Trialkylamine Derivatives Containing a Triazole Moiety as Promising Ergosterol Biosynthesis Inhibitor: Design, Synthesis, and Antifungal Activity

Guoqing Sui; Wen Zhang; Kun Zhou; Yulin Li; Bingyu Zhang; Dan Xu; Yong Zou; Wenming Zhou

doi:10.1248/cpb.c16-00732

Abstract

As a part of our continuing research on amine derivative antifungal agents, 19 novel target compounds containing 1,2,4-triazole and tertiary amine moieties were designed and synthesized, and their in vitro antifungal activities against six phytopathogenic fungi (Magnaporthe grisea, Alternaria solani, Fusarium solani, Curvularia lunata, A. alternata, F. graminearum) were assayed. All target compounds were elucidated by means of ¹H-NMR, ¹³C-NMR, high resolution (HR)-MS, and IR analysis. The results showed that most of the derivatives exhibited obvious activity against each of the fungi at 50 µg/mL. Among them, compounds 7f, l, and o displayed excellent activity against A. solani with median effective concentration values (EC₅₀) of 2.88, 8.20, and 1.92 µg/mL. 7o in particular was superior to tebuconazole (EC₅₀=2.03 µg/mL), a commercial fungicide. Furthermore, compounds 7j, k, and m also showed good activity against F. graminearum with EC₅₀ values of 11.60, 5.14, and 16.24 µg/mL, and the value of 7k was extremely close to that of tebuconazole (EC₅₀=3.13 µg/mL). The preliminary analysis of the structure–activity relationship (SAR) demonstrated that combination of the active structure of 1,2,4-triazole with the tertiary amine group containing benzene rings effectively increased the antifungal activities. Generally, introducing halogen atoms obviously improved activities against most of the test fungi to varying degrees, while the presence of OMe decreased the activities. Thus, the results strongly indicate that the newly synthesized derivatives should be lead compounds for the development of novel antifungal agents for the effective control of phytopathogenic fungi.

Plant fungal pathogens have significant impacts on a wide range of crops, often resulting in severe yield losses and quality decrease of agricultural products. More severely, many of the phytopathogenic fungi can produce mycotoxins harmful to animal and human health.^1–3) To date, the application of plant fungicides is considered as an efficient measure for preventing and controlling fungal diseases.^4,5) Thus, it is necessary to develop new fungicides with novel molecular framework and action mechanisms. Ergosterol biosynthesis inhibitors are a type of fungicides which can inhibit the process of ergosterol biosynthesis in fungi.⁶⁾ Ergosterol is a common sterol in fungi, whose structure is different from that of sterol in plants and animals. Therefore, because of its efficiency, broad spectrum, low toxicity and environmental compatibility, ergosterol biosynthesis inhibitors have been extensively studied since they appeared for the first time.^7,8)

As a class of C-14 demethylase inhibitor, triazole fungicides could effectively inhibit the biosynthesis of ergosterol through hindering C-14 demethylation in the process of ergosterol biosynthesis.^6,9) In addition, in our previous research, we found triazole fungicides and tertiary amine fungicides, the important classes of ergosterol biosynthesis inhibitors, are of great significance to protect a variety of crops, and have achieved a unique position in the chemical control of fungal diseases since their discovery.^10,11) The representatives of triazole fungicides are triadimefon, myclobutanil, tebuconazole, and triadimenol, among others¹²⁾ (Fig. 1). As to tertiary amine fungicides, some typical products are tridemorph, fenpropimorph, fenpropidin, and spiroxamine¹³⁾ (Fig. 2).

Fig. 1. Typical Products of C-14 Demethylase Inhibitor (Triazole Fungicide)

Fig. 2. Typical Products of Δ¹⁴ Reductase/Δ⁸→Δ⁷ Isomerase Inhibitor (Tertiary Amine Fungicide)

However, intensive application of all kinds of fungicides for a long time has caused the appearance of resistant strains against triazole fungicides.¹⁴⁾ In order to search for novel antifungal agents for the more effective control of phytopathogenic fungi, especially against resistant fungi, this study used an intermediate derivatization method (IDM) to design and synthesize a series of target compounds containing 1,2,4-triazole and tertiary amine moieties (Fig. 3) through combining the active structure of triazole fungicides with the active trialkylamine structure of Δ14-reductase/Δ8→Δ7 isomerase inhibitors.

Fig. 3. Structure of Target Compounds

Herein, 1,2,4-triazol-1-ylpinacolin, an important intermediate for synthesizing diverse triazole fungicides, was chosen as a starting material based on the relevant structure–activity relationship (SAR) which has been established through enormous research on various triazole fungicides.¹³⁾

Results and Discussion

Chemistry

The synthetic route of target compounds 7a to s is outlined in Chart 1, and their substitution patterns for each of the target compounds are listed in Table 1. Commercially available 1,2,4-triazol-1-ylpinacolin (1) was used as a starting material to react with trimethylsulfoxonium iodide (TMSOI), known as sulfur ylide,¹⁵⁾ in the presence of tetrabutylammonium iodide (TBAI; phase transfer catalyst), and 50% potassium hydroxide (KOH) aqueous solution (reaction catalyst) in toluene to prepare compound 2 in 71% yield. Next, compound 2 was reacted with sodium azide in N,N-dimethylformamide (DMF) in the presence of NH₄Cl to give compound 3, followed by the treatment with triphenylphosphine in dichloromethane to afford compound 4 in 82% yield. Then, compound 5 as a key intermediate was obtained in 90% yield through hydrolyzing 4 using sodium hydroxide aqueous solution (2 mol/L) in methanol. The preparation of 6a to s from 5 was a key step. Compound 5 was reacted with a series of benzaldehydes or 1-naphthaldehyde in absolute ethanol, followed by the addition of solid sodium borohydride to obtain the desired 6a to s in 47–77% yield. Finally, compound 6a to s was treated with 88% formic acid and 37% formaldehyde aqueous solution, respectively, to obtain the target compounds 7a to s.

