Azidothymidine "clicked" into 1,2,3-Triazoles: First Report on Carbonic Anhydrase-Telomerase Dual-Hybrid Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c00636

Cancer cells rely on the enzyme telomerase (EC 2.7.7.49) to promote cellular immortality. Telomerase inhibitors (i.e., azidothymidine) can represent promising antitumor agents, although showing high toxicity when administered alone. Better outcomes were o

Full text of DOI:10.1021/acs.jmedchem.0c00636

Subscriber access provided by University of Texas Libraries
Article
Azidothymidine (AZT) “clicked” into 1,2,3-Triazoles: First Report
on Carbonic Anhydrase-Telomerase Dual Hybrid Inhibitors
Emanuela Berrino, Andrea Angeli, Dmitry D. Zhdanov, Anna Pavlovna Kiryukhina, Andrea
Milaneschi, Alessandro De Luca, Murat Bozdag, Simone Carradori, Silvia Selleri, Gianluca
Bartolucci, Thomas S. Peat, Marta Ferraroni, Claudiu T. Supuran, and Fabrizio Carta
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00636 • Publication Date (Web): 28 May 2020
Downloaded from pubs.acs.org on May 29, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Azidothymidine (AZT) “clicked” into 1,2,3-Triazoles: First Report on
Carbonic Anhydrase-Telomerase Dual Hybrid Inhibitors
1
1
2, 3
2
Emanuela Berrino , Andrea Angeli , Dmitry D. Zhdanov , Anna P. Kiryukhina , Andrea
1
1
1
4
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Milaneschi , Alessandro De Luca , Murat Bozdag , Simone Carradori , Silvia Selleri , Gianluca
1
5
6
1
1
Bartolucci , Thomas S. Peat , Marta Ferraroni , Claudiu T. Supuran * and Fabrizio Carta *
1
Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo
Schiff 6, 50019, Sesto Fiorentino (Florence), Italy
2
3
4
5
6
Institute of Biomedical Chemistry, Pogodinskaya st. 10/8, 119121, Moscow, Russia
Peoples Friendship University of Russia (RUDN University), Miklukho-Maklaya st. 6, 117198, Moscow, Russia.
Department of Pharmacy "G. d'Annunzio" University of Chieti-Pescara Via dei Vestini 31, 66100, Chieti. Italy.
CSIRO, 343 Royal Parade, Parkville, Victoria, 3052, Australia.
Università di Firenze, Dipartimento di Chimica “Ugo Schiff”, Via della Lastruccia 3-13, 50019, Sesto Fiorentino
Florence), Italy.
(
*
corresponding authors
Abstract. Cancer cells rely on the enzyme Telomerase (EC 2.7.7.49) to promote cellular immortality.
Telomerase inhibitors (i.e. azidothymidine-AZT) can represent promising antitumor agents, although
showing high toxicity when administered alone. Better outcomes were observed within a multi
pharmacological approach instead. In this context we exploited the validated antitumor targets
Carbonic Anhydrases (CAs; EC 4.2.1.1) IX and XII to attain the first proof of concept on CA-
Telomerase dual hybrid inhibitors. Compounds 1b, 7b, 8b and 11b showed good in vitro inhibition
potency against the CAs IX and XII, with K values in the low nanomolar range, and strong
I
antitelomerase activity in PC-3 and HT-29 cells (IC values ranging from 5.2 to 9.1 M). High
5
0
resolution X-ray crystallography on selected derivatives in adduct with hCA II as model study
allowed to determine their binding modes and thus to set the structural determinants necessary for
further development of compounds selectively targeting the tumoral cells.
Keywords: Carbonic Anhydrase Inhibitors; AZT; Telomerase Inhibitors; Cancer; Molecular hybrids;
Click Chemistry; Triazole.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
INTRODUCTION
Eukaryotic cells do possess limited replicative potential as progressive shortage of the
1
chromosome ends (i.e. the telomeres) takes place after every duplication cycle. Once the critical
2
physical limits are reached, cellular senescence programs, i.e. apoptosis, are triggered. Such an effect
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
is properly referred as the “Hayflick limit”, who firstly reported experimentally the finite capacity of
normal cells to replicate.1,3
State of-the-art knowledge on telomeres accounts for rather complicated and highly dynamic
4
structures which are evolutionarily conserved among the eukaryotic cells. Human telomeres are
composed by repetitive, non-coding hexameric nucleotide repeats in complex with the telomere
associated proteins (i.e. the shelterin proteins) and the telomerase.5-8 The former are mainly
responsible in maintaining the telomere structure and its signalling functions, whereas the latter in
synthetizing new telomeric DNA strands from its own RNA template.4,5 This enzyme is normally
highly active in adult germline and stem cells, whereas it is poorly or not expressed at all in the
somatic ones.9,10 Besides the canonical function of telomere elongation, the telomerase enzymes (EC
2
.7.7.49) were also found to act as transcriptional regulators of the Wnt/-catenin signalling pathway,
thus suggesting a role in determining cell growth, differentiation and apoptosis via a non-telomeric
dependent manner too.11-13
The majority of malignant tumors in humans were demonstrated to depend from the
telomerase activity, which resulted increased when compared to the non-tumorigenic counterpart
1
4
cells. As a matter of fact the catalytic subunit of the telomerase enzyme (i.e. hTERT) was found
over-expressed in several tumors 15-18 and its regulatory role in metastatic events was also proved.19
20
In light of such data the telomerase is properly considered a tumor marker, and still it is taken into
consideration as rational target for developing potent and effective anticancer drugs.15,20-22
By making use of the DNA polymerase activity of the telomerase, nucleoside and nucleotide
23
analogues have been extensively investigated as potential inhibitors. In particular chain-terminator
2
3
reverse transcriptase inhibitors have been explored as antitumor agents. The first study of this type
2
ACS Paragon Plus Environment

Page 3 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
was conducted by Blackburn in 1994 on the ciliated protozoan Tetrahymena thermophila which is
2
4
quite rich in telomeres. Such studies revealed that azidothymidine (AZT) was able to decrease the
2
4
de novo telomere addition, thus resulting in telomeres shortening. Further studies showed that in
spite of the low affinity of AZT for mammalian DNA polymerases, its triphosphate derivative (AZT-
TP) was incorporated into telomeric region of eukaryotic genome through a process mediated by the
telomerases.25,26 The efficiency of AZT in affecting tumor growth was properly assessed 27-29 and its
association with cisplatin, paclitaxel or 5-fluorouracil showed synergistic interactions. 30,31 Although
such promising results were obtained, AZT was dropped as antitumor drug for its potential
tumorigenic properties and to the tardiness of the drug to be fully functional, which may expose
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
patients to dangerous side effects. Various drawbacks are associated with the use of telomerase
33
inhibitors for cancer therapy. The tardiness to take action is the most critical issue, as cellular
senescence is induced only when telomeres have reached their critical length, and thus implying that
such agents do require appropriate time to become effective.32,33 Induction of cellular senescence by
telomeric dysfunction may also result in activation of oncogenes and/or silencing of tumor suppressor
3
4
genes, thus promoting malignant transformations to occur instead. In addition, the use of inhibitors
of the telomerases may interfere with highly proliferative cells such germlines and stem cells.10,22 For
all these reasons the use of telomerase inhibitors (i.e. AZT, Imetelstat, BIBR1532 and antisense
molecules) for the management of cancer is better envisaged within a polypharmacologically based
approach and the metalloenzyme Carbonic Anhydrase (CA; EC 4.2.1.1) IX is well suited.35-37 CA IX
(and marginally CA XII) is selectively over-expressed in hypoxic solid tumors, it actively participates
in a complex pH regulation machinery tuned to warrant cancer cells survival within a metabolic
driven pH dysregulated environment.37-40 The paramount importance of CA IX in regulating proton
dynamics by means of the equation 1 was conclusively demonstrated and that allowed to validate
such an enzyme as druggable target for the management of hypoxic tumors.38,39
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
-
+
CO + H O
H CO
HCO + H
3
2
2
2
3
Equation 1
A recent contribution on the active involvement of CA IX in tumor physiology demonstrated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
such an enzyme to provide the H ions needed by the matrix metalloproteinase 14 (MMP14) to
4
1
perform proteolytic cleavage of collagen which in turn determines tumor invasiveness. In this
context, during the last years great interests have been turned to the CA IX “interactome”.42-45
A
significant study conducted on HEK-293 cells showed that the ARM and/or HEAT-repeat domains
4
5
are a feature of CA IX interacting partners. The majority of such proteins belong to the nuclear-
cytoplasmatic trafficking machinery, such as XPO1 exportin and TNPO 1 importin, and were found
4
5
to interact with the CA IX C terminal region. These results strongly suggested that CA IX may take
a role as cell-surface signal transducer by undergoing nuclear translocation. This is in agreement with
confocal immunofluorescence spectroscopy experiments which showed nuclear distribution of CA
IX in several cell lines, with a marked localization when experimental hypoxic conditions were
established.45
In consideration of the robust antitumor effects observed when the Telomerase and the CA IX
were targeted, the research herein reported is aimed to obtain CA-Telomerase dual small molecule
inhibitors (CAI-TI) able to: i) efficiently bind to the CA IX (XII) enzymes which are assumed as
discriminant feature between tumor and normal cells; ii) exert their antitumoral activity by inhibition
of both the CA IX (or XII) and Telomerase. As consequence appropriate CAI-TI molecules will have
the potential to achieve therapeutic performances far superior to the ones reached when co-
administration of single therapeutic agents are considered. To the best of our knowledge this is the
first report on CA-TI dual hybrid compounds designed to target two crucial players in cancer
progression.
4
ACS Paragon Plus Environment

