CRBOC Journal

1.       Introduction

Urea derivatives have played a pivotal role in pesticide chemistry due to their significant biological activities, such as herbicidal activities [1-2], antimicrobial [3], and antiviral [4-6]. There were many structural optimization studies on changing both sides of carbamide bridge’s amines. It has been reported that substituted N-nitroanilines have a borad range of biological effects, including herbicidal properties [7-8], plant growth regulating activities and antifungal effects [9-10].

Additionally, in recent years, amino acid derivatives have received considerable attention because of their applications in pharmacological, food additives and agricultural field [11-12]. While the activities depended mainly on the effect of the amino acid groups [13], and it was also mentioned that attachment of an amino acid to a drug enhances its cellular uptake [14]. Furthermore, amino acid ester derivatives have wide market potential according to Chinese custom export products related statistics and have more and more extensive applications [15]. Our group are actively engaged in studying on the synthesis of N-nitrourea derivatives for a long time [16-17].

 3.1.  Aim of the Work 

As a part of our extensive research, and in order to look for higher biological activity compounds, we herein introduce the amino acid ester group to the NH-CO-NH linkage with a nitro. We have linked the active group N-nitro aryl amine with plant growth regulating and herbicidal activities, amino acid ester group to urea substructure, retained a class of novel N-nitro amino acid ethyl ester urea derivatives.

2.                   Experimental

All reagents and chemicals were commercially available from Across, Aldrich, Shanghai or Beijing chemical company and all solvents and liquid reagents were dried by standard methods and distilled before use.

Melting points (uncorrected) were determined on an XRC-1 apparatus (Sichuan University Scientific Instruments Factory, Chengdu, China). MS were measured on a Finnigan Trace MS spectrometer. IR spectra were recorded on a PE-983 infrared spectrometer as KBr pellets with absorption in cm^-1. NMR were recorded in CDCl₃ or DMSO-d₆ on a Bruker DPX 600 spectrometer and resonances are given in ppm (d) relative to TMS. Elementary analyses were taken on a Vario EL III elementary analysis instrument.

2.1    General Synthetic Procedure for 2,4,6-trichlorophenylnitramine 2

The key intermediate 2,4,6-trichlorophenylnitramine 2 was obtained according to the reference [9]. Fuming nitric acid (1.01 mL, 24 mmol) was added dropwise to the stirred acetic anhydride (2.27 mL, 24 mmol) at 10 ~ 12^oC. After the addition, the temperature of the reaction mixture was maintained between 10 and 12^oC for 40 min. And a crude product of acetyl nitrate was obtained and was used directly for the next step. The obtained acetyl nitrate was added dropwise to a solution of substituted aniline (20 mmol) in dry acetic acid (20 mL) and acetic anhydride (2 mL) at 16 ~ 18^oC. The reaction mixture was stirred for a further 45 ~ 90 min. The resulting purple solution was then poured into 60 mL of water (0 ^oC), and the resulting precipitate was filtered, washed with water (1L), and dissolved in aqueous 10% sodium carbonate, then acidified with ice-cold 2N hydrochloric acid to precipitate the substituted Phenylnitramine 2, and recrystallized from cyclohexane. The Physico-chemical spectral data and melting point of compounds 2 was in agreement with the data reported in the literature [9]. 

Compounds 5 were prepared according to the reference [18]: SOCl₂ (1.8 ml, 26 mmol) was added dropwise to a solution of respective α-L-amino acids (20 mmol) 4 in EtOH (30 mL) at -8 ~ 10^oC. After the addition, the mixture was refluxed for 5 ~ 7 h, and the solvent was evaporated under reduced pressure. The resulting residue was re dissolved in ethyl ether and the solvents were evaporated again to remove EtOH completely. The intermediate 5 were formed and were used in the next step without further purification.

2.2    General Procedure for the Preparation of the Target Compounds 6a-w 

A solution of trisubstituted phenylnitramine 2 (10 mmol) in 50 mL of anhydrous toluene with 2 mL triethylamine was added dropwise to the solution of triphosgene (3.6 mmol) in dry toluene (50 mL) over a period of 2 h at 0 ~ 5^oC. After a further 1 h of stirring at room temperature and 2 h at 50^oC. The intermediate 3 was formed after quenching the unreacted phosgene with dry nitrogen, then a mixture of 10 mmol of triethylamine (2 or 3 molar equivalents) and a suitable α-amino acid ethyl ester hydrochloride 5 in CH₂Cl₂(or CH₃CN or CHCl₃ or DMF, details are in Table 1) (10 mL) was added. The mixture was stirred for 2 ~ 7 h at 30 ~ 80^oC. After cooling to room temperature, the mixture was evaporated to dryness under reduced pressure, diluted with 80 ml ethyl acetate, washed with saturated salt solution (50 mL × 3) ，dried over anhydrous Na₂SO₄, filtered, the solvent was removed to yield the crude product 6a-w, Crude products were purified by recrystallization with acetone/H₂O or on a silica gel column using petroleum/ethyl acetate (6:1 ~ 3:1) as a solvent system. 