Chart 1. Synthesis of Trialkylamine Derivatives Containing a Triazole Moiety

Table 1. Substitution Patterns of Compounds and Their Preliminary Antifungal Activity at 50 µg/mL (72 h)

Compounds		Average inhibition rate±S.D. (%) (n=3)
No.	R	M. grisea	A. solani	F. graminearum	F. solani	C. lunata	A. alternata
7a	H	−1.0±0.1ij	74.3±1.1a^a⁾	63.2±1.7cde	55.5±2.4cde	42.9±0.6gh	13.0±0.2efgh
7b	2-Me	14.1±0.6fgh	28.2±0.9bc	54.5±0.9efg	46.0±1.3efgh	37.3±2.1h	31.6±1.1d
7c	3-Me	71.0±2.1b	23.2±0.7bc	70.4±1.7bcd	35.2±1.5ghi	46.0±0.8fgh	87.7±1.8a
7d	4-Me	19.3±0.9f	−1.0±1.3cde	53.0±0.7efg	32.3±2.4hi	48.1 ±1.2efgh	44.1±0.8c
7e	2-F	14.7±1.4fgh	58.1±2.7ab	69.3±1.1bcde	46.5±2.3efg	22.3±0.5i	18.5±1.5ef
7f	3-F	3.3±0.4 hi	82.5±0.8a	68.1±0.3bcde	42.1±1.3fgh	41.7±1.9gh	15.0±1.4efg
7g	4-F	−8.1±1.3j	40.3±1.2bc	59.6±2.7def	39.0±1.5fgh	19.0±2.3ij	7.2±2.6efg
7h	2-Cl	20.0±0.6f	17.1±1.8bcd	64.9±0.1cde	48.6±0.6def	41.2±0.7gh	35.3±2.8fgh
7i	3-Cl	44.3±1.8cd	26.0±1.3bc	57.3±1.0ef	55.2±2.5cde	48.0±0.4defg	60.5±0.8b
7j	4-Cl	23.5±1.7ef	81.5±2.1a	86.0±1.9ab	59.7±0.4bcd	54.4±1.7cdef	36.3±1.6cd
7k	2,4-diCl	54.0±1.2 c	26.3±0.5bc	80.4±1.2bc	68.2±2.8b	58.1±2.5bc	61.4±2.5b
7l	3,4-diCl	71.4±2.7b	86.4±1.2a	36.5±2.1g	67.3±2.0b	64.2±1.9b	69.0±2.1b
7m	2-Br	24.2±1.8ef	67.0±0.9ab	80.2±1.2bc	59.3±1.5bcd	43.5±2.4gh	38.1±0.4cd
7n	3-Br	47.0±2.9c	48.1±2.1ab	58 .3± 1.6ef	43.1±0.4fgh	56.2±1.7cd	60.5±1.1b
7o	4-Br	55.3±1.9c	80.7±1.3a	77.2±1.1bcd	62.0±1.0bcd	54.1±2.6cdef	44.3±0.7c
7p	2-OMe	5.2±1.4ghi	33.2±0.5abc	−4.0±0.5h	2.1±1.7j	8.7±1.0k	6.2±0.4gh
7q	3-OMe	16.1±1.5fg	−1.1±0.9bcde	44.3±1.8fg	40.4±0.3fgh	22.6±1.2i	43.0±2.4c
7r	4-OMe	7.2±0.6ghi	−42.0±1.8de	38.5±1.5g	25.2±0.3i	11.3±0.5jk	21.4±1.3e
7s		34.3±1.7de	37.6±0.6abc	61.0±0.3def	38.1±1.1fgh	55.0±1.9cde	66.3±1.6b
5		6.7±0.6fgh	−53.0±1.6e	−2.2±1.2h	−3.0±0.8j	1.4±0.6L	2.0±0.5h
Tebuconazole		100±0.0a	68.2±2.4ab	98.1±0.4a	94.7±0.2a	87.0±1.5a	95.5±2.0a

a) The differences between data with the different lowercase letters within a column are significant for the tested fungus (p<0.05), which were carried out by Duncan’s multiple comparison.

Antifungal Activity

According to a reported mycelium linear growth rate method,¹⁶⁾ the in vitro antifungal activities against six plant pathogenic fungi (Alternaria solani, Magnaporthe grisea, A. alternate, Curvularia lunata, Fusarium graminearum, F. solani) of synthetic compounds 7a to s at the concentration of 50 µg/mL were assayed.^17–19) Tebuconazole (≥ 99.1%), a commercial fungicide, was used as the positive control. Compound 7a was regarded as the reference compound. The results are summarized in Table 1.

It can clearly be seen from Table 1 that most of the target compounds (7b–s) displayed better activities against all test fungi than intermediate compound 5 and unsubstituted reference compound 7a at 50 µg/mL. Compound 7c exhibited the highest activity among all the tested compounds against A. alternata with an inhibition rate of 87.7%, which was much higher than that of 7a (13.0%) (p<0.05). Compounds 7f, j, l, and o in particular showed excellent activity against A. solani with inhibition rates of 82.5, 81.5, 86.4, and 80.7%, respectively, which were signiﬁcantly higher than that of tebuconazole (68.2%) (p<0.05), a commercial fungicide. In addition, compared with 7a (63.2%) (p<0.05), compounds 7j, k, and m also showed higher activity against F. graminearum with inhibition rates of 86.0, 80.4, and 80.2%, respectively. Compared with other target compounds, compound 7l exhibited a broader antifungal spectrum, which showed inhibitory activities against M. grisea, A. solani, F. solani, C. lunata, and A. alternata with inhibition rates of 71.4, 86.4, 67.3, 64.2 and 69.0%, respectively.

Antifungal Toxicity

The excellent activities of 7c, f, j, k, l, m, and o (inhibition rates>80%) in Table 1 encouraged us to further determine their median effective concentrations (EC₅₀) against three corresponding strains of fungi. The assay method was the same as that described above. Tebuconazole was used as the positive control. Toxicity regression equations for the concentration–effect of the compounds and their corresponding EC₅₀ values are listed in Tables 2–4.

