N-[(2,6-Dimethylphenoxy)alkyl]aminoalkanols - Their physicochemical and anticonvulsant properties

Article abstract of DOI:10.1016/j.bmc.2015.06.045

Twenty four new N-[(dimethylphenoxy)alkyl]aminoalkanols have been synthesized and evaluated for anticonvulsant activity in a series of in vivo tests: the maximum electroshock (MES), 6 Hz, and subcutaneous metrazole (ScMet). The compounds were also evaluated for possible neurotoxicity in the rotarod test. The majority of the achieved compounds exhibit quantified anticonvulsant activity. The most active compound 4: R-(-)-2N-[(2,6-dimethylphenoxy)ethyl]aminopropan-1-ol is active in MES with ED₅₀ = 5.34 (male mice, ip), 22.28 (female mice, ip), 51.19 (male mice, po), 7.43 (rats, ip), and 28.60 (rats, po). Thermal analysis proved that its hydrochloride (4a) can exist in polymorphic forms. The compound binds to σ, 5-HT_1A, and α₂ receptors as well as 5-HT transporter and it does not exhibit mutagenic properties.

Full text of DOI:10.1016/j.bmc.2015.06.045

Accepted Manuscript
N-[(2,6-Dimethylphenoxy)alkyl]aminoalkanols - Their Physicochemical and
Anticonvulsant Properties
Anna M. Waszkielewicz, Marek Cegła, Ewa Żesławska, Wojciech Nitek,
Karolina Słoczyńska, Henryk Marona
PII:
S0968-0896(15)00539-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.06.045
BMC 12401
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
26 March 2015
12 June 2015
18 June 2015
Please cite this article as: Waszkielewicz, A.M., Cegła, M., Żesławska, E., Nitek, W., Słoczyńska, K., Marona, H.,
N-[(2,6-Dimethylphenoxy)alkyl]aminoalkanols - Their Physicochemical and Anticonvulsant Properties,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.06.045
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N-[(2,6-Dimethylphenoxy)alkyl]aminoalkanols - Their
Physicochemical and Anticonvulsant Properties
a
b
c
d
Anna M. Waszkielewicz *, Marek Cegła , Ewa Żesławska , Wojciech Nitek , Karolina
a,e
a
Słoczyńska , and Henryk Marona
a
Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy,
Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
b
Department of Organic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy,
Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
c
Department of Chemistry, Institute of Biology, Pedagogical University, Podchorążych 2, 30-
0
84 Krakow, Poland
d
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland
Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University
e
Medical College, Medyczna 9, 30-688 Krakow, Poland
*
e-mail address awaszkie@cm-uj.krakow.pl
Tel. +4812 620 55 76
Fax +4812 620 54 05
ABSTRACT
Twenty four new N-[(dimethylphenoxy)alkyl]aminoalkanols have been synthesized and
evaluated for anticonvulsant activity in a series of in vivo tests: the maximum electroshock
(
MES), 6 Hz, and subcutaneous metrazole (ScMet). The compounds were also evaluated for
possible neurotoxicity in the rotarod test. The majority of the achieved compounds exhibit
quantified anticonvulsant activity. The most active compound 4: R-(-)-2N-[(2,6-
dimethylphenoxy)ethyl]aminopropan-1-ol is active in MES with ED50 =5.34 (male mice, i.p.),
2
2.28 (female mice, i.p.), 51.19 (male mice, p.o.), 7.43 (rats, i.p.), and 28.60 (rats, p.o.).
Thermal analysis proved that its hydrochloride (4a) can exist in polymorphic forms. The
2
compound binds to σ, 5-HT1A, and α receptors as well as 5-HT transporter and it does not
exhibit mutagenic properties.
1

Keywords: 6 Hz, Ames test, aminoalkanols, anticonvulsant activity, crystal structure, DSC,
MES, mutagenicity, rotarod, seizures, TG, TOX
2

1. Introduction
Epilepsy is one of the most common neurological disorders, characterized by
hipersynchronous activity of neurons. Being caused by multiple factors such as genes, trauma,
neurodegeneration, or intoxication, it concerns over 50 million people worldwide.
Approximately 35% seizures are considered pharmacoresistant, therefore, there exist premises
for further drug discovery process for this indication. Search for new drugs is continuously
based on in vivo screening, basically performed within the Antiepileptic Drug Development
1
program (ADD, National Institute of Neurological Disorders and Stroke, Bethesda, USA).
Due to the fact that all current antiepileptic drugs have been associated with this program, in
order to improve effectiveness of new drugs, the program has evolved for epileptogenesis and
psychomotor seizure in vivo tests (6 Hz). Up to date, many papers on new anticonvulsant
2
-4
agents base on the program, concerning various chemical entities.
Anticonvulsant properties of cardiovascular drugs containing aroxyalkyl or aminoalkanol
5-6
moieties, such as mexiletine and propranolol, respectively, have been premise for our
research. On this basis we have found some promising anticonvulsant activity among
7
-11
aroxyalkyl (and also aroxyacetyl) derivatives of aminoalkanols.
We already described
synthesis and evaluation of pharmacological properties of appropriate derivatives of 4-
7
-9
10
methylphenol, 2,6-dimethylphenol, 4-chlor-3-methylphenol and 2-chlor-5-methylphenol,
1
1
as well as 4-chlor-2-methylphenol.
One of them, e.g. (S)-(+)-2N-[(2,6-
dimethylphenoxy)ethyl]aminobutan-1-ol hydrochloride displayed protection against MES-
8
induced seizures and low toxicity (mice, i.p.) with an ED50=7.57 mg / kg and PI=4.55.
Another compound, (D,L)-trans-2N-[(2,6-dimethylphenoxy)ethyl]aminocyclohexan-1-ol
7
exhibited ED50 = 7.73 mg/kg b.w. and PI (MES, mice, i.p.) = 3.90 (Figure 1). Both protective
indices in the MES test in mice are higher than that of valproate (PI=1.7) and similar to that of
carbamazepine (PI=4.9). From the preliminary assay data, it was ascertained that
anticonvulsant activity was associated with aminoalkanol type and configuration. Such
activity drew our attention onto influence of position and type of substituents within the
phenyl ring as well as length of the alkyl linker between the phenoxy and the aminoalkanol
groups. In case of active racemates, enantiomeric structures have been achieved.
3

2
-3, 7-8
Figure 1. Chemical structures of reference compounds.
We herein report results of evaluation of biological activity of novel derivatives of 2,6-
dimethylphenol, where the linker between phenoxyl and aminoalkanol moieties contains 2-4
methylene groups. Some of the presented compounds – 7, 9, and 12, 15, 17, and 19-20 – have
9
been published for evaluation of their lipophilicity and screening results. This article
completes the documentation concerning their physicochemical data, activity profile and
structure-activity relationship. Compounds 3-6, 8, 10, 12-15 and 17-22 were subject to
intellectual property rights protection as a group of potential anticonvulsant drug candidates,
12
in our team, and therefore they were assigned CAS numbers.
The choice of substituents in the phenyl ring was based on our previous results and it seemed
optimal to use two methyl substituents in positions 2,6. Within the group of 2,6-
dimethylphenoxyalkyl derivatives, the chosen aminoalkanol moieties represent examples of
our experience as well – the 1,2-aminoalkanol configuration has proved the optimal for
anticonvulsant activity. However, exceptions also have been achieved and evaluated, e.g.
derivatives of 3-amino-1-propanol (compound 2), 5-amino-1-pentanol (8), or D,L-valinol
(
10). Moreover, it was decided to choose as simple moiety as possible, in order to keep
lipophilicity in the required range of clogP=2-3, for possible distribution in the nervous
system instead of cardiovascular one. Therefore, any potential of adverse events related with
similar mechanisms of action would be minimized.
4

