Theoretical study on structural and mechanistic aspects of synthesis of a 3-aminopyrazole derivative

Article abstract of DOI:10.1016/j.tet.2010.05.093

The structure of 5-hydroxy-3,5-dimethyl-1-S-methylisothiocarbamoyl-2- pyrazolinium iodide (HDMCPI), a cyclic intermediate for a 3-aminopyrazole derivative, was determined by means of X-ray analysis and spectroscopic techniques. In a treatment of HDMCPI in alkaline aqueous solution, 4-acetyl-3(5)-amino-5(3)-methylpyrazole (AAMP) was unexpectedly yielded. The reaction of HDMCPI was monitored by ¹H and ¹³C NMR spectroscopy. It was shown that keto-imine tautomer appears as the only tautomeric form. Density functional theory explained the spontaneous formation of keto-imine tautomer, whose existence is the main condition for generating a carbanion in alkaline medium. The carbanion further undergoes cyclization and elimination of MeSH, thus yielding AAMP. In the reaction of acetylacetone with thiosemicarbazide instead of S-methylisothiosemicarbazide, there were no traces of AAMP. This result can be attributed to the absence of keto-imine form in the tautomeric equilibrium, which would provide the formation of a carbanion for a nucleophilic attack and further cyclization.

Full text of DOI:10.1016/j.tet.2010.05.093

Tetrahedron 66 (2010) 6205e6211
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Theoretical study on structural and mechanistic aspects of synthesis
of a 3-aminopyrazole derivative
a
a
,
b
c
a
Svetlana Markovi ꢀc , Milan D. Joksovi ꢀc , Petra Bombicz , Vukadin M. Leovac , Violeta Markovic ,
*
ꢀ
a
Ljubinka Joksoviꢀc
a
Faculty of Sciences, University of Kragujevac, R. Domanovi ꢀc a 12, 34000 Kragujevac, Serbia
Institute of Structural Chemistry, Chemical Research Center, Hungarian Academy of Sciences, PO Box 17, H-1525 Budapest, Hungary
Faculty of Sciences, University of Novi Sad, Trg D. Obradovi ꢀc a 3, 21000 Novi Sad, Serbia
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure of 5-hydroxy-3,5-dimethyl-1-S-methylisothiocarbamoyl-2-pyrazolinium iodide (HDMCPI),
Received 2 March 2010
Received in revised form 6 May 2010
Accepted 24 May 2010
a cyclic intermediate for a 3-aminopyrazole derivative, was determined by means of X-ray analysis and
spectroscopic techniques. In a treatment of HDMCPI in alkaline aqueous solution, 4-acetyl-3(5)-amino-5
1
(
3)-methylpyrazole (AAMP) was unexpectedly yielded. The reaction of HDMCPI was monitored by H and
Available online 4 June 2010
1
3
C NMR spectroscopy. It was shown that keto-imine tautomer appears as the only tautomeric form.
Density functional theory explained the spontaneous formation of keto-imine tautomer, whose existence
is the main condition for generating a carbanion in alkaline medium. The carbanion further undergoes
cyclization and elimination of MeSH, thus yielding AAMP. In the reaction of acetylacetone with thio-
semicarbazide instead of S-methylisothiosemicarbazide, there were no traces of AAMP. This result can be
attributed to the absence of keto-imine form in the tautomeric equilibrium, which would provide the
formation of a carbanion for a nucleophilic attack and further cyclization.
Keywords:
Crystal structure
Reaction mechanism
Keto-imine tautomer
DFT
3-Aminopyrazole
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Although the synthesis of 4-acetyl-3(5)-amino-5(3)-methyl-
11
pyrazole (AAMP) has been reported about two decades ago, the
recent discoveries in the field of 3-aminopyrazole derivatives and
their pharmacological and medicinal importance prompted us to
shed much more light on the mechanism of formation of AAMP,
starting from relatively simple precursors. In the present study we
have focused our attention on theoretical and mechanistical as-
pects of the synthesis of this 3-aminopyrazole derivative, as a high
versatile precursor for preparation of a number of potential kinase
inhibitors bearing in mind the great possibilities of transformation
of acetyl group linked to 3-aminopyrazole scaffold.
