Substituted tetrahydroisoquinolines: synthesis, characterization, antitumor activity and other biological properties

Article abstract of DOI:10.1016/j.ejmech.2017.12.098

This work deals with the molecular design, synthesis and biological activity of a series of tetrahydro[1,4]dioxanisoquinolines and dimethoxyisoquinoline analogues. This study describes the synthesis strategy of these potential antitumor compounds, their multi-step synthesis and their optimization. A series of tetrahydroisoquinolines was synthesized and their cytotoxicity evaluated. Some of these tetrahydroisoquinolines showed promising KRas inhibition, antiangiogenesis activity and antiosteoporosis properties. Molecular modeling studies showed that compound 12 bind in the p1 pocket of the KRas protein making interactions with the hydrophobic residues Leu56, Tyr64, Tyr71 and Thr74 and hydrogen bonds with residues Glu37 and Asp38.

Full text of DOI:10.1016/j.ejmech.2017.12.098

Accepted Manuscript
Substituted tetrahydroisoquinolines: synthesis, characterization, antitumor activity and
other biological properties
A. Sergi Capilla, Richard Soucek, Laura Grau, Manel Romero, Jaime Rubio-Martínez,
Daniel H. Caignard, Maria Dolors Pujol
PII:
S0223-5234(17)31129-7
10.1016/j.ejmech.2017.12.098
EJMECH 10076
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 September 2017
Revised Date: 29 December 2017
Accepted Date: 30 December 2017
Please cite this article as: A.S. Capilla, R. Soucek, L. Grau, M. Romero, J. Rubio-Martínez, D.H.
Caignard, M.D. Pujol, Substituted tetrahydroisoquinolines: synthesis, characterization, antitumor
activity and other biological properties, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.12.098.
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ACCEPTED MANUSCRIPT
Substituted tetrahydroisoquinolines: synthesis, characterization, antitumor activity and
other biological properties
A. Sergi Capilla, Richard Soucek, Laura Grau, Manel Romero, Jaime Rubio-Martínez, Daniel H.
Caignard, and Maria Dolors Pujol

ACCEPTED MANUSCRIPT
Highlights
•
18 Dioxigenated tetradydroisoquinoline compounds were synthesized from the
corresponding aldehyde.
•
•
•
Compounds 12, 13, 14, 15, 16, 18, 20 and 21 exhibited significant cytotoxic activity.
Isoquinoline 14 presents the best KRas activity profile on RKO KRasSL.
Molecular modeling studies showed that the tetrahydroisoquinoline 12 binds directly
to the p1 pocket of the KRas protein.
Substituted tetrahydroisoquinolines: synthesis, characterization,
antitumor activity and other biological properties
A. Sergi Capilla,# Richard Soucek,# Laura Grau,# Manel Romero,# Jaime Rubio-Martínez,§ Daniel H.
Caignard,¥ and Maria Dolors Pujol#*
# Laboratori de Química Farmacèutica (Unitat associada al CSIC). Facultat de Farmàcia, Universitat
de Barcelona, Spain.
§
Department of Physical Chemistry, Faculty of Chemistry, University of Barcelona and the Institut
de Recerca en Química Teòrica i Computacional (IQTCUB), Barcelona, Spain.
¥
Les laboratoires Servier. 1 rue Carle Hébert-92415 Courbevoie cedex. 92200 Neuilly-sur Seine
(France)
Keywords: Isoquinolines, Dioxigenated compounds, Antitumor, KRas, Molecular Dynamics,
Allosteric Sites, Drug Design.
Abstract. This work deals with the molecular design, synthesis and biological activity of a series of
tetrahydro[1,4]dioxanisoquinolines and dimethoxyisoquinoline analogues. This study describes the
synthesis strategy of these potential antitumor compounds, their multi-step synthesis and their
optimization. A series of tetrahydroisoquinolines was synthesized and their cytotoxicity evaluated.

Some of these tetrahydroisoquinolines showed promising KRas inhibition, antiangiogenesis activity
ACCEPTED MANUSCRIPT
and antiosteoporosis properties. Molecular modeling studies showed that compound 12 bind in the
p1 pocket of the KRas protein making interactions with the hydrophobic residues Leu56, Tyr64,
Tyr71 and Thr74 and hydrogen bonds with residues Glu37 and Asp38.
1. Introduction
Substituted N-heterocycles systems like indole, azaindole, quinolone, isoquinoline and piperidine
analogues are structural subunits present in numerous natural and synthetic compounds exhibiting a
wide range of biological activities [1]. Among them the tetrahydroisoquinoline core in particular is
widely studied as a scaffold for the preparation of potential therapeutically active compounds [2].
This heterocycle can be found in several drugs, such as noscapine, a natural benzyl
tetrahydroisoquinoline alkaloid considered antitussive and also exhbits antitumor and antiisquemic
properties [3], solifenacin, an antimuscarinic agent that is used for the treatment of overactive
bladder [4] and EDL-155 showed an anti-glioma profile (Figure 1) [5].
The tetrahydroisoquinoline heterocycle is a privileged scaffold in the antitumor agents [6] that may
be introduced in the structures with potential antitumor activity by KRas inhibition [6]. It is known
that KRas inhibition plays an important role in the treatment of diverse cancer diseases such as
leukemia, lung adenocarcinoma, pancreas and colorectal adenocarcinoma. As a consequence, the
preparation of small molecules that bind to the KRas proteins has stimulated considerable interest. A
key feature of this research is the preparation of small structures possessing an isoquinoline nucleus
condensed with the 1,4-dioxan ring which must contribute with two sites of modest polarity.
Figure 1. Bioactive isoquinolines
2. Results and discussion

2.1. Chemistry
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Classical methods for preparing tetrahydroisoquinolines consist of intramolecular electrophilic
aromatic cyclization of arylethylamides under Bischler Napieralski conditions followed by reduction
[7]. A while ago, we developed a direct access to tetrahydroisoquinolines by cyclization of an imine
intermediate under acid catalysis, affording acylated tetrahydroisoquinolines in good to high yields
[8].
Target tetrahydroisoquinolines were synthesized as racemic mixtures according to the sequence of
reactions indicated in Schemes 1-4.
Reagents and conditions: i) CH3NO2 (10 mL), CH3COONH4 (3.5 eq), 100 °C, 2 h, 99%. ii) Pd/C (10% w/w),
H2 (7 atm), MeOH/EtOAc 1:3, 16 h, 53% or LiAlH4 (4 eq), THF, rt, 16 h, 61%.
Scheme 1. Synthesis of compound 3
The aldehyde 1 reacted with nitromethane and ammonium acetate according to the literature
procedure to afford 2 in excellent yield [9]. Reduction of the nitroethene 2 under hydrogen stream in
the presence of Pd/C led mostly to the vinyl amine and only traces of the amine 3. Hydrogenation of
2 (6-8 atm) with Pd/C slightly improved the reduction to 3 in 53% yield. Finally, the reduction of 2
with LiAlH4 under classical conditions afforded the amine 3 in acceptable yield (61%) (Scheme 1).
Using the last reduction procedure the overall yield of these 2-steps reaction was 61%.
The tetrahydroisoquinolines 8-9 were synthesized following our previously reported procedure [8].
Amination of 3,4,5-trimethoxybenzaldehyde (4) with the arylethylamine 3 using PTSA (p-
toluensulfonic acid) or CSA (canforsulfonic acid) in toluene did not yield the corresponding
isoquinoline due to solubility problems. However, treatment of the aldehyde 4 with the amine 3 in
EtOH at pH 6 in presence of molecular sieves 4 Å, followed by an intramolecular cyclization
reaction of the resulting imine with TFA (trifluoroacetic acid) and TFAA (trifluoroacetic anhydride)
gave the intermediate 6 which after hydrolysis with NaOH 2N gave the dioxine-isoquinoline 8 in
97% yield.

An alternative was tried to improve the yield of 8. Amination of the 3,4,5-trimethoxybenzaldehyde 4
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with the arylethylamine 3 in a Dean Stark apparatus, followed by addition of H3PO4 (85% in aqueous
solution) afforded the isoquinoline 8 in 32% yield (Scheme 3). From the phenethylamine 3 and the 4-
pyridinyl carbaldehyde 5 following the same procedure the isoquinoline 9 was obtained in acceptable
yield [8]. The oxidation of 8 with Pd in quinoline gave the isoquinoline 10 in moderate yield.
Reagents and conditions: i) toluene, PTSA, Dean Stark, 16 h, reflux. ii) TFA, TFAA, rt, 16 h, 33%. iii) NaOH
2N/MeOH 7:3, reflux, 16 h, 97% from 6. iii) NaOH 2N, MeOH, reflux, 16 h, 98% from 7. iv) Pd, quinoline,
200 ºC.
Alternative method: v) 3,4,5-trimethoxybenzaldehyde (1.1 eq), benzene, Dean-Stark, 110 °C, 3 h. vi) H3PO4
(2 mL of a 85% aqueous solution), Dean-Stark, 110 °C, 4 h, 32% yield.
Scheme 2. Synthesis of the dioxinisoquinolines 8-10
The isoquinoline 8 was involved in an alkylation process using classical conditions. In a general
procedure, 8 and 9 were treated with the corresponding alkyl halide, Et3N or K2CO3, KI and DMF to
obtain the desired compounds in low to satisfactory yields.
Reagents and conditions: a) R-X (25 eq over 7 days), Et3N (8 eq), KI (0.1 eq), rt, DMF. b) PdCl2((o-
tolyl)3P)2, (±)-BINAP and Cs2O3

