Study of ADMET Descriptors of Novel Chlorinated N-Arylcinnamamides ^†

Abstract

(2E)-3-(3,4-Dichlorophenyl)-N-arylprop-2-enanilides were designed as potential bioactive agents. Six compounds were mono- and di-chlorinated also on the anilide ring. Since the biological activities of molecules are influenced by lipophilicity, the hydro-lipophilic characteristics of these compounds were experimentally studied. In addition, the overall ADMET (absorption, distribution, metabolism, excretion, toxicity) profiles of these molecules were studied to establish whether they comply with the Lipinski’s rule of five and thus meet the “druglikeness” requirement. All the compounds were analyzed using the reversed-phase high performance liquid chromatography method. The procedure was carried out under isocratic conditions and a C₁₈ stationary reversed-phase column. The structure–lipophilicity relationships of the investigated compounds are discussed.

1. Introduction

The ADMET (an abbreviation for “absorption, distribution, metabolism, excretion, and toxicity”) properties of compounds characterizing pharmacokinetics are as important as the biological effect of a drug [1,2,3,4]. Physicochemical properties affecting the permeability and bioaccumulation of cells belong to the area of quantitative structure–property relationships (QSPR) and are influenced by chemical composition [5,6,7]. In this context, lipophilicity was recognized more than a hundred years ago as the most important parameter influencing ADMET and bioactivity (e.g., lipoid theory of narcosis formulated by Meyer and Overton) [3,4]. The lipophilicity parameter is also part of Lipinski’s Rule of Five (Ro5) or Carr’s rule of three (Ro3) [6,7,8,9]. The issue of lipophilicity was also addressed by Hansch et al. who derived a set of empirical lipophilicity descriptors, so-called π-values [10].

Lipophilicity is the affinity of a molecule for a lipophilic environment and is determined by the distribution behavior in a two-phase system; liquid-liquid or solid-liquid. In general, it is a thermodynamic parameter describing the partitioning of a compound between an aqueous and an organic phase and can be characterized by the partition coefficient (log P). Log P is defined as a logarithm of the partition coefficient of the compound between n-octanol and water at a pH where all of the compound molecules are in the in the neutral form [3,4]. As classical methods for determining lipophilicity are time consuming, reversed-phase high performance liquid chromatography (RP-HPLC) has become the methodology used. Most often, retention times are measured under isocratic conditions with varying amounts of organic modifier in the mobile phase using end-capped non-polar C₁₈ stationary RP columns and the logarithm of the capacity factor k is then calculated [3,11].

Because most drugs are weak bases or acids that are ionized under physiological conditions, another parameters describing lipophilicity can be found, namely the distribution coefficient D_pH and its log D_pH, which is the logarithm of the distribution coefficient of the compound between n-octanol and an aqueous phase (buffer) at a specified pH. A portion of the compound molecules may be in the ionic form and a portion may be in the neutral form [3,4,12]. The distribution coefficient is important because it takes into account ionization. It is most often determined for physiological values, for example, for pH 7.4 (log D_7.4 values). Likewise, from the point of view of absorption after oral administration, the partition coefficient at pH 6.5 (log D_6.5) is important, because it is the pH in the small intestine [3,4,13,14].

Recently, a large series of ring-substituted N-arylcinnamanilides together with their biological activities were published [15,16,17,18]. Since early prediction of physicochemical properties, i.e., “druglikeness”, is important for identification of a suitable candidate at the early drug discovery stage, several compounds from the new series of chlorinated N-arylcinnamanilides were investigated in relation to their ADMET profile and structure–lipophilicity relationships.

2. Results and Discussion

The reaction of 3,4-dichlorocinnamic acid with phosphorus trichloride and aniline in dry chlorobenzene in a microwave reactor provided a set of N-arylcinnamamides 1–7, Scheme 1 and

Table 1. Structure of ring-substituted (2E)-3-(3,4-dichlorophenyl)-N-arylprop-2-enamides 1–7, calculated lipophilicities (log P/Clog P), and experimentally determined log k, log D_7.4, and log D_6.5 values of investigated compounds.

