Design, physico-chemical properties and biological evaluation of some new N-[(phenoxy)alkyl]- and N-{2-[2-(phenoxy)ethoxy]ethyl}aminoalkanols as anticonvulsant agents

Bioorganic & Medicinal Chemistry 24 (2016) 1793–1810

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Design, physico-chemical properties and biological evaluation

of some new N-[(phenoxy)alkyl]- and N-{2-[2-(phenoxy)ethoxy]

ethyl}aminoalkanols as anticonvulsant agents

a

b

c

A. M. Waszkielewicz ^a,, A. Gunia-Krzyzak , B. Powroznik , K. Słoczynska , E. Pe˛kala , M. Walczak ,

⇑

_

´

d

e

f

a

_

M. Bednarski , E. Zesławska , W. Nitek , H. Marona

^aDepartment of Bioorganic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland

^bDepartment of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland

^cDepartment of Toxicology, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland

^dLaboratory of Pharmacological Screening, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland

e

_

Department of Chemistry, Institute of Biology, Pedagogical University, Podchora˛zych 2, 30-084 Krakow, Poland

^fFaculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland

a r t i c l e i n f o

a b s t r a c t

Article history:

A series of thirty N-(phenoxy)alkyl or N-{2-[2-(phenoxy)ethoxy]ethyl}aminoalkanols has been designed,

synthesized and evaluated for anticonvulsant activity in MES, 6 Hz test, and pilocarpine-induced status

epilepticus. Among the title compounds, the most promising seems R-(ꢀ)-2N-{2-[2-(2,6-dimethylphe-

noxy)ethoxy]ethyl}aminopropan-1-ol hydrochloride (22a) with proved absolute conﬁguration with X-

ray analysis and enantiomeric purity. The compound is effective in MES test with ED₅₀= 12.92 mg/kg

b.w. and its rotarod TD₅₀= 33.26 mg/kg b.w. The activity dose is also effective in a neurogenic pain

model—the formalin test. Within high throughput proﬁle assay, among eighty one targets, the strongest

Received 28 January 2016

Revised 1 March 2016

Accepted 2 March 2016

Available online 3 March 2016

Dedicated to the memory of Professor Jan

Krzek

afﬁnity of the compound is observed towards

not bind to hERG. It also does not exhibit mutagenic properties in the Vibrio harveyi test. Moreover, mur-

ine liver microsomal assay and pharmacokinetics proﬁle (mice, i , p.o., ip) indicate that the liver is the

primary site of biotransformation of the compound, suggesting that both 22a and its metabolite(s) are

active, compensating probably low bioavailability of the parent molecule.

r receptors and 5-HT transporter and the compound does

Keywords:

v

Aminoalkanols

Anticonvulsant activity

Seizures

Sigma receptors

5-HT

MES

Metabolic stability

Mutagenicity

Vibrio harveyi

Pharmacokinetics

1. Introduction

are still premises for search of new anticonvulsant compounds

which will be both effective and safe.²

Epilepsy constitutes one of major neurological disorders—it is a

set of disorders having in common hypersynchronous discharges

in neurons. Overall incidence of epilepsy is estimated 50 per

100,000/year. The variety of types of seizures causes situation that

particular patients suffer from drug resistant seizures, which con-

stitute about 30% of cases. Moreover, toxicity and adverse reactions

of anticonvulsants are a signiﬁcant cause for self-discontinuation

of treatment by patients, especially during pregnancy.¹Therefore,

despite large progress in antiepileptic drug development, there

Antiepileptic drugs belong to many chemical groups, exhibit

various activity, and therefore are used in various types of seizures.

Signiﬁcant anticonvulsant activity has been observed also for pro-

pranolol and mexiletine—well known antiarrhythmic drugs which

exert properties of stabilizing neuronal cellular membrane poten-

tial and therefore exhibit some anticonvulsant activity.^3–5They

both contain structural features which drew our attention to the

chemical group of aminoalkanol derivatives.

Aminoalkanol derivatives and their anticonvulsant activity have

been subject of our interest for many years, and among this group a

couple drug candidates have been selected. For instance, (2,6-

dimethylphenoxy)ethyl derivatives of 1,2-aminoalkanols (e.g.

⇑

Corresponding author.

E-mail address: awaszkie@cm-uj.krakow.pl (A.M. Waszkielewicz).

http://dx.doi.org/10.1016/j.bmc.2016.03.006

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A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

S-(+)-2-aminobutan-1-ol, R-(ꢀ)-2-aminopropan-1-ol,

D

,

L

-trans-2-

The synthesis of 1–30 (Scheme 1) was a multistage process and

the ﬁnal reaction constituted N-alkylation of appropriate aminoalka-

nols with use of formerly synthesized appropriate phenoxyalkyl or

[2-(phenoxy)ethoxy]ethyl bromides which had been formerly pre-

pared according to published procedures.^9,6,12Two compounds have

been characterized as hydrochlorides (22a, 23a), which were

obtained from base in an acetone solution of gaseous HCl.

The absolute conﬁguration of the structure 22a was conﬁrmed

with use of X-ray structure analysis (the crystals were obtained

from a mixture of acetone and n-decane (1:1) by slow evaporation

of the solvent at room temperature). The asymmetric unit of 22a

with the atom numbering scheme of non-hydrogen atoms is

shown in Figure 2.

The crystal structure of 22a conﬁrms R conﬁguration at carbon

atom C2. The bond lengths and bond angles have typical values.

The arrangement of the ether oxygen atoms (O3) and (O2) shows

synclinal conformation (gauche). Furthermore, the conformation

of respective pairs of atoms (O2) and (N1), (N1) and (O1) is also

synclinal. The same conformation was observed in crystal struc-

tures of similar compounds R-2-[2-(2,6-dimethylphenoxy)ethyl]

aminopropan-1-ol hydrochloride⁸and mexiletine.¹³The methyl

substituents at phenoxy moiety are coplanar with benzene ring.

The deviation of C14 and C15 from the benzene plane is 0.007

(5) Å and 0.050(5) Å, respectively. The carbon (C4, C5, C6, C7)

and oxygen (O2) atoms of ethoxyethyl moiety lie in one plane with

the rms deviation of the atoms 0.061 Å, which is almost parallel to

benzene plane, with the interplanar angle 1.8(3)°.

aminocyclohexan-1-ol) have proved the highest activity in cur-

rent-evoked seizures so far (Fig. 1).^6–8It is interesting that among

synthesized compounds the conﬁguration in the aminoalkanol

moiety is important—one of the enantiomers is usually more active

than the other—but it also depends on the whole structure, which

enantiomer will prove more potent. Within our synthetic research

we have already done modiﬁcation of ethyl or propyl linker

between amine and phenoxyl group into ethoxyethyl in case of

antidepressant-like activity, and in that case such modiﬁcation

proved successful.⁹

The aim of our study was to see if modiﬁcation of the alkyl lin-

ker between amine and phenoxyl group into ethoxyethyl improves

also anticonvulsant activity in the title group of compounds. The

premise to use the same linker was common etiology and comor-

bidity of depression and epilepsy as well as certain similarity of

stabilization of neuronal membrane potential of both anticonvul-

sant and antidepressant drugs.¹⁰Another purpose of the study

was to examine whether substitution in the phenoxyl moiety stays

optimal for 2,6-(CH₃)₂also the case of ethoxyethyl derivatives.

Therefore, we aimed to modify the most active compounds in

terms of substitution in the phenyl ring in order to ﬁnd optimal

structures in the title group.

The studies resulted in ﬁling of some promising compounds for

a European patent.¹¹Moreover, pharmacological output of the

compounds resulting in probable mechanism of action, drew our

attention towards preliminary safety, metabolic stability, and

pharmacokinetics studies concerning the most active compounds.

The geometry of 2-aminopropan-1-ol moiety present in the

crystal structure of 22a molecule and R-2N-[2-(2,6-dimethylphe-

noxy)ethyl]aminopropan-1-ol hydrochloride was compared. The

main difference is observed for the orientation of hydroxyl group.

The respective torsion angle N1–C2–C1–O1 in 22a is ꢀ76.47°,

whereas in the compared structure is 45.99°.⁸

2. Results and discussion

2.1. Chemistry

The arrangement of 22a molecules in the crystal lattice is dom-

inated by hydrogen bonds with chlorine anion (Table 2). Each chlo-

rine anion interacts with two molecules forming hydrogen bond

with N1–HN1 and O1–H1 of one molecule and N1–HN2 of the sec-

ond one (Fig. 3). Furthermore, chlorine anion is involved in C–H

interactions. The crystal structure of 22a is also stabilized by weak

C–Hꢁ ꢁ ꢁO interactions.

Chemical structures of the tested compounds are shown in

Table 1. The title compounds included substituted (phenoxy)ethyl-

(1–6, 12–18), (phenoxy)propyl- (7–11), and [2-(phenoxy)ethoxy]

ethylaminoalkanols (19–30). Substitutions in the phenyl ring con-

stituted 2,3-dimethylphenol (1–11, 19–20), 2,4-dimethylphenol

(12–13), xylenol (21–28), and 2,4,6-trimethylphenol (14–18, 29–

30). The aminoalkanols were used the same as formerly proved

optimal for anticonvulsant activity: 2-aminopropan-1-ol,

2-aminobutan-1-ol, 2- or 4-aminocyclohexan-1-ol. Moreover, for

structure–activity relationship observations, 2-amino-2-methyl-

propan-1-ol, 2-amino-1-phenylethan-1-ol, as well as 3- or

4-piperidinol were also used. In case of activity of a racemate,

enantiomers were synthesized as well. Two compounds have been

achieved also as hydrochlorides (22a, 23a).

2.2. Enantiomeric purity

The examples of the representative chromatographic traces

resulted for enantiomeric forms of studied aminoalkanols deriva-

tives (22, 23 and 22a) under the optimized method conditions are

presented in Figure 4. Well separated chromatographic peaks are

seen, corresponding to the enantiomers of R,S-2N-{2-[2-(2,6-

dimethylphenoxy)ethoxy]ethyl}aminopropan-1-ol

(compound

NH₂

CH₃

H₃C

O

*

21): R-(ꢀ)-2N-{2-[2-(2,6-dimethylphenoxy)ethoxy]ethyl}amino-

propan-1-ol hydrochloride (compound 22a) and S-(+)-2N-{2-[2-

(2,6-dimethylphenoxy)ethoxy]ethyl}aminopropan-1-ol (compound

23). The studied compounds were obtained with enantiomeric

excess as high as 98%.

H

N

O

OH

CH₃

H₃C

CH₃

Mexiletine

I

ED₅₀=20 mg/kg

ED₅₀=7.57 mg/kg (6.206-9.002)

TD₅₀=34.45 mg/kg (28.83-39.24) (rotarod)

2.3. Pharmacology

The screening results in mice are presented in Table 1. Within

the title group of compounds, ethoxyethyl modiﬁcation of com-

pound II—compound 22a exhibits similar activity of the reference

compound. ED₅₀in MES, mice, ip is observed 5.34 (3.50–7.40) for

hydrochloride of compound II⁸and 12.92 (8.49–19.01) for com-

pound 22a (Table 3). It should be noticed that the conﬁdence inter-

vals do not cover which suggests that the compounds may have

different pharmacological proﬁles. However, at this point it seems

that the difference in activity of the enantiomers is slight.

H

N

O

OH

HO

CH₃

H₃C

CH₃CH₃

H₃C

III

II

ED₅₀=5.34 mg/kg (3.50-7.40)

TD₅₀=29.48 mg/kg (21.76-37.87) (rotarod)

ED₅₀=7.73 mg/kg

TD₅₀=28.36 mg/kg (rotarod)

Figure 1. Reference compounds and their activity in MES, mice, ip.^5–8

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

1795

Table 1

Structures and screening results of compounds 1–30 (mice, ip)

O

Z

O

Z

n

R

Comp. 19-30

Comp. 1-18

Compound

R

n

Z

Conﬁguration

Dose (mg/kg)

Time (h)

ScMet^a

MES^a

1.0

TOX^b

0.25

0.5

4.0

0.5

4.0

0.25

0.5

1.0

4.0

H

N

30

100

300

0/1

3/3

0/1

0/3

0/1

0/4

7/8

4/4

0/2

0/4

OH

1

2

2,3-(CH₃)₂

2

R,S

CH₃

H

N

30

100

300

0/1

3/3

0/1

0/3

0/1

0/4

7/8

4/4

0/2

0/4

CH₃

OH

—

CH₃

H

N

30

100

300

0/1

3/3

0/1

0/3

0/1

0/4

8/8

4/4

0/2

0/4

OH

3

R,S

CH₃

N

H

HO

4

5

100

4/4

0/4

4/4

0/4

D,L-trans

N

OH

—

OH

30

100

300

0/1

0/3

0/1

0/3

0/1

1/5

0/1

0/4

4/8

3/4

0/2

1/4

0/2

H

N

6

7

8

R,S

H

N

30

100

300

0/1

3/3

0/1

0/3

0/1

0/4

8/8

4/4

0/2

0/4

OH

3

CH₃

H

N

30

100

300

0/1

3/3

0/1

0/3

0/1

0/4

8/8

4/4

0/2

0/4

CH₃

OH

30

100

3/4

4/4

3/4

4/4

0/4

4/4

0/4

3/4

0/4

3/4

0/4

9

—

N

OH

10

100

4/4

1/4

0/4

3/4

3/3

1/4

0/4

3/3

0/4

OH

30

100

300

0/1

0/3

1/1

0/1

0/3

0/1

1/4

6/8

4/4

1/2

3/4

2/2

H

N

0/3

11

R,S

H

N

OH

12

13

14

2,4-(CH₃)₂

2

R,S

R

CH₃

HO

H

N

H

N

30

100

0/1

3/3

0/1

0/3

0/1

0/4

8/8

0/2

0/4

OH

2,4,6-(CH₃)₃

R,S

300

4/4(1)

H

N

3

10

0/4

OH

30

100

300

1/1

3/3

0/1

0/3

0/1

0/4

8/8

4/4(1)

0/2

0/4

15

R

3

10

0/4

1/4

0/4

1/4

N

H

30

100

300

1/1

0/1

0/4

8/8(6)

4/4(4)

0/2

1/1

16

17

D,L-trans

OH

30

100

0/1

2/2

0/1

0/3

0/1

0/4

2/8(1)

0/2

0/3

N

H

trans

300

4/4(1)

(continued on next page)

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A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

Table 1 (continued)

Compound

R

n

Z

Conﬁguration

Dose (mg/kg)

Time (h)

ScMet^a

MES^a

1.0

TOX^b

0.25

0.5

4.0

0.5

4.0

0.25

0.5

1.0

4.0

OH

30

100

300

0/1

3/3

1/1

0/1

0/3

1/1(1)

0/1

0/4

0/8

3/4

0/2

0/4

0/2

H

N

18

R,S

H

N

30

100

300

0/1

0/4

8/8(7)

4/4(3)

0/2

0/1

OH

19

20

21

2,3-(CH₃)₂

2,6-(CH₃)₂

—

R,S

CH₃

OH

N

H

trans

R,S

H

N

30

100

300

0/1

0/4

8/8

4/4

0/2

0/1

OH

CH₃

H

N

3

10

30

100

300

0/4

1/4

1/1

0/4

8/8(8)

4/4(4)

0/1

0/2

22

R

S

H

N

3

10

30

100

300

0/4

1/4

1/1

0/4

8/8(8)

4/4(4)

OH

0/2

22a

23

CH₃

x HCl

H

N

3

10

30

100

300

0/4

2/4

1/1

0/4

1/4

8/8(8)

4/4(4)

OH

H

N

3

10

30

100

300

0/4

1/1

0/4

8/8(8)

4/4(4)

OH

23a

24

S

x HCl

H

N

30

100

300

0/1

1/1

0/1

0/4

8/8(7)

4/4(1)

0/2

R,S

R

CH₃

H

N

3

10

30

100

300

0/4

1/1

0/4

0/1

0/4

7/8(7)

4/4(4)

0/4

0/2

0/1

OH

CH₃

0/1

25

H

N

3

10

30

100

300

0/4

1/1

0/4

8/8(8)

4/4(4)

OH

CH₃

0/1

0/2

26

S

30

100

300

0/1

3/3

0/1

0/3

0/1

0/4

0/8

4/4(4)

0/2

0/4

N

H

HO

27

28

D,L-trans

N

OH

-

30

0/1

0/4

0/2

100

300

7/8(6)

4/4(4)

H

30

100

300

0/1

3/3

0/1

1/3

0/1

0/4

4/8

4/4(4)

0/2

0/4

N

OH

29

30

2,4,6-(CH₃)₃

—

R,S

CH₃

HO

N

H

D,L-trans

a

No. of animals protected/no. of animals tested.

