Substituent effects on the basicity (p: K a) of aryl guanidines and 2-(arylimino)imidazolidines: Correlations of pH-metric and UV-metric values with predictions from gas-phase ab initio bond lengths

Article abstract of DOI:10.1039/c7nj02497e

The dissociation constants of two related series of 2-(arylimino)imidazolidine and aryl guanidine α₂-adrenoceptor antagonists (35 compounds in total) were measured by potentiometric titrations and by UV-spectrophotometry using the 96-well microtitre plate method. The experimental values obtained using both methods were quite consistent and showed a very good agreement with the pK_a values calculated using the AIBLHiCoS methodology, which uses only a single bond length obtained using ab initio calculations at a low level of theory. The prediction power of the imidazolidine and guanidine set of compounds was very good with deviations typically <0.30 and <0.24 pK_a units, and a mean absolute error (MAE) of 0.23 and 0.29, respectively. The study of the quantitative effect of diverse substituents on the basicity of aryl guanidine and 2-(arylimino)imidazolidine derivatives is useful for medicinal chemists working with biologically relevant guanidine-containing molecules.

Full text of DOI:10.1039/c7nj02497e

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Substituent effects on the basicity (pKa) of aryl
guanidines and 2-(arylimino)imidazolidines:
correlations of pH-metric and UV-metric values with
predictions from gas-phase ab initio bond lengths†
Cite this: New J. Chem., 2017,
41, 11016
Christophe Dardonville, *a Beth A. Caine,bc Marta Navarro de la Fuente,a
bc
Guillermo Martın Herranz,a Beatriz Corrales Mariblancaa and Paul L. A. Popelier
´
The dissociation constants of two related series of 2-(arylimino)imidazolidine and aryl guanidine
a2-adrenoceptor antagonists (35 compounds in total) were measured by potentiometric titrations and by
UV-spectrophotometry using the 96-well microtitre plate method. The experimental values obtained
using both methods were quite consistent and showed a very good agreement with the pKa values
calculated using the AIBLHiCoS methodology, which uses only a single bond length obtained using
ab initio calculations at a low level of theory. The prediction power of the imidazolidine and guanidine
set of compounds was very good with deviations typically o0.30 and o0.24 pKa units, and a mean
absolute error (MAE) of 0.23 and 0.29, respectively. The study of the quantitative effect of diverse
substituents on the basicity of aryl guanidine and 2-(arylimino)imidazolidine derivatives is useful for
medicinal chemists working with biologically relevant guanidine-containing molecules.
Received 10th July 2017,
Accepted 28th July 2017
DOI: 10.1039/c7nj02497e
rsc.li/njc
clonidine (i.e. N-(2,6-dichlorophenyl)-4,5-dihydro-1H-imidazol-
2-amine) used, among other applications, as an antihyperten-
1. Introduction
The guanidine group of the amino acid arginine is ubiquitous sive drug and for controlling neuropathic pain (Fig. 1).21 As both
in Nature. In its protonated state, several resonance forms that the guanidine and 2-aminoimidazoline groups are strong bases,
delocalize the cationic charge of the guanidinium cation over they are protonated at physiological pH. Thus, this physico-
the entire functional group contribute to the high basicity chemical property may impair the absorption and/or limit the
of guanidine (pKaH = 13.6 in water).1 The H-bonding ability of uptake of guanidine-containing molecules through biological
guanidine makes this group a valuable tool for the design of membranes, including the blood–brain barrier,22,23 a drawback
compounds for different pharmacological applications such as that is highly relevant for centrally acting drugs.
DNA binding drugs (e.g. anticancer and antiprotozoal agents),2–11
The protonation state of a drug molecule determines its
analgesic compounds,12–14 a1-noradrenaline receptors (e.g. a1-AR lipophilicity, solubility in biological fluids, ability to cross biological
antagonists for the treatment of benign prostate hyperplasia),15 membranes, degree of protein binding, and its capacity to bind to
or a2-AR modulators for neuropsychiatric therapies.16–19
biological targets. Hence, knowing the dissociation constants
Centrally acting a2-adrenoceptor antagonists are potentially (pKa) of drugs is crucial because this parameter affects the key
useful pharmacological tools for the treatment of depression and pharmacokinetic properties such as administration, distribution,
schizophrenia.16 Among these, arylguanidines and 2-(arylimino)- metabolism and excretion (ADME).24
imidazolidines represent an important family of a-adrenoceptor
One of the objectives of this work is to understand the
modulators.16–20 The prototypical example of a (phenylimino)- quantitative effect of diverse substituents on the aqueous basicity
imidazolidine drug is a2-AR and imidazoline receptor agonist of pharmacologically important families of compounds: aryl
guanidines and 2-(arylimino)imidazolidines. This knowledge
a
is useful to assist in the design of new biologically relevant
guanidine-containing molecules with improved pharmacokinetic
properties.22 Another objective is to compare the experimental pKa
values of these series with those obtained by ab initio calculations
using the AIBLHiCoS method.25,26 The Ab Initio Bond Lengths
High Correlation Subsets protocol works on the basis that, for a
´
´
Instituto de Quımica Medica, IQM-CSIC, Juan de la Cierva 3, E-28006 Madrid,
Spain. E-mail: dardonville@iqm.csic.es; Fax: +34 915644853; Tel: +34 912587490
b Manchester Institute of Biotechnology (MIB), 131 Princess Street, Manchester,
M1 7DN, UK
c School of Chemistry, Univ. of Manchester, Oxford Road, Manchester M13 9PL, UK
† Electronic supplementary information (ESI) available: Excel sheet template for
the data analysis of UV-metric pKa determination. Tables S1–S6 and Fig. S1–S4. 1
and 13C NMR spectra of compounds 1a and 11a–15a. See DOI: 10.1039/c7nj02497e series of electronically congeneric compounds, chemical space
H
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Correlation Subset (HiCoS). In this work, using a specific bond
length and tautomeric form in the neutral state, we have
constructed one predictive model with a training set of 13
guanidines and a second model with a training set of 23
2-(phenylimino)imidazolidines. This method is a valuable tool
that has already proven its power for the accurate determination
of pKas of other guanidine-containing drugs in addition to a
number of other functional groups.25,27–29 However, this method
has not been tested, until now, with the medically relevant
2-(phenylimino)imidazolidine scaffold. The predictive models
built with these series show that the AIBLHiCoS method is an
accurate pKa prediction tool with this pharmacologically important
family of compounds.
