Amidino substituted 2-aminophenols: biologically important building blocks for the amidino-functionalization of 2-substituted benzoxazoles

Article abstract of DOI:10.1039/d1ob00235j

Unlike the closely related and widely investigated amidino-substituted benzimidazoles and benzothiazoles with a range of demonstrated biological activities, the matching benzoxazole analogues still remain a largely understudied and not systematically evaluated class of compounds. To address this challenge, we utilized the Pinner reaction to convert isomeric cyano-substituted 2-aminophenols into their amidine derivatives, which were isolated as hydrochlorides and/or zwitterions, and whose structure was confirmed by single crystal X-ray diffraction. The key step during the Pinner synthesis of the crucial carboximidate intermediates was characterized through mechanistic DFT calculations, with the obtained kinetic and thermodynamic parameters indicating full agreement with the experimental observations. The obtained amidines were subjected to a condensation reaction with aryl carboxylic acids that allowed the synthesis of a new library of 5- and 6-amidino substituted 2-arylbenzoxazoles. Their antiproliferative features against four human tumour cell lines (SW620, HepG2, CFPAC-1, HeLa) revealed sub-micromolar activities on SW620 for several cyclic amidino 2-naphthyl benzoxazoles, thus demonstrating the usefulness of the proposed synthetic strategy and promoting amidino substituted 2-aminophenols as important building blocks towards biologically active systems.

Full text of DOI:10.1039/d1ob00235j

Organic &
Biomolecular Chemistry
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PAPER
Amidino substituted 2-aminophenols: biologically
important building blocks for the amidino-
functionalization of 2-substituted benzoxazoles†
Cite this: Org. Biomol. Chem., 2021,
19, 2784
a
Lucija Ptiček,a Lucija Hok, b Petra Grbčić,c Filip Topić,d Mario Cetina,
Kari Rissanen, d Sandra Kraljević Pavelić,e Robert Vianello *b and Livio Racané
*
a
Unlike the closely related and widely investigated amidino-substituted benzimidazoles and benzothiazoles
with a range of demonstrated biological activities, the matching benzoxazole analogues still remain a
largely understudied and not systematically evaluated class of compounds. To address this challenge, we
utilized the Pinner reaction to convert isomeric cyano-substituted 2-aminophenols into their amidine
derivatives, which were isolated as hydrochlorides and/or zwitterions, and whose structure was confirmed
by single crystal X-ray diffraction. The key step during the Pinner synthesis of the crucial carboximidate
intermediates was characterized through mechanistic DFT calculations, with the obtained kinetic and
thermodynamic parameters indicating full agreement with the experimental observations. The obtained
amidines were subjected to a condensation reaction with aryl carboxylic acids that allowed the synthesis
of a new library of 5- and 6-amidino substituted 2-arylbenzoxazoles. Their antiproliferative features
against four human tumour cell lines (SW620, HepG2, CFPAC-1, HeLa) revealed sub-micromolar activities
on SW620 for several cyclic amidino 2-naphthyl benzoxazoles, thus demonstrating the usefulness of the
proposed synthetic strategy and promoting amidino substituted 2-aminophenols as important building
blocks towards biologically active systems.
Received 5th February 2021,
Accepted 2nd March 2021
DOI: 10.1039/d1ob00235j
rsc.li/obc
African trypanosomiasis, although treatment with this com-
pound is associated with serious toxicities.1,2 In order to
Introduction
Aromatic and heteroaromatic diamidines have been extensively search for safer and more effective drugs, great efforts have
studied because of their broad spectrum of antimicrobial been made in the synthesis and biological evaluation of new
activity. Compounds such as pentamidine, berenil, and 4′,6-di- dicationic molecules. In particular, diamidino-substituted ben-
amidino-2-phenylindole (DAPI) have been known to be zimidazoles, as analogues of DAPI, have emerged as important
efficient in the treatment of trypanosomiasis and leishmania- pharmacophores in the development of antimicrobial agents.
sis. Pentamidine, which was discovered over 60 years ago, is Binding in the minor groove of DNA at AT-rich sites is thought
still one of the most used drugs for the first stage of human to be a key step in the mode of action of these types of dicatio-
nic systems, which likely leads to the inhibition of DNA-depen-
dent enzymes or a direct inhibition of transcription.2 Positively
aDepartment of Applied Chemistry, Faculty of Textile Technology, University of
charged extremities are not the sole structural elements for
Zagreb, Prilaz baruna Filipovića 28a, 10000 Zagreb, Croatia.
strong DNA binding, but the three-dimensional organization
of the molecules is also crucial for sequence-selective DNA
binding. One direction in the development of DNA sequence-
specific agents is modification of the diamidine structure by
including a variety of nitrogen heterocycles into molecules of
different shapes.3–5 With the recent approval of the minor
groove binding agent trabectedin for the treatment of patients
with soft tissue sarcomas, there has been renewed interest in
small molecules as antitumor agents that target the DNA as
well.6,7
E-mail: livio.racane@ttf.unizg.hr
bDivision of Organic Chemistry and Biochemistry, Ruđer Bošković Institute,
Bijenička cesta 54, 10000 Zagreb, Croatia. E-mail: robert.vianello@irb.hr
cDepartment of Biotechnology, Centre for high-throughput technologies,
University of Rijeka, Radmile Matejčić 2, 51000 Rijeka, Croatia
dUniversity of Jyvaskyla, Department of Chemistry, P.O. Box 35, 40014 Jyväskylä,
Finland
eFaculty of Health Studies, University of Rijeka, Viktora Cara Emina 5, 51000 Rijeka,
Croatia
†Electronic supplementary information (ESI) available: Experimental and com-
putational details; single crystal X-ray diffraction analysis; 1H and 13C NMR
spectra; Cartesian coordinates for all computed systems and their output files.
