Structure-based discovery of pyrazolobenzothiazine derivatives as inhibitors of hepatitis C virus replication

Article abstract of DOI:10.1021/jm301643a

The NS5B RNA-dependent RNA polymerase is an attractive target for the development of novel and selective inhibitors of hepatitis C virus replication. To identify novel structural hits as anti-HCV agents, we performed structure-based virtual screening of our in-house library followed by rational drug design, organic synthesis, and biological testing. These studies led to the identification of pyrazolobenzothiazine scaffold as a suitable template for obtaining novel anti-HCV agents targeting the NS5B polymerase. The best compound of this series was the meta-fluoro-N-1-phenyl pyrazolobenzothiazine derivative 4a, which exhibited an EC₅₀ = 3.6 μM, EC₉₀ = 25.6 μM, and CC₅₀ > 180 μM in the Huh 9-13 replicon system, thus providing a good starting point for further hit evolution.

Full text of DOI:10.1021/jm301643a

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Article
Structure-based discovery of pyrazolobenzothiazine
derivatives as inhibitors of hepatitis C virus replication
Maria Letizia Barreca, Giuseppe Manfroni, Pieter LEYSSEN, Johan Winquist, Neerja
Kaushik-Basu, Jan Paeshuyse, Ramalingam Krishnan, Nunzio iraci, Stefano Sabatini,
Oriana Tabarrini, Amartya Basu, U. Helena Danielson, Johan Neyts, and Violetta Cecchetti
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301643a • Publication Date (Web): 14 Feb 2013
Downloaded from http://pubs.acs.org on February 17, 2013
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Structure-based discovery of
pyrazolobenzothiazine derivatives as inhibitors of
hepatitis C virus replication
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Maria Letizia Barreca,*,† Giuseppe Manfroni,*,† Pieter Leyssen,‡ Johan Winquist,# Neerja Kaushikꢀ
Basu,§ Jan Paeshuyse,‡ Ramalingam Krishnan,§ Nunzio Iraci,† Stefano Sabatini,† Oriana
Tabarrini,† Amartya Basu,§ U. Helena Danielson,# Johan Neyts,‡ and Violetta Cecchetti†
†Dipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Farmaceutica II,
Università degli Studi di Perugia, Via del Liceo 1, 06123 Perugia, Italy
‡Rega Institute for Medical Research, Katholieke Universiteit Leuven, Bꢀ3000 Leuven, Belgium
#Department of Chemistry – BMC, Uppsala University, Box 576, SEꢀ751 23 Uppsala, Sweden.
§Department of Biochemistry and Molecular Biology UMDNJꢀNew Jersey Medical School 185
South Orange Avenue, New Jersey 07103, USA
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The NS5B RNAꢀdependent RNA polymerase is an attractive target for the development of novel
and selective inhibitors of hepatitis C virus replication. In order to identify novel structural hits as
antiꢀHCV agents, we performed structureꢀbased virtual screening of our inꢀhouse library followed
by rational drug design, organic synthesis and biological testing. These studies led to the
identification of pyrazolobenzothiazine scaffold as a suitable template for obtaining novel antiꢀHCV
agents targeting the NS5B polymerase. The best compound of this series was the metaꢀfluoroꢀNꢀ1ꢀ
phenyl pyrazolobenzothiazine derivative 4a, which exhibited an EC50= 3.6 ꢁM, EC90= 25.6 ꢁM and
CC50> 180 ꢁM in the Huh 9ꢀ13 replicon system, thus providing a good starting point for further hit
evolution.
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Keywords. Hepatitis C virus, RNAꢀdependent RNA polymerase, structureꢀbased drug discovery,
virtual screening, NS5B inhibitors, pyrazolobenzothiazines.
Introduction
Hepatitis C virus (HCV) is a small, enveloped RNA virus that belongs to the Flaviviridae
family. Worldwide, over 170 million people have become chronically infected with HCV;1 the
overall prevalence is ~3%, with rates up to 20%, for example in Egypt.2 The virus may remain
undetected for >15 years, a period during which transmission can occur. Due to intensive donor
screening, transfusionꢀassociated cases have become very rare, and most of the new infections can
be attributed to high risk drugꢀrelated behavior (60%) and unsafe injection practices. Following a
usually mild and subclinical acute infection, a chronic stage develops in 70ꢀ85% of the cases.2
Chronic HCV infection is one of the leading causes of liver cirrhosis and hepatocellular carcinoma
(primary liver cancer), and the first indication for liver transplantation in industrialized countries.3
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The standard treatment of HCV infection used to be a regimen of pegylated interferon alpha
(pegIFNꢀα) in combination with ribavirin (RBV). During treatment, a high number of patients
suffer from severe adverse effects and the sustained viral response rate unfortunately is rather low
for individuals infected with genotype 1, the most prevalent genotype in North America, Europe
and Asia. Recently, the first two directꢀacting antiviral agents (DAAs) targeting the NS3/4A
protease, telaprevir and boceprevir, have been approved by the Food and Drug Administration and
European Medicines Agency for the treatment of patients infected with HCV genotype 1.4 Any of
these two drugs, combined with pegINFꢀα and RBV in a triple combination therapy, induce a
significantly improved sustained virological response rate both in treatmentꢀnaïve patients as well
as those that have a treatment history.5 However, despite improved response rates, combination
treatment including either boceprevir or telaprevir shows longꢀterm toxic effects, is associated with
high costs, results in an increased pill burden on the patient, suffers from many drug interactions,
and has a low barrier to resistance if used as monotherapy.5 Therefore, there is still a need for
alternative treatment options, which are preferably pegINFꢀα and RBVꢀfree and which target
multiple steps of the virus replication cycle or different sites on a viral protein. In principle, every
step of the HCV lifecycle, from receptor binding and endocytosis, to fusion, uncoating, translation,
polyprotein processing, RNA replication, virion assembly, maturation, transport and release, can be
a target for the development of new and selective antiꢀHCV drugs.6
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Since the elucidation of the molecular genetics of the virus, the nonꢀstructural HCV proteins
NS3/4A protease and NS5B polymerase have drawn most attention as drug targets. In contrast to
the protease, drugs targeting the NS5B polymerase have yet to reach the market. NS5B is a RNAꢀ
dependentꢀRNA polymerase which catalyzes the synthesis of progeny viral RNA strands, and
because of its apparent sequence and structural difference with human DNA and RNA polymerases,
poses an attractive target for the development of selective inhibitors.7,8 To date, a variety of
nucleoside and nonꢀnucleoside HCV polymerase inhibitors have been reported.9,10 The nonꢀ
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nucleoside inhibitors (NNIs) can be divided into five groups based on the distinct allosteric binding
site on the HCV polymerase they target, namely thumb site I (TSI), thumb site II, (TSII), palm site I
(PSI), palm site II (PSII), and palm site III (PSIII).11 Because of the number of drugable pockets on
NS5B and the lack of crossꢀresistance between inhibitors that target these different binding sites,
multiple NS5Bꢀtargeting drugs may likely become part of future combination therapy. For many of
these pockets, proteinꢀligand crystal structures, together with biochemical evidence, are described in
literature, thus offering a solid base to set up exploratory projects aimed at identifying novel antiꢀ
NS5B chemotypes.
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In continuation of our efforts towards identification of new chemical entities that inhibit HCV
replication,12,13 in this study we have focused our attention on the allosteric PSI, located at the
junction of the thumb and palm domains and in close proximity to the active site. Several
chemotypes that bind at this site have been reported in literature;10,11 among them,
hydroxyquinolineꢀbenzothiadiazine (BTDZ) and hydroxyquinolineꢀbenzothiazine (BTZ) classes
represented by derivatives 1 and 2, respectively (Figure 1), are the most investigated PSIꢀNNIs with
promising candidates in clinical development.14
Figure 1. Chemical structures of 1 and 2 as representative of BTDZ and BTZ PSIꢀNNIs,
respectively.