No. of animals exhibiting neurotoxicity/no. of animals tested in rotarod, (deaths).

b

The ED₅₀was also determined for compound 23 and it can be

b.w. (Table 3) while its lead ED₅₀is 8.59 (7.23–10.03) mg/kg

b.w.⁸Moreover, the compound is active after intraperitoneal

administration to rats (Table 4). Its hydrochloride shows the same

potency in the assay.

compared to the S enantiomer of compound II, taking into account

the whole conﬁdence intervals of the active dose. The ethoxyethyl

derivative exhibits ED₅₀(MES, mice, ip) 11.3 (9.39–13.3) mg/kg

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

1797

1 11 19-20

R - 2,3-(CH₃)₂for

2,4-(CH₃)₂for 12-13

21 28

-

,

Br

n

O

Z

2,6-(CH₃)₂for

-

a

2,4,6-(CH₃)₃for 14-18, 29-30

R

Z - aminoalkanol

n=2 for 1-6, 12-18

n=3 for 7-11

1 18

-

CH₃

*

O

OH

x HCl

Br

O

Z

O

N

H

H₃C

CH₃

a

b

R

22a 23a

,

19-30

Scheme 1. Synthetic routes to ﬁnal compounds. Reagents: (a) Z-aminoalkanol, K₂CO₃/toluene or acetone (TEBA); (b) gaseous HCl/acetone.

Since many registered drugs were developed with use of MES

test screening, and efﬁcacy of these drugs is not satisfactory,

another screening test has been introduced in the Antiepileptic

Drug Development program, i.e. the 6 Hz test (for epileptogenesis,

psychomotor seizures, etc.).¹⁴Compounds 4–5, 9–10, 12, 19, 22–

23a, and 30 were subject to such screening at various doses and

their activity was observed in time (Table 5). Comparing these

results, compounds 4, 9–10, 19, 23, 23a were tested in both MES

and 6 Hz tests and TOX (Tables 1 and 5).

Considering activity of the most active compounds 22 and 23,

both of them could be compared in 6 Hz test (Table 5)—compound

22 exhibits interesting activity at 50 mg/kg for 1 h after adminis-

tration and after 4 h, which presumes activity of a metabolite as

well. For comparison, compound 23 tested at 40 mg/kg exhibits

similar activity until 4 h after administration. Results of the

hydrochloride 23a are consistent with efﬁcacy of the base 23.

The interesting pharmacological results of compound 22 drew

our attention to probability of activity of either the compound

itself or its metabolite, since the activity in the 6 Hz test was

observed until 1 h after administration. Then, after 4 h some activ-

ity appeared again, which suggests positive effects of a metabolite.

Similar effect is observed for compounds 5 and 23a.

Compounds 22a and 23 have been also evaluated in pilo-

carpine-induced status epilepticus and the results are presented

in Table 6. On one hand, deaths are observed at very high doses

short after administration of relatively (for rats) high doses of the

tested compound. However, status epilepticus is itself risk of life,

therefore, it is often observed in results of this test that animals

die, and in the case of 22a at a dose of 400 mg/kg no animal died.

Within the Anticonvulsant Drug Development program ADD,¹⁵

due to promising anticonvulsant activity of 22a, the compound

was advanced to evaluation of analgesic activity in neurogenic pain

in the formalin test (mice, ip). The premise for such considerations

Figure 2. The molecular structure of 22a showing the atom numbering scheme.

Displacement ellipsoids are drawn at the 50% probability level.

Table 2

Intermolecular interaction for 22a

D–Hꢁ ꢁ ꢁA

Hꢁ ꢁ ꢁA (Å) Dꢁ ꢁ ꢁA (Å) D–H–A (°) Symmetry code

N1–HN2ꢁ ꢁ ꢁCl1 2.34(5)

N1–HN1ꢁ ꢁ ꢁCl1 2.21(4)

3.232(2)

3.127(2)

3.136(2)

3.743(3)

3.773(3)

3.640(3)

3.175(3)

160(3)

178(3)

174(4)

143.8

161.8

149.5

117.9

ꢀx + 1, y ꢀ 1/2, ꢀz + 2

O1–H1ꢁ ꢁ ꢁCl1

C1–H1Aꢁ ꢁ ꢁCl1

C3–H3Cꢁ ꢁ ꢁCl1

C4–H4Bꢁ ꢁ ꢁCl1

C6–H6Bꢁ ꢁ ꢁO1

2.32(5)

2.92

2.85

2.77

2.60

ꢀx, y ꢀ 1/2, ꢀz + 2

x, y ꢀ 1, z

ꢀx + 1, y + 1/2, ꢀz + 2

In terms of the ethoxyethyl analog of compound III—compound

7, its activity was not strong enough to determine ED₅₀. Anticon-

vulsant properties are observed in rats after 1 h of oral administra-

tion, which may suggest activity of its metabolite (Table 4).

Figure 3. Packing of molecules in the unit cell of the crystalline 22a projected along [010] direction. Dashed lines indicate hydrogen bonds.

1798

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

A

B

C

XIC of +MRM (10 pairs): 268.200/250.300 Da from Sample 27 (2_14) of DataMW.wiff (Turbo Spray)

9.04

Max. 8070.0 cps.

8000

7500

7000

6500

6000

5500

5000

4500

4000

3500

3000

2500

2000

1500

1000

500

0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

Time, min

11.0

12.0

13.0

14.0

15.0

16.0

17.0

18.0

19.0

XIC of +MRM (10 pairs): 268.200/250.300 Da from Sample 28 (3_14) of DataMW.wiff (Turbo Spray)

Max. 8880.0 cps.

9.21

8880

8500

8000

7500

7000

6500

6000

5500

5000

4500

4000

3500

3000

2500

2000

1500

1000

500

0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

Time, min

11.0

12.0

13.0

14.0

15.0

16.0

17.0

18.0

19.0

XIC of +MRM (10 pairs): 268.200/250.300 Da from Sample 23 (7_14) of DataMW.wiff (Turbo Spray)

Max. 3890.0 cps.

6.61

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

Time, min

11.0

12.0

13.0

14.0

15.0

16.0

17.0

18.0

19.0

Figure 4. The mass chromatograms of 22 (A), 23 (B) and 22a (C).

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

1799

Table 3

Table 5

ED₅₀and TD₅₀in mice, ip at 0.25 h

Anticonvulsant activity of selected compounds in the 6 Hz and TOX tests (mice, ip)

Compound MES ED₅₀(mg/kg)

(conﬁdence interval)

ScMet ED₅₀

(mg/kg)

TD₅₀(mg/kg)

(conﬁdence interval)

Compound

Test

Dose (mg/kg)

Time^a(h)

1.0

0.25

0.5

2.0

4.0

22a

23

12.92 (8.49–19.01)

11.3 (9.39–13.3)

5.34 (3.50–7.40)

>45.0^D

>43.0

>50

33.26 (31.33–35.71)

39.24 (32.27–43.72)

29.48 (21.76–37.87)

4

6 Hz^a

TOX^b

50

2/4

1/4

0/4

II⁸

5

6 Hz

TOX

100

3/4

1/4

2/4

0/4

1/4

0/4

D

Death.

9

6 Hz

TOX

100

2/4

0/4

are, as formerly stated—stabilization of neuronal membrane poten-

tial exhibited by many anticonvulsants. The results of the test are

presented in Figure 5 and Table 7. Within the acute phase of the

test (10 min after injection of formaldehyde), the analgesic activity

is signiﬁcant. Activity in the inﬂammatory phase (the remaining

time of the 40-min test) is not signiﬁcant, however, some effect

is visible.

Due to the above considerations, compounds 22a and 23a were

subject to search for mechanism of action with use of radioligand

binding studies. Therefore, standard high throughput proﬁle (Cerep,

France) was performed for them. In order to extract maximum

information regarding safety e.g. at intravenous administration, a

high concentration 10^ꢀ5M was chosen as preliminary. The results

are presented in Table 8, and they indicate that the compounds look

as quite ‘‘clean pharmacophores” since the strongest binding is

10

12

19

22

23

6 Hz

TOX

100

3/4

2/4

3/4

2/4

1/4

0/4

6 Hz

TOX

100

2/4

0/4

2/4

0/4

6-Hz

TOX

100

4/4

3/4

4/4

0/4

6-Hz

TOX

50

3/4

4/4

3/4

2/4

0/4

2/4

0/4

6-Hz

TOX

20

40

20

40

0/4

3/4

0/4

4/4

0/4

4/4

0/4

2/4

0/4

2/4

0/4

1/4

0/4

1/4

0/4

23a

30

6-Hz

TOX

30

2/4

0/4

2/4

0/4

2/4

0/4

6-Hz

TOX

50

2/4

3/4

2/4

0/4

observed only for

for the less interesting 23a, some binding is observed for sodium

channels, norepinephrine transporter, D₃, , NK₁. Therefore, due to

r, 5-HT transporter, 5-HT_2B, 5-HT_1A. Nevertheless,

a

No. of animals protected/no. of animals tested.

No. of animals exhibiting neurotoxicity/no. of animals tested in rotarod, ‘—’

b

j

more promising results of 22a, hERG binding was also determined

compound was not tested in these conditions.

Table 4

Anticonvulsant activity of selected compounds in the MES and TOX tests (rats)

Compound

Route of

Test

Dose

Time^a(h)

administration

(mg/kg)

0.25

0.5

1.0

2.0

4.0

9

ip

MES^a

TOX^b

MES

TOX

30

3/4

0/4

3/4

0/4

1/4

0/4

p.o.

10

ip

MES

TOX

MES

TOX

MES

TOX

30

3/4

0/4

3/4

0/4

3/4

0/4

3/4

0/4

1/4

0/4

1/4

0/4

ip

p.o.

14

16

17

18

23

p.o.

MES

TOX

30

0/4

1/4

0/4

MES

TOX

30

1/4

0/4

1/4

0/4

MES

TOX

30

0/4

MES

TOX

30

0/4

2/4

0/4

2/4

0/4

2/4

0/4

ip

MES

TOX

MES

TOX

30

4/4

0/4

4/4

0/4

3/4

0/4

p.o.

23a

ip

MES

TOX

MES

TOX

30

4/4

0/4

4/4

0/4

3/4

0/4

p.o.

26

27

p.o.

MES

TOX

30

0/4

MES

TOX

30

0/4

1/4

0/4

a

No. of animals protected/no. of animals tested.

No. of animals exhibiting neurotoxicity /no. of animals tested in rotarod, ‘—’ compound was not tested in these conditions.

b

1800

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

Table 6

Activity in pilocarpine-induced status epilepticus test (rats, ip)

Compound

Dose (mg/kg)

Time^a(h)

1.0 h

Dose

(mg/kg b.w.)

Time^a(h)

Effect^c

Average weight change

(g) SEM^b

0.25 h

0.5 h

2.0 h

4.0 h

22a

23

30

100

0/2

0/1

0/2

0/1

0/2

0/1

0/2

0/1

65

400

0

0.5

0/8^a

6/8^S

1/2^D

17.5 5.2

25.0 5.0

30

100

1/2^A

2/2^D

0/2

a

b

c

Post ﬁrst stage 3 seizure.

Weight change 24 h post ﬁrst stage 3 seizure.

No. of animals protected/no. of animals tested.

Sedation.

Ataxia.

Death.

S

A

D

0.5% methylcellulose

13 mg/kg 22a

and 23) for information regarding inﬂuence of metabolism on the

activity of the compounds. The results are presented in Table 12.

According to them, compounds 22a, 23 and 29 exhibited mod-

erate in vitro metabolic stability. Noteworthy, compound 29 being

inactive in former in vivo studies, demonstrated Cl_intvalues lying

in the lower limit of the medium category range (13 lL/mg/min).

On the other hand, active compounds 22a and 23, as enantiomers

were characterized with Cl_intvalues lying at the border between

medium and high intrinsic clearance values (47 lL/mg/min). Thus,

it is probable that metabolites of compounds 22a and 23 may con-

tribute to the previously observed in vivo activity of parent com-

pounds. Moreover, since 29 is racemic 4-methyl derivative of 22,

it can be deduced that the presence of methyl moiety in position

4 probably diminishes pharmacological activity of the compound

and inhibits its metabolism.

80

60

40

20

0

10

20

30

40

50

Time [min]

Figure 5. Analgesic properties of 22a in the formalin test, mice, ip.

Table 7

2.6. Pharmacokinetic proﬁle

Results of formalin test for compound 22a (mice, ip)

Dose (mg/

kg b.w.)

Phase

Area under the curve (AUC)

The results of the microsomal assay became premises to evalua-

tion of pharmacokinetic proﬁle of the most promising compound

22a, after its intravenous (1 mg/kg), intragastric (12.92 mg/kg) and

intraperitoneal (12.92 mg/kg) administration in mice. The concen-

tration of target compound in plasma was quantiﬁed using LC/ESI-

MS/MS system, and the method was thoroughly validated. The mean

plasma concentrations versus time proﬁles are depicted in Figure 6,

whereas the pharmacokinetic parameters are given in Table 13.

After the intravenous, intragastric or intraperitoneal adminis-

tration of the studied compound in mice the terminal half-lives

were rather short, 51.5 min, 29.1 min, and 77.2 min, respectively,

with relatively rapid elimination of the molecule from the body.

The pharmacokinetic results indicate that the absorption of 22a

was rapid, with the peak concentration occurring at 5 min after

an intragastric or intraperitoneal administration of target com-

pound, and with maximum concentration found to be 950 ng/mL

or 1190 ng/mL, respectively. Unfortunately, the absolute bioavail-

ability estimated based on the AUC_0?1calculated from zero time

to inﬁnity was very low, and yielded only the values of 2.98% for

intragastric or 6.78% for intraperitoneal administration. The main

reason of the low bioavailability of the studied compound might

be on one hand meaningful metabolism by the liver, and on the

other hand, possible poorly absorption by intestine or its interac-

tion with drug transporters. It is worth noting that the target com-

pound has high binding afﬁnity to mice brain (Fig. 7 and Tables 14

and 15).

Control 22a

% of

SEM

P

control

13.0

Acute

Inﬂammatory 819.6

237.47

130.39 54.9

665.07 81.14

11.52 <0.05

10.43 >0.05

at 10^ꢀ4M for veriﬁcation of preliminary cardiac safety, which is

obligatory for all drug candidates (Table 8).