CCDC 2060884–2060894. For ESI and crystallographic data in CIF or other elec-
tronic format see DOI: 10.1039/d1ob00235j
Amidino-substituted benzimidazole derivatives are a well-
studied class of compounds with some new recent examples
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associated with prominent antibacterial,8 antiparasitic,9 anti- nophenols, while the key step during the Pinner synthesis of
fungal,10 and antiproliferative activities.11 Besides the benzimi- the crucial carboximidate intermediate was characterized
dazole core, over the last decade, our scientific focus has been through mechanistic DFT calculations. Additionally, in order
placed on benzothiazole compounds bearing different to explore the biological potential of a new small library of
amidino substituents with an emphasis on their synthesis and amidino-substituted 2-arylbenzoxazoles, the antiproliferative
the study of anticancer activities and DNA binding activities of some selected compounds were tested on four
properties.12–20 The obtained results revealed that 2-aryl/ human tumour cell lines.
heteroaryl benzothiazole derivatives substituted with the cyclic
amidino substituent, namely the 2-imidazolinyl group, had
the most significant influence on the antiproliferative activity
and its enhancement with IC50 values in the submicromolar
range of concentrations.
Results and discussion
Synthesis of amidino-substituted 2-aminophenols
Surprisingly, in contrast to a great number of amidino-sub-
stituted benzimidazoles and benzothiazoles, structurally
related benzoxazoles are still rare. There are only a few reports
that deal with the synthesis of amidino-substituted benzoxa-
zoles and their antitumor21 and antimicrobial22,23 activities as
well as their DNA binding properties.24 One reason is the lack
of a general method for their preparation, which could be
based on the condensation reaction of amidino-substituted
2-aminophenols with aldehydes, carboxylic acids or carboxylic
acid derivatives since this method is largely and efficiently
used for the preparation of the corresponding benzimidazole
and benzothiazole derivatives, from amidino-substituted
phenylenediamines and 2-aminothiophenols, respectively
(Scheme 1).
The most common method for amidine preparation is the
nucleophilic addition of amines or ammonia to suitably acti-
vated carboxylate equivalents such as imidates, thioimidates,
and imidoyl chlorides. Imidates can generally be prepared
either by a base-catalysed or acid-catalysed (Pinner synthesis)
addition of alcohol to nitrile. The addition of dry hydrochloric
acid to a mixture of nitrile and alcohol leads to the hydro-
chloride salt of an imidate. This salt can further react with
ammonia or an amine to form amidine.25 The key step in the
Pinner synthesis is the formation of the imidate functionality
which has been recently exploited for the syntheses of
diverse classes of N-heterocycles via C–N annulation reactions
under acid/base/metal-catalysed or radical-mediated reaction
conditions.26
Commercially available 4-hydroxybenzonitrile (1a) and
3-hydroxybenzonitrile (1b) were used as starting substrates for
the preparation of isomeric cyano-substituted 2-aminophenols
in the nitration reaction with fuming nitric acid in glacial
acetic acid (Scheme 2).
The nitration reaction of 4-hydroxybenzonitrile (1a) was
carried out according to the described procedure27 and
4-hydroxy-3-nitrobenzonitrile (2a) was obtained in 95% yield.
Our computations confirm the presence of a single product by
showing that the stability of the crucial carbocation intermedi-
ate is the highest upon the attack of the active nitronium ion
(NO2 ) on position 3, being −17.7 kcal mol−1 more stable than
+
isolated reactants, while in the case of position 2, it is only
−0.9 kcal mol−1, thus rationalizing why only 2a was isolated.
In addition, kinetic aspects further support this conclusion as
the activation barrier to approach position 3 is only ΔG‡
=
1.6 kcal mol−1, being 5.5 kcal mol−1 lower than for position 2.
Due to the direction of the electrophilic aromatic substitution
conditioned by the hydroxy and cyano substituents on the
benzene ring, in the nitration reaction of 3-hydroxybenzoni-
trile (1b), more products were formed. An analogous compu-
tational analysis revealed that the stability of the carbocation
intermediate is −17.6, −17.0 and −21.3 kcal mol−1 lower than
+
those of the isolated reactants, when NO2 approaches posi-
tions 2, 4 and 6 in 1b, respectively, while it is only −3.3 kcal
mol−1 for the attack on position 5. This confirms approxi-
mately equal distribution of products 2b–2d, and the absence
of the potential 5-nitro derivative. Still, only those regioisomers
in which the hydroxy and nitro groups are on the adjacent C
In this work, we present a novel synthesis of 5- and
6-amidino-substituted 2-arylbenzoxazoles through the conden-
sation reaction of different amidino-substituted 2-aminophe-
nols as the key building blocks, which were unknown to date.
We describe the efficient synthetic protocols and structural
analysis of the obtained isomeric amidino-substituted 2-ami-
Scheme 1 Retrosynthetic strategy for amidino-functionalization of
2-substituted benzazoles.