Comp.	R	log k	log D7,4	log D6.5	log P a	log P/Clog P b
1	H	0.6199	0.6354	0.6669	4.42	4.30/4.9700
2	2-Cl	0.7764	0.8019	0.8203	5.10	4.86/5.0906
3	3-Cl	0.9071	0.8735	0.9453	5.31	4.86/5.9406
4	4-Cl	0.9009	0.8660	0.9381	5.19	4.86/5.9406
5	2,4-Cl	1.0932	1.0565	1.0985	5.68	5.41/5.8938
6	2,5-Cl	1.0840	1.0474	1.0887	5.72	5.41/5.8938
7	3,5-Cl	1.3043	1.3080	1.3336	5.90	5.41/6.7438

^a ACD/Percepta ver. 2012, ^b ChemBioDraw Ultra 13.0.

.

The log P/Clog P data of all the investigated chlorinated N-arylcinnamamide derivatives were predicted using commercially available programs ChemBioDraw Ultra 13.0 and ACD/Percepta ver. 2012. The lipophilicity of the compounds was also examined by the RP-HPLC determination of capacity factors k followed by calculation of log k and the determination of distribution coefficients D_7.4 and D_6.5 with the subsequent calculation of log D_7.4 and log D_6.5. All the results are shown in Table 1. The HPLC procedure was performed under isocratic conditions with methanol as an organic modifier in the mobile phase using end-capped non-polar C18 stationary RP columns.

Parameters predicted by the ChemBioDraw software (log P and Clog P) for individual positional isomers are not distinguished. Therefore, these values are listed only in Table 1 without other discussion. On the other hand, lipophilicity data log P for compounds 1–7 predicted by ACD/Percepta showed high consensus with all the experimentally determined values log k, log D_7.4, and log D_6.5, as can be seen in graphs in Figure 1; the correlation coefficients r for n = 7 are as follows: 0.9609, 0.9420, and 0.9513, respectively. The mutual consensus of all the experimental parameters is also very high (r = 0.9931, 0.9981, and 0.9952, respectively), see Figure 2. Thus, based on the experimental and predicted results, it can be stated that unsubstituted (2E)-3-(3,4-dichlorophenyl)-N-phenylprop-2-enamide (1) is the least lipophilic compound, while (2E)-N-(3,5-dichlorophenyl)-3-(3,4-dichlorophenyl)prop-2-enamide (7) is the most lipophilic. The only difference between all experimental and predicted values was observed for compounds 5 (R = 2,4-Cl) and 6 (R = 2,5-Cl), where compound 5 actually shows a higher lipophilicity than compound 6, which was predicted by the software vice versa: log P = 5.68 (5) and log P = 5.72 (6). This is caused by specific intra- and intermolecular interactions of the substituent in the ortho position with other spatially close moieties/fragments and the polar medium as was described recently [15,18,19,20,21,22]. These results confirm that the log P values generated by ACD/Percepta are in good agreement with the experimental values. The question remains what will be the inaccuracies in the prediction for anilide substituents capable of forming mainly hydrogen bonds (e.g., -F, -CF₃, -OCH₃) with the surrounding aqueous/buffered medium.

Distribution parameters π [23,24] were introduced to characterize the lipophilic contribution of individual substituents to the scaffold and they are calculated according to the relationship π = log k_S - log k_U, where log k_S is the determined logarithm of the capacity factor of the compound, and log k_U indicates the determined logarithm of the capacity factor of unsubstituted derivative 1, whose π value is 0. The same applies to the values of the distribution coefficient D_pH. The π values of individual substituted anilide rings (π_Ar) of drivatives 1–7 are mentioned in

Table 2. Comparison of determined distributive parameters π calculated from log k and log D_pH and parameters π of individual substituted anilide rings predicted by ACD/Percepta.

