Novel Trifluoromethylcoumarinyl Urea Derivatives: Synthesis, Characterization, Fluorescence, and Bioactivity

A series of novel trifluoromethylcoumarinyl urea derivatives were designed, synthesized, and characterized by 1H-NMR, 13C-NMR, and HR-ESI-MS. The fluorescence spectra of the target compounds were recorded. The spectra show that most of the title compounds glow green with λmaxem of 500–517 nm, while compounds 5r, 5s, 5u, and 5l (compounds named by authors) glow violet with λmaxem of 381–443 nm. Moreover, the herbicidal and antifungal activities of the synthesized compounds were evaluated for their potential use as pesticides. The results indicate that compound 5f against the caulis of Amaranthus retroflexus and compounds 5j and 5l against the taproot of Digitaria sanguinalis are equivalent to the commercial herbicide Acetochlor. Nine of the title compounds are more antifungal than commercial fungicide Carbendazim against Botrytis cinerea.

On the other hand, coumarin is a subunit of many natural products and a wide range of synthetic compounds, constituting an important class of heterocycles.These kinds of compounds are widely used in the food, cosmetic, and perfume industries.Coumarins are also an important class of fluorescents and are used as laser dyes, brighteners, fluorescent labels, and other organic optical materials, such as emission layers of organic light-emitting diodes (OLED) [21][22][23][24].Additionally, they are sometimes used as probes to monitor certain physiological process and trace harmful substances in environment [25][26][27].It has been noted that the photophysical and spectroscopic properties of coumarin can be easily adjusted by the introduction of different substituents into the core, which leads them to be more flexible and fit well in various applications [28].It is reported that the electron-withdrawing groups on 3-or 4-position and electron-releasing groups on 6-or 7-position of the coumarin skeleton could enhance the band intensity of fluorescence [29].Moreover, coumarins have been extensively studied for their use as biological agents with anti-depressant, anticoagulant, anti-inflammatory, antibacterial, antifungal, antimalarial, moluscicidal, and anthelmintic activities [30][31][32][33][34][35][36].Much research has been focused on coumarin derivatives for their pesticidal activities too.One of the representative compounds is 7-hydroxyl-4-methylcoumarin, which exhibited interesting pesticidal activities of allelopathy and antifungal, and it is reported that the C7 hydroxyl group and C4 methyl substitution contributed significantly to the activity [37,38].Moreover, brominated derivatives of the compound showed remarkable larvicidal and ovicidal activities against vector mosquitoes [39].Derivatives of 7-hydroxyl-4-methylcoumarin are also reported to show anthelmintic and acaricidal activities [40,41].
In addition, fluorine is the most electronegative of all elements, which endows it with unique properties.Indeed, it is commonly accepted that introducing a fluorine-containing group onto the molecule scaffold often leads to improvement of the molecular chemical, physical, and biological properties [42].To date, the trifluoromethyl (CF 3 ) group has appeared as the archetypal and most sought-after fluorine-containing group, and there have been reported a huge and ever-increasing number of trifluoromethylated compounds in the literature [43,44].The CF 3 group is very important in medicinal chemistry [45] due to its high lipophilicity, easy transportation, and hard degradation in vivo [46,47].Thus, the introduction of a CF 3 group into bioactive molecules has become a preferable approach in pharmaceutical studies [48,49].However, the literature about CF 3 -substituted coumarins is limited, and the reports about these compounds are mainly focused on their use as substrate probes to detect the physiology [50][51][52].Also, in view of these observations and in continuation of our research program on the synthesis of fluorinated coumarins [53], we herein report the synthesis and characterization of some novel 4-trifluoromethyl-6-substituted-ureido-7-methoxylcoumarins, whose fluorescent properties and antifungal and herbicidal activities are also evaluated.

Chemistry
The synthesis of target compounds was carried out according to Scheme 1. Compound 1 was prepared from the reaction of resorcinol with ethyl trifluoroacetoacetate catalyzed by Con.(concentrated) H 2 SO 4 .Compound 2 was synthesized by methylation of compound 1 with dimethyl sulfate using anhydrous K 2 CO 3 as catalyst in acetone.Compound 3 was synthesized by nitration of compound 2 with guanidine nitrate in Con.H 2 SO 4 .Compound 4 was obtained by reduction of compound 3 with Fe powder in aqueous solution of NH 4 Cl.The target compounds 5a-5u were furnished by addition of compounds 4 to a series of isocyanates catalyzed by triethylamine in CH 2 Cl 2 (DCM).The structures of all target compounds were well characterized by 1 H-NMR, 13 C-NMR, and HR-ESI-MS.The spectrograms were listed in the supplementary materials.

