The unsubstituted ortho-amidino benzoic acid: crystal structure, characterization and pK a determination

Article abstract of DOI:10.1007/s11224-016-0822-x

The simplest o-amidino benzoic acid—2-(amino(imino)methyl)benzoic acid—has been isolated in 23?% yield as a side product of the reaction of phthalonitrile and ammonia along with desired 1-imino-1H-isoindol-3-amine. This amidino acid was fully characterized by nuclear magnetic resonance, IR spectroscopy, mass spectrometry; pK_a values were also determined. X-ray diffraction study and quantum chemical calculations revealed that in the solid state it exists as a zwitterion that is stabilized by intermolecular hydrogen bonds.

Full text of DOI:10.1007/s11224-016-0822-x

Struct Chem
DOI 10.1007/s11224-016-0822-x
ORIGINAL RESEARCH
The unsubstituted ortho-amidino benzoic acid: crystal structure,
characterization and pK determination
a
1
1
1
•
Olga V. Hordiyenko
Roman I. Zubatyuk
•
Angelina V. Biitseva
2,3
•
Yuliya Yu. Kostina
4
2
1
•
Oleg V. Shishkin
•
Ulrich M. Groth
•
Mikhail Yu. Kornilov
Received: 23 April 2016 / Accepted: 8 August 2016
Ó Springer Science+Business Media New York 2016
Abstract The simplest o-amidino benzoic acid—2-
Introduction
(
amino(imino)methyl)benzoic acid—has been isolated in
3 % yield as a side product of the reaction of phthaloni-
trile and ammonia along with desired 1-imino-1H-isoindol-
-amine. This amidino acid was fully characterized by
2
Reaction of phthalonitrile and ammonia is one of the
preparations of 1-imino-1H-isoindol-3-amine (1) com-
monly known as 1,3-diiminoisoindoline, one of the most
important phthalogens [1–5]. The process could be carried
out by heating phthalonitrile with an excess of ammonia in
methanol at 90–100°C in autoclave [4, 6], by the elemental
sulfur-catalyzed reaction of phthalonitrile with ammonia
[7] or by passing ammonia through a refluxing solution of
phthalonitrile in methanol containing traces of sodium
methoxide [8, 9]. The yield of 1 in the last case [9] is
almost quantitative.
3
nuclear magnetic resonance, IR spectroscopy, mass spec-
trometry; pKa values were also determined. X-ray
diffraction study and quantum chemical calculations
revealed that in the solid state it exists as a zwitterion that
is stabilized by intermolecular hydrogen bonds.
Keywords Phthalonitrile Á 2-
(
Amino(imino)methyl)benzoic acid Á 1-Imino-1H-isoindol-
3
-amine Á Solid state structure Á Zwitterion
In order to obtain 1-imino-1H-isoindol-3-amine 1 by the
method [8], we succeeded in isolation along with the desired
compound 1 an unexpected product of this reaction. It
crystallized from mother methanolic liquor as rather large
colorless prisms in 23 % yield. The compound has been
identified as the first example of unsubstituted ortho-ami-
dino benzoic acid, namely 2-(amino(imino)methyl)benzoic
acid (2) (Scheme 1).
Dedicated to the memory of Prof. Oleg V. Shishkin and Prof. Ulrich
M. Groth.
Oleg V. Shishkin and Ulrich M. Groth: Deceased.
Electronic supplementary material The online version of this
article (doi:10.1007/s11224-016-0822-x) contains supplementary
material, which is available to authorized users.
Despite the simplicity of the molecule 2, it was not
described to date. Only a few examples of substituted
derivatives of 2: in benzene ring [10], in amidine function
[11, 12] and on the carboxyl group (methyl, ethyl, propyl,
isopropyl and tert-butyl esters) [13, 14] could be found in
the literature. Meanwhile, isomeric m- and p-amidino acids
were also described [15, 16].
&
Olga V. Hordiyenko
ov_hordiyenko@univ.kiev.ua
1
Department of Chemistry, Taras Shevchenko National
University of Kyiv, 64/13 Volodymyrs’ka str., Kyiv 01601,
Ukraine
2
3
4
SSI ‘‘Institute for Single Crystals’’ National Academy of
Science of Ukraine, 60 Lenina ave., Kharkiv 61001, Ukraine
Experimental section
Department of Inorganic Chemistry, V. N. Karazin Kharkiv
National University, 4 Svobody sq., Kharkiv 61122, Ukraine
Melting points were determined with a Boetius microscope
hot plate apparatus. Elemental analyses (C, H, N) were
conducted with a Vario Micro Cube. NMR spectra were
Fachbereich Chemie, Universit a¨ t Konstanz, 78457 Konstanz,
Germany
123

Struct Chem
O
Anal. calc. for C H N O : C, 58.53; H, 4.91; N, 17.06.