Scheme 3. Synthesis of the dioxinisoquinolines 11-24
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The results of a number of alkylation attempts of 8 carried out revealed better yields when performed
at room temperature despite of an increased reaction time. Indeed, alkylation of 8 at 80 °C was faster
but favored the oxidation of the tetrahydroisoquinoline 8 to the isoquinoline 10.
The acylation of 8 under classical conditions gave the chloroformate 11 in acceptable yield (79%).
The tetrahydroisoquinoline 8 was alkylated with 2-chloroethanol under classical conditions in the
presence of Et3N and KI providing the alcohol 12 in 32% yield after purification.
Alkylation of 8 using chloroacetonitrile gives 13 in a satisfactory yield (84%). The nitrile group of
13 was reduced with LiAlH4 to yield 14 in acceptable yield.
The tetrahydroisoquinoline 8 was involved in an N-alkylation reaction under the same reaction
conditions as indicated above with 2-dimethylaminoethyl chloride, Et3N and KI to afford 15 in 54%
yield. The hyrolysis of 17 with aqueous NaOH solution provided the caraboxylic acid 18 in
acceptable yield. Using the same conditions 15 and 16 were obtained from 8 in 25 and 46%
respectively. The acetal 19 was synthesized by N-alkylation of 8 with the corresponding alkyl halide
in low yield.
The preparation of the tetrahydroisoquinolines 20 and 21 was accomplished by N-arylation of the
tetrahydroisoquinoline 8 following a general procedure described in the literature [10]. The
tetrahydroisoquinoline 8 reacted with 1-bromo-4-nitrobenzene in the presence of PdCl2((o-tolyl)3P)2,
(±)-BINAP and Cs2O3 in toluene yielding 20 in 33% yield. In the same way, the N-arylation of 8 with
4-bromobenzonitrile afforded 21 in 26% yield (Scheme 3). The low yields should be attributed to the
hindrance provided by the trimethoxyphenyl substituent at the C-1 position as well as the high
temperature required for this N-arylation that favored the formation of the aromatized isoquinoline
10.
The pyridine derivatives 22-24 were obtained using similar conditions to those employed for the
preparation of 11, 17 and 18 respectively.
In order to determine if the substitution of the 1-4-dioxane nucleus by a dimethoxy group results in
an increase or decrease in biological activity, two dimethoxy-isoquinoline analogues were
synthesized from the commercially available 2-(3,4-dimethoxyphenyl)ethylamine 29 following the

same experimental protocol used for the preparation of the isoquinoline 8. 3,4,5-
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Trimethoxybenzaldehyde (4) and 2-(3,4-dimethoxyphenyl)ethylamine (29) were coupled under
amination reaction conditions, followed by intramolecular cyclization of the corresponding imine
with TFA and TFAA to afford the amide 26 in moderate yield. The tetrahydroisoquinoline 27 was
prepared from hydrolysis of 28 with NaOH 2N in satisfactory yield (Scheme 4).
Reagents and conditions: i) toluene, PTSA, Dean Stark, 16 h, reflux. ii) TFA, TFAA, rt, 16 h, 33%. iii) NaOH
2N/MeOH 7:3, reflux, 16 h, 97%. iv) 2-chloroethanol (25 eq over 7 days), Et3N (8 eq), KI (0.1 eq), rt, DMF,
10 days, 16%. v) 1) (±)-epichlorohydrin (9 eq over 2 days), Et3N (8 eq), KI (0.1 eq), rt, DMF, 2 days, 45%. 2)
NaOH 2N/1,4-dioxane 5:2, rt, 2 days, 48%.
Scheme 4. Synthesis of isoquinolines 25 and 26
The dimethoxyisoquinoline 27 was alkylated giving two new compounds. First, treatment of 27 with
2-chloroethanol under classical conditions led to the alcohol 25 in low yield (Scheme 4). The
reaction was performed at room temperature because of the same stability problems than the amine
8.
Similarly, the alkylation of 27 with (±)-epichlorohydrin in the presence of Et3N and KI led to the
intermediate epoxide which was hydrolyzed with NaOH 2N to afford the diol 26 in moderate yield
(Scheme 4).
2.2. Biological evaluation

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2.2.1. In vitro cytotoxic activity on L1210 murine leukemia cell line
In vitro assay was used to screen all the newly synthesized isoquinolines for their effects on tumor
cell lines. In this study, we focused on the isoquinoline substituents to evaluate their effect over the
biological activities.
Isoquinolines 8 and 10-26 synthesized during this study were tested on L1210 murine leukemia cell
line and the compounds 12-16, 18, and 20-21 exhibited biologically significant anticancer activity.
The cytotoxicity showed by compound 14 was of great interest and also its action over the phase G1
of the cell cycle was remarkable, whereas compounds 8, 10, 25 and 26 were much less active and
isoquinolines 11, 17, 19 and 22-24 were inactive (Table 1). The results show that compounds 25 and
26 showed poor or no inhibition of cancer cell lines growth on the three studied lines.
The structure-activity-relationships between 12 and 25 show that the dimethoxide groups result in a
decrease of biological activity compared to the 1,4-benzodioxin moiety. The structure-activity-
relationships between 25 and 26 reveals that a diol side chain results in an increase of antitumor
activity compared to a terminal alcohol side chain. The presence of amino ethyl group (Table 1,
compounds 14 and 15) as isoquinoline substituent gives selectivity by the phase G1, whereas a
hydroxyethyl substituent favors the action over the 8N subphase (Table 1, compound 12). An aryl as
N-substituent maintains a good cytotoxic activity (Table 1, compounds 20 and 21). Curiously the
nitrobenzene group (compound 20) facilitates the action over de phase G1 and the benzonitrile
(compound 21) on the 8N subphase. With respect to the substituent at the C-1 of the
tetrahydroisoquinoline, the 3,4,5-trimethoxyphenyl group contributes to a better therapeutic profile
than 4-pyridinyl.
Table 1. In vitro cytotoxity activity of compounds 8 and 10-26
Compound
Cycle (L1210)
IC50 (µM, L1210)
21.1±3.40
8
G1 (+100 µM)
G2M+++100 µM
Not tested
25.1±3.20
10
>10
11
12
2.6±0.87
70% 8N 5 µM

4.2±0.78
13
14
15
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G1 (+100 µM)
2.0±0.72
3.7±0.66
G1 (+25 µM)
G1 (++25 µM)
Not specific
Not tested
4.1±0.89
>10
16
17
18
19
20
9.4±1.20
>10
Not specific
Not tested
4.6±075
G1 (+25 µM)
3.6±0.69
21
70% 8N 10 µM
Not tested
>10
22
>10
Not tested
23
>10
Not tested
24
23.4±2.56
15.3±2.43
21,0±2.01
Not specific
Not specific
G2M (++50 µM)
25
26
Acronycine
Results are expressed as the mean (n = 3) of L1210 inhibition and expressed by IC
50
2.2.2. KRas inhbition
The isoquinolines 12, 13, 14, 20 and 21 were selected for testing at the Eli Lilly Laboratories [11]
(Indianapolis, USA) to estimate their antitumor activities and to study their mode of action. The
KRas activity of the five tested compounds were carried out on four different colon cancer cell lines
(HCT KRasSL, RKO KRasSL, Colo 320 KRasSL and SNU-C1 KRasSL) in three different
concentrations (0.2 µM, 2 µM and 20 µM) (Table 2).
Table 2. KRas inhibition of the tetrahydroisoquinoline analogues
Products
Assays

ACCEPT1E2D.1 MANUSCRIPT
18
0
16.2
56.9
0
0
19.3
55.9
17.9
32.9
35.9
16.0
45.2
95.8
7.7
% In. 0.2 µM
% In. 2 µM
% In. 20 µM
% In.0.2 µM
% In. 2 µM
12.1
46.5
0
17.0
49.1
0
6.3
16.3
10.1
76.7
12.2
0
79.2
0
92.7
0
70.5
0
66.5
0
% In. 20 µM
% In. 0.2 µM
% In. 2 µM
8.2
20.1
4
22.5
*SN
U-
78.2
74.7
85.3
92.5
89.9
% In. 20 µM
12.3
11.5
10.0
6.0
12.2
2.4
17.7
35.0
6.6
% In. 0.2 µM
% In. 2 µM
C1,
Colo
n
32.5
%
Inhib 20
19.7
59.8
57.1
42.7
43.5
µM
320, RKO and HCT are colon cancer cell lines.
The results show that all of the studied compounds have a higher overall KRas inhibition.
Surprisingly, the alcohol 12 displayed a lower KRas inhibition than the other isoquinoline analogues
(see Table 2). The amine isoquinoline 14 presents the best KRas activity profile on RKO KRasSL of
the tested tetrahydroisoquinoline analogues, which reveals that a terminal ionic interaction leads to
an increase of KRas inhibition. The N-arylisoquinoline 20 and 21 have the highest KRas inhibition
on Colon 320 KRas SL cell line, which suggests that a low electron density aromatic side chain
structure results in a higher KRas activity for this type of cancer cells. The isoquinoline 21 shows a
surprisingly high KRas activity at 0.2 µM concentration (RKO KRasSL 95.8% inhib., HCT KRasSL
35.9% inhib.) which suggests that its activity is not dose-dependent for these types of cancer cell
lines.
2.2.3. Angiogenesis and antiosteoporosis activities
The antiangiogenesis evaluation was carried out for 12, 13, 14, 20 and 21. The results show that the
isoquinolines 13 and 14 possess an interesting antiangiogenic activity while 20 and 21 demonstrated
moderate activity and 12 was inactive (Table 3). The nitrile isoquinoline 13 was the most potent

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antiangiogenic in this study and shows the highest antiangiogenesis (IC50 = 2.9 µM) and
antiosteoporosis activity, which suggests that a dipolar lipophilic terminal group such as a nitrile
group results in an increase of antiangiogenesis and antiosteoporosis activity (Table 3).
These results suggest that a functionalized group on the side chain bound to the isoquinoline nitrogen
atom in general, improves the antitumor properties significantly.
Table 3. Antiangiogenesis and antiosteoporosis activity of the tetrahydroisoquinoline analogues
Products
Assays
0
15
8.5
6
16.6
55.3
% In. 2 µM
%
In. 10
6.7
76.9
58.6
37.5
µM
N.A
N.A
2.9 µM
6.5 µM
>10 µM
>10 µM
>10 µM
>10 µM
IC50
IC50
>10 µM
>10 µM
%Stim
µM
2
0
4.6
0
0.3
4,3
27.2
14,2
%Stim 10
µM
2,9
51,8
25,4
*Angiogenesis nuclear area, β-catenin osteoporosis and angiogenesis tube area (tracheal tube area)
3. Molecular Modeling Studies
Because the potency of the compounds described in Tables 2 is different depending on the biological
assay and also for having a minimal antiangiogenic effect (Table 3) compound 12 was selected as
representative member of the diverse tetrahydroisoquinoline analogues described above for the
determination of its most probable binding site to KRas. Bearing this in mind and aimed at exploring
the conformational space of the KRas-Lig system [12] thoroughly, ten KRas-Lig8 different
complexes were prepared and subjected to a 100 ns of MD trajectory, as explained in the Methods
section. Visual inspection of the MD trajectories shows different possible situations. In some of the
calculations none of the eight ligands remains bound to the KRas protein in the last ten nanoseconds;
in others, only one ligand is bounded to the KRas protein during the last ten nanoseconds, although

they are found in different pockets; and finally, in some of the calculations more than one ligand
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remained bound to the KRas protein during the last ten nanoseconds [13]. In the latter case, ligands
were bound, in general, in different pockets but found close together due to van der Waals attractive
inter-ligands interactions. Once the multiple copies MD calculations were analyzed and aimed at
achieving a better understanding of the binding process, avoiding any bias due to the presence of
multiple-ligands in the calculations, eighteen complexes with only one ligand, extracted from these
calculations (see Molecular Modeling Methods) were used to analyze the behavior of the complex
along the molecular dynamics with different starting points having the ligand bound to different
sites.
For a clearer discussion about the interactions of the ligand with the KRas protein, Figure 2 shows its
most relevant structural motifs together with the different allosteric sites explored in this study. The
stating position and the behavior of the ligand along the MD in each of the eighteen studied
complexes are described in Table S1 of Supporting Information. Thus, six MD starts at the p1 pocket
(MD 1 to 6). However, two of them after different production times leave the protein (MD 3 and 4).
The MD 5 and 6 leave the protein but finally return to the protein at different sites. Molecular
dynamics 1 and 2 remain at the p1 pocket being the first the most stable of all. Three MD start at the
p2 pocket but two of them leave the protein. Four MD start at p4 and they all go. However, two of
them return to the hypervariable zone. Also four MD start between helices α-3 and α-4. None of
them remain at this position. Finally, one MD start between β2 and β3 strands but go out and return
to another place.
In order to determine simulations convergence as well as the stability of the different complexes, the
total binding free energy calculated by the MMGBSA approach was plotted as a function of the
trajectory length. The plot for the most stable KRas-Ligand complex (MD 1) is showed in Figure 3.
As it can be seen, the system remains stable during the last 300 ns with an average value of ∆G
binding = -27.4 kcal/mol for the last 20 ns. The complex starts at the p1 pocket with the three
methoxide group inserted in the pocket and the fused rings exposed to the solvent. However, around
200 ns and during 50 ns the ligand changes its orientation doing a complete rotation to put the fused
rings inside the p1 pocket and two of the methoxide group exposed to the solvent.

