Hydrophobicity-oriented drug design (HODD) of new human 4-hydroxyphenylpyruvate dioxygenase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.01.032

Involved in the tyrosine degradation pathway, 4-hydroxyphenylpyruvate dioxygenase (HPPD) is an important target for treating type I tyrosinemia. To discover novel HPPD inhibitors, we proposed a hydrophobicity-oriented drug design (HODD) strategy based on the interactions between HPPD and the commercial drug NTBC. Most of the new compounds showed improved activity, compound d23 being the most active candidate (IC₅₀ = 0.047 μM) with about 2-fold more potent than NTBC (IC₅₀ = 0.085 μM). Therefore, compound d23 is a potential drug candidate to treat type I tyrosinemia.

Full text of DOI:10.1016/j.ejmech.2019.01.032

Accepted Manuscript
Hydrophobicity-oriented drug design (HODD) of new human 4-hydroxyphenylpyruvate
dioxygenase inhibitors
Ferdinand Ndikuryayo, Wei-Ming Kang, Feng-Xu Wu, Wen-Chao Yang, Guang-Fu
Yang
PII:
S0223-5234(19)30042-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.032
EJMECH 11039
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 November 2018
Revised Date: 13 January 2019
Accepted Date: 13 January 2019
Please cite this article as: F. Ndikuryayo, W.-M. Kang, F.-X. Wu, W.-C. Yang, G.-F. Yang,
Hydrophobicity-oriented drug design (HODD) of new human 4-hydroxyphenylpyruvate dioxygenase
inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.01.032.
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ACCEPTED MANUSCRIPT

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Hydrophobicity-Oriented Drug Design (HODD) of New Human 4-
Hydroxyphenylpyruvate dioxygenase Inhibitors
Ferdinand Ndikuryayoa, Wei-Ming Kanga, Feng-Xu Wua, Wen-Chao Yang*a, Guang-Fu Yang*a,b
aKey Laboratory of Pesticide & Chemical Biology of Ministry of Education, International Joint
Research Center for Intelligent Biosensor Technology and Health, and Chemical Biology Center,
College of Chemistry, Central China Normal University, Wuhan 430079, P.R. China;
bCollaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071, P.R. China.
*Wen-Chao Yang, e-mail: tomyang@mail.ccnu.edu.cn; Tel: +86-27-67867706; Fax: +86-27-7867141;
Guang-Fu Yang, e-mail: gfyang@mail.ccnu.edu.cn; Tel: +86-27-67867800; Fax: +86-27-67867141.

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Abstract
Involved in the tyrosine degradation pathway, 4-hydroxyphenylpyruvate dioxygenase (HPPD) is an
important target for treating type I tyrosinemia. To discover novel HPPD inhibitors, we proposed
a hydrophobicity-oriented drug design (HODD) strategy based on the interactions between HPPD and
the commercial drug NTBC. Most of the new compounds showed improved activity, compound d23
being the most active candidate (IC50 = 0.047 µM) with about 2-fold more potent than NTBC (IC50 =
0.085 µM). Therefore, compound d23 is a potential drug candidate to treat type I tyrosinemia.
Keywords: Hydrophobicity-oriented drug design (HODD), 4-hydroxyphenylpyruvate dioxygenase,
inhibitors, type I tyrosinemia, alkaptonuria, hawkinisinuria.

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1. Introduction
Existing in all aerobic organisms except some gram-positive bacteria [1,2], 4-
hydroxyphenylpyruvate dioxygenase (HPPD, EC:1.13.11.27) converts 4-hydroxyphenylpyruvic acid
(HPPA) into homogentisic acid (HGA) in the tyrosine breakdown pathway [3]. As shown in Scheme
1, four of the five enzymes involved in this crucial pathway are associated with human diseases [4].
Scheme 1. Simplified tyrosine breakdown pathway and associated diseases (red). Enzymes (blue) and
substrates are abbreviated as following: TAT, tyrosine aminotransferase; HPPA,
hydroxyphenylpyruvic acid; HPPD, hydroxyphenylpyruvate dioxygenase; HGA, homogentesic acid;
HGD, homogentisate 1,2-dioxygenase; MAA, maleylacetoacetate; MAAI, maleylacetoacetate
isomerase. The red “stop” signs indicate the enzyme failure.
The deficiency of tyrosine aminotransferase results in type II tyrosinemia whose symptoms
comprise mental retardation, photophobia, keratitis, painful corneal eruptions, and painful
palmoplantar hyperkeratosis [5]. The weakness of HPPD could cause either hawkinsinuria or type III
tyrosinemia [6]. Characterized by failure to grow, permanent metabolic acidosis, and fine and sparse
hair, hawkinsinuria evolves from an autosomal dominant N241S mutation that produces a variant

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enzyme. Instead of the native homogentisic acid (HGA), the variant HPPD produces an active
quinolacetic acid that reacts with cellular thiols to generate a two-electron oxidized form of hawkinsin
[7]. Type III tyrosinemia, the rarest deficiency of tyrosine degradation pathway, is due to pathogenic
mutations in HPPD gene whose dysfunction results in mental retardation or neurological symptoms
[8]. A defective homogentisate 1,2-dioxygenase (HGD) leads to alkaptonuria, a disease associated
with an increased level of plasma HGA whose accumulation and polymerization in cartilage results in
osteoarthropathy [9]. Type I tyrosinemia, the most severe deficiency of the tyrosine breakdown
pathway, is due to the failure of fumarylacetoacetase (FAA) that induces the accumulation of
fumarylacetoacetate and maleylacetoacetate (Scheme 1). These two metabolites can then be converted
into succinylacetone, the cause of liver failure, painful neurologic crises, rickets, and hepatocarcinoma
[10]. Fumarylacetoacetate and succinylacetone are the most harmful metabolites. For example, besides
being mutagenic, fumarylacetoacetate could induce apoptosis, and prevent DNA repair, whereas
succinylacetone prevents the heme synthesis [4].
Initially staked on the liver transplantation, the treatment of type I tyrosinemia is currently based
on NTBC, a synthetic compound that inhibits human HPPD [11,12]. Blocking the pathway at this step
made HPPD a favorite target to treat associated diseases because it prevents the formation of the
aforesaid harmful metabolites. Interestingly, this blockage did not induce the liver damage [6]. Besides
the treatment of type I tyrosinemia, NTBC could also alleviate the symptoms of alkaptonuria and
hawkinisinuria [7,13]. However, recent studies have reported NTBC drawbacks. For example, NTBC
could not induce a response in one of ten treated children, whereas another developed hepatic
dysplasia associated with poor quality of life [14]. Next to this finding, recent studies have shown that
NTBC might cause a significant decrease in intellectual quotient (IQ) in patients and corneal lesions in
rats [15,16]. At present, it is uncertain if this unique medication will be appropriate to prevent

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problems for a long-term period of time, therefore, suggesting the need for the discovery and
development of new drugs.
As known to all, computer-aided drug design (CADD) is classified into ligand-based and protein
structure-based design, and has become an essential part of the drug discovery pipeline [17]. Whereas
ligand-based design consists in the building of 3D pharmacophore models based on existing
compounds, structure-based drug design involves the design of small molecules based on the shape
and chemical features of a known three-dimensional structure of a target enzyme [17]. We performed
the molecular docking of NTBC into human HPPD active pocket, and found an empty hydrophobic
region surrounded by Phe347 residue at the active site (Figure 1B). Thus, we hypothesized that a π-π
stacking interaction between Phe347 and inhibitor may increase the hydrophobic interaction with
HPPD pocket, and thus improves the inhibitory activity. Therefore, we proposed a hydrophobicity-
oriented drug design (HODD) strategy to design new HPPD inhibitors. Guided by this rationale, we
introduced a benzyloxy substituent at the meta position of the benzene ring of NTBC, and prepared a
series of new 2-(3-(benzyloxy)benzoyl)-3-hydroxycyclohex-2-en-1-one analogs as novel human
HPPD inhibitors.
Figure 1. Identification of the free region for the optimal filling of the human HPPD active site. While
(A) shows the binding mode of NTBC (red), (B) depicts the empty region surrounded by hydrophobic
residues (blue).

