Design, synthesis and molecular docking of amide and urea derivatives as Escherichia coli PDHc-E1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.04.003

By targeting the ThDP binding site of Escherichia coli PDHc-E1, two new 'open-chain' classes of E. coli PDHc-E1 inhibitors, amide and urea derivatives, were designed, synthesized, and evaluated. The amide derivatives of compound 6d, with 4-NO₂ in the benzene ring, showed the most potent inhibition of E. coli PDHc-E1. The urea derivatives displayed more potent inhibitory activity than the corresponding amide derivatives with the same substituent. Molecular docking studies confirmed that the urea derivatives have more potency due to the two hydrogen bonds formed by two NH of urea with Glu522. The docking results also indicate it might help us to design more efficient PDHc-E1 inhibitors that could interact with Glu522.

Full text of DOI:10.1016/j.bmc.2014.04.003

Accepted Manuscript
Design, synthesis and molecular docking of amide and urea derivatives as Es-
cherichia coli PDHc-E1 inhibitors
Jun-Bo He, Yan-Liang Ren, Qiu-Shuang Sun, Ge-Yun You, Li Zhang, Peng
Zou, Ling-Ling Feng, Jian Wan, Hong-Wu He
PII:
S0968-0896(14)00247-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.04.003
BMC 11498
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 March 2014
2 April 2014
4 April 2014
Please cite this article as: He, J-B., Ren, Y-L., Sun, Q-S., You, G-Y., Zhang, L., Zou, P., Feng, L-L., Wan, J., He,
H-W., Design, synthesis and molecular docking of amide and urea derivatives as Escherichia coli PDHc-E1
inhibitors, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.04.003
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Design, synthesis and molecular docking of amide and urea derivatives
as Escherichia coli PDHc-E1 inhibitors
Jun-Bo He a,b,1, Yan-Liang Ren1, Qiu-Shuang Sun, Ge-Yun You, Li Zhang, Peng Zou, Ling-Ling Feng, Jian Wan*,
Hong-Wu He*
a
Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, Department of Chemistry
Central China Normal University, Wuhan 430079, China
b College of Food Science & Engineering, Wuhan Polytechnic University, Wuhan 430023, China
* To whom correspondence should be addressed. Tel./fax: +86 (0)27 67867960
E-mail addresses: he1208@mail.ccnu.edu.cn (H. He), jianwan@mail.ccnu.edu.cn (J. Wan)
1 These authors equally contributed to this work

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Abstract: By targeting the ThDP binding site of E. coli PDHc-E1, two new “open-chain” classes of E. coli
PDHc-E1 inhibitors, amide and urea derivatives, were designed, synthesized, and evaluated. The amide
derivatives of compound 6d, with 4-NO2 in the benzene ring, showed the most potent inhibition of E. coli
PDHc-E1. The urea derivatives displayed more potent inhibitory activity than the corresponding amide derivatives
with the same substituent. Molecular docking studies confirmed that the urea derivatives have more potency due
to the two hydrogen bonds formed by two NH of urea with Glu522. The docking results also indicate it might help
us to design more efficient PDHc-E1 inhibitors that could interact with Glu522.
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Keywords: PDHc-E1 inhibitors, amide and urea derivatives, molecular docking
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1. Introduction
The pyruvate dehydrogenase complex (PDHc) is an exquisite machine that catalyzes the irreversible oxidative of
pyruvate to acetyl CoA.1 The fundamental reactions of this complex are carried out by three enzymatic
components: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide
dehydrogenase (E3).2 The pyruvate dehydrogenase complex E1 component (PDHc-E1) catalyzes the first
inreversible step of the multistep process, using thiamine diphosphate (ThDP) (Figure 1) and Mg2+ as cofactors.3-5
Therefore, the PDHc-E1 has been reported as promising target for herbicide6-8 and fungicide.9
So far, a number of studies on small molecular inhibitors of PDHc-E1 have been reported, such as pyruvate
analogs10-12 and phosphonate analogs of pyruvate.6 Specially, the ThDP analogs have been studied greatly,13-16 for
their high binding affinities against PDHc-E1 and more modification sites at ThDP. However, because of the
structural complexity and highly charged pyrophosphate, there are no commercial potential inhibitors of ThDP
analogs that occupy the binding site of ThDP in PDHc-E1. In the present, it is an utmost need for the development
of practical small molecules as inhibitors of PDHc-E1.

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In our early efforts to design novel PDHc-E1 inhibitors, series of novel PDHc-E1 inhibitors (Figure 2) were
reported17-19 and showed potent Escherichia coli (E. coli) PDHc-E1 inhibitory activity and antifungal activity18.
Structure-activity relationship (SAR) indicated the introduction of iodine into the 5-position of 1,2,3-triazole of
title compounds could further increase the inhibitory activity against E. coli PDHc-E1 and antifungal activity18.
These results convinced us that the linker plays a vital role in the biological activity of these compounds. Aiming
to explore more optimizing linkages, the amide, which has been reported as bioisostere of 1,4-substituted
1,2,3-triazole,20, 21 is introduced to the structure I as a “open-chain”22 linker. Furthermore, the amide was replaced
by urea for its ability to simultaneously donate two hydrogen bonds compared with amide. Therefore, two new
structural classes of PDHc-E1 inhibitors were formed (Figure 3).
Herein, the chemical synthesis of these new amide and urea derivatives as E. coli PDHc-E1 inhibitors is described
in details. The inhibitory activities on E. coli PDHc-E1 are presented along with their structure-activity
relationship (SAR) analysis as follows. We also performed molecular docking studies leading to identify the
critical binding sites of the target PDHc-E1.
2. Chemistry
The synthesis of amide derivatives (6a-6n) was depicted in Scheme 1. The critical intermediate of
5-(aminomethyl)-2-methylpyrimidin-4-amine (2) was synthesized by Pd/C-catalyzed reduction of
5-(azidomethyl)-2-methylpyrimidin-4-amine (1), which was prepared readily from thiamine hydrochloride
according to literature.23 The substituted phenoxyacetic acids 3 were prepared by condensation of corresponding
substituted phenols with chloroacetic acid in the presence of sodium hydroxide. However, the yield of the reaction
of chloroacetic acid with strong electron-withdrawing substituted phenols in the presence of sodium hydroxide
was very poor. Therefore, the substituted phenoxyacetic acids 5 were prepared in satisfactory yields by reaction of
corresponding substituted phenols with ethyl bromoacetate in the presence of K2CO3 in DMSO followed by

