Design, synthesis and biological evaluations of quaternization harman analogues as potential antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2018.10.012

Thirty-three new quaternization harman analogues were synthesized and their antibacterial activity against four Gram-positive and two Gram-negative bacteria were evaluated. The structure–activity relationships were summarized and compounds 4f, 4i, 4l, 4u,

Full text of DOI:10.1016/j.ejmech.2018.10.012

Accepted Manuscript
Design, synthesis and biological evaluations of quaternization harman analogues as
potential antibacterial agents
Jiangkun Dai, Wenjia Dan, Siyu Ren, Congguo Shang, Junru Wang
PII:
S0223-5234(18)30874-2
10.1016/j.ejmech.2018.10.012
EJMECH 10798
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 January 2018
Revised Date: 16 September 2018
Accepted Date: 5 October 2018
Please cite this article as: J. Dai, W. Dan, S. Ren, C. Shang, J. Wang, Design, synthesis and biological
evaluations of quaternization harman analogues as potential antibacterial agents, European Journal of
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.10.012.
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Design, synthesis and biological evaluations of quaternization
harman analogues as potential antibacterial agents
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Jiangkun Dai, Wenjia Dan, Siyu Ren, Congguo Shang, and Junru Wang*
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College of Chemistry & Pharmacy, Northwest A&F University, 22 Xinong Road,
Yangling 712100, Shaanxi, China.
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*Corresponding author
E-mail: wangjunru@nwafu.edu.cn (J.R. Wang)
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ABSTRACT
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Thirty-three new quaternization harman analogues were synthesized and their
antibacterial activity against four Gram-positive and two Gram-negative bacteria were
evaluated. The structure–activity relationships were summarized and compounds 4f,
4i, 4l, 4u, 4w, 4x and 5c showed excellent antibacterial activity, low cytotoxicity,
good thermal stability and “drug-like” properties. In particular, compound 4x
exhibited better bactericidal effect (4-fold superiority against methicillin-resistant
Staphylococcus aureus) than standard drugs fosfomycin sodium and ampicillin
sodium (minimum inhibitory concentration = 50 nmol/mL). Scanning electron
microscopy revealed morphological changes of the bacterial cell surface and the
docking evaluation provided a good total score (6.4952) for 4x which is close to the
score of ciprofloxacin (6.9723). The results indicated that the quaternization harman
analogues might exert their bactericidal effect by damaging bacterial cell membrane
and wall, and disrupting the function of type II topoisomerase. In addition, the in vivo
antibacterial assay with a protective efficacy of 81.3% further demonstrated the
potential of these derivatives as new bactericides and antibiotics.
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Keywords: harman; quaternization; derivatization synthesis; bactericides; structure–
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activity relationships; molecular docking.
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1. Introduction
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Infections caused by bacterial pathogens are a major cause of morbidity and
mortality worldwide. Although the successful treatment of such infections by
antibiotic drugs is widely regarded as a major medical breakthrough of the 20th
century, this achievement may not be sustainable in the future, as bacteria have
counteracted antibiotic pressure and developed or acquired resistances that render
formerly efficacious drugs inactive [1,2]. In recent years, the emergence of the
antibiotic crisis has brought great challenges to normal production and life such as
healthcare systems, farming, and the food production industry [3–5]. For example, the
misuse and overuse of current antibiotics has resulted in a situation that higher dose is
required to successfully treat the bacterial infection [6]. Moreover, the intensive
antibacterial discovery effort has seen a dramatic decline in the large pharmacy
industry in the last two decades [7]. “The cost in terms of lost global production
between now and 2050 would be an enormous 100 trillion USD if we do not take
action,” as a UK Government report states [8]. Therefore, we need to develop new
drugs to replace the ones that no longer work.
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Natural products are an important source of antibacterial agents [9]. The seeds of
Peganum harmala, containing about 2–6% pharmacologically active alkaloids which
are mostly β-carbolines such as harman L1 (Fig. 1), were used as antibacterial drugs
many years ago in North Africa and Middle East [10]. Canthin-6-one L4, a subclass
of β-carboline alkaloids with an additional D ring, isolated from Allium neapolitanum,
showed good antibacterial activity (MIC = 8–64 µg/mL) against S. aureus [11].
Eudistomin U L6, β-carboline with an additional indole ring at position 1, isolated
from several species of marine ascidians, also exhibited good antibacterial activity
(IC50 = 6.4 µg/mL) [12]. In our previous work, we also reported the antibacterial
activity of some modified β-carbolines L2–L5 [13,14].
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With the aim of finding more promising antibacterial harman analogues, we
report here a strategy to generate the bioactive compounds. Our idea and design are
relatively simple (Fig. 1). Briefly, considering the antibacterial activity data of L2 and
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L3, we speculate that the substituents at position 1 are favorable. Simultaneously, the
activity data of L1 and L6 indicate that the aromatic substituents are more potent. So
we deduce that an additional aromatic substituent at position 1 might improve the
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bactericidal effect of harman. Our previous work showed that some N -substituted
canthin-6-ones L5 exhibited approximate 32-fold advantage against S. aureus (MIC =
0.98 µg/mL) over canthin-6-one L4 (MIC = 31.25 µg/mL) [13]. The result indicates
that quaternization derivatives might be promising. During the course of our initial
efforts to improve the antibacterial activity of harman L1, we confirmed the main
skeleton of the leading compound L7 based on the vital factors mentioned. In order to
enrich the chemical diversity as well as to further study the structure–activity
relationships, the quaternization harman analogues incorporating the methoxy group
at position 6 were also synthesized. The synthesized compounds were characterized
and tested for their in vitro and in vivo antibacterial activity. In addition, their
structure–activity relationships, cytotoxicity, thermal stability, “drug-like” properties
and preliminary antibacterial mechanism were studied.
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2. Results and Discussion
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2.1.
Chemistry
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The initial synthetic aim was to obtain a convenient route for the preparation of
quaternization harman analogues. So, it was decided to begin with the preparation of
2a–2h (yield: 92%–96%) through the Pictet–Spengler reaction [15]. Compounds 3a–
3h (yield: 62%–73%) were then easily obtained using a palladium catalyst [16].
Subsequently, the quaternization reaction was carried out at room temperature [17].
The synthesis of the target compounds 4a–4x (yield: 67%–96%) is shown in Fig. 2. In
order to clarify the influence of different substituted benzyl groups on activity,
compounds 5a–5i (yield: 72%–95%) were synthesized as shown in Fig. 3. Nuclear
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magnetic resonance (NMR) spectroscopy ( H NMR, C NMR, DEPT 135,
heteronuclear multiple-bond correlation, heteronuclear single quantum correlation,
and homonuclear correlation spectroscopy) of 4q confirmed that the benzylation
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position is N (Fig. S1). The successful synthesis of the intermediates 3a–3h and the
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quaternization harman derivatives (4a–4x and 5a–5i) was further confirmed by crystal
structures of the compounds 3e and 5f (Fig. 4). Moreover, these two crystal structures
further guided the structural confirmation of all the 41 compounds. The x-ray data
block (bond lengths, bond angles etc.) of compounds 3e and 5f has been presented in
Supplementary data.
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All spectral and analytical data were consistent with the assigned structures. In
the NMR spectra of compound 3a, the signals of the methyl group were detected
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around δ = 3.94 ppm ( H NMR) and δ = 52.2 ppm ( C NMR). The signal of the
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imino hydrogen atom was observed at δ = 8.81 ppm ( H NMR) and the signals of the
phenyl hydrogen atoms appeared in the aromatic region. Moreover, the signal of the
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carbonyl carbon atom was detected around δ = 166.8 ppm ( C NMR). After
quaternization, the signals of the methylene group were detected around δ = 5.82 ppm
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( H NMR) and δ = 60.9 ppm ( C NMR) in the NMR spectra of compound 4a, which
indicated that the quaternization harman derivatives were successfully synthesized. In
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addition, the signals of the carbonyl could be found at 1718 and 1712 cm in the IR
spectra of compounds 3a and 4a, respectively.
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2.2.
Antibacterial activity
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Bacteria could be divided into two categories by Gram staining, Gram-positive
and Gram-negative bacteria. S. aureus and MRSA are the leading causes of bacterial
infections in humans with symptoms ranging from simple skin infections to severe
necrotizing fasciitis and pneumonia [18]. Bacillus cereus and Escherichia coli could
cause food poisoning, such as a diarrheal syndrome and an emetic syndrome, both
through the production of distinct toxins [19]. Bacillus subtilis and Ralstonia
solanacearum are major components of plant pathogens [20]. So four Gram-positive
bacteria (S. aureus CGMCC 1.8721, MRSA ATCC 43300, B. cereus CGMCC 1.1846,
and B. subtilis 769) and two Gram-negative bacteria (E. coli CGMCC 1.1636 and R.
solanacearum CGMCC 1.12711) were selected as the tested bacteria in this work.
Compounds (3a–3h, 4a–4x and 5a–5i) were evaluated for their in vitro antibacterial
activity through double dilution method, with fosfomycin sodium, ampicillin sodium,
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and cefotaxime sodium as the positive controls.
The antibacterial results (Table 1) revealed that most of the quaternization
harman analogues displayed good in vitro biological activity against all of the tested
bacterial strains except for E. coli. Although most of the intermediates were inactive
under the tested concentrations, compound 3h exhibited weak activity (MIC = 200
nmol/mL) against MRSA and S. aureus. Compared with the positive control
fosfomycin sodium, compounds 4a–4l, 4n–4o and 4q–4x were found to be the most
potent compounds against S. aureus, with peak MICs of lower than 25 nmol/mL. Six
compounds (4f, 4i, 4l, 4u, 4w and 4x) displayed equal or superior activity against
MRSA compared with the positive controls. Fifteen compounds exhibited better
activity against B. cereus than ampicillin sodium. Specifically, the MIC of 4f and 4x
(25 nmol/mL) was equal to that of the positive controls fosfomycin sodium and
cefotaxime sodium. Nine compounds (4f, 4h, 4i, 4k, 4l, 4t, 4u, 4w and 4x) showed a
potential MIC (25 nmol/mL) against B. subtilis. As shown in Table 1, eight
compounds (4f, 4h, 4i, 4k, 4l, 4u, 4w and 4x) exhibited better activity (MIC = 25
nmol/mL) against R. solanacearum than fosfomycin sodium (MIC = 50 nmol/mL).
The activity of different substituted benzyl derivatives was tested (Table 2), and
compound 5c was considered to be the compound with the most potential.
Interestingly, other quaternization harman analogues also showed similar effects on
different
bacteria.
For
example,
the
MIC
of
the
compound
2,6-dimethyl-9H-pyrido[3,4-b]indol-2-ium iodide against MRSA was 4 µg/mL [21].
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2.3.
Structure–activity relationships
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Based on the antibacterial activity data, the structure–activity relationships were
carefully investigated (Fig. 5). The derivatives containing a methoxy group at position
6 were practically the same or higher than that derivatives without this group, with the
exception of pyridine. For example, the MIC of compound 4f was 25 nmol/mL
against S. aureus, which was better than 4c (MIC = 50 nmol/mL). Compared with
harman L1 (MIC = 1000 µg/mL, ≈ 5494 nmol/mL), compound 3h displayed better
activity (MIC = 200 nmol/mL) against S. aureus, which indicates that an aromatic
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group at position 1 is effective. Comprehensive analysis indicated that the
benzo[d][1,3]dioxole group is the most effective substituent at position 1, and the
furan group is better than the methyl benzoate group. For example, the MIC against
MRSA of compound 4x was 12.5 nmol/mL, which was better than compounds 4f (25
nmol/mL), 4l (25 nmol/mL) and 4r (100 nmol/mL). Compared with compound 4c
(MIC = 50 nmol/mL against R. solanacearum), with a methyl benzoate group
substituent at position 1, the MIC of compound 4i (furan group at position 1, MIC =
25 nmol/mL against R. solanacearum) is better.
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Quaternization is clearly beneficial for improving antibacterial potency. Various
substituted benzyl groups have a significant effect on the activity. The influence of
different groups is easily seen in Table 1 and Table 2: CF > Br > Cl ≈ CH > F ≈ NO .
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For example, the MIC of compound 4f was 25 nmol/mL against all the tested bacteria
except for E. coli. In addition, the MICs of compounds 5c (25 nmol/mL against S.
aureus), 5f (75 nmol/mL against S. aureus) and 5g (50 nmol/mL against S. aureus)
indicate the activity sequence for substituents at different positions: para > meta >
ortho. Interestingly, similar findings have been reported by others, which supported
our SAR results (para substituents are better) and confirmed the advantages of
trifluoromethyl group [22].
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2.4.
Cell toxicity and molecular physicochemical properties
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Many natural and synthetic agents with the ability to interact with DNA often
have relatively low therapeutic index, most likely due to the unspecific manner of
