Synthesis, bioactivity, theoretical and molecular docking study of 1-cyano-N-substituted-cyclopropanecarboxamide as ketol-acid reductoisomerase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2007.04.003

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes the second common step in branched-chain amino acid biosynthesis. The catalyzed process consists of two stages, the first of which is an alkyl migration from one carbon atom to its neighbouring ato

Full text of DOI:10.1016/j.bmcl.2007.04.003

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3784–3788
Synthesis, bioactivity, theoretical and molecular docking study of
1-cyano-N-substituted-cyclopropanecarboxamide as ketol-acid
reductoisomerase inhibitor
Xing-Hai Liu, Pei-Quan Chen, Bao-Lei Wang, Yong-Hong Li,
Su-Hua Wang and Zheng-Ming Li*
The Research Institute of Elemento-Organic Chemistry, The State-Key Laboratory of Elemento-Organic Chemistry,
National Pesticide Engineering Research Center, Nankai University, Tianjin 300071, China
Received 4 December 2006; revised 15 March 2007; accepted 2 April 2007
Available online 6 April 2007
Abstract—Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes the second common step in branched-chain amino acid
biosynthesis. The catalyzed process consists of two stages, the first of which is an alkyl migration from one carbon atom to its
neighbouring atom. The likely transition state is a cyclopropane derivative, thus a series of new cyclopropane derivatives, such
1
as 1-cyano-N-substituted-cyclopropanecarboxamide, were designed and synthesized. Their structures were verified by H NMR,
FTIR spectrum, MS and elemental analysis. The Ki values of active compounds 2, 4b against rice KARI were 95.30 13.71,
207.9 21.99 lM, respectively. The X-ray crystal structure of compound 4a was also determined. Auto-Dock was used to predict
the binding mode of 4a. This was done by analyzing the interaction of the compounds 4a with the active sites of spinach KARI. This
result was in accord with the result analyzed by the frontier molecular orbital theory.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Plants and most micro-organisms have biosynthetic
ability which allows them to survive on relatively simple
nutrients. For this reason, plants and microorganisms
contain numerous enzymes that are potential targets
for designing bioactive compounds such as herbicides
and antibiotics. Enzymes involved in the biosynthesis
of the branched chain amino acids are one such exam-
ple. Isoleucine and valine are synthesized in a parallel
set of four reactions while an extension of the valine
pathway results in leucine.1
of two steps,7,8 an alkyl migration followed by a
NADPH dependent reduction. Both steps require a
divalent metal ion, such as Mg2+, Mn2+ or Co2+, but
the alkyl migration is highly specific for Mg2+. HOE
7049 and IpOHA10 are potent competitive inhibitors of
the enzyme (Scheme 1).
A transition state being a cyclopropane is postulated
and mimicked by Gerwick et al.11 They showed that
cyclopropane-1,1-dicarboxylate (CPD) can inhibit Esch-
erichia coli KARI. They also showed that application of
CPD to various plant tissues caused the accumulation of
the substrate 2-acetolactate; in vivo data strongly sug-
gest that the CPD can inhibit the activity of KARI
Scheme 1.12
This pathway is the target for the sulfonylureas,2 the
imidazolinones3 and a variety of other herbicides,1
which all inhibit the first enzyme, acetohydroxyacid syn-
thase. The success of these herbicides has stimulated re-
search into inhibitors of other enzymes in the pathway,
including the second enzyme in the common pathway,4
ketol-acid reductoisomerase (KARI; EC 1.1.1.86), and
two enzymes in the leucine extension.5,6 The reaction
catalyzed by KARI is shown in Scheme 1 which consists
The first step in the KARI catalyzed process involves an
alkyl migration from one carbon atom to its neighboring
atom. The likely transition state is a cyclopropane deriv-
ative. For this reason, some new cyclopropane derivatives
were synthesized in our laboratory (Scheme 2).
Keywords: Cyclopropanecarboxamide; Synthesis; Structure; Molecular
docking; Theoretical study; KARI.
Biological studies revealed that some of these com-
pounds inhibit ketol-acid reductoisomerase in vivo
effectively.
