Harmaline Analogs as Substrate-Selective Cyclooxygenase-2 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00555

We report the design, synthesis, and evaluation of a series of harmaline analogs as selective inhibitors of 2-arachidonylglycerol (2-AG) oxygenation over arachidonic acid (AA) oxygenation by purified cyclooxygenase-2 (COX-2). A fused tricyclic harmaline analog containing a CH3O substituent at C-6 and a CH3 group at the C-1 position of 4,9-dihydro-3H-pyrido[3,4-b]indole (compound 3) was the best substrate-selective COX-2 inhibitor of those evaluated, exhibiting a 2AG-selective COX-2 inhibitory IC50 of 0.022 μM as compared to >1 μM for AA. The 2.66 ? resolution crystal complex of COX-2 with compound 3 revealed that this series of tricyclic indoles binds in the cyclooxygenase channel by flipping the side chain of L531 toward the dimer interface. This novel tricyclic indole series provides the foundation for the development of promising substrate-selective molecules capable of increasing endocannabinoid (EC) levels in the brain to offer new treatments for a variety of diseases, from pain and inflammation to stress and anxiety disorders.

Full text of DOI:10.1021/acsmedchemlett.9b00555

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Letter
Harmaline Analogs as Substrate-Selective Cyclooxygenase-2 Inhibitors
Md. Jashim Uddin, Shu Xu, Brenda C Crews, Ansari M. Aleem,
Kebreab Ghebreselasie, Surajit Banerjee, and Lawrence J. Marnett
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00555 • Publication Date (Web): 14 Feb 2020
Downloaded from pubs.acs.org on February 16, 2020
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Harmaline Analogs as Substrate-Selective Cyclooxygenase-2 Inhibitors
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Md. Jashim Uddin , Shu Xu , Brenda C. Crews , Ansari M. Aleem , Kebreab Ghebreselasie , Surajit
⁑
‖
†
Banerjee , and Lawrence J. Marnett *
†
A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry and
Pharmacology, Vanderbilt Institute of Chemical Biology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University
School of Medicine, Nashville, Tennessee 37232, USA
⁑Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA
‖Northeastern Collaborative Access Team, Argonne National Laboratory, Argonne, IL 60439, USA
KEYWORDS Cyclooxygenase-2, 2-arachidonylglycerol, arachidonic acid, substrate-selective inhibition.
ABSTRACT: We report the design, synthesis, and evaluation of a series of harmaline analogs as selective inhibitors of 2-
arachidonylglycerol (2-AG) oxygenation over arachidonic acid (AA) oxygenation by purified cyclooxygenase-2 (COX-2). A
fused tricyclic harmaline analog containing a CH O–substituent at C-6 and a CH -group at the C-1 position of 4,9-dihydro-3H-
3 3
pyrido[3,4-b]indole (compound 3) was the best substrate–selective COX-2 inhibitor of those evaluated, exhibiting a 2AG-
selective COX-2 inhibitory IC50 of 0.022 M as compared to >1 M for AA. The 2.66 Å resolution crystal complex of COX-2 with
compound 3 revealed that this series of tricyclic indoles binds in the cyclooxygenase channel by flipping the side chain of
L531 towards the dimer interface. This novel tricyclic indole series provides the foundation for the development of promising
substrate-selective molecules capable of increasing endocannabinoid (EC) levels in the brain to offer new treatments for a
variety of diseases, from pain and inflammation to stress and anxiety disorders.
Cyclooxygenases (COXs) are key enzymes that
modulate the physiology and pathophysiology of many
disease processes. These enzymes catalyze the
cancer cells.5-16 Furthermore, as both 2-AG and AEA are
high affinity endogenous ligands for the cannabinoid
receptors (CB1 and CB2) oxygenation of these lipids by
COX-2 may serve as an important mechanism by which
cannabinoid signaling is terminated. Thus, COX-2-
dependent endocannabinoid oxygenation may serve
important physiological or pathophysiological functions.
Defining these functions in vivo has remained elusive due
to the difficulty of differentiating the effects of AA
oxygenation from endocannabinoid oxygenation.