Scheme 2 Synthesis of nitro- and amino-substituted 2-aminophenols.
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Table 1 Synthesis of carboximidates 5a–6c
atoms were of interest for further synthesis. The reaction was
optimized according to the more favourable ratio of cyano-sub-
stituted 2-nitrophenol of interest, where the reaction tempera-
ture proved to be the parameter with the most influence on
the total yield and the ratio of the resulting products. A reac-
tion temperature of 30–35 °C gave a high total yield of nitrated
products where 3-hydroxy-4-nitrobenzonitrile (2b) and
3-hydroxy-2-nitrobenzonitrile (2c) were isolated by chromato-
graphy in 23% and 21% yields, respectively, after the isolation
of isomer 2d in 23% yield by simple crystallization. The next
step of the synthesis was the reduction of nitro to the amino
group on all prepared 2-nitrophenol isomers of interest, 2a, 2b
and 2c (Scheme 2), by our previously optimized method using
tin(II) chloride in an ethanol/hydrochloric acid media.28 Since
nitro derivatives 2a, 2b and 2c were soluble in water, the reac-
tion was carried out in dilute hydrochloric acid at reflux with 4
Nitrile
R
Imidate
Isolated yield
3a
CH3
5a
5b
5c
6a
90%
Quantitative
95%
NDa
CH2CH2OCH3
CH2CH2OCH2CH2OCH2CH3
CH3
CH2CH2OCH3
CH2CH2OCH2CH2OCH2CH3
CH3
3b
3c
6b
98%
6c
NDa
NRb
NRb
—
—
CH2CH2OCH3
a Not determined. b No reaction.
equivalents of tin(II) chloride. All amino derivatives 3a, 3b and of a viscous hygroscopic liquid which was difficult to handle.
3c were isolated in excellent yields of 90–97%. The structures However, from the reaction carried out in 2-methoxyethanol
of the previously reported nitro derivatives 2a, 2b and 2d and (Table 1), carboximidate dihydrochloride 6b was isolated in
1
amino derivatives 3a and 3b were confirmed by H NMR and the form of a stable white solid in an almost quantitative
LC-MS. The unknown nitro 2c and amino 3c isomers were yield of 98% with very good purity confirmed by 1H NMR
fully characterized. In addition, the structure of compound 3c spectroscopy.
was unequivocally determined by single crystal X-ray diffrac-
tion (Fig. S1†).
It is known from the literature data that the formation of
imidates for ortho-substituted aromatic nitriles is often
The classical Pinner reaction was then used for the prepa- impossible.31 Due to the higher available amounts of o-sub-
ration of amidino-substituted 2-aminophenols. In the first stituted cyano-derivative 3c, an attempt was made to prepare
step, the nitrile was converted to the corresponding carboximi- carboximidates in the first step of the Pinner reaction using
date hydrochloride by the addition of alcohol, catalysed by dry methanol (4a) and 2-methoxyethanol (4b) (Table 1). It was
hydrogen chloride. For this reaction, it is common to use equi- hypothesized that the use of a primary alcohol with a higher
molar ratios of methanol or ethanol and nitrile in a dry solvent molecular weight such as 2-(2′-ethoxyethoxy)ethanol (4c)
or an excess of absolute alcohol in which the corresponding could lead to steric disturbances during alcohol addition;
imidates are obtained in high yields.25,26 Various primary alco- therefore we performed the reaction in alcohols 4a and 4b.
hols can be used including different alkoxy- and trihalogen- The result of the reaction showed no conversion of benzo-
substituted ethanoles,29 as well as secondary alcohols such as nitrile 3c even with a prolonged reaction time up to 10 days,
isopropanol.30 In the second step of the reaction, the alkoxide and we regenerated only the starting substrate 3c. It was con-
group was substituted with an amine to form an amidine cluded that the formation of imidate from ortho-substituted
group. In order to investigate the reaction yield of this key step aromatic nitrile 3c was indeed unsuccessful. Following the
of the Pinner reaction, as well as the purity and stability of the described investigation of the first step of the Pinner reac-
corresponding carboximidate hydrochlorides, the use of three tion, 2-methoxyethanol proved to be the best choice due to
primary alcohols was investigated: methanol (4a), 2-methox- the obtained quantitative yields of crude carboximidate
yethanol (4b) and 2-(2′-ethoxyethoxy)ethanol (4c), while the hydrochlorides 5b and 6b with very good purity. Also, they
mechanistic details and the rationalization of the product dis- were always obtained as solids which are easy to dry and
tribution were obtained using DFT computations (see later).
stable to handle within a few days.
The conversion of benzonitrile 3a to the corresponding imi-
The second step of the Pinner reaction was performed with
dates 5a–5c (Table 1) was achieved with all alcohols in excel- freshly prepared carboximidates in absolute ethanol with an
lent yields higher than 89%, with imidate 5b being obtained excess of ammonia at room temperature for 2 days or an
quantitatively with very good purity.
excess of primary amine at reflux temperature for 2–4 h (TLC-
Although imidate 5c was also isolated as a white solid in controlled). Thus, from 2-aminophenol-4-imidate 5a, 5b or 5c,
an almost quantitative yield, it proved to be highly hygro- in reaction with gaseous ammonia (7a), ethylene-diamine (7b),
scopic and sensitive to moisture. The structures of 5a–5c and 1,3-propanediamine (7c) and isopropylamine (7d), 4-amidino-
1
their purity were confirmed by H NMR spectroscopy. Due to substituted 2-aminophenols were isolated as hydrochlorides
their reactivity, they have not been additionally purified and 8a, 8b and 8d and/or zwitterions 8a′, 8b′, 8c′ and 8d′ in moder-
fully characterised. The first step of the Pinner reaction for ate to good yields (Table 2).