these agents in causing DNA damage both in lesions and in highly proliferative
normal tissues. Similarly, the frequency of utilization of β-carbolines as therapeutic
agents is greatly reduced because of their cytotoxic activity toward mammalian cells
(low selectivity) [23]. We determined the cytotoxic effects of the highly bioactive
quaternization harman derivatives 4f, 4i, 4l, 4u, 4w, 4x and 5c on MARC 145 cells
(normal monkey kidney cells) and L02 cells (normal human hepatocyte) [24].
Simultaneously, the activity under mass concentration was also tested. As shown in
Table 3, the cell viabilities of all the tested compounds were more than 87% on
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MARC 145 cells and more than 71% on human hepatocyte L02 cells. The results
suggest that these molecules are of low toxicity. Interestingly, compound 4x exhibited
excellent viability (103%) on MARC 145 cells under an effective antibacterial dose (8
µg/mL).
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In 1997, Lipinski et al. published what is widely regarded as the key paper
defining physicochemical and structural properties profiles for the optimal oral
availability of drugs [25]. In 2003, Clarke and Delaney reported that most herbicides
and fungicides also adhere to the Lipinski rule [26]. As we know, for good oral
bioavailability an ideal molecule should have good intestinal absorption and reduced
molecular flexibility [27]. The molecular properties of the preferred ionic derivatives
are shown in Table 4. For all the preferred compounds, the log P values were lower
than 5, the number of hydrogen bond acceptors were lower than 10, the number of
rotatable bonds were lower than 10, and the number of hydrogen bond donors were
lower than 5. Their physicochemical data therefore conform to the Lipinski rule
without considering the molecular weight, which demonstrate they might have good
“drug-like” properties. Further, the topological polar surface area (TPSA), a
parameter defined as the surface sum over all polar atoms and used extensively in
medicinal chemistry to predict absorption and optimize a compound's membrane
permeability, was calculated for the potent compounds. It is suggested that
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compounds with a TPSA greater than 140ꢀÅ tend to be poor at permeating cell
membranes [27]. Based on the predicted data mentioned above, the calculated TPSA
values for the highly bioactive quaternization harman derivatives would indicate a
possible good bioavailability.
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2.5.
Thermal stability of compound 4x in vitro [28,29]
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Considering the poor thermal stability of antibiotics, it was decided to further
investigate the bioactive compound 4x using an in vitro assay, to consider whether its
thermal stability had improved compared with the control. As depicted in Fig. S2,
ampicillin sodium began to degrade after being kept at 37 °C for 6 h. After being kept
at 65 °C for 6 h, very little ampicillin sodium residue could be found. Compound 4x
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did not degrade any further under the same conditions.
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2.6.
Scanning electron microscopy analysis
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SEM of MRSA and R. solanacearum revealed morphological changes in the
bacterial cell surface (Fig. 6). The surfaces of cells in the untreated group (Fig. 6A and
6C) were relatively smooth and regular, whereas when treated with compound 4x (Fig.
6B and 6D) there was shrinkage. Increased permeabilization of the membrane may
explain the leakage of cytoplasmic material. Additionally, bacteria exposed to the
compound 4x experienced cell wall disintegration. These results indicated that
compound 4x may exert its bactericidal effect by damaging bacterial cell membrane
and wall.
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2.7.
Molecular docking study
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The Surflex-Dock scoring function is a weighted sum of non-linear functions
based on the binding affinities of protein-ligand complexes coupled with their
crystallographically determined structures. Surflex-Dock scores are expressed in
-log (K ) units to represent binding affinities [30,31]. Type II topoisomerase present
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in bacteria is attractive target for antibacterial drug discovery, which is independently
essential for bacterial DNA replication [32]. Mechanism of topoisomerases inhibition
is known to occur in two ways: these inhibitors may bind with topoisomerase directly
or they may bind to DNA and alter its structure, so that it cannot be recognized by
topoisomerases [33]. A previous study demonstrated that large planar surface areas
could function at the DNA level via intercalation between base pairs. Further, it was
shown that DNA intercalation of molecules could disrupt the function of
topoisomerases, ultimately leading to cell death [18]. For quaternization harman
analogues, β-carboline core per se is a planar molecule. In some extent, their
structures are also similar to the commercial antibiotic ciprofloxacin which targets to
the type II topoisomerase. So we selected a crystal of bacterial type II topoisomerase
complex (PDB ID: 5IWM) as a possible target.
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A docking investigation based on the Surflex-Dock program in the Sybyl-X 2.0
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package was undertaken to explore the possible mechanism. Firstly, we performed a
re-docking of the extracted co-crystallized ligand present in the type II topoisomerase
complex. The RMSD (root-mean-square deviation) value was 0.923Å (< 2.0 Å),
which indicated the docking protocol was feasible [34, 35]. The highly active
compound 4x gave a good total score (6.4952) which was close to the score of the
standard compound ciprofloxacin (6.9723). Interestingly, the MIC value of
ciprofloxacin against S. aureus (4 µg/mL) further supported the rationality of the
docking protocol. As shown in Fig. 7, compound 4x mainly bound to DNA area.
There are many π–π stacked interactions between the plane structure of the small
molecule and the DNA base (DA10, DA11, DT10, and DT11). The hydrogen atom of
the methoxy group is adjacent to the DNA base (DC12), forming two carbon–
hydrogen bonds (2.65 and 2.69 Å). The trifluoromethyl fragment of compound 4x is
adjacent to the ASP83 residue of the target, forming a strong halogen bond with a
length of 2.73 Å. Simultaneously, two carbon–hydrogen bonds (2.32 and 2.50 Å) are
formed between the trifluoromethyl group and the SER84 residue. Interestingly, the
interaction of the trifluoromethyl group confirms the activity and structure–activity
relationship finding that trifluoromethyl fragments are beneficial for improving
antibacterial activity.
As we all known, there are two types of topoisomerase, namely, type I
topoisomerase and type II topoisomerase [36]. In order to check the selectivity of the
titled compounds against type II topoisomerase over type I topoisomerase, a docking
investigation between compound 4x and type I topoisomerase complex (PDB ID:
4RUL) was also undertaken. The total score was 4.0326 which indicated that
compound 4x showed approximately 290-fold selectivity against type II
topoisomerase over type I topoisomerase (Total score = -log (K ), K ×K = 1, K
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d
d
represent dissociation constant, K represent binding constant, selectivity = K (type II
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a
topoisomerase) / K (type I topoisomerase)).
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Considering with the SEM results, we deduced that compound 4x might attack
and cause damage to the bacterial cell membrane and wall. The consequent increased
permeability of the bacterial cell membrane will then allow compound 4x to enter the
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cells. The 4x in the cytoplasm will then disrupt the function of type II topoisomerase,
which in turn caused cell death.
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2.8.
Protective effect in vivo[37,38]
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The in vivo antibacterial activity (protective effect) of the highly bioactive
compound 4x was tested against R. solanacearum on eggplant leaf (Fig. 8).
Compound 4x was found to have a good preventative effect against R. solanacearum,
with a protective efficacy of 81.3%. This demonstrates the potential of the
quaternization harman derivatives as new bactericides.
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3. Conclusions
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In summary, 41 compounds, including 33 new quaternization harman derivatives,
were prepared via a convenient synthetic route and their antibacterial activity was
evaluated. The structure–activity relationships were summarized which provided
some important guidance for the development of antibacterial harman agents. The
highly bioactive compounds 4f, 4i, 4l, 4u, 4w, 4x and 5c, with a peak MIC of 4
µg/mL, showed low cytotoxicity, good thermal stability and “drug-like” properties.
Further, the SEM analysis and molecular docking evaluations suggested multiple
possible antibacterial targets of quaternization harman analogues, namely membrane,
wall and type II topoisomerase. As expected, the in vivo antibacterial assay
demonstrated the potential of the quaternization harman agents as new bactericides
and antibiotics. Overall, this work enriched the types of candidate antibiotics and
provided more options for solving the current antibiotic crisis.
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Chirality of drugs is an eternal topic in medicinal chemistry. For some highly
active quaternization harman molecules, chiral axis is existed at position 1. In order to
separate the isomers, we have used twelve different separation conditions
(Supplementary data). Regrettably, we did not get the isomers. Here, we are to appeal
to researchers to further study this issue to promote the development and application
of quaternization harman antibiotics.
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4. Experimental
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4.1.
Materials
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H NMR and C NMR spectroscopy was carried out using an Avance
spectrometer (Bruker, Billerica, MA, USA) at 500 and 125 MHz. Chemical shifts
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were measured relative to the residual solvent peaks of CD OD ( H, δ = 3.31 ppm;
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C, δ = 49.00 ppm) or dimethyl sulfoxide (DMSO-d ) ( H, δ = 2.50 ppm; C, δ =
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39.52 ppm) with tetramethylsilane as the internal standard. High-resolution mass
+
spectroscopy (HRMS) was undertaken using an AB SCIEX Triple TOF 5600
spectrometer. Liquid chromatography–electrospray ionization–tandem mass
spectrometry
(LC-ESI-MS/MS)
analysis
was
accomplished
using
ultra-high-performance liquid chromatography (UHPLC) (Nexera UHPLC LC-30A)
+
coupled to an AB SCIEX Triple TOF 5600 spectrometer, with the system equipped
with a C18 trap column (Shim-pack XR-ODS, 2.0 mm i.d. × 100 mm). Elemental
analyses were performed on a Heraeus CHN-O-Rapid instrument. Compounds 3e and
5f were collected at 100 K on a Rigaku Oxford Diffraction Supernova Dual Source,
Cu at Zero equipped with an AtlasS2 CCD using Cu Kα radiation. Data reduction was
carried out with the diffractometer's software Scanning electron microscopy (SEM)
analysis was carried out on Nova nano SEM450 instrument. Molecular docking
evaluation was conducted with the Sybyl-X 2.0 software and Discovery Studio 2017
client. Fourier transform infrared (FT-IR) spectra were obtained on a TENSOR37
–
1
spectrometer (Bruker) using KBr pellets, and the absorptions are reported in cm .
The reaction progress was monitored by thin-layer chromatography (TLC) on silica
gel GF254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) with ultraviolet
detection. Reagents were purchased from commercial sources (Bodi Chemical Co.,
Ltd., Tianjin, China; Aladdin Industrial Co., Ltd., Shanghai, China), and used as
received.
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4.2.
Synthesis
4.2.1. Synthesis of intermediates 3a–3h
A solution of tryptamine (1.6 g, 10 mmol) or 5-methoxytryptamine (1.9 g, 10
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mmol), aldehyde (methyl 4-formylbenzoate, furfural, pyridine-2-carboxaldehyde, or
piperonyl aldehyde) (11 mmol), and trifluoroacetic acid (100 µL) in dichloromethane
(50 mL) was stirred at room temperature for 36 h. Dichloromethane (30 mL) and
ammonia water (5%, 30 mL) were added, and the organic layer was separated. Next,
the water phase was extracted with dichloromethane, and the organic phases were
combined and dried over sodium sulfate and filtered. The solvent was then evaporated,
and the crude product purified by column chromatography (ethyl acetate as the eluent)
to give the products 2a–2h in 92–96% yield. Pd/C (10%, 0.1 eq) was then added to
the solution of 2a–2h in xylene (50 mL), and the solution was heated at 145 °C and
monitored by TLC. On completion, the resulting mixture was filtered and evaporated.
The residue was purified by silica gel column chromatography with petroleum ether
and ethyl acetate (v/v = 2:1) to produce 3a–3h in 62–73% yield.
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4.2.1.1. methyl 4-(9H-pyrido[3,4-b]indol-1-yl)benzoate (3a): white solid; yield, 65%;
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m.p. 147.4–148.3 C; H NMR (500 MHz, CDCl ) δ 8.81 (s, 1H), 8.58 (d, J = 2.5 Hz,
3
1H), 8.19–8.17 (m, 3H), 8.02 (d, J = 5 Hz, 2H), 7.97 (d, J = 5 Hz, 1H), 7.59–7.53 (m,
1
3
2H), 7.33 (t, J = 7.5 Hz, 1H), 3.94 (s, 3H); C NMR (125 MHz, CDCl ) δ 166.8,
3
142.9, 142.8, 141.5, 140.5, 139.6, 133.6, 130.4, 130.2, 128.8, 128.1, 121.9, 121.8,
–
1
120.5, 114.4, 111.6, 52.2. IR (KBr, cm ) 3362, 3065, 2957, 1718, 1637, 1619, 1582,
1526, 1497, 1469, 1438, 1326, 1294, 1279, 1223, 1164, 1128, 1067, 1026, 1015, 825,
792, 769, 726. Elemental anal. calcd for C H N O : C, 75.48; H, 4.67; N, 9.27;
1
9
14
2
2
+
found C, 75.44; H, 4.69; N, 9.31. HRMS (ESI) m/z calcd for C H N O [M+H]
19
14
2
2
303.1128, found 303.1127.
336
337
338
339
340
341
342
4.2.1.2. methyl 4-(6-methoxy-9H-pyrido[3,4-b]indol-1-yl)benzoate (3b): yellow solid;
o
1
yield, 62%; m.p. 124.1–125.3 C; H NMR (500 MHz, CDCl ) δ 8.84 (s, 1H), 8.57 (d,
3
J = 5 Hz, 1H), 8.19 (d, J = 10 Hz, 2H), 8.05 (d, J = 10 Hz, 2H), 7.96 (d, J = 2.5 Hz,
1H), 7.63 (s, 1H), 7.48 (d, J = 10 Hz, 1H), 7.26 (d, J = 10 Hz, 1H), 3.99 (s, 3H), 3.97
1
3
(s, 3H); C NMR (125 MHz, CDCl ) δ 166.8, 154.5, 142.9, 141.7, 139.1, 135.5,
3
134.3, 130.3, 130.2, 130.1, 128.1, 122.1, 118.8, 114.3, 112.5, 103.6, 56.0, 52.2. IR
–
1
(KBr, cm ) 3064, 2998, 2951, 2832, 1722, 1609, 1584, 1495, 1468, 1436, 1400, 1286,
1
3

