New 1 H-pyrazole-4-carboxamides with antiplatelet activity

Article abstract of DOI:10.1002/ardp.200800181

Nine title compounds were synthesized and investigated in the Born test for their antiplatelet activities against collagen, ADP, adrenaline, and platelet activating factor (PAF) as inducers of the aggregation. Using collagen three compounds with IC₅₀

Full text of DOI:10.1002/ardp.200800181

Arch. Pharm. Chem. Life Sci. 2009, 342, 27–33
K. Rehse et al.
27
Full Paper
New 1H-Pyrazole-4-Carboxamides with Antiplatelet Activity
Klaus Rehse1, Joscha Kotthaus2, and Laleh Khadembashi1
1 Institut fꢀr Pharmazie, Freie Universitꢁt Berlin, Berlin, Germany
2 Pharmazeutisches Institut, Universitꢁt Kiel, Kiel, Germany
Nine title compounds were synthesized and investigated in the Born test for their antiplatelet
activities against collagen, ADP, adrenaline, and platelet activating factor (PAF) as inducers of
the aggregation. Using collagen three compounds with IC50 values below 100 lM were found
(3b, 3e, 3i). Activities in nanomolar concentrations were observed against ADP (3b, IC50 = 9.4 nM),
adrenaline (3i, IC50 = 5.8 nM), and platelet activating factor (3e, IC50 = 0.45 nM).
Keywords: ADP / Adrenaline / Antiplatelet properties / 1H-Pyrazole-4-carboxamides / PAF antagonism /
Received: March 14, 2008; accepted: September 29, 2008
DOI 10.1002/ardp.200800181
Introduction
In a number of previous publications, we were able to
show that the substitution of heterocycles rich in nitro-
gen like purines [1], indazoles [2], triazoles [3], oxadia-
zoles [4], imidazoles [5], pyrimidocinnolines [6], phthala-
zines [7], or thiazoles [8] with a carboxamide partial struc-
ture in addition to basic groups leads to a wide variety of
compounds with antiplatelet activities in micromolar
concentrations. In this paper, we wish to report a num-
ber of pyrazole derivatives fulfilling these structural
requirements and, consequently, were promising to
show remarkable antiplatelet activities.
Results and discussion
Chemistry
Scheme 1. Synthesis of type 3 pyrazole-4-carboxamides.
The synthesis of the title pyrazole-4-carboxamides is
shown in Scheme 1. Starting material is the commer-
cially available ethyl 5-amino-1-phenyl-1H-4-pyrazolecar-
boxylate 1. This compound is converted with 4-chloro-
phenylsulfonic acid chloride to the sulfonamide 2. Ami-
nolysis with suitable amines R-NH2 yielded the type 3 title
compounds.
Correspondence: Klaus Rehse, Institut fꢀr Pharmazie, Freie Universitꢁt
Berlin, D-14195 Berlin, Germany.
E-mail: rehiwer@zedat.fu-berlin.de
Biology
The antiplatelet effects obtained in the Born test with
compounds 3a–3i are summarized in Table 1. As
inducers of the platelet aggregation, we used collagen,
adenosindiphosphate (ADP), adrenaline, or platelet acti-
vating factor (PAF), respectively. The rationale for design-
Fax: + 49 30 838-53251
Abbreviations: NECA = 5-(N-ethylcarboxamido) adenosine; platelet acti-
vating factor (PAF); platelet-rich plasma (PRP); platelet-poor plasma
(PPP)
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K. Rehse et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 27–33
Table 1. Inhibition of platelet aggregation induced by collagen, adrenaline, ADP, or PAF by selected type 3 pyrazole-4-carboxa-
mides.
Compound
R
Collagen
IC50 (lM) ADP
Adrenaline
PAF
3a
3b
3c
3d
3e
3f
3g
3h
H3C-NH-(CH2)2-
H5C2-NH-(CH2)2-
H9C4-NH-(CH2)2-
H13C6-NH-(CH2)2-
Cyclohexyl-NH-(CH2)3-
Ph-NH-(CH2)2-
Ph-CH2-piperidin-4-yl
H3CO-(CH2)2-
H3CO-(CH2)3-
–
110
> 300
95
> 300
28
120
125
105
50
175
–
0.5
0.094
215
30
94
45
10
> 300
240
0.58
1.25
30
14
75
0.12
1.05
0.0058
–
–
2
7.5
55
0.00045
56
56
65
125
0.54
–
32
12.5
3i
Asa
–
–
–
NECA§
–
–
–
1
Phentolamine
Apafant (WEB-2086)
–
–
–
–
–
0.6
Incubation time 209, Standard deviation f10%; § NECA = 5-(N-Ethylcarboxamido)-adenosine.
