untitled European Journal of Chemistry 7 (2) (2016) 225‐229 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2016 Atlanta Publishing House LLC ‐ All rights reserved ‐ Printed in the USA http://dx.doi.org/10.5155/eurjchem.7.2.225‐229.1423 European Journal of Chemistry Journal webpage: www.eurjchem.com Synthesis and HPLC resolution of isomers of novel phosphorus fluorinated 2,4,6‐trimethylphenylazo pyridines Zineb Hacini 1,* and Lakhdar Sekhri 2 1 Department of Chemistry, University of Hadj Lakhdar, Batna, 05000, Algeria 2 Department of Chemistry, University of Kasdi Merbah, Ouargla, 30000, Algeria * Corresponding author at: Department of Chemistry, University of Hadj Lakhdar, Batna, 05000, Algeria. Tel.: +213.7.76213615. Fax: +213.29.711936. E‐mail address: zn.hacini@gmail.com (Z. Hacini). ARTICLE INFORMATION ABSTRACT DOI: 10.5155/eurjchem.7.2.225‐229.1423 Received: 05 March 2016 Accepted: 17 April 2016 Published online: 30 June 2016 Printed: 30 June 2016 New compounds of phosphorus fluorinated 2,4,6‐trimethylphenylazo pyridines (4a‐c) have been synthesized in high yields via adding n‐butyl lithium in hexane to a stirred solution of methyldiphenylphosphine oxide in dry THF at 0 °C, then cooled to around ‐78 °C, treated with azo‐pyridines(2a‐c) and then allowed to warm at room temperature over 2 h. The isomers of (E)‐((5‐chloro‐3,6‐difuoro‐4‐(mesityldiazenyl)pyridin‐2‐yl)methyl)diphenyl phosphine oxide (4b) and (E)‐((3,6‐difuoro‐4‐(mesityldiazenyl)5‐methoxypyridin‐2‐ yl)methyl)diphenyl phosphine oxide (4c) can be separated on analytical HPLC: Chiralcel OD‐H column, hexane:2‐PrOH, (9:1, v:v) mobile phase, flow‐rate, 1.0 mL/min, 25 °C, λ = 254 nm and polarimetric detection, 20 µL injection volume. The resolution of this isomers were 2.12, 1.84, respectively. KEYWORDS Isomers Synthesis HPLC resolution Chiralcel OD‐H column Phosphorus fluorinated X‐Ray single crystal structure Cite this: Eur. J. Chem. 2016, 7(2), 225‐229 1. Introduction The fluorinated organic phosphorus is a heterogeneous class of products widely used for the treatment of a number of cardiovascular diseases, including congestive heart failure and coronary heart disease. A combination of fluorinated and diphenylphosphinoyl groups in azo‐compounds will hopefully make them very interesting biological active compounds. Since phosphorus substituents regulate important biological functions [1‐3] and fluorine containing compounds play important role in organic synthesis and in medicinal chemistry [4‐10]. Since the literature contains little or no information on phosphorus fluorinated azo type (Figure 1), in this article, we describe the synthesis, isolation of the novel isomers of the phosphorus fluorinated 2,4,6‐trimethylphenylazo pyridines obtained by introducing a diphenylphosphinium ring on the terminal fluorine group of azo‐pyridines (2a‐c) (Scheme 1) [11]. The resulting of this reaction gave the structural isomers of phosphorus fluorinated azo, HPLC will be used to separate the structural isomers. 2. Experimental 2.1. Instrumentation Melting points were determined with a capillary apparatus (Büchi 540) and are uncorrected. IR spectra were obtained with a Perkin‐Elmer 983 G spectrometer on KBr disks. 1H NMR spectra were run on a Bruker AC 300 spectrometer, 1H NMR spectra was recorded using CHCl3 as internal standard. 19F spectra, chemical shifts were measured relative to trifluoro‐ acetic acid (TFA) as an external interchange reference unless otherwise stated. Fast atom bombardment (FAB) spectra were recorded on a Kratos MS 50, using a meta‐nitrobenzyl alcohol matrix. Figure 1. The structure chemical of phosphorus fluorinatedazo. 