ACCEPTED MANUSCRIPT
343
344
345
1218, 1177, 1132, 1116, 1037, 1014, 818, 768. Elemental anal. calcd for C H N O :
2
0
16
2
3
C, 72.28; H, 4.85; N, 8.43; found C, 72.26; H, 4.83; N, 8.47. HRMS (ESI) m/z calcd
+
for C H N O [M+H] 333.1234, found 333.1230.
2
0
16
2
3
346
347
348
349
350
351
352
353
354
4.2.1.3. 1-(furan-2-yl)-9H-pyrido[3,4-b]indole (3c): brown solid; yield, 66%; m.p.
o
1
127.1–128.6 C; H NMR (500 MHz, CDCl ) δ 9.54 (s, 1H), 8.50 (d, J = 2.5 Hz, 1H),
3
8.15 (d, J = 5 Hz, 1H), 7.89 (d, J = 2.5 Hz, 1H), 7.71 (s, 1H), 7.60 (s, 2H), 7.35 (s,
1
3
2H), 6.67 (s, 1H); C NMR (125 MHz, CDCl ) δ 154.4, 142.8, 140.5, 138.8, 133.4,
3
–
1
131.3, 130.3, 128.6, 121.7, 121.2, 120.1, 113.7, 112.3, 111.6, 108.8. IR (KBr, cm )
3458, 1625, 1556, 1493, 1452, 1425, 1378, 1366, 1318, 1301, 1284, 1252, 1235, 1210,
1164, 998, 749, 458. Elemental anal. calcd for C H N O: C, 76.91; H, 4.30; N,
15
10
2
11.96; found C, 76.87; H, 4.35; N, 11.94. HRMS (ESI) m/z calcd for C H N O
1
5
10
2
+
[M+H] 235.0866, found 235.0863.
355
356
357
358
359
360
361
362
363
364
4.2.1.4. 1-(furan-2-yl)-6-methoxy-9H-pyrido[3,4-b]indole (3d): brown solid; yield,
o
1
68%; m.p. 66.4–67.8 C; H NMR (500 MHz, CDCl ) δ 9.30 (s, 1H), 8.41 (d, J = 5
3
Hz, 1H), 7.80 (d, J = 5 Hz, 1H), 7.67 (d, J = 2.5 Hz, 1H), 7.53 (d, J = 5 Hz, 1H), 7.46
(d, J = 10 Hz, 1H), 7.29 (d, J = 2.5 Hz, 1H), 7.22–7.20 (m, 1H), 6.64–6.63 (m, 1H),
13
3.93 (s, 3H); C NMR (125 MHz, CDCl ) δ 154.5, 154.2, 142.7, 138.3, 135.3, 133.6,
3
–
1
131.9, 130.0, 121.6, 118.6, 113.5, 112.4, 112.3, 108.7, 103.4, 56.0. IR (KBr, cm )
2923, 1606, 1585, 1556, 1489, 1462, 1437, 1379, 1287, 1253, 1215, 1163, 1125, 1020,
810, 772, 738, 622. Elemental anal. calcd for C H N O : C, 72.72; H, 4.58; N, 10.60;
1
6
12
2
2
+
found C, 72.68; H, 4.60; N, 10.58. HRMS (ESI) m/z calcd for C H N O [M+H]
1
6
12
2
2
265.0972, found 265.0970.
365
366
367
368
369
370
371
4.2.1.5. 1-(pyridin-2-yl)-9H-pyrido[3,4-b]indole (3e): white solid; yield, 70%; m.p.
o
1
139.4–141.2 C; H NMR (500 MHz, CDCl ) δ 11.27 (s, 1H), 8.71–8.69 (m, 2H),
3
8.50 (d, J = 5 Hz, 1H), 8.09 (d, J = 5 Hz, 1H), 7.93 (d, J = 5 Hz, 1H), 7.83–7.80 (m,
1
3
1H), 7.56–7.50 (m, 2H), 7.24–7.22 (m, 2H); C NMR (125 MHz, CDCl ) δ 158.0,
3
148.2, 140.6, 138.1, 138.1, 136.8, 134.8, 130.5, 128.4, 122.9, 121.7, 121.3, 121.1,
–
1
119.8, 115.4, 111.9. IR (KBr, cm ) 3395, 3291, 3057, 1625, 1588, 1564, 1484, 1446,
1414, 1362, 1316, 1280, 1234, 1146, 1091, 1065, 798, 743, 622, 597. Elemental anal.
1
4

ACCEPTED MANUSCRIPT
3
72
73
calcd for C H N : C, 78.35; H, 4.52; N, 17.13; found C, 78.33; H, 4.55; N, 17.10.
1
6
11
3
+
3
HRMS (ESI) m/z calcd for C H N [M+H] 246.1026, found 246.1025.
1
6
11
3
374
375
376
377
378
379
380
381
382
383
4.2.1.6. 6-methoxy-1-(pyridin-2-yl)-9H-pyrido[3,4-b]indole (3f): white solid; yield,
o
1
73%; m.p. 131.4–132.2 C; H NMR (500 MHz, CDCl ) δ 11.09 (s, 1H), 8.67 (d, J =
3
10 Hz, 1H), 8.64 (d, J = 5 Hz, 1H), 8.44 (d, J = 5 Hz, 1H), 7.84 (d, J = 5 Hz, 1H),
7.80–7.76 (m, 1H), 7.48 (d, J = 2.5 Hz, 1H), 7.40 (d, J = 10 Hz, 1H), 7.23–7.19 (m,
13
1H), 7.16–7.14 (m, 1H), 3.86 (s, 3H); C NMR (125 MHz, CDCl ) δ 157.9, 154.0,
3
148.1, 138.1, 137.5, 136.7, 135.6, 135.3, 130.2, 122.8, 121.4, 121.2, 118.3, 115.3,
–
1
112.6, 103.5, 55.9. IR (KBr, cm ) 3347, 1583, 1564, 1490, 1455, 1439, 1414, 1362,
1292, 1280, 1221, 1176, 1139, 1118, 1033, 1020, 837, 823, 798, 742, 630. Elemental
anal. calcd for C H N O: C, 74.17; H, 4.76; N, 15.26; found C, 74.15; H, 4.79; N,
1
7
13
3
+
15.30. HRMS (ESI) m/z calcd for C H N O [M+H] 276.1131, found 276.1128.
1
7
13
3
384
385
386
387
388
389
390
391
392
393
4.2.1.7. 1-(benzo[d][1,3]dioxol-5-yl)-9H-pyrido[3,4-b]indole (3g): white solid; yield,
o
1
66%; m.p. 167.5–168.9 C; H NMR (500 MHz, CDCl ) δ 8.63 (s, 1H), 8.57 (d, J = 5
3
Hz, 1H), 8.20 (d, J = 10 Hz, 1H), 7.95 (d, J = 5 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H), 7.55
(d, J = 10 Hz, 1H), 7.51–7.49 (m, 2H), 7.36 (t, J = 7.5 Hz, 1H), 7.05–7.03 (m, 1H),
13
6.09 (s, 2H); C NMR (125 MHz, CDCl ) δ 148.5, 148.2, 142.6, 140.3, 139.5, 133.31,
3
132.8, 129.8, 128.5, 122.0, 121.8, 120.3, 113.5, 111.5, 108.8, 108.7, 103.1, 101.4. IR
–
1
(KBr, cm ) 3054, 2884, 1626, 1565, 1502, 1471, 1452, 1415, 1320, 1283, 1243, 1223,
1126, 1039, 935, 919, 825, 812, 747. Elemental anal. calcd for C H N O : C, 74.99;
1
8
12
2
2
H, 4.20; N, 9.72; found C, 74.95; H, 4.18; N, 9.76. HRMS (ESI) m/z calcd for
+
C H N O [M+H] 289.0972, found 289.0970.
18
12
2
2
394
395
396
397
398
399
400
4.2.1.8. 1-(benzo[d][1,3]dioxol-5-yl)-6-methoxy-9H-pyrido[3,4-b]indole (3h): yellow
o
1
solid; yield, 72%; m.p. 96.5–97.2 C; H NMR (500 MHz, CDCl ) δ 8.91 (s, 1H), 8.44
3
(d, J = 5 Hz, 1H), 7.82 (d, J = 5 Hz, 1H), 7.54 (d, J = 5 Hz, 1H), 7.40–7.39 (m, 2H),
7.35 (d, J = 10 Hz, 1H), 7.17–7.16 (m, 1H), 6.87–6.86 (m, 1H), 5.94 (s, 2H), 3.92 (s,
1
3
3H); C NMR (125 MHz, CDCl ) δ 154.3, 148.4, 148.1, 142.9, 138.7, 135.5, 134.1,
3
132.7, 129.7, 122.3, 121.9, 118.5, 113.4, 112.5, 108.7,108.6, 103.5, 101.3, 56.0. IR
–
1
(KBr, cm ) 1565, 1501, 1470, 1447, 1285, 1248, 1209, 1158, 1113, 1038, 814.
1
5

