# Catalytic Synthesis and Antifungal Activity of New Polychlorinated Natural Terpenes

Authors: Hana Ighachane; Brahim Boualy; Mustapha Ait Ali; My. H. Sedra; Larbi El Firdoussi; Hassan Bihi Lazrek
Journal: Advances in Materials Science and Engineering
 (2017)

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## Abstract

Various unsaturated natural terpenes were selectively converted to the corresponding polychlorinated products in good yields using iron acetylacetonate in combination with nucleophilic cocatalyst. The synthesized compounds were evaluated for their in vitro antifungal activity. The antifungal bioassays showed that2c and2d possessed significant antifungal activity againstFusarium oxysporum f. sp.albedinis (Foa),Fusarium oxysporum f. sp.canariensis (Foc), andVerticillium dahliae (Vd).


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## Body

## 1. Introduction

Terpenes are naturally occurring molecules derived from isoprene units that have demonstrated therapeutic activities with a wide spectrum of biological activity, including anti-inflammatory, antitumor, and fungicidal properties [1, 2]. They are considered as convenient starting materials for the synthesis of a large and diverse collection of bioactive compounds [3]. Furthermore, they are commercially available, inexpensive, and generally accessible in enantiomeric forms [4].Catalytic functionalization is a well-established process in organic synthesis and an efficient method for the preparation of high added value products [5–8], among which is catalyzed Kharasch addition or atom transfer radical addition (TMC ATRA) which is an effective tool for carbon-carbon bond formation and has gained considerable interest in organic synthesis during the last 50–60 years [9–11]. On the other hand, it is a synthetically useful process for functionalizing organic compounds by means of halogen derivatives with a high level of atom economy. Kharasch addition is typically catalyzed by transition metal complexes of Cu, Ru, Fe, or Ni [10, 12–14]. However, although the Kharasch addition of CCl4 to simple alkenes has been extensively studied [12–14], to our knowledge no works have been devoted to the corresponding reactions using natural terpenic alkenes.On the other hand, by 2050 food request is expected to increase up to 98%; this is due to the global population that has quadrupled over the last century [15]. Thus more attention is required to protect agricultural product from different deleterious factors such as the contamination caused byVerticillium dahliae Kleb. (Vd) which is a broadly distributed vascular soil-borne pathogen that causesVerticillium wilt and it has a large host range, with over 300 plant species known to be susceptible to this fungal pathogen [16, 17].Fusarium oxysporum f. sp.albedinis (Foa) is the most injurious fungus species affecting date palm (Phoenix dactylifera) especially in Morocco and Algeria [18] andFusarium oxysporum f. sp.canariensis (Foc) that infect date palm (Phoenix canariensis) has now spread worldwide [19]. These phytopathogenic fungi are hard to control. Thus there is growing interest for discovering and developing a new antifungal agent especially using natural compound for plant protection.In the course of our studies on monoterpenes and their derivatives, we have recently reported an extremely efficient method for the preparation of various allylic terpenic chlorides [14, 20–23]. Following our catalysis objective, we became interested in the preparation of a new class of polychlorinated natural product using Kharasch addition of CCl4 catalyzed by iron acetylacetonate and to evaluate their inhibition capacity of the growth of the three pathogenic microorganisms.


## 2. Materials and Methods

NMR studies were performed on a Bruker Avance 300 spectrometer in CDCl3; chemical shifts are given in ppm relative to external TMS and coupling constant (J) in Hz. Mass spectra were recorded on AMD 402 spectrometer (70 eV, EI). The reaction mixtures were analyzed on a Trace GC Thermo Finnigan chromatograph equipped with an FID detector (flame ionization detector). GC parameters for capillary columns BP (25 m × 0.25 mm, SGE) were as follows: injector 250°C; detector 250°C; oven 70°C for 5 minutes and then 3°C/minute until 250°C for 30 minutes; column pressure 20 kPa, column flow 6.3 mL/minute; linear velocity 53.1 cm/s; total flow 138 mL/minute. Liquid chromatography was performed on silica gel (Merck 60, 220–440 mesh; eluent: hexane). Analytical thin-layer chromatography (TLC) was conducted on Merck aluminium plates with 0.2 mm of silica gel 60F-254. All the reagents and solvents used in the experiments were purchased from commercial sources and used as received without further purification (Aldrich, Acros).


### 2.1. General Procedure for Atom Transfer Radical Addition

In a 15 mL rotaflow Schlenk, we mixed 1 mol% of Fe(acac)2 (0.0414 mmol), 5 mol% of NEt3 (20.9 mg, 0.207 mmol), olefin (4.14 mmol), and CCl4 (2.55 g, 16.56 mmol). The solvent was added to bring the total volume to 5 mL. The resulting solution was stirred at 80°C, and the reaction was monitored by TLC. The reaction was quenched by the slow addition of saturated aqueous Na2SO3. The layers were separated and the aqueous layer was extracted with CH2Cl2 (2×10 mL). The combined organic layer was dried over anhydrous Na2SO4. The solvent was removed under reduced pressure. Pure chlorinated products were obtained by column chromatography over silica gel. All isolated pure products were fully characterized by 1H and 13C NMR and MS.


### 2.2. Spectral Data for the Products

#### 2.2.1. 4-(1-Chloro-1-methyl-ethyl)-1-(2,2,2-trichloro-ethyl)-cyclohexene2a [22]

1H NMR (300 MHz, CDCl3) δ: 5.71 (m, 1H), 3.28 (s, 2H), 2.29 (m, 3H), 1.97 (m, 2H), 1.65 (m, 1H), 1.53 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 131.03 (Cq), 130.53 (CH=C), 99.06 (CCl3), 73.07 (CCl), 62.02 (CH2-CCl3), 45.73 (CH), 30.61 (CH2) 29.83 (CH3), 28.19 (CH3), 24.56 (CH2). ESI-MSm/z: [M + H]+ 292.03.