2.3    Ethyl 2-(3-Nitro-3-(2,4,6-Trichlorophenyl) Ureido) Acetate (6a) 

White needle crystal. IR (KBr, cm^-1) ν: 3366(N-H), 1726(C=O ester), 1645(C=O urea), 1585(C=C Phenyl), 1257(N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 7.67 (s, 2H, ArH), 6.71 (s, 1H, NH), 4.09 (q, J =10.5 Hz, 2H, OCH₂), 3.83 (d, J = 7.8 Hz, 2H, NCH₂), 1.19 (t, J = 9.9 Hz, 3H, CH₃). ¹³C NMR (DMSO-d₆, 150 MHz) δ 14.0, 41.7, 60.2, 128.0, 131.2, 133.2, 134.7, 154.7, 170.4. Anal. calcd for C₁₁H₁₀Cl₃N₃O₅:C 35.65, H 2.72, N 11.34; found C 35. 73, H 2.85，N 11.27.

2.4    Ethyl 2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)-3-Phenylpropanoate (6b) 

White solid. IR (KBr, cm^-1) ν: 3339(N-H), 2974, 2938(-CH₂), 1732(C=O ester), 1646(C=O urea), 1566 (C=C Phenyl), 1225(N-NO₂). ¹H NMR (CDCl₃, 600 MHz) δ 7.36-7.10 (m, 7H, ArH), 6.39 (s, 1H, NH), 4.82-4.76 (m,1H, NCH), 4.17 (d, J = 10.2 Hz, 2H, OCH₂), 3.18-3.12 (m, 2H, PhCH₂), 1.26 (s, 3H, CH₃). ¹³C NMR (CDCl₃, 150 MHz) δ 14.1, 38.2, 54.1, 61.6, 127.0, 128.4, 128.5, 129.5, 131.4, 133.0, 134.6, 135.9, 153.9, 172.1; Anal. calcd for C₁₈H₁₆Cl₃N₃O₅:C 46.93, H 3.50, N 9.12; found C 46.98, H 3.57, N 9.03.  

2.5    Ethyl 2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)Propanoate (6c) 

White crystal. IR (KBr, cm^-1) ν: 3327(N-H), 2997(-CH₃), 1731(C=O ester), 1643(C=O urea), 1572(C=C Phenyl), 1226(N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 7.67 (s, 2H, ArH), 6.82 (d, J = 7.2 Hz, 1H, NH), 4.17-4.14 (m, 1H, NCH), 4.07 (q, J = 6.9 Hz, 2H, OCH₂), 1.27 (d, J = 7.2 Hz, 3H, CH₃), 1.16 (t, J = 7.2 Hz, 3H, CH₃). Anal. calcd for C₁₂H₁₂Cl₃N₃O₅:C 37.47, H 3.14, N 10.93; found C 37.55, H 3.18, N 10.81. 

2.6    Ethyl 3-Methyl-2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)Butanoate (6d)

White solid. IR (KBr, cm^-1) ν: 3345(N-H), 2964(-CH₃), 1735(C=O ester), 1646(C=O urea), 1577 (C=C Phenyl), 1202(N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ7.69 (s, 2H, ArH), 6.79 (s, 1H, NH), 4.12-4.08 (m, 3H, OCH₂, NCH), 2.03 (q, J = 6.0 Hz, 1H, CH), 1.18 (t, J = 7.2 Hz, 3H, CH₃), 0.86 (t, J = 6.9 Hz, 6H, 2CH₃). Anal. calcd for C₁₄H₁₆Cl₃N₃O₅:C 40.75, H 3.91, N 10.18; found C 40.81, H 3.97, N 10.13. 

2.7    Ethyl 4-(Methylthio)-2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)Butanoate (6e) 

White crystal. IR (KBr, cm^-1) ν: 3346(N-H), 1718(C=O ester), 1649(C=O urea), 1572 (C=C Phenyl), 1241(N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 7.67 (s, 2H, ArH), 6.88 (d, J = 7.8 Hz, 1H, NH), 4.27 (q, J = 6.6 Hz, 1H, NCH), 4.08 (q, J =6.6 Hz, 2H, OCH₂), 2.02 (s, 3H, SCH₃), 1.96-1.80 (m, 4H, CH₂, SCH₂), 1.17 (t, J = 6.9 Hz, 3H, CH₃). Anal. calcd for C₁₄H₁₆Cl₃N₃O₅S:C 37.81, H 3.63, N 9.45; found C 37.87, H 3.68, N 9.34.

