N-Alkyl Carbamoylimidazoles as Versatile Synthons for the Synthesis of Urea-Based PSMA Inhibitors

Abstract

We describe N-alkyl carbamoylimidazoles as readily available and highly versatile synthons for synthesizing urea-based prostate-specific membrane antigen (PSMA) inhibitors. Urea formation proceeded in high yields (>80%) at room temperature under aqueous conditions. All novel compounds were tested for their PSMA inhibitory potency in a cell-based radiometric binding assay. Compound 17 was identified as a novel high-affinity PSMA inhibitor (IC₅₀ = 0.013 μM) suitable for developing an ¹⁸F-labeled radioligand for PET imaging of PSMA in prostate cancer.

Prostate cancer is the most common malignancy in men in Western countries and is the fifth leading cause of cancer-related death in men.¹ Early and accurate diagnosis of high-risk prostate cancer is critical for improved outcomes and quality of life, leading to increased overall survival.² Prostate-specific membrane antigen (PSMA) has evolved as a robust and reliable biomarker for the diagnosis of primary and metastatic prostate cancer using nuclear medicine imaging techniques, positron emission tomography (PET) and single-photon emission computed tomography (SPECT). Expression of PSMA correlates with cancer biomarker prostate-specific antigen (PSA) and is linked to tumor aggressiveness and metastasis.³⁻⁶ The past decade has witnessed the development and evaluation of many PSMA-targeting small-molecule inhibitors. Several urea-based small molecules, initially developed by Kozikowski et al. as glutamate carboxypeptidase II (GCPII) inhibitors, have emerged as high-affinity PSMA radiopharmaceuticals for nuclear medicine imaging and radioligand therapy of prostate cancer.⁷ Most compounds contain a Lys-urea-Glu (KuE) motif as the PSMA binding pharmacophore as a result of several structural refinements to optimize binding to the PSMA binding pocket consisting of Zn²⁺ active sites and S1 and S1′ pockets.⁸ The latter pocket (S1′) is typically occupied by the glutamate portion of the PSMA ligand, whereas the S1 pocket offers more flexibility for structural modifications. Prominent examples of KuE radiopharmaceuticals for PET imaging (labeled with ¹⁸F or ⁶⁸Ga) and radioligand therapy (labeled with ¹⁷⁷Lu or ²²⁵Ac) currently tested in the clinic are given in Figure 1.

Figure 1.

Examples of clinically used KuE radioligands for the imaging and therapy of prostate cancer.

Typical synthesis strategies for the preparation of urea-based PSMA inhibitors involve (1) the formation of acylimidazole derivatives and their conversion into activated methylimidazolium salts for the subsequent reaction with protected amino acids like H-Lys(Z)-O^tBu; (2) the preparation of isocyanates as reactive intermediates for the reaction protected amino acids like H-Lys(Z)-O^tBu; and (3) cross-coupling reactions between protected amino acids using 4-nitrophenyl chloroformate or N,N′-disuccinimidyl carbonate for the activation of α-amine groups of one of the protected amino acids. The reactions are usually performed in solution, but solid-phase syntheses have also been reported.⁹

N-Alkyl carbamoylimidazoles have become a green and sustainable alternative to highly toxic and moisture-sensitive phosgene and phosgene substitutes like triphosgene, and isocyanate equivalents for the synthesis of ureas, carbamates and thiocarbamates.¹⁰N-Alkyl carbamoyl-imidazoles can easily be prepared from primary amines using 1,1′-carbonyldiimidazole (CDI) in both aqueous¹¹ and organic solvents¹² to form crystalline, readily storable and water-stable compounds (Scheme 1).

Scheme 1. Preparation of N-Alkyl Carbamoylimidazoles as Phosgene Substitutes and Isocyanate Equivalents for the Synthesis of Ureas (I), Carbamates (II), and Thiocarbamates (III).

N-Alkyl carbamoylimidazoles were previously used in synthesizing unsymmetrical ureas, which required activation with methyl triflate or a Brönsted acid before the reaction with another amine.¹⁰ Batey and co-workers developed an improved protocol for the synthesis of N-methyl carbamoylimidazoles as methyl isocyanate equivalents for the preparation of unsymmetrical ureas.¹² A further extension of this methodology yielded N-alkyl carbamoylimidazoles directly from alkyl amine hydrochloride salts without forming highly reactive isocyanate intermediates. Although this methodology was published a decade ago, to the best of our knowledge, this synthesis route has not yet been explored to its full potential for preparing urea-based PSMA inhibitors beyond two recently published reports.^13,14

Herein, we report on the facile synthesis of several urea-based PSMA inhibitors using N-alkyl carbamoylimidazoles as key synthons. For the first time, we demonstrate the use of unprotected amino acids to prepare the corresponding ureas from N-alkyl carbamoyl imidazoles. In addition to that, we also show that the proposed chemistry can be performed in a one-pot reaction without isolating the intermediate, which avoids tedious purification and leads to high isolated yields (95% for two steps).

