Fragment based drug design and diversity-oriented synthesis of carboxylic acid isosteres

Article abstract of DOI:10.1016/j.bmc.2020.115731

The medicinal chemist toolbox is plenty of (bio)isosteres when looking for a carboxylic acid replacement. However, systematic assessment of acid surrogates is often time consuming and expensive, while prediction of both physicochemical properties (logP and logD) as well as acidity would be desirable at early discovery stages for a better analog design. Herein in this work, to enable decision making on a project, we have synthesized by employing a Diversity-Oriented Synthetic (DOS) methodology, a small library of molecular fragments endowed with acidic properties. By combining in-silico and experimental methodologies these compounds were chemically characterized and, particularly, with the aim to know their physicochemical properties, the aqueous ionization constants (pKa), partition coefficients logD and logP of each fragment was firstly estimated by using molecular modeling studies and then validated by experimental determinations. A face to face comparison between data and the corresponding carboxylic acid might help medicinal chemists in finding the best replacement to be used. Finally, in the framework of Fragment Based Drug Design (FBDD) the small library of fragments obtained with our approach showed good versatility both in synthetic and physico-chemical properties.

Full text of DOI:10.1016/j.bmc.2020.115731

Bioorganic&MedicinalChemistry28(2020)115731
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fragment based drug design and diversity-oriented synthesis of carboxylic
acid isosteres
Martina Ferria,1, Manuel Alunnoa,1, Francesco Antonio Grecob, Andrea Mammolia,
⁎
Giorgio Salutic, Andrea Carottia, Roccaldo Sardellaa, Antonio Macchiaruloa, Emidio Camaionia, ,
Paride Lisciob
a Department of Pharmaceutical Sciences, University of Perugia, via del Liceo 1, 06123 Perugia, Italy
b TES Pharma, via P. Togliatti 22bis, 06073 Terrioli, Corciano, Italy
c Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche, via G. Salvemini, 1, 06126 Perugia, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
The medicinal chemist toolbox is plenty of (bio)isosteres when looking for a carboxylic acid replacement.
However, systematic assessment of acid surrogates is often time consuming and expensive, while prediction of
both physicochemical properties (logP and logD) as well as acidity would be desirable at early discovery stages
for a better analog design. Herein in this work, to enable decision making on a project, we have synthesized by
employing a Diversity-Oriented Synthetic (DOS) methodology, a small library of molecular fragments en-
dowed with acidic properties. By combining in-silico and experimental methodologies these compounds were
chemically characterized and, particularly, with the aim to know their physicochemical properties, the aqu-
eous ionization constants (pKa), partition coefficients logD and logP of each fragment was firstly estimated by
using molecular modeling studies and then validated by experimental determinations. A face to face com-
parison between data and the corresponding carboxylic acid might help medicinal chemists in finding the best
replacement to be used. Finally, in the framework of Fragment Based Drug Design (FBDD) the small library of
fragments obtained with our approach showed good versatility both in synthetic and physico-chemical
properties.
Carboxylic acid isosteres, pKa determination
Molecular modeling studies
Diversity-oriented synthesis, fragment based
drug design
1. Introduction
discovery process. Consequently, a systematic assessment of the ioni-
zation state of a potential replacement as well as of its physicochemical
According to literature data, the carboxylic acid pharmacophore is
widely represented among all marketed drugs accounting for a per-
centage higher than 13% of total New Molecular Entities (NMEs) ap-
proved by FDA from 2015 to 2018 with a record of approvals in 2015
(27% of total approved drugs).1–3 However, the ability of this func-
tional group to make strong polar and ionic interactions within the
protein target may often be translated in poor permeability across
biological membranes with undesirable metabolic pathways. In this
context, prodrug (e.g. ester prodrugs) strategies or carboxylic acid (bio)
isosteres were usually taken from the medicinal chemist toolbox to
overcome these limitations, on the route towards improved analogs
design. Considering both the plethora of acid surrogates available to
date and as well as the specific properties for a selected bioisostere, a
screening panel of different acidic fragments is generally required
during the lead optimization phase, slowing down the entire drug
properties (e.g. as partition coefficient, logD) would be desirable in the
early stage of drug discovery to enable prioritization during analog
design.4,5 However, the pursuit of an accurate and all-encompassing
pKa prediction method is still a key challenge6,7 especially when charge
delocalization occurs in five membered heterocycles.
Relying on small molecular fragments (MW < 300) as versatile
templates for hit compound discovery in the Fragment Based Drug
Design (FBDD) field,8 in this work in silico and synthetic skills coupled
to experimental measurements of pKa and logD gave the readers access
to a systematic assessment of a focused library of fragments endowed
with acidic properties. To this end, a Diversity-Oriented Synthetic
(DOS) approach was applied, starting from common chemical building
blocks in order to enable rapid decisions on the potential replacement
to be used.
⁎ Corresponding author.
E-mail address: emidio.camaioni@unipg.it (E. Camaioni).
1 The two authors contributed equally to this work.
https://doi.org/10.1016/j.bmc.2020.115731
Received 6 April 2020; Received in revised form 28 July 2020; Accepted 19 August 2020
Availableonline28August2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.

M. Ferri, et al.
Bioorganic&MedicinalChemistry28(2020)115731
N
2. Results and discussion
n
CN
n
OH
a
NH2
2.1. Fragment design and synthesis
3, n = 0
4, n = 1
5, n = 0
6, n = 1
With the aim to prepare a library of aromatic carboxylic acid bioi-
sosteres, as reported in Fig. 1, in the early phase of the design, the 3-
methylbenzoic acid (1) and the homologue 3-methylphenylacetic acid
(2) were chosen as acidic reference fragments.
Scheme 1. Synthesis of benzamidine 5 and 6. Reagent and conditions: (a) H2O,
NH2OH, NaHCO3, 80 °C, 4 h.
N
The acidic chain stands for the modified moiety to yield the desired
bioisosteres, while the methyl substituent in the aromatic portion re-
presents a modifiable group useful for further elaboration of the frag-
ments (linking, elongation, etc).8 In the next synthetic step, as a fruitful
strategy, the DOS methodology was employed starting from a common
building block and obtaining through a small number of steps a library
of compounds.9 In our case, starting from the suitable cyano derivative
3-methylbenzonitrile (3), the derivatives 7, 9, 11, 13, 15 and 17 were
planned (Fig. 2). Moreover, in order to evaluate the influence on the
physicochemical properties of the fragments of a methylene linker be-
tween the aromatic ring and the heterocyclic system, also the deriva-
tives 8, 10, 12, 14, 16 and 18 were designed and synthesized but
starting from 3-methylphenylacetonitrile (4, Fig. 2).
n
a
b
O
O
HN
7, n = 0
8, n = 1
N
n
O
N
N
n
OH
CF3
9, n = 0
10, n = 1
NH2
5, n = 0
6, n = 1
N
n
c
S
HN
11, n = 0
12, n = 1
O
N
d
n
O
The synthetic approach, as depicted in the scheme 1, started from
the two commercially available building blocks 3 and 4. As first, the N-
hydroxybenzamidine intermediates 510 and 6, were synthesized in
quantitative yield by reacting 3 and 4 with hydroxylamine hydro-
chloride in basic aqueous environment. The reaction was so clean that
the obtained derivatives 5 and 6 were directly used for the next steps
without any further purification after the usual work-up.
