Integrated synthesis and testing of substituted xanthine based DPP4 inhibitors: Application to drug discovery

Article abstract of DOI:10.1021/ml400171b

A novel integrated discovery platform has been used to synthesize and biologically assay a series of xanthine-derived dipeptidyl peptidase 4 (DPP4) antagonists. Design, synthesis, purification, quantitation, dilution, and bioassay have all been fully integrated to allow continuous automated operation. The system has been validated against a set of known DPP4 inhibitors and shown to give excellent correlation between traditional medicinal chemistry generated biological data and platform data. Each iterative loop of synthesis through biological assay took two hours in total, demonstrating rapid iterative structure-activity relationship generation.

Full text of DOI:10.1021/ml400171b

Letter
pubs.acs.org/acsmedchemlett
Integrated Synthesis and Testing of Substituted Xanthine Based
DPP4 Inhibitors: Application to Drug Discovery
Werngard Czechtizky,‡ Juergen Dedio,‡ Bimbisar Desai,† Karen Dixon,† Elizabeth Farrant,† Qixing Feng,†
̈
Trevor Morgan,† David M. Parry,† Manoj K. Ramjee,† Christopher N. Selway,† Thorsten Schmidt,‡
Gary J. Tarver,*,† and Adrian G. Wright†
†Cyclofluidic Ltd., Biopark, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AX, U.K.
‡Sanofi-Aventis, Industriepark Hochst, Gebaude K703, 65929 Frankfurt, Germany
̈
̈
S
* Supporting Information
ABSTRACT: A novel integrated discovery platform has been
used to synthesize and biologically assay a series of xanthine-
derived dipeptidyl peptidase 4 (DPP4) antagonists. Design,
synthesis, purification, quantitation, dilution, and bioassay have
all been fully integrated to allow continuous automated
operation. The system has been validated against a set of
known DPP4 inhibitors and shown to give excellent
correlation between traditional medicinal chemistry generated
biological data and platform data. Each iterative loop of
synthesis through biological assay took two hours in total,
demonstrating rapid iterative structure−activity relationship generation.
KEYWORDS: Closed loop drug design, flow synthesis, automated drug discovery, DPP4
the next compound for synthesis and test. This offers the
edicinal chemistry is an iterative process of synthesis and
Mscreening of novel molecules to optimize both biological
and physical properties to ultimately yield a candidate molecule
for the clinic. We have previously reported1 a closed loop
integrated approach to elements of medicinal chemistry
whereby the synthesis process is fully integrated with the
biological assay enabling the rapid and automated generation of
structure−activity relationship (SAR) data. This approach
enables a true closed loop approach to medicinal chemistry
to be undertaken where molecules are designed for synthesis
and screening based upon the emerging SAR in a true serial
iterative manner.
advantage of fully automated, fast serial generation of SAR for
drug discovery with each design iteration taking no more than
two hours from the start of synthesis to biological readout.
The integrated optimization platform (Figure 1) comprises a
reagent autosampler and flow synthesis apparatus connected to
a commercial high-performance liquid chromatography
(HPLC) mass spectrometer. Liquid chromatography mass
spectrometry (LC−MS) is used to purify and characterize
synthesized material with an evaporative light-scattering
detector (ELSD) to establish sample concentration.6 Purified
material is subsequently reformatted to the correct concen-
tration for biological assay using proprietary hardware.
Biological IC50 determination is achieved using a bespoke
liquid handler coupled to a fluorescence plate reader. The IC50
is determined by fitting a curve to the measured data using in
house software written in Matlab.7 The integrated optimization
platform is controlled from a workstation using an in-house
designed software package.
Data published in this letter demonstrates both the
reproducibility and consistency of automated SAR generation
for a series of compounds with known activity against
dipeptidyl peptidase 4 (DPP4).