In this study, we have focused our attention on novel fluorinated small-molecule PSMA inhibitors as potential leads for developing fluorine-18 (¹⁸F, t_1/2 = 108.9 min)-labeled radioligands for PET imaging.

First, we explored N-alkyl carbamoylimidazoles as synthons for the preparation of PSMA ligands derived from 2-aminoadipic acid 1, a building block that Kozikowski and co-workers initially reported for the synthesis of high-affinity PSMA inhibitors with IC₅₀ values in the low nM and sub-nanomolar range.¹⁵

We envisioned increasing the synthetic efficiency by optimizing protection and deprotection steps while decreasing the number of reaction steps. The synthesis of 2-aminoadipic acid-based PSMA inhibitors commenced by protecting both the α-amine and carboxylic acid group of 2-aminoadipic acid 1 with 9-borabicyclononane (9-BBN)¹⁶ to yield the amino acid boron complex 2 for subsequent chemoselective side-chain modification. The carboxylic acid side chain in boron-containing amino acid 2 was coupled with fluorine-containing amines (4-fluorobenzylamine, 6-fluorofluorobenzpyridine-3-amine), followed by removal of 9-BBN to give amino acids 4a and 4b.¹⁷ Treatment of tert-butyl ester-protected glutamic acid with CDI afforded N-alkyl carbamoyl-imidazole 5, which was reacted with amino acids 4a and 4b in aqueous solutions to yield unsymmetrical ureas 6a and 6b after ester hydrolysis. The syntheses of ureas 6a and 6b as novel PSMA inhibitors are summarized in Figure 2.

Figure 2.

Synthesis of PSMA ligands derived from 2-aminoadipic acid (R-NH₂ = 4-fluorobenzylamine; 6-fluoropyridine-3-amine).

A second direction aims to replace glutamic acid in KuE PSMA inhibitors with a 2-amino-thiophene-3,4-dicarboxylic acid (ATDA) as a planar nonchiral β,γ-amino acid mimicking glutamic acid. The synthesis of KuATDA PSMA inhibitors started from the reaction of diethyl 2-amino-thiophene-3,4-dicarboxylate 7 with the N-alkyl carbamoylimidazole derivative of lysine 8 in the presence of NaH in DMF to give protected nonsymmetrical urea 9. Our initial attempts to deprotect the benzyloxy carbamate (Cbz) protecting groups from 8 using hydrogenation with Pd/C or Pd(OH)₂/C failed, presumably due to catalyst poisoning with sulfur, originating from the thiophene moiety. However, the use of HBr/AcOH resulted in clean Cbz deprotection, and we observed the hydrolysis of two ester groups in 9, which was treated with fluorine-containing active ester motifs (10a or 10b) under basic conditions. Further hydrolysis with LiOH provided the target compounds 11a and 11b. The syntheses of KuATDA PSMA inhibitors 11a and 11b are summarized in Figure 3.

Figure 3.

Synthesis of KuATDA PSMA inhibitors 11a and 11b.

Finally, we tested the potential of N-alkyl carbamoylimidazoles as synthons for widely studied KuE-based PSMA ligands in a straightforward one-pot reaction. tert-Butyl glutamate hydrochloride 12 was treated with CDI to form the corresponding carbamoylimidazole compound 5, which was directly further reacted with H-Lys(Cbz)-O^tBu 14 under basic conditions to give the desired urea 15 (Figure 4).

Figure 4.

One-pot synthesis of KuE PSMA inhibitors.

