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. 2010 Mar 11;1(2):80–84. doi: 10.1021/ml100005j

Synthesis and In Vitro Evaluation of Imidazo[1,2-b]pyridazines as Ligands for β-Amyloid Plaques

Fanxing Zeng †, David Alagille §, Gilles D Tamagnan §, Brian J Ciliax ‡, Allan I Levey ‡, Mark M Goodman †,*
PMCID: PMC4007799  PMID: 24900181

Abstract

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A series of imidazo[1,2-b]pyridazine derivatives were synthesized and evaluated for binding to amyloid plaques in vitro using synthetic aggregates of Aβ1−40. Binding affinities of these compounds were found to range from 11.0 to >1000 nM, depending on the various substitution patterns in the 6-position and 2-position. 2-(4′-Dimethylaminophenyl)-6-(methylthio)imidazo[1,2-b]pyridazine (4) showed high binding affinity (Ki = 11.0 nM) and might be useful for the development of novel positron emission tomography radiotracers for imaging Aβ plaques.

Keywords: Imidazo[1,2-b]pyridazine; β-amyloid plaque; structure−activity relationship; positron emission tomography

Alzheimer's disease (AD) is a common cause of dementia, which is clinically characterized by progressive loss of memory and decline of cognitive function. The neuropathologic signature of AD generally revealed on postmortem brain examination is the massive deposit of β-amyloid plaques (Aβ) and neurofibrillary tangles (NFTs).1−3 Therefore, amyloid-specific imaging agents would likely prove useful to identify and follow individuals at risk for AD and to assist in evaluating the efficacy of new therapeutic approaches that act early on the neurodegenerative progress and inhibit the accumulation of amyloid plaques and NFTs.4−7

The development of amyloid-specific imaging agents is generally based on conjugated dyes, such as Congo red, Chrysamine G, and thioflavin-T, which have been used in fluorescent staining of plaques and tangles in postmortem AD brain sections. Several radioligands for positron emission tomography (PET) and single photon emission computerized tomography (SPECT), including [11C]PIB (Pittsburgh compound B),8−10 [11C]SB-13 (4-N-methylamino-4′-hydroxystilbene),11 [11C]AZD2184 (2-[6-(methylamino)pyridin-3-yl]-1,3-benzothiazol-6-ol),12 [18F]FDDNP (2-(1-{6-[(2-fluoroethyl)-(methyl)amino]-2-naphthyl}ethylidene)malononitrile),13,14 [18F]GE067 (2-(3′-fluoro-4′-(methylamino)phenyl)-6-hydroxybenzothiazole),15 [18F]BAY94-9172 (4-(N-methylamino)-4′-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)stilbene),16 [18F]AV-45 ((E)-4-(2-(6-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-methyl benzenamine),17,18 and [123I]IMPY (2-(4′-dimethylaminophenyl)-6-iodoimidazo[1,2-a]pyridine),19 have been tested in clinical trials and demonstrated the potential utility as in vivo imaging agents for Aβ plaque deposition in the living human brain (Figure 1).

Figure 1.

Figure 1

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Radioligands for Aβ plaque imaging with PET and SPECT in clinical trials.

In an attempt to develop more effective amyloid-specific imaging agents, we have chosen to investigate a novel series of imidazo[1,2-b]pyridazine derivatives. Imidazo[1,2-b]pyridazines are designed to be isosteric analogues of IMPY, of which CH adjacent to N on the six-membered heterocyclic ring was replaced with N. We reasoned that substitution of aryl CH by an imino group might be effective in reducing ligand lipophilicity, thus reducing ligand nonspecific binding. Additionally, an appropriately substituted pyridazine ring lends itself well to easy attachment of different groups via heteroaromatic nucleophilic displacement reaction. We report here the synthesis and in vitro evaluation of imidazo[1,2-b]pyridazines as potential imaging agents for detecting Aβ plaques.

The synthesis of imidazo[1,2-b]pyridazine derivatives is shown in Schemes 1 and 2. In all cases, the formation of imidazo[1,2-b]pyridazine backbone was accomplished through a condensation reaction between an α-bromoketone and a 3-amino-6-halopyridazine under a mild basic condition such as sodium bicarbonate. In fact, the successful formation of imidazo[1,2-b]pyridazine rings in good yield was due to the introduction of a halogen in the pyridazine ring. In 3-aminopyridazine, the ring nitrogen that is not adjacent to the amino group is the most nucleophilic. Thus, alkylation by the α-bromoketone takes place preferentially at this site, hampering an effective synthesis of the desired bicyclic product. This was overcome by placing a halogen in the ring, adjacent to the offending nitrogen, thus greatly reducing the nucleophilicity of this nitrogen and returning the preferential site of alkylation to the ring nitrogen adjacent to the amino function.

