Synthesis of σ Receptor Ligands with a Spirocyclic System Connected with a Tetrahydroisoquinoline Moiety via Different Linkers

Abstract With the aim to develop new σ2 receptor ligands, spirocyclic piperidines or cyclohexanamines with 2‐benzopyran and 2‐benzofuran scaffolds were connected to the 6,7‐dimethoxy‐1,2,3,4‐tetrahydroisoquinoline moiety by variable linkers. In addition to flexible alkyl chains, linkers containing an amide as functional group were synthesized. The 2‐benzopyran and 2‐benzofuran scaffold of the spirocyclic compounds were synthesized from 2‐bromobenzaldehyde. The amide linkers were constructed by acylation of amines with chloroacetyl chloride and subsequent nucleophilic substitution, the alkyl linkers were obtained by LiAlH4 reduction of the corresponding amides. For the development of σ2 receptor ligands, the spirocyclic 2‐benzopyran scaffold is more favorable than the ring‐contracted 2‐benzofuran system. Compounds bearing an alkyl chain as linker generally show higher σ affinity than acyl linkers containing an amide as functional group. A higher σ1 affinity for the cis‐configured cyclohexanamines than for the trans‐configured derivatives was found. The highest σ2 affinity was observed for cis‐configured spiro[[2]benzopyran‐1,1′‐cyclohexan]‐4′‐amine connected to the tetrahydroisoquinoline system by an ethylene spacer (cis‐31, K i (σ2)=200 nM; the highest σ1 affinity was recorded for the corresponding 2‐benzofuran derivative with a CH2C=O linker (cis‐29, K i (σ1)=129 nM).


