Utilization of DmbNHNH2 in the synthesis of amino-substituted 4-((3,5-diamino-1H-pyrazol-4-yl)diazenyl)phenols

Article abstract of DOI:10.1016/j.tet.2012.03.063

(2,4-Dimethoxybenzyl)hydrazine (DmbNHNH₂) was utilized in the synthesis of Dmb-protected 4-((3,5-diamino-1H-pyrazol-4-yl)diazenyl)phenols. This unambiguous protection of the pyrazole endocyclic nitrogen atom enabled the preparation of amino-sub

Full text of DOI:10.1016/j.tet.2012.03.063

Tetrahedron 68 (2012) 3996e4002
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Utilization of DmbNHNH2 in the synthesis of amino-substituted 4-((3,5-diamino-
1H-pyrazol-4-yl)diazenyl)phenols
b
c
d
b
€
ꢀ a
ꢁ
ꢁ
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Eva Schutznerova , Igor Popa , Vladimír Krystof , Hiroyuki Koshino , Zdenek Travnícek ,
a
,
ꢁa
*
Pavel Hradil , Petr Cankar
a
ꢀ
Department of Organic Chemistry, Faculty of Science, Palacky University, 17. listopadu 1192/12, 771 46 Olomouc, Czech Republic
Regional Centre of Advanced Technologies and Materials, Department of Inorganic Chemistry, Faculty of Science, Palacky University, 17. listopadu 1192/12, 771 46 Olomouc,
b
ꢀ
Czech Republic
c
ꢁ
ꢀ
ꢂ
Laboratory of Growth Regulators, Institute of Experimental Botany AS CR & Palacky University, Slechtitelu 11, 783 71 Olomouc, Czech Republic
d Molecular Characterization Team, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
(2,4-Dimethoxybenzyl)hydrazine (DmbNHNH2) was utilized in the synthesis of Dmb-protected 4-((3,5-
diamino-1H-pyrazol-4-yl)diazenyl)phenols. This unambiguous protection of the pyrazole endocyclic
nitrogen atom enabled the preparation of amino-substituted 4-((3,5-diamino-1H-pyrazol-4yl)diazenyl)
phenols that were inaccessible through direct substitution. The Boc group could be selectively cleaved in
the presence of the Dmb group.
Received 28 November 2011
Received in revised form 27 February 2012
Accepted 19 March 2012
Available online 28 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
(2,4-Dimethoxybenzyl)hydrazine
Dmb-protecting group
Nitrogen heterocycles
Regioselective acylation
CDK inhibitors
1. Introduction
Protein kinases are the most important drug targets in the
pharmaceutical industry for the treatment of proliferative disor-
ders, such as cancer and inflammation.1 A few years ago, derivatives
of a 4-(phenyldiazenyl)-1H-pyrazole-3,5-diamine core were iden-
tified as ATP-competitive inhibitors of the cyclin-dependent ki-
nases CDK2 and CDK9, which control cell proliferation and
transcription, respectively.1b,2 The X-ray structural analysis of CDK2
complexed with 4-((3,5-diamino-1H-pyrazol-4-yl)diazenyl)phenol
1 (CAN508, Scheme 1)3 and the recently published structural data
for CDK9 provide useful information about the structural sur-
roundings of the enzyme active site.2 It was believed that modifi-
cation of the amino groups in 1 could provide derivatives with
biological activity on the same level as previously reported.3
Scheme 1. Acylation of the unprotected pyrazole 1.
regioselective substitution.4 We first attempted to carry out a ben-
zoylation of the unprotected pyrazole, 1, with benzoyl chloride in
pyridine (Scheme 1). Unexpectedly, the benzoylation proceeded
entirely on the endocyclic nitrogen atom in the pyrazole ring,
affording derivative 2a. This result led us to attempt to protect the
endocyclic nitrogen atom where benzoylation occurred using a Boc
group based on reports of the successful Boc protection of the
endocyclic nitrogen atom in related aminopyrazole systems.5 Sev-
eral attempts to unequivocally obtain 15a, a regioisomer of 2a,
through the benzoylation and subsequent deprotection of the
Boc-protected pyrazole 1 formed a mixture of regioisomers, which
2. Results and discussion
The amino groups attached to the pyrazole ring are engaged in
a
relatively complex tautomeric system that complicates
ꢁ
* Corresponding author. E-mail address: cankar@orgchem.upol.cz (P. Cankar).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.03.063

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again included 2a. The Boc protection of 1 proved to be non-
regioselective.
These preliminary experiences indicated that the substitutions
on the 3,5-diaminopyrazole moiety were strongly influenced by the
nature of the reactants and other reaction conditions. To avoid
problems with regioselectivity, a different strategy was chosen. We
decided to introduce the protecting group earlier in the synthesis
via the cycloaddition of a hydrazine derivative, already bearing the
protecting group, to hydrazone 3 (Scheme 2), which forms pyr-
azoles unequivocally protected at the 1 position. The choice of the
protecting group was limited to those that both form a pyrazole
ring during the cycloaddition reaction and cleave under acidic
conditions. Because a cycloaddition reaction of tert-butyl hydrazi-
necarboxylate with a hydrazone similar to 3 has not been reported6
and our initial attempt failed, we turned our attention to (4-
methoxybenzyl)hydrazine (PMBNHNH2),7 which was more con-
venient for cycloaddition with 3 (Scheme 2).
Scheme 3. Synthesis of DmbNHNH2 10.
In this communication, we report the synthesis of both 10 and
several Dmb-protected 4-((3,5-diamino-1H-pyrazol-4-yl)diazenyl)
phenol derivatives. Furthermore, a preliminary study on the acyl-
ation reactions of both Dmb-protected and unprotected 3,5-
diaminopyrazole moieties is presented and illustrates the re-
activity of these heterocyclic systems.
First, it was necessary to develop a synthesis for 10. After initial
trials, an unoptimized three-step synthesis (Scheme 3) starting
from 2,4-dimethoxybenzaldehyde, 7, was found to be expedient.
It is possible to prepare 10 without first isolating the hydrazone,
8, formed by the condensation with acetohydrazide. Subsequent
catalytic hydrogenation forms a hydrazine derivative, 9, that is di-
rectly treated with hydrazine hydrate to yield the desired product,
10, as a free base. Because of the low stability of the free base,
conversion into the corresponding hydrochloride salt is necessary
for storage. Alternatively, crude 10 could be immediately used in
cycloaddition reaction with 3 to form a stable pyrazole, 11,
(Scheme 4).
Scheme 2. Cleavage study of the PMB protecting group.
A PMB-protected pyrazole derivative, 4, was di-benzoylated to
afford the amino-substituted pyrazole, 5, which was studied by
HPLC-MS to determine the cleavage conditions of the PMB group.
The protecting group was almost entirely cleaved by neat TFA after
48 h at 60 ꢀC. Although insufficient for complete deprotection, the
elevated temperature and prolonged reaction time yielded not only
the expected deprotected pyrazole, 6, but also an almost equal
amount of an undesired trifluoroacetylated impurity. Since the
reaction led to a mixture the products were not isolated but only
detected via HPLC-MS. This result indicates that another methoxy
group, this time in the ortho position of the protective group, was
needed to both promote cleavage under milder conditions and
eliminate the formation of impurities.
The 2,4-dimethoxybenzyl protecting group (Dmb) is generally
introduced to a hydroxy group via a reactive benzyl derivative, such
as the chloride8 or trichloroacetimidate.9 Although Dmb-N-
protection via both reactive benzyl derivatives10 and a Mitsunobu
reaction using 2,4-dimethoxybenzyl alcohol is also possible,11 the
predominantly used protection strategy is the reductive alkylation
of 2,4-dimethoxybenzaldehyde.12 Another common method for the
regioselective introduction of Dmb is the use of 2,4-
dimethoxybenzylamine for the synthesis of N-protected function-
alities13 or heterocyclic systems.14 Bis(2,4-dimethoxybenzyl)amine
was similarly applied during the synthesis of bis(Dmb)-N-protected
functionalities.15 To unequivocally prepare Dmb-protected 3,5-
diaminopyrazole derivatives, we searched the literature for a syn-
thesis of DmbNHNH2, 10 (Scheme 3). To the best of our knowledge,
neither a preparation procedure nor the compound itself has been
reported, although some commercial companies offer it for sale.
