Organocatalytic synthesis and sterol 14α-demethylase binding interactions of enantioriched 3-(1H-1,2,4-triazol-1-yl)butyl benzoates

Article abstract of DOI:10.1016/j.bmcl.2009.03.028

1H-1,2,4-Triazole reacted with 2-butenal in the presence of diaryl prolinol silyl ether 3 and benzonic acid to give 3-(1H-1,2,4-triazol-1-yl)butanal 4, which was subsequently reduced and then treated with various acyl chloride to generate enantioriched 3-

Full text of DOI:10.1016/j.bmcl.2009.03.028

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3938–3940
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Organocatalytic synthesis and sterol 14
a
-demethylase binding interactions of
enantioriched 3-(1H-1,2,4-triazol-1-yl)butyl benzoates
b
b
Zhi-Hui Ming a, Sheng-Zhen Xu a, Lei Zhou a, Ming-Wu Ding a, , Jiao-Yan Yang , Shao Yang ,
*
Wen-Jing Xiao a,
*
a Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, Central China Normal University, Wuhan 430079, PR China
b College of Life Sciences, Central China Normal University, Wuhan 430079, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
1H-1,2,4-Triazole reacted with 2-butenal in the presence of diaryl prolinol silyl ether 3 and benzonic acid
to give 3-(1H-1,2,4-triazol-1-yl)butanal 4, which was subsequently reduced and then treated with vari-
ous acyl chloride to generate enantioriched 3-(1H-1,2,4-triazol-1-yl)butyl benzoates 6. Some of triazoles
Received 23 February 2009
Revised 9 March 2009
Accepted 10 March 2009
Available online 14 March 2009
6 exhibited strong binding interactions with the cytochrome P450-dependent sterol 14
a
-demethylase
M.
(CYP51). For example, compound (R)-6f showed the best binding activity with Kd 0.3381
l
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Organocatalysis
Triazole
Michael addition
CYP51
Because of their lower application doses, high selectivity, re-
duced undesired environmental impact and high therapeutic in-
dex, azoles are the most widely used and studied class of
antifungal agents in both agricultural and medicinal usage.1 The
biochemical site of action of the fungicidal azoles is the cyto-
lanosterol 14
a
-demethylase (CYP51) for chiral triazole pesticides
are therefore of particular interest in this study.
Since the rediscovery of the proline-catalyzed aldol reaction,5
organocatalysis has expanded widely within the last few years.6
Many chiral secondary amines are effective catalysts for enantiose-
chrome P450-dependent lanosterol 14
a
-demethylase (CYP51), en-
lective b addition to
case of these ,b-unsaturated systems, the catalyst activates the
substrate through the iminium-ion mechanism, thereby facilitat-
ing the addition of the nucleophile to the b-carbon atom. Recently,
several organocatalyzed nucleophilic nitrogen addition reactions of
a
,b-unsaturated carbonyl compounds.7 In the
coded by the ERG11 gene in fungal cells.2 This haem protein binds
tetracyclic steroid molecules, inserting one oxygen atom into a C–
H bond of the C-14 methyl group.3 Azole antifungal agents inhibit
CYP51 by the mechanism in which the heterocyclic nitrogen atom
(N-3 of imidazole and N-4 of triazole) binds to the heme iron atom
in the binding site of the enzyme. Azoles are fungistatic against
yeasts, but unfortunately, the broad usage of these compounds
leads to the development of resistance. Therefore, the invention
of new and effective antifungal agents is of great importance.