ACCEPTED MANUSCRIPT
401
402
403
Elemental anal. calcd for C H N O : C, 71.69; H, 4.43; N, 8.80; found C, 71.67; H,
19
14
2
3
+
4.41; N, 8.85. HRMS (ESI) m/z calcd for C H N O [M+H] 319.1077, found
19
14
2
3
319.1074.
404
4.2.2. Synthesis of 4a–4x and 5a–5i
405
406
407
408
409
A solution of 3a–3h (1 mmol) and substituted benzyl bromide (5 mmol) in
acetonitrile (15 mL) was stirred at room temperature. The reaction was monitored by
TLC. On completion, the resulting mixture was directly evaporated, and the residue
was purified by silica gel column chromatography with dichloromethane and
methanol (v/v = 20:1) to produce 4a–4h and 5a–5i in 67–96% yield.
410
411
412
413
414
415
416
417
418
419
420
4.2.2.1. 2-benzyl-1-(4-(methoxycarbonyl)phenyl)-9H-pyrido[3,4-b]indol-2-ium
o
1
bromide (4a): yellow solid; yield, 87%; m.p. 158.8–159.6 C; H NMR (500 MHz,
CD OD) δ 8.82 (dd, J = 15 HZ, 5 Hz, 2H), 8.49 (d, J = 10 Hz, 1H), 8.27 (d, J = 10 Hz,
3
2H), 7.82–7.78 (m, 1H), 7.69–7.65 (m, 3H), 7.52–7.49 (m, 1H), 7.32–7.27 (m, 3H),
1
3
6.95–6.93 (m, 2H), 5.82 (s, 2H), 4.00 (s, 3H); C NMR (125 MHz, CD OD) δ165.9,
3
145.2, 139.2, 135.8, 135.6, 134.4, 134.1, 132.8, 132.5, 132.4, 130.2, 130.0, 128.8,
–
1
128.6, 127.0, 123.1, 122.1, 119.8, 117.0, 112.6, 60.9, 51.7. IR (KBr, cm ) 3397, 3058,
2948, 1712, 1629, 1580, 1528, 1496, 1454, 1434, 1403, 1341, 1329, 1281, 1107, 1014,
767, 757, 705. Elemental anal. calcd for C H BrN O : C, 65.97; H, 4.47; N, 5.92;
2
6
21
2
2
+
found C, 65.94; H, 4.51; N, 5.95. HRMS (ESI) m/z calcd for C H BrN O [M–Br]
2
6
21
2
2
393.1598, found 393.1592.
421
422
423
424
425
426
427
428
4.2.2.2. 1-(4-(methoxycarbonyl)phenyl)-2-(4-methylbenzyl)-9H-pyrido[3,4-b]indol-2-
o
1
ium bromide (4b): yellow solid; yield, 96%; m.p. 156.7–157.9 C; H NMR (500 MHz,
CD OD) δ 8.82 (dd, J = 15 H , 5 Hz, 2H), 8.47 (d, J = 10 Hz, 1H), 8.26 (d, J = 10 Hz,
3
Z
2H), 7.81–7.77 (m, 1H), 7.67–7.63 (m, 3H), 7.51–7.48 (m, 1H), 7.31–7.27 (m, 2H),
13
6.95–6.93 (m, 2H), 5.82 (s, 2H), 4.00 (s, 3H), 2.27 (s, 3H); C NMR (125 MHz,
CD OD) δ 165.5, 143.3, 139.2, 135.7, 135.6, 134.4, 134.1, 132.6, 132.3, 132.1, 130.0,
3
129.7, 127.6, 125.9, 123.0, 121.7, 119.6, 118.8, 116.9, 112.6, 60.9, 50.9, 22.3. IR
–
1
(KBr, cm ) 3401, 3016, 2950, 1720, 1605, 1587, 1531, 1505, 1464, 1442, 1401, 1327,
1
6

ACCEPTED MANUSCRIPT
429
430
431
1275, 1108, 1010, 769, 756. Elemental anal. calcd for C H BrN O : C, 66.54; H,
27
23
2
2
4.76; N, 5.75; found C, 66.52; H, 4.77; N, 5.78. HRMS (ESI) m/z calcd for
+
C H BrN O [M–Br] 407.1754, found 407.1754.
27
23
2
2
432
433
434
435
436
437
438
439
440
441
442
443
444
4.2.2.3. 1-(4-(methoxycarbonyl)phenyl)-2-(4-(trifluoromethyl)benzyl)-9H-pyrido[3,4-
o
1
b]indol-2-ium bromide (4c): yellow solid; yield, 92%; m.p. 159.2–160.3 C; H NMR
(500 MHz, CD OD) δ 8.93 (d, J = 5 Hz, 1H), 8.87 (d, J = 10 Hz, 1H), 8.51 (d, J = 5
3
Hz, 1H), 8.28 (d, J = 10 Hz, 2H), 7.82 (t, J = 5 Hz, 1H), 7.76–7.74 (m, 2H), 7.69 (d, J
= 10 Hz, 1H), 7.62 (d, J = 10 Hz, 2H), 7.52 (t, J = 7.5 Hz, 1H), 7.18 (d, J = 10 Hz,
1
3
2H), 5.99 (s, 2H), 4.02 (s, 3H); C NMR (125 MHz, CD OD) δ 165.9, 145.3, 139.2,
3
138.8, 135.7, 134.4, 134.4, 133.0, 132.5, 132.4, 130.5, 130.3, 130.0, 127.7, 125.7,
125.6, 125.6, 125.6, 124.9, 123.2, 122.8, 122.1, 119.8, 117.3, 112.7, 60.3, 51.8. IR
–
1
(KBr, cm ) 3347, 2360, 1581, 1565, 1490, 1455, 1439, 1414, 1362, 1291, 1280, 1221,
1176, 1139, 1119, 1090, 1034, 1020, 837, 824, 799, 768, 742, 701, 630, 543.
Elemental anal. calcd for C H BrF N O : C, 59.90; H, 3.72; N, 5.17; found C, 59.87;
2
7
20
3
2
2
+
H, 3.76; N, 5.12. HRMS (ESI) m/z calcd for C H BrF N O [M–Br] 461.1471,
27
20
3
2
2
found 461.1469.
445
446
447
448
449
450
451
452
453
454
455
4.2.2.4. 2-benzyl-6-methoxy-1-(4-(methoxycarbonyl)phenyl)-9H-pyrido[3,4-b]indol-2
o
1
-ium bromide (4d): yellow solid; yield, 89%; m.p. 150.5–152.0 C; H NMR (500
MHz, DMSO-d6) δ 12.15 (s, 1H), 8.94 (t, J = 5 Hz, 2H), 8.18–8.16 (m, 2H), 8.11 (d, J
= 5 Hz, 1H), 7.78 (d, J = 5 Hz, 2H), 7.59 (d, J = 10 Hz, 1H), 7.45–7.43 (m, 1H), 7.31–
1
3
7.25 (m, 3H), 6.93 (d, J = 5 Hz, 2H), 5.80 (s, 2H), 3.95 (s, 3H), 3.92 (s, 3H); C
NMR (125 MHz, DMSO-d6) δ 166.1, 155.3, 140.6, 139.6, 136.0, 135.3, 134.1, 133.1,
133.0, 132.4, 131.0, 130.3, 129.2, 128.9, 127.6, 124.0, 120.5, 118.1, 114.6, 104.2,
–
1
60.5, 56.3, 53.1. IR (KBr, cm ) 3401, 1720, 1612, 1578, 1499, 1437, 1278, 1222,
1113, 1024, 821, 767, 738, 704. Elemental anal. calcd for C H BrN O : C, 64.42; H,
2
7
23
2
3
4.61; N, 5.57; found C, 64.40; H, 4.64; N, 5.56. HRMS (ESI) m/z calcd for
+
C H BrN O [M–Br] 423.1703, found 423.1698.
27
23
2
3
4
56
57
4.2.2.5. 6-methoxy-1-(4-(methoxycarbonyl)phenyl)-2-(4-methylbenzyl)-9H-pyrido[3,4
o
1
4
-b]indol-2-ium bromide (4e): yellow solid; yield, 96%; m.p. 147.2–148.1 C; H NMR
1
7

ACCEPTED MANUSCRIPT
458
459
460
461
462
463
464
465
466
(500 MHz, CD OD) δ 8.74 (dd, J = 10 Hz, 5 Hz, 2H), 8.27 (d, J = 10 Hz, 2H), 7.93 (d,
3
J = 2.5 Hz, 1H), 7.68 (d, J = 10 Hz, 2H), 7.56 (d, J = 10 Hz, 1H), 7.45–7.42 (m, 1H),
7.11 (d, J = 5 Hz, 2H), 6.83 (d, J = 5 Hz, 2H), 5.74 (s, 2H), 4.01 (s, 3H), 3.97 (s, 3H),
1
3
2.30 (s, 3H); C NMR (125 MHz, CD OD) δ 166.0, 155.8, 140.6, 139.3, 138.8, 135.7,
3
133.4, 133.1, 132.8, 132.6, 131.4, 130.2, 129.9, 129.4, 127.0, 124.1, 120.2, 117.0,
–1
113.6, 102.6, 60.6, 55.0, 51.7, 19.7. IR (KBr, cm ) 3407, 3016, 2951, 1721, 1609,
1581, 1498, 1442, 1281, 1223, 1115, 1026, 826, 779, 707, 653. Elemental anal. calcd
for C H BrN O : C, 65.00; H, 4.87; N, 5.41; found C, 64.97; H, 4.86; N, 5.44.
2
8
25
2
3
+
HRMS (ESI) m/z calcd for C H BrN O [M–Br] 437.1860, found 437.1854.
2
8
25
2
3
467
468
469
470
471
472
473
474
475
476
477
478
4.2.2.6. 6-methoxy-1-(4-(methoxycarbonyl)phenyl)-2-(4-(trifluoromethyl)benzyl)-9H-
o
pyrido[3,4-b]indol-2-ium bromide (4f): yellow solid; yield, 81%; m.p. 168.7–169.6 C;
1
H NMR (500 MHz, CD OD) δ 8.83 (s, 2H), 8.27 (d, J = 10 Hz, 2H), 7.94 (d, J = 2.5
3
Hz, 1H), 7.72 (d, J = 10 Hz, 2H), 7.60 (d, J = 10 Hz, 2H), 7.56 (d, J = 10 Hz, 1H),
1
3
7.42–7.39 (m, 1H), 7.16 (d, J = 5 Hz, 2H), 5.96 (s, 2H), 4.00 (s, 3H), 3.96 (s, 3H); C
NMR (125 MHz, CD OD) δ 167.3, 157.1, 142.0, 140.6, 140.3, 137.1, 135.0, 134.9,
3
134.3, 133.8, 131.9, 131.6, 131.4, 129.0, 127.0, 127.0, 126.9, 126.3, 125.5, 124.1,
–
1
121.6, 118.7, 115.0, 104.1, 61.5, 56.5, 53.1. IR (KBr, cm ) 3362, 3065, 2957, 1718,
1659, 1637, 1619, 1582, 1526, 1497, 1469, 1326, 1294, 1279, 1223, 1164, 1128, 1067,
1015, 870, 825, 792, 769, 726, 660, 645, 596. Elemental anal. calcd for
C H BrF N O : C, 58.86; H, 3.88; N, 4.90; found C, 58.90; H, 3.87; N, 4.85. HRMS
28
22
3
2
3
+
(ESI) m/z calcd for C H BrF N O [M–Br] 491.1577, found 491.1573.
2
8
22
3
2
3
479
480
481
482
483
484
485
486
4.2.2.7. 2-benzyl-1-(furan-2-yl)-9H-pyrido[3,4-b]indol-2-ium
bromide
(4g):
o
1
red-brown solid; yield, 83%; m.p. 141.1–142.7 C; H NMR (500 MHz, CD OD) δ
3
8.75–8.70 (m, 2H), 8.43 (d, J = 10 Hz, 1H), 8.12 (d, J = 2.5 Hz, 1H), 7.83–7.77 (m,
2H), 7.50–7.47 (m, 1H), 7.36–7.34 (m, 3H), 7.27 (d, J = 2.5 Hz, 1H), 7.13–7.11 (m,
1
3
2H), 6.88 (dd, J = 5 Hz, 2.5 Hz, 1H), 6.09 (s, 2H); C NMR (125 MHz, CD OD) δ
3
148.7, 146.5, 141.8, 136.4, 136.1, 135.8, 135.7, 133.8, 130.8, 130.3, 130.0, 128.2,
–
1
124.3, 123.5, 121.1, 120.0, 118.2, 114.2, 113.9, 62.8. IR (KBr, cm ) 3399, 3055,
2924, 1627, 1580, 1500, 1453, 1335, 1270, 1249, 1201, 1153, 1018, 762, 742, 701.
1
8

