Discovery of 2-aminothiazolyl berberine derivatives as effectively antibacterial agents toward clinically drug-resistant Gram-negative Acinetobacter baumanii

Article abstract of DOI:10.1016/j.ejmech.2018.01.038

Aminothiazolyl berberine derivatives as potentially antimicrobial agents were designed and synthesized in an effort to overcome drug resistance. The antimicrobial assay revealed that some target compounds exhibited significantly inhibitory efficiencies toward bacteria and fungi including drug-resistant pathogens, and the aminothiazole and Schiff base moieties were helpful structural fragments for aqueous solubility and antibacterial activity. Especially, aminothiazolyl 9-hexyl berberine 9c and 2,4-dichlorobenzyl derivative 18a exhibited good activities (MIC = 2 nmol/mL) against clinically drug-resistant Gram-negative Acinetobacter baumanii with low cytotoxicity to hepatocyte LO2 cells, rapidly bactericidal effects and quite slow development of bacterial resistance toward A. baumanii. Molecular modeling indicated that compounds 9c and 18a could bind with GLY-102, ARG-136 and/or ALA-100 residues of DNA gyrase through hydrogen bonds. It was found that compounds 9c and 18a were able to disturb the drug-resistant A. baumanii membrane effectively, and molecule 9c could not only intercalate but also cleave bacterial DNA isolated from resistant A. baumanii, which might be the preliminary antibacterial action mechanism of inhibiting the growth of A. baumanii strain. In particular, the combination use of compound 9c with norfloxacin could enhance the antibacterial activity, broaden antibacterial spectrum and overcome the drug resistance.

Full text of DOI:10.1016/j.ejmech.2018.01.038

Accepted Manuscript
Discovery of 2-aminothiazolyl berberine derivatives as effectively antibacterial agents
toward clinically drug-resistant Gram-negative Acinetobacter baumanii
Wei-Wei Gao, Lavanya Gopala, Rammohan R. Yadav Bheemanaboina, Guo-Biao
Zhang, Shuo Li, Cheng-He Zhou
PII:
S0223-5234(18)30051-5
10.1016/j.ejmech.2018.01.038
EJMECH 10115
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 November 2017
Revised Date: 9 January 2018
Accepted Date: 11 January 2018
Please cite this article as: W.-W. Gao, L. Gopala, R.R. Yadav Bheemanaboina, G.-B. Zhang, S. Li,
C.-H. Zhou, Discovery of 2-aminothiazolyl berberine derivatives as effectively antibacterial agents
toward clinically drug-resistant Gram-negative Acinetobacter baumanii, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.038.
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Graphical Abstract
Discovery of 2-aminothiazolyl berberine derivatives as effectively antibacterial agents toward clinically
drug-resistant Gram-negative Acinetobacter baumanii
1
1,†
1,‡
1
2,*
Wei-Wei Gao , Lavanya Gopala , Rammohan R. Yadav Bheemanaboina , Guo-Biao Zhang , Shuo Li ,
1
,*
Cheng-He Zhou
1
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing
Municipality, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R.
China.
2
School of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, P. R. China.
†
Postdoctoral researcher from Sri Venkateswara University, India
‡
Postdoctoral researcher from CSIR-Indian Institute of Integrative Medicine (IIIM), India
*
Corresponding Address:
Tel.: +86-23-68254967; fax: +86-23-68254967; E-mail: zhouch@swu.edu.cn (Cheng-He Zhou);
lishuo@cqut.edu.cn (Shuo Li).

ACCEPTED MANUSCRIPT
Title page
Title:
Discovery of 2-aminothiazolyl berberine derivatives as effectively antibacterial agents toward clinically
drug-resistant Gram-negative Acinetobacter baumanii
Author Names and Affiliations:
1
1,†
1,‡
1
2,*
Wei-Wei Gao , Lavanya Gopala , Rammohan R. Yadav Bheemanaboina , Guo-Biao Zhang , Shuo Li ,
1
,*
Cheng-He Zhou
1
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing
Municipality, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R.
China.
2
School of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, P. R. China.
†
Postdoctoral researcher from Sri Venkateswara University, India
‡
Postdoctoral researcher from CSIR-Indian Institute of Integrative Medicine (IIIM), India
*
Corresponding Address:
Tel.: +86-23-68254967; fax: +86-23-68254967; zhouch@swu.edu.cn (Cheng-He Zhou);
lishuo@cqut.edu.cn (Shuo Li).
Abstract:
Aminothiazolyl berberine derivatives as potentially antimicrobial agents were designed and synthesized in
an effort to overcome drug resistance. The antimicrobial assay revealed that some target compounds
exhibited significantly inhibitory efficiencies toward bacteria and fungi including drug-resistant pathogens,
and the aminothiazole and Schiff base moieties were helpful structural fragments for aqueous solubility and
antibacterial activity. Especially, aminothiazolyl 9-hexyl berberine 9c and 2,4-dichlorobenzyl derivative
18a exhibited good activities (MIC = 2 nmol/mL) against clinically drug-resistant Gram-negative
Acinetobacter baumanii with low cytotoxicity to hepatocyte LO2 cells, rapidly bactericidal effects and
quite slow development of bacterial resistance toward A. baumanii. Molecular modeling indicated that
compounds 9c and 18a could bind with GLY-102, ARG-136 and/or ALA-100 residues of DNA gyrase
through hydrogen bonds. It was found that compounds 9c and 18a were able to disturb the drug-resistant A.
1

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baumanii membrane effectively, and molecule 9c could not only intercalate but also cleave bacterial DNA
isolated from resistant A. baumanii, which might be the preliminary antibacterial action mechanism of
inhibiting the growth of A. baumanii strain. In particular, the combination use of compound 9c with
norfloxacin could enhance the antibacterial activity, broaden antibacterial spectrum and overcome the drug
resistance.
Keywords:
Antimicrobial; Cytotoxicity; Drug combination; DNA; Membrane permeabilization; Thiazole
2