The chemistry of 3-aminopyrazole has remained largely
underdeveloped until the discovery of its derivatives as antitumor
agents with their great therapeutic potential against various
proliferative disorders in the role of chemical inhibitors of cyclin-
dependent kinases (CDKs), a family of enzymes involved in
1
e4
controlling normal cell proliferation.
The frequent deregulation
of cell cycle progression in cancer has intensified search for kinase
inhibitors with high affinity and specificity over the past several
years.5
e7
PHA-739358, a small molecule of 3-aminopyrazole
derivative with strong activity against Aurora kinases and cross-
reactivities with some receptor tyrosine kinases, exhibits
significant antitumor activity in a wide range of cancers and shows
2. Results and discussion
8
a favorable pharmacokinetic and safety profile. Recently, a series of
3
-aminopyrazole based Aurora kinase inhibitors with a pyrimidine
2.1. The crystal structure of 5-hydroxy-3,5-dimethyl-1-S-
scaffold led to a class of very potent inhibitors of cellular
proliferation in treatment of chronic myelogenous leukemia or
Philadelphia chromosome-positive acute lymphoblastic leukemia.
methylisothiocarbamoyl-2-pyrazolinium iodide (HDMCPI)
9
The crystal data, details of structure determination and
refinement of HDMCPI are given in the Supplementary data.
The iodide anion is hydrogen bonded to the organic moiety by the
O5eH5/I1 and N7eH7A/I1 interactions (Fig. 1a, Table 1). The
packing coefficient is 65.7%, there is no residual solvent accessible
void. The basic building unit of the crystal is a dimer organized by
the symmetry centre (Fig. 1b, Table 1). H7B is a bifurcated hydrogen
Very recently, a selective 3-aminopyrazole MK2 kinase inhibitors
were discovered and profiled to show potent inhibition MK2
10
activity and reasonable cellular activity.
*
Corresponding author. E-mail address: mjoksovic@kg.ac.rs (M.D. Joksovi ꢀc ).
0
040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.05.093

6206
S. Markovi ꢀc et al. / Tetrahedron 66 (2010) 6205e6211
atom, taking part in both intramolecular and intermolecular
12
hydrogen bonds to O5 oxygen atoms. The graph set description of
2
2
the ring formed by the hydrogen bonds is R (6). The neighboring
dimers are kept together by weak CeH/O interactions. A herring
bone arrangement can be observed from the crystallographic a di-
rection in the packing diagram (Supplementary data).
The structure of HDMCPI was reproduced using density func-
tional theory (DFT), in order to test the suitability of the applied
computational method. The obtained bond lengths, bond angles,
and dihedral angles are provided in Tables S5eS7 of Supplementary
data. The average relative errors for bond angles and dihedral an-
gles amount 0.89 and 0.45%, respectively. The lengths of the bonds
including hydrogen are overestimated, whereas the average rela-
tive error for bond lengths among heavy atoms equals 0.99%. Such
agreement between the experimental and computed structure of
HDMCPI confirms the applicability of the used DFT method.
2.2. Mechanistic studies
The synthesis of 4-acetyl-3(5)-amino-5(3)-methylpyrazole (AAMP)
starting from acetylacetone and S-methylisothiosemicarbazide
11
hydrogen iodide has been already described, but the intermediate
was incorrectly presented, only on the basis of elemental analysis, as
acetylacetone mono-S-methylisothiosemicarbazone. Now, we have
obtained single crystals suitable for X-ray analysis, and using spectro-
scopic methods completely characterized this compound as the cyclic
compound 5-hydroxy-3,5-dimethyl-1-S-methylisothiocarbamoyl-2-
pyrazolinium iodide (HDMCPI) (Scheme 1).
