Structural and conformational studies on carboxamides of 5,6-diaminouracils-precursors of biologically active xanthine derivatives

Article abstract of DOI:10.3390/molecules24112168

8-Arylethynylxanthine derivatives are potent, selective adenosine A_2A receptor antagonists, which represent (potential) therapeutics for Parkinson's disease, Alzheimer's dementia, and the immunotherapy of cancer. 6-Amino-5-amidouracil derivatives are important precursors for the synthesis of such xanthines. We noticed an unexpected duplication of NMR signals in many of these uracil derivatives. Here, we present a detailed analytical study of structurally diverse 6-amino-5-carboxamidouracils employing dynamic and two-dimensional NMR spectroscopy, density functional theory calculations, and X-ray analysis to explain the unexpected properties of these valuable drug intermediates.

Full text of DOI:10.3390/molecules24112168

molecules
Article
Structural and Conformational Studies on
Carboxamides of 5,6-Diaminouracils—Precursors of
Biologically Active Xanthine Derivatives
Daniel Marx 1,2, Gregor Schnakenburg 3, Stefan Grimme 4 and Christa E. Müller 1,2,
*
1
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, D-53121 Bonn, Germany;
dmarx@uni-bonn.de
Pharma Center Bonn, University of Bonn, D-53121 Bonn, Germany
Department of Chemistry, Institute of Inorganic Chemistry, University of Bonn, D-53121 Bonn, Germany;
gregor.schnakenburg@uni-bonn.de
Mulliken Center for Theoretical Chemistry, Institute of Physical and Theoretical Chemistry,
University of Bonn, D-53115 Bonn, Germany; grimme@thch.uni-bonn.de
Correspondence: christa.mueller@uni-bonn.de; Tel.: +49-0228-732-301
2
3
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: F. Javier Luque
Received: 2 May 2019; Accepted: 4 June 2019; Published: 9 June 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: 8-Arylethynylxanthine derivatives are potent, selective adenosine A2A receptor antagonists,
which represent (potential) therapeutics for Parkinson’s disease, Alzheimer’s dementia, and the
immunotherapy of cancer. 6-Amino-5-amidouracil derivatives are important precursors for
the synthesis of such xanthines. We noticed an unexpected duplication of NMR signals in
many of these uracil derivatives. Here, we present a detailed analytical study of structurally
diverse 6-amino-5-carboxamidouracils employing dynamic and two-dimensional NMR spectroscopy,
density functional theory calculations, and X-ray analysis to explain the unexpected properties of
these valuable drug intermediates.
Keywords: amide; DFT calculation; dynamic NMR; rotamers/conformers; uracil; xanthine; X-ray
crystallography
1. Introduction
The nucleoside adenosine is an important (patho)physiological modulator in the brain as well
as in peripheral tissues and organs [1]. It activates G protein-coupled adenosine receptors (ARs)
which comprise A1–, A2A–, A2B–, and A3AR subtypes. The A2AAR subtype has become a major
(potential) drug target. A2AAR antagonists have been developed for neurodegenerative diseases such as
Parkinson’s [
of cancer has been discovered [
(1,3,7-trimethylxanthine, ) and theophylline (1,3-dimethylxanthine,
2
] and Alzheimer’s disease [
]. The first identified AR antagonists were the natural alkaloids caffeine
) (Figure 1) [ ]. After discovering
3], and, recently, their great potential for the immunotherapy
4
1
2
5
these alkylxanthines as moderately potent, non-selective AR antagonists, several groups tried to modify
the xanthine scaffold to obtain more potent and selective compounds. N7-methylation, as in caffeine,
combined with coplanar aromatic substitution at the C8-position of xanthines as in the styrylxanthines
3
and
4 (Figure 1) led to high affinity and selectivity for the A2AAR [6–8]. A disadvantage of the
styrylxanthine structure is its photosensitivity in solid form as well as in dilute solutions [9].
Molecules 2019, 24, 2168; doi:10.3390/molecules24112168
www.mdpi.com/journal/molecules

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Figure 1. Examples of adenosine receptor antagonists with a xanthine scaffold. Shown is the
development of the xanthine derivatives caffeine and theophylline towards potent and selective
adenosine A2A receptor antagonists.
Replacement of the styryl moiety present in
3 and 4, by an arylethynyl residue, as in 6, resulted
in a new class of photochemically stable A2AAR antagonists (Figure 1) [10]. Their 6-amino-5-
carboxamidouracil precursors (e.g., compound
but directly converted to the final xanthine derivatives [11]. Upon close examination of H- and
13C-NMR spectra of 6-amino-5-arylethynylcarboxamidouracil 8a (Scheme 1), obtained by coupling of
5
, Figure 1) were often not fully characterized,
1
5,6-diamino-3-ethyl uracil
7 with phenylethynyl carboxylic acid, we observed a duplication of almost
all signals (see Figure 2). However, this phenomenon was not observed for its styryl analog containing
a double bond instead of the triple bond in 8a. Thus, we performed a comprehensive analytical study
of 6-amino-5-arylethynylcarboxamidouracil (8a) in order to rationalize these unexpected observations.
Additionally, different N1,N3-substituted 6-amino-5-carboxamidouracil derivatives were synthesized
and analyzed.

