Design, synthesis, and biological evaluation of novel oxadiazole- and thiazole-based histamine H3R ligands

Article abstract of DOI:10.1016/j.bmc.2018.06.028

Histamine H₃ receptor (H₃R) is largely expressed in the CNS and modulation of the H₃R function can affect histamine synthesis and liberation, and modulate the release of many other neurotransmitters. Targeting H₃R with antagonists/inverse agonists may have therapeutic applications in neurodegenerative disorders, gastrointestinal and inflammatory diseases. This prompted us to design and synthesize azole-based H₃R ligands, i.e. having oxadiazole- or thiazole-based core structures. While ligands of oxadiazole scaffold were almost inactive, thiazole-based ligands were very potent and several exhibited binding affinities in a nanomolar concentration range. Ligands combining 4-cyanophenyl moiety as arbitrary region, para-xylene or piperidine carbamoyl linkers, and/or pyrrolidine or piperidine basic heads were found to be the most active within this series of thiazole-based H₃R ligands. The most active ligands were in silico screened for ADMET properties and drug-likeness. They fulfilled Lipinski's and Veber's rules and exhibited potential activities for oral administration, blood–brain barrier penetration, low hepatotoxicity, combined with an overall good toxicity profile.

Full text of DOI:10.1016/j.bmc.2018.06.028

Accepted Manuscript
Design, Synthesis, and Biological Evaluation of Novel Oxadiazole- and Thia-
zole-Based Histamine H3R Ligands
Mohammad A. Khanfar, David Reiner, Stefanie Hagenow, Holger Stark
PII:
S0968-0896(18)30764-8
https://doi.org/10.1016/j.bmc.2018.06.028
BMC 14420
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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8 June 2018
20 June 2018
Please cite this article as: Khanfar, M.A., Reiner, D., Hagenow, S., Stark, H., Design, Synthesis, and Biological
Evaluation of Novel Oxadiazole- and Thiazole-Based Histamine H3R Ligands, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.06.028
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Design, Synthesis, and Biological Evaluation of Novel Oxadiazole-
and Thiazole-Based Histamine H3R Ligands
Mohammad A. Khanfar1,2,3, David Reiner1, Stefanie Hagenow1, Holger Stark1*
1Heinrich Heine University Düsseldorf, Institute of Pharmaceutical and Medicinal
Chemistry, Universitaetsstr. 1, 40225 Duesseldorf, Germany
2Faculty of Pharmacy, University of Jordan, P.O Box 13140, Amman 11942, Jordan.
3College of Pharmacy, Alfaisal University, Al Takhassusi Rd, Riyadh, 11533, Saudi
Arabia
*Corresponding author: Prof. Holger Stark, Tel.: +49 211 81-10478, Fax: +49 211 81-
13359, email: stark@hhu.de
1

Abstract
Histamine H3 receptor (H3R) is largely expressed in the CNS and modulation of the
H3R function can affect histamine synthesis and liberation, and modulate the release
of many other neurotransmitters. Targeting H3R with antagonists/inverse agonists
may have therapeutic applications in neurodegenerative disorders, gastrointestinal and
inflammatory diseases. This prompted us to design and synthesize azole-based H3R
ligands, i.e. having oxadiazole- or thiazole-based core structures. While ligands of
oxadiazole scaffold were almost inactive, thiazole-based ligands were very potent and
several exhibited binding affinities in a nanomolar concentration range. Ligands
combining 4-cyanophenyl moiety as arbitrary region, para-xylene or piperidine
carbamoyl linkers, and/or pyrrolidine or piperidine basic heads were found to be the
most active within this series of thiazole-based H3R ligands. The most active ligands
were in silico screened for ADMET properties and drug-likeness. They fulfilled
Lipinski’s and Veber’s rules and exhibited potential activities for oral administration,
blood-brain barrier penetration, low hepatotoxicity, combined with an overall good
toxicity profile.
Keywords: histamine; H3 receptor; ligands; ADMET; thiazole; oxadiazole
2

1. Introduction
Histamine belongs to biogenic amines, which influence several intracellular
pathways, including its neurotransmission activities.1-4 Histamine’s regulatory
character in cellular activities is attributed to its binding to four subtypes of G-protein-
coupled receptors (GPCRs); H1, H2, H3, and H4 that are differentially expressed in
several cell types.5, 6
Histamine H3 receptor (H3R) is a member of transmembrane class A of GPCR
family.7, 8 H3R is largely expressed on the histaminergic neurons of the CNS, located
pre- and postsynaptically.9 It plays an essential role in the biosynthesis and release of
histamine as well as in the modulation the release of different neurotransmitters (e.g.,
11
dopamine, serotonin, acetylcholine, noradrenaline, GABA, and glutamate.)10,
Peripherally, H3R is marginally distributed in the peripheral nervous system and
regulates the sympathetic effector systems and pain sensation.12 Consequently,
modulation of the H3R function can potentially affect histaminergic
neurotransmission. Therefore, targeting H3R with antagonists/inverse agonists may
have therapeutic applications in neurodegenerative disorders, such as Parkinson's
disease, Alzheimer’s disease, as well as in depression, epilepsy, schizophrenia and in
sleep disorders. 14-19
Recently, a number of H3R antagonists/inverse agonists have entered late clinical
trials for the treatment of several CNS disorders.15, 20, 21 In March 2016, the European
Medicines Agency authorized marketing of pitolisant (Wakix®) as the first H3R
inverse agonist for the treatment of narcolepsy, designated as an orphan drug since
narcolepsy belongs to the rare diseases.22-24 Pitolisant is also currently in phase III
clinical trials for the treatment of hypersomnia.25
3

