New derivatives of 3,4-dihydroisoquinoline-3-carboxylic acid with free-radical scavenging, d-amino acid oxidase, acetylcholinesterase and butyrylcholinesterase inhibitory activity

Molecules 2014, 19, 15866-15890; doi:10.3390/molecules191015866

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molecules

ISSN 1420-3049

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Article

New Derivatives of 3,4-Dihydroisoquinoline-3-carboxylic

Acid with Free-Radical Scavenging, D-Amino Acid Oxidase,

Acetylcholinesterase and Butyrylcholinesterase

Inhibitory Activity

Jolanta Solecka ^1,*, Adam Guśpiel ¹, Magdalena Postek ¹, Joanna Ziemska ¹, Robert Kawęcki ^2,*,

Katarzyna Łęczycka ², Agnieszka Osior ², Bartłomiej Pietrzak ², Krzysztof Pypowski ²and

Agata Wyrzykowska ²

1

National Institute of Public Health—National Institute of Hygiene, Chocimska 24, Warsaw 00-791,

Poland; E-Mails: aguspiel@pzh.gov.pl (A.G.); mpostek@pzh.gov.pl (M.P.);

jziemska@pzh.gov.pl (J.Z.)

2

Institute of Chemistry, Siedlce University, 3.Maja 54, Siedlce 80-110, Poland;

E-Mails: katarzyna.leczycka@icho.edu.pl (K.L.); agnieszka.osior@icho.edu.pl (A.O.);

bartlomiej.pietrzak@gmail.com (B.P.); pypowski@uph.edu.pl (K.P.);

agatawyrzykowska@gmail.com (A.W.)

* Authors to whom correspondence should be addressed; E-Mails: jsolecka@pzh.gov.pl (J.S.);

rkaw@uph.edu.pl (R.K.); Tel./Fax: +48-22-5421387 (J.S.); Tel.: +48-25-6431143 (R.K.).

External Editor: Derek J. McPhee

Received: 14 August 2014; in revised form: 17 September 2014 / Accepted: 22 September 2014 /

Published: 30 September 2014

Abstract: A series of 3,4-dihydroisoquinoline-3-carboxylic acid derivatives were synthesised

and tested for their free-radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl

radical (DPPH^·), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS^·+),

·−

superoxide anion radical (O₂) and nitric oxide radical (^·NO) assays. We also studied D-amino

acid oxidase (DAAO), acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)

inhibitory activity. Almost each of newly synthesised compounds exhibited radical scavenging

capabilities. Moreover, several compounds showed moderate inhibitory activities against

DAAO, AChE and BuChE. Compounds with significant free-radical scavenging activity

may be potential candidates for therapeutics used in oxidative-stress-related diseases.

Molecules 2014, 19

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Keywords: free radicals; scavenger; DAAO; AChE; BuChE; inhibitor; 3,4-dihydroisoquinoline

1. Introduction

3,4-Dihydroisoquinoline derivatives have several important biological properties and are

in the area of interest of the pharmaceutical industry. Published activities include antimetastatic [1],

anti-inflammatory and analgesic [2], antiarrhythmic, and antiaggregatory [3] antihypertensive [4], and

antibacterial [5]. They also inhibit catechol O-methyltransferase [6] and kinase JNK3 [7]. Most of

investigated 3,4-dihydroisoquinoline derivatives have substituents at C1.

We have recently described the isolation, synthesis and antimicrobial activity of 6,7-dihydroxy-3,4-

dihydroisoquinoline-3-carboxylic acid (5e), a C1-unsubstituted secondary metabolite of the

Streptomyces sp. 8812 fermentation process [8,9]. This prompted us to prepare a series of hydroxy- and

halogeno-substituted 3,4-dihydroisoquinoline-3-carboxylic acids and investigate their biological activity.

We also included in our investigation 2-oxo-1,4,5,9b-tetrahydro-2H-azeto[2,1-a]isoquinoline analogue,

as well as a 4,5-dihydro-3H-2-benzazepine analogue.

Isoquinoline alkaloid derivatives (benzyltetrahydroisoquinolines (±)-coclaurine and (±)-norarmepavine)

also present antioxidative properties [10]. Berberine, with widely demonstrated antioxidative activities,

exhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities,

as well [11].

Our main interests are concerned with the determination of antioxidative properties due to the fact

that phenolic compounds often present potent free radical scavenging activities. Several known, potent

free-radical scavengers such as flavonols (e.g., quercetin, kaempferol), flavones (e.g., luteolin, apigenin),

flavanols (e.g., catechin) and isoflavones (e.g., genistein) contain phenols and polyphenolic moiety [12].

Previous studies have shown that polyphenolic structures are more potent free-radical scavengers than

those with only one hydroxyl group [13]. Also, the ortho location of two hydroxyl groups increases the

antioxidative properties [13,14]. The free-radical scavenging potential of polyphenolic compounds is

associated with their reducing (hydrogen- or electron-donating) activities [12,15].

Oxidative stress is a process that arises when free radicals and oxidants are produced in excess and

cells cannot destroy them properly. In other words, an imbalance between the formation and neutralisation

of free radicals can occur, owing to a depletion of antioxidants or the accumulation of reactive oxygen

species (ROS), and can result in the appearance of oxidative stress [16–19]. The latter may lead to the

change of cell components such as proteins, lipids, lipoproteins, nucleic acids and so forth [18–20].

Oxidative stress plays a crucial role in the development of various diseases such as cardiovascular

disorders; atherosclerosis; hypertension or heart failure; diabetes mellitus; cancer; rheumatoid arthritis;

ocular diseases; neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease (AD), as well

as psychic impairments such as schizophrenia and is linked to aging [15–18].

The synthesised compounds were investigated for their free-radical scavenging activity against

2,2-diphenyl-1-picrylhydrazyl radical (DPPH^·), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

·−

radical (ABTS^·+), superoxide anion radical (O₂) and nitric oxide radical (^·NO). Additionally, we

evaluated compounds for some activities associated with oxidative stress–inhibitory activity against

Molecules 2014, 19

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D-amino acid oxidase (DAAO, a potential target in schizophrenia treatment) and for the inhibition of

two enzymes important in pathophysiology of Alzheimer’s disease: acetylcholinesterase (AChE) and

butyrylcholinesterase (BuChE).

2. Results and Discussion

2.1. Chemistry

Derivatives of the isoquinoline 5 have been obtained in a multistep synthesis using the

Bischler-Napieralski reaction [21] as a cyclization step (Scheme 1, Table 1). Benzyl alcohols 1 can be

easily obtained by known methods from commercially available hydroxy- or methoxy-substituted

benzaldehydes. We have used benzyl protection for hydroxyl groups. Some other protective groups

like allyl, carbonate or acetal groups were not stable in the cyclization step. Reduction of the carbonyl

group and chlorination with thionyl chloride gave benzyl chlorides 2. Alkylation of dibenzyl

formamidomalonate with 2 gave Bischler-Napieralski reaction substrates 3. Cyclization was conducted

under standard conditions using POCl₃in acetonitrile at 70–95 °C. Higher reaction temperatures

resulted in extensive formation of polymers and lowered the yield. Two isomers can form in the case

of 2,6-unsubstituted derivatives 3b, d, g and h (R¹= H). Usually only one product was obtained. For

example cyclization of compound 3b, gives predominantly 8-benzyloxy-7-bromo-3,4-dihydroisoquinoline

instead of the corresponding 6-benzyloxy-7-bromo derivative. Apparently the C2 carbon atom in

formamidomalonate 3b is more nucleophilic than C6. The stereochemistry of 4d, g and h has been

determined on the basis of HMBC correlation spectra, and derivative 4b showed a vicinal coupling

constant between C5-H and C6-H. In the last step of the synthesis, all benzyl groups were removed

with BBr₃[22] and decarboxylation took place gently to give acid 5 (Table 2). We have also used

hydrogenation on Pd/C with 1,4-cyclohexadiene as hydrogen donor. This is very mild method which

selectively removes benzyl groups in the presence of C=N bond. However formation of isoquinoline

derivatives due to aromatization of the ring was noticed. More difficult was removal of methyl groups

with BBr₃. In the case of trimethoxy-substituted dihydroisoquinoline we have obtained mixture of

isomers, where 5i was the major one.

Scheme 1. Synthesis of 3,4-dihydroisoquinoline-3-carboxylic acid derivatives.

Reagents and conditions: (a) SOCl₂, Py, CH₂Cl₂; (b) K₂CO₃, dibenzyl formamidomalonate, acetone;

(c) POCl₃, MeCN; (d) BBr₃, CH₂Cl₂.

Molecules 2014, 19

Table 1. Formamidomalonates 3 and 3,4-dihydroisoquinoline derivatives 4.

15869

3 (Yield,%) 4 (Yield,%) R¹

R²

H

R³

H

R⁴

OBn

I

3a (63)

3b (95)

3c (77)

3d (65)

3f (66)

3g (65)

3h (57)

3i (70)

3j (75)

3k (88)

3l (56)

4a (31)

4b (26)

4c (94)

4d (94)

4f (58)

4g (94)

4h (95)

4i (95)

4j (85)

4k (94)

4l (86)

Br

H

Br

H

OBn

OMe OBn

OMe OMe

H

Br

H

OMe OMe

Br

Cl

H

OMe OMe OMe

H

OBn

Br

H

OBn

H

Cl

OMe OMe

Table 2. 3,4-Dihydroisoquinoline-3-carboxylic acid derivatives.

5 (Yield,%) R¹

R²

H

R³

H

R⁴

OH

I

5a (22)

5b (87)

5c (62)

5d (22)

5e ^a

Br

H

Br

H

OH

OH OH

H

5f (21)

5g (34)

5h (15)

5i (60)

5j (53)

5k (17)

5l (8)

OH OH

H

Br

Cl

H

OH OH

OH OMe OH

OH

Br

H

OH

H

Cl OH

Cl OH OH

^aReference [9].

The most problematic was purification of polyhydroxylated derivatives of 3,4-dihydroisoquinoline-

3-carboxylic acid 5. The products are sensitive to air and bases giving aromatic derivatives of

isoquinolines. Due to the very low solubility in organic solvents and high polarity of the products, we

had to use chromatography on ion exchange and polyamide columns. In a similar way, we obtained

4,5-dihydro-3H-2-benzazepin-3-carboxylic acid 9 from 4-(2-iodoethyl)-1,2-dimethoxybenzene

(Scheme 2). Optically active 3-methyl derivative of 3,4-dihydroisoquinoline-3-carboxylic acid 13 was

obtained using L-methyl-DOPA by formylation with formyloxyacetonitrile and Bischler-Napieralski

cyclization. We also synthesised a β-lactam derivative of dihydroisoquinoline 15 by the Bose method

using [2+2] cycloaddition with chloroketene generated in situ from chloroacetyl chloride [23]. This

type of reaction is usually highly stereoselective. Examination of the coupling constant of C1-H

(1.7 Hz) suggests that we have obtained trans stereoisomer. The benzyl groups in 14 were removed by

hydrogenolysis to give β-lactam 15 (Scheme 3).