2.4. Mutagenic or antimutagenic activity

Compounds 21, 22a, 23, and 25–30 have been also subject to

preliminary evaluation of mutagenic properties in the Vibrio har-

veyi test and the results are presented in Table 9. It is clearly visi-

ble, that not only the most interesting compounds 22a and 23, but

all evaluated samples, are not mutagenic. Due to accessibility of

the test, additionally antimutagenic properties have been tested.

The results are presented in Tables 10 and 11. The latter one serves

as a conclusion of such properties of the tested compounds—none

of them is mutagenic. Compounds 21, 27, and 29 exhibit signiﬁcant

although unexpected antimutagenic potential which could be fur-

ther explored.

2.5. Metabolic stability

Compounds 22a and 23 were chosen for further investigation of

metabolic stability with use of mice liver microsomes, in order to

determine which compound of either pair 22a or 23 or their

metabolites are responsible for long lasting activity in rodents.

Moreover, one very similar but inactive compound was added to

the assay—compound 29 (racemic 4-methyl derivative of 22a

3. Conclusions

The presented research resulted in 30 new compounds, among

which new active structures have been found. Additionally, for

compound 22a, absolute conﬁguration has been determined with

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

1801

Table 8

Receptor binding results for 22a and 23a at 10^ꢀ5M concentration

Receptor

Mean %

inhibition of

control

Study conditions

Radioligand

Reference data

speciﬁc

binding^a

22a

23a

Origin of receptor

Radioligand

type

Compound

IC₅₀[M]

A₁

A_2A

ꢀ32.2 ꢀ6.9

Human recombinant CHO cells [³H]DPCPX

Human recombinant HEKꢀ293 [³H]CGS 21680

cells

Antagonist

Agonist

DPCPX

NECA

5.0 ꢂ 10^ꢀ10

3.7 ꢂ 10^ꢀ8

ꢀ0.8

ꢀ4.0

A₃

ꢀ1.8

3.0

Human recombinant HEK-293

cells

[

¹²⁵I]ABMECA

Agonist

IB-MECA

6.0 ꢂ 10^ꢀ10

a₁

a₂

b₁

32.3

42.8

4.8

28.3

35.5

11.1

Rat cerebral cortex

Human recombinant HEK-293

cells

[³H]prazosin

Antagonist

Agonist

Prazosin

Yohimbine

Atenolol

2.5 ꢂ 10^ꢀ10

9.6 ꢂ 10^ꢀ8

2.1 ꢂ 10^ꢀ7

[³H]RX 821002

[³H](ꢀ)CGP12177

b₂

AT₁

2.0

5.7

Human recombinant CHO cells [³H](ꢀ)CGP12177

Agonist

Antagonist

ICI 118551

Saralasin

7.4 ꢂ 10^ꢀ10

4.0 ꢂ 10^ꢀ10

ꢀ27.4 ꢀ28.9 Human recombinant HEK-293

[

¹²⁵I] [Sar¹,Ile⁸]-ATII

¹²⁵I]CGP42112A

cells

AT₂

6.4

11.7

Human recombinant HEK-293

cells

[

Agonist

Angiotensin-II

8.0 ꢂ 10^ꢀ11

BZD (central)

BZD (peripheral)

BB (non-selective)

B₂

CGRP

CB₁

CCK₁(CCK_A)

CCK₂(CCK_B)

D₁

ꢀ11.0 ꢀ22.4 Rat cerebral cortex

[³H]ﬂunitrazepam

[³H]PK 11195

Agonist

Antagonist

Agonist

Antagonist

Diazepam

PK 11195

Bombesin

NPC 567

hCGRPalpha

CP 55940

CCK-8s

SCH 23390

(+)butaclamol

6.8 ꢂ 10^ꢀ9

1.6 ꢂ 10^ꢀ9

3.4 ꢂ 10^ꢀ10

1.4 ꢂ 10^ꢀ8

3.2 ꢂ 10^ꢀ11

9.0 ꢂ 10^ꢀ10

1.3 ꢂ 10^ꢀ10

1.2 ꢂ 10^ꢀ10

ꢀ3.9

ꢀ1.4

ꢀ3.9

ꢀ7.5

12.6

ꢀ3.6

ꢀ6.7

ꢀ5.4

1.6

ꢀ7.4

11.1

2.2

Rat heart

Rat cerebral cortex

[

¹²⁵I][Tyr⁴]bombesin

Human recombinant CHO cells [³H]bradykinin

Human recombinant CHO cells

[

¹²⁵I]hCGRP

a

Human recombinant CHO cells [³H]CP 55940

Human recombinant CHO cells

[

¹²⁵I]CCK-8s

ꢀ16.4 ꢀ32.0 Human recombinant CHO cells

10

12.4

12.0

16.2

14.7

Human recombinant CHO cells [³H]SCH23390

Human recombinant HEK-293

3.1 ꢂ 10ꢀ

D_2S

[³H]methylspiperone

1.1 ꢂ 10^ꢀ9

cells

D₃

D_4.4

D₅

ET_A

44.1

40.9

10.1

ꢀ3.4

57.4

35.8

5.2

ꢀ4.4

Human recombinant CHO cells [³H]methylspiperone

Human recombinant GH4 cells [³H]SCH23390

Antagonist

Agonist

(+)butaclamol

Clozapine

SCH 23390

Endothelin-1

Endothelin-3

GABA

1.6 ꢂ 10^ꢀ9

4.1 ꢂ 10^ꢀ8

5.6 ꢂ 10^ꢀ10

2.3 ꢂ 10^ꢀ11

6.3 ꢂ 10^ꢀ12

2.1 ꢂ 10^ꢀ8

7.9 ꢂ 10^ꢀ11

Human recombinant CHO cells

[

¹²⁵I]endothelin-1

ET_B

ꢀ15.6 ꢀ24.2 Human recombinant CHO cells

GABA (non-selective)

GAL₁

0.8

17.7

Rat cerebral cortex

Human recombinant HEK-293

cells

[³H]GABA

ꢀ0.7

ꢀ0.4

[

¹²⁵I]galanin

Agonist

Galanin

GAL₂

PDGF

CXCR2 (IL-8B)

ꢀ16.5 ꢀ11.1 Human recombinant CHO cells

[

¹²⁵I]galanin

¹²⁵I]PDGF BB

¹²⁵I]IL-8

Agonist

Galanin

PDGF BB

IL-8

2.6 ꢂ 10^ꢀ9

4.7 ꢂ 10^ꢀ11

5.1 ꢂ 10^ꢀ11

ꢀ12.5 ꢀ5.6

2.9 2.7

Balb/c 3T3 cells

Human recombinant HEK-293

cells

CCR1

ꢀ11.2 ꢀ14.2 Human recombinant HEK-293

[

¹²⁵I]MIP-1

¹²⁵I]TNF-

a

Agonist

MIP-1alpha

2.1 ꢂ 10^ꢀ11

cells

U-937 cells

TNF-

H₁

a

5.1

4.6

[

a

Agonist

Antagonist

TNF-alpha

Pyrilamine

9.9 ꢂ 10^ꢀ11

1.4 ꢂ 10^ꢀ9

22.7

14.1

Human recombinant HEK-293

cells

Human recombinant CHO cells

[³H]pyrilamine

H₂

MC₄

MT₁(ML_1A

M₁

M₂

M₃

M₄

M₅

NK₁

NK₂

NK₃

Y₁

0.6

5.3

17.1

9.0

[

¹²⁵I]APT

Antagonist

Agonist

Antagonist

Agonist

Antagonist

Agonist

Cimetidine

NDP-alpha-MSH

Melatonin

Pirenzepine

Methoctramine

4-DAMP

[Sar9,Met(O2)11]-SP

[Nleu10]-NKA (4–10)

SB 222200

NPY

Neurotensin

DPDPE

U 50488

DAMGO

1.0 ꢂ 10^ꢀ6

1.5 ꢂ 10^ꢀ10

1.1 ꢂ 10^ꢀ10

2.1 ꢂ 10^ꢀ8

2.2 ꢂ 10^ꢀ8

4.1 ꢂ 10^ꢀ10

4.9 ꢂ 10^ꢀ10

3.2 ꢂ 10^ꢀ10

1.1 ꢂ 10^ꢀ10

3.5 ꢂ 10^ꢀ9

6.7 ꢂ 10^ꢀ9

2.1 ꢂ 10^ꢀ10

2.8 ꢂ 10^ꢀ11

4.9 ꢂ 10^ꢀ10

1.3 ꢂ 10^ꢀ9

6.4 ꢂ 10^ꢀ10

4.3 ꢂ 10^ꢀ10

¹²⁵I]NDP-

a-MSH

)

ꢀ12.2 ꢀ7.5

¹²⁵I]2-iodomelatonin

6.0

20.8

8.5

22.8

37.2

4.4

Human recombinant CHO cells [³H]pirenzepine

Human recombinant CHO cells [³H]AF-DX384

Human recombinant CHO cells [³H]4-DAMP

11.2

8.6

15.7

5.7

17.3

2.2

8.0

51.7

41.2

15.0

U-373MG cells (endogenous)

Human recombinant CHO cells

[

¹²⁵I]BH-SP

¹²⁵I]NKA

Human recombinant CHO cells [³H]SR142801

ꢀ34.5 ꢀ29.6 SK-N-MC cells (endogenous)

[

¹²⁵I]peptideYY

Y₂

ꢀ5.1

ꢀ4.2

ꢀ1.7

29.3

23.6

ꢀ14.5 KAN-TS cells

¹²⁵I]peptideYY

NTS₁(NT₁)

d₂(DOP)

ꢀ10.5 Human recombinant CHO cells

¹²⁵I]Tyr3-neurotensin

ꢀ2.2

54.0

40.2

Human recombinant CHO cells [³H]DADLE

Rat recombinant CHO cells

Human recombinant HEK-293

cells

Human recombinant HEK-293

cells

Agonist

j

l

(KOP)

(MOP)

[³H]U 69593

[³H]DAMGO

NOP (ORL1)

1.5

0.2

[³H]nociceptin

Agonist

Nociceptin

1.3 ꢂ 10^ꢀ9

PAC₁(PACAP)

PPAR

PCP

EP₂

ꢀ38.2 ꢀ12.1 Human recombinant CHO cells

[

¹²⁵I]PACAP1-27

Agonist

Antagonist

Agonist

PACAP1-38

Rosiglitazone

MK 801

4.0 ꢂ 10^ꢀ11

1.0 ꢂ 10^ꢀ8

4.6 ꢂ 10^ꢀ9

2.6 ꢂ 10^ꢀ9

ꢀ12.7 ꢀ26.0 Human recombinant (E. coli)

[³H]rosiglitazone

[³H]TCP

c

9.6

3.4

5.8

11.2

Rat cerebral cortex

Human recombinant HEK-293

cells

[³H]PGE₂

PGE2

(continued on next page)

1802

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

Table 8 (continued)

Receptor

Mean %

inhibition of

control

Study conditions

Reference data

speciﬁc

binding^a

22a

23a

Origin of receptor

Radioligand

type

Compound

Iloprost

IC₅₀[M]

EP₄

14.9

3.0

Human recombinant HEK-293

cells

Human recombinant HEK-293

cells

Rat urinary bladder

Rat cerebral cortex

Human recombinant HEK-293

cells

[³H]PGE2

Agonist

IP (PGI₂)

ꢀ11.5 ꢀ6.0

[³H]iloprost

1.0 ꢂ 10^ꢀ8

P2X

P2Y

5-HT_1A

3.5

9.4

70.6

4.8

9.4

63.9

[³H]

³⁵S]dATP

[³H]8-OHDPAT

a,b-MeATP

Agonist

alpha,beta-MeATP

dATPalpha S

8-OH-DPAT

1.9 ꢂ 10^ꢀ9

2.9 ꢂ 10^ꢀ8

5.3 ꢂ 10^ꢀ10

[

aS

5-HT_1B

5-HT_2A

ꢀ1.8

16.5

32.1

Rat cerebral cortex

[

¹²⁵I]CYP (+30

lM

Antagonist

Serotonin

4.9 ꢂ 10^ꢀ9

3.5 ꢂ 10^ꢀ10

isoproterenol)

22.6

Human recombinant HEK-293

cells

[³H]ketanserin

Ketanserin

5-HT_2B

5-HT_2C

89.4

34.7

78.8

47.4

Human recombinant CHO cells

Human recombinant HEK-293

cells

[

¹²⁵I]( )DOI

[³H]mesulergine

Agonist

Antagonist

( )DOI

RS 102221

2.5 ꢂ 10^ꢀ9

1.5 ꢂ 10^ꢀ9

5-HT₃

5-HT_5A

29.4

4.2

12.3

10.7

Human recombinant CHO cells [³H]BRL43694

Antagonist

Agonist

MDL 72222

Serotonin

6.9 ꢂ 10^ꢀ9

1.8 ꢂ 10^ꢀ7

Human recombinant HEK-293

cells

[³H]LSD

5-HT₆

5-HT₇

6.7

15.4

24.6

95.6

ꢀ2.0

8.3

Human recombinant CHO cells [³H]LSD

Agonist

Serotonin

1.2 ꢂ 10^ꢀ7

2.5 ꢂ 10^ꢀ10

1.7 ꢂ 10^ꢀ8

7.4 ꢂ 10^ꢀ11

3.8 ꢂ 10^ꢀ9

1.1 ꢂ 10^ꢀ10

24.5

92.9

ꢀ5.5

31.3

r

(non-selective)

Jurkat cells (endogenous)

AtT-20 cells

IM-9 cells (cytosol)

[³H]DTG

Haloperidol

Somatostatin-14

Dexamethasone

VIP

sst (non-selective)

GR

VPAC₁(VIP₁)

V_1a

[

¹²⁵I]Tyr11-somatostatin-14 Agonist

[³H]dexamethasone

Agonist

ꢀ11.9 ꢀ17.8 Human recombinant CHO cells

[

¹²⁵I]VIP

ꢀ3.2

ꢀ0.8

Human recombinant CHO cells [³H]AVP

[³H]D888

[d(CH2)51,Tyr(Me)2]- 9.9 ꢂ 10^ꢀ10

AVP

D 600

Ca²⁺channel (L, verapamil

site)

K_Vchannel

43.6

65.9

Rat cerebral cortex

Antagonist

1.5 ꢂ 10^ꢀ8

ꢀ20.9 ꢀ15.9 Rat cerebral cortex

[

¹²⁵I]

¹²⁵I]apamin

a

-dendrotoxin

Antagonist

Alpha-dendrotoxin

Apamin

Veratridine

Picrotoxinin

Protriptyline

5.7 ꢂ 10^ꢀ10

1.6 ꢂ 10^ꢀ11

4.0 ꢂ 10^ꢀ6

7.7 ꢂ 10^ꢀ8

2.8 ꢂ 10^ꢀ9

SK_Cachannel

ꢀ11.1 ꢀ0.9

Rat cerebral cortex

Na⁺channel (site 2)

Cl^ꢀchannel (GABA-gated)

Norepinephrine

transporter (h)

54.9

ꢀ7.6

52.6

79.0

4.6

34.5

[³H]batrachotoxinin

[

³⁵S]TBPS

Human recombinant CHO cells [³H]nisoxetine

Dopamine transporter

5-HT transporter

hERG (at 10^ꢀ4M)

24.5

96.5

3.6

16.2

91.4

ꢀ

Human recombinant CHO cells [³H]BTCP

Antagonist

BTCP

Imipramine

2.8 ꢂ 10^ꢀ9

8.8 ꢂ 10^ꢀ10

Human recombinant CHO cells [³H]imipramine

Human recombinant HEK-293

cells

[³H]astemizole

The bold values show >50% binding.

a

n = 2.

