Privileged scaffolds in medicinal chemistry: Studies on pyrazolo[1,5-a]pyrimidines on sulfonamide containing Carbonic Anhydrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2021.128309

We report for the first time a small series of compounds endowed in vitro with inhibitory properties for the human (h) expressed Carbonic Anhydrase (CAs, E.C. 4.2.1.1) enzymes of physiological interest (i.e. I, II, VA, IX and XII) and bearing the pyrazolo[1,5-a]pyrimidine (PP) scaffold at the tail section. Among the series reported, 1a-3a, 7a, 8a, 1b and 2b resulted effective ligands and with good selectivities for the hCAs II, IX or XII. In consideration of the nearly matching K_I values of 7a for both the hCA II and IX (i.e. 26.4 and 23.0 nM respectively) we explored its binding mode within the CA IX mimic isoform by means of X-ray crystal experiments on the corresponding adduct.

Full text of DOI:10.1016/j.bmcl.2021.128309

Bioorg. Med. Chem. Lett. 49 (2021) 128309
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Privileged scaffolds in medicinal chemistry: Studies on pyrazolo[1,5-a]
pyrimidines on sulfonamide containing Carbonic Anhydrase inhibitors
,
*
Arzu Gumusa, Murat Bozdagb, Andrea Angelib, Thomas S. Peatc, Fabrizio Cartab
,
,
*
Claudiu T. Supuranb, Silvia Sellerib
a Balıkesir University, Faculty of Science and Art, Department of Chemistry, 10145 Balıkesir, Turkey
b NEUROFARBA Department, Pharmaceutical and Nutraceutical Section, University of Florence, Via Ugo Schiff 6, 50019 Sesto Fiorentino (FI), Italy
c CSIRO, 343 Royal Parade, Parkville, Victoria 3052, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords:
We report for the first time a small series of compounds endowed in vitro with inhibitory properties for the human
(h) expressed Carbonic Anhydrase (CAs, E.C. 4.2.1.1) enzymes of physiological interest (i.e. I, II, VA, IX and XII)
and bearing the pyrazolo[1,5-a]pyrimidine (PP) scaffold at the tail section. Among the series reported, 1a-3a, 7a,
8a, 1b and 2b resulted effective ligands and with good selectivities for the hCAs II, IX or XII. In consideration of
the nearly matching KI values of 7a for both the hCA II and IX (i.e. 26.4 and 23.0 nM respectively) we explored its
binding mode within the CA IX mimic isoform by means of X-ray crystal experiments on the corresponding
adduct.
Pyrazolopyrimidines
Carbonic Anhydrase inhibitors
Privilege scaffolds
X-ray crystallography
The pyrazolo[1,5-a]pyrimidine (PP) refers to a class of hetero bicy-
clic compounds containing a pyrazole fused to a pyrimidine ring which
has been identified in EGFR tyrosine kinase inhibitors,1 cyclin-
dependent kinase inhibitors including CDK2,1–3 CDK54 and CDK9,1–3
aurora-A kinase inhibitors,4 anti-hepatitis C drugs,5 DPP-4 inhibitors
endowed various properties.6 PPs are also present within currently used
drugs such as the GABAA subtype receptor ligand Zaleplon7 and its de-
rivatives Indiplon8,9 and Lorediplon10 which were under clinical
investigation for the treatment of insomnia. Ocinaplon is clinically used
as anxiolytic11 and Pyrazophos is commercially available as fungicide12
(Fig. 1).
interaction with diverse and valuable biological targets, they are prop-
erly classified in Medicinal Chemistry as privileged scaffolds.19 In light
of such a consideration along with our previous experience on the syn-
thesis of PPs,20–23 we thought to include this moiety as tail on sulfon-
amide based molecules potentially useful as inhibitors of the human (h)
expressed Carbonic Anhydrase (CA; EC 4.2.1.1) enzymes and thus to
explore the in vitro kinetic outcomes on selected isoforms. The chemical
strategy reported in this study relies on the synthesis of CAIs possessing
the benzenesulfonamide warhead connected to the 7-methylpyrazolo
[1,5-a]pyrimidine-6-carboxylate tails in the form of ethyl esters (i.e.
compounds 1a-9a in Scheme 1) or free acids (i.e. compounds (1b, 3b,
5b, 6b and 8b) upon hydrolysis of selected compounds as reported in
Scheme 1.
