Design, Synthesis, and in vitro Evaluation of Tubulin-Targeting Dibenzothiazines with Antiproliferative Activity as a Novel Heterocycle Building Block

Article abstract of DOI:10.1002/cmdc.202100383

We prepared a series of free NH and N-substituted dibenzonthiazines with potential anti-tumor activity from N-aryl-benzenesulfonamides. A biological test of synthesized compounds (59 samples) was performed in vitro measuring their antiproliferative activity against a panel of six human solid tumor cell lines and its tubulin inhibitory activity. We identified 6-(phenylsulfonyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide and 6-tosyl-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide as the best compounds with promising values of activity (overall range of 2–5.4 μM). Herein, we report the dibenzothiazine core as a novel building block with antiproliferative activity, targeting tubulin dynamics.

Full text of DOI:10.1002/cmdc.202100383

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Design, Synthesis, and in vitro Evaluation of Tubulin-
Targeting Dibenzothiazines with Antiproliferative Activity
as a Novel Heterocycle Building Block
Walter D. Guerra,[a] Daniel Lucena-Agell,[b] Rafael Hortigüela,[b] Roberto A. Rossi,[a]
J. Fernando Díaz,[b] José M. Padrón,*[c] and Silvia M. Barolo*[a]
We prepared a series of free NH and N-substituted dibenzon-
thiazines with potential anti-tumor activity from N-aryl-benze-
nesulfonamides. A biological test of synthesized compounds
(59 samples) was performed in vitro measuring their antiproli-
ferative activity against a panel of six human solid tumor cell
lines and its tubulin inhibitory activity. We identified 6-(phenyl-
sulfonyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide and 6-tosyl-
6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide as the best compounds
with promising values of activity (overall range of 2–5.4 μM).
Herein, we report the dibenzothiazine core as a novel building
block with antiproliferative activity, targeting tubulin dynamics.
Introduction
stabilizing drugs (MSAs) bind to tubulin of the microtubule and
stabilize it, preventing its depolymerization; these agents bind
to taxanes and laulimalide/peloruside A sites.[6,8,9]
Cancer is one of the leading causes of mortality around the
world.[1] The development of safer and more effective chemo-
therapeutic agents remains on top of the biggest challenges in
medicinal chemistry.[2] Drugs targeting microtubules (micro-
tubule-targeting agents, MTAs) are among the most successful
agents. Many types of cancers have been successfully treated
with MTAs including breast, lung, head and neck tumors, as
well as lymphomas and melanomas.[3]
Microtubules are tubular dynamic structures which are
lengthened or shortened by polymerization (addition) or
depolymerization (removal) of dimers of α,β-tubulin,
respectively.[4] In this sense, MTAs bind into seven regulatory
sites and they are classified into two main groups, microtubule-
destabilizing agents (MDAs) and microtubule-stabilizing agents
(MSAs), according to their effects on microtubule polymer mass
at high concentrations.[5,6] MDAs act by inhibiting the formation
of contacts between tubulin dimers or hindering the curved-to-
straight conformational change of tubulin necessary for micro-
tubule formation;[7] these drugs bind to the colchicine, vinca,
gatorbulin, maytansine or pironetin site. On the other hand,
Sulfonamides, also known as “sulfa drugs”, have the general
formula RSO2NR1R2 where R is the corresponding substituent
(aliphatic, aromatic or heteroaromatic chain, among others).
Depending on the number of substituents in the nitrogen, they
are referred to as primary (R1 and R2 equal to H), secondary (R1
or R2 equal to H) or tertiary (R1 and R2 different from H)
sulfonamides. This organic-type structures are recognized for
their biological relevance[10] since it is an important building
block when designing biological active compounds.[11,12] In this
context, there are drugs containing sulfonamides in clinical use,
including antibacterial,[13] antifungal,[14] anti-inflammatory,[15]
antihypertensive,[16]
applications.[17,18]
and
other
pharmacological
Sulfonamides have also been explored as anti-cancer agents
due to their promising in vitro and in vivo activity.[19] The
mechanism of action described in anti-tumor sulfonamides
include either inhibition of several enzymes like carbonic
anhydrase (CA) (most of them are aromatic or heteroaromatic
primary sulfonamides),[20,21] histone deacetylase (HDAC) (secon-
dary sulfonamides),[22] aromatase (secondary[23–25] and tertiary[23]
sulfonamides), indoleamine 2,3-dioxygenase 1 (IDO1) (secon-
dary sulfonamides),[26] matrix metalloproteinase (MMP) (tertiary
sulfonamides),[27] or tubulin polymerization inhibition (secon-
dary and tertiary sulfonamides).[28] Regarding to antitubulin
sulfonamides, a major breakthrough was the discovery of E7010
(N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-meth-
[a] Dr. W. D. Guerra, Prof. R. A. Rossi, Dr. S. M. Barolo
Instituto de Investigaciones en Físico Química de Córdoba
Departamento de Química Orgánica, Facultad de Ciencias Químicas
Universidad Nacional de Córdoba, X5000HUA Córdoba (Argentina)
E-mail: sbarolo@fcq.unc.edu.ar
[b] Dr. D. Lucena-Agell, R. Hortigüela, Dr. J. Fernando Díaz
Centro de Investigaciones Biológicas Margarita Salas (CIB-MS, CSIC)
Ramiro de Maeztu 9, 28040 Madrid (Spain)
oxybenzenesulfonamide or ABT-751, A in Figure 1).[29,30] This
compound showed consistent growth-inhibitory activities
against a broad panel of human tumor cell lines and was under
clinical trial for lung type cancer, although it was declined due
to lack of efficiency.[31] The mechanism of action of E7010
involve rapid binding to tubulin and competition with
colchicine for binding.[31,32] E7010 has inspired many research
projects to develop analogues with improved efficiency.[28] Most
[c] Prof. J. M. Padrón
BioLab, Instituto Universitario de Bio-Orgánica Antonio González (IUBO-AG)
Universidad de La Laguna
C/Astrofísico Francisco Sánchez 2, 38206 La Laguna (Spain)
E-mail: jmpadron@ull.es
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100383
This article belongs to the Special Collection “Medicinal Chemistry in Latin
America”.
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Figure 1. Chemical structure of E7010 (A) and fused analogues (B and C) with anti-tumor activity. Target molecules in this work, N-substituted
dibenzothiazines.
of these analogues described in literature are related to acyclic
and/or non-fused sulfonamides having aromatic or heterocyclic
chemical motifs.
presents a wide variety of biological activities, including anti-
hepatitis C virus (HCV),[39,40] 11β-HSD1 inhibitors,[41,42] anti-HIV
and anticancer.[43,44]
In contrast, for fused sulfonamides with potential anti-tumor
activity there are only a few examples, one of them described
the synthesis and evaluation of benzopyridothiadiazepines (B,
Figure 1), as conformationally restricted analogues of E7010
(A).[33] This concept of molecular restriction is widely used in
medicinal chemistry to maximize biological activity.[34–36] Addi-
tionally, the study of benzothiadiazine derivatives (C, Fig-
ure 1)[37] as a novel class of anticancer agents that effectively
inhibit tubulin polymerization had also been described. In this
context, we proposed a novel organic scaffold: the dibenzothia-
zines (Figure 1) as a new class of tricyclic fused sulfonamides
(like B and C) and as a potentially cytotoxic compound that
could inhibit tubulin polymerization.
The aim of this work is the design and synthesis of several
(di)benzothiazine derivatives to explore these scaffolds as
building blocks of potentially biological active compounds. The
biological activity was evaluated against a panel of human
tumor cell lines using the SRB antiproliferative/cytotoxicity
assay,[45–47] as well as in vitro to determine their cellular target.
Overall, we report dibenzothiazine as a novel scaffold to achieve
compounds with good values of antiproliferative activity
through tubulin inhibition. Our results were rationalized in a
preliminary structure-activity relationship (SAR) to highlight the
most active compounds.
Regarding to the synthesis of the dibenzothiazines, we Chemistry
recently described a general protocol to obtain this scaffold
with a large variety of substituents under mild conditions.[38]
Applying this methodology, we transformed the flexible
conformation of the N-aryl-benzenesulfonamides into a fused
aromatic system, resulting in a structurally rigid analogue. On to
other hand, the skeleton of the dibenzothiazines is constituted
by the 1,2 benzothiazine 1,1-dioxide building block, which
All synthetic pathways employed in this work are presented in
Schemes 1–8, highlighting N-aryl-benzene-sulfonamides 3a, 3e,
3m, 3n and 3s-v, dibenzothiazines 6m and 6t, dibenzothia-
zines derivatives 6z, aa–ac and 7a–n, and 3-alkyl-benzothia-
zines 8a–b prepared for this work. Synthetic procedure, as well
as structural characterization for all these compounds are
Scheme 1. General protocol for the preparation of functionalized dibenzothiazines.
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Scheme 2. Reagents and conditions, yields in %. i) Method A: 1a–b,d, aniline (2a,d,g,j), pyridine, in CH2Cl2 at rt for 18 h, 30–97%.
°
Scheme 3. Reagents and conditions, yields in %: i) Method B: 4b, aryl halides (5a–b), CuI, DMEDA, K2CO3 in MeCN 18 h at 90 C, 30% for 3s, 3t was used in
°
the next step without purification; ii) 3s–t, PhB(OH)2, Pd(PPh3)2Cl2, PPh3, NaHCO3 in THF-water (1:1) at 100 C overnight, 54–59%.
Scheme 4. Reagents and conditions, t-BuOK, DMSO at rt, 5 h under
irradiation (metal iodide HPIÀ T 400 W lamps).
Scheme 5. Reagents and conditions: i) CÀ C cyclization, t-BuOK, DMSO at rt,
5 h under irradiation (metal iodide HPIÀ T 400 W lamps). Compounds were
Scheme 6. Synthesis of dibenzothiazines derivatives, 6z-ac. Reagents and
obtained as previously reported in Ref. [38].
conditions, yields in %: i) 6b, HNO3/AcOH, rt 2 h, 35%. ii) 6b or 6c, NBS,
NH4OAc in MeCN at rt overnight, 30–50%. iii) 6aa, PhB(OH)2, Pd(PPh3)2Cl2,
°
PPh3, NaHCO3 in THF-water (1:1) at 100 C overnight, 25%.
described in the experimental section. Additionally, compounds
3(b–d, f–l, o–r, w–y) and 6(b–d, f–l, o–r, w–y) were obtained as
previously reported.[38]
Our initial goal focused on the synthesis of the N-aryl-
benzene-sulfonamides (3a-v) where we explored two ap-
proaches (Scheme 1): reaction between benzenesulfonyl chlor-
ides (1a–d) and several anilines (2a–j) (Method A),[48] and Cu–
Catalyzed N-arylation of aryl-halides (5a–f) with 2-choloroben-
zenesulfonamide (4a) (Method B).[49–51] A complete description
of the synthetic protocols employed for all the benzenesulfona-
The overall synthetic strategy to obtain the final functional-
ized dibenzothiazines 7a–n involves three steps (Scheme 1): 1)
construction of N-aryl-benzene-sulfonamides (Method A and B),
2) photoinduced CÀ C cyclization to obtain the fused sulfona-
mides core, and 3) CÀ H or NÀ H derivatization reaction to obtain
functionalized dibenzothiazines.