Table 1a. Parameters for the rule-of-five for pyrazoles 3a–3i.
Compound
Molecular weight
(g/mol)
clogP
Number of H-bond Number of H-bond Chance of good oral
acceptors (N/O)
donors (NH/OH)
biovailability proposed
3a
3b
3c
3d
3e
3f
3g
3h
3i
433.9
447.9
476.0
504.1
516.1
496.0
536.1
434.9
448.9
2.14
2.16
3.22
4.23
4.31
3.83
4.47
2.33
2.61
8
8
8
8
8
8
8
8
8
3
3
3
3
3
3
2
2
2
yes
yes
yes
yes
yes
yes
yes
yes
yes
Lipohilicity was calculated using the software ”molinspiration property calculator” (Molinspiration Cheminformatics, Slovensky
Grob, Slovak Republic). Violations of the rule-of-five are illustrated in bold style.
ing compounds 3a–3e was to check the influence of R = specific adrenaline antagonist. In contrast, 3e (IC50
(cyclo)alkylaminoalkyl. In 3f and 3g, the presence of an 0.45 nM) is a potent and specific PAF antagonist.
=
aromatic moiety in R was investigated. Compounds 3h
Since this is our last paper in the series of antiplatelet
and 3i were synthesized to test the suitablility of a meth- agents, a comprehensive view on structure-activity rela-
ylether function with different spacers instead of alkyl- tionships appears indicated. As this has already been
amino groups for antiplatelet activities.
done for collagen as inducer of the platelet aggregation
In general, we observe only small effects against colla- [8], the effects against ADP, adrenaline, and PAF (platelet
gen. It is, however, interesting that 3e with a cyclohexyla- activating factor) will be discussed. Therefore, Figure 1
minopropyl function which was here selected from the summarizes the most active compounds for each hetero-
thiazole derivatives [8] is as well the most active com- cyclic system identified in our previous investigations
pound with respect to collagen.
including the characteristic positions of the substituents.
A very thrilling pattern of effects was observed with The standard inhibitors are NECA = 5-(N-ethylcarbox-
inducers other than collagen. When ADP is used, com- amido) adenosine, IC50 = 1 lM against ADP, phentol-
pound 3b shows an IC50 = 94 nM. Compounds 3a and 3i amine (IC50 = 2 lM against adrenaline), or apafant (WEB
still show an activity a1 lM. The other compounds show 2086) IC50 = 0.6 lM against PAF.
smaller effects between IC50 = 30–215 lM. As 3b addition-
The important structural features are an aromatic het-
ally has an IC50 = 0.58 lM and 3i an IC50 = 5.8 nM against erocycle with several nitrogen and sulfur atoms. This het-
adrenaline both compounds can be defined as mixed erocycle has two or three substituents with the following
ADP / adrenaline antagonists. Compound 3g appears as properties: (i) A basic moiety especially –NH-(CH2)3-NH-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 27–33
1H-Pyrazole-4-Carboxamides
29
Figure 1. Structure of the most active compounds in each class of heterocycles.
cyclohexyl or 1-pyrrolidinylpropyl-amino either bound
The first two rests are essential while the third one is
directly or via a carboxamido group to the heterocycle; an additional option (see Figure 1, purines, pyrazole,
(ii) an aromatic moiety bound via a sulfonamido or car- imidazole).
boxamido group or connected directly to the heterocycle.
Using this rationale, a large number of antiplatelet
This shows that these groups are suitable spacers but not agents were obtained. For each inducer of the platelet
essential for the antiplatelet activity. (iii) A hydrophobic aggregation (ADP, adrenaline, PAF) the four most power-
group, i. e. a phenyl or benzyl rest.
ful inhibitors so far found are listed in Tables 2/2a, 3/3a,
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K. Rehse et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 27–33
Table 2. Inhibition of platelet aggregation induced by ADP.
Rank
Ref.
Hetero-cyclus
Amide moiety Aromatic moiety
Basic moiety
IC50 (lM)
1
2
3
4
[1]
[8]
[1]
[8]
purine
thiazole
purine
CONH
CONH, SO2NH 4-chloro-phenyl
CONH 4-(R-NH-SO2)phenyl
CONH, SO2NH 4-chloro-phenyl
3-cyano-phenyl
1-pyrrolidinyl-(CH2)3-
cyclohexyl-NH-(CH2)2-
1-pyrrolidinyl-(CH2)2-
cyclohexyl-NH-(CH2)4-
0.00045
0.0022
0.0035
0.0053
thiazole
Standard inhibitor NECA = 5-(N-ethylcarboxamido)-adenosine, IC50 = 1 lM.
