545
Acta Chim. Slov. 2023, 70, 545–559
Gršič et al.:   Synthesis and Cholinesterase Inhibitory Activity of Selected   ...
DOI: 10.17344/acsi.2023.8460
Scientific paper
Synthesis and Cholinesterase Inhibitory Activity of Selected 
Indole-Based Compounds
Marija Gršič,
1
 Anže Meden,
2
 Damijan Knez,
2
 Marko Jukič,
3
 Jurij Svete,
1
  
Stanislav Gobec
2
 and Uroš Grošelj*
,1
1
 University of Ljubljana, Faculty of Chemistry and Chemical Technology, Večna pot 113, SI-1000 Ljubljana, Slovenia.
2
 University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, SI-1000 Ljubljana, Slovenia.
3
 University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova ulica 17, SI-2000 Maribor, Slovenia.
* Corresponding author: E-mail: uros.groselj@fkkt.uni-lj.si
Received: 09-20-2023
Abstract
Synthesis and anticholinesterase activity of 18 previously unpublished indole- and tryptophan-derived compounds are 
disclosed. These compounds containing an indole structural unit exhibit selective submicromolar inhibition of human 
butyrylcholinesterase (hBChE). The structures of the newly synthesized compounds were confirmed by 
1
H and 
13
C 
NMR, IR spectroscopy, and high-resolution mass spectrometry.
Keywords: Indole derivatives, Tryptophan derivatives, Amidations, Cholinesterase (ChE) inhibitors, 1,1’-Carbonyldiim-
idazole (CDI), Rearrangements, Catalytic hydrogenation
1. Introduction
Indole is a privileged scaffold found in countless nat-
ural products that have diverse biological activities and 
functions.
1–6
 In addition to the well-known skatole 
(3-methylindole), serotonin, L-tryptophan, tryptamine, 
the plant growth hormone 3-indoleacetic acid, and others, 
indole-containing alkaloids represent one of the most im-
portant alkaloid subgroups.
7,8
 Indoles exhibit diverse bio-
logical activities ranging from antitumor to antibacterial 
activity.
9–12
 Commercially available drugs with indole 
moiety include ajmaline
13
 (antiarrhythmic agent), phys-
ostigmine
14
 (for the treatment of glaucoma and anticho-
linergic poisoning), and vincristine
15
 (antitumor agent), 
among others.
1,16,17
Dementia is a serious neurological condition that 
severely affects patient’s quality of life. It is estimated 
that Alzheimer’s disease (AD), the most common form 
of dementia, affects more than 50 million people world-
wide, and this number is expected to triple by 2050, 
mainly due to the aging of the population.
18
 Selective 
human butyrylcholinesterase (hBChE) inhibitors im-
proved cognitive functions, memory, and learning abili-
ty in a scopolamine mice model of cognitive deficit with-
out causing peripheral cholinergic side effects typical of 
acetylcholinesterase (AChE) inhibitors.
19,20
 These data 
suggest that hBChE may be considered a promising 
therapeutic target to improve cognitive functions in 
late-stages of AD.
21
 Recently, we have disclosed a hit-to-
lead development of a new series of tryptophan-derived 
selective hBChE inhibitors with nanomolar inhibitory 
potencies, which were developed from (+)-isocampho-
lenic acid-derived tryptophan amide hit A
22
 by a medic-
inal chemistry-based approach (Figure 1).
23,24
 Lead 
compounds B and C inhibited hBChE in the low nano-
molar range with high selectivity over AChE, and pos-
sessed advantageous physicochemical properties for 
high blood-brain barrier permeability. Furthermore, 
compound B showed beneficial effects on fear-motivat-
ed long-term memory and spatial long-term memory 
retrieval in a scopolamine AD mouse model, with no 
adverse peripheral cholinergic side effects.
23–25
While the structure-activity relationships (SAR) has 
been explored, several of the indole products were not in-
cluded in our earlier reports. Therefore, we report here the 
synthesis and cholinesterase inhibitory activity of those 
unpublished indole-based compounds.

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Gršič et al.:   Synthesis and Cholinesterase Inhibitory Activity of Selected   ...
2. Experimental
2. 1. Materials and Measurements
Solvents for extractions and chromatography were of 
technical grade and were distilled prior to use. Extracts 
were dried over technical grade anhydrous Na
2
SO
4
. Melt-
ing points were determined on a Kofler micro hot stage 
and using the SRS OptiMelt MPA100 – Automated Melt-
ing Point System (Stanford Research Systems, Sunnyvale, 
CA, USA). NMR spectra were recorded on a Bruker Ul-
traShield 500 plus (Bruker, Billerica, MA, USA) at 500 
MHz for the 
1
H nucleus and 126 MHz for the 
13
C nucleus, 
using CDCl
3
 with TMS as the internal standard, as sol-
vents. Mass spectra were recorded using an Agilent 6224 
Accurate Mass TOF LC/MS (Agilent Technologies, Santa 
Clara, CA, USA), IR spectra using a Perkin-Elmer Spec-
trum BX FTIR spectrophotometer (Perkin-Elmer, 
Waltham, MA, USA). Column chromatography was per-
formed on silica gel (silica gel 60, particle size: 0.035–0.070 
mm (Sigma-Aldrich, St. Louis, MO, USA)). All commer-
cial chemicals used were purchased from Sigma-Aldrich 
(St. Louis, MO, USA). Catalytic hydrogenation was per-
formed in a Parr Pressure Reaction Hydrogenation Appa-
ratus (Moline, IL, USA). Microanalyses were performed 
by combustion analysis on a Perkin-Elmer Series II 
CHNS/O Analyser (Perkin-Elmer, Waltham, MA, USA).
General procedure 1 (GP1) – amide formation. To 
a solution/suspension of acid (1 equivalent) in anhydrous 
MeCN under argon at room temperature was added CDI 
(1.20 equivalents). The resulting reaction mixture was 
stirred at room temperature for 1 h, then amine (1.13 
equivalents) was added. After stirring the reaction mixture 
at room temperature for 16 h, the volatile components 
were evaporated in vacuo and the residue was purified by 
column chromatography on silica gel 60. The fractions 
containing the pure product (amide) were combined and 
the volatiles were evaporated in vacuo.
General procedure 2 (GP2) – Boc-deprotection 
and double bond isomerization. To a solution of the 
starting compound in anhydrous CH
2
Cl
2
 at 0 °C was add-
ed trifluoroacetic acid (TFA). The resulting reaction mix-
ture was stirred at 0 °C for 30 minutes and then stirred at 
room temperature for 2 h. Volatile components were evap-
orated in vacuo. The residual trifluoroacetic acid was re-
moved by azeotropic evaporation with anhydrous toluene.
General procedure 3 (GP3) – acetamidation. To a 
solution of the trifluoroacetate salt in anhydrous CH
2
Cl
2
 