#### 2.2.2. 1-Methyl-4-(1,3,3,3-tetrachloro-1-methyl-propyl)-7-oxa-bicyclo[4.1.0] heptane2c

1H NMR (300 MHz, CDCl3) δ: 3.22 (m, 1H), 2.91 (s, 2H), 2.12 (m, 2H), 1.65 (m, 1H), 1.27 (s, 3H), 1.13 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 131.03 (Cq), 130.53 (CH=C), 99.06 (CCl3), 73.07 (CCl), 62.02 (CH2-CCl3), 45.73 (CH), 30.61 (CH2) 29.83 (CH3), 28.19 (CH3), 24.56 (CH2). ESI-MSm/z: [M + H]+ 307.07.


#### 2.2.3. 2-Methyl-5-(1,3,3,3-tetrachloro-1-methyl-propyl)-cyclohex-2-enone2d [23]

1H NMR (300 MHz, CDCl3) δ: 6.77 (m, 1H), 3.42 (s, 2H), 2.30–2.80 (m, 6H), 1.90 (s, 3H), 1.79 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 195.50 (C=O), 143.52 (CH=C), 135.50 (C=C), 94.46 (CCl3), 73.42 (CCl), 62.37 (CH2-CCl3), 45.88 (CH), 39.50 (CH2) 27.72 (CH2), 27.32 (CH3), 15.50 (CH3). ESI-MSm/z: [M + H]+ 289.10.


#### 2.2.4. 1-Methyl-4-(1,3,3,3-tetrachloro-1-methyl-propyl)-cyclohexene2e

1H NMR (300 MHz, CDCl3) δ: 3.4 (m, 1H), 3.3 (s, 2H), 2.2–1.95 (m, 7H), 1.8 (s, 3H), 1.65 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 131.5 (Cq), 118.4 (CH=C), 73.8 (CCl3), 63.0 (CCl), 46.7 (CH2-CCl3), 44.2 (CH), 30.6 (CH2) 27.2 (CH2), 25.1 (CH3), 24.2 (CH2), 23.2 (CH3). ESI-MSm/z: [M + H]+ 291.08.


#### 2.2.5. 4a,5-Dimethyl-3-(1,3,3,3-tetrachloro-1-methyl-propyl)-1,2,3,4,4a,5,6,7-octahydro-naphthalene2f

1H NMR (300 MHz, CDCl3) δ: 5.35 (m, 1H), 3.34 (s, 2H), 2.29 (m, 3H), 1.97 (m, 2H), 1.65 (m, 1H), 1.53 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 142.20 (Cq), 120.71 (CH=C), 94.75 (CCl3), 75.28 (CCl), 62.41 (CH2–CCl3), 46.40 (–CH–), 45.00 (Cq), 41.11 (–CH–), 40.99 (CH2), 33.24 (CH2), 29.83 (CH2), 27.70 (CH3), 27.00 (CH2), 25.80 (CH2), 18.50 (CH3), 16.20 (CH3). ESI-MSm/z: [M + H]+ 359.20.


### 2.3. Antifungal Assays

#### 2.3.1. Experimental Protocol

In 250 mL Erlenmeyer flasks, we prepared various solution under aseptic and sterilized condition. 1.25, 2.5, 5, and 7.5μg/mL of each compound were previously dissolved in 0.1% dimethyl sulfoxide (DMSO); the solution was added to 100 mL of Czapek solid medium at 45°C and then poured into Petri dishes.Pelt (trademark: Procida Groups Roussel Uclaf) which is a systemic fungicide containing 70% methyl thiophanate was used as a standard antifungal compound and was also prepared with the same concentrations. The control water is used under the same conditions.Mycelial discs of 5 mm diameter, from young cultures ofFusarium oxysporum f. sp.albedinis (Foa),Fusarium oxysporum f. sp.canariensis (Foc), orVerticillium dahliae (Vd) which were provided by Laboratory LPGLI of INRA Marrakech, Morocco (Centre Regional de Recherche Agronomique), and maintained in Czapek medium, were disposed in the middle of the Petri dishes. Incubation was done at 28°C under continuous light. Mycelial growth of colonies was estimated every 2 days for 8 days of incubation by the average of two perpendicular diameters [24, 25]. The values represent the average of 5 independent experiments.For measuring sporulation on different media, a single block of 5 mm diameter was cut out from the fungal colony near the margin by sterilized cork borer and was transferred to 100 mL sterile distilled water in a test tube, where it was mixed thoroughly to make a uniform spore suspension. Two dilutions of 10−5 and 10−6 were prepared from the mother solution. The fungal suspension was then stirred using a vortex for 20 seconds to release spores conidiophores. For each dilution we took 1 mL of the solution being distributed in a Petri dish. These experiments were repeated five times.


#### 2.3.2. Evaluation of Mycelial Growth

The inhibition rate of mycelial growth of three fungi was calculated using the following formula [24, 25]:(1)Growth inhibition rate=Dco-DtDco×100,where Dco = colony diameter of the control (water) and Dt = colony diameter of the test plate.


#### 2.3.3. Evaluation of Sporulation

We proceeded to count the total number of spores using a Malassez cell. The inhibition percentage of the sporulation is calculated using the following formula [24, 26]:(2)Sporulation inhibition rate%=Nco-NtNco×100,where Nco = estimated number of germinated spores of the control (water) and Nt = estimated number of germinated spores of the test plate.


#### 2.3.4. Statistical Analysis

Calculations and comparisons of treatment means for each experiment were conducted using analysis of variance (ANOVA) and the SPSS 20.0 software; means were separated by Tukey Honestly Significant Difference (Tukey HSD) test atP=0.05.