2. Results and discussion
2.1. Synthesis
Fig. 1 Resonance forms of the guanidinium and 2-iminoimidazolidinium
cations responsible for the high basicity of these functional groups.
Structure of the archetypal 2-(arylimino)imidazolidine drug clonidine.
Trifluoroacetate salts of phenylguanidines (1b, 2b, 3b, 6b, 7b,
and 24–27b) and 2-(phenylimino)imidazolidines (2a–10a, and
may be partitioned according to a linear free energy relationship 23a) were synthesized from their Boc-protected precursors using
(LFER) between the calculated gas-phase equilibrium bond lengths CF3CO2H/CH2Cl2 as reported (Scheme 1).7 These Boc-protected
and aqueous pKa values. The equations resulting from highly precursors were obtained by a reaction of the corresponding
correlated, local linear relationships for structurally similar mole- anilines with N,N0-bis-(tert-butoxycarbonyl)imidazolidin-2-thione
cules are then used as predictive models for calculating the pKa or N,N0-bis(tert-butoxycarbonyl)thiourea and HgCl2/Et3N/DMF.7
values of other compounds belonging to the appropriate High The pyridino derivatives 17–22 were synthesized as hydrochloride
Scheme 1 Synthesis of 2a–10a, 23a, 1b–3b, 6b, 7b, and 24b–27b.
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salts by Rozas and co-workers following a procedure similar to next section. The mean absolute error (MAE) was found to be
that shown in Scheme 1.30
0.23 with a standard deviation of 0.22 for the error values using
Compounds 1a and 11a–15a were synthesized as shown in the model which returned the highest validation statistics
Scheme 2 following the procedure of Mundla et al.31
during calibration (C–N(im), Fig. S1, ESI†). This may be compared
to the MAE value for ChemAxon predictions using the imino
tautomer, which was found to be 0.77, with a standard deviation
of 0.47 (Table S1 in the ESI†). The prediction power with the
guanidine set of compounds was slightly lower with an MAE of
0.29, and most deviations r0.24 pKa units except for compounds
17b (ꢀ0.55), 24b (+0.70) and one very high error for 22b (+1.05),
which will also be discussed in the next section (Table S2, ESI†).
Finally, with respect to the UV-metric pKa values, deviations
r0.52 pKa units were observed against the predictions of
AIBLHiCoS (and r2 = 0.947) with the exception of compounds
5a (+0.58), 13a (ꢀ0.94), 15a (ꢀ0.58), 21a (+0.56) and 22a (ꢀ0.59)
(Fig. 2C).
2.2. Correlation between experimental pH-metric, UV-metric,
and calculated pKa
The pKa values of two sets of 23 2-(arylimino)imidazolidines
(1a–23a) and 12 arylguanidines (1b, 2b, 3b, 6b, 7b, 17b, 18b,
22b–27b), most of them developed by Rozas and co-workers as
a2-adrenoceptor ligands,16–20 were measured by potentiometric
titration (i.e. pH-metric) at 25 1C using the Sirius T3 apparatus,
and by spectrophotometry (i.e. UV-metric) using the 96-well
microtitre plate method developed in our group.32 The pKa
values determined with the T3 apparatus were highly consistent
(SD o 0.03, n = 3) whereas the 96-well microtitre plate method
gave somewhat less precise results (SD = 0.02–0.45, n Z 3)
(Table 1).
2.3. Validation of the AIBLHiCoS prediction method applied
to arylguanidines and 2-(arylimino)imidazolidines
A good correlation (r2 = 0.993) was observed between the pKa
values measured using spectrophotometry and potentiometry The bond length vs. pKa model used for most of the imidazo-
(Fig. 2A). The pKa differences observed between both experimental lidine predictions in this work was chosen on the basis of its
techniques were generally r0.3 pKa units except for compounds superior r2, leave-one-seventh-out-q2 and Root Mean Error of
1a (+0.67), 4a (+0.37), 5a (+0.57), 20a (+0.51), 21a (+0.47), and 22b Estimation (RMSEE) values compared to a number of other
(+0.41).
candidate bond length models. For the 22 compounds of the
It is known that low aqueous solubility and chromophore training set (listed 1i–22i in Table S3, ESI†), the highest validation
strength can affect the accuracy of experimental pKa determination statistics correspond to a model constructed using a C–N bond
for certain bases.34 As all the compounds with greater discrepancies length of the imidazolidine ring, when compounds were
between UV-metric and pH-metric pKa values have weak chromo- represented as the imino tautomer ‘‘T3’’. This model is labelled
phores, this could explain the lack of accuracy with these C–N(im) in Fig. S1 (ESI†) and will be referred to as such
compounds. In contrast, if we take the nitro derivatives 11a–15a throughout the following discussion. The second best validation
as an example of compounds with a strong chromophore and a statistics were observed for the N–C(Ph) bond, which is also
smooth transition from the protonated (lmax E 298 nm at pH 3) to labelled as such in Fig. S1 (ESI†). Predictions using both models
the neutral form (lmax E 364 nm at pH 12), we observe an are also listed in Table 1.
excellent correlation between the UV-metric and pH-metric values
(Table 1).
For guanidines, superior validation statistics for the training
set (listed g1–g13 in Table S4, ESI†) were obtained using the
The pKa values calculated using the AIBLHiCoS methodology CQN bond length with compounds represented as amino
correlated well (r2 = 0.965) with the experimental results obtained tautomer ‘‘A’’, as illustrated in Fig. S2 (ESI†). All predictions
using pH-metric titrations (see Fig. 2B) with deviations o0.30 pKa listed in Table 1 and Table S2 (ESI†) were obtained using
units for the phenyl-imidazolidine derivatives. However, some this model.
deterioration was observed for pyridinyl compounds (o0.58) and
A high degree of prediction accuracy in modern terms is
two compounds containing nitro groups, 13a (ꢀ0.79) and 15a considered to be within 1 pKa unit per prediction, with a mean
(ꢀ0.62), were also poorly predicted and are discussed in the absolute error (MAE) for a test set of less than 0.5 units.35 As the
Scheme 2 Synthesis of 1a and 11a–15a.