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Scheme 1.
phenylethynylcarboxylic acid. (
coupling reaction of
Potential products obtained by coupling of 5,6-diamino-3-ethyluracil
) Possible regioisomers 8a and that could result from an amide
with phenylethynylcarboxylic acid. ( ) Possible formation of tautomers 8b 8c,
7 with
A
9
7
B
,
and 8d of 8a. (C) Possible cis- and trans-amide bond conformers 8a and 8e.
CH3 (trans)
Harom (trans)
Harom (cis)
CH3 (cis)
DMF
NHCO (trans)
N1-H (trans)
N1-H(cis)
NH2 (trans)
NH2 (cis)
CH2
NHCO (cis)
Figure 2. 1H-NMR spectra of compound 8a in DMSO-d6.
Different explanations for the duplication of peaks in the NMR spectra of
6-amino-5-arylethynylcarboxamides are conceivable. A mixture of regioisomers 8a and appeared to
be likely due to possible amide coupling with the 5- or the 6-amino group of 5,6-diamino-3-ethyluracil (
Scheme 1A). On the other hand, the presence of amide–iminol tautomers might be possible (Scheme 1B,
9
7
,

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structures 8a
,
8b
,
8c, and 8d). Moreover, amides can exist as different stereoisomers with s-cis (cisoid) or
16].
s-trans (transoid) conformation (see Scheme 1C), a phenomenon that is well known for peptides [12
–
In the present study, we performed dynamic and 2D-NMR studies and X-ray crystallography,
and additionally employed computational methods to elucidate the structures of 5/6-amino-5/6-
carboxamidouracil derivatives such as 8/9 (Scheme 1). Different analogs of compound 8a were
synthesized and analyzed in detail in order to explore if different N1,N3-substituents or different
linkers affect the structures leading to a duplication of NMR signals, as observed for compound 8.
2. Results and Discussion
2.1. Chemistry
Condensation of 5,6-diaminouracil derivatives
7, 10, and 11 with different carboxylic
acid derivatives in the presence of (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-
morpholinocarbenium hexafluorophosphate (COMU) yielded the desired 6-amino-5-carboxamido
derivatives
8 and 12–17 (Scheme 2). The amide coupling reaction was performed by the
recently described optimized procedure using COMU as a coupling reagent in the presence of
diisopropylethylamine (DIPEA) as a base in dimethylformamide (DMF) at room temperature [17].
The products were precipitated by the addition of water, filtered off, and dried; no further purification
was required. These precursors can be further converted to the corresponding xanthine derivatives by
a dehydrative cyclization reaction [9,18,19].
Scheme 2. Synthesis of 6-amino-5-carboxamidouracil derivatives. Reagents and conditions:
(i) R3-COOH, COMU, DIPEA, DMF, rt, 5 min, H2O (79%–90%). The synthesis of the starting material
was performed according to procedures described in the literature (Scheme S1 and S2, Supplementary
Information) [9].
2.2. Analytical Studies
In order to explain the duplication of signals in the NMR spectra of compound 8a (Figure 2)
different analytical experiments were performed. Yang et al. [20] described the formation of two
regioisomers during the condensation reaction of 5,6-diamino-1,3-dimethyluracil with 2-hydroxyacetic
acid. Furthermore, Poulsen et al. [21] proposed the formation of a 6-carboxamidouracil derivative
during the condensation reaction of 5,6-diamino-1,3-dimethyluracil with glutamic anhydride. However,
unambiguous evidence for the acylation of the 6-amino group was not provided by these authors.
High performance liquid chromatography (HPLC) of 8a pointed to only a single product, a first
indication that different regioisomers were likely not present, and the formation of 9 could be excluded
(Scheme 1A). Additionally, compound 12 was selected for dynamic HPLC (DHPLC) experiments to
explore the compound’s behavior at different temperatures. DHPLC measurements were performed at
5, 15, 25, and 40 ◦C, but in all cases one single peak was observed. Only a slight shift in the retention
times and peak broadening was visible upon decreasing of the temperature (Figure S16, Supporting
Information). To further elucidate whether regioisomers, tautomers, or conformational isomers were

