,
DMSO)
d
7.73 (s, 1H), 7.65e7.57 (m, 2H), 7.34 (d, J ¼ 7.8 Hz, 1H),
3.99e3.77 (m, 4H), 3.60 (s, 3H),1.69 (dd, J ¼ 14.4, 7.2 Hz, 4H), 0.87 (t,
J ¼ 7.2 Hz, 6H). 13C NMR (101 MHz, DMSO)
d 187.05, 156.10, 153.83,
139.20, 132.46, 131.21, 128.95, 122.65, 107.84, 107.48, 102.96, 42.08,
32.99, 21.24, 11.03. HRMS (ESIþ) cal. for [MþKþ]: cal. 381.1329,
found. 381.1315. Anal. Calcd. For C18H22N4O3: C, 63.14; H, 6.48; N,
16.36. Found: C, 63.01; H, 6.30; N, 16.48.
4.1.66. 5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-1-
isopropyl-3-methyl-1H- benzo[d] imidazol-2(3H)-one 10e
Write solid, Yield: 48%. m.p. 151e153 ꢀC. 1H NMR (600 MHz,
CDCl3)
4.88e4.64 (m, 1H), 3.66 (s, 3H), 3.45 (s, 3H), 2.14 (s, 3H), 1.57 (s, 3H),
1.56 (s, 3H). 13C NMR (100 MHz, CDCl3)
191.63, 160.11, 153.83,
d
7.41 (d, J ¼ 7.8 Hz, 1H), 7.31 (s, 1H), 7.19 (d, J ¼ 8.4 Hz, 1H),
4.1.61. 5-(5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-1-
isopropyl-3-propyl-1H-benzo[d] imidazol-2(3H)-one 9r
Yellow solid, Yield: 47%. m.p. 137e139 ꢀC. 1H NMR (600 MHz,
d
146.41, 131.36, 130.76, 130.00, 122.35, 107.63, 107.21, 102.12, 45.31,
32.37, 27.04, 20.13, 15.78. HRMS (ESIþ) cal. for [MþHþ]: cal.
329.1614, found. 329.1619. Anal. Calcd. For C17H20N4O3: C, 62.18; H,
6.14; N, 17.06. Found: C, 62.48; H, 6.01; N, 16.98.
CDCl3)
d
7.83 (s, 1H), 7.73 (d, J ¼ 8.4 Hz, 1H), 7.58 (s, 1H), 7.24 (d,
J ¼ 8.4 Hz, 1H), 4.85e4.69 (m, 1H), 3.89 (t, J ¼ 7.2 Hz, 2H), 3.75 (s,
3H), 1.87e1.73 (m, 2H), 1.57 (d, J ¼ 7.0 Hz, 6H), 0.99 (t, J ¼ 7.2 Hz,
3H). 13C NMR (100 MHz, DMSO)
d
187.04, 156.07, 153.15, 139.24,
4.1.67. 1-ethyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-
carbonyl)-3-methyl-1H-benzo[d] imidazol-2(3H)-one 10f
Write solid, Yield: 45%. m.p. 189e191 ꢀC. 1H NMR (600 MHz,
131.24, 130.95, 129.13, 122.44, 108.36, 107.83, 103.00, 44.72, 41.88,
32.99, 21.25, 19.84, 11.12. HRMS (ESIþ) cal. for [MþNaþ]: cal.
365.1590, found. 365.1595. Anal. Calcd. For C18H22N4O3: C, 63.14; H,
6.48; N, 16.36. Found: C, 62.98; H, 6.73; N, 16.39.
CDCl3)
d
7.44 (d, J ¼ 7.8 Hz, 1H), 7.32 (s, 1H), 7.06 (d, J ¼ 8.4 Hz, 1H),
4.00 (q, J ¼ 7.2 Hz, 2H), 3.68 (s, 3H), 3.47 (s, 3H), 2.14 (s, 3H), 1.38 (t,
J ¼ 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d 191.51, 159.90, 154.01,
4.1.62. 5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-3-
methyl-1-propyl-1H-benzo[d] imidazol-2(3H)-one 10a
146.35, 132.03, 131.11, 129.80, 122.61, 107.17, 106.23, 102.07, 36.00,
32.28, 27.03, 15.64, 13.43. HRMS (ESIþ) cal. for [MþHþ]: cal.
315.1457, found. 315.1465. Anal. Calcd. For C16H18N4O3: C, 61.13; H,
5.77; N, 17.82. Found: C, 61.22; H, 5.99; N, 17.98.