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1
. Introduction
The drug resistance for antibiotics and clinically synthetic antibacterial agents is becoming a major global
issue and threatens to overburden healthcare systems because of the increasing occurrence of multi-drug
resistant (MDR) pathogens worldwide [1−4]. Acinetobacter baumannii as one of the ESKAPE pathogens
is an important cause for severe infections, particularly in immunocompromised patients [5,6], and has
acquired resistance to a wide spectrum of antibiotics used in clinical practice, which often makes treatment
extremely difficult and the eradication of this bacterium from the healthcare environment almost
impossible [7,8]. A. baumannii has been listed by the World Health Organization first on their global
priority list of MDR pathogens currently threatening human health [9]. Therefore, an increasingly urgent
need is to discover new antibacterial compounds with novel or multi-target activity against drug resistant
strains. It is well-known that the discovery of new scaffolds derived from natural sources can open the way
to alternative classes of antibiotics with probably new mechanisms of action.
Berberine is a natural isoquinoline quaternary alkaloid from many kinds of naturally medicinal plants
such as Coptis chinensis (a traditional Chinese herb Huanglian), Hydrastis canadensis, Berberis aristata,
Coptis japonica, Phellondendron amurense etc [10,11]. It has paid extensive attention for the large clinical
potential in treating a number of diseases including infection, Alzheimer’s disease, hyperlipidemia,
diabetes, metabolic syndrome, obesity, fatty liver disease and so on [12,13]. In particular, berberine as a
validated antibacterial agent against gastroenteritis, abdominal pain and diarrhea has been used in China
for more than 2000 years [14,15]. Despite of the long term clinical use, the genotoxic, cytotoxic or
mutagenic effects, and drug-resistance are seldom reported [16]. However, berberine is present at a very
low level in blood and its bioavailability in vivo is less than 1% [17,18], which seriously limits its useful
profile. It has been found that the structural changes such as disruption of the symmetry or molecular
planarity would be efficient strategies to improve the intrinsic aqueous solubility [19,20].
Tetrahydroprotoberberrubine as one of the berberine derivatives can improve the molecular flexibility to a
certain extent, which may be conducive to enhancing the water solubility and bioavailability, and is a valid
structural alternative with widely medicinal potentiality [21]. Thus, it is important and worthy to further
develop tetrahydroprotoberberrubine-based agents for their medicinal value with broader spectrum of
antimicrobial activity and more effective for infectious treatments.
Azole compounds like imidazoles [22], triazoles [23,24], tetrazoles [25,26], thiazoles [27,28] and
benzene-fused derivatives benzimidazoles [29,30], benzotriazoles [31] and benzothiazoles [32,33] are one
of the most important kinds of N-heterocycles with various bioactivities and have been extensively
employed in the development of antimicrobial agents, especially the aminothiazole derivatives.
Aminothiazole moiety is present in a variety of clinical drugs, such as antibacterial sulfathiazole and
cephalosporins cefodizime, cefmenoxime, anticancer dasatinib, and antiinflammatory meloxicam [34−36].
The successful development of many clinical aminothiazole-based drugs has been motivating more and
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more efforts to develop new bioactive molecules on the basis of this fragment [37]. The structural
modifications of clinical drugs by 2-aminothiazole is one of the most convenient and rewarding methods to
exploit new medicinal agents [38]. Very recently, the introduction of 2-aminothiazole moiety into the
3
-position of antibacterial quinolone skeleton was found to not only improve antimicrobial potency but
also afford multi-targeting agents with both membrane active and DNA intercalating potency [39−41].
Aminothiazole fragment can easily interact with DNA, enzymes and other biomacromolecules through
noncovalent interactions such as hydrogen bonds, π−π stacking, coordination, ion dipole, hydrophobic
effect as well as van der Waals force, because of the unique structural fragment aminothiazole with both
electron-donating groups (-NH- or -NH , -S-) and the electron-accepting C=N group [42,43]. Therefore,
2
the aminothiazole modified derivatives were endowed to possess multi-targeting binding with multiple
biological active sites in biological system, thus possibly overcoming the severe resistance and exhibiting
strong bioactivity [44].
Our previous research revealed that the introduction of azoles such as imidazoles [45], benzimidazoles
[
46,47] and triazoles [48] into the natural berberine scaffold not only improved biological potencies but
also broadened the antimicrobial spectrum, including against methicillin-resistant Staphylococcus aureus
MRSA). However, to our surprise, aminothiazole, an important structural fragment which is extensively
(
present in a large number of clinical drugs and candidates, so far has not been observed to modify the
naturally antibacterial berberine. As continuous efforts in discovering and developing novel small molecule
compounds with new or multiple targets effectively inhibiting resistant bacterial strains, herein we have
overwhelming interest in combining the naturally antibacterial berberine backbone and the important
antibacterial structural aminothiazole to develop a novel series of potentially antibacterial hybrids. The
new hybrids of aminothiazole and berberine derivative are expected to exert multi-targeting properties
including the validated berberine targeting DNA and membrane activity via multiple binding to biological
system by using electron-donating groups (-NH- or -NH , -S-), electron-accepting bond (C=N) and
2
aromatic moiety in aminothiazole and berberine fragments. More importantly, the aminothiazole functional
group may also be helpful for enhancing the water solubility of target molecules via forming hydrogen
bonds by using its amino and imino groups. Reasonably, the designed target compounds may have larger
potential in treating antimicrobial infections including clinically drug-resistant strains because of potential
multi-targeting. Therefore, in this work, a series of aminothiazolyl berberine derivatives were constructed.
In order to investigate the structure-activity relationships, various substituents, including halophenyl
groups and aliphatic chains like alkyl, hydroxyalkyl, alkenyl, alkynyl and cyano ones for regulating the
molecular rigidity and flexibility with the validated remarkable influence in biological activities, were
introduced to modify the aminothiazolyl berberine backbone. The Schiff base moiety was employed as a
bridge linker between aminothiazolyl fragment and berberine backbone because it is a validated fragment
to beneficially improve the water solubility [49,50] (Fig. 1).
Fig. 1
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The antimicrobial activities in vitro for the target 2-aminothiazolyl berberine derivatives and their new
precursor compounds were evaluated against clinically Gram-positive and Gram-negative bacteria and
fungi including drug-resistant strains. The aqueous solubility of compounds was investigated to helpfully
explain the differences in antimicrobial activities. To identify the safety profile, the cytotoxic test for the
highly active compound was done against normal human cells. The further experimental studies including
bactericidal kinetic assay, drug resistance development, bacterial membrane permeabilization and
theoretical exploration of molecular modeling was also performed to verify the research values and
foreground of the derivatives. Moreover, the genomic DNA was isolated from the sensitive resistant strains
in order to explore the possible antibacterial mechanism by the use of UV-visible absorption spectra and
along with Agarose gel electrophoresis. Finally, the combination use of the most active molecule with
clinically antibacterial drugs was evaluated to further excavate its potential in enhancing the antimicrobial
efficiency and overcoming drug resistance.
2
. Results and discussion
2
.1. Chemistry
The target 2-aminothiazolyl berberine derivatives were conveniently synthesized from natural berberine
according to the routes outlined in Schemes 1−3. Commercially available berberine chloride 1 was easily
converted into tetrahydroprotoberberrubine-12-carbaldehyde 4 through demethylation, reduction and
formylation in an excellent yield of 82.0% (Scheme 1). The condensation of compound 4 with
hydrazinecarbothioamide in anhydrous alcohol conveniently afforded carbothioamide derivative 5, and
then further cyclization with 2-chloroacetaldehyde efficiently produced hydroxyl aminothiazolyl berberine
derivative 6 in 45.0% yield.
Scheme 1
The desired 2-aminothiazolyl berberine derivative 6 was further structurally modified with an attempt to
investigate the effects of various substituents on bioactivities. Much effort failed in modifying the
9
-hydroxyl group of compound 6. An alternative strategy had to start from 9-hydroxyl berberine-derived
aldehyde 4. The synthetic route was shown in Scheme 2.
Scheme 2
The substitution of compound 4 with a series of aliphatic bromides or chlorides in
N,N-dimethylformamide (DMF) at 80 °C using potassium carbonate as the base gave the 9-position
substituted berberine derivatives including alkyl one 7, alkenyl one 10, alkynyl or cyano one 13. These
prepared intermediates were further condensed with hydrazinecarbothioamide to afford aliphatic
carbothioamide precursors 8, 11 and 14, respectively, which were further reacted with
2
-chloroacetaldehyde in anhydrous ethanol at 80 °C to produce the desirable aliphatic aminothiazolyl
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berberine derivatives 9, 12 and 15 in 30.1−45.8% yields, respectively.
The target phenyl series of aminothiazolyl berberine derivative 18 was also obtained in yields ranging
from 33.3% to 42.1% under the similar reaction condition starting from intermediate 4, the substituted
benzyl chlorides, hydrazinecarbothioamide and 2-chloroacetaldehyde (Scheme 3).
Scheme 3
1
13
The structures of all the new compounds were confirmed by H NMR, C NMR and HRMS spectra.
The spectral analyses were in accordance with the assigned structures, and all the spectral data were listed
in the Experimental section. The HRMS for each new target compounds gave a major fragment of [M +
+
H] according to their molecular formula.
2
.2. Biological Activity
All the new compounds including berberine thioamide intermediates (5, 8a-g, 11, 14a-b and 17a-i) and
target 2-aminothiazolyl berberine derivatives (6, 9a-g, 12, 15a-b and 18a-i) were evaluated for biological
activities in vitro toward a panel of bacteria (standard S. aureus ATCC 25923, B. subtilis ATCC 21216, M.
luteus ATCC 4698, E. coli DH52, P. aeruginosa ATCC 27853; MRSA (Methicillin-resistant
Staphylococcus. aureus (N315), drug-resistant E. faecalis, E. coli, P. aeruginosa, K. pneumonia, A.
baumanii) and fungi (standard C. albicans ATCC 76615, C. utilis ATCC 9950, C. mycoderma ATCC
9
6918, A. flavus ATCC 204304, S. cerevisiae ATCC 9763, C. parapsilosis ATCC 22019; drug-resistant C.
albicans C. tropicals, A. fumigates). The MIC (minimum inhibitory concentration) values were determined
by using a protocol according to the Clinical and Laboratory Standard Institute (CLSI) guidelines [51].
2
.2.1. Antibacterial activity
The antibacterial data in Tables S1 and S2 revealed that some intermediates berberine thioamide
derivatives displayed better inhibitory activity against the tested standard and clinically drug-resistant
bacterial strains than natural berberine. The alkyl berberine thioamides 8c and 8e not only gave good
activity against standard E. coli DH52 with MIC value of 0.03 µmol/mL, but also displayed 53 times
stronger inhibitory effect toward drug-resistant E. coli than norfloxacin. Moreover, the clinically
drug-resistant A. baumanii was sensitive to most of the berberine thioamides such as 2,4-dichlorobenzyl
intermediate 17a (MIC = 3 nmol/mL). These results suggested that the modified berberine backbone was
favorable to antibacterial potencies, especially for resistant strains.
Table 1 showed that some target aminothiazolyl berberine derivatives possessed moderate to good
efficacies in inhibiting the growth of standard bacterial strains. Hexyl derivative 9c gave potent activities
with MIC values ranging from 0.03 to 0.06 µmol/mL against S. aureus ATCC 25923, M. luteus ATCC 4698,
E. coli DH52 and P. aeruginosa ATCC 27853, which exerted almost equivalent effects to chloromycin. The
replacement of hexyl group with long alkyl chain to yield dodecyl derivative 9f resulted in decreased
activities against standard strains (MIC = 0.03−0.83 µmol/mL), which might be attributed to the excessive
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lipophilicity. Comparably, the short alkyl chain modified molecule 9b gave better inhibitory potencies with
MIC values ranging from 0.06 to 0.25 µmol/mL. Aminothiazolyl berberine derivative 9a incorporating
ethyl group exhibited profound efficiency to obviously suppress the growth of S. aureus ATCC 25923, P.
aeruginosa ATCC 27853 and E. coli DH52 with MIC value of 0.07 µmol/mL, which was more active than
ethoxyl compound 9g with the same length chain, and this indicated that the hydroxyl group was
unfavorable for the antibacterial activity. The unsaturated substituents modified derivatives 12 and 15
displayed no obvious enhancement in inhibitory effect.
Table 1
In comparison with aliphatic modified compounds, most benzyl derivatives 18a−i showed relatively
lower activities in inhibiting the growth of standard bacterial strains. Compound 18h with unsubstituted
phenyl moiety did not show apparent inhibition even though the concentration was up to 0.95 µmol/mL.
The introduction of halogen atom significantly improved the antibacterial effect. Chlorobenzyl molecules
1
8b−d gave good activities against S. aureus ATCC 25923, B. subtilis ATCC 21216 and E. coli DH52 with
MIC value of 0.11 µmol/mL. Especially, 4-chlorobenzyl derivative 18d displayed better inhibitory
potencies (MIC = 0.11−0.45 µmol/mL) than berberine (MIC = 0.34−0.69 µmol/mL), which indicated that
the position of chlorine atom on benzyl group would lead to different bioactivities. Compounds 18a and
1
8e with the increased chlorine atom did not remarkably improved effect. The displacement of
chlorobenzyl moiety by fluorobenzyl group would afford aminothiazolyl berberine derivatives 18f and 18g
with similar inhibitory efficiency (MIC = 0.06 µmol/mL) against S. aureus ATCC 25923 to chloromycin
(MIC = 0.05 µmol/mL) and 12-fold greater anti-M. luteus ATCC4698 activity than berberine, respectively.
Noticeably, Gram-negative P. aeruginosa (ATCC 27853) was sensitive to nitrobenzyl compound 18i with
MIC value of 0.05 µmol/mL, 14- and 2-fold more potent than berberine and chloromycin, respectively,
which showed that the electron-withdrawing group was useful for improving antibacterial potentiality.
Fig. 2a and 2b showed the antibacterial data of aminothiazolyl berberine compounds toward
drug-resistant strains, it was revealed that the most potent inhibitors were derivatives 9c and 18a against
the drug-resistant A. baumanii with a low nanomolar MIC value of 2 nmol/mL. Moreover, E. faecalis,
MRSA and P. aeruginosa were also sensitive to compound 9c with MICs of 0.03, 0.03 and 0.06 µmol/mL,
respectively. Alkyl derivatives 9a and 9b gave MIC values ranging from 0.06 to 0.07 µmol/mL against
MRSA and drug-resistant E. coli, respectively. Moreover, 3-chlorobenzyl derivative 18c (MIC = 0.01
µmol/mL) and benzyl one 18h (MIC = 0.02 µmol/mL) displayed more potent antibacterial efficiencies
against A. baumanii than their precursors berberine thioamides 17c and 17h, which strongly manifested
that the aminothiazole moiety could optimize the inhibitory activities against drug-resistant strains,
particularly A. baumanii.
Fig. 2
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The antibacterial evaluation showed that drug-resistant A. baumanii was sensitive to most of the
aminothiazolyl berberines, especially to derivatives 9c and 18a, but almost all the target compounds could
not effectively inhibit the growth of drug-resistant K. pneumonia. Hydroxyl compound 6 showed slightly
weaker activity against the tested strains than derivatives modified by aliphatic chains or substituted benzyl
groups, which suggested that the discernible inhibitory benefit of substituents on the antibacterial activity.
2
.2.2. Antifungal activity
The antifungal evaluation in Tables S3 and S4 revealed that most of the non-thiazole intermediates
exhibited weak inhibition activities against the tested fungi including drug-resistant ones. However, the
formation of aminothiazole ring would lead to aminothiazolyl berberine derivatives with good antifungal
potencies. Compound 9c (MIC = 4 nmol/mL) showed comparable inhibition against the growth of C.
parapsilosis ATCC 22019 to fluconazole (MIC = 7 nmol/mL). It also conferred about 14-fold anti-A.
flavus ATCC 204304 activity than fluconazole. Particularly, compound 18a as the most potent bacterial
inhibitor also displayed large anti-A. fumigates potencies (MIC = 0.03 µmol/mL).
All the antimicrobial results implied that the antimicrobial efficacies should be closely related to
aminothiazole ring and the substituents at 9-position of berberine skeleton to some extent. In particular,
aminothiazolyl berberine compounds 9c with a hexyl chain and 18a with a 2,4-dichlorobenzyl group
exhibited unprecedentedly effective inhibition toward drug-resistant A. baumanii with the same low MIC
value of 2 nmol/mL, and should be deserved to be further investigated as potentially antimicrobial agents.
The antibacterial activities of aminothiazolyl berberine derivatives were commonly stronger than those of
thioamide intermediates, proper length of the aliphatic chains and halophenyl groups were also important
for the activity. In general, halo- or nitro-substituted phenyl derivatives seemed to be less active than those
of short aliphatic chain ones, which might be due to a relative decrease in water solubility.
2
.3. Aqueous solubility
Aqueous solubility is also a governing property for small molecules interacting with biomolecule in living
systems, which plays a critical role in every stage of drug development and discovery.[52] Therefore, the
water solubility of all aminothiazolyl berberine derivatives was tested at physiological pH, natural
berberine as the control, and the obtained results were depicted in Fig. 3. Generally, most of the
aminothiazolyl compounds were more soluble in water than berberine (0.086 mg/mL), except for decyl
derivative 9e (0.061 mg/mL). For aliphatic ethyl molecule 9a, butyl one 9b, hydroxyethyl one 9g, allyl one
1
2, and cyano one 15b, they were more soluble in water (7.02−10.2 mg/mL) than the phenyl ones 18a-i
(0.374−3.19 mg/mL) and hydroxyl one 6 (1.66 mg/mL). In particular, compound 9a showed the best water
solubility of 10.2 mg/mL in the three series, 118 times higher than berberine. Whereas, the highly active
compounds 9c and 18a possessed the aqueous solubility of 1.42 and 1.90 mg/mL, respectively. These
obtained results were almost consistent with the antibacterial activities that the compounds with proper
aqueous solubility and lipophilicity showed high inhibitory efficiency against bacterial strains. Moreover,
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the insertion of 2-aminothiazolyl and Schiff base moieties might be beneficial for the improvement of
aqueous solubility.
Fig. 3
2
.4. Cytotoxicity
Despite of the long term clinical use of berberine as a validated antibacterial drug, the genotoxicity or
cytotoxicity has been seldom reported. The cytotoxicity is one of the most essential criteria to evaluate the
potential of antimicrobial agents. In order to identify the safety profile, the highly active aminothiazolyl
berberine derivatives 9c and 18a were further evaluated for their toxicities against normal human
hepatocyte LO2 cells using the colorimetric cell proliferation MTT assay. The cell viability of compounds
9
c and 18a against LO2 cells was more than 70.7% and 80.5%, respectively, which suggested that they
exhibited low toxicities to the cells even though the concentration up to 100 µg/mL (Fig. 4). Also, it might
be concluded that the antibacterial activities exhibited by aminothiazolyl berberine derivatives were not
due to the cytotoxic effects.
Fig. 4
Upon the basis of in vitro antimicrobial and cytotoxic results, hexyl derivative 9c and 2,4-dichlorobenzyl
one 18a were selected for detailed evaluation, including bactericidal kinetics, drug resistance development,
bacterial membrane permeabilization and molecular docking studies. Furthermore, the preliminary action
mechanism of compound 9c was also investigated.
2
.5. Bactericidal kinetics
There is always a need to discover rapidly bactericidal agents to combat antibacterial resistance, and time
kill kinetics is an important method to judge the efficacy of investigational antimicrobial agents [53]. To
determine whether the aminothiazolyl berberine derivatives have bactericidal efficiency, the viability of
exponentially growing drug-resistant A. baumanii was checked toward the highly active molecules 9c and
1
8a by time-kill kinetics experiment.
In the time kill assay, the colony forming units (CFUs) of the A. baumanii were rapidly decrease after
3
treatment with both compounds 9c and 18a at the concentration of 8 nmol/mL, and more than 10
CFU/mL reduction in the number of viable bacteria within 2 h. Therefore, it could be interpreted that
derivatives 9c and 18a gave a similar bactericidal efficacy and had potent killing effects against
drug-resistant A. baumanii (Fig. 5).
Fig. 5
2
.6. Drug resistance development
Resistance to antibiotics has been increasing in recent years and becoming a serious and global challenge
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to the drug discovery. The mutagenic properties of bacteria raise the possibility of the resistance even in
smaller populations of bacteria exposed to low concentration of antibacterial drugs [54]. Therefore, it is
significantly vital to study the resistance of the potent compound against bacterial strains. The highly
active compounds 9c and 18a were employed to investigate the drug resistance of A. baumanii. Fig. 6
showed that the slowly developing resistance of A. baumanii to aminothiazolyl berberine derivatives 9c
and 18a even after 10 passages.
Fig. 6
2
.7. Bacterial membrane permeabilization
Bacterial membrane has been considered as a significant and intriguing antibacterial target, and
membrane-active nature induces low propensity for bacteria to develop resistance [55,56]. Therefore, it is
reasonable to evaluate the membrane permeabilization of compounds 9c and 18a against drug-resistant A.
baumanii via fluorescence spectra using propidium iodide (PI), a common dye that can only pass through
the membrane of dead and apoptotic cells and fluoresces upon forming the PI-DNA complex [57,58].
There is an increase of fluorescence intensity within 75 min in Fig. 7, which should be due to the formed
PI-DNA complex as the gradual damage of bacterial membrane in the presence of derivatives 9c and 18a
at 0.024 µmol/mL (12 times MIC). The results manifested that compounds 9c and 18a could also
effectively disturb the membrane of A. baumanii.
Fig. 7
2
.8. Molecular docking study
Molecular docking is considered as one of the convenient and effective theoretical methods in binding
studies [59]. Thus, a docking investigation was undertaken to explore the inhibitory effects of
2
-aminothiazolyl berberine derivatives against DNA gyrase. DNA gyrase catalyzes changes in DNA
topology by breaking and rejoining double stranded DNA. In particular, DNA gyrase introduces negative
supercoils in DNA in front of the replication fork [60]. Thus, DNA gyrase is involved in very important
processes during DNA replication and are essential for cell viability. The crystal of DNA gyrase B was
selected as a representative target in the Protein Data Bank, and compounds 9c and 18a were used to dock
with DNA gyrase B. The docking evaluation gave good binding energy of 6.31 and 5.26 kcal/mol for
derivatives 9c and 18a against DNA gyrase B, respectively, which should also rationalize the antibacterial
mechanism of 2-aminothiazolyl berberine derivatives.
As shown in Fig. 8, the amino group at 2-position of the thiazole ring in molecule 9c was adjacent to
GLY-102 residue, forming a hydrogen bond with a distance of 2.4 Å. The oxygen atom of methoxyl
fragment at 10-position of the berberine skeleton and ARG-136 residue could also form a hydrogen bond
with a distance of 2.7 Å. For compound 18a, a similar hydrogen bond was formed between the amino
group at 2-position of the thiazole ring and residue ALA-100 with a distance of 2.5 Å. All of these
1
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hydrogen bonds could be favorable to stabilize the 2-aminothiazolyl berberine derivative-enzyme complex,
which might be responsible for the good inhibitory efficacy of compounds 9c and 18a against the tested
strains and the importance of 2-aminothiazolyl pharmacophore.
Fig. 8
2
.9. Preliminary antibacterial mechanism study
The good antimicrobial efficiency, no apparent cytotoxicity, rapid killing of A. baumanii pathogens, low
drug resistance development and bacterial membrane disruptive properties for compounds 9c and 18a
prompted us to examine the preliminary mechanism of action. It is known that the biological targets of
many antibacterial drugs are DNA, cell membrane, enzymes, etc. Especially, a growing investigation
directs toward the interaction of small drug molecules with DNA for the rational design and construction
of new and efficient agents [61]. To explore the preliminary antibacterial action mechanism, the most
active hexyl aminothiazolyl berberine derivative 9c was presumed to exhibit activity due to targeting
drug-resistant A. baumanii DNA by using UV-vis spectroscopic method.
2
.9.1. Interaction between compound 9c and drug-resistant A. baumanii DNA
Berberine and its derivatives could effectively interact with DNA through various binding modes.
Therefore, the binding behavior of compound 9c with A. baumanii DNA was studied on molecular level
using neutral red (NR) dye as a spectral probe. Genomic DNA was isolated from drug-resistant A.
baumanii bacteria which were the most sensitive to 9c, based on the reported procedure and assay
evaluated [62]. According to the Bouguer−Lambert−Beer law, the concentration of A. baumanii DNA in
stock solution was calculated on the basis of UV absorption value at 260 nm and the molar absorption
−
1
−1
coefficient (ξ260 = 6600 L•mol •cm ) expressed as molarity of phosphate groups. The purity of A.
baumanii DNA was checked by gel electrophoresis (Fig. 10a) and monitoring the ratio of the absorbance at
2
60 nm to that at 280 nm. A ratio of > 1.8 at A260/A280 was given, which indicated that the DNA was
completely free from protein in the solution.
The absorption spectroscopy as one of the most important methods is extensively used in DNA-binding
studies [63]. The important spectral characteristics to distinguish the change of DNA double-stranded
structure in absorption spectroscopy are hyperchromism and hypochromism. A large hypochromism will
be observed because of the strong interaction between the electronic states of DNA bases and that of the
intercalating chromophore, which strongly indicates a close proximity of the aromatic chromophore to
-4
DNA bases. With a fixed DNA concentration of 8.12 × 10 mol/L, UV-vis absorption spectra were
recorded with the increasing amount of compound 9c (Fig. 9a). The maximum absorption peak of DNA at
2
60 nm exhibited proportional increase with the gradually increasing concentration of derivative 9c.
Furthermore, a weak hypochromism existed between DNA and compound 9c because the absorption value
of the 9c-DNA complex was lower than that of simply sum of free DNA and free molecule 9c. This
1
1

