CRHR1 Receptor binding and lipophilicity of pyrrolopyrimidines, potential nonpeptide corticotropin-releasing hormone type 1 receptor antagonists

https://doi.org/10.1016/S0968-0896(01)00261-9Get rights and content

Abstract

A series of compounds related to N-butyl-N-ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidin-4-yl]amine (1, antalarmin, Fig. 1) have been prepared and evaluated for their CRHR1 binding affinity as the initial step in the development of selective high affinity hydrophilic nonpeptide corticotropin-releasing hormone type 1 receptor (CRHR1) antagonists. Calculated log P (Clog P) values were used to evaluate the rank order of hydrophilicity for these analogues. Introducing oxygenated functionalities (δ-hydroxy or bis-β-ethereal) into 1 gave more hydrophilic compounds, which had good affinity for the receptor. Introducing an amino group or shortening the alkyl side chain was detrimental to CRHR1 affinity. The alcohol 4-[ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidin-4-yl]amino]butan-1-ol (3), bearing a terminal hydroxyl group on an N-alkyl side-chain, showed the highest CRHR1 binding affinity among these compounds (Ki=0.68 nM), and is one of the highest affinity CRHR1 ligands known. Compounds 35, and 8, which are likely to be less lipophilic than 1, have high CRHR1 affinity and may be valuable probes to further study the CRH system.

The synthesis of a less lipophilic high affinity CRHR1 ligand is reported.

  1. Download : Download full-size image

Introduction

Corticotropin-releasing hormone (CRH) was isolated in 1981 from ovine hypothalamus extracts and characterized as a 41-amino acid neuropeptide.1 CRH stimulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis through the release of adrenocorticotropic hormone (ACTH),2, 3 coordinating the overall response of the body to stress. It mediates pituitary ACTH release, which in turn triggers the secretion of the adrenal steroid, cortisol. In addition, CRH is found throughout the central nervous system where it acts as a peptide neurotransmitter.4, 5, 6 The actions of CRH as a neurotransmitter are not fully understood but diverse neuropsychiatric and neurodegenerative diseases show changes in CRH secretion and action. In addition to its central effects, CRH also acts in the periphery as an immune cytokine. CRH acts in concert with cortisol to regulate inflammatory responses found in both infection and autoimmune disorders.7, 8, 9, 10, 11

To date, there are two classes of G-protein-coupled CRH receptors (CRHR1 and CRHR2) that have been characterized and cloned from mouse, rat, and human,12, 13, 14, 15, 16, 17 and there is a distinct regional distribution for them in the central nervous system (CNS). The broad central distribution of CRH and CRHR1 and CRHR2 supports CRH as an important peptide neurotransmitter within the CNS.4, 5, 6 Overproduction of CRH in the brain has been associated with mental disorders such as anxiety,18 depression,19 and substance abuse.20 Treatment of depressed patients with antidepressant or electroconvulsive therapy resulted in decreased cerebrospinal fluid CRH as well as in improvement in the clinical conditions of these patients. Overproduction of CNS CRH may also underlie some of the symptoms of affective and addictive disorders. Selective CRHR1 antagonists may be efficacious for the treatment of these disorders by blocking the effects of excess CRH on CRHR1.21 Furthermore, CRHR1 antagonists may prove to be useful research tools to probe CRH and stress related disorders.22

Several peptide CRHR antagonists including α-helical CRH (9–41), [D-Phe12, Nle22]CRH (12–41), and astressin have been synthesized and studied extensively.23, 24, 25 However, their potential clinical utility is limited because of poor CRHR1 selectivity and poor penetration through the blood–brain barrier (BBB). Therefore, a potent and selective nonpeptide CRHR1 antagonist with good BBB penetration could be a versatile tool to further investigate the biological effects of CRH and CRHR1. In addition, such compounds would aid the discovery and development of future CRH-specific medications.

