Pharmacophore reassignment for induction of the immunosurveillance cytokine TRAIL

Article abstract of DOI:10.1002/anie.201402133

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an immunosurveillance cytokine that kills cancer cells but demonstrates little toxicity against normal cells. While investigating the TRAIL-inducing imidazolinopyrimidinone TIC10, a misassignment of its active structure was uncovered. Syntheses of the two isomers, corresponding to the published and reassigned structures, are reported. The ability of each to induce TRAIL expression in macrophages was investigated and it was found that only the compound corresponding to the reassigned structure shows the originally reported activity; the compound corresponding to the published structure is inactive. Importantly, this structural reassignment has furnished a previously unknown antitumor pharmacophore. Setting the record straight: An investigation of an imidazolinopyrimidinone (TIC10) reported to induce expression of the immunosurveillance cytokine TRAIL led to a constitutional reassignment of the active pharmacophore. Analysis of TRAIL induction in macrophages revealed the reported structure to be inactive. The structural identity of the active compound was established. The active compound was then synthesized and its activity confirmed.

Full text of DOI:10.1002/anie.201402133

Angewandte
Chemie
DOI: 10.1002/anie.201402133
Antitumor Agents
Pharmacophore Reassignment for Induction of the
Immunosurveillance Cytokine TRAIL**
Nicholas T. Jacob, Jonathan W. Lockner, Vladimir V. Kravchenko, and Kim D. Janda*
Abstract: Tumor necrosis factor (TNF)-related apoptosis-
inducing ligand (TRAIL) is an immunosurveillance cytokine
that kills cancer cells but demonstrates little toxicity against
normal cells. While investigating the TRAIL-inducing imid-
azolinopyrimidinone TIC10, a misassignment of its active
structure was uncovered. Syntheses of the two isomers,
corresponding to the published and reassigned structures, are
reported. The ability of each to induce TRAIL expression in
macrophages was investigated and it was found that only the
compound corresponding to the reassigned structure shows the
originally reported activity; the compound corresponding to
the published structure is inactive. Importantly, this structural
reassignment has furnished a previously unknown antitumor
pharmacophore.
for studying the complex immunosurveillance signaling
system.
A recent report highlighted imidazolinopyrimidinone
1 (Figure 1), obtained from the NCI, which was able to
induce TRAIL expression in a variety of cell lines.[12] This
compound, designated TIC10 (TRAIL-inducing com-
C
ancer immunosurveillance relies on various effector func-
tions of the immune system that can modify both induced and
spontaneous carcinogenesis.[1,2] TRAIL is an immunosurveil-
lance cytokine that plays a critical role in this process through
its ability to selectively induce apoptosis in cancer cells over
normal cells.[2–6] The TRAIL gene is expressed in a variety of
tissues and cells,[3] including dendritic cells, natural killer
(NK) cells, and monocytes/macrophages.[7] Its expression is
controlled by several transcriptional regulators, such as the
transcription factors NF-kB and p53.[8] The reduction of
TRAIL expression by neutralizing antibodies and the abla-
tion of TRAIL expression in mice lacking the TRAIL gene
results in the development of carcinogen-induced fibrosarco-
mas, sarcomas, and lymphomas; especially in p53-deficient
mice.[9,10] These data are also consistent with observations that
a change in TRAIL expression in immune cells is associated
with TRAIL resistance in cancer cells.[11] The effectors of
TRAIL production in immune cells are thus of clinical
relevance[7] and could also be used to achieve a model system
Figure 1. Structures of the imidazolinopyrimidinones synthesized in
this study. The tricyclic core of each structure is highlighted in bold.
The compound with an angular structure (2) and not that with a linear
structure (1) induces TRAIL expression.
pound 10), was demonstrated to be thermally stable and
orally bioavailable, thus surmounting liabilities inherent in
recombinant TRAIL therapies. It was also able to induce
tumor regression in several xenograph models with no
observed toxicity at four times the effective dose.
It is important to note that the effective dose of TIC10
(25 mgkgÀ1) was as a stand-alone therapy and thus suffers
from the same resistance mechanisms observed in other
TRAIL therapies.[13] We have reported how quorum-sensing
signaling molecules (acylhomoserine lactones) can overcome
tumor-cell TRAIL resistance through the inhibition of NF-
kB.[14] In terms of antitumor therapy, the combination of these
two treatments could address the fundamental shortcomings
of TRAIL as a therapeutic.[15] As a logical starting point, 1 was
required; yet despite a wealth of biological data, no chemical
synthesis was reported.
[*] N. T. Jacob,[+] Dr. J. W. Lockner,[+] Prof.Dr. V. V. Kravchenko,
Prof. Dr. K. D. Janda
Department of Chemistry
Dept. of Immunology and Microbial Science
The Skaggs Institute for Chemical Biology
The Worm Institute of Research and Medicine
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
E-mail: kdjanda@scripps.edu
TIC10 (1) was prepared in four steps from 4-chloronico-
tinic acid (Scheme 1). Briefly, acylation of an activated
carboxylic acid followed by a double displacement reaction,
subsequent hydrogenation, and reductive amination afforded
1 in 52% overall yield. This structure was confirmed by mass
spectrometric, NMR spectroscopic, and X-ray crystallo-
graphic analyses (Figure 1).