ACCEPTED MANUSCRIPT
hypochromism effect might be attributed to the formed binary complexes between compound 9c to DNA
bases. More importantly, the intercalation of the aromatic chromophore of 9c into the DNA double helix
and the strong overlap of π-π* states in the π-conjugated system with DNA bases electronic states were
agreement with the observed spectral changes [64].
Fig. 9
On the basis of the variations in the absorption spectra of A. baumanii DNA upon binding to derivative
3
9
c, equation (S1) was adopted to calculate the binding constant K = 2.54 × 10 L/mol, R = 0.999, SD =
0
.01 (R is the correlation coefficient. SD is standard deviation).
2
.9.2. Interaction between NR and drug-resistant A. baumanii DNA
It has been demonstrated that the binding of NR, a planar phenazine dye, with DNA is an intercalation
mode in recent years [65]. Therefore, NR was employed as the spectral probe to investigate the binding
mode of derivative 9c with A. baumanii DNA in the present work.
The absorption peak of the NR around 460 nm was gradual decrease with the continuous addition of A.
baumanii DNA (Fig. S5). Due to the formation of the DNA-NR complex, a new band emerged at 530 nm.
2
.9.3. Competitive interaction of compound 9c and NR with drug-resistant A. baumanii DNA
As shown in Fig. 9b, there was a competitive binding between derivative 9c and NR with A. baumanii
DNA. An obvious intensity increase was observed in the developing band around 460 nm with the
increasing concentration of 9c. Moreover, the absorbance at 460 nm exhibited the reverse process in
comparison with that of around the same wavelength of free NR in the presence of the increasing
concentration of DNA in Fig. S5. The results indicated that compound 9c could intercalate into A.
baumanii DNA double helix through displacement of NR in the DNA-NR complex. Furthermore, the
increase of absorbance at 276 nm provided another evidence for compound 9c intercalating into DNA.
2
.9.4. Cleavage of drug-resistant A. baumanii DNA
The effective cleavage of biological DNA is a quite important strategy to treat various diseases [66]. An
increasing effort is to develop the DNA cleavage agents. In order to investigate the different action modes
of small drug molecule with DNA, the cleaving activity of compound 9c toward A. baumanii DNA was
o
studied under physiological conditions (T = 37 C and pH = 7.4). As shown in Fig. 10b, there was no DNA
cleavage for the control in which without derivative 9c (Lane 1 and Lane 2). However, the apparent DNA
cleaving evidence was observed in the presence of 9c (Lane 3). It suggested that molecule 9c might inhibit
the growth of A. baumanii by dual action modes of both intercalating and cleaving DNA.
Fig. 10
2
.10. Drug combination use of compound 9c with norfloxacin
1
2

ACCEPTED MANUSCRIPT
The combination therapy currently is an increasingly prevalent method to treat severely microbial
infections, such as the combination use of colistin, auranofin and ceftazidime toward MDR K. pneumoniae,
A. baumannii and P. aeruginosa [67]. Along with the rapid emergence of resistance toward clinically
antibacterial drugs, more and more effort has been witnessed to develop the drug combination treatment of
bacterial infections. A lot of work showed that the combination use of new structural agents with clinically
antimicrobial drugs could improve the efficiency, reduce or eliminate side effects and even combat drug
resistance via different modes of action [68]. In our present work, the drug combination use between the
active molecule 9c and the clinically used norfloxacin was investigated toward the drug-resistant bacteria
including MRSA, E. faecalis, E. coli, P. aeruginosa, K. pneumonia and A. baumanii.
The fraction inhibitory concentration (FIC) index was designated for combination study of 9c with
norfloxacin in inhibiting bacterial growth. The results in Table 2 indicated that 9c showed significant
synergism (FIC < 0.50) with norfloxacin against drug-resistant bacteria, except for against E. faecalis (0.50
<
FIC < 1.00). Particularly, compound 9c in combination use resulted in enhancing antibacterial efficacy of
norfloxacin by 4-fold (MIC from 0.025 to 0.006 µmol/mL) toward MRSA. At the same time, MIC value of
c was lowered by 4-fold in this combination (MIC from 0.030 to 0.007 µmol/mL). It was also worthy to
9
note that the combination could effectively inhibit the growth of drug-resistant A. baumanii. These results
manifested that the combination use of molecule 9c with clinical norfloxacin opened up an efficacious
approach to combat drug resistance with different action modes. This also represented a dynamic strategy
to improve antibacterial potency.
Table 2
3
. Conclusion
In this work, a series of aminothiazolyl berberine derivatives as potentially antimicrobial agents were
developed via convenient procedures from natural berberine. All the structures of the new compounds were
characterized by NMR and HRMS spectra. The biological evaluation in vitro indicated that some prepared
compounds showed moderate to good antibacterial and antifungal activities, and the insertion of
2
-aminothiazolyl and Schiff base moieties was beneficial to improve antibacterial efficiency and aqueous
solubility. Hexyl aminothiazolyl berberine derivative 9c and 2,4-dichlorobenzyl one 18a gave strong
inhibitory activities against drug-resistant Gram-negative A. baumanii with low MIC values of 2 nmol/mL,
low cytotoxicity to human hepatocyte LO2 cells and rapidly bactericidal effects, also displayed quite slow
resistance development toward A. baumanii and were found to be active toward drug-resistant A. baumanii
membrane. Molecular docking showed that hydrogen bonds existed in the supramolecular interaction
between DNA gyrase and the active molecule 9c or 18a. The preliminary exploration showed that
compound 9c could intercalate and/or cleave bacterial DNA from sensitive A. baumanii strains and thus
might inhibit A. baumanii growth. The combination use of the active molecule 9c with antibacterial
norfloxacin could enhance the efficiency, broaden antimicrobial spectrum and effectively combat drug
1
3

ACCEPTED MANUSCRIPT
resistance. It might be concluded that aminothiazole modified berberine derivative 9c should have great
potential in new antibacterial drug development.
4
. Experimental
4
.1. General methods
All starting material chemicals and solvents were achieved from commercial sources unless otherwise
indicated and used without any additional purification. The masses were weighed on a microbalance with a
resolution of 0.0001 g. Thin-layer chromatography (TLC) was made using glass plates precoated with
silica gel (HG/T2354-92) impregnated with a fluorescent indicator (254 nm). Visualization on TLC was
acquired using UV light (254 nm) and also staining with iodine. Column chromatography was undertaken
on silica gel (500-600 mesh size). Melting point (mp) was measured on a melting point apparatus (X-6
type). High resolution mass spectra (HRMS) were obtained with IonSpec FT-ICR mass spectrometer with
1
13
ESI-TOF method. H and C NMR spectra were recorded on Bruker AVANCE III 600 MHz spectrometer
using DMSO-d or CDCl as solvent, or TMS as internal standard. Proton and carbon chemical shifts were
6
3
expressed in parts per million (δ, ppm) and were referenced to NMR solvent DMSO-d , δ 2.50, 39.5 ppm;
6
CDCl , δ 7.26, 77.0 ppm. The following abbreviations were used to describe peak patterns: s = singlet, d=
3
doublet, t = triplet, q = quadruplet, m = multiplet. Coupling constants (J) were expressed in hertz unit (Hz).
The abbreviations of THPB and Ph were adopted to assign the berberine skeleton and aryl group,
respectively.
4
.1.1. Synthesis of 9-hydroxy-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin
7-ium chloride (2), 10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]
isoquinolin-9-ol (3) and 9-hydroxy-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino
3,2-a]isoquinoline-12-carbaldehyde (4)
-
[
The intermediates 2−4 were prepared according to the previously reported procedures [69].
4
.1.2. Synthesis of 2-((9-Hydroxy-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino
[
3,2-a]isoquinolin-12-yl)methylene)hydrazine-1-carbothioamide (5)
A mixture of compound 4 (1.0 g, 2.8 mmol), hydrazinecarbothioamide (0.28 g, 3.1 mmol) and glacial
o
acetic acid (0.15 eq) as the catalyst in anhydrous alcohol (20 mL) was stirred at 80 C under N protection
2
for 6 h. Upon completion of the reaction, the solvent was evaporated. The residue was washed with
methanol, and further purified by silica gel column chromatography (eluent, chloroform/acetone (3/1, V/V))
o
1
to afford the desired compound 5 (0.94 g, 78.8%) as yellow solid. Mp: 196−197 C; H NMR (600 MHz,
DMSO-d ) δ 11.16 (s, 1H, NH), 9.81 (s, 1H, OH), 8.33 (s, 1H, N=CH), 8.17 (s, 1H, NH), 8.03 (s, 1H, NH),
6
7
.64 (s, 1H, THPB-11-H), 6.92 (s, 1H, THPB-1-H), 6.67 (s, 1H, THPB-4-H), 6.01 (d, J = 3.0 Hz, 2H,
OCH O), 4.67 (s, 1H, THPB-8-H), 4.43 (d, J = 14.5 Hz, 1H, CH), 4.30–4.26 m, 1H, THPB-8-H),
2
1
4

ACCEPTED MANUSCRIPT
4
.11–4.07 (m, 1H, THPB-13-H), 3.91 (s, 3H, OCH ), 3.86 (s, 1H, THPB-13-H), 3.42 (s, 1H, THPB-5-H),
3
13
3
.34–3.29 (m, 1H, THPB-5-H), 3.06 (s, 1H, THPB-6-H), 2.90 (d, J = 11.4 Hz, 1H, THPB-6-H) ppm; C
NMR (151 MHz, DMSO-d ) δ 178.1, 150.4, 147.3, 146.1, 145.7, 144.8, 141.1, 131.3, 128.5, 127.7, 125.8,
6
1
24.0, 118.5, 109.1, 108.3, 106.3, 101.8, 68.2, 59.1, 56.5, 49.1, 33.8, 29.5 ppm.
4
.1.3. Synthesis of 10-Methoxy-12-((2-(thiazol-2-yl)hydrazono)methyl)-5,8,13,13a-tetrahydro-6H-[1,3]
dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-9-ol (6)
A mixture of compound 5 (0.31 g, 0.72 mmol) and 2-chloroacetaldehyde (0.08 g, 1.1 mmol) in anhydrous
o
alcohol (20 mL) was stirred at 80 C under N protection for 3 h. Upon completion of the reaction, the
2
solvent was evaporated. The residue was washed with methanol, and further purified by silica gel column
chromatography (eluent, chloroform/acetone (2/1, V/V)) to afford the desired compound 6 (0.15 g, 45.0%)
o
1
as light yellow solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 11.40 (s, 1H, NH), 9.83 (s, 1H, OH),
6
8
.29 (s, 1H, N=CH), 7.32 (s, 1H, THPB-11-H), 7.26 (d, J = 3.1 Hz, 1H, thiazolyl-4-H), 7.19 (s, 1H,
thiazolyl-5-H), 6.84 (s, 2H, THPB-1-H, THPB-4-H), 6.05 (d, J = 3.0 Hz, 2H, OCH O), 4.68 (s, 1H,
2
THPB-8-H), 4.58 (d, J = 15.6 Hz, 1H, CH), 4.30 (dd, J = 15.3, 7.7 Hz, 1H, THPB-8-H), 4.09–4.05 (m, 1H,
THPB-13-H), 3.89 (s, 3H, OCH ), 3.85 (s, 1H, THPB-13-H), 3.43 (d, J = 6.8 Hz, 1H, THPB-5-H),
3
3
.37–3.31 (m, 1H, THPB-5-H), 3.06 (dd, J = 16.9, 12.4 Hz, 1H, THPB-6-H), 2.90 (d, J = 15.8 Hz, 1H,
1
3
THPB-6-H) ppm; C NMR (151 MHz, DMSO-d ) δ 169.4, 150.3, 147.4, 146.2, 145.9, 144.9, 141.2, 131.4,
6
1
28.1, 127.9, 125.6, 124.2, 117.5, 109.0, 108.6, 106.4, 101.8, 68.2, 59.1, 56.5, 49.1, 33.7, 29.4 ppm;
+
HRMS (ESI) calcd. for C H N O S [M + H] , 451.1440; found, 451.1441.
2
3
22
4
4
4
.1.4. Synthesis of 9-Ethoxy-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]
isoquinoline-12-carbaldehyde (7a)
To a solution of compound 4 (2.0 g, 5.7 mmol) in DMF (50 mL) was added potassium carbonate (1.2 g, 8.5
o
mmol), the mixture was stirred at 80 C for 1 h and bromoethane (0.93 g, 8.5 mmol) was then added and
again the temperature maintained at 80 °C for 6 h, then diluted with chloroform (3 × 20 mL). The organic
phase was washed with saturated sodium chloride, dried over anhydrous sodium sulphate and further
purified by silica gel column chromatography (eluent, petroleum ether/chloroform (20/1, V/V)) to afford
o
1
the desired compound 7a (1.75 g, 80.4%) as yellow solid. Mp: 190−191 C; H NMR (600 MHz, CDCl ) δ
3
1
2
1
3
0.21 (s, 1H, CHO), 7.32 (s, 1H, THPB-11-H), 6.79 (s, 1H, THPB-1-H), 6.59 (s, 1H, THPB-4-H), 5.93 (s,
H, OCH O), 4.28–4.21 (m, 2H, CH CH ), 4.17 (dd, J = 9.5, 7.1 Hz, 1H, CH), 3.90 (s, 3H, OCH ), 3.85 (s,
2
2
3
3
H, THPB-8-H), 3.51 (d, J = 15.6 Hz, 2H, THPB-8-H, THPB-13-H), 3.22–3.16 (m, 1H, THPB-13-H),
.12 (s, 1H, THPB-5-H), 3.07–2.99 (m, 1H, THPB-5-H), 2.70–2.59 (m, 2H, THPB-6-H), 1.40 (t, J = 7.0
1
3
Hz, 3H, CH CH ) ppm; C NMR (151 MHz, CDCl ) δ 190.7 (C=O), 150.2, 149.5, 146.3, 146.1, 131.7,
2
3
3
1
30.4, 129.9, 129.0, 127.7, 113.7, 108.4, 105.6, 100.8, 68.7, 59.2, 55.9, 54.2, 51.3, 50.7, 33.8, 29.5, 15.9
ppm.
4
.1.5. Synthesis of 9-Butoxy-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]
1
5