Additional valuable drugs containing the PP core are the tropomy-
osin kinase receptor (TrkA, TrkB and TrkC) inhibitor Larotrectinib,
approved by the FDA for the treatment of solid tumors by NTRK gene
fusion13,14 as well as its 2th generation derivative Selitrectinib currently
under clinical investigation for the same disease,15,16 and Dinaciclib
which was developed as CDK inhibitor for anticancer purposes.17 In
addition several studies have demonstrated the usefulness of [11C]DPA-
713 and [18F]DPA-714 PET in quantifying neuroinflammation in
different diseases, including multiple sclerosis (MS), Parkinson’s disease
(PD), and Alzheimer disease (AD) both in animal and human studies (Fig
2).18
The ethyl-7-methyl-3-nitropyrazolo[1,5-a]pyrimidine-6-carboxylate
1 was synthesized according to previously reported procedures24 and
then it was subjected to Fe reduction of the nitro group to give the
compound 2 which in turn was used as substrate for a variety of
chemical manipulations including: amide (1a, 2a and 9a), ureido (3a,
4a) and thioureido insertions (5a-8a) by means of different synthetic
approaches (Scheme 1). As for the cinnamic acid derivative 9a a
multistep synthetic procedure was applied to insert the primary sul-
fonamide within the cinnamic scaffold (i.e. compound 13). Among the
Since PPs are endowed with a multitude of activities by means of
7-methyl-3-nitropyrazolo[1,5-a]pyridine-6-carboxylate
esters,
* Corresponding authors.
E-mail addresses: fabrizio.carta@unifi.it (F. Carta), silvia.selleri@unifi.it (S. Selleri).
https://doi.org/10.1016/j.bmcl.2021.128309
Received 24 May 2021; Received in revised form 19 July 2021; Accepted 3 August 2021
Available online 6 August 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

A. Gumus et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128309
Fig. 1. Chemical structures of drugs containing the pyrazolo[1,5-a]pyrimidine core.
Fig. 2. Chemical structures of Dinaciclib, Larotrectinib and Selitrectinib.
compounds 1a, 3a, 5a, 6a and 8a as representatives of each class, were
treated with LiOH to afford the corresponding carboxylic acids 1b, 3b,
5b, 6b and 8b (Scheme 2).
differentiated in length by a single carbon atom induced consid-
erable effects on the hCA I inhibition potency such as in 3a and 4a
being the former 9.2-fold more potent (KIs of 82.9 and 766.3 nM
respectively). Such an effect was suppressed when the ureido
moiety as in 3a and 4a was replaced with the thioureido group
instead to afford 7a and 8a (KIs of 97.1 and 81.7 nM respec-
tively). Interestingly elongated alkenyl spacers as in the case of
derivative 9a resulted detrimental for the inhibition potency of
this isozyme (KI of 940.9 nM).
The synthesized compounds 1a-9a, 1b, 3b, 5b, 6b and 8b were
investigated for their inhibitory effects against five physiological rele-
vant isoforms, i.e., hCA I, II, VA, IX and IX, by means of a stopped flow
CO2 hydrase assay using Acetazolamide (AAZ) as a reference drug
(Table 1).25
Based on the data in Table 1, the following structure–activity re-
lationships (SARs) can be drawn and are discussed below:
(ii) Overall, the compounds considered in this study showed more
potent inhibition properties for the hCA II although a similar
trend with the hCA I isozyme may be glimpsed (Table 1). Com-
pound 1a resulted the most potent among the series with a KI
value of 0.7 nM. Again the insertion of the chlorine atom in para
and the sulfonamide group in meta within the 1a scaffold to
afford 2a resulted detrimental for the inhibition potency (KI of
162.5 nM). The same effect was also reported for the regioiso-
meric couple 5a and 6a being the former 8.5-fold more potent
when compared to the latter. Of interest is the tether effect which
resulted particularly evident for the ureido derivatives 3a and 4a.