Also, for a better description of the behavior of the different complexes along the molecular
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dynamics progression, the ∆Gbinding calculated using the MMGBSA methodology for each of the
remaining seventeen complexes is reported in Figure S2 of the Supporting Information.
Residues of KRas that contribute to the binding free energy of the KRas-Ligand complex were
analyzed during the last 20ns of the most stable molecular dynamics (MD 1). A 3D representation of
the binding site and the interacting residues is showed in Figure 4 (3D structure in PDB format
available as supporting information). Also, the averaged numerical values are reported in Figure S3
of the Supporting Information. From Figure 4 it can be seen that the ligand inserts in a deeper pocket
where the fused rings interact with the hydrophobic residues Leu56, Glu63, Tyr64, Tyr71 and Thr74.
However, the picture shows the possibility to increase the size of this ligand on the left of the pocket
by modifications in the 1,4-dioxane group and in one of the methoxide groups. Also, the complex
makes two stable hydrogen bonds (HB) with the protein (see Figure 4c). The first HB is established
between the oxygen carbonyl of residue Glu37 and the NH of the isoquinoline nucleus. As it can be
seen in Figure S4 of Supporting Information, this hydrogen bond is stable along the last 20 ns of MD
(86.4% of occupation). The second hydrogen bond is formed between the OH of the ligand and both
oxygens of the Glu37 residue. Figure S5 of Supporting Information shows the evolution of both
distances along the time. We can see that this HB exists during the mayor part of the time (59.3% of
occupation) but some time disappears. Thus, another hydrogen donor able to make stronger
interactions with Glu37 should improve the ligand affinity. This fact can explain the better activity
of compound 14 where the OH group is replaced by a NH2 group.

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Figure 2. The most relevant structural motifs of the KRas protein are indicated. The functionally important
switch regions Sw1 and Sw2 are highlighted in blue and orange respectively. The location of the four allosteric
ligand-binding sites studied in this article is showed in colored mesh (p1 to p4). The GTP cofactor is
represented in sticks.
10
MD_1
0
-10
-20
-30
-40
-50
-60
0
100
200
300
400
500
600
700
time (ns)
Figure 3. Evolution of ∆Gbinding versus time for the most stable KRas-Ligand complex (MD_1) using the
MMGBSA algorithm.

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Figure 4. A Spatial representation of the complex KRas-Ligand for the most stable MD (MD_1). a)
Description of the p1 pocket with the Ligand. b) Spatial representation of the most important residues that
interact with the Ligand. c) The KRas-Ligand complex showing the hydrogen bonds in green. The 4DSN
structure was used as starting point for the complexes.
4. Conclusion
In conclusion, the synthesis of substituted isoquinolines was accomplished through a process of
aldehyde amination followed by cyclization and N-alkylation or N-arylation under classical
conditions. The synthesized isoquinolines were tested for its biological activities. However, a
rational design of novel compounds with increased inhibitory activity against KRas could be difficult
without the knowledge of the binding site and therefore the interactions between the synthetized
compounds and the KRas protein. In this sense, molecular modeling can help in obtaining important
information and contributing to explain the in vitro results. The amine 14 and the alcohol 12 exhibit
in vitro promising antileukemic activity (IC50 = 2.0 and 2.6 µM on L1210 cells respectively) and an
investigation of cytotoxic mechanisms suggest the involvement of KRas inhibition in both without
additional anti-angiogenic effects in the case of compound 12.
5. Experimental section.
5.1. Chemistry
Melting points were obtained on an MFB-595010M Gallenkamp apparatus in open capillary tubes
and are corrected. IR spectra were obtained using a FTIR Perkin-Elmer 1600 Infrared
Spectrophotometer. Only noteworthy IR absorptions are listed (cm-1). 1H and 13C NMR spectra were
recorded on a Varian Gemini-200 (200 and 50.3 MHz respectively) or Varian Gemini-300 (300 and

75.5 MHz) or Varian Gemini-400 (400 and 100.6 MHz) Instrument using CDCl as solvent with
ACCEPTED MANUSCRIPT
3
tetramethylsilane as internal standard or (CD3)2CO. Other 1H NMR spectra and heterocorrelation 1H-
13C (HMQC and HMBC) experiments were recorded on a Varian VXR-500 (500 MHz). Mass spectra
were recorded on a Helwett-Packard 5988-A. Column chromatography was performed with silica gel
(E. Merck, 70-230 mesh). Reactions were monitored by TLC using 0.25 mm silica gel F-254 (E.
Merck). Microanalysis was determined on a Carlo Erba–1106 analyzer. All reagents were of
commercial quality or were purified before use. Organic solvents were of analytical grade or were
purified by standard procedures. Commercial products were obtained from Sigma-Aldrich.
Elemental analysis was used to ascertained purity of >95% for all compounds of this work for which
biological activities were determined.
5.1.1. (E)-6-(2-Nitrovinyl)-2,3-dihydro[1,4]benzodioxine (2). 2,3-Dihydro[1,4]benzodioxin-6-
carbaldehyde (1) (1 g, 6.1 mmol) and ammonium acetate (123 mg, 1.57 mmol) were dissolved in
nitromethane (10 mL) in a flame-dried round-bottom flask under argon and refluxed under stirring
for 16 h. Then, TLC of the crude mixture (CH2Cl2 / hexane 7:3) indicated formation of a bright
yellow compound (Rf = 0.55) and complete consumption of the starting material (Rf = 0.35). The
crude mixture was filtered and concentrated in vacuo to afford (E)-6-(2-nitrovinyl)-2,3-
dihydro[1,4]benzodioxine (2) (1.25 g, 99% yield) as a bright yellow solid. Rf = 0.55 (CH2Cl2/hexane
o
1
7:3). M.p. 148-150 C (EtOAc). NMR H (CDCl3, 300 MHz) δ (ppm): 4.30 (s, 4H, CH2-O (x 2)),
6.91 (d, J = 6.0 Hz, 1H, H-8), 7.05 (dd, J = 3.0 Hz, J = 6.0 Hz, 1H, H-7), 7.07 (d, J = 3.0 Hz, 1H, H-
5), 7.47 (d, J = 13.5 Hz, 1H, CH=CH-NO2), 7.90 (d, J = 13.5 Hz, 1H, CH=CH-NO2). NMR 13C
(CDCl3, 75.5 MHz) δ (ppm): 64.6 (CH2 (x 2)), 117.7 (CH, C-5), 117.9 (CH, C-8), 122.0 (C, C-6),
122.0 (CH, C-7), 130.2 (CH, CH-NO2), 142.7 (C, C-8a), 143.9 (C, C-4a), 151.1 (CH, CH=CH-NO2).
5.1.2.
2-(2,3-Dihydro[1,4]benzodioxin-6-yl)ethylamine
(3).
(E)-6-(2-Nitrovinyl)-2,3-
dihydro[1,4]benzodioxine (2) (1.6 g, 7.68 mmol) was dissolved in EtOAc (80 mL) and MeOH (5
mL). 10% Palladium on charcoal catalyst (268 mg, 10% w/w) was added and the mixture was put
under a 7 atm hydrogen atmosphere under stirring for 2 days. After 16 h, TLC of the crude mixture
(hexane/EtOAc) indicated formation of a new compound and complete consumption of starting
material (Rf = 0.80). The mixture was filtered and concentrated in vacuo to afford the
phenethylamine 3 (852 mg, 53% yield as a brown oil. Rf = 0.55 (hexane/EtOAc 2:8). IR (film) ν cm-

1
1
ACCEPTED MANUSCRIPT
: 3100 (NH), 2910 (CH), 1200 (C-O). NMR H (CDCl3, 300 MHz) δ (ppm): 2.63 (t, J = 7.6 Hz, 2H,
Ar-CH2-CH2), 2.91 (t, J = 7.6 Hz, 2H, CH2-CH2-NH2), 4.24 (s, 4H, CH2-O (x 2)), 6.66 (dd, J = 2 Hz,
8.2 Hz, 1H, H-7), 6.70 (d, J = 2 Hz, 1H, H-5), 6.80 (d, J = 8.2 Hz, 1H, H-8). NMR 13C (CDCl3, 75.5
MHz) δ (ppm): 38.7 (CH2, CH2-Ar), 43.1 (CH2, CH2-N), 63.8 (CH2, CH2-O), 63.9 (CH2, CH2-O),
116.7 (CH, C-5), 116.9 (CH, C-8), 121.2 (CH, C-7), 132.5 (C, C-6), 141.4 (C, C-4a), 142.9 (C, C-
8a).
5.1.3.
2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)ethylamine
(3).
(E)-6-(2-Nitrovinyl)-2,3-
dihydro[1,4]benzodioxine (2) (1 g, 4.82 mmol) was dissolved in THF (20 mL). LiAlH4 (740 mg,
19.5 mmol) was added portion wise. The reaction was stirred at rt for 20 h and TLC of the reaction
mixture (hexane/EtOAc 1:1) indicated formation of a new compound and complete consumption of
starting material (Rf = 0.60). The crude mixture was quenched dropwise with water (1 mL) and
filtered. The solid residue was extracted with CH2Cl2 (3 x 20 mL) and the combined organic phases
were dried (Na2SO4), filtered and concentrated in vacuo to afford 2-(2,3-dihydro[1,4]benzodioxin-6-
yl)ethylamine (3) (530 mg, 61% yield) as a pale brown oil. This material was identical in all respects
with that previously described.
5.1.4. 2-(2,3-Dihydro[1,4]benzodioxin-6-yl)ethylamine (3). 6-(2-benzylamineethyl)-2,3-dihydro
benzo[1,4]dioxine (200 mg, 1.01 mmol) was dissolved in EtOAc (80 mL) and MeOH (5 mL). 10%
Palladium on charcoal catalyst (268 mg) and HCl (30 µL) were added and the mixture was put under
hydrogen atmosphere under stirring for 2 days. The crude mixture was concentrated in vacuo. The
residue was purified by silica gel flash column chromatography to afford 2-(2,3-
dihydro[1,4]benzodioxin-6-yl)ethylamine (3) (71 mg, 53% yield) as a brown oil. Analytical data was
identical with the previously described compound.
5.1.5.
(6-(3,4,5-Trimethoxyphenyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxino[2,3-g]isoquinolin-7-yl)
2,2,2-trifluoroacetate (6). To a solution of 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethylamine 3
(250 mg, 1.39 mmol) in ethanol (20 mL) was added HCl (0.1 mL), molecular sieves (50 mg) and
3,4,5-trimethoxybenzaldehyde 4 (410 mg, 2.09 mmol) in a flame-dried round-bottom flask under
argon. Et3N was added until pH 6-6.5 was reached and the mixture was heated to reflux under
stirring for 16 h. The mixture was concentrated in vacuo and the residue was dissolved in EtOAc (20
mL), washed with NaOH (3 x 20 mL of a 2N solution), dried (Na2SO4), filtered and concentrated in