Triazole derivatives such as diniconazole, tebuconazole, hexac-
onazole, triadimefon, triadimenol, and so on represent the most
important category of fungicides to date. These compounds have
excellent protective, curative, and eradicant power toward a wide
spectrum of crop diseases. Structurally, they inherently have a chi-
ral centre which results in two enantiomers. It is well known that
both stereoisomers of above fungicides would display different
fungicidal activities.4 The questions concerning the synthetic
method of active enantiomers and the influence of stereochemistry
upon binding constants (Kd) of the cytochrome P450-dependent
a
N-heterocyclic compounds to
a,b-unsaturated carbonyl com-
pounds have been impressively presented with high enantioselec-
tivities using chiral organocatalysts.8 We extend this
organocatalytic strategy to the design and synthesis of potential
triazole-based antifungal agents.
In previous study, we have carried out cloning, expression, and
inhibition of CYP51 from Magnaporthe grisea and Penicillium digit-
atum. The structural characteristics of the interaction between het-
erologous CYP51 and commercial azoles were also analysed by
binding assay.9 In this Letter, we describe the synthesis of some
new triazole compounds and their binding interactions with the
cytochrome P450-dependent lanosterol 14a-demethylase.
The reaction of 1H-1,2,4-triazole (1) with 2-butenal (2) has been
performed in the presence of racemic, (R)-, and (S)-310 and benzoic
acid, respectively, to prepare racemic and both enantiomeric 4. The
result showed that full conversion to adduct 4 was achieved in tol-
uene at room temperature for 2 h. The product 3-(1H-1,2,4-triazol-
1-yl)butanal (4) was reduced with NaBH4 subsequently to give 3-
* Corresponding authors. Tel.: +86 27 67862041 (W.-J.X.).
E-mail address: wxiao@mail.ccnu.edu.cn (W.-J. Xiao).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.028

Z.-H. Ming et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3938–3940
3939
N
N
N
a
N
b
N
N
+
N
CH3
N
O
N
H
CH3
O
HO
CH3
1
2
4
5
N
N
Ar
c
N
Ar
N
O
H
OTMS
Ar
O
CH3
Ar = 3,5-(CF3)2C6H3
6
3
Scheme 1. Preparation of 3-(1H-1,2,4-triazol-1-yl)butyl benzoates 6. Reagents and
conditions: (a) 10 mol % 3, 10 mol % PhCO2H, toluene, rt; (b) NaBH4, methanol, 70%
in two steps; (c) Acyl chloride, NEt3 (1.5 equiv.), 10 mol % DMAP, dichloromethane.
(1H-1,2,4-triazol-1-yl)butanol (5).11 Further reaction of 5 with acyl
chloride in the presence of NEt3/DMAP produced 3-(1H-1,2,4-tria-
zol-1-yl)butyl benzoates 6 in 53–91% total yields (Scheme 1).12 By
the use of this three-step reaction procedure, the reaction under-
went with good enantioselectivities (72–82% ee) and in high yields
(Table 1).
The binding interactions of triazoles 6 with CYP51 were inves-
tigated with the commercial fungicides triadimefon and Dinico-
nazole as standards.13 Similar to our previous results, the
purified CYP51 revealed a typical absolute absorbance spectrum
of oxidized P450 with Soret maximum at 417 nm and the reduced
CO spectrum had a maximum at 449 nm. UV–visible absorption
spectroscopy provided a simple and accurate method for the deter-
mination of the level of binding of substrates and inhibitors to
P450s. Compound (S)-6c binding with the purified CYP51 was ini-
tially examined at 25 °C and induced a typical type II spectrum
with a spectral peak located at 425 nm and a trough located at
398 nm, respectively (Fig. 1). Higher concentrations of (S)-6c pro-
duced higher absorbance of different values, D425-398, accord-
ingly. These spectra resulted from an interaction of the triazole
N-4 of (S)-6c with the heme of the P450 causing a shift towards
the high-spin state.14 This exhibited the displacement of the native
sixth ligand of the heme iron by the nitrogen atom in the triazole
ring of (S)-6c. The spectral results showed that the affinity of (S)-
6c with the purified CYP51 was tight.