In analogy to the hCA I isoform also in this case the shorter benzyl
3a resulted more effective in inhibiting the enzyme hydration
activity when compared to 4a (KIs of 171.8 and 586.3 nM
respectively). Substitution of the ureido group in 3a/4a with a
thioureido moiety instead to afford 7a and 8a reverted the inhi-
bition profile for the hCA II being the longer derivative up to 33-
fold more potent and with a KI value of 0.8 nM and thus almost
identical to 1a which resulted the most potent inhibitor within
the series (see Table 1). Introduction of the trans-cinnamic tether
as in compound 9a was detrimental for inhibition of the hCA II
isoform (KI of 423.6 nM). As for the acid derivatives 1b, 3b, 5b,
6b and 8b they all showed kinetic values higher when compared
(i) The hCA I was inhibited by the compounds 1a-9a and 1b-8b with
KIs ranging between 62.7 and 959.6 nM. Among the ethyl esters
series (i.e. 1a-9a) the thioureido 5a and the amide 1a resulted the
most potent inhibitors (i.e. KI values of 62.7 and 64.5 nM
respectively), followed by the acid derivative 8b (KI 67.9 nM)
which resulted 1.2-fold more potent when compared to its ethyl
ester precursor 8a (i.e. KI of 81.7 nM; see Table 1). This is the only
case among the compounds tested with the acid being more
potent hCA I inhibitor compare to its ester counterpart (Table 1).
This is the only case among the compounds tested with the acid
being more potent hCA I inhibitor of to its ester counterpart
(Table 1). Deeper SAR considerations for the hCA I isozyme ac-
count for a dramatic reduction of the compound’s inhibition
potency when the primary sulfonamide moiety is switched from
para to meta position such as for 1a and 2a (KIs of 64.5 and 954.5
nM respectively) or for the regioisomers 5a and 6a (KIs of 62.7
and 779.2 nM respectively). Since in both cases the magnitude of
the regioisomeric effect resulted almost superimposable, we can
reasonably speculate that the role of the chlorine atom in 2a to
the enzyme binding is marginal and thus no appreciable effects
on the measured kinetics were observed. Linear tethers
2

A. Gumus et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128309
Scheme 1. Synthesis of compounds 1a-9a.
3

A. Gumus et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128309
Scheme 2. Synthesis of acids 1b, 3b, 5b, 6b and 8b.
(iv) Quite interesting kinetic profile was found for the primary tumor
associated hCA IX isoform. Manipulation of the chemical groups
on the benzyl ring in 1a to afford 2a, determined remarkable
enhancement of the inhibition potency up to 21.8-fold (KIs of
141.6 and 6.5 nM respectively). On the contrary, simple shift of
the sulfonamide moiety from para to meta (i.e. as in regioisomers
5a and 6a) determined a limited effect on kinetics being the latter
just 2.2-fold more hCA IX effective inhibitor when compared to
the formed 6a (KIs of 679.8 and 312.1 nM respectively). The ef-
fect of the tether elongation on the hCA IX kinetic profile was
particular evident for derivatives 3a and 4a being the former up
to 100.5-fold (KIs of 9.7 and 974.6 nM respectively). In analogy
the same effect on kinetics was determined for derivatives 7a and
8a although with reduced magnitude (i.e. 7a was 40.0-fold more
potent when compared to 8a). Finally, among the ester series the
cinnamic derivative 9a proved high nanomolar hCA IX inhibitor
with a KI value of 843.0 nM. Among the acid derivatives, 1b
resulted the most potent with a KI value of 142.3 nM, followed by
8b which resulted 1.5-fold less effective inhibitor (KI 212.1 nM).
As for the remaining ones (i.e. 3b, 5b and 6b) the KI values
spanned between 522.8 and 850.7 nM.