2.8    Ethyl 1-(Nitro(2,4,6-Trichlorophenyl)Carbamoyl)Pyrrolidine-2-Carboxylate (6f) 

Brown solid. IR (KBr, cm^-1) ν: 2978(-CH₂), 1754(C=O ester), 1648(C=O urea), 1513 (C=C Phenyl), 1395(N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 7.68 (s, 2H, ArH), 4.26 (m, 1H, NCH), 4.03 (q, J =7.5 Hz, 2H, OCH₂), 3.54-3.46(m, 2H, NCH₂), 2.20-2.15 (m, 2H, CH₂), 1.95-1.92 (m, 2H, CH₂), 1.86-1.82 (m, 2H, CH₂), 1.14 (t, J = 6.9 Hz, 3H, CH₃). Anal. calcd for C₁₄H₁₄Cl₃N₃O₅:C 40.95, H 3.44, N 10.23; found C 41.02, H 3.48, N 10.17. 

2.9    Diethyl 2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)Succinate (6g) 

White crystal. IR (KBr, cm^-1) ν: 3356(N-H), 1741, 1720(C=O ester), 1654(C=O urea), 1570 (C=C Phenyl), 1299(N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 7.68 (s, 2H, ArH), 6.89 (d, J = 8.4 Hz, 1H, NH), 4.54 (q, J = 6.9 Hz, 1H, NCH), 4. 05-4.01 (m, 4H, 2OCH₂), 2.78 (d, J = 5.4 Hz, 2H, CH₂), 1.16 (q, J = 6.0 Hz, 6H, 2CH₃). Anal. calcd for C₁₅H₁₆Cl₃N₃O₇:C 39.45, H 3.53, N 9.20; found C 39.49, H 3.58, N 9.12. 

2.10  Diethyl 2-(3-Nitro-3-(2,4,6-Trichlorophenyl) Ureido) Pentanedioate (6h) 

White needle crystal. IR (KBr, cm^-1) ν: 3337(N-H), 1734, 1718(C=O ester), 1647(C=O urea), 1583 (C=C Phenyl), 1263(N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 7.68 (s, 2H, ArH), 6.86 (d, J = 8.4 Hz, 1H, NH), 4.19 (q, J = 6.9 Hz, 1H, NCH), 4.05 (dd, J = 6.3 Hz, 2H, 2OCH₂), 2.36 (t, J = 7.2 Hz, 2H, CH₂), 1.98-1.79 (m, 2H, CH₂), 1.18~1.12 (m, 6H, 2CH₃). Anal. calcd for C₁₆H₁₈Cl₃N₃O₇:C 40.83, H 3.85, N 8.93; found C 40.88, H 3.89, N 8.87. 

2.11 Ethyl 3-Methyl-2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)Pentanoate (6i) 

White solid. IR (KBr, cm^-1) ν: 3345(N-H), 2966(-CH₃), 1735(C=O ester), 1646(C=O urea), 1577 (C=C Phenyl), 1260(N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 7.69 (s, 2H, ArH), 6.81 (d, J = 7.8 Hz, 1H, NH), 4.19-4.16 (m, 1H, NCH), 4.07 (q, J = 6.6 Hz, 2H, OCH₂), 1.69-1.63 (m, 1H, CH), 1.52-1.47 (m, 2H, CH₂), 1.16 (t, J = 6.9 Hz, 3H, CH₃), 0.92~0.80 (m, 6H, 2CH₃). Anal. calcd for C₁₅H₁₈Cl₃N₃O₅:C 42.22, H 4.25, N 9.85; found C 42.28, H 4.31, N 9.81. 

2.12 Ethyl 4-Methyl-2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)Pentanoate (6j) 

White solid. IR (KBr, cm^-1) ν: 3314(N-H), 2952(-CH₃), 1728(C=O ester), 1641(C=O urea), 1562 (C=C Phenyl), 1224 (N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 7.66 (s, 2H, ArH), 6.78 (d, J = 7.8 Hz, 1H, NH), 4.15-4.07 (m, 3H, OCH₂, NCH), 1.69-1.63 (m, 1H, CH), 1.41-1.36 (m, 2H, CH₂), 1.17 (t, J = 8.4 Hz, 3H, CH₃), 0.86 (d, J = 7.2 Hz, 6H, 2CH₃). Anal. calcd for C₁₅H₁₈Cl₃N₃O₅:C 42.22, H 4.25, N 9.85; found C 42.27, H 4.31, N 9.80. 