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Table 1 pKa values of aryl/pyridinyl guanidines and iminoimidazolidines calculated using AIBLHiCos and determined experimentally by potentiometric
and spectrophotometric methods
b
c
Cmpd
Structure
Predicted pKa C–N(im)a pKa N–C(Ph)d
pH-metric pKa
10.24 ꢁ 0.001
UV-metric pKa
10.91 ꢁ 0.10
10.54
9.90
1a
10.34
9.89
2a
10.49 ꢁ 0.01
—
10.16
9.78
3a
4a
5a
6a
10.29 ꢁ 0.01
10.42 ꢁ 0.001
10.50 ꢁ 0.01
10.44 ꢁ 0.01
10.40 ꢁ 0.13
10.79 ꢁ 0.10
11.07 ꢁ 0.15
10.27 ꢁ 0.17
10.33
9.89
10.49
9.94
10.54
9.97
10.76
10.26
7a
8a
10.78 ꢁ 0.01
10.62 ꢁ 0.01
10.91 ꢁ 0.09
10.80 ꢁ 0.13
10.67
10.16
10.16
9.73
9a
10.17 ꢁ 0.01
9.11 ꢁ 0.001
10.28 ꢁ 0.45
9.42 ꢁ 0.02
8.90
8.79
10a
8.54
8.26
11a
12a
13a
14a
15a
8.52 ꢁ 0.001
7.29 ꢁ 0.07e
8.07 ꢁ 0.001
7.41 ꢁ 0.001
7.80 ꢁ 0.001
8.37 ꢁ 0.06
7.33 ꢁ 0.03
8.06 ꢁ 0.07
7.41 ꢁ 0.02
7.69 ꢁ 0.06
7.28
7.48
7.28
8.01
7.60
7.32
7.18
7.89
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Table 1 (continued)
Predicted pKa C–N(im)a pKa N–C(Ph)d
pH-metric pKa
UV-metric pKa
b
c
Cmpd
Structure
9.50
9.30
8.99 ꢁ 0.001
3.05 ꢁ 0.01f
8.99 ꢁ 0.24
3.11 ꢁ 0.17
16a
9.95
9.54
9.37 ꢁ 0.001
4.36 ꢁ 0.001f
9.57 ꢁ 0.32
4.37 ꢁ 0.19
17a
18a
10.63
10.02
10.33 ꢁ 0.001
4.97 ꢁ 0.001f
10.35 ꢁ 0.42
4.96 ꢁ 0.08
9.17
7.65
19a
20a
21a
9.45 ꢁ 0.001g
9.47 ꢁ 0.01g
9.68 ꢁ 0.001g
9.60 ꢁ 0.06
9.98 ꢁ 0.05
10.15 ꢁ 0.15
9.46
7.81
9.59
7.91
8.51
7.30
22a
23a
8.97 ꢁ 0.001g
9.08 ꢁ 0.01e
9.10 ꢁ 0.09
9.26 ꢁ 0.06
8.83
9.14
1b
2b
11.01
11.22
10.90 ꢁ 0.001h
11.36 ꢁ 0.01
—
—
3b
11.10
11.15
11.24
10.60
10.98 ꢁ 0.001
10.98 ꢁ 0.001
11.35 ꢁ 0.01
10.95 ꢁ 0.25
6b
—
—
7b
10.05 ꢁ 0.001
4.44 ꢁ 0.001f
N/A
4.13 ꢁ 0.35
17b
11.01 ꢁ 0.01
4.94 ꢁ 0.01f
N/A
4.98 ꢁ 0.09
18b
10.77
22b
24b
10.52
11.31
10.47 ꢁ 0.001g
10.88 ꢁ 0.08
12.01 ꢁ 0.03
3.63 ꢁ 0.001
—
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Table 1 (continued)
b
c
Cmpd
Structure
Predicted pKa C–N(im)a pKa N–C(Ph)d
pH-metric pKa
11.68 ꢁ 0.01
UV-metric pKa
25b
11.79
11.70 ꢁ 0.09
11.23 ꢁ 0.15
11.12 ꢁ 0.15
26b
10.89
11.03
11.03 ꢁ 0.01
11.01 ꢁ 0.01
27b
a
b
Calculated using the AIBLHiCos approach with the C–N(im) bond model of the imino tautomer. pH-metric determination at 25 1C using Sirius
T3 apparatus. Mean of 3 titrations ꢁ SD. Determined by UV-spectrophotometry at 30 1C using the 96-well microtiter plate method.32 Mean of Z 3
c
d
titrations ꢁ SD. Bond length model built from the same imidazolidine training set with compounds in the imino tautomeric form, but with the
e
C–N(Ph) bond length connecting 2-iminoimidazolidine to benzene. psKa measured with MeOH as a co-solvent (Yasuda–Shedlowsky extrapolation
in H2O). pKa value of the pyridin-3-yl nitrogen. The pKa value of the pyridin-2-yl nitrogen could not be worked out. Literature: pKa = 10.77.33
f
g
h
training sets consist of pKa values taken from various sources, that compound 7i, (2-(2,6-Cl2,4-NO2-phenylimino)imidazolidine),
and therefore contain measurements obtained using different is present within the training set used to form the predictive
techniques, noise within the experimental datasets is to be HiCoS, and 11a, 12a and 14a of the test set fall in the expected
expected. Small variations in conditions are also possible, i.e. region of the trend line for the LFER, as illustrated by their low
solvent purity, sample purity and lab temperature, which can prediction errors. According to the LFER exhibited between the
also have an effect on the values measured. According to the C–N(im) bond length and pKa, a shorter equilibrium bond
above conditions, the error statistics for both test sets show that length simply corresponds to a lower pKa. In the case of 13a
both the guanidine and imidazolidine AIBLHiCoS models and 15a, we therefore observe an anomalously short C–N(im)
perform well. On only one occasion does any error exceed bond, resulting in lower pKa values than we would expect
1 unit, and the MAE values for both test sets are well below according to the LFER exhibited by most other congeners.
