Synthesis and antifungal activity of a series of N-substituted [2-(2,4-dichlorophenyl)-3-(1,2,4-triazol-1-yl)]propylamines

Article abstract of DOI:10.1021/jf071631g

A series of N-mono- or N,N-disubstituted [2-(2,4-dichlorophenyl-3-(1,2,4- triazol-1-yl)]propylamines and N-[2-(2,4-dichlorophenyl-3-(1,2,4-triazol-1-yl) propyl]amides were synthesized and tested for their fungicidal activity in vitro and in vivo against a group of plant pathogenic fungi. Some compounds exhibited a fairly good in vitro activity. The replacement of the ether group of tetraconazole with a secondary or tertiary amino group leads to compounds that maintain the antifungal activity on several phytopathogenic fungi, provided that the substituents are not too bulky or lipophilic. The allyl, propargyl, and cyclopropyl groups appear particularly suitable. Although these compounds have some structural similarities with terbinafine and naftifine, which act as squalene epoxidase inhibitors, they maintain the usual mechanism of action of the other triazoles.

Full text of DOI:10.1021/jf071631g

J. Agric. Food Chem. 2007, 55, 8187–8192 8187
Synthesis and Antifungal Activity of a Series of
N-Substituted
[2-(2,4-Dichlorophenyl)-3-(1,2,4-triazol-1-yl)]propylamines
ANNA ARNOLDI,† SABRINA DALLAVALLE,*,† LUCIO MERLINI,† LOANA MUSSO,†
§
GANDOLFINA FARINA,‡ MADDALENA MORETTI,‡ AND LALITH JAYASINGHE
Dipartimento di Scienze Molecolari Agroalimentari and Istituto di Patologia Vegetale, Università di
Milano, Via Celoria 2, 20133 Milano, Italy, and Institute of Fundamental Studies, Hantana Road,
Kandy, Sri Lanka
A series of N-mono- or N,N-disubstituted [2-(2,4-dichlorophenyl-3-(1,2,4-triazol-1-yl)]propylamines
and N-[2-(2,4-dichlorophenyl-3-(1,2,4-triazol-1-yl)propyl]amides were synthesized and tested for their
fungicidal activity in vitro and in vivo against a group of plant pathogenic fungi. Some compounds
exhibited a fairly good in vitro activity. The replacement of the ether group of tetraconazole with a
secondary or tertiary amino group leads to compounds that maintain the antifungal activity on several
phytopathogenic fungi, provided that the substituents are not too bulky or lipophilic. The allyl, propargyl,
and cyclopropyl groups appear particularly suitable. Although these compounds have some structural
similarities with terbinafine and naftifine, which act as squalene epoxidase inhibitors, they maintain
the usual mechanism of action of the other triazoles.
KEYWORDS: Triazole; ergosterol biosynthesis inhibitors; fungicide; synthesis; tetraconazole
INTRODUCTION
Although 1 is characterized by an efficacy against biotrophes
higher than that of most commercial compounds (7, 10), only
a few analogues of this fungicide have been synthesized and
tested for their activity (11).
The agrochemical industry is continuously searching for new
active pesticide compounds. The main goal of this research is
to develop substances with lower application doses, increased
selectivity and reduced undesired ecological impact (1).
Azole fungicides were synthesized for the first time in the
early 1970s and quickly gained a great importance in the
protection of various crops, because they represented a signifi-
cant progress in the chemical control of fungal diseases. In fact,
this class includes several excellent systemic fungicides with
long protective and curative activity against a broad spectrum
of foliar, root, and seedling diseases caused by many Asco-
mycetes, Basidiomycetes, and Deuteromycetes (1). Their mech-
anism of action is related to the inhibition of the sterol
biosynthesis (2, 3) in fungi. In the past decades this class of
fungicides has been thoroughly explored and many active
structures have been reported (4–6).
Tetraconazole (1) (Eminent, 2-(2,4-dichlorophenyl)-3-(1H-
1,2,4-triazol-1-yl)-propyl-1,1,2,2-tetrafluoroethylether) is cur-
rently among the most employed agricultural fungicides. The
mechanism of action of 1 has been studied in detail (refs 7–9
and preceding papers quoted therein), but little is known about
the structure–activity relationships of tetraconazole analogues.
In a previous paper, we reported studies of a series of ethers
(2) related to tetraconazole (11). A QSAR study showed that
for this class of compounds lipophilicity was a major positive
parameter affecting the activity. To gain a further insight into
the most relevant physicochemical features affecting fungitox-
icity in tetraconazole analogues, we planned to replace the ether
oxygen with an amino group. The advantage of such a
replacement could be the functionalization of the nitrogen atom
with either one or two alkyl and arylalkyl chains differing in
size, shape, and lipophilicity. In addition, as the designed amines
showed structural similarity with the antifungal allylamines, such
as terbinafine or naftifine (12), which act as inhibitors of
squalene epoxidase, we investigated whether our compounds
might also exhibit such a mechanism of inhibition.
* To whom correspondence should be addressed. Phone: +39-2-
50316818. Fax: +39-2-50316801. E-mail: sabrina.dallavalle@unimi.it.
† Dipartimento di Scienze Molecolari Agroalimentari.
‡ Istituto di Patologia Vegetale.
Moreover, the presence of the amino group allowed us to
prepare some representative amido derivatives and to evaluate
the effect of this moiety on the fungicidal activity. Here we
§ Institute of Fundamental Studies.
10.1021/jf071631g CCC: $37.00
2007 American Chemical Society
Published on Web 10/03/2007

8188 J. Agric. Food Chem., Vol. 55, No. 20, 2007
Arnoldi et al.