1
oxygenation of arachidonic acid (AA) into prostaglandins
17
(PGs) and thromboxane. Of the two COX isoforms,
constitutively expressed, COX-1 is found in nearly all
normal tissues, whereas COX-2 is induced in response to
biologically active endogenous and pathogen-associated
macromolecules – such as, cytokines, growth factors,
tumor promoters, and bacterial lipopolysaccharide – so
that inflammatory, preneoplastic, and neoplastic tissues
We have discovered that weak, competitive inhibitors of
AA oxygenation by COX-2 (e.g., ibuprofen, mefenamic
acid) are potent non-competitive inhibitors of 2-AG
2
express elevated levels of this enzyme. Thus, non-
selective COX inhibitors or selective COX-2 inhibitors
exhibit anti-inflammatory, analgesic, and anticancer
18
oxygenation. We proposed a mechanism to explain this
3
activities.
substrate-selective inhibition based on accumulating
evidence that the COX enzymes are structural
homodimers that behave as functional heterodimers with
one subunit serving as the active site while the second
In addition to their distinct expression patterns, the COX
isoforms also differ with regard to substrate specificity.
For example, COX-2 oxygenates neutral ester and amide
derivatives of AA that are poor substrates for COX-1. Of
19-27
serves an allosteric function.
Current data support the
particular
interest
in
this
regard
are
the
hypothesis that substrate-selective inhibitors bind with
high affinity in the allosteric site of COX-2 and alter the
endocannabinoids, 2-arachidonoylglycerol (2-AG) and
arachidnoylethanolamide (AEA), which are converted to
PG glyceryl ester (PG-G) and ethanolamide (PG-EA)
structure of the active site so that 2-AG oxygenation is
20, 25
inhibited but AA oxygenation is not.
Inhibition of AA
4
derivatives, respectively. The PG-Gs originating from
oxygenation requires lower affinity, reversible binding of a
18, 28
oxygenation of 2-AG exhibit biological properties distinct
from those of free acid PGs produced from AA in a range
of cell types, including neuronal, inflammatory, and
second molecule of inhibitor in the active site.
Substrate-selective inhibition of COX-2 offers a potential
means to elucidate the biological impact of
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endocannabinoid oxygenation in vivo; however, inhibitors
that display substrate-selectivity in vitro have failed to do
so in vivo due, at least in part, to inadequate selectivity or
29
metabolic instability. To address this failure, we now
describe the design and structure-activity relationship
(SAR) studies of a series of compounds obtained from the
derivatization of 4,9-dihydro-3H-pyrido[3,4-b]indole to
optimize an effective and metabolically stable substrate-
selective COX-2 inhibitor.
Key determinants of the substrate-selectivity of COX-2
inhibitors are unknown, which makes the design of such
molecules particularly challenging. The X-ray crystal
structure of 1-AG (the thermodynamically stable isomer of
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2-AG) in complex with COX-2 reveals that there is a small
hydrophobic pocket adjacent to Leu-531, Val-116, and
Val-349, all of which are located above a constriction that
demarcates the opening of the COX-2 active site. The
30
Scheme 1. Synthesis of 4,9-dihydro-3H-pyrido[3,4-b]indole derivatives
-6. (a) CH COCl, TEA, DCM, r.t. 5h; POCl , toluene reflux 7 h, (b) NaH,
-Br, 0 C 1 h, (c) t-butyl 2-chloro-2-
oxoacetate, TEA, DCM, r.t. 5 h; POCl , toluene, reflux 7h, (d) TFA, r.t. 1
glycerol moiety of 1-AG inserts into this pocket. This
information suggests that compounds containing a
relatively small functional group capable of binding in the
hydrophobic pocket might selectively inhibit the
oxygenation of 2-AG by COX-2.
1
3
3
o
o
6 4 2
DMF 0 C 1 h; 4-Cl-C H -CH
3
o
o
6 4 2
h, (e) NaH, DMF 0 C 1 h; 4-Cl-C H -CH -Br, 0 C 1 h.