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Given the scenario of PSIꢀNNIs as highly effective antiviral agents, we herein describe the
rational design, synthesis, antiꢀNS5B activity and inhibitory effect on HCV replication of novel
pyrazolobenzothiazineꢀbased compounds in a quest to discover new selective antiꢀHCV agents.
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Results and Discussion
Structure-based identification of the pyrazolobenzothiazine scaffold
The first goal of this study was to investigate inꢀdepth the binding requirements for potent NS5B
inhibitory activity at PSI. This aspect was important to gain useful information regarding the
identification of new potential PSIꢀNNIs. Towards this, we focused our attention on the BTDZ and
BTZ classes (e.g., compounds 1 and 2) (Figure 1) which, beside being potent inhibitors in both
NS5B polymerase and cellular replicon system assays, are well characterized from the structureꢀ
activity relationships (SAR) standpoint.14 Several studies have reported detailed structural analysis
of the ligand/NS5B contacts for different inhibitors of the BTDZ and BTZ series.14 All these
inhibitors share similar contacts with the NS5B residues, although the more potent compounds
establish some additional hydrogen bond interactions. Of note, all of the available Xꢀray structures
reveal some highly conserved water molecules which mediate the proteinꢀinhibitor hydrogenꢀbond
interactions.
Using as reference the coꢀcrystal structure of NS5B with compound 2,15 we have summarized the
key binding requirements as follows (Figure 2): i) a hydrophobic group (HYDꢀ1) occupying a
pocket mainly defined by residues Met414, Gly410, Gln446, Asn411 and Tyr415; ii) a Nꢀalkyl/aryl
lipophilic tail (HYDꢀ2) filling a deep pocket shaped by Tyr448, Cys366, Pro197, Tyr415, Leu384,
Arg200 and Ser368; iii) a hydrogen bond acceptor group (HBAꢀ1) involved in a direct interaction
with the backbone NꢀH of Tyr448 and in a waterꢀmediated interaction with Gly449; iv) one of the
sulfone oxygens of the BTZ/BTDZ moiety (HBAꢀ2) establishing a waterꢀmediated interaction with
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Ser556 or Ser288; v) the phenyl portion of the benzothiadiazine ring system (ETF) making an edgeꢀ
toꢀface aromatic interaction with Phe193; and vi) the methylsulfonamide group (HBDꢀHBA)
making strong interactions with Asn291 and Asp318. Moreover, one of the sulfonamide oxygen
atoms of the methylsulfonamide moiety forms a third Hꢀbond network with residue Ser288 via the
structural water molecule mentioned in point iv.
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Figure 2. Schematic representation of the interactions between NS5B polymerase and compound 2
(PDB ID 3G86) as representative of BTZ/BTDZ inhibitors. NS5B residues lying within a distance
of 4 Å from the bound ligand are shown and color coded as follows: redꢀacidic; greenꢀhydrophobic;
purpleꢀbasic; cyanꢀpolar; whiteꢀGly. Waters are displayed as gray circles, while interactions
between ligand atoms and protein residues are marked with lines: solid pink, Hꢀbonds to the protein
backbone; dotted pink, Hꢀbonds to protein side chains; green, πꢀπ stacking interactions. The six
chemical features of PSIꢀNNIs are highlighted as well: HBA, hydrogen bond acceptor; HBD,
hydrogen bond donor; HYD, hydrophobic regions; ETF, group involved in edgeꢀtoꢀface interaction.
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Taking into account the above observations, structureꢀbased drug discovery (SBDD) approaches
were performed to discover novel antiꢀNS5B chemotypes from an inꢀhouse library of 2692
molecules synthesized and/or published by our research group. The compound collection was
prepared for docking using the LigPrep process16 and submitted to a structureꢀbased virtual
screening workflow against the crystal structure of NS5B in complex with inhibitor 2 (PDB ID
3G86) by means of the Glide software.17 All water molecules found in the crystal structure were
removed, with the exception of the conserved water molecules involved in direct interactions with
the ligand (Figure 2). In order to validate the docking performance, test calculations using 2 were
carried out, extracting the ligand from the corresponding NS5B complex and then docking it back
into the allosteric pocket of the enzyme crystal structure. The best docking pose of 2 (XP GScore =
ꢀ9.4 kcal/mol) agreed well with its experimental binding conformation, with a rootꢀmean square
(rms) deviation value of 0.3 Å.
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The topꢀranked compounds were visually inspected searching for virtual hits endowed with high
docking score and also able to create the key interactions with the PSI residues. Unfortunately, none
of the inꢀhouse compounds possessed both these characteristics. However, our attention was
attracted by the pyrazolobenzothiazineꢀ3ꢀcarboxymethyl ester derivative 3a18 (rank position #94,
XP GScore = ꢀ5.8 kcal/mol) (Figure 3), a compound already reported by our research group as an
intermediate in the preparation of antiꢀinflammatory agents.19
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Figure 3. A) Simplified 2D representation of the predicted interactions between compound 3a and
NS5B amino acids (color legend as in Figure 2). B) Docking pose of compound 3a (green) together
with the experimental position of BTZ derivative 2 (violet). Water molecules are displayed in cyan,
whereas hydrogen bonds are represented as yellow dashed lines. Both pictures show protein
residues within 4 Å of the docked compound.
Interestingly, compound 3a was predicted to bind in a fashion somewhat similar to that of known
PSIꢀNNIs (Figure 3), establishing some of the already mentioned key ligandꢀNS5B interactions
(Figure 2). The sulfonyl function of the molecule, corresponding to the HBAꢀ1 feature, was
engaged in a direct hydrogen bonding interaction with Tyr448; the same moiety also established a
waterꢀmediated interaction with Gly449. The previously described hydrophobic features HYDꢀ1
and HYDꢀ2, were represented here by the phenyl ring of the pyrazolobenzothiazine nucleus and the
Nꢀphenyl tail, respectively. Of note, the pyrazole ring showed an additional π−π interaction with the
side chain of Tyr448.
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Computer-driven design of pyrazolobenzothiazine derivatives
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The interaction between compound 3a and NS5B polymerase was evaluated using surface
plasmon resonance (SPR), while its effect on the catalytic activity of the enzyme was determined in
a functional assay and its effect on transcription was evaluated in a cellꢀbased HCV subgenomic
replicon system. The analysis of the interaction with NS5B (Conꢀ1 strain, genotype 1b) indicated
that the affinity was very low with a dissociation equilibrium constant KD > 300 ꢁM. In the
functional antiꢀNS5B (C∆21, genotype 1b) assay, 50 ꢂM of the compound resulted in 11%
inhibition. A preliminary screening analysis for its ability to inhibit HCV replication (genotype 1b),
using a HCV subgenomic replicon containing human hepatoma cells (Huh 5ꢀ2), showed that
compound 3a exhibited ~40% inhibition of HCV RNA replication at nonꢀtoxic concentration.
Albeit the modest biological results obtained for 3a did not allow to classify it as hit, the interesting
information raised from the proposed binding conformation suggested that this chemotype was
worthy of further optimization. Therefore, taking into account the key chemical features of PSIꢀ
NNIs (Figure 2), we planned suitable chemical modifications of derivative 3a with the objective of
obtaining potent NS5B smallꢀmolecule inhibitors.
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Since it was not straight forward to synthesize molecules possessing the six desired features at the
same time, we decided to design a series of pyrazolobenzothiazineꢀbased compounds (4a-j, 5a and
6a), (Scheme 1 and Table 1) which could potentially contain all the binding requirements except
HBAꢀ2. In particular, superimposition of the docked conformation of compound 3a on the BTZ
inhibitor 2ꢀNS5B coꢀcrystal structure (Figure 3) suggested replacement of methyl with paraꢀ
methanesuphonamidephenyl moiety from the Cꢀ3 carboxylic group. Thus, derivative 4a was
designed where the ester linkage was replaced by the more chemically stable amide bridge.