vacuo to afford brown oil. CF COOH (3 mL, excess amount) and (CF CO) O (3 mL, excess amount)
ACCEPTED MANUSCRIPT
3
3
2
were added and the crude mixture was refluxed under stirring for 16 h. Then, TLC of the crude
mixture (EtOAc/hexane 1:1) indicated presence of a new compound (Rf = 0.75) and uncomplete
consumption of the 3,4,5-trimethoxybenzaldehyde (Rf = 0.80). The mixture was dissolved in EtOAc
(20 mL), washed with NaOH (3 x 30 mL of a 2N solution), dried (Na2SO4) filtered and concentrated
in vacuo. The crude residue was purified by silica gel flash column chromatography (hexane/EtOAc
7:3) to afford the desired isoquinoline 6 (102 mg, 16% yield) as a brown oil. Rf = 0.75
(hexane/EtOAc 1:1). IR (film) ν cm-1: 1686 (C=O), 1504 (Ar-H), 1299 (Ar-O), 1127 (C-O). NMR 1H
(CDCl3, 300 MHz) δ (ppm): 2.75-2.82 (m, 1H, CH2-CH2-N), 2.94-3.05 (m, 2H, CH2-CH2-N, CH2-
CH2-N), 3.18-3.25, (m, 1H, CH2-CH2-N), 3.77 (s, 6H, CH3-O (x 2)), 3.83 (s, 3H, CH3-O), 4.20-4.25
(m, 4H, CH2-O), 6.45 (s, 2H, H-2’, H-6’), 6.57 (s, 1H, H-5), 6.62 (s, 1H, H-6), 6.70 (s, 1H, H-10).
NMR 13C (CDCl3, 75.5 MHz) δ (ppm): 28.4 (CH2, CH2N), 39.5 (CH2, CH2-Ar), 56.2 (CH, C-6), 60.8
(CH3, OCH3 (x 2)), 64.4 (CH3, OCH3), 64.6 (CH2, CH2-O(x 2)), 106.2 (CH, C-2’, C-6’), 116.5 (CH,
C-5), 116.3 (C, J = 288 Hz, CF3), 116.8 (CH, C-10), 126.0 (C, C-1’), 136.5 (C, C-5a), 137.8 (C, C-
9a), 142.3 (C, C-10a), 142.9 (C, C-4a), 152.9 (C, C-4’), 153.0 (C, C-3’, C-5’), 156.4 (C, C=O). MS
(EI) (m/z, %): 453 (M+, 71), 438 (M+-CH3, 100), 286 (M+-C9H11O3, 32). (C9H11O3 = 3,4,5-
trimethoxyphenyl. MS EI m/z (%): 453 (M+, 12), 355 (M+-C2F3O, 100).
5.1.6. 6-(3,4,5-Trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-g]isoquinoline (8). The
isoquinolin-trifluoroacetate 6 (285 mg, 0.63 mmol) was dissolved in MeOH (15 mL), then NaOH 2N
(45 mL) was added and the reaction was heated to reflux overnight under stirring. Then, TLC of the
crude mixture (EtOAc/hexane 1:1) showed total consumption of SM (Rf = 0.80) and formation of the
desired product (Rf = 0.10). The methanol was evaporated in vacuo and the aqueous phase was
extracted with diethyl ether (3 x 20 mL). The combined organic phases were dried (Na2SO4), filtered
and concentrated in vacuo to afford the substituted isoquinoline 8 (218 mg, 97% yield) as a brown
solid. Rf = 0.30 (EtOAc). M.p. 131-132 oC (diethyl ether). IR (KBr) ν cm-1: 3100 (NH), 1589 (C=C),
1
1297 (Ar-O), 1125 (C-O). RMN H (CDCl3, 300 MHz) δ (ppm): 2.54-2.65 (m, 1H, CH2-CH2-N),
2.83-3.03 (m, 2H, CH2-CH2-N, CH2-CH2-N), 3.18-3.25 (m, 1H, CH2-CH2-N), 3.75 (s, 6H, CH3-O (x
2)), 3.77 (s, 3H, CH3-O), 4.13 (m, 4H, CH2-O (x 2)), 4.81 (s, 1H, H-6), 6.22 (s, 1H, H-5), 6.45 (s,

13
ACCEPTED MANUSCRIPT
2H, H-2’, H-6’), 6.56 (s, 1H, H-10). RMN C (CDCl3, 75.5 MHz) δ (ppm): 28.9 (CH2, C-9), 43.1
(CH2, CH2N), 56.0 (CH3, OCH3 (x 2)), 60.7 (CH3, OCH3), 62.4 (CH, C-6), 64.3 (CH2, OCH2), 64.2
(CH2, OCH2), 105.6 (CH, C-2’, C-6’), 115.9 (CH, C-5), 116.6 (CH, C-10), 128.1 (C, C-5a), 131.3 (C,
C-9a), 140.1 (C, C-1’), 141.4 (C, C-4a), 141.9 (C, C-10a), 152.8 (C, C-4’), 152.9 (C, C-3’, C-5’).
HRMS ESI(+) m/z for C20H24NO5 [M+H]+ calcd. 358.1654, found: 358.1651.
5.1.7.
6-(3,4,5-Trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-g]isoquinoline
(8)
(alternative synthesis). The phenethylamine
3
(228 mg, 0.84 mmol) and 3,4,5-
trimethoxybenzaldehyde (4) (274 mg, 0.92 mmol) were dissolved in benzene (15 mL) in a flame-
dried round-bottom flask under argon. The reaction mixture was heated to reflux under stirring in a
o
Dean Stark reaction for 4 h. The solution was cooled at 0 C and H3PO4 (2 mL of 85% aqueous
solution) was added. The reaction mixture was refluxed under stirring in a Dean Stark reaction for 3
h and TLC of the crude mixture (EtOAc) indicated presence of the tetrahydroisoquinoline desired
product (Rf 0.30) and uncomplete consumption of the aldehyde (Rf = 0.80). The reaction mixture was
o
cooled at 0 C and quenched with NaOH (5 mL of a 2N aqueous solution). Then the mixture was
dissolved in CH2Cl2 (20 mL), washed with NaOH 2N (3 x 30 mL) and the organic phases were re-
extracted with CH2Cl2 (3 x 20 mL). The combined organic phases were dried (Na2SO4), filtered and
concentrated in vacuo. The residue was purified by silica gel flash column chromatography
(hexane/EtOAc 3:7) to afford the tetrahydroisoquinoline 8 (200 mg, 40% yield) as a brown solid.
The reaction was scaled up using 2 g of phenethylamine 3 to afford 8 (2.4 g, 55% yield) as a brown
solid. Analytical data was identical with the previously described compound.
5.1.8. 6-(3,4,5-Trimethoxyphenyl)-2,3-dihyhydro[1,4]dioxino[2,3-g]isoquinoline (10). 6-(3,4,5-
Trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-g]isoquinoline (8) was oxidized to
isoquinoline 10 using Pd/C in decahydronaphthalene [8a].
5.1.9. General N-alkylation procedure to tetrahydroquinolines. The tetrahydroisoquinoline (150
mg, 0.42 mmol) was dissolved in DMF (7 mL) and put in a flame-dried round-bottom flask under
argon. The alkylating agent (0.11 mL, 1.66 mmol), KI (cat) and Et3N (0.47 mL, 3.36 mmol) were
added under argon. The reaction was stirred at rt and the corresponding alkylating agent (0.11 mL,
1.66 mmol) and KI (cat) were added every day under argon for 7 days. Water (20 mL) was added and
the crude mixture was extracted with diethyl ether (3 x 20 mL). The combined organic phases were