The length of the linker between both moieties was also an important parameter, since using
ethylene group resulted in good activity and acceptable lipophilicity, and then propylene
linker diminished activity and enlarged neurotoxicity at higher lipophilicity values. Next, use
of butylene linker was dictated by interest in structure-activity relationship for the most active
compounds in the investigated group. It was also interesting for the fact that clogP did not
differ much for both propylene and butylene derivatives.
2. Results and discussion
2
.1. Chemistry
Twenty four new compounds have been achieved by N-alkylation of appropriate (2,6-
7
-
dimethylphenoxy)ethyl, -propyl or -butyl bromides and the procedures have been published.
8
, 13
The structures and clogP are presented in Table 1. It can be observed that with this group
of derivatives the lipophilicity parameter clogP ranges 2.31-4.03 which is acceptable for
1
4
anticonvulsants.
Table 1
Chemical structures of the title compounds
n
2
Compd.
Z
Configuration clogP
1
-
2.74
x HCl
2
3
-
2.39
2.40
R,S
a)
3a
R,S
2.40
x HCl
x HCl
4
R
R
2.40
a)
4a
2.40
5

5
S
S
2.40
a)
5a
2.40
3.05
x HCl
x HCl
a)
6
7
R,S
R,S
2.93
a)
8
9
-
-
2.69
x HCl
2.45
10
D,L
3.10
1
1
2
2.31
3.66
1
R,S
13
14
15
R
S
3.66
3.66
a)
R,S
4.31
x HCl
16
L-threo
3.57
3
17
R,S
R,S
2.76
2.76
18
1
9
0
-
3.16
3.29
2
R,S
6

a)
2
1
2
R,S
R,S
3.29
x HCl
2
4.03
4
23
R,S
R
2.74
2.74
24
a)
Parameter calculated for base with use of ChemBioDraw Ultra 12.0 computer program.
During synthetic work, the pure compound 4a was achieved by saturation of 4 in acetone
solution with gaseous HCl, resulting in crystals with melting point 107-109 °C. However,
double recrystallization resulted in achievement of crystals with melting point 115-117 °C.
The higher melting point was also achieved if the base was saturated with gaseous HCl in
ethyl acetate/EtOH (4:1) solution instead of acetone. For this reason, the salts are marked as
4
a1 for the lower mp. and 4a2 for the higher one and they have been evaluated separately.
However, it was observed, that 4a2 during storage at room temperature can reform into 4a1.
The same was observed for 5a – the melting points ranged 106-108 °C and 115-117 °C (5a1
and 5a2, respectively). Due to the above observations, thermal analysis was performed for 3a-
5
a. The premise was that polymorphism – the ability of a molecule to crystallize into more
than one crystal form, influences many parameters such as solubility, shelf life, and
formulation processes. Pseudopolymorphism resulting from solvatation is related with
different amount of solvent molecules included in the crystal, and this fact may influence
1
5
accuracy of drug dosing. Differential scanning calorimetry (DSC) and thermogravimetry
TG) was performed for compounds 3a-5a. The results for 3a and 4a2 are presented in Fig. 2-
, respectively. It can be noticed from the DSC curve that 3a can exist in one crystalline form
(
3
since two separate endothermic peaks exist: one at the temperature corresponding to melting
point measured with criometer, and one more at ca. 300 °C indicating deterioration in these
conditions, confirmed by thermogravimetry (TG) and its derivative (DTG) curves. The results
obtained for 4a and 5a indicate that they both can exist in polymorphic forms. The thermal
curves for 4a and 5a indicate that they both deteriorate at ca. 300 °C. None of the
polymorphic forms of 4a contains water whereas both polymorphic forms of 5a contain small
amount of water. The mass of 5a1 during heating to 120 °C diminished by 1.68% and for 5a2
the mass diminished by 5.5%. The DSC curve for enantiomer 5a contains two endothermic
7

peaks: for 5a1 the peak at 109.9 °C correlates with melting point whereas the other
endothermic peak indicates transformation of 5a1 to 5a2 with mp. 115-117 °C (Fig. 5a2).
Figure 2. DSC, TG, and TDG curves of compound 3a.
Figure 3. DSC, TG, and TDG curves of compound 4a2.
8

Crystal structure of 4a2
An overview of the asymmetric unit of 4a2 with the atom numbering is presented in Fig.7.
The crystal structure of 4a2 confirms R configuration at C2. The bond lengths and bond
angles have typical values. The geometry of this molecule was compared to the earlier
1
6
determined crystal structure of mexiletine.
Both compounds contain 2-(2,6-
dimethylphenoxy)ethylamine moiety. 4a2 has longer side chain than mexiletine and its
conformation is bent. The arrangement of the ether oxygen atom (O2) and the protonated
nitrogen atom shows synclinal conformation (gouche). This conformation is also observed in
mexiletine. In 4a2 the hydroxyl oxygen atom (O1) and nitrogen atom also has synclinal
conformation. The comparison of selected parameters is shown in Table 2. The nitrogen atom
of 4a2 has different orientation in comparison to the known structure of mexiletine.
Figure 4. The molecular structure of 4a2 showing the atom numbering scheme. Displacement
ellipsoids are drawn at the 50% probability level.
Table 2
The selected bond lengths (Å), bond and torsion angles (°) in the crystal structure of 4a2 and
mexiletine
1
7
4
a2
Mexiletine
C6-O2-C5
111.52
-169.22
57.11
114.69
- 174.49
-60.55
56.24
C6-O2-C5-C4
O2-C5-C4-N1
O2-C5-C4-H4B
-63.22
9

The unit cell consists of two molecules of 4a2. The packing of the molecules in the unit cell
can be characterized by the intermolecular interaction listed in Table 3. The chlorine anion
connects two molecules via hydrogen bonds between protonated nitrogen atom of one
molecule and hydroxyl group of another molecule (Fig. 8). The crystal structure is also
stabilized by weak C-H··O and C-H··Cl interactions.
Table 3
Intermolecular interactions for 4a2
D-H···A
H···A (Å) D···A (Å) D-H-A (°) Symmetry code
N1-HN2B···Cl1
O1-H1···Cl1
C2-H2···O1
C3-H3B···Cl1
C4-H4B···O1
2.29(4)
2.23(4)
2.58
2.91
2.60
3.127(2)
3.034(2)
3.462(3)
3.858(3)
3.450(3)
174(3)
176(4)
147.5
163.0
144.5
-x+1, y-1/2, -z+1
-x+1, y-1/2, -z+1
-x, y-1/2, -z+1
-x+1, y-1/2, -z+1
10

