Synthesis and SAR assessment of novel Tubathian analogs in the pursuit of potent and selective HDAC6 inhibitors

Article abstract of DOI:10.1039/c5ob02625c

The synthesis of novel isoform-selective HDAC inhibitors is considered to be an important, emerging field in medicinal chemistry. In this paper, the preparation and assessment of thirteen selective HDAC6 inhibitors is disclosed, elaborating on a previously developed thiaheterocyclic Tubathian series. All compounds were evaluated in vitro for their ability to inhibit HDAC6, and a selection of five potent compounds was further screened toward all HDAC isoforms (HDAC1-11). The capability of these Tubathian analogs to inhibit α-tubulin deacetylation was assessed as well, and ADME/Tox data were collected. This thorough SAR evaluation revealed that the oxidized, para-substituted hydroxamic acids can be recognized as valuable lead structures in the pursuit of novel potent and selective HDAC6 inhibitors.

Full text of DOI:10.1039/c5ob02625c

View Article Online
View Journal
Organic &
Biomol ec ular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: R. De Vreese, Y.
Depetter, T. Verhaeghe, T. De Smet, V. Benoy, W. Haeck, L. Van den Bosch, S. Shuttleworth, S. Kerry and
M. D'hooghe, Org. Biomol. Chem., 2016, DOI: 10.1039/C5OB02625C.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/obc

Page 1 of 17
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB02625C
Synthesis and SAR assessment of novel Tubathian analogs in the pursuit of
potent and selective HDAC6 inhibitors
a
a
b
b
c
c
Rob De Vreese, Yves Depetter, Tom Verhaeghe, Tom Desmet, Veronick Benoy, Wanda Haeck, Ludo Van
c
d
d
a,
Den Bosch, Stephen J. Shuttleworth, Simon Kerry and Matthias D’hooghe *
a
Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
b
Centre for Industrial Biotechnology and Biocatalysis, Faculty of Bioscience Engineering, Ghent University, Coupure Links
6
53, B-9000 Ghent, Belgium
c
Laboratory of Neurobiology and Vesalius Research Center (VIB), Research Institute for Neuroscience and Disease (LIND), KU
Leuven
d
Karus Therapeutics Ltd, Innovation Drive Milton Park 93, Abingdon Oxfordshire OX14 4RZ, United Kingdom
Abstract: The synthesis of novel isoform-selective HDAC inhibitors is considered to be an important, emerging
field in medicinal chemistry. In this paper, the preparation and assessment of thirteen selective HDAC6
inhibitors is disclosed, elaborating on a previously developed thiaheterocyclic Tubathian series. All compounds
were evaluated in vitro for their ability to inhibit HDAC6, and a selection of five potent compounds was further
screened toward all HDAC isoforms (HDAC1-11). The capability of these Tubathian analogs to inhibit α-tubulin
deacetylation was assessed as well, and ADME/Tox data were collected. This thorough SAR evaluation revealed
that the oxidized, para-substituted hydroxamic acids can be recognized as valuable lead structures in the
pursuit of novel potent and selective HDAC6 inhibitors.
Introduction
The interplay between histone acetyltransferases (HATs) and histone deacetylases (HDACs)
1
represents an important epigenetic regulatory mechanism in biochemistry of life processes. This
epigenetic interaction controls the structural transformation of DNA between a compact, inactivated
2
form and a loosely bound, activated form, and thus plays a major role in the functioning of cells.
Besides the regulation of histone acetylation, HATs and HDACs mediate the acetylation of other
proteins as well, and therefore these enzymes are more correctly referred to as lysine
3
acetyltransferases (KATs) and deacetylases (KDACs). The KDAC family can be divided into four
4
classes (I, IIa/IIb, III and IV), existing of 18 proteins. Selective inhibition of these isoforms could
significantly contribute to our knowledge on this family of epigenetic erasers (enzymes known to
catalyze the removal of epigenetic marks), and potentially lead to new drugs. One of these proteins,
defined as HDAC6, emerged in recent years as a valuable target in drug design and belongs to the
class IIb HDACs. Because of its cytoplasmic location, HDAC6 has many interaction partners other than
histones, and this feature renders it an interesting protein to study the acetylation status of proteins
3
in cells. The use of small molecule inhibitors of HDAC6 has been proposed as an efficient strategy to
block its catalytic activity, and is therefore considered to be a valuable new approach in
5
6
7
neurodegenerative diseases, cancer and immunology research.
A milestone achievement in the quest for selective HDAC6 inhibitors concerned the development of
Tubastatin A (1) in 2010, a molecule with a good ‘drug-likeness’ profile that showed great promise in
8
vitro and in vivo. This discovery, together with the growing interest of academia and industry in the
design of small molecule inhibitors, prompted us to pursue new analogs of this lead compound with
1

Organic & Biomolecular Chemistry
Page 2 of 17
View Article Online
DOI: 10.1039/C5OB02625C
possibly enhanced pharmacological properties. Based on available structure-activity relationships
SAR), sulfur analogs 2 of Tubastatin A (1) were constructed recently by our group, as shown in Figure
(
9
1
, and tested for their ability to inhibit HDAC6 in vitro. Within this thiaheterocyclic series, sulfone
derivatives 2c and 2d - designated as Tubathians - exhibited the most pronounced activity and
selectivity toward HDAC6.
The first major objective of the present study comprised a full and thorough biological evaluation of
this Tubathian family 2 to shed more light on their potential as lead structures for HDAC6 inhibitor
design. Furthermore, in view of the promising preliminary results of these Tubathian molecules, an
expansion of compound library 2 to general structures 3 was envisioned as a second major objective
to study structure-activity relationships in more detail. Guided by in-house docking studies and by
the advancing progress made in the literature with regard to selective HDAC6 inhibitor
1
0
development, three main structural modifications of template molecules 2 were proposed. First,
modification of the ring size of the non-aromatic C-ring (a six-membered versus a five-membered
thiaheterocyclic ring) was pursued. A second key modification of the core scaffold molecule
comprised assessment of the oxidation state of the sulfur atom, implying the selective synthesis of
sulfides, sulfoxides and sulfones. Recently, also meta-substituted benzohydroxamic acids have been
1
1
studied and showed dual HDAC6/8 selectivity. Hence, the third structural variation involved the
synthesis and evaluation of the meta-substituted counterparts of the Tubathian core structure. Once
in hand, this set of compounds 2 and 3 will then be subjected to an elaborate biological investigation
of their medicinal relevance as potential efficient and selective HDAC6 inhibitors.
Figure 1. Expanded SAR of sulfur analogs of Tubastatin A (1).
Results and Discussion
In silico docking studies of the proposed compounds 3 using a homology model of HDAC6 revealed
that all theoretical structures fit the binding pocket quite well and thus represent compounds worth
to be studied (Figure 2). In general, the sulfone derivatives proved to have slightly higher predicted
binding energies (better binding) than the corresponding sulfides, due to additional interactions of
the sulfone group with surrounding residues. The para-substituted compounds resulted in better
binding energies than the meta-substituted ones, and phenyl substitution (R = Ph) on the aromatic
ring seemed to be preferred because of π-stacking interactions with the side chain of a phenylalanine
amino acid.
2

Page 3 of 17
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB02625C
However, it must be emphasized that the differences in calculated binding energies of these virtual
complexes were small, pointing to the necessity of lab synthesis and detailed biological evaluation in
vitro (for more details on these docking studies, see ESI).
Figure 2. Docking of selected molecules from class 3 (left: 3a; right: 3l) in a homology model of HDAC6 (green:
carbon, blue: nitrogen, red: oxygen, yellow: sulfur).
The synthesis of compounds 2 has been reported before by us, and the same approach was used
9
here for the preparation of molecules 3 (Scheme 1, Figure 3). First, the tricyclic indole-containing
‘
cap’-group was synthesized via a bismuth-nitrate catalyzed Fisher-indole synthesis between
1
2
aromatic hydrazines 4 and sulfur-containing cyclic ketones 5. The obtained tricycles 6 were
modified through selective oxidation of the sulfur atom employing meta-chloroperbenzoic acid, with
or without the addition of boron trifluoride, leading to the corresponding sulfoxides 7 and sulfones 8,
respectively.
7
-Bromo-2,3-dihydrothieno[3,2-b]indole 6f (R = Br, n = 0) appeared hard to purify because it
contained the corresponding sulfoxide as a side product, which could not be removed by means of
column chromatography. Therefore, this compound was used as an intermediate toward direct
sulfoxidation, resulting in the synthesis of sulfoxide 7c (R = Br, n = 0). Phenyl-containing sulfone 8f (R
=
1
Ph, n = 1) was obtained through full oxidation of sulfide 6c (R = Br, n = 1) to sulfone 8c (R = Br, n =
), followed by a Suzuki-Miyaura cross coupling. The obtained thiaheterocycles 6, 7 and 8 were N-
deprotonated with sodium hydride and the resulting anion subsequently quenched with methyl 4-
bromomethyl)benzoate or methyl 3-(bromomethyl)benzoate 9 to give methyl esters 10. In the final
(
step, esters 10 were converted to hydroxamic acids 3 upon treatment with a large excess of hydroxyl
amine, which were subsequently used for pharmacological evaluation.
As can be seen from Figure 3, no five-membered cyclic thioether-containing hydroxamic acids (with n
=
0 and x = 0) were obtained, which was due to the fact that reaction of compound 6d (R = H, n = 0)
with sodium hydride and methyl 3-(bromomethyl)benzoate or methyl 4-(bromomethyl)benzoate
always resulted in complex reaction mixtures. To circumvent this problem, an alternative approach
toward the synthesis of these molecules was attempted, in which the synthesis of the ‘cap’-group
1
3
was postponed to a later stage in the reaction pathway, however without any success. In total, a
set of thirteen novel hydroxamic acids 3a-m was prepared and, together with the four earlier
discovered Tubathian HDAC6 inhibitors 2a-d, evaluated for their ability to selectively inhibit HDAC6.
3

Organic & Biomolecular Chemistry
Page 4 of 17
View Article Online
DOI: 10.1039/C5OB02625C
Scheme 1. Synthesis of the expanded Tubathian library 3.
Conditions: a: 1 equiv. ketone 5, 0.2 equiv. Bi(NO ·5H O, MeOH, ꢀ, 3.5 h (30-90%, 6a-f). b: 1 equiv. mCPBA
, 2 h (41-83%, 7a-c). c: 3 equiv. mCPBA (≤77%), THF, 0°C to rt, 2 h
52-80%, 8a-e). d: 2 equiv. phenylboronic acid, Na CO (7 equiv), 0.04 equiv. Pd(PPh , toluene/ethanol/H
2/1/1), ꢀ, N , 8 h (60%, 8f). e: 1) 1 equiv. NaH, DMF, rt, N , 0.5 h 2) 1 equiv. methyl (bromomethyl)benzoate 9,
.01 equiv. KI, DMF, 80°C, N , 2 h (21-80%, 10a-m). f: 100 equiv. NH OH (50% in H O), 50 equiv. KOH (4M in
MeOH), THF, rt or ꢀ, 10 min (5-70%, 3a-m).
3
)
3
2
(
(
(
3 2 5 2 2
≤77%), 4 equiv. BF ·O(C H ) , THF, -20°C, N
2
3
3
)
4
2
O
2
2
0
2
2
2
4