[2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propyl]prop-2-ynyl-
amine (7b). Eluted with dichloromethane/methanol 95:5, oil (yield
33%). 1H NMR (300 MHz, CDCl3): δ 2.23–2.20 (m, 1H, -C)CH),
2.90– 3.10 (m, 2H, H-1), 3.43 (m, 2H, C H2C)CH), 3.90 (m, 1H,
H-2), 4.48 (dd, 1H, J ) 6.75, 15.00 Hz, H-3A), 4.58 (dd, 1H, J ) 7.50,
15.00 Hz, H-3B), 7.02 (d, 1H, J ) 7.50 Hz, H-6′), 7.21 (dd, 1H, J )
3.75, 7.50 Hz, H-5′), 7.43 (d, 1H, J ) 3.75 Hz, H-3′), 7.80 (s, 1H,
triazole), 7.90 (s, 1H, triazole). MS m/z (%) 311 (6), 309 (9), 243 (51),
241 (77), 206 (27) 174 (50), 172 (70), 68 (100). Anal. Calcd for
C14H14Cl2N4: C, 54.38; H, 4.56; N, 18.12. Found: C, 54.27; H, 4.50;
N, 18.01.
(E)-[2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propyl]-(3-phen-
ylallyl)amine (7c). Eluted with ethyl acetate, oil (yield 27%). 1H NMR
(300 MHz, CDCl3): δ 2.90–3.10 (m, 2H, H-1), 3.45 (d, 2H, J ) 8.00
Hz, -NC H2CH)CH2), 3.90–4.00 (m, 1H, H-2), 4.50 (dd, 1H, J ) 6.00,
14.00 Hz, H-3A), 4.62 (dd, 1H, J ) 8.00, 14.00 Hz, H-3B), 6.25 (dt,
1H, J ) 8.00, 16.00 Hz, -C H)CHPh), 6.50 (d,1H, J ) 16.00 Hz,
-CH)C HPh), 6.95 (d, J ) 8.80 Hz, H-6′), 7.20–7.50 (m, 7H, 5Ar +
H-5′ + H-3′), 7.80 (s, 1H, triazole), 7.90 (s, 1H, triazole). MS m/z
(%): 387 (24), 385 (37), 316 (5), 262 (97), 172 (9), 117 (100), 91 (26).
Anal. Calcd for C20H20Cl2N4:C, 62.02; H, 5.20; N, 14.47. Found: C,
62.11; H, 5.14; N, 14.41.
report the synthesis and antifungal activity of this new series
of amines and amides.
MATERIALS AND METHODS
Chemistry. All reagents and solvents were reagent grade or were
purified by standard methods before use. Melting points were determined
in open capillaries on a melting point apparatus and are uncorrected.
Column chromatography was carried out on flash silica gel (Merck
230–400 mesh). TLC analysis was conducted on silica gel plates (Merck
60F254). 1H NMR spectra were recorded at 300 MHz. Chemical shifts (δ
values) and coupling constants (J values) are given in ppm and Hz,
respectively. Mass spectra were recorded on a Finnigan-MAT TSQ70
spectrometer. Solvents were routinely distilled prior to use. All reactions
requiring anhydrous conditions were performed under a positive nitrogen
flow, and glassware were oven-dried and/or flame-dried. All of the
compounds synthesized had satisfactory elemental analysis data.
2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propylamine (4). 2-(2,4-
Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propionitrile (3) (kindly provided
by Isagro, Milano) (2.18 g) was dissolved in 50 mL of methanol by
gentle heating, and then CoCl2 ·6H20 (3.56 g, 14.98 mmol) and NaBH4
(2.83 g, 74.9 mmol) were added in portions. The mixture was stirred
for 2 h, and the pH was adjusted to 2 with 2 N HCl. The solvent was
evaporated, and the aqueous phase was extracted with dichloromethane.
The aqueous phase was then basified with ammonia and extracted with
dichloromethane. Evaporation of dichloromethane gave a brown oil
(1.64 g). The crude material was purified by chromatography on silica
gel with dichloromethane/methanol 9:1 to give 1.04 g of an oil (yield
52%). 1H NMR (CDCl3): δ 2.95–3.10 (m, 2H, H-1), 3.75–3.80 (m,
1H, H-2), 4.45–4.55 (m, 2H, H-3), 6.98 (d, 1H, J ) 8.19 Hz, H-6′),
7.25 (dd, 1H, J ) 2.23, 8.19 Hz, H-5′), 7.40 (d, 1H′, J ) 2.23 Hz,
H-3), 7.76 (s, 1H, triazole), 7.85 (s, 1H, triazole).
[2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propyl]dimethyl-
amine (6). Compound 3 (0.5 g, 1.84 mmol) was stirred with formic
acid (0.47 g, 10 mmol) and 37% aqueous formaldehyde (0.35 g, 4.3
mmol) at 120 °C. After 6 h, the mixture was acidified with 1 mL of 1
N HCl and extracted with dichloromethane. The aqueous phase was
basified with 2 N NaOH and extracted with dichloromethane. The
organic extracts were dried, filtered, and evaporated to give a crude
that was purified by flash chromatography on silica gel (dichlo-
romethane/methanol 9:1) to give 250 mg (45%) of an oil (6). 1H NMR
(CDCl3): δ 2.29 (s, 6H, N(CH3)2), 2.45–2.55 (m, 2H, H-1), 3.85–4.00
(m, 1H, H-2), 4.41 (dd, 1H, J ) 13.02, 7.50 Hz, H-3A), 4.55 (dd, 1H,
J ) 13.02, 5.80 Hz, H-3B), 6.92 (d, 1H, J ) 8.19 Hz, H-6′), 7.19 (dd,
1H, J ) 2.23, 8.19 Hz, H-5′), 7.36 (d, 1H, J ) 2.23 Hz, H-3′), 7.70 (s,
1H, triazole), 7.88 (s, 1H, triazole). MS m/z (%): 301 (1), 299 (2), 204
(2), 58 (100). Anal. Calcd for C13H16Cl2N4: C, 52.19; H, 5.39; N, 18.73.