## 2.1. General Procedure for Atom Transfer Radical Addition

In a 15 mL rotaflow Schlenk, we mixed 1 mol% of Fe(acac)2 (0.0414 mmol), 5 mol% of NEt3 (20.9 mg, 0.207 mmol), olefin (4.14 mmol), and CCl4 (2.55 g, 16.56 mmol). The solvent was added to bring the total volume to 5 mL. The resulting solution was stirred at 80°C, and the reaction was monitored by TLC. The reaction was quenched by the slow addition of saturated aqueous Na2SO3. The layers were separated and the aqueous layer was extracted with CH2Cl2 (2×10 mL). The combined organic layer was dried over anhydrous Na2SO4. The solvent was removed under reduced pressure. Pure chlorinated products were obtained by column chromatography over silica gel. All isolated pure products were fully characterized by 1H and 13C NMR and MS.


## 2.2. Spectral Data for the Products

### 2.2.1. 4-(1-Chloro-1-methyl-ethyl)-1-(2,2,2-trichloro-ethyl)-cyclohexene2a [22]

1H NMR (300 MHz, CDCl3) δ: 5.71 (m, 1H), 3.28 (s, 2H), 2.29 (m, 3H), 1.97 (m, 2H), 1.65 (m, 1H), 1.53 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 131.03 (Cq), 130.53 (CH=C), 99.06 (CCl3), 73.07 (CCl), 62.02 (CH2-CCl3), 45.73 (CH), 30.61 (CH2) 29.83 (CH3), 28.19 (CH3), 24.56 (CH2). ESI-MSm/z: [M + H]+ 292.03.


### 2.2.2. 1-Methyl-4-(1,3,3,3-tetrachloro-1-methyl-propyl)-7-oxa-bicyclo[4.1.0] heptane2c

1H NMR (300 MHz, CDCl3) δ: 3.22 (m, 1H), 2.91 (s, 2H), 2.12 (m, 2H), 1.65 (m, 1H), 1.27 (s, 3H), 1.13 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 131.03 (Cq), 130.53 (CH=C), 99.06 (CCl3), 73.07 (CCl), 62.02 (CH2-CCl3), 45.73 (CH), 30.61 (CH2) 29.83 (CH3), 28.19 (CH3), 24.56 (CH2). ESI-MSm/z: [M + H]+ 307.07.


### 2.2.3. 2-Methyl-5-(1,3,3,3-tetrachloro-1-methyl-propyl)-cyclohex-2-enone2d [23]

1H NMR (300 MHz, CDCl3) δ: 6.77 (m, 1H), 3.42 (s, 2H), 2.30–2.80 (m, 6H), 1.90 (s, 3H), 1.79 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 195.50 (C=O), 143.52 (CH=C), 135.50 (C=C), 94.46 (CCl3), 73.42 (CCl), 62.37 (CH2-CCl3), 45.88 (CH), 39.50 (CH2) 27.72 (CH2), 27.32 (CH3), 15.50 (CH3). ESI-MSm/z: [M + H]+ 289.10.


### 2.2.4. 1-Methyl-4-(1,3,3,3-tetrachloro-1-methyl-propyl)-cyclohexene2e

1H NMR (300 MHz, CDCl3) δ: 3.4 (m, 1H), 3.3 (s, 2H), 2.2–1.95 (m, 7H), 1.8 (s, 3H), 1.65 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 131.5 (Cq), 118.4 (CH=C), 73.8 (CCl3), 63.0 (CCl), 46.7 (CH2-CCl3), 44.2 (CH), 30.6 (CH2) 27.2 (CH2), 25.1 (CH3), 24.2 (CH2), 23.2 (CH3). ESI-MSm/z: [M + H]+ 291.08.


### 2.2.5. 4a,5-Dimethyl-3-(1,3,3,3-tetrachloro-1-methyl-propyl)-1,2,3,4,4a,5,6,7-octahydro-naphthalene2f

1H NMR (300 MHz, CDCl3) δ: 5.35 (m, 1H), 3.34 (s, 2H), 2.29 (m, 3H), 1.97 (m, 2H), 1.65 (m, 1H), 1.53 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 142.20 (Cq), 120.71 (CH=C), 94.75 (CCl3), 75.28 (CCl), 62.41 (CH2–CCl3), 46.40 (–CH–), 45.00 (Cq), 41.11 (–CH–), 40.99 (CH2), 33.24 (CH2), 29.83 (CH2), 27.70 (CH3), 27.00 (CH2), 25.80 (CH2), 18.50 (CH3), 16.20 (CH3). ESI-MSm/z: [M + H]+ 359.20.


## 2.2.1. 4-(1-Chloro-1-methyl-ethyl)-1-(2,2,2-trichloro-ethyl)-cyclohexene2a [22]

1H NMR (300 MHz, CDCl3) δ: 5.71 (m, 1H), 3.28 (s, 2H), 2.29 (m, 3H), 1.97 (m, 2H), 1.65 (m, 1H), 1.53 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 131.03 (Cq), 130.53 (CH=C), 99.06 (CCl3), 73.07 (CCl), 62.02 (CH2-CCl3), 45.73 (CH), 30.61 (CH2) 29.83 (CH3), 28.19 (CH3), 24.56 (CH2). ESI-MSm/z: [M + H]+ 292.03.


## 2.2.2. 1-Methyl-4-(1,3,3,3-tetrachloro-1-methyl-propyl)-7-oxa-bicyclo[4.1.0] heptane2c

1H NMR (300 MHz, CDCl3) δ: 3.22 (m, 1H), 2.91 (s, 2H), 2.12 (m, 2H), 1.65 (m, 1H), 1.27 (s, 3H), 1.13 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 131.03 (Cq), 130.53 (CH=C), 99.06 (CCl3), 73.07 (CCl), 62.02 (CH2-CCl3), 45.73 (CH), 30.61 (CH2) 29.83 (CH3), 28.19 (CH3), 24.56 (CH2). ESI-MSm/z: [M + H]+ 307.07.


