Synthesis and activity against mycobacterium tuberculosis of olivacine and oxygenated derivatives

Article abstract of DOI:10.3390/molecules23061402

The tetracyclic pyrido[4,3-b]carbazole olivacine and four of its oxygenated derivatives have been synthesized by a late-stage palladium-catalyzed Heck-type cyclization of the pyrrole ring as a key step. In a test for the inhibition of the growth of Mycobacterium tuberculosis, 9-methoxyolivacine showed the most significant inhibitory activity against Mycobacterium tuberculosis, with an MIC₉₀ value of 1.5 μM.

Full text of DOI:10.3390/molecules23061402

molecules
Article
Synthesis and Activity against Mycobacterium
tuberculosis of Olivacine and Oxygenated Derivatives
Ulrike Schmidt 1, Gabriele Theumer 1, Anne Jäger 1, Olga Kataeva 2, Baojie Wan 3,
Scott G. Franzblau 3 and Hans-Joachim Knölker 1,
*
ID
1
Faculty of Chemistry, Technische Universität Dresden, Bergstraße 66, 01069 Dresden, Germany;
ulrike.schmidt@chemie.tu-dresden.de (U.S.); Gabriele.Theumer@tu-dresden.de (G.T.);
anne.jaeger@chemie.tu-dresden.de (A.J.)
A. M. Butlerov Chemistry Institute, Kazan Federal University, Kremlevskaya Str. 18, Kazan 420008, Russia;
olga-kataeva@yandex.ru
Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago,
833 S. Wood St., MC 964, Chicago, IL 60612-7231, USA; baojie@uic.edu (B.W.); sgf@uic.edu (S.G.F.)
Correspondence: hans-joachim.knoelker@tu-dresden.de; Fax +49-351-463-37030
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 30 April 2018; Accepted: 6 June 2018; Published: 9 June 2018
Abstract: The tetracyclic pyrido[4,3-b]carbazole olivacine and four of its oxygenated derivatives have
been synthesized by a late-stage palladium-catalyzed Heck-type cyclization of the pyrrole ring as
a key step. In a test for the inhibition of the growth of Mycobacterium tuberculosis, 9-methoxyolivacine
showed the most significant inhibitory activity against Mycobacterium tuberculosis, with an MIC90
value of 1.5 µM.
Keywords: inhibitory activity; catalysis; cyclization; olivacine; palladium; pyrido[4,3-b]carbazoles
1. Introduction
The pyrido[4,3-b]carbazole alkaloid olivacine (
Schmutz et al. [ ], and its structural assignment was confirmed by total synthesis only two years
later [ ]. The tetracyclic alkaloid and many structurally related compounds, for example the isomeric
natural product ellipticine ( ), show useful biological activities, such as anti-tumor activity, based on
DNA intercalation, topoisomerase II inhibition, and antimalarial activity [ ]. Since the 1980s, A-ring
oxygenated derivatives of ellipticine ( ) have attracted much attention because of their anti-tumor
activity [ ]. Elliptinium acetate ( ) has reached the status of a licensed drug for the treatment of advanced
breast cancer [ ]. Diverse total syntheses of olivacine ( ) have been reported [10 18]. Surprisingly,
) and its oxygenated derivatives (for example and ) has
1, Figure 1) was first isolated in 1958 by
1
2
1
2
3–7
2
8
3
9
1
–
the pharmacological potential of olivacine (
1
4
5
been much less investigated [19].
Figure 1. Pyrido[4,3-b]carbazole alkaloids and oxygenated derivatives.
Molecules 2018, 23, 1402; doi:10.3390/molecules23061402
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1402
2 of 16
Although 9-hydroxyolivacine (
of olivacine ( ) [
extensively in the literature [
usually involve the annulation of an isoquinoline or a pyridine at an indole or carbazole framework [8,10,11]
5
) is the main derivative produced by the metabolic conversion
1
3
], derivatives of olivacine (
1) with A-ring substitution have been not described
3
,11,13,20]. This may be because the syntheses of pyrido[4,3-b]carbazoles
.
Thus, a facile variation of the substitution pattern at ring A is not easy to accomplish. Herein, we
present a novel route for the synthesis of the tetracyclic pyrido[4,3-b]carbazole framework [21].
2. Results and Discussion
For a convergent access to various A-ring substituted derivatives, we envisaged a late-stage
B-ring construction of the pyrido[4,3-b]carbazole framework. Therefore, we applied the two-step
sequence of palladium-catalyzed reactions developed by our group for carbazole assembly: synthesis of
a diarylamine via Buchwald–Hartwig coupling of appropriate anilines
followed by oxidative cyclization to the pyrido[4,3-b]carbazoles (Scheme 1) [11]. The isoquinoline
would be available by Bischler–Napieralski cyclization of the arylethylamine via the corresponding
acetamide. Henry reaction of an appropriately substituted benzaldehyde 10 and subsequent reduction
should afford the arylethylamine . As the Bischler–Napieralski reaction works best on electron
7 with a substituted isoquinoline
8
6
8
9
9
rich aromatic systems, we decided to start from the commercially available methoxy-substituted
benzaldehyde 11 (Scheme 2) and to transform the methoxy group into a suitable leaving group at
a later stage of our synthesis.
Scheme 1. Retrosynthetic analysis for the pyrido[4,3-b]carbazole olivacine and its A-ring derivatives.
2.1. Total Synthesis
Starting from the commercial benzaldehyde 11, which can also be obtained in one step and 87% yield
from the much cheaper m-anisaldehyde [22], amide 12 is prepared by a three-step sequence of Henry
reaction, lithium aluminum hydride reduction, and N-acetylation (Scheme 2) [23]. Bischler–Napieralski
cyclization using phosphorus oxychloride led to the corresponding dihydroisoquinoline, which was fully
aromatized to 6-methoxy-1,5-dimethylisoquinoline (13) by dehydrogenation with palladium on charcoal
in the presence of cyclohexene as additive. Cleavage of the methyl ether afforded the isoquinolinol, which
on reaction with trifluoromethanesulfonic anhydride provided the known isoquinolinyl triflate 14 [24]
in 58% yield over seven steps.
Scheme 2. Synthesis of the triflate 14. Reagents and conditions: (
110 min, 77%; (
) LiAlH4, THF, 0 ◦C to reflux, 19.5 h, 92%; ( ) Ac2O, cat. DMAP, pyridine, 0 ◦C, 4 h,
99%; ( ) POCl3, reflux, 1 h, 99%; ( ) Pd/C (10%), cyclohexene, PhMe, reflux, 1.5 h, 100%; ( ) pyridinium
chloride, microwave (300 W), 155 C, 30 min, 96%; (g) Tf2O, pyridine, MeCN, 0 C, 20 h, 87%.
a
) MeNO2, NH4OAc, AcOH, 80 ◦C,
b
c
d
e
f
◦
◦

Molecules 2018, 23, 1402
3 of 16
Buchwald–Hartwig coupling [25] of the triflate 14 and aniline (15) provided the diarylamine
16 (Scheme 3). However, the oxidative cyclization to afford the pyrido[4,3-b]carbazole framework
proved to be very difficult [26]. Several attempts to optimize this reaction failed, which included using
different reaction temperatures, different solvents (HOAc, HOPiv, dioxane, toluene), catalytic amounts
of palladium(II) acetate in the presence of different re-oxidants, as well as stoichiometric amounts of
palladium(II) acetate [27
–29]. All of these experiments resulted to a large extent in decomposition,
and led to olivacine (1) in only low to moderate yields with poor reproducibility.
Scheme 3. Synthesis of olivacine (
cat. XPhos, Cs2CO3, PhMe, reflux, 48 h, 100%; (
argon, 9–49%.
1
) via oxidative cyclization. Reagents and conditions: (
a
) cat. Pd(OAc)2,
b
) 1.1 equiv. Pd(OAc)2, AcOH, 80–100 ◦C, 24 h,
Therefore, we decided to apply a Heck-type cyclization for the formation of the crucial
carbon–carbon bond of the central pyrrole ring. This approach was already described by Sakamoto et al.
in 1999 [30]. Buchwald–Hartwig coupling of the triflate 14 with the commercially available o-chloroanilines
17a–c led to the corresponding diarylamines 18a–c in 83–94% yield (Scheme 4). Compound 18a was
structurally confirmed by an X-ray analysis (Figure 2).
Scheme 4. Synthesis of the pyrido[4,3-b]carbazoles
1,
4
and
5. Reagents and conditions: (a) cat. Pd(OAc)2,
cat. XPhos, Cs2CO3, PhMe, reflux, 1–5 h, 83–94% (18a
DMF, 140 C, 20–35 min, 62–71% (1, 19b, 19c), 3–12% (20a–c); (c) HBr(aq), reflux, 24 h, 70–84% (4, 5).
–
c
); (
b
) cat. Pd(OAc)2, P(tBu)3
·
HBF4, K2CO3,
◦

Molecules 2018, 23, 1402
4 of 16
Figure 2. Molecular structure of the diarylamine 18a in the crystal (ORTEP plot showing thermal
ellipsoids at the 50% probability level).
The cyclization reaction of the diarylamine 18a with catalytic amounts of palladium(II) acetate in
the presence of P(tBu)3 ,32] proceeded
HBF4 and K2CO3 in DMA at 110 ◦C or in DMF at 120 ◦C [31
·
very slowly and gave the product in only moderate yields after 1–2 days (Table 1, entries 1 and 4).
Hydrodehalogenation leading to compound 16 was the major side reaction. Using only slightly higher
temperatures (130–140 ◦C), the reaction proceeded much faster, and the yields for olivacine (
1
) were
significantly better (Table 1, entries 2, 5, and 6). Finally, using larger amounts of the catalyst combined
with shorter reaction times, olivacine ( ) was obtained in 71% yield. The structure of was confirmed
by an X-ray crystal structure determination (Figure 3).
1
1
Table 1. Optimization of the Heck-type cyclization of 18a to olivacine (1).
◦
Pd(OAc)2 (Equiv.) Ligand 1 (Equiv.)
K2CO3 (Equiv.)
Solvent Temp. ( C) Time (h)
Yield (%)
RSM 2 (%)
1
2
3
0.1
0.1
0.2
0.5
0.2
0.3
0.2
0.2
0.4
1.0
0.4
0.6
2
2
4
10
4
4
DMA
DMA
DMA
DMF
DMF
DMF
110
130
120
120
140
140
24
1.5
3.0
45
3.0
0.5
11
46
35
46
62
71
60
18
35
31
7
4 3
5
6
–
1
P(tBu)3
·
HBF4 was used as ligand; 2 RSM = reisolated starting material; 3 reagents added in portions of 0.1 equiv.
Pd(OAc)2, 0.2 equiv. ligand, 2 equiv. K2CO3 after 0, 1, 3, 6, 30 h of reaction time.
Figure 3. Molecular structure of olivacine (1) in the crystal (ORTEP plot showing thermal ellipsoids at
the 50% probability level).
Application of these conditions to the cyclization of the diarylamines 18b and 18c provided
8-methoxyolivacine (19b) and 9-methoxyolivacine (19c) in 65% and 62% yield, respectively (Scheme 4).