8
8 2 2
NH2
N
-
Found: C, 59.06, H, 5.28, N, 17.47. IR (KBr, cm ): 1580,
1
CN
CN
NH3
O
NH2
–
1539, 1383 (COO ), 3090, 2949 (NH). H NMR (D O,
1
+
2
MeONa
400 MHz, 25°C): d 7.38 (d, J = 7.6 Hz, 1H, 3-H), 7.43 (t,
J = 7.6 Hz, 1H, 4-H), 7.50 (t, J = 7.6 Hz, 1H, 5-H), 7.59
NH
NH
2
1
major
2 minor
1
3
d, J = 7.6 Hz, 1H, 5-H). C NMR (D O, 100 MHz,
(
2
-
?
Scheme 1 Reaction of phthalonitrile with ammonia and the isolated
products 1 and 2
25°C): d 171.5 (COO ), 166.9 [C(-NH ) = NH ], 135.0
C-1), 129.8, 127.8, 126.7, 125.9 (C-2), 125.8.
2
2
(
Hydrate of 2-(amino(imino)methyl)benzoic acid (2b)
was obtained on crystallization of acid 2 from distilled
water. Mp 179–181 °C. Anal. calc. for C H N O 9 1.2
recorded on a Mercury (Varian) 400 spectrometer
1 13
400 MHz H and 100 MHz for C) with tetramethylsi-
(
8
8 2 2
lane as an internal standard and D O as a solvent.
2
H O: C, 51.72; H, 5.64; N, 15.08. Found: C, 51.79, H, 5.25,
2
-
1
1
N, 14.92. IR (KBr, cm ): 3399 br, 1673, 1581, 1393. H
ESI–MS analysis was performed with an SCIEX 3200
Q-Trap mass spectrometer (Applied Biosystems, Concord,
Ontario, Canada) equipped with a Turbospray ion MS
conditions: Scan type, Enhanced MS; Positive ion mode,
mass range 100-500 amu; declustering potential 30 V;
entrance potential 10 V; curtain gas 20 PSI; ion source gas
NMR (D O, 400.13 MHz, 25°C): d 7.42 (d, J = 7.6 Hz,
2
1H, 3-H), 7.46 (t, J = 7.6 Hz, 1H, 4-H), 7.53 (t,
J = 7.6 Hz, 1H, 5-H), 7.63 (d, J = 7.6 Hz, 1H, 5-H). MS
?
ESI, amu): m/z = 329 ([2M?H] ), 165 ([M?H] ), 148
?
(
(
?
?
?
[M?H] -17), 147 ([M ? H] -18), 130 [C H (CN)CO] .
6
4
0
PSI; ion spray voltage 5000 V; source temperature
Method of the preparation of 1-imino-1H-isoindol-3-
ambient C; sample flow rate 5 lL/min. Samples were
dissolved in 50 % MeOH solution and infused in the ESI
amine (1) with NaOH. Phthalonitrile (100 g, 0.78 mol) was
mixed with methanol (500 mL). NaOH (0.92 g, 0.023 mol)
was added, and ammonia was bubbled through the stirred
suspension for 45 min at room temperature. The mixture
was brought to reflux and maintained at this temperature
for 3 h while stirring and passing ammonia. Phthalonitrile
dissolved and compound 1 precipitated. Upon cooling 50 g
of bluish crystals of 1 were filtered off. The mother liquor
was evaporated under reduced pressure to afford yellowish
crude crystals. Methanol (200 mL) was added, and the
mixture was heated to dissolve the crystals. After cooling
and standing overnight, an additional amount of 1 (67 g, by
Ò
source by Dionex UltiMate 3000 HPLC.
The pH-metric titrations were performed at 25 °C using
a MOLSPIN 1000 automatic titration system with a
microcombined glass-Ag/AgCl electrode Rusell CMA
W711. SuperQuad 5.2 [17] program was used for calcu-
lation of pK values. To determine the pK values, HNO
3
-
4 9 10 mol dm ) and NaOH (0.1 mol dm ) solu-
3
-3
-3
(
tions were used. Sample concentrations were on the order
-3
-
3
of 2 9 10
mol dm
(2.338 mg of acid 2). Stability
constants were calculated from titrations carried out using
3
total volumes of 2 cm . Equivalence point was determined
1
H NMR) was filtered off making a total yield of 80 %.
at 0.0882 in volume units.