UV Absorption and Fluorescence Characteristics
The λ max ab (maximum absorption wavelength), ε max (molar absorptivity), λ max em (maximum emission wavelength), ∆λ (Stokes shift) and Φ f (fluorescence quantum yield) of the title compounds are listed in Table 1.The λ max ab of all the title compounds are 360-374 nm, and the ε max are 5.24-7.67× 10 3 L•mol −1 •cm −1 .Most of the title compounds glow green fluorescence with λ max em of 500-517 nm when illuminated by ultraviolet light, while compounds 5r, 5s, 5u, and 5l glow violet fluorescence with λ max em of 381-443 nm (Figure 1).However compounds 5o and 5t do not fluoresce at the concentration.The values of Φ f indicate that all the title compounds are weaker fluorescent compare to the standard reference compound quinine sulfate.Table 1 shows that alkyl substituted compounds 5a-5i have higher fluorescence quantum yield than the phenyl substituted compounds 5k-5p and 5r-5u, but compounds 5j and 5q with high Φ f were the exceptions.From the phenomenon, it can be concluded that electron-donating groups may favor the fluorescence of the compounds.

Compd
Synthetic route of title compounds.-no fluorescence was detected.

Herbicidal Activities
The herbicidal activities of the 21 title compounds of 20 mg•L −1 and 100 mg•L −1 against the taproot and caulis growth of dicotyledonous weed A. retroflexus and monocotyledonous weed D. sanguinalis were assayed.The inhibition of the compounds with effectiveness of greater than 50% to at least one organ of the test weeds at 100 mg•L −1 are displayed in Figure 2. It indicates that compounds 5j and 5s show medium inhibition against the taproot growth of A. retroflexus, and compounds 5f, 5s, 5k and 5n show medium to high inhibition against the caulis growth of A. retroflexus.Moreover, compounds 5j and 5l show medium inhibition against the taproot growth of D. sanguinalis, and compounds 5f, 5q, and 5r show medium inhibition against the caulis growth of D. sanguinalis.Furthermore, the herbicidal activities of 5f against the caulis of A. retroflexus, 5j and 5l against the taproot of D. sanguinalis are equivalent to the commercial herbicide Acetochlor.It also shows that only compounds 5j and 5s are both active to the two test herbs; the other compounds display certain selectivity between the test herbs.Contrary to the fluorescence, the phenyl substituted compounds show higher herbicidal activity than the alkyl substituted compounds except compound 5f substituted by cyclopentyl.All the compounds substituted by phenyls with chlorine (5j-5l) exhibit better activity.Compounds 5o (3-CH 3 Ph-), 5r (2-CH 3 OPh-) and 5s (3-CH 3 OPh-) substituted by phenyls with electron donating groups are more active.

Herbicidal Activities
The herbicidal activities of the 21 title compounds of 20 mg•L −1 and 100 mg•L −1 against the taproot and caulis growth of dicotyledonous weed A. retroflexus and monocotyledonous weed D. sanguinalis were assayed.The inhibition of the compounds with effectiveness of greater than 50% to at least one organ of the test weeds at 100 mg•L −1 are displayed in Figure 2. It indicates that compounds 5j and 5s show medium inhibition against the taproot growth of A. retroflexus, and compounds 5f, 5s, 5k and 5n show medium to high inhibition against the caulis growth of A. retroflexus.Moreover, compounds 5j and 5l show medium inhibition against the taproot growth of D. sanguinalis, and compounds 5f, 5q, and 5r show medium inhibition against the caulis growth of D. sanguinalis.Furthermore, the herbicidal activities of 5f against the caulis of A. retroflexus, 5j and 5l against the taproot of D. sanguinalis are equivalent to the commercial herbicide Acetochlor.It also shows that only compounds 5j and 5s are both active to the two test herbs; the other compounds display certain selectivity between the test herbs.Contrary to the fluorescence, the phenyl substituted compounds show higher herbicidal activity than the alkyl substituted compounds except compound 5f substituted by cyclopentyl.All the compounds substituted by phenyls with chlorine (5j-5l) exhibit better activity.Compounds 5o (3-CH3Ph-), 5r (2-CH3OPh-) and 5s (3-CH3OPh-) substituted by phenyls with electron donating groups are more active.