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responsible for the signal duplication in the NMR spectra, dynamic NMR and 2D-NMR experiments
were performed.
2.2.1. Dynamic NMR Experiments of 8a at High Temperatures
The most deshielded signal, that of the N1-H, resonates as a broad singlet at 10.43 ppm measured
in DMSO-d6, and the second signal of the corresponding isomer is slightly shifted downfield to
10.48 ppm. The amide proton resonates at 9.17 ppm as a broad singlet, and the second signal shows a
large upfield shift to 8.50 ppm. The amino group at position six resonates as a broad singlet at 6.14 ppm
with an integration of two, while the corresponding second signal is shifted downfield to 6.39 ppm,
showing an integration of 0.67. Dynamic NMR experiments were performed to study whether different
tautomers of 8a were present. The temperature should have a measurable influence on the ratio
between tautomers. At a higher temperature, a higher percentage of the thermodynamically more
stable tautomer would be expected [22]. 1H- and 13C-NMR spectra were recorded in DMF-d7 and
DMSO-d6 from 223 to 378 K with 5–10 K intervals. The ratio of the isomers determined by integration
of the 1H-NMR signals was found to be 73:27. Unexpectedly, in both solvents, no change in the ratio
between the two species could be observed upon raising of the temperature (Figure 3). Since the
dynamic NMR experiments showed that the existence of two tautomers next to each other in solution
could be excluded (Scheme 1B), a third explanation was investigated in more detail. To confirm the third
hypothesis of different conformers, 2D-NMR experiments in DMSO-d6 and DMF-d7 were performed.
Figure 3. Dynamic NMR experiments of carboxamidouracil 8a at high temperatures. The 1H-NMR
spectra from 5.0 to 12.0 ppm of 8a were measured in DMSO-d6 at different temperatures, between 298
and 373 K, to analyze the signals of the 6-amino group, the amide NH, and the N1-H in detail.
2.2.2. Two-Dimensional NMR Studies
Nuclear Overhauser enhancement and exchange spectroscopy (NOESY and EXSY) experiments,
showed the presence of cross peaks with the same phase as the diagonal peaks (experimental
mixing time D8 of 300 ms, Figure 4) for almost all 1H signals, which points to a chemical exchange
mechanism [12,23]. This takes place if a nucleus is transferred from one magnetic environment to
another. As an example, the EXSY cross peaks of the cis-amide NH at 8.50 ppm and the trans-amide
NH at 9.20 ppm are shown in Figure 4 in the same sign of the diagonal peaks (shown in red). Chemical

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exchange with the solvent could be excluded by the fact that we observed a cross-correlation also
for non-exchangeable protons. Cross-correlation in phase of the signal of one conformer with the
corresponding signal from the other conformer was observed as a proof for conformer equilibrium
(Figure 4).
trans amide-NH (8a)
cis amide-NH (8e)
cis amide-NH (8e)
trans amide-NH (8a)
Figure 4. Exchange spectroscopy (EXSY)-NMR spectrum of 8a in DMSO-d6 indicating cross-correlation
in phase between the two amide-NH protons of conformer 8a and 8e. EXSY cross peaks of the cis-amide
NH at 8.50 ppm and the trans-amide NH at 9.20 ppm are shown.
These results clearly showed that the duplication of peaks in the NMR spectra of 8a (and related
amidouracil derivatives) was due to the presence of different conformational isomers, such as 8a and
8e (Scheme 1C).
2.2.3. Analysis of Differently Substituted 5-Amino-6-Carboxamidouracil Derivatives
We subsequently synthesized and investigated different 6-amino-5-carboxamidouracil derivatives
with various carboxylic acid residues (Table 1) by dynamic and multidimensional NMR experiments.
The equilibrium of their conformers and the energy of their rotational barrier were analyzed. In order
to explore if the N1-substitution or the N3-substitution has any influence on the conformer equilibrium,
compounds 12 (3-propargyl) and 13 (1,3-dimethyl) were synthesized (Table 1). Neither the change
of the substitution pattern at the N1- or N3-position nor diethoxy substitution at the phenylethynyl
residue changed the cis–trans ratio significantly (Table 1; compare 8a, 12, and 13).

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Table 1. Synthesized 6-amino-5-carboxamidouracil derivatives, yields, and experimental trans/cis ratios
from 1H-NMR in DMSO-d6.
trans/cis % Ratio b
Compound
Structure
Yield a
8a
12
85%
73:27
79%
88%
72:28
77:23
13
O
H
N
H3C
N
14
80%
90%
95:5
89:11
100:0
O
O
N
H
NH2
O
H
N
H3C
N
15
O
NH2
O
N
H
16
17
87%
80%
O
H
N
H3C
N
91:9
O
NH2
O
N
H
a
Isolated yields of the amide coupling reaction of 5,6-diaminouracil derivatives (
7,
10, and 11) with different
b
carboxylic acid derivatives. The ratio of the cis- and trans-amide conformers of 8a and 12–17 were determined by
integration of their 1H-NMR signals.
Finally, derivatives containing different linkers were prepared and analyzed. In the case of
8-arylethynyl derivatives the amount of the less stable conformer was between 20% and 30% in all three
investigated cases (8a
, 12, and 13). Mainly a single conformer was observed for the styryl-substituted
derivative 14, while with a single-bond linker in phenylethyl derivative 15 the ratio of trans- and