226 Hacini and Sekhri / European Journal of Chemistry 7 (2) (2016) 225‐229 Scheme 1 Desorption chemical ionization (DCI) of the isomers (4a‐c) were obtained with a Finnigan‐Mat 8400 spectrometer using iso‐butane as reacting gas. Flash column chromato‐graphy was performed on silica gel (Merck Kieselgel 60, 230‐400 mesh ASTM) using the indicated eluents. Petroleumether 40‐60 °C (PE) was used as eluent. The progress of the reactions was followed by thin‐layer chromatography (TLC) on 5×3×20 cm plates with a layer thickness of 0.25 mm. When necessary, they were developed with diphenylamine reagent. Anhydrous magnesium sulfate was used as a drying agent for the organic phases. Organic solvents were removed under vacuum at room temperature. Column chromatography was conducted using silica gel, pore size 60 Å, 230‐400 mesh particle size (Merck & Co).Thin layer chromatography (TLC) was conducted on pre‐ coated aluminum sheets (60F254) with a 0.2 mm thickness (Aldrich Chemical Co.). Elemental analysis of the target compounds was performed by REDOX (Monza) and the results are represented in full. Analytical isomers HPLC experiments on Chiralcel OD‐H column (250×4.6 mm, 5 µm) (Daicel Co., Tokyo) were performed with LaChrom1 (Merck) screening unit equipped with an L‐7100 pump, an L‐7200 autosampler, an L‐7360 oven which accommodates 12 columns alimented by a Valco positions valve, an L‐7400 UV detector, and a Jasco OR‐1590 polarimeter detector. Analyses were performed at 1 mL/min, at a controlled temperature (25 °C) with UV (254 nm) and polarimetric detection. Retention times (Rt) in minutes, retention factor ki = (Rti‐Rt0)/Rt0 and enantio selectivity factor α = k2/k1 are given. Semi preparative separations were performed with a Merck‐Hitachi LiChrograph Model L‐6000 HPLC pump, and a Merck‐Hitachi LiChrograph L‐4000 UV detector (254 nm). For semi‐prepa‐ rative separations, a Chiralcel OD (250×10 mm, 10 µm) was used. The solvents were HPLC grade from SDS (Peypin, France) and were filtered on a Millipore membrane of 0.45µm and degassed before use. The optical rotations were measured on a 241 MC Perkin‐Elmer polarimeter with a sodium lamp and a double‐jacketed cell at 25 °C. All reagents were commercially available. 2.2. Synthesis of phosphorus fluorinated 2,4,6‐trimethyl phenylazopyridine (4a‐c) 2.2.1. Synthesis of (E)‐diphenyl((3,5,6‐trifuoro‐4‐(mesityl diazenyl)pyridin‐2‐yl)methyl)phosphine oxide (4a) A dry 500 mL flask equipped with a magnetic stirring bar was charged with 6.0 g (27.6 mmol) of methyldiphenyl phosphine oxide, capped with a rubber septum, and flushed with nitrogen. Anhydrous tetrahydrofuran (175 mL) was then added to the flask via cannula, and the resulting solution cooled in an ice bath to 0 °C. A solution of n‐butyllithium in hexane (11.04 mL, 27.6 mmol, 2.5 M) was added dropwise via a syringe over a 5 min period. The solution turned deep red. The resulting red solution was stirred for 30 min at 0 °C, then cooled to around ‐78 °C in an acetone‐solid carbon dioxide cooling bath. Fresh solution compound 2a (8.2 g, 27.6 mmol) was added in one portion by syringe. After the addition was complete, the red color of the anion had disappeared. The resulting pale yellow solution was stirred for 15 min at ‐78 °C, then allowed to warm to ambient temperature over 2 hrs. Water (40 mL) was added and the bulk of the tetrahydrofuran and hexane removed on a rotary evaporator (Bath temp.: 25‐ 30 °C). Brine and dilute hydrochloric acid (200 mL) was added to the aqueous residue and extracted with dichloromethane (3 × 100 mL). The combined organic extracts were dried over magnesium sulfate and evaporated to dryness under reduced pressure on a rotavapor, and the residue, light yellow oil, was placed in a refrigerator overnight to crystallize. The resulting solid was recrystallized from ethylacetate to give compound 4a (Scheme 1). Yield: 81%. Color: White. M.p.: 167‐169 °C. FT‐ IR (KBr, , cm‐1): 1650‐1565 (N=N), 1260‐1454 (Ar‐F), 1049 (P=O). 