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alkaline hydrolysis.7 The final compounds 6a-6n were synthesized easily by coupling reaction of compound 2
with mixed anhydrides, which were generated from substituted phenoxyacetic acid 3 and 5 in THF at -5 oC by the
addition of ethyl chloroformate in the presence of triethylamine.
The synthesis of urea derivatives 8a-8e was depicted in Scheme 2. Acyl azides were prepared from NaN3 with
mixed anhydries, which could be easily generated from acid 3 and 5 with ethyl chloroformate. Then acyl azides
undergo Curtius rearrangement24 in toluene to give isocyanates, which were trapped by amine 2 leading to the
formation of urea derivatives 8a-8e in 57-82% yields.
3. Results and discussion
3.1. In vitro inhibition of E. coli PDHc-E1
The synthesized amide and urea derivatives (6a-6n and 8a-8e) were evaluated for their inhibitory activities against
PDHc-E1 from E. coli. The IC50 values were summarized in Table 1. It was observed that most compounds
exhibited good inhibitory activity (IC50 < 20 µM). The structure-activity relationships (SAR) in these compounds
were investigated by introducing substituents on the benzene ring, and changing the linker with amide and urea
linkages.
We started our work focusing on the amide linkage. As shown in Table 1, it was noticed that the inhibitory activity
is highly related to the nature of the substituents on benzene ring, and electron-withdrawing groups are beneficial
for PDHc-E1 inhibitory activity. Comparing the data of compounds 6b, 6k, 6l, and 6m, the activity sequence is
2-Cl-4-NO2 (6l) > 2-Cl-4-F (6k) > 2,4-diCl (6b) > 2-CH3-4-Cl (6m), which indicates the stronger of the
electron-withdrawing groups, the more potent inhibitory activity. We also notice that compound 6d, with 4-NO2 on
the benzene ring, possesses the most potent inhibitory activity with IC50 value of 3.58 0.52 µM. Based on the
recently publication,17 we suppose that the nitryl group on benzene ring presented a special case as it can form
hydrogen bonds and coordinate bond with Mg2+ in the active site. It should be noted that compound 6d (IC50
=

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3.58 0.52 µM) showed slightly improved inhibitory activity, compared with compound 6l (IC50 = 4.90 0.81
µM). This suggests that the 2-position group on benzene ring is not good for the PDHc-E1 inhibitory activity
because of the steric effect. Compared with compound 6h (2-Br), compound 6i (2-Cl) showed more potent
inhibitory activity, which can be explained that compound 6i (2-Cl) has less steric hindrance effect. In conclusion,
the amide derivatives 6a-6n exhibited higher inhibitory activity than structure I.18
Encouraged by the improved potency of compounds 6a-6n versus structure I, we continued our optimization effort
by changing the amide linkage with urea linkage. As shown in Table 2, all the compounds 8a-8e displayed
improved inhibitory activity versus the corresponding amide derivatives with the same substituents. We notice that
compound 8e (4-Cl, IC50 = 3.67 0.38 µM) exhibited the same potency with compound 6d (4-NO2), and showed
3-folds increase than compound 6c. The results suggest that the urea linkage should play an important role in the
PDHc-E1 inhibitory activity. We also tried to synthesize the urea derivative with 4-NO2 in the benzene ring, but
failed.
Based on the aforementioned results, the “open-chain” linkers, amide and urea linkages, do have a beneficial
effect on the E. coli PDHc-E1 inhibition. Compared to 1,2,3-triazole derivatives, the “open-chain” linker
derivatives can be easily functionalized into more new classes of inhibitors.
3.2 Molecular docking studies
To explore the interaction modes of amide and urea derivatives with the active site of PDHc-E1, several molecular
docking simulation studies were carried out by using SURFLEX module of SYBYL package version. Based on
the in vitro inhibition results, we selected compounds 6d and 8e, our best PDHc-E1 inhibitors in present study
(IC50 = 3.58 0.52 µM and 3.67 0.38 µM, respectively), as ligand examples.
The binding modes of compound 6d and 8e were shown in Figure 4A and 4B, respectively. As depicted in Figure
4A, 4-aminopyrimidine moiety formed two hydrogen bonds with residues Val192 and Glu571, and π-π stacking

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interaction with Phe602. For benzene ring part, the nitryl group can form three hydrogen bonds with Gly231,
Asn260, and Lys392, furthermore, the coordination of nitryl group with Mg2+ in the active site also plays an
important role in increasing the bind interaction. The binding modes of these two parts, 4-aminopyrimidine and
benzene ring, were in perfect agreement with the recently publication.17 The amide, was used as the bioisostere of
1,4-substituted 1,2,3-triazole, can form a strong hydrogen bond with Glu522 which is an important residue in the
stabilization of the enzyme-bound LThDP.25, 26 We also investigated how the two NH of urea bind in the active site
of E. coli PDHc-E1. Figure 4B showed the binding mode of compound 8e, it can be seen that the binding mode of
4-aminopyrimidine of compound 8e is almost same to those of compound 6d, but the two NH of urea really
formed two strong hydrogen bonds with Glu522. Interestingly, the oxygen atom of phenoxy also formed a
hydrogen bong with Leu264, which maybe because the chain length is increased by introduction of urea. The
docking results provided us a reasonable explanation for why compound 8e has more potent E. coli PDHc-E1
inhibitory activity. The docking results also provide us a new method to design new PDHc-E1 inhibitors that can
interact with Glu522 based on amide and urea linkages.
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4. Conclusion
In the present study, two new “open-chain” classes of E. coli PDHc-E1 inhibitors, amide and urea derivatives,
were designed and synthesized. SAR analyses indicated that the inhibitory potency against E. coli PDHc-E1 of
title compounds could be increased by introducing amide and urea linkages. The amide derivatives with 4-NO2 in
the benzene ring exhibited much more inhibitory potency against E. coli PDHc-E1 than other compounds.
Moreover, optimizing the amide linker with urea linker, the E. coli PDHc-E1 inhibitory activity could be further
increased. Molecular docking was performed to study the inhibitor-PDHc-E1 protein interactions. Analysis of
compounds 6d’s and 8e’s binding modes in the active binding site demonstrated that the two hydrogen bonds,
formed by two NH of urea with Glu522, really plays an important role in increasing the inhibitory potency of urea