Scheme 4. Synthesis of the Dmb-protected pyrazole 11 and its amino-substituted
derivatives.
Before the Dmb-protected pyrazole 11 can be mono-acylated
with acyl chloride, it is necessary to protect the hydroxy group,
which tends to acylate in pyridine along with the amino group. To
this end, di-tert-butyl dicarbonate was used to give pyrazole 12.
Then, the 3-amino group of pyrazole 12 was acylated in pyridine
with the corresponding acyl chloride to afford pyrazoles 13aee in
high yield. The positions of both the acyl and protecting groups
were unequivocally confirmed by the X-ray structure analysis of
13b (Fig. 1).
The direct synthesis of the desired unprotected amino-acylated
pyrazoles, 15aee, from 13aee required the use of neat TFA. Four
hours at room temperature is sufficient to remove both acid-labile
groups. It is also possible to selectively cleave the Boc group from

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the acylation of the exocyclic amino group increased the inhibitory
potency of all tested compounds. Inhibitor 15a was the most active
with a 2- and 3-fold increase in potency over 1 for CDK2 and MCF7,
respectively. These results were expected from the accepted CDK
pharmacophore model, which accounts for hydrophobic sub-
stituents in positions analogous to those in this study.1b
3. Conclusions
In conclusion, the protection of the endocyclic pyrazole nitrogen
atom with Dmb was found to be crucial for obtaining the amino-
acylated pyrazoles 15aee, and a novel, unambiguous, protection
strategy utilizing DmbNHNH2, 10, was developed for this purpose.
It was necessary to protect the hydroxy group with Boc to prevent
the formation of a bis-acylated pyrazoles when the Dmb-protected
pyrazole 11 was acylated. Treatment of the pyrazoles 13aee with
dilute TFA selectively cleaved the Boc group in presence of the acid-
labile Dmb group. Acylation of the unprotected pyrazole 1 yielded
only pyrazoles 2aee. This report indicates that 10 could potentially
be used to protect related heterocyclic systems and possibly even
hydrazine functionalities.
4. Experimental section
4.1. General
All starting materials are commercially available. Commercial
reagents were used without purification. Melting points were de-
termined on a Boetius stage and are uncorrected. The infrared (IR)
spectra were obtained on an ATI Mattson Genesis Series FT-IR
spectrometer (KBr discs). Flash column chromatography was per-
ꢂ
formed on silica gel (pore size 60 A, 40e63
mm particle size). The
LC/MS analyses were carried out on a UHPLC-MS system consisting
of UHPLC chromatography Accela with photodiode array detector
and triple quadrupole mass spectrometer TSQ Quantum Access
(both Thermo Scientific, CA, USA), using Nucleodur Gravity C18
Fig. 1. X-ray structure of 13b (CCDC 837640).
13aee using dilute TFA at room temperature. After 1 h, pyrazoles
14aee were isolated and dissolved in neat TFA to yield 15aee after
several hours, as expected.
Finally, several other acyl derivatives of 1 (Scheme 1) were in-
vestigated, and it was found that only the endocyclic nitrogen could
be acylated, which formed the pyrazoles 2bee.
column at 30 ꢀC and flow rate of 800
2.6
mL/min (Kinetex, Phenomenex,
m
m, 2.1ꢁ50 mm, USA). Mobile phase was (A) 0.01 M ammonium
acetate in water, and (B) acetonitrile, linearly programmed from
10% to 80% B over 2.5 min, kept for 1.5 min. The column was re-
equilibrated with a 10% of solution B for 1 min. The APCI source
13C NMR spectra of most prepared pyrazoles can evince some
discrepancies. Some pyrazole quaternary carbon signals were not
observed. These pyrazoles exist in tautomeric forms with slow
exchange on the NMR time scale.7,16 As a result, the signal shape is
very broadened, the intensity is very low. Usually, these signals are
difficult to be identified.
operated at discharge current of 5 mA, vaporizer temperature of
400 ꢀC, and capillary temperature of 200 ꢀC. The HRMS analyses
were carried out on HRMSdExactive (Orbitrap) MS, Thermo, USA.
The purification of compounds 13e and 15e was carried out on
semi-preparative chromatograph (Agilent Technologies, 1200 Se-
ries) using SunFire Prep C18 OBD column 19ꢁ100 mm, 5
mm par-
ticles, gradient was formed from MeCN and water both HPLC grade,
flow rate 15 mL/min. The 1H and 13C NMR spectra of compounds
2aee, 4, 5, 10, 13a, 13d, 13e, 14aee, 15aee were measured in DMSO-
d6 at 20 ꢀC on a Bruker Avance 300 FT NMR spectrometer. The 1H
and 13C NMR spectra of compounds 12, 13b, 13c were measured in
DMF-d7 at 25 and 65 ꢀC on a Varian 400 FT NMR spectrometer. Rf
values were determined only for pyrazoles, which were purified by
chromatography. A single crystal X-ray data collection of 13b was
performed on an XcaliburÔ2 diffractometer (Oxford Diffraction
The biological activity of selected pyrazoles was assayed as
previously described.3 Based on the binding mode of 1 within the
CDK2 active site,3 the substitution of a key CDK-interacting motif in
2a and 2e substantially decreased their activity (Table 1). However,
Table 1
Inhibitory activity on recombinant CDK2/cyclin E kinase and cytotoxicity against the
MCF7 breast carcinoma cell line by selected pyrazoles
Ltd.) with Sapphire2 CCD detector, and with Mo K
a (Mono-
chromator Enhance, Oxford Diffraction Ltd.) and at 100 K. Addi-
tional details regarding structure determination, such as crystal
data and structure refinement, selected bond lengths and angles of
covalent as well as non-covalent contacts are summarized in
Supplementary data.
Compound
IC50
(
m
M)
CDK2
MCF7
1 (CAN508)
2a
2e
15a
15b
15c
15e
3.5 ꢂ 0.7
23 ꢂ 7.2
100 ꢂ 16
1.7 ꢂ 0.5
5.2 ꢂ 1.3
4.3 ꢂ 1.4
2.6 ꢂ 0.7
33 ꢂ 5
>25
70 ꢂ 11
10.5 ꢂ 4.2
>25
>12.5
13.9 ꢂ 3.3
4.1.1. General procedure for acylation of 4-((3,5-diamino-1H-pyrazol-
4-yl)diazenyl)phenol. To a solution of 4-((3,5-diamino-1H-pyrazol-
4-yl)diazenyl)phenol 1 (218 mg, 1.0 mmol) in dry pyridine (5 mL)

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diluted acyl chloride (1.0 mmol in 0.5 mL of DCM) was added
dropwise at 2e5 ꢀC. The reaction mixture was allowed to stir at
2e5 ꢀC for 30 min and then at room temperature for 18 h. After that
the solvent was evaporated under reduced pressure and the residue
was diluted with methanol (10 mL). The solution was added
dropwise to ice water (50 mL). The precipitate was filtered-off,
dried in the air to afford the acyl derivate 2aee.
151.3, 152.0 (br s), 158.0 ppm; HRMS (HESI, m/z) calcd for
C
12H14N6O3 (290.28) [MþH]þ 291.1200, found 291.1200.
4.1.2. 4-((3,5-Diamino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)dia-
zenyl)phenol (4). To a suspension of (4-hydroxyphenyl)carbon-
ohydrazonoyl dicyanide 33 (508 mg, 2.73 mmol) in methanol
(3 mL)
a solution of (4-methoxybenzyl)hydrazine (831 g,
5.45 mmol) in methanol (3 mL) was added at room temperature.
The solution was heated at reflux for 4 h. After cooling to room
temperature, the solvent was evaporated under reduced pressure.
