Identification of selective 8-(piperidin-4-yloxy)quinoline sulfone and sulfonamide histamine H1 receptor antagonists for use in allergic rhinitis

Article abstract of DOI:10.1016/j.bmcl.2017.09.020

A series of potent, selective and long-acting quinoline-based sulfonamide human H₁ histamine receptor antagonists, designed for once-daily intranasal administration for the treatment of rhinitis were developed. Sulfonamide 33b had a slightly lower affinity for the H₁ receptor than azelastine, had low oral bioavailability in the rat and dog, and was turned over to five major metabolites. Furthermore, 33b had longer duration of action than azelastine in guinea pigs, lower rat brain-penetration, and did not cause time dependent inhibition of CYP2D6 or CYP3A4. The clinical dose in humans is expected to be low (approximately 0.5 mg per day) based on the clinical dose used for azelastine and a comparison of efficacy data from animal models for 33b and azelastine.

Full text of DOI:10.1016/j.bmcl.2017.09.020

Accepted Manuscript
Identification of selective 8-(piperidin-4-yloxy)quinoline sulfone and sulfona-
mide histamine H1 receptor antagonists for use in allergic rhinitis.
Panayiotis A. Procopiou, Alison J. Ford, Paul M. Gore, Ashley P. Hancock,
Simon T. Hodgson, Duncan S. Holmes, Brian E. Looker, Sadie Vile, Kenneth
L. Clark, Ken A. Saunders, Robert J. Slack, Clarissa J. Watts
PII:
S0960-894X(17)30915-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.09.020
BMCL 25284
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
22 August 2017
8 September 2017
Please cite this article as: Procopiou, P.A., Ford, A.J., Gore, P.M., Hancock, A.P., Hodgson, S.T., Holmes, D.S.,
Looker, B.E., Vile, S., Clark, K.L., Saunders, K.A., Slack, R.J., Watts, C.J., Identification of selective 8-(piperidin-4-
yloxy)quinoline sulfone and sulfonamide histamine H1 receptor antagonists for use in allergic rhinitis., Bioorganic
& Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.09.020
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Identification of selective 8-(piperidin-4-yloxy)quinoline sulfone and sulfonamide
histamine H1 receptor antagonists for use in allergic rhinitis.
Panayiotis A. Procopiou,*a Alison J. Ford,b Paul M. Gore,a Ashley P. Hancock,a Simon T.
Hodgson,a Duncan S. Holmes,a Brian E. Looker,a Sadie Vile,a Kenneth L. Clark,b Ken A.
Saunders,b Robert J. Slack,b Clarissa J. Wattsc
a Medicinal Chemistry, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road,
Stevenage, Hertfordshire, SG1 2NY, United Kingdom
b Respiratory Biology, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road,
Stevenage, Hertfordshire, SG1 2NY, United Kingdom
c Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Medicines Research Centre,
Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom
•
•
Corresponding author. Tel.: +44 1438 790496; fax: +44 1438 768302
E-mail address: pan.a.procopiou@gsk.com (P.A. Procopiou)
Keywords: Histamine H1 receptor antagonist, allergic rhinitis, once-daily dosing, topical
application, quinoline, sulfone, sulfonamide.

Abstract
A series of potent, selective and long-acting quinoline-based sulfonamide human H1
histamine receptor antagonists, designed for once-daily intranasal administration for the
treatment of rhinitis were developed. Sulfonamide 33b had a slightly lower affinity for the
H1 receptor than azelastine, had low oral bioavailability in the rat and dog, and was turned
over to five major metabolites. Furthermore, 33b had longer duration of action than
azelastine in guinea pigs, lower rat brain-penetration, and did not cause time dependent
inhibition of CYP2D6 or CYP3A4. The clinical dose in humans is expected to be low
(approximately 0.5 mg per day) based on the clinical dose used for azelastine and a
comparison of efficacy data from animal models for 33b and azelastine
.

Introduction
Allergic rhinitis is a condition that affects a large number of people, approximately 25% of
the global population, with high prevalence in the industrialised world, and a near
quadrupling of medical care consultations over the last 50 years.1,2 Symptoms include
irritation and repetitive sneezing, rhinorrhoea, pruritus, headache, epiphora, nasal congestion,
irritation of the throat, and oedema. Nasal congestion may lead to breathing through the
mouth, snoring,3 and hyposmia.4 Allergic rhinitis is mainly treated with antihistamines and
corticosteroids,5 with H1 receptor antagonists (antihistamines) being the most frequently used
medication.6 In addition to oral antihistamines intranasal treatments, such as azelastine7 and
olopatadine (Chart 1) have gained popularity because the dose for topical treatments is
generally lower, and hence their side-effects are fewer. Treatments destined for intranasal
dosing must be delivered in a small volume, have high potency, and also have low oral
absorption because a significant portion of the dose is swallowed and becomes available for
absorption through the gastrointestinal track. Azelastine and olopatadine have comparable
efficacy and duration of action (12 h), however, both suffer from dysgeusia, headache and
epistaxis.8
Chart 1 Representative intranasal H1 receptor antagonists
Our group has published on selective histamine H3 receptor antagonists,9 on dual H1H3
antagonists,10,11 and on selective H1 antagonists.12,13 More recently we have focussed our