Table 2a. Parameters for the Rule-of-Five.
Compound
Rank
Molecular
weight (g/mol)
clogP
Number of H-bond Number of H-bond Chance of good oral
acceptors (N/O)
donors (NH/OH)
biovailability
1
2
3
4
480.6
457.0
592.7
485.1
4.34
3.65
3.66
4.12
9
7
12
7
2
3
3
3
yes
yes
no
yes
Inhibition of platelet aggregation induced by ADP.
Table 3. Inhibition of platelet aggregation induced by adrenaline.
Rank
Ref.
Hetero-cyclus
Amide moiety Aromatic moiety
Basic moiety
IC50 (lM)
1
2
3
4
[1]
[8]
[8]
Tab. 1
purine
CONH
CONH, SO2NH 4-fluoro-phenyl
CONH, CONH 4-fluoro-phenyl
CONH, SO2NH 4-chloro-phenyl
3-cyano-phenyl
cyclohexyl-NH-(CH2)3-
0.00018
0.0027
0.0028
0.0058
thiazole
thiazole
pyrazole
cyclohexyl-NH-(CH2)3-
cyclohexyl-NH-(CH2)3-
H3CO-(CH2)3-
Standard inhibitor phentolamine, IC50 = 2 lM.
Table 3a. Parameters for the Rule-of-Five.
Compound
Rank
Molecular weight
(g/mol)
clogP
Number of H-bond Number of H-bond Chance of good oral
acceptors (N/O)
donors (NH/OH)
biovailability
1
2
3
4
508.6
454.6
418.5
448.9
5.60
3.41
3.53
2.61
9
7
6
8
3
3
3
2
no
yes
yes
yes
Inhibition of platelet aggregation induced by adrenaline.
4/4a. Their chemical structure is given in columns to
In Table 4 finally, a majority of purine derivatives is
allow better comparison. Table 2 shows that only two concentrated. The only new heterocycle is a pyrazole
heterocycles, i. e. purine and thiazole, are the structural derivative which inhibits PAF with an IC50 = 0.45 nM.
basis for peak activities in low nanomolar concentration
During the evaluation of this paper it has been sug-
ranges. The basic moieties are similar to each other. The gested to determine the suitability for anticoagulant pur-
aromatic moieties are substituted with electron with- poses where a good absorption after oral administration
drawing residues. The binding to the central heterocycle is obligatory. This should be performed by the calcula-
is performed by amide bonds or directly. Table 3 offers tion of the lipophilicity (clogP) and extended to the
the adrenaline inhibitors. In addition to Table 2, now examination of the Rule-of-Five developed by Lipinski et
highly active pyrazole derivatives are found. Surpris- al. [11].
ingly, the methoxypropyl derivative 3i is active in spite of
It was recognized in Table 1a that, in the pyrazole ser-
lacking a basic moiety.
ies dealt with in this paper, all compounds (3a–3i) are
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Arch. Pharm. Chem. Life Sci. 2009, 342, 27–33
1H-Pyrazole-4-Carboxamides
31
Table 4. Inhibition of platelet aggregation induced by PAF.
Rank
Ref.
Hetero-cyclus
Amide moiety Aromatic moiety
Basic moiety
IC50 (lM)
1
2
3
4
Tab. 1
[1]
[1]
pyrazole
purine
purine
purine
SO2NH, CONH 4-chloro-phenyl
cyclohexyl-NH-(CH2)3-
cyclohexyl-NH-(CH2)3-
1-pyrrolidinyl-(CH2)3-
1-pyrrolidinyl-(CH2)3-
0.00045
0.001
0.035
CONH
CONH
CONH
3-cyano-phenyl
3-cyano-phenyl
2-furyl
[1]
0.074
Standard inhibitor Apafant (WEB 2086) IC50 = 0.6 lM.
Table 4a. Parameters for the Rule-of-Five.
Compound
Rank
Molecular weight
(g/mol)
clogP
Number of H-bond Number of H-bond Chance of good oral
acceptors (N/O)
donors (NH/OH)
biovailability
1
2
3
4
516.1
508.6
480.6
445.5
4.31
5.60
4.34
3.87
8
9
9
9
3
3
2
2
yes
no
yes
yes
Inhibition of platelet aggregation induced by PAF.
