Angiotensin II AT2 receptor ligands with phenylthiazole scaffolds

The syntheses and the AT 1 R and AT 2 R binding data of a series of new small molecule ligands are reported. These ligands comprise a phenylthiazole scaffold rather than the biphenyl or phenylthiophene scaffolds found in essentially all of the previously described ligands originating from the nonselective AT 1 R/AT 2 R ligand L-162,313 and the AT 2 R selective agonist C21, the latter now in Phase II/III clinical trials. A phenylthiazole rather than the phenylthiophene scaffold that is present in the AT 2 R selective agonist C21 and in the AT 2 R selective antagonist C38 had a deleterious effect on the affinity to AT 2 R. Nevertheless, a significant improvement could be accomplished by introduction of a small bulky alkyl group in the 2-position of the imidazole ring attached through a methylene group bridge to the phenylthiazole scaffold. Hence, a combination of a 2-tert -butyl or a 2-isopropyl group and a butoxycarbonyl furnished potent AT 2 R selective ligands. Furthermore, a high affinity ligand derived from L-162,313 and exhibiting a > 35 fold selectivity for AT 1 R was identified ( 10 ). The ligand 21 with the 2-tert -butyl group and ~ 35 fold selectivity for AT 2 R, demonstrated high stability in human, rat and mouse liver microsomes and a very attractive profile with regard to the inhibition of common drug-metabolizing CYP enzymes. Thus, very low levels of inhibition of CYP 3A (5%), 2D6 (12%), 2C8 (26%), 2C9 (23%) and 2B6 (24%) were observed with the 2-tert -butyl derivative comprising the methoxycarbonyl sulfonamide function, levels that are significantly lower than those obtained with C21 under the same experimental conditions.


Introduction
] Likewise, the structurally related isobutyl biphenyl derivative L-162,782 (2) acts as an AT 1 R agonist (Figure 1).4] The three ligands demonstrated essentially the same affinity to AT 1 R and AT 2 R. Later, our group demonstrated that the thiophene derivative L-162,313 (1) is serving not only as an AT 1 R agonist but also as an agonist at the AT2 receptor, as deduced from both in vitro and in vivo data. 5This finding encouraged a systematic search for drug-like AT 2 R selective agonists as potential pharmaceutical agents and valuable research tools, since such agonists had hitherto not been disclosed.Subsequently, after structural modifications of 1, the first nonpeptide AT 2 R selective agonist C21 (4) was discovered at our laboratories. 6This compound is now in phase II/III clinical trials and aimed for treatment of idiopathic pulmonary fibrosis and Covid-19. 71][12][13][14][15][16] These drug-like molecules are in general more suitable as pharmaceuticals than the peptide ligands activating the AT 2 R. [17][18] Migration of the methylene imidazole group of C21 from the para position to the meta position delivered the AT 2 R selective ligand C38 (5)  that acts as an antagonist at the receptor [19][20] (Figure 1).Furthermore, an improved metabolic stability in liver microsomes could be achieved and the receptor affinity for the most part maintained after replacement of the butoxycarbonyl for a methoxycarbonyl sulfonamide group in some meta substituted derivatives related to C38. 21he C21 and C38 analogues reported to date, with very few exceptions comprise either a phenylthiophene or a biphenyl scaffold.Herein, we report the synthesis and AT 1 R and AT 2 R affinities of a series of new ligands, 6-39 encompassing an alternative skeleton, a phenylthiazole scaffold.Ligands with high AT 2 R affinity and demonstrating high stability in liver microsomes and with a low capability to inhibit common drug metabolizing CYP 450 enzymes were identified.

