Modified carbazoles destabilize microtubules and kill glioblastoma multiform cells

Article abstract of DOI:10.1016/j.ejmech.2018.09.026

Small molecules that target microtubules (MTs) represent promising therapeutics to treat certain types of cancer, including glioblastoma multiform (GBM). We synthesized modified carbazoles and evaluated their antitumor activity in GBM cells in culture. Modified carbazoles with an ethyl moiety linked to the nitrogen of the carbazole and a carbonyl moiety linked to distinct biaromatic rings exhibited remarkably different killing activities in human GBM cell lines and patient-derived GBM cells, with IC₅₀ values from 67 to >10,000 nM. Measures of the activity of modified carbazoles with tubulin and microtubules coupled to molecular docking studies show that these compounds bind to the colchicine site of tubulin in a unique low interaction space that inhibits tubulin assembly. The modified carbazoles reported here represent novel chemical tools to better understand how small molecules disrupt MT functions and kill devastating cancers such as GBM.

Full text of DOI:10.1016/j.ejmech.2018.09.026

Accepted Manuscript
Modified carbazoles destabilize microtubules and kill glioblastoma multiform cells
Philippe Diaz, Eric Horne, Cong Xu, Ernest Hamel, Michael Wagenbach, Ravil R.
Petrov, Benjamin Uhlenbruck, Brian Haas, Parvinder Hothi, Linda Wordeman, Rick
Gussio, Nephi Stella
PII:
S0223-5234(18)30798-0
10.1016/j.ejmech.2018.09.026
EJMECH 10737
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 May 2018
Revised Date: 7 September 2018
Accepted Date: 9 September 2018
Please cite this article as: P. Diaz, E. Horne, C. Xu, E. Hamel, M. Wagenbach, R.R. Petrov, B.
Uhlenbruck, B. Haas, P. Hothi, L. Wordeman, R. Gussio, N. Stella, Modified carbazoles destabilize
microtubules and kill glioblastoma multiform cells, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.09.026.
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ACCEPTED MANUSCRIPT

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Modified carbazoles destabilize microtubules
and kill glioblastoma multiform cells
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Philippe Diaz*1 ,2, Eric Horne3, Cong Xu4, Ernest Hamel5, Michael Wagenbach4, Ravil R. Petrov1,
Benjamin Uhlenbruck1, Brian Haas4, Parvinder Hothi6, Linda Wordeman4, Rick Gussio5
and Nephi Stella*3, 4
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1Department of Biomedical and Pharmaceutical Sciences, The University of Montana, 32 Campus Drive,
Missoula, MT 59812, USA.
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2DermaXon LLC, 32 Campus Drive, Missoula, MT 59812, USA.
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3Stella Therapeutics, Inc., Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122, USA.
4Department of Pharmacology (CX, BH and NS), Department of Physiology and Biophysics (MW and LW),
Department of Psychiatry and Behavioral Sciences (NS), The University of Washington, Seattle WA 98195, USA.
5Screening Technologies Branch (EH) and Computational Drug Development Group (RG), Developmental
Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for Cancer
Research, National Cancer Institute, National Institutes of Health, Frederick MD 21702, USA.
6Ivy Center for Advance Brain Tumor Treatment, Swedish Neuroscience Institute, 550 17th Ave, Seattle, WA
98122, USA.
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*corresponding author(s):
For Chemistry : Philippe Diaz: Philippe.diaz@umontana.edu
For Biology: Nephi Stella: nstella@uw.edu
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ABSTRACT (146 words)
Small molecules that target microtubules (MTs) represent promising therapeutics to treat certain types of
cancer, including glioblastoma multiform (GBM). We synthesized modified carbazoles and evaluated their
antitumor activity in GBM cells in culture. Modified carbazoles with an ethyl moiety linked to the nitrogen of the
carbazole and a carbonyl moiety linked to distinct biaromatic rings exhibited remarkably different killing
activities in human GBM cell lines and patient-derived GBM cells, with IC50 values from 67 to > 10,000 nM.
Measures of the activity of modified carbazoles with tubulin and microtubules coupled to molecular docking
studies show that these compounds bind to the colchicine site of tubulin in a unique low interaction space that
inhibits tubulin assembly. The modified carbazoles reported here represent novel chemical tools to better
understand how small molecules disrupt MT functions and kill devastating cancers such as GBM.
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KEYWORDS: Microtubules, colchicine, carbazole, gliomas.
Abbreviations1
1GBM:
MOA:
MTs:
MTAs:
NC:
glioblastoma multiform
mechanisms of action
microtubules
microtubule-targeting agents
not converged
ND:
not determined
PDB:
protein data bank
PD-GBM: patient-derived GBM
OD:
TGI:
optical density
total growth inhibition
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33 1. Introduction
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Many subtypes of cancers are successfully treated with microtubule-targeting agents (MTAs), including breast,
lung and head and neck tumors, lymphoma and melanoma [1]. MTAs act by binding to tubulin or MTs and
disrupting MT assembly, disassembly and dynamics, all of which impairs the precise orchestration of mitosis and
triggers cell death by engaging the MT spindle check-point [2]. Starting in the 2000s, evidence suggested that
GBM, the most common, devastating and incurable type of brain cancer, is particularly sensitive to MTAs [3-6].
The sensitivity of GBM cells likely results from the expression of mutated proteins that erroneously control MT
dynamics and their interactions with kinetochores [7, 8]. Accordingly, several studies have shown that GBM cells
treated with MTAs undergo death through MT spindle check-point arrest and ensuing apoptosis [4, 5]. Human
clinical trials showed that MTA treatment reduces tumor burden in patients diagnosed with GBM [9], and the
recent advent of tumor-treating fields for the treatment of patients diagnosed with GBM provide additional
evidence that GBMs are sensitive to the disruption of MT function [10]. Specifically, the polarizing field
established by tumor-treating fields affects tubulin assembly into MTs, disrupts mitosis and triggers the spindle
check-point and ensuing cell death [11]. Together, these genetic, pharmacological and tumor-treating field
findings show that GBM cells are particularly sensitive to alterations in MT function and highlights the need to
develop novel MTAs for the treatment of this devastating disease.
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50
51
52
53
54
There are several classes of MTAs that disrupt MT function through distinct mechanisms of action (MOA).
Three of the most extensively studied classes of MTAs are vinca alkaloids, taxanes and colchicinoids, which bind
to distinct sites on tubulin and differentially affect MT dynamics [1, 12]. Prototypical MTAs that bind to the
colchicine sites include combretastatin A-4 (1), the carbazole-based analogues of combretastatin (2) that weakly
inhibit tubulin polymerization and yet triggers pronounced apoptosis, and nocodazole (3), which rapidly and
fully inhibits tubulin polymerization and triggers apoptosis in cancer cells (Figure 1) [13, 14].
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56
57
Figure 1. Microtubule targeting agents that bind to the colchicine site and chemical strategy for the synthesis of modified
carbazoles. Combretastatin A-4 (1), carbazole-based analogues of combretastatin (2), nocodazole (3) and scaffold of
modified carbazoles (4).
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MeO
O
OMe
OMe
O
O
MeO
MeO
O
O
NH
NH
N
S
R3
NH
S
OMe
OH
N
OMe
N
R1
O
R2
1
2
3
4
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59
60
61
62
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64
65
66
67
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In a previous study, we identified a series of indole-based MTAs that bind to the colchicine site of tubulin,
reduce MT polymerization in vitro, reduce MT dynamics in GBM cell lines in culture and kill GBM cells by
apoptosis when tested in an orthotopic syngeneic mouse model [14]. Here, we used these results as the starting
point for the development of a series of carbazole-based MTAs (4) and studied their antitumor activity in both
GBM cell lines and patient-derived GBM cells in culture. We also studied how the antitumor activity of carbazole
analogues relate to their ability to disrupt MT. Our studies emphasize the importance of substituting the
carbonyl linker with either carbinol or thiocarbonyl functional groups, as well as the importance of substitutions
at the nitrogen atom of the carbazole moiety (4). Finally, we provide a modeling study of the docking of the
most potent modified carbazoles to the colchicine site and outline its unique binding mode and how it affects
MT assembly.
69 2. Chemistry
70
The route for the synthesis of the carbazole derivatives is outlined in Scheme 1.
O
R3
N
R1
R2
b
c
a
O
O
N
OH
N
H
R1
R1
R3
R2
d
5: R1 = H, R2 = Et
: R = H, R = nPr
15
1
2
N
R2
R1
N
R1
: R = H, R = CH -CF
17
3
1
2
2
R2
29: R1 = OMe, R2 = H
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Scheme 1: Reagents and conditions: a. Cs2CO3, alkylbromide, DMF, r.t.; b. acyl chloride, AlCl3, benzene 0 °C to
r.t.; c. i. POCl3, DMF, μW, 1 h, 100 °C; ii. KMnO4, water/acetone, reflux; d. EDC, DMAP, DIPEA, amine, DMF.
