Lipophilicity in drug
discovery
Klara Valko
Bio-Mimetic ChromatographyConsultancy for Successful Drug Discovery
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What is lipophilicity?
IUPAC definition
Lipophilicity represents the affinity of a
molecule or a moiety for a lipophilic
environment.
Hydrophobicity measures the association of
non-polar groups or molecules in an aqueous
environment which arises from the tendency
of water to exclude non-polar molecules.
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Why do we need to measure
lipophilicity?
A compound partitioning between aqueous and organic phase can model
compound partitioning in vivo
Compound
partitions
between two
immiscible
solvent
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Lipophilicity is measured by
partition coefficient
Partition coefficient is the quotient of the compound
concentration in an aqueous and a non-miscible solvent
under equilibrium condition.
It is expressed by the quotient of the compound
concentrations in the two phases under equilibrium
condition.
It depends on the nature of the two phases, the
compound properties, pH, and the temperature.
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Procedure of measuring
octanol/water partition coefficients
Add the compound into the octanol saturated
aqueous buffer
Add various amounts of water saturated octanol
to the constant volumes of buffer solution
containing the compound
Equilibrate with shaking/rolling
Measure the peak area of the compound in the
buffer phase by injecting the same amounts from
each vial
Calculate the partition coefficient
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Roller-Vial method for the
measurement of octanol/water
partition coefficients.
Inject from the bottom layer (aqueous phase) to the HPLC for the
concentration determination of the compound.www.bio-mimetic-chromatography.com
Concentration determination by
measuring HPLC peak areas from the
water phase
at is the peak area obtained from the aqueous phase without octanol
aa is the peak area obtained from the aqueous phase after equilibration with octanol
Voctanol volume of the octanol phase; Vwater volume of the aqueous phase
𝑷 =𝒂𝒕 − 𝒂𝒂𝒂𝒂
∗𝑽𝒘𝒂𝒕𝒆𝒓𝑽𝒐𝒄𝒕𝒂𝒏𝒐𝒍
at
aa1
aa2
aa3
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Measuring lipophilicity by HPLC
Cs. Horváth, W. Melander, I. Molnár, J. Chromatogr. 125 (1976) 129.
Measuring bio-relevant association constants has
great impact on drug discovery!
Based on the solvophobic theory the interaction between the solute
and the stationary phase is considered as a reversible association of
the solute molecules with the stationary phase moiety
(hydrocarboneous, membrane, or protein). Accordingly solute
retention is governed by the dynamic equilibrium constant.
Vs = volume of the
stationary phase
Vm = volume of the mobile phase
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Chromatographic
Hydrophobicity Index (CHI)
Approximates the organic phase concentration (%) when the compound elutes from a reversed
phase column using linear gradient. CHI gives a straight line with the gradient retention time.
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LunaC18(2) 50 x 3 mm; 1.00 ml/min; Mobile phase A 50 mM ammonium acetate pH 7.4 and B is 100%
acetonitrile. Gradient: 0 - 2.5 min 0 - to 100% B; 2.5 - 2.7 min 100% B.
Calibration of CHI at pH7.4
y = 54.329x - 71.702
R2 = 0.9972
0.00
20.00
40.00
60.00
80.00
100.00
120.00
1.4 1.9 2.4 2.9 3.4
Compound CHI7.4
at pH 7.4
CHI2
at pH 2
CHI10.5
at pH 10.5
Theophylline 18.4 17.9 5.0
Phenyltetrazole 23.6 42.2 16.0
Benzimidazole 34.3 6.3 30.6
Colchicine 43.9 43.9 43.9
Phenyltheophylline 51.7 51.7 51.7
Acetophenone 64.1 64.1 64.1
Indole 72.1 72.1 72.1
Propiophenone 77.4 77.4 77.4
Butyrophenone 87.3 87.3 87.3
Valerophenone 96.4 96.4 96.4
Ultrahigh Performance Liquid
Chromatography (uPLC) for CHI
CHI TM1
Time0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80
AU
0.0
5.0e-2
1.0e-1
1.5e-1
2.0e-1
2.5e-1
3.0e-1
CHI TM1_LunapH74 Diode Array 254
Range: 3.969e-1
1.02
0.86
0.64
0.590.72
0.92
1.27
1.16
1.10
1.37
The CHI test mix is separated in less than 90 sec
Now we can determine a compound’s lipophilicity in 90 sec
using various starting mobile phase pH
Courtesy of Shenaz Bunally at GSKwww.bio-mimetic-chromatography.com
Parallel measurement of a compound’s
retention at various pH to reveal acid/base
characterTypical 4-way chromatograms of a base
pH2
pH7.4
pH 10.5
IAM 7.4
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CHIs measured at 3 pHs provide an automatic way of grouping molecules
according to acid/base character without structural information.