Starting from compound 4a, the deep hydrophobic cavity occupied by the chemical feature HYDꢀ
2 was explored by moving the fluorine atom from meta to ortho (4b) and para (4c) positions, or by
designing the unsubstitued Nꢀ1 phenyl derivative 4d. The fluorine atom was also replaced with
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strong electron withdrawing (i.e. NO2, 4e-g) or electron donating (i.e. NH2, 4h-j) substituents.
Finally, the methanesuphonamide group was substituted by an amino group obtaining compound
5a. This change was aimed at assessing the impact of the mesyl moiety in this series of compounds.
Finally, the direct optimized version of compound 3a, where the methyl fragment of the Cꢀ3
carboxylic group was substituted by the paraꢀmethanesuphonamidephenyl moiety, was also
planned (6a).
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Before starting with the chemical synthesis of the new pyrazolobenzothiazineꢀbased compounds,
we sought to validate our hypothesis by performing automated docking studies of the designed
derivatives using the computational protocol described above. The docking results predicted that
4a-j, 5a and 6a may potentially interact with the NS5B residues of PSI in a fashion similar to
known NNIs, as discussed below. For instance, the proposed binding mode of
pyrazolobenzothiazine derivative 4a (XP GScore = ꢀ7.4 kcal/mol) is shown in Figure 4, where a
schematic representation of the predicted NS5Bꢀcompound 4a interactions is also illustrated.
Figure 4. A) Schematic representation of the predicted interactions between compounds 4a and the
NS5B residues (color legend as in Figure 2). B) Docked conformation of compound 4a (green)
together with the Xꢀray conformation of BTZ derivative 2 (violet). Water molecules are displayed
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in cyan, while hydrogen bonds are represented as yellow dashed lines. Both figures illustrated
NS5B residues lying within a distance of 4 Å from the docked compound.
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Beside the ligandꢀNS5B interactions already highlighted for the parent compound 3a (Figure 3),
derivative 4a was able to establish the additional desired π−π (EFT) and threeꢀpoint (HBDꢀHBA)
interactions through its paraꢀmethanesuphonamide phenyl group, in agreement with the SBDD.
Further, since NS5B harbors multiple allosteric sites, we next investigated the binding mode of the
rationally designed pyrazolobenzothiazine derivatives at the other pockets. For this purpose,
molecular docking studies at PSII (PDB ID 3FQL),20 PSIII (PDB ID 3LKH),21 TSI (PDB ID
2BRK)12b,22 and TSII (PDB ID 3FRZ)12b,23 were performed. At first, the ability of the docking setup
to reproduce the coꢀcrystallized pose of each inhibitor within the respective NS5B site was
validated. The pyrazolobenzothiazine derivatives were then docked into the allosteric pockets, and
the visual inspection of the results revealed their inability to properly occupy those binding sites.
Together, this data suggested that the newly designed compounds 4aꢀj, 5a and 6a may bind at
PSI, thus providing a rationale to synthesize and test these molecules as potential inhibitors of
NS5B polymerase and HCV replication.
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Synthesis of pyrazolobenzothiazine derivatives
The synthetic route employed for the preparation of targets compounds 4aꢀj, 5a and 6a is
depicted in Scheme 1. Starting from key intermediate 7,19 the pyrazolobenzothiazineꢀ3ꢀ
carboxymethyl esters 3a,19 8b-c, 8d,19 8eꢀg were prepared by condensation with the appropriate
commercially available phenylhydrazines hydrochloride, in MeOH at reflux. According to our
previous works19,24 this reaction led to the formation of the sole Nꢀ1 phenylpyrazolobenzothiazine
regioisomers. In fact, in all 1H NMR spectra of derivatives 3a, and 8bꢀg the presence of Hꢀ9 proton
shielded by the anisotropic effect of Nꢀ1 phenyl ring is a proof consistency of the assigned
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structure. In order to unambiguously demonstrate the reaction regioselectivity, a bidimensional
NOESY spectrum was performed for derivative 8e, as an exhaustive example. The results indicated
two diagnostic interactions: the first one was observed between the Hꢀ9 proton of
pyrazolobenzothiazine nucleus and the Hꢀ2' proton of the Nꢀ1 phenyl ring whereas the second one
occurred between the same Hꢀ9 proton and the Hꢀ4' proton (Figure 5). No interactions were found
between the Nꢀ1 phenyl ring and the Cꢀ3 methyl ester.
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The esters intermediates 3a,19 and 8bꢀg were hydrolyzed under basic conditions at reflux to give
the pyrazolobenzothiazineꢀ3ꢀcarboxylates 9aꢀc, 9d,19 and 9eꢀg. They were then converted in the
corresponding carbonyl chloride, under reflux of SOCl2, and immediately reacted with Nꢀ(4ꢀ
aminophenyl)methanesulfonamide,25 using Et3N as scavenger, to afford the target amide derivatives
4aꢀg. On the other hand, nitro derivatives 4eꢀg were reduced to the corresponding amino targets 4hꢀ
j employing H2 flux and RaneyꢀNi as catalyst.
Using the same procedures, the carbonyl chloride of 9a was reacted with Nꢀ(4ꢀ
hydroxyphenyl)methanesulfonamide26 to give the target ester derivative 6a; intermediate 9a was
also converted into amino derivative 5a by reaction with 4ꢀnitroaniline followed by catalytic
reduction with H2/RaneyꢀNi.
NO2
H4'
H2'
N
H9
N
CO2Me
N
S
Me
O2
Figure 5. NOESY experiments for 8e showing two main interactions: Hꢀ9→Hꢀ2' and Hꢀ9→Hꢀ4'.
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Scheme 1a
4
5
6
R
7
8
OH
O
N
N
CO2Me
9
i
CO2Me
Me
10
11
12
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N
N
S
O2
S
O2
Me
7
3a, 8b-g
ii
R
R
N
F
N
N
N
N
N
iii
iii
O
O
CO2H
from 9a
HN
Me
HN
Me
N
N
N
S
Me
RI
S
S
O2
NHSO2Me
O2
O2
10a RI= NO2
9a-g
4a-g
4h-j
iv
from 4e-g iv
5a RI= NH2
iii
from 9a
F
3, 4, 8 and 9
R
a
b
c
d
e
f
g
h
i
3-F
2-F
4-F
H
3-NO2
2-NO2
4-NO2
3-NH2
2-NH2
4-NH2
N
N
O
O
N
S
O2
Me
NHSO2Me
6a
j
aReagents and conditions: (i) ArNHNH2 HCl, MeOH, reflux; (ii) 10% acq NaOH, MeOH, reflux;
(iii) a) SOCl2, reflux; b) ArNH2 or ArOH, Et3N, dry DMF, 40 °C; (iv) H2, RaneyꢀNi, DMF, rt, atm
pressure.
Biochemical and biological studies
The synthesized compounds 4aꢀj, 5a and 6a were evaluated by a combination of three
biochemical and biological methods to determine the binding affinity for NS5B polymerase, the
ability to inhibit the enzyme and the antiꢀHCV activity. The SPR biosensor analysis is a direct
binding method that provides information on the interaction between the compounds and the
enzyme; the affinity is an important SAR characteristic, but does not provide the functional
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information that is obtained from the enzyme activityꢀbased inhibition or replicon assays. The latter
is the most complex assay of the three, providing a measure not only of effects on polymerase
activity but other features related to viral replication. Ideally, a good compound shows a low KD,
IC50 and EC50 values. In recent years, ligand efficiency indices have been shown to be useful tools
in reviewing the potency data, where potency is either a measure of binding (KD) or biological
activity (Ki, IC50 or EC50). Ligand efficiency (LE)27 refers to the potency of a compound averaged
by its nonꢀhydrogen atom count and is used to try to avoid unnecessary increases in molecular
weight as a lever to increase potency. Although LE is probably the most widely used and intuitive
metric, it does not take into account lipophilicity. Considering the crucial role of lipophilicity in
drug discovery, the concept of ligandꢀlipophilic efficiency (LLE or LipE) has recently become a
popular measure linking potency and lipophilicity in an attempt to estimate druglikeness.28 For a
given compound, LLE is defined as pPotency – logP or logD, where pPotency is –log(Potency),
logP is the octanol/water partition coefficient and logD is the distribution coefficient. LogP value of
a substance is most appropriate for neutral (unꢀionized) compounds, while logD (the pHꢀdependent
lipophilicity descriptor) should be used when working with ionizable compounds. Since our
pyrazolobenzothiazines did not contain a functionality likely to be ionized at the pH of the used
assays, logP value was chosen as the relevant parameter. In this study, LLE values were calculated
to support the SAR understanding and to evaluate the druglikeness of the compounds by employing
logP calculated using the program QikProp (see also Supporting Information Table 1S).29
These data are reported in Table 1.