X-ray Crystallography for structure 2a. Intensity data
were collected at 100 K on the ‘‘Xcalibur-3’’ diffrac-
tometer with graphite monochromated MoKa radiation, x-
scans, 2hmax = 60°. Crystal data: C H N O ÁCH OH
2
-(Amino(imino)methyl)benzoic acid (2) and solvate
with methanol 2a. Phthalonitrile (100 g, 0.78 mol) was
added to dry methanol (500 mL). Sodium methoxide
8
8
2
2
3
(
1.25 g, 0.023 mol) was then added to the mixture, and
˚
Mr = 196.21), triclinic, a = 7.0293(5) A, b = 7.4905(5)
(
anhydrous ammonia was bubbled through the stirred sus-
pension for 45 min at room temperature. The mixture was
brought to reflux and maintained at this temperature for 3 h
while stirring and passing ammonia. Phthalonitrile dis-
solved and the green-gray crystals of 1-imino-1H-isoindol-
˚
˚
A, c = 10.5106(7) A, a = 70.977(6)°, b = 86.476(5)°,
3
˚
ꢀ
c = 65.214(6)°, V = 473.17(6) A , space group P 1,
Z = 2, l(MoKa) = 0.11 mm , d = 1.377 g sm . 4953
reflections were measured, 2750 were unique,
Rint = 0.016. The structure was solved by the direct
methods and refined by full-matrix least squares techniques
-1
-1
Á
3
-amine 1 precipitated. Upon cooling 37 g of bluish crys-
tals of 1 were filtered off. The mother liquor was evapo-
rated under reduced pressure to give yellowish crude
crystals. Then methanol (200 mL) was added, and the
mixture was heated to dissolve the crystals. After cooling
and standing overnight, an additional amount of 1 (37 g)
was filtered off. The next day large colorless prisms of
2
on all F data using SHELX package [18]. All non-hy-
drogen atoms were refined with anisotropic thermal
parameters. Hydrogen atoms were located in Fourier dif-
ference maps and freely refined. The refinement converged
at wR = 0.111 (all data), R1 = 0.037 (2232 reflections
2
with F [ 4r(F)), S = 1.06.
2
-(amino(imino)methyl)benzoic acid as solvate with
X-ray Crystallography for structure 2b. Intensity data
were collected at 298 K on the ‘‘Xcalibur-3’’ diffrac-
tometer with graphite monochromated MoKa radiation, x-
methanol 2a suitable for X-ray study precipitated from the
filtrate. After filtering and drying, the colorless powder
crystals of 2 (30 g, 23 %) were obtained, mp 179–180 °C.
1
23

Struct Chem
scanning, 2hmax = 52°. Crystal data: C H N O ÁH O
all cases geometry optimizations were followed by fre-
quency calculations to ensure that stationery point found on
potential energy surface is either minima or transition state.
Intrinsic reaction coordinate scan was performed as mass-
weighted coordinates using Gonzalez-Schlegel method
[21].
8
8
2
2
2
˚
A,
(
Mr = 182.18),
monoclinic,
a = 4.2889(6)
˚
˚
b = 23.215(2) A, c = 8.969(1) A, b = 96.70(1)°,
3
V = 886.9(2) A , space group P2 /c, Z = 4,
˚
1
-
1
-1
l(MoKa) = 0.106 mm
reflections were measured, Rint = 0.103. The structure was
,
d = 1.364 gÁsm
.
1744
solved by the direct methods and refined by full-matrix
2
least squares techniques on all F data using SHELX
package [18]. Positions of the hydrogen atoms were located
in Fourier difference maps. C–H atoms were refined using
riding model with Uiso = 1.2Ueq of the carrier atom,
whereas positions and Uiso of the N–H and O–H hydrogen
atoms were refined independently. Signs of non-merohe-
dral twinning due to rotation around (1 0 0) by 180° were
observed for the crystal structure. Final refinement was
done against partially overlapping set of reflections (HKLF
Results and discussion
Probable formation pathways
The described metha- and para- parent amidino benzoic
acids [16, 22–24] were prepared from 3- or 4-cyanoben-
zoic acids, respectively, through iminoester hydrochloride
and subsequent conversion to amidine hydrochloride via
treatment with ammonia in ethanol according to Pinner
protocol [25]. The most similar to that observed in the
transformation of phthalonitrile is the recently reported by
Nocera et al. [26–28] method of 3-amidinium benzoate
preparation from corresponding 3-cyanobenzoic acid by
base-catalyzed ammonolysis of the nitrile group. Com-
pared to the previous approaches, this method consists of
converting the nitrile group into free base imidate ester
carboxylate and following treatment with an excess of
5
) generated with TWINROTMAT program of PLATON
software [19]. Refined weight of the main twin component
weight is 0.757(3). The refinement converged at
wR = 0.117 (all data), R1 = 0.061 (861 reflections with
2
F [ 4r(F)), S = 0.927.
