Identification of 4-isopropyl-thiotropolone as a novel anti-microbial: Regioselective synthesis, NMR characterization, and biological evaluation

Article abstract of DOI:10.1039/c8ra06297h

We have synthesized and separated tosylated thujaplicin isomers for the first time, and elucidated their structures using 1D, 2D-NMR techniques and X-ray crystallography. The tosylated isomers were used to synthesize 4-isopropyl-thiotropolone and 6-isopro

Full text of DOI:10.1039/c8ra06297h

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Identification of 4-isopropyl–thiotropolone as
a novel anti-microbial: regioselective synthesis,
NMR characterization, and biological evaluation†
Cite this: RSC Adv., 2018, 8, 29967
ab
a
c
d
Mohamed Elagawany, Lamees Hegazy, Feng Cao, Maureen J. Donlin,
e
f
ag
Nigam Rath, John Tavis and Bahaa Elgendy
*
We have synthesized and separated tosylated thujaplicin isomers for the first time, and elucidated their
structures using 1D, 2D-NMR techniques and X-ray crystallography. The tosylated isomers were used to
1
synthesize 4-isopropyl–thiotropolone and 6-isopropyl–thiotropolone in a regioselective manner. H and
13
C Chemical shifts of synthesized isomers were fully assigned using several NMR experiments, and their
isotropic magnetic shielding was calculated using the GIAO (Gauge Including Atomic Orbitals) method
and the B3LYP def2-TZVPP level of theory. The calculated chemical shift values were in a good
agreement with the experimental results. The biological activity of all synthesized compounds was
evaluated against the fungal pathogen Cryptococcus neoformans and four different bacterial strains
Received 25th July 2018
Accepted 20th August 2018
(Staphylococcus aureus (ATCC 29213), E. coli (ATCC 35218), Acinetobacter baumannii and Pseudomonas
aeruginosa (ATCC 27853)). 4-Isopropyl–thiotropolone was found to inhibit Staphylococcus aureus in
a low micro molar range and exhibit good therapeutic index and ADME properties. This compound can
be used for future lead optimization to design inhibitors against Staphylococcus aureus (ATCC 29213).
DOI: 10.1039/c8ra06297h
rsc.li/rsc-advances
9
familial Mediterranean fever since the time of ancient Egyp-
tians. Hinokitiol (b-thujaplicin) (2) is among the known tropo-
Introduction
10
The troponoid core is well known in hundreds of natural prod- lone derivatives that showed inhibitory effects against Chlamydia
ucts. The tropone ring has a unique structure and properties and trachomatis and has clinical potential to be used as a topical drug
11
despite their non-benzenoid nature, they are planar and possess to prevent sexually transmitted diseases.
aromatic character. Natural and synthetic compounds that have
Thiotropolones have not been widely explored compared to
the troponoid core have profound biological activities and have structurally related hydroxytropolones. Some examples exist in
1
–3
4
been used as antitumor,
antifungal, and antimicrobial literature that shows anticancer activity such as thiocolchicin
12 13
5
agents. The biological activities of these compounds has been (3), which can act as tubulin or topoisomerase II inhibitor.
mainly attributed to their inhibitory effect on bimetallic Thiotropocin (4) is another structurally related antibiotic that
enzymes. Colchicine (1) is the most recognized member of this was isolated from Pseudomonas sp. CB-104 in 1984.
6,7
14,15
This
8
family because it has been used for the treatment of gout and antibiotic was found to exhibit antibacterial, antifungal, and
antiprotozoal activities in vitro. Recently, Donlin and co-
4
a
workers reported that thiotropolone 5 can inhibit the growth
of the fungus Cryptococcus neoformans with a minimal 80%
Departments of Pharmacology and Physiology, Saint Louis University School of
Medicine, St. Louis, MO, USA
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Damanhour inhibitory concentration (MIC80) of 0.25 mM. In our effort to
University, Damanhour, Egypt
design and synthesize new thiotropolones to treat pathogenic
fungi and multidrug resistant bacteria, we used the natural
product b-thujaplicin (2) as starting point (Fig. 1).
c
John Cochran Division, Department of Veteran's Affairs Medical Center, 915 North
Grand Blvd., St. Louis, MO 63106, USA
d
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis
Here, we report for the rst time the isolation of the
University School of Medicine, St. Louis, Missouri, USA
e
intractable mixture of tosylated thujaplicines, 8 and 9, their
full NMR assignments and the X-ray structure of 8. Moreover,
Department of Molecular Microbiology and Immunology, The Saint Louis University we report the synthesis, full NMR chemical shi assignments
Department of Chemistry and Biochemistry, Center for Nanoscience, University of
Missouri – St. Louis, One University Boulevard, St. Louis, USA
f
Liver Center, Saint Louis University School of Medicine, St. Louis, Missouri, USA
g
of 4-isopropyl and 6-isopropyl-2-mercaptocyclohepta-2,4,6-
trien-1-one, 10 and 11, and their anti-Cryptococcus neofor-
mans and antibacterial activity. Moreover, we have evaluated
the ADME (adsorption, distribution, metabolism, excretion
and toxicity) properties of tested compounds.
Chemistry Department, Faculty of Science, Benha University, Benha 13518, Egypt.
E-mail: belgendy@fsc.bu.edu.eg
†
Electronic supplementary information (ESI) available. CCDC 1856450. For ESI
and crystallographic data in CIF or other electronic format see DOI:
0.1039/c8ra06297h
1
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Fig. 1 Examples of biologically active tropolones.