Table 1
Inhibition data of hCA I, II, VA, IX and XII with compounds 1a-9a and 1b, 3b,
5b, 6b, 8b and AAZ by a stopped-flow CO2 hydrase assay.25
KI (nM)*
Cmp
hCA I
hCA II
hCA VA
hCA IX
hCA XII
1a
2a
3a
4a
5a
6a
7a
8a
9a
1b
3b
5b
6b
8b
AAZ
64.5
0.7
77.1
96.8
90.6
447.2
93.6
87.4
89.4
355.2
84.2
61.6
94.3
60.4
86.9
388.5
63.0
141.6
6.5
9.1
761.8
321.8
84.9
77.2
79.8
853.8
64.1
49.7
846.1
802.8
56.7
45.9
83.3
5.7
954.5
82.9
162.5
171.8
586.3
52.5
444.2
26.4
0.8
9.7
766.3
62.7
974.6
679.8
312.1
23.0
779.2
97.1
81.7
919.1
843.0
142.3
703.5
850.7
522.8
212.1
25.8
940.9
91.8
423.6
33.1
3240
67.5
2141
28.0
12.1
353.5
86.4
959.6
67.9
250
* Mean from 3 different assays, by a stopped flow technique (errors were in the
range of ± 5–10 % of the reported values).
(v) The hCA XII kinetic profile for the compounds considered in this
study resulted opposite when compared to the hCA IX. For
instance the KI inhibition data of 1a and 2a revealed the former
being 83.7-fold more effective inhibitor when compared to the
latter (KIs of 9.1 and 761.8 nM respectively). Likewise, the tether
effect on 3a/4a and 7a/8a showed the longer derivatives being
far more effective inhibitors than their shorter counterparts (i.e.
3.8- and 13.3-fold for 4a and 8a). On the other hand, the
regioisomeric effect clearly observed for 5a/6a resulted almost
suppressed in the case of the hCA XII (KIs of 77.2 and 79.8 nM
respectively). The cinnamic derivative 9a resulted hCA XII
effective inhibitor when compared to the other enzymatic iso-
forms discussed being a medium nanomolar inhibitor (KI 49.7
nM) and the second within the ester series best after 1a (Table 1).
As for the acid derivatives, 1b and 3b showed high nanomolar KI
values (i.e. 846.1 and 802.1 nM respectively), whereas the
remaining ones resulted medium nanomolar hCA XII inhibitors
with the compound 6b being the most potent (KI of 45.9 nM)
to their corresponding ester precursors and comprised between
28.0 and 3240 nM thus far less effective inhibitors of the refer-
ence AAZ (KI 12.1 nM).
(iii) The constitutive mitochondrial expressed hCA VA was inhibited
from the compounds considered in this study with KI values
comprised within the medium nanomolar range. Deeper SAR
considerations showed that the structural modifications on 1a to
afford the derivative 2a didn’t result in remarkable effects on the
kinetics (i.e. KIs of 77.1 and 96.8 nM respectively). The effect of
the linker elongation as for 3a/4a and 7a/8a was clearly reported
in table 1 and in both cases the longer derivatives (i.e. 4a and 8a)
resulted far less efficient hCA VA inhibitors (KIs of 447.2 and
355.2 nM respectively). Switch of the sulfonamide warhead from
para as in compound 5a to the adjacent meta position to afford 6a
determined only slight decrease of the inhibition potency (KIs of
93.6 and 87.4 nM respectively) whereas for the previously dis-
cussed isoforms hCAs I and II a significative worsening inhibition
was observed. Finally among the ester series, the cinnamic de-
rivative 9a reported a medium nanomolar inhibition potency
which resulted only slightly less potent when compared to the
most potent hCA VA inhibitor 1a (KIs of 84.2 and 77.1 nM
respectively). Among the acid series compounds 1b and 5b
resulted 1.26- and 1.54-fold respectively more potent when
compared to their corresponding ester counterparts 1a and 5a. As
for the remaining ones the acid 6b showed an almost matching KI
value when compared to its ester precursor 6a, whereas all the
others (i.e. 3b and 8b) resulted less effective hCA VA inhibitors
(see Table 1).
