Conformations, conformational preferences, and conformational exchange of N′-substituted N -acylguanidines: Intermolecular interactions hold the key

Article abstract of DOI:10.1021/ja103756y

Guanidine and acylguanidine groups are crucial structural features of numerous biologically active compounds. Depending on the biological target, acylguanidines may be considered as considerably less basic bioisosteres of guanidines with improved pharmacokinetics and pharmacodynamics, as recently reported for N′-monoalkylated N-acylguanidines as ligands of G-protein-coupled receptors (GPCRs). The molecular basis for enhanced ligand-receptor interactions of acylguanidines is far from being understood. So far, only a few and contradictory results about their conformational preferences have been reported. In this study, the conformations, conformational preferences, and conformational exchange of four unprotonated and seven protonated monoalkylated acylguanidines with up to six anions and with bisphosphonate tweezers are investigated by NMR. Furthermore, the effects of the acceptor properties in acylguanidine salts, of microsolvation by dimethylsulfoxide, and of varying acyl and alkyl substituents are studied. Throughout the whole study, exclusively two out of eight possible acylguanidine conformations were detected, independent of the compound, the anion, or the solvent used. For the first time, it is shown that the strength and number of intermolecular interactions with anions, solvent molecules, or biomimetic receptors decide the conformational preferences and exchange rates. One recently presented and two new crystal structures resemble the conformational preferences observed in solution. Thus, consistent conformational trends are found throughout the structurally diverse compound pool, including two potent GPCR ligands, different anions, and receptors. The presented results may contribute to a better understanding of the mechanism of action at the molecular level and to the prediction and rational design of these biologically active compounds.

Full text of DOI:10.1021/ja103756y

Published on Web 07/23/2010
Conformations, Conformational Preferences, and
Conformational Exchange of N′-Substituted N-Acylguanidines:
Intermolecular Interactions Hold the Key
Roland Kleinmaier,‡ Max Keller,† Patrick Igel,† Armin Buschauer,† and
Ruth M. Gschwind*,‡
Institut fu¨r Organische Chemie and Institut fu¨r Pharmazie, UniVersita¨t Regensburg,
UniVersita¨tsstrasse 31, D-93053 Regensburg, Germany
Received May 3, 2010; E-mail: ruth.gschwind@chemie.uni-regensburg.de
Abstract: Guanidine and acylguanidine groups are crucial structural features of numerous biologically active
compounds. Depending on the biological target, acylguanidines may be considered as considerably less
basic bioisosteres of guanidines with improved pharmacokinetics and pharmacodynamics, as recently
reported for N′-monoalkylated N-acylguanidines as ligands of G-protein-coupled receptors (GPCRs). The
molecular basis for enhanced ligand-receptor interactions of acylguanidines is far from being understood.
So far, only a few and contradictory results about their conformational preferences have been reported. In
this study, the conformations, conformational preferences, and conformational exchange of four unprotonated
and seven protonated monoalkylated acylguanidines with up to six anions and with bisphosphonate tweezers
are investigated by NMR. Furthermore, the effects of the acceptor properties in acylguanidine salts, of
microsolvation by dimethylsulfoxide, and of varying acyl and alkyl substituents are studied. Throughout the
whole study, exclusively two out of eight possible acylguanidine conformations were detected, independent
of the compound, the anion, or the solvent used. For the first time, it is shown that the strength and number
of intermolecular interactions with anions, solvent molecules, or biomimetic receptors decide the
conformational preferences and exchange rates. One recently presented and two new crystal structures
resemble the conformational preferences observed in solution. Thus, consistent conformational trends are
found throughout the structurally diverse compound pool, including two potent GPCR ligands, different
anions, and receptors. The presented results may contribute to a better understanding of the mechanism
of action at the molecular level and to the prediction and rational design of these biologically active
compounds.
Y receptors.9-11 Especially in the field of medicinal chemistry,
acylguanidines are greatly appreciated because they show
Introduction
Guanidine compounds are highly valued in various biological,
biochemical, and medical applications,1-3 including inhibition
of enzymes4 and the Na+-H+ ion exchanger (NHE),5,6 as
substances with positive inotropic effects,7 as anticoagulants,8
and as highly active ligands of transmembrane receptors from
various families, e.g., aminergic and peptidergic G-protein-
coupled receptors (GPCRs) such as histamine and neuropeptide
improved pharmacokinetic properties compared to those of the
corresponding guanidines, while retaining or even improving
the biological activity.9-11 Acylation of guanidine reduces its
basicity by 4-5 orders of magnitude, which improves the
pharmacokinetic properties, but retains its general ability to
interact with acidic functions of the biological target.9 Indeed,
the increased acidity of the acylguanidinium NH protons leads
to stronger binding to carboxylates compared to that observed
for simple guanidinium cations,12 e.g., in supramolecular
assemblies.13
‡ Institut fu¨r Organische Chemie.
† Institut fu¨r Pharmazie.
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K. V. J. Org. Chem. 2004, 69, 309–313.
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1448.
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Schnell, D.; Bernhardt, G.; Dove, S.; Zabel, M.; Elz, S.; Seifert, R.;
Buschauer, A. J. Med. Chem. 2008, 51, 7193–7204.
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Rep. 2008, 25, 919–954.
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(5) Lee, S.; Kim, T.; Lee, B. H.; Yoo, S.; Lee, K.; Yi, K. Y. Bioorg.
Med. Chem. Lett. 2007, 17, 1291–1295.
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Gross, D.; Wolfbeis, O. S.; Bernhardt, G.; Buschauer, A. ChemBio-
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9
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J. AM. CHEM. SOC. 2010, 132, 11223–11233 11223

A R T I C L E S
Kleinmaier et al.
Regarding structural and conformational investigations of
acylguanidines, extensive theoretical studies on the potassium-
sparing monoacylguanidine diuretic amiloride and its derivatives
in the solid state and in solution have been published.14-17 In
addition, the crystal structure of a protein-amiloride complex
proved the formation of charge-assisted H-bonds between the
planar acylguanidine moiety and an aspartate side chain,18,19
which are much stronger than H-bonds between neutral
species.20,21 These charge-assisted H-bonds are similar to the
famous arginine-carboxylate salt bridges,22,23 which are crucial
to the tertiary structure of many proteins24 and have been subject
of numerous theoretical studies, too.13,24-26 For N-alkylated
acylguanidines, a conformational study of rotationally restricted,
i.e., mostly cyclized, acylguanidine compounds is available,27,28
but it is limited to compounds which are at least N′,N′′-
bisalkylated. No compounds are included with a free NH2 group
as observed in the biologically active compounds cited above.9-11
However, to our knowledge, for the class of N′-monosubstituted
N-acylguanidines (further on referred to as monoalkylated
acylguanidines, see Figure 1), conformational studies are still
missing.
Figure 1. Possible conformations of monoalkylated acylguanidines with
intramolecular H-bonds in the unprotonated (I-III) and protonated states
4
(I+-III+). Zigzag pathways leading to JH,H scalar couplings between
H-atoms are indicated for the protonated forms.
In principle, the acylguanidine moiety of both protonated and
uncharged monoalkylated acylguanidines can adopt eight planar
conformations. Five of these conformations are energetically
unfavorable due to severe steric hindrance and/or the lack of
an intramolecular H-bond (see Supporting Information for
details). Each of the remaining three conformations (see Figure
1) shows an intramolecular H-bond and similar contributions
to steric hindrance, suggesting small energy differences between
the conformations I, II, and III or I+, II+, and III+.