2.13  Ethyl 3-(1H-Indol-3-Yl)-2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)Propanoate (6k)

White solid. IR (KBr, cm^-1) ν: 3363(N-H), 2921(-CH₂), 1725(C=O ester), 1647(C=O urea), 1560, 1544(C=C Phenyl), 1202 (N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 10.93 (s, 1H, indole NH), 7.67 (s, 2H, ArH), 7.47 (d, J = 8.4 Hz, 1H, ArH), 7.32 (d, J = 8.4 Hz, 1H, ArH), 7.13 (s, 1H, indole NCH), 7.04 (t, J = 7.5 Hz, 1H, ArH), 6.95 (t, J = 7.2 Hz, 1H, ArH), 6.69 (d, J = 7.8 Hz, 1H, NH), 4.47 (d, J = 6.6 Hz, 1H, NCH), 4.01 (q, J = 3.6 Hz, 2H, OCH₂), 3.12 (t, J = 3.0 Hz, 2H, CH₂), 1.09 (t, J = 7.2 Hz, 3H, CH₃). Anal. calcd for C₂₀H₁₇Cl₃N₄O₅:C 48.07, H 3.43, N 11.21; found C 48.12, H 3.49, N 11.14. 

2.14  Ethyl 3-(4-Hydroxyphenyl)-2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)Propanoate (6l) 

White solid. IR (KBr, cm^-1) ν: 3355(N-H), 2977(-CH₂), 1726(C=O ester), 1654(C=O urea), 1570, 1513(C=C Phenyl), 1231 (N-NO₂). ¹H NMR (DMSO-d₆,600 MHz) δ 9.27 (s, 1H, OH), 7.70 (s, 2H, ArH), 6.99 (d, J = 8.4 Hz, 2H, ArH), 6.68 (t, J = 8.4 Hz, 3H, NH, ArH), 4.38 (q, J = 6.8 Hz, 1H, NCH), 4.08 (d, J = 7.2 Hz, 2H, OCH₂), 2.90 (q, J = 5.3 Hz, 2H, PhCH₂),1.16 (t, J = 7.2 Hz, 3H, CH₃). Anal. calcd for C₁₈H₁₆Cl₃N₃O₆:C 45.35, H 3.38, N 8.81; found C 45.39, H 3.45, N 8.72. 

2.15  Ethyl 3-Hydroxy-2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)Butanoate (6m)

White solid. IR (KBr, cm^-1) ν: 3475(-OH), 3328(N-H), 2984(-CH₃), 1718(C=O ester), 1653(C=O urea), 1576(C=C Phenyl), 1281 (N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 7.66 (s, 2H, ArH), 6.61 (d, J = 6.8 Hz, 1H, NH), 5.10 (d, J = 4.8 Hz, 1H, OH), 4.13-4.05 (m, 4H, OCH₂, CH, NCH), 1.17 (t, J = 6.9 Hz, 3H, CH₃), 1.09 (d, J = 6.0 Hz, 3H, CH₃). Anal. calcd for C₁₃H₁₄Cl₃N₃O₆: C 37.66, H 3.40, N 10.13; found C 37.74, H 3.45, N 10.09. 

2.16  Ethyl 4-Amino-2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)-4-Oxobutanoate (6n) 

White solid. IR (KBr, cm^-1) ν: 3356(N-H), 1741(C=O amide), 1735(C=O ester), 1648(C=O urea), 1560(C=C Phenyl), 1299(N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 7.68 (s, 2H, ArH), 6.90 (s, 1H, NH), 4.59-4.50 (m, 1H, NCH), 4.10-4.04 (m, 4H, OCH₂, NH₂), 2.78 (s, 2H, CH₂), 1.18 (t, J = 7.2 Hz, 3H, CH₃). Anal. calcd for C₁₃H₁₃Cl₃N₄O₆:C 36.51, H 3.06, N 13.10; found C 36.57, H 3.03, N 13.02. 

2.17  Ethyl 5-Amino-2-(3-Nitro-3-(2,4,6-Trichlorophenyl)Ureido)-5-Oxopentanoate (6o) 

White solid. IR (KBr, cm^-1) ν: 3345(N-H), 1735(C=O ester), 1718(C=O amide), 1647(C=O urea), 1577(C=C Phenyl), 1264 (N-NO₂). ¹H NMR (DMSO-d₆, 600 MHz) δ 7.68 (s, 2H, ArH), 6.85 (d, J = 7.8 Hz, 1H, NH), 4.19 (d, J = 5.4 Hz, 1H, NCH), 4.09- 4.02 (m, 4H, NH₂, OCH₂), 2.36 (d, J = 7.2 Hz, 2H, CH₂), 2.01-1.79 (m, 2H, CH₂), 1.18 (t, J = 7.2 Hz, 3H, CH₃). Anal. calcd for C₁₄H₁₅Cl₃N₄O₆:C 38.07, H 3.42, N 12.69; found C 38.11, H 3.47, N 12.64. 