efforts in identifying potent and selective human H1 receptor antagonists with low oral
absorption and long duration of action, suitable for once-daily intranasal administration. Due
to allergic rhinitis’ close links to other inflammatory diseases such as allergic conjuctivitis,
rhinosinusitis and asthma, we envisaged using a novel H1 receptor antagonist in combination
with the long-acting glucocorticoid fluticasone furoate. We have very recently reported our
efforts in identifying phthalazinone 1 as a preclinical candidate for rhinitis, which fulfils all
of the above requirements.13 In this publication we describe our efforts in identifying another
candidate as a back-up to 1, which is derived from a non-phthalazinone scaffold.
Azelastine which has a phthalazinone core has a bitter taste and we wished to avoid this
problem, if possible. We considered starting our investigations from the 8-(piperazin-1-
yl)quinoline scaffold 2 (Figure 1),11 however, we opted for the 8-(piperidin-4-yloxy)quinoline
scaffold 3 that we briefly examined previously as part of our dual H1H3 antagonist project.
Scaffold 3 was slightly less potent at the histamine H1 receptor, however, it was significantly
more selective than equivalent piperazines across a range of aminergic GPCRs, particularly
α1A. We were also interested in the introduction of the strongly electron-withdrawing sulfone
or sulfonamide groups in substituent R in order to reduce the basicity of the piperidine amino
group of 3, and concurrently reduce any hERG channel liability associated with strongly
basic and lipophilic compounds. Our strategy was to optimise potency by investigating the
chain-length between the piperidine nitrogen and the sulfone/sulfonamide groups and also the
substituent on these groups. We considered that a compound with H1 receptor affinity close
to that of azelastine was a good target to aim for in order to achieve the small volume – low
dose requirement for topical administration. Increasing the duration of action to twenty-four
hours was hoped to be achievable from SAR optimisation of analogues with duration in vitro
of at least as long as azelastine.
Figure 1 Scaffolds 2 and 3

Chemistry
The synthesis of target sulfones commenced from 6-bromo-8-fluoroquinoline 4 with the
introduction of the C6 substituent using a selective Suzuki reaction to provide the cross-
coupled product 5 as outlined in Scheme 1. The Suzuki reaction utilised tributylborane and
was catalysed by [1,1’-bis(diphenylphosphino)ferrocene palladium (II)] chloride
[Pd(dppf)Cl2] to give 5 in 67% yield. Fluoride displacement with the alkoxide of N-Boc-4-
hydroxypiperidine in N-methylpyrrolidone (NMP) provided ether 6 in 81% yield, which was
then deprotected with TFA to give the piperidine 7 in quantitative yield. This compound was
a common intermediate for the preparation of all target sulfones and sulfonamides. The ethyl
sulfone with the two-carbon chain 8 was obtained in 70% yield by heating 7 with ethyl vinyl
sulfone in DMF at 100°C under microwave irradiation. The analogous ethyl sulfone with the
three-carbon chain 9a was prepared in 61% yield from 7 and the tosylate 10 in the presence
of NaI, NaHCO3 in DMF at 100°C. The tosylate 10 was prepared from commercially
available 3-(ethylthio)propanol 11 which was converted to the tosylate 12 (24% yield) and
then oxidised with mCPBA to provide 10 in 99% yield. Alternatively, compound 9a and the
homologues n-Pr, iso-Pr and tert-Bu sulfones 9b-d were prepared by alkylating 7 in a similar
way (NaI, NaHCO3 in DMF at elevated temperature) using the halides 13, which in turn were
made from 1-bromo-3-chloropropane 14 by reaction with the appropriate sodium thiolate in
DMF, followed by mCPBA oxidation of the resulting sulfide 15 to the corresponding sulfone.
The halides 13 and 15 were obtained as mixtures of chlorides and bromides (variable ratios