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derivatives against E. coli PDHc-E1. Furthermore, the docking results also give us a new direction to design new
PDHc-E1 inhibitors that can interact with Glu522. The above-mentioned results of SAR analysis and molecular
docking study may allow the rational design of more efficient PDHc-E1 inhibitors.
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5. Experiments
5.1. Chemistry
Melting points (mp) were measured on an electrothermal melting point apparatus and were uncorrected. 1H NMR
and 13C NMR spectra were obtained at 400 MHz or 600 MHz, in CDCl3 or DMSO-d6 solution on a Varian
Mercury-Plus 400 or 600 sepctrometer and chemical shifts were recorded in parts per million (ppm) with TMS as
the internal reference. Mass spectra (MS) were run on a QTRSP LC/MS/MS system (API2000; Applied
Biosystems, Foster City, CA, USA) or a TraceMS 2000 organic mass spectrometry, and signals were given in m/z.
Elemental analyses (EA) were measured on a Vario ELIII CHNSO elemental analyzer. Unless otherwise noted,
reagents were purchased from commercial suppliers and used without further purification. All the reactions were
monitored by thin-layer chromatography (TLC) on pre-coated silica gel G plates at 254 nm under a UV lamp.
5-(azidomethyl)-2-methylpyrimidin-4-amine (1) was synthesized according to the existing method.23 Substituted
phenoxyacetic acid 3 and 5 were prepared according to the methods previously reported.7
5.2. 5-(aminomethyl)-2-methylpyrimidin-4-amine (2)
To a solution of 1 (4.0 g) in methanol (100 mL) was added 10% Pd/C (0.4 g), then the flask was fit tightly over
with a balloon filled with hydrogen gas. The mixture was stirred at room temperature for 12 h. The reaction
mixture was filtered through a pad of Celite and the solvent were evaporated under reduced pressure to afford
1
compound 2 as white solid (3.34 g, 99%). H NMR (CDCl3, 400 MHz): δ 1.85 (s, 2H, NH2), 2.48 (s, 3H, CH3),
3.93 (s, 2H, CH2), 6.04 (s, 2H, NH2), 7.92 (s, 1H, pyrimidine CH).
5.3. General procedure for preparation of compounds 6a-6n

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To a solution of corresponding substituted phenoxyacetic acid 3 or 5 (1.5 mmol) and Et3N (0.16 g, 1.6 mmol) in
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THF (15 mL) was added ethyl chlorocarbonate (0.17 g, 1.6 mmol) in THF (2 mL) slowly at -5 C. After the
addition was complete, the cold mixture was stirred for an additional 15 min. A solution of 2 (0.22 g, 1.6 mmol) in
DMF (2 mL) was added dropwise while the temperature was kept at -5 oC. After the addition was complete, the
mixture was stirred at room temperature for 12 h. It was poured into water (30 mL), and the precipitate was
collected by filtration and dried in the atmospheric pressure. Recrystallization with appropriate solvent afforded
the desired solid compounds 6a-6n.
5.3.1. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-phenoxyacetamide (6a)
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White solid, yield: 92%, mp 120-121; H NMR (CDCl3, 400 MHz): δ 2.47 (s, 3H, CH3), 4.38 (d, 2H, J = 9.6 Hz,
CH2), 4.53 (s, 2H, CH2), 5.95 (s, 2H, NH2), 6.89 (d, 1H, J = 12.0 Hz, Ar-H), 7.03 (t, 2H, J = 10.8 Hz, Ar-H), 7.12
(s, 1H, NH), 7.31 (t, 2H, J = 12.0 Hz, Ar-H), 7.98 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.17, 36.45,
66.85, 110.63, 114.77, 121.30, 129.51, 154.94, 157.60, 161.52, 165.64, 168.59. EI-MS m/z (%): 273.3 (M++1,
3.62), 272.2 (M+, 35.15). Anal. Calcd. For C14H16N4O2: C, 61.75; H, 5.92; N, 20.58. Found: C, 61.48; H, 5.82; N,
20.64.
5.3.2. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(2,4-dichlorophenoxy)acetamide (6b)
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Light yellow solid, yield: 67%, mp 201-203; H NMR (DMSO-d6, 600 MHz): δ 2.29 (s, 3H, CH3), 4.10 (s, 2H,
CH2), 4.68 (s, 2H, CH2), 6.73 (s, 2H, NH2), 7.04 (s, 1H, Ar-H), 7.35 (s, 1H, Ar-H), 7.60 (s, 1H, Ar-H), 7.89 (s, 1H,
CH), 8.48 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): δ 25.12, 36.49, 67.88, 110.49, 115.50, 122.65, 125.29,
128.03, 129.41, 152.41, 154.70, 161.51, 165.69, 167.83. EI-MS m/z (%): 342.2 (M++2, 10.06), 340.2 (M+, 10.13).
Anal. Calcd. For C14H14Cl2N4O2: C, 49.28; H, 4.14; N, 16.42. Found: C, 49.78; H, 4.29; N, 16.74.
5.3.3. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(4-chlorophenoxy)acetamide (6c)
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White solid, yield: 92%, mp 192-194; H NMR (CDCl3, 600 MHz): δ 2.47 (s, 3H, CH3), 4.38 (d, 2H, J = 7.2 Hz,