The main difference of conformation I/I+ compared to
conformations II/II+ and III/III+ is the formation of the
intramolecular H-bond to the RNH or the NH2 group, respec-
tively. In complexes of non-acylated arginines with bisphos-
phonate tweezers, energetic preferences were calculated for
intermolecular H-bonds to the RNH group in quantum chemical
calculations29 and molecular dynamics simulations.30 Thus, also
in acylguanidines, a preferred formation of H-bonds to the NH
group might be expected (conformation I/I+). Indeed, the only
crystal structure of a monoalkylated acylguanidine published
so far, to our knowledge, which is the precursor of a histamine
H2 receptor ligand crystallized as free base, shows conformation
I with an intramolecular H-bond to the NH group (see Figure
2a).9,31
In contrast, the only existing NMR study about the H-bond
pattern of an acylguanidine moiety, which was performed on a
complex of an arginine derivative with bisphosphonate tweezers,
reports the existence of conformation II+ in this bioorganic
model complex in solution (see Figure 2b).32 Also a docking
study of an acylguanidine-type agonist in the binding pocket
of the guinea pig H2 receptor suggests conformation II+ (see
Figure 2c).9 Interestingly, the structure used in this docking study
is a detritylated and protonated form of a close analogue of the
ligand, the precursor of which adopts conformation I in the solid
state, as mentioned above (Figure 2a).
The deviations among these results from theoretical calcula-
tions and NMR, X-ray, and docking studies show that monoalky-
lated acylguanidines may not have one generally preferred
conformation but suggest that the substituents and the proton-
ation state of the acylguanidine and/or the geometry and the
H-bond acceptor qualities of the receptor may substantially
influence the conformational preferences. Considering the
general importance of pre-organization effects for the binding
constants of complexes,33 e.g., via H-bonding,34 and the recent
theoretical results about facilitated self-assembly of rigidified
guanidines,35 the elucidation and prediction of the conforma-
(14) Noel, J.; Germain, D.; Vadnais, J. Biochemistry 2003, 42, 15361–
15368.
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Crystallogr. 2001, C57, 1217–1219.
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C. A. J. Am. Chem. Soc. 1994, 116, 1502–1513.
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S.; Stuerzebecher, J.; Jacob, U. J. Mol. Biol. 2000, 301, 465–475.
(19) Zeslawska, E.; Oleksyn, B.; Stadnicka, K. Struct. Chem. 2004, 15,
567–572.
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(21) Steiner, T. Angew. Chem. 2002, 114, 50–80.
(22) Schlereth, K.; Beinoraviciute-Kellner, R.; Zeitlinger, M. K.; Bretz,
A. C.; Sauer, M.; Charles, J. P.; Vogiatzi, F.; Leich, E.; Samans, B.;
Eilers, M.; Kisker, C.; Rosenwald, A.; Stiewe, T. Mol. Cell 2010, 38,
356–368.
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M.; Kessler, H. Angew. Chem., Int. Ed. 2005, 44, 5247–5251.
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J. F. R.; Magalhaes, A. L.; Maigret, B.; Nascimento, M. C.; Reuter,
N. J. Mol. Struct. (Theochem) 1999, 463, 81–90.
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2004, 126, 10228–10229.
(31) CCDC 686506 contains the supplementary crystallographic data for
the compound shown in Figure 2 (available free of charge from the
Cambridge Crystallographic Data Centre viawww.ccdc.cam.ac.uk/
data_request/cif).
(25) Condic-Jurkic, K.; Perchyonok, V. T.; Zipse, H.; Smith, D. M.
J. Comput. Chem. 2008, 29, 2425–2433.
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Chem. Soc. 2008, 130, 16846–16847.
(26) Zheng, Y.-J.; Ornstein, R. L. J. Am. Chem. Soc. 1996, 118, 11237–
11243.
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(27) Greenhill, J. V.; lsmail, M. J.; Bedford, G. R.; Edwards, P. N.; Taylor,
P. J. J. Chem. Soc., Perkin Trans. 2 1985, 1265–1274.
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P. J. J. Chem. Soc., Perkin Trans. 2 1985, 1255–1264.
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11224 J. AM. CHEM. SOC. VOL. 132, NO. 32, 2010

Conformations of N′-Substituted N-Acylguanidines
A R T I C L E S
Figure 2. (a) Crystal structure representation9,31 (ORTEP diagram) of the free base of trityl-protected acylguanidine in conformation I. (b) Bioorganic
model complex of [15Nη ]-Bz-Arg-(Nη-propionyl)-OEt (conformation II+) with bisphosphonate tweezers.32 (c) Computational docking model9 of the gpH2R
2
binding site for protonated N-acyl-N′-imidazolylpropylguanidine with H-bond, indicating conformation II+.
tional preferences of monoalkylated acylguanidines may con-
siderably support the rational development of this pharmaco-
logically important class of ligands.
Therefore, in this study, the conformations, conformational
preferences, and conformational exchange of four unprotonated
and seven protonated monoalkylated acylguanidines with up to
six anions and with bisphosphonate tweezers are investigated
by NMR. The effect of the acceptor properties in acylguanidine
salts, the influence of microsolvation by dimethylsulfoxide
(DMSO), and the contribution of varying acyl and alkyl
substituents to the conformational equilibria and their exchange
rates are presented. Consistent conformational trends are derived
depending on the strengths of the intermolecular interactions,
which are additionally corroborated by two new crystal structures.
Results and Discussion
Compound Pool. For this conformational study of acylguani-
dines, seven model compounds were selected (Figure 3). First,
[15Nη ]-Bz-Arg-(Nη-propionyl)-OEt (1), the acylguanidine used
2
in the previous NMR complexation study, was chosen to enable
a comparison between the acylguanidine conformations in the
free state and bound to an artificial receptor molecule.32 Second,
two highly potent acylguanidine-type GPCR ligands were
investigated, the histamine H4 receptor agonist UR-PI29436 (2)
Figure 3. Compounds investigated in this work: [15Nη ]-Bz-Arg-(Nη-
2
and the arginine-derived neuropeptide Y (NPY) Y1 receptor
selective antagonist UR-MK50 (3), which is an acylated
derivative of the first potent and selective Y1 receptor antagonist,
BIBP 3226.37 Third, N-propionyl-N′-butylguanidine (4a) and
N-cinnamoyl-N′-butylguanidine(4b)werechosenasacylguanidines
with very simple alkyl substituents and strongly differing acyl
substituents to eliminate any interference from additional
functional groups in the alkyl moiety. Finally, to further
investigate the influence of structural and electronic variations
in the acyl substituents, the para-substituted benzoic acid
derivatives N-p-dimethylaminobenzoyl-N′-butylguanidine (4c)
and N-p-methoxybenzoyl-N′-butylguanidine (4d) were used.
The syntheses of 138 and 236 have already been reported,
while the syntheses of 3 and 4a-d are described in the
Experimental Section, and further details can be found in the
Supporting Information.
propionyl)-OEt (1), N-propionyl-N′-(3-imidazol-4-ylpropyl)guanidine (2),
UR-MK50 (3), the simple n-butyl chain analogues N-propionyl-N′-bu-
tylguanidine (4a) and N-cinnamoyl-N′-butylguanidine (4b), and benzoic acid
derivatives N-p-dimethylaminobenzoyl-N′-butylguanidine (4c) and N-p-
methoxybenzoyl-N′-butylguanidine (4d). The free bases 4a-d were pro-
tonated with one or several of the acids shown to give the 1:1 salts 4a-d·X.
NMR Studies of Protonated Acylguanidines in Solution.
Conformational Equilibria and Conformational Assignment.
Organic solvents were chosen for all NMR investigations of
acylguanidines, because recently Limbach et al. showed that
the hydrophobic environment of the active site in the interior
of an enzyme, i.e., in the absence of bulk water, may be modeled
better by an aprotic solvent than by water.39 In addition, these
solvents strengthen the H-bonds and allow for low-temperature
measurements to detect conformational equilibria.
To optimize the NMR investigations of protonated monoalky-
lated acylguanidines and to identify dynamic equilibria between
the different possible conformations, first a screening of the
(36) Igel, P.; Schneider, E.; Schnell, D.; Elz, S.; Seifert, R.; Buschauer, A.
J. Med. Chem. 2009, 52, 2623–2627.
(37) Rudolf, K.; Eberlein, W.; Engel, W.; Wieland, H. A.; Willim, K. D.;
Entzeroth, M.; Wienen, W.; Beck-Sickinger, A. G.; Doods, H. N. Eur.