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Table 1. Scope of photoinduced CÀ C intramolecular coupling. Synthesis of
dibenzothiazines 6b–d, f–m, o–r and t.
Entry
R1
R2
X
3
6
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
H
F
F
CF3
CF3
H
H
H
H
H
4-CF3
4-F
4-Cl
H
3a
–
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3b[a]
3c[a]
3d[a]
3e
6b[a]
6c[a]
6d[a]
nd[b]
6f[a]
6g[a]
6h[a]
6i[a]
4-CN
4-C(O)OEt
2-Ph
2-Pyrrolyl
H
4-Me
4-Me
2-Ph
2-(2-ClÀ Ph)
4-Me
4-OMe
3,5-di-OMe
4-Ph
3f[a]
3g[a]
3h[a]
3i[a]
3j[a]
3k[a]
3l[a]
3m
3n
9
10
11
12
13
14
15
16
17
18
19
20
21
22
6j[a]
6k[a]
6l[a]
6m
nd[b]
6o[a]
6p[a]
6q[a]
6r[a]
nd[b]
6t
3o[a]
3p[a]
3q[a]
3r[a]
3s
3t
3u
3v
H
H
Br
Ph
Br
Ph
H
H
4-CF3
4-CF3
ni[c]
nd[b]
[a] Compounds 3(b–d, f–l, o–r) and 6(b–d, f–l, o–r) were obtained as
previously reported in Ref. [38]. [b] The corresponding product of the
reaction was not detected after the reaction and the starting benzene-
sulfonamide 3 was recovered. [c] Compound was not isolated and used in
the next step without purification.
Scheme 7. Synthesis of dibenzothiazines derivatives, 7a-n. Reagents and
conditions, yields in %: i) For 6b–c, 6m and 6q–r, t-BuOK, and RX (Me2SO4,
ClÀ Bn, Cl–CH2-C6H4-p-CF3) in DMSO at rt overnight, 30–100%. ii) For 6b, NaH,
RX (ClÀ C(O)Me, ClÀ Ts) in THF rt overnight, 65–89%. iii) For 6b–c and 6r
NaOH, RX (ClÀ Ts, Cl-SO2Ph) in CH2Cl2 at rt overnight, 50–89%.
obtain the corresponding N-aryl-benzenesulfonamides (3s–t).
Both compounds (3s–t) underwent a Suzuki-Miyaura cross-
coupling reaction to achieve the corresponding N-aryl-biphen-
yl-sulfonamides 3u and 3v in good overall yields.
All synthesized N-aryl-benzenesulfonamides 3b–y, were
used to evaluate the scope of the photoinduced CÀ C intra-
molecular coupling and consequently obtained the fused
sulfonamides core (Scheme 4). The results regarding to the
cyclization reaction of N-aryl-benzenesulfonamides 3a–v are
presented in Table 1 where is important to highlight the novel
compounds prepared. Accordingly, dibenzothiazines 6m and
6t were isolated in 90 and 35% yields respectively and gave
further functionalities over the dibenzothiazine core previously
accomplished.[38] Despite the generality of the photo-induced
cyclization, a few benzenesulfonamides 3(e, n, s and v) did not
provide the expected dibenzothiazines 6e, 6n, 6s and 6v.
In order to synthesize tetracyclic benzothiazines, N-aryl-
benzenesulfonamides 3w–y were prepared following the same
protocols already described (see SI section, Scheme S1).[38]
Photoinduced CÀ C intramolecular coupling of 3w–y success-
fully gave the corresponding tetracyclic benzothiazines 6w–y
(Scheme 5).
Scheme 8. Synthesis of 3-alkyl-benzothiazines, 8a–b. Reagents and condi-
°
tions, yields in %: i) t-BuOK, NH3 at À 33 C, 3 h under irradiation (metal
iodide HPIÀ T 400 W lamps), 47–78%.
mides 3a–v is presented in Table S1 in the Supporting
Information (SI), section 1.1. Additionally, all chemical structures
of the synthesized benzenesulfonamides (3a–v) are listed in
Scheme 4 and Table 1 of this manuscript, as starting materials
for the CÀ C cyclization.
Regarding to benzenesulfonamides prepared for this work,
compounds 3a, 3e, 3m and 3n were successfully obtained
according to Method A (Scheme 2). Furthermore, a sequence of
Cu-catalyzed N-arylation and Suzuki-Miyaura cross-coupling
reaction[52,53] was employed to synthesize benzenesulfonamides
3u–v (Scheme 3). In this sense, 2-chloro-4-bromobenzenesulfo-
namide (4b) reacted under Cu-catalyzed conditions with
iodobenzene (5a) or bromo-4-(trifluoromethyl)-benzene (5b) to
Next, and with the purpose of expanding the functionalities
over the dibenzothiazine core, a series of CÀ H or NÀ H
derivatization reactions were performed (Schemes 6 and 7).
Initially, 6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (6b) reacted
under nitration conditions[54] to obtain regioselectively the 7-
nitrated derivative 6z (Scheme 6). Bromination of 6b and 6c
employing NBS and NH4OAc in MeCN[55] achieved the bromi-
nated dibenzothiazines 6aa and 6ab in good yields. Both
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brominated compounds (6aa and 6ab) were used as aryl
halides in a Suzuki-Miyaura cross-coupling reaction with PhB
(OH)2.[52,53] Only dibrominated dibenzothiazine 6aa was reactive
in those conditions and gave the corresponding diphenyl-
dibenzothiazine 6ac, achieving a double CÀ C substitution
(Scheme 6).
Furthermore, N-substitution of free NH of the dibenzothia-
zine core was performed. As a result, dibenzothiazines 6b–c,
6m and 6q–r were effectively N-substituted[56,57] with different
electrophiles (Me2SO4, ClÀ Bn, Cl–CH2-C6H4-p-CF3, ClÀ C(O)Me,
ClÀ Ts or Cl-SO2Ph) to achieve N-substitution of fused sulfona-
mides 7a–n (Scheme 7).
Finally, to compare the biological activity of benzothiazines
vs dibenzothiazines, we evaluated the synthesis of 2H-1,2-
benzothiazine 1,1-dioxides 8a–b under intermolecular photo-
induced unimolecular radical-nucleophilic substitution (SRN1)
type reactions with 2-chlorobenzenesulfonamide 4a and alkyl
methyl ketones (Scheme 8).[58] Despite our efforts, we only
obtained alkyl derivatives (R=t-butyl and adamantyl) 8a–b
with moderate to good yields. Other aryl methyl ketones were
tested in this reaction without success.
Table 2. For the complete list of all compounds tested refer to
the SI (Tables S2–S5).
The effect on the antiproliferative activity of the wide range
of previously synthesized compounds was made to rationalize
the results and seek initial structure-activity relationships (SAR).
This knowledge is useful to search potential leading com-
pounds for further development in future studies.[60]
Due to the fact that non-fused sulfonamides are well known
for their anti-tumor activity,[28] the description and understand-
ing of the SAR is made with compounds carrying the
benzothiazine core. The corresponding SAR description for N-
aryl-benzenesulfonamides (3a–y) is presented in Figure S3 and
described in section 1.2.2. in the SI.
First analysis comparing benzothiazines 8 and dibenzothia-
zines 6 showed no inhibition (GI50 >100 μM) for compounds
8a–b which emphasized the development of dibenzothiazines
derivatives, and the remaining analysis was made only with
families 6 and 7.
Evaluation of free NH-dibenzothiazines 6 (Table 2) exhibited
that the tricyclic sulfonamide 6b or tetracyclic sulfonamides
6w–y (without any substituent) showed no antiproliferative
activity in any of the cell lines employed. However, introduction
of a substituent(s) results in positive effects and generates
compounds with moderate activity.
Results and Discussion
Among the substituted NH-dibenzothiazines synthetically
obtained, substitution pattern could be divided in mono-
substitution (ring A or ring B, Figure 2) or di-substitution
(mono-substitution in ring A+mono-substitution in ring B or
disubstitution in ring B, Figure 2). In this manner, mono-
substitution in ring A (compounds 6j (R1 =F) and 6t (R1 =Ph))
has a positive effect over the activity only when the substituent
The compounds synthesized were evaluated for their in vitro
antiproliferative activity against human cancer cell lines A549
(lung), HeLa (cervical), HBL-100 (breast), SW1573 (lung), T-47D
(breast), and WiDr (colon). The results, expressed as 50%
growth inhibition (GI50),[59] of selected dibenzothiazines (free
NH) and N-substituted dibenzothiazines are summarized in
Table 2. Antiproliferative activity (GI50 in μM) against human solid tumor cell lines of selected compounds (6 and 7).
Comp.
A549
HBL-100
HeLa
SW1573
T-47D
WiDr
6c
6h
6i
6l
6m
6r
6t
6aa
6ab
39�2.4
37�5.9
43�3.8
44�5.3
22�0.8
34�3.2
13�2.3
28�2.9
24 �10.9
28�4.0
68�18
25�1.1
33�4.5
>100
52�4.8
48�6.8
51�8.1
83�1.8
46�4.4
39�4.9
16�3.4
47.0�0.1
36�0.5
44�2.9
40�2.5
41�4.1
52�7.2
27�1.1
34�2.1
16�4.8
30.0�7.5
25�4.7
26�1.8
42�7.2
29�4.7
25�5.5
>100
41�1.6
39�6.0
43�5.1
57�7.1
23�6.4
35�3.5
18�1.1
28�2.9
28�9.3
52�7.7
45�3.7
48�5.9
78�1.9
27�0.3
41�4.5
15.0�0.1
30�0.2
61�12
48�8.3
59�5.7
>100
31�4.2
45�6.1
19�2.6
33�0.4
37�4.9
23�3.6
33�9.3
64�5.6
28�1.9
27�1.2
50�5.8
3.4�0.7
8.1�2.1
5.5�0.5
2.1�0.5
2.0�0.7
29.0�0.5
6ac
7a
7c
7d
7e
7f
7g
7i
7j
45�5.9
21�3.5
37�4.5
>100
>100
>100
51�12
49�0.1
39�2.8
14�2.5
>100
39�16
36�8.7
>100
30�4.5
33�2.0
54�6.2
5.6�0.6
>100
6.5�0.1
3.4�0.3
11�3.6
9.7�3.8
3.1�0.2
2.6�0.5
18.0�0.5
36�6.3
4.4�0.2
>100
>100
12.0�0.5
5.4�0.8
4.7�0.9
31.0�0.9
36�4.1
4.9�0.2
1.5�0.3
n.d.
33�14
3.2�0.6
3.8�0.3
72�3.3
71�3.3
5.7�1.2
4.7�0.8
4.2�0.9
47�1.0
93�6.1
3.0�0.4
14�1.5
4.5�1.7
13.0�0.4
2.7�0.3
3.1�0.4
30�7.7
30�7.7
7k
7l
7m
36�15
Cisplatin
Etoposide
Paclitaxel*
1.9�0.2
2.0�0.3
15�2.3
22�5.5
0.15�0.02
26�5.3
1.4�0.1
3.3�1.6
23�3.1
0.015�0.004
0.0045�0.0012
0.32�0.02
n.d.: Not determine. For all compounds, GI50 values are given in μM and are means of two to four experiments (Mean�SD). For paclitaxel, GI50 values are
given in nM.
