Tetrahydroisoquinoline-Based Non-Peptidomimetic Plasmepsin Inhibitors

Article abstract of DOI:10.1007/s10593-020-02623-6

[Figure not available: see fulltext.] A series of tetrahydroisoquinoline derivatives containing different aryl substituents were designed and synthesized using Pictet–Spengler reaction as the key step. The synthesized tetrahydroisoquinoline derivatives di

Full text of DOI:10.1007/s10593-020-02623-6

DOI 10.1007/s10593-020-02623-6
Chemistry of Heterocyclic Compounds 2020, 56(1), 60–66
Tetrahydroisoquinoline-based non-peptidomimetic
plasmepsin inhibitors
Linda Kinena1*, Vita Ozola1
1 Latvian Institute of Organic Synthesis,
21 Aizkraukles St., Riga LV-1006, Latvia; е-mail: linda@osi.lv
Published in Khimiya Geterotsiklicheskikh Soedinenii,
2020, 56(1), 60–66
Submitted December 13, 2019
Accepted December 20, 2019
A series of tetrahydroisoquinoline derivatives containing different aryl substituents were designed and synthesized using Pictet–Spengler
reaction as the key step. The synthesized tetrahydroisoquinoline derivatives displayed micromolar inhibitory activity against plasmepsins I and II.
Keywords: plasmepsins, tetrahydroisoquinoline, malaria, Pictet–Spengler reaction, X-ray analysis.
Malaria is a potentially life-threatening disease caused
by Plasmodium falciparum parasite. It is transmitted
through the bite of an infected mosquito. The increasing
resistance of the malaria parasite to currently available
drugs requires urgent development of new antimalarial
agents.1–3 Malarial aspartic proteases – plasmepsins (Plm I,
Plm II, Plm IV) are involved in hemoglobin degradation
and are potential drug targets.4,5 There are two kinds of
plasmepsin inhibitors: peptidomimetic and non-peptido-
mimetic inhibitors. Peptidomimetic plasmepsin inhibitors
usually show high inhibitory activity, but low selectivity
against human aspartic proteases.6 Several studies have
been performed to discover new non-peptidomimetic
plasmepsin inhibitors in order to overcome selectivity
issues.7–9 Scientists from Actelion have published first non-
peptidomimetic Plm II inhibitor A based on the amino-
piperidine scaffold (Fig. 1). Aminopiperidine-based inhibi-
tors have several drawbacks – they display adverse
physicochemical properties such as high ClogP and low
solubility.10,11 Therefore, we decided to design a new series
of inhibitors by rescaffolding Actelion aminopiperidine-
type inhibitor A (Fig. 1).
H-bonding interaction with the catalytic Asp 34), biphenyl
substituent (occupies S1 subpocket), and n-pentyl chain
(placed in the flap pocket) (Fig. 2).12
A series of tetrahydroisoquinoline derivatives B were
designed by introducing bond between C-3 carbon atom of
aromatic ring and C-3 carbon atom of piperidine and
opening piperidine cycle. According to the docking studies,
the newly designed tetrahydroisoquinoline inhibitor B
binds to the enzyme similarly to aminopiperidine derivative
A. Our design retains the key pharmacophoric elements
needed for inhibitory activity: amino moiety (makes ionic
interaction with Asp 214 residue and water-bridged
Figure 1. Design strategy of tetrahydroisoquinoline-based inhibi-
tors B by rescaffolding Actelion aminopiperidine-type inhibitors A.
0009-3122/20/56(1)-0060©2020 Springer Science+Business Media, LLC
60

Chemistry of Heterocyclic Compounds 2020, 56(1), 60–66
Figure 3. The molecular (a) and chemical (b) structure of methyl
ester (R)-6a with atoms represented by thermal vibration ellip-
soids of 50% probability.
After hydrolysis and deprotection of amino function in
compound 5a, acid 6 was obtained (Scheme 2). To
determine the absolute configuration of carboxylic acid
(R)-6 it was converted to methyl ester (R)-6a. Single crystal
X-ray analysis of methyl ester (R)-6a confirmed
(R) absolute configuration of stereogenic center (Fig. 3).
For further modifications, only acid (R)-6 was used.
Next, amino function was protected with Boc moiety,15
yielding tetrahydroisoquinoline which was reacted with
diethylamine in the presence of HOBt leading to
tetrahydroisoquinoline derivative (R)-7 (Scheme 2). After
cleavage of the N-Boc protecting group, the amide moiety
was reduced with LiAlH4 to give amine. The latter was
acylated with 4-pentylbenzoyl chloride in the presence of
DIPEA yielding tetrahydroisoquinoline (R)-8. This was
used as the key building block. Suzuki–Miyaura cross-
coupling reaction between bromide (R)-8 and boronic acids
in the presence of Pd(PPh3)4 was used for the synthesis of
diaryl derivatives (R)-9a,b. Demethylation of tetrahydro-
isoquinoline derivative (R)-9b was accomplished with
NaH/1-dodecanethiol in DMF16 to yield hydroxy group-
containing derivative (R)-9c. Buchwald–Hartwig amination
reaction17 was used for the introduction of 4-methoxy-
phenylamino substituent yielding tetrahydroisoquinoline
derivative (R)-10 (Scheme 2).
Figure 2. Binding mode of tetrahydroisoquinoline-based inhibitor
B to the active site of Plm II. Docking was performed using the
AutoDock Vina software package18 on the crystal structure of
Plm II solved in complex with an aminopiperidine-based Plm II
inhibitor (Protein Data Bank ID 2IGX11).