Fig. 2 (A) b-Thujaplicin anions. (B) Tosyloxycolchicides 6 and 7.
Results and discussion
Tosylation is usually the rst step toward functionalization of
hydroxytropolones to prepare biologically active derivatives.
Under standard conditions of tosylation, where base is used in
excess, the formed anion of unsymmetrical hydroxytropolones
(Fig. 2A) undergoes isomerization to give a mixture of isomers.
During their attempt to synthesize O-alkyl derivatives of 4-iso-
propyl–tropolone as ribonucleotide reductase inhibitors, Crozet
Scheme 1 Synthesis of compounds 8 and 9.
16
and his co-workers observed the formation of a mixture of 4-
and 6-isopropyl derivatives (1 : 1 mixture). They were only able
to obtain sufficient amounts of the 4-isopropyl derivatives in
a pure form suitable for testing. Similarly, tosylation of colchi-
cine (1) gave the biologically inactive 9-tosyloxycolchicide
isomer (6) as the major product (z74%) while the biologically
active 10-tosyloxycolchicide isomer (7) was the minor product
by hydrogenation using Pd/C (10%) in presence of anhydrous
sodium acetate. Nevertheless, we were able to separate the two
isomers using a Reveleris X2 ash chromatography system. The
structures of 8 and 9 (Scheme 1) were elucidated using 1D and 2D
NMR spectroscopy and single crystal X-ray diffraction.
19
Compound 8 was obtained as colorless sharp needles. Its
17
(z26%).
18 4
molecular formula C17H O S was determined by HRESIMS
Tosylated thujaplicines, 8 and 9, are important synthetic
+
1
data (m/z 341.0816 [M + Na] , calc. 341.0818). The analysis of H,
C, COSY, HSQC, and HMBC spectra revealed the presence of
a singlet proton H-7 (d /d 137.7/7.05, C-7). This proton shows
intermediates in the synthesis of many biologically active tro- 13
polones. For example, they were used as an intermediate in the
preparation of copper aminotropones, which were found to be
C
H
two bond correlations with C-1 (d
strong three bond correlations with C-2 (d
8.3). Furthermore, the one methine septet H-8 (d
C-8) shows two bond correlations to C-6 and three bond corre-
lations to C-7. The two-methyl doublet H -9 and H -10 (d /d
2.6/1.17, C-9 and C-10) shows a strong three bond correlation
to C-6. H-4 (d /d 129.1/6.88, C-4) shows strong three bond
C
179.1) and C-6 (d
154.5) and C-8 (d
/d 38.3/2.73,
C
157.3) and
more effective than a commercial toothpaste formulation in
C
C
18
inhibiting plaque formation. The inability to separate 8 and 9
and their use as a mixture of isomers complicates the puri-
cation of the desired ligands. Obtaining such mixture of
isomers is undesirable, and selective tosylation or facile sepa-
3
C
H
3
3
C
H
2
18
ration of isomers is crucial from the synthetic point of view.
C
H
Hinokitiol (b-thujaplicin) (2) was treated with p-toluenesulfonyl
chloride in presence of pyridine in dichloromethane (DCM) at
room temperature to give a mixture of 5-isopropyl-7-oxocyclohepta-
correlations to C-2 and C-6, which further conrms the location
of tosyl and isopropyl groups (Table 1).
1
13
We have calculated both H and C isotropic chemical
shielding of 8 at the B3LYP/def2-TZVPP level of theory. Corre-
lation plots between experimental and calculated chemical
shis shows very good correlations with correlation coefficients
1,3,5-trien-1-yl-4-methylbenzenesulfonate (8) and 3-isopropyl-7-
oxocyclohepta-1,3,5-trien-1-yl-4-methylbenzenesulfonate (9) in
z1 : 1 ratio. The isolation of this mixture was reported to be
19
intractable. To obtain 9 (the isomer of the natural product)
exclusively, it has to be through multistep synthesis. The rst step
involves iodination of 2 in position 7 followed by tosylation of
hydroxyl group at position 2. The nal step is to remove the iodine
2
13
1
(
R ) of 0.9946 and 0.9755 for C and H NMR chemical shis,
respectively (Fig. S50†).
Compound 9 was obtained as a white powder. Its molecular
formula C17
H
18
O
4
S was determined by HRESIMS data (m/z
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13
Table 1 Experimental and calculated H and C isotropic chemical
a
shifts (ppm) of compounds 8 and 9
Fig. 3 X-ray crystal structure of compound 8 showing the atom-
numbering scheme.
Atom
d
Exp
8
d
Calc
8
d
Exp
9
Calc
d 9
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
179.1
154.5
129.2
129.6
136.6
157.3
137.7
38.3
188.9
173.1
140.9
142.1
143.4
167.1
155.5
51.2
178.9
154.6
131.7
152.4
130.5
137.0
138.9
38.0
189.1
171.4
145.6
167.8
138.2
144.5
152.4
48.1
group is in position 6 of the tropolone ring. Compound 8 was
crystallized from ethyl acetate (0.2 mL), ether (0.5 mL), and
hexanes (10 mL) by solvent evaporation. Crystallographic data is
given in Table S1.† The crystals of compound 8 crystallize in the
monoclinic space group P2 /c with four molecules in the unit
1
˚
cell (Z ¼ 4) of dimensions a ¼ 5.62, b ¼ 28.31, c ¼ 9.51 A and b ¼
ꢀ
˚
3
C-9 and C-10
C-11
C-12 and C-16
C-13 and C-15
C-14
C-17
H-3
H-4
H-5
22.6
28.9
22.7
28.3
92.4 . V ¼ 1512.31(11) A .