Page 5 of 17
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB02625C
Figure 3. Overview of the newly synthesized hydroxamic acids 3.
A preliminary in vitro screening of their inhibitory potential toward HDAC6 at a concentration of 10
µM learned that the meta-substituted compounds 3a-h only moderately inhibited HDAC6 (34-74%
inhibition, relative to the control; see ESI for data). This stands in sharp contrast to all the para-
substituted compounds 2a-d and 3i-m, as these molecules completely inhibited HDAC6 at this
concentration (99-100% inhibition, relative to the control; see ESI for data). It must be noted that
within the meta-substituted series, phenyl-decorated compound 3e showed the highest inhibition
percentage (74%), as predicted by the docking studies. Subsequently, the IC50-values of the five new
para-substituted compounds 3i-m were determined and compared with the previously obtained
9
results for compounds 2a-d (Table 1). All molecules exhibited low nanomolar IC50-values toward
HDAC6 (≤ 22 nM), and the 6-membered sulfones 2c, 2d and 3k displayed the highest HDAC6
9
inhibitory activity (1.9, 3.7 and 3.4 nM, respectively). As noted in our previous communication, this
could be explained (and confirmed in silico) through hydrogen bond formation of both oxygen atoms
on the sulfone moiety with surrounding residues.
Table 1. In vitro enzyme inhibition data: IC50-values toward HDAC6
Compound
2a
2b
2c
2d
3i
3j
3k
3l
3m
HDAC6 IC50 (nM) 15
22
1.9
3.7
14
9.4
3.4
5.2
16
The selectivity toward HDAC6 was assessed on the enzymatic level through a full-panel HDAC1-11
screening of representative compounds 2b, 2c, 2d, 3j and 3l. Compounds 2b, 2d and 3j were selected
2 2 2 2
to compare the influence of the oxidation state of sulfur (R S, R SO and R SO ) on the selectivity. The
influence of the ring size (thiolane vs. thiane) on the inhibitory selectivity was studied by selection of
hydroxamic acids 2c and 3l. The data in Table 2 reveal that all screened compounds display a similar
selectivity profile. These molecular entities inhibit HDAC1, 2, 3, 10 and 11 at IC -values higher than 5
50
µM, except for the five-membered sulfone 3j, which also shows a reasonable affinity for HDAC11
(IC50 = 0.52 µM). All compounds inhibit HDAC4, 5 and 8 with IC50-values around 1 µM, and HDAC7 and
5

Organic & Biomolecular Chemistry
Page 6 of 17
View Article Online
DOI: 10.1039/C5OB02625C
9
at IC50-values between 0.1 and 1 µM. In all cases, the lowest values can be observed with respect to
HDAC6 inhibition, with IC -values <30 nM. After this in-depth selectivity screening, it can be stated
5
0
that this set of Tubathian and related compounds selectively inhibit HDAC6 in a potent and
pronounced way, but also display some moderate affinity for class IIa HDACs (HDAC4, 5, 7 and 9) and
HDAC8.
a,b
Table 2. HDAC1-11 screening of selected compounds 2b, 2c, 2d, 3j and 3l (IC50-values in µM)
Compound HDAC1 HDAC2 HDAC3 HDAC4 HDAC5 HDAC6 HDAC7 HDAC8 HDAC9 HDAC10 HDAC11
2
2
2
3
b
c
d
j
21
11
12
9.4
12
NC
26
23
29
26
24
NC
1.5
1.6
1.9
1.2
0.6
1.8
0.5
0.5
1.3
0.4
0.0220 0.2
0.0019 0.1
0.0037 0.1
0.0094 0.2
0.0082 0.1
2.8
1.7
0.9
2.4
1.9
0.8
0.3
0.5
0.3
0.2
21
9.7
NC
NC
0.5
17
7.7
5.9
6.4
13
29
>30
>30
3l
a
Reference compound: Trichostatin A (HDAC6 IC50 = 0.0093 µM)
b
8a
Tubastatin A HDAC6 inhibition IC50 = 0.015 µM
NC : IC50-value not calculable. Concentration-response curve shows less than 25% effect at the highest
validated testing concentration (30 µM). >30: IC50 value above the highest test concentration. Concentration-
response curve shows less than 50 % effect at the highest validated testing concentration (30 µM). Dark-grey:
IC50 > 5 µM, light-grey: 5 µM > IC50 > 1 µM, white: IC50 < 1 µM.
Next, the potency and selectivity of compounds 2a-d, 3i-m together with control substance
Tubastatin A was evaluated on a cellular level (Neuro-2a cells) by determining their ability to modify
the acetylation level of α-tubulin (a specific HDAC6 substrate) and histones via Western Blots. First,
all the compounds were tested at 1 µM for both assays. From Figure 4 it can be seen that the HDAC6
inhibitors clearly hyperacetylate α-tubulin at this concentration and do not affect the acetylation
status of histones. Second, Tubathians 2a-d and control molecule Tubastatin A were tested at a range
of concentrations (10, 50, 100, 500 and 1000 nM), revealing that these compounds already
presented a maximal acetyl α-tubulin/α-tubulin ratio at a concentration of 100 nM. Finally, also the
newly synthesized Tubathian analogs 3i-m were tested at a lower concentration of 10 nM, pointing
to the conclusion that compound 3k (together with control substance Tubastatin A) demonstrated an
even more pronounced activity than the other compounds (for more details, see ESI).
Figure 4a and 4b. Comparison of α-tubulin and histone hyperacetylation of compounds 2a-d, 3i-m and control
substance Tubastatin A (Neuro-2a cells, 1 µM).
6

Page 7 of 17
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB02625C
With a strong HDAC inhibition profile for this Tubathian family in hand, the following step involved
acquirement of in vitro ‘ADME’ (Absorption, Distribution, Metabolism, and Excretion) and ‘Tox’
(toxicity) data to know whether further optimization of these compounds in the framework of drug
development is appropriate. Therefore, molecules 2a-d as representative Tubathian ‘mother
structures’ were preliminary screened for their capability to inhibit cytochrome P450 (cytochrome
P450 inhibition can cause unanticipated adverse reactions or therapeutic failures), hERG safety (to
exclude potential cardiotoxicity, as inhibition of the hERG might result in fatal ventricular
tachyarrhythmia), microsomal stability in mouse and human (to measure in vitro intrinsic clearance),
and plasma protein binding in mouse and human (the less bound a compound is to proteins in blood
plasma, the more efficiently it can diffuse or traverse cell membranes) (Table 3). Apparently, whereas
sulfides 2a and 2b were shown to inhibit the cytochrome P450 enzymes at low micromolar
concentrations, which is harmful for possible drug-drug interactions in vivo, the sulfones 2c and 2d
scored much better in this regard. The same can be stated for the hERG safety, showing sulfides 2a
and 2b to be inferior as compared to sulfones 2c and 2d. In the microsomal stability assay and the
plasma protein binding assay, sulfides 2a and 2b seemed difficult to detect, this in contrast to
sulfones 2c and 2d which showed acceptable values in both assays. In summary, sulfones 2c and 2d
clearly demonstrated a much better preliminary ADME/Tox profile than sulfides 2a and 2b and might
thus be considered as potential lead compounds for further elaboration in future research.
Table 3. Preliminary ADME/Tox screening of representative compounds 2a-d
compound
CYP1A2
CYP2CP
CYP2C19
CYP2D6
CYP3A4
hERG
(
IC50 µM)
(IC50 µM)
(IC50 µM)
(IC50 µM)
(IC50 µM)
(IC50 µM)
2
2
2
2
a
b
c
>50
>50
>50
>50
4.7
1.3
9.6
2.1
9.1
3.7
1.3
9.5
1.8
15
5.1
>50
11.8
19.9
>50
>50
>50
>11
>11
d
9.7
compound
Mouse MS
µl/min/mg)
Mouse MS
t1/2 (min)
Human MS
(µl/min/mg)
Human MS
t1/2 (min)
Mouse PPB
(%)
Human PPB
(%)
(
2
2
2
a
b
c
<LOQ
58.3
16.2
22.6
<LOQ
<LOQ
<LOQ
<LOQ
<LOQ
<LOQ
62.7
11.9
42.7
30.6
28.7
26.5
47.0
24.1
26.1
14.7
<LOQ
69.8
58.3
2
d
52.5
<LOQ: peak areas below limit of quantification
Ina final assay, the genotoxicity of six-membered sulfone 2c and five-membered sulfone 3l as
representative examples was evaluated, bearing in mind the known potential mutagenicity
1
4,15
associated with hydroxamic acids.
The Ames fluctuation test toward four strains of Salmonella
typhimurium (TA98, TA100, TA1535 and TA1537), with and without metabolic activation by using rat
liver S9 fraction, revealed that both compounds were only mutagenic toward strain TA1537, with and
without S9, starting at a concentration of 50 µM (for more details, see ESI). No mutagenicity was
detected toward the other strains.
7