Table 9

Table 10

Mutagenic activity of the test compounds, assessed by use of the V. harveyi test

Antimutagenic activity of the test compounds, assessed by use of the V. harveyi test

Compound^aBB7

BB7X BB7M BB7XM

Number of colonies: mean SD (inhibition rate %)^b

Compound

BB7

BB7X

BB7M

BB7XM

Number of colonies (mean SD)^b

CTR1^a

CTR2^a

NQNO

13

20

21

22a

23

25

26

27

13

14

37

22

24

20

15

20

28

29

22

25

20

3

2

4

5

3

2

5

8

2

5

8

15

16

31

17

27

29

25

22

26

27

25

16

24

15

3

1

4

7

4

2

5

4

5

14

13

45

26

25

27

22

26

20

26

18

19

10

2

5

4

7

9

5

6

7

2

4

7

5

14

33

23

20

19

18

27

22

26

24

26

4

5

1

3

4

6

8

3

4

5

4

5

3

7

CTR1

CTR2

NQNO

21

22a

23

25

26

27

28

16

12

38

11

9

11

4

5

7

6

7

15

3

4

2

3

2

3

2

1

2

3

11

13

31

1

2

5

3

4

1

3

7

14

16

45 11

11

14

4

2

14

31

16

18

4

1

3

4

(70) 23

(75) 29

(70) 29

(81) 14

(88) 24

(82) 15

(84) 27

(81) 19

(62) 59

(27)

(7)

(6)

1

3

2

(75)

(69)

(48)

(41)

(69) 29 12 (5)

(56) 20 22 (55)

22

27

21

23

18

34

4

3

6

2

3

(30)

(15)

(33)

(26)

(43)

(0)

(22)

(52)

(12)

(38)

(0)

22

11

30

17

33

2

5

4

5

(52)

(75)

(34)

(62)

(27)

29

30

28

29

30

a

CTR1 (H₂O)—negative control; CTR2 (DMSO)—negative control; NQNO (nitro-

quinoline-N-oxide, 40 ng/mL)—positive control.

a

b

CTR1 (H₂O)—negative control; CTR2 (DMSO)—negative control; NQNO (nitro-

Mean values from three experiments standard deviation (SD) are presented.

quinoline-N-oxide, 40 ng/mL)—positive control.

The values in parenthesis are the inhibition rates (%) of mutagenicity; 25–40%

inhibition: moderate antigenotoxicity, 40% or more inhibition: strong antigeno-

toxicity, 25% or less inhibition: no antigenotoxicity.

b

Mean values from three experiments standard deviation (SD) are presented.

use of crystallography and compared with the reference

compounds.

(22a) (the ethoxyethyl analog of the reference compound II),

exhibiting interesting anticonvulsant activity in MES and 6 Hz test

(mice, ip), pilocarpine-induced status epilepticus (rats, ip) as well

The most active compound is R-(ꢀ)-2N-{2-[2-(2,6-

dimethylphenoxy)ethoxy]ethyl}aminopropan-1-ol hydrochloride

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

1803

Table 11

Summary of mutagenicity and antimutagenicity of tested compounds

Compound

Mutagenicity^a

BB7M

Antimutagenicity^b

BB7M

BB7

BB7X

BB7XM

BB7

BB7X

BB7XM

21

22a

23

25

26

27

28

29

30

—

S

M

0

S

W

S

W

M

0

S

M

S

M

S

0

M

W

M

S

0

a

Positive result of the mutagenicity assay (+) indicates at least twofold increase in the number of neomycin-resistant colonies relative to the control experiment and/or the

number of colonies higher than that observed in the experiment with a known mutagen (NQNO). Otherwise, the result is considered negative (ꢀ).

b

Abbreviations are as follows: S—strong antimutagenic properties (>40% inhibition of mutagenicity of NQNO); M—moderate antimutagenic properties (25–40% inhibition

of mutagenicity of NQNO); W—weak antimutagenic properties (10–25% inhibition of mutagenicity of NQNO); 0—no antimutagenic properties (<10% inhibition of muta-

genicity of NQNO).

Table 12

Metabolic stability screen of compounds 22a, 23 and 29 in mouse liver microsomes (MLMs) and major metabolites characteristics

Compound

t_1/2[min]

Molecular

Retention

Cl_int

(lL/mg/min)

Metabolites

ions (m/z)

time (min)

ID

Retention

time (min)

% content among

metabolites

Molecular ions (m/z)

22a

23

37

268

3.81

3.80

47

M1

M2

2.81

2.73

72

28

284 [M+16]⁺

300 [M+32]⁺

M1

M2

M3

2.81

2.50

2.72

70

17

13

284 [M+16]⁺

300 [M+32]⁺

29

136

282

4.31

13

M1

M2

M3

2.57

3.34

4.17

61

20

19

298 [M+16]⁺

224 [Mꢀ58]⁺

Imipramine¹⁶

Fentanyl¹⁶

11

39

—

126

89

Table 13

Basic pharmacokinetic parameters of compound 22a after its single intravenous,

intragastric, or intraperitoneal administration in mice

Parameters

Administration

iv

p.o.

ip

C₀(ng/mL)

C_max(ng/mL)

t_max(min)

2205

—

950

5

—

1190

5

t_1/2(min)

MRT (min)

AUC_0?1(ng min/mL)

AUMC_0?1(ng min²/mL)

Cl_t(mL/min/kg)

V_ss(mL/kg)

51.5

55.6

61,832.7

3,439,871

16.2

899.7

—

29.1

40.3

23,769.6

958,477

—

77.2

85.2

54,175.6

4,614,684

—

2.98

—

6.78

F (%)

C₀: the initial concentration, C_max: the maximum plasma concentration; t_max: time

to reach the maximum plasma concentration; t_1/2: the terminal half-life; MRT: the

mean residence time; AUC_0?1: the area under the concentration–time curve from

zero up to inﬁnitive time; AUMC_0?1: the area under the ﬁrst-moment curve from

zero up to inﬁnitive time; Cl_t: the total clearance; V_ss: the volume of distribution at

steady state; F: the absolute bioavailability.

Figure 6. Plasma concentration–time proﬁles of compound 22a following its

intravenous, intragastric or intraperitoneal administration in mice (semi-logarith-

mic plots).

as analgesic activity in a neurogenic pain model—the formalin test.

The probable mechanism of action results from binding to some of

undertaken efforts (metabolic stability and pharmacokinetic pro-

ﬁle) prove that compound 22a is metabolized mostly by the liver

(rather than intestines), since the bioavailability after ip adminis-

tration is not much higher (6.78%) compared to bioavailability after

intragastric administration (2.98%). Taking into account the high

activity of the compound in mice, we suppose that the formation

of the active metabolites play an important role in the mechanism

of action, compensating low bioavailability of the parent molecule.

r

, 5-HT_2B, 5-HT_2Areceptors, or 5-HT transporter. The compound

exhibits safety in Vibrio harveyi test tor mutagenesis. In terms of

aim of the study—incorporation of ethoxyethyl linker instead of

ethyl between phenoxyl and aminoalkanol resulted in interesting

pharmacological proﬁle.

During the study further questions arose, regarding metabolism

and pharmacokinetics of the selected molecules. The results of

1804

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

to distillation under reduced pressure, until oily residue. Then

50 mL of water and l0% HCl was added until acidic pH, and the mix-

ture was reﬂuxed with activated carbon. Then the suspension was

ﬁltered off and 10% NaOH was added for alkalization of the ﬁltrate

and precipitation of the base. The oily product was extracted to

toluene and the organic phase was dried with anhyd Na₂SO₄over-

night and the solvent was distilled off. The oily residue was recrys-

tallized with use of n-hexane or petroleum ether for achievement

of required crystal forms. The yield of N-alkylation ranged

45–75%. The yield of the method of N-alkylation with use of TEBA

in acetone for several compounds ranged 35–60%.

In cases of 22a or 23a the bases were transformed into

hydrochlorides with use of acetone solution of gaseous HCl. The

obtained hydrochlorides were recrystallized from mixture

acetone/MeOH (3:1).

Melting points (mp) were determined using a Büchi SMP-20

apparatus and are uncorrected. Analyses of C, H, N were within

0.4% of the theoretical values. Analytical TLC was carried out on

precoated plates (silica gel, 60 F-254 Merck). Spots were visualized

with UV light. ¹H NMR and ¹³C NMR spectra for compounds ¹H

NMR and ¹³C NMR spectra for compounds 1–3, 6–8, and 11,

20–21 were recorded at Faculty of Chemistry, Jagiellonian Univer-

sity (Krakow, Poland), on a Bruker AVANCE III 600 (resonance

frequencies 600.20 MHz for ¹H and 150.94 MHz for ¹³C) equipped

a 5-mm probehead: PABBO with z-gradient or TBI with XYZ gradi-

ents. The ¹H spectra were recorded with 16 scans, 1 s relaxation

delay, 4 s acquisition time, 128 kW FID size, with 16,234 Hz spec-

tral width. The ¹³C spectra were recorded with WALTZ-16 ¹H

broadband decoupling, a few thousands scans, 2 s relaxation delay,

0.9 s acquisition time, 64 kW FID size, 36,057 Hz spectral width.

Standard pulse sequences from Bruker library were used for 2D

spectra. Gradient enhanced sequences were used for the homo-

and heteronuclear 2D experiments. All processing and analysis

were performed using Bruker’s TopSpin 3.0 software suite.

¹H NMR spectra for compounds ¹H NMR spectra for compounds

4–5, 9–10, 12–19, 22–30 were recorded in CDCl₃and 15a and

23a—in DMSO-d₆were recorded at Faculty of Pharmacy, Jagiel-

lonian University Medical College (Krakow, Poland) with a Varian

Mercury-VX 300 NMR spectrometer at 29 °C. Chemical shifts were

referenced against solvent lock signal. Standard Varian pulse

sequences were used for 2D experiments.

Figure 7. Distribution of 22a to mice brain after its intravenous (1 mg/kg),

intragastric (12.92 mg/kg), or intraperitoneal (12.92 mg/kg) administration.

Table 14

Distribution of 22a in mice brain after intravenous, intragastric, or intraperitoneal

administration

Parameters

iv

po

ip

AUC_0?1(ng min/g)

MRT (min)

C_max(ng/g)

309,009

50.1

11,527

5

73,322

73.8

1640

5

129,540

83.1

2360

15

t_max(min)

Table 15

Ratio of 22a mean concentrations in brain and plasma after single intravenous,

intragastric, or intraperitoneal administration

Time (min)

Ratio of 22a concentrations in brain versus

plasma

iv

po

ip

5

5.27

5.36

5.41

3.58

6.66

2.98

1.73

2.67

2.12

5.95

3.18

1.13

2.55

4.25

1.91

2.86

1.94

15

30

60

120

240

Results are presented in the following format: chemical shift d

(ppm), multiplicity, J values in Hertz (Hz), number of protons, pro-

tons’ position. Multiplicities are showed as the abbreviations: s

(singlet), br s (broad singlet), d (doublet), dd (doublet of doublets),

dt (doublet of triplets), t (triplet), tt (triplet of triplets), m

(multiplet).

4. Experimental protocols

4.1. Chemistry

The aminoalkanols used for synthesis of compounds 1, 3–4, 6–8,

11–12, 14, 16, 18–19, 21, 24, 27, 30 were racemic (Table 1).

The IR spectra were recorded on a Jasco FT/IR 410 spectrometer

(KBr pellets). Measurement of optical rotation ([a]₅₈₉) was carried

D

,

L

-trans-2-Aminocyclohexan-1-ol was achieved from cyclohexene

out using Jasco 2000 (k = 589 nm). For mass spectrometry analysis

samples were prepared in acetonitrile/water (10/90 v/v) mixture.

The LC/MS system consisted of a Waters Acquity UPLC, coupled

to a Waters TQD mass spectrometer (electrospray ionization mode

ESI-tandem quadrupole). All the analyses were carried out usingan

Acquity UPLC BEH C18, 1.7 lm, 2.1 ꢃ 100 mm column. A ﬂow rate

of 0.3 mL/min and a gradient of (5–95)% B over 10 min and then

100% B over 2 min was used. Eluent A: water/0.1% HCO₂H; eluent

B: acetonitrile/0.1% HCO₂H. LC/MS data were obtained by scanning

the ﬁrst quadrupole in 0.5 s in a mass range from 50 to 1000 Da;

eight scans were summed up to produce the ﬁnal spectrum.

oxide according to formerly published procedures.^7,17All other

reagents were purchased from Alfa Aesar GmbH & Co KG (Karlsruhe,

Germany) and Merck Sp. z o.o. (Warszawa, Poland) and solvents

were commercially available materials of reagent grade.

4.1.1. Synthesis of the title compounds

0.025 mole of appropriate bromide (i.e. (2,3-dimethylphenoxy)

ethyl bromide, (2,3-dimethylphenoxy)propyl bromide, (2,4-

dimethylphenoxy)ethyl bromide, (2,4,6-trimethylphenoxy)ethyl

bromide, (2,3-dimethylphenoxy)ethoxyethyl bromide, (2,6-

dimethylphenoxy)ethoxyethyl bromide, or (2,4,6-trimethylphe-

noxy)ethoxyethyl bromide) was placed in a 250 mL ﬂask and

0.022 mole of appropriate aminoalkanol was added with

0.02 mol anhyd K₂CO₃as proton acceptor. The mixture was

reﬂuxed in 50 mL toluene for 5 h, and afterwards the reaction

mixture was ﬁltered off (KBr/K₂CO₃) and the ﬁltrate was subject

4.1.1.1. R,S-2N-[(2,3-Dimethylphenoxy)ethyl]aminopropan-1-ol

(1). White solid. Yield = 60%. M = 223.31; C₁₃H₂₁NO₂; mp 76–78 °C

[%^calcd

(KBr, cm^ꢀ1

2601, 2361, 2116, 1909, 1587, 1461, 1266, 771; ¹H NMR (d

/

_analyzed] C^69.92

/

_69.45, H^9.48

/

N^6.27

/_6.24; R_f= 0.47 (CH₃OH); IR

9.73

)

v: 3735, 3649, 3295, 3127, 2964, 2931, 2886, 2833,

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

1805

ppm): 7.01 (dd, J = 7.9, J = 7.5, 1H, H-5); 6.77 (d, J = 7.9, 1H, H-4);

6.74 (d, J = 7.5, 1H, H-6); 4.54 (t, J = 5.2, 1H, OH); 4.03–3.97 (m,

1H, CHH–OAr); 3.97–3.91 (m, 1H, CHH–OAr); 3.27–3.24 (m, 1H,

CHH–OH); 3.24–3.17 (m, 1H, CHH–OH); 2.97–2.90 (m, 1H, CHH–

N); 2.90–2.83 (m, 1H, CHH–N); 2.71–2.62 (m, 1H, CH); 2.20 (s,

3H, CH₃–Ar (2)); 2.07 (s, 3H, CH₃–Ar (3)); 1.89 (br s, 1H, NH);

0.92 (d, J = 6.4, 3H, CH₃).

122.01 (C-4), 109.29 (C-6), 71.61 (CH), 67.93 (Ar–O–CH₂–), 57.54

(N–CH₂–), 48.17 (–CH₂–N), 19.69 (CH₃–Ar (3)), 11.37 (CH₃–Ar (2)).