Figure 5. Packing of molecules in the unit cell of the crystalline 4a2 projected along [100]
direction. Dashed lines indicate hydrogen bonds.
2
.2. Pharmacology
The anticonvulsant activity and neurotoxicity of all synthesized compounds have been
evaluated according to standard protocols within the National Institutes of Neurological
1
Disorders and Stroke (NINDS, Rockville, USA). Currently three tests are used for screening
the compounds for their anticonvulsant activity: MES (maximal electroshock seizures), ScMet
(
subcutaneous metrazol), 6 Hz (6 Hertz test). MES represents grand-mal epilepsy, ScMet –
petit-mal epilepsy, and 6 Hz – psychomotor seizures and epileptogenesis. The results are
confronted with rotarod test used for evaluation of neurotoxicity (TOX).
Comparing the screening results of compounds 1-24 (Table 4), it can be noticed that 2-amino-
1
-propanol derivatives exhibit the strongest activity. Moreover, R enantiomers have proved
better potency than S, which is not consistent with reference structure II. In particular, 2N-
11

(
2,6-dimethylphenoxy)ethyl]amino-1-propanol (3-5) seems the optimal compound of all title
group, especially the R enantiomer (4), active at 10 mg/kg b.w. 0.5 h after administration. It is
interesting that the racemate (3) is more toxic than any of the enantiomers (4, 5). The N-
methyl derivative (6) is visibly more toxic than its analog 4a.
Table 4
Screening results of the tested compounds in mice, i.p.
c)
Compd.
Dose
mg/kg
b.w.]
Time [h]
ASP
1
[
a)
a)
b)
MES
.5
ScMet
TOX
0.5 4
0
4
0.5
4
3
3
0/4
0/4
1/1 0/1
0/4
0/4
1
3
0
0
0/1 0/1 2/4 0/2
1
3
00
00
3
0
0
8/8
4/4
0/4
0/4
3
a
0/4
0/4
1/1 0/1
1/1
4
1
1
3
0/1
0/1
3/4 0/2
D
1
3
00
00
3
8/8
4/4
0/4
D
4
0/4
1/4
1/1 0/1
2/2 1/2
0/4
0/4
1
3
0
0
0/1 0/1 0/4 0/2
D
100
0/1 0/1 8/8 D 1/3
300
4/4
0/1 0/1 0/4 0/2
0/1 0/1 8/8 0/4
4/4 1/1
4
a2
5
30
0/1 0/1
3/3 0/3
1
1
1
3
00
00
3
0/4
0/4
1
3
0
0
2/4
1/1 0/1
0/4
0/1 0/1 0/4 0/2
D
1
3
00
00
3
8/8
4/4
D
6
8
0/4
0/4
1/1 0/1
0/4
0/4
4
1
3
0
0
0/1 0/1 1/4 0/2
1
3
30
00
00
1/1 0/1
8/8 0/4
4/4
0/1 0/1
3/3 0/1
0/1 0/1
1
1
1
1
3
00
00
0/1 0/1 7/8
0/1 4/4 1/1
0/1 0/1 0/4 0/2
1
0
3
30
0/1 0/1
2/2 0/2
D
D
1
3
00
00
3
0/1
7/8
0/2
4/4
0/4
0/4
1
0/4
0/4
1/1 0/1
1
3
0
0
0/1 0/1 0/4 0/2
D
1
3
00
00
3
8/8
4/4
D
1
4
0/4
0/4
0/4
0/4
1
1
0
12

3
0
1/1 0/1
3/3 0/3
0/1 0/1 0/4 0/2
D
100
00
0/1
7/8
4/4
0/3
D
3
1
1
6
8
30
0/1 0/1
0/1 0/1 0/4 0/2
-
D
D
D
D
1
3
00
00
3
0/1
0/1
7/8
3/4
0/4
0/4
1/1 0/1
3/3 0/1
0/4
0/4
1
1
3
0
0
0/1 0/1 2/4 0/2
0/1 0/1 8/8 1/3
1
3
00
00
3
4/4
0/4
0/4
2
2
1
2
0/4
0/4
1/1 0/1
3/3 0/2
1
4
1
3
0
0
0/1 0/1 0/4 0/2
D
1
3
00
00
0/1 8/8 D 0/3
4/4
D
30
0/1 0/1
1/1 0/1
0/1 1/1 2/4 0/2
D
1
3
00
00
0/1
8/8
0/1
4/4
a)
b)
Number of animals protected/number of animals tested in the MES and ScMet tests; Number of
c)
animals displaying motor impairment/number of animals used in the rotarod test; ASP classification:
1
– anticonvulsant activity at doses 100 mg/kg or less; 2 – anticonvulsant activity at doses greater than
1
00 mg/kg; 3 – compound inactive at 300 mg/kg; 4 – compound either active or inactive but toxic at
doses of 30 mg/kg; “-“ the compound was not tested in the particular case.;* number of animals which
D
died during test are stated in brackets; - death.
The most active compounds have been advanced to observations of the activity and
neurotoxicity in time in order to reach time of peak effect (TPE) (Table 5), which was needed
for evaluation of ED50 at the particular time – for most compounds it was 15 min after
administration. Then, ED50s and TD50s with proper confidence intervals (CI) for activity or
neurotoxicity doses have been determined (Table 6). It can be observed that among 2-amino-
1
-propanol derivatives of (2,6-dimethylphenoxy)butyl (3-5), that racemate’s ED50 in MES,
mice, i.p., seems larger than any of the enantiomers’ activity; however, the confidence
interval is broader.
13

Table 5
Observation of anticonvulsant activity and neurotoxicity in mice
Compd.
Route of
administration
i.p.
Test
Dose
Time [h]
TPE
[h]
0.25
0.25
0.25
[mg/kg] 0.25 0.5 1.0 2.0 4.0 6.0 24.0
2
4
6 Hz (32 mA)
TOX
100
100
15
4/4
4/4
8/8
4/4
4/4
4/4
8/8
8/8
4/4
6/8
4/4
5/8
2/4
4/4
3/4
4/4
0/8
6/8
4/4
4/4
4/8
3/4
0/4
0/4
3/4
3/4
7/8
4/4
4/4
3/4
4/8
7/8
3/4
5/8
2/4
0/8
2/4
4/4
3/4
4/4
0/8
0/8
4/4
0/4
3/4
0/4
0/4
0/4
0/4
0/4
i.p.
MES
20
0/4
2/4
0/4
1/8
0/4
1/4
0/4
1/8
0/4
0/4
0/4
0/8
6
6
Hz (32 mA)
Hz (44 mA)
TOX
55
50
50
0.25
0.25
0.25
0/8
1/8
55
p.o.
i.p.
i.p.
MES
TOX
MES
TOX
MES
40
200
15
40
25
0/4
3/8
0/4
0/8
0/4
3/4
0/4
2/4
0/8
0/8
0/4
0/4
0/4
0/8
0/4
0/8
0.25
0.25
0.25
0.25
0.25
0/8
0/4
5
1
1
50
0/4
0/4
0/4
0/8
0/8
0/4
0/4
0/4
0/4
0/4
0/4
0/8
0/4
0/4
6
Hz (32 mA)
TOX
50
00
50
0.25
0.25
1
85
1
00
1
2
2
2
3
4
i.p.
i.p.
i.p.
6 Hz (32 mA)
30
0.25
60
6 Hz (32 mA)
TOX
50
50
30
2/3
1/3
0/4
0/4
0/4
0/4
3/3
0/4
4/4
0/4
0/4
0/4
1/4
0.25
0.5
6 Hz (32 mA)
0.25
1
00
30
00
4/4
0/4
4/4
3/3
0/4
4/4
3/4
0/4
0/3
1/4
TOX
0.25
1
a)
b)
Number of animals protected / number of animals tested; number of animals exhibiting
toxicity / number of animals tested in the rotarod test TPE – time of peak effect.
14