First H3R antagonists/inverse agonists reported in literature were imidazole-based
compounds followed by non-imidazoles in the next generation. They share a general
26, 27
pharmacophore generated from numerous chemical scaffolds (Figure 1).15,
Prototype H3R antagonists/inverse agonists pharmacophore consist of basic moiety
(mainly tertiary amine electrostatically interacting with Asp114 within H3R binding
site) separated by a spacer to a central core (mainly electron-rich moiety)28. The
distance between the basic head and the electron rich moiety is approximately 4-5 Å,
equivalent to 4 bonds in linear arrangement. The central core is usually connected to
an arbitrary region, mainly lipophilic, but polar or basic and even acidic moieties are
also tolerable.29 This arbitrary region modulates potency and pharmacokinetic
properties of H3R antagonists/inverse agonists.27, 30
Figure 1. Schematic presentation of the pharmacophore model of H3R
antagonists/inverse agonists.
Oxadiazole and thiazole nuclei have attracted a wide attention in the search for new
therapeutic molecules. They are widely exploited in various applications because of
their versatile applicability in multiple therapeutic agents and their blood-brain barrier
32
(BBB) permeability;31,
Five-membered heterocycles as core motifs in non-
4

imidazole H3R ligands with structural resemblance to the presented structures were
explored in earlier research projects resulting in affine compounds (Figure 2).33, 34
Among them ADS-531 that recently underwent in vivo examination upon effects on
food-intake and neurotransmitter systems35, 36 The former progress in the design of
potent oxadiazole- and thiazole-based H3R ligands prompted our research group to
explore 2-arylthiazole-4-ylethers as analogues to ST-979 but differing in the
arrangement of the substituents.37 Furthermore, N-aryl-1,3,4-oxadiazole-2-amines and
3-phenyl-1,2,4-oxadiazole-5-carboxamides (cf. GSK247246 and ST-1095) were
designed to increase structural and linkage diversity in the H3R central core motif ,
basically linked to our previous work on variations of polar azole core motifs in H3R
ligands (Figure 2).37-40 Our synthetic effort combines various aliphatic and aromatic
spacers of variable length, and arbitrary region of electronically and sterically variable
substituents. Several H3R analogues with binding affinities in low nanomolar
concentrations were obtained within this project.
Figure 2. Selected oxadiazole- and thiazole-based H3 receptor ligands serving as a
basis for design of new H3R ligands aKi values calculated from published pKi values.
2. Results and Discussions
2.1 Hit-Identification and Hit-to-Lead-Optimization
5

Synthetic design strategy was driven by an initial structural hypothesis for various
azole-based H3R ligands fitting the H3R-pharmacophoric map (Figure 1).37-39 The
following structural modification strategies were applied to design potent H3R
ligands: (1) employing oxadiazole (1,2,4 and 1,3,4) or thiazole rings as a polar,
electron-rich central core; (2) several cyclic and noncyclic amines were examined as a
basic head of the designed H3R ligands including: pyrrolidine, piperidine, piperazine
and noncyclic aliphatic tertiary amines; (3) variable spacers of different chain length
connecting the central core and the basic head were examined using aliphatic and
aromatic linkers. Linkers of 2 to 5 elements of cyclic or noncyclic structure were
tested for optimal length; (4) substituted and unsubstituted aromatic and
heteroaromatic rings of variable size and electronic properties were placed on the
arbitrary region. Substantial modifications can be conducted on the arbitrary region to
modulate potency, solubility and pharmacokinetic properties. Initially, only a few
ligands of each compound series without changes in the arbitrary region were
synthesized and evaluated for hit identification. Hit-to-lead optimization was
conducted for those structures with promising results.
2.2 Chemistry.
An efficient one-pot synthesis of substituted 2-anilino-1,3,4-oxadiazoles was applied.
A carboxylic acid (1.0 eq.) and 4-phenyl-3-thiosemicarbazide (1.0 eq.) were mixed in
DCM at room temperature with three equivalents of N-(3-dimethylaminopropyl)-N1-
ethylcarbodiimide (EDCI) as coupling reagent, producing the corresponding 2-
anilino- 1,3,4-oxadiazoles (2a-m) (Scheme 1). The thiosemicarbazides 1a-e were
readily prepared by reacting the corresponding isothiocyanate with hydrazine under
reflux in MeOH.
6

Scheme 1. Synthesis of 2-Anilino-1,3,4-oxadiazoles. Reagents and Conditions:
(a) (1) N2H4, MeOH, rt, 1 h. (2) 65-80 oC, 20 min (88-96%); (b) EDCI (3 eq), CH2Cl2, rt, 12 h
(50-84%). Structure of compounds 2a – 2m are listed in Table 1.
The synthesis of 1,2,4-oxadiazole derivatives started from commercially available
nitriles that were reacted with hydroxylamine in EtOH/H2O in the presence of
NaHCO3 to give the corresponding phenylamidoximes 3a–b in good yields. The latter
were suspended in DCM and then refluxed for 5 h in pyridine with ethyl
chlorooxalate to provide 3-aryl-1,2,4-oxadiazole-5-carboxylates 4a–b. Then , the
obtained esters were eadily converted into the desired compounds 5a-c by reaction
with 3-substituted amine-1-yl propan-1-amine (Scheme 2).
Scheme 2. Synthesis of 3-phenyl-1,2,4-oxadiazole derivatives. Reagents and Conditions: (a)
NH4OH, EtOH, NaHCO3, reflux, 3 h (67-77%); (b) CHCl3, ethyl chlorooxalate, rt for 30 min, then
refluxed for 5 h (79-82%); (c) EtOH, alkyl amine, reflux for 8 h (73-87%). Structure of compounds
5a – 5c are listed in Table 1.
The synthesis of 5-methyl thiazole analogues (7 - 36) was based on literature
procedure where the aryl / heteroaryl nitrile, α-mercaptocarboxylic acid, and pyridine
were reacted to form the corresponding hydroxymethylthiazole (6).41 Subsequently,
the hydroxymethiazole was O-alkylated following a Williamson-type etherification
7

with K2CO3 as the base to afford the final compounds (7 – 36) (Scheme 3). Analogues
39-42 were synthesized by first refluxing 4-hydroxythiazole with 4 equivalents of
α,α′-dibromo-p-xylene in acetone and K2CO3. After column chromatography, the
monoether products (38a-b) were reacted with either piperidine or pyrrolidine to
furnish the corresponding final compounds (39-42) (Scheme 4). The 5-ethyl thiazole
analogues (44-46) were synthesized by first mixing the corresponding
phenylthioamide, ethyl 2-bromobutanoate and pyridine under argon and slowly
heating up to 100-110 °C for 2 h to form hydroxyethylthiazole (43), which was O-
alkylated to afford the final compounds (44 – 46) (Scheme 5).42
Scheme 3. Synthesis of 5-methyl thiazole derivatives. Reagents and Conditions:
(a) pyridine, 100 oC for 2 h (69-96%); (b) DMF (or acetone), Cs2CO3 (or K2CO3) (3.0
eq.), KI (1.0 eq.), alkyl halide (0.9 eq.) (57-95%). Structure of compounds 7 – 37 are
listed in Table 2.
8