Introduction
σ Receptors, initially classified as class of opioid receptors, are well established as unique class of receptors without any homology to opioid receptors or NMDA receptors. [1] Based on the results of comprehensive radioligand binding studies and biochemical analysis, the class of σ receptors was further divided into two distinct subtypes, which were termed σ 1 and σ 2 receptor. [2] The σ 1 receptor has been cloned from different species, including human, rat, mouse, and guinea pig. The crystal structure of the human σ 1 receptor was recently reported by Kruse et al. [3,4] In contrast to the σ 1 receptor, details concerning the σ 2 receptor have been rather vague for many years. As a result from photoaffinity labeling studies a molecular weight of 21.5 kDa was postulated for the σ 2 receptor. [5] Xu and coworkers utilized a photoaffinity probe to label σ 2 receptors in rat liver and proposed that the σ 2 receptor binding site resides within the progesterone receptor membrane component 1 (PGRMC1) complex. [6] During the following years, the correlation between the σ 2 receptor and PGRMC1 protein complex was considered controversial. [7] In 2017, the σ 2 receptor was isolated from calf liver tissue and identified as the endoplasmic reticulum (ER)-resident membrane protein TMEM97, which is also described as MAC30 (meningioma-associated protein 30). Subsequent molecular cloning and binding experiments confirmed this result. Mutagenesis studies identified two aspartate residues as crucial for binding of [ 3 H]DTG, a radioligand frequently used in σ 2 receptor binding assays. Furthermore, it was demonstrated that the TMEM97 ligands elacridar (1; Figure 1) and Ro 48-8071 showed the same K i values towards cell membranes from Sf9 cells overexpressing the TMEM97 protein and σ 2 receptor overexpressing MCF-7 cells. [8] According to these findings, the σ 2 receptor is now often termed σ 2 receptor/TMEM97. In 2018, Riad et al. demonstrated that the σ 2 receptor/TMEM97 protein, the PGRMC1 protein and the LDL receptor form a ternary complex, which is necessary for the rapid internalization of LDL. [9] In contrast to the σ 1 receptor, no crystal structure of the σ 2 receptor protein has been published so far.
In a previous study, we have reported that the spirocyclic 2benzopyran derivatives trans-6 and cis-6 bearing the 6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline residue without linker show medium to high affinity to the σ 2 receptor. [34] (Figure 2) However, the selectivity over the σ 1 subtype is moderate and has room for improvement. Therefore, it was envisaged to synthesize a new set of σ 2 selective ligands by introducing a linker between the spirocyclic 2-benzopyran scaffold and the isoquinoline ring system. To exploit further structure affinity relationships, not only alkyl chains were planned as linkers, but also amides with variable chain lengths and different positions of the carbonyl group were designed. Moreover, a ring contraction of the spirocyclic 2-benzopyran to the spirocyclic 2benzofuran ring system was planned as this compound class is also known for its high σ affinity from previous studies. An overview of the structure modifications is presented in Figure 3.
The final compounds were obtained by nucleophilic substitution of the terminal chloride in the side chain of the amides 15-20. The acylated spirocyclic 2-benzopyrans 16-18 and 2benzofurans 19 and 20 were reacted with the tetrahydroisoquinoline 14, while the acylated isoquinoline 15 underwent a nucleophilic substitution with the spirocyclic amines 10-13. In Table 1, the products and yields of these transformations are summarized.
The nucleophilic substitution of the 2-chloroacetylated isoquinoline derivative 15 with spirocyclic amines 10-13 in DMF with TBAI as catalyst resulted in the formation of the desired compounds 21-24 in satisfactory yields. Due to purification problems, the benzopyran-based spirocyclic compound trans-22 could not be isolated in pure form for testing. S N 2 reaction of spirocyclic chloroacetamides 16, 17 and 20 with the tetrahydroisoquinoline 14 provided the amides 25, 26 and 29 in 62-86 % yields. The pure spirocyclic benzofuran 28 was obtained in only 44 % yield, due to purification problems. While the nucleophilic substitution of the 2-chloroacetylated compounds 16, 17, 19, and 20 with tetrahydroisoquinoline 14 led to clean conversions, he corresponding 4-chlorobutyramide 18 reacted slower to produce cis-27, which was isolated in only 19 % yield (Table 1).
During the reaction to obtain the secondary amines trans-22, cis-22, trans-24 and cis-24, formation of tertiary amines as side-products was observed (double nucleophilic substitution). The R f values of the tertiary amines was almost identical to the R f value of the secondary amines, rendering the fc purification of the desired products very difficult. Although the isolation and purification of the secondary amines cis-22, trans-24 and cis-24 was successful, trans-22 could not be isolated in sufficient purity.
As not only linkers bearing a carbonyl group were planned, derivatives 30, trans-31 and cis-31 with an ethylene linker between the amino moiety of the spirocyclic benzopyran and the tetrahydroisoquinoline were synthesized. (Scheme 3) This type of compounds features two basic amino moieties instead of one and can therefore adopt different orientations within the binding pocket of both σ receptor subtypes. Additionally, the effect of the carbonyl moiety on the binding affinity and selectivity can be studied.
At first, a direct alkylation of the tetrahydroisoquinoline 14 was envisaged. For this purpose, 2-bromoethanol was oxidized with Dess-Martin perioidinane to afford 2-bromoacetaldehyde. The aldehyde should be attached to the isoquinoline 14 in a reductive alkylation with NaBH(OAc) 3 . Unfortunately, after 4 h reaction time the alkylated isoquinoline could not be isolated. Next, a nucleophilic substitution with 1,2-dibromoethane and K 2 CO 3 in CH 3 CN was performed. But even after a reaction time of 18 h the desired product could not be obtained. The reaction conditions which led to a successful acylation of the isoquinoline 14 (DMF, Et 3 N and TBAI) also didn't lead to the formation of the alkylated product. Finally, the desired alkylated amines 30, trans-31 and cis-31 were synthesized by reduction of the corresponding amides 25, trans-26 and cis-26 with LiAlH 4 . (Scheme 3) The piperidine derivative 30 was obtained in 63 % yield after 2 h heating to reflux. trans-31 and cis-31 were isolated after 22 h in 86 and 76 % yield, respectively.