J. Pharmacol. 1994, 271, R11–R13.
(39) Sharif, S.; Fogle, E.; Toney, M. D.; Denisov, G. S.; Shenderovich, I.;
Buntkowsky, G.; Tolstoy, P. M.; Huot, M. C.; Limbach, H.-H. J. Am.
Chem. Soc. 2007, 129, 9558–9559.
(38) Kleinmaier, R.; Gschwind, R. M. J. Label. Compd. Radiopharm. 2009,
52, 29–32.
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A R T I C L E S
Kleinmaier et al.
Figure 4. Generally observed dynamic behavior of protonated acylguanidines
for the example of 4d·DCA in CD2Cl2/DMSO-d6 (9:1): (a) at 285 K four
NH signals are observed; (b) upon cooling to 210 K the four signals split
into eight NH signals, indicating the two conformations I+ and II+, which
exchange fast on the NMR time scale at elevated temperatures (for labeling
see Figure 5).
spectroscopic properties of 1-3·TFA, 4b·X (X ) HCl, TFA,
DCA, AcOH, Boc-Asp-OBn, TMBA), 4a·X, (X ) HCl, TFA,
DCA, AcOH), 4c·X (X ) TFA, Boc-Asp-OBn), and 4d·X (X
) HCl, TFA, DCA, Boc-Asp-OBn) was performed in a
temperature range between 300 and 200 K in CD2Cl2/DMSO-
d6 (9:1). This survey revealed, for all protonated monoalkylated
Figure 5. NH section of a 1H,1H COSY spectrum of 4b·TMBA in CD2Cl2
and DMSO-d6 (9:1) at 200 K. For the major conformation, the detection of
4
the two JH,H long-range couplings W2 and W3 allow the assignment to
1
acylguanidines in this solvent mixture, one general H signal
II+; for the minor conformation, W1 allows the identification of I+.
pattern with a similar temperature-dependent behavior, which
is shown for the example of 4d·DCA in Figure 4. At elevated
temperatures, four NH signals are detected, which fit perfectly
to the four NH protons expected for the acylguanidine moiety
(Figure 4a). However, upon cooling these NH signals broaden,
show coalescence effects, and split into eight NH resonances,
indicating two conformations at low temperatures (Figure 4b).
These two conformations, separable at low temperature, are
denoted as I+ and II+ in Figure 4b and exchange fast on the
NMR time scale at elevated temperature (Figure 4a). This
general 1H pattern and this trend in the temperature dependence
were found for all protonated acylguanidines investigated in this
study in this solvent mixture. The individual coalescence
temperatures, conformational ratios, and absolute chemical shift
values of each complex, however, show slight variations and
depend on the anion, microsolvation, and substitution pattern
applied (see discussion below).
In the case of several protonated acylguanidines, which show
not only well-separated NH signals but also small line widths
at low temperatures, the two conformations I+ and II+ could be
unambiguously assigned as described elsewhere.40 Generally
1H,1H COSY and 1H,13C HMBC spectra allow the identification
of NHalk and NHamide (for labeling see Figure 5). In addition,
differentiation among conformations I+, II+, and III+ (see Figure
1) is possible using the spatial information of so-called
w-couplings within the acylguanidine moiety. These long-range
4JH,H scalar couplings are only detectable in a zigzag arrange-
ment of two NH protons (see marking in Figures 1 and 5) and
therefore allow the identification of the spatial arrangement of
pairs of NH protons, resulting in the assignment of the two
conformations I+ and II+. This is exemplarily shown on the
NH section of a 1H,1H COSY spectrum of 4b·TMBA at 200 K
in Figure 5. For the major conformation, two w-couplings are
detected, one to the NHamide (W3) and one to the NHalk (W2).
This combination of w-couplings allows identification of the
major conformation as II+. For the minor conformation only
one w-coupling to NHalk (W1) is detected, which allows
assignment of this conformation to I+. These assignments are
also in accordance with the relative chemical shifts of the NH
resonances, which are influenced by the formation of strong
H-bonds to the anion (see below).
Interaction with Anions: A Conformational Exchange Brake.
To investigate the influence of the anions on the exchange
dynamics between the two conformations of protonated
acylguanidines, 4b in CD2Cl2 and DMSO-d6 (9:1) was selected,
because the NMR survey showed for this combination the best-
resolved spectra within a wide range of anions. With strong
acids, four sharp guanidinium NH signals are detected for 4b
at elevated temperatures, indicating fast exchange between the
two conformations on the NMR time scale. This is shown in
Figure 6a for the 1H spectrum of 4b·TFA at 300 K. In addition,
the conformational exchange of 4b·TFA is so fast that the
coalescence point is even lower than 210 K (see Figure 6b). In
1
contrast, for 4b·AcOH, the H spectra of the acylguanidine
moieties change drastically. At 300 K, only broad signals are
detected, which are close to the coalescence point (Figure 6c),
(40) Kleinmaier, R.; Gschwind, R. M. Magn. Reson. Chem. 2010, in press
(published online Jul 18, doi: 10.1002/mrc.2648).
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Conformations of N′-Substituted N-Acylguanidines
A R T I C L E S
Figure 6. NH sections of the 1H NMR (600 MHz) spectra of 4b·TFA
(a,b) and 4b·AcOH (c,d) in CD2Cl2 and DMSO-d6 (9:1) at 300 K (a,c) and
at 210 K (b,d). The conformational exchange is significantly faster with
TFA than with AcOH.
whereas at 210 K, the signals sharpen and the typical chemical
shift pattern of the conformations I+ and II+ is observed (Figure
6d).
These spectra suggest that the exchange rate between the two
conformations can be gradually adjusted by the strength of the
interaction with the anion. Therefore, salts of 4b derived from
six different acids (HCl, TFA, DCA, AcOH, Boc-Asp-OBn, and
1
TMBA) were investigated at 210 K, and their H spectra are
shown in Figure 7, ordered by decreasing acidity. With Cl- as
anion, only one broad and unresolved 1H signal is detected for
all six amine protons of both conformations, indicating medium-
fast exchange above the coalescence point. With decreasing
acidity, i.e., increasing H-bond acceptor strength, the NH signal
dispersion becomes better while the resonances are still broad
until six separate and rather sharp signals are detected. This
indicates reduced conformational exchange with increasing
interaction strength between the cationic acylguanidine and its
anion.41 In addition, the signals with the highest chemical shift,
the NHamide signals of II+ and I+, NHalk(II+), and NHhb(I+),
experience a strong further increase in chemical shift with
decreasing acidity of the acid, whereas the chemical shifts of
the remaining NH signals are nearly unaffected by the change
of the anion. These selective low-field shifts in the chemical
shift pattern can be attributed to the formation of strong H-bonds
to the anion,42 and thus the main positions of the anions in
conformations I+ and II+ are derived as depicted in Figure 5.43
Both the reduced chemical exchange and the higher chemical
shift dispersion of specific NH protons indicate specific inter-
molecular H-bonds to the anion. In addition, the conformational
Figure 7. Modulation of the conformational exchange rate and the NH
signal dispersion by the H-bond acceptor properties of the anion. Shown
1
are the stacked NH sections of H spectra of 4b in CD2Cl2 and DMSO-d6
(9:1) at 210 K, protonated with six different acids whose anions possess
increasing electron densities in the carboxylate, going from lowest at the
top (a) to highest at the bottom (f). The exchange rate is reduced and the
signal separation is enhanced with reduced acid strength.
exchange rate is modulated by the H-bond strength.44,45 It can
be seen in Figure 7 that the higher the H-bond acceptor strength,
the stronger are the H-bonds between anion and cation and the
lower is the conformational exchange rate. Thus, a strong
interaction with the anion acts like a brake for the conforma-
tional exchange.
(41) Simulations of the conformational exchange rates between I+ and II+
for 4b·Boc-Asp-OBn and 4b·TFA showed that the broadening of the
signals in Figure 7 is dominated by the exchange rate and that the
different chemical shift dispersions affect the exchange rates only
marginally. According to these simulations, at 210 K the rate constant
for the conformational exchange is increased by a factor of 30 when
going from the strong H-bond acceptor Boc-Asp-OBn to the much
weaker TFA as anions.