Scheme 4. Synthesis of xylene linker thiazole derivatives. Reagents and conditions:
o
(a) K2CO3, acetone, reflux, 12 h (74-86%); (b) alicyclic amine, K2CO3, DMF, 60 C,
12 h (84-90%).
Scheme 5. Synthesis of 5-ethyl thiazole derivatives. Reagents and Conditions:
o
(a) pyridine (1.0 eq.), 100-110 C, 3 h (76-98%); (b) DMF, K2CO3 (3.0 eq.), KI (1.0
eq.), alkyl halide (0.9 eq.) (62-94%). Ligand structures of 44 – 46 are listed in Table
2.
2.3 Biological Evaluation
All final synthesized compounds were tested for their in vitro H3R binding affinity in
a binding assay by competitive displacement of [3H]-Nα-methylhistamine as
radioligand. Their corresponding Ki values with 95% confidence intervals are listed in
Table 1 and Table 2.
2.3.1 Oxadiazole-based ligands
As it was previously demonstrated that oxadiazoles, e.g. linked either via oxy-
/thioethers or carboxamides to the basic amine moiety, are accepted as central core in
H3R ligands, a small series of 1,2,4- and 1,3,4-oxadiazole derivatives were
designed.38, 43
9

Analogues of 1,3,4-oxadiazole scaffold showed low binding affinities at H3R (Ki >
1000 nM), thus, permitting only limited conclusions for structural variations to
improve H3R affinity. The prototypes (2a - 2d) without modifications at the anilino
substructure resulted in ligand 2d showing weak affinity. Only a few variations of
these ligands were constructed to examine possibilities for improving this binding
behavior, resulting in 2e – 2m. Ligands with alicyclic basic head and 2-3 carbon
linkers showed comparable activity with 2d exhibiting low micromolar Ki values (2c
2g, 2j, 2k, and 2m). However, analogues with the same linker length but rigid linker
and basic head have negligible binding affinity (2b, 2f, 2i). This behavior implies the
necessity of flexibility to position the basic head in favorable orientation for salt
bridge formation with Asp114, described as crucial for H3R antagonists/inverse
agonists binding.44 Moreover, ligands with noncyclic basic head and/or shorter linker
(2a, 2h, 2l) appeared inactive, except the 4-chlorine derivative showing micromolar
affinity (2e). These data suggest the need of a defined distance between the central
core and the cyclic basic head for these compounds. Nevertheless, the modifications
performed here were not able to improve affinity substantially, limiting any SAR
interpretations in this series.
The smaller series of 1,2,4-oxadiazole-5-carboxamides analogues structurally related
to previously described compounds FUB 654, ST-1095 or GSK247246were inactive
38,
(5a, 5b and 5c, Table 1).33,
39Therefore, they are considered as unsuitable
fundamental structure for further synthetic efforts, compared to 1,2,4-oxadiazole FUB
654 (Figure 2), anon-imidazole analogue of the highly affine Glaxo Wellcome
compound GR175737 (Ki = 2.5 nM).33, 45Compared to previously published more
potent alkyl-/thioether-linked oxadiazole derivatives (Figure 2),38 N-phenyl-1,3,4-
oxadiazole-2-amines and 1,2,4-oxadiazole-5-carboxamides seem to bear a less
10

promising or more restricted core motif on substitution and linkage, respectively.
Subsequently, the more promising thiazole class has been followed in more detail.
Table 1. Oxadiazole-based ligands along with H3R binding affinities.
Compd
No.
H3R Ki (nM)a
Scaffold
I
R1
H
n
3
R2
[95% CI] a; (n)b
> 5,000; (3)
> 5,000; (2)
2a
2b
I
I
H
H
0
2
2,000 [100; 72,200]; (2)
2c
I
I
I
H
3
3
0
1,300 [700; 2,500]; (2)
5,700 [300; 112,800]; (3)
> 5,000; (2)
2d
2e
2f
4-Cl
4-Cl
I
I
I
4-Cl
2
1
0
1,600 [100; 55,200]; (2)
> 5,000; (2)
2g
2h
2i
4-OCH3
4-OCH3
> 5,000; (2)
I
I
4-OCH3
4-OCF3
2
2
1,600 [700; 3,800]; (2)
2j
1,300 [100; 34,800]; (2)
11
2k

3,5-
dimethoxy
I
I
1
2
> 5,000 (2)
2l
3,5-
dimethoxy
1,100 [1,000; 1,200]; (2)
2m
II
II
II
H
H
3
3
3
> 5,000 (2)
> 5,000 (4)
> 5,000; (2)
5a
5b
5c
OCH3
aBinding affinity values (Ki) are expressed as mean with 95% confidence intervals;
bNumber of experiments.
2.3.2 Thiazole-based ligands
The synthetic approach for thiazole-based ligands was supported by the previously
published structure, ST-979 (Figure 1).37 They can be considered as analogues were
the substituents were shifted by one carbon atom yielding 11 as initial structure with
moderate affinity. The resulting analogues exhibited superior binding affinities with
Ki values ranging from low micromolar to nanomolar concentrations. Similar to 1,3,4-
oxadiazole analogues, optimum binding activities are achieved with pyrrolidine or
piperidine basic heads. For example, compound 21 with pyrrolidine basic head and
three-carbon chain linker demonstrated a Ki value of 4 nM. Yet, analogues with para-
xylene (e.g., 41 and 42 with Ki values 7.0 nM and 72 nM, respectively) or piperidine
carbamoyl (e.g., 14 , 20, 23 with Ki values 5.4 nM, 54 nM and 42 nM, respectively)
linkers showed high binding affinities. Interestingly, the methylpiperazine carbamoyl
substituted thiazole 13 showed only weak affinity (Ki = 5,800 nM) compared to the
piperidine carbamoyl linked analogue 14 being more active by more than three orders
of magnitude. Different affinities of 2- and 3-carbon linked pyrrolidine or piperidine-
analogues were detected between the corresponding structures of (pyrrolidine-1-
12

yl)alkoxy linked 2-phenylthiazoles with Ki of 47 nM and 520 nM for dimethylen 8
and trimethylen analogue 10, respectively. Comparable binding behavior was detected
between the corresponding 4-chlorophenylthiazole analogues 16 and 18 as well as
between 17 and 19, both carrying a (piperidin-1-yl)alkoxy linker instead (Table
2).The arbitrary region (i.e. the phenyl moiety) showed wide electronic and steric
tolerability. Replacing the phenyl ring with pyridine was tolerable (or slightly less
active) but not with pyrimidine (except when it is coupled with para-xylene linker in
41 and 42). A cyano group in para-position generated the most active analogues (e.g.,
21, 22), which may indicate the favorable positioning of hydrogen bond acceptor at
this site presumably interacting with the Threonine residue identified by Roche et al.46
Replacing the 5-methyl by an 5-ethyl substituent showed inconsistent pattern; it
improves the binding affinity as with 46 (Ki = 200 nM), which is about five fold more
active than its methyl counterpart (19, Ki = 930 nM), but not with 44 and 45.
Table 2. Thiazole-based ligands along with H3R binding affinities.
Compd
No.
H3R Ki [nM]
X
Y
Z
R1
H
H
H
n
3
2
2
R2
R3
[95% CI]a, (n)b
4,100
[1,400; 12,200]; (3)
CH CH CH
CH CH CH
CH CH CH
CH3
CH3
CH3
7
8
9
47
[20; 110]; (3)
200
[90; 490]; (3)
520
[340; 780]; (3)
CH CH CH
CH CH CH
H
H
3
3
CH3
CH3
10
11
870
[130; 5,710]; (5)
13