Figure 1. CYP51 type II binding spectrum in the presence of compound (S)-6c. Type
II spectral response to (S)-6c and the concentrations of the (S)-6c added to the
reaction mixture were 0.1, 0.2, 0.4, 0.8, 1, 2, 3 and 8 lM.
The Kd values of some of the compounds 6 are highlighted in Ta-
ble 2. The results showed that some of them exhibited strong bind-
ing interactions with CYP51. As indicated in Table 2, most of the
compounds showed strong associative interactions with CYP51.
Compounds (R)-6f showed the best binding activities with Kd value
of 0.3381
lM. Although these compounds displayed less binding
activities than Diniconazole did (Kd 0.2473
lM), some samples
(e.g., (S)-6b, (R)-6b, (RS)-6c, (S)-6c, (R)-6d, (S)-6e, and (R)-6f) did
indeed exhibit stronger binding activities than triadimefon (Kd
0.9355 lM). The presence of the groups like 3-Cl ((S)-6c), 2,4-Cl2
((S)-6e) and 4-Me ((R)-6f) on the benzene ring plays a significant
role in imparting binding activity. The absolute configuration of tri-
azoles 6 also shows influence on the binding activity. For example,
(S)-6c (Kd 0.4724
2.3568 M), whereas (R)-6f (Kd 0.3381
ger binding activity to CYP51 than (S)-6f (Kd 10.4903
l
M) is much more active than (R)-6c (Kd
M) displays much stron-
M) does.
l
l
l
In conclusion, we have synthesized some enantioriched 3-(1H-
1,2,4-triazol-1-yl)butyl benzoates 6 via organocatalytic Michael
addition and subsequent reduction and functionalization. The pre-
Table 1
Preparation of 3-(1H-1,2,4-triazol-1-yl)butyl benzoates 6
Compd
Ar
Yield (%)
eea (%)
(RS)-6a
(S)-6a
(R)-6a
(RS)-6b
(S)-6b
(R)-6b
(RS)-6c
(S)-6c
(R)-6c
(RS)-6d
(S)-6d
(R)-6d
(RS)-6e
(S)-6e
(R)-6e
(RS)-6f
(S)-6f
Ph
Ph
Ph
72
69
75
61
53
64
84
81
71
82
87
68
86
76
77
91
66
78
80
60
88
74
74
85
76
82
Table 2
In vitro binding constants (Kd, lM) of compounds 6
2-Cl–C6H4
2-Cl–C6H4
2-Cl–C6H4
3-Cl–C6H4
3-Cl–C6H4
3-Cl–C6H4
4-Cl–C6H4
4-Cl–C6H4
4-Cl–C6H4
2,4-Cl2–C6H3
2,4-Cl2–C6H3
2,4-Cl2–C6H3
4-Me–C6H4
4-Me–C6H4
4-Me–C6H4
4-F–C6H4
4-F–C6H4
4-F–C6H4
4-MeO–C6H4
4-MeO–C6H4
4-MeO–C6H4
Compd
Kd
81
78
(R,S)-6a
(S)-6a
(R)-6a
(R,S)-6b
(S)-6b
(R)-6b
(R,S)-6c
(S)-6c
(R)-6c
(R,S)-6d
(S)-6d
(R)-6d
(R,S)-6e
(S)-6e
(R)-6e
(R,S)-6f
(S)-6f
(R)-6f
1.1824
1.4173
1.6183
1.1163
0.5600
0.5801
0.7429
0.4724
2.3568
1.3759
0.9881
0.6824
0.9734
0.4524
/
77
79
79
77
81
77
76
79
(R)-6f
(RS)-6g
(S)-6g
(R)-6g
(RS)-6h
(S)-6h
(R)-6h
82
81
1.7392
10.4903
0.3381
0.9355
0.2473
78
72
Triadimefon
Diniconazole
a
Determined by chiral HPLC.

