Carboxylate binding in polar solvents using pyridylguanidinium salts

Article abstract of DOI:10.1039/b700988g

A series of thiourea and guanidinium derivatives have been prepared and their ability to bind a carboxylate group has been investigated. Guanidinium 33, featuring two additional amides and a pyridine moiety, proved to be the most potent carboxylate bindin

Full text of DOI:10.1039/b700988g

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Carboxylate binding in polar solvents using pyridylguanidinium salts†
Richard J. Fitzmaurice, Francesca Gaggini, Natarajan Srinivasan and Jeremy D. Kilburn*
Received 23rd January 2007, Accepted 2nd April 2007
First published as an Advance Article on the web 25th April 2007
DOI: 10.1039/b700988g
A series of thiourea and guanidinium derivatives have been prepared and their ability to bind a
carboxylate group has been investigated. Guanidinium 33, featuring two additional amides and a
pyridine moiety, proved to be the most potent carboxylate binding site and was able to bind acetate in
aqueous solvent systems (Kass = 480 M−1 in 30% H2O–DMSO). The pyridine moiety is critical to
obtaining strong binding, and comparison with the binding properties of analogous compounds in
which the pyridine is replaced by a benzene ring provides a striking example of enthalpy–entropy
compensation.
Introduction
The carboxylate group is a very common functional group
in biological molecules and there is considerable interest in
developing binding motifs for carboxylate functionality1 which
canthenbe incorporated into larger receptor structures for binding
of complex biological molecules such as amino acids and peptides,
particularly in aqueous solvents.2,3
Thioureas and guanidinium salts have been popular as carboxy-
late recognition motifs,1 providing a pair of suitably aligned hy-
drogen bond donors to interact with the carboxylate oxygens, and,
in the case of a guanidinium cation, providing a complementary
electrostatic interaction with the carboxylate anion.4 However,
simple thioureas and guanidinium salts have limited binding
properties in competitive solvents, and particularly in aqueous
media. Increased binding can be achieved by incorporating further
hydrogen bonds to interact with the carboxylate guest and a
number of approaches have been adopted to achieve this. Thus,
for example, we have studied5 the binding properties of thioureas
1 and 2 (Fig. 1), with the amides providing hydrogen bonds
to a carboxylate guest in addition to those from the thiourea.
Incorporating a pyridine in 1 has the potential to help preorganise
the binding cavity into the desired conformation by weak hydrogen
bonds between the pyridine N-lone pair and NH1 and NH2,
but this preorganisation is not available in the benzo-analogue
Fig. 1 Thiourea and guanidinium derivatives as carboxylate binding sites.
2. A pyridine dicarboxamide motif has been used elsewhere to
preorganise macrocyclic and simple acyclic receptors,6 but the
benefit of this preorganisation for the complexation of anions
Schmuck et al. have studied8 a range of acylated guanidinopy-
rroles 3 and 4 and found that the combination of additional hydro-
can be outweighed by the unfavourable electrostatic interaction
gen bonding and the enhanced hydrogen bonding potential of the
between the pyridine N-lone pair and the guest anion.7 In the
acylated guanidinium produces particularly effective carboxylate
case of 1 and 2 the binding constants with phenylacetate as guest
were very similar in 10% DMSO-d6–CDCl3 indicating that the
benefits of preorganisation in 1 were effectively balanced by the
unfavourable electrostatic interaction in 2.
receptors (e.g. with the picrate salt of 3, R = Et, using Ac–L–Ala–
O− as a guest in 40% H2O–DMSO (dimethylsulfoxide):8d Kass
=
770 M−1), althoughacylationalsohas the effectofloweringthe pKa
of the guanidinium. Schmuck and Machon have also described an
analogous binding site 5, again with an acylated guanidinium, but
using a pyridine in place of the pyrrole moiety.8b These receptors
also bound carboxylates in 40% H2O–DMSO solvent mixtures,
but less strongly than the pyrrole analogues.
School of Chemistry, University of Southampton, Southampton, UK SO17
1BJ
† Electronic supplementary information (ESI) available: The synthesis and
characterisation data of compounds 6–8, 20, 24, 29 and 31. NMR and ITC
titration data for all binding studies reported in Tables 1 and 2 of this paper.
See DOI: 10.1039/b700988g
In other work we have successfully used a combinatorial
approach to prepare tweezer receptors able to bind peptides
with a free carboxylate terminus in aqueous solvents, using
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a guanidinium group as the binding site for the carboxylate
terminus.3b,9 Although the combinatorial approach provides a
powerful method for identifying suitable receptors it was clear
that we had not necessarily optimised the receptor to maximise
the binding interaction with the carboxylate, or to preorganise the
side arms of the ‘tweezer’ to interact with the peptide backbone.
We therefore set out to prepare thiourea and guanidinium
derivatives with additional hydrogen bonding functionality, de-
signed to optimise the binding affinity with a carboxylate. At
the same time we wanted to provide a functionalised scaffold
which could be readily synthesised and incorporated into more
sophisticated structures in the future. In particular, examination
of molecular models suggested that such a scaffold could be used
to produce tweezer receptors with peptidic side arms correctly
orientated to form b-sheet interactions with peptide guests (Fig. 2).
or two methylene units, and/or with an additional amide on the
other side separated by a benzyl or pyridyl moiety. Although
these compounds were designed to provide a binding cleft for
carboxylates (Fig. 1), it is clear that they may exist in solution in
a variety of conformations stabilised by intramolecular hydrogen
bonds, and some of these conformations may not be suitable for
strong carboxylate binding. As outlined above, the pyridyl moiety
has the potential to help preorganise the binding cavity into the
desired conformation for carboxylate binding whilst potentially
introducing an unfavourable electrostatic interaction between the
pyridine N-lone pair and the guest anion.
The thiourea derivatives were prepared by coupling an amine
with an isothiocyanate. Thus 3-cyanobenzoic acid was converted
to the corresponding benzamide 6, the nitrile group was reduced
to give amine 7 and then converted to the isothiocyanate 8
(Scheme 1). Thiourea 9 was prepared directly from amine 7 by cou-
pling with benzylisothiocyanate, and coupling of isothiocyanate
8 with the amines 14 and 15 (Scheme 2) gave thioureas 10 and
11 respectively. Similarly, thioureas 12 and 13 were prepared by
coupling amines 14 and 15 with benzylisothiocyanate.
Scheme 1 i) a) SOCl2, MeOH; b) 40% MeNH2–H2O, MeOH; ii) H2, Pd,
Fig. 2 New guanidinium and thiourea derivatives as carboxylate recep-
+
=
C; iii) Cl2C S, K2CO3, H2O, MeOH; iv) 14 or 15, CH2Cl2; v) amine 14 or
tors and scaffolds for peptide tweezer receptors: X = N, CH; Y = S, NH2
;
15, CH2Cl2.
n = 1,2.
Pyrido ester 19 was prepared from pyridine dicarboxylate 18 as
previously described10 and on treatment with excess methylamine
led to aminolysis of the ester, with concomitant removal of the
phthalimide protecting group to give amine 20. Amine 20 could
not, however, be cleanly converted to the corresponding isothio-
cyanate, and instead was coupled with benzylisothiocyanate to
give 21 or with isothiocyanates 16 or 17 to give thioureas 22 and
23 respectively.