(44) We correlate here the H-bond acceptor strength of the anion with the
electron density available for H-bonding in the carboxylate moiety,
which is typically higher for more basic anions, i.e., when they are
derived from weaker acids. This concept of ordering the anions by
their electron densities has been checked and confirmed by an analysis
of the 13C chemical shifts of the carboxylic carbons of alkali salts of
the acids used here (see Supporting Information).
(42) Sharif, S.; Denisov, G. S.; Toney, M. D.; Limbach, H.-H. J. Am. Chem.
Soc. 2007, 129, 6313–6327, and references therein.
(43) DOSY spectra of 4a·DCA at 300 K and 4d·DCA at 270 K showed
identical diffusion coefficients for the anion and the cation. Due to
the fast exchange of the NH protons in acylguanidines, the positions
of the anions could not be verified by NOESY cross peaks.
(45) Gojło, E.; Smiechowski, M.; Panuszko, A.; Stangret, J. J. Phys. Chem.
B 2009, 113, 8128–8136.
9
J. AM. CHEM. SOC. VOL. 132, NO. 32, 2010 11227

A R T I C L E S
Kleinmaier et al.
Figure 8. Effect of microsolvation by DMSO on the conformational distribution and exchange rate of protonated acylguanidines. The conformational shift
from I+ to II+ upon microsolvation by DMSO is shown on the 1H sections of 4d·Boc-Asp-OBn at 225 K: (a) 95/5% I+/II+ in pure CD2Cl2 and (b) 50/50%
1
I+/II+ after addition of 10% DMSO-d6. The acceleration of conformational exchange upon microsolvation by DMSO is demonstrated on the H sections of
4d·HCl at 195 K: (c) in pure CD2Cl2, both conformations are detected (70/30% I+/II+), whereas (d) after addition of 3% DMSO-d6, one conformationally
averaged set of signals is observed.
Microsolvation by DMSO. Because of severe solubility prob-
lems of protonated monoalkylated acylguanidines in apolar
solvents, so far exclusively solvent mixtures (CD2Cl2 and
DMSO-d6, 9:1) have been used. In terms of H-bond networks,
microsolvation by DMSO means a considerable H-bond accep-
tor competition between the DMSO solvent molecules and the
anion. Additionally, the electrostatic attraction between the anion
and the acylguanidinium cation is weakened by the high
susceptibility of DMSO. Therefore, the interaction between
anion and acylguanidinium cation should be considerably
stronger in solvents with reduced susceptibility and without
H-bond acceptor properties, such as pure CD2Cl2. The com-
pound pool was screened for solubility in pure CD2Cl2, and
4d·Boc-Asp-OBn, 4d·HCl, and 4c·Boc-Asp-OBn were found
to be sufficiently soluble in pure CD2Cl2 to conduct DMSO-d6
titration experiments at 225 and 195 K, i.e., temperatures at
which the conformational exchange is slow on the NMR time
scale. Starting from pure CD2Cl2, increasing amounts of DMSO
were added and the spectral changes monitored, as shown for
4d·Boc-Asp-OBn and 4d·HCl in Figure 8. The spectra of
4d·Boc-Asp-OBn (Figure 8a,b) show impressively that micro-
solvation with DMSO not only affects the conformational
exchange rate, as expected from the results about the interaction
with the anions (see above), but also shifts drastically the ratio
of the two conformations. In pure CD2Cl2, conformation I+ is
strongly predominant over conformation II+, with a ratio of 95:5
(Figure 8a). However, upon addition of about 10% DMSO-d6,
the portion of the conformation II+ grows to 50% (Figure 8b).
Further addition of DMSO changes the conformational distribu-
tion only marginally but leads to significant line broadening,
indicating increased conformational exchange (data not shown).
Interestingly, the conformational preference for I+ over II+ (70:
30) is significantly smaller for 4d · HCl (see Figure 8c), and
already addition of less than 5% DMSO (see Figure 8d) leads
to one set of four NH signals, i.e., fast exchange between the
two conformations on the NMR time scale.
strongest H-bond interaction between acylguanidinium moiety
and anion, i.e., with a strong H-bond acceptor (Boc-Asp-OBn)
and without competing DMSO molecules, the highest preference
for conformation I+ is observed. Upon weakening this interac-
tion either through microsolvation by DMSO or through
changing the anion properties, i.e., by replacing Boc-Asp-OBn
with Cl-, the conformational preference for I+ is significantly
reduced. By further weakening the interaction with the anion,
e.g., by raising the percentage of DMSO, the exchange between
the two conformations becomes too fast to be observed by NMR.
As a result, all data hint at a conformational preference of I+
induced by a strong interaction with the anion. However, for
both conformations, forklike H-bonds to two guanidinium
protons are proposed and confirmed by the chemical shift
patterns of I+ and II+ (see Figure 5 and discussion above), and
in both conformations, three NH protons are involved in
H-bonds. Thus, at first glance the interaction patterns look quite
similar. However, in I+ the NH2 group is involved in the
intermolecular H-bond, whereas in II+ the NH2 group is involved
in the intramolecular H-bond. Therefore, we suggest an entropic
contribution from the rotation of the NH2 group inside the
H-bond network as the reason for the preference of conformation
I+ in pure CD2Cl2. In a partially flexible salt bridge to the anion,
the rotational barrier of the NH2 group is expected to be lower
than in an intramolecular H-bond to the carbonyl within a rather
rigid six-membered ring. In pure CD2Cl2, these H-bonds are
strong enough to produce a sufficient difference in total energy,
which makes I+ the preferred conformation. (For a further
reasoning and spectral evidence for a NH2 rotation, see
Supporting Information.) Additional H-bond acceptors such as
DMSO, reduced electrostatic attraction because of higher
susceptibility of the solvent, or anions with reduced interaction
strength are expected to lead to lower rotational barriers for the
NH2 group in conformation I+ and thus to a reduced preference
for I+, as observed experimentally.
Impact of Alkyl and Acyl Substituents. The next step was to
investigate whether varying substituents on the acylguanidine
moiety affect the conformational preferences or exchange rates.
First, the influence of different alkyl substituents was investi-
gated. As evident from the compound pool shown in Figure 3,
The described trend in the conformational preferences
depending on the amount of microsolvation with DMSO and
the anion properties now fit perfectly to the interactions with
the anions discussed above. For the combination with the
9
11228 J. AM. CHEM. SOC. VOL. 132, NO. 32, 2010

Conformations of N′-Substituted N-Acylguanidines
A R T I C L E S
substituted aromatic ring in 4c is a better mesomeric electron
donator than the p-methoxy-substituted one in 4d. Therefore,
in 4c the positive charge of the guanidine unit may be attenuated
more than in 4d. This makes the acylguanidine unit of 4c a
weaker H-bond donator, which manifests in lower chemical
shifts of the NH protons affected by H-bonds.39 Consequently,
the most acidic proton, i.e., NHamide, shows the greatest chemical
shift change.
These comparisons show that structural changes in remote
parts of the alkyl substituent do not affect the structural
properties of the acylguanidine moiety significantly. In contrast,
acyl substituents that change the electronic or mesomeric
properties of the acylguanidine moiety can change the strength
of the H-bond network.
Conformations of the Free Base. In addition to the protonated
acylguanidines, the spectral and conformational properties of the
free acylguanidine bases were investigated to elucidate the effect
of protonation on preferred conformations and conformational
exchange. For this purpose, the free bases of 4a-d were synthe-
sized and investigated NMR spectroscopically. In accordance with
previous reports that the acylimino tautomer was the preferred
conformation of unprotonated acylguanidines,17,46,47 in our spectra
of 4a-d, no hint of protonation of the amide nitrogen was found.
Therefore, in Figure 1 and in the following, only conformations
of the acylimino tautomer are discussed.