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75
76
77
78
79
80
81
82
83
8: R1 = H, R2 = ethyl, R3 = 4-methylnaphthalenyl;
9: R1 = H, R2 = ethyl, R3 = 4-methylpiperazinyl;
10: R1 = H, R2 = ethyl, R3 = 4-chlorophenyl;
11: R1 = H, R2 = ethyl, R3 = 4-fluorophenyl;
12: R1 = H, R2 = ethyl, R3 = p-tolyl;
13: R1 = H, R2 = ethyl, R3 = methylbenzene;
14: R1 = H, R2 = ethyl, R3 = naphthalenyl;
16: R1 = H, R2 = propyl, R3 = 4-methylnaphthalenyl;
17: R1 = H, R2 = 2,2,2-trifluoroethyl, R3 = 4-methylnaphthalenyl
31: R1 = MeO-, R2 = ethyl, R3 = 4-methylnaphthalenyl;
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85
86
87
88
89
90
Compounds 8, 10, 11, 12, 13, 14, 16, 17, 25 and 31 were prepared from commercially available carbazoles via
consecutive substitution with the corresponding n-alkyl bromide under alkaline conditions, followed by a
Friedel-Crafts reaction using the corresponding acyl chloride derivatives (Scheme 1). Carbazole 28 was obtained
by alkylation of carbazole with 4-methylnaphthalene-1-carbonyl chloride under basic conditions. Bromine-
lithium exchange of bromo-carbazole derivative (t-BuLi, -78 °C), followed by the addition of quinolone aldehyde
derivatives, furnished the corresponding alcohols, 19 and 21, which were oxidized to form 20 and 22 (Scheme
2).
HO
O
R3
R3
Br
Br
a
b
c
R3-CHO
N
H
N
N
N
19: R3 = 5-quinolinyl
21: R3 = 8-quinolinyl
20: R3 = 5-quinolinyl
22: R3 = 8-quinolinyl
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92
Scheme 2: Reagents and conditions: a. Cs2CO3, ethylbromide, DMF; b. t-BuLi, THF, -78 to 0 ⁰C; c. PDC, CH2Cl2,
93
Molecular sieves r.t.
94
95
Quinoline derivatives 23 and 24 were conveniently prepared after reduction of compounds 20 and 22,
respectively.
96
97
The thione and the carbinol derivatives (26 and 27) were obtained by heating amide 8 with Lawesson’s
reagent under microwave conditions and by reducing its carbonyl with NaBH4, respectively (Scheme 3).
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S
O
N
a
b
26
HO
N
8
N
27
98
99
Scheme 3: Reagents and conditions: a. Lawesson’s reagent, toluene; b. NaBH4, MeOH.
100
101
To validate the importance of the planar carbazole ring, we synthesized the carboline derivative 35 (Scheme
4) from phenylhydrazine and ethyl-4-oxocyclohexanecarboxylate.
O
O
O
O
O
O
O
R1
N
N
N
N
O
b
a
c,d
+
N
H
N
N
NH
H2N
32
33
35
102
103
Scheme 4: Reagents and conditions: a. HCl, EtOH; b. NaH, alkylbromide, DMF; c. KOH, H2O, EtOH, reflux; d. EDC, DMAP,
104
DIPEA, amine, DMF.
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35: R1 = H, R2 = ethyl, R3 = 4-methylnaphthalenyl
106 3. Results and Discussion
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3.1. Antitumor activity of reference MTAs and modified carbazoles in the human GBM cell line T98G.
The two prototypical, colchicine site targeting agents, combretastatin A-4 (1) and nocodazole (3), killed human
GBM cells line, T98G, with the expected antitumor EC50 values (0.6 and 29 nM, respectively), but only killed T98G
cells by a maximum of 30.4% ± 2.1 (at 10 nM) and 45.6% ± 1.7 (at 10 µM), respectively (Figure 2A).
Supplementary Figure 1 shows that antitumor activity of 3 additional references MTAs also only partially killed
T98G cells: EC50 and maximal killing values for colcemide were 113 nM and 35.9% ± 6.4 at 10 µM, for vinblastine
were 184 nM and 36.0% ± 2.3 and for paclitaxel were 148 µM and 63.2% ± 2.5 at 30 µM. These results show that
reference MTAs that both inhibit assembly and destabilize MTs exhibit moderate antitumor activities in T98G
cells.
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Figure 2: Antitumor activity of reference MTAs that target the colchicine site, as well as select modified-carbazoles, in
T98G GBM cells in culture. T98G cells in culture were treated with increasing concentrations of A) two MTAs acting through
the colchicine site of tubulin, combretastatin A-4 (1) and nocodazole (3), B) compounds 8, 20 and 27, and C) compounds 23
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and 25. Cell viability was measured 72 h following treatment using WST-1. Dotted line shows 100% vehicle control. Data are
the mean ± SEM of at least three independent experiments performed in triplicate.
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We then treated T98G cells with the 19 modified-carbazoles synthesized for this study and report the EC50
values and maximal killing activity values of their antitumor activities in Tables 1 and 2.
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Table 1: Antitumor activity of 18 modified carbazoles in T98G cells in culture. Cells were treated with increasing
concentrations of the compounds and cell viability was measured 72 h following initiation of treatment using WST-1. EC50
values were calculated using Prism® software from curves shown in Figure 1 and Supplementary Figure 2. When the curves
could not be extrapolated, the EC50 could not be calculated and values are indicated as > 10 µM. Unless otherwise noted,
maximal killing activities were measured at 10 µM. Data are the mean of at least three independent experiments
performed in triplicate.
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Antitumor activity
Compound
R1
R2
R3
Maximal killing
(% ± SEM)
EC50
O
8
H
Ethyl
87 nM
45.3 ± 4.1
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O
9
H
H
Ethyl
Ethyl
> 10 µM
1.5 µM
13.2 ± 1.7
71.1 ± 5.3
N
N
O
O
10
Cl
11
H
Ethyl
8 µM
54.8 ± 4.0
F
O
83.6 ± 2.2
(at 3 µM)
12
13
H
H
Ethyl
Ethyl
993 nM
> 10 µM
O
14.0 ± 2.1
72.0 ± 5.7
O
14
16
18
H
H
H
Ethyl
1.7 µM
> 10 µM
5.6 µM
O
Propyl
16.3 ± 2.2
O
35.6 ± 9.4
CF3
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O
74.4 ± 1.7
(at 1 µM)
20
H
Ethyl
184 nM
N
O
22
23
H
H
Ethyl
8.8 µM
60.0 ± 4.9
N
O
-17.0 ± 10.8
(at 3 µM)
Ethyl
> 10 µM
NH
O
-18.0 ± 5.2
(at 100 nM)
24
H
Ethyl
> 10 µM
HN
O
N
89.0 ± 0.7
25
26
27
H
H
H
Ethyl
Ethyl
Ethyl
757 nM
> 10 µM
560 nM
(at 1 µM)
S
-17.0 ± 10.8
(at 3 µM)
OH
72.8 ± 7.6
(at 3 µM)
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O
28
31
H
H
957 nM
89.1 ± 0.7
O
MeO-
Ethyl
2.6 µM
71.6 ± 6.1
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Table 2: Antitumor activity of modified pyridoindole 35 in T98G cells in culture. Cells were treated with increasing
concentrations of compound 35 and cell viability was measured 72 h following treatment initiation using WST-1. Since with
35 a curve of the concentration-dependent antitumor activity could not be extrapolated, the EC50 is indicated as > 10 µM.
Data are the mean of at least 3 independent experiments performed in triplicate.
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Antitumor activity
Compound
R1
R2
R3
Maximal killing
(mean ± SEM)
EC50
O
45.1 ± 9.9
(at 3 µM)
35
H
Ethyl
> 10 µM
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Figure 2B and 2C show the antitumor activities of five modified carbazoles (8, 20, 23, 25 and 27) that hold
key SAR characteristics. The antitumor activities for the remaining compounds are in Supplementary Figure 2.
Thus, a comparison of the chemical structures of the modified carbazoles and their antitumor activities
expressed as EC50 values (potency) and maximal killing activities (efficacy) allows for multiple conclusions around
the SAR of this response.
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First, we found that fourteen modified carbazoles that all have an ethyl moiety linked to the nitrogen of the
carbazole and a carbonyl moiety linked to distinct biaromatic rings exhibited remarkably different EC50s.
Specifically, five such compounds killed T98G cells with EC50 values below 1 µM [8 (87 nM) < 20 (184 nM) < 27
(560 nM) < 25 (757 nM) < 12 (993 nM)], two compounds killed T98G cells with EC50s between 1 and 2 µM [10
(1.5 µM) and 14 (1.7 µM)] and seven compounds were only weakly active at concentrations above five
micromolar or essentially inactive [9, 13, 16, 18, 23, 24 and 26]. Note that the replacement of the
methylnaphthyl group present in the most potent compound 8 (87 nM) by either toluyl (as in 12, 993 nM) or a
non-substituted naphthyl moiety (as in 14, 1.7 µM) or a benzyl moiety (as in 13, inactive), increased EC50s values
by 11-fold, 20-fold and more than 115-fold, respectively.
Second, modified carbazoles and carboline exhibited remarkably different maximal killing activities.
Specifically, nine compounds killed T98G cells by more than 50% (i.e. more than the maximal killing activities
reached by reference MTAs). Thus, the rank order of these compounds is: 25 (89%) > 12 (84%) > 20 (74%) > 27
(73%) > 14 (72%) = 31 (72%) > 10 (71%) > 22 (60%) > 11 (55%). Three compounds killed T98G cells by 35-45% (8,
18 and 35) and six compounds did not significantly affect T98G cell viability (9, 13, 16, 23, 24 and 26). Note that
the position of the nitrogen atom in the quinoline portion of modified carbazoles controls their maximal killing
activities, as exemplified by the reduction in maximal killing activity when moving the nitrogen atom from
position 1 (as in 25, 89% killing) to either position 5 (as in 20, 60% killing) or position 8 (as in 22, 60% killing).