Herein are described the results of these studies which have
led to the development of a potent carboxylate binding cleft
and have also revealed a striking example of enthalpy–entropy
compensation in the binding properties of closely related receptor
structures.
Results
Attempts to convert the various thiourea derivatives directly
to guanidiniums via the S-methyl thiouroniums were largely
unsuccessful. However, this difficulty was circumvented using a
carbodiimide mediated coupling of an amine with a carbamoyl
activated thiourea.11 Hence amine 7 was converted to thiourea 24
and then coupled with amine 14 or 15 to give the Cbz-protected
Synthesis
In order to probe the optimal structure for a carboxylate
binding site scaffold a series of thioureas, 9–13 and 21–23,
and guanidinium salts, 27, 28 and 33, have been prepared
incorporating an additional amide on one side separated by one
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Amine 20 could also be converted to the corresponding
carbamoyl thiourea 29 but attempted carbodiimide mediated
coupling of this product with amines 14 or 15 led only to the
imidazo[1,5-a]pyridine derivative 30 (Scheme 4).12
Scheme 4 i) CbzNCS, CH2Cl2; ii) 14 or 15, EDC, DIPEA, CH2Cl2.
However, amine 15 could be converted to Cbz activated thiourea
31 and hence coupled with amine 20 to give the corresponding
Cbz-protected guanidine 32 (Scheme 5). Removal of the Cbz
group and treatment with HPF6 gave guanidinium 33 as the
hexafluorophosphate salt. Analogous conversion of amine 14 to
the corresponding guanidinium could not be successfully carried
out.
=
Scheme 2 i) Cl2C S, NaHCO3, CH2Cl2, H2O; ii) 40% MeNH2–H2O;
iii) PhCH2NCS, CH2Cl2; iv) 16 or 17, CH2Cl2.
guanidines 25 and 26 (Scheme 3). Removal of the Cbz-group and
treatment with HPF6 gave the guanidiniums 27 and 28 as the
hexafluorophosphate salts.
Scheme
5
i) CbzNCS, CH2Cl2; ii) 20, EDC, DIPEA, CH2Cl2;
iii) a) H2/Pd/C, MeOH; b) HPF6, H2O.
All of the thiourea and guanidinium derivatives were freely
soluble in DMSO. Analysis of the 1H NMR spectra (in DMSO-d6)
revealed that for the pyrido derivatives the signals for the various
NH were generally downfield relative to those for the analogous
benzo derivatives, with the effect being most significant (Dd ≈
0.25 ppm) for NH1 (Fig. 2). These observations are consistent
with a weak intramolecular hydrogen bond interaction of NH1
and NH2 with the pyridine nitrogen, and the anticipated partial
preorganisation of the pyridyl series, but may also indicate stronger
hydrogen bond interactions with solvent molecules.
Binding studies
The binding properties of the various thioureas were investigated
using both NMR titration experiments and isothermal calorime-
try (ITC), with tetrabutyl ammonium actetate as the guest, and
Scheme 3 i) CbzNCS, CH2Cl2; ii) 14 or 15, EDC, DIPEA, CH2Cl2;
iii) a) H2/Pd/C, MeOH; b) HPF6, H2O.
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Table 1 Binding data for thiourea derivatives with tetrabutylammonium acetate in DMSO
1H NMR titrationa
Isothermal calorimetry
Thiourea
K
ass/M−1 b
DG/kJ mol−1
K
ass/M−1 a
DG/kJ mol−1
DH/kJ mol−1
TDS/kJ mol−1
9
10
11
12
13
21
22
23
260
290
200
230
230
370
950
1740
−13.8
−14.0
−13.1
−13.5
−13.5
−14.7
−17.0
−18.5
440
230
270
370
260
370
980
2200
−15.3
−13.5
−13.9
−14.7
−13.7
−14.7
−17.1
−19.1
−6.9
−6.3
−8.7
−4.8
−6.6
−9.7
−3.8
−4.3
8.4
7.2
5.2
9.9
7.1
5.0
13.3
14.8
a Data derived by monitoring the shift of the NH protons during titration. b Errors in association constants were estimated as ∼10%.
using DMSO as the solvent (Table 1). Dilution experiments were
conducted to confirm that these receptors do not form dimers
(as thioureas are prone to do in less polar solvents) at the
concentrations used for the binding studies.
and gave a large 1 : 1 binding constant and a much smaller 1 : 2
(receptor : acetate) binding constant (Kass < 150 M−1) in each
1 : 2
case (Table 2).
Binding studies were also conducted using ITC with 27, 28 and
33 in H2O–DMSO solvent mixtures. In these more competitive
solvent mixtures the data for the pyridoguanidinium 33 could
be fitted to a simple 1 : 1 binding model with no evidence of
appreciable 1 : 2 binding. Binding of acetate was significant even
in 30% H2O–DMSO (the limit of solubility of the receptor), but
the presence of water effectively prevented binding by the benzo
analogues 27 and 28.
Data from the 1H NMR titrations could be fitted to a
1 : 1 (host : guest) binding isotherm using the NMRTit HG
software,13,14 and 1 : 1 binding stoichiometry was confirmed using
Job plots.15 Isothermal calorimetric binding data16 could also be
fitted using a one-site binding model, consistent with a 1 : 1 binding
stoichiometry. Notably the association constants obtained with
these two distinct methods were in good agreement, although the
values obtained by ITC were generally slightly larger than those
obtained from the 1H NMR titrations.
Discussion
1
In the H NMR titration experiments, addition of tetrabuty-
lammonium acetate to the thioureas led to significant downfield
shifts (Dd ≈ 1–2 ppm) of both thiourea protons H2 and H3
(for atom labels see Fig. 2) in each case, consistent with strong
hydrogen bonding interactions between the acetate and these
protons. Smaller shifts of the amide protons H1 and H4 were
observed, the most significant of these (Dd > 0.6 ppm) being for
methyl amide proton H1 in the pyridyl series 21–23.
The binding properties of the guanidiniums 27, 28 and 33 were
initially investigated using both NMR titration experiments and
ITC,17 with tetrabutylammonium acetate as the guest, and using
DMSO as the solvent. In the 1H NMR titration experiments,
addition of acetate to the guanidiniums led to significant downfield
shifts of the various NH protons (Dd up to ∼1 ppm) but also
substantial broadening in most cases and as a consequence the
data from the NMR titration experiments could not be reliably
fitted to give binding constants.