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CH2), 4.50 (s, 2H, CH2), 5.94 (s, 2H, NH2), 6.83 (d, 2H, J = 9.0 Hz, Ar-H), 7.09 (s, 1H, NH), 7.26 (d, 2H, J = 9.0
Hz, Ar-H), 7.98 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.15, 36.45, 67.14, 110.58, 116.55, 125.09,
129.28, 154.88, 156.45, 161.54, 165.73, 168.37. EI-MS m/z (%): 308.3 (M++2, 5.16), 307.3 (M++1, 2.87), 306.3
(M+, 17.32),. Anal. Calcd. For C14H15ClN4O2: C, 54.82; H, 4.93; N, 18.26. Found: C, 54.46; H, 5.28; N, 17.91.
5.3.4. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(4-nitrophenoxy)acetamide (6d)
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Yellow solid, yield: 79%, mp 239-241; H NMR (DMSO-d6, 600 MHz): δ 2.29 (s, 3H, CH3), 4.11 (s, 2H, CH2),
4.73 (d, 2H, J = 4.8 Hz, CH2), 6.71 (s, 2H, NH2), 7.16 (d, 2H, J = 8.4 Hz, Ar-H), 7.23 (d, 2H, J = 9.0 Hz,
Ar-H),7.89 (s, 1H, CH), 8.68 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): δ 25.13, 36.46, 67.14, 110.49, 115.36,
125.82, 141.33, 154.76, 161.50, 162.79, 165.73, 167.72. EI-MS m/z (%): 318.2 (M++1, 3.87), 317.3 (M+, 25.46).
Anal. Calcd. For C14H15N5O4: C, 52.99; H, 4.76; N, 22.07. Found: C, 52.68; H, 4.38; N, 19.78.
5.3.5. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(p-tolyloxy)acetamide (6e)
White solid, yield: 57%, mp 172-174; 1H NMR (CDCl3, 600 MHz): δ 2.29 (s, 3H, CH3), 2.48 (s, 3H, CH3), 4.38 (d,
2H, J = 6.6 Hz, CH2), 4.49 (d, 2H, J = 7.2 Hz, CH2), 5.95 (s, 2H, NH2), 6.78 (d, 1H, J = 8.4 Hz, Ar-H), 7.09 (d, 3H,
J = 8.4 Hz, Ar-H + NH), 7.99 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 20.08, 25.14, 36.44, 67.11, 110.66,
114.63, 129.86, 130.11, 154.91, 155.53, 161.57, 165.70, 168.81. EI-MS m/z (%): 286.2 (M+, 3.62). Anal. Calcd.
For C15H18N4O2: C, 62.92; H, 6.34; N, 19.57. Found: C, 62.81; H, 6.62; N, 19.47.
5.3.6. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(4-bromophenoxy)acetamide (6f)
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White solid, yield: 63%, mp 209-210; H NMR (CDCl3, 600 MHz): δ 2.48 (s, 3H, CH3), 4.38 (d, 2H, J = 7.2 Hz,
CH2), 4.50 (s, 2H, CH2), 5.91 (s, 2H, NH2), 6.78 (d, 1H, J = 9.0 Hz, Ar-H), 7.02 (s, 1H, NH), 7.41 (d, 2H, J = 9.6
Hz, Ar-H), 7.99 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.16, 36.43, 67.03, 110.56, 112.78, 117.07,
132.16, 154.87, 156.90, 161.51, 165.70, 168.31. EI-MS m/z (%): 352.1 (M++2, 10.61), 350.2 (M+, 11.10). Anal.
Calcd. For C14H15BrN4O2: C, 47.88; H, 4.31; N, 15.95. Found: C, 47.58; H, 4.39; N, 15.57.

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5.3.7. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(4-methoxyphenoxy)acetamide (6g)
White solid, yield: 67%, mp 177-179; 1H NMR (CDCl3, 400 MHz): δ 2.48 (s, 3H, CH3), 3.76 (s, 3H, OCH3), 4.38
(d, 2H, J = 10.8 Hz, CH2), 4.48 (s, 2H, CH2), 5.93 (s, 2H, NH2), 6.84 (d, 2H, J = 12.6 Hz, Ar-H), 7.03 (d, 2H, J =
10.8 Hz, Ar-H), 7.26 (s, 1H, NH), 7.99 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.16, 36.44, 55.37, 67.72,
110.68, 114.62, 115.83, 151.64, 153.96, 154.98, 161.58, 165.74, 168.93. EI-MS m/z (%): 302.3 (M+, 13.45). Anal.
Calcd. For C15H18N4O3: C, 59.59; H, 6.00; N, 18.53. Found: C, 59.55; H, 6.43; N, 18.71.
5.3.8. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(2-bromophenoxy)acetamide (6h)
White solid, yield: 63%, mp 196-198; 1H NMR (CDCl3, 400 MHz): δ 2.48 (s, 3H, CH3), 4.41 (d, 2H, J = 10.2 Hz,
CH2), 4.57 (s, 2H, CH2), 5.89 (s, 2H, NH2), 6.86 (d, 1H, J = 7.2 Hz, Ar-H), 6.93 (t, 1H, J = 7.2 Hz, Ar-H), 7.30 (d,
1H, J = 6.8 Hz, Ar-H), 7.55 (d, 1H, J = 8.0 Hz, Ar-H), 8.04 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.16,
36.47, 67.91, 110.52, 111.30, 122.89, 128.95, 133.11, 154.15, 154.74, 161.50, 165.70, 168.07. EI-MS m/z (%):
352.1 (M++2, 6.39), 350.2 (M+, 5.11). Anal. Calcd. For C14H15BrN4O2: C, 47.88; H, 4.31; N, 15.95. Found: C,
48.24; H, 4.70; N, 16.36.
5.3.9. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(2-chlorophenoxy)acetamide (6i)
1
White solid, yield: 65%, mp 188-190; H NMR (CDCl3, 400 MHz): δ 2.47 (s, 3H, CH3), 4.38 (d, 2H, J = 9.6 Hz,
CH2), 4.50 (s, 2H, CH2), 5.92 (s, 2H, NH2), 6.83 (d, 2H, J = 4.4 Hz, Ar-H), 7.07 (s, 1H, NH), 7.26 (t, 2H, J = 4.0
Hz, Ar-H), 7.98 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.18, 36.48, 67.16, 110.65, 116.62, 125.14,
129.34, 154.89, 156.49, 161.58, 165.78, 168.46. EI-MS m/z (%): 308.2 (M++2, 6.53), 306.2 (M+, 22.59). Anal.
Calcd. For C14H15ClN4O2: C, 54.82; H, 4.93; N, 18.26. Found: C, 54.57; H, 5.28; N, 18.35.
5.3.10. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(4-fluorophenoxy)acetamide (6j)
White solid, yield: 79%, mp 165-167; 1H NMR (CDCl3, 400 MHz): δ 2.48 (s, 3H, CH3), 4.38 (d, 2H, J = 10.2 Hz,
CH2), 4.49 (s, 2H, CH2), 5.93 (s, 2H, NH2), 6.83-6.86 (m, 2H, Ar-H), 7.00 (t, 2H, J = 12.6 Hz, Ar-H), 7.07 (s, 1H,