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cis-amide was approximately 89:11. A shorter linker shifted the equilibrium to a single conformer (16
,
17). However, this might also be due to a fast equilibrium between these two isomers, which cannot be
distinguished by NMR spectroscopy. Due to the crystal structure and density functional theory (DFT)
calculation of compound 8a and the NOESY spectra of compound 17, the trans-amide conformer is
concluded to be the more stable rotational conformer.
2.2.4. NMR Studies at Low Temperature
When further dynamic NMR experiments were performed for amide
8 at low temperature
(213–283 K), a different conformer equilibrium was observed. NOESY spectra at 223 K showed the
separation of the amino groups of both amide conformers 8a and 8e (Figure 5). A cross-correlation for
the amino group of conformer A (compound 8a) was found between two peaks at 6.76 and 7.89 ppm.
The same cross peak was found for the amino group of conformer B (compound 8e) at 6.58 and
7.58 ppm. The peak corresponding to the amino group of conformer B could only be observed with
NOESY NMR because it was covered by the aromatic signals. The latter was expected to slow rotation
of the Csp2–N bond [24]. The coalescence temperature of this amino group could be observed between
228 and 233 K (Figure 6). Using the simplified Eyring equation (Equation (4)), compound 8a showed a
rotational barrier for the amino group, ∆G# = 46.4 ± 0.42 kJ·mol−1
.
Figure 5. EXSY cross-correlation in DMSO-d6 suggesting a low rotational barrier of the Csp2–N bond
in amide 8a and 8e. Cross-correlations for the amino groups of conformer A and conformer B were
found at 6.76 and 7.89 ppm, and at 6.58 and 7.58 ppm, respectively.

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Figure 6. Proton dynamic NMR experiments of the carboxamidouracil isomers 8a and 8e in DMF-d7
at low temperature. Different NMR spectra for amide were recorded from 213–283 K, with 5–10 K
intervals. Coalescence of the 6-amino group was detected between 228 K and 233 K.
8
However, the Eyring equation to calculate
of the two conformers is equal. To determine
∆
∆
G# can only be used if the thermodynamic stability
G#, quantum chemical calculations are required.
A line-shape analysis was not possible due to the fact that the coalescence temperature was above
105 ◦C (see Figure 3). In order to predict the 3D structures of these conformers, density functional
theory (DFT) calculations in liquid phase and X-ray crystallography for the solid state of 8a were
subsequently performed (Figures 7 and 8).
2.2.5. DFT Calculations
The DFT computed free energy difference between the two conformers is
∆
G# =
1 kcal, which
−
corresponds to a ktrans/kcis ratio of 84:16 (Figure 7) at room temperature. This is consonant with
the NMR results, which showed a ratio of 73:27 both indicating a higher stability of the s-trans
conformer. The rotational barrier in DMSO solution is calculated to be
in accordance with the results from the NMR experiments.
∆
G# = 20.0 kcal
mol−1, which is
·
Figure 7. Geometry-optimized amide bond rotational conformers 8a and 8e. The conformers are shown
in stick models (carbon atoms colored grey, oxygen atoms in red, the nitrogen atoms in blue, and the
hydrogen atoms connected to nitrogen in white).

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2.2.6. X-ray Crystal Structure
Several solvents were tried to obtain single crystals, but finally we only obtained suitable crystals
(triclinic space group P1) of compound 8a from DMSO solution. The resulting crystal structure showed
exclusively the more stable regioisomer 8a (Figure 8). No intramolecular hydrogen bonding and no
π
-stacking between the molecules was observed. The crystals were found to be mainly formed by
intermolecular hydrogen bonding as shown in Figure 8.
Figure 8. Results of the single X-ray crystallography of compound 8. X-ray crystal structure (A)
and chemical structure (
crystal (colored in cyan). (
interactions in the crystal structure. For color coding see Figure 7.
B
) of compound 8a. (
C
) Intermolecular hydrogen bond interactions in the
D) Crystal unit cell of the P1 space group. (
E) Distances of intermolecular
3. Conclusions
6-Amino-5-carboxamidouracil derivatives, which are important intermediates in the synthesis of
pharmaceutically important 8-substituted xanthine derivatives, were observed to show a more or less
pronounced duplication of NMR signals, depending on their carboxylic acid residue. In order
to understand this phenomenon, selected 6-amino-5-carboxamidouracils were analyzed using
dynamic and 2D NMR-experiments, DFT calculations, and single-molecule X-ray crystallography.
The duplication of NMR signals could be correlated with a partial double bond character of the amide
bond and a low rotational barrier of this bond depending on the carboxylic acid residue. According to
DFT calculations, in the case of 5-ethynylcarboxamidouracils, the triple bond appears to stabilize the
thermodynamically less stable cis conformer. This could be observed in solution, while the obtained
crystal structure consisted solely of the more stable trans conformer.