Comparing the binding data for the thioureas (Table 1) it is clear
that the binding constants for all of the benzo derivatives 9–13 are
essentially the same and indeed are little different from literature
values for binding simple carboxylates with unfunctionalised
thioureas in DMSO.18 The results indicate that the incorporation
of additional hydrogen bonding interactions in this series is not
effective in producing more potent carboxylate binders. In the
pyrido series however it is apparent that binding of acetate can
be improved by incorporation of additional hydrogen bonding
functionality and both 22 and 23 are stronger receptors (DDG =
3–4 kJ mol−1) than any of the other thioureas tested. Notably
the enthalpic contribution to binding in the pyrido compounds
22 (DH = −3.8 kJ mol−1) and 23 (DH = −4.3 kJ mol−1) is in
fact smaller than that for the analogous benzo compounds 10
(DH = −6.3 kJ mol−1) and 11 (DH = −8.7 kJ mol−1). However,
the entropic contribution to binding is much greater for the
pyrido compounds (TDS = 13–15 kJ mol−1) than for the benzo
compounds (TDS = 5–7 kJ mol−1). The improvement in binding
Isothermal calorimetric binding data obtained using DMSO as
the solvent could, however, be fitted using a two site model,17d
Table 2 Binding data for guanidinium salt derivatives with tetrabutylammonium acetate in various solvents
b
Guanidinium salta
Solvent
Kass1 : 1/M−1
DG/kJ mol−1
DH/kJ mol−1
TDS/kJ mol−1
27
DMSO
DMSO
DMSO
10% H2O–DMSO
30% H2O–DMSO
DMSO
6900c
5300c
22 000c
3900d
480d
−21.9
−21.2
−24.8
−20.5
−15.3
−20.0
−19.6
−19.4
−8.0
2.3
1.8
16.8
13.1
13.3
−1.8
28
33
33
−7.4
33
−2.0
Dibenzyl guanidinium
3100c
−21.8
a All guanidinium salts were used as the hexafluorophosphate salts. b Errors in association constants were estimated as ∼10%. c ITC data were fitted using
a two site model, giving the reported 1 : 1 binding constants and much smaller 1 : 2 binding constants (Kass < 150 M−1 in each case). d ITC data was
1 : 2
fitted using a one site (1 : 1) binding model.
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for the pyrido compounds, perhaps surprisingly, is more marked
with the longer and more flexible b-alanine derived side-arm.
Notably the improvement is only observed with incorporation
of additional functionality on both arms of the thiourea (thiourea
21 is only a marginally stronger receptor than, for example, 9, and
neither 12 nor 13 is any stronger than simple, unfunctionalised
thioureas) suggesting a significant cooperative effect for 22 and
23.19
As with the thiourea series, the pyridoguanidinium 33 proved to
be a much more effective receptor than the benzo compounds 27
and 28. Indeed 27 and 28 appear to be only marginally better car-
boxylate receptors than unfunctionalised dibenzylguanidinium.
Receptor 33, however, proved to be sufficiently good at carboxylate
binding so that a reasonably strong complex (Kass = 480 M−1)
could be formed with acetate even in a 30% H2O–DMSO solvent
mixture, which compares favourably with systems developed by
Schmuck et al.8 However, neither 27 nor 28 gave a measurable
binding constant in 10% or 30% H2O–DMSO solvent mixtures
using ITC. Notably binding by the benzo derivatives 27 and
28 in neat DMSO was highly exothermic (i.e. almost entirely
enthalpically driven) as was the case with dibenzyl guanidinium.
Previous studies of guanidinium–carboxylate complexation using
isothermal calorimetry have similarly found that binding is
generally strongly exothermic.17 Binding by 33, however, was
largely entropically driven in DMSO, and almost entirely so in
the more competitive H2O–DMSO mixtures.
thioureas 22 and 23) introduction of a pyridine moiety has a very
beneficial effect in preorganising an otherwise very flexible binding
pocket, which outweighs the detrimental electrostatic interaction
between pyridine nitrogen lone pair and anionic guest. Isothermal
calorimetry experiments show that binding a carboxylate by
33 in aqueous solvents is driven almost entirely by entropy,
and comparison of the enthalpic and entropic contributions to
binding of acetate by the series of benzo and pyrido derivatives
provides a particularly striking example of enthalpy–entropy
compensation.19
Experimental
General experimental
Reactions requiring a dry atmosphere were conducted in oven-
dried glassware under nitrogen. Where degassed solvents were
used, a stream of nitrogen was passed through them immediately
prior to use, unless otherwise stated. Solvents were of commercial
grade and were used without further purification unless otherwise
stated. Dichloromethane was distilled over calcium hydride, as
was petroleum ether where the fraction boiling between 40 ◦C
and 60 ◦C was used. TLC analysis was carried out using foil-
backed sheets coated with silica gel (0.25 mm) and containing the
fluorescent indicator UV254. Flash column chromatography was
performed, on Sorbsil C60, 40–60 mesh silica.
Presumably in the pyrido series of compounds, the intramolec-
ular hydrogen bonds between the pyridine nitrogen lone pair
and adjacent amide and thiourea/guanidinium NH’s create
a preorganised binding pocket that binds tightly to solvent
molecules. Hence the entropic cost of organizing the receptor into
a productive conformation for binding is reduced, and there is a
significant entropic gain on binding a carboxylate from the release
of tightly bound solvent molecules. The enthalpic contribution
to binding may be relatively small because of the unfavourable
electrostatic interaction between the pyridine nitrogen lone pair
and the guest anion, and because strong hydrogen bonds to the
solvent have to be broken. The benzo derivatives, on the other
hand, lack any preorganisation, so there is little contribution to
binding from the amide moieties, and solvent is less tightly bound.
Hence, on binding acetate there is less entropic gain from release
of solvent, but greater enthalpic gain from the formation of strong
hydrogen bonds, largely involving the thiourea/guanidinium
NH’s, and the carboxylate, as also observed with unfunctionalised
dibenzylguanidinium.
Instrumentation
Proton NMR spectra were obtained at 300 MHz on a Bruker AC
300 and at 400 MHz on a Bruker DPX 400 spectrometer. Carbon
NMR spectra were recorded at 75 MHz on a Bruker AC 300
spectrometer and at 100 MHz on a Bruker DPX 400 spectrometer.
Chemical shifts are reported in ppm on the d scale relative to the
signal of the solvent used. Coupling constants (J) are given in
Hz. Signal multiplicities were determined using the distortionless
enhancement by phase transfer (DEPT) spectral editing technique.
For certain compounds quaternary carbons were not observed
due to long relaxation times; these are stated in the appropriate
sections.
Infra-red spectra were recorded on a BIORAD Golden Gate
FTS 135. All samples were run either as neat solids or as oils.
Melting points were determined in open capillary tubes using
a Gallenkamp Electrothermal melting point apparatus and are
uncorrected.
Mass spectra were obtained on a VG analytical 70–250 SE nor-
mal geometry double focusing mass spectrometer. All electrospray
(ES) spectra were recorded on a Micromass Platform quadrupole
mass analyser with an electrospray ion source using acetonitrile
as the solvent. High resolution accurate mass measurements were
carried out at 10 000 resolution on a Bruker Apex III FT-ICR mass
spectrometer. Microanalyses were performed by MEDAC Ltd.,
Surrey. Calorimetry experiments were performed on an isothermal
titration calorimeter from Microcal Inc., USA.
Conclusion
Tight binding of guest molecules is generally best achieved
using highly preorganised receptors which often require complex
synthesis (for example to give constrained macrocyclic systems).