4.1.1.7. R,S-2N-[(2,3-Dimethylphenoxy)propyl]aminopropan-1-

ol (7). White solid. Yield = 64%; M = 237.34; C₁₄H₂₃NO₂; mp 88–

90 °C; [%^calcd

/

]

C^70.85

/

,

H^9.77

/

,

N^5.90

/ ;

5.86

R_f= 0.24

analyzed

70.89

10.02

(CH₃OH); IR (KBr, cm^ꢀ1

) v: 3260, 3110, 2957, 2924, 2824, 2599,

2359, 1593, 1481, 1257, 1091, 768; ¹H NMR (d ppm): 7.00 (dd,

J = 8.2, J = 7.5, 1H, H-5); 6.76 (d, J = 8.5, 1H, H-4); 6.73 (d, J = 7.5,

1H, H-6); 4.45 (br s, 1H, OH); 3.91 (t, J = 6.2, 2H, CH₂–OAr);

3.28–3.18 (m, 2H, CH₂–OH); 2.80–2.70 (m, 2H, CH₂–N); 2.69–

2.55 (m, 1H, CH); 2.20 (s, 3H, CH₃–Ar (2)); 2.07 (s, 3H, CH₃–Ar

(3)); 1.87–1.80 (m, 2H, R–CH₂–R); 1.56 (br s, 1H, NH); 0.90

(d, J = 6.4, 3H, CH₃).

4.1.1.2. 2N-[(2,3-Dimethylphenoxy)ethyl]amino-2-methylpropan-

1-ol (2). White solid. Yield = 62%; M = 237.34; C₁₄H₂₃NO₂; mp 75–

77 °C; [%^calcd

/

_analyzed] C^70.85

/

_70.70, H^9.77

/

_9.90, N^5.90

/

_5.72; R_f= 0.44 (CH₃OH);

IR (KBr, cm^ꢀ1

)

v: 3295, 3154, 2986, 2971, 2926, 2877, 2827, 2756,

1

2557, 2359, 1584, 1464, 1263, 1068, 768; H NMR (d ppm): 7.05 (dd,

J = 8.0, J = 7.5, 1H, H-5); 6.80 (d, J = 8.0, 1H, H-6); 6.78 (d, J = 7.5, 1H,

H-4); 4.68 (t, J = 3.1, 1H, OH); 3.98 (t, J = 5.7, 2H, CH₂–OAr); 3.24 (d,

J = 3.1, 2H, CH₂–OH); 2.86 (t, J = 5.7, 2H, CH₂–N); 2.24 (s, 3H, CH₃–Ar

(3)); 2.12 (s, 3H, CH₃–Ar (2)); 1.82 (br s, 1H, NH); 1.01 (s, 6H, 2ꢃ CH₃).

4.1.1.8. R,S-2N-[(2,3-Dimethylphenoxy)propyl]aminobutan-1-ol

(8). White solid. Yield = 60%; M = 251.37; C₁₅H₂₅NO₂; mp 82–

84 °C; [%^calcd

/

_analyzed] C^71.67

/

_71.43, H^10.02

/

_10.01, N^5.57

/

_5.53; R_f= 0.24

4.1.1.3. R,S-2N-[(2,3-Dimethylphenoxy)ethyl]aminobutan-1-ol

(3). White solid. Yield = 55%; M = 237.34; C₁₄H₂₃NO₂; mp 55–

(CH₃OH); IR (KBr, cm^ꢀ1

) v: 3258, 3100, 2964, 2922, 2863, 2815,

1910, 1584, 1464, 1255, 1096, 768; ¹H NMR (d ppm): 7.00 (dd,

J = 8.2; J = 7.6, 1H, H-5); 6.77 (d, J = 8.2, 1H, H-6); 6.70 (d, J = 7.6,

1H, H-4); 4.40 (br s, 1H, –OH); 3.99 (t, J = 6.2, 2H, Ar–O–CH₂–);

3.39–3.21 (m, 2H, CH₂–OH); 2.69 (t, J = 6.8, 2H, –CH₂–N);

2.41–2.31 (m, 1H, CH); 2.20 (s, 3H, CH₃–Ar (3)); 2.07 (s, 3H, CH₃–

Ar (2)); 1.84 (tt, J = 6.8, J = 6.2, 2H, R–CH₂–R); 1.48 (br s, 1H, –

NH); 1.39–1.28 (m, 2H, –CH₂–CH₃); 0.83 (t, J = 7.5, 3H, R–CH₃);

¹³C NMR (d ppm): 156.40 (C-1), 137.02 (C-3), 125.77 (C-5),

124.03 (C-2), 121.75 (C-4), 109.07 (C-6), 65.96 (Ar–O–CH₂–),

62.47 (CH₂–OH), 60.22 (CH), 43.42 (–CH₂–N), 29.93 (R–CH₂–R),

23.60 (CH₃–CH₂–), 19.64 (CH₃–Ar(3)), 11.29 (CH₃–Ar(2)), 9.94

(CH₃–CH₂–).

56 °C; [%^calcd

/

_analyzed] C^70.86

/

_70.76, H^9.77

/

_9.76, N^5.90

/

_5.82; R_f= 0.51 (CH₃-

OH); IR (KBr, cm^ꢀ1

)

v: 3295, 3158, 2964, 2931, 2875, 2837, 1900,

1585, 1470, 1263, 1109, 1062, 762; ¹H NMR (d ppm): 7.01 (dd,

J = 8.3, J = 7.7, 1H, H-5); 6.77 (d, J = 8.3, 1H, H-6); 6.74 (d, J = 7.7,

1H, H-4); 4.46 (t, J = 4.4, 1H, OH); 3.98 (dt, J = 9.5, J = 5.5, 1H,

CHH–OAr); 3.94 (dt, J = 9.5, J = 5.5, 1H, CHH–OAr); 3.43–3.20 (m,

2H, CH₂–OH); 2.90 (t, J = 5.5, 2H, CH₂–N); 2.49–2.41 (m, 1H, CH);

2.20 (s, 3H, CH₃–Ar (2)); 2.07 (s, 3H, CH₃–Ar (3)); 1.81 (br s, 1H,

NH); 1.41–1.31 (m, 2H, CH₂–CH₃); 0.85 (t, J = 7.3, 3H, CH₂–CH₃).

4.1.1.4.

hexan-1-ol (4). White solid. Yield = 57%; M = 263.38; C₁₆H₂₅NO₂;

mp 95–97 °C; [%^calcd_analyzed] C^72.97H^9.57N^5.32

R_f= 0.54 (CH₃OH/ethyl acetate 1:1); IR (KBr, cm^ꢀ1

D,L-trans-2N-[(2,3-Dimethylphenoxy)ethyl]aminocyclo-

/

,

/

,

/ ;

5.23

4.1.1.9. N-[(2,3-Dimethylphenoxy)propyl]piperidin-3-ol (9). White

72.71

9.26

)

v

: 3298, 3113,

solid. Yield = 53%; M = 263.38; C₁₆H₂₅NO₂; mp 86–88 °C; [%^calcd

/

]

analyzed

2929, 2858, 2816, 2365, 1585, 1461, 1264, 1111; ¹H NMR (d

ppm): 6.97–7.09 (m, 1H, Ar–H5); 6.78 (d, J = 7.69, 1H, Ar–H4);

6.71 (d, J = 8.21, 1H, Ar–H6); 4.05 (t, J = 5.26, 2H, O–CH₂–CH₂–

NH); 3.33 (br s, 1H, NH); 3.27–3.10 (m, 2H, O–CH₂–CH₂–NH);

2.89 (dt, J = 12.50, J = 4.78, 1H, CH–OH); 2.27 (s, 3H, CH₃–Ar (2));

2.34–2.22 (m, 1H, CH–NH); 2.15 (s, 3H, CH₃–Ar (3)); 2.13–2.00

(m, 2H, cyclohex); 1.80–1.67 (m, 2H, cyclohex); 1.35–1.17 (m,

3H, cyclohex); 1.09–0.92 (m, 1H, cyclohex).

C^72.97

/

_72.48, H^9.57

/

_9.45, N^5.32

/

_5.25; R_f= 0.54 (CH₃OH/ethyl acetate 1:1);

cLogP = 2.76; IR (KBr, cm^ꢀ1

)

v: 3149, 2947, 2867, 2826, 2775, 2513,

2149, 1914, 1584, 1467, 1068; ¹H NMR (d ppm): 7.08–6.99 (m, 1H,

Ar–H5); 6.77 (d, J = 7.44, 1H, Ar–H4); 6.70 (d, J = 8.21, 1H, Ar–H6);

3.99 (t, J = 6.28, 2H, O–CH₂–CH₂–CH₂–NH); 3.82 (br s, 1H, NH); 2.61–

2.45 (m, 5H, O–CH₂–CH₂–CH₂–NH, CH–OH, pip-H); 2.42 (br s, 1H,

OH); 2.36–2.28 (m, 1H, pip-H); 2.27 (s, 3H, CH₃–Ar (2)); 2.14 (s, 3H,

CH₃–Ar (3)); 2.04–1.90 (m, 2H, O–CH₂–CH₂–CH₂–NH); 1.88–1.72 (m,

1H, pip); 1.69–1.46 (m, 3H, pip).

4.1.1.5. N-[(2,3-Dimethylphenoxy)ethyl]piperidin-4-ol (5). White

solid. Yield = 53%; M = 249.35; C₁₅H₂₃NO₂; mp 71–72 °C; [%^calcd

/

]

4.1.1.10.

(10). White solid. Yield = 56%; M = 263.38; C₁₆H₂₅NO₂; mp 68–

70 °C; [%^calcd_analyzed] C^72.97_73.15, H^9.57_9.42, N^5.32

_5.29; R_f= 0.39 (CH₃-

OH/ethyl acetate 1:1); cLogP = 2.74; IR (KBr, cm^ꢀ1

: 3133, 2959,

1N-[(2,3-Dimethylphenoxy)propyl]piperidin-4-ol

analyzed

C^72.25

/

_72.44, H^9.30

/ /_5.59; R_f= 0.47 (CH₃OH/ethyl acetate 1:1); IR

_9.10, N^5.62

(KBr, cm^ꢀ1

)

v

: 3153, 2974, 2943, 2912, 2866, 2798, 2688, 2599, 2503,

/

1

1581, 1459, 1254, 998; H NMR (d ppm): 7.04 (t, J = 7.9, 1H, Ar–H5);

6.78 (d, J = 7.8, 1H, Ar–H4); 6.70 (d, J = 8.2, 1H, Ar–H6); 4.08 (t, J = 5.9,

2H, Ar–O–CH₂); 3.74–3.68 (m, 1H, CH–OH); 2.93–2.88 (m, 2H, pip);

2.84 (t, J = 5.6, 2H, CH₂–N); 2.37–2.29 (m, 2H, pip); 2.27 (s, 3H, CH₃–

Ar); 2.14 (s, 3H, CH₃–Ar); 1.95–1.87 (m, 2H, pip); 1.68–1.54 (m, 2H, pip).

) v

2874, 2818, 2802, 2748, 2362, 2140, 1900, 1582, 1468, 1261; ¹H

NMR (d ppm): 7.08–6.99 (m, 1H, Ar–H5); 6.76 (d, J = 7.44, 1H,

Ar–H4); 6.70 (d, J = 8.21, 1H, Ar–H6); 3.99 (t, J = 6.16, 2H, O–CH₂–

CH₂–CH₂–NH); 3.71 (m, 1H, CH–OH); 2.91–2.73 (m, 2H, pip);

2.61–2.47 (m, 2H, O–CH₂–CH₂–CH₂–NH); 2.26 (s, 3H, CH₃–Ar

(2)); 2.22–2.15 (m, 2H, pip); 2.14 (s, 3H, CH₃–Ar (3)); 2.07–1.84

(m, 4H, pip); 1.66–1.52 (m, 2H, O–CH₂–CH₂–CH₂–NH); 1.47 (br s,

1H, OH).

4.1.1.6. R,S-2N-[(2,3-Dimethylphenoxy)ethyl]amino-1-pheny-

lethan-1-ol (6). White solid. Yield = 67%; M = 285.38; C₁₈H₂₃NO₂;

mp 118–120 °C; [%^calcd

/

_analyzed] C^75.75

/

_75.83, H^8.12

/

_8.28, N^4.91

/

;

4.82

R_f= 0.62 (CH₃OH); cLogP = 3.03; IR (KBr, cm^ꢀ1

) v: 3310, 3064,

3033, 2975, 2921, 2899, 2871, 2836, 2735, 2519, 2364, 1892,

1588, 1453, 1266, 1108, 762; ¹H NMR (d ppm): 7.39–7.18 (m,

5H, H-2⁰-H6⁰); 7.01 (dd, J = 8.2, J = 7.6, 1H, H-5); 6.76 (d, J = 8.2,

1H, H-6); 6.73 (d, J = 7.6, 1H, H-4); 5.30 (br s, 1H, –OH); 4.65 (t,

J = 6.3, 1H, CH); 3.97 (t, J = 5.5, 2H, Ar–O–CH₂–); 2.92 (t, J = 5.5,

2H, –CH₂–N); 2.73 (d, J = 6.3, 2H, N–CH₂–); 2.20 (s, 3H, CH₃–Ar

(3)); 2.03 (s, 3H, CH₃–Ar (2)); 1.92 (br s, 1H, –NH); ¹³C NMR (d

ppm): 156.37 (C-1), 144.54 (C-1⁰), 137.15 (C-3), 127.90 (C-3⁰, C-

5⁰), 126.75 (C-4⁰), 125.89 (C-2⁰, C-6⁰), 125.85 (C-5), 124.19 (C-2),

4.1.1.11. R,S-2N-[(2,3-Dimethylphenoxy)propyl]amino-1-phe-

nylethan-1-ol (11). White solid. Yield = 62%; M = 299.42;

C

₁₉H₂₅NO₂; mp 111–112 °C; [%^calcd

/

_analyzed] C^76.22

/

_76.21, H^8.42

/

,

8.43

N^4.68_4.63; R_f= 0.38 (CH₃OH); IR (KBr, cm^ꢀ1

/ ) v: 3333, 3322, 3063,

3030, 2946, 2922, 2895, 2844, 2754, 1953, 1582, 1256, 1102,

1

703; H NMR (d ppm): 7.38–7.16 (m, 5H, H-2⁰H-6⁰); 7.01 (dd,

J = 8.1, J = 7.6, 1H, H-5); 6.75 (d, J = 8.1, 1H, H-6); 6.73 (d, J = 7.6,

1H, H-4), 5.24 (br s, 1H, –OH); 4.62 (t, J = 6.5, 1H, CH); 3.97 (t,

J = 6.3, 2H, Ar–O–CH₂–); 2.72 (t, J = 6.9, 2H, –CH₂–N); 2.64 (d,

1806

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

J = 6.5, 2H, N–CH₂–); 2.20 (s, 3H, CH₃–Ar (3)); 2.06 (s, 3H, CH₃–Ar

(2)); 1.85 (tt, J = 6.9, J = 6.3, 2H, R–CH₂–R); 1.72 (br s, 1H, –NH);

¹³C NMR (d ppm): 156.36 (C-1), 144.58 (C-1⁰), 137.01 (C-3),

127.78 (C-3⁰, C-5⁰), 126.62 (C-4⁰), 125.78 (C-2⁰, C-6⁰), 125.75 (C-5),

124.02 (C-2), 121.75 (C-4), 109.04 (C-6), 71.41 (CH), 65.91 (Ar–

O–CH₂–), 57.73 (N–CH₂–), 45.86 (–CH₂–N), 29.43 (R–CH₂–R),

19.63 (CH₃–Ar (3)), 11.31 (CH₃–Ar (2)).

