2-Aminopyrimidine-Containing H4R Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6579
histamine receptor family. Mol. Pharmacol. 2001, 59, 427–433. (c)
Zhu, Y.; Michalovich, D.; Wu, H.-L.; Tan, K. B.; Dytko, G. M.;
Mannan, I. J.; Boyce, R.; Alston, J.; Tierney, L. A.; Li, X.; Herrity,
N. C.; Vawter, L.; Sarau, H. M.; Ames, R. S.; Davenport, C. M.;
Hieble, J. P.; Wilson, S.; Bergsma, D. J.; Fitzgerald, L. R. Cloning,
expression, and pharmacological characterization of a novel human
histamine receptor. Mol. Pharmacol. 2001, 59, 434–441. (d) Liu, C.;
Ma, X.-J.; Jiang, X.; Wilson, S. J.; Hofstra, C. L.; Blevitt, J.; Pyati,
J.; Li, X.; Chai, W.; Carruthers, N.; Lovenberg, T. W. Cloning and
pharmacological characterization of a fourth histamine receptor (H4)
expressed in bone marrow. Mol. Pharmacol. 2001, 59, 420–426.
combined CH2Cl2 layers were dried (MgSO4), filtered, concentrated,
and crystallized from EtOAc to provide 14.9 g (71%) of the title
compound. 1H NMR (CDCl3, δ): 2.34 (s, 3H), 2.45 (m, 4H), 3.60
(m, 4H), 4.82 (s, 2H), 5.96 (s, 1H). MS (DCI/NH3) m/z: 228 (M +
H)+. Anal. Calcd for (C9H14ClN5 ·0.06CH2Cl2): C, H, N.
Example of Suzuki Conditions. 4-(4-Methylpiperazin-1-yl)-
6-p-tolylpyrimidin-2-ylamine (64). A vial containing compound
46 (0.10 g, 0.44 mmol) was treated with 4-methylphenylboronic
acid (90 mg, 0.66 mmol), Pd(PPh3)4 (51 mg, 0.044 mmol), 1,2-
dimethoxyethane (1.3 mL), and 2 M Na2CO3 (0.48 mL, 0.97 mmol)
and was degassed with a stream of N2 for 1 min. The vial was
sealed and was heated to 90 °C for 16 h, was cooled, was treated
with 1 M NaOH, and was extracted with CH2Cl2 (2 × 25 mL).
The combined CH2Cl2 layers were dried (MgSO4), filtered, and
concentrated. The residue was purified by preparative HPLC on a
Waters Symmetry C8 column (40 × 100 mm2, 7 µm particle size)
by the use of a gradient of 10-100% acetonitrile and 0.1% aqueous
TFA over 12 min (15 min run time) at a flow rate of 70 mL/min.
The purified compound was converted to the free base by
chromatography on silica gel by elution with 2, 3.5, and 5% MeOH/
concd NH4OH (9:1) in CH2Cl2 to provide 94 mg (75%) of the title
(3) (a) Zhang, M.; Thurmond, R. L.; Dunford, P. J. The histamine H4
receptor: a novel modulator of inflammatory and immune disorders.
Pharmacol. Ther. 2007, 113, 594–606. (b) Damaj, B. B.; Becerra,
C. B.; Esber, H. J.; Wen, Y.; Maghazachi, A. A. Functional expression
of H4 histamine receptor in human natural killer cells, monocytes,
and dendritic cells. J. Immunol. 2007, 179, 7907–7915.
(4) (a) Dunford, P. J.; Williams, K. N.; Desai, P. J.; McQueen, D.;
Karlsson, L.; Thurmond, R. Histamine H4 receptor antagonists are
superior to traditional antihistamines in the attenuation of experimental
pruritus. J. Allergy Clin. Immunol. 2007, 119, 176–183. (b) Rees, J.;
Murray, C. S. Itching for progress. Clin. Exp. Dermatol. 2005, 30,
471–473. (c) Thurmond, R. L.; Gelfand, E. W.; Dunford, P. J. The
role of histamine H1 and H4 receptors in allergic inflammation: the
search for new antihistamines. Nat. ReV. Drug DiscoVery 2008, 7,
41–53.
(5) (a) Ling, P.; Ngo, K.; Nguyen, S.; Thurmond, R. L.; Edwards, J. P.;
Karlsson, L.; Fung-Leung, W. P. Histamine H4 receptor mediates
eosinophil chemotaxis with cell shape change and adhesion molecule
upregulation. Br. J. Pharmacol. 2004, 142, 161–171. (b) Buckland,
K. F.; Williams, T. J.; Conroy, D. M. Histamine induces cytoskeletal
changes in human eosinophils via the H4 receptor. Br. J. Pharmacol.
2003, 140, 1117–1127.
(6) Jablonowski, J. A.; Grice, C. A.; Chai, W.; Dvorak, C. A.; Venable,
J. D.; Kwok, A. K.; Ly, K. S.; Wei, J.; Baker, S. M.; Desai, P. J.;
Jiang, W.; Wilson, S. J.; Thurmond, R. L.; Karlsson, L.; Edwards,
J. P.; Lovenberg, T. W.; Carruthers, N. I. The first potent and selective
non-imidazole human histamine H4 receptor antagonists. J. Med. Chem.
2003, 46, 3957–3960.