A small series of tetrahydroisoquinoline derivatives
(R)-9a–c, (R)-10 with different aryl substituents was prepared
to evaluate the inhibitory activity against Plm I, Plm II, and
Plm IV isoforms. Tetrahydroisoquinoline-based inhibitors
(R)-9a–c, (R)-10 were synthesized from ester 4 and
paraformaldehyde using Pictet–Spengler reaction as the
key step. The synthesis was started by C-alkylation of diethyl
2-acetamidomalonate with meta-bromobenzyl bromide (1)
in presence of NaOEt.13 Subsequent hydrolysis of ester and
deprotection of the amino group in compound 2 was
followed by decarboxylation to afford amino acid 3.13 The
Pictet–Spengler reaction involving protected amino acid 4
and paraformaldehyde14 resulted in the formation of tetra-
hydroisoquinoline derivative as a mixture of regioisomers
5a and 5b. Enantiomerically pure tetrahydroisoquinoline
(R)-5a was obtained by initial separation of regioisomers
by preparative column chromatography, followed by separa-
tion of enantiomers on a chiral stationary phase (Scheme 1).
Scheme 1
61

Chemistry of Heterocyclic Compounds 2020, 56(1), 60–66
Scheme 2
O
NEt2
O
O
O
Br
Br
Br
OH
R
OMe
OEt
i
iii
ii
NH2
Cl–
N
Ot-Bu
N
44%
81%
84%
(R)-6
(R)-7
(R)-5a
O
NEt2
O
NEt2
C5H11
Br
C5H11
iv
N
N
(R)-8
O
(R)-9a (47%)
(R)-9b (49%)
(R)-9c
vi
55%
v
58%
NEt2
O
H
N
C5H11
N
MeO
(R)-10
i: 33% HBr in AcOH, rt, 84 h, then aq 6 M HCl, 70°C, 18 h
ii: (Boc)2O, NaOH, t-BuOH–H2O, 1:1, rt, 18 h, then Et2NH, DCC, HOBt·H2O, DMF, rt, 16 h
iii: 4 M HCl in dioxane, rt, 16 h, then LiAlH4, THF, rt, 2 h, then 4-pentylbenzoyl chloride, DIPEA, CH2Cl2, rt, 16 h
iv: RB(OH)2, Pd(PPh3)4 (3 mol %), aq 2 M Na2CO3, 1,4-dioxane, 105°C, 16 h
v: 1-dodecanethiol, NaH, DMF, 0°C, then 130°C, 2 h
vi: 4-methoxyaniline, Pd2(dba)3 (2 mol %), X-Phos (8 mol %), NaOt-Bu, PhMe, 90°C, 16 h
The synthesized tetrahydroisoquinoline derivatives
In summary, we have developed a new series of tetra-
hydroisoquinoline-based non-peptidomimetic Plm inhibitors.
Synthesis of the inhibitors was performed using Pictet–
Spengler reaction as the key step. Plm I, Plm II, and Plm IV
inhibiting properties of the synthesized tetrahydroiso-
quinoline derivatives were tested. The best tetrahydroiso-
quinoline derivatives show inhibitory activity toward plas-
mepsins I and II at micromolar level. However, inhibition
potency of tetrahydroisoquinoline derivatives is lower than
Actelion aminopiperidine-type inhibitor. Hence, the per-
formed rescaffolding does not allow the molecule to adopt
the bioactive conformation in the active site of enzyme.
(R)-9a–c, (R)-10 were tested for their Plm I, Plm II, and
Plm IV inhibiting properties (Table 1). Tetrahydroiso-
quinoline 9a displayed micromolar inhibitory activity
against Plm I, but hardly any activity against Plm II and
Plm IV was observed. Inhibitor 9b with p-OMe substituent
at the phenyl ring showed the highest activity against Plm I,
Plm II, and Plm IV. In contrast, p-OH-substituted and amino-
linker-containing inhibitors 9c and 10 showed 2–3 times
lower inhibitory activity against Plm I, Plm II, Plm IV
(Table 1).
Table 1. Plm I, Plm II, and Plm IV inhibitory activity
of compounds 9a–c, 10, A
Experimental
1H and 13C NMR spectra (400 and 100 MHz, respec-
tively) were recorded on a Bruker 400 spectrometer in
CDCl3, DMSO-d6, or CD3OD; TMS was used as internal
standard. HRMS (ESI) spectra were recorded on a Waters
Acquity UPLC H-Class apparatus with a time-of-flight
(TOF) mass analyzer Waters Synapt G2 Si TOF MS.
Analytical thin-layer chromatography (TLC) was performed
on precoated Merck silica gel F-254 plates.
IC50*, µM
Com-
R
pound
Plm I
22
Plm II
Plm IV
~100
–**
9a
1.9 0.09
7.3 0.3
16.2 0.8
46.6
2
9b
9c
Unless otherwise noted, all chemicals were used as
obtained from commercial sources and all reactions were
performed under nitrogen or argon atmosphere in glass-
ware dried in an oven (120°C) and cooled under a stream
of argon. Dry PhMe, THF, CH2Cl2, and Et2O were
obtained by passing commercially available anhydrous
solvents through activated alumina columns.
Diethyl (acetylamino)(3-bromobenzyl)propanedioate
(2). A pressure tube was charged with diethyl 2-acet-
amidomalonate (1.74 g, 8.00 mmol) and NaOEt (2.59 g,
8.00 mmol), then anhydrous EtOH (10 ml) was added and
light-yellow suspension formed. A solution of m-bromo-
40
2
~100
7
0.3
0.4
45.5
2
73.6
0.92
3
10
A***
0.42
* Plm I, Plm II, and Plm IV inhibitory activity was determined by enzy-
matic FRET assay in triplicate experiments.
