Influence of intramolecular charge transfer and nuclear quantum effects on intramolecular hydrogen bonds in azopyrimidines

Article abstract of DOI:10.1021/acs.joc.7b01810

Intramolecular hydrogen bonds (IMHBs) in 5-azopyrimidines are investigated by NMR spectroscopy and DFT computations that involve nuclear quantum effects. A series of substituted 5-phenylazopyrimidines with one or two hydrogen bond donors able to form IMHB

Full text of DOI:10.1021/acs.joc.7b01810

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Article
Influence of intramolecular charge transfer and nuclear quantum
effects on intramolecular hydrogen bonds in azopyrimidines
Katerina Bartova, Lucie #echová, Eliska Prochazkova, Ondrej Socha, Zlatko Janeba, and Martin Dracinsky
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b01810 • Publication Date (Web): 22 Aug 2017
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Influence of intramolecular charge transfer and nuclear quantum effects on intramolecular
hydrogen bonds in azopyrimidines
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Kateřina Bártová,†,‡ Lucie Čechová,†,§ Eliška Procházková,† Ondřej Socha,† Zlatko Janeba,†
Martin Dračínský*,†
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†Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Flemingovo nam. 2, 166 10, Prague, Czech Republic
‡Faculty of Science, Charles University, Prague, Czech Republic
§Department of Chemistry of Natural Compounds, University of Chemistry and Technology
Prague, Technická 5, 166 28 Prague, Czech Republic
Eꢀmail: dracinsky@uochb.cas.cz
Abstract
Intramolecular hydrogen bonds (IMHBs) in 5ꢀazopyrimidines are investigated by NMR
spectroscopy and DFT computations that involve nuclear quantum effects. A series of substituted
5ꢀphenylazopyrimidines with one or two hydrogen bond donors able to form IMHBs with the azo
group is prepared by azo coupling. The barrier of interconversion between two rotamers of the
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compounds with two possible IMHBs is determined by variable temperature NMR spectroscopy
and it is demonstrated that the barrier is significantly affected by intramolecular charge transfer.
Throughꢀhydrogenꢀbond scalar coupling is investigated in 15N labelled compounds and the
stability of the IMHBs is correlated with experimental NMR parameters and rationalized by path
integral molecular dynamics simulations that involve nuclear quantum effects. Detailed
information on the hydrogen bond geometry upon hydrogenꢀtoꢀdeuterium isotope exchange is
obtained from a comparison of experimental and calculated NMR data.
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Introduction
Hydrogen bonding is an enormously important and fascinating type of weak bonding that
is crucial, for example, for maintaining the shape and function of proteins and nucleic acids.1ꢀ2
However, despite its ubiquitous involvement in biomolecular processes, the detailed
understanding of hydrogen bonding in terms of energies, geometric preferences and dynamics is
still rather limited.3 The biggest obstacle in hydrogen bond investigation stems from the fact that
common experimental techniques for structural analysis cannot provide hydrogen atom positions
with high precision. A prominent example is Xꢀray crystallography, which is the most widely
used technique for determination of structures with atomic resolution, but hydrogen atoms are
very difficult to characterize by this technique. Furthermore, hydrogen atoms may also be
problematic for common computational methods, because nuclear quantum effects (NQEs),
which may be important for light hydrogen nuclei, are not commonly involved in the
calculations.
Intramolecular hydrogen bonds (IMHBs) affect molecular properties significantly; for
example, molecules become more lipophilic and may exhibit enhanced membrane permeability
upon the formation of IMHBs.4 Molecules containing pseudoring formed by an IMHB have been
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proposed as bioisosteres of several biologically important molecules and it has been proven that
sixꢀmembered hydrogen bonded pseudorings effectively mimic an aromatic ring.5ꢀ11
Exceptionally stable IMHBs have recently been observed in polysubstituted 5ꢀnitrosopyrimidine
derivatives.12ꢀ14 When two hydrogen bond donors were in the neighboring positions to the nitroso
group, two stable rotamers of the nitroso group were observed and even separated in several
cases.15 The extraordinary stability of hydrogen bonds in these systems has been explained in
terms of resonanceꢀassisted hydrogen bonding (RAHB)16ꢀ19 and intramolecular charge transfer
(push–pull interactions).20
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NMR spectroscopy is one of the most powerful tools to study the strength and geometry
of hydrogen bonds, because all observable NMR parameters are affected by the formation of
hydrogen bonds.21 A direct evidence of the formation of a hydrogen bond can be obtained, for
example, by the observation of a scalar coupling across the hydrogen bonds (hJ), allowing
simultaneous identification of hydrogenꢀbond donor and acceptor.22 Since their first observation
in 1998,23ꢀ24 scalar interactions across hydrogen bonds have become an important tool for the
structural elucidation of biological macromolecules.3, 25ꢀ27
Here, we present the results of our synthetic, NMR and computational study of substituted
5ꢀphenylazopyrimidines with one (1a–1c) and with two (2aꢀ2c) hydrogen bond donors in
positions 4 and 6 (Figure 1). These compounds can form two different sixꢀmembered pseudorings
with IMHBs. We demonstrate that the nature of the substituents has a large effect on the stability
of the two forms of the molecules. Furthermore, 15N labelling of the compounds allowed us
determination of throughꢀhydrogenꢀbond NMR coupling constants, which are correlated to the
hydrogen bond strengths. A combination of experimental NMR data with path integral molecular
dynamics (PIMD) simulations, that involve NQEs, is shown to provide intimate details about the
IMHBs in these systems.
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5ꢀAzopyrimidine nucleosides with the same structural motif as the molecules investigated
here have recently been suggested as nucleodyes – visibly colored nucleoside analogs with
potential application in studying nucleic acids and their cellular interactions.28 5ꢀAzopyrimidines
have also been proposed as prodrugs of pyrimidine derivatives with antioxidant activity.29
Generally, azocompounds have found multitude of applications ranging from synthetic chemistry
to medicine and nanorobotics.30ꢀ45
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Figure 1. The structure of the studied compounds.
Results and Discussion
Synthesis
The key reaction step in the synthesis of the target compounds 1 and 2 is azo coupling
between properly substituted pyrimidine and aromatic diazonium salt formed from aniline
bearing a substituent in para position of the phenyl ring (pꢀNO2, pꢀOMe). Thus, azo coupling of
2ꢀaminoꢀ4,6ꢀdihydroxypyrimidine (3) with benzenediazonium chlorides 4a, 4b and 4bꢀ15N1
(Scheme 1) gave azo compounds 5a, 5b and 5bꢀ15N1, respectively, as solids that precipitated
from the reaction mixtures. The products 5 were chlorinated with VilsmeierꢀHaackꢀArnold
reagent to obtain 4,6ꢀdichloropyrimidine derivatives 6. The conventional heating of compounds 6
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with ethanolic ammonia or methanolic 15N ammonia solutions afforded compounds 1 and
subsequent heating of compounds 1 with ethanolic methylamine solution or with 15N
methylamine hydrochloride in isopropanol under microwave conditions gave desired products 2
(Scheme 1). Similarly, azo coupling of 2,4ꢀdiaminoꢀ6ꢀchloropyrimidine (7, Scheme 1) with pꢀ
nitrobenzenediazonium chloride 4c led to the formation of azo compound 1c, which upon
treatment with ethanolic methylamine solution afforded final derivative 2c. Purification of final
azo compounds 1 and 2 was quite laborious and multiple reverse phase chromatographies were
necessary to obtain pure products.
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Scheme 1. Synthesis of target azo compounds 1 and 2.