under argon at room temperature was added N,N-diiso-
propylethylamine (DIPEA) followed by CH
3
COCl. After 
stirring the reaction mixture at room temperature for 12 h, 
the volatiles were evaporated in vacuo. The residue was 
dissolved in EtOAc (50 mL) and washed with NaHSO
4
 (1 
M in H
2
O, 2×5 mL), NaHCO
3
 (aq. sat, 2 × 5 mL), and Na-
Cl (aq. sat, 2 × 5 mL). The organic phase was dried under 
anhydrous Na
2
SO
4
, filtered, and the volatiles were evapo-
rated in vacuo. The residue was purified by column chro-
matography on silica gel 60. The fractions containing the 
pure product were combined and the volatiles were evapo-
rated in vacuo.
General procedure 4 (GP4) – amine N-Boc protec-
tion. To a solution of the trifluoroacetate salt in anhydrous 
CH
2
Cl
2
 under argon at room temperature were added 
Et
3
N and Boc
2
O. After stirring the reaction mixture at 
room temperature for 16 h, the volatile components were 
evaporated in vacuo. The residue was purified by column 
chromatography on silica gel 60. The fractions containing 
the pure product were combined and the volatiles were 
evaporated in vacuo.
General procedure 5 (GP5) – alkene hydrogena-
tion. To a solution of alkene in MeOH, Pd–C (10% Pd on 
C; 20% by mass reagent was used) was added under argon. 
The resulting reaction mixture was thoroughly purged 
with hydrogen and shaken on a Parr apparatus under H
2
 (4 
bar) at room temperature. The reaction mixture was fil-
tered through a short pad of Celite® on a ceramic frit and 
Celite® was washed with MeOH. The volatiles were evapo-
rated in vacuo. If necessary, the product was additionally 
purified by column chromatography on silica gel 60. The 
fractions containing the pure product were combined and 
the volatiles were evaporated in vacuo.
N-(Cycloheptylmethyl)-3-(1H-indol-3-yl)propanamide (8)
Following GP1. Prepared from 3-(1H-indol-3-yl)
propanoic acid (1) (283 mg, 1.50 mmol), MeCN (3 mL), 
CDI (280 mg, 1.73 mmol), cycloheptylmethanamine (5) 
Figure 1. Hit compound A and selected lead compounds B and C.

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Gršič et al.:   Synthesis and Cholinesterase Inhibitory Activity of Selected   ...
(250 μL, 1.74 mmol); column chromatography (EtOAc/
petroleum ether = 1:1). Yield: 348 mg (1.17 mmol, 78%) of 
white solid; mp 80.9–85.0 °C. Anal. Calcd for C
19
H
26
N
2
O: 
C, 76.47; H, 8.78; N, 9.39. Found: C, 76.38; H, 8.85; N, 9.31. 
ESI-HRMS Calcd for C
19
H
27
N
2
O: m/z 299.2118 (MH
+
). 
Found: m/z 299.2119 (MH
+
). IR ν
max
 3258, 3087, 2914, 
2848, 1612, 1564, 1492, 1455, 1429, 1350, 1274, 1217, 1181, 
1102, 1065, 1008, 979, 790, 767, 733, 698 cm
–1
. 
1
H NMR 
(500 MHz, DMSO-d
6
): δ 1.00–1.11 (m, 2H), 1.28–1.64 (m, 
11H), 2.44 (dd, J = 6.9, 8.4 Hz, 2H), 2.84–2.96 (m, 4H), 
6.93–6.99 (m, 1H), 7.02–7.09 (m, 2H), 7.32 (dt, J = 0.9, 8.1 
Hz, 1H), 7.52 (dd, J = 1.0, 7.9 Hz, 1H), 7.79 (t, J = 5.8 Hz, 
1H), 10.73 (s, 1H). 
13
C NMR (126 MHz, DMSO-d
6
): δ 
21.15, 25.87, 27.99, 31.63, 36.36, 45.11, 111.28, 113.89, 
118.09, 118.37, 120.85, 122.08, 127.07, 136.25, 171.87 (one 
signal missing).
tert-Butyl (R)-(1-((2-Cyclohexylethyl)amino)-3-(1H-in-
dol-3-yl)-1-oxopropan-2-yl)carbamate (9)
Following GP1. Prepared from (tert-butoxycarbon-
yl)-D-tryptophan (2) (182 mg, 0.598 mmol), MeCN (2 
mL), CDI (112 mg, 0.691 mmol), 2-cyclohexy-
lethan-1-amine (6) (100 μL, 0.677 mmol); column chro-
matography (EtOAc). Yield: 221 mg (0.534 mmol, 89%) of 
white solid; mp 103.9–106.2 °C. Anal. Calcd for 
C
24
H
35
N
3
O
3
: C, 69.70; H, 8.53; N, 10.16. Found: C, 69.77; 
H, 8.60; N, 10.08. ESI-HRMS Calcd for C
24
H
36
N
3
O
3
: m/z 
414.2751 (MH
+
). Found: m/z 414.2762 (MH
+
). IR ν
max
 