The values of selected geometrical parameters are given
in Table 1. The final atomic coordinates and crystallo-
graphic data for molecules 2a and 2b have been deposited
with the Cambridge Crystallographic Data Centre, 12
Union Road, CB2 1EZ, UK (fax: ?44-1223-336033;
e-mail: deposit@ccdc.cam.ac.uk) and are available on
request quoting the deposition numbers CCDC 1487525 for
NH Cl.
4
Phthalonitrile is known to form diiminoester dihy-
drochloride in the reaction with dry ethanolic HCl in
chloroform, and diiminoester dihydrochloride gives
monohydrochloride of 1 with an excess of ammonia in a
dry alcohol [6]. An attempted crystallization from n-bu-
tanol turns it into a compound identified by Elvidge and
Linstead as hydrochloride of 3-iminoisoindolin-1-one (5)
2
a and CCDC 1440890 for 2b.
Computational details. Quantum chemical calculations
of amidino acid 2, its polyhydrated complex and dimers
were performed at B3LYP/6-311?G** level of density
functional theory using PC GAMESS 7.0 software [20]. In
(Scheme 2). It is also possible to isolate a semi-product of
the ethanol addition to one of the phthalonitrile cyano
groups by carrying out the reaction in ethanol/chloroform
mixture saturated with gaseous HCl and staying at 0 °C
during 2 weeks [29]. The more important observation is
that at least one nitrile group of 1,3-dicyanobenzene could
be transformed into amidine function leading to
Table 1 Selected geometrical parameters for acid 2 according to
X-ray diffraction study
Structure 2a
Structure 2b
˚
Distances (A)
N(1)–C(8)
1.3066(12)
1.3129(12)
1.2677(11)
1.2474(11)
1.5160(13)
1.4905(12)
1.307(4)
1.304(4)
1.244(3)
1.263(3)
1.516(4)
1.490(3)
3-cyanobenzamidinium chloride [28]. The reaction pro-
N(2)–C(8)
ceeds at the same conditions used for 3-amidinium ben-
zoate preparation.
O(1)–C(7)
O(2)–C(7)
Inasmuch as 3-methoxy-1H-isoindol-1-imine (3) [4, 30]
or 1,1-dimethoxy-1H-isoindol-3-amine (4) [31] are con-
sidered as intermediates in the sodium methoxide catalyzed
reaction of phthalonitrile and ammonia, and they can be
formed through the corresponding sodium derivatives
C(1)–C(7)
C(2)–C(8)
Angles (°)
C(6)–C(1)–C(7)
C(2)–C(1)–C(7)
C(3)–C(2)–C(8)
C(1)–C(2)–C(8)
C(2)–C(1)–C(7)–O(2)
C(1)–C(2)–C(8)–N(2)
120.16(9)
121.67(8)
115.84(9)
123.80(9)
-9.4(1)
118.6(3)
122.5(3)
117.8(3)
121.8(3)
27.2(5)
[
32, 33]; therefore, one of the possible routes of amidino
acid formation may consists of partial hydrolysis of such
species. Moreover, compound 3 may be considered as the
cyclic tautomeric form of the mono imidate ester derived
from 1,2-dicyanobenzene.
96.3(1)
-124.5(4)
123

Struct Chem
NH
N
NH2
N
CN
CN
MeOH
or
H O
2
MeONa
OMe
O
MeO
4
OCH3
3
O
NH2
NH2
NH
N
O
2
OH
OH
H O
2
NH
NH
NH3
NH2
1
5
Scheme 2 Plausible formation pathways of amidino acid 2
It seems possible to assume that formation of acid 2
could be a result of 1-imino-1H-isoindol-3-amine 1
hydrolysis via isoindolinone 5 intermediate under catalytic
action of NaOH, which can be present in the reaction
mixture due to traces of moisture. Compound 1 is known to
be quite stable in water at r.t. but is hydrolyzed to the
corresponding iminoimide
Scheme 2).
This pathway may be supported by the fact that similar
5 by short boiling [5]
(
NH2
N,N’-di(p-tolyl)substituted derivative of amidino acid 2
was recently isolated from the reaction between phtha-
laldehyde and p-toluidine [34], and its formation was
supposed to occur through isoindolinone 5 type interme-
diate that was expected to be hydrolyzed by water.