The product was purified by flash column chromatography (CHCl3/
MeOH 20:1) to give phenol 4 (882 mg, 96% yield of crude product;
569 mg, 62% yield of pure product): yellow solid; mp¼190e192 ꢀC;
Rf¼0.18; IR (cmꢃ1) 3380, 3153, 2925, 1600, 1567, 1513, 1496, 1452,
1403, 1362, 1240, 1174, 1024, 836, 821, 754, 672, 535, 511; 1H NMR
4.1.1.1. (3,5-Diamino-4-((4-hydroxyphenyl)diazenyl)-1H-pyrazol-
1-yl)(phenyl) methanone (2a). Yield 260 mg, 81%: yellow solid; mp
194e196 ꢀC; IR (cmꢃ1) 3291, 3210, 3170, 3059, 1689, 1660, 1626,
1570,1496,1414,1368,1280,1217, 840, 816, 695; 1H NMR (300 MHz,
DMSO-d6)
(d, J¼8.4 Hz, 2H), 7.62e7.46 (m, 3H), 6.84 (d, J¼8.4 Hz, 2H), 6.18 (br
s, 2H) ppm; 13C NMR (101 MHz, DMSO-d6)
d¼9.82 (s, 1H), 8.08 (br s, 2H), 7.96 (d, J¼7.0 Hz, 2H), 7.71
d
¼112.3, 115.4, 122.7,
127.6, 130.0, 131.6, 133.3, 145.8, 146.1, 152.5, 158.1, 169.0 ppm; HRMS
(HESI, m/z) calcd for C16H14N6O2 (322.33) [MþH]þ 323.1251, found
323.1252.
(300 MHz, DMSO-d6)
(d, J¼8.4 Hz, 2H), 6.89 (d, J¼8.4 Hz, 2H), 6.78 (d, J¼8.6 Hz, 2H), 5.55
d
¼9.54 (br s, 1H), 7.55 (d, J¼8.6 Hz, 2H), 7.19
(br s, 1H), 4.87 (s, 2H), 3.72 (s, 3H) ppm; 13C NMR (75 MHz, DMSO-
d6)
d
¼48.7, 55.1, 112.9, 113.7, 115.3, 121.9, 128.9, 129.5, 146.5, 156.9,
4.1.1.2. (3,5-Diamino-4-((4-hydroxyphenyl)diazenyl)-1H-pyrazol-
1-yl)(4-nitrophenyl)methanone (2b). Yield 359 mg, 98%: orange
solid; mp 238e240 ꢀC; IR (cmꢃ1) 3297, 3175, 3114, 3041, 2386,
2285, 1662, 1632, 1591, 1571, 1518, 1498, 1477, 1415, 1349, 1273, 840,
158.5, 161.9 ppm; HRMS (HESI, m/z) calcd for C17H18N6O2 (338.37)
[MþH]þ 339.1564, found 339.1565.
4.1.3. N,N0-(4-((4-Hydroxyphenyl)diazenyl)-1-(4-methoxybenzyl)-
1H-pyrazole-3,5-diyl)dibenzamide (5). To a solution of 4-((3,5-
diamino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)diazenyl)phenol 4
813, 712; 1H NMR (300 MHz, DMSO-d6)
d
¼9.82 (s, 1H), 8.33 (d,
J¼8.8 Hz, 2H), 8.13 (d, J¼8.8 Hz, 2H), 7.72 (d, J¼8.8 Hz, 2H), 6.83 (d,
J¼8.8 Hz, 2H), 6.27 (br s, 2H) ppm; 13C NMR (75 MHz, DMSO-d6)
(338 mg, 1.0 mmol) in dry pyridine (3 mL) benzoyl chloride (256 mL,
d
¼112.3, 115.5, 122.8, 122.9, 131.0, 139.5, 146.1, 148.8, 152.9, 158.3,
2.20 mmol) was added at 2e5 ꢀC. The reaction mixture was allowed
to stir at room temperature for 18 h. After that the solvent was
evaporated under reduced pressure and the residue was diluted
with methanol (3 mL). The solution was added dropwise to ice
water (15 mL). The precipitate was filtered-off, dried in the air to
afford the crude dibenzamide 5, which was purified by flash col-
umn chromatography (CHCl3/MeOH 60:1) to give pure product
(464 mg, 85% yield of crude product; 372 mg, 68% yield of pure
product): yellow solid; mp 82e84 ꢀC; Rf¼0.27; IR (cmꢃ1) 3313,
2360, 2341,1731,1680,1624,1601,1577,1544,1514,1412,1389,1267,
167.4 ppm; HRMS (HESI, m/z) calcd for C16H13N7O4 (367.33)
[MþH]þ 368.1102, found 368.1117.
4.1.1.3. (3,5-Diamino-4((4-hydroxyphenyl)diazenyl)-1H-pyrazol-
1-yl)(thiophene-2-yl)methanone (2c). The product was purified by
flash column chromatography (CHCl3/MeOH 40:1) to give meth-
anone 2c (325 mg, 99% yield of crude product; 134 mg, 41% yield of
pure product): yellow solid; mp 220e222 ꢀC; Rf¼0.34; IR (cmꢃ1
)
3411, 1649, 1618, 1590, 1566, 1502, 1409, 1381, 1227, 839, 812, 729,
607; 1H NMR (300 MHz, DMSO-d6)
d
¼9.81 (br s, 1H), 8.31 (dd,
1248, 1192, 1178, 1062, 707; 1H NMR (300 MHz, DMSO-d6)
d
¼10.53
J¼3.9, 1.3 Hz, 1H), 8.10 (dd, 5.0, 1.3 Hz, 1H), 8.09 (br s, 2H), 7.71 (d,
J¼8.9 Hz, 2H), 7.26 (dd, J¼5.0, 3.9 Hz, 1H), 6.83 (d, J¼8.9 Hz, 2H),
(br s, 1H), 8.14 (d, J¼7.1 Hz, 2H), 7.98 (d, J¼6.6 Hz, 2H), 7.75 (d,
J¼6.8 Hz, 2H), 7.65e7.50 (m, 6H), 7.41 (br s, 1H), 7.36 (d, J¼8.6 Hz,
2H), 7.27 (d, J¼7.7 Hz, 2H), 6.94 (d, J¼7.9 Hz, 2H), 6.70 (br s, 1H), 5.14
6.34 (br s, 2H) ppm; 13C NMR (75 MHz, DMSO-d6)
d¼112.3, 115.5,
122.8, 127.2, 133.0, 137.0, 137.9, 146.1, 152.5 (br s), 158.2, 160.8 ppm;
HRMS (HESI, m/z) calcd for C14H12N6O2S (328.35) [MþH]þ
329.0815, found 329.0812.
(br s, 2H), 3.74 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
d
¼49.2, 55.1,
113.9, 121.7, 122.5, 127.6, 128.6, 128.7, 128.9, 129.0, 129.8, 131.8,
134.0, 134.1, 150.1, 150.8, 158.7, 164.6. HRMS (HESI, m/z) calcd for
C31H26N6O4 (546.59) [MþH]þ 547.2088, found 547.2086.
4.1.1.4. 1-(3,5-Diamino-4-((4-hydroxypheny)diazenyl)-1H-pyr-
azol-1-yl)ethanone (2d). The product was purified by flash column
chromatography (CHCl3/MeOH 30:1) to give ethanone 2d (146 mg,
56% yield of crude product; 938 mg, 36% yield of pure product):
yellow solid; mp 228e230 ꢀC; Rf¼0.17; IR (cmꢃ1) 3423, 3291, 3208,
1678, 1598, 1576, 1499, 1422, 1266, 1220, 1158, 1137, 1099, 842, 810;
4.1.4. (2,4-Dimethoxybenzyl)hydrazine hydrochloride (10). To a so-
lution of acetic acetohydrazide (659 mg, 8.0 mmol) in methanol
(40 mL), 2,4-dimethoxy-benzaldehyde (1.33 g, 8.0 mmol) was
added at room temperature. The solution was heated at reflux for
2
h. After cooling
to room temperature, N0-(2,4-
1H NMR (300 MHz, DMSO-d6)
(d, J¼8.4 Hz, 2H), 6.82 (d, J¼8.4 Hz, 2H), 6.15 (br s, 2H), 2.42 (s,
3H) ppm; 13C NMR (75 MHz, DMSO-d6)
d
¼9.76 (br s, 1H), 7.88 (br s, 2H), 7.67
dimethoxybenzylidene)acetohydrazide 8 started to precipitate.