The values of bond lengths between the carbon and two nitro-
gens of S-methylisothiocarbamoyl group indicate delocalization of
positive charge through all three atoms in the solid state. In addi-
tion, the natural bond orbital (NBO) analysis of HDMCPI shows
strong donation of density from the p orbitals on N7 and S to the
p
*
0
.45
antibonding N1eC6 orbital, and from the sp
antibonding C6eN7 orbital, thus confirming the delocalization
in the S-methylisothiocarbamoyl group. However, in DMSO-d so-
eMe and
eMe) and one AB system for the pyrazoline ring protons, shows
orbital on S to the
s
*
6
1
lution, the H NMR spectra, except for two singlets (C
3
C
5
Figure 1. ORTEP representation13 of HDMCPI at 50% probability level, heteroatoms are
two singlets for SeMe protons, as well as two groups of signals at
lower field. The first, two broad singlets, one bigger at 7.73 ppm and
a smaller one at 7.52 ppm of the total relative intensity 1, can be
attributed to two kinds of hydroxyl protons. The second set of three
broad peaks, one bigger at 8.80 ppm and two smaller at 8.96 and
shaded. (b)The dimer formed in the structure of HDMCPI and the connected iodide
organized by the symmetry centre.14
Table 1
9
.42 ppm of the total relative intensity 2 can be assigned to the
nitrogen protons. Three smaller peaks were in an intensity ratio of
:1:1 while bigger signals at 8.80 ppm and 7.73 ppm were in an
intensity ratio 2:1, respectively. These facts suggest that not only
one form of HDMCPI was present in the DMSO-d solution. In order
Intermolecular interactions in the crystal structure of HDMCPI
DeH/A
Type
DeH H/A D/A
DeH/A Symmetry
1
ꢁ
ꢁ
ꢁ
ꢀ
[A]
[A]
[A]
[ ]
operation
N7eH7B/O5
O5eH5/I1
Intra
0.86 2.37 2.903 (3) 121
6
In asym 0.82 2.62 3.399 (3) 159
unit
1
to consider an explanation of this H NMR complexity, we must
take into an account the existence of other distinct tautomers with
possibly different NMR spectra whose concentration will vary with
selected deuterated solvent. For example, a proton could be moved
from the exocyclic nitrogen onto ring nitrogen atom giving
a structure with three chemically nonequivalent protons with the
same intensities (Scheme 2).
N7eH7A/I1
N7eH7B/O5
C51eH51A/I1 Inter
Inter
Inter
0.86 2.83 3.612 (3) 153
0.86 2.23 3.003 (3) 149
0.96 3.05 3.993 (4) 167
x, 1þy, z
1ꢁx, 1ꢁy, 1ꢁz
ꢁ1/2þx, 1/2ꢁy,
ꢁ
1/2þz
C8eH8A/I1 Inter
0.96 3.05 3.886 (5) 146
1/2ꢁx, 1/2þy,
/2ꢁz
3
Scheme 1. Synthesis of HDMCPI (I).

S. Markovi ꢀc et al. / Tetrahedron 66 (2010) 6205e6211
6207
Scheme 2. Two possible tautomeric forms of HDMCPI in DMSO-d
6
solution (the ratio of tautomers is determined by integration of 1H NMR signals).
Addition of D
2
O to the DMSO-d
6
solution caused the collapse of
respectively. The G298 values are in agreement with a simple
chemical reasoning, based on the fact that B and C are stabilized
with strong hydrogen bonds. On the basis of these facts, the pres-
ence of tautomer A is not expected as the only form in the tauto-
meric equilibrium.
the AB system to a singlet and disappearance of the NeH and the
OeH protons because of fast exchange of these protons with D O.
Simultaneously, one singlet attributable to the SeMe protons was
observed instead of two singlets in DMSO-d solution. Finally, in
pure D O solution all signals appeared only as singlets.
2
6
2
To explain this unexpected experimental finding, we examined
possible interactions between the HDMCP cation and hydroxide
anion. All examined pathways are here presented. Our inves-
tigation did not reveal any transition state for a reaction pathway
between the HDMCP cation and hydroxide anion. On the other
hand, our attempts to optimize a structure consisting of the HDMCP
cation and hydroxide anion resulted in a spontaneous transfer of
a proton (i.e., without activation barrier) from the hydroxyl group of
Dissolving HDMCPI in an alkaline aqueous solution (pH>13)
unexpectedly affords AAMP after about 15 h of standing at room
temperature, in the forms of white needle-like crystals. In order to
slow this reaction for H NMR monitoring, we dissolved HDMCPI in
2
D O and adjusted pD to 9.5 by careful addition of NaOD. Bearing in
mind that thiosemicarbazones of 1,3-dicarbonylic compounds are
complex multitautomeric systems, we could expect an equilibrium
mixture of several open-chain tautomeric forms in the solution, in-
cluding the cyclic one with predominance of keto-enamine form.