NH2
O
O
Fig. 1 Molecular structure of compound 2 according to X-ray
diffraction study. Thermal ellipsoids of non-hydrogen atoms are
shown at 50 % probability level
In our attempts to model plausible experimental condi-
tions of acid 2 formation, phthalonitrile ammonolysis was
carried out in MeOH taken without a drying step in the
presence of catalytic amounts of NaOH instead of NaOMe.
Nevertheless, no acid 2 has been isolated, and instead,
˚
˚
double C = N bonds (1.336 A and 1.279 A, respectively
35]).
It is well known that natural amino acids exist as
[
1
-imino-1H-isoindol-3-amine 1 was obtained in 80 %
yield. Thus, the method of 1-imino-1H-isoindol-3-amine 1
preparation can be scaled up and can be significantly
simplified compare to the common procedures [8, 9].
zwitterions in solution or in crystals, but prefer non-ionic
canonical form in gas phase [36–38]. Therefore, it is
essential to check either the ability of acid 2 to exist in
zwitterionic form is an intrinsic property of this molecule
or this form is stabilized by some crystal packing effects.
First-principles quantum chemical calculations demon-
strate that zwitterion 2 observed in the crystal is unstable in
the case of the isolated molecule. Moreover, the conforma-
tion of the molecule observed in crystal, with carboxyl group
lying almost in the plane of benzene ring and amidine group
oriented almost orthogonal to this plane, corresponds to a
transition state of the isolated molecule (Fig. 2).
X-ray and quantum chemical calculations
In crystal phase, the compound 2 was obtained as a solvate
with methanol in equimolar ratio (structure 2a) or as a
hydrate (structure 2b) on crystallization from water. The
acid molecule 2 exists in zwitterionic form in both struc-
tures as shown in Fig. 1. This is undoubtedly confirmed by
the positions of hydrogen atoms, which were refined
without any constraints for 2a, and also by the values of the
This transition state connects two almost isoenergetic
0
minima on the potential energy surface (the DG is only
C–N and C–O bond lengths: the C–O bond lengths
˚
(
Table 1) are very close to mean value of 1.254 A for
1.2 kcal/mol), in none of which molecule has zwitterionic
carboxylate anions [35] and the C–N bond lengths are also
intermediate between mean values for single C–N and
structure. One minimum corresponds to canonical structure
of amidino acid with the proton of amidine group moved to
1
23

Struct Chem
Fig. 2 Intrinsic reaction
coordinate scan for proton
transfer in acid 2 (B3LYP/6-
3
11?G**)
the oxygen atom of carboxylic group. In another minimum,
other one new C-O bond is formed and resulting molecule
has a hypothetical structure of 3,3-diamino-2-benzofuran-
This unambiguously confirms that the zwitterion of 2 is
stabilized by intermolecular hydrogen bonds. It is inter-
esting to note that the carboxyl group in the complex with
seven water molecules is rotated against the plane of
benzene ring by about 60°. Such orientation of the carboxyl
group prevents hindering of one of the oxygen atoms from
formation of hydrogen bonds with solvate molecule. Most
probably, in solution the molecule will have the similar
conformation. Such conformation of the molecule is closer
to the left minimum shown in Fig. 2. Taking into account
rather high energy of zwitterionic transition state for the
isolated molecule, one can draw a conclusion that when the
molecule is derived from a solution in protic solvent, it will
be converted into canonical form of amino acid 2, rather
than into diaminobenzofuranic form 6.
1(3H)-one (6) (Scheme 3). The value of energy barrier
between these minima is 17.8 kcal/mol.
The stabilization of acid 2 zwitterionic form in crystals
a and 2b is probably provided by the formation of a three-
2
dimensional network of intermolecular hydrogen bonds as
shown in Fig. 3. The geometrical parameters of the
H-bonds are listed in Table 2. Each of the oxygen atoms of
the carboxyl group forms two intermolecular hydrogen
bonds, and each nitrogen atom of amidine group is a proton
donor for two H-bonds.
Similarly, a formation of intermolecular hydrogen bonds
can lead to the stabilization of the zwitterionic form in
solution. In order to evaluate this hypothesis, we have
performed quantum chemical calculations of the hydrated
zwitterions of 2. Initially, the structure of the complex was
built starting from experimental geometry with all proton
donors and acceptors sites surrounding molecule in a
crystal replaced by water molecules. In the optimized
complex, the molecule has the zwitterionic form (Fig. 4).
Arginine amino acid which contains carboxyl and
guanidine groups is known to possess a high propensity to
form clusters when electrosprayed into the gas phase
[39, 40]. Quantum chemical calculations of such clusters
performed at DFT level of theory indicate that the arginine
dimers are stabilized due to formation of intermolecular
hydrogen bonds between carboxyl and guanidine fragments
[41]. The dimer of zwitterions of arginine is more stable than
the dimer of molecules in canonical form by 9.3 kcal/mol.