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Reagents and conditions: (a) acetic buffer (pH 7), rt, overnight; (b) VilsmeierꢀHaackꢀArnold
reagent, CHCl3, reflux, 1–3 h, 59–78 %; (c) NH3 (2M ethanolic solution), 60 °C (conventional
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heating), 1–12 h, 20–60 %; (d) N NH3 (methanolic solution), 60 °C (conventional heating), 12
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h, 33 %; (e) methylamine (33% ethanolic solution), 150 °C (MW conditions), 10–30 min, 27–40
%; (f) 15N methylamine hydrochloride, iPrOH, 150 °C (MW conditions), 1 h, 20 %.
NMR Spectroscopy
One set of signals is observed in NMR spectra of chloro derivatives 1a–1c. The formation of
a sixꢀmembered pseudoring with IMHB can be inferred from the chemical shifts of protons in the
NH2 group in position 4; the two amino hydrogen atoms are not equivalent and the hydrogen
atom involved in IMHB resonates at higher chemical shift values (1–1.2 ppm downfield than the
other hydrogen atom).
Compounds 2a–2c with two hydrogen bond donors in positions 4 and 6 may form two
stable IMHBs (rotamer A and rotamer B, Figure 1), which can lead to observation of two sets of
signals in NMR spectra. However, variable temperature NMR experiments with compounds 2a–
2c revealed remarkable differences in the interconversion barriers between these two rotamers. In
1H NMR spectrum of compound 2c with nitro substituent, two sets of signals are well resolved at
room temperature and, for example, the coalescence of the methyl signals (Figure 2) is observed
at 310 K. On the other hand, the coalescence of this signal in the spectra of compounds 2b and 2a
is observed at lower temperature (260 K and 240 K, respectively). The rotational barriers
(barriers of interconversion between rotamer A and B) were estimated from lineshape analysis of
the variable temperature proton spectra and from the coalescence temperature (Table 1 and Table
S1 in Supporting Information). The observed rotamer ratio is almost independent on the
substituent in the phenyl ring and temperature. Rotamer A with the azo group heading towards
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the methylamino group is always more stable and the rotamer ratio is ca 6:4. For comparison, we
have also prepared compound 2d analogous to 2c with the nitro substituent in meta position to
the azo group and determined the interconversion barrier (SI). The push–pull interaction is
evidently suppressed in this compound.
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Figure 2. Variable temperature H NMR spectra of compounds 2a–2c. Only the region with
methyl signals of the methylamino group is depicted.
Table 1. Experimental and calculated interconversion barriers (kcal/mol) between two rotamers
of compounds 2a–2c.
Coalescence T /
Compound Substituent
ꢁG#(exp)b ꢁG#(calc)
Ka
pꢀOMe
–
pꢀNO2
240
260
320
12.5±0.3
13.4±0.3
16.2±0.3
14.7
15.9
20.6
2a
2b
2c
aCoalescence of methylamino CH3 signals; calculated at coalescence temperature using the
equation k = 2.22*ꢁν, where k is the rate constant and ꢁν is the difference of resonance
frequency of methyl hydrogens in rotamer A and B, full details are given in Table S1 in the SI.
b
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The differences in the heights of the rotational barriers in compounds 2a–2c may be
caused either by differences in the stability of the IMHB induced by the substituents or by
increased bond order of the C5–N bond caused by intramolecular charge transfer (push–pull
interaction). The influence of the substituent on the hydrogen bond strength might be deduced
from the changes of chemical shifts of amino and methylamino NH protons. The contribution of
the electronic nature of the paraꢀsubstituent to magnetic shielding of amino hydrogens in position
4 and methylamino hydrogen in position 6 should be identical, because it is transferred via the
same structural fragment. Chemical shifts of the (methyl)amino NH protons in rotamer A of
compounds 2a–2c measured at 210 K depend on the substituent, but those involved and not
involved in IMHB change evenly (SI, Table S2). Therefore, the hydrogen bond strength is
probably not affected by the substituent in the phenyl ring.
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Two sets of signals with 6:4 ratio corresponding to rotamers A and B can also be observed
15
in N NMR spectrum of compound 2bꢀ15N3. The signal assignment was done with the help of
compounds 1bꢀ15N1 and 1bꢀ15N2 with one and two 15N labels, respectively. Azo nitrogen (Figure
S1, SI) in proton decoupled 15N spectrum appears as a doublet close to 400 ppm for both
rotamers; the splitting of the signal is caused by throughꢀhydrogen bond interaction (2hJN,N) with
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the N amino nitrogen in position 6 or 4. These amino nitrogens are observed in the region of
70–95 ppm. The signals of amino nitrogen atoms involved in the hydrogen bond appear at higher
chemical shifts (close to 90 ppm) and are split by the above mentioned throughꢀhydrogen bond
coupling in protonꢀdecoupled spectra (Figure 3). Further splitting to triplet (NH2 group, rotamer
B) or doublet (NHMe group, rotamer A) is observable in protonꢀcoupled spectra due to oneꢀbond
1JN–H interaction. The signals of nitrogen atoms not involved in the hydrogen bond appear at
1
lower chemical shifts (ca 15 ppm upfield). Hydrogen bonding also influences the JN–H coupling
constant between the hydrogenꢀbond donor and the hydrogen atom. It has been documented that
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strong hydrogen bonding results in a decrease of the JN–H coupling constant across the
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corresponding N–H covalent bond.26, 46 Indeed, slightly smaller JN–H interaction is observed for
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amino groups involved in hydrogen bonds, i.e. in NH2 group of rotamer B and NHMe group in
rotamer A.
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Figure 3. Amino and methylamino region of protonꢀcoupled (blue) and protonꢀdecoupled (black)
15N NMR spectra of compound 2bꢀ15N3.
Throughꢀhydrogenꢀbond coupling may also be detected between the amino/methylamino
hydrogen atom and the azo nitrogen atom. This interaction leads to observation of throughꢀ
hydrogen bond crossꢀpeaks in H,NꢀHMBC spectra of compound 2bꢀ15N3 and to splitting of the
1
NH signal in H NMR spectrum (Figure 4). All relevant H–N and N–N couplings observed in
1h
2h
compound 2bꢀ15N3 are summarized in Figure 5. The JN,H and JN,N throughꢀhydrogen bond
couplings have values typically found in systems with N–HꢂꢂꢂN hydrogen bonds.3 It can be seen
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that both observable throughꢀhydrogenꢀbond couplings (1hJN,H and 2hJN,N) are larger in rotamer A,
which corresponds to stronger hydrogen bond interaction of the methylamino group leading also
to higher relative concentration of rotamer A.
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Figure 4. Left: part of H,NꢀHMBC spectrum of compound 2bꢀ15N3 measured at 230K with
throughꢀhydrogen bond interactions. Right: Methylaminoꢀhydrogen region of proton NMR
spectrum of compound 2bꢀ15N3 acquired at 230 K (black) and the same spectrum with selective
homodecoupling of the CH3 protons (blue).
6.3
3.0
HB HA
N
HB HA
N
N
91.1
90.8
91.1 N
90.8 N
N
N
H
N
4.0
H
H2N
N
N
H2N
N
N
7.4
95.1
92.5
rotamer A
rotamer B
Figure 5. Selected N–H (red) and N–N scalar coupling constant values observed in both rotamers
of compound 2bꢀ15N3.
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It has recently been shown that hydrogen bonding interactions in RAHBs are closely
related to nuclear quantum effects (NQEs), particularly to the delocalization of hydrogen
nucleus.19 One of the consequences of NQEs is the effect of deuterium isotope substitution on the
hydrogen bond properties. Therefore, we prepared compound 2bꢀ15N3 with ca 1:1 hydrogen to
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deuterium isotope exchange of the amino and methylamino hydrogens and acquired N NMR
spectra. The effect of deuteration on NMR spectra is twofold: first, 15N signals are shifted due to
deuterium isotope shift (Table 2) and second, the value of 2hJN,N throughꢀhydrogen bond coupling
in rotamer A is slightly reduced (7.4 → 7.3 Hz).