133.4
128.4
129.2
145.3
21.7
7.29
6.88
6.98
151.9
141.4
138.5
157.3
27.3
8.12
7.69
7.73
133.4
128.5
129.6
145.6
21.8
7.36
—
6.93
7.14
7.02
2.80
1.21
7.90
7.33
2.43
151.7
141.4
138.7
157.2
27.4
8.31
—
7.68
7.94
8.044
3.64
2.05
10.07
8.28
The 7-membered ring is disordered over 2 orientations
(80 : 20%). The disorder was modelled with partial occupancy
atoms and it indicates that the 7-membered ring was
“breathing” by moving from less hindered to more hindered
conrmation which facilitate the migration of tosyl group in
substitution reactions. The tosyl group in 8 and 9 were shown to
be uxional and undergo thermally-induced shi between the
two thujaplicin oxygen atoms. Molecular mechanics calcula-
tions by the MMFF94 force eld suggests that this process takes
H-6
H-7
H-8
—
—
7.05
2.73
1.17
7.89
7.31
2.42
8.23
3.47
2.01
10.15
8.27
3.35
20
H-9 and H-10
H-12 and H-16
H-13 and H-15
H-17
place most likely through bipyramidal intermediate.
-Isopropyl–thiotropolone (10) and 4-isopropyl–thio-
tropolone (11) were readily accessible in good yields by reacting
and 9 with sodium hydrosulde in ethanol at room temper-
6
3.3445
8
a 13
1
C assignments were based on HSQC and HMBC spectra.
H
ature (Scheme 2). The structures of 10 and 11 were fully
assigned using 1D and 2D NMR spectroscopy.
1
assignments were based on 1D- H NMR and COSY spectra.
2-Hydroxy-2,4,6-cycloheptatrien-1-one
(tropolone
12)
+
1
13
possesses hydroxyl group alpha to the carbonyl group and is
expected to exhibit keto–enol tautomerism. Interestingly, this
compound exists almost exclusively in the enol form (12A),
which possess resonance stabilization because of its aroma-
ticity. Density functional theory calculations of the keto–enol
3
41.0818 [M + Na] , calc. 341.0815). The analysis of H, C,
COSY, HSQC, and HMBC spectra revealed the presence of
a singlet proton (d /d 131.7/7.36, C-3). This proton shows two
bond correlations with C-2 (d 154.5) and C-4 (d 152.4) and
strong three bond correlations with C-1 (dC 178.9) and C-8 (dC
8.0). Furthermore, the one methine septet H-8 (d /d 38.0/2.80,
C
H
C
C
3
C
H
C-8) shows two bond correlations to C-4 (dC 152.4) and three
bond correlations to C-3. The two-methyl doublet H -9 and H
0 (d /d 22.7/1.21, C-9 and C-10) shows a strong three bond
correlation to C-6. H-6 (d /d 137.0/7.14, C-6) shows strong three
bond correlations to C-1 and C-4, which further conrms the
location of carbonyl and isopropyl groups. Besides, H-7 (d /d
38.9/7.02, C-7) shows a strong three bond correlation to C-2,
which conrms the position of tosyl group (Table 1).
3
3
-
1
C
H
C
H
C
H
1
1
13
We have calculated both H and C isotropic chemical
shielding of 9 at the B3LYP/def2-TZVPP level of theory. Corre-
lation plots between experimental and calculated chemical
2
shis shows very good correlations with R of 0.9962 and 0.9789
1
3
1
for C and H NMR chemical shis, respectively (Fig. S51†).
Our NMR assignments were further conrmed by obtaining
an X-ray crystal structure of compound 8 (Fig. 3). The X-ray
shows that the tosyl group is in position 2 while isopropyl
Scheme 2 Synthesis of 6-isopropyl–thiotropolone (10) and 4-iso-
propyl–thiotropolone (11).
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Fig. 4 Hydroxytropolone tautomers (12A–C) and mercapto tropolone
tautomers (13A and B).
1
1
Fig. 5 Key H– H COSY and HMBC correlations of 10A and 11A.
equilibrium constants of 2-hydroxy-2,4,6-cycloheptatrien-1-one
(
1
12A) and 3,5- and 3,6-cycloheptadiene-1,2-dione (12B and
Compound 10 was obtained as a dark red oil. Its molecular
+
2C) showed that the equilibrium is tilted in favour of the enol formula (C H OS) Na was determined by HRESIMS data (m/z
1
0
11
2
+
21
1
(12A) (Fig. 4).