The suspension was diluted with a mixture of methanol (20 mL)
and THF (20 mL). Then to the solution 10% Pd(C) (100 mg) was
added and hydrazone 8 was reduced with hydrogen under vigorous
stirring at room temperature and atmospheric pressure. After 24 h
the reaction mixture was filtered through a pad of Celite. The fil-
trate was evaporated under reduced pressure to afford N0-(2,4-
dimethoxybenzyl)acetohydrazide 9 as a colorless oil. To crude N0-
(2,4-dimethoxybenzyl)acetohydrazide 9 98% hydrazine hydrate
(8 mL, 165 mmol) was added. The reaction mixture was heated at
100 ꢀC for 30 h, cooled down to room temperature, and water
(0.5 mL) was added. The aqueous hydrazine layer was extracted
with ethyl ether (3ꢁ8 mL). The combined organic layers were
washed with brine (2 mL), dried over anhydrous Na2SO4, and
concentrated under reduced pressure to afford crude (2,4-
dimethoxy-benzyl)hydrazine 10 as a viscous oil, that could be
d
¼22.9, 112.4, 115.4, 122.7,
144.7 (br s), 146.0, 152.0 (br s), 158.1, 172.5 ppm; HRMS (HESI, m/z)
calcd for C11H12N6O2 (260.26) [MþH]þ 261.1095, found 261.1093.
4.1.1.5. Ethyl
3,5-diamino-4-((4-hydroxyphenyl)diazenyl)-1H-
pyrazole-1-carboxylate (2e). The product was purified by flash
column chromatography (CHCl3/MeOH 20:1) to give ethyl carbox-
ylate 2e (264 mg, 91% yield of crude product; 187 g, 65% yieldꢃo1f
pure product): yellow solid; mp 234e236 ꢀC; Rf¼0.25; IR (cm
)
3488, 3416, 3395, 3375, 3290, 1720, 1620, 1601, 1555, 1499, 1467,
1449, 1428, 1375, 1311, 1287, 1138, 841, 750, 537; 1H NMR (300 MHz,
DMSO-d6)
2H), 6.06 (br s, 2H), 4.33 (q, J¼7.1 Hz, 2H), 1.31 (t, J¼7.1 Hz, 3H) ppm;
13C NMR (75 MHz, DMSO-d6)
¼14.1, 63.0, 112.2, 115.4, 122.6, 146.0,
d
¼9.75 (br s, 1H), 7.66 (d, J¼8.6 Hz, 3H), 6.82 (d, J¼8.6 Hz,
d

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4000
directly used for the next reaction step resulting in 4-((3,5-
diamino-1-(2,4-dimethoxybenzyl)-1H-pyrazol-4-yl)diazenyl)phe-
nol 11. The compound 10 was isolated as a hydrochloride salt after
addition of a methanolic solution of hydrochloric acid (3 mL, concn
3.29 mol/L) to the combined organic layers. The precipitated 10$HCl
was filtered-off, washed with ethyl ether, and dried in a desiccator
over phosphorus pentaoxide as a white solid (862 mg, 49% over
three steps); mp 138e140 ꢀC; IR (cmꢃ1) 3265, 3156, 2965, 2840,
2812, 2769, 2595, 2441, 1613, 1587, 1508, 1445, 1321, 1287, 1273,
1211, 1157, 1133, 1042, 925, 834; 1H NMR (300 MHz, DMSO-d6)
pyridine (6 mL), acyl chloride (for 13a: benzoyl chloride (0.082 mL,
0.706 mmol) diluted with DCM (1 mL); for 13b: 4-nitrobenzoyl
chloride (131 mg, 0.708 mmol); for 13c: thiophene-2-carbonyl
chloride (0.075 mL, 0.701 mmol) diluted with DCM (0.5 mL); for
13d: acetyl chloride (0.090 mL, 1.26 mmol) diluted with DCM
(1.5 mL); for 13e: ethyl carbonochloridate (0.067 mL, 0.704 mmol)
diluted with DCM (6 mL)) was added (dropwise for 13a, 13c, 13d,
13e) at 2e5 ꢀC. The reaction mixture was allowed to stir at room
temperature for 18 h. After that the solvent was evaporated under
reduced pressure and the residue was diluted with methanol
(3 mL). The solution was added dropwise to ice water (15 mL). The
precipitate was filtered-off, dried in the air to afford product 13aee.
d
¼7.77 (br s, 2H), 7.28 (d, J¼8.3 Hz, 1H), 6.58 (d, J¼2.3 Hz, 1H), 6.52
(dd, J¼2.3, 8.3 Hz, 1H), 3.99 (s, 2H), 3.79 (s, 3H), 3.76 (s, 3H) ppm;
13C NMR (75 MHz, DMSO-d6)
d
¼48.5, 55.3, 55.6, 98.3, 104.7, 113.2,
131.8, 158.6, 161.0 ppm; HRMS (HESI, m/z) calcd for C9H14N2O2
4.1.7.1. 4-((5-Amino-3-benzamido-1-(2,4-dimethoxybenyl)-1H-
pyrazol-4-yl)diazenyl)phenyl tert-butyl carbonate (13a). Yield
349 mg, 95%: yellow solid; mp 110e112 ꢀC; IR (cmꢃ1) 3316, 2979,
2837, 1757, 1689, 1614, 1575, 1540, 1391, 1273, 1210, 1147, 1035, 824,
(182.22) [MþH]þ 183.1128, found 183.1130.
4.1.5. 4-((3,5-Diamino-1-(2,4-dimethoxybenzyl)-1H-pyrazol-4-yl)di-
azenyl) phenol (11). To a suspension of (4-hydroxyphenyl) car-
bonohydrazonoyl dicyanide 33 (500 mg, 2.69 mmol) in methanol
(6 mL) a solution of DmbNHNH2$HCl (734 mg, 3.36 mmol) and TEA
(3 mL, 21.5 mmol) in methanol (6 mL) was added dropwise at room
temperature. The solution was heated at reflux for 4 h. After cooling
to room temperature, the solvent was evaporated under reduced
pressure and the residue was diluted in methanol (10 mL). The
solution was added dropwise to ice water (50 mL). The precipitate
was filtered-off, dried in the air to afford phenol 11 as a yellow solid
(920 mg, 93%); mp 108e110 ꢀC; IR (cmꢃ1) 3576, 3439, 3345, 3080,
2390, 2284, 1610, 1563, 1498, 1460, 1393, 1356, 1265, 1209, 1157,
704; 1H NMR (300 MHz, DMSO-d6)
d
¼10.49 (br s, 1H), 7.96 (d,
J¼7.1 Hz, 2H), 7.69 (d, J¼8.8 Hz, 2H), 7.63e7.50 (m, 3H), 7.28 (br s,
1H), 7.25 (d, J¼8.8 Hz, 2H), 6.84 (d, J¼8.3 Hz, 1H), 6.61 (d, J¼2.0 Hz,
1H), 6.51 (dd, J¼2.0, 8.3 Hz, 1H), 5.06 (s, 2H), 3.84 (s, 3H), 3.75 (s,
3H), 1.49 (s, 9H) ppm; 13C NMR (75 MHz, DMSO-d6)
d
¼27.2, 45.0,
55.3, 55.6, 83.3, 98.4, 104.6, 116.6, 116.8, 121.6, 121.9, 127.6, 128.5,
129.0, 129.3, 131.8, 134.0, 141.9 (br s), 150.1, 150.7, 151.1, 157.5,
160.2 ppm; HRMS (HESI, m/z) calcd for C30H32N6O6 (572.63)
[MþH]þ 573.2456, found 573.2453.
4.1.7.2. 4-((5-Amino-1-(2,4-dimethoxybenzyl)-3-(4-nitrobenzam-
1110, 840; 1H NMR (300 MHz, DMSO-d6)
d
¼9.55 (br s, 1H), 7.56 (d,
ido)-1H-pyrazol-4-yl)diazenyl)phenyl tert-butyl carbonate (13b).