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As expected, the inhibitory activity of the tested compounds was in nanomolar range against
recombinant human HPPD. A number of compounds exhibited improved activity, and compound d23
(IC50 = 0.047 µM), the most active candidate, showed about 2-fold more potency than NTBC (IC50 =
0.085 µM).
2. Results and discussion
2.1. Molecular modelling and lead compound design
Molecular modeling is used to simulate the binding modes of small molecules in a target enzyme.
Thus, in this study, NTBC was docked into the human HPPD active site to explore its binding mode
(Figure 2). Because there was no available NTBC-bound human HPPD crystal structure, the HPPD
crystal structure derived from rat HPPD (PDB ID: 1SQI) was selected as template to model human
HPPD three-dimensional structure using human HPPD sequence (NCBI GI: 258588704). By
analyzing the binding mode, we located an empty region in the human HPPD active site.
Figure 2. Design strategy of the 2-(3-(benzyloxy)benzoyl)-3-hydroxycyclohex-2-en-1-one
analogs as new human HPPD inhibitors.
Given that HPPD is competitively inhibited, we hypothesized that the optimal filling of the active
site may increase the enzyme inhibition. It was then crucial to characterize the chemical properties of

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the empty region. To this end, the Arabidopsis thaliana HPPD crystal in complex with NTBC (PDB
ID: 5CTO) was superimposed with human HPPD. As result, important residues including Leu287,
Phe336, Pro339, and Phe347 were located in the empty region. Based on the hydrophobic character of
these residues, we concluded that the empty region was hydrophobic (Figure 1B). We further
hypothesized that a π-π stacking interaction between Phe347 and inhibitor may increase the inhibitory
activity. We then added a virtual hydrophobic fragment to NTBC-derived scaffold and docked the
resultant structure into human HPPD active site to generate the structure of the title compound.
The binding mode of the lead compound in the active site showed beneficial features for further
optimization. Beside the Fe2+-chelation found in all triketone-containing HPPD inhibitors (Figure 3A)
[18], π-π stacking interaction was formed between the benzene ring of the newly added fragment and
Phe347 as expected (Figure 3B). The displacement of the middle benzene ring in favor of face-to-face
(sandwich) π-π interaction with both Phe336 and Phe364 was also observed (Figure 3C). For the sake
of structure optimization, a series of analogs were synthesized and tested against recombinant human
HPPD.

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Figure 3. Simulated binding modes of selected compounds in human HPPD active site. Red and
blue-dashed lines represent the Fe2+-chelating and other interactions, respectively. Fe2+ is depicted by
pink sphere, and key residues in green. Beside Fe2+-chelating and sandwich interactions of bound
NTBC (A), compound a1 (B) showed additional π-π interaction with Phe347 along with displacement
of the middle benzene ring in favor of face-to-face π-π interaction with both Phe336 and Phe364 (C).
The decreased activity of compound b12 was due to steric hindrance (D) while the high potency of
compound d23 was attributed to hydrogen bond (E) that adjusted its binding mode (F).
2.2. Chemistry
The preparation of the title compound 6 was shown in Scheme 2. The synthesis followed a six-
step synthetic route starting from the commercially available 3-hydroxybenzoic acid.
3-
hydroxybenzoic acid was esterified to afford compound 1, which was then treated with p-bromobenzyl
bromide in the presence of anhydrous potassium carbonate (K2CO3) in N,N-dimethylformamide
(DMF) to produce the compound 2. This product was hydrolyzed using sodium hydroxide (NaOH) in

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methanol to yield the product 3 that reacted with oxalyl chloride in the presence of DMF as catalyst to
speed up the reaction that yielded compound 4. Intermediates 3 (3a-k) were synthesized and
characterized as shown in Supporting Information. The reaction was followed by esterification with
cyclohexane-1,3-dione using triethylamine as a base in dichloromethane to get the key enol ester 5.
Finally, acetone cyanohydrin catalyzed Fries-type rearrangement of enolesther 5 was performed by
using triethylamine in anhydrous acetonitrile at room temperature to afford tittle compound 6 in good
yield (61-69%). Intermediates 5 (5a-p) were synthesized and characterized as shown in Supporting
1
Information. All the structures of target compounds were characterized by H NMR and high-
resolution mass spectral data whereas 13C NMR was only applied to target compounds.
Reagents and conditions: (a) SOCl2, CH3OH, 60 °C; (b) K2CO3, DMF, C7H6Br2, 80 °C; (c) NaOH,
CH3OH: H2O (1:1), reflux; (d) Oxalyl chloride, CH2Cl2, -5°C, DMF; (e) Substituted1,3-
cyclohexanedione, Et3N, CH2Cl2, -5 °C; (f) Acetone cyanohydrin, Et3N, CH3CN, rt.
Scheme 2. Synthetic route of the title compound
In the final step of the rearrangement reaction, the reaction system was simple and complete. The
target compound was extracted using column chromatography. However, the reaction yield was low
because the target compound not only had a certain water solubility but also was adsorbed on the
column.

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2.3. Enzyme inhibition and structure-activity relationship (SAR) study
Using the coupled enzyme reaction, the inhibitory activity of synthesized compounds was
evaluated, using NTBC as the positive control. For this purpose, the recombinant human HPPD was
purified using Ni-NTA column chromatography. The enzyme purity was validated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 4), and was estimated to be 92%
using ImageJ software (ImageJ 1.52a, Bethesda, MD, USA).
Figure 4. Validation of the over-expression of recombinant human HPPD by SDS-PAGE. M, marker;
lane 1, cell-free supernatant, lanes 2 to 5, elution with 0, 10, 30 and 250 mM imidazole, respectively.
Lane 5 represents the purified enzyme.
The structure-activity relationship (SAR) of synthesized analogs was analyzed. Based on the
position and the type of substituents on the title compound 6, all the compounds were divided into 4
groups for the sake of clarity. With R1 = R2 = R4 = H, the introduction of electron withdrawing (F, Cl
and Br) groups at the 4-position of the aryloxyphenyl ring considerably increased the inhibitory
activities. For example, Br, the most active group (a2, IC50 = 0.091 µM), showed a 4-fold increase in
activity compared with unsubstituted parent a1 (IC50 = 0.335 µM). However, the 4-Br-induced
improvement in activity was compromised by introducing methyl substituents on 5-position (R4) of the
benzene ring of triketone moiety. We observed for example, in comparison with compound a2, at least

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a 2-fold decrease in inhibitory activity in compounds b11, b12 and b13. Compared with their
respective counterparts c19, d29 and d31, a similar trend was also observed in compounds c20, d30
and d32, suggesting the presence of a steric hindrance that might significantly reduce the potency
[19,20]. Methylation of the middle benzene ring (R1 = CH3) of the halogenated compounds
significantly decreased the inhibitory activity (c14-c20). Despite the disadvantageous effect of
methylation at R1, the chlorination of the same benzene ring (R2 = Cl) (c22 and c23) resulted in
improved activity, therefore, suggesting that introducing electron withdrawing groups at R2 may afford
more potent human HPPD inhibitors. Interestingly, nitration of the middle benzene ring (R1 = NO2)
considerably increased the inhibitory activity. For example, seven compounds (d23, d24, d26, d27,
d29, d31 and d33) exhibited similar to higher inhibitory activity compared with NTBC, compound
d23 (IC50 = 0.047 µM) being the most active, about 2-fold higher than NTBC (IC50 = 0.085 µM)
(Table 1).
Table 1. Chemical structures of new compounds and their biological activity against recombinant
human HPPD.
compound R1
R2
R3
R4
IC50 (µM)
NTBC
0.085±0.005
H
4-Br
4-Cl
4-F
H
H
H
H
H
H
H
H
H
H
0.335±0.016
0.091±0.003
0.217±0.019
0.264±0.013
0.274±0.020
0.266±0.016
0.143±0.007
0.422±0.019
0.121±0.005
0.147±0.006
a1
a2
a3
a4
a5
a6
a7
a8
a9
a10
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3-F
4-CH3
2,4-diCl
2-F
2-F-4-Br
2-F-4-Cl