(KBr, cm^ꢀ1

) v: 3411, 3298, 3142, 2927, 2857, 2833, 2360, 1606,

1465, 1213, 1148, 1069857, 626; ¹H NMR (d ppm): 6.81 (s, 2H,

H–Ar); 3.89 (t, J = 5.26, 2H, CH₂–O); 3.70–3.57 (m, 1H, CH–OH);

3.03 (t, J = 5.26, 2H, CH₂–NH); 2.68–2.54 (m, 1H, CH–NH); 2.24

(s, 9H, CH₃–Ar); 2.07–1.96 (m, 5H, cyclohex, OH, NH); 1.44–1.17

(m, 4H, cyclohex).

4.1.1.18. R,S-4N-[(2,4,6-Trimethylphenoxy)ethyl]amino-1-phe-

nylethan-1-ol (18). White solid. Yield 58%; M = 299.47;

4.1.1.12. R,S-2N-[(2,4-Dimethylphenoxy)ethyl]aminopropan-1-

ol (12). White solid. Yield = 70%; M = 223.31; C₁₃H₂₁NO₂; mp 60–

₁₉H₂₅NO₂; mp 120–121 °C; [%^calcd

/

_analyzed] C^76.20

/

_76.26, H^8.41

/

,

61 °C; [%^calcd

/

_analyzed] C^69.92

/

_70.14, H^9.48

/

_9.42, N^6.27

/

_6.22; R_f= 0.44 (CH₃-

OH/CHCl₃1:9), R_f= 0.49 (CH₃OH/ethyl acetate 1:1); IR (KBr, cm^ꢀ1

: 3296, 3119, 3018, 2961, 2917, 2838, 2729, 2603, 1505, 1461,

C

8.65

N^4.70_4.81; R_f= 0.58 (CH₃OH/benzene 1:5); ¹H NMR (d ppm): 7.40–

/

)

7.28 (m, 4H, H–Ar(2,3,5,6)); 7.26–7.20 (m, 1H, H–Ar(4)); 6.80 (s,

2H, H–Ar); 5.33 (d, 1H, J = 2.6, OH); 4.66 (d, 1H, J = 2.5, CH–OH);

3.73 (t, 2H, J = 5.3, Ar–O–CH₂); 2.84–2.82 (m, 2H, CH₂–N); 2.72

(d, 2H, J = 6.3, N–CH₂); 2.17 (s, 3H, CH₃–Ar(4)); 2.16 (s, 6H, CH₃–

Ar(2,6)); 2.11 (br s, 1H, NH).

v

1252, 1223, 1050, 791; ¹H NMR (d ppm): 6.95–6.92 (m, 2H, H–Ar

(3,5)); 6.72 (d, J = 7.9, 1H, H–Ar (6)); 4.07–4.00 (m, 2H, Ar–O–

CH₂); 3.60 (dd, J = 4.1, J = 10.5, 1H, CHH–OH); 3.27 (dd, J = 6.9,

J = 10.5, 1H, CHH–OH); 3.17–3.09 (m, 1H, CHH–N); 2.96–2.82 (m,

2H, CH + CHH–N); 2.26 (s, 3H, CH₃–Ar); 2.19 (s, 3H, CH₃–Ar);

1.09 (d, J = 6.4, 3H, CH–CH₃).

4.1.1.19.

aminopropan-1-ol (19). White solid. Yield = 45%; M = 267.36;

₁₅H₂₅NO₃; mp 50–52 °C; [%^calcdC^67.38H^9.42

R,S-2N-{2-[2-(2,3-Dimethylphenoxy)ethoxy]ethyl}

4.1.1.13. R-(ꢀ)-2N-[(2,4-Dimethylphenoxy)ethyl]aminopropan-

1-ol (13). White solid. Yield = 70%; M = 223.31; C₁₃H₂₁NO₂; mp 69–

71 °C; R_f= 0.40 (CH₃OH/CHCl₃1:9), R_f= 0.50 (CH₃OH/ethyl acetate

C

/

]

/

,

/

,

analyzed

67.42

9.36

N^5.24_5.11; R_f= 0.13 (CH₃OH/ethyl acetate 1:1); IR (KBr, cm^ꢀ1):

/

3255, 3095, 2962, 2918, 2853, 2692, 2585, 1584, 1479, 1260,

1107, 760; ¹H NMR (d ppm): 7.04 (t, J = 7.7; 1H, Ar–H5); 6.78 (d,

J = 7.4; 1H, Ar–H4); 6.72 (d, J = 8.2. 1H, Ar–H6); 4.11 (d, J = 4.6;

2H, Ar–O–CH₂); 3.87–3.82 (m, 2H, Ar–O–CH₂–CH₂); 3.72–3.65

(m, 2H, O–CH₂–CH₂–NH); 3.58 (dd, J = 10.5; J = 4.1; 1H, CHH–

OH); 3.22 (dd, J = 10.5; J = 6.7; 1H, CHH–OH); 2.99–2.90 (m, 1H,

O–CH₂–CHH–NH); 2.82–2.68 (m, 2H, O–CH₂–CHH–NH + NH–CH);

2.27 (s, 3H, CH₃–Ar); 2.16 (s, 3H, CH₃–Ar); 1.05 (d, J = 6.7; 3H,

1:1); IR (KBr, cm^ꢀ1

) v: 3299, 3170, 2969, 2923, 2842, 2733, 1613,

1506, 1255, 1048; ¹H NMR (d ppm): 6.95–6.92 (m, 2H, H–Ar

(3,5)); 6.72 (d, J = 7.9, 1H, H–Ar(6)); 4.07–4.00 (m, 2H, Ar–O–

CH₂); 3.61 (dd, J = 4.1, J = 10.5, 1H, CHH–OH); 3.17–3.09 (m, 1H,

CHHN); 2.96–2.82 (m, 2H, CH–CH₃, CHH–N); 2.27 (dd, J = 6.9,

J = 10.5, 1H, CHH–OH); 2.25 (s, 3H, CH₃–Ar); 2.19 (s, 3H, CH₃–Ar);

1.09 (d, J = 6.4, 3H, CH–CH₃); LC–MS [M+H]⁺m/z:8 224.33,

99.34%; [

a]

²⁰= ꢀ15.75° (CH₃OH, c 2%).

13

CH–CH₃); C NMR (d ppm): 156.31 (C-1⁰), 137.16 (C-3⁰), 125.78

D

(C-5⁰), 124.34 (C-2⁰), 122.09 (C-4⁰), 109.55 (C-6⁰), 70.63 (C-5),

68.88 (C-3), 67.67 (C-2), 65.49 (C-9), 54.37 (C-8), 46.26 (C-6),

19.62 (CH₃-3⁰), 17.16 (CH₃-8), 11.37 (CH₃-2⁰).

4.1.1.14. R,S-2N-[(2,4,6-Trimethylphenoxy)ethyl]aminopropan-

1-ol (14). White solid. Yield = 64%; M = 237.33; C₁₄H₂₃NO₂; mp

80–82 °C; [%^calcd_analyzed] C^70.85_69.95, H^9.77_9.52, N^5.90

/ / / /_5.98; R_f= 0.41

(EtOH/ethyl acetate 1:1); ¹H NMR (d ppm): 6.80 (s, 2H, Ar–H),

4.56 (br s, 1H, OH), 3.78–3.69 (m, J = 5.5, J = 5.5, J = 2.6, 2H, Ar–

O–CH₂), 3.43–3.16 (m, 3H, CH₂–OH + NH–CH), 2.94–2.78 (m, 2H,

CH₂–N), 2.73–2.60 (m, 1H, NH), 2.17 (s, 9H, CH₃–Ar), 0.93 (d,

J = 6.4 Hz, 3H, CH–CH₃).

4.1.1.20.

aminocyclohexan-1-ol

M = 307.43; C₁₈H₂₉NO₃; mp 68–70 °C; [%^calcd

H^9.50_9.12, N^{4.56 1}H NMR (d ppm): 7.07 (dd, J = 8.1; J = 7.5; 1H,

trans-4N-{2-[2-(2,3-Dimethylphenoxy)ethoxy]ethyl}

(20). White solid. Yield = 65%;

_analyzed] C^70.32

/

,

70.69

/

;

4.33

Ar–H5); 6.78 (d, J = 8.1; 1H, Ar–H6); 6.75 (d, J = 7.5; 1H, Ar–H4);

4.43 (br s, 1H, –OH); 4.04 (ddd, J = 6.2; J = 4.7; J = 3.2; 2H, Ar–O–

CH₂); 3.71 (ddd, J = 6.2; J = 4.7; J = 3.2; 2H, Ar–O–CH₂–CH₂); 3.51

(t, J = 5.7; 2H, O–CH₂–CH₂–NH); 3.36–3.30 (m, 1H, CH–OH); 2.66

(t, J = 5.7; 2H, O–CH₂–CH₂–NH); 2.29 (tt, J = 10.6; J = 3.5; 1H, CH–

NH); 2.20 (s, 3H, CH₃–Ar); 2.07 (s, 3H, CH₃–Ar); 1.80–1.73 (m,

4H, cyclohex); 1.40 (br s, 1H, ꢀNH); 1.16–1.07 (m, 2H, cyclohex);

1.01–0.92 (m, 2H, cyclohex). ¹³C NMR (d ppm): 156.30 (C-1),

137.19 (C-3), 125.81 (C-5), 124.27 (C-2), 122.09 (C-4), 109.53 (C-

6), 70.45 (–O–CH₂–CH₂–NH–), 68.83 (Ar–O–CH₂–CH₂–O–), 68.78

(CH–OH (cyclohex)), 67.62 (Ar–O–CH₂–CH₂–O–), 55.61 (CH–NH

(cyclohex)), 46.10 (–O–CH₂–CH₂–NH–), 33.78 (C-3⁰, C-5⁰), 30.90

(C-2⁰, C-6⁰), 19.66 (CH₃–Ar (3)), 11.39 (CH₃–Ar (2)).

4.1.1.15.

R-(ꢀ)-2N-[(2,4,6-Trimethylphenoxy)ethyl]amino-

(15). White solid. Yield = 60%; M = 237.33;

_analyzed] C^70.85_71.15, H^9.77

propan-1-ol

5.90

C

₁₄H₂₃NO₂; mp 91–92 °C; [%^calcd

/

/_9.86, N

/

;

5.92

R_f= 0.40 (EtOH/ethyl acetate 1:1); ¹H NMR (dmso,300 MHz):

d (ppm) 6.78 (s, 2H, Ar–H), 4.54 (t, J = 5.3 Hz, 1H, OH), 3.64–3.79

(m, 2H, –O–CH₂–), 3.26–3.33 (m, 2H, –CH₂–OH), 3.16–3.25

(m, 1H, CH), 2.80–2.88 (m, 1H, –CHH–NHꢀ), 2.65 (q, J = 5.1x(3)

Hz, 1H, NH), 2.48 (dt, J = 3.7, 1.7 Hz, 1H, –CHH–NHꢀ), 2.15

(s, 9H, Ar–CH₃), 0.91 (d, J = 6.4 Hz, 3H, CH–CH₃); [

(c 1%, CHCl₃); LC–MS [M+H]⁺m/z: 238.36, 100.0%.

a]

²⁰= ꢀ34.4°

D

4.1.1.16.

clohexan-1-ol (16). White solid. Yield = 74%; M = 277.46;

₁₇H₂₇NO₂; mp 90–91 °C; [%^calcdC^73.58H^9.81

D,L-trans-2N-[(2,4,6-Trimethylphenoxy)ethyl]aminocy-

C

/

]

/

,

/

,

analyzed

72.80

9.87

N^5.07_4.95; R_f= 0.55 (CH₃OH/benzene 1:5); ¹H NMR (d ppm): 6.80

/

4.1.1.21.

aminopropan-1-ol (21). White solid. Yield = 62%; M = 267.36;

₁₅H₂₅NO₃; mp 46–48 °C; [%^calcdC^67.38H^9.42

R,S-2N-{2-[2-(2,6-Dimethylphenoxy)ethoxy]ethyl}

(s, 2H, Ar–H); 4.63 (d, 1H, J = 5.1, OH); 3.80–3.69 (m, 2H, Ar–O–

CH₂); 3.15–3.08 (m, 1H, CH–OH); 2.93–2.87 (m, 1H, CHH–N);

2.84–2.77 (m, 1H, CHH–N); 2.44 (br s, 1H, NH); 2.26–2.19 (m,

1H, N–CH); 2.17 (s, 6H, CH₃–Ar(2,6)); 2.17 (s, 3H, CH₃–Ar(4));

1.96–1.87 (m, 1H, cyclohex); 1.85–1.76 (m, 1H, cyclohex); 1.85–

1.54 (m, 2H, cyclohex); 1.27–1.10 (m, 3H, cyclohex); 0.98–0.88

(m, 1H, cyclohex).

C

/

]

/

,

/

,

analyzed

67.21

9.66

N^5.24_5.12; IR (KBr, cm^ꢀ1): 3429, 3295, 2918, 2872, 1631, 1474,

/

1204, 1134, 1059, 756; ¹H NMR (d ppm): 7.00 (d, J = 7.4; 2H, Ar–

H3); 6.92 (dd, J = 8.1; J = 6.2, 1H, Ar–H4); 3.98–3.92 (m, 2H, Ar–

O–CH₂); 3.84–3.78 (m, 2H, Ar–O–CH₂–CH₂); 3.74–3.67 (m, 2H,

O–CH₂–CH₂–NH); 3.61 (dd, J = 10.5; J = 3.7; 1H, CHH–OH); 3.30

(dd, J = 11.2; J = 7.2; 1H, CHH–OH); 3.06–2.96 (m, 1H, O–CH₂–

CHH–NH); 2.91–2.74 (m, 1H, O–CH₂–CHH–NH); 2.91–2.74 (m,

1H, NH–CH); 2.41 (br s, 2H, OH, NH); 2.29 (s, 6H, CH₃–Ar); 1.09

(d, J = 6.7; 3H, CH–CH₃), LC–MS [M+H]⁺m/z: 268.34, 97.92%.