Antifungal Activities
The in vitro antifungal activities of the title compounds of 20 mg•L −1 and 100 mg•L −1 against the mycelium growth of the phytopathogens Valsa mali, B. cinerea, Colletotrichum glecosporioides, and Fusarium oxysporum were assayed.The inhibition of the synthesized compounds with effectiveness of greater than 50% at 100 mg•L −1 are exhibited in Figure 3.This indicates that more compounds exhibit stronger inhibition against B. cinerea than the other three plant disease fungi (the inhibitory rates of all the title compounds against F. oxysporum are less than 50%, and they do not appear in Figure 3).There are only four title compounds showing medium inhibition against V. mali, and only one compound having an inhibitory rate of more than 50% to C. glecosporioides.Meanwhile, 12 of the title compounds exhibit medium to high activity against B. cinerea, and nine of them are more antifungal than the commercial fungicide Carbendazim.Based on the above description, it seems that the phenyl substitutes are of more benefit to the antifungal activity.Compounds 5j, 5l, and 5o-5t with substitutes of -Cl, -CH 3 , -CF 3 , or -OCH 3 on phenyls have more potential.

Antifungal Activities
The in vitro antifungal activities of the title compounds of 20 mg•L −1 and 100 mg•L −1 against the mycelium growth of the phytopathogens Valsa mali, B. cinerea, Colletotrichum glecosporioides, and Fusarium oxysporum were assayed.The inhibition of the synthesized compounds with effectiveness of greater than 50% at 100 mg•L −1 are exhibited in Figure 3.This indicates that more compounds exhibit stronger inhibition against B. cinerea than the other three plant disease fungi (the inhibitory rates of all the title compounds against F. oxysporum are less than 50%, and they do not appear in Figure 3).There are only four title compounds showing medium inhibition against V. mali, and only one compound having an inhibitory rate of more than 50% to C. glecosporioides.Meanwhile, 12 of the title compounds exhibit medium to high activity against B. cinerea, and nine of them are more antifungal than the commercial fungicide Carbendazim.Based on the above description, it seems that the phenyl substitutes are of more benefit to the antifungal activity.Compounds 5j, 5l, and 5o-5t with substitutes of -Cl, -CH3, -CF3, or -OCH3 on phenyls have more potential.

General Procedures
All chemicals were obtained from commercial sources and used without further purification.Glass plates pre-coated with silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) were used for analytical thin layer chromatography (TLC).An uncorrected WRS-1B digital melting-point apparatus (Shanghai Precision Optical Instrument Co., Ltd., Shanghai, China) was used to determine the melting points.A Bruker Avance III HD 500 MHz nuclear magnetic resonance instrument (Bruker, Fällanden, Switzerland) was used to record the 1 H-and 13 C-NMR spectra in CDCl3 or DMSO-d6 ( 1 H at 500 MHz and 13 C at 126 MHz) using tetramethylsilane (TMS) as the internal standard.A maXis Q-TOF high-resolution mass spectra instrument (Bruker, Karlsruhe, Germany) was used to measure the HR-ESI-MS.An F-4600 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) was used to record the fluorescence.

Synthesis of 4-Trifluoromethyl-7-hydroxycoumarin (1)
Concentrated sulfuric acid (2 mL) and trifluoroacetoacetate (3.7 g, 10 mmol) were added dropwise in turn to the solution of resorcinol (2.2 g, 10 mmol) in ethanol (4 mL) with stirring.Then the mixture was heated to maintain at 80 °C until the end of the reaction was detected by TLC.The