washed with H O (5 x 30 mL), dried (Na SO ), filtered and concentrated in vacuo. The crude residue
ACCEPTED MANUSCRIPT
2
2
4
was purified by silica gel flash column chromatography to afford the corresponding isoquinoline.
5.1.10. 7-Etoxycarbonil-2-[6-(3,4,5-trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-g]
isoquinoline (11). From the tetrahydroisoquinoline 8 (55 mg, 0.15 mmol) and ethyl chloroformate
(0.05 mL, 0.23 mmol, d 1.125), and following the general procedure described above the title
compound was obtained (53 mg, 79% yield) as yellow oil. Rf = 0.52 (EtOAc/Hex 5:5). IR (film) ν
cm-1: 1856-1920 (C-H), 1654, 1502, 1465, 1222 (Ar-0), 1082 (Ar-O). NMR 1H (CDCl3, 200 MHz) δ
(ppm): 1.28 (t, J = 7Hz, 3H, CH3), 2.60-2.85 (m, 2H, H-9), 3.13-3.27 (m, 2H, H-8), 3.79 (s, 6H,
CH3-O (x 2)), 3.80 (s, 3H, CH3-O), 4.19 (q, J = 7 Hz, 4H, CH2-O), 4.36 (s, 1H, H-1), 6.43 (s, 2H, H-
2’, H-6’), 6.54 (s, 1H, H-10), 6.66 (s, 1H, H-5). NMR 13C (CDCl3, 50.4 MHz) δ (ppm): 14.8 (CH3),
27.8 (CH2), 38.2 (CH2), 56.1 (CH3-O x 2), 57.1 (CH), 60.6 (CH3-O), 61.5 (CH2-O), 64.3 and 64.4
(CH2-O x 2), 105.6 (C-2’and C-6’), 116.6 (C-5 and C-10), 127.9 (C-9a), 137.1 (C-5a, C-4’), 138.5
(C-1’), 141.8 (C-4a), 142.5 (C-10a), 152.8 (C-3’and C-5’), 155.3 (CO). HRMS ESI(+) m/z for
C23H28NO7 [M+H]+ calcd. 430.1866, found: 430.1854.
5.1.11. 2-[6-(3,4,5-Trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-g]isoquinolin-7-yl)]
ethanol (12). From the tetrahydroisoquinoline 8 (150 mg, 0.42 mmol) and 2-chloroethanol (0.11 mL,
1.66 mmol), and following the general procedure described above the title compound was obtained
o
(54 mg, 32% yield) as a white solid. Rf = 0.50 (EtOAc/MeOH 9:1). M.p. 120-122 C (CH2Cl2). IR
(film) ν cm-1: 3527 (OH), 1852-1920 (C-H), 1593, 1502, 1465, 1422 (Ar-H), 1302, 1127 (Ar-O),
1
1062 (Ar-O). NMR H (CDCl3, 300 MHz) δ (ppm): 2.36-2.43 (m, 1H, Ar-CH2-CH2-N), 2.52-2.61
(m, 1H, Ar-CH2-CH2-N), 2.71-2.85 (m, 2H, N-CH2-CH2-OH), 2.95-3.05 (m, 1H, Ar-CH2-CH2-N),
3.21-3.28 (m, 1H, Ar-CH2-CH2-N), 3.40-3.47 (m, 1H, CH2-OH), 3.65-3.73 (m, 1H, CH2-OH), 3.81
(s, 6H, CH3-O (x 2)), 3.84 (s, 3H, CH3-O), 4.15-4.25 (m, 4H, O-CH2-CH2-O), 4.40 (s, 1H, H-6), 6.26
(s, 1H, H-5), 6.45 (s, 2H, H-2’, H-6’), 6.63 (s, 1H, H-10). NMR 13C (CDCl3, 75.5 MHz) δ (ppm):
28.6 (CH2, Ar-CH2-CH2-N). 47.8 (CH2, Ar-CH2-CH2-N), 55.6 (CH2, N-CH2-CH2-OH), 56.5 (CH3,
CH3-O (x 2)), 58.5 (CH2, Ar-CH2-CH2-OH), 61.2 (CH3, CH3-O), 64.7 (CH2, O-CH2-CH2-O), 64.7
(CH2, O-CH2-CH2-O), 69.4 (CH, C-6), 106.7 (CH, C-2’, C-6’), 116.4 (CH, C-5), 117.1 (CH, C-10),
127.7 (C, C-5a), 131.3 (C, C-9a), 139.6 (C, C-1’), 141.9 (C, C-4a), 142.4 (C, C-10a), 153.5 (C, C-3’,

C-4’, C-5’). MS (EI) (m/z, %): 401 (M+, 6), 370 (M+-CH OH, 100), 234 (M+-C H O , 78). (C H O
3
ACCEPTED MANUSCRIPT
2
9
12
3
9
11
= 3,4,5-trimethoxyphenyl). HRMS ESI(+) m/z for C22H28NO6 [M+H]+ calcd. 402.1917, found:
402.1912.
5.1.12. 2-(6-(3,4,5-Trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-g]isoquinolin-7-yl)
acetonitrile (13). From the tetrahydroisoquinoline 8 (150 mg, 0.42 mmol) and chloroacetonitrile
(0.08 mL, 1.26 mmol), and following the general procedure described above the title compound was
obtained (140 mg, 84% yield) as a brown solid. Rf = 0.85 (EtOAc). M.p. 144-146 oC
(hexane/EtOAc). IR (film) ν cm-1: 2925 (C-H), 2363 (CN), 1588, 1503, 1455, 1417 (Ar-H), 1295,
1
1233 (Ar-O), 1122, 1066 (C-O). NMR H (CDCl3, 300 MHz) δ (ppm): 2.65-2.75 (m, 1H, Ar-CH2-
CH2-N), 2.85-3.00 (m, 1H, Ar-CH2-CH2-N), 3.01-3.35 (m, 2H, Ar-CH2-CH2-N), 3.43 (s, 2H, CH2-
CN), 3.80 (s, 6H, OCH3 (x 2)), 3.82 (s, 3H, OCH3), 4.09-4-26 (m, 4H, O-CH2-CH2-O), 4.45 (s, 1H,
H-6), 6.18 (s, 1H, H-5), 6.52 (s, 2H, H-2’, H-6’), 6.58 (s, 1H, H-10). NMR 13C (CDCl3, 75.5 MHz) δ
(ppm): 29.0 (CH2, Ar-CH2-CH2-N), 44.3 (CH2, CH2-N), 50.5 (Ar-CH2-CN), 56.5 (CH3, CH3-O (x
2)), 61.1 (CH3, CH3-O), 64.6 (CH2, O-CH2-CH2-O), 64.7 (CH2, O-CH2-CH2-O), 67.6 (CH, C-6),
106.3 (CH, C-2’, C-6’), 115.1 (C, CN), 116.4 (CH, C-5), 116.9 (CH, C-10), 126.8 (C, C-5a), 130.7
(C, C-9a), 137.7 (C, C-1’), 137.9 (C, C-1’), 142.0 (C, C-4a), 142.5 (C, C-10a), 153.4 (C, C-4’), 153.8
(C, C-3’, C-5’). MS (EI) (m/z, %): 396 (M+, 33), 229 (M+-C9H12O3, 100). (C9H11O3 = 3,4,5-
trimethoxyphenyl). HRMS ESI(+) m/z for C22H25NO5 [M+H]+ calcd. 397.1763, found: 397.1764
5.1.8. 2-(6-(3,4,5-Trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-g]isoquinolin-7-yl)
ethylamine (14). The isoquinoline 13 (115 mg, 0.29 mmol) was dissolved in THF (10 mL) in a
flame-dried round-bottom flask under argon, and LiAlH4 (35 mg, 0.86 mmol) was added. The
reaction was stirred at rt for 16 h and TLC of the crude mixture (EtOAc/MeOH, 7:3) showed
formation of a new compound (Rf = 0.25) and complete consumption of SM (0.95). The crude
mixture was quenched dropwise with water and filtered. The solid residue was extracted with CH2Cl2
(3 x 20 mL) and the combined organic phases were dried (Na2SO4), filtered and concentrated in
vacuo. The residue was purified by silica gel flash column chromatography (EtOAc/MeOH, 2:8) to
afford the isoquinoline 14 (60 mg, 52% yield) as a yellow solid. Rf = 0.25 (EtOAc/MeOH 7:3). M.p.
55-60 °C (hexane/EtOAc). IR (film) ν cm-1: 3300-3100 (NH2), 2923, 2834 (C-H), 1588, 1503, 1456,
1418 (Ar-H), 1296, 1232 (Ar-O) 1122, 1066 (C-O). NMR 1H (CDCl3, 300 MHz) δ (ppm): 2.40-2.56

(m, 2H, CH -Ar), 2.59-2.85 (m, 4H, CH -N (x 2), 2.96-3.06 (m, 1H, CH -N), 3.15-3.22 (m, 1H, CH -
ACCEPTED MANUSCRIPT
2
2
2
2
N), 3.81 (s, 6H, CH3-O (x 2)), 3.84 (s, 3H, CH3-O), 4.14-4.20 (m, 4H, O-CH2-CH2-O), 4.27 (s, 1H,
H-6), 6.25 (s, 1H, H-5), 6.51 (s, 2H, H-2’, H-6’), 6.61 (s, 1H, H-10). NMR 13C (CDCl3, 75.5 MHz) δ
(ppm): 28.9 (CH2, Ar-CH2-CH2-N), 39.4 (CH2, N-CH2-CH2-NH2), 48.4 (CH2, Ar-CH2-CH2-N), 56.4
(CH3, OCH3 (x 2)), 57.4 (CH2, 2H, N-CH2-CH2-NH2) , 61.1 (CH3, OCH3), 64.6 (CH2, O-CH2-CH2-
O), 64.7 (CH2, O-CH2-CH2-O), 69.9 (CH, C-6), 106.7 (CH, C-2’, C-6’), 116.4 (CH, C-5), 117.0 (CH,
C-10) , 127.9 (C, C-5a), 131.8 (C, C-9a), 140.2 (C, C-1’), 141.8 (C, C-4a), 142.2 (C, C-10a), 153.4
(C, C-3’, C-4’, C-5’). MS EI m/z (%): 400 (M+, 1), 370 (M+-CH5N, 64) 355 (M+-C2H7N, 57). HRMS
ESI(+) m/z for C22H29N2O5 [M+H]+ calcd. 401.2076, found: 401.2082.
5.1.9.
N,N-Dimethyl-(6-(3,4,5-trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-
g]isoquino lin-7-yl))ethylamine (15). From the tetrahydroisoquinoline 8 (100 mg, 0.28 mmol) and
2-dimethylaminoethyl chloride (121 mg, 0.84 mmol), and following the general procedure described
above the title compound was obtained (67 mg, 54% yield) as a yellow oil. Rf = 0.25 (EtOAc/MeOH
8:2). IR (film) ν cm-1: 2935, 2800 (C-H), 1589, 1505, 1418 (Ar-H), 1290, 1235, 1124 (Ar-O), 1067
1
(C-O). NMR H (CDCl3, 300 MHz) δ (ppm): 2.31 (s, 6H, CH3-N (x 2)), 2.64 (t, J = 6 Hz, 2H, N-
CH2-CH2-N), 2.69 (t, J = 6 Hz, 2H, N-CH2-CH2-N), 2.80-2.95 (m, 2H, Ar-CH2-CH2-N), 3.20-3.29
(m, 2H, Ar-CH2-CH2-N), 3.78 (s, 6H, OCH3 (x 2)), 3.82 (s, 3H, OCH3), 4.20-4.30 (m, 4H, O-CH2-
CH2-O), 4.28 (s, 1H, H-6), 6.45 (s, 2H, H-2’, H-6’), 6.57 (s, 1H, H-5), 6.68 (s, 1H, H-10). NMR 13C
(CDCl3, 75.5 MHz) δ (ppm): 38.7 (CH3, CH3-N x 2), 46.1 (CH2, Ar-CH2), 56.5 (CH2, N-CH2), 57.73
(CH, C-6), 57.8 (CH3, O-CH3), 58.5 (CH2, N-CH2), 61.2 (CH3, OCH3 (x 2)), 63.8 (CH2, N-CH2),
64.7 (CH2, O-CH2-), 64.8 (CH2, CH2-O), 106.1 (CH, C-2’, C-6’), 116.9 (CH, C-5), 117.1 (CH, C-
10), 128.4 (C, C-5a), 137.7 (C, C-9a), 138.8 (C, C-1’) 142.2 (C, C-4a), 142.9 (C, C-10a), 153.2 (C,
C-3’, C-4’, C-5’). MS (EI) (m/z, %): 428 (M+, 21). HRMS ESI(+) m/z for C24H33N2O5 [M+H]+ calcd.
429.2389, found: 429.2385.
5.1.10.
N,N-Diethyl-(6-(3,4,5-trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-g]
isoquinolin-7-yl))ethylamine (16). From the tetrahydroisoquinoline 8 (53 mg, 0.14 mmol) and 2-
dietilaminoethyl chloride (51 mg, 0.28 mmol), and following the general procedure described above
the title compound was obtained (16 mg, 25% yield) as a yellow oil. Rf = 0.27 (EtOAc/MeOH 8:2).
1
IR (film) ν cm-1: 2939, 2812 (C-H), 1586, 1500, 1186 (Ar-O), 1098 (C-O). NMR H (CDCl3, 200