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Figure 2. SAR of dibenzothiazines (6b-d, 6f-m, 6o-r, 6x-z, 6aa-ac and 7a-n).
in position 2 is Ph, with a GI50 range of 13–19 μM (Table 2).
Regarding monosubstitution in ring B, compounds with CF3
(6c) or Ph (6r) group in position 9, and compounds with Ph
(6h) or pyrrolyl (6i) in position 7 showed a moderate growth-
inhibitory activity (34–61 μM, Table 2). In the case of disubstitu-
tion, when both R groups were in ring B, compounds 6aa–6ac
gave moderate activity (overall range 21–47 μM). The best result
obtained for this series was compound 6ac (7,9-di-Ph) with an
activity of 21 μM over the lung cancer cells SW1573. Finally,
disubstitution coming from substitution in both rings A and B
(compounds 6k–m) went from no activity (6k) to some activity
(6l) to moderate activity in compound 6m (R1 =CF3 and R2 =7-
Ph) with a GI50 range of 22–46 μM. In summary, the best result
across all the free NH-dibenzothiazines 6 was obtained for
compound 6t (R1 =Ph and R2 =H) in the lung cancer cell type
A549 with an activity of 13 μM.
The results for the family of N-substituted dibenzothiazines
(7a–n, Table 2), showed that this family of compounds is the
most active considering all the compound families synthesized
and analyzed in this work (see Tables S2-S5 in section 1.2.1. in
the SI). In consequence, different substituents like methyl,
acetyl, benzyl, tosyl, among others were attached. Particularly,
the large increase in antiproliferative activity was found after N-
substitution of the dibenzothiazine core (R1 =R2 =H) with Bn
(7f), p-CF3-C6H4CH2 (7i), SO2-Ph (7j) or Ts (7k) (Figure 2).
Compounds 7j (N-SO2-Ph) and 7k (N-Ts) showed the best
growth inhibition values against all cell lines (2.0–5.4 μM)
(Figure 2). On the other hand, 7j and 7k showed GI50 values
comparable to the reference compounds, cisplatin and
etoposide.[61] In addition to cisplatin and etoposide, we used
the tubulin binding agent paclitaxel as a reference drug. As
shown in Table 2, GI50 values for paclitaxel were in the nano-
molar range for SW1573 cells and in the sub nanomolar range
for the other cells tested. Paclitaxel and other naturally
occurring antimitotic drugs (colchicine and vinca alkaloids)
have shown in multiple clinical trials a limited therapeutic
window due to toxicities to tissues as a result of their extremely
high potencies.[61] Therefore, our compounds might represent
alternative drug candidates with a balanced activity-potency.
Finally, when N-substitution and CÀ H substitution in ring B
were combined, the antiproliferative activity did not improve.
For instance, compound 6c (R2 =9-CF3 and R1 =R3 =H) slightly
increases the activity from 39–61 μM to 25–49 μM for 7d (N-
methyl derivative). In contrast, compounds with Ts in the N
together with CF3 or Ph group in position 9 in the cyclic
sulfonamide core were less or non-active (6c vs 7m and 6r vs
7n). However, compound 7g (R1 =H, R2 =CF3) with a benzyl
group in the N resulted in a very potent agent against HeLa
and T-47D cell lines with activity values in the range 8.1–11 μM.
From the initial SAR of these novel dibenzothiazines
scaffolds, compounds 7f, 7j–k presented the best antiprolifer-
ative activities with values in an overall range of 2–14 μM.
Regarding these compounds, a chemical space inspection was
made (Table S6, section 2 in the SI) showing suitable values of
molecular descriptors for a future lead optimization program.
With the aim of understanding the molecular mechanism of
action, compounds 7j and 7k (GI50 range of 2–5.4 μM) were
initially tested for their effect on the cell cycle. Antiproliferative
cancer chemotherapy frequently takes advantage of cell cycle
checkpoints to alter cell division and ultimately induce cell
death. In this sense, DNA duplication or DNA segregation are
two broad targets for cell cycle anti-tumor drugs. For the first
class, DNA damage agents[62] or induction of replication stress[63]
are used while for the second type, agents are aimed towards
impeding chromosome segregation (i.e. antitubulin, antitopoi-
somerase II)[64] or interfering with the spindle assembly
checkpoint (i.e. Aurora B, Plk1).[65] Those compounds targeting
DNA segregation stop cell cycle in the G2/M phase. Both
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compounds studied arrest cell cycle in the G2/M phase
(Figure 3), indicating a blocking of the DNA segregation
(antimitotic) way of action.
tubulin assembly inhibition mechanisms, through two known
sites (pironetin/maytansine and colchicine). Ligands that bind
to pironetin[67] and maytansine[68] sites poison microtubules by
blocking the longitudinal interfaces without preventing the
curve-to-straight transition needed to form the lateral contacts
with non-poisoned subunits. These drugs are extremely potent
in inhibiting microtubule assembly at extremely low concen-
trations (sub nM).[69] On the other hand, colchicine site drugs
use a blocking mechanism binding at the intradimer interface,
preventing the curve-to-straight transition needed for assembly
and creating a set of inactive subunits which bind with lower
affinity to the microtubule end (because they cannot form the
lateral contacts with straight tubulin subunits) increasing the
critical concentration required for assembly; such kind of
inhibitors require a significant percent of tubulin molecules
bound to the drug to observe assembly inhibition[70] and
therefore inhibit microtubule assembly at concentrations closer
to this of tubulin i.e. μM. Our data suggest that the compounds
explored use a blocking mechanism for tubulin assembly
inhibition, pointing towards the colchicine site; however also a
maytansine site drug with a low affinity (in the order of 104–
105 MÀ 1 range)[71] would behave in the same way, so a poisoning
mechanism cannot be discarded.
Tubulin is one of the targets for antimitotic agents, and
those compounds targeting tubulin modulate its ability to
polymerize forming microtubules. As described in the introduc-
tion section, tubulin modulators either inhibit (MDAs) or
enhance (MSAs) tubulin polymerization, for their ability to
inhibit tubulin polymerization in vitro. In this sense, compounds
7j and 7k were tested for their ability to modulate tubulin
assembly and selected results of these experiments are
presented in Figure 4. Results show that these compounds were
able to fully inhibit tubulin assembly (Figure 4A). In addition, 7j
and 7k show the best antiproliferative values against all cell
lines (2.0–5.4 μM, Table 2). Additionally, tubulin polymerization
assay has also been carried out with compounds 7e–f and N-
aryl-benzenesulfonamides 3b, 3n, 3h to further inspect these
compounds and, the results are presented in Figure S4, section
1.2.3 in the SI. It should be mentioned that tubulin assembly
inhibition showed a good correlation with the observed growth
inhibition values.[66]
Tubulin assembly inhibition by these compounds requires a
significant stoichiometry of drug to tubulin to be effective
(Figure 4B and 4 C), like the compound podophyllotoxin (Fig-
ure S5, section 1.2.4. in the SI) suggesting a blocking mecha-
nism (bound subunits are unable to assemble) instead of a
poisoning one (bound subunits prevent the addition of un-
bound ones poisoning the microtubule). There are two kinds of
Tubulin is a promiscuous protein with up to 6 well known
pharmacologically regulation sites (paclitaxel, laulimalide, pir-
onetin, vinca, maytansine and colchicine)[7] and a seventh one
just recently described, gatorbulin.[6] Binding of ligands to four
of these sites results in tubulin assembly inhibition (vinca,
Figure 3. Flow cytometry analysis of cell cycle distribution of A549 cells treated for 24 h with different concentrations of 7j (A), 7k (B) and podophyllotoxin
(C). All experiments were performed with 0.5% of DMSO. All experiments were carried out at least three times.
Figure 4. Inhibition of tubulin assembly activity by selected compounds. All experiments were performed in triplicate. (A) Time course of assembly of 25 μM
Tubulin in GAB in the presence of vehicle (DMSO) black line, 7k (green), 7j (pink) or podophyllotoxin (blue). (B) Dose response inhibition of tubulin assembly
by 7j. Time course of assembly of 25 μM Tubulin in GAB in the presence of vehicle (DMSO) black line, 1 μM (red), 5 μM (green), 10 μM (yellow), 15 μM (blue),
20 μM (pink), 25 μM (cyan) or 27.5 μM (orange) of 7j. (C) Dose response inhibition of tubulin assembly by 7k. Time course of assembly of 25 μM Tubulin in
GAB in the presence of vehicle (DMSO) black line, 1 μM (red), 5 μM (green), 10 μM (yellow), 15 μM (blue), 20 μM (pink), 25 μM (cyan) or 27.5 μM (orange) of
7k.
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gatorbulin, maytansine and colchicine). Due to this, fluorescent
bona fide probes have been developed for evaluating the
binding features to the three classical MDAs sites (colchicine,
vinca and maytansine).[72–74] These tests are based either on
fluorescence changes upon compound immobilization or on
the higher anisotropy of the fluorescence exerted by the probe
when it is bound to a larger molecule,[75] which allows the
binding evaluation by competition between the fluorescent
probes and the problem ligands for the site.[76] Among the
available probes, we selected those available for the vinca and
maytansine site, Eribulin-A488[73] and Fc-Maytansine.[74] Further-
more, for the colchicine site we selected MTC (2-methoxy-5-
(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one) because
of its mild affinity for the colchicine binding site (Ka 4.7×
105 MÀ 1 Kd 2.1 μM);[71] in this way, a mild affinity ligand should
permit detection of a weaker competitor.
substrates. Some preliminary SAR were described with the
in vitro antiproliferative activity against six human cancer cell
lines to rationalize the results obtained and highlight the most
active compounds. These compounds arrest the cell cycle in the
G2/M phase indicating an antimitotic way of action. Additional
biochemical experiments demonstrate that the cellular target of
these compounds is tubulin and that they act inhibiting its
polymerization at substoichiometric concentrations, showing a
minimal affinity in the μM range. However, no competition with
the three canonical tubulin inhibition sites (colchicine, vinca
and maytansine) is detected even at 50 μM concentrations of
the most active compounds. Our studies suggest that inter-
action with tubulin does not occur through colchicine, vinca or
maytansine sites. As a result, we are currently studying the
mechanism of action of these novel building blocks targeting
tubulin dynamics.
Unexpectedly, compounds 7j and 7k, which were the most
potent in the in-cell and tubulin polymerization tests failed in
displacing both Eribulin-A488 from vinca site[73] and Fc- Experimental Section
Maytansine from maytansine site[74] at concentrations of up to
50 μM, indicating that their interaction with tubulin does not
occur through these sites. More surprisingly, they also fail to
displace MTC from the colchicine site.[77] In the conditions
employed for the assay (10 μM of tubulin and MTC and 50 μM
of the ligands), a displacement (10%) would be expected if the
binding affinity is over Ka 5×104 MÀ 1 Kd 0.2 mM. Considering
that the compounds produce significant assembly inhibition at
1 μM concentrations (and the fact that inhibitory effect could
be observed only if the compound is bound to its site) their
binding constants for tubulin have to be in the 105–106 MÀ 1
(Kd =1–10 μM) range. Therefore, if the compounds require a
Kd =1–10 μM to inhibit tubulin assembly in the assay and they
do not displace MTC at 50 μM concentrations, it is straightfor-
ward to deduce that they do not bind at the colchicine site.