Yellow solid, Yield: 58%. m.p. 160e162 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.43 (d, J ¼ 7.8 Hz, 1H), 7.32 (s, 1H), 7.05 (d, J ¼ 8.4 Hz, 1H),
3.89 (t, J ¼ 7.2 Hz, 2H), 3.66 (s, 3H), 3.47 (s, 3H), 2.12 (s, 3H), 1.81 (dd,
J ¼ 14.4, 6.6 Hz, 2H), 0.99 (t, J ¼ 7.8 Hz, 3H). 13C NMR (100 MHz,
4.1.68. 1-tert-butyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-
carbonyl)-3-methyl-1H-benzo [d] imidazol-2(3H)-one 10g
Yellow solid, Yield: 48%. m.p. 132e134 ꢀC. 1H NMR (600 MHz,
CDCl3) d 191.81,160.22,154.41,146.42,132.55,131.14,129.83,122.60,
107.17, 106.47, 102.04, 42.88, 32.42, 27.15, 21.64, 15.90, 11.19. HRMS
(ESIþ) cal. for [MþHþ]: cal. 329.1614, found. 329.1629. Anal. Calcd.
For C17H20N4O3: C, 62.18; H, 6.14; N, 17.06. Found: C, 62.53; H, 6.16;
CDCl3)
d
7.46 (d, J ¼ 9.0 Hz, 1H), 7.34 (d, J ¼ 8.4 Hz, 1H), 3.65 (s, 3H),
3.40 (s, 3H), 2.14 (s, 3H), 1.82 (s, 9H). 13C NMR (100 MHz, CDCl3)

436
Y.-L. Xu et al. / European Journal of Medicinal Chemistry 92 (2015) 427e438
d
191.32, 159.98, 154.15, 146.32, 132.29, 130.303, 130.154, 121.76,
4.1.74. 1-ethyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-
carbonyl)-3-propyl-1H-benzo[d] imidazol-2(3H)-one 10m
Write solid, Yield: 69%. m.p. 205e207 ꢀC. 1H NMR (600 MHz,
110.27, 106.71, 102.01, 58.60, 32.20, 29.13, 26.63, 15.71. HRMS (ESIþ)
cal. for [MþNaþ]: cal. 365.1590, found. 365.1595. Anal. Calcd. For
C
18H22N4O3: C, 63.14; H, 6.48; N, 16.36. Found: C, 63.25; H, 6.06; N,
DMSO)
d
7.53 (s, 1H), 7.49 (s, 1H) 7.27 (d, J ¼ 7.8 Hz, 1H), 3.92 (q,
16.47.
J ¼ 6.6 Hz, 2H), 3.84 (t, J ¼ 7.2 Hz, 2H), 3.48 (s, 3H), 2.09 (s, 3H),
1.75e1.59 (m, 2H), 1.23 (t, J ¼ 7.2 Hz, 3H), 0.87 (t, J ¼ 7.2 Hz, 3H). 13
C
NMR (100 MHz, CDCl3)
d 191.75, 160.05, 153.91, 146.39, 132.23,
4.1.69. 5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-3-
methyl-1-p-tolyl-1H-benzo[d] imidazol-2(3H)-one 10h
131.03, 129.32, 122.50, 107.37, 106.41, 102.15, 42.73, 36.02, 32.40,
21.68, 15.70, 13.52, 11.17. HRMS (ESIþ) cal. for [MþKþ]: cal. 381.1329,
found. 381.1304. Anal. Calcd. For C18H22N4O3: C, 63.14; H, 6.48; N,
16.36. Found: C, 62.95; H, 6.76; N, 16.33.
Yellow solid, Yield: 54%. m.p. 275e277 ꢀC. 1H NMR (600 MHz,
CDCl3)
J ¼ 7.8 Hz, 2H), 7.09 (d, J ¼ 7.8 Hz, 1H), 3.66 (s, 3H), 3.54 (s, 3H), 2.44
(s, 3H), 2.11 (s, 3H). 13C NMR (101 MHz, CDCl3)
191.80, 160.24,
d
7.41 (d, J ¼ 8.4 Hz, 2H), 7.38 (d, J ¼ 6.0 Hz, 2H), 7.35 (d,
d
4.1.75. 5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-1,3-
dipropyl-1H-benzo[d] imidazol-2(3H)-one 10n
153.65, 146.49, 138.10, 132.62, 131.98, 131.30, 130.09, 129.99, 125.79,
122.85, 107.56,107.40, 102.17, 32.48, 27.38, 21.15,15.89. HRMS (ESIþ)
cal. for [MþHþ]: cal. 377.1614, found. 377.1611. Anal. Calcd. For
Write solid, Yield: 74%. m.p. 190e192 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.42 (d, J ¼ 7.2 Hz, 1H), 7.31 (s, 1H), 7.06 (d, J ¼ 8.4 Hz, 1H),
C
21H20N4O3: C, 67.01; H, 5.36; N, 14.88. Found: C, 66.86; H, 5.31; N,
3.89 (dd, J ¼ 15.6, 7.2 Hz, 4H), 3.66 (s, 3H), 2.12 (s, 3H), 1.90e1.66 (m,
14.60.