4.1.1.17. trans-4N-[(2,4,6-Trimethylphenoxy)ethyl]aminocyclo-

hexan-1-ol (17). White solid. Yield = 55%; M = 277.41; C₁₇H₂₇NO₂;

mp 100–102 °C; [%^calcd_analyzed] C^73.58_73.54, H^9.81_9.83, N^5.07

/ / / /_4.98; IR

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

1807

4.1.1.22. R-(ꢀ)-2N-{2-[2-(2,6-Dimethylphenoxy)ethoxy]ethyl}

aminopropan-1-ol (22). White solid. Yield = 60%; M = 267.36;

N^4.98_4.91; IR (KBr, cm^ꢀ1): 3294, 3131, 3021, 2964, 2922, 2870,

/

2834, 2570, 2398, 1459, 1203, 1129, 1064, 762; ¹H NMR (d

ppm): 7.05–6.97 (m, 2H, Ar–H3); 6.96–6.87 (m, 1H, Ar–H4);

3.97–3.91 (m, 2H, Ar–O–CH₂); 3.83–3.77 (m, 2H, Ar–O–CH₂–

CH₂); 3.72–3.64 (m, 2H, O–CH₂–CH₂–NH); 3.61 (dd, J = 10.6;

J = 4.0; 1H, CHH–OH); 3.29 (dd, J = 10.8; J = 6.4; 1H, CHH–OH);

2.99–2.88 (m, 1H, O–CH₂–CHH–NH); 2.81–2.71 (m, 1H, O–CH₂–

CHH–NH); 2.62–2.51 (m, 1H, NH–CH); 2.29 (s, 6H, CH₃–Ar);

1.56–1.36 (m, 2H, CH–CH₂–CH₃); 0.92 (t, J = 7.4; 3H, CH–CH₂–

C

₁₅H₂₅NO₃; mp 46–48 °C; [%^calcd

/

]

C^67.38

/

,

H^9.42

/

,

analyzed

67.31

9.70

N^5.24_5.04; IR (KBr, cm^ꢀ1): 3423, 3301, 2915, 2869, 1475, 1203,

/

1137, 1061, 756; ¹H NMR (d ppm): 7.00 (d, J = 7.4; 2H, Ar–H3);

6.92 (dd, J = 8.1; J = 6.2, 1H, Ar–H4); 3.98–3.92 (m, 2H, Ar–O–

CH₂); 3.84–3.78 (m, 2H, Ar–O–CH₂–CH₂); 3.74–3.67 (m, 2H, O–

CH₂–CH₂–NH); 3.61 (dd, J = 10.5; J = 3.7; 1H, CHH–OH); 3.30 (dd,

J = 11.2; J = 7.2; 1H, CHH–OH); 3.06–2.96 (m, 1H, O–CH₂–CHH–

NH); 2.91–2.74 (m, 1H, O–CH₂–CHH–NH); 2.91–2.74 (m, 1H, NH–

CH); 2.41 (br s, 2H, OH, NH); 2.29 (s, 6H, CH₃–Ar); 1.09 (d,

CH₃); [

a]

D

²⁰= ꢀ11.719° (CH₃OH, c 1%).

J = 6.7; 3H, CH–CH₃); [

a

]

²⁰= ꢀ13.25° (CH₃OH, c 1%).

4.1.1.26.

S-(+)-2N-{2-[2-(2,6-Dimethylphenoxy)ethoxy]ethyl}

D

4.1.1.22.1. R-(ꢀ)-2N-{2-[2-(2,6-Dimethylphenoxy)ethoxy]ethyl}

aminobutan-1-ol (26). White solid. Yield = 75%; M = 281.39;

aminopropan-1-ol hydrochloride (22a). White solid. Yield = 85%;

C

₁₆H₂₇NO₃; mp 51–53 °C; [%^calcdC^68.29H^9.67

/

]

/

,

/

,

analyzed

67.83

9.78

M = 303.82;

C

₁₅H₂₆NO₃Cl;

/

mp

98–100 °C;

[%^calcd

/

]

N^4.98

/

;

¹H NMR (d ppm): 7.00 (d, J = 7.6; 2H, Ar–H3); 6.92 (dd,

analyzed

4.91

C^59.29_58.98, H^8.63

/

_8.68, N^4.53_4.59; IR (KBr, cm^ꢀ1): 3255, 3095, 2962,

/

J = 8.6; J = 6.5; 1H, Ar–H4); 3.96–3.92 (m, 2H, Ar–O–CH₂); 3.86–

3.81 (m, 2H, Ar–O–CH₂–CH₂); 3.72 (t, J = 4.9; 2H, O–CH₂–CH₂–

NH); 3.67 (dd, J = 11.4; J = 4.0; 1H, CHH–OH); 3.37 (dd, J = 11.2;

J = 6.7; 1H, CHH–OH); 3.00 (m, 1H, O–CH₂–CHH–NH); 2.90 (dt,

J = 12.5; J = 4.5; 1H, O–CH₂–CHH–NH); 2.76–2.66 (m, 1H, NH–

2919, 2853, 2692, 2585, 1479, 1260; ¹H NMR (d ppm): 8.50 (br s,

2H, NH⁺₂); 7.01–6.99 (m, 2H, Ar–H); 6.92–6.87 (m, 1H, Ar–H);

5.36 (t, J = 5.1; 1H, OH); 3.89–3.86 (m, 2H, Ar–O–CH₂); 3.79–3.74

(m, 4H, Ar–O–CH₂–CH₂–O–CH₂–CH₂–NH); 3.64–3.58 (m, 1H,

CHH–OH); 3.52–3.44 (m, 1H, CHH–OH); 3.32–3.23 (m, 1H, NH–

CH); 3.14 (t, 2H, J = 5.6. O–CH₂–CH₂–NH); 2.20 (s, 6H, CH₃–Ar);

1.70 (d, J = 6.7; 3H, CH–CH₃), LC–MS [M+H]⁺m/z: 268.34, 100%.

CH); 2.58 (br s, 2H, OH, NH); 2.29 (s, 6H, CH₃–Ar); 1.64–1.45 (m,

20

2H, CH–CH₂–CH₃); 0.95 (t, J = 8.2; 3H, CH–CH₂–CH₃); [

+11.660° (CH₃OH, c 1%).

a]

=

D

[

a]

D

²⁰= ꢀ6.64° (CH₃OH, c 2%).

4.1.1.27.

ethyl}aminocyclohexan-1-ol (27). White solid. Yield = 60%;

M = 307.43; C₁₈H₂₉NO₃; mp 47–49 °C; [%^calcd_analyzed] C^70.32

H^9.51_9.53, N^4.56_4.51; IR (KBr, cm^ꢀ1): 3297, 3178, 3022, 2954, 2921,

D,L-trans-2N-{2-[2-(2,6-Dimethylphenoxy)ethoxy]

4.1.1.23.

aminopropan-1-ol (23). White solid. Yield = 70%; M = 267.36;

₁₅H₂₅NO₃; mp 46–48 °C; [%^calcd_analyzed] C^67.38H^9.42

N^{5.24 1}H NMR (d ppm): 7.00 (d, J = 7.4; 2H, Ar–H3); 6.92 (dd,

S-(+)-2N-{2-[2-(2,6-Dimethylphenoxy)ethoxy]ethyl}

/

,

70.98

C

/

,

/

,

/

67.23

9.75

/

;

2852, 2832, 2664, 1591, 1477, 1205, 1119, 1083, 775, 405; ¹H

NMR (d ppm): 7.01–6.89 (m, 3H, Ar–H); 3.95–3.92 (m, 2H, Ar–O–

CH₂); 3.81–3.79 (m, 2H, Ar–O–CH₂–CH₂); 3.69–3.66 (m, 2H, CH₂–

CH₂–NH); 3.18–3.12 (m, 1H, CH–OH); 3.12–3.03 (m, 1H, CHH–

NH); 2.72–2.65 (m, 1H, O–CH₂–CHH–NH); 2.29 (s, 6H, CH₃–Ar);

2.27–2.19 (m, 1H, NH); 2.11–2.02 (m, 2H, cyclohex); 1.74–1.69

(m, 2H, cyclohex); 1.24–1.29 (m, 4H, cyclohex).

4.99

J = 8.1; J = 6.2; 1H, Ar–H4); 3.98–3.92 (m, 2H, Ar–O–CH₂); 3.84–

3.78 (m, 2H, Ar–O–CH₂–CH₂); 3.74–3.67 (m, 2H, O–CH₂–CH₂–

NH); 3.61 (dd, J = 10.5; J = 3.7; 1H, CHH–OH); 3.30 (dd, J = 11.2;

J = 7.2; 1H, CHH–OH); 3.06–2.96 (m, 1H, O–CH₂–CHH–NH); 2.91–

2.74 (m, 1H, O–CH₂–CHH–NH); 2.91–2.74 (m, 1H, NH–CH); 2.41

(br s, 2H, OH, NH); 2.29 (s, 6H, CH₃–Ar); 1.09 (d, J = 6.7; 3H, CH–

CH₃); [

4.1.1.23.1.

aminopropan-1-ol hydrochloride (23a). White solid. Yield = 80%;

M = 303.82; C₁₅H₂₆NO₃Cl; mp 97–99 °C; [%^calcd_analyzed] C^59.29

H^8.63_8.80, N^4.53_4.60; IR (KBr, cm^ꢀ1): 3411, 2927, 1628, 1476, 1202,

a]

²⁰= +13.07° (CH₃OH, c 2%).

D

S-(+)-2N-{2-[2-(2,6-Dimethylphenoxy)ethoxy]ethyl}

4.1.1.28. 1N-{2-[2-(2,6-Dimethylphenoxy)ethoxy]ethyl}piperi-

din-4-ol (28). White solid. Yield = 55%; M = 293.40; C₁₇H₂₇NO₃;

/

,

mp 51–53 °C; [%^calcd_analyzed] C^69.59_69.41, H^9.28_9.23, N^4.77

/ / / /_4.75; IR

59.34

/

(KBr, cm^ꢀ1): 3434, 3288, 2947, 2930, 2888, 2870, 2810, 2360,

2341, 1475, 1201, 1079, 781, 766; ¹H NMR (d ppm): 7.02–6.97

(m, 2H, Ar–H3); 6.94–6.88 (m, 1H, Ar–H4); 3.96–3.89 (m, 2H, Ar–

O–CH₂); 3.81–3.76 (m, 2H, Ar–O–CH₂–CH₂); 3.70 (t, J = 5.7; CHH–

CH₂–NH); 3.72–3.68 (m, 1H, CH–OH); 2.91–2.79 (m, 2H, N–CH₂(-

pip)); 2.64 (t, 2H, J = 5.7; CH₂–CH₂–NH); 2.28 (s, 6H, CH₃–Ar);

2.27–2.17 (m, 2H, N–CH₂(pip)); 1.96–1.84 (m, 2H, CH₂(pip));

1.69–1.53 (m, 2H, CH₂(pip)).

1128, 1052, 770; ¹H NMR (d ppm): 8.77 (br s, 1H, NHH⁺); 8.54

(br s, 1H, NHH⁺); 7.00 (d, J = 7.4; 1H, Ar–H3); 6.89 (dd, J = 8.5;

J = 6.5; 1H, Ar–H4); 5.37 (t, J = 5.3; 1H, OH); 3.90–3.87 (m, 2H,

Ar–O–CH₂); 3.81–3.74 (m, 4H, Ar–O–CH₂–CH₂–O–CH₂–CH₂–NH);

3.63 (dt, J = 11.8; J = 4.3; 1H, O–CH₂–CHH–NH); 3.50 (dt, J = 11.8;

J = 5.4; O–CH₂–CHH–NH); 3.28 (br s, 1H, NH–CH); 3.15 (br s, 2H,

CH₂–OH); 2.21 (s, 6H, CH₃–Ar); 1.19 (d, J = 6.7; 3H, CH–CH₃).

[a]

²⁰= +6.36° (CH₃OH, c 2%).

D

4.1.1.29.

aminopropan-1-ol (29). White solid. Yield = 50%; M = 281.39;

C₁₆H₂₇NO₃; mp 53–55 °C; [%^calcdC^68.29H^9.67

R,S-2N-{2-[2-(2,4,6-Trimethylphenoxy)ethoxy]ethyl}

4.1.1.24.

aminobutan-1-ol (24). White solid. Yield = 65%; M = 281.39;

₁₆H₂₇NO₃; mp 48–50 °C; [%^calcd_analyzed] C^68.29H^9.67

N^4.98_4.88; IR (KBr, cm^ꢀ1): 3422, 3297, 3155, 2963, 2914, 2871,

R,S-2N-{2-[2-(2,6-Dimethylphenoxy)ethoxy]ethyl}

/

]

/

,

/

,

analyzed

68.29

9.61

C

/

,

/

,

N^4.98_4.86; IR (KBr, cm^ꢀ1): 3429, 3293, 3158, 2962, 2912, 2835,

/

68.40

10.07

/

1637, 1459, 1216, 1135, 1062, 883; ¹H NMR (d ppm): 6.81 (s, 2H,

Ar–H); 3.97–3.86 (m, 2H, Ar–O–CH₂); 3.83–3.75 (m, 2H, Ar–O–

CH₂–CH₂); 3.72–3.63 (m, 2H, O–CH₂–CH₂–NH); 3.58 (dd, J = 10.5;

J = 4.1; 1H, CHH–OH); 3.25 (dd, J = 10.6; J = 6.8; 1H, CHH–OH);

3.05–2.89 (m, 1H, O–CH₂–CHH–NH); 2.86–2.68 (m, 2H, O–CH₂–

CHH–NH + CH–CH₂–OH); 2.25 (s, 6H, CH₃–Ar); 2.23 (s, 3H, CH₃–

Ar); 1.05 (d, J = 6.7; 3H, CH–CH₃).

2830, 2360, 1629, 1593, 1461, 1205, 1136, 1062, 763; ¹H NMR (d

ppm): 7.00 (d, J = 7.6; 2H, Ar–H3); 6.92 (dd, J = 8.6; J = 6.5; 1H,

Ar–H4); 3.96–3.92 (m, 2H, Ar–O–CH₂); 3.86–3.81 (m, 2H, Ar–O–

CH₂–CH₂); 3.76 (t, J = 4.9; 2H, O–CH₂–CH₂–NH); 3.71 (dd,

J = 11.4; J = 4.0; 1H, CHH–OH); 3.43 (dd, J = 11.1; J = 6.5; 1H,

CHH–OH); 3.05 (dt, J = 12.0; J = 5.5; 1H, O–CH₂–CHH–NH); 2.90

(dt, J = 12.9; J = 4.6; 1H, O–CH₂–CHH–NH); 2.76–2.66 (m, 1H,

NH–CH); 2.58 (br s, 2H, OH, NH); 2.29 (s, 6H, CH₃–Ar); 1.64–1.45

(m, 2H, CH–CH₂–CH₃); 0.95 (t, J = 7.7; 3H, CH–CH₂–CH₃).

4.1.1.30.

ethyl}aminocyclohexan-1-ol (30). White solid. Yield = 55%;

M = 321.45; C₁₉H₃₁NO₃; mp 54–56 °C; [%^calcd_analyzed] C^70.93

H^9.72_9.53, N^4.36_4.35; IR (KBr, cm^ꢀ1): 3424, 3300, 3158, 3001, 2928,

D,L-trans-2N-{2-[2-(2,4,6-Trimethylphenoxy)ethoxy]

/

,

70.90

4.1.1.25. R-(ꢀ)-2N-({2-[2-(2,6-Dimethylphenoxy)ethoxy]ethyl}

aminobutan-1-ol (25). White solid. Yield = 65%; M = 281.39;

/

2858, 2362, 1636, 1484, 1456, 1217, 1122, 1060, 904, 856,

C

₁₆H₂₇NO₃; mp 50–52 °C; [%^calcd

/

_analyzed] C^68.29

/

,

H^9.67

/

,

590; ¹H NMR (d ppm): 6.81 (s, 2H, Ar–H); 3.94–3.87 (m, 2H,

68.14

9.66

1808

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

Ar–O–CH₂); 3.81–3.75 (m, 2H, Ar–O–CH₂–CH₂); 3.70–3.62 (m, 2H,

O–CH₂–CH₂–NH); 3.44 (br s, 1H, NH); 3.23–3.11 (m, 1H, CH–OH);

3.11–3.00 (m, 1H, O–CH₂–CHH–NH); 2.67 (dt, J = 12.2; J = 4.6; 1H,

O–CH₂–CHH–NH); 2.25 (s, 6H, CH₃–Ar); 2.23 (s, 3H, CH₃–Ar);

2.22–2.16 (m, 1H, CH–NH); 2.12–1.98 (m, 2H, cyclohex); 1.77–

1.66 (m, 2H, cyclohex); 1.35–1.17 (m, 3H, cyclohex); 1.06–0.91

(m, 1H, cyclohex).