Molecules 2018, 23, 1402
5 of 16
The structure of 8-methoxyolivacine (19b) was additionally confirmed by an X-ray analysis of single
crystals (Figure 4). 9-Methoxyolivacine (19c) is a natural product that was isolated in 1967 from
the bark of the coastal Venezuelan tree Aspidosperma vargasii A. DC. [33], and has been synthesized
previously [
carbon atom were obtained as byproducts of the cyclization reactions of the diarylamines 18a
to 12% yield. The structural assignments for the 11bH-pyrido[3,4-c]carbazoles 20a were supported by
two-dimensional (2D) NMR (COSY, HMBC, HSQC, NOESY) spectroscopic studies (see Supplementary
Materials). The compounds 20a result from an attack at the C5 carbon atom of the isoquinoline moiety.
Cleavage of the methyl ether of 19b and 19c provided 8-hydroxyolivacine ( ) and 9-hydroxyolivacine
) [ ] in 84% and 70% yield, respectively. For biological testing, the products were additionally
purified by HPLC.
3,
13
,20]. Interestingly, the 11bH-pyrido[3,4-c]carbazoles 20a
–
c
containing a quaternary
–c
in up
–
c
–
c
4
(5
3
Figure 4. Molecular structure of 8-methoxyolivacine (19b) in the crystal (ORTEP plot showing thermal
ellipsoids at the 50% probability level).
2.2. Biological Activity
A weak inhibitory activity against Mycobacterium tuberculosis was described in early reports for
some simple tricyclic carbazole alkaloids [34
activity of a range of oxygenated carbazole alkaloids and their derivatives, and found very promising
results for several compounds [37 39]. Therefore, we also tested olivacine ( ) and its oxygenated
derivatives 4, 5, 19b, and 19c for their inhibition of M. tuberculosis (Table 2).
–36]. Based on that work, we investigated the inhibitory
–
1
Table 2. Inhibitory activity against M. tuberculosis of olivacine (
19b, and 19c.
1) and its oxygenated derivatives 4, 5,
Compound
MIC90 (µM) 1
IC50 (µM) 2
SI 3
Olivacine (1)
8-Hydroxyolivacine (4)
9-Hydroxyolivacine (5)
8-Methoxyolivacine (19b)
9-Methoxyolivacine (19c)
3-Methoxy-2-methyl-
carbazole-1,4-quinone 5
Isoniazid 5
4.7
n.d.4
n.d.4
n.d.4
1.5
18.05
n.d.
n.d.
n.d.
24.5
3.8
–
–
–
16.3
4.0
>50
>12.5
0.24
0.02
>50
>50
>208
>2500
Rifampicin 5
1
Minimum inhibitory concentrations (
(MABA); values are the mean of three replicate experiments; n.d. = not determined. Cytotoxicity corresponding to
the concentration ( M) effecting a 50% decrease in tetrazolium dye reduction by Vero cells (African green monkey
µM) against M. tuberculosis H37Rv in the microplate Alamar blue assay
2
µ
kidney cells); values are the mean of three replicate experiments; for experiments giving a value higher than the
3
4
max. concentration used, >50
µ
M is denoted. Selectivity index: SI = IC50/MIC90
.
These compounds showed no
5
significant inhibition in a preliminary assay. 3-Methoxy-2-methylcarbazole-1,4-quinone, isoniazid, and rifampicin
(rifampin) were used as positive control; solvent was used as negative control.

Molecules 2018, 23, 1402
6 of 16
In a preliminary activity test against M. tuberculosis, only two of the five pyrido[4,3-b]carbazoles,
namely olivacine ( ) and 9-methoxyolivacine (19c), showed significant effects and have been studied
further. The minimum concentrations effecting a 90% inhibition of growth (MIC90) of the M. tuberculosis
strain H37Rv were determined by the microplate Alamar blue assay (MABA) [40 41]. The in vitro
1
,
cytotoxicity towards mammalian (vero) cells was determined as described previously [40,42].
The MIC90 value for 3-methoxy-2-methylcarbazole-1,4-quinone served as a benchmark for
comparison with the inhibitory activities of carbazoles that were found in our previous studies [39].
Although olivacine (
considerably lower (SI = 3.8) due to its toxicity. However, 9-methoxyolivacine (19c) exhibits a strong
inhibition of M. tuberculosis (MIC90 = 1.5 M) combined with a lower cytotoxicity towards mammalian
1) shows an activity comparable to our benchmark compound, the SI value is
µ
cells, which leads to a very good selectivity index (SI = 16.3).
3. Materials and Methods
3.1. General
All of the reactions were carried out in oven-dried glassware using anhydrous solvents under
an argon atmosphere, unless stated otherwise. CH2Cl2, THF, and toluene were dried using a solvent
purification system (MB-SBS, M. Braun Inertgas-Systeme GmbH, Garching, Germany). The petroleum
◦
ether that was used refers to the hydrocarbon mixture with a boiling range of 40–65 C. Pd(OAc)2 was
recrystallized from glacial AcOH. All of the other chemicals were used as received from commercial
sources. A microwave reactor (CEM Discover, CEM GmbH, Kamp-Lintfort, Germany) was utilized
for reactions taking place under microwave irradiation. Flash chromatography was performed using
silica gel (0.035–0.070 mm) from Acros Organics (Thermo Fisher Scientific, Waltham, MA, USA).
Alox N (aluminum oxide 90 active neutral) was obtained from Merck (Merck KGaA, Darmstadt,
Germany). TLC was performed with TLC plates (silica gel 60 F254) from Merck (Merck KGaA,
Darmstadt, Germany) using UV light for visualization. Melting points were measured on a Gallenkamp
MPD 350 melting point apparatus (A. Gallenkamp & Co. Ltd, London, UK). Ultraviolet spectra were
recorded on a PerkinElmer 25 UV/Vis spectrometer (Perkin Elmer, Waltham, MA, USA). Fluorescence
spectra were obtained using a Varian Cary Eclipse spectrometer (Agilent, Santa Clara, CA, USA).
IR spectra were recorded on a Thermo Nicolet Avatar 360 FT-IR spectrometer (Thermo Fisher Scientific,
Waltham, MA, USA) using the ATR method (Attenuated Total Reflectance). NMR spectra were
recorded on Bruker DRX 500 (Bruker Corp., Billerica, MA, USA) and Avance III 600 (Bruker Corp.,
Billerica, MA, USA) spectrometers. Chemical shifts δ are reported in parts per million (ppm) with the
solvent signal as an internal standard. Standard abbreviations were used to denote the multiplicities
of the signals. MS and HRMS (EI) were recorded on a Finnigan MAT-95 spectrometer (electron
impact, 70 eV, Thermo Fisher Scientific, Waltham, MA, USA) or by GC/MS-coupling using an Agilent
Technologies 6890 N GC System equipped with a 5973 Mass Selective Detector (electron impact,
70 eV, Agilent Technologies, Santa Clara, CA, USA). ESI-MS spectra were recorded on an Esquire
LC with an ion trap detector from Bruker (Bruker Corp., Billerica, MA, USA). Positive and negative
ions were detected. ESI-HRMS were recorded using a Q-TOF 6538 (Agilent Technologies, Santa Clara,
CA, USA). Elemental analyses were measured on an EuroVector EuroEA3000 elemental analyzer
(Eurovector Srl, Pavia, Italy). X-ray crystal structure analyses were performed with a Bruker-Nonius
Kappa CCD (Bruker Corp., Billerica, MA, USA) that was equipped with a 700 series Cryostream
low-temperature device from Oxford Cryosystems (Oxford Cryosystems Ltd, Long Hanborough,
UK). SHELXS-97 [43], SADABS version 2.10 [44], SHELXL-97 [45], POV-Ray for Windows version
3.7.0.msvc10.win64 (Persistence of Vision Raytracer Pty. Ltd, Williamstown, Australia), and ORTEP-3
for Windows [46] (University of Glasgow, Glasgow, UK) were used as software.