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SPR interaction analysis.
The interaction between the synthesized compounds and HCVꢀNS5B genotype 1b was assayed
using a SPRꢀbased biosensor as reported in Experimental Section.
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No compounds were found to affect the immobilized enzyme irreversibly or linger in the
instrument flow system to potentially interfere with subsequent measurements. An appropriate level
of structural integrity and functionality of the detection surface was maintained throughout the
experiments, as determined by injections of a positive control substance (filibuvir, PF00868554).23
The apparent affinities were determined for all compounds except two, i.e. 4c and 4g. The results
from derivative 4c suggested that this compound might interact with the enzyme, but possible
solubility issue prevented a proper determination of the affinity. On the other hand, there was no
detectable interaction between compound 4g and the target.
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Starting from pyrazolobenzothiazine 3a, the introduction of the paraꢀmethanesuphonamidephenyl
moiety at the Cꢀ3 position permitted an increased affinity to be observed for almost all the
synthesized compounds (KD values ranged from 14 to 165 ꢀM and corresponding LLE parameters
from 0.99 to 3.41). In particular, the analogues bearing an amide or ester linkage (compounds 4a
and 6a, respectively) exhibited increased NS5B affinity when compared to the parent compound 3a.
Deletion of the methansulfonyl fragment negatively affected the binding affinity, as highlighted by
comparing both KD and LLE values of derivative 4a (KD = 75 ꢀM, LLE = 1.85) and 5a (KD = 161
ꢀM and LLE = 0.99). Both these observations were in agreement with the rationale of our design.
NS5B affinity was also influenced by the position of the substituent on the Nꢀ1 phenyl ring. Indeed,
SPR data combined with lipophilic efficiency index analysis revealed that in the fluoro and nitro
subsets, the meta substitution (4a and 4e) was preferred over the ortho substitution (4b and 4f). In
particular, metaꢀnitro Nꢀ1ꢀphenyl derivative 4e (KD = 14 ꢁM and LLE = 3.41) displayed both the
best affinity and LLE value within this pyrazolobenzothiazine series.
NS5B polymerase assay.
The antiꢀNS5B RdRp activity of all the synthesized compounds was evaluated by the standard
primerꢀdependent elongation reaction employing poly rA/U12 templateꢀprimer and recombinant
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HCV NS5BC∆21 according to previously described procedures.30 Compound 131 was included as a
reference NS5B inhibitor and yielded an IC50 value of 0.085 ± 0.006 ꢂM, consistent with previously
reported data.32 All compounds were first screened at 50 ꢂM concentration to identify NS5B
inhibitor candidates exhibiting ≥50% inhibition of NS5B RdRp activity at this concentration. This
investigation led to the identification of eight compounds (4aꢀ4d, 4f, 4g, 4i and 4j) satisfying this
criterion, while four compounds (4e, 4h, 5a and 6a) exhibited, at the same concentration, ~21ꢀ46%
inhibition of NS5B RdRp activity. The eight selected compounds were further screened for their
NS5B inhibition potency and yielded IC50 values ranging from 3.9 ꢂM to 40 ꢂM, with
corresponding LLE values ranging from 2.4 to 3.8 (Table 1). The enzymatic data, in agreement with
the SPR analysis, thus highlighted that the rational introduction of the paraꢀ
methanesuphonamidephenyl moiety at the Cꢀ3 position was a valid strategy to increase the
inhibitory activity of the starting compound 3a; which did not reach 50% inhibition but only
showed 11% inhibition at 50 ꢀM. Furthermore, the enzyme inhibition results, similarly to that
observed by the SPR data analysis, clearly showed how the deletion of the methansulfonyl resulted
detrimental in this series of pyrazolonbenzothiazines (compare 4a with an IC50 value of 21 ꢀM
versus 5a with 31.5% inhibition at 50 ꢀM).
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Looking at both the NS5B inhibition data and LLE values, further considerations can be
proposed. The position of the substituent on the Nꢀ1 phenyl ring influenced the antiꢀNS5B activity.
In particular, in this series the substitution at the para position (4c, 4g and 4j) was preferred over
the ortho (4b, 4f and 4i) and meta (4a, 4e and 4h) positions. For instance, paraꢀfluoro Nꢀ1ꢀphenyl
pyrazolobenzothiazine 4c displayed the most potent activity in this series with an IC50 of 3.9 ꢂM
and LLE of 3.11, while its ortho (4b) and meta (4a) analogues showed IC50 of 14.2 ꢂM and 21 ꢂM,
and LLE of 2.66 and 2.40, respectively. These data clearly suggest that, although our compounds
exhibited lower antiꢀNS5B potency compared to the representative BTDZ 1, the
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pyrazolonbenzothiazine nucleus merits further investigation representing a new chemical scaffold
unrelated to any known class of NS5B NNIs.
6
7
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9
Anti-HCV activity.
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The selective antiviral activity of the target compounds (4aꢀj, 5a, and 6a) was evaluated in the
HCV genotype 1b subgenomic replicon system (Huh 5ꢀ2). From the doseꢀresponse curves, the
concentration of the compound that inhibits virus replication by 50% (i.e., EC50) as well as the
concentration of compound that reduces host cell metabolism by 50% (i.e., CC50) was derived.
These values in turn allowed us to calculate the selectivity index (i.e., SI = CC50/EC50), a measure
for the therapeutic potential of the compound in the assay system (Table 1).
A preliminary analysis of the data obtained from the replicon assay apparently showed that, with
the exception of derivatives 4g, 4h, and 4j, the pyrazolobenzothiazines herein reported did not
possess any significant cytotoxicity. Furthermore, the nontoxic compounds were able to inhibit
HCV replication in a micromolar range.
However, even though an EC50 may be obtained from the doseꢀresponse curve, following
interpretation of the doseꢀresponse curve, compounds were only considered as selective inhibitors
in the replicon assay when significant inhibition (>70%) of virus RNA replication was observed at
concentrations that did not exert an adverse effect on host cell metabolism. The antiviral response
observed for the compounds that did not match these criteria most likely had to be attributed to a
pleiotropic or aspecific effect of the compound on the host cell metabolism. In addition, the cell and
monolayer morphology of the selected compounds was checked microscopically, as the microtiter
plate readout data did not necessarily allow to identify minor changes that can be detected visually.
Even though the doseꢀresponse curves (data not shown) of several compounds (derivatives 4aꢀc and
5a) matched the hit selection criteria, following microscopic inspection, it was apparent that the
compounds induced changes at the level of cell and monolayer morphology. Even though these
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observations pointed towards an aspecific or pleiotropic effect being responsible for the observed
antiꢀHCV activity, it however cannot be excluded from this assay that the compounds in addition
did not inhibit HCV replication as well.
9
Taking into account the above general considerations, some preliminary SAR can be proposed. A
good activity coupled with no apparent cytotoxic effects was maintained within the Nꢀ1ꢀ
fluorophenyl subset, with compound 4a being the most interesting derivative (EC50 = 7.5 ꢁM) since
notably it was also able to reach 90% inhibition of HCV replication at nonꢀtoxic concentration
(EC90 = 42 ꢁM). To better evaluate compound 4a as true antiꢀHCV agent, it was validated at the
level of viral RNA replication in the Huh 9ꢀ13 replicon system by means of realꢀtime quantitative
RTꢀPCR. In this assay, derivative 4a showed a selective antiviral effect (EC50 = 3.6 ꢁM; EC90 =
25.6 ꢁM) without any cytostatic effect (CC50 > 180 ꢁM, SI > 50). A doseꢀresponse curve matching
the hit selection criteria was obtained, suggesting selective antiꢀHCV activity. However, based on
the microscopic observations, no final conclusion can be drawn.
