High Throughput Screening of Compound Lipophilicity (log P Values) Using Multiplexed, Absorbance-Based Capillary Electrophoresis Kit-sum Wong, Jeremy Kenseth, and Dennis Tallman CombiSep, Inc., 2711 South Loop Drive, Suite 4200 Ames, IA 50010 USA

OBJECTIVES To investigate the application of multiplexed, absorbancebased, vacuum assisted capillary electrophoresis towards the high throughput screening of compound octanol-water partition coefficients (log Pow values), including:

Validation of MMEEKC by LFER Analysis of Tested Solutes

RESULTS

Shake-Flask (613 Solutes)

Representative 24-Capillary MMEEKC log Pow Data

logPOW = 0.088 + 0.562R2 − 1.054π 2H + 0.034 ∑ α 2H − 3.460 ∑ β 2 + 3.481Vx N = 613, R = 0.997, R2 = 0.995, s = 0.116, F = 223162

Shake-Flask (37 Tested Solutes) logPOW = 0.133 (± 0.057) + 0.523 (± 0.049) R2 − 1.072 (± 0.055)π 2H

+ 0.051(± 0.106) ∑ α 2H − 3.431 (± 0.062) ∑ β 2 + 3.849 (± 0.071)Vx

• Comparisons between literature values and values obtained using multiplexed microemulsion electrokinetic chromatography (MMEEKC)

N = 37, R = 0.997, R2 = 0.995, s = 0.126, F = 1142

MMEEKC (37 Tested Solutes) MMEEKC log POW = 0.345 (± 0.121) + 0.474 (± 0.104) R 2 − 1.062 (± 0.118)π 2H + 0.014 (± 0.225) ∑ α 2H − 3.136 (± 0.133) ∑ β 2 + 3.569 (± 0.150)V x N = 37, R = 0.986, R2 = 0.972, s = 0.269, F = 214

• Evaluation of the run-to-run reproducibility of MMEEKC log Pow screening

System Constant Ratios Normalized to Vx Term

• Validation of the MMEEKC log Pow method via a Linear Free Energy Relationship (LFER) analysis

System Octanol-water (n = 613) Octanol-water (n = 37) MMEEKC

• Order of migration in each capillary is DMSO, Solute, Dodecylbenzene • Standards: 1. Pyrazine, 2. Benzamide, 3. Nicotine, 4. Quinoline, 5. Naphthalene, 6. Imipramine

r/v 0.15 0.14 0.13

s/v -0.28 -0.27 -0.3

a/v 0.01 0.01 0.00

b/v -0.91 -0.89 -0.88

v 1 1 1

Reference 9 This work This work

INSTRUMENTATION Typical Calibration Plot Constructed from a MMEEKC Run Long Term (> 8 months) Reproducibility of log Pow Values

MCE 2000™ System (CombiSep, Ames, IA USA)

• 96-capillary array CE instrument (standard microplate compatibility) • Near universal UV detection at 214 nm • Detection of up to 96 individual CE separations simultaneously • Averaged (n = 4) log k’ values for the six standards were used to construct the calibration plot

cePRO 9600™ System (CombiSep, Ames, IA USA)

MMEEKC log k’ b

EXPERIMENTAL

• Microemulsion Buffer: 6.6% (w/v) 1-butanol, 0.8% (w/v) n-heptane, 3.3% (w/v) sodium dodecyl sulfate, with 68 mM CAPS buffer (pH 10.3) made up to volume. • Various drug-like compounds with a wide range of known log Pow values and chemical structures were selected to evaluate method accuracy and ruggedness. • A standard mixture containing 6 compounds with a range of known log Pow values was used to calibrate the system. Standards used were (with literature log P value): Pyrazine (-0.26), Benzamide (0.64), Nicotine (1.17), Quinoline (2.03), Naphthalene (3.30), Imipramine (4.42). • The standard mixture and other test solutes were dissolved in microemulsion buffer with DMSO (EOF marker) and dodecylbenzene (microemulsion marker). • Capacity factors (log k’ values) were calculated for the six standards and test solutes using Equation 1: t s − t eof k' = (1)

t eof (1 − t s /t me )

anthracene benzamide benzylamine bifonazole butylbenzene caffeine carbamazepine chloramphenicol 4-chloroaniline chlorpromazine chlorthalidone coumarin 3,5-dimethylaniline estradiol ethyl p-aminobenzoate ethylbenzene ethylbenzoate fluoranthene hydrocortisone hydrocortisone-21-acetate hydroquinine imipramine indazole lidocaine 2,4-lutidine 3,5-lutidine α-methylbenzylamine 2-methylbenzylamine 3-methylbenzylamine metronidazole naphthalene nefopam nicotine nifedipine nitrobenzene nitrofurazone pentoxifylline phenanthrene phenyl acetate procaine propylbenzene pyrazine pyrene pyrilamine pyrimidine quinidine quinine quinoline tetracaine toluene a. b. c. d. e. f. g. h. i.

