Synthesis and evaluation of various heteroaromatic benzamides as analogues of –ylidene-benzamide cannabinoid type 2 receptor agonists

Article abstract of DOI:10.1016/j.tetlet.2019.151019

The CB₂ receptor is an attractive target for the treatment of a wide range of diseases and pathological conditions. Compounds that selectively activate the CB₂ receptor are desirable as this avoids CB₁-mediated psychoactive effects. Heteroarylidene-benzamides have demonstrated efficacy as selective CB₂ receptor agonists. We aimed to expand the structure-activity relationship studies of this series of compounds by investigating the heteroaromatic core via the synthesis and in vitro evaluation of a small library of various heteroaromatic benzamide analogues. As heteroaromatic amides are privileged scaffolds in drug design, methods to synthesise them are of interest. Concise and reliable synthetic strategies were developed to access these novel analogues. The –ylidene-benzamide moiety is shown to be essential for CB activity as all amide derivatives exhibit no functional activity at either CB₂ or CB₁ receptors.

Full text of DOI:10.1016/j.tetlet.2019.151019

Journal Pre-Proof
Synthesis and Evaluation of Various Heteroaromatic Benzamides as Analogues
of –Ylidene-Benzamide Cannabinoid Type 2 Receptor Agonists
Michael Moir, Rochelle Boyd, Hendra Gunosewoyo, Andrew P. Montgomery,
Mark Connor, Michael Kassiou
PII:
S0040-4039(19)30776-2
DOI:
https://doi.org/10.1016/j.tetlet.2019.151019
TETL 151019
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 June 2019
1 August 2019
5 August 2019
Please cite this article as: Moir, M., Boyd, R., Gunosewoyo, H., Montgomery, A.P., Connor, M., Kassiou, M.,
Synthesis and Evaluation of Various Heteroaromatic Benzamides as Analogues of –Ylidene-Benzamide
Cannabinoid Type 2 Receptor Agonists, Tetrahedron Letters (2019), doi: https://doi.org/10.1016/j.tetlet.
2019.151019
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Synthesis and Evaluation of Various Heteroaromatic
Benzamides as Analogues of Ylidene-Benzamide
Cannabinoid Type 2 Receptor Agonists
Michael Moir,a Rochelle Boyd,b Hendra Gunosewoyo,c Andrew P. Montgomery,a
Mark Connor,b and Michael Kassiou*a
a School of Chemistry, The University of Sydney, NSW 2006 Australia
b Department of Biomedical Sciences, Macquarie University, NSW 2109, Australia
c
School of Pharmacy and Biomedical Sciences, Curtin University, WA, 6102,
Australia
Corresponding Author
#E-mail: michael.kassiou@sydney.edu.au. Phone: +612 9351 2745
The authors declare no competing financial interest
1

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Abstract
The CB2 receptor is an attractive target for the treatment of a wide range of diseases
and pathological conditions. Compounds that selectively activate the CB2 receptor are
desirable as this avoids CB1-mediated psychoactive effects. Heteroarylidene-
benzamides have demonstrated efficacy as selective CB2 receptor agonists. We aimed
to expand the structure-activity relationship studies of this series of compounds by
investigating the heteroaromatic core via the synthesis and in vitro evaluation of a
small library of various heteroaromatic benzamide analogues. As heteroaromatic
amides are privileged scaffolds in drug design, methods to synthesise them are of
interest. Concise and reliable synthetic strategies were developed to access these
novel analogues. The ylidene-benzamide moiety is shown to be essential for CB
activity as all amide derivatives exhibit no functional activity at either CB2 or CB1
receptors.
Keywords: Cannabinoid receptor, CB2, heterocyclic chemistry, synthesis.