Table 2. Experimental deuterium isotope induced changes of nitrogen chemical shifts (ppm) and
throughꢀhydrogenꢀbond coupling (Hz) in compound 2bꢀ15N3 and calculated isotopeꢀinduced
changes of N–H bond distances (Å).
Rotamer A
ꢁδ / ꢁJa
1.05
Rotamer B
ꢁδ / ꢁJa
0.62
ꢁd(N6–H)b
ꢁd(N6–H)b ꢁd(N4–HA)b
N6
N4
0.007
0.004
0.55
~0.7c
Nꢀazo
–0.74
0.1
0.006
0.008
~–0.5c
0.005
2h
J
N6,Nꢀazo
aCalculated as the difference between protonated and deuterated compound, i.e. positive value
b
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means upfield shift; calculated from the experimental deuteriumꢀinduced N chemical shift and
2h
J
changes and DFT calculated dependence of these NMR parameters on N–H bond
N6,Nꢀazo
distance; csignal broadening due to 0, 1 or 2 hydrogen atom exchange in NH2 group.
Calculations
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To better understand the intramolecular hydrogen bonding and its influence on the
structure and properties of the studied molecules, we performed a number of density functional
theory (DFT) calculations. First, we optimized the geometries of both rotamers of compounds
2a–2c and calculated magnetic shielding of the nuclei and scalar coupling values. Excellent
correlation between the calculated shielding values and experimental chemical shifts was
observed (SI, Figure S2), which confirmed our signal assignment. The calculated coupling
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1h
2h
constant values reproduce well the trends observed in compound 2bꢀ15N3, i.e. JH,N and JN,N
1
throughꢀhydrogen bond couplings are larger in rotamer A of compound 2b and JN–H coupling
constants decrease upon hydrogen bond formation (Table S3). Furthermore, the calculations
revealed that the throughꢀhydrogen bond couplings are almost independent on the substitution on
the phenyl ring. This confirms our conclusion, inferred from experimental proton chemical shifts,
that the substitution does not affect the hydrogen bond strength.
The calculated barriers of rotamer interconversion reproduce the trends observed by
variableꢀtemperature NMR experiments, i.e. the highest barrier is in compound 2c followed by
2b and 2a. Given that the hydrogen bond strength is similar in all three compounds, the
differences in rotamer interconversion barriers must be caused by pushꢀpull interactions of the
substituents. For example, electronꢀdonating substituents in the pyrimidine ring of compound 2c
push electron density towards the electronꢀwithdrawing nitro substituent in the phenyl ring. This
is well reflected in the calculated electrostatic potential plots (Figure S3) bond distances in
compounds 2a–2c (Table S4), where the C5–N distance is significantly smaller in compound 2c
(1.348 Å) than in compound 2b (1.365 Å) and 2a (1.370 Å). Smaller bond distance corresponds
to higher bond order and hence to higher rotational barrier.
To estimate the geometry changes in the IMHBs upon hydrogenꢀtoꢀdeuterium isotope
2h
exchange, the experimental isotopeꢀinduced changes of nitrogen chemical shifts and
J
N,N
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coupling were compared with a DFT calculated dependence of nitrogen shieldings and throughꢀ
hydrogenꢀbond couplings on the 6N–H and 4N–H bond distances. The calculations revealed that
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lengthening of N–H distance leads to smaller shielding of the NH nitrogen atom, larger shielding
1h
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of the hydrogenꢀbonded azo nitrogen and larger throughꢀhydrogenꢀbond couplings
J
N,H
and
2h
J
N,N
.The comparison of the calculated distance dependence of shieldings and couplings with
experimental isotopeꢀexchange induced changes was used to estimate average deuteriumꢀinduced
bondꢀlength shortening of 0.006–0.008 Å in the case of N6ꢀH in rotamer A (involved in IMHB)
and to a smaller shortening of N6–H in rotamer B (not involved in IMHB) and of N4–H in
rotamer B (involved in a weaker IMHB, see Table 2).
It has recently been demonstrated that an incorporation of NQEs is important for an
accurate prediction of hydrogen bond properties, because hydrogen atom possess the lightest
nucleus and effects, such as tunneling or delocalization of nuclear positions, become more
important than for heavier nuclei.19, 47ꢀ51 One way of incorporation of NQEs into quantumꢀ
chemical calculations is path integral molecular dynamics (PIMD). PIMD simulations use a
decomposition of nuclei into a set of “beads” connected with harmonic oscillators; the force
constant of the oscillator depends on the nuclear mass and temperature. Light nuclei and low
temperatures lead to more delocalized nuclei.
We applied PIMD simulations to compounds 1a–1c and to both rotamers of compound 2b
and we analyzed the simulations by plotting probability distributions of bond distances between
atoms involved in the IMHBs. The probability distribution of N–H distances in compounds 1a–
1c shows that the amino N–H bond involved in the IMHB is longer and the probability
distribution of this N–H distance is broader (the hydrogen is more delocalized) than for the N–H
bond not involved in the IMHB. The N–H and HꢂꢂꢂN distances are almost identical for all three
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compounds 1a–1c (SI, Figure S4), which confirms our conclusion that the substituent on the
phenyl ring does not affect the hydrogen bond strength.
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On the other hand, the probability distributions found for compound 2b depend
significantly on the conformation (rotamer A or B, see Figure 6). The 6N–H of rotamer A
(involved in IMHB) probability distribution is slightly broader and shifted towards higher
distances than the 4N–HA of rotamer B (also involved in IMHB) distribution. The distance
between hydrogen bond donor and the hydrogen atom has been correlated to hydrogen bond
strength.52 These probability distributions thus reflect the stability of the two rotamers: rotamer A
is more abundant because the hydrogen atom in the IMHB is more delocalized and hence, the
hydrogen bond in this rotamer is more stable. The distance probabilities of N–H bonds not
involved in IMHBs are significantly narrower and shifted to shorter values. Significantly
narrower distribution is also observed for N–D distances in deuterated compound 2b. Similar
information about the geometry of the IMHBs can be obtained from probability distributions of
distances between the hydrogen atom and the hydrogen bond acceptor (azo nitrogen). In rotamer
A, the HꢂꢂꢂN distance probability is significantly shorter than in rotamer B and deuteration leads
to lengthening of the HꢂꢂꢂN distance (Figure S5). All relevant average distances between atoms
involved in the IMHBs obtained from the PIMD simulations are listed in the SI (Table S7).
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0.00
A: 6N-H
B: 4N-HA
A: 4N-HA
A-D: 6N-H
A-D: 4N-HA
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0.9
1.0
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1.2
1.3
N-H bond distance / angstrom
Figure 6. Probability distribution of selected N–H distances in rotamers A and B of compound 2b
and of deuterated rotamer A (AꢀD) found in PIMD simulations.
Conclusions
Intramolecular hydrogen bonds in substituted 5ꢀphenylazopyrimidines were studied by
NMR spectroscopy and DFT calculations. Both experimental chemical shifts, and computed N–H
bond distances and throughꢀhydrogenꢀbond scalar couplings indicate that the hydrogenꢀbond
strength is not affected by the substitution in para position of the phenyl ring. The substituents
have, however, a large effect on the C5–N rotational barrier, which was explained in terms of
push–pull interactions of the substituents. Electronꢀdonating substituents (amino groups) in the
pyrimidine ring push electron density through the azo nitrogens to the phenyl ring. This
intramolecular charge transfer is more effective when an electronꢀaccepting (nitro) substituent is
in para position of the phenyl ring. The intramolecular charge transfer increases the C5–N bond
order and, hence, also the C5–N rotational barrier.