Table 6
Quantitative evaluation of activity in mice, i.p.
Compd.
Route of
Test
Dose
Activity at
TPE
TPE
ED50 [mg/kg] (Confidence
Interval)
a)
administration
[mg/kg]
c)
3
i.p.
MES
2
4
8
0/8
3/8
3/8
5/8
7/8
1/8
3/8
7/8
8/8
TPE=0.5 h
ED50=10.08 (5.62-18.64)
1
3
6
2
4
i.p.
i.p.
MES
2.5
5
TPE=0.25 h
ED50=5.34 (3.50-7.40)
1
1
0
5
c)
n
MES
5
0/8
TPE=0.25 h
ED50=22.28 (19.55-24.7)
1
2
2
3
5
0
5
0
0/8
2/8
6/8
8/8
0/8
3/8
5/8
6/8
0/2
0/2
0/2
0/2
0/8
1/8
9/16
8/8
p.o.
i.p.
i.p.
MES
ScMET
6 Hz
20
TPE=0.25 h
ED50=51.19 (36.52-70.16)
4
5
7
0
8
5
6.3
2.5
ED50>50
1
2
5
30
37
4
6
5
0
TPE=0.25 h
ED50=19.5 (10.3-30.5)
(22 mA)
5
0
c)
i.p.
i.p.
6 Hz
TPE=0.25 h
ED50=7.06 (2.25-15.81)
(
22 mA)
6 Hz
32 mA)
23
27
0/8
4/16
3/8
13/16
8/8
0/8
TPE=0.25 h
ED50=30.90 (28.81-33.48)
(
3
3
5
1
5
5
a)
i.p.
6 Hz
12.5
25
TPE=0.25 h
ED50=30.02 (24.38-33.81)
(
44 mA)
2/8
3
3
5
15
30
0
5
0
3/8
13/16
15/16
0/8
c)
i.p.
i.p.
6 Hz
TPE=0.25 h
ED50=41.3 (31.24-51.29)
(
44 mA)
2/8
4
6
2.5
5
0
3/8
8/8
1/8
FRINGS
TPE=0.25 h
15

5
2/8
6/8
8/8
1/8
2/8
0/8
1/8
4/8
2/8
4/8
1/8
2/8
5/8
8/8
0/8
1/8
12/16
8/8
0/8
1/8
3/8
5/8
6/8
ED50=5.39 (3.71-6.98)
7.5
1
0
i.p.
i.p.
BIC
PIC
24
TPE=0.25h
ED50>75
4
7
0
5
25
TPE=0.25h
ED50>75
4
5
7
0
7
5
i.p.
i.p.
p.o.
TOX
15
TPE=0.25 h
TD50=29.48 (21.76-37.87)
2
3
5
5
8
0
b) c)
TOX
15
TPE=0.25 h
TD50=38.71 (31.67-43.53)
3
4
6
0
5
0
TOX
70
TPE=0.25 h
TD50=144.28 (120.69-185.38)
1
00
20
50
00
1
1
2
4
a2
5
i.p.
i.p.
MES
MES
TPE=0.25 h
ED50(MES)=22.28
TD50=38.71
TPE=0.25 h
6
0/8
4/8
5/8
6/8
8/8
0/8
0/8
0/3
0/3
0/8
3/8
5/8
8/8
1/8
0/8
6/8
5/8
5/8
8/8
0/8
0/8
ED50=8.59 (7.23-10.03)
7.5
9
1
1
2
5
i.p.
i.p.
i.p.
ScMET
TOX
20
ED50>52
3
4
5
0
0
2
30
TPE=0.25 h
TD50=37.66 (34.67-42.36)
3
4
5
5
0
2
1
1
MES
8
TPE=0.25 h
ED50=17.31 (13.47-21.62)
1
1
2
2
4
2
7
0
5
0
i.p.
ScMET
60
TPE=0.25 h
ED50>70.0
7
0
16

i.p.
i.p.
TOX
50
0/8
3/8
4/8
6/8
7/8
TPE=0.25 h
TD50=71.26 (62.05-81.71)
6
7
8
0
0
5
D
1
00
12
6Hz
32 mA)
MES
30
0/8M
6
0
4/8
(
1
8
Carbamazepine i.p.
ED50=7.81 (6.32-8.45)
Max. 75% protection at 40 and
6
Hz
8
0
FRINGS
TOX
MES
ED50=11.2 (7.73-16.2)
TD50=45.4 (32.9-54.4)
ED50=15-20
6
Propranolol
i.p.
Allopregnanolone i.p.
MES
TPE=0.17 h
ED50>100
1
9
ScMET
TPE=0.17 h
ED50=13.7 (10.1-18.7)
6
Hz
TPE=0.17 h
ED50=14.2 (10.3-19.4)
(
32 mA)
a)
b)
Number of animals protected/number of animals tested; Number of animals displaying motor
impairment/number of animals used in the rotarod test; female mice; BIC – bicuculline; PIC –
c)
n
M
picrotoxin; - not included in probit; – minimal motor impairment.
Taking a closer look at the activity of 4, it is active in MES, 6 Hz (22-42 mA), and audiogenic
seizures, and the protective indices vs. rotarod test (TD50/ED50) are 5.5 (MES and Frings,
mice, i.p.) or >17 (MES, rats, p.o.). Activity in 6 Hz tests is observed at higher doses than
MES, from 10.3 mg/kg b.w. up to 30 mg/kg b.w. which is the very TD50 value (Table 6).
Similar procedure as in mice has been repeated in rats mainly after oral administration with
some adjustments. For compounds first active in mice, observation of anticonvulsant activity
at various doses in time has been performed (Table 7). The experiments resulted in
preliminary dose range and TPE, which was used for determination of ED50 or TD50, for
relevant compounds and tests (Table 8).
Also in the above procedure, the superior activity of 2-amino-1-propanol derivatives (3-5) is
clearly visible. Among them, the R enantiomer (4) is more active than the racemate 3 – the CI
of activity in MES is 24.84-70.47 mg/kg for 3 (higher values can be relevant to S-enantiomer
in the racemate) and 20.44-34.64 mg/kg for 4.
In both mice and rats, the peak effect is observed 15 min after i.p. administration. Oral
administration elongates onset of peak activity to 30 min for rats only (Table 7). In terms of
activity profile, the confidence intervals of ED50s cover for MES and audiogenic seizures
(
mice, i.p.). Moreover, considering the ratio between oral and intraperitoneal ED50, it can be
17