ACCEPTED MANUSCRIPT
isoquinoline-12-carbaldehyde (7b)
Compound 7b was prepared according to the procedure described for compound 7a, starting from
compound 4 (2.0 g, 5.7 mmol), 1-bromobutane (1.2 g, 8.5 mmol) and potassium carbonate (1.2 g, 8.5
o
1
mmol). The pure product 7b (1.91 g, 81.7%) was obtained as light yellow solid. Mp: 168−169 C; H
NMR (600 MHz, CDCl ) δ 10.20 (s, 1H, CHO), 7.31 (s, 1H, THPB-11-H), 6.78 (s, 1H, THPB-1-H), 6.59
3
(
s, 1H, THPB-4-H), 5.92 (s, 2H, OCH O), 4.23 (d, J = 15.9 Hz, 1H, CH), 4.20–4.15 (m, 1H,
2
CHCH CH CH ), 4.09 (dt, J = 9.3, 6.7 Hz, 1H, CHCH CH CH ), 3.89 (s, 3H, OCH ), 3.85 (dd, J = 16.9,
2
2
3
2
2
3
3
3
.4 Hz, 1H, THPB-8-H), 3.50 (d, J = 15.7 Hz, 2H, THPB-8-H, THPB-13-H), 3.18 (dd, J = 10.3, 4.5 Hz,
H, THPB-13-H), 3.15–3.07 (m, 1H, THPB-5-H), 3.03 (dd, J = 16.7, 11.3 Hz, 1H, THPB-5-H), 2.70–2.59
1
(
m, 2H, THPB-6-H), 1.80–1.73 (m, 2H, CH CH CH CH ), 1.54–1.47 (m, 2H, CH CH CH CH ), 0.99 (t, J
2 2 2 3 2 2 2 3
1
3
=
7.4 Hz, 3H, CH CH CH CH ) ppm; C NMR (151 MHz, CDCl ) δ 190.6 (C=O), 150.1, 149.7, 146.3,
2 2 2 3 3
1
2
46.1, 131.8, 130.5, 129.9, 129.0, 127.7, 113.8, 108.4, 105.7, 100.8, 72.7, 59.2, 56.0, 54.2, 51.4, 33.9, 32.5,
9.5, 19.1, 13.8 ppm.
4
.1.6. Synthesis of 9-(Hexyloxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino
[
3,2-a]isoquinoline-12-carbaldehyde (7c)
Compound 7c was prepared according to the procedure described for compound 7a, starting from
compound 4 (2.0 g, 5.7 mmol), 1-bromohexane (1.4 g, 8.5 mmol) and potassium carbonate (1.2 g, 8.5
o
1
mmol). The pure product 7c (1.96 g, 78.7%) was obtained as yellow solid. Mp: 64−65 C; H NMR (600
MHz, CDCl ) δ 10.20 (s, 1H, CHO), 7.31 (s, 1H, THPB-11-H), 6.79 (s, 1H, THPB-1-H), 6.59 (s, 1H,
3
THPB-4-H), 5.92 (s, 2H, OCH O), 4.23 (d, J = 15.9 Hz, 1H, CH), 4.16 (dd, J = 11.5, 4.6 Hz, 1H,
2
CH(CH ) CH ), 4.08 (dd, J = 11.4, 4.6 Hz, 1H, CH(CH ) CH ), 3.89 (s, 3H, OCH ), 3.84 (d, J = 4.2 Hz,
2
4
3
2 4
3
3
1
H, THPB-8-H), 3.50 (d, J = 15.6 Hz, 2H, THPB-8-H, THPB-13-H), 3.17 (dd, J = 10.2, 4.9 Hz, 1H,
THPB-13-H), 3.11 (s, 1H, THPB-5-H), 3.02 (dd, J = 16.8, 11.3 Hz, 1H, THPB-5-H), 2.69–2.60 (m, 2H,
THPB-6-H), 1.77 (dd, 14.5, 7.2 Hz, 2H, CH CH (CH ) CH ), 1.58–1.51 (m, 1H,
J
=
2
2
2 3
3
(
CH ) CH (CH ) CH ), 1.50–1.43 (m, 2H, (CH ) CH CH CH ), 1.29 (d, J = 10.6 Hz, 2H, (CH ) CH CH ),
2 2 2 2 2 3 2 3 2 2 3 2 4 2 3
1
3
0
1
3
.92 (t, J = 6.6 Hz, 3H, (CH ) CH ) ppm; C NMR (151 MHz, CDCl ) δ 190.7 (C=O), 150.1, 149.7, 146.3,
2 5 3 3
46.1, 131.7, 130.4, 129.8, 128.9, 127.6, 113.8, 108.4, 105.6, 100.8, 73.1, 62.9, 59.1, 55.9, 54.2, 51.3, 33.8,
1.6, 29.4, 25.6, 22.6, 14.0 ppm.
4
.1.7. Synthesis of 10-Methoxy-9-(octyloxy)-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino
[
3,2-a]isoquinoline-12-carbaldehyde (7d)
Compound 7d was prepared according to the procedure described for compound 7a, starting from
compound 4 (2.0 g, 5.7 mmol), 1-bromooctane (1.6 g, 8.5 mmol) and potassium carbonate (1.2 g, 8.5
o
1
mmol). The pure product 7d (2.12 g, 79.9%) was obtained as yellow solid. Mp: 97−98 C; H NMR (600
MHz, CDCl ) δ 10.20 (s, 1H, CHO), 7.32 (s, 1H, THPB-11-H), 6.79 (s, 1H, THPB-1-H), 6.60 (s, 1H,
3
THPB-4-H), 5.93 (s, 2H, OCH O), 4.23 (d, J = 15.9 Hz, 1H, CH), 4.19–4.14 (m, 1H, CH(CH ) CH ), 4.08
2
2 6
3
1
6

ACCEPTED MANUSCRIPT
(
d, J = 9.3 Hz, 1H, CH(CH ) CH ), 3.90 (s, 3H, OCH ), 3.85 (s, 1H, THPB-8-H), 3.51 (d, J = 15.7 Hz, 2H,
2
6
3
3
THPB-8-H, THPB-13-H), 3.18 (dd, J = 10.3, 4.7 Hz, 1H, THPB-13-H), 3.15–3.09 (m, 1H, THPB-5-H),
.01 (dd, J = 15.9, 4.8 Hz, 1H, THPB-5-H), 2.67 (d, J = 16.9 Hz, 1H, THPB-6-H), 2.65–2.60 (m, 1H,
THPB-6-H), 1.81–1.74 (m, 2H, CH CH (CH ) CH ), 1.46 (dd, 14.7, 7.2 Hz, 2H,
3
J
=
2
2
2 5
3
13
(
CH ) CH (CH ) CH ), 1.29 (d, J = 5.9 Hz, 8H, (CH ) (CH ) CH ), 0.89 (s, 3H, (CH ) CH ) ppm;
C
2
2
2
2 4
3
2 3
2 4
3
2 7
3
NMR (151 MHz, CDCl ) δ 190.7 (C=O), 150.1, 149.7, 146.3, 146.1, 131.6, 130.4, 129.8, 128.9, 127.6,
3
113.9, 108.4, 105.6, 100.8, 73.0, 62.9, 59.1, 55.9, 54.2, 51.3, 36.4, 32.8, 31.4, 30.4, 29.3, 25.7, 22.6, 14.0
ppm.
4
.1.8. Synthesis of 9-(Decyloxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino
[
3,2-a]isoquinoline-12-carbaldehyde (7e)
Compound 7e was prepared according to the procedure described for compound 7a, starting from
compound 4 (2.0 g, 5.7 mmol), 1-bromodecane (1.9 g, 8.5 mmol) and potassium carbonate (1.2 g, 8.5
o
1
mmol). The pure product 7e (2.28 g, 81.1%) was obtained as yellow solid. Mp: 50−51 C; H NMR (600
MHz, CDCl ) δ 10.20 (s, 1H, CHO), 7.31 (s, 1H, THPB-11-H), 6.79 (s, 1H, THPB-1-H), 6.59 (s, 1H,
3
THPB-4-H), 5.93 (s, 2H, OCH O), 4.23 (d, J = 15.8 Hz, 1H, CH), 4.19–4.14 (m, 1H, CH(CH ) CH ),
2
2 8
3
4
.10–4.05 (m, 1H, CH(CH ) CH ), 3.89 (s, 3H, OCH ), 3.85 (s, 1H, THPB-8-H), 3.51 (d, J = 15.2 Hz, 2H,
2 8 3 3
THPB-8-H, THPB-13-H), 3.21–3.16 (m, 1H, THPB-13-H), 3.12 (s, 1H, THPB-5-H), 3.03 (d, J = 4.5 Hz,
1
H, THPB-5-H), 2.70–2.60 (m, 2H, THPB-6-H), 1.80–1.74 (m, 2H, CH CH (CH ) CH ), 1.46 (dd, J =
2 2 2 7 3
1
4.5, 7.2 Hz, 2H, (CH ) CH (CH ) CH ), 1.31 (d, J = 37.5 Hz, 12H, (CH ) (CH ) CH ), 0.89 (d, J = 5.3
2
2
2
2 6
3
2 3
2 6
3
1
3
Hz, 3H, (CH ) CH ) ppm; C NMR (151 MHz, CDCl ) δ 190.7 (C=O), 150.1, 149.7, 146.3, 146.1, 131.7,
2
9
3
3
1
3
30.4, 129.7, 128.9, 127.6, 113.8, 108.4, 105.6, 100.8, 73.1, 63.0, 59.1, 55.9, 54.2, 51.3, 50.6, 33.7, 32.8,
1.9, 30.4, 29.3, 25.9, 25.7, 22.6, 14.0 ppm.
4
.1.9. Synthesis of 9-(Dodecyloxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino
[
3,2-a]isoquinoline-12-carbaldehyde (7f)
Compound 7f was prepared according to the procedure described for compound 7a, starting from
compound 4 (2.0 g, 5.7 mmol), 1-bromododecane (2.1 g, 8.5 mmol) and potassium carbonate (1.2 g, 8.5
o
1
mmol). The pure product 7f (2.26 g, 76.0%) was obtained as yellow solid. Mp: 75−76 C; H NMR (600
MHz, CDCl ) δ 10.20 (s, 1H, CHO), 7.31 (s, 1H, THPB-11-H), 6.79 (s, 1H, THPB-1-H), 6.59 (s, 1H,
3
THPB-4-H), 5.92 (d, J = 8.6 Hz, 2H, OCH O), 4.23 (d, J = 15.9 Hz, 1H, CH), 4.16 (d, J = 9.2 Hz, 1H,
2
CH(CH ) CH ), 4.11–4.04 (m, 1H, CH(CH ) CH ), 3.89 (s, 3H, OCH ), 3.84 (s, 1H, THPB-8-H), 3.50 (d,
2
10
3
2 10
3
3
J = 15.7 Hz, 2H, THPB-8-H, THPB-13-H), 3.21–3.15 (m, 1H, THPB-13-H), 3.12 (s, 1H, THPB-5-H), 3.03
dd, J = 16.6, 11.4 Hz, 1H, THPB-5-H), 2.69–2.60 (m, 2H, THPB-6-H), 1.82–1.72 (m, 2H,
CH CH (CH ) CH ), 1.50–1.42 (m, 2H, (CH ) CH (CH ) CH ), 1.32 (d, J = 28.7 Hz, 16H,
(
2
2
2 9
3
2 2
2
2 8
3
1
3
(
CH ) (CH ) CH ), 0.88 (t, J = 6.9 Hz, 3H, (CH ) CH ) ppm; C NMR (151 MHz, CDCl ) δ 190.7
2 3 2 8 3 2 11 3 3
(C=O), 150.1, 149.7, 146.3, 146.1, 131.7, 130.4, 129.8, 128.9, 127.7, 113.8, 108.4, 105.6, 100.8, 73.1, 63.0,
17

ACCEPTED MANUSCRIPT
5
9.2, 55.9, 54.2, 51.3, 50.6, 33.8, 32.8, 31.9, 30.4, 29.7, 29.5, 29.3, 25.9, 25.7, 22.6, 14.0 ppm.
4
.1.10. Synthesis of 9-(2-Hydroxyethoxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinoline-12-carbaldehyde (7g)
Compound 7g was prepared according to the procedure described for compound 7a, starting from
compound 4 (1.3 g, 3.7 mmol), 2-bromoethan-1-ol (0.69 g, 5.5 mmol) and potassium carbonate (0.76 g,
o
1
5
.5 mmol). The pure product 7g (1.07 g, 73.1%) was obtained as yellow solid. Mp: 116−117 C; H NMR
(
600 MHz, CDCl ) δ 10.22 (s, 1H, CHO), 7.33 (s, 1H, THPB-11-H), 6.78 (s, 1H, THPB-1-H), 6.59 (s, 1H,
3
THPB-4-H), 5.93 (s, 2H, OCH O), 4.30–4.25 (m, 2H, OCH CHOH, CH), 4.16 (ddd, J = 10.6, 5.7, 3.2 Hz,
2
2
1
3
3
H, OCH CHOH), 3.91 (s, 3H, OCH ), 3.89 (ddd, J = 9.3, 6.7, 3.8 Hz, 3H, THPB-8-H, OCH CH OH),
2
3
2
2
.53 (d, J = 15.5 Hz, 2H, THPB-8-H, THPB-13-H), 3.18 (ddd, J = 10.8, 5.2, 1.7 Hz, 1H, THPB-13-H),
13
.14–3.08 (m, 1H, THPB-5-H), 3.06–3.01 (m, 1H, THPB-5-H), 2.69–2.62 (m, 2H, THPB-6-H) ppm;
C
NMR (151 MHz, CDCl ) δ 190.7 (C=O), 150.2, 149.5, 146.3, 146.1, 131.7, 130.4, 129.9, 129.0, 127.7,
3
113.7, 108.4, 105.6, 100.8, 68.7, 59.2, 55.9, 54.2, 51.3, 50.7, 33.8, 29.5 ppm.
4
.1.11. Synthesis of 9-(Allyloxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino
[
3,2-a]isoquinoline-12-carbaldehyde (10)
Compound 10 was prepared according to the procedure described for compound 7a, starting from
compound 4 (1.3 g, 3.7 mmol), 3-bromoprop-1-ene (0.67 g, 5.5 mmol) and potassium carbonate (0.76 g,
o
1
5
.5 mmol). The pure product 10 (1.05 g, 72.5%) was obtained as yellow solid. Mp: 88−89 C; H NMR
(
600 MHz, CDCl ) δ 10.21 (s, 1H, CHO), 7.33 (s, 1H, THPB-11-H), 6.78 (s, 1H, THPB-1-H), 6.60 (s, 1H,
3
THPB-4-H), 6.07 (ddd, J = 17.0, 5.8, 4.6 Hz, 1H, CH CH=CH ), 5.93 (s, 2H, OCH O), 5.38 (dd, J = 17.1,
2
2
2
1
.4 Hz, 1H, CH CH=CH), 5.25 (d, J = 10.3 Hz, 1H, CH CH=CH), 4.72 (dd, J = 12.3, 5.9 Hz, 1H,
2 2
CHCH=CH), 4.63 (dd, J = 12.4, 5.9 Hz, 1H, CHCH=CH), 4.27 (d, J = 15.5 Hz, 1H, CH), 3.91 (s, 3H,
OCH ), 3.87 (d, J = 15.0 Hz, 1H, THPB-8-H), 3.56 (s, 2H, THPB-8-H, THPB-13-H), 3.20 (s, 1H,
3
THPB-13-H), 3.13 (s, 1H, THPB-5-H), 3.06 (s, 1H, THPB-5-H), 2.69 (d, J = 15.5 Hz, 2H, THPB-6-H)
13
ppm; C NMR (151 MHz, CDCl ) δ 190.7 (C=O), 150.1, 149.0, 146.3, 146.1, 131.7, 130.4, 129.8, 128.9,
3
1
27.7, 118.1, 113.8, 108.4, 105.6, 100.9, 73.6, 73.1, 63.0, 58.4, 55.9, 33.8, 31.6, 29.3 ppm.
4
.1.12. Synthesis of 10-Methoxy-9-(prop-2-yn-1-yloxy)-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinoline-12-carbaldehyde (13a)
Compound 13a was prepared according to the procedure described for compound 7a, starting from
compound 4 (1.3 g, 3.7 mmol), 3-bromoprop-1-yne (0.66 g, 5.5 mmol) and potassium carbonate (0.76 g,
o
1
5
.5 mmol). The pure product 13a (1.02 g, 70.6%) was obtained as yellow solid. Mp: 89−90 C; H NMR
(
600 MHz, CDCl ) δ 10.21 (s, 1H, CHO), 7.37 (s, 1H, THPB-11-H), 6.74 (s, 1H, THPB-1-H), 6.61 (s, 1H,
3
THPB-4-H), 5.94 (s, 2H, OCH O), 4.76 (d, J = 2.0 Hz, 2H, CH C≡CH), 4.23 (d, J = 15.9 Hz, 1H, CH),
2
2
3
.89 (s, 3H, OCH ), 3.50 (s, 1H, CH C≡CH), 3.45–3.41 (m, 2H, THPB-8-H), 3.23–3.19 (m, 2H,
3 2
THPB-13-H), 3.07 (d, J = 6.6 Hz, 1H, THPB-5-H), 2.95 (d, J = 19.9Hz, 1H, THPB-5-H), 2.60 (d, J = 15.5
1
8