Organic & Biomolecular Chemistry
Page 8 of 17
View Article Online
DOI: 10.1039/C5OB02625C
Conclusions
Thirteen novel Tubathian analogs were synthesized and, together with four previously developed
analogs, evaluated in depth as HDAC6 inhibitors. The nine para-substituted compounds showed the
best HDAC6 IC50-values and proved to be selective inhibitors in cells. A detailed study of five selected
representatives revealed that these Tubathian analogs preferentially inhibit HDAC6, although also a
moderate affinity for class IIa HDACs (especially HDAC7 and 9) should be recognized. ADME/Tox
evaluation demonstrated that sulfone analogs 2c and 2d display a much better preliminary
ADME/Tox profile than the corresponding sulfides 2a and 2b, and therefore further research should
be focused on the oxidized analogs as valuable lead structures in the pursuit of novel selective
HDAC6 inhibitors.
Experimental Section
1
13
19
Chemistry. General Methods. H NMR, C NMR and F NMR spectra were recorded at 400, 100.6 or 376.5
MHz (Bruker Avance III) with CDCl or [D ]DMSO as the solvent and tetramethylsilane as the internal standard.
3
6
Mass spectra were obtained with a mass spectrometer Agilent 1100, 70 eV. IR spectra were measured with a
Spectrum One FT-IR spectrophotometer. High resolution electron spray (ES) mass spectra were obtained with
an Agilent Technologies 6210 series time-of-flight instrument. Melting points of crystalline compounds were
measured with a Kofler Bench, type WME Heizbank of Wagner & Munz. The purity of all tested compounds was
1
assessed by H-NMR analysis and/or HPLC analysis, confirming a purity of ≥95%.
Representative procedure for the synthesis of sulfides 6a-f. To a solution of phenyl hydrazine hydrochloride
4
a (12 mmol) and 4,5-dihydro-3(2H)-thiophenone 5b (12 mmol) in methanol (50 mL), was added Bi(NO
2.4 mmol). After being stirred for 3.5 h under reflux, the reaction mixture was poured into water (100 mL), and
bismuth nitrate was removed through filtration over celite. The crude product was extracted with ethyl acetate
100 mL), washed with saturated NaHCO (100 mL), brine (100 mL) and dried over anhydrous MgSO . Filtration
3 3 2
) ·5H O
(
(
3
4
of the drying agent and removal of the solvent in vacuo afforded the crude cyclic thioether 6c, which was
purified by means of column chromatography (EtOAc/PE 1/5) to provide pure 2,3-dihydrothieno[3,2-b]indole
6
c (3.7 mmol, 31%). The synthesis of 1,2,4,9-tetrahydro-3-thia-9-azafluorene 6a and 6-fluoro-1,2,4,9-
9
tetrahydro-3-thia-9-azafluorene 6b has already been described previously. 7-Bromo-2,3-dihydrothieno[3,2-
b]indole 6f was not easily purified because it contained the corresponding sulfoxide as a side product and was
therefore used as an intermediate for further transformation.
1
6
-Bromo-1,2,4,9-tetrahydro-3-thia-9-azafluorene 6c (reaction time 22h). H NMR (400 MHz, CDCl
3
): δ 3.00-
3
.02 (m, 4H), 3.80 (s, 2H), 7.16 (d, J = 8.5 Hz, 1H), 7.23 (d × d, J = 8.5, 1.9 Hz, 1H), 7.57 (d, J = 1.9 Hz, 1H), 7.83
1
3
(
bs, 1H). C NMR (100.6 MHz, CDCl
3
): δ 22.5, 25.2, 25.6, 106.7, 111.9, 112.8, 120.3, 124.4, 128.7, 133.1, 134.6.
+
+
MS (70 eV): m/z (%) = 268/70 ([M + 1] , 58). HRMS (ESI) m/z for C11
H
11BrNS [M + H] calcd 269.9770, found
2
2
7
1
69.9769. Yellow powder. Recrystallisation from EtOH, yield 43%. Mp = 169.5°C.
1
,3-Dihydrothieno[3,2-b]indole 6d. H NMR (400 MHz, CDCl
3
): δ 3.20 and 3.83 (2 × t, J = 7.9 Hz, 2 × 2H), 7.07-
13
.15 (m, 2H), 7.28-7.31 (m, 1H), 7.36-7.38 (m, 1H), 7.97 (bs, 1H). C NMR (100.6 MHz, CDCl
3
): δ 28.4, 37.6,
-
11.6, 113.1, 118.7, 120.0, 121.4, 123.1, 137.1, 140.5. MS (70 eV): m/z (%) = 174 ([M - 1] , 100). HRMS (ESI) m/z
+
for C10
H
10NS [M + H] calcd 176.0529, found 176.0526. Brown-orange powder. Column chromatography R
f
(
SiO ) = 0.23, EtOAc/PE (1/5), yield 31%. Mp = 144.0°C.
2
1
7
-Fluoro-2,3-dihydrothieno[3,2-b]indole 6e. H NMR (400 MHz, CDCl
3
): δ 3.18 and 3.81 (2 × t, J = 7.9 Hz, 2 ×
2
H), 6.86 (t × d, J = 9.0, 2.5 Hz, 1H), 7.02 (d × d, J = 9.4, 2.5 Hz, 1H), 7.18 (d × d, J = 9.0, 4.3 Hz, 1H), 7.95 (bs, 1H).
1
3
C NMR (100.6 MHz, CDCl
3
): δ 28.4, 37.6, 103.8 (d, J = 24.3 Hz), 109.5 (d, J = 26.3 Hz), 112.1 (d, J = 9.7 Hz),
8

Page 9 of 17
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB02625C
1
9
1
1
1
3
13.2 (d, J = 4.4 Hz), 123.2 (d, J = 10.5 Hz), 136.9, 139.3, 157.9 (d, J = 234.9 Hz). F NMR (376.5 MHz, CDCl ): δ (-
-
-
23.90)-(-123.84) (m). MS (70 eV): m/z (%) = 192 ([M - 1] , 100). HRMS (ESI) m/z for C10
7
H FNS [M - H] , calcd
92.0289, found 192.0291. Brown-orange powder. Column chromatography R (SiO ) = 0.20, EtOAc/PE (1/4),
f
2
yield 30%. Mp = 133.5°C.
Representative procedure for the synthesis of sulfoxides 7a-c. To a solution of 1,2,4,9-tetrahydro-3-thia-9-
azafluorene 6a (5 mmol) in tetrahydrofuran (50 mL) was added boron trifluoride diethyl etherate (20 mmol) at
-
20 °C under nitrogen atmosphere. Then m-chloroperbenzoic acid was added (5 mmol) at -20 °C and the
mixture was stirred at -20 °C for 2 h. After two hours the reaction mixture was poured into a saturated solution
of NaHCO (100 mL) and subsequently extracted with ethyl acetate (100 mL). The organic phase was washed
with water (2 × 50 mL), brine (50 mL) and dried over anhydrous MgSO . Filtration of the drying agent and
3
4
removal of the solvent in vacuo afforded the crude cyclic sulfoxide 7a, which was purified by recrystallization
from EtOH to provide pure 1,2,4,9-tetrahydro-3-thia-9-azafluorene-3-oxide 7a (4.15 mmol, 83%).
1
1
,2,4,9-Tetrahydro-3-thia-9-azafluorene-3-oxide 7a. H NMR (400 MHz, d
6
-DMSO): δ 3.03-3.34 (m, 4H), 3.93
and 4.21 (2 × d, J = 15.1 Hz, 2 × 1H), 6.98 (t, J = 7.5 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.30 (d, J = 7.5 Hz, 1H), 7.42
1
3
(
d, J = 7.5 Hz, 1H), 11.10 (bs, 1H). C NMR (100.6 MHz, d
21.5, 127.8, 132.3, 136.1. MS (70 eV): m/z (%) = 206 ([M + 1] , 90). HRMS (ESI) m/z for C11
calcd 206.0634, found 206.0638. Beige powder. Recrystallization from EtOH, yield 83%. Mp > 260.0°C.
6
-DMSO): δ 17.7, 44.5, 45.1, 98.9, 111.3, 117.7, 119.1,
+
+
1
H12NOS [M + H]
1
6
4
7
4
1
-Fluoro-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3-oxide 7b. H NMR (400 MHz, d
6
-DMSO): δ 3.02-3.33 (m,
H), 3.93 and 4.16 (2 × d, J = 15.2 Hz, 2 × 1H), 6.89 (t × d, J = 9.0, 2.6 Hz, 1H), 7.22 (d × d, J = 9.9, 2.6 Hz, 1H),
1
3
.29 (d × d, J = 9.0, 4.5 Hz, 1H), 11.14 (bs, 1H). C NMR (100.6 MHz, d -DMSO): δ 17.5, 44.2, 44.8, 99.4 (d, J =
6
.4 Hz), 102.8 (d, J = 23.6 Hz), 109.3 (d, J = 25.9 Hz), 112.2 (d, J = 9.7 Hz), 128.2 (d, J = 10.0 Hz), 132.7, 134.5,
1
9
57.3 (d, J = 231.4 Hz). F NMR (376.5 MHz, d -DMSO): δ (-125.11)-(-125.05) (m). MS (70 eV): m/z (%) = 224
6
+
+
(
[M + 1] , 100). HRMS (ESI) m/z for C H FNOS [M + H] calcd 224.0540, found 224.0548. Yellow powder.
11 11
Recrystallization from EtOH, yield 68%. Mp = 242.0°C.
1
7
-Bromo-2,3-dihydrothieno[3,2-b]indole-1-oxide 7c. H NMR (400 MHz, d -DMSO): δ 3.15-3-22, 3.28-3.33,
6
3
.59-3.67 and 3.96-4.03 (4 × m, 4 × 1H), 7.33 (d × d, J = 8.6, 1.9 Hz, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.83 (d, J = 1.9
1
3
Hz, 1H), 12.07 (bs, 1H). C NMR (100.6 MHz, d -DMSO): δ 24.0, 59.0, 113.9, 115.0, 120.8, 120.9, 124.5, 125.3,
6
+
+
140.1, 153.4. MS (70eV): m/z (%) 270/2 ([M + 1] , 100). HRMS (ESI) m/z for C H BrNOS [M + H] calcd
10 9
269.9583, found 269.9593. Black powder. Recrystallization from EtOH, yield 41%. Mp = 191.0°C.
Representative procedure for the synthesis of sulfones 8a-e. To a solution of 6-bromo-1,2,4,9-tetrahydro-3-
thia-9-azafluorene 6c (5 mmol) in tetrahydrofuran (50 mL) was added m-chloroperbenzoic acid in
tetrahydrofuran (15 mmol) at 0°C. The mixture was stirred at room temperature for 2 h. The solvent was
removed in vacuo and the residue was dissolved in ethyl acetate (100 mL). The solution was washed with
4
saturated aqueous sodium sulfite (30 mL), water (30 mL), brine (2 × 30 mL), and dried over anhydrous MgSO .
Filtration of the drying agent and removal of the solvent in vacuo afforded the crude cyclic sulfone 8c, which
was purified by recrystallization from EtOH to provide pure 6-bromo-1,2,4,9-tetrahydro-3-thia-9-azafluorene-
3
,3-dioxide 8c (3.05 mmol, 61%). The synthesis of 1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 8a and 6-
9
fluoro-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 8b has been described previously.
1
6
-Bromo-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 8c. H NMR (400 MHz, d
6
-DMSO): δ 3.27 and
3
.48 (2 × t, J = 6.1 Hz, 2 × 2H), 4.44 (s, 2H), 7.20 (d × d, J = 8.6, 1.5 Hz, 1H), 7.30 (d, J = 8.6 Hz, 1H), 7.65 (d, J = 1.5
1
3
6
Hz, 1H), 11.36 (bs, 1H). C NMR (100.6 MHz, d -DMSO): δ 23.1, 47.1, 48.6, 101.9, 111.9, 113.4, 120.3, 124.3,
+
-
1
2
2
2
29.1, 132.4, 135.2. MS (70eV): m/z (%) 322/4 (M +23, 55). HRMS (ESI) m/z for C11
2
H10BrNO S [M - H] calcd
97.9543, found 297.9541. Brown powder. Recrystallization from EtOH, yield 61%. Mp = 215.0°C.
1
,3-Dihydrothieno[3,2-b]indole-1,1-dioxide 8d. H NMR (400 MHz, d
× 2H), 7.17-7.21 (m, 1H), 7.24-7.28 (m, 1H), 7.48-7.50 (m, 1H), 7.54-7.56 (m, 1H), 12.00 (bs, 1H). C NMR
-DMSO): δ 21.4, 57.6, 113.3, 116.1, 118.4, 119.4, 121.9, 123.7, 140.8, 147.7. MS (70 eV): m/z (%)
6
-DMSO): δ 3.44 and 3.90 (2 × t, J = 6.6 Hz,
1
3
(
100.6 MHz, d
6
+
+
+
=
208 ([M + 1] , 30), 225 [M + NH
4 2
], 100). HRMS (ESI) m/z for C10H10NO S [M + H] calcd 208.0427, found
208.0429. Beige powder. Recrystallization from EtOH, yield 52%. Mp = 260.0°C.
9