c

MMEEKC log P OW 1.74 ± 0.01 2.29 ± 0.01 0.40 ± 0.01 0.91 ± 0.02 4.63 ± 0.14 0.78 ± 0.01 1.10 ± 0.02 4.21 ± 0.15 4.35 ± 0.11 0.08 ± 0.01 2.34 ± 0.01 1.39 ± 0.02 2.16 ± 0.08 4.74 ± 0.03 1.17 ± 0.01 1.44 ± 0.02 2.09 ± 0.03 3.15 ± 0.04 2.02 ± 0.02 3.54 ± 0.06 2.72 ± 0.02 4.47 ± 0.06 2.04 ± 0.01 2.17 ± 0.07 3.24 ± 0.12 4.13 ± 0.02 1.69 ± 0.04 2.65 ± 0.01 1.58 ± 0.01 1.74 ± 0.01 1.45 ± 0.01 1.62 ± 0.02 1.82 ± 0.02 -0.05 ± 0.02 3.52 ± 0.02 3.02 ± 0.01 1.39 ± 0.00 2.66 ± 0.01 1.75 ± 0.02 0.74 ± 0.01 0.67 ± 0.01 4.37 ± 0.08 1.35 ± 0.02 2.20 ± 0.01 3.85 ± 0.04 -0.50 ± 0.00 4.40 ± 0.07 3.12 ± 0.01 -0.67 ± 0.01 2.77 ± 0.05 2.89 ± 0.03 1.98 ± 0.01 3.46 ± 0.01 2.77 ± 0.04

0.40 ± 0.00 0.72 ± 0.01 -0.42 ± 0.01 -0.10 ± 0.01 2.14 ± 0.08 -0.18 ± 0.00 0.01 ± 0.01 1.89 ± 0.03 1.98 ± 0.02 -0.60 ± 0.01 0.76 ± 0.01 0.19 ± 0.01 0.64 ± 0.05 2.21 ± 0.02 0.06 ± 0.01 0.21 ± 0.01 0.60 ± 0.02 1.25 ± 0.02 0.56 ± 0.01 1.49 ± 0.03 0.99 ± 0.01 2.07 ± 0.04 0.59 ± 0.01 0.67 ± 0.04 1.31 ± 0.07 1.83 ± 0.01 0.36 ± 0.02 0.94 ± 0.01 0.30 ± 0.01 0.40 ± 0.01 0.21 ± 0.01 0.33 ± 0.01 0.45 ± 0.01 -0.68 ± 0.01 1.47 ± 0.01 1.17 ± 0.01 0.18 ± 0.00 0.96 ± 0.01 0.40 ± 0.02 -0.21 ± 0.01 -0.25 ± 0.01 1.92 ± 0.05 0.16 ± 0.01 0.67 ± 0.01 1.66 ± 0.03 -0.96 ± 0.01 2.00 ± 0.04 1.22 ± 0.01 -1.06 ± 0.00 1.03 ± 0.03 1.10 ± 0.02 0.54 ± 0.01 1.43 ± 0.01 1.02 ± 0.02

e

• Microemulsion electrokinetic chromatography (MEEKC) was employed for indirect log Pow evaluation.

Literature log P OW 1.71 2.25 0.49 0.9 4.45 0.64 1.09 4.77 4.38 -0.07 2.45 1.14 1.88 5.19

d

f

0.85 1.39

g

2.17 4.01 1.86 3.15 2.64 5.16 1.61 2.19

g

3.43 h 4.42 1.77 2.26 g

1.90 1.78 1.49g g 1.62 g 1.62 -0.02 3.3 g

3.05 1.17

i

3.17 1.85 0.23 0.29 4.46 1.49 1.92 3.72

h

-0.26 4.88 3.27 -0.4 2.88 2.64 2.03 3.73 2.73

∆log P OW 0.03 0.04 -0.09 0.01 0.18 0.14 0.01 -0.56 -0.03 0.15 -0.11 0.25 0.28 -0.45 0.32 0.05 -0.08 -0.85 0.16 0.39 0.08 -0.69 0.43 -0.02 -0.19 -0.29 -0.08 0.39 -0.32 -0.04 -0.05 0 0.2 -0.03 0.22 -0.03 0.22 -0.51 -0.1 0.51 0.38 -0.14 -0.01 0.28 0.13 -0.24 -0.48 -0.15 -0.27 -0.11 0.25 -0.05 -0.27 0.04

Average of four replicates ± standard deviation in a single run. Calculated from Equation 1. Calculated from Equation 2. From Reference 1 unless otherwise noted. Average of three replicates ± standard deviation in a single run. From reference 2. Calculated value from reference 3. From reference 4 From reference 5.

log POW = A × log k’ + B

(2)

where A is the slope and B is the y-intercept. • The log k’ values of test solutes were calculated from the MMEEKC data and entered into Equation 2, calculating their corresponding log Pow values. • Sample injection: -0.2 psi, 20-25 sec. CE Run: 6.5 kV (86 V/cm), –0.1 psi vacuum, 90 min run.