Molecules 2014, 19

Scheme 2. Synthesis of 4,5-dihydro-3H-2-benzazepin-3-carboxylic acid 9.

15870

Reagents and conditions: (a) NaH, dibenzyl formamidomalonate, DMF, 43%; (b) POCl₃, MeCN, 52%;

(c) BBr₃, CH₂Cl₂, 58%.

Scheme 3. Synthesis of the 3-methyl derivative of 3,4-dihydroisoquinoline-3-carboxylic

acid 13 and β-lactam derivative 15.

Reagents and conditions: (a) OHCOCH₂CN, DMSO, 64%; (b) BnBr, K₂CO₃, MeCN, 48%; (c) POCl₃, MeCN,

88%; (d) BBr₃, CH₂Cl₂, 88%; (e) ClCH₂COCl, Et₃N, CH₂Cl₂, 47%; (f) H₂, Pd/C, EtOH, 85%.

2.2. Biological Activities

2.2.1. Free-Radical Scavenging Activity

All the synthesised derivatives were assayed in vitro for their antioxidant and free-radical

scavenging activity. In these assays, the compounds were tested for their ability to scavenge DPPH^·,

·−

·

ABTS^·+, O₂and NO. The EC₅₀values are summarized in Table 3. In general, almost all of the

described compounds were potent scavengers on at least one free radical used in our study. Lower

EC₅₀value indicates higher radical-scavenging activity. Free-radical scavenging activities of

synthesised derivatives were compared to the properties shown by naturally obtained compound 5e.

Compounds 5e, 5g and 9 showed the best EC₅₀value, better than ascorbic acid on DPPH^·

scavenging. Compounds 5l, 5f and 5d also presented high DPPH^·scavenging activity, comparable to

the standard (Table 3).

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Table 3. Biological activities of synthesised compounds.

Scavenging Activity [EC₅₀] ^a

DAAO

Inhibition

[IC₅₀] ^c/[%] ^d

8.46 ^c

13.88 ^d

1.66 ^c

3.35 ^c

7.52 ^d

2.06 ^c

2.00 ^c

1.74 ^c

3.28 ^c

11.50 ^d

7.27 ^d

1.29 ^c

0.55 ^c

7.89 ^d

2.02 ^c

-

AChE

BuChE

Tested

Inhibition Inhibition

DPPH^·ABTS^·+

O₂

^·NO

·−

Compound

[IC₅₀] ^c

-

N.A. ^e

N.A.

-

IC₅₀] ^c

-

5a

351.60 29.62

402.62

N.D. ^b

N.D.

4.53

2196.69

901.25

2459.99

330.61

386.38

337.07

402.82

3219.46

2236.12

540.23

979.18

1644.66

N.D.

5b

1103.08 280.47

N.A.

-

5c

520.32

39.45

23.26

36.07

26.22

52.69

63.15

269.19

494.52

32.98

27.03

56.01

84.62

42.11

-

16.22

22.58

7.19

5d

5e

35.96

9.54

0.56

0.54

N.A.

0.82

N.A.

0.47

0.59

N.A.

2.08

1.78

N.A.

-

0.37

N.A.

0.64

1.42

0.17

N.A.

0.62

1.75

-

5f

13.49

11.74

21.48

23.73

113.71

28.85

9.97

5g

14.23

116.09

35.54

869.76

49.21

5.42

5h

5i

5j

5k

5l

9

12.02

18.76

10.62

28.25

-

5.92

13

203.61

4.40

65.05

15

1054.32

-

Ascorbic acid

Trolox

98.77

-

161.37

-

Benzoic acid

Galanthamine

-

2.28 ^c

-

0.001

0.009

hydrobromide

a

b

c

The values were expressed as μM concentration; N.D.—not determined; The values were expressed as

mM concentration; ^d% of DAAO inhibition at 3.57 mM concentration; ^eN.A.—not active.

Compounds 5h and 13, also showed good activity (slightly lower than standard). The presence of

two hydroxyl groups at 6- and 7-position (5e, 5g, 5l, 5d, 5h, 13) and respectively at 7- and 8-position

for compound 9 resulted in significant DPPH radical scavenging activity. Meta location of hydroxyl

groups (compounds 5i, 5j, 5k, 5c) did not result in better activities in comparison to 5e (ortho

location). Among compounds with meta location of hydroxyl groups (compounds 5i, 5j, 5k, 5c),

compound 5i with an electron-donating group at 7-position presented better activity than others.

Introduction of a halogen atom at 5- or 8-position of a compound, with two hydroxyl groups at 6- and

7-positions (5g, 5l, 5d, 5h), did not potentiate scavenging activity on DPPH^·in comparison to 5e. DPPH

radical was scavenged by compounds 5e, 5g, 9, 5l and ascorbic acid in a concentration-dependent

manner (Figure 1).

Compound 5e, was the most active scavenger on ABTS^·+. The EC₅₀value of the above-mentioned

compound was approximately one quarter of that of ascorbic acid. Also, 5l, 15, 5g, 9, 5f, 5c, 13, 5h,

5d, 5i and 5k showed very potent activities. The presence of two hydroxyl groups at the 6- and

7-positions (5e, 5l, 5g, 13, 5h, 5d) and respectively at 7- and 8-positions in 15 and 9, resulted in

significant ABTS^·+scavenging activity of the compounds. Halogen atoms (chlorine or bromine) at the

5- and 8-positions did not significantly influence the activity in comparison to 5e. ABTS^·+was

Molecules 2014, 19

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scavenged by compounds 5e, 5l, 15, 5g, 9, 5f, 5c, 13 and ascorbic acid in a concentration-dependent

manner (Figure 2).

Figure 1. DPPH^·scavenging of the most active compounds.

Figure 2. ABTS^·+scavenging of the most active compounds.

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·−

We obtained nine compounds (15, 5d, 5l, 9, 5f, 5g, 5i, 5e, 5k) with superoxide anion radical (O₂)

scavenging activities better than the standard (ascorbic acid). Compounds 15, 5d, 5l, 9, and 5f

presented very high superoxide anion radical scavenging activities. Their EC₅₀values were 22-times

lower (15 and 5d) and more than 10-times lower (5l, 9, 5f) than the EC₅₀value for ascorbic acid. The

chloroazetidinone moiety at [2,1a]-position in compound 15 and methyl group at the 4-position

improved the activity in comparison to compound 5e. Also, the presence of two hydroxyl groups at the

6- and 7-positions and iodine at the 8-position (5d) or chlorine at the 5-position (5l) caused significant

·−

O₂scavenging activity. Ortho position of two hydroxyl groups in the tested compounds contributed

to significant superoxide anion radical scavenging activity. A bromine atom at the 8-position

·−

(compound 5g) enhanced O₂scavenging activity of the compound, in comparison to a chlorine

atom at the same position (compound 5h). The EC₅₀value for compound 5g was about sevenfold

lower than the EC₅₀value for ascorbic acid. This data suggests that substitution of phenolic

compounds with a halogen atom (I > Br > Cl) stabilizes the superoxide anion radical. Compound 9,

with a dihydrobenzoazepine ring, showed 6-times greater O₂^·−scavenging activity in comparison to the

similar compound, 5e, with a dihydropyridine ring. Tested compounds were able to scavenge

superoxide anion radicals in a concentration-dependent manner (Figure 3).

Figure 3. O₂^·−scavenging of the most active compounds.

·

Compound 13 was the most potent NO scavenger. Its EC₅₀value was lower than the standard

(Trolox). All of the other tested compounds showed rather moderate scavenging activities on the nitric

oxide radical. Comparing the results of all above assays, in general, compounds with two hydroxyl

groups (5c–l, 9, 13, 15) were more potent free-radical scavengers than phenolic compounds (5a, 5b).

Also, an ortho position of hydroxyl groups determined more significant free radical scavenging

activity than the meta position.

Molecules 2014, 19

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2.2.2. DAAO Inhibitory Activity

Similarly, all newly synthesised compounds were screened for DAAO inhibitory activity.

Compound 9 was the most active inhibitor of DAAO (Table 3). It showed a fourfold lower IC₅₀value

than the standard benzoic acid. Other compounds, including 5l, 5c, 5h, 5g, 15 and 5f, showed

comparable or better DAAO inhibitory activities than the standard. The presence of two hydroxyl

groups at the meta position in the benzene ring (5c) as well as the ortho location of hydroxyl groups

with additional chlorine or bromine atom at 5- or 8-positions (5g, 5h, 5l, 5f) exhibited good activity.

Comparison of the compound with two hydroxyl groups at 6- and 7-positions (5e) and compounds

with an additional halogen atom at the 8-position (5g, 5h, 5d) showed that halogen atoms enhanced

DAAO inhibition. Moreover, the more electronegative the substituent (Cl > Br > I) at the 8-position,

the stronger the resulting DAAO inhibitor. Compounds 5a–5l and 9 are obtained as racemic mixtures.

Biological activities of separated enantiomers may be higher (even two times). However, the activity

would remain in milimolar concentration range. Synthesised derivatives inhibited DAAO in a

concentration-dependent manner (Figure 4).

Figure 4. DAAO inhibition of the most active compounds.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

2.2.3. AChE Inhibitory Activity

The effect of the synthesised compounds on AChE activity was evaluated, using a selective AChE

inhibitor, galanthamine hydrobromide as a standard. Only a few of the synthesised compounds (5j, 5f,

5e, 5k) showed rather weak activity against AChE. None of the tested compounds were more potent

than the standard. The results are presented in Table 3.

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2.2.4. BuChE Inhibitory Activity

The reported compounds were tested for their BuChE inhibitory activity (IC₅₀values are

summarised in Table 3). They showed moderate activities (5l, 5e, 13, 5j) against BuChE.

3. Experimental Section

3.1. General Information

D-Amino acid oxidase from porcine kidney (DAAO), D-alanine, nitroblue tetrazolium chloride

(NBT), 2,2-diphenyl-1-picrylhydrazyl (DPPH^·), 2,2'-azino bis(3-ethylbenzothiazoline-6-sulfonic acid)

(ABTS^·+), potassium persulphate, sodium nitroprusside, Griess reagent, acetylthiocholine iodide,

acetylcholinesterase from Electrophorus electricus (AChE), 5,5-dithiobis-2-nitrobenzoic acid (DTNB),

galanthamine hydrobromide, butyrylcholinesterase from equine serum (BuChE) and butyrylthiocholine

iodide were obtained from Sigma-Aldrich (Poznań, Poland) and 2,4-dinitro phenylhydrazine was

purchased from POCH S.A. (Gliwice, Poland). Dibenzyl formamidomalonate has been prepared

analogously to the procedure described for diethyl derivative [24]. All others reagents were of standard

analytical grade.