Organic & Biomolecular Chemistry
Page 10 of 17
View Article Online
DOI: 10.1039/C5OB02625C
1
7
=
1
-Fluoro-2,3-dihydrothieno[3,2-b]indole-1,1-dioxide 8e. H NMR (400 MHz, d
6
-DMSO): δ 3.44 and 3.89 (2× t, J
6.6 Hz, 2 × 2H), 7.12 (t × d, J = 9.1, 2.5 Hz, 1H), 7.33 (d × d, J = 9.3, 2.5 Hz, 1H), 7.51 (d × d, J = 9.1, 4.5 Hz, 1H),
13
6
2.10 (bs, 1H). C NMR (100.6 MHz, d -DMSO): δ 21.5, 57.6, 103.8 (d, J = 25.1 Hz), 111.7 (d, J = 25.7 Hz), 114.6
1
9
(
(
d, J = 9.8 Hz), 116.2 (d, J = 4.4 Hz, Cquat,arom), 119.6 (d, J = 11.3 Hz), 137.4, 149.3, 158.3 (d, J = 235.3 Hz). F NMR
+
376.5 MHz, d
FNO S [M + H] calcd 226.0333, found 226.0334. Pink powder. Recrystallization from EtOH, yield 70%.
Mp > 260.0°C.
6 4
-DMSO): δ (-121.35)-(-121.29) (m). MS (70 eV): m/z (%) = 243 ([M + NH ], 100). HRMS (ESI) m/z
+
for C10
H
9
2
Representative procedure for the synthesis of 6-phenyl-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide
f. 6-Bromo-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 8c (2 mmol) was dissolved in toluene (15 mL),
and to this solution an aqueous solution of sodium carbonate (7 mL, 2M) and a solution of phenylboronic acid
4 mmol) in ethanol (7 mL) were added. This mixture was then flushed with nitrogen gas for 10 minutes before
8
(
tetrakis(triphenylphosphine)palladium(0) (0.08 mmol) was added, and the reaction mixture was heated under
reflux for 8 hour. The reaction mixture was then poured into brine (20 mL) and extracted with EtOAc (3 × 20
4
mL). The combined organic fraction was washed with brine (3 × 15 mL), dried (MgSO ), filtered and evaporated
under vacuum. Purification through recrystallization from EtOH yielded 6-phenyl-1,2,4,9-tetrahydro-3-thia-9-
azafluorene-3,3-dioxide 8f (1.2 mmol, 60%) as a light-brown powder.
1
6
-Phenyl-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 8f. H NMR (400 MHz, d
6
-DMSO): δ 3.29 and
3
.50 (2 × t, J = 6.2 Hz, 2 × 2H), 4.51 (s, 2H), 7.30 (t, J = 7.4 Hz, 1H), 7.39-7.43 (m, 2H), 7.45 (t, J = 7.4 Hz, 2H), 7.68
13
(
d, J = 7.4 Hz, 2H), 7.74 (s, 1H), 11.21 (bs, 1H). C NMR (100.6 MHz, d
6
-DMSO): δ 23.2, 47.2, 48.9, 102.5, 111.8,
+
116.1, 121.2, 126.8, 127.1, 127.9, 129.2, 131.4, 131.9, 136.2, 142.1. MS (70eV): m/z (%) 298 ([M + 1] , 40).
+
HRMS (ESI) m/z for C H NO S [M + H] calcd 298.0896, found 298.0902. Light brown powder. Recrystallization
1
7
16
2
from EtOH, yield 60%. Mp = 237.0°C.
Representative procedure for the synthesis of N-methoxycarbonylbenzyl-1,2,4,9-tetrahydro-3-thia-9-
azafluorenes 10a-m. 1,2,4,9-Tetrahydro-3-thia-9-azafluorene 6a (6 mmol) and sodium hydride (60 wt % in
mineral oil, 6 mmol) were placed under nitrogen and dissolved in DMF (10 mL). After stirring for 30 minutes,
methyl 3-(bromomethyl)benzoate 9 (6 mmol) and potassium iodide (0.06 mmol) were added. The mixture was
heated to 80 °C for 2 h, after which it was quenched with water (30 mL), followed by addition of ethyl acetate
(
30 mL). The aqueous layer was extracted with ethyl acetate (2 × 10 mL) and the combined organic layers were
washed with water (2 × 20 mL) and brine (15 mL), dried (MgSO ) and concentrated in vacuo. Purification by
4
means of column chromatography (EtOAc/PE 1/10, Rf = 0.18) afforded pure N-(3-methoxycarbonylbenzyl)-
1
,2,4,9-tetrahydro-3-thia-9-azafluorene 10a (2.46 mmol, 41%).
N-(3-Methoxycarbonylbenzyl)-1,2,4,9-tetrahydro-3-thia-9-azafluorene 10a. H NMR (400 MHz, CDCl
and 3.04 (2 × t, J = 5.8 Hz, 2 × 2H), 3.93 (s, 3H), 3.95 (s, 2H), 5.33 (s, 2H), 7.03-7.05 (m, 1H), 7.13-7.20 (m, 2H),
1
3
): δ 2.92
1
3
7
2
1
3
.24-7.26 (m, 1H), 7.33 (t, J = 7.7 Hz, 1H), 7.52-7.55 (m, 1H), 7.90-7.95 (m, 2H). C NMR (100.6 MHz, CDCl
3
): δ
3.1, 24.1, 25.9, 46.0, 52.3, 107.3, 108.9, 117.7, 119.5, 121.7, 126.8, 127.4, 128.7, 129.1, 130.5, 130.6, 134.5,
+
+
2
35.7, 138.2, 166.8. MS (70 eV): m/z (%) = 338 ([M + 1] , 90). HRMS (ESI) m/z for C20H20NO S [M + H] calcd.
38.1209, found 338.1219. White-yellow powder. Column chromatography R
f
(SiO
2
) = 0.18, EtOAc/PE (1/10),
yield 41%. Mp = 115.0°C.
1
N-(3-Methoxycarbonylbenzyl)-6-fluoro-1,2,4,9-tetrahydro-3-thia-9-azafluorene 10b. H NMR (400 MHz,
CDCl
H), 6.99-7.01 (m, 1H), 7.08-7.15 (m, 2H), 7.32 (t, J = 7.7 Hz, 1H), 7.84 (s, 1H), 7.90-7.93 (m, 1H). C NMR (100.6
MHz, CDCl ): δ 22.9, 24.2, 25.8, 46.2, 52.3, 103.0 (d, J = 23.6 Hz), 107.4 (d, J = 4.4 Hz), 109.5 (d, J = 9.6 Hz), 109.7
d, J = 26.0 Hz), 127.1 (d, J = 9.7 Hz), 127.3, 128.8, 129.2, 130.4, 130.8, 132.2, 136.3, 137.9, 157.9 (d, J = 235.0
3
): δ 2.89 and 3.00 (2 × t, J = 5.7 Hz, 2 × 2H), 3.85 (s, 2H), 3.90 (s, 3H), 5.27 (s, 2H), 6.87 (t × d, J = 9.1, 2.5 Hz,
1
3
1
3
(
1
9
+
Hz), 166.7. F NMR (376.5 MHz, CDCl
HRMS (ESI) m/z for C20 S [M + H] calcd. 356.1115, found 356.1131. White-yellow powder. Column
chromatography R (SiO ) = 0.16, EtOAc/PE (1/10), yield 50%. Mp = 97.0°C.
N-(3-Methoxycarbonylbenzyl)-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 10c. H NMR (400 MHz, d
DMSO): δ 3.21 and 3.51 (2 × t, J = 6.2 Hz, 2 × 2H), 3.82 (s, 3H), 4.51 (s, 2H), 5.53 (s, 2H), 7.06-7.10 (m, 1H), 7.14-
3
): δ (-134.64)-(-124.52) (m). MS (70 eV): m/z (%) = 356 ([M + 1] , 100).
+
H19FNO
2
f
2
1
6
-
1
0