*
Corresponding author. Tel.: +86 22 23503732; fax: +86 22 2350
5948; e-mail: nkzml@vip.163.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.003

X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3784–3788
3785
CH3
P
CH3
H
C
-
-
H3C
C
N
C
O
CO2
IpOHA
H3C
CO2
HOE 704
H
OH
O
OH
Acetohydroxyacid
Methylhydroxyketolacid
Dihydroxyacid
R
R
R
CH2
CH2
M2+
CH2
H
Mg2+
-
-
-
H3C
C
C
O
CO2
H3C
C
O
C
CO2
H3C
C
C
CO2
R
OH
OH OH
OH
NADP+
NADPH
+ H+
H2C
-
H3C
C
O
C
CO2
O
Transition state
H
Scheme 1. Reaction catalyzed by KARI.
Table 1. Inhibition rate (%) of compounds 4a–p against rice KARI at
200 ppm in vitro
reflux
O
COOCH2CH3
CN
Cl
NC
Cl
O
Compound
R
KARI activity
1
1
0
1M NaOH
EtOH
SOCl2
PhH
CN
CN
2
100
61.21
100
32.23
69.81
0
COCl
COOH
4a
4b
4c
4d
4e
4f
4g
4h
4i
p-CH3C6H4–
2-CHCl2C2N2S–
p-BrC6H4–
2
3
CN
Aryl, Heterocyclic
Alkyl
NH
R
2-CH3C3HNS–
2,4,5-Cl3C6H2–
m-BrC6H4–
O
4a~4p
17.25
77.23
97.04
100
93.92
98.92
0
R=p-CH3C6H4, 2-CHCl2C2N2S, p-BrC6H4, 2-CH3C3HNS,
2,4,5-Cl3C6H2, m-BrC6H4, C6H5, 2,4-Cl2C6H3, o-CH3C6H4
p-ClC6H4, OHCH2CH2, p-CF3C6H4, m-ClC6H4
o-CF3C6H4, m-CF3C6H4, p-OCH3C6H4
C6H5–
2,4-Cl2C6H3–
o-CH3C6H4–
p-ClC6H4–
4j
Scheme 2. Synthesis route for compounds 4a–p.
4k
4l
OHCH2CH2–
p-CF3C6H4–
m-ClC6H4–
The 1-cyan-1-cyclopropane carboxylic acid, prepared
from 1,2-dichloroethane and ethyl cyanacetate was cyc-
lized for 16 h at refluxing temperature. In order to opti-
mize the reaction time, microwave assisted irradiation
was applied which shortened the reaction time to
40 min. Compound 3 reacted with substituted anilines,
heterocyclic amine or alkyl amines in the presence of
inorganic base to yield substituted cyclopropanecarbox-
amides as shown in Scheme 2.13
4m
4n
4o
4p
0
o-CF3C6H4–
m-CF3C6H4–
p-OCH3C6H4–
0
0
3.95
In order to study the structure–activity relationship, the
single-crystal structure of 4a was determined15 by X-ray
crystallography16 as illustrated in Figure 1 in which
three C–N bond lengths C(5)–N(2), C(6)–N(2) and
N(1)–C(1) are 0.135, 0.142, and 0.114 nm, respectively,
which are all longer than that of 0.134 nm in the single
heterocycle ring.17 In 4a, the bond length of C(1)–N(1)
is 0.1396 nm, which is longer than the double C–N
bond.18 Based on the computal results by Gaussian,19,20
it was seen that DFT, HF and MP2 have good coher-
ence with the crystal diffraction, for example it can be
observed that C(2)–C(5) > C(1)–C(2) > N(2)–C(6) >
N(2)–C(5) >O(1)–C(5) > N(1)–C(1) in crystal structure,
which is accordance with the order of C(2)–C(5)
>C(1)–C(2) > N(2)–C(6) > N(2)–C(5) > O(1)–C(5) >
N(1)–C(1) in all calculation structures.
The KARI activities in vitro of these compounds were
determined.14 The results for compound, 1, 2 and com-
pounds 4a–p are summarized in Table 1.