HN
13, n = 0
14, n = 1
S
Scheme 2. Synthesis of oxadiazole and thiadiazole derivatives. Reagents and
conditions: (a) 1) ethylchloroformate, acetone 0 °C 1 h, then NaOH (5%) 3 h; 2)
Pyridine, toluene, reflux 16 h (50–76%); (b) trifluoroacetic anhydride, pyridine,
2 h, 0–25 °C; then HCl 1 N, 0 °C (31–48%); (c) 1) 1,1′-thiocarbonyldiimidazole,
THF, r.t., 1 h; 2) BF3·OEt2, THF, r.t., 3 h, (24–71%); (d) 1,1′-thiocarbonyldii-
midazole, DBU, dioxane 110 °C, 5 h. (40–83%).
In the next step, starting from 5 and 6, the fragments 7–14 were
synthesized as reported in the Scheme 2. That is undoubtedly the reason
why DOS synthesis shines through many other synthetic methods when
it is necessary to build a focused and heterogeneous library from a few
precursors. In particular, cyclization of 5 and 6 with ethylchloroformate
furnished the oxadiazolones 711 and 8 in 76% and 50% yield respec-
tively. The neutral derivatives 9 and 10, synthesized to confirm and
expand the versatility of our DOS approach, were obtained in good
yield (48% and 31% respectively) by cyclization of benzamidine 5 and
6 with trifluoroacetic anhydride. For the synthesis of thiadiazolones 11
and 12, a known procedure reported in literature was followed.12 In
this case, the cyclization reaction took place in two steps from 5 and 6
with thiocarbonyldiimidazole in the presence of BF3(OEt2) afforded the
desired compounds 1113 and 12 in 71% and 24% yield, respectively.
With a synthetic procedure similar to the one used above for 11 and
12, the derivatives 1314 and 14 were obtained in 83% and 40% yield,
respectively.
Fig. 1. Reference fragments and work strategy.
N
O
n
n
N
N
H2N
NH
15, n = 0
17, n = 0
18, n = 1
16, n = 1
The two building blocks 3 and 4 were also used for the synthesis of
the other fragments 15–18 (Scheme 3). Indeed, reaction of the two
nitrile derivatives 3 and 4 with sodium azide afforded the corre-
sponding tetrazole derivatives 1510,15 and 16,16,17 in high yield (85%
and 76%, respectively). Interestingly, the amide derivatives 1715 and
1818 were obtained by using microwave irradiation of 3 and 4, in basic
n
CN
3, n = 0
4, n = 1
N
N
N
n
n
n
OH
O
O
O
N
NH
HN
HN
n
N
NH2
a
b
N
S
7, n = 0
8, n = 1
13, n = 0
14, n = 1
5, n = 0
6, n = 1
15, n = 0
16, n = 1
n
CN
O
n
3, n = 0
4, n = 1
H2N
17, n = 0
18, n = 1
N
N
n
n
S
O
HN
N
O
CF3
11, n = 0
12, n = 1
9, n = 0
10, n = 1
Scheme 3. Synthesis of tetrazoles and amides. Reagent and conditions: (a)
sodium azide, Et3N, toluene, reflux, 16 h (76–85%); (b) potassium carbonate,
water, MW, 200 PSI, 130 °C, 30 min (18–50%).
Fig. 2. Fragment based library obtained with a DOS-like synthetic strategy.
2

M. Ferri, et al.
Bioorganic&MedicinalChemistry28(2020)115731
H
N
O
fragment were first estimated in-silico by using the software MoKa that
implements an algorithm based on descriptors derived from GRID
molecular interaction fields, checking also the tautomers.20 Indeed,
many of our studied fragments can behave under different tautomeric
rearrangements affecting the results of the cheminformatics tools that
estimates physical–chemical properties. To cope with this issue we also
employed ab-initio calculations. All the molecules were built in all the
desired tautomeric states and submitted to a deep conformational
analysis. All the conformers were then energetically optimized by the
ORCA software21 (see details in the Experimental section). This pro-
cedure allowed us to identify the conformer endowed with the
minimum energy (global minima) of each tautomer and to retrieve the
Δ energy among the different tautomers, as reported in Table 1 (Δ(ΔG),
thus indicating the relative stability.
S
a
b
O
O
OH
19
HO
O
O
2
O
22
Scheme 4. Synthesis of the derivatives 19 and 22. Reagents and conditions: (a)
CDI, DCM, 30 min; then Et3N, methanesulfonamide, r.t., 12 h (72%); (b) 1)
ethylchloroacetate, THF, Et3N, reflux, 16 h; 2) sodium tert-butoxide, DMF, 0 °C,
1 h, r.t. 5 h (60%).
aqueous medium, in 50% and 18% yield, respectively.
Several methods have been reported for the determination of pKa,22
and the most common and accurate is still the potentiometric half-ti-
tration volumetric technique (using a glass electrode). Indeed, the ex-
perimental pKas were then determined by potentiometric titration im-
plemented in the Sirius T3 apparatus that has been reported to be very
sensitive and accurate although its working range is limited to a pKa
values ranging from 2 up to 12.23 The octanol–water partition coeffi-
cient (logP) was measured through potentiometric titration in an im-
miscible biphasic system by using the same Sirius T3 apparatus. In
particular, multiple titrations were performed in the aqueous phase at
different volumes of water and octanol, in order to evaluate the beha-
vior of the analyte in the partitioning equilibrium. Some of the analyzed
fragments were not soluble in water at the used concentration so the
stock solution was prepared in DMSO (see experimental details).
Our studied fragments cover a portion of the chemical space (Fig. 3)
spanning from 132 to 227 of MW, from 2.64 to over 12 of pKa, and from
0.97 to 2.6 of logP values. Considering m-toluic acid 1 as the reference
compound, in our hands, fragments bearing respectively the oxadiazol-
5(4H)-thione (13, 14) and the 5H-furan-2-one moiety (22) work rather
below the acidity of the starting acid 1, showing experimental values of
pKa below 4. Examples of fragments with acidity closest to the starting
reference 1 are of note m-methylphenylylacetic acid 2 and the two
tetrazole derivatives 15 and 16. Interesting values of pKa were obtained
with oxadiazol-5(4H)-ones 7 and 8 which resulted to be near 5 log unit.