** The assay was performed at five concentrations of the inhibitor (0.01–
100 µM). At this range of concentrations inhibitor 9a did not show any effect.
*** Reference compound, see Figure 1.
62

Chemistry of Heterocyclic Compounds 2020, 56(1), 60–66
benzyl bromide (1) (2.00 g, 8.00 mmol) in anhydrous
(3H, s, CH3); 3.15–2.98 (2H, m, CH2CH); 1.23 (3H, t,
J = 7.1, CH2CH3). 13C NMR spectrum (CDCl3), δ, ppm:
171.9; 155.9; 138.4; 132.5; 130.4; 130.2; 128.0; 122.7;
61.4; 54.7; 52.5; 38.1; 14.7. Found, m/z: 330.0331 [M+H]+.
C13H17BrNO4. Calculated, m/z: 330.0341.
EtOH (10 ml) was added, and the resulting orange solution
was heated at 85°C for 16 h. After cooling to room
temperature, orange suspension was concentrated under
reduced pressure and the residue was partitioned between
EtOAc (50 ml) and H2O (50 ml). Water phase was
extracted with EtOAc (3×50 ml), combined extracts were
washed with brine, dried over Na2SO4, and evaporated
under reduced pressure. The residue was purified by
column chromatography on silica gel (100 g), eluent
hexane–EtOAc, gradient from 10 to 100% EtOAc. Yield
2.76 g (89%), light-yellow solid. Analytical data of the
compound were identical to the literature data.13
2-Ethyl 3-methyl (R)-6-bromo-3,4-dihydroisoquinoline-
2,3(1H)-dicarboxylate (5a) and 2-ethyl 3-methyl 8-bromo-
3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate (5b).
AcOH (7.4 ml), H2SO4 (2.5 ml), and paraformaldehyde
(198 mg, 6.60 mmol) were added to methyl
3-(3-bromophenyl)-2-[(ethoxycarbonyl)amino]propanoate (4)
(1.98 g, 6.00 mmol), and the resulting light-brown
suspension was stirred at room temperature for 20 h, then the
second portion of paraformaldehyde (198 mg, 6.60 mmol)
was added and stirring was continued for 20 h more. The
light-brown solution was poured into ice water (25 ml). After
20 min, the suspension was extracted with EtOAc (3×50 ml).
The combined extracts were washed with saturated
NaHCO3 solution (2×50 ml), brine (50 ml), dried over
Na2SO4, and evaporated under reduced pressure. The
residue was purified by column chromatography on silica
gel (100 g), eluent hexane–EtOAc, gradient from 25 to
100% EtOAc. The crude product was obtained as a mixture
of regioisomers 5a:5b in ratio 2.9:1. The regioisomers 5a
and 5b were separated by preparative chromatography
(Sunfire™ Prep Silica OBD™ 5μm, 30 × 100 column),
eluent hexane–EtOAc, 9:1, flow rate 40 ml/min, detector
UV 230 nm, 260 nm. Yield of compound 5a 717 mg
(35%), yield of compound 5b 247 mg (12%). Enantio-
merically pure material (R)-5a was obtained by preparative
HPLC on chiral stationary phase (DAICEL, Chiralpak-IC),
eluent hexane–EtOAc, 5:1, flow rate 36 ml/min, detector
UV 230 nm, 260 nm. Yield of compound (R)-5a 351 mg
2-Amino-3-(3-bromophenyl)propanoic acid hydro-
chloride (3). Diethyl (acetylamino)(3-bromobenzyl)propane-
dioate (2) (2.95 g, 7.66 mmol) was dissolved in a mixture
of concentrated AcOH (7.6 ml) and concentrated aqueous
HCl (23 ml). The light-yellow solution was heated at 100°C
for 18 h. The light-brown suspension was concentrated
under reduced pressure, the precipitate was filtered off and
washed with petroleum ether, dried over P2O5 at 60°C for
18 h. The product was used in a subsequent step without
purification. Yield 1.75 g (82%), light-brown solid.
1H NMR spectrum (DMSO-d6), δ, ppm: 14.22–13.52 (1H,
+
br. s, COOH); 8.78–8.32 (3H, m, NH3 ); 7.56–7.51 (1H, m,
H Ar); 7.51–7.45 (1H, m, H Ar); 7.35–7.27 (2H, m, H Ar);
4.25–4.13 (1H, m, CH2CH); 3.16 (2H, d, J = 6.3, CH2CH).
13C NMR spectrum (DMSO-d6), δ, ppm: 170.1; 137.9; 132.3;
130.6; 130.1; 128.7; 121.8; 52.9; 35.1. Found, m/z: 243.9971
[M+H]+. C9H11BrNO2. Calculated, m/z: 243.9973.