Comp.	R	πAr (exp. log k)	πAr (exp. log D7.4)	πAr (exp. log D6.5)	πAr (ACD/Percepta)
1	H	0	0	0	1.76
2	2-Cl	0.16	0.17	0.15	2.23
3	3-Cl	0.29	0.24	0.28	2.32
4	4-Cl	0.28	0.23	0.27	2.33
5	2,4-Cl	0.47	0.42	0.43	2.82
6	2,5-Cl	0.46	0.41	0.42	2.73
7	3,5-Cl	0.68	0.67	0.67	2.90

, where there are differences (mutual order of values) between experimental and calculated π_Ar values of compounds 5 (R = 2,4-Cl) and 6 (R = 2,5-Cl). The differences between π_Ar values calculated by ACD/Percepta are due to the failure to include possible interactions of substituents in the ortho position with a spatially close carboxamide, while π_Ar values based on experimental log k/log D_pH data contain these interactions. The π_Ar values calculated from the experimentally determined parameters have insignificant differences.

The Ro5 [8,9] is one of the most important rules used for drug design [25]. Ro5 contains the limits of specific molecular descriptors (see

Table 3. Values of parameters characterizing physicochemical properties calculated using ACD/Percepta ver. 2012 in relation to Lipinski’s Rule of Five (Ro5).

Comp.	R	MW	log P	HBD	HBA	RB	TPSA	Parachor
1	H	292.16	4.42	1	2	3	29.10	581.26
2	2-Cl	326.60	5.10	1	2	3	29.10	617.13
3	3-Cl	326.60	5.31	1	2	3	29.10	617.13
4	4-Cl	326.60	5.19	1	2	3	29.10	617.13
5	2,4-Cl	361.05	5.68	1	2	3	29.10	653.00
6	2,5-Cl	361.05	5.72	1	2	3	29.10	653.00
7	3,5-Cl	361.05	5.90	1	2	3	29.10	653.00
Ro5		<500	<5	<5	<10	–	–	–

Molecular weight (MW), lipophilicity (log P), number of H-bond donors (HBD), number of H-bond acceptors (HBA), number of rotatable bonds (RB), topological polar surface area (TPSA).

) determined on the basis of experimentally and statistically obtained results. A biologically active compound that meets these criteria has a higher chance of becoming a drug. Table 3 lists the parameters contained in Ro5 plus some of the other most used. Based on the data presented in Table 3, it can be stated that in general, the investigated compounds meet the Ro5 requirements. However, it should be mentioned that a suitable drug-like profile does not ensure that the molecule will become a drug and vice versa [26]. In this context, compounds 2–7 have a slightly higher lipophilicity (log P values) than recommended by Ro5. In addition to higher lipophilicity, the individual substituents on both the phenyl acid core and the anilide ring are characterized by electron-withdrawing properties (electronic σ parameters of anilide substituents ranged from 0.75 to 1.22 [27]), making them potentially interesting chemotherapeutics as well as agrochemicals [28]. On the other hand, these higher lipophilic compounds showed a log D_7.4 slightly higher than 1, indicating that the compounds are expected to have good solubility, good intestinal absorption (good balance of solubility and passive diffusion permeability), and minimized metabolism (lower binding to metabolic enzymes) [3,4].

3. Experimental

3.1. General

All reagents were purchased from Merck (Sigma-Aldrich, St. Louis, MO, USA) and Alfa (Alfa-Aesar, Ward Hill, MA, USA). Reactions were performed using an Anton-Paar Monowave 50 microwave reactor (Graz, Austria). The melting points were determined on a Kofler hot-plate apparatus HMK (Franz Kustner Nacht KG, Dresden, Germany) and are uncorrected. Infrared (IR) spectra were recorded on a Nicolet iS5 IR spectrometer (Thermo Scientific, West Palm Beach, FL, USA). The spectra were obtained by the accumulation of 256 scans with 2 cm⁻¹ resolution in the region of 4000–450 cm⁻¹. All ¹H- and ¹³C-NMR spectra were recorded on a JEOL JNM-ECA 600II device (600 MHz for ¹H and 150 MHz for ¹³C, JEOL, Tokyo, Japan) in dimethyl sulfoxide-d₆ (DMSO-d₆). ¹H and ¹³C chemical shifts (δ) are reported in ppm.