In the crystal phase molecules 2 form such centrosym-
metric dimers which are bonded by four N–H…O inter-
molecular hydrogen bonds. Quantum chemical calculations
indicate that in the gas phase two dimers of molecules 2 are
possible (Fig. 5). One of them has C2h symmetry, and its
structure is very similar to observed in the crystal. Another
O
O
O
NH2
O
NH2
H N
2
2
NH2
6
Scheme 3 Hypothetical cyclization of amidino acid 2 into benzofu-
ranone 6
dimer has C symmetry, and the conformation of monomers
2
123

Struct Chem
Fig. 3 Intermolecular hydrogen
bonds (dashed lined) formed in
the crystal of 2a (on the left)
and 2b (on the right)
Table 2 Geometrical
˚
D-H…A
Symmetry code
Distance
Angle
D-H…A, °
parameters (A, °) of hydrogen
bonds in the crystal of 2a and 2b
H…A, A˚
D…A, A˚
Structure 2a
N1-H1A…O2
N1-H1B…O1
N2-H2A…O2
N2-H2B…O1S
O1S-H1S…O1
(1-x,1-y,1-z)
(1-x, -y,1-z)
(1-x,1-y,1-z)
2.183(15)
1.918(14)
1.969(15)
1.966(15)
1.839(19)
2.9238(12)
2.8164(12)
2.8029(12)
2.8301(13)
3.3236(15)
141.8(13)
172.1(13)
152.0(13)
177.3(14)
132.2(10)
(-x,1-y,1-z)
Structure 2b
N1-H1A…O2
N1-H1B…O1
N2-H2A…O1
N2-H2B…O1S
O1S-H1SA…O2
O1S-H1SB…O2
(x, 1.5-y, -0.5?z)
(1?x, y, z)
2.02(3)
1.93(3)
1.86(3)
2.10(4)
1.88(6)
1.96(5)
2.940(3)
2.841(4)
2.795(4)
2.866(4)
2.762(4)
2.841(4)
171(3)
163(3)
176(4)
143(3)
167(4)
170(4)
(x, 1.5-y, -0.5?z)
(x, y, z-1)
(x-1, y, z)
is similar to those predicted for polyhydrated amidino acid
. The second dimer is more stable by 10.5 kcal/mol,
2
probably, due to a formation of two intramolecular hydrogen
bonds. Also, absence of bifurcated intermolecular hydrogen
bonds in the second dimer leads to higher value for the
interaction energy (-63.2 kcal/mol in second dimer as
compared to -54.7 kcal/mol for the first dimer).
Thus, the theoretical calculations lead to a conclusion
that zwitterionic form of compound 2 is stabilized in the
crystal due to the formation of intermolecular hydrogen
bonds. The formation of similar hydrogen bonds in solution
can also stabilize the zwitterion of 2. In the absence of
intermolecular hydrogen bonds, the molecule can exist in
either canonical non-ionic form or as benzofuranone 6.
Probably, this also explains the insolubility of acid 2 in
most of organic solvents, its moderate solubility in DMSO
and a rather high water solubility as well as a high melting
point. Acid 2 crystallizes from water without decomposi-
tion in the form of a long prism hydrate 2b.
Fig. 4 Optimized structure of the complex of acid 2 with seven water
molecules (B3LYP/6-311?G**)
1
23

Struct Chem
Fig. 5 Structure of dimers of
acid 2 optimized using B3LYP/
6
-311?G** method
The ability of amidino acid 2 to form dimers is an
It gives unequivocal evidence of the parent ion (m/z 329)
relation with its daughter one at 165 m/z. The further
isolation of the ion at 165 m/z and its subsequent frag-
mentation in MS/MS mode gives the ions at 148, 147 and
130 m/z. It seems that these ions correspond to the parent
ion 165 m/z daughter fragments and transformation
according to the Scheme 4. We suggest that the parent ion
fragmentation undergoes two cleavage paths: the first fol-
attractive feature of the molecule as a potential substrate,
which depending on conditions could selectively bind to
complementary centers of other molecules. Amidinium–
carboxylate salt bridge is considered as a simple model of
Arg–Asp interactions that used for PCET (Proton-Coupled
Electron Transfer) studies in biological systems [42]. The
arginine–aspartate (Arg-Asp) or the arginine–glutamate
(
Arg-Glu) salt bridges which involve guanidinium–car-
lowed by elimination of NH gives rise peak of 148 ion
3
boxylate interactions play an important role in three-di-
mensional structure of proteins [43], and positively charged
guanidinium group of an arginine is definitive binding site
of the enzymes [44]. This explains the design of amidines
to mimic the function of the arginine moiety in pep-
tidomimetics such as thrombin, factor Xa or VIIa inhibi-
tors, integrin receptor antagonists, nitric oxide synthase
inhibitors [45].