1H NMR (300 MHz, CDCl3, δ, ppm): 2.52 (s, 3H, 4‐CH3), 2.32 (s, 6H, 2,6‐CH3), 3.33 (s, 2H, CH2PO), 6.89 (s, 2H, Ar‐H), 7.05 (m, 10H, Ar‐H). MS (FAB, m/z (%)): 493 (M++1, 100), 374 (C18H12F3PON3+, 40), 292 (80.1), 278 (10.2), 215 (6.2), 201 (20), 119 (60). Anal. calcd. for C27H23ON3F3P: C, 65.72; H, 4.70; N, 8.52. Found: C, 65.80; H, 4.80; N, 8.50%. 2.2.2. Synthesis of (E)‐((5‐chloro‐3,6‐difuoro‐4‐(mesityl diazenyl)pyridin‐2‐yl)methyl)diphenyl phosphine oxide (4b) isomers Compound 4b was prepared from methyldiphenyl phosphine oxide (6.0 g, 27.6 mmol), and compound 2b (8.65 g, 27.6 mmol) in a similar way to compound 4a. Work‐up of the reaction product gave a white crystalline solid of compound 4b isomers (Scheme 1). Yield: 76%. Color: White. FT‐IR (KBr, , cm‐1): 1600‐1565 (N=N), 1436 (Ar‐F), 1250‐1070 (Ar‐Cl), 1181 (P=O). 19F NMR (54.6 MHz, CDCl3, δ, ppm): ‐84.3 (1F, d, J = 25.6.6 Hz, F‐5), ‐14.5 (1F, d, J = 25.6 Hz, F‐2). MS (FAB, m/z (%)): 474 (M++ 1‐Cl, 100), 215 (6.0), 201 (20), 147 (17), 119 Hacini and Sekhri / European Journal of Chemistry 7 (2) (2016) 225‐229 227 (60). Anal. calcd. for C27H23ON3F2ClP: C, 63.60; H, 4.55; N, 8.24. Found: C, 63.70; H, 4.50; N, 8.20 %. Isomers purities (4b1, 4b2) were assessed on analytical HPLC: Chiralcel OD‐H column, hexane:2‐PrOH, 9:1, v:v mobile phase, flow‐rate 1.0 mL/min, 25 °C, λ = 254 nm and polarimetric detection, 20 µL injection volume (first eluted peak: k = 2.01; second peak: k = 2.36), α = 1.18, Rs = 2.12. 2.2.3. Synthesis of (E)‐((3,6‐difluoro‐4‐(mesityldiazenyl)‐5‐ methoxypyridin‐2‐yl)methyl)diphenyl phosphine oxide (4c) isomers Compound 4c was prepared from methyldiphenyl phosphine oxide (1.0 g, 4.6 mmol), and compound 2c (0.5 g, 1.61 mmol) in a similar way to compound 4a. The white solid resulted was recrystallized from ethyl acetate to give a solid of compound 4c isomers (Scheme 1). Yield: 89%. Color: White. FT‐IR (KBr, , cm‐1): 1600 (N=N), 1436 (Ar‐F), 1173 (P=O), 1203‐1277 (C‐O asym. stretch), 1042 (C‐O sym. stretch). 19F NMR (54.6 MHz, CDCl3, δ, ppm): ‐82.5 (1F, d, J = 25.3 Hz, F‐5), ‐ 11.2 (1F, d, J = 25.3 Hz, F‐2). 1H NMR (300 MHz, CDCl3, δ, ppm): 2.30 (s, 6H, 2,6‐CH3), 2.45 (s, 3H, 4‐CH3), 3.32 (s, 2H, CH2PO), 3.95 (s, 3H, O‐CH3), 6.85 (s, 2H, Ha), 7.02 (m, 10H, Ha). MS (FAB, m/z (%)): 490 (M++1‐Me, 100), 304 (78.5), 290 (18.5), 215 (6.0), 201 (22), 147 (18), 119 (63). Anal. calcd. for C28H26O2N3F2P: C, 66.53; H, 5.18; N, 8.31. Found: C, 66.70; H, 5.10; N, 8.30 %. The composition isomers 4c1 and 4c2 were assessed on analytical HPLC: Chiralcel OD‐H column, hexane:2‐PrOH, 9:1, v:v mobile phase, flow‐rate 1.0 mL/min, 25 °C, λ = 254 nm and polarimetric detection, 20 µL injection volume (first eluted peak: k = 3.25; second peak: k = 3.65), α = 1.12, Rs =1.84. 