Molecules 2018, 23, 1402
7 of 16
3.2. Procedures
1-Methoxy-2-methyl-3-(2-nitrovinyl)benzene. Nitromethane (427 mg, 6.99 mmol) and freshly sublimated
ammonium acetate (433 mg, 5.62 mmol) were added to a solution of 3-methoxy-2-methylbenzaldehyde
(
11,◦ 800 mg, 5.33 mmol) in acetic acid (645 mg, 10.74 mmol), and the mixture was stirred at
80 C for 1 h 50 min. After cooling to room temperature, the precipitate was dissolved by adding
ethyl acetate. The mixture was transferred to a separatory funnel, and then washed twice with
water and brine. The aqueous layer was extracted with ethyl acetate, the combined organic layers
were dried (magnesium sulfate), and the solvent was evaporated. Purification of the residue by
column chromatography (silica gel, petroleum ether/ethyl acetate, 1% to 15% ethyl acetate) provided
◦
1-methoxy-2-methyl-3-(2-nitrovinyl)benzene (791 mg, 4.09 mmol, 77%) as yellow crystals. M.p. 97–98 C;
UV (MeOH):
1627, 1594, 1573, 1541, 1498, 1477, 1450, 1331, 1260, 1244, 1102, 1080, 1007, 968, 893, 873, 844, 806,
777, 725, 693 cm–1; 1H-NMR (500 MHz, CDCl3):
= 2.33 (s, 3H), 3.86 (s, 3H), 6.96 (d, J = 8.2 Hz, 1H),
7.10 (d, J = 7.8 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.48 (d, J = 13.5 Hz, 1H), 8.33 (d, J = 13.5 Hz, 1H);
13C-NMR (125 MHz, CDCl3):
= 11.96 (CH3), 55.84 (CH3), 113.12 (CH), 119.25 (CH), 127.11 (CH),
λ = 205, 228, 251, 317 nm; IR (ATR): ν = 3116, 2959, 2920, 2838, 1901, 1820, 1697, 1653,
δ
δ
128.40 (C), 130.13 (C), 137.25 (CH), 138.20 (CH), 158.29 (C); MS (EI): m/z (%) = 193 (100, [M]+), 178 (6),
161 (7), 146 (70), 131 (52), 115 (54), 103 (67), 91 (37), 77 (47), 63 (18), 51 (18); HRMS: calcd. for C10H11NO3:
193.0738, found: 193.0733; elemental analysis: calcd. for C10H11NO3: C: 62.17, H: 5.74, N: 7.25; found:
C: 62.16, H: 5.77, N: 7.50.
2-(3-Methoxy-2-methylphenyl)ethanamine. Over a period of 1 h, a solution of 1-methoxy-2-methyl-3(2-
nitrovinyl)benzene (6.79 g, 35.2 mmol) in THF (95 mL) was added to a suspension of lithium aluminum
hydride (6.83 g, 180 mmol) in THF (360 mL) at 0 ◦C. The cooling bath was removed, and the mixture
was heated for 30 min at room temperature and 18 h at reflux. A second portion of lithium aluminum
hydride (0.35 g, 9.1 mmol) was added to the slightly reddish-colored solution, and the mixture was
heated at reflux for an additional hour. After cooling to room temperature, the reaction mixture was
carefully quenched with saturated aqueous ammonium chloride, and the pH value was adjusted to
nine. Diethyl ether was added, and the mixture was transferred into a separatory funnel. Still under
argon, the layers were separated, and the aqueous layer was extracted three times with diethyl ether.
The combined organic layers were washed with water and brine, dried (magnesium sulfate), and the
solvent was evaporated to provide 2-(3-methoxy-2-methylphenyl)ethanamine (5.33 g, 32.3 mmol, 92%)
as a yellow oil. UV (MeOH):
2065, 1658, 1604, 1581, 1510, 1463, 1395, 1293, 1256, 1194, 1171, 1149, 1122, 1096, 1006, 953, 875, 789,
776, 763, 719 cm–1; 1H-NMR (600 MHz, CDCl3):
= 2.22 (s, 3H), 3.10–3.19 (m, 4H), 3.82 (s, 3H), 6.77
(d, J = 8.2 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 7.13 (t, J = 7.9 Hz, 1H); 13C-NMR (150 MHz, methanol-d4):
= 11.47 (CH3), 32.26 (CH2), 40.35 (CH2), 55.49 (CH3), 109.10 (CH), 121.76 (CH), 125.07 (C), 126.59
(CH), 135.84 (C), 158.03 (C); MS (ESI, +10 V): m/z = 149.0 [M
NH3 + H]+, 166.0 [M + H]+, 331.2 [2M +
H]+; HRMS: calcd. for C10H15NO: 165.1153, found: 165.1144.
λ = 204, 218, 273, 280 nm; IR (ATR): ν = 3402, 2989, 2923, 2848, 2659, 2480,
δ
δ
−
2-(3-Methoxy-2-methylphenyl)ethyl acetamide (12). DMAP (14 mg, 0.11 mmol) was added to a solution
of 2-(3-methoxy-2-methylphenyl)ethanamine (230 mg, 1.14 mmol) in pyridine (4.5 mL), and the
mixture was cooled to 0 ◦C. Acetic anhydride (140
µL, 15 mmol) was added dropwise over
a period of five minutes, and the reaction mixture was stirred for 4 h. The solvent was evaporated,
and the raw material was purified by chromatography (Alox N, 5% H2O; ethyl acetate) to provide
2-(3-methoxy-2-methylphenyl)ethyl acetamide (12, 235 mg, 1.13 mmol, 99%) as a light yellow solid.
M.p. 84–85 ◦C; UV (MeOH):
λ
= 205, 219, 229, 271, 279 nm; IR (ATR):
2009, 1976, 1716, 1659, 1630, 1564, 1508, 1489, 1472, 1459, 1435, 1396, 1370, 1298, 1285, 1247, 1201, 1180,
1110, 1092, 1037, 1013, 896, 812, 776, 748, 723, 701, 651, 606 cm–1; 1H-NMR (500 MHz, CDCl3):
= 1.95
(s, 3H), 2.19 (s, 3H), 2.84 (t, J = 6.9 Hz, 2H), 3.46 (q, J = 6.9 Hz, 2H), 3.82 (s, 3H), 5.46 (br s, 1H), 6.76
(d, J = 7.9 Hz, 2H), 7.12 (t, J = 7.9 Hz, 1H); 13C-NMR (125 MHz, CDCl3):
= 11.53 (CH3), 23.51 (CH3),
ν = 3267, 3085, 2932, 2836, 2030,
δ
δ

Molecules 2018, 23, 1402
8 of 16
33.38 (CH2), 39.87 (CH2), 55.64 (CH3), 108.61 (CH), 121.89 (CH), 125.24 (C), 126.36 (CH), 138.38 (C),
158.09 (C), 170.21 (C=O); MS (ESI, +10 V): m/z = 208.0 [M + H]+, 415.1 [2M + H]+, 437.1 [2M + Na]+;
HRMS: calcd. for C12H17NO2: 207.1259, found: 207.1248; elemental analysis: calcd. for C12H17NO2: C:
69.54, H: 8.27, N: 6.76; found: C: 69.04, H: 8.73, N: 6.78.
6-Methoxy-1,5-dimethyl-3,4-dihydroisoquinoline. Phosphorus oxychloride (1.9 mL, 21 mmol) was added
to a refluxing solution of acetamide 12 (433 mg, 2.09 mmol) in freshly distilled chloroform (23 mL),
and the mixture was stirred for one hour. Subsequently, solvent and excess phosphorus oxychloride
were removed under vigorous stirring by a nitrogen stream through a pair of soda lye-filled gas
washing bottles. The remaining oily raw product was dissolved in ethyl acetate. Soda lye (10%) was
added, and the pH value was adjusted to 8–9 using saturated aqueous ammonium chloride. The layers
were separated, and the aqueous layer was extracted with ethyl acetate. The combined organic
layers were washed with water and brine, and then dried (magnesium sulfate), and the solvent
was evaporated. Purification of the crude product by chromatography (Alox N, 5% H2O; ethyl
acetate + 3% triethylamine) afforded 6-methoxy-1,5-dimethyl-3,4-dihydroisoquinoline (393 mg,
2.08 mmol, 99%) as a yellow solid. M.p. 57–58 ◦C (subl.); UV (MeOH):
= 3002, 2939, 2838, 1735, 1699, 1629, 1594, 1576, 1539, 1507, 1482, 1435, 1368, 1291, 1258, 1184, 1149,
1101, 1015, 922, 901, 873, 805, 751, 700, 666, 637 cm–1; 1H-NMR (500 MHz, CDCl3):
= 2.15 (s, 3H), 2.35
(t, J = 1.4 Hz, 3H), 2.64 (t, J = 7.4 Hz, 2H), 3.63 (tq, J = 7.4, 1.4 Hz, 2H), 3.85 (s, 3H), 6.75 (d, J = 8.5 Hz, 1H),
7.37 (d, J = 8.5 Hz, 1H); 13C-NMR (125 MHz, CDCl3):
= 11.08 (CH3), 23.32 (CH2), 23.62 (CH3), 46.91
λ = 229, 274, 319 nm; IR (ATR):
ν
δ
δ
(CH2), 55.61 (CH3), 107.50 (CH), 123.06 (C), 123.40 (C), 124.76 (CH), 137.70 (C), 159.47 (C), 164.80 (C);
MS (EI): m/z (%) = 189 (95, [M]+), 174 (100), 158 (16), 144 (23), 131 (22), 115 (31), 105 (23), 91 (22), 77 (29),
63 (17), 51 (20); HRMS: calcd. for C12H15NO: 189.1154, found: 189.1147; elemental analysis: calcd. for
C12H15NO: C: 76.16, H: 7.99, N: 7.40; found: C: 76.25, H: 7.98, N: 7.46.
6-Methoxy-1,5-dimethylisoquinoline (13). A flask filled with 6-methoxy-1,5-dimethyl-3,4-dihydroisoquinoline
(90.3 mg, 0.48 mmol) and palladium on charcoal (10%, 93.6 mg) was evacuated under vigorous stirring
for 15 min, and then filled with argon. Toluene (3.6 mL) and cyclohexene (1.3 mL, 13 mmol) were
added, and the mixture was heated at reflux until full conversion was detected (TLC: Alox N; ethyl
acetate/isohexane, 2:1 + 1 drop of ethanol). The catalyst was removed by filtration (ethyl acetate),
and the crude product was purified by chromatography (Alox N, 5% H2O; petroleum ether/ethyl
acetate, 5:1) to provide 6-methoxy-1,5-dimethylisoquinoline (13, 90 mg, 0.48 mmol, 100%) as a beige
solid. M.p. 99–102 ◦C; UV (MeOH):
1608, 1563, 1542, 1495, 1457, 1401, 1344, 1324, 1267, 1179, 1153, 1116, 1078, 1009, 984, 913, 848, 814, 774,
698, 673, 648, 581, 528 cm–1; 1H-NMR (600 MHz, CDCl3):
= 2.50 (s, 3H), 2.97 (s, 3H), 4.00 (s, 3H), 7.35
(d, J = 9.2 Hz, 1H), 7.63 (d, J = 6.2 Hz, 1H), 8.05 (d, J = 9.2 Hz, 1H), 8.32 (d, J = 6.2 Hz, 1H); 13C-NMR
(150 MHz, CDCl3): = 10.49 (CH3), 22.17 (CH3), 56.39 (CH3), 113.71 (CH), 115.83 (CH), 118.97 (C),
λ = 203, 236, 301 nm; IR (ATR): ν = 3058, 3015, 2965, 2940, 2847,
δ
δ
123.04 (C), 125.66 (CH), 127.47 (C), 136.96 (C), 140.72 (CH), 158.57 (C); MS (EI): m/z (%) = 187 (100,
[M]+), 172 (26), 156 (16), 144 (80), 128 (19), 115 (43), 103 (21), 89 (11), 77 (30), 63 (24), 51 (22); HRMS:
calcd. for C12H13NO: 187.0997, found: 187.0986; elemental analysis: calcd. for C12H13NO: C: 76.98, H:
7.00, N: 7.48; found: C: 76.43, H: 7.04, N: 7.53.
1,5-Dimethylisoquinolin-6-ol. For small amounts: In a microwave tube, a mixture of 6-methoxy-1,5-
dimethylisoquinoline (13, 45 mg, 0.24 mmol) and pyridinium chloride (1 g, 8 mmol) was irradiated
◦
at 155 C (300 W) for 30 min. After cooling to room temperature, the mixture was dissolved in
water and ethyl acetate, and neutralized with a saturated aqueous solution of sodium bicarbonate.
The layers were separated, and the aqueous layer was carefully extracted with ethyl acetate.
The combined organic layers were dried (magnesium sulfate), and the solvent was evaporated to give
1,5-dimethylisoquinolin-6-ol (40 mg, 0.23 mmol, 96%) as a brownish solid.
For larger amounts: Freshly distilled hydrobromic acid (22 mL, 0.19 mol) was carefully added at 0 ◦C
to 6-methoxy-1,5-dimethylisoquinoline (13, 3.01 g, 16.1 mmol). After the addition was completed,