Conversely, ease of synthesis may come at the price that the
receptor is poorly preorganised and gives only weak binding. In the
case of guanidinium 33, however, an appropriate balance between
ease of synthesis and degree of preorganisation appears to have
been achieved since 33 is able to bind a carboxylate in competitive
solvent, while the synthesis is sufficiently straightforward to
suggest that it may be readily incorporated into more sophisticated
architectures such as tweezer receptors (Fig. 2). For 33 (and
1H NMR titration experiments14
1
All H NMR titration experiments were conducted on a Bru¨ker
AM 300 spectrometer at 298 K, unless otherwise stated. A sample
of host was dissolved in the deuterated solvent. A portion of this
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solution was used as the host NMR sample and the remainder
used to dissolve a sample of the guest, so that the concentration
of the host remained constant throughout the titration. Guest
stock solutions were typically prepared such that 10 lL of that
solution contained 0.1 equivalents of guest with respect to host.
Successive aliquots of the guest solution were added to the host
NMR sample and 1H NMR sample recorded after each addition.
The changes in chemical shifts of various signals as a function
of guest concentration were analysed using the NMRTit HG
software,13 assuming a 1 : 1 or a 1 : 2 binding stoichiometry.
(found: C, 61.40; H, 6.04; N, 14.81; C19H22N4O2S requires C,
61.60; H, 5.99; N, 15.12%); mmax (solid)/cm−1 3293m, 3064w,
2935w, 1727w, 1655s, 1638s, 1545s; dH (400 MHz, DMSO-d6) 2.77
(3 H, d, J 4, CH3NH), 4.17 (2 H, d, J 6, CH2C(O)), 4.31 (2 H, d,
J 6, NHCH2Ph), 4.71 (2 H, br, ArCH2NHC(S)), 7.20–7.34 (5 H,
m, Ar), 7.37–7.45 (2 H, m, Ar), 7.67–7.71 (2 H, m, Ar and
NHCH2C(O)), 7.75 (1 H, s, Ar), 8.27 (1 H, br, ArCH2NHC(S)),
8.39 (1 H, q, J 4, CH3NH), 8.48 (1 H, br, NHCH2Ph); dC
(100 MHz, DMSO-d6) 24.7 (CH3), 40.6 (CH2), 45.5 (CH2),
47.1 (CH2), 123.9 (CH), 124.6 (CH), 125.3 (CH), 125.7 (CH),
126.71 (CH), 126.73 (CH), 128.4 (CH), 133.1 (C), 137.7 (C),
165.1 (C), 167.3 (C); m/z (ES) 393 ((M + Na)+, 100%); HRMS
(ES) C19H22N4O2SNa (M + Na)+ requires calc. 393.1355, found
393.1356.
Isothermal calorimetry titrations14
All experiments were performed using an isothermal titration
calorimeter from Microcal Inc. (Northampton, MA). In a typical
experiment a 1–2 mM receptor solution was added to the
calorimetric cell. A solution of tetrabutylammonium acetate (48–
78 mM) was introduced in 50 injections of 5 lL, to a total of
250 lL of added guest. The solution was continuously stirred to
ensure rapid mixing and kept at 25 ◦C, through the combination
of an external cooling bath and an internal heater. Dilution effects
were determined by performing a blank experiment by adding
the same guest solution to the pure solvent and subtracting this
from the raw titration to produce the final binding curve. Binding
parameters were determined by applying either one-site or two-
sites models, using the Origin software provided. These methods
rely on standard nonlinear least-squares regression (Levenberg–
Marquard method) to fit the curves, taking into account the
change in volume that occurs during the calorimetric titration.
N -1-Methyl-3-[([3-(benzylamino)-3-oxopropyl]aminocarbo-
thioyl)amino]methyl benzamide 11. Isothiocyanate 8 (64 mg,
0.31 mmol) and amine 15 (61 mg, 0.34 mmol) were stirred in
CH2Cl2 (4 cm3) for 24 h. The solvent was removed in vacuo and
purification by column chromatography (5% CH3OH–CH2Cl2)
gave thiourea 11 (86 mg, 0.22 mmol, 71%) as a white solid. Mp
147–150 ◦C; (found: C, 62.71; H, 6.18; N, 14.30; C20H24N4O2S
requires C, 62.47; H, 6.29; N, 14.57%); mmax (solid)/cm−1 3274m,
3074w, 2921w, 1638s, 1543s; dH (400 MHz, DMSO-d6) 2.77 (2 H,
t, J 7, NHCH2CH2), 2.77 (3 H, d, J 5, CH3), 3.65 (2 H, br,
NHCH2CH2), 4.27 (2 H, d, J 6, NHCH2Ph), 4.69 (2 H, br,
ArCH2NHC(S)), 7.19–7.33 (5 H, m, Ar), 7.35–7.43 (2 H, m, Ar),
7.56 (1 H, br, NHCH2CH2), 7.68 (1 H, d, J 7, Ar), 7.74 (1 H, s,
Ar), 8.01 (1 H, br, ArCH2NHC(S)), 8.38 (1 H, br, CH3NH), 8.40
(1 H, br, NHCH2Ph); dC (100 MHz, DMSO-d6) 1 × CH2 obscured
by solvent, 25.7 (CH3), 34.3 (CH2), 40.0 (CH2), 41.5 (CH2), 124.8
(CH), 125.7 (CH), 126.2 (CH), 126.7 (CH), 127.6 (CH), 127.7
(CH), 129.3 (CH), 134.1 (C), 138.9 (C), 139.0 (C), 166.1 (C), 170.1
(C), 176.8 (C); m/z (ES) 407 ((M + Na)+, 100%), 385 (27); HRMS
(ES) for C20H24N4O2SNa (M + Na)+ requires 407.1512, found
407.1519.
Synthesis
Amines 14 and 15 were prepared by hydrazinolysis of the corre-
sponding N-phthalimidoyl amides.20 The synthesis and character-
isation data of compounds 6–8, 20, 24, 29 and 31 are described in
the electronic supplementary information.†
N-1-Methyl-3-([(benzylamino)carbothioyl]aminomethyl) benza-
mide 9. Amine 7 (58 mg, 0.27 mmol) and benzyl isothiocyanate
(0.036 cm3, 0.27 mmol) were stirred in CH2Cl2 (2 cm3) for 20 h.
The solvent was removed in vacuo and purification by column
chromatography (20–60% EtOAc–CH2Cl2) gave thiourea 9 (66 mg,
0.21 mmol, 78%) as a white foam. Mp 78–81 ◦C; (found: C, 65.30;
H, 6.08; N, 13.21; C17H19N3OS requires C, 65.15; H, 6.11; N,
13.41%); mmax (solid)/cm−1 3284m, 3062w, 3033w, 2935w, 1739m,
1642s, 1536s; dH (400 MHz, DMSO-d6) 2.78 (3 H, d, J 4, CH3),
4.69 (2 H, br, CH2), 4.72 (2 H, br, CH2), 7.21–7.35 (5 H, m, Ar),
7.37–7.44 (2 H, m, Ar), 7.69 (1 H, d, J 7, Ar), 7.77 (1 H, s, Ar), 8.00
(2 H, br, thiourea-NH), 8.40 (1 H, q, J 4, CH3NH); dC (100 MHz,
CDCl3) 28.0 (CH3), 49.4 (CH2), 49.9 (CH2), 126.9 (CH), 127.2
(CH), 128.9 (CH), 129.1 (CH), 130.0 (CH), 130.1 (CH), 132.1
(CH), 135.8 (C), 139.0 (C), 140.0 (C), 170.2 (C), 184.3 (C); m/z
(ES) 336 ((M + Na)+, 100%).