53.2; H, 4.2; N, 10.6. 1H-NMR (DMSO-d6): d (ppm) = 1.19 (t, J =
7.0 Hz, 3H, OCH2CH3), 4.01 (q, J = 7.1 Hz, 2H, OCH2), 7.40 (m, 9H,
arom.), 7.89 (s, 1H, pyrazole-3H). IR (KBr): k (cm– 1) = 3427; 3063;
2905; 2565; 2260; 1905; 1717 (CO); 1651; 1499; 1392; 1235; 1118;
976; 756; 693; 604. MS (1308C): m/z (%) = 405 (17) [M+9], 359 (34),
230 (13), 184 (100), 175 (41), 111 (38), 77 (68).
promising for a good oral bioavailability. Exeptions are
only previewed for compound 3 (Table 2a), compound 1
(Table 3a), and compound 2 (Table 2a) where 2 of 4 crite-
ria are violated, respectively. The remaining nine most
active compounds in Tables 2a, 3a, and 4a fulfill the crite-
ria for good absorption.
The correspondence to the Rule-of-Five may be an
important advantage of the newly discovered 1H-pyra-
zole-4-carboxamides. Altogether, the development of this
new class of anticoagulant compounds with the possibil-
ity of good oral bioavailabilities is promising.
General procedure for the synthesis of type-3
carboxamides
2.0 g 2 are suspended in 10–15 mL of the desired amine and
heated at 80–1008C with stirring for a time between three days
and three weeks. The progress of the reaction is controlled by
TLC on SIL G/UV254 plates (Augram1, Machery-Nagel, Germany)
with CH2Cl2 / ethylacetate / methanol saturated with NH3: 5 / 3 / 2
as eluent. After cooling to room temperature the product is dis-
solved in CH2Cl2 and extracted with water. The organic layer is
purified by column chromatography on silica gel (63–200 lm,
Merck, Germany). If the product remains in the aqueous layer, it
is precipitated either at pH = 7 or at pH = 1–2. If no precipitate
forms, the aqueous layer is extracted with ethyl acetate, dried
with Na2SO4, and concentrated in vacuo. After keeping it for
some days in the refrigerator at 58C, the crystals formed are
sucked off and washed with small amounts of ethyl acetate. If
necessary, the product is recrystallized from the solvent stated.
The authors have declared no conflict of interest.
Experimental
Chemistry
M.p. (uncorr.), Linstrꢀm apparatus (Bꢁhler, Tꢁbingen, Germany),
Elemental analysis: Elementar vario EL (Elementaranalysen Sys-
teme, Hanau, Germany), NMR: Bruker DPX 400 (Bruker Bio-
science, Billerica, MA, USA), EI-MS: CH-7A-Varian MAT (70 eV; Var-
ian Inc., Palo Alto, CA, USA), FAB-MS: CH-5-DF-MAT-Varian (cf.
references 1-8 for further details).
5-(4-Chlorophenylsulfonylamino)-N-(2-methylamino-
ethyl)-1-phenyl-1H-pyrazole-4-carboxamide 3a
From 4.0 g (10 mmol) 2, brown crystals (dichloromethane), m.p.:
5-(4-Chlorophenylsulfonylamino)-1-phenyl-1H-pyrazole-
4-carboxylicacidethylester 2
2028C, yield: 2.8 g (65%). – Anal. calcd. for C19H20ClN5O3S (433.7):
C, 52.6; H, 4.6; N, 16.1. Found C, 52.5; H, 4.5; N, 16.3. H-NMR
1
20 mmol of 1 are dissolved in 50 mL freshly destillated pyridine.
While stirring 20 mmol (9.1 g) 4-chlorobenzene-sulfonic acid-
chloride are added in portions. The mixture is heated to 1008C,
kept at this temperature for 72 h and then pored on cold water.
A pH = 1-2 is obtained with 20% hydrochloric acid. The precipi-
tate is sucked off, washed with water, and recrystallized from
ethanol. Brown crystals, m.p.: 1918C, yield: 11.2 g (70%). Anal.