Third, introduction of a nitrogen atom in position 6 of the carbazole scaffold increases the EC50 value by
more than 115-fold as exemplified when comparing the response of 8 (87 nM) and the carboline analogue 35 (>
10 µM). A possible explanation for this change in EC50 value is that both the carbazole moiety and the aromatic
moiety attached to the carbonyl will impact the positioning of the lipophilic moiety attached to the carbonyl.
Thus, this result suggest a need for modified carbazoles to be planar for optimal activity [15]. Importantly, the
nitrogen atom in position 6 of the carbazole scaffold only affect the EC50 values of 8 and 35, as the maximal
killing reached by both compounds was 45%, indicating that a nitrogen atom in position 6 of the carbazole
scaffold will control the EC50 value of the compound independently of the maximal killing activity.
Fourth, the carbonyl linker determined the compound’s EC50 values as evidenced by comparing 8 (87 nM)
with both the carbinol 27 (560 nM) and the thiocarbonyl compound 26 (> 10 µM), which results in 6- and 115-
fold increase in EC50 values, respectively. This result can be explained by a loss of a hydrogen bond interaction
since thiocarbonyl derivatives are known to destabilize hydrogen bond formation through higher steric demands
that are imposed by both the larger sulfur atom and the lower electronegativity compared with the oxygen
atom [15]. Remarkably, changes in the carbonyl linker had an opposite effect on the maximal killing activity of
these compound, as the carbinol 27 killed more T98G cells than the carbonyl 8 (72% versus 45%, respectively).
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Fifth, the importance of the chain born from the endocyclic nitrogen is evident by comparing the EC50 values
of the ethyl compound 8 (87 nM) with both the trifluoroethyl compound 18 (5.6 µM) and the propyl compound
16 (> 10 µM), corresponding to 64- and 115-fold increases in the EC50s values, respectively. Remarkably, the
chain linked to the endocyclic nitrogen has less impact on the maximal killing activity: 8 (45%) and 18 (36%). This
result suggests a critical interaction site between the chain borne by the endocyclic nitrogen of modified
carbazoles and the target ((i.e. colchicine site of tubulin) that controls antitumor activity.
Sixth, a small methoxy R1 substituent in position 7 of the carbazole moiety affects the EC50 value by 30-fold
(as exemplified by 8 (87 nM) and 31 (2.6 µM)), suggesting an essential interaction site between the R1
substituent in position 7 of the carbazole and the target. Remarkably, the R1 substituent in position 7 of the
carbazole moiety had an opposite effect on their maximal killing activities: 31 (72%) killed more T98G cells
compared with 8 (45%). Note that the positioning of the methylnaphthyl moiety has the inverse impact on the
EC50 values (8 (87 nM) and 28 (957 nM)) and the maximal killing activities (8 (45%) and 28 (89%)). Together,
these results show that the antitumor EC50 values (potency) and maximal killing activities (efficacy) of modified
carbazoles can be mechanistically separated by specific chemical substitutions.
In conclusion, the SAR analysis of the antitumor activities of novel modified carbazoles measured in T98G
cells identified chemical features that independently control their antitumor EC50s values and maximal killing
activities, including a carbonyl moiety that links the carbazole to aromatic moieties, the ethyl moiety linked to
the nitrogen atom of the carbazole and select chemical modifications in the second aromatic moiety.
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3.2. Killing activity of select modified carbazoles in patient-derived GBM cells and the human liver
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200
201
202
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cell line HepG2
To explore the therapeutic potential of the modified carbazoles, we measured their antitumor activity in human
patient-derived GBM (PD-GBM) cells in culture. Specifically, these cells represent a recognized preclinical model
system for testing for the efficacy of novel treatments of GBM as they are categorized in three subtypes based
on their mutation profile (i.e., proneural, mesenchymal and classical) that differentially respond to treatment
[16-20]. To test for potential liver toxicity, we treated the human liver cell line HepG2, a commonly used cell
culture model system [21-24]. Therefore, we treated the PD-GBM cells in culture with the four most potent
modified carbazoles (8, 20, 27 and 25) from our initial screen and found that they kill the PD-GBM cells with EC50
values and maximal efficacy values that both mirrored their activity on T98G cells and remained within similar
values irrespective of the PD-GBM subtype (Table 3). Importantly, the four compounds exhibited lower cell-
killing activity in HepG2 cells, reaching only 8-41% maximal killing activities as compared with 44-99% maximal
killing activities reached in PD-GBM cells (Table 3).
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Table 3: Cell-killing activity of selected modified carbazoles in patient-derived GBM and liver HepG2 cells. PD-GBM and
HepG2 cells in culture were treated with increasing concentrations of compound. Cell viability was measured using WST-1
72 h after treatment. EC50 values were calculated using Prism® software using the curves shown in Supplementary Figure 3.
Maximal killing activity was measured at 5.6 µM. Data are the mean of at least three independent experiments performed
in triplicate. Not converged (NC).
210
Cell Killing Activity
Compounds
PD-GBM
(proneural)
Maximal
PD-GBM
(classical)
Maximal
PD-GBM
(mesenchymal)
Maximal
HepG2 (liver)
Maximal
Killing
EC50
EC50
EC50
EC50
Killing
Killing
Killing
(mean ± SEM)
(mean ± SEM)
(mean ± SEM)
(mean ± SEM)
(nM)
542
(nM)
698
(nM)
268
(nM)
1689
53.3% ± 10.9
(at 5.6 µM)
44.8% ± 10.5
(at 1 µM)
47.9% ± 6.5
(at 3 µM)
64.3% ± 1.3
(at 1 µM)
25.0% ± 3.8
(at 5.6 µM)
28.6% ± 13.9
(at 0.6 µM)
8.3% ± 8.5
(at 5.6 µM)
30.8% ± 13.3
(at 10 µM)
8
53.9% ± 4.2
(at 1 µM)
58.9% ± 1.4
(at 1 µM)
20
25
27
316
613
630
407
846
633
335
1823
386
66
NC
101
98.9% ± 0.1
(at 5.6 µM)
44.0% ± 14.2
(at 1 µM)
98.6% ± 0.9
(at 5.6 µM)
56.0% ± 3.8
(at 3 µM)
97.2% ± 0.8
(at 10 µM)
50.6% ± 6.2
(at 3 µM)
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216
217
218
219
220
221
The preferential sensitivity of PD-GBM cells to the killing activity of modified carbazoles compared with
HepG2 cells suggests a promising therapeutic index (i.e., the ratio of the amount of agent that causes the
therapeutic effect to the amount that causes toxicity). One compound, 25, demonstrated a clear-cut result by
killing 95-100% of the PD-GBM cells when applied at 1-10 µM while remaining inactive at these concentrations
in HepG2 cells (Table 3 and Supplementary Figure 3). Another advantage of the modified carbazoles developed
here is that they kill all PD-GBM subtypes independent of their genetic make-up. This addresses one of the most
thorny and extensively highlighted issues concerning the selectivity of targeting GBM tumors, which are known
to rapidly become heterogeneous in their genetic make-up, often encompassing all subtypes (proneural,
mesenchymal and classical) [25, 26]. Therefore, the equally potent and efficacious killing activities of modified
carbazole measured in PD-GBM subtypes shows their potential to kill the majority of GBM cells forming the
heterogeneous tumor mass.
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3.3. Cell-killing activities and MOA of modified carbazoles in the NCI-60 cancer cell line panel
We worked with The Developmental Therapeutics Program’s NCI 60 cell screen to study the MOA by which the
three most potent modified carbazoles (8, 20 and 27) kill cancer cell lines and included the inactive analogue 23
as negative control. This screening platform measures the antitumor activities of compounds expressed as
percent growth inhibition, which differentiates the compound’s activity at inhibiting cell proliferation and
reducing cell number (i.e. killing cells) [27]. As expected, the inactive compound 23 did not significantly affect
the viability and proliferation of any of the cell lines (growth inhibition: Mean 102.05; Delta 10.42, and Range
28.77) (Supplementary Figure 4). By contrast, the three active compounds 8, 20 and 27 killed 4 cell lines (13% to
60% loss in cell number), halted the proliferation of 11 cell lines (from 9% to -1% growth inhibition) and reduced
the proliferation rate of 41 cell lines (from 67% to 10% percent growth inhibition) (Supplementary Figure 4). Of
note, two GBM cells lines that are included in the NCI-60 panel, SF-295 and SF-539, were among the top ten cell
lines that were the most susceptible to 8, 20 and 27 treatments (Figure 3 and Supplementary Table 1). The
respective EC50s of the compounds measured in SF-295 and SF-530 cells mirrored their EC50s measured in T98G
cells. Additional cell lines among the top ten cell lines that were sensitive to these three compounds were three
cell lines of colon cancer origin: Colo205, HT-29 and HCC-2998 (Supplementary Figure 5).
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Figure 3: Cell-killing activity of select modified-carbazoles in SF-539 and SF-295 GBM cells in culture. A) SF-539 cells in
culture and B) SF-295 cells in culture were treated with increasing concentrations of compounds 8, 20 and 27. Cell viability
was measured 48 h following treatment using Alamar blue and is expressed as optical density (O.D.). Data are gathered at
seeding and after 48 h, providing an index of the inhibition of cell proliferation (between both dotted lines) and triggering
of cell death (below Seed dotted line). Parameters of cell-killing activity (GI50, Total Growth Inhibition [TGI] and LC50) are in
Supplementary Table 1.