2.18  Ethyl 2-(3-(2,6-Dibromo-4-Fluorophenyl)-3-Nitroureido)Acetate (6p) 

White solid. IR (KBr, cm^-1) ν: 3355 (N-H), 2979 (CH₂), 1724 (C=O ester), 1637 (C=O urea), 1515 (C=C Phenyl), 1210 (N-NO₂) cm^-1; ¹H NMR (DMSO-d₆, 600 MHz) δ 7.72 (d, J = 7.8 Hz, 2H, ArH), 6.66 (s, 1H, NH), 4.11 (q, J = 7.1 Hz, 2H, OCH₂), 3.83 (d, J = 4.1 Hz, 2H, NCH₂), 1.20 (t, J = 7.2 Hz, 3H, CH₃). Anal. calcd for C₁₁H₁₀Br₂FN₃O₅: C 29.82, H 2.28, N 9.48; found C 29.67, H 2.35，N 9.30. 

2.19  Ethyl 2-(3-Nitro-3-(2,6-Dibromo-4-Fluorophenyl)Ureido)-3-Phenylpropanoate (6q)

White solid. IR (KBr, cm^-1) ν: 3347 (N-H), 2976, 2936 (CH₂), 1734 (C=O ester),1645 (C=O urea), 1551 (C=C Phenyl), 1223 (N-NO₂) cm^-1; ¹H NMR (DMSO-d₆, 600 MHz) δ 7.70 (d, J = 8.4 Hz, 2H, ArH), 7.33-7.29 (m, 2H, ArH), 7.25-7.21 (m, 3H, ArH), 6.69 (s, 1H, NH), 4.47 (d, J = 6.6 Hz, 1H, NCH), 4.08 (t, J = 7.2 Hz, 2H, OCH₂), 3.02 (q, J = 5.5 Hz, 2H, PhCH₂), 1.16 (t, J = 7.2 Hz, 3H, CH₃). Anal. calcd for C₁₈H₁₆Br₂FN₃O₅:C 40.55, H 3.02, N 7.88; found C 40.63, H 3.11, N 7.76.  

2.20  Ethyl 2-(3-(2,6-Dibromo-4-Fluorophenyl)-3-Nitroureido)Propanoate (6r) 

White solid. IR (KBr) ν: 3333 (N-H), 2992 (CH₃), 1727 (C=O ester), 1641 (C=O urea),1570 (C=C Phenyl), 1223 (N-NO₂) cm^-1; ¹H NMR (DMSO-d₆, 600 MHz) δ 7.71 (d, J = 8.4 Hz, 2H, ArH), 6.76 (s, 1H, NH), 4.19 (t, J = 7.5 Hz, 1H, NCH), 4.13-4.08 (m, 2H, OCH₂), 1.30 (d, J = 7.2 Hz, 3H, CH₃), 1.20 (t, J = 7.2 Hz, 3H, CH₃). Anal. calcd for C₁₂H₁₂Br₂FN₃O₅:C 31.53, H 2.65, N 9.19; found C 31.45, H 2.55, N 9.31. 

2.21  Ethyl 2-(3-(2,6-Dibromo-4-Fluorophenyl)-3-Nitroureido)-4-(Methylthio)Butanoate (6s) 

White solid. IR (KBr, cm^-1) ν: 3328 (N-H), 1718 (C=O ester), 1647 (C=O urea), 1556 (C=C Phenyl), 1206 (N-NO₂) cm^-1; ¹H NMR (DMSO-d₆, 600 MHz) δ 7.72 (d, J = 8.4 Hz, 2H, ArH), 6.83 (s, 1H, NH), 4.20 (q, J = 4.1 Hz, 1H, NCH), 4.12 (q, J =6.0 Hz, 2H, OCH₂), 2.06 (s, 3H, SCH₃), 1.97 (t, J = 6.0 Hz, 2H, SCH₂), 1.92-1.89 (m, 2H, CH₂), 1.21 (t, J = 7.2 Hz, 3H, CH₃). Anal. calcd for C₁₄H₁₆Br₂FN₃O₅S:C 32.51, H 3.12, N 8.13; found C 32.70, H 3.19, N 8.02. 

