Source: Yakudo Cyber
The University of Florence is the first to report a series of compounds that may be used in the treatment of oxaliplatin-induced neuropathy that modulate human carbonic anhydrase isoform (hCA) as well as transient receptor potential vanillin 1 (TRPV1) activity. All compounds exhibit potent in vitro inhibitory activity against major hCAs involved in such pathologies, while the compounds selected from them have a moderate agonist effect of TRPV1. In vivo evaluations of the most potent compounds (R)-12a, (R)-37a, and the two enantiomers (R)-39a, (S)-39b, were found to induce durable analgesic effects in a mouse model of neuropathy with maximum efficacy 30 minutes post-dose.
Figure 1.Compound discovery and molecular optimization process with dual activities of CAs inhibition and TRPV1 agonization
Platinum-based chemotherapy (i.e., cisplatin, carboplatin, and oxaliplatin) is an effective treatment for tumors, especially advanced metastatic cancers such as colorectal, ovarian, breast, and lung cancers. However, there are certain toxic and side effects in the clinical use of platinum drugs, especially dose-limiting toxicity, nephrotoxicity, ototoxicity, bone marrow suppression and neurotoxicity are the main toxic side effects that people are concerned about. For example, peripheral neurotoxicity causes paresthesias or obtundation, which then becomes chronic sensory neurotoxicity. In addition, chronic pain symptoms can seriously affect quality of life. To date, there are no effective treatments for oxaliplatin-induced neuropathy, and only nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids are available to relieve pain. Although the pathophysiology of neuropathy is unknown, many reports suggest that homeostatic dysfunction in dorsal root ganglia (DRG) neurons may precipitate neuropathy.
This hypothesis agrees that DRGs are located outside the central nervous system and are therefore not protected by the blood-brain barrier. An important piece of evidence for this theory is that in patients treated with oxaliplatin, certain subtypes of the transient receptor potential (TRP) ion channel family (e.g., TRPM8, TRPV1, and TRPA1) are disrupted by chelating calcium ions through oxaliplatin metabolites (i.e., oxaliplatin oxalate), causing neuropathy symptoms. Based on this phenomenon, the University of Florence began to study TRPV1 as a potential analgesic target, as it can participate in the transmission of nociceptive stimuli by triggering the influx of important calcium ions. Desensitization of TRPV1 receptors by stimulating TRPV1 receptors is an effective strategy for pain treatment. It has been reported in the literature that capsaicin is regarded as a "molecular scalpel", which will lead to the prolongation of the opening time of TRPV1 ion channels, and the cytotoxic effect only significantly affects the sensory neurons expressing TRPV1. TRPV1 partial agonists are also effective in relieving pain.
Another class of drugs for pain relief is TRPV1 antagonists, such as capsazepine in Fig1 B or SB-705498. Although TRPV1 antagonists can reverse pain, they have side effects such as high fever and accidental burns, so TRPV1 antagonists cannot be further developed. Neuropathy is often associated with uncontrolled intracellular acidification of DRG neurons, resulting in the formation of adducts of metals (e.g., platinum) with hemoglobin. In the same literature, it was reported that uncontrolled pH fluctuations due to the removal of the primary pH buffer system may be reversed by inhibition of the carbonic anhydrase (CA, EC 4.2.1.1) subtype (i.e., carbonic anhydrase II, hCAII). Based on the groundbreaking research by Potenzieri et al., researchers at the University of Florence have attempted to intervene in neuropathic pH imbalance by using compounds capable of inhibiting metalloenzymes and activating TRPV1 receptors. In addition to pH effects, inhibition of carbonic anhydrases (i.e., II, VII, and XII isoforms) expressed in a large number of central nervous systems may lead to a decrease in bicarbonate-dependent depolarization of GABAA receptors when KCC2 is impaired in peripheral nerve injury.
Design ideas
The University of Florence began to use the potent central nervous system permeability and selective antagonism of TRPV1 of SB-705498 as a starting compound for molecular modification, based on which the compound introduced the least functional group for molecular optimization to obtain compounds with hCA inhibitory activity.
The synthesis strategy adopted takes into account:
1. Replace the CF3 substituent on SB-705498 with a pharmacophore inhibited by carbonic anhydrase, such as sulfonamide, and at the same time, replace different substituents on the benzene ring to find compounds that act on two targets at the same time;
2. Study the replacement of bromine on the benzene ring with meta or para-substitution in SB-705498, and consider replacing CF3 with other substituents (such as NO2 or H). Finally, the stereoisomers of the modified compounds were investigated to see if they had an effect on the affinity of different carbonic anhydrase isoforms and TRPV1 receptors.