0
10
20
30
40
50
60
70
80
90
100
Neutral
(Zwitterionic)
Strong acid Weak acid Strong base Weak base Amphoteric
pH2
pH7.4
pH10.5
The change of CHI values by changing the pHCHI
CHI values at pH 2, pH 7.4 and pH 10.5
reveal acid/base character of compounds
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Bio-mimetic HPLC measurement of Human Serum
Albumin (HSA), α-1acidglycoprotein (AGP) and
Immobilized Artificial Membrane (IAM) partition
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HSA AGP IAM
pH 7.4 aqueous mobile phases
IPA IPA ACN
Bio-Mimetic Stationary
phases
Biomimetic lipophilicity measurements
(Membrane partition) using Immobilised
Artificial Membrane stationary phase
Stationary phaseIAM calibration y = 26.647x - 37.653
R2 = 0.9966
0
10
20
30
40
50
60
1 1.5 2 2.5 3 3.5gtR
CHI
Typical calibration
Compound gtR CHI IAM
Octanophenone 3.269 49.4
Heptanophenone 3.145 45.7
Hexanophenone 3.001 41.8
Valerophenone 2.822 37.3
Butyrophenone 2.601 32
Propiophenone 2.341 25.9
Acetophenone 2.013 17.2
Acetanilide 1.83 11.5
Paracetamol 1.591 2.9
Column: IAM PC2 (CH2)12 150 x 4.6
Mobile Phase flow rate: 2 ml/min
Gradient: 0 to 3 min 0 to 80% acetonitrile
3 to 3.5 min 80% acetonitrile
3.5 to 3.7 min 0% acetonitrile
Cycle time: 5 min
K. Valko et al. J. Pharm. Sci.89 (2000) 1085-1096www.bio-mimetic-chromatography.com
Calibration ploty = 2.177x + 0.1304
R2 = 0.9612
-0.50
0.00
0.50
1.00
1.50
2.00
2.50
-0.40 -0.20 0.00 0.20 0.40 0.60 0.80 1.00
logtR
logK
litera
ture
Calculate %Binding
logK = slope * log(tR) + int
K = %B / (101-%B)
y = 0.9309x - 0.3329
R2 = 0.879
0
20
40
60
80
100
0 20 40 60 80 100
HSA Column
Lite
ratu
re %
bin
ding
n=71
K. Valko et al. J. Pharm. Sci. 92 (2003) 2236
Column: HSA 50 x 3 mm (Chrom Tech, Chiral Technologies)
Flow rate: 1.8 ml/min at 300C
Mobile phase: 50 mM ammonium acetate pH7.4
Gradient: 0 - 3 min 0 to 30% 2-propanol;
3 to 10 min 30% 2-propanol;
10 to 10.5 min 0% 2-propanol
Cycle time: 15 min
Serum albumin binding measurement using
chemically bonded serum albumin stationary
phases
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Retention time of compounds can be
converted to % binding or K association
constant
)(logexplog HSAKHSAkHSAke
2 min
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AGP binding measurement by
HPLC
Same principle as HSA
binding measurements:
AGP column
2-propanol gradient
pH 7.4 ammonium acetate
Calibration with AGP binding
data derived from published
% AGP bound values
Typical AGP column calibration ploty = 2.7976x - 0.5289
R2 = 0.9744
-0.400
-0.200
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70
log tR
logK
AG
P
Calibration set of compounds: Nizatidine,
Bromazepam, Warfarin, Propranolol, Imipramin,
Nicardipine, Chlorpromazine
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References
C. Hansch, A. Leo, ρ-σ-π analysis. A method for correlation of biological activity and chemical structure. J. Amer.
Chem.Soc. 86(1964) 1616-1624
Horvath, Cs., Melander, W., Molnar, I. Solvophobic interactions in liquid chromatography with non-polar stationary
phases, Journal of Chromatography, 125 (1976) 129-156
Valko, K.; Snyder, L.R.; Glajch, G.L. Retention in reversed-phase liquid chromatography as a function of mobile-
phase composition. J. Chromatogr. A 656, (1993) 501–520.
Harnisch, M., Mockel, H. J., Shulze, G. J., Relationship between log Pow, shake flask values and capacity factors
derived from reversed-phase high performance liquid chromatography for n-alkylbenzenes and some OECD
reference substances. Journal of Chromatography 282 (1983) 315-332
Tomlinson, E., Chromatographic hydrophobicity parameters in correlation analysis of structure – activity
relationships. Journal of Chromatography 113 (1975) 1-45
Giaginis, A. Tsantili-Kakoulidou, Current state of the art in HPLC Methodology for lipophilicity assessment of basic
drugs (Review) Journal of Liquid Chromatography & Related Technologies, 31: (2008) 79–96.
Lombardo, F., Shalaeva, M. Y., Tupper, K. A., Gao, F., ElogDoct: A tool for Lipophilicity Determination in Drug
Discovery. 2. Basic and neutral compounds. (2001) 2490-2497
Gocan, S., Cimpan, G., Comer, J., Lipophilicity measurements by liquid chromatography in Advances in
Chromatography, Eds: E. Grushka, N. Grinberg, 44 (2005) 79-176, Taylor & Francis Group, 1574447343
Valko, K., Bevan, C., Reynolds, D., Chromatographic hydrophobicity index by fast-gradient RP-HPLC: A high
throughput alternative to log P/log D. Analytical Chemistry 69 (1997) 2022-2029
Valko, K. Measurements of lipophilicity and acid/base character using HPLC methods. In “Pharmaceutical
profiling in drug discovery for lead selection” Eds. Borchardt, R., Kerns, E., AAPS (2004)Arlington, VA 127-
182
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