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The COMPARE and CellMiner web applications allow for both the statistical analysis of the antitumor
activities of drugs tested in the NCI-60 cell line panel and the identification of other compounds that similarly
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impact the proliferation and viability of NCI 60 cells [27, 28]. Using these applications, we found three MTAs
(paclitaxel, thalicarpine and maytansine) among the top eight compounds that exhibit antitumor activities in the
NCI-60 cell line panel that correlate with the antitumor activities of 8, 20 and 27 (Supplementary Table 2). While
this result suggests that 8, 20 and 27 kill cancer cells by disrupting MTs, the respective correlation parameters
were relatively low, pointing to a somewhat different MAO as compared to that of paclitaxel, thalicarpine and
maytansine, and all other MTAs tested on NCI 60 cells. Based on these results, it was reasonable to expect that
the modified carbazoles would interact with tubulin or MTs through a different MOA.
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3.4. Activity of modified carbazoles with tubulin and microtubules
First, we determined whether modified carbazoles compete for [3H]colchicine binding to native purified tubulin
as previously described [29]. In this assay, the reference MTAs 1 (5 µM) and 3 (5 µM) competed for binding by
98% and 74%, respectively (Table 4).
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Table 4: Activity of selected modified carbazole with tubulin and microtubules. Colchicine binding to tubulin and tubulin
assembly (as assessed by turbidity development of tubulin solutions) were inhibited in a dose dependent manner by
combretastatin A-4 (1) and nocodazole (3) (positive controls) and by the selected modified carbazoles. Results are the mean
± standard deviation (SD) of at least three independent experiments. MT and free tubulin partitioning was shifted toward
free tubulin by all compounds indicating MT destabilization. EC50 values are the results from representative experiments
that were repeated three times with comparable results. Not determined (ND).
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Tubulin
Colchicine Binding
Assembly
MT-Tubulin Partitioning
% Inhibition
EC50 (µM)
IC50 ( M)
µ
Compounds
mean ± SD
mean ± SD
mean
at 5 µM
at 1 µM
98 ± 0.1
74 ± 0.8
4 ± 1
89 ± 3
23 ± 0.8
ND
0.64 ± 0.01
0.48 ± 0.01
> 20
0.6
2.4
> 20
5.3
2.1
6.4
3.4
1
3
23
25
8
ND
41 ± 4
53 ± 3
70 ± 1
91 ± 1
4.4 ± 0.4
1.4 ± 0.2
1.6 ± 0.01
0.89 ± 0.04
ND
ND
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20
61 ± 4
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Under these experimental conditions, modified carbazoles competed for colchicine binding with increasing
affinity (23 << 25 < 8 < 27 < 20) that mirrored their respective EC50s at killing GBM cells. We next tested their
ability to inhibit the assembly of purified tubulin using an in vitro turbidity assay as previously described [29]. In
this assay, where tubulin is present at 10 µM in 0.8 M glutamate with 0.4 mM GTP and the extent of assembly is
measured after 20 min at 30 °C, 1 and 3 have IC50s of 0.6 µM and 0.5 µM, respectively (Table 4). Under these
experimental conditions, 8, 20 and 27 exhibited IC50s of 1.4, 0.9 and 1.6 µM, respectively, and 23 was inactive at
concentrations up to 20 µM (Table 4). To build on these results, we measured the ability of modified carbazoles
to influence the partitioning of MT dimers and polymers, an index of MT assembly and disassembly as previously
described [14]. In this assay, compounds are incubated with sheared MTs at 37°C and the resulting amounts of
tubulin dimers (free tubulin) and polymer (MT) after 15 min is measured on polyacrylamide gels [30]. Under
these experimental conditions, 1 and 3 have IC50s of 0.6 and 2.4 µM, respectively; 8, 20 and 27 have IC50s of 2.1,
3.4 and 6.6 µM, respectively; and 23 was inactive when tested up to 20 µM (Table 4). Thus, the IC50s values of
these modified carbazoles to bind to the colchicine site on tubulin parallels their EC50s to kill GBM cells, whereas
their IC50s to trigger MT disassembly does not correlate as well and remained in the micromolar range as
measured in two in vitro assays.
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The analysis of the SARs of modified carbazoles to bind to the colchicine site, disrupt MT assembly and favor
disassembly provide initial mechanistic insights on how their binding to the colchicine site in tubulin might favor
conformational changes in this protein that might affect MT dynamics. The potency of modified carbazole
derivatives are in the micromolar range when affecting MT assembly and disassembly. A possible explanation is
that the affinity of modified carbazoles for the colchicine site depends on specific interactions with a subset of
amino acid within the binding pocket, whereas the conformational changes in tubulin that are stabilized by the
modified carbazoles are only reached at high concentrations. Thus, the modified carbazoles that we developed
here may represent new chemical tools to study how binding to the colchicine site might affect tubulin
conformation and how this affects MT end dynamics.
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3.5. Gas Phase Pharmacophore Study
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To understand the structural basis of the MT-disrupting and antitumor activities of the modified carbazole
series, we initiated a gas phase study to determine common pharmacophoric features with sterically-relevant
colchicine site agent. The structure of 20, the most potent of the series to bind to the colchicine site, was used
to compare complementary features and volumetric space filling models of ligand co-crystal structures that
currently exist in the Protein Data Bank (PDB) for the colchicine site on tubulin. Two MTAs that target the
colchicine site, colchicine and podophyllotoxin, were found to have the most steric, hydrophilic and hydrophobic
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spatial similarity with the carbazole analogues. Using methodology employed previously [31, 32], the X-ray
based configurations and conformations of the two chiral molecules (colchicine and podophyllotoxin, taken
from the X-ray co-crystals 402B [31], and 1SA1 [33], respectively) were employed as templates for the
translational and conformational overlap modeling of 20. Figure 4 displays the results of the gas phase
pharmacophore overlap study for colchicine, podophyllotoxin and 20.
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Figure 4: Gas phase pharmacophore overlap model of colchicine, podophyllotoxin, and 20. Panel A illustrates a three-
dimensional stick rendering of the gas phase pharmacophoric overlap model for colchicine (carbons in magenta),
podophyllotoxin (carbons in cyan), and 20 (carbons in yellow). For purposes of visual clarity, oxygen atoms are colored red
in podophyllotoxin and deeper red for both colchicine and 20. In all three structures, nitrogen atoms are colored blue. The
red circles correspond to the two 20 polar hydrogen bond acceptor features in common with colchicine or podophyllotoxin.
Panels B, C and D are two-dimensional illustrations of the pharmacophore overlap model depicted in Panel A. Panel B
illustrates colchicine. Panel C illustrates podophyllotoxin and Panel D illustrates 20. In Panels B-D, the bolded blue atoms
correspond to the steric overlaps of the colchicine and podophyllotoxin atoms that are in common with 20; otherwise,
atoms in Panels B-D are colored magenta for colchicine, cyan for podophyllotoxin and yellow for 20. As with Panel A, the
red circles in Panels B-D correspond to polar hydrogen bond acceptor features of compound 20 that are in common with
colchicine or podophyllotoxin.
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The results from the gas-phase pharmacophore overlap study identified the common steric features
between 20, podophyllotoxin and colchicine. The atoms of 20 are almost completely subsumed within the steric
space of podophyllotoxin and approximately 80% of the steric space of colchicine. The common steric overlap
between these three molecules is largely composed of hydrophobic atoms. However, the two key polar atoms of
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20 are the basic quinoline N and its ketone O. The quinoline N atom of 20 shows remarkable spatial consistency
with the central methoxy O’s of the trimethoxylated-aryl moieties of podophyllotoxin and colchicine (Figure 4),
whereas the ketone O of 20 is isosteric with the lactone carbonyl of podophyllotoxin. Unique to the 20 scaffold
is the relatively strong hydrogen bond acceptor quinoline N in spatial proximity to the comparatively much
weaker hydrogen bond accepting ether O’s of podophyllotoxin and colchicine. Furthermore, the 10-membered
aromatic quinoline ring system of 20 offers unique topology to satisfy the binding requirements of the subsite
normally occupied by the trimethoxylated-aryl systems of podophyllotoxins and colchicine. An additional unique
feature of the 20 scaffold is the ethyl moiety attached to the carbazole N (Figure 4). The consistent polar,
hydrophobic and steric overlap identified by the gas phase pharmacophore study provides a template to
conduct molecular docking studies to determine a detailed, all-atom rationalization of the modified carbazole
series’ structure activity relationships in the colchicine site.
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3.5. Molecular Docking Studies
Given the close steric congruency with podophyllotoxin, we employed the alpha and beta tubulin subunits of the
recently solved 2.30
Å
co-crystal of the podophyllotoxin analogue: 4β-(1,2,4-triazol-3-ylthio)-4-
deoxypodophyllotoxin (PDB entry code =5JCB) [34] as the initial geometry to conduct the molecular docking
studies of the modified carbazole series. Maestro [34] protein preparation tools (utilizing the OPLS force field)
were utilized for X-ray co-crystal comparisons and to prepare the structure for molecular mechanics energy
refinement simulations. The 5JCB coordinates were optimized in a stepwise fashion (hydrogens first, followed by
side chain and backbone) resulting in an all-atom energy-refined structure used as the starting structure for the
carbazole molecular docking studies. Superimposition of the 5JCB X-ray coordinates onto the resulting energy
refined structure revealed only a 0.98 Å RMS deviation between the 13,646 heavy atoms. Of the 6880 backbone
atoms compared, only a 0.74 Å deviation was observed. These data signify that very close geometries were
obtained between the X-ray and energy refined structures that are well within the expected thermodynamic
variation and resolution of the 2.3 Å 5JCB X-ray structure.