J¼8.6 Hz, 2H), 6.84e6.71 (m, 3H), 6.57 (d, J¼2.1 Hz, 1H), 6.46 (dd,
J¼2.1, 8.3 Hz, 1H), 5.58 (br s, 1H), 4.81 (s, 2H), 3.81 (s, 3H), 3.73 (s,
Yield 380 mg, 96%: orange-brown solid; mp 126e128 ꢀC; IR (cmꢃ1
)
3437, 3341, 3109, 2976, 2359, 2340, 1756, 1726, 1639, 1616, 1603,
3H) ppm; 13C NMR (75 MHz, DMSO-d6)
d
¼44.3, 55.3, 55.5, 98.2,
1581, 1541, 1389, 1344, 1273, 1209, 1145, 820, 782, 711; 1H NMR
104.5, 112.9, 115.3, 117.4, 121.9, 128.7, 146.5, 152.2 (br s), 156.9, 157.3,
159.9 ppm; HRMS (HESI, m/z) calcd for C18H20N6O3 (368.40)
[MþH]þ 369.1670, found 369.1666.
(400 MHz, DMF-d7)
d
¼10.86 (br s, 1H), 8.46 (m, 2H), 8.31 (m, 2H),
8.02 (s, 1H), 7.81 (d, J¼8.4 Hz, 2H), 7.40 (s, 1H), 7.31 (d, J¼8.2 Hz, 2H),
6.93 (d, J¼8.2 Hz,1H), 6.66 (s,1H), 6.54 (dd, J¼2.4, 8.4 Hz,1H), 5.16 (s,
2H), 3.90 (s, 3H), 3.81 (s, 3H), 1.54 (s, 9H) ppm; 13C NMR (101 MHz,
4.1.6. tert-Butyl (4-((3,5-diamino-1-(2,4-dimethoxybenzyl)-1H-pyr-
azol-4-yl)diazenyl)phenyl) carbonate (12). To a solution of com-
pound 11 (1.00 g, 2.71 mmol) in dry pyridine (10 mL), di-tert-butyl
dicarbonate (0.934 mL, 4.07 mmol) diluted in dry pyridine (1 mL)
was added dropwise at 2e5 ꢀC. The reaction mixture was allowed
to stir at room temperature for 18 h. After that the solvent was
removed under reduced pressure and the residue was purified by
flash column chromatography (CHCl3/MeOH 80:1) to give the car-
bonate 12 (1.20 g, 94% yield of crude product; 890 mg, 70% yield of
pure product): yellow solid; mp 172e174 ꢀC; Rf¼0.14 (80:1); IR
(cmꢃ1) 3419, 3338, 3285, 2967, 2923, 1745, 1621, 1593, 1575, 1560,
1509, 1488, 1422,1384, 1341, 1272, 1257, 1209, 1149,1120, 1045, 828;
DMF-d7)
d
¼27.2, 45.5, 55.3, 55.5, 83.5, 98.5, 104.8, 117.1, 122.1, 124.0,
129.1, 129.5, 131.1, 137.2, 140.6, 142.9, 149.9, 151.0, 151.4, 151.9, 158.1,
161.0, 164.2, 166.2 ppm; HRMS (HESI, m/z) calcd for C30H31N7O8
(617.62) [MþH]þ 618.2307, found 618.2308.
4.1.8. 4-((5-Amino-1-(2,4-dimethoxybenzyl)-3-(thiophene-2-
carboxamido)-1H-pyrazol-4-yl)diazenyl)phenyl tert-butyl carbonꢃat1e
(13c). Yield 336 mg, 91%: yellow solid; mp 110e112 ꢀC; IR (cm
)
3315, 3222, 3098, 2978, 2936, 2836, 2354, 1757, 1673, 1615, 1574,
1542, 1509, 1422, 1390, 1371, 1273, 1209, 1147, 1034, 841, 823, 737,
573, 534; 1H NMR (400 MHz, DMF-d7)
d
¼10.62 (br s, 1H), 8.10 (d,
J¼3.5 Hz, 1H), 7.91 (dd, J¼1.1, 5.1 Hz, 1H), 7.81 (d, J¼8.6 Hz, 2H), 7.40
(br s, 1H), 7.38 (br s, 1H), 7.32 (d, J¼8.8 Hz, 2H), 7.28 (m, 1H), 6.93 (d,
J¼8.6 Hz,1H), 6.65 (d, J¼2.5 Hz,1H), 6.54 (dd, J¼2.4, 8.5 Hz,1H), 5.14
(s, 2H), 3.91 (s, 3H), 3.82 (s, 3H), 1.54 (s, 9H) ppm; 13C NMR
(101 MHz, DMF-d7)¼27.2, 44.9, 55.3, 55.6, 83.3, 98.4, 104.6, 116.5,
117.0, 121.6, 121.9, 128.2, 129.0, 129.4, 131.9, 139.3, 141.4 (br s), 142.4
(br s), 150.1, 150.7, 151.1, 157.5, 160.3 ppm; HRMS (HESI, m/z) calcd
for C28H30N6O6S (578.65) [MþH]þ 579.2020, found 579.2047.
1H NMR (400 MHz, DMF-d7)
d
¼7.78 (dd, J¼2.0, 8.9 Hz, 2H), 7.38 (br
s, 1H), 7.27 (dd, J¼2.0, 8.9 Hz, 2H), 6.91 (d, J¼8.4 Hz, 1H), 6.68 (br s,
1H), 6.63 (d, J¼2.5 Hz, 1H), 6.52 (dd, J¼2.5, 8.4 Hz, 1H), 6.04 (br s,
2H), 5.38 (br s, 2H), 4.94 (br s, 2H), 3.89 (s, 3H), 3.81 (s, 3H), 1.55 (s,
9H) ppm; 13C NMR (101 MHz, DMF-d7)
d
¼27.2, 45.0, 55.2, 55.5, 83.3,
98.4, 104.7, 114.9, 117.7, 121.4, 121.9, 129.0, 139.8, 146.9, 149.0, 149.9,
152.0, 152.2, 153.9, 157.9, 160.8 ppm; 1H NMR (400 MHz, DMF-d7)
(65 ꢀC)
d
¼7.74 (d, J¼8.8 Hz, 2H), 7.24 (d, J¼8.8 Hz, 2H), 7.00 (d,
J¼8.1 Hz, 1H), 6.75 (br s, 2H), 6.64 (d, J¼2.2 Hz, 1H), 6.54 (d,
J¼8.1 Hz,1H), 5.52 (br s, 2H), 4.94 (br s, 2H), 3.91 (s, 3H), 3.83 (s, 3H),
4.1.9. 4-((3-Acetamido-5-amino-1-(2,4-dimethoxybenzyl)-1H-pyr-
azol-4-yl)diazenyl)phenyl tert-butyl carbonate (13d). Yield 285 mg,
87%: yellow solid; mp 92e94 ꢀC; IR (cmꢃ1) 3410, 3303, 2980, 2938,
2837, 1758, 1689, 1616, 1572, 1537, 1509, 1387, 1372, 1273, 1210, 1147,
1.57 (s, 9H) ppm; 13C NMR (101 MHz, DMF-d7) (65 ꢀC)
d
¼27.4, 45.0,
55.4, 55.7, 83.3, 98.8, 105.2, 115.1, 117.9, 121.5, 121.7, 129.5, 143.3,
150.2, 151.5, 152.0, 152.3, 158.2, 161.0 ppm; HRMS (HESI, m/z) calcd
for C23H28N6O5 (468.52) [MþH]þ 469.2194, found 469.2188.
1034, 842, 825, 782, 578, 535; 1H NMR (300 MHz, DMSO-d6)
d
¼9.77
(br s, 1H), 7.76 (d, J¼8.6 Hz, 2H), 7.27 (d, J¼8.4 Hz, 4H), 6.76 (d,
J¼8.4 Hz, 1H), 6.59 (d, J¼2.0 Hz, 1H), 6.48 (dd, J¼2.0, 8.4 Hz, 1H),
5.00 (s, 2H), 3.82 (s, 3H), 3.74 (s, 3H), 2.05 (s, 3H), 1.50 (s, 9H) ppm;
4.1.7. General procedure for acylation of tert-butyl (4-((3,5-diamino-
1-(2,4-dimethoxybenzyl)-1H-pyrazol-4-yl)diazenyl)phenyl)
carbo-
13C NMR (75 MHz, DMSO-d6)
d¼23.2, 27.3, 44.9, 55.3, 55.6, 83.4,
nate. To a solution of compound 12 (300 mg, 0.640 mmol) in dry
98.4, 104.6, 116.2, 116.6, 121.8, 121.9, 128.8, 140.6 (br s), 143.4 (br s),

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E. Schutznerova et al. / Tetrahedron 68 (2012) 3996e4002
4001
150.1, 150.8, 151.2, 157.5, 160.2, 168.6 (br s) ppm; HRMS (HESI, m/z)
calcd for C25H30N6O6 (510.55) [MþH]þ 511.2300, found 511.2298.