from 2:1 to 2:3), and were used without any further purification. The four-carbon tert-butyl
sulfone 16 was made from 7 and the bromide 17 using the same alkylation conditions (NaI,
NaHCO3, DMF, 150°C, microwave irradiation) in 46% yield. The bromide 17 was prepared
from 1,4-dibromobutane 18 and tert-butyl thiolate to give sulfide 19 (21% yield), which was
then oxidised to the sulfone (71% yield). Finally, the branched four-carbon chain ethyl
sulfone 20 was prepared from 7 and the mesylate 21. The mesylate 21 synthesis commenced
with LiAlH4 reduction of commercially available ethyl ester 22 to give the alcohol 23
(quantitative yield), convertion to mesylate 24 (92% yield), and finally oxidation to sulfone
(95% yield). The racemic sulfone 20 was resolved using preparative HPLC on a Chiralpak
AD column eluting with 15% ethanol-heptane containing 0.1% trifluoroacetic acid. The
enantiomer eluting first off the column was labelled 20a, and the enantiomer eluting last was
labelled 20b.
Scheme 1 Synthesis of sulfones 8, 9a-d, 16, 20a and 20b

Reagents and Conditions: i) n-Bu3B solution in THF, Pd(dppf)Cl2, DMF, 75°C, 67%; ii) N-
Boc-4-hydroxypiperidine, tert-BuONa, NMP, 140°C, 81%; iii) TFA, DCM, 100%; iv) ethyl
vinyl sulfone, NaHCO3, DMF, microwave, 100°C, 15 min, 70%; v) TsCl, pyridine, 24%; vi)
m-CPBA, DCM; vii) RSNa, (R=Et-, n-Pr-, iso-Pr-, tert-Bu-), DMF; viii) LiAlH4, THF,
100%; ix) MsCl, DCM, 0°C, 92%.
The sulfonamide series were prepared from intermediate 7 which was alkylated with 2-
phthalimidoethyl bromide, 3-(Boc-amino)propyl bromide and 4-(Boc-amino)butyl bromide to
give the protected amines 25, 26 and 27 in 70, 78 and 94% yield respectively (Scheme 2).
Amine 25 was deprotected with hydrazine monohydrate to give the amine 28 (100%),

whereas 26 and 27 were deprotected by treatment with HCl to give 29 and 30 in 76 and 88%
yield respectively. The amines 28 and 29 were sulfonylated with ethanesulfonyl chloride to
give 31 and 32 (38 and 67% yield respectively). The butylamine 30 was similarly treated
with a number of sulfonyl chlorides to give sulfonamides 33a-f. The sulfonamide 33b was
alkylated with methyl iodide in the presence of sodium hydride to give the N-
methylsulfonamide 34.
Scheme 2. Synthesis of sulfonamides 31, 32, 33a-f and 34.
n-Bu
n-Bu
n-Bu
i
iii
ii
N
N
7
N
O
O
O
O
N
N
NH2
NHSO2Et
N
N
28
31
25
O
n-Bu
n-Bu
NH2
n-Bu
iii
v
iv
N
N
N
7
O
O
O
N
NHBoc
N
NHSO2Et
N
26
32
29
n-Bu
vi
n-Bu
n-Bu
v
vii
7
N
N
N
O
O
O
N
N
N
NHBoc
NH2
NHSO2R
30
27
n-Bu
33
a = Me
=
R
= Et
= n-Pr
= iso-Pr
= iso-Bu
= c-C6H11
b
c
d
e
f
viii
N
33b
O
N
SO2Et
N
34

Reagents and Conditions: i) 2-phthalimidoethyl bromide, K2CO3, 2-butanone, 80°C, 70%;
ii) NH2NH2.H2O, EtOH, 80°C, 100%; iii) EtSO2Cl, Et3N, DCM, 38% for 31 and 67% for 32;
iv) 3-(Boc-amino)propyl bromide, K2CO3, 2-butanone, 80°C, 78%; v) 4M HCl, dioxane,
76% for 29 and 88% for 30; vi) 4-(Boc-amino)butyl bromide, K2CO3, 2-butanone, 80°C,
94%; vii) RSO2Cl, Et3N, DCM, 36% for 33a, 53% for 33b, 32% for 33c, 39% for 33d, 18%
for 33e, 23% for 33f; viii) NaH, MeI, DMF, 52%.
The reverse sulfonamide analogues were prepared by alkylation of piperidine 7 with 2-
chloro-N-(1,1-dimethylethyl)ethanesulfonamide 35, 3-chloro-N-(1,1-dimethylethyl)-1-
propanesulfonamide 36 and 4-chloro-N-propyl-1-butanesulfonamide 37 to give 38, 39 and 40
respectively. The alkylating agents 35, 36 and 37 were prepared by treatment of the
commercially available chloroalkylsulfonyl chlorides with one equivalant of tert-butylamine
or n-propylamine.
Scheme 3 Synthesis of reverse sulfonamides 38, 39 and 40