Found: C, 52.32; H, 5.30; N, 18.64.
[2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propyl]benzyl-
amine (7d). Eluted with dichloromethane/methanol 98:2, oil (yield
1
20%) H NMR (CDCl3): δ 2.85–3.05 (m, 2H, H-1), 3.78 (d, 2H, J )
5.09 Hz, CH2Ph), 3.85–3.98 (m, 1H, H-2), 4.45 (dd, 1H, J ) 6.24,
13.87 Hz, H-3A), 4.59 (dd, 1H, J ) 6.24, 13.87 Hz, H-3B), 6.96 (d,
1H, J ) 8.21 Hz, H-6′), 7.20 (dd, 1H, J ) 2.08, 8.21 Hz, H-5′),
7.25–7.39 (m, 5H, 5Ar), 7.39 (d, 1H, H-3′, J ) 2.08 Hz), 7.69 (s, 1H,
triazole), 7.87 (s, 1H, triazole). Anal. Calcd for C18H18Cl2N4: C, 59.84;
H, 5.02; N, 15.51. Found: C, 59.77; H, 5.10; N, 15.39.
Synthesis of Compounds 8a,b,c,e. To a solution of 4 (1.84 mmol)
in dry ethanol (10 mL)were added K2CO3 (11 mmol) and the appropriate
bromide (3.68 mmol). The mixture was refluxed for 5 h and filtered at
a reduced pressure. The filtrate was purified by flash chromatography
on silica gel.
[2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propyl]diallyl-
amine (8a). Eluted with dichloromethane/methanol 98:2. Yield 16%.
1H NMR (300 MHz, CDCl3): δ 2.55–2.72 (m, 2H, H-1), 3.0–3.12 (m,
4H, -N(C H2CH)CH2)2), 3.85–3.95 (m, 1H, H-2), 4.30 (dd, 1H, J )
7.50, 15.00 Hz, H-3B), 4.55 (dd, 1H, J ) 5.00, 15.00 Hz, H-3A),
5.07–5.12 (m, 4H, -CH)C H2), 5.65–5.75 (m, 2H, -C H)CH2), 6.92
(d,1H, J ) 8.19 Hz, H-6′), 7.17 (dd, 1H, J ) 2.23, 8.19 Hz, H-5′),
7.27 (d, 1H, J )2.23 Hz, H-3′), 7.62 (s, 1H, triazole), 7.75 (s, 1H,
triazole). MS m/z (%) 355 (35), 353 (94), 351 (100), 258 (22), 256
(26), 97 (46), 91 (13). Anal. Calcd for C17H20Cl2N4: C, 58.13; H, 5.74;
N, 15.95. Found: C, 58.19; H, 5.66; N, 16.06.
[2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propyl]-di-prop-2-
ynylamine (8b). Eluted with dichloromethane/methanol 97:3, oil (yield
44%). 1H NMR (300 MHz, CDCl3): δ 2.22 (m, 1H, -C)CH), 2.89 (m,
2H, H-1), 3.40–3.51 (m, 4H, -NC H2C)CH), 3.92 (m, 1H, H-2), 4.47
(dd, 1H, J ) 6.75, 14.25 Hz, H-3B), 4.60 (dd, 1H, J ) 5.25, 14.25 Hz,
H-3A), 6.95 (d, 1H, J ) 7.44 Hz, H-6′), 7.19 (dd, 1H, J ) 2.23, 7.44
Hz, H-5′), 7.36 (s, 1H, J ) 2.23 Hz, H-3′), 7.72 (s, 1H, triazole), 7.86
(s, 1H, triazole). MS m/z (%) 349 (8), 347 (18), 311 (7), 266 (10), 264
(15), 106 (100). Anal. Calcd for C17H16Cl2N4: C, 58.80; H, 4.64; N,
16.13. Found: C, 58,72; H, 4,66; N, 16,18.
(E)-[2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propyl]-bis(3-
phenylallyl)amine (8c). Eluted with dichloromethane/methanol 97:3,
oil (yield 36%). 1H NMR (300 MHz, CDCl3): δ 2.75–2.85 (m, 2H,
H-1), 3.45 (d, 4H, J )8.00 Hz, -NC H2CH)CH2), 4.00–4.10 (m, 1H,
H-2), 4.40–4.65 (m, 2H, H-3), 6.19 (m, 2H, -C H)CHPh), 6.48 (d,2H,
J ) 16.00 Hz, -CH)C HPh), 6.95 (d, J ) 8.50 Hz, H-6′), 7.12 (dd, J
) 2.23, 8.50 Hz, H-5′), 7.20–7.50 (m, 11H, 10Ar + H-3′), 7.80 (s,
1H, triazole), 7.90 (s, 1H, triazole). MS CI m/z (%) 505 (5), 503 (7),
387 (4), 154 (48), 117 (9), 91 (100). Anal. Calcd for C29H28Cl2N4: C,
69.18; H, 5.61; N, 11.13. Found: C, 69.08; H, 5.67; N, 11.19.