Table 2. Toxicity Regression Equations for Concentration–Effect and EC₅₀ Values of Compounds against A. alternata

Compd.	Regression equation^a⁾	R²	EC₅₀ (µg/mL)	95% CI^b⁾	Linear scope (µg/mL)
7c	y=1.8194x+2.8545	0.926	15.11	7.89–28.94	0.78–50
Tebuconazole	y=1.0776x+4.8058	0.993	1.51	1.39–1.65	0.39–25

a) y: Probability of average inhibition rate; x: lg[concentration(µg/mL)]. b) Confidence interval of EC₅₀ (µg/mL) at 95% probability.

Table 3. Toxicity Regression Equations for Concentration–Effect and EC₅₀ Values of Compounds against F. graminearum

Compd.	Regression equation^a⁾	R²	EC₅₀ (µg/mL)	95% CI^b⁾	Linear scope (µg/mL)
7j	y=1.3878x+3.5230	0.989	11.60	10.38–12.95	0.78–50
7k	y=2.0228x+3.5627	0.975	5.14	3.92–6.63	0.78–50
7m	y=2.1769x+2.3644	0.942	16.24	10.30–9.38	0.78–50
Tebuconazole	y=1.7666x+4.1259	0.983	3.13	2.57–3.80	0.39–25

a) y: Probability of average inhibition rate; x: lg[concentration (µg/mL)]. b) Confidence interval of EC₅₀ (µg/mL) at 95% probability.

Table 4. Toxicity Regression Equations for Concentration–Effect and EC₅₀ Values of Compounds against A. solani

Compd.	Regression equation^a⁾	R²	EC₅₀ (µg/mL)	95% CI^b⁾	Linear scope (µg/mL)
7f	y=1.2049x+4.4469	0.984	2.88	2.23–3.58	0.78–50
7j	y=1.1219x+3.7652	0.982	12.61	9.72–16.94	0.78–50
7l	y=0.8149x+4.2553	0.983	8.20	6.13–10.97	0.78–50
7o	y=0.7530x+4.7874	0.991	1.92	1.43–2.43	0.78–50
Tebuconazole	y=1.0764x+4.6687	0.966	2.03	1.23–3.34	0.39–25

a) y: Probability of average inhibition rate; x: lg[concentration (µg/mL) ]. b) Confidence interval of EC₅₀ (µg/mL) at 95% probability.

From Table 2, it is clear that the tested compound 7c displayed marked activity against A. alternata with EC₅₀ values of 15.11 µg/mL, and its activity was much lower than that of tebuconazole (EC₅₀=1.51 µg/mL), the positive control. As for F. graminearum, Table 3 indicated that all of the three tested compounds (7j, k, m) exhibited good activities, and their EC₅₀ values were 5.14–16.24 µg/mL. Among them, compound 7k (EC₅₀=5.14 µg/mL) was extremely close to that of tebuconazole (EC₅₀=3.13 µg/mL), the positive control. Regarding A. solani, all four tested compounds (7f, j, l, o) showed excellent activities in Table 4, and their EC₅₀ values ranged from 1.92 to 12.61 µg/mL. Compound 7o in particular exhibited the most potent activity with an EC₅₀ value of 1.92 µg/mL, superior to that of tebuconazole (EC₅₀=2.03 µg/mL), the positive control. The results described above indicate that most of the tested compounds could be promising lead compounds for the development of novel antifungal agents.

SAR

By comparing the results of the EC₅₀ values in Table 2 to 4, with the inhibition rates of all the target compounds at 50 µg/mL in Table 1, it was observed that almost all of the trialkylamine derivatives (7a–s) possessed higher activity against most of the test fungi than their key intermediate 5, which implied that the antifungal activities of the compounds were effectively increased by combining the active structure of 1,2,4-triazole with a tertiary amine group containing benzene rings. Furthermore, the results described above revealed that introduction of different substituents to the benzene ring significantly influenced the antifungal activities. Compared with unsubstituted 7a, the introduction of 4-Cl (7j) or 4-Br (7o) was beneficial for improvement of the activities against all the tested fungi. In addition, the effects of other substituents on the activity depended on their positions on the benzene ring and the species of fungi (Fig. 4).

Fig. 4. Structure–Activity Relationship of Compounds 7b–r

According to the antifungal data against M. grisea, compared with 7a, the introduction of the other substituents to the benzene ring had active influences upon the antifungal activity, except 4-F (7g). However, the introduction of a 3-Me (7c) or 3,4-diCl (7l) group remarkably improved the activity. As for the result against A. solani, introducing electron-donating substituents like Me (7b–d) or OMe (7p–s) remarkably decreased the activity. On the contrary, the presence of 3-F (7f), 4-Cl (7j), 3,4-diCl (7l), or 4-Br (7o) remarkably increased the activity. Regarding the data against F. graminearum, on the whole, the introduction of halogen atoms (7e–o) enhanced the activity, and the 4-chlorinated compound 7j showed the highest activity. However, in most cases, the introduction of the above substituents had little effect on the activity against F. solani compared with the corresponding result of the unsubstituted 7a, and similar conditions were observed in the result against C. lunata. On the contrary, with the exception of compounds with 4-F (7g) and 2-OMe (7p), introducing the substituents described above generally improved the activity against A. alternate, and 3-methyl compound 7c with the highest inhibition rate (87.7%) especially exhibited a contrast with the data of other synthesized derivatives.

Conclusion

The present study used an intermediate derivatization method (IDM) to design and synthesize 19 novel trialkylamine derivatives containing a triazole moiety, and subsequent evaluation of their antifungal activities in vitro against six plant pathogenic fungi (M. grisea, A. solani, F. solani, C. lunata, A. alternata, F. graminearum). Most of the title compounds displayed higher antifungal activities against almost all the test fungi than their intermediate compound 5 and unsubstituted compound 7a at 50 µg/mL. Among them, target compounds 7f, l, and o showed excellent activity against A. solani with EC₅₀ values of 2.88, 8.20, and 1.92 µg/mL, respectively. Compound 7o was superior to tebuconazole (EC₅₀=2.03 µg/mL), a commercial fungicide. Furthermore, the title compounds 7j, k, and m also displayed higher antifungal activities against F. graminearum with EC₅₀ values of 11.60, 5.14, and 16.24 µg/mL. The value of compound 7k was very close to that of tebuconazole (EC₅₀=3.13 µg/mL). The SAR demonstrated that combination of the active structure of 1,2,4-triazole with a tertiary amine group containing benzene rings effectively increased the antifungal activities. Generally, introducing halogen atoms improved the activities against most of the test fungi to varying degrees, while the presence of OMe decreased the activities. Therefore, the present results strongly suggest that the newly synthesized target compounds are potential candidates for the development of new antifungal agents for the effective control of plant fungal pathogens.