Scheme 1.

Scheme 1

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Scheme 2.

Scheme 2

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The synthesis of 6-substituted-2-phenylimidazo[1,2-b]pyridazines is shown in Scheme 1. 3-Amino-6-chloropyridazine20 and 3-amino-6-fluoropyridazine21 were prepared from 3,6-dichloro- and 3,6-difluoropyridazine by reaction with aqueous ammonia at 130 °C, respectively. While 3-amino-6-iodopyridazine could also be prepared from 3,6-diiodopyridazine using the same method, the yield was very low, and 3,6-diaminopyridazine was the major product. Alternatively, 3-amino-6-iodopyridazine was obtained in 81% yield by refluxing 3-amino-6-chloropyridazine in 57% HI solution.22 α-Bromoketone 1 was prepared from 4′-dimethylaminoacetophenone through dibromination and debromination procedures reported previously.23 Nucleophilic substitution of the 6-chloro derivative 2b by methoxide, thiomethoxide, and deprotonated fluoroethanol, fluoropropanol, 2-mercaptoethanol, and 3-mercaptopropanol afforded 3−8 in good to excellent yields. 2-Fluoroethylthio and 3-fluoropropylthio compounds (9 and 10) were obtained by the treatment of 7 and 8 with DAST, respectively.

The free amino derivatives, 12a−c, were readily prepared from 11a−c by reduction with SnCl2. Conversion of 12a and 12c to their N-methylamino derivatives was achieved by a selective monomethylation reaction using sodium methoxide and paraformaldehyde, followed by a reduction reaction with NaBH4. However, the 6-chloro group of 12b was also converted to methoxyl group under the same condition to give 13b as the only product. Alternatively, 14 was prepared by methylation by MeI in the present of K2CO3, while dimethylation product 2b was also observed in this reaction.

The synthesis of 6-halogen-2-pyridinylimidazo[1,2-b]pyridazines and 6-halogen-2-thiophenylimidazo[1,2-b]pyridazines, based on the similar method shown above for 6-substituted-2-phenylimidazo[1,2-b]pyridazines, is outlined in Scheme 2. α-Bromoketone 15 was prepared by α-bromination of 1-(5-(dimethylamino)pyridin-2-yl)ethanone with pyridinium tribromide. α-Bromoketone 17 was prepared by bromination of 2-acetyl-5-nitrothiophene with copper(I) bromide.24

In vitro binding affinity of imidazo[1,2-b]pyridazines for Aβ plaques was determined via the binding competition with [3H]BTA-1 (2-(4′-(dimethylamino)phenyl)benzothiazole) using synthetic aggregates of Aβ1−40. IMPY and PIB were also screened under the same assay system for comparison. Initially, we prepared 6-halogen N,N-dimethylamino, N-methylamino, and primary amino analogues. Comparison of the Ki values (Table 1) reveals that the tertiary amino analogues typically have a higher affinity than their secondary amino analogues (2a vs 13a, 2b vs 14, and 2c vs 13c), in accord with the previous data on the tertiary and secondary amino analogues of IMPY,25 while the corresponding primary amino analogues show a much lower affinity. Therefore, we decided to focus our efforts on tertiary analogues and synthesized several 6-substituent N,N-dimethylamino analogues. The 6-iodo N,N-dimethylamino analogue has a higher affinity than the 6-chloro or 6-fluoro analogue, and this trend was repeated in the corresponding secondary amino and primary amino analogues (2c vs 2b vs 2a, 12c vs 12b, and 13c vs 14 vs 13a). This indicates that the size and electronegativity of the halogen atom presumably influence the binding of these ligands. Substitution of aryl CH of IMPY by an imino group as in 2c is effective in reducing ligand lipophilicity (compare cLog P value with that of IMPY) but slightly reduced binding affinity. The 6-methylthio analogue (4) shows a higher affinity over the 6-methoxyl analogue (3). Incorporation of ω-fluoroethyl or ω-fluoropropyl group, as reflected in 5, 6, 9, and 10, decreases binding affinity. To further study the structure−activity relationship (SAR) of imidazo[1,2-b]pyridazines, we synthesized 16a, 16b, 16c, 20a, and 20b with the replacement of phenyl ring with pyridinyl or thiophenyl ring. Unfortunately, these ligands show significantly reduced binding affinity, indicating that the phenyl ring might be required to retain high binding affinity. Among all of the ligands tested, the 6-methylthio analogue (4), which exhibits a high binding affinity (Ki = 11.0 nM) comparable to the values for PIB and IMPY, might be of interest for evaluation as a prospective PET radioligand if labeled with carbon-11 in N-methyl or S-methyl position. Four is also expected to have a higher target-to-background ratio for plaque labeling of an AD brain with regard to its reduced lipophilicity, as compared to IMPY and its derivatives. It is interesting to mention that the 6-methylthio IMPY derivative was reported to have a low retention to Aβ in AD patients.26

Table 1. Inhibition Constant (Ki Values) of Imidazo[1,2-b]pyridazines for Binding Synthetic Amyloid Plaques Aβ 1−40.