Reagents and Conditions: i) NaI, K2CO3, DMF, 4-33%
Results and discussion
The H1 receptor affinity of compounds was evaluated in vitro using recombinant human
histamine H1 receptor in intact CHO cells which provided apparent pA2 values. The hERG
activity was measured in [3H]-dofetilide radioligand binding assay, and the data from all the
above screens are summarised in Table 1. Azelastine was used as a reference compound and
exhibited high affinity for both the H1 receptor (pA2 = 9.7) and the hERG channel (pIC50 =
7.0). Similarly, the data for our phthalazinone candidate 1 is also included in Table 1 for
comparison (pA2 = 9.7 and hERG pIC50 = 6.4). The experimental details on the assays were
reported in our earlier publications.9,10 Duration of action in vitro was determined in the
CHO cell assay by incubation with antagonist for 30 min, followed by washing, and then by
repeat histamine challenges at intervals of 90 and 270 min at 37°C. The duration of action in
vitro for these analogues is expressed as faster, slower or no-difference wash-out time relative
to azelastine. Slower wash-out equated to longer duration of action than azelastine, whereas
faster wash-out to shorter duration.
The ethyl sulfones with the two-carbon (8) and branched four-carbon (20) chains had lower
affinity for the H1 receptor (Table 1), however, sulfone 8 which was the least basic compound
tested (measured pKa 6.5) had also the lowest hERG affinity (pIC50 5.8). The three-carbon
linked sulfones were more potent than the other chains tested. The terminal alkyl group of
these sulfones did not influence the H1 activity with the ethyl (9a), iso-propyl (9c) and tert-
butyl (9d) sulfones being equipotent (pA2 9.5, 9.4 and 9.6 respectively), and also maintaining
lower affinity for the hERG channel (pIC50 7.1, 7.0 and 6.3 respectively). The duration of
action in vitro for 9a was longer than azelastine, whereas for 9c and 9d was the same, making
all three compounds worthy of further investigation in pharmacokinetic studies. The

lipophilic (clogP = 4.4) four-carbon chain tert-butyl sulfone 16 was less potent than the
analogous three-carbon chain sulfone 9d (pA2 8.85) and was rejected, although its hERG
affinity was low (pIC50 6.5) and its duration of action not different from azelastine.
For the sulfonamide series the optimal linker was the four-carbon chain when comparing the
ethylsulfonamides 31, 32 and 33b (pA2 8.7, 8.9 and 9.3 respectively). Investigation of the
sulfonamide nitrogen substituent in the four-carbon chain series (33a-f) identified three
potent analogues 33a-c. Compound 33a was however rejected due to its higher hERG
activity. The calculated pKa for analogues 33a-f was 8.42, however a drop in the hERG
activity was observed with increasing substitution from the methyl 33a to isopropyl
analogues 33d and then an increase with increasing lipophilicity for the isobutyl 33e and
cyclohexyl 33f analogues. The duration of action in vitro of sulfonamide 33c was longer than
azelastine’s, whereas for 33b was similar. Both 33b and 33c were progressed for further
investigation. The H1 affinity of the N-methyl sulfonamide 34 was reduced slightly by
comparison to 33b and was rejected.
The reverse sulfonamides 38-40 with the two-, three- and four-carbon linker chains had lower
H1 receptor affinity and were also rejected. The hERG activity of 38, which was less basic
than both 39 and 40, was lower. The more branched tert-butyl 39 had lower hERG activity
than the n-propyl analogue 40, despite the fact that both 39 and 40 had the same basicity
(calc. pKa 8.1).
Pharmacokinetic studies
All animal studies were ethically reviewed and carried out in accordance with Animals
(Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of
Animals. Experimental procedures for the PK studies in vivo and rat CNS penetration were
reported in our earlier publications and outlined in the suplementary information section.9,10

From the three sulfones and two sulfonamides selected for further investigation 9a, 33b and
33c had low CNS penetration in the rat, assessed in samples taken 5 min after an intravenous
bolus dose of 1 mg/kg (mean brain concentration of 62, 26 and 41 ng/g of tissue
respectively), and low brain-blood ratios (0.09, 0.10 and 0.16 respectively). Sulfone 9d had
higher levels of concentration in the brain 133 ng/g (presumably due to its higher
lipophilicity, clogP 4.7), and a higher brain-blood ratio of 0.77 and was therefore rejected.
The sulfones (9a and 9c) had higher systemic exposure in the rat after oral administration at 3
mg/kg (mean AUC0-last 568 and >361 h.ng/mL respectively) than the sulfonamides (33b and
33c, mean AUC0-last 62 and 63 h.ng/mL respectively). The sulfones 9a and 9c were therefore
rejected as the aim of this project was to identify compounds with low systemic exposure,
suitable for intranasal dosing. Sulfonamide 33b having the lower AUC was progressed to
pharmacokinetic studies in vivo in both the male CD Sprague Dawley rat and Beagle dog and
the data are shown in Table 2. The compound was dosed as a solution in
H2O−PEG200−DMSO (50:45:5) at 1 mg/kg (both species) iv, at 3 mg/kg for the rat po, and 1
and 2 mg/kg for the dog po studies. In addition one dog was also dosed subcutaneously. The
blood clearance was moderate compared to liver blood flow (means of 38 and 20 mL/min/kg
in rat and dog respectively) and the volume of distribution was also moderate (means of 4.2
and 4.5 L/kg in rat and dog respectively). The resultant elimination half-life was moderate
(means of 1.8 and 3.8 h in rat and dog respectively). Elimination of parent compound via
urine was low following oral and intravenous administration of 33b to the dog (<0.1%
following oral administration and <1% following intravenous administration). Following
oral administration of 33b bioavailability was low using low doses (9 and 5% for rat and dog
respectively). Rat absorption of 33b into the hepatic portal vein following oral administration
was low (13 to 16% using a 3 mg/kg dose). Sulfonamide 33b was also dosed orally to rats at
10 mg/kg using a hydroxypropylmethyl cellulose formulation. The animals dosed at 10