Furthermore, we found that some of the determined pKa values fall
over the working range as predicted in the case of amides 17 and 18,
but also the two hydroxyamidines 5 and 6 show very weak acidic
properties (pKa > 12). Furthermore, 5 and 6 can act as bases. Indeed,
we found that these two fragments can be protonated in acidic condi-
Although our prototypes 1 and 2 could be synthesizable from 3 and
4, they are commercially available and can be easily exploited for the
synthesis of other interesting fragments. An example is reported in
Scheme 4.
Indeed, the acidic sulfonamide 19 was obtained in 72% yield by
using a coupling reaction between the carboxylic acid 2 and the me-
thanesulfonamide in the presence of CDI. Finally, starting from 2, the
lactone 22 was synthesized. In detail, esterification of 2 with ethyl-
chloroacetate provided the intermediate 2-ethoxy-2-oxoethyl 2-(m-
tolyl)acetate (21) in quantitative yield. Lactonization of the latter in
basic conditions provided the desired product 22,19 in 60% yield
(Scheme 5).
The synthesized fragments were also in silico filtered for PAINS (pan-
assay interference compounds) activity showing no structural alerts.
Furthermore, these fragments are non fluorescent under UV irradiation.
2.2. LogP-LogD/pKa calculations and experimental determinations
These compounds were chemically characterized and, particularly,
some of the physico-chemical properties such as their aqueous ioniza-
tion constants (pKa) and logP/logD were first estimated and then va-
lidated by experimental determinations. Beside logP, distribution
coefficient (logD) is helpful for the compound characterization during
the drug discovery. Conversely to logP, which is referred to the parti-
tioning of the unionized or neutral species between the organic and
water layers, logD is concerned to the partitioning of both ionized and
unionized species between the two phases at a specific pH values (i.e. at
pH 3 and 8). These two parameters describe the same physical property
(lipophilicity) but logD accounts for the pH dependence of a molecule
in aqueous solution and it is the appropriate descriptor for lipophilicity
of ionizable compounds. Thus, physico-chemical properties of each
tions with a pKa of 4.97
0.03 for 5 and 5.27
0.01 for 6, re-
spectively. We have also observed a significant increasing in the pKa
values when
a
methylene spacer is placed between the acidic
OH
O
O
a
+
Cl
O
O
O
O
100%
O
2
20
21
b
60%
OH
O
O
22
Scheme 5. Synthesis of the derivative 22. Reagents and conditions: (a) ethylchloroacetate, THF, Et3N, reflux, 16 h; (b) sodium tert-butoxide, DMF, 0 °C, 1 h and then
r.t. 5 h.
3

M. Ferri, et al.
Bioorganic&MedicinalChemistry28(2020)115731
Table 1
Calculated and experimental properties of test compounds.
#
Structures
MW
Orca Δ(ΔG)
clogP
logP
clogD
logD
cpKa
pKa
Kcal/mola
MoKa
SiriusT3
MoKa
SiriusT3
Mokab
SiriusT3
pH = 3 pH = 8
pH = 3 pH = 8
1
136.1
–
2.3
2.21–1.14
1.40
0.01–0.65
0.02
0.01
3.94 (OH)
3.99
0.03
0.01
2
150.1
150.2
–
1.8
2.0
1.98
n.d.
0.04
1.80–1.43
1.35
n.d.
0.01–0.70
4.39 (OH)
4.13
5a
0.0
−1.80 0.64
12.39 (OH)
> 12
5b
+8.9
9.48 (OH)
6a
6b
180.2
176.2
0.0
2.0
2.5
n.d.
−2.18 1.00
2.50 0.41
n.d.
12.66 (OH)
> 12
5.12
+9.6
9.68 (OH)
7a
7b
7c
+14.3
0.0
1.14
0.04
0.80
0.01–1.11
0.02
5.90 (OH)
7.68 (NH)
–
0.04
+10.6
8a
8b
8c
190.2
192.2
+15.0
0.0
2.6
2.6
1.63
2.22
0.02
0.08
2.30 1.50
2.60 1.00
1.39
2.04
0.01–0.42
0.02 0.34
0.01
0.04
7.27 (OH)
8.13 (NH)
–
5.59
5.99
0.01
12.3
11a
11b
11c
+12.9
0.0
6.41 (OH)
9.01 (NH)
–
0.09c
+6.3
12a
12b
12c
206.3
192.2
+13.3
0.0
2.4
2.7
2.15
2.65
0.02
0.03
2.39 1.97
2.66–0.65
2.02
1.44
0.01 0.42
0.01–0.70
0.01
0.01
7.78 (OH)
9.46 (NH)
–
6.35
2.64
0.01c
0.05c
+7.9
13a
13b
13c
+8.9
0.0
4.14 (SH)
6.88 (NH)
–
+12.8
14a
14b
14c
206.3
+9.7
0.0
2.5
2.39
0.02
2.47–0.78
1.42
0.01–0.70
0.01
4.36 (SH)
7.33 (NH)
–
3.19
0.02c
+14.4
(continued on next page)
4

M. Ferri, et al.
Bioorganic&MedicinalChemistry28(2020)115731
Table 1 (continued)
#
Structures
MW
Orca Δ(ΔG)
clogP
MoKa
logP
clogD
logD
cpKa
pKa
Kcal/mola
SiriusT3
MoKa
SiriusT3
Mokab
SiriusT3
pH = 3 pH = 8
pH = 3 pH = 8
15a
15b
160.2
+2.8
0.0
2.3
1.75
0.14
0.03
2.25–0.78
1.10
0.01–0.95
0.01
0.01
4.32 (NH)
–
4.07
0.02c
0.03c
16a
16b
17
174.2
135.2
2.3
0.0
–
2.1
1.2
1.51
–
2.13 0.55
1.20 1.20
1.07
n.d.
0.01–0.96
5.24 (NH)
–
4.73
12.56 (NH2)
> 12
18
19
149.2
227.3
–
–
1.1
0.9
–
1.05 1.05
0.87–1.69
n.d.
13.09 (NH2)
5.40 (NH)
> 12
4.63
0.97
0.03
0.02
0.44
0.11–1.58
0.02–0.87
0.14
0.03
0.02c
0.06c
22a
22b
190.2
0.0
1.1
2.32
−0.88–3.80
1.26
2.56 (OH)
3.18 (CH2)
2.95
+5.6
a
b
Δ(ΔG) in Kcal/mol was estimated at ab-initio PBE0/TZVP level of theory and is displayed only for fragments making tautomers.