Figure 9. Influence of the electronic properties of the acyl substituent on
the strengths of the H-bond network, shown on the 1H sections of (a)
4a·TFA, (b) 4b·TFA, (c) 4c·TFA, and (d) 4d·TFA at 215 K in CD2Cl2
and DMSO-d6 (9:1). Exchange is much faster, leading to four sharp lines
in (c) and (d), because the H-bond network is weakened due to delocalization
of the charge.
with 1, 2, and 4a, three different alkyl residues are available
with identical acyl moieties. In addition, 3 exhibits only an
n-butyl instead of an ethyl group in the acyl part, which is
expected to induce only marginal changes. Therefore, 3 was
also included in the comparison. All four of these compounds
have an alkyl chain directly attached to the guanidine moiety.
In addition, two amino acid scaffolds are present in 1 and 3, as
well as an imidazolium moiety with a second positive charge
in 2. Comparisons of the 1H spectra of the TFA salts of 1, 2, 3,
and 4a show astonishingly similar signal patterns of the
guanidine NH signals and only a slightly reduced exchange for
4a (see Supporting Information for spectra). This shows that,
for protonated acylguanidines, remote changes in the structure,
i.e., the presence of additional functional groups potentially
capable of forming additional but weaker H-bonds, do not affect
the structural properties of the acylguanidine moiety significantly.
Second, the influence of structural changes in the acyl moiety
was investigated on 4a-d, which have identical alkyl group
but deviating acyl substituents. The 1H spectra of the NH region
of the TFA salts of 4a-d at 215 K are shown in Figure 9.
Interestingly, the spectra of 4a with an ethyl group and 4b with
a styryl group are very similar (see Figure 9a,b), aside from
small low-field shifts for 4b. This indicates the formation of
slightly stronger H-bonds in 4b, supported by the additional
π-system, which do not significantly affect the exchange rates.
In contrast, for 4c and 4d, much higher exchange rates are
observed. The TFA salts of 4c and 4d (see Figure 9c,d) show
only four separate peaks with quite narrow line widths,
indicating that conformational exchange is already fast on the
NMR time scale. As discussed above, this faster conformational
exchange can be slowed down by using lower amounts of
DMSO (see Supporting Information for spectra). A possible
reason for the increased exchange rate is the reduced strength
of the intramolecular H-bond caused by the para-substituted
phenyl rings acting as electron donators. Additionally, delocal-
ization of the positive charge of the guanidinium carbon over
the π-system may weaken the electrostatic interactions with the
anions and thus reduce the salt-bridge character of the inter-
molecular H-bonds. This trend is also evident from the increased
chemical shift when comparing 4c and 4d: the p-dimethylamino-
NMR investigations of 4a-c revealed again the existence of
two conformations at low temperatures, which show confor-
mational exchange at elevated temperatures and a shift in the
conformational preferences upon microsolvation by DMSO.
Both effects are exemplarily shown on the 1H spectra of 4d in
Figure 10. Again, the NHalk resonances of both conformations
were unambiguously assigned on the basis of 1H,1H COSY cross
peaks to the alkyl substituents. Regarding the NH2 protons, in
the spectra of conformation II of 4d, a 2JNH splitting of 4.5 Hz
2
was observed, confirming the assignment. However, in contrast
to the protonated acylguanidines, for none of the unprotonated
acylguanidines were 4JH,H scalar couplings detected, which was
attributed to the reduced delocalization of the double bond.
Therefore, the further conformational assignment was based on
the interpretation of chemical shift differences and dimerization
trends measured by 1H,1H DOSY spectra. Interestingly, for the
two sets of NH signals, very similar diffusion coefficients were
measured, indicating dimers for both conformations in pure
CD2Cl2 (for details, see Supporting Information). As evident
from Figure 1, conformations I and II exhibit both structurally
typical and very similar dimerization sites, with the unprotonated
amide nitrogen and one NH proton on the same side. In contrast,
conformation III does not possess such an H-bond donor/
acceptor pair, and intermolecular interactions with the amide
nitrogen are hampered by severe steric hindrance. These
structural features of III should lead to a significantly reduced
aggregation tendency compared to I and II. Therefore, on the
basis of the nearly equal diffusion coefficients of the two
observed conformations, conformation III was excluded.
The remaining assignment starts from the two unambiguously
assigned NHalk signals. In I, the NHalk proton is in the
intramolecular H-bond and will exert the highest chemical shift.
This is found for the conformation with the broad line widths
(see Figure 10b-d). In contrast, in the second set of signals,
(46) Skawinski, W. J.; Ofsievich, A.; Venanzi, C. A. Struct. Chem. 2002,
13, 73–80.
(47) Venanzi, C. A.; Plant, C.; Venanzi, T. J. J. Comput. Chem. 1991, 12,
850–861.
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J. AM. CHEM. SOC. VOL. 132, NO. 32, 2010 11229

A R T I C L E S
Kleinmaier et al.
1
Figure 10. Unprotonated acylguanidines. Again, increased temperature leads to conformational exchange, as shown in the NH sections of the H spectra
of 4d in pure CD2Cl2 at (a) 285 and (b) 210 K, and again, a conformational shift from I to II upon microsolvation by DMSO is observed, as shown in the
1H spectra of 4d at 225 K in (c) pure CD2Cl2 and (d) CD2Cl2 and DMSO-d6 (9:1).
NHalk has the lowest chemical shift value, in accordance with
conformation II. For further interpretations of the line widths
and chemical shift patterns of I and II, see the Supporting
Information.
significant contributions from any chemical exchange or rota-
tional movements within the H-bond network.
In addition, the complex of the NPY Y1 receptor antagonist
3 with T1 (see Figure 11) was investigated. This highly
biologically active ligand contains various additional functional
1
The H spectra of 4d in pure CD2Cl2 (Figure 10c) and after
1
addition of 10% DMSO (Figure 10d) now reveal that, also in
the unprotonated acylguanidines, conformation I is highly
preferred (about 93/7% I/II) and that microsolvation with 10%
DMSO shifts the preferred conformation to II (about 42/58%
I/II). Similar to the protonated acylguanidines, the conforma-
tional preference for I in pure CD2Cl2 might be driven by
intermolecular interactions, which are weakened upon micro-
solvation by DMSO, allowing the portion of conformation II
to grow. In contrast to the protonated acylguanidines, in which
the interaction with the anion determines the conformational
preference, one can speculate that, in unprotonated acylguanidines,
the dimerization properties of the conformations may be crucial
for the conformational preference. Besides stronger H-bonds
in the dimer of I, which are suggested by the chemical shift
pattern and geometry-optimized structure of the dimer (see
Supporting Information for details), again the entropic properties
seem to promote conformation I in the dimer. This is strongly
suggested by the broad line widths of the NH signals in I,
indicating conformational flexibility within the dimer, even in
pure CD2Cl2 (see Figure 10b,c).
groups that are expected to form additional H-bonds. The H
spectra of this complex show one major conformation (∼60%)
with sharp line widths and one minor conformation with mainly
extremely broad NH signals (for spectra and details, see
Supporting Information). The major conformation could be
4
assigned to II+ on the basis of long-range JH,H couplings as
2h
described above, and four
J
scalar couplings could be
H,P
detected, indicating intermolecular H-bonds (see Figure 11a and
Supporting Information for spectra). For the minor conformation,
no structural information could be detected due to the broad
line widths. Therefore, it cannot be distinguished whether this
minor conformation shows deviations in the acylguanidine
conformation or different modes of interaction or conformations
in the part of 3 remote from the acylguanidine.