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H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
2-F-4-Br
4-Br
4-Br
5-CH3
0.200±0.009
b11
b12
b13
c14
c15
c16
c17
c18
c19
c20
c21
c22
d23
d24
d25
d26
d27
d28
d29
d30
d31
d32
d33
5,5-diCH3 0.558±0.049
5-CH3
H
H
0.176±0.009
0.274±0.015
0.266±0.005
0.374±0.021
0.302±0.003
0.280±0.013
0.257±0.010
0.447±0.022
0.128±0.005
0.102±0.006
0.047±0.004
0.070±0.004
0.128±0.005
0.070±0.003
0.068±0.005
0.168±0.006
0.068±0.003
0.105±0.004
0.063±0.005
0.106±0.007
0.095±0.005
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
4-Br
4-Cl
4-CH3
4-F
3-Br
2-F-4-Cl
2-F-4-Cl
H
4-F
4-Br
H
H
H
H
H
H
5-CH3
H
H
H
H
H
H
H
5-CH3
H
5-CH3
H
4-CH3
4-F
4-CH(CH3)2
3,5-diCH3
4-CH3
2-F-4-Br
2-F-4-Br
2,4-diCl
2,4-diCl
2,4-diF
5-CH3
H
To understand the structure-activity relationship (SAR) at the atomic level, selected compounds
(a1, a2, b12, and d23) and NTBC were docked into human HPPD active site, and their respective
binding modes and affinities were generated accordingly. As depicted in Figure 3A, NTBC was bound
in human HPPD active site by Fe2+-chelation in which five-coordinate were made by residues
(Glu349, His183, and His266) and 5' and 7' oxygens of NTBC. The former chelating interaction was
consolidated by Phe364, which in combination with Phe336, sandwiches the NTBC phenyl ring [21].
The binding mode of the parent compound a1 also showed a similar pattern (Figure 3B) with a
reduced Fe2+-chelation distance. In comparison with reported human HPPD inhibitors, for example,
while compound a1 was located at 2.0 Å and 1.8 Å of Fe2+ (Figure 3B), the distance between
pyarazole-benzimidazolone hybrid and Fe2+ was 2.5 Å and 2.7 Å [22]. Moreover, this distance was
reported to be 1.9 Å and 3.4 Å for pyrazolone-quinazolone hybrid [19]. Interestingly, superimposition
of NTBC and a1 (Figure 3C) confirmed the formation of π-π stacking interaction between the newly

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added benzene ring of a1 and Phe347. Also, a displacement of the middle benzene ring of a1 in favor
of face-to-face (sandwich) π-π interaction with both Phe336 and Phe364 was confirmed (Figure 3C).
Altogether, the aforementioned interactions may significantly contribute to the stabilization of a1 and
analogs in human active site.
The presence of dimethyl group on the benzene ring of triketone moiety (b12) resulted in more
than 5-fold decrease in activity due to steric hindrance with Ser226 (Figure 3D). On another hand,
compared with a1, the remarkable activity of compound a2 might come from the presence of Br group
on the benzene ring that improved the interaction. This Br-induced activity was consolidated by the
nitration of the middle benzene ring (R1 = NO2) that resulted in an additional hydrogen bond (N-H···O
interaction) between His266 and -NO2 group (Figure 3E). As shown by superimposition of d23 and a2
(Figure 3F), the resultant interaction stabilized d23 by adjusting its binding mode. This assumption
was corroborated by the binding energy of d23 (-10.73 Kcal/mol) that has more contribution for the
binding compared with others (Table 2).
Table 2. Binding free energy calculated for the compounds (Kcal/mol)
H-bonda Electrostaticb VDWc
Conformation entropyd Desolvatione
Cpd
a1
a2
b12
c14
d23
NTBC
Binding free energy
-7.27
-6.89
-6.75
-7.06
-7.66
-7.34
-0.01
-0.01
-0.01
-0.01
-0.02
-0.02
-1.88
-1.77
-1.46
-1.85
-1.89
-1.64
-9.29
-9.06
-9.29
-9.11
-10.73
-9.68
1.79
1.79
1.79
1.76
2.08
1.49
2.13
2.16
2.22
2.16
2.88
2.51
b
c
d
aHydrogen bonding term. Electrostatic energies term. van der Waals term. conformation entropy
contribution. edesolvation contribution
In the light of our findings, beside the great potential of 2-(3-(benzyloxy)benzoyl)-3-
hydroxycyclohex-2-en-1-one analogs for the discovery of more potent human HPPD inhibitors,
compound d23, with about 2-fold more potent than NTBC, could be used as potential drug for the
treatment of type I tyrosinemia, alkaptonuria and hawkinsinuria. Using various electronic withdrawing

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groups, much more optimization of the title compound 6, particularly at R2, may afford more potent
human HPPD inhibitors.
3. Conclusion
In summary, a new strategy of HODD was developed for the structure-based design of HPPD
inhibitors, and thus a series of 2-(3-(benzyloxy)benzoyl)-3-hydroxycyclohex-2-en-1-one analogs were
synthesized and tested as human HPPD inhibitors. On the basis of the bioevaluation assay, most of the
newly synthesized compounds exhibited significant human HPPD inhibitory activity and some
compounds showed similar to higher efficiency in comparison with NTBC (IC50 = 0.085 µM).
Moreover, compound d23 was discovered to be the most active candidate (IC50 = 0.047 µM) with
about 2-fold increase in activity. Threrfore, analogs of 2-(3-(benzyloxy)benzoyl)-3-hydroxycyclohex-
2-en-1-one could serve as a novel lead scaffold for discovering new human HPPD inhibitors, while
compound d23 is a potential drug candidate to treat type I tyrosinemia, alkaptonuria, and
hawkinsinuria.
4. Experimental section
4.1 Methods and synthesis
All chemical reagents were commercially available and treated with standard methods before use.
13
1H NMR and C NMR were recorded on Varian NMR 400 MHz and 600 MHz NMR (Varian, Palo
Alto, CA), Bruker NMR 500 MHz NMR (Bruker, Madison, WI) with TMS as the internal standard,
and deuterated chloroform (CDCl3), deuterated-dimethyl sulfoxide (DMSO-d6) as solvents. The
following abbreviations are used to designate multiplicities: s=singlet, d=doublet, t=triplet,
m=multiplet, dd=doublet of doublets, dt=doublet of triplets. Mass spectrometry (MS) and high
resolution mass spectrometry (HRMS) data were obtained using DSQII GC-MS mass spectrometer
(Thermo Fisher, Austin, TX) and 6224 TOF LC/MS Mass spectrometer (Agilent Technologies, Santa

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Clara, CA). Melting points were taken on a Buchi B-545 without correction. Column chromatography
silica gel (200-300 mesh) and TLC thin-layer chromatography silica gel plate were purchased from
Qingdao Ocean Chemical Plant. The boiling range of petroleum ether was 60-90 °C.
4.1.1. General procedure for intermediate 1
3-Hydroxybenzoic acid (10 mmol) and anhydrous methanol (20 mL) were added to a 100 mL dry
eggplant-shaped flask. With stirring, thionyl chloride (25 mmol) was added dropwise 5-12 h at 60 °C,
and the reaction progress was tracked using TLC. After the reaction completion, excess thionyl
chloride was removed under reduced pressure, and the system was slowly poured into 100 mL of ice
water. The mixture was extracted with ethyl acetate (30 mL×3), the combined organic layer was dried
over Na2SO4, concentrated under vaccum. The crude product 1 was used in the next step without
further purification.
4.1.2. General procedure for intermediate 2
Compound 1 (10 mmol), anhydrous potassium carbonate (20 mmol, K2CO3) and DMF (20 mL) were
added to a 100 mL-dry eggplant flask and stirred at room temperature for 30 min prior to addition of
p-bromobenzyl bromide (12 mmol). The resulting mixture was maintained at 80 °C for 3-8 h, and the
reaction course was monitored using TLC. Upon the reaction completion, the mixture was cooled to
room temperature before the addition of 100 mL of water, and the system was extracted with ethyl
acetate (30 mL×3). The organic phase was dried over anhydrous Na2SO4 and concentrated using rotary
evaporator. The crude product 2 was used in the next step without further purification.
4.1.3. General procedure for intermediate 3
Compound 2 (10 mmol), NaOH (20 mmol), methanol (10 mL) and water (10 mL) were added to a 100
mL clean eggplant flask. The reaction mixture was heated at reflux for 1-2 h, and monitored by TLC.
After the reaction was completed, methanol was removed from the system under reduced pressure