NMR spectra were recorded using Varian 400 MR (400 MHz) spectrometer. Chemical shifts of

13

aqueous solutions were referenced against solvent signal (4.75 ppm). Most of the C-NMR chemical

shifts of deprotected phenols 5 could not be determined due to low solubility and deuteriation in the

ring. IR spectra were recorded on Shimadzu IRAffinity-1S spectrophotometer using single reflection

ATR (ZnSe). Mass spectra were obtained using LTQ Orbitrap Velos (Thermo Scientific) and QToF

Premier (Waters), HR and LR respectively. The radical scavenging activity and enzymes inhibitory

activity were assessed by UV-VIS spectrophotometer (FLUOstar Omega BMG LABTECH microplate

reader). Copies of ¹H and ¹³C-NMR spectra are available in the Supplementary Information file.

3.2. Synthesis and Characterisation

3.2.1. General Procedure for the Synthesis of Benzyl Chlorides 2

Pyridine (0.36 mL) and thionyl chloride (0.37 mL) were added dropwise to a solution of protected

benzyl alcohol 1 (4.78 mmol) in CH₂Cl₂(15 mL). The resultant mixture was then stirred at rt for 2.5 h.

The reaction was quenched with 10 mL of water and stirred for 0.5 h to destroy excess of thionyl

chloride. The organic layer was removed, and the aqueous layer was extracted twice with CH₂Cl₂

(3 × 15 mL). The combined organic layers were dried over MgSO₄and evaporated. The products were

used in the next step without purification.

3.2.2. General Procedure for Alkylation of Dibenzyl Formamidomalonate 3a–3l

Dibenzyl formamidomalonate (315 mg, 0.96 mmol) followed by K₂CO₃(1.76 g, 12.75 mmol) and

KI (478 mg, 2.88 mmol) were added to a solution of respective benzyl chlorides (0.96 mmol) in

acetone (20 mL). The reaction mixture was stirred under reflux (oil bath, 60 °C) for 20 h. When

Molecules 2014, 19

15876

complete, the reaction mixture was cooled and filtered through Celite. The solvent was evaporated to

give a solid.

Dibenzyl 2-(2-bromo-5-benzyloxybenzyl)-2-formamidomalonate (3a). The compound was purified by

1

chromatography on silica gel (EtOAc-hexane, 1:3). White crystals. Yield 0.363 g (63%). H-NMR

(CDCl₃) δ: 3.84 (s, 2H, CH₂); 4.98 (s, 2H, OCH₂Ph); 5.12 and 5.16 (AB, J = 14, 4H, CH₂Ph); 6.58 (bs,

1H, NH); 6.69 (d, J = 3.3 Hz, 1H, C6-H); 6.76 (dd, J = 3.3, 8.8 Hz, 1H, C4-H); 7.40 (d, J = 8.8 Hz;

1H, C3-H); 7.16–7.38 (m, 15H, Ar); 7.94 (d, J = 1.3 Hz, 1H, CHO). ¹³C-NMR (CDCl₃) δ: 37.6; 65.5;

68.5; 70.0; 116.0; 116.1; 118.7; 127.2; 128.1; 128.3; 128.5; 128.55; 128.6; 133.7; 134.5; 135.4; 136.5;

157.6; 160.1; 166.8. HR MS ESI calculated for C₃₂H₂₉BrNO₆(M+H) 602.1173. Found 602.1170.

Dibenzyl 2-(3-benzyloxy-4-bromobenzyl)-2-formamidomalonate (3b). This compound was used in the

next step without purification. Beige solid. Yield 95%. ¹H-NMR (CDCl₃) δ: 3.84 (s, 2H, CH₂); 4.98 (s,

2H, OCH₂Ph); 5.12 and 5.16 (AB, J = 12, 4H, CH₂Ph); 6.76 (dd, J = 2.9 Hz, J = 8.8, 1H, C6-H); 6.89

(d, J = 2.9, 1H, C2-H); 6.60 (bs, 1H, NH); 7.14–7.38 (m, 15H, Ar); 7.40 (d, J = 8.8, 1H, C5-H); 7.93

13

(d, J = 0.8 Hz, 1H, CHO). C-NMR (CDCl₃) δ: 37.6; 65.5; 68.4; 69.9; 116.04; 116.05 118.6; 127.2;

128.0; 128.3; 128.5; 128,6; 133.7; 134.4; 135.4; 136.5; 157.6; 160.1, 166.8. HR MS ESI calculated for

C₃₂H₂₉BrNO₆(M+H) 602.1172. Found 602.1170.

Dibenzyl (3,5-dibenzyloxybenzyl)-2-formamidomalonate (3c). The product was purified on silica gel

1

(EtOAc-hexane 1:2.5). White solid. Yield 77%. H-NMR (CDCl₃) δ: 3.59 (s, 2H, CH₂); 4.93 (s, 4H,

OCH₂Ph); 5.08 and 5.12 (AB, J = 12 Hz, 4H, CH₂Ph); 6.17 (d, J = 2.1 Hz, 2H, C2-H, C6-H); 6.52

(t, J = 2.1 Hz, 1H, C4-H); 6.55 (bs, 1H, NH); 7.16–7.44 (m, 20H, Ar); 7.91 (d, J = 0.8 Hz, 1H, CHO).

¹³C-NMR (CDCl₃) δ: 38.2; 66.7; 68.3; 69.9; 101.2; 109.2; 127.3; 127.9; 128.3; 128.5; 128.61; 128.62;

134.5; 136.7; 136.8; 159.7; 159.8; 166.6. HR MS ESI calculated for C₃₉H₃₆NO₇(M+H) 630.2486.

Found 630.2466.

Dibenzyl 2-(3-iodo-4-benzyloxy-5-methoxybenzyl)-2-formamidomalonate (3d). This compound was

1

used in the next step without purification. Beige solid. Yield 65%. H-NMR (CDCl₃) δ: 3.63 (s, 2H,

CH₂); 3.67 (s, 3H, OCH₃); 4.97 (s, 2H, OCH₂Ph); 5.13 and 5.20 (AB, J = 12.1 Hz, 4H, CH₂Ph); 6.51

(d, J = 1.8 Hz, 1H, C6-H); 6.80 (bs, 1H, NH); 6.99 (d, J = 1.8 Hz, 1H, C2-H); 7.24–7.40 (m, 13H, Ar);

7.53–7.57 (m, 2H, Ar); 8.20 (d, J = 1.1 Hz, 1H, CHO). ¹³C-NMR (CDCl₃) δ: 37.2; 55.91; 55.94; 66.8;

68.5; 74.4; 92.8; 114.8; 128.0; 128.3; 128.37; 128.4; 128.69; 128.74; 131.6; 132.7; 134.4; 137.0;

147.2; 152.4; 159.9; 166.5. HR MS ESI calculated for C₃₃H₃₀INO₇(M+H) 680.1139. Found 680.1131.

Dibenzyl 2-(5-bromo-2,3-dimethoxybenzyl)-2-formamidomalonate (3f). The product was purified on

1

silica gel (EtOAc-hexane 1:2). White solid. Yield 66%. H-NMR (CDCl₃) δ: 3.66 (s, 2H, CH₂); 3.66

(s, 3H, OCH₃); 3.82 (s, 3H, OCH₃); 5.13 and 5.17 (AB, J = 12 Hz, 4H, OCH₂Ph); 6.67 (bs, 1H, NH);

6.75 (d, J = 2.5 Hz, 1H, C6-H); 6.93 (d, J = 2.5 Hz, 1H, C4-H); 7.21–7.38 (m, 10H, Ar); 8.12 (d,

J = 1.3 Hz, 1H, CHO). ¹³C-NMR (CDCl₃) δ: 33.0; 55.9; 60.6; 65.8; 68.4; 115.2; 116.1; 126.3; 128,2;

128.5; 128.6; 130.1; 134.6; 147.3; 153.3; 160.2; 166.9. HR MS ESI calculated for C₂₇H₂₇BrNO₇

(M+H) 556.0965. Found 556.0960.

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Dibenzyl 2-(3-bromo-4,5-dimethoxybenzyl)-2-formamidomalonate (3g). The product was purified on

1

silica gel (EtOAc-hexane 1:2). White solid. Yield 65%. H-NMR (CDCl₃) δ: 3.62 (s, 2H, CH₂); 3.66

(s, 3H, OCH₃); 3.81 (s, 3H, OCH₃); 5.12 and 5.20 (AB, J = 12 Hz, 4H, OCH₂Ph); 6.44 (d, J = 2.1 Hz,

1H, C2-H); 6.71 (d, J = 2.1 Hz, 1H, C6-H); 6.76 (bs, 1H, NH); 7.24–7.38 (m, 10H, Ar); 8.20

(d, J = 0.8 Hz, 1H, CHO). ¹³C-NMR (CDCl₃) δ: 37.4; 55.9; 60.5; 66.8; 68.5; 113.6; 117.5; 125.8;

128.4; 128.7; 128,8; 131.7; 134.4; 145.8; 153.4; 159.9; 166.5. HR MS ESI calculated for C₂₇H₂₇BrNO₇

(M+H) 558.0965. Found 558.0935.

Dibenzyl 2-(3-chloro-4,5-dimethoxybenzyl)-2-formamidomalonate (3h). This compound was purified

1

on silica gel (EtOAc-hexane 1:2.5). White solid. Yield 57%. H-NMR (CDCl₃) δ: 3.62 (s, 2H, CH₂);

3.67 (s, 3H, OCH₃); 3.82 (s, 3H, OCH₃); 5.12 and 5.20 (AB, J = 12.1 Hz, 2H, OCH₂Ph); 6.40 (d,

J = 1.7 Hz, 1H, C6-H); 6.52 (d, J = 1.7 Hz, 1H, C2-H); 6.76 (s, 1H, NH); 7.25–7.37 (m, 10H, Ar);

8.20 (d, J = 1.3 Hz, 1H, CHO). ¹³C-NMR (CDCl₃) δ: 37.5; 56.0; 60.6; 66.7; 68.5; 112.9; 123.0; 128.1;

128.4; 128.7; 128.8; 131.0; 134.4; 144.8; 153.5; 159.9; 166.6. HR MS ESI calculated for C₂₇H₂₇ClNO₇

(M+H) 512.1470. Found 512.1467.

Dibenzyl 2-(3,4,5-trimethoxybenzyl)-2-formamidomalonate (3i). This compound was purified on silica

1

gel (EtOAc-hexane 1:1.5). White solid. Yield 70%. H-NMR (CDCl₃) δ: 3.63 (s, 6H, OCH₃); 3.67 (s,

2H, CH₂); 3.80 (s, 3H, OCH₃); 5.10 and 5.20 (AB, J = 12.1 Hz, 4H, OCH₂PH); 6.16 (s, 2H, C2-H,

C6-H); 6.73 (bs, 1H, NH); 7.23–7.36 (m, 10H, Ar); 8.19 (d, J = 1.2 Hz, 1H, CHO). ¹³C-NMR (CDCl₃)

δ: 38.3; 55.9; 60.8; 66.9; 68.3; 106.9; 128.2; 128.67; 128.68; 130.1; 134.5; 137.3; 153.0; 159.8; 166.8.