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Introduction
Since the identification of the cannabinoid (CB) receptors and their endogenous
ligands, there has been great interest in the endocannabinoid system and its regulatory
functions in health and disease. The cannabinoid type 1 (CB1) receptor is found in
high levels in the central nervous system, but also in a number of peripheral tissues,
while the cannabinoid type 2 (CB2) receptor is expressed primarily on cells of the
immune system such as astrocytes and microglia.1 The CB2 receptor has become a
promising target for a number of diseases that contain an inflammatory component.2
One of the caveats of cannabinoid-based drug design is that activation of the CB1
receptor results in undesirable psychoactive effects. Therefore there is a need to
develop compounds that selectively activate the CB2 receptor to avoid CB1 mediated
side effects. There have been a number of potent and selective CB2 receptor agonists
developed by academic laboratories and pharmaceutical companies.3 Despite this, no
drugs that target the CB2 receptor have progressed through clinical trials and there is
still an unmet need to develop novel CB2 receptor agonists.4
A class of compounds originally developed by Taisho Pharmaceuticals Co. Ltd.5-6 and
then Abbott Laboratories7-8 are heteroarylidene-benzamide derivatives such as 1.
These compounds are potent agonists of the CB2 receptor and generally exhibit no
measureable activity at the CB1 receptor. Extensive structure-activity relationship
studies (SARs) of the pendant groups are summarised in Figure 1, A.8 We aimed to
expand on the minimal SAR focused on the core of this class of compounds by
modifying the heteroaromatic unit. As the –ylidene-benzamide moiety is only
applicable to certain heterocycles we decided to explore isosteric amides. In light of
results from previous SAR studies,8-9 simplified pendent groups (achiral
tetrahydropyran and 4-trifluoromethylbenzamide) were also chosen (Figure 1, B).
The study aimed to investigate the number, position and identity of heteroatoms as
well as the size of the aromatic ring. Synthetic strategies have been developed to
access these compounds, many of which do not have reliable established syntheses,
and their in vitro activity at the cannabinoid receptors evaluated.
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Figure 1: (A) Summary of SAR of heteroarylidene-benzamide derivatives such as 1; (B) Strategy to
explore the heterocyclic core of known heteroarylidene-benzamide agonists (2-9).
Results and Discussion
Synthesis Heteroaromatic Amides
The synthesis of lead compound 1 followed modified procedures reported by Carroll
and co-workers (Scheme 1).8 Starting with commercially available chiral alcohol,
(R)-(tetrahydrofuran-2-yl)methanol, a Mitsunobu reaction using di-Boc protected
hydrazine as the nucleophile afforded the requisite protected monosubstituted
hydrazine 10. Deprotection under acidic conditions followed by condensation with
4,4-dimethyl-3-oxopentanenitrile yielded amino-pyrazole 11. Amide coupling via the
requisite acid chloride and methylation under neutral conditions using methyl triflate
afforded pyrazolylidene-benzamide derivative 13. Finally, a nucleophilic aromatic
substitution reaction provided the lead compound 1 in good overall yield.
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Scheme 1: Synthesis of lead compound 1. Reagents and conditions: (a) Di-tert-butyl hydrazine-1,2-
dicarboxylate, PPh3, di-tert-butyl azodicarboxylate, THF, 0 °C-rt, 16 h, 72%; (b) i) HCl, dioxane, rt, 16
h, ii) 4,4-dimethyl-3-oxopentanenitrile, EtOH, reflux,
4
h, 75%; (c) i) 2-Fluoro-5-
(trifluoromethyl)benzoic acid, oxalyl chloride, DMF, CH2Cl2, rt, 3 h, ii) (iPr)2EtN, CH2Cl2, rt, 4 h,
67%; (d) MeOTf, MePh, 100 °C, 16 h, 72%; (e) 2-Methylpropane-1,2-diol, KOtBu, 0 °C-rt, 2 h, 60%.
The synthesis of the heteroaromatic amide derivatives (2-9) was achieved via two
main approaches: either direct synthesis of the requisite amino-heterocycle followed
by amide coupling, or synthesis of the corresponding carboxylic acid followed by a
Curtius rearrangement and quenching of the isocyanate with an aryl Grignard reagent
to afford the desired heteroaromatic benzamides.
Pyrroles are notoriously capricious heterocycles and are prone to undergo degradation
under a variety of conditions. The presence of an electron-donating group on an
already electron-rich nucleus makes 2-aminopyrroles unstable unless the ring is
substituted with electron withdrawing groups.10 The known instability of electron-rich
pyrroles meant that access to the requisite 2-aminopyrrole was not possible. An
alternative approach was taken whereby a Curtius rearrangement of a stable pyrrole-
2-carboxylic acid precursor could afford the challenging molecular synthon.