2.22  Diethyl 2-(3-(2,6-Dibromo-4-Fluorophenyl)-3-Nitroureido)Succinate (6t) 

White solid. IR (KBr, cm^-1) ν: 3356 (N-H), 2980 (CH₂), 1735, 1718 (C=O ester), 1647 (C=O urea),1572 (C=C Phenyl), 1293 (N-NO₂) cm^-1; ¹H NMR (DMSO-d₆, 600 MHz) δ 7.70 (d, J = 7.8 Hz, 2H, ArH), 6.83 (s, 1H, NH), 4.56-4.52 (m, 1H, NCH), 4.09-4.05 (m, 4H, 2OCH₂), 2.78 (d, J = 6.0 Hz, 2H, CH₂), 1.18-1.15 (m, 6H, 2CH₃). Anal. calcd for C₁₅H₁₆Br₂FN₃O₇:C 34.05, H 3.05, N 7.94; found C 34.17, H 3.11, N 7.80.

2.23  Ethyl 2-(3-(2,6-Dibromo-4-Fluorophenyl)-3-Nitroureido)-3-(4-Hydroxyphenyl)Propanoate (6u) 

White solid. IR (KBr, cm^-1) ν: 3355 (N-H), 2979 (CH₂), 1724 (C=O ester), 1654 (C=O urea), 1589,1513 (C=C Phenyl), 1210 (N-NO₂) cm^-1; ¹H NMR (DMSO-d₆, 600 MHz) δ 9.23 (s, 1H, OH), 7.69 (d, J = 7.8 Hz, 2H, ArH), 6.97 (d, J = 8.4 Hz, 2H, ArH), 6.66 (d, J = 8.4 Hz, 2H, ArH), 6.58 (s, 1H, NH), 4.39-4.34 (m, 1H, NCH), 4.05 (d, J = 7.2 Hz, 2H, OCH₂), 2.89-2.86 (m, 2H, PhCH₂), 1.13 (t, J = 7.2 Hz, 3H, CH₃). Anal. calcd for C₁₈H₁₆Br₂FN₃O₆:C 39.37, H 2.94, N 7.65; found C 39.48, H 3.01, N 7.42.

2.24  Ethyl 2-(3-(2,6-Dibromo-4-Fluorophenyl)-3-Nitroureido)-3-Hydroxybutanoate (6v)

White solid. IR (KBr, cm^-1) ν: 3466 (OH), 3332 (N-H), 2984 (CH₃), 1715 (C=O ester), 1655 (C=O urea), 1576 (C=C Phenyl), 1286 (N-NO₂) cm^-1; ¹H NMR (DMSO-d₆, 600 MHz) δ 7.72 (d, J = 7.8 Hz, 2H, ArH), 6.58 (s, 1H, NH), 5.12 (s, 1H, OH), 4.18-4.09 (m, 4H, OCH₂, CH, NCH), 1.21 (t, J = 6.9 Hz, 3H, CH₃), 1.13 (d, J = 6.6 Hz, 3H, CH₃). Anal. calcd for C₁₃H₁₄Br₂FN₃O₆:C 32.06, H 2.90, N 8.63; found C 32.18, H 2.97, N 8.42.

2.25  Ethyl 4-Amino-2-(3-(2,6-Dibromo-4-Fluorophenyl)-3-Nitroureido)-4-Oxobutanoate (6w) 

White solid. IR (KBr, cm^-1) ν: 3355 (N-H), 1735 (C=O ester), 1720 (C=O amide), 1640 (C=O urea), 1547 (C=C Phenyl), 1192 (N-NO₂) cm^-1; ¹H NMR (DMSO-d₆, 600 MHz) δ 7.71 (d, J = 8.4 Hz, 2H, ArH), 6.84 (s, 1H, NH), 4.56-4.52 (m, 1H, NCH), 4.11-4.05 (m, 4H, OCH₂, NH₂), 2.77 (d, J = 6.0 Hz, 2H, CH₂), 1.17 (t, J = 7.2 Hz, 3H, CH₃). Anal. calcd for C₁₃H₁₃Br₂FN₄O₆:C 31.22, H 2.62, N 11.20.; found C 31.13, H 2.45, N 11.32. 