calcd. for C18H16ClN3O4S (405.1): C, 53.3; H, 3.9; N, 10.4. Found: C,
(DMSO-d6): d (ppm) = 2.59 (s, 3H, H3CNH), 3.02 (t, J = 5.7 Hz, 2H,
CH3NHCH2CH2), 3.39 (dt, J = 5.8/5.8 Hz, 2H, CONHCH2CH2), 7.12
(m, 1H, 4-ph-H), 7.14 (“d”, J = 8.4 Hz, 2H, 3,5 ph-H), 7.18 (“d”, J =
7.8 Hz, 2H, 3,5-suph-H), 7.23 (“d”, J = 8.5 Hz, 2H, 2,6-ph-H), 7.44
(“d”, J = 7.5 Hz, 2H, 2,6-suph-H), 7.68 (s, 1H, pyrazole-3H), 8.30 (t,
J = 5,8 Hz, 1H, CONHCH2). IR (KBr): k (cm– 1) = 3434; 3013; 2746;
1953; 1834; 1650 (CO); 1542; 1458; 1370; 1232; 1127; 1014; 916;
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32
K. Rehse et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 27–33
816; 756; 705; 654. MS (2308C): m/z (%) = 433 (2) [M+.], 186 (71), 57
(63), 44 (100), 30 (13).
exchange, CONHCH2), 10.89 (brs, 1H, D2O exchange, SO2NH). IR
(KBr): m (cm– 1) = 3410; 2939; 2434; 1635 (CO); 1499; 1393; 1257;
1032; 943; 756; 620. MS (358C): m/z (%) = 515 (5) [M+9], 186 (23),
158 (100), 112 (18).
5-(4-Chlorophenylsulfonylamino)-N-(2-ethylaminoethyl)-
1-phenyl-1H-pyrazole-4-carboxamide 3b
From 4.0 g (10 mmol) 2, brown crystals (dichloromethane), m.p.:
5-(4-Chlorophenylsulfonylamino)-N-(2-phenylamino-
ethyl)-1-phenyl-1H-pyrazole-4-carboxamide 3f
2178C, yield: 2.7 g (60%). Anal. calcd. for C20H22ClN5O3S (447.5): C,
1
53.7; H, 4.9; N, 15.6. Found: C, 53.5; H, 4.9; N, 15.5. H-NMR
From 4 g (10 mmol) 2, light brown crystals (ethanol), m.p.:
2198C, yield: 3.0 g (60%). Anal. calcd. for C24H22ClN5O3S (495.1): C,
58.1; H, 4.4; N, 14.1. Found: C, 58.0; H, 4.5; N, 14.3. 1H-NMR
(DMSO-d6): d (ppm) = 3.19 (m, 4H, -CH2CH2NHCO), 6.79 (m, 3H,
arom.), 7.2 (m, 2H arom.), 7.41 (m, 9H arom.), 8.04 (s, 1H, pyra-
zole-3H), 8.14 (t, J = 5,7 Hz, 1H, D2O exchange, CONHCH2). IR
(KBr): m (cm– 1) = 3313; 2928; 2602; 1647 (CO); 1499; 1391; 1254;
1032; 894; 693. MS (2208C): m/z (%) = 495 (10) [M+9], 186 (52), 119
(100).
(DMSO-d6): d (ppm) = 1.17 (t, J = 7.2 Hz, 3H, H3CCH2), 2.97 (q, J =
7.2 Hz, 2H, CH2CH3), 3.03 (t, J = 5.8 Hz, 2H, CH2NHCH2CH2), 3.41
(dt, J = 5.8/5.8 Hz, 2H, CONHCH2CH2), 7.13 (m, 7H, 3,5 ph-H, 4-ph-
H, 3,5-suph-H, 2,6-ph-H), 7.43 (“d”, J = 7.5 Hz, 2H, 2,6-suph-H),
7.68 (s, 1H, pyrazole-3H), 8.23 (t, J = 5.8 Hz, 1H, partly D2O
exchange, CONHCH2), 8.62 (brs, 2H, D2O exchange, CH2N+H2CH2).
IR (KBr): m (cm– 1) = 3092; 2851; 2523; 2359; 2057; 1906; 1647
(CO); 1495; 1389; 1255; 1129; 1013; 916; 820; 693; 635. MS
(2108C): m/z (%) = 447 (2) [M+.], 186 (60), 71 (75), 58 (100), 30 (21).