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In compounds with two hydrogenꢀbond donors in neighboring positions to the azo group,
two different IMHBs can exist, both leading to the formation of a sixꢀmembered pseudo ring. The
formation of the IMHBs in these two structures was confirmed, for example, by the observation
of throughꢀhydrogenꢀbond scalar couplings. Rotamer A with the pseudoring formed with the
methylamino group was more stable than rotamer B in all studied compounds. Higher stability of
rotamer A is reflected in larger shielding of the hydrogenꢀbond acceptor (azoꢀnitrogen) and larger
throughꢀhydrogenꢀbond couplings in this rotamer. Higher stability of the IMHB in rotamer A was
also confirmed by PIMD simulations, where larger delocalization of the hydrogen atom in the
IMHB in rotamer A was observed. This larger delocalization of the hydrogen atom also leads to
larger deuteriumꢀisotopeꢀinduced changes of nitrogen chemical shifts, which were confirmed
experimentally.
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The potential applications of the studied compounds range from biocompatible azodyes to
orthogonal molecular switches with switching either the IMHB (rotamer A or B) or the cis/trans
isomers of the azo group, which is a widely exploited type of isomerism in many applications.
The work presented here demonstrates that fine tuning of the switching barrier between the
rotamers A and B is possible by introducing proper substituents in the phenyl ring. These
substituents do not influence the hydrogen bond strengths, which are controlled by the nature of
the hydrogen bond donors. Relative strengths of the IMHBs can easily be determined by NMR
spectroscopy. The combination of experimental NMR data with DFT calculations enables to
obtain intimate details about IMHBs, such as the changes of hydrogen bond geometry upon
hydrogenꢀtoꢀdeuterium isotope exchange.
EXPERIMENTAL SECTION
General. Instrumentation and calculations.
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Unless otherwise stated, solvents were evaporated at 40 °C/2 kPa, and the compounds
were dried over P2O5 at 2 kPa. Analytical TLC was performed on silica gel preꢀcoated aluminium
plates with fluorescent indicator (Merck 5554, 60 F254). Spots were visualized with UV light (254
nm). Flash reversedꢀphase chromatography was carrried out on CombiFlash® Rf+ (TELEDYNE
ISCO) with RediSep RF Gold® C18 Aq column (50 g, 20ꢀ40 ꢀm, TELEDYNE ISCO). Mass
spectra were measured on a QꢀTof micro (Waters) and HR MS were taken on a LTQ Orbitrap XL
(Thermo Fisher Scientific) spectrometer. Melting points were measured on a Stuart
SMP3Melting Point Apparatus
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The microwaveꢀassisted reactions were carried out in CEM Discover (Explorer)
microwave aparatus, 24ꢀposition system for 10ꢀmL vessels sealed with Teflon septum. It was
operated at a frequency of 2.45 GHz with continuous irradiation power from 0 to 300W. The
solutions were steadily stirred during the reaction. The temperature was measured with an IR
sensor on the outer of the process vessel. The vials were cooled to ambient temperature with gas
jet cooling system. The pressure was measured with an inboard CEM Explorer pressure control
system (0ꢀ21 bar).
1H and 13C NMR spectra were recorded on a 400, 500 and/or 600 MHz NMR
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spectrometer (1H at 400 MHz and C at 100.6 MHz, or H at 500 MHz, C at 125.7 MHz and
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15N at 50.7 MHz, or H at 600.1 MHz and C at 150.9 MHz) in DMSOꢀd6 (referenced to the
solvent signal δ = 2.50 and 39.70 ppm, respectively) and DMFꢀd7 (referenced to the solvent
signal δ = 2.75 (1H), 104.9 (15N) and 163.15 ppm (13C), respectively). Complete signal
assignment is based on heteronuclear correlation experiments HSQC and HMBC. The 15N
chemical shifts were determined using 1D experiment with direct detection of nitrogens and
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gated decoupling of hydrogen nuclei. For the assignment of N signals based on coupling with
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directly attached hydrogen atoms, proton coupled nitrogen NMR spectra were also measured.
Chemical shifts (δ) are in ppm and coupling constants (J) in Hz. Partly deuterated compound 2bꢀ
15N3 was prepared by adding a drop of D2O into the DMF solution of the compound and
evaporating to dryness under reduced pressure.
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The studied structures were subjected to geometry optimization at DFT level, using B3LYP
functional,53ꢀ54 standard 6ꢀ31+G(d,p) basis set and polarizable continuum model used for implicit
DMF solvation.55ꢀ56 NMR shielding values and scalar coupling constants were calculated for the
optimized structures. The Gaussian09 program package was used throughout this study.57 The
QST3 optimization method58ꢀ59 was applied in the search for the transition state structures of the
rotamer interconversion, that is the structures of the reactant, product, and estimated transition
state were used as input for the TS search. The vibrational frequencies and free energies were
calculated for all of the optimized structures, and the stationaryꢀpoint character (a minimum or a
firstꢀorder saddle point) was thus confirmed.
The dependence of nitrogen shieldings and throughꢀhydrogenꢀbond couplings on the 6N–H
and 4N–H bond distances was calculated by manually adjusting the bond distance in the range
0.9–1.3 Å (with a 0.1 Å step). The calculated distance dependence of the shielding and coupling
values was fitted to a straight line.
Path integral molecular dynamics (PIMD) simulations were run in the CASTEP program,60
which is a DFTꢀbased code, using an NVT ensemble maintained at a constant temperature of 300
K using a Langevin thermostat, a 0.5 fs integration time step, simulation length of 5 ps, ultrasoft
pseudopotentials,61 a planewave cutoff energy of 300 eV, and with integrals taken over the
Brillouin zone using a MonkhorstꢀPack62 grid of a minimum kꢀpoint sampling of 0.1 Å–1.
Electronꢀcorrelation effects were modelled using the generalized gradient approximation of
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Perdew, Burke, and Ernzerhof.63 The atomic positions were optimized at the same computational
level prior to the PIMD runs. Compounds 1a–1c and both rotamers of compound 2b were
modelled as isolated molecules in a cubic periodic box of 16 x 16 x 16 Å3. The path integral was
used on top of the DFTꢀMD simulations, with a Trotter decomposition of all nuclei into P = 16
beads. For the evaluation of deuterium isotope effects, new PIMD simulations were performed
with the mass of all exchangeable protons (all N–H protons) adjusted to the mass of deuterium.
Probability distributions of the N4–H and N6–H bond distances were plotted with 0.04 Å step.
The PIMD distance probabilities were determined independently for all 16 replicas and then
averaged.
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General procedure A. Substituted aniline (1.2 eq) was dissolved in 1M HCl (5 mL), the
solution was cooled to 0 °C and sodium nitrite (1.3 eq) was added. The reaction mixture was
stirred for 30 min at 0 °C. The solution of formed diazonium salt 4 was then added dropwise to a
solution of 2ꢀaminoꢀ4,6ꢀdihydroxypyrimidine (3, 1 eq) in acetic buffer and the mixture was
stirred overnight. Precipitated solid was filtreted and dried to obtain 4,6ꢀdihydroxypyrimidine
intermediates 5. Compounds 5 were consequently treated with VilsmeierꢀHaackꢀArnold reagent
(4.0 eq) in CHCl3 (40 mL) at reflux. The solvent was evaporated to dryness. The formed 2ꢀ
(dimethylamino)methylene)amino substituted pyrimidine was dissolved in water (40 mL) with
catalytic amount of concentrated HCl. The reaction mixture was stirred overnight and
precipitated solid was filtered and dried. Reversedꢀphase flash chromatography (MeOH/H2O, 0ꢀ
100 %) gave desired 4,6ꢀdichloropyrimidine products 6.
