Design and Catalyzed Activation of Tak-242 Prodrugs for Localized Inhibition of TLR4-Induced Inflammation

Article abstract of DOI:10.1021/acsmedchemlett.9b00518

Tak-242 (resatorvid), a Toll-like Receptor 4 (TLR4) inhibitor, has been identified as a potent suppressor of innate inflammation. As a strategy to target Tak-242 to select tissue, four TLR4-inactive prodrugs were synthesized for activation via two different release mechanisms. Two nitrobenzyl Tak-242 prodrugs released the parent drug upon exposure to the exogenous enzyme nitroreductase, while the two propargyl prodrugs were converted to Tak-242 in the presence of Pd⁰.

Full text of DOI:10.1021/acsmedchemlett.9b00518

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Letter
Design and Catalyzed Activation of Tak-242 Prodrugs
for Localized Inhibition of TLR4-induced Inflammation
Michael A. Plunk, Alyssa Alaniz, Olatunde P. Olademehin,
Thomas L Ellington, Kevin L Shuford, and Robert R Kane
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00518 • Publication Date (Web): 03 Jan 2020
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Design and Catalyzed Activation of Tak-242 Prodrugs for Localized
Inhibition of TLR4-induced Inflammation
Michael A. Plunk†, Alyssa Alaniz‡, Olatunde P. Olademehin†, Thomas L. Ellington†, Kevin L. Shuford†,
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and Robert R. Kane*,†,‡
†Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76798, United States
‡Institute of Biomedical Studies, Baylor University, Waco, TX 76798, United States
ABSTRACT: Tak-242 (resatorvid), a Toll-like Receptor 4 (TLR4) inhibitor, has been identified as a potent suppressor of innate
inflammation. As a strategy to target Tak-242 to select tissue, four TLR4-inactive prodrugs were synthesized for activation via two
different release mechanisms. Two nitrobenzyl Tak-242 prodrugs released the parent drug upon exposure to the exogenous enzyme
nitroreductase, while the two propargyl prodrugs were converted to Tak-242 in the presence of Pd0.
KEYWORDS: prodrug, enzyme prodrug therapy, bioorthogonal organometallic activation, nitroreductase, TLR4
We have recently demonstrated that the potent toll-like
As an alternative strategy for site-specific prodrug
2
receptor 4 (TLR4) inhibitor Tak-2421, can be utilized to
significantly improve outcomes in islet transplantation. While
the free drug protects pancreatic islets from TLR4-mediated
innate inflammation during their isolation,3 the conjugation of
a Tak-242 prodrug to islet surfaces using a slow-release linker
provides localized and sustained protection of the islets after
transplantation.4 Due to the promising outcomes from those
studies we have continued to explore strategies for the targeted
delivery of Tak-242, and herein report our work on the
synthesis and characterization of two classes of Tak-242
prodrugs for localized delivery.
Prodrugs are inactive compounds that are converted to
active drugs in vivo, and are commonly developed to address
suboptimal chemical or pharmacokinetic properties in the
parent drugs.5, 6 Site-selective prodrug activation, which can
take advantage of inherent differences in the tissue of interest
or rely upon the selective delivery of exogeneous prodrug
activators, is able to provide drug localization and limit off-
target effects. Two examples of prodrug targeting utilizing
Figure 1. Co-transplantation of transplant tissue and immobilized
catalysts provides drug activation localized to the vicinity of
transplanted tissue.
activation we chose to explore catalysis using Pd0
nanoparticles immobilized on TentaGel® resins (Rapp
Polymere GmbH). These catalysts have been used for the in
vivo activation of prodrugs containing propargyl, allyl, and
benzyl groups.22, 23 Propargyl prodrugs have been reported to
efficiently undergo hydrolysis to release the parent drug along
with hydroxyacetone (Scheme 1B).24, 25
exogeneous activators are Directed Enzyme Prodrug Therapy
7-13
(DEPT)
and Bioorthogonal Organometallic (BOOM) drug
activation.14-17 In DEPT, a prodrug-activating enzyme (with
activity not found in ‘normal’ tissue) is localized at the target
tissue, often using antibodies. Unciti-Broceta’s BOOM
strategy involves drug activation by localized biocompatible
metal catalysts. Implanting solid-supported enzymes (DEPT)
or metal catalysts (BOOM) in the vicinity of transplanted
tissue has the potential to afford local prodrug activation with
minimal systemic exposure (Figure 1).