Carbonic anhydrase inhibitor effect
All synthetic compounds (7a, b-22a, b, 24a, 25a-b, 35a-46a, 37b-40b, 43b-46b) were tested for the inhibitory activity of hCA isoforms I, II, IV, VII, IX, and XIII, as shown in Table 1.
The structure-activity relationship of the inhibitory activity of carbonic anhydrase isoforms is as follows:
- Compounds 7-22a,b inhibitive activity Ki against cytoplasmic hCA II ranges from 12.1 nM to 818 nM. Affinity for hCA I is similar to that of subtype II. Compound 13a has the lowest potency (Ki of 937.6 nM for hCA I). The same kinetic trend was observed for subtypes I and II. Notably, all R-configuration enantiomers (7-22a, 24a, 25a, and 35-46a) have stronger inhibitory activity than S-configurations containing 7-22b, 25b, 37-40b, and 43-46b. Among them, the potency of compound 7a on both carbonic anhydrase isoforms is nearly 10 times stronger than that of its S configuration enantiomer 7b. The anti-hCAI activity of compound 11a (Ki of 37.0 nM) is 13-fold stronger than that of compound 11b (Ki of 475.8 nM). Opposite inhibition tendencies were observed for compounds 10a-b, 16a,b, and 20a,b. (S)-10b, (S)-16b, and (S)-20b are more potent hCAI and II inhibitors (i.e., (R)-10b, (R)-16b, and (R)-20b) compared to the R configuration. It is speculated that this reversal activity between the R and S configurations may be due to the CF3 substituent. In addition, the introduction of large volumes of substituents into compounds 13a,b and 17a,b eliminates the effect of chiral centers. For hCA II, the introduction of a series of sulfonamide substituents (35-46) on the benzene ring found that para- or meta-substitution of sulfonamide in thiourea derivatives 37-40a,b could increase the potency of the enantiomers of the R configuration. In addition, when sulfonamides are in para positions (e.g., 43a and 45a), urea derivatives exhibit the same trend.
- The Ki value of the compound for the membrane protein hCA IV was in the micromolar range. Notably, substitution of urea with sulfonylurea resulted in a substantial increase in the potency of compound 24a to nanomol levels (Ki of 36.6 nM). The introduction of a chlorine atom (25a,b) in the ortho position of sulfonylurea reduces the potency by a factor of 20.
- Almost all compounds have strong inhibitory activity against the brain-associated subform hCA VII, with Ki at the sub-nanomo level (12b Ki reaches 0.9 nM). The results showed that the compounds had a different inhibition tendency for isoform VII compared to isoforms I and II, and most of the enantiomers of the S configuration were more effective than the R configurations. In contrast, compounds that replaced urea with thiourea (7a,b and 8a,b) observed an activity flip of the corresponding enantiomers. Compared to the S configuration (7b, Ki of 12.1 nM), the R configuration has 2-fold higher potency of 7a (Ki of 6.6 nM). In contrast, the thiourea derivative S configuration 8b (Ki of 2.6 nM) shows a 3-fold higher than the R configuration (8a with a Ki of 8.6 nM). Halogen pro-atom pro-location appears to be critical for S-configuration selectivity, such as derivatives 11a,b and 21a,b, which are 10- and 80-fold selective compared to compounds 12a,b. The substitution position of the sulfonyl group in compounds 37-46 plays an important role in the selectivity of enantiomers. For example, in the metameric series, the increased selectivity of the R configuration relative to the S configuration.
- All compounds potently inhibit tumor-associated hCA IX and XII isoforms and show Ki values ranging from 1.3 nM to 971.3 nM. The selectivity of enantiomers for both isoforms is mainly influenced by the substituents on the benzene ring rather than the pyridine ring. As shown in Table 1, the results show that the enantiomers of compounds 9, 14, 15, 17, 18, and 21 have reverse selectivity between hCAI X and XII. The potency of compounds 11a,b and 21a,b against hCAIX, replacing the urea group of the former with the thiourea group of the latter, resulted in increased selectivity of the S configuration (11b was 13.9-fold more active than 11a; 21b was 52.5-fold more active than 11a). For hCA XII, the R configuration has higher inhibitory activity, such as compound 21a,b. The S-configuration enantiomers of compounds 37-46 were observed to be the best inhibitors, especially the derivatives 38a, b showed more than 100-fold selectivity for enantiomers.