Page 11 of 17
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB02625C
1
3
7
6
.18 (m, 1H), 7.22-7.25 (m, 1H), 7.45-7.52 (m, 3H), 7.78 (s, 1H), 7.85 (d, J = 7.9 Hz, 1H). C NMR (100.6 MHz, d -
DMSO): δ 22.4, 45.9, 46.7, 48.4, 52.7, 102.8, 110.3, 118.3, 120.0, 122.5, 126.6, 127.7, 128.6, 129.8, 130.5,
+
+
1
31.5, 131.6, 137.1, 139.3, 166.4. MS (70 eV): m/z (%) = 370 ([M + 1] , 7), 387 ([M + NH
4
], 100). HRMS (ESI)
+
m/z for C20
SiO
N-(3-Methoxycarbonylbenzyl)-6-fluoro-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 10d. H NMR (400
MHz, d -DMSO): δ 3.21 and 3.50 (2 × t, J = 6.1 Hz, 2 × 2H), 3.83 (s, 3H), 4.48 (s, 2H), 5.54 (s, 2H), 7.01 (t × d, J =
.2, 2.5 Hz, 1H), 7.22 (d, J = 7.9 Hz, 1H), 7.33 (d × d, J = 9.3, 2.5 Hz, 1H), 7.47 (t, J = 7,9 Hz, 1H), 7.51 (d × d, J =
H20NO
4
S [M + H] , calcd 370.1108, found 370,1111. Yellow powder. Column chromatography R
f
(
2
) = 0.26, EtOAc/PE (4/5), yield 21%. Mp = 200.0°C.
1
6
9
9
5
1
3
.2, 4.3 Hz, 1H), 7.77 (s, 1H), 7.86 (d, J = 7.9 Hz, 1H). C NMR (100.6 MHz, d
6
-DMSO): δ 22.5, 46.1, 46.6, 48.3,
2.7, 103.1 (d, J = 4.6 Hz), 103.5 (d, J = 24.0 Hz), 110.4 (d, J = 25.9 Hz), 111.5 (d, J = 9.6 Hz), 126.9 (d, J = 10.3
1
9
Hz), 127.7, 128.7, 129.8, 130.5, 131.6, 133.5, 133.8, 139.1, 157.7 (d, J = 233.0 Hz), 166.4. F NMR (376.5 MHz,
+
d
6
-DMSO): δ (-124.05)-(-123.99) (m). MS (70 eV): m/z (%) = 405 ([M + NH
4
], 100). HRMS (ESI) m/z for
+
C
20 4
H19FNO S [M + H] calcd 388.1013, found 388.1019. Beige powder. Recrystallization from EtOH, yield 67%.
Mp = 214.0 °C.
1
N-(3-Methoxycarbonylbenzyl)-6-phenyl-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 10e. H NMR
(
400 MHz, d
6
-DMSO): δ 3.24 and 3.52 (2 × t, J = 5.9 Hz, 2 × 2H), 3.83 (s, 3H), 4.57 (s, 2H), 5.57 (s, 2H), 7.26 (d, J =
7
.7 Hz, 1H), 7.32 (t, J = 7.3 Hz, 1H), 7.44-7.50 (m, 4H), 7.58 (d, J = 8.6 Hz, 1H), 7.70 (d, J = 7.3 Hz, 2H), 7.81-7.83
1
3
(
m, 2H), 7.87 (d, J = 7.7 Hz, 1H). C NMR (100.6 MHz, d
6
-DMSO): δ 22.5, 46.1, 46.7, 48.5, 52.7, 103.4, 110.7,
1
16.6, 121.7, 126.9, 127.2, 127.7, 128.6, 129.3, 129.8, 130.5, 131.6, 132.3, 132.6, 136.7, 139.3, 141.8, 166.4.
+
+
MS (70eV): m/z (%) 446 ([M + 1] , 80). HRMS (ESI) m/z for C26
27 2 4 4
H N O S [M + NH ] calcd. 463.1686, found
4
63.1694. Light brown powder. Recrystallization from EtOH, yield 60%. Mp = 201.0°C.
1
N-(3-Methoxycarbonylbenzyl)-2,3-dihydrothieno[3,2-b]indole-1,1-dioxide 10f. H-NMR (400 MHz, d -DMSO):
6
δ 3.44 (t, J = 6.5 Hz, 2H), 3.84 (s, 3H), 3.95 (t, J = 6.5 Hz, 2H), 5.57 (s, 2H), 7.23-7.32 (m, 2H), 7.42-7.45 (m, 1H),
1
3
7
5
.49-7.53 (m, 1H), 7.59-7.62 (m, 2H), 7.88-7.91 (m, 2H). C-NMR (100.6 MHz, d -DMSO): δ 21.0, 46.9, 52.7,
6
7.4, 112.3, 116.2, 118.8, 119.3, 122.5, 124.0, 128.2, 129.0, 129.9, 130.6, 132.3, 137.9, 141.0, 148.5, 166.4. MS
+
+
(
70 eV): m/z (%) = 373 ([M + NH ], 100). HRMS (ESI) m/z for C H NO S [M + H] calcd. 356.0951, found
4 19 18 4
3
56.0958. White powder. Recrystallization from EtOH, yield 72%. Mp = 210.5°C.
1
N-(3-Methoxycarbonylbenzyl)-7-fluoro-2,3-dihydrothieno[3,2-b]indole-1,1-dioxide 10g. H NMR (400 MHz,
d -DMSO): δ 3.44 (t, J = 6.5 Hz, 2H), 3.84 (s, 3H), 3.95 (t, J = 6.5 Hz, 2H), 5.57 (s, 2H), 7.18 (t × d, J = 9.2, 2.5 Hz,
6
1
3
1
H), 7.39-7.44 (m, 2H), 7.49-7.53 (m, 1H), 7.63 (d × d, J = 9.2, 4.3 Hz, 1H), 7.89-7.91 (m, 2H). C NMR (100.6
MHz, d -DMSO): δ 21.1, 47.1, 52.7, 57.3, 104.3 (d, J = 25.2 Hz), 112.1 (d, J = 26.0 Hz), 113.7 (d, J = 9.9 Hz), 116.2
6
(
d, J = 4.4 Hz), 119.5 (d, J = 11.2 Hz), 128.2, 129.1, 130.0, 130.6, 132.3, 137.6, 137.7, 150.1, 158.7 (d, J = 236.9
1
9
+
Hz), 166.4. F NMR (376.5 MHz, d
00). HRMS (ESI) m/z for C19
Recrystallization from EtOH, yield 60%. Mp = 222.0°C.
N-(3-Methoxycarbonylbenzyl)-7-bromo-2,3-dihydrothieno[3,2-b]indole-1-oxide 10h. H NMR (400 MHz,
CDCl ): δ 3.01-3-07 (m, 1H), 3.45-3.51 (m, 1H), 3.61-3.69 (m, 1H), 3.91-4.00 (m, 1H), 3.91 (s, 3H), 5.29 and 5.35
2 × d, J = 16.4 Hz, 2 × 1H), 7.13 (d, J = 8.8 Hz, 1H), 7.19 (d, J = 7.7 Hz, 1H), 7.34 (d × d, J = 8.8, 1.8 Hz, 1H), 7.40
6
-DMSO): δ (-120.57)-(-120.50) (m). MS (70 eV): m/z (%) = 391 ([M + NH
4
],
+
1
H17FNO S [M + H] calcd. 374.0857, found 374.0857. White powder.
4
1
3
(
(
1
3
t, J = 7.7 Hz, 1H), 7.90 (s, 1H), 7.96 (d, J = 1.8 Hz, 1H), 7.99 (d, J = 7.7 Hz, 1H). C NMR (100.6 MHz, CDCl
3
): δ
2
1
4
5
3.8, 48.9, 52.4, 58.3, 112.0, 115.5, 120.9, 122.1, 124.3, 126.3, 127.8, 129.5, 129.6, 130.9, 131.2, 135.8, 140.1,
+
+
3
52.4, 166.4. MS (70eV): m/z (%) 418/20 ([M + 1] , 100). HRMS (ESI) m/z for C19H17BrNO S [M + H] calcd.
18.0107, found 418.0125. Brown powder. Column chromatography R
4%. Mp = 187.5°C.
f
(SiO
2
) = 0.23, acetone/PE (1/1), yield
1
N-(4-Methoxycarbonylbenzyl)-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3-oxide 10i. H NMR (400 MHz, d
6
-
DMSO): δ 2.98-3.16 (m, 3H), 3.28-3.38 (m, 1H), 3.82 (s, 3H), 4.03 and 4.23 (2 × d, J = 15.4 Hz, 2 × 1H), 5.53 (s,
2
2
1
H), 7.05-7.14 (m, 2H), 7.17 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 8.3 Hz,
1
3
H). C NMR (100.6 MHz, d
6
-DMSO): δ 16.6, 44.0, 44.8, 45.9, 52.6, 99.8, 110.1, 118.1, 119.8, 122.0, 127.1,
+
27.6, 129.1, 130.1, 133.2, 136.7, 144.2, 166.4. MS (70 eV): m/z (%) = 354 ([M + 1] , 70). HRMS (ESI) m/z for
+
C
20
H
20NO
3
S [M + H] calcd. 354.1158, found 354.1153. Brown powder. Recrystallization from EtOH, yield 47%.
Mp = 78.0°C.
1
1

Organic & Biomolecular Chemistry
Page 12 of 17
View Article Online
DOI: 10.1039/C5OB02625C
1
N-(4-Methoxycarbonylbenzyl)-6-fluoro-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3-oxide 10j. H NMR (400
MHz, d -DMSO): δ 2.97-3.14 (3H, m), 3.32-3.36 (m, 1H), 3.82 (s, 3H), 4.02 and 4.18 (2 × d, J = 15.3 Hz, 2 × 1H),
.53 (s, 2H), 6.96 (t × d, J = 9.0, 2.5 Hz, 1H), 7.16 (d, J = 8.3 Hz, 2H), 7.33 (d × d, J = 9.7, 2.5 Hz, 1H), 7.45 (d × d, J
6
5
=
1
3
6
9.0, 4.3 Hz, 1H), 7.90 (d, J = 8.3 Hz, 2H). C NMR (100.6 MHz, d -DMSO): δ 16.5, 43.7, 44.6, 46.1, 52.6, 100.1
(
d, J = 4.4 Hz), 103.3 (d, J = 23.8 Hz), 109.8 (d, J = 26.0 Hz), 111.2 (d, J = 9.6 Hz), 127.1, 128.0 (d, J = 10.2 Hz),
1
9
1
6
29.1, 130.1, 133.4, 135.3, 143.9, 157.7 (d, J = 232.6 Hz), 166.4. F NMR (376.5 MHz, d -DMSO): δ (-124.41)-(-
+
+
1
3
24.34) (m). MS (70 eV): m/z (%) = 372 ([M + 1] , 77). HRMS (ESI) m/z for C20H19FNO S [M + H] calcd. 372.1064,
found 372.1065. Brown powder. Recrystallization from EtOH, yield 42%. Mp = 135.5°C.
N-(4-Methoxycarbonylbenzyl)-6-bromo-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 10k. H NMR
400 MHz, CDCl ): δ 3.25-3.28 and 3.30-3.33 (2 × 2H, 2 × m, CH CH S), 3.90 (3H, s, CH O), 4.37 (2H, s, CquatCH S),
.32 (2H, s, CH N), 7.01 (2H, d, J = 8.5 Hz, 2 × CHarom), 7.09 (1H, d, J = 8.7 Hz, CHarom), 7.30 (1H, d × d, J = 8.7, 1.8
Hz, CHarom), 7.57 (1H, d, J = 1.8 Hz, CHarom), 7.97 (2H, d, J = 8.5 Hz, 2 × CHarom). C NMR (100.6 MHz, CDCl
1
(
3
2
2
3
2
5
2
13
3
): δ
2
1
1
2 2 2 2 2 2 3
2.2 (CH CH S), 46.7 (CH N), 47.2 (CH CH S), 48.7 (Carom,quatCH S), 52.3 (CH O), 102.2 (Carom,quat), 111.0 (CHarom),
13.8 (Carom,quat), 120.4, 125.8 and 125.9 (4 × CHarom), 128.0 and 130.0 (2 × Carom,quat), 130.5 (2 × CHarom), 131.4,
+
35.9 and 141.3 (3 × Carom,quat), 166.4 (C=O). MS (70eV): m/z (%) 448/50 ([M + 1] , 70). HRMS (ESI) m/z for
+
C
20
H
22BrN
2
O
4
S [M + NH
4
] calcd 465.0478, found 465.0473. Light brown powder. Column chromatography R
f
(
SiO
2
) = 0.25, EtOAc/PE (1/1), yield 80%. Mp = 191.0°C.
1
N-(4-Methoxycarbonylbenzyl)-2,3-dihydrothieno[3,2-b]indole-1,1-dioxide 10l. H NMR (400 MHz, d
6
-DMSO):
δ 3.44 (t, J = 6.5 Hz, 2H), 3.83 (s, 3H), 3.95 (t, J = 6.5 Hz, 2H), 5.57 (s, 2H), 7.23-7.31 (m, 2H), 7.35 (d, J = 8.4 Hz,
1
3
2
1
+
H), 7.55-7.62 (m, 2H), 7.94 (d, J = 8.4 Hz, 2H). C NMR (100.6 MHz, d
6
-DMSO): δ 20.9, 47.0, 52.6, 57.4, 112.2,
16.3, 118.8, 119.3, 122.5, 124.0, 127.8, 129.5, 130.2, 141.0, 142.5, 148.6, 166.3. MS (70 eV): m/z (%) = 356 ([M
+
+
+
1] , 41), 373 ([M + NH ], 100). HRMS (ESI) m/z for C H NO S [M + H] calcd. 356.0951, found 356.0954.
4
19 18
4
White powder. Recrystallization from EtOH, yield 60%. Mp = 226.5°C.
1
N-(4-Methoxycarbonylbenzyl)-7-fluoro-2,3-dihydrothieno[3,2-b]indole-1,1-dioxide 10m. H NMR (400 MHz,
d6-DMSO): δ 3.44 (t, J = 6.5 Hz, 2H), 3.84 (s, 3H), 3.95 (t, J = 6.5 Hz, 2H), 5.57 (s, 2H), 7.16 (t × d, J = 9.2, 2.5 Hz,
1
2
H), 7.34 (d, J = 8.3 Hz, 2H), 7.41 (d × d, J = 9.1, 2.5 Hz, 1H), 7.59 (d × d, J = 9.2, 4.3 Hz, 1H), 7.94 (d, J = 8.3 Hz,
13
H). C NMR (100.6 MHz, d -DMSO): δ 21.0, 47.2, 52.7, 57.3, 104.3 (d, J = 25.1 Hz), 112.1 (d, J = 25.8 Hz), 113.7
6
(
d, J = 9.9 Hz), 116.3 (d, J = 4.5 Hz), 119.5 (d, J = 11.3 Hz), 127.8, 129.6, 130.2, 137.6, 142.2, 150.1, 158.7 (d, J =
1
9
2
36.9 Hz), 166.3. F NMR (376.5 MHz, d -DMSO): δ (-120.57)-(-120.51) (m). MS (70 eV): m/z (%) = 391 ([M +
6
+
+
NH ], 100). HRMS (ESI) m/z for C H FNO S [M + H] calcd 374.0857, found 374.0852. White powder.
4
19 17
4
Recrystallization from EtOH, yield 80%. Mp = 243.0°C.
Representative procedure for the synthesis of hydroxamic acids 3a-m. N-(3-methoxycarbonylbenzyl)-1,2,4,9-
tetrahydro-3-thia-9-azafluorene 10a (1 mmol) was dissolved in THF (10 mL), and to this solution was added
hydroxylamine (100 mmol) and subsequently potassium hydroxide in methanol (4M, 50 mmol). The resulting
mixture was stirred for an additional 10 minutes at room temperature, before it was poured into a saturated
aqueous solution of NaHCO
which the combined organic fractions were washed with water (10 mL) and a saturated brine solution (10 mL),
dried (MgSO ), filtered and evaporated. Purification through recrystallization from EtOH yielded N-(3-
3
(10 mL). This aqueous solution was extracted two times with ethyl acetate, after
4
hydroxycarbamoylbenzyl)-1,2,4,9-tetrahydro-3-thia-9-azafluorene 3a (0.57 mmol, 57%) as a white-yellow
powder. Note: the mixture was stirred at reflux temperature for the synthesis of hydroxamic acids 3d, 3g and
3
m.
1
N-(3-Hydroxycarbamoylbenzyl)-1,2,4,9-tetrahydro-3-thia-9-azafluorene 3a. H NMR (400 MHz, d
6
-DMSO): δ
.90 and 3.00 (2 × t, J = 5.5 Hz, 2 × 2H), 3.84 (s, 2H), 5.42 (s, 2H), 7.01-7.05 (m, 1H), 7.07-7.11 (m, 2H), 7.36 (t, J
7.7 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.54 (s, 1H), 7.58 (d, J = 7.7, 1H), 9.00 (bs, 1H),
2
=
1
1
13
1.19 (bs, 1H). C NMR (100.6 MHz, d
6
-DMSO): δ 22.7, 24.0, 25.6, 45.7, 106.8, 109.9, 118.0, 119.4, 121.5,
+
25.8, 125.9, 126.7, 129.2, 129.5, 133.7, 135.4, 135.7, 139.3, 164.6. MS (70 eV): m/z (%) = 339 ([M + 1] , 100).
+
HRMS (ESI) m/z for C19
19 2 2
H N O S [M + H] calcd 339.1162, found 339.1159. White-yellow powder. Crystallization
from EtOH, yield 57%. Mp = 124.5°C.
1
2

