The Discovery of LML134, a Histamine H3 Receptor Inverse Agonist for the Clinical Treatment of Excessive Sleep Disorders

Article abstract of DOI:10.1002/cmdc.201900176

Histamine H3 receptor (H3R) inverse agonists that have been in clinical trials for the treatment of excessive sleep disorders, have been plagued with insomnia as a mechanism-based side effect. We focused on the identification of compounds that achieve high receptor occupancy within a short time, followed by rapid disengagement from the receptor, a target profile that could provide therapeutic benefits without the undesired side effect of insomnia. This article describes the optimization work that led to the discovery of 1-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)piperidin-4-yl 4-cyclobutylpiperazine-1-carboxylate (18 b, LML134).

Full text of DOI:10.1002/cmdc.201900176

Accepted Article
Title: The discovery of LML134, a histamine H3 receptor inverse
agonist for the clinical treatment of excessive sleep disorders
Authors: Thomas Troxler, Dominik Feuerbach, Xuechun Zhang,
Charles Yang, Bharat Lagu, Mark Perrone, Tei-Lin Wang,
Karin Briner, Mark Bock, and Yves P. Auberson
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To be cited as: ChemMedChem 10.1002/cmdc.201900176
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ChemMedChem
FULL PAPERS
DOI: 10.1002/cmdc.200((will be filled in by the editorial staff))
The discovery of LML134, a histamine H3
receptor inverse agonist for the clinical
treatment of excessive sleep disorders
Thomas Troxler,[a] Dominik Feuerbach,[a] Xuechun Zhang,[b] Charles R. Yang,[c] Bharat
Lagu,[d] Mark Perrone,[d] Tie-Lin Wang,[b] Karin Briner,[d] Mark G. Bock,[d] Yves P.
Auberson*[a]
Histamine 3 receptor (H3R) inverse agonists that have been in clinical
trials for treatment of excessive sleep disorders, have been plagued
with insomnia as a mechanism based side effect. We focussed on
identifying compounds that achieve high receptor occupancy within a
short time, followed by fast disengagement from the receptor, a target
profile that could provide the therapeutic benefits without the undesired
insomnia side effect. The article describes the optimization work that
led to the discovery of 1-(1-methyl-6-oxo-1,6-dihydropyridazin-3-
yl)piperidin-4-yl 4-cyclobutylpiperazine-1-carboxylate (18b, LML134).
with a high frequency of side effects, from nausea to anxiety,
hallucinations and anaphylaxis. In the United States, it is classified
as a schedule IV controlled substance due to its addictive potential.
Finally, sodium oxybate (γ-hydroxybutyrate, GBH) is an effective
alternative.[8] Due to its short half-life, it must however be dosed
twice nightly. It is also associated with numerous unwanted side
effects, including suicidal ideation and depression, and has a black
box warning related to risks resulting from excessive central
nervous system depression. So far, these treatments are all
associated with substantial residual sleepiness, and no drug works
in all patients. There is still a strong medical need for improved
treatments, both in terms of efficacy and safety.
Introduction
Excessive sleepiness is often simply the consequence of not
getting enough sleep. It is frequently a result of lifestyle choices, or
due to work and other activities interfering with normal sleeping
periods. Independently, underlying diseases affecting sleep quality
such as depression, sleep apnea, restless legs syndrome or
narcolepsy induce the same condition. Additionally, narcolepsy is
associated with sudden episodes of muscle weakness (cataplexy)
and an abnormal sleep architecture.[1] This disease is thought to
be autoimmune in nature, T cell-mediated, and is associated with
a selective loss of hypothalamic orexin-secreting neurons.[2]
Autoreactive lymphocytes have recently been detected in the blood
and cerebrospinal fluid of narcoleptic patients, both as orexin-
specific CD4+ (helper) and CD8+ (killer) T cells,[3] strongly
reinforcing the assumption of an autoimmune origin of narcolepsy.
The biological effect of histamine and its relationship to
narcolepsy are well understood.[9] Orexin cells in the hypothalamus
are responsible for the sleep/wake cycle: they activate
histaminergic neurons in the tubero-mamillary nucleus, which
release histamine towards histamine receptors on excitatory
neurons, increasing wakefulness (Scheme 1). The released
histamine also diffuses out of the synapse, reaching extra-synaptic
H3 receptors (H3R). These provide a feedback loop that limits
release of excessive histamine.
During the day, wakefulness is maintained by activating
neurotransmitters such as noradrenaline, dopamine, orexin and
histamine. Therefore several treatments targeting their associated
receptors were developed to increase wakefulness.[4] Initially,
narcolepsy has been treated with amphetamines. These act on
noradrenergic and dopaminergic circuits and are effective, but
often lead to unwanted side effects and development of
tolerance.[5] They were later replaced by modafinil as first-line
treatment. This drug has multiple mechanisms of action and is
effective in 60-80% of patients.[6] Among others, modafinil acts on
the dopaminergic system and reduces the GABAergic outflow from
the ventrolateral preoptic area (VLPO). The VLPO is one of the
diurnal pacemakers and projects both to orexinergic neurons and
to the wake center in the tubero-mamillary nucleus (TMN).[7] The
net result is a decrease of inhibition on the TMN, thereby promoting
arousal. While safer than amphetamines, modafinil is associated
[a]
[b]
Dr. Y. P. Auberson, Dr. T. Troxler, Dr. D. Feuerbach
Novartis Institutes for BioMedical Research
Novartis Pharma AG, Klybeckstrasse 141, 4057 Basel, Switzerland
E-mail: yves.auberson@novartis.com
Dr.X.Zhang, Dr. C. Yang, Dr. T.-L. Wang
ChemPartner, 998 Halei Road, Zhangjiang Hi-Tech Park Pudong
New Area, Shanghai China, 201203
[c]
[d]
Dr. C. Yang, ShangPharma Innovation Inc., 280 Utah Avenue,
South San Francisco, CA 94080, USA.
Dr. B. Lagu, Dr. M. Perrone, Dr. K. Briner, Dr. M. G. Bock
Novartis Institutes for BioMedical Research
Novartis Pharma AG, 250 Massachusetts Avenue, Cambridge,
MA 02139, United States
1
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10.1002/cmdc.201900176
ChemMedChem
In narcolepsy patients, orexin cells degenerate and fail to
stimulate histaminergic neurons adequately, resulting in decreased
histamine release and insufficient activation of excitatory neurons.
This leads to increased sleepiness, and to narcolepsy symptoms.
We initially explored ergoline derivatives as H3R inverse
agonists,[12] and showed that their use was limited by low brain
penetration. Further optimization and opening of the core structure
led to the discovery of a series of indole-based inverse agonists.
These efforts ultimately led to compound 2,[14] which was
considered a promising candidate for further development until
evaluation of its in vitro rate of metabolism across species indicated
a significantly slower clearance in human liver microsomes (T
=
½
50 min), compared to rat liver microsomes (T = 8.2 min). We
½
presumed that the efficacy of 2 might not be short enough for
treating excessive sleepiness without causing insomnia on the
following night. We discontinued its development in favor of the
drug candidate described hereafter.
The lead for a completely new series of inverse agonists,
compound 3, was selected from the patent literature.[15] This
molecule has a high affinity for H3R (Table 1) and reasonable drug-
like properties (MW = 395.5, PSA = 85 Å2, logP = 3.3). It
unfortunately suffers from a very limited brain penetration (rat
brain/blood ratio = 0.09, one hour after 10 mg/kg p.o.), and of
Figure 1. Wakefulness control in the healthy brain. HA: histamine; H1R:
histamine receptor type 1; TMN: tubero-mamillary nucleus; PH: posterior
hypothalamus.
potential cardiovascular toxicity (hERG IC50
=
11 µM).