(parent-17) 2A that is isomeric to probable benzofuranone
6. The second one consists of the loss of 18 units (H O
2
molecule) and formation of m/z 147 2B—an open form of
possible cyclic iminoisoindoline 3 (Scheme 2). Both of
these ions undergo further fragmentation. Loss of water
from m/z 148 ion results in a 130 m/z peak of acyl cation
2C. Fragmentation pathway of m/z 147 ion consists of
ammonia elimination and results in the same base peak
1
30 mass units of 2C. Its structure is similar to those
observed for the corresponding 2-cyanopyridine-3-car-
boxylic acid [46].
Mass spectrometric analysis
Mass spectrum of acid 2 (see Supporting Information)
exhibits the weaker ion peak at m/z 329 amu which cor-
responds to the dimer of 2. The spectrum also contains the
peaks at m/z 165, 148, 147 and 130. The ion m/z 329 was
isolated by the entrance quadrupole with further fragmen-
tation at low and high collision energies in MS/MS mode.
Mass spectrum of hydrate 2b exhibits the most intensive
ion peak of dimer of 2 at m/z 329 amu and less intensive at
m/z 165. Its subsequent fragmentation in MS/MS mode
gives the same ions at 165, 148, 147 and 130 m/z. Thus,
mass spectrometry provides evidence for stability of
zwitterionic form of acid 2 in the gas phase due to the
Scheme 4 Fragmentation of
amidino acid 2 ion
O
O
OH
O
-
NH3
- H O
2
O
O
N
NH
NH2
OH
NH2
2A m/z 148
2
m/z 165
NH2
2C m/z 130
O
O
-
NH3
-
H O
2
NH
NH2
NH
NH2
2B m/z 147
123

Struct Chem
formation of intermolecular hydrogen bonds where proto-
nated amidinium group of one zwitterion 2 coordinates to
carboxylate of another similar to arginine species [39, 40].
Two consequent deprotonation steps were observed
upon decreasing the acidity. The first equilibration
(pK = 3.14) involves the carboxylic group deprotonation
1
that is the most acidic site. While its pK value is of the
same order as calculated for 3- and 4-isomers, though slight
increase of acidity is observed.
NMR and IR spectra
D O is a solvent of choice for NMR spectra recording due
2
The second dissociation step (pK = 11.48) occurs at
2
to the best solubility of the acid 2 although the exchange-
1
able protons could not be observed. H NMR spectrum
the protonated amidinium group. This group is less acidic
and thus more basic than those from 3- (pK = 10.90) and
2
exhibits ABCD spin system with resonances at d 7.38 (d),
7
4-amidino benzoic acids (pK = 10.17). pK value of acid
2
2
1
.43 (t), 7.50 (t) and 7.59 (d) ppm. The C spectrum
3
2 and hence its basicity are comparable to those of ben-
zamidine (pK = 11.6 in water) [47]. It is a result of
reveals characteristic signals at d = 171.5 of carboxylic
a
–
COO ) and 166.9 ppm of amidine group [C(-
(
amidine group is permitted to twist out of the aryl ring
plane (Fig. 1). As one can assume, the electron-with-
drawing carboxylic group in the ortho-position with respect
to amidine moiety has no influence on basic properties of
the molecule 2.
?
NH ) = NH ].
2
2
-
In IR acid 2 exhibited characteristic COO group
-
1
absorption bands at 1580, 1539 and 1383 cm resulted in
symmetric and asymmetric of C–O stretching vibrations.
NH protons revealed intensive absorption bands at 3090 na
Isoelectric point at which all species of the molecule 2
are present in zwitterion form was calculated on the basis
of its pKs:
-
1
2
949 cm
which means that they are involved in a
hydrogen bond.
pK þ pK
1
2
pH ¼
¼ 7:31
pKs determination
i
2
To determine the pKs values of acid 2 protolytic dissoci-
ation, potentiometric titration was employed. The pKs
determined are reported in Table 3 and are compared with
calculated values of 3- and 4-(amino(imino)methyl)ben-
zoic acids.