Carbazole structures were built and initially optimized with the MM2 force field and transferred into the
cff91/cvff force fields for potential assignment [35]. For molecular docking studies, we used methodology
employed previously with added refinements [36]. Briefly, the carbazoles were docked into the energy refined
5JCB X-ray co-crystal structure to examine all translation and rotational steric complementarity between the
most potent ligand of the carbazole series, 20, its conformational isomers, and the side chains of the energy-
refined podophyllotoxin binding site. The resulting all-atom model was derived from iterative constrained
optimizations to refine a maximum complementarity between the atoms of 20 and its contacts with the amino
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acid side chains of the colchicine site. Figure 5 shows the optimally docked conformation of 20 and its overlap
with the triazole-podophyllotoxin analogue in the model.
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Figure 5: Comparison of modeling and X-ray poses. Panel A displays the view of the optimized 20 (carbons in yellow) in the
colchicine site from the molecular docking studies In Panel B, the pose of the podophyllotoxin-triazole analog (carbons in
cyan) from the X-ray 5JCB is shown with 20 overlapped from molecular docking studies. In addition to the high degree of
steric congruency with the podophyllotoxin-triazole analog, the ketone oxygens of both 20 and podophyllotoxin-triazole are
isosteric. Unique to t 20, compared to podophyllotoxin and colchicine, is the relatively strong hydrogen bond acceptor
quinoline N located in the trimethoxy-aryl subsite (near β-Cys-241). 20 can form a direct hydrogen bond (blue dashed lines
in both panels) with the side chain S-H of β-Cys-241. The quinoline N of 20 can form a stronger hydrogen bond with a water
bridge to polar backbone atoms compared to the ether O’s of the central methoxy groups of podophyllotoxin and
colchicine (also see Supplementary Figure 6).
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An overlap that includes colchicine can be found in Supplementary Figures 6 and 7. The optimally docked
model of 20 served as a template for molecular docking studies with the rest of the carbazole series to
rationalize the protein-atom ligand contacts’ SARs. Constrained optimizations were performed in an iterative
fashion to eliminate bad contacts and biochemically unfeasible hydrophobic-polar interactions as identified by
the HINT program [37]. When available, the in vitro binding data were prioritized to rank-order the carbazoles;
otherwise the percent inhibition of the rank-ordered congeneric series served as the biological activity data for
the SAR. A total of twenty structures (including the R and S enantiomers of 27) provided a structure-based
explanation that rationalized the SAR of the carbazole series.
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The modified carbazoles described herein exemplify a new chemotype for tubulin depolymerizers that
interact at the colchicine site. Our modeling studies indicate that the 20 scaffold is almost entirely subsumed by
the steric space of podophyllotoxin and approximately 80% within the steric space of colchicine. Potent
carbazoles bind more efficiently (requiring fewer atoms) in the same steric space than podophyllotoxin because
of the conformational restriction imposed by the 13-membered (carbazole) and 10-membered
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(quinoline/napthalene) rings systems when both are bridged by a methanone. The methanone carbonyl, which
functions as a strong hydrogen bond acceptor, is important for potent activity in the carbazole series, as
evidenced by a substantial loss of activity when the carbonyl is replaced by a thiocarbonyl (26). Thus, 26 forms a
weaker hydrogen bond r and imposes the bulkier S atom in the sterically restricted space where the lactone
carbonyl O of podophyllotoxins and carbazole Os overlap.
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Although most of the atoms that are sterically congruent with podophyllotoxin and colchicine are
hydrophobic, 20 introduces several unique structural elements to its colchicine site interactions. 20 poses a 10-
membered quinoline aromatic ring system in the colchicine subsite normally occupied by the
colchicine/podophyllotoxin trimethoxy-aryl system. With respect to enthalpic interactions, two hydrogen bond
acceptors: the methanone O, and the quinoline N account for most of the potency of 20. Specifically, the
methanone O can hydrogen bond with the backbone N-H of β-Asp-251, and the quinoline N located at the 5
position enables the formation of a strong can hydrogen bond with the S-H of β-Cys-241) (Supplementary Figure
6). Perhaps this occurs as the tubulin dimers polymerize and could therefore play a significant role in the
prevention of MT assembly. By contrast, 22 possesses the quinoline N in the 8 position which greatly reduces
activity due to its inability to form the hydrogen bonds characteristic of 20, podophyllotoxin, and colchicine
(Supplementary Figure 7).
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Our modeling studies indicate that the 8-quinoline N enables the formation a water-mediated
intramolecular hydrogen bond that stabilizes a sterically-dissimilar conformation from both 20 and
podophyllotoxin, which potentially also contributes to a loss of activity. Removal of the N atom altogether (as in
in 8) to form a naphthalene, also reduces activity due to the loss of the hydrogen bonding N altogether. Our
docking studies further indicate that the 4-methyl group of 8 forces the naphthalene deeper into the
trimethoxy-aryl subsite to avoid unfavorable hydrophobic-polar contacts with backbone polar atoms. This then
places the methanone O slightly further from the ketone O in 20’s optimal orientation. The net effect is that 8’s
methanone O is less conformationally available to function as a strong hydrogen bond acceptor. This is also
supported by 8’s close congener 14, which only differs structurally by the lack of the methyl at the napthalene 4
position, and 14 is markedly less active due to loss of a hydrogen bond acceptor. Conversely, adding a
hydrophobic bulk to the carbazole ring system reduces activity, as exemplified by the 7-methoxy substituted
carbazole of 31 where the methyl portion of its methoxy forms unfavorable hydrophobic-polar contacts with the
backbone N-H atoms of β−Lys-352, β-Val-351-349 and β−Asn-350 (Supplementary Figure 8, Panel A).
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A second unique structural feature of the most potent analogue 20 is the ethyl group attached to the
carbazole. Both conformational analysis and docking studies indicate that this ethyl group helps stabilize the
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scaffold’s conformation through steric hindrance during binding. An additional attribute of the ethyl group is
that it forms favorable hydrophobic contacts with the aliphatic component of the β-Lys-352 side chain
(Supplementary Figure 8, Panel A). Accordingly, the less active compound 18, in which the hydrophobic distal
carbon of the ethyl is replaced with an isosteric polar trifluoromethyl group, is closer to the hydrocarbon
component of β−Lys-352 side chain (Supplementary Figure 8, Panel B). Moreover, replacement of this ethyl
group with a bulkier hydrophobic propyl group (as with 16) reduces activity because of unfavorable
hydrophobic-polar liabilities with the cationic NH3 of the β−Lys-352 side chain (Supplementary Figure 8, Panel B).
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Compound 25 features a 10-membered system linked to the methanone by a N atom. Since it is a non-
planar dihydroquinoline derivative, it also lacks ring aromaticity in this part of the molecule. There are several
possible explanations for the reduced activity of 25. First, it could be due to the formation of a more rigid amide-
like linkage that produces a less optimal orientation of the hydrogen bond accepting O due to the rigidity of the
amide. Second, it could be due to the bulkier unsaturated ring that induces a twisted binding mode that
potentially introduces an unfavorable hydrophobic-polar interaction liability near the side chain group of β-Ala-
250. Third, it could be due to a loss of a hydrogen bond with β−Cys-241 (Supplementary Figure 9).
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Several conclusions can be drawn from several less active compounds. For example, a feature to highlight is
how removal of one the aromatic rings from the naphthalene/quinoline systems of the carbazoles reduces
activity. Specifically, despite its p-tolyl methyl group, the single ring of 12 sterically occupies almost half of the
trimethoxy-aryl subsite compared with colchicine, podophyllotoxins and 20 and yet it is less potent. An
implication for this finding could be that binding to the colchicine site has a minimum steric requirement of
occupation at the trimethoxy-aryl subsite to incur significant activity. However, replacement of the 10-
membered ring with a 6-membered ring (as in 12 and 10) also increases the number of conformational isomers,
which reduces conformational access compared to the more active carbazoles possessing double aromatic ring
systems. Another example is 13, which also loses activity by reducing conformational access while increasing
conformation isomerism as it extends the single ring linkage to the methanone by a methylene group. Thus,
replacement of the p-tolyl methyl of 12 with a hydrophobic and isosteric Cl atom (as in 10) reduces activity by
introducing similar disadvantages. The poor activity of 9 is due to the presence of a positive charge introduced
by the likely protonated piperazinyl single ring system, reinforcing the finding that the trimethoxy-aryl subsite
favors hydrophobic occupation with carefully positioned hydrogen bond acceptors. Accordingly, both 23 and 24
contain hydrogen bond donating N-Hs that are potentially charged.
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Finally, our molecular docking studies indicate that the structural configuration of 28 requires a unique
binding mode to form a biochemically-feasible complementarity with the colchicine site that accounts for its
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reduced biological activity compared to 20 (Supplementary Figure 10, Panel A). Specifically, the 4-tolyl-
naphthalene methanone of 28 is attached directly to the carbazole N, which causes the carbazole moiety of 28
to bind less congruently with the dioxolane component of podophyllotoxin’s tetracycle. Instead, one of the
carbazole’s aromatic rings finds steric overlap with the hydrophobic portion of the 5-membered lactone ring of
the podophyllotoxin analog. Compared to the other carbazoles, 28 forms unique, favorable hydrophobic
contacts with the side chain of β-Leu-248. Interestingly, the methanone O of 28 is still positioned close to both
the other methanone O’s of the more potent modified carbazole and the lactone carbonyl O’s of
podophyllotoxin (Supplemental Figure 10, Panel B). The significance of this novel binding mode is that 28 can be
modified to include key features from the optimal carbazole 20, while retaining its novel interactions with the
colchicine site.