The putative polarizable functional groups are reported in brackets. The estimated more populated tautomer is highlighted in bold. pKa determinations were
performed in water or in.
c
DMSO (50 mM stock solution). n.d. not determinable, n.i. not ionizable.
functionality and the phenyl ring.
account the experimental pKas and thus, experimental logP values for
compounds 5, 6, 17 and 18 were not measured. As a result, all the
fragments showed moderate lipophilicity, with the logP values ranging
from 0.97 to 2.65, and overall < 3, a value generally accepted as a
Concerning the lipophilicity, almost all compounds demonstrated
experimental logP value comparable to the predicted one. The logP
measurement protocol is based on a titration method taking into
H
Me
N
S
O
O
O
N
S
HN
O
H
N
N
N
=
N
Less
Acidic
CO2
OH
H
O
More
Acidic
Less
Lipophilic
More
Lipophilic
Fig. 3. Chemical space of test fragments represented by size (MW), lipophobicity (logP) and charge in terms of dissociation constant (pKa). For fragments 5, 6, 17 and
18 predicted values were plotted.
5

M. Ferri, et al.
Bioorganic&MedicinalChemistry28(2020)115731
good cut-off for fragments as starting hits.24 Regarding the logD values,
compound 19 displayed from moderate to good accordance between
the in silico and the experimental values at both pH (Table 1). Frag-
ments 13 and 14 experimental and predicted logD at pH 8 registered
both a value of −0.70. Differently, in the case of free carboxylic acids,
the MoKa logD overestimated the pH effect, recording a range between
the predictions at pH 8 and 3 higher of 1 unit with respect of the Sir-
iusT3 ones, precisely 3.35 vs 2.05 and 3.23 vs 2.05 for parent com-
pounds 1 and 2, respectively. An unsatisfactory prediction performance
was also recorded in almost all the rest of the cases. More in general, it
should be noted that most of these fragments contains heterocycles that
are not usually well parametrized and often needs to be studied by
calculations at an ab-initio level, thus it was expected to be challenging
to predict logD values. Moreover, in the case of prediction systems
based on derived empirical models, it is important often the presence of
analog compounds in the training set. On the opposite, it must be noted
that MoKa achieved a higher performance in the case of pKa estimation,
indicating that probably a new model should be trained to obtain more
accurate logD predictions.
synthetic approach starting from m-tolunitrile or m-tolylacetonitrile, we
reported herein a rapid experimental evaluation of the acidity and li-
pophilicity of a small series of acidic fragments. Empirical methods such
as implemented in MoKa packages possessed fast and predictive cap-
abilities in computational estimation of pKa and logP, while the logD
estimation resulted poor. The advanced and automated potentiometric
method, Sirius T3, displayed to be very accurate and sensitive in the
determination of the pKa, logP and logD values. Finally, fragments
obtained with this approach showed good versatility both in synthetic
and physico-chemical properties providing low nanomolar ACMSD in-
hibitor.10
In conclusion, when searching for an acid surrogate the medicinal
chemist toolbox is plenty of isosteric replacements which might allow a
fine tuning, during the drug discovery process, of the physico-chemical
properties of the new molecules without loss of potency.
4. Experimentals
Derivatives, with a pKa values ranging in close proximity to the
neutrality, such as the thiadiazol-5(4H)-ones 11 and 12 could be con-
sidered as the best compromises of choice between acidity and lipo-
philicity when searching for carboxylic acid surrogates or bioisosteres.
The versatility of our fragments could be exploited during the
elongation part of the FBDD process. Indeed, the methyl group in meta
position of the substituted phenyl ring could be modified in the corre-
sponding versatile alkylbromide derivatives by using the classical ra-
dical reaction with NBS/AIBN (Fig. 4).
4.1. Chemistry
1H NMR spectra were recorded at 200 and 400 MHz and 13C NMR
spectra were recorded at 100.6 MHz using the solvents indicated below.
Chemical shifts were reported in parts per million (ppm). The ab-
breviations used are as follows: s, singlet; d, doublet; dd, doublet of
doublets; ddd, doublet of doublets of doublets; t, triplet; dt, doublet of
triplets; q, quartet; qui, quintet. The final products were purified by
flash chromatography on silica gel 60 (0.040–0.063 mm). TLC were
performed on aluminum backed silica plates (silica gel 60 F254). All the
reaction were performed under nitrogen atmosphere using distilled
solvent. All reagents were from commercial sources. The HPLC analy-
Thus, this approach was validated by some of the authors in a work
devoted to the discovery of new inhibitors of the enzyme ACMSD (α-
amino-β-carboxymuconate-ε-semialdehyde decarboxylase).10 For in-
stance, as depicted in Scheme 6, fragment 15 was reacted in AcCN with
NBS in the presence of catalytic amount of AIBN to obtain the corre-
sponding tetrazole benzylbromide derivative 23, in moderate yield. 23
was then coupled with the scaffold 4-oxo-6-thiophen-2-yl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carbonitrile (24) to obtain the final
derivative 25 that was endowed with a very high inhibitory potency (Ki
about 0.003 μM) against the targeted enzyme.10
tical scale measurements were carried out on
a Shimadzu LC
Workstation Class LC-10 equipped with SCL-10 A VP system controller,
a LC-10AT VP high pressure binary gradient delivery system, an SPD-
10° VP variable-wavelenght UV–vis detector, and a Rheodyne 7725i
injector with a 20 μL stainless steel loop. The chromatographic profile
was obtained with EZ Start software. All analytical runs were performed
by employing a H2O/CH3CN/TFA (60/40/0.1 v/v/v) solution as the
mobile phase. HPLC grade water was obtained from a tandem Milli-Ro /
Milli-Q apparatus. A Grace Smart C18 250 × 4.6 mm i.d. 5 μm 100 Å
analytical column was used after previous conditioning by passing
through the column the selected mobile phase for at least 45 min. The
UV detection wavelengths were set at 254 and 210 nm. Samples for
analytical-scale analyses were prepared in approximate concentration
between 0.1 and 0.5 mg/mL in filtered mobile phase components with
auxiliary DMSO, if necessary, and sonicated until completely dissolved.
The GC–MS analyses were performed on an Agilent 7890A GC coupled
to a triple quadrupole mass spectrometer Agilent 7000 MS system
(Agilent Technologies, Waldbronn, Germany). 1 μL of the powder so-
lubilized in ethylacetate was injected in a programmable temperature
vaporization (PTV) inlet. The chromatographic separation was achieved
The high versatility of benzyl bromide derivatives toward different
types of reaction could make this series of acidic fragments a highly
desirable building block set for fragment-based drug discovery.