The inhibitory potencies of the synthesized compounds
against purified human COX-2 or ovine COX-1 were
determined by a thin layer chromatography- (TLC)-based
Harmaline is a member of the harmala alkaloid family of
naturally occurring organic compounds containing a
tricyclic indole nucleus. Studies have shown that
harmaline is psychoactive in humans, and rutaecarpine,
a natural polycyclic analog of harmaline, binds to COX
enzymes with varying degrees of isoform selectivity when
14
assay that measures the conversion of [1- C]-AA to
34
radiolabeled PGs. Briefly, reaction mixtures of 200 L
contained hematin-reconstituted protein in 100 mM Tris-
14
HCl, pH 8.0, 500 M phenol, and [1- C]AA (5 M, ~55-
57 mCi/mmol, Perkin Elmer). Reactions were terminated
by solvent extraction in diethyl ether:methanol:1 M citrate
buffer, pH 4.0 (30:4:1). The phases were separated by
centrifugation at 2000 rpm for 2 min, and the organic
phase was spotted on a TLC plate (EMD Kieselgel 60,
VWR). Following development in ethyl acetate:methylene
31-33
tested using AA as a substrate.
We hypothesized that
un-natural analogs of harmaline would selectively inhibit
oxygenation of 2-AG by COX-2, thereby resulting in
increased endocannabinoid (EC) levels in the brain and a
concomitant reduction in stress and anxiety. To test this
hypothesis, we synthesized and evaluated a series of
harmaline derivatives as substrate-selective inhibitors of
COX-2.
o
chloride:glacial acetic acid (75:25:1) at 4 C, radiolabeled
products were quantified with a radioactivity scanner
(Bioscan, Inc., Washington, D.C.).
We used the Bischler-Napieralski intramolecular
electrophilic aromatic substitution reaction for the
Compounds were next evaluated for substrate-selective
COX-2 inhibitory activity using a mass spectrometry-
(MS)-based assay. The final assay reaction mixture
contained 50 nM purified murine COX-2, 100 nM heme, 5
synthesis
of
substituted-4,9-dihydro-3H-pyrido[3,4-
b]indoles from the cyclization of substituted-N-
acetyltriptamine catalyzed by phosphorus oxychloride
(POCl
,9-dihydro-3H-pyrido[3,4-b]indoles was performed using
substituted benzyl bromide in good yields (compounds 1-
, Scheme 1, and see Supporting Information (SI) for
synthetic details).
3
) in refluxing toluene. Benzylation of substituted-
µM AA or 2-AG,
1
µM 5-phenyl-4-pentenyl-1-
4
hydroperoxide (PPHP), and inhibitor or vehicle (DMSO at
a final concentration of 5%) in a buffer of 50 mM Tris-HCl,
pH 8.0, 0.5 mM phenol. Following addition of inhibitor, the
mixture was incubated for 15-min, and then the reaction
was initiated by the addition of AA or 2-AG. After 30s, the
reaction was quenched by addition of ethyl acetate or
6
acetonitrile containing internal standards (PGE
2 4
-d and
PGE G-d ) at 0.3 µM. The samples were injected onto a
2
5
C18 5 X 0.2 cm, 3 µ M particle size column connected to
a Shimadzu LC system coupled with an ABSCIEX MS.
Elution solvents were solvent A (5 mM ammonium
acetate, pH 3.6) and solvent B (94% acetonitrile with 6%
solvent A) applied in a gradient of 30% to 100% B over
1
.5 min followed by 100% B for 1 min. Analytes of interest
were detected by selected reaction monitoring MS/MS
using the following transitions: PGE /D m/z 370→317;
PGE -d m/z 374→321; PGE /D -G m/z 444→391; PGE
G-d m/z 449→396. Analyte peak areas were normalized
2
2
2
4
2
2
2
-
5
to those of their deuterated internal standards for the
2
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Table 2. Substrate-selective Inhibition of COX-2 by 1-6.
quantification of product formation and inhibition. Note
that PPHP was included in this assay to eliminate
possible effects of inhibitors on peroxide-mediated
activation of COX-2, a mechanism that has been
implicated in substrate-selective inhibition of 2-AG
No.