0.5 (0.23 for imidazolidines and 0.29 for guanidines). For
Looking to the optimised structures for 13a and 15a to
predictions made using the pKa predictor plug-in within the explain these short bond lengths reveals the presence of an
Marvin Suite by ChemAxon,36 with the imino tautomer as the interaction between the Oꢀ atom of the p-nitro group, and the
input structure (Tables S1 and S2, ESI†), nine prediction errors m-Cl (13a) and m-F (15a) atoms. As a result of this interaction,
exceed 1.0 pKa units and the MAE values are in excess of the nitro group is seen to adopt a geometry whereby it is no
0.5 units for both imidazolidines and guanidines (0.77 and longer co-planar with benzene. It may be asserted that the
0.54). AIBLHiCoS therefore finds its place here as a useful tool presence of this substituent interaction and deviation in nitro
in instances where the empirical tools with a wider applicability geometry, which is absent in all remaining p-nitro compounds,
radius struggle, as is the case for these guanidine-type ionisable may be the cause of the anomalously short C–N(im) bond lengths.
groups.
The internal and external validation statistics for the chosen
2.3.1. Imidazolidines. A particular instance of where AIBL- model (C–N(im)) are superior to any other bond length vs. pKa
HiCoS performs notably well compared to ChemAxon is for the plot (r2 = 0.97, q2 = 0.96 and RMSEE = 0.23). The next best
imidazolidine compounds 11a–15a, all of which contain nitro model according to these validation statistics is observed for
groups at the para position of the phenyl ring. ChemAxon the same imidazolidine training set with compounds in the
underestimates the acidity of these compounds by +1.23, imino tautomeric form, but for the C–N bond length connecting
+1.64, +1.18, +1.23 and +1.41 units, whereas the AIBLHiCoS benzene to 2-iminoimidazolidine (marked as N–C(Ph) within the
model provides errors of +0.02, +0.01, ꢀ0.79, +0.19 and ꢀ0.62. structure labelled ‘‘T3’’ in Fig. S1, ESI†). This model has an r2
However, two of the largest prediction errors of the imidazolidine value of 0.96, a q2 value of 0.96 and a RMSEE value of 0.25. When
test set correspond to those for 13a and 15a, 2-(3-chloro-4-nitro- the linear equation for this alternative model is used to predict
phenylamino) and 2-(3-fluoro-4-nitro-phenylamino)imidazolidine. for the whole test set 1a–23a, the resultant MAE is significantly
Errors for the pH-metric measurements are ꢀ0.79 and ꢀ0.62, and worse than the original model we chose to implement based on
compared to the UV-metric values they also give errors of ꢀ0.94 and its superior statistics (the MAE is now 0.57 vs. 0.23).
ꢀ0.58. The resonance-induced strong electron-withdrawing effect of
For the 3-pyridyl compounds synthesized in this work,
nitro groups is known to perturb active bond lengths to such an 16a–18a, we observe errors of +0.51, +0.58 and +0.30 for the
extent that compounds containing these groups often form their pH-metric measurements and ꢀ0.51, +0.38 and +0.28 for the
own separate HiCoS.28,29 Contrary to the previous results, the UV-metric values. Despite the increase in MAE when the whole
partitioning of the imidazolidine set into more structurally specific set is considered, use of the alternative N–C(Ph) model is seen
subsets may not be defined in this case by the presence of a to reduce the errors for 16a and 17a to ꢀ0.31 and +0.17. This
nitro group at the para-position. This is explained by the fact improvement of predicted values can also be seen for the
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problematic nitro compounds, 13a and 15a, for which the errors
decrease dramatically when predicted using the N–C(Ph) model
(ꢀ0.26, +0.19, +0.06, ꢀ0.09 and +0.09 for 11a–15a).
We now take a moment to briefly introduce the Interacting
Quantum Atoms (IQA) energy partitioning scheme in accordance
with the Quantum Theory of Atoms in Molecules (QTAIM).37,38
By partitioning the total energy of a system into intra- and
interatomic terms, it has been shown that we may derive a
quantitative measure of covalent-like interactions between
atoms in molecules. This comes in the form of the exchange–
correlation potential energy V AxcB, which is the sum of the
exchange energy V AxB and the correlation energy V AcB. The former
term dominates V AxcB, and expresses the Fock–Dirac exchange, a
consequence of the Pauli Principle, which describes the ever
reducing probability of finding two electrons of the same spin as
they approach each other (i.e. the Fermi hole). The latter term
V AcB is associated with the Coulomb hole, which corresponds to
the electrostatic repulsion between electrons. The absolute value
AB
xc
of V between two atoms can be taken as the extent of
delocalization of electrons between them, a factor that also
determines the bond distance.
For the training set compounds, for C–N(im) and N–C(Ph),
the VCxcN values (Table S5, ESI†) for the bonded atoms are found
to have r2 values of 0.990 and 0.992 with the corresponding
bond lengths (Fig. S3(a) and (d), ESI†). However, the analogous
plots for other bonds (marked ‘‘b’’ and ‘‘c’’ in Fig. S1 and (b and
c) in Fig. S3, ESI†) have weaker correlations (r2 = 0.939 and
0.897). Therefore, we can conclude that the bond lengths that
correlate most highly with aqueous pKa are those which are also
most accurately reflecting the extent of delocalization between
CN
xc
the two bonded atoms. Including the V vs. bond lengths for
our outliers, 13a, 15a, 16a and 17a, shows that they do not
diverge substantially from the highly correlated lines of the best
fit for either C–N(im) or N–C(Ph). Correspondingly, these four
compounds remain as outliers for the plot of VCxcN values vs. pKa
for C–N(im), and as observed for the bond lengths, fall within
the expected region of the plot for VCxcN values vs. pKa for N–C(Ph)
(Fig. S4, ESI†).