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4. Materials and Methods
Chemicals were purchased from Merck (Darmstadt, Germany), ABCR (Karlsruhe, Germany),
or TCI (Eschborn, Germany). Thin layer chromatography (TLC) was performed on TLC plates F254
(Merck) and analyzed using UV light. High-resolution mass spectra (HR-MS) were recorded on a
micrOTOF-Q mass spectrometer (Bruker, Billerica, MA, USA), further mass spectra were performed on
1
an API 2000 (Applied Biosystems, Foster City, CA, USA) mass spectrometer. H- and 13C-NMR spectra
were recorded in CDCl3, DMSO-d6, or DMF-d7 on a Bruker Ascend 600 MHz NMR-spectrometer
(Bruker) operating at 600.18 MHz (1H) or 150.93 MHz (13C), respectively. Chemical shifts (
δ) are
reported in ppm and are referenced to the chemical shifts of the residual solvent proton(s) present
1
in CHCl3 (7.26 ppm for the H-NMR spectra and 77.16 ppm for the 13C-NMR spectra), in DMSO
(2.50 ppm for the 1H-NMR spectra and 39.52 ppm for the 13C-NMR spectra), and in DMF (2.75 ppm
for the 1H-NMR spectra and 29.76 ppm for the 13C-NMR spectra). Multiplicity: s, singlet; d, doublet;
q, quartet; m, multiplet. Coupling constants (J) are shown in Hertz (Hz). Dynamic HPLC analyses
were performed on “Knauer GmbH” (Berlin, Germany) systems of the “PLATINblue” series with a P-1
pump, a column oven, a PDA-1 diode array UV-detector, and a Knauer Eurospher II 100-2 C18, (2 mm,
100 × 2.0 mm; number 19040303147) column. The solvents were of HPLC grade.
4.1. Eyring Equation
If rotation around an amide bond is slow on the NMR timescale, and the rotational barrier is
between 3 and 19 kcal/mol, the observation of two conformers is permitted due to the partial double
bond character of the amide C–N bond [25]. Under this assumption, one sharp peak for the duplicated
should be observed at the coalescence temperature in the 1H-NMR spectrum [12
signals of 8 ,26–28].
Thus, the rotational barrier could be determined using the Eyring equations. Before the coalescence
temperature is reached, the interconversion rate constant corresponds to the following equation:
1
t
∆ν
√
k = ꢀ π
(1)
2
where k is interconversion rate constant (s−1), t is interconversion time (s), and ∆ν is the NMR shift
(Hz) separation of the signals at low temperatures when exchange does not occur.
Heating leads to a faster exchange rate relative to the NMR timescale and only one averaged
signal becomes detectable. At the coalescence temperature, the equation of the interconversion rate
constant is
∆ν
k = π
Tc
(2)
√
2
where kTc is the rate constant (s−1).
The free Gibbs activation energy
Eyring equations:
∆
G# of rotation can be calculated using the following
#
−∆G
kB×T
− ∆G# = K
e
(3)
(4)
RT
h
Tc
∆ν
∆G#= 4.57 × 10−3 T (9.972+ log
)
where R is the universal gas constant (1.0872 cal ), kB is the Boltzmann constant (3.2998
×
10−24 cal ), h is
K·mol
K
the Planck constant (1.584 × 10−34 cal·s), and TC is the coalescence temperature (K) [12].
4.2. DFT Calculation
Most of the quantum chemical calculations were carried out with the TURBOMOLE and ORCA
programs [29 30]. All structures were fully optimized at the dispersion-corrected DFT level using the
,
PBEh-3c DFT [31]. This composite method contains a modified Perdew–Burke–Ernzerhof (PBE)-based
hybrid function together with an efficient valence double-zeta AO basis set. The method also involves