3.       Results and Discussion 

3.1    Synthesis of Novel N-Nitro Urea Derivatives Incorporating Amino Acid Ethyl Esters 6a-w 

The reported substituted Phenylnitramines which exhibit varieties of bioactivities and stability, are often trisubstituted (ortho- and para-position) phenylnitramines. The preferred substituents commonly are halogen, with the proviso that no more than two of the groups may represent iodine [9]. We preciously synthesized a series of unsymmetrical aryl ureas which contain 2,4,6-trisubstituted phenylnitramine. In continuation of our effort to develop N-nitro urea derivatives and in favor of forceful contrast, the synthetic route of title compounds is shown in Scheme 1. To synthesize urea derivatives, there are several known methods. Our choice is based on BTC (triphosgene), which was reported to possess advantages of easy quantitative and controlled, safe, mild reaction conditions, provide a convenient and safe ‘one-pot’ procedure for the synthesis of N, N’ -unsymmetrically substituted urea [19-21]. 

The trisubstituted phenylnitramines 2 are prepared from 1 with acetyl nitrate by react with nitric acid in the presence of acetic anhydride. Then the key intermediates 2 were treated with Triphosgene to obtain various N-nitro-2,4,6-trisubstituted phenyl carbamic chloride 3, which were directly reacted with various L-amino acid ethyl ester in different solvent to afford target compounds 6a-w in satisfied yields (55-80%).

Scheme 1: General synthetic route for amino-acid containing derivatives 6a-w. Reagents and conditions: ( i ) CH₃COOH, Ac₂O, nitric acid, 10 ~ 12^°C, 40 min, then 16 ~ 18^°C, 45 ~ 90 min; ( ii ) Triphosgene, toluene, NEt₃, 0 ~ 5^°C, 2h, then 25 ~ 50^°C, 3h; ( iii ) SOCl₂, EtOH, reflux, 5-7h; ( iv ) Substituted amino acid esters, NEt₃, CH₂Cl₂(or CH₃CN or CHCl₃ or DMF), 30 ~ 80^°C, 2-7h. 

Amino acid is hardly dissolved in non-polarity solvents. However, the esters have preferable dissolution in organic solvents which are in favor of the synthesis. Amino acid ethyl ester hydrochloride was prepared from the esterification of corresponding amino acid (Gly; L-Phe; L-Ala; L-Val; L-Met; L-Pro; L-Asp; L-Glu; L-Ile; L-Leu; L-Trp; L-Tyr; L-Thr; L-Asn; L-Gln.) and dry ethanol in the presence of excess SOCl₂ [20]. Most of the esters were obtained as crystalline solids in excellent yield. We prepared amino acid ethyl esters as the starting materials for further reaction, which used in different solvents for respective amino acid ester due to the different R group (Table 1). The structures of these compounds were confirmed by spectral techniques. For example, the ¹H NMR spectrum data of 6g showed the signals of the two CH₃ at 1.164 ppm as quaternary absorption and the signals of the two CH₂ at 4.063 ppm as mutiplets absorption due to the CH₃. The other signals appeared at δ 2.78 (d, J = 5.4 Hz, 2H, CH₂), 4.54 (q, 1H, J = 5.7 Hz, NCH), 6.89 (d, J = 8.4 Hz, 1H, NH), 7.68 (s, 2H, ArH). 

3.2    Biological Activity Evaluation 

The herbicidal activities of the title compound 6a-w against Echinochloa crusgalli and Amaranthus albus have been investigated at the dosages of 50 mg/L compared to distilled water and the commercially available herbicide Diuron according to the method that reported in our previous literatures [20-21]. The preliminary results of bioassay (Table 2) showed that most of the target compounds possessed higher herbicidal activity against hypocotyl than that of root to Echinochloa crusgalli. Respectively, compound 6q exhibited the highest herbicidal activity against Amaranthus albus, which is close to the commercial herbicide Diuron. Compounds 6c, 6p, 6s, and 6t exhibited higher herbicidal activity against hypocotyls to Echinochloa crusgalli than Diuron. These data show that the presence of the electron-withdrawing group fluorine may increase their herbicidal activity. 

The plant growth regulatory activity of title compounds against rice was also evaluated at the concentration of 10 mg/L, and the results are shown in Table 2. From Table 2, we can find that the compounds 6a, 6c, 6d, 6k, 6l, 6r, and 6s exhibited higher plant growth regulating activity than the others.

4.       Conclusion 

Twenty-three novel N-nitro amino acid ethyl ester urea compounds were synthesized. The target compounds were confirmed by IR, ¹H NMR and elemental analyses. The preliminary result of biological activity test showed that the target compounds possesses moderate inhibitory activities on E. crusgalli and A. albus at test concentration. 

5.       Acknowledgements 

This project is supported by Natural Science Foundation of China (Grant No. 31500562), Fundamental Research Funds for the Central Universities (Grant No. 2662016PY122) and Hubei 2011 Cooperative Innovation Center Foundation (Grant No. 2011JH-2014CXTT02).