On the basis of previous work, we suspect that for compounds
with m-halogen and p-nitro substituents (13a and 15a), the
halo-nitro interaction perturbs the charge distribution of the
guanidine fragment to such a degree that the relationship
between C–N(im) and pKa is no longer reflected in the general
trend observed for most other congeners. If more pKa data were
available, it is proposed that we could form a new HiCoS
consisting of only compounds of this type. Currently, in the
absence of an adequate quantity of pKa data, a separate
predictive HiCoS using the C–N(im) bond lengths cannot be
constructed. Given that the N–C(Ph) bond lengths and corres-
ponding V CxcN values do not diverge from the general trend as a
result of the halo-nitro interaction, the C–N(Ph) model should
be used to predict compounds of this type. For two of three
3-pyridyl compounds (16a–18a), an explanation for their 40.5
prediction errors is not immediately accessible in terms of their
structure. This is due to the fact that one 3-pyridyl containing
compound, 18a, has a relatively low error of +0.30 predicted
Fig. 2 (A) Correlation (r2 = 0.993) between experimental pH-metric and
UV-metric pKa values for compounds 3a–23a, 3b, 17b, 18b, 22b, 26b, and
27b. (B) Correlation (r2 = 0.965) between pH-metric pKas and AIBLHiCoS
predicted pKa values for compounds 1a–23a and 1b–27b. (C) Correlation
(r2 = 0.947) between UV-metric pKas and AIBLHiCoS predicted pKa values
for compounds 3a–23a, 3b, 17b, 18b, 22b, 26b, and 27b.
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using the C–N(im) model. We can therefore assert that the bond lengths of their optimised structures are included in our
presence of the heteroatom at the 3-position is not the sole CQN vs. pKa plot using tautomer A (shown in Fig. S2 of the
cause of the higher pKa values we predict, based on their longer ESI,† bond labelled ii), they also appear below the line of the
C–N(im) bond lengths. Once again, in the absence of an best fit, near the data point for 22b. When predicted using
adequate amount of experimental data for like compounds at the model equation, errors of +1.51 and +1.22 units are
the time of writing, we cannot prove indefinitely that these observed. It appears that the presence of the heteroatom at
compounds would form their own subset. We therefore suggest the 2-position of the aryl group causes further partitioning of
that future predictions for all 3-pyridyl compounds be made via the set once again. A new HiCoS cannot be constructed using
implementation of the N–C(Ph) model equation.
only two data points, and so a predictive equation which would
2.3.2. Guanidines. For the guanidine test set, the only better describe the LFER for these compounds cannot be
AIBLHiCoS prediction above 1 unit corresponds to 22b, 1-(5- formulated until more pKa data become available.
chloro-2-pyridinyl)guanidine. Our model gives an error of +1.05 units
Boltzmann weighting of tautomers according to either internal
whereas ChemAxon gives an error of ꢀ0.89. There are no pyridinyl- or Gibbs energies at the level of theory used to obtain the bond
type compounds within the training set, but two 3-pyridinyls, 17b lengths for the AIBLHiCoS protocol reveals a general prevalence
and 18b, return respectable errors of +0.55 and ꢀ0.24 using of the ‘‘imino’’ tautomer for both sets of guanidines and
the CQN model. pKa data for 1-(2-pyridinyl)guanidine and imidazolidines. This tautomer is characterised by a CQN double
1-(6-methyl-2-pyridinyl)guanidine are available, and when the bond between the central carbon atom of the Y-shaped guanidine
Fig. 3 Substituent effects on the pKa of 2-(arylimino)imidazolidines and arylguanidines. The pKa values were determined using potentiometric titrations
at 25 1C. DpKa is the difference in pKa of the substituted compound with respect to the unsubstituted compound (1a, 1b, 19a, and 16a, respectively).
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ꢀ
fragment and the nitrogen adjacent to the phenyl group. For imidazolidine nitrogen (2a, 2b, 24b), sp values were used to
imidazolidines, the optimal model emerges from one of the C–N get a good correlation. In contrast, compounds with an amide
bond lengths of the 5-membered ring of this most stable tautomer. group (9a) in para position correlated poorly with the Hammett
The emergence of the optimal model from the most stable constant (red points in Fig. 4).
tautomer is therefore congruent with it being the dominant
Amino substituents (18a, 18b, 24b) in the para-position to
species in solution, confirmed by the energetics. In terms of the the guanidine/imidazoline group yield the strongest pKa
ChemAxon predictions, using the most stable tautomer as an increase (DpKa = +1.11 to 1.34), with the exception of the
input structure allows for better prediction accuracy for both the aminophenyl group (2a, 2b), the electron donating capacity of
imidazolidines and guanidines. However, for AIBLHiCoS the best which is much reduced by the presence of the phenyl ring
internal validation statistics for guanidines are revealed using (DpKa = +0.25 to 0.46). Expectedly, the pKa of the pyridine
equilibrium bond lengths from a specific conformation of a higher nitrogen is influenced to a larger extent by the presence of
energy tautomeric form, which is not in agreement with the energy amino and alkyl electron-donating substituents (DpKa = +1.31
rankings. Regardless of that these results corroborate those of our and +1.92 for 17a and 18a, respectively). Alkoxy substituents
previous work,27 which states that in the case of guanidines (8a, 26b, 27b) have a smaller effect on the pKa increase
conformational commonality undermines stability; that is to say, (DpKa = +0.06 to 0.38) with respect to the phenoxy group (25b:
as long as the compound of interest containing the guanidine DpKa = +0.78). For electron-withdrawing substituents, the pKa
group is optimised from this specific form, a very high degree of decrement follows this order: 4-PhNHCO o 4-Ac o 4-NO2 o (3-
prediction accuracy may be obtained.
Cl,4-NO2) o (3-F,4-NO2) o (2-F,4-NO2) o (2Cl,4-NO2). The
addition of one halogen atom on the aryl ring results in pKa
decrements of 0.45 (3-Cl), 0.72 (3-F), 1.11 (2-F), and 1.23 (2-Cl).