N-(2-butylaminoethyl)-5-(4-chlorophenylsulfonylamino)-
1-phenyl-1H-pyrazole-4-carboxamide 3c
5-(4-Chlorophenylsulfonylamino)-1-phenyl-N-(1-phenyl-
methyl-4-piperidinyl)-1H-pyrazole-4-carboxamide
dihydrate 3g
From 5.2 g (13 mmol) 2, crystals (dichloromethane), m.p.: 2298C,
yield: 3.0 g (50%). Anal. calcd. for C22H26ClN5O3S (475.8): C, 55.5;
H, 5.5; N, 14.7. Found: C, 55.5; H, 5.4; N, 14.7. 1H-NMR (DMSO-d6):
d (ppm) = 0.87 (t, J = 7.2 Hz, 3H,CH3CH2), 1.31 (tq, J = 7.2/7.2 Hz,
2H, CH3CH2CH2), 1.55 (m, J = 7.8 Hz, 2H, CH3CH2CH2), 2.92 (t, J =
7.6 Hz, 2H, CONHCH2), 3.03 (t, J = 5.7 Hz, 2H, CH3CH2CH2-
CH2NHCH2), 3.42 (dt, J = 5.8 Hz, 2H, CONHCH2CH2), 7.12 (t, 1H, 4-
ph-H), 7.14 (“d”, J = 8.5 Hz, 2H, 3,5-ph-H), 7.18 (m, 6H, arom. 3,5-
suph-H, 2,6-ph-H, 2,6-suph-H), 7.69 (s, 1H, pyrazole-3H), 8.25 (t, J =
5.8 Hz, 1H, CONHCH2). IR (KBr): m (cm– 1) = 3059; 2934; 2783;
1956; 1637 (CO); 1526; 1451; 1259; 1087; 972; 822; 695; 614. MS
(408C): m/z (%) = 475 (2) [M+9], 359 (10), 186 (42), 86 (100), 30 (75).
From 5.0 g (12.5 mmol) 2, light yellow crystals (ethanol), m.p.:
1178C, yield: 1.7 g (25%). Anal. calcd. for C28H32ClN5O5S (585.2): C,
57.4; H, 5.5; N, 12.0. Found: C, 57.6; H, 5.2; N, 11.7. 1H-NMR
(DMSO-d6): d (ppm) = 1.33 (m, 2H, piperidine-3-H), 1.46 (m, 2H,
piperidine-5-H), 1.72 (m, 2H, piperidine-2-H), 1.83 (m, 2H, piperi-
dine-6-H), 2.05 (m, 1H, piperidine-4-H), 2.81 (m, 2H, CH2-piperi-
dine), 7.24 (m, 14H, arom.), 7.64 (s, 1H, pyrazole-3H), 8.26 (d, J =
7.3 Hz, 1H, CONH-pip). IR (KBr): m (cm– 1) = 3287 cm– 1; 2943; 2677;
2358; 1954; 1608 (CO); 1474; 1344; 1129; 1014; 911; 781; 674;
625. MS (2508C): m/z (%) = 549 (5) [M+9], 374 (26), 173 (31), 90 (100),
82 (46).
5-(4-Chlorophenylsulfonylamino)-N-(2-hexylaminoethyl)-
1-phenyl-1H-pyrazole-4-carboxamide monohydrate 3d
From 5.5 g (14 mmol) 2, crystals (dichloromethane), m.p.: 1888C,
yield: 0.3 g (5%). Anal. calcd. for C24H39ClN5O4S (521.8): C, 55.2; H,
6.2; N, 13.4. Found: C, 55.1; H, 5.9; N, 13.5. 1H-NMR (DMSO-d6): d
(ppm) = 0.83 (t, J = 6.8 Hz, 3H, CH3CH2), 1.27 (m, 6H, CH2CH2CH2-
CH2CH2CH3), 1.56 (m, J = 7.3 Hz, 2H, CH2NHCH2CH2CH2CH2CH2-
CH3), 2.93 (t, J = 7.7 Hz, 2H, CH2NHCH2(CH2)4CH3), 3.03 (t, J =
5-(4-Chlorophenylsulfonylamino)-N-(2-methoxyethyl)-1-
phenyl-1H-pyrazole-4-carboxamide semihydrate 3h
From 4.0 g (10 mmol) 2, brown crystals, m.p.: 1778C, yield: 2.7 g
(60%). Anal. calcd. for C19H20ClN4O4.5S (443.7): C, 51.4; H, 4.5; N,
12.6. Found: C, 51.3; H, 4.3; N, 12.3. 1H-NMR (DMSO-d6): d (ppm) =
3.13 (t, J = 5.7 Hz, 2H, CONHCH2CH2), 3.27 (s, 3H, OCH3), 3.29 (dt, J
= 5.9/5.9 Hz, 2H, CONHCH2CH2), 7.40 (m, 9H, arom.), 7.93 (t, J =
5.5 Hz, 1H, D2O exchange, CONHCH2), 8.02 (s, 1H, pyrazole-3H).
IR (KBr): m (cm– 1) = 3379; 2932; 2760; 1952; 1636 (CO); 1500;
1390; 1260; 1120; 968; 829; 662. MS (1508C): m/z (%) = 433 (14)
[M+9], 360 (49), 229 (16), 184 (100), 158 (100), 77 (30).