Methyl 3-(3-bromophenyl)-2-[(ethoxycarbonyl)amino]-
propanoate (4). SOCl2 (1.13 ml, 15.66 mmol) was added
to cooled (0°C) anhydrous MeOH (20 ml), and the
resulting solution was stirred for 10 min. Hydrochloride 3
(1.75 g, 6.27 mmol) was then added in portions, and the
light-brown solution was stirred at 60°C for 3 h. The
volatiles were removed under reduced pressure, and
anhydrous PhMe (2×20 ml) was added and evaporated
under reduced pressure. The precipitate was dried over
P2O5 at 40°C and used in subsequent step without purifi-
cation. Yield 1.82 g (98%), light-brown powder. Pyridine
(1.50 ml, 18.54 mmol) was added dropwise to a solution of
methyl 2-amino-3-(3-bromophenyl)propanoate hydro-
chloride from the previous step (1.82 g, 6.18 mmol) in
anhydrous CH2Cl2 (20 ml). The light-brown solution was
cooled to 0°C, and ethyl chloroformate (737 mg, 649 µl,
6.80 mmol) was added dropwise. After stirring at room
temperature for 18 h, the light-brown suspension was
concentrated under reduced pressure. The residue was
dissolved in EtOAc (50 ml), washed with aqueous 1 M HCl
solution (30 ml), saturated aqueous NaHCO3 solution
(50 ml), and brine (30 ml). The organic layer was
evaporated under reduced pressure, and product 4 was used
in a subsequent step without purification. Yield 1.98 g
(96%), light-brown oil. 1H NMR spectrum (CDCl3),
δ, ppm: 7.38 (1H, ddd, J = 7.9, J = 2.0, J = 1.1, H Ar); 7.30–
7.26 (1H, m, H Ar); 7.16 (1H, dd, J = 7.9, J = 7.9, H Ar);
7.08–7.04 (1H, m, H Ar); 5.15 (1H, d, J = 8.2, NH); 4.68–
4.56 (1H, m, CH2CH); 4.11 (2H, q, J = 7.1, CH2CH3); 3.72
20
(17%), light-yellow oil, mixture of rotamers, [α]D –45.7
(c 1.03, CHCl3).
1
Compound 5a. H NMR spectrum (CDCl3), δ, ppm:
7.36–7.28 (2H, m, H Ar); 7.02 (0.45H, d, J = 8.0, H Ar);
6.98 (0.55H, d, J = 8.0, H Ar); 5.18 (0.55H, dd, J = 6.1,
J = 2.9, CH); 4.96 (0.45H, dd, J = 6.1, J = 4.2, CH); 4.77–
4.67 (1H, m, CH2N); 4.56–4.42 (1H, m, CH2N); 4.32–4.13
(2H, m, CH2CH3); 3.63 (3H, s, OCH3); 3.33–3.06 (2H, m,
CH2CH); 1.33 (1.65H, t, J = 7.1, CH2CH3); 1.26 (1.35H, t,
J = 7.1, CH2CH3). 13C NMR spectrum (CDCl3), δ, ppm:
171.7; 171.5; 156.3; 155.7; 134.0; 132.2; 131.6; 131.5;
131.1; 130.2; 130.1; 128.2; 128.0; 120.5; 62.2; 62.1; 53.3;
52.6; 44.1; 44.0; 31.3; 31.0; 14.8; 14.7. Found, m/z:
342.0327 [M+H]+.C141H17BrNO4. Calculated, m/z: 342.0341.
Compound 5b. H NMR spectrum (CDCl3), δ, ppm:
7.46–7.40 (1H, m, H Ar); 7.14–7.08 (1H, m, H Ar); 7.08–
7.02 (1H, m, H Ar); 5.25 (0.6H, dd, J = 6.3, J = 2.5, CH);
5.06 (0.4H, dd, J = 6.3, J = 3.2, CH); 4.86 (0.4H, d,
J = 17.6, CH2N); 4.79 (0.6H, d, J = 17.6, CH2N); 4.45
(0.6H, d, J = 17.6, CH2N); 4.43 (0.4H, d, J = 17.6, CH2N);
4.31–4.16 (2H, m, CH2CH3); 3.64 (3H, s, OCH3); 3.33–
3.13 (2H, m, CH2CH); 1.35 (1.8H, t, J = 7.1, CH2CH3);
1.28 (1.2H, t, J = 7.1, CH2CH3). 13C NMR spectrum
(CDCl3), δ, ppm: 171.5; 171.4; 156.4; 134.1; 133.9; 132.3;
131.8; 131.2; 131.1; 128.1; 128.0; 127.6; 122.6; 122.5;
62.3; 62.1; 53.1; 52.6; 52.4; 45.2; 31.6; 31.2; 14.8; 14.7.
63

Chemistry of Heterocyclic Compounds 2020, 56(1), 60–66
(R)-6-Bromo-1,2,3,4-tetrahydroisoquinoline-3-carboxy-
graphy on silica gel (25 g), eluent hexane–EtOAc, gradient
from 15 to 85% EtOAc. Yield 250 mg (84%), light-yellow
lic acid hydrochloride (6). 2-Ethyl 3-methyl (R)-6-bromo-
3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate (5a) (351 mg,
1.03 mmol) was dissolved in 33% HBr in AcOH (4.1 ml,
24.6 mmol) and the orange solution was stirred at room
temperature for 84 h. Volatiles were removed under
reduced pressure, and the residue was treated with aqueous
6 M HCl solution (10 ml). The light-brown suspension was
stirred at 70°C for 18 h. The formed precipitate was filtered
off, dried over P2O5 at 50°C. Yield 244 mg (81%), light-
20
oil, mixture of rotamers, [α]D 27.9 (c 0.86, CHCl3).
1H NMR spectrum (CDCl3), δ, ppm: 7.33–7.26 (2H, m,
H Ar); 7.04–6.91 (1H, m, H Ar); 5.33–5.24 (0.6H, m,
CH2CH); 5.00–4.84 (0.8H, m, CH2N, CH2CH); 4.78 (0.6H,
d, J = 16.6, CH2N); 4.35 (0.6H, d, J = 16.6, CH2N); 4.25
(0.4H, d, J = 16.2, CH2); 3.56–3.16 (4H, m, (CH2CH3)2);
3.14–2.92 (2H, m, CH2CH); 1.47 (9H, s, C(CH3)3); 1.31–
1.19 (3H, m, CH2CH3); 1.14–1.01 (3H, m, CH2CH3).