HR MS ESI calculated for C₂₈H₃₀NO₈(M+H) 508.1965. Found 508.1945.

Dibenzyl 2-(4-bromo-3,5-dibenzyloxybenzyl)-2-formamidomalonate (3j). This compound was purified

on silica gel (EtOAc-hexane 1:4). Beige solid. Yield 75%. ¹H-NMR (CDCl₃) δ: 3.58 (s, 2H, CH₂); 5.00

(s, 4H, OCH₂Ph); 5.02 and 5.05 (AB, J = 12.1 Hz, 4H, CH₂Ph); 6.17 (s, 2H, C2-H, C6-H); 6.34 (bs,

13

1H, NH); 7.14–7.42 (m, 20H, Ar); 7.68 (d, J = 1.2 Hz, 1H, CHO). C-NMR (CDCl₃) δ: 38.2; 66.6;

68.3; 70.6; 101.7; 108.5; 126.7; 127.8; 128.1; 128.5; 128.6; 128.7; 134.4; 134.9; 136.5; 155.9; 159.9;

166.4. HR MS ESI calculated for C₃₉H₃₅BrNO₇(M+H) 708.1591. Found 708.1593.

Dibenzyl (2-chloro-3,5-dibenzyloxybenzyl)-2-formamidomalonate (3k). This compound was used in

1

the next step without purification. Beige solid. Yield 88%. H-NMR (CDCl₃) δ: 3.87 (s, 2H, CH₂);

4.94 (s, 2H, OCH₂Ph); 5.07 (s, 2H, OCH₂Ph); 5.11 and 5.16 (AB, J = 12.5 Hz, 4H, CH₂Ph); 6.30 (d,

J = 2.9 Hz, 1H, C4-H); 6.56 (br, 1H, NH); 6.57 (d, J = 2.9 Hz, 1H, C6-H); 7.18–7.45 (m, 20H, Ar);

13

7.88 (d, J = 1.3, 1H, CHO). C-NMR (CDCl₃) δ: 35.7; 65.5; 68.4; 70.1; 70.9; 101.7; 109.7; 116.3;

127.1; 127.2; 128.0; 128.1; 128.3; 128.5; 128.56; 128.59; 128.6; 134.47; 134.52; 136.2; 136.5; 155.0;

157.3; 160.1; 166.9. HR MS ESI calculated for C₃₉H₃₅ClNO₇(M+H) 664.2096. Found 664.2079.

Dibenzyl 2-(2-chloro-3,4-dimethoxybenzyl)-2-formamidomalonate (3l). This compound was purified

on silica gel (EtOAc-hexane 1:2.5). White solid. Yield 56%. ¹H-NMR (CDCl₃) δ: 3.81 (s, 3H, OCH₃);

3.82 (s, 2H, CH₂); 3.83 (s, 3H, OCH₃); 5.15 (s, 4H, CH₂Ph); 6.63 and 6.70 (AB, J = 8.3 Hz, 2H, C6-H

and C5-H); 6.66 (br, 1H, NH); 7.21–7.34 (m, 10H, Ar); 8.13 (s, 1H, CHO). ¹³C-NMR (CDCl₃) δ: 35.1;

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55.9; 60.5; 65.7; 68.4; 110.3; 125.4; 126.9; 128.4; 128.6; 129.6; 134.5; 145.4; 152.9; 154.4; 160.1;

166.9. HR MS ESI calculated for C₂₇H₂₇ClNO₇(M+H) 512.1470. Found 512.1448.

3.2.3. General Procedure for Bischler-Napieralski Cyclization. Preparation of Dibenzyl 3,4-Dihydro-

isoquinolin-3,3-dicarboxylates 4a–4l

POCl₃(0.23 mL) was added to a stirred solution of the respective dibenzyl formamidomalonate 3a–3l

(0.83 mmol) in acetonitrile (10 mL) and the mixture was heated under various conditions depending on

the compound. The solvent was evaporated and residue was made alkaline with aqueous NaHCO₃

(5 mL). The mixture was extracted with CH₂Cl₂(3 × 10 mL). The combined organic layers were dried

with MgSO₄and concentrated to give the product.

Dibenzyl 5-bromo-8-benzyloxy-3,4-dihydroisoquinolin-3,3-dicarboxylate (4a). The reaction mixture

was heated at 80 °C (temp. of oil bath) for 8 h. The compound was purified by chromatography on

1

silica gel (EtOAc-hexane, 1:3). Brown oil. Yield 0.150 g (31%). H-NMR (CDCl₃) δ: 3.44 (s, 2H

C4-H); 5.12 (s, 2H, OCH₂Ph); 5.10 and 5.20 (AB, J = 12.1 Hz, 4H, CH₂PH); 6.76 (d, J = 8.7 Hz, 1H,

C7-H); 7.48 (d, J = 8.7 Hz, 1H, C6-H); 7.14–7.43 (m, 15H, Ar); 8.95 (s, 1H, C1-H). ¹³C-NMR

(CDCl₃) δ: 30.8; 67.8; 70.3; 70.6; 113.2; 114.5; 118.8; 126.9; 127.1; 128.2; 128.3; 128.4; 128.5; 128.7;

134.4; 135.1; 135.8; 136.5; 155.7; 157.7; 168.4. MS ESI calculated for C₃₂H₂₇BrNO₅(M+H) 586.1.

Found 586.1.

Dibenzyl 8-benzyloxy-7-bromo-3,4-dihydroisoquinolin-3,3-dicarboxylate (4b). The reaction mixture

was heated at 73 °C for 15 h. The product was purified on silica gel (EtOAc-hexane 1:6). Brown oil.

1

Yield 100 mg (26%). H-NMR (CDCl₃) δ: 3.44 (s, 2H C4-H); 5.11 (s, 2H, OCH₂Ph); 5.10 and 5.21

(AB, J = 12.5 Hz, 4H, CH₂PH); 6.76 (d, J = 8.7 Hz, 1H, C5-H); 7.19–7.40 (m, 15H, Ar); 7.47 (d,

J = 8.7 Hz, 1H, C6-H); 8.94 (s, 1H, C1-H). ¹³C-NMR (CDCl₃) δ: 30.8; 67.8; 70.3; 70.6; 113.2; 114.5;

118.8; 127.1; 128.0; 128.2; 128.3; 128.4; 128.7; 134.4; 135.1; 135.8; 136.5; 155.7; 157.7; 168.4. MS

ESI calculated for C₃₂H₂₇NO₅Br (M+H) 584.1. Found 583.9.

Dibenzyl 6.8-dibenzyloxy-3,4-dihydroisoquinolin-3,3-dicarboxylate (4c). The reaction mixture was

heated at 70 °C for 8 h. Product was purified by extraction with hot cyclohexane. Orange solid. Yield

94%. ¹H-NMR (CDCl₃) δ: 3.31 (s, 2H C4-H); 5.00 (s, 2H, OCH₂Ph); 5.07 (s, 2H, OCH₂Ph); 5.10 and

5.19 (AB, J = 12.5 Hz, 4H, CH₂Ph); 6.33 (d, J = 2.1 Hz, 1H, C8-H); 6.42 (d, J = 2.1 Hz, 1H, C5-H);

7.20–7.44 (m, 20H, Ar); 8.87 (s, 1H, C1-H). ¹³C-NMR (CDCl₃) δ: 31.7; 67.8; 68.3; 70.3; 99.1; 106.0;

109.1; 127.1; 127.6; 128.0; 128.2; 128.4; 128.7; 128.74; 134.4; 135.1; 135.8; 136.9; 158.0; 158.1. HR

MS EI calculated for C₃₉N₃₃NO₆611.2308. Found 611.2292.

Dibenzyl 6-methoxy-7-benzyloxy-8-iodo-3,4-dihydroisoquinolin-3,3-dicarboxylate (4d). The reaction

mixture was heated at 80 °C for 9 h. The product was purified on silica gel (EtOAc-hexane 1:3).

1

Brown oil. Yield 94%. H-NMR (CDCl₃) δ: 3.30 (s, 2H, C4-H); 3.86 (s, 3H, OCH₃); 4.97 (s, 2H,

OCH₂Ph); 5.14 and 5.19 (AB, J = 12.1 Hz, 4H, CH₂Ph); 6.63 (s, 1H); 7.17–7.21 (m, 4H, Ar); 7.26–7.29

13

(m, 5H, Ar); 7.34–7.42 (m, 4H); 7.55–7.58 (m, 2H); 8.65 (s, 1H, C1-H). C-NMR (CDCl₃) δ: 31.3;

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56.1; 67.8; 70.3; 74.5; 98.3; 111.6; 122.4; 128.0; 128.18; 128.2; 128.3; 128.43; 128.44; 132.6; 135.1;

136.7; 147.0; 155.4; 165.2; 168.6. HR MS EI calculated for C₃₃H₂₉INO₆662.1034. Found 662.1037.

Dibenzyl 8-bromo-5,6-dimethoxy-3,4-dihydroisoquinolin-3,3-dicarboxylate (4f). The reaction mixture

was heated at 95 °C for 4 h. The product was purified on silica gel (EtOAc-hexane 1:3). Beige oil.

Yield 58%. ¹H-NMR (CDCl₃) δ: 3.36 (s, 2H, C4-H); 3.67 (s, 3H, OCH₃); 3.88 (s, 3H, OCH₃); 5.14 and

5.17 (AB, J = 12.1 Hz, 4H, CH₂Ph); 6.95 (s, 1H, C7-H); 7.18–7.30 (m, 10H, Ar); 8.72 (s, 1H, C1-H).

¹³C-NMR (CDCl₃) δ: 25.4; 56.0; 60.6; 67.7; 70.1; 114.9; 119.0; 119.7; 128.0; 128.2; 128.4; 129.3;

135.1; 145.2; 155.9; 160.8; 168.5. MS ESI calculated for C₂₇H₂₅BrNO₆(M+H) 538.1. Found 538.1.

Dibenzyl 8-bromo-6,7-dimethoxy-3,4-dihydroisoquinolin-3,3-dicarboxylate (4g). The reaction mixture

was heated at 80 °C for 9 h. Product was used in the next step without purification. Brown oil. Yield

94%. ¹H-NMR (CDCl₃) δ: 3.30 (s, 2H, C4-H); 3.83 (s, 3H, OCH₃); 3.86 (s, 3H, OCH₃); 5.14 and 5.19

(AB, J = 12.1 Hz, 4H, CH₂Ph); 6.59 (s, 1H, C5-H); 7.15–7.36 (m, 10H, Ar); 8.78 (s, 1H, C1-H).