(rotarod), noted at 30 min and 4 h after administration.²²The MES

were elicited by 60 Hz alternating current at 50 mA (mice) or

150 mA (rats) delivered for 0.2 s via corneal electrodes. A drop of

0.9% NaCl solution was placed into each eye prior to applying the

electrodes. Protection in the MES test was deﬁned as the abolition

of the hindlimb tonic extension component of the seizure. The

ScMet was conducted by administration 85 mg/kg of pentylenete-

trazole dissolved in 0.9% NaCl solution into the posterior midline of

mice. A minimal time of 30 min subsequent to subcutaneous

administration of pentylenetetrazole was used for seizure detec-

tion. A failure to observe even a threshold seizure (a single episode

of clonic spasm of at least 5 s in duration) was regarded as protec-

tion. Neurological deﬁcit was measured in mice by the rotarod test.

The mouse was placed on a 1 in. diameter knurled plastic rod rotat-

ing at 6 rpm. Neurotoxicity was indicated by the inability of the

animal to maintain equilibrium on the rod for at least 1 min in each

of the three trials. In rats, neurological deﬁcit was indicated by

ataxia and loss of placing response and muscle tone.

The 6 Hz model test was carried out according to the protocol

originally described by Brown et al.²³and more recently by Barton

et al.¹⁴and Kaminski et al.²⁴Corneal stimulation (0.2 ms duration

monopolar rectangular pulses at 6 Hz for 3 s) was delivered by a

constant current device. During the stimulation, mice were manu-

ally restrained and released into the observation cage immediately

after application of current. The seizures were often preceded by a

brief period of intense locomotor agitation. The animals then

exhibited a ‘stunned’ posture associated with rearing, forelimb

automatic movements and clonus, twitching of the vibrissae, and

Straub-tail. The duration of the seizure activity ranged from 60 to

120 s in untreated animals. In the end of the seizure, animals

resumed their normal exploratory behavior. The experimental

endpoint was protection against the seizure. The animal was con-

sidered to be protected if it resumed its normal exploratory behav-

ior within 10 s from the stimulation. The pilocarpine test involves

determination whether the investigated compound can halt acute

Data for single crystal were collected at 120 K using the Oxford

Diffraction SuperNova four circle diffractometer, equipped with

the Cu (1.54184 Å) K

a radiation source, graphite monochromator

and Oxford CryoJet system for measurements at low temperature.

The structure was solved by direct methods using SIR-97¹⁸and all

non–hydrogen atoms were reﬁned anisotropically using weighted

full-matrix least-squares on F². Reﬁnement and further calcula-

tions were carried out using Shelxl-97.¹⁹The hydrogen atoms

bonded to carbon atoms were included in the structure at idealized

positions and were reﬁned using a riding model with U_iso(H) ﬁxed

at 1.2 U_eqof C and 1.5 U_eqfor methyl groups. Hydrogen atoms

attached to oxygen and nitrogen atoms were found from the differ-

ence Fourier map and reﬁned without any restraints. For molecular

graphics ORTEP²⁰and MERCURY²¹programs were used.

C

₁₂H₂₆ClNO₃, M_r= 308.82, crystal size = 0.45 ꢃ 0.40 ꢃ 0.27 mm³,

monoclinic, space group P2₁, a = 7.7258(1) Å, b = 7.2259(1) Å,

c = 14.792(2) Å, V = 812.56(2) Å³, Z = 2, T = 120(2) K, 9314 reﬂec-

tions collected, 3031 unique reﬂections (R_int= 0.0333), R1 = 0.0343,

wR2 = 0.0938 [I > 2r(I)].

CCDC 1431730 contains the supplementary crystallographic

data. These data can be obtained free of charge from The Cam-

bridge Crystallographic Data Centre via www.ccdc.cam.ac.

uk/data_request/cif.

The liquid chromatography separation of the analytes was done

using an Agilent 1100 (Agilent Technologies, Waldbronn, Ger-

many) LC system. The mass spectrometric analyses were per-

formed on an Applied Biosystems MDS Sciex (Concord, Ontario,

Canada) API 2000 triple quadrupole mass spectrometer equipped

with an electrospray ionization interface and operated in the pos-

itive ionization mode. The data acquisition and processing were

done by using the Applied Biosystems Analyst software, version

pilocarpine-induced status.

A challenge dose of pilocarpine

(50 mg/kg) is administered ip and animals are observed until the

ﬁrst convulsive (e.g., stages 3, 4, or 5) seizure (time zero). The sei-

zure severity is determined using the Racine scale.²⁵At this point a

minimally toxic dose of the candidate drug is administered to a

group of 8 male albino Sprague Dawley rats (150–180 g) ip Efﬁcacy

is deﬁned by the ability of an investigational drug to halt the fur-

ther expression of pilocarpine induced convulsive seizures (e.g.

stages 3, 4, or 5). Anticonvulsant quantiﬁcation, that is the doses

of drug required to cause the biological responses in 50% of animals

(ED₅₀), and the respective 95% conﬁdence intervals, were deter-

mined for selected compounds displaying sufﬁcient anticonvulsant

activity and low neurotoxicity in the above primary evaluations, by

means of a computer program using probit analysis.

Formalin test is conducted in mice. The test covers the adminis-

tration of formalin to hindlimb of the animal, which causes the lick-

ing of the place of administration. The total time of licking is

considered as corresponding to the intensity of pain sensation

experienced by the animal. It is characteristic, that there are two

phases of the test. The ﬁrst phase (acute) is about 10 min long

and corresponds to the direct stimulation of peripheral sensor

and pain neurons. The second phase (inﬂammatory) develops as

the response of liberation of inﬂammatory mediators from

impaired tissues and nerves’ endings. The activity in that phase cor-

responds with beneﬁcial effect in neuropathic pain. Simultaneously

control tests are performed which involve administration to other

animals solvent alone (e.g. methylcellulose). The shortening of the

time of hindlimb licking is scored as analgesic activity.²⁶

1.4.2. The injected volume of the analytes solutions were 10

at the concentration of 250 ng/mL in methanol.

lL

The chromatographic separation of the analytes was carried out

m)

on a Chiralcel OD-RH analytical column (250 mm ꢃ 4.6 mm, 5

l

from Daicel Chemical Industries (Tokyo, Japan). The optimized sep-

aration resolution for the compounds was achieved at 30 °C col-

umn temperature with a ﬂow rate of the mobile phase 0.4 mL/

min containing acetonitrile and water (60:40, v/v) with an addition

of 0.01% formic acid.

The ion source parameters were set to: 5500 V ion spray volt-

age, 40 psi the nebulizer gas pressure, 35 psi the turbo gas pres-

sure, 400 °C the heated nebulizer temperature, and 10 psi the

curtain gas pressure. With the unit mass resolution, the mass spec-

trometer was operated in the multiple reaction monitoring mode

(MRM), by monitoring the transitions of m/z 268.2 ? 250.3.

4.2. Pharmacology

Antiepileptic activity and neurological toxicity assays were car-

ried out by the Antiepileptic Drug Development Program, Epilepsy

Branch, National Institute of Neurological and Communicative

Disorders and Stroke, National Institute of Health in Rockville,

USA.¹⁵Compounds were injected as suspensions in 0.5% methyl-

cellulose at three dosage levels (30, 100 and 300 mg/kg b.w.)

intraperitoneally (ip) into mice, and orally. The preliminary evalu-

ation was a qualitative assay which used small groups of animals

(1–8) and included three tests: maximal electroshock seizure

(MES), subcutaneous pentylenetetrazol (ScMet), and neurotoxicity

Receptor binding studies for 22a and 23a have been performed

as standard high throughput proﬁle at Cerep (France). Results

showing an inhibition (or stimulation for assays run in basal

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

1809

conditions) higher than 50% are considered to represent signiﬁcant

4.5. Pharmacokinetics

effects of the test compounds. 50% is the most common cut-off

value for further investigation (determination of IC₅₀or EC₅₀values

from concentration–response curves) that would be recom-

mended. Results showing an inhibition (or stimulation) between

25% and 50% are indicative of weak to moderate effects. Results

showing an inhibition (or stimulation) lower than 25% are not con-

sidered signiﬁcant and mostly attributable to variability of the sig-

nal around the control level. Low to moderate negative values have

no real meaning and are attributable to variability of the signal

around the control level. High negative values (P50%) that are

sometimes obtained with high concentrations of test compounds

are generally attributable to non-speciﬁc effects of the test com-

pounds in the assays. On rare occasion they could suggest an allos-

teric effect of the test compound.

The LC/ESI-MS/MS analysis were performed on an Applied

Biosystems/MDS Sciex (Concord, Ontario, Canada) API 2000 tri-

ple quadrupole mass spectrometer equipped with an electro-

spray ionization interface. This instrument was coupled to an

Agilent 1100 (Agilent Technologies, Waldbronn, Germany) LC

system. Data acquisition and processing were accomplished

using ABSciex Analyst 1.4.2 data collection and integration

software.

Using an original LC/ESI-MS/MS method, a pharmacokinetic

study for 22a was carried out following its intravenous (1 mg/

kg), intragastric (12.92 mg/kg) or intraperitoneal (12.92 mg/kg)

administration to male CD1 mice. Mice were kept under conditions

of constant temperature (21–25 °C) and relative humidity of

approximately 40–65% with a standard light/dark cycle. Animals

were housed in stainless steel cages with suspended wire-mesh

ﬂoors (maximum of 10 mice per cage). Animals were fasted over-

night and then weighed. Mice had free access to water throughout

the experimental period. Animals were anesthetized with keta-

mine (100 mg/kg)/xylasine (10 mg/kg) and sacriﬁced at the follow-

ing time intervals: at control (0 min) and 5, 15, 30, 60, 120 and

240 min after compound administration. Blood samples were col-

lected into heparinized microfuge tubes. The samples were imme-

diately centrifuged at 3500 rpm for 10 min. The plasma was

separated into clean tubes and frozen at ꢀ30 °C prior to analysis,

whereas brains were stored at ꢀ80 °C. Mice were sampled four

at a time and one sample per animal was collected. The plasma

concentrations of 22a at different time points were expressed as

mean SD, and the mean concentration–time curves were plotted.

All animal experiments were performed in accordance with the

institutional guidelines and were approved by the Animal Care

and Ethics Committee of the Jagiellonian University.

4.3. Mutagenicity or antimutagenicity assay

Mutagenicity assay was performed according to previously

described procedure.^27,28Brieﬂy, for the V. harveyi mutagenicity

test, 10 lL of test compound was added to exponentially growing

cultures (A₆₀₀= 0.1, 30 °C) and incubation was continued until

the OD₆₀₀had increased to 0.3–0.4. Then, 5 ꢃ 10⁶bacterial cells

were spread onto BOSS agar plates containing neomycin at a ﬁnal

concentration of 100 lg/mL. Plates were incubated for 48 h at

30 °C and neomycin-resistant colonies were counted manually.

All the assays were carried out in triplicate.

In the antimutagenicity assay performed with the V. harveyi

strains, 10

ing cultures (A₆₀₀= 0.1, 30 °C) and incubation was continued for

15 min. Then, 10 L of NQNO was added to samples with tested

lL of test compound was added to exponentially grow-

l

compounds and cultivation was continued till A₆₀₀between 0.3

and 0.4 to mid-log phase of growth. Next, 5 ꢃ 10⁶bacterial cells

grown in BOSS medium were transferred onto BOSS plates. Follow-

ing 48 h incubation at 30 °C, neomycin-resistant colonies were

counted.²⁹All experiments were analyzed in three independent

repetitions.

All data in pharmacokinetic experiments were processed with

the pharmacokinetic software Phoenix WinNonlin (Certara, USA).

The non-compartmental pharmacokinetic parameters of t_1/2

,

MRT, AUC, Cl_tand V_sswere calculated based on moment meth-

ods. First order elimination rate constant (k_z) was calculated by

linear regression of time versus log concentration according to

the Eq. 1.

4.4. Metabolic stability

k_z¼ ꢀ2:303 a

where a is a slope of a line.

ð1Þ

Mouse liver microsomes (MLMs), glucose-6-phosphate, NADP,

glucose-6-phosphate dehydrogenase and levallorphan were sup-

plied by Sigma Aldrich.

Mouse liver microsomal incubations were conducted in dupli-

cate. Incubations were composed of test compound (20 lM), MLMs

The terminal half-life (t_1/2) was calculated as:

0:693

k_z

t₁₌₂

¼

ð2Þ

(0.4 mg/mL), NADPH-regenerating system and potassium phos-

phate buffer (100 mM, pH 7.4). NADPH-regenerating system con-

For each compound, the area under the mean serum and tissue

tained

NADP,

glucose-6-phosphate,

glucose-6-phosphate

drug concentration versus time curve extrapolated to inﬁnity

(AUC_0?1) was estimated using the log-linear trapezoidal rule

(Eq. 3), where C_nis the concentration of last sampling.

dehydrogenase and potassium phosphate buffer (100 mM, pH

7.4). Firstly, the mixtures containing microsomes, test compound

and buffer were preincubated at 37 °C for 15 min before the addi-

tion of NADPH-regenerating system. The resulting mixture was

incubated for different time points (5, 15, 30, 60 and 90 min) at

ꢀ

ꢁ

n

X

C_iþ C_iþ1

C_n

k_z

AUC_0!1

¼

ꢁ ðt_iþ1ꢀ t_iÞ þ

ð3Þ

2

i¼1

37 °C. Next, levallorphan (20 lM, internal standard) was added,

and the reaction was quenched by addition of perchloric acid

(69–72%, by volume). The solution was then centrifuged to sedi-

ment the precipitates. Supernatant analysis was performed using

UPLC/MS (Waters Corporation, Milford, MA, USA). For control sam-

ples NADPH-regenerating system was replaced with phosphate

buffer.^30–32The in vitro half times (t_1/2) for test compounds were

determined from the slope of the linear regression of ln % parent

compound remaining versus incubation time. The calculated t_1/2

was incorporated into the following equation to obtain intrinsic

The area under the ﬁrst-moment curve (AUMC_0?1) was esti-

mated by calculation of the total area under the ﬁrst-moment

curve and extrapolated area using the Eq. 4, where t_nis the time

of last sampling.

ꢀ

ꢁ

n

X

t_iꢁ C_iþ t_iþ1ꢁ C_iþ1

t_nꢁ C_nC_n

AUMC_0!1

¼

ꢁ ðt_iþ1ꢀ t_iÞ þ

þ

ð4Þ

k_z²

2

k_z

i¼1

Mean residence time (MRT) was calculated as:

AUMC_0!1

clearance (Cl_int) = (volume of incubation [lL]/protein in the incu-

16

MRT ¼

ð5Þ

bation [mg]) ꢃ 0.693/t_1/2

.

AUC_0!1

1810

A. M. Waszkielewicz et al. / Bioorg. Med. Chem. 24 (2016) 1793–1810

Total clearance (Cl_t) was calculated as:

D_i

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3386.

Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.bmc.2016.03.006.

These data include MOL ﬁles and InChiKeys of the most important

compounds described in this article.

Article Doi

Design, physico-chemical properties and biological evaluation of some new N-[(phenoxy)alkyl]- and N-{2-[2-(phenoxy)ethoxy]ethyl}aminoalkanols as anticonvulsant agents

DOI: 10.1016/j.bmc.2016.03.006

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Full text of DOI:10.1016/j.bmc.2016.03.006

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