F. Rancati et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127975
Table 2
In vivo profile of 12 and 13. See text for definition of terms.
MABA
Dose (nmol / kg)
% inhibition of bronchoconstriction
at 1 h post dose
at 24 h post dose
M
3 (n)
β2 (n)
MABA (n)
M3 (n)
β2 (n)
MABA (n)
12
12
13
13
1.0
3.0
1.0
3.0
73 ± 7 (6)
73 ± 7 (4)
33 ± 6 (10)
78 ± 6 (7)
27 ± 8 (8)
63 ± 6 (6)
20 ± 8 (7)
67 ± 9 (6)
73 ± 11 (6)
86 ± 6 (4)
64 ± 6 (10)
91 ± 4 (8)
51 ± 7 (6)
44 ± 10 (5)
54 ± 7 (6)
20 ± 5 (6)
42 ± 2 (6)
35 ± 7 (6)
64 ± 7 (6)
71 ± 11 (5)
77 ± 8 (7)
n = number of replicates.
governed by the reduction in clogP associated with 2, and the expected
improved solubility and reduced non-specific tissue binding associated
with 5, forasmuch as permeability through cell membranes is strongly
dependent on the molecule physicochemical properties, among which
lipophilicity plays a key role. 5 reaches an adequate balance between
hydrophobicity, needed to effectively cross the hydrophobic phospho-
lipid bilayer, and hydrophilicity, necessary to achieve solubility in
aqueous body fluids before being absorbed. Unfortunately, the duration
of action previously observed with the nonyl 2 was lost upon truncating
the linker to hexyl (5).
selective β2-antagonist, ICI118,55118. The data from these experiment
is shown in Table 2.
Compound 12 showed a dose dependent inhibition of bronchocon-
striction in the β-arm of the experiment. When tested at the higher dose
(3 nmol/kg), 12 showed a balanced efficacy profile across both M3 and
β2 receptors at one hour post dosing. However, when the efficacy was
determined 24 h after dosing, the inhibition against acetylcholine-
induced bronchoconstriction was heavily biased towards the anti-
muscarinic effect.
In contrast, a clear dose response effect was observed for 13 in all
three arms of the experiment when examined one hour after dosing. A
balance between the two pharmacologies, in agreement with that pre-
viously observed in the GPT assay, was observed at this time point.
When the 24 h efficacy of 13 was determined, no significant differ-
ence in efficacy was observed between the two doses, with the indi-
vidual M3 and β2 efficacies balanced across both doses. This finding
fulfils one of our original objectives of delivering a MABA that showed a
balanced efficacy against both pharmacological targets over 24 h.
We have shown that compound 13 already displayed high plasma
protein binding, which should minimize the free levels of 13 available to
interact with either receptor. To further understand the potential sys-
temic fate of 13, the compound was profiled in a panel of in vitro ADME
assays (Table 3). Compound 13 displayed low permeability, consistent
with the high tPSA, suggesting that systemic exposure from the swal-
lowed portion of the dose would be minimal. As a surrogate for lung
stability, 13 was assessed in an isolated lung S9 assay, with 13 dis-
playing a long half-life in all three species tested. Low to modest clear-
ance was determined for 13 in both isolated microsomes and
hepatocytes.
MABAs 1 to 6 were initially prepared as a mixture of di-
astereoisomers at the benzhydryl carbon; however, the identification of
5 allowed for investigation into the effect that chirality at this position
had on the pharmacological profile of our MABAs.
The individual isomers of MABA 5 were prepared as 8 and 9 and their
profiles shown in Table 1. As expected from the proximity of the chiral
centre to the muscarinic “head-group,” a significant difference in affinity
for the M3 receptor was observed, with the (S)-isomer 9 showing a 30-
fold increase over the corresponding (R)-isomer. Less expected, due to
the distance separating the resolved stereogenic center and the β2
pharmacophore was the finding that there was a 10-fold difference in
the β2 affinity for the two isomers, with the (R)-isomer unfortunately
displaying the higher affinity. In light of this finding, further exploration
of the linker was undertaken, whilst maintaining the optimal (S)-benz-
hydrylamine isomer, since changes proximal to the β-head group would
be expected to heavily influence the β2 affinity of the newly prepared
MABAs.
Using MABA 9 as a starting point, a wide range of linkers were
prepared, with a selection shown in Table 3. No significant impact on the
affinity of the newly prepared MABAs at the M3 receptor was observed;
however, as predicted, the nature of the linker had a large effect on the
affinity profile at the β2 receptor. This finding was highlighted by the
high affinity observed with 10, which displayed a 30-fold increase in
affinity over 9. The aim of generating MABAs with high plasma protein
binding was realized with 12 and 13; unfortunately, this high binding
was at the expense of a good translation from binding affinity to func-
tional efficacy in the GPT assay.
In conclusion, we have shown that muscarinic chemotype A can be
converted, following the switch to (R)-3-quinuclinyl amine in the
muscarinic “head-group”, into MABAs that show a desired balance of
efficacy across the interactions with the muscarinic and β-adrenergic
receptors in pre-clinical in vivo models over a 24 h period, making them
suitable for a one-daily inhaled administration. This remarkably
involved, at the time, a novel means of linking the individual muscarinic
and β-adrenergic moieties through the lipophilic portion of the musca-
rinic antagonist differently from previous state-of-the-art, leading to a
groundbreaking and unexpected structural finding.
MABAs 12 and 13 were next evaluated in an in vivo model of bron-
choconstriction.29 The compounds were dosed as liquid formulation
(solution) via intra-tracheal (IT) administration to an anesthetized
guinea-pig and their impact on bronchoconstriction induced by either
acetylcholine (M3 and β2 effect) or histamine (β2 effect only) assessed
using a modified Konzett-Roessler methodology.30 In analogy with the
GPT assay, the antimuscarinic effect was assessed by challenging the
animals with acetylcholine, silencing the β2-mediated response with a
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Table 3
In vitro ADME profile of 13.
MABA
Hepatocyte clearance*
Microsome clearance†
Lung S9 stability t (min)
PPB#
H
Caco-2 permeability‡
½
H
GP
R
H
R
H
R
GP
GP
99
R
A-B
B-A
ER
13
6
17
25
16
10
216
200
70
98
98
2.0
3.1
1.5
*
μ
L/min/106 cells. †
μ
L/min/mg prot. #Plasma Protein Binding %. ‡ x 10-6 cm /sec; A-B = apical to basal; B-A = basal to apical; ER = efflux ratio. H = human; GP =
guinea pig; R = rat.
4