presumed that bioavailability could range 10% in mice (Table 6) and 25% in rats (Table 8).
These presumptions should be confirmed with pharmacokinetic studies. Moreover, it can be
observed that the confidence intervals in the same tests but for various sex of animals do not
cover: in MES CI for males is 3.50-7.40, for females ranges 19.55-24.7 mg/kg. The CI for 6
Hz (22 mA) in males is 40.1-48.29 and in females is 2.25-15.81 mg/kg. At 42 mA the CI for
males is 24.38-33.81 and for females 31.24-51.29 mg/kg. Such differences in sex for
neurological activity could be explained by possible interactions with sex hormones,
19
especially that allopregnanolone is known for its activity in MES (Table 6).
Table 7.
Observation of anticonvulsant activity and neurotoxicity in rats
Compd.
Route of
Administration
Test
MES
TOX
Dose
Time [h]
TPE
[mg/kg 0.25 0.5 1.0 2.0 4.0 6.0 8.0 24.0
b.w.]
10
3
i.p.
3/4 2/4 1/4 1/4 1/4
3/4 4/4 1/4 1/4 0/4
1/8 7/8
0.5 h
1
3
2
2
12
0/4 0/4 0/4 0/4 0/4
5
0
1/8 1/8 1/8
|
|
1
30
6
30
00
8/8 8/8 8/8 8/8 7/8 7/8
1/4 0/4 1/4 1/4 0/4
1/4 1/4 2/4 0/4 0/4
0/4 0/4 0/4 0/4 0/4
7/8
1/8
p.o.
MES
TOX
1 h
TD50<500
0
400
2/8 2/8 0/8 0/8 4/8
4/8
500
4/5 4/5 3/5 1/5 2/5
2/5 2/5 1/5
D
4
i.p.
MES
TOX
12
4/4 3/4 2/4 2/4 2/4
4/4 4/4 4/4 4/4 2/4
0/4 0/4 0/4 0/4 0/4
0.25 h
5
0
12
5
0
4/4 3/4 2/4 0/4 0/4
D
100
8/8
8/8 8/8 8/8 8/8
1/4 1/4 0/4 0/4 1/4
3/4 4/4
7/8 7/8 7/8
p.o.
MES
TOX
30
6
8
30
0.5 h
0
0
3/4 3/4 2/4 1/4 1/4
0/4 0/4 0/4 0/4 0/4
TD50>500
6
1
2
5
2.5
0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2
0/2 0/2 0/2
0/2 0/2 0/2
0/2 0/2 0/2
25
50
00
8
0/2
0/2
0/2
5
i.p.
MES
TOX
3/4 4/4 0/4 0/4
0.5 h
1
12
5
12.5
12.5
30
30
10
2
3/4 3/4 3/4 2/4 0/4
0/4 0/4 0/4 0/4 0/4
5/8 1/8 0/8 0/8 0/8
1/4 2/4 2/4 2/4 2/4
0/4 0/4 0/4 0/4 0/4
0/4 0/4 0/4 0/4 0/4
0/4 0/4 0/4 0/4 0/4
0/2 0/2 0/2 0/2 0/2
0.25 h
0
0/8
0/8
p.o.
p.o.
i.p.
MES
TOX
MES
TOX
TOX
6
7
18

3
0
1/2 0/2 0/2 0/2 0/2
D
100
2/2
p.o.
MES
TOX
12.5
12.5
2/4 2/4 1/4 1/4 1/4
0/4 0/4 0/4 0/4 0/4
1 h
TD50>500
1
1
1
0
1
2
p.o.
i.p.
i.p.
p.o.
MES
TOX
MES
TOX
TOX
30
30
30
30
30
0/4 0/4 0/4 0/4 1/4
0/4 0/4 0/4 0/4 0/4
4/4 3/4 2/4 1/4 0/4
0/4 0/4 0/4 0/4 0/4
0/2 0/2 0/2 0/2 0/2
2/2 2/2 2/2 1/2 0/2
0/2 0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2 0/2
0/4 0/4 0/4 1/4 0/4
0.25 h
100
TOX
62.5
1 h
TD50>500
125
250
500
1
8
p.o.
MES
TOX
30
30
0/4 0/4 0/4 0/4 0/4
a)
b)
Number of animals protected / number of animals tested; number of animals exhibiting toxicity /
number of animals tested in the rotarod test; – death; TPE – time of peak effect.
D
Table 8
Quantitative analysis in rats
Compd.
Route of
Test
Dose
[mg/kg
b.w.]
Activity
TPE
administration
ED50 [mg/kg b.w.]
(Confidence
Interval)
3
i.p.
MES
2
4
8
0/8
3/8
3/8
5/8
7/8
2/8
2/8
4/8
6/8
7/8
TPE=0.5 h
ED50=10.08
(5.62-18.64)
1
3
6
2
p.o.
MES
20
TPE=1 h
ED50=47.03
40
60
70
80
(24.84-70.47)
4
i.p.
MES
MES
TPE=0.5 h
ED50=7.43
(3.76-11.7)
p.o.
12.5
0/8
3/8
6/8
8/8
0/2
0/2
1/10
0/2
TPE=0.5 h
ED50=28.60
25
(20.44-34.64)
37.5
50
ScMET
MES
31.3
2.5
25.0
TPE=0.5 h
ED50>250.0
6
1
2
50
5
i.p.
2.5
5
1/8
2/8
4/8
TPE=0.5 h
ED50=6.50
(4.30-10.33)
7
.5
19

1
7.8
0
7/8
0/2
0/2
0/2
0/2
1/8
2/8
5/8
i.p.
i.p.
ScMET
TOX
TPE=0.5 h
ED50>62.5
1
3
6
5.6
1.3
2.5
12.5
TPE=0.25 h
TD50=35.81
(22.28-58.09)
25
50
D
1
00
6/6
7
ED50=34.86
1
2
p.o.
MES
7.5
2/8
2/8
4/8
6/8
6/8
0/2
0/2
0/2
0/2
TPE=1 h
ED50=21.06
(9.51-41.57)
15
23
30
45
ScMET
MES
31.3
ED50>250
ED50=5.35
62.5
1
2
25
50
Carbama
zepine
p.o.
(3.26-7.62)
1
8
ScMET
TOX
ED >250
5
0
[
]
TD50=364
223-500)
(
a)
b)
Number of animals protected / number of animals tested; number of animals exhibiting
toxicity / number of animals tested in the rotarod test; – death; TPE – time of peak effect.
D
Compound 4 was also evaluated for activity in the murine model of status epilepticus –
pilocarpine test (Table 9). As a result, TD95 was determined as 64.99 mg/kg i.p.
Table 9
Pilocarpine test results in mice, i.p.
Compd. Test
Dose
mg/kg b.w.]
65
Time [h]
0.25 0.5 1.0 2.0 4.0
TD95
[mg/kg b.w.]
64.99
[
0.0
1/8
a)
a)
4
2
TOX
TOX
4
300
0/2
0/2 1/2 2/2 2/2
a)
Number of animals exhibiting toxicity / number of animals tested in the rotarod test.
Some anticonvulsant drugs cause higher susceptibility of patients to seizures. Therefore,
compound 4 has been tested for its potential proconvulsant activity in the intravenous
metrazol test (ivMet) in mice, after intraperitoneal administration. Typically, metrazol causes
seizures after timed intravenous administration (time and dose are measured). If compound
administered intraperitoneally prior to the test diminishes the dose (or shortens the time), its
20