## 2.2.3. 2-Methyl-5-(1,3,3,3-tetrachloro-1-methyl-propyl)-cyclohex-2-enone2d [23]

1H NMR (300 MHz, CDCl3) δ: 6.77 (m, 1H), 3.42 (s, 2H), 2.30–2.80 (m, 6H), 1.90 (s, 3H), 1.79 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 195.50 (C=O), 143.52 (CH=C), 135.50 (C=C), 94.46 (CCl3), 73.42 (CCl), 62.37 (CH2-CCl3), 45.88 (CH), 39.50 (CH2) 27.72 (CH2), 27.32 (CH3), 15.50 (CH3). ESI-MSm/z: [M + H]+ 289.10.


## 2.2.4. 1-Methyl-4-(1,3,3,3-tetrachloro-1-methyl-propyl)-cyclohexene2e

1H NMR (300 MHz, CDCl3) δ: 3.4 (m, 1H), 3.3 (s, 2H), 2.2–1.95 (m, 7H), 1.8 (s, 3H), 1.65 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 131.5 (Cq), 118.4 (CH=C), 73.8 (CCl3), 63.0 (CCl), 46.7 (CH2-CCl3), 44.2 (CH), 30.6 (CH2) 27.2 (CH2), 25.1 (CH3), 24.2 (CH2), 23.2 (CH3). ESI-MSm/z: [M + H]+ 291.08.


## 2.2.5. 4a,5-Dimethyl-3-(1,3,3,3-tetrachloro-1-methyl-propyl)-1,2,3,4,4a,5,6,7-octahydro-naphthalene2f

1H NMR (300 MHz, CDCl3) δ: 5.35 (m, 1H), 3.34 (s, 2H), 2.29 (m, 3H), 1.97 (m, 2H), 1.65 (m, 1H), 1.53 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 142.20 (Cq), 120.71 (CH=C), 94.75 (CCl3), 75.28 (CCl), 62.41 (CH2–CCl3), 46.40 (–CH–), 45.00 (Cq), 41.11 (–CH–), 40.99 (CH2), 33.24 (CH2), 29.83 (CH2), 27.70 (CH3), 27.00 (CH2), 25.80 (CH2), 18.50 (CH3), 16.20 (CH3). ESI-MSm/z: [M + H]+ 359.20.


## 2.3. Antifungal Assays

### 2.3.1. Experimental Protocol

In 250 mL Erlenmeyer flasks, we prepared various solution under aseptic and sterilized condition. 1.25, 2.5, 5, and 7.5μg/mL of each compound were previously dissolved in 0.1% dimethyl sulfoxide (DMSO); the solution was added to 100 mL of Czapek solid medium at 45°C and then poured into Petri dishes.Pelt (trademark: Procida Groups Roussel Uclaf) which is a systemic fungicide containing 70% methyl thiophanate was used as a standard antifungal compound and was also prepared with the same concentrations. The control water is used under the same conditions.Mycelial discs of 5 mm diameter, from young cultures ofFusarium oxysporum f. sp.albedinis (Foa),Fusarium oxysporum f. sp.canariensis (Foc), orVerticillium dahliae (Vd) which were provided by Laboratory LPGLI of INRA Marrakech, Morocco (Centre Regional de Recherche Agronomique), and maintained in Czapek medium, were disposed in the middle of the Petri dishes. Incubation was done at 28°C under continuous light. Mycelial growth of colonies was estimated every 2 days for 8 days of incubation by the average of two perpendicular diameters [24, 25]. The values represent the average of 5 independent experiments.For measuring sporulation on different media, a single block of 5 mm diameter was cut out from the fungal colony near the margin by sterilized cork borer and was transferred to 100 mL sterile distilled water in a test tube, where it was mixed thoroughly to make a uniform spore suspension. Two dilutions of 10−5 and 10−6 were prepared from the mother solution. The fungal suspension was then stirred using a vortex for 20 seconds to release spores conidiophores. For each dilution we took 1 mL of the solution being distributed in a Petri dish. These experiments were repeated five times.


### 2.3.2. Evaluation of Mycelial Growth

The inhibition rate of mycelial growth of three fungi was calculated using the following formula [24, 25]:(1)Growth inhibition rate=Dco-DtDco×100,where Dco = colony diameter of the control (water) and Dt = colony diameter of the test plate.


### 2.3.3. Evaluation of Sporulation

We proceeded to count the total number of spores using a Malassez cell. The inhibition percentage of the sporulation is calculated using the following formula [24, 26]:(2)Sporulation inhibition rate%=Nco-NtNco×100,where Nco = estimated number of germinated spores of the control (water) and Nt = estimated number of germinated spores of the test plate.


### 2.3.4. Statistical Analysis

Calculations and comparisons of treatment means for each experiment were conducted using analysis of variance (ANOVA) and the SPSS 20.0 software; means were separated by Tukey Honestly Significant Difference (Tukey HSD) test atP=0.05.


## 2.3.1. Experimental Protocol

In 250 mL Erlenmeyer flasks, we prepared various solution under aseptic and sterilized condition. 1.25, 2.5, 5, and 7.5μg/mL of each compound were previously dissolved in 0.1% dimethyl sulfoxide (DMSO); the solution was added to 100 mL of Czapek solid medium at 45°C and then poured into Petri dishes.Pelt (trademark: Procida Groups Roussel Uclaf) which is a systemic fungicide containing 70% methyl thiophanate was used as a standard antifungal compound and was also prepared with the same concentrations. The control water is used under the same conditions.Mycelial discs of 5 mm diameter, from young cultures ofFusarium oxysporum f. sp.albedinis (Foa),Fusarium oxysporum f. sp.canariensis (Foc), orVerticillium dahliae (Vd) which were provided by Laboratory LPGLI of INRA Marrakech, Morocco (Centre Regional de Recherche Agronomique), and maintained in Czapek medium, were disposed in the middle of the Petri dishes. Incubation was done at 28°C under continuous light. Mycelial growth of colonies was estimated every 2 days for 8 days of incubation by the average of two perpendicular diameters [24, 25]. The values represent the average of 5 independent experiments.For measuring sporulation on different media, a single block of 5 mm diameter was cut out from the fungal colony near the margin by sterilized cork borer and was transferred to 100 mL sterile distilled water in a test tube, where it was mixed thoroughly to make a uniform spore suspension. Two dilutions of 10−5 and 10−6 were prepared from the mother solution. The fungal suspension was then stirred using a vortex for 20 seconds to release spores conidiophores. For each dilution we took 1 mL of the solution being distributed in a Petri dish. These experiments were repeated five times.