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MHz) δ (ppm): 0.94 (t, J = 7 Hz, 6H, CH3- x 2), 1.98-2.04 (m, 4H, CH2 x2), 2.45 (q, J = 7Hz, 4H, N-
CH2 x2), 2.67 (t, J = 7 Hz, 2H, N-CH2), 2.82-2.95 (m, 2H, Ar-CH2), 3.21-3.28 (m, 2H, Ar-CH2), 3.82
(s, 6H, OCH3 (x 2)), 3.83 (s, 3H, OCH3), 4.18-4-19 (m, 4H, O-CH2-CH2-O), 4.29 (s, 1H, H-6), 6.22
(s, 1H, H-5), 6.53 (s, 2H, H-2’, H-6’), 6.62 (s, 1H, H-10). NMR 13C (CDCl3, 50.4 MHz) δ (ppm):
11.2 (CH3, CH3-N (x 2)), 28.7 (CH2, Ar-CH2), 47.2 (CH2, N-CH2 x 2), 49.3 (CH2, N-CH2), 50.3 (CH2,
N-CH2), 51.9 (CH2, N-CH2), 56.1 (CH3, OCH3 (x 2)), 60.9 (CH3, O-CH3), 64.3 (CH2, O-CH2-), 64.4
(CH2, -CH2-O), 69.6 (CH-Ar), 106.2 (CH, C-2’, C-6’), 116.0 (CH, C-5), 116.6 (CH, C-10), 122.1 (C,
C-9a), 127.5 (C, C-5a), 130.3 (C-1’), 139.8 (C-4’), 141.3 (C-10a), 141.8 (C-4a), 152.9 (C-3’, C-5’).
HRMS ESI(+) m/z for C26H37N2O5 [M+H]+ calcd. 457.2702, found: 457.2712.
5.1.11. Ethyl 6-(6-(3,4,5-trimethoxyphenyl)-2,3,8,9-tetrahydro-[1,4]dioxino[2,3-g]isoquinolin-
7(6H)-yl)hexanoate (17). From the tetrahydroisoquinoline 8 (100 mg, 0.27 mmol) and methyl 6-
bromohexanoate (87 mg, 0.41 mmol), and following the general procedure described above the title
compound was obtained (62 mg, 46% yield) as a yellow oil. Rf = 0.40 (EtOAc/hexane 8:2). IR (film)
1
ν cm-1: 2938, 1736, 1589, 1512, 1298 (Ar-O), 1125 (C-O). NMR H (CDCl3, 300 MHz) δ (ppm):
1.43-1.58 (m, 9H, CH2- x 3 and CH3-), 2.14-2.26 (m, 2H, CH2-CO), 2.40-2.42 (m, 2H, CH2-N ),
2.67-2.75 (m, 1H, N-CH2), 2.80-2.96 (m, 2H, Ar-CH2), 3.21-3.28 (m, 1H, -CH2), 3.82 (s, 6H, OCH3
(x 2)), 3.83 (s, 3H, OCH3), 4.18-4-19 (m, 6H, O-CH2-CH2-O and CH2-O), 4.29 (s, 1H, H-6), 6.25 (s,
13
1H, H-5), 6.51 (s, 2H, H-2’, H-6’), 6.61 (s, 1H, H-10). NMR C (CDCl3, 50.4 MHz) δ (ppm): 24.9
(CH2), 26.4 (CH2), 26.9 (CH2), 28.6 (CH2), 34.0 (CH2-COO-), 48.0 (CH2), 51.2 (CH, N-CH), 54.2
(CH2, N-CH2), 56.0 (CH3, OCH3 (x 2)), 60.8 (CH3, O-CH3), 64.3 (CH2, O-CH2-), 64.4 (CH2, -CH2-
O), 69.3 (CH-Ar), 106.1 (CH, C-2’, C-6’), 116.0 (CH, C-5), 116.6 (CH, C-10), 124.7 (C, C-9a),
127.7 (C, C-5a), 131.7 (C-1’), 139.9 (C-4’), 141.2 (C-10a), 141.6 (C-4a), 152.8 (C-3’, C-5’), 174.0
(C=O). HRMS ESI(+) m/z for C28H38NO7 [M+H]+ calcd. 500.2648, found: 500.2646.
5.1.12. 6-(6-(3,4,5-Trimethoxyphenyl)-2,3,8,9-tetrahydro-[1,4]dioxino[2,3-g]isoquinolin-7(6H)-
yl)hexanoic acid (18). The tetrahydroisoquinoline 17 (40 mg, 0.82 mmol) was disolved in 2N NaOH
solution (10 mL). The reaction mixture was vigorously stirred at room temperature for 6 h and TLC
of the crude reaction (hexane/EtOAc 5:5) indicated formation of a new compound (Rf = 0.1). HCl
2N (15 mL) was added to the crude of reaction, and the mixture was extracted with EtOAc (2 x 10
mL). The organic fraction was dried over Na2SO4. After concentration the resulting yellowish solid

was purified by silica gel column chromatography using EtOAc/MeOH as eluent and the carboxylic
ACCEPTED MANUSCRIPT
acid was obtained (32 mg, 85% yield) as white solid. Rf = 0.1 (EtOAc/hexane 8:2). M.p. 63-64 ºC.
1
IR (film) ν cm-1: 2928, 1725, 1589, 1504, 1299 (Ar-O), 1126 (C-O). NMR H (CDCl3, 200 MHz) δ
(ppm): 1.15-1.25 (m, 2H, CH2-), 1.51-1.58 (m, 4H, CH2-), 2.24 (t, J = 7 Hz, 2H, CH2-), 2.44-2.47
(m, 2H, N-CH2), 2.80-2.83 (m, 1H, CH2), 2.97-3.08 (m, 2H, -CH2), 3.22-3.31 (m, 1H, CH2), 3.80 (s,
6H, OCH3 (x 2)), 3.84 (s, 3H, OCH3), 4.18-4-19 (m, 4H, O-CH2-CH2-O), 4.41 (s, 1H, H-6), 6.26 (s,
13
1H, H-5), 6.51 (s, 2H, H-2’, H-6’), 6.61 (s, 1H, H-10). NMR C (CDCl3, 50.4 MHz) δ (ppm): 24.9
(CH2), 25.5 (CH2), 26.9 (CH2), 27.4 (CH2), 29.7 (CH2), 46.9 (CH2-COO-), 53.8 (CH2), 56.1 (CH3,
OCH3 (x 2)), 60.9 (CH3, O-CH3), 64.3 (CH2, O-CH2-), 64.4 (CH2, -CH2-O), 68.4 (CH-Ar), 106.6
(CH, C-2’, C-6’), 116.1 (CH, C-5), 116.7 (CH, C-10), 127.1 (C, C-9a), 130.1 (C, C-5a), 137.0 (C-1’),
138.0 (C-4’), 141.5 (C-10a), 142.0 (C-4a), 152.9 (C-3’, C-5’), 178.1 (C=O). HRMS ESI(+) m/z for
C26H34NO7 [M+H]+ calcd. 472.2335, found: 472.2353.
5.1.13. N-(3,3-(Diethoxy)propyl)-6-(3,4,5-trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino
[2,3-g]isoquinoline (19). From the tetrahydroisoquinoline 8 (200 mg, 0.56 mmol) and 3-
chloropropionaldehydediethyl acetal (0.14 mL, 0.84 mmol), and following the general procedure
described above the title compound was obtained (80 mg, 30% yield) as a white oil. Rf = 0.80
(EtOAc). IR (film) ν cm-1: 2949, 2929 (C-H), 1586, 1505, 1457 (Ar-H), 1300, 1122 (Ar-O), 1062 (C-
O). NMR 1H (CDCl3, 300 MHz) δ (ppm): 1.05 (t, J = 6.9 Hz, 3H, O-CH2-CH3), 1.13 (t, J = 6.9 Hz,
3H, O-CH2-CH3), 1.70-1.85 (m, 2H, N-CH2-CH2-CH), 2.20-2.35 (m, 1H, Ar-CH2-CH2-N), 2.40-2.80
(m, 6H, Ar-CH2-CH2-N, N-CH2-CH2-CH), 2.90-3.10 (m, 2H, Ar-CH2-CH2-N), 3.14-3.33 (m, 2H, O-
CH2-CH3), 3.37-3.60 (m, 2H, O-CH2-CH3), 3.81 (s, 6H, OCH3 (x 2)), 3.83 (s, 3H, OCH3), 4.12-4.22
(m, 4H, O-CH2-CH2-O), 4.26 (s, 1H, H-6), 4.45 (t, J = 5.1 Hz, 1H, CH2-CH2-CH), 6.24 (s, 1H, H-5),
6.51 (s, 2H, H-2’, H-6’), 6.59 (s, 1H, H-10). NMR 13C (CDCl3, 75.5 MHz) δ (ppm): 25.9 (CH3,
OCH3), 28.9 (CH3, O-CH2-CH3), 31.2, (CH2, CH2-CH), 48.6 (CH2, Ar-CH2), 50.5 (CH2, Ar-CH2-
CH2-N), 56.4 (CH3, OCH3 (x 2)), 57.2 (CH3, OCH3), 60.7 (CH2, N-CH2-CH2-CH), 61.1 (CH2, O-
CH2-CH3), 61.6 (CH2, CH3-CH2-O), 63.1 (CH2, O-CH2-CH2-O), 64.7 (CH2, O-CH2-CH2-O), 69.4
(CH, C-6), 101.7 (CH, CHOO), 106.7 (CH, C-2', C-6'), 116.3 (CH, C-5), 116.5 (CH, C-10), 127.9
(C-5a), 132.9 (C-4a), 137.3 (C-1’), 141.7 (C, C-9a), 142.1 (C, C-10a), 153.3 (C, C-3’, C-5’), 153.9