Experimental

Materials

Tebuconazole (≥99.1%), a commercial fungicide, was purchased from Yi Fang Biotechnology Co., Ltd. (Zhejiang, China). The starting material, 3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-butan-2-one, was purchased from Jiangsu Yancheng Chemical Factory (Jiangsu, China). Other chemicals used in the present study were purchased locally and were analytical grade and used without further purification.

Apparatus

Melting points (mp) were determined on an XT-4 micro-mp apparatus and uncorrected. IR were obtained on a Bruker TENSOR 27 spectrometer with KBr disks. 1H- and ¹³C-NMR spectra were recorded on a Bruker Avance III 500 MHz NMR with tetramethylsilane (TMS) as an internal standard. Electrospray ionization (ESI)-MS were carried out with a Thermo Fisher LCQ Fleet instrument. High resolution (HR)-MS images were obtained with a Thermo Scientific LTQ Orbitrap instrument.

Synthesis of Compound 2

According to a reported method,^5,20) an aqueous solution of potassium hydroxide (50%, 50 mL) was slowly added to a mixture of 3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-butan-2-one (1) (36.7 g, 0.22 mol), TMSOI; 55 g, 0.25 mol, TBAI; 0.5 g and toluene (120 mL) with stirring, and heated to 70°C for several hours until the reaction was completed according to TLC detection. After cooling to room temperature, the mixture was extracted with 120 mL ethyl acetate. The aqueous phase was also extracted with ethyl acetate (3×60 mL). The combined organic phase was washed with saturated saline solution (3×50 mL) and then dried over anhydrous Na₂SO₄. After filtration, the solvent was removed under vacuum to yield 29.4 g of yellow oil, followed by adding diethyl ether (25 mL), and then recrystallized to yield compound 2.

2-t-Butyl-2-(1H-1,2,4-triazol-1-yl)methyl Oxirane (2): Colorless crystal in 71% yield, mp 49.7–50.6°C (lit.⁵⁾ 49.9–50.7°C). ¹H-NMR (500 MHz, CDCl₃) δ: 8.08 (1H, s), 7.90 (1H, s), 4.66 (1H, d, J=15.1 Hz), 4.48 (1H, d, J=15.1 Hz), 2.71 (1H, d, J=3.6 Hz), 1.88 (1H, d, J=3.6 Hz), 1.04 (9H, s). ¹³C-NMR (125 MHz, CDCl₃) δ: 151.5, 144.8, 62.2, 48.9, 47.5, 32.8, 25.9.

Synthesis of Compound 3

Based on a previously reported method,⁵⁾ compound 2 (18.0 g, 0.1 mol), sodium azide (11.7 g, 0.18 mol) and ammonium chloride (6.4 g, 0.12 mol) were completely dissolved in 100 mL DMF. Afterwards, the mixture was stirred and heated slowly to 60–70°C for 1 h or so until the reaction was completed according to TLC detection, and then cooled to room temperature. The solution was then poured into water (200 mL), and extracted with chloroform (3×80 mL). The combined organic layer was washed with water (3×150 mL), and finally dried over anhydrous Na₂SO₄. After filtration, the solvent was removed under vacuum to yield 1-azido-2-(1,2,4-triazol-1-yl)methyl-3,3-dimethyl-2-butanol (3) as a pale yellow oil. The crude intermediate 3 was directly used in the following step.

Synthesis of Compound 4

A solution of compound 3 (22.4 g, 0.1 mol) and triphenylphosphine (26.2 g, 0.1 mol) in dichloromethane (100 mL) was stirred at room temperature until the reaction was completed according to TLC detection. After removal of the solvent, the residue was recrystallized in ethyl acetate.

1-(Triphenylphosphoranylidene)amino-3,3-dimethyl-2-[(1H-1,2,4-triazol-1-yl)methyl]-2-butanol (4): White crystal in 82% yield, mp 150.3–151.8°C (lit.⁵⁾ 150.7–151.8°C). ¹H-NMR (500 MHz, CDCl₃) δ: 8.30 (1H, s), 7.62 (1H, s), 7.43–7.51 (15H, m), 6.14 (1H, s), 4.36 (1H, d, J=13.9 Hz), 4.30 (1H, d, J=13.9 Hz), 3.07 (1H, t, J=11.3 Hz), 2.96–2.99 (1H, m), 0.90 (9H, s). ¹³C-NMR (125 MHz, CDCl₃) δ: 150.1, 144.6, 132.3, 131.5, 130.5, 128.5, 73.3, 55.2, 45.3, 37.2, 25.3.

Synthesis of Compound 5

A solution of compound 4 (27.4 g, 0.06 mol) and sodium hydroxide aqueous solution (2 mol/L, 80 mL) in methanol (200 mL) was refluxed for approximately 3 h until the reaction was completed according to TLC detection, and then cooled to room temperature. After removal of the solvent under vacuum, toluene (100 mL) and water (100 mL) were added to the residue, followed by acidification with hydrochloric acid (10%) to pH 2–3. After the mixture was partitioned, the aqueous phase was collected and washed with toluene (3×80 mL), alkalized to pH 11–12 with anhydrous sodium carbonate, extracted with dichloromethane (3×80 mL), and then dried over anhydrous Na₂SO₄. After filtration and removal of the solvent, the residue was dissolved with ethyl acetate and recrystallized in a mixed solution of ethyl acetate and n-hexane to yield compound 5.