ACCEPTED MANUSCRIPT
1
3
Hz, 1H, THPB-6-H), 2.46 (d, J = 11.0 Hz, 1H, THPB-6-H) ppm; C NMR (151 MHz, CDCl ) δ 190.6
3
(C=O), 150.1, 149.7, 146.3, 146.1, 131.6, 130.2, 129.8, 128.9, 127.6, 118.0, 113.8, 108.4, 105.6, 100.9,
7
9.5, 73.6, 63.0, 58.4, 57.8, 56.1, 33.7, 29.6 ppm.
4
.1.13. Synthesis of 2-((12-Formyl-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino
[
3,2-a]isoquinolin-9-yl)oxy)acetonitrile (13b)
Compound 13b was prepared according to the procedure described for compound 7a, starting from
compound 4 (1.3 g, 3.7 mmol), 2-bromoacetonitrile (0.42 g, 5.5 mmol) and potassium carbonate (0.76 g,
o
1
5
.5 mmol). The pure product 13b (1.09 g, 75.0%) was obtained as yellow solid. Mp: 164−165 C; H NMR
(
600 MHz, CDCl ) δ 10.24 (s, 1H, CHO), 7.36 (s, 1H, THPB-11-H), 6.76 (s, 1H, THPB-1-H), 6.60 (s, 1H,
3
THPB-4-H), 5.93 (s, 2H, OCH O), 5.01 (d, J = 16.0 Hz, 1H, CHC≡N), 4.92 (d, J = 16.0 Hz, 1H, CHC≡N),
2
4
1
3
.26 (d, J = 16.0 Hz, 1H, CH), 3.94 (d, J = 6.1 Hz, 3H, OCH ), 3.86–3.82 (m, 1H, THPB-8-H), 3.59 (d, J =
3
6.0 Hz, 1H, THPB-8-H), 3.55 (dd, J = 10.9, 3.1 Hz, 1H, THPB-13-H), 3.20–3.16 (m, 1H, THPB-13-H),
.13–3.08 (m, 1H, THPB-5-H), 3.03 (dd, J = 16.9, 11.2 Hz, 1H, THPB-5-H), 2.66 (t, J = 12.5 Hz, 2H,
1
3
THPB-6-H) ppm; C NMR (151 MHz, CDCl ) δ 190.7 (C=O), 150.1, 149.7, 146.3, 146.1, 131.7, 130.4,
3
1
29.8, 128.9, 127.7, 118.1, 113.8, 108.4, 105.6, 100.9, 73.6, 73.1, 63.0, 58.4, 57.6, 55.9, 33.5, 29.4 ppm.
4
.1.14. Synthesis of 2-((9-Ethoxy-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino
[
3,2-a]isoquinolin-12-yl)methylene)hydrazine-1-carbothioamide (8a)
Compound 8a was prepared according to the procedure described for compound 5, starting from
compound 7a (2.0 g, 5.2 mmol), hydrazinecarbothioamide (0.52 g, 5.7 mmol) and glacial acetic acid (0.15
o
1
eq). The pure product 8a (1.20 g, 50.8%) was obtained as brown solid. Mp: 201−202 C; H NMR (600
MHz, CDCl ) δ 10.16 (s, 1H, NH), 8.32 (s, 1H, N=CH), 7.20 (s, 1H, THPB-11-H), 7.12 (s, 1H, NH), 6.90
3
(s, 1H, THPB-1-H), 6.60 (s, 1H, THPB-4-H), 6.51 (s, 1H, NH), 5.95 (s, 1H, OCHO), 5.92 (s, 1H, OCHO),
4
3
2
.23 (d, J = 15.7 Hz, 1H, CH), 4.15 (d, J = 7.1 Hz, 1H, CHCH ), 4.09 (d, J = 7.1 Hz, 1H, CHCH ), 3.88 (s,
3
3
H, OCH ), 3.48 (s, 3H, THPB-8-H, THPB-13-H), 3.20 (s, 1H, THPB-13-H), 3.12 (s, 1H, THPB-5-H),
3
.79 (t, J = 13.8 Hz, 1H, THPB-5-H), 2.67 (d, J = 16.0 Hz, 1H, THPB-6-H), 2.61 (s, 1H, THPB-6-H), 1.39
1
3
(
t, J = 7.0 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 178.2 (C=S), 150.4, 146.2, 146.0,
2
3
6
1
45.9, 141.2, 131.4, 129.4, 128.1, 128.0, 127.3, 108.6, 108.3, 106.1, 101.1, 68.2, 59.2, 56.5, 53.9, 51.1,
3.7, 29.4, 16.1 ppm.
3
4
.1.15. Synthesis of 2-((9-Butoxy-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino
[
3,2-a]isoquinolin-12-yl)methylene)hydrazine-1-carbothioamide (8b).
Compound 8b was prepared according to the procedure described for compound 5, starting from
compound 7b (0.33 g, 0.81 mmol), hydrazinecarbothioamide (0.08 g, 0.90 mmol) and glacial acetic acid
o
1
(
(
(
0.15 eq). The pure product 8b (0.30 g, 77.4%) was obtained as brown solid. Mp: 148−149 C; H NMR
600 MHz, DMSO-d ) δ 11.14 (s, 1H, NH), 8.43 (s, 1H, N=CH), 8.18 (s, 1H, NH), 8.01 (s, 1H, NH), 7.61
6
s, 1H, THPB-11-H), 6.93 (s, 1H, THPB-1-H), 6.68 (s, 1H, THPB-4-H), 5.96 (d, J = 18.6 Hz, 2H, OCH O),
2
1
9

ACCEPTED MANUSCRIPT
4
.05 (d, J = 15.7 Hz, 1H, CH), 3.98 (dt, J = 13.3, 6.7 Hz, 1H, CHCH CH CH ), 3.95–3.91 (m, 1H,
2
2
3
CHCH CH CH ), 3.85 (d, J = 9.8 Hz, 3H, OCH ), 3.46 (d, J = 15.6 Hz, 1H, THPB-8-H), 3.42–3.34 (m,
2
2
3
3
2
H, THPB-8-H, THPB-13-H), 3.08 (d, J = 6.5 Hz, 1H, THPB-13-H), 2.91 (t, J = 11.1 Hz, 1H, THPB-5-H),
.61 (d, J = 15.5 Hz, 1H, THPB-5-H), 2.49–2.41 (m, 2H, THPB-6-H), 1.70–1.63 (m, 2H,
2
CH CH CH CH ), 1.49–1.41 (m, 2H, CH CH CH CH ), 0.94 (t, J = 7.3 Hz, 3H, CH CH CH CH ) ppm;
2
2
2
3
2
2
2
3
2
2
2
3
1
3
C NMR (151 MHz, DMSO-d ) δ 178.2 (C=S), 150.6, 146.2, 146.0, 145.9, 141.2, 131.4, 129.2, 128.1,
6
1
28.0, 127.4, 108.6, 108.4, 106.1, 101.1, 72.2, 59.1, 56.5, 54.1, 51.1, 33.7, 32.4, 29.4, 19.2, 14.2 ppm.
4
.1.16.
Synthesis
of
2-((9-(Hexyloxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinolin-12-yl)methylene)hydrazine-1-carbothioamide (8c).
Compound 8c was prepared according to the procedure described for compound 5, starting from
compound 7c (1.0 g, 2.3 mmol), hydrazinecarbothioamide (0.23 g, 2.5 mmol) and glacial acetic acid (0.15
o
1
eq). The pure product 8c (0.64 g, 54.1%) was obtained as brown solid. Mp: 138−139 C; H NMR (600
MHz, CDCl ) δ 10.31 (s, 1H, NH), 8.36 (s, 1H, N=CH), 7.20 (s, 1H, THPB-11-H), 7.12 (s, 1H, NH), 6.92
3
(s, 1H, THPB-1-H), 6.59 (s, 1H, THPB-4-H), 6.54 (s, 1H, NH), 5.95 (s, 1H, OCHO), 5.91 (s, 1H, OCHO),
4
.23 (d, J = 15.7 Hz, 1H, CH), 4.07 (dd, J = 11.4, 4.5 Hz, 1H, CH(CH ) CH ), 4.01–3.96 (m, 1H,
2
4
3
CH(CH ) CH ), 3.87 (s, 3H, OCH ), 3.47 (t, J = 13.6 Hz, 3H, THPB-8-H, THPB-13-H), 3.21–3.15 (m, 1H,
2
4
3
3
THPB-13-H), 3.11 (s, 1H, THPB-5-H), 2.78 (t, J = 14.2 Hz, 1H, THPB-5-H), 2.67 (d, J = 16.1 Hz, 1H,
THPB-6-H), 2.61 (s, 1H, THPB-6-H), 1.76 (dd, J = 14.6, 7.3 Hz, 2H, CH CH (CH ) CH ), 1.50–1.43 (m,
2
2
2 3
3
2
H, (CH ) CH (CH ) CH ), 1.38–1.32 (m, 4H, (CH ) (CH ) CH ), 0.92 (t, J = 6.6 Hz, 3H, (CH ) CH )
2 2 2 2 2 3 2 3 2 2 3 2 5 3
13
ppm; C NMR (151 MHz, CDCl ) δ 178.1 (C=S), 150.4, 147.0, 146.2, 146.1, 143.2, 131.4, 128.4, 127.8,
3
1
25.8, 109.2, 108.4, 105.7, 100.8, 73.0, 62.9, 59.1, 55.9, 54.2, 51.3, 36.4, 32.8, 31.4, 30.4, 29.3, 25.7, 22.6,
4.0 ppm.
1
4
.1.17.
Synthesis
of
2-((10-Methoxy-9-(octyloxy)-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinolin-12-yl)methylene)hydrazine-1-carbothioamide (8d)
Compound 8d was prepared according to the procedure described for compound 5, starting from
compound 7d (1.9 g, 4.0 mmol), hydrazinecarbothioamide (0.40 g, 4.4 mmol) and glacial acetic acid (0.15
o
1
eq). The pure product 8d (1.13 g, 52.6%) was obtained as brown solid. Mp: 157−158 C; H NMR (600
MHz, CDCl ) δ 11.15 (s, 1H, NH), 8.43 (s, 1H, N=CH), 8.11 (s, 1H, NH), 8.03 (s, 1H, NH), 7.36 (s, 1H,
3
THPB-11-H), 6.78 (s, 1H, THPB-1-H), 6.62 (s, 1H, THPB-4-H), 5.98 (s, 2H, OCH O), 4.23 (d, J = 15.9
2
Hz, 1H, CH), 4.17–4.12 (m, 1H, CH(CH ) CH ), 4.06 (d, J = 9.2 Hz, 1H, CH(CH ) CH ), 3.89 (s, 3H,
2
6
3
2 6
3
OCH ), 3.86 (s, 1H, THPB-8-H), 3.52 (d, J = 15.7 Hz, 2H, THPB-8-H, THPB-13-H), 3.19 (d J = 10.3 Hz,
3
1
H, THPB-13-H), 3.14–3.06 (m, 1H, THPB-5-H), 3.03 (dd, J = 15.9, 4.8 Hz, 1H, THPB-5-H), 2.79–2.75
(
(
m, 1H, THPB-6-H), 2.65–2.60 (m, 1H, THPB-6-H), 1.81–1.75 (m, 2H, CH CH (CH ) CH ), 1.48–1.44
2
2
2 5
3
13
m, 2H, (CH ) CH (CH ) CH ), 1.29–1.25 (m, 8H, (CH ) (CH ) CH ), 0.89 (s, 3H, (CH ) CH ) ppm; C
2
2
2
2 4
3
2 3
2 4
3
2 7
3
NMR (151 MHz, CDCl ) δ 178.2 (C=S), 150.6, 149.7, 147.0, 146.2, 146.0, 143.2, 141.2, 131.4, 130.4,
3
2
0