3. Conclusion
The high versatility of the carboxylic acid functional group in drug
design lies behind its intrinsic acidity and relies on its ability to es-
tablish strong electrostatic interactions and hydrogen-bonds with a
target protein thus ensuring also relatively high water solubility to the
drug-like molecule. However, the same properties could be also re-
sponsible of the “Janus Face” of this versatile functional group, such as
low the permeability across biological membranes. From this stand-
point, the quest to quickly find additional groups working as acid bio
(isosteres) with improved physicochemical properties drove us in this
work, with the aim to help medicinal chemists in the choice and in the
using
a
Restek
Rtx®-5MS
column
(Bellefonte,
PA)
(30 m × 0.25 mm × 0.25 µm) as follows: 2 min hold at 70 °C, ramp to
300 °C at 10 °C min−1, 3 min hold at 300 °C. The mass spectrometric
acquisition was performed in Full Scan mode (m/z 50–230).
prioritization of compounds synthesis. Indeed, exploiting
a DOS
Fig. 4. General approach of FBDD with our
Sn2 Fragment Elongation Example
synthesized fragments.
n
n
R
n
R
R1
Br
S
R
n = 0 or 1
6

M. Ferri, et al.
Bioorganic&MedicinalChemistry28(2020)115731
Scheme 6. Synthesis of the 1,6-dihydro-6-
oxo-2-[[[3-(2H-tetrazol-5yl)phenyl]methyl]
thio]-(2-thienyl)-5-pyrimidinecarbonitrile.
Reagents and conditions: (a) NBS, AIBN,
AcCN, reflux, 16 h. (b) DIPEA, DMSO, rt,
16 h.
S
S
N
N
N
N
N
N
NC
O
NC
O
a
b
N
N
N
NH
N
+
N
Br
H
N
H
N
N
H
24
S
N
S
H
H
15
23
25
4.2. General procedure A
1H NMR (400 MHz, DMSO‑d6) δ 2.40 (s, 3H), 7.39 (d, J = 7.5 Hz,
1H), 7.48 (t, J = 7.6 Hz, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.8 (s, 1H). 13
C
To an aqueous solution (7 mL) of hydroxylamine hydrochloride
(1.42 g, 20.5 mmol) and sodium bicarbonate (1.72 g, 20.5 mmol), a
solution the appropriate nitrile derivative (2 mL, 17 mmol) in ethanol
(13 mL) was slowly added dropwise. The reaction mixture was heated
to reflux (80 °C) for 4 h. When the reaction was ended the solvent was
removed under reduced pressure and the crude was poured into water
and extracted with ethyl acetate (3 × 30 mL). The combined organic
layers were washed with saturated aqueous sodium chloride and dried
over Na2SO4 then concentrated under reduced pressure.
NMR (100.6 MHz, DMSO‑d6): δ 21.3, 124.4, 127.7, 129.7, 132.2, 139.2,
155.7.
GC–MS: RT 16.3 min; m/z 132.0 (100), 117.0 (68), 116.0 (50),
104.1 (34), 160.1 (M+., 15)
HPLC: RT = 5.43 min (> 98%)
Analytical data are in accordance with those reported in litera-
ture.10,15
4.7. 5-[(3-Methylphenyl)methyl]-1H-tetrazole (16)
4.3. N-hydroxy-3-methyl-benzamidine (5)
Starting from 2-(3-methylphenyl)acetonitrile (4, 2 mL, 15.25 mmol)
and following the general procedure F the title compound was obtained
as a white solid (2.05 g, yield 76%). m.p.: 132–136 °C.
1H NMR (400 MHz, DMSO-d6) δ 2.26 (s, 3H), 7.04–7.08 (m, 2H),
7.31 (t, 1H, J = 7.51 Hz)
Starting from 3-methylbenzonitrile (2 mL, 17 mmol) and following
the general procedure A the title compound was obtained as a thick oil
which gradually turned into a white crystalline solid (2.5 g, quantita-
tive yield). m.p.: 86–89 °C
13C NMR (100.6 MHz, DMSO-d6) δ 21.33, 126.10, 128.04, 129.03,
129.64, 136.20, 138.29.
1H NMR (400 MHz, DMSO-d6) δ 2.31 (s, 3H), 5.75 (s, 2H),
7.16–7.26 (m, 2H), 7.44–7.48 (m, 2H), 9.6 (s, 1H).
GC–MS: RT 16.9 min; m/z 105.0 (100), 131.0 (23), 174.1 (M+, 18),
116.0 (17).
13C NMR (100.6 MHz, DMSO‑d6): δ 21.48, 122.97, 126.39, 128.39,
129.87, 133.70, 137.55, 151.28.
HPLC: RT = 5.59 min (> 98%).
GC–MS: RT 7.2 min; m/z 117.0 ([M−NH2OH]+, 100), 116.0 (65).
HPLC: RT = 3.04 min (> 96%)
Analytical data are in accordance with those reported in litera-
ture.16,17
Analytical data are in accordance with those reported elsewhere.10
4.8. General procedure C
4.4. N-Hydroxy-2-(3-methylphenyl)-acetamidine (6)
In a microwave sealed tube, the appropriate nitrile derivative
(1.54 mL, 12.8 mmol) and potassium carbonate (707 mg, 5.12 mmol)
were suspended in water (10 mL). The mixture was irradiated at 130 °C
at 200 PSI and 200 W for 30 min. The crystalline solid obtained was
collected by filtration. The water phase was extracted with EtOAc
(3 × 20 mL) and the combined organic layers were washed with NaCl
saturated aqueous solution and dried over Na2SO4. The solvent was
evaporated under reduced pressure affording the desired product
combined with the crystalline solid isolated by filtration.
Starting from 2-(3-methylphenyl)acetonitrile (0.6 mL, 4.57 mmol)
and following the general procedure A the title compound was obtained
as a yellow oil (688 mg, quantitative yield).
1H NMR (400 MHz, DMSO‑d6) δ 2.31 (s, 3H), 3.20 (s, 2H), 5.36 (s,
2H), 7.-7.18 (m, 4H), 8.87 (s, 1H).
13C NMR (100.6 MHz, DMSO‑d6) δ 21.41, 37.52, 126.19, 127.23,
128.39, 129.71, 137.45, 138.26, 152.36.
GC–MS: RT 9.3 min; m/z 131.0 (M−NH2OH]+, 100), 116.0 (90),
104.0 (76).
HPLC: RT = 3.27 min (> 95%).