3413, 3324, 2920, 2849, 1682, 1650, 1521, 1455, 1390, 1366, 
1247, 1166, 1090, 1065, 1046, 1011, 857, 795, 736 cm
–1
. [α]
D
r.t.
 = –14.4 (c = 1.1 mg/mL, CH
2
Cl
2
). 
1
H NMR (500 MHz, 
CDCl
3
): δ 0.71–0.85 (m, 2H), 0.99–1.21 (m, 6H), 1.43 (s, 
9H), 1.51–1.68 (m, 5H), 3.04–3.20 (m, 3H), 3.31 (dd, J = 
5.0, 14.3 Hz, 1H), 4.38 (s, 1H), 5.19 (s, 1H), 5.55 (s, 1H), 
7.05 (d, J = 2.3 Hz, 1H), 7.11–7.16 (m, 1H), 7.18–7.23 (m, 
1H), 7.36 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 7.9 Hz, 1H), 8.15 
(s, 1H, NH). 
13
C NMR (126 MHz, CDCl
3
): δ 26.26, 26.60, 
28.47, 28.78, 33.12, 33.18, 35.23, 36.80, 37.36, 55.43, 80.08, 
111.17, 111.30, 119.14, 119.95, 122.47, 123.18, 127.56, 
136.37, 155.60, 171.47.
tert-Butyl (S)-(1-((2-Cyclohexylethyl)amino)-3-(1-me-
thyl-1H-indol-3-yl)-1-oxopropan-2-yl)carbamate (10)
Following GP1. Prepared from N
α
-(tert-butoxycar-
bonyl)-1-methyl-L-tryptophan (3) (159 mg, 0.499 mmol), 
MeCN (2 mL), CDI (99.6 mg, 0.614 mmol), 2-cyclohexy-
lethan-1-amine (6) (83.3 μL, 0.564 mmol); column chro-
matography (EtOAc/petroleum ether = 1:1). Yield: 198 mg 
(0.463 mmol, 93%) of white solid; mp 118.2–123.8 °C. 
Anal. Calcd for C
25
H
37
N
3
O
3
: C, 70.23; H, 8.72; N, 9.83. 
Found: C, 70.15; H, 8.89; N, 9.76. ESI-HRMS Calcd for 
C
25
H
38
N
3
O
3
: m/z 428.2908 (MH
+
). Found: m/z 428.2910 
(MH
+
). IR ν
max
 3341, 3320, 2921, 2852, 1679, 1655, 1543, 
1514, 1486, 1463, 1452, 1420, 1391, 1369, 1321, 1291, 1238, 
1206, 1165, 1123, 1093, 1062, 1045, 1024, 1001, 963, 924, 
887, 867, 783, 767, 734 737 cm
–1
. [α]
D
r.t.
 = –1.03 (c = 2.8 
mg/mL, CH
2
Cl
2
). 
1
H NMR (500 MHz, CDCl
3
): δ 0.73–
0.85 (m, 2H), 0.98–1.21 (m, 6H), 1.43 (s, 9H), 1.51–1.70 
(m, 5H), 3.03–3.23 (m, 3H), 3.30 (dd, J = 5.2, 14.5 Hz, 1H), 
3.74 (s, 3H), 4.37 (s, 1H), 5.18 (s, 1H), 5.59 (s, 1H), 6.90 (s, 
1H), 7.10–7.14 (m, 1H), 7.21–7.25 (m, 1H), 7.29 (dt, J = 
0.9, 8.3 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H). 
13
C NMR (126 
MHz, CDCl
3
): δ 26.24, 26.57, 28.45, 28.61, 32.81, 33.12, 
33.18, 35.27, 36.86, 37.33, 55.41, 80.04, 109.36, 119.20, 
119.38, 121.96, 127.99, 137.07, 155.61, 171.49 (one signal 
missing).
tert-Butyl ((S)-1-((2-((R)-2,2-Dimethyl-3-methylenecy-
clopentyl)ethyl)amino)-3-(1-methyl-1H-indol-3-yl)-1-
oxopropan-2-yl)carbamate (11)
Following GP1. Prepared from N
α
-(tert-butoxycar-
bonyl)-1-methyl-L-tryptophan (3) (318 mg, 0.999 mmol), 
MeCN (3 mL), CDI (188 mg, 1.16 mmol), (R)-2-(2,2-di-
methyl-3-methylenecyclopentyl)ethan-1-amine (7)
22
 (182 
µL, 1.13 mmol); column chromatography (EtOAc/petrole-
um ether = 1:2). Yield: 183 mg (0.403 mmol, 40%) of yel-
low oil. ESI-HRMS Calcd for C
27
H
40
N
3
O
3
: m/z 454.3064 
(MH
+
). Found: m/z = 454.3068 (MH
+
). IR ν
max
 3306, 3068, 
2958, 2932, 2867, 1651, 1524, 1473, 1436, 1390, 1365, 1325, 
1240, 1166, 1046, 1013, 878, 780, 737 cm
–1
. [α]
D
r.t.
 = +5.40 
(c = 1.8 mg/mL, CH
2
Cl
2
). 
1
H NMR (500 MHz, CDCl
3
): δ 
0.72 (s, 3H), 0.95 (s, 3H), 1.00–1.08 (m, 1H), 1.10–1.20 (m, 
1H), 1.21–1.31 (m, 1H), 1.31–1.39 (m, 1H), 1.43 (s, 9H), 
1.64–1.79 (m, 1H), 2.16–2.28 (m, 1H), 2.35–2.45 (m, 1H), 
3.03–3.22 (m, 3H), 3.30 (dd, J = 5.2, 14.5 Hz, 1H), 3.73 (s, 
3H), 4.38 (s, 1H), 4.72 (t, J = 2.5 Hz, 1H), 4.74 (t, J = 2.2 Hz, 
1H), 5.18 (s, 1H), 5.67 (s, 1H), 6.91 (s, 1H), 7.09–7.14 (m, 
1H), 7.20–7.25 (m, 1H), 7.28 (d, J = 8.2 Hz, 1H), 7.64 (d, J 
= 8.0 Hz, 1H). 
13
C NMR (126 MHz, CDCl
3
): δ 23.43, 26.51, 
28.04, 28.45, 28.57, 29.71, 30.68, 32.82, 38.94, 44.00, 47.92, 
55.43, 80.13, 103.20, 109.38, 119.19, 119.40, 122.01, 127.96, 
128.00, 137.08, 155.64, 162.07, 171.54 (one signal miss-
ing).
tert-Butyl ((R)-1-((2-((R)-2,2-Dimethyl-3-methylenecy-
clopentyl)ethyl)amino)-3-(1-methyl-1H-indol-3-yl)-1-
oxopropan-2-yl)carbamate (12)
Following GP1. Prepared from N
α
-(tert-butoxycar-
bonyl)-1-methyl-D-tryptophan (4) (200 mg, 0.628 mmol), 
MeCN (3 mL), CDI (123 mg, 0.759 mmol), (R)-2-(2,2-di-
methyl-3-methylenecyclopentyl)ethan-1-amine (7)
22
 (114 
µL, 0.707 mmol); column chromatography (EtOAc/petro-
leum ether = 1:1). Yield: 140 mg (0.309 mmol, 49%) of or-
ange oil. ESI-HRMS Calcd for C
27
H
40
N
3
O
3
: m/z 454.3064 
(MH
+
). Found: m/z 454.3048 (MH
+
). IR ν
max
 3307, 2958, 
1651, 1523, 1474, 1365, 1325, 1240, 1166, 1046, 1013, 877, 
781, 737 cm
–1
. [α]
D
r.t.
 = +4.21 (c = 1.4 mg/mL, CH
2
Cl
2
). 
1
H 
NMR (500 MHz, CDCl
3
): δ 0.73 (s, 3H), 0.96 (s, 3H), 
0.99–1.10 (m, 1H), 1.13–1.22 (m, 1H), 1.28–1.37 (m, 2H), 
1.43 (s, 9H), 1.70–1.78 (m, 1H), 2.19–2.29 (m, 1H), 2.37–
2.45 (m, 1H), 3.01–3.25 (m, 3H), 3.31 (dd, J = 5.2, 14.3 Hz, 
1H), 3.74 (s, 3H), 4.37 (s, 1H), 4.73 (t, J = 2.5 Hz, 1H), 4.75 