Protection of freshly distilled pyrrole with a benzene sulfonyl group allowed
regioselective aluminium chloride mediated Friedel-Crafts alkylation to install the
tert-butyl group at the 3-positon (Scheme 2).11 Removal of the protecting group using
magnesium under ultrasonic irradiation,12 followed by alkylation with tetrahydro-2H-
pyran-4-yl)methyl tosylate afforded N-alkylpyrrole 16. Reaction with trichloroacetyl
chloride followed by hydrolysis under basic conditions gave the 2-substituted
carboxylic acid 18. The amide was formed via a Curtius rearrangement using a three-
step procedure. Formation of the acyl azide with diphenylphosphoryl azide followed
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by heating at 90 °C for 1.5 hours gave clean conversion to the corresponding
isocyanate. The isocyanate was quenched with 4-trifluoromethylmagnesium chloride
to afford the desired amide 2 in moderate yield (45% over 3 steps).
Scheme 2: Synthesis of pyrrole derivative 2. Reagents and conditions: (a) Bu4NHSO4, benzene
sulfonyl chloride, NaOH (aq.), CH2Cl2, rt, 24 h, 99%; (b) t-BuCl, AlCl3, CH2Cl2, rt, 2 h, 81%; (c) i)
Mg, NH4Cl, MeOH, 1 h, ii) NaH, Bu4NBr, tetrahydro-2H-pyran-4-yl)methyl tosylate, DMF, 0-50 °C,
16 h, 52%; (d) Trichloroacetyl chloride, pyridine, THF, rt, 6 h, 97%; (e) NaOH (aq.), MeOH, reflux, 16
h, 92%; (f) i) DPPA, Et3N, CH2Cl2, rt, 16 h, ii) MePh, 90 °C, 1.5 h, iii) (4-
(Trifluoromethyl)phenyl)magnesium chloride, 0 °C-rt, 1 h, 45%.
Pyrazole derivative 3 was synthesised via amino pyrazole 22 (Scheme 3). Mono-
substituted hydrazine 21 was by synthesised by hydrazone formation from tetrahydro-
2H-pyran-4-carbaldehyde and tert-butyl carbazate followed by sodium
cyanoborohydride reduction and Boc group deprotection.13 Reaction of the hydrazine
hydrochloride salt 21 and 4,4-dimethyl-3-oxopentanenitrile afforded 5-aminopyrazole
22. Finally, amide coupling with 4-trifluoromethylbenzoyl chloride yielded the
desired amide 3.
Scheme 3: Synthesis of pyrazole derivative 3. Reagents and conditions: (a) t-Butyl carbazate, MgSO4,
MeOH, rt, 16 h, 63%; (b) NaCNBH3, TsOH.H2O, THF, rt, 16 h, 87%; (c) HCl, dioxane, MeOH rt, 16
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h, 100%; (d) 4,4-Dimethyl-3-oxopentanenitrile, EtOH, reflux, 16 h, 52%; (d) i) 4-
Trifluoromethylbenzoic acid, oxalyl chloride, DMF, CH2Cl2, rt, 4 h, ii) (iPr)2EtN, CH2Cl2, rt, 16 h,
72%.
Imidazole derivative 4 was synthesised via amino imidazole 25 (Scheme 4).
Bromination of pinacolone gave α-bromoketone 23 which was added slowly to an
excess of 4-methanamine tetrahydropyran at -78 °C to afford the secondary amine 24
in moderate yield.14 Condensation with cyanamide gave the requisite 2-
aminoimidazole 25, which was converted to amide 4 via an acid chloride amide
coupling.
Scheme 4: Synthesis of imidazole derivative 4. Reagents and conditions: (a) Br2, MeOH, -40 °C-rt, 30
min, 83%; (b) 4-methanamine tetrahydropyran, diethyl ether, -78 °C-rt, 16 h, 43%; (c) Cyanamide,
EtOH, reflux, 16 h, 94%; (d) 4-Trifluoromethylbenzoic acid, oxalyl chloride, DMF, CH2Cl2, rt, 4 h, ii)
iPr2EtN, CH2Cl2, rt, 16 h, 65%.
In a similar fashion triazole 5 was synthesised via the amino species 28 (Scheme 5).