Synthesis
A general protocol used for the synthesis of thiazole-carbamate analogs is shown in Scheme 1.The method of synthesizing these molecules is indirect to some extent, mainly because of two stages.The first step is the preparation of thiazole key intermediate and the second is the synthesis of different substituted imidazole head groups.The thiazole intermediates were synthesized from the corresponding 2,4dibromothiazole (40), via a selective alkylation (41), 22 followed by sulfonation (42), chlorination (43) and finally the protection with tertbutylamine to afford the 2-alkylated thiazole-sulfonamide key intermediate (44).Following the literature procedures, various substituted imidazole head groups were synthesized (47) and subjected to Nalkylation reactions with bromobenzylbromides.4] Finally, the deprotection of the N-tert-butyl sulfonamide functionality using trifluoroacetic acid (TFA) afforded the corresponding primary sulfonamides (51), which following reactions with chloroformates furnished the desired compounds (6-39) in moderate yields.

Results and discussion
We examined the phenylthiazole scaffold as a substitute for the phenylthiophene or biphenyl scaffolds present in the known AT 2 R ligands.Hence, the phenylthiazole scaffold was incorporated to replace the biaryls a) in the nonselective AT 1 R/AT 2 R ligands 1-3, b) in the AT 2 R selective agonist C21 (4, K i = 0.4 nM 6 and with a K i value of 1.8 nM in the HEK-293 cell assay applied herein) and c) in the AT 2 R antagonist C38 (5, K i = 19 nM 19 , K i = 270 nM 21 ).The binding affinity of ligands with the acidic butoxycarbonyl sulfonamide function were compared with those with a methoxycarbonyl sulfonamide function.Furthermore, the imidazole ring of phenylthiazole analogues related to C21 was substituted in the 2-position with either alkyl groups or a thiazole heterocycle since recently a such maneuver furnished ligands with a significantly improved AT 2 R affinity. 25.
G. Gopalan et al.  nM) 1 for a thiazole heterocycle has a significant impact on the AT 1 R and AT 2 R affinity (Fig 2).The phenylthiazole derivative 6 exhibits a low AT 2 R affinity (K i = 480 nM) while the affinity to AT 1 R is 20-fold better (K i = 26 nM).
A methoxycarbonyl rather than a butoxycarbonyl sidechain results in an even higher receptor selectivity and preference for AT 1 R, although the affinity of 7 to the receptor is lower (K i = 91 nM).The n-propylthiazole derivatives 8 and 9 exhibit very similar AT 1 R/AT 2 R ratio and affinity data as 6 and 7, i.e. the butoxycarbonyl compound 8 is binding 4-fold better to AT 1 R than 9. Notably, a terminal trifluoromethyl group improved the affinity to both AT 2 R and AT 1 R and the n-propylthiazole compound 10 exhibited both high AT 1 R selectivity and AT 1 R affinity (AT 1 R K i = 8.5 nM and AT 2 R K i = 320 nM).Hence, in summary a comparison with L-162,313 (1) suggests that replacement of the thiophene for a thiazole heterocycle strongly favors binding to AT 1 R. Furthermore, an n-propyl rather than an isobutyl group render compounds with similar AT 1 R affinity while the binding to AT 2 R is disfavored by an n-propyl group, c.f. 6 and 8.
A methylene imidazole heterocycle attached to a phenylthiophene or biphenyl scaffold is known to result in high preference for AT 2 R both in ligands with para and meta substitution patterns, e.g.C21 (4) 6 and C38 (5) 19 respectively.7] Surprisingly, the incorporation of the phenylthiazole scaffold led to a dramatic loss of affinity to not only the AT1 receptor but also the AT2 receptor despite the imidazole heterocycle present, as demonstrated with the C21 analogues 11-15 and the C38 analogues 16-19 (Figure 3).The C21 analogue 11 exhibited the strongest AT 2 R affinity in the series (K i = 210 nM), which is still 100-fold lower than the affinity of the parent compound C21 (4).The loss of binding affinity to AT 2 R observed after incorporation of thiazole to replace the thiophene ring of C21 is difficult to explain.Possibly the free electron pair of the nitrogen atom makes 11 less prone to adopt a proper conformation and allow efficient binding into the lipophilic cavity of AT 2 R that C21 (4) binds to, according to modeling and molecular dynamic simulations. 