Page 5 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
RESULT AND DISCUSSION
Design and Synthesis of Compounds.
The hybridization strategy was performed by exploiting the versatile “click chemistry”
approach, which allows to merge efficiently single chemical entities and thus granting easy access to
wide molecular diversities.46,47 In this study we performed a Copper-catalyzed Azide–Alkyne
Cycloaddition (CuAAC) between the azide of the reverse transcriptase inhibitor AZT with the
terminal alkyne pendant installed on various CAIs scaffolds (Figure 1). Our interest in establishing
such a chemical connection was mainly based on: i) the rapid and regioselective formation of the 1,4-
disubstituted-1,2,3-triazole ring under mild reaction conditions;47,48 ii) the 1,2,3-triazole is among the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
most commonly used scaffolds in Medicinal Chemistry in the last decade, since it is a bioisostere of
the amide group and it shows good tolerance to metabolic processes as well as to pH fluctuations.49,50
In addition, the abundancy of electrons within the triazole ring allows it to establish H-bonds and π-
π stacking interactions with biological targets and thus ensuring additional stabilization of the adducts
formed.49,50
Figure 1. Schematic representation of the synthetized hybrids consisting of a CAI portion linked to
AZT through the 1,2,3-triazole ring.
The synthesis and characterization data of the appropriate alkyne precursors 1a-3a, 5a-10a,
1
4a-20a and 4c, reported in Table 1, are descripted within the Experimental Section. Both classical
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
i.e. sulfonamides) and non-classical (i.e. coumarins and sulfocoumarins) CAIs have been included
in our study. In particular, sulfonamide-based compounds 6a, 9a, 10a and coumarin-based
compounds 14a, 18a and 19a are new.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SO NH2
SO NH2
SO2NH2
SO NH2
2
2
2
OH
S
2
NH2
NH2
1
2
A
5
SO NH2
SO2NH2
2
SO NH2
2
Cl
O
O
O
N
H
OH
O
OH
9: 3-SO NH2
2
6
7
10: 4-SO NH2
2
OH
O
HO
HO
S O
O
O
O
O
HO
O
O
O
14
15
17
20
Figure 2. Substrates for the synthesis of alkynes 1b-3b, 5b-10b, 14b-20b, 4d and 13e.
Table 1. Reagents and conditions for the synthesis of compounds 1a-3a, 5a-10a, 14a-20a, 4c, 13d.
Substratea X-Alkynyl
Product (%)b
Substratea X-Alkynyl
Product (%)b
SO NH2
2
SO NH
2
2
O
O
1
X=Br
X=Cl
10
14
X=NH2
X=Cl
N
N
H
H
10a (60)
1
a (47)
a (83)
O
SO NH2
2
2
S
O
O
2
1
4a (80)
SO NH
2
2
O
N
H
A
X=Br
15
X=Br
O
O
3
a (33)
15a (65)
6
ACS Paragon Plus Environment

Page 7 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
SO NH
O
2
2
N
O
O
A
X=Br
X=Br
15
17
X=Cl
X=Br
4
c (10)
16a (79)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SO NH
2
2
O
O
O
N
5
1
7a (73)
5
6
a (70)
Cl
O
O
O
O
6
X=OH
SO2NH2
17
X=Cl
O
a (40)
18a (71)
SO NH
2
2
O
O
O
O
7
7
9
X=OH
X=NH2
X=NH2
17
20
X=Cl
X=Br
O
7a (38)
1
9a (80)
SO NH
O
2
2
H
N
S O
O
O
O
8
a (82)
2
0a (85)
SO NH2
2
O
N
N
H
H
9
a (65)
a
b
Reported in Figure 2; Yields refer to isolated products.
Compounds 13d, here reported for the first time, was obtained through a multistep synthetic
approach, reported in Scheme 1.
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
O
SO NH
O
2
2
O
S
H
N
N
N
H
N
SO NH2
Cl
THF
Cl
2
O
O
O
O
1
3b (41%)
NH2
O
DMF
O
OH
Br
1
3a
13
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(83%)
K CO DMF
2
4,
O
O
1
.5M HCl
in MeOH
N
N
NH2
S
S
N
O
N
O
O
O
O
O
1
3c
13d
(90%)
(55%)
Scheme 1. Synthesis of compounds 1b-3b, 5b-10b, 14b-20b, 4d and 13e. Yields are reported in
brackets.
The final compounds 1b-3b, 5b-12b, 14b-20b, 4d and 13e, reported in Figure 3, were
obtained by performing the CuAAC by using Cu (0) nanosized, tetramethyl ammonium chloride
(
TMACl) as a phase transfer agent in tBuOH/H O 1/1 as solvent at 40 °C (Scheme 2).
2
O
O
NH
Cu(0) nanosized (0.1 eq)
TMACl (1 eq)
NH
N
O
HO
N
O
HO
N
O
N
O
H O / tBuOH (1:1)
2
1a-3a
40°C
1b-3b
5a-10a
14a-20a
4c, 13d
N
5b-10b
14b-20b
4d, 13e
N
N
N
Scheme 2. Synthesis of compounds 1b-3b, 5b-10b, 14b-20b, 4d and 13e. Yields are reported in
brackets.
8
ACS Paragon Plus Environment