proconvulsant activity can be assumed. For compound 4 administered at doses ca. ED50
MES, mice, i.p.) 5 mg/kg and TD50 (rotarod, mice, i.p.) 30 mg/kg, the doses of Met causing
(
seizures are 31.18 (negative control), 30.11 (at ED50) and 29.26 mg/kg (at TD50) (Table 10).
Table 10
Influence on seizure threshold – i.v. metrazol in mice, i.p.
Compd.
Time Dose
Weight [g] Time to
Dose at
twitch
[mg/kg
b.w.]
Time to
clonus
± SEM
Dose at
clonus
[mg/kg
b.w.]
[
h]
[mg/kg
b.w.]
± SEM
twitch
[min]
±
SEM
±
SEM
± SEM
35.67 ±
0.91
4
0.25
0.25
0.5
0
5
27.3 ± 0.2 30.07 ±
.23
27.3 ± 0.2 29.04 ±
.30
27.6 ± 0.3 28.50 ±
.46
27.3 ± 0.3 29.93 ±
.87
27.0 ± 0.2 25.87 ±
.91
27.0 ± 0.1 25.57 ±
.00
31.18 ±
1.18
34.38 ±
0.94
1
30.11 ±
1.26
29.26 ±
1.55
31.11 ±
1.04
27.14 ±
0.92
26.82 ±
1.02
29.44 ±
0.75
31.26 ±
1.43
29.70 ±
1.29
33.06 ±
1.23
32.70 ±
1.28
33.75 ±
0.96
30.82 ±
0.95
29.28 ±
0.86
36.54 ±
0.92
36.96 ±
1.58
35.95 ±
2.04
46.2 ±
1.5
48.6 ±
4.2
42.7 ±
2.1
54.5 ±
3.8
43.4 ±
1.8
45.0 ±
2.3
55.2 ±
1.9
34.32 ±
1.25
33.62 ±
1.53
35.05 ±
1.05
32.32 ±
0.90
30.71 ±
0.86
34.18 ±
0.81
34.54 ±
1.49
34.18 ±
1.82
1
3
0
6
3
0
2
4
0
1
2
4
0
6
1
0
1
7
0
0
3
1
1
2
30.3 ±
.38
30.4 ±
.41
29.8 ±
.54
31.45 ±
0.75
33.43 ±
1.48
31.23 ±
1.44
0
9
6
0
0
Carbamazepine 0.25
35.2 ±
0.7
36.1 ±
1
8
[
]
1
8
8
1
.3
33.5 ±
.8
36.5 ±
.1
0
1
Valproic acid
0.25
34.8 ±
1.8
37.1 ±
1
8
[
]
0
1
.1
40.4 ±
.0
20
2
Data is average of 10 animals in group; p>0.05.
Another model for psychomotor seizures is hippocampal kindling model in rats i.p., where
compound 4 exhibits activity at 30 mg/kg b.w. (Table 11-12). Some activity is also visible in
21

a rat model of drug-resistant seizures – the lamotrigine-resistant amygdala kindling test
already at 30 mg/kg (i.p.) where the compound diminishes seizures into the state referring to
aura in humans - stage 1 in the Racine’s scale (Table 13).
Table 11
Results of preliminary hippocampal kindling screen in rats, i.p.
Compd. Dose Time Animal Seizure score
Afterdischarge
duration [s]
TPE
[h]
[
mg/kg
[h]
No.
b.w.]
Pre-drug Drug Pre-drug Drug
4
30
0
Rat #1
Rat #2
Rat #1
Rat #2
Rat #1
Rat #2
Rat #1
Rat #2
Rat #1
Rat #2
5
5
5
5
5
5
5
5
5
35
100
38
90
43
140
81
65
0
1
2
4
.25
5
5
48
5
7
1
10
20
30
0.25- Rat #1
.25 Rat #2
0.25- Rat #1
4-5
5
5
4-5
5
4-5
4-5
5
5
2
5
5
5
5
5
5
5
4
1
50-82
37-75
68-77
79-100
46-137
43-95
53-88
57-67
44
33
14
42
57
40
62
57
55
56
57
17
2
2.25
Rat #2
Rat #1
Rat #2
Rat #1
Rat #2
Rat #1
Rat #2
Rat #1
Rat #2
Rat #1
Rat #2
Rat #1
Rat #2
4-5
2
0
0.75
0
1
2
4
.25
50
2.25
5
5
70-74
49-70
Seizures are scored according to the following criteria (Racine’s scale): stage 1 – mouth and
facial clonus; stage 2 – stage 1 plus head nodding; stage 3 – stage 2 plus forelimb clonus;
2
0 #
stage 4 – stage 3 plus rearing; stage 5 – stage 4 plus repeated rearing and falling ;
significantly different from control.
22

Table 12
Activity in hippocampal kindling in rats, i.p.
Compd. Dose
Time
[h]
Seizure score ± Duration [s] ±
No.
animals
ED50
(Confidence
a)
[
mg/kg
SEM
SEM
b.w.]
protected/ interval)
tested
[mg/kg b.w.]
4
30
0
4.88 ± 0.13
4.00 ± 0.33
4.75 ± 0.16
4.63 ± 0.18
4.25 ± 0.62
4.88 ± 0.13
4.9 ± 0.1
74.57 ± 8.29
63.00 ± 7.11
62.88 ± 4.43
84.75 ± 10.99
60.75 ± 11.29
82.50 ± 4.89
49 ± 4
TPE=0.25
ED50>30
#
0
0
1
1
2
0
0
0
1
1
2
.25
.75
.25
.75
.25
60
#
.25
.75
.25
.75
.25
2.7 ± 0.6
3.6 ± 0.2
3.6 ± 0.4
3.7 ± 0.5
41 ± 4
35 ± 1
#
#
#
43 ± 3
55 ± 4
58 ± 3
#
4.4 ± 0.2
1
2
12.5
0.75
0.75
0.75
4.67 ± 0.21
2.50 ± 0.76
2.00 ± 0.63
60.50 ± 5.25
30.75 ± 7.51
28.50 ± 9.00
0/6
4/8
6/8
30.63
(18.52-53.46)
#
#
#
2
5
5
0
#
a)
#
Racine’s scale as in Table 11; significantly different from control
Table 13
Lamotrigine-resistant amygdala kindling in rats, i.p.
Compd. Dose
Time
Seizure score ± Duration [s] ±
No. animals protected/
tested
a)
[
mg/kg [h]
SEM
SEM
b.w.]
4
Control 0.5
0.5
Control 0.5
0.5
5.0 ± 0.0
3.6 ± 0.6
5.0 ± 0.0
1.4 ± 0.6
104.60 ± 23.57
158.40 ± 21.60
109.40 ± 19.56
124.80 ± 32.05
0/5
3/5
0/5
5/5
3
0
#
45
a)
#
Racine’s scale as in Table 11; Statistically significant
An interesting pharmacological profile can be observed also for the 4-piperidinol derivative –
compound 11. It has undergone procedures involving 4 h observations of activity at 6 Hz,
MES and TOX. The activity is observed already in 25 mg/kg in MES and in 50 mg/kg in 6
Hz, both at 0.25 h after administration (Table 5). Its ED50 in MES is 17.31 and TD50 is 71.26
mg/kg (mice, i.p.), and the confidence intervals do not cover (Table 6). Activity in corneal
kindling of mice is observed with ED50 57.52 mg/kg (Table 14). Compound 11 is also most
active in rats, 15 min after i.p. administration.
23