[+] These authors contributed equally to this work.
[**] We thank Josh Foote for the artwork appearing in the Table of
Contents entry for this communication. We are grateful to Prof.
Arnold Rheingold and Dr. Curtis E. Moore (UCSD) for X-ray
crystallographic analyses.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201402133.
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of the imidazoline ring and the
carbonyl carbon atom. Mean-
while, significant correlation
was observed between the ben-
zylic protons of the 2-methyl-
benzyl moiety and the carbonyl
carbon atom. This data is more
consistent with a constitutional
isomer of TIC10 possessing an
angular (rather than linear) tri-
cyclic core (2). This critical
structural difference between
1 and 2 might also explain why
Scheme 1. Synthesis of the reported structure of TIC10 (1). Reagents and conditions: a) SOCl2, 908C, 1 h,
then 2-methylthio-2-imidazoline hydroiodide, Et3N, CH2Cl2, 08C to RT, 19 h, 96%; b) 2-methylbenzylamine,
K3PO4, N,N-dimethylacetamide, reflux, 1 h, 79%; c) 45 psi H2(g), PtO2, MeOH/TFA, RT, 5 h, 80%;
d) PhCHO, Na(OAc)3BH, AcOH, CH2Cl2, RT, 4 h, 87%; e) methyl chloroformate, Et3N, CH2Cl2, 08C to RT,
44 h, 97%; f) 2-methylbenzylamine, MeOH/AcOH, reflux, 45 h, 87%; g) NaOMe, MeOH, reflux, 18 h,
97%. TFA=trifluoroacetic acid.
The biological activity of 1 was measured by RT-PCR
analysis of TRAIL mRNA expression in the murine macro-
phage cell line RAW 264.7. Surprisingly, no change in TRAIL
mRNA expression over controls was observed, even at doses
as high as 10 mm (Figure 2a) or with prolonged exposure
(Figure 2b). In order to confirm the disparity between our
the imidazoline protons in the 1H NMR spectrum for 2 appear
as a congested multiplet (d ꢀ 3.9 ppm), whereas as imidazo-
line protons in the 1H NMR spectrum for 1 are cleanly
separated into two triplets (d ꢀ 4.1 and 3.8 ppm) owing to
a marked difference in shielding by the conjugated system
(Figure 3). In addition to the equivocality of the NMR data,
the structure of the repository TIC10 (2) was ultimately
confirmed by X-ray crystallographic analysis (Figure 1).
Our new target, angular imidazolinopyrimidinone 2, was
prepared in three steps in 82% yield (Scheme 2). Guanidine 7
was obtained in two steps from commercially available 2-
methylthio-2-imidazoline
hydroiodide.[17]
Subsequently,
a mixture of 7 and 1-benzyl-4-oxopiperidine-3-carboxylate
hydrochloride (8) in refluxing methanol and sodium meth-
Figure 2. Angular fusion of the tricyclic core is required for imidazoli-
nopyrimidinone-mediated induction of TRAIL. a) Induction of TRAIL
mRNA in RAW cells treated for 48 h with the indicated dose of linear
(1) or angular (2a) TIC10. b) Induction of TRAIL mRNA following
treatment with 1 or 2a (5 mm) for the indicated times. c) Dose-
dependent response to repository (2a) or synthesized (2b) angular
imidazolinopyrimidinone after 48 h exposure. d) Dose-dependent
response to angular (2b) or commercial (9) imidazolinopyrimidinone.
results when using 1 and those reported by Allen et al.,[12]
a sample of TIC10 (2a) was acquired from the NCI.[16]
In sharp contrast to our results with 1, this material was
able to significantly induce TRAIL mRNA expression over
controls at doses as low as 2.5 mm (Figure 2a) and within 12 h
of exposure (Figure 2b). The induction appeared linear over
48 h at 5 mm (Figure 2b).
Figure 3. 1H NMR overlay of 1, 2, and 9. The imidazoline ring protons
have distinct patterns corresponding to the linear or angular core
structure.
Because of the stark dis-
crepancy in the biological activ-
ities of these materials, an in-
depth structural analysis of 2a
was initiated. The material
obtained from the repository
was examined through hetero-
nuclear multiple-bond correla-
tion (HMBC) NMR spectrosco-
Scheme 2. Synthesis of the actual structure of TIC10 (2). Reagents and conditions: a) SOCl2, 908C, 1 h,
then 2-methylthio-2-imidazoline hydroiodide, Et3N, CH2Cl2, 08C to RT, 19 h, 96%; b) 2-methylbenzylamine,
K3PO4, N,N-dimethylacetamide, reflux, 1 h, 79%; c) 45 psi H2(g), PtO2, MeOH/TFA, RT, 5 h, 80%;
d) PhCHO, Na(OAc)3BH, AcOH, CH2Cl2, RT, 4 h, 87%; e) methyl chloroformate, Et3N, CH2Cl2, 08C to RT,
44 h, 97%; f) 2-methylbenzylamine, MeOH/AcOH, reflux, 45 h, 87%; g) NaOMe, MeOH, reflux, 18 h,
py,
revealed
which,
no
unexpectedly,
correlation
between the methylene protons 97%.