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NH), 7.99 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.15, 36.43, 67.49, 110.60, 115.78, 116.01, 116.13,
116.21, 153.92, 154.85, 161.51, 165.69, 168.54. EI-MS m/z (%): 291.3 (M++1, 3.99), 290.2 (M+, 28.62). Anal.
Calcd. For C14H15FN4O2: C, 57.92; H, 5.21; N, 19.30. Found: C, 58.24; H, 5.54; N, 18.98.
5.3.11. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(2-chloro-4-fluorophenoxy)acetamide (6k)
1
White solid, yield: 71%, mp 167-169; H NMR (CDCl3, 400 MHz): δ 2.48 (s, 3H, CH3), 4.40 (d, 2H, J = 9.6 Hz,
CH2), 4.54 (s, 2H, CH2), 5.91 (s, 2H, NH2), 6.84-6.87 (m, 1H, Ar-H), 6.93-6.96 (m, 1H, Ar-H), 7.14-7.19 (m, 2H,
Ar-H + NH), 8.03 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.13, 36.49, 68.34, 110.51, 114.75, 115.40,
115.48, 117.11, 122.28, 122.39, 150.0, 154.77, 161.51, 165.70, 168.01. EI-MS m/z (%): 326.3 (M++2, 2.97), 324.2
(M+, 13.84). Anal. Calcd. For C14H14ClFN4O2: C, 51.78; H, 4.35; N, 17.25. Found: C, 51.98; H, 3.98; N, 17.65.
5.3.12. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(2-chloro-4-nitrophenoxy)acetamide (6l)
1
Light yellow solid, yield: 57%, mp 201-202; H NMR (CDCl3, 400 MHz): δ 2.49 (s, 3H, CH3), 4.43 (d, 2H, J =
9.6 Hz, CH2), 4.68 (s, 2H, CH2), 5.84 (s, 2H, NH2), 6.99 (d, 1H, J = 13.8 Hz, Ar-H), 7.05 (s, 1H, NH), 8.05 (s, 1H,
CH), 8.19 (d, 1H, J = 10.2 Hz, Ar-H), 8.34 (s, 1H, Ar-H); 13C NMR (DMSO-d6, 100 MHz): δ 25.16, 36.60, 67.85,
110.43, 113.77, 122.05, 124.39, 125.48, 141.14, 154.71, 158.61, 161.51, 165.79, 167.22. EI-MS m/z (%): 353.2
(M++2, 4.66), 352.2 (M++1, 2.89), 351.2 (M+, 18.48). Anal. Calcd. For C14H14ClN5O4: C, 47.80; H, 4.01; N, 19.91.
Found: C, 47.61; H, 4.31; N, 20.05.
5.3.13. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(4-chloro-2-methylphenoxy)acetamide (6m)
1
White solid, yield: 83%, mp 188-189; H NMR (DMSO-d6, 600 MHz): δ 2.29 (s, 3H, CH3), 2.35 (s, 3H, CH3),
4.17 (d, 2H, J = 5.4 Hz, CH2), 4.62 (s, 2H, CH2), 6.81 (s, 2H, NH2), 6.90 (d, 1H, J = 8.4 Hz, Ar-H), 7.23 (d, 1H, J
= 8.4 Hz, Ar-H), 7.31 (s, 1H, Ar-H), 7.94 (s, 1H, CH), 8.48 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): δ 15.95,
25.14, 36.49, 67.42, 110.57, 113.21, 124.61, 126.35, 128.74, 130.07, 154.66, 161.49, 165.64, 168.46. EI-MS m/z
(%): 322.1 (M++2, 3.29), 321.1 (M++1, 3.62), 320.2 (M+, 35.15). Anal. Calcd. For C15H17ClN4O2: C, 56.16; H,