2.3. Single crystal structure determination In order to establish the absolute configuration of the newly created isomers, the structure of the major product of compound 4b and 4c were elucidated by X‐ray single crystal diffraction analysis (Figure 2‐5). Figure 2. Molecular structure of compound 4b1. Figure 3. Molecular structure of compound 4b2. The crystals used for the X‐ray single crystal diffraction study were grown by routine recrystallization from aceto‐ nitrile and ethyl acetate for compound 4b and 4c, respectively. The crystal data are given in Table 1. The unit cell dimensions were determined by least‐squares using 25 for compound 4b and 15 for compound 4c centered reflections using graphite monochromated Cu‐Kα radiation. Data were corrected for Lorentz and polarization effects. A correction for secondary extinction was applied (coefficient = 0.53319×10‐5 for 4b, 0.878954×10‐5 for compound 4c). The structures for compound 4b and 4c were solved by direct methods. The non‐ hydrogen atoms (for compound 4b and 4c) were refined anisotropically. All other hydrogens were located on difference Fourier maps and were refined isotropically. The molecule of compound 4c crystallizes with the solvent ethyl acetate in the proportion 2:1. The ethyl acetate molecule is disordered over two orientations about the two‐fold axis. Each orientation corresponds to 50% occupancy. No atoms are on the axis but the methylene and carbonyl carbons are close. The carbonyl carbon was refined isotropically because of its proximity to the two‐fold axis. Hydrogen atoms in the solvent molecule were not included in the model. Figure 4. Molecular structure of compound 4c1. Figure 5. Molecular structure of compound 4c2. Table 1. Crystal data of compound 4b and 4c. Parameters 4b 4c Chemical formula C27H23ON3F2ClP C28H26O2N3F2P Crystal system Orthorhombic Orthorhombic Cell dimension, Å a = 9.940(2) a = 12.773(2) b = 25.077(7) b = 6.694(3) c = 7.654(2) c = 11.890(2) Cell volume, Å3 1907.7(3) 1006.1(5) Space group P2/c (no. 13) P21/c (no. 14) Z 4 4 ρ(calc.), g/cm‐3 1.13 1.11 µ, cm‐1 2.0 1.7 3. Results and discussion Phosphorus fluorinated 2,4,6‐trimethylphenylazo pyridines (4a‐c) were synthesized according to the procedures reported in Scheme 1. The general procedure involves drop wise addition of an equimolar quantity of a solution of n‐BuLi in hexane to a stirred solution of methyldiphenylphosphine oxide in dry THF at 0 °C, then cooled to around ‐78 °C. Fresh solution azo‐pyridines (2a‐c) [11] was added in one portion. After the addition was complete, the red color of the anion had disappeared. The resulting pale yellow solution was stirred for 15 min at ‐78 °C, then allowed to warm to ambient tempe‐ rature over 2 h. Work‐up of the reaction product gave the corresponding phosphorus fluorinated compound 4a‐c in high yields ranging from 76 to 89% after purification by column chromatography. The obtained products were identified by elemental analysis and spectral data. 228 Hacini and Sekhri / European Journal of Chemistry 7 (2) (2016) 225‐229 Table 2. Selected bond lengths (Å). Compound 4b1 C(26)‐C(27) 1.3401(6) C(22)‐C(23) 1.5090(16) P‐C(10) 1.8560(17) C(24)‐C(25) 1.3340(8) C(23)‐N(1) 1.2600(10) P‐C(16) 1.8560(17) C(2)‐C(8) 1.4970(24) C(27)‐N(1) 1. 2600(7) P‐C(22) 1.8560(15) C(4)‐C(9) 1.4970(23) C(24)‐F(1) 1.3920(2) C(3)‐N(3) 1.3430(4) C(6)‐C(7) 1.4970(23) C(27)‐F(2) 1.3920(9) C(25)‐N(2) 1.3430(3) Compound 4b2 C(26)‐C(27) 1.5090(28) C(22)‐C(23) 1.3340(30) P‐C(10) 1.8560(25) C(24)‐C(25) 1.3340(32) C(22)‐N(1) 1.2600(36) P‐C(16) 1.8560(17) C(2)‐C(8) 1.4970(21) C(26)‐N(1) 1.2600(36) P‐C(27) 1.8560(19) C(4)‐C(9) 1.4970(21) C(22)‐F(2) 1.3920(30) C(3)‐N(3) 1.2600(36) C(6)‐C(7) 1.4970(22) C(23)‐F(1) 1.3920(33) C(24)‐N(2) 1.2600(29) Compound 4c1 C(26)‐C(27) 1.4170(36) C(22)‐C(23) 1.5090(25) P‐C(10) 1.8560(24) C(24)‐C(25) 1.4170(36) O(1)‐C(28) 1.5090(36) P‐C(16) 1.8560(16) C(2)‐C(8) 1.4970(24) C(26)‐O(1) 