Page 9 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
2
O
O
O
O
O
O
O
O
O
S
S
S
S
S
H N
H2N
H2N
H2N
H2N
N
O
S
N
H
N
1b (84%)
2b (37%)
3b (21%)
4d (32%)
5b (82%)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
S
NH2
O
O
S
O
O
O
O
Cl
S
O
S
H2N
H2N
NH2
O
O
O
O
HN
HN
O
HN
6
b (21%)
7b (30%)
8b (20%)
9b (70%)
O
N
NH
O
O
O
O
O
2
O
O
O
HO
S
S
S
O
N
H2N
H2N
O
S
H N
O
NH2
S
N
S
HN
N
N
N
H
HN
O
1
b-3b
b-12b
4b-20b
d, 13e
H
HN
Se
O
HN
Se
5
1
4
N
10b (47%)
11b (15%)
12b (60%)
13e (53%)
O
O
O
O
O
O
O
O
O
O
O
O
1
4b (45%)
15b (91%)
16b (27%)
17b (91%)
O
O
S
O
O
O
O
O
O
O
O
1
8b (21%)
19b (21%)
20b (78%)
Figure 3. Chemical structures of compounds 1b-3b, 5b-12b, 14b-20b, 4d and 13e. Yields are
reported in brackets and are referred to the final coupling reaction.
S
Cu(0) nanosized
0.1 eq)
TMACl (1 eq)
NH2
NH2
Se
NH2
Se
C
(
N
Br
CH CN
NaBH4
3
11b: 3
2b: 4-SO NH (60%)
-SO NH (15%)
2
2
1
EtOH
H O / tBuOH (1:1)
2
2
2
over night
40°C
SO NH
2 2
SeCN
D
E
11a: 3-SO NH
12a: 4-SO NH
C
2
2
(79%)
(20%)
2
2
Scheme 3. Synthesis of compounds 11b and 12b. Yields are reported in brackets.
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The synthesis of compounds 11b and 12b are reported separately (Scheme 3), as for these
compound CuAAC was not performed as last reaction step. The synthesis started with the preparation
of compound D, bearing the terminal alkyne pendant, obtained reducing 4-selenocyanatoaniline C
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
with NaBH and treating it in situ with propargyl bromide. The CuAAC reaction between the azide
4
of AZT and the terminal alkyne of D was then performed, to afford the common intermediate E,
which was subsequently reacted with 3-isothiocyanatobenzenesulfonamide or 4-
isothiocyanatobenzenesulfonamide, to afford compound 11b and 12b, respectively. All final
compounds were obtained in good yields and with high purity grade (i.e. ≥ 95 %) as determined by
HPLC. Structural characterization of both intermediates and final compounds was assessed by means
1
13
of H NMR and C NMR as well as high resolution mass spectra (HRMS).
To the best of our knowledge the AZT-coumarin derivative 17b was previously reported in
the literature as part of a set of compounds intended to be used for their fluorescent properties. No
5
1
biological applications were reported in such a study. In addition ester-triazole-linked triterpenoid–
5
2
AZT conjugates were also reported. Cytotoxic analysis of these hybrids and their triterpenoid
precursors revealed moderate to good cytotoxic activities against two human tumor cell lines (KB,
5
2
Hep-G2). However, no detailed studies on the specific targets responsible for the anticancer effects
were conducted.
Carbonic Anhydrase Inhibition Profiling.
The library of compounds obtained 1b-3b, 5b-12b, 14b-20b, 4d and 13e was evaluated for
the inhibition properties against the human expressed (h) CAs I, II, VA, VB, VII, IX and XII isoforms
5
3
by means of the stopped flow technique applied to the CO hydrase assay. The inhibition data,
2
compared to those of the standard sulfonamide inhibitor acetazolamide (AAZ) are reported in Table
2
.
1
0
ACS Paragon Plus Environment

Page 11 of 58
Journal of Medicinal Chemistry
1
2
3
Table 2. Inhibition data of hCA I, hCA II, hCA VA, hCA VB, hCA VII, hCAIX and hCA XII with
compounds 1b-3b, 5b-12b, 14b-20b, 4d and 13e and the standard sulfonamide inhibitor
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
acetazolamide (AAZ) by a Stopped flow CO hydrase assay.53
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
K (nM)*
I
hCA I hCA II hCAVA hCAVB hCAVII
hCA IX
6.2
hCA XII
78.9
61.6
74.4
74.0
74.0
40.4
7.0
1b
2b
4666.7
4037.5
>10000
>10000
>10000
85.5
9.3
7.7
59.1
57.3
141.3
52.6
52.6
45.9
42.9
22.6
54.2
29.0
47.8
37.9
45.0
46.7
28.0
44.0
161.0
40.1
151.5
42.7
43.9
54.6
54.0
51.6
31.0
329.8
383.5
281.1
688.5
48.5
9.5
653.3
488.6
6557.1
8047.1
240.1
3.7
3
4
5
6
7
8
9
b
d
b
b
b
b
b
32.9
8.5
64.6
57.3
70.7
7.7
59.4
3217.2
4795.3
1469.3
3243.0
437.8
3972.2
6258.9
3651.5
1725.8
57.8
28.0
1.3
483.0
289.7
93.1
13.4
6.3
85.5
7.8
9.4
>10000
267.6
373.2
>10000
>10000
>10000
6557.1
21.2
8.4
1
0b
8.2
9.4
38.9
9.0
11b
12b
13e
92.8
73.2
5.6
66.0
21.8
38.0
0.7
62.3
7.1
8771.0
21.3
8.1
1
1
1
1
1
1
2
4b
5b
6b
7b
8b
9b
0b
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
8.7
9.3
3.6
666.8
179.4
301.8
531.2
172.4
63.0
0.7
9.4
9.4
4885.7
2948.3
>10000
5852.3
25.8
3.5
0.6
40.4
8.9
0.6
10.5
2.5
2.8
AAZ
250.0
12.1
5.7
*
Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 % of
the reported values).
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
As reported above in Table 2 the compound series was investigated on the most relevant hCA
isoforms such as the ubiquitous hCAs I and II, the mitochondrial expressed hCAs VA and VB, the
abundantly central nervous system (CNS) expressed hCA VII and tumor associated hCA IX and XII.
The structure-activity relationships (SARs) for the titled compounds are below discussed:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
i)
Overall the compound series screened in vitro against the ubiquitous hCAs I and II showed
preferential inhibition in the low nanomolar range for the latter. In both cases the coumarin and
sulfocoumarin based derivatives (i.e. 14b-20b) resulted ineffective (i.e. K s > 10000 nM) as in
I
agreement with previously reported data.54,55 The isosteric ethers 1b and 2b resulted low micromolar
inhibitors of the hCA I with the latter being just 1.2-fold more potent inhibitor (K s of 4666.7 and
I
4037.5 nM respectively). Interestingly the same kinetic profile for both compounds was retained for
the hCA II isoform too, although the kinetic data were in the low nanomolar range (K s of 9.3 and 7.7
I
nM, respectively). Further manipulations on the scaffold of the type reported in compounds 3b, 4d
and 5b resulted detrimental for the hCA I (K s > 10000 nM). As for the isoform II the introduction of
I
a N atom as in 3b and 5b determined enhancement of the K values (32.9 and 70.7 nM, respectively)
I
which were re-aligned to the previous ones when the N,N-bis-substituted aniline moiety was
introduced instead (K of 4d 8.5 nM). Compound 7b was the most potent inhibitor among the series
I
against both the hCA I and II (K s of 28.0 and 1.3 nM, respectively). Variations of the sulfonamide
I
position (i.e. 6b) or of the linker connection (i.e. 8b) badly affected the potencies (see Table 2).
Noteworthy the switch of the sulfonamide moiety from 3- to 4-position as in 9b to 10b and 11b to
12b resulted in decrease of the inhibition values for the hCA I. As for the hCA II a similar profile
was observed only for 11b and 12b, whereas the opposite was obtained for the regioisomers 9b and
1
0b (i.e K s of 6.3 and 8.2 nM, respectively). Finally, compound 13e showed excellent discrimination
I
between the isoforms tested, being 411.8-fold more potent against the hCA II over the I.
ii) Despite the high degree of similarity between the mitochondrial expressed hCAs VA and VB,
the kinetic profile of the majority of the tested compounds 1b-20b accounted for the preferential
inhibition of the latter. The ether derivative 1b was the only sulfonamide bearing compound among
1
2
ACS Paragon Plus Environment

