Synthesis of a Carbazole Intermediate, a Dihydronaphthalene
For Analysis of Antimicrobial and Anti-tumor Effects
Tiffany Layport, Department of Biology, York College
Glycosmis Mauritiana. Eflora.org
The compound that was synthesized is a
derivative of dihydronaphthalene, a naturally
occurring carbazole intermediate. This key
intermediate of Glycomaurrol can be extracted
from the bark of Glycosmis mauritiana. The
dihydronaphthalene was synthesized in order
to test for anti-tumor and antimicrobial
properties present at the intermediate level.
The key intermediate was synthesized via a
Diels-Alder strategy following preparation of
the diene and dienophile. The target
dihydronaphthalene derivative was
synthesized in 5 steps. The final product was
then exposed to E. coli, S.aureus, P. mirabilis,
B. cereus, P. stuartii, C. albicans, S. cerevisiae
and U87-MG Human Glioblastoma cells to
determine medicinal benefits. The
dihydronaphthalene derivative had no effect on
microbial growth inhibition, however it showed
85% U87-MG cell death at maximum
concentration.
Results
Methods
Part I
Scheme 1: Preparation of diene
MgCl
•Effect of synthetic dihydronaphthalene
derivative, an intermediate of Glycomaurrol,
in antimicrobial and anti-tumor bioassays is
unknown.
•This study examines the antimicrobial and
anti-tumor activity of the
dihydronaphthalene derivative at varying
concentrations.
Hypotheses
Antimicrobial Assays
H0: The dihydronaphthalene derivative will
have no effect on microbial growth inhibition.
HA: The dihydronaphthalene derivative with a
concentration range of 0-25mg/mL will show a
difference in microbial growth inhibition from
the untreated organisms.
Anti-tumor Assays
H0: There is no difference between the effect
of the dihydronaphthalene derivative with a
concentration range of 0-0.044M and the
untreated U87-MG cells on cell viability.
HA: There is a difference between the effect
of the dihydronaphthalene derivative within a
concentration range of 0-0.044M and the
untreated U87-MG cells on cell viability.

Scheme 2: Preparation of dienophile
NH2
NHTs
NTs
TsCl
NaIO4
H2O/THF
SiO2
OH
OH
Minimum Inhibitory Concentration
DHN
DMSO
Untreated
AMP
MPA
OH
O
Figure 2. U87-MG Human Glioblastoma cells treated with the
dihydronaphthalene derivative in rows A, B, C, D, and E, columns 1-11. Cells
were treated with DMSO in row E and F, columns 1-11. Cells were treated with
cyclohexamine in row G, columns 1-4. Column 12 contained untreated cells.
Row G, columns 5-12 were blank.
no growth inhibition
no growth inhibition
no growth inhibition
>0.0098mg/mL inhibits growth
>0.001mg/mL inhibits growth
Conclusions
O
Table 2. Percent of viable U87-MG Human
Glioblastoma cells after exposure to a
dihydronaphthalene derivative at varying
concentrations.
Scheme 3: Diels-Alder Reaction
NHTs
NTs
% Viable Cellsa
Concentration (M)
DBU
nb
SEMc
SDd
+
CH2Cl2
•Naturally occurring carbazoles isolated
from the stem bark of Glycosmis mauritiana
have demonstrated antimicrobial and antitumor activity. (Kumar et al. 1989)
•Medicinal benefits of the synthetic
carbazoles are unknown.
Organism
Control
PTSA
Grignard
Introduction
•Regiocontrolled carbazole synthetic
strategies have been developed for cost
efficiency of product yield. Several
carbazoles, Glycomaurrol and its
derivatives Eustifolines A-D have been
successfully synthesized. (Lebold and Kerr
2007)
Table 1. Minimum concentration of Dihydronaphthalene,
DMSO, Ampicillin, Mycophenolic Acid, and no treatment
required to inhibit organismal growth.