ACCEPTED MANUSCRIPT
1
29.2, 128.1, 128.0, 127.4, 125.8, 108.6, 108.4, 106.1, 101.1, 72.2, 59.1, 56.5, 54.1, 51.1, 33.7, 32.4, 29.4,
9.2, 14.2 ppm.
1
4
.1.18.
Synthesis
of
2-((9-(Decyloxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinolin-12-yl)methylene)hydrazine-1-carbothioamide (8e)
Compound 8e was prepared according to the procedure described for compound 5, starting from
compound 7e (1.9 g, 3.9 mmol), hydrazinecarbothioamide (0.39 g, 4.2 mmol) and glacial acetic acid (0.15
o
1
eq). The pure product 8e (1.12 g, 50.8%) was obtained as brown solid. Mp: 118−119 C; H NMR (600
MHz, CDCl ) δ 10.20 (s, 1H, NH), 8.33 (s, 1H, N=CH), 7.20 (s, 1H, THPB-11-H), 7.12 (s, 1H, NH), 6.91
3
(s, 1H, THPB-1-H), 6.60 (s, 1H, THPB-4-H), 6.51 (s, 1H, NH), 5.95 (s, 1H, OCHO), 5.91 (s, 1H, OCHO),
4
1
.23 (d, J = 15.6 Hz, 1H, CH), 4.07 (dt, J = 13.8, 6.8 Hz, 1H, CH(CH ) CH ), 3.98 (dd, J = 15.8, 6.8 Hz,
2 8 3
H, CH(CH ) CH ), 3.87 (s, 3H, OCH ), 3.48 (d, J = 8.1 Hz, 2H, THPB-8-H), 3.47–3.43 (m, 1H,
2
8
3
3
THPB-13-H), 3.18 (s, 1H, THPB-13-H), 3.11 (s, 1H, THPB-5-H), 2.78 (t, J = 13.8 Hz, 1H, THPB-5-H),
.67 (d, J = 16.0 Hz, 1H, THPB-6-H), 2.62 (d, J = 10.9 Hz, 1H, THPB-6-H), 1.76 (dt, J = 13.9, 6.8 Hz, 2H,
CH CH (CH ) CH ), 1.45 (dd, J = 14.5, 7.0 Hz, 2H, (CH ) CH (CH ) CH ), 1.39–1.26 (m, 12H,
2
2
2
2 7
3
2 2
2
2 6
3
1
3
(
CH ) (CH ) CH ), 0.88 (t, J = 6.8 Hz, 3H, (CH ) CH ) ppm; C NMR (151 MHz, CDCl ) δ 178.1 (C=S),
2 3 2 6 3 2 9 3 3
1
50.4, 147.0, 146.2, 146.1, 143.3, 131.4, 130.4, 128.4, 127.8, 125.8, 109.2, 108.4, 105.8, 100.8, 73.0, 59.2,
6.0, 54.2, 51.5, 50.6, 34.6, 32.8, 31.9, 30.4, 29.6, 29.4, 26.0, 25.7, 22.7, 14.1 ppm.
5
4
.1.19. Synthesis of 2-((9-(Dodecyloxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinolin-12-yl)methylene)hydrazine-1-carbothioamide (8f)
Compound 8f was prepared according to the procedure described for compound 5, starting from compound
7
f (1.9 g, 3.6 mmol), hydrazinecarbothioamide (0.36 g, 4.0 mmol) and glacial acetic acid (0.15 eq). The
o
1
pure product 8f (1.16 g, 54.0%) was obtained as brown solid. Mp: 119−120 C; H NMR (600 MHz,
CDCl ) δ 10.22 (s, 1H, NH), 8.34 (s, 1H, N=CH), 7.20 (s, 1H, THPB-11-H), 7.12 (s, 1H, NH), 6.91 (s, 1H,
3
THPB-1-H), 6.60 (s, 1H, THPB-4-H), 6.52 (s, 1H, NH), 5.95 (s, 1H, OCHO), 5.91 (s, 1H, OCHO), 4.23 (d,
J = 15.7 Hz, 1H, CH), 4.07 (dd, J = 15.1, 7.2 Hz, 1H, CH(CH ) CH ), 3.98 (dd, J = 15.3, 6.9 Hz, 1H,
2
10
3
CH(CH ) CH ), 3.87 (s, 3H, OCH ), 3.49 (s, 2H, THPB-8-H), 3.45 (s, 1H, THPB-13-H), 3.18 (d, J = 5.3
2
10
3
3
Hz, 1H, THPB-13-H), 3.11 (s, 1H, THPB-5-H), 2.78 (t, J = 13.9 Hz, 1H, THPB-5-H), 2.67 (d, J = 16.0 Hz,
1
H, THPB-6-H), 2.61 (t, J = 10.3 Hz, 1H, THPB-6-H), 1.79–1.73 (m, 2H, CH CH (CH ) CH ), 1.45 (dd, J
2 2 2 9 3
=
14.2, 7.0 Hz, 2H, (CH ) CH (CH ) CH ), 1.38–1.26 (m, 16H, (CH ) (CH ) CH ), 0.88 (t, J = 6.6 Hz, 3H,
2 2 2 2 8 3 2 3 2 8 3
1
3
(
CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 178.1 (C=S), 150.6, 149.7, 146.2, 146.0, 141.1,
2 11 3 6
1
3
31.3, 129.2, 128.0, 127.9, 127.4, 113.8, 108.6, 108.3, 106.1, 101.1, 72.4, 59.2, 56.5, 54.1, 51.2, 50.6, 33.7,
2.8, 31.8, 30.3, 29.7, 29.5, 29.2, 25.9, 25.7, 22.6, 14.4 ppm.
4
.1.20. Synthesis of 2-((9-(2-Hydroxyethoxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinolin-12-yl)methylene)hydrazine-1-carbothioamide (8g)
Compound 8g was prepared according to the procedure described for compound 5, starting from
2
1

ACCEPTED MANUSCRIPT
compound 7g (0.20 g, 0.50 mmol), hydrazinecarbothioamide (0.050 g, 0.55 mmol) and glacial acetic acid
o
1
(
(
(
0.15 eq). The pure product 8g (0.079 g, 33.4%) was obtained as brown solid. Mp: > 250 C; H NMR
600 MHz, DMSO-d ) δ 11.15 (s, 1H, NH), 8.44 (s, 1H, N=CH), 8.19 (s, 1H, NH), 8.01 (s, 1H, NH), 7.61
6
s, 1H, THPB-11-H), 6.93 (s, 1H, THPB-1-H), 6.68 (s, 1H, THPB-4-H), 5.96 (d, J = 18.0 Hz, 2H, OCH O),
2
4
.76 (t, J = 5.3 Hz, 1H, OCHCH OH), 4.34 (t, J = 5.0 Hz, 1H, OCHCH OH), 4.19 (d, J = 15.9 Hz, 1H,
2 2
CH), 4.07 (dt, J = 10.1, 5.4 Hz, 1H, OCH CHOH), 3.96 (dt, J = 10.3, 5.1 Hz, 1H, OCH CHOH), 3.85 (s,
2
2
3
1
H, OCH ), 3.49–3.44 (m, 2H, THPB-8-H), 3.38 (dd, J = 23.7, 13.5 Hz, 2H, THPB-13-H), 3.10–3.05 (m,
3
H, THPB-5-H), 2.95–2.87 (m, 1H, THPB-5-H), 2.62 (d, J = 15.7 Hz, 1H, THPB-6-H), 2.48–2.43 (m, 1H,
1
3
THPB-6-H) ppm; C NMR (151 MHz, DMSO-d ) δ 178.2 (C=S), 150.4, 146.2, 145.9, 141.2, 131.4, 129.4,
6
1
28.0, 127.3, 108.6, 108.3, 106.1, 101.1, 79.6, 74.6, 60.9, 59.2, 56.5, 55.3, 53.9, 51.1, 49.1, 33.7, 29.4
ppm.
4
.1.21.
Synthesis
of
2-((9-(Allyloxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinolin-12-yl)methylene)hydrazine-1-carbothioamide (11)
Compound 11 was prepared according to the procedure described for compound 5, starting from
compound 10 (0.31 g, 0.79 mmol), hydrazinecarbothioamide (0.079 g, 0.87 mmol) and glacial acetic acid
o
1
(
(
(
0.15 eq). The pure product 11 (0.12 g, 32.1%) was obtained as brown solid. Mp: 151−152 C; H NMR
600 MHz, DMSO-d ) δ 11.16 (s, 1H, NH), 8.43 (s, 1H, N=CH), 8.20 (s, 1H, NH), 8.03 (s, 1H, NH), 7.62
6
s, 1H, THPB-11-H), 6.93 (s, 1H, THPB-1-H), 6.68 (s, 1H, THPB-4-H), 6.04 (dd, J = 11.0, 5.4 Hz, 1H,
CH CH=CH ), 5.96 (d, J = 18.7 Hz, 2H, OCH O), 5.35 (d, J = 17.2 Hz, 1H, CH CH=CH), 5.21 (d, J =
2
2
2
2
1
0.3 Hz, 1H, CH CH=CH), 4.57–4.47 (m, 2H, CH CH=CH ), 4.06 (d, J = 15.9 Hz, 1H, CH), 3.86 (s, 3H,
2 2 2
OCH ), 3.45 (dd, J = 17.1, 10.9 Hz, 2H, THPB-8-H), 3.42–3.34 (m, 2H, THPB-13-H), 3.08 (d, J = 6.8 Hz,
3
1
H, THPB-5-H), 2.90 (d, J = 11.2 Hz, 1H, THPB-5-H), 2.61 (d, J = 15.5 Hz, 1H, THPB-6-H), 2.47–2.42
1
3
(
m, 1H, THPB-6-H) ppm; C NMR (151 MHz, DMSO-d ) δ 178.2 (C=S), 150.5, 146.2, 146.0, 145.5,
6
1
41.1, 135.0, 131.3, 129.3, 128.0, 127.6, 118.0, 108.6, 108.2, 106.1, 101.1, 79.6, 73.2, 59.1, 56.5, 54.2,
1.1, 33.7, 29.4 ppm.
5
4
.1.22. Synthesis of 2-((10-Methoxy-9-(prop-2-yn-1-yloxy)-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinolin-12-yl)methylene)hydrazine-1-carbothioamide (14a)
Compound 14a was prepared according to the procedure described for compound 5, starting from
compound 13a (0.55 g, 1.4 mmol), hydrazinecarbothioamide (0.14 g, 1.6 mmol) and glacial acetic acid
o
1
(
(
(
0.15 eq). The pure product 14a (0.33 g, 50.1%) was obtained as brown solid. Mp: 174−175 C; H NMR
600 MHz, DMSO-d ) δ 11.18 (s, 1H, NH), 8.43 (s, 1H, N=CH), 8.22 (s, 1H, NH), 8.06 (s, 1H, NH), 7.64
6
s, 1H, THPB-11-H), 6.94 (s, 1H, THPB-1-H), 6.69 (s, 1H, THPB-4-H), 5.98 (s, 1H, OCHO), 5.95 (s, 1H,
OCHO), 4.78 (d, J = 1.9 Hz, 2H, CH C≡CH), 4.14 (d, J = 15.8 Hz, 1H, CH), 3.87 (s, 3H, OCH ), 3.51 (s,
2
3
1
H, CH C≡CH), 3.45 (dd, J = 11.9, 6.7 Hz, 4H, THPB-8-H, THPB-13-H), 3.08 (d, J = 6.7 Hz, 1H,
2
THPB-5-H), 2.92 (dd, J = 19.9, 8.8 Hz, 1H, THPB-5-H), 2.62 (d, J = 15.5 Hz, 1H, THPB-6-H), 2.45 (d, J
2
2

ACCEPTED MANUSCRIPT
1
3
=
11.1 Hz, 1H, THPB-6-H) ppm; C NMR (151 MHz, DMSO-d ) δ 178.2 (C=S), 150.4, 146.2, 146.0,
6
1
45.5, 141.1, 134.8, 131.3, 128.9, 128.1, 119.6, 108.6, 106.4, 101.8, 100.0, 79.7, 76.3, 60.0, 59.0, 56.5,
5.3, 51.7, 33.5, 29.8 ppm.
5
4
.1.23. Synthesis of 2-((9-(Cyanomethoxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinolin-12-yl)methylene)hydrazine-1-carbothioamide (14b)
Compound 14b was prepared according to the procedure described for compound 5, starting from
compound 13b (0.66 g, 1.7 mmol), hydrazinecarbothioamide (0.17 g, 1.9 mmol) and glacial acetic acid
o
1
(
(
(
0.15 eq). The pure product 14b (0.55 g, 69.2%) was obtained as brown solid. Mp: 219−220 C; H NMR
600 MHz, DMSO-d ) δ 11.21 (s, 1H, NH), 8.44 (s, 1H, N=CH), 8.25 (s, 1H, NH), 8.10 (s, 1H, NH), 7.68
6
s, 1H, THPB-11-H), 6.95 (s, 1H, THPB-1-H), 6.69 (s, 1H, THPB-4-H), 5.98 (d, J = 0.8 Hz, 1H, OCHO),
5
3
.95 (d, J = 0.7 Hz, 1H, OCHO), 5.05 (d, J = 3.3 Hz, 2H, CH C≡N), 4.11 (d, J = 15.7 Hz, 1H, CH), 3.90 (s,
2
H, OCH ), 3.47–3.43 (m, 4H, THPB-8-H, THPB-13-H), 3.10 (d, J = 7.3 Hz, 1H, THPB-5-H), 2.95–2.88
3
(m, 1H, THPB-5-H), 2.63 (d, J = 15.5 Hz, 1H, THPB-6-H), 2.47 (d, J = 12.8 Hz, 1H, THPB-6-H) ppm;
1
3
C NMR (151 MHz, DMSO-d ) δ 178.2 (C=S), 150.5, 146.3, 146.1, 131.7, 130.4, 129.8, 128.9, 127.7,
6
118.1, 113.8, 108.4, 105.6, 101.1, 100.8, 73.6, 73.1, 63.0, 58.4, 57.6, 55.9, 33.8, 29.4 ppm.
4
.1.24. Synthesis of 9-Ethoxy-10-methoxy-12-((2-(thiazol-2-yl)hydrazono)methyl)-5,8,13,13a-tetrahydro
-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinoline (9a)
Compound 9a was prepared according to the procedure described for compound 6, starting from
compound 8a (0.31 g, 0.69 mmol) and 2-chloroacetaldehyde (0.08 g, 1.1 mmol). The pure product 9a (0.15
o
1
g, 45.8%) was obtained as brown solid. Mp: > 250 C; H NMR (600 MHz, CDCl ) δ 8.10 (s, 1H, N=CH),
3
7
.30 (s, 1H, THPB-11-H), 7.26–7.24 (m, 1H, thiazolyl-4-H), 6.83 (s, 1H, THPB-1-H), 6.70 (s, 1H,
thiazolyl-5-H), 6.61 (s, 1H, THPB-1-H), 5.95 (d, J = 15.5 Hz, 2H, OCH O), 4.26 (d, J = 14.6 Hz, 1H, CH),
2
4
.18–4.12 (m, 1H, CHCH ), 4.10–4.04 (m, 1H, CHCH ), 3.90 (s, 3H, OCH ), 3.52 (d, J = 15.2 Hz, 3H,
3 3 3
THPB-8-H, THPB-13-H), 3.22 (s, 1H, THPB-13-H), 3.14 (d, J = 11.6 Hz, 1H, THPB-5-H), 2.86 (t, J =
2.5 Hz, 1H, THPB-5-H), 2.67 (dd, J = 24.0, 13.9 Hz, 2H, THPB-6-H), 1.39 (t, J = 6.9 Hz, 3H, CH CH )
1
2
3
13
ppm; C NMR (151 MHz, DMSO-d ) δ 169.4, 150.3, 147.4, 146.2, 145.9, 144.9, 141.2, 131.4, 128.1,
6
1
27.9, 126.0, 124.2, 112.0, 109.0, 108.5, 106.5, 101.1, 68.2, 59.3, 56.1, 53.9, 51.1, 33.7, 29.4, 16.2 ppm;
+
HRMS (ESI) calcd. for C H N O S [M + H] , 479.1753; found, 479.1752.
2
5
26
4
4
4
.1.25. Synthesis of 9-Butoxy-10-methoxy-12-((2-(thiazol-2-yl)hydrazono)methyl)-5,8,13,13a-tetrahydro
-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinoline (9b)
Compound 9b was prepared according to the procedure described for compound 6, starting from
compound 8b (0.14 g, 0.29 mmol) and 2-chloroacetaldehyde (0.034 g, 0.44 mmol). The pure product 9b
o
1
(
0.061 g, 41.6%) was obtained as brown solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 11.76 (s,
6
1
H, NH), 8.28 (s, 1H, N=CH), 7.36 (s, 1H, THPB-11-H), 7.24 (s, 1H, thiazolyl-4-H), 7.19 (s, 1H,
thiazolyl-5-H), 6.84 (s, 2H, THPB-1-H, THPB-4-H), 6.05 (s, 2H, OCH O), 4.66 (s, 1H, CH), 4.59 (d, J =
2
2
3