Molecules 2018, 23, 1402
9 of 16
the cooling bath was removed, and the mixture was heated at reflux for five hours. Then, the excess of
hydrobromic acid was removed under vacuo. The brownish raw material was completely dissolved in
water (115 mL, ultrasound), filtered, and neutralized by the dropwise addition of a saturated aqueous
solution of sodium bicarbonate. The resulting solid was carefully washed with water and dried in
vacuo to provide 1,5-dimethylisoquinolin-6-ol (2.49 g, 14.4 mmol, 89%) as a brownish solid. M.p.
248–250 ◦C (sublimation); UV (MeOH):
λ = 234, 279, 301, 328, 382 nm; IR (ATR): ν = 2920, 2850, 2475
(br), 1808 (br), 1617, 1599, 1564, 1479, 1423, 1385, 1356, 1337, 1279, 1202, 1057, 1006, 939, 813, 774, 718,
1
672, 660 cm–1; H-NMR (500 MHz, methanol-d4):
δ = 2.43 (s, 3H), 2.84 (s, 3H), 7.22 (d, J = 9.1 Hz,
1H), 7.64 (d, J = 6.2 Hz, 1H), 7.95 (d, J = 9.1 Hz, 1H), 8.12 (d, J = 6.2 Hz, 1H); 13C-NMR (125 MHz,
methanol-d4):
δ = 10.13 (CH3), 21.33 (CH3), 116.26 (C), 116.72 (CH), 119.91 (CH), 123.50 (C), 126.19
(CH), 138.79 (C), 140.60 (CH), 157.91 (C), 158.92 (C); MS (ESI, +10 V): m/z = 174.0 [M + H]+; HRMS:
calcd. for C11H11NO: 173.0841, found: 173.0851; elemental analysis: calcd. for C11H11NO: C: 76.28, H:
6.40, N: 8.09; found: C: 76.00, H: 6.47, N: 8.21.
1,5-Dimethylisoquinolin-6-yl trifluoromethanesulfonate (14). Pyridine (1.1 mL, 12 mmol) was added
◦
to a suspension of 1,5-dimethylisoquinolin-6-ol (0.60 g, 3.5 mmol) in acetonitrile (66 mL) at 0 C.
Subsequently, trifluoromethanesulfonic anhydride (0.87 mL, 5.2 mmol) was added dropwise, and the
reaction mixture was stirred at this temperature for 20 h. Ethyl acetate and water were added, and the
layers were separated. The aqueous layer was extracted three times with ethyl acetate. The combined
organic layers were washed with water and brine, and then dried (sodium sulfate). The solvent was
evaporated, and the residue was purified by column chromatography (silica gel, pentane/ethyl acetate,
1:1) to provide 1,5-dimethylisoquinolin-6-yl trifluoromethanesulfonate (14, 0.92 g, 3.0 mmol, 87%) as
a beige solid. M.p. 67–67.5 ◦C; UV (MeOH):
2995, 2927, 2856, 1612, 1564, 1522, 1473, 1459, 1414, 1375, 1350, 1245, 1207, 1170, 1132, 1038, 994, 933,
861, 826, 815, 767, 663, 621 cm–1; 1H-NMR (500 MHz, CDCl3):
= 2.68 (s, 3H), 3.00 (s, 3H), 7.48 (d,
J = 9.3 Hz, 1H), 7.71 (d, J = 6.1 Hz, 1H), 8.10 (d, J = 9.3 Hz, 1H), 8.52 (d, J = 6.1 Hz, 1H); 13C-NMR
(125 MHz, CDCl3): = 12.24 (CH3), 22.88 (CH3), 116.23 (CH), 118.77 (q, JC,F = 321 Hz, CF3), 120.75
(CH), 126.42 (CH and C), 126.64 (C), 136.97 (C), 143.40 (CH), 147.91 (C), 159.50 (C); 19F-NMR (282 MHz,
λ = 198, 219, 274, 308, 321 nm; IR (ATR): ν = 3088, 3031,
δ
δ
CDCl3):
δ
=
−
73.58 (s, 3F); MS (EI): m/z (%) = 305 (1, [M]+), 172 (8), 144 (48), 128 (7), 115 (19), 103 (13),
89 (5), 77 (18), 69 (100), 63 (10), 51 (12); MS (ESI, +10 V): m/z = 306.0 [M + H]+; elemental analysis:
calcd. for C12H10F3NO3S: C: 47.21, H: 3.30, N: 4.59, S: 10.50; found: C: 47.09, H: 3.02, N: 4.58, S: 10.45.
1,5-Dimethyl-N-phenylisoquinolin-6-amine (16). Aniline (15, 0.1 mL, 1.2 mmol) was added dropwise to a
solution of 1,5-dimethylisoquinolin-6-yl trifluoromethanesulfonate (14, 0.235 g, 0.770 mmol), palladium(II)
acetate (13 mg, 58 µmol), XPhos (55 mg, 0.12 mmol), and cesium carbonate (0.35 g, 1.1 mmol) in toluene
(20 mL). The mixture was heated at reflux for 48 h. After cooling to room temperature, the reaction
mixture was filtered over a short pad of Hyflo (ethyl acetate), and the solvent was evaporated.
Purification of the residue by column chromatography (silica gel, dichloromethane/ethyl acetate 1:3
+ 1% methanol) provided 1,5-dimethyl-N-phenylisoquinolin-6-amine (16, 0.19 g, 0.77 mmol, 100%)
as a yellow solid. M.p. 175 ◦C (decomp.); UV (MeOH):
= 3207, 3163, 3090, 3012, 2985, 2919, 2860, 1632, 1615, 1594, 1562, 1526, 1492, 1439, 1397, 1380, 1310,
λ = 223, 250, 280, 325, 358 (sh) nm; IR (ATR):
ν
1286, 1174, 1151, 1060, 990, 938, 864, 844, 819, 788, 748, 695, 678 cm–1; H-NMR (500 MHz, CDCl3):
= 2.50 (s, 3H), 2.91 (s, 3H), 5.83 (br s, 1H), 7.01 (t, J = 7.4 Hz, 1H), 7.05 (d, J = 7.7 Hz, 2H), 7.29–7.33 (m,
2H), 7.53 (d, J = 9.1 Hz, 1H), 7.60 (d, J = 6.1 Hz, 1H), 7.92 (d, J = 9.1 Hz, 1H), 8.35 (d, J = 6.1 Hz, 1H);
13C-NMR (125 MHz, CDCl3):
= 12.38 (CH3), 22.66 (CH3), 115.32 (CH), 118.78 (C), 118.88 (2 CH), 119.96
1
δ
δ
(CH), 121.99 (CH), 123.77 (C), 124.80 (CH), 129.63 (2 CH), 136.93 (C), 141.95 (C), 142.27 (CH), 142.91 (C),
158.59 (C); MS (EI): m/z (%) = 248 (100, [M]+), 233 (16), 171 (17); MS (ESI, +10 V): m/z = 249.1 [M + H]+;
HRMS (ESI): calcd. for C17H16N2: 248.1313, found: 248.1310.
N-(2-Chlorophenyl)-1,5-dimethylisoquinolin-6-amine (18a). 2-Chloroaniline (17a, 78
added dropwise to a solution of 1,5-dimethylisoquinolin-6-yl trifluoromethanesulfonate (14, 0.15 g,
0.49 mmol), palladium(II) acetate (8.3 mg, 37 mol), XPhos (35 mg, 74 mol), and cesium carbonate
µL, 0.74 mmol) was
µ
µ