O

0.044
0.022
0.011
0.006
0.003
0.001
0.0007
0.0003
0.0002
0.00009
0.00004
OH
Dihydronaphthalene Derivative
The synthesis of the dihydronaphthalene was monitored via thin
layer chromatography (TLC) and purified via flash column
chromatography (FCC). The success of the reactions was
confirmed by Fourier Transform Infrared Spectroscopy (FTIR)
and Nuclear Magnetic Resonance Spectroscopy (NMR).
Part II
Antimicrobial and anti-tumor bioassays were
performed in a 96 well microtiter plate.
Antimicrobial
•100 uL of appropriate media was placed in all rows for columns
2-12.
•200 uL of DHN was placed in rows A, B, E, and F for column 1
only.
•200 uL of DMSO was placed in rows D and H for column 1
only.
•200 uL of specified control (AMP or MPA) was placed in rows C
and G for column 1 only.
•Each row was diluted via a 2-fold serial dilution in columns 110.
•100 uL of appropriate organism was placed in all rows from
column 1-11.
•Column 11 contained organism and media only.
•Column 12 contained media only.
•Plate was allowed to incubate at 37oC for 48 hours before
results were recorded.
Anti-tumor
•150 uL U87-MG cells were placed in all rows for columns 1-11.
•The cells set for 24 hours to allow reattachment.
•100 uL of DHN was placed in rows A, B, C, and D for column 1
only.
•100 uL of DMSO was placed in rows E and F for column 1 only.
•100 uL of cycloheximide was placed in rows G and H for
column 1 only.
•Each row was diluted via a 2-fold serial dilution in columns 111.
•50 uL of U87-MG cell media was placed in all rows for columns
1-11.
•Column 12 contained U87-MG cells and media only.
•The cells were allowed to incubate at 37oC for 48 hours.
•Celltiter 96 aqueous reagent proliferation assay was added to
all wells.
•The absorbance for each well was recorded at 490nm.
15.025
8.550
55.725
47.275
64.125
91.0
99.225
100.0
98.5
97.95
87.35
5
5
5
5
5
5
5
5
5
5
5
5.262
5.128
8.390
9.390
7.256
9.0
0.775
0.0
1.5
2.05
748
10.524
10.256
16.779
18.780
14.53
18.0
1.55
0.0
3.0
4.1
9.496
a
mean
b sample size
c standard error of the mean
d standard deviation
Antimicrobial
•The results showed that the
dihydronaphthalene derivative had no effect on
the growth inhibition for the organisms being
tested. Therefore, the null hypothesis must be
accepted, stating that there is no significant
difference between the effect of the
dihydronaphthalene derivative and the
untreated organisms that were used as a
control group. It is implied that the minimum
inhibitory concentration if any is greater than the
tested range of drug concentration.
Anti-tumor
•The P value= 0.0045 indicating that there is a
significant difference in the mean percent of cell
viability between maximum dihydronaphthalene
derivative concentration and the untreated cells.
Therefore, the null hypothesis can be rejected.
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Table 3. Percent of viable cells present at minimum
and maximum concentrations of a dihydronaphthalene
derivative.
Concentration (M)
% Viable Cellsa
Minimum (0.0)
Maximum (0.044)
87.3
15.025
SDb
SEMc
9.5
4.75
10.524 5.262
mean
b standard deviation
c standard error of the mean
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DM SO
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a
Mean Percent Viable
Glioblastoma Cells (%)
Abstract
0.01
0.02
0.03
0.04
0.05
Concentration of Dihydronaphthalene Derivative (M)
Figure 1. Mean percent viable U87-MG Human
Glioblastoma cells from absorbance values at 490nm
after exposure to a dihydronaphthalene derivative at
varying concentrations of a 2-fold serial dilution. Data
are means and standard errors from five replicates
for each concentration. P= 0.0045.
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Acknowledgements
I would like to thank Dr. Thompson and Dr. Halligan for mentoring this
research.
I would also like to thank Dr. Singleton and Dr. Mathur for their guidance and
contributions.
Thank you to the Biology Department and faculty.