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followed by the addition of 50 mL of water, and the mixture was washed with diethyl ether (20
mL×2). The aqueous phase was acidified to pH=1-2, the white solid was precipitated. The obtained
solid was filtered and dried to give pure compounds 3. The characterization of important intermediates
(3 and 5) and the spectra of representative compounds were displayed in the Supporting Information
(Figures S1-S7).
4.1.4. General procedure for intermediate 4
Compound 3 (5 mmol) and dichloromethane (10 mL) were added to 50 mL dry eggplant-shaped flask
and stirred at 0 °C for 5 min. The redistilled oxalyl chloride (12.5 mmol) was added dropwise followed
by the addition of a catalytic amount of DMF (3-5 drops). The mixture was then stirred for 3-5 h, and
the progress of the reaction was monitored by TLC. After the completion of the reaction, the
dichloromethane is removed under reduced pressure to obtain the acid chloride 4.
4.1.5. General procedure for intermediate 5
Cyclohexanedione (6 mmol), dichloromethane (15 mL) and triethylamine (10 mmol) were added to a
100 mL eggplant-shaped flask, stirred for 10 min at 0°C. Subsequently, the intermediate 4 was
dissolved in 15 mL of dry dichloromethane before addition to the above system. The reaction was
carried out for 0.5-1 h and monitored by TLC. Upon the completion of the reaction, the reaction
system was washed with water (20 mL), saturated sodium bicarbonate solution (20 mLx2), and brine
(20 mL). The organic phase was then dried over anhydrous Na2SO4 and concentrated by rotary
evaporation. The residue was purified via flash column chromatography to give 5.
4.1.6. General procedure for target compounds
Compounds 5 (2 mmol), acetonitrile (20 mL), triethylamine (4 mmol) and acetone cyanohydrin (0.2
mmol) were added to a 100 mL eggplant-shaped flask, stirred for 20 h at room temperature, TLC was
used to track the reaction progress until the complete disappearance of 5. The system was then diluted

ACCEPTED MANUSCRIPT
17
with aqueous HCl solution (30 mL, 1 M) and stirred for 5 min. The obtained solid was filtered, dried
and then recrystallized in methanol to give title compounds 6 that were classified into four series (a, b,
c and d).
4.1.7. 2-(3-(Benzyloxy)benzoyl)-3-hydroxycyclohex-2-en-1-one (a1)
1
Light yellow solid, Yield: 64%. m.p. 80.3-81.8 °C. H NMR (600 MHz, CDCl3) δ 7.43 (d, J=7.2 Hz,
2H), 7.39 (t, J=7.2 Hz, 2H), 7.35-7.28 (m, 2H), 7.15-7.07 (m, 3H), 5.07 (s, 2H), 2.78-2.69 (m, 2H),
2.53-2.44 (m, 2H), 2.06 (dt, J=13.2, 6.6 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ 198.77, 195.86,
194.19, 158.24, 139.49, 136.60, 128.78, 128.57, 128.03, 127.58, 120.97, 118.48, 114.16, 113.34,
70.13, 38.01, 32.25, 19.10. HRMS (ESI): calcd for C20H18O4 [M+H]+ 323.12779, found 323.12799.
4.1.8. 2-(3-((4-Bromobenzyl)oxy)benzoyl)-3-hydroxycyclohex-2-en-1-one (a2)
1
Light yellow solid, Yield: 61%. m.p. 112.1-113.3 °C. H NMR (600 MHz, CDCl3) δ 11.87 (s, 1H),
7.60 (d, J=8.4 Hz, 2H), 7.45-7.37 (m, 3H), 7.35 (d, J=7.2 Hz, 1H), 7.31 (s, 1H), 7.23 (d, J=6.6 Hz,
13
1H), 5.14 (s, 2H), 2.45 (s, 4H), 1.97 (s, 2H). C NMR (150 MHz, CDCl3) δ 198.71, 194.10, 157.85,
139.56, 135.64, 131.68, 129.13, 128.83, 121.92, 121.15, 118.43, 114.11, 113.31, 69.36, 38.02, 32.26,
19.10. HRMS (ESI): calcd for C20H17BrO4 [M+H]+ 401.03830, found 401.03729.
4.1.9. 2-(3-((4-Chlorobenzyl)oxy)benzoyl)-3-hydroxycyclohex-2-en-1-one (a3)
1
Light yellow solid, Yield: 67%. m.p. 111.2-112.4 °C. H NMR (400 MHz, CDCl3) δ 16.79 (s, 1H),
7.36 (s, 4H), 7.31 (t, J=8.2 Hz, 1H), 7.09 (dd, J=7.8, 3.6 Hz, 3H), 5.04 (s, 2H), 2.75 (t, J=6.0 Hz, 2H),
13
2.49 (t, J=6.6 Hz, 2H), 2.12-2.03 (m, 2H). C NMR (150 MHz, CDCl3) δ 198.71, 195.90, 194.15,
157.86, 139.56, 135.12, 133.77, 128.84, 128.73, 121.14, 118.43, 114.11, 113.30, 69.33, 38.01, 32.23,
19.09. HRMS (ESI): calcd for C20H17ClO4 [M+H+] 357.08881, found 357.08857.
4.1.10. 2-(3-((4-Fluorobenzyl)oxy)benzoyl)-3-hydroxycyclohex-2-en-1-one (a4)

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Light yellow solid, Yield: 68%. m.p. 84.6-85.7 °C. 1H NMR (600 MHz, CDCl3) δ 7.40 (dd, J=8.4, 5.4
Hz, 2H), 7.30 (t, J=7.8 Hz, 1H), 7.12-7.05 (m, 4H), 5.02 (s, 2H), 2.74 (t, J=6.0 Hz, 2H), 2.54-2.44 (m,
13
2H), 2.07 (dt, J=13.2, 6.6 Hz, 2H). C NMR (150 MHz, CDCl3) δ 198.74, 195.90, 194.14, 163.29,
161.66, 158.06, 139.55, 132.37, 129.48, 129.42, 128.81, 121.09, 118.43, 115.55, 115.40, 114.09,
113.31, 69.43, 38.02, 32.24, 19.09. HRMS (ESI): calcd for C20H17FO4 [M+H]+ 341.11836, found
341.11783
4.1.11. 2-(3-((3-Fluorobenzyl)oxy)benzoyl)-3-hydroxycyclohex-2-en-1-one (a5)
1
Light yellow solid, Yield: 65%. m.p. 79.8-81.4 °C. H NMR (600 MHz, DMSO-d6) δ 7.47-7.42 (m,
1H), 7.42-7.38 (m, 1H), 7.36 (d, J=7.2 Hz, 1H), 7.31 (d, J=9.0 Hz, 3H), 7.25 (d, J=7.2 Hz, 1H), 7.17 (t,
J=7.8 Hz, 1H), 5.18 (s, 2H), 2.46 (s, 4H), 1.98 (dd, J=11.3, 5.3 Hz, 2H). 13C NMR (150 MHz, CDCl3)
δ 198.69, 195.86, 194.07, 163.70, 162.07, 157.83, 139.57, 139.19, 130.15, 130.09, 128.84, 122.80,
121.18, 118.41, 114.93, 114.78, 114.34, 114.19, 114.13, 69.28, 38.02, 32.24, 19.10. HRMS (ESI):
calcd for C20H17FO4 [M+H]+ 341.11836, found 341.11805.
4.1.12. 3-Hydroxy-2-(3-((4-methylbenzyl) oxy)benzoyl)cyclohex-2-en-1-one (a6)
Light yellow solid, Yield: 65%. m.p. 83.6-84.8 °C; 1H NMR (400 MHz, CDCl3) δ 16.72 (s, 1H), 7.32-
7.27 (m, 2H), 7.26 (d, J=7.8 Hz, 1H), 7.17 (d, J=7.8 Hz, 2H), 7.11-7.04 (m, 3H), 5.00 (s, 2H), 2.73 (s,
13
2H), 2.48 (s, 2H), 2.35 (s, 3H), 2.10-2.01 (m, 2H). C NMR (150 MHz, CDCl3) δ 198.71, 195.69,
194.02, 158.20, 139.36, 137.74, 133.44, 129.17, 128.66, 127.66, 120.79, 118.37, 114.06, 69.94, 37.82,
32.13, 21.12, 19.01. HRMS (ESI): calcd for C21H20O4 [M+H]+ 337.14344, found 337.14309.
4.1.13. 2-(3-((2,4-Dichlorobenzyl)oxy)benzoyl)-3-hydroxycyclohex-2-en-1-one (a7)
Light yellow solid, Yield: 69%. m.p. 119.6-121.1 °C. 1H NMR (600 MHz, CDCl3) δ 7.50 (d, J=8.2 Hz,
1H), 7.42 (d, J=1.8 Hz, 1H), 7.32 (t, J=7.8 Hz, 1H), 7.28 (dd, J=8.4, 1.8 Hz, 1H), 7.11 (t, J=7.8 Hz,
13
3H), 5.14 (s, 2H), 2.75 (t, J=6.0 Hz, 2H), 2.54-2.45 (m, 2H), 2.11-2.03 (m, 2H). C NMR (150 MHz,