The pharmaceutical industry has led the development of CRHR1 antagonists as medications.26 The structure–activity relationships (SAR) for nonpeptide CRHR1 antagonists in a wide variety of structural classes have been investigated.26, 27, 28, 29, 30, 31, 32, 33, 34 Among them, the (4-dialkylamino)pyrrolo[2,3-d]pyrimidines, CP-154,526 (Fig. 1) and antalarmin (1, first described by Chen at Pfizer),35 show high affinity and selectivity for CRHR1 in binding assays and significant pharmacological effects in animal behavioral studies.30, 36, 37, 38, 39, 40, 41 Non-peptide CRHR1 receptor ligands, such as antalarmin41 and its analogue, CP154, 526,29 have been identified which are selective antagonists for CRHR1 receptors; they inhibit CRH-stimulation of cAMP or CRH-stimulated ACTH release from cultured rat anterior pituitary cells. However, these pyrimidines have poor solubility, possibly due to their high lipophilicity, which restricts their practical use as medications and as radiotracers. Our efforts42 to develop a positron emission tomography (PET) radiotracer for CRHR1 resulted in the synthesis of a fluoro-substituted analogue of 1 (Fig. 1). This analogue (2) showed subnanomolar affinity for CRHR1 (Ki=0.91 nM).42 However, only a small fraction of this compound rapidly penetrated the BBB, as shown by studies with [3H]2.43 As the initial step towards the important goal of obtaining CRHR1-selective antagonists capable of crossing the BBB, we have synthesized a number of new ligands in the pyrrolopyrimidine series and examined their affinity for the CRHR1 receptor.

High affinity ligands have been obtained in the pyrrolopyrimidines series, but attempts to decrease their lipophilicity resulted in lowering receptor affinity.28 In order to find high affinity CRHR1 antagonists, which were less lipophilic, we examined several of our formerly prepared pyrrolopyrimidine intermediate compounds, 3, 4,42 5,43 and 67.42 These, except for 6, had not been previously evaluated as CRHR1 ligands, and none were previously described chemically. We also synthesized a more oxygenated compound (8) that subsequently appeared in the literature.28 Further, several compounds in this series which we have synthesized (Figure 2, Scheme 1) have been mentioned, and their NMR data listed, in a patent,35 but neither their other chemical properties, nor any pharmacological data were mentioned therein.

We believed that if a sufficiently hydrophilic compound could be obtained it would be better able to cross the BBB than antalarmin (1), and if it also had comparable affinity with 1 for the CRHR1 receptor it might, then, be a useful template for further investigation. We hoped to find compounds with decreased lipophilicity while still retaining good affinity for the CRHR1 receptor, and to do that we assumed that the relative lipophilicities of the compounds were related to their calculated log P values (Clog P); these calculated values were used as a rough numerical descriptor to indicate the trend in lipophilicity for the various molecules. In our comparison of the lipophilicities of the compounds in Table 1, we anticipated that an increase in Clog P would indicate that we had obtained a more lipophilic compound, and a decrease would imply that we had prepared a more hydrophilic compound.

Section snippets

Chemistry

4-Chloro-2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidine (12) was prepared by modification of the route previously described by Chen.35 The amino alcohols, 4-ethylamino-1-butanol (13), 4-allylamino-1-butanol (14), and 3-(cyclopropylmethyl)amino-1-propanol (15), were prepared in 80–90% yield as previously described.42, 43

The preparation of β-hydroxyantalarmin Figure 2, Scheme 1 is shown in Scheme 1. Treating 12 with an excess amount of (2-butylamino)ethanol in dimethylformamide

Pharmacology and Discussion

The CRHR1 binding affinity and the calculated log P (Clog P) values of these compounds are listed in Table 1. The effect of a hydroxyl group on binding to the CRHR1 receptor was determined by its position on the nitrogen atom's side-chain. Thus, compound Figure 2, Scheme 1,35 the N-2-hydroxyethyl relative of antalarmin 1, had little or no affinity, and the isomeric N-4-hydroxybutyl compound 342 had the highest affinity (Ki=0.68 nM) of all of the evaluated compounds. Appropriate placement of the

Melting points (Table 1) were determined on a Thomas–Hoover capillary melting point apparatus and are uncorrected. Elemental analyses were performed by Atlantic Microlabs, Atlanta. Chemical ionization mass spectra (CIMS) were obtained using a Finnigan 1015 mass spectrometer. Electron ionization mass spectra (EIMS) and high-resolution mass measurements (HR-MS) were obtained using a V. G. Micro Mass 7070F mass spectrometer. Optical rotations were obtained using a Perkin–Elmer 341 polarimeter and

Acknowledgements

We thank Noel Whittaker and Wesley White of the Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, for the mass spectral data.