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5.34; N, 17.47. Found: C, 56.01; H, 5.44; N, 17.54.
5.3.14. N-((4-amino-2-methylpyrimidin-5-yl)methyl)-2-(4-cyanophenoxy)acetamide (6n)
1
White solid, yield: 71%, mp 232-233; H NMR (DMSO-d6, 600 MHz): δ 2.28 (s, 3H, CH3), 4.09 (d, 2H, J = 4.2
Hz, CH2), 4.67 (s, 2H, CH2), 6.72 (s, 2H, NH2),7.11 (d, 2H, J = 7.8 Hz, Ar-H), 7.79 (d, 2H, J = 8.4 Hz, Ar-H), 7.87
(s, 1H, CH), 8.66 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): δ 25.16, 36.43, 66.83, 103.58, 110.51, 115.86,
119.04, 134.20, 154.79, 161.01, 161.504, 165.73, 167.88. EI-MS m/z (%): 298.16 (M++1, 6.34), 297.1 (M+, 41.81).
Anal. Calcd. For C15H15N5O2: C, 60.60; H, 5.09; N, 23.56. Found: C, 60.59; H, 4.95; N, 23.54.
5.4. General procedure for preparation of compounds 8a-8e
To a solution of corresponding substituted phenoxyacetic acid 3 or 5 (4 mmol) and Et3N (0.48 g, 4.8 mmol) in
acetone (15 mL) was added ethyl chlorocarbonate (0.51 g, 4.8 mmol) in acetone (5 mL) at -5 oC. After the addition
was complete, the cold mixture was stirred for an additional 15 min. A solution of sodium azide (0.52 g, 8 mmol)
in water (2 mL) was added over 5 min while the temperature was kept at -5 oC. The mixture was stirred for 30 min
longer at this temperature, poured into ice water (50 mL), and shaken with toluene (30 mL x 3). The combined
toluene extracts were dried over MgSO4. The toluene solution was heated cautiously at 70 oC for 1 h, and then the
solution was cooled to room temperature, which was used for next step directly.
To the solution of isocyanate 7 in toluene was added compound 2 (0.21 g, 1.5 mmol) in DMF (2 mL) at 0 oC over
5 min, and then the solution was stirred at room temperature. After 4 h, the toluene was removed under reduced
pressure to give a residue. The residue was poured into cold water (30 mL), and the precipitate was collected by
filtration and dried to afford the desired compounds 8a-8e.
5.4.1. 1-((4-amino-2-methylpyrimidin-5-yl)methyl)-3-(phenoxymethyl)urea (8a)
1
White solid, yield: 82%, mp 158-160; H NMR (DMSO-d6, 600 MHz): δ 2.27 (s, 3H, CH3), 4.01 (d, 2H, J = 6.0
Hz, CH2), 5.09 (d, 2H, J = 6.6 Hz, CH2), 6.71 (s, 1H, NH), 6.82 (s, 2H, NH2), 6.91-6.94 (m, 3H, Ar-H), 7.26 (t, 2H,

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J = 7.2 Hz, Ar-H), 7.33 (t, 1H, J = 6.6 Hz, NH), 7.84 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.17, 37.18,
69.34, 111.96, 115.25, 120.73, 129.40, 129.49, 154.43, 156.86, 157.72, 161.72, 165.62. EI-MS m/z (%): 287.35
(M+, 0.50). Anal. Calcd. For C14H17N5O2: C, 58.52; H, 5.96; N, 24.37. Found: C, 58.39; H, 5.80; N, 24.23.
5.4.2. 1-((4-amino-2-methylpyrimidin-5-yl)methyl)-3-((4-methylphenoxy)methyl)urea (8b)
White solid, yield: 57%, mp 172-174 oC; 1H NMR (DMSO-d6, 600 MHz): δ 2.22 (s, 3H, CH3), 2.28 (s, 3H, CH3),
4.02 (d, 2H, J = 6.0 Hz, CH2), 5.06 (d, 2H, J = 6.6 Hz, CH2), 6.68 (s, 1H, NH), 6.80 (s, 2H, NH2), 6.83 (s, 2H,
Ar-H), 7.06 (t, 2H, J = 6.0 Hz, Ar-H), 7.27 (s, 1H, NH), 7.85 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ
20.11, 25.14, 37.13, 69.45, 111.96, 115.19, 129.40, 129.82, 154.37, 154.70, 157.72, 161.71, 165.57. EI-MS m/z
(%): 301.49 (M+, 0.86). Anal. Calcd. For C15H19N5O2: C, 59.79; H, 6.36; N, 23.24. Found: C, 59.60; H, 6.47; N,
24.43.
5.4.3. 1-((4-amino-2-methylpyrimidin-5-yl)methyl)-3-((4-methoxyphenoxy)methyl)urea (8c)
White solid, yield: 67%, mp 177-179 oC; 1H NMR (DMSO-d6, 600 MHz): δ 2.27 (s, 3H, CH3), 3.68 (s, 3H, CH3),
4.01 (d, 2H, J = 6.0 Hz, CH2), 5.01 (d, 2H, J = 7.2 Hz, CH2), 6.67 (s, 1H, NH), 6.83 (d, 4H, J = 7.8 Hz, Ar-H +
NH2), 6.87 (d, 2H, J = 8.4 Hz, Ar-H), 7.27 (t, 1H, J = 6.6 Hz, NH), 7.84 (s, 1H, CH); 13C NMR (DMSO-d6, 100
MHz): δ 25.15, 37.15, 55.34, 70.02, 222.99, 114.60, 116.57, 150.74, 153.62, 154.37, 157.75, 161.72, 165.59.
EI-MS m/z (%): 317.26 (M+, 0.40). Anal. Calcd. For C15H19N5O3: C, 56.77; H, 6.03; N, 22.07. Found: C, 56.93; H,
5.95; N, 21.91.
5.4.4. 1-((4-amino-2-methylpyrimidin-5-yl)methyl)-3-((4-fluorophenoxy)methyl)urea (8d)
White solid, yield: 79%, mp 165-167; 1H NMR (DMSO-d6, 600 MHz) δ 2.27 (s, 3H, CH3), 4.01 (d, 2H, J = 6.0 Hz,
CH2), 5.07 (d, 2H, J = 6.6 Hz, CH2), 6.76 (s, 1H, NH), 6.80 (s, 2H, NH2), 6.96 (d, 2H, J = 8.4 Hz, Ar-H), 7.08 (t,
2H, J = 8.4 Hz, Ar-H), 7.36 (t, 1H, J = 6.6 Hz, NH), 7.84 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz) δ 25.13,
37.13, 70.03, 111.91, 115.71, 115.86, 116.70, 116.76, 153.17, 154.40, 155.87, 157.68, 161.68, 165.58. EI-MS m/z