ACCEPTED MANUSCRIPT
487
488
489
Elemental anal. calcd for C H BrN O: C, 65.20; H, 4.23; N, 6.91; found C, 65.17; H,
22
17
2
+
4.25; N, 6.93. HRMS (ESI) m/z calcd for C H BrN O [M–Br] 325.1335, found
22
17
2
325.1332.
490
491
492
493
494
495
496
497
498
499
4.2.2.8. 1-(furan-2-yl)-2-(4-methylbenzyl)-9H-pyrido[3,4-b]indol-2-ium bromide (4h):
o
1
red-brown solid; yield, 85%; m.p. 156.6–157.8 C; H NMR (500 MHz, CD OD) δ
3
8.79–8.75 (m, 2H), 8.45 (d, J = 10 Hz, 1H), 8.08 (d, J = 2.5 Hz, 1H), 7.84–7.78 (m,
2H), 7.64 (d, J = 5 Hz, 2H), 7.50 (t, J = 5 Hz, 1H), 7.30–7.26 (m, 3H), 6.88–6.86 (m,
1
3
1H), 6.19 (s, 2H); C NMR (125 MHz, CD OD) δ 149.1, 146.9, 141.9, 140.6, 136.7,
3
136.3, 134.3, 132.0, 131.1, 129.1, 127.4, 127.4, 124.8, 124.0, 121.5, 120.5, 118.7,
–
1
114.5, 114.3, 62.6, 31.0. IR (KBr, cm ) 3409, 3146, 3049, 2925, 2855, 1629, 1583,
1505, 1452, 1422, 1327, 1279, 1164, 1127, 1067, 1017, 753. Elemental anal. calcd for
C H BrN O: C, 65.88; H, 4.57; N, 6.68; found C, 65.85; H, 4.59; N, 6.67. HRMS
23
19
2
+
(ESI) m/z calcd for C H BrN O [M–Br] 339.1492, found 339.1490.
2
3
19
2
500
501
502
503
504
505
506
507
508
509
510
4.2.2.9. 1-(furan-2-yl)-2-(4-(trifluoromethyl)benzyl)-9H-pyrido[3,4-b]indol-2-ium
o
1
bromide (4i): red-brown solid; yield, 78%; m.p. 185.6–187.2 C; H NMR (500 MHz,
CD OD) δ 8.80–8.75 (m, 2H), 8.45 (d, J = 2.5 Hz, 1H), 8.08 (s, 1H), 7.84–7.79 (m,
3
2H), 7.65 (d, J = 10 Hz, 2H), 7.50 (t, J = 5 Hz, 1H), 7.31–7.27 (m, 3H), 6.87 (dd, J =
1
3
12.5 Hz, 2.5 Hz, 1H), 6.2 (s, 2H); C NMR (125 MHz, CD OD) δ 148.8, 146.6,
3
141.6, 140.2, 136.5, 136.4, 136.0, 134.0, 131.9, 131.7, 130.8, 128.7, 127.1, 127.1,
127.1, 127.0, 126.3, 124.4, 124.2, 123.6, 122.0, 121.2, 120.2, 118.4, 114.2, 114.0,
–
1
62.2. IR (KBr, cm ) 3410, 3048, 1629, 1511, 1502, 1327, 1280, 1164, 1127, 1067,
1018, 753. Elemental anal. calcd for C H BrF N O: C, 58.37; H, 3.41; N, 5.92;
2
3
16
3
2
+
found C, 58.35; H, 3.43; N, 5.89. HRMS (ESI) m/z calcd for C H BrF N O [M–Br]
2
3
16
3
2
393.1209, found 393.1202.
511
512
513
514
515
4.2.2.10. 2-benzyl-1-(furan-2-yl)-6-methoxy-9H-pyrido[3,4-b]indol-2-ium
o
1
bromide (4j): red-brown solid; yield, 92%; m.p. 194.8–196.1 C; H NMR (500 MHz,
DMSO-d6) δ 12.43 (s, 1H), 8.88 (dd, J = 10 Hz, 5 Hz, 2H), 8.24 (d, J = 2.5 Hz, 1H),
8.07 (d, J = 5 Hz, 1H), 7.73 (d, J = 10 Hz, 1H), 7.49–7.47 (m, 1H), 7.35–7.32 (m, 4H),
13
7.08–7.06 (m, 2H), 6.95 (dd, J = 5 Hz, 2.5 Hz, 1H), 6.07 (s, 2H), 3.92 (s, 3H); C
1
9

ACCEPTED MANUSCRIPT
516
517
518
519
520
521
NMR (125 MHz, DMSO-d6) δ155.1, 148.7, 146.5, 141.8, 136.4, 136.1, 135.8, 133.8,
130.8, 130.3, 130.0, 128.2, 124.3, 123.5, 121.1, 120.0, 118.2, 114.2, 113.9, 62.8, 56.4.
–
1
IR (KBr, cm ) 3409, 3048, 2917, 1626, 1588, 1516, 1462, 1327, 1266, 1230, 1199,
1142, 1008, 767, 722. Elemental anal. calcd for C H BrN O : C, 63.46; H, 4.40; N,
2
3
19
2
2
6.44; found C, 63.45; H, 4.43; N, 6.40. HRMS (ESI) m/z calcd for C H BrN O
2
2
3
19
2
+
[M–Br] 355.1441, found 355.1437.
522
523
524
525
526
527
528
529
530
531
532
533
4.2.2.11. 1-(furan-2-yl)-6-methoxy-2-(4-methylbenzyl)-9H-pyrido[3,4-b]indol-2-iu
o
1
m bromide (4k): red-brown solid; yield, 81%; m.p. 191.6–193.2 C; H NMR (500
MHz, CD OD) δ 8.66 (dd, J = 10 Hz, 5 Hz, 2H), 8.10 (d, J = 5 Hz, 1H), 7.89 (d, J = 5
3
Hz, 1H), 7.69 (d, J = 10 Hz, 1H), 7.47–7.45 (m, 1H), 7.26 (d, J = 5 Hz, 1H), 7.16 (d, J
= 5 Hz, 2H), 6.99 (d, J = 10 Hz, 2H), 6.88 (dd, J = 5 Hz, 2.5 Hz, 1H), 6.00 (s, 2H),
1
3
3.96 (s, 3H), 2.30 (s, 3H); C NMR (125 MHz, CD OD) δ 157.3, 148.6, 141.9, 141.8,
3
140.2, 136.5, 135.1, 135.0, 132.8, 130.9, 128.2, 125.5, 121.6, 119.8, 118.1, 115.7,
–1
115.2, 113.9, 104.0, 62.5, 56.4, 21.1. IR (KBr, cm ) 3424, 3047, 2998, 2953, 1632,
1605, 1582, 1512, 1498, 1469, 1434, 1351, 1311, 1295, 1221, 1184, 1126, 1023, 836,
807, 758. Elemental anal. calcd for C H BrN O : C, 64.15; H, 4.71; N, 6.23; found
2
4
21
2
2
+
C, 64.19; H, 4.70; N, 6.19. HRMS (ESI) m/z calcd for C H BrN O [M–Br]
2
4
21
2
2
369.1598, found 369.1596.
534
535
536
537
538
539
540
541
542
543
544
4.2.2.12. 1-(furan-2-yl)-6-methoxy-2-(4-(trifluoromethyl)benzyl)-9H-pyrido[3,4-b]
o
1
indol-2-ium bromide (4l): red-brown solid; yield, 76%; m.p. 198.5–199.6 C; H
NMR (500 MHz, CD OD) δ 8.72 (dd, J = 10 Hz, 5 Hz, 2H), 8.07 (d, J = 2.5 Hz, 1H),
3
7.92 (d, J = 2.5 Hz, 1H), 7.71–7.64 (m, 3H), 7.49–7.47 (m, 1H), 7.30–7.25 (m, 3H),
1
3
6.86 (dd, J = 5 Hz, 2.5 Hz, 1H), 6.17 (s, 2H), 3.97 (s, 3H); C NMR (125 MHz,
CD OD) δ 157.1, 148.5, 141.7, 141.4, 140.1, 136.3, 135.3, 135.0, 130.6, 128.4, 126.9,
3
126.8, 126.8, 126.8, 126.1, 125.4, 121.4, 120.7, 119.8, 118.2, 118.1, 115.0, 113.7,
–
1
103.7, 61.8, 56.2. IR (KBr, cm ) 3435, 3152, 3045, 1658, 1640, 1620, 1611, 1582,
1512, 1501, 1468, 1328, 1297, 1223, 1166, 1127, 1068, 1019. Elemental anal. calcd
for C H BrF N O : C, 57.27; H, 3.60; N, 5.57; found C, 57.22; H, 3.65; N, 5.55.
2
4
18
3
2
2
+
HRMS (ESI) m/z calcd for C H BrF N O [M–Br] 423.1315, found 423.1311.
2
4
18
3
2
2
2
0