838, 719, 622, 538, 481; 1H NMR (300 MHz, DMSO-d6)
d
¼10.39 (s,
1H), 9.73 (s,1H), 7.94 (d, J¼3.8 Hz,1H), 7.84 (d, J¼4.8 Hz,1H), 7.54 (d,
J¼8.6 Hz, 2H), 7.22 (dd, J¼4.8, 3.8 Hz, 1H), 7.03 (s, 2H), 6.84e6.77
(m, 3H), 6.60 (d, J¼2.2 Hz, 1H), 6.49 (dd, J¼2.2, 8.4 Hz, 1H), 5.03 (s,
2H), 3.83 (s, 3H), 3.75 (s, 3H) ppm; 13C NMR (75 MHz, DMSO-d6)
4.1.10. Ethyl (5-amino-4-((4-((tert-butoxycarbonyl)oxy)phenyl)dia-
zenyl)-1-(2,4-dimethoxybenzyl)-1H-pyrazole-3-yl)carbamate
(13e). The purification of carbamate 13e was carried out on semi-
preparative HPLC (319 mg, 92% yield of crude product; 128 mg,
d
¼44.9, 55.3, 55.6, 98.4, 104.6, 115.5, 116.3, 116.7, 122.4, 128.2, 129.0,
129.3, 131.7, 139.4, 141.0, 141.8, 145.9, 157.5, 158.0, 160.1, 160.2 ppm;
HRMS (HESI, m/z) calcd for C23H22N6O4S (478.53) [MþH]þ
479.1496, found 479.1495.
37% yield of pure product): yellow solid; mp 86e88 ꢀC; IR (cmꢃ1
)
3566, 3413, 2980, 2949, 1756, 1618, 1558, 1273, 1209, 1146, 1044,
892, 781, 630; 1H NMR (300 MHz, DMSO-d6)
d
¼9.31 (s, 1H), 7.75
(d, J¼8.8 Hz, 2H), 7.27 (d, J¼8.8 Hz, 4H), 6.76 (d, J¼8.4 Hz, 1H),
6.59 (d, J¼1.8 Hz, 1H), 6.48 (dd, J¼2.0, 8.4 Hz, 1H), 4.99 (s, 2H),
4.06 (q, J¼7.0 Hz, 2H), 3.82 (s, 3H), 3.74 (s, 3H), 1.50 (s, 9H), 1.18 (t,
4.1.11.4. N-(5-Amino-1-(2,4-dimethoxybenzyl)-4-((4-hydroxyph-
enyl)diazenyl)-1H-pyrazol-3-yl)acetamide (14d). Yield 143 mg, 69%:
yellow solid; mp 136e138 ꢀC; IR (cmꢃ1) 3420, 3205, 3024, 2934,
2838, 2408, 2283, 1680, 1613, 1573, 1537, 1508, 1454, 1395, 1294,
1267, 1237, 1209, 1157, 1119, 1035, 937, 841, 674, 538; 1H NMR
J¼7.0 Hz, 3H) ppm; 13C NMR (75 MHz, DMSO-d6)
¼14.5, 27.3,
d
44.8, 55.3, 55.6, 60.3, 83.3, 98.4, 104.6, 116.2, 116.6, 121.7, 121.9,
122.1, 128.8, 150.0, 150.8, 151.2, 153.7, 157.5, 160.0, 160.2 ppm;
HRMS (HESI, m/z) calcd for C26H32N6O7 (540.58) [MþH]þ
541.2405, found 541.2404.
(300 MHz, DMSO-d6)
d
¼9.73 (s, 1H), 7.59 (d, J¼8.8 Hz, 2H), 7.00 (br
s, 2H), 6.82 (d, J¼8.8 Hz, 2H), 6.73 (d, J¼8.4 Hz, 1H), 6.59 (d,
J¼2.2 Hz, 1H), 6.47 (dd, J¼2.2, 8.4 Hz, 1H), 4.98 (s, 2H), 3.82 (s, 3H),
3.74 (s, 3H), 2.03 (s, 3H) ppm; 13C NMR (75 MHz, DMSO-d6)
d
¼23.1,
4.1.11. General procedure for deprotection of Boc protective group.-
Compound 13 (0.5 mmol) was dissolved in 10% TFA/DCM (15 mL).
The reaction mixture was allowed to stir at room temperature. After
1 h the solvent was evaporated under reduced pressure. To the
residue water (20 mL) was added and pH was adjusted with am-
monia to pH 10. The suspension was allowed to stir at room tem-
perature for 2 h. The crude product 14aee was filtered-off and dried
in the air.
44.8, 55.3, 55.6, 98.4, 104.6, 115.5, 116.8, 122.5, 128.7, 140.3 (br s),
142.8, 146.0, 155.9 (br s), 157.4, 157.9, 160.1, 168.7 (br s) ppm; HRMS
(HESI, m/z) calcd for C20H22N6O4 (410.44) [MþH]þ 411.1775, found
411.1777.
4.1.11.5. Ethyl (5-amino-1-(2,4-dimethoxybenzyl)-4-((4-hydroxy-
phenyl) diazenyl)-1H-pyrazol-3-yl) carbamate (14e). Yield 207 mg,
94%: yellow solid; mp 118e120 ꢀC; IR (cmꢃ1) 3430, 3337, 2933,
2836, 2360, 1727, 1614, 1568, 1507, 1465, 1399, 1294, 1267, 1209,
4.1.11.1. N-(5-Amino-1-(2,4-dimethoxybenzyl)-4-((4-hydroxyph-
enyl) diazenyl)-1H-pyrazol-3-yl)benzamide (14a). Yield 234 mg,
99%: orange-brown solid; mp 140e142 ꢀC; IR (cmꢃ1) 3650, 3465,
3180, 2941, 2834, 2391, 2285, 1671, 1582, 1544, 1507, 1455, 1393,
1293, 1267, 1208, 1157, 1137, 1030, 838, 698, 536; 1H NMR
1157,1041, 840, 738, 539, 438; 1H NMR (300 MHz, DMSO-d6)
d¼9.73
(s, 1H), 9.19 (s, 1H), 7.58 (d, J¼8.6 Hz, 2H), 6.98 (br s, 2H), 6.82 (d,
J¼8.6 Hz, 2H), 6.73 (d, J¼8.4 Hz, 1H), 6.59 (d, J¼2.0 Hz, 1H), 6.47 (dd,
J¼2.1, 8.3 Hz, 1H), 4.97 (s, 2H), 4.05 (q, J¼7.1 Hz, 2H), 3.82 (s, 3H),
3.74 (s, 3H), 1.17 (t, J¼7.0 Hz, 3H) ppm; 13C NMR (75 MHz, DMSO-d6)
(300 MHz, DMSO-d6)
d
¼10.45 (s, 1H), 9.74 (s, 1H), 7.96 (d, J¼7.1 Hz,
d
¼14.5, 44.7, 55.3, 55.6, 60.3, 98.4, 104.6, 115.5, 115.5, 116.8, 122.4,
2H), 7.61e7.50 (m, 5H), 7.02 (br s, 2H), 6.81 (d, J¼8.4 Hz, 3H), 6.60
128.7, 140.5, 142.4, 146.0, 153.9, 157.4, 157.9, 160.1 ppm; HRMS
(HESI, m/z) calcd for C21H24N6O5 (440.46) [MþH]þ 441.1883, found
441.1881.
(d, J¼2.2 Hz, 1H), 6.50 (dd, J¼2.2, 8.4 Hz, 1H), 5.04 (s, 2H), 3.83 (s,
3H), 3.75 (s, 3H) ppm; 13C NMR (75 MHz, DMSO-d6)
d¼44.9, 55.3,
55.6, 98.4, 104.6, 115.5, 116.0, 116.8, 122.3, 127.5, 128.5, 128.9, 131.7,
134.1, 141.5, 142.0, 145.9, 157.5, 157.9, 160.2165.3 ppm; HRMS (HESI,
m/z) calcd for C25H24N6O4 (472.51) [MþH]þ 473.1932, found
473.1932.