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Acta Chim. Slov. 2023, 70, 545–559
Gršič et al.:   Synthesis and Cholinesterase Inhibitory Activity of Selected   ...
(t, J = 2.3 Hz, 1H), 5.17 (s, 1H), 5.66 (s, 1H), 6.92 (s, 1H), 
7.10–7.14 (m, 1H), 7.21–7.25 (m, 1H), 7.29 (dt, J = 0.9, 8.3 
Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H). 
13
C NMR (126 MHz, 
CDCl
3
): δ 23.47, 26.54, 28.06, 28.51, 28.45, 29.71, 30.70, 
32.84, 39.03, 44.01, 47.97, 55.49, 80.14, 103.21, 109.37, 
119.20, 119.41, 122.00, 127.98, 128.04, 137.08, 155.61, 
162.09, 171.53 (one signal missing).
(R)-1-((2-Cyclohexylethyl)amino)-3-(1H-indol-3-yl)-1-
oxopropan-2-aminium 2,2,2-Trifluoroacetate (13)
Following GP2. Prepared from Boc-amine 9 (199 
mg, 0.481 mmol), CH
2
Cl
2
 (2 mL), TFA (1.8 mL); the prod-
uct 13 was thoroughly dried in high vacuum. Yield: 187 
mg (0.437 mmol, 91%) of orange oil. ESI-HRMS Calcd for 
C
19
H
28
N
3
O: m/z 314.2227 (MH
+
). Found: m/z = 314.2242 
(MH
+
). IR ν
max
 3293, 2923, 2851, 1448, 1661, 1341, 1180, 
1135, 1011, 838, 799, 741, 722 cm
–1
. [α]
D
r.t.
 = –36.4 (c = 1.8 
mg/mL, CH
2
Cl
2
). 
1
H NMR (500 MHz, DMSO-d
6
): δ 0.75–
0.87 (m, 2H), 1.04–1.25 (m, 6H), 1.52–1.69 (m, 5H), 2.97–
3.22 (m, 4H), 3.84–3.97 (m, 1H), 6.98–7.03 (m, 1H), 7.07–
7.11 (m, 1H), 7.19 (d, J = 2.5 Hz, 1H), 7.37 (d, J = 8.1 Hz, 
1H), 7.62 (d, J = 7.8 Hz, 1H), 8.15 (s, 3H), 8.38 (t, J = 5.6 
Hz, 1H), 11.05 (d, J = 2.5 Hz, 1H). 
13
C NMR (126 MHz, 
DMSO-d
6
): δ 25.67, 26.09, 27.43, 32.52, 32.56, 34.31, 
36.13, 36.45, 52.90, 107.00, 111.47, 118.39, 121.11, 124.73, 
127.07, 136.27, 158.13 (q, J = 31.9 Hz), 168.08 (one signal 
missing).
(S)-3-(1-Methyl-1H-indol-3-yl)-1-oxo-1-((2-(2,3,3-tri-
methylcyclopent-1-en-1-yl)ethyl)amino)pro-
pan-2-aminium 2,2,2-Trifluoroacetate (14)
Following GP2. Prepared from Boc-amine 11 (98.5 
mg, 0.217 mmol), CH
2
Cl
2
 (2 mL), TFA (1 mL); the prod-
uct 14 was thoroughly dried in high vacuum. Yield: 92.9 
mg (0.199 mmol, 92%) of dark brown oil. ESI-HRMS Cal-
cd for C
22
H
32
N
3
O: m/z 354.2540 (MH
+
). Found: m/z 
354.2541 (MH
+
). IR ν
max
 3056, 2951, 2934, 2862, 1779, 
1662, 1549, 1474, 1435, 1378, 1359, 1330, 1199, 1175, 1134, 
1014, 965, 837, 798, 739, 722 cm
–1
. [α]
D
r.t.
 = +6.52 (c = 2.3 
mg/mL, CH
2
Cl
2
). 
1
H NMR (500 MHz, CDCl
3
): δ 0.86 (s, 
3H), 0.89 (s, 3H), 1.34–1.36 (m, 3H), 1.43–1.56 (m, 2H), 
1.88–2.06 (m, 4H), 2.95–3.11 (m, 2H), 3.24 (d, J = 7.2 Hz, 
2H), 3.70 (s, 3H), 4.16 (t, J = 7.3 Hz, 1H), 6.71 (t, J = 5.5 Hz, 
1H), 7.00–7.07 (m, 2H), 7.11–7.20 (m, 1H), 7.22–7.30 (m, 
1H), 7.51 (d, J = 7.9 Hz, 1H), 7.94 (s, 3H). 
13
C NMR (126 
MHz, CDCl
3
): δ 9.26, 26.44, 26.47, 27.72, 27.99, 32.06, 
32.78, 38.36, 38.76, 46.92, 54.42, 106.28, 109.75, 118.54, 
119.65, 122.26, 127.41, 129.05, 129.30, 138.02, 142.05, 
161.69 (q, J = 36.9 Hz), 168.64 (one signal missing).
(R)-3-(1-Methyl-1H-indol-3-yl)-1-oxo-1-((2-(2,3,3-tri-
methylcyclopent-1-en-1-yl)ethyl)amino)pro-
pan-2-aminium 2,2,2-Trifluoroacetate (15)
Following GP2. Prepared from Boc-amine 12 (99.7 
mg, 0.220 mmol), CH
2
Cl
2
 (2 mL), TFA (1 mL); the product 
15 was thoroughly dried in high vacuum. Yield: 89.2 mg 
(0.191 mmol, 87%) of dark orange oil. ESI-HRMS Calcd for 
C
22
H
32
N
3
O: m/z 354.2540 (MH
+
). Found: m/z 354.2535 
(MH
+
). IR ν
max
 3061, 2951, 2862, 1779, 1663, 1550, 1473, 
1435, 1378, 1359, 1330, 1251, 1172, 1135, 1013, 960, 836, 
798, 739 cm
–1
. [α]
D
r.t.
 = –9.0 (c = 1.9 mg/mL, CH
2
Cl
2
). 
1
H 
NMR (500 MHz, CDCl
3
): δ 0.86 (s, 3H), 0.89 (s, 3H), 1.36 
(t, J = 2.2 Hz, 3H), 1.44–1.57 (m, 2H), 1.89–2.07 (m, 4H), 
2.96–3.13 (m, 2H), 3.24 (d, J = 7.3 Hz, 2H), 3.71 (s, 3H), 
4.18 (t, J = 7.3 Hz, 1H), 6.64 (t, J = 5.5 Hz, 1H), 7.02 (s, 1H), 
7.03–7.07 (m, 1H), 7.13–7.19 (m, 1H), 7.23–7.28 (m, 1H), 
7.50 (d, J = 7.9 Hz, 1H), 7.85 (s, 3H). 
13
C NMR (126 MHz, 
CDCl
3
): δ 9.27, 26.44, 26.47, 27.76, 27.97, 32.05, 32.81, 
38.39, 38.76, 46.94, 54.43, 106.13, 109.80, 118.47, 119.71, 
122.33, 127.34, 129.05, 129.24, 138.02, 142.18, 161.60 (q, J = 
37.6 Hz), 168.61 (one signal missing).
(R)-2-Acetamido-N-(2-cyclohexylethyl)-3-(1H-indol-3-
yl)propanamide (19)
Following GP3. Prepared from trifluoroacetate salt 
13 (160 mg, 0.374 mmol), CH
2
Cl
2
 (2 mL), DIPEA (196 μL, 
1.13 mmol), CH
3
COCl (32.1 μL, 0.450 mmol); column 
chromatography (EtOAc/petroleum ether = 1:1). Yield: 78 
mg (0.219 mmol, 59%) of white solid; mp 195.7–198.6 °C. 
ESI-HRMS Calcd for C
21
H
30
N
3
O
2
: m/z 356.2333 (MH
+
). 
Found: m/z 356.2347 (MH
+
). IR ν
max
 3407, 3287, 2914, 
2849, 1636, 1561, 1539, 1455, 1370, 1287, 1243, 1091, 1023, 
1012, 813, 779, 741 cm
-1
. [α]
D
r.t.
 = –15.3 (c = 1.2 mg/mL, 
CH
2
Cl
2
). 
1
H NMR (500 MHz, DMSO-d
6
): δ 0.74–0.88  
(m, 2H), 1.04–1.25 (m, 6H), 1.54–1.67 (m, 5H), 1.78  
(s, 3H), 2.87 (dd, J = 8.5, 14.5 Hz, 1H), 2.96–3.11 (m, 3H), 
4.42–4.50 (m, 1H), 6.94–6.98 (m, 1H), 7.02–7.07 (m, 1H), 
7.10 (d, J = 2.3 Hz, 1H), 7.31 (dt, J = 0.9, 8.1 Hz, 1H), 7.57 
(d, J = 7.8 Hz, 1H), 7.84 (t, J = 5.6 Hz, 1H), 7.99 (d, J = 8.4 
Hz, 1H), 10.77 (s, 1H, NH). 
13
C NMR (126 MHz,  
DMSO-d
6
): δ 22.57, 25.72, 26.09, 28.07, 32.60, 34.50, 
36.24, 36.41, 53.47, 110.26, 111.19, 118.09, 118.42, 120.76, 
123.36, 127.32, 136.00, 168.88, 171.25.
(R)-2-Acetamido-3-(1H-indol-3-yl)-N-(2-(2,3,3-tri-
methylcyclopent-1-en-1-yl)ethyl)propanamide (20)
Following GP3. Prepared from trifluoroacetate salt 
16
23
 (267 mg, 0.589 mmol), CH
2
Cl
2
 (3.5 mL), DIPEA (307 
μL, 1.76 mmol), CH
3
COCl (50.3 μL, 0.705 mmol); column 
chromatography (EtOAc/petroleum ether = 1:1). Yield: 
148.6 mg (0.389 mmol, 66%) of yellowish solid; mp 78.2–
83.8 °C. ESI-HRMS Calcd for C
23
H
32
N
3
O
2
: m/z 382.2489 
(MH
+
). Found: m/z 382.2489 (MH
+
). IR ν
max
 3282, 3079, 
2951, 2861, 1636, 1533, 1457, 1435, 1372, 1358, 1287, 1234, 
1204, 1138, 1043, 1011, 908, 838, 801, 732 cm
–1
. [α]
D
r.t.
 = 
–4.9 (c = 2.3 mg/mL, CH
2
Cl
2
). 
1
H NMR (500 MHz,  
CDCl
3
): δ 0.86 (s, 3H), 0.87 (s, 3H), 1.33 (t, J = 2.1 Hz, 3H), 
1.44–1.54 (m, 2H), 1.91 (s, 3H), 1.94–2.02 (m, 4H), 3.05–
3.17 (m, 3H), 3.23 (dd, J = 5.9, 14.5, Hz, 1H), 4.68 (q, J = 
7.3 Hz, 1H), 6.02 (t, J = 5.6 Hz, 1H), 6.79 (d, J = 7.8 Hz, 
1H), 6.98 (d, J = 2.4 Hz, 1H), 7.05–7.10 (m, 1H), 7.12–7.19 
(m, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 