A Pinner reaction with pivalonitrile afforded the imidate salt which reacted with
cyanamide to form cyanoimidate 27 under buffered conditions.15 Reaction with
hydrazine 21 afforded the poorly nucleophilic 5-aminotriazole 28, which required
deprotonation with sodium hydride and elevated temperatures to react with 4-
trifluoromethylbenzoyl chloride to yield amide 5.
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Scheme 5: Synthesis of triazole derivative 5. Reagents and conditions: (a) EtOH, AcCl, 0 °C-rt, 16 h,
79%; (b) Cyanamide, Na(OH)2PO2.H2O, Na2HPO4.7H2O, MeCN, rt, 72 h, 51%; (c) Hydrazine 21,
DBU, MeOH, reflux, 16 h, 78%; (d) 4-Trifluoromethylbenzoic acid, oxalyl chloride, DMF, CH2Cl2, rt,
4 h, ii) NaH, MePh, reflux, 3 h, 75%.
Oxazole 6 and thiazole 7 could be formed from a common intermediate 34 (Scheme
6). A Horner-Wadsworth-Emmons olefination of dihydro-2H-pyran-4(3H)-one with
2-oxopropyl dimethyl ester phosphonic acid followed by reaction of the enolate with
Mander’s reagent afforded the β-ketoester 31 after selective hydrogenation of the
alkene under hydrogen transfer conditions.16 Oxime formation using sodium nitrite
followed by hydrogenation under acidic conditions afforded the stable amine
hydrochloride salt which reacted cleanly with pivalic anhydride to yield the amide
common intermediate 34. Cyclodehydration of the β-ketoamide using
triphenylphosphine, iodine and triethylamine17 afforded substituted oxazole 35 while
the thiazole 36 was formed by reaction with Lawesson’s reagent under elevated
temperatures.18 Basic ester hydrolysis followed by reaction with diphenylphosphoryl
azide afforded the requisite acyl azides, which underwent Curtius rearrangements at
elevated temperatures. Quenching with the appropriate Grignard reagent afforded
amides 6 and 7.
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Scheme 6: Synthesis of oxazole and thiazole derivatives 6 and 7. Reagents and conditions: (a) KOH,
2-oxopropyl dimethyl ester phosphonic acid, EtOH, 0 °C-rt, 5 h, 95%; (b) LDA, methyl cyanoformate,
THF, -78 to -40 °C, 1 h, 58%; (c) NH4HCO2, Pd/C, MeOH, rt, 3 h, 95%; (d) i) NaNO2, AcOH, H2O, -5
°C-rt, 2 h, ii) HCl, Pd/C, H2, EtOH, rt, 16 h, iii) Pivalic anhydride, Et3N, CH2Cl2, rt, 16 h; (e) PPh3, I2,
Et3N, CH2Cl2, rt, 3 h, to give 35 60% over 4 steps; (f) Lawesson’s reagent, THF, reflux, 3 h, to give 36
40% over 4 steps; (g) NaOH (aq.), THF, MeOH, rt, 4 h, 78-95%; (h) i) DPPA, Et3N, CH2Cl2, rt, 16 h,
ii) MePh, 90 °C, 1.5 h, iii) (4-(Trifluoromethyl)phenyl)magnesium chloride, 0 °C-rt, 1 h, 22-30%.
For the synthesis of phenyl derivative 8 it was envisioned that the tetrahydropyran
group could be incorporated by cross-coupling chemistry (Scheme 7). Bromination of
commercially available 4-(tert-butyl)aniline, amide coupling and finally a Miyaura
borylation afforded cross-coupling partner 41. A Suzuki cross-coupling with 4-
(bromomethylene)tetrahydro-2H-pyran (42), which was formed by a Wittig-type
reaction of dihydro-2H-pyran-4(3H)-one with (bromomethyl)triphenylphosphonium
bromide,19 achieved the key carbon-carbon bond formation. Hydrogenation of the
alkene gave the final product 8.