¹³C-NMR (CDCl₃) δ: 31.2; 56.1; 60.6; 67.8; 70.2; 110.5; 119.7; 119.8; 128.0; 128.2; 128.4; 132.1;

135.1; 145.7; 156.3; 160.8; 168.6. HR MS EI calculated for C₂₇H₂₄BrNO₆537.0787. Found 537.0774.

Dibenzyl 8-chloro-6,7-dimethoxy-3,4-dihydroisoquinolin-3,3-dicarboxylate (4h). The reaction mixture

was heated at 70 °C for 8 h. Product was used in the next step without purification. Brown oil. Yield

95%. ¹H-NMR (CDCl₃) δ: 3.31 (s, 2H, C4-H); 3.84 (s, 3H, OCH₃); 3.87 (s, 3H, OCH₃); 5.14 and 5.20

(AB, J = 12.1 Hz, 4H, CH₂Ph); 6.56 (s, 1H, C5-H); 7.19–7.38 (m, 10H, Ar); 8.85 (s, 1H, C1-H).

¹³C-NMR (CDCl₃) δ: 31.1; 56.1; 60.7; 67.8; 70.2; 109.8; 118.4; 128.0; 128.2; 128.3; 128.4; 131.4;

135.1; 144.6; 156.5; 158.5; 168.6. HR MS ESI calculated for C₂₇H₂₅ClNO₆(M+H) 494.1365.

Found 494.1360.

Dibenzyl 6,7,8-trimethoxy-3,4-dihydroisoquinolin-3,3-dicarboxylate (4i). The reaction mixture was

heated at 70 °C for 9 h. Product was used in the next step without purification. Brown oil. Yield 95%.

¹H-NMR (CDCl₃) δ: 3.66 (s, 2H, C4-H); 3.83 (s, 3H, OCH₃), 3.99 (s, 3H, OCH₃); 4.17 (s, 3H, OCH₃);

5.20 and 5.27 (AB, J = 12.1 Hz, 4H, CH₂Ph); 6.53 (s, 1H, C5-H); 7.21–7.39 (m, 10H, Ar); 9.02 (s, 1H,

C1-H). ¹³C-NMR (CDCl₃) δ: 32.4; 57.3; 61.5; 61.6; 66.3; 69.9; 107.4; 110.3; 128.6; 128.7; 128.9;

133.2; 133.8; 139.7; 157.0; 161.5; 163.8; 165.0. HR MS ESI calculated for C₂₈H₂₈NO₇(M+H)

490.1860. Found 490.1841.

Dibenzyl 7-bromo-6,8-dibenzyloxy-3,4-dihydroisoquinolin-3,3-dicarboxylate (4j). The reaction mixture

was heated at 80 °C for 7 h. Product was used in the next step without purification. Brown oil. Yield

85%. ¹H-NMR (CDCl₃) δ: 3.29 (s, 2H C4-H); 4.84 (s, 2H, OCH₂Ph); 5.15 (s, 2H, OCH₂Ph); 5.15 and

5.18 (AB, J = 12.5 Hz, 4H, CH₂Ph); 6.55 (s, 1H, C5-H); 7.10–7.55 (m, 20H, Ar); 8.69 (s, 1H, C1-H).

¹³C-NMR (CDCl₃) δ: 31.4; 53.4; 67.8; 70.3; 71.1; 106.5; 108.5; 116.4; 127.0; 128.1; 128.26; 128.3;

128.4; 128.44; 128.57; 128.61; 128.7; 135.0; 135.1; 135.6; 135.8; 156.1; 157.8; 159.0; 168.6. HR MS

ESI calculated for C₃₉H₃₃BrNO₆(M+H) 690.1485. Found 690.1464.

Dibenzyl 5-chloro-6,8-dibenzyloxy-3,4-dihydroisoquinolin-3,3-dicarboxylate (4k). The reaction mixture

was heated at 75 °C for 10 h. Product was used in the next step without purification. Brown oil. Yield

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1

of crude product 94%. H-NMR (CDCl₃) δ: 3.75 (s, 2H, C4-H); 5.18 and 5.26 (AB, J = 12.1 Hz, 4H,

CH₂Ph); 5.20 (s, 2H, OCH₂Ph); 5.21 (s, 2H, OCH₂Ph); 6.49 (s, 1H, C7-H); 7.22–7.46 (m, 20H, Ar);

9.07 (s, 1H, C1-H). HR MS EI calculated for C₃₉H₃₃ClNO₆646.1991. Found 646.1978.

Dibenzyl 5-chloro-6,7-dimethoxy-3,4-dihydroisoquinolin-3,3-dicarboxylate (4l). The reaction mixture

was heated at 75 °C for 10 h. Product was used in the next step without purification. Brown oil. Yield

1

of crude product 86%. H-NMR (CDCl₃) δ: 3.44 (s, 2H, C4-H); 3.88 (s, 3H, OCH₃); 3.89 (s, 3H,

OCH₃); 5.09 and 5.22 (AB, J = 12.1 Hz, 2H, CH₂Ph); 6.84 (s, 1H, C8-H); 7.15–7.40 (m, 10H, Ar);

8.43 (s, 1H, C1-H). HR MS ESI calculated for C₂₇H₂₅ClNO₆(M+H) 494.1365. Found 494.1352.

3.2.4. General Procedure for Removal of Benzylic Groups. Preparation of 3,4-Dihydroisoquinolin-3-

carboxylic Acids 5a–5l

BBr₃(0.2 mL, 2.13 mmol) was added dropwise under argon to a solution of respective dibenzyl

3,4-dihydroisoquinolin-3,3-dicarboxylate 4a–4l (0.17 mmol) in CH₂Cl₂(10 mL) at −20 °C. The

reaction mixture was stirred for 0.5 h at the same temperature and quenched with H₂O (25 mL). The

resultant mixture was then stirred at rt for 0.5 h. The water layer was separated and washed 3 times

with CH₂Cl₂. The solution was concentrated under vacuum at rt and the residue was loaded on a

column packed with ion exchange resin Dowex 50W X4 (ca. 30 mL, pretreated subsequently with

water, 2 M HCl, and water to pH 6–7). The column was washed with water (200 mL), and the product

was eluted with 2 M aqueous NH₃. Brown to yellow colored fractions were collected. Ammonia was

removed under water pump vacuum at rt and the solution was evaporated (water bath temp. 35 °C).

The residue was acidified with 2 M aqueous HCl to pH 2, and purified on polyacrylamide (Biogel P-2)

using 0.1% aqueous TFA.

5-Bromo-8-hydroxy-3,4-dihydroisoquinolin-3-carboxylic acid (5a). Beige powder. Yield 10 mg (22%).

¹H-NMR (D₂O/DCl) δ: 3.25 and 3.35 (ABX, J = 7.1 Hz, 8.8 Hz, 17.5 Hz, 2H, C4-H); 4.48 (t, J = 7.1 Hz,

1H, C3-H); 6.58 (d, J = 9.6 Hz, 1H, C7-H); 7.59 (d, J = 9.6 Hz, 1H, C6-H); 8.73 (s, 1H, C1-H). MS

ESI calculated for C₁₀H₉NO₃Br (M+H) 269.9. Found 269.9.

7-Bromo-8-hydroxy-3,4-dihydroisoquinolin-3-carboxylic acid (5b). Beige powder. Yield 87%.

¹H-NMR (D₂O/DCl) δ: 3.27 and 3.36 (ABX, J = 7.1 Hz, 8.8 Hz, 17.5 Hz, 2H, C4-H), 4.50 (t, J = 7.9 Hz,

C3-H); 6.62 (d, J = 9.1, 1H, C5-H), 7.62 (d, J = 9.1, 1H, C6-H), 8.77 (s, 1H, C1-H). IR: 579; 617;

665; 698; 723; 750; 781; 804; 841; 897; 937; 993; 1040; 1084; 1136; 1184; 1234; 1277; 1292; 1310;

1344; 1364; 1400; 1452; 1601; 1634; 2250–3500 br; 2806; 3044; 3129. MS ESI calculated for

C₁₀H₉NO₃Br (M+H) 270.0. Found 269.9.

1

6,8-Dihydroxy-3,4-dihydroisoquinolin-3-carboxylic acid (5c). Beige powder. Yield 62%. H-NMR

(D₂O-H₂O/DCl) δ: 3.19 and 3.31 (ABX, J = 7.0 Hz, 9.2 Hz, 16.9 Hz, 2H, C3-H); 4.44 (m, 1H, C3-H);

6.13 (d, J = 1.5 Hz, 1H, C7-H); 6.26 (s, 1H, C6-H); 8.64 (s, 1H, C1-H). IR: 586; 619; 633; 662; 704;

733; 770; 808; 843; 870; 901; 957; 986; 1015; 1069; 1113; 1169; 1188; 1202; 1250; 1314; 1348; 1385;

1404; 1504; 1541; 1582; 1609; 2250–3300 br; 2519; 2612; 2689; 2722; 2793; 3071; 3250. MS ESI

calculated for C₁₀H₁₀NO₄(M+H) 208.0. Found 208.0.

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6,7-Dihydroxy-8-iodo-3,4-dihydroisoquinolin-3-carboxylic acid (5d). The reaction mixture was kept at

rt for 24 h. Beige powder. Yield 22%. ¹H-NMR (DMSO) δ: 3.23 and 3.31 (ABX, J = 7.5; 8.3; 17.5 Hz,

2H, C4-H); 4.82 (t, J = 7.9 Hz, 1H, C3-H); 6.59 (s, 1H, C5-H); 8.48 (s, 1H, C1-H); 11.9

(bs, 1H, OH). IR: 598; 648; 667; 692; 741; 768; 795; 822; 870; 885; 908; 943; 966; 1013; 1032; 1057;

1128; 1177; 1192; 1219; 1256; 1281; 1298; 1371; 1485; 1508; 1557; 1587; 1634; 1721; 2250–3650 br;

2826; 2911; 2959; 3146. MS ESI calculated for C₁₀H₉INO₄(M+H) 333.9. Found: 333.9.

6,7-Dihydroxy-3,4-dihydroisoquinolin-3-carboxylic acid (5e). This compound was obtained from

1

L-DOPA according to published procedure [9]. H-NMR (D₂O-H₂O/DCl) δ: 3.28 and 3.38 (ABX,

J = 17.0, 8.6, 7.7, Hz, 1H, C4-H); 4.56 (dd, J = 8.6 Hz, 7.7 Hz, 1H, C3-H); 6.91 (s, 1H, C5-H); 7.28

(s, 1H, C8-H); 8.64 (s, 1H, C1-H). IR: 586; 629; 648; 691; 725; 752; 773; 802; 843; 853; 883; 908;

937; 982; 1013; 1130; 1177; 1196; 1258; 1288; 1371; 1393; 1429; 1531; 1557; 2250–3500 br; 3038.