Molecules 2018, 23, 1402
10 of 16
(224 mg, 0.688 mmol) in toluene (12 mL). The mixture was heated at reflux for 1.5 h. After cooling to room
temperature, the reaction mixture was filtered over a short pad of Hyflo (ethyl acetate), and the solvent was
evaporated. Purification of the residue by column chromatography (silica gel, dichloromethane/ethyl
acetate, 9:1 to 0:1, each + 1% ethanol) provided N-(2-chlorophenyl)-1,5-dimethylisoquinolin-6-amine
(
18a, 0.130 g, 0.460 mmol, 94%) as brownish crystals. M.p. 194–198 ◦C; UV (MeOH):
320 nm; fluorescence (MeOH): λex = 221, λem = 229 (sh), 334 nm; IR (ATR): = 3189, 3078, 2955, 2919,
2850, 1589, 1567, 1542, 1501, 1474, 1452, 1396, 1367, 1307, 1294, 1267, 1225, 1198, 1129, 1058, 1033, 996,
933, 862, 843, 822, 793, 751, 706 cm–1; 1H-NMR (500 MHz, CDCl3):
= 2.35 (s, 3H), 2.93 (s, 3H), 6.15 (br
λ = 221, 249, 278,
ν
δ
s, 1H), 6.85 (td, J = 7.7, 3.0 Hz, 1H), 6.95 (dd, J = 8.2, 1.4 Hz, 1H), 7.10–7.14 (m, 1H), 7.40 (dd, J = 8.0,
1.4 Hz, 1H), 7.51 (d, J = 9.0 Hz, 1H), 7.63 (d, J = 6.1 Hz, 1H), 7.96 (d, J = 9.0 Hz, 1H), 8.39 (d, J = 6.1 Hz,
1H); 13C-NMR (125 MHz, CDCl3):
δ = 12.58 (CH3), 22.58 (CH3), 115.47 (CH), 116.05 (CH), 120.90 (CH),
121.87 (C), 122.07 (CH), 122.83 (C), 124.52 (C), 124.73 (CH), 127.51 (CH), 129.80 (CH), 136.78 (C), 140.07
(C), 140.19 (C), 142.35 (CH), 158.61 (C); MS (EI): m/z (%) = 282 (100, [M]+), 247 (74), 232 (29), 204 (15),
171 (12), 115 (10), 75 (11); MS (ESI, +25 V): m/z = 283.2 [M + H]+.
Crystal data: C17H15ClN2, crystal size 0.22
space group: Cc, a = 11.700(2), b = 9.117(2), c = 14.024(3) Å,
ρcalcd. = 1.342 g cm−3 = 0.264 mm−1, T = 198(2) K,
= 0.71073 Å, θ
×
0.20
×
0.06 mm3, M = 282.76 g mol−1, monoclinic,
= 110.73(3)◦, V = 1399.1(5) Å3, Z = 4,
range: 3.11–27.00◦, 20817
β
,
µ
λ
reflections collected, 3047 independent (Rint = 0.0534), 187 parameters. The structure was solved by
direct methods and refined by the full-matrix least-squares method on F2; 2296 reflections observed,
R1 = 0.0407, wR2 = 0.0805 [I > 2 σ(I)]; maximal residual electron density: 0.276 e Å−3. CCDC 1838728.
N-(2-Chloro-5-methoxyphenyl)-1,5-dimethylisoquinolin-6-amine (18b). 2-Chloro-5-methoxyaniline (17b, 92
0.74 mmol) was added dropwise to a solution of 1,5-dimethylisoquinolin-6-yl trifluoromethanesulfonate
14, 0.15 g, 0.49 mmol), palladium(II) acetate (8.3 mg, 37 mol), XPhos (35 mg, 74 mol), and cesium
µL,
(
µ
µ
carbonate (224 mg, 0.688 mmol) in toluene (12 mL). The mixture was heated at reflux for five
hours. After cooling to room temperature, the reaction mixture was filtered over a short pad
of Hyflo (ethyl acetate), and the solvent was evaporated. Purification of the residue by column
chromatography (silica gel, dichloromethane/ethyl acetate, 1:1 to 0:1, each + 1% ethanol) provided
N-(2-chloro-5-methoxyphenyl)-1,5-dimethylisoquinolin-6-amine (18b, 0.141 g, 0.451 mmol, 92%) as
a beige solid. M.p. 135–138 ◦C; UV (MeOH):
λem = 301 (sh), 336 nm; IR (ATR): = 3416, 3068, 2998, 2929, 2853, 1596, 1508, 1447, 1421, 1383, 1343,
1312, 1287, 1230, 1207, 1170, 1138, 1069, 1027, 924, 820, 732, 671, 640 cm–1; 1H-NMR (500 MHz, CDCl3):
= 2.53 (s, 3H), 2.94 (s, 3H), 3.68 (s, 3H), 6.13 (br s, 1H), 6.40 (dd, J = 8.8, 2.8 Hz, 1H), 6.47 (d, J = 2.8 Hz,
1H), 7.28 (d, J = 8.8 Hz, 1H), 7.53 (d, J = 9.0 Hz, 1H), 7.63 (d, J = 6.1 Hz, 1H), 7.97 (d, J = 9.0 Hz,
1H), 8.39 (d, J = 6.1 Hz, 1H); 13C-NMR (125 MHz, CDCl3):
= 12.66 (CH3), 22.61 (CH3), 55.46 (CH3),
λ = 224, 277, 322 nm; fluorescence (MeOH): λex = 224,
ν
δ
δ
101.82 (CH), 105.94 (CH), 113.40 (C), 115.53 (CH), 122.55 (CH), 123.53 (C), 124.67 (C), 124.76 (CH), 130.02
(CH), 136.78 (C), 139.77 (C), 141.06 (CH), 142.38 (C), 158.64 (C), 159.20 (C); MS (EI): m/z (%) = 312
(100, [M]+), 277 (80), 262 (76), 247 (13), 233 (18), 219 (12), 139 (10), 117 (16), 63 (10); MS (ESI, +10 V):
m/z = 313.3 [M + H]+.
N-(2-Chloro-4-methoxyphenyl)-1,5-dimethylisoquinolin-6-amine (18c). A solution of 2-chloro-4-methoxyaniline
(
17c, 127 mg, 0.806 mmol) in toluene (4 mL) was added dropwise to a solution of 1,5-dimethylisoquinolin-6-yl
trifluoromethanesulfonate (14, 164 mg, 0.537 mmol), palladium(II) acetate (9 mg, 0.04 mmol), XPhos (38 mg,
81 mol), and cesium carbonate (245 mg, 0.752 mmol) in toluene (10 mL). The mixture was heated at
µ
reflux for one hour. After cooling to room temperature, the reaction mixture was filtered over a short
pad of Hyflo (ethyl acetate), and the solvent was evaporated. Purification of the residue by column
chromatography (silica gel, dichloromethane/ethyl acetate, 9:1 to 0:1, each + 1% ethanol) provided
N-(2-chloro-4-methoxyphenyl)-1,5-dimethylisoquinolin-6-amine (18c, 139 mg, 0.444 mmol, 83%) as
a beige solid. M.p. 104–107 ◦C; UV (MeOH):
λem = 422 nm; IR (ATR): = 3229, 3074, 2993, 2948, 2832, 1731, 1633, 1606, 1562, 1485, 1451, 1436, 1387,
1341, 1308, 1283, 1211, 1182, 1112, 1046, 936, 894, 864, 822, 789, 773, 689, 664 cm–1; 1H-NMR (500 MHz,
λ = 226, 255, 318 nm; fluorescence (MeOH): λex = 255,
ν

Molecules 2018, 23, 1402
11 of 16
CDCl3):
(d, J = 2.8 Hz, 1H), 7.09 (d, J = 8.9 Hz, 1H), 7.29 (d, J = 9.1 Hz, 1H), 7.62 (d, J = 6.2 Hz, 1H), 7.90 (d,
J = 9.1 Hz, 1H), 8.31 (d, J = 6.2 Hz, 1H); 13C-NMR (125 MHz, CDCl3):
= 12.01 (CH3), 21.92 (CH3),
δ = 2.51 (s, 3H), 2.93 (s, 3H), 3.80 (s, 3H), 5.87 (br s, 1H), 6.79 (dd, J = 8.7, 2.8 Hz, 1H), 7.02
δ
55.84 (CH3), 113.76 (CH), 115.29 (CH), 115.33 (CH), 117.69 (C), 118.89 (CH), 122.01 (CH), 123.18 (C),
124.98 (CH), 126.33 (C), 132.26 (C), 136.87 (C), 140.79 (CH, HSQC), 142.90 (C, HMBC), 155.61 (C), 158.06
(C); MS (EI): m/z (%) = 312 (100, [M]+), 297 (44), 277 (12), 262 (14), 233 (17), 169 (12), 155 (11), 128 (14),
116 (15); MS (ESI, +10 V): m/z = 313.2 [M + H]+; elemental analysis: calcd. for C18H17ClN2O: C: 69.12,
H: 5.48, N: 8.96; found: C: 68.62, H: 5.72, N: 9.30.
Olivacine (1). N-(2-Chlorophenyl)-1,5-dimethylisoquinolin-6-amine (18a, 20 mg, 71
µmol), palladium(II)
acetate (4.8 mg, 21
µmol), tri-tert-butylphosphonium tetrafluoroborate (8.1 mg, 42
µmol),
and potassium carbonate (39.1 mg, 0.283 mmol) were dissolved in DMF (0.5 mL). The reaction mixture
◦
was placed in a preheated oil bath at 140 C and stirred for 30 min. After filtration over a short
pad of Celite (CH2Cl2), the halogenated solvent was evaporated, and the residue was dissolved
in ethyl acetate, and washed three times with water, and then with brine. The aqueous layer
was extracted with ethyl acetate, and the combined organic layers were dried (sodium sulfate).
The solvent was evaporated, and the residue was purified by column chromatography (silica gel,
dichloromethane/ethyl acetate, 9:1 to 0:1, each + 5% ethanol) to provide olivacine (
mol, 71%) as brown crystals. M.p. 320–324 ◦C; UV (MeOH):
= 223, 237, 275, 285, 292, 327, 342, 374,
391 nm; fluorescence (MeOH): λex = 285, λem = 431 nm; IR (ATR): = 3058, 2965, 2909, 2765, 1674,
1597, 1479, 1467, 1407, 1334, 1311, 1280, 1252, 1222, 1196, 1150, 1108, 1064, 942, 862, 813, 765, 739, 695,
640 cm–1; 1H-NMR (500 MHz, methanol-d4):
= 2.85 (s, 3H), 3.07 (t, JHD = 1.1 Hz) and 3.09 (s, 3H),
7.24–7.27 (m, 1H), 7.49–7.54 (m, 2H), 7.89 (d, J = 6.3 Hz, 1H), 8.18 (d, J = 6.3 Hz, 1H), 8.27–8.29 (m,
1H), 8.87 (s, 1H); 13C-NMR (125 MHz, methanol-d4):
= 12.42 (CH3), 22.35 (CH3), 111.86 (CH), 112.42
1, 12.4 mg, 50.3
µ
λ
ν
δ
δ
(C), 116.05 (CH), 116.64 (CH), 120.58 (CH), 122.14 (CH), 123.57 (C), 124.41 (C), 127.25 (C), 128.93 (CH),
134.41 (C), 138.84 (CH), 142.80 (C), 144.34 (C), 160.29 (C); MS (EI): m/z (%) = 246 (100, [M]+), 229 (7),
217 (7), 204 (9), 123 (7); MS (ESI, +10 V): m/z = 247.1 [M + H]+; HRMS (ESI): calcd. for C17H14N2:
246.1157, found: 246.11537; elemental analysis: calcd. for C17H14N2: C: 82.90, H: 5.73, N: 11.37; found:
C: 83.20, H: 5.81, N: 11.42.
Crystal data: C17H14N2
orthorhombic, space group: Pbca, a = 4.860(1), b = 21.337(5), c = 28.048(6) Å, V = 2908.5(11) Å3,
Z = 8, ρcalcd. = 1.271 g cm−3 = 0.080 mm−1, T = 198(2) K, range: 3.48–25.40◦, 60682
= 0.71073 Å,
·
CH3OH, crystal size 0.45
×
0.12
×
0.07 mm3, M = 278.34 g mol−1
,
,
µ
λ
θ
reflections collected, 2656 independent (Rint = 0.0501), 198 parameters. The structure was solved by
direct methods and refined by the full-matrix least-squares method on F2; 1934 reflections observed,
R1 = 0.0463, wR2 = 0.1044 [I > 2 σ(I)]; maximal residual electron density: 0.204 e Å−3. CCDC 1838729.
4,11b-Dimethyl-11bH-pyrido[3,4-c]carbazole (20a, 2.1 mg, 8.5
product. UV (MeOH): = 250, 282 (sh), 359 nm; fluorescence (MeOH): λex = 250, λem = 417 nm;
IR (ATR):
= 3348, 2924, 2853, 2487, 1630, 1594, 1545, 1446, 1200, 1116, 950, 811, 772, 749, 679 cm–1;
1H-NMR (600 MHz, methanol-d4):
= 1.65 (s, 3H), 2.76 (s, 3H), 7.01 (d, J = 10.0 Hz, 1H), 7.49 (t, J = 7.4 Hz,
1H), 7.55 (t, J = 7.5 Hz, 1H), 7.63 (d, J = 10.0 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.84 (d, J = 5.1 Hz, 1H),
8.09 (d, J = 7.3 Hz, 1H), 8.40 (d, J = 5.1 Hz, 1H); 13C-NMR (150 MHz, methanol-d4):
= 21.70 (CH3),
µmol, 12%), dark brown oil, less polar side
λ
ν
δ
δ
33.28 (CH3), 59.3 (C, HMBC), 119.63 (CH), 122.46 (CH), 123.20 (CH), 125.25 (CH), 127.3 (C, HMBC),
127.89 (CH), 129.93 (CH), 136.11 (CH), 140.89 (C), 149.15 (CH), 153.38 (C), 155.0 (C, HMBC), 158.0
(C, HMBC), 184.5 (C, HMBC); MS (EI): m/z (%) = 246 (100, [M]+), 231 (24), 204 (12), 176 (7); MS (ESI,
+50 V): m/z = 247.1 [M + H]+, 493.5 [2M + H]+.
8-Methoxyolivacine (19b). N-(2-Chloro-5-methoxyphenyl)-1,5-dimethylisoquinolin-6-amine (18b, 14 mg,
45
27
µmol), palladium(II) acetate (3.0 mg, 13 µmol), tri-tert-butylphosphonium tetrafluoroborate (5.1 mg,
µmol) and potassium carbonate (24.7 mg, 0.179 mmol) were dissolved in DMF (0.5 mL). The reaction
◦
mixture was placed in a preheated oil bath at 140 C and stirred for 20 min. After filtration over a short