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Scheme 7: Synthesis of phenyl derivative 8. Reagents and conditions: (a) NBS, DMF, 0 °C-rt, 16 h,
78%; (b) 4-Trifluoromethylbenzoic acid, oxalyl chloride, DMF, CH2Cl2, rt, 4 h, ii) (iPr)2EtN, CH2Cl2,
rt, 16 h, 75%; (c) KOAc, Pd(dppf)Cl2, (Bpin)2, MePh, 90 °C, 16 h, 79%; (d) i) CH2Br2, PPh3, MePh,
reflux, 16 h, ii) KOtBu, THF, -78 ° to -40 °C, 3 h, 37%; (e) K2CO3, Pd(PPh3)4, DMF, H2O, 80 °C, 4 h,
92%; (f) Pd/C, H2, EtOAc, 16 h, 86%.
tert-Butyl-substituted aminopyridine 45 is not readily available and hence required
synthesis to access pyridine derivative 9 (Scheme 8). Reaction of Boc-protected 2-
amino-5-bromopyridine with an excess of tert-butyl magnesium chloride and copper
cyanide afforded the desired alkylated product 44 in low yield.8 Unlike in the case of
the phenyl derivative 8, it was found that the Suzuki cross-coupling reaction was most
successful when the coupling partners were reversed and performed before the amide
was formed. Optimised conditions using Pd(dppf)Cl2 and aqueous potassium
hydroxide as the base yielded the final product 9 after hydrogenation of the alkene.
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Scheme 8: Synthesis of pyridine derivative 9. Reagents and conditions: (a) t-BuMgCl, CuCN, THF, -
78 °C-rt, 16 h, 19%; (b) TFA, CH2Cl2, rt, 2h, 100%; (c) NaOAc, Br2, AcOH, rt, 3 h, 69%; (d) t-BuLi,
isopropyl pinacol borate, THF, -78 °C-rt, 2.5 h, 63%; (e) KOH (aq.), Pd(dppf)Cl2, THF, 60 °C, 16 h,
69%; (f) (i) 4-Trifluoromethylbenzoic acid, oxalyl chloride, DMF, CH2Cl2, rt, 4 h, ii) NaOH (aq.),
CH2Cl2, 0 °C-rt, 2 h, iii) Bu4N, THF, rt, 16 h, 87%; (g) Pd/C, H2, EtOAc, 16 h, 91%.
The amide analogue of the lead compound 1 could be accessed from previously
synthesised intermediate 12 by a simple nucleophilic aromatic substitution reaction
using methylpropane-1,2-diol to afford 50 (Scheme 9).
Scheme 9: Synthesis of amide derivative of lead compound 50. Reagents and conditions: (a) 2-
Methylpropane-1,2-diol, KOtBu, 0 °C-reflux, 4 h, 72%.
Functional activity at the CB1 and CB2 receptors
The heteroaromatic amides were assessed for their ability to activate the CB1 and CB2
receptors using a Fluorescence Imaging Plate Reader (FLIPR) membrane potential
assay in AtT-20 cells expressing human CB1 or CB2 receptors as previously
described.20-21 The assay measures activation of endogenously expressed G protein-
gated inwardly rectifying K+ channels (GIRKs) at the CB1 or CB2 receptors.22
Compounds displaying more than 40% activation at 10 μM in the assay were
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evaluated further in dose−response studies. Their half maximal effective
concentrations (EC50) and maximal effect relative to high efficacy CB1/CB2 receptor
agonist CP 55,940 (Emax),23-24 which produced a maximal decrease in fluorescence at
a concentration of 1 μM, corresponding to cellular hyperpolarization, were calculated
as listed in Table 1. Further experimental details are provided in the Supplementary
Information.
The lead pyrazoylidene-benzamide 1 exhibited potent and selective activation of the
CB2 receptor as expected. About a ten-fold decrease in potency was observed
compared to the data reported for rat CB2 receptor activation (Figure 1, A).
Unfortunately, the heteroaromatic amide derivatives displayed no activity at the either
of the CB receptors. Pyrrole derivative 2 was the only exception, possessing
micromolar activity at CB2. We consequently decided to investigate the structural
basis for the observed loss of activity. We assumed the dramatic loss of activity was
due to either substitution of the –ylidene-benzamide functionality for a benzamide or
the particular combination of pendant groups used. We therefore synthesised the
direct amide analogue of lead compound 1 with identical pendant groups to probe the
importance of the linkage between the heterocycle and substituted phenyl group.