4H), 0.99 (dd, J ¼ 12, 7.2 Hz, 6H). 13C NMR (100 MHz, DMSO)
d
188.62, 153.81, 147.32, 132.11, 132.08, 128.45, 123.10, 108.32,
106.87, 103.26, 42.03, 41.86, 32.12, 21.17, 14.17, 10.92. HRMS (ESIþ)
4.1.70. Benzyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-
carbonyl)-3-methyl-1H-benzo[d] imidazol-2(3H)-one 10i
Yellow solid, Yield: 38%. m.p. 169e171 ꢀC. 1H NMR (600 MHz,
CDCl3)
1H), 5.12 (s, 2H), 3.65 (s, 3H), 3.51 (s, 3H), 2.08 (s, 3H). 13C NMR
(100 MHz, DMSO) 188.67, 154.05, 147.45, 136.79, 132.59, 131.75,
129.33, 128.64, 127.54, 127.41, 123.43, 108.34, 107.09, 103.32, 43.95,
32.10, 27.14, 14.27. HRMS (ESIþ) cal. for [MþHþ]: cal. 377.1614,
found.377.1614. Anal. Calcd. For C21H20N4O3: C, 67.01; H, 5.36; N,
14.88. Found: C, 66.91; H, 5.21; N, 14.87.
cal. for [MþHþ]: cal. 357.1927, found. 357.1940. Anal. Calcd. For
C
19H24N4O3: C, 64.03; H, 6.79; N, 15.72. Found: C, 63.94; H, 6.46; N,
d
7.38e7.31 (m, 6H), 7.30e7.27 (m, 1H), 6.93 (d, J ¼ 7.8 Hz,
15.83.
d
4.1.76. 5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-1-
isopropyl-3-propyl-1H-benzo[d] imidazol-2(3H)-one 10o
Write solid, Yield: 48%. m.p. 202e204 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
7.40 (d, J ¼ 8.4 Hz, 1H), 7.31 (s, 1H), 7.20 (d, J ¼ 7.8 Hz, 1H),
4.86e4.70 (m, 1H), 3.86 (t, J ¼ 7.2 Hz, 2H), 3.68 (s, 3H), 2.16 (s, 3H),
1.86e1.73 (m, 2H), 1.57 (d, J ¼ 7.2 Hz, 6H), 0.99 (t, J ¼ 7.8 Hz, 3H). 13
C
NMR (100 MHz, CDCl3)
d 191.85, 160.30, 153.70, 146.44, 131.43,
4.1.71. 3-ethyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-
carbonyl)-1-propyl-1H-benzo[d] imidazol-2(3H)-one 10j
Write solid, Yield: 41%. m.p. 168e170 ꢀC. 1H NMR (600 MHz,
130.68, 129.46, 122.16, 107.80, 107.37, 102.09, 45.19, 42.71, 32.46,
21.68, 20.19, 15.90, 11.22. HRMS (ESIþ) cal. for [MþKþ]: cal.
395.1485, found. 395.1496. Anal. Calcd. For C19H24N4O3: C, 64.03; H,
6.79; N, 15.72. Found: C, 64.40; H, 6.99; N, 15.89.
CDCl3)
d
7.43 (d, J ¼ 7.2 Hz, 1H), 7.33 (s, 1H), 7.07 (d, J ¼ 6.6 Hz, 1H),
4.05e3.94 (m, 2H), 3.89 (t, J ¼ 7.2 Hz, 2H), 3.68 (s, 3H), 2.15 (s, 3H),
1.82 (dd, J ¼ 11.4, 6.0 Hz, 2H), 1.37 (t, J ¼ 6.6 Hz, 3H), 1.00 (t,
J ¼ 5.4 Hz, 3H). 13C NMR (150 MHz, DMSO)
d 188.72, 153.53,
4.2. Bioassay
147.56 132.26, 132.23, 128.05, 123.29, 108.48, 107.07, 103.39, 42.14,
35.37, 32.12, 21.37, 14.30, 13.70, 11.12. HRMS (ESIþ) cal. for [MþKþ]:
cal. 381.1329, found. 381.1319. Anal. Calcd. For C18H22N4O3: C, 63.14;
H, 6.48; N, 16.36. Found: C, 63.09; H, 6.68; N, 16.47.