N-1-Benzyl-2-[(benzylamino)carbothioyl]aminoacetamide 12.
Amine 14 (75 mg, 0.42 mmol) and benzyl isothiocyanate
(0.051 cm3, 0.38 mmol) were stirred in CH2Cl2 (2 cm3) for
24 h. The solvent was removed in vacuo and purification by
column chromatography (20% EtOAc–CH2Cl2 to neat EtOAc)
gave thiourea 9 (85 mg, 0.26 mmol, 68%) as a white solid. Mp
146–149 ◦C; (found: C, 65.27; H, 5.97; N, 13.32; C17H19N3OS
requires C, 65.15; H, 6.11; N, 13.41%); mmax (solid)/cm−1 3286m,
3062w, 3036w, 2937w, 1738m, 1642s, 1537s; dH (400 MHz,
DMSO-d6) 4.15–4.18 (2 H, br, CH2C(O)), 4.29–4.32 (2 H, m,
C(O)NHCH2Ph), 4.66 (2 H, br, PhCH2NHC(S)), 7.22–7.33
(10 H, m, Ar), 7.64 (1 H, br, NHCH2C(O)), 8.21 (1 H, br,
ArCH2NHC(S)), 8.45 (1 H, br, C(O)NHCH2Ph); dC (100 MHz,
1 : 1 CD3OD–CDCl3) 1 × CH2 obscured by solvent, 43.8 (CH2),
48.1 (CH2), 127.8 (CH), 128.0 (CH), 128.0 (CH), 128.1 (CH),
129.1 (2 × CH), 138.6 (C), 170.9 (C); m/z (ES) 336 ((M +
Na)+, 100%); HRMS (ES) C17H19N3OSNa (M + Na)+ requires
336.1141, found 336.1147.
N-1-Methyl-3-[([2-(benzylamino)-2-oxoethyl]aminocarbothioyl)-
amino]methyl benzamide 10. Isothiocyanate
8
(57 mg,
0.28 mmol), amine 14 (91 mg, 0.56 mmol) and triethylamine
(TEA) (0.010 cm3, 0.14 mmol) were stirred in CH2Cl2 (3 cm3)
for 18 h. The solvent was removed in vacuo and purification by
column chromatography (2–10% CH3OH–CH2Cl2) gave thiourea
10 (68 mg, 0.18 mmol, 64%) as a white solid. Mp 168–171 ◦C;
N-1-Benzyl-3-[(benzylamino)carbothioyl]aminopropanamide 13.
Amine 15 (35 mg, 0.21 mmol) and benzyl isothiocyanate
(0.035 cm3, 0.27 mmol) were stirred in CH2Cl2 (2 cm3) for 24 h.
CH2Cl2 (5 cm3) was added and the mixture was washed with H2O
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(5 cm3) and brine (5 cm3), dried (MgSO4) and the solvent was
removed in vacuo. Purification by column chromatography (2–6%
CH3OH–CH2Cl2) and recrystallisation (EtOAc–petroleum ether)
gave thiourea 13 (46 mg, 0.15 mmol, 71%) as a white solid. Mp 143–
147 ◦C; (found: C, 66.31; H, 6.58; N, 12.61; C18H21N3OS requires
C, 66.02; H, 6.46; N, 12.83%); mmax (solid)/cm−1 3289m, 3062w,
3034w, 2936w, 1739m, 1643s, 1522s; dH (400 MHz, DMSO-d6)
2.43 (2 H, t, J 7, NHCH2CH2), 3.64 (2 H, br, NHCH2CH2), 4.27
(2 H, d, J 6, C(O)NHCH2Ph)), 4.64 (2 H, br, PhCH2NHC(S)),
7.34–7.22 (10 H, m, Ar), 7.52 (1 H, br, NHCH2CH2)), 7.96 (1 H,
br, PhCH2NHC(S)), 8.41 (1 H, t, J 6, C(O)NHCH2Ph); dC
(100 MHz, DMSO-d6) 2 × CH2 obscured by solvent, 34.4 (CH2),
42.0 (CH2), 126.7 (CH), 126.8 (CH), 127.2 (CH), 127.3 (CH), 128.2
(2 × CH), 139.4 (2 × C), 170.6 (C), 179.1 (C); m/z (ES) 328 ((M +
H)+, 100%); HRMS (ES) for C18H21N3OSNa (M + Na)+ requires
350.1297, found 350.1294.
m/z (ES) 372 ((M + H)+, 100%); HRMS (ES) C18H21N5O2SNa
(M + H)+ requires 394.1308, found 394.1308.
N -2-Methyl-6-[([3-(benzylamino)-3-oxopropyl]aminocarbo-
thioyl)amino]methyl-2-pyridine carboxamide 23. Amine 1520
(89 mg, 0.50 mmol) was stirred in CH2Cl2 (5 cm3) at 0 ◦C and
sat. NaHCO3 (2.5 cm3) was added. Thiophosgene (0.076 cm3,
1.00 mmol) was added to the organic phase and the reaction
was stirred vigorously for 20 min. CH2Cl2 (5 cm3) was added, the
organic phase was washed with 2 M HCl (5 cm3) and brine (5 cm3),
and dried (MgSO4) and the solvent was removed in vacuo to give
isothiocyanate 17 which was used without further purification.
Isothiocyanate 17 was added to amine 20 (42 mg, 0.25 mmol) in
CH2Cl2 (3 cm3) and the mixture was stirred for 48 h. The solvent
was removed in vacuo and purification by column chromatography
(1–5% CH3OH–CH2Cl2) gave thiourea 23 (30 mg, 0.08 mmol,
31% overall) as a brown waxy solid. (Found: C, 59.34; H, 5.84;
N, 18.37; C19H23N5O2S requires C, 59.20; H, 6.01; N, 18.17%);
mmax (solid)/cm−1 3298br, 3087w, 2941w, 1648s, 1593w, 1536s; dH
(400 MHz, DMSO-d6) 2.47 (2 H, t, J 6, NHCH2CH2), 2.96 (3 H,
d, J 5, CH3), 3.70 (2 H, br, NHCH2CH2), 4.29 (2 H, d, J 6,
CH2Ph), 4.81 (2 H, br, pyrCH2), 7.18–7.32 (5 H, m, Ph), 7.47
(1 H, d, J 7, pyr), 7.83 (1 H, br, NHCH2CH2), 7.87–7.96 (2 H,
m, pyr), 8.16 (1 H, br, pyrCH2NH), 8.43 (1 H, br, NHCH2Ph),
8.73 (1 H, q, J 5, CH3NH); dC (100 MHz, CD3OD) 26.4 (CH3),
36.4 (CH2), 41.6 (CH2), 44.1 (CH2), 54.8 (CH2), 121.5 (CH), 125.6
(CH), 128.1 (CH), 128.4 (CH), 129.49 (CH), 129.51 (CH), 139.4
(C), 139.9 (C), 150.3 (C), 158.0 (C), 167.1 (C), 173.8 (C); m/z (ES)
386 ((M + H)+, 100%); HRMS (ES) C19H23N5O2SNa (M + Na)+
requires 408.1464, found 408.1467.