3.2. Synthesis

General procedure for synthesis of target compounds 1–8: 3,4-Dichlorocinnamic acid (0.9 mM) was suspended in dry chlorobenzene (6 mL) at ambient temperature and phosphorus trichloride (0.45 mM, 0.5 eq.), and the corresponding substituted aniline (0.9 mM, 1 eq.) was added dropwise. The reaction mixture was transferred to the microwave reactor, where the synthesis was performed (40 min, 130 °C). Then, the mixture was cooled to 40 °C, and then the solvent was removed to dryness under reduced pressure. The residue was washed with hydrochloride acid and water. The crude product was recrystallized from ethanol.

(2E)-3-(3,4-Dichlorophenyl)-N-phenylprop-2-enamide (1). Yield 63%; Mp 140–143 °C; IR (cm⁻¹): 3251, 3126, 3038, 1654, 1618, 1597, 1551, 1533, 1497, 1486, 1469, 1444, 1391, 1290, 1240, 1197, 1183, 1129, 1076, 1031, 1004, 969, 947, 922, 894, 868, 814, 784, 751, 735, 693, 684, 677, 661, 590, 564, 508, 485; ¹H-NMR (DMSO-d₆) δ: 10.23 (s, 1H), 7.90 (d, J = 2.1 Hz, 1H); 7.71–7.69 (m, 3H), 7.62 (dd, J = 8.9 Hz, J = 2.1 Hz, 1H); 7.57 (d, J = 15.1 Hz, 1H); 7.35–7.32 (m, 2H), 7.09–7.06 (m, 1H), 6.89 (d, J = 15.8 Hz, 1H); ¹³C-NMR (DMSO-d₆), δ: 163.02, 139.08, 137.42, 135.67, 131.87, 131.74, 131.13, 129.61, 128.83, 127.37, 124.64, 123.51, 119.24.

(2E)-N-(2-Chlorophenyl)-3-(3,4-dichlorophenyl)prop-2-enamide (2). Yield 68%; Mp 154–156 °C; IR (cm⁻¹): 3293, 1659, 1626, 1592, 1533, 1468, 1441, 1387, 1337, 1289, 1276, 1242, 1198, 1183, 1148, 1127, 1058, 1034, 1026, 1001, 966, 956, 938, 915, 888, 865, 826, 743, 721, 713, 697, 679, 659, 615, 589, 532, 497, 461; ¹H-NMR (DMSO-d₆), δ: 9.66 (s, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.93 (d, J = 1.4 Hz, 1H), 7.72–7.71 (m, 1H), 7.64 (dd, J = 8.2 Hz, J = 2.1 Hz, 1H), 7.59 (d, J = 15.1 Hz, 1H), 7.52 (dd, J = 7.6 Hz, J = 1.4 Hz, 1H), 7.36 (td, J = 7.6 Hz, J = 1.4 Hz, 1H), 7.21 (d, J = 1.4 Hz, 1H), 7.19 (dd, J = 7.9 Hz, 1.7 Hz, 1H); ¹³C-NMR (DMSO-d₆), δ: 163.45, 138.15, 135.63, 134.84, 132.01, 131.75, 131.14, 129.58, 129.52, 127.63, 127.49, 126.12, 125.51, 125.21, 124.15.