Page 13 of 58
Journal of Medicinal Chemistry
1
2
3
the series which showed selective inhibition of the hCA VA over the VB up to 2.4-fold. The
substitution of the ethereal oxygen in 1b with a sulphur or a nitrogen instead, as in compounds 2b
and 3b respectively, suppressed any isoform selectivity, that was maintained when N,N-disubstitution
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(i.e. 4d) or elongation (i.e. 5b) was applied (see Table 2). As for the remaining sulfonamide
derivatives 6b-12b and 13e, their K values against the hCA VA were all in the micromolar range
I
with compound 10b being the most potent among them (K of 437.8 nM). The same compounds were
I
more effective in inhibiting the second mitochondrial expressed hCA as they showed medium
nanomolar K values. The derivatives 6b, 13e and 8b were the most effective against the hCA VB
I
and their K values resulted up to 2.4-fold lower when compared to the reference AAZ (see Table 2).
I
Interesting kinetic data were observed for the coumarin containing CAIs. The 4-alkyl substituted
derivative 14b resulted quite effective in inhibiting the hCA VB with a Selectivity Index (SI; K hCA
I
VA/ hCA VB) of 39.2. Relocation of the chain to 7-position of the coumarin ring as in 19b didn’t
change the kinetic profile but heavily reduced the SI for the preferential inhibition of the VB isoform
(see Table 2). Regioisomeric effects on kinetics were also evident for compounds 15b and 17b. As
reported in Table 2 the 6-methylenesubstituted coumarin derivative 15b resulted 2.8-fold stronger
inhibitor of the hCA VA over the VB. The preferential inhibition for the former was lost when the
chain in 15b was moved to the adjacent 7-position as in 17b (See Table 2). Interestingly the same
swapping position as in compounds 16b and 18b did not altered the SI, which was in favour of the
hCA VB for both derivatives, affected its intensity as it resulted halved. Finally, the sulfocoumarin
prodrug 20b also reported preferential inhibition for the hCA VB isoform with K values of 172.4 and
I
5
4.6 nM, respectively.
iii) As for the central nervous system (CNS) expressed hCA VII, the majority of the
compounds tested resulted low nanomolar inhibitors. On considering the SARs it is worth noting that
the ethers 1b and 2b showed K values within the medium nanomolar range (51.6 and 31.0 nM,
I
respectively). The introduction of a nitrogen atom instead (i.e. compounds 3b and 4d) or a tertiary
amine with an alkyl spacer (i.e. compound 5b) spoiled the inhibition potency against the hCA VII
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and thus raising the inhibition values up to the high nanomolar range (See Table 2). Interestingly the
ester linkage seems to affect the inhibition potency for this isoform as demonstrated by the kinetic
data for both compounds 6b and 7b. As a matter of fact the insertion of the amide, as in compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
b, or the ureido linker (i.e. 9b and 10b) resulted in a sensible enhancement of the hCA VII inhibition
potency as reported in Table 2 for the corresponding K values which are all comprised in the low
I
nanomolar range (i.e. 9.5, 9.42 and 9.4 nM for 8b-10b, respectively). Interesting results were obtained
for the seleno containing compounds 11b and 12b as the regioisomer effect on kinetics was clearly
observed. As reported in Table 2 the para substituted benzenesulfonamide derivative 12b was 3.0-
fold more potent inhibitor of the hCA VII when compared to the meta one 11b (K s of 21.8 and 66.0
I
nM, respectively). Finally, among the sulfonamide containing CAIs is the 2-oxo-2,3-
dihydrobenzo[d]oxazole derivative 13e which resulted a medium hCA VII nanomolar inhibitor with
a K value of 38.0 nM. As for the coumarin containing CAI moieties, the regioisomeric substitution
I
seems to be ineffective on the kinetic profile of such compounds against the hCA VII isoform. As
reported in Table 2, compounds 14b-19b resulted low nanomolar inhibitors and among them the 6-
and 7-methylene substituted derivatives 15b and 17b were the less effective when compared to
compounds bearing longer alkyne chain between the CAI portion and the AZT scaffold (14b, 16b,
18b and 19b).
iv)
A very interesting inhibitory profile can be observed for all the synthetized compounds
1
b-12b, 14b-20b and 13e against the tumor associated isoforms hCA IX and XII. In general, all of
them acted as low nanomolar inhibitors of CA XII, with K values ranging from 2.8 to 78.9 nM. As
I
for CA IX, the different CAI moiety inserted within the scaffold (sulfonamide or coumarin) as well
as the substitution patterns, both turned out to deeply influence the inhibition potency against this
isoform. Three main groups can be delineated on the basis of the observed K values against CA IX.
I
To the first group belong compounds which efficiently inhibit both tumor associated isoforms, such
as compounds 1b, 7b, 8b and 16b (K values < 100 nM against CA IX) and compounds 2b, 3b, 6b,
I
1
0b and 11b (K values < 1000 nM against CA IX). Except for compound 16b, which is a 6-
I
1
4
ACS Paragon Plus Environment

Page 15 of 58
Journal of Medicinal Chemistry
1
2
3
substituted coumarin derivative, all the compounds belonging to this group are sulfonamide based
derivatives, in which only one AZT moiety is present within the scaffold. In the second group (KI
values < 10000 nM against CA IX) we can include di-substituted sulfonamide based compounds 4d
and 5b, in which two AZT moieties were “clicked” to the di-propargyl aminobenzensulfonamide and
ethylaminobenzensulfonamide, respectively. In particular, ethylaminobenzensulfonamide derivative
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
b proved to be 1.23 fold less potent against CA IX then the shorter analogue 4d (Table 2). In the
second group we can also enumerate coumarin based compounds 15b, 17b and 18b and
sulfocoumarin compound 20b, which inhibited CA IX in the micromolar range (K values ranging
I
from 2948.3 nM to 6557.1 nM). Interestingly, these compounds strongly inhibited CA XII in the low
nanomolar range (Table 2). Finally, in the third group (K values > 10000 nM against CA IX) we can
I
find compounds which selectively inhibited CA XII over CA IX. In particular, 4 and 7-substituted
coumarins 14b and 19b, both bearing a four methylene alkyne chain between the coumarin scaffold
and the AZT moiety, showed to be ineffective against CA IX in the concentration range considered,
whereas a strong inhibition of CA XII can be observed (K values of 8.7 nM and 8.9 nM for 14b and
I
1
9b, respectively). Meta-substituted ureido compound 9b, para-substituted thioureido compound 12b
and 2-oxo-2,3-dihydrobenzo[d]oxazole-5-sulfonamide compound 13e proved to be inactive in CA
IX inhibition too (K values > 10000 nM). Again, a strong CA XII inhibition can be observed for all
I
the compounds. Noteworthy, comparing homologous compounds such as meta- and para-substituted
ureido compounds 9b and 10b and thioureido compounds 11b and 12b, the crucial impact of the
regioisomery on the inhibition potency against CA IX can be appreciated, one isomer being about 30
fold more potent than the other. In particular, the meta-substituted ureido compound 10b proved to
be more potent than the para-analogue 9b, whereas for the seleno-containing thioureido compounds
1
1b and 12b, the meta analogue 11b showed to be the most potent.
Co-Crystallographic Studies.
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In light of the promising K values observed against the tumor associated isoforms CA IX and
I
XII, the binding modes of compounds 1b and 3b within the hCA II, used as a model study, was
determined by means of X-ray experiments. The electron density maps of both 1b-hCA II and 3b-
hCA II adducts accounted for both ligands placed well-ordered within the enzymatic cleft with their
sulfonamide moieties deep buried up to the bottom of the cavity and coordinated to the zinc (II) ion
in the canonical tetrahedral geometry. Again the additional interaction between the sulfonamidic
oxygen with the T199 residue was conserved (Figures 4 and 5).56
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Inhibitor 1b bound within the active site of hCA II at 1.1 Å resolution and showing the σA-
weighted |Fo−Fc| map contoured at 2.5 σ. Ligand 1b is shown in cyan. Hydrogen bonds, van der
Waals interactions and Water Bridges are shown and labelled in red, blue and green, respectively.
Residues involved in the binding of inhibitors are also shown. PDB access code 6YPW.
1
6
ACS Paragon Plus Environment