4.1.12. General procedure for deprotection of Dmb and Boc protecting
groups. Compound 13 (0.5 mmol) was dissolved in neat TFA
(15 mL). The reaction mixture was allowed to stir at room tem-
perature. After 4 h the solvent was evaporated under reduced
pressure. To the residue water (20 mL) was added and pH was
adjusted with aqueous ammonia (25%) to pH 10. The suspension
was allowed to stir at room temperature for 2 h. Then the crude
product 15aee was filtered-off and dried in the air.
4.1.11.2. N-(5-Amino-1-(2,4-dimethoxybenzyl)-4-((4-hydroxyph-
enyl)diazenyl)-1H-pyrazol-3-yl)-4-nitrobenzamide (14b). The crude
4-nitrobenzamide 14b was purified by flash column chromatogra-
phy (CHCl3/MeOH 40:1) to give product 14b (204 mg, 79% yield of
crude product; 134 mg, 52% yield of pure product): orange solid;
mp 150e152 ꢀC; Rf¼0.28; IR (cmꢃ1) 3412, 3177, 2950, 2836, 2391,
2291, 1673, 1602, 1582, 1540, 1525, 1510, 1452, 1347, 1294, 1272,
1209,1158,1033, 841, 831, 705, 613, 533; 1H NMR (300 MHz, DMSO-
4.1.12.1. N-(5-Amino-4-((4-hydroxyphenyl)diazenyl)-1H-pyrazol-
3-yl)benzamide (15a). The benzamide 15a was purified by column
chromatography (CHCl3/MeOH 10:1) to give pure product (159 mg,
99% yield of crude product; 95.1 mg, 59% yield of pure product):
yellow solid; mp 262e264 ꢀC; Rf¼0.20; IR (cmꢃ1) 3317, 3221, 3110,
2924, 2847, 1686, 1634, 1580, 1448, 1506, 1394, 1239, 1203, 1037,
d6)
d
¼10.69 (s, 1H), 9.75 (s, 1H), 8.37 (d, J¼8.8 Hz, 2H), 8.16 (d,
J¼8.4 Hz, 2H), 7.53 (d, J¼8.8 Hz, 2H), 7.05 (s, 2H), 6.80 (d, J¼8.6 Hz,
3H), 6.60 (d, J¼1.8 Hz, 1H), 6.49 (dd, J¼2.3, 8.3 Hz, 1H), 5.04 (s, 2H),
3.83 (s, 3H), 3.74 (s, 3H) ppm; 13C NMR (75 MHz, DMSO-d6)
d¼44.9,
833, 695, 531; 1H NMR (300 MHz, DMSO-d6)
d
¼11.79 (br s, 1H),
55.3, 55.6, 98.4, 104.6, 115.5, 116.1, 116.7, 122.5, 123.7, 129.0, 129.1,
139.8, 141.1, 141.8, 145.8, 149.3, 157.5, 158.0, 160.2, 164.2 ppm; HRMS
(HESI, m/z) calcd for C25H23N7O6 (517.51) [MþH]þ 518.1783, found
518.1783.
10.58 (br s, 1H), 9.76 (br s, 1H), 7.98 (d, J¼7.0 Hz, 2H), 7.66e7.50 (m,
5H), 6.81 (d, J¼8.6 Hz, 2H), 6.59 (br s, 2H) ppm; 13C NMR
(75.47 MHz, DMSO-d6)
d
¼115.5, 115.9, 122.4, 127.6, 128.6, 131.9,
133.8, 145.8, 158.0, 165.3 ppm; HRMS (HESI, m/z) calcd for
C16H14N6O2 (322.33) [MþH]þ 323.1251, found 323.1251.
4.1.11.3. N-(5-Amino-1-(2,4-dimethoxybenzyl)-4-((4-hydroxyph-
enyl)diazenyl)-1H-pyrazol-3-yl)thiophene-2-carboxamide (14c). The
crude carboxamide 14c was purified by flash column chromatog-
raphy (CHCl3/MeOH 40:1) to give product 14c (233 mg, 98% yield of
crude product; 134 mg, 56% yield of pure product): yellow solid;
mp 132e134 ꢀC; Rf¼0.15; IR (cmꢃ1) 3549, 3469, 3413, 2409, 2279,
1637, 1618, 1578, 1543, 1508, 1422, 1397, 1293, 1268, 1209, 1157, 964,
4.1.12.2. N-(5-Amino-4-((4-hydroxyphenyl)diazenyl)-1H-pyrazol-
3-yl)-4-nitrobenzamide (15b). The 4-nitrobenzamide 15b was pu-
rified by column chromatography (CHCl3/MeOH 10:1) to give pure
product (182 mg, 99% yield of crude product; 102 mg, 56% yield of
pure product): yellow solid; mp 232e234 ꢀC; Rf¼0.20; IR (cmꢃ1
)
3712, 3346, 3003, 2924, 2834, 2361, 2344, 1676, 1587, 1572, 1508,

€
ꢀ
E. Schutznerova et al. / Tetrahedron 68 (2012) 3996e4002
4002
1467, 1340, 1315, 1301, 1278, 1203, 1038, 842, 814, 704, 532; 1H NMR
(300 MHz, DMSO-d6)
Supplementary data
d
¼11.83 (br s, 1H), 10.76 (br s, 1H), 9.75 (s, 1H),
8.39 (d, J¼8.4 Hz, 2H), 8.19 (d, J¼8.4 Hz, 2H), 7.54 (d, J¼8.4 Hz, 2H),
Experimental details, spectroscopic data, and screening of dif-
ferent reaction conditions for the synthesis of 2ae15e. Crystallo-
graphic data for 13b have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication no.
CCDC 837640. Copies of the data can be obtained free of charge on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax:
(þ44) 1223 336 033; e-mail: deposit@ccdc.cam.ac.uk). Crystal data
and refinement, selected bond lengths and angles of covalent as
well as non-covalent contacts are also summarized. Supplementary
data associated with this article can be found, in the online version,
at doi:10.1016/j.tet.2012.03.063.
6.80 (d, J¼8.8 Hz, 2H), 6.67 (br s, 2H) ppm; 13C NMR (75 MHz,
DMSO-d6)
d
¼116.0, 116.7, 123.03, 124.3, 124.5, 129.7, 129.9, 140.2,
146.4, 149.9, 158.6, 164.7 ppm; HRMS (HESI, m/z) calcd for
C16H13N7O4 (367.33) [MþH]þ 368.1102, found 368.1101.
4.1.12.3. N-(5-Amino-4-((4-hydroxyphenyl)diazenyl)-1H-pyrazol-
3-yl)thiophene-2-carboxamide (15c). Yield 142 mg, 87%: yellow-
brown solid; mp 284e286 ꢀC; IR (cmꢃ1) 3311, 3219, 3113, 2994,
2950, 2833, 2390, 2284, 1673, 1632, 1577, 1545, 1506, 1396, 1296,
1243, 1203, 1037, 835, 726, 530; 1H NMR (300 MHz, DMSO-d6)
d
¼11.83 (br s, 1H), 10.48 (br s, 1H), 9.75 (br s, 1H), 7.98 (d,
J¼4.0 Hz, 1H), 7.87 (d, J¼4.7 Hz, 1H), 7.55 (d, J¼8.4 Hz, 2H), 7.24
(dd, J¼4.7, 4.0 Hz, 1H), 6.81 (d, J¼8.4 Hz, 2H), 6.64 (br s, 2H) ppm;
References and notes
13C NMR (75 MHz, DMSO-d6)
d
¼115.5, 116.3, 122.5, 128.3, 129.6,
1. (a) Fabbro, D.; Ruetz, S.; Buchdunger, E.; Cowan-Jacob, S. W.; Fendrich, G.;
Liebetanz, J.; Mestan, J.; O’Reilly, T.; Traxler, P.; Chaudhuri, B.; Fretz, H.; Zim-
mermann, J.; Meyer, T.; Caravatti, G.; Furet, P.; Manley, P. W. Pharmacol. Ther.
2002, 93, 79e98; (b) Johnson, L. N. Q. Rev. Biophys. 2009, 42, 1e40; (c) Cohen, P.