3940
Z.-H. Ming et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3938–3940
Evaporate the solvent to provide the alcohol which was used in next step
direcltly.
liminary investigation on the biological activities of 6 show that
some of them exhibited strong binding interactions with the cyto-
12. Preparation of 3-(1H-1,2,4-triazol-1-yl)butyl benzoates 6: To 3-(1H-1,2,4-triazol-
1-yl)butanol 5 (3 mmol) in CH2Cl2 (24 mL) was added acyl chloride (4.5 mmol),
Et3N (4.5 mmol) and DMAP (10 mol %, 0.04 g, 0.3 mmol) at 0 °C. The reaction
mixture was stirred overnight and was quenched with satd NaHCO3. The
mixture was extracted with CH2Cl2 several times and dried over K2CO3/MgSO4.
The solvent was evaporated and the pure product was then obtained by flash
chromatography (eluent:PE–Et2O = 3:1 first, and then neat Et2O).3-(1H-1,2,4-
Triazol-1-yl)butyl benzoate 6a: 1H NMR (CDCl3, 400 MHz): d (ppm) 8.13 (s, 1H,
triazolyl-5-H), 7.98 (s, 1H, triazolyl-3-H), 8.06–7.46 (m, 5H, Ar–H), 4.68–4.63
(m, 1H, NCH), 4.36–4.02 (m, 2H, OCH2), 2.44–2.28 (m, 2H, CH2), 1.62 (d,
J = 6.8 Hz, 3H, CH3). 13C NMR (CDCl3, 100 MHz): d (ppm) 165.9, 151.7, 141.9,
132.9, 129.5, 129.2, 128.1, 60.9, 53.1, 35.1, 20.7 MS m/z (%): 246 (M++1, 49), 177
(8), 123 (26), 105 (100), 77 (67). Anal. Calcd for C13H15N3O2: C, 63.66; H, 6.16;
N, 17.13. Found: C, 63.41; H, 6.28; N, 17.21.3-(1H-1,2,4-Triazol-1-yl)butyl 2-
chlorobenzoate 6b: 1H NMR (CDCl3, 600 MHz): d (ppm) 8.14 (s, 1H, triazolyl-5-
H), 7.98 (s, 1H, triazolyl-3-H), 7.78–7.34 (m, 4H, Ar–H), 4.72–4.68 (m, 1H,
NCH), 4.45–4.03 (m, 2H, OCH2), 2.43–2.27 (m, 2H, CH2), 1.61 (d, J = 6.6 Hz, 3H,
CH3). 13C NMR (CDCl3, 150 MHz): d (ppm) 165.2, 151.7, 142.1, 133.1, 132.5,
131.2, 130.8, 129.6, 126.5, 61.5, 53.0, 35.0, 20.7.MS m/z (%): 244 (M+-Cl, 6), 231
(5), 139 (100), 111 (88). Anal. Calcd for C13H14ClN3O2: C, 55.82; H, 5.04; N,
15.02. Found: C, 55.97; H, 4.97; N, 15.27.3-(1H-1,2,4-Triazol-1-yl)butyl 3-
chlorobenzoate 6c: 1H NMR (CDCl3, 600 MHz): d (ppm) 8.12 (s, 1H, triazolyl-5-
H), 7.96 (s, 1H, Ar–H), 7.96 (s, 1H, triazolyl-3-H), 7.87–7.40 (m, 3H, Ar–H),
4.69–4.60 (m, 1H, NCH), 4.34–4.09 (m, 2H, OCH2), 2.48–2.25 (m, 2H, CH2), 1.62
(d, J = 6.6 Hz, 3H, CH3). 13C NMR (CDCl3, 150 MHz): d 164.5, 151.6, 141.8, 134.0,
132.7, 131.1, 129.4, 129.0, 127.2, 61.3, 53.0, 34.9, 20.7 MS m/z (%): 156 (3), 139
(24), 111 (77), 69 (84), 42 (100). Anal. Calcd for C13H14ClN3O2: C, 55.82; H,
5.04; N, 15.02. Found: C, 55.61; H, 4.90; N, 15.16.3-(1H-1,2,4-Triazol-1-yl)butyl
4-chlorobenzoate 6d: 1H NMR (CDCl3, 600 MHz): d 8.12 (s, 1H, triazolyl-5-H),
7.97 (s, 1H, triazolyl-3-H), 7.93–7.42 (m, 4H, Ar–H), 4.67–4.61 (m, 1H, NCH),
4.36–4.12 (m, 2H, OCH2), 2.45–2.26 (m, 2H, CH2), 1.62 (d, J = 6.8 Hz, 3H, CH3).