220
[70; 650]; (8)
CH CH CH
CH CH CH
CH CH CH
CH CH CH
CH CH CH
H
H
1
0
0
3
2
CH3
CH3
CH3
CH3
CH3
12
13
14
15
16
5,800
[1,200; 27,000]; (4)
5.4
H
[2.2; 13.2]; (3)
280
4-Cl
4-Cl
[70; 1,170]; (4)
22
[5; 98]; (3)
44
CH CH CH
CH CH CH
CH CH CH
4-Cl
4-Cl
4-Cl
2
3
3
CH3
CH3
CH3
17
18
19
[12; 167]; (3)
450
[180; 1,150]; (3)
930
[570; 1,540]; (4)
54
CH CH CH
CH CH CH
4-Cl
0
3
CH3
CH3
20
21
22
[22; 131]; (3)
4.2
4-CN
[2.6; 6.6]; (3)
21
CH CH CH
CH CH CH
CH CH CH
CH CH CH
CH CH CH
CH CH CH
4-CN
4-CN
3-Cl
3-Cl
3-Cl
3
0
2
3
3
CH3
CH3
CH3
CH3
CH3
[2; 201]; (3)
42
23
24
25
[15; 122]; (4)
600
[310; 1,150]; (3)
430
[120; 1,550]; (3)
300
[130; 700]; (4)
26
27
28
29
98
3-Cl
H
0
3
2
CH3
CH3
CH3
[56; 170]; (6)
110
[40; 300]; (4)
CH
CH
N
N
CH
CH
100
[30; 380]; (4)
H
14

57
CH
CH
CH
N
N
N
CH
CH
CH
H
H
H
2
3
3
CH3
CH3
CH3
30
31
32
[42; 78]; (3)
410
[170; 1,010]; (4)
130
[20; 820]; (3)
5,900
[2,800; 12,600]; (4)
N
N
N
N
CH
CH
CH
CH
N
N
N
N
H
H
H
H
3
2
3
0
CH3
CH3
CH3
CH3
33
34
35
36
2,100
[800; 6,100]; (4)
1,100
[500; 2,400]; (6)
> 10,000
87
CH CH CH
CH CH CH
CH CH CH
3
1
1
1
1
3
3
3
CH3
CH3
37
39
40
41
42
44
45
46
[16; 462]; (5)
450
4-CN
4-CN
H
[190; 1,100]; (4)
190
[130; 280]; (3)
CH3
7.0
N
N
CH
CH
N
N
CH3
[2.6; 18.4]; (4)
72
H
CH3
[11; 492]; (4)
380
[180; 810]; (3)
CH CH CH
CH CH CH
CH CH CH
H
CH2CH3
CH2CH3
CH2CH3
170
[30; 870]; (4)
4-Cl
200
[100; 370]; (4)
4-Cl
aBinding affinity values (Ki) are expressed as average along with 95% confidence
intervals; bNumber of experiments.
Accordingly, analogues combining optimum moieties, i.e. 4-cyanophenyl moiety as
arbitrary region, para-xylene or piperidine carbamoyl linkers, and/or pyrrolidine or
15

piperidine basic heads were found to be the most active within this series of thiazole-
based H3R ligands.
2.6 In silico Molecular and ADMET properties of designed thiazole ligands.
Prediction of how much a ligand would have proper pharmacokinetics (ADME) and
pharmacodynamics (e.g., toxicological) properties (drug-like) is of great importance
during pre-clinical evaluation to assist drug discovery/development process, to guide
the optimization from a lead compound to a successful drug candidate, and to reduce
attrition rates during clinical trials, hence increasing the chance of reaching the
market.47
Some important chemical descriptors correlate well with ADMET properties such as
polar surface area (PSA) and low molecular weight (MW) for high oral absorption.48
Likewise, rapid renal clearance is correlated with hydrophilic and small compounds.
The hepatic metabolism of most drugs is associated with large and hydrophobic
compounds. Higher lipophilicity of compounds leads to increased metabolism and
poor absorption, along with an increased probability of binding to undesired
hydrophobic macromolecules, thereby increasing the potential for toxicity.49
Table 3. Compliance of the most active ligands to Lipinski’s and Veber’s rules.
Compound AlogP
MW
(˂ 500)
386.5
421.0
314.4
411.5
421.0
367.5
No. HBAa No. HBDb No. rotatable Molecular PSAc
(˂ 5)
(≤ 10)
(≤ 5)
bonds (≤ 10)
(˂ 140 Å)
2.33
3.00
1.80
2.21
3.00
1.56
5
5
4
6
5
5
1
1
1
1
1
1
4
4
5
4
4
6
75
75
79
99
75
81
14
20
21
23
27
41
aNumber of Hydrogen-bond acceptors (HBA); bNumber of Hydrogen-bond donors
(HBD); cPolar Surface Area (PSA).
16