Page 13 of 17
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB02625C
1
6
N-(3-Hydroxycarbamoylbenzyl)-6-fluoro-1,2,4,9-tetrahydro-3-thia-9-azafluorene 3b. H NMR (400 MHz, d -
DMSO): δ 2.89 en 2.99 (2 × t, J = 5.4 Hz, 2 × 2H), 3.81 (s, 2H), 5.42 (s, 2H), 6.92 (t × d, J = 9.1, 2.5 Hz, 1H), 7.07 (d,
J = 7.7 Hz, 1H), 7.26 (d × d, J = 9.7, 2.5 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.44 (d × d, J = 9.1, 4.4 Hz, 1H), 7.51 (s,
1
3
1
H), 7.59 (d, J = 7.7 Hz, 1H), 9.04 (bs, 1H), 11.14 (bs, 1H). C NMR (100.6 MHz, d
6
-DMSO): δ 22.6, 24.2, 25.5,
4
5.9, 103.1 (d, J = 23.4 Hz), 107.1 (d, J = 4.4 Hz), 109.3 (d, J = 25.8 Hz), 110.9 (d, J = 9.8 Hz), 125.7, 125.9, 127.0
19
(
6
d, J = 10.1 Hz), 129.2, 129.4, 132.4, 133.7, 137.5, 139.0, 157.5 (d, J = 232.0 Hz), 164.5. F NMR (376.5 MHz, d -
+
2 2
DMSO): δ (-124.92)-(-124.86) (m). MS (70 eV): m/z (%) = 357 ([M + 1] , 100). HRMS (ESI) m/z for C19H18FN O S
+
[
M + H] calcd. 357.1068, found 357.1062. Yellow powder. Crystallization from diethyl ether, yield 63%. Mp =
90.0°C.
N-(3-Hydroxycarbamoylbenzyl)-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 3c. H NMR (400 MHz, d
DMSO): δ 3.24 and 3.51 (2 × t, J = 6.0 Hz, 2 × 2H), 4.50 (s, 2H), 5.47 (s, 2H), 7.06-7.11 (m, 2H), 7.14-7.18 (m, 1H),
1
1
6
-
1
3
7
d
1
.37 (t, J = 7.9 Hz, 1H), 7.48-7.51 (m, 2H), 7.59-7.61 (m, 2H), 9.04 (bs, 1H), 11.22 (bs, 1H). C NMR (100.6 MHz,
6
-DMSO): δ 22.4, 46.2, 46.7, 48.4, 102.7, 110.4, 118.2, 120.0, 122.4, 126.0, 126.1, 126.6, 129.3, 129.5, 131.5,
+
+
19 2 4
33.8, 137.1, 138.9, 164.5. MS (70 eV): m/z (%) = 371 ([M + 1] , 95). HRMS (ESI) m/z for C19H N O S [M + H]
calcd. 371.1060, found 371,1066. Yellow powder. Crystallization from diethyl ether, yield 32%. Mp = 229.5°C.
1
N-(3-Hydroxycarbamoylbenzyl)-6-fluoro-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 3d. H NMR (400
MHz, d -DMSO): δ 3.23 and 3.50 (2 × t, J = 5.9 Hz, 2 × 2H), 4.48 (s, 2H), 5.47 (s, 2H), 7.00 (t × d, J = 9.1, 2.5 Hz,
H), 7.08 (d, J = 7.5 Hz, 1H), 7.32 (d × d, J = 9.7, 2.5 Hz, 1H), 7.34-7.38 (m, 1H), 7.50 (d × d, J = 9.1, 4.3 Hz, 1H),
6
1
7
4
1
1
3
.58 (s, 1H), 7.61 (d, J = 7.5 Hz, 1H), 9.05 (bs, 1H), 11.21 (bs, 1H). C NMR (100.6 MHz, d
6
-DMSO): δ 22.5, 46.4,
6.6, 48.3, 102.9 (d, J = 4.6 Hz), 103.4 (d, J = 24.0 Hz), 110.3 (d, J = 26.1 Hz), 111.5 (d, J = 9.5 Hz), 125.9, 126.1,
19
26.9 (d, J = 10.2 Hz), 129.2, 129.4, 133.5, 133.7, 133.8, 138.6, 157.7 (d, J = 232.9 Hz), 164.3. F NMR (376.5
+
MHz, d -DMSO): δ (-124.15)-(-124.09) (m). MS (70 eV): m/z (%) = 389 ([M + 1] , 100). HRMS (ESI) m/z for
6
+
C H FN O S [M + H] calcd. 389.0966, found 389.0967. White powder. Crystallization from EtOH, yield 5%. Mp
19
18
2
4
=
237.0°C.
N-(3-Hydroxycarbamoylbenzyl)-6-phenyl-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 3e. H NMR
400 MHz, d -DMSO): δ 3.25 and 3.52 (2 × t, J = 5.8 Hz, 2 × 2H), 4.57 (s, 2H), 5.49 (s, 2H), 7.10 (d, J = 7.4 Hz, 1H),
1
(
6
7
1
1
.30-7.36 (m, 2H), 7.44-7.48 (m, 3H), 7.57 (d, J = 8.6 Hz, 1H), 7.61-7.63 (m, 2H), 7.70 (d, J = 7.2 Hz, 2H), 7.81 (s,
1
3
H) 9.02 (bs, 1H), 11.17 (bs, 1H). C NMR (100.6 MHz, d -DMSO): δ 22.5, 46.4, 46.7, 48.5, 103.2, 110.8, 116.5,
6
21.6, 125.7, 126.0, 126.9, 127.1, 127.2, 129.1, 129.2, 129.3, 132.3, 132.5, 134.2, 136.7, 138.6, 141.8, 164.0.
+
+
MS (70eV): m/z (%) 447 ([M + 1] , 85). HRMS (ESI) m/z for C H N O S [M + H] calcd. 447.1373, found
25
23
2
4
4
47.1361. White powder. Crystallization from CH Cl , yield 63%. Mp = 214.0°C.
2 2
1
N-(3-Hydroxycarbamoylbenzyl)-2,3-dihydrothieno[3,2-b]indole-1,1-dioxide 3f. H NMR (400 MHz, d
6
-DMSO):
δ 3.47 and 3.96 (2 × t, J = 6.5 Hz, 2 × 2H), 5.50 (s, 2H), 7.22-7.34 (m, 3H), 7.43 (t, J = 7.9 Hz, 1H), 7.58-7.61 (m,
1
3
2
H,), 7.65-7.66 (m, 2H), 9.04 (bs, 1H), 11.24 (bs, 1H). C NMR (100.6 MHz, d
6
-DMSO): δ 21.0, 47.1, 57.4, 112.3,
1
16.1, 118.7, 119.3, 122.5, 123.9, 126.3, 126.6, 129.4, 130.3, 133.8, 137.4, 141.0, 148.5, 164.3. MS (70 eV): m/z
+
+
(
%) = 357 ([M + 1] , 100). HRMS (ESI) m/z for C18
powder. Crystallisation from ethanol, yield 25%. Mp = 236.5°C.
N-(3-Hydroxycarbamoylbenzyl)-7-fluoro-2,3-dihydrothieno[3,2-b]indole-1,1-dioxide 3g. H NMR (400 MHz,
-DMSO): δ 3.47 and 3.96 (2 × t, J = 6.5 Hz, 2 × 2H), 5.51 (s, 2H), 7.17 (t × d, J = 9.2, 2.5 Hz, 1H), 7.33 (d, J = 7.8
17 2 4
H N O S [M + H] calcd. 357.0904, found 357.0907. White
1
d
6
1
3
C
Hz, 1H), 7.38-7.45 (m, 2H), 7.61 (d × d, J = 9.2, 4.3 Hz, 1H), 7.65-7.67 (m, 2H), 9.04 (bs, 1H), 11.23 (bs, 1H).
NMR (100.6 MHz, d -DMSO): δ 21.1, 47.3, 57.3, 104.2 (d, J = 25.4 Hz), 112.0 (d, J = 25.8 Hz), 113.8 (d, J = 9.7
Hz), 116.1 (d, J = 4.4 Hz), 119.5 (d, J = 11.2 Hz), 126.3, 126.7, 129.5, 130.3, 133.9, 137.2, 137.6, 150.1, 158.7 (d, J
6
1
9
=
1
6
236.6 Hz), 164.3. F NMR (376.5 MHz, d -DMSO): δ (-120.64)-(-120.58) (m). MS (70 eV): m/z (%) = 375 ([M +
+
+
2 4
] , 87). HRMS (ESI) m/z for C18H16FN O S [M + H] calcd. 375.0809, found 375.0810. White powder.
Crystallisation from diethyl ether, yield 40%. Mp > 260.0°C.
N-(3-Hydroxycarbamoylbenzyl)-7-bromo-2,3-dihydrothieno[3,2-b]indole-1-oxide 3h. H NMR (400 MHz, d
DMSO): δ 3.24-3-31 (m, 1H), 3.36-3.43 (m, 1H), 3.57-3.64 (m, 1H), 4.00-4.07 (m, 1H), 5.51 and 5.57 (2 × d, J =
6.3 Hz, 2 × 1H), 7.31 (d, J = 7.9 Hz, 1H), 7.38 (d × d, J = 8.8, 1.9 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.56 (d, J = 8.8
1
6
-
1
13
Hz, 1H), 7.64-7.66 (m, 2H), 7.90 (d, J = 1.9 Hz, 1H), 9.04 (bs, 1H), 11.23 (bs, 1H). C NMR (100.6 MHz, d
6
-
DMSO): δ 23.9, 48.5, 58.5, 114.1, 114.6, 120.4, 121.2, 124.4, 125.5, 126.3, 126.6, 129.4, 130.2, 133.9, 137.4,
1
3