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Together, the structure-activity relationships of these carbazoles signify that there are key features for them
to retain optimal activity in the colchicine site: 1) steric occupation of a moiety of similar size to a naphthyl,
quinolinyl or trimethoxy-aryl system with preferably a hydrogen bond interacting with the β-Cys-241 sulfhydral,
2) lack of cationic ionizability for moieties that occupy the trimethoxy-aryl subsite, 3) reduction of
conformational isomerism by either intramolecular steric hindrance or inherent rigidity, and 4) a strong
hydrogen bond acceptor with an overlapping pose of the methanone O of 20 and the lactone carbonyl O of
podophyllotoxins. Thus, the most potent 20 appears to share similar binding features with both
podophyllotoxins and colchicine but provides better atom economy to achieve a comparable potency. In 20, the
larger quinoline aromatic system fills the trimethoxy-aryl subsite in a sterically more compact manner than
colchicine and podophyllotoxin. Moreover, the quinoline N serves as a strong hydrogen bond acceptor capable
of forming diverse hydrogen bonding interactions either directly with the side chain of β-Cys-241
(Supplementary Figure 6) or via water bridges (models not shown). We have shown that the short N-ethyl
substituent on the carbazole part of 20 is involved in key hydrophobic interactions with the side chain of β-Lys-
352 (Supplementary Figure 8, Panel A). Our molecular docking studies provide a unified structure-based
explanation for both positive and negative substitutions according to the SAR of the carbazole analogs.
460 4. Conclusion
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We report here a novel series of modified carbazoles that destabilize MTs by binding to the colchicine site of
tubulin in a similar mode to a podophyllotoxin analogue and appears to interact with a unique low interaction
binding space. Several modified carbazoles trigger marked cell death in multiple GBM model systems while
exhibiting a much less lower activity in the HepG2 liver cells, suggesting a promising therapeutic index. The use
of the heterocyclic carbazole scaffold provides several advantages when considering the future optimization of
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small-molecule for therapeutic use. For example, this scaffold can be modified in a variety of ways using readily
available and simple starting materials, and their chemical modification typically requires only as few steps of
well-established chemistry, thereby providing a versatile scaffold for medicinal chemistry optimization. In
summary, this work increases our understanding of how targeting the colchicine site by small molecules affects
tubulin assembly and disrupts MT function, and provides a reference design approach to develop the next
generation of MTAs for treating devastating cancers, such as GBM.
472 5. Experimental Section
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General experimental methods. Moisture sensitive reactions were performed in an inert, dry atmosphere of Ar
in flame-dried glassware. Air sensitive liquids were transferred via syringe or cannula through rubber septa.
Reagent grade solvents were used for extraction and flash chromatography. THF was distilled from
Na/benzophenone under Ar. All commercially obtained reagents and solvents were used directly without further
purification. The progress of reactions was checked by analytical thin-layer chromatography (Silica G TLC plates
w/UV 254). Flash column chromatography was performed using prepacked Biotage SNAP/ZIP cartridges on a
Biotage Isolera One instrument. The solvent compositions reported for all chromatographic separations are on a
1
volume/volume (v/v) basis. H NMR spectra were recorded at 400 MHz and are reported in parts per million
(ppm) on the δ scale relative to tetramethylsilane as an internal standard. 13C NMR spectra were acquired at 101
1
MHz operating with H decoupling and are reported in parts per million (ppm) on the δ scale relative to CDCl3.
Melting points were determined on a Stuart melting point apparatus from Bibby Scientific Limited and are
uncorrected. LC/MS and HRMS analyses were obtained on a Waters ACQUITY UPLC-series liquid
chromatography system equipped with a diode array detector and coupled to a LCT PREMIER XETM time of flight
(TOF) mass spectrometer with electrospray ionization (ESI). The liquid chromatography conditions were as
follows: a Phenomenex column (NX, 3µ, C18, 110A, 50.0x4.60 mm) was used, and bound compounds were
eluted with the following gradient over 15 min at a rate of 0.4mL/min (water (0.1% formic acid)/acetonitrile):
(90/10 to 2/98, 0-6.6 min), (2/98 isocratic, 6.6-13 min), (2/98 to 90/10, 13-15 min). Compound purity was
assigned based on 254 nM detection data assessed by comparing relative peak areas of the signals. All final
compounds were more than 95% pure.
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9-ethyl-9H-carbazole (5). Under Ar, a solution of carbazole (5.0 g, 29.9 mmol), bromoethane (4.45 mL, 35.9
mmol), and Cs2CO3 (14.6 g, 44.8 mmol) in DMF (20 mL) was stirred at 80⁰C for 16 h. The reaction mixture was
cooled, diluted with EtOAc (50 mL), and filtered. The organic solvents were evaporated in vacuo. The resultant
dark oil was purified by column chromatography on silica gel using heptanes/EtOAc in different proportions to
1
afford the title compound as a light-yellow oil (5.075 g, 87%). H NMR (500 MHz, CDCl3) δ ppm 8.18 (d, J=7.79
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Hz, 2H), 7.54 (t, J=7.66 Hz, 2H), 7.46 (d, J=8.06 Hz, 2H), 7.26 - 7.35 (m, 2H), 4.40 (q, J=7.25 Hz, 3H), 1.41 - 1.52 (m,
3H) 13C NMR (126 MHz, CDCl3) δ ppm 139.85, 125.54, 122.85, 120.37, 118.68, 108.37, 37.42, 13.75.
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9-ethyl-9H-carbazole-3-carbaldehyde (6). POC13 (1.4 mL, 10.2 mmol) was added, over a period of 10 min, to ice-
cooled, stirred DMF (3.2 mL, 40.8 mmol) under Ar. The reddish solution was stirred at room temperature for 1 h.
9-ethyl-9H-carbazole 5 (1.0 g, 5.1 mmol) was added over 10 min, and the mixture was subjected to microwave
irradiation at 100 °C for 1 h. The reaction mixture was cooled and then poured into crushed ice. After warming
to room temperature, the resultant product was extracted with EtOAc. The organic phase was washed with
water, and brine, dried (MgSO4), filtered, and evaporated in vacuo. The residue was purified by column
chromatography on silica gel using heptanes/EtOAc in different proportions to afford the title compound as a
1
white solid (1.054 g, 93%); H NMR (400 MHz, CDCl3) δ ppm 10.03 (s, 1H), 8.57 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H),
7.94 (d, J = 8.5 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 8.6 Hz, 2H), 7.30 (t, J = 7.4 Hz, 1H), 4.26 (t, J=7.2 Hz,
13
2H), 1.45 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl3) δ ppm 191.83, 144.18, 141.27, 128.56, 127.23, 126.82,
124.06, 123.14, 123.08, 120.84, 120.40, 109.53, 109.05, 42.76, 13.91.
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
9-ethyl-9H-carbazole-3-carboxylic acid (7). To an ice-cold solution of 9-ethyl-3-carbaldehyde 6 (1.0 g, 4.5 mmol)
in water/acetone (50 mL, 1:1) was added dropwise with stirring a solution of potassium permanganate (711 mg,
4.5 mmol) in acetone (25 mL). The mixture was heated 3 h at reflux and then allowed to cool to room
temperature. After that, the reaction mixture was quenched with ethanol (20 mL) and then stirred for 30 min at
reflux. After cooling to room temperature, the mixture was filtered through a pad of Celite© and concentrated in
vacuo. The concentrated solution was diluted with water (100 mL), basified with NaOH to pH ca. 10, and
extracted with heptane/ether (4:1, 50 mL x 3) to remove the unreacted starting material. The aqueous solution
was cooled on an ice-water bath and then acidified with an ice-cold solution of 2 N HCl to pH ca. 2. The resultant
precipitate was extracted with EtOAc (150 mL). The organic layer was washed with brine (30 mL), dried over
MgSO4, filtered, and concentrated in vacuo. The precipitated product was collected by filtration, washed with
heptanes (20 mL), and dried overnight to produce the title compound 3 (527.2 mg, 49%) as a yellow oil; 1H NMR
(500 MHz, CDCl3) δ ppm 12.34 (s, 1H), 8.89 (d, J = 1.6 Hz, 1H), 8.31 (dd, J = 8.6, 1.6 Hz, 1H), 8.12 (d, J = 7.7 Hz,
1H), 7.51 (ddd, J = 8.3, 7.1, 1.3 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.28 (ddd, 7.9, 6.9, 1 Hz,
13
1H), 4.32 (t, J = 7.2 Hz, 2H), 1.45 (t, J = 7.2 Hz, 3H). C NMR (126 MHz, CDCl3) δ ppm 173.30, 143.47, 141.17,
128.13, 126.62, 123.87, 123.14, 122.67, 120.88, 120.33, 119.82, 109.34, 108.45, 42.57, 14.01.