Nevertheless, 3 was deemed an interesting starting point, and a
chemistry program was initiated that initially focused on opening its
5,6,7,8-tetrahydro-1,6-naphthyridin core.
Understanding this biological pathway also defines a treatment
option for narcolepsy: H3R have high levels of constitutive activity
and their functional level can therefore be decreased using H3R
inverse agonists. This mechanism reduces H3R signaling and
consequently limits inhibition of histamine release, increasing
synaptic histamine concentration, and correcting the weak signal
generated by dysfunctional orexin cells. Interestingly, H3 receptors
are not only present at neurons that control arousal: they are also
found at neurons that direct motor control, explaining why
cataplectic attacks are also reduced by H3 inverse agonists.
The optimization and characterization of novel derivatives built
on the experience acquired with the previous series of H3R inverse
agonists.[12,14] We profiled new drug candidates by simultaneous
determination of their pharmacokinetic (PK) properties,
pharmacodynamic (PD) effect, and brain receptor occupancy (RO).
In particular, we used changes in brain tele-methylhistamine
(tMeHA) and RO levels one hour after oral administration as
indicators of oral absorption, brain penetration, and receptor
kinetics. We then studied the time course of target engagement in
more detail for the most promising compounds. Receptor
occupancy is easily translatable to human pharmacology, using
values determined in patients by positron emission tomography
(PET). Several clinically validated imaging agents allowing H3R
occupancy determination have been described[16] and are available
for clinical use. It is known that achieving a RO > 80% is required
for a robust increase of waking activity.[17]
Clinical trials with pitolisant (1, Figure 2), a high-affinity H3R
inverse agonist, validated this principle as a therapeutic approach.
These studies confirmed that elevating brain histaminergic
neurotransmission enhances wakefulness and decreases
cataplexy in narcolepsy patients.[10] Pitolisant has been approved
for the treatment of narcolepsy, however with a plasma half-life in
humans of 11h, it has too long a duration of action and causes
insomnia during the night following administration.[11]
We defined the target profile for our potential drug candidate
as having rapid pharmacokinetics, leading to a short duration of
action with a high initial H3R RO (>80%), and a residual occupancy
below 20%, six hours after oral dosing at 10 mg/kg in rats.
O
N
N
O
N
N
Cl
2
1
O
NH2
Results and Discussion
N
N
N
3
O
N
Chemistry
Figure 2. Structure of pitolisant (1), optimized indole 2 and lead compound 3
Reaction of 6-chloronicotinamide with tert-butyl 4-aminopiperidine-
1-carboxylate and deprotection afforded amine 4 (Scheme 1),
which was transformed into carbamate 5 using a suitably
functionalized chloroformate. In an alternative sequence, 6-
chloronicotinamide was reacted with piperidinol to yield alcohol 6.
The carbamate functionality was then introduced using
carbonyldiimadazole and the desired amine substituent, leading to
compounds 8 and 9a-e.
A number of alternative candidates have been evaluated
clinically. They all display too long a plasma half-life,[12] and too
long a duration of receptor occupancy, leading to insomnia.[13] Our
aim was thus to identify a safe and efficacious H3R inverse agonist
with rapid receptor disengagement, achieving therapeutic efficacy
without mechanism-related unwanted effects.
2
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10.1002/cmdc.201900176
ChemMedChem
O
Scheme 2. Reagents and conditions: a) Piperidin-4-ol, Bu4NI, K2CO3, DMF, 140°,
3 h, 71%; b) triphosgene, DMAP, 1-isopropylpiperazine, CH2Cl2, RT, 5 h, 54%;
c) LiOH, H2O2, H2O, RT, 5 h, 27%; d) triphosgene, DMAP, 1-isopropylpiperazine,
CH2Cl2, RT, 5 h, 99%; e) TFA, CH2Cl2, RT, 5 h (100%); f) 2-(benzyloxy)-5-
bromopyridine, t-BuOK, BINAP, Pd2(dba)3 , toluene, 120°, 16 h, 20%; g) H2, Pd/C,
MeOH, RT, 10 min, 94%.
NH2
O
O
N
N
O
a, b
NH2
c
O
N
N
H
NH2
N
N
4
5
Cl
N
H2N
d
O
Finally, pyridazinone derivatives of pyridinone 15 were
prepared from 6-chloro-pyridazin-3(2H)-ones. Initial coupling with
4-hydroxypiperidine led to 16a,b (Scheme 3). These were either
transformed into 4-isopropyl (17a,b) or 4-cyclobutyl piperazine
carbamates (18a,b) using methods similar to those described
above. Compound 18a was further used to prepare derivatives
19a,b using sodium hydride and 2-iodoisopropane or iodoethane,
respectively.
O
O
NH2
NH2
f
NH2
O
N
N
O
N
N
N
N
e
HN
N
O
N
O
HO
7
8
6
N
h
O
g
NH2
9a R=iPr
O
N
N
9b R=cPr
9c R=cBu
9d R=cPent
O
N
O
O
R
b, c
or d
N
N
O
R
R
O
N
N
R
a
9e R=CH
2CH2OH
N
N
N
N
N
O
N
Cl
N
HO
16a R=H
18a R=H
Scheme 1. Reagents and conditions: a) tert-butyl 4-aminopiperidine-1-
carboxylate, DIPEA, MeCN, rflx, 48 h, 98%; b) HCl/EtOAc, RT, 4 h, 94%; c) 1-
isopropylpiperidin-4-ol, 4-nitrophenyl chloroformate, Et3N, CH2Cl2, 0° to RT, 16h;
then 4, Et3N, DMF, RT, 48 h, 11%; d) piperidin-4-ol, DIPEA, 110°, 8 h, 75%; e)
carbonyldiimidazole, THF, 65°, 24 h, quant.; f) 4-amino-1-isopropylpiperidine, N-
methylmorpholine (NMM), N-hydroxysuccinimide (NHS), THF, 65°, 19 h, 55%; g)
carbonyldiimidazole, NHS, 1-alkylpiperazine, NMM, THF, 65°, 63% for 9a, 34%
for 9b; h) NHS, 1-alkylpiperazine, NMM, THF, 65°, 53% for 9c, 56% for 9d, 41%
for 9e.
R=Me
16b
18b R=Me
g or h
e or f
19a
19b
(R = Et)
(R = iPr)
O
R
N
O
N
N
N
O
N
17a
17b
R=H
R=Me
Scheme 3. Reagents and conditions: a) 4-Hydroxypiperidine, n-butanol, 140°,
214 h, 67% for 16a, 41% for 16b; b) 4-nitrophenyl chloroformate, DIPEA, pyridine,
30°, 2 h, 46%; c) 1-cyclobutylpiperazine, Et3N, CH2Cl2, 30°, 2 h, 93%; d)
carbonyldiimidazole, N-hydroxysuccinimide, N-cyclobutylpiperazine dihydro-
chloride, NMM, CH2Cl2, RT, 42 h, 85%; e) 4-nitrophenyl chloroformate, DIPEA,
pyridine, 30°, 2 h, then 1-isopropylpiperazine, Et3N, CH2Cl2, 30°, 2 h, 4%; f)
carbonyldiimidazole, NHS, N-isopropylpiperazine, NMM, THF, 65°, 138 h, 31%;
g) NaH, DMF, RT, 1 h, then 2-iodopropane, RT, 1 h, 52%; h) NaH, DMF, RT, 1
h, then iodoethane, RT, 1 h, 33%.