AA
2-AG
IC50 (µM)
> 1
IC50 (µM)a
a
1
2
3
4
5
6
> 1
> 1
> 1
> 1
> 1
> 1
> 1
0.022
> 1
35
oxygenation by some inhibitors. The presence of PPHP
tends to increase the IC50 for inhibition of both AA and 2-
AG oxygenation; however, its effect is greater in the case
of 2-AG because higher peroxide tone is required for
> 1
36
activation in the case of that substrate.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
0
1
2
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4
5
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9
0
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2
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7
8
9
0
0.8
Compounds 1-6 possessed a tricyclic indole core (4,9-
dihydro-3H-pyrido[3,4-b]indole) containing a methoxy
substitution at the C-6 or C-7 position. The p-chlorobenzyl
substitution was introduced at C-9 position of compounds
aIC50 values were determined by incubating five concentrations of
inhibitor and a solvent control in DMSO with purified COX-2 (40 nM) for
3
min followed by addition of arachidonic acid (AA) or 2-
arachidonylglycerol (2-AG) 5 μM at 37 °C for 30 s.
3-4 and 6 in order to achieve improved in vitro and in vivo
metabolic stability. In the purified COX inhibition assay,
compounds 1 and 2 (harmaline) showed no COX
inhibitory activity. However, 9-(4-chlorobenzyl)-6-
methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole
(compound 3) was a selective COX-2 inhibitor (IC50 0.2
μM), whereas its regioisomer 9-(4-chlorobenzyl)-7-
methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole
To explore the structural basis for substrate-selective
inhibition of COX-2 by compound 3, we obtained a 2.66 Å
resolution X-ray crystal structure of the inhibitor in
complex with the protein (PDB ID 6V3R). Statistics of X-
ray data collection and structure refinement is described
in the SI section (Table 1s). As seen in Fig. 1, compound
3
binds in the cyclooxygenase active site of COX-2,
(compound 4) exhibited COX-2 selectivity with a poor
resting above a “constriction” designated by Arg-120, Tyr-
355, and Glu-524, as is the case for the vast majority of
COX inhibitors. Notably, however, steric hindrance
between the tricyclic indole core and Leu-531 induces a
movement of the side chain of this amino acid relative to
the position it occupies in most COX-inhibitor crystal
structures. A similar movement of Leu-531 has been
observed in other COX-2:ligand complexes, including
potency (IC50 2.1 μM). Although, the carboxylic acid-
containing compound 5 showed no COX inhibitory
activity,
pyrido[3,4-b]indole-1-carboxylic acid (compound 6, Table
) exhibited mild inhibitory activity against COX-1 with an
9-(4-chlorobenzyl)-6-methoxy-4,9-dihydro-3H-
1
IC50 value of 2.9 μM.
Table 1. Biochemical properties of 4,9-dihydro-3H-pyrido[3,4-b]indole
derivatives 1-6.
COX-2:AA (PDB files 1CVU and 3HS5, unproductive
37, 38
binding mode),
COX-2:eicosapentaenoic acid (PDB
38
30
No.
oCOX-1
IC50 (µM)
> 4
mCOX-2
IC50 (µM)
file 3HS6), COX-2:1-AG (PDB file 3MDL), COX-
a
a
21
2:3(S)- methylarachidonic acid (PDB file 4RUT), and
1
2
3
4
5
6
> 4
complexes with inhibitors of the oxicam class (PDB files
39
> 4
> 4
> 4
> 4
2.9
> 4
0.2
2.1
> 4
> 4
4M10, 4M11, and 4O1Z). In each case, displacement
aIC50 values were determined by incubating several concentrations of
inhibitors or DMSO vehicle with purified murine COX-2 (63 nM) or ovine
14
COX-1 (22.5 nM) for 20 min, followed by addition of [1- C]-AA (5 M) at
7 ºC for 30 s. Assays were run in triplicate.
3
We evaluated all the compounds for their ability to inhibit
-AG oxygenation selectively over AA oxygenation by
2
COX-2. The evaluation was performed using an MS-
based assay, as described above with added inhibitor
concentrations up to 1 μM. Under these conditions, no
compound reached its IC50 for COX-2-dependent AA
oxygenation. Only compounds
3 and 6 exhibited
Figure 1. Stereo-diagram of the X-Ray co-crystal structure of compound
substrate-selective inhibitory activity against 2-AG
oxygenation, with IC50 values of 0.022 μM and 0.8 μM,
respectively (Table 2). In addition, we evaluated
compound 3 in presence of both AA and 2-AG (see SI for
details), where it showed substrate-selective COX-2
inhibitory activity against 2-AG oxygenation with an IC50
value of 0.145 μM.