525
526
527
9-ethyl-3-[(4-methylnaphthalen-1-yl)carbonyl]-9H-carbazole (8). Under Ar, AlCl3 (199 mg, 1.5 mmol) was added
to a solution of 9-ethyl-9H-carbazole 7 (300 mg, 1.25 mmol) in anhydrous benzene (30 mL), and the solution was
cooled by an ice bath for 20 min. 4-methyl-1-naphthoyl chloride (282 mL, 2.43 mmol) was added dropwise via a
24

ACCEPTED MANUSCRIPT
528
529
530
531
532
533
534
535
536
syringe to the solution, and the reaction mixture was stirred for 16 h while warming at room temperature. The
reaction mixture was cooled on an ice-water bath, then poured onto a mixture of ice and a 4 M NaOH solution
(50 mL) and extracted with ethyl acetate (150 mL). The obtained residue was purified by column
chromatography on silica gel eluting with EtOAc/heptanes in different proportions to yield the target product
1
(296.3 mg, 64%) as a light yellow solid: H NMR (400 MHz, CDCl3) δ ppm 8.63 (s, 1H), 8.00 - 8.18 (m, 4H), 7.37 -
13
7.61 (m, 7H), 7.24 – 7.27 (m, 1H), 4.41 (q, J = 7.28 Hz, 2H), 2.81 (s, 3H), 1.47 (t, J = 7.28 Hz, 3H) C NMR (101
MHz, CDCl3) δ ppm 197.9, 142.9, 140.7, 137.3, 136.2, 132.9, 131.3, 129.8, 128.6, 126.9, 126.6, 126.6, 126.5,
126.2, 125.3, 124.4, 124.3, 123.3, 122.7, 120.9, 120.0, 109.0, 108.1, 37.9, 19.9, 13.9. HRMS calcd for C26H22NO
(M+H)+: 364.1684, found HRMS: 364.1701.
537
538
539
540
541
542
543
544
545
546
547
548
549
9-ethyl-3-(4-methylpiperazine-1-carbonyl)-9H-carbazole (9). 9-ethyl-9H-carbazole-3-carboxylic acid 7 (200 mg,
0.84 mmol), 1-methylpiperazine (171 mg, 1.29 mmol), DIPEA (284 µL, 1.7 mmol), and DMAP (122 mg, 1 mmol)
were added to DCM (26 mL) under N2. The obtained solution was cooled on an ice-water bath. EDC (275 mg,
1.44 mmol) was added to the solution, and the reaction mixture was then allowed to warm to room
temperature and stirred for 16 h. The solvent was removed in vacuo, and the obtained residue was extracted
into ethyl acetate (100 mL). The organic layer was washed consecutively with 5% citric acid solution (50 mL × 3),
concentrated NaHCO3 (50 mL × 3), brine (50 mL), dried over MgSO4, filtered, and concentrated in vacuo. The
residue was purified on silica gel using heptanes/ethyl acetate in different proportions to afford 199 mg (74%) of
9 as a yellow glass. 322.1952 1H NMR (400 MHz, CDCl3) δ ppm 8.24 (s, 1H), 8.10 (d, J=7.45 Hz, 1H), 7.50 - 7.59 (m,
2H), 7.41 - 7.48 (m, 2H), 7.29 (t, J=7.53 Hz, 1H), 4.40 (q, J=7.19 Hz, 2H), 3.72 (br s, 1H), 2.52 (br s, 1H), 2.44 (s,
13
3H), 2.17 (s, 1H), 1.40 - 1.49 (m, 4H) C NMR (101 MHz, CDCl3) δ ppm 167.78, 141.17, 140.49, 126.63, 125.27,
122.83, 122.51, 120.72, 120.66, 119.79, 108.93, 108.46, 53.47, 43.58, 37.79, 26.89, 13.78. HRMS calcd for
C20H24N3O (M+H)+: 322.1913, found: 322.1952.
550
551
552
553
554
555
556
557
558
3-(4-chlorobenzoyl)-9-ethyl-9H-carbazole (10). Under Ar, AlCl3 (1.13 g, 8.44 mmol) was added to a solution of 5
(1.5 g, 7.68 mmol) in anhydrous benzene (20 mL), and the solution was cooled by an ice bath for 20 min. 4-
chlorobenzoic acid (1.12 mL, 8.83 mmol) was added dropwise via a syringe to the solution, which was tightly
capped in a microwave vessel and subjected to microwave irradiation at 100 °C for 1 h and then cooled to room
temperature. The reaction mixture was cooled on an ice-water bath, then poured onto a mixture of ice and a 4
M NaOH solution (50 mL) and extracted with ethyl acetate (150 mL). The organic phase was washed with
saturated aqueous NaHCO3 and brine, dried (MgSO4), filtered and evaporated in vacuo. The residue was purified
by column chromatography on silica gel eluting with EtOAc/heptanes in different proportions to yield the target
product (1.374 g, 54%) as a white solid: 1H NMR (400 MHz, CDCl3) δ ppm 8.63 (d, J=1.30 Hz, 1H), 8.16 (d, J=7.77
25

ACCEPTED MANUSCRIPT
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Hz, 1H), 8.04 (dd, J=8.58, 1.67 Hz, 1H), 7.87 - 7.98 (m, 2H), 7.56 (td, J=7.64, 1.03 Hz, 1H), 7.45 - 7.53 (m, 2H), 7.29
13
- 7.37 (m, 1H), 7.17 - 7.28 (m, 2H), 4.43 (q, J=7.23 Hz, 2H), 1.51 (t, J=7.23 Hz, 3H) C NMR (101 MHz, CDCl3) δ
ppm 195.06, 166.11, 163.60, 142.43, 140.57, 135.12, 135.09, 132.36, 132.28, 128.25, 128.22, 126.46, 123.76,
123.04, 122.51, 120.68, 119.92, 115.30, 115.08, 108.95, 107.99, 37.75, 13.74. ESI: m/z 334.1 (M + H)+. HRMS
calcd for C21H17ClNO (M + H)+ 334.0999, found 334.0981.
564
565
566
567
568
569
570
571
9-ethyl-3-(4-fluorobenzoyl)-9H-carbazole (11). Using 9-ethyl-9H-carbazole 5 (1.57 g, 8.05 mmol) and 4-
fluorobenzoic acid (1.1 mL, 9.26 mmol), as starting compounds, the title compound was prepared by the
1
procedures described in the preparation of compound 10 to yield 1.07 g (42%) of 11 as an orange oil. H NMR
(400 MHz, CDCl3) δ ppm 8.56 (d, J = 1.7 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.97 (dd, J = 8.6, 1.7 Hz, 1H), 7.86 (dd, J =
8.6, 5.6 Hz, 2H), 7.50 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.43 - 7.40 (m, 2H), 7.33 – 7.23 (m, 1H), 7.17 (t, J = 8.6 Hz,
13
2H), 4.36 (q, J = 7.2 Hz, 2H), 1.44 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl3) δ ppm 195.28, 142.56, 140.62,
137.93, 137.30, 131.28, 128.60, 128.45, 128.26, 128.06, 126.53, 123.89, 123.08, 122.58, 120.75, 120.01, 109.00,
108.07, 37.83, 26.87, 13.79. HRMS calcd for C21H17FNO (M + H)+ 318.1294, found 318.0766.
572
573
574
575
576
577
578
579
9-ethyl-3-(4-methylbenzoyl)-9H-carbazole (12). Using 9-ethyl-9H-carbazole 5 (325 mg, 1.67 mmol) and p-toluoyl
chloride (246 mg, 2.00 mmol) as starting compounds, the title compound was prepared by the procedures
1
described in the preparation of compound 10 to yield 241 mg (46%) of 12 as a yellow glass. H NMR (400 MHz,
CDCl3) δ ppm 8.63 (d, J=1.30 Hz, 1H), 8.16 (d, J=7.77 Hz, 1H), 8.04 (dd, J=8.58, 1.67 Hz, 1H), 7.64 - 7.73 (m, 2H),
7.56 (td, J=7.64, 1.03 Hz, 1H), 7.29 - 7.37 (m, 1H), 7.17 - 7.28 (m, 4H), 4.43 (q, J=7.23 Hz, 2H), 2.31 (s, 3H) 1.51 (t,
J=7.23 Hz, 3H) 13C NMR (101 MHz, CDCl3) δ ppm 197.02, 143.46, 141.52, 135.19, 135.07, 131.36, 131.28, 128.15,
128.12, 126.46, 123.76, 122.64, 122.22, 120.66, 119.72, 115.90, 115.68, 108.95, 107.54, 36.98, 21.43, 13.13.
HRMS calcd for C22H20NO (M+H)+: 314.1539, found: 314.1545.
580
581
582
583
584
585
586
587
1-(9-ethyl-9H-carbazol-3-yl)-2-phenylethan-1-one (13). Using 9-ethyl-9H-carbazole 5 (325 mg, 1.67 mmol) and
phenylacetyl chloride (246 mg, 2.00 mmol) as starting compounds, the title compound was prepared by the
procedures described in the preparation of compound 10 to yield 299 mg (57%) of 13 as a yellow glass. 1H NMR
(400 MHz, CDCl3) δ ppm 8.81 (d, J=1.30 Hz, 1H), 8.24 (d, J=7.77 Hz, 1H), 8.09 (dd, J=8.58, 1.67 Hz, 1H), 7.59 - 7.69
(m, 2H), 7.29 - 7.37 (m, 2H), 7.23 - 7.36 (m, 5H), 4.43 (q, J=7.23 Hz, 2H), 4.12 (s, 2H), 2.31 (s, 3H), 1.51 (t, J=7.23
Hz, 3H) 13C NMR (101 MHz, CDCl3) δ ppm 195.38, 141.72, 135.66, 129.74, 129.13, 128.19, 128.42, 126.35,
124.32, 123.64, 122.22, 120.63, 119.79, 115.57, 115.35, 108.78, 107.54, 44.43, 36.98, 13.13. HRMS calcd for
C22H20NO (M-H)- 314.1539, found: 314.1530.