1-Amino-3,3-dimethyl-2-(1H-1,2,4-triazol-1-yl)methyl-2-butanol (5): White granular crystal in 90% yield,⁵⁾ mp 59.5–60.8°C. ¹H-NMR (500 MHz, CDCl₃) δ: 8.23 (1H, s), 7.92 (1H, s), 5.13 (1H, s), 4.26–4.32 (2H, m), 2.94 (1H, d, J=13.4 Hz), 2.73 (1H, d, J=13.4 Hz), 1.01 (9H, s). ¹³C-NMR (125 MHz, CDCl₃) δ: 151.5, 145.4, 72.7, 55.2, 41.3, 37.1, 25.2. HR-MS [M+H]⁺: Calcd for C₉H₁₉N₄O⁺ 199.1553. Found 199.1552.

General Procedure for the Synthesis of Compounds 6a–s

To a solution of compound 5 (0.4 g, 2 mmol) in 10 mL absolute ethanol benzaldehyde or 1-naphthaldehyde (2.4 mmol) was added dropwise under stirring, and the mixture stirred for 8 h at room temperature. The reaction was detected by TLC. Afterwards, solid NaBH₄ (0.09 g, 2.4 mmol) was added to the resulting solution, and the mixture was stirred for 2 h at room temperature. Ice water was added to the residue, followed by acidification with hydrochloric acid (10%) to pH≈1, and excess solvent was removed under vacuum. Water (15 mL) was then added to the mixture, and the mixture alkalized to pH 8–9 with 10% aqueous sodium hydroxide before it was extracted with dichloromethane (3×20 mL). The combined organic layer was washed with water (3×20 mL) and dried over anhydrous Na₂SO₄. After filtration and removal of the solvent, the desired compounds (6a–s) were obtained as a colorless oil.²¹⁾ The crude intermediates 6a–s were extremely unstable, so 6a–s were directly used in the following step.

General Procedure for the Synthesis of Target Compounds 7a–s

Secondary amine compounds 6a–s (1 mmol) and a 37% formaldehyde aqueous solution (0.15 mL, 2 mmol) were added to 10 mL 88% formic acid. The mixture was heated at reflux for 10 h under stirring until the secondary amine was no longer detected by TLC. After cooling, the mixture was added to water (15 mL), and then alkalized to pH≈12 with 10% aqueous sodium hydroxide, followed by extraction with dichloromethane (3×20 mL). The combined organic layer was washed with water (3×20 mL), and dried over anhydrous Na₂SO₄. After filtration and removal of the solvent, the residue was purified by column chromatography on silica gel by using dichloromethane–ethyl acetate (4 : 1, v/v) as eluent to obtain compounds 7a–s.²²⁾

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-(benzyl(methyl)amino)-3,3-dimethylbutan-2-ol (7a)

Colorless granular crystal in 59% yield, mp 107.1–108°C. IR (KBr) cm⁻¹: 3424, 2959, 1503, 1476. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.51 (s, 1H), 7.95 (s, 1H), 7.29–7.32 (m, 2H), 7.23–7.25 (m, 3H), 4.71 (s, 1H), 4.34 (s, 2H), 3.54 (d, 1H, J=12.9 Hz), 3.28 (d, 1H, J=12.9 Hz), 2.63 (q, 2H, J=14.2 Hz), 1.97 (s, 3H), 0.86 (s, 9H). ¹³C-NMR (125 MHz, dimethyl sulfoxide (DMSO)-d₆) δ: 151.0, 145.8, 139.4, 129.2, 128.7, 127.4, 75.5, 63.3, 58.7, 54.1, 43.6, 37.7, 25.6. HR-MS [M+H]⁺: Calcd for C₁₇H₂₇N₄O⁺ 303.2179. Found 303.2180.

2-((1H-1,2,4-Triazol-1-yl)methyl)-3,3-dimethyl-1-(methyl(2-methylbenzyl)amino)butan-2-ol (7b)

Colorless granular crystal in 43% yield, mp 81.6–83.0°C. IR (KBr) cm⁻¹: 3396, 2958, 1504, 1462. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.45 (s, 1H), 7.94 (s, 1H), 7.21–7.22 (m, 1H), 7.13–7.15 (m, 3H), 4.61 (s, 1H), 4.26 (q, 2H, J=14.0 Hz), 3.56 (d, 1H, J=13.2 Hz), 3.29 (d, 1H, J=13.3 Hz), 2.63 (s, 2H), 2.26 (s, 3H), 2.02 (s, 3H), 0.80 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 150.9, 145.7, 137.4, 137.0, 130.5, 129.8, 127.4, 126.1, 75.7, 61.3, 59.3, 53.9, 44.1, 37.8, 25.5, 19.4. HR-MS [M+H]⁺: Calcd for C₁₈H₂₉N₄O⁺ 317.2336. Found 317.2333.

2-((1H-1,2,4-Triazol-1-yl)methyl)-3,3-dimethyl-1-(methyl(3-methylbenzyl)amino)butan-2-ol (7c)

Colorless oil in 33% yield, IR (KBr) cm⁻¹: 3434, 2959, 1505, 1478. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.51 (s, 1H), 7.96 (s, 1H), 7.19 (t, 1H, J=7.5 Hz), 7.01–7.05 (m, 3H), 4.72 (s, 1H), 4.33 (s, 2H), 3.49 (d, 1H, J=12.4 Hz), 3.24 (t, 1H, J=13.0 Hz), 2.62 (q, 2H, J=14.3 Hz), 2.28 (s, 3H), 1.95 (s, 3H), 0.86 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 151.0, 145.8, 139.3, 137.7, 129.8, 128.6, 128.1, 126.3, 75.4, 63.2, 58.7, 54.1, 43.6, 37.7, 25.6, 21.5. HR-MS [M+H]⁺: Calcd for C₁₈H₂₉N₄O⁺ 317.2336. Found 317.2335.