13C NMR (CDCl3, 150 MHz): d 164.7, 151.4, 141.7, 138.9, 130.4, 128.2, 127.7,
61.0, 52.9, 34.8, 20.5. MS m/z (%): 280 (M++1, 2), 156 (4), 139 (82), 111 (100), 75
(75). Anal. Calcd for C13H14ClN3O2: C, 55.82; H, 5.04; N, 15.02. Found: C, 55.94;
H, 5.17; N, 14.93.3-(1H-1,2,4-Triazol-1-yl)butyl 2,4-dichlorobenzoate 6e: 1H NMR
(CDCl3, 600 MHz): d 8.12 (s, 1H, triazolyl-5-H), 7.97 (s, 1H, triazolyl-3-H), 7.97–
7.33 (m, 3H, Ar–H), 4.67–4.62 (m, 1H, NCH), 4.43 - 4.04 (m, 2H, OCH2), 2.43–
2.23 (m, 2H, CH2), 1.61 (d, J = 6.8 Hz, 3H, CH3). 13C NMR (CDCl3, 150 MHz): d
164.0, 151.4, 141.8, 138.0, 134.2, 132.2, 130.5, 127.5, 126.7, 61.6, 52.9, 34.8,
20.6.MS m/z (%): 173 (5), 145 (6), 109 (19), 69 (62), 42 (100). Anal. Calcd for
C13H13ClN3O2: C, 49.70; H, 4.17; N, 13.38. Found: C, 49.51; H, 4.25; N, 13.52.3-
(1H-1,2,4-Triazol-1-yl)butyl 4-methylbenzoate 6f: 1H NMR (CDCl3, 400 MHz): d
8.14 (s, 1H, triazolyl-5-H), 7.97 (s, 1H, triazolyl-3-H), 7.88–7.25 (m, 4H, Ar–H),
4.65–4.62 (m, 1H, NCH), 4.34–4.07 (m, 2H, OCH2), 2.47–2.27 (m, 5H, CH2 and
CH3), 1.62 (d, J = 6.6 Hz, 3H, CH3). 13C NMR (CDCl3, 100 MHz): d 166.0, 151.7,
143.7, 142.0, 129.3, 128.9, 126.7, 60.7, 53.1, 35.1, 21.4, 20.8.MS m/z (%): 260
(M++1, 59), 191 (8), 119 (100), 91 (52). Anal. Calcd for C14H17N3O2: C, 64.85; H,
6.61; N, 16.20. Found: C, 64.91; H, 6.57; N, 16.43.3-(1H-1,2,4-Triazol-1-yl)butyl
4-fluorobenzoate 6g: 1H NMR (CDCl3, 600 MHz): d 8.14 (s, 1H, triazolyl-5-H),
7.97 (s, 1H, triazolyl-3-H), 8.04–7.12 (m, 4H, Ar–H), 4.64–4.62 (m, 1H, NCH),
4.34–4.08 (m, 2H, OCH2), 2.44 -2.24 (m, 2H, CH2), 1.62 (d, J = 6.6 Hz, 3H, CH3).
13C NMR (CDCl3, 150 MHz): d 166.1, 164.5, 151.4, 141.8, 131.6, 125.6, 115.1,
61.0, 53.1, 34.9, 20.5.MS m/z (%): 264 (M++1, 71), 195 (8), 123 (100), 95 (78).