ACCEPTED MANUSCRIPT
1
5.5 Hz, 1H, CHCH CH CH ), 4.41 (d, J = 13.6 Hz, 1H, CHCH CH CH ), 4.05 (dd, J = 16.7, 10.7 Hz, 3H,
2
2
3
2
2
3
THPB-8-H, THPB-13-H), 3.87 (s, 3H, OCH ), 3.40 (s, 3H, THPB-5-H, THPB-13-H), 3.13–3.05 (m, 1H,
3
THPB-6-H), 2.88 (d, J = 12.7 Hz, 1H, THPB-6-H), 1.75–1.67 (m, 2H, CH CH CH CH ), 1.46 (dd, J =
2
2
2
3
1
3
1
3.7, 6.9 Hz, 2H, CH CH CH CH ), 0.96 (t, J = 7.3 Hz, 3H, CH CH CH CH ) ppm; C NMR (151 MHz,
2 2 2 3 2 2 2 3
DMSO-d ) δ 169.3, 150.7, 147.4, 147.1, 145.9, 144.8, 141.2, 131.4, 128.1, 127.4, 126.0, 124.2, 112.0,
6
1
09.0, 108.8, 106.4, 101.8, 72.6, 58.9, 56.4, 51.5, 50.4, 49.1, 32.4, 32.2, 19.1, 14.2 ppm; HRMS (ESI)
+
calcd. for C H N O S [M + H] , 507.2066; found, 507.2064.
2
7
30
4
4
4
.1.26.
Synthesis
of
9-(Hexyloxy)-10-methoxy-12-((2-(thiazol-2-yl)hydrazono)methyl)-5,8,13,13a-
tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinoline (9c)
Compound 9c was prepared according to the procedure described for compound 6, starting from
compound 8c (0.20 g, 0.39 mmol) and 2-chloroacetaldehyde (0.046 g, 0.59 mmol). The pure product 9c
o
1
(
0.090 g, 43.2%) was obtained as brown solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 11.71 (s,
6
1
1
4
1
H, NH), 8.24 (s, 1H, N=CH), 7.25 (s, 1H, THPB-11-H), 7.21 (d, J = 3.4 Hz, 1H, thiazolyl-4-H), 7.00 (s,
H, THPB-1-H), 6.81 (s, 1H, thiazolyl-5-H), 6.69 (s, 1H, THPB-4-H), 5.97 (d, J = 7.0 Hz, 2H, OCH O),
2
.09 (d, J = 15.9 Hz, 1H, CH), 3.98 (dd, J = 11.1, 4.6 Hz, 1H, CH(CH ) CH ), 3.94 (dd, J = 11.1, 4.6 Hz,
2
4
3
H, CH(CH ) CH ), 3.82 (s, 3H, OCH ), 3.59 (d, J = 14.1 Hz, 2H, THPB-8-H), 3.30 (d, J = 7.6 Hz, 2H,
2
4
3
3
THPB-13-H), 3.10 (s, 1H, THPB-5-H), 2.97–2.88 (m, 1H, THPB-5-H), 2.63 (d, J = 16.1 Hz, 1H,
THPB-6-H), 2.56 (d, J = 16.0 Hz, 1H, THPB-6-H), 1.68 (dd, J = 14.3, 6.9 Hz, 2H, CH CH (CH ) CH ),
2
2
2 3
3
1
3
1
3
5
.43 (d, J = 6.6 Hz, 2H, (CH ) CH (CH ) CH ), 1.34–1.31 (m, 4H, (CH ) (CH ) CH ), 0.89 (d, J = 6.6 Hz,
2 2 2 2 2 3 2 3 2 2 3
1
3
H, (CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 169.4, 150.7, 147.4, 145.9, 144.8, 140.0, 131.4,
2
5
3
6
29.3, 128.1, 127.4, 124.2, 112.1, 108.8, 106.4, 101.8, 73.0, 62.9, 59.1, 56.4, 54.2, 49.1, 45.2, 36.4, 31.5,
+
0.1, 29.3, 25.5, 22.5, 14.4 ppm; HRMS (ESI) calcd. for C H N O S [M + H] , 535.2379; found,
2
9
34
4
4
35.2378.
4
.1.27.
Synthesis
of
10-Methoxy-9-(octyloxy)-12-((2-(thiazol-2-yl)hydrazono)methyl)-5,8,13,13a-
tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinoline (9d)
Compound 9d was prepared according to the procedure described for compound 6, starting from
compound 8d (0.20 g, 0.37 mmol) and 2-chloroacetaldehyde (0.043 g, 0.56 mmol). The pure product 9d
o
1
(
0.084 g, 40.2%) was obtained as brown solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 11.83 (s,
6
1
H, NH), 8.48 (s, 1H, N=CH), 7.49 (s, 1H, THPB-11-H), 7.38 (s, 1H, thiazolyl-4-H), 7.19 (s, 1H,
THPB-1-H), 7.00 (s, 1H, thiazolyl-5-H), 6.84 (s, 1H, THPB-4-H), 6.05 (d, J = 6.2 Hz, 2H, OCH O), 4.68
2
(
s, 1H, CH), 4.59 (d, J = 15.6 Hz, 1H, CH(CH ) CH ), 4.42 (d, J = 14.1 Hz, 1H, CH(CH ) CH ), 4.04 (s,
2 6 3 2 4 3
2
2
H, THPB-8-H), 3.89 (s, 3H, OCH ), 3.41 (s, 2H, THPB-13-H), 3.14–3.07 (m, 1H, THPB-5-H), 2.89 (s,
3
H, THPB-5-H, THPB-6-H), 2.73 (s, 1H, THPB-6-H), 1.73 (s, 2H, CH CH (CH ) CH ), 1.43 (s, 2H,
2
2
2 5
3
13
(
CH ) CH (CH ) CH ), 1.30 (d, J = 20.0 Hz, 8H, (CH ) (CH ) CH ), 0.88 (s, 3H, (CH ) CH ) ppm;
C
2
2
2
2 4
3
2 3
2 4
3
2 7
3
NMR (151 MHz, DMSO-d ) δ 169.3, 150.7, 147.4, 147.1, 145.9, 144.8, 141.2, 131.4, 128.1, 127.4, 125.9,
6
2
4

ACCEPTED MANUSCRIPT
1
2
25.5, 124.2, 112.1, 108.6, 108.4, 106.3, 101.8, 73.0, 58.9, 56.5, 54.1, 49.1, 36.3, 31.7, 31.3, 30.2, 29.1,
+
5.8, 22.6, 14.4 ppm; HRMS (ESI) calcd. for C H N O S [M + H] , 563.2692; found, 563.2693.
3
1
38
4
4
4
.1.28.
Synthesis
of
9-(Decyloxy)-10-methoxy-12-((2-(thiazol-2-yl)hydrazono)methyl)-5,8,13,13a-
tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinoline (9e)
Compound 9e was prepared according to the procedure described for compound 6, starting from
compound 8e (0.50 g, 0.88 mmol) and 2-chloroacetaldehyde (0.10 g, 1.3 mmol). The pure product 9e (0.21
o
1
g, 41.3%) was obtained as brown solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 11.91 (s, 1H,
6
NH), 8.26 (s, 1H, N=CH), 7.34 (s, 1H, THPB-11-H), 7.23 (d, J = 3.0 Hz, 1H, thiazolyl-4-H), 7.13 (d, J =
3
9.4 Hz, 1H, thiazolyl-5-H), 6.82 (s, 2H, THPB-1-H, THPB-4-H), 6.04 (s, 2H, OCH O), 4.57 (s, 2H,
2
CH (CH ) CH ), 4.39 (s, 1H, CH), 4.04–3.97 (m, 3H, THPB-8-H, THPB-13-H), 3.86 (s, 3H, OCH ), 3.33
2
2 8
3
3
(s, 3H, THPB-5-H, THPB-13-H), 3.05 (s, 1H, THPB-6-H), 2.88 (d, J = 12.7 Hz, 1H, THPB-6-H),
1
.74–1.69 (m, 2H, CH CH (CH ) CH ), 1.42 (d, J = 6.7 Hz, 2H, (CH ) CH (CH ) CH ), 1.27 (s, 12H,
2
2
2 7
3
2 2
2
2 6
3
1
3
(
CH ) (CH ) CH ), 0.86 (t, J = 6.6 Hz, 3H, (CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 169.4,
2 3 2 6 3 2 9 3 6
1
50.6, 147.4, 147.1, 145.9, 144.8, 141.2, 131.4, 130.4, 128.1, 127.4, 126.0, 124.2, 112.0, 109.0, 108.7,
1
06.5, 101.7, 72.9, 59.2, 56.7, 56.4, 50.6, 49.1, 34.6, 32.8, 31.8, 30.2, 29.5, 29.2, 25.8, 22.5, 14.4 ppm;
+
HRMS (ESI) calcd. for C H N O S [M + H] , 591.3005; found, 591.3006.
3
3
42
4
4
4
.1.29. Synthesis of 9-(Dodecyloxy)-10-methoxy-12-((2-(thiazol-2-yl)hydrazono)methyl)-5,8,13,13a-
tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinoline (9f)
Compound 9f was prepared according to the procedure described for compound 6, starting from compound
8
4
8
f (0.30 g, 0.50 mmol) and 2-chloroacetaldehyde (0.059 g, 0.76 mmol). The pure product 9f (0.13 g,
o
1
0.5%) was obtained as brown solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 11.84 (s, 1H, NH),
6
.47 (s, 1H, N=CH), 7.48 (s, 1H, THPB-11-H), 7.37 (s, 1H, thiazolyl-4-H), 7.19 (s, 1H, THPB-1-H), 6.99
(
s, 1H, thiazolyl-5-H), 6.84 (s, 1H, THPB-4-H), 6.05 (d, J = 6.4 Hz, 2H, OCH O), 4.68 (s, 1H,
2
CH(CH ) CH ), 4.59 (d, J = 15.6 Hz, 1H, CH), 4.41 (d, J = 14.7 Hz, 1H, CH(CH ) CH ), 4.05–4.01 (m,
2
10
3
2 10
3
2
H. THPB-8-H), 3.89 (s, 3H, OCH ), 3.42 (d, J = 12.5 Hz, 2H, THPB-13-H), 3.14–3.07 (m, 1H,
3
THPB-5-H), 2.88 (d, J = 10.7 Hz, 2H, THPB-6-H), 2.73 (s, 1H, THPB-5-H), 1.72 (d, J = 6.5 Hz, 2H,
CH CH (CH ) CH ), 1.42 (s, 2H, (CH ) CH (CH ) CH ), 1.26 (s, 16H, (CH ) (CH ) CH ), 0.86 (t, J = 6.4
2
2
2 9
3
2 2
2
2 8
3
2 3
2 8
3
1
3
Hz, 3H, (CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 169.3, 150.6, 147.4, 147.1, 145.9, 144.8,
2
11
3
6
1
41.2, 131.4, 128.1, 127.4, 126.0, 125.5, 124.0, 113.0, 108.6, 108.3, 106.4, 101.8, 73.0, 58.9, 56.5, 54.1,
5
1.5, 49.1, 36.2, 33.7, 31.8, 30.2, 29.7, 29.5, 29.2, 25.8, 25.7, 22.5, 14.4 ppm; HRMS (ESI) calcd. for
+
C H N O S [M + H] , 619.3318; found, 619.3317.
35
46
4
4
4
.1.30. Synthesis of 2-((10-Methoxy-12-((2-(thiazol-2-yl)hydrazono)methyl)-5,8,13,13a-tetrahydro-6H-[1,3]
dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-9-yl)oxy)ethan-1-ol (9g).
Compound 9g was prepared according to the procedure described for compound 6, starting from
compound 8g (0.10 g, 0.21 mmol) and 2-chloroacetaldehyde (0.025 g, 0.32 mmol). The pure product 9g
2
5