13C NMR spectrum (CDCl3), δ, ppm: 170.3; 154.7; 135.5;
135.3; 132.6; 131.2; 130.6; 129.6, 129.3; 127.7; 127.4;
120.4; 120.2; 80.9; 51.5; 49.5; 44.3; 43.7; 42.0; 40.6; 31.4;
30.8; 28.6; 14.6; 13.0. Found, m/z: 433.1086 [M+Na]+.
C19H27BrNaN2O3. Calculated, m/z: 433.1103.
20
brown solid, [α]D 66.5 (c 1.05, MeOH). 1H NMR
spectrum (CD3OD), δ, ppm: 7.55–7.50 (1H, m, H Ar); 7.46
(1H, dd, J = 8.3, J = 2.1, H Ar); 7.20 (1H, d, J = 8.3, H Ar);
4.50–4.34 (3H, m, CH2N, CH2CH); 3.48 (1H, dd, J = 17.4,
J = 5.3, CH2CH); 3.23 (1H, dd, J = 17.4, J = 11.6, CH2CH).
13C NMR spectrum (CD3OD), δ, ppm: 170.7; 134.2; 132.9;
131.7; 129.6; 128.1; 122.9; 55.0; 45.2; 29.3. Found, m/z:
255.9970 [M+H]+. C10H11BrNO2. Calculated, m/z: 255.9973.
Methyl (R)-6-bromo-1,2,3,4-tetrahydroisoquinoline-
3-carboxylate hydrochloride (6a). SOCl2 (12 ml,
0.17 mmol) was added to the cooled (0°C) anhydrous
MeOH (1 ml), and resulting solution was stirred for
10 min. Hydrochloride 6 (20 mg, 0.068 mmol) was then
added, and the light-brown solution was stirred at 60°C for
3 h. The volatiles were removed under reduced pressure,
and anhydrous PhMe (2×1 ml) was added and evaporated
under reduced pressure. The precipitate was dried over
P2O5 at 40°C. Yield 20 mg (95%), light-brown powder,
(R)-{6-Bromo-3-[(diethylamino)methyl]-3,4-dihydroiso-
quinolin-2(1H)-yl}(4-pentylphenyl)methanone (8). 4 M HCl
in 1,4-dioxane (1.52 ml, 6.10 mmol) was added dropwise
to a solution of amide (R)-7 (251 mg, 0.61 mmol) in anhydrous
1,4-dioxane (4 ml). The light-yellow solution was stirred at
room temperature for 16 h, then solvent was evaporated
under reduced pressure to give unprotected tetrahydroiso-
quinoline (207 mg, 98%) which was used in the next step
without purification. LiAlH4 (2.4 M solution in THF,
496 µl, 1.19 mmol) was added dropwise to a cooled
solution (0°C) of crude product from previous step
(207 mg, 0.59 mmol) in anhydrous THF (6 ml). After
stirring at room temperature for 2 h, the light-yellow
solution was cooled to 0°C and quenched by sequential
(within intervals of 10 min) addition of H2O (45 µl),
aqueous 4 M NaOH solution (90 µl), and more H2O
(135 µl). Ten minutes after addition of the final amount of
H2O, the white suspension was filtered. The filter cake was
washed with EtOAc (20 ml). The filtrate was evaporated to
dryness to yield 162 mg (92%) of (R)-N-[(6-bromo-1,2,3,4-
tetrahydroisoquinolin-3-yl)methyl]-N-ethylethanamine as a
light-yellow oil, which was used in subsequent step without
purification. DIPEA (283 µl, 211 mg, 1.64 mmol) was
added to a cooled (0°C) solution of amine from previous
step (162 mg, 0.54 mmol) in anhydrous CH2Cl2 (7 ml)
followed by 4-pentylbenzoyl chloride (122 µl, 126 mg,
0.60 mmol). The light-yellow solution was stirred at room
temperature for 16 h, then evaporated under reduced
pressure. The residue was partitioned between EtOAc
(10 ml) and H2O (10 ml) and extracted with EtOAc
(3×10 ml). The combined extracts were washed with brine,
dried over Na2SO4, and evaporated under reduced pressure.
The residue was purified by column chromatography on
silica gel (15 g), eluent hexane–EtOAc, gradient from 10 to
100% EtOAc. Yield 113 mg (44% in three steps), light-
20
1
[α]D 55.2 (c 0.55, MeOH). H NMR spectrum (CD3OD),
δ, ppm: 7.55–7.49 (1H, m, H Ar); 7.47 (1H, dd, J = 8.3,
J = 2.0, H Ar); 7.20 (1H, d, J = 8.3, H Ar); 4.56–4.34 (3H,
m, CH2N, CH2CH); 3.91 (3H, s, OCH3); 3.47 (1H, dd,
J = 17.4, J = 5.2, CH2CH); 3.28–3.17 (1H, m, CH2CH).
13C NMR spectrum (CD3OD), δ, ppm: 169.9; 133.8; 132.9;
131.7; 129.6; 128.0; 122.9; 55.1; 54.0; 45.3; 29.1. Found, m/z:
270.0141 [M+H]+. C11H13BrNO2. Calculated, m/z: 270.0130.
tert-Butyl (R)-6-bromo-3-(diethylcarbamoyl)-3,4-di-
hydroisoquinoline-2(1H)-carboxylate (7). NaOH (70 mg,
1.76 mmol) was added to a suspension of hydrochloride 6
(215 mg, 0.84 mmol) in t-BuOH–H2O, 1:1 (6 ml). The
reaction mixture was stirred till clear solution was formed,
then Boc2O (183 mg, 0.84 mmol) was added. The light-
yellow solution was stirred at room temperature for 16 h.