Picoline amide derivative 12 was prepared from 5-
chloropicolinonitrile, using a similar reaction as above to introduce
the piperidinol ring, yielding 10 (Scheme 2). Compound 10 was
subsequently transformed into a carbamate with triphosgene and
1-isopropylpiperazine, then hydrolyzed into amide 12. Pyridinone
15 was obtained from Boc-protected piperidin-4-ol and 1-
isopropylpiperazine using triphosgene, followed by deprotection,
coupling to 2-(benzyloxy)-5-bromopyridine and hydrogenolysis of
the benzyl protective group.
CN
CN
N
CN
N
O
a
b
N
N
N
N
O
Cl
Lead optimization
10
12
11
N
HO
O
The first question we addressed was whether the 5,6,7,8-
tetrahydro-1,6-naphthyridine core of 3 could be replaced with a
monocyclic moiety (Scheme 4). We found that replacement of the
2-alkoxy pyridine system by a carbamate (5) retained some affinity
for H3R, although with a 170-fold drop in Ki value (Table 1). As a
first step towards recovering affinity, reversal of the carbamate
bond increased it back tenfold (8). Replacement of the piperidinyl
carbamate by a piperazine-1-carboxylate led to 9a, which has
sufficient affinity to serve as a new starting point for optimization.
The physicochemical characterization of 9a indicates decreased
logP (2.6 vs 3.3) and pKa (7.7 vs 9.5) values compared to 3, which
translates into a weaker interaction with hERG channels (IC50 > 30
µM vs 11 µM). Compound 9a shows only 11% of the effect of the
reference compound amiodarone in a phospholipidosis induction
assay.
NH2
N
c
N
O
N
O
N
OBn
O
NH
N
Boc
O
N
f
d, e
N
N
O
N
O
N
HO
N
14
13
O
g
NH
O
N
N
O
N
15
3
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10.1002/cmdc.201900176
ChemMedChem
O
O
and the amide group. As a result, permeability and brain
penetration improved noticeably, while affinity for H3R and
functional potency increased about fivefold. This leads to a higher
RO and a stronger effect on t-MeHA brain concentration. Its
duration of target engagement is however too long, and remains at
63% after 6h.
NH2
NH2
N
O
N
N
N
N
N
O
N
H
O
N
5
3
O
O
NH2
NH2
The replacement of picolinamides by pyridinones or pyridazinones
in H3R ligands has been illustrated previously.[18] Applying this
transformation to the current series led to structures 15 and 17a,
respectively. Interestingly, while both derivatives have similar in
vitro potencies, they display very different behaviors in vivo.
Compound 15 fails to occupy H3R to any significant extent 1h after
a 10 mg/kg oral dose, while 17a reaches 56% RO. We naturally
focused on the latter molecule for further optimization. Replacing
the isopropyl group of 17a by a cyclobutyl (18a) provided a
marginally increased affinity and brain exposure, and a high RO
after 0.5 h, which then rapidly faded. To improve brain exposure,
we then aimed to decrease the number of hydrogen bond donors
and methylated the pyridazinone nitrogen. This provided 17b,
which also has a high affinity for H3R and no significant hERG
binding. Compared to 17a, the brain exposure and brain/plasma
ratio of 17b are significantly better, leading to improved RO and a
strong effect on tMeHA concentration.
O
N
N
N
O
N
N
N
O
N
N
H
O
9a
8
Scheme 4. Transformation of 3 into 9a, a carbamate derivative of similar potency
at H3R.
Overall, the physicochemical properties of 9a proved encouraging
(logD7.4 = 0.9; MW = 375), with high passive transcellular
permeability as determined in Caco-2 cells (Papp x 10-6 cm/s = 19.7),
and good solubility at pH = 6.8 of 122 µM (high-throughput
equilibrium solubility assay using a miniaturized shake-flask
system and HPLC analysis). The selectivity profile of 9a with
regard to histamine H1, H2 and H4 receptors is excellent (IC50s >
30 µM), while a panel of 56 additional targets routinely tested for
safety profiling did not reveal further affinities. In vivo, 9a shows a
clear effect on tMeHA brain concentration, but only reaches a
moderate brain concentration, leading to a RO of 27%, 1h after a
10 mg/kg oral dose. Combined with a long terminal half-life in rat
Combining both modifications afforded 18b, a very potent
analogue with high brain exposure and high RO after 1h, which
also disengages from the receptor rapidly, leading to a remaining
occupancy of only 17%, 6h after oral administration. Figure 3
shows a full comparison of the time-course of RO of 18b and (4-
isopropylpiperazin-1-yl)(4-(morpholinomethyl)phenyl)methanone
(bavisant),[19] at that time one of the most advanced H3R inverse
agonist in clinical development. Both compounds achieve the
same level of RO (ca. 90%) at an early time point. After 5 h
however, the RO of bavisant remained at 50%, while the RO value
for 18b was clearly lower, around 20%.
(T = 3.3 h), this makes 9a unsuitable for the intended use, and its
½
properties had to be optimized further.
Table 1. In vitro and in vivo properties of H3R inverse agonists
#
hH3R
cAMP/ bdg
Ki [nM][a]
0.4/12.6
84/2163
67/221
2.8/37
5.5/52
1.4/33
1.7/101
182/nd
0.6/9.3
1.6/12
hERG
Brain
Brain/
tMeHA
induction
RO at
0.5/1/6h
[b]
levels[c]
plasma
[%]
-
[ng/g]
-
-
-
411
-
-
-
-
ratio[d]
[%][e]
-
-
-
58
-
-
-
[%][f]
-
-
-
3
5
8
9a
9b
9c
9d
9e
12
15
-
-
-
-
-
7
13
19
37
25
8
The SAR around the pyridazinone was explored further, and
confirmed that a methyl substituent is optimal, with the ethyl (19a)
and isopropyl derivatives (19b) showing slightly decreased RO
values. Based on the above, we selected 18b for further
characterization.
0.25
-
-
-
-
-/27/-
-
-
-
-
-
3180
-
1.55
-
119
-
87/91/63
0.3
-
17a
17b
18a
18b
19a
19b
1.7/42
0.8/24
0.8/17
0.3/12
0.2/6
0.2/12
5
2
13
6
-
587
2679
806
1309
-
0.31
0.99
0.4
0.93
-
115
138
72
76
-
17/56/22
80/69/30
80/70/44
89/90/17
68
-
-
-
-
62
[a] Ki values in the cAMP and binding assays were determined from 2-3
samples; [b] patch-clamp assay, % inhibition at 10 µM; [c] brain concentration.
in ng/g, 1h after oral administration of 10 mg/kg; [d] brain to plasma
concentration ratio, 1h after oral administration of 10 mg/kg. iv.; [e] % increase
of tMeHA brain concentration 1h after oral administration of 10 mg/kg,
compared to vehicle group (n=5 rats per dose group); [f] Receptor occupancy
after 10 mg/kg, determined in an ex vivo radioligand binding assay of brain
homogenate with [3H]-N-α-methylhistamine.
Figure 3. Time course of receptor occupancy of 18b and bavisant, in male
As a next step, we explored variations of the isopropyl group on
the piperazine ring. Replacing it with small cyclic alkyl substituents
(9b-d) did not have a marked effect, while introducing a
hydroxyethyl group was detrimental (9e). Modification of the
aminopyridine ring proved more fruitful, with the simple structural
change resulting from moving the ring nitrogen by one position, to
form 12. In contrast to the original nicotinamide, picolinamide 12 is
able to form an internal hydrogen bond between the ring nitrogen
Sprague-Dawley rats, measured from brain homogenate in
methylhistamine binding assay.
a
[3H]-N-α-
Compound 18b has excellent drug-like properties.[20] It is water-
soluble (95 µM at pH 6.8) and has good permeability (Papp x 10-6
cm/s = 19.0) in the Madin Darby canine kidney (MDCK) cell line
transfected with the human multidrug resistance (MDR1) gene. It
4
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10.1002/cmdc.201900176
ChemMedChem
shows no efflux in this assay (efflux ratio = 1.28). These values are Conclusion
predictive of a good brain penetration.