8-Bromo-5,6-dihydroxy-3,4-dihydroisoquinolin-3-carboxylic acid (5f). The reaction mixture was kept

1

at rt for 24 h. Beige powder. Yield 21%. H-NMR (D₂O-H₂O/DCl) δ: 3.13 and 3.21 (ABX,

J = 16.9, 8.8, 7.0 Hz, 2H, C4-H); 4.30 (t, J = 8.1 Hz, 1H, C3-H) 6.73 (s, 1H, C7-H); 8.42 (s, 1H,

C1-H). ¹³C-NMR (CDCl₃) δ: 23.9; 54.9; 108.8; 120.1; 121.4; 126.2; 143.2; 158.4; 175.9. IR: 615; 669;

694; 735; 770; 797; 824; 853; 891; 962; 1009; 1096; 1128; 1202; 1219; 1234; 1298; 1348; 1377; 1404;

1420; 1474; 1499; 1560; 1595; 1719; 2250–3500 br; 2970; 3038; 3134. MS ESI calculated for

C₁₀H₈BrNO₄(M+H) 286.0. Found 286.0.

8-Bromo-6,7-dihydroxy-3,4-dihydroisoquinolin-3-carboxylic acid (5g). The reaction was kept at rt for

1

24 h. Beige powder. Yield 34%. H NMR (D₂O-H₂O/DCl) δ: 3.32 and 3.43 (ABX, J = 16.5, 8.8,

7.0 Hz, 2H, C4-H); 4.53 (t, J = 8.8 Hz, 1H, C3-H); 6.59 (s, 1H, C5-H); 8.64 (s, 1H, C1-H). ¹³C-NMR

(CDCl₃) δ: 32.7; 114.1; 114.9; 115.8; 133.9; 142.4; 155.7; 163.9; 170.3. IR: 600; 667; 696; 721; 762;

799; 839; 893; 1040; 1088; 1136; 1182; 1236; 1258; 1279; 1292; 1344; 1368; 1393; 1557; 1593;

2250–3500 br; 2806; 3044; 3125. MS ESI calculated for C₁₀H₈BrNO₄(M+H) 286.0. Found 286.0.

8-Chloro-6,7-dihydroxy-3,4-dihydroisoquinolin-3-carboxylic acid (5h). The reaction mixture was kept

1

at rt for 16 h. Beige powder. Yield 15%. H-NMR (D₂O-H₂O/DCl) δ: 3.15 and 3.25 (ABX,

J = 7.1 Hz, J = 9.2 Hz, J = 17.1 Hz, 2H, C4-H); 4.37 (t, J = 7.9 Hz, 1H, C3-H); 6.41 (s, 1H, C5-H);

8.50 (s, 1H, C1-H). IR: 600; 640; 665; 696; 719; 735; 824; 851; 876; 908; 943; 968; 989; 1032; 1047;

1105; 1134; 1179; 1194; 1215; 1234; 1256; 1265; 1325; 1379; 1393; 1454; 1470; 1487; 1557; 1595;

1611; 1682; 1730; 2851; 2916; 2957; 3225; 3294. HR MS ESI calculated for C₁₀H₉ClNO₄(M+H)

242.0215. Found: 242.0213.

6,8-Dihydroxy-7-methoxy-3,4-dihydroisoquinolin-3-carboxylic acid (5i). The reaction mixture was

kept at rt for 24 h. Product 5i could not be purified from 6- and 8-methoxy isomers. Beige powder.

1

Yield 61 mg. Major isomer (60%). H-NMR (D₂O-H₂O/DCl) δ: 3.10 (m, 2H, C4-H), 3.82 (s, 3H,

OCH₃), 4.30 (m, 1H, C3-H), 6.33 (s, 1H, C5-H), 8.39 (s, 1H, C1-H). IR: 554; 600; 635; 671; 756; 777;

800; 901; 949; 991; 1080; 1098; 1138; 1202; 1265; 1304; 1354; 1373; 1454; 1522; 1589; 2250–3650

br; 2835; 3030; 3179. HR MS ESI calculated for C₁₁H₁₂NO₅(M+H) 238.0709. Found: 238.0702.

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7-Bromo-6,8-dihydroxy-3,4-dihydroisoquinolin-3-carboxylic acid (5j). Beige powder. Yield 53%.

¹H-NMR (D₂O-H₂O/DCl) δ: 2.99 and 3.11 (ABX, J = 6.7, 9.6, 16.2 Hz, 2H, C4-H); 4.25 (dd, J = 9.6,

13

6.7 Hz; 1H, C3-H); 6.00 (s, 1H, C5-H); 8.33 (s, 1H, C1-H). C-NMR (D₂O-H₂O/DCl) δ: 27.7; 53.8;

97.5; 106.4; 109.0; 137.8; 159.6; 159.7; 164.5; 171.3. IR: 590; 660; 691; 746; 764; 797; 826; 854; 872;

945; 1011; 1030; 1061; 1096; 1123; 1173; 1229; 1246; 1283; 1329; 1369; 1514; 1589; 2250–3500 br;

2970; 3144. MS ESI calculated for C₁₀H₉NO₄Br (M+H) 286.0. Found 285.9.

5-Chloro-6,8-dihydroxy-3,4-dihydroisoquinolin-3-carboxylic acid (5k). The reaction mixture was kept

1

at −20 °C for 1 h. Beige powder. Yield 17%. H-NMR (D₂O-H₂O/DCl) δ: 3.22 and 3.29 (ABX,

J = 7.8, J = 8.6, 18.0 Hz, 2H, C4-H); 4.69 (ddd, J = 1.3, 7.8, 8.6 Hz, 1H, C3-H); 6.25 (s, 1H, C7-H);

8.68 (d, J = 1.3 Hz, 1H, C1-H). IR: 598; 633; 650; 671; 743; 781; 829; 858; 939; 1001; 1036; 1094;

1186; 1215; 1248; 1285; 1319; 1337; 1398; 1420; 1504; 1531; 1557; 2250–3650 br; 2602; 2745; 3065.

MS ESI calculated for C₁₀H₉ClNO₄(M+H) 242.0. Found: 241.9.

5-Chloro-6,7-dihydroxy-3,4-dihydroisoquinolin-3-carboxylic acid (5l). The reaction mixture was kept

1

at rt for 21 h. Beige powder. Yield 8%. H-NMR (D₂O-H₂O/DCl) δ: 2.77 and 2.92 (ABX. J = 7.9 Hz,

J = 10.0, 17.5 Hz, 2H, C4-H); 4.42 (ddd, J = 7.9, 10.0, 1.7 Hz, 1H, C3-H); 6.64 (s, 1H, C8-H); 8.20

(d, J = 1.5 Hz, 1H, C1-H). ¹³C-NMR (D₂O-H₂O/DCl) δ: 53.4; 114.8; 119.9; 127.6; 143.8; 150.6;

150.7; 164.3; 169.7. IR: 606; 631; 654; 689; 718; 731; 758; 800; 826; 853; 868; 907; 930; 951; 976;

1020; 1142; 1171; 1254; 1265; 1308; 1342; 1396; 1429; 1454; 1472; 1514; 1557; 1593; 1639;

2250–3500 br; 2731; 2920; 3063; 3146; 3566. MS ESI calculated for C₁₀H₉ClNO₄(M+H) 242.0.

Found 241.9.

3.2.5. Preparation of Dibenzyl 2-[2-(3,4-Dimethoxyphenyl)ethyl]-2-formamidomalonate (7)

Solution of dibenzyl formamidomalonate (1.629 g, 4.98 mmol) in anh. DMF (15 mL) was added to

a suspension of NaH (299 mg, 7.46 mmol, 60% in oil) in anh. DMF (10 mL). The reaction mixture

was stirred for 1 h at rt. Next, a solution of 4-(2-iodoethyl)-1,2-dimethoxybenzene (1.53 g, 4.98 mmol)

in anh. DMF (10 mL) was added and the reaction mixture was heated at 60 °C for 20 h. The solvent

was distilled off under vacuum. Water (100 mL) was carefully added and the mixture was extracted

with CH₂Cl₂(3 × 50 mL). Collected extracts were dried over MgSO₄and evaporated. Product was

purified by crystallization from diethyl ether cooling the solution to −20 °C. Orange solid. Yield 1.05 g

(43%). ¹H-NMR (CDCl₃) δ: 2.34 (m, 2H, ArCH₂CH₂); 2.70 (m, 2H, ArCH₂CH₂); 3.79 (s, 3H, OCH₃);

3.83 (s, 3H, OCH₃); 5.08 and 5.14 (AB, J = 12.1 Hz, 4H, CH₂Ph); 6.51–6.55 (m, 2H, C2,6-H); 6.72 (d,

J = 8.0 Hz, C5-H); 6.95 (s, 1H, NH); 7.22–7.35 (m, 10H, Ar), 8.18 (d, J = 1.3 Hz, 1H, CHO). ¹³C-NMR

(CDCl₃) δ: 29.4; 33.9; 55.8; 55.9; 65.9; 68.3; 111.1; 111.6; 120.3; 128.3; 128.5; 128.6; 132.6; 134.6;

147.4; 148.8; 159.7; 167.2. MS ESI calculated for C₂₈H₂₉NO₇(M+H) 492.1. Found: 492.2.

3.2.6. Preparation of Dibenzyl 7,8-Dimethoxy-4,5-dihydro-3H-2-benzazepine-3,3-dicarboxylate (8)

This compound was obtained according to the procedure 3.2.3. The reaction mixture was heated at

80 °C for 20 h. The product was purified by dissolving in boiling cyclohexane and cooling to 10 °C.

1

Brown oil. Yield 52%. H-NMR (CDCl₃) δ: 2.68 (m, 2H, C5-H); 2.85 (m, 2H, C4-H); 3.86 (s, 3H,

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OCH₃); 3.87 (s, 3H, OCH₃); 5.09 and 5.18 (AB, J = 12.5 Hz, 4H, CH₂Ph); 6.58 (s, 1H, C7-H); 6.84 (s,

1H, C10-H); 7.20–7.30 (m, 10H, Ar); 8.41 (s, 1H, C1-H). ¹³C-NMR (CDCl₃) δ: 30.6; 33.1; 55.9; 56.0;

67.5; 75.8; 112.2; 116.4; 125.4; 128.0; 128.1; 128.3; 134.8; 135.2; 147.2; 150.8; 163.7; 169.1. HR MS

EI calculated for C₂₈H₂₇NO₆473.1838. Found: 473.1852.