General Procedures
All chemicals were obtained from commercial sources and used without further purification.Glass plates pre-coated with silica gel 60 GF 254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) were used for analytical thin layer chromatography (TLC).An uncorrected WRS-1B digital melting-point apparatus (Shanghai Precision Optical Instrument Co., Ltd., Shanghai, China) was used to determine the melting points.A Bruker Avance III HD 500 MHz nuclear magnetic resonance instrument (Bruker, Fällanden, Switzerland) was used to record the 1 H-and 13 C-NMR spectra in CDCl 3 or DMSO-d 6 ( 1 H at 500 MHz and 13 C at 126 MHz) using tetramethylsilane (TMS) as the internal standard.A maXis Q-TOF high-resolution mass spectra instrument (Bruker, Karlsruhe, Germany) was used to measure the HR-ESI-MS.An F-4600 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) was used to record the fluorescence.Dimethyl sulfate (2.5 g, 20 mmol) was added dropwise to the suspension of compound 1 (5.8 g, 25 mmol) and anhydrous K 2 CO 3 (4.1 g, 30 mmol) in 66 mL of acetone with stirring.Then the mixture was heated to reflux for 8 h.After K 2 CO 3 was filtered out, the filtrate was rotary evaporated to 1/3 of the total volume.Then the residue was allowed to cool to room temperature until a white solid precipitated.The solid product was filtered out and washed with cold water to neutral pH to get compound 2. Yield 97.8%, m.p. 110-112 • C (lit.111-112 • C) [55].

Synthesis of 4-Trifluoromethyl-6-nitro-7-methoxycoumarin (3)
Guanidine nitrate (1.4 g, 11.6 mmol) was added batch-wise to the solution of 2 (2.6 g, 10.7 mmol) in 16 mL Conc.H 2 SO 4 with stirring at 0-5 • C. The mixture was stirred at room temperature for 3 h until the end of the reaction.Then the reaction mixture was poured into 40 mL of ice water with stirring to obtain a pale yellow solid.The solid product was filtered and dried to give compound 3. Yield 97.7%, m.p. 118-119 • C (as reported) [53].

Synthesis of 4-Trifluoromethyl-6-amino-7-methoxycoumarin (4)
The mixture of compound 3 (2.9 g, 10 mmol), iron powder (5.0 g, 90 mmol), NH 4 Cl (1.1 g, 20 mmol) and 50 mL water was mechanically stirred at room temperature until the end of the reaction.The mixture was extracted with ethyl acetate (50 mL × 3).The extract was dried over anhydrous sodium sulfate, then filtered and rotary evaporated to get a dark yellow crystal of compound 4. Yield 70%, m.p. 179-180 • C (as reported) [53].The compound is unstable and needs to be used as soon as possible for the next step.

General Procedure for Synthesis of 4-Trifluoromethyl-6-amino-7-methoxycoumarinyl Urea Derivatives (5a-5u)
To a cool suspension of 4 (0.65 g, 2.5 mmol) in 3.5 mL of anhydrous dichloromethane, isocyanate (3 mmol) was added dropwise with stirring.The mixture was allowed to return to room temperature until the end of the reaction.The solid produced was filtered and chromatographed on a flash silica gel column (ethyl acetate/petroleum ether = 1:3) to give the target compounds 5a-5u.The yield, appearance, m.p., NMR and HR-ESI-MS (MS) date of the synthesized compounds are listed below.

Determination of UV Absorption and Fluorescence Spectrum
The title compounds were dissolved in methanol to prepare 1 and 10 µg•mL −1 solutions, and the same concentrations of the reference compound, quinine sulfate, were obtained using 0.1 mol•L −1 H 2 SO 4 aqueous solution as the solvent.The 10 µg•mL −1 solutions of all the compounds were used to record UV absorption spectra.The intersection point wavelength of UV spectrums of title compound and quinine sulfate that lay between 300 to 400 nm was selected as the excitation wavelength to record the fluorescence spectrum.The 1 µg•mL −1 solutions were used to determine the fluorescence to ensure the absorbance was less than 0.05.The refraction indexes (n) of the 1 µg•mL −1 solutions were also measured to calculate the Φ f .∆λ and Φ f were calculated according to the following formulas [56]: ∆λ = λ max em − λ max ab (1) where n x , n std -refraction index of title compound and reference compound, respectively; D x , D std -fluorescence peak area of title compound and reference compound, respectively; Φ fstd -fluorescence quantum efficiency of reference compound with value of 0.55.

Table 1 .
Properties of UV absorption and fluorescence of title compounds.

Table 1 .
Properties of UV absorption and fluorescence of title compounds.

Table 1 .
Properties of UV absorption and fluorescence of title compounds.