We disclose here the discovery of the clinical candidate 18b
(LML134), an H3R inverse agonist currently under investigation for
the treatment of excessive sleep disorders. From a medicinal
chemistry point-of-view, this concludes an optimization program
that first focused on ergoline derivatives. These were very
efficacious compounds, however limited in brain penetration.
Further optimization of this series by opening the ergoline scaffold
gave access to a number of highly potent and selective indole-
based H3R inverse agonists. This optimization effort allowed the
identification of indole 2, whose characterization confirmed
encouraging properties, until its metabolism was shown to be much
slower in human than rat liver microsomes, and therefore of a
potentially undesirable, long duration of action in patients.
A screen of 137 targets relevant for pharmacology or safety,
including hERG channels, as well as H1, H2 and H4 receptors, did
not detect any significant activity (all IC50 values > 30 µM; results
not shown), confirming the high selectivity of 18b for H3R. In
addition, 18b did not inhibit cytochrome P450 enzymes at 10 µM,[21]
was inactive in a phospholipidosis induction assay, and were
negative in the Ames and micronucleus genotoxicity assays.
The pharmacokinetic profile of 18b in rats (Table 2) indicates rapid
oral absorption, with a Tmax of 0.5 h and a fraction absorbed of
44%, as well as a rapid clearance, with a terminal half-life after
intravenous administration of 0.44 h. The low plasma protein
binding observed in rat (Fu =39.0%) was confirmed in dog (57.6%)
and human plasma (33.6%).
Finally, an independent optimization program focused on the
modification of 3, a compound described in the patent literature.
Extensive modification of this lead structure improved its
pharmacokinetic properties and cardiosafety profile, culminating in
the discovery of 18b. This drug candidate penetrates the brain
rapidly, leading to high H3R occupancy, and disengages its target
with a fast kinetic profile, indicative of a low potential for
mechanism-related insomnia on the day following administration.
Compound 18b completed a Phase I study to assess the safety,
tolerability and pharmacokinetics of single and multiple doses in
healthy volunteers.[22] It is currently undergoing further clinical
studies to assess its wakefulness promoting effect in shift work
disorder.[23] These results will be reported separately.
Table 2. Pharmacokinetic properties of 18b in male Sprague-Dawley rat
Rat i.v. PK (1 mg/kg) [a]
Rat oral PK (10 mg/kg) [a]
CL
Vss
T
28 mL/min/kg
Tmax
Cmax
0.5 h
0.79 L/kg
0.44 h
1127 ng/mL
1.54 h
T
½, z
½, z
AUC∞
MRT∞
601 h∙ng/mL
0.47 h
AUC∞
F
2979 h∙ng/mL
44.3 %
[a] CL: total blood clearance; VSS: apparent volume of distribution at steady
state; T½, z: apparent terminal half-life for elimination; AUC∞: area under the
curve (extrapolated to infinity); MRT∞: mean residence time (extrapolated to
infinity); Tmax: time of peak blood concentration after oral administration;
Cmax: maximal blood concentration after oral administration; F: fraction
absorbed.
Our previous experience with the optimization of compound 2
showed that long-lived active metabolites could influence the RO
time curve significantly.[14] As it is difficult to predict the extent of
this effect across species, we studied the metabolism of 18b in vitro,
looking for the presence of active metabolites.
Experimental Section
Chemistry
O
O
N
NH
2.1%
N
N
N
N
O
O
General methods. All reagents and anhydrous solvents were obtained
from commercial sources and used as received. 1H-NMR spectra were
recorded on a Bruker Advance III 400 MHz or a Bruker Advance III 500
MHz instrument in the solvent indicated, with tetramethylsilane as an
internal standard. Coupling constants (J) are in Hertz (Hz). Liquid
chromatography-mass spectrometry (LC/MS) analyses were run by
one of the following methods: Method A (tRA = retention time A): Agilent
1200 HPDMPALC / 6110 SQ system; mobile phase: water + 10 mM
NH4HCO3 (A), acetonitrile (B). Gradient: 5% B for 0.2 min, increase to
95% B within 1.2 min, and after 1.5 min back to 5% B within 0.01 min;
flow rate: 1.8 ml/min; column: XBridge C18, 4.6 x 50 mm, 3.5 μm; oven
temperature: 50°C. Method B (tRB = retention time B): Agilent 1200
HPLC / 6110 SQ system; Mobile Phase: water + 0.05% TFA (A),
acetonitrile +0.05% TFA (B); gradient: 5% B for 0.2 min, increase to
95% B within 1.2 min, after 1.6 min back to 5% B within 0.01 min; flow
rate: 1.8 mL/min; column: Sunfire, 3.5 μm, 50 x 4.6mm; oven
temperature: 50°C. Method C (tRC = retention time C): Acquity
UPLC/SQD MS (ESI) system; Mobile Phase: water + 0.05 % formic
acid + 3.75 mM ammonium acetate (A), acetonitrile + 0.04 % formic
acid (B); gradient: from 5 to 98 % B in 1.4 min; flow rate: 1.0 mL/min;
column: ACQUITY UPLC® HSS T3 1.8μm; oven temperature: 60°C.
Compounds were purified by silica gel chromatography and monitored
N
N
O
O
N
N
18a
2%
18b
8.8%
O
O
N
N
N
N
N
N
O
O
-O
N
N
O
O
HN
N+
21
20
Scheme 5. Metabolites of 18b, after 30 min. exposure to rat liver microsomes.
Exposure of 18b to rat liver microsomes for 30 min. led to the
formation of three main metabolites (Scheme 5). The main path of
biotransformation is N-dealkylation, leading to 18a and 20; N-
oxydation additionally leads to 21. In contrast to 20 and 21,
compound 18a is active on H3R. While its RO profile indicates that
it disengages slightly slower than the parent compound, 18a has a
lower brain penetration and the small amounts produced are
therefore not expected to interfere with the pharmacological activity
of 18b. This conclusion however needs validation in higher species.
5
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10.1002/cmdc.201900176
ChemMedChem
at 254 nm. Preparative HPLC conditions: XBridge Prep C18 10 μm
OBD, 19x250 mm. Mobile phase: A: water (0.1% NH3); B: acetonitrile;
gradient: 25% to 55% B in 10 min.
resulting in a white suspension. All volatiles were evaporated under
reduced pressure, and the residue used without further purification.
LC/MS: tRC = 0.54 min, m/z = 316.3 [M+H]+.
tert-Butyl (1-(5-carbamoylpyridin-2-yl)piperidin-4-yl)carbamate: 4-
1-(5-Carbamoylpyridin-2-yl)piperidin-4-yl (1-isopropylpiperidin-4-
N-Boc-piperidinamine (270 mg, 1.34 mmol) and DIPEA (530 mg, 3.83
yl)carbamate (8): 4-Amino-1-isopropylpiperidine (0.100 mL, 0.634
mmol) were added to a stirred solution of 6-chloronicotinamide (200 mg, mmol) was dissolved in THF (30 mL) under argon. NMM (0.209 mL,
1.28 mmol) in MeCN (5 ml). The reaction mixture was stirred at reflux
for 48 h. The solid was collected by filtration and washed with MeCN.
After drying in vacuo, the title compound was obtained as a white solid
(400 mg, 98%). LC/MS tRA =1.71 min, [M+H]+= 321.