These values are consistent with the pKa decrements measured
for a series of chloro- and fluoro-substituted bis-2-(arylimino)-
imidazolidines reported previously.22
Timmermans et al. have shown that the presence of two
chlorine atoms in the position ortho to the 2-imino nitrogen
reduces the pKa value of the molecule by 1.66 units (e.g. 4-nitro-
clonidine: pKa = 6.86 at 20 1C).41 According to the pKa values
reported by Rozas and co-workers for a series of symmetric and
unsymmetrical bisguanidines and bis-2-(phenylimino)imidazo-
lidines,42,43 the addition of a 4-imidazoline or 4-guanidine group on
the phenyl substituent of 2a–b and 3a–b results in a pKa decrement
of approximately ꢀ0.50 units with respect to 2a–b and 3a–b. The
values of DpKa with respect to 1a–b are shown in Fig. 5.
2.4. Quantitative effect of aryl substituents on the pKa of aryl
guanidines and 2-(arylimino)imidazolidines
According to a survey by Manallack on the pKa distribution of
drugs,24 no CNS drug has a pKa of above 10.5. Thus, phenyl-
guanidine (1b: pKa = 10.90), the reference compound in our
study, is unlikely to have good CNS activity. Replacing the
guanidine group of 1b by a 2-iminoimidazolidine group (1a:
pKa = 10.24) leads to a drop in the basicity of the molecule
(DpKa = ꢀ0.66). Changing the phenyl ring of 1a for a 2-pyridinyl
(19a) or a 3-pyridinyl (16a) group has a strong effect on the pKa
decrement (DpKa = ꢀ0.79 and ꢀ1.25, respectively). The effect
of electron-donating and electron-withdrawing groups on
the pKa of 2-(arylimino)imidazolidines and arylguanidines is
summarized in Fig. 3 and Table S6 (ESI†). As expected, these
effects depend mainly on the capacity of these substituents to
donate or withdraw electrons from the aryl ring, resulting in higher
or lower electron density on the 2-imino nitrogen, respectively.
A linear relationship was found between the pKa values and
the Hammett s values of meta and para substituents as shown
in Fig. 4.39,40 For para-amino substituents conjugated with the
3. Conclusions
Arylguanidines and 2-(arylimino)imidazolidines whose pKa values
are reported here belong to a pharmacologically important class of
molecules with promising applications as centrally active a2-AR
modulators. As the ionisation constant is a key physicochemical
parameter that governs the membrane permeability of drugs (e.g.
the BBB permeability in the case of CNS active compounds), the
data of substituent effects on the basicity of arylguanidines and
2-(arylimino)imidazolidines can be useful for the design of new
molecules of this class.
The dissociation constants of 23 arylguanidines and 12
2-(arylimino)imidazolidines measured using the spectrophoto-
metry method were consistent with those measured using
potentiometric titrations, further validating the 96-well micro-
titre plate method32 as a useful tool for the medium throughput
determination of pKa of series of molecules.
Finally, we have shown that the AIBLHiCoS method is useful
to predict the pKa values of aryl guanidines and 2-(arylimino)-
imidazolidines with low RMSD values, using only a single bond
length obtained at a low level of theory. It must be noted that
compound 12a is an excellent example of the predictive power
Fig. 4 Relationship between experimental pKa (determined by potentio-
metric titrations at 25 1C) and S Hammett s constants39 for meta and para
substituents of 2-(arylimino)imidazolidines (K) and arylguanidines (m).
Compounds that deviate significantly from the bottom (right) linear rela-
tionship appear in red (9a).
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Fig. 5 Substituent effects on the pKa of bis(2-(phenylimino)imidazolidines) and bisguanidines. Reported pKa values determined using potentiometric42
or UV-spectrophotometric43 titrations at 25 1C. DpKa is the difference in pKa of the 4-substituted compound with respect to 1a or 1b.
of the AIBLHiCoS model. The initial (pH-metric) pKa value we scale: 5 to 25 mg) was dissolved in CH2Cl2 (1 mL) and CF3CO2H
were working with was 8.13, meaning that our prediction of (0.5 mL) was added. The reaction was stirred for 4 h at room
7.28 was 0.85 units out, whilst for 14a, the 2-fluoro analogue, temperature and the volatiles were evaporated. Et2O was added
the prediction error was only 0.19. Re-measurement of the pKa to the crude residue and evaporation was repeated. The TFA
value for 12a then revealed the first to be erroneous, for reasons salts were obtained by precipitation from hexane/Et2O (guanidines)
that are not clear, and the new pKa value was observed to be or hexane (2-aminoimidazolines). The precipitates were rinsed
7.29, only 0.01 units out from the prediction. This value was with hexane and dried under high vacuum over P2O5 to yield the
also in excellent agreement with the UV-metric pKa value (7.33). TFA salts as brownish hygroscopic solids. 1H NMR spectra were
consistent with the reported data.7 Most of the compounds were
Z95% pure, or 490% (4a, 5a, 6a, 8a, and 6b, 7b) as checked
using HPLC-MS.
Synthesis of compounds 1a and 11a–15a. A Kimax tube
charged with the aniline reagent (0.4 mmol, 1 equiv.) and
4. Experimental
Chemistry
Melting points were measured in open capillary tubes using a methyl 2-(methylthio)-4,5-dihydro-1H-imidazole-1-carboxylate31
Stuart Scientific SMP3 apparatus or a Mettler Toledo MP70 (0.5 mmol, 1.25 equiv.) in glacial acetic acid (1.2 mL) was heated
melting point system and are uncorrected. LC-MS spectra were at 70 1C (1 day for 1a and 13a, 3 days for 11a and 15a) or 90 1C
recorded on a WATERS apparatus integrated with a HPLC (4 days, 12a and 14a), until the formation of the methyl
separation module (2695), a PDA detector (2996) and a Micro- 2-(phenylamino)-4,5-dihydro-1H-imidazole-1-carboxylate inter-
mass ZQ spectrometer. Analytical HPLC was performed with a mediate. Then, methanol was added (1 mL) and the reaction was
SunFire C18–3.5 mm column (4.6 mm ꢂ 50 mm). Mobile phase heated at 100 1C for 2 days (1a, 11a, 13a, 15a) or 80 1C for
A: CH3CN + 0.08% formic acid and B: H2O + 0.05% formic acid. 4 days (12a, 14a). The solvent was evaporated under vacuum and
UV detection was carried over 190 to 440 nm. Accurate mass the acetate salt of the product was isolated using silica chromato-
was measured with an Agilent Technologies Q-TOF 6520 spec- graphy (Isolute prepacked column, 2g). The non-polar starting
1
trometer using electrospray ionisation. H NMR and 13C NMR materials were first eluted with 100% CH2Cl2, then the products
spectra were registered on Bruker Avance-300, Varian Inova-400, were obtained with CH2Cl2 :MeOH (10:1) (compounds 1a, 11a, 13a,
and Varian-system-500 spectrometers. Chemical shifts of the 15a) or CH2Cl2 :MeOH (15:1 - 10 : 1) (compounds 12a and 14a).