Page 17 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Inhibitor 3b bound within the active site of hCA II at 1.3 Å resolution and showing the σA-
weighted |Fo−Fc| map contoured at 2.5 σ. Ligand 3b is shown in magenta. Hydrogen bonds, van der
Waals interactions are shown and labelled in red and blue. Residues involved in the binding of
inhibitors are also shown. PDB access code 6WKA.
The ligands backbones of 1b and 3b are stabilized within the hCA II cavity site by means of
a network of hydrogen bonds and van der Waals interactions with substantial differences of the tails
orientations as clearly showed when superposition of two structures was performed as in Figure 6.
Figure 6. Superposition of inhibitors 1b and 3b bound in the active site of hCA II. Ligand 1b is
shown in cyan and 3b in magenta.
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The diverse spatial orientations of the tail sections must be ascribed to the replacement of the
ethereal oxygen in 1b with the nitrogen atom instead as in 3b which is the only structural difference
among them. The tail in 1b is located towards the hydrophobic half of the catalytic cleft with the
F131 residue acting as the major clipping point. The adduct is further stabilized by a network of
hydrogen bonds which connects the inner face of the inhibitor to the opposite hydrophilic half of the
enzymatic cleft by means of bridged water molecules (Figures 4 and 6). As for the compound 3b tail,
it resulted laid towards the hydrophilic section of the enzymatic cavity and directly stabilized by
means of hydrogen bonds to the aminoacidic residues N67, E69 and Q92 (Figures 5 and 6). Such
results were in agreement with the previously discussed CA kinetic data which showed the strongly
stabilized compound 1b being 3.7-fold more potent inhibitor against the hCA II when compared to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
b.
Telomerase Activity Assay.
As mentioned above, AZT is known to be a potent telomerase inhibitor.57,58 To check whether
our compounds can affect telomerase, we incubated PC3 and HT-29 cells with the most potent CA
IX and XII CAI-TI compounds 1b, 7b, 8b or 11b and measured telomerase activity. Results of
Telomerase Repeated Amplification Protocol (TRAP) assay showed that all the tested compounds
suppressed telomerase in both PC3 and HT-29 cells (Figure 7A and B). Telomerase activity in PC3
cells was higher than in HT-29. Compounds 1b and 11b demonstrated the strongest antitelomerase
activity, while 7b and 8b appeared to be less potent.
1
8
ACS Paragon Plus Environment

Page 19 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Suppression of telomerase activity by CAI-TI compounds. (A) Representative TRAP gel
electrophoresis for PC3 or HT-29 cells incubated with 20 µM CAI-TI for 48 h. (B) Quantification of
TRAP for living cells. (C) hTERT expression in incubated PC3 or HT-29 cells. Levels of hTERT
mRNA were normalized relative to the levels of reference 18S RNA. (D, F, H, J) Representative
TRAP gel electrophoresis for cell lysates treated with different concentrations of CAI-TI. (E, G, I,
K) Quantification of TRAP for treated cell lysates. One representative TRAP gel of total four for
each of experiment is shown. The results are presented as the mean±standard error of the mean
(SEM). Con., control intact cells.
Telomerase activity is strongly regulated by the expression of its catalytic subunit hTERT and
inhibition of its expression can be one of the ways of how CAI-TI suppress telomerase in cells.59
We investigated hTERT expression in cells incubated with CAI-TI. In general, hTERT expression
in PC3 cells was higher than in HT-29, which corresponds to increased telomerase activity in such
cells (Figure 7C). We found that the compounds have no effect on hTERT gene expression in both
types of cells. Another possible way of telomerase inhibition is the binding of substance to hTERT
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
60
protein subunit. As it shown in Figure 7A, PC3 cells have more active telomerase, that is why their
lysates were used for telomerase testing in cell-free experiments. All the compounds demonstrated
dose-dependent activity to inhibit telomerase within the rage of concentrations 0.1 – 100 µM (Figure
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
D–K). IC and IC values for each compound are shown in Тable 3. Compounds 1b and 11b had
50 90
the lowest IC , that is in accordance to telomerase inhibition in living cell.
5
0
Table 3. Determined IC and IC values for telomerase inhibitors (CAI-TI).
5
0
90
IC , µM*
IC , µM*
5
0
90
1
b
b
5.2
6.0
9.1
5.6
40.0
31.8
60.3
42.8
7
8b
1b
1
* Mean from 4 different assays, by a RTQ-TRAP (errors were in the range of  5 % of the reported
values).
CONCLUSIONS
To the best of our knowledge this work is the proof-of-concept study about the concomitant
use of CAIs and TIs merged within the same molecular scaffold and able to act on two validated
targets for the management of cancer. Molecular hybridization is a powerful tool in Medicinal
6
1
Chemistry, with extensive and several successful applications reported so far. Herein, a series of 20
CAI-TI of the AZT type compounds has been synthetized and fully characterized. Then, inhibition
potencies against the two designed targets have been assessed. CA inhibition data against seven hCA
isoforms revealed that all the titled compounds 1b-3b, 5b-12b, 14b-20b, 4d and 13e strongly inhibit
hCA XII, whereas some of them (1b-3b, 6b-8b, 10b, 11b, and 16b) showed medium-high inhibition
potency against hCA IX.
2
0
ACS Paragon Plus Environment

Page 21 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
The evaluation of telomerase activity in cell lysates or in cells incubated with the CA IX and
XII most potent inhibitors 1b, 7b, 8b and 11b showed their strong antitelomerase properties, which
rely on the ability to suppress processivity of the enzyme rather than the suppression of hTERT
expression.
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
High resolution X-ray crystallography on compounds 1b and 3b in adduct with hCA II as
model study allowed to properly assess their binding mode. In particular we: i) highlighted the crucial
role played by a single heteroatom in determining CA isoform selectivity by means of diverse space
orientation of the tail; ii) firstly determined molecular features of the CAI-TI molecules which may
be useful to address CA selectivity once proper chemical manipulation is operated.
Overall the preliminary results obtained in this study fully sustained our strategy and gave us
a strong background to further proceed in developing ad hoc designed CAI-TI molecules which will
be considered in appropriate tumor cell lines.
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EXPERIMENTAL PROTOCOLS
Chemistry.
Anhydrous solvents and all reagents were purchased from Sigma-Aldrich (Milan, Italy), Alfa
Aesar (Milan, Italy) and TCI (Milan, Italy). All reactions involving air- or moisture-sensitive
compounds were performed under a nitrogen atmosphere using dried glassware and syringes
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
13
techniques to transfer solutions. Nuclear magnetic resonance spectra ( H-NMR: 400 MHz; C-NMR:
00 MHz) were recorded in DMSO-d using an Avance III 400 MHz spectrometer (Bruker, Milan,
1
6
Italy). Chemical shifts are reported in parts per million (ppm) and the coupling constants (J) are
expressed in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet;
q, quadruplet; m, multiplet; brs, broad singlet; dd, double of doublets. The assignment of
exchangeable protons (OH and NH) was confirmed by the addition of D O.
2
The purity of the final compounds was determined in high purity grade (i.e. ≥ 95 %) by HPLC using
an Agilent 1200 liquid chromatography system composed by autosampler, binary pumps, column
oven and diode-array detector (LC-DAD) operating in UV range (210-400 nm). The operating
conditions were reported within the Supporting Information file.
The solvents used in MS measures were acetone, acetonitrile (Chromasolv grade), purchased from
Sigma–Aldrich and mQ water 18 MΩ cm, obtained from Millipore’s Simplicity system (Milan, Italy).
The high resolution mass spectrometry (HRMS) analysis was performed with a Thermo Finnigan
LTQ Orbitrap mass spectrometer equipped with an electrospray ionization source (ESI). The accurate
-1
mass measure was carried out by introducing, via syringe pump at 10 L min , the sample solution
-1
(
1.0 g mL in mQ water: acetonitrile 50:50), and the signal of the positive ions was acquired. The
proposed experimental conditions allowed to monitoring the protonated molecules of studied
+
compounds ([M+H] species), that they were measured with a proper dwell time to achieve 60,000
units of resolution at Full Width at Half Maximum (FWHM).
Synthesis of final compounds 1b-3b, 5b-12b, 14b-20b, 4d and 13e and their intermediates.
2
2
ACS Paragon Plus Environment