28e recently reported that smaller but bulky groups attached to the 2position of benzimidazoles related to C21 (4) could significantly improve the affinity to the AT2 receptor. 25A tert-butyl group was therefore introduced in the 2-position of 11 to furnish 20 and by this operation a 10-fold improvement of the AT 2 R affinity was achieved (Figure 4).A negligible change in AT 1 R affinity was registered.Moreover, by shortening the butoxycarbonyl chain of 20 (K i = 18 nM), the methoxycarbonyl compound 21, that was found to still exhibit a fair AT 2 R affinity (K i = 29 nM) and high selectivity versus AT 1 R (K i = 1000 nM), was obtained.However, a lower affinity to the AT2 receptor was observed with an n-propyl group rather than an isobutyl group attached to the 2-position of the thiazole ring of the scaffold, c.f. 22 and 23.
The positive impact of the tert-butyl group on the AT 2 R affinity encouraged synthesis and evaluation of new ligands encompassing smaller groups in the 2-position of the imidazole heterocycle.Hence, the 2-isopropyl imidazole derivatives 24-27 and the 2-cyclopropyl imidazole derivatives 28-31 were prepared and examined as AT 2 R ligands.(Figure 4).The 2-isopropyl analogue 24 (K i = 5.8 nM) and the cyclopropyl analogue 28 (K i = 9.8) exhibited the highest affinity to AT 2 R of all compounds reported in the present study.These two phenylthiazole derivatives comprise an isobutyl sidechain attached to the scaffold as in C21 and C38 and a butoxycarbonyl sulfonamide function as carboxylic acid bioisoster.
Moreover, ligands with a thiazole ring attached at the 2-positon of the imidazole heterocycle, 32-35, were assessed.Of those ligands only 32 demonstrated a fair affinity to the receptor (K i = 41 nM) (Figure 4).In previous efforts to find alternatives to the imidazole ring, anticipated to contribute to the inhibition of CYP 450 enzymes observed for C21 (4), an oxazole heterocycle was introduced as a substitute.This replacement resulted in an almost 10-fold lower binding affinity to the AT 2 R, but a somewhat better CYP inhibition profile. 13The phenylthiazole derivatives 36-39, with an oxazole head group were synthesized and examined.This operation was not fruitful and the affinity to AT 2 R dropped dramatically (Figure 4).
The metabolic stability of seven representative compounds in liver microsome assays were compared to determine the influence of a butoxycarbonyl versus a methoxycarbonyl function on the stability.Please note, that the metabolic stability data of C21 depicted in Figure 5 in this manuscript were conducted under specific conditions for this study and is slightly different from previously reported values. 21The methoxycarbonyl sulfonamide 21 exhibited a t ½ of > 60 min in human liver microsomes (HLM), t ½ > 60 min in rat liver microsomes (RLM) and t ½ > 60 min in mice liver microsomes (MLM).These data are in sharp contrast to the data obtained with the butoxycarbonyl sulfonamide 20 in identical microsome assays.Thus, 20 degraded faster and a t ½ of 45 min in HLM, a t ½ of 12 min in RLM and a t ½ of 7 min in MLM were observed.The same trend to decompose was seen after comparing the inactive methoxycarbonyl compound 12 with the phenylthiazole analog of C21 compound 11, although less pronounced.Thus, the latter butoxycarbonyl compound 11 exhibited a t ½ > 60 min in HLM, 32 min in RLM and a t ½ of 57 min in MLM, while t ½ values > 60 min were experienced in all three assays with 12. Hence, a replacement of the butoxycarbonyl chain for the shorter methoxycarbonyl chain seems to have a positive influence on the metabolic stability (Figure 5).The 2-isopropyl and 2cyclopropyl phenylthiazoles 24 and 28, like the butoxycarbonyl derivatives C21 and compound 11 exhibited t ½ > 60 min in HLM.