benzonitrile 3b, carried out in alcohols 4a and 4c (Table 1),
Reactions of imidate 5c with ammonia 7a afforded ami-
did not give a satisfactory result for carboximidate dihy- dines 8a and 8a′, isolated as hydrochloride and a zwitterion,
drochlorides 6a and 6c because they were isolated in the form respectively. It was shown that it is possible to isolate both
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Table 2 Synthesis of 4-amidino-substituted 2-aminophenols
Imidate Amine
Amidine
Product Isolated yield
5c
5c
5b
7a; R = H
8a
44%
64%
41%
7a; R = H
8a’
8b
Scheme 3 Reaction of imidate 5b with 1,4-butanediamine 7e.
7b; R = (CH2)2NH2
5c
7b; R = (CH2)2NH2
8b’
78%
reaction time offer N,N‘-disubstituted and cyclic amidines.25
The 1H NMR spectra of 8e′ and 8f′ were very similar in the
chemical shift and multiplicity of signals and could not be
unambiguously attributed to the structure of the product.
The key data for determining their structures were obtained
from the mass spectra, regarding the values of m/z for the
molecular ion of structures 8e′ (206.2) and 8f′ (357.2). To
further confirm the formation of the zwitterion 8e′, a reac-
tion with methanesulfonic acid was carried out in ethanol
from which dimethanesulfonate 8e was isolated, whose
molecular structure was confirmed by single-crystal X-ray
diffraction.
5a
5b
7c; R = (CH3)2NH2
7d; R = CH(CH3)2
8c’
75%
46%
8d
5a
7d; R = CH(CH3)2
8d’
51%
forms of amidines, though the isolation of the product was
different, as well as the yield of the overall reaction.
Hydrochloride 8a was isolated in a moderate yield while
zwitterion 8a′ was isolated in a greater yield after dilution of
the reaction mixture with water and basification to pH
10–11 (Table 2). A similar pattern of behaviour was observed
in the reaction with 5 eq. of diamine 7b, where 2-imidazoli-
nyl-substituted 2-aminophenols were prepared as hydro-
chloride 8b and zwitterion 8b′ via imidate 5b or 5c, respect-
ively. Zwitterion 8b′ was isolated in a much greater yield
than hydrochloride 8b, which was isolated in a moderate
yield. In the reaction of imidate 5a with 4 eq. of diamine 7c,
2-pyrimidinyl-substituted 2-aminophenol 8c′ in a zwitter-
ionic form was prepared in a good yield. From imidates 5b
and 5a, in the reaction with 2.5 eq. of amine 7d,
N-isopropylamidino-substituted 2-aminophenol as hydro-
chloride 8d and zwitterion 8d′ were obtained in similar
moderate yields. According to these results, it was prefer-
able to isolate the products in the zwitterionic form, since
higher yields and cleaner products were obtained with no
need for purification.
From the isomeric 2-aminophenol-5-carboximidates 6a–6c
in reaction with ammonia 7a, diamines 7b and 7c and amine
7d, 5-amidino-substituted 2-aminophenols were isolated as
hydrochlorides 8g, 8h, and 8j and/or zwitterions 8h′ and 8i′ in
moderate to good yields (Table 3).
After the reaction of imidate 6b with 7a and removal of
excess of ammonia from the reaction mixture, only precipi-
Table 3 Synthesis of 5-amidino-substituted 2-aminophenols
Imidate
Amine
Amidine
Product
Isolated yield
72%
6b
7a; R = H
8g
Unexpectedly, the reaction of imidate 5b with 1,4-butane-
diamine (7e) gave two different products depending on the
amine concentration in solution. Thus, with a higher excess
and a higher concentration of amine 7e (c = 1.25 mol dm−3),
the targeted seven-membered cyclic 1,3-diazepinyl amidine 8e′
was isolated as a zwitterion in a good yield (Scheme 3).
6b
6c
7a; R = H
8g’
0%
7b; R = (CH2)2NH2
8h
60%a
6b
7b; R = (CH2)2NH2
8h’
81%
In the reaction with a reduced excess and a lower concen-
tration of amine 7e (c = 0.25 mol dm−3), only the product of
the intermolecular Pinner reaction, bis(butane-1,4-diimida-
mide) 8f′, was isolated in a modest yield, while 8e′ was not
observed by qualitative TLC analysis. This agrees with the
literature that a large excess of amines and a prolonged
6a
6b
7c; R = (CH3)2NH2
7d; R = CH(CH3)2
8i’
75%a
59%
8j
a Yields were calculated from nitrile 3b.