(C, C-4’). MS (EI) (m/z, %): 487 (M+, 4), 370 (M+-C H O , 65), 356 (M+-C H O ], 94) 320 (M-
ACCEPTED MANUSCRIPT
7
16
2
6
14
2
C9H12O3, 100). (C9H11O3 = 3,4,5-trimethoxyphenyl). HRMS ESI(+) m/z for C27H38NO7 [M+H]+
calcd. 488.2648, found: 488.2645.
5.1.14. N-(4-Nitrophenyl)-6-(3,4,5-trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-g]
isoquinoline (20). The tetrahydroisoquinoline 8 (120 mg, 0.34 mmol) and 1-bromo-4-nitrobenzene
(81 mg, 0.4 mmol) were dissolved in toluene (5 mL) in a flame-dried round-bottom flask under
argon. Cs2CO3 (219 mg, 0.67 mmol), (±)-BINAP (cat) and PdCl2((o-tolyl)3P)2 (cat) were added under
argon. The reaction was heated at 130 °C for 24 h and TLC of the crude mixture (hexane/EtOAc 1:1)
indicated formation of a yellow product (Rf = 0.55) and complete consumption of starting material
(Rf = 0.10). The crude mixture was evaporated in vacuo and purified by silica gel flash column
chromatography (hexane/EtOAc 8:2) to afford the N-arylated isoquinoline 20 (52 mg, 33% yield) as
a bright yellow solid. Rf = 0.55 (hexane/EtOAc 1:1). M.p. 78-80 oC (hexane/EtOAc). IR (film) ν cm-
1
1: 2934 (C-H), 1591, 1502, 1460, 1414 (Ar-H), 1290, 1112 (Ar-O), 1066 (C-O). NMR H (CDCl3,
300 MHz) δ (ppm): 2.83-2.98 (m, 2H, Ar-CH2-CH2-N), 3.52-3.61 (m, 2H, Ar-CH2-CH2-N), 3.74 (s,
6H, OCH3 (x 2)), 3.80 (s, 3H, OCH3), 4.26 (s, 4H, CH2-O (x 2)), 5.74 (s, 1H, H-6), 6.41 (s, 2H, H-2’,
H-6’), 6.72 (s, 1H, H-5), 6.77 (d, J = 8 Hz, 2H, H-2’’, H-6’’), 6.89 (s, 1H, H-10), 8.12 (d, J = 8Hz,
2H, H-3’’, H-5’’). NMR 13C (CDCl3, 75.5 MHz) δ (ppm): 27.6 (CH2, Ar-CH2-), 45.2 (CH2, CH2-N),
56.6 (CH3, CH3-O (x 2)), 61.1 (CH3, CH3-O), 62.3 (CH, C-6), 64.7 (CH2, CH2-O (x 2)), 104.2 (CH,
C-2’, C-6’), 111.6 (CH, C-3’’, C-5’’), 116.5 (CH, C-5), 116.9 (CH, C-10), 126.4 (CH, C-2’’, C-6’’),
128.2 (C, C-5a), 130.1 (C, C-9a), 137.3 (C, C-1’), 138.1 (C, C-4’’), 142.5 (C, C-4a), 143.3 (C, C-
10a), 153.7 (C, C-3’, C-4’, C-5’), 153.9 (C, C-1’’). MS (EI) (m/z, %): 479 (M+, 7), 311 (M+-
C9H12O3, 100). (C9H11O3 = 3,4,5-trimethoxyphenyl). HRMS ESI(+) m/z for C26H27N2O7 [M+H]+
calcd. 479.1818, found: 479.1824.
5.1.15. N-(4-Cyanophenyl)-6-(3,4,5-trimethoxyphenyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-
g]isoquinolone (21). The tetrahydroisoquinoline 8 (120 mg, 0.34 mmol) and 1-bromo-4-
cyanobenzene (92 mg, 0.50 mmol) were dissolved in toluene (5 mL) in a flame-dried round-bottom
flask under argon. Cs2CO3 (2.44 mg, 0.67 mmol), (±)-BINAP (cat) and PdCl2((o-tolyl)3P)2 (cat) were
added under argon. The reaction was heated at 150 °C under stirring for 72 h and TLC of the reaction
mixture (hexane/EtOAc 1:1) indicated formation of a yellow product (Rf = 0.60) and uncomplete

consumption of SM. The crude mixture was concentrated in vacuo and purified by silica gel flash
ACCEPTED MANUSCRIPT
column chromatography (hexane/EtOAc 8:2) to afford the desired isoquinoline 21 (92 mg, 60%
o
yield) as a yellow solid. Rf = 0.60 (hexane/EtOAc 1:1). M.p. 72-78 C (hexane/EtOAc). IR (film) ν
cm-1: 2924, 2851 (C-H), 2212 (CN), 1603, 1503, 1461, 1413 (Ar-H), 1235 (Ar-O), 1178, 1066 (C-
O). NMR 1H (CDCl3, 300 MHz) δ (ppm): 2.84-2.91 (m, 2H, Ar-CH2-CH2-N), 3.46-3.54 (m, 2H, Ar-
CH2-CH2-N), 3.74 (s, 6H, OCH3 (x 2)), 3.80 (s, 3H, OCH3), 4.26 (s, 6H, CH2-O (x 3)), 5.66 (s, 1H,
H-6), 6.41 (s, 2H, H-2’, H-6’), 6.70 (s, 1H, H-5), 6.79 (d, J = 8 Hz, 2H, H-2’’, H-6’’), 6.86 (s, 1H, H-
10), 7.47 (d, J = 8 Hz, 2H, H-3’’, H-5’’). NMR 13C (CDCl3, 75.5 MHz) δ (ppm): 27.6 (CH2, Ar-CH2-
CH2-N), 44.7 (CH2, CH2-N), 56.6 (CH3, CH3-O (x 2)), 61.1 (CH, C-6), 62.2, (CH3, CH3-O), 64.7
(CH2, CH2-O (x 2)), 99.0 (C, CN) 104.2 (CH, C-2’, C-6’), 112.8 (CH, C-2’’, C-6’’), 116.4 (CH, C-5),
116.8 (CH, C-10), 120.7 (C, C-4’’), 128.4 (C, C-5a), 130.3 (C, C-9a), 133.8 (CH, C-3’’, C-5’’), 137.6
(C, C-1’), 142.4 (C, C-4a), 143.2 (C, C-10a), 152.2 (C, C-1’’), 153.6 (C, C-3’, C-4’, C-5’). MS (EI)
(m/z, %): 458 (M+, 23), 291 (M+-C9H12O3, 100). (C9H11O3 = 3,4,5-trimethoxyphenyl). HRMS ESI(+)
m/z for C27H26N2O5 [M+H]+ calcd. 459.1920, found: 459.1918.
5.1.16.
Ethyl
6-(6-(pyridinyl)-2,3,8,9-tetrahydro-[1,4]dioxino[2,3-g]isoquinolin-7(6H)-
yl)carboxylate (22). The title compound was prepared, using a similar procedure to that described
for the synthesis of 11 starting from the isoquinoline 9 (60 mg, 0.22 mmol), Et3N (0.1 mL, 0.67
mmol) and ethyl chloroformate (0.05 mL, 0.24 mmol) dissolved in CH2Cl2 (12 mL) in 43% yield (33
mg) as a white solid. M.p. 114-115 ºC. IR (film) ν cm-1: 2931, 1695, 1504, 1296 (Ar-O), 1067 (C-O).
1
NMR H (CDCl3, 200 MHz) δ (ppm): 1.26 (t, J = 7 Hz, 3H, CH3-), 2.44-2.47 (m, 1H, CH2), 2.62-
2.65 (m, 1H, CH2), 2.80-2.83 (m, 1H, -CH2), 3.22-3.31 (m, 1H, CH2), 4.18 (s, 1H, H-6), 4.19-4.25
(m, 4H, O-CH2-CH2-O), 6.47 (s, 1H, H-5), 6.70 (s, 1H, H-10), 7.14 (d, J = 6 Hz, 2H, pyridine), 8.50
(d, J = 6 Hz, 2H, pyridine). NMR 13C (CDCl3, 50.4 MHz) δ (ppm): 14.7 (CH2), 27.6 (CH2), 39.0
(CH2), 56.4 (CH-Ar), 61.8 (CH2, O-CH2-CH3), 64.3 (CH2, -CH2-O), 64.4 (CH2, -CH2-O), 116.4 (CH,
C-5), 116.9 (CH, C-10), 122.8 (CH x 2, pyridine), 128.0 (C, C-9a), 130.1 (C, C-5a), 141.5 (C-10a),
142.0 (C-4a), 149.7 (CH x 2, pyridine), 151.2 (C-1’ pyridine), 178.1 (C=O). HRMS ESI(+) m/z for
C19H21N2O4 [M+H]+ calcd. 341.1501, found: 341.1498.
5.1.17.
Methyl
6-(6-(pyridinyl)-2,3,8,9-tetrahydro-[1,4]dioxino[2,3-g]isoquinolin-7(6H)-

yl)hexanoate (23). The title compound was prepared, using a similar procedure to that described for
ACCEPTED MANUSCRIPT
the synthesis of 17 starting from the isoquinoline 9 (150 mg, 0.56 mmol), Et3N (0.3 mL, 2.01 mmol)
and methyl bromohexanoate (175 mg, 0.84 mmol) dissolved in DMF (10 mL) in 42% yield (93 mg)
as a white solid. M.p. 111-112 ºC. IR (film) ν cm-1: 2933, 1734, 1504, 1299 (Ar-O), 1072 (C-O).
1
NMR H (CDCl3, 200 MHz) δ (ppm): 1.27 (t, J = 7 Hz, 2H, CH2-), 1.49-1.54 (m, 2H, CH2-), 2.21-
2.29 (m, 4H, CH2-), 2.36-2.46 (m, 2H, N-CH2), 2.60-2.62 (m, 1H, CH2), 2.90-2.98 (m, 2H, -CH2),
3.02-3.18 (m, 1H, CH2), 3.65 (s, 3H,CH3O), 4.15-4.18 (m, 4H, O-CH2-CH2-O), 4.40 (s, 1H, H-6),
6.16 (s, 1H, H-5), 6.27 (s, 1H, H-10), 7.21 (d, J = 6.2 Hz, 2H, Ar), 8.52 (d, J = 6.2Hz, 2H, Ar). NMR
13C (CDCl3, 50.4 MHz) δ (ppm): 24.8 (CH2), 26.3 (CH2), 26.6 (CH2), 28.0 (CH2), 29.7 (CH2), 46.9
(CH2-COO-), 54.1 (CH2), 64.2 (CH2, O-CH2-), 64.3 (CH2, -CH2-O), 67.3 (CH-Ar), 116.4 (CH, C-5),
116.6 (CH, C-10), 124.9 (CH x 2, pyridine), 127.8 (C, C-9a), 129.3 (C, C-5a), 141.5 (C-10a), 142.0
(C-4a), 148,3 (CH x 2, pyridine), 154.9 (C-1’ pyridine), 177.2 (COOH). HRMS ESI(+) m/z for
C24H31N2O4 [M+H]+ calcd. 411.2284, found: 411.2288.
5.1.18. 6-(6-(Pyridin-4-yl)-2,3,8,9-tetrahydro-[1,4]dioxino[2,3-g]isoquinolin-7(6H)-yl)hexanoic
acid (24). The title compound was prepared, using a similar procedure to that described for the
synthesis of 18 starting from the isoquinoline 23 (30 mg, 0.10 mmol) and NaOH in 74% yield (35
mg) as a white solid. M.p. 57-58 ºC. IR (film) ν cm-1: 2927, 1716, 1603, 1505, 1299 (Ar-O), 1066
1
(C-O). NMR H (CDCl3, 200 MHz) δ (ppm): 1.25 (t, J = 7 Hz, 2H, CH2-), 1.49 (qt, J = 7 Hz, 2H,
CH2-), 2.24-2.29 (m, 4H, CH2-), 2.38-2.47 (m, 2H, N-CH2), 2.80-2.82 (m, 1H, CH2), 2.91-3.09 (m,
2H, -CH2), 3.12-3.21 (m, 1H, CH2), 4.13-4.20 (m, 4H, O-CH2-CH2-O), 4.43 (s, 1H, H-6), 6.12 (s,
1H, H-5), 6.62 (s, 1H, H-10), 7.29 (d, J = 6.2 Hz, 2H, Ar), 7.98 (bs, 2H, Ar); 8.42 (bs, 1H, COOH).
NMR 13C (CDCl3, 50.4 MHz) δ (ppm): 24.8 (CH2), 26.6 (CH2), 26.7 (CH2), 27.9 (CH2), 34.0 (CH2),
46.9 (CH2-COO-), 54.2 (CH2), 64.3 (CH2, O-CH2-), 64.4 (CH2, -CH2-O), 67.2 (CH-Ar), 116.3 (CH,
C-5), 116.7 (CH, C-10), 124.4 (CH x 2, pyridine), 127.9 (C, C-9a), 129.5 (C, C-5a), 141.5 (C-10a),
142.0 (C-4a), 149,5 (CH x 2, pyridine), 153.7 (C-1’ pyridine), 173.9 (COOH). HRMS ESI(+) m/z for
C22H27N2O4 [M+H]+ calcd. 383.1971, found: 383.1973.
5.1.19.
2-(6,7-Dimethoxy-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydroisoquinoline)ethanol
(25). From the tetrahydroisoquinoline 27 (150 mg, 0.42 mmol) and 2-bromoethanol (0.06 mL, 0.83