549
Acta Chim. Slov. 2023, 70, 545–559
Gršič et al.:   Synthesis and Cholinesterase Inhibitory Activity of Selected   ...
8.61 (s, 1H, NH). 
13
C NMR (126 MHz, CDCl
3
): δ 9.30, 
23.14, 26.42, 26.46, 28.27, 28.62, 32.10, 37.94, 38.73, 46.86, 
54.35, 110.60, 111.41, 118.70, 119.68, 122.19, 123.22, 
127.51, 129.58, 136.28, 141.83, 170.52, 171.47.
tert-Butyl (R)-(3-(1H-Indol-3-yl)-1-oxo-1-((2-(2,3,3-
trimethylcyclopent-1-en-1-yl)ethyl)amino)propan-2-
yl)carbamate (21)
Following GP4. Prepared from trifluoroacetate salt 
17
23
 (382 mg, 0.842 mmol), CH
2
Cl
2
 (5 mL), Et
3
N (500 μL, 
3.59 mmol), Boc
2
O (377 mg, 1.73 mmol); column chro-
matography (EtOAc/petroleum ether = 1:2). Yield: 245 mg 
(0.557 mmol, 66%) of colorless oil. ESI-HRMS Calcd for 
C
26
H
38
N
3
O
3
: m/z 440.2908 (MH
+
). Found: m/z 440.2903 
(MH
+
). IR ν
max
 3307, 2931, 2861, 1698, 1654, 1493, 1457, 
1436, 1391, 1365, 1246, 1163, 1102, 1065, 1046, 1011, 909, 
857, 780, 735 cm
–1
. [α]
D
r.t.
 = –11.1 (c = 2.2 mg/mL, 
CH
2
Cl
2
). 
1
H NMR (500 MHz, CDCl
3
): δ 0.84 (s, 3H), 0.88 
(s, 3H), 1.32 (s, 3H), 1.42 (s, 9H), 1.46–1.52 (m, 2H), 1.92–
2.02 (m, 4H), 3.05–3.20 (m, 3H), 3.34 (d, J = 14.7 Hz, 1H), 
4.39 (s, 1H), 5.06 (s, 1H), 5.63 (s, 1H), 7.04 (d, J = 2.5 Hz, 
1H), 7.10–7.15 (m, 1H), 7.17–7.22 (m, 1H), 7.36 (d, J = 8.0 
Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 8.24 (s, 1H, NH). 
13
C 
NMR (126 MHz, CDCl
3
): δ 9.32, 26.42, 26.55, 28.23, 28.43, 
28.64, 32.07, 37.68, 38.78, 46.93, 55.37, 80.14, 110.89, 
111.30, 119.08, 119.91, 122.45, 123.21, 127.63, 129.72, 
136.36, 142.12, 155.52, 171.47.
tert-Butyl (R)-(3-(1H-Indol-3-yl)-1-oxo-1-(((2,3,3-tri-
methylcyclopent-1-en-1-yl)methyl)amino)propan-2-yl)
carbamate (22)
Following GP4. Prepared from trifluoroacetate salt 
18
22
 (216 mg, 0.491 mmol), CH
2
Cl
2
 (5 mL), Et
3
N (386 µL, 
2.77 mmol), Boc
2
O (350 mg, 1.60 mmol); column chro-
matography (EtOAc/petroleum ether = 1:2). Yield: 109 mg 
(0.256 mmol, 52%) of colorless oil. ESI-HRMS Calcd for 
C
25
H
36
N
3
O
3
: m/z 426.2751 (MH
+
). Found: m/z 426.2751 
(MH
+
). IR ν
max
 3303, 2929, 2860, 1691, 1655, 1492, 1457, 
1437, 1390, 1365, 1246, 1163, 1099, 1056, 1011, 859, 739 
cm
–1
. [α]
D
r.t.
 = +1.92 (c = 1.9 mg/mL, CH
2
Cl
2
). 
1
H NMR 
(500 MHz, CDCl
3
): δ 0.89 (s, 3H), 0.90 (s, 3H), 1.34–1.46 
(m, 12H), 1.47–1.57 (m, 2H), 1.84–2.02 (m, 2H), 3.09–3.22 
(m, 1H), 3.23–3.36 (m, 1H), 3.73 (s, 2H), 4.33–4.49 (m, 
1H), 5.05–5.28 (m, 1H), 5.54–5.75 (m, 1H), 7.03 (d, J = 2.5 
Hz, 1H), 7.09–7.15 (m, 1H), 7.17–7.21 (m, 1H), 7.35 (d, J 
= 8.1 Hz, 1H), 7.65 (d, J = 7.9 Hz, 1H), 8.41 (s, 1H, NH). 
13
C NMR (126 MHz, CDCl
3
): δ 9.45, 26.31, 28.41, 28.63, 
31.07, 37.93, 38.61, 47.08, 55.34, 80.11, 110.81, 111.35, 
118.98, 119.79, 122.31, 123.26, 127.49, 128.86, 136.39, 
143.51, 155.59, 171.63 (one signal missing).
tert-Butyl (S)-(3-(1-Methyl-1H-indol-3-yl)-1-oxo-1-((2-
(2,3,3-trimethylcyclopent-1-en-1-yl)ethyl)amino)pro-
pan-2-yl)carbamate (23)
Following GP4. Prepared from trifluoroacetate salt 
14 (62.9 mg, 0.134 mmol), CH
2
Cl
2
 (3 mL), Et
3
N (170 µL, 
1.22 mmol), Boc
2
O (203 mg, 0.930 mmol); column chro-
matography (EtOAc/petroleum ether = 1:4). Yield: 45.2 
mg (0.0996 mmol, 74%) of yellow oil. ESI-HRMS Calcd 
for C
27
H
40
N
3
O
3
: m/z 454.3064 (MH
+
). Found: m/z = 
454.3068 (MH
+
). IR ν
max
 3305, 2950, 2931, 2861, 1652, 
1523, 1474, 1365, 1325, 1241, 1166, 1121, 1045, 1013, 859, 
779, 737 cm
–1
. [α]
D
r.t.
 = +6.95 (c = 1.7 mg/mL, CH
2
Cl
2
). 
1
H 
NMR (500 MHz, CDCl
3
): δ 0.82 (s, 3H), 0.87 (s, 3H), 1.31 
(s, 3H); 1.42 (s, 9H), 1.44–1.51 (m, 2H), 1.90–1.97 (m, 
2H), 2.00 (t, J = 6.9 Hz, 2H), 3.04–3.22 (m, 3H), 3.33 (d, J 
= 11.9 Hz, 1H), 3.74 (s, 3H), 4.37 (s, 1H), 5.04 (s, 1H), 5.63 
(s, 1H), 6.91 (s, 1H), 7.09–7.13 (m, 1H), 7.20–7.25 (m, 
1H), 7.28 (dt, J = 1.0, 8.3 Hz, 1H), 7.61 (d, J = 7.9 Hz, 1H). 
13
C NMR (126 MHz, CDCl
3
): δ 9.28, 26.36, 26.51, 28.16, 
28.42, 31.98, 32.79, 37.62, 38.76, 46.91, 55.48, 80.13, 
109.24, 109.34, 119.17, 119.39, 122.00, 127.91, 128.14, 
129.74, 137.08, 142.11, 155.51, 171.48.
tert-Butyl (R)-(3-(1-Methyl-1H-indol-3-yl)-1-oxo-1-
((2-(2,3,3-trimethylcyclopent-1-en-1-yl)ethyl)amino)
propan-2-yl)carbamate (24)
Following GP4. Prepared from trifluoroacetate salt 
15 (61.8 mg, 0.132 mmol), CH
2
Cl
2
 (3 mL), Et
3
N (97.6 μL, 
0.700 mmol), Boc
2
O (110 mg, 0.504 mmol); column chro-
matography (EtOAc/petroleum ether = 1:3). Yield: 45.7 
mg (0.101 mmol, 76%) of orange oil. ESI-HRMS Calcd for 
C
27
H
40
N
3
O
3
: m/z 454.3064 (MH
+
). Found: m/z 454.3067 
(MH
+
). IR ν
max
 3305, 3052, 2950, 2931, 2861, 1652, 1522, 
1474, 1365, 1325, 1242, 1165, 1046, 1013, 923, 860, 778, 
734 cm
–1
. [α]
D
r.t.
 = +28.7 (c = 1.7 mg/mL, CH
2
Cl
2
). 
1
H 
NMR (500 MHz, CDCl
3
): δ 0.82 (s, 3H), 0.87 (s, 3H), 1.32 
(t, J = 2.1 Hz, 3H), 1.42 (s, 9H), 1.45–1.52 (m, 2H), 1.88–
1.97 (m, 2H), 2.00 (t, J = 7.0 Hz, 2H), 3.04–3.22 (m, 3H), 
3.34 (d, J = 14.8 Hz, 1H), 3.74 (s, 3H), 4.36 (s, 1H), 5.03 (s, 
1H), 5.62 (s, 1H), 6.91 (s, 1H), 7.10–7.14 (m, 1H), 7.20–
7.25 (m, 1H), 7.28 (dt, J = 1.0, 8.3 Hz, 1H), 7.61 (d, J = 7.9 
Hz, 1H). 
13
C NMR (126 MHz, CDCl
3
): δ 9.29, 26.36, 26.52, 
28.17, 28.43, 31.99, 32.80, 37.62, 38.77, 46.92, 55.47, 80.11, 
109.25, 109.34, 119.18, 119.40, 122.01, 127.91, 128.15, 
129.74, 137.09, 142.12, 155.53, 171.49.
2-(1H-Indol-3-yl)-N-(2-((1R,3R)-2,2,3-trimethylcyclo-
pentyl)ethyl)acetamide (cis-27) and 2-(1H-Indol-3-yl)-
N-(2-((1R,3S)-2,2,3-trimethylcyclopentyl)ethyl)aceta-
mide (trans-27)
Following GP5 Prepared from alkene 25
23
 (126 mg, 
0.406 mmol), MeOH (20 mL), Pd–C (29 mg); column 
chromatography (EtOAc/petroleum ether = 1:2). The 
product was isolated and characterized as a diastereomer 
mixture of cis-27:trans-27 = 86:14. Yield: 83.9 mg (0.268 
mmol, 66%) of orange oil. ESI-HRMS Calcd for 
C
20
H
29
N
2
O: m/z 313.2274 (MH
+
). Found: m/z 313.2277 
(MH
+
). IR ν
max
 3407, 3273, 2949, 2867, 1639, 1526, 1456, 
1435, 1365, 1339, 1252, 1227, 1186, 1126, 1099, 1009, 925, 
878, 778, 738 cm
–1
. [α]
D
r.t.
 = +9.78 (c = 1.7 mg/mL, 
CH
2
Cl
2
). 
1
H NMR (500 MHz, CDCl
3
) for cis-27: δ 0.41 (s, 

