Measurement of atropisomer racemization kinetics using segmented flow technology

Article abstract of DOI:10.1021/ml2003108

When stable atropisomers are encountered by drug discovery teams, they can have important implications due to potential differences in their biological activity, pharmacokinetics, and toxicity. Knowledge of an atropisomer's activation parameters for interconversion is required to facilitate informed decisions on how to proceed. Herein, we communicate the development of a new method for the rapid measurement of atropisomer racemization kinetics utilizing segmented flow technology. This method leverages the speed, accuracy, low sample requirement, safety, and semiautomated nature of flow instrumentation to facilitate the acquisition of kinetics data required for experimentally probing atropisomer activation parameters. Measured kinetics data obtained for the atropo isomerization of AMPA antagonist CP-465021 using segmented flow and traditional thermal methods were compared to validate the method.

Full text of DOI:10.1021/ml2003108

Technology Note
pubs.acs.org/acsmedchemlett
Measurement of Atropisomer Racemization Kinetics Using
Segmented Flow Technology
Jennifer E. Davoren,* Mark W. Bundesmann, Qi T. Yan, Elizabeth M. Collantes, Scot Mente,
Deane M. Nason, and David L. Gray
Neuroscience Chemistry, Pfizer Global Research and Development, Groton, Connecticut 06340, United States
S
* Supporting Information
ABSTRACT: When stable atropisomers are encountered by
drug discovery teams, they can have important implications due
to potential differences in their biological activity, pharmacoki-
netics, and toxicity. Knowledge of an atropisomer's activation
parameters for interconversion is required to facilitate informed
decisions on how to proceed. Herein, we communicate the
development of a new method for the rapid measurement of
atropisomer racemization kinetics utilizing segmented flow
technology. This method leverages the speed, accuracy, low
sample requirement, safety, and semiautomated nature of flow instrumentation to facilitate the acquisition of kinetics data
required for experimentally probing atropisomer activation parameters. Measured kinetics data obtained for the atropo
isomerization of AMPA antagonist CP-465021 using segmented flow and traditional thermal methods were compared to validate
the method.
KEYWORDS: Atropisomer, Reaction kinetics, Segmented flow chemistry
he incidence of axial chirality known as atropisomerism1
zolepropionic acid (AMPA) receptor antagonist whose syn-
thesis and resolution are reported elsewhere.6−9 This molecule
Tin chemical research is steadily increasing due to the
has an axial element of chirality due to hindered rotation of the
N-aryl bond. Previously, serial measurements of the chiral
stability of this molecule were conducted in a thermally
controlled oil bath in 3-methylbutan-1-ol at 120 °C. These
experiments suggest that interconversion of CP-465021 occurs
advent of new chemical methodologies, which have made
sterically encumbered bonds more facile to form.2 Their
existence in a drug discovery setting can have important
implications due to possible differences in their biological
activity, pharmacokinetics, and toxicity.3 Knowledge of
atropisomer activation parameters facilitates informed decisions
about appropriate handling, storage, and reaction conditions.
These data can also influence preclinical development strategy.4
Herein, we report the rapid measurement of atropisomer
racemization kinetics utilizing segmented flow technology. This
method leverages the speed, accuracy, low sample requirement,
safety, and semiautomated nature of flow instrumentation5 to
facilitate convenient acquisition of the kinetics data required for
experimentally probing activation parameters.
via a planar, nonionic transition state with a rate constant krac
=
1.07 × 10−2 min−1.10
Segmented flow technology11 is uniquely suited for
measuring thermal interconversion kinetics and has several
advantages over traditional methods. First, the reaction
temperature can be maintained safely above the boiling point
of solvent due to the instrument's ability to contain pressure,
thus accelerating reaction times and data acquisition. Second,
the thermal mass of the system is much greater than the
reaction fluid permitting near instantaneous heating and
cooling, which translates into more accurate data (Figure 2).
Last, many flow systems feature programmable methods and
automated sample collection for convenient hands-off “over-
night” acquisition.