(2E)-N-(3-Chlorophenyl)-3-(3,4-dichlorophenyl)prop-2-enamide (3). Yield 63%; Mp 186–188 °C; IR (cm⁻¹): 3277, 3127, 1664, 1626, 1597, 1536, 1484, 1469, 1426, 1407, 1396, 1341, 1295, 1249, 1239, 1198, 1184, 1131, 1100, 1074, 1026, 1002, 996, 973, 923, 905, 881, 863, 818, 814, 784, 776, 729, 682, 677, 592, 575, 557, 498, 451; ¹H-NMR (DMSO-d₆) δ: 10.42 (s, 1H), 7.93–7.91 (m, 2H), 7.70 (d, J = 8.2 Hz, 1H), 7.63 (dd, J = 8.6 Hz, J = 1.7 Hz, 1H), 7.58 (d, J = 15.8 Hz, 1H), 7.51 (dd, J = 8.2 Hz, J = 1.4 Hz, 1H), 7.36 (t, J = 7.9 Hz, 1H), 7.13 (dd, J = 8.2 Hz, 1.4 Hz, 1H), 6.85 (d, J = 15.8 Hz, 1H); ¹³C-NMR (DMSO-d₆), δ: 163.34, 140.52, 138.07, 135.49, 133.15, 132.07, 131.77, 131.15, 130.54, 129.75, 127.46, 124.13, 123.22, 118.70, 117.66.

(2E)-N-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)prop-2-enamide (4). Yield 71%; Mp 158–160 °C; IR (cm⁻¹): 3291, 1660, 1623, 1590, 1554, 1528, 1489, 1473, 1397, 1338, 1294, 1282, 1244, 1203, 1181, 1135, 1092, 1030, 1012, 997, 973, 949, 904, 853, 818, 813, 788, 726, 709, 667, 637, 627, 560, 524, 509, 479, 442; ¹H-NMR (DMSO-d₆), δ: 10.37 (s, 1H), 7.90 (d, J = 2.1 Hz, 1H), 7.73–7.70 (m, 2H), 7.70 (d, J = 8.2 Hz, 1H), 7.62 (dd, J = 8.6 Hz, J = 1.7 Hz, 1H), 7.57 (d, J = 15.8 Hz, 1H), 7.40–7.37 (m, 2H), 6.85 (d, J = 15.8 Hz, 1H); ¹³C-NMR (DMSO-d₆), δ: 163.14, 138.04, 137.79, 135.57, 131.99, 131.76, 131.14, 129.68, 128.75, 127.42, 127.08, 124.29, 120.77.

(2E)-N-(2,4-Dichlorophenyl)-3-(3,4-dichlorophenyl)prop-2-enamide (5). Yield 64%; Mp 190–192 °C; IR (cm⁻¹): 3276, 1658, 1626, 1579, 1553, 157, 1467, 1381, 1336, 1301, 1287, 1197, 1184, 1143, 1128, 1100, 1052, 1029, 1005, 963, 948, 920, 882, 868, 856, 831, 817, 797, 754, 720, 700, 684, 666, 610, 571, 558, 509, 472; NMR (DMSO-d₆) δ: 9.72 (s, 1H), 8.01 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 2.1 Hz, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 2.1 Hz, 1H), 7.63 (dd, J = 8.6 Hz, J = 1.7 Hz, 1H), 7.59 (d, J = 15.8 Hz, 1H), 7.44 (dd, J = 8.9 Hz, J = 2.1 Hz, 1H), 7.19 (d, J = 15.8 Hz, 1H); ¹³C-NMR (DMSO-d₆), δ: 163.54, 138.44, 135.55, 134.04, 132.09, 131.76, 131.14, 129.60, 129.08, 128.93, 127.64, 127.61, 126.24, 126.02, 123.88.

(2E)-N-(2,5-Dichlorophenyl)-3-(3,4-dichlorophenyl)prop-2-enamide (6). Yield 72%; Mp 203–205 °C; IR (cm⁻¹): 3398, 3115, 1696, 1633, 1581, 1554, 1512, 1474, 1444, 1408, 1329, 1308, 1259, 1236, 1201, 1159, 1133, 1091, 1047, 1026, 997, 975, 962, 923, 903, 873, 824, 802, 732, 685, 582, 571, 557, 548, 495, 458; ¹H-NMR (DMSO-d₆), δ: 9.74 (s, 1H), 8.15 (d, J = 2.7 Hz, 1H), 7.94 (d, J = 1.4 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.64 (dd, J = 8.2 Hz, J = 2.1 Hz, 1H), 7.60 (d, J = 15.8 Hz, 1H), 7.56 (d, J = 8.9 Hz, 1H), 7.26 (dd, J = 8.6 Hz, J = 2.4 Hz, 1H), 7.24 (d, J = 15.8 Hz, 1H); ¹³C-NMR (DMSO-d₆), δ: 163.71, 138.73, 136.10, 135.51, 132.18, 131.78, 131.63, 131.18, 130.87, 129.65, 127.71, 125.50, 123.84, 123.46.