4,6ꢀDichloroꢀ5ꢀ((4ꢀmethoxyphenyl)diazenyl)pyrimidinꢀ2ꢀamine (6a). Treatment of pꢀanisinide
(145 mg, 1.2 mmol) with sodium nitrite (88 mg, 1.3 mmol) by procedure A afforded diazonium
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salt 4a, which reacted with 2ꢀaminoꢀ4,6ꢀdihydroxypyrimidine (3, 127 mg, 1 mmol) to give 2ꢀ
aminoꢀ5ꢀ((4ꢀmethoxyphenyl)diazenyl)pyrimidineꢀ4,6ꢀdiol (5a). Treatment of 5a (1 g, 3.8 mmol)
with VilsmeierꢀHaackꢀArnold reagent (1.946 g, 15.2 mmol) for 3 h gave 6a (940 mg, 78 %) as an
1
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orange solid, m. p. 220–230 °C. H NMR (DMSOꢀd6, 298 K): 7.97 (2H, bs, NH2), 7.82 (2H, m,
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H2’), 7.14 (2H, m, H3’); C NMR (DMSOꢀd6, 298 K): 162.7 (C4’), 160.3 (C2), 154.9 (C4 and
C6), 146.6 (C1’), 130.5 (C5), 124.7 (C2’), 114.9 (C3’), 55.9 (OCH3); ESI MS, m/z (%): 298.1 [M
+ H]+; HRMS (ESI) calcd for C11H10Cl2N5O [M + H]+ 298.0257, found 298.0257.
4,6ꢀDichloroꢀ5ꢀ(phenyldiazenyl)pyrimidinꢀ2ꢀamine (6b). Treatment of aniline (0.2 mL, 2.4
mmol) with sodium nitrite (176 mg, 2.6 mmol) by procedure A gave diazonium salt 4b, which
reacted with 2ꢀaminoꢀ4,6ꢀdihyroxypyrimidine (3, 254 mg, 2 mmol) to give 2ꢀaminoꢀ5ꢀ
(phenyldiazenyl)pyrimidineꢀ4,6ꢀdiol (5b). Treatment of 5b (100 mg, 0.4 mmol) with Vilsmeierꢀ
HaackꢀArnold reagent (205 mg, 1.6 mmol) for 1h gave 6b (73 mg, 68 %) as a red solid, m. p .
160ꢀ173 °C. 1H NMR (DMSOꢀd6): 8.10 (2H, bs, NH2), 7.82 (2H, m, H2’), 7.63ꢀ7.56 (3H, m, H3’
and H4’); 13C NMR (DMSOꢀd6): 160.5 (C2), 155.5 (C4 and C6), 152.3 (C1’), 132.1 (C4’), 130.3
(C5), 129.8 (C3’), 122.5 (C2’); ESI MS, m/z (%): 268.0 [M + H]+; HRMS (ESI) calcd for
C10H8Cl2N5 [M + H]+ 268.0151, found 268.0149.
4,6ꢀDichloroꢀ5ꢀ(phenyldiazenylꢀ2ꢀ15N)pyrimidinꢀ2ꢀamine (6b-15N1). Treatment of anilineꢀ15N
(0.12 mL, 1.2 mmol) with sodium nitrite (74 mg, 1.3 mmol) by procedure A gave diazonium salt
4b-15N1, which reacts with 2ꢀaminoꢀ4,6ꢀdihydroxypyrimidine (3, 127 mg, 1 mmol) to give 2ꢀ
aminoꢀ5ꢀ(phenyldiazenylꢀ2ꢀ15N)pyrimidineꢀ4,6ꢀdiol (5bꢀ15N1). Treatment of 5b-15N1 (100 mg,
0.6 mmol) with VilsmeierꢀHaackꢀArnold reagent (307 mg, 2.4 mmol) for gave 6b-15N1 (95 mg,
59 %) as red solid. 1H NMR (DMSOꢀd6): 8.10 (2H, bs, NH2), 7.81 (2H, m, H2’), 7.62ꢀ7.56 (3H,
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m, H3’ and H4’); C NMR (DMSOꢀd6): 160.5 (C2), 155.5 (d, JCꢀCꢀNꢀN = 2.0, C4 and C6), 152.3
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(d, JCꢀN = 2.5, C1’), 132.1 (C4’), 130.3 (d, JCꢀNꢀN = 5.2, C5), 129.8 (d, JCꢀCꢀCꢀN = 1.9, C3’), 122.5
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(d, JCꢀCꢀN = 4.0, C2’); ESI MS, m/z (%): 269.0 [M + H]+; HRMS (ESI) calcd for C10H8N4 NCl2
[M + H]+ 269.0122, found 269.0123.
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6ꢀChloroꢀ5ꢀ((4ꢀnitrophenyl)diazenyl)pyrimidineꢀ2,4ꢀdiamine (1c). Sodium nitrite (88 mg, 1.3
mmol) was added to the cooled (0 °C) solution of pꢀnitroaniline (166 mg, 1.2 mmol) in 1M HCl
(5 mL) and the reaction mixture was stirred for 30 min at 0 °C to give a solution of diazonium
salt 4c. To this solution, a solution of 2,4ꢀdiaminoꢀ6ꢀhydroxypyrimidine (3, 144 mg, 1 mmol) in
acetic buffer was added dropwise and the reaction mixture was stirred overnight. Precipitated
solid was filtered and reversedꢀphase flash chromatography (0ꢀ100 % MeOH in water) gave 1c
(188 mg, 64 %) as an orange solid, m. p. > 300 °C. 1H NMR (DMSOꢀd6): 9.40 (1H, d, JGEM= 3.3,
4ꢀNHa), 8.41 (1H, d, JGEM= 3.3, 4ꢀNHb), 8.35 (2H, m, H3’), 7.98 (2H, m, H2’), 7.74 (1H, bs, 2ꢀ
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NHb), 7.55 (1H, bs, 2ꢀNHa); C NMR (DMSOꢀd6): 166.0 (C6), 161.1 (C2), 156.5 (C1’), 156.0
(C4), 146.8 (C4’), 125.2 (C3’), 122.3 (C2’), 119.9 (C5); ESI MS, m/z (%): 294.1 [M + H]+;
HRMS (ESI) calcd for C10H9ClN7O2 [M + H]+ 294.0501, found 294.0503.
General procedure B. Starting 4,6ꢀdichloropyrimidine derivative 6 was dissolved in ammonia
(2.5 M ethanolic solution) and the reaction mixture was heated at 60 °C. Water (50 mL) was
added and obtained solution was extracted with EtOAc (3 x 40 mL). Organic layers were
collected, dried over MgSO4 and filtered. Solution was evaporated under vacuum and pure
product was obtained by reversedꢀphase flash chromatography (0ꢀ100 % MeOH in water).
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6ꢀChloroꢀ5ꢀ((4ꢀmethoxyphenyl)diazenyl)pyrimidineꢀ2,4ꢀdiamine (1a). Treatment of 6a (50 mg,
0.2 mmol) with ammonia (10 mL) for 4 h by procedure B gave 1a (10 mg, 20 %) as a yellow
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solid, m. p. 250–253 °C. H NMR (DMSOꢀd6): 9.21 (1H, d, JGEM= 3.7, 6ꢀNHa), 7.98 (1H, d,
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GEM= 3.7, 6ꢀNHb), 7.77 (2H, m, H2’), 7.30 and 7.15 (2H, bs, 2ꢀNH2), 7.06 (2H, m, 3’), 3.82 (3H,
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s, 4’ꢀOꢀCH3); C NMR (DMSOꢀd6): 136.8 (C4), 161.1 (C2), 160.6 (C4’), 156.2 (C6), 145.7
(C1’), 123.2 (C2’), 118.5 (C5), 114.7 (C3’), 55. 7 (OCH3); ESI MS, m/z (%): 279.1 [M + H]+;
HRMS (ESI) calcd for C11H12N6OCl [M + H]+ 279.0756, found 279.0756.