Molecules 2018, 23, 1402
12 of 16
pad of Celite (CH2Cl2), the halogenated solvent was evaporated, and the residue was dissolved in
ethyl acetate, washed three times with water, and then with brine. The aqueous layer was extracted
with ethyl acetate, and the combined organic layers were dried (sodium sulfate). The solvent was
evaporated and the residue was purified by column chromatography (silica gel, dichloromethane/ethyl
acetate, 9:1 to 0:1, each + 5% eth◦anol) to provide 8-methoxyolivacine (19b, 8.0 mg, 29
µmol, 65%) as
λ = 227, 271, 281, 300, 316, 351 nm; fluorescence
yellow crystals. M.p. 280–283 C; UV (MeOH):
(MeOH): λex = 300, λem = 430, 515 nm; IR (ATR):
1493, 1472, 1460, 1412, 1388, 1335, 1315, 1297, 1267, 1216, 1197, 1160, 1137, 1099, 1068, 1030, 996, 942,
916, 870, 810, 753 cm–1; 1H-NMR (500 MHz, DMSO-d6):
= 2.79 (s, 3H), 3.01 (s, 3H), 3.89 (s, 3H), 6.85
(dd, J = 8.6, 2.2 Hz, 1H), 7.00 (d, J = 2.2 Hz, 1 H), 7.78 (d, J = 6.1 Hz, 1H), 8.23 (d, J = 6.1 Hz, 1H), 8.24
(d, J = 8.6 Hz, 1 H), 8.77 (s, 1H), 11.26 (s, 1H); 13C-NMR (125 MHz, DMSO-d6):
= 12.36 (CH3), 22.97
ν = 3141, 3046, 2993, 2886, 2821, 2713, 1622, 1595, 1563,
δ
δ
(CH3), 55.32 (CH3), 94.84 (CH), 107.55 (CH), 110.68 (C), 113.55 (CH), 114.78 (CH), 116.19 (C), 122.00 (C),
122.28 (CH), 125.00 (C), 131.74 (C), 139.10 (CH), 140.79 (C), 144.13 (C), 158.26 (C), 159.96 (C); MS (EI):
m/z (%) = 276 (100, [M]+), 261 (14), 233 (49), 138 (8), 116 (10); MS (ESI, +10 V): m/z = 277.1 [M + H]+;
HRMS (ESI): calcd. for C18H16N2O: 276.1263, found: 276.1261.
Crystal data: C18H16N2O
orthorhombic, space group: Pbca, a = 4.9253(3), b = 21.4925(15), c = 29.523(2) Å, V = 3125.3(4) Å3, Z = 8,
ρcalcd. = 1.311 g cm−3 = 0.685 mm−1, T = 150(2) K, range: 2.993–68.188◦, 31382
= 1.54178 Å,
·
CH3OH, crystal size 0.160
×
0.080
×
0.060 mm3, M = 308.37 g mol−1
,
,
µ
λ
θ
reflections collected, 2811 independent (Rint = 0.0544), 230 parameters. The structure was solved by
direct methods and refined by the full-matrix least-squares method on F2; 2283 reflections observed,
R1 = 0.0387, wR2 = 0.1001 [I > 2 σ(I)]; maximal residual electron density: 0.221 e Å−3. CCDC 1838730.
9-Methoxy-4,11b-dimethyl-11bH-pyrido[3,4-c]carbazole (20b, 1.1 mg, 4.0
side product. UV (MeOH): = 221, 300, 325 nm; fluorescence (MeOH): λex = 221, λem = 296, 339 nm;
IR (ATR): = 3414, 3058, 2924, 2855, 1734, 1655, 1632, 1593, 1535, 1484, 1459, 1437, 1377, 1334, 1276,
1231, 1182, 1149, 1129, 1074, 935, 826, 740, 683 cm–1; 1H-NMR (600 MHz, methanol-d4):
= 1.62 (s, 3H),
µmol, 9%), brown oil, less polar
λ
ν
δ
2.74 (s, 3H), 3.94 (s, 3H), 6.98 (d, J = 10.0 Hz, 1H), 7.04 (dd, J = 8.2, 1.7 Hz, 1H), 7.26 (s, 1H), 7.61
(d, J = 10.0 Hz, 1H), 7.78 (d, J = 4.9 Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H), 8.38 (d, J = 4.9 Hz, 1H); 13C-NMR
(150 MHz, methanol-d4):
δ = 21.67 (CH3), 33.43 (CH3), 56.12 (CH3), 58.80 (C), 108.13 (CH), 113.67 (CH),
119.73 (CH), 123.16 (CH), 125.46 (CH), 127.23 (C), 132.78 (C, HMBC), 136.16 (CH), 149.15 (CH), 153.81
(C), 156.57 (C, HMBC), 158.04 (C), 162.22 (C), 186.18 (C); MS (EI): m/z (%) = 276 (85, [M]+), 261 (100),
233 (25), 218 (52), 190 (16); MS (ESI, +50 V): m/z = 277.2 [M + H]+.
9-Methoxyolivacine (19c). N-(2-Chloro-4-methoxyphenyl)-1,5-dimethylisoquinolin-6-amine (18c, 55.0 mg,
176
106
µmol), palladium(II) acetate (11.8 mg, 53 µmol), tri-tert-butylphosphonium tetrafluoroborate (20.1 mg,
µmol), and potassium carbonate (97.2 mg, 0.703 mmol) were dissolved in DMF (1.4 mL).
◦
The reaction mixture was placed in a preheated oil bath at 140 C and stirred for 35 min. After filtration
over a short pad of Celite (CH2Cl2), the halogenated solvent was evaporated, and the residue was
dissolved in ethyl acetate, and then washed three times with water, and then with brine. The aqueous
layer was extracted with ethyl acetate, and the combined organic layers were dried (sodium sulfate).
The solvent was evaporated, and the residue was purified by column chromatography (silica gel,
dichloromethane/ethyl acetate, 9:1 to 0:1, each + 5% ethanol) to provide 9-methoxyolivacine (19c
30.1 mg, 109 = 224, 242, 272, 296, 332,
mol, 62%) as a yellow solid. M.p. 273–274 ◦C; UV (MeOH):
394 nm; fluorescence (MeOH): λex = 296, λem = 471 nm; IR (ATR): = 3143, 2914, 1632, 1600, 1485, 1436,
1405, 1380, 1330, 1306, 1265, 1206, 1175, 1104, 1030, 935, 879, 862, 838, 809, 767, 735, 698 cm–1; 1H-NMR
(500 MHz, DMSO-d6): = 2.80 (s, 3H), 3.04 (s, 3H), 3.90 (s, 3H), 7.14 (dd, J = 10.0, 2.5 Hz, 1H), 7.44 (d,
J = 8.7 Hz, 1H), 7.79 (d, J = 6.1 Hz, 1H), 8.01 (d, J = 2.5 Hz, 1H), 8.24 (d, J = 6.1 Hz, 1H), 8.96 (s, 1H),
11.16 (s, 1H); 13C-NMR (125 MHz, DMSO-d6):
= 12.80 (CH3), 23.45 (CH3), 56.11 (CH3), 104.87 (CH),
,
µ
λ
ν
δ
δ
111.36 (C), 112.00 (CH), 115.18 (CH), 115.71 (CH), 117.04 (CH), 122.00 (C), 123.65 (C), 125.36 (C), 132.64
(C), 137.53 (C), 139.69 (CH), 141.61 (C), 153.78 (C), 159.21 (C); MS (EI): m/z (%) = 276 (100, [M]+), 261