mg/kg showed a proportional increase in Cmax compared to the 3 mg/kg data and
bioavailability was estimated to be 4%. Following subcutaneous administration to the rat
bioavailability was estimated to be 45%. The in vitro plasma protein binding for 33b was
determined using an ultrafiltration technique and was found to be high across all species
tested (means of 97.9, 91.1, 97.7 and 98.2% for rat, guinea pig, dog and human respectively
at 10 µg/mL). Plasma protein binding did not appear to be concentration dependent across
the three concentrations tested, 0.5 or 1 µg/mL and 10 µg/mL. The in vitro blood cell
binding was low to moderate at nominal concentrations of 1 and 10 µg/mL in all species
tested (means of 27, 42, 49 and 23% for rat, guinea pig, dog and human respectively at 1
µg/mL). The in vitro rate of metabolism for 33b was moderate using rat and human liver
microsomes (2.9 and 1.4 mL/min/g tissue respectively); moderate to high using dog liver
microsomes (3.6 mL/min/g) and high using mouse liver microsomes (3.7 mL/min/g). The rat
and dog in vitro microsomal clearance data is in keeping with the in vivo clearance data for
rat and dog. Incubation of 33b with cryopreserved human hepatocytes resulted in extensive
metabolism of the compound with at least 35 metabolites being formed. Five major
metabolites, labelled M11, M15, M19, M24 and M26 were observed, which were identified
by mass spectrometry as M11 and M15 hydroxylation products on the C6-butyl group
(M+16), M19 oxidation product of either M11 or M15 (M+14), M24 N-dealkylation of the
piperidine nitrogen (M-163) and M26 oxidative N-dealkylation of the sulfonamide nitrogen
(M-77). Sulfonamide 33b had an acceptable profile (IC50 >4 µM) against human CYP450
enzymes (CYP1A2, >100 µM; CYP2C9, 31 µM; CYP2C19, >40 µM; CYP2D6, 15 µM and
CYP3A4, 4 µM). Furthermore, 33b did not cause time-dependent inhibition with CYP2D6
or CYP3A4 (less than two-fold change in IC50 values over a 30 min incubation).
The MDCK permeability of 33b was low (52 nm/s; n=3), however, the compound was found
to be a P-glycoprotein (PgP) substrate. The brain levels of 33b determined 30 min post a 300

mg/kg oral dose in rat were found to be very high (14,480 ng/g) with high brain-blood ratio
of 11.0. This increase in brain penetration observed at higher doses is thought to be a
consequence of 33b being a PgP substrate.
Effect of 33b on histamine-induced nasal congestion in guinea pigs.
We have used the previously reported Buxco whole body plethysmography technique to
investigate the effect of intranasally dosed 33b on histamine-induced nasal congestion in
conscious, unrestrained guinea pigs, which were previously sensitised with ovalbumin and
aluminum hydroxide intranasally over a 3 week period prior to the study (further information
in SI section). Recording PenH (enhanced pause) AUC over 40 min following bilateral
histamine challenge (10 mM, 25 µL/nostril, under light isoflurane anaesthesia) allows for the
assessment of efficacy and duration of action of intranasally dosed antihistamines.13,14
Significant inhibition of histamine-induced congestion, compared to a vehicle-
pretreated/histamine challenged control group, at both 3 and 24 h after intranasal
administration of a 1 mg/mL solution of 33b (25 µL/nostril). In contrast, azelastine failed to
show a similar duration of action when administered at the same concentration (Figure 2).
Arterial blood samples taken immediately following evaluation of nasal congestion (4 or 25 h
after intranasal dosing) show negligible systemic exposure following 1 mg/mL 33b,
suggesting a low bioavailability by this route and a local mechanism of action. A separate
study was performed in order to investigate the onset of action in vivo. Significant inhibition
of histamine-induced congestion 1 h after intranasal administration of a 1 mg/mL solution (25
µL/nostril, data not shown) which suggests that an early onset time could be clinically
achievable. Neither 33b nor azelastine produced a pro-congestant response at any time-point
studied (effect on baseline PenH – data not shown).