These results seem to suggest that additional strong H-bond
acceptors do not reverse the conformational trends described
above for the free acylguanidine salts but rather support the
preference and stability of conformation II+ in complex H-bond
networks. This trend is also in agreement with a docking study
of the histamine H2 GPCR ligand in the binding pocket of the
guinea pig H2 receptor (see Figure 2c), which results in
conformation II+ for the complex receptor surrounding in the
active site of the transmembrane protein.9
Conformational Preferences in Crystal Structures. In addition
to the NMR studies, further conformational information about
monoalkyl-substituted acylguanidines was collected from X-ray
structures. Besides the one previously published crystal structure
of an unprotonated imidazolylpropylguanidine,9 which adopts
conformation I (see Figure 2a), two further crystal structures
were obtained from our compound pool, 4b and 4d·HCl (see
Conformational Preferences in More Complex Receptors. With
regard to the relevance of acylguanidines as ligands for
pharmacologically important receptor proteins, the conforma-
tional preferences in a more complex receptor system were
studied. One example, recently published by our group,32 is the
additional complexation of 1·TFA by a bisphosphonate tweezers
molecule, which resembles an artificial arginine fork (see Figure
2a). This complex was also measured in CD2Cl2/DMSO-d6 (9:
1). Interestingly, the acylguanidine was found to adopt exclu-
sively conformation II+ at 220 K. The NMR detection of scalar
couplings across two H-bonds in combination with the signifi-
cantly reduced line widths of the NH signals allow us to exclude
(48) Spartan; Wavefunction, Inc.: Irvine, CA, 1991-2007.
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11230 J. AM. CHEM. SOC. VOL. 132, NO. 32, 2010

Conformations of N′-Substituted N-Acylguanidines
A R T I C L E S
2h
Figure 11. Complex between 3 and bisphosphonate tweezers T1 (green). (a) Schematic representation adapted to the H-bond pattern observed.
J
H,P
scalar
couplings (dotted arrows) indicate the experimentally determined intermolecular H-bonds shown by dashed lines (magenta). (b) Geometry-optimized 3D
representation showing the four experimentally detected plus one additional intermolecular H-bonds (dashed lines, yellow), with H · · ·O distances as calculated
by Spartan48 (for details, see Supporting Information).
Figure 12. Crystal structures of (a) unprotonated 4b showing conformation I and (b) 4d·HCl adopting conformation II+ in a complex H-bond network
between antiparallel strands of 4d·HCl (see text).
Figure 12). All attempts to crystallize further compounds failed,
especially those to obtain crystals of carboxylate salts.
In the crystal structure of the unprotonated 4b, the intramo-
lecular H-bond is found to be highly probable between the NHalk
group and the carbonyl oxygen, indicating conformation I (see
Figure 12a). Thus, in both crystal structures of monoalkylated
acylguanidines known so far, conformation I is observed, which
agrees nicely with the major conformation in pure CD2Cl2 found
in the NMR studies (see Figure 10b,c).
In contrast, the crystal structure of the protonated 4d·HCl
shows conformation II+, with a complex network of H-bonds
(see Figure 12b). The chloride is in H-bonding distance (<2.5
Å) to three NH protons and interconnects two parallel planes
in the crystal, which are formed by antiparallel strands of
4d·HCl. These strands themselves are interconnected via
H-bonds between two antiparallel acylguanidinium units. In-
terestingly, the observed conformation II+ is again closely
related to the NMR observations. First, the NMR spectra of
HCl salts of acylguanidines in pure CD2Cl2 contain considerable
amounts of conformation II+ (e.g., 30% II+ for 4d · HCl see
Figure 8). Second, the complex H-bond network observed in
the crystal structure might resemble the complex H-bonds
induced upon DMSO microsolvation in solution and further
supports the preference for conformation II+. Thus, the three
crystal structures of monoalkylated acylguanidines known so
far not only adopt exclusively the conformations found in our
NMR studies but in addition resemble the principal conforma-
tional preferences observed in solution.
Conclusion
Throughout the whole study of monoalkylated acylguanidines,
exclusively two (I/I+ and II/II+) out of eight possible acylguani-
dine conformations were detected in both the unprotonated state
and the protonated state, independent of the compound, the
anion, or the solvent used. The intermolecular interactions
between the monoalkylated acylguanidines and the anions,
9
J. AM. CHEM. SOC. VOL. 132, NO. 32, 2010 11231

A R T I C L E S
Kleinmaier et al.
romethane (DCM) (5 mL) at rt, and butylamine (0.69 mL, 7.10
mmol) was added in one batch via syringe. The mixture was stirred
for 3 h and then washed once with saturated aqueous NaHCO3
solution, water, and brine, respectively. The organic phase was dried
over Na2SO4, and the solvents were evaporated under reduced
pressure. The colorless oily residue was purified by column
chromatography over silica gel to yield pure N-Boc-N′-propionyl-
N′′-butylguanidine (0.30 g, 78%). 1H NMR (300 MHz, CDCl3, 300
K): δ 0.90 (t, 3H), 1.18 (t, 3H), 1.35 (m, 2H), 1.48 (s, 9H), 1.51
(m, 2H), 2.42 (q, 2H), 3.39 (q, 2H), 8.95 (as, 1H), 12.43 (s, 1H).
N-Propionyl-N′-butylguanidine (4a). TFA and DCA Salts. N-Boc-
N′-propionyl-N′′-butylguanidine was dissolved in dry DCM with
50% TFA or dichloroacetic acid (DCA), respectively, upon cooling
with an ice bath and then allowed to warm to rt. After 3 h the
solvents were evaporated in vacuo, and the product was purified.
The oily residue was subjected to column chromatography over
silica gel to yield the pure TFA or DCA salt.
solvent molecules, or biomimetic receptors decide both the
conformational preferences and exchange rates. Also, in the
presence of receptor molecules forming additional strong
H-bonds to protonated acylguanidines, conformation II+ was
found exclusively for two different acylguanidines in NMR
studies with bisphosphonate tweezers and also in a previous
docking study with a membrane protein. Even the crystal
structures of acylguanidines known so far resemble the con-
formational trend identified in this study.
In summary, this study gives for the first time detailed
insight into the conformational preferences of monoalkylated
acylguanidines.Consideringthepotentialoftheseacylguanidines
in medicinal chemistry and the fact that, in the past, such
known preferences often led to ligands with greatly increased
affinity through the rigidization of the ideal binding
conformation,49-51 the presented work may contribute to the
prediction and rational design of biologically active com-
pounds containing monoalkylated acylguanidines.
TFA Salt: 0.30 g (1.11 mmol) of starting compound gave 0.21 g
1
of product (80%). H NMR (300 MHz, CDCl3, 300 K): δ 0.96 (t,
3H, 3J ) 7.31 Hz), 1.16 (t, 3H, 3J ) 7.49 Hz), 1.42 (m, 2H), 1.65
3
(m, 2H), 2.55 (q, 2H, J ) 7.49 Hz), 3.30 (m, 2H), 7.34 (s, 1H),
Experimental Section
9.74 (s, 1H), 9.86 (as, 1H), 13.07 (s, 1H).
DCA Salt: 0.09 g (0.33 mmol) of starting compound gave 0.05 g
Routine NMR measurements were conducted on Bruker Avance
300, 400, and 600 MHz spectrometers equipped with 5 mm BBI
or TBI probeheads. The variable-temperature experiments were
conducted on a Bruker Avance 600 MHz spectrometer equipped
with a BVT 3000 variable-temperature unit. Deuterated NMR
solvents were purchased from Aldrich, Deutero, and Merck.
Starting materials were obtained from Sigma-Aldrich and Merck.
Solvents were distilled prior to use.
1
of product (50%). H NMR (300 Hz, CDCl3, 300 K): δ 0.96 (t,
3H, 3J ) 7.31 Hz), 1.16 (t, 3H, 3J ) 7.49 Hz), 1.43 (m, 2H), 1.67
(m, 2H), 2.55 (q, 2H), 3.38 (m, 2H), 5.89 (s, 1H, DCA), 7.30 (s,
1H), 9.82 (s, 1H), 10.06 (s, 1H), 13.40 (s, 1H).
Liberation of Free Base of 4a. N-Boc-N′-propionyl-N′′-bu-
tylguanidine (0.10 mg, 0.37 mmol) was deprotected with 50% TFA
in dry DCM. The crude product (oily residue) was subjected to
column chromatography over silica gel in the presence of 5%
triethylamine (TEA) to yield the pure free base after evaporation
of solvents (0.05 g, 78%). 1H NMR (300 MHz, CDCl3, 300 K): δ
For clarity, please refer to the Supporting Information for a
synthetic scheme.