Molecules 2019, 24, 2168
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an approximate counterpoise correction for basis set superposition errors (BSSE), as well as three-body
dispersion effects [31]. Conformational searches [32], pre-optimizations, as well as the calculation
of the Hessian to start transition state searches were conducted at the semi-empirical tight-binding
level (GFN-xTB method) [33]. The GFN-xTB-optimized structures were used as input for subsequent
full PBEh-3c optimizations. Reaction paths were obtained with the growing-string method (GSM)
of Zimmerman [34] which was interfaced to our in-house XTB code [35]. Single-point gas phase
energies were computed with the large polarized triple-zeta (def2-TZVPP) sets by Weigend et al. [36
]
in combination with the very accurate DSD-BLYP double hybrid functional [37]. The atom pairwise
D3 correction with Becke–Johnson (BJ) damping to account for intra- and intermolecular London
dispersion interactions was included in all treatments [38]. Note, that the original D2 treatment
in DSD-BLYP is replaced by the D3 version (with damping parameters s6 = 0.57, a1 = 0, s8 = 0,
a2 = 5.4) [39]. This functional performs excellently on the huge GMTKN55 thermochemical database
(i.e., is practically the best out of 200+ tested DFT approximations) [40]. The combined level of theory
used for electronic gas phase energies is denoted DSD-BLYP/TZ//PBEh-3c in standard notation. In all
DFT treatments, the resolution-of-the-identity approximation [41] has been used for the two-electron
integrals to speed up the computations. The numerical quadrature grid m4 (grid 5 in ORCA) was
employed for the integration of the exchange-correlation contribution. Gibbs free energies at 298.15 K
in DMSO were reported as a solvent (termed ∆G). The ro-vibrational corrections to the free energy
are obtained from a modified rigid rotor, harmonic oscillator statistical treatment [42] based on scaled
harmonic frequencies obtained at the (gas phase) HF-3c [43] level. For the entropy, all frequencies
with wavenumbers below 100 cm−1 were treated as mixed rigid rotors and harmonic oscillators.
Solvent effects on the thermochemical properties have been obtained by the COSMO-RS method [44
]
]
(COSMOtherm software package [44], parametrization from 2016) based on BP86/TZVP [36 45
,
single-point calculations. The PBEh-3c as well as GFN-xTB optimizations were run consistently in a
continuum solvent. For GFN-xTB the built-in GBSA solvation model 23 is employed; while for PBEh-3c,
the DCOSMO-RS method is used [46]. The solvation contributions to free energies at 298.15 K in DMSO
solution are computed at those structures (i.e., PBEh-3c[DCOSMO-RS]) The computed free energies
are obtained by ∆G = ∆E + ∆GRRHO + ∆δGCOSMO-RS, where the last two terms refer to the above
mentioned ro-vibrational/translational and solvation contributions, respectively, to the free energy.
The final theory level is denoted as DSD-BLYP/TZ[COSMO-RS(BP86/TZVP)]//PBEh-3c[DCOSMO-RS]
where the abbreviations in square brackets denote the level of the solvation treatment.
4.3. General Procedures
4.3.1. Synthesis of 5,6-Diaminouracil Derivatives
The different N-substituted 5,6-diaminouracils where synthesized in analogy to procedures
described in the literature [9].
4.3.2. General Procedure for Amide Formation
To a solution of the respective carboxylic acid (1.0 equiv.) and COMU (1.1 equiv.), dissolved in a
minimum amount of DMF, a mixture of diaminouracil derivative (1.1 equiv.) and DIPEA (2.0 equiv.),
dissolved in a minimum amount of DMF, was added dropwise. The reaction was stirred for 5–10 min
at room temperature. Then, water was added, and the resulting precipitate was filtered off, washed
with water, and dried under reduced pressure.
4.3.3. NMR Spectra
N-(6-Amino-3-ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-3-phenylpropiolamide (
8
). Yield: 85%
1
(white solid); mp = 270–272 ◦C. Major isomer: H-NMR (600 MHz, DMSO-d6)
δ
10.45 (s, 1H, N1-H),
9.16 (s, 1H, CONH), 7.62–7.56 (m, 2H, Harom), 7.53–7.45 (m, 3H, Harom), 6.14 (s, 2H, NH2), 3.79–3.67
(m, 2H, N1-CH2), 1.04 (t, J = 7.0 Hz, 2H, CH2CH3). 13C-NMR (126 MHz, DMSO-d6)
160.0 (CON),
δ