We demonstrate the utility of this method by employing it to
generate kinetics data for CP-465021 (Figure 1), a potent and
selective noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxa-
Operationally, programmed segments of blank system
solvent and solubilized substrate are alternatingly delivered to
a small diameter tube reactor as shown schematically in Figure
3. The segments are separated by an immiscible perfluor-
odecalin spacer that acts as a physical barrier to minimize
Received: December 29, 2011
Accepted: March 12, 2012
Published: March 12, 2012
Figure 1. AMPA antagonist CP-465021 and its atropoenantiomer CP-
465022.
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Technology Note
Figure 2. Representation of flow heating vs thermal heating. With flow
heating, the relative thermal mass of the reactant compared to the
heating block permits near instantaneous heating of a sample
compared to a traditional system.
Figure 4. Eyring plot for the atropo isomerization of CP-465021 in 3-
methyl-butane-1-ol.
a t1/2 of approximately 4 years.17 An identical ΔG⧧ was
obtained from the thermally derived data points out to three
significant figures (Table 2).
Table 2. Extrapolated Activation Parameters for CP-465021
and BINOL
Figure 3. Schematic of a reaction segmented.
diffusion. As the sample segment exits the reactor, it passes a
UV detector, which registers its residence time and triggers
collection. The degree of racemization is then measured by an
appropriate analytical method.12
kcal/mol
solute
T (°C) flow ΔG⧧ thermal ΔG⧧ extrapolated t1/2 (years)
CP-465021
S-BINOL
37
37
29.9
37.1
29.9
ND
4
The interconversion of CP-46502113 was measured in 3-
methyl-butane-1-ol with 3% toluene added to assist UV
detection.10 Because flow technology is automated and
programmable, samples for six time points were collected in
duplicate at four temperatures (125, 130, 135, and 140 °C) in a
single overnight run (Table 1, entries 1−5). The chiral purity
460 000
Encouraged with the strong correlation of the thermal and
flow kinetics data obtained from CP-465021, we applied the
method to two compounds whose measured t1/2 values at 37
°C were between 4 and 7 h. For these compounds, collectively,
the R2 values were lower than those obtained from CP-465021.
Background epimerization of the stock solution over the course
of the experiment and during the analytical phase would
account for some of the observed drift. There was still excellent
correlation between the data obtained from segmented flow
and that obtained thermally with R2 in the generated Eyring
plots typically >0.97. Therefore, while we believe there are still
some automation and accuracy benefits to using the flow
system for measuring rapidly interconverting atropisomers, they
are less pronounced.
To complete our study, S-1,1′-bi-2-naphthol (S-BINOL), a
compound with a much higher barrier to interconversion, was
also tested.18 For this substrate, we measured 6−7 time points
in duplicate at four temperatures (280, 285, 290, and 295 °C)
using diglyme as solvent. From the Eyring equation, we
obtained an extrapolated ΔG⧧ of 37.1 kcal/mol at 37 °C (Table
2), which corresponds to a t1/2 of nearly 500 000 years. The R2
value for the Eyring plot generated from S-BINOL data was
0.98, lending further support that segmented flow technology is
a robust method for measuring reaction kinetics.
Ignoring atropisomerism can have deleterious consequences
in drug development settings. Measured activation parameters
combined with powerful computational models19 can guide the
use of compounds with this unique form of chirality. We have
adopted segmented flow as a reproducible and convenient
method for generating the samples necessary for measuring
high temperature atropo isomerization kinetics. Because of its
programmable semiautomated nature, once appropriate tem-
peratures are identified, a run can potentially be completed
overnight without intervention. It is compatible with a wide
range of solvents over a broad range of pH values (1−12),
temperatures (−20 to 300 °C), and pressure (up to 2500 psi),
Table 1. Measured First-Order Rate Constants for the
Racemization of CP-465021 in 3-Methyl-butane-1-ol
entry
T (°C)
krac (min−1
)
R2
0.997
t1/2 (min)
1
2
140
135
135
130
125
7.20 × 10−2
4.64 × 10−2
4.81 × 10−2
2.93 × 10−2
1.80 × 10−2
1.07 × 10−2
5.89 × 10−3
3.91 × 10−3
9.60
14.9
14.4
23.7
38.7
64.5
118
0.969
a
3
0.970
4
5
6
7
8
0.962
0.993
b
120
not reported
0.994
c
115
c
110
0.989
177
a
Performed on different day and reaction block to assess
reproducibility. Literature value.10 Data obtained via conventional
b
c
thermal flask method.