It was found from Table 1 that compounds 2, 4b, 4h, 4i,
4j and 4k have favourable inhibitory activity against
KARI. The data given in Table 1 indicated that the
change of substituent at phenyl ring affects the KARI
activity. When the benzene ring is substituted by CF3
group, the compounds generally have no KARI bioac-
tivity, as 4l, 4n, 4o. While for heterocyclic and alkane
substituents, their inhibitory activities increase for 4b
and 4k. For the compounds, 2, 4a and 4b, further bioas-
say was conducted and their Ki values against KARI
were 95.30 13.71, >300 and 207.9 21.99 lM, respec-
tively. Hence, these identified cyclopropane derivatives
could be useful for further optimization work in finding
the potential KARI inhibitors.
According to the frontier molecular orbital theory,
HOMO and LUMO are two most important factors
which affect the bioactivities of compounds. HOMO
has the priority to provide electrons, while LUMO
accepts electrons first.21,22 Thus, study on the frontier

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X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3784–3788
Figure 1. Molecular structure of 4a.
orbital energy can provide some useful information for
the active mechanism. Taking HF (Hatree–Fork)
results, the geometry of the frame of 4a is hardly influ-
enced by the introduction of either the cyano group or
the cyclopropane ring from Figure 2. The HOMO of
4a is mainly located on aromatic ring and the amide
group. On the other hand, the LUMO of 4a contains
aromatic ring, the amido group, the cyano group and
the cyclopropane ring. The fact that 4a has strong affin-
ity suggests the importance of the frontier molecular
orbital in the p–p stacking or hydrophobic interactions.
This also implies that the orbital interaction between 4a
and the rice KARI amino acid residues is dominated by
p–p or hydrophobic interaction between the frontier
molecular orbitals.
To make the results predicted by our frontier molecular
orbital model more relevant to the active sites of the en-
zyme and to further explore a probable binding site in
the KARI, the compound 4a was docked21,22 into the
active sites of KARI.23
Figure 3. Binding modes of compound 4a in the active sites of spinach
KARI: (a) p–p stacking interaction between the His 226 side chain and
phenyl ring; (b) hydrogen bond and hydrophobic interaction between
4a and the rice KARI amino acid residues.
Visual inspection of the conformation of 4a docked into
the KARI binding site revealed that the phenyl rings are
hosted in the pocket of KARI and are oriented to estab-
lish p–p stacking interactions with the His 226 side
chains (Fig. 3a). Moreover, two hydrogen bonds be-
tween the amino groups of 4a and the carbonyl oxygen
of Glu 319 and Asp 315 side chain are also observed.
Furthermore, the cyclopropane ring and aromatic ring
are embedded in a large hydrophobic pocket formed
by Ser 225, His 226, Glu 496, Leu 323, Ser 518, Glu
319 and Asp 315 (Fig. 3b).
Acknowledgments
This work was supported by the National Basic
Research Key Program of China (Grant No. 2003
CB114406), the National Natural Science Foundation
Figure 2. Frontier molecular orbitals of compound 4a: (a) HOMO of
compound 4a; (b) LUMO of compound 4a.

X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3784–3788
3787
810 cmÀ1, ESI-MS (41 eV): m/z: 199.6, 65.96.
Key Project of China (Grant No. 20432010) and the
High Performance Computing Project of Tianjin Minis-
try of Science and Technology of China (Grant No.
043185111-5).
Compound 4b. A white solid, yield 82%; mp 109–117 ꢁC;
1HNMR (CDCl3, 300M) 1.73–1.80 (m, 4H, CH2), 6.26 (s,
1H, hetero-H), 7.02 (s, 1H, NH); IR (cmÀ1) 3336, 3184,
3077, 2991, 2268, 1731. ESI-MS: 273.48, 230.08, 204.43,
141.20.
Compound 4c. A white solid, yield 93%; mp 106–108 ꢁC;
1HNMR (CDCl3, 300 M) 1.59–1.72 (m, 4H, CH2), 7.15–
7.39 (m,J = 8.784 Hz, 4H, ArH), 8.05 (s, 1H, NH); IR
(cmÀ1) 3347, 3093, 2236, 1692, 1598, 1525, 1489, 814. ESI-
MS: 264.98, 197.95, 116.02, 80.99. Elemental analysis: C,
49.80; H, 3.28; N, 10.58; calculated from C12H9BrN2O.
Observed: C, 49.84; H, 3.42; N, 10.57.