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152.7 (C6), 150.2 (CO), 149.5 (CO), 132.0 (2C, Carom), 130.2 (Carom), 129.0 (2C, Carom), 120.0 (Carom), 85.7
(C5 or Calkyne), 84.9 (C5 or Calkyne), 83.6 (C5 or Calkyne), 34.4 (N1-CH2), 13.2 (CH2 H3). Minor isomer:
1H-NMR (600 MHz, DMSO-d6)
10.49 (s, 1H, N1-H), 8.51 (s, 1H, CONH), 7.54–7.51 (m, 1H, Harom),
7.43–7.39 (m, 2H,Harom), 7.35–7.32 (m, 2H, Harom), 6.40 (s, 2H, NH2), 3.74–3.78 (m, 2H, N1-CH2), 1.01 (t,
J = 7.0 Hz, 3H, CH2CH3). 13C-NMR (151 MHz, DMSO-d6)
162.3 (CON), 157.6 (C6), 151.5 (CO), 149.5
(CO), 132.0 (2C, Carom), 130.3 (Carom), 129.0 (2C, Carom), 120.0 (Carom), 87.5 (C5 or Calkyne), 85.8 (C5 or
C
δ
δ
Calkyne), 83.2 (C5 or Calkyne), 34.4 (N1-CH2), 13.3 (CH2CH3). High resolution mass spectra (HRMS)
(electrospray ionization-quadrupole-time-of-flight) (ESI-QTOF)) calculated for C15H14N4O3 [M + H]+:
299.1139; found: 299.1139.
N-(6-Amino-2,4-dioxo-3-(prop-2-yn-1-yl)-1,2,3,4-tetrahydropyrimidin-5-yl)-3-(3,4-diethoxyphenyl)
1
propiolamide (12). Yield: 79% (white solid); mp = 185–183 ◦C. Major isomer: H-NMR (600 MHz,
DMSO-d6)
J = 2.1 Hz, 1H, Harom), 7.02 (d, J = 8.3 Hz, 1H, Harom), 6.25 (s, 2H, NH2), 4.41 (d, J = 2.4 Hz, 2H,
Hpropargyl), 4.09–4.01 (m, 4H, 2 OCH2), 3.02 (t, J = 2.4 Hz, 1H, Hpropargyl), 1.34–1.31 (m, 6H, 2
OCH2CH3). 13C-NMR (151 MHz, DMSO-d6)
159.3, 153.0, 150.7, 150.2, 149.1, 147.9, 125.8 (Carom),
116.2 (Carom), 113.1 (Carom), 111.5 (Carom), 85.5 (C5 or Calkyne), 84.7 (C5 or Calkyne), 83.7 (C5 or Calkyne),
79.9 (Cpropargyl), 72.5 (Cpropargyl), 63.8 (2C, OCH2), 28.9 (N1-CH2), 14.6 (2C, OCH2 H3). Minor isomer:
1H-NMR (600 MHz, DMSO-d6)
10.72 (s, 1H, N1-H), 8.45 (s, 1H, CONH), 6.97–6.90 (m, 2H, Harom),
6.82 (d, J = 1.8 Hz, 1H, Harom), 6.50 (s, 2H, NH2), 4.51–4.44 (m, 2H, Hpropargyl), 4.05–4.02 (m, 2H, OCH2)
3.98 (q, J = 7.4 Hz, 2H, OCH2), 3.02–3.01 (m, 1H, Hpropargyl), 1.32–1.28 (m, 6H, OCH2C
3). 13C-NMR
(151 MHz, DMSO-d6) 160.3, 157.8, 157.8, 151.9, 150.2, 149.1, 147.8, 126.0 (Carom), 116.2 (Carom), 113.0
(Carom), 111.4 (Carom), 87.4 (C5 or Calkyne), 87.1 (C5 or Calkyne), 81.8 (C5 or Calkyne), 79.9 (Cpropargyl),
δ 10.64 (s, 1H, N1-H), 9.09 (s, 1H, CONH), 7.14 (dd, J = 8.4, 1.9 Hz, 1H, Harom), 7.09 (d,
×
×
δ
C
δ
H
δ
72.4 (Cpropargyl), 63.9 (2C, OCH2), 28.9 (N1-CH2), 14.5 (2C, OCH2CH3). HRMS (ESI-QTOF) calculated
for C20H20N4O5 [M + H]+: 397.1504; found: 397.1506.
N-(6-Amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-3-(3,4-diethoxyphenyl)
1
propiolamide (13). Yield: 88% (white solid); mp = 220–223 ◦C. Major isomer: H-NMR (600 MHz,
DMSO-d6)
7.03 (d, J = 8.8 Hz, 2H, Harom), 6.76 (s, 2H, NH2), 4.07 (dq, J = 16.9, 7.0 Hz, 4H, 2
CH3), 3.12 (s, 3H, CH3), 1.34 (td, J = 7.0, 2.7 Hz, 6H, 2 OCH2C δ
3). 13C-NMR (151 MHz, DMSO-d6)
δ
9.08 (s, 1H, CONH), 7.16 (dd, J = 8.3, 2.0 Hz, 1H, Harom), 7.11 (d, J = 1.9 Hz, 1H, Harom),
×
OCH2), 3.31 (s, 3H,
×
H
158.9, 153.3, 152.1, 150.5, 150.2, 147.9, 125.8 (Carom), 116.2 (Carom), 113.1 (Carom), 111.6 (Carom), 86.1 (C5
or Calkyne), 84.5 (C5 or Calkyne), 83.9 (C5 or Calkyne), 63.9 (2C, OCH2), 30.0 (CH3), 27.5 (CH3), 14.6 (2C,
1
OCH2CH3). Minor isomer: H-NMR (600 MHz, DMSO-d6)
δ
8.46 (s, 1H, CONH), 7.04 (s, 1H, Harom),
6.96 (d, J = 8.4 Hz, 1H, Harom), 6.85 (dd, J = 8.3, 1.9 Hz, 1H, Harom), 6.75 (s, 2H, NH2), 4.07–4.05 (m,
2H, OCH2), 3.95 (q, J = 6.9 Hz, 2H, OCH2), 3.34 (s, 3H, CH3), 3.15 (s, 3H, CH3), 1.32–1.28 (m, 6H, 2
OCH2CH3). 13C-NMR (151 MHz, DMSO-d6)
160.0, 158.0, 153.2, 150.5, 150.2, 147.9, 125.8 (Carom),
116.1 (Carom), 113.1 (Carom), 111.5 (Carom), 88.1 (C5 or Calkyne), 86.9 (C5 or Calkyne), 82.1 (C5 or Calkyne),
×
δ
63.8 (2C, OCH2), 30.1 (CH3), 27.6 (CH3), 14.5 (2C, OCH2CH3). HRMS (ESI-QTOF) calculated for
C19H22N4O5 [M + H]+: 387.1663; found: 387.1668.
N-(6-Amino-3-ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)cinnamamide (14). Yield: 80%
(off-white solid); mp > 320 ◦C; 1H-NMR (500 MHz, DMSO-d6)
10.43 (s, 1H, N1-H), 8.68 (s, 1H, CONH),
δ
7.58 (d, J = 7.4 Hz, 2H, Harom), 7.50–7.37 (m, 4H, Harom + Hvinyl), 6.83 (d, J = 15.9 Hz, 1H, Hvinyl), 5.99
(s, 2H, NH2), 3.74 (q, J = 6.5 Hz, 2H, CH2), 1.06 (t, J = 6.7 Hz, 3H, CH3). 13C-NMR (DMSO-d6, 126 MHz)
δ
164.9 (CON), 160.3 (C6), 149.7 (CO), 149.5 (CO), 138.8 (Cvinyl or Carom), 135.0 (Cvinyl or Carom), 129.4
(Cvinyl or Carom), 129.0 (2C, Carom), 127.4 (2C, Carom), 122.4 (Cvinyl or Carom), 87.4 (C5), 34.4 (N3-CH2),
13.2 (CH3). HRMS (ESI-QTOF) calculated for C15H16N4O3 [M + H]+: 301.1295; found: 301.1294.
N-(6-Amino-3-ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-3-phenylpropanamide (15). Yield:
90% (white solid); mp > 320 ◦C; 1H-NMR (500 MHz, DMSO-d6)
δ
10.38 (s, 1H, N1-H), 8.39 (s, 1H,
CONH), 7.28 (t, J = 7.4 Hz, 2H, Harom), 7.24 (d, J = 6.9 Hz, 2H, Harom), 7.18 (t, J = 7.1 Hz, 1H, Harom),