ACCEPTED MANUSCRIPT
545
546
547
548
549
550
551
552
553
554
555
4.2.2.13.
2-benzyl-1-(pyridin-2-yl)-9H-pyrido[3,4-b]indol-2-ium bromide (4m):
o
1
yellow solid; yield, 67%; m.p. 137.6–138.9 C; H NMR (500 MHz, DMSO-d6) δ
12.42 (s, 1H), 9.05–8.92 (m, 2H), 8.93–8.92 (m, 1H), 8.58 (d, J = 5 Hz, 1H), 8.12 (td,
J = 5 Hz, 2.5Hz, 1H), 7.87 (d, J = 5 Hz, 1H), 7.82 (td, J = 5 Hz, 2.5Hz, 1H), 7.77–
7.75 (m, 1H), 7.71 (d, J = 5 Hz, 1H), 7.50 (td, J = 10 Hz, 5Hz, 1H), 7.28–7.22 (m,
13
3H), 6.94 (dd, J = 10 Hz, 2.5 Hz, 2H), 5.97 (s, 2H); C NMR (125 MHz, DMSO-d6)
δ 150.6, 147.0, 144.8, 138.2, 137.3, 135.0, 134.7, 134.7, 133.9, 132.4, 128.6, 128.4,
–
1
127.2, 127.0, 126.0, 123.7, 121.9, 119.4, 117.9, 113.1, 59.9. IR (KBr, cm ) 3414,
3056, 2780, 1629, 1574, 1459, 1330, 1270, 1243, 745. Elemental anal. calcd for
C H BrN : C, 66.36; H, 4.36; N, 10.09; found C, 66.33; H, 4.39; N, 10.12. HRMS
23
18
3
+
(ESI) m/z calcd for C H BrN [M–Br] 336.1495, found 336.1494.
2
3
18
3
556
557
558
559
560
561
562
563
564
565
566
4.2.2.14.
2-(4-methylbenzyl)-1-(pyridin-2-yl)-9H-pyrido[3,4-b]indol-2-ium
o
1
bromide (4n): yellow solid; yield, 70%; m.p. 133.4–135.1 C; H NMR (500 MHz,
CD OD) δ 8.95 (dt, J = 10 Hz, 2.5 Hz, 1H), 8.78 (dd, J = 10 Hz, 5 Hz, 2H), 8.45 (d, J
3
= 5 Hz, 1H), 8.12 (td, J = 10 Hz, 2.5 Hz, 1H), 7.81–7.75 (m, 3H), 7.67 (d, J = 10 Hz,
1H), 7.48 (td, J = 10 Hz, 5 Hz, 1H), 7.07 (d, J = 10 Hz, 2H), 6.87 (d, J = 10 Hz, 2H),
1
3
5.90 (s, 2H), 2.26 (s, 3H); C NMR (125 MHz, CD OD) δ 152.2, 148.7, 146.8, 140.2,
3
139.6, 138.7, 136.8, 136.1, 135.5, 133.9, 132.6, 130.7, 128.7, 128.2, 127.5, 124.5,
–1
123.4, 121.0, 118.7, 114.0, 61.9, 21.1. IR (KBr, cm ) 3404, 3050, 3008, 2779, 1628,
1573, 1518, 1461, 1430, 1332, 1267, 1143, 793, 753. Elemental anal. calcd for
C H BrN : C, 66.98; H, 4.68; N, 9.76; found C, 66.94; H, 4.71; N, 9.79. HRMS
24
20
3
+
(ESI) m/z calcd for C H BrN [M–Br] 350.1652, found 350.1649.
2
4
20
3
567
568
569
570
571
572
573
4.2.2.15.
1-(pyridin-2-yl)-2-(4-(trifluoromethyl)benzyl)-9H-pyrido[3,4-b]indol-2-i
o
1
um bromide (4o): yellow solid; yield, 69%; m.p. 194.3–196.0 C; H NMR (500 MHz,
CD OD) δ 8.91 (d, J = 5 Hz, 1H), 8.86 (dd, J = 10 Hz, 5 Hz, 2H), 8.50 (d, J = 10 Hz,
3
1H), 8.09 (td, J = 10 Hz, 2.5 Hz, 1H), 7.84–7.79 (m, 2H), 7.75–7.73 (m, 1H), 7.69 (d,
J = 5 Hz, 1H), 7.56 (d, J = 5 Hz, 2H), 7.52 (t, J = 7.5 Hz, 1H), 7.18 (d, J = 10 Hz, 2H),
13
6.09 (s, 2H); C NMR (125 MHz, CD OD) δ 152.2, 148.5, 146.9, 140.1, 139.6, 138.8,
3
136.9, 136.5, 135.9, 134.1, 131.9, 129.2, 128.3, 127.6, 126.9, 126.9, 126.9, 126.8,
2
1

ACCEPTED MANUSCRIPT
–
1
574
575
576
577
126.3, 124.6, 124.1, 123.6, 121.1, 118.9, 114.1, 61.4. IR (KBr, cm ) 3347, 3011,
1629, 1330, 1274, 1165, 1112, 1067, 756. Elemental anal. calcd for C H BrF N : C,
2
4
17
3
3
59.52; H, 3.54; N, 8.68; found C, 59.49; H, 3.53; N, 8.71. HRMS (ESI) m/z calcd for
+
C H BrF N [M–Br] 404.1369, found 404.1367.
24
17
3
3
578
579
580
581
582
583
584
585
586
587
588
4.2.2.16.
2-benzyl-6-methoxy-1-(pyridin-2-yl)-9H-pyrido[3,4-b]indol-2-ium
o
1
bromide (4p): yellow solid; yield, 79%; m.p. 157.2–158.6 C; H NMR (500 MHz,
DMSO-d6) δ 8.99 (dd, J = 15 Hz, 5 Hz, 2H), 8.91 (d, J = 5 Hz, 1H), 8.13–8.10 (m,
2H), 7.87 (d, J = 5 Hz, 1H), 7.77–7.74 (m, 1H), 7.63 (d, J = 10 Hz, 1H), 7.44–7.42 (m,
1
3
1H), 7.27–7.21 (m, 3H), 6.94 (d, J = 5 Hz, 2H), 5.96 (s, 2H), 3.91 (s, 3H); C NMR
(125 MHz, DMSO-d6) δ 154.8, 150.6, 147.0, 140.3, 138.2, 137.4, 135.1, 134.7, 133.8,
133.2, 128.6, 128.4, 127.3, 127.0, 126.0, 123.6, 119.8, 117.8, 114.1, 103.8, 59.7, 55.8.
–
1
IR (KBr, cm ) 3409, 3039, 2958, 1608, 1571, 1515, 1499, 1472, 1454, 1435, 1358,
1348, 1296, 1274, 1147, 1132, 1017, 824, 791, 730. Elemental anal. calcd for
C H BrN O: C, 64.58; H, 4.52; N, 9.41; found C, 64.55; H, 4.54; N, 9.43. HRMS
24
20
3
+
(ESI) m/z calcd for C H BrN O [M–Br] 366.1601, found 366.1593.
2
4
20
3
589
590
591
592
593
594
595
596
597
598
599
600
4.2.2.17.
6-methoxy-2-(4-methylbenzyl)-1-(pyridin-2-yl)-9H-pyrido[3,4-b]indol-2-
o
1
ium bromide (4q): yellow solid; yield, 75%; m.p. 164.8–165.6 C; H NMR (500 MHz,
DMSO-d6) δ 12.30 (s, 1H), 8.96–8.93 (m, 3H), 8.17–8.12 (m, 2H), 7.90 (d, J = 10 Hz,
2.5 Hz, 1H), 7.79–7.76 (m, 1H), 7.63 (d, J = 10 Hz, 1H), 7.46–7.44 (m, 1H), 7.05 (d,
1
3
J = 10 Hz, 2H), 6.85 (d, J = 10 Hz, 2H), 5.89 (s, 2H), 3.92 (s, 3H), 2.22 (s, 3H); C
NMR (125 MHz, DMSO-d6) δ 154.8, 150.6, 147.0, 140.1, 138.2, 137.9, 137.4, 135.0,
133.7, 133.1, 131.7, 129.2, 127.3, 127.0, 126.0, 123.6, 119.8, 117.8, 114.1, 103.8,
–
1
59.5, 55.8, 40.0, 39.8, 39.6, 39.5, 39.3, 39.1, 39.0, 20.6. IR (KBr, cm ) 3383, 3053,
3008, 1632, 1613, 1576, 1521, 1498, 1466, 1293, 1224, 1142, 1020, 825, 787.
Elemental anal. calcd for C H BrN O: C, 65.22; H, 4.82; N, 9.13; found C, 65.20; H,
25
22
3
+
4.80; N, 9.17. HRMS (ESI) m/z calcd for C H BrN O [M–Br] 380.1757, found
25
22
3
380.1752.
6
01
02
4.2.2.18.
6-methoxy-1-(pyridin-2-yl)-2-(4-(trifluoromethyl)benzyl)-9H-pyrido[3,4-
o
1
6
b]indol-2-ium bromide (4r): yellow solid; yield, 70%; m.p. 144.8–145.7 C; H NMR
2
2

ACCEPTED MANUSCRIPT
603
604
605
606
607
608
609
610
611
612
(500 MHz, CD OD) δ 8.89 (d, J = 5 Hz, 1H), 8.83 (dd, J = 10 Hz, 5 Hz, 2H), 8.37 (d,
3
J = 10 Hz, 1H), 8.17 (td, J = 10 Hz, 2.5 Hz, 1H), 7.89–7.81 (m, 1H), 7.72–7.69 (m,
1H), 7.61 (d, J = 5 Hz, 1H), 7.47 (d, J = 5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 1H), 7.18 (d, J
1
3
= 10 Hz, 2H), 6.07 (s, 2H), 2.11 (s, 3H); C NMR (125 MHz, CD OD) δ 153.4, 148.5,
3
146.8, 140.1, 139.6, 138.7, 137.0, 136.5, 135.9, 134.1, 131.9, 129.7, 128.0, 127.5,
127.1, 126.9, 126.9, 126.9, 126.6, 124.5, 124.0, 123.6, 121.0, 119.0, 114.2, 60.3, 20.1.
–
1
IR (KBr, cm ) 3409, 3006, 2958, 1611, 1578, 1516, 1499, 1472, 1438, 1296, 1224,
1139, 792. Elemental anal. calcd for C H BrF N O: C, 58.38; H, 3.72; N, 8.17;
2
5
19
3
3
+
found C, 58.34; H, 3.75; N, 8.20. HRMS (ESI) m/z calcd for C H BrF N O [M–Br]
2
5
19
3
3
434.1475, found 434.1470.
613
614
615
616
617
618
619
620
621
622
623
4.2.2.19. 1-(benzo[d][1,3]dioxol-5-yl)-2-benzyl-9H-pyrido[3,4-b]indol-2-ium
o
1
bromide (4s): yellow solid; yield, 83%; m.p. 152.8–153.4 C; H NMR (500 MHz,
CD OD) δ 8.76–8.72 (m, 2H), 8.40 (d, J = 10 Hz, 1H), 7.75 (dd, J = 15 Hz, 5 Hz, 1H),
3
7.67 (d, J = 5 Hz, 1H), 7.67 (t, J = 5 Hz, 1H), 7.32–7.31 (m, 3H), 7.12 (d, J = 5 Hz,
13
1H), 7.08–7.03 (m, 4H), 6.16 (dd, J = 10 Hz, 2.5 Hz, 2H), 5.85 (s, 2H); C NMR
(125 MHz, CD OD) δ 151.9, 150.3, 146.3, 141.6, 137.3, 136.0, 135.2, 134.9, 133.4,
3
130.1, 129.9, 128.5, 125.6, 124.4, 123.2, 122.4, 121.2, 118.1, 114.0, 110.8, 110.4,
–1
103.6, 61.9. IR (KBr, cm ) 3401, 3022, 2930, 1605, 1492, 1430, 1331, 1229, 1140,
1109, 1032, 767. Elemental anal. calcd for C H BrN O : C, 65.37; H, 4.17; N, 6.10;
2
5
19
2
2
+
found C, 65.34; H, 4.15; N, 6.13. HRMS (ESI) m/z calcd for C H BrN O [M–Br]
2
5
19
2
2
379.1441, found 379.1439.
624
625
626
627
628
629
630
631
4.2.2.20. 1-(benzo[d][1,3]dioxol-5-yl)-2-(4-methylbenzyl)-9H-pyrido[3,4-b]indol-
o
1
2-ium bromide (4t): yellow solid; yield, 83%; m.p. 141.2–142.6 C; H NMR (500
MHz, CD OD) δ 8.73–8.67 (m, 2H), 8.40 (d, J = 5 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H),
3
7.65 (d, J = 5 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.14–7.12 (m, 3H), 7.09–7.07 (m, 1H),
7.04 (d, J = 2.5 Hz, 1H), 6.93 (d, J = 5 Hz, 2H), 6.18 (d, J = 10 Hz, 2H), 5.79 (s, 2H),
1
3
2.28 (s, 3H); C NMR (125 MHz, CD OD) δ 151.9, 150.3, 146.3, 141.5, 140.1, 137.2,
3
135.1, 134.7, 133.4, 133.0, 130.7, 128.7, 125.6, 124.3, 123.2, 122.5, 121.2, 118.0,
–1
114.0, 110.8, 110.4, 103.6, 61.7, 21.1. IR (KBr, cm ) 3387, 3012, 2921, 1627, 1482,
2
3