4.9. 3-Methylbenzamide (17)
4.5. General procedure B
Starting from 3 (1.54 mL, 12.8 mmol) and following the general
procedure C the desired product 17 was obtained as opalescent crystals
(870 mg, Yield 50%). m.p.: 92–94 °C
To a solution of the appropriate nitrile derivative (1 g, 8.53 mmol)
in toluene (15 mL) NaN3 (564 mg, 9,4 mmol) and Et3N HCl (1.86 g,
9.4 mmol) were added. The mixture was refluxed for 16 h. Then the
reaction was quenched with a mixture of ice/water and stirred for
30 min. The organic phase was separated from aqueous one and the
1H NMR (400 MHz, DMSO‑d6) δ 2.34 (s, 3H), 7.31–7,33 (m, 3H),
7.64 (s, 1H), 7.69 (s, 1H), 7.91 (s, 1H)
13C NMR (100.6 MHz, DMSO‑d6) δ 21.36, 124.98, 128.49 (2C),
132.17, 134.62, 137.83, 168.41.
latter was acidified with HCl
1
N
and extracted with EtOAc
GC–MS: RT 12.0 min; m/z 119.0 (100), 135.1 (M+., 65)
HPLC: RT = 4.23 min (> 98%)
(3 × 15 mL). The combined organic layers were washed with NaCl
saturated aqueous solution and dried over sodium sulfate and con-
centrated under vacuum. The title compound was crystallized from
Et2O.
Analytical data are in accordance with the literature ones.15
4.10. 3-Methylphenylacetamide (18)
4.6. 5-m-Tolyl-1H-tetrazole (15)
Starting from 2-(3-methylphenyl)acetonitrile (4, 0.3 mL, 2.3 mmol)
and following the general procedure C the desired compound 18 was
obtained as an opalescent crystalline solid (60 mg, yield 18%). m.p.:
146–148 °C.
Starting from 3-methylbenzonitrile (3, 1.0 g, 8.53 mmol) and fol-
lowing the general procedure B the desired product 15 was obtained as
a white crystalline solid (1.0 g, yield 85%). m.p.: 145–147 °C.
1H NMR (400 MHz, DMSO‑d6) δ 2.27 (s, 3H), 3.34 (s, 2H), 6.85 (s,
7

M. Ferri, et al.
Bioorganic&MedicinalChemistry28(2020)115731
1H), 7.03–7.06 (m, 2H), 7.16 (s, 1H), 7.43 (s, 1H).
13C NMR (100.6 MHz, DMSO‑d6) δ 21.39, 42.63, 126.53, 127.29,
128.46, 130.10, 136.77, 137.53, 172.68.
solvent was removed under reduced pressure and the crude was pur-
ified by flash chromatography eluting with DCM/MeOH (from 0% to
1% of MeOH).
GC–MS: RT 12.6 min; m/z 106.1 (100), 149.1 (M+., 38).
HPLC: RT = 4.53 min (> 98%)
4.15. 3-(3-Methylphenyl)-4,5-dihydro-1,2,4-oxadiazol-5-thione (13)
Analytical data are in accordance with literature ones.18
Starting from the N-Hydroxybenzamidine (5, 600 mg, 4 mmol) and
following the general procedure E the desired compound 13 was ob-
tained as a yellow solid (640 mg, yield 83%) m.p.: 153.6–159.2 °C
1H NMR (400 MHz, DMSO‑d6) δ 2.39 (s, 3H), 7.39–7.6 (m, 4H),
14.45 (bs, 1H).
4.11. General procedure D
To a solution of amidine (2.5 g, 17 mmol) in dry acetone (10 mL)
ethylchloroformate (1,8 mL, 18.72 mmol) solution in acetone (10 mL)
was added dropwise at 0 °C. The reaction was stirred for 1 h under these
conditions. Then an aqueous solution of sodium hydroxide (5%, 15 mL)
was added at room temperature and the mixture was stirred for addi-
tional 3 h. The formation of a white precipitate was observed. Then the
solvent was removed under reduced pressure and the crude was parti-
tioned between water and ethylacetate (3 × 30 mL). The combined
organic layers were washed with saturated solution of NaCl and dried
over Na2SO4 then concentrated under vacuum. The product was pur-
ified by flash chromatography eluting with PET/EtOAc (from 0 to 40%
of EtOAc) affording the intermediate as a white solid (3.58 g,
16.12 mmol). The latter was solubilized in toluene (90 mL) and pyr-
idine (13.03 mL, 161.12 mmol) was added dropwise to the mixture. The
reaction mixture was heated to reflux (110 °C) and stirred at this
temperature for 16 h. Then the mixture was diluted with HCl 1 N
(100 mL) and extracted with EtOAc (3 × 40 mL). The combined or-
ganic layers were washed with saturated solution of NaCl and dried
over Na2SO4 then concentrated under vacuum. The crude was purified
by flash chromatography eluting with PET/EtOAc (from 0% to 40% of
EtOAc) affording the desired product.
13C NMR (100.6 MHz, DMSO‑d6) δ 21.24, 122.07, 124.43, 127.59,
129.71, 133.68, 139.33, 159.44, 187.81.
GC–MS: RT 18.0 min; m/z 117.0 (100), 149.0 (90), 116.0 (65),
192.0 (M+., 53).
HPLC: RT = 11.93 min (> 98%)
Other analytical data are known in literature.14
4.16. 3-[(3-Methylphenyl)methyl]-4,5-dihydro-1,2,4-oxadiazol-5-thione
(14)
Starting from 6 (400 mg, 2.44 mmol) and following the general
procedure E the desired product 14 was obtained as a brown solid
(200 mg, yield 40%). m.p: 85–90 °C.
1H NMR (400 MHz, DMSO‑d6) δ 2.29 (s, 3H), 3.96 (s, 2H),
7.12–7.08 (m, 2H), 7.26 (s, 1H), 14.35 (bs, 1H)
13C NMR (100.6 MHz, DMSO‑d6) δ 21.32, 29.70, 126.42, 128.47,
129.06, 129.96, 133.91, 138.36, 161.12, 187.40.
GC–MS: RT 18.0 min; m/z 105.0 (100), 206.1 (M+., 85), 131.0 (80),
116.0 (75).
HPLC: RT = 12.25 min (> 98%).
4.12. 3-(3-methylphenyl)-4,5-dihydro-1,2,4-oxadiazol-5-one (7)
4.17. General procedure F
Starting from 5 (2.5 g, 17 mmol) and following the general proce-
dure D the desired product 7 was obtained as a white solid (2.3 g, yield
76%). m.p.: 138–140 °C
To a solution of the appropriate amidine (1.0 g, 6.7 mmol) in dry
THF (10 mL) 1,1-thiocarbonyldiimidazole (1.8 g, 10.1 mmol) was
added. The reaction was stirred at room temperature for 1 h. The
mixture was poured into water and extracted with DCM (3 × 30 mL).