N -2-Methyl-6-([(benzylamino)carbothioyl]aminomethyl)-2-
pyridine carboxamide 21. Amine 20 (76 mg, 0.46 mmol) and
benzyl isothiocyanate (0.055 cm3, 0.46 mmol) were stirred in
CH2Cl2 (2 cm3) for 18 h. The solvent was removed in vacuo and
purification by column chromatography (EtOAc) gave thiourea
21 (96 mg, 0.30 mmol, 73%) as a white foam. (Found: C, 61.30;
H, 6.03; N, 17.81; C16H18N4OS requires C, 61.12; H, 5.77; N,
17.82%); mmax (solid)/cm−1 3288m, 3061w, 3031w, 2939w, 1739m,
1643s, 1537s; dH (400 MHz, DMSO-d6) 2.85 (3 H, d, J 5, CH3), 4.72
(2 H, br, CH2), 4.86 (2 H, d, J 5, CH2), 7.23–7.36 (5 H, m, Ar), 7.46
(1 H, br, pyr), 7.88–7.97 (2 H, m, Ar), 8.13 (1 H, t, J 5, thiourea-
NH), 8.21 (1 H, br, thiourea-NH), 8.65 (1 H, q, J 5, CH3NH);
dC (100 MHz, CDCl3) 26.10 (CH3), 48.7 (CH2), 49.4 (CH2), 120.8
(CH), 124.9 (CH), 127.7 (2 × CH), 128.8 (CH), 137.6 (C), 138.3
(CH), 148.7 (C), 155.8 (C), 165.2 (C), 183.0 (C); m/z (ES) 337
((M + Na)+, 100%), 315 (61); HRMS (ES) C16H18N4OSNa (M +
Na)+ requires 337.1093, found 337.1097.
Benzyl N-{[2-(benzylamino)-2-oxoethyl]amino(3-[(methylamino)-
carbonyl]benzylamino)methylene}carbamate 25. Thiourea 24
(70 mg, 0.20 mmol), amine 14 (64 mg, 0.39 mmol) and diisopropy-
lethylamine (DIPEA, 0.070 cm3, 0.39 mmol) were stirred in dry
CH2Cl2 (4 cm3) and dimethylformamide (DMF, 0.5 cm3) under N2
N-2-Methyl-6-[([2-(benzylamino)-2-oxoethyl]aminocarbothioyl)-
amino]methyl-2-pyridine carboxamide 22. Amine 1420 (100 mg,
0.61 mmol) was dissolved in CH2Cl2 (5 cm3) at 4 ◦C and
sat. NaHCO3 (2.5 cm3) was added. Thiophosgene (0.093 cm3,
1.21 mmol)◦was added to the organic phase and the reaction was
stirred at 4 C for 10 min. CH2Cl2 (5 cm3) was added, the phases
were separated, the organic phase was washed with 2 M HCl
(5 cm3) and brine (5 cm3), and dried (MgSO4) and the solvent
was removed in vacuo to give isothiocyanate 16 which was used
without further purification. Crude isothiocyanate 16 (37 mg,
0.18 mmol) was added to amine 20 (30 mg, 0.18 mmol) in CH2Cl2
(3 cm3) and the mixture was stirred for 18 h. The solvent was
removed in vacuo and purification by column chromatography
(1–6% CH3OH–CH2Cl2) gave thiourea 22 (37 mg, 0.10 mmol,
55%) as a brown waxy solid. (Found: C, 58.46; H, 5.76; N,
18.97; C18H21N5OS requires C, 58.20; H, 5.70; N, 18.85%); mmax
(solid)/cm−1: 3294br, 3086w, 2924w, 1656s, 1592w, 1543s; dH
(400 MHz, DMSO-d6) 2.86 (3 H, d, J 4, CH3), 4.20 (2 H, br,
CH2C(O)), 4.33 (2 H, d, J 6, NHCH2Ph), 4.84 (2 H, br, pyrCH2),
7.20–7.33 (5 H, m, Ph), 7.51 (1 H, d, J 8, pyr), 7.88–7.97 (3 H,
m, pyr and NHCH2C(O)), 8.43 (1 H, br, pyrCH2NH), 8.58 (1 H,
t, J 6, NHCH2Ph), 8.76 (1 H, q, J 4, CH3NH); dC (100 MHz,
DMSO-d6) 26.0 (CH3), 42.1 (CH2), 47.1 (CH2), 48.7 (CH2), 120.0
(CH), 123.9 (CH), 126.8 (CH), 127.2 (CH), 128.3 (CH), 138.3 (C),
139.3 (CH), 149.2 (C), 156.8 (C), 158.7 (C), 164.2 (C), 168.8 (C);
◦
at 4 C. 1-Ethyl-3-(3ꢀ-dimethylaminopropyl)carbodiimide (EDC,
74 mg, 0.39 mmol) was added and the reaction was stirred at
◦
4 C for 1 h and at room temperature for 48 h. The solvent was
removed in vacuo and purification by column chromatography (1–
4% CH3OH–CH2Cl2) gave guanidine 25 (71 mg, 0.15 mmol, 75%)
as a white solid. Mp 173–176 ◦C; mmax (solid)/cm−1 3309w, 1635s,
1605s, 1556s; dH (400 MHz, DMSO-d6) 9.25 (1 H, br, NH), 8.45
(1 H, br, NH), 8.40 (2 H, m, 2 × NH), 7.80 (1 H, s, Ar), 7.71
(1 H, d, J 8, Ar), 7.38–7.48 (2 H, m, Ar), 7.18–7.38 (10 H, m,
Ar), 4.96 (2 H, s, OCH2Ph), 4.50 (2 H, d, J 6, CH2), 4.31 (2 H,
d, J 6, CH2), 3.92 (2 H, br, CH2), 2.76 (3 H, d, J 5, CH3); dC
(100 MHz, DMSO-d6): 1 × CH2 obscured by solvent, 26.2 (CH3),
42.1 (CH2), 43.7 (CH2), 65.6 (CH2), 125.9 (CH), 126.7 (CH), 127.1
(CH), 127.5 (CH), 127.8 (CH), 128.2 (CH), 129.7 (CH), 134.5 (C),
137.8 (C), 142.1 (C), 159.9 (C × 2), 166.5 (C), 170.9 (C); m/z
(ES) 510 ((M + Na)+, 100%); HRMS (ES) C27H30N5O4 (M + H)+
requires 488.2293, found 488.2295.