TRPV1 experiment
The selected R configurations enantiomers 7a, 9a-16a, 18a-22a, 24a, 35a-46a, and S configurations enantiomers 39b and 45b were evaluated to regulate TRPV1 receptor activity, and the specific results are shown in Table 2.
Although the designed series of compounds are derived from the TRPV1 antagonist SB-705498, the results of the tests in Table 2 indicate that the compounds have a significant agonistic effect. Even minor chemical structural modifications may lead to agonist-antagonistic switching in the regulation of TRPV1 activity.
Compounds 10a, 37a, 38a, 39ab, 40a, 45a, b, and 46a exhibit moderate agonistic effects with EC50 values ranging from 3.1 nM to 74.5 μM. Most of the sulfonamide compounds are introduced on the pyridine ring and the activity is lost. Compound 10a has a weak agonistic activity (10a EC50 of 74.5 μM), and the agonistic activity is significantly restored when 2-position chloride substitution is introduced in compound 12a. Conversely, most benzene rings show agonistic activity when they have sulfonamide substituents on them, with EC50 values in the low micromothy range, such as 8.0 μM and 3.1 μM for compounds 37a and 45b, respectively. In some cases, the configuration of the stereocentric center does not affect activity or potency, such as the enantiomers 39a and 39b with a TRPV1 EC50 of 12.5 μM. Conformational selectivity has been reported for the potency of (R)-45a and (S)-45b, with the latter being 9-fold more active.
X-ray crystal structure
In order to elucidate the molecular basis of compound inhibition of CA, the X-ray structure of hCA II complexes with enantiomers (R)-37a and (S)-37b was determined at resolutions of 1.3Å and 1.6Å, respectively (Fig3). Electron density mapping analysis (Figure S1 in Supporting Information (SI)) shows the density of inhibitor (R)-37a, entering the catalytic fissure, which is fully compatible with the ligand. As expected, the sulfonamide moiety interacts directly with zinc ions and Thr199 residues to form hydrogen bonds, thus showing a typical binding pattern for such inhibitors. In addition, typical hydrophobic interactions were established between the benzenesulfonamide moiety and the side chains of Val121 and Leu198 and helped to enhance the complex within the active site. The proximal nitrogen atom of the thiourea-based moiety of (R)-37a forms a water bridge with Thr200. A valuable additional hydrophobic interaction was observed between Leu198 and Pro202 and the hydrophobic portion of the main scaffold, which is responsible for adhering the entire ligand within the hydrophobic region of the active site (Fig3A). A second inhibitor (S)-37b that binds to hCAII also reveals interesting structural features (Fig3B). First, the thiourea-based moiety was observed to be biconformed.
In addition, interesting features were observed in the tail section of the derivative (S)-37b. In fact, the S-configuration stereocenter of the pyrrolidine ring moves this section to the other side of Phe131, where it undergoes hydrophobic interaction with this residue. This different position of the tail of (S)-37b is also stabilized by the water bridge between the nitrogen of the pyridine ring and Glu69 and the hydrophobic interaction with Ile91. A structural comparison between the two enantiomers (R)-37a and (S)-37b (Fig3C) also revealed similar features, such as the typical interaction of benzenesulfonamide with catalytic zinc atoms and Thr199, and on the other hand, the stereocentric center was able to influence the tail conformation of two molecules that occupy two different hydrophobic pockets divided by Phe131 residues. However, this structural diversity does not significantly affect the level of inhibition of this subtype by the two inhibitors.
Validation of in vivo pain relief
Based on the CA and TRPV1 profiles obtained in vitro, the University of Florence selected the most suitable compound to perform an in vivo mouse model of neuropathic pain caused by repeated treatment of oxaliplatin. Compounds considered include:
(i) (R)-36a and (R)-43a as potent CA inhibitors lacking TRPV1 activity;
(ii) (R)-12a and (R)-37a, which are effective against both targets;
(iii) Two enantiomers, (R)-39a and (S)-39b, showed similar efficacy against CA II and TRPV1. The results are shown in Fig4. In vivo experiments with animal licking latency after oral administration of the selected compound (concentration increased to 100 mg/kg) were evaluated. Dose-dependent relationships have been observed with:
- Compounds (R)-36a and (R)-43a, which have no activity against TRPV1, show dose-dependent efficacy peaking at 45 minutes post-dose, followed by a rapid decline in effect, and inhibition at 75 minutes (as shown in Fig 4).