Lipinski’s49 and Veber’s50 rules are one of the most important measures to evaluate
the drug-likeness and to predict if a compound of certain chemical properties would
be orally bioavailable. In spite of some observed exceptions to Lipinski’s and Veber’s
rules, the vast majority (90%) of the orally bioavailable compounds are within their
cut-off limits. Lipinski's rule of five states that, generally, an orally bioavailable
compound should not violate the following criteria: ≤ 5 hydrogen bond donors
(HBD); ≤ 10 hydrogen bond acceptors (HBA); MW ˂ 500; and logP value of ˂ 5.
Alternatively, Veber’s findings described the role of PSA and number of rotatable
bonds as criteria to estimate the oral bioavailability. Veber’s rule stated that for a
compound to be orally bioavailable it should have either: a PSA ≤ 140 Å and ≤ 10
rotatable bonds, or ≤ 12 HBD and HBA in total and ≤ 10 rotatable bonds. Clearly
from Table 3, the most potent ligands (those with Ki ˂ 100 nM) followed all
Lipinski’s and Veber’s rules without any exception, which highlight their drug-
likeness properties and their potential to pass the drug development process.
The most active ligands were in silico screened for predicted pharmacokinetics
properties (BBB penetration, absorption, solubility (Sw), hepatotoxicity, inhibition of
CYP2D6, and plasma protein binding (PPB)) being summarized in Table 4. The
intestinal absorption and BBB penetration were predicted by developing an ADME
model using descriptors 2D PSA and AlogP98 that include 95% and 99% confidence
ellipses. These ellipses define regions where well-absorbed compounds are expected
to be found. All active ligands showed medium (brain-blood ratio between 0.3:1 and
1:1) to high (brain-blood ratio between 1:1 and 5:1) penetration of BBB and good
intestinal absorption (at least 90% absorbed into bloodstream) within 95% confidence
ellipses (Table 4 and Figure 3). These results are therapeutically crucial for H3R
ligands to be effective for targeting neurodegenerative diseases and their potential for
17

oral administration. Moreover, these ligands showed low (-6.0 ˂ logSw ˂ -4.0) to
good (-4.0 ˂ logSw ˂ -2.0) solubility, non-hepatotoxicity, and variable properties for
inhibition of CYP2D6 and PPB (Table 4). However, the compounds with highest
affinity in the series (14, 21 and 41) provide some deficiencies regarding BBB
permeability and display high PPB (PPB > 95% as with 14 and 41).
Table 4. Predicted ADME profiles of the most active ligands.
Compound
BBBa
Absorptionb Solubilityc Hepatotoxicityd
CYP2D6e
inhibitor
non-inhibitor
non-inhibitor
non-inhibitor
non-inhibitor
non-inhibitor
PPBf
2
1
2
2
1
2
0
0
0
0
0
0
3
2
3
3
2
3
nontoxic
nontoxic
nontoxic
nontoxic
nontoxic
nontoxic
2 (˃ 95%)
0 (˂ 90%)
1 (˃ 90%)
0 (˂ 90%)
0 (˂ 90%)
2 (˃ 95%)
14
20
21
23
27
41
aPredicts ability of the compound to penetrate the blood brain barrier (BBB). Levels 0, 1, 2, 3, or 4
b
correspond to very high, high, medium, low, or undefined penetration, respectively; Predicts human
intestinal absorption after oral administration. Levels 0, 1, 2, or 3 correspond to good, moderate, poor,
c
or very poor absorption, respectively; Predicts the solubility of each compound in water at 25oC.
Levels 0, 1, 2, 3, 4, 5, or 6 correspond to extremely low, very low, low, good, optimal, too soluble, or
d
unknown solubility, respectively; Predicts the occurrence of dose-dependent human hepatotoxicity.
f
ePredicts cytochrome P450, 2D6 inhibition. Predicts the likelihood that a compound will be highly
bound to carrier proteins on the blood (PPB, plasma protein binding). Levels 0, 1, or 2 correspond to
binding ˂ 90%, binding ˃ 90%, or binding ˃ 95%, respectively.
As ligand efficiency measures emerge as increasingly important parameters in hit-to-
lead optimization, we calculated ligand efficiency (LE) and lipophilic ligand
efficiency (LLE) according to literature, based on in silico AlogP-values and in vitro
binding affinities.51 LE values were within the commonly accepted limits with LE >
0.3 kcal per mole per non-H-atoms (nHA) (0.42, 0.50 and 0.41 kcal / mole / nHA for
compounds 14, 21 and 41 respectively).52 LLE values conform to the acceptance
criteria of LLE > 5, being 5.9, 6.6 and 6.6 for 14, 21 and 41, respectively.53
18

Figure 3. Plot of PSA vs AlogP (calculated via “Calculate Molecular Properties”
module of Discovery Studio 2.5.5) for the most active ligands showing 95% and 99%
confidence limit ellipses corresponding to penetration of BBB and intestinal
absorption.
The United States Food and Drug Administration (US FDA) standard toxicity risk
predictor software TOPKAT (Discovery Studio, Accelrys, USA) locates fragments
within the compound that indicate a potential threat to toxicity risk. TOPKAT
(TOxicity Prediction by Komputer Assisted Technology) employs robust and cross-
validated Quantitative Structure Toxicity Relationship (QSTR) models for assessing
various measures of toxicity and utilizing the patented Optimal Predictive Space
validation method to assist in interpreting the results. Toxicity screening results of
TOPKAT for the most active H3R ligands showed no risk of AMES mutagenicity, and
tolerable ocular and skin irritation (Table 5); however, they possess potential US FDA
rodent carcinogenicity, except against female mouse. Compound 14 is the only non-
skin sensitizing agent among the three most active compounds and appears as single-
carcinogen in male mouse and female rat, whereas the cyano-containing ligands 21
and 41 differentiate as skin sensitizing and multi-carcinogenic agents. Moreover,
particular attention was paid to the risk of aerobic biodegradability as 2-
phenylthiazoles are known to produce hepatotoxic and/or nephrotoxic thioamides and
19

55
1,2-dicarbonyl-species upon oxidative metabolism.54,
Thus, exclusively in 5-
position alkylated thiazole derivatives were synthesized to achieve reduced oxidative
ring-scission and biodegradable probability, confirmed by the TOPKAT screening
results.56
Besides the biological evaluation, this in silico screening provided additional
information that could be useful for further lead optimization of the presented
compounds (cf. additional in silico ADMET and toxicity information on compounds
8, 16, 17, 22, 30, 37, and 42 is provided in Supplementary Material).
20

Table 5. Toxicity profile of the most active ligands using toxicity prediction, extensible protocol of Accelrys Discovery studio 2.5.5.
Carcinogenicity_USFDAd
Aerobic
AMES
Ocular
Skin
Skin
Compound
BioDegradabilitya
Mutagenicityb
Irritancyc
Irritancyc
Sensitizerc
Female Mouse Male Mouse
Female rat
Male Rat
-
-
-
-
-
-
+
+
+
+
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
+
-
-
-
14
20
21
23
27
41
-
++
+
+
-
++
++
-
++
++
-
-
-
++
+
+
+
+
++
aPrediction of oxidative degradability-properties of compounds. Indicators -, or + correspond to non-biodegradability, or degradability by bioorganisms, respectively;
bPredicted mutagenicity in AMES mutagenicity test. Indicators -, or + correspond to non-mutagenicity, or mutagenicity, respectively; Prediction of organic toxicity.
c
d
Indicators -, +, or ++ correspond to none, mild, or severe / strong toxic properties, respectively; Prediction of carcinogenic properties. Indicators -, +, or ++ correspond to
classification as Non-Carcinogen, Single-Carcinogen, or Multi-Carcinogen, respectively.
21