1.4912(33) P‐C(22) 1.8560(15) C(4)‐C(9) 1.4970(23) C(24)‐F(1) 1.3920(17) C(3)‐N(3) 1.4170(33) C(6)‐C(7) 1.4970(23) C(27)‐F(2) 1.3920(19) C(25)‐N(2) 1.4170(28) Compound 4c2 C(22)‐C(23) 1.4200(15) C(25)‐C(26) 1.4200(13) P‐C(10) 1.8560(11) C(26)‐C(27) 1.4200(16) O(1)‐C(28) 1.3960(19) P‐C(16) 1.8560(16) C(2)‐C(8) 1.4970(14) C(25)‐O(1) 1.3550(21) P‐C(27) 1.8560(13) C(4)‐C(9) 1.4970(14) C(22)‐F(2) 1.3920(17) C(3)‐N(3) 1.4560(10) C(6)‐C(7) 1.4970(14) C(23)‐F(1) 1.3920(19) C(24)‐N(2) 1.4560(13) Table 3. Selected bond angles (°). Compound 4b1 C(10)‐P‐C(16) 109.5200(11) C(8)‐C(2)‐C(3) 121.4000(12) P‐C(22)‐C(23) 109.4618(11) C(9)‐C(4)‐C(3) 121.4000(12) F(1)‐C(24)‐(23) 120.0000(16) C(24)‐C(25)‐C(26) 114.5118(14) F(1)‐C(24)‐(25) 120.0000(16) Cl‐C(26)‐C(27) 122.7441(18) F(2)‐C(27)‐N(1) 117.2559(16) Cl‐C(26)‐C(25) 122.7441(18) Compound 4b2 C(10)‐P‐C(16) 109.5200(10) C(8)‐C(2)‐C(3) 121.4000(12) P‐C(27)‐ C(26) 109.4618(10) C(9)‐C(4)‐C(3) 121.4000(12) F(1)‐C(23)‐C(22) 115.0000(10) C(24)‐C(25)‐C(26) 117.2559(12) F(1)‐C(23)‐C(24) 120.0000(16) Cl‐C(25)‐C(26) 122.7441(18) F(2)‐C(22)‐N(1) 116.5000(11) Cl‐‐C(25)‐C(24) 122.7441(18) Compound 4c1 C(10)‐P‐C(16) 109.4618(10) C(8)‐C(2)‐C(3) 121.4000(12) C(10)‐P‐C(22) 109.5200(17) C(9)‐C(4)‐C(3) 121.4000(12) F(1)‐C(24)‐C(23) 120.0000(16) C(24)‐C(25)‐C(26) 120.0000(11) F(1)‐C(24)‐C(25) 120.0000(14) C(24)‐C(25)‐N(2) 117.2559(10) F(2)‐C(27)‐N(1) 120.0000(14) C(26)‐C(27)‐N(1) 125.4882(17) Compound 4c2 C(10)‐P‐C(16) 109.5200(10) C(8)‐C(2)‐C(3) 121.4000(12) C(10)‐P‐C(27) 109.5000(10) C(9)‐C(4)‐C(3) 121.4000(12) F(1)‐C(23)‐C(24) 120.0000(16) C(25)‐C(26)‐C(27) 121.4000(12) F(1)‐C(23)‐C(22) 115.0000(10) C(23)‐C(22)‐N(1) 120.0000(12) F(2)‐C(22)‐N(1) 116.5000(11) C(27)‐C(26)‐N(1) 125.4882(20) The IR spectrum of the compounds showed characteristic P=O stretching at frequency in the region (1040‐1180 cm‐1). The proton coupled 1H NMR of compound 4b and 4c showed a singly centered at δ 3.33 and 3.32 ppm corresponding to the CH2 group. The mass spectrum of compound 4a, 4b and 4c showed the base peaks at m/z 493, 474 and 490, respectively, and clearly showed the presence of (Ph)2P=O and C9H11 in the chemical structure. Crystallization of compound 4a from ethylacetate gave single crystals in 81% yield. Thus, attack by nucleophilic at the position 2 (or 6) has a faster rate than that at the 3‐ (or 5‐) position. The preferential substitution at the 2‐ or 6‐position because the result was by attained the essence compound. The substitution in these positions was preferred comparison with the 3‐ or 5‐position because they offered a stable position (Scheme 1). We have succeeded in achieving the nucleophilic aromatic substitution of the fluoro group in compound 2b with (Ph)2‐ POCH2‐ anion at 2‐ and 6‐position, thus obtaining compound 4b isomers. The substitution at the 2‐ or 6‐positionin exchange for the 5‐position because they are crowd in this position. Compound 4b and 4c were resolved into the corres‐ ponding isomers (Scheme 1) by chiral chromatography using a Chiralcel OD column in a high degree of optical purity. These separations were amenable to semi‐preparative scale. Special care was taken during all the semi preparative experiments and the isolated isomers were kept covered by the solvent of elution during the concentration step to minimize the explosive hazard. However, as the alcohol, propan‐2‐ol, decreased, there solutions (Rs) was all steadily increased, suggesting that the polar interaction (mainly hydrogen‐ bonding interaction) between solute and stationary phase was not only the primary factor for solute retention