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tation of ammonium chloride from ethanol occurred and the
expected hydrochloride remained in the mother liquor. An
attempt to precipitate the product as zwitterion 8g′ by dilution/
basification was also unsuccessful, because of its great solubi-
lity in water. Therefore, in a second run, after the completion
of the reaction of 6b with 7a by saturating the ethanolic
mother liquor with dry gaseous hydrogen chloride, the
product was precipitated as dihydrochloride 8g and isolated in
a good yield (Table 3). In the reaction of imidate 6c with 2.5
eq. of diamine 7b, the hydrochloride of 2-imidazolinyl substi-
tuted 2-aminophenol 8h was afforded in 60% yield. The corres-
ponding zwitterion 8h′ was prepared from imidate 6b in the
reaction with 2.5 eq. of diamine 7b and isolated in a very good
yield. From imidate 6a, in the reaction with 4 eq. of diamine
7c, the zwitterionic form of 2-pyrimidinyl-substituted 2-amino-
phenol 8i′ was obtained in 71% yield. After heating the reac-
tion mixture of imidate 6b and 2.5 eq. of amine 7d,
N-isopropylamidino-substituted 2-aminophenol hydrochloride
8j was obtained in a moderate yield without the need for a
purification step.
Fig. 1 Molecular structures of (a) 8a I, (b) 8d and (c) 8a’, with atom
numbering schemes. Displacement ellipsoids for non-hydrogen atoms
are drawn at the 40% probability level. Only the major component of the
disordered chloride ion is shown for 8a I. Solvent molecules are omitted
for clarity.
The structures of all newly prepared amidino-substituted
2-aminophenols were fully characterised by 1H and 13C NMR
spectroscopy, UPLC-MS spectrometry and elemental analysis.
NMR analysis based on the values of H–H coupling constants
and chemical shifts in the 1H spectra confirmed the structures
of compounds. Furthermore, 13C chemical shifts were consist-
ent with the suggested structures. In addition, we succeeded
in obtaining nine crystals of 2-aminophenols suitable for
X-ray analysis which unequivocally confirm their molecular
structures.
pure zwitterionic form crystallizing as hydrates. The C7–N2
and C7–N3 bonds of the amidinium moiety as well as the C1–
O1− bond in these three structures are all of approximately the
same lengths as those of the equivalent ones in previously
described zwitterionic salt solvate structures (Table S1†). X-ray
crystallographic study also confirmed that 8g and 8e crystal-
lized as dications with two chloride or methanesulfonate
anions, respectively (Fig. S6†).
Computational studies
Computational analysis was performed to interpret the reactiv-
ity differences among the systems in the Pinner reaction with
a particular focus on rationalizing the higher reactivity of 3a
and 3b towards alcohols relative to 3c, by calculating the
kinetic and thermodynamic parameters for the matching
reactions.
Our initial efforts started with neutral un-ionized 3a–3c and
considered the feasibility of solvent-mediated transformations
into the corresponding imidates, taking 3a and MeOH as
illustrative examples. As seen in Fig. 2, the reaction starts with
Single crystal X-ray studies
Amidino-substituted aminophenols 8a, 8a′, 8b, 8b′, 8c′, 8d, 8e,
8g and 8h crystallized as salts, solvates or salt solvates. X-ray
crystallographic study of 8a (two polymorphic forms, 8a I and
8a II, see the ESI†) shows that this compound exists in a
zwitterionic form, with the C7–N2 and C7–N3 bonds in the
amidinium moiety approximately equal (Table S1†). Such a
form is also corroborated by the C1–O1− bond distance being
ca. 0.02–0.03 Å shorter than in similar structures where the
phenol group is not deprotonated (phenol C–O bond length
ca. 1.36 Å). Crystallizing as a hemihydrochloride monohydrate,
the structure of 8a contains dimeric cations formed by two
molecules joined by an O−–H+–O− hydrogen bond bridge
(Fig. 1a and Fig. S2†). The structure of 8b (Fig. S3†), where the
amidinium moiety is a part of a dihydroimidazolium ring,
exhibits the same cation : anion : water ratio as that of 8a. The
C7–N2, C7–N3 and C1–O1− bonds are of the same lengths as
the corresponding ones in 8a. In contrast, 4-amidino-substi-
tuted derivative 8d (Fig. 1b) and 5-amidino-substituted deriva-
tive 8h (Fig. S4†) did not contain zwitterions forming cationic
dimers, but un-ionized phenol groups, resulting in singly
charged cations. The C1–O1 bond in 8d and the C2–O1 bond
in 8h are typical of a phenol C–O bond (Table S1†). Isolation of
4-amidino-substituted 2-aminophenols 8a′ (Fig. 1c), 8b′ and
Fig. 2 Reaction mechanism involving neutral and monoprotonated 3a
with methanol. Numbers indicate relative solution-phase Gibbs free
energies obtained by the (SMD)/M06-2X/6-31+G(d) model (in kcal
8c′ (Fig. S5†) from water at pH 10–11 afforded structures in a mol−1).
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the reactive complex SP1, where methanol is aligned for the gives the imidate product SP2 in a single step. Here, the reac-
nucleophilic attack on the cyano C-atom, with the relevant tion becomes exergonic (ΔrG = −2.3 kcal mol−1); however the
O(methanol)–Ccyano (3a) bond distance of 3.31 Å. In the tran- process is kinetically still very much unfeasible given only a
sition state TS1, the mentioned bond is reduced to 1.60 Å, modest reduction in the activation barrier to ΔG‡ = 53.4 kcal
while the vibration leading to products (1871i cm−1) involves mol−1. This indicates that even the protonation of the adjacent
both the O–C bond formation and the proton transfer from amine does not improve the electrophilicity of the –CN
the methanol –OH group to the cyano nitrogen. This immedi- moiety to make it susceptible to the nucleophilic approach of
ately gives the imidate product, with thermodynamic para- MeOH.