ACCEPTED MANUSCRIPT
632
633
634
1434, 1334, 1233, 1144, 1113, 1033, 753. Elemental anal. calcd for C H BrN O : C,
2
6
21
2
2
65.97; H, 4.47; N, 5.92; found C, 65.95; H, 4.49; N, 5.90. HRMS (ESI) m/z calcd for
+
C H BrN O [M–Br] 393.1598, found 393.1595.
26
21
2
2
635
636
637
638
639
640
641
642
643
644
645
646
4.2.2.21.
1-(benzo[d][1,3]dioxol-5-yl)-2-(4-(trifluoromethyl)benzyl)-9H-pyrido[3,
o
1
4-b]indol-2-ium bromide (4u): yellow solid; yield, 89%; m.p. 156.8–157.2 C; H
NMR (500 MHz, CD OD) δ 8.82–8.79 (m, 2H), 8.44 (d, J = 10 Hz, 1H), 7.76 (t, J =
3
7.5 Hz, 1H), 7.68 (d, J = 10 Hz, 1H), 7.61 (d, J = 10 Hz, 2H), 7.45 (t, J = 7.5 Hz, 1H),
7.23 (d, J = 10 Hz, 1H), 7.11–7.05 (m, 3H), 6.15 (dd, J = 15 Hz, 2.5 Hz, 2H), 6.00 (s,
1
3
2H); C NMR (125 MHz, CD OD) δ 157.1, 151.3, 150.2, 141.0, 139.5, 137.8, 135.0,
3
134.2, 131.4, 130.5, 129.1, 128.0, 127.8, 127.1, 126.8, 126.3, 124.9, 124.3, 123.9,
–1
121.7, 121.7, 117.9, 114.8, 110.6, 110.2, 103.7, 102.6, 56.3. IR (KBr, cm ) 3402,
3015, 2780, 1628, 1484, 1435, 1325, 1236, 1166, 1118, 1065, 1036, 821, 753.
Elemental anal. calcd for C H BrF N O : C, 59.22; H, 3.44; N, 5.31; found C, 59.19;
2
6
18
3
2
2
+
H, 3.47; N, 5.34. HRMS (ESI) m/z calcd for C H BrF N O [M–Br] 447.1315,
26
18
3
2
2
found 447.1313.
647
648
649
650
651
652
653
654
655
656
657
4.2.2.22. 1-(benzo[d][1,3]dioxol-5-yl)-2-benzyl-6-methoxy-9H-pyrido[3,4-b]indol
o
1
-2-ium bromide (4v): yellow solid; yield, 81%; m.p. 167.6–168.4 C; H NMR (500
MHz, CD OD) δ 8.68–8.63 (m, 2H), 7.90 (d, J = 2.5 Hz, 1H), 7.59 (d, J = 10 Hz, 1H),
3
7.45–7.42 (m, 1H), 7.34–7.32 (m, 3H), 7.10 (d, J = 5 Hz, 1H), 7.04–7.01(m, 4H), 6.15
1
3
(d, J = 5 Hz, 2H), 5.81 (s, 2H), 3.97 (s, 3H); C NMR (125 MHz, CD OD) δ 157.2,
3
152.0, 150.3, 141.9, 141.8, 137.5, 136.2, 134.3, 134.3, 130.2, 129.9, 128.4, 125.5,
–
125.2, 122.5, 121.7, 118.0, 115.0, 110.7, 110.4, 104.0, 103.7, 61.7, 56.4. IR (KBr, cm
1
) 3394, 3060, 3023, 2955, 2903, 2836, 2790, 1609, 1580, 1487, 1443, 1344, 1313,
1248, 1223, 1156, 1112, 1033, 928, 819. Elemental anal. calcd for C H BrN O : C,
2
6
21
2
3
63.81; H, 4.33; N, 5.72; found C, 63.82; H, 4.30; N, 5.74. HRMS (ESI) m/z calcd for
+
C H BrN O [M–Br] 409.1547, found 409.1544.
26
21
2
3
658
659
660
4.2.2.23.
1-(benzo[d][1,3]dioxol-5-yl)-6-methoxy-2-(4-methylbenzyl)-9H-pyrido[3
o
1
,4-b]indol-2-ium bromide (4w): yellow solid; yield, 88%; m.p. 166.5–167.2 C; H
NMR (500 MHz, CD OD) δ 8.68–8.62 (m, 2H), 7.89 (d, J = 2.5 Hz, 1H), 7.58 (d, J =
3
2
4

ACCEPTED MANUSCRIPT
661
662
663
664
665
666
667
668
2.5 Hz, 1H), 7.42–7.40(m, 1H), 7.16–7.03 (m, 5H), 6.92 (d, J = 10 Hz, 2H), 6.16 (d, J
1
3
= 5 Hz, 2H), 5.76 (s, 2H), 3.95 (s, 3H), 2.30 (s, 3H); C NMR (125 MHz, CD OD) δ
3
157.1, 151.9, 150.3, 141.8, 141.7, 140.1, 137.4, 134.2, 134.2, 133.1, 130.8, 128.5,
125.5, 125.1, 122.6, 121.7, 118.0, 115.0, 110.8, 110.4, 104.0, 103.7, 61.6, 56.4, 21.1.
–
1
IR (KBr, cm ) 3442, 3058, 3025, 2963, 2892, 2835, 1612, 1578, 1523, 1485, 1439,
1344, 1317, 1299, 1224, 1188, 1113, 1038, 934, 822, 784. Elemental anal. calcd for
C H BrN O : C, 64.42; H, 4.61; N, 5.57; found C, 64.39; H, 4.57; N, 5.60. HRMS
27
23
2
3
+
(ESI) m/z calcd for C H BrN O [M–Br] 423.1703, found 423.1696.
2
7
23
2
3
669
670
671
672
673
674
675
676
677
678
679
4.2.2.24.
1-(benzo[d][1,3]dioxol-5-yl)-6-methoxy-2-(4-(trifluoromethyl)benzyl)-9
H-pyrido[3,4-b]indol-2-ium bromide (4x): yellow solid; yield, 89%; m.p. 181.5–182.7
o
1
C; H NMR (500 MHz, CD OD) δ 8.72 (dd, J = 10 Hz, 5 Hz, 2H), 7.91 (d, J = 5 Hz,
3
1H), 7.64–7.58 (m, 3H), 7.43–7.41 (m, 1H), 7.20 (d, J = 5 Hz, 2H), 7.09–7.03 (m,
1
3
3H), 6.14 (d, J = 15 Hz, 2H), 5.95 (s, 2H), 3.96 (s, 3H); C NMR (125 MHz, CD OD)
3
δ 157.1, 152.0, 150.3, 141.9, 141.9, 140.6, 137.5, 134.6, 134.5, 131.8, 131.6, 129.0,
127.0, 127.0, 126.9, 126.9, 126.4, 125.5, 125.3, 124.2, 122.4, 121.7, 118.2, 115.0,
–1
110.7, 110.4, 104.0, 103.7, 61.2, 56.4. IR (KBr, cm ) 3422, 3018, 2784, 1616, 1579,
1490, 1439, 1326, 1227, 1164, 1118, 1068, 1036, 936, 821. Elemental anal. calcd for
C H BrF N O : C, 58.18; H, 3.62; N, 5.03; found C, 58.16; H, 3.59; N, 5.06. HRMS
27
20
3
2
3
+
(ESI) m/z calcd for C H BrF N O [M–Br] 477.1421, found 477.1419.
2
7
20
3
2
3
680
681
682
683
684
685
686
687
688
689
4.2.2.25.
2-(4-fluorobenzyl)-6-methoxy-1-(4-(methoxycarbonyl)phenyl)-9H-pyrido
o
1
[3,4-b]indol-2-ium bromide (5a): yellow solid; yield, 73%; m.p. 158.9–156.3 C; H
NMR (500 MHz, DMSO-d6) δ 12.18 (s, 1H), 8.93 (dd, J = 10 Hz, 5 Hz, 2H), 8.18 (d,
J = 5 Hz, 2H), 8.10 (d, J = 2.5 Hz, 1H), 7.78 (d, J = 10 Hz, 2H), 7.58 (d, J = 10 Hz,
1H), 7.45–7.43 (m, 1H), 7.10 (t, J = 10 Hz, 2H), 7.00–6.97 (m, 2H), 5.78 (s, 2H), 3.95
1
3
(s, 3H), 3.92 (s, 3H); C NMR (125 MHz, DMSO-d6) δ 165.6, 162.8, 160.8, 154.8,
140.2, 139.1, 135.5, 133.5, 132.5, 131.9, 131.0, 130.4, 129.9, 129.5, 123.5, 119.9,
–
1
117.5, 115.7, 114.1, 103.7, 59.4, 55.7, 52.6. IR (KBr, cm ) 3407, 3063, 2953, 1716,
1636, 1607, 1580, 1510, 1498, 1468, 1436, 1351, 1296, 1278, 1221, 1163, 1120, 1042,
1029, 1013, 825, 786, 762. Elemental anal. calcd for C H BrFN O : C, 62.20; H,
27
22
2
3
2
5