1.903 mmol) was added and the colorless solution stirred for a few
minutes. NHS (261 mg, 2.220 mmol) and 1-(5-carbamoylpyridin-2-
yl)piperidin-4-yl 1H-imidazole-2-carboxylate (303 mg, 0.634 mmol)
were added, and the mixture stirred for 19 h at 65°C resulting in a white
suspension. All volatiles were evaporated and the residue purified by
column chromatography (CH2Cl2/MeOH 9:1) to afford the title
compound (154 mg, 55%) as a white solid. LC/MS: tRC = 0.40 min, m/z
= 390.4 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J = 2.2 Hz,
1H), 7.93 (dd, J = 9.0, 2.3 Hz, 1H), 7.72 (s, 1H), 7.06 (d, J = 7.9 Hz,
2H), 6.84 (d, J = 9.0 Hz, 1H), 4.74 (s, 1H), 4.04 (d, J = 12.8 Hz, 2H),
3.30 (s, 3H), 2.75 - 2.56 (m, 3H), 2.09 (t, J = 10.8 Hz, 2H), 1.88 (d, 2H),
1.69 (d, J = 10.3 Hz, 2H), 1.46 (d, J = 9.1 Hz, 2H), 1.32 (q, J = 11.2 Hz,
2H), 0.92 (d, J = 6.5 Hz, 6H).
6-(4-Aminopiperidin-1-yl)nicotinamide hydrochloride (4): tert-Butyl
1-(5-carbamoylpyridin-2-yl)piperidin-4-ylcarbamate (400 mg, 1.25
mmol) was added to a stirred solution of HCI in EtOAc (9 ml, 3N), and
the mixture stirred at RT for 4 h. The resulting precipitate was collected
by filtration and washed with EtOAc to give a white solid (300 mg, 94%).
LC/MS tRA =0.85 min, [M+H]+= 221.1.
1-(5-Carbamoylpyridin-2-yl)piperidin-4-yl 4-isopropylpiperazine-
1-carboxylate (9a): Carbonyldiimidazole (220 mg, 1.356 mmol) was
dissolved in THF (20ml). 6-(4-hydroxypiperidin-1-yl)nicotinamide (200
mg, 0.904 mmol) was added to the solution and heated to 65°C. NHS
(372 mg, 3.16 mmol) was added. To this mixture, a solution of 1-
isopropylpiperazine (132 µl, 0.904 mmol) and NMM (298 µl, 2.71 mmol)
in THF (15ml) was added, and stirring at 65°C was continued for 47 h.
All volatiles were evaporated and the residue purified by column
chromatography (CH2Cl2/MeOH 9:1) to afford the title compound (224
mg, 63%) as a white solid. LC/MS: tRC = 0.39 min, m/z = 376.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 2.2 Hz, 1H), 7.93 (dd, J =
9.0, 2.4 Hz, 1H), 7.72 (s, 1H), 7.09 (s, 1H), 6.85 (d, J = 9.0 Hz, 1H),
4.86 - 4.76 (m, 1H), 3.91 - 3.78 (m, 2H), 3.55 - 3.44 (m, 2H), 3.33 (s,
4H), 2.69 - 2.60 (m, 1H), 2.37 (s, 4H), 1.90 - 1.80 (m, 2H), 1.59 - 1.48
(m, 2H), 0.94 (d, J = 6.5 Hz, 6H).
1-Isopropylpiperidin-4-yl (1-(5-carbamoylpyridin-2-yl)piperidin-4-
yl)carbamate (5): A solution of 1-isopropylpiperidin-4-ol (360 mg, 2.5
mmol) in CH2Cl2 (5 mL) was cooled to 0°C. 4-Nitrophenyl chloroformate
(404 mg, 2 mmol) was added, followed by Et3N (260 mg, 2.5 mmol).
After stirring at RT for 16 h, the mixture was diluted with CH2Cl2 (40
mL), washed with water, dried and concentrated under reduced
pressure to yield 1-isopropylpiperidin-4-yl (4-nitrophenyl) carbonate as
a white solid (600 mg, crude). LC/MS tRA = 1.90 min, m/z 309.1 [M+H]+.
This intermediate was dissolved in DMF (5 mL), and treated with 6-(4-
aminopiperidin-1-yl)nicotinamide hydrochloride (586 mg, 2 mmol) and
Et3N (600 mg, 6 mmol). The mixture was stirred at RT for 2 days. The
precipitate was collected by filtration and purified by preparative HPLC
to afford the title compound (109 mg, 11%) as a white solid. LC/MS tRA
= 1.45 min, m/z 390.2 [M+H]+. 1H NMR (500MHz, DMSO-d6) δ = 8.58
(d, J = 2.0 Hz, 1H), 7.92 (d, J1 = 9.0 Hz, J2 = 2.0 Hz, 1H), 7.72 (br, 1H),
7.13 (d, J = 7.5 Hz, 1H), 7.09 (br, 1H), 6.83 (d, J = 9.0 Hz, 1H), 4.47-
4.46 (m, 1H), 4.31-4.28 (m, 2H), 3.57-3.56 (m, 1H), 3.01-3.00 (m, 2H),
2.68-2.65 (m, 3H), 2.23-2.19 (m, 2H), 1.82-1.77 (m, 4H), 1.48-1.45 (m,
2H), 1.34-1.24 (m, 2H), 0.94 (d, J = 6.0 Hz, 6H).
1-(5-Carbamoylpyridin-2-yl)piperidin-4-yl 4-(2-hydroxyethyl)piper-
azine-1-carboxylate (9b): N-(2-hydroxyethyl)-piperazin (0.079 mL,
0.634 mmol) was dissolved in THF (30 mL). NMM (0.209 mL, 1.903
mmol) and NHS (261 mg, 2.220 mmol) were added, followed by 1-(5-
carbamoylpyridin-2-yl)piperidin-4-yl 1H-imidazole-2-carboxylate (303
mg, 0.634 mmol). The resulting yellow solution was stirred for 18 h at
65°C. A second portion of N-(2-hydroxyethyl)-piperazin (0.079 mL,
0.634 mmol) was added and stirring continued for another 6 h. All
volatiles were evaporated and the residue purified by column
chromatography (CH2Cl2/MeOH 9:1) to afford the title compound (107
mg, 41%) as a white solid. LC/MS: tRC = 0.35 min, m/z = 378.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 2.3 Hz, 1H), 7.93 (dd, J =
9.0, 2.4 Hz, 1H), 7.72 (s, 1H), 7.09 (s, 1H), 6.85 (d, J = 9.1 Hz, 1H),
4.86 - 4.76 (m, 1H), 4.42 - 4.34 (m, 1H), 3.91 - 3.79 (m, 2H), 3.55 - 3.42
(m, 4H), 3.34 (s, 4H), 2.41 - 2.28 (m, 6H), 1.91 - 1.78 (m, 2H), 1.61 -
1.47 (m, 2H).
6-(4-Hydroxypiperidin-1-yl)nicotinamide (6): Piperidin-4-ol (517 mg,
5.1 mmol) was added to a solution of 6-chloronicotinamide (800 mg,
5.1 mmol) in DIPEA (4 mL), and the resulting mixture stirred at 110°C
for 8 h. After cooling to RT, the reaction mixture was concentrated
under reduced pressure and the residue purified by silica gel column
chromatography (CH2Cl2/MeOH, 10:1) to afford the title compound
(850 mg, 75%) as a pale yellow solid. LC/MS: tRA = 1.27 min, m/z =
222.1 [M+H]+.