Compound 4d. A white solid, yield 93%; mp 106–108 ꢁC;
1HNMR (CDCl3, 300 M)1.26(s,3H,CH3), 1.73–1.94(m,
4H, CH2), 7.81(d,1H, Heterocycle); IR (cmÀ1) 3450,
3077, 2991, 2855, 2268, 1731.ESI-MS: 206.37.
References and notes
1. Duggleby, R. G.; Pang, S. S. J. Biochem. Molec. Biol.
2000, 33, 1.
2. Chaleff, R. S.; Mauvais, C. J. Science 1984, 224, 1443.
3. Shaner, D. L.; Anderson, P. C.; Stidham, M. A. Plant
Physiol. 1984, 76, 545.
4. Dumas, R.; Biou, V. F.; Douce, H. R.; Duggleby, R. G.
Acc. Chem. Res. 2001, 34, 399.
5. Wittenbach, V. A.; Teaney, P. W.; Rayner, D. R.; Schl
oss, J. V. Plant Physiol. 1993, 102, 50.
Compound 4e. A white solid, yield 93%; mp 106–108 ꢁC;
1HNMR (CDCl3, 300M) 1.62–1.84 (m, 4H, CH2), 7.52 (s,
6. Hawkes, T. R.; Cox, J. M.; Fraser, T. E. M.; Lewis, T. Z.
Naturforsch. 1993, C 48, 364.
1H, ArH), 8.50 (s, 1H, ArH), 8.68(s, 1H, NH); IR (cmÀ1
)
3369, 3122, 3020, 2229, 1692, 1576, 1496, 1456, 879. ESI-
MS: 287.22, 219.95, 253.84, 222.10, 183.72. Elemental
analysis: C, 45.82; H, 2.21; N, 9.41; calculated from
C11H7Cl3N2O. Observed: C, 45.63; H, 2.44; N, 9.67.
Compound 4f. A white solid, yield 93%; mp 106–108 ꢁC;
1HNMR (CDCl3, 300M) 1.61–1.83 (m, 4H, CH2), 7.15 (d,
J = 7.573 Hz, 1H, ArH), 7.31 (d, J = 3.893 Hz, 1H, ArH),
7.66 (d, J = 1.938 Hz, 1H, ArH), 8.50 (s, 1H, ArH), 8.68(s,
7. Dumas, R.; Butikofer, M. C.; Job, D.; Douce, R.
Biochemistry 1995, 34, 6026.
8. Chunduru, S. K.; Mrachko, G. T.; Calvo, K. C. Biochem-
istry 1989, 28, 486.
9. Schulz, A.; Sponemann, P.; Kocher, H.; Wengenmayer, F.
FEBS Lett. 1988, 238, 375.
10. Aulabaugh, A.; Schloss, J. V. Biochemistry 1990, 29, 2824.
11. Gerwick, B. C.; Mireles, L. C.; Eilers, R. J. Weed Technol.
1993, 7, 519.
1H, NH); IR (cmÀ1
) 3320, 3195,3115, 2244, 1680,
1590,1527, 1476, 883,810,766,656.
12. Lee, Y. T.; Ta, H. T.; Duggleby, R. G. Plant Sci. 2005,
168, 1035.
Compound 4g. A white solid, yield 93%; mp 106–108 ꢁC;
1HNMR (CDCl3, 300M) 1.59–1.81 (m, 4H, CH2), 7.18 (t,
J = 6.848 Hz, 1H, ArH), 7.36 (t, J = 7.933 Hz, 2H, ArH),
7.50 (d, J = 8.293 Hz, 2H, ArH), 8.03 (s, 1H, NH); IR
(cmÀ1) 3249, 3191,3127, 2239, 1680, 1595,1536, 1488,
751,693. ESI-MS: 185.41, 65.97. Elemental analysis: C,
70.94; H, 5.41; N, 15.39; calculated from C11H10N2O.
Observed: C, 70.95; H, 5.41; N, 15.04.
