J. J. Harnett et al. / Bioorg. Med. Chem. Lett. 25 (2015) 88–91
89
chemical series to generate potent antioxidants.16,23,24 The BHT
pharmacophore ring is depicted in red in Figure 1. This novel lead
therefore takes advantage of the combination of its potential
antioxidant activity with the hCB2 ligation. Compound 1 thus
represents a powerful lead for further optimization. Since the lead
1 already includes a BHT moiety, our optimization program was
firstly set up to focus on the tetrahydrothiopyran, the amine sub-
stitution and the 5-membered heterocycle replacement. Hence
the BHT antioxidant moiety was kept constant. Compounds 1–11
were prepared according to the synthetic pathway shown in
Scheme 1.
our optimization program by introducing a range of different
spirocyclic entities to replace the THTP moiety. When the THTP
of 1 is replaced by a spirocyclic tetrahydropyranyl (R1,R2 = THP,
3), binding is improved by 2.8 fold while selectivity is retained.
The 6-membered ring THP (3), is favoured over a 5-membered tet-
rahydrofuran (THF) ring in 4 (13 fold loss in hCB2 binding).
Having determined the best spirocyclic moiety (R1,R2 = THP, 3)
we turned our attention to the importance of the dimethyl tertiary
(3°) amine moiety. Table 1 shows the trend of hCB2 binding activ-
ity, clearly favouring the 3°-amines, where the 3° (3, 7) >2° (6) >>1°
(5b). Replacing the nitrogen by other heteroatoms leads to a
decrease in hCB2 binding (results not shown). Heterocyclic amines
such as piperidine (8) and morpholine (9) provided selective hCB2
ligands accompanied by a loss in affinity for the receptor, however
less marked for the lipophilic piperidine.
Keeping the 3° amine and the THP moiety constant the azole
ring was modified and the importance of the hydroxyl group (R5)
on the aromatic ring examined. Replacing the thiazole in 3 by an
oxazole20 10, was accompanied by a slight loss (ꢀ1.8 fold) in selec-
tivity and binding. On the other hand, the presence of a hydroxyl
group (R5) between the two bulky tert-butyl substituants was
found to be critical to the hCB2 affinity, 3 being 4 times more active
than 11. Characterization of the pharmacology of this novel class of
cannabinoid ligands was carried out on the most potent com-
pounds (hCB2<100 nM), Table 2.
Readily available N-Fmoc protected
a-amino acids were con-
verted to the corresponding amides using HOBT-NH3.18 The amides
were then treated with phosphorous pentasulphide in dimethoxy-
ethane under basic conditions to yield the corresponding
thioamides.
The BHT antioxidant moiety was introduced in the following
manner, the thioamides and amides reacted with the 2,6-di-tert-
butyl bromoacetophenone, to afford, respectively, the thiazoles
(X = S) 1a–9a, 11a and the oxazole analog 10a (X = O). With the
antioxidant pharmacophore in place, the Fmoc protecting groups
of oxazole & thiazoles 1a–11a were removed under basic condi-
tions using diethylamine to afford the corresponding primary
(1°) amines 1b–11b. The secondary (2°) and tertiary (3°) aliphatic
amine 1–4, 6–7, 10–11 were prepared from the corresponding 1°
amines using either the Eschweiler–Clarke reaction under micro-
wave conditions19 or reductive amination using either sodium
borohydride or sodium triacetoxyborohydride.
As can be seen from Table 2, compounds tested 1, 3, 7, 8 and 10
showed selective full agonist activity for the hCB2 receptor versus
hCB1. Compound 3 which has the same affinity (Table 1) as the ref-
erence compound AM124121 for hCB2 behaved slightly differently
in the functional assay. Although, 3 has an EC50 for hCB2 2.6 times
greater than AM1241 (Table 2), in the end they both displayed the
same level of selectivity ratio, 64 versus 63.
The bioassay used exploits the negative coupling of the hCB
receptor to adenylate cyclase (AC), the assay measures the inhibi-
tion of forskolin stimulated cAMP production.15
When –N(R3R4) is a piperidine (8) or morpholine (9) these com-
pounds were prepared from the 1° amine 5b reacting with the suit-
able di-bromides via an intermolecular–intramolecular one-pot
nucleophilic di-substitution reaction to provide the desired cyclic
compounds.
A potentially metabolic soft spot in lead compound 1 is the
presence of the tetrahydrothiopyranyl (THTP) spirocyclic ring
system which may be prone to oxidative metabolism. However,
as can be seen from Table 1, removal of the THTP system, com-
pound 2, results in a 76 fold loss in binding. We therefore began
This chemical series was built around the BHT moiety and
compounds possessing this chemical fragment were systemically
demonstrated by our group to possess potent antioxidant
O
O
S
H
N
X
H
N
v
H
N
ii
5
H
N
i
iv
R
HO
Fmoc
H2N
Fmoc
N
R1 R2
H2N
Fmoc
R1 R2
Fmoc
R1 R2
R1 R2
1a-11a
Amides
Thioamides
Acids
iii
R3
N
X
R5
X
N
R5
vi
NH2
R1 R2
R4
N
1
R
R2
1b-11b
1-7
10, 11
vii
S
Y
HO
N
N
O
8: Y= CH2
9: Y= O
Scheme 1. Synthetic pathway25 for compounds 1–11.
Harnett, Jeremiah J.