Bis(4-tert-butyl-benzyl)-[2-(2,4-dichlorophenyl)-3-[1,2,4]triazol-
1-yl-propyl]amine (8e). Eluted with hexane/ethyl acetate 1:1, waxy
solid (yield 66%), mp 84–85 °C. 1H NMR (300 MHz, CDCl3): δ 1.41
(s, 18H, tBut), 2.60–2.70 (m, 2H, H-1), 3.60 (m, 4H, -CH2Ph),
Synthesis of Compounds 7a–d. To a solution of 4 (1.84 mmol) in
dry ethanol (10 mL) were added K2CO3 (11 mmol) and the appropriate
bromide (3.68 mmol). The mixture was stirred at room temperature
for 5–20 h and filtered at a reduced pressure. The filtrate was purified
by flash chromatography on silica gel.
[2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propyl]allylamine (7a).
Eluted with dichloromethane/methanol 98:2, oil (yield 33%). 1H NMR
(300 MHz, CDCl3): δ 2.85–2.95 (m, 2H, H-1), 3.18–3.30 (m, 2H, -NC
H2CH)CH2), 3.95–4.01 (m, 1H, H-2), 4.44 (dd, 1H, J ) 6.48, 14.11
Hz, H-3A), 4.58 (dd, 1H, J ) 6.48, 14.11 Hz, H-3B), 5.06–5.20 (m,
2H, -CH2CH)C H2), 5.74–5.90 (m, 1H, -CH2C H)CH2), 7.00 (d, 1H,
J ) 8.77 Hz, H-6′), 7.19 (dd, 1H, J ) 2.29, 8.77 Hz, H-5′), 7.39 (d,
1H, J ) 2.29 Hz, H-3′), 7.77 (s, 1H, triazole), 7.87 (s, 1H, triazole).
MS m/z (%): 313 (2), 311 (4), 243 (66), 241 (100), 174 (35), 172 (53).
Anal. Calcd for C14H16Cl2N4: C, 54.03; H, 5.18; N, 18.00. Found: C,
54.11; H, 5.27; N, 17.91.

J. Agric. Food Chem., Vol. 55, No. 20, 2007 8189
3.90–4.40 (m, 3H, H-2 + H-3), 6.70 (d, 1H, J ) 8.50 Hz, H-6′),
7.00–7.60 (m, 11H, Ar + H3′ + H-5′ triazole), 7.80 (s, 1H, triazole).
MS CI m/z (%) 564(4), 565 (17), 563 (36), 415 (11), 322 (100), 147
(49). Anal. Calcd for C33H40Cl2N4: C, 70.32; H, 7.15; N, 9.94. Found:
C, 71.04; H, 7.11; N, 10.03.
at a reduced pressure to give the crude product. Purification by flash
chromatography (dichloromethane/methanol 9:1) afforded 151 mg (yield
71%) of the title compound as a sticky solid. 1H NMR (300 MHz,
CDCl3): δ 3.70–3.86 (m, 2H, H-1), 3.95–4.15 (m, 1H, H-2), 4.50 (d,
2H, J ) 5.95 Hz, H-3), 6.86 (t, 1H, J ) 6.33 Hz, NH), 6.96 (d, 1H, J
) 8.56 Hz, H-6′), 7.17 (dd, 1H, J ) 2.23, 8.56 Hz, H-5′), 7.38–7.58
(m, 3H, 3Ar), 7.71 (d, 2H, J ) 8.93 Hz, 2Ar), 7.85 (s, 1H, triazole),
7.90 (s, 1H, triazole). Anal. Calcd for C18H16Cl2N4O: C, 57.61; H, 4.30;
N, 14.93. Found: C, 57.74; H, 4.27; N, 14.99.
But-2-enoic Acid [2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-
propyl]amide (5c). Compound 4 (150 mg; 0.55 mmol) was allowed
to react with triethylamine (58 mg; 0.557 mmol; 81 µL) and croto-
noylchloride (60.3 mg; 0.577 mmol; 55 µL). The mixture was stirred
at room temperature for 3 h, and then water (10 mL) was added. The
organic layer was separated, dried with Na2SO4, and evaporated at a
reduced pressure to give the crude product. Purification by flash
chromatography (dichloromethane/methanol 9:1) gave 155 mg (yield
64%) of pure compound 5c. 1H NMR (300 MHz, CDCl3): δ 1.85 (dd,
3HJ ) 1.49 Hz, CH3-,), 3.54–3.74 (m, 2H, H-1), 3.90–4.10 (m, 1H,
H-2), 4.45 (d, 2H, J ) 5.95 Hz, H-3), 5.75 (dd, 1H, J ) 1.49, 15.26
Hz, CH)), 6.80 (dq, 1H, J ) 15.26, 6.70 Hz, CH)), 6.91 (d, 1H, J )
8.56 Hz, H-6′), 7.19 (dd, 1H, J ) 1.86, 8.56 Hz, H-5′), 7.40 (d, 1H, J
) 1.86 Hz, H-3′), 7.83 (s, 1H, triazole), 7.90 (s, 1H, triazole). Anal.
Calcd for C15H16Cl2N4O: C, 53.11; H, 4.75; N, 16.52. Found: C, 53.02;
H, 4.70; N, 16.61.
Partition Coefficients. The log P (where P is the octanol–water
partition coefficient) was obtained as described in ref 13, using reversed-
phase chromatography (14) by comparison with seven reference
compounds whose log P are known (15). Retention times were
determined on a Hewlett-Packard HPLC equipped with a quaternary
pump HP-1050 with a Rheodyne injector (20 L loop) with an UV–vis
detector HP-1050 or a Waters Lambda Max Model 481. Methanol for
HPLC was purchased from Baker, and water for HPLC was produced
with a Milli-Q Water purification system (Millipore). Analyses were
performed on a Merck column LiChrospher 100 RP18 (250 mm × 4
mm, 5 m), the flow rate was 1 mL/min, using methanol/ammonium
phosphate buffer at pH 7.0 80:20 as eluent.