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CDCl3) δ 198.65, 195.93, 194.10, 157.65, 139.64, 134.11, 133.06, 133.03, 129.67, 129.13, 128.90,
127.32, 121.34, 118.35, 114.18, 113.30, 66.64, 38.01, 32.25, 19.10. HRMS (ESI): calcd for
C20H16Cl2O4 [M+H]+ 391.04984, found 391.04933.
4.1.14. 2-(3-((2-Fluorobenzyl)oxy)benzoyl)-3-hydroxycyclohex-2-en-1-one (a8)
Light yellow solid, Yield: 63%. m.p. 64.4-65.9 °C. 1H NMR (600 MHz, CDCl3) δ 7.51 (s, 1H), 7.31 (t,
J=7.8 Hz, 2H), 7.17 (s, 1H), 7.15 (s, 1H), 7.11 (dd, J=15.0, 8.4 Hz, 3H), 5.15 (s, 2H), 2.74 (s, 2H),
2.49 (s, 2H), 2.11-2.04 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 198.71, 195.84, 194.10, 161.18,
159.54, 157.86, 139.54, 129.69, 129.64, 128.83, 124.28, 123.72, 121.14, 118.39, 115.37, 115.23,
114.12, 113.34, 103.83, 63.73, 38.01, 32.25, 19.10. HRMS (ESI): calcd for C20H17FO4 [M+H]+
341.11836, found 341.11758.
4.1.15. 2-(3-((4-Bromo-2-fluorobenzyl)oxy)benzoyl)-3-hydroxycyclohex-2-en-1-one (a9)
Light yellow solid, Yield: 68%. m.p. 87.6-88.5 °C. 1H NMR (400 MHz, CDCl3) δ 16.79 (s, 1H), 7.40
(t, J=7.8 Hz, 1H), 7.35-7.26 (m, 3H), 7.11 (dd, J=7.2, 5.4 Hz, 3H), 5.09 (s, 2H), 2.75 (s, 2H), 2.49 (s,
2H), 2.12-2.03 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 198.64, 195.90, 194.05, 160.84, 159.17,
157.69, 139.63, 130.79, 130.76, 128.87, 127.66, 127.64, 123.13, 123.04, 122.08, 121.32, 119.06,
118.90, 118.33, 114.07, 113.28, 63.26, 63.24, 38.01, 32.25, 19.09. HRMS (ESI): calcd for C20H16BrO4
[M+H]+ 419.02888, found 419.02825.
4.1.16. 2-(3-((4-Chloro-2-fluorobenzyl)oxy)benzoyl)-3-hydroxycyclohex-2-en-1-one (a10)
Light yellow solid, Yield: 65%. m.p. 81.0-81.9 °C. 1H NMR (400 MHz, CDCl3) δ 16.79 (s, 1H), 7.45
(t, J=8.0 Hz, 1H), 7.32 (t, J=8.0 Hz, 1H), 7.17 (d, J=8.4 Hz, 1H), 7.15-7.11 (m, 3H), 7.09 (s, 1H), 5.10
(s, 2H), 2.75 (s, 2H), 2.50 (s, 2H), 2.13-2.02 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 198.65, 195.90,
194.04, 160.87, 159.21, 157.71, 139.63, 134.68, 134.61, 130.51, 130.48, 128.86, 124.73, 124.71,

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122.60, 122.51, 121.31, 118.33, 116.22, 116.06, 114.06, 113.31, 63.23, 63.20, 38.02, 32.25, 19.10.
HRMS (ESI): calcd for C20H16ClFO4 [M+H]+ 375.07939, found 375.07910.
4.1.17.
(b11)
2-(3-((4-Bromo-2-fluorobenzyl)oxy)benzoyl)-3-hydroxy-5-methylcyclohex-2-en-1-one
Light yellow solid, Yield: 63%. m.p. 82.0-83.4 °C. 1H NMR (400 MHz, CDCl3) δ 16.79 (s, 1H), 7.39
(t, J=7.6 Hz, 1H), 7.35-7.27 (m, 3H), 7.10 (s, 3H), 5.08 (s, 2H), 2.79 (d, J=17.6 Hz, 1H), 2.57 (d,
J=16.2 Hz, 1H), 2.46 (dd, J=17.8, 10.6 Hz, 1H), 2.33 (dd, J=10.6, 4.2 Hz, 1H), 2.19 (dd, J=16.2, 11.4
Hz, 1H), 1.14 (d, J=6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 198.54, 195.52, 194.10, 161.34,
158.84, 157.77, 139.66, 130.87, 128.96, 127.76, 123.26, 122.24, 121.38, 119.19, 118.95, 118.41,
114.14, 112.92, 63.36, 63.32, 46.41, 40.22, 26.74, 20.86. HRMS (ESI): calcd for C21H18BrFO4
[M+H]+ 433.04453, found 443.04517.
4.1.18. 2-(3-((4-Bromobenzyl)oxy)benzoyl)-3-hydroxy-5,5-dimethylcyclohex-2-en-1-one (b12)
1
Light yellow solid, Yield: 65%. m.p. 89.8-90.6 °C. H NMR (600 MHz, CDCl3) δ 7.51 (d, J=8.4 Hz,
2H), 7.30 (d, J=8.0 Hz, 3H), 7.07 (d, J=7.8 Hz, 3H), 5.01 (s, 2H), 2.63 (s, 2H), 2.37 (s, 2H), 1.14 (s,
6H). 13C NMR (150 MHz, CDCl3) δ 197.86, 195.13, 193.90, 157.82, 139.32, 135.56, 131.57, 129.03,
128.72, 121.79, 121.01, 118.27, 113.95, 112.21, 69.20, 51.98, 45.79, 30.92, 28.08. HRMS (ESI): calcd
for C22H21BrO4 [M+H]+429.06960, found 429.06931.
4.1.19. 2-(3-((4-Bromobenzyl)oxy)benzoyl)-3-hydroxy-5-methylcyclohex-2-en-1-one (b13)
1
Light yellow solid, Yield: 66%. m.p. 86.0-87.0 °C. H NMR (600 MHz, CDCl3) δ 7.51 (d, J=8.4 Hz,
2H), 7.31 (d, J=8.4 Hz, 3H), 7.09 (s, 3H), 5.02 (s, 2H), 2.79 (d, J=16.8 Hz, 1H), 2.57 (d, J=15.0 Hz,
1H), 2.49-2.42 (m, 1H), 2.33 (s, 1H), 2.19 (d, J=16.8 Hz, 1H), 1.14 (d, J=6.6 Hz, 3H). 13C NMR (150
MHz, CDCl3) δ 198.50, 195.42, 194.07, 157.83, 139.50, 135.66, 131.67, 129.12, 128.82, 121.90,