Molecules 2018, 23, 1402
13 of 16
(90), 233 (27), 116 (10); MS (ESI, +10 V): m/z = 277.1 [M + H]+; HRMS (ESI): calcd. for C18H16N2O:
276.1263, found: 276.1269.
10-Methoxy-4,11b-dimethyl-11bH-pyrido[3,4-c]carbazole (20c, 1.4 mg, 5.0
µmol, 3%), brown oil, less polar
side product. UV (MeOH): = 260, 291 (sh), 381 nm; fluorescence (MeOH): λex = 260, λem = 349 (sh),
λ
434 nm; IR (ATR):
ν
= 3389, 2924, 2854, 1733, 1655, 1624, 1590, 1536, 1466, 1434, 1380, 1335, 1295,
1275, 1240, 1218, 1165, 1065, 1030, 952, 865, 822, 744, 677 cm–1; H-NMR (600 MHz, methanol-d4):
1
δ
= 1.63 (s, 3H), 2.74 (s, 3H), 4.00 (s, 3H), 6.95 (d, J = 10.0 Hz, 1H), 7.10 (dd, J = 8.5, 2.1 Hz, 1H), 7.55
(d, J = 10.0 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 2.1 Hz, 1H), 7.83 (d, J = 5.1 Hz, 1H), 8.38 (d,
J = 5.1 Hz, 1H); 13C-NMR (150 MHz, methanol-d4):
= 21.69 (CH3), 33.33 (CH3), 56.44 (CH3), 59.25 (C),
δ
112.13 (CH), 114.60 (CH), 119.52 (CH), 122.93 (CH), 123.26 (CH), 127.5 (C, HMBC), 134.87 (CH), 142.7
(C, HMBC), 147.3 (C, HMBC), 148.89 (CH), 153.2 (C, HMBC), 157.8 (C, HMBC), 160.73 (C), 182.4 (C,
HMBC); MS (EI): m/z (%) = 276 (100, [M]+), 261 (42), 246 (24), 233 (46), 218 (31), 190 (13); MS (ESI, +50
V): m/z = 277.2 [M + H]+.
8-Hydroxyolivacine (4). 8-Methoxyolivacine (19b, 17.0 mg, 61.5 µmol) was dissolved in 48% aqueous HBr
(1.1 mL), and the mixture was heated at reflux for 24 h. After cooling to room temperature, the mixture
was carefully neutralized using a 25% aqueous solution of ammonia. The mixture was extracted with
ethyl acetate until the aqueous layer was completely colorless. Evaporation of the organic solvent led to
a yellow solid, which was purified by chromatography (Alox N, 5% H2O, CH2Cl2/methanol, 1:1) to
provide 8-hydroxyolivacine (
by preparative HPLC provided very pure
(MeOH): = 239, 301, 317 nm; fluorescence (MeOH): λex = 301, λem = 434, 520 nm; IR (ATR):
3279, 3198, 2827, 1660, 1619, 1474, 1433, 1407, 1341, 1190, 1166, 1138, 1102, 840, 800, 722, 633 cm–1;
1H-NMR (500 MHz, methanol-d4):
= 2.94 (s, 3H), 3.34 (s, 3H), 6.90 (dd, J = 8.5, 2.1 Hz, 1H), 7.01
(d, J = 2.1 Hz, 1H), 8.18 (d, J = 7.0 Hz, 1H), 8.20 (d, J = 8.5 Hz, 1H), 8.37 (d, J = 7.0 Hz, 1H), 9.00 (s, 1H);
13C-NMR (125 MHz, methanol-d4):
= 12.41 (CH3), 18.63 (CH3), 98.31 (CH), 111.38 (CH), 113.55 (C),
4
, 13.5 mg, 51.5
µ
mol, 84%) as a yellow solid. An additional purification
(8.5 mg, 32
mol) for biological testing. M.p. 239 ◦C; UV
= 3505,
4
µ
λ
ν
δ
δ
115.93 (C), 116.93 (CH), 119.58 (CH), 121.73 (C), 123.95 (CH), 127.12 (CH), 130.23 (C), 134.44 (C), 146.42
(2C), 157.30 (C), 161.11 (C); MS (EI): m/z (%) = 262 (100, [M]+), 180 (10); MS (ESI, +10 V): m/z = 263.1
[M + H]+, 547 [2M + Na]+; HRMS (ESI): calcd. for C17H14N2O: 262.1106, found: 262.1104.
9-Hydroxyolivacine (5). 9-Methoxyolivacine (19c, 38.0 mg, 138 µmol) was dissolved in 48% aqueous
HBr (2.3 mL), and the mixture was heated at reflux for 24 h. After cooling to room temperature,
the mixture was carefully neutralized using a 25% aqueous solution of ammonia. The mixture was
extracted with ethyl acetate until the aqueous layer was colorless. The combined organic layers were
washed with water and brine, and then dried (sodium sulfate), and the solvent was evaporated.
The residue was purified by column chromatography (silica gel, CH2Cl2/THF, 4:1 to 2:3) to provide
9-hydroxyolivacine (
preparative HPLC provided very pure
(MeOH): = 245, 274, 311, 355, 375 nm; fluorescence (MeOH): λex = 311, λem = 482 nm; IR (ATR):
= 3220, 2921, 2853, 1734, 1666, 1611, 1425, 1328, 1288, 1185, 1127, 975, 840, 799, 721 cm–1; 1H-NMR
(500 MHz, methanol-d4): = 2.96 (s, 3H), 3.36 (s, 3H, HSQC), 7.22 (dd, J = 8.6, 2.3 Hz, 1H), 7.51
(d, J = 8.6 Hz, 1H), 7.82 (d, J = 2.3 Hz, 1H), 8.19 (d, J = 7.1 Hz, 1H), 8.38 (d, J = 7.1 Hz, 1H), 9.17 (s, 1H);
13C-NMR (125 MHz, methanol-d4):
= 12.42 (CH3), 18.67 (CH3), 107.97 (CH), 113.13 (CH), 113.96
5, 25.2 mg, 96.1
µ
mol, 70%) as a yellow solid. An additional purification by
5
(6.1 mg, 23
µ
mol) for biological testing. M.p. 249 ◦C; UV
λ
ν
δ
δ
(C), 119.43 (CH), 119.48 (CH), 119.54 (CH), 121.00 (C), 124.35 (C), 127.40 (CH), 129.67 (C), 134.68 (C),
138.24 (C), 146.56 (C), 153.52 (C), 158.18 (C); MS (EI): m/z (%) = 262 (100, [M]+), 131 (12); MS (ESI,
+10 V): m/z = 263.1 [M + H]+; HRMS (ESI): calcd. for C17H14N2O: 262.1106, found: 262.1107.
4. Conclusions
In conclusion, we have developed a straightforward synthesis of olivacine (
oxygenated pyrido[4,3-b]carbazole derivatives via Buchwald–Hartwig coupling of the isoquinolinyl
triflate 14 and the ortho-chloroarylamine 18a followed by a Heck-type cyclization. In a test for
1) and four of its
–
c

Molecules 2018, 23, 1402
14 of 16
the inhibition of the growth of M. tuberculosis (strain H37Rv), 9-methoxyolivacine (19c) proved to
be the most active compound, with an MIC90 value of 1.5 M and a relatively low toxicity for
µ
a mammalian cell line. These initial results indicate that the pyrido[4,3-b]carbazoles are a promising
class of compounds for our ongoing search for a carbazole-based tuberculosis drug candidate.
Supplementary Materials: The following data are available online. Copies of the 1H-NMR, 13C-NMR and 2D
NMR spectra.
Author Contributions: U.S. and H.-J.K. conceived and designed the experiments; U.S. and G.T. performed the
chemical synthesis and characterized the compounds; A.J. and O.K. performed the X-ray analyses; B.W. and S.G.F.
designed and performed the study for inhibition of M. tuberculosis; U.S. and H.-J.K. wrote the paper.
Funding: This research received no external funding.
Acknowledgments: We are grateful to Thomas Hopfmann and Erik Troschke for their experimental contributions.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Schmutz, J.; Hunziker, F. Die Alkaloide von Aspidosperma olivaceum M. Arg. Aspidosperma-Alkaloide, 3.
Mitteilung. Pharm. Acta Helv. 1958, 33, 341–347.
Wittwer, H.; Schmutz, J. Die Synthese von Olivacin, Dihydro-olivacin, Tetrahydro-olivacin, N-Methyl-
tetrahydro-olivacin, und die Konstitution von u-Alkaloid D. Helv. Chim. Acta 1960, 43, 793–799.
Maftouh, M.; Besselievre, R.; Monsarrat, B.; Lesca, P.; Meunier, B.; Husson, H.P.; Paoletti, C. Synthesis
and cytotoxic activity of hydroxylated derivatives of olivacine in relation with their biotransformation.
J. Med. Chem. 1985, 28, 708–714. [CrossRef] [PubMed]
4.
5.
Stiborová, M.; Sejbal, J.; Borˇek-Dohalská, L.; Aimová, D.; Poljaková, J.; Forsterová, K.; Rupertová, M.;
Wiesner, J.; Hudecˇek, J.; Wiessler, M.; et al. The anticancer drug ellipticine forms covalent DNA adducts,
mediated by human cytochromes P450, through metabolism to 13-hydroxyellipticine and ellipticine N2-oxide.
Cancer Res. 2004, 64, 8374–8380. [CrossRef] [PubMed]
Rocha e Silva, L.F.; Montoia, A.; Amorim, R.C.N.; Melo, M.R.; Henrique, M.C.; Nunomura, S.M.; Costa, M.R.F.;
Andrade Neto, V.F.; Costa, D.S.; Dantas, G.; et al. Comparative in vitro and in vivo antimalarial activity of
the indole alkaloids ellipticine, olivacine, cryptolepine and a synthetic cryptolepine analog. Phytomedicine
2012, 20, 71–76. [CrossRef] [PubMed]
6.
7.
Deane, F.M.; O'Sullivan, E.C.; Maguire, A.R.; Gilbert, J.; Sakoff, J.A.; McCluskey, A.; McCarthy, F.O. Synthesis
and evaluation of novel ellipticines as potential anti-cancer agents. Org. Biomol. Chem. 2013, 11, 1334–1344.
[CrossRef] [PubMed]
Montoia, A.; Rocha e Silva, L.F.; Torres, Z.E.; Costa, D.S.; Henrique, M.C.; Lima, E.S.; Vasconcellos, M.C.;
Souza, R.C.Z.; Costa, M.R.F.; Grafov, A.; et al. Antiplasmodial activity of synthetic ellipticine derivatives and
an isolated analog. Bioorg. Med. Chem. Lett. 2014, 24, 2631–2634. [CrossRef] [PubMed]
Miller, C.M.; McCarthy, F.O. Isolation, biological activity and synthesis of the natural product ellipticine and
related pyridocarbazoles. RSC Adv. 2012, 2, 8883–8918. [CrossRef]
8.
9.
Rouëssé, J.; Spielmann, M.; Turpin, F.; Le Chevalier, T.; Azab, M.; Mondésir, J.M. Phase II study of elliptinium
acetate salvage treatment of advanced breast cancer. Eur. J. Cancer 1993, 29, 856–859. [CrossRef]
10. Gribble, G.W. Approaches to the synthesis of the antitumor pyridocarbazole alkaloids. Synlett 1991, 289–300.
[CrossRef]
11. Schmidt, A.W.; Reddy, K.R.; Knölker, H.-J. Occurrence, biogenesis, and synthesis of biologically active
carbazole alkaloids. Chem. Rev. 2012, 112, 3193–3328. [CrossRef] [PubMed]
12. Bergman, J.; Carlsson, R. Conversion of diindolyl methanes to 3-vinylindoles. A simple synthesis of the
indole alkaloid olivacine. Tetrahedron Lett. 1978, 4055–4058. [CrossRef]
13. Besselièvre, R.; Husson, H.-P. Syntheses in the ellipticine-olivacine series, a possible biogenetic model.
Tetrahedron 1981, 37 (Suppl. 1), 241–246. [CrossRef]
14. Kutney, J.P.; Noda, M.; Lewis, N.G.; Monteiro, B.; Mostowicz, D.; Worth, B.R. Dihydropyridines in synthesis
and biosynthesis. V. Synthesis of pyridocarbazole alkaloids: olivacine and ( )-guatambuine. Can. J. Chem.
±
1982, 60, 2426–2430. [CrossRef]

