Synthesis and evaluation of aporphinoid 5-HT_7AR ligands as inhibitors of PC3 prostate cancer cell growth

Abstract

Aporphines are a class of isoquinoline alkaloids that are endowed with a range of biological activities. The 5-HT7R is an emerging biological target for prostate cancer therapeutics. In this manuscript, we report the synthesis and evaluation of aporphine enantiomers as 5-HT7R ligands, as well as their activity in inhibiting the proliferation of prostate cancer cells (specifically, PC3).

The (S)-enantiomers displayed higher affinity at the 5-HT7R than the racemates and the (R)-enantiomer counterparts. The (S)-enantiomers were found to be antagonists at the 5-HT7R. Racemates as well as their respective enantiomers were selective for the 5-HT7R receptor over other serotonin and dopamine receptors evaluated. In the anticancer activity assays, the compounds showed more potent cytotoxic effects than the selective 5-HT7R antagonist control SB269970. However, no correlation was observed between the 5-HT7R affinity or 5-HT7R antagonist activity and anticancer potency, suggesting that other non-5-HT7R mechanisms play a role in the anticancer effects of the compounds. Compounds (R)-1 and (R)-4 were identified as the most potent antiproliferative compounds and will be useful as lead molecules for prostate cancer therapeutic development in future studies.

Prostate cancer (PC) is the most common cancer in men in the United States¹ and the fifth leading cancer-related death worldwide,² with its incidence increasing every year. It was estimated that in 2025, about 313,780 new PC cases would be diagnosed and ~36,000 patients would succumb to the disease.³ Studies show that patients with localized prostate cancer have a 5-year survival of 100 % but this reduces to about 30 % in metastatic PC patients.⁴ Androgen deprivation therapy (ADT) is the main treatment given to PC patients on initial diagnosis. ADT is responsible for the reduction of serum androgens and inhibits androgen receptors (AR). However, about 20–30 % of PC patients do not respond to this treatment due to its progression to the more aggressive metastatic castrate-resistant prostate cancer (mCRPC).⁵ Although there are treatment options for mCRPC, such as radium-23, chemotherapy, and radioimmunotherapy, mCRPC remains incurable, hence the need for novel therapeutics and biomarkers for PC.⁵

5Hydroxytryptamine-7 receptors (5-HT₇Rs) are the most recently studied serotonergic subfamily of the seven known 5-hydroxytryptamine (5-HT) receptors.⁶ There are three isoforms of human 5-HT₇ receptors, and these are 5-HT_7AR, 5-HT_7BR, and 5-HT_7DR. The 5-HT_7AR is a full-length membrane receptor made up of 445 amino acid residues and is the most abundant in humans. The 5-HT_7BR is a truncated variant with 432 amino acid residues whilst 5-HT_7DR is a unique isoform with 479 amino acid residues.^7,8 5-HT₇Rs are known to be highly expressed in the central nervous system (CNS) and gastrointestinal tract (GIT) and are also expressed in other peripheral tissues. The 5-HT₇R has recently emerged as a potential therapeutic target for prostate cancer. In support of this, a recent study reported that 5-HT₇R mRNA expression is approximately 200-fold higher in PC cells as compared to healthy ones.⁹ Furthermore, treatment with the selective 5-HT₇R antagonist SB-269970, inhibited proliferation and induced apoptosis in PC3 cells.⁹ These findings underscore the potential therapeutic value of targeting 5-HT₇R in the context of PC.

Aporphine alkaloids are endowed with a range of biological activities and are recognized as “privileged structures” for drug discovery.¹⁰ Extensive research has established their activity as ligands for dopamine D₁ and D₂ receptors (D₁R and D₂R respectively)^11–15 as well as for serotonin receptors, particularly 5-HT_1A and 5-HT_2A receptor (5-HT_1AR and 5-HT_2AR)^14,16–19 subtypes. In prior work, we identified a number of novel racemic aporphine derivatives exhibiting high affinity for the 5-HT_7AR.⁸ Given the emerging therapeutic relevance of 5-HT₇R antagonism as a potential therapeutic strategy for PC, we sought to investigate the 5-HT_7AR antagonist properties of their individual enantiomers and to determine their anti-proliferative effects in PC3 cells.

As alluded to above, racemic aporphines 1–4 (Fig. 1) were previously identified as high affinity and selective ligands for the 5-HT_7AR.⁸ As an extension to this work, we first evaluated the 5-HT_7AR functional effects of compounds 1, 2 and 4. Pharmacological characterization revealed that compounds 1, 2 and 4 functioned as 5-HT_7AR antagonists with IC₅₀ values of 3.5, 8.7 and 11 μM respectively (see Supporting Information). No 5-HT_7AR agonist activity was observed for these compounds.

Fig. 1.

Structures of 5-HT_7AR aporphine ligands from our previous study.