Biological Assays. Compounds were tested in vitro and in vivo
against a number of phytopathogenic fungi. The results were compared
with those obtained with tetraconazole, used as a standard compound.
Test Pathogens. Botrytis cinerea Pers., Cercospora beticola Sacc.,
Cercosporella herpotricoides Fron., Fusarium roseum Link., and
Helminthosporium teres Sacc. of the IPV Collection (Milan, Italy) were
maintained on potato dextrose agar Difco (PDA); Ustilago maydis (DC)
Corda, strain ATCC 14826 was cultured on Czapek Agar Difco (CZA)
and Coursen and Sisler broth.
Erysiphe graminis DC f. sp. triticiMarchel and Puccinia recondita
Rob. ex Desm. f. sp. tritici were grown on stock plants. Phytophtora
infestans (Mont.) De Bary kindly provided by Isagro was maintained
on V8 juice agar and on tomato stock plants.
Among the pathogens selected, some were chosen because they are
typically controlled by azoles, others because they are generally less
sensitive.
Plants. Lycopersicon esculentum L. cv Marmande and Triticum
aestiVum L. cv Gemini used in this study were grown in plastic pots
(diameter 11 cm) in a growth room or greenhouse at 21 ( 1 °C and 70
( 10 % RH.
In Vitro Fungicidal Activity. The activity of the new compounds
against B. cinerea, C. beticola, C. herpotricoides, F. roseum, H. teres,
and U. maydis was determined in vitro on PDA or CZA as inhibition
of the radial growth at different concentrations (100–0.1 mg L-1).
Growth inhibition was calculated from the mean difference between
treated and control cultures as a percentage of the latter. Percentage
growth inhibition was plotted on a probit scale against compound doses
in order to obtain EC50 values (50% inhibitory concentration).
Compound 7 was also tested on U. maydis broth cultures (7). Growth
was estimated by cell count at the end of the treatment, and the results
were expressed as percentage inhibition compared to the control. These
cultures were used for sterol and squalene analysis.
Synthesis of [2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propyl]-
dibenzylamine (8d). Compound 4 (1.84 mmol) was dissolved in N,N′-
dimethylpropyleneurea (5 mL, DMPU) and treated with K2CO3 (3.68
mmol) and the appropriate bromide (3.68 mmol). The mixture was
heated and stirred for 4 h at 100 °C under nitrogen. The cooled mixture
was diluted with water (15 mL) and extracted with ethyl acetate. The
organic extracts were purified by chromatography on silica gel eluting
with a solution of dichloromethane/methanol 98:2. Yield 47%. 1H NMR
(300 MHz, CDCl3): δ 2.70 (d, 2H, J ) 6.00 Hz, H-1), 3.54 (s, 4H,
-CH2Ph), 3.90–4.00 (m, 1H, H-2), 4.15 (dd, 1H, J ) 9.00, 15.00 Hz,
H-3A), 4.55 (dd, 1H, J ) 6.00, 15.00 Hz, H-3B), 6.70 (d, 1H, J ) 8.25
Hz, H-6′), 7.06 (dd, 1H, J ) 2.25, 8.25 Hz, H-5′), 7.20–7.40 (m, 11H,
-CH2Ph + H-3′), 7.42 (s, 1H, triazole), 7.80 (s, 1H, triazole). MS m/z
(%) 455 (3), 453 (19), 451 (33), 210 (100), 91 (56). Anal. Calcd for
C25H24Cl2N4: C, 66.52; H, 5.36; N, 12.41. Found: C, 66.01; H, 5.30;
N, 12,98.
Synthesis of Compounds 9a and 9b. To a solution of the amine 4
(5.8 mmol) in methanol (10 mL) were added acetic acid (5.8 mmol),
1-ethoxy-1-trimethylsilylcyclopropane (2.32 mmol), and sodium cy-
anoborohydride (2.32 mmol). The mixture was stirred and refluxed for
5 h. The solvent was removed at a reduced pressure to give a dense oil
that was partitioned between ethyl acetate and a 2 N NaOH solution.
The organic phase was dried and evaporated to obtain a crude that
was purified by flash column chromatography on silica gel (eluent, ethyl
acetate).
[2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propyl]dicyclopropyl-
amine (9a). Yield 32%. 1H NMR (300 MHz, CDCl3): δ 0.38–0.58
(m, 8H, cyclopropyl), 1.85–1.95 (m, 2H, cyclopropyl-N), 3.10–3.20
(m, 2H, H-1), 4.05–4.20 (m, 1H, H-2), 4.30–4.50 (m, 2H, H-3), 6.98
(d, 1H, H-6′, J ) 8.53 Hz), 7.10 (dd, 1H, J ) 2.23, 8.53 Hz, H-5′),
7.30 (d, 1H, J ) 2.23 Hz, H-3′), 7.61 (s, 1H, triazole), 7.82 (s, 1H,
triazole). MS m/z (%): 353 (3), 351 (5), 317 (18), 315 (43), 270 (43),
268 (76), 256 (19), 218 (26), 186 (18), 159 (26), 110 (100). Anal. Calcd
for C17H20Cl2N4: C, 58.13; H, 5.74; N, 15.95. Found: C, 58.01; H, 5.66;
N, 16.08.