Even though compounds with similar molecular weights,
compared to compounds presented in this work, interact with
the colchicine binding site, it should be noted that there are
examples of compounds that interact with other binding sites
on tubulin such as paclitaxel,[66] pironetin,[78] and vinca.[79]
Despite our efforts, the binding site of compounds 7j and 7k
was not detected, although the results suggest that interaction
with tubulin does not occur through the binding sites of
colchicine, vinca or maytansine. Consequently, the study of
these compounds’ mechanism of action is still under inves-
tigation.
Chemistry
Purification of reaction products was carried out by flash chroma-
tography using column chromatography on silica gel. Structural
characterization of all compounds was made by H NMR (400 MHz)
1
and 13C NMR (100.62 MHz) spectra employing CDCl3, dimethyl
sulfoxide-d6 (CD3SOCD3) or acetone-d6 (CD3COCD3) as solvent.
Coupling constants are given in Hz and chemical shifts are reported
in δ values in ppm. Data are reported as followed: chemical shift,
multiplicity (s=singlet, s br=broad singlet, d=doublet, t=triplet,
dd=double doublet, dt=double triplet, ddd=double doublet,
m=multiplet), coupling constants (Hz), and integration. Copies of
1H NMR and 13C NMR spectra are provided in SI. All new products
were further characterized by HRMS. HRMS analyses were carried
out using a TOF-MS instrument with an ESI source. Photoinduced
reactions were conducted in a reactor equipped with two Philips
HPIÀ T 400 W lamps of metallic iodide (cooled with water). DMSO,
THF and CH2Cl2 as solvents were stored under molecular sieves
(4 Å). All solvents were analytical grade. Silica gel (0.063–0.200 mm)
was used in column chromatography. Unless otherwise stated, all
reagents were purchased from commercial suppliers and used
without further purification. Synthetic procedures of all synthesized
compounds are described in next session as well as purification and
characterization of unknown compounds.
Synthesis of N-aryl-benzenesulfonamides (3a–y). We employed
two methodologies (Method A and Method B) to synthetize the
family of N-aryl-benzenesulfonamides, 3a–y (see Table S1). Addi-
tionally, compounds 3b and 3c were obtained employing both
methodologies.
Method A. Employing the reaction between benzenesulfonyl
chlorides and anilines,[48] compounds 3a-n and 3w were obtained.
As an example of this methodology the synthesis of N-phenyl-
benzenesulfonamide (3a) is described next. In a Schlenk tube
equipped with a nitrogen inlet and magnetic stirred was charged
with aniline 2a (1 equiv, 1 mmol) dissolved in dry CH2Cl2 (1.2 mL),
the solution was treated with sulfonyl chloride (1a, 1.2 equiv,
1.2 mmol) and pyridine (3 equiv, 3 mmol). The mixture was stirred
at rt for 18 h, diluted with H2O (15 mL) and extracted with CH2Cl2
(3×25 mL). The combined organic layers were washed with 1 M
HCl, brine, dried over Na2SO4, and concentrated in vacuum. After
removal of volatile components, the resulting crude product was
purified by column chromatography on silica gel eluting with
Conclusion
We presented the design, synthesis, and biological evaluation
of a wide number of fused and non-fused sulfonamides. This is
the first report of dibenzothiazine core as a novel building block
to obtain anti-tumor agents. The best results were found in the
N-substituted dibenzothiazines 7j and 7k, which inhibited cell
proliferation with GI50 values in the range 2–5.4 μM. These are
simple and robust molecules, which can be easily obtained in
just three reaction steps from commercial and accessible
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°
pentane/EtOAc (100:0!80:20). N-phenylbenzenesulfonamide (3a)
25 C, TMS): δ=135.3, 134.2, 133.0, 132.3, 130.5, 129.5, 128.2, 126.0,
121.6 ppm. HRMS (TOF, ESI+) m/z calcd for C12H9BrClNO2S+Na+:
367.9118 [M+Na]+; found: 367.9173.
1
was isolated as a white solid in 97% yield (0.97 mmol, 226 mg). H
°
NMR (400 MHz, CDCl3, 25 C, TMS): δ= 7.80–7.78 (m, 2H), 7.56–7.48
(m, 1H), 7.45–7.41 (m, 2H), 7.25–7.21 (m, 2H), 7.13–7.00 ppm (m,
4H). Resonances of NÀ H is overlap with the ArÀ H signals and no
assignment was made in this region. 13C NMR (101 MHz, CDCl3,
Compound 2-chloro-N-phenylbenzenesulfonamide (3b) was iso-
lated in 78% yield employing the first approach (Method A) and
92% using the second approach (Method B). In the same manner,
2-chloro-N-(4-(trifluoromethyl)phenyl)benzenesulfonamide (3c) was
isolated in a 51% with Method A and in 86% employing Method B.
°
25 C, TMS): δ= 139.1, 136.4, 133.0, 129.3, 129.0, 127.2, 125.5,
121.8 ppm.[80]
2-Chloro-N-(4-chlorophenyl)benzenesulfonamide (3e).[81] Titled
compound was obtained according to previously described
methodology and purified by column chromatography on silica gel
eluting with pentane/EtOAc (100:0!80:20). White solid was
obtained in 90% yield (163 mg, 0.54 mmol). 1H NMR (400 MHz,
Derivatization of N-aryl-benzenesulfonamides. Synthesis of com-
pounds 3t and 3v. 3-Chloro-N-phenyl-[1,1’-biphenyl]-4-sulfona-
mide (3t) was synthesized starting with 3s (as an aryl halide) and
phenylboronic acid using a previously reported Suzuki-Miyaura
procedure.[52] A solution of 4-bromo-2-chloro-N-phenylbenzenesul-
fonamide (3s) (34.7 mg, 0.1 mmol), phenylboronic acid (14.6 mg,
0.12 mmol), Pd(PPh3)2Cl2 (3.5 mg, 0.005 mmol), PPh3 (2.6 mg,
0.01 mmol) and NaHCO3 (25.2 mg, 0.3 mmol) in DME (2 mL) was
stirred at room temperature for 5 min. H2O (2 mL) was added, and
the resulting mixture was slightly degassed, sealed, and stirred at
°
CDCl3, 25 C, TMS): δ=7.86 (dd, J=7.6, 0.8 Hz, 1H), 7–51–7.45 (m,
2H), 7.36–7.32 (m, 1H), 7.19–7.16 (m, 2H), 7.07–7.05 ppm (m, 3H).
Resonances of NÀ H is overlap with the ArÀ H signals and no
assignment was made in this region.
N-([1,1’-Biphenyl]-2-yl)-2-bromo-4-(trifluoromethyl)-benzenesul-
fonamide (3m). The product was purified by column chromatog-
raphy on silica gel eluting with pentane/EtOAc (100:0!95:5).
Colorless oil was obtained in 30% yield (68.4 mg, 0.15 mmol). 1H
°
120 C overnight. After being cooled to room temperature, the
mixture was extracted with EtOAc. The extracts were combined,
dried over Na2SO4, and filtered. After removal of volatile compo-
nents, the resulting crude product was purified by column
chromatography on silica gel eluting with pentane/EtOAc (100:0!
°
NMR (400 MHz, CDCl3, 25 C): δ= 8.12 (d, J=8.4 Hz, 1H; ArÀ H), 7.79
(br.s, 1H; NH), 7.62 (d, J=8.4 Hz, 1H; ArÀ H), 7.54 (d, J=8.4 Hz, 1H;
ArÀ H), 7.41–7.39 (m, 3H; ArÀ H), 7.30–7.25 (m, 2H; ArÀ H), 7.15–
80:20). White solid was obtained in 59% yield (20.3 mg,
13
1
°
°
7.14 ppm (m, 4H; ArÀ H). C NMR (101 MHz, CD3COCD3, 25 C): δ=
0.059 mmol). H NMR (400 MHz, CDCl3, 25 C, TMS): δ= 8.04 (d, J=
142.1, 137.4, 135.5 (q, J=33 Hz, 1 C), 134.2, 132.8, 132.4 (q, J=4 Hz,
1 C), 132.3, 130.9, 129.4, 129.2, 128.8, 128.5, 125.5, 124.6 (q, J=4 Hz,
8.4 Hz, 1H), 7.70 (d, J=1.2 Hz, 1H), 7.55–7.42 (m, 7H), 7.26–7.05 ppm
(m, 5H). Resonances of NÀ H is overlap with the ArÀ H signals and no
assignment was made in this region. 13C NMR (101 MHz, CDCl3,
19
°
1 C), 123.8, 121.1, 121.0 ppm. F NMR (377 MHz, CD3COCD3, 25 C):
δ=À 63.27 ppm. HRMS (TOF, ESI+) m/z calcd for C19H13BrF3NO2S+
Na+: 477.9695 [M+Na]+; found: 477.9729.
25 C, TMS): δ= 147.2, 137.7, 135.8, 134.4, 132.4, 131.6, 129.9, 129.4,
°
129.1, 129.1, 127.2, 125.8, 125.6, 121.6 ppm. HRMS (TOF, ESI+) m/z
calcd for C18H14ClNO2S+Na+: 366.0326 [M+Na]+; found: 366.0304.
2-Chloro-N-(2’-chloro-[1,1’-biphenyl]-2-yl)benzenesulfonamide
(3n). The product was purified by column chromatography on silica
gel eluting with pentane/EtOAc (100:0!70:30). Light yellow oil
was obtained in 50% yield (113.5 mg, 0.3 mmol). 1H NMR (400 MHz,
3-Chloro-N-(4-(trifluoromethyl)phenyl)-[1,1’-biphenyl]-4-sulfona-
mide (3v) was obtained as a sequence of two steps. First, Cu–
Catalyzed N-arylation between 4-bromo-2-chlorobenzenesulfona-
mide (4a, 95.8 mg, 0.5 mmol) and 4-(trifluoromethyl)-1-bromoben-
zene (5b, 272 mg, 1 mmol) lead us obtain the corresponding 4-
bromo-2-chloro-N-(4-(trifluoromethyl)-phenyl)-benzenesulfonamide
(3u). This last compound (3u) was obtained using the same
procedure used for 3s, but the resulting crude product was used in
the next step without purification. HRMS (TOF, ESI+): calcd for 3u,
C13H8BrClF3NO2S+Na (M+Na)+: 435.8992; found: 435.8977. Finally,
3r was obtained after the Suzuki-Miyaura cross-coupling between
3u and phenylboronic acid using the same procedure followed for
3t. The product was purified by column chromatography on silica
gel eluting with hexane/EtOAc (100:10!80:20). Brown oil was
°
CDCl3, 25 C, TMS): δ= 7.93 (d, J=7.6 Hz, 1H), 7.63 (d, J=8.0 Hz, 1
H), 7.48–7.16 (m, 8H), 7.07 (d, J=7.2 Hz, 1H), 6.93–6.91 ppm (m, 2H).