meters suggesting a likely process (ΔrG = 0.1 kcal mol−1). Still,
the reaction has a very high activation barrier of ΔG‡
The second protonation occurs exclusively on the cyano
=
nitrogen (Table 4) and gives dications 3a++–3c++. It turns out
55.0 kcal mol−1, which renders it unfeasible under these con- that this process activates the –CN group, in line with the lit-
ditions. In other words, in neutral derivatives, the matching erature reports on acidic conditions as an important prerequi-
–CN group does not possess enough electrophilicity to allow site for the Pinner reaction.34,35 The reactive complexes feature
the Pinner reaction.
shorter O(solvent)–Ccyano(3++) bonds, being 2.85, 2.79 and
Given that experiments are performed in excess gaseous 2.80 Å for 3a++–3c++ in methanol, respectively, while 2.95, 2.82
HCl that allows sequential protonation in solution, we pro- and 2.70 Å in the same order in 2-methoxyethanol. These are
ceeded by inspecting the relative stabilities of various protona- shortest in 3c++, since the neighbouring ortho-NH3 group pro-
+
tion forms of 3a–3c (Table 4), while evaluating their influence motes the hydrogen bond formation with the solvent
on the Pinner reaction outcomes. In all systems, regardless of (Fig. S7†), which brings the latter closer to the reacting cyano
the solvent, the first protonation preferentially occurs on the moiety. Yet, it is precisely this hydrogen bonding motif in 3c++
amino nitrogen (N2), being consistent with the typically higher that will somewhat hinder an efficient solvent approach
basicity of anilines relative to both phenols and aromatic towards the –CN group, as the former needs to overcome this
nitriles.32 The difference between N2 protonation and other stabilizing interaction to initiate the Pinner reaction. Indeed,
sites is the highest in 3a, where the –CN group is in the meta- Fig. 3 shows that the formation of the O(solvent)–Ccyano(3++)
position to the protonated amine, and where it least affects its bond occurs through
a transition state TS1 requiring
basicity. Specifically, in 3b and 3c, the –CN group is at the 7.5–8.8 kcal mol−1 in MeOH and 7.1–8.5 kcal mol−1 in 2-meth-
para- and ortho-positions, where it lowers the aniline basicity oxyethanol, being always the highest for 3c++. It is interesting
via resonance participation,33 and thus reduces the energy to note that the reactivity for this step follows the trend 3b++
>
difference between different protonation sites.
3a++ > 3c++ in both solvents, while the reaction is kinetically
Building on that, we analysed the mechanistic aspects of always more feasible in 2-methoxyethanol. This process offers
the Pinner reaction starting from the monocationic 3a+ an intermediate with the formed O(solvent)–Ccyano bond and
(Fig. 2), and again taking MeOH as a typical case. The reaction the reaction free energies around neutrality. In MeOH, this
proceeds through a similar transition state TS1 and, again, first step is most favourable for 3b++, while in 2-methoxyetha-
Table 4 Relative stability of different protonation forms of 3a–3c in methanol and 2-methoxyethanol solutions (all values in kcal mol−1). Third pro-
tonation always occurs on the hydroxyl oxygen atom O1
Methanol
2-Methoxyethanol
System
Protonation step
N1
N2
0.0
O1
N1
N2
O1
1st protonation (3a → 3a+)
22.4
0.0
30.5
14.0
21.4
0.0
0.0
32.3
18.0
2
nd protonation (3a+ → 3a++
)
1st protonation (3b → 3b+)
nd protonation (3b+ → 3b++
17.4
0.0
0.0
0.0
26.6
11.0
16.7
0.0
0.0
0.0
28.6
15.6
2
)
1st protonation (3c → 3c+)
nd protonation (3c+ → 3c++
17.7
0.0
24.5
5.2
17.2
0.0
26.5
8.8
2
)
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Fig. 3 Reaction mechanism involving diprotonated 3a++–3c++ with methanol (in round brackets, R = −CH3) and 2-methoxyethanol (in square
brackets, R = −CH2CH2OCH3). Numbers indicate relative solution-phase Gibbs free energies obtained by the (SMD)/M06-2X/6-31+G(d) model (in
kcal mol−1).
nol, it is for 3a++, with a consistent conclusion that the reac- the possibility of its participation in the reaction. For 3a++, this
tion is thermodynamically again least favourable for 3c++.
is possible, but the activation barrier is by 0.9 kcal mol−1
The next step involves the 1,3-proton transfer from the higher and the reaction free energy is by 4.8 kcal mol−1 less
solvent –OH group to the imino N-atom (Fig. 3). Interestingly, exergonic than for the primary hydroxyl O-atom. Even more so,
this step is kinetically more demanding than the previous one such a route gives an intermediate that hinders further reac-
and defines the rate-limiting part of the overall transform- tions, making this pathway very unlikely. Also, knowing that
ation. Still, the process is more feasible by 2–3 kcal mol−1 for HCl is used in excess, we studied the reaction of the tricationic
3a++ and 3b++ than for 3c++, being in excellent agreement with 3a+++, with the protonated –OH group, in MeOH. The reaction
experiments. Moreover, the obtained results suggest that the is thermodynamically more favourable than for 3a++ by
reaction proceeds faster in 2-methoxyethanol, which again 2.8 kcal mol−1, but is kinetically less feasible by 2.0 kcal
agrees with experiments. For the latter, we did not observe any mol−1, which likely dominates, or might also contribute
notable structural or electronic effect that would help in inter- towards high reaction yields.