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Highlights from the LLE analysis
The introduction of the concept of LE has been useful as it provides a measure of the binding
energy of the ligand per atom. This has been extended to also account for lipophilic contributions,
in the LLE metric. The magnitude of LLE is thought to be correlated with druggability and it can be
used to select and prioritize compounds with high affinity per atom and low lipophilicity. The new
pyrazolobenzothiazine derivatives were therefore evaluated using this metric. The LLE calculated
from KD and IC50 values was useful for ranking and identified compounds 4e (3.4) and 4g (3.80) as
the ones with the highest LLE, respectively (see also Supporting Information Figure S1 and S2).
These results can be compared to the calculated mean LLE values for a large and diverse data set
representing different stages of drug discovery recently published by Tarcsay et al.33 Although LLE
greater than 5 is usually considered optimal for a promising drug candidate, LLE values of 2.5 were
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found for both fragment and HTS hits, while fragment and HTS leads showed higher values of 3.9
and 3.6, respectively. The study further reported LLE values greater than 5 for compounds that
entered phase II trials and marketed drugs. In our work, several synthesized derivatives had LLE
values greater than 2.5 and low logP values (see Supporting Information Figure S1 and S2), thereby
suggesting that the pyrazolobenzothiazine chemotype is noteworthy of further investigation.
Another surprising finding was that, although 4a was the antiꢀHCV hit compound validated by the
strict criteria of the replicon system, curiously this pyrazolobenzothiazine did not rank among the
derivatives which exhibited the highest LLE values based on calculations using either KD or IC50. In
contrast, both compound 4e and 4g that showed the highest LLE values were not detected as
genuine antiꢀHCV agents in the replicon system. This may be an effect of the higher complexity of
the replicon assay compared to the SPR and enzyme inhibition assays. It is influenced by the
pharmacokinetic properties of a compound (e.g. the ability to enter the cell) and its capability to
interact also with a multitude of other cell components. Even though the concept of LLE was
originally introduced specifically using pIC50 and pKi presumably to avoid the complication due to
the use of cell system, it was later expanded to include pEC50.34 LLE for 4a using its EC50 data
determined by RTꢀPCR assay was 3.21, thus ranging between the LLE values for hit compounds
(mean value of 2.5) and lead compounds (mean values of 3.6ꢀ3.9).
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Additional pyrazolobenzothiazine analogues will have to be explored to identify the features that
are associated with high potency in the replicon system and that give higher LLE.
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Table 1. Biological results obtained for derivatives 3a, 4aꢀj, 5a, and 6a.
9
R
N
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N
O
RI
N
S
Me
O2
NS5B
functional
assay
SPR on
NS5B
Replicon assay on Huh 5ꢀ2 cells
LLE
LLE
cpds
R
R'
(KD)g
(IC50)h
KD
IC50
EC50
EC90
CC50
SIf
2
(ꢁM)a
(ꢁM)b
(ꢁM)c
(ꢁM)d
(ꢁM)e
NDi,j
172 ± 10
7.5 ± 2.5
6.4 ± 0.2
23 ± 13
NDi
42 ± 2
NDi
>323
>370
>231
>231
ꢀ
ꢀ
OMe
3a
4a
3ꢀF
3ꢀF
2ꢀF
4ꢀF
>300
75±12
162±63
NDi
HN
NHSO2Me
NHSO2Me
NHSO2Me
NHSO2Me
NHSO2Me
NHSO2Me
NHSO2Me
NHSO2Me
NHSO2Me
NHSO2Me
NH2
21.0±2.8
14.2±0.6
3.9±0.3
>49
1.85
1.61
ꢀ
2.40
2.66
3.11
4b
4c
4d
4e
4f
HN
HN
HN
HN
HN
HN
HN
HN
HN
>36
>10
>17
≥1
NDi
H
53±6
14±6
19.8±3.6
NDi,j
14 ± 1
>220
NDi
>220
NDi
NDi
NDi
NDi
NDi
NDi
NDi
>239
>220
>220
55 ± 8
20 ± 6
>232
25 ± 5
>270
>185
2.18
3.41
2.84
ꢀ
2.61
ꢀ
3ꢀNO2
2ꢀNO2
4ꢀNO2
3ꢀNH2
2ꢀNH2
4ꢀNH2
3ꢀF
35±3
20±0.4
7.7±1.3
NDi,j
17.8 ± 0.1
7.9 ± 1.0
9.2 ± 0.9
26 ± 2
>12.4
7.0
3.09
3.80
ꢀ
4g
4h
4i
NDi
163±8
106±42
165±16
161±103
63±8
2.2
2.52
2.66
2.53
0.99
1.87
39.9±1.7
18.2±2.1
NDi,j
>9
3.08
3.48
ꢀ
4j
12 ± 1
2
5a
6a
HN
19 ± 2
>14
>2
3ꢀF
O
NHSO2Me
NDi,j
88 ± 10
ꢀ
a
KD= Dissociation equilibrium constant. At this concentration 50% of all binding site are occupied. Values as averages and standard deviation (SD) from
b
duplicate or triplicate runs. IC50 = concentration of compound that inhibits 50% enzyme activity in vitro. The reported values represent the means ± SD of
data derived from two independent experiments performed in duplicate. c EC50 = the effective concentration required to inhibit virus induced cytopathic effect
by 50%. The reported values represent the means ± SD of data derived at least from three independent experiments. d EC90 = the effective concentration
required to inhibit virus induced cytopathic effect by 90%. The reported value represents the means ± SD of data derived at least from three independent
e
experiments. CC50 = is the concentration required to reduce the bioreduction of MTS (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀcarboxymethoxyꢀphenyl)ꢀ2ꢀ(4ꢀ
sulfophenyl)ꢀ2Hꢀtetrazolium) into formazan by 50%. The reported value represents the means ± SD of data derived at least from three independent
experiments. f SI = selectivity index (ratio of CC50 to EC50). g LLE(KD) = pKD – predicted logP. h LLE(IC50) = pIC50 – predicted logP. i ND = not determined. j
Compounds that did not reach the 50% inhibition and show a maximum % inhibition @ 50 ꢁM: 3a 10.6%, 4e 33.7%, 4h 45.8%, 6a 21.4%, 5a 31.5%.
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Conclusions
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Although a wide variety of compounds have been reported as NS5B inhibitors, drugs active
against this enzyme have not yet been approved by the FDA. Thus, there is still a compelling need
to identify new antiꢀHCV chemotypes acting on NS5B polymerase. In this context, structureꢀbased
virtual screening of our inꢀhouse library led to the identification of the pyrazolobenzothiazine 3a as
a suitable starting point for the rational design of new potential NS5B inhibitors. A simple synthetic
route for novel pyrazolobenzothiazines bearing a paraꢀmethanesuphonamidephenyl moiety at Cꢀ3
position has been developed, leading to the preparation of twelve derivatives. Biochemical and
biological testing confirmed that our strategy was successful in the search for a new antiꢀHCV
chemotype most likely targeting the NS5B polymerase. In summary, the results showed that 4a
represented the best hit within this first series of pyrazolobenzothiazines, having relatively good
affinity and inhibitory effects in both enzymatic and replicon assays. Further investigations are
warranted and are currently in progress to develop pyrazolobenzothiazine derivatives with
improved activity and to better elucidate their antiviral mechanism of action.