Several groups have implemented online screening of drug-
like compounds. For example, Kool et al. used online bioaffinity
analysis to screen a library of fragments for activity against
acetylcholine binding protein, and Guetzoyan et al. used frontal
affinity chromatography to screen a range of γ-aminobutyric
acid (GABA) agonists synthesized in flow.2,3 In addition, the
opportunity to improve the speed, efficiency, and quality of
medicinal chemistry hypothesis testing by the direct integration
of compound synthesis with biology and design algorithms has
been recognized by several groups.4,5 The integrated platform
described in this letter is designed to select a compound from a
drug-like chemical space and synthesize, purify, and biologically
test the compound. The SAR generated is then used to select
In order to further validate the integrated synthesis and
screening approach, a joint project was undertaken to replicate
an existing data set in a blinded experiment. Sanofi-Aventis
provided a series of compounds with known inhibitory activity
against DPP48−10 for evaluation on the platform. The SAR
Received: May 7, 2013
Accepted: June 9, 2013
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Figure 3. Example curve of human DPP4 inhibition.
Figure 1. Cyclofluidic optimization platform.
Scheme 1. General Synthesis of DPP4 Inhibitors
Figure 4. Flow reactor setup for diamine reaction.
Scheme 2. Synthesis of Diamino DPP4 Inhibitors
Table 2. Results of Diamine Synthesis and Testing
Table 1. Repeated Synthesis and Assay of Compound 3
loop number DPP4 (human) nM column 1 DPP4 (human) nM column 2
1
2
3
4
5
6
7
8
9
10
76
115
104
83
84
86
120
103
76
110
72
128
105
158
131
128
82
166
122
124
Figure 2. Graph of IC50 results for compound 3.
generated would then be unblinded by Sanofi-Aventis to
provide validation of the fully integrated approach.
The compounds to be synthesized are shown in Scheme 1.
The first step of the synthesis used either boc-protected
diamines or amino alcohols to displace 8-bromoxanthines. The
in situ generated boc-protected intermediates were then
deprotected to give the compound of interest for testing.
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Scheme 3. Synthesis of Amino-Alcohol Modified Xanthines
Table 3. Results of Amino-Alcohol Synthesis and Testing
Figure 5. Reaction setup for amino-alcohol reaction.
The first evaluation was to establish consistency between
cycles by repeatedly synthesizing and screening a single
compound 3 (Scheme 2) ten times on the integrated platform.
For each individual compound synthesis, the sample was
assayed twice in alternate assay plate columns against human
DPP4 to assess variance in the assay. By way of control, a
discrete solid sample was assayed manually to provide
comparator data.
The data from the experiment is displayed in Table 1, and
Figure 2 clearly indicates that, with over 20 hours and ten cycles
of both synthesis and screening on the platform, the data is
consistent within normal error; the activity for a solid sample
was 106 nM. This clearly illustrates that as well as giving very
little variance, the IC50 is in complete agreement with the
discrete sample determined manually.
An example IC50 curve for compound 3 generated during the
run is shown in Figure 3 showing excellent fit.
With reproducibility now confirmed, a series of diamine
modified xanthines were synthesized via a two-step synthetic
protocol using a Vapourtec R4 flow chemistry system. An
example reaction is shown in Scheme 2.
The first synthetic displacement was conducted on a 2 mL
volume stainless steel coil heated at 150 °C with injections of
250 μL of the 8-bromosubstituted xanthine in N-methyl-2-
pyrrolidone (NMP) and 250 μL solution of two equivalents of
the desired boc-protected diamine in NMP. The flow rate for
step 1 was 50 μL/min per pump to give a residence time of 20
min. Next, the boc-protected intermediate was mixed with 500
μL of 30% methanesulfonic acid in water added at 50 μL/min
to deprotect the boc group. This reaction was conducted in a
second 2 mL coil at 90 °C and the product subsequently run
through a 20 μL injection loop into the LC−MS purification
system switched at the point of maximum concentration. NMP
was used as the system and wash solvent. A schematic of the
setup is shown in Figure 4.
a
b
Synthesis failed. Mass spec. failed to give expected molecular ion.