381.0951 [M + Na] , calc. 381.0953). The H NMR showed that
2
The corresponding thiotropolones have seven possible this compound exist in CDCl as two tautomers 10A (z87%)
3
tautomers. The tautomeric equilibrium of all tautomers of thi- and 10B (z13%). We were able to fully assign the chemical
1
otropolone was studied using Hartree Fock (HF) and density shis of the major tautomer 10A (Table 2). The analysis of H,
22
13
functional theory (B3LYP) at different levels of theory. Calcu-
lating electronic energy and Gibbs free energy showed that the a singlet proton H-3 (d /d 119.6/7.45, C-3). This proton shows
C, COSY, HSQC, and HMBC spectra revealed the presence of
C
H
enol forms were more stable than the keto forms because of its two bond correlations with C-2 (d 172.5) and C-4 (d 161.2) and
C
C
aromaticity. This observation was supported by calculating the strong three bond correlations with C-1 (d 183.4), C-5 (d 132.1)
C
C
nucleus independent chemical shis of NMR chemical shis of and C-8 (d 38.8). Furthermore, the one methine septet H-8 (d /
C
C
22
the studied thiotropolones. Compound 13A (2-hydroxy-2,4,6- dH 38.8/2.91, C-8) shows two bond correlations to C-4, C-9 and C-
cycloheptatriene-1-thione) was the most stable followed by 10 (d 23.4) and three bond correlations to C-3 and C-5. The two-
C
compound 13B (2-mercapto-2,4,6-cycloheptatrien-1-one). The methyl doublet H -9 and H -10 (d /d 23.4/1.27, C-9 and C-10)
3
3
C
H
low energy of these two isomers is attributed to the favourable shows two bond correlations to C-8 and a strong three bond
2
2
interactions between H5-S1 in 13A and H5-O1 in 13B.
C H
correlation to C-4. H-5 (d /d 132.1/7.21, C-5) shows two bond
23
Nozoe et al. was the rst to synthesize 2-mercaptotropone correlations to C-6, and strong three bond correlations to C-3, C-
and it was suggested to exist in two tautomeric forms. Sulfur- 7 and C-8. H-6 (d /d 133.8/7.14, C-3) shows strong three bond
C
H
substituted derivatives were obtained upon alkylation and correlations to C-1 and C-4. H-7 (d /d 143.0/8.45, C-7) shows
C
H
acylation of this compound, which suggests that 2-mercapto- strong three bond correlations to C-2 and C-5 (Fig. 5).
tropone (13B) is the prevailing tautomer. However, studying the
physicochemical properties of 2-mercaptotropone and its
structural analogues 6-isopropyl-2-mercaptotropone (10) and 4-
isopropyl-2-mercaptotropone (11) in depth suggested that the
24
major tautomer is the 2-hydroxyl tropothione (13A).
1
13
Table 2 Experimental and calculated H and C NMR chemical shifts
ppm) of compound 10
(
Calc Calc
d d
Atom
d
Exp 10A
d
Calc 10A
d
Calc 10B Atom
dExp 10A 10A 10B
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
183.4
172.5
119.6
161.2
132.1
133.8
143.0
38.8
210.3
179.0
118.2
164.9
132.6
130.9
159.8
47.8
180.0
185.9
141.2
164.1
133.9
136.8
130.8
47.9
H-3
H-4
H-5
H-6
H-7
H-8
7.45
—
6.71 7.48
—
—
7.21
7.14
8.45
2.91
1.27
1.27
6.98 7.07
6.88 7.18
8.98 8.08
2.62 2.91
1.46 1.42
1.45 1.47
H
H
3
-9
-10
3
1
3
23.4
23.4
26.9
24.3
25.2
24.0
OH or SH 9.50
8.98 4.04 Fig. 6 (A) Experimental C chemical shifts (d) of 10A (vs.) calculated.
1
(B) Experimental H chemical shifts (d) of 10A (vs.) calculated.
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Table 3 Experimental and calculated H and C NMR chemical shifts (ppm) of compound 11
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1
13
Atom
d
Exp 11A
d
Calc 11A
d
Calc 11B
Atom
d
Exp 11A
dCalc 11A
dCalc 11B
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
184.1
172.2
118.2
137.9
133.0
156.5
144.0
39.0
202.0
179.8
117.1
142.0
134.3
161.1
152.8
47.8
181.5
185.5
138.3
140.4
131.1
163.3
135.2
47.7
H-3
H-4
H-5
H-6
H-7
H-8
7.34
7.35
7.19
—
8.62
2.88
1.27
1.27
9.47
7.50
7.54
7.34
—
9.32
2.94
1.47
1.51
7.29
7.51
7.07
—
8.27
2.91
1.51
1.44
4.10
H
H
3
-9
-10
3
23.1
23.1
26.4
25.4
24.0
28.1
OH or SH
11.46
1
13
We have calculated both H and C isotropic chemical
shielding of 10A and 10B at the B3LYP/def2-TZVPP level of
theory. Correlation plots between experimental and calculated
chemical shis shows very good correlations in case of 10A with
2
13
1
R
of 0.9828 and 0.99 for C and H NMR chemical shis,
respectively (Fig. 6). The calculated chemical shi values of
isomer 10B correlated to less extent with the experimental
values (see Fig. S52†).