Allyl-cyclopropyl-[2-(2,4-dichlorophenyl)-3-[1,2,4]triazol-1-yl-
propyl]amine (9b). Obtained from compound 7a. Yield 39%. 1H NMR
(300 MHz, CDCl3): δ 0.38–0.58 (m, 4H, cyclopropyl), 1.75–1.85 (m,
1H, cyclopropyl-N), 3.18–3.32 (m, 2H, H-1), 4.05–4.18 (m, 1H, H-2),
4.40 (dd, 1H, J ) 6.70, 13.80 Hz, H-3B), 4.54 (dd, 1H, J ) 4.50, 13.80
Hz, H-3A), 5.10–5.20 (m, 2H, -CH)C H2), 5.75–5.90 (m, 1H, -C
H)CH2), 6.99 (d, 1H, J ) 8.19 Hz, H-6′), 7.18 (dd, 1H, J ) 2.23,
8.19 Hz, H-5′), 7.36 (d, 1H, J ) 2.23 Hz, H-3′), 7.65 (s, 1H, triazole),
7.85 (s, 1H, triazole). MS m/z (%): 353 (1), 351 (3), 317 (12), 315
(29), 270 (14), 268 (24), 256 (19), 254 (32), 240 (12) 218 (11), 176
(15), 159 (17), 110 (100). Anal. Calcd for C17H20Cl2N4: C, 58.13; H,
5.74; N, 15.95. Found: C, 58.24; H, 5.59; N, 15.14.
N-[2-(2,4-Dichlorophenyl)-3-[1,2,4]triazol-1-yl-propyl]aceta-
mide (5a). Compound 4 (150mg, 0.55 mmol) was dissolved in pyridine
(1 mL) and acetic anhydride (1 mL) The solution was stirred overnight
at room temperature and then evaporated to dryness. The residue was
purified by flash chromatography with dichloromethane as eluent to
1
give 168 mg of the title compound.Yield 98%. H NMR (300 MHz,
CDCl3): δ 1.96 (s, 3H, Ac), 3.50–3.56 (m, 2H, H-1), 3.95–4.01 (m,
1H, H-2), 4.45 (d, 2H, J ) 6.33 Hz, H-3), 5.78 (brs, 1H, NH), 6.92 (d,
1H, J ) 8.19 Hz, H-6′), 7.20 (dd, 1H, J ) 2.23, 8.19 Hz, H-5′), 7.41
(d, 1H, J ) 2.23 Hz, H-3′), 7.82 (s, 1H, triazole), 7.91 (s, 1H, triazole).
Anal. Calcd for C13H14Cl2N4O: C, 49.86; H, 4.51; N, 17.89. Found: C,
49.77; H, 4.61; N, 18.02.
N-[2-(2,4-Dichloro-phenyl)-3-[1,2,4]triazol-1-yl-propyl]-benza-
mide (5b). A solution of freshly distilled benzoyl chloride (81 mg,
0.58 mmol, 66 µL) in dichloromethane (2 mL) was added dropwise to
a stirred solution of 4 (150 mg, 0.55 mmol) and triethylamine (58 mg,
0.577 mmol, 81 µL) at 0 °C under nitrogen. The resulting mixture was
stirred at room temperature for 3 h, and then water (10 mL) was added.
The organic layer was separated, dried with Na2SO4, and evaporated
In Vivo Fungicidal Activity. Compounds 7c, 8a, 8b, and 9a were
assayed in preventive application tests. They were dissolved in acetone/

8190 J. Agric. Food Chem., Vol. 55, No. 20, 2007
Arnoldi et al.
Scheme 1a
a Reagents and conditions: (a) NaBH4, CoCl2, MeOH, 2 h, rt, 52%; (b) acetic anhydride, Py, rt, overnight, quantitative; PhCOCl or crotonylchloride, TEA,
CH2Cl2, rt, 3 h; (c) HCOOH, HCHO, 120 °C, 6 h, 45%; (d) K2CO3, R-Br, EtOH, rt, 5–20 h; (e) K2CO3, R-Br, EtOH, reflux, 5 h or DMPU, K3CO3, benzyl bromide,
100 °C 4 h (47% for 8d); (f) 4 or 7a, methanol, glacial acetic acid, (1-Ethoxycyclopropoxy)trimethylsilane, NaCNBH3, reflux, 5 h.
distilled water (1:4, v/v) containing Tween 20 (0.3 mg mL-1) and
sprayed on wheat (7 days old) and tomato plant (six leaves stage) until
moist, at the standard concentration of 125 µg mL-1. One day later,
wheat plants were inoculated with a water suspension of P. recondita
conidia (1 mg mL-1 plus 0.1 % Tween 20), and tomato plants with a
suspension of P. infestans zoosporangia (5 × 104 mL-1). After
inoculation, wheat and tomato plants were kept 24 h in an incubation
cabin at 20 ( 1 °C and 100% RH and then transferred to a greenhouse
(23 ( 1 °C and 70 ( 10 % RH). Inoculation of wheat plants with E.
graminis was obtained by strongly stirring infected plants with abundant
sporification on the test plants, then moving the plants to a growth
room (20 ( 1 °C and 70 ( 10 % RH). The fungicidal activity was
evaluated on wheat plants 10 days and on tomato plants 7 days after
inoculation. The area of inoculated treated leaves covered by disease
symptoms was assessed and compared with that of nontreated ones to
determine the percentage inhibition of disease.
Squalene and Sterol Analysis of U. maydis Sporidia. Cultures of
U. maydis grown in Coursen and Sisler broth amended with different
concentrations of compound 8a (0.01–100 mg L-1) were used for sterol
and squalene analysis. After 18 h treatment, sporidia were harvested
by centrifugation and added with a known amount of dihydrocholesterol,
and then heated with 2 mL of 20% KOH in 60% ethanol for 1 h at 90
°C. The unsaponifiable fraction was extracted with heptane (3 × 3 mL),
dried with anhydrous sodium sulfate, and concentrated in vacuo. A
known amount of cholestane was added as a second internal standard
before derivatizing samples as trimethylsilyl ethers with a 1:1 mixture
of pyridine/N,O-bis(trimethylsilyl)trifluoroacetamide (1:1, v/v). Gas
chromatographic analyses were performed on a DANI 3800 gas
chromatograph equipped with a FID detector and PTV injector. A
capillary column SPB5 Supelco, 30 m × 0.25 mm i.d., 0.25-µm film
thickness, was used. Analysis conditions were carrier gas, helium; flow,
0.75 mL/min; oven temperature, 275 °C; injector from 30 to 290 °C in
30 s; splitless injection; after 30 s split 30 mL min-1; detector 300 °C.