ACCEPTED MANUSCRIPT
6
90
91
4.25; N, 5.37; found C, 62.17; H, 4.29; N, 5.34. HRMS (ESI) m/z calcd for
+
6
C H BrFN O [M–Br] 441.1609, found 441.1605.
27
22
2
3
692
693
694
695
696
697
698
699
700
701
4.2.2.26.
2-(4-chlorobenzyl)-6-methoxy-1-(4-(methoxycarbonyl)phenyl)-9H-pyrido
o
1
[3,4-b]indol-2-ium chloride (5b): yellow solid; yield, 86%; m.p. 157.7–159.2 C; H
NMR (500 MHz, DMSO-d6) δ 12.13 (s, 1H), 8.89 (s, 2H), 8.18 (d, J = 10 Hz, 2H),
8.08 (d, J = 2.5 Hz, 1H), 7.77 (d, J = 5 Hz, 2H), 7.60 (d, J = 10 Hz, 1H), 7.46–7.44 (m,
1H), 7.33 (d, J = 10 Hz, 2H), 7.33 (d, J = 5 Hz, 2H), 5.77 (s, 2H), 3.96 (s, 3H), 3.93 (s,
–
1
3H). IR (KBr, cm ) 3383, 3062, 2951, 2837, 1715, 1633, 1610, 1580, 1496, 1468,
1453, 1436, 1405, 1350, 1295, 1279, 1223, 1188, 1120, 1089, 1029, 1015, 826, 811,
782. Elemental anal. calcd for C H Cl N O : C, 65.73; H, 4.49; N, 5.68; found C,
2
7
22
2
2
3
+
65.71; H, 4.47; N, 5.70. HRMS (ESI) m/z calcd for C H Cl N O [M–Cl] 457.1313,
27
22
2
2
3
459.1284, found 457.1311, 459.1280.
702
703
704
705
706
707
708
709
710
711
4.2.2.27. 2-(4-bromobenzyl)-6-methoxy-1-(4-(methoxycarbonyl)phenyl)-9H-pyrido
o
1
[3,4-b]indol-2-ium bromide (5c): yellow solid; yield, 91%; m.p. 160.9–162.4 C; H
NMR (500 MHz, DMSO-d6) δ 12.16 (s, 1H), 8.91 (dd, J = 10 Hz, 5 Hz, 2H), 8.19 (d,
J = 5 Hz, 2H), 8.19 (d, J = 2.5 Hz, 1H), 7.79 (d, J = 10 Hz, 2H), 7.59 (d, J = 10 Hz,
1H), 7.48–7.44 (m, 3H), 6.90 (d, J = 10 Hz, 2H), 5.75 (s, 2H), 3.95 (s, 3H), 3.92 (s,
–
1
3H). IR (KBr, cm ) 3374, 3148, 3062, 2951, 1716, 1635, 1610, 1580, 1527, 1497,
1469, 1453, 1436, 1405, 1294, 1280, 1223, 1186, 1111, 1071, 1029, 1013, 826, 807,
782, 770. Elemental anal. calcd for C H Br N O : C, 55.69; H, 3.81; N, 4.81; found
2
7
22
2
2
3
+
C, 55.67; H, 3.83; N, 4.78. HRMS (ESI) m/z calcd for C H Br N O [M–Br]
2
7
22
2
2
3
501.0808, 503.0788, found 501.0806, 503.0785.
712
713
714
715
716
717
718
4.2.2.28. 2-(2-fluorobenzyl)-6-methoxy-1-(4-(methoxycarbonyl)phenyl)-9H-pyrido
o
1
[3,4-b]indol-2-ium bromide (5d): yellow solid; yield, 72%; m.p. 155.8–156.9 C; H
NMR (500 MHz, DMSO-d6) δ 12.18 (s, 1H), 8.94 (dd, J = 10 Hz, 5 Hz, 2H), 8.16 (d,
J = 5 Hz, 2H), 8.11 (d, J = 2.5 Hz, 1H), 7.77 (d, J = 10 Hz, 2H), 7.59 (d, J = 10 Hz,
1H), 7.46–7.44 (m, 1H), 7.39–7.35 (m, 1H), 7.14–7.07 (m, 2H), 6.90 (t, J = 7.5 Hz,
1
3
1H), 5.88 (s, 2H), 3.95 (s, 3H), 3.92 (s, 3H); C NMR (125 MHz, DMSO-d6) δ 165.6,
160.5, 158.5, 154.8, 140.2, 139.1, 135.4, 133.8, 132.7, 132.4, 131.9, 130.9, 130.9,
2
6

ACCEPTED MANUSCRIPT
719
720
721
722
723
724
130.3, 129.8, 129.6, 129.6, 124.8, 123.6, 121.8, 121.7, 119.9, 117.4, 115.5, 115.3,
–
1
114.1, 103.7, 55.8, 54.9, 52.6. IR (KBr, cm ) 3409, 3046, 3015, 2954, 1723, 1608,
1580, 1497, 1454, 1439, 1295, 1278, 1225, 1183, 1119, 1102, 1028, 828, 764.
Elemental anal. calcd for C H BrFN O : C, 62.20; H, 4.25; N, 5.37; found C, 62.17;
27
22
2
3
+
H, 4.21; N, 5.41. HRMS (ESI) m/z calcd for C H BrFN O [M–Br] 441.1609,
27
22
2
3
found 441.1597.
725
726
727
728
729
730
731
732
733
734
4.2.2.29. 2-(3-fluorobenzyl)-6-methoxy-1-(4-(methoxycarbonyl)phenyl)-9H-pyrido
o
1
[3,4-b]indol-2-ium bromide (5e): yellow solid; yield, 79%; m.p. 147.2–149.0 C; H
NMR (500 MHz, DMSO-d6) δ 12.20 (s, 1H), 8.92 (t, J = 5 Hz, 2H), 8.17 (d, J = 10
Hz, 2H), 8.10 (d, J = 5 Hz, 1H), 7.78 (d, J = 10 Hz, 2H), 7.59 (d, J = 5 Hz, 1H), 7.46–
7.43 (m, 1H), 7.33–7.29 (m, 1H), 7.13 (td, J = 10 Hz, 2.5 Hz, 1H), 6.83 (d, J = 10 Hz,
–
1
1H), 6.75 (d, J = 10 Hz, 1H), 5.80 (s, 2H), 3.95 (s, 3H), 3.92 (s, 3H). IR (KBr, cm )
3397, 3006, 2952, 2835, 1722, 1635, 1611, 1579, 1526, 1498, 1467, 1452, 1437, 1403,
1348, 1279, 1223, 1184, 1131, 1118, 1030, 1013, 826, 779. Elemental anal. calcd for
C H BrFN O : C, 62.20; H, 4.25; N, 5.37; found C, 62.18; H, 4.23; N, 5.39. HRMS
27
22
2
3
+
(ESI) m/z calcd for C H BrFN O [M–Br] 441.1609, found 441.1607.
2
7
22
2
3
735
736
737
738
739
740
741
742
743
4.2.2.30.
2-(2-bromobenzyl)-6-methoxy-1-(4-(methoxycarbonyl)phenyl)-9H-pyrido
o
1
[3,4-b]indol-2-ium bromide (5f): yellow solid; yield, 93%; m.p. 249.8–251.1 C; H
NMR (500 MHz, DMSO-d6) δ 12.21 (s, 1H), 8.94–8.81 (m, 2H), 8.15 (d, J = 10 Hz,
2H), 8.11 (d, J = 2.5 Hz, 1H), 7.76 (d, J = 10 Hz, 2H), 7.61–7.58 (m, 2H), 7.48–7.45
(m, 1H), 7.32–7.26 (m, 2H), 6.84 (d, J = 5 Hz, 1H), 5.81 (s, 2H), 3.93 (s, 3H), 3.93 (s,
–
1
3H). IR (KBr, cm ) 3009, 2986, 2951, 1720, 1608, 1577, 1496, 1437, 1297, 1268,
1229, 1133, 1118, 1104, 1029, 828, 765. Elemental anal. calcd for C H Br N O : C,
2
7
22
2
2
3
55.69; H, 3.81; N, 4.81; found C, 55.66; H, 3.84; N, 4.79. HRMS (ESI) m/z calcd for
+
C H Br N O [M–Br] 501.0808, 503.0788, found 501.0806, 503.0785.
27
22
2
2
3
744
745
746
747
4.2.2.31.
2-(3-bromobenzyl)-6-methoxy-1-(4-(methoxycarbonyl)phenyl)-9H-pyrido
o
1
[3,4-b]indol-2-ium bromide (5g): yellow solid; yield, 95%; m.p. 150.3–152.1 C; H
NMR (500 MHz, DMSO-d6) δ 12.19 (s, 1H), 8.93 (dd, J = 10 Hz, 5 Hz, 2H), 8.17 (d,
J = 10 Hz, 2H), 8.09 (d, J = 5 Hz, 1H), 7.76 (d, J = 10 Hz, 2H), 7.58 (d, J = 10 Hz,
2
7

ACCEPTED MANUSCRIPT
748
749
750
751
752
753
1H), 7.49–7.43 (m, 2H), 7.21 (t, J = 7.5 Hz, 1H), 7.13 (t, J = 2.5 Hz, 1H), 6.91 (d, J =
–
1
10 Hz, 1H), 5.79 (s, 2H), 3.95 (s, 3H), 3.92 (s, 3H). IR (KBr, cm ) 3409, 3046, 3015,
2954, 2837, 1723, 1608, 1580, 1497, 1454, 1439, 1349, 1295, 1279, 1225, 1183, 1151,
1133, 1119, 1102, 1028, 1012, 828, 764. Elemental anal. calcd for C H Br N O : C,
2
7
22
2
2
3
55.69; H, 3.81; N, 4.81; found C, 55.67; H, 3.84; N, 4.83. HRMS (ESI) m/z calcd for
+
C H Br N O [M–Br] 501.0808, 503.0788, found 501.0807, 503.0786.
27
22
2
2
3
754
755
756
757
758
759
760
761
762
763
4.2.2.32.
2-(2,4-difluorobenzyl)-6-methoxy-1-(4-(methoxycarbonyl)phenyl)-9H-py
o
rido[3,4-b]indol-2-ium bromide (5h): yellow solid; yield, 87%; m.p. 142.3–144.3 C;
1
H NMR (500 MHz, DMSO-d6) δ 12.14 (s, 1H), 8.93 (dd, J = 10 Hz, 5 Hz, 2H), 8.19
(d, J = 5 Hz, 2H), 8.10 (d, J = 2.5 Hz, 1H), 7.78 (d, J = 10 Hz, 2H), 7.58 (d, J = 5 Hz,
1H), 7.46–7.44 (m, 1H), 7.23–7.19 (m, 1H), 7.02–6.96 (m, 2H), 5.84 (s, 2H), 3.96 (s,
–
1
3H), 3.92 (s, 3H). IR (KBr, cm ) 3069, 3034, 3002, 2960, 1711, 1609, 1577, 1532,
1501, 1472, 1435, 1409, 1318, 1286, 1227, 1184, 1136, 1110, 1086, 1035, 1014, 960,
827, 767. Elemental anal. calcd for C H BrF N O : C, 60.12; H, 3.92; N, 5.19;
2
7
21
2
2
3
found C, 60.15; H, 3.90; N, 5.15. HRMS (ESI) m/z calcd for C H BrF N O [M–
27
21
2
2
3
+
Br] 459.1515, found 459.1513.
764
765
766
767
768
769
770
771
772
773
774
4.2.2.33. 6-methoxy-1-(4-(methoxycarbonyl)phenyl)-2-(4-nitrobenzyl)-9H-pyrido[
o
1
3,4-b]indol-2-ium chloride (5i): yellow solid; yield, 83%; m.p. 162.1–162.8 C; H
NMR (500 MHz, DMSO-d6) δ 12.34 (s, 1H), 9.01 (dd, J = 10 Hz, 5 Hz, 2H), 8.15–
8.12 (m, 5H), 7.78 (d, J = 5 Hz, 2H), 7.64 (d, J = 10 Hz, 1H), 7.50–7.47 (m, 1H), 7.23
1
3
(d, J = 10 Hz, 2H), 6.00 (s, 2H), 3.97 (s, 3H), 3.96 (s, 3H); C NMR (125 MHz,
DMSO-d6) δ 165.5, 154.8, 147.1, 142.3, 139.3, 137.0, 135.7, 133.7, 133.6, 132.9,
132.7, 132.4, 131.9, 130.4, 129.8, 128.1, 123.6, 120.0, 117.6, 103.6, 59.3, 55.7, 52.5.
–
1
IR (KBr, cm ) 2998, 2952, 1715, 1610, 1580, 1522, 1500, 1434, 1405, 1345, 1318,
1285, 1229, 1135, 1110, 829. Elemental anal. calcd for C H ClN O : C, 64.35; H,
27
22
3
5
4.40; N, 8.34; found C, 64.31; H, 4.42; N, 8.33. HRMS (ESI) m/z calcd for
+
C H ClN O [M–Cl] 468.1554, found 468.1551.
27
22
3
5
775
4.2.3. X-ray structures of compounds 3e and 5f
2
8