Resonances of NÀ H is overlap with the ArÀ H signals and no
assignment was made in this region. 13C NMR (101 MHz, CDCl3,
°
25 C, TMS): δ=136.9, 136.0, 133.8, 133.7, 133.4, 132.0, 132.0, 131.7,
131.3 (x2), 130.7, 130.0, 129.9, 129.1, 127.2, 126.9, 125.3, 122.8 ppm.
HRMS (TOF, ESI+) m/z calcd for C18H13Cl2NO2S+Na+: 399.9936 [M+
Na]+; found: 399.9967.
Method B. Using Cu–Catalyzed N-arylation of aryl-halides with 2-
halobenzenesulfonamides,[38] compounds 3b-c, 3o-r and 3x-y were
synthetized. As an example of this methodology, 4-bromo-2-chloro-
N-phenylbenzenesulfonamide (3s) is described next. In a Schlenk
tube equipped with a nitrogen inlet and magnetic stirred was
charged with 4-bromo-2-chlorobenzenesulfonamide 4a (162 mg,
0.6 mmol), copper iodide (11.4 mg, 0.06 mmol), aryl halide (iodo-
benzene, 5a, 244.48 mg, 1.2 mmol), K2CO3 (248.8 mg, 1.8 mmol),
acetonitrile (MeCN) (4 mL), and N,N-dimethylethane-1,2-diamine
obtained in an overall 54% (111.2 mg, 0.27 mmol) yield after two
steps. H NMR (400 MHz, CDCl3, 25 C, TMS): δ= 8.15 (d, J=8.4 HZ,
1H), 7.70 (d, J=1.6 Hz, 1H), 7.59–7.40 (m, 10H), 7.27–7.25 ppm (m,
1H; ArÀ H). Resonances of NÀ H is overlap with the ArÀ H signals and
no assignment was made in this region. 13C NMR (101 MHz, CDCl3,
1
°
°
25 C, TMS): δ= 147.8, 139.2, 137.5, 134.0, 132.5, 131.8, 130.2, 129.3,
129. 2, 127.3, 126.7 (q, J=4 Hz, 1 C), 125.7, 123.8 (q, J=270 Hz, 1 C),
°
(DMEDA, 26.4 mg, 0.3 mmol). The tube was then heated to 90 C for
119. 8 ppm. 19F NMR (377 MHz, CDCl3, 25 C, TMS): δ=À 62.29 ppm.
°
18 h. The reaction mixture was then cooled to room temperature,
2 M HCl (10 mL) was added slowly, followed by EtOAc extraction
(3×15 mL). The organic layers were combined, dried over Na2SO4,
and filtered. After removal of volatile components, the resulting
crude reaction product was purified by column chromatography on
silica gel eluting with pentane/EtOAc (100:0!80:20). 4-Bromo-2-
chloro-N-phenylbenzenesulfonamide (3s) was obtained as a white
HRMS (TOF, ESI+) m/z calcd for C19H13ClF3NO2S+Na+: 434.0200 [M
+Na]+; found: 434.0207.
Synthesis of dibenzothiazines 6: Intramolecular photoinduced
arylation of N-aryl-benzenesulfonamides. Compounds 6b–d, 6f–l,
6o–r and 6w–y were obtained employing developed procedures.[38]
In this family, compounds 6m and 6t were not described before
and the procedure is describe next exemplified for 2-phenyl-6H-
dibenzo[c,e][1,2]thiazine 5,5-dioxide (6t). The reaction was carried
out in a Schlenk tube equipped with a nitrogen inlet and magnetic
stirred. DMSO (5 mL) was deoxygenated and t-BuOK (3.0 equiv,
50.5 mg, 0.45 mmol) was added and after 5 min the corresponding
1
solid in 30% yield (62.4 mg, 0.18 mmol). H NMR (400 MHz, CDCl3,
°
25 C, TMS): δ=7.84 (d, J=8.4 Hz, 1H), 7.65 (d, J=2.0 Hz, 1H), 7.45
(dd, J=8.4, 2.0 Hz, 1H), 7.26–7.21 (m, 2H), 7.12–7.10 ppm (m, 4H).
Resonances of NÀ H is overlap with the ArÀ H signals and no
assignment was made in this region. 13C NMR (101 MHz, CDCl3,
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3-chloro-N-phenyl-[1,1’-biphenyl]-4-sulfonamide
(3t,
1 equiv,
(100:0!80:20). White solid was obtained in 30% yield (11.7 mg,
1
°
0.15 mmol, 51.6 mg) was added and the reaction mixture was
irradiated for 5 h. The reaction was quenched with ammonium
nitrate in excess and the residue was extracted with EtOAc (3×
30 mL), organic extracted was washed with water, dried with
anhydrous Na2SO4 and concentrated to remove the volatile fraction.
The product was purified by column chromatography on silica gel
eluting with pentane/EtOAc (100:10!80:20). White solid was
obtained in 90% yield (41.5 mg, 0.135 mmol). 1H NMR (400 MHz,
0.03 mmol). H NMR (400 MHz, CD3SOCD3, 25 C): δ= 10.59 (s br,
1H; NH), 8.02 (s, 1H; ArÀ H), 7.87 (d, J=7.6 Hz, 1H; ArÀ H), 7.61 (s, 1H;
ArÀ H), 7.50 (d, J=7.2 Hz, 1H; ArÀ H), 7.38–7.30 ppm (m, 2H; ArÀ H). 13
°
C NMR (101 MHz, CD3SOCD3, 25 C): δ= 135.4, 135.4, 134.2, 132.8,
130.5, 130.1, 128.0, 127.8, 125.1, 121.6, 118.7, 118.2 ppm. MS (EI,
70 eV) m/z (%): 391 (40), 390 (27), 389 (57), 387 (46), 325 (11), 246
(21), 244 (35), 166 (13), 165 (100), 164 (61), 138 (28), 137 (22), 82
(60), 69 (31), 63 (12). HRMS (TOF, ESIÀ ) m/z calcd for C12H7Br2NO2S-
HÀ : 385.8491 [MÀ H]À ; found: 385.8487.
°
CD3COCD3, 25 C, TMS): δ= 9.94 (s br, 1H; NH), 8.39 (d, J=8.0 Hz,
2H; ArÀ H), 8.05 (d, J=8.0 Hz, 1H; ArÀ H), 7.94–7.92 (m, 1H; ArÀ H),
7-Bromo-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-di-
oxide (6ab). Titled compound was synthesized according to the
bromination procedure described above and employing 9-
(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (6c) as
starting material. The product was purified by column chromatog-
raphy on silica gel eluting with pentane/EtOAc (100:10!80:20).
White solid was obtained in 50% yield (18.9 mg, 0.05 mmol). 1H
7.84 (d, J=7.6 Hz, 2H; ArÀ H), 7.56–7.45 (m, 4H; ArÀ H), 7.37–
7.32 ppm (m, 2H; ArÀ H). 13C NMR (101 MHz, CD3COCD3, 25 C, TMS):
°
δ=146.0, 140.4, 138.0, 135.2, 133.9, 131.3, 129.9, 129.4, 128.4, 127.8,
126.6, 125.1, 124.9, 123.4, 123.0, 120.8 ppm. HRMS (TOF, ESI+) m/z
calcd for C18H13NO2S+Na+: 330.0559 [M+Na]+; found: 330.0535.
7-Phenyl-2-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-di-
oxide (6m). The product was obtained according to the above
°
NMR (400 MHz, CDCl3, 25 C, TMS): δ= 8.06 (d, J=8.0 Hz, 1H; ArÀ H),
procedure
employing
N-([1,1’-biphenyl]-2-yl)-2-bromo-4-
7.94 (d, J=8.0 Hz, 1H; ArÀ H), 7.84 (s, 1H; ArÀ H), 7.80–7.76 (m, 1H;
(trifluoromethyl)benzenesulfonamide (3m) as starting material and
purified by column chromatography on silica gel eluting with
ArÀ H), 7.70–7.66 (m, 1H; ArÀ H), 7.59 ppm (m, 2H; ArÀ H and NH). 13
C
°
NMR (101 MHz, CDCl3, 25 C, TMS): δ= 145.1, 134.9, 132.9, 130.9,
129.8, 126.2, 125.9, 124.7, 122.2, 120.4 (q, J=257 Hz, 1 C), 117.6,
pentane/EtOAc (100:0!90:10). White solid was obtained in 35%
1
19
°
°
yield (19.7 mg, 0.052 mmol). H NMR (400 MHz, CD3COCD3, 25 C,
114.2 ppm. F NMR (377 MHz, CDCl3, 25 C, TMS): δ=À 58.13 ppm.
TMS): δ= 9.29 (s br, 1H; NH), 8.63 (s, 1H; ArÀ H), 8.43 (dd, J=7.2,
2.4 Hz, 1H; ArÀ H), 8.20 (d, J=8.0 Hz; 1H; ArÀ H), 8.06 (d, J=8.0 Hz,
1H; ArÀ H), 7.59–7.44 ppm (m, 7H; ArÀ H). 13C NMR (101 MHz,
HRMS (TOF, ESI+) m/z calcd for C13H7BrF3NO2S+K+: 415.8965+ [M
+K]; found: 415.8970.
The compound 7,9-diphenyl-6H-dibenzo[c,e][1,2]thiazine 5,5-di-
°
CD3COCD3, 25 C, TMS): δ= 139.1, 137.7, 136.1, 133.9, 133.7, 133.5
oxide (6ac) was synthesized employing
a
Suzuki-Miyaura
(q, J=32 Hz, 1 C), 132.5, 129.6, 128.8, 127.9, 125.7, 125.3, 125.3 (q,
J=4 Hz, 1 C), 124.5, 123.7 (q, J=271 Hz, 1 C), 123.6 (q, J=4 Hz, 1 C),
procedure[52] and 7,9-dibromo-6H-dibenzo[c,e][1,2]thiazine 5,5-diox-
ide (6aa) previously isolated and characterized as an aromatic
halide. A solution of 6u (39 mg, 0.1 mmol), phenylboronic acid
(36.5 mg, 0.3 mmol), Pd(PPh3)2Cl2 (3.5 mg, 0.005 mmol), PPh3
(2.6 mg, 0.01 mmol) and NaHCO3 (25.2 mg, 0.3 mmol) in DME
(2 mL) was stirred at room temperature for 5 min. H2O (2 mL) was
added, and the resulting mixture was slightly degassed, sealed, and
123.0 ppm. 19F NMR (377 MHz, CD3COCD3, 25 C, TMS): δ=
°
À 63.38 ppm. HRMS (TOF, ESI+) m/z calcd for C19H12F3NO2S+Na+:
398.0433 [M+Na]+; found: 398.0456.