ACCEPTED MANUSCRIPT
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121.12, 118.43, 114.10, 112.83, 69.34, 46.31, 40.13, 26.66, 20.78. HRMS (ESI): calcd for C21H19BrO4
[M+H]+ 415.05395, found 415.05316.
4.1.20. 2-(3-((4-Bromobenzyl)oxy)-2-methylbenzoyl)-3-hydroxycyclohex-2-en-1-one (c14)
1
Light yellow solid, Yield: 61%. m.p. 108.7-109.9 °C. H NMR (400 MHz, CDCl3) δ 17.70 (s, 1H),
7.51 (d, J=7.8 Hz, 2H), 7.32 (d, J=7.8 Hz, 2H), 7.18 (t, J=7.8 Hz, 1H), 6.91 (d, J=8.0 Hz, 1H), 6.73 (d,
J=7.4 Hz, 1H), 5.03 (s, 2H), 2.78 (t, J=5.8 Hz, 2H), 2.49-2.38 (m, 2H), 2.15 (s, 3H), 2.09-1.99 (m, 2H).
13C NMR (150 MHz, CDCl3) δ 199.99, 198.33, 193.55, 156.31, 140.41, 136.07, 131.59, 128.79,
126.28, 123.20, 121.62, 118.03, 114.03, 112.28, 69.26, 38.04, 33.05, 19.05, 12.90. HRMS (ESI): calcd
for C21H19BrO4 [M+H]+ 415.05395, found 415.05289.
4.1.21. 2-(3-((4-Chlorobenzyl)oxy)-2-methylbenzoyl)-3-hydroxycyclohex-2-en-1-one (c15)
1
Light yellow solid, Yield: 58%. m.p. 112.3-113.0 °C. H NMR (400 MHz, CDCl3) δ 17.70 (s, 1H),
7.37 (d, J=1.8 Hz, 4H), 7.18 (t, J=7.8 Hz, 1H), 6.91 (d, J=8.2 Hz, 1H), 6.73 (d, J=7.6 Hz, 1H), 5.05 (s,
13
2H), 2.78 (t, J=6.4 Hz, 2H), 2.44 (t, J=6.4 Hz, 2H), 2.15 (s, 3H), 2.04 (m, J=6.4 Hz, 2H). C NMR
(150 MHz, CDCl3) δ 199.99, 198.31, 193.52, 156.33, 140.41, 135.65, 133.48, 128.65, 128.48, 126.28,
123.21, 118.02, 114.03, 112.29, 69.24, 38.06, 33.06, 19.06, 12.90. HRMS (ESI): calcd for C21H19ClO4
[M+H]+ 371.10446, found 371.10428.
4.1.22. 3-Hydroxy-2-(2-methyl-3-((4-methylbenzyl)oxy)benzoyl)cyclohex-2-en-1-one (c16)
Yield, 61%；light yellow solid；m.p. 110.1-110.8 °C；1H NMR (600 MHz, CDCl3) δ 7.33 (d, J=6.4
Hz, 2H), 7.18 (dd, J=15.8, 7.4 Hz, 3H), 6.95 (d, J=8.0 Hz, 1H), 6.71 (d, J=7.4 Hz, 1H), 5.04 (s, 2H),
13
2.77 (s, 2H), 2.44 (s, 2H), 2.37 (s, 3H), 2.15 (s, 3H), 2.04 (s, 2H). C NMR (150 MHz, CDCl3) δ
200.08, 198.28, 193.50, 156.67, 140.24, 137.48, 134.13, 129.14, 127.29, 126.21, 123.23, 117.76,

ACCEPTED MANUSCRIPT
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112.35, 103.83, 69.94, 38.07, 33.09, 21.20, 19.06, 12.93. HRMS (ESI): calcd for C22H22O4 [M+H]+
351.15909, found 351.15887.
4.1.23. 2-(3-((4-Fluorobenzyl)oxy)-2-methylbenzoyl)-3-hydroxycyclohex-2-en-1-one (c17)
1
Light yellow solid, Yield: 57%. m.p. 112.1-113.0 °C. H NMR (400 MHz, CDCl3) δ 17.71 (s, 1H),
7.41 (dd, J=8.0, 5.6 Hz, 2H), 7.19 (t, J=7.8 Hz, 1H), 7.08 (t, J=8.4 Hz, 2H), 6.93 (d, J=8.0 Hz, 1H),
6.73 (d, J=7.4 Hz, 1H), 5.04 (s, 2H), 2.78 (t, J=6.0 Hz, 2H), 2.45 (s, 2H), 2.15 (s, 3H), 2.05 (s, 2H). 13C
NMR (150 MHz, CDCl3) δ 200.02, 193.52, 163.14, 161.50, 156.42, 140.38, 132.90, 132.88, 129.00,
128.94, 126.27, 123.22, 117.87, 115.44, 115.30, 114.07, 112.32, 103.83, 69.36, 38.05, 33.04, 19.05,
12.90. HRMS (ESI): calcd for C21H19FO4 [M+H]+ 355.13401, found 355.13342.
4.1.24. 2-(3-((3-Bromobenzyl)oxy)-2-methylbenzoyl)-3-hydroxycyclohex-2-en-1-one (c18)
1
Light yellow solid, Yield: 62%. m.p. 107.1-107.8 °C. H NMR (400 MHz, CDCl3) δ 17.71 (s, 1H),
7.60 (s, 1H), 7.46 (d, J=7.8 Hz, 1H), 7.37 (d, J=7.6 Hz, 1H), 7.28 (s, 1H), 7.24 (s, 1H), 7.19 (t, J=7.8
Hz, 1H), 6.91 (d, J=7.8 Hz, 1H), 6.74 (d, J=7.6 Hz, 1H), 5.05 (s, 2H), 2.79 (t, J=6.4 Hz, 2H), 2.45 (t,
J=6.4 Hz, 2H), 2.17 (s, 3H), 2.09-2.01 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 199.97, 198.28,
193.52, 156.28, 140.43, 139.49, 130.83, 130.09, 130.02, 126.30, 125.58, 123.25, 122.54, 118.01,
112.29, 69.13, 38.05, 33.05, 19.06, 12.92. HRMS (ESI): calcd for C21H19BrO4 [M+H]+ 415.05395,
found 415.05364.
4.1.25.
(c19)
2-(3-((4-Chloro-2-fluorobenzyl)oxy)-2-methylbenzoyl)-3-hydroxycyclohex-2-en-1-one
1
Light yellow solid, Yield: 52%. m.p. 102.8-104.0 °C. H NMR (400 MHz, CDCl3) δ 17.74 (s, 1H),
7.52 (t, J=8.0 Hz, 1H), 7.25 (d, J=7.8 Hz, 1H), 7.22 (d, J=7.8 Hz, 1H), 7.18 (d, J=9.6 Hz, 1H), 6.99 (d,
J=8.0 Hz, 1H), 6.79 (d, J=7.4 Hz, 1H), 5.16 (s, 2H), 2.83 (t, J=6.0 Hz, 2H), 2.49 (t, J=6.2 Hz, 2H), 2.20
13
(s, 3H), 2.14-2.05 (m, 2H). C NMR (150 MHz, CDCl3) δ 199.95, 198.32, 193.56, 160.75, 159.08,

ACCEPTED MANUSCRIPT
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156.09, 140.46, 134.28, 130.07, 126.34, 124.66, 124.64, 123.28, 118.27, 116.04, 115.98, 112.28,
63.33, 63.31, 38.04, 33.05, 19.05, 12.86. HRMS (ESI): calcd for C21H18ClFO4 [M+H]+ 389.09504,
found 389.09498.
4.1.26. 2-(3-((4-Chloro-2-fluorobenzyl)oxy)-2-methylbenzoyl)-3-hydroxy-5-methylcyclo-hex-2-
en-1-one (c20)
1
Light yellow solid, Yield: 59%. m.p. 106.4-107.2 °C. H NMR (400 MHz, CDCl3) δ 17.74 (s, 1H),
7.44 (t, J=8.0 Hz, 1H), 7.24 (m, J=15.2, 8.8 Hz, 3H), 7.01 (d, J=8.2 Hz, 1H), 6.80 (d, J=7.6 Hz, 1H),
5.17 (s, 2H), 2.88 (dd, J=18.0, 2.4 Hz, 1H), 2.56 (dd, J=18.8, 11.0 Hz, 2H), 2.37 (s, 1H), 2.21 (s, 3H),
1.19 (d, J=6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 199.63, 197.77, 193.45, 160.74, 159.08,
156.07, 140.39, 134.28, 130.02, 126.35, 124.66, 123.27, 123.12, 123.03, 118.25, 116.04, 115.97,
112.28, 46.35, 40.90, 40.88, 26.53, 20.84, 12.84. HRMS (ESI): calcd for C22H20ClFO4 [M+H]+
403.11069, found 403.11021.
4.1.27. 2-(3-(Benzyloxy)-4-chloro-2-methylbenzoyl)-3-hydroxycyclohex-2-en-1-one (c21)
Light yellow oil, Yield: 47%. 1H NMR (600 MHz, CDCl3) δ 7.45 (d, J=6.0 Hz, 2H), 7.43 (t, J=7.2 Hz,
2H), 7.31 (d, J=8.4 Hz, 1H), 6.86 (d, J=6.0 Hz, 1H), 5.00 (s, 2H), 2.80 (t, J=6.6 Hz, 2H), 2.47 (t, J=6.6
Hz, 2H), 2.20 (d, J=3.0 Hz, 3H), 2.07 (t, J=6.0 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 199.00,
198.37, 193.69, 153.15, 139.54, 136.95, 130.44, 129.60, 128.56, 128.22, 128.03, 127.62, 122.29,
114.00, 74.58, 38.05, 32.97, 19.09, 13.57. HRMS (ESI): calcd for C21H19ClO4 [M+Na]+ 393.08641
found 393.08688.
4.1.28.
(c22)
2-(4-Chloro-3-((4-fluorobenzyl)oxy)-2-methylbenzoyl)-3-hydroxycyclohex-2-en-1-one
1
Light yellow solid, Yield: 51%. m.p. 87.6-88.9 °C. H NMR (600 MHz, CDCl3) δ 7.42-7.48 (m, 2H),
7.30 (d, J=9.0 Hz, 1H), 7.10 (t, J=8.4 Hz, 2H), 6.85 (d, J=8.4 Hz, 1H), 4.95 (s, 2H), 2.80 (t, J=6.0 Hz,