The solution was evaporated under reduced pressure to 1/3
of volume, then acidified with aqueous 5% KHSO4 solu-
tion to pH 3 and extracted with EtOAc (3×15 ml). The
combined extracts were washed with brine (20 ml), dried
over Na2SO4, and evaporated under reduced pressure.
The crude product (258 mg, 0.72 mmol) was dissolved
in anhydrous DMF (4 ml) and HOBt·H2O (144 mg,
0.94 mmol) was added, followed by DCC (194 mg,
0.94 mmol). The resulting light-yellow solution was stirred
at 0°C for 1 h, then diethylamine (79 mg, 112 µl, 1.09 mmol)
was added and stirring was continued at room temperature
for 16 h. The light-yellow solution was diluted with H2O
(10 ml) and extracted with EtOAc (3 × 10 ml). The
combined extracts were washed with H2O (15 ml), brine
(15 ml), dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by column chromato-
20
yellow oil, mixture of rotamers, [α]D 10.2 (c 0.98,
1
CHCl3). H NMR spectrum (CDCl3), δ, ppm: 7.38–7.28
(4H, m, H Ar); 7.24–7.17 (2H, m, H Ar); 7.11–7.03 (1H,
m, H Ar); 5.23 (1H, d, J = 17.9, CH2N); 4.57–4.12 (2H, m,
CH2N, CH2CH); 3.20–2.97 (1H, m, CH2CH); 2.89–2.75 (1H,
m, CH2CH); 2.69–2.45 (4H, m, 1-CH2, CH2CH3); 2.44–
2.10 (4H, m, CHCH2NEt2, CH2CH3); 1.62 (2H, quint, J =
7.3, 2-CH2); 1.42–1.20 (4H, m, 3,4-CH2); 1.01–0.93 (3H,
m, CH2CH3); 0.89 (3H, t, J = 6.9, 5-CH3); 0.82–0.63 (3H,
64

Chemistry of Heterocyclic Compounds 2020, 56(1), 60–66
m, CH2CH3). 13C NMR spectrum (CDCl3), δ, ppm: 172.3;
(1H, m, CHCH2NEt2); 2.34–2.13 (3H, m, CHCH2NEt2,
CH2CH3); 1.63 (2H, quint, J = 7.4, 2-CH2); 1.41–1.23 (4H,
m, 3,4-CH2); 1.08–0.94 (3H, m, CH2CH3); 0.90 (3H, t,
J = 6.8, 5-CH3); 0.76 (3H, t, J = 7.1, CH2CH3). 13C NMR
spectrum (CDCl3), δ, ppm: 172.3; 159.3; 144.7; 139.3;
134.2; 133.5; 132.6; 130.8; 128.6; 128.1; 127.7; 127.0;
126.2; 125.0; 114.3; 55.5; 54.5; 52.1; 47.4; 47.2; 41.9;
35.9; 31.5; 31.1; 22.7; 14.2; 11.8. Found, m/z: 499.3322
[M+H]+. C33H43N2O2. Calculated, m/z: 499.3325.
144.9; 134.6; 133.9; 132.3; 131.4; 129.7; 128.6; 128.3;
126.9; 120.3; 54.3; 51.7; 47.3; 45.1; 41.7; 35.9; 31.5; 31.4;
31.1; 22.7; 14.2; 11.8. Found, m/z: 471.2007 [M+H]+.
C26H36BrN2O. Calculated, m/z: 471.2011.
(R)-{3-[(Diethylamino)methyl]-6-(pyridin-3-yl)-3,4-di-
hydroisoquinolin-2(1H)-yl}(4-pentylphenyl)methanone
(9a). A vial was charged with bromide (R)-8 (20 mg,
0.042 mmol), pyridin-3-ylboronic acid (7.8 mg, 0.064 mmol),
and Pd(PPh3)4 (1.5 mg, 0.0013 mmol), then 1,4-dioxane
(0.5 ml) was added, followed by aqueous 2 M Na2CO3
solution (42 µl, 0.084 mmol). After stirring at 105°C for
16 h, the orange suspension was cooled to room tempe-
rature and diluted with H2O (5 ml) and EtOAc (5 ml). The
organic layer was decanted, and the aqueous layer was
extracted with EtOAc (3×5 ml). The combined organic
layers were washed with brine, dried over Na2SO4, and
evaporated under reduced pressure. The residue was puri-
fied by column chromatography on silica gel (15 g), eluent
hexane–EtOAc, gradient from 50 to 100% EtOAc, then
EtOAc–MeOH, 9:1. Yield 9 mg (47%), light-yellow oil,
(R)-{3-[(Diethylamino)methyl]-6-(4-hydroxyphenyl)-
3,4-dihydroisoquinolin-2(1H)-yl)}(4-pentylphenyl)-
methanone (9c). A vial was charged with NaH (60%
suspension in mineral oil) (7.4 mg, 0.184 mmol) and
washed with anhydrous Et2O (3×1 ml), then anhydrous
DMF (1 ml) was added. The suspension was cooled to 0°C,
and 1-dodecanethiol (44 µl, 0.184 mmol) was added
dropwise (Caution! Gas evolution!). After stirring at room
temperature for 10 min, solution of isoquinoline derivative
(R)-(9b) (23 mg, 0.046 mmol) in anhydrous DMF (0.5 ml)
was added to the white suspension. The yellow solution was
stirred at 130°C for 2 h, then evaporated, and H2O (3 ml)
was added to the residue and extracted with EtOAc (3 × 3 ml).