Compound 4h. A white solid, yield 93%; mp 106–108 ꢁC;
1HNMR (CDCl3, 300M) 1.63–1.82 (m, 4H, CH2), 7.28 (d,
J = 2.270 Hz, 1H, ArH), 7.44 (d, J = 2.297 Hz, 1H, ArH),
7.28 (d, J = 8.888 Hz, 1H, ArH), 8.67(s, 1H, NH); IR
(cmÀ1) 3398, 3115, 2236, 1699, 1583, 1510, 959, 923, 821,
727. ESI-MS: 253.29, 185.98, 149.81, 114.05. Elemental
analysis: C, 51.70; H, 3.21; N, 10.79; calculated from
C11H8Cl2N2O. Observed: C, 51.79; H, 3.16; N, 10.98.
Compound 4i. A white solid, yield 93%; mp 106–108 ꢁC;
1HNMR (CDCl3, 300M) 1.59–1.81 (m, 4H, CH2), 7.08 (t,
J = 6.815 Hz, 2H, ArH), 7.18 (d,J = 5.187 Hz, 1H, ArH),
7.77 (d, J = 8.998 Hz, 1H, ArH), 7.99 (s, 1H, NH); IR
(cmÀ1) 3435, 3105,3019, 2225, 1695, 1588, 1531,1459, 758.
ESI-MS: 200.15, 171.18, 106.16, 77.11.
Compound 4j. A white solid, yield 93%; mp 106–108 ꢁC;
1HNMR (CDCl3, 300 M) 1.62–1.81 (m, 4H, CH2), 7.32 (d,
J = 8.794 Hz, 2H, ArH), 7.47 (d, J = 8.856 Hz, 2H, ArH),
8.03 (s, 1H, NH); IR (cmÀ1) 3331, 3120,2941, 2247, 1667,
1593, 1534,1490, 832.ESI-MS: 219.42, 152.06, 116.04.
Elemental analysis: C, 59.66; H, 3.96; N, 12.58; calculated
from C11H9ClN2O. Observed: C, 59.88; H, 4.11; N, 12.70.
Compound 4k. A white solid, yield 93%; m.p. 106–108 ꢁC;
1HNMR (CDCl3, 300 M) 1.24 (t, 1H, OH), 1.51–1.71(m,
4H, CH2), 3.62 (t, 2H, J = 5.380 Hz, NH–CH2), 4.17 (q,
13. General procedure: ethyl cyanacetate (22.6 g, 0.2 mol),
1,2-dichloroethane (160 g, 0.2 mol), potassium carbonate
(220 g, 1.6 mol) and catalytic amount of Bu4NHSO4
(1.0 g) were vigorously refluxed in 1,2-dichloroethane for
6 h after which the reaction mixture was poured into water
(800 mL). The product was extracted with ether
(5· 100 mL), combined extracts were dried over MgSO4
and then the solvent was removed on a rotary evaporator
and the residue was distilled under pressure: bp 115–118/
15 mmHg. Yield 85%. 1H NMR(d, CDCl3):1.30–1.34 (t,
J = 7.14 Hz, 3H, CH3), 1.59–1.69 (m, J = 3.27 Hz, 4H,
cyclopropane–CH2), 4.21–4.27 (q, J = 7.13 Hz, 2H, CH2).
An ester (0.03 mol) was added to a ca. 15% aqueous
solution containing 3 mol equivalents of sodium hydrox-
ide and the suspension was vigorously stirred at ambient
temperature for 2 days until a homogeneous solution was
formed. The solution was extracted with ether (2· 50 mL)
to remove traces of unreacted ester, the water phase was
acidified with concentrated hydrochloric acid and a free
acid was extracted with ether (3· 100 mL). The combined
extracts were dried over MgSO4 and then the solvent was
removed on a rotary evaporator. Yields 51%. Mp 88–
90 ꢁC.
To a benzene solution (25 mL) of cyanocyclopropane-
carboxylic acid (7.50 mmol) was added thionyl chloride
(30 mmol) and the mixture was refluxed for 2 h to give
acid chloride.