Molecules 2018, 23, 1402
15 of 16
15. Miki, Y.; Tsuzaki, Y.; Hibino, H.; Aoki, Y. Synthesis of 3-methoxyolivacine and olivacine by Friedel-Crafts
reaction of indole-2,3-dicarboxylic anhydride with 2,4,6-trimethoxypyridine. Synlett 2004, 12, 2206–2208.
[CrossRef]
16. Bennasar, M.L.; Roca, T.; Ferrando, F. Regioselective 6-endo cyclizations of 2-indolylacyl radicals: total
synthesis of the pyrido[4,3-b]carbazole alkaloid guatambuine. J. Org. Chem. 2006, 71, 1746–1749. [CrossRef]
[PubMed]
17. Ramkumar, N.; Nagarajan, R. Total synthesis of ellipticine quinones, olivacine, and calothrixin B. J. Org. Chem.
2013, 79, 736–741. [CrossRef] [PubMed]
18. Itoh, T.; Abe, T.; Choshi, T.; Nishiyama, T.; Yanada, R.; Ishikura, M. Concise total syntheses of
pyrido[4,3-b]carbazole alkaloids using copper-mediated 6π-electrocyclization. Eur. J. Org. Chem. 2016, 13,
2290–2299. [CrossRef]
19. Pierré, A.; Atassi, G.; Devissaguet, M.; Bisagni, E. Novel olivacine and ellipticine derivatives: S-16020-2 and
related compounds as potential antitumor agents. Drugs Future 1997, 22, 53–59. [CrossRef]
20. Jasztold-Howorko, R.; Croisy, A.; Carrez, D. An alternative way of the synthesis of 1-substituted
9-methoxy-5-methyl-6H-pyrido[4,3-b]carbazole derivatives. Acta Pol. Pharm. 2005, 62, 207–212. [PubMed]
21. Brütting, C.; Schmidt, A.W.; Kataeva, O.; Knölker, H.-J. First total synthesis of 7-isovaleryloxy-8-
methoxygirinimbine. Synthesis 2018, 50. [CrossRef]
22. Comins, D.L.; Brown, J.D. Ortho substitution of m-anisaldehyde via
J. Org. Chem. 1989, 54, 3730–3732. [CrossRef]
α-amino alkoxide directed lithiation.
23. Bur, D.; Grisostomi, C.; Kimmerlin, T.; Remen, L.; Siendt, H.; Vercauteren, M.; Welford, R. Tricyclic piperidine
compounds. Patent WO2016177690A1, 10 November 2016.
24. Becknell, N.C.; Dandu, R.R.; Dorsey, B.D.; Gotchev, D.B.; Hudkins, R.L.; Weinberg, L.; Zificsak, C.A.;
Zulli, A.L. 1,4-Substituted piperidine derivatives. Patent WO2016205633A1, 22 December 2016.
25. Ruiz-Castillo, P.; Buchwald, S.L. Applications of palladium-catalyzed C–N cross-coupling reactions.
Chem. Rev. 2016, 116, 12564–12649. [CrossRef] [PubMed]
26. Miller, R.B.; Moock, T. A general synthesis of 6-H-pyrido[4,3-b]carbazole alkaloids. Tetrahedron Lett. 1980, 21,
3319–3322. [CrossRef]
27. Åkermark, B.; Eberson, L.; Jonsson, E.; Pettersson, E. Palladium-promoted cyclization of diphenyl ether,
diphenylamine, and related compounds. J. Org. Chem. 1975, 40, 1365–1367. [CrossRef]
28. Krahl, M.P.; Jäger, A.; Krause, T.; Knölker, H.-J. First total syntheis of the 7-oxygenated carbazole alkaloids
clauszoline-K, 3-formyl-7-hydroxycarbazole, clausine M, clausine N and the anti-HIV active siamenol using
a highly efficient palladium-catalyzed approach. Org. Biomol. Chem. 2006, 4, 3215–3219. [CrossRef] [PubMed]
29. Liégault, B.; Lee, D.; Huestis, M.P.; Stuart, D.R.; Fagnou, K. Intramolecular Pd(II)-catalyzed oxidative biaryl
synthesis under air: reaction development and scope. J. Org. Chem. 2008, 73, 5022–5028. [CrossRef] [PubMed]
30. Iwaki, T.; Yasuhara, A.; Sakamoto, T. Novel synthetic strategy of carbolines via palladium-catalyzed
amination and arylation reaction. J. Chem. Soc. Perkin Trans. 1 1999, 11, 1505–1510. [CrossRef]
31. Campeau, L.C.; Parisien, M.; Jean, A.; Fagnou, K. Catalytic direct arylation with aryl chlorides, bromides,
and iodides: intramolecular studies leading to new intermolecular reactions. J. Am. Chem. Soc. 2006, 128,
581–590. [CrossRef] [PubMed]
32. Queiroz, M.-J.R.P.; Ferreira, I.C.F.R.; Gaetano, Y.D.; Kirsch, G.; Calhelha, R.C.; Estevinho, L.M. Synthesis
and antimicrobial activity studies of ortho-chlorodiarylamines and heteroaromatic tetracyclic systems in the
benzo[b]thiophene series. Bioorg. Med. Chem. 2006, 14, 6827–6831. [CrossRef] [PubMed]
33. Burnell, R.H.; Della Casa, D. Alkaloids of Aspidosperma vargasii A. DC. Can. J. Chem. 1967, 45, 89–92.
[CrossRef]
34. Sunthitikawinsakul, A.; Kongkathip, N.; Kongkathip, B.; Phonnakhu, S.; Daly, J.W.; Spande, T.F.; Nimit, Y.;
Rochanaruangrai, S. Coumarins and carbazoles from Clausena excavata exhibited antimycobacterial and
antifungal activities. Planta Med. 2003, 69, 155–157. [CrossRef] [PubMed]
35. Okunade, A.L.; Elvin-Lewis, M.P.F.; Lewis, W.H. Natural antimycobacterial metabolites: current status.
Phytochemistry 2004, 65, 1017–1032. [CrossRef] [PubMed]
36. Ma, C.; Case, R.J.; Wang, Y.; Zhang, H.-J.; Tan, G.T.; Hung, N.V.; Cuong, N.M.; Franzblau, S.G.; Soejarto, D.D.;
Fong, H.H.S.; et al. Anti-tuberculosis constituents from the stem bark of Micromelum hirsutum. Planta Med.
2005, 71, 261–267. [CrossRef] [PubMed]

Molecules 2018, 23, 1402
16 of 16
37. Choi, T.A.; Czerwonka, R.; Fröhner, W.; Krahl, M.P.; Reddy, K.R.; Franzblau, S.G.; Knölker, H.-J. Synthesis
and activity of carbazole derivatives against Mycobacterium tuberculosis. Chem. Med. Chem. 2006, 1, 812–815.
[CrossRef] [PubMed]
38. Choi, T.A.; Czerwonka, R.; Forke, R.; Jäger, A.; Knöll, J.; Krahl, M.P.; Krause, T.; Reddy, K.R.; Franzblau, S.G.;
Knölker, H.-J. Synthesis and pharmacological potential of carbazoles. Med. Chem. Res. 2008, 17, 374–385.
[CrossRef]
39. Börger, C.; Brütting, C.; Julich-Gruner, K.K.; Hesse, R.; Kumar, V.P.; Kutz, S.K.; Rönnefahrt, M.; Thomas, C.;
Wan, B.; Franzblau, S.G.; Knölker, H.-J. Anti-tuberculosis activity and structure—activity relationships of
oxygenated tricyclic carbazole alkaloids and synthetic derivatives. Bioorg. Med. Chem. 2017, 25, 6167–6174.
[CrossRef] [PubMed]
40. Pauli, G.F.; Case, R.J.; Inui, T.; Wang, Y.; Cho, S.; Fischer, N.H.; Franzblau, S.G. New perspectives on natural
products in TB drug research. Life Sci. 2005, 78, 485–494. [CrossRef] [PubMed]
41. Cho, S.; Lee, H.S.; Franzblau, S. Microplate alamar blue assay (MABA) and low oxygen recovery assay
(LORA) for Mycobacterium tuberculosis. In Mycobacteria Protocols; Parish, T., Roberts, D.M., Eds.; Springer:
New York, NY, USA, 2015; pp. 281–292.
42. Falzari, K.; Zhu, Z.; Pan, D.; Liu, H.; Hongmanee, P.; Franzblau, S.G. In vitro and in vivo activities of
macrolide derivatives against Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 2005, 49, 1447–1454.
[CrossRef] [PubMed]
43. Sheldrick, G.M. SHELXS-97, Programs for Crystal Structure Solution; University of Göttingen: Göttingen,
Germany, 1997.
44. Sheldrick, G.M. SADABS, v. 2.10, Bruker/Siemens Area Detector Absorption Correction Program; Bruker AXS Inc.:
Madison, WI, USA, 2002.
45. Sheldrick, G.M. SHELXL-97, Programs for Crystal Structure Refinement; University of Göttingen: Göttingen
Germany, 1997.
46. Farrugia, L.J. ORTEP-3 for Windows—A version of Ortep-III with a graphical user interface (GUI).
J. Appl. Crystallogr. 1997, 30, 565. [CrossRef]
Sample Availability: Not available.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).