Benzyl
N-{[3-(benzylamino)-3-oxopropyl]amino(3-[(methyl
amino)carbonyl]benzylamino)methylene}carbamate 26. Thiourea
24 (115 mg, 0.31 mmol), amine 15 (100 mg, 0.61 mmol) and
dry TEA (0.083 cm3, 0.61 mmol) were stirred in dry CH2Cl2
(5 cm3). EDC (117 mg, 0.61 mmol) was added and the reaction
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was stirred for 8 h at room temperature. The solvent was removed
in vacuo and purification by column chromatography (EtOAc to
10% CH3OH–CH2Cl2) gave guanidine 26 (115 mg, 0.21 mmol,
69%) as a white foam. Mp 162–166 ◦C; (found: C, 66.98; H, 6.22;
N, 13.71; C28H31N5O4 requires C, 67.05; H, 6.23; N, 13.96%);
mmax (solid)/cm−1 3285m, 3064w, 2930w, 1738w, 1640s, 1537s; dH
(400 MHz, DMSO-d6) 2.45 (2 H, br, NHCH2CH2), 2.77 (3 H, d,
J 4, CH3), 3.47 (2 H, dt, J 6 and 6, NHCH2CH2), 4.27 (2 H, d, J
5, NHCH2Ph), 4.46 (2 H, d, J 6, ArCH2NHC(S)), 4.96 (2 H, s,
OCH2Ph), 7.19–7.35 (11 H, m, Ar and NH), 7.36–7.41 (2 H, m,
Ar), 7.69 (1 H, d, J 8, Ar), 7.76 (1 H, s, Ar), 8.40 (1 H, q, J 4,
CH3NH), 8.47 (1 H, t, J 5, NHCH2Ph), 9.01 (1 H, br, NH); dC
(100 MHz, DMSO-d6) 1 × C not observed, 1 × CH2 obscured
by solvent, 26.2 (CH3), 35.1 (CH2), 37.3 (CH2), 42.1 (CH2), 65.5
(CH2), 118.0 (C), 126.0 (CH), 126.7 (CH), 127.2 (2 × CH), 127.4
(CH), 127.7 (CH), 128.2 (3 × CH), 129.6 (CH), 137.8 (C), 139.3
(2 × C), 166.6 (2 × C), 170.3 (C); m/z (ES) 524 ((M + Na)+,
100%); HRMS (ES) C28H32N5O4 (M + H)+ requires 502.2449,
found 502.2451.
6, NHCH2CH2), 4.29 (2 H, d, J 6, NHCH2Ph), 4.43 (2 H, d, J 6,
ArCH2NHC(N)), 7.21–7.34 (5 H, m, Ar), 7.41–7.46 (2 H, m, Ar),
7.51 (3 H, br, NH2 and NHCH2CH2), 7.75 (1 H, d, J 7, Ar), 7.79
(1 H, s, Ar), 7.94 (1 H, br, ArCH2NHC(N)), 8.45 (1 H, q, J 4,
CH3NH), 8.52 (1 H, t, J 6, NHCH2Ph); dC (100 MHz, CD3OD)
27.0 (CH3), 36.0 (CH2), 39.0 (CH2), 44.2 (CH2), 45.6 (CH2), 127.1
(CH), 127.5 (CH), 128.2 (CH), 128.5 (CH), 129.5 (CH), 130.1
(CH), 131.4 (CH), 136.0 (C), 138.3 (C), 139.7 (C), 157.7 (C), 170.3
(C), 173.2 (C); m/z (ES) 368 ((M − PF6)+, 100%); HRMS (ES)
C20H26N5O2 (M − PF6)+ requires 368.2081, found 368.2079.
Benzyl N-5-[(methylamino)carbonyl]imidazo[1,5-a]pyridin-3-yl-
carbamate 30. Thiourea 29 (74 mg, 0.21 mmol) and N-1-benzyl-
2-aminoacetamide 14 (68 mg, 0.41 ◦mmol) were stirred in dry
CH2Cl2 (3 cm3) and DMF (1 cm3) at 4 C. EDC (78 mg, 0.41 mmol)
was added and the reaction was stirred at room temperature
for 8 h. The solvents were removed in vacuo, the residue was
dissolved in CHCl3 (10 cm3) and the solution was extracted
with 1% HCl (3 × 5 cm3). The aqueous phases were combined,
basified (2 M NaOH, pH ≈ 12) and extracted with EtOAc (3 ×
10 cm3). The combined EtOAc phases were dried (MgSO4), the
solvent was removed in vacuo and the product was purified by
column chromatography (CH2Cl2 to 4% CH3OH–CH2Cl2) to give
imidazole 30 (27 mg, 0.08 mmol, 40%) as a yellow foam. mmax
(solid)/cm−1 3251w, 3048w, 2933w, 1732m, 1717m, 1652m, 1625m,
1542s; dH (400 MHz, DMSO-d6) 2.78 (3 H, d, J 3, CH3NH), 5.12
(2 H, s, OCH2Ph), 6.79–6.88 (2 H, m, Ar), 7.36–7.46 (5 H, m, Ph),
7.48 (1 H, s, NCH), 7.68 (1 H, dd, J 9 and 1, Ar), 8.77 (1 H, q,
J 3, CH3NH), 9.42 (1 H, br, CbzNH); dC (100 MHz, DMSO-d6)
26.5 (CH3), 66.7 (CH2), 114.2 (C), 118.5 (CH), 118.9 (C), 120.2
(CH), 128.3 (CH), 128.5 (CH), 128.9 (CH), 130.4 (CH), 130.5 (C),
130.9 (CH), 137.0 (C), 155.1 (C), 163.6 (C); m/z (ES) 325 ((M +
H)+, 100%); HRMS (ES) C17H17N4O3 (M + H)+ requires 325.1295,
found 325.1295.
N-1-Methyl-3-[(ammonio[2-(benzylamino)-2-oxoethyl]amino-
methyl)amino]methylbenzamide hexafluorophosphate 27. Guani-
dine 25 (71 mg, 0.15 mmol) and 10% Pd/C (cat.) in CH3OH (3 cm3)
and CH2Cl2 (1 cm3) were stirred under a hydrogen atmosphere
(1 atm) for 2 h. The palladium residues were removed by filtration
and washed with CH3OH (10 cm3). The filtrate was concentrated
in vacuo and the residue was dissolved in H2O (4 cm3), 60%
HPF6–H2O (10 drops) was added and the aqueous solution was
extracted with EtOAc (3 × 5 cm3). The combined organic phases
were washed with brine (3 cm3), and dried (MgSO4), and the
solvent was removed in vacuo to give guanidinium salt 27 (51 mg,
0.10 mmol, 70%) as a white foam. (Found: C, 39.64; H, 4.22; N,
12.46; C19H24N5O2PF6 requires C, 39.70; H, 4.21; N, 12.18%); mmax
(solid)/cm−1 3357w, 1737w, 1631s, 1556s; dH (400 MHz, CD3CN)
3.00 (3 H, s, CH3), 4.01 (2 H, br, CH2), 4.43 (1 H, d, J 6, CH2), 4.51
(2 H, d, J 5, CH2), 6.51 (2 H, br, NH2), 6.69 (1 H, br, NH), 7.22
(1 H, br, NH), 7.28–7.48 (5 H, m, Ar), 7.52–7.61 (3 H, m, Ar and
NH), 7.71 (1 H, d, J 8, Ar), 7.76 (1 H, s, Ar), 8.39 (1 H, br, NH);
dC (100 MHz, CD3OD) 27.0 (CH3), 44.3 (CH2), 44.9 (CH2), 45.7
(CH2), 126.8 (CH), 127.6 (CH), 128.4 (CH), 128.6 (CH), 129.6
(CH), 130.2 (CH), 131.4 (CH), 136.0 (C), 138.2 (C), 139.4 (C),
158.5 (C), 169.7 (C), 170.3 (C); m/z (ES) 354 ((M − PF6)+, 100%);
HRMS (ES) C19H24N5O2 (M − PF6)+ requires 354.1934, found
354.1929.