Preparation of compound 4a. Dropwised the acid chloride
to substituted p-toluidine (7.50 mmol), then vigorously
stirred at ambient temperature for 4 h. The yield was 84%
with mp (106–108) ꢁC; 1H NMR (CDCl3, 300 MHz) d:
1.58–1.79 (m, 4H, CH2), 2.92 (s, 3H, CH3), 7.15–7.39 (m,
4H, ArH), 7.96 (s, 1H, NH); 13C NMR (CDCl3, 300 MHz)
d: 14.35 (s, 2C, CH2), 18.44 (s, 1C, C), 21.13 (s, 1C, CH3),
120.30 (s, 1C, CN), 120.81 (s, 2C, ArC), 129.82 (s, 2C,
ArC), 163.48 (s, 1C, CO), 135.40 (d, 1C, ArC); IR (KBr) m:
3338 (–NH), 3107, 3035, 2920 (cyclopropane, CH), 2243
(C ꢀ N), 1898 (C@O), 1689,1602, 1523 (Ar C–C),
2H, J = 5.239 Hz, OH–CH2), 6.88 (s, 1H, NH); IR (cmÀ1
3356, 3195,3115, 2251, 1698, 1588, 1534,1486, 832.
Compound 4l. A white solid, yield 93%; mp 106–108 ꢁC;
1HNMR (CDCl3, 300 M) 1.64–1.84 (m, 4H, CH2), 7.60 (d,
J = 9.086 Hz, 2H, ArH), 7.65 (d, J = 9.071 Hz, 2H, ArH),
8.17 (s, 1H, NH); IR (cmÀ1) 3500,3327, 3120, 2239, 1674,
1617, 1567,1495, 808. ESI-MS: 287.34(M+Na), 160.33.
)

3788
X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3784–3788
Compound 4m. A white solid, yield 93%; mp 106–108 ꢁC;
of the title compound. Vibration analysis showed that the
optimized structures were in accordance with the mini-
mum points on the potential energy surfaces, which means
no virtual frequencies, proving that the obtained opti-
mized structures were stable. All the convergent precisions
were the system default values, and all the calculations
were carried out on the Nankai Stars supercomputer at
Nankai University.
1HNMR (CDCl3, 300 M) 1.63–1.83 (m, 4H, CH2), 7.47
(dd, J = 7.530 Hz, 2H, ArH), 7.65 (d, J = 7.631 Hz, 1H,
ArH), 7.89 (s, 1H, ArH), 8.12 (s, 1H, NH); IR (cmÀ1
)
3313, 3198,3120, 2261, 1681, 1602, 1538,1474, 872,
765,672. ESI-MS: 219.33, 152.02.
Compound 4n. A white solid, yield 93%; mp 106–108 ꢁC;
1HNMR (CDCl3, 300 M) 1.61–1.82 (m, 4H, CH2), 7.55 (d,
J = 7.361 Hz, 1H, ArH), 7.65 (dd, J = 7.793 Hz, 1H,
ArH), 7.94 (d, J = 8.150 Hz, 1H, ArH), 8.48 (d,
20. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G.
E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A.
Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.;
Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.;
Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda,
R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.;
Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.;
Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.;
Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A.
J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.;
Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J.
J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain,
M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.;
Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.;
Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.;
Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P.-
M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez,
C.; Pople, J. A. Gaussian 03, Revision C. 01, Gaussian,
Inc., Wallingford CT, 2004.
J = 8.065 Hz, 1H, ArH), 6.64 (s, 1H, NH); IR (cmÀ1
)
3312, 3188,3115, 2244, 1680, 1593, 1527,1476, 766. ESI-
MS:285.63(M+Na), 194.94, 117.05.
Compound 4o. A white solid, yield 93%; mp 106–108 ꢁC;
1HNMR (CDCl3, 300 M) 1.61–1.81 (m, 4H, CH2), 7.15
(m, J = 7.626 Hz, 1H, ArH), 7.30(dd, J = 7.894 Hz, 1H,
ArH), 7.51 (d, J = 8.841 Hz, 1H, ArH), 7.66 (d,
J = 1.914 Hz, 1H, ArH), 8.05 (s, 1H, NH); IR (cmÀ1
)
3334, 3127,3062, 2247, 1688, 1602, 1531,1476, 844.ESI-
MS:253.22,186.20, 166.02, 145.82. Elemental analysis: C,
59.41; H, 4.12; N, 12.13; calculated from C11H9ClN2O.
Observed: C, 59.88; H, 4.11; N, 12.70.