G. Gopalan et al.
0] The prototype AT 2 R agonist C21 (4) was reported to be an efficient inhibitor of some of the important drug metabolizing CYP 450 enzymes.Hence, in the case of inhibition of CYP 2C9, 2C19 and CYP 3A4 the inhibition was very high, 94%, 62% and 82%, respectively, at 10 μM of the inhibitor (C21), while the corresponding value for CYP 2D6 was less pronounced, 41%. 13To assess whether the phenylthiazole analogue of C21 i.e. compound 11 exhibits a similar profile as C21 with regard to CYP inhibition and to examine if a bulky group in the 2-position of the imidazole heterocycle could possibly suppress a potential interaction between CYP enzymes and the imidazole ring, 11 and 12 were compared with 20 and 21, respectively.
The results are presented in Table 1.In this assay employed herein, the inhibitory capacity of C21 was shown to be more pronounced than previously reported 13 and the important drug metabolizing CYP enzymes were inhibited to a higher degree than previously reported; Thus, the inhibition by C21 of CYP 2C9, 2C19 and of CYP 3A was 99%, 96% and 93%, respectively.The capacity to inhibit CYP 2D6 was under the conditions applied in the present study also significantly higher (90%).The phenylthiazole analogue of C21 compound, 11 exhibited similarly a high level of inhibition, with the exception for CYP 2B6 (31%) and CYP 2D6 (49%).A methoxycarbonyl rather than a butoxycarbonyl group had a major impact on inhibition of some CYP enzymes and the inhibition of CYP 2B6 (14%), CYP 2D6 (27%) and CYP 3A (41%) were further reduced.
As apparent from Table 1, introduction of a tert-butyl group in the 2position of the imidazole heterocycle had a large impact on the ability of the ligands to inhibit the enzymes.Thus, considering 20 and with the exception of CYP 2C8 (70%), CYP 2C9 (68%) and CYP 2C19 (66%) a very low CYP inhibition was observed.Moreover, by replacement of the lipophilic butoxy group of 20 for a methoxy group to furnish 21, a further suppression of the enzyme inhibition could be achieved.Hence, the methoxycarbonyl sulfonamide derivative 21 exhibited a negligible inhibition of CYP 3A (5%) and a very low tendency to inhibit CYP 2D6 (12%), 2C8 (26%), 2C9 (23%) and 2B6 (24%).Although 2C19 is inhibited by 38%, it is still a significant improvement compared to the 2C19 inhibition data obtained with C21 (96%), 11 (93%), 12 (80%) and 20 (66%).
In all CYP inhibition assays employed herein, a replacement of a butoxycarbonyl sulfonamide for a methoxycarbonyl sulfonamide function group suppressed the CYP enzyme inhibition and a combination with a tert-butyl group attached to the 2-position of the imidazole heterocycle seems to render an optimal outcome in this series of selective AT 2 R ligands.

Conclusion
In summary, we report the synthesis and AT 1 R and AT 2 R binding data of a series of new ligands, 6-39 derived from L-162,313 (1) and from the selective AT 2 R agonist C21 (4).All ligands comprise a phenylthiazole scaffold rather than the biphenyl or phenylthiophene scaffolds found in essentially all of the previously reported AT 2 R ligands.The affinity to AT 2 R were generally lower with a phenylthiazole scaffold than with the corresponding biphenyl or phenylthiophene scaffolds.Hence, the nonselective AT 1 R/AT 2 R ligand phenylthiophene L-162,313 (1) exhibited a 2-fold preference for AT 1 R in comparison to its phenylthiazole analogue 6 with an 18-fold and 8 with a 50-fold preference for AT 1 R.A potent AT 1 R selective ligand (10) exhibiting an AT 1 R K i of 8.5 nM and AT 2 R K i of 320 nM was identified.
Notably, a phenylthiazole rather than the phenylthiophene scaffold in the AT 2 R selective C21 (4) and C38 (5) reduced the affinity to AT 2 R significantly.Nevertheless, a significant improvement could be accomplished through the right choice of substituent in the 2-position of the imidazole heterocycle.A tert-butyl or an isopropyl group provided AT 2 R selective ligands with low K i values, 18 nM (20) and 5.8 nM (24), respectively.Furthermore, replacement of the butoxycarbonyl for a methoxycarbonyl resulted in the equipotent and AT 2 R selective ligands 21 (AT 2 R K i = 29 nM) and 25 (AT 2 R K i = 27 nM).The stability of 21 in liver microsomes was examined.The tert-butyl compound exhibited a high stability in the microsome assays and in addition presented a very attractive CYP inhibition profile.Phenylthiazole 21 was selected for further in depth in vivo studies.Hence, ligands with high AT 2 R affinity and demonstrating high stability in liver microsomes and with a low capability to inhibit the most common drug metabolizing CYP 450 enzymes were identified.