It has been known that the tautomeric equilibria are very sensitive to
1
ꢁ
the HDMCP cation to the OH anion. This rearrangement includes
15,16
a ring opening, and formation of the structure D (Fig. 2), with the
stabilization of the system of 143.2 kJ/mol. It is worth pointing out
that structure D is actually a hydrated tautomeric form A. The
tautomer A can be transformed into B via transition state TS1, re-
quiring an activation barrier of 185.0 kJ/mol, whereas B can rear-
range into C via transition state TS2, requiring an activation energy
17
1
pH and the polarity of the solvent. However, after analyzing the H
13
and C NMR spectra at pD¼9.5 we have observed surprisingly that
this compound is present in D O only in the form of keto-imine tau-
tomer A (Scheme 3). The presence of the keto-imine tautomer A in
2
Scheme 3. Tautomeric forms of 5-hydroxy-3,5-dimethyl-1-S-methylisothiocarbamoyl-2-pyrazolinium cation (HDMCP).
solution is unambiguously indicated by the 13C NMR spectrum, in
which a resonance signal at the high value of 214.3 ppm is in the
of 14.8 kJ/mol (Fig. 2). A reaction path for a transformation of A into
C was not revealed. One can conclude, on the basis of the presented
activation energies, that the only pathway for transformation of the
HDMCP cation in alkaline medium is the formation of the tautomer
A. In this way the presence of A as the only form in tautomeric
equilibrium is explained.
d
range characteristic of aliphatic ketones, not that of keto-enamines or
enol-imine forms.18 Further evidences, confirming the structure of A
result from a systematic pattern of isomer peaks of the methyl group
typical for S-alkylated thiosemicarbazones due to cis/trans isomerism
19
13
from the C]N double bond. The C NMR spectrum also has com-
Our investigation shows that in alkaline medium tautomer A can
undergo pathways that lead to the formation of carbanions. Namely,
the acidic hydrogens of A can delocalize the charge of the hydroxide
anion, forming structure E in Scheme 4. Three reaction pathways
wererevealed for thetransformation of E, wherethe carbanions CA1,
CA2, and CA3 wereformed. The activation energies for the formation
of CA1, CA2, and CA3 amount 4.9, 8.8, and 31.3 kJ/mol, respectively.
plex broad signal at 50.1 ppm corresponding to the carbon between
1
13
carbonylandiminogroup. Otherdetailsof the Hand CNMRspectra
agree with keto-imine tautomeric form.
This finding motivated us to investigate the mechanism of the
formation of A from the HDMCP cation using DFT calculations. If
298
Gibbs free energy (G ) is taken as a measure of tautomer stability,
it turns out that A is less stable than B and C by 18.1 and 2.8 kJ/mol,
2
98
As expected, G
of CA2 is lower than those of CA1 and CA3 by 27.1

6208
S. Markovi ꢀc et al. / Tetrahedron 66 (2010) 6205e6211
Figure 2. Optimized structures of hydrated tautomer A (D), and transition states TS1 and TS2.
Scheme 4. Mechanism of formation of carbanions CA1, CA2, and CA3, via transition states TS3, TS4, and TS5, respectively.
and 61.3 kJ/mol, respectively. Taking into account low activation
energy and pronounced stability of CA2, we assume that the main
reaction path for the transformation of A is the formation of CA2.
Thus, the existence of tautomeric form A is the main condition for
generating the carbanion CA2 in alkaline medium.
The natural resonance analysis of CA2 revealed the possibility of
its resonance stabilization. The major resonance structures of CA2
are presented in Scheme 5. The numbers below the resonance
structures denote the resonance weights in the equilibrium geom-
etry of CA2. It is obvious that the resonance structure with the 25.3%

S. Markovi ꢀc et al. / Tetrahedron 66 (2010) 6205e6211
6209
Scheme 5. Possible mechanism of formation of AAMP.
resonance weight dominates in the resonance hybrid, thus pro-
viding the conditions for nucleophilic attack and further cyclization.
The carbanion structure (that with the highest resonance
weight) gives the possibility for intramolecular nucleophilic attack
on the very electrophilic carbon of the C]N group, leading to the
formation of the corresponding cyclic pyrazoline compound. This
step of the reaction proceeds via transition state TS6 (Scheme 5),
and requires activation barrier of 115.7 kJ/mol. The elimination of
MeSH takes place easily in an alkaline medium, followed by an
unpleasant mercaptane odor, yielding the tautomeric equilibrium
carbon atom of the conjugated carbonyl group at 183.04 ppm.