## 2.3.2. Evaluation of Mycelial Growth

The inhibition rate of mycelial growth of three fungi was calculated using the following formula [24, 25]:(1)Growth inhibition rate=Dco-DtDco×100,where Dco = colony diameter of the control (water) and Dt = colony diameter of the test plate.


## 2.3.3. Evaluation of Sporulation

We proceeded to count the total number of spores using a Malassez cell. The inhibition percentage of the sporulation is calculated using the following formula [24, 26]:(2)Sporulation inhibition rate%=Nco-NtNco×100,where Nco = estimated number of germinated spores of the control (water) and Nt = estimated number of germinated spores of the test plate.


## 2.3.4. Statistical Analysis

Calculations and comparisons of treatment means for each experiment were conducted using analysis of variance (ANOVA) and the SPSS 20.0 software; means were separated by Tukey Honestly Significant Difference (Tukey HSD) test atP=0.05.


## 3. Results and Discussion

### 3.1. Chemical Result

The synthesis of the target compounds was accomplished using our previously optimized catalytic system for the Kharasch addition of tetrachloromethane to olefins under mild reaction conditions [14]. Using various transition metal acetylacetonates M(acac)n (M = Zn, Co, V, Ni, Mo, Mn, Fe, and Cr) and triethylamine as cocatalyst and iron complex highlighting its potential as an effective catalyst, the reactions offer the halogen derivatives in moderate to good yields. Encouraged by these results, we were interested to apply this catalytic system to some mono- and sesquiterpenes in order to synthesize a new series of polyorganochloride products. The Kharasch addition reaction requires either a free radical precursor as the promoter or a metal complex as the catalyst. The classical free radical chain mechanism is operative not only with initiators such as light, heat, and peroxides [27–29] but also with a variety of metal complexes (Scheme 1) [27].Scheme 1
Proposed mechanism for Kharasch addition of CCl4 to olefins catalyzed by Fe(acac)2.The addition of tetrachloromethane to unsaturated terpenes can be obtained by treatment with CCl4, Fe(acac)2 as catalyst, and NEt3 in dry acetonitrile at 80°C. A possible mechanism for Kharasch reaction is shown in Scheme 1. As proposed by Murai et al. [30] and Kameyama and Kamigata [31] in ruthenium catalyzed Kharasch reaction, we can assume that low-valent iron species probably react in a similar way as RuII catalysts. At this stage, we cannot exclude a multiple-step process that involves radical intermediates as described by Oldroydg et al. [29].In a typical experiment, NEt3 as cocatalyst [14] was added to a solution of Fe(acac)2 in toluene at 80°C and after 15 min CCl4 was added. The reaction mixture was stirred for 30 min and then terpene1a–f was added. The progress of the reaction was monitored by TLC and GC–MS. The final mixture was filtered through anhydrous K2CO3 and the filtrate was evaporated under vacuum. The polyorganochlorides2a–f were then purified by flash column chromatography. Reaction times, chemical yields, and conversions are reported in Table 1. The addition of tetrachloromethane to β-pinene1a and α-pinene1b led to the formation of the adduct2a in excellent yields (86% and 85%, resp.) after opening the cyclobutane ring. Limonene oxide1c reacted selectively with CCl4 and led to the corresponding adduct2c in 63% yield.Table 1
Synthesis of polyorganochloride2a–f.
Entry
Olefins
Products
Conversiona (%)
Yieldb (%)
1
96
86
2
95
85
3
73
63
4
83
68
5
90
77
6
93
74
Reaction conditions. Olefin/CCl4 (0.25), Fe(acac)2 (0.01 equiv.), solvent (toluene), triethylamine (0.05 equiv.), and temperature (80°C), for 24 h.aConversion based on GC. bIsolated yield.In order to investigate the competition reaction between two carbon-carbon double bonds present in1d and1e, Kharasch addition was then carried out on limonene1e to lead selectively towards the monoadduct product2e in 77% yield, corresponding to the activation of the exocyclic double bond. Likewise, the reaction with carvone1d leads to2d in 68% yield (Table 1). The reaction of sesquiterpene was tested using valencene1f and led to the corresponding product2f in 74% yield. We characterized the structures of all products by 1H-NMR, 13C-NMR, and mass spectrometry. In addition, the structure of2d was confirmed by X-ray crystallographic analysis [22, 23].The reaction between iron complex and CCl4 leads to the formation of radical CCl3 which reacts with β-pinene. The reaction occurs through a “ligand-transfer” mechanism (inner-sphere electron transfer) (Scheme 2) [26–29].Scheme 2
Proposed mechanism for the cyclobutane opening ring ofβ-pinene in Kharasch addition of CCl4 catalyzed by Fe(acac)2.