Samples (n = 23 per plate) and the standard mixture were loaded in 4 consecutive wells of a 96-well plate:

MMEEKC log k' avg. ± SD

%RSD

MMEEKC log Pow avg. ± SD

%RSD

Lit. log P OW

∆ log P OW

0.41 ± 0.03 0.71 ± 0.03 -0.41 ± 0.01 -0.12 ± 0.02 2.09 ± 0.10 -0.17 ± 0.02 -0.59 ± 0.02 0.62 ± 0.03 2.21 ± 0.04 0.07 ± 0.02 0.22 ± 0.02 0.57 ± 0.03 0.40 ± 0.09 1.49 ± 0.01 0.97 ± 0.04 1.26 ± 0.06 1.86 ± 0.08 0.38 ± 0.03 0.89 ± 0.04 0.31 ± 0.01 0.42 ± 0.02 0.24 ± 0.03 0.34 ± 0.02 0.47 ± 0.02 1.36 ± 0.07 1.14 ± 0.05 0.18 ± 0.02 0.40 ± 0.02 1.92 ± 0.06 0.18 ± 0.02 -0.96 ± 0.01 2.21 ± 0.23 1.18 ± 0.06 -1.05 ± 0.02 0.54 ± 0.03 1.42 ± 0.07

7.32 4.23 2.44 16.67 4.78 11.76 3.39 4.84 1.81 28.57 9.09 5.26 22.50 0.67 4.12 4.76 4.30 7.89 4.49 3.23 4.76 12.50 5.88 4.26 5.15 4.39 11.11 5.00 3.13 11.11 1.04 10.41 5.08 1.90 5.56 4.93

1.80 ± 0.04 2.31 ± 0.03 0.41 ± 0.01 0.90 ± 0.02 4.54 ± 0.17 0.81 ± 0.02 0.11 ± 0.07 2.16 ± 0.04 4.74 ± 0.06 1.22 ± 0.05 1.48 ± 0.05 2.04 ± 0.05 1.78 ± 0.15 3.54 ± 0.01 2.75 ± 0.04 3.23 ± 0.10 4.23 ± 0.08 1.75 ± 0.08 2.62 ± 0.03 1.60 ± 0.03 1.77 ± 0.03 1.48 ± 0.04 1.65 ± 0.03 1.86 ± 0.04 3.40 ± 0.09 3.04 ± 0.04 1.40 ± 0.02 1.79 ± 0.04 4.29 ± 0.11 1.41 ± 0.03 -0.51 ± 0.03 4.75 ± 0.38 3.11 ± 0.05 -0.67 ± 0.03 2.00 ± 0.04 3.52 ± 0.10

2.22 1.30 2.44 2.22 3.74 2.47 63.64 1.85 1.27 4.10 3.38 2.45 8.43 0.28 1.45 3.10 1.89 4.57 1.15 1.88 1.69 2.70 1.82 2.15 2.65 1.32 1.43 2.23 2.56 2.13 5.88 8.00 1.61 4.48 2.00 2.84

1.71 2.25 0.49 0.9 4.45 0.64 -0.07 1.88 5.19 0.85 1.39 2.17 1.86 3.15 2.64 3.43 4.42 1.77 2.26 1.9 1.78 1.49 1.62 1.62 3.3 3.05 1.17 1.85 4.46 1.49 -0.26 4.88 3.27 -0.4 2.03 3.73

0.09 0.06 -0.08 0 0.09 0.17 0.18 0.28 -0.61 0.37 0.09 -0.13 -0.08 0.39 0.11 -0.2 -0.19 -0.02 0.36 -0.3 -0.01 -0.01 0.03 0.24 0.1 -0.01 0.23 -0.06 -0.17 -0.08 -0.25 -0.13 -0.16 -0.3 -0.03 -0.21

Plot of Residuals vs. Literature log P Value for Tested Solutes

SUMMARY • Multiplexed, absorbance-based CE offers an attractive approach for the high throughput screening of log Pow values • Excellent run-to-run accuracy and reproducibility can be achieved for log Pow screening • Multiplexed MEEKC-based evaluation of log Pow values requires microgram amounts of sample with lower stringency on sample purity as compared to nonseparation techniques • LFER analysis of the MEEKC system suggests it can be used to model the shake-flask system for log Pow measurements • Utilizing the MCE 2000™ System, one can complete up to 96 log Pow analyses in a single run • The current MMEEKC method works for neutral compounds and weak bases (pKa < 9.5); a method for acidic compounds is in development

where ts, teof, and tme are the migration times of the solute, EOF marker (DMSO), and microemulsion marker (dodecylbenzene), respectively.