(2E)-N-(3,5-Dichlorophenyl)-3-(3,4-dichlorophenyl)prop-2-enamide (7). Yield 59%; Mp 169–171 °C; IR (cm⁻¹): 3449, 3182, 3114, 3083, 1659, 1620, 1587, 1544, 1476, 1442, 1410, 1387, 1341, 1300, 1269, 1193, 1151, 1139, 1116, 1097, 1032, 1012, 973, 953, 939, 865, 849, 815, 785, 724, 702, 675, 666, 602, 581, 554, 530, 467,3 454; ¹H-NMR (DMSO-d₆) δ: 10.56 (s, 1H), 7.90 (d, J = 2.1 Hz, 1H), 7.73–7.72 (m, 2H), 7.69 (d, J = 8.9 Hz, 1H), 7.62 (dd, J = 8.9 Hz, J = 2.1 Hz, 1H), 7.59 (d, J = 15.8 Hz, 1H), 7.28–7.27 (m, 1H), 6.79 (d, J = 15.8 Hz, 1H); ¹³C-NMR (DMSO-d₆), δ: 163.59, 141.37, 138.67, 135.29, 134.15, 132.24, 131.79, 131.15, 129.84, 127.52, 123.64, 122.71, 117.36.

3.3. Lipophilicity Determination by HPLC

A HPLC separation module Waters Alliance 2695 XE equipped with a Waters Dual Absorbance Detector 2486 (Waters Corp., Milford, MA, USA) was used. A chromatographic column Symmetry^® C18 5 μm, 4.6 × 250 mm, Part No. W21751W016 (Waters Corp., Milford, MA, USA) was used. The HPLC separation process was monitored by Empower^® 3 Chromatography Manager Software (Waters Corp.). Isocratic elution by a mixture of MeOH p.a. (72%) and H₂O-HPLC Mili-Q grade (28%) as a mobile phase was used for the determination of capacity factor k. Isocratic elution by a mixture of MeOH p.a. (72%) and acetate buffered saline (pH 7.4 and pH 6.5) (28%) as a mobile phase was used for the determination of distribution coefficient expressed as D_7.4 and D_6.5. The total flow of the column was 1.0 mL/min, injection 20 μL, column temperature 40 °C, and sample temperature 10 °C. The detection wavelength of 210 nm was chosen. A KI methanolic solution was used for determination of the dead times (t_D). Retention times (t_R) were measured in minutes. The capacity factors k were calculated according to the formula k = (t_R - t_D)/t_D, where t_R is the retention time of the solute, and t_D is the dead time obtained using an unretained analyte. The distribution coefficients D_pH were calculated according to the formula D_pH = (t_R - t_D)/t_D. Each experiment was repeated three times. The log k values of individual compounds are shown in Table 1.

3.4. Lipophilicity Calculations

Log P, i.e., the logarithm of the partition coefficient for n-octanol/water, was calculated using the programs ACD/Percepta (Advanced Chemistry Development. Inc., Toronto, ON, Canada, 2012) and ChemBioDraw Ultra 13.0 (CambridgeSoft, PerkinElmer Inc., MA, USA). Clog P values (the logarithm of n-octanol/water partition coefficient based on established chemical interactions) were calculated using ChemBioDraw Ultra 13.0 (CambridgeSoft) software. The results are shown in Table 1. The distributive parameters π_Ar of individual substituted anilide rings of individual compounds were predicted using ACD/Percepta and are shown in Table 2, while other physicochemical and topological descriptors are mentioned in Table 3.