Compound 11 was obtained as orange microcrystals. Its
+
molecular formula (C10
data (m/z 381.0950 [M
exists in CDCl as a mixture of two tautomers 11A (z96%) and
1B (z4%). We were able to fully assign the chemical shis of
H
11OS)
2
Na was determined by HRESIMS
+
2
+ Na] , calc. 381.0953). This compound
3
1
1
13
the major tautomer 11A (Table 3). The analysis of H, C, COSY,
HSQC, and HMBC spectra revealed the presence of a singlet
proton H-7 (d /d 144.0/8.62, C-7). This proton shows two bond
C
H
correlations with C-1 (d
three bond correlations with C-2 (d
Furthermore, the one methine septet H-8 (d
shows two bond correlations to C-6 (d 156.5) and three bond
correlations to C-7. The two-methyl doublet H -9 and H -10 (d /d
3.1/1.27, C-9 and C-10) shows two bond correlations to C-8 and
a strong three bond correlation to C-6. H-5 (d /d 133.0/7.19, C-5)
C
184.1) and C-6 (d
172.2) and C-5 (d
/d 39.0/2.88, C-8)
C
156.5) and strong
C
C
133.0).
C
H
C
3
3
C H
2
C
H
13
Fig. 7 (A) Experimental C chemical shifts (d) of 11A (vs.) calculated.
(
shows two bond correlations to C-6, strong three bond correla-
tions to C-3 and C-8, and four bond correlations to C-2. H-3 (d /d
18.2/7.34, C-3) shows two bond correlations to C-2 and C-4 and
strong three bond correlations to C-1 and C-5. H-4 (d /d 137.9/
.35, C-4) shows two bond correlations to C-5 and strong three
1
B) Experimental H chemical shifts (d) of 11A (vs.) calculated.
C
H
1
C
H
chemical shi values of isomer 11B correlated to less extent
with the experimental values (see Fig. S53†).
7
bond correlations to C-2 and C-6 which further conrms the
location of the isopropyl groups (Fig. 5).
1
13
H and C isotropic chemical shielding of 11A and 11B Computational details
were calculated at the B3LYP/def2-TZVPP level of theory
Table 3). Correlation plots between experimental and
calculated chemical shis shows very good correlations in
2
5,26
Electronic structure calculations were performed using ORCA.
(
Crystal structures of compounds 8 and 9 were optimized at the
DFT theory BLYP with basis sets de2-SVP and RI approximation.
Geometries of compounds 10 and 11, and their isomers were
2
13
1
the case of 11A, with R of 0.994 and 0.9861 for C and H
NMR chemical shis, respectively (Fig. 7). The calculated
27
modelled using the Avogadro program. Their geometries were
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Table 4 MIC80 and CC50 values against E. coli, S. aureus, A. baumannii, P. aeruginosa, and C. neoformans
Bacteria MIC80 (mM)
CC50
S. aureus
E. coli
A. baumannii
P. aeruginosa
C. neoformans
CC50
Comp.
(29213)
(35218)
(from a patient)
(27853)
MIC80
(neutral red)
CC50 (MTS)
4
2
66.7
>100
8.8
>100
16
44.4
51.2
>100
>100
>100
>100
>100
21 (ref. 4)
na
66.4
33.6
34.2
95
8
9
>100
>100
>100
>100
>100
>100
>100
>100
24
24
50
24
31.8
19.5
39.7
30.4
1
1
0
1
64.2
Table 5 Calculated molecular descriptors for prediction of ADME properties for each compound. Recommended values or range for 95% of
known drugs is shown in parenthesis
e
%
Human
a
b
c
d
f
g
h
i
j
k
Comp.
mol_MW
QP log Po/w
HBD
HBA oral absorption
PSA
Qp log S
QPPCaco
#Metab
Qp log BB
QPPMDCK
2
8
9
164.204
318.387
318.387
180.264
180.264
180.264
180.264
1.443
2.1
2.216
2.351
2.4
1
0
0
0.8
1
2.75
6.5
6.5
2.5
2.25
2.5
89.394
92.748
94.321
100
100
100
50.125 ꢁ2.193
68.778 ꢁ2.742
1040.346
975.826
2
2
2
2
2
2
2
ꢁ0.396
ꢁ0.615
ꢁ0.591
0.207
0.168
0.195
516.318
490.424
555.56
4437.612
3962.203
4334.35
3935.7
68.77
ꢁ2.952
1095.291
3286.638
3010.296
3222.618
2998.094
1
1
1
1
0A
0B
1A
1B
28.102 ꢁ2.794
29.939 ꢁ2.881
28.166 ꢁ2.843
2.36
2.398
0.8
1
2.25
100
30.01
ꢁ2.884
0.166
a
c
b
Molar weight in Daltons (130–725). Logarithm of partitioning coefficient between n-octanol and water phases (range for 95% of drugs: ꢁ2 to 6).
d
e
Number of hydrogen bonds donors (0–6). Number of hydrogen bond acceptors (2–20). Predicted human oral absorption on a 0–100% scale,
f
g
based on a multiple linear regression model (<25% low, >80% high). Polar surface area (7–200). Predicted aqueous solubility, log S. S in mol
ꢁ3
h
ꢁ1
dm (ꢁ6.5 to 0.5). Predicted apparent Caco-2 cell permeability in nm s as a model for the gut-blood barrier (<25 poor, >500 excellent).
i
j
k
Number of possible metabolic reactions (2–8). Predicted brain/blood partition coefficient (ꢁ3 to 1.2). Predicted apparent MDCK cell
ꢁ
1
permeability in nm s as a mimic for blood/brain barrier (<25 poor, >500 excellent). Qikrop predictions are for non-active transport.
28,29
30
optimized at the B3LYP functional
and def2-TZVPP basis set.