but also playing some roles in isomeric recognition. The phosphorus and fluorinated azo compounds 4b1 (M.p.: 194‐196 °C) was identified by elemental analysis and spectroscopic methods. The 1H NMR spectrum showed four absorptions. The 19F NMR spectrum showed two doublet absorption bands of equal intensity at δ ‐14.5(F2) and ‐84.3 (F5) ppm which suggests that the (Ph)2‐POCH2‐ group lies in position 6 not 2, the X‐ray single crystal diffraction analysis showed that the conformation of this bond (Figure 2). The bond distances and bond angles are given in Table 2 and 3. The mass spectrum of compound 4b1 showed a molecular ion at m/z 119 and 201 clearly showed the presence of C9H11 and (Ph)2‐P=O in the chemical structure and a base peak at m/z 474. The other novel isomer compound 4b2 (M.p.: 190‐192 °C) was also identified by elemental analysis and spectros‐ copically. The IR, 1H NMR and the mass spectrum were very Hacini and Sekhri / European Journal of Chemistry 7 (2) (2016) 225‐229 229 similar to that of compound 4b1. It’s 19F NMR spectrum showed two doublets absorptions of equal intensity at δ ‐9.4 and ‐79.0 ppm, which suggests that the (Ph)2‐POCH2 group lies in position 2 not 6, the new structure of compound 4b2 present in Figure 3 was elucidated by single crystal X‐ray diffraction analysis. The bond distances and bond angles are listed in Table 2 and 3. Isomers 4c was separated by on analytical HPLC to give compound 4c1 and 4c2 (Scheme 1). The structure of compound 4c1 and 4c2 presented in Figure 4 and 5 was elucidated by single crystal X‐ray diffraction analysis and reveals new carbon atom bonded together, C(25)‐C(26) for compound 4c1, new carbon atom C(22)‐C(23) for compound 4c2 for compound and the distance between them (Table 2). The novel phosphorus and fluorinated azo compounds 4c1 (M.p.: 202‐204 °C) possessed satisfactory elemental compo‐ sition. It’s 1H NMR spectrum showed four absorption bands The 19F NMR spectrum showed two doublets absorptions bands of intensity ‐11.2 and ‐82.5 ppm which clearly the position of (Ph)2‐POCH2 group the mass spectrum showed a molecular ion at 260, 201, 119 and base peak at 490 m/z. The isomer 4c2 (M.p.: 201‐203 °C) was identified by comparison of its IR and 19F‐, 1H NMR spectra. The 19F NMR of compound 4c2 exhibits showed two doublets absorptions bands of intensity ‐ 8.1 and ‐72.2 ppm, the mass spectrum were very similar to that of compound 4c1. 4. Conclusions In conclusion the preparation of phosphorus fluorinated 2,4,6‐trimethylphenylazo pyridines are easily achieved by the condensation of 2,4,6‐trimethylphenylazopyridines with methyldiphenylphosphineoxide. The success and yields of the reaction are affected by the following factors: the stability and nucleophilicity of the fluorinated and the other is the electrophilicity of the group substitution, then we have developed a new method for the synthesis of phosphorus fluorinated 2,4,6‐trimethylphenylazo pyridines. The identity of the isomers was then confirmed with the established HPLC method, the resolution of their structural isomers was superior. The methods have potential applications in the determination of this isomer. Furthermore, since ChiraSphe column is characterized by its high stability and high loading capacity, this column can be used for semi‐preparative separation of phosphorus and fluorinated azo compounds isomers and therefore this method could be useful for further pharmacological investigation of the individual isomer of phosphorus and fluorinated azo compounds. 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