5.7 Hz, 2H, CONHCH2CH2), 3.42 (dt,
J = 5.7/5.7 Hz, 2H,
CONHCH2CH2), 7.13 (m, 7H, arom. 3,5-ph-H, 3,5-suph-H, 4-ph-H,
2,6-ph-H), 7.43 (“d”, J = 7.4 Hz, 2H, 2,6-suph-H), 7.69 (s, 1H, pyra-
zole-3H), 8.24 (t, J = 5.9 Hz, 1H, partly D2O exchange, CONHCH2),
8.32 (brs, 2H, D2O exchange, CH2N+H2CH2). IR (KBr): m (cm– 1) =
3032; 2857; 1943; 1626 (CO); 1496; 1376; 1170; 1014; 908; 783;
674. MS (408C): m/z (%) = 503 (1) [M+9], 333 (11), 186 (24), 158 (58),
99 (100), 30 (41).
5-(4-Chlorophenylsulfonylamino)-N-(3-methoxypropyl)-1-
phenyl-1H-pyrazole-4-carboxamide 3i
5-(4-Chlorophenylsulfonylamino)-N-(3-cyclohexylamino-
propyl)-1-phenyl-1H-pyrazole-4-carboxamide 3e
From 4.8 g (12 mmol) 2, light brown crystals, m.p.: 1508C, yield:
3.4 g (65%). Anal. calcd. for C20H21ClN4O4S (448.1): C, 53.5; H, 4.7;
N, 12.5. Found: C, 53.2; H, 4.9; N, 12.3.1H-NMR (DMSO-d6): d (ppm)
= 1.59 (quint, J = 6.6 Hz, 2H, CH2CH2CH2NHCO), 3.02 (dt, J = 6.5/
6.5 Hz, 2H CONHCH2), 3.24 (s, 3H, OCH3), 3.35 (t, J = 6.2 Hz, 2H,
OCH2), 7.39 (m, 9H, arom.), 7.86 (t, J = 5.5 Hz, 1H, D2O exchange,
CONHCH2), 7.99 (s, 1H, pyrazole-3H). IR (KBr): m (cm– 1) = 3019;
2930; 2754; 1839; 1626 (CO); 1499; 1388; 1248; 1092; 895; 693.
MS (1408C): m/z (%) = 448 (6) [M+], 359 (26), 230 (17), 184 (100), 158
(100), 77 (27).
From 4.9 g (12 mmol) 2, crystals by column chromatography,
m.p.: 1458C, yield: 0.2 g (3%). Anal. calcd. for C25H30ClN5O3S
(575.8): C, 52.3; H, 5.3; N, 12.3. Found: C, 52.7; H, 5.3; N, 12.3. 1H-
NMR (DMSO-d6): d (ppm) = 1.11 (m, 1H, 4a-cyhex-H), 1.26 (m, 4H,
2a, 3a, 5a, 6a-cyhex-H), 1.59 (m, 1H, 4e-cyhex-H), 1.78 (m, 4H, 2e,
3e, 5e, 6e-cyhex-H), 2.04 (m, 2H, CH2CH2CH2), 2.95 (brs, 3H, 1-
cyhex-H, NHCH2), 3.13 (dt, J = 6.2/6.2 Hz, 2H, CONHCH2CH2), 7.40
(m, 9H arom.), 8.04 (s, 1H, pyrazole-3H), 8.30 (t, J = 5.7 Hz, D2O
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2009, 342, 27–33
1H-Pyrazole-4-Carboxamides
33
test cuvette is below 0.3%, as in higher concentrations DMSO
itself is a platelet aggregation inhibitor. By dilution of the stem
solutions of the test compound in 1 : 1 steps with hepes buffer,
its concentration is bisected in each step and measured again so
that the percentage of aggregation as function of the concentra-
tion of the test compound is obtained. These values are plotted
in a semilogarithmic scale [% = f (lg c)] and the corresponding
aggregation curves obtained. Drawing a line at the 50% value
parallel to the x-axis yields an intersection with the aggregation
curve. At this point, a perpendicular is raised from the x-axis and
the IC50 value can be read off directly the x-axis. The standard
deviation is determined from the standard inhibitors with n =
10 and is found generally to be f10 rel.%. The assay with the test
compounds was mostly run in duplicate only, provided that the
difference of the values obtained was below 10%. The IC50 value
for the asa lysinate is 175 l 20 lM.