The mercapto derivative of 8, compound 10, has no anti-
The RijCosX approximation and de2/J auxiliary basis set were bacterial activity against all four tested bacteria. The activity of
1
used during optimization. The absolute H NMR chemical shis 10 against C. neoformans (MIC80 ¼ 50 mM) is >2-fold less than
1
3
H C
(d ), C NMR chemical shis (d ) for all compounds were the corresponding hydroxy tropolone 2. The mercapto deriva-
calculated with the GIAO (Gauge Including Atomic Orbitals) tive of 9, compound 11, which is the direct isomer of 2, showed
3
1–33
method
at B3LYP def2-TZVPP level of theory. The TMS promising antibacterial activity against S. aureus (MIC80 ¼ 16
shielding was calculated at the same level of theory and relative mM), but no activity against the other tested bacteria.
chemical shis were then estimated by subtracting the calculated Compound 11 showed similar activity as 2, 8, and 9 against C.
1
13
chemical shi from TMS. The calculated H and C isotropic neoformans (MIC80 ¼ 24 mM) (Table 4).
chemical shielding of TMS are 31.942/183.923.
We tested the cytotoxicity of the synthesized compounds in
hepatoblastoma cells using an MTS cytotoxicity assay that
measures mitochondrial function and a neutral red assay that
measures lysosomal function and calculated the CC50, which is
the concentration of inhibitor required to reduce cell viability
Biological evaluation
The broad anti-bacterial activity of b-thujaplicin (Hinokitiol, 2)
was reported several decades ago.
34,35
Compound 2 could
50% relative to untreated cells (Table 4). Compound 10 have the
modestly inhibit S. aureus, E. coli, and A. baumannii in our
experimental conditions, with MIC80 values of 66.7, 44.4 and 51.2
mM, respectively. Compound 8, which is the rearranged tosyl
isomer of the natural product 2, has no activity against all tested
bacteria (MIC80 > 100 mM). However, this compound showed
similar activity to 2 against C. neoformans (MIC80 ¼ 24 mM for 8 vs.
MIC80 ¼ 21 mM for 2). Interestingly, compound 9, which is the
lowest toxicity among all synthesized compounds in both
assays. The toxicity of compound 11 is relatively low, and
comparable to the natural product b-thujaplicin (2).
In silico ADME evaluation
tosylated derivative of the natural product 2 showed 8-fold We calculated a set of physical descriptors and pharmaceutical
increase in the antibacterial activity against S. aureus, but properties of all tested compounds using Qikprop to predict
diminished activity against the Gram-negative bacteria E. coli, A. their absorption, distribution, metabolism, and excretion
baumannii, and P. aeruginosa. Compound 9 has similar activity to (ADME) properties (Table 5). All compounds are in compliance
36
37
2
and 8 against C. neoformans (MIC80 ¼ 24 mM) (Table 4).
with Lipinski's rule of ve and Jorgensen's rule of three and
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are predicted to have good oral bioavailability and excellent 3H), 7.08 (s, 1H), 7.01 (d, J ¼ 11.4 Hz, 1H), 6.91 (t, J ¼ 10.3 Hz,
1
3
absorption through cell membranes.
1H), 2.82–2.67 (m, 1H), 2.44 (s, 3H), 1.20 (d, J ¼ 6.7 Hz, 6H);
NMR (100 MHz, chloroform-d) d 179.2, 157.5, 154.6, 145.4,
37.8, 136.7, 133.5, 129.7, 129.6, 129.3, 128.6, 38.4, 22.7, 21.8;
C
1
Conclusions
+
+
LC/MS m/z: 319.1 [M + H] , 341 [M + Na] . HRESIMS m/z
+
+
Tosylated thujaplicin isomers 8 and 9 were separated for the rst 341.0816 [M + Na] (calcd for C17
18 4
H O SNa , 341.0818).
time and their structures were fully characterized. Isomers 8 and
9
were used to synthesize the two mercapto isomers of b-thuja- 4-(1-Methylethyl)-2-[[(4-methylphenyl)sulfonyl]oxy]-2,4,6-
1
13
plicin, 10 and 11, in a regioselective manner. H and
C
cycloheptatrien-1-one (9)
Chemical shis of synthesized isomers were fully assigned using
several NMR experiments, and their isotropic magnetic shielding
was calculated computationally. The calculated chemical shi
values were in a good agreement with the experimental results.
When the biological activity of the synthesized compounds was
evaluated against C. neoformans and four different bacterial
strains, 4-isopropyl–thiotropolone (11) displayed good potency
against Staphylococcus aureus (ATCC 29213). The relatively non-
toxic nature of this compound and excellent predicted ADME
properties make it a potential lead compound to develop potent
inhibitors against Staphylococcus aureus infection.
1
White powder (43%), R
(
3
f
¼ 0.483 (EtOAc/hexanes 1 : 1). H NMR
400 MHz, chloroform-d) d 7.93 (d, J ¼ 7.6 Hz, 2H), 7.41–7.32 (m,
H), 7.17 (dd, J ¼ 12.1, 8.7 Hz, 1H), 7.04 (d, J ¼ 12.3 Hz, 1H), 6.96
(
d, J ¼ 8.6 Hz, 1H), 2.89–2.75 (m, 1H), 2.46 (s, 3H), 1.24 (d, J ¼
13
6
1
2
.8 Hz, 6H); C NMR (100 MHz, chloroform-d) d 178.9, 154.5,
52.4, 145.5, 139.0, 136.9, 133.4, 131.8, 130.5, 129.6, 128.6, 38.1,
2.8, 21.8; LC/MS m/z: 319.1 [M + H] , 341 [M + Na] . HRESIMS
m/z 341.0815 [M + Na] (calcd for C17H O SNa , 341.0818).