3
bound in the active site of COX-2 (PDB ID 6V3R). An omit map is
contoured at 3  in black mesh. The key residues are illustrated with a
stick representation of the final model (carbon in yellow, oxygen in red,
nitrogen in blue), and compound 3 is colored in green.
of the Leu-531 side chain helps to accommodate a bulky
ligand or otherwise unfavorable binding pose. This finding
suggests that movement of Leu-531 may play a role in
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substrate-selective inhibition, but such a displacement
was not observed in the crystal structures of other such
40
inhibitors [e.g., ibuprofen (PDB file 4RSO)
and
ABBREVIATIONS
35
mefenamic acid (PDB file 5IKR) ]. Furthermore, the
oxicams induce a similar displacement of Leu-531, but
they are not substrate-selective inhibitors. It is also
notable that in the crystal structure, compound 3 is bound
to both subunits of COX-2. Thus, the structure does not
provide support for the hypothesis that substrate-
selective inhibition results from binding of inhibitor to the
allosteric site only. This may be the result of constraints
present during crystallization, however, and may not
reflect the behavior of the enzyme in the cellular
environment.
Cyclooxygenases (COXs), arachidonic acid (AA), 2-
arachidonylglycerol
prostaglandin glyceryl esters (PG-Gs), prostaglandin E2
(PGE ), prostaglandin -G (PGE -G), cannabinoid
receptors (CB1 and CB2), arachidnoylethanolamide (AEA),
(2-AG),
prostaglandins
(PGs),
2
E
2
2
prostaglandin ethanolamide (PG-EA)
EC), phosphorus oxychloride (POCl
endocannabinoid
(
3
), and 5-phenyl-4-
0
1
2
3
4
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1
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4
5
6
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9
0
1
2
3
4
5
6
7
8
9
0
pentenyl-1-hydroperoxide (PPHP).
REFERENCES
1.
Dubois, R. N.; Abramson, S. B.; Crofford, L.;
In conclusion, we have described the design and
synthesis of a novel series of harmaline analogs that are
derived from 4,9-dihydro-3H-pyrido[3,4-b]indole core.
These compounds have been evaluated for their COX
inhibitory activity with respect to both isoform- and
substrate-selectivity. The SAR identified a fused tricyclic
rigid indole derivative, compound 3, as a promising
substrate-selective COX-2 inhibitor. The crystal complex
of COX-2 with 3 revealed a movement of Leu-531 in the
COX active site to accommodate inhibitor binding,
suggesting that subtle structural changes in this region
may contribute to its substrate-selectivity. This molecule
will serve as a useful tool compound for further
exploration of the biological relevance of COX-2-
dependent endocannabinoid oxygenation.
Gupta, R. A.; Simon, L. S.; Van De Putte, L. B.; Lipsky, P.
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ASSOCIATED CONTENTS
Supporting Information
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Alhouayek, M.; Masquelier, J.; Cani, P. D.;
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available free of charge via the Internet at
http://pubs.acs.org.
6
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Khasabova, I. A.; Uhelski, M.; Khasabov, S. G.;
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AUTHOR INFORMATION
Corresponding Authors
133 (18), 1989-1998.
*Md. Jashim Uddin, Ph.D., phone: 615-484-8674, e-mail:
jashim.uddin@vanderbilt.edu, and Lawrence J. Marnett,
Ph.D., e-mail: larry.marnett@vanderbilt.edu
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Funding Sources
This work was supported by research grants from (i) the
National Institutes of Health CA89450 (LJM), and (ii)
Northeastern Collaborative Access Team beamlines (funded
by NIGMS P30 GM124165, S10 RR029205) located in
Advanced Photon Source (DE-AC02-06CH11357).
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ACKNOWLEDGMENTS
Deoxy-Delta(1)(2),(1)(4)-prostaglandin J(2)-glycerol, a
putative metabolite of 2-arachidonyl glycerol and a
peroxisome proliferator-activated receptor gamma ligand,
modulates nuclear factor of activated T cells. J Pharmacol
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Spectroscopic analysis of all new molecules was conducted
in the Small Molecule NMR Facility and Mass Spectrometry
Research Center at the Vanderbilt Institute of Chemical
Biology. In addition, we are grateful to Dr. Carol A. Rouzer of
the Vanderbilt Basic Sciences for critical reading and editing
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