The combined extracts were washed with H2O (7 ml), brine
(7 ml), dried, and evaporated. The mixture was purified by
column chromatography on silica gel (10 g), eluent
CH2Cl2, gradient to CH2Cl2–MeOH, 96:4. Yield 13 mg
20
mixture of rotamers, [α]D 7.0 (c 0.62, CHCl3). 1H NMR
spectrum (CD3OD), δ, ppm: 8.84–8.76 (1H, m, H Ar); 8.55–
8.47 (1H, m, H Ar); 8.15–8.04 (1H, m, H Ar); 7.59–7.48
(3H, m, H Ar); 7.46–7.36 (3H, m, H Ar); 7.36–7.28 (2H,
m, H Ar); 5.24 (1H, d, J = 18.5, CH2N); 4.65–4.24 (2H, m,
CH2N, CH2CH); 3.30–3.17 (1H, m, CH2CH); 3.07–2.94
(1H, m, CH2CH); 2.76–2.53 (4H, m, 1-CH2, CH2CH3);
2.52–2.13 (4H, m, CHCH2NEt2, CH2CH3); 1.66 (2H,
quintet, J = 7.5, 2-CH2); 1.45–1.23 (4H, m, 3,4-CH2); 1.03
(3H, t, J = 7.1, CH2CH3); 0.96–0.84 (3H, m, 5-CH3); 0.77
(3H, t, J = 7.1, CH2CH3). 13C NMR spectrum (CD3OD),
δ, ppm: 174.4; 148.8; 148.3; 138.2; 137.2; 136.4; 134.8;
133.5; 133.1; 133.0; 130.0; 129.9; 129.8; 129.2; 128.5;
128.1; 126.4; 125.5; 55.2; 53.6; 49.3; 48.5; 43.1; 36.7;
32.4; 32.3; 23.6; 14.4; 12.1. Found, m/z: 470.3164 [M+H]+.
C31H40N3O. Calculated, m/z: 470.3171.
20
(58%), light-yellow oil, [α]D 9.8 (c 1.16, CHCl3).
1H NMR spectrum (CDCl3), δ, ppm: 7.46–7.34 (4H, m,
H Ar); 7.34–7.17 (5H, m, H Ar); 7.00–6.82 (2H, m, H Ar);
5.32 (1H, d, J = 18.0, CH2N); 4.66–4.17 (2H, m, CH2N,
CH2CH); 3.31–3.06 (1H, m, CH2CH); 2.96–2.84 (1H, m,
CH2CH); 2.76–2.54 (4H, m, 1-CH2, CH2CH3); 2.50–2.37
(1H, m, CHCH2NEt2); 2.35–2.15 (3H, m, CHCH2NEt2,
CH2CH3); 1.71–1.54 (2H, m, 2-CH2); 1.44–1.23 (4H, m,
3,4-CH2); 1.12–0.96 (3H, m, CH2CH3); 0.90 (3H, t, J = 6.7,
5-CH3); 0.76 (3H, t, J = 6.9, CH2CH3). 13C NMR spectrum
(CDCl3), δ, ppm: 172.6; 156.0; 144.9; 139.5; 133.8; 133.0;
132.4; 130.5; 128.6; 128.2; 127.6; 127.3; 127.0; 126.2;
125.0; 116.0; 54.5; 52.2; 47.3; 42.0; 35.9; 31.5; 31.1; 22.7;
14.2; 11.7. Found, m/z: 514.3428 [M+H]+. C33H44N3O2.
Calculated, m/z: 514.3434.
(R)-{3-[(Diethylamino)methyl]-6-(4-methoxyphenyl)-
3,4-dihydroisoquinolin-2(1H)-yl}(4-pentylphenyl)-
methanone (9b). A vial was charged with bromide (R)-8
(50 mg, 0.106 mmol), (4-methoxyphenyl)boronic acid (24 mg,
0.159 mmol), and Pd(PPh3)4 (3.7 mg, 0.0032 mmol), then
1,4-dioxane (1 ml) was added, followed by aqueous 2 M
Na2CO3 solution (106 µl, 0.212 mmol). After stirring at
105°C for 16 h, the orange suspension was cooled to room
temperature and diluted with H2O (5 ml) and EtOAc
(5 ml). The organic layer was decanted, and the aqueous
layer was extracted with EtOAc (3×5 ml). The combined
organic layers were washed with brine, dried over Na2SO4,
and evaporated under reduced pressure. The residue was
purified by column chromatography on silica gel (15 g),
eluent hexane–EtOAc, gradient from 10 to 100% EtOAc.
(R)-{3-[(Diethylamino)methyl]-6-[(4-methoxyphenyl)-
amino]-3,4-dihydroisoquinolin-2(1H)-yl}(4-pentylphenyl)-
methanone (10). A vial was charged with Pd2(dba)3
(0.78 mg, 0.0009 mmol), X-Phos (0.81 mg, 0.0017 mmol),
then anhydrous PhMe (1 ml) was added and the solution
was heated to 60°C. After 10 min, bromide (R)-8 (20 mg,
0.042 mmol), 4-methoxyaniline (6.3 mg, 0.051 mmol), and
NaOt-Bu (5.7 mg, 0.059 mmol) were added. After stirring
at 90°C for 16 h, the yellow suspension was cooled to room
temperature and diluted with H2O (5 ml) and EtOAc
(5 ml). The organic layer was decanted, and the aqueous
layer was extracted with EtOAc (3×5 ml). The combined
organic layers were washed with brine, dried over Na2SO4,
and evaporated under reduced pressure. The residue was
purified by column chromatography on silica gel (15 g),
eluent hexane–EtOAc, gradient from 50 to 100% EtOAc.