3. Conclusions
A novel series of oxadiazole and thiazole derivatives as H3R ligands are explored in
the present study. Oxadiazole ligands showed mediocre affinities unlike the thiazole,
which showed potent H3R affinities in low nanomolar concentration range. The
highest affinities are observed with ligands of pyrrolidine or piperidine basic heads
and 2 – 3 carbon linkers. However, thiazole ligands of para-xylene or piperidine
carbamoyl linkers showed high binding affinities, too. The examined compounds
show excellent drug-like properties in compliance with Lipinski’s and Veber’s rules
and satisfactory in silico ADMET properties. The results from this study will be
helpful for further improvements of potent H3R ligands.
22

4. Experimental
4.1. Chemistry
Reagents and solvents for synthesis were purchased from Sigma-Aldrich, VWR
Chemicals, Fisher Scientific, Alfa Aesar and Chemsolute and were used without
1
further purifications. H-NMR and 13C-NMR were recorded on a Bruker AMX
spectrometer (Bruker, Germany) at 300 and 75 MHz respectively, where CDCl3 or
DMSO-d6 was used as a solvent. Tetramethylsilane was used as standard and
chemical shifts are reported in part per million (ppm). Spin multiplicities are given as
s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quintet) or m
(multiplet). Approximate coupling constants (J) in Hertz (Hz). Number and
assignment of protons (ax, axial; eq, equatorial; ph, phenyl; pip, piperidine; pyra,
pyrazine; pyr, pyridine; pyrr, pyrrolidine). Elementary analyses (C, H, N) were
measured on a CHN-Rapid (Heraeus, Germany) and all final compounds were within
0.4 % of the theoretical values. Electrospray ionization mass spectrometry (ESI-MS)
was performed on an amaZon speed (Bruker, Germany) in positive polarity. Data are
listed as mass number ([M+H+]). High-resolution mass spectra (HRMS) were run in
electrospray ionization (ESI) mode. Melting points (m.p., uncorrected) were
determined on a M-564 Büchi melting point apparatus (Büchi, Germany). Preparative
column chromatography was performed on silica gel 60 M, 0.04-0.063 mm
(Macherey-Nagel, Germany) and thin-layer chromatography (TLC) was carried out
using pre-coated silica gel 60 with fluorescence indicator at UV 254 nm (Macherey-
Nagel, Germany).
4.1.1. General Reaction Procedures.
General Procedure (A). Preparation of 4-Phenyl-3-thiosemicarbazide. The
phenylisothiocyanate (30 mmol) was added, dropwise, over a period of 1 h to a stirred
23

o
solution of hydrazine (30 mmol) in methanol (8 mL) at 65-80 C. The reaction
mixture was stirred for 20 min more at the same temperature. The solvent was
removed by evaporation in vacuo and the precipitate was collected by filtration and
washed with petroleum ether. The compound was used without further purification.
General Procedure (B). Preparation of 2-Anilino-5-alkyl-1,3,4-oxadiazole. A
carboxylic acid (0.33 mmol), a 4-phenyl-3-thiosemicarbazide (0.33 mmol), and EDCI
(182 mg, 1.0 mmol) were mixed in CH2Cl2 (15 mL), and the reaction mixture was
stirred at room temperature for 12 h. The organic layer was washed three times with
saturated NaHCO3, dried over MgSO4, filtered, and concentrated. Chromatography
(5:95 MeOH/DCM, silica gel) of the residue afforded the title compound.
General Procedure (C). Preparation of Phenyl amidoximes. To a stirred solution
of phenyl nitrile (0.1 mol) in ethanol (100 mL), a solution of hydroxylamine (0,6 mol)
in H2O (150 mL) was slowly added followed by NaHCO3 (0.3 mol). The resulting
mixture was refluxed for 3h. The solvent was evaporated in vacuo and the resulting
residue was poured into cold water. The formed precipitate was filtered off, washed
with water and dried in vacuo. The obtained product was recrystallized from
methanol.
General Procedure (D). Preparation of 1,2,4-Oxadiazole-5-carboxylates. Pyridine
(0.015 mol) was added to a solution of phenyl amidoxime (0.01 mol) dissolved in dry
CHCl3 (100 ml). Ethyl chloroxalate (0.025 mol) was then added to the solution under
vigorous stirring. The mixture was stirred at room temperature for 30 min, then
refluxed for 5 h, cooled to rt, washed with water and HCl (5%). Organic layer was
dried under MgSO4, concentrated in vacuo and the formed residue was purified by
flash-chromatography on silica gel.
24

General Procedure (E). Preparation of 1,2,4-Oxadiazole-5-carboxamides. A
primary amine (0.5 mmol.) was added to the carboxylate (0.5 mmol) dissolved in dry
ethanol (5 mL). The reaction mixture was refluxed for 12 h, and then cooled to rt. The
organic solvent was dried in vacuo and the crude product was purified by flash-
chromatography on silica gel to give the target compounds
General Procedure (F). Preparation of 4-Hydroxy-5-methylthiazole. Pyridine
(0.005 mol) was added to a mixture of thiolactic acid (0.02 mol) and nitrile (0.0.2
O
mol) at 23 C under argon. The reaction mixture was then heated at 100 OC for 2 h.
After cooling, the precipitate was collected, washed with absolute ethanol, and
recrystallized from methanol to afford the product.
General procedure (G). O-Alkylations of Hydroxy thiazoles
To a solution of thiazole (1.0 eq.) dissolved in DMF (4 mL), K2CO3 (3.0 eq.) was
added, and the reaction mixture was left for 30 min at 60 °C. Then KI (1.0 eq.) and
the alkyl halide (0.9 eq.) was added and the reaction was left for an additional 8 h.
The reaction mixture was taken up in DCM, washed with Na2CO3 (3x) and dried over
MgSO4 and concentrated in vacuo. The concentrate was purified by flash
chromatography yielding the desired compound.
General procedure (H). Preparation of 2-Phenyl-4-hydroxy-5-ethylthiazole.
Pyridine (5.83 mmol) was added to a mixture of ethyl 2-bromobutanoate (6.41 mmol)
O
and thiobenzamide (5.83 mmol) at 23 C under argon. The reaction mixture was then
heated at 100 OC for 2 h. After cooling, the precipitate was collected and washed with
absolute ethanol, and the recrystallized from methanol to afford the desired product.
General procedure (I). Preparation of Bromoethyl benzyl thiazole. 1,4-
Bis(bromomethyl)benzen (4.0 mmol), 4-hydroxythiazole (1.0 mmol) and K2CO3 (2.0
mmol) dissolved in 50 mL acetone was refluxed for 12 h. After cooling, acetone was
25