Experimental section
Molecular Modeling
The crystal structure of NS5B RdRP complexed with inhibitor 2 (PDB ID 3G86) was retrieved
from the RCSB Protein Data Bank and used for our modeling studies on PSI. Water molecules,
except those involved in mediating hydrogen bonds between the ligands and the protein were
deleted. Schrödinger Protein Preparation Wizard,35 a workflow designed to ensure chemical
correctness and to optimize a protein structure for further analysis, was then used to obtain a
satisfactory starting structure. In particular, hydrogen atoms were added, and bond orders and
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charges were assigned; the orientation of hydroxyl groups on Ser, Thr and Tyr, the side chains of
Asn and Gln residues, and the protonation state of His residues were optimized. Steric clashes were
relieved by performing a small number of minimization steps, not intended to minimize the system
completely. In our study, the minimization (OPLS 2500 force field) was stopped when the RMSD
of the nonꢀhydrogen atoms reached 0.30 Å.
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Docking studies were performed using the software Glide version 5.6.17 The prepared protein
structure was used to generate the receptor grid, which was centered on the crystallographic
positions of PSIꢀNNI 2.
Prior to docking experiments, the inꢀhouse library and the designed compounds (4aꢀj, 5a and 6a)
were built using the Schrödinger Maestro interface36 and then submitted to the LigPrep utility,16
which rapidly produces low energy 3D structures taking into account ionization states, tautomers,
sterochemistries, and ring conformations at the desired pH. For our study, a pH range of 6ꢀ8 was
set.
The PSIꢀNNI 2 was instead extracted from the complex with NS5B, and its crystallographic
coordinates were transformed in order to avoid biasing the docking toward the known NS5Bꢀ bound
conformation. After modeling the ligand enolic moiety as anion due to its ionization at
physiological pH, the compound was submitted to Polak–Ribiere conjugate gradient minimization
[0.0005 kJ/(Å mol) convergence].
All compounds were flexibly docked in a stepwise manner with Glide SP (Standard Precision)
and XP (Extra Precision) as scoring functions. Docking experiments were performed using a 0.80
factor to scale the VdW radii of the nonpolar ligand atoms with partial atomic charge less than 0.15
(absolute value). Poses were discarded as duplicates when rms deviation in the ligandꢀall atoms was
less than 1.5 Å and maximum atomic displacement was less than 2.0 Å. Three bound conformations
per ligand were retained from SP docking, and these were refined, rescored and minimized using
GlideXP. The best XP scoring bound conformation for each ligand was retained. Docking
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experiments at PSII (PDB ID 3FQL),20 PSIII (PDB ID 3LKH),12b,21 TSI (PDB ID 2BRK)12b,22 and
TSII (PDB ID 3FRZ)23 were performed using the same protocol described for PSI.
Figure 2, 3A and 4A were generated using the Ligand Interaction Diagram tool of Maestro GUI,36
whereas Figure 3B and 4B were prepared using the software PyMOL.37
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LogP values of the pyrazolobenzothiazines were computed by using Schrödinger QikPro
program29 (descriptor QPlogPo/w), and the LLE calculations were performed by using Excel
spreadsheet.
Chemistry
All starting materials were commercially available, unless otherwise indicated. Reagents and
solvents were purchased from common commercial suppliers and were used as such. Organic
solutions were dried over anhydrous Na2SO4 and concentrated with a rotary evaporator at low
pressure. All reactions were routinely checked by thinꢀlayer chromatography (TLC) on silica gel
60F254 (Merck) and visualized by using UV or iodine. Column chromatography separations were
carried out on Merck silica gel 60 (mesh 70ꢀ230), flash chromatography on Merck silica gel 60
(mesh 230ꢀ400). Melting points were determined in capillary tubes (Büchi Electrotermal model
1
9100) and are uncorrected. Yields were of purified products and were not optimized. H NMR
spectra were recorded at 200 or 400 MHz (Bruker Avance DRXꢀ200 or 400, respectively) while 13C
1
NMR spectra were recorded at 100 MHz (Bruker Avance DRXꢀ400) as well as 2D H NMR
NOESY run in phase sensitive mode. Chemical shifts are given in ppm (δ) relative to TMS. Spectra
were acquired at 298 K. Data processing was performed with standard Bruker software XwinNMR
and the spectral data are consistent with the assigned structures. Elemental analyses were performed
on a Fisons elemental analyzer, model EA1108CHN, and data for C, H, and N are within 0.4% of
the theoretical values.
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General procedure for condensation of key intermediate 7 with phenylhydrazines
hydrochloride. Method A. To a refluxing solution of key intermediate 719 (1 mmol) in MeOH (20
mL), the appropriate hydrazine hydrochloride (1.2 mmol), dissolved in hot MeOH (2 mL), was
added at once. The reaction was maintained at reflux for 3ꢀ15 h, then concentrated under reduced
pressure to half volume and cooled to room temperature The precipitated formed was filtered under
vacuum to give the pyrazolobenzothiazineꢀ3ꢀcarboxymethyl esters 8b-g, pure by TLC
(CHCl3/MeOH 98:2).
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Methyl
1-(2-fluorophenyl)-4-methyl-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazine-3-
carboxylate 5,5-dioxide (8b). Following the general procedure method A and using the 2ꢀ
(fluorophenyl)hydrazine hydrochloride, compound 8b was obtained in 71% yield (reaction time 5
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h) as yellowish crystalline solid: mp 174ꢀ175 °C. H NMR (200 MHz, acetoneꢀd6): δ 3.30 (s, 3H,
NCH3), 4.00 (s, 3H, OCH3), 7.10 (d, J = 7.8 Hz, 1H, Hꢀ9), 7.50ꢀ7.90 (m, 6H, Hꢀ7, Hꢀ8, Hꢀ3', Hꢀ4',
Hꢀ5' and Hꢀ6'), 8.05 (dd, J = 1.5 and 7.6 Hz, 1H, Hꢀ6).
Methyl
1-(4-fluorophenyl)-4-methyl-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazine-3-
carboxylate 5,5-dioxide (8c). Following the general procedure method A and using the 4ꢀ
(fluorophenyl)hydrazine hydrochloride, compound 8c was obtained in 86% yield (reaction time 10
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h) as white crystalline solid: mp 215ꢀ216 °C. H NMR (200 MHz, CDCl3): δ 3.35 (s, 3H, NCH3),
4.05 (s, 3H, OCH3), 7.00 (dd, J = 0.7 and 7.8 Hz, 1H, Hꢀ9), 7.25ꢀ7.35 (m, 2H, Hꢀ3' and Hꢀ5'), 7.45ꢀ
7.70 (m, 4H, Hꢀ7, Hꢀ8, Hꢀ2' and Hꢀ6'), 8.10 (dd, J = 1.4 and 7.8 Hz, 1H, Hꢀ6).
Methyl
4-methyl-1-(3-nitrophenyl)-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazine-3-
carboxylate 5,5-dioxide (8e). Following the general procedure method A and using the 3ꢀ
(nitrophenyl)hydrazine hydrochloride, compound 8e was obtained in 90% yield (reaction time 12 h)
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as yellow woolly solid: mp 283ꢀ284 °C. H NMR (200 MHz, DMSOꢀd6): δ 3.20 (s, 3H, NCH3),
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4.00 (s, 3H, OCH3), 7.15 (d, J = 7.6 Hz, 1H, Hꢀ9), 7.65 (dt, J = 1.5 and 7.9 Hz, 1H, Hꢀ8), 7.80 (dt, J
= 1.5 and 7.9 Hz, 1H, Hꢀ7), 7.90 (t, J = 8.1 Hz, 1H, Hꢀ5'), 8.00ꢀ8.10 (m, 2H, H4' and Hꢀ6), 8.45 (t, J
= 1.9 Hz, 1H, Hꢀ2') 8.55 (m, 1H, Hꢀ6').