Figure 2.
Duration of action of 33b and azelastine in a conscious guinea pig
model of histamine-induced nasal congestion. Animals were exposed to histamine at
the time indicated after an intranasal dose of 1 mg/mL 33b or azelastine. Mean
SEM (n = 11-22 per group as indicated). (*p<0.05 compared to time-matched histamine
control group; # p<0.05 compared to vehicle/PBS control group. Bar indicates p<0.05
individual comparison as indicated. ANOVA with post-hoc Hochberg analysis)
Predicted human dose
The clinical dose of 33b is expected to be appoximately 0.5 mg per day, based on the clinical
dose used for azelastine and a comparison of efficacy data from animal models for 33b and
azelastine. Using the low oral bioavailability in rat and dog, moderate blood clearance and a
low clinical dose the systemic exposure in humans is expected to be very low. The
concentration of 33b was less than 7 nM (3 ng/mL) in all assayed blood samples taken from
guinea pigs showing a pharmacological response in the histamine induced nasal congestion
model. The pharmacokinetic profiles in preclinical species following intravenous
administration were used to give estimates of the maximum systemic exposure in humans if

most of the intranasal dose was swallowed and bioavailability was complete. A standard
absorption rate was assumed, the volume of distribution was kept constant and the
elimination rate was scaled based on liver blood flow. Using these simulations the maximum
estimates for exposure in humans following a 0.5 mg dose are a total drug Cmax of 0.89
ng/mL and 0.99 ng/mL (based on rat and dog data respectively) and an AUC0-24 of 10.5
h.ng/mL and 7.4 h.ng/mL (based on rat and dog data respectively).
Specificity profiling of 33b
The specificity profiles for both 33b and azelastine were evaluated in the CEREP Specificity
Screen at 50 different receptors, ion channels and transporters). Off-target activity was
observed with 5-HT2B (antagonist, pKi 6.1), 5-HT2C (antagonist, pKi 5.7), α1A (antagonist, pKi
5.7) and M2 (antagonist, pIC50 6.2). The data for azelastine against these targets were 5-HT2B
(antagonist, pKi 7.7), 5-HT2C (antagonist, pKi 6.3), α1A (antagonist, pKi 7.3) and M2
(antagonist, pIC50 5.2). For all of these targets relative to histamine H1 receptor (pA2=9.3)
33b shows >1000-fold specificity. As mentioned earlier 33b also showed significant affinity
(pIC50 7.1) for the hERG channel, having a similar potency to azelastine (pIC50 = 7.0) in the
hERG dofetilide binding assay. Evaluation in rabbit hearts in vitro (SCREENIT model)
suggests that the risk of 33b-induced QT prolongation is low at therapeutically relevant
predicted exposure levels.
The mutagenic and clastogenic potential of 33b was assessed in the AMES test, Mouse
Lymphoma screen and DEREK-structure activity relationship in silico analysis). No
evidence of mutagenic, clastogenic or toxic potential was detected in these assays up to the
maximum concentrations tested.
Conclusion

A series of quinoline sulfone and sulfonamide human H1 histamine receptor antagonists were
prepared using a four- and six-step linear synthesis respectively. The sulfone series although
highly potent were orally absorbed and were not progressed any further. In contrast the
sulfonamides were not orally absorbed, and had low brain penetration. The optimal chain-
length between the piperidine nitrogen and the sulfonamide group was the four-carbon chain,
and the optimal sulfonamide nitrogen substituents were methyl, ethyl or n-propyl group. The
ethyl analogue 33b had slightly lower potency than azelastine, however, it had longer
duration of action in a nasal congestion model in vivo. The compound was metabolised in
human hepatocytes to 5 major metabolites, and at least another 30 minor ones. The main
routes of metabolism were oxidation in the butyl substituent (hydroxylation and oxidation to
ketone), and N-dealkylation of the piperidine or sulfonamide nitrogens. Sulfonamide 33b
was selective for the H1 receptor over 50 other different receptors, ion channels and
transporters with >1000-fold selectivity, and did not have any issues with P450 inhibition. It
showed similar affinity to azelastine for the hERG channel and evaluation in rabbit hearts in
vitro suggested that the risk of 33b-induced QT prolongation is low at therapeutically
relevant predicted exposure levels. The predicted dose for humans is 0.5 mg per day. 33b
may have increased brain penetration in human if administered with a PgP inhibitor. The low
clinical dose and low bioavailability reduces the potential for significant clinical effects. In
summary, 33b is suitable for progression as a back-up to our phthalazinone intra-nasal
candidate for the treatment for allergic rhinitis.
Acknowledgements: We thank Alasdair J Carey, Kam Gohil and David Gomez Ulloa for
technical assistance, Anne Jones for the metabolite identification work, Christopher
Browning and Linda J. Russell for the duration studies.
Conflicts of Interest: none