Table 1: Preparation of compounds 6a-w derivatives.

Table 2: Herbicidal and plant growth regulating activities of targeted compounds 6a-w.

1.       Yonova PA, Stoilkova GM (2005) Synthesis and biological activity of urea and thiourea derivatives from 2-aminoheterocyclic compounds. J Plant Growth Regul 23: 280-291.

2.       Groszek G (2002) A Convenient Method of Synthesis of Unsymmetrical Urea Derivatives. Org Process Res Dev 6: 759-761.

3.       Pandreti V, Hasti S, Naga RC (2017) Synthesis and antimicrobial activity of new urea and thiourea derivatives of (2′-(1H-tetrazol-5-yl) biphenyl-4-yl) methanamine . Res Chem Intermed 43: 3251-3263.

4.       Dzimbeg G, Zorc B, Kralj M, Ester K, Pavelic K, et al. (2008) The novel primaquine derivatives of N-alkyl, cycloalkyl or aryl urea: Synthesis, cytostatic and antiviral activity evaluations. Eur J Med Chem 43: 1180-1187.

5.       Manjula SN, Noolvi NM, Parihar KV, Manohara Reddy SA, Ramani V, et al. (2009) Synthesis and antitumor activity of optically active thiourea and their 2-aminobenzothiazole derivatives: A novel class of anticancer agents. Eur J Med Chem 44: 2923-2929.

6.       Khan KM, Saeed S, Ali M, Gohar M, Zahid J, et al. (2009) Unsymmetrically disubstituted urea derivatives: A potent class of antiglycating agents. Bioorganic & Medicinal Chemistry 17: 2447-2451.

7.       Cross B, Gastrock WH (1974) Phenylnitramine herbicides. Patent, US 3844762.

8.       Jones RJ, Metcalfe TP, Sexton WA (1954) The relationship between the constitution and the effect of chemical compounds on plant growth. V.-aromatic nitro‐compounds and nitramines. J Sci Food Agric 5: 38-43.

9.       O'Neal TD, Bhalla PR, Cross B (1987) Method for the control of stem growth and stem stiffness of graminaceous crops. Patent, US 4643755.

10.    Cross B, Dawe DH (1978) Patent US 4130645.

11.    Sun BH (2018) Patent, CN 108451943.

12.    Farhangian H, Moghadam ME, Divsalar A, Rahiminezhad A (2017) Anticancer activity of novel amino acid derivative of palladium complex with phendione ligand against of human colon cancer cell line. J Biol Inorg Chem 22: 1055-1064.

13.    Hussein MA,  El-Sayed W, Tomader ARG (2007) Synthesis, Pesticidal Activity And Quantitative Structure-Activity Relationships of Aseries of N-(2-oxido-1,3,2-benzodioxaphosphol-2-yl) Amino Acid Ethyl or Diethyl Esters. Aust J Basic & Appl Sci 1: 593-599.

14.    Ali HM, Ali MK (2000) Quantitative structure-activity relationships (QSAR) of two series of O-aryl or N-aryl O-ethyl phosphoramidate and phosphorodiamidate fungicides incorporating amino acid ethyl esters. Bull Environ Contam Toxicol 65: 415-420.

15.    Men BQ (2005) Master’s Degree Dissertation of East South University.

16.    Xiong JP, Chen CS, Li XG, Xu HT (2008) Synthesis and Bifunctional Activity of N-Nitro-N,N'-Diphenyl Urea Derivatives[J]. Chin J of Applied Chem 25: 17-21.

17.    Xu HT, Shen XX, Cao D, Chen CS (2009) Synthesis and Antibacterial Activity of N-Nitro-N,N'-Diphenyl Urea Derivatives. Chin J of Applied Chem 26: 507-510.

18.    Almansa R, Guijarro D,Yus M (2007) Enantioselective addition of dialkylzinc reagents to N-(diphenylphosphinoyl)imines catalyzed by β-aminoalcohols with the prolinol skeleton. Tetrahedron: Asymmetry 18: 2828-2840.

19.    Li XG, Chen CS, Xie JG (2003) Chin Fine Chem Intermed 33: 14-15.

20.    Bai ZY, Wei ZJ, Wang YC, Xu SZ, Li XG, et al. (2011) Cycloalkyl substituted N-nitrourea derivatives: a convenient synthesis and biological evaluation. Res Chem Intermed 37: 859-868.

Wang YC, Bai ZY, Wei ZJ, Xu SZ, Li XG, et al. (2011) Facile synthesis and biological evaluation of novel fluorine-containing N-nitro-N′-substituted phenylurea. Res Chem Intermed 37: 1029-1039.