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Methyl
4-methyl-1-(2-nitrophenyl)-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazine-3-
carboxylate 5,5-dioxide (8f). Following the general procedure method A and using the 2ꢀ
(nitrophenyl)hydrazine hydrochloride (reaction time 15 h); after flash column chromatography
purification (CHCl3/MeOH 99:1), compound 8f was obtained in 15% yield as yellow woolly solid:
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mp 277ꢀ278 °C. H NMR (400 MHz, DMSOꢀd6): δ 3.10 (s, 1H, NCH3), 3.75 (s, 1H, OCH3), 6.90
(d, J = 7.8 Hz, 1H, Hꢀ9), 7.60 (dt, J = 1.3 and 7.8 Hz, 1H, Hꢀ8), 7.70 (dt, J = 1.0 and 7.8 Hz, 1H, Hꢀ
7), 7.80ꢀ7.85 (m, 1H, Hꢀ4'), 7.90ꢀ8.00 (m, 3H, Hꢀ6, Hꢀ5' and Hꢀ6'), 8.30ꢀ8.35 (m, 1H, Hꢀ3').
Methyl
4-methyl-1-(4-nitrophenyl)-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazine-3-
carboxylate 5,5-dioxide (8g). Following the general procedure method A and using the 4ꢀ
(nitrophenyl)hydrazine hydrochloride, compound 8g was obtained in 40% yield (reaction time 6 h)
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as yellow solid: mp 275ꢀ276 °C. H NMR (400 MHz, DMSOꢀd6): δ 3.10 (s, 3H, NCH3), 3.90 (s,
3H, OCH3), 7.10 (d, J = 7.8 Hz, 1H, Hꢀ9), 7.65 (dt, J = 1.3 and 7.8 Hz, 1H, Hꢀ8), 7.70 (dt, J = 1.1
and 7.8 Hz, 1H, Hꢀ7), 7.80ꢀ7.85 (m, 2H, Hꢀ2' and Hꢀ6'), 8.00 (dd, J = 1.1 and 7.8 Hz, 1H, Hꢀ6),
8.40ꢀ8.45 (m, 2H, Hꢀ3' and Hꢀ5').
General procedure of basic hydrolysis. Method B. A stirred mixture of appropriate
pyrazolobenzothiazine methyl esters 3a,19 8bꢀc, 8d,19 and 8e-g (1 mmol) in acq. 10% NaOH (6 mL)
and MeOH (6 mL) was refluxed for 1 h and then concentrated to oneꢀthird of the volume under
reduced pressure. The mixture was poured into ice/water and acidified with 2N HCl (pH = 3). The
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precipitated formed was then filtered under vacuum, washed with Et2O and dried to give the
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pyrazolobenzothiazineꢀ3ꢀcarboxylic acids 9aꢀg used as it in the next reaction step.
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1-(3-Fluorophenyl)-4-methyl-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazine-3-carboxylic
acid 5,5-dioxide (9a). Following the general procedure method B, compound 9a was obtained from
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3a,19 in 88% yield as white solid: mp 243ꢀ245 °C. H NMR (200 MHz, acetoneꢀd6): δ 3.30 (s, 3H,
NCH3), 7.20 (dd, J = 1.2 and 7.7 Hz, 1H, Hꢀ9), 7.45ꢀ7.60 (m, 3H, Hꢀ7, Hꢀ8 and Hꢀ6'), 7.65ꢀ7.85 (m,
3H, Hꢀ2', Hꢀ4' and Hꢀ5'), 8.05 (dd, J = 1.4 and 7.7 Hz, 1H, Hꢀ6).
1-(2-Fluorophenyl)-4-methyl-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazine-3-carboxylic
acid 5,5-dioxide (9b). Following the general procedure method B, compound 9b was obtained from
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8b, in 89% yield as white solid: mp 224ꢀ225 °C. H NMR (200 MHz, acetoneꢀd6): δ 3.30 (s, 3H,
NCH3), 7.10 (d, J = 7.3 Hz, 1H, Hꢀ9), 7.45ꢀ7.95 (m, 6H, Hꢀ7, Hꢀ8, Hꢀ3', Hꢀ4', Hꢀ5' and Hꢀ6'), 8.05
(dd, J = 1.4 and 7.7 Hz, 1H, Hꢀ6).
1-(4-Fluorophenyl)-4-methyl-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazine-3-carboxylic
acid 5,5-dioxide (9c). Following the general procedure method B, compound 9c was obtained from
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8c, in 88% yield as white solid: mp 248ꢀ250 °C. H NMR (200 MHz, acetoneꢀd6): δ 3.30 (s, 3H,
NCH3), 7.20 (dd, J = 1.0 and 7.5 Hz, 1H, Hꢀ9), 7.45ꢀ7.55 (m, 2H, Hꢀ7 and Hꢀ8), 7.60ꢀ7.80 (m, 4H,
Hꢀ2', Hꢀ3', Hꢀ5' and Hꢀ6'), 8.05 (dd, J = 1.4 and 7.6 Hz, 1H, Hꢀ6).
4-Methyl-1-(3-nitrophenyl)-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazine-3-carboxylic acid
5,5-dioxide (9e). Following the general procedure method B, compound 9e was obtained from 8e,
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in 99% yield as yellow solid: mp 184ꢀ185 °C. H NMR (200 MHz, DMSOꢀd6): δ 3.05 (s, 3H,
NCH3), 7.10 (d, J = 7.8 Hz, 1H, Hꢀ9), 7.65 (dt, J = 1.2 and 7.8 Hz, 1H, Hꢀ8), 7.75 (dt, J = 1.2 and
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7.8 Hz, 1H, Hꢀ7), 7.90 (t, J = 8.1 Hz, 1H, Hꢀ5'), 8.00ꢀ8.10 (m, 2H, Hꢀ6 and Hꢀ4'), 8.40 (t, J = 2.0
Hz, 1H, Hꢀ2'), 8.50 (m, 1H, Hꢀ6'), 13.80 (bs, 1H, CO2H). NMR NOESY spectra showed two
relevant NOE crossꢀpeaks: Hꢀ9→Hꢀ2'; Hꢀ9→Hꢀ4'.
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4-Methyl-1-(2-nitrophenyl)-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazine-3-carboxylic acid
5,5-dioxide (9f). Following the general procedure method B, compound 9f was obtained from 8f, in
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60% yield as yellow solid: mp 220ꢀ221 °C. H NMR (400 MHz, DMSOꢀd6): δ 3.05 (s, 3H, NCH3),
6.85 (d, J = 7.5 Hz, 1H, Hꢀ9), 7.60 (t, J = 7.5 Hz, 1H, Hꢀ8), 7.70 (t, J = 7.5 Hz, 1H, Hꢀ7), 7.80 (dd,
J = 2.0 and 9.0 Hz, 1H, Hꢀ6'), 7.85ꢀ8.00 (m, 3H, Hꢀ6, Hꢀ4' and Hꢀ5'), 8.30 (dd, J = 2.1 and 7.5 Hz,
1H, Hꢀ3').
4-Methyl-1-(4-nitrophenyl)-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazine-3-carboxylic acid
5,5-dioxide (9g). Following the general procedure method B, compound 9g was obtained from 8g,
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in 98% yield as yellow solid: mp 226ꢀ228°C. H NMR (200 MHz, DMSOꢀd6): δ 3.05 (s, 3H,
NCH3), 7.15 (dd, J = 1.0 and 6.7 Hz, 1H, Hꢀ9), 7.65ꢀ7.80 (m, 2H, Hꢀ7 and Hꢀ8), 7.85ꢀ7.95 (m, 2H,
Hꢀ2' and Hꢀ6'), 8.05 (dd, J = 1.4 and 6.9 Hz, 1H, Hꢀ6), 8.45ꢀ8.55 (m, 2H, Hꢀ3' and Hꢀ5').
General procedure for amidation reaction with N-(4-aminophenyl)methanesulfonamide.