Anal. Calcd for C13H14FN3O2: C, 59.31; H, 5.36; N, 15.96. Found: C, 59.57; H,
6.25; N, 15.89.3-(1H-1,2,4-Triazol-1-yl)butyl 4-methoxybenzoate 6h: 1H NMR
(CDCl3, 400 MHz): d 8.12 (s, 1H, triazolyl-5-H), 7.97 (s, 1H, triazolyl-3-H), 8.00–
6.93 (m, 4H, Ar–H), 4.72–4.54 (m, 1H, NCH), 4.33–4.03 (m, 2H, OCH2), 3.87 (s,
3H, OCH3), 2.42–2.26 (m, 2H, CH2), 1.62 (d, J = 6.8 Hz, 3H, CH3). 13C NMR
(CDCl3, 150 MHz): d 165.3, 162.9, 151.4, 141.7, 131.0, 121.6, 113.1, 60.4, 54.8,
52.9, 34.9, 20.5.MS m/z (%): 275 (M+, 5), 152 (17), 135 (100), 92 (19). Anal.
Calcd for C13H17N3O3: C, 61.08; H, 6.22; N, 15.26. Found: C, 61.00; H, 6.10; N,
15.32.
chrome P450-dependent lanosterol 14a-demethylase.
Acknowledgements
We are grateful to the Program for Academic Leader in Wuhan
Municipality (200851430486), the National Science Foundation of
China (20772041) and the Program for New Century Excellent Tal-
ents in University (NCET-05-0672) for support of this research.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.03.028.
References and notes
1. (a) Sheehan, D. J.; Hitchcock, C. A.; Sibley, C. M. Clin. Microbiol. Rev. 1999, 12, 40;
(b) Berg, D. In Fungicide Chemistry: Advances and Practical Applications; Green,
M. B., Spilker, D. A., Eds.; ACS Symposium Series 304; American Chemical
Society: Washington, DC, 1986; pp 25–51.
2. (a) Sheng, C.; Zhang, W.; Ji, H.; Zhang, M.; Song, Y.; Xu, H.; Zhu, J.; Miao, Z.;
Jiang, Q.; Yao, J.; Zhou, Y.; Zhu, J.; Lu, J. J. Med. Chem. 2006, 49, 2512; (b) Xiao, L.;
Madison, V.; Chau, A. S.; Loebenberg, D.; Palermo, R. E.; McNicholas, P. M.
Antimicrob. Agents Chemother. 2004, 48, 568; (c) Chen, S. H.; Sheng, C. Q.; Xu, X.
H.; Zhang, W. N.; He, C. Biol. Pharm. Bull. 2007, 30, 1246.
3. Popkov, S. V.; Kovalenko, L. V.; Bobylev, M. M.; Molchanov, O. Y.; Krimer, M. Z.;
Tashchi, V. P.; Putsykin, Y. G. Pestic. Sci. 1997, 49, 125.
4. (a) Upadhayaya, R. S.; Sinha, N.; Jain, S.; Kishore, N.; Chandra, R.; Arora, S. K.
Bioorg. Med. Chem. 2004, 12, 2225; (b) Deas, A. H. B.; Carter, G. A.; Clark, T.;
Clifford, D. R. Pestic. Biochem. Physiol. 1986, 26, 10.
5. List, B.; Lerner, R. A.; Barbas, C. F., III J. Am. Chem. Soc. 2000, 122, 2395.
6. (a) Dalko, P. I.; Moisan, L. Angew. Chem., Int. Ed. 2004, 43, 5138; (b)Asymmetric
Organocatalysis; Berkessel, A., GrGger, H., Eds.; Wiley-VCH: Weinheim,
Germany, 2005; (c)Enantioselective Organocatalysis; Dalko, P. I., Ed.; Wiley-
VCH: Weinheim, Germany, 2007.