C-H Derivatization of dibenzothiazines. Synthesis of compounds
6z-ac. 7-Nitro-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (6z). Titled
compound was obtain employing a nitration procedure previously
reported.[54] A solution of 2 mL of nitric acid in acetic acid (1:5) was
added dropwise to a solution of 6a (34.7 mg, 0.15 mmol) in acetic
acid (1 mL). After 60 min., the yellow precipitate was removed by
filtration and dissolved in 1 M NaOH (5 mL). The pH level of a final
solution was acidified at neutral pH, followed by EtOAc extraction
(3×15 mL). The organic extracted was washed with water and dried
with anhydrous Na2SO4. After removal of volatile components from
the filtrate, the resulting crude product was purified by column
chromatography on silica gel eluting with pentane/EtOAc (100:0!
°
stirred at 120 C overnight. After being cooled to room temperature,
the mixture was extracted with EtOAc (3×15 mL). The extracts were
combined, dried over Na2SO4, and filtered. After removal of volatile
components, the resulting crude product was purified by column
chromatography on silica gel eluting with pentane/EtOAc (100:0!
90:10). Light-yellow oil of 6u was isolated in 25% yield (9.6 mg,
1
°
0.025 mmol). H NMR (400 MHz, CD3SOCD3, 25 C): δ= 10.28 (s br,
1H; NH), 8.48 (d, J=8.0 Hz, 1H; ArÀ H), 8.44 (s, 1H; ArÀ H), 7.94–7.82
(m, 4H; ArÀ H), 7.73–7.65 (m, 4H; ArÀ H), 7.56–7.40 ppm (m, 6H;
ArÀ H). 13C NMR (101 MHz, CD3SOCD3, 25 C): δ= 139.2, 138.1, 137.9,
°
60:40). A yellow solid of 6t was obtained in 35% yield (14.5 mg,
137.5, 135.8, 132.6, 132.4, 129.9, 129.7, 129.0, 129.0, 128.4, 127.8,
127.7, 127.2, 127.0, 126.7, 123.1, 121.6 ppm. HRMS (TOF, ESI+) m/z
calcd for C24H17NO2S+Na+: 406.0872 [M+Na]+; found: 406.0834.
1
°
0.052 mmol). H NMR (400 MHz, CD3SOCD3, 25 C): δ= 8.62–8.60
(m, 1H, ArÀ H), 8.31 (d, J=8.0 Hz, 1H; ArÀ H), 8.16 (dd, J=8.0, 1.2 Hz,
1H; ArÀ H), 7.99 (dd, J=7.2, 0.8 Hz, 1H; ArÀ H), 7.91–7.87 (m, 1H;
ArÀ H), 7.78 (t, J=7.2 Hz, 1H; ArÀ H), 7.58 ppm (t, J=8.0 Hz, 1H;
Synthesis of N-substituted dibenzothiazines (7a–n). In order to
achieve the N-substitution of some of the previously obtained
dibenzothiazines we employed three methodologies that are
described next. Method A. In a dried Schlenk tube, equipped with a
nitrogen inlet and a magnetic bar, was prepared a solution of t-
BuOK (33.7 mg, 0.3 mmol) and the corresponding dibenzothiazine
6 (0.1 mmol) in anhydrous and degassed DMSO (2 mL). The mixture
was stirred for 1 h at rt. and then the halide was added (0.2 mmol
of Me2SO4, ClÀ Bn or Cl–CH2-C6H4-p-CF3). After 2 h of reaction, the
crude was extracted with water (30 mL) and EtOAc (3×15 mL). The
organic layers were combined, washed with water, dried with
anhydrous Na2SO4, filtered and evaporated under reduced pressure.
The residue was purified by chromatographic column on silica gel
eluting with pentane/EtOAc. This methodology was used to
synthesized compounds 7a–d, 7f and 7h–i.
ArÀ H). Resonance of NÀ H was not observed is this solvent probably
13
°
due to rapid exchange. C NMR (101 MHz, CD3SOCD3, 25 C): δ=
143.0, 135.6, 133.4, 131.3, 131.2, 130.4, 130.3, 127.4, 126.6, 126.4,
125.4, 122.0 ppm. HRMS (TOF, ESI+) m/z calcd for C12H8N2O4S+Na+
: 299.0097 [M+Na]+; found: 299.0113.
Brominated compounds 6aa and 6ab, were prepared following a
methodology already reported.[55] The general procedure for is
exemplified next for the synthesis of 7,9-Dibromo-6H-dibenzo
[c,e][1,2]thiazine 5,5-dioxide (6aa).
A solution of 6H-dibenzo
[c,e][1,2]tiazina 5,5-dioxide (6b, 1 equiv, 23.1 mg, 0.1 mmol) in dry
MeCN (2 mL) was added NH4OAc (0.5 equiv, 3.8 mg, 0.05 mmol)
and NBS (5 equiv, 89.0 mg, 0.5 mmol). After the mixture was stirred
for 24 h at rt, the reaction mixture was extracted with EtOAc (3×
15 ml) and the organic layers were washed with water, dried with
anhydrous Na2SO4 and concentrated. The product was purified by
column chromatography on silica gel eluting with pentane/EtOAc
6-Methyl-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (7a). The prod-
uct was purified by column chromatography on silica gel eluting
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with pentane/EtOAc (100:0!70:30). White solid was obtained in
ArÀ H), 7.19 À 7.09 (m, 5H; ArÀ H), 5.19 ppm (s, 2H; CH2). 13C NMR
1
°
°
96% yield (23.5 mg, 0.096 mmol). H NMR (400 MHz, CDCl3, 25 C,
(101 MHz, CD3SOCD3, 25 C): δ= 145.2, 145.2, 136.6, 135.5, 134.5,
TMS): δ= 8.00 (d, J=7.6 Hz, 2H; ArÀ H), 7.95 (d, J=8.0 Hz, 1H; ArÀ H),
133.0, 130.3, 129.7, 128.4, 127.6, 127.2, 126.8, 126.2, 123.5, 123.1,
1
1
°
7.69 (t, J=7.6 Hz, 1H; ArÀ H), 7.56 (t, J=7.6 Hz, 1H; ArÀ H), 7.49 (t, J=
121.7, 118.8, 50.8 ppm. H/ H COSY NMR (CD3SOCD3, 25 C): δH/δH =
7.6 Hz, 1H; ArÀ H), 7.29–7.34 (m, 2H; ArÀ H), 3.44 ppm (s, 3H; CH3). 13
C
8.24-8.22/7.84, 8.24-8.22/7.54, 8.02/7.76, 7.84/7.76, 7.62/7.54, 7.19/
1
13
°
°
NMR (101 MHz, CDCl3, 25 C, TMS): δ= 139.6, 134.3, 132.4, 132.4,
130.4, 128.2, 125.5, 125.4, 124.7, 124.0, 122.5, 119.5, 32.8 ppm.
HRMS (TOF, ESI+) m/z calcd for C13H11NO2S+Na+: 268.0403 [M+
Na]+; found: 268.0425.
7.19, 5.19/5.19 ppm. H/ C HSQC NMR (CD3SOCD3, 25 C): δH/δC =
8.24-8.22/118.8, 8.24-8.22/126.8, 8.02/121.7, 7.84/133.0, 7.76/129.7,
7.62/123.5, 7.54/123.1, 7.19-7.09/127.2, 7.19-7.09/127.6, 7.19-7.09/
1
13
°
128.4, 5.19/50.8 ppm. H/ C HMBC NMR (CD3SOCD3, 25 C): δH/δC =
8.24-8.22/121.7, 8.24-8.22/123.1, 8.24-8.22/126.2, 8.24-8.22/130.3,
8.24-8.22/134.5, 8.24-8.22/136.6, 8.24-8.22/145.2, 8.02/130.3, 8.02/
133.0, 7.84/121.7, 7.84/130.3, 7.76/126.8, 7.76/134.5, 7.62/126.2,
7.62/145.2, 7.54/118.8, 7.62/136.6, 7.62/145.2, 7.19-7.09/127.2, 7.19-
8,10-Dimethoxy-6-methyl-6H-dibenzo[c,e][1,2]thiazine 5,5-diox-
ide (7b). Titled compound was purified by column chromatography
on silica gel eluting with pentane/EtOAc (100:10!70:30). White
solid was obtained in 65% yield (19.8 mg, 0.065 mmol). 1H NMR
7.09/128.4, 7.19-7.09/135.5, 5.19/127.2, 5.19/136.6 ppm. 19F NMR
°
(400 MHz, CDCl3, 25 C, TMS): δ= 8.45 (d, J=8.4 Hz, 1H; ArÀ H),
+
°
(376 MHz, CD3SOCD3, 25 C): δ=À 56.9 ppm. HRMS (TOF, ESI ) m/z
7.95–7.93 (m, 1H; ArÀ H), 7.61–7.57 (m, 1H; ArÀ H), 7.44 (t, J=7.6 Hz,
calcd for C20H14F3NO2S+K+ : 428.0329 [M+K]+; found: 428.0340.
1H; ArÀ H), 6.46 (d, J=2.0 Hz, 2H; ArÀ H), 3.94 (s, 3H; OCH3), 3.89 (s,
13
°
3H; OCH3), 3.36 ppm (s, 3H; CH3). C NMR (101 MHz, CDCl3, 25 C,
6-Benzyl-7-phenyl-2-(trifluoromethyl)-6H-dibenzo[c,e][1,2]
thiazine 5,5-dioxide (7h). Titled compound was purified by column
chromatography on silica gel eluting with pentane/EtOAc
TMS): δ= 161.3, 159.2, 142.0, 133.3, 131.4, 131.0, 128.9, 126.5, 122.0,
107.8, 97.1, 95.9, 55.9, 55.5, 33.3 ppm. HRMS (TOF, ESI+) m/z calcd
for C15H15NO4S+Na+: 328.0614 [M+Na]+; found: 328.0637.
(100:10!80:20). Yellow oil was obtained in 30% (14 mg,
1
°
0.03 mmol). H NMR (400 MHz, CDCl3, 25 C, TMS): δ= 7.93 (d, J=
6-Methyl-9-phenyl-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (7c).
Titled compound was purified by column chromatography on silica
gel eluting with pentane/EtOAc (100:0!90:10). White solid was
quantitively obtained (32.1 mg, 0.1 mmol). 1H NMR (400 MHz,
8.0 Hz, 1H; ArÀ H), 7.80–7.78 (m, 2H; ArÀ H), 7.72 (dd, J=7.2, 2.0 Hz,
1H; ArÀ H), 7.66–7.62 (2H, m; ArÀ H), 7.57–7.45 (m, 5H; ArÀ H), 6.88–
6.85 (m, 1H; ArÀ H), 6.71–6.67 (m, 2H; ArÀ H), 6.38–6.36 (m, 2H;
ArÀ H), 4.64 (d, J=14.4 Hz, 1H; CH2), 3.70 ppm (d, J=14.8 Hz, 1H;
°
CDCl3, 25 C, TMS): δ= 8.18 (d, J=1.6 Hz, 1H; ArÀ H), 8.03 (d, J=
13
°
CH2). C NMR (101 MHz, CDCl3, 25 C, TMS): δ= 139.6, 139.3, 138.0,
7.6 Hz, 2H; ArÀ H), 7.73–7.69 (m, 2H; ArÀ H), 7.63–7.56 (m, 3H; ArÀ H),
135.5, 134.1, 133.8 (q, J=33 Hz, 1 C), 132.8, 131.6, 129.3, 129.1,
128.9, 128.8, 128.3, 128.2, 127.8, 127.3, 124.7 (q, J=4 Hz, 1 C), 124.5,
124.1, 123.1 (q, J=279 Hz, 1 C), 123.0 (q, J=4 Hz, 1 C), 54.5 ppm.