6ꢀChloroꢀ5ꢀ(phenyldiazenyl)pyrimidineꢀ2,4ꢀdiamine (1b) Treatment of 6b (50 mg, 0.2 mmol)
with ammonia (10 mL) overnight by procedure B gave 1b (30 mg, 60 %) as a yellow solid, m. p.
1
230–245 °C (decomp.). H NMR (DMSOꢀd6): 9.29 (1H, d, JGEM= 3.6, 6ꢀNHa), 8.11 (1H, d,
JGEM= 3.6, 6ꢀNHb), 7.78 (2H, m, H2’), 7.51 (2H, m, H3’), 7.44 and 7.26 (2H, bs, 2ꢀNH2), 7.40
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(1H, m, H4’); C NMR (DMSOꢀd6): 164.6 (C4), 161.3 (C2), 156.2 (C6), 152.5 (C1’), 129.5
(C4’), 129.4 (C3’), 121.6 (C2’), 118.8 (C5); ESI MS, m/z (%): 249.1 [M + H]+; HRMS (ESI)
calcd for C10H10ClN6 [M + H]+ 249.0650, found 249.0650.
6ꢀChloroꢀ5ꢀ(phenyldiazenylꢀ2ꢀ15N)pyrimidineꢀ2,4ꢀdiamine (1b-15N1). Treatment of 6b-15N1 (100
mg, 0.4 mmol) with ammonia (10 mL) for 6 h by procedure B gave 1b-15N1 (60 mg, 60 %) as a
yellow solid, m. p. 248–250 °C. 1H NMR (DMSOꢀd6, 298 K): 9.29 (1H, m, 4ꢀNHA), 8.05 (1H, d,
JGEM = 3.9, 4ꢀNHB), 7.77 (2H, m, H2’), 7.50 (2H, m, H3’), 7.39 (1H, m, H4’); 13C NMR (DMSOꢀ
d6, 298 K): 164.8 (d, 3JCꢀN = 3.1, C4), 161.4 (C2), 156.3 (C6), 152.6 (C1’), 129.8 (C4’), 129.7 (d,
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3JCꢀN = 2.0, C3’), 121.8 (d, JCꢀN = 3.6, C2’), 118.9 (d, JCꢀN = 4.0, C5); ESI MS, m/z (%): 250.1
15
[M + H]+; HRMS (ESI) calcd for C10H10N5 NCl [M + H]+ 250.0620, found 250.0620.
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6ꢀChloroꢀ5ꢀ(phenyldiazenylꢀ2ꢀ15N)pyrimidineꢀ2,4ꢀdiamineꢀ15N (1b-15N2). Ammoniaꢀ15N (0.1 mL,
2 M methanolic solution) was added to the solution of compound 6b-15N1 (200 mg, 0.7 mmol) in
MeOH (10 mL) and the reaction mixture was heated at 60 °C for 6 h. Water (50 mL) was added
and obtained solution was extracted with EtOAc (3 x 40 mL). Organic layers were collected,
dried over MgSO4 and filtered. Solution was evaporated under vacuum and reversedꢀphase flash
chromatography (0ꢀ100 % MeOH in water) gave 1b-15N2 (150 mg, 33 %) as a yellow solid, m. p.
234–236 °C. 1H NMR (DMSOꢀd6, 298.5 K): 9.30 (1H, dt, JHꢀN = 90.1, JGEM = JHꢀN = 3.8, 4ꢀNHa),
8.12 (1H, dd, JHꢀN = 90.7, JGEM = 3.9, 4ꢀNHb), 7.78 (2H, m, Hꢀ2’), 7.50 (2H, m, Hꢀ3’), 7.40 (1H,
m, Hꢀ4’); 13C NMR (DMSOꢀd6, 298.5 K): 164.7 (dd, 3JCꢀN = 3.1, 1JCꢀN = 1.3, C4), 161.3 (d, 3JCꢀN
= 3.6, C2), 156.2 (d, 3JCꢀN = 19.3, C6), 152.5 (C1’), 129.5 (C4’), 129.4 (d, 3JCꢀN = 2.0, C3’), 121.7
(2JCꢀN = 3.7, C2’), 118.8 (dd, 2JCꢀ1’N = 4.1, 2JCꢀ4N = 1.2, C5); 15N NMR (DMSOꢀd6, 298.5 K): 96.4
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2h
2h
(d, JNꢀN = 5.6, N4), 444.8 (d, JNꢀN = 5.6, N1’); ESI MS, m/z (%): 251.1 [M + H]+; HRMS
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(ESI) calcd for C10H10ClN4 N2 [M + H]+ 251.0591, found 251.0591.
General procedure C. Methylamine (3 eq, 33 % ethanolic solution) was added to the solution of
compound 1 in iPrOH (15 mL) and the reaction mixture was heated under MW conditions at 150
°C. Water (30 mL) was added and the solution was extracted wit EtOAc (3 x 20 mL). Organic
layers were collected, dried over MgSO4 and filtered. The solution was evaporated under vacuum.
Product was purified by reversedꢀphase flash chromatography (0ꢀ100 % MeOH in water).
5ꢀ((4ꢀMethoxyphenyl)diazenyl)ꢀN4ꢀ
methylpyrimidineꢀ2,4,6ꢀtriamine (2a). Treatment of
1a (200 mg, 0.7 mmol) with methylamine (0.1 mL,
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2.1 mmol) for 30 min by procedure C gave 2a (70 mg, 37 %) as a yellow solid, m. p. 193–195
°C. 1H NMR (DMFꢀd7, 230 K): 10.43 (1H, q, JNHꢀCH3 = 4.8, 6ꢀNH, A), 9.76 (1H, d, JGEM = 4.5, 4ꢀ
NHA, B), 7.88ꢀ7.95 (5H, m, H2’, A and B, 4ꢀNHb, B), 7.80 (1H, q, JNHꢀCH3 = 4.6, 6ꢀNH, B), 7.54
and 7.34 (2H, 4ꢀNH2, A), 6.98ꢀ7.10 (8H, m, 2ꢀNH2, A and B, H3’, A and B), 3.84 (5H, s, OCH3,
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A and B), 2.99 (3H, d, JCH3ꢀNH = 4.8, NHCH3, A), 2.97 (3H, d, JCH3ꢀNH = 4.6, NHCH3, B); C
NMR (DMFꢀd7, 230 K): 164.6 (C4, A), 163.8 (C2,A and C2, B), 162.8 (C6, B), 159.2 (C4’, B),
159.1 (C4’, A), 156.6 (C6, A), 155.4 (C4, B), 147.8 (C1’, B), 147.5 (C1’, A), 122.6 (C2’, A and
B), 114.1 (C3’, A and B), 109.7 (C5, A and B), 55.3 (OCH3, A and B), 27.4 (NHCH3, B), 26.5
(NHCH3, A); ESI MS, m/z (%): 274.1 [M + H]+; HRMS (ESI) calcd for C12H16N7O [M + H]+
274.1411, found 274.1411.