Supplementary Information
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2017. These include experimental details for the synthesis
of compounds 5, 6, 7, 27, 30, 33b and 33b.2HCl, and for the pharmacokinetic and
pharmacodynamic studies.

Table 1. Antagonist pA2 Affinity at the Human H1 Receptor, in vitro Duration, dofetilide
hERG binding affinity, calculated logP and pKa, and measured pKa for the piperidine nitrogen
e
Compounda
Wash-
outc
hERG
pIC50
(n)
clogPd
cpKa
pKa
pA2 SEMb
(n)
Ref.
S
4.0
3.7
3.9
4.0
4.5
4.3
4.7
4.4
4.3
4.3
4.4
4.5
3.8
4.3
4.8
4.6
8.9
7.3
6.7
6.9
6.9
6.9
6.9
7.0
7.2
7.2
7.2
8.0
8.4
8.4
8.4
8.4
Azelastine
1
9.7 0.1
(19)
7.0 0.0
(114)
9.7 0.1
(20)
6.4 0.0
(16)
8
-
6.5
8.43 0.01
(2)
5.78 0.01
(2)
S
7.9
9a
9.46 0.04
(13)
7.12 0.04
(5)
-
-
9b
9.13 0.06
(3)
7.54 0.04
(4)
ND
ND
ND
-
-
9c
9.4 0.1
(16)
7.04 0.02
(4)
8.0
9d
9.58 0.09
(6)
6.33 0.03
(5)
16
-
8.85 0.08
(12)
6.52 0.01
(4)
-
20a
20b
31
8.7 0.2
(4)
6.7 0.0
(2)
-
-
8.6 0.2
(4)
6.88 0.02
(2)
ND
-
-
8.68 0.08
(13)
7.12 0.03
(4)
8.5
32
8.9 0.4
(2)
7.2 0.1
(2)
33a
33b
33c
33d
ND
ND
S
-
-
-
-
9.4 0.1
(10)
7.7 0.2
(6)
9.27 0.08
(18)
7.11 0.02
(9)
9.7 0.2
(16)
7.06 0.02
(6)
-
8.7 0.2
(3)
6.89 0.05
(4)

S
5.3
5.8
4.6
5.1
5.2
4.9
8.4
8.4
8.4
7.2
8.1
8.1
-
-
-
-
-
-
33e
33f
34
9.08 0.08
(13)
7.36 0.00
(2)
-
9.0 0.2
(3)
7.74 0.06
(4)
-
9.0 0.2
(3)
6.6 0.1
(4)
F
38
8.7 0.1
(8)
6.20 0.04
(4)
ND
-
39
9.2 0.1
(14)
6.55 0.03
(4)
40
9.0 0.1
(3)
7.02 0.07
(4)
a
All compounds were tested as dihydrochloride salts except for 9d, 38 and 40 which were
mono formate salts, 16 which was a diformate salt, azelastine was the hydrochloride salt, and
1 was the free base, b All pA2 values calculated from curve shifts generated at 30 min
incubation time and at 100 nM antagonist concentration. Table 1 shows mean pA2 SEM
for n<3 the SEM is the SD. n = number of experiments, c the duration of action in vitro for
these analogues is expressed as faster (F), slower (S) or no-difference (ND) wash-out time
relative to azelastine, d calculated logP value from Biobyte v4.3, e calculated pKa from
Chemaxon v5.4.1.1

Table 2. Pharmacokinetic parameters for 33b.2HCl in the rat and dog.
CD Rat
(n=4 iv, n=3 po)
Beagle Dog
(n=4 iv, n=4 po)
Species
Clb (mL/min/kg) (% LBF)
Vss (L/kg)
38 (45)
4.2
20 (65)
4.5
T1/2 (h) (IV)
1.8
9a,b
3.8
5c
Oral bioavailability (%)
Calculated fraction of oral dose
absorbed.
13 to 16%
a 33b was also dosed sub-cutaneously to one rat at 2 mg/kg formulated in DMSO-PEG 200-
Water (5:45:50). Bioavailability was determined to be at least 45% and the Cmax was
approximately six-fold higher than achieved following oral administration at 3 mg/kg in the
same study.
b 33b was also dosed orally to 3 rats at 10 mg/kg formulated in 0.5% (hydroxylpropylmethyl
cellulose). Bioavailability was determined to be at least 4%, the terminal phase was not
sufficiently well defined to give a more accurate value.
c Mean of data from n=2 dosed at 1 mg/kg and n=2 dosed at 2 mg/kg.