Page 23 of 58
Journal of Medicinal Chemistry
1
2
3
General procedure A: The proper alkyl halide (1.2 eq.) was added to a suspension of either 1, 14,
5, 17-20 (0.5 g, 1.0 eq.) and K CO (2.0 eq.) in dry DMF (4 mL) under N atmosphere. The mixture
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
2
was stirred at r.t or 60° or 100°C, depending on the alkyl halide, until consumption of starting material
5 h, TLC monitoring). The reaction mixture was cooled at r.t. and quenched with slush. The mixture
was extracted with EtOAc (x 3) and the combined organic layers were washed with H O and brine
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2
solution, then dried over Na SO , filtered-off and concentrated under vacuum.
2
4
General procedure B. The proper carbamate derivative 9 or 10 (0.5 g, 1 eq.) was dissolved in EtOH
and propargylamine (1.2 eq.) was added. The reaction was refluxed for 16h, then cooled at r.t. and
quenched with slush. The mixture was extracted with EtOAc (x 3), dried over Na SO , filtered-off
2
4
and concentrated under vacuum, to afford a solid that was purified by silica gel column
chromatography, eluting with 60% EtOAc/Hx.
General Procedure C. To a suspension of azidonucleoside AZT (1.1 eq. or 2.2 eq.) in H O/t-BuOH
2
1
/1 (4 mL) the appropriate alkyne (0.12 g, 1.0 eq.) was added at r.t., followed by copper(0) nanosized
(0.1 eq.) and TMACl (1.0 eq.). The suspension was stirred at 40°C until starting materials were
consumed (TLC monitoring), then diluted with MeOH (20 mL), and filtered through Celite 521®.
The solvent was evaporated, affording to a residue that was triturated from EtOAc, to give a white
powder.
4
-(Prop-2-ynyloxy)benzenesulfonamide (1a). Compound 1a was synthetized according to the
general procedure A using 4-hydroxybenzenesulfonamide 1 and propargyl bromide 80% in toluene
at 60°C. Purified by silica gel column chromatography eluting with 50% ethyl acetate in n-hexane to
afford the titled compound 1a as a white powder. 47% yield; δ (400 MHz, DMSO-d ) 3.67 (1H, br
H
6
s, CH), 4.94 (2H, br s, CH ), 7.17 (2H, d, J = 7.2, Ar-H), 7.28 (2H, s, exchange with D O, SO NH ),
2
2
2
2
8
.01 (2H, d, J = 7.2, Ar-H). Experimental in agreement with reported data.62
4
-((1-(2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-4-yl)methoxy)benzenesulfonamide (1b). Compound
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
b was obtained according to the general procedure A using 1a as starting material, to afford the
title compound 1b as a light yellow solid: 84% yield; δ (400 MHz, DMSO-d ): 1.85 (3H, s, CH ),
H
6
3
2.73 (2H, m, CH ), 3.70 (2H, m, CH ), 4.27 (1H, q, J= 3.5, CH), 5.29 (2H, s, CH ), 5.34 (1H, br t,
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
exchange with D O, OH), 5.45 (1H, m, CH), 6.46 (1H, t, J= 6.5, CH), 7.24 (4H, m, overlapped signals,
2
2
x ArH, exchange with D O, SO NH ), 7.80 (2H, d, J= 8.8, ArH), 7.86 (1H, s, CH), 8.50 (1H, s,
2 2 2
CH), 11.36 (1H, br s, exchange with D O, NH); δ (100 MHz, DMSO-d ):13.1, 38.0, 55.2, 60.3, 62.0,
2
C
6
84.5, 85.4, 110.5, 115.6, 125.4, 128.5, 137.1, 137.4, 143.2, 151.3, 161.2, 164.6; ESI-HRMS (m/z)
+
calculated for [M+H] ion species C H N O S= 479.1343, found 479.1336.
1
9
23
6
7
4
-(Prop-2-ynylthio)benzenesulfonamide (2a). NaBH (23 mg, 0.60 mmol, 3.0 eq.) was added
4
portion-wise to a freshly prepared solution of solution of 4,4′-disulfanediyldibenzenesulfonamide 2
(75 mg, 0.20 mmol, 1.0 eq.) in EtOH (2 mL) at r.t. under a N atmosphere. After 2 h, propargyl
2
chloride (0.42 mmol, 2.1 eq.) was slowly added and the reaction mixture was stirred at r.t. for 3 h,
until complete consumption of the starting material was observed by TLC. The reaction was quenched
by addition of saturated NH Cl aqueous solution (2 mL) and diluted with EtOAc (5 mL), The layers
4
were separated and the aqueous layer was extracted with EtOAc (2 × 5 mL), dried over Na SO ,
2
4
filtered and concentrated under vacuum. The crude material was purified by silica gel flash
chromatography to afford the titled compound 2a as a white solid. 83% yield; δ (400 MHz, DMSO-
H
d ): 3.22 (1H, t, J = 2.6, CH), 4.02 (2H, d, J = 2.6, CH ), 7.37 (2H, s, exchange with D O, SO NH ),
6
2
2
2
2
7
.56 (2H, dd, J = 2.0, 6.7, Ar-H), 7.79 (2H, dd, J = 2.0, 6.7, Ar-H). Experimental in agreement with
reported data.63
4
-(((1-(2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-4-yl)methyl)thio)benzenesulfonamide
(2b).
Compound 2b was obtained according to the general procedure C using 2a as starting material, to
afford the title compound 2b as a white solid: 37% yield; δ (400 MHz, DMSO-d ): 1.84 (3H, s,
H
6
CH ), 2.69 (2H, m, CH ), 3.64 (2H, m, CH ), 4.19 (1H, q, J= 3.5, CH), 4.45 (2H, s, CH ), 5.38 (2H,
3
2
2
2
2
4
ACS Paragon Plus Environment