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(%): 306.51 (M+, 0.75). Anal. Calcd. For C14H16FN5O2: C, 55.08; H, 5.28; N, 22.94. Found: C, 55.17; H, 5.34; N,
22.77.
5.4.5. 1-((4-amino-2-methylpyrimidin-5-yl)methyl)-3-((4-chlorophenoxy)methyl)urea (8e)
Yellow solid, yield: 67%, mp 192-194; 1H NMR (DMSO-d6, 600 MHz): δ 2.27 (s, 3H, CH3), 4.01 (d, 2H, J = 6.0
Hz, CH2), 5.09 (d, 2H, J = 7.2 Hz, CH2), 6.73 (s, 1H, NH), 6.82 (s, 2H, NH2), 6.97 (d, 2H, J = 9.0 Hz, Ar-H), 7.30
(d, 2H, J = 8.4 Hz, Ar-H), 7.35 (t, 1H, J = 6.6 Hz, NH), 7.84 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ
25.17, 37.18, 69.83, 111.91, 117.07, 124.49, 129.24, 154.43, 155.73, 157.64, 161.71, 165.63. EI-MS m/z (%):
321.20 (M+, 0.65). Anal. Calcd. For C14H16ClN5O2: C, 52.26; H, 5.01; N, 21.77. Found: C, 52.60; H, 5.45; N,
21.44.
5.5. Evaluation of inhibitory activity of PDHc E1
The expressing plasmid pMal-C2X-PDHc-E1 was transformed into E. coli stain TB1 and inoculated in
Luria-Bertani (LB) broth containing 2% glucose and 30 mg/ml ampicillin at 37 °C until reaching a cell density to
A600 of 0.6–0.8. Then cells were induced with
a final concentration of 0.5 mM isopropyl
β-D-1-thiogalactopyranoside (IPTG) for 7 h at 25 °C before harvesting. Purification of the fusion protein was
carried out using a maltose-binding protein (MBP) affinity column attached to an AKTA purifier 10 (UPC-F920,
GE Healthcare Life Sciences). The concentrations of purified proteins were determined by the method of
Bradford27 using bovine serum albumin (Tiangen) as standard. The final purify (> 95 %) of the sample was
verified by SDS–PAGE and then the purified protein was stored in 50 % (v/v) glycerol at -20 °C.
The inhibitory activities of synthesized compounds were measured by the enzymatic assay. PDHc-E1 activity was
assayed by a modified methods of N. Nemeria,14 and measured by monitoring the reduction of 2,
6-DichloroPhenolindophenol (2,6-DCPIP) at 600 nm using a microplate reader (BioTek Synergy2, USA). The
total volume of 100 µL reaction mixture contained 50 mM K3PO4, pH 7.2, 2.0 mM sodium pyruvate as substrate,

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0.8 mM 2,6-DCPIP, 7.1 µM enzyme and different concentration of inhibitors. The reaction mixtures were
incubated for 3 min at 37 °C, then added different concentration of ThDP (ranging from 0 to 200 µM) to initial
reaction. To determine the inhibitor concentration of synthesized compounds at 50% inhibition (IC50), initial rate
data taken at saturating substrate, fixed effectors, and systematically varied inhibitor concentrations were fit to Hill
equation, V = V0 – (V0- V∞)/((IC50/I)n + 1),28 Where V, V0, and V∞ are the velocity, maximum velocity (at I = 0),
and the limiting velocity (at I saturating); n is the Hill coefficient associated with the inhibitor; and IC50 is the
inhibition concentration of synthesized compounds at 50% inhibition. All kinetic data were fit to the
Growth/sigmoidal model from origin 7.0 software. One unit of activity is defined as the amount of 2,6-DCPIP
reduced (µmol/min/mg of PDHc-E1).
5.6. Molecular docking
For docking purposes, the crystallographic coordinates of the PDHc-E1 with bound ThDP from E. coli (PDB code:
1L8A) were obtained from Brookhaven Data Bank. Hydrogen atoms were added to the structure allowing for
appropriate ionization at physiological pH. The protonated state of several important residues, such as His106,
His142, Tyr599, Glu751 and His640, were adjusted by using SYBYL7.3 (Tripos, St. Louis, USA) in favor of
forming reasonable hydrogen bond with the ligand. Molecular docking analysis was carried out by the SURFLEX
module of SYBYL package to explore the interaction model for the active site of PDHc-E1 with its ligand. All
atoms located within the range of 6.5 Å from any atom of the cofactor ThDP were selected into the active site, and
the corresponding amino acid residue was, therefore, involved into the active site if only one of its atoms was
selected. Other default parameters were adopted in the SURFLEX-docking calculations. All calculations were
performed
on
a
CCNUGrid-based
computational
environment
(CCNUGrid
website
http://www.202.114.32.71:8090/ccnu/chem/platform.xml).