To investigate the influence of chirality on 5-HT_7AR affinity, we engaged an enantioselective synthesis for the enantiomers of 1–4. The enantiomers were synthesized via an adaptation of our previously published racemic routes. The key step here was an enantioselective reduction of the dihydroisoquinoline 5 as depicted in Scheme 1 (in contrast to reduction of 5 with sodium borohydride in the racemic synthesis).⁸ Thus, the chiral tetrahydroisoquinolines (R)-7 and (S)-7 were obtained upon Noyori reduction of 5 with catalyst (S,S)-6 and (R, R)-6, respectively. Thereafter, (R)-7 and (S)-7 were transformed into the enantiomeric analogues (R)-1 to (R)-4 and (S)-1 to (S)-4, via synthetic sequences as previously reported in the racemic syntheses.

Scheme 1.

Synthesis of enantiomers (R)-1 to (R)-4 and (S)-1 to (S)-4

Enantiomeric analogues (R)-1 to (R)-4 and (S)-1 to (S)-4 were then evaluated for affinity across a range of serotonin and dopamine receptors. The results of these assays are compiled in Table 1. The compounds as a group showed low or no affinity at the dopamine receptor subtypes examined. Similarly, among the serotonin receptor subtypes, low or no affinity was observed consistently for the analogues at the 5-HT₆R; here, the highest affinity of 601 nM was observed for compound (S)-4. Affinities at the 5-HT_2AR were are also generally low (K_is ranging from 541 to 7234 nM). Unsurprisingly, the highest affinities were observed at the 5-HT_7AR (Kis ranging from 2.2 to 46.4 nM). Individual compounds maintained moderate selectivity for 5-HT_7AR over the 5-HT1AR, with compound (R)-4 showing the highest such selectivity (approximately 32-fold). A notable aspect of the structure-affinity relationships was the stereochemical preference of the (S)-enantiomers for binding at the 5-HT_7AR. For each enantiomeric pair, the (S)-enantiomer showed at least 3-fold stronger 5-HT_7AR affinity than their (R) counterparts.

Table 1.

Affinity of (R)-1 to (R)-4 and (S)-1 to (S)-4 at serotonin and dopamine receptors.

	Ki (nM)a
Cmpd #	5-HT1ARb	5-HT2AR	5-HT6R	5-HT7AR	D1Rc	D2Rd	5-HT1AR/5-HT7AR
(R)-1	71.7	4838	nae	10.7	na	na	6.7
(S)-1	11.4	969	na	2.2	na	na	5.2
(R)-2	282	7234	na	28.2	na	na	10
(S)-2	27	541	3106	3.1	1677	1829	8.7
(R)-3	138	4749	na	14.9	na	na	9.3
(S)-3	33.4	1791	2845	4.5	2553	na	7.4
(R)-4	1471	1117	1736	46.4	762	na	31.7
(S)-4	85.0	1718	601	7.4	808	na	11.5
Clozapine		9.41	12.1	12.3
SB-269970				1.3f

^aExperiments carried out in triplicate – SEM values are within 13 % of reported K_i.

^b8-OH-DPAT used as control (K_i = 1.2 nM).

^c(+)-Butaclamol used as control (K_i = 2.0 nM).

^dHaloperidol used as control (K_i = 10.4 nM).

^ena = no affinity – % inhibition <50 % at 10 μM.

^fBased on reported pKi of 8.9 ± 0.1 nM.²⁰

Given that the (S)-enantiomers exhibited higher 5-HT_7AR binding affinities, subsequent functional activity studies were carried out exclusively on these enantiomers. Thus, we next assessed the functional activities of (S)-2, (S)-3 and (S)-4 at the 5-HT_7AR. All three compounds showed antagonist activity with IC₅₀ values of 20.0, 39.8 and 67.6 nM respectively and were devoid of agonist effects. The significantly enhanced antagonist potency of the (S)-enantiomers, particularly for compounds (S)-2 and (S)-4 (>160-fold improvement versus racemic forms, Table 2), establishes a clear SAR where stereochemistry critically influences both receptor binding and functional antagonism.

Table 2.

5-HT_7AR antagonist IC₅₀ values of the (S)-enantiomers and fold improvement over the racemates.

Cmpd #	IC50 (μM)a	Fold improvement over racemate
(S)-2	20	435×
(S)-3	39.8	na
(S)-4	67.6	163×

^aExperiments carried out in triplicate – SEM values are within 13 % of reported IC₅₀.

The PC3 cell line lacks androgen receptors making it a valuable cell line for studying androgen-independent cancers such as mCRPC.²¹ As mentioned previously, the selective 5-HT₇R antagonist SB-269970 exhibited anticancer activity against the PC3 prostate cancer cell line, which is known to express 5-HT₇R.⁹ To enable direct comparisons with these findings as well as to evaluate SAR trends among our compounds, compounds 1–4 and their enantiomers were tested for anti-proliferative activity versus PC3 cells in MTS assays, using SB-269970 as a reference control. The results of these assays are displayed in Table 3 (for all compounds) and Fig. 2 (for compounds 1–4; please see Supporting Information for the IC₅₀ curves for the enantiomeric analogues).