Page 25 of 58
Journal of Medicinal Chemistry
1
2
3
m, overlapped signals, 1 x CH, exchange with D O, 1 x OH), 6.43 (1H, t, J= 6.5, CH), 7.36 (2H, s,
2
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
exchange with D O, SO NH ), 7.57 (2H, d, J= 8.4, ArH), 7.76 (2H, d, J= 8.4, ArH), 7.85 (1H, s, CH),
2
2
2
8.29 (1H, s, CH), 11.35 (1H, br s, exchange with D O, NH); δ (100 MHz, DMSO-d ): 13.1, 27.0,
2
C
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7.9, 60.2, 61.6, 84.8, 85.5, 110.5, 124.2, 127.1, 127.7, 137.1, 141.9, 142.1, 144.1, 151.3, 164.6; ESI-
+
HRMS (m/z) calculated for [M+H] ion species C H N O S = 495.1115, found 495.1118.
1
9
23
6
6 2
4
-(Prop-2-ynylamino)benzenesulfonamide (3a). Propargyl bromide 80% in toluene (1.2 eq.) was
added to a suspension of sulfanilamide A (0.5 g, 1.0 eq.) and pyridine (1.2 eq.) in dry DMF (2 ml)
under N atmosphere and the mixture was stirred at 70°C o.n. (TLC monitoring). The reaction was
2
quenched with H O (10 ml) and extracted with EtOAc (3 x 15 ml). The combined organic layers were
2
washed with H O (3 x 15 ml) and brine (3 x 15 ml), dried over anhydrous Na SO , filtered-off and
2
2
4
concentrated under vacuum to give a solid that was purified by silica gel column chromatography
eluting with 50% ethyl acetate in n-hexane to afford the desired product 65 as a yellow solid. 33 %
yield; δ (400 MHz, DMSO-d ): 3.13 (1H, t, J = 2.4, CH), 3.97 (2H, dd, J= 2.4, 6.0, CH ), 6.73 (2H,
H
6
2
d, J = 8.8, Ar-H), 6.76 (1H, br t, exchange with D O, NH), 6.98 (2H, br.s, exchange with D O,
2
2
6
4
SO NH ), 7.59 (2H, d, J = 8.8, Ar-H). Experimental in agreement with reported data.
2
2
4-(((1-(2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)benzenesulfonamide
(3b).
Compound 3b was obtained according to the general procedure C using 3a as starting material, to
afford the title compound 3b as a white solid. 21% yield; δ (400 MHz, DMSO-d ): 1.84 (3H, s,
H
6
CH ), 2.69 (2H, m, CH ), 3.68 (2H, m, CH ), 4.23 (1H, q, J= 3.5, CH), 4.40 (2H, d, J= 5.7, CH ),
3
2
2
2
5
.32 (1H, br t, 1H, exchange with D O, OH), 5.38 (1H, m, CH), 6.45 (1H, t, J= 6.5, CH), 6.74 (2H,
2
d, J= 8.8, 2x Ar-H), 6.92 (1H, t, J=5.7, exchange with D O, NH), 6.98 (2H, s, exchange with D O,
2
2
SO NH ), 7.55 (2H, d, J= 8.8, 2x Ar-H), 7.84 (1H, s, CH), 8.24 (1H, s, CH), 11.4 (1H, br s, exchange
2
2
with D O, NH); δ (100 MHz, DMSO-d ):12.2, 36.9, 37.9, 59.1, 60.7, 83.8, 84.5, 110.1, 111.2, 122.6,
2
C
6
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
1
27.2, 130.4, 136.3, 145.2, 150.4, 150.9, 163.7; ESI-HRMS (m/z) calculated for [M+H] ion species
C H N O S= 478.1503, found 478.1508.
1
9
24
7
6
4
-(diProp-2-ynylamino)benzenesulfonamide (4c). Sulfanilamide A (0,5 g, 1.0 eq.) was dissolved
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
in DMF and the solution was cooled to 0°C. Then, dimethoxy-N,N-dimethylmethanamine (1.2 eq.)
was added. The solution was stirred at r.t. until consumption of starting material (2h). The reaction
was quenched with DCM and precipitate formed was filtered-off, dried to afford N'-((4-
aminophenyl)sulfonyl)-N,N-dimethylformimidamide 4a which was used for the next step without
further purification. N'-((4-aminophenyl)sulfonyl)-N,N-dimethylformimidamide 4a (1.0 eq.) was
solubilized in dry DMF and K CO (3.0 eq.) was added. Then, propargyl bromide 80% in toluene (4.0
2
3
eq.) was added and the mixture was stirred at 80°C o.n. until consumption of starting material. Then
the reaction was quenched with H O (20 ml) and extracted with EtOAc (3 x 15 ml). The combined
2
organic layers were washed with H O (3 x 15 ml) and brine(3 x 15 ml), dried over Na SO , filtered-
2
2
4
off and concentrated under vacuum to give a residue (4b) that was suspended in isopropylamine in a
sealed tube and stirred at r.t. o.n. The solvent was removed in vacuo obtaining a residue that was
purified by silica gel column chromatography eluting with 50% ethyl acetate in n-hexane to afford a
sticky residue which was triturated from Et O to afford the titled compound 4c as a white powder:
2
1
0% yield; δ (400 MHz, DMSO-d ): 3.21 (2H, t, J = 2.4, 2x CH), 4.29 (4H, d, J = 2.4, 2 x CH ),
H 6 2
7.03 (2H, d, J = 8.8, Ar-H), 7.08 (2H, s, exchange with D O, SO NH ), 7.70 (2H, d, J = 8.8, Ar-H).
2
2
2
Experimental in agreement with reported data.65
4
-(bis((1-(2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)benzenesulfonamide
(4d).
Compound 4d was obtained according to the general procedure C using 4c as starting material, to
afford the title compound 4d as a light yellow solid. 32% yield; δ (400 MHz, DMSO-d ): 1.84 (6H,
H
6
s, 2 x CH ), 2.69 (4H, m, 2 x CH ), 3.68 (4H, m, 2 x CH ), 4.21 (2H, q, J= 3.8, 2 x CH), 4.79 (4H, s,
3
2
2
2
x CH ), 5.35 (2H, br t, exchange with D O, 2 x OH), 5.40 (2H, m, 2 x CH), 6.45 (2H, t, J= 6.4, 2 x
2 2
2
6
ACS Paragon Plus Environment