### 3.2. Screening of Antifungal Activity In Vitro

The compounds2a–f and the reference drug were screened for their potential in vitro antifungal activity against three plant pathogenic fungi (Fusarium oxysporum f. sp.canariensis (Foc),Fusarium oxysporum f. spalbedinis (Foa), andVerticillium dahliae (Vd)). The results are listed in Tables 2 and 3.Table 2
Linear growth inhibition of (Foc, Foa, and Vd) under the effect of different concentrations of the polyorganochlorine derivatives (2a–f) in comparison with the control, after 8 days of incubation at 28°C on Czapek medium.
Concentrations (µg/mL)
Mycelial growth inhibitory rates (means %)∗∗
2a
2c
2d
2e
2f
Pelt
Foc
1.25
−1.69c
15.82b
1.56c
−0.72c
−12.18d
94.73a
2.5
2.4d
52.7b
10.62c
7.95c
0.62d
100a
5
11.67f
75b
41c
33.33d
21.67e
100a
7.5
23.8f
91.2b
57c
39.4d
34.5d
100a
Foa
1.25
−18.34e
24.03b
8.65c
−0.75d
−24.82e
94a
2.5
−0.93e
58.67b
47.01c
10d
−2.72e
100a
5
4.62e
86b
73c
30.77d
3.08e
100a
7.5
10.3d
100a
85b
42.9c
10d
100a
Vd
1.25
−22.92e
6.07b
0.54c
−6.93d
−7.72d
92.86a
2.5
−4.68e
44.75b
29.08c
0d
0d
100a
5
8.38d
63.59b
52.1c
14.06d
13.29d
100a
7.5
18.75e
84.38b
71.25c
34.38d
31.25d
100a
Note. Foc: Fusarium oxysporum f. sp. canariensis, Foa: Fusarium oxysporum f. sp albedinis, and Vd: Verticillium dahliae. ∗∗Data in the table are the averages of five replicates. Data followed by different lower case letters show statistically significant differences at P<0.05 according to Tukey’s HSD.Table 3
Inhibition of the spore germination of Foc, Foa, and Vd under the effect of different concentrations of the polyorganochlorine derivatives (2a–f) in comparison with the control, after 48 h of incubation at 28°C.
Concentrations (µg/mL)
Spore germination inhibitory rates (means %)∗∗
2a
2c
2d
2e
2f
Pelt
Foc
1.25
−8.35e
12.11b
3.73c
−2.93c
−14.73d
100a
2.5
8.83e
43.51b
24.36c
14.42d
2.83f
100a
5
21.36e
60.77b
46.68c
35.3d
18.7e
100a
7.5
30.4e
69b
52c
42.93d
23.15f
100a
Foa
1.25
−35.35d
32.64b
22.83c
−62.29e
−54.41e
100a
2.5
−18.83d
53.95b
44.36c
−44.82e
−39.33e
100a
5
−0.68d
69.82b
56.21c
−21.51e
−15.67e
100a
7.5
3.53c
100a
63.95b
−1.48c
0.34c
100a
Vd
1.25
−35.35e
2.37b
−4.83c
−12.67d
−15.23d
100a
2.5
−28.47d
13.63b
8.36b
−4.85c
−9.35c
100a
5
−17.27e
42.79b
33.68c
19.19d
10.42d
100a
7.5
−5.53e
68.74b
53.95c
35.33d
30.17d
100a
Note. Foc: Fusarium oxysporum f. sp. canariensis, Foa: Fusarium oxysporum f. sp albedinis, and Vd: Verticillium dahliae. ∗∗Data in the table are the averages of five replicates. Data followed by different lower case letters show statistically significant differences at P<0.05 according to Tukey’s HSD.As shown in Table2, almost all the title compounds2a–f displayed activities in varying degrees against the tested fungi. We noticed also that the compounds are more active against theFocandFoa thanVd.The rate of the inhibition varied among the synthesized molecules. The first bioassay using mycelia growth rate method indicated the following result.For theF.o. f. sp.canariensis (Foc), we observed that compounds2c and2d had the highest activities at the concentration of 7.5 μg/mL, the inhibition rate was around 90% and 57% (P<0.001), respectively, and at the concentration of 5 μg/mL the inhibition rate was 75% and 41%, respectively, while we noticed that for the concentrations 2.50 μg/mL and 1.25 μg/mL some of the compounds did not have any effect.On the other hand, it seems from the results reported in Table2 that compounds2c and2d also showed the greater inhibition rates (86%, 73% and 100%, 85%) (P<0.001) at 5 μg/mL and 7.5 μg/mL, respectively, againstF.o.f. sp.albedinis (Foa). Compound2c exhibited parallel activity as pelt which is in our case a positive control. As for the evaluation of the in vitro activity of the compounds againstVerticillium dahliae (Vd), the results, therefore, suggest that compounds2c and2d at the concentration of 7.5 μg/mL exhibited the highest inhibition.The second bioassay was designed to measure fungistatic and fungicidal effects of the compounds by using a sporulation inhibition test. The results of the bioassay were summarized in the tables (Table3). It confirmed that almost all the compounds exhibited fungistatic actions against the tested fungi, the range of inhibition was from −35 to 100%, and the most potent activity was demonstrated against (Foa). This result provided useful information to study the structure-activity relationship for these new structures, shown in Table 1.Thus, the findings demonstrate that compound2c exhibited the highest fungicidal effect against all the fungus followed by compound2d (Table 3). This can be due to the presence of oxygen atom in common limonene oxide2c as an epoxy group and carvone2d as a carbonyl group; hence the nature of the group in the monoterpene could lead to highest interaction with the biological target. For instance, the evaluation of antifungal activity for diterpene2f reveals that this structure significantly decreases antifungal activity and also the inhibition of sporulation.A large number of studies have been done in recent years on terpenoids as active components showing antifungal activities against various fungi [32]. For instance, polygodial was found to exhibit a fungicidal activity against a food spoilage yeast [33]; the diterpenoids 16α-hydroxy-cleroda-3,13-(14)-Z-diene-15,16-olide and 16-oxo-cleroda-3,13-(14)-E-diene-15-oic acid also demonstrated significant antifungal activity [34], while methyl angolensate and 1,3,7-trideacetylkhivorin displayed the highest antifungal activity, with 62.8 and 64% mycelial growth inhibition at 1000 mg/L, respectively, against the plant pathogenic fungusBotrytis cinerea [35, 36]. The results from the present study indicate the antifungal nature of new polychlorinated natural terpenes which showed significant inhibition in linear growth and sporulation against the three fungi (Foc,Foa, andVd). The antifungal activity of these new compounds was observed for the first time; however, detailed studies regarding its mode of action and efficacy under field condition are needed to be carried out.