Overall, the data reported in table 1 accounted for 1a-3a, 7a and 8a
as the most effective hCA inhibitors among the series. In particular 1a
resulted particularly effective inhibitor of the hCA II and XII isoforms
with KIs of 0.7 and 9.1 nM, whereas 2a and 3a were both potent in-
hibitors of the tumor associated hCA IX with KI values of 6.5 and 9.7 nM
respectively and thus 4- and 2.7-fold respectively more potent when
compared to the reference AAZ (KI 25.8 nM). The thioureido derivative
7a showed almost matching KI values for the housekeeping hCA II and
tumor associated IX of 26.4 and 23.0 nM respectively, thus far lower
when compared to the remaining isoforms considered (see Table 1). As
for 8a, such compound resulted a potent hCA II inhibitor with a KI values
4

A. Gumus et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128309
Fig. 3. A) |Fo-Fc| electron density maps of compound 7a; B) Active site region of the hCA IX-mimic/7a complex (PDB: 7MU3). Hydrogen bonds (red), van der Waals
interactions (blue) are also shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
almost identical to 1a (i.e. 0.8 and 0.7 nM respectively)
kinetic outcome was preserved for its acidic derivative 8b although with
a value 25-fold higher. It is therefore clear that small structural changes
in our series heavily affected the kinetic profile. By means of X-ray
crystallographic experiments on the 7a/hCA IX mimic adduct we
assessed the main structural interactions within the enzymatic cleft
which were well defined only for the phenylsulfonamide warhead.
Although lack of electronic density only allowed to approximately
define the PP moiety allocation within the enzyme, we set a first round
of knowledge useful for future investigations in the field based on such a
privileged moiety.
The interesting KI values of compound 7a for the fast hCA II and the
tumor-associated hCA IX isoforms, induced us to determine its binding
mode within the hCA IX mimic isoform by means of X-ray experiments
of the corresponding adduct (Fig. 3).
The |Fo-Fc| electron density maps after the initial rounds of refine-
ment accounted for a 70% occupancy of the compound which was
buried within the enzymatic cleft (Fig. 3A). Clear electron densities
accounted for the primary sulfonamide directly interacting with the zinc
(II) ion as in agreement with the canonical hydrogen bond coordination
cluster.26 The phenyl ring is further stabilized by means of van der Waals
interactions with Val121 and Leu198 residues. Although the electron
density was less defined from the thioureido moiety up to the hetero-
cyclic tail of the ligand, the main identified conformation showed in-
teractions with Gln92 by means of a water bridge hydrogen bond which
involved the warhead proximal nitrogen, with the distal heteroatom also
interacting directly with the same amino acid residue. A rather disor-
dered conformation was attributed to the PP group which established a
water bridge mediated interaction by means of its N1 with Gln67 twisted
upwards facing the rim of the catalytic cleft.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
A.G. is grateful to TUBITAK (The Scientific and Technological
Research Council of Turkey) which supported her stay in Florence
during this project. Fabrizio Carta (F⋅C.) is grateful to “Bando di Ateneo
To the best of our knowledge this is the first report on pyrazolo[1,5-
a]pyrimidines (PPs) containing CAIs dealing with hCAs of physiological
interest (i.e. hCAs I, II, VA, IX and XII). The data reported in this
manuscript retrieved the main structural determinants which endowed
such compounds of selective inhibitory features. Overall, the in vitro
kinetic data accounted for our compounds as medium–high nanomolar
CAIs. As for 1a and 2a the former resulted highly efficient in inhibiting
both hCAs II and XII (KIs of 0.7 and 9.1 nM respectively) whereas the
latter was only on the primarily tumor associated hCA IX (KI of 6.5 nM).
The drastic change on the kinetic profile for 2a may be ascribed to a
combined regioisomeric and halogen depended effect. Interestingly the
preferential inhibition of the hCA IX isoform was retained also from the
ureido containing derivative 3a which resulted only 1.5-fold less effec-
tive. The insertion of the thioureido moiety in 3a dramatically affected
the kinetics as both the II and IX isoforms resulted inhibited with very
close KI values (26.4 and 23.0 nM respectively). However simple elon-
gation of the aliphatic tether of a single carbon unit, as in 8a, induced
selective inhibition of the hCA II with a KI value almost superimposable
to the one reported for 1a (KIs of 0.8 and 0.7 nM respectively). The same
per il Finanziamento di Progetti Competitivi per Ricercatori a Tempo
′′
`
Determinato (RTD) dell’Universita di Firenze” 2020–2021 , and Fon-
dazione Cassa di Risparmio di Firenze (Grant Number ECR2018.1001)
for partially supporting this work. We thank Janet Newman and the C3
Crystallisation Centre for all crystals, the Australian Synchrotron for
beamtime and the beamline scientists for help with data collection.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128309.
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