18,21
These data agree with the spectra of many enamines.
It is
evident that the absence of the keto-imine form (F) in the tauto-
meric equilibrium does not allow any possibility of the formation
of a carbanion for a nucleophilic attack and further cyclization.
Also, we detected 3,5-dimethylpyrazole-1-thiocarboxamide as
a consequence of the dehydration of pyrazolinol compound. The
structure of this compound was confirmed by comparison with
authentic sample. It is notable that cyclic pyrazolinol (Scheme 6) is
a
very unstable compound even in the solid state. Its
2
0
of corresponding pyrazoles.
In order to confirm our hypothesis about the crutial role of
keto-imine tautomer, we repeated the same experiment with
decomposition begins after several hours and all material was
transformed into 3,5-dimethylpyrazole-1-thiocarboxamide after
two weeks of standing.
Scheme 6. Tautomeric forms of 5-hydroxy-3,5-dimethyl-1-thiocarbamoyl-2-pyrazoline. The ratio of tautomer (E), 3,5-dimethylpyrazole-1-thiocarboxamide and starting pyrazoline
compound was determined by integration of 1H NMR signals of the methyl groups.
thiosemicarbazide instead of S-methylisothiosemicarbazide in the
3. Conclusion
reaction with acetylacetone preparing its cyclic analogue 5-hydroxy-
3
,5-dimethyl-1-thiocarbamoyl-2-pyrazoline (Scheme 6) according to
The agreement between experimentally obtained and calcu-
lated bond distances, bond angles, and torsion angles of 5-hydroxy-
3,5-dimethyl-1-S-methylisothiocarbamoyl-2-pyrazolinium iodide
(HDMCPI) is very good, confirming that the selected computational
method is suitable for investigating the reactions of HDMCPI. In this
work, the transformation of 5-hydroxy-3,5-dimethyl-1-S-methyl-
isothiocarbamoyl-2-pyrazolinium cation (HDMCP) in alkaline
aqueous solution was investigated using experimental and com-
putational methods.
15
the known procedure.
After 15 h of standing in highly alkaline solution (pH>13) there
were no traces of AAMP. Then we adjusted pD at 9.5, and the
1
almost quantitatively formed white solid was analyzed by H and
13
C NMR in the same conditions for comparison with S-methylated
derivative. We established that the main form in the state of the
equilibrium in DMSO-d solution is the starting cyclic compound
Scheme 6) The keto-enamine form (G) in the NMR spectrum of
the mixture was presented as vinyl proton signal at 6.48 ppm as
well as the -carbon of the vinyl group at 84.98 and a signal for the
6
(
It was shown that the reaction between the HDMCP cation and
hydroxyde anion proceeds smoothly, without activation barrier,
a

6210
S. Markovi ꢀc et al. / Tetrahedron 66 (2010) 6205e6211
and with significant stabilization of the system. In this way the
hydrated keto-imine tautomer A is formed, at 100% yield. The ac-
tivation energy for the isomerization of A into keto-enamine tau-
tomer B is significantly higher in comparison to the formation of
carbanion CA2 in alkaline medium. CA2 undergoes further cycli-
zation and elimination of MeSH, thus yielding 4-acetyl-3(5)-amino-
4.3. X-ray data collection and structure refinement
A light yellow prism shape single crystal of the compound 5-
hydroxy-3,5-dimethyl-1-S-methylisothiocarbamoyl-2-pyrazolinium
iodide (HDMCPI) was selected and mounted on glass fiber. Dif-
fraction data were collected on an OXFORD DIFFRACTION KM4 four-
5
(3)-methylpyrazole (AAMP).
The absence of AAMP as a product of the reaction between
circle goniometer equipped with SAPPHIRE CCD detector using
ꢁ
graphite monochromated Mo K
a
X-radiation (
l
¼0.71073 A) at 293
ꢀ
acetylacetone with thiosemicarbazide instead of S-methyl-
isothiosemicarbazide can be explained with the absence of keto-
imine form in the tautomeric equilibrium, which would allow
the formation of a carbanion for further cyclization. This fact con-
firms the crucial role of the keto-imine tautomer A in the formation
of AAMP.