(R)-Nr-(2,2-Diphenylacetyl)-N-(4-hydroxybenzyl)-Nω-pentanoyl-
argininamide (3, UR-MK50). Pentanoic anhydride (97.6 mg, 0.52
mmol, 1.1 equiv) was added to a solution of (R)-N-(4-tert-
butoxybenzyl)-Nω-tert-butoxycarbonyl-NR-(2,2-diphenylacetyl)argin-
inamide (300 mg, 0.48 mmol, 1 equiv) and NEt3 (48 mg, 66 µL,
0.48 mmol, 1 equiv) in CH2Cl2 (10 mL). The mixture was stirred
at room temperature (rt) for 5 h. Trifluoroacetic acid (TFA) (10
mL) was added, and stirring was continued at rt for 2 h. MeOH
(30 mL) was added, followed by evaporation under reduced
pressure. Purification with preparative HPLC (column: Eurospher-
100 C18, 250 × 32 mm, 5 µm; Knauer, Berlin, Germany) and
lyophilization afforded the product as a white fluffy solid (190 mg,
3
3
0.89 (t, 3H, J ) 7.31 Hz), 1.06 (t, 3H, J ) 7.49 Hz), 1.37 (m,
2H), 1.55 (m, 2H), 2.23 (q, 2H, J ) 7.49 Hz), 3.10 (m, 2H). 13C
NMR: δ 10.0, 13.6, 20.0, 30.8, 33.4, 40.8, 161.1, 186.9. HR-MS
(EI): m/z calcd 171.1372, found 171.1369.
3
HCl Salt. The free base was treated with a mixture of 10%
concentrated aqeuous HCl and 90% MeCN at rt and stirred for 5
min. The solvents were evaporated to yield the colorless solid
product quantitatively. 1H NMR (600 MHz, CD2Cl2/10% (CD3)2SO,
3
285 K, ref TMS internal): δ 0.95 (t, 3H, J ) 7.31 Hz), 1.16 (t,
3
3H, 3J ) 7.49 Hz), 1.42 (m, 2H), 1.62 (m, 2H), 2.53 (q, 2H, J )
7.49 Hz), 3.29 (m, 2H), 8.66 (s, 1H), 8.80 (s, 1H), 9.27 (as, 1H),
12.58 (s, 1H). For complete 13C data (assignment via HSQC,
HMBC), see Supporting Information.
1
0.28 mmol, 59%), mp > 118 °C (decomp.). H NMR (400 MHz,
3
CD3OD, COSY): δ (ppm) 0.93 (t, 3H, J ) 7.35 Hz, CH3), 1.37
AcOH Salt. Prepared in situ by titration of the free base with
AcOH, both dissolved in the required NMR solvent, until integration
proved a 1:1 ratio. For spectral data, see Supporting Information.
(m, 2H, CH2-CH3), 1.48-1.76 (bm, 5H, CH-CH2-CH2, CH2-CH2-
CH3), 1.83 (m, 1H, CH-CH2-CH2), 2.45 (t, 2H, 3J ) 7.44 Hz, CH2-
CO), 3.23 (m, 2H, CH2-CH2-NH), 4.2 (d, 1H, 2J ) 14.6 Hz, CH2-
N-Cinnamoyl-N′-butylguanidine (4b). 1H-Pyrazol-1-carboxa-
midine Hydrochloride. Aminoguanidinium hydrogen carbonate
(13.60 g, 100 mmol) was dissolved in water (25 mL) upon addition
of concentrated HCl (17 mL). To the resulting clear solution was
added 1,1,3,3-tetramethoxypropane (17.29 mL, 105 mmol) over 15
min using a dropping funnel. The mixture was warmed to 45 °C
and stirred for 3 h at that temperature. Evaporation of the solvent
to the beginning of crystallization and completion upon resting in
the refrigerator yielded large colorless crystals of 1H-pyrazol-1-
carboxamidine hydrochloride, which were suctioned off to obtain
the clean product. Yield: 10.3 g, 70.3 mmol, 70.3%. 1H NMR (300
2
ArOH), 4.26 (d, 1H, J ) 14.59 Hz, CH2-ArOH), 4.43 (m, 1H,
3
CHR), 5.07 (s, 1H, CH-(Ph)2), 6.7 (d, 2H, J ) 8.6 Hz, AA′BB′),
3
7.04 (d, 2H, J ) 8.62 Hz, AA′BB′), 7.16-7.31 (m, 10H, Ph).
RP-HPLC (210 nm): 99% (tR ) 16.6 min, k ) 5.1). HR-MS (FAB+,
MeOH/glycerin): m/z calcd for [C32H39N5O4 + H]+ 558.3080, found
558.3080; C32H39N5O4 × C2HF3O2, 671.7.
N-Propionyl-N′-butylguanidine (4a). The preparation of [15N2]-
N-Boc-N′-propionyl-S-methylisothiourea has been published else-
where.38 According to that protocol, the unlabeled compound
N-Boc-N′-propionyl-S-methylisothiourea was prepared and reacted
with an excess of butylamine to yield N-Boc-N′-propionyl-N′′-
butylguanidine, which was Boc-deprotected to obtain 4a.
N-Boc-N′-propionyl-N′′-butylguanidine. N-Boc-N′-propionyl-S-
methylisothiourea (0.35 g, 1.42 mmol) was dissolved in dichlo-
3
3
MHz, CD3OD, 300 K): δ 6.75 (dd, 1H, J ) 1.45 Hz, J ) 3.05
3
Hz, pyrazol-4-H), 7.98 (d, 1H, J ) 1.45 Hz, pyrazol-H), 8.48 (d,
3
1H, J ) 3.05 Hz, pyrazol-H).
N-Cinnamoyl-1H-pyrazol-1-carboxamidine. 1H-Pyrazol-1-car-
boxamidine hydrochloride (1.00 g, 6.84 mmol) was dissolved in
DCM (15 mL), and TEA (1.94 mL, 13.68 mmol) was added.
Cinnamoyl chloride (1.14 g, 6.84 mmol) dissolved in DCM (10
mL) was added upon cooling in an ice bath through a dropping
funnel. After 3 h of stirring, the mixture was washed once with
(49) Boeckler, F.; Gmeiner, P. Pharmacol. Therapeut. 2006, 112, 281–
333.
(50) Boeckler, F.; Gmeiner, P. Biochim. Biophys. Acta 2007, 1768, 871–
887.
(51) Kubinyi, H. Pharm. Acta HelV. 1995, 69, 259–269.
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11232 J. AM. CHEM. SOC. VOL. 132, NO. 32, 2010

Conformations of N′-Substituted N-Acylguanidines
A R T I C L E S
saturated aqueous NaHCO3 solution, water, and brine, respectively.
The organic phase was dried over Na2SO4 and the solvent
evaporated under reduced pressure. Column chromatography in
DCM over silica gel (Rf ) 0.5) was necessary to isolate the pure
product (0.90 g, 55%). 1H NMR (400 MHz, CDCl3, 300 K): δ 6.46
(dd, 1H, 3J ) 1.59 Hz, 3J ) 2.76 Hz), 6.75 (d, 1H, 3J ) 15.91 Hz),
7.32-7.43 (m, 3H, Ph), 7.55-7.63 (m, 2H, Ph), 7.72 (1H pyrazol
evaporated, and a solution of the oily product mixture in diethyl
ether was prepared, from which the pure product was precipitated
as the sulfate salt by addition of 1 M aqueous H2SO4. The free
base was liberated by addition of 1 M aqueous NaOH to the
crystalline salt covered by ethyl acetate. After phase separation,
1
the pure free base was obtained by evaporation of the solvent. H
3
NMR (600 MHz, CD2Cl2, 300 K): δ 0.95 (t, 3H, J ) 7.35 Hz),
3
3
+1H NH), 7.85 (d, 1H, J ) 15.91 Hz), 8.57 (d, 1H, J ) 2.76
1.42 (m, 2H), 1.61 (m, 2H), 2.99 (s, 3H, -NMe2), 3.20 (bs, 2H),
6.65 (m, 2H, ar), 8.01 (m, 2H, ar). HR-MS (EI): m/z calcd 262.1794,
found 262.1789.