2-((1H-1,2,4-Triazol-1-yl)methyl)-3,3-dimethyl-1-(methyl(4-methylbenzyl)amino)butan-2-ol (7d)

Colorless granular crystal in 51% yield, mp 75.0–76.1°C. IR (KBr) cm⁻¹: 3443, 2959, 1507, 1476. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.53 (s, 1H), 7.97 (s, 1H), 7.13 (s, 4H), 4.75 (s, 1H), 4.35 (s, 2H), 3.50 (d, 1H, J=12.7 Hz), 3.24 (d, 1H, J=13.0 Hz), 2.63 (q, 2H, J=14.0 Hz), 1.97 (s, 3H), 0.89 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 151.0, 145.8, 136.5, 136.3, 129.3, 129.2, 75.4, 62.9, 58.6, 54.1, 43.4, 37.7, 25.6, 21.2. HR-MS [M+H]⁺: Calcd for C₁₈H₂₉N₄O⁺ 317.2336. Found 317.2334.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((2-fluorobenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7e)

Colorless granular crystal in 45% yield, mp 67.3–67.9°C. IR (KBr) cm⁻¹: 3448, 2961, 1505, 1457. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.49 (s, 1H), 7.95 (s, 1H), 7.37 (td, 1H, J=7.7 Hz, 1.6 Hz), 7.29–7.34 (m, 1H), 7.14–7.19 (m, 2H), 7.20 (td, 1H, J=7.8 Hz, 1.7 Hz), 4.64 (s, 1H), 4.32 (s, 2H), 3.64 (d, 1H, J=13.8 Hz), 3.38 (d, 1H, J=13.2 Hz), 2.65 (q, 2H, J=14.2 Hz), 2.01 (s, 3H), 0.84 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 161.2, 151.0, 145.8, 132.0, 129.6, 125.8, 124.7, 115.7, 75.6, 58.7, 56.1, 53.8, 43.7, 37.7, 25.5. HR-MS [M+H]⁺: Calcd for C₁₇H₂₆FN₄O⁺ 321.2085. Found 321.2085.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((3-fluorobenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7f)

Colorless granular crystal in 35% yield, mp 92.7–93.2°C. IR (KBr) cm⁻¹: 3447, 2961, 1507, 1483. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.53 (s, 1H), 7.96 (s, 1H), 7.35 (q, 1H, J=7.7 Hz), 7.06–7.10 (m, 3H), 4.64 (s, 1H), 4.35 (s, 2H), 3.59 (d, 1H, J=13.5 Hz), 3.28 (d, 1H, J=13.5 Hz), 2.63 (q, 2H, J=14.4 Hz), 1.99 (s, 3H), 0.86 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 162.7, 151.0, 145.8, 142.7, 130.6, 125.1, 115.6, 114.2, 75.8, 62.7, 58.9, 54.0, 43.8, 37.6, 25.6. HR-MS [M+H]⁺: Calcd for C₁₇H₂₆FN₄O⁺ 321.2085. Found 321.2087.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((4-fluorobenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7g)

Colorless granular crystal in 35% yield, mp 94.1–94.9°C. IR (KBr) cm⁻¹: 3435, 2961, 1508, 1477, 1221. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.52 (s, 1H), 7.96 (s, 1H), 7.27–7.29 (m, 2H), 7.13 (t, 2H, J=8.9 Hz), 4.67 (s, 1H), 4.34 (s, 2H), 3.53 (d, 1H, J=13.1 Hz), 3.28 (d, 1H, J=13.1 Hz), 2.62 (q, 2H, J=14.1 Hz), 1.97 (s, 3H), 0.86 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 161.7, 151.0, 145.8, 135.6, 131.1, 115.4, 75.7, 62.4, 58.7, 54.0, 43.6, 37.7, 25.6. HR-MS [M+H]⁺: Calcd for C₁₇H₂₆FN₄O⁺ 321.2085. Found 321.2084.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((2-chlorobenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7h)

Colorless oil in 52% yield. IR (KBr) cm⁻¹: 3422, 2960, 1505, 1474. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.51 (s, 1H), 7.98 (s, 1H), 7.49 (dd, 1H, J=7.6 Hz, 1.6 Hz), 7.44 (dd, 1H, J=7.8 Hz, 1.3 Hz), 7.35 (td, 1H, J=7.4 Hz, 1.3 Hz), 7.30 (td, 1H, J=7.6 Hz, 1.8 Hz), 4.63 (s, 1H), 4.34 (q, 2H, J=14.4 Hz), 3.75 (d, 1H, J=14.0 Hz), 3.49 (d, 1H, J=14.8 Hz), 2.71 (q, 2H, J=14.2 Hz), 2.07 (s, 3H), 0.84 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 151.0, 145.8, 136.8, 133.5, 131.4, 129.7, 129.1, 127.6, 75.9, 60.3, 59.2, 53.8, 44.1, 37.7, 25.5. HR-MS [M+H]⁺: Calcd for C₁₇H₂₆ClN₄O⁺ 337.1790. Found 337.1791.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((3-chlorobenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7i)

Colorless granular crystal in 48% yield, mp 60.3–61.0°C. IR (KBr) cm⁻¹: 3392, 2961, 1505, 1476. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.53 (s, 1H), 7.96 (s, 1H), 7.29–7.34 (m, 3H), 7.22 (d, 1H, J=7.4 Hz), 4.63 (s, 1H), 4.35 (s, 2H), 3.59 (d, 1H, J=13.6 Hz), 3.33 (s, 1H), 2.63 (q, 2H, J=14.2 Hz), 1.99 (s, 3H), 0.86 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 151.0, 145.8, 142.2, 133.4, 130.5, 128.8, 127.8, 127.4, 75.8, 62.7, 58.9, 54.0, 43.8, 37.6, 25.6. HR-MS [M+H]⁺: Calcd for C₁₇H₂₆ClN₄O⁺ 337.1790. Found 337.1787.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((4-chlorobenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7j)

Colorless granular crystal in 64% yield, mp 88.5–90.0°C. IR (KBr) cm⁻¹: 3424, 2960, 1489, 1015, 804, 678. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.52 (s, 1H), 7.96 (s, 1H), 7.37 (d, 2H, J=8.4 Hz), 7.27 (d, 2H, J=8.4 Hz), 4.65 (s, 1H), 4.35 (s, 2H), 3.56 (d, 1H, J=13.6 Hz), 3.31 (s, 1H, J=14.1 Hz), 2.62 (q, 2H, J=14.1 Hz), 1.97 (s, 3H), 0.86 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 151.0, 145.8, 138.5, 131.9, 131.0, 128.6, 75.7, 62.5, 58.9, 54.0, 43.7, 37.7, 25.6. HR-MS [M+H]⁺: Calcd for C₁₇H₂₆ClN₄O⁺ 337.1790. Found 337.1789.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((2,4-dichlorobenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7k)