References
1.
Bousquet J, Van Cauwnberge P, Khaltaev N. Allergic rhinitis and impact on asthma.
J Allergy Clin Immunol. 2001: 108 (5 Suppl); S147-334.
2.
Greiner AN, Hellings, PW, Rotiroti G, et al. Allergic rhinitis. The Lancet, 2011: 378;
2112-2122.
3.
Mandhane SN, Shah JH, Thennati R. Allergic rhinitis: An update on disease, present
treatments and future prospects. Int Immunopharm. 2011: 11; 1646-1662.
4.
Berger WE. Overview of allergic rhinitis. Ann. Allergy Asthma Immunol. 2003: 90;
7-12.
5.
Bernstein DI, Schwartz G, Bernstein, JA. Allergic rhinitis. Mechanisms and
treatment. Immunol Allergy Clin N Am. 2016: 36; 261-278.
6. Simons FER, Simons KJ. Histamine and H1-antihistamines: Celebrating a century of
progress J Allergy Clin. Immunol. 2011: 128; 1139-1150.
7. Lee C, Corren J. Review of Azelastine nasal spray in the treatment of allergic and
non-allergic rhinitis. Exp Opin Pharmacother. 2007: 8; 701-709.
8.
Hoyte FCL, Katial RK. Antihistamine therapy in allergic rhinitis. Immunol Allergy
Clin N Am. 2011: 31; 509-543.
9.
Procopiou, PA, Ancliff RA, Bamford MJ, Browning C, Connor H, Davies S, Fogden
YC, Hodgson ST, Holmes DS, Looker, BE, Morriss KLM, Parr NA, Pickup EA, Sehmi SS,
White GV, Watts CJ, Wilson DM, Woodrow MD. 4-Acyl-1-(4-aminoalkoxyphenyl)-2-
ketopiperazines as a Novel Class of Non-Brain-Penetrant Histamine H3 Receptor
Antagonists. J Med Chem. 2007: 50; 6706–6717.

10.
Procopiou PA, Browning C, Buckley JM, Clark, KL, Fechner L, Gore PM,
Hancock AP, Hodgson ST, Holmes DS, Kranz M, Looker BE, Morriss KLM, Parton
DL, Russell LJ, Slack RJ, Sollis SL, Vile S, Watts CJ. The discovery of phthalazinone
based human H1 and H3 single-ligand antagonists suitable for intranasal administration for the
treatment of allergic rhinitis. J Med Chem. 2011: 54; 2183-2195.
11.
Procopiou PA, Ancliff RA, Gore PM, Hancock AP, Hodgson ST, Holmes DS,
KeelingSP, Looker BE, Parr NA, Rowedder JE, Slack RJ. The discovery of quinoline based
single-ligand human H1 and H3 receptor antagonists. Bioorg Med Chem Lett. 2016: 26; 5855-
5859.
12.
Procopiou PA, Browning C, Gore PM, Lynn SM, Richards SA, Slack RJ, Sollis SL.
Synthesis and pharmacological investigation of azaphthalazinone human histamine H1
receptor antagonists. Bioorg Med Chem. 2012: 20; 6097-6108.
13.
Procopiou PA, Ford AJ, Gore PM, Looker BE, Hodgson ST, Holmes DS, Vile S,
Clark KL, Saunders KA, Slack RJ, Rowedder JE, Watts CJ. Design of phthlazinone amide
histamine H1 receptor antagonists for use in rhinitis. ACS Med Chem Lett. 2017: 8; 577-581.
14.
Slack RJ, Russell LJ, Hall DA, Luttmann MA, Ford AJ, Saunders KA, Hodgson ST,
Connor HE, Browning C, Clark KL. Pharmacological characterization of GSK1004723, a
novel long-acting antagonist at histamine H1 and H3 receptors. Br J Pharmacol. 2011: 164;
1627-1641.

Graphical Abstract
Identification of selective 8-(piperidin-4-yloxy)quinoline sulfone and sulfonamide histamine H1 receptor
antagonists for use in allergic rhinitis.
Panayiotis A. Procopiou,*a Alison J. Ford,b Paul M. Gore,a Ashley P. Hancock,a Simon T. Hodgson,a Duncan S.
Holmes,a Brian E. Looker,a Sadie Vile,a Kenneth L. Clark,b Ken A. Saunders,b Robert J. Slack,b Clarissa J.
Wattsc
Selective human H1 histamine receptor antagonist pA2 = 9.3 with 24 h duration of action