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ligand R1 R2 R3 Ar Ki (nM)a cLog Pb
2a F CH3 CH3 phenyl 41.4 ± 1.8 3.00
2b Cl CH3 CH3 phenyl 34.2 ± 1.2 3.57
2c I CH3 CH3 phenyl 18.4 ± 0.4 3.98
3 OCH3 CH3 CH3 phenyl 18.3 ± 3.3 3.06
4 SCH3 CH3 CH3 phenyl 11.0 ± 1.2 3.54
5 O(CH2)2F CH3 CH3 phenyl 32.0 ± 1.8 3.32
6 O(CH2)3F CH3 CH3 phenyl 47.5 ± 1.8 3.54
9 S(CH2)2F CH3 CH3 phenyl 35.5 ± 9.9 3.80
10 S(CH2)3F CH3 CH3 phenyl 38.8 ± 1.2 4.03
12b Cl H H phenyl 470 ± 10 1.68
12c I H H phenyl 280 ± 46 2.66
13a F CH3 H phenyl 91.4 ± 4.2 2.42
13c I CH3 H phenyl 34.0 ± 1.1 3.40
14 Cl CH3 H phenyl 78.8 ± 2.5 2.99
16a F CH3 CH3 pyridinyl >1000 2.24
16b Cl CH3 CH3 pyridinyl 280 ± 6 2.81
16c I CH3 CH3 pyridinyl 233 ± 6 3.22
20a F CH3 CH3 thiophenyl 550 ± 23 2.93
20b Cl CH3 CH3 thiophenyl 336 ± 11 3.50
PIB         11.0 ± 0.2 3.99
IMPY         9.0 ± 0.9 4.91
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a

Measured in at least three independent experiments, each performed in triplicate with results given as the mean ± SD.

b

Calculated in ChemDraw Ultra 11 (CambridgeSoft, Cambridge, MA).

In summary, we have synthesized a series of imidazo[1,2-b]pyridazines with various substitution patterns in the 6-position and 2-position. The SAR studies of these compounds with regard to Aβ showed that 2-N,N-dimethylaminophenyl moiety might be a requirement for these compounds to exhibit desirable binding affinities. The Ki values of 2-dimethylaminophenyl imidazo[1,2-b]pyridazines with different substituents in the 6-position range from 10 to 50 nM, indicating moderate tolerance for modification at this location. The 6-methylthio analogue (4) exhibited high binding to Aβ plaques in vitro and might be interest for evaluation as a prospective PET radioligand if labeled with carbon-11 in the N-methyl or S-methyl position.

Abbreviations

AD, Alzheimer's disease; Aβ, β-amyloid plaques; NFTs, neurofibrillary tangles; PET, positron emission tomography; SPECT, single photon emission computerized tomography; PIB, Pittsburgh compound B; SB-13, 4-N-methylamino-4′-hydroxystilbene; FDDNP, 2-(1-{6-[(2-fluoroethyl)-(methyl)amino]-2-naphthyl}ethylidene)malononitrile; GE067, 2-(3′-fluoro-4′-(methylamino)phenyl)-6-hydroxybenzothiazole; BAY94-9172, 4-(N-methylamino)-4′-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)stilbene; AV-45, (E)-4-(2-(6-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-methyl benzenamine; IMPY, 2-(4′-dimethylaminophenyl)-6-iodoimidazo[1,2-a]pyridine; AZD2184, 2-[6-(methylamino)pyridin-3-yl]-1,3-benzothiazol-6-ol; BTA-1, 2-(4′-(dimethylamino)phenyl)benzothiazole; SAR, structure−activity relationship.

Supporting Information Available

Procedure for the preparation of new ligands, analytical data, and in vitro binding assay. This material is available free of charge via the Internet at http://pubs.acs.org.

Supplementary Material

ml100005j_si_002.pdf (77.8KB, pdf)

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Associated Data

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Supplementary Materials

ml100005j_si_002.pdf (77.8KB, pdf)

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