For enzymatic activation we chose to explore the use of
nitroreductase, a bacterial enzyme commonly utilized in
DEPT.18, 19 This enzyme efficiently reduces aryl nitro groups,
and p-nitrobenzyl-modified prodrugs can undergo this
reduction followed by a 1,6-elimination to release active drug
along with p-aminobenzylalcohol (Scheme 1A).20, 21
Scheme 1. Release of active drugs: a) a p-nitrobenzyl prodrug and
nitroreductase (NTR) and b) a propargyl prodrug and Pd(0).
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Scheme 2. Synthesis of racemic Tak-242 prodrugs 2-5. i.) p-nitrobenzyl chloroformate, sodium carbonate (aq), EtOAc, 55% yield. ii.) p-nitrobenzyl chloride,
TEA, DCM, 47% yield. iii.) propargyl bromide, potassium carbonate, MeCN, 83% yield. iv.) propargyl chloroformate, TEA, DMAP, DCM, 54% yield.
Since biologically inactive carbamates of Tak-242 could be
readily synthesized from chloroformates,4 and as carbamates
has been widely utilized in prodrug design,26 carbamate
prodrugs 2 and 5 were chosen as the initial synthetic targets.
Additionally, due to the relatively acidic sulfonamide N-H
bond of Tak-242, we hypothesized that the drug itself could
also serve as a leaving group without the assistance of a
carbamate linkage and selected the alkylated compounds 3 and
4 as additional targets. In the event, racemic Tak-242 1 was
readily converted to to p-nitrobenzyl prodrugs 2 and 3 and
propargyl prodrugs 4 and 5 (Scheme 2) with reasonable
yields.
Initial examination of the proton NMR (20 °C; CDCl3) of
prodrug 2 revealed two distinct sets of peaks at a ~2:1 ratio
(Scheme 3), which is not unexpected for this complex
carbamate.27, 28 In DMSO-d6 the proton NMR of compound 2
had broad peaks, while at 55 °C the proton NMR spectrum
coalesced to a single set of clean signals (Figure 2). The
proton NMR of carbamate 5 also exhibited two sets of peaks
at an approximate 2:1 ratio at room temperature in CDCl3, but
heating this NMR sample to 70 °C in DMSO-d6 only resulted
in partial coalescence of the signals. The proton NMR
spectrum of non-carbamate prodrug 3 only revealed one set of
peaks, with the quartet and triplet associated with the ethyl
ester showing up as broad singlets that resolve to the expected
patterns at elevated temperature.
Figure 2. Stacked proton NMR spectra for compound 2 run in a)
chloroform-d at 27 °C, b) DMSO-d6 at 27 °C, c) DMSO-d6 at 40 °C, and
d) DMSO-d6 at 55 °C.
In order to better understand the conformational energetics
of these prodrugs (2-5), each compound was probed through a
set of relaxed torsional energy scans and subsequent full
geometry optimizations and harmonic vibrational frequency
computations using the hybrid B3LYP29-31 density functional
in conjunction with a split-valence triple- quality 6-
311G(2df, 2pd) basis set32-35 within the Gaussian 09 software
package.36 The cross-section of the potential energy surfaces
of prodrugs 2 and 5 exhibit clear minima and relatively large
energetic barriers of rotation (Erot = 13.41 kcal mol-1 and
12.99 kcal mol-1, respectively). Along with the similar
energies calculated for the low energy rotamers of these
carbamate prodrugs (E< 2 kcal mol-1), these barriers are
consistent with the observation of two distinct conformers in
the experimental NMR spectra.37 As an example, the complete
cross section of the potential energy surface for 2 is shown in
Figure 3; the full results from these quantum chemical
computations for all four compounds are found in the
Supporting Information.
Scheme 3. Rotation of the N-C bond results in the existence of two unique
conformers of Tak-242 carbamate prodrug 2.
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Figure 3. Cross section of the potential energy surface about torsional angle  (C-N-C-O)º and the energetic barriers of rotation (∆Erot and ∆Grot) between
the two minima of prodrug 2 computed at the B3LYP/6-311G(2df,2pd) level of theory. The green markers denote the fully optimized stationary points
identified by the relaxed torsional energy scan.