Data were recorded by a Shimadzu CR2A integrator. Gas chromatog-
raphy–mass spectrometry analyses were performed on a Finnigan
TSQ70 instrument, equipped with the same column. Analysis conditions
were oven, 265 °C; injector, 280 °C; transfer line, 280 °C; electron
energy, 70 eV; electromultiplier, 900V. The results are the average of
two replicates per test.
RESULTS AND DISCUSSION
Chemistry. The key intermediate for the preparation of all
new compounds was amine 4 (Scheme 1).
It was obtained by reduction of the corresponding nitrile (3)
(kindly provided by Isagro, Milano) with NaBH4 and CoCl2 ·6H2O.
This procedure, despite the formation of stable emulsions that
complicated the workup, gave better yields than diborane without
catalysts. Standard acetylation methods gave the amides 5a–c. The
N,N′-dimethylderivative 6 was prepared by Leuckart methylation
of 4 with formic acid and formaldehyde. The secondary amines
7a–d were obtained by alkylation of 4 with the corresponding
alkylbromides. The reactions were performed at room temperature
in the presence of potassium carbonate as a base. The corresponding
tertiary amines 8a–c and 8e were obtained after refluxing the
ethanolic solutions for about 5 h. Compound 8d was obtained with
a better yield using N,N-dimethylpropyleneurea (DMPU) as a
solvent, as reported by Juaristi et al. (16).
As far as the synthesis of cyclopropyl derivatives is con-
cerned, a survey of the literature revealed few methods to

J. Agric. Food Chem., Vol. 55, No. 20, 2007 8191
Table 1. In Vitro Fungicidal Activity Expressed as 50% Effective
Concentration (EC50, µg mL-1
)
B. C.
C.
F.
H.
U.
log P
cinerea beticola herpotricoides roseum teres maydis
4
1.92a >100
1.85a >100
3.75a ∼100
2.90a >100
2.54 75.0
3.13 >100
>100
>100
>100
>100
80.3
>100
6.5
91.3
11.0
7.5
23.4
3.6
>100
>100
>100
>100
>100
>100
>100
26.0
>100
>100
>100
>100
70.30
60.00
1.90
20.70
2.50
19.9
5a
5b
5c
6
2.5
11.5
>100
42.0
>100
38.0
12.0
60.5
8.0
31.7
4.0
2.4
2.7
2.6
1.1
12.5
4.0
7.0
0.4
0.2
7a
7b
7c
7d
8a
8b
8c
8d
8e
9a
9b
2.15
3.67
38.0
42.0
1.30
1.40
4.17 >100
30.3 11.3
3.92
3.48
10.0
4.9
6.5
2.1
6.0
1.9
0.22
2.8
9.0
0.14
0.65
0.09
9.00
0.02
0.01
5.00 >100
4.60 6.0
9.69 >100
>100
17.0
>100
>100
>100
>100
55.0
55.0
4.00
>100
Figure 1. Growth inhibition of U. maydis by count of sporidia versus
percentage composition of unsaponifiable fraction after incubation with
increasing concentrations of compound 8a.
2.86
2.86
7.5
4.4
1.2
0.8
0.45
5.5
3.2
Tetrab 3.99a
0.04
0.12
0.40 0.25
a Calculated (ChemDraw 7.0 package). b Tetraconazole.
activity is mainly correlated to log P. When the introduction of
further alkyl groups leads to compounds with an intermediate
log P (see 7a,b,d versus 8a,b,d), the activity increases going
from a secondary to the corresponding tertiary amine. On the
contrary, when the introduction of the third alkyl group leads
to compounds having a log P higher than 4.60 (see 7c versus
8c), a drop in the biological activity is observed, with the only
exception of H. teres and U. maydis.
cyclopropylate primary or secondary amines (17–20). Moreover,
these routes could not be extended to aliphatic amines, which
were of interest to us. To the best of our knowledge, only
Gillaspy et al. reported an efficient, one-step method to
cyclopropylate aliphatic amines, based on the reductive ami-
nation with (1-ethoxycyclopropoxy)trimethylsilane and NaC-
NBH3 (21). Following this procedure we obtained cyclopropy-
lamines 9a and 9b in fair yield. Attempts to isolate the
monosubstituted cyclopropyl derivative of 4 failed, as the
reaction gave only the disubstituted derivative 9a.
In Vitro Assays. The in vitro fungitoxicity of the new
compounds expressed as EC50 is reported in Table 1 with
tetraconazole used as a standard.
Some of the compounds (6; 7b–d; 8a,b,d; 9a,b) showed fairly
good activity, in particular against U. maydis, which seems to
be the most sensitive one among the tested fungi. In fact, EC50
values of compounds 9a,b, 8b, and 8d against this pathogen
are lower than the EC50 of the standard tetraconazole. C. beticola
and H. teres growth was also inhibited by various compounds
at low concentrations.