Organic & Biomolecular Chemistry
Page 14 of 17
View Article Online
DOI: 10.1039/C5OB02625C
+
+
1
40.2, 154.6, 166.3. MS (70eV): m/z (%) 419/21 ([M + 1] , 100). HRMS (ESI) m/z for C18
H16BrN
2
O
3
S [M + H]
calcd. 419.0060, found 419.0056. White powder. Recrystallization from CH
N-(4-Hydroxycarbamoylbenzyl)-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3-oxide 3i. H NMR (400 MHz, d
DMSO): δ 3.02-3.16 (m, 3H), 3.32-3.38 (m, 1H), 4.03 and 4.22 (2 × d, J = 15.2 Hz, 2 × 1H), 5.48 (s, 2H), 7.04-7.15
2 2
Cl , yield 70%. Mp = 218.5°C.
1
6
-
1
3
(
m, 4H), 7.44 (d, J = 8.1 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.66 (d, J = 8.2 Hz, 2H), 9.00 (bs, 1H), 11.14 (bs, 1H). C
6
NMR (100.6 MHz, d -DMSO): δ 16.6, 43.9, 44.8, 45.9, 99.7, 110.1, 118.1, 119.8, 122.0, 126.8, 127.6, 127.7,
+
1
19 2 3
32.3, 133.2, 136.7, 141.7, 164.4. MS (70 eV): m/z (%) = 355 ([M + 1] , 100). HRMS (ESI) m/z for C19H N O S [M
+
+
H] calcd 355.1111, found 355.1112. White powder. Crystallisation from EtOH, yield 13%. Mp = 258.5°C.
1
N-(4-Hydroxycarbamoylbenzyl)-6-fluoro-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3-oxide 3j. H NMR (400
MHz, d -DMSO): δ 3.01-3.15 (m, 3H), 3.28-3.38 (m, 1H), 4.02 and 4.17 (2 × d, J = 15.6 Hz, 2 × 1H), 5.44 (s, 2H),
.95 (t × d, J = 9.0, 2.5 Hz, 1H), 7.09 (d, J = 8.2 Hz, 2H), 7.32 (d × d, J = 9.7, 2.5 Hz, 1H), 7.45 (d × d, J = 9.0, 4.3 Hz,
6
6
1
4
1
3
H), 7.67 (d, J = 8.2 Hz, 2H), 9.00 (bs, 1H), 11.15 (bs, 1H). C NMR (100.6 MHz, d
6
-DMSO): δ 16.5, 43.7, 44.6,
6.0, 100.0 (d, J = 4.3 Hz), 103.3 (d, J = 23.7 Hz), 109.8 (d, J = 26.1 Hz), 111.2 (d, J = 9.7 Hz), 126.8, 127.8, 128.0
19
(
6
d, J = 10.0 Hz), 132.4, 133.3, 135.3, 141.5, 157.7 (d, J = 231.9 Hz), 164.4. F NMR (376.5 MHz, d -DMSO): δ (-
+
+
124.45)-(-124.39) (m). MS (70 eV): m/z (%) = 373 ([M + 1] , 100). HRMS (ESI) m/z for C19
H
18FN
2
O
3
S [M + H]
calcd. 373.1017, found 373.1014. White powder. Crystallisation from EtOH, yield 28%. Mp = 244.5°C.
1
N-(4-Hydroxycarbamoylbenzyl)-6-bromo-1,2,4,9-tetrahydro-3-thia-9-azafluorene-3,3-dioxide 3k.
H NMR
(
400 MHz, d
6
-DMSO): δ 3.22 and 3.51 (2 × t, J = 5.9 Hz, 2 × 2H), 4.51 (s, 2H), 5.50 (s, 2H), 7.07 (d, J = 8.2 Hz, 2H),
7
1
1
.27 (d × d, J = 8.7, 1.7 Hz, 1H), 7.47 (d, J = 8.7 Hz, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.75 (d, J = 1.7 Hz, 1H), 9.01 (bs,
1
3
H), 11.16 (bs, 1H). C NMR (100.6 MHz, d
6
-DMSO): δ 22.4, 46.2, 46.5, 48.2, 102.7, 112.5, 112.7, 120.8, 124.8,
+
26.8, 127.8, 128.3, 132,5, 133.3, 135.8, 141.2, 164.3. MS (70eV): m/z (%) 449/51 ([M + 1] , 5). HRMS (ESI) m/z
+
for C H BrN O S [M + H] calcd. 449.0165, found 449.0148. White powder. Crystallisation from CH Cl , yield
1
9
18
2
4
2
2
7
0%. Mp = 230.0°C.
1
N-(4-Hydroxycarbamoylbenzyl)-2,3-dihydrothieno[3,2-b]indole-1,1-dioxide 3l. H NMR (400 MHz, d -DMSO):
6
δ 3.46 and 3.96 (2 × t, J = 6,3 Hz, 2 × 2H), 5.51 (2H, s, CH N), 7.22-7.30 (m, 4H), 7.56-7.61 (m, 2H), 7.71 (d, J = 8.2
2
1
3
Hz, 2H), 9.03 (bs, 1H), 11.17 (bs, 1H). C NMR (100.6 MHz, d -DMSO): δ 20.9, 47.0, 57.4, 112.3, 116.1, 118.7,
6
+
1
19.3, 122.5, 123.9, 127.5, 127.9, 132.7, 140.1, 140.9, 148.5, 164.2. MS (70 eV): m/z (%) = 357 ([M + 1] , 33),
+
+
3
74 ([M + NH ], 100). HRMS (ESI) m/z for C H N O S [M + H] calcd. 357.0904, found 357.0900. White
4
18 17
2
4
powder. Crystallisation from EtOH, yield 25%. Mp = 198.0°C.
1
N-(4-Hydroxycarbamoylbenzyl)-7-fluoro-2,3-dihydrothieno[3,2-b]indole-1,1-dioxide 3m. H NMR (400 MHz,
d6-DMSO): δ 3.47 and 3.96 (2 × t, J = 6.5 Hz, 2 × 2H), 5.51 (s, 2H), 7.16 (t × d, J = 9.2, 2.5 Hz, 1H), 7.29 (d, J = 8.2
Hz, 2H), 7.40 (d × d, J = 9.1, 2.5 Hz, 1H), 7.60 (d × d, J = 9.2, 4.3 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 9.03 (bs, 1H),
1
3
1
1.19 (bs, 1H). C NMR (100.6 MHz, d
6
-DMSO): δ 21.0, 47.2, 57.3, 104.3 (d, J = 25.3 Hz), 112.0 (d, J = 26.2 Hz),
1
13.7 (d, J = 9.8 Hz), 116.2 (d, J = 5.3 Hz), 119.5 (d, J = 11.1 Hz), 127.5, 127.9, 132.8, 137.6, 139.9, 150.1, 158.7
19
(
(
6
d, J = 235.5 Hz), 164.2. F NMR (376.5 MHz, d -DMSO): δ (-120.60)-(-120.54) (m). MS (70 eV): m/z (%) = 392
+
+
4 3 4 4
[M + NH ], 100). HRMS (ESI) m/z for C18H19FN O S [M + NH ] calcd. 392.1075, found 392.1075. White
powder. Crystallisation from diethyl ether, yield 5%. Mp = 240.5°C.
Docking studies (performed at the Centre for Industrial Biotechnology and Biocatalysis). All manipulations were
1
6
performed with the molecular modelling program YASARA and the YASARA/WHATIF twinset, and the figure
17
was created with PyMol v1.3. The HDAC6 sequence was obtained from the UniProt database
(
www.uniprot.org, UniProt entry Q9UBN7). To increase the accuracy of the model, the sequence was limited to
the major functional domain of HDAC6 (Gly482-Gly800). Possible templates were identified by running 3 PSI-
BLAST iterations to extract a position specific scoring matrix (PSSM) from UniRef90, and then searching the PDB
for a match. To aid the alignment of the HDAC6 sequence and templates, and the modelling of the loops, a
secondary structure prediction was performed, followed by multiple sequence alignments. All side chains were
ionised or kept neutral according to their predicted pKa values. Initial models were created from different
templates (pdb entry 2VQW, 2VQQ and 3C10), each with several alignment variations and up to hundred
conformations tried per loop. After the side-chains had been built, optimised and fine-tuned, all newly
modelled parts were subjected to a combined steepest descent and simulated annealing minimisation, i.e. the
1
4