N4ꢀMethylꢀ5ꢀ(phenyldiazenyl)pyrimidineꢀ2,4,6ꢀ
triamine (2b). Treatment of 1b (75 mg, 0.3 mmol)
with methylamine (0.03 mL, 0.9 mmol) for 30 min
by procedure C gave 2b (20 mg, 27 %) as a yellow
solid, m. p. 183–187 °C. H NMR (DMFꢀd7, 230 K): 10.60 (1H, q, JNH,CH3= 4.9, 6ꢀNHa), 9.92
1
(1H, d, JGEM= 5.3, 4ꢀNHa, B), 8.02 (1H, d, JGEM= 5.3, 4ꢀNHb, B), 7.60ꢀ7.87 (5H, m, H2’, A and
B) 7.63 (1H, s, 4ꢀNHa, A), 7.42 (4H, m, H3’, A and B), 7.30 (1H, s, 4ꢀNHb, A), 7.28ꢀ7.22 (2H, m,
H4’, A and B), 7.21ꢀ7.10 (4H, m, 2ꢀNH2, A and B), 3.02 (3H, d, JCH3NH = 4.8, NHꢀCH3, A), 2.99
(3H, d, JCH3NH = 4.8, NHꢀCH3, B); 13C NMR (DMFꢀd7, 230 K): 165.6 (C4, A), 164.8 (C2, A and
B), 163.6 (C6, B), 156.4 (C6, A), 156.4 (C4, B), 154.3 (C1’, B), 154.2 (C1’, A), 129.9 (C3’ and
C5’, B), 129.8 (C3’ and C5’, A), 127.8 (C4’, B), 127.7 (C4’, A), 121.9 (C2’ and C6’, A and B),
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111.1 (C5, A and B), 28.1 (CH3NH, B), 27.1 (CH3NH, A); N NMR (DMFꢀd7, 230 K): 399.2
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(N1’, B), 391.8 (N1’, A), 92.0 (N4, B), 89.2 (N6, A), 78.6 (N4, A), 73.4 (N6, B); ESI MS, m/z
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(%): 244.1 [M + H]+; HRMS (ESI) calcd for C11H14N7 [M + H]+ 244.1305, found 244.1305.
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N4ꢀMethylꢀ5ꢀ(phenyldiazenylꢀ2ꢀ15N)pyrimidineꢀ
2,4,6ꢀtriamineꢀN4ꢀ15N2 (2b-15N3). Treatment of 1b-
15N2 (200 mg, 0.8 mmol) with methylamineꢀ15N
hydrochloride (109 mg, 1.6 mmol) for 1 h by
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procedure C gave 2b-15N3 (40 mg, 20 %) as a yellow solid, m. p. 178–181 °C. H NMR (DMFꢀ
d7, 230 K): 10.60 (1H, dp, JHꢀN = 92.9, JNHꢀCH3 = 4.7, 1h JHꢀN = 4.0, 6ꢀNH, A), 9.92 (1H, dm, JHꢀN
89.0, 4ꢀNHa, B), 8.13ꢀ7.87 (5H, m, H2’, A and B, 4ꢀNHB, B), 7.88 (1H, dq, JHꢀN = 95.3, JNHꢀCH3
=
=
4.8, 6ꢀNH, B), 7.61 (1H, dd, JHꢀN = 91.5, JGEM = 2.5, 4ꢀNHA, A) 7.46ꢀ7.22 (6H, m, H3’, A and B,
H4’, A and B, 4ꢀNHB, A), 7.18ꢀ7.08 (4H, m, 2ꢀNH2, A and B), 3.02 (3H, dd, JCH3ꢀNH = 4.9, JCH3ꢀN
= 1.2, CH3NH, A), 2.99 (2.1 dd, JCH3ꢀNH = 4.9, JCH3ꢀN = 1.2, CH3NH, B); 13C NMR (DMFꢀd7, 234
K): 165.6 (d, JCꢀN = 21.1, C4, A), 164.8 (t, 3JCꢀ6N = 3JCꢀ4N = 3.4, C2, A and B), 163.6 (m, C6, B),
156.4 (d, JCꢀN = 18.3, C6, A), 156.3 (d, JCꢀN = 18.0, C4, B), 154.4 (C1’, B), 154.2 (C1’, A), 129.9
(C3’, B), 129.9 (C3’, A), 127.8 (C4’, B), 127.7 (C4’, A), 121.9 (d, 2JCꢀN = 3.5, C2’, A), 111.1 (m,
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C5, A and B), 28.1 (d, JCꢀN = 11.9, CH3NH, B), 27.1 (d, JCꢀN = 10.9, CH3NH, A); N NMR
(DMFꢀd7, 230 K): 99.16 (d, 2JNꢀN = 6.3, N1’, B), 91.87 (d, 2JNꢀN = 7.5, N1’, A), 92.03 (td, JN.Ha
=
JNꢀHb = 90.8, 2hJNꢀN = 6.3, N4, B), 89.22 (t, JNꢀH = 91.1, N4, A), 73.45 (d, JNꢀH = 95.1, N6, B); ESI
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MS, m/z (%): 247.2 [M + H]+; HRMS (ESI) calcd for C11H14N4 N3 [M + H]+ 247.1216, found
247.1217.
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N4ꢀMethylꢀ5ꢀ((4ꢀnitrophenyl)diazenyl)pyrimidineꢀ
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2,4,6ꢀtriamine (2c). Treatment of 1c (200 mg, 0.7
mmol) with methylamine (0.08 mL, 2.1 mmol) for
10 min by procedure C gave 2c (80 mg, 40 %) as a
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red solid, m. p. > 300 °C. H NMR (DMSOꢀd6):
10.69 (1H, bs, 6ꢀNH, A), 9.87 (1H, bs, 4ꢀNHA, B), 8.18ꢀ8.22 (4H, m, H3’, A and H3’, B), 7.93ꢀ
8.03 (4H, m, H2’, A and H2’, B), 7.84 (1H, bs, 4ꢀNHB, B), 7.73 (1H, bs, 6ꢀNH, B), 7.36 and 7.04
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(2ꢀNH2, A), 2.99 (3H, bs, CH3NH, A), 2.94 (3H, bs, (CH3NH, B); C NMR (DMSOꢀd6): 164.6
(C4, A), 163.9ꢀ164.0 (m, C2, A and B), 162.6 (C6, B), 158.1 (C1’, A and B), 155.5 (C4, B),
155.4 (C6, A), 144.3 (C4’, A and B), 124.9 (C3’, A and B), 121.1 (C2’, A and B), 112.7 (C5, A
and B), 27.8 (CH3, B), 27.0 (CH3, A); ESI MS, m/z (%): 289.1 [M + H]+; HRMS (ESI) calcd for
C11H13N8O2 [M + H]+ 289.1156, found 289.1156.
Acknowledgment
The work has been supported by the Czech Science Foundation (grant no. 15ꢀ11223S).
Supporting Information
Synthesis of compound 2d, additional NMR spectra, additional figures and tables, 1H and 13C
spectra of newly prepared compounds, Cartesian coordinates of transitionꢀstate structures.
This material can be downloaded free of charge via the internet at http://pubs.acs.org.
References
1.
Hobza, P.; Havlas, Z. BlueꢀShifting Hydrogen Bonds. Chem. Rev. 2000, 100, 4253ꢀ4264.
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1
2
3
4
5
6
7
8
9
2.
2597ꢀ2625.
3. Grzesiek, S.; Cordier, F.; Jaravine, V.; Barfield, M. Insights into Biomolecular Hydrogen
Grabowski, S. J. What Is the Covalency of Hydrogen Bonding? Chem. Rev. 2011, 111,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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27
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Bonds from Hydrogen Bond Scalar Couplings. Prog. Nucl. Magn. Reson. Spectrosc. 2004, 45,
275ꢀ300.
4.