Conclusions
-(Amino(imino)methyl)benzoic acid was isolated as a by-
2
product in phthalonitrile base-catalyzed ammonium addi-
tion. This amidino acid behaves like a bifunctional mole-
cule possessing both carboxylic and amidinic groups very
weakly influenced by one another due to their almost
orthogonal space arrangement as it follows from single
crystal X-ray diffraction study of two, its crystallosolvates
with methanol and water. Quantum chemical calculations
of the zwitterions clearly indicate their stabilization by
intermolecular hydrogen bonds, which could occur either
in crystal or in solution. In the solid state acid 2 exists as
zwitterionic form that is stabilized by the intermolecular
hydrogen bonds. Two acid–base dissociation steps of acid
Table 3 Dissociation
constants
pKs
of
isomeric
amino(imino)methylbenzoic acids obtained from potentiometric
titration and calculated
Compound
pKa1
pKa2
O
3.14(3)
11.48(3)
3
.20 ± 0.36*
OH
NH2
NH
O
3.66 ± 0.10*
10.90 ± 0.20*
2 were detected, and appropriate pKs and pH were
i
determined.
OH
An amidino acid 2 is a new type of rare class of amidino
acids, which by themselves or being modified both on
aromatic ring and at functional groups, will be able to
manifest itself in discovery of arginine mimetics, molecu-
lar recognition, self-organization or the formation of
supramolecular structures.
HN
HN
NH2
O
3
.40 ± 0.10*
10.17 ± 0.20*
OH
It is worth mentioning that the substituted benzamidines
do play a role in medicinal chemistry: among them are
antiparasitic agents [48], DNA binders [49, 50], inhibitors
of plasmin and d-plasmin [51] etc. Moreover, benzamidine
derivatives have also found an application as ligands in
NH2
Ò
Calculated data; taken from SciFinder (Advanced Chemistry
Development (ACD/Labs) Software V11.02 (Ó 1994-2015 ACD/
Labs)
1
23

Struct Chem
coordination chemistry and their metal complexes have
been used as catalysts for cross-coupling reactions [52–54],
e-caprolactone polymerization [55, 56], etc. Additionally,
the derivatives of m- and p-isomeric amidino benzoic acids
have shown biological activities as fibrinogen receptor
antagonists [57] and selective factor Xa inhibitors [58, 59].
In this context, we believe that amidino acid 2 will become
useful in materials science, organic synthesis, and medical
research as promising building block. Practical procedure
for the synthesis of compound 2 is now under progress and
will be published soon.
19. Spek AL (2009) Acta Cryst. D65:148–155
2
2
2
0. Granovsky AA http://classic.chem.msu.su/gran/gamess
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8:2247–2252
3
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1
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Supporting information
29. Young MB, Barrow JC, Glass KL, Lundell GF, Newton CL,
Pellicore JM, Rittle KE, Selnick HG, Stauffer KJ, Vacca JP,
Williams PD, Bohn D, Clayton FC, Cook JJ, Krueger JA, Kuo
LC, Lewis SD, Lucas BJ, McMasters DR, Miller-Stein C, Pietrak
BL, Wallace AA, White RB, Wong B, Yan Y, Nantermet PG
(2004) J Med Chem 47:2995–3008
For this article that contains the copies of NMR, IR and
mass-spectra, potentiometric titration and X-ray diffraction
data is available online to authorized users.
3
3
0. Borodkin VF, Postnikov VI (1974) Chem Abstr 81:105273
1. Baumann F, Bienert B, R o¨ sch G, Vollmann H, Wolf W (1956)
Angew Chem Int Edit 68:133–150
2. Leznoff CC, Lever ABP (1996) Phthalocyanines: properties and
applications. VHC Publishers, New York
3. Borodkin VF (1958) Zh Prikl Khim (USSR) 5:813–816
4. Takahashi I, Nishiuchi K, Miyamoto R, Hatanaka M, Uchida H,
Isa K, Sakushima A, Hosoi S (2005) Lett Org Chem 2:40–43
5. Orpen AG, Brammer I, Allen FH, Kennard O, Watson DG,
Taylor R (1994) In: Burgi H-B, Dunitz JD (eds) Structure cor-
relation. Wiley-VCH, Weinheim
Acknowledgments We thank Konstanz University for visiting pro-
fessorship (to O. V. H.) and Dr. Nikolay Youhnovski for providing
ESI mass spectrum measurements. We are grateful to Dr. Agnieszka
3
Dobosz (Wroclaw Medical University, Poland) for carrying out pK
determination. We would like to thank Dr. Svetlana Shishkina for
X-ray and computational discussion, and Dr. Andrei Esaulenko
a
3
3
(
ALSI-CHROM, Kyiv) for ESI–MS analysis discussion.
3
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