1H NMR spectra were referenced to the residual proton reso-
N-Phenyl-4,5-dihydro-1H-imidazol-2-amine acetate salt (1a).
nance of the deuterated solvents: D2O (d 4.79) and CD3OD Yellowish gum (88%). HPLC (UV) 492%. dH (300 MHz, D2O) d
(d 3.31). Chemical shifts of the 13C NMR spectra were internally 7.44 (dq, J 2.9, 4.3, 6.0, 2H), 7.39–7.30 (m, 1H), 7.29–7.21 (m,
referenced for D2O and CD3OD (d 49.0). Coupling constants J are 2H), 3.71 (s, 4H), 1.86 (s, 3H, AcOꢀ). dC (75 MHz, D2O) d 181.8
given in hertz (Hz).
Synthesis of phenylguanidines (1b, 2b, 3b, 6b, 7b, and (ESI+) m/z 162 (M + H)+.
(AcOꢀ), 159.2, 135.4, 130.2, 127.8, 124.7, 43.0, 23.6 (AcOꢀ). MS
24–27b) and 2-(phenylimino)imidazolidines (2a–10a, and 23a)
N-(4-Nitrophenyl)-4,5-dihydro-1H-imidazol-2-amine acetate
as trifluoroacetate salts. The di-Boc-protected precursor7 (typical salt (11a). Yellow-orangish solid (14%). Mp 190.2–193.1 1C.
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HPLC (UV) 495%. dH (300 MHz, D2O) d 8.30 (d, J 9.1, 2H), 7.43 6.19, 6.42, 6.62, and 6.78), (e) 25 mM ethanolamine buffer (pH 8.88,
(d, J 9.1, 2H), 3.80 (s, 4H), 1.90 (s, 3H, AcOꢀ). dC (75 MHz, D2O) d 9.17, 9.23, 9.66, 9.74, 10.13, and 10.32). The pH of the buffer
181.8 (AcOꢀ), 158.6, 145.1, 143.6, 125.9, 123.2, 43.1, 23.6 solutions was measured at 30 1C (i.e. working temperature).
(AcOꢀ). MS (ESI+) m/z 207 (M + H)+. HRMS (ESI+) m/z
The raw UV data were processed using the Excel program
206.0805 (C9H10N4O2 requires 206.0804).
(see the Excel sheet template in the ESI†) and the pKa values
N-(2-Chloro-4-nitrophenyl)-4,5-dihydro-1H-imidazol-2-amine were determined by linear regression from the total absorbance
acetate salt (12a). Yellow solid (5%). Mp 4195 1C. HPLC (UV) vs. pH curve as reported.32 The pKa values are the mean of 3 or
495%. dH (400 MHz, D2O) d 8.54 (d, J 2.5, 1H), 8.30 (dd, J 2.5, 4 experiments (expressed as Ka values) ꢁ SD.
8.8, 1H), 7.73 (d, J 8.8, 1H), 3.85 (s, 4H), 1.95 (s, 3H, AcOꢀ). MS
(ESI+) m/z 241, 243 (M + H)+. HRMS (ESI+) m/z 240.0413
Gas-phase ab initio calculations
(C9H9ClN4O2 requires 240.0414).
The initial structures of each member of the respective training
N-(3-Chloro-4-nitrophenyl)-4,5-dihydro-1H-imidazol-2-amine sets (guanidines and 2-(phenylamino)imidazolidines) were
acetate salt (13a). Yellow solid (26%). Mp 156.5–160.3 1C. HPLC built using the program GaussView in each available tautomeric
(UV) 495%. dH (300 MHz, D2O) d 8.10 (d, J 8.8, 1H), 7.55 (d, form. This equates to three tautomers for the imidazolidines,
J 2.4, 1H), 7.35 (dd, J 2.4, 8.8, 1H), 3.81 (s, 4H), 1.89 (s, 3H, labelled T1–T3 in Fig. S1 (ESI†) and five ‘‘tautomers’’ for
AcOꢀ). dC (75 MHz, D2O) d 181.7 (AcOꢀ), 158.3, 144.4, 141.6, 128.8, guanidines, labelled A–E in Fig. S2 (ESI†). For compounds with
128.2, 125.4, 121.6, 43.1, 23.6 (AcOꢀ). MS (ESI+) m/z 241, 243 (M + higher degrees of conformational freedom, several starting
H)+. HRMS (ESI+) m/z 240.0417 (C9H9ClN4O2 requires 240.0414).
structures were generated using the ‘‘Conformers’’ plug-in
N-(2-Fluoro-4-nitrophenyl)-4,5-dihydro-1H-imidazol-2-amine within Marvin Sketch by ChemAxon.35 Geometry optimization
(14a). Yellow solid (28%). Mp 220–226 1C. HPLC (UV) 495%. dH in the gas-phase was then performed for each conformer of each
(500 MHz, methanol-d4) d 7.95 (dd, J 2.5, 8.7, 1H), 7.92 (dd, compound in each of the tautomeric forms using the GAUSSIAN09
J 2.5, 10.7, 1H), 7.20 (t, J 8.7, 1H), 3.55 (s, 4H). dC (126 MHz, program.44 Calculations were initially carried out at the B3LYP/6-
methanol-d4) d 161.9, 156.2 (d, J 246.6), 146.9 (d, J 11.5), 142.7 31G(d,p) level but the basis set was changed to 6-311G(d,p) for
(d, J 8.7), 125.7 (d, J 3.3), 121.4 (d, J 3.0), 112.6 (d, J 25.9), 43.5. reasons that will be explained below. For biguanide derivatives
MS (ESI+) m/z 225 (M + H)+. HRMS (ESI+) m/z 224.0706 included in the guanidine training set, various tautomeric
(C9H9FN4O2 requires 224.0710).