HRMS (TOF, ESI+) m/z calcd for C26H18F3NO2S+Na+: 488.0903 [M+
Na]+; found: 488.0910.
7.50–7.46 (m, 2H; ArÀ H), 7.41–7.36 (m, 2H; ArÀ H), 3.48 ppm (s, 3H;
13
°
CH3). C NMR (101 MHz, CDCl3, 25 C, TMS): δ= 140.0, 138.6, 137.8,
134.3, 132.4, 129.2, 129.0, 128.4, 127.6, 127.1, 125.4, 124.2, 124.2,
32.7 ppm. HRMS (TOF, ESI+) m/z calcd for C19H15NO2S+Na+:
344.0716 [M+Na]+; found: 344.0734.
6-(4-(Trifluoromethyl)benzyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-di-
oxide (7i). Titled compound was purified by column chromatog-
raphy on silica gel eluting with pentane/EtOAc (100:0!80:20).
Light yellow oil was obtained in 30% yield (11.7 mg, 0.03 mmol). 1H
6-Methyl-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-di-
oxide (7d). Titled compound was purified by column chromatog-
raphy on silica gel eluting with pentane/EtOAc (100:0!85:15).
1
White solid was obtained in 92% yield (28.8 mg, 0.092 mmol). H
°
NMR (400 MHz, CD3COCD3, 25 C, TMS): δ= 8.18–8.16 (m, 1H; ArÀ H),
°
NMR (400 MHz, CDCl3, 25 C, TMS): δ= 8.27 (s, 1H; ArÀ H), 8.06–8.00
8.11 (d, J=8.0 Hz, 1H; ArÀ H), 7.99 (dd, J=7.8, 0.8 Hz, 1H; ArÀ H),
7.83–7.79 (m, 1H; ArÀ H), 7.70 (td, J=7.6, 0.8 Hz, 1H; ArÀ H), 7.56–
7.37 (m, 7H; ArÀ H), 5.28 ppm (s, 2H; CH2). 13C NMR (101 MHz,
(m, 2H; ArÀ H), 7.79–7.75 (m, 2H; ArÀ H), 7.64 (t, J=7.6 Hz, 1H; ArÀ H),
7.39 (d, J=8.8 Hz, 1H; ArÀ H), 3.53 ppm (s, 3H; CH3). 13C NMR
°
(101 MHz, CDCl3, 25 C, TMS): δ= 141.9, 134.4, 132.7, 131.2, 129.1,
°
CD3COCD3, 25 C, TMS): δ= 141.8, 139.3, 136.4, 133.6, 133.2, 131.3,
127.0 (q, J=4 Hz, 1 C), 126.5 (q, J=33 Hz, 1 C), 125.6, 124.3 (q, J=
130.1 (q, J=32 Hz, 1 C), 129.5, 129.0, 127.0, 126.6, 126.4, 126.1 (q,
270 Hz, 1 C), 123.8, 122.7 (q, J=4 Hz, 1 C), 122.5, 118.8, 118.9 ppm.
J=4 Hz, 2 C), 125.2 (q, J=270 Hz, 1 C), 122.9, 122.6, 52.1 ppm. 19F
19
°
F NMR (377 MHz, CDCl3, 25 C, TMS): δ=À 62.15. HRMS (TOF,
°
NMR (377 MHz, CD3COCD3, 25 C, TMS): δ=À 63.08 ppm. HRMS
ESI+) m/z calcd for C14H10F3NO2S+Na+: 336.0277 [M+Na]+; found:
336.0285.
(TOF, ESI+) m/z calcd for C20H14F3NO2S+Na+: 412.0590+ [M+Na]+;
found: 412.0600.
6-Benzyl-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (7f). Titled com-
pound was purified by column chromatography on silica gel eluting
with pentane/EtOAc (100:10!80:20). Yellow oil was obtained in
60% yield (19.3 mg, 0.06 mmol). 1H NMR (400 MHz, CD3COCD3,
Method B. In a Schlenk tube, equipped with a magnetic bar and a
nitrogen inlet a solution of NaH (4.8 mg, 0.2 mmol) and the
corresponding dibenzothiazine (0.1 mmol) in anhydrous THF (2 mL)
was prepared. The mixture was stirred for 1 h at rt. and then the
corresponding halide was added (0.2 mmol of ClÀ C(O)CH3 or
ClÀ S(O)2C6H4-p-Me). After 2 h of reaction, the crude was extracted
with water (30 mL) and EtOAc (3×15 mL). The organic layers were
combined, washed with an additional portion of water, dried with
anhydrous Na2SO4, filtered and evaporated under reduced pressure.
The residue was purified by chromatographic column on silica gel.
This methodology was used to synthesized compounds 7e and 7k.
°
25 C): δ= 8.13 (d, J=7.2 Hz, 1H; ArÀ H), 8.09 (d, J=8 Hz, 1H; ArÀ H),
7.99 (dd, J=8, 0.8 Hz, 1H; ArÀ H), 7.79 (td, J=8, 1.2 Hz, 1H; ArÀ H),
7.69 (td, J=7.6, 1.2 Hz, 1H; ArÀ H), 7.48–7.42 (m, 2H; ArÀ H), 7.37–
7.33 (m, 1H; ArÀ H), 7.18–7.16 (m, 5H; ArÀ H), 5.17 ppm (s, 2H; CH2).
13
°
C NMR (101 MHz, CD3COCD3, 25 C): δ= 138.6, 136.2, 135.7, 132.5,
132.4, 130.1, 128.5, 128.3, 127.5, 126.0, 125.6, 125.5, 125.2, 121.9,
121.8, 51.4 ppm. HRMS (TOF, ESI+) m/z calcd for C19H15NO2S+Na+:
344.0716 [M+Na]+; found: 344.0684.
1-(5,5-Dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)ethenone (7e). Ti-
tled compound was purified by column chromatography on silica
gel eluting with pentane/EtOAc (100:0!60:40). White solid was
obtained in 65% yield (17.8 mg, 0.065 mmol). 1H NMR (400 MHz,
6-Benzyl-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-diox-
ide (7g). Titled compound was purified by column chromatography
on silica gel eluting with pentane/EtOAc (95:5!90:10). White solid
was obtained in 87% yield (67.7 mg, 0.174 mmol). 1H NMR
°
CDCl3, 25 C, TMS): δ= 7.99 (d, J=8.0 Hz, 1H; ArÀ H), 7.92–7.90 (m,
°
(400 MHz, CD3SOCD3, 25 C): δ= 8.24–8.22 (m, 2H; ArÀ H), 8.02 (d,
2H; ArÀ H), 7.76–7.71 (m, 1H; ArÀ H), 7.58–7.48 (m, 4H; ArÀ H),
J=7.6 Hz, 1H; ArÀ H), 7.84 (t, J=7.4 Hz, 1H; ArÀ H), 7.76 (t, J=7.5 Hz,
1H; ArÀ H), 7.62 (d, J=9.0 Hz, 1H; ArÀ H), 7.54 (d, J=8.8 Hz, 1H;
2.64 ppm (s, 3H; CH3). 13C NMR (101 MHz, CDCl3, 25 C, TMS): δ=
°
169.3, 136.4, 134.5, 133.7, 133.0, 129.7, 128.8, 128.5, 128.5, 127.4,
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126.2, 125.7, 123.3, 25.9 ppm. HRMS (TOF, ESI+) m/z calcd for
C14H11NO3S+Na+: 296.0352 [M+Na]+; found: 296.0373.
ArÀ H), 7.77–7.75 (m, 1H; ArÀ H), 7.66–7.60 (m, 4H; ArÀ H), 7.55–7.42
(m, 4H; ArÀ H), 7.34–7.30 (m, 3H; ArÀ H), 6.88 (d, J=8.0 Hz, 2H; ArÀ H),
13
°
2.24 ppm (s, 3H; CH3). C NMR (101 MHz, CDCl3, 25 C, TMS): δ=
6-Tosyl-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (7k). Titled com-
pound was purified by column chromatography on silica gel eluting
with pentane/EtOAc (100:0!90:10). White solid was obtained in
145.1, 143.0, 139.5, 134.9, 133.5, 132.9, 132.9, 132.78, 131.7, 129.1,
129.0, 128.4, 128.3, 128.2, 127.3, 125.8, 124.5, 124.2, 21.4 ppm.
HRMS (TOF, ESI+) m/z calcd for C25H19NO4S2 +K+: 500.0387 [M+
K]+; found: 500.0399.
89% yield (34.3 mg, 0.09 mmol). 1H NMR (400 MHz, CDCl3, 25 C,
°
TMS): δ= 7.84–7.82 (m, 1H; ArÀ H), 7.77–7.75 (m, 1H; ArÀ H), 7.60–
7.49 (m, 5H; ArÀ H), 7.25–7.32 (m, 3H; ArÀ H), 6.86 (d, J=8.0 Hz, 2H;
Synthesis of 2H-benzo[e][1,2]thiazine 1,1-dioxide (8a-b). Benzo-
thiazines 10a-b were prepared following a previously described
method.[58] The procedure is described next exemplified with 3-
(tert-butyl)-2H-benzothiazine 1,1-dioxide (8a). Distilled liquid
ammonia (150 mL), previously dried over Na metal, was charged in
a three-necked (250 mL) round bottom flask equipped with a cold-
finger condenser charged with dry ice-ethanol, a magnetic stirrer
and a nitrogen inlet. Subsequently, t-BuOK (0.337 g, 3.0 mmol) and
3,3-dimethylbutan-2-one (0.250 mL, 2.0 mmol) were added to the
stirred ammonia. After 15 min, 2-chlorobenzenesulfonamide (4a,
0.095 g, 0.5 mmol) was added, and the reaction mixture was
irradiated for 3 h. After that, the reaction was quenched with an
excess of ammonium nitrate. The residue was extracted with EtOAc
(3×30 mL) and the organic extracted was washed with water and
dried with anhydrous Na2SO4. The solvent was removed under
reduced pressure. The product was purified by flash column
chromatography on silica gel eluting with petroleum ether/EtOAc
(90:10!60:40). White solid of 8a was obtained in 78% yield
ArÀ H), 2.23 ppm (s, 3H; CH3). 13C NMR (101 MHz, CDCl3, 25 C, TMS):
°
δ= 145.1, 134.8, 134.0, 133.4, 133.0, 132.7, 131.4, 130.2, 129.8,
128.9, 128.3, 128.2, 128.0, 125.8, 125.6, 124.5, 21.4 ppm. HRMS
(TOF, ESI+) m/z calcd for C19H15NO4S2+Na+: 408.0335 [M+Na]+;
found: 408.0374.