Compound 4p. A white solid, 93% yield; mp 106–108 ꢁC;
1HNMR (CDCl3, 300M) 1.57–1.81 (m, 4H, CH2), 3.80 (s,
3H, CH3), 6.88 (d, J = 9.015 Hz, 2H, ArH), 7.04(d,
J = 9.001 Hz, 2H, ArH), 7.94 (s, 1H, NH); IR (cmÀ1
)
3334, 3195,3115, 2251, 1680, 1600, 1534,1490, Elemental
analysis: C, 66.59; H, 5.51; N, 12.96; calculated from
C12H12N2O2. Observed: C, 66.65; H, 5.59; N, 12.96.
14. KARI activity was measured by following the decrease in
A340 at 30 ꢁC in solutions containing 0.2 mM NADPH,
1 mM MgCl2, substrate (2-acetolactate or hydroxypyru-
vate) and CPD or other inhibitors as required, in 0.1 M
Tris–HCl, pH 8.0. The reaction was started by adding the
enzyme except for inhibitor preincubation experiments,
where the substrate was added last.
21. (a) Ma, H. X.; Song, J. R.; Xu, K. Z.; Hu, R. Z.; Zhai, G.
H.; Wen, Z. Y.; Yu, K. B. Acta Chem. Sinica 2003, 61,
1819; (b) Ma, H. X.; Song, J. R.; Hu, R. Z.; Li, J. Chin. J.
Chem. 2003, 21, 1558.
22. All docking procedures were done in NanKai Stars
supercomputer at Nankai University. The automated
molecular docking calculations were carried out using
AutoDock 3.05. The AUTOTORS module of AutoDock
defined the active torsions for each docked compound.
The active sites of the protein were defined using
AutoGrid centred on the IpOHA in the crystal struc-
ture. The grid map with 60 · 60 · 60 points centred at
the center of mass of the KARI and a grid spacing of
15. Crystal data of 4a. C12H12N2O, M = 200.24, Monoclinic,
a = 7.109(4),
˚
c = 11.505(6) A,
b = 13.758(7),
˚
b =
3
102.731(8)ꢁ, V = 1097.6(9) A , T = 294(2) K, space group
P2(1)/c, Z = 4, Dc = 1.212 g/cm3,
l
(Mo–Ka) =
0.71073 mmÀ1, F (000) = 424. 6109 reflections measured,
2290 unique (Rint = 0.0294), which were used in all
calculation. Fine R1 = 0.0490, wR (F2) = 0.1218 (all data).
Full crystallographic details of 4a have been deposited at
the Cambridge Crystallographic Data Center and allo-
cated the deposition number CCDC 612888.
˚
0.375 A was calculated using the AutoGrid program to
evaluate the binding energies between the inhibitors and
the protein. The Lamarckian genetic algorithm (LGA)
was used as a searching method. Each LGA job
consisted of 50 runs, and the number of generation in
each run was 27000 with an initial population of 100
˚
individuals. The step size was set to 0.2 A for translation
and 5ꢁ for orientation and torsion. The maximum
number of energy evaluations was set to 15,00,000.
Operator weights for cross-over, mutation and elitism
were 0.80, 0.02 and 1, respectively. The docked com-
plexes of the inhibitor-enzyme were selected according to
the criterion of interaction energy combined with
geometrical and electronic matching quality.
16. (a) Bruker, SMART., Bruker AXS Inc., Madison, Wis-
consin, USA, 1998; (b) Sheldrick G.M., SHELXS97 and
SHELXL97., University of Go¨ttingen, Germany, 1997; (c)
Bruker, SAINT and SHELXTL. Bruker AXS Inc., Mad-
ison, Wisconsin, USA, 1999.
17. Dmitry, S.; Yufit, M. J. T.; Judith, A. K. H. Acta Cryst.
2006, E, 62, o1237.
18. Zhao, G. L.; Feng, Y. L.; Liu, X. H. Chin. J. Inorg. Chem.
2005, 21, 598.
19. According to the above crystal structure, a crystal unit was
selected as the initial structure, while HF/6-31G (d,p),
DFT-B3LYP/6-31G(d,p) and MP2/6-31G(d,p) methods in
Gaussian 03 package were used to optimize the structure
23. Biou, V.; Dumas, R.; Cohen-Addad, C.; Douce, R.; Job,
D.; Pebay-Peyroula, E. EMBO J. 1997, 16, 3405.