The combined organic layers were washed with saturated solution of
NaCl and dried over Na2SO4 and concentrated under reduced pressure.
Then the crude was dissolved in dry THF (15 mL) and of boron tri-
fluoride diethyl etherate 46,5% solution (6.14 mL, 20.1 mmol) was
added dropwise to the mixture. The reaction was stirred at room tem-
perature for 3 h and an orange precipitate was observed. The mixture
was then diluted with water and extracted with EtOAc (3 × 30 mL),
washed with saturated solution of NaCl and dried over Na2SO4. The
crude was purified by flash chromatography eluting with PET/EtOAc
(from 0% to 15% of EtOAc).
1H NMR (400 MHz, DMSO‑d6) δ 2.38 (s, 3H), 7.41–7.49 (m, 2H),
7.59–7.69 (m, 2H), 12.93 (bs, 1H).
13C NMR (100.6 MHz, DMSO-d6) δ 21.25, 123.65, 126.77, 129.60,
133.26, 139.18, 157.82, 160.37.
GC–MS: RT 17.8 min; m/z 117.0 (100), 116.0 (65), 133.0 (26),
176.1 (M+., 18).
HPLC: RT = 7.00 min (> 98%)
Analytical data are in accordance with literature ones.11
4.13. 3-[(3-methylphenyl)methyl]–2H-1,2,4-oxadiazol-5-one (8)
Starting from 6 (688 mg, 4.19 mmol) and following the general
procedure D the desired product 8 was obtained as a yellowish solid
(485 mg yield 50%). m.p.: 106–109 °C
4.18. 3-(3-Methylphenyl)-1,2,4-thiadiazol-5-one (11)
1H NMR (400 MHz, DMSO‑d6) δ 2.29 (s, 3H), 3.82 (s, 2H),
7.07–7.12 (m, 3H), 7.24 (t, 1H, J = 7.4 Hz), 12.30 (bs, 1H).
13C NMR (100.6 MHz, DMSO‑d6) δ 21.31, 30.96, 126.28, 128.37,
129.04, 129.82, 134.08, 138.34, 159.45, 160.23.
Starting from 5 (1.0 g, 6.7 mmol) and following the general pro-
cedure F the desired product 11 was obtained as a white solid (918 mg,
yield 71%). m.p.: 168–172 °C.
GC–MS: RT 17.8 min; m/z 131.0 (100), 116.0 (76), 104.0 (70),
190.1 (M+., 12).
1H NMR (400 MHz, DMSO‑d6) δ 2.36 (s, 3H), 7.36–7.43 (m, 2H),
7.71–7.78 (m, 2H), 13.36 (bs, 1H)
HPLC: RT = 7.12 min (> 95%).
13C NMR (100.6 MHz, DMSO‑d6) δ 21.31, 124.07, 127.35, 128.83,
129.26, 132.42, 138.76, 155.17, 179.94.
4.14. General procedure E
GC–MS: RT 18.1 min; m/z 149.0 (100), 117.0 (70), 116.0 (55),
192.0 (M+., 53).
To a solution of the amidine (600 mg, 4 mmol) in dioxane (20 mL)
treated 1–1-thiocarbonyldiimidazole (1.07 g, 6 mmol) was added fol-
lowed by 1,5-diazabicyclo(5.4.0)undec-5-ene (DBU, 2.42 mL,
16 mmol). The resulting mixture was heated at 110 °C for 5 h. Then the
solvent was evaporated under vacuum and the crude was diluted with
DCM (50 mL) and washed with HCl 1 N (3 × 60 mL). The organic
HPLC: RT = 9.16 min (> 98%).
Analytical data are in accordance with literature ones.13
4.19. 3-[(3-Methylphenyl)methyl]-1,2,4-thiadiazol-5-one (12)
Starting from 6 (1.0 g, 6.1 mmol) and following the general
8

M. Ferri, et al.
Bioorganic&MedicinalChemistry28(2020)115731
procedure F the desired compound 12 was obtained as a white solid
(300 mg, Yield 24%). m.p.: 112–114 °C. 1H NMR (400 MHz, DMSO‑d6)
δ 2.28 (s, 3H), 3.8 (s, 2H), 7.05–7.09 (m, 3H), 7.2 (s, 1H), 12.86 (bs,
1H).
experimental method were reported elsewhere.25 SiriusT3 instrument
works in a pH range from 2 to 12 and the protocol employed takes into
account only the compounds with pKa value into the range. The octa-
nol–water partition coefficient (logP) was measured through potentio-
metric titration in an immiscible biphasic system using a SiriusT3 ap-
paratus. Thus, potentiometric logPs (pH-metric medium logP) were
recordered at 25 °C in a constant ionic strength (0.15 M KCl). This
analysis required the pKa values determined in the previous analysis
using the same instrument. LogD values are also related to logP taking
into account the pKa of each compound and the pH values were fixed at
3 and 8. SiriusT3 allowed us to determine experimentally logP and pKa
for all the compounds when the pKa value is included in the pKa
working range of 2–12. Applying the following equation,
13C NMR (100.6 MHz, DMSO‑d6) δ 21.36, 37.14, 126.33, 128.07,
128.90, 129.88, 135.31, 138.13, 157.81, 179.71.
GC–MS: RT 16.9 min; m/z 105.0 (100), 131.0 (23), 174.1 (M+., 18),
116.0 (17).
HPLC: RT = 8.07 min (> 98%).
4.20. 2-(3-methylphenyl)-N-methylsulfonylacetamide (19)
To a solution of m-tolyl acetic acid (500 mg, 3.33 mmol) in DCM
(15 mL), 1-carbonyldiimidazole (CDI, 589 mg, 3.63 mmol) was added.
The resulting solution was stirred at room temperature for 30 min. After
this time Et3N (0.93 mL, 6.66 mmol) and methylsulfonamide (634 mg,
6.66 mmol) were added to the mixture and stirring was continued at
room temperature for 12 h. The reaction was cooled to 0 °C then KHPO4
buffer solution (50 mL, pH = 2.4) was added. The aqueous layer was
separated and extracted with EtOAc (3 × 25 mL). The combined or-
ganic layers were washed with brine and dried over Na2SO4 and con-
centrated under vacuum. The title compound was obtained as a col-
ourless oil which crystallized slowly into a white solid (549 mg, yield
72%). m.p. = 105.3–108.2 °C. 1H NMR (400 MHz, DMSO-d6) δ 2.28 (s,
3H), 3.09 (s, 3H), 3.47 (s, 2H), 7.03–7.05 (m, 3H), 7.19 (t, J = 7.49 Hz,
1H).
pK
)
logD(pH) = logP log 1 + 10(pH
a
[
]
logD values were determined for each acidic fragments focusing the
pH values to 3 and 8.