Table 14
Corneal kindling in mice, i.p.
Compd. Dose
Time
[h]
Individual
seizure
scores
Average seizure ED50
[
mg/kg
score
[mg/kg b.w.]
a)
b.w.]
11
a)
17
0.25
3, 5, 0, 3
2.75
57.52
Racine’s scale as in Table 11.
The final interesting compound in this group is an R,S-2-amino-1-phenylethanol derivative –
compound 12. In subsequent assays it proved higher potency than its enantiomers 13 (R) and
1
4 (S) which were not advanced to further assays after screening (Table 4). ED50 of 12 in rats
in MES after oral administration is 21.06 mg/kg (Table 8) and the compound does not have a
clear influence on seizure threshold in mice (Table 10). Taking into account activity in
psychomotor seizures reflected by hippocampal kindling in rats (i.p.) – the activity is
documented with ED50=30.63 mg/kg (Table 12).
Among the title compounds, 4 has been selected for verification of possible mechanism of
action, with use of radioligand binding studies as the high throughput profile at Cerep,
2
1
France. The study included binding of the compound to 82 targets at a single concentration.
First, it was chosen to examine the base – compound 4, at a large concentration 100 µM, in
order to receive an overview of possible local adverse reactions after injection of its solution.
The result is presented in Table 15. However, 50% binding at such concentration may show
more of adverse events than of the activity, therefore, IC50 values were measured as well. In
order to receive the exact data of the chosen form, the assay was performed for the
hydrochloride – 4a2. The results are presented in Table 16. As it can be observed, 4a2 binds
-6
to four targets at 10 M: σ, 5-HT1A, 5-HT transporter, and α . Among the observed
2
pharmacological profile, the activity can be attributed to these receptors and, as a result, some
possible adverse reactions could be further explored due to the shown binding. Moreover,
functional assays may be another step in understanding the observed activity.
24

Table 15
Receptor binding of compound 4
-
4
Receptor
Binding at 10 M of compound 4
Study conditions
Radioligand
Reference
Compound
%
Inhibition Mean / SEM %
Origin of receptor
Radioligand
type
IC50 [M]
of Control
Specific
Binding
% of
Control
Control
Specific
Binding
3
-10
A
A
A
1
-33
133.1
2.1
human recombinant [ H]DPCPX
CHO cells
Antagonist
Agonist
DPCPX
5.0*10
3.7*10
6.0*10
2.5*10
3
-8
2A
3
30
69.8
9.0
4.2
1.0
human recombinant [ H]CGS 21680
NECA
HEK-293 cells
1
25
-10
-10
-13
73
113.0
26.6
human recombinant [ I]ABMECA
Agonist
IB-MECA
prazosin
HEK-293 cells
rat cerebral cortex
3
α
1
[ H]prazosin
Antagonist
Antagonist
3
-8
-7
α
β
2
70
15
29.9
85.2
1.8
10.7
rat cerebral cortex
[ H]RX 821002
yohimbine
atenolol
9.6*10
2.1*10
3
1
human recombinant [ H](-)CGP12177 Agonist
HEK-293 cells
3
-10
-10
-11
β
2
8
91.9
3.9
3.2
0.8
human recombinant [ H](-)CGP12177 Agonist
ICI 118551
saralasin
7.4*10
4.0*10
8.0*10
CHO cells
1
25
1
8
AT
AT
1
2
-33
-3
132.7
102.8
human recombinant [ I] [Sar ,Ile ]-
antagonist
HEK-293 cells ATII
1
25
human recombinant [ I]CGP42112A agonist
angiotensin-II
HEK-293 cells
rat cerebral cortex
rat heart
3
-9
-9
BZD (central) -10
109.5
107.6
2.7
3.0
[ H]flunitrazepam Agonist
diazepam
PK 11195
6.8*10
1.6*10
3
BZD
peripheral)
-8
[ H]PK 11195
Antagonist
(
1
25
4
-10
BB (non-
selective)
4
96.5
1.5
rat cerebral cortex
[ I][Tyr ]bombes Agonist
in
bombesin
3.4*10
2
5

3
-8
B
2
-12
-21
-3
112.3
121.3
103.3
121.2
119.2
78.2
4.4
1.2
5.7
7.7
1.5
1.2
0.6
2.6
2.3
5.6
1.9
2.1
2.3
6.6
6.4
human recombinant [ H]bradykinin
Agonist
Agonist
Agonist
Agonist
Agonist
Antagonist
NPC 567
1.4*10
3.2*10
9.0*10
1.3*10
1.2*10
3.1*10
1.1*10
1.6*10
4.1*10
5.6*10
2.3*10
6.3*10
2.1*10
7.9*10
2.6*10
CHO cells
1
25
-11
-10
-10
-10
-10
-9
CGRP
CB
human recombinant [ I]hCGRPα
hCGRPalpha
CP 55940
CCK-8s
CHO cells
3
1
human recombinant [ H]CP 55940
CHO cells
1
25
CCK
CCK
1
2
(CCK
(CCK
A
)
-21
-19
22
36
58
55
27
-39
-19
5
human recombinant [ I]CCK-8s
CHO cells
1
25
B
)
human recombinant [ I]CCK-8s
CHO cells
CCK-8s
3
D
D
D
D
D
1
human recombinant [ H]SCH23390
SCH 23390
(+)butaclamol
(+)butaclamol
clozapine
SCH 23390
endothelin-1
endothelin-3
GABA
CHO cells
3
2S
3
64.0
human recombinant [ H]methylspipero Antagonist
HEK-293 cells
human recombinant [ H]methylspipero Antagonist
CHO cells ne
human recombinant [ H]methylspipero antagonist
CHO cells ne
ne
3
-9
42.2
3
-8
4.4
5
44.7
3
-10
-11
-12
-8
73.2
human recombinant [ H]SCH23390
Antagonist
GH4 cells
1
25
ET
ET
A
B
139.3
119.3
94.9
Human recombinant [ I]endothelin-1 Agonist
CHO cells
1
25
human recombinant [ I]endothelin-1 Agonist
CHO cells
rat cerebral cortex
3
GABA (non-
selective)
GAL
[ H]GABA
Agonist
Agonist
Agonist
1
25
-11
-9
1
-9
108.5
96.2
human recombinant [ I]galanin
HEK-293 cells
galanin
1
25
GAL
2
4
human recombinant [ I]galanin
CHO cells
galanin
1
25
-11
-11
PDGF
CXCR2 (IL-
7
-16
93.0
116.0
10.8
4.5
Balb/c 3T3 cells
[ I]PDGF BB
Agonist
Agonist
PDGF BB
IL-8
4.7*10
5.1*10
1
25
human recombinant [ I]IL-8
2
6