## 3.1. Chemical Result

The synthesis of the target compounds was accomplished using our previously optimized catalytic system for the Kharasch addition of tetrachloromethane to olefins under mild reaction conditions [14]. Using various transition metal acetylacetonates M(acac)n (M = Zn, Co, V, Ni, Mo, Mn, Fe, and Cr) and triethylamine as cocatalyst and iron complex highlighting its potential as an effective catalyst, the reactions offer the halogen derivatives in moderate to good yields. Encouraged by these results, we were interested to apply this catalytic system to some mono- and sesquiterpenes in order to synthesize a new series of polyorganochloride products. The Kharasch addition reaction requires either a free radical precursor as the promoter or a metal complex as the catalyst. The classical free radical chain mechanism is operative not only with initiators such as light, heat, and peroxides [27–29] but also with a variety of metal complexes (Scheme 1) [27].Scheme 1
Proposed mechanism for Kharasch addition of CCl4 to olefins catalyzed by Fe(acac)2.The addition of tetrachloromethane to unsaturated terpenes can be obtained by treatment with CCl4, Fe(acac)2 as catalyst, and NEt3 in dry acetonitrile at 80°C. A possible mechanism for Kharasch reaction is shown in Scheme 1. As proposed by Murai et al. [30] and Kameyama and Kamigata [31] in ruthenium catalyzed Kharasch reaction, we can assume that low-valent iron species probably react in a similar way as RuII catalysts. At this stage, we cannot exclude a multiple-step process that involves radical intermediates as described by Oldroydg et al. [29].In a typical experiment, NEt3 as cocatalyst [14] was added to a solution of Fe(acac)2 in toluene at 80°C and after 15 min CCl4 was added. The reaction mixture was stirred for 30 min and then terpene1a–f was added. The progress of the reaction was monitored by TLC and GC–MS. The final mixture was filtered through anhydrous K2CO3 and the filtrate was evaporated under vacuum. The polyorganochlorides2a–f were then purified by flash column chromatography. Reaction times, chemical yields, and conversions are reported in Table 1. The addition of tetrachloromethane to β-pinene1a and α-pinene1b led to the formation of the adduct2a in excellent yields (86% and 85%, resp.) after opening the cyclobutane ring. Limonene oxide1c reacted selectively with CCl4 and led to the corresponding adduct2c in 63% yield.Table 1
Synthesis of polyorganochloride2a–f.
Entry
Olefins
Products
Conversiona (%)
Yieldb (%)
1
96
86
2
95
85
3
73
63
4
83
68
5
90
77
6
93
74
Reaction conditions. Olefin/CCl4 (0.25), Fe(acac)2 (0.01 equiv.), solvent (toluene), triethylamine (0.05 equiv.), and temperature (80°C), for 24 h.aConversion based on GC. bIsolated yield.In order to investigate the competition reaction between two carbon-carbon double bonds present in1d and1e, Kharasch addition was then carried out on limonene1e to lead selectively towards the monoadduct product2e in 77% yield, corresponding to the activation of the exocyclic double bond. Likewise, the reaction with carvone1d leads to2d in 68% yield (Table 1). The reaction of sesquiterpene was tested using valencene1f and led to the corresponding product2f in 74% yield. We characterized the structures of all products by 1H-NMR, 13C-NMR, and mass spectrometry. In addition, the structure of2d was confirmed by X-ray crystallographic analysis [22, 23].The reaction between iron complex and CCl4 leads to the formation of radical CCl3 which reacts with β-pinene. The reaction occurs through a “ligand-transfer” mechanism (inner-sphere electron transfer) (Scheme 2) [26–29].Scheme 2
Proposed mechanism for the cyclobutane opening ring ofβ-pinene in Kharasch addition of CCl4 catalyzed by Fe(acac)2.