Benzyl N-[3-(benzylamino)-3-oxopropyl]amino[(6-[(methylamino)-
carbonyl]-2-pyridylmethyl)amino]methylenecarbamate 32. EDC
(111 mg, 0.58 mmol) was added to a mixture of thiourea
31 (106 mg, 0.29 mmol), N-2-methyl-6-(aminomethyl)-2-
pyridinecarboxamide 20 (81 mg, 0.49 mmol) and dry TEA
(0.082 cm3, 0.58 mmol) in dry CH2Cl2 (5 cm3) and the reaction was
stirred for 5 h. The solvent was removed in vacuo and purification
by column chromatography (5% CH3OH–CH2Cl2) gave guanidine
◦
32 (111 mg, 0.22 mmol, 76%) as a white solid. Mp 118–120 C;
N-1-Methyl-3-[(ammonio[3-(benzylamino)-3-oxopropyl]amino-
methyl)amino]methylbenzamide hexafluorophosphate 28. Guani-
dine 26 (52 mg, 0.10 mmol) and 10% Pd/C (cat.) in CH3OH (3 cm3)
was stirred under a hydrogen atmosphere (1 atm) for 2 h. The
palladium residues were removed by filtration and washed with
CH3OH (10 cm3). The filtrate was concentrated in vacuo and the
residue was dissolved in H2O (4 cm3), 60% HPF6–H2O (10 drops)
was added and the aqueous solution was extracted with EtOAc
(3 × 5 cm3). The combined organic phases were washed with
brine (3 cm3), and dried (MgSO4), and the solvent was removed
in vacuo to give guanidinium salt 28 (44 mg, 0.09 mmol, 83%) as a
white foam. (Found: C, 40.98; H, 4.33; N, 11.76; C20H26N5O2PF6
requires C, 40.80; H, 4.45; N, 11.89%); mmax (solid)/cm−1 3275w,
2969w, 1738s, 1630s, 1543s; dH (400 MHz, DMSO-d6) 1 × CH2
obscured by solvent, 2.78 (3 H, d, J 4, CH3NH), 3.42 (2 H, q, J
(found: C, 64.58; H, 6.05; N, 16.45; C27H30N6O4 requires C, 64.53;
H, 6.02; N, 16.71%); mmax (solid)/cm−1 3349w, 2945w, 1738m,
1667m, 1620s, 1597s, 1542s; dH (400 MHz, DMSO-d6) 1 × CH2
obscured by solvent, 2.83 (3 H, d, J 3, CH3NH), 3.52 (2 H, d, J
6, NHCH2CH2), 4.28 (2 H, d, J 4, NHCH2Ph), 4.57 (2 H, d, J 4,
pyr-CH2), 4.97 (2 H, br, OCH2Ph), 7.16–7.36 (10 H, m, Ar), 7.48
(1 H, m, pyr), 7.84–8.00 (2 H, m, pyr), 8.50 (1 H, br, NHCH2Ph),
8.72 (1 H, br, CH3NH), 9.03 (1 H, br, guanidine-NH), 10.14 (1 H,
br, guanidine-NH); dC (100 MHz, CD3OD) 1 × C not observed,
26.5 (CH3), 37.3 (CH2), 38.8 (CH2), 44.2 (CH2), 46.4 (CH2), 67.8
(CH2), 121.6 (CH), 125.6 (CH), 128.1 (CH), 128.5 (CH), 128.7
(CH), 128.8 (CH), 129.4 (CH), 129.5 (CH), 139.0 (2 × C), 139.7
(C), 139.9 (C), 143.3 (CH), 165.0 (C), 173.8 (2 × C); m/z (ES) 503
((M + H)+, 100%); HRMS (ES) C27H31N6O4 (M + H)+ requires
503.2411, found 503.2402.
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N -2-Methyl-6-[(ammonio[3-(benzylamino)-3-oxopropyl]amino
methyl)amino]methyl-2-pyridinecarboxamide hexafluorophosphate
33. Guanidine 32 (44 mg, 0.17 mmol) and 10% Pd/C (cat.) in
CH3OH (3 cm3) was stirred under a hydrogen atmosphere (1 atm)
for 2 h. The palladium residues were removed by filtration and
washed with CH3OH (10 cm3). The filtrate was concentrated
in vacuo and the residue was dissolved in H2O (4 cm3), 60%
HPF6–H2O (10 drops) was added and the aqueous solution was
extracted with EtOAc (3 × 5 cm3). The combined organic phases
were washed with brine (3 cm3), and dried (MgSO4), and the
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◦
0.11 mmol, 91%) as a white foam. Mp 173 C (dec.); (found: C,
38.74; H, 4.39; N, 14.41; C19H25N6O2PF6 requires C, 38.69; H,
4.27; N, 14.25%); mmax (solid)/cm−1 3468w, 3395w, 3336w, 3205w,
2970w, 1738w, 1641s, 1540m; dH (400 MHz, DMSO-d6) 1 × CH2
obscured by solvent, 2.81 (3 H, d, J 5, CH3), 3.40 (2 H, dt, J 6
and 6, NHCH2CH2), 4.25 (2 H, d, J 6, CH2Ph), 4.51 (2 H, d,
J 6, pyrCH2), 7.15–7.28 (5 H, m, Ph), 7.45 (1 H, d, J 8, pyr),
7.55 (3 H, br, NHCH2CH2 and NH2), 7.84 (1 H, br, pyrCH2NH),
7.90–7.99 (2 H, m, pyr), 8.49 (1 H, t, J 6, NHCH2Ph), 8.61 (1 H,
q, J 5, CH3NH); dC (100 MHz, CD3OD) 26.4 (CH3), 36.0 (CH2),
39.2 (CH2), 44.3 (CH2), 46.9 (CH2), 122.3 (CH), 125.7 (CH), 128.3
(CH), 128.5 (CH), 129.5 (CH), 139.8 (C), 140.0 (CH), 150.7 (C),
155.6 (C), 158.1 (C), 166.9 (C), 173.3 (C); m/z (ES) 369 ((M −
PF6)+, 100%); HRMS (ES) C19H25N6O2 (M − PF6)+ requires
369.2033, found 369.2031.
11 B. R. Linton, A. J. Carr, B. P. Orner and A. D. Hamilton, J. Org. Chem.,
2000, 65, 1566–1568.
12 J. Bourdais, A. Omar and M. E. Mohsen, J. Heterocycl. Chem., 1980,
17, 555–558.
13 A. P. Bisson, C. A. Hunter, J. C. Morales and K. Young, Chem.–Eur. J.,
1998, 4, 845.
14 All titration data is provided in the electronic supplementary
information†.
15 K. A. Connors, Binding Constants, John Wiley and Sons, New York,
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1714 | Org. Biomol. Chem., 2007, 5, 1706–1714
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