550
Acta Chim. Slov. 2023, 70, 545–559
Gršič et al.:   Synthesis and Cholinesterase Inhibitory Activity of Selected   ...
3H), 0.71 (s, 3H), 0.78 (d, J = 6.8 Hz, 3H), 0.99–1.21 (m, 
4H), 1.33–1.47 (m, 2H), 1.62–1.68 (m, 2H), 3.08–3.24 (m, 
2H), 3.74 (s, 2H), 5.66 (t, J = 5.9 Hz, 1H), 7.13–7.18 (m, 
2H), 7.22–7.26 (m, 1H), 7.41 (dt, J = 0.9, 8.3 Hz, 1H), 7.56 
(d, J = 7.9 Hz, 1H), 8.45 (s, 1H, NH). 
1
H NMR (500 MHz, 
CDCl
3
) for trans-27: δ 0.76 (d, J = 7.2 Hz, 1H). 
13
C NMR 
(126 MHz, CDCl
3
) for cis-27: δ 13.95, 14.45, 25.53, 28.06, 
30.24, 30.53, 33.61, 39.20, 42.39, 45.02, 48.37, 109.30, 
111.53, 118.93, 120.22, 122.76, 123.88, 127.17, 136.58, 
171.45.
tert-Butyl ((S)-3-(1-Methyl-1H-indol-3-yl)-1-oxo-1-((2-
(2,3,3-trimethylcyclopentyl)ethyl)-amino)propan-2-yl)
carbamate (28)
Following GP5 Prepared from alkene 23 (55.0 mg, 
0.121 mmol), MeOH (20 mL), Pd–C (19.7 mg); column 
chromatography (EtOAc/petroleum ether = 1:2). The 
product was isolated and characterized as a diastereomer 
mixture in a ratio of 89:11. Yield: 38.4 mg (0.0843 mmol, 
70%) of yellow oil. ESI-HRMS Calcd for C
27
H
42
N
3
O
3
: m/z 
456.3221 (MH
+
). Found: m/z 456.3222 (MH
+
). IR ν
max
 