1-(5-Carbamoylpyridin-2-yl)piperidin-4-yl 1H-imidazole-1-carbo-
xylate (7): Carbonyldiimidazole (879 mg, 5.42 mmol) was dissolved in
THF (50 mL). 6-(4-hydroxypiperidin-1-yl)nicotinamide (800 mg, 3.62
mmol) was added, and the colorless solution heated to 65°C for 24 h,
6
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10.1002/cmdc.201900176
ChemMedChem
1-(5-Carbamoylpyridin-2-yl)piperidin-4-yl 4-cyclopropylpiperazi-
ne-1-carboxylate (9c): Carbonyldiimidazole (220 mg, 1.356 mmol)
and 6-(4-hydroxypiperidin-1-yl)nicotinamide (200 mg, 0.904 mmol)
were dissolved in THF (20 ml) and the resulting suspension stirred at
RT for 5 h. This mixture was heated to 65°C and stirred for another 18
h. NHS (372 mg, 3.16 mmol) was added and the mixture was stirred
for another 30 min at 65°C. The resulting suspension was cooled to RT,
and a solution of 1-cyclopropylpiperazine (114 mg, 0.904 mmol) and
NMM (0.298 mL, 2.71 mmol) in THF (15 ml) was added dropwise within
15 min. The resulting mixture was heated to reflux for 5 days. Another
portion of NHS (372mg, 3.16mmol) was added and stirring continued
for another 2 days. All volatiles were evaporated and the residue
purified by column chromatography (CH2Cl2/MeOH 9:1) to afford the
title compound (118 mg, 34%) as a white solid. LC/MS: tRC = 0.42 min,
m/z = 374.4 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 2.2
Hz, 1H), 7.93 (dd, J = 9.1, 2.4 Hz, 1H), 7.72 (s, 1H), 7.09 (s, 1H), 6.86
(d, J = 9.2 Hz, 1H), 4.87 - 4.77 (m, 1H), 3.92 - 3.81 (m, 2H), 3.54 - 3.43
(m, 2H), 3.30 (s, 4H), 2.48 (s, 4H), 1.91 - 1.80 (m, 2H), 1.64 - 1.47 (m,
3H), 0.40 (d, J = 6.4 Hz, 2H), 0.33 - 0.26 (m, 2H).
140°C for 3 h. After cooling to RT, the solid was removed by filtration.
The filtrate was concentrated to 50 mL and the residue poured into
water (300 mL). The precipitate was collected by filtration and the cake
washed with water (2 x 50 mL), then dried under reduced pressure to
yield the title compound as a pale yellow solid (14.4 g, 71%). LC/MS:
tRA = 1.22 min, m/z = 204.1 [M+H]+.
1-(6-Cyanopyridin-3-yl)piperidin-4-yl
4-isopropylpiperazine-1-
carboxylate (11): DMAP (2.9 g, 23.7 mmol) and, portionwise,
triphosgene (3.5 g, 11.9 mmol) were added to a solution of 5-(4-
hydroxypiperidin-1-yl)picolinonitrile (2.41 g, 11.9 mmol) in CH2Cl2 (30
mL). After stirring at RT for 1 h, 1-isopropylpiperazine (3.04 g, 23.7
mmol) was added and the resulting mixture stirred at RT for an
additional 5 h. The reaction was quenched with sat. aq. NH4Cl (50 mL)
and extracted with CH2Cl2 (3 x 50 mL). The combined organic extracts
were dried, concentrated and purified by silica gel column
chromatography (CH2Cl2/MeOH, 20:1) to afford the title compound as
a pale yellow solid (2.3 g, 54%). LC/MS: tRA = 1.52 min, m/z = 358.3
[M+H]+.
1-(5-Carbamoylpyridin-2-yl)piperidin-4-yl 4-cyclobutylpiperazine-
1-carboxylate (9d): N-Cyclobutylpiperazine dihydrochloride (136 mg,
0.637 mmol) and NMM (0.210 mL, 1.912 mmol) were suspended in
THF (30 ml). NHS (262 mg, 2.230 mmol) and 1-(5-carbamoylpyridin-2-
yl)piperidin-4-yl 1H-imidazole-2-carboxylate (427.5 mg, 0.637 mmol)
were added, and the resulting yellowish suspension was stirred for 65
h at 65°C. All volatiles were evaporated and the residue purified by
column chromatography (CH2Cl2/MeOH 9:1) to afford the title
compound (134 mg, 53%) as a white solid. LC/MS: tRC = 0.41 min, m/z
= 388.4 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 2.3 Hz,
1H), 7.93 (dd, J = 9.0, 2.5 Hz, 1H), 7.72 (s, 1H), 7.09 (s, 1H), 6.85 (d,
J = 9.0 Hz, 1H), 4.86 - 4.76 (m, 1H), 3.91 - 3.80 (m, 2H), 3.49 (dd, J =
11.3, 7.1 Hz, 2H), 3.33 (d, J = 8.5 Hz, 4H), 2.66 (p, J = 7.6 Hz, 1H),
2.17 (s, 4H), 1.99 - 1.79 (m, 4H), 1.79 - 1.68 (m, 2H), 1.66 - 1.47 (m,
4H).
1-(6-Carbamoylpyridin-3-yl)piperidin-4-yl 4-isopropylpiperazine-
1-carboxylate
(12):
1-(6-cyanopyridin-3-yl)piperidin-4-yl
4-
isopropylpiperazine-1-carboxylate (2.3 g, 6.4 mmol) and 30% H2O2 (15
mL) were added to a solution of LiOH (310 mg, 12.9 mmol) in water (5
mL). The reaction mixture was stirred at RT for 5 h. After cooling to 0°
C, the precipitate was collected by filtration and purified by silica gel
column chromatography (CH2Cl2/MeOH, 10:1) to give the title
compound as a white solid (650 mg, 27%). LC/MS: tRB = 1.02 min, m/z
= 376.3 [M+H]+. 1H NMR (400 MHz, CDCl3) δ = 8.20 (d, 1 H, J = 3 Hz),
8.04 (d, 1 H, J = 8.5 Hz), 7.61 (br, 1 H), 7.21-7.27 (m, 1 H), 5.49 (br,
1H), 4.92-4.96 (m, 1 H), 3.49-3.59 (m, 6 H), 3.27-3.32 (m, 2 H), 2.72 (s,
1 H), 2.49 (br, 4 H), 2.02-2.07 (m, 2 H), 1.79-1.86 (m, 2 H), 1.05 (d, 6
H, J = 6.5 Hz).
1-(5-Carbamoylpyridin-2-yl)piperidin-4-yl
4-cyclopentylpiper-
1-(tert-Butoxycarbonyl)piperidin-4-yl
4-isopropylpiperazine-1-
azine-1-carboxylate (9e): 1-Cyclopentylpiperazine (0.099 mL, 0.637
mmol) and NMM (0.210 mL, 1.912 mmol) were dissolved in THF (30
ml). NHS (262 mg, 2.230 mmol) and 1-(5-carbamoylpyridin-2-
yl)piperidin-4-yl 1H-imidazole-2-carboxylate (427.5 mg, 0.637 mmol)
were added and the off-white suspension was stirred for 64 h at 65°C.