2
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oxide afforded 2 almost exclusively; a trace amount of 1 was
detected by 1H NMR following work-up of this reaction but it
was removed by subsequent purification. We rationalize this
result by considering that the imidazoline ring nitrogen atoms
of 7 possess both statistical and steric advantages over the
benzylic nitrogen atom of 7. Initial attack by nitrogen at the
ketone carbonyl of 8 affords an aminocarbinol intermediate,
which undergoes intramolecular cyclocondensation and dehy-
dration to provide 2. Its structure was confirmed by mass
spectrometric and NMR spectroscopic analyses. Our syn-
thetic product 2b was able to induce TRAIL mRNA
expression with similar potency to the material obtained
through the repository (2a; Figure 2c).
Figure 5. A plausible rationale for undesired condensation and alkyla-
tion outcomes during imidazolinopyrimidinone synthesis.
During the preparation of this manuscript, linear imid-
azolinopyrimidinone (TIC10; 1) purportedly became com-
mercially available.[18] This commercial material (9) also
proved unable to induce TRAIL expression in RAW cells
(Figure 2d). X-ray crystallographic analysis revealed this
compound to be a third constitutional isomer (Figure 4). It
has the same linear tricyclic core as 1, but the 2-methylbenzyl
substituent is attached to the adjacent (imidazoline ring)
nitrogen atom.
a plethora of literature precedent for linear fused pyrimidi-
nones akin to 1, there is a paucity of literature precedent for
angular fused pyrimidinones akin to 2. Finally, one may
speculate that compound
9 may have arisen through
attempted N-alkylation of a linear fused precursor en route
to 1 (the originally reported TRAIL-inducing compound),
where N-alkylation of the wrong nitrogen atom[20] afforded 9
(Figure 5, path d) instead of 1.
The report by Allen et al.[12] extolled the clinical signifi-
cance of TIC10, a fact further borne out by its recent
commercial availability and that it is featured in an investiga-
tional new drug (IND) application.[21] However, our findings
imply that the angular fusion of the tricyclic core (as in 2
rather than 1) is a required feature of this pharmacophore for
TRAIL induction. Given the structural misassignment
explained herein, this drug development pursuit must thus
be questioned.
With a concise synthesis for the active material (2) and
irrefutably structural confirmation through X-ray crystallo-
graphic analysis, further evaluation of this angular scaffold
with respect to TRAIL induction can be undertaken. It will
now also be possible to examine with confidence the
structure–activity relationships (SAR) of this scaffold,
explore its mechanism of action, proceed with subsequent
optimization, and initiate studies into possible combination
therapies. Finally, this angular imidazolinopyrimidinone has
not been previously disclosed and thus represents a promising
new pharmacophore for further exploration as a therapeutic.
Figure 4. The structure of a commercially available imidazolinopyrimi-
dinone. The tricyclic core is highlighted in bold. This compound
proved unable to induce TRAIL expression.
In view of these discrepancies, a brief discussion of the
heterocyclic chemistry of 1, 2, and 9 is warranted. With regard
to the cyclocondensation step, depending on the reaction
conditions used to form the carbinol, it may be possible to
obtain either product isomer (1 or 2). Indeed, it has been
reported that the K2CO3-mediated reaction of a b-keto ester
with a 2-amino-2-oxazoline (a type of unsymmetrical 1,1-
diamine) affords a mixture of linear and angular products.[19]
Empirical and theoretical evidence supported the notion that
“the endocyclic nitrogen atom is the most nucleophilic and
attacks the most electrophilic carbon of the biselectrophile. A
ring closure between the exocyclic nitrogen atom and the
second electrophilic center concludes the bicyclic heterocycle
synthesis.”[19] This is consistent with our own observations of
the synthesis of 2. One may speculate that the original
synthesis of 1 unexpectedly produced 2 instead (Figure 5,
path a). In the absence of rigorous structure elucidation
through correlation NMR spectroscopy and/or X-ray crystal-
lographic analysis, misassignment is possible. Only with full
spectral data (and X-ray crystal structures) in hand can the
discrepancies really be appreciated. Also, whereas there is
Received: February 5, 2014
Revised: March 7, 2014
Published online: && &&, &&&&
Keywords: cancer therapy · drug discovery ·
.
structure elucidation · TIC10 · TRAIL
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Chemie
Communications
Antitumor Agents
Setting the record straight: An investiga-
tion of an imidazolinopyrimidinone
(TIC10) reported to induce expression of
the immunosurveillance cytokine TRAIL
led to a constitutional reassignment of
the active pharmacophore. Analysis of
TRAIL induction in macrophages revealed
the reported structure to be inactive. The
structural identity of the active compound
was established. The active compound
was then synthesized and its activity
confirmed.
N. T. Jacob, J. W. Lockner,
V. V. Kravchenko,
K. D. Janda*
&&&& — &&&&
Pharmacophore Reassignment for
Induction of the Immunosurveillance
Cytokine TRAIL
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
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