18 4
+
+
+
+
General procedure for the synthesis of 10 and 11
NaSH (0.122 g, 2.17 mmol) was added portion wise to a solution
of 8 or 9 (0.150 g, 0.54 mmol) in ethanol (10 mL). The reaction
mixture was stirred for 2 h at room temperature and was
monitored by LCMS and TLC till completion. Ethanol was
evaporated, and the resulting solid was dissolved in water and
acidied with HCl (2 M) then extracted with DCM (3 ꢂ 20 mL) to
yield the desired product. Purication was performed by ash
chromatography using a mixture of EtOAc/hexanes to obtain the
desired compound.
Experimental section
All materials were purchased from commercial suppliers and
used without further purication. Purication was done by Rev-
eleris X2 ash chromatography. The purities of the nal
compounds were characterized by high-performance liquid
chromatography (HPLC) using a gradient elution program
(
9
Ascentis Express Peptide C18 column, acetonitrile/water 5/95 /
5/5, 5 min, 0.05% triuoracetic acid) and UV detection (254 nM).
The purities of nal compounds were 95% or greater. NMR
2
-Hydroxy-4-isopropylcyclohepta-2,4,6-triene-1-thione (10)
1
spectra was recorded on a Bruker NMR 400 MHz Avance III Red dark oil, R ¼ 0.876 (EtOAc/hexanes 1 : 1). H NMR (400
f
1
spectrometer operating at 400 MHz for H NMR and 100 MHz for MHz, chloroform-d) d 9.71 (s, 1H), 8.46 (d, J ¼ 10.6 Hz, 1H), 7.47
1
3
C NMR. Chemical shis are given in part per million (ppm) (s, 1H), 7.23 (d, J ¼ 9.7 Hz, 1H), 7.16 (t, J ¼ 10.2 Hz, 1H), 2.99–
1
3
relative to tetramethylsilane (TMS), coupling constants J are given 2.87 (m, 1H), 1.29 (d, J ¼ 6.9 Hz, 7H); C NMR (101 MHz, CDCl )
3
in Hertz. High-resolution mass spectra were obtained using d 183.5, 172.5, 161.4, 143.1, 134.0, 132.2, 119.7, 38.9, 23.5; LC/
+
+
electrospray ionization on a Bruker 12 T APEX-Qe FTICR-MS with MS m/z: 181 [M + H] . HRESIMS m/z 381.0951 [M + Na]
an Apollo II ion source at the COSMIC Laboratory facility at Old (calcd for (C H OS) Na , 381.0953).
2
+
1
0
11
2
Dominion University, VA.
7-Hydroxy-3-isopropylcyclohepta-2,4,5-triene-1-thione (11)
Synthesis of compounds 8 and 9
1
Orange crystal, R ¼ 0.793, (EtOAc/hexanes 1 : 1). H NMR (400
f
p-Toluenesulfonyl chloride (2.3 g, 12.2 mmol) was added to MHz, chloroform-d) d 9.86 (s, 1H), 8.63 (s, 1H), 7.39–7.26 (m, 2H),
1
3
a solution of tropolone (2 g, 12.2 mmol) and DIPEA (4.3 mL, 24.4 7.26–7.11 (m, 1H), 2.94–2.83 (m, 1H), 1.27 (d, J ¼ 6.9 Hz, 6H);
C
mmol) in DCM (50 mL), and the reaction mixture was stirred NMR (101 MHz, CDCl ) d 184.3, 172.2, 156.6, 144.1, 138.0, 133.1,
3
+
overnight. The resulting mixture was successively washed with 118.3, 39.1, 23.5; LC/MS m/z: 181 [M + H] . HRESIMS m/z
+
+
1
M HCl (30 mL), water (30 mL) and brine (30 mL). The organic 381.0950 [M + Na] (calcd for (C H OS) Na , 381.0953).
2 10 11 2
layer was dried over Na SO , ltered and the solvent was evap-
2
4
orated in vacuo yielding a mixture of the two isomers, which
were separated by ash chromatography using (EtOAc/hexanes
X-ray structure determination of 8
A crystals of approximate dimensions 0.544 ꢂ 0.254 ꢂ 0.132
ꢁ1
3
: 7) with a ow rate 10 mL min to obtain each isomer in
3
mm was mounted on a MiTeGen cryoloop in a random orien-
a pure form.
tation. Preliminary examination and data collection were per-
formed using a Bruker X8 Kappa Apex II Charge Coupled Device
6
-(1-Methylethyl)-2-[[(4-methylphenyl)sulfonyl]oxy]-2,4,6-
cycloheptatrien-1-one (8)
White needles (46%), R
(CCD) Detector system single crystal X-ray diffractometer
equipped with an Oxford Cryostream LT device. All data were
1
f
¼ 0.655 (EtOAc/hexanes 1 : 1). H NMR collected using graphite monochromated Mo K radiation
˚
(400 MHz, chloroform-d) d 7.92 (d, J ¼ 7.3 Hz, 2H), 7.38–7.26 (m, (¼0.71073 A) from a ne focus sealed tube X-ray source.