Page 27 of 58
Journal of Medicinal Chemistry
1
2
3
CH), 7.03 (4H, m, overlapped signals, 2 x ArH, exchange with D O, SO NH ), 7.60 (2H, d, J= 8.9,
2
2
2
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
x ArH), 7.85 (2H, s, 2 x CH), 8.28 (2H, s, 2 x CH), 11.38 (2H, br s, exchange with D O, 2 x NH).
2
δ (100 MHz, DMSO-d ): 12.2, 37.9, 45.4, 59.1, 60.7, 83.8, 84.5, 110.1, 111.2, 122.6, 127.2, 130.4,
C
6
36.3, 145.2, 150.4, 150.9, 163.7; ESI-HRMS (m/z) calculated for [M+H]+ ion species
C H N O S= 783.2627, found 783.2632.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
2
39 12 10
4
-(2-(di-Prop-2-ynylamino)ethyl)benzenesulfonamide (5a). Propargyl bromide (80% in toluene)
2 eq.) and DIPEA (1.7 eq.) were added to a stirred solution of 4-(2-aminoethyl) benzensulfonamide
5 (0.5 g, 1.0 eq.) in CH CN (8 ml) under N atmosphere. The mixture was stirred at r.t. o.n. until
(
3
2
consumption of starting material (TLC monitoring). The solvent was removed under reduced pressure
and the obtained residue was portioned between H O and EtOAc followed by extraction with EtOAc
2
(3 x 15 ml). The combined organic layers were washed with H O(3 x 15 ml) and brine(3 x 15 ml),
2
then dried over Na SO , filtered-off and concentrated under vacuum to afford compound 5a as a dark
2
4
oil. 70 % yield; δ (400 MHz, DMSO-d ): 2.75 (2H, t, J = 6.8, CH ), 2.84 (2H, t, J = 6.8, CH ), 3.21
H
6
2
2
(2H, br t, 2 x CH), 3.44 (4H, d, J = 2.0, 2 x CH ), 7.37 (2H, s, exchange with D O, SO NH ), 7.45
2 2 2 2
(2H, d, J = 8.4, Ar-H), 7.76 (2H, d, J = 8.4, Ar-H); δ (100 MHz, DMSO-d ): 32.6, 41.5, 53.3, 75.8,
C 6
+
7
2
9.1, 125.6, 129.1, 141.9, 144.4; ESI-HRMS (m/z) calculated for [M+H] ion species C H N O S=
14 17 2 2
77.1005, found 277.1009. Experimental in agreement with reported data.66
-(2-(bis((1-(2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
4
yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethyl)benzenesulfonamide (5b).
Compound 5b was obtained according to the general procedure C using 5a as starting material, to
afford the title compound 5b as a white solid. 82% yield; δ (400 MHz, DMSO-d ): 1.85 (6H, s, 2 x
H
6
CH ), 2.72 (6H, m, overlapped signals, 3 x CH ), 2.93 (2H, t, J= 7.2, CH ), 3.70 (4H, m, 2 x CH ),
3
2
2
2
3
.80 (4H, s, 2 x CH ), 4.24 (2H, q, J= 3.5, 2 x CH), 5.40 (4H; m, overlapped signals, 2 x CH, exchange
2
with D O, 2 x OH), 6.48 (2H, t, J= 6.4, 2 x CH), 7.32 (2H, br s, exchange with D O, SO NH ), 7.41
2
2
2
2
(2H, d, J= 8.3, 2x Ar-H ), 7.75 (2H, d, J= 8.3, 2x Ar-H), 7.87 (2H, s, 2 x CH), 8.22 (2H, s, 2 x CH),
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1.40 (2H, br s, exchange with D O, 2 x NH). δ (100 MHz, DMSO-d ): 13.1, 33.4, 37.9, 48.2, 54.7,
2 C 6
6
0.1, 61.6, 84.8, 85.5, 110.5, 124.5, 126.5, 130.0, 137.2, 142.6, 144.6, 145.7, 151.4, 164.6; ESI-
+
HRMS (m/z) calculated for [M+H] ion species C H N O S= 811.2940, found 811.2951.
3
4
43 12 10
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Prop-2-yn-1-yl 4-chloro-3-sulfamoylbenzoate (6a). To a stirring solution of 4-chloro-3-
sulfamoylbenzoic acid 6 (1 eq.) in dry DMF, EDC HCl (1.2 equiv) was added at 0°C. After 30 min,
propargyl alcohol (1.2 eq.) and DMAP (1.2 eq.) were added. The mixture was stirred at room
temperature under N for an additional 3 h, until consumption of starting material. The reaction was
2
quenched with slush and extracted with EtOAc (x3). The organic extract was washed with saturated
aqueous NaHCO , water and brine, dried over Na SO , filtered-off and concentrated under vacuum.
3
2
4
The crude was purified by flash silica chromatography (40% EtOAc/Hx) to afford the title compound
6a as a white solid. 40% yield; δ (400 MHz, DMSO-d ): 3.68 (1H, t, J= 2.4, CH), 5.05 (2H, d, J=
H
6
2
.5, CH ), 7.86 (3H, m, 1x Ar-H, 2x SO NH ), 8.16 (1H, dd, J= 2.2, 8.2, Ar-H), 8.56 (1H, d, J= 2.1,
2 2 2
Ar-H); δ (100 MHz, DMSO-d ): 53.6, 79.0, 79.3, 129.0, 130.6, 133.4, 134.5, 136.7, 142.4, 165.5;
C
6
+
ESI-HRMS (m/z) calculated for [M+H] ion species C H N O = 270.1237, found 270.1237.
15 16
3
2
(
1-((2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl 4-chloro-3-sulfamoylbenzoate
6b). Compound 6b was obtained according to the general procedure C using 6a as starting material,
to afford the title compound 6b as a white solid. 21% yield; δ (400 MHz, DMSO-d ): 1.83 (3H, s,
(
H
6
CH ), 2.69 (2H, m, CH ), 3.67 (2H, m, CH ), 4.22 (1H, q, J= 3.9, CH), 4.76 (2H, s, CH ), 5.29 (1H,
3
2
2
2
br t, 1H, exchange with D O, OH), 5.38 (1H, dt, J= 8.3, 5.29, CH), 6.43 (1H, t, J= 6.5, CH), 7.47
2
(2H, s, exchange with D O, SO NH ), 7.86 (2H, m, 2x Ar-H), 7.98 (1H, s, CH), 8.10 (1H, s, Ar-H),
2 2 2
8.38 (1H, s, CH), 11.3 (1H, br s, exchange with D O, NH). δ (100 MHz, DMSO-d ):13.1, 27.5, 38.0,
2
C
6
6
1
5
0.4, 61.6, 84.8, 85.3, 110.5, 111.6, 116.9, 123.1, 124.6, 137.1, 142.0, 143.2, 151.3, 153.8, 163.2,
+
64.6, 167.1; ESI-HRMS (m/z) calculated for [M+H] ion species C H ClN O S= 541.0903, found
2
0
22
6
8
41.0899.
2
8
ACS Paragon Plus Environment

Page 29 of 58
Journal of Medicinal Chemistry
1
2
3
Prop-2-ynyl 4-sulfamoylbenzoate (7a). To a stirring solution of 4-sulfamoylbenzoic acid 7 (2.0 g,
.9 mmol) in dry DMF (40 mL) were successively added propargyl alcohol (1.17 mL, 19.8 mmol,
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
2.0 eq.), Et N (2.8 mL, 19.9 mmol, 2.0 eq.), and EDC HCl (1.9 g, 9.9 mmol, 1.0 eq.). The solution
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was stirred at room temperature under N for an additional 4 h. The mixture was then concentrated
2
under reduced pressure and ethyl acetate (40 mL) was added. The organic extract was washed with
saturated aqueous NaHCO (40 mL) and back-extracted with ethyl acetate (40 mL). The organic
3
layers were combined and washed with brine (40 mL), dried over Na SO , filtered, and evaporated.
2
4
The crude oil was purified by flash silica chromatography (50% EtOAc/Hx) to afford the title
compound 7a as a white crystalline solid. 38% yield; δ (400 MHz, DMSO-d ): 3.63 (1H, t, J= 2.4,
H
6
CH), 4.97 (2H, d, J= 2.8, CH ), 7.55 (2H, brs, exchange with D O, SO NH ), 7.98 (4H, m, 4x ArH);
2
2
2
2
δ (100 MHz, DMSO-d ): 53.0, 78.1, 78.3, 126.2, 130.0, 131.7, 148.3, 164.0. Experimental in
C
6
agreement with reported data.67
1-(2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
(
yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-4-yl)methyl 4-sulfamoylbenzoate (7b). Compound 7b
was obtained according to the general procedure C using 7a as starting material, to afford the title
compound 7b as a white solid. 30% yield; δ (400 MHz, DMSO-d ): 1.84 (3H, s, CH ), 2.74 (2H, m,
H
6
3
CH ), 3.71 (2H, m, CH ), 4.28 (1H, m, CH), 5.32 (1H, t, J= 5.1, exchange with D O, OH), 5.42 (1H,
2
2
2
m, CH), 5.50 (2H, s, CH ), 6.46 (1H, t, J= 6.6, CH), 7.60 (2H, s, exchange with D O, SO NH ), 7.85
2
2
2
2
(1H, s, CH), 8.00 (2H, d, J= 8.4, 2x Ar-H), 8.18 (2H, d, J= 8.5, 2x Ar-H), 8.49 (1H, s, CH), 11.4 (1H,
br s, exchange with D O, NH); δ (100 MHz, DMSO-d ): 13.3, 38.0, 59.3, 60.5, 61.7, 84.8, 85.3,
2
C
6
110.5, 125.7, 127.3, 131.2, 133.0, 137.1, 142.8, 149.1, 151.3, 164.6, 165.4; ESI-HRMS (m/z)
+
calculated for [M+H] ion species C H N O S= 507.1293, found 507.1294.
2
0
23
6
8
N-(Prop-2-ynyl)-4-sulfamoylbenzamide (8a). To a stirring solution of 4-sulfamoylbenzoic acid 7
2.0 g, 9.9 mmol) and propargylamine (0.64 mL, 9.9 mmol, 1.0 eq.) in dry DMF (40 mL) were
successively added N-hydroxybenzotriazole monohydrate (0.94 g, 6.6 mmol, 0.6 eq.),
(
2
9
ACS Paragon Plus Environment