ACCEPTED MANUSCRIPT
o
1
(
0.041 g, 39.4%) was obtained as brown solid. Mp: 236−237 C; H NMR (600 MHz, DMSO-d ) δ 11.85
6
(s, 1H, NH), 8.33 (s, 1H, N=CH), 7.43 (s, 1H, THPB-11-H), 7.25 (s, 1H, thiazolyl-4-H), 7.06 (s, 1H,
THPB-1-H), 6.95 (s, 1H, thiazolyl-5-H), 6.84 (s, 1H, THPB-4-H), 6.02 (d, J = 18.6 Hz, 2H, OCH O), 4.77
2
(
s, 1H, OCHCH OH), 4.71 (s, 1H, OCHCH OH), 4.53 (d, J = 15.9 Hz, 1H, CH), 4.23–4.19 (m, 1H,
2
2
OCH CHOH), 4.17–4.13 (m, 1H, OCH CHOH), 3.89 (s, 3H, OCH ), 3.45 (d, J = 15.8 Hz, 2H,
2
2
3
THPB-8-H), 3.38 (dd, J = 23.3, 13.5 Hz, 2H, THPB-13-H), 3.05 (d, J = 6.1 Hz, 1H, THPB-5-H), 2.92 (t, J
=
11.1 Hz, 1H, THPB-5-H), 2.62 (d, J = 15.5 Hz, 1H, THPB-6-H), 2.49–2.45 (m, 1H, THPB-6-H) ppm;
1
3
C NMR (151 MHz, DMSO-d ) δ 169.4, 150.4, 146.2, 146.0, 145.9, 141.2, 131.4, 129.4, 128.0, 127.3,
6
112.1, 108.6, 108.2, 106.1, 101.1, 79.6, 74.6, 60.9, 59.2, 56.5, 54.0, 51.1, 49.1, 33.7, 29.5 ppm; HRMS
+
(
ESI) calcd. for C H N O S [M + H] , 495.1702; found, 495.1704.
2
5
26
4
5
4
.1.31.
Synthesis
of
9-(Allyloxy)-10-methoxy-12-((2-(thiazol-2-yl)hydrazono)methyl)-5,8,13,13a-
tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinoline (12)
Compound 12 was prepared according to the procedure described for compound 6, starting from
compound 11 (0.10 g, 0.22 mmol) and 2-chloroacetaldehyde (0.026 g, 0.33 mmol). The pure product 12
o
1
(
0.032 g, 30.1%) was obtained as brown solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 11.85 (s,
6
1
H, NH), 8.32 (s, 1H, N=CH), 8.25 (s, 1H, THPB-11-H), 7.32 (s, 1H, THPB-1-H), 7.23 (d, J = 3.6 Hz, 1H,
thiazolyl-4-H), 6.82 (d, J = 3.3 Hz, 1H, thiazolyl-5-H), 6.78 (s, 1H, THPB-4-H), 6.07 (dd, J = 11.1, 5.8 Hz,
H, CH CH=CH ), 6.02 (s, 2H, OCH O), 5.38 (d, J = 17.2 Hz, 1H, CH CH=CH), 5.24 (d, J = 9.5 Hz, 1H,
1
2
2
2
2
CH CH=CH), 4.58 (d, J = 5.4 Hz, 3H, CH CH=CH , CH), 4.34 (s, 1H, THPB-8-H), 3.89 (s, 1H,
2
2
2
THPB-8-H), 3.87 (s, 3H, OCH ), 3.44 (dd, J = 13.9, 6.9 Hz, 2H, THPB-13-H), 3.32 (s, 3H, THPB-5-H,
3
1
3
THPB-6-H), 3.17 (s, 1H, THPB-6-H) ppm; C NMR (151 MHz, DMSO-d ) δ 169.3, 150.5, 146.2, 146.0,
6
1
5
4
45.5, 141.1, 134.8, 131.3, 129.3, 128.3, 127.6, 118.5, 112.0, 108.6, 106.5, 101.5, 79.7, 73.4, 59.1, 56.6,
+
6.5, 55.4, 54.2, 49.1, 33.7, 29.4 ppm; HRMS (ESI) calcd. for C H N O S [M + H] , 491.1753; found,
2
6
26
4
4
91.1756.
4
.1.32. Synthesis of 10-Methoxy-9-(prop-2-yn-1-yloxy)-12-((2-(thiazol-2-yl)hydrazono)methyl)-5,8,13,13a-
tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinoline (15a)
Compound 15a was prepared according to the procedure described for compound 6, starting from
compound 14a (0.18 g, 0.39 mmol) and 2-chloroacetaldehyde (0.045 g, 0.58 mmol). The pure product 15a
o
1
(
0.067 g, 35.2%) was obtained as brown solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 11.73 (s,
6
1
H, NH), 8.33 (s, 1H, N=CH), 7.42 (s, 1H, THPB-11-H), 7.27 (s, 1H, thiazolyl-4-H), 7.19 (s, 1H,
THPB-1-H), 6.87 (s, 1H, thiazolyl-5-H), 6.84 (s, 1H, THPB-4-H), 6.05 (s, 2H, OCH O), 4.67 (d, J = 14.2
2
Hz, 2H, CH C≡CH), 4.45 (d, J = 15.2 Hz, 1H, CH), 4.08 (d, J = 16.0 Hz, 1H, THPB-8-H), 3.90 (s, 3H,
2
OCH ), 3.85 (s, 1H, THPB-8-H), 3.55 (s, 1H, THPB-13-H), 3.44 (dd, J = 13.6, 6.6 Hz, 2H, CH C≡CH,
3
2
THPB-13-H), 3.40 (d, J = 16.0 Hz, 2H, THPB-5-H), 3.14–3.08 (m, 1H, THPB-6-H), 2.89 (d, J = 15.6 Hz,
1
3
1
H, THPB-6-H) ppm; C NMR (151 MHz, DMSO-d ) δ 169.3, 150.5, 147.4, 146.2, 146.0, 145.5, 141.1,
6
2
6

ACCEPTED MANUSCRIPT
1
34.8, 131.3, 128.9, 127.6, 118.5, 112.0, 108.6, 106.4, 101.8, 100.0, 79.7, 60.0, 59.0, 56.5, 55.3, 51.7, 49.1,
+
3
2.1, 26.8 ppm; HRMS (ESI) calcd. for C H N O S [M + H] , 489.1597; found, 489.1598.
2
6
24
4
4
4
.1.33. Synthesis of 2-((10-Methoxy-12-((2-(thiazol-2-yl)hydrazono)methyl)-5,8,13,13a-tetrahydro-6H-[1,3]
dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-9-yl)oxy)acetonitrile (15b)
Compound 15b was prepared according to the procedure described for compound 6, starting from
compound 14b (0.18 g, 0.39 mmol) and 2-chloroacetaldehyde (0.046 g, 0.58 mmol). The pure product 15b
o
1
(
0.063 g, 33.1%) was obtained as brown solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 11.85 (s,
6
1
H, NH), 8.46 (s, 1H, N=CH), 7.55 (s, 1H, THPB-11-H), 7.37 (s, 1H, thiazolyl-4-H), 7.21 (s, 1H,
THPB-1-H), 6.99 (s, 1H, thiazolyl-5-H), 6.85 (s, 1H, THPB-4-H), 6.06 (d, J = 5.8 Hz, 2H, OCH O), 5.15
2
(
d, J = 7.2 Hz, 2H, CH C≡CN), 4.71 (d, J = 14.9 Hz, 2H, THPB-8-H), 4.47 (d, J = 15.2 Hz, 1H, CH), 4.11
2
(
d, J = 15.7 Hz, 1H, THPB-13-H), 3.95 (s, 3H, OCH ), 3.86 (d, J = 7.1 Hz, 1H, THPB-13-H), 3.40 (d, J =
3
1
6.4 Hz, 2H, THPB-5-H), 3.12 (d, J = 15.9 Hz, 1H, THPB-6-H), 2.91 (d, J = 15.3 Hz, 1H, THPB-6-H)
13
ppm; C NMR (151 MHz, DMSO-d ) δ 169.4, 150.1, 147.4, 146.2, 146.0, 142.8, 131.7, 130.4, 129.8,
6
1
28.9, 127.7, 117.2, 112.1, 109.6, 108.67, 105.6, 101.8, 79.7, 73.6, 63.0, 58.9, 58.0, 56.7, 33.5, 29.4 ppm;
+
HRMS (ESI) calcd. for C H N O S [M + H] , 490.1549; found, 490.1548.
2
5
23
5
4
4
.1.34. Synthesis of 9-((2,4-Dichlorobenzyl)oxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinoline-12-carbaldehyde (16a)
Compound 16a was prepared according to the procedure described for compound 7a, starting from
compound 4 (2.0 g, 5.7 mmol), 2,4-dichloro-1-(chloromethyl)benzene (1.7 g, 8.5 mmol) and potassium
carbonate (1.2 g, 8.5 mmol). The pure product 16a (2.34 g, 80.1%) was obtained as yellow solid. Mp:
o
1
1
1
00−101 C; H NMR (600 MHz, CDCl ) δ 10.21 (s, 1H, CHO), 7.55 (d, J = 8.2 Hz, 1H, Ph-3-H), 7.35 (s,
3
H, THPB-11-H), 7.30 (dd, J = 8.3, 1.7 Hz, 1H, Ph-5-H), 7.25 (d, J = 1.8 Hz, 1H, Ph-6-H), 6.77 (s, 1H,
THPB-1-H), 6.59 (s, 1H, THPB-4-H), 5.93 (s, 2H, OCH O), 5.27 (d, J = 12.5 Hz, 1H, Ph-1-CH), 5.19 (d, J
2
=
12.5 Hz, 1H, Ph-1-CH), 4.19 (d, J = 15.8 Hz, 1H, CH), 3.92 (s, 3H, OCH ), 3.84 (d, J = 17.0 Hz, 1H,
3
THPB-8-H), 3.48 (s, 1H, THPB-8-H), 3.44 (s, H, THPB-13-H), 3.10 (s, 2H, THPB-5-H), 3.06–2.99 (m,
1
3
1
H, THPB-13-H), 2.65 (d, J = 15.7 Hz, 1H, THPB-6-H), 2.58 (s, 1H, THPB-6-H) ppm; C NMR (151
MHz, CDCl ) δ 190.7 (C=O), 150.0, 148.5, 146.3, 137.0, 134.5, 133.9, 133.7, 133.0, 130.4, 129.5, 129.3,
3
1
29.0, 127.5, 127.3, 113.8, 108.4, 105.6, 100.9, 70.7, 61.9, 59.1, 56.0, 54.0, 51.3, 33.5, 29.2 ppm.
4
.1.35. Synthesis of 9-((2-Chlorobenzyl)oxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinoline-12-carbaldehyde (16b)
Compound 16b was prepared according to the procedure described for compound 7a, starting from
compound 4 (2.0 g, 5.7 mmol), 1-chloro-2-(chloromethyl)benzene (1.4 g, 8.5 mmol) and potassium
carbonate (1.2 g, 8.5 mmol). The pure product 16b (2.13 g, 78.2%) was obtained as yellow solid. Mp:
o
1
4
8−49 C; H NMR (600 MHz, CDCl ) δ 10.20 (s, 1H, CHO), 7.60 (dd, J = 7.4, 1.7 Hz, 1H, Ph-3-H), 7.34
3
(d, J = 1.2 Hz, 1H, THPB-11-H), 7.31–7.29 (m, 1H, Ph-4-H), 7.26 (s, 1H, Ph-6-H), 7.22 (d, J = 1.7 Hz, 1H,
27

ACCEPTED MANUSCRIPT
Ph-5-H), 6.77 (s, 1H, THPB-1-H), 6.58 (s, 1H, THPB-4-H), 5.92 (s, 2H, OCH O), 5.31 (t, J = 12.7 Hz, 1H,
2
Ph-1-CH), 5.24 (d, J = 12.2 Hz, 1H, Ph-1-CH), 4.23 (d, J = 15.9 Hz, 1H, CH), 3.93 (s, 3H, OCH ), 3.84
3
(dd, J = 16.9, 3.2 Hz, 1H, THPB-8-H), 3.50 (s, 1H, THPB-8-H), 3.46 (s, 1H, THPB-13-H), 3.11 (s, 2H,
THPB-5-H), 3.05 (d, J = 12.4 Hz, 1H, THPB-13-H), 2.65 (d, J = 15.8 Hz, 1H, THPB-6-H), 2.58 (d, J =
1
3
1
1.0 Hz, 1H, THPB-6-H) ppm; C NMR (151 MHz, CDCl ) δ 190.7 (C=O), 150.2, 148.9, 146.3, 146.2,
3
1
38.4, 135.3, 133.2, 132.7, 129.7, 129.4, 129.3, 128.7, 127.0, 113.8, 108.4, 105.6, 100.8, 71.3, 62.7, 59.0,
5
8.4, 56.1, 54.0, 51.1, 31.6, 26.9 ppm.
4
.1.36. Synthesis of 9-((3-Chlorobenzyl)oxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinoline-12-carbaldehyde (16c)
Compound 16c was prepared according to the procedure described for compound 7a, starting from
compound 4 (1.5 g, 4.2 mmol), 1-chloro-3-(chloromethyl)benzene (1.0 g, 6.4 mmol) and potassium
carbonate (0.88 g, 6.4 mmol). The pure product 16c (1.56 g, 77.5%) was obtained as yellow solid. Mp:
o
1
4
5−46 C; H NMR (600 MHz, CDCl ) δ 10.22 (s, 1H, CHO), 7.47 (s, 1H, Ph-2-H), 7.36 (s, 1H,
3
THPB-11-H), 7.32 (s, 2H, Ph-5,6-H), 7.22 (d, J = 7.2 Hz, 1H, Ph-4-H), 6.77 (s, 1H, THPB-1-H), 6.59 (s,
H, THPB-4-H), 5.93 (s, 2H, OCH O), 5.20 (d, J = 11.6 Hz, 1H, Ph-1-CH), 5.08 (d, J = 11.6 Hz, 1H,
1
2
Ph-1-CH), 4.20 (d, J = 15.7 Hz, 1H, CH), 3.94 (s, 3H, OCH ), 3.85 (d, J = 5.2 Hz, 1H, THPB-8-H), 3.53 (s,
3
1
H, THPB-8-H), 3.45 (s, 1H, THPB-13-H), 3.13 (s, 2H, THPB-5-H), 3.08–3.01 (m, 1H, THPB-13-H),
1
3
2
.67 (d, J = 16.0 Hz, 1H, THPB-6-H), 2.62 (d, J = 11.4 Hz, 1H, THPB-6-H) ppm; C NMR (151 MHz,
CDCl ) δ 190.6 (C=O), 150.1, 148.7, 146.4, 139.4, 134.4, 129.8, 129.5, 128.3, 128.1, 127.6, 126.9, 126.0,
3
1
24.8, 113.7, 108.4, 105.6, 100.9, 73.6, 64.4, 59.0, 58.4, 56.0, 54.1, 51.2, 31.6, 26.9 ppm.
4
.1.37. Synthesis of 9-((4-Chlorobenzyl)oxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinoline-12-carbaldehyde (16d)
Compound 16d was prepared according to the procedure described for compound 7a, starting from
compound 4 (2.0 g, 5.7 mmol), 1-chloro-4-(chloromethyl)benzene (1.4 g, 8.5 mmol) and potassium
carbonate (1.2 g, 8.5 mmol). The pure product 16d (2.07 g, 76.0%) was obtained as yellow solid. Mp:
o
1
6
2
6−67 C; H NMR (600 MHz, CDCl ) δ 10.19 (s, 1H, CHO), 7.34 (d, J = 3.9 Hz, 2H, Ph-3,5-H), 7.32 (s,
3
H, Ph-2,6-H), 7.26 (s, 1H, THPB-11-H), 6.76 (s, 1H, THPB-1-H), 6.58 (s, 1H, THPB-4-H), 5.92 (s, 2H,
OCH O), 5.19 (d, J = 11.5 Hz, 1H, Ph-1-CH), 5.07 (d, J = 11.5 Hz, 1H, Ph-1-CH), 4.14 (d, J = 15.7 Hz,
2
1
1
H, CH), 3.93 (s, 3H, OCH ), 3.86–3.81 (m, 1H, THPB-8-H), 3.52–3.47 (m, 1H, THPB-8-H), 3.40 (d, J =
3
5.7 Hz, 1H, THPB-13-H), 3.09 (d, J = 8.2 Hz, 2H, THPB-5-H), 3.04–2.97 (m, 1H, THPB-13-H), 2.65 (d,
1
3
J = 15.7 Hz, 1H, THPB-6-H), 2.58 (d, J = 10.3 Hz, 1H, THPB-6-H) ppm; C NMR (151 MHz, CDCl ) δ
3
1
90.7 (C=O), 150.1, 148.8, 146.3, 146.2, 139.4, 135.9, 134.1, 133.3, 129.4, 129.3, 128.7, 128.6, 128.2,
1
27.6, 113.6, 108.4, 105.6, 100.9, 73.6, 64.5, 59.0, 56.0, 54.2, 51.2, 33.6, 29.3 ppm.
4
.1.38. Synthesis of 9-((3,4-Dichlorobenzyl)oxy)-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinoline-12-carbaldehyde (16e)
2
8