3.2.7. Preparation of 7,8-Dihydroxy-4,5-dihydro-3H-2-benzazepine-3-carboxylic Acid (9)

This compound was obtained according to the procedure 3.2.4. The reaction mixture was kept at rt

1

for 24 h. Brown solid. Yield 58%. H-NMR (D₂O-H₂O/DCl) δ: 2.07–2.18 (m, 2H, C4-H); 2.70 and

2.86 (m, 2H, C5-H); 4.62 (t, J = 5.3 Hz, 1H, C3-H); 6.61 (s, 1H, C6-H); 6.99 (s, 1H, C9-H); 8.11 (s,

1H, C1-H). IR: 552; 565; 594; 667; 694; 752; 804; 843; 881; 941; 1038; 1103; 1138; 1171; 1238;

1252; 1281; 1373; 1456; 1506; 1558; 1576; 1601; 2250–3500 br; 2805; 2870; 3042; 3123. MS ESI

calculated for C₁₁H₁₂NO₄(M+H) 222.0. Found 222.0.

3.2.8. Preparation of (S)-Benzyl 2-(3,4-Dibenzyloxy)benzyl-2-formamidopropanoate (11)

Formylation of L-methyl-DOPA. Formyloxyacetonitrile (1.326 g, 15.6 mmol) was added to a

solution of L-methyl-DOPA hydrate, (1.38 g, 5.8 mmol) in DMSO (10 mL) and the mixture was stirred

at rt for 3 days. Solvent was removed under vacuum and the residue was dissolved in acetonitrile

(5 mL). Dichloromethane (150 mL) and hexane (10 mL) were added to precipitate product. After

1

filtration white solid was obtained (0.88 g). Yield 64%. H-NMR (DMSO) δ: 1.32 (s, 3H, CH₃), 2.94

and 2.98 (AB, J = 13.3 Hz, 2H, CH₂), 6.36 (dd, J₁=1.9, 7.9 Hz, 1H, C6-H), 6.51 (d, J = 1.9 Hz, 1H,

C2-H), 6.60 (d, J = 7.9 Hz, 1H, C5-H), 7.90 (br, 1H, NH), 7.93 (d, J = 1.7 Hz, 1H, CHO); 8.70 (br, 1H,

OH). ¹³C-NMR (DMSO) δ: 22.7; 58.9; 115.1; 117.8; 117.8; 121.2; 127.2; 144.0; 144.7; 160.8; 174.6.

Benzylation of L-N-formylmethyl-DOPA. K₂CO₃, (12.4 g) and benzyl bromide (10.74 g, 62.8 mmol)

were added to a solution of L-N-formylmethyl-DOPA (4.29 g, 17.9 mmol) in anh. acetonitrile (80 mL).

The reaction mixture was stirred and heated at 60 °C for 16 h. The solvent was evaporated and the

water was added to the residue. The mixture was extracted with CH₂Cl₂(3 × 20 mL). Combined extracts

were dried with Na₂SO₄and evaporated. Product was purified on silica gel (EtOAc-CH₂Cl₂1:1).

1

White solid. Yield 244 mg (48%). H-NMR (CDCl₃) δ: 1.68 (s, 3H, CH₃), 3.10 (d, J = 13.8 Hz, 1H,

CH₂), 3.45 (d, J = 13.8 Hz, 1H, CH₂), 5.05–5.14 (m, 6H, CH₂Ph), 6.06 (bs, 1H, NH), 6.47 (dd, J = 8.3,

2.1 Hz, 1H, C6-H), 6.63 (d, J = 2.1 Hz, 1H, C2-H), 6.75 (d, J = 8.3 Hz, 1H, C5-H), 7.28–7.46 (m,

15H, Ar), 7.87 (d, J = 2.1 Hz, 1H, CHO). ¹³C-NMR (CDCl₃) δ: 23.5; 40.9; 61.4; 67.6; 71.0; 71.2; 114.7;

117.0; 122.7; 127.1; 127.2; 127.3; 127.7; 127.8; 128.3; 128.4; 128.6; 128.7; 129.1; 134.9; 137.3;

148.1; 148.3; 160.4; 173.2. HR MS ESI calculated for C₃₂H₃₂NO₅(M+H) 510.2275. Found 510.2252.

3.2.9. Preparation of (S)-Benzyl 6,7-Dibenzyloxy-3-methyl-3,4-dihydroisoquinolin-3-carboxylate (12)

This compound was obtained according to the procedure 3.2.3. The reaction mixture was heated at

60 °C (oil bath) for 1.5 h. The product was purified on silica gel (first CH₂Cl₂then CH₂Cl₂-EtOAc

1:1). Orange oil. Yield 1.479 g (88%). ¹H-NMR (CDCl₃) δ: 1.45 (s, 3H, CH₃), 2.75 (d, 1H, J = 16.3 Hz,

C4-H), 3.16 (d, 1H, J = 16.3 Hz, C4-H), 5.15 (s, 2H, CH₂Ph); 5.17 (s, 2H, CH₂Ph); 5.18 (s, 2H,

CH₂Ph); 6.72 (s, 1H, C5-H); 6.92 (s, 1H, C8-H); 7.25–7.45 (m, 15H, Ar); 8.22 (s, 1H, C1-H).

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¹³C-NMR (CDCl₃) δ: 23.6; 33.8; 62.7; 66.8; 71.0; 71.6; 113.8; 114.5; 120.7; 127.2; 127.3; 127.9; 127.

94; 128.0; 128.0; 128.2; 128.4; 128.5; 128.6; 135.9; 136.5; 136.9; 147.8; 152.0; 159.0; 174.5. HR MS

ESI calculated for C₃₂H₃₀NO₄(M+H) 492.2169. Found 492.2169.

3.2.10. Preparation of (S)-6,7-Dihydroxy-3-methyl-3,4-dihydroisoquinolin-3-carboxylic Acid (13)

This compound was obtained according to the procedure 3.2.4. Green powder. Yield 88%. ¹H-NMR

(D₂O-H2O/DCl) δ: 1.62 (s, 3H, CH₃); 3.12 and 3.36 (AB, J = 17.1 Hz, 2H, C4-H); 6.79 (s, 1H, C5-H);

13

7.16 (s, 1H, C8-H); 8.57 (s, 1H, C1-H). C-NMR (D₂O-H₂O/DCl) δ: 21.8; 34.0; 60.8; 115.2; 116.0;

120.7; 131.7; 144.1; 155.8; 164.2; 173.8. IR: 554; 596; 627; 650; 719; 750; 797; 835; 883; 941; 966;

1126; 1180; 1233; 1277; 1331; 1381; 1447; 1516; 1557; 1568; 1614; 1634; 1643; 1667; 1732;

2250–3500 br; 2787; 2945; 3123; 3196. HR MS ESI calculated for C₁₁H₁₁NO₄Na (M+Na) 244.0580.

Found 244.0589.

3.2.11. Preparation of Benzyl 1-Chloro-7,8-dibenzyloxy-4-methyl-2-oxo-1,4,5,9b-tetrahydro-2H-

azeto[2,1-a]isoquinoline-4-carboxylate (14)

Et₃N (654 mg, 6.48 mmol) and solution of chloroacetyl chloride (549 mg, 4.86 mmol) in CH₂Cl₂

(2 mL) were added to a stirred solution of benzyl 6,7-dibenzyloxy-3,4-dihydroisoquinolin-3-methyl-3-

carboxylate (400 mg, 0.81 mmol) in CH₂Cl₂(10 mL). The reaction mixture was stirred at rt for 20 h

and quenched with aqueous NaHCO₃(2 mL). Stirring was continued for next 1 h. Water layer was

separated and extracted with CH₂Cl₂. Organic layers were dried over MgSO₄, evaporated and purified

1

by chromatography on silica gel (acetone/CH₂Cl₂150:1). Brown oil. Yield 140 mg (47%). H-NMR

(CDCl₃) δ: 1.88 (s, 3H, CH₃); 2.94 and 3.14 (AB, J = 15.8, 2H, C5-H); 4.41 (d, J = 1.7, 1H, C1-H);

4.69 (br, 1H, C9b-H); 5.09 (¹/₂AB, J = 12.1, 1H, CH₂Ph); 5.10 (s, 2H, OCH₂Ph); 5.12 (s, 2H,

OCH₂Ph); 5.13 (¹/₂AB, J = 12.1, 1H, CH₂Ph); 6.64 (s, 1H, C6-H); 6.75 (s, 1H, C9-H); 7.13–7.46 (m,

13

15H, Ar). C-NMR (CDCl₃) δ: 22.3; 39.3; 60.3; 60.4; 61.2; 67.3; 71.2; 71.4; 111.6; 115.4; 124. 2;

124.9; 127.2; 127.4; 127.8; 127.9; 128.0; 128.4; 128.5; 135.1; 136.6; 136.7; 148.5; 149.0; 162.9;

171.3. HR MS ESI calculated for C₃₄H₃₁ClNO₅(M+H) 568.1813. Found: 568.1887.

3.2.12. Preparation of 1-Chloro-7,8-dihydroxy-4-methyl-2-oxo-1,4,5,9b-tetrahydro-2H-azeto[2,1-a]-

isoquinoline-4-carboxylic Acid (15)

Pd/C (10%, 15 mg) was added to a solution of benzyl 1-chloro-7,8-dibenzyloxy-4-methyl-2-oxo-

1,4,5,9b-tetrahydro-2H-azeto[2,1-a]isoquinoline-4-carboxylate 14 (90 mg, 0.16 mmol) in EtOH (7 mL)

and a balloon with hydrogen was connected. The suspension was stirred for 3 h. The catalyst was

centrifuged and washed two times with EtOH (3 mL). Collected solution were evaporated and dried

under vacuum. White solid. Yield 40 mg (85%). ¹H-NMR (acetone-d₆) δ: 1.80 (s, 3H, CH₃); 2.94 and

3.13 (AB, J = 15.4 Hz, 2H, C5-H); 4.70 (br, 1H, C9b-H); 4.74 (d, J = 2.1 Hz, 1H, C1-H); 6.69 (s, 1H,

C6-H); 6.77 (s, 1H, C9-H); 8.10 (br, 1H, OH); 8.15 (br, 1H, OH). ¹³C-NMR (acetone-d₆) δ: 22.5; 39.5;

60.7; 61.1; 62.1; 112.8; 116.8; 124.1; 124.7; 145.5; 146.1; 163.3; 173.3. HR MS ESI calculated for

C₁₃H₁₂ClNO₅(M+H) 298.0404. Found: 298.0478.

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3.3. Free-Radical Scavenging Properties

3.3.1. DPPH Radical Scavenging Assay

The radical-scavenging activity of tested compounds against DPPH^·was determined by Williams’

method [25]. Tested compounds dissolved in water or methanol (30 μL) were added at various

concentrations (to final concentration of 2, 4, 8, 16, 32, 64, 128, 256, 512 μg/mL) to freshly prepared

0.22 mM DPPH^·in methanol (120 μL). The absorbance was measured against the control in the

microplate reader at 517 nm after 30 min of incubation at room temperature (rt). The control contained

all reagents without the tested compound. The corresponding blank reading was also taken and the

remaining DPPH^·was calculated. Each sample was replicated three times. Ascorbic acid was used

as a standard.