Table 3.

Mean effect of compounds on viability in prostate cancer cell line PC3.

Cmpd #	IC50 (μM)a	5-HT7AR Ki (nM)b
1	36.0 ± 1.9	6.5c
2	82.2 ± 4.6	4.5c
3	43.9 ± 6.4	8.8c
4	24.7 ± 2.3	9.9c
(R)-1	16.2 ± 0.1****	10.7
(S)-1	21.5 ± 2.7****	2.2
(R)-2	38.5 ± 3.1***	28.2
(S)-2	33.5 ± 4.1****	3.1
(R)-3	30.6 ± 0.5****	14.9
(S)-3	36.7 ± 0.4****	4.5
(R)-4	16.9 ± 0.5****	46.4
(S)-4	31.7 ± 1.6****	7.4
Doxorubicin	2.8 ± 0.8****	ndd
SB-269970	52.1 ± 6.5	1.3e

^aExperiments performed in triplicate.

^bExperiments carried out in triplicate – SEM values are within 13 % of reported K_i.

^cData from previous publication.

^dnd means not determined;

^*p < 0.05,

^**p < 0.01,

^***p < 0.001 and

^****p < 0.0001 compared to the 5-HT₇R antagonist, SB-269970.

^eBased on reported pKi of 8.9 ± 0.1 nM.²⁰

Fig. 2.

PC3 cell viability with racemic 5-HT_7AR aporphine ligands 1–4 in MTS assay.

Among the racemic compounds (1–4), compound 4 exhibited the highest anticancer activity, despite having the lowest 5-HT_7AR affinity. Also, as compared to compound 2, compound 4 had a lower 5-HT_7AR antagonist potency (i.e. 8.7 μM vs 11 μM for 2 and 4 respectively), yet it showed a more potent anticancer effect. Thus, among the racemates, no correlation was observed between 5-HT_7AR affinity or functional activity with anticancer activity.

The (R)-enantiomers were more active than their corresponding (S)-enantiomers in most cases, except for the enantiomers of compound 2, where marginally better anticancer activity was seen for the (S)-enantiomer [38 vs 33 μM for the (R)- and (S)-enantiomers respectively]. Individual enantiomers generally showed more potent anticancer effects than their racemic congeners. For example, in the case of compound 3 the racemate shows an anticancer IC₅₀ of approximately 44 μM, whereas the individual enantiomers show IC₅₀ values of approximately 31 and 37 μM for the (R)- and (S)-enantiomers respectively. The only exception to this general trend was compound (S)-4, where its IC₅₀ (approximately 32 μM) was higher than that of the racemate (approximately 25 μM).

As compared to the known selective 5-HT₇R antagonist SB-269970, all enantiomeric compounds showed more potent anticancer activity. The racemic compounds generally also exhibited higher anticancer potency than SB-269970, with compound 2 being the only exception to this general trend.

In conclusion, we investigated the 5-HT_7AR binding and activity as well as the anticancer effects of racemic and enantiomeric aporphines in PC3 cells. We found that the (S)-enantiomers had higher affinities for 5-HT_7AR than corresponding (R)-enantiomers. However, no correlation was observed between 5-HT_7AR affinity and anticancer activity in either the racemic or enantiomeric series of compounds. Furthermore, among the compounds examined, the potency of 5-HT_7AR functional antagonism did not correlate with the observed anticancer activities. Thus, while 5-HT_7AR antagonism likely plays a role in the anticancer effects, it is evident that there are also other non-5-HT_7AR mechanisms that contribute to the anticancer effects of the compounds.

Aporphines have been reported to possess anti-proliferative and cytotoxicity properties in various cancer models such as breast cancer,²² gliomas,²³ neuroblastoma²³ and colorectal cancer.²⁴ There is a paucity in the literature of studies on aporphines with anticancer activity in prostate cancer cell lines.²⁵ This study is the first to report the antiproliferative effects of aporphines in PC3 cells, expanding their known activity in other cancers.

The most potent compounds identified in the anticancer assays were compounds (R)-1 (IC₅₀ = 16.24 μM) and (R)-4 (IC₅₀ = 16.89 μM), each representing an approximately 3-fold higher potency as compared to SB-269970. This enhanced activity underscores their potential as lead molecules in the development of therapeutics for targeting PC. More detailed mechanistic investigations on these promising compounds to delineate the biological pathways contributing to their anticancer effects is warranted at this time. Understanding these mechanisms will inform the rational design of more potent analogs that incorporate 5-HT₇R antagonism as a key targeting strategy. This study lays the groundwork for future explorations of aporphines as potential PC therapeutics in that regard.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bmcl.2025.130328.

Data availability

Data will be made available on request.