(2) K in the range 3.45 ꢂ
were 45.0 mm. Crystal data:
0.69ꢃ0.37ꢃ0.23 mm, monoclinic, space group P2
qꢂ26.37 . The crystals to detector distances
C
7
H
14IN
3
OS, Fwt.: 315.17, size:
/n, a¼12.204
1
ꢁ
ꢁ
ꢁ
ꢀ
ꢁ
3
(5) A b¼6.417(5) A, c¼15.995(5) A,
b
¼110.402(5) , V¼1174(1) A ,
ꢁ
3
ꢁ1
T¼293(2) K, Z¼4, F(000)¼616, D
x
¼1.783 Mg m
,
m¼2.876 mm
.
Cell parameters were determined by least-squares of the setting
ꢀ
angles of 5004 (3.05ꢂ
q
ꢂ29.1 ) reflections. A total of 4625 re-
flections were collected of which 2366 were unique [R(int)¼0.0118,
R( (I). Completeness to
)¼0.0183]; 1998 reflections were >2
¼0.989. The data were reduced using the Oxford Diffraction
4
4
. Experimental section
s
s
2q
.1. General methods
pro 22
program CrysAlis
based upon the intensities of equivalent reflections was applied
the minimum and maximum transmission factors were 0.53295
.
A semiempirical absorption-correction
IR spectrum was recorded on a PerkineElmer Spectrum One
(
1
13
FT-IR spectrometer with a KBr disc. H and C NMR spectra were
obtained using a Varian Gemini 200 spectrometer. Melting point
was determined on a Mel-Temp capillary melting points apparatus,
model 1001.
and 1.000), and the data were corrected for Lorentz, polarization,
and background effects. Neutral atomic scattering factors, together
with anomalous-dispersion corrections, were taken from the In-
2
3
ternational Tables for X-ray Crystallography. The structures were
solved by direct methods (SIR-92) using program package
2
4
25
4
.2. Procedure for the preparation of HDMCPI
WinGX, and the figures were drawn using ORTEP and MER-
14
CURY. Anisotropic full-matrix least-squares refinement (SHEXL-
2
6
2
4
.2.1. 5-Hydroxy-3,5-dimethyl-1-S-methylisothiocarbamoyl-2-pyr-
97) on F for all non-hydrogen atoms yielded R1¼0.0199 and
azolinium iodide (HDMCPI). A mixture of thiosemicarbazide (9.10 g,
00 mmol) and methyl iodide (15.05 g, 106 mmol) in absolute
wR2¼0.0447 for 1998 [I>2 (I)] and R1¼0.0275 and wR2¼0.0464
s
1
for all (2366) intensity data (goodness-of-fit¼1.064; the maximum
and mean shift/esd 0.002 and 0.000). Number of parameters¼122.
The maximum and minimum residual electron density in the final
ethanol (40 mL) was refluxed for 45 min. After 12 h, the separated
white crystals of S-methylisothiosemicarbazide hydrogen iodide
were filtered, washed with ethanol and used in the next step
without further purification. Yield: 15.1 g (65%). A mixture of S-
methylisothiosemicarbazide hydrogen iodide (11.65 g, 50 mmol)
and acetylacetone (19.50 g, 195 mmol) in absolute ethanol (20 mL)
was refluxed for 5 min. After cooling to room temperature, the
crystallization was induced by rubbing the walls of the vessel with
a glass stick. The light yellow crystals were filtered, washed three
ꢁ
ꢁ3
difference map was 0.302 and ꢁ0.385 e A . All hydrogen atomic
positions could be located in the difference maps. The final re-
finement included atomic positional and displacement parameters
for all non-H atoms. The isotropic displacement parameters of the
hydrogen atoms were approximated from the U(eq) value of the
atom they were bonded. The weighting schemes applied was w¼1/
2
2
2
2
2
[s
(F
o
)þ(0.0247P) þ0.00648P] in 3, where P¼(F
o
þ2F
c
)/3.
times with 5 mL of ether, and dried in air. Yield: 10.0 g (63%). Mp
Crystallographic data (excluding structure factors) have been
ꢀ
157 C. The suitable single crystals of HDMCPI for X-ray analysis
deposited with the Cambridge Crystallographic Data Centre as
supplementary publication number CCDC 755236. Copies of this
information may be obtained free of charge from The Director,
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: þ44 1223
336033; e-mail: deposit@ccdc.cam.ac.uk or www: http://www.
ccdc.cam.ac.uk).
were obtained by recrystallization from ethanol.