Several of the compounds have repeated values to further demonstrate
consistency.
The total synthesis time per compound to purification was
50 min, and the total cycle time per sample was 120 min.
All 12 compounds were successfully synthesized and tested
in 24 h total platform time. Yields varied between 3% and 38%.
It should be noted that even low yields provided enough
material for the compounds to be purified and assayed.
The results are displayed in Table 2.
Next, the synthesis and testing of a series of amino alcohols
was undertaken. An example synthetic route is shown in
Scheme 3.
In this case, the initial reaction was very fast using KOtBu as
base and could be conducted in a 32 cm long 1 mm ID tube in
∼3 min. Subsequent deprotection of the boc group was again
achieved using 30% methanesulfonic acid in water at 90 °C
with yields for the two step process of between 0% and 23%.
The overall synthesis success for this process was a respectable
82%.
A schematic of the reaction setup is shown in Figure 5.
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ACS Medicinal Chemistry Letters
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porcine DPP4 (Sigma) or 34 U/mL human DPP4 (Sigma).
Residual enzyme activity was monitored by adding substrate
(H-Gly-Pro-AMC; Bachem) to give a final concentration
equivalent to the KaMpp (KaMpp = 25 μM for human DPP4 and
KaMpp = 35 μM for porcine DPP4). Data was analyzed by
nonlinear regression using a four parameter logistic variable
slope model for IC50 determinations.
Table 4. Comparison of Sanofi-Aventis and Integrated
Platform Data
a
The integrated platform data was compared against Sanofi-
Aventis data as shown in Table 4. The porcine data matched
closely validating the technique (Figure 6). The historical
human DPP4 data was limited, but where available, the
platform data is also in agreement with previously reported
values.
Overall, the data obtained by the platform is fully consistent
with reference data provided by Sanofi-Aventis using traditional
means.
The synthesis was generally robust, and the integrated
approach enables biological data to be measured from low
yielding reactions. This additionally minimizes the optimization
time needed to achieve a reliable, generic, flow synthesis
method and ensures results are obtained from reagents with a
range of reactivity.
The biological assay has also been shown to be both robust
and stable over an extended period of time, essential for an
automated environment.
A plot of the porcine (closed circle) and human (open
CYC
square) Sanofi-Aventis (log IC50SA) and platform (log IC50
)
inhibition data presented in Table 4 is shown in Figure 6. The
lines represent the best fit linear regression analysis of the data.
In conclusion, a series of DPP4 inhibitors have been
successfully synthesized, purified, and tested using the platform
with an overall chemistry success rate of 93%. Each compound
was synthesized and tested in two hours total. It took less than
three days to synthesize and test the total of 29 compounds.
Close correlation between integrated platform data and data
generated within the corresponding traditional medicinal
chemistry approach has validated the technique (R2 = 0.9253
and 0.9893 for porcine and human data, respectively; Figure 6).
Further work on a series of synthetic routes and various target
classes is currently underway.
a
(P) = porcine data; (H) = human data.
ASSOCIATED CONTENT
* Supporting Information
■
S
General synthetic and analytical purity determination proce-
dures for all synthesized compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*(G.J.T.) Tel: +44 (0)1707358673. E-mail: gary.tarver@
cyclofluidic.co.uk.
Funding
Cyclofluidic is grateful for support from the UK Technology
Strategy Board, UCB, and Pfizer.
Notes
Figure 6. Correlation between Sanofi-Aventis and platform data.
The authors declare no competing financial interest.
The results from 21 iterations of the system are shown in
Table 3.
Biological assays were carried out in 40 mM tris·HCl, pH 7.4
at 25 2 °C, in a total assay volume of 50 μL using 384-well
plates (Costar 3574). Routinely, compounds were titrated in
assay buffer to which was added either enzyme (0.82 mU/mL
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