All volatiles were evaporated and the residue purified by column
chromatography (CH2Cl2/MeOH 9:1) to afford the title compound (148
mg, 56%) as a white solid. LC/MS: tRC = 0.44 min, m/z = 402.5 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 2.3 Hz, 1H), 7.93 (dd, J =
9.0, 2.5 Hz, 1H), 7.72 (s, 1H), 7.09 (s, 1H), 6.85 (d, J = 9.1 Hz, 1H),
4.85 - 4.76 (m, 1H), 3.91 - 3.79 (m, 2H), 3.55 - 3.44 (m, 2H), 3.41 - 3.31
(m, 4H), 2.47 - 2.39 (m, 1H), 2.33 (d, J = 4.7 Hz, 4H), 1.90 - 1.80 (m,
2H), 1.79 - 1.68 (m, 2H), 1.62 - 1.50 (m, 4H), 1.50 - 1.40 (m, 2H), 1.35
- 1.20 (m, 2H).
carboxylate: To solution of tert-butyl 4-hydroxypiperidine-1-
a
carboxylate (4.4 g, 21.9 mmol) in CH2Cl2 (100 mL) was carefully added
DMAP (5.3g, 43.8mmol) and triphosgene (3.2g, 10.95 mmol)
portionwise. After stirring at RT for 2 h, 1-isopropylpiperazine (3.3g, 26
mmol) was added and the reaction mixture was stirred at RT for 5 h.
The reaction was quenched with saturated NH4Cl solution (100 mL)
and the mixture was extracted with CH2Cl2 (3x100 mL). The combined
organic layers were washed sequentially with saturated NH4Cl (2x100
mL) and brine (50mL), dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure to afford the title compound as a
white solid (7.7 g, 99%). LC/MS: TRA=1.67min, m/z 356.3 [M+H]+.
Piperidin-4-yl 4-isopropylpiperazine-1-carboxylate (13): To
a
solution of 1-(tert-butoxycarbonyl)piperidin-4-yl 4-isopropyl piperazine-
1-carboxylate (7.7 g, 21.7 mmol) in CH2Cl2 (30 mL) was added TFA (10
mL) and the reaction mixture was stirred at RT for 5 h. The solvent was
removed under reduced pressure and the residue was re-dissolved in
CH2Cl2/MeOH 10/1 (100 mL). Then solid Na2CO3 was added and the
mixture was stirred at RT for 2 h. Excess Na2CO3 was removed by
5-(4-Hydroxypiperidin-1-yl)picolinonitrile (10): K2CO3 (41.4 g, 300
mmol) and tetrabutylammonium iodide (740 mg, 2 mmol) were added
to a solution of 5-chloropicolinonitrile (13.8 g, 100 mmol) and piperidin-
4-ol (12.1 g, 120 mmol) in DMF (50 mL). The mixture was stirred at
7
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10.1002/cmdc.201900176
ChemMedChem
filtration and the cake was washed with CH2Cl2 (2x100 mL). The
combined filtrates were concentrated under reduced pressure to afford
the desired compound as yellow oil (5.5 g, 100%). LC/MS: tRA =1.12
min, m/z 256.2 [M+H]+.
compound as a white solid (3.3 g, 46%). LC/MS: tRA =1.47 min, m/z
361.1 [M+H]+.
1-(6-Oxo-1,6-dihydropyridazin-3-yl)piperidin-4-yl
4-cyclobutyl-
piperazine-1-carboxylate (18a): Et3N (616 mg, 6.1 mmol) and 1-
1-(6-(Benzyloxy)pyridin-3-yl)piperidin-4-yl 4-isopropylpiperazine-
1-carboxylate (14): BINAP (248 mg, 0.4 mmol) and t-BuOK (336 mg,
3 mmol) were added to a solution of 2-(benzyloxy)-5-bromopyridine
(133 mg, 0.5 mmol) and piperidin-4-yl 4-isopropylpiperazine-1-
carboxylate (101 mg, 0.4 mmol) in toluene (30 mL). The mixture was
degassed with N2 for 3 min before addition of Pd2(dba)3 (140 mg, 0.4
mmol). The reaction mixture was heated to 120°C and stirred for 16 h.
After cooling to RT, the reaction mixture was diluted with EtOAc (40
mL), washed with water (20 mL) and concentrated to dryness. The
residue was taken up into 1 N aq. HCl (20 mL) and extracted with
EtOAc (3 x 40 mL). The aqueous phase was basified with Na2CO3 to
pH 10 and extracted with CH2Cl2 (3 x 50 mL). The extracts were
combined, dried over anhydrous Na2SO4, concentrated, and the
residue purified by silica gel chromatography (EtOAc/MeOH, 50:1) to
give the title compound as a white solid (50.0 mg, 20%). LC/MS: tRA =
2.41 min, m/z = 439.1 [M+H]+.
cyclobutylpiperazine (388 mg, 1.83 mmol) were added to a solution of
4-nitrophenyl
1-(6-oxo-1,6-dihydropyridazin-3-yl)piperidin-4-yl
carbonate (440 mg, 1.22 mmol) in CH2Cl2 (20 mL). The resulting
mixture was stirred at 30°C for 2 h before concentration to dryness.
Silica gel chromatography (CH2Cl2/MeOH=50:1 to 5:1) afforded the title
compound as a white solid (410 mg, 93%). LC/MS: tRA = 1.36 min, m/z
362.3 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 11.20 (s, 1H), 7.20 (d, J=10
Hz, 1H), 6.87 (d, J=10 Hz, 1H), 4.87~4.92 (m, 1H), 3.45~3.50 (m, 6H),
3.16~3.23 (m, 2H), 2.68~2.76 (m, 1H), 2.29 (br, 4H), 1.72~2.07 (m,
10H).
6-(4-Hydroxypiperidin-1-yl)-2-methylpyridazin-3(2H)-one (16b): 6-
chloro-2-methylpyridazin-3(2H)-one (75 mg, 0.519 mmol) was
dissolved in n-butanol (2.5 mL). 4-Hydroxypiperidin (107 mg, 1.038
mmol) was added and the resulting solution was stirred at 140°C for
214 h to give a brown solution. All volatiles were removed under
reduced pressure and the residue purified by column chromatography
(CH2Cl2/MeOH 9:1) to afford the title compound as a slightly brownish
solid (46 mg, 41%). tRC = 0.43min, m/z = 210.2 [M+H]+. 1H NMR (400
MHz, DMSO-d6) δ 7.48 (d, J = 10.0 Hz, 1H), 6.77 (d, J = 10.0 Hz, 1H),
4.67 (d, J = 4.2 Hz, 1H), 3.64 - 3.56 (m, 3H), 3.46 (s, 3H), 2.88 (dtd, J
= 13.0, 10.1, 3.0 Hz, 2H), 1.79 - 1.71 (m, 2H), 1.43 - 1.32 (m, 2H).
1-(6-Oxo-1,6-dihydropyridin-3-yl)piperidin-4-yl 4-isopropylpiper-
azine-1-carboxylate (15): A solution of 1-(6-(Benzyloxy)pyridin-3-
yl)piperidin-4-yl 4-isopropylpiperazine-1-carboxylate (200 mg, 0.46
mmol) in MeOH (10 mL) was treated with 10% Pd/C (20 mg) and the
suspension was stirred at RT for 10 min under an H2 atmosphere. The
catalyst was then removed by filtration, the filtrate concentrated under
reduced pressure and the residue purified by preparative HPLC to give
the title compound as a pale yellow solid (150 mg, 94%). LC/MS tRA =
1
1.37 min, m/z 349.2 [M+H]+. H NMR (400 MHz, MeOH-d4) δ = 7.50
1-(1-Methyl-6-oxo-1,6-dihydropyridazin-3-yl)piperidin-4-yl
4-
(dd, J1 = 10Hz, J2 = 3.2Hz, 1H), 6.81 (d, J = 3.2Hz,1H), 6.41 (d, J =
10Hz, 1H), 4.68 (m, 1H), 3.45 (br, 4H), 3.00 (m, 2H), 2.75 (m, 3H), 2.59
(m, 4H), 1.90 (m, 2H), 1.70 (m, 2H), 1.02 (d, J = 6.4Hz, 6H).