Molecules 2019, 24, 2168
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5.82 (s, 2H, NH2), 3.73 (q, J = 6.9 Hz, 2H, N3-CH2), 2.91–2.80 (m, 2H, CH2), 2.53 (dd, J = 9.2, 7.0 Hz,
2H, CH2), 1.04 (t, J = 7.0 Hz, 3H, CH3). 13C-NMR (DMSO-d6, 126 MHz)
171.7 (CON), 160.4 (C6),
δ
149.9 (CO), 149.6 (CO), 141.5 (Carom), 128.3 (2C, Carom), 128.1 (2C, Carom), 125.8 (Carom), 87.2 (C5), 36.8
(CH2), 34.3 (N3-CH2), 30.9 (CH2), 13.2 (CH3). HRMS (ESI-QTOF) calculated for C15H18N4O3 [M + H]+:
303.1452; found: 303.1454.
N-(6-Amino-3-ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)benzamide (16). Yield: 87% (yellowish
solid; mp > 320 ◦C; 1H-NMR (500 MHz, DMSO-d6)
δ
10.38 (s, 1H, N1-H), 8.86 (s, 1H, CONH), 7.99–7.91
(m, 2H, Harom), 7.56–7.51 (m, 1H, Harom), 7.47 (t, J = 7.5 Hz, 2H, Harom), 6.06 (s, 2H, NH2), 3.75 (q,
J = 7.0 Hz, 2H, N3-CH2), 1.06 (t, J = 7.0 Hz, 3H, CH3). 13C-NMR (126 MHz, DMSO-d6)
166.4 (CON),
δ
160.5 (C6), 150.4 (CO), 149.7 (CO), 134.5 (Carom), 131.1 (Carom), 128.0 (Carom), 127.8 (Carom), 87.1 (C5),
34.4 (N3-CH2), 13.3 (CH3). HRMS (ESI-QTOF) calculated for C13H14N4O3 [M + H]+: 275.1139; found:
275.1142.
N-(6-Amino-3-ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-2-phenylacetamide (17). Yield: 80%
(white solid); mp > 320 ◦C; 1H-NMR (500 MHz, DMSO-d6)
δ 10.39 (s, 1H, N1-H), 8.58 (s, 1H, CONH),
7.35–7.31 (m, 2H, Harom), 7.28 (m, 2H, Harom), 7.23–7.19 (m, 1H, Harom), 5.90 (s, 2H, NH2 ), 3.71
(q, J = 7.0 Hz, 2H, N3-CH2), 3.56 (s, 2H, CH2), 1.03 (t, J = 7.0 Hz, 3H, CH3). 13C-NMR (DMSO-d6,
126 MHz)
δ 170.6 (CON), 160.5 (C6), 150.1 (CO), 149.7 (CO), 136.6 (Carom), 129.4 (Carom), 128.2 (Carom),
126.3 (Carom), 87.5 (C5), 42.1 (COCH2), 34.5 (N3-CH2), 13.4 (CH3). HRMS (ESI-QTOF) calculated for
C14H16N4O3 [M + H]+: 289.1295; found: 289.1304.
Supplementary Materials: The following data are available online. Figure S1–S15: NMR spectra, Figure S16:
DHPLC analyses of 12 at 205 nm, Figure S17: UV-spectrum of compound 12. Table S1–S3: Crystal data and
structure refinement for compound 8. Scheme S1–S2: Synthesis of diaminouracil derivatives.
Author Contributions: D.M. synthesized the compounds and analyzed the NMR spectra. S.G. performed the
DFT calculations. G.S. measured the X-ray crystal structure. C.E.M. supervised the experiments. D.M. and C.E.M.
wrote the manuscript, all coauthors contributed to writing.
Funding: D.M. and C.E.M. were supported by the BMBF (German Federal Ministry for Education and Research)
within the “Neuroallianz” consortium of the BioPharma initiative.
Acknowledgments: We thank Andreas Schneider for the DHPLC measurements, Jörg Hockemeyer for helpful
discussions, and Senada Nozinovic, Marion Schneider, Sabine Terhart-Krabbe, and Annette Reiner for recording
NMR and MS data.
Conflicts of Interest: No conflicts of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).