1
compound. H NMR (CDCl3, δ): 2.34 (s, 3H), 2.39 (s, 3H), 2.48
(t, J ) 5.4 Hz, 4H), 3.68 (t, J ) 5.1 Hz, 4H), 4.83 (bs, 2H), 6.33
(s, 1H), 7.23 (d, J ) 8.1 Hz, 2H), 7.80 (d, J ) 8.5 Hz, 2H). MS
(DCI/NH3) m/z: 284 (M + H)+. Anal. Calcd for (C16H21N5 ·
0.06CH2Cl2): C, H, N.
Example of Ullmann Conditions. 4-(4-Methylpiperazin-1-yl)-
6-(4-phenylimidazol-1-yl)pyrimidin-2-ylamine (95). A mixture of
compound 15 (160 mg, 0.5 mmol), 4-phenylimidazole (87 mg, 0.6
mmol), CuI (25 mg, 0.13 mmol), and K2CO3 (90 mg, 0.65 mmol)
in DMF (0.5 mL) was heated to 135 °C overnight, was cooled,
was treated with 1 M NaOH (10 mL), and was extracted with
CH2Cl2 (3 × 25 mL). The combined CH2Cl2 layers were dried
(MgSO4), filtered, and concentrated. The residue was purified by
preparative HPLC on a Waters Symmetry C8 column (40 × 100
mm2, 7 µm particle size) by the use of a gradient of 10-100%
acetonitrile and 0.1% aqueous TFA over 12 min (15 min run time)
at a flow rate of 70 mL/min. The purified compound was converted
to the free base via chromatography (2, 3.5, and 5% MeOH/NH4OH
(9:1) in CH2Cl2) to provide 87 mg (52%) of the title compound.
1H NMR (CDCl3, δ): 2.36 (s, 3H), 2.50 (t, J ) 5.1 Hz, 4H), 3.69
(t, J ) 4.7 Hz, 4H), 4.88 (bs, 2H), 5.93 (s, 1H), 7.27 (m, 1H), 7.40
(t, J ) 7.5 Hz, 2H), 7.81-7.87 (m, 3H), 8.34 (d, J ) 1.4 Hz, 1H).
MS (DCI/NH3) m/z: 336 (M + H)+. Anal. Calcd for (C18H21N7 ·0.25
CH2Cl2 ·0.5CH3OH): C, H, N.
(7) Thurmond, R. L.; Desai, P. J.; Dunford, P. J.; Fung-Leung, W. P.;
Hofstra, C. L.; Jiang, W.; Nguyen, S.; Riley, J. P.; Sun, S.; Williams,
K. N.; Edwards, J. P.; Karlsson, L. A potent and selective histamine
H4 receptor antagonist with anti-inflammatory properties. J. Pharmacol.
Exp. Ther. 2004, 309, 404–413.
(8) Intracellular calcium responses were measured in cell lines that
coexpressed Gqi5 and human, rat, or mouse H4 receptors by the use of
FLIPR methods, as previously described, with modifications.8a Briefly,
we modified the FLIPR assay by setting the FLIPR temperature to 37
°C, and cell plates and drug plates were prewarmed in a 37 °C
incubator for 10 min before being placed in the FLIPR because
responses in these cell lines were dramatically increased at this
temperature compared with ambient temperature. Chlorpheniramine
(10 uM) was also added to the wash buffer to mask any H1-receptor-
mediated responses. We obtained agonist potencies (EC50) and
efficacies (percent max response) of H4R ligands by measuring peak
responses, which were expressed as a percentage of the maximal
response that was observed for 100 mM histamine. We obtaned
antagonist potencies (Kb) by measuring the inhibition of a reference
response (1000, 300, and 100 nM histamine for rat, mouse, and human
H4 cell lines, respectively). Antagonists were added 5 min prior to
the addition of histamine. Peak response values for the antagonist wells
were expressed as a percentage of the reference-peak response for
histamine in the absence of H4-receptor antagonists. We performed
experiments in duplicate wells, and we analyzed data by using
GraphPad Prism to obtain EC50, percent max, and IC50 values. The
generalized Cheng-Prusoff equation8b,c was used to determine Kb
values, which are presented as the-log10(mean) ( SEM (standard error
of the mean). (a) Esbenshade, T. A.; Krueger, K. M.; Miller, T. R.;
Kang, C. H.; Denny, L. I.; Witte, D. G.; Yao, B. B.; Fox, G. B.; Faghih,
R.; Bennani, Y. L.; Williams, M.; Hancock, A. A. Two novel and
selective nonimidazole histamine H3 receptor antagonists A-304121
and A-317920: I. In vitro pharmacological effects. J. Pharmacol. Exp.
Ther. 2003, 305, 887–896. (b) Cheng, Y. C.; Prusoff, W. Relationship
between the inhibition constant Ki and the concentration of inhibitor
which causes 50% inhibition (IC50) of an enzymatic reaction. Biochem.
Pharmacol. 1973, 22, 2099–2108. (c) Leff, P.; Dougall, I. G. Further
concerns over Cheng-Prusoff analysis. Trends Pharmacol. Sci. 1993,
14, 110–112.
Acknowledgment. We thank Marina I. Strakhova, Tracy L.
Carr, Arlene M. Manelli, Rahul Sharma, David J. Anderson,
Anthony V. Daza, Eric Johnson, Matthias Mayrer, and Gilbert
J. Diaz for their in vitro analysis; and Anita K. Salyers, Prasant
Chandran, Madhavi Pai, and Chang Z. Zhu for their in vivo
analysis of compound 4.
Supporting Information Available: Combustion analysis, full
synthetic experimental section, and description of biological
methods. This material is available free of charge via the Internet
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