ACCEPTED MANUSCRIPT
24
13
2H), 2.46 (t, J=9.0 Hz, 2H), 2.16 (s, 3H), 2.07 (p, J=6.0 Hz, 2H). C NMR (100 MHz, CDCl3) δ
198.94, 198.35, 193.62, 163.93, 161.48, 152.92, 139.58, 132.72, 130.35, 130.14, 130.06, 129.50,
127.66, 122.32, 115.54, 115.33, 113.98, 73.87, 38.07, 32.96, 19.09, 13.56. HRMS (ESI): calcd for
C21H18ClFO4 [M+Na]+ 411.07699 found 411.07910.
4.1.29. 2-(3-((4-Bromobenzyl)oxy)-2-nitrobenzoyl)-3-hydroxycyclohex-2-en-1-one (d23)
1
Light yellow solid, Yield: 60%. m.p. 154.5-155.4 °C. H NMR (400 MHz, CDCl3) δ 16.47 (s, 1H),
7.41 (t, J=9.0 Hz, 3H), 7.33 (d, J=8.0 Hz, 2H), 7.13 (d, J=7.8 Hz, 1H), 6.86 (d, J=6.8 Hz, 1H), 5.17 (s,
13
2H), 2.77 (t, J=6.0 Hz, 2H), 2.43 (t, J=6.2 Hz, 2H), 2.05 (p, J=6.0 Hz, 2H). C NMR (100 MHz,
CDCl3) δ 196.31, 193.83, 150.98, 137.76, 137.13, 134.41, 132.74, 131.88, 128.72, 122.26, 119.05,
115.69, 113.02, 70.65, 37.60, 31.95, 19.12. HRMS (ESI): calcd for C20H16BrNO6 [M+Na]+ 468.00532
found 468.00683.
4.1.30. 3-Hydroxy-2-(3-((4-methylbenzyl)oxy)-2-nitrobenzoyl)cyclohex-2-en-1-one (d24)
Light yellow solid, Yield: 53%. m.p. 142.4-143.8 °C. 1H NMR (600 MHz, CDCl3) δ 7.46 (t, J=7.8 Hz,
1H), 7.33 (d, J=6.0 Hz, 2H), 7.19 (d, J=7.8 Hz, 2H), 7.16 (d, J=6.0 Hz, 1H), 6.83 (d, J=7.8 Hz, 1H),
13
5.19 (s, 2H), 2.76 (t, J=6.6 Hz, 2H), 2.43 (t, J=6.0 Hz, 2H), 2.35 (s, 3H), 2.04 (m, J=6.6 Hz, 2H). C
NMR (100 MHz, CDCl3) δ 196.41, 196.34, 193.88, 151.33, 138.02, 137.79, 136.93, 132.62, 132.37,
129.42, 127.23, 118.71, 115.90, 113.06, 71.42, 37.62, 31.97, 21.27, 19.13. HRMS (ESI): calcd for
C21H19NO6 [M+Na]+ 404.11046 found 404.11040.
4.1.31. 2-(3-(1-(4-Fluorophenyl)ethoxy)-2-nitrobenzoyl)-3-hydroxycyclohex-2-en-1-one (d25)
1
Light yellow solid, Yield: 63%. m.p. 166.4-167.4 °C. H NMR (400 MHz, CDCl3) δ 16.53 (s, 1H),
7.45-7.39 (m, 2H), 7.36 (t, J=8.0 Hz, 1H), 7.09 (t, J=8.6 Hz, 2H), 6.95 (d, J=8.4 Hz, 1H), 6.81 (d,
J=7.6 Hz, 1H), 5.46 (d, J=6.4 Hz, 1H), 2.81 (t, J=8.0 Hz, 2H), 2.47 (t, J=6.0 Hz, 2H), 2.08 (p, J=6.4
Hz, 2H), 1.69 (d, J=6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 196.40, 196.35, 193.86, 163.57,

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161.12, 150.29, 137.40, 136.70, 132.17, 127.35, 127.27, 118.63, 116.97, 115.98, 115.76, 113.05,
37.61, 31.98, 24.39, 19.11. HRMS (ESI): calcd for C21H18FNO6 [M+Na]+ 422.10104 found
422.10142.
4.1.32. 3-Hydroxy-2-(3-((4-isopropylbenzyl)oxy)-2-nitrobenzoyl)cyclohex-2-en-1-one (d26)
Light yellow solid, Yield, 58%；；m.p. 116.5-117.8°C；1H NMR (600 MHz, CDCl3) δ 7.47 (t, J=9.0
Hz, 1H), 7.37 (d, J=7.8 Hz, 2H), 7.26 (s, 2H), 7.18 (d, J=8.4 Hz, 1H), 6.84 (d, J=7.8 Hz, 1H), 5.19 (s,
2H), 2.94-2.89 (m, 1H), 2.77 (t, J=6.0 Hz, 2H), 2.43 (t, J=6.6 Hz, 2H), 2.04 (p, J=6.6 Hz, 2H), 1.25 (d,
J=6.0 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 196.41, 196.30, 193.84, 151.38, 149.10, 136.94,
132.73, 132.62, 127.33, 126.82, 118.70, 115.87, 113.07, 77.43, 77.11, 76.79, 71.44, 37.62, 33.93,
31.97, 24.02, 19.14. HRMS (ESI): calcd for C23H23NO6 [M+Na]+ 432.14176 found 432,14119.
4.1.33. 2-(3-((3,5-Dimethylbenzyl)oxy)-2-nitrobenzoyl)-3-hydroxycyclohex-2-en-1-one (d27)
1
Light yellow solid, Yield: 52%. m.p. 153.4-154.9 °C. H NMR (400 MHz, CDCl3) δ 16.50 (s, 1H),
7.46 (t, J=8.0 Hz, 1H), 7.15 (d, J=8.4 Hz, 1H), 7.05 (s, 2H), 6.96 (s, 1H), 6.83 (d, J=7.6 Hz, 1H), 5.15
(s, 2H), 2.76 (t, J=6.0 Hz, 2H), 2.43 (t, J=6.4 Hz, 2H), 2.33 (s, 6H), 2.04 (m, J=6.2 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ 196.45, 196.27, 193.80, 151.47, 138.36, 137.79, 136.97, 135.31, 132.65, 129.98,
124.95, 118.71, 115.97, 113.06, 71.69, 37.64, 31.98, 21.37, 19.15. HRMS (ESI): calcd for C22H21NO6
[M+Na]+ 418.02611 found 418.02616.
4.1.34.
(d28)
3-Hydroxy-5-methyl-2-(3-((4-methylbenzyl)oxy)-2-nitrobenzoyl)cyclohex-2-en-1-one
1
Light yellow solid, Yield: 49%. m.p. 112.3-113.6 °C. H NMR (400 MHz, CDCl3) δ 16.46 (s, 1H),
7.46 (t, J=8.0 Hz, 1H), 7.32 (d, J=7.6 Hz, 2H), 7.17 (t, J=10.0 Hz, 3H), 6.82 (d, J=7.6 Hz, 1H), 5.18 (s,
2H), 2.85-2.71 (m, 1H), 2.49 (t, J=12.0 Hz, 2H), 2.35 (s, 4H), 2.14 (dd, J=12.0, 8.0 Hz, 1H), 1.11 (d,