- (R)-12a and (R)-37a peak at 30 minutes post-dose and are effective for up to 45 minutes. The potency and activity of (R)-37a is more potent than that of (R)-12a. This effect may be plausible to the fact that (R)-37a inhibits CA II more effectively than (R)-12a (i.e., 10.4-fold), also considering that DRG neurons are particularly rich in this subtype.
- (R)-39a and (S)-39b were significantly effective at 30 and 100 mg/kg, completely recovering oxaliplatin allergy at higher doses. Based on the close in vitro activity of CA II and TRPV1 of the two enantiomers (as shown in Tables 1 and 2), the slightly better (S)-39a may be due to different in vivo metabolic processes.
conclusion
This is the first time that a dual-targeted molecule has been reported in the literature on its ability to alleviate neuropathy by simultaneously activating TRPV1 and inhibiting CAs enzymes. Preliminary SAR analysis of the effects of the two targets was performed by aromatic ring substitution, biological isometric transitions between urea and thiourea linkers, and the introduction of stereocentric centers. The presence of (R)- or (S)-stereocentric centers in the synthesized compound group does not appear to have a correlated effect on the activity of either target. Of particular interest is the case of (R)-37a and (S)-37b (i.e., CA II Ki 6.7 and 4.9 nM, respectively), as their eutectic structure with CA II shows an enzymatic hydrophobic moiety of the molecular tail at the active site and occupies different subpockets separated by Phe131 residues.
The method of introducing the sulfonamide moiety of the CA warhead into the TRPV1 antagonist modulator SB-705498 results in a reversal of activity to moderate agonism. The observed in vitro effects of the stereocentric center of the molecule on TRPV1 are varied. For example, (R)-39a and (S)-39b (i.e., EC50 of 12.5 μM for both compounds) do not cause any change in potency, whereas for compound 45, the agonistic activity of the (S)-configuration is 9-fold higher than that of its corresponding (R)-configuration (EC50). (R)-45 and (S)-45 are 29.5 and 3.1 μM, respectively.
Select the most valuable in vitro performance compounds (i.e., (R)-12a, (R)-36a, (R)-37a, (R)-39a, (S)-39b, and (R)-43a) to explore their effects on OINP in vivo mouse models. All compounds with CAII or TRPV1 activity induce a durable analgesic effect and exert maximum efficacy 30 minutes after dosing. In contrast, only compounds (R)-36a and (R)-43, which have activity against CA, have reported moderate and short response outcomes, demonstrating the important contribution of the TRPV1 agonist portion of the reported molecule to the biological model. It is very interesting to note that the enantiomers (R)-39a and (S)-39b differ significantly in inducing biochemical reactions in in vivo models, with the former being more potent and durable than their (S)-configurations. Small molecules with both moderately active TRPV1 agonists and strong inhibitors of CA are effective and worthwhile strategies to be developed to minimize neuropathy-induced symptoms (e.g., pain).
Article source:
https://doi.org/10.1021/acs.jmedchem.2c01911
Please click here to watch the detailed tutorial on how to use the CyberSAR system
1. Combined with drug design ideas, Yaodu CyberSAR excavates the active structure reported in the literature and patents, and uses CyberSAR to facilitate and quickly obtain the target structure of interest of R&D personnel for developing ideas, and the following examples are given for TRPV1 agonists:
2. Select the cascade "Cluster Structure View" tab under the "Chemical Space" option tab in the target interface, and the literature and patents included in the CyberSAR platform can display the molecules with experimental test activity related to TRPV1 in the form of "parent nuclear structure clustering". Among them, the "green font highlighted" is the active molecular structure of EC50<100nM in the in vitro enzyme and cell activity testing experiments reported in the literature, the specific experiments, the experimental results and the experimental sources.
3. Select the cascading "Raw Structure View" tab under the "Chemical Space" option tab in the target interface, and the molecules with TRPV1-related experimental activity included in the CyberSAR platform can be displayed in the form of "R&D stage timeline". Among them, "data mining" highlighted in green font is potential Hit.