preting the higher reactivity in 2-methoxyethanol over metha-
Lastly, given that our analysis was based on inspecting
nol. What likely dominates is a markedly lower dielectric con- geometric and electronic parameters in the implicit sol-
stant of 2-methoxyethanol (ε = 17.2) relative to methanol (ε = vation, we also considered a potential effect of the bulk sol-
32.6), which likely favours a less polar transition state TS2 with vation on the reaction outcomes. For that, we performed a
a delocalized charge, compared to the preceding SP2 (Fig. 3) set of classical MD simulations with 3a++ and 3c++ in explicit
which, then, lowers the barrier. The latter stationary point has solvents. The obtained RDF graphs for the corresponding
a more localized and exposed positive charge, thus being more O(solvent)–Ccyano(3++) bonds (Fig. S8†) show that, in both
favoured in a more polar MeOH. Such a notion could poten- cases, 2-methoxyethanol solution allows for a closer solvent
tially suggest that alcohols with further lower polarities would approach towards the reactive –CN group, which is likely an
provide even more efficient environments for the reaction; yet additional effect contributing towards its higher reactivity
this has to be considered with certain care, since the utiliz- over MeOH. Moreover, the obtained peaks in the RDF graphs
ation of other solvents could exert some structural or elec- have the first maximum around 3 Å, which puts our SP1 geo-
tronic effects that would predominate over a simple solvent metries presented earlier in good qualitative agreement with
polarity effect, and, perhaps, reveal different reactivity trends.
these results, thus further confirming the validity of mechan-
Following TS2, the reaction offers the desired imidates that istic calculations.
are considerably more stable than the initial reactants, thus
Synthesis and antiproliferative activity of amidino-substituted
2-arylbenzoxazoles
making the overall reactions highly exergonic and feasible.
This is again by 4–5 kcal mol−1 more evident in 2-methox-
yethanol, which also contributes towards making it a better In order to explore the biological potential of 2-arylbenzoxa-
solvent over methanol, in agreement with experiments.
zoles functionalized with amidines, we performed the syn-
In concluding this section, computations consistently thesis of a new small library of systems. Previously, we devel-
predict a higher reactivity of 3a and 3b over 3c in both sol- oped robust methods for the preparation of a number of
vents, while confirming 2-methoxyethanol as a better environ- 6-amidino-2-aryl/heteroaryl-benzothiazole
derivatives
by
ment for the Pinner reaction, likely due to its lower dielectric merging 5-amidino-substituted 2-aminothiophenols with car-
constant, particularly suitable for a non-polar transition state boxylic acids in polyphosphoric acid (PPA).13,14,18 This method
during the rate-limiting step. To further validate these con- is commonly used for condensation of 2-aminophenols with
clusions, we performed several additional calculations. Given carboxylic acids, in which cyclization and formation of a ben-
that 2-methoxyethanol also contains a secondary O-atom, we zoxazole ring occur.36 Thus, novel amidino-substituted 2-aryl-
inspected its nucleophilicity towards the –CN group to exclude benzoxazoles 10a–10l were prepared from amidino-substituted
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Table 5 Synthesis of amidino-substituted 2-arylbenzoxazoles
Amidino-substituted 2-arylbenzoxazoles 10a–10l showed
strong antiproliferative activity on all tested cell lines at micro-
molar concentrations, stronger than or comparable to a stan-
dard chemotherapeutic 5-fluorouracil. None of the tested
systems revealed a significant selectivity towards particular cell
lines. The highest activity was observed for 10h, with a pyrimi-
dinium amidinic substituent at the C-6 position of the benzox-
azole skeleton, toward metastatic colorectal adenocarcinoma
(SW620) cells with an IC50 value of 0.16 μM. In general, ben-
zoxazole 10g with an imidazolinium amidinic substituent at
position C-6 and substituted with naphthalene at position C-2
proved to be the most active on all tested cell lines (IC50
=
0.82–2.8 μM). On the other hand, 2-arylbenzoxazoles with the
unsubstituted amidinium moiety at the C-5 and C-6 positions
had the weakest activity. Comparing the C-5 and C-6 positions
of the corresponding amidine substituents on the benzoxazole
backbone between derivatives 10a and 10b, 10c and 10d, 10e
and 10f, and 10i and 10j, no difference in position-dependent
antiproliferative activity was observed.
2-aminophenols 8a′–8c′, 8g and 8h′ and aromatic carboxylic
acids 9a–9c in PPA (Table 5). Since it was previously deter-
mined that methanesulfonates of amidine derivatives of 2-phe-
nylbenzothiazoles were more stable and soluble in water than
chlorides,16 all amidine benzoxazole derivatives were isolated
in the form of methanesulfonates by a simple two-step pro-
cedure in moderate overall yields.
Antiproliferative activity was tested in vitro on four human
tumour cell lines of metastatic colorectal adenocarcinoma
(SW620), hepatocellular liver cancer (HepG2), ductal pancrea-
tic adenocarcinoma (CFPAC-1), and cervical cancer (HeLa) by
MTT assay. The results are expressed as concentrations of com-
pounds required to inhibit cell growth by 50% (IC50) in
Table 6.