was measured by chiral supercritical fluid chromatography
(SFC), and first-order rate constants, krac, for the rate of
racemization were obtained from the plots of ln(% ee) vs
time.14 As a control, rate constants were similarly derived from
four thermal runs performed at two temperatures, 110 and 115
°C, in duplicate (Table 1, entries 7 and 8). In every case, the
reactions were monitored for between one and three
interconversion half-lives.15,16 The rate constants and R2 values
from these experiments are shown in Table 1.
Figure 4 is an overlay of the Eyring plots for the flow and
thermal methods, showing excellent agreement. Using the four
flow points, the Eyring expression gives ΔH⧧ = 29.5 kcal/mol
and ΔS⧧ = −1.07 × 10−3 kcal/mol K. From these parameters,
we calculate Gibbs energy of activation ΔG⧧ to be 29.9 kcal/
mol at physiological temperature (37 °C). This corresponds to
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ACS Medicinal Chemistry Letters
Technology Note
diethylaminomethylpyridin-2-yl)vinyl]-6-fluoroquinazolin-4(3H)-one.
J. Chem. Soc., Perkin Trans. 2 2001, No. 6, 961−963.
(11) For information on segmented flow, see http://www.
accendocorporation.com/.
(12) Ward, T. J. Chiral separations. Anal. Chem. 2006, 78 (12),
3947−3956.
(13) The chiral purity on the day of kinetics experiments was
determined to be 97% ee.
(14) Anslyn, E. V.; Dougherty, D. A. Modern Physical Organic
Chemistry; University Science Books: Sausalito, CA, 2006; Vol. 1, p
388.
(15) At equlibrium, a mixture of atropoenantiomers will approximate
0% ee; therefore, t1/2 is defined as the time that it takes a sample of a
single enantiomer to reach 50% ee.
(16) The latter requirement is to confirm that the interconversion
approximates a first-order reaction.
(17) Alternatively, the Arrhenius equation can be used to calculate
the relationship between the rate constant (k) to the activation energy
(Ea) and the preexponential factor (A).
(18) Pu, L. 1,1-Binaphthyl-Based Chiral Materials: Our Journey;
Imperial College Press: London, 2009; p 368.
(19) LaPlante, S. R.; Edwards, P. J.; Fader, L. D.; Jakalian, A.; Hucke,
O. Revealing atropisomer axial chirality in drug discovery.
ChemMedChem 2011, 6 (3), 505−513.
(20) Shimizu, H.; Nagasaki, I.; Saito, T. Recent advances in biaryl-
type bisphosphine ligands. Tetrahedron 2005, 61 (23), 5405−5432.
(21) Berthod, M.; Mignani, G.; Woodward, G.; Lemaire, M. Modified
BINAP: The how and the why. Chem. Rev. 2005, 105 (5), 1801−1836.
(22) Tang, W.; Zhang, X. New chiral phosphorus ligands for
enantioselective hydrogenation. Chem. Rev. 2003, 103 (8), 3029−
3069.
making it useful not only for pharmaceutical examples but also
for chiral ligands, which must stand up to high temperatures for
prolonged periods of time to convey their chirality.20−22
A
report detailing the use of experimentally and computationally
derived activation parameters to guide decisions around
atropisomers in drug discovery is currently in preparation.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for kinetics experiments, SFC
chromatograms, raw kinetics data, and Eyring analyses for
CP-465021 and S-BINOL. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: jennifer.e.davoren@pfizer.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Eric V. Anslyn, Mike Brodney, Todd Butler, Xinjun
Hou, Jan Hughes, Kjell Johnson, Terry Long, Laurence
Philippe, Bruce Rogers, Patrick Verhoest, and Anabella
Villalobos for excellent discussions and support.
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