Similarly, urea 15 was also prepared in a one-pot reaction starting from the carbamoylimidazole derivative of H-Lys(Cbz)-O^tBu, followed by treatment with tert-butyl glutamate hydrochloride 12. The obtained yields were comparable for both reactions. Next, urea 15 was subjected to hydrogenolysis to release the ε-amino group and provide compound 16 as a suitable intermediate for further functionalization to prepare KuE PSMA inhibitors. Recently, Kung and co-workers described the synthesis of a new high-affinity ⁶⁸Ga-labeled PSMA radioligand ([⁶⁸Ga]Ga-P16-093).¹⁸ The novel PSMA radioligand consists of a novel O-(carboxymethyl)-l-tyrosine linker. Clinical trials with prostate cancer patients demonstrated higher tumor uptake and better tumor detection capability of [⁶⁸Ga]Ga-P16-093 PET/CT than with [⁶⁸Ga]Ga-PSMA-617 PET/CT, suggesting that radioligand with [⁶⁸Ga]Ga-P16-093 holds promise for diagnosis and staging of primary prostate cancer patients.¹⁹ We envisioned the replacement of the HBED-CC chelator in compound [⁶⁸Ga]Ga-P16-093 with a 4-fluorobenzylideneamino-oxyacetyl motif, which can easily be prepared through oxime formation between 4-fluorobenzaldehyde and an amino-oxy group to prepare compound 17 (Scheme 2).

Scheme 2. Proposed Replacement of ⁶⁸Ga HBED-CC Chelator (Blue) with a 4-Fluorobenzylidene-aminooxy-acetyl motif (red).

Moreover, 4-[¹⁸F]fluorobenzaldehyde is readily available and can be used to prepare the corresponding ¹⁸F-labeled PSMA radioligand for PET imaging. The synthesis of PSMA inhibitor 17 is illustrated in Figure 5. Dipeptide 20 was prepared in quantitative yield by reacting Z-Gly-OH 19 with H-Tyr-O^tBu 18. The phenol group in compound 20 was alkylated with methyl bromoacetate to afford compound 21 in an 85% yield. The methyl ester of 21 was hydrolyzed with LiOH to give compound 22 in a 61% yield. Compound 16 (Figure 4) was coupled to Z-Phe-OH 23 to prepare compound 24. The Cbz group was removed with Pd/C and H₂ to give free amine 25. Amine 25 and dipeptide 22 were coupled using HATU to yield Cbz-protected amine 26 in 90% yield. Hydrogenolysis of compound 26 followed by coupling with acid 28 and global deprotection gave compound 17 in a 21% yield in three steps.

Figure 5.

Synthesis of PSMA inhibitor 17.

All compounds were tested for their PSMA inhibitory potency in an in vitro competitive binding assay using PSMA-expressing LNCaP cells with [¹⁸F]PSMA-1007 as the radioligand according to a modified procedure published by our research group.^20,21 2-(Phosphonomethyl)pentanedioic acid (2-PMPA) and PSMA-1007 as high-affinity PSMA inhibitors were used as reference compounds. Results of the in vitro competition binding assay are summarized in Table 1.

Table 1. In Vitro Inhibition Potencies (IC₅₀ Values) of Control (2-PMPA and PSMA-1007) and Fluorine-Containing PSMA Inhibitors.

Compound	IC50 [μM]
2-PMPA	0.065 ± 0.009
PSMA-1007	0.004 ± 0.001
6a	1.5 ± 0.8
6b	0.09 ± 0.01
11a	n.i.a
11b	n.i.
17	0.013 ± 0.004

^an.i. = no inhibition in the tested concentration range (10^–10 – 10^–4 M).

The analysis of the binding assay resulted in no inhibition in the tested concentration range for KuATDA PSMA inhibitors 11a and 11b and low to moderate inhibitory potency of 1.5 and 0.09 μM for 2-aminoadipic acid-containing PSMA ligands 6a and 6b, respectively.

The highest inhibitory potency of 0.013 μM was found for compound 17. The results clearly show that introducing ATDA as a planar nonchiral β,γ-amino acid mimicking glutamic acid resulted in the formation of inactive inhibitors, as demonstrated with compounds 11a and 11b. The result is consistent with the apparent importance of the glutamic acid residue for favorable binding to the PSMA binding pocket. The observed low to moderate affinities of PSMA inhibitors 6a and 6b were somewhat surprising, as comparable compounds containing a 2-aminoadipic acid residue and various fluorophenyl and fluoropyridyl motifs displayed IC₅₀ values in the low or subnanomolar range.¹⁸ Compound 6b, containing a fluoropyridine motif, was reported in the literature as a high-affinity PSMA inhibitor displaying an IC₅₀ value of 1 nM.¹⁸ In contrast, we determined an IC₅₀ value of 0.4 μM for the compound in our assay (data not shown). However, we were pleased to see the high inhibitory potency of compound 17 (IC₅₀ = 0.013 μM), confirming the favorable PSMA binding properties of urea-PSMA ligands containing an O-(carboxymethyl)-l-tyrosine linker, as demonstrated with ⁶⁸Ga-labeled PSMA ligand [⁶⁸Ga]Ga-P16-093.^18,19 PSMA ligand 17 displayed affinity similar to those of PSMA-11 (IC₅₀ = 0.017 μM) and [^natGa]Ga-P16-093 (IC₅₀ = 0.016 μM).¹⁸ The comparable PSMA binding affinity of fluorine-containing PSMA ligand 17 warrants the development and testing of a corresponding ¹⁸F-labeled PSMA radioligand for PET imaging. In a clinical setting, ¹⁸F-labeled PSMA radioligands can be prepared in multiple patient doses, whereas ⁶⁸Ga-labeled PSMA radioligands such as PSMA-11 and [⁶⁸Ga]Ga-P16-093 can only support 1–2 patient PET scans. The longer half-life of ¹⁸F and the possibility of preparing multipatient doses represent a critical advantage of ¹⁸F-labeled PSMA ligands over ⁶⁸Ga-labeled compounds. Moreover, ¹⁸F-labeled PSMA ligands can be shipped and distributed to other centers for clinical PET studies, thus improving access to PET imaging capacity.