Method C. Employing previously described conditions,[56] in a round
bottom flask equipped with a magnetic stirred, the corresponding
dibenzothiazine (0.1 mmol) in anhydrous CH2Cl2 is treated with an
excess of NaOH (4.8 mg, 0.12 mmol) and the desired sulfonyl
chloride (0.15 mmol of ClÀ S(O)2C6H4-p-Me or ClÀ S(O)2Ph) at rt. The
resulting mixture was stirred overnight (18 h) at rt. After that
period, the suspension was filtered and the filtrated cake was
washed with copious amount of CH2Cl2 (100 mL). After evaporation
the reaction crude was analyzed with TLC, GC and isolated with
column chromatography over silica gel. This methodology was
employed to synthesized compounds 7j and 7l-n.
(92.5 mg, 0.39 mmol). This solid was recrystallized from petroleum
6-(Phenylsulfonyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (7j).
Titled compound was purified by column chromatography on silica
gel eluting with pentane/EtOAc (100:10!80:20). White solid was
obtained in 80% yield (29.7 mg, 0.08 mmol). 1H NMR (400 MHz,
1
°
ether/ EtOAc as white solid. H NMR (400 MHz, CD3SOCD3, 25 C):
δ= 10.38 ppm (s, 1H; NH), 7.80–7.72 (m, 1H; ArÀ H), 7.67–7.59 (m,
1H; ArÀ H), 7.53 (d, J=7.3 Hz, 1H; ArÀ H), 7.47 (td, J=7.6, 1.3 Hz, 1H;
ArÀ H), 6.25 (s, 1H; CH=C), 1.23 ppm (s, 9H; CH3). 13C NMR (101 MHz,
°
CDCl3, 25 C, TMS): δ= 7.86–7.76 (m, 2H; ArÀ H), 7.60–7.48 (m, 5H;
ArÀ H), 7.41–7.26 (m, 4H; ArÀ H), 7.11–7.07 ppm (m, 2H; ArÀ H). 13C
°
CD3SOCD3, 25 C): δ= 149.8, 134.0, 132.3, 131.2, 127.6, 127.2, 120.7,
101.8, 35.8, 28.3 ppm. MS (EI, 70 eV) m/z (%): 238 (M+1, 15), 237
(M+, 100), 223 (12.5), 158 (39), 143 (42), 137 (15), 130 (14), 117 (21),
115 (15).[58]
°
NMR (101 MHz, CDCl3, 25 C, TMS): δ= 136.3, 134.5, 133.9, 133.8,
133.3, 132.5, 131.3, 130.3, 129.9, 128.5, 128.4, 128.2, 127.9, 125.8,
125.6, 124.3 ppm. HRMS (TOF, ESI+) m/z calcd for C18H13NO4S2 +
Na+: 394.0178 [M+Na]+; found: 394.0198.
3-(Adamantan-1-yl)-2H-benzo[e][1,2]thiazine 1,1-dioxide (8b). Ti-
tled compound was purified by column chromatography on silica
gel eluting with petroleum ether/EtOAc (90:10!0:100). Light
yellow was obtained in 47% yield (74.1 mg, 0.235 mmol). This solid
was recrystallized from CH2Cl2 as white solid. 1H NMR (400 MHz,
6-(Phenylsulfonyl)-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]
thiazine 5,5-dioxide (7l). Titled compound was purified by column
chromatography on silica gel eluting with pentane/EtOAc
(100:10!80:20). White solid was obtained in 65% yield (28.5 mg,
1
°
CD3SOCD3, 25 C): δ= 10.32 (s, 1H; NH), 7.74 (d, J=8.0 Hz, 1H;
°
0.065 mmol). H NMR (400 MHz, CDCl3, 25 C, TMS): δ= 7.83 (d, J=
ArÀ H), 7.61 (td, J=8.0, 1.2 Hz, 1H; ArÀ H), 7.51 (d, J =7.2 Hz, 1H;
8.8 Hz, 1H; ArÀ H), 7.55–7.47 (m, 4H; ArÀ H), 7.37–7.26 (m, 5H; ArÀ H),
7.08–7.04 ppm (m, 2H; ArÀ H). 13C NMR (101 MHz, CDCl3, 25 C, TMS):
ArÀ H), 7.45 (td, J=8.0, 1.2 Hz, 1H; ArÀ H), 6.17 (s, 1H; CH=C), 2.02–
°
2.05 (m, 3H; CH), 1.85 (d, J=2.8 Hz, 6H; CH2), 1.70 ppm (t, J=
δ= 149.8, 136.2, 134.6, 134.1, 133.5, 133.1, 132.0, 131.3, 129.62,
129.4, 128.5, 128.3, 125.9, 124.4, 122.2, 120.2 (q, J=257 Hz, 1 C),
13
°
12.2 Hz, 6H; CH2). C NMR (101 MHz, CD3SOCD3, 25 C): δ= 149.6,
117.7 ppm. 19F NMR (377 MHz, CDCl3, 25 C): δ=À 57.7 ppm. HRMS
133.6, 131.8, 130.9, 127.2, 126.6, 120.2, 101.4, 39.0, 36.8, 35.9,
27.9 ppm.
°
(TOF, ESI+) m/z calcd for C19H12F3NO4S2 +K+: 477.9791 [M+K]+;
found: 477.9815.
6-Tosyl-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-diox-
ide (7m). Titled compound was purified by column chromatog-
raphy on silica gel eluting with pentane/EtOAc (100:10!80:20).
White solid was obtained in 50% yield (22.7 mg, 0.05 mmol). 1H
Antiproliferative tests
The antiproliferative activity of the reported compounds was
studied using our implementation of the NCI protocol[82] against
human solid tumor cell lines (A549, HBL-100, HeLa, SW1573, T-47D,
WiDr). Cell line suspensions were counted with Moxi Z and diluted
to reach the appropriate cell densities (A549, HBL-100, HeLa and
SW1573 2500 cells/well; T-47D and WiDr 5000 cells/well) for
inoculation onto 96-well plates. After 24 h, pure compounds were
added. The results are expressed as GI50 and were determined for
an exposure time of 48 h. In this cytotoxicity experiments the
maximal DMSO concentration is 0.25% v/v (see ref [59]), as
recommended by the NCI in its NCI60 protocol.
°
NMR (400 MHz, CDCl3, 25 C, TMS): δ= 7.81 (d, J=8.8 Hz, 1H; ArÀ H),
7.56–7.45 (m, 4H; ArÀ H), 7.35–7.19 (m, 4H; ArÀ H), 6.83 (d, J=8.0 Hz,
2H, ArÀ H), 2.18 ppm (s, 3H; CH3). 13C NMR (101 MHz, CDCl3, 25 C,
°
TMS): δ= 145.5, 134.8, 133.3, 133.2, 132.2, 131.5, 129.7, 129.1, 128.3,
125.9, 124.6, 122.1, 120.3 (q, J=257 Hz, 1 C), 116.5, 21.4 ppm. 19F
°
NMR (377 MHz, CDCl3, 25 C, TMS): δ=À 57.7 ppm. HRMS (TOF,
ESI+) m/z calcd for C20H14F3NO4S2 +K+: 491.9948 [M+K]+; found:
491.9977.
9-Phenyl-6-tosyl-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (7n). Ti-
tled compound was purified by column chromatography on silica
gel eluting with pentane/EtOAc (100:10!80:20). White solid was
obtained in 50% yield (23 mg, 0.05 mmol). 1H NMR (400 MHz,
°
CDCl3, 25 C, TMS): δ= 7.94 (s, 1H; ArÀ H), 7.90 (d, J=8.0 Hz, 1H;
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Cell cycle analysis
fellowship from CONICET. J. M. P. thanks the Spanish Government
for financial support through project PGC2018-094503-B-C22
(MCIU/AEI/FEDER, UE). The biochemical work was supported by
grants Ministerio de Ciencia e Innovación PID2019-10454RB-I00/
AEI/10.13039/501100011033, PIEs 201920E111 and 202020E301
from Consejo Superior de Investigaciones Científicas, grant “Bases
moleculares de la regulación de microtúbulos y sus implicaciones
en la neurotoxicidad producida por fármacos” from Fundación
TATIANA and European Union H2020-MSCA-ITN-2019 860070
TUBINTRAIN grant to JFDP and a generous donation from Club
Deportivo Escuela Hungaresa de Pontevedra.
Progression through the cell cycle was assessed by flow cytometry
DNA determination with propidium iodide. To do so, 1.5×105 cells
per well of the A549 cell line (non-small cell lung carcinoma) were
seeded in
a 24-well plate and cultured in DMEM medium
supplemented with 10% fetal calf serum, 2 mM L-glutamine,
40 mg/mL gentamycin, 100 IU/mL penicillin, and 100 mg/mL
streptomycin. Cells were left to attach to the substrate for 24 h at
°
37 C and in a 5% CO2 air atmosphere. Then, drugs were added to
the cells and incubated for another 24 h. Finally, cells were washed
°
in PBS and fixed with 70% ethanol at 4 C for 1 h, washed twice in
phosphate-buffered saline (PBS), and resuspended in 500 μL of PBS
containing 60 μg/mL DNase-free RNase A and 50 μg/mL propidium
°
iodide. Samples were incubated at 37 C for 30 min and analyzed
with a Coulter Epics XL flow cytometer.
Conflict of Interest
Biochemistry
The authors declare no competing financial interest.
Calf brain tubulin was purified as described in Ref. [83]. MTC[72] was
a kind gift from Prof. Wei-Shuo Fang (Institute of Materia Medica,
Beijing). Eribulin-A488[73] was a gift of Michel Steinmetz (PSI,
Vilingen) synthesized by Eisai chemist Daniel Custar.
FcMaytansine[74] was a kind gift from Prof. Karl-Heinz Altmann (ETH,
Zürich). The compounds were diluted in 99.8% CD3SOCD3 (Merck,
Darmstadt, Germany) to a final concentration of 10 mM and stored
Keywords: Antiproliferation · dibenzothiazines · drug design ·
sulfonamides · tubulin inhibitors
°
at À 80 C. For the biochemical experiments, the maximal DMSO
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Acknowledgements
This study was supported by Agencia Córdoba Ciencia, Consejo
Nacional de Investigaciones Científicas y Técnicas (CONICET),
Secretaría de Ciencia y Tecnología, Universidad Nacional de
Córdoba (SECyT), and Agencia Nacional de Promoción Científica y
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© 2021 Wiley-VCH GmbH
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ChemMedChem
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Revised manuscript received: June 27, 2021
Accepted manuscript online: July 6, 2021
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FULL PAPERS
Building blocks for disassembly: We
synthesized a series of NH-unsubsti-
tuted and N-substituted dibenzothia-
zines, and we evaluated the antiproli-
ferative activity against six human
solid tumor cell lines. The more
active compounds exhibit activity in
the submicromolar range and
involve targeting tubulin assembly.
We highlight the dibenzothiazine
core as a novel organic building
block to prepare potential antitumor
agents with tubulin as a cellular
target.
Dr. W. D. Guerra, Dr. D. Lucena-Agell,
R. Hortigüela, Prof. R. A. Rossi, Dr. J.
Fernando Díaz, Prof. J. M. Padrón*,
Dr. S. M. Barolo*
1 – 15
Design, Synthesis, and in vitro Eval-
uation of Tubulin-Targeting Diben-
zothiazines with Antiproliferative
Activity as a Novel Heterocycle
Building Block