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Preliminary unit cell constants were determined with a set of 36 Inhibition of C. neoformans growth
narrow frame scans. Typical data sets consist of combinations
of and f scan frames with typical scan width of 0.5 and counting
time of 10 seconds per frame at a crystal to detector distance of
Compounds were tested C. neoformans var grubii, KN99 (sero-
type A, MATa) in a limiting dilution assay with a starting optical
density (650 nM) of 0.001 in YNB-02 (0.67% yeast nitrogen base,
4.0 cm. The collected frames were integrated using an orienta-
0
.2% dextrose, pH 7.0 with 50 mM MOPs) + 1% DMSO. Cells
tion matrix determined from the narrow frame scans. Apex II
and SAINT soware packages (Bruker Analytical X-ray, Madison,
WI, 2016) were used for data collection and data integration.
Analysis of the integrated data did not show any decay. Final cell
constants were determined by global renement of 7781
reections harvested from the complete data set. Collected data
were corrected for systematic errors using SADABS (Bruker
Analytical X-ray, Madison, WI, 2016) based on the Laue
symmetry using equivalent reections.
ꢀ
were incubated without shaking for 48 hours at 35 C and cell
density was measured at 650 nM. The minimal inhibitory
concentration was determined using compound concentrations
from 0.19 to 50 mM of the compound in YNB-02 + 1% DMSO.
Each assay was done in triplicate and all values are the average
of two or more independent assays. The data are presented as
the average cell density as a percent of DMSO-only treated cells.
MICs are reported as the minimal concentration needed to
inhibit 80% of C. neoformans growth relative to vehicle-treated
controls.
Crystal data and intensity data collection parameters are
listed in Table 1S.† Structure solution and renement were
40
carried out using the SHELXTL-PLUS soware package. The
structure was solved and rened successfully in the monoclinic
Cytotoxicity in hepatoma cells
38
4
HepDES19 cells (1.0 ꢂ 10 cells per well) were seeded in 96-
well plates and incubated in Dulbecco's Modied Eagle
Medium (DMEM) with 10% FBS plus 1% penicillin/
streptomycin solution, 1% nonessential amino acids, and 1%
glutamine. The compounds were diluted in the medium to the
desired concentrations at a nal concentration of 1% DMSO
and added to the cells 48 h aer plating, with each concentra-
tion tested in triplicate.
space group P2 /c. Full matrix least-squares renements were
1
2
2 2
carried out by minimizing w(Fo ꢁ Fc ) . The non-hydrogen
atoms were rened anisotropically to convergence. The 7-
membered ring is disordered, and the two orientations were
rened with geometrical restraints to 82 : 18%. All hydrogen
atoms were treated using appropriate riding model (AFIX m3).
The nal residual values and structure renement parameters
are listed in Table 1S.†
Complete listings of positional and isotropic displacement
coefficients for hydrogen atoms, anisotropic displacement MTS cytotoxicity assay (CC )
5
0
coefficients for the non-hydrogen atoms and other geometrical
parameters are listed as ESI (Tables 2S to 7S†). Table of calcu-
lated and observed structure factors are available in electronic
format.
Soluble MTS reagent [3-(4,5-dimethylthiazol-2-yl)-5-(3-carbox-
ymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Promega]
was added 72 h aer incubation, the cultures were incubated for
90 min, and absorbance was read at 490 nm. The CC50 was
calculated as the concentration of inhibitor required to reduce
cell viability 50% relative to untreated cells. The data are plotted
as log[inhibitor] versus response and t to a variable slope
Bacterial strains
Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC
3
4
model using GraphPad Prism (v6, www.graphpad.com).
5218), and Pseudomonas aeruginosa (ATCC 27853) were ob-
tained from the American Type Culture Collection (ATCC). Aci-
netobacter baumannii was collected from the microbiology Neutral red assay (CC )
5
0
laboratory at the John Cochran division of the St. Louis VA
Medical Center (STLVAMC) under a Subcommittee on Research
Safety (SRS)-approved protocol.
Cells were incubated with the compound for 72 hours and
cytotoxicity was measured using a neutral red assay. The data
were transformed to log[inhibitor] and t to a 4-variable slope
curve using GraphPad Prism. The concentration at which 50%
of cells were inhibited relative to vehicle-treated control is re-
ported as the CC50 value.
39
Determination of the minimum inhibitory concentration
MIC80s were determined by the broth microdilution method
recommended by the Clinical and Laboratory Standards Insti-
tute (CLSI) in cation-adjusted Mueller–Hinton II broth
Conflicts of interest
(CAMHB). A 1.5-fold dilution series of the compounds was
prepared in CAMHB. Overnight bacterial culture was added to There are no conicts to declare.
the diluted compounds in a 96 well plate aer adjusting the
5
ꢁ1
bacterial concentration to 5 ꢂ 10 CFU mL in nal concen-
Acknowledgements
ꢀ
ꢀ
tration. Aer 16–24 hours incubation at 35 C ꢃ 2 C, the plates
were read at 600 nM in microplate reader. The MIC80 was This work was nancially supported by Saint Louis University
dened as the concentration of an antibacterial agent that Start up fund (No. 203437). Funding from the National Science
inhibited bacteria growth $80% compared to vehicle-treated Foundation (MRI, CHE-0420497) for the purchase of the Apex-II
control cultures. All values were determined at least twice diffractometer is acknowledged. We would like to thank Ms
independently, and the average number is reported.
Ashley Anderson for help with acquiring NMR.
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