Surprisingly amide 50 exhibited no functional activity at both cannabinoid receptors,
confirming the –ylidene functional group is key for functional activity at the CB2
receptor for this class of compounds. It is possible that amide 50 and its simplified
analogues 2-9 may still bind to the CB2 receptor but not have functional activity in the
FLIPR membrane potential assay. To investigate the molecular basis for the differing
functional activity at the CB2 receptor of lead compound 1 and direct amide analogue
50 we performed docking simulations. For the docking study we used the first and
only reported crystal structure of the CB2 receptor, recently published by Li and co-
workers.25 A caveat to the simulations performed is that the inactive cannabinoid
receptor structure, solved with an antagonist bound, is not ideal for predicting agonist
interactions. Similar docking scores and near identical binding poses were observed
for both –ylidene-benzamide 1 and amide 50 (see Supplementary Information, Figure
S1). More in depth molecular dynamics and mutagenesis studies would be required to
elucidate the molecular basis for the lack of functional activity of amide 50 despite its
structural similarity to the lead compound 1.26 This finding has led us to further
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explore the subtle SAR of this class of compounds, the results of which will be
disclosed in due course.
Table 1: Agonist activities of lead compound (1) and heteroaromatic amide derivatives (2-9 and 50) in
AtT-20 cells expressing human CB1 or CB2 receptors by a FLIPR membrane potential assaya
CB1
pEC50 ± SEM
CB2
pEC50 ± SEM
Compound
Emax (%)
Emax (%)
(EC50 nM)
(EC50 nM)
1
2
NA
NA
ND
ND
7.72 ± 0.08 (19)
93
45
5.61 ± 0.24
(2470)
3
NA
ND
ND
ND
ND
ND
ND
ND
ND
99
NA
ND
ND
ND
ND
ND
ND
ND
ND
101
4
NA
NA
5
NA
NA
6
NA
NA
7
NA
NA
8
NA
NA
NA
NA
9
50
NA
NA
CP 55,940
7.67 ± 0.04 (21)
7.50 ± 0.05 (32)
aSee Supplementary Information for more details. Data represent mean values ± SEM from at least
three independent experiments each performed in duplicate, with CP 55,940 used as a positive control.
c
bNA: Not active, defined as <40% activation at 10 μM in the assay. ND: Not determined; for
compounds defined as not active, their maximal effects were not determined.
Conclusion
We report herein the design and synthesis of a small library of heteroaromatic amides
as proposed CB2 receptor agonists. Heteroaromatic amides are privileged scaffolds in
drug design and the synthetic strategies developed here offer concise and reliable
methods to access these products. The absence of functional activity at the
cannabinoid receptors observed for these compounds has highlighted the importance
of the –ylidene-benzamide moiety within this series of compounds. Preliminary
docking studies were unsuccessful in determining the molecular basis for the lack of
functional activity of the amide derivatives in this study. We have therefore refocused
our efforts on further investigating the –ylidene-benzamide moiety and why it is
crucial for functional activity.
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Acknowledgements
This work was supported in part by the National Health and Medical Research
Council (APP1107088).
Appendix A. Supplementary data
Supplementary data to this article can be found online
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JOURNAL PRE-PROOF
25.
Li, X.; Hua, T.; Vemuri, K.; Ho, J.-H.; Wu, Y.; Wu, L.; Popov, P.; Benchama, O.;
Zvonok, N.; Locke, K. a.; Qu, L.; Han, G. W.; Iyer, M. R.; Cinar, R.; Coffey, N. J.; Wang,
J.; Wu, M.; Katritch, V.; Zhao, S.; Kunos, G.; Bohn, L. M.; Makriyannis, A.; Stevens, R.
C.; Liu, Z.-J., Crystal Structure of the Human Cannabinoid Receptor CB2. Cell
2019, 176 (3), 459-467.
26.
Feng, Z.; Alqarni, M. H.; Yang, P.; Tong, Q.; Chowdhury, A.; Wang, L.; Xie, X.
Q., Modeling, molecular dynamics simulation, and mutation validation for
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J. Chem. Inf. Model. 2014, 54 (9), 2483-99.
Highlights
 Synthetic routes to access heterocyclic benzamides are developed.
 Functional activity at the cannabinoid receptors is evaluated.
 Ylidene benzamide moiety is determined to be imperative for activity.
16