4
.2.1.1. Spectral data for HDMCPI. IR (KBr, cmꢁ1): 3331, 3266,
3191, 3161, 3132, 2980, 1635, 1629, 1557, 1427, 1372, 1328, 1254,
1
1113, 733;
H
NMR (200 MHz, DMSO-d
eCeOH), 2.04 (3H, s, CH eC]N), 2.55 and 2.62 (3H, two s,
eS), ¼3.14 (2H, AB system, JAB 19.7 Hz, CH ),
¼3.24 and
6
): d 1.68 (3H, s,
CH
CH
3
3
3
d
A
d
B
2
7.52 and 7.73 (1H, two br s, OH), 8.80, 8.96, and 9.42 (2H, three br
4.4. Computational methods
13
s, nitrogen protons); C NMR (DMSO-d
6
):
eCeOH), 52.8 (C-4), 96.2 (C-5), 164.9
eC(N)eS), 165.3 (C-3).
3
d 14.4 (CH eS), 16.7
(
(
CH
NH
3
eC]N), 24.7 (CH
3
All calculations were carried out using the Gaussian 03 pro-
2
7
28e30
2
gram using the B3LYP functional.
The 6-311þþG(d,p) basis
set was used for C, O, N, S, and H, whereas 6-311G(d,p) was
employed for I. The geometrical parameters of all stationary points
4
.2.1.2. NMR data for HDMCPI in D
1.83 (3H, s, CH eCeOH), 2.13 (3H, s, CH
CH eS), 3.32 (2H, s, CH
eC¼N), 26.8 (CH eCeOH), 55.2 (C-4), 98.9 (C-5), 168.5 (NH
N)eS), 168.7 (C-3).
2
O. 1H NMR (200 MHz, D
eC]N), 2.65 (3H, s,
16.3 (CH eS), 18.5
eC
2
O):
d
3
3
and transition states were optimized in water (
e
¼78.36), using the
13
31,32
3
2
); C NMR (D
2
O):
d
3
conductor-like solvation model (CPCM).
All calculated struc-
(CH
3
3
2
tures were confirmed to be local minima (all positive eigenvalues)
for ground state structures, or first-order saddle points (one
negative eigenvalue) for transition state structures, by frequency
calculations. The intrinsic reaction coordinates (IRCs), from the
transition states down to the two lower energy structures, were
traced using the IRC routine in Gaussian in order to verify that each
saddle point is linked with two putative minima. The results of the
IRC calculations for two crucial transition states, TS4 and TS6, are
presented in Figs S2 and S3 of Supplementary data. Evolution of
(
4
.2.1.3. NMR data for HDMCPI at pD¼9.5 (tautomeric form A). 1H
2 3
NMR (200 MHz, NaOD/D O): d 1.95 and 2.07 (3H, two s, CH eC]N),
2
.17 and 2.30 (3H, two s, CH
3
eCO), 2.38 and 2.44 (3H, two s,
15.2 and 15.4 (CH eS), 19.9 and
eC]N), 31.2 and 32.6 (CH eCO), 50.1 (complex signal,
), 163.4 (CH eC]N), 163.8 (NH eC(N)eS), 214.3 (CO).
CH
2
CH
3
eS); 13C NMR (NaOD/D
2
O):
d
3
4.8 (CH
3
3
2
3
2

S. Markovi ꢀc et al. / Tetrahedron 66 (2010) 6205e6211
6211
relevant bonds along the reaction pathway was estimated using the
natural bond orbital analysis.
S.; Pierard, F.; Pinder, J.; Pollard, J.; Ramaya, S.; Robinson, D.; Rutherford, A.;
Studley, J.; Westcott, J. Bioorg. Med. Chem. Lett. 2009, 19, 3586e3592.
0. Velcicky, J.; Feifel, R.; Hawtin, S.; Heng, R.; Huppertz, C.; Koch, G.; Kroemer, M.;
3
3
1
Moebitz, H.; Revesz, L.; Scheufler, C.; Schlapbach, A. Bioorg. Med. Chem. Lett.
2
009, , doi:10.1016/j.bmcl.2009.10.138
Acknowledgements
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Theworkwas financed bythe Ministry for Science of the Republic
of Serbia (Grants N 142028 and 142025) and the Provincial Secre-
tariat for Science and Technological Development of Vojvodina.
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