evaporated and the crude product was constituted in DCM. The organic layer was
washed with NaHCO3 solution (3x) and dried over Na2SO3. The final compound was
purified with flash chromatography.
General procedure (J). Nucleophilic substitution of Bromoethyl benzyl thiazole.
The bromoethyl benzyl thiazole (1.0 eq.) was dissolved in DMF (4 mL) and K2CO3
(3.0 eq.) was added. The reaction mixture was left for 30 min at 60 °C, then the
piperidine or pyrolidine (0.9 eq.) was added and the reaction was left for an additional
8 h. The reaction mixture was taken up in DCM, washed with Na2CO3 (3x) and dried
over MgSO4 and concentrated in vacuo. The concentrate was purified by flash
chromatography yielding the desired compound.
5-Methyl-2-phenyl-4-(2-(pyrrolidin-1-yl)ethoxy)thiazole (8). Compound 8 was
prepared according to procedure G from 5-methyl-2-phenylthiazol-4-ol and 1-(2-
1
chloroethyl)pyrrolidine to afford 62 mg of yellow solid (70%). H NMR (CDCl3, 300
MHz) δ 7.77 (dd, J = 8.1, 1.8 Hz, 2H, ph-2,6H), 7.38 – 7.23 (m, 3H, ph-3,4,5H), 4.45
(t, J = 6.0 Hz, 2H, OCH2), 2.87 (t, J = 6.0 Hz, 2H, OCH2CH2N), 2.73 – 2.50 (m, 4H,
13
pyrr-2,5H2), 2.22 (s, 3H, CH3), 1.77 (p, J = 1.8 Hz, 4H, pyrr-3,4H2). C NMR (151
MHz, CDCl3) δ 9.40, 23.53, 54.67, 55.32, 69.26, 107.09, 125.34, 128.78, 129.29,
134.00, 159.47, 159.53. HRMS m/z 289.1384 [M+H+] (calcd for C16H21N2OS,
289.1375). Anal. Calcd for C16H20N2OS: C 66.63, H 6.99, N 9.51, S 11.12. Found:
C 66.82, H 7.24, N 9.18, S 11.51.
5-Methyl-2-phenylthiazol-4-yl [1,4'-bipiperidine]-1'-carboxylate (14). Compound
14 was prepared according to procedure G from 5-methyl-2-phenylthiazol-4-ol and
1
[1,4'-bipiperidine]-1'-carbonyl chloride to afford 74 mg of yellow solid (80%). H
NMR (CDCl3, 300 MHz) δ 7.79 – 7.74 (dd, J = 2.1, 1.8 Hz,, 2H, ph-2,6H), 7.31 (d, J
26

= 1.8 Hz, 1H, ph-4H), 7.29 (d, J = 2.1 Hz, 2H, ph-3,5H), 4.27 (dd, J = 30.2, 12.8 Hz,
2H, pip1-2,6Heq), 2.85 (m, 2H, pip1-2,6Hax), 2.55 – 2.32 (m, 5H, pip1-4H, pip2-
2,6H2ax/eq), 2.22 (s, 3H, CH3), 1.94 – 1.72 (m, 2H, pip1-3,5Heq), 1.67 – 1.42 (m, 6H,
pip1-3,5Hax, pip2-3,5H2ax/eq), 1.42 – 1.27 (m, 2H, pip2-4H2ax/eq). 13C NMR (75 MHz,
CDCl3) δ 10.03, 24.63, 26.21, 28.10, 44.40, 50.20, 62.36, 117.88, 125.72, 128.76,
129.81, 133.37, 151.79, 152.31, 161.20. HRMS m/z 386.1910 [M+H+] (calcd for
C21H28N3O2S, 386.1902). Anal. Calcd for C21H27N3O2S: C 65.43, H 7.06, N 10.90,
S 8.32. Found: C 66.18, H 7.12, N 10.84, S 8.38.
2-(4-Chlorophenyl)-5-methyl-4-(2-(pyrrolidin-1-yl)ethoxy)thiazole
(16).
Compound 16 was prepared according to procedure G from 2-(4-chlorophenyl)-5-
methylthiazol-4-ol and 1-(2-chloroethyl)pyrrolidine to afford 79 mg of yellow solid
1
(91%). H NMR (CDCl3, 300 MHz) δ 7.66 (d, J = 8.6 Hz, 2H, ph-2,6H), 7.24 (d, J =
8.6 Hz, 2H, ph-3,5H), 4.40 (t, J = 6.0 Hz, 2H, OCH2), 2.81 (t, J = 6.0 Hz, 2H,
OCH2CH2N), 2.63 – 2.47 (m, 4H, pyrr-2,5H2), 2.19 (s, 3H, CH3), 1.72 (p, J = 3.0 Hz,
4H, pyrr-3,4H2). 13C NMR (75 MHz, DMSO) δ 9.28, 23.42, 54.59, 55.26, 69.42,
107.31, 126.37, 128.82, 132.38, 134.89, 157.85, 159.64. HRMS m/z 323.0991
[M+H+] (calcd for C16H19ClN2OS, 323.0985). Anal. Calcd for C16H20ClN2OS: C
59.52, H 5.93, N 8.68, S 9.93. Found: C 59.88, H 6.07, N 8.41, S 10.37.
2-(4-Chlorophenyl)-5-methyl-4-(2-(piperidin-1-yl)ethoxy)thiazole (17). Compound
17 was prepared according to procedure G from 2-(4-chlorophenyl)-5-methylthiazol-
1
4-ol and 1-(3-chloroethyl)piperidine to afford 43 mg of yellow solid (57%). H NMR
(CDCl3, 300 MHz) δ 7.69 (d, J = 8.5 Hz, 2H, ph-2,6H), 7.28 (d, J = 8.5 Hz, 2H, ph-
3,5H), 4.44 (t, J = 6.0 Hz, 3H, OCH2), 2.76 (t, J = 6.0 Hz, 3H, OCH2CH2N), 2.62 –
2.47 (m, 4H, pip-2,6H2ax/eq), 2.21 (s, 3H, CH3), 1.72 – 1.52 (m, 4H, pip-3,5H2ax/eq),
27