The introduction of an amide moiety is detrimental to the
activity (compounds 5a and 5c). It is likely that the increase in
polarity makes it difficult for the compounds to be carried
through the cell membrane. In fact some activity is restored in
the less polar 5b. All of these results corroborate the hypothesis
that lipophilicity plays an important role in determining anti-
fungal activity. This is in agreement with the results we obtained
in our previous work with a similar series of compounds. (11)
The compounds were also tested in vitro against a series of
human fungal pathogens, such as some strains of Candida
albicans, Cryptococcus neoformans, Aspergillus niger, Tricho-
phyton mentagrophytes, and Microsporum canis. No remarkable
activity was found except for compounds 7c and 8b, which were
active against C. albicans strain SKF2270 (MIC 0.13 and 0.25
µg mL-1, respectively, compared with the standard fluconazole
0.25 µg mL-1) and against a strain of fluconazole- and
On the contrary, F. roseum was the pathogen less sensitive
to this group of compounds, followed by B. cinerea and C.
herpotricoides.
The most active compounds were 7c, 8b, an 9a,b, followed
by 8d. Compound 8e seemed to act selectively against C.
beticola and U. maydis. Interestingly, compound 8b showed a
significant activity against F. roseum, having an EC50 value
comparable to that of the standard tetraconazole, despite the
generally very low efficacy of triazole fungicides against this
pathogen. This is a promising result, as rot and wilt agents of
many economically important crops belong to the genus
Fusarium. Some species are also responsible for mycotoxin
contamination of cereals and cereal-derived foods and feeds.
When the amino moiety is involved in an amidic bond (5a–c),
there is a dramatic decrease of activity.
To test the effect of lipophilicity on the antifungal activity,
the log P for most compounds was measured with a HPLC
method (Table 1). The most active compounds showed a log P
in the range 2.54–3.92. Compounds 8c,d and 8e, characterized
by log P values higher than 4.60, showed a decrease in activity.
On the other hand, the unsubstituted amine 4 (log P ) 1.85)
was substantially inactive.
itraconazole-resistant C. albicans (MIC 2.0 and 16 µg mL-1
,
respectively).
In Vivo Assays. Some of the compounds most active in vitro
(7c, 8a, 8b, 9a) were also assayed for their preventive activity
in vivo on two pathogen–host combinations, i.e., Erysiphe
graminis f. sp . tritici–Triticum aestiVum (wheat powdery
mildew) and Puccinia recondita f. sp . tritici–Triticum aestiVum
(wheat brown rust). As already mentioned, our compounds show
a structure similar to that of allylamines, well-known inhibitors
of squalene biosynthesis. For this reason, we selected a further
pathogen–host combination (Phytophthora infestans–Lycoper-
sicon esculentum) to rule out squalene biosynthesis inhibition
as a possible side mechanism of action for this class of
compounds. In fact, the last pathogen does not synthesize sterols
and is therefore insensitive to triazole fungicides.
Results are reported in Table 2. All compounds were more
active in protecting wheat plants from powdery mildew than
from rust. Compound 9a induced the highest inhibition, control-
ling powdery mildew at 60% at 125 µg mL-1. As expected,
the tested compounds were completely inactive against tomato
late blight.
A comparison of the disubstituted amines (7a–d) with the
corresponding trisubstituted ones (8a–d) revealed that the

8192 J. Agric. Food Chem., Vol. 55, No. 20, 2007
Arnoldi et al.
Table 2. In Vivo Protectant Activity Expressed as Disease Percentage
Inhibition at One Standard Concentration (125 µg mL-1
on Ustilago maydis. Pestic. Biochem. Physiol. 1991, 40, 274–
283.
)
(8) Carelli, A.; Farina, G.; Gozzo, F.; Merlini, L. Interaction of
tetraconazole and its enantiomers with cytochrome P450 from
Ustilago maydis. Pestic. Sci. 1992, 35, 167–170.
(9) Gozzo, F.; Carelli, A.; Carzaniga, R.; Farina, G.; Arnoldi, A.;
Lamb, D.; Kelly, S. L. Stereoselective interaction of tetraconazole
with 14R-demethylase in fungi. Pestic. Biochem. Physiol. 1995,
53, 10–22.
P. infestans/
E. graminis/
T. aestivum
P. recondita/
T.aestivum
compound
7c
8a
8b
L. esculentum
0
0
0
0
0
25
30
35
60
20
25
15
10
9a
tetraconazole
100
100
(10) Gozzo, F.; Garavaglia, C.; Mirenna, L. Enantiomers and racemate
of M14360: investigation of their antifungal activity. Tagungsber.
Akad. Landwirtschaftswis., DDR 1990, 291, 87–94.
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Squalene and Sterol Analysis. Broth cultures of U. maydis
treated with different concentrations of compound 8a were used
to check the activity of this amine as a squalene epoxidase
inhibitor, as shown by some related molecules (allylamines) with
antifungal activity (22). Interestingly, squalene percentage
decreased as long as growth inhibition increased (Figure 1).
Therefore the squalene epoxidase inhibition could be ruled out
as a possible side mechanism of action for this compound.
Furthermore, growth inhibition was strictly related to an increase
in the 14-methyl sterols percentage, due to the inhibition of the
14R-demethylase, a key enzyme of the ergosterol biosynthesis
pathway. The inhibition of this enzyme is the typical mechanism
of action for triazoles in general. (23)
In conclusion, the replacement of the ether group of tetra-
conazole with a secondary or tertiary amino group maintains
the antifungal activity on several phytopathogenic fungi,
provided that the substituents are not too bulky or lipophilic.
The allyl, propargyl, and cyclopropyl groups appear particularly
suitable. Although these compounds have some structural
similarities with terbinafine and naftifine, which act as squalene
epoxidase inhibitors, they maintain the usual mechanisms of
action of the other triazoles.
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ACKNOWLEDGMENT
We are indebted to Dr. L. Mirenna (ISAGRO Research) for
the in vivo tests and Lepetit (then Dow), Gerenzano, for the
tests against human pathogenic strains.
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