To further study the binding of compound 17 to the PSMA binding pocket, we performed molecular docking studies to examine the binding mode of compound 17 in the PSMA-binding pocket. Compound 17 was docked into the PSMA-binding pocket of the protein structure (PDB 5O5T, 1.43 Å) using recently published computational analysis to estimate its binding interactions with active amino acid residues (Figure 6).²¹ The docking studies indicated that the KuE scaffold of compound 17 was able to enter the PSMA active-site tunnel (E_{intermolecular} = −7.50 kcal mol^–1) and establish H-bonding interactions with R534 (2.3 Å), R536 (2.4 Å) and R463 (2.6 and 2.1 Å). The COOH group of compound 17 also showed two H-bonding interactions with R511 (2.6 and 2.1 Å). The 4-fluorophenyl substituent of compound 17 also showed cation−π interactions with R511 (4.07 Å). Comparative docking studies with crystallized ligand PSMA-1007 revealed that the KuE scaffold of compound 17, despite several H-bonding interactions with the key amino acid residues, does not reach the deep vicinity of the PSMA active binding site (Supporting Information Figure S1).

Figure 6.

Molecular docking results for compound 17 in the PSMA binding site. A) Molecular orientation of compound 17 (carbons are displayed as orange spheres) in the PSMA tunnel. B) Close-up view to highlight the molecular interactions observed between compound 17 and key active-site amino acid residues. For clarity, selected active-site residues (gray lines) and zinc ions (gray spheres) are displayed. The H-bonding interactions are indicated by dashed red lines.

In conclusion, we have demonstrated the use of N-alkyl carbamoylimidazoles as versatile synthons for the facile preparation of a variety of PSMA ligands, including 2-aminoadipic acid-C(O)-glutamates (6a and 6b), Lys-C(O)-thiophene-3,4-dicarboxylates (11a and 11b) and widely studied KuE inhibitors like compound 17. All compounds were tested in an in vitro competitive binding assay in PSMA-expressing LNCaP cells. Results clearly showed that the substitution of glutamic acid with a planar 2-aminothiophene-3,4-dicaboxylic acid motif results in the loss of inhibitory potency, confirming the importance of the glutamic acid residue in urea-based PSMA inhibitors for favorable binding to the PSMA binding pocket.

We have also designed and synthesized a fluorinated derivative of Ga-P16-093 (compound 17) to further study and understand binding to PSMA, especially with PSMA inhibitors such as 17 containing the novel O-(carboxymethyl)-l-tyrosine linker.

Replacement of the HBED-CC chelator with a 4-fluorobenzylidene-amino-oxy motif did not reduce the PSMA binding affinity of compound 17 compared to clinically used PSMA ligands [⁶⁸Ga]Ga-P16-093 and PSMA-11. Therefore, we propose that compound 17 represents a new lead compound for the development of an ¹⁸F-labeled PSMA radioligand using oxime formation chemistry between the respective amino-oxy compound and readily available 4-[¹⁸F]fluorobenzaldehyde.²²

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00087.

• Synthetic procedures, analytical data of compounds and ¹H and ¹³C NMR spectra (PDF)

Author Contributions

N.R.G. conceived the idea and designed, synthesized and characterized all compounds. J.D., J.K. and M.W. designed and performed in vitro and cell-based experiments. A.B. performed computational studies. N.R.G. and F.W. wrote the manuscript and compiled the Supporting Information. N.R.G., M.W. and F.W. analyzed the data, discussed the results and contributed to the editing of the manuscript. All authors have approved the final version of the manuscript.

The authors declare no competing financial interest.