References (48)

  • C.P. Chang et al.

    Neuron.

    (1993)
  • N. Vita et al.

    FEBS Lett.

    (1993)
  • M.R. Irwin et al.

    Brain Behav. Immun.

    (1987)
  • D.T. Chalmers et al.

    Trends Pharmacol. Sci.

    (1996)
  • F. Holsboer

    J. Psychiatric Res.

    (1999)
  • Y.L. Chen et al.

    J. Med. Chem.

    (1997)
  • J.P. Beck et al.

    Bioorg. Med. Chem. Lett.

    (1999)
  • J.P. Beck et al.

    Bioorg. Med. Chem. Lett.

    (1999)
  • J. Lundkvist et al.

    Eur. J. Pharmacol.

    (1996)
  • L.-W. Hsin et al.

    Bioorg. Med. Chem. Lett.

    (2000)
  • A.J. Cocuzza et al.

    Bioorg. Med. Chem. Lett.

    (1999)
  • P.J. Gilligan et al.

    Bioorg. Med. Chem.

    (1999)
  • W. Vale et al.

    Science

    (1981)
  • Rivier, C.; Plotsky, P. M. In Annual Review of Physiology; Berne, R. M., Ed.; Academic Press: Palo Alto, 1986; Vol. 48,...
  • G.P. Chrousos

    N. Engl. J. Med.

    (1995)
  • Y. Kishimoto et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (1995)
  • E.L. Webster et al.

    Molec. Psychiatry

    (1997)
  • Koob, G. F.; Heinrichs, S. C.; Pich, E. M.; Menzaghi, F.; Baldwin, H.; Miczek, K.; Britton, K. T. In Proceedings of the...
  • M.J. Owens et al.

    Pharmacol. Rev.

    (1991)
  • Brown, M. R. In Stress, Neurobiology and Neuroendocrinology; Brown, M. R.; Koob, G. F., Eds.; Marcel Dekker: New York,...
  • R. Chen et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (1993)
  • M. Perrin et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (1995)
  • T.W. Lovenberg et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (1995)
  • M.H. Perrin et al.

    Endocrinology

    (1993)
  • Cited by (36)

    • Corticotropin-releasing hormone receptor-1 and 2 activity produces divergent resistance against stress-induced pulmonary Streptococcus pneumoniae infection

      2011, Journal of Neuroimmunology
      Citation Excerpt :

      In support of previous studies linking CRH to inflammatory disease etiology, studies have not only documented CRH receptor expressed by stromal inflamed tissues, but have also identified the expression of CRH and its receptors by immune cell populations (Webster et al., 1990; Cao et al., 2005; Gonzales et al., 2008; Zheng et al., 2009). With the identification and development of CRH receptor 1 and 2 antagonists (Slominski et al., 2001; Grammatopoulos and Chrousos, 2002; Hsin et al., 2002; Richard et al., 2002), studies have begun to uncover CRH's direct influence on the regulation of inflammatory processes (Wlk et al., 2002; Gao et al., 2007) For example, Wlk et al. (2002), showed that blockade of CRH-R1 abrogated disease pathogenesis in Toxin A-induced intestinal inflammation. In addition, CRH-R2 signaling has also been shown to alleviate inflammatory responses in the intestine and pulmonary tissues (Kokkotou et al., 2006; Moffatt et al., 2006; Poon et al., 2008).

    View all citing articles on Scopus

    Current address: School of Pharmacy, College of Medicine, National Taiwan University, No. 1 Sec. 1, Room 1336, Jen-Ai Road, Taipei, Taiwan.

    Current address: Procter & Gamble Pharmaceuticals, 8700Mason Montgomery Rd., Mason, OH 45040, USA.

    §

    Current address: University of North Carolina, Chapel Hill, NC, USA.

    View full text