Chemistry
All experiments were performed under a nitrogen atmosphere in oven-dried glassware, employing standard techniques for handling air and moist sensitive materials, unless otherwise stated.All the purchased solvents and chemicals were used as without further purification.For Suzuki cross couplings the solvents were degassed prior to use.Microwave heating was performed in a Biotage single-mode microwave reactor producing controlled irradiation at 2450 MHz with a power of 0-400 W. The reaction temperature was determined and controlled using the built-in online IR-sensor and reactions were performed in septum sealed Biotage vials.Analytical TLC was performed on silica gel 60F-254 plates and visualized with UV light (λ = 254 nm).Automated Fig. 5.The stability of some of the selected the carbamates (t ½ in minutes) in mouse liver microsomes (MLM), rat liver microsomes (RLM) and human liver microsomes (HLM).

General procedure for deprotection of the sulfonamide
The Suzuki coupled product (50) (0.15 mmol) was stirred in trifluoroacetic acid (1.5 mL) at 40 • C overnight.The reaction was diluted with water (10 mL) and the product was extracted with dichloromethane (2 × 20 mL).The combined organic layers were washed with brine (15 mL), dried over MgSO 4 and concentrated.The sulfonamide was passed through a silica plug (0-5% with MeOH in CH 3 CN) and used in the following carbamate formation reactions without further purification.

Synthesis of substituted imidazoles
Synthesis of 2-ethyl-5,7-dimethyl-3H-imidazo [4,5-b]pyridine 31 .To a solution of KOH (1.47 g, 26.1 mmol, 1.2 equiv.) in MeOH (50 mL) in a two-neck round bottom flask was added 3-amino-3-imino-propanamide;hydrochloride (3.00 g, 21.8 mmol, 1 equiv.).After stirring at ambient temperature for 5 min, 2,4-pentadione (2.24 mL, 21.8 mmol, 1 equiv.)was added dropwise to the formed milky suspension and the reaction mixture was stirred at room temperature for 20 h.MeOH (50 mL) was added along with a solution of KOH (3.06 g, 54.5 mmol, 2.5 equiv.) in MeOH (20 mL).After stirring for further 30 min, the reaction mixture was cooled to − 5 • C, and (diacetoxyiodo)benzene (7.02 g, 21.8 mmol, 1 equiv.)was added.The mixture was allowed to reach room temperature over 3 h and stirred for additional 15 h.The solid formed in the reaction mixture was collected through filtration and washed with MeOH (200 mL) to yield a colorless solid.To this solid (2.42 g, 14.8 mmol, 1 equiv.)was added propionic acid (20.8 mL, 279 mmol, 19 equiv.)and propionic anhydride (20.8 mL, 162 mmol, 11 equiv.)and this reaction mixture was stirred at ambient temperature for 5 min.MgCl 2 (1.4 g, 14.7 mmol, 1 equiv.)was added and the resulting suspension refluxed overnight.After cooling to 60 • C MeOH (25 mL) was added.After partial evaporation of the solvent, the pH of the solution was adjusted to 10 using NH 4 OH (25 % aq.).This process was done carefully maintaining a temperature below 40 • C. Precipitate was formed while stirring this mixture at 0 • C for 90 min.The precipitate was collected through filtration, washed with cold water and dried in vacuo to afford the title compound as pale brown solid (2.58 g, 78%).
[Sar1Ile8]-angiotensin II (Sarile) acts as a nonselective AT2R agonist. 18he affinity was determined using a seven-point dose-response curve and the measurements were performed in duplicates.The AT2R selective antagonist PD-123,319 was used as reference compound.The compounds were also evaluated for inhibition of [125I][Sar1Ile8]angiotensin II binding to human AT1R expressed in HEK-293 cells.For AT1R the percent inhibition was determined at 10 μM, in duplicates, with the endogenous ligand (angiotensin II) used as reference.