20
Yield 26 mg (49%), light-yellow oil, [α]D 13.3 (c 1.03,
1
CHCl3). H NMR spectrum (CDCl3), δ, ppm: 7.54–7.46
(2H, m, H Ar); 7.44–7.28 (4H, m, H Ar); 7.25–7.19 (3H,
m, H Ar); 7.01–6.93 (2H, m, H Ar); 5.31 (1H, d, J = 18.9,
CH2N); 4.61–4.16 (2H, m, CH2N, CH2CH); 3.85 (3H, s,
OCH3); 3.29–3.07 (1H, m, CH2CH); 2.95–2.83 (1H, m,
CH2CH); 2.72–2.49 (4H, m, 1-CH2, CH2CH3); 2.47–2.37
20
Yield 12 mg (55%), light-yellow oil, [α]D 11.5 (c 0.81,
CHCl3). 1H NMR spectrum (CDCl3), δ, ppm: 7.40–7.33
65

Chemistry of Heterocyclic Compounds 2020, 56(1), 60–66
(2H, m, H Ar); 7.25–7.18 (2H, m, H Ar); 7.10–6.98 (3H,
Docking studies. Docking studies were performed using
m, H Ar); 6.90–6.83 (2H, m, H Ar); 6.82–6.66 (2H,
m, Ar); 5.43 (1H, s, NH); 5.19 (1H, d, J = 17.4, CH2N);
4.47–4.06 (2H, m, CH2N, CH2CH); 3.80 (3H, s, OCH3);
3.17–2.96 (1H, m, CH2CH); 2.72 (1H, d, J = 16.2,
CH2CH); 2.67–2.49 (4H, m, 1-CH2, CH2CH3); 2.44–2.33
(1H, m, CHCH2NEt2); 2.31–2.12 (3H, m, CHCH2NEt2,
CH2CH3); 1.71–1.54 (2H, m, 2-CH2); 1.44–1.19 (4H, m,
3,4-CH2); 1.03–0.94 (3H, m, CH2CH3); 0.93–0.86 (3H, m,
5-CH3); 0.75 (3H, t, J = 7.1, CH2CH3). 13C NMR spectrum
(CDCl3), δ, ppm: 172.2; 155.4; 144.6; 143.8; 136.1; 134.3;
133.2; 128.5; 127.6; 127.2; 127.0; 122.4; 122.1; 116.2;
114.8; 55.7; 54.6; 52.1; 47.4; 41.7; 35.9; 31.6; 31.2; 27.7;
22.7; 14.2; 11.9. Found, m/z: 514.3428 [M+H]+. C33H44N3O2.
Calculated, m/z: 514.3434.
the AutoDock Vina software package.18
This work was supported by the European Regional
Development Fund (agreement No. 1.1.1.1/16/A/290).
Authors thank Dr. S. Belyakov for X-ray crystallographic
analysis and Dr. I. Domraceva for IC50 measurements.
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Inhibitory activity assays of compounds 9a–c, 10.
A fluorescence resonance energy transfer (FRET) assay
was performed to evaluate the ability of compounds to
inhibit Plm I, Plm II, and Plm IV. Km of the substrate was
determined for each enzyme: Plm I – 2.7 0.3 µM, Plm II –
2
0.2 µM, Plm IV – 2.8 0.2 µM. A solution of each
compound for testing (concentration 0.01–100 µM) was
added to the enzyme (Plm I, Plm II, or Plm IV) in buffer
(0.1 M NaOAc, pH 4.5, 10% glycerol) on 96-well plate.
The mixture was incubated for 30 min at 37°C. Substrate
(DABCYL-Glu-Arg-Nle-Leu-Ser-Phe-Pro-EDANS, AnaSpec
Inc.) was then added to reach the final concentration of
5 µM. Hydrolysis of the substrate was detected as an
increase in fluorescence (Em 490 nm, Ex 336 nm) at 37°C.
The data points were collected every 1 min within 8–15 min.
For the rate calculation, only the linear interval was used,
which was slightly different for each enzyme. Compounds
were tested in triplicate experiments. IC50 values were
calculated using the software Graph Pad Prism 5.0.
Pepstatin A (IC50, nM: 0.42 0.02 (Plm II), 0.9 0.02
(Plm I), 0.3 0.04 (Plm IV)) and compound A were used
as positive control.
X-ray structural analysis of compound (R)-6a. Single
crystals of C11H13BrClNO2 were investigated on a Rigaku,
XtaLAB Synergy, Dualflex, HyPix diffractometer. The
crystal was kept at 150.0(1) K during data collection. Using
Olex2,19 the structure was solved with the ShelXT20
structure solution program using Intrinsic Phasing and
refined with the olex2.refine21 refinement package using
Levenberg–Marquardt minimization. Crystal data for
C11H13BrClNO2 (M 306.59 g/mol): triclinic, space group
P1; a 7.0731(4), b 7.9997(4), c 12.2609(4) Å; α 77.412(4),
β 74.682(4), γ 68.708(5)°; V 617.68(6) Å3; Z 1; μ(CuKα)
6.417 mm–1; dcalc 1.6483g/cm3. 16651 reflections measured
(2Θ ≤ 155.0°), 4805 unique (Rint 0.0475, Rsigma 0.0349)
which were used in all calculations. The final R1 was 0.0438
(I > 2σ(I)) and wR2 was 0.1318 (all data). The complete
crystallographic dataset was deposited at the Cambridge
Crystallographic Data Center (deposit CCDC 1953912).
21. Bourhis, L. J.; Dolomanov, O. V.; Gildea, R. J.; Howard, J. A. K.;
Puschmann, H. Acta Crystallogr., Sect. A.: Found. Adv. 2015,
A71, 59.
66