Hz), 10.00 (bs, 1H, NH).
N-Cinnamoyl-N′-butylguanidine (4b). N-Cinnamoyl-1H-pyrazol-
1-carboxamidine (0.50 g, 2.08 mmol) was dissolved in DCM (5
mL), and TEA (0.29 mL, 2.08 mmol) was added. Butylamine (1.05
mL, 10.4 mmol) was added via syringe and the mixture stirred at
rt for 1 h. The solvents were evaporated, the oily residue was taken
up in ethyl acetate, and petroleum ether was added. Upon resting
in the refrigerator, the clean product precipitated (0.31 g, 61%).
Recrystallization following the same procedure yielded crystals of
4b suitable for X-ray crystallography. 1H NMR (600 MHz, CDCl3,
300 K): δ 0.96 (t, 3H, 3J ) 7.42 Hz), 1.44 (m, 2H), 1.64 (m, 2H),
N-p-Methoxybenzoyl-N′-butylguanidine (4d). N-p-Methoxyben-
zoyl-1H-pyrazol-1-carboxamidine. 1H-Pyrazol-1-carboxamidine hy-
drochloride (1.00 g, 6.84 mmol) was dissolved in CHCl3 (15 mL),
and TEA (1.94 mL, 13.68 mmol) was added. p-Methoxybenzoyl
chloride (1.14 g, 7.52 mmol) dissolved in CHCl3 (5 mL) was added
upon cooling in an ice bath through a dropping funnel. The mixture
was allowed to warm to rt, stirred for 2 h, and then washed once
with saturated aqueous NaHCO3 solution, water, and brine,
respectively. The organic phase was dried over Na2SO4 and the
solvent evaporated under reduced pressure. Column chromatography
in ethyl acetate/petroleum ether (2:5) over silica gel (Rf ) 0.5) was
necessary to isolate the pure product (1.50 g, 90%). 1H NMR (300
3
3.18 (m, 2H), 6.60 (d, 1H, J ) 15.91 Hz), 7.28-7.38 (m, 3H,
Ph), 7.62 (d, 1H, 3J ) 15.91 Hz). HR-MS (EI): m/z calcd 245.1528,
found 245.1534.
Preparation of Salts. HCl Salt. The free base was treated with
a mixture of 10% concentrated aqueous HCl and 90% MeCN at rt
and stirred for 5 min. The solvents were evaporated to yield the
colorless solid product quantitatively. For complete 1H and 13C data
(assignment via HSQC, HMBC), see Supporting Information.
TFA Salt. The free base was treated with 5% TFA in MeCN at
rt and stirred for 5 min. The solvents were evaporated to yield the
colorless solid product quantitatively.
Salts with DCA and AcOH. The free base was titrated in situ
with a carboxylic acid, both dissolved in the required NMR solvent,
until integration proved a 1:1 ratio. For spectral data, see Supporting
Information.
3
MHz, CDCl3, 300 K): δ 3.88 (s, 3H, -OMe), 6.49 (dd, 1H, J )
3
1.59 Hz, J ) 2.76 Hz), 6.95 (m, 2H), 7.75 (1H pyrazol + 1H
NH), 8.26 (m, 2H), 8.65 (as, 1H), 10.08 (bs, 1H, NH).
N-p-Methoxybenzoyl-N′-butylguanidine (4d). N-p-Methoxyben-
zoyl-1H-pyrazol-1-carboxamidine (0.75 g, 3.07 mmol) was dis-
solved in CHCl3 (5 mL), butylamine (1.56 mL, 15.4 mmol) was
added via syringe, and the mixture was stirred at rt for 1 h. The
solvents were evaporated, and crude column chromatography in
ethyl acetate/petroleum ether yielded the crude product with pyrazol
impurity. The pure HCl salt crystallized after addition of 1 M
aqueous HCl to a solution of the oily product mixture in diethyl
ether. This yielded crystals of the HCl salt suitable for X-ray
crystallography. The free base was liberated from the crystalline
salt by dissolution in CHCl3 and addition of 1 M aqueous NaOH.
Salts with Boc-L-Asp-OBn and 3,4,5-Trimethoxybenzoic Acid.
The free base and the respective acid were weighed to 1:1
stoichiometry and dissolved in the required NMR solvent. For
spectral data, see Supporting Information.
1
Characterization data are given for the HCl salt. H NMR (600
N-p-Dimethylaminobenzoyl-N′-butylguanidine (4c). N-p-Di-
methylaminobenzoyl-1H-pyrazol-1-carboxamidine. p-Dimethy-
laminobenzoyl chloride was prepared by refluxing p-dimethylami-
nobenzoic acid (2 g, 12.1 mmol) in 15 mL of CHCl3 with 0.5 mL
of dimethylformamide and thionyl chloride (1.73 g, 14.5 mmol)
for 1 h. This reaction solution was added in one batch to a solution
of 1H-pyrazol-1-carboxamidine hydrochloride (2.13 g, 14.5 mmol)
in CHCl3 (15 mL) and TEA (8.4 mL, 60.5 mmol) while cooling
with an ice bath. The mixture was allowed to warm to rt, stirred
for 2 h, and then washed once with saturated aqueous NaHCO3
solution, water, and brine, respectively. The organic phase was dried
over Na2SO4 and the solvent evaporated under reduced pressure.
The product crystallized from CHCl3 pure enough for the next step
(2.90 g, 93%). 1H NMR (300 MHz, CDCl3, 300 K): δ 3.06 (s, 3H,
-NMe2), 6.46 (dd, 1H, 3J ) 1.59 Hz, 3J ) 2.76 Hz), 6.69 (m, 2H),
7.5-7.75 (s, 1H pyrazol + bs, 1H NH), 8.20 (m, 2H), 8.63 (d, 1H,
2.76 Hz), 10.04 (bs, 1H, NH).
MHz, CDCl3, 300 K): δ 0.97 (t, 3H, 3J ) 7.42 Hz), 1.49 (m, 2H),
1.73 (m, 2H), 3.48 (m, 2H), 3.86 (s, 3H, -OMe), 6.99 (m, 2H, ar),
7.77 (s, 1H, NH), 8.29 (m, 2H, ar), 9.53 (s, 1H, NH), 10.03 (s, 1H,
NH), 11.89 (s, 1H, NH). HR-MS (EI): m/z calcd 249.1477, found
249.1476.
Computational Details. Equilibrium geometry calculations at
ground state were carried out by means of the Spartan ‘0648 program
package using the standard Hartree-Fock 3-21G basis set.
Acknowledgment. This work was supported by the Gra-
duiertenkolleg GRK 760 “Medicinal Chemistry: Molecular Rec-
ognition s Ligand-Receptor Interactions” of the DFG. We thank
Dr. S. Joseph for invaluable aid in the graphical representation of
the X-ray structures.
Supporting Information Available: Spectra, complete ref 4,
crystallographic data (.cif) files for 4b (CCDC 772555) and
4d·HCl (CCDC 772556), as well as synthetic and computational
details. This material is available free of charge via the Internet
at http://pubs.acs.org.
N-p-Dimethylaminobenzoyl-N′-butylguanidine (4c). N-p-Dim-
ethylaminobenzoyl-1H-pyrazol-1-carboxamidine (1.0 g, 3.89 mmol)
was dissolved in CHCl3 (5 mL), butylamine (1.90 mL, 19.5 mmol)
was added via syringe, and the mixture was stirred at rt for 1 h.
Since TLC analysis showed no conversion, the reaction mixture
was heated under refluxing conditions for 3 h. The solvents were
JA103756Y
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J. AM. CHEM. SOC. VOL. 132, NO. 32, 2010 11233