7. (a) Anniina, E.; Inkeri, M.; Petri, M. P. Chem. Rev. 2007, 107, 5416; (b) Prieto, A.;
Halland, N.; Jørgensen, K. A. Org. Lett. 2005, 7, 3897; (c) Knudsen, K. R.; Mitchell,
C. E. T.; Ley, S. V. Chem. Commun. 2006, 66; (d) Horstmann, T. E.; Guerin, D. J.;
Miller, S. J. Angew. Chem., Int. Ed. 2000, 39, 3635; (e) Brandau, S.; Landa, A.;
Franzén, J.; Marigo, M.; Jørgensen, K. A. Angew. Chem., Int. Ed. 2006, 45, 4305; (f)
Gotoh, H.; Masui, R.; Ogino, H.; Shoji, M.; Hayashi, Y. Angew. Chem., Int. Ed.
2006, 45, 6853; (g) Marigo, M.; Schulte, T.; Franzén, J.; Jørgensen, K. A. J. Am.
Chem. Soc. 2005, 127, 15710; (h) Bertelsen, S.; Dinér, P.; Johansen, R. L.;
Jørgensen, K. A. J. Am. Chem. Soc. 2007, 129, 1536.
8. (a) Chen, Y. K.; Yoshida, M.; MacMillan, D. W. C. J. Am. Chem. Soc. 2006, 128,
9328; (b) Gandelman, M.; Jacobsen, E. N. Angew. Chem., Int. Ed. 2005, 44, 2393;
(c) Dinér, P.; Nielsen, M.; Marigo, M.; Jørgensen, K. A. Angew. Chem., Int. Ed.
2007, 46, 1983; (d) Carlson, E. C.; Rathbone, L. K.; Yang, H.; Collett, N. D.; Carter,
R. G. J. Org. Chem. 2008, 73, 5155.
9. (a) Zhao, L.; Liu, D.; Zhang, Q.; Zhang, S.; Wan, J.; Xiao, W. FEMS Microbiol. Lett.
2007, 277, 37; (b) Yang, J.; Zhang, Q.; Liao, M.; Xiao, M.; Xiao, W.; Wan, J.; Yang,
S. Pest. Manag. Sci. 2009, 65, 260.
10. Palomo, C.; Mielgo, A. Angew. Chem., Int. Ed. 2006, 45, 7876.
11. Preparation of 3-(1H-1,2,4-triazol-1-yl)butanol 5: A mixture of 2-butenal (2)
(0.6 mL, 7.5 mmol), catalyst 3 (10 mol %, 0.5 mmol, 298.8 mg), toluene (50 mL),
and benzoic acid (10 mol %, 0.5 mmol, 61.1 mg) were introduced into a sample
vial equipped with a magnetic stirring bar. The mixture was stirred for 30 min
at ambient temperature, 1H-1,2,4-triazole (1) (345.4 mg, 5 mmol) was then
added. After 2 h (monitored by TLC), the reaction was diluted with pre-cooled
MeOH (10 mL) and NaBH4 (0.3 g, 8 mmol) was added. After 1 h, the reaction
was quenched with NH4Cl (satd). The mixture was extracted with CH2Cl2
several times and dried over MgSO4. The solvent was evaporated and the
product was purified by flash chromatography (first Et2O and then EtOAc).
13. The E. coli membrane fragments containing expressed fungal P. digitatum
CYP51 were used for binding spectra analysis. Binding assays to P. digitatum
CYP51 were repeated three times. Azole binding spectra were recorded on an
S3100 UV–visible scanning spectrophotometer (Sinco, Korea) as described by
the following reference: Venkateswarlu, K.; Denning, D. W.; Manning, N. J.;
Kelly, S. L. Antimicrob. Agents Chemother 1996, 40, 1382.
14. Yoshida, Y.; Aoyama, Y. Biochem. Pharmacol. 1987, 36, 229.