3429, 3305, 2950, 2868, 2243, 1651, 1523, 1501, 1472, 1365, 
1325, 1244, 1166, 1047, 1013, 908, 860, 779, 734 cm
–1
. [α]
D
r.t.
 = +7.6 (c = 2.4 mg/mL, CH
2
Cl
2
). 
1
H NMR (500 MHz, 
CDCl
3
) for the major diastereomer: δ 0.41 (s, 3H), 0.75 (s, 
3H), 0.80 (d, J = 6.9 Hz, 3H), 0.91–1.05 (m, 2H), 1.06–1.18 
(m, 2H), 1.28–1.38 (m, 2H), 1.43 (s, 9H), 1.57–1.89 (m, 
2H), 2.96–3.17 (m, 3H), 3.30 (dd, J = 5.2, 14.6 Hz, 1H), 
3.74 (s, 3H), 4.39 (s, 1H), 5.19 (s, 1H), 5.61 (s, 1H), 6.92 (s, 
1H), 7.10–7.14 (m, 1H), 7.20–7.25 (m, 1H), 7.29 (d, J = 8.2 
Hz, 1H), 7.65 (d, J = 7.9 Hz, 1H). 
13
C NMR (126 MHz, 
CDCl
3
) for the major diastereomer: δ 13.94, 14.47, 25.59, 
28.03, 28.46, 28.65, 30.26, 30.36, 32.83, 39.18, 42.39, 44.98, 
48.39, 55.42, 80.07, 109.36, 119.21, 119.41, 121.99, 127.97, 
137.09, 155.57, 171.47 (two signals missing).
N -(2-((1R ,3R )-2,2,3-Trimethylcyclopentyl)
ethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxamide 
(cis-29), N-(2-((1R,3S)-2,2,3-Trimethylcyclopentyl)
ethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxamide 
(trans-29) and N-(2-((1R,3R)-2,2,3-Trimethylcyclopen-
tyl)ethyl)octahydro-1H-indole-2-carboxamide (cis-30), 
N-(2-((1R,3S)-2,2,3-Trimethylcyclopentyl)ethyl)oc-
tahydro-1H-indole-2-carboxamide (trans-30)
Following GP5 Prepared from alkene 26
23
 (148 mg, 
0.499 mmol), MeOH (20 mL), Pd–C (45 mg); column 
chromatography (1. EtOAc/petroleum ether = 1:1 for the 
elution of the cis-29/trans-29 mixture; 2. EtOAc/MeOH = 
1:1 for the elution of the cis-30/trans-30 mixture).
The mixture cis-29/trans-29 = 88:12 elutes first from 
the column. The aniline was isolated and characterized as 
a mixture of two diastereomers. Yield: 14.8 mg (0.0489 
mmol, 10%) of dark orange oil. ESI-HRMS Calcd for 
C
19
H
31
N
2
O: m/z 303.2431 (MH
+
). Found: m/z 303.2429 
(MH
+
). IR ν
max
 3231, 2930, 2865, 1614, 1585, 1539, 1465, 
1411, 1365, 1322, 1266, 1246, 1148, 1132, 1058, 981, 929, 
835, 816, 761, 711 cm
–1
. [α]
D
r.t.
 = +15.1 (c = 1.1 mg/mL, 
CH
2
Cl
2
). 
1
H NMR (500 MHz, CDCl
3
) for cis-29: δ 0.51 (s, 
3H), 0.83 (d, J = 6.8 Hz, 3H), 0.86 (s, 3H), 1.13–1.36 (m, 
4H), 1.38–1.55 (m, 2H), 1.63–1.92 (m, 6H), 2.49 (t, J = 6.0 
Hz, 2H), 2.60 (t, J = 6.1 Hz, 2H), 3.27–3.48 (m, 2H), 5.72 
(s, 1H), 6.26 (d, J = 2.4 Hz, 1H), 9.15 (s, 1H). 
13
C NMR 
(126 MHz, CDCl
3
) for cis-29: δ 14.00, 14.56, 22.89, 22.92, 
23.19, 23.72, 25.72, 28.33, 30.36, 31.15, 39.06, 42.56, 45.14, 
48.67, 107.42, 118.90, 123.97, 131.53, 161.45.
The mixture cis-30/trans-30 = 90:10 elutes second 
from the column. The product pyrrolidine was isolated 
and characterized as a mixture of diastereomers. Yield: 
58.0 mg (0.189 mmol, 38%) of dark orange oil. ESI-HRMS 
Calcd for C
19
H
35
N
2
O: m/z 307.2744 (MH
+
). Found: m/z 
307.2751 (MH
+
). IR ν
max
 3210, 3064, 2929, 2865, 2675, 
1672, 1559, 1449, 1366, 1300, 1270, 1249, 1186, 1136, 1079, 
1031, 981, 944, 917, 903, 844, 811, 729 cm
–1
. [α]
D
r.t.
 = +25.8 
(c = 1.9 mg/mL, CH
2
Cl
2
). 
1
H NMR (500 MHz, CDCl
3
) for 
the mixture of diastereomers: δ 0.51 (s, 3H), 0.83 (d, J = 6.8 
Hz, 3H), 0.86 (d, J = 2.6 Hz, 3H), 1.13–1.54 (m, 9H), 1.55–
1.94 (m, 8H), 2.27–2.38 (m, 1H), 2.55–2.66 (m, 1H), 3.12–
3.25 (m, 1H), 3.27–3.40 (m, 1H), 3.61–3.76 (m, 1H), 4.49 
(s, 1H), 8.50 (s, 1H). 
13
C NMR (126 MHz, CDCl
3
) for the 
mixture of diastereomers: δ 13.99, 14.58, 22.00, 22.09, 
25.73, 25.76, 26.06, 26.52, 28.09, 28.16, 30.31, 30.37, 30.51, 
34.74, 37.79, 37.81, 39.70, 42.51, 42.53, 45.19, 45.22, 48.49, 
48.59, 58.68, 58.72, 58.84, 58.86, 170.59.
2. 2.  Biological Evaluation – Inhibition of 
Cholinesterases
The inhibitory potencies of the compounds against 
the ChEs were determined using the method of Ellman fol-
lowing the procedure described previously.
19
 Briefly, com-
pound stock solutions in DMSO were incubated with Ell-
man’s reagent and the ChEs (final concentrations: 370 μM 
Ellman’s reagent, 1 nM or 50 pM hBChE or murine (m)
AChE, respectively) in 0.1 M phosphate buffer pH 8.0 for 5 
min at 20 °C. mAChE was chosen as the surrogate for 
hAChE as they are structurally highly conserved in the 
composition of active site amino acid residues.
26
 The reac-
tions were started by the addition of the substrate (final 
concentration, 500 μM butyrylthiocholine iodide or acet-
ylthiocholine iodide for hBChE and mAChE, respectively). 
The final content of DMSO was always 1%. The increase in 
absorbance at 412 nm was monitored for 2 min using a 
96-well microplate reader (Synergy H4, BioTek Instru-
ments, VT, USA). The initial velocities in the presence (v
i
) 
and absence (v
o
) of the test compounds were calculated. 
The inhibitory potencies were expressed as the residual ac-
tivities, according to RA = (v
i
 – b) / (v
o
 – b), where b is the 
blank value using phosphate buffer without ChEs. For IC
50
 
determinations, at least seven different concentrations of 
each compound were used. The IC
50
 values were obtained 
by plotting the residual ChE activities against the applied 
inhibitor concentrations, with the experimental data fitted 

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to a four-parameter logistic function (GraphPad Prism 8.0, 
GraphPad Software, Boston, MA, USA). Tacrine and done-
pezil were used as positive controls.
3. Results and Discussion
3. 1. Synthesis and ChE Inhibitory Activity
The synthesis of the products is not presented in a 
linear fashion, as the individual sequences range from one 
to four steps. Instead, the synthesis is divided into amida-
tions with 1,1'-carbonyldiimidazole (CDI), N-Boc depro-
tection and isomerization with trifluoroacetic acid (TFA), 
N-Boc protection and acetylation, and catalytic hydro-
genation (Schemes 1–4).
3-(1H-Indol-3-yl)propanoic acid (1) and tryptophan 
derivatives 2–4 were activated with CDI activating reagent 
before coupling with cycloalkyl-alkane-amines 5–7. 
Amides 8–12 were obtained in 40–93% yields after isola-
tion by column chromatography (Scheme 1).
Scheme 1. Synthesis of amides 8–12.