Metabolic studies in liver microsomes
Human, mouse and rat liver microsomes were used to assess the metabolic stability of the compounds.Metabolic stability was determined in 0.5 mg/mL human, mouse or rat liver microsomes at a compound concentration of 1 µM in 100 mM potassium phosphate buffer (pH 7.4) in a total incubation volume of 500 µL by Eurofins.The assay matrix was: human liver microsomes: mixed gender and pool of 50, rat liver microsomes: male, Sprague-Dawley, pool of 100 or more and mouse liver microsomes: male, CD-1, pool of 250 or more.Final microsomal protein concentration: 0.1 mg/mL.The test concentration was 0.1 μM with 0.01% DMSO, 0.25% acetonitrile and 0.25% methanol by default.The experimental protocol was as following: the test compound was pre-incubated with pooled liver microsomes in phosphate buffer (pH 7.4) for 5 min in a 37 • C shaking waterbath.The reaction was initiated by adding NADPH-generating system and was incubated for 0, 15, 30, 45, and 60 min.The reaction was stopped by transferring the incubation mixture to acetonitrile/methanol.Samples were then mixed and centrifuged.Four reference compounds were tested in each assay.Propranolol and imipramine are relatively stable, whereas verapamil and terfenadine are readily metabolized in human liver microsomes.Samples were analyzed by HPLC-MS/MS using selected reaction monitoring.The HPLC system consisted of a binary LC pump with autosampler, a C-18 column, and a gradient.Peak areas corresponding to the test compound were recorded.The compound remaining was calculated by comparing the peak area at each time point to time zero.The half-life was calculated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming first order kinetics.

CYP inhibition studies
The following procedure was designed to assess if a test compound inhibits the activity of each of the common cytochrome P450 (CYP) enzymes in pooled human liver microsomes in 96-well plate format.The compounds were tested at a single concentration (10 μM) with 0.1% DMSO.The test compound was pre-incubated with substrate and human liver microsomes (mixed gender, pool of 50 donors, 0.1 mg/mL) in phosphate buffer (pH 7.4) for 5 min in a 37 • C shaking waterbath.The reaction was initiated by adding a NADPH-generating system.The reaction was allowed for 10 min and stopped by transferring the reaction mixture to acetonitrile/methanol.Samples were mixed and centrifuged.Supernatants were used for HPLC-MS/MS of the respective metabolite.The activity of human cytochrome P450 (CYP) enzymes were determined by following the formation of a metabolite of the probe substrate.The following enzymes were evaluated for inhibition using the listed reference substrates and reference inhibitors: CYP 1A -phenacetin (reference inhibitor furafylline), CYP 2B6 -bupropion (reference inhibitor clopidogrel), CYP 2C8 -amodiaquine (reference inhibitor montelukast), CYP 2C9 -diclofenac (reference inhibitor sulfaphenazole), CYP2C19 -omeprazole (reference inhibitor oxybutynin), CYP 2D6 -dextromethorphan (reference inhibitor quinidine), and CYP 3A -midazolam (reference inhibitor ketoconazole).The reference inhibitor were tested in each assay at multiple concentrations to obtain an IC 50 value.Peak areas corresponding to the metabolite were recorded.The percent of control activity was calculated by comparing the peak area in the presence of the test compound to the control samples containing the same solvent.Subsequently, the percent inhibition was calculated by subtracting the percent control activity from 100.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 4 .
Fig. 4. Phenylthiazole analogues of C21 substituted in the 2-position of the imidazole heterocycle and phenylthiazole analogues of C21 featuring an oxazole ring instead of the imidazole head group.The compounds 20-39 are AT 2 R selective with K i (AT 1 R) values > 1000 nM.