1.45 – 1.32 (m, 2H, pip-4H2ax/eq). 13C NMR (75 MHz, CDCl3) δ, 9.73, 24.63, 26.34,
55.33, 58.66, 68.65, 107.37, 126.48, 128.93, 132.48, 134.99, 158.01, 159.78. HRMS
m/z 337.1148 [M+H+] (calcd for C17H22ClN2OS, 337.1141). Anal. Calcd for
C17H21ClN2OS: C 60.61, H 6.28, N 8.32, S 9.52. Found: C 61.08, H 6.51, N 8.41,
S 9.85.
2-(4-Chlorophenyl)-5-methylthiazol-4-yl [1,4'-bipiperidine]-1'-carboxylate (20).
Compound 20 was prepared according to procedure G from 2-(3-chlorophenyl)-5-
methylthiazol-4-ol and [1,4'-bipiperidine]-1'-carbonyl chloride to afford 84 mg of
yellow solid (89%). 1H NMR (CDCl3, 300 MHz) δ 7.69 (d, J = 8.6 Hz, 2H, ph-2,6H),
7.27 (d, J = 8.6 Hz, 2H, ph-3,5H), 4.27 (dd, J = 28.1, 13.3 Hz, 2H, pip1-2,6Heq), 2.86
(m, 2H, pip2-2,6Heq), 2.62 – 2.34 (m, 5H, pip1-4H, pip2-2,6H2ax/eq), 2.22 (s, 3H, CH3),
1.96 – 1.74 (m, 2H, pip1-3,5Heq), 1.68 – 1.45 (m, 6H, pip1-3,5Hax, pip2-3,5H2ax/eq),
13
1.44 – 1.29 (m, 2H, pip2-4H2). C NMR (75 MHz, CDCl3) δ 10.02, 24.46, 25.96,
27.40, 27.97, 44.04, 44.33, 50.16, 62.39, 118.40, 126.90, 128.99, 131.86, 135.69,
151.88, 152.22, 159.79. HRMS m/z 420.1514 [M+H+] (calcd for C21H26ClN3O2S,
420.1513). Anal. Calcd for C21H27ClN3O2S: C 60.06, H 6.24, N 10.01, S 7.63. Found:
C 60.12, H 6.20, N 10.18, S 7.66.
4-(5-Methyl-4-(2-(pyrrolidin-1-yl)ethoxy)thiazol-2-yl)benzonitrile
(21).
Compound 21 was prepared according to procedure G from 2-(3-cyanophenyl)-5-
methylthiazol-4-ol and 1-(2-chloroethyl)pyrrolidine to afford 67 mg of yellow solid
(80%). 1H NMR (CDCl3, 300 MHz) δ 7.74 (d, J = 8.7 Hz, 2H, ph-2,6H), 7.49 (d, J =
8.7 Hz, 2H, ph-3,5H), 4.38 (t, J = 5.4 Hz, 2H, OCH2), 2.84 (t, J = 5.5 Hz, 2H,
OCH2CH2N), 2.66 – 2.55 (m, 4H, pyrr-2,5H2), 2.16 (s, 3H, CH3), 1.80 – 1.64 (m, 4H,
28

pyrr-3,4H2). 13C NMR (75 MHz, CDCl3) δ 9.36, 31.25, 36.37, 54.98, 68.92, 109.48,
111.85, 118.48, 125.35, 132.44, 137.43, 156.31, 160.10. HRMS m/z 314.1332
[M+H+] (calcd for C17H20N3OS, 314.1327). Anal. Calcd for C17H19N3OS: C 66.03,
H 6.46, N 12.83, S 9.79. Found: C 66.18, H 6.51, N 12.79, S 9.72.
4-(5-Methyl-4-(3-(piperidin-1-yl)propoxy)thiazol-2-yl)benzonitrile
(22).
Compound 22 was prepared according to procedure G from 2-(3-cyanophenyl)-5-
methylthiazol-4-ol and 1-(3-chloropropyl)piperidine to afford 75 mg of yellow solid
(83%). 1H NMR (CDCl3, 300 MHz) δ 7.81 (d, J = 8.7 Hz, 2H, ph-2,6H), 7.55 (d, J =
8.7 Hz, 2H, ph-3-5H), 4.29 (t, J = 6.4 Hz, 2H, OCH2), 2.45 – 2.40 (m, 2H,
OCH2CH2), 2.39 – 2.31 (m, 4H, pip-2,6H2ax/eq), 2.21 (s, 3H, CH3), 2.03 – 1.82 (m, 2H,
OCH2CH2CH2N), 1.68 – 1.48 (m, 4H, pip-3,5H2ax/eq), 1.45 – 1.27 (m, 2H, pip-
4H2ax/eq). 13C NMR (75 MHz, CDCl3) δ 9.40, 24.31, 25.80, 26.97, 54.54, 55.91, 69.11,
109.34, 112.00, 118.62, 125.45, 132.53, 137.66, 156.33, 160.53. HRMS m/z 342.1645
[M+H+] (calcd for C19H24N3OS, 342.1640). Anal. Calcd for C19H23N3OS: C 66.83,
H 6.79, N 12.31, S 9.39. Found: C 66.79, H 6.80, N 12.31, S 9.44.
2-(4-Cyanophenyl)-5-methylthiazol-4-yl [1,4'-bipiperidine]-1'-carboxylate (23).
Compound 23 was prepared according to procedure G from 4-(4-hydroxy-5-
methylthiazol-2-yl)benzonitrile and [1,4'-bipiperidine]-1'-carbonyl chloride to afford
1
91 mg of yellow solid (91%). H NMR (CDCl3, 300 MHz) δ 7.85 (d, J = 8.7 Hz, 2H,
ph-2,6H), 7.59 (d, J = 8.2 Hz, 2H, ph-3,5H), 4.26 (dd, J = 31.1, 13.0 Hz, 2H, pip1-
2,6Heq), 2.84 (m, 2H, pip2-2,6Heq), 2.56 – 2.44 (m, 5H, pip1-4H, pip2-2,6H2ax/eq), 2.26
(s, 3H, CH3), 2.01 – 1.76 (m, 2H, pip1-3,5Heq), 1.66 – 1.47 (m, 6H, pip1-3,5Hax, pip2-
3,5H2ax/eq), 1.45 – 1.30 (m, 2H, pip2-4H2). 13C NMR (75 MHz, CDCl3) δ 10.08, 24.41,
29