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The work was supported in part by the National Basic Research Program of China (2010CB126100), the National
Natural Science Foundation of China (No. 20372023, 21172090).
References and notes
1. Koike, M.; Reed, L. J.; Carroll, W. R. J. Biol. Chem. 1960, 235, 1924.
2. Wei, W.; Li, H.; Nemeria, N.; Jordan, F. Protein Expr. Purif. 2003, 28, 140.
3. Dobritzsch, D.; König, S.; Schneider, G.; Lu, G. J. Biol. Chem. 1998, 273, 20196.
4. Alvarez, F. J.; Ermer, J.; Huebner, G.; Schellenberger, A.; Schowen, R. L. J. Am. Chem. Soc. 1991, 113, 8402.
5. Kern, D.; Kern, G.; Neef, H.; Tittmann, K.; Killenberg-Jabs, M.; Wikner, C.; Schneider, G.; Hübner, G. Science
1997, 275, 67.
6. Baillie, A.; Wright, K.; Wright, B.; Earnshaw, C. Pestic. Biochem. Physiol. 1988, 30, 103.
7. He, H.-W.; Yuan, J.-L.; Peng, H.; Chen, T.; Shen, P.; Wan, S.-Q.; Li, Y.; Tan, H.-L.; He, Y.-H.; He, J.-B.; Li, Y. J.
Agric. Food Chem. 2011, 59, 4801.
8. He, H.-W.; Peng, H.; Wang, T.; Wang, C.-B.; Yuan, J.-L.; Chen, T.; He, J.-B.; Tan, X.-S. Agric. Food Chem.
2013, 61, 2479.
9. Birkenstock, T.; Liebeke, M.; Winstel, V.; Krismer, B.; Gekeler, C.; Niemiec, M. J.; Bisswanger, H.; Lalk, M.;
Peschel, A. J. Biol. Chem. 2012, 287, 2887.
10. Lowe, P. N.; Perham, R. N. Biochemistry 1984, 23, 91.
11. Flournoy, D. S.; Frey, P. A. Biochemistry 1989, 28, 9594.
12. Bisswanger, H. Biochem. Biophys. Res. Commun. 1980, 95, 513.
13. Lowe, P. N.; Leeper, F. J.; Perham, R. N. Biochemistry 1983, 22, 150.

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14. Nemeria, N.; Yan, Y.; Zhang, Z.; Brown, A. M.; Arjunan, P.; Furey, W.; Guest, J. R.; Jordan, F. J. Biol. Chem.
2001, 276, 45969.
15. Nemeria, N.; Baykal, A.; Joseph, E.; Zhang, S.; Yan, Y.; Furey, W.; Jordan, F. Biochemistry 2004, 43, 6565.
16. Arjunan, P.; Sax, M.; Brunskill, A.; Chandrasekhar, K.; Nemeria, N.; Zhang, S.; Jordan, F.; Furey, W. J. Biol.
Chem. 2006, 281, 15296.
17. Ren, Y.; He, J.; Feng, L.; Liao, X.; Jin, J.; Li, Y.; Cao, Y.; Wan, J.; He, H. Bioorg. Med. Chem. 2011, 19, 7501.
18. He, J.; Feng, L.; Li, J.; Tao, R.; Wang, F.; Liao, X.; Sun, Q.; Long, Q.; Ren, Y.; Wan, J. Bioorg. Med. Chem.
2012, 20, 1665.
19. He, J.-B.; Feng, L.-L.; Li, J.; Tao, R.-J.; Ren, Y.-L.; Ren, Y.-L.; Wan, J.; He, H.-W. Bioorg. Med. Chem. 2014,
22, 89.
20. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128.
21. Appendino, G.; Bacchiega, S.; Minassi, A.; Cascio, M. G.; De Petrocellis, L.; Di Marzo, V. Angew. Chem. Int.
Ed. 2007, 119, 9472.
22. Agyei‐Owusu, K.; Leeper, F. J. FEBS J. 2009, 276, 2905.
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Table 1. Structures and E. coli PDHc-E1 inhibitory activities of amide derivatives 6a-6n
Compounds
X
Y
IC50 (µM)
6a
6b
6c
6d
6e
6f
H
H
45.42 4.12
9.45 1.34
12.98 1.86
3.58 0.52
21.59 4.89
11.14 1.07
16.78 0.95
60.81 9.08
16.55 1.64
17.43 4.96
7.60 1.84
4.90 0.81
21.92 3.44
13.42 2.02
2-Cl
H
4-Cl
4-Cl
4-NO2
4-CH3
4-Br
4-OCH3
H
H
H
H
H
6g
6h
6i
2-Br
2-Cl
H
H
4-F
6j
6k
6l
2-Cl
2-Cl
2-CH3
H
4-F
4-NO2
4-Cl
4-CN
6m
6n

Table 2. Structures and E. coli PDHc-E1 inhibitory activity of urea derivatives 8a-8e
Compounds
X
H
H
H
H
H
Y
IC50 (µM)
8a
8b
8c
8d
8e
H
12.88 1.33
9.23 0.80
6.30 0.75
10.33 1.36
3.67 0.38
4-CH3
4-OCH3
4-F
4-Cl

Figure 1. Mechanism of pyruvate dehydrogenase complex (E1) component

Figure 2. Structures of known PDHc-E1 inhibitors
Figure 3. Design of the new amide and urea derivatives as E. coli PDHc-E1 inhibitors

A
B
Figure 4. Binding modes of compound 6d (A) and 8e (B) target into active site of E. coli PDHc-E1, in which
PDHc-E1 is shown in ribbon, ligands and some key residues are shown in stick, both coordination bonds and
hydrogen bonds are shown in dashed lines (green).

o
Scheme 1. Reagents and conditions: (a) NaN3, Na2SO3, H2O, 60-65 C, 6 h; (b) Pd/C, H2, methanol, rt; (c)
o
o
ClCH2COOH, 40% NaOH, 100-110 C, 2-3 h; (d) BrCH2CO2Et, K2CO3, DMSO, 70 C, 5-6 h; (e) 2 N NaOH,
acetone, rt, 2-3 h; (f) 3 or 5, ClCO2Et, Et3N, THF/DMF, rt, 12 h.

Scheme 2. Reagents and conditions: (a) ClCO2Et, Et3N, acetone, -5 oC, 15 min; then NaN3, H2O, -5 oC; 1 h; (b)
toluene, 70 oC, then 2, DMF, 0 oC, 12 h.

Graphical Abstract