mmol), and following the general procedure described above the title compound was obtained (82
ACCEPTED MANUSCRIPT
o
mg, 49% yield) as a white solid. Rf = 0.65 (EtOAc/MeOH 8:2). M.p. 77-80 C (EtOAc). IR (film) ν
cm-1: 3516 (OH), 2937, 2834 (C-H), 1591, 1516, 1423 (Ar-H), 1257, 1221 (Ar-O), 1121 (C-O).
NMR 1H (CDCl3, 300 MHz) δ (ppm): 2.40-2.52 (m, 1H, CH2-Ar), 2.52-2.65 (m, 1H, CH2-Ar), 2.70-
2.85 (m, 2H, CH2-N), 2.85-3.00 (m, 1H, CH2-N), 3.10-3.25 (m, 1H, CH2-N), 3.40-3.55 (m, 1H, CH2-
OH), 3.61 (s, 3H, CH3-O), 3.62-3.70 (m, 1H, CH2-OH), 3.76 (s, 6H, CH3-O (x 2)), 3.81 (s, 3H, CH3-
O), 3.82 (s, 3H, CH3-O), 4.45 (s, 1H, H-1), 6.22 (s, 1H, H-5), 6.41 (s, 2H, H-2’, H-6’), 6.58 (s, 1H,
H-8). NMR 13C (CDCl3, 75.5 MHz) δ (ppm): 28.1 (CH2, CH2-Ar), 46.9 (CH2, CH2-N), 55.4 (CH2,
CH2-N), 56.1 (CH3, OCH3), 56.2 (CH3, OCH3), 56.5 (CH3, OCH3 (x 2)), 58.5 (CH2, CH2-OH), 61.2
(CH3, OCH3), 68.6 (CH, C-1), 106.7 (CH, C-2’, C-6’), 111.1 (CH, C-5), 111.8 (CH, C-8), 126.8 (C,
C-4a), 129.4 (C, C-8a), 139.6 (C, C-1’), 147.4 (C, C-7), 147.9 (C, C-6), 153.4 (C, C-3’, C-4’, C-5’).
MS EI m/z (%): 403 (M+, 11), 372 (M+-C2H8, 100), 236 (M+-C9H12O3, 77). (C9H11O3 = 3,4,5-
trimethoxyphenyl). HRMS ESI(+) m/z for C22H30NO6 [M+H]+ calcd. 404.2073, found: 404.2076.
5.1.20. 3-(6,7-Dimethoxy-1-(3,4,5-trimethoxyphenyl)isoquinolin-2-yl)-1,2-propanediol (26).
First step: preparation of the intermediate 6,7-Dimethoxy-N-(oxirane-2-yl-methyl)-1-(3,4,5-
trimethoxyphenyl)-1,2,3,4-tetrahydroisoquinoline. From the tetrahydroisoquinoline 27 (100 mg, 0.28
mmol) and epichlorohydrin (0.2 mL, 2.55 mmol), and following the classical procedure for the N-
alkylation, agitation of the mixture at room temperature during 24 h the title compound was obtained
(52 mg, 45% yield) as an orange solid. Rf = 0.30 (hexane/EtOAc 2:8). IR (film) ν cm-1: 2920, 2849
(C-H), 1586, 1503, 1450, 1410 (Ar-H), 1221 (Ar-O), 1120, 1002 (C-O). NMR 1H (CDCl3, 300 MHz)
δ (ppm): 2.50-3.10 (m, 9H, CH2-CH2-N, CH2-N (x 2), CH-O (x 2), CH2-O), 3.65 (s, 3H, CH3-O),
3.81 (s, 6H, CH3-O (x 2)), 3.85 (s, 3H, CH3-O), 3.87 (s, 3H, CH3-O), 4.60 (s, 1H, H-1), 6.25 (s, 2H,
H-2’, H-6’), 6.48 (s, 1H, H-5), 6.63 (s, 1H, H-8).
The oxirane (50 mg) was treated with NaOH 2N in 1,4-dioxane (10 and 4 mL respectively) and the
solution was stirred 48 h at room temperature. The crude mixture was extracted with dichoromethane
(3 x 10 mL), dried, filtered and concentrated in vacuo. The residue was purified by silica gel flash
column chromatography (hexane/EtOAc 8:2) to afford the diol 26 as a brown solid (48% yield). Rf =
0.60 (hexane/EtOAc 8:2). M.p. 70-73 ºC (hexane/EtOAc). IR (film) ν cm-1: 3700-3050 (OH), 2919,

2849 (C-H), 1505, 1461, 1452, 1415 (Ar-H), 1215 (Ar-O), 1120 (C-O). NMR 1H (CDCl , 300 MHz)
ACCEPTED MANUSCRIPT
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δ (ppm): 2.60-3.25 (m, 9H, CH2-CH2-N, CH2-N (x 2), CH-O (x 2), CH2-O), 3.73 (s, 3H, CH3-O),
3.81 (s, 6H, CH3-O (x 2)), 3.84 (s, 3H, CH3-O), 3.87 (s, 3H, CH3-O), 4.18 (s, 1H, H-1), 6.58 (s, 1H,
H-5); 6.80 (s, 2H, H-2’, H-6’), 6.85 (s, 1H, H-8). NMR 13C (CDCl3, 75.5 MHz) δ (ppm): 30.0 (CH2,
Ar-CH2), 47.9 (CH2, CH2-N), 54.3 (CH2); 56.1 (CH3, CH3-O), 56.4 (CH3, OCH3), 56.5 (CH3, OCH3
(x 2)), 61.1 (CH3, OCH3), 65.0 (CH2), 75.8 (CH, C-1), 76.7 (CH, CH-OH), 104.2 (CH, C-2’, C-6’),
111.6 (CH, C-5), 126.9 (C, C-8a), 128.7 (C, C-4a), 140.7 (C, C-1’), 147.8 (C, C-7), 148.7 (C, C-6),
153.5 (C, C-4’), 153.3 (C, C-3’, C-5’). HRMS ESI(+) m/z for C23H31NO7 [M+H]+: calcd. 433.2101,
found: 433.2156.
5.1.21. 6,7-Dimethoxy-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydroisoquinoline (27). The acyl-
isoquinoline 28 (870 mg, 1.91 mmol) was dissolved in MeOH (15 mL). NaOH 2N (45 mL) was
added and the reaction was heated to reflux for 16 h. Then, TLC of the crude mixture (hexane/EtOAc
1:1) showed total consumption of Starting material (Rf = 0.75) and formation of a new compound (Rf
= 0.15). The methanol was evaporated in vacuo. The aqueous phase was extracted with CH2Cl2 (3 x
20 mL) and the combined organic phases were dried (Na2SO4), filtered and concentrated in vacuo.
The crude residue was purified by flash column chromatography (hexane/EtOAc 1:1) to afford the
desired isoquinoline 27 (480 mg, 70% yield) as a white solid. Rf = 0.15 (hexane/EtOAc 1:1). M.p.
o
95-97 C (EtOAc). IR (film) ν cm-1: 3310 (NH), 3002-2828 (C-H), 1736 (C=O), 1590, 1515, 1504,
1
1461, 1420 (Ar-H), 1251, 1226, 1214 (Ar-O), 1123, 1111 (C-O). NMR H (CDCl3, 300 MHz) δ
(ppm): 2.65-2.78 (m, 1H, CH2-CH2-N), 2.90-3.10 (m, 2H, H-C-4, H-C-3) 3.20-3.30 (m, 1H, CH2-N),
3.68 (s, 3H, CH3-O), 3.81 (s, 6H, CH3-O (x 2)), 3.85 (s, 3H, CH3-O), 3.88 (s, 3H, CH3-O), 4.97 (s,
1H, H-1), 6.31 (s, 1H, H-5), 6.49 (s, 2H, H-2’, H-6’), 6.63 (s, 1H, H-8). NMR 13C (CDCl3, 75.5
MHz) δ (ppm): 29.0 (CH2, Ar-CH2), 39.7 (CH2, CH2-N), 56.2 (CH3, CH3-O), 56.3 (CH3, OCH3), 56.5
(CH3, OCH3 (x 2)), 56.8 (CH3, OCH3), 61.1 (CH, C-1), 106.6 (CH, C-2’, C-6’), 111.3 (CH, C-5),
111.4 (CH, C-8), 125.2 (C, C-4a), 126.1 (C, C-8a), 136.8 (C, C-1’), 148.2 (C, C-7), 148.9 (C, C-6),
153.5 (C, C-3’, C-4’, C-5’). HRMS ESI(+) m/z for C20H26NO5 [M+H]+: calcd. 360.1811, found:
360.1814.
5.1.22.
(6,7-Dimethoxy-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydroisoquinolin-2-yl)2,2,2-