• The log k’ values for the standard mixture compounds were plotted against their corresponding literature log Pow values to calibrate the CE run via Equation 2:

n

42 37 34 36 6 50 35 36 7 38 26 15 36 6 38 42 52 46 36 12 14 8 12 9 53 32 53 35 13 36 53 8 35 36 53 38

Summary of MCE 2000™ MEEKC log Pow Valuesa acebutolol 1-aminonaphthalene 2-aminopyridine aniline

• Second generation 96-capillary array CE instrument • Near universal UV detection at 214 nm • Increased automation providing unattended operation • Robotic interfacing capabilities

Solute

acebutolol 1-aminonaphthalene 2-aminopyridine aniline anthracene benzamide caffeine 4-chloroaniline chlorpromazine chlorthalidone coumarin 3,5-dimethylaniline ethyl pethylbenzene ethylbenzoate hydroquinine imipramine indazole lidocaine 2,4-lutidine 3,5-lutidine α-methylbenzylamine 2-methylbenzylamine 3-methylbenzylamine naphthalene nefopam nicotine nitrobenzene phenanthrene phenylacetate pyrazine pyrene pyrilamine pyrimidine quinoline tetracaine

Solvation Descriptors of Tested Solutes for LFER Analysis Solute

R2

1-aminonaphthalene aniline anthracene benzamide benzylamine bifonazole butylbenzene caffeine carbamazepine chloramphenicol 4-chloroaniline coumarin estradiol ethyl p-aminobenzoate ethylbenzene ethylbenzoate fluoranthene hydrocortisone hydrocortisone-21-acetate imipramine indazole 3,5-lutidine metronidazole naphthalene nicotine nifedipine nitrobenzene nitrofurazone pentoxifylline phenanthrene phenylacetate propylbenzene pyrazine pyrene pyrimidine quinoline toluene

1.67 0.955 2.29 0.99 0.829 2.41 0.6 1.5 2.15 1.85 1.06 1.06 1.8 1.04 0.613 0.689 2.377 2.03 1.89 1.48 1.18 0.659 1.05 1.34 0.865 1.5 0.871 1.65 1.64 2.055 0.661 0.604 0.629 2.808 0.606 1.268 0.601

π 2H 1.26 0.94 1.34 1.5 0.88 2.25 0.51 1.6 2.07 0.72 1.13 1.79 1.77 1.52 0.51 0.85 1.53 3.49 2.88 1.75 1.35 0.79 1.6 0.92 0.75 2.45 1.11 1.79 2.28 1.29 1.13 0.5 0.95 1.71 1 0.97 0.52

∑ α 2H

∑ β2

0.2 0.26 0 0.49 0.1 0 0 0 0.52 0.34 0.3 0 0.86 0.32 0 0 0 0.71 0.46 0 0.54 0 0.18 0 0 0.23 0 0.4 0 0 0 0 0 0 0 0 0

0.57 0.5 0.26 0.67 0.72 1.12 0.15 1.33 1.13 2.09 0.35 0.46 1.1 0.64 0.15 0.46 0.2 1.9 2.16 1.19 0.3 0.44 1.03 0.2 1.14 1.45 0.28 1.08 1.84 0.26 0.54 0.15 0.61 0.29 0.65 0.51 0.14

VX

Reference

1.185 0.816 1.454 0.973 0.987 2.501 1.28 1.363 1.811 2.073 0.939 1.062 2.199 1.313 0.998 1.214 1.585 2.798 3.095 2.402 0.905 0.957 1.192 1.085 1.371 2.495 0.891 1.264 2.083 1.454 1.072 1.139 0.634 1.585 0.634 1.044 0.857

6 6 6 6 7 5 6 5 5 5 6 8 5 7 6 6 6 5 5 8 7 7 5 6 8 5 6 5 5 6 8 6 9 6 9 7 9

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ACKNOWLEDGEMENTS The authors would like to thank Dr. Ho-ming Pang, Kathryn Gossett, and Bruce Boeke for development of the log P analysis software, and Kevin Kennedy, Tom Kurt, and Karen Strodtman for hardware assistance.