σ 1 and σ 2 receptor affinity
Competitive binding assays with tritiated radioligands were utilized to determine the σ 1 and σ 2 receptor affinity of the synthesized compounds. In the σ 1 binding assay, [ 3 H]-(+)-pent-azocine was used as radioligand and homogenates of guinea pig brains served as receptor material. The σ 2 assay was performed with the radioligand [ 3 H]-di(o-tolyl)guanidine ([ 3 H] DTG) and homogenates of rat liver were used as receptor material. The nonselective properties of DTG was compensated by masking σ 1 receptors with an excess of non-tritiated (+)-pentazocine. [40] In Table 2, the receptor affinities of the synthesized compounds are summarized. In comparison to the lead compounds trans-7 and cis-7, the 2-benzopyran derivatives with an acetyl linker generally show a lower σ 2 affinity. The highest σ 2 affinity was observed for cis-26 with a K i value of 371 nM. In this compound, the acyl group is located at the spirocyclic ring system. When the acyl moiety is located at the isoquinoline ring system (cis-22), the σ 2 affinity is reduced (11 % inhibition of radioligand binding). A similar trend was observed for the corresponding piperidine derivatives 25 and 21 with K i values of 534 nM and 19 % inhibition of radioligand binding, respectively.
The σ 1 affinity of the piperidine derivative 21 is higher than that of the corresponding cyclohexanamine derivative cis-22. A general observation is that the σ 1 affinity is higher for the compounds bearing the acyl group at the isoquinoline ring. For the development of σ 1 ligands with the 2-benzoypran scaffold, it can therefore be concluded that the basic center at the spirocyclic ring system should be retained.
For the derivatives with the 2-benzofuran scaffold similar observations were made in terms of σ 2 affinity. The introduction of an acetyl spacer led to loss of σ 2 affinity, independent of the position of the acyl moiety (e. g., trans-24, cis-24, 28). In contrast to the 2-benzopyrans, the σ 1 affinity of the piperidine derivatives of the spirocyclic 2-benzofurans was not higher than the respective cyclohexanamines. A notable exception is cis-29 with a K i value of 129 nM at the σ 1 receptor. This compound even represents a σ 1 receptor selective ligand despite the 6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline structural element.
The elongation of the acyl linker also led to a decrease in σ 2 affinity, while the σ 1 affinity was slightly increased. For the butyramide cis-27 a K i value of 2100 nM at the σ 2 receptor and 712 nM at the σ 1 receptor was observed.
For the derivatives 30, cis-31 and trans-31 with an ethylene linker an increased σ 2 affinity in comparison to the corresponding amides (e. g., 21, cis-22) was found. The σ 1 receptor affinity of the cyclohexanamines cis-31 and trans-31 was also increased, resulting in a loss of σ 2 preference of cis-31. The piperidine 30 shows a slight preference for the σ 2 receptor (K i values of 348 nM and 608 nM, respectively).

Conclusion
The introduction of a spacer between the spirocyclic 2benzopyran and 2-benzofuran scaffold and the tetrahydroisoquinoline system was envisaged to study structure affinity relationships and evaluate possibilities to optimize selectivity of the lead compounds trans-7 and cis-7. A set of compounds with amide and alkyl spacers was synthesized and pharmacologically evaluated in competitive binding assays. Although the introduction of the linker generally resulted in a loss of σ affinity in comparison to the lead compounds 7 without linker, some interesting observations could be made. Compounds containing the 2-benzopyran scaffold showed a higher affinity than the corresponding 2-benzofurans. Compounds 30, trans-31 and cis-31 with an ethylene linker displayed higher affinity than compounds with an amide in the side chain. The introduction of the linker in compounds 21 and cis-29 resulted in an unexpected selectivity for the σ 1 receptor. In conclusion, the combination of wo promising σ 2 pharmacophoric elements, that is, the connection of an O-containing spirocyclic system with the tetrahydroisoquinoline moiety by different spacers, did not provide high-affinity σ 2 selective ligands. However, the synthesized σ ligands allow an interesting insight into the limitations of acyl chains as linker between the two pharmacophoric elements. cis-31 and trans-31 could serve as a starting point for further structural modifications resulting in higher σ 2 affinity and selectivity.

Experimental Section
Chemistry, General

Synthetic procedures
The synthesis of the spirocyclic piperidines 10 and 12 has been reported in the literature. [35,36] The synthesis of exocyclic primary amines trans-11 and cis-11 was described in ref. [34]. The synthesis  protons is not observed in the spectrum. 13