lipophilicity in drug discovery · derived from reversed-phase high performance liquid...

Lipophilicity in drug

discovery

Klara Valko

Bio-Mimetic ChromatographyConsultancy for Successful Drug Discovery

www.bio-mimetic-chromatography.com

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.


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


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.


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


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


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


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.


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


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


Bio-mimetic HPLC measurement of Human Serum

Albumin (HSA), α-1acidglycoprotein (AGP) and

Immobilized Artificial Membrane (IAM) partition


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


Retention time of compounds can be

converted to % binding or K association

constant

)(logexplog HSAKHSAkHSAke

2 min


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


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-

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