Page 15 of 17
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB02625C
backbone atoms of aligned residues were kept fixed to preserve the folding, followed by a full unrestrained
simulated annealing minimisation for the entire model. The final model was obtained as a hybrid model of the
best parts of the initial models, and checked once more for anomalies like incorrect configurations or colliding
side chains. Furthermore, it was structurally aligned with known HDAC crystal structures to check if the
chelating residues and the zinc atom were arranged correctly. The HDAC inhibitor structures were created with
1
8
YASARA Structure and energy minimised with the AMBER03 force field. The grid box used for docking had a
dimension of 25 x 25 x 25 angstrom, and comprised the entire catalytic cavity including the zinc ion and the
1
9
outer surface of the active site entrance. Docking was performed with AutoDock VINA and default
parameters. Ligands were allowed to freely rotate during docking. The first conformer from the cluster that has
its zinc binding group in the vicinity of the zinc ion, was selected as the binding mode for analysis.
Enzyme inhibition assay (performed by Eurofins Cerep Panlabs). In vitro determination of IC50-values by using
2
0
human recombinant HDAC1-11 and fluorogenic HDAC substrate. For more details, see ESI.
Western blots (performed at the Laboratory of Neurobiology and Vesalius Research Center, VIB). Values
represent the normalized ratio Acetyl α-Tubulin/α-Tubulin and Acetyl Histone 3/Histone 4 against Tubastatin A
(
Tub A) in an established neuronal cell line (Neuro-2a cells: ATCC N° CCL-131). Neuro-2a cells are treated
overnight with different concentrations of the HDAC6 inhibitors and the effect on the acetylation level of α-
tubulin is determined by using Western blot. For more details, see ESI.
Cell culture. Mouse neuroblastoma (Neuro-2a) cells were grown in a 1:1 mix of D-MEM (Dulbecco's Modified
Eagle Medium) and F12 medium supplemented with glutamax (Invitrogen), 100 μg/ml streptomycin, 100 U/ml
penicillin (Invitrogen), 10% fetal calf serum (Greiner Bio-one), 1% non-essential amino acids (Invitrogen) and
1
.6% NaHCO (Invitrogen) at 37 °C and 7.5% CO . To split the cells, cells were washed with Versene (Invitrogen)
3 2
and dissociated with 0.05% Trypsine-EDTA (Invitrogen). The Neuro-2a cells were treated overnight at 37°C with
dosages ranging from 10 nM up to 1 µM of either Tubastatin A (Asclepia, Destelbergen, Belgium) or the
candidate HDAC6 inhibitors.
Western Blot. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, transfected
cells were collected using the EpiQuik Total Histone Extraction Kit (EpiGentek) according to manufacturer’s
instructions. Protein concentrations were determined using microBCA kit (Thermo Fisher Scientific Inc.,
Pittsburgh, PA, USA) according to manufacturer’s instructions. Before resolving the samples on a 12% SDS–
PAGE gel, samples containing equal amounts of protein were supplemented with reducing sample buffer
(
Thermo Scientific) and boiled at 95 °C for 5 min. After electrophoresis, the proteins were transferred to a
polyvinylidene difluoride (PVDF) membrane (Millipore Corp.). The non-specific binding was blocked by
incubation of the membrane in 5% bovine serum albumin (BSA), diluted in Tris Buffered Saline Tween (TBST, 50
mM TRIS, 150 mM NaCl, 0,1% Tween-20 (Applichem, Darmstadt, Duitsland) for 1h at room temperature
followed by incubation with primary antibodies overnight. The antibodies, diluted in TBS-T, were directed
against α-tubulin (1/5000, Sigma-Aldrich), acetylated α-tubulin (1/5000, monoclonal, Sigma-Aldrich) and gapdh
(
1/1000, Ambion). The secondary antibodies, coupled to alkaline phosphatase (anti-mouse or anti-rabbit,
/5000, Sigma-Aldrich) were used. Blots were visualized by adding the ECF substrate (Enhanced Chemical
1
Fluorescence, GE Healthcare, Uppsala, Sweden) and imaged with the ImageQuant_LAS 4000. A mild reblotting
buffer (Millipore) was applied to strip the blots. ImageQuant TL version 7.0-software was used to quantify the
blots.
Ames fluctuation assay (performed by Eurofins Cerep Panlabs). Wells that displayed bacteria growth due to
the reversion of the histidine mutation (as judged by the ratio of OD430/OD570 being greater than 1.0) are
counted and recorded as positive counts. The significance of the positive counts between the treatment (in the
presence of test compound) and the control (in the absence of test compound) are calculated using the one-
tailed Fisher's exact test. Three significance levels are reported as follows: weak positive, if 0.01 ≤ p < 0.05,
1
5

Organic & Biomolecular Chemistry
Page 16 of 17
View Article Online
DOI: 10.1039/C5OB02625C
denoted as "+", strong positive, if 0.001 ≤ p < 0.01, denoted as "++" , very strong positive, if p < 0.001, denoted
as "+++". For more details, see ESI.
Acknowledgements
This research is supported by the Ghent University Special Research Fund (BOF) and the Ghent
University Industrial Research Fund (IOF, F2014 IOF StarTT 029). V. Benoy, W. Haeck and L. Van Den
Bosch are supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen), the
University of Leuven, the Belgian government (Interuniversity Attraction Poles of the Belgian Federal
Science Policy Office), the Association Belge contre les Maladies neuro-Musculaires (ABMM), the ALS
Association (ALSA), the ALS Therapy Alliance, the Muscular Dystrophy Association (MDA), and the
European Community’s Health Seventh Framework Programme (FP7/2007-2013 under grant
agreement 259867). V. Benoy is supported by the Agency for Innovation by Science and Technology
in Flanders (IWT-Vlaanderen).
References
1
2
3
.
.
.
C. H. Arrowsmith, C. Bountra, P. V. Fish, K. Lee and M. Schapira, Nat. Rev. Drug. Discov., 2012, 11, 384-
00.
A. J. M. De Ruijter, A. H. Van Gennip, H. N. Caron, S. Kemp and A. B. P. Van Kuilenburg, Biochem. J., 2003,
70, 737-749.
4
3
a) C. Scholz, B. T. Weinert, S. A. Wagner, P. Beli, Y. Miyake, J. Qi, L. J. Jensen, W. Streicher, A. R. McCarthy,
N. J. Westwood, S. Lain, J. Cox, P. Matthias, M. Mann, J. E. Bradner and C. Choudhary, Nat. Biotechnol.,
2
015, 33, 415-423. b) X.-J. Yang and E. Seto, Nat. Rev. Mol. Cell Biol., 2008, 9, 206-218. c) G. Giannini, W.
Cabri, C. Fattorusso and M. Rodriquez Future Med. Chem., 2012, 4, 1439-1460.
I. V. Gregoretti, Y. M. Lee and H. V. Goodson, J. Mol. Biol., 2004, 338, 17-31.
4
5
6
.
.
.
L. Van Helleputte, V. Benoy and L. Van Den Bosch, Res. Rep. Biol., 2014, 5, 1-13.
a) G. I. Aldana-Masangkay and K. M. Sakamoto, J. Biomed. Biotechnol., 2011, 875824. b) S. Dallavalle, C.
Pisano and F. Zunino, Biochem. Pharmacol., 2012, 84, 756-765.
7
8
.
.
a) M. R. Shakespear, M. A. Halili, K. M. Irvine, D. P. Fairlie and M. J. Sweet, Trends Immunol., 2011, 32,
3
35-343; b) J. H. Kalin and J. A. Bergman, J. Med. Chem., 2013, 56, 6297-6313.
a) K. V. Butler, J. Kalin, C. Brochier, G. Vistoli, B. Langley and A. P. Kozikowski, J. Am. Chem. Soc., 2010, 132,
0842-10846; b) K. V. Woan, M. Lienlaf, P. Perez-Villaroel, C. Lee, F. Cheng, T. Knox, D. M. Woods, K.
1
Barrios, J. Powers, E. Sahakian, H. W. Wang, J. Canales, D. Marante, K. S. Smalley, J. Bergman, E. Seto, A.
Kozikowski, J. Pinilla-Ibarz, A. Sarnaik, E. Celis, J. Weber, E. M. Sotomayor and A. Villagra, Mol. Oncol.,
2
015, 9, 1447-1457; c) J. H. Kalin, K. V. Butler, T. Akimova, W. W. Hancock and A. P. Kozikowski, J. Med.
Chem., 2012, 55, 639-651.
9
1
1
.
R. De Vreese, T. Verhaeghe, T. Desmet and M. D'hooghe, Chem. Commun., 2013, 49, 3775-3777.
0. F. Thaler and C. Mercurio, ChemMedChem, 2014, 9, 523-536.
1. D. E. Olson, F. F. Wagner, T. Kaya, J. P. Gale, N. Aidoud, E. L. Davoine, F. Lazzaro, M. Weiwer, Y. L. Zhang
and E. B. Holson, J. Med. Chem., 2013, 56, 4816-4820.
1
2. A. Sudhakara, H. Jayadevappa, H. N. H. Kumar and K. M. Mahadevan, Lett. Org. Chem. 2009, 6, 159-164.
3. M. V. Kozlov, A. A. Kleymenova, K. A. Konduktorov and S. N. Kochetkov, Russ. J. Bioorg. Chem. 2013, 39,
1
1
02-105.
14. a) C. Y. Wang, Mutat. Res., 1977, 56, 7-12. b) P. L. Skipper, S. R. Tannenbaum, W. G. Thilly, E. E. Furth and
W. W. Bishop, Cancer Res., 1980, 40, 4704-4708.
1
6

Page 17 of 17
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB02625C
15. For a full account on the nonclinical safety assessment of Vorinostat, see: S. Galloway, A. Lagrutta, M.
Armstrong, T. Miller, V. M. Richon and P. A. Andrews, Int. J. Toxicol., 2010, 29, 3-19.
16. a) E. Krieger, G. Koraimann and G. Vriend, Proteins, 2002, 47, 393-402; b) G. Vriend, J. Mol. Graph., 1990,
8
, 52-56.
17. L. Schrodinger, 2010.
1
8. Y. Duan, C. Wu, S. Chowdhury, M. C. Lee, G. M. Xiong, W. Zhang, R. Yang, P. Cieplak, R. Luo, T. Lee, J.
Caldwell, J. M. Wang and P. Kollman, J. Comput. Chem., 2003, 24, 1999-2012.
9. O. Trott and A. J. Olson, J. Comput. Chem., 2010, 31, 455-461.
0. B. D.Strahl and C. D. Allis, Nature, 2000, 403, 41-45.
1
2
1
7