Colorless oil in 28% yield. IR (KBr) cm⁻¹: 3423, 2960, 1505, 1472. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.52 (s, 1H), 7.98 (s, 1H), 7.59 (d, 1H, J=2.1 Hz), 7.53 (d, 1H, J=8.4 Hz), 7.44 (dd, 1H, J=8.4 Hz, 2.1 Hz), 4.60 (s, 1H), 4.35 (q, 2H, J=14.4 Hz), 3.74 (d, 1H, J=14.4 Hz), 3.49 (d, 1H, J=14.4 Hz), 2.71 (q, 2H, J=14.3 Hz), 2.07 (s, 3H), 0.84 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 151.0, 145.8, 136.1, 134.3, 132.6, 132.5, 129.1, 127.8, 76.1, 59.8, 59.3, 53.7, 44.2, 37.7, 25.6. HR-MS [M+H]⁺: Calcd for C₁₇H₂₅Cl₂N₄O⁺ 371.1400. Found 371.1400.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((3,4-dichlorobenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7l)

Colorless granular crystal in 36% yield, mp 115.9–116.1°C. IR (KBr) cm⁻¹: 3441, 2961, 1505, 1471, 1027, 678, 655. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.53(s, 1H), 7.96 (s, 1H), 7.57 (d, 1H, J=8.5 Hz), 7.52 (d, 1H, J=1.9 Hz), 7.26 (dd, 1H, J=8.2 Hz, 1.9 Hz), 4.61 (s, 1H), 4.36 (s, 2H), 3.60 (d, 1H, J=13.7 Hz), 3.38 (s, 1H), 2.63 (q, 2H, J=15.0 Hz), 2.00 (s, 3H), 0.86 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 151.1, 145.8, 141.0, 131.3, 130.9, 130.8, 129.8, 129.3, 76.0, 62.0, 59.0, 53.9, 43.9, 37.6, 25.6. HR-MS [M+H]⁺: Calcd for C₁₇H₂₅Cl₂N₄O⁺ 371.1400. Found 371.1398.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((2-bromobenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7m)

Colorless oil in 48% yield. IR (KBr) cm⁻¹: 3422, 2959, 1505, 1469, 1023. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.49 (s, 1H), 7.95 (s, 1H), 7.58 (dd, 1H, J=7.9 Hz, 1.1 Hz), 7.47 (dd, 1H, J=7.9 Hz, 1.6 Hz), 7.37 (td, 1H, J=7.5 Hz, 1.1 Hz), 7.20 (td, 1H, J=7.8 Hz, 1.7 Hz), 4.58 (s, 1H), 4.32 (q, 2H, J=14.4 Hz), 3.72 (d, 1H, J=14.1 Hz), 3.47 (d, 1H, J=14.2 Hz), 2.69 (q, 2H, J=14.2 Hz), 2.07 (s, 3H), 0.81 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 151.0, 145.8, 138.4, 133.0, 131.4, 129.4, 128.2, 124.2, 75.9, 62.8, 59.2, 53.8, 44.2, 37.7, 25.5. HR-MS [M+H]⁺: Calcd for C₁₇H₂₆BrN₄O⁺ 381.1284. Found 381.1283.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((3-bromobenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7n)

Colorless oil in 30% yield. IR (KBr) cm⁻¹: 3422, 2960, 1505, 1474. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.55 (s, 1H), 7.99 (s, 1H), 7.45–7.49 (m, 2H), 7.29–7.30 (m, 2H), 4.66 (s, 1H), 4.38 (s, 2H), 3.60 (d, 1H, J=13.4 Hz), 3.35 (s, 1H), 2.64 (q, 2H, J=14.0 Hz), 2.01 (s, 3H), 0.88 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 151.0, 145.8, 142.5, 131.7, 130.9, 130.3, 128.2, 122.1, 75.9, 62.6, 58.9, 53.9, 43.8, 37.7, 25.6. HR-MS [M+H]⁺: Calcd for C₁₇H₂₆BrN₄O⁺ 381.1284. Found 381.1286.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((4-bromobenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7o)

Colorless oil in 26% yield. IR (KBr) cm⁻¹: 3445, 2960, 1506, 1482. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.52(s, 1H), 7.96 (s, 1H), 7.50 (d, 2H, J=8.4 Hz), 7.22 (d, 2H, J=8.4 Hz), 4.64 (s, 1H), 4.35 (s, 2H), 3.54 (d, 1H, J=12.9 Hz), 3.29 (d, 1H, J=13.9 Hz), 2.62 (q, 2H, J=14.2 Hz), 1.97 (s, 3H), 0.86 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 151.0, 145.8, 139.0, 131.6, 131.3, 120.4, 75.8, 62.5, 58.9, 54.0, 43.7, 37.7, 25.6. HR-MS [M+H]⁺: Calcd for C₁₇H₂₆BrN₄O⁺ 381.1284. Found 381.1285.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((2-methoxybenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7p)

Colorless oil in 24% yield. IR (KBr) cm⁻¹: 3439, 2958, 1496, 1463, 1244, 1026. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.49 (s, 1H), 7.93 (s, 1H), 7.24 (td, 1H, J=8.0 Hz, 1.7 Hz), 7.19 (dd, 1H, J=7.4 Hz, 1.5 Hz), 6.97 (d, 1H, J=8.1 Hz), 6.89 (t, 1H, J=7.3 Hz), 4.80 (s, 1H), 4.29 (q, 2H, J=14.4 Hz), 3.76 (s, 3H), 3.53 (d, 1H, J=12.9 Hz), 3.23 (d, 1H, J=13.0 Hz), 2.66 (s, 2H), 1.94 (s, 3H), 0.85 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 157.8, 151.0, 145.8, 131.0, 128.9, 126.8, 120.6, 111.3, 75.2, 58.5, 58.0, 55.7, 54.0, 43.6, 37.7, 25.5. HR-MS [M+H]⁺: Calcd for C₁₈H₂₉N₄O₂⁺ 333.2285. Found 333.2285.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((3-methoxybenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7q)

Colorless oil in 39% yield. IR (KBr) cm⁻¹: 3439, 2959, 1505, 1463, 1244, 1028. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.51 (s, 1H), 7.95 (s, 1H), 7.21 (t, 1H, J=7.6 Hz), 6.79–6.81 (m, 3H), 4.72 (s, 1H), 4.34 (s, 2H), 3.73 (s, 3H), 3.51 (d, 1H, J=12.9 Hz), 3.26 (d, 1H, J=13.6 Hz), 2.62 (q, 2H, J=14.2 Hz), 1.98 (s, 3H), 0.87 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 159.7, 151.0, 145.8, 141.1, 129.7, 121.3, 114.7, 112.8, 75.5, 63.3, 58.8, 55.4, 54.1, 43.7, 37.7, 25.6. HR-MS [M+H]⁺: Calcd for C₁₈H₂₉N₄O₂⁺ 333.2285. Found 333.2284.