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revealed that while the alkylated compounds 3 and 4 are stable
under these conditions, the sulfonamide bonds of carbamates 2
and 5 slowly underwent hydrolysis (Scheme 4). This
reactivity resembles the previously reported4 carbamate-
functionalized prodrug/linker, and confirms our suspicion that
adding a carbamate functionality to the TAK-242 sulfonamide
destabilizes it towards hydrolysis. While only ~25% of 5
remained after one day of incubation, ~85% of 2 remained at
In order to determine whether compounds 2-5 would
function as inactive prodrugs, a TLR4 reporter cell assay was
performed. All four racemic prodrugs demonstrated a
significant reduction of TLR4 antagonism compared to the
active drug (racemic Tak-242; Figure 4). Perhaps
unsurprisingly, the simple propargyl-alkylated prodrug 4 was
most Tak-242-like, inhibiting the TLR4 response to LPS
stimulation with an IC50 approximately 10X the parent drug.
Scheme 4. Aqueous hydrolysis of sulfonamide bonds in carbamate
prodrugs 2 and 5.
this point, presumably due to the increased steric bulk (see
Supporting Information, Figure S14).
The catalyzed conversion of each prodrug to the parent drug
was then examined. Exposure of the nitrobenzyl prodrugs 2
and 3 to nitroreductase (from E. coli, 2 units/mL) and the
reducing cofactor -nicotinamide adenine dinucleotide
(NADH; 1 mg/mL) in PBS (5% DMSO) resulted in the rapid
consumption of the prodrugs and the efficient release of the
parent drug 1 (Figure 5). The putative reduced amine
intermediate of carbamate 2 was not observed, presumably
due a rapid 1,6-elimination and loss of CO2. Under the same
activation conditions, the alkylated nitro-benzyl prodrug 3 was
rapidly converted to the amine intermediate (visible in HPLC)
which slowly underwent 1,6-elimination and release of
compound 1 (Figure S13).
Figure 4. TLR4 reporter cell assay. HEK TLR4 reporter cells were treated
with compounds 1-5 at various concentrations and stimulated with LPS.
Complete suppression of TLR4 was defined as the optical density at
650nm of cells incubated with racemic Tak-242 (1) at 15 M.
(Table 1).
Table 1. IC Values of Racemic Tak-242 and Racemic Tak-242
Prodrugs.
Compound
1
2
3
4
5
0.040
2.701
1.785
0.334
3.897
IC50 (M)
Similarly, the Pd0 catalyzed unmasking of propargyl
prodrugs 4 and 5 was evaluated. Incubating the prodrugs in
PBS (5% DMSO) with Pd0 immobilized on amino terminated
The aqueous stabilities of the Tak-242 prodrugs were then
determined by incubation in phosphate buffered saline (PBS,
pH 7.4 containing 5% DMSO) at 37 °C. Analysis by HPLC
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polystyrene beads17 (30 m diameter beads, 1 mg/mL;
provided by Asier Unciti-Broceta, University of Edinburgh)
release of parent drug, and TLR4 Reporter Cell Assay. The
Supporting Information is available free of charge on the ACS
Publications website.
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AUTHOR INFORMATION
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Corresponding Author
* Bob_Kane@baylor.edu
Author Contributions
9
MAP synthesized and characterized the prodrugs, performed
the kinetic analyses, and produced the manuscript. AA
performed the reporter-cell assays and contributed to the
manuscript preparation. OPO, TLE, and KLS computed the
rotamer energetics and contributed to the manuscript
preparation. RRK guided the experimentation and edited the
manuscript. All authors have given approval to the final
version of the manuscript.
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Figure 5. Tak-242 prodrugs 2 and 3 were incubated with nitroreductase
(from E. coli ; 2 units/mL) and NADPH (1 mg/mL) in PBS (pH 7.4)
containing 5% DMSO and monitored by HPLC.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This research was supported by JDRF (Grant 1-INO-2019- 787-S-
B) and Baylor University (Faculty Research Investment Program
and the Vice Provost for Research). OPO, TLE, and KLS were
supported by the Chemical Sciences, Geosciences, and
Biosciences Division, Office of Basic Energy Sciences, Office of
Science, U.S. Department of Energy under Award Number DE-
SC0019327
The authors thank Professor Asier Unciti-Broceta for helpful
conversations and for providing Pd0-resins.
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