forms of the guanidine fragment present within the R group
N-(3-Fluoro-4-nitrophenyl)-4,5-dihydro-1H-imidazol-2-amine were considered, whilst maintaining the specific tautomeric
acetate salt (15a). Yellow solid (24%). Mp 197.3–200 1C. HPLC form of the guanidine fragment under consideration. Frequency
(UV) 495%. dH (300 MHz, D2O) d 8.20 (t, J 8.7, 1H), 7.30 (dd, calculations were carried out on optimised structures again using
J 2.4, 12.5, 1H), 7.25–7.15 (m, 1H), 3.82 (s, 4H), 1.88 (d, J = 3.8, GAUSSIAN09, at the same level of theory. The output files were
3H). dC (75 MHz, D2O) d 181.7 (AcOꢀ), 158.4, 156.5 (d, J 242.5), then inspected to confirm the absence of negative eigenvalues
143.8 (d, J 13.1), 134.0 (d, J 9.4), 128.4, 118.0, 111.6 (d, J 24.6), after diagonalization of the Hessian matrix, so that geometries
43.1, 23.5 (AcOꢀ). MS (ESI+) m/z 225 (M + H)+. HRMS (ESI+) m/z are confirmed as true energy minima. Where a number of
224.0707 (C9H9FN4O2 requires 224.0710).
starting conformers of a tautomer were generated, the most
Potentiometric pKa determination. Titrations were carried stable conformer was chosen by comparing the total energies
out at 25 ꢁ 0.5 1C in 0.15 M aqueous KCl solution under a of each optimised structure. At this point we noticed that the chosen
nitrogen atmosphere using a SiriusT3 apparatus (Sirius Analytical basis set was causing an issue in terms of ranking conformer
Instruments Ltd, East Sussex, UK) equipped with an Ag/AgCl stability between 2-(2-halogen-phenylimino)imidazolidine geo-
double junction reference pH electrode and a turbidity sensor. metries with and without IHBs. These IHBs are seen to exist
Standardised 0.5 M KOH and 0.5 M HCl were used as titration between an N–H group on imidazolidine and the o-halogen
reagents. The KOH solution was standardized by potassium acid atom on benzene, and are ubiquitous throughout the most
phthalate. The pKa values are the mean of 3 titrations ꢁSD except stable conformations of all compounds containing this substituent
otherwise noted.
for tautomer T3, with the exception of 2-(2,4-Cl-phenylimino) and
Spectrophotometric pKa determination. The UV-spectra 2-(2,5-Cl-phenylimino)imidazolidine. This observation prompted
were recorded at 30 1C (i.e. room temperature) with a Thermo the re-optimisation of all T3 training set compounds at the B3LYP/
Multiskan spectrum apparatus using the 96-well microtiter 6-311G(d,p) level of theory. Using the valence triple zeta basis set
plate method as described,32 with the following modifications: revealed a consistent picture, where the presence of an IHB is a
(1) compounds’ stock solutions were prepared in DMSO at stabilising feature for all compounds. At this point all other
C = 5 mM to ensure that the maximum absorbance of the calculations for T1 and T2 and tautomers A–E of the guanidine
compound was below 1.5 AU (i.e. the final concentration of the training set were also repeated for consistency. All analysis
compound in the well was 0.1 mM). (2). The buffer solutions of corresponds to the results of the calculations using the 6-311G(d,p)
constant ionic strength (0.1 M KCl) were prepared according to basis set.
http://www.biomol.net/en/tools/buffercalculator.htm: (a) 25 mM
Seven bond lengths were extracted from each imidazolidine
phosphate buffer (pH 2.01, 2.54, 7.0, 7.27, 7.61, 7.94, 11.50, 11.80, compound, corresponding to each of the three CN bonds of the
and 12.40), (b) 25 mM citrate buffer (pH 3.23), (c) 25 mM acetate guanidine fragment, in addition to the N–C(Ph) bond connecting
buffer (pH 4.17 and 4.61), (d) 25 mM MES buffer (pH 5.12, 5.66, 6.01, imidazolidine to benzene and the N–C, C–C and C–N bonds of
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`
the imidazolidine ring. These are labelled a–g in Fig. S1 of the We thank Chansele Jourdan for her assistance with the UV-metric
ESI.† Five bond lengths from within the guanidine moiety were pKa measurements. Dr Rozas is gratefully acknowledged for the
also extracted, which correspond to the two CN single and one gift of the pyridino derivatives 16–22 and the Boc-protected
CN double bond of the guanidine group, the N–H bond attached precursors of compounds 1–10 and 23–27.
to the imine nitrogen in A, B, D and E, and one N–H bond of a
primary amine group. These are labelled i–v, in Fig. S2 (ESI†). The
five bond lengths i–v within tautomers A–E were then regressed
against the pKa values for the set of guanidines. The seven bond
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dines. The squared correlation coefficient (r2), Root-Mean-Squared
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Conflicts of interest
16 B. Kelly, M. McMullan, C. Muguruza, J. E. Ortega, J. J. Meana,
L. F. Callado and I. Rozas, J. Med. Chem., 2015, 58, 963–977.
17 C. Muguruza, F. Rodriguez, I. Rozas, J. J. Meana, L. Uriguen
and L. F. Callado, Neuropharmacology, 2013, 65, 13–19.
There are no conflicts of interest to declare.
Acknowledgements
´
18 F. Rodrıguez, I. Rozas, J. E. Ortega, A. M. Erdozain, J. J. Meana
This work was supported by the Spanish Ministerio de Economia y
and L. F. Callado, J. Med. Chem., 2008, 51, 3304–3312.
Competitividad (Grant SAF2015-66690-R). B. A. Caine thanks 19 F. Rodriguez, I. Rozas, J. E. Ortega, J. J. Meana and
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the EPSRC for funding through the award of an Established Career 20 F. Rodriguez, I. Rozas, J. E. Ortega, A. M. Erdozain, J. J. Meana
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