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J=8.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 196.21, 195.73, 193.80, 151.32, 138.00, 136.88,
132.63, 132.38, 129.42, 127.23, 118.73, 115.91, 71.42, 45.80, 39.68, 26.71, 21.28, 20.81. HRMS
(ESI): calcd for C22H21NO6 [M+Na]+ 418.02666 found 418.02582.
4.1.35. 2-(3-((4-Bromo-2-fluorobenzyl)oxy)-2-nitrobenzoyl)-3-hydroxycyclohex-2-en-1-one (d29)
1
Light yellow solid, Yield: 60%. m.p. 158.0-159.8 °C. H NMR (400 MHz, CDCl3) δ 16.46 (s, 1H),
7.42 (t, J=6.8 Hz, 1H), 7.40-7.46 (m, 1H), 7.35 (m, J=8.0, 1H), 7.31-7.27 (m, 1H), 7.19 (d, J=8.4 Hz,
1H), 6.88 (d, J=7.6 Hz, 1H), 5.23 (s, 2H), 2.78 (t, J=6.0 Hz, 2H), 2.43 (t, J=6.0 Hz, 2H), 2.05 (m,
J=6.0, Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 196.32, 196.24, 193.86, 160.89, 158.39, 150.79,
137.28, 132.90, 130.47, 130.42, 128.07, 128.04, 119.31, 119.10, 118.86, 115.56, 113.00, 64.74, 37.60,
31.96, 19.13. HRMS (ESI): calcd for C20H15BrFNO6 [M+Na]+ 485.99590 found 485.99506.
4.1.36. 2-(3-((4-Bromo-2-fluorobenzyl)oxy)-2-nitrobenzoyl)-3-hydroxy-5-methylcyclohex-2-en-1-
one (d30)
1
Light yellow solid, Yield: 63%. m.p. 140.9-142.8 °C. H NMR (400 MHz, CDCl3) δ 16.45 (s, 1H),
7.43 (dt, J=16.0, 8.0 Hz, 2H), 7.37 (d, J=8.2 Hz, 1H), 7.32 (s, 1H), 7.22 (d, J=8.4 Hz, 1H), 6.91 (d,
J=8.0 Hz, 1H), 5.26 (s, 2H), 2.83 (dd, J=16.0, 4.0 Hz, 1H), 2.52 (dd, J=16.0, 12.0 Hz, 2H), 2.19 (d,
J=10.2 Hz, 1H), 1.60 (s, 1H), 1.15 (d, J=4.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 196.03, 195.72,
193.76, 160.88, 158.39, 150.75, 137.21, 132.90, 130.43, 128.03, 119.32, 119.08, 118.84, 115.55,
112.53, 64.69, 45.77, 39.65, 26.70, 20.79. HRMS (ESI): calcd for C21H17BrFNO6 [M+Na]+ 500.01155
found 500.01368.
4.1.37. 2-(3-((2,4-Dichlorobenzyl)oxy)-2-nitrobenzoyl)-3-hydroxycyclohex-2-en-1-one (d31)
1
Light yellow solid, Yield: 55%. m.p. 150.4-152.1 °C. H NMR (400 MHz, CDCl3) δ 16.48 (s, 1H),
7.62 (d, J=8.4 Hz, 1H), 7.43 (t, J=8.0 Hz, 1H), 7.42 (d, J=2.0 Hz, 1H), 7.32 (d, J=8.0, 1H), 7.19 (d,
J=8.0 Hz, 1H), 6.89 (d, J=8.0 Hz, 1H), 5.27 (s, 2H), 2.78 (t, J=6.0 Hz, 2H), 2.43 (t, J=6.0 Hz, 2H), 2.05

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13
(m, J=6.0 Hz, 2H). C NMR (100 MHz, CDCl3) δ 196.30, 196.27, 193.86, 150.80, 137.39, 134.46,
133.00, 132.41, 131.86, 129.38, 129.11, 127.72, 119.28, 115.51, 112.99, 67.89, 37.61, 31.96, 19.14.
HRMS (ESI): calcd for C20H15Cl2NO6 [M+Na]+ 458.01686 found 458.01734.
4.1.38. 2-(3-((2,4-Dichlorobenzyl)oxy)-2-nitrobenzoyl)-3-hydroxy-5-methylcyclohex-2-en-1-one
(d32)
1
Light yellow solid, Yield: 51%. m.p. 114.5-116.2 °C. H NMR (400 MHz, CDCl3) δ 16.47 (s, 1H),
7.65 (d, J=8.0 Hz, 1H), 7.47 (t, J=8.0 Hz, 1H), 7.45 (s , 1H), 7.35 (dd, J=8.0, 1H), 7.22 (d, J=8.4 Hz,
1H), 6.92 (d, J=7.6 Hz, 1H), 5.30 (s, 2H), 2.84 (dd, J=16.0, 4.0 Hz, 1H), 2.58-2.48 (m, 2H), 2.33 (dt,
13
J=8.0, 6.0 Hz, 1H), 2.18 (dd, J=16.0, 12.0 Hz, 1H), 1.15 (d, J=6.4 Hz, 3H). C NMR (100 MHz,
CDCl3) δ 196.06, 150.77, 137.61, 137.37, 134.45, 133.02, 132.40, 131.87, 129.37, 129.10, 127.72,
119.31, 115.51, 112.54, 67.88, 26.72, 20.82. HRMS (ESI): calcd for C21H17Cl2NO6 [M+Na]+
472.03251 found 472.03186.
4.1.39. 2-(3-((2,4-Difluorobenzyl)oxy)-2-nitrobenzoyl)-3-hydroxycyclohex-2-en-1-one (d33)
1
Light yellow solid, Yield: 57%. m.p. 130.7-132.5 °C. H NMR (400 MHz, CDCl3) δ 16.47 (s, 1H),
7.48 (t, J=8.0 Hz, 1H), 7.42 (t, J=7.8 Hz, 1H), 7.21 (d, J=8.4 Hz, 1H), 6.94 (t, J=8.0 Hz, 1H), 6.88 (d, J
=4.0 Hz, 1H), 6.84 (d, J=8.6 Hz, 1H), 5.24 (s, 2H), 2.77 (s, 2H), 2.43 (s, 2H), 2.05 (t, J=6.0 Hz, 2H).
13C NMR (100 MHz, CDCl3) δ 196.25, 164.24, 161.45, 150.86, 137.78, 137.19, 132.84, 132.52,
130.65, 119.24, 115.66, 113.00, 111.98, 111.76, 104.08, 103.83, 103.58, 64.75, 38.86, 35.01, 19.11.
HRMS (ESI): calcd for C20H15F2NO6 [M+Na]+ 426.07596 found 426.07518.
4.2. Bioassay
Human HPPD (hHPPD) and human homogentisate 1,2-dioxygenase (hHGD) were required to
determine the inhibitory activity using the coupled enzyme reaction. Starting from cDNA, pET-28a-
hHPPD and pET-28a-hHGD plasmids were constructed as previously described [22,23] to express the

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aforesaid enzymes. The recombinant enzymes were over-expressed in E. coli BL21 cells (DE3) for
which cells were incubated at 37 °C in Luria broth media containing appropriate antibiotics. When the
E. coli growth reached an A600 of ~0.6, the temperature was decreased to 20 °C, and the cells were
then grown for additional 14 h (hHPPD) and 40h (hHGD). The cells were collected by centrifugation
(8000g, 5 min), and washed twice with (150 mM NaCl, 20 mM HEPES, pH 7.0) before disruption by
sonication. The mixture was then centrifuged at 13500g for 45 min to get the crude enzyme. The crude
supernatant containing recombinant enzyme was then loaded onto Ni-NTA column (Invitrogen)
equilibrated with 150 mM NaCl and 20 mM HEPES, pH 7.0, and enzyme was purified with imidazole
gradient and eluted with 250 mM imidazole. The overexpression and the relative molecular weight of
recombinant HPPDs were validated by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE), while the purity was estimated using ImageJ software (ImageJ 1.52a, Bethesda, MD,
USA). As HGD is highly active compared with HPPD, the crude enzyme was directly used for in vitro
inhibitory activity of synthetic compounds against hHPPD. Indeed, the inhibitory activity was
evaluated by monitoring the formation of maleylacetoacetate at 318 nm in 96-well plates at 30 °C
using a UV/visible plate reader (Biotech, Winooski, VT, USA). The volume of the reaction mixture
was 200 µL containing 50 µM HPPA, 2 mM sodium ascorbate, 20 mM HEPES buffer (pH 7.0), 100
µM FeSO4, and appropriate amounts of HGD and HPPD. To ensure that the reaction was efficiently
coupled, the amount of HGD was set to be in large excess compared with HPPD by setting the rate
constant k at 0.1 and 0.01, respectively. The reaction components were equilibrated at 30 °C for about
10 min before the measurement, whereas the inhibitors were dissolved in dimethyl sulfoxide (DMSO)
and diluted with testing buffer before use. Fitted curve and IC50 values were obtained using OriginPro
7.5 software (OriginLab, Northampton, MA).
4.3. Computational method