## 3.2. Screening of Antifungal Activity In Vitro

The compounds2a–f and the reference drug were screened for their potential in vitro antifungal activity against three plant pathogenic fungi (Fusarium oxysporum f. sp.canariensis (Foc),Fusarium oxysporum f. spalbedinis (Foa), andVerticillium dahliae (Vd)). The results are listed in Tables 2 and 3.Table 2
Linear growth inhibition of (Foc, Foa, and Vd) under the effect of different concentrations of the polyorganochlorine derivatives (2a–f) in comparison with the control, after 8 days of incubation at 28°C on Czapek medium.
Concentrations (µg/mL)
Mycelial growth inhibitory rates (means %)∗∗
2a
2c
2d
2e
2f
Pelt
Foc
1.25
−1.69c
15.82b
1.56c
−0.72c
−12.18d
94.73a
2.5
2.4d
52.7b
10.62c
7.95c
0.62d
100a
5
11.67f
75b
41c
33.33d
21.67e
100a
7.5
23.8f
91.2b
57c
39.4d
34.5d
100a
Foa
1.25
−18.34e
24.03b
8.65c
−0.75d
−24.82e
94a
2.5
−0.93e
58.67b
47.01c
10d
−2.72e
100a
5
4.62e
86b
73c
30.77d
3.08e
100a
7.5
10.3d
100a
85b
42.9c
10d
100a
Vd
1.25
−22.92e
6.07b
0.54c
−6.93d
−7.72d
92.86a
2.5
−4.68e
44.75b
29.08c
0d
0d
100a
5
8.38d
63.59b
52.1c
14.06d
13.29d
100a
7.5
18.75e
84.38b
71.25c
34.38d
31.25d
100a
Note. Foc: Fusarium oxysporum f. sp. canariensis, Foa: Fusarium oxysporum f. sp albedinis, and Vd: Verticillium dahliae. ∗∗Data in the table are the averages of five replicates. Data followed by different lower case letters show statistically significant differences at P<0.05 according to Tukey’s HSD.Table 3
Inhibition of the spore germination of Foc, Foa, and Vd under the effect of different concentrations of the polyorganochlorine derivatives (2a–f) in comparison with the control, after 48 h of incubation at 28°C.
Concentrations (µg/mL)
Spore germination inhibitory rates (means %)∗∗
2a
2c
2d
2e
2f
Pelt
Foc
1.25
−8.35e
12.11b
3.73c
−2.93c
−14.73d
100a
2.5
8.83e
43.51b
24.36c
14.42d
2.83f
100a
5
21.36e
60.77b
46.68c
35.3d
18.7e
100a
7.5
30.4e
69b
52c
42.93d
23.15f
100a
Foa
1.25
−35.35d
32.64b
22.83c
−62.29e
−54.41e
100a
2.5
−18.83d
53.95b
44.36c
−44.82e
−39.33e
100a
5
−0.68d
69.82b
56.21c
−21.51e
−15.67e
100a
7.5
3.53c
100a
63.95b
−1.48c
0.34c
100a
Vd
1.25
−35.35e
2.37b
−4.83c
−12.67d
−15.23d
100a
2.5
−28.47d
13.63b
8.36b
−4.85c
−9.35c
100a
5
−17.27e
42.79b
33.68c
19.19d
10.42d
100a
7.5
−5.53e
68.74b
53.95c
35.33d
30.17d
100a
Note. Foc: Fusarium oxysporum f. sp. canariensis, Foa: Fusarium oxysporum f. sp albedinis, and Vd: Verticillium dahliae. ∗∗Data in the table are the averages of five replicates. Data followed by different lower case letters show statistically significant differences at P<0.05 according to Tukey’s HSD.As shown in Table2, almost all the title compounds2a–f displayed activities in varying degrees against the tested fungi. We noticed also that the compounds are more active against theFocandFoa thanVd.The rate of the inhibition varied among the synthesized molecules. The first bioassay using mycelia growth rate method indicated the following result.For theF.o. f. sp.canariensis (Foc), we observed that compounds2c and2d had the highest activities at the concentration of 7.5 μg/mL, the inhibition rate was around 90% and 57% (P<0.001), respectively, and at the concentration of 5 μg/mL the inhibition rate was 75% and 41%, respectively, while we noticed that for the concentrations 2.50 μg/mL and 1.25 μg/mL some of the compounds did not have any effect.On the other hand, it seems from the results reported in Table2 that compounds2c and2d also showed the greater inhibition rates (86%, 73% and 100%, 85%) (P<0.001) at 5 μg/mL and 7.5 μg/mL, respectively, againstF.o.f. sp.albedinis (Foa). Compound2c exhibited parallel activity as pelt which is in our case a positive control. As for the evaluation of the in vitro activity of the compounds againstVerticillium dahliae (Vd), the results, therefore, suggest that compounds2c and2d at the concentration of 7.5 μg/mL exhibited the highest inhibition.The second bioassay was designed to measure fungistatic and fungicidal effects of the compounds by using a sporulation inhibition test. The results of the bioassay were summarized in the tables (Table3). It confirmed that almost all the compounds exhibited fungistatic actions against the tested fungi, the range of inhibition was from −35 to 100%, and the most potent activity was demonstrated against (Foa). This result provided useful information to study the structure-activity relationship for these new structures, shown in Table 1.Thus, the findings demonstrate that compound2c exhibited the highest fungicidal effect against all the fungus followed by compound2d (Table 3). This can be due to the presence of oxygen atom in common limonene oxide2c as an epoxy group and carvone2d as a carbonyl group; hence the nature of the group in the monoterpene could lead to highest interaction with the biological target. For instance, the evaluation of antifungal activity for diterpene2f reveals that this structure significantly decreases antifungal activity and also the inhibition of sporulation.A large number of studies have been done in recent years on terpenoids as active components showing antifungal activities against various fungi [32]. For instance, polygodial was found to exhibit a fungicidal activity against a food spoilage yeast [33]; the diterpenoids 16α-hydroxy-cleroda-3,13-(14)-Z-diene-15,16-olide and 16-oxo-cleroda-3,13-(14)-E-diene-15-oic acid also demonstrated significant antifungal activity [34], while methyl angolensate and 1,3,7-trideacetylkhivorin displayed the highest antifungal activity, with 62.8 and 64% mycelial growth inhibition at 1000 mg/L, respectively, against the plant pathogenic fungusBotrytis cinerea [35, 36]. The results from the present study indicate the antifungal nature of new polychlorinated natural terpenes which showed significant inhibition in linear growth and sporulation against the three fungi (Foc,Foa, andVd). The antifungal activity of these new compounds was observed for the first time; however, detailed studies regarding its mode of action and efficacy under field condition are needed to be carried out.


## 4. Conclusion

The catalytic Kharasch reaction ofα-pinene, β-pinene, limonene, limonene oxide, and valencene was studied using the iron acetylacetonate as catalyst and triethylamine as cocatalyst. The catalytic system showed to be efficient for the addition of carbon tetrachloride to all terpenes used. Regarding biological control of plant disease management, the antifungal bioassays showed that compounds2c and2d could constitute new lead compounds for further studies to develop antifungal agent againstFusarium oxysporum f. sp.albedinis,Fusarium oxysporum f. sp.canariensis, andVerticillium dahliae.



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*Source: 2784303-2017-07-09.xml*