Starting from cDNA of human HPPD, the plasmid of pET-28a
containing HPPD was constructed with sequential PCR, enzyme
digestion and ligation. Then the HPPD DNA sequences of the pos-
itive clones obtained from Escherichia coli (E. coli) XL-10 trans-
formation were confirmed by DNA sequencing with Shanghai
SANGON Company. The confirmed HPPD was over-expressed in
4.1.72. 1,3-diethyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-
carbonyl)-1H-benzo[d] imidazol-2(3H)-one 10k
E. coli BL-21(DE3) cells under
1
mM isopropyl
b-D-1-
Write solid, Yield: 41%. m.p. 199e201 ꢀC. 1H NMR (600 MHz,
thiogalactopyranoside (IPTG) induction at 23 ꢀC for 14 h. Then
the cells were sonicated and centrifuged to yield the crude enzyme
solution. Similarly, the human HGA dioxygenase as the coupling
enzyme was cloned and expressed in E. coli BL-21(DE3) cells under
0.5 mM IPTG induction at 15 ꢀC for almost 40 h. Since crude HGA
dioxygenase is highly active and needs a 100-fold dilution for the
coupling assay, we used the crude enzyme without further purifi-
cation. The purification of His6-tagged recombinant HPPD was
DMSO)
3.91 (q, J ¼ 6.6 Hz, 4H), 3.46 (s, 3H), 2.11 (s, 3H), 1.22 (q, J ¼ 6.0Hz,
6H). 13C NMR (100 MHz, CDCl3)
191.75, 160.05, 153.91, 146.39,
132.23, 131.03, 129.32, 122.50, 107.37, 106.41, 102.15, 42.73, 36.02,
32.40, 21.68, 15.70, 13.52, 11.17. HRMS (ESIþ) cal. for [MþNaþ]: cal.
351.1433, found. 351.1391. Anal. Calcd. For C17H20N4O3: C, 62.18; H,
6.14; N, 17.06. Found: C, 61.99; H, 6.42; N, 17.29.
d
7.54 (s, 1H), 7.49 (d, J ¼ 6.6 Hz, 1H), 7.27 (d, J ¼ 8.4 Hz, 1H),
d
carried out following
a commonly used method. The over-
4.1.73. 3-ethyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-
carbonyl)-1-isopropyl-1H-benzo[d] imidazol-2(3H)-one 10l
Write solid, Yield: 60%. m.p. 159e161 ꢀC. 1H NMR (600 MHz,
expression and the relative molecular weight of recombinant hu-
man HPPD were validated by both sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie
Brilliant Blue-R250 and Simple Western analysis using SimonTM
instrumentation (ProteinSimple, CA), which is consistent with the
previous reports [35].
We established methods of coupled enzyme assays in vitro ac-
tivity against human 4-HPPD that were measured by a modification
of methods previously reported [36]. Assays were performed in 96-
well plates at 30 ꢀC using a UV/visible plate reader. The reaction
CDCl3)
d
7.41 (d, J ¼ 7.8 Hz, 1H), 7.32 (s, 1H), 7.20 (d, J ¼ 8.4 Hz, 1H),
4.85e4.71 (m, 1H), 3.97 (q, J ¼ 7.2 Hz, 2H), 3.66 (s, 3H), 2.15 (s, 3H),
1.57 (d, J ¼ 7.2 Hz, 6H), 1.36 (t, J ¼ 7.2 Hz, 3H). 13C NMR (100 MHz,
DMSO)
d
188.63, 152.80, 147.49, 131.84, 130.99, 128.22, 123.03,
108.40, 107.89, 103.34, 44.67, 35.25, 32.07, 19.89, 14.25, 13.59. HRMS
(ESIþ) cal. for [MþHþ]: cal. 343.1770, found. 343.1780. Anal. Calcd.
For C18H22N4O3: C, 63.14; H, 6.48; N, 16.36. Found: C, 63.13; H, 6.72;
N, 16.52.
mixture is in a total assay volume of 200 mL contained appropriate

Y.-L. Xu et al. / European Journal of Medicinal Chemistry 92 (2015) 427e438
much is enough? Eur. J. Pediatr. 169 (2010) 689e693.
437
amounts of HPPA, 100 mM FeSO4, 2 mM sodium ascorbate, 20 mM
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tightly coupled. Each experiment was repeated at least three times
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