J. Med. Chem. 2010, 53, 2601ꢀ2611.
5. Furet, P.; Caravatti, G.; Guagnano, V.; Lang, M.; Meyer, T.; Schoepfer, J. Entry into a
Kuhn, B.; Mohr, P.; Stahl, M. Intramolecular Hydrogen Bonding in Medicinal Chemistry.
New Class of Protein Kinase Inhibitors by Pseudo Ring Design. Bioorg. Med. Chem. Lett. 2008,
18, 897ꢀ900.
6.
Hodge, C. N.; Pierce, J. A Diazine Heterocycle Replaces a 6ꢀMembered Hydrogenꢀ
Bonded Array in the ActiveꢀSite of Scytalone Dehydratase. Bioorg. Med. Chem. Lett. 1993, 3,
1605ꢀ1608.
7.
Furet, P.; Bold, G.; Hofmann, F.; Manley, P.; Meyer, T.; Altmann, K. H. Identification of
a New Chemical Class of Potent Angiogenesis Inhibitors Based on Conformational
Considerations and Database Searching. Bioorg. Med. Chem. Lett. 2003, 13, 2967ꢀ2971.
8.
Menear, K. A.; Adcock, C.; Alonso, F. C.; Blackburn, K.; Copsey, L.; Drzewiecki, J.;
Fundo, A.; Le Gall, A.; Gomez, S.; Javaid, H.; Lence, C. F.; Martin, N. M. B.; Mydlowski, C.;
Smith, G. C. M. Novel Alkoxybenzamide Inhibitors of Poly(ADPꢀRibose) Polymerase. Bioorg.
Med. Chem. Lett. 2008, 18, 3942ꢀ3945.
9.
Lord, A. M.; Mahon, M. F.; Lloyd, M. D.; Threadgill, M. D. Design, Synthesis, and
Evaluation in Vitro of Quinolineꢀ8ꢀCarboxamides, a New Class of Poly(AdenosineꢀDiphosphateꢀ
Ribose)Polymeraseꢀ1 (PARPꢀ1) Inhibitor. J. Med. Chem. 2009, 52, 868ꢀ877.
ACS Paragon Plus Environment

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Page 28 of 34
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2
3
4
10.
Osmialowski, B.; Kolehmainen, E.; Kowalska, M. 2ꢀAcylaminoꢀ6ꢀPyridones: Breaking of
5
6
7
8
an Intramolecular Hydrogen Bond by SelfꢀAssociation and Complexation with Double and Triple
Hydrogen Bonding Counterparts. Uncommon Steric Effect on Intermolecular Interactions. J.
Org. Chem. 2012, 77, 1653ꢀ1662.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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34
35
36
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11.
Hydrogen Bond in Polysubstituted 5ꢀNitrosopyrimidines. J. Org. Chem. 2013, 78, 10121ꢀ10133.
12. Olivella, M.; Marchal, A.; Nogueras, M.; Melguizo, M.; Lima, B.; Tapia, A.; Feresin, G.
Procházková, E.; Čechová, L.; Janeba, Z.; Dračínský, M. A Switchable Intramolecular
E.; Parravicini, O.; Giannini, F.; Andujar, S. A.; Cobo, J.; Enriz, R. D. A New Series of
Antibacterial Nitrosopyrimidines: Synthesis and StructureꢀActivity Relationship. Arch. Pharm.
2015, 348, 68ꢀ80.
13.
IllánꢀCabeza, N. A.; GarcíaꢀGarcía, A. R.; MorenoꢀCarretero, M. N. Theoretical
Investigations on the Structure and Relative Stabilities of the Tautomeric Forms of 6ꢀAminoꢀ5ꢀ
Nitrosouracil and Violuric Acid Derivatives (PM3ꢀCosmo Calculation). Comput. Theor. Chem.
2011, 964, 83ꢀ90.
14.
IllánꢀCabeza, N. A.; GarcíaꢀGarcía, A. R.; MorenoꢀCarretero, M. N. Complexes with 6ꢀ
Aminoꢀ5ꢀNitrosoꢀ2ꢀThiouracil and Violuric Acid Derivatives Containing the facꢀReI(CO)3 Core:
Synthesis, XRD Structural and Photoluminescence Characterization. Inorg. Chim. Acta 2011,
366, 262ꢀ267.
15.
Čechová, L.; Procházková, E.; Císařová, I.; Dračínský, M.; Janeba, Z. Separation of
Planar Rotamers through Intramolecular Hydrogen Bonding in Polysubstituted 5ꢀ
Nitrosopyrimidines. Chem. Commun. 2014, 50, 14892ꢀ14895.
16.
Bolvig, S.; Hansen, P. E. Isotope Effects on Chemical Shifts as an Analytical Tool in
Structural Studies of Intramolecular Hydrogen Bonded Compounds. Curr. Org. Chem. 2000, 4,
19ꢀ54.
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2
3
17.
Sobczyk, L.; Grabowski, S. J.; Krygowski, T. M. Interrelation between HꢀBond and Piꢀ
4
5
6
Electron Delocalization. Chem. Rev. 2005, 105, 3513ꢀ3560.
7
8
9
18.
Hydrogen Bonds in Crystals. Russ. Chem. Bull. 2006, 55, 1ꢀ15.
19. Dračínský, M.; Čechová, L.; Hodgkinson, P.; Procházková, E.; Janeba, Z. Resonanceꢀ
Lyssenko, K. A.; Antipin, M. V. The Nature and Energy Characteristics of Intramolecular
10
11
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51
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53
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55
56
57
58
59
60
Assisted Stabilisation of Hydrogen Bonds Probed by NMR Spectroscopy and Path Integral
Molecular Dynamics. Chem. Commun. 2015, 51, 13986ꢀ13989.
20.
Interactions in 5ꢀNitrosopyrimidines. J. Org. Chem. 2016, 81, 3780ꢀ3789.
21. Dračínský, M. The Chemical Bond: The Perspective of NMR Spectroscopy. Annu. Rep.
NMR Spectrosc. 2017, 90, 1ꢀ40.
22. Löhr, F.; Mayhew, S. G.; Ruterjans, H. Detection of Scalar Couplings across NHꢂꢂꢂOP and
OHꢂꢂꢂ OP Hydrogen Bonds in a Flavoprotein. J. Am. Chem. Soc. 2000, 122, 9289ꢀ9295.
Procházková, E.; Čechová, L.; Tarábek, J.; Janeba, Z.; Dračínský, M. Tunable PushꢀPull
23.
Dingley, A. J.; Grzesiek, S. Direct Observation of Hydrogen Bonds in Nucleic Acid Base
Pairs by Internucleotide 2JNN Couplings. J. Am. Chem. Soc. 1998, 120, 8293ꢀ8297.
24.
Pervushin, K.; Ono, A.; Fernandez, C.; Szyperski, T.; Kainosho, M.; Wüthrich, K. NMR
Scaler Couplings across WatsonꢀCrick Base Pair Hydrogen Bonds in DNA Observed by
Transverse Relaxation Optimized Spectroscopy. Proc. Natl. Acad. Sci. USA 1998, 95, 14147ꢀ
14151.
25.
Scalar Couplings in ProteinꢀNucleotide Complexes. J. Am. Chem. Soc. 2001, 123, 11079ꢀ11080.
26. Alkorta, I.; Elguero, J.; Denisov, G. S. A Review with Comprehensive Data on
Czernek, J.; Brüschweiler, R. Geometric Dependence of 3hJ(31P–15N) and 2hJ(31P–1H)
Experimental Indirect Scalar NMR SpinꢀSpin Coupling Constants across Hydrogen Bonds.
Magn. Reson. Chem. 2008, 46, 599ꢀ624.
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