The compounds were collected in stock solutions 50 mM in DMSO
solvent, and for each sample the analysis has been carried out using
150 μL of stock solution.
5. Computational methods
The molecules were built in all the tautomeric states of interest,
using the Maestro (version 12, Schrödinger, LLC, New York, NY, 2019)
interface present in the Schrödinger Suite 2019-2. Next, a conforma-
tional search was performed with MacroModel (version 12.4,
Schrödinger, LLC, New York, NY, 2019) and the geometries of each
conformer energetically optimized with ORCA 4.1.2 software,21 leading
to the identification of the global minimum. In particular, the con-
formational analysis was performed using the OPLS3e force field and
the “Mixed torsional/Low-mode sampling”, executing 1000 steps, re-
taining per each molecule all the conformers lying in a window of
10.0 kcal mol−1 from the global minimum and using water as the
solvent. The torsional sampling involves multiple Monte Carlo
minimum searches for global exploration, and the low mode con-
formational search allows for automatic local exploration. The con-
formers were considered to be different if the maximum atom deviation
for any pair of corresponding atoms exceeds the threshold of 0.5 Å. All
the options were set as default with the exception of the “Torsional
sampling options” that were changed from “Intermediate” to “Ex-
tended” in order to improve the conformational space searched. This
step produced a total of 333 conformers that were all submitted to a
quantum mechanical energy optimization by using the PBE0 func-
tional26 that was used in combination with the TZVP basis set27 and the
conductor-like polarizable continuum model (CPCM keyword)28 to
keep in consideration the water environment. Finally the level of ac-
curacy was set to tightscf. The resulting conformer of each tautomeric
state was used to identify the global minimum of each compound. This
value was then used to compute the energy difference (ΔΔG) among
different tautomeric states of the same molecule, thus allowing to
identify the preferred state(s) of that species in water.
13C NMR (100.6 MHz, DMSO‑d6) δ 9.15, 21.38, 40.50, 41.11, 43.54,
46.10, 126.79, 127.58, 128.52, 130.38, 135.48, 137.62, 172.30.
GC–MS: RT 17.1; m/z 105.0 (100), 132.0 (95), 227.1 (M+., 17).
HPLC: RT = 6.47 min (> 96%).
4.21. 4-Hydroxy-3-m-tolyl-5H-furan-2-one (22)
To a solution of m-toluic acid (2 g, 13.3 mmol) and ethyl-
chloroacetate (2.14 mL, 19.9 mmol) in THF (10 mL) was added Et3N
(2.8 mL, 19.9 mL). Stirring was continued at reflux for 16 h. A white
solid was formed. The solid was filtered through a sintered pad. The
filter cake was washed with THF (3 × 5 mL). The solvent was removed
under reduced pressure. The intermediate 2-ethoxy-2-oxoethyl 2-(m-
tolyl)acetate was obtained as a yellow oil quantitative yield and used
directly for the next step (3.7 g). To solution of the starting intermediate
(3.7 g, 15.6 mmol) in DMF (15 mL) sodium tert-butoxide (3.5 g,
31.3 mmol) was added portionwise. Stirring was continued at 0 °C for
1 h and for additional 5 h at room temperature. The mixture was di-
luted with water, extracted with EtOAc (3 × 20 mL). the combined
organic layers were washed with NaCl saturated aqueous solution and
dried over Na2SO4 and concentrated under vacuum. 800 mg The de-
sired compound 24 was obtained as a white solid after shredding with
Et2O (800 mg, yield 30%). m.p.: 208–210 °C.
1H NMR (400 MHz, DMSO‑d6): δ 2.39 (s, 3H, CH3); 4.88 (s, 2H,
CH2); 7.42–7.46 (m, 1H, ArH); 7.72–7.85 (m, 3H, ArH); 12.58 (s, 1H,
OH).
13C NMR (100.6 MHz, DMSO‑d6) δ 21.68, 66.40, 97.75, 123.94,
127.22, 127.43, 128.38, 130.83, 137.36, 173.37, 175.35.
GC–MS: RT 16.9; m/z 190.1 (M+., 100), 132.0 (75), 103.0 (68),
117.0 (42), 161.0 (38).
The global minima conformers were used as input for the MoKa
v3.1.1 software20 to estimate the pKa and the logP/logD values. Simi-
larly, the same input was used in the Canvas program (Schrödinger
Release 2019–2: Canvas, version 4.0, Schrödinger, LLC, New York, NY
(USA)) to check for PAINS compounds by applying the relative filter.29
HPLC: tR = 7.15 min (> 98%).
Analytical data are in accordance with those reported by Wang
et al.19
6. Notes
4.22. LogPs and pKas determination
All co-authors have agreed with the submission of the final manu-
script and participated in the research.
Experimental pKa values were determined by means of a SiriusT3
instrument (Sirius Analytical Instruments Ltd., Forest Row, E. Sussex,
UK). Analyzed compounds were dissolved in water or in DMSO pre-
paring stock solutions with a concentration of 50 mM. Details of the
Funding
University of Perugia (Fondo Ricerca di Base 2018) is gratefully
acknowledged for the financial support.
9

M. Ferri, et al.
Bioorganic&MedicinalChemistry28(2020)115731
Declaration of Competing Interest
1,2,4-thiadiazoles from amidoximes. J Heterocycl Chem. 2000;37:1419–1423.
https://doi.org/10.1002/jhet.5570370603.
[14]. Sonawane AD, Rode ND, Nawale L, et al. Synthesis and biological evaluation of
1,2,4-triazole-3-thione and 1,3,4-oxadiazole-2-thione as antimycobacterial agents.
Chem Biol Drug Des. 2017;90:200–209. https://doi.org/10.1111/cbdd.12939.
[15]. Maiti Bag S, Jayarajan R, Dutta U, Chowdhury R, Mondal R, Maiti D. Remote meta-
C-H cyanation of arenes enabled by a pyrimidine-based auxiliary. Angew Chem Int
Ed Engl. 2017;56:12538–12542. https://doi.org/10.1002/anie.201706360.
[16]. Satyanand K, Shristy D, Nisha S, Kumar AS. (NH4)2Ce(NO3)6 as an inexpensive,
eco-friendly, efficient catalyst for the synthesis of 5-substituted 1-H tetrazoles from
nitriles. Tetrahedron Lett. 2014;55:6034–6038. https://doi.org/10.1016/j.tetlet.
2014.09.010.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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