Conclusions
In summary, we developed an efficient synthetic protocol for
isomeric amidino-substituted 2-aminophenols from accessible
cyano-substituted 2-aminophenols by the Pinner reaction. The
key step during the synthesis of the crucial carboximidate
intermediate was characterized through mechanistic DFT cal-
culations, which offered insight into the electronic and geo-
metrical aspects determining the higher reactivity of 3a and 3b
over 3c, while confirming 2-methoxyethanol as a better solvent
over methanol. It also justified the necessity to perform the
reaction under excess gaseous HCl, thus affording substrate
dications with the protonated cyano group, which allows the
conversion into the desired imidates.
X-ray crystal structure study shows that amidino-substituted
aminophenols 8a′, 8b′ and 8c′ crystallized in a pure zwitter-
ionic form, while 8a and 8b exist in a zwitterionic form as
hemihydrochlorides. On the other side, amidino-substituted
derivatives 8d and 8h consist of one independent cation and
one chloride anion and did not crystallize as zwitterions, as
well as two dications, 8e and 8g, which contain two methane-
sulfonate and chloride anions, respectively.
Table 6 Antiproliferative activity in vitro of amidino-substituted 2-aryl-
benzoxazole methanesulphonates. 5-Fluorouracil (5-FU) was used as a
standard chemotherapeutic compound (internal laboratory control)
IC50a (μM)
Compound
SW620
HepG2
CFPAC-1
HeLa
The applicability of the described 2-aminophenols as new
building blocks for the preparation of 2-substituted-benzoxa-
zoles by a simple condensation reaction was demonstrated
through the synthesis of a small library of amidino-substituted
2-arylbenzoxazoles. Their antiproliferative activities against
four human tumour cell lines in micro- and submicro-molar
concentrations revealed the biological potential of this thus far
vaguely studied class of compounds. The synthesis of a larger
library of amidino-functionalized mono- and bis-2-substituted-
benzoxazoles by the presented method, together with the
evaluation of their antitumor, antitrypanosomal and DNA/RNA
binding activities, is currently in progress, and the results will
be published in due course.
10a
10b
10c
10d
10e
10f
10g
10h
10i
25 6.7
26 6.2
4.6 1.5
8.3 1.6
3.2 0.65
4.3 0.02
0.82 0.58
0.16 0.19
2.8 0.16
2.0 0.81
0.22 0.12
0.18 0.11
6.4 0.22
25 10
21 10
19 1.9
49 1.7
25 4.2
22 0.59
24 12
4.7 0.41
5.2 0.54
2.8 1.1
4.5 3.3
3.7 0.37
3.4 0.26
1.5 0.72
3.4 1.0
6.45 0.66
27 6.8
16 7.6
9.1 0.21
9.1 0.37
3.2 1.8
2.7 0.27
0.88 0.37
3.7 0.41
2.2 0.72
2.1 0.63
0.50 0.06
2.1 0.53
47.52 1.93
18 0.24
2.7 0.42
3.0 1.4
1.6 0.15
3.5 0.04
2.2 0.98
2.2 1.3
1.4 0.26
1.8 0.73
55.22 15.0
10j
10k
10l
5-FU
a IC50 values are concentrations that cause 50% inhibition of cancer
cell growth.
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M.-H. David-Cordonnier, K. Pavelić and G. Karminski-
Zamola, J. Med. Chem., 2010, 53, 2418–2432.
Conflicts of interest
There are no conflicts to declare.
13 L. Racané, S. Kraljević Pavelić, I. Ratkaj, V. Stepanić,
K. Pavelić, V. Tralić-Kulenović and G. Karminski-Zamola,
Eur. J. Med. Chem., 2012, 55, 108–116.
14 L. Racané, S. Kraljević Pavelić, R. Nhili, S. Depauw, C. Paul-
Constant, I. Ratkaj, M.-H. David-Cordonnier, K. Pavelić,
V. Tralić-Kulenović and G. Karminski-Zamola, Eur. J. Med.
Chem., 2013, 63, 882–891.
Acknowledgements
We greatly appreciate the financial support of the Croatian
Science Foundation under the project HRZZ-IP-2018-01-4379
entitled “Exploring the antioxidative potential of benzazole 15 L. Racané, M. Kralj, L. Šuman, R. Stojković, V. Tralić-
scaffold in the design of novel antitumor agents” and
University of Rijeka research grant UNIRI-biomed-18-133. L.H.
Kulenović and G. Karminski-Zamola, Bioorg. Med. Chem.,
2010, 18, 1038–1044.
wishes to thank the Croatian Science Foundation for a doctoral 16 L. Racané, R. Stojković, V. Tralić-Kulenović, H. Cerić,
stipend through the Career Development Project for Young
Researchers. L.H. and R.V. would like to thank the Zagreb
M. Đaković, K. Ester, A. Mišir Krpan and M. Radić
Stojković, Eur. J. Med. Chem., 2014, 86, 406–419.
University Computing Centre (SRCE) for granting compu- 17 L. Racané, M. Sedić, N. Ilić, M. Aleksić, S. Kraljević Pavelić
tational resources on the ISABELLA cluster.
and G. Karminski-Zamola, Anti-Cancer Agents Med. Chem.,
2017, 17, 57–66.
18 L. Racané, L. Ptiček, M. Sedić, P. Grbčić, S. Kraljević
Pavelić, B. Bertoša, I. Sović and G. Karminski-Zamola, Mol.
Divers., 2018, 22, 723–741.
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