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TFA was used for the N-Boc protecting group cleav-
age of carbamates 9, 11, and 12, and the corresponding 
trifluoroacetate salts 13–15 were obtained in 87–92% 
yields (Scheme 2). The Boc protecting group cleavage of 
carbamates 11 and 12 was accompanied by isomerization 
of the exocyclic double bond
22
 by initial protonation of the 
double bond, 1,2-methyl shift, followed by deprotonation, 
giving cyclopentenes 14 and 15.
Treatment of trifluoroacetate salts 13 and 16 with 
acetyl chloride in the presence of DIPEA gave acetamides 
19 and 20 in 59% and 66% yield, respectively. Similarly, 
treatment of 14, 15, 17, and 18 with Boc
2
O in the presence 
of Et
3
N in dichloromethane gave Boc-protected amines 
21–24 in 52–76% yield (Scheme 3).
Finally, catalytic hydrogenation of alkene 25 with 
Pd–C in methanol gave an inseparable mixture of cyclopen-
tanes cis-27 and trans-27 in an 86:14 ratio and 66% yield 
(Scheme 4). The formation of the major cyclopentane cis-27 
was explained by the approach of the reagent from the less 
hindered side of the exocyclic double bond. Similarly, re-
duction of the endocyclic alkene 23 afforded an inseparable 
mixture of two diastereomers of product 28 in the relative 
ratio 89:11 in 70% yield. The absolute configuration of the 
newly formed stereocentres could not be determined; a rel-
ative cis-configuration on cyclopentane is shown for both 
isomers. Catalytic hydrogenation of the indole-2-carboxylic 
acid-derived amide 26 was not chemoselective and afforded 
a separable mixture of pyrrole derivatives cis-29/trans-29 
and pyrrolidine derivatives cis-30/trans-30. The pyrroles 
cis-29/trans-29 were isolated in 10% yield as an inseparable 
mixture of geometric isomers in the ratio 88:12. Similarly, 
the pyrrolidines cis-30/trans-30 were isolated in 38% yield 
as an inseparable mixture of several isomers, with a cis/trans 
cyclopentanes ratio of 90:10 (Scheme 4).
The structures of novel compounds were confirmed 
by spectroscopic methods (
1
H and 
13
C NMR, IR, and 
high-resolution mass spectrometry) and by elemental 
analyses for C, H, and N. Figure 2 shows the most typical 
proton shifts and multiplicities of compounds with substi-
tuted cyclopentene or cyclopentane structural motif. The 
germinal protons of the exocyclic alkene of compound 11 
appear at 4.72 and 4.74 ppm as two triplets with a coupling 
constant of 2.2 and 2.5 Hz. The methyl groups of the cyclo-
pentene moiety appear as singlet at 0.72 and 0.95 ppm. Af-
ter acid-catalyzed rearrangement of the double bond and 
methyl group migration, a tetrasubstituted endocyclic 
bond is formed as shown in compound 20. The two germi-
nal methyl groups appear as singlet at 0.86 and 0.87 ppm, 
Scheme 2. N-Boc deprotection – synthesis of ammonium salts 13–15.

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Scheme 3. N-Boc protection and acetylation – synthesis of compounds 19–24.

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while the methyl group on the endocyclic double bond ap-
pears as triplet at 1.33 ppm (J = 2.1 Hz). Similar chemical 
shifts and multiplicities are found for the related com-
pounds 14, 15, 21–24. Finally, catalytic hydrogenation of 
either the exocyclic or endocyclic double bond leads to 
substituted cyclopentane, yielding two diastereomers (see 
Scheme 4). As in compound cis-29 (the major diastere-
omer), the two germinal methyl groups appear as singlet at 
0.51 and 0.86 ppm, while the third methyl group appears 
as doublet at 0.83 ppm (J = 6.8 Hz). A very similar pattern 
of methyl groups is also observed for compound cis-27 
(Figure 2). The proton spectra of the compounds contain-
ing a substituted cyclopentene/cyclopentane structural 
motif are consistent with the previously reported com-
pounds containing the same structural elements.
22
All synthesized compounds were tested for inhibito-
ry activity on human (h)BChE and murine (m)AChE (Ta-
ble 1). Compounds 13, 20, 21, and 24 showed selective 
submicromolar inhibition of hBChE, with compounds 13 
(IC
50
 = 617 nM) and 21 (IC
50
 = 501 nM) being the most 
potent inhibitors of the series.
4. Conclusion
We report on 18 new compounds with indole 
structural motif that were synthesized and fully charac-
terized. Additionally, inhibitory potencies of the synthe-
sized compounds against hBChE (human butyrylcho-
linesterase) and mAChE (murine acetylcholinesterase) 
Scheme 4. Reduction of alkenes 23, 25, 26 – synthesis of com-
pounds 27–30.

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Figure 2. Representative proton shifts and multiplicities of products with substituted cyclopentene and cyclopentane motif.
Table 1. In vitro ChE inhibition.
Entry Compound hBChE mAChE
  RA at 100 µM [% ± SD] or
  IC
50
 [nM] ±SEM
a 
1  1687.6 ± 126.7 71.3 ± 8.7%
   Not active
2  7653.3 ± 1141.1 87.4 ± 18.0%
   Not active
3  54.8 ± 8.5% 40981.3 ± 14812.5
  Not active 
4 11 50.8 ± 2.7% 11512.1 ± 2469.4
  Not active 
5  9733.3 ± 2037.7 52.9 ± 8.5%
   Not active

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Entry Compound hBChE mAChE
  RA at 100 µM [% ± SD] or
  IC
50
 [nM] ±SEM
a 
6  617.3 ± 11.0 99.1 ± 6.2%
   Not active
7  1004.5 ± 103.2 81009.0 ± 9059.0
8  1151.9 ± 150.1 72.3 ± 10.2%
   Not active
9  4175.6 ± 245.1 96.7 ± 6.2%
   Not active
10  988.4 ± 111.2 86.7 ± 4.5%
   Not active
11  501.1 ± 46.7 51.4 ± 2.2%
   Not active
12  3588.9 ± 715.5 35458.6 ± 7236.4

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Entry Compound hBChE mAChE
  RA at 100 µM [% ± SD] or
  IC
50
 [nM] ±SEM
a 
13  42.6 ± 11.7% 53.5 ± 0.6%
  Not active Not active
14  952.5 ± 228.5 67.6 ± 13.7%
   Not active
15
b 
 1888.7 ± 123.2 7479.9 ± 2297.0
16
b 
 49.3 ± 13.5% 12625.0 ± 3926.5
  Not active
 
17
b 
 2948.9 ± 653.3 21002.0 ± 4815.0

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was determined by the method of Ellman. The highest 
selective submicromolar inhibition of hBChE was 
achieved with compounds 13 (IC
50
 = 617 nM) and 21 
(IC
50
 = 501 nM).
Supplementary Material
Copies of 
1
H and 
13
C NMR and MS spectra of the 
products are presented in the supporting information.
Acknowledgement
This research was funded by the Slovenian Research 
and Innovation Agency (ARIS), Research Core Funding 
No. P1-0179, P1-0208 and L1-8157.
Conflicts of interest
There are no conflicts to declare.
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Entry Compound hBChE mAChE
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 [nM] ±SEM
a 
18
b,c 
 16179.7 ± 1108.0 84.7 ± 1.5%
   Not active
a 
RA – residual activity expressed as percentage ± standard deviation (SD) of one independent 
measurement performed in triplicate, SEM – standard error of the mean, IC
50
 values are average 
of two independent measurements; 
b 
prepared as an inseparable cis/trans-mixture; 
c 
obtained as 
a mixture of diastereomers.

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Povzetek
V članku je opisana sinteza in antiholinesterazna aktivnost 18 doslej neobjavljenih spojin, derivatov indola in triptofana. 
Spojine, ki vsebujejo indolni strukturni fragment, izkazujejo selektivno submikromolarno zaviranje človeške butirilholin 
esteraze (hBChE). Strukture na novo sintetiziranih spojin so bile potrjene z 
1
H in 
13
C NMR, IR spektroskopijo in masno 
spektrometrijo visoke ločljivosti.
Except when otherwise noted, articles in this journal are published under the terms and conditions of the  
Creative Commons Attribution 4.0 International License
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