3.3.2. ABTS Radical Scavenging Assay

The radical-scavenging activity of tested compounds against ABTS^·+and the generation of the

ABTS^·+radical was performed according to methods described previously [26,27]. Briefly, an ABTS^·+

solution was prepared by mixing 2 mM ABTS (50 mL) with 70 mM potassium persulphate (200 μL),

both in deionized water, and used after 2 h of being kept in the dark at rt. The ABTS^·+solution (40 μL)

was added to 0.1 M phosphate buffer pH 7.4 (60 μL) and tested compounds (25 μL) were dissolved in

water or methanol at various concentrations (to final concentration of 2, 4, 8, 16, 32, 64, 128, 256,

512 μg/mL). The absorbance was measured against the control in the microplate reader at 734 nm after

30 min of incubation at rt. The control contained all reagents without the tested compound. The

corresponding blank reading was also taken and the remaining ABTS^·+was calculated. Each sample

was replicated three times. Ascorbic acid was used as a standard.

3.3.3. Superoxide Anion Radical Scavenging Assay

·−

Superoxide scavenging assay was based on inhibition of the formazan dye formation. O₂was

generated in alkaline DMSO by the addition of sodium hydroxide to air-saturated pure DMSO [28].

The generated superoxide reacts with NBT to give coloured diformazan. To the reaction mixture

containing NBT (10 μL; 1 mg/mL solution in DMSO) and the various concentration of scavenger

(30 μL; to final concentration of 2, 4, 8, 16, 32, 64, 128, 256, 512 μg/mL), alkaline DMSO (100 μL)

was added to give a final volume of 140 μL per well in a microplate. The absorbance was measured in

the microplate reader at 560 nm after 30 min of incubation at rt [29]. The same procedure was repeated

for the control in which DMSO was added instead of scavenger solution. Each sample was replicated

three times and the corresponding blank reading was also taken. Ascorbic acid was used as a standard.

3.3.4. Nitric Oxide Radical Scavenging Assay

Nitric oxide was generated from sodium nitroprusside and measured by the Griess reaction [26].

Scavengers of ^·NO compete with oxygen leading to reduced production of nitric oxide. 10 mM sodium

nitroprusside in 0.1 M phosphate buffer pH 7.4 (30 μL) was mixed with tested compounds dissolved in

water or methanol (25 μL) at various concentrations (to final concentration of 2, 4, 8, 16, 32, 64, 128,

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256, 512 μg/mL) and incubated at 25 °C for 5 h. After incubation, Griess reagent (50 μL) was added.

The absorbance was measured in the microplate reader at 546 nm against a control (without tested

compounds) treated in the same way with Griess reagent. The corresponding blank reading was also

taken. Each sample was replicated three times. Trolox was used as a standard.

3.3.5. Effective Concentration of Scavengers (EC₅₀)

·

The percentage of radical scavenging was calculated using Equation (1) (DPPH^·, ABTS^·+and NO

scavenging assays). The absorbance for the control is A_cand the absorbance in the presence of the

compounds or other scavenger is A_s:





A_s





Radical scavenging

[

%

]

= 1−

×100

(1)





A

c



·−

The percentage of O₂scavenging was calculated using Equation (2). The absorbance for the

control is A_cand the absorbance in the presence of the compounds or other scavenger is A_s:



_c

A





Superoxide scavenging

[

%

]

= 1−

×100

(2)





A_s



The amount of tested compound needed to scavenge free radicals by 50%, EC₅₀(effective

concentration), was calculated by the linear regression between scavenger concentration and percentage

of scavenging and expressed as μM.

3.4. DAAO Inhibitory Activity

Inhibition of DAAO was based on the determination of the α-keto acid obtained from the reaction

between a DAAO and D-Ala according to the described method [30]. The method was adapted to

microtitre plates and a total volume of 210 μL per well. Inhibitor solution in DMSO (15 μL), 5 mM

D-Ala in 0.2 M Tris-HCl buffer pH 8.2 (121 μL) and 10 U/mL DAAO solution in 0.2 M Tris-HCl

buffer pH 8.2 (5 μL) were mixed and incubated at 37 °C for 30 min. After incubation, 1 mM

2,4-dinitro phenylhydrazine dissolved in 1 M HCl (14 μL) was added to the well, mixed and further

incubated at 37 °C for 10 min. Next, 1.5 M NaOH (55 μL) was added and incubated for 10 min. The

absorbance was read at 445 nm against a blank sample consisting the same assay mixture but without

the substrate (using Tris-HCl buffer instead of D-Ala). The positive control of the enzyme activity

consisted of DMSO (15 μL) instead of inhibitor solution and the analogous blank contained Tris-HCl

buffer instead of D-Ala. Sample, control and related blanks were carried out under the same condition.

Benzoic acid was used as a standard. Eight different solutions of inhibitor were examined: 0.0005,

0.0025, 0.005, 0.00625, 0.0125, 0.025, 0.0375 and 0.05 M. For each of these inhibitor concentrations

percentage of inhibition was calculated by using Equation (3). The absorbance for the control is A_cand

the absorbance in the presence of the compounds or other inhibitor is A_i:





A

i





DAAO inhibition

[

%

]

= 1−

×100

(3)





A

c



Molecules 2014, 19

15887

The inhibitor concentration needed to inhibit DAAO by 50% (IC₅₀) was calculated by the linear

regression between inhibitor concentration and percentage of inhibition. Each sample was replicated

three times.

3.5. AChE Inhibitory Activity

The assay was based on Ellman’s method [31] with modifications. AChE activity was measured by

the determination of a yellow colour produced from acetylthiocholine iodide when it reacted with the

dithiobisnitrobenzoate ion. The assay was performed using microtitre plates and a total volume of

100 µL per well. In a 96-well plate, 0.1 M pH 7.4 phosphate buffer (32 µL), a serially diluted solution

of tested compounds (5 µL), 0.59 U/mL AChE solution (4 µL) and 0.3 mM DTNB (42 µL) were

mixed and pre-incubated at 25 °C for 2 min. Then, the substrate, 0.4mM acetylthiocholine iodide

(17 µL), was added to the well and mixed. The enzymatic reaction was followed for 10 min, with a

measurement every 12 s. Changes in the absorbance at 412 nm were detected in the microplate reader.

The absorbance was measured against a blank sample containing the same assay mixture but without

an enzyme (using phosphate buffer instead of AChE). The positive control of the enzyme activity

consisted of DMSO (1.5 μL) instead of inhibitor solution. The sample, control and related blanks were

measured under the same conditions. Galanthamine hydrobromide was used as a standard. Each

sample was replicated three times. The concentration of the compounds that caused 50% inhibition of

AChE activity (IC₅₀) was calculated using linear regression analysis. At least three concentrations of

tested compounds were assayed. For each of these inhibitor concentrations, the percentage of inhibition

was calculated using Equation (4). The difference in absorbance for the inhibitor at t = 10 min and t = 0

is denoted ΔA_i. The difference in absorbance for the control at t = 10 min and t = 0 is denoted ΔA_c:





ΔA

(4)

i





Cholinesterase inhibition

[%

]

= 1−

×100





ΔA

c



3.6. BuChE Inhibitory Activity

The assay was based on Ellman’s method [31] with modifications. BuChE activity was measured

by the determination of the yellow colour produced from butyrylthiocholine iodide when it reacted

with the dithiobisnitrobenzoate ion. The assay was performed using microtitre plates and a total

volume of 100 µL per well. In a 96-well plate, 0.1 M pH 7.4 phosphate buffer (35 µL), a serially

diluted solution of tested compounds (5 µL), 1.485 U/mL BuChE solution (5 µL) and 0.3 mM DTNB

(42 µL) were mixed and pre-incubated at 25 °C for 2 min. Then, the substrate, 0.6 mM butyrylthiocholine

iodide (13 µL), was added to the well and mixed. The enzymatic reaction was followed for 10 min,

with a measurement every 12 s. Changes in absorbance at 412 nm were detected in the microplate

reader. The absorbance was measured against a blank sample containing the same assay mixture, but

without an enzyme (using phosphate buffer instead of BuChE). The positive control for enzyme

activity contained DMSO (1.5 μL) instead of inhibitor solution. The sample, control and related blanks

were measured under the same conditions. Galanthamine hydrobromide was used as a standard. Each

sample was replicated three times. The concentration of the compounds that caused 50% inhibition of

BuChE activity (IC₅₀) was calculated using linear regression analysis. At least three concentrations of

Molecules 2014, 19

15888

the tested compounds were assayed. For each of these inhibitor concentrations, the percentage of

inhibition was calculated using Equation (4).

4. Conclusions

We investigated newly synthesised compounds (hydroxy- and halogeno-substituted derivatives

of 3,4-dihydroisoquinoline-3-carboxylic acid, 2-oxo-1,4,5,9b-tetrahydro-2H-azeto[2,1-a]isoquinoline

analogue and 4,5-dihydro-3H-2-benzazepine analogues) for their free-radical-scavenging activity and

DAAO, AChE, BuChE inhibitory activity. The obtained dihydroxy-substituted derivatives of

3,4-dihydroisoquinoline-3-carboxylic acid were found to be more potent scavengers of DPPH^·,

·−

·

ABTS^·+, O₂and NO than monohydroxy-substituted derivatives. The presence of ortho-dihydroxy

structures determined higher free-radical scavenging activities than the meta location of these

substituents. These compounds were shown to be potent free-radical scavengers on at least one free

radical used in this study. Obtained results are significant due to the fact that the 3,4-dihydroisoquinoline

derivatives have not been described so far as potent free radical scavengers.

In conclusion, the potent free-radical scavengers obtained in the study may be potential candidates

for therapeutics used in oxidative-stress-related diseases. The synthesised inhibitors of evaluated

enzymes may deserve attention in the design of new antipsychotic drugs. Despite rather low inhibitory

activities against DAAO, AChE and BuChE, it seems that compounds with potent free-radical scavenging

properties may play a supporting role in the treatment of neurodegenerative and psychiatric diseases.

Supplementary Materials

Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/10/15866/s1.

Acknowledgments

Financial support by the European Union within the European Regional Development Fund

(grant number UDA-POIG.01.03.01-14-136/09/08) is gratefully acknowledged.

Author Contributions

J.S. and R.K. designed research; J.S., R.K., A.G., M.P., J.Z., K.L., A.O., B.P., K.P. and A.W.

performed research, analyzed and interpreted the data; J.S, R.K., J.Z., A.G. and M.P wrote the paper.

All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Sample of the compound 5e is available from the authors.

distributed under the terms and conditions of the Creative Commons Attribution license

(http://creativecommons.org/licenses/by/4.0/).

Article Doi

New derivatives of 3,4-dihydroisoquinoline-3-carboxylic acid with free-radical scavenging, d-amino acid oxidase, acetylcholinesterase and butyrylcholinesterase inhibitory activity

DOI: 10.3390/molecules191015866

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Article abstract of DOI:10.3390/molecules191015866

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