8
B)
HEK-293 cells
human recombinant [ I]MIP-1α
1
25
-11
CCR1
-2
102.0
3.5
Agonist
MIP-1alpha
2.1*10
HEK-293 cells
U-937 cells
human recombinant [ H]pyrilamine
HEK-293 cells
human recombinant [ I]APT
1
25
-11
-9
TNF-α
H
1
1
40
98.5
60.2
5.4
0.8
[ I]TNF-α
Agonist
antagonist
TNF-alpha
pyrilamine
9.9*10
1.4*10
3
1
25
-6
H
2
-4
4
104.2
96.1
73.1
88.9
98.8
81.9
79.6
71.4
105.3
104.9
96.7
118.9
3.5
2.0
3.9
4.4
1.3
3.6
1.2
2.0
0.5
0.2
1.8
1.6
Antagonist
cimetidine
1.0*10
1.5*10
1.1*10
2.1*10
CHO cells
1
25
-10
-10
-8
MC
4
human recombinant [ I]NDP-α-MSH Agonist
CHO cells
human recombinant [ I]2-
CHO cells iodomelatonin
human recombinant [ H]pirenzepine
CHO cells
human recombinant [ H]AF-DX384
CHO cells
human recombinant [ H]4-DAMP
CHO cells
human recombinant [ H]4-DAMP
CHO cells
human recombinant [ H]4-DAMP
CHO cells
U-373MG cells
NDP-alpha -
MSH
melatonin
1
25
MT
1
(ML1A
)
27
11
1
Agonist
3
M
M
M
M
M
1
2
3
4
5
Antagonist
Antagonist
Antagonist
Antagonist
Antagonist
Agonist
pirenzepine
3
-8
methoctramine 2.2*10
3
-10
-10
-10
-10
-9
18
20
29
-5
-5
3
4-DAMP
4-DAMP
4-DAMP
4.1*10
4.9*10
3.2*10
3
3
1
25
NK
NK
NK
1
2
3
[ I]BH-SP
[Sar9,Met(O2)1 1.1*10
1]-SP
[Nleu10]-NKA 3.5*10
(
endogenous)
1
25
human recombinant [ I]NKA
Agonist
CHO cells
human recombinant [ H]SR142801
CHO cells
(4-10)
3
-9
Antagonist
Agonist
SB 222200
NPY
6.7*10
2.1*10
1
25
-10
Y
Y
1
-19
SK-N-MC cells
endogenous)
KAN-TS cells
human recombinant [ I]Tyr3-
[ I]peptideYY
(
1
25
-11
-10
2
-26
-12
126.1
112.1
13.2
11.8
[ I]peptideYY
Agonist
Agonist
NPY
neurotensin
2.8*10
4.9*10
1
25
NTS
1
(NT
1
)
2
7

CHO cells
human recombinant [ H]DADLE
CHO cells
rat recombinant
CHO cells
human recombinant [ H]DAMGO
HEK-293 cells
human recombinant [ H]nociceptin
HEK-293 cells
human recombinant [ I]PACAP1-27 Agonist
CHO cells
neurotensin
3
-9
δ
2
(DOP)
2
97.7
66.7
84.0
98.7
99.7
93.1
2.3
1.2
3.3
1.6
3.8
7.5
Agonist
Agonist
Agonist
Agonist
DPDPE
1.3*10
6.4*10
4.3*10
1.3*10
4.0*10
1.0*10
3
-10
-10
-9
κ (KOP)
33
16
1
[ H]U 69593
U 50488
3
µ (MOP)
DAMGO
nociceptin
PACAP1-38
rosiglitazone
3
NOP (ORL1)
1
25
-11
-8
PAC
1
(PACAP) 0
3
PPAR
γ
7
human recombinant [ H]rosiglitazone Agonist
(
E. coli)
3
-9
-9
PCP
26
5
74.2
95.3
4.1
5.6
rat cerebral cortex
human recombinant [ H]PGE
[ H]TCP
Antagonist
agonist
MK 801
PGE2
4.6*10
2.6*10
3
EP
2
2
HEK-293 cells
human recombinant [ H]iloprost
HEK-293 cells
rat urinary bladder
3
-8
-9
IP (PGI
2
)
-1
-9
101.3
108.6
1.3
1.2
Agonist
Agonist
iloprost
1.0*10
1.9*10
3
P2X
[ H]α,β-MeATP
alpha ,beta -
MeATP
dATPalpha S
3
5
-8
P2Y
4
94
96.5
6.2
1.3
0.5
rat cerebral cortex
human recombinant [ H]8-OHDPAT
HEK-293 cells
[ S]dATPαS
Agonist
Agonist
2.9*10
5.3*10
3
-10
5
5
5
5
5
5
-HT1A
-HT1B
-HT2A
-HT2B
-HT2C
8-OH-DPAT
1
25
-9
15
25
85
39
6
85.5
75.3
15.3
61.4
94.2
5.1
9.1
1.2
5.7
2.7
rat cerebral cortex
[ I]CYP (+30
Antagonist
Antagonist
Agonist
serotonin
ketanserin
(±)DOI
4.9*10
3.5*10
2.5*10
1.5*10
6.9*10
µM isoproterenol)
3
-10
-9
human recombinant [ H]ketanserin
HEK-293 cells
human recombinant [ I](±)DOI
CHO cells
human recombinant [ H]mesulergine
HEK-293 cells
1
25
3
-9
Antagonist
Antagonist
RS 102221
MDL 72222
3
-9
-HT
3
human recombinant [ H]BRL43694
2
8

CHO cells
human recombinant [ H]LSD
HEK-293 cells
human recombinant [ H]LSD
CHO cells
human recombinant [ H]LSD
CHO cells
Jurkat cells
(endogenous)
AtT-20 cells
3
-7
5
5
5
-HT5A
13
-11
57
99
15
-17
-19
3
87.1
110.8
42.6
1.1
9.1
0.2
0.6
4.5
1.5
10.5
7.2
7.3
2.9
Agonist
Agonist
Agonist
Agonist
Agonist
serotonin
serotonin
serotonin
haloperidol
1.8*10
1.2*10
2.5*10
1.7*10
3
-7
-HT
-HT
6
7
3
-10
-8
3
σ (non-
selective)
sst (non-
selective)
GR
[ H]DTG
1
25
-11
-9
84.9
116.7
119.0
96.7
67.9
[ I]Tyr11-
somatostatin-14 7.4*10
dexamethasone 3.8*10
somatostatin-14
3
IM-9 cells
[ H]dexamethason Agonist
e
(
cytosol)
1
25
-10
-10
-8
VPAC
1
(VIP
1
)
human recombinant [ I]VIP
CHO cells
human recombinant [ H]AVP
CHO cells
rat cerebral cortex
Agonist
VIP
1.1*10
3
V
1a
Agonist
[d(CH2)51,Tyr( 9.9*10
Me)2]-AVP
D 600
2
+
3
Ca channel
L, verapamil
site)
channel
32
[ H]D888
1.5*10
(
Antagonist
1
25
-10
K
V
-3
103.4
2.8
rat cerebral cortex
[ I]α-
antagonist
antagonist
alpha-
dendrotoxin
apamin
5.7*10
dendrotoxin
1
25
-11
-6
SKCa channel
Na channel
4
60
95.9
40.2
2.2
4.5
rat cerebral cortex
rat cerebral cortex
[ I]apamin
1.6*10
4.0*10
+
3
[ H]batrachotoxini antagonist
veratridine
(
site 2)
n
-
35
-8
-9
Cl channel
GABA-gated)
-2
102.1
76.3
2.6
3.1
rat cerebral cortex
[ S]TBPS
antagonist
antagonist
picrotoxinin
protriptyline
7.7*10
2.8*10
(
3
norepinephrine 24
transporter (h)
human recombinant [ H]nisoxetine
CHO cells
Antagonist
(
antagonist
radioligand)
dopamine
3
-9
58
42.0
1.7
human recombinant [ H]BTCP
antagonist
BTCP
2.8*10
2
9