cyclobutylpiperazine-1-carboxylate (18b): Carbonyldiimidazole
(27.6 mg, 0.165 mmol) was dissolved in CH2Cl2 (1ml) under argon. 6-
(4-hydroxypiperidin-1-yl)-2-methylpyridazin-3(2H)-one (25 mg, 0.110
mmol) was added and the resulting brownish solution was stirred for 2
h. NHS (45.2 mg, 0.385 mmol) was added, and the mixture stirred for
another 1 h. To this mixture, a solution of NMM (0.069 mL, 0.627 mmol)
and N-cyclobutylpiperazine dihydrochloride (23.43 mg, 0.110 mmol) in
CH2Cl2 (1 ml) was added dropwise, and the resulting mixture was
stirred at RT for 16 h. A second portion of NHS (25.8mg, 0.220 mmol)
was added, and stirring continued for 26 h. All volatiles were removed
under reduced pressure and the residue purified by column
chromatography (CH2Cl2/MeOH 9:1) to afford the title compound (35
mg, 85%) as an off-white solid. tRC = 0.47 min, m/z = 376.4 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) δ 7.50 (d, J = 10.0 Hz, 1H), 6.80 (d, J =
10.0 Hz, 1H), 4.78 - 4.70 (m, 1H), 3.47 (s, 3H), 3.38 - 3.28 (m, 6H),
3.20 - 3.11 (m, 2H), 2.67 (s, 1H), 2.17 (s, 4H), 1.98 - 1.82 (m, 4H), 1.79
- 1.71 (m, 2H), 1.65 - 1.53 (m, 4H); mp (free base): 121.0-122.0°C
(crystallized from iPrOH/MeOtBu).
6-(4-Hydroxypiperidin-1-yl)pyridazin-3(2H)-one (16a): 6-chloro-
pyridazin-3(2H)-one (250 mg, 1.82 mmol) was dissolved in n-butanol
(2.5 mL). 4-Hydroxypiperidin (376 mg, 3.64 mmol) was added and the
mixture stirred at 140°C for 214 h to give a brown suspension. All
volatiles were removed under reduced pressure and the residue
purified by column chromatography (CH2Cl2/MeOH 9:1) to afford the
title compound as a slightly yellow solid (258 mg, 67%). tRC = 0.33min,
1
m/z = 196.1 [M+H]+. H NMR (400 MHz, DMSO-d6) δ 12.00 (s, 1H),
7.47 (d, J = 10.1 Hz, 1H), 6.72 (d, J = 10.1 Hz, 1H), 4.66 (d, J = 4.2 Hz,
1H), 3.67 - 3.50 (m, 3H), 2.85 (ddd, J = 13.0, 10.1, 2.9 Hz, 2H), 1.80 -
1.68 (m, 2H), 1.44 - 1.31 (m, 2H).
4-Nitrophenyl
1-(6-oxo-1,6-dihydropyridazin-3-yl)piperidin-4-yl
carbonate: DIPEA (3.87g, 30.0 mmol) and 4-nitrophenyl chloroformate
(6.03 g, 30 mmol) were added to a solution of 6-(4-hydroxypiperidin-1-
yl)pyridazin-3(2H)-one (3.9 g, 20 mmol) in pyridine (10 mL), and the
resulting mixture was stirred at 30°C for 2 h. After concentration under
reduced pressure, the residue was purified by silica gel
chromatography (CH2Cl2 to CH2Cl2/MeOH = 20/1) to afford the title
1-(6-Oxo-1,6-dihydropyridazin-3-yl)piperidin-4-yl
4-isopropyl-
piperazine-1-carboxylate (17a): Following a similar protocol as for
18a but using 1-isopropylpiperazine as starting material, the title
compound was obtained as yellow oil (258 mg, 4%). LC/MS tRA = 1.40
min, m/z 350.2 [M+H]+. 1H NMR (400 MHz,CDCL3) δ = 10.87 (s, 1H),
7.15-7.12 (d, 1H), 6.82-6.80 (d, 1H), 4.86 (m, 1H), 3.69 (m, 6H), 3.16-
8
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10.1002/cmdc.201900176
ChemMedChem
3.10 (m, 2H), 2.67-2.64 (m, 1H), 2.42 (s, 4H), 1.93-1.88 (m, 2H), 1.74-
MDCK-MDR1 assays were carried out according to protocols outlined
in detail in our previous article.[14] The phospholipidosis assay was
conducted as previously described.[24] Screening assays, including
Ames and micronucleus genotoxicity assays were conducted at
Eurofins according to published protocols.[25]
1.67（m, 2H), 0.99-0.97 (d, 6H).
1-(1-Methyl-6-oxo-1,6-dihydropyridazin-3-yl)piperidin-4-yl
4-
isopropylpiperazine-1-carboxylate (17b): Carbonyldiimidazole (57.5
mg, 0.344 mmol) was dissolved in THF (10 mL) under argon. 6-(4-
hydroxypiperidin-1-yl)-2-methylpyridazin-3(2H)-one (50 mg, 0.229
mmol) was added and the mixture was heated to 65°C for 117 h. NHS
(94 mg, 0.803 mmol) was added and stirring continued for another 21
h. NMM (0.076 mL, 0.688 mmol) and 1-isopropylpiperazine (0.033 mL,
0.229 mmol) were added and stirring was continued for another 24 h.
All volatiles were removed under reduced pressure and the residue
purified first by column chromatography (CH2Cl2/MeOH 9:1) and then
preparative SFC to afford the title compound (26 mg, 31%) as a
colorless oil. tRC = 0.45 min, m/z = 364.4 [M+H]+. 1H NMR (400 MHz,
Chloroform-d) δ 7.10 (d, J = 9.9 Hz, 1H), 6.84 (d, J = 9.9 Hz, 1H), 4.93
- 4.84 (m, 1H), 3.64 (s, 3H), 3.62 - 3.53 (m, 4H), 3.52 - 3.43 (m, 2H),
3.21 - 3.12 (m, 2H), 2.90 - 2.78 (m, 1H), 2.65 - 2.51 (m, 4H), 2.02 - 1.92
(m, 2H), 1.81 - 1.69 (m, 2H), 1.11 (d, J = 6.5 Hz, 6H).
Acknowledgements
The authors would like to acknowledge Yi-Ying Li, Dou Yin, Xu-
Zhao Zhou, Wei-Min Qu and Professor Zhi-Li Huang, Department
of Pharmacology, Shanghai Medical College, Fudan University, for
EEG measurements, as well as Dushan Zhang, Chunxiu Wang,
Xiaoxia Shen, Lijun Lei, Yu-Chih Liu and Sebastian Schwarz for
synthetic support.
Keywords: Histamine, H3 receptor, carbamate,
neurotransmitters, medicinal chemistry.
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9
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Received: ((will be filled in by the editorial staff))
Published online: ((will be filled in by the editorial staff))
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Entry for the Table of Contents
FULL PAPERS
Thomas Troxler, Dominik Feuerbach]
Xuechun Zhang, Charles R. Yang,
Bharat Lagu, Mark Perrone, Tie-Lin
Wang, Karin Briner, Mark G. Bock, Yves
P. Auberson* Page No. – Page No.
Wake up, enjoy the day and sleep
again! The histamine H3 receptor
inverse agonist LML134 (18b)
achieves rapid oral absorption and
brain penetration, leading to strong
wakefulness-enhancing effects in rats.
It also rapidly disengages from the
receptor, a desirable profile to limit the
risk of insomnia during the night
following treatment. Clinical trials are
ongoing to confirm its clinical efficacy
in humans.
The discovery of LML134, a histamine
H3 receptor inverse agonist for the
clinical treatment of excessive sleep
disorders
11
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