Method C. A mixture of intermediates 9aꢀg (1 mmol) in an excess of SOCl2 was refluxed under
stirring for 1 h. The solvent was distilled under reduced pressure and the residues was washed three
time with dry benzene. The corresponding pyrazolobenzothiazineꢀ3ꢀcarbonyl chloride was
immediately dissolved in dry DMF (2 mL) and added dropwise, under nitrogen flux, to a solution of
Nꢀ(4ꢀaminophenyl)methanesulfonamide25 (2 mmol) and Et3N (2 mmol) in dry DMF (7 mL). The
mixture was heated at 40 °C for 24 h. The solvent was then concentrated to half volume and the
mixture was poured into iceꢀwater, acidified with 2N HCl (pH = 3) and the precipitated formed was
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filtered under vacuum. The solid obtained was crystallized by EtOH/DMF (2:1) to give the target
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pyrazolobenzothiazineꢀ3ꢀcarboxyamides 4aꢀg.
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dihydropyrazolo[4,3-c][1,2]benzothiazine-3-carboxamide 5,5-dioxide (4a). Following the
general procedure method C, compound 4a was obtained from 9a, in 70% yield as white solid: mp
285ꢀ287 °C. 1H NMR (400 MHz, DMSOꢀd6): δ 3.00 (s, 3H, SO2CH3), 3.15 (s, 3H, NCH3), 7.05 (d,
J = 7.6 Hz, 1H, Hꢀ9), 7.20ꢀ7.25 (m, 2H, Hꢀ3" and Hꢀ5"), 7.40ꢀ7.50 (m, 1H, Hꢀ7), 7.55 (dt, J = 1.7
and 8.5 Hz, 1H, Hꢀ8), 7.65ꢀ7.80 (m, 6H, Hꢀ2', Hꢀ4', Hꢀ5', Hꢀ6', Hꢀ2" and Hꢀ6"), 8.00 (dd, J = 1.1
and 6.6 Hz, 1H, Hꢀ6), 9.60 (s, 1H, SO2NH), 10.40 (s, 1H, CONH). 13C NMR (100 MHz, DMSOꢀ
d6), δ 39.15, 39.36, 113.89 (d, JCꢀF = 20 Hz, Cꢀ2'), 117.50 (d, JCꢀF = 20 Hz, Cꢀ4'), 121.28, 122.13,
122.45 (d, JCꢀF = 2 Hz, Cꢀ6'), 123.35, 124.90, 125.16, 126.77, 130. 62, 130.74, 130.86, 132.29 (d,
JCꢀF = 10 Hz, Cꢀ5'), 133.47, 134.70, 135.03, 139.71, 140.52 (d, JCꢀF = 10 Hz, Cꢀ1'), 158.72, 162.73
(d, JCꢀF = 245 Hz, Cꢀ3'). Anal. Calcd (%) for C24H20FN5O5S2: C 53.13, H 3.72, N 12.93. Found C,
53.30; H, 3.60; N 12.88.
1-(2-Fluorophenyl)-4-methyl-N-{4-[(methylsulfonyl)amino]phenyl}-1,4-
dihydropyrazolo[4,3-c][1,2]benzothiazine-3-carboxamide 5,5-dioxide (4b). Following the
general procedure method C, compound 4b was obtained from 9b, in 40% yield as white solid: mp
264ꢀ266 °C. 1H NMR (400 MHz, DMSOꢀd6): δ 3.00 (s, 3H, SO2CH3), 3.30 (s, 3H, NCH3), 6.95 (d,
J = 7.7 Hz, 1H, Hꢀ9), 7.20ꢀ7.25 (m, 2H, Hꢀ3" and Hꢀ5"), 7.55ꢀ7.60 (m, 2H, Hꢀ7 and Hꢀ8), 7.65ꢀ7.80
(m, 5H, Hꢀ4', Hꢀ5', Hꢀ6', Hꢀ2" and Hꢀ6"), 7.95 (t, J = 7.8 Hz 1H, Hꢀ3'), 8.00 (d, J = 7.5 Hz, 1H, Hꢀ
6), 9.60 (s, 1H, SO2NH), 10.40 (s, 1H, CONH). 13C NMR (100 MHz, DMSOꢀd6): δ 39.26, 39.38,
117.62 (d, JCꢀF = 37 Hz, Cꢀ3'), 121.15, 122.08, 123.24, 123.30, 125.22, 126.03, 126.59 (bs, Cꢀ6'),
127.10 (d, JCꢀF = 20 Hz, Cꢀ1'), 129.61 (bs, Cꢀ5'), 130.74, 130.89, 131.97, 133.29 (d, JCꢀF = 10 Hz,
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Cꢀ4'), 133.71, 134.60, 134.95, 140.31, 156.34 (d, JCꢀF = 490 Hz, Cꢀ2'), 158.55. Anal. Calcd (%) for
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C24H20FN5O5S2: C, 53.13; H, 3.72; N, 12.93. Found C, 52.97; H, 3.80; N, 13.01.
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dihydropyrazolo[4,3-c][1,2]benzothiazine-3-carboxamide 5,5-dioxide (4c). Following the
general procedure method C, compound 4c was obtained from 9c, in 40% yield as white solid: mp
298ꢀ300 °C. 1H NMR (400 MHz, DMSOꢀd6): δ 3.00 (s, 3H, SO2CH3), 3.30 (s, 3H, NCH3), 7.00 (d,
J = 7.7 Hz, 1H, Hꢀ9), 7.20ꢀ7.25 (m, 2H, Hꢀ3" and Hꢀ5"), 7.45ꢀ7.55 (m, 2H, Hꢀ7 and Hꢀ8), 7.65ꢀ7.80
(m, 6H, Hꢀ2', Hꢀ3', Hꢀ5', Hꢀ6', Hꢀ2" and Hꢀ6"), 8.00 (d, J = 7.5 Hz, 1H, Hꢀ6), 9.60 (s, 1H, SO2NH),
10.40 (s, 1H, CONH). 13C NMR (100 MHz, DMSOꢀd6): δ 39.23, 39.34, 117.50 (d, JCꢀF = 20 Hz, Cꢀ
3' and Cꢀ5'), 121.28, 122.12, 123.45, 124.76, 125.19, 126.51, 128.82 (d, JCꢀF = 10 Hz, Cꢀ2' and Cꢀ
6'), 130.64, 130.76, 130.90, 133.49, 134.65, 135.09, 135.72 (d, JCꢀF = 3 Hz, Cꢀ1'), 139.55, 158.80,
162.89 (d, JCꢀF = 250 Hz, Cꢀ4'). Anal. Calcd (%) for C24H20FN5O5S2: C, 53.13; H, 3.72; N, 12.93.
Found C, 53.02; H, 3.86; N, 12.90.
N-{4-[(Methylsulfonyl)amino]phenyl}-1-phenyl-1,4-dihydropyrazolo[4,3-
c][1,2]benzothiazine-3-carboxamide 5,5-dioxide (4d). Following the general procedure method
C, compound 4d was obtained from 9d,19 in 70% yield as pale pink solid: mp 280ꢀ283 °C. 1H NMR
(400 MHz, DMSOꢀd6): δ 3.00 (s, 3H, SO2CH3), 3.30 (s, 3H, NCH3), 6.95 (d, J = 7.8 Hz, 1H, Hꢀ9),
7.20ꢀ7.25 (m, 2H, Hꢀ3" and Hꢀ5"), 7.65ꢀ7.80 (m, 9H, Hꢀ7, Hꢀ8, Hꢀ2', Hꢀ3', Hꢀ4', Hꢀ5', Hꢀ2" and Hꢀ
6"), 8.00 (d, J = 7.70 Hz, 1H, Hꢀ6), 9.60 (s, 1H, SO2NH), 10.40 (s, 1H, CONH). 13C NMR (100
MHz, DMSOꢀd6): δ 39.26, 39.38, 121.32, 122.15, 123.61, 124.75, 125.20, 126.36, 126.62, 130.57,
130.61, 130.89, 130.92, 133.39, 134.67, 135.14, 139.35, 139.54, 158.89. Anal. Calcd (%) for
C24H21N5O5S2: C, 55.05; H, 4.04; N, 13.38. Found C, 54.97; H, 3.89; N, 13.61.
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ACS Paragon Plus Environment