588
589
9-ethyl-3-(naphthalene-1-carbonyl)-9H-carbazole (14). Using 9-ethyl-9H-carbazole 4 (100 mg, 0.40 mmol) and
1-naphthoyl chloride (74 mL, 0.71 mmol) as starting compounds, the title compound was prepared following the
26

ACCEPTED MANUSCRIPT
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594
procedures described in the preparation of compound 10. A yellowish viscous oil was obtained. Yield: 141.1 mg
(41.5%). 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 8.16(d, 1H), 8.04 (t, 2H), 7.96 (d, 1H), 7.51(d, 2H), 7.42(m, 2H),
7.33 (m, 4H), 7.18(t, 1H), 4.18 (q, 2H), 1.45 (t, 3H). 13C NMR (101 MHz, CDCl3) δ 197.79, 143.02, 140.73, 137.27,
133.77, 130.44, 128.64, 128.37, 126.56, 126.38, 125.97, 124.58, 124.33, 120.89, 120.12, 109.05, 108.13, 37.92,
13.87. HRMS calcd for C25H19NO (M+H)+: 350.1466, found: 350.1536.
595
596
597
598
599
600
9-propyl-9H-carbazole (15). Using carbazole (550 mg, 2.52 mmol) and 1-bromopropane (369 mg, 3 mmol), as
the starting compounds, the title compound was prepared by the procedures described in the preparation of
1
compound 10 to yield 410 mg (74%) of 15 as a yellow oil. H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 7.8 Hz, 2H),
7.44 (ddd, J = 8.2, 6.9, 1.2 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H), 7.21 (ddd, J = 8.0, 6.8, 1.1 Hz, 2H), 4.24 (t, J = 7.2 Hz,
2H), (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 140.71, 125.75, 123.00, 120.53, 118.90, 108.89,
44.80, 22.51, 12.02.
601
602
603
604
605
606
607
608
609
9-propyl-3-[(4-methylnaphthalen-1-yl)carbonyl]-9H-carbazole (16). Using 9-propyl-9H-carbazole (350 mg, 1.67
mmol) and 4-methyl-1-naphthoyl chloride (246 mg, 2.00 mmol) as starting compounds, the title compound was
prepared by the procedures described in the preparation of compound 10 to yield 327 mg (52%) of 16 as a
yellow glass. 1H NMR (400 MHz, CDCl3) δ ppm 8.62 (s, 1H), 8.14 (d, J=8.53 Hz, 1H), 8.04 (t, J=9.03 Hz, 2H), 7.96 (d,
J=7.78 Hz, 1H), 7.47 - 7.55 (m, 2H), 7.38 - 7.47 (m, 2H), 7.27 - 7.38 (m, 3H), 7.18 (t, J=7.40 Hz, 1H), 4.18 (t, J=7.03
Hz, 2H), 2.73 (s, 3H), 1.78 - 1.90 (m, 2H), 0.97 (t, J=7.40 Hz, 3H) 13C NMR (101 MHz, CDCl3) δ ppm 197.63, 143.30,
141.06, 137.08, 132.71, 129.58, 128.35, 126.76, 126.41, 126.04, 125.42, 124.24, 122.97, 122.41, 120.53, 119.84,
118.56, 109.16, 108.57, 44.62, 60.38, 22.72, 19.67, 11.83. HRMS calcd for C27H24NO (M+H)+: 378.1852, found:
378.1851. .
610
611
612
613
614
615
9-(2,2,2-trifluoroethyl)-9H-carbazole (17). Using carbazole (300 mg, 1.80 mmol) and 2-chloro-1,1,1-
trifluoroethane (0.25 mL, 1.28 mmol) as starting compounds, the title compound was prepared by the
1
procedures described in the preparation of compound 10 to yield 372 mg (83%) of 17 as a yellow oil. H NMR
(500 MHz, CDCl3) δ ppm 8.17 (d, J=7.86 Hz, 2H), 7.53 (t, J=7.63 Hz, 2H), 7.37 (d, J=7.86 Hz, 2H), 7.22 - 7.32 (m,
2H), 4.35 (s, 2H), 13C NMR (126 MHz, CDCl3) δ ppm 140.14, 127.44, 122.91, 120.53, 118.77, 111.23, 108.37,
72.14, 19.7.
616
617
618
619
620
3-(4-methylnaphthalene-1-carbonyl)-9-(2,2,2-trifluoroethyl)-9H-carbazole (18). Using 17 (350 mg, 1.41 mmol)
and 4-methyl-1-naphthoyl chloride (246 mg, 2.00 mmol) as starting compounds, the title compound was
prepared by the procedures described in the preparation of compound 10 to yield 363 mg (62%) of 18 as a
yellow glass.1H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 8.00 - 8.19 (m, 4H), 7.36 - 7.59 (m, 7H), 7.24 – 7.27 (m, 1H),
4.37 (s, 2H), 2.79 (s, 3H), 13C NMR (101 MHz, CDCl3) δ ppm 197.6, 142.8, 139.7, 136.6, 136.1, 133.2, 131.2, 129.5,
27

ACCEPTED MANUSCRIPT
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622
128.8, 126.7, 126.9, 126.7, 126.5, 126.2, 125.2, 124.9, 124.6, 123.3, 122.4, 120.9, 120.0, 112.7 109.8, 108.6,
71.8, 19.7. HRMS calcd for C26H19F3NO (M+H)+: 418.1413, found: 418.1345.
623
624
625
626
627
628
629
630
631
632
633
634
635
636
(9-ethyl-9H-carbazol-3-yl)(quinolin-5-yl)methanol (19). A solution of 1.5 M tert-buthyllithium in pentane (9 mL)
was added dropwise in 20 min. at -78 °C to a solution of 3-bromo-9-ethyl-9H-carbazole (1.9 g; 0.007 mol)
dissolved in dry THF (50 mL). The solution was stirred at -78 °C for 1 h. A brownish precipitate formed. TLC in
cyclohexane/DCM 9/1: 100 % conversion. A solution of quinoline-5-carbaldehyde (1.1 g; 0.007 mol) dissolved in
50 mL of dry THF was added dropwise at -78 °C in 15 min. The resulting solution was stirred at -78 °C for 1.5 h.
220 mL of a saturated NH4Cl solution was added. The product was then extracted with EtOAc (200 mL and then
50 mL). The organic phases were combined and washed with water (2X100 mL) and dried over MgSO4. The
solution was concentrated under reduced pressure. The residue was purified by column chromatography on
silica gel eluting with EtOAc/cyclohexanes to afford the pure product as a pale yellow solid. Yield: 1.54 g (62.4%).
1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 4.0, 1H), 8.40 (d, J = 8.6, 1H), 8.12 (s, 1H), 8.06 (d, J = 8.4, 1H), 8.02 (d, J =
7.8, 1H), 7.82 (d, J = 7.1, 1H), 7.78 – 7.67 (m, 1H), 7.45 (t, J = 7.6, 1H), 7.39 (td, J = 4.7, 2.3, 2H), 7.31 (d, J = 8.5,
13
1H), 7.23 (d, J =4.2, 1H), 7.20 (t, J = 7.3, 1H), 6.65 (s, 1H), 4.32 (q, J = 7.2, 2H), 1.39 (t, J = 7.2, 3H). C NMR (101
MHz, CDCl3) δ 149.94, 139.74, 139.66, 133.53, 132.82, 129.69, 128.90, 125.94, 124.93, 124.89, 120.80, 120.53,
119.17, 119.01, 108.70, 108.61, 74.18, 37.65, 13.83.
637
638
639
640
641
642
643
644
645
646
9-ethyl-3-(quinoline-5-carbonyl)-9H-carbazole (20). A suspension of 19 (1.4g; 3.97 mmol) dissolved in DCM (20
mL), PDC (2.95 g, 7.84 mmol) and molecular sieves 4 Å (2.95 g) was stirred for 2 h at r.t. The resulting solution
was filtered over a pad of SiO2 and eluted with AcOEt. 350 mL of solvent (AcOEt and DCM) was collected, and
the solutions were combined and concentrated under reduced pressure to afford the pure product as a pale
yellow solid. Yield: 995 mg (78.7%). 1H NMR (400 MHz, CDCl3) δ 9.04 – 8.92 (m, 1H), 8.62 (s, 1H), 8.49 (d, J = 8.5,
1H), 8.31 (d, J = 8.3, 1H), 8.06 (d, J = 7.4, 2H), 7.87 – 7.77 (m, 1H), 7.75 (d, J = 6.9, 1H), 7.52 (t, J = 7.6, 1H), 7.49 –
13
7.43 (m, 2H), 7.43 – 7.39 (m, 1H), 7.31 – 7.25 (m, 2H), 4.42 (q, J = 7.2, 2H), 1.48 (t, J = 7.2, 3H). C NMR (101
MHz, CDCl3) δ 196.38, 150.91, 148.35, 143.07, 140.74, 137.72, 134.31, 132.13, 129.16, 128.54, 128.02, 127.78,
126.69, 126.63, 124.37, 123.19, 122.84, 121.95, 120.87, 120.24, 109.11, 108.24, 37.96, 13.86. HRMS calcd for
C24H19N2O (M+H)+: 351.1491, found: 351.1487.
647
648
649
650
651
(9-ethyl-9H-fluoren-3-yl)(quinolin-7-yl)methanol (21). To an oven-dried round-bottom flask flushed with N2 was
added 3-bromo-9-ethyl-9H-carbazole (508 mg, 1.85 mmol) in 13 mL dry THF. The mixture was cooled to -78
and tert-BuLi (2M in heptane) (1.85 mL, 3.7 mmol) was added dropwise. The mixture was stirred at -78 for 1
hr, then 8-Quinolinecarboxaldehyde (291 mg, 1.85 mmol) was added. The resulting mixture was stirred at -78
for 1.5 hr, then allowed to warm to 0 . 50 mL sat. NHꢀCl was added, then organics were extracted with EtOAc
℃
℃
℃
℃
28