Biological assays
Born test
Preparation of the blood plasma
Freshly drawn venous human citrated blood (1 pt sodium citrate
solution 3, 13%, Fa. Eifelfango, Neuenahr, Germany, 9 pts blood)
from healthy subjects, who had not taken acetylsalicylic acid or
other drugs with antiplatelet activity for ten day, was centri-
fuged (Micro 20, A. Hettich GmbH, Tutlingen, Germany) with
100 g (800 rpm) for platelet-rich plasma (PRP) or 2000 g (12500
rpm; Biofuge A, Haereus, Hanau, Germany) for platelet-poor
plasma (PPP).
Platelet aggregation procedures are explicitly described in
references 4, 9, and 10.
Platelet aggregation induced by collagen
At first, the concentration of collagen fibrils which induce maxi-
mum aggregation of the platelets is determined. To 200 lL of
PRP, 20 lL of hepes buffer, i. e. 2-[4-(2-hydroxyethyl)-piperazin-1-
yl]-ethane-sulfonic acid 0.001 M (238.3 mg/L; Fa. Sigma-Alrich,
Germany) (without test compound) are added and the mixture
incubated 4 min at 37.48C. Now, the cuvette is put in the chan-
nel of the APACT aggregometer (Automated Platelet Aggregation
and Coagulation Tracer, Biochemica GmbH, Flacht, Germany)
with software APACT professional version 1.1. While automati-
cally stirred by a small magnet, 20 lL of the aggregation inducer
collagen Hormm, (Nycomed Pharma GmbH, Konstanz, Germany),
in hepes buffer which contain 0.25 lg fibrils are added. The solu-
tion is obtained by dilution of the stock solution containing
1 mg fibrils/mL with hepes. Now the change in light transmis-
sion is recorded and generally the maximum aggregation
response observed. To assure this, the procedure is repeated
with 0.32 lg fibrils/20 lL. On the other hand, the procedure is
repeated with 0.125 lg fibrils/20 lL or 0.16 lg fibrils/20 lL. To
check the correct function of the test system, the influence of a
standard aggregation inhibitor namely DL-lysine monoacetylsali-
cylate (Bayer, Germany) on the platelet aggregation is deter-
mined as if it were a test compound.
Platelet aggregation with other inducers
The final concentration of the inducer in the test cuvette and
the IC50 values of the standard inhibitors are (lM): ADP 0.5 – 1.0 /
NECA 1.0; adrenaline 0.1–1.0 / phentolaminemesylate 2.0; PAF
0.25 – 1.0/apafant (WEB 2086) 0,6.
References
[1] K. Mꢂrschenz, K. Rehse, Arch. Pharm. Chem. Life Sci. 2006,
339, 115–122.
[2] A. K. Yildiz, K. Rehse, J.-P. Stasch, E. Bischoff, Arch. Pharm.
Pharm. Med. Chem. 2004, 337, 311–316.
[3] A. Cwiklicki, K. Rehse, Arch. Pharm. Pharm. Med. Chem.
2004, 337, 156–163.
[4] K. Bethge, H. H. Pertz, K. Rehse, Arch. Pharm. Chem. Life Sci.
2005, 338, 78–86.
[5] K. Rehse, J. Steege, Arch. Pharm. Chem. Life Sci. 2005, 338,
539–547.
[6] K. Rehse, H. Gonska, Arch. Pharm. Chem. Life Sci. 2005, 338,
590–597.
The test compound (or the standard inhibitor) is dissolved in
hepes buffer. Then 20 lL of the test solution is given to 200 lL
PRP in the test cuvette and incubated 4 min at 37.48C. Then,
20 lL of the inducer in the concentration determined above is
added and the change in light transmission recorded. The per-
centage of aggregation is determined as the ratio of heights of
the aggregation curves with and without the test compound.
Each curve is corrected automatically for the light absorption of
platelet-poor plasma (PPP) of the same donor. If the test com-
pound is not totally soluble in hepes buffer, DMSO is added. It is
carefully assured that the final concentration of DMSO in the
[7] M. Johnsen, K. Rehse, H. Pertz, J.-P. Stasch, E. Bischhoff,
Arch. Pharm. Med. Chem. 2003, 336, 1–7.
[8] K. Rehse, T. Baselt, Arch. Pharm. Chem. Life Sci. Accepted for
publication.
[9] G. V. R. Born, Nature (London) 1962, 194, 927–929.
[10] F. Seuter, Haemostasis 1976, 5, 85–95.
[11] C. A. Lipinski, F. Lombardo, B. W. Domini, P. J. Feeney, Adv.
Drug Deliv. Rev. 1997, 23, 3–25.
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com