2-((1H-1,2,4-Triazol-1-yl)methyl)-1-((4-methoxybenzyl)(methyl)amino)-3,3-dimethylbutan-2-ol (7r)

Colorless oil in 57% yield. IR (KBr) cm⁻¹: 3434, 2959, 1505, 1463, 1236, 1025. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.49 (s, 1H), 7.95 (s, 1H), 7.14 (d, 2H, J=9.1 Hz), 6.86 (d, 2H, J=8.6 Hz), 4.72 (s, 1H), 4.32 (s, 2H), 3.72 (s, 3H), 3.44 (d, 1H, J=12.3 Hz), 3.19 (d, 1H, J=12.3 Hz), 2.60 (q, 2H, J=14.2 Hz), 1.94 (s, 3H), 0.86 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 158.8, 151.0, 145.8, 131.2, 130.4, 114.1, 75.3, 62.5, 58.5, 55.5, 54.2, 43.4, 37.7, 25.5. HR-MS [M+H]⁺: Calcd for C₁₈H₂₉N₄O₂⁺ 333.2285. Found 333.2286.

2-((1H-1,2,4-Triazol-1-yl)methyl)-3,3-dimethyl-1-(methyl(naphthalen-1-ylmethyl)amino)butan-2-ol (7s)

Colorless oil in 38% yield. IR (KBr) cm⁻¹: 3439, 2959, 1505, 1476. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.45 (s, 1H), 8.25 (d, 1H, J=7.9 Hz), 7.94 (s, 1H), 7.90–7.92 (m, 1H), 7.83–7.85 (m, 1H), 7.45–7.54 (m, 4H), 6.97 (d, 1H, J=8.1 Hz), 4.67 (s, 1H), 4.29 (d, 1H, J=14.4 Hz), 4.20 (d, 1H, J=14.4 Hz), 4.10 (d, 1H, J=13.1 Hz), 3.78 (d, 1H, J=13.2 Hz), 2.73 (q, 2H, J=14.1 Hz), 2.10 (s, 3H), 0.75 (s, 9H). ¹³C-NMR (125 MHz, DMSO-d₆) δ: 151.0, 145.7, 135.2, 133.9, 132.3, 128.8, 128.2, 127.8, 126.3, 126.2, 125.8, 124.9, 76.0, 61.6, 59.4, 53.7, 44.5, 37.7, 25.5. HR-MS [M+H]⁺: Calcd for C₂₁H₂₉N₄O⁺ 353.2336. Found 353.2334.

Pharmacology. Assay of Antifungal Activity in Vitro

The in vitro antifungal activity of compounds 5, and 7a–s against six phytopathogenic fungi (A. solani, M. grisea, A. alternate, C. lunata, F. graminearum, F. solani) were investigated using the mycelium linear growth rate method.^2,3,5) The test fungi, provided by the Center of Pesticide Research, Northwest A&F University, China, were maintained on potato dextrose agar (PDA) medium slants and were subcultured for 48 h in Petri dishes prior to testing and used for inoculation of fungal strains on PDA plates. All the test compounds and the tebuconazole (the positive control) were completely dissolved in 0.5 mL DMSO and the solution was added to 9.5 mL of sterile water. The resulting solution was added to 90 mL of melted PDA medium at a temperature below 50°C. After rapid and complete mixing, the medium containing the compounds at a concentration of 50 µg/mL was poured into sterilized Petri dishes for screening. A solution of DMSO without any compounds mixed with PDA served as the blank control. When the medium in the plate was partially solidified, a 5 mm thick and 4 mm diameter disc of fungus cut beforehand from subcultured Petri dishes was placed at the centre of the semi-solid medium. The dishes were kept in an incubator at 28°C for 72 h. Three replicates were performed for each experiment. The growth inhibitory rates were calculated according to the following formula and expressed as the mean±standard deviation (S.D.).

where d_o is the diameter of the fungus cut, d_c is the diameter of a fungal colony in the blank test, and d_s is the diameter of a fungal colony in the compound-treated test.

Antifungal Toxicity Assay

Based on the above results of in vitro antifungal activity, the most effective compounds 7c, f, j, k, l, m, and o were selected to determine their median effective concentration (EC₅₀) according to the same method described above. Each stock solution was respectively mixed with the autoclaved PDA medium to prepare a set of mediums containing 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 µg/mL of the tested compounds. Meanwhile, 0.5% DMSO in culture medium was used as a blank control. Each experiment was performed in triplicate. The concentration (µg/mL) of the compound was transformed to the corresponding logarithm value (lg C). Lg C values for each compound and its corresponding probit values were used to establish a toxicity regression equation by the linear least-square fitting method. EC₅₀ values and their confidence interval at 95% probability (95% CI) were calculated from the toxicity regression equations by using PRISM software ver. 5.0 (GraphPad Software Inc., San Diego, CA, U.S.A.).

Statistical Analysis

PRISM 5.0 statistical software was used to analyze the data and establish toxicity regression equations. Duncan’s multiple comparison test was performed on the data to evaluate significant differences between the activities of various compounds at the same concentration.

Acknowledgment

This work was supported by the Natural Science Research Program of Shaanxi Province of China (No. 2015JM3120).

Conflict of Interest

The authors declare no conflict of interest.

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