Nat. Rev. Drug Discovery 2002, 1, 309e315.
132.0, 139.2 (br s), 145.9, 158.0, 160.1 ppm; HRMS (HESI, m/z)
calcd for C14H12N6O2S (328.35) [MþH]þ 329.0815, found
329.0813.
2. Baumli, S.; Lolli, G.; Lowe, E. D.; Troiani, S.; Rusconi, L.; Bullock, A. N.; De-
breczeni, J. E.; Knapp, S.; Johnson, L. N. EMBO J. 2008, 27, 1907e1918.
4.1.12.4. N-(5-Amino-4-((4-hydroxyphenyl)diazenyl)-1H-pyrazol-
3-yl)acetamide (15d). The acetamide 15d was purified by column
chromatography (CHCl3/MeOH 10:1) to give product (97.3 mg,
75% yield of crude product; 51.5 mg, 40% yield of pure product):
yellow-brown solid; mp 158e160 ꢀC; Rf¼0.16; IR (cmꢃ1) 3128,
2925, 2834, 2387, 2284, 1674, 1603, 1594, 1552, 1506, 1454, 1294,
1268, 1240, 1203, 1039, 841, 663, 633, 526; 1H NMR (300 MHz,
ꢁ
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3. Krystof, V.; Cankar, P.; Frysova, I.; Slouka, J.; Kontopidis, G.; Dzubak, P.; Hajduch,
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M.; Srovnal, J.; Azevedo, W. F., Jr.; Orsag, M.; Paprskarova, M.; Rolcík, J.; Latr, A.;
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5. (a) Kusakiewicz-Dawid, A.; Gorecki, L.; Masiukiewicz, E.; Rzeszotarska, B. Synth.
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Commun. 2009, 39, 4122e4132; (b) Seelen, W.; Schafer, M.; Ernst, A. Tetrahedron
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ashi, K.; Kawanishi, N.; Fukasawa, K.; Takaki, T.; Ikeura, C.; Ikuta, M.; Suzuki-
Takahashi, I.; Hayama, T.; Nishimura, S.; Morishima, H. J. Med. Chem. 2001, 44,
4628e4640; (d) Pask, C. M.; Camm, K. D.; Kilner, C. A.; Halcrow, M. A. Tetra-
hedron Lett. 2006, 47, 2531e2534 Via an oxidationereduction sequence of the
exocyclic amino group, see: (e) Orsini, P.; Traquandi, G.; Sansonna, P.; Pevarello,
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DMSO-d6)
J¼8.8 Hz, 2H), 6.82 (d, J¼8.8 Hz, 2H), 6.26 (br s, 2H), 2.12 (s,
3H) ppm; 13C NMR (75 MHz, DMSO-d6)
d
¼11.71 (br s, 1H), 10.25 (br s, 1H), 9.75 (s, 1H), 7.63 (d,
d
¼23.1, 115.4, 115.6, 122.6,
123.6, 143.3 (br s), 146.0, 158.0, 169.1 (br s) ppm; HRMS (HESI, m/
z) calcd for C11H12N6O2 (260.26) [MþH]þ 261.1095, found
261.1093.
6. Stanovnik, B.; Svete, J. Sci. Synth. 2002, 12, 15e225.
7. Eller, G. A.; Holzer, W. Heterocycles 2004, 63, 2537e2555.
8. (a) Ueda, M.; Yamaura, M.; Ikeda, Y.; Suzuki, Y.; Yoshizato, K.; Hayakawa, I.;
Kigoshi, H. J. Org. Chem. 2009, 74, 3370e3377; (b) Imagawa, H.; Saijo, H.;
Kurisaki, T.; Yamamoto, H.; Kubo, M.; Fukuyama, Y.; Nishizawa, M. Org. Lett.
2009, 11, 1253e1255.
9. Esumi, T.; Mori, T.; Zhao, M.; Toyota, M.; Fukuyama, Y. Org. Lett. 2010, 12,
888e891.
10. Gude, M.; Hubschwerlen, C.; Rueedi, G.; Surivet, J.; Zumbrunn Acklin, C. WO
2008126024, 2008; Chem. Abstr. 2008, 149, 493644.
4.1.12.5. Ethyl (5-amino-4-((4-hydroxyphenyl)diazenyl)-1H-pyr-
azol-3-yl) carbamate (15e). The purification of carbamate 15e was
carried out on semi-preparative HPLC (92.7 mg, 64% yield of crude
product; 29.5 mg, 20% yield of pure product): yellow solid; mp
170e172 ꢀC; IR (cmꢃ1) 3180, 3108, 2939, 2834, 2390, 2284, 1731,
1589, 1565, 1506, 1452, 1296, 1270,1222, 1204, 1172, 1096, 1037, 837,
11. Niida, A.; Tomita, K.; Mizumoto, M.; Tanigaki, H.; Terada, T.; Oishi, S.; Otaka, A.;
Inui, K.; Fujii, N. Org. Lett. 2006, 8, 613e616.
815, 530; 1H NMR (300 MHz, DMSO-d6)
d
¼11.57 (br s, 1H), 9.61 (br
s, 2H), 7.59 (d, J¼8.4 Hz, 2H), 6.81 (d, J¼8.4 Hz, 2H), 6.51 (br s, 2H),
12. For examples see: (a) Park, C. M. J. Org. Chem. 2006, 71, 413e415; (b) Gutierrez,
C. D.; Bavetsias, V.; McDonald, E. Tetrahedron Lett. 2005, 46, 3595e3597; (c)
Suginome, M.; Tanaka, Y.; Hasui, T. Synlett 2006, 1047e1050.
13. For examples see: (a) Chern, C.-Y.; Huang, Y.-P.; Kan, W. M. Tetrahedron Lett.
2003, 44, 1039e1041; (b) Conroy, T.; Jolliffe, K. A.; Payne, R. J. Org. Biomol. Chem.
2010, 8, 3723e3733; (c) Thompson, M. J.; Chen, B. Tetrahedron Lett. 2008, 49,
5324e5327.
4.10 (q, J¼7.0 Hz, 2H), 1.21 (t, J¼7.0 Hz, 3H); 13C NMR (75 MHz,
DMSO-d6)
d
¼14.5, 60.5, 115.4, 115.5, 122.5, 142.4 (br s), 146.0, 153.8,
157.9; HRMS (HESI, m/z) calcd for C12H14N6O3 (290.28) [MþH]þ
291.1200, found 291.1199.
14. For examples see: (a) Watson, D. J.; Dowdy, E. D.; Li, W.-S.; Wang, J.; Polniaszek,
R. Tetrahedron Lett. 2001, 42, 1827e1830; (b) Llauger, L.; Bergami, C.; Kinzel, O.
D.; Lillini, S.; Pescatore, G.; Torrisi, C.; Jones, P. Tetrahedron Lett. 2009, 50,
172e177; (c) Klein, J. E. M. N.; Perry, A.; Pugh, D. S.; Taylor, R. J. K. Org. Lett. 2010,
12, 3446e3449.
Acknowledgements
ꢁ
We would like to thank to Dr. Michal Malon for his assistance in
measuring and interpreting the NMR spectra. This work was sup-
ported by the Ministry of Education, Youth and Sports of the Czech
Republic (grants: MSM6198959216, MSM6198959218, ME09057,
CZ.1.07/2.3.00/09.027, CZ.1.05/2.1.00/03.0058, and CZ.1.07/2.3.00/
15. (a) Katrizky, A. R.; Zhao, X.; Hitchings, G. J. Synthesis 1991, 703e708; (b) Hill, B.;
Liu, Y.; Taylor, S. D. Org. Lett. 2004, 6, 4285e4288.
ꢀ
ꢀ
16. (a) Claramunt, R. M.; García, M. A.; Lopez, C.; Trofimenko, S.; Yap, G. P. A.; Al-
korta, I.; Elguero, J. Magn. Reson. Chem. 2005, 43, 89e91; (b) Litchman, W. M. J.
Am. Chem. Soc. 1979, 101, 545e547; (c) Chenon, M. T.; Coupry, C.; Grant, D. M.;
Pugmire, R. J. J. Org. Chem. 1977, 42, 659e661.
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20.0017) and Palacky University (grant: PrF_2011_019).