Abstract
Accordingly, embodiments herein disclose a compound and method of small molecule inhibitors or ligands for diagnosis and treatment of cancers such as prostate, brain, breast, etc., and neurodegenerative diseases. A new class of PSMA inhibitors called as aminoacetamide, 1, has been designed by extensive in silico studies. A simple, mild and high yielding synthetic methodology is developed for 1 and shown to have high affinity for PSMA protein. Fluorescent conjugates 22 and 25 derived from 1 show selective uptake in prostate cancer cell lines and can be used for surgical removal of tumors during intra-operative surgery. Conjugates 31 and 34 for tagging 99mTc radioisotope were synthesized. Macrocyclic chelating cores such as DOTA, NOTA or prosthetic groups can be introduced to tag radionuclides 68Ga, 64Cu, 18F and 177Lu for diagnosis and treatment of PCa, incurable mCRPC and neurodegenerative diseases such as ALS, schizophrenia and neuropathic pain that over-express PSMA protein.
Claims
1) A conjugate comprising: a) a ligand; b) a spacer; and c) a drug; wherein the ligand is a compound of Formula I ##STR00010## and stereoisomers thereof, wherein A and B are independently selected from a group consisting of hydrogen, optionally substituted C.sub.1-C.sub.7 alkyl, and optionally substituted aryl groups; X and Y are selected from the groups comprising of H, OH, and COOH groups, and Z is one of O or S groups.
2) The conjugate as claimed in claim 1, wherein A and B are independently selected from a group consisting of hydrogen, C.sub.1-C.sub.3 alkyl, and aryl groups; X and Y are selected from the groups comprising of H, OH, and COOH groups, Z is a 0 group.
3) The conjugate as claimed in claim 1, wherein stereochemical configuration of the stereocenter 1 and 2 of the compound of Formula I is of S configuration.
4) The conjugate as claimed in claim 1, wherein the spacer is a peptide comprising at least 2-20 amino acids.
5) The conjugate as claimed in claim 1, wherein the spacer comprises at least two phenylalanine residues, each of which is optionally substituted.
6) The conjugate as claimed in claim 1, wherein the spacer comprises amino caprylic acid.
7) The conjugate as claimed in claim 1, wherein the drug is at least one of imaging agents, anticancer drug or a radionuclide.
8) The conjugate as claimed in claim 1, wherein the ligand is selected from a group consisting of: ##STR00011## ##STR00012##
9) The conjugate of claim 7, wherein the imaging agent is a radioactive isotope of a metal coordinated to a chelating group, where the radioactive isotope is selected from a group consisting of .sup.99mTc, .sup.68Ga, .sup.18F and .sup.177Lu.
10) The conjugate as claimed in claim 9, wherein the chelating group has a formula II ##STR00013## wherein *indicates the site of attachment to the spacer
11) The conjugate as claimed in claim 1, having a formula III ##STR00014##
12) The conjugate as claimed in claim 1, having a formula IV ##STR00015##
13) The conjugate as claimed in claim 1, having a formula V ##STR00016##
14) The conjugate as claimed in claim 1, having a formula VI ##STR00017##
15) The conjugate as claimed in claim 1, having a formula VII ##STR00018##
16) A pharmaceutical composition comprising a therapeutically effective amount of the conjugate as claimed in claim 1, and at least one component selected from a group consisting of carriers, diluents, excipients and combinations thereof.
Description
BRIEF DESCRIPTION OF FIGURES
[0042] This method is illustrated in the accompanying drawings, throughout which like reference letters indicate corresponding parts in the various figures. The embodiments herein will be better understood from the following description with reference to the drawings, in which:
[0043] FIG. 1a illustrates four components or modules, according to an embodiment as disclosed herein;
[0044] FIG. 1b illustrates molecular modelling method for the inhibitors, according to an embodiment as disclosed herein;
[0045] FIG. 1c illustrates chemical synthesis of designed inhibitor, according to an embodiment as disclosed herein;
[0046] FIG. 1d illustrates design of bioconjugate handle in the linker for attaching cargos such as near-infrared fluorescent agents or anticancer drugs, according to an embodiment as disclosed herein;
[0047] FIG. 1e illustrates design of hydrophobic peptidic spacer for enhanced binding affinity to PSMA protein along with chelating moieties for tagging nuclear radioisotopes or nanoparticles that can be used for imaging and therapy, according to an embodiment as disclosed herein;
[0048] FIG. 2 illustrates design of lead aminoacetamide peptidomimetic 1 for PSMA enzyme inhibition, according to an embodiment as disclosed herein;
[0049] FIG. 3 illustrates design of library of peptidomimetics based on amino acetamide scaffold, according to an embodiment as disclosed herein;
[0050] FIG. 4 illustrates molecular docking study of JB7 with GCPII protein (PDB id-4NGM), according to an embodiment as disclosed herein;
[0051] FIG. 5 illustrates molecular docking study of ligand 1 designated as AAPT at GCPII active cavity (PDB id-4NGM), according to an embodiment as disclosed herein;
[0052] FIG. 6 illustrates computational docking of aminoacetamide inhibitor 3 in the active site of GCPII (PDB=4NGM), according to an embodiment as disclosed herein;
[0053] FIG. 7 illustrates hydrogen bonding interactions of aminoacetamide inhibitor 2 in the active site of GCPII (PDB=4NGM) in comparison to 3, according to an embodiment as disclosed herein;
[0054] FIG. 8 illustrates synthesis of precursors 15a-j for acetamide based GCPII ligands 1-10, according to an embodiment as disclosed herein;
[0055] FIG. 9 illustrates list of acetamide based GCPII inhibitor precursors 15a-j, according to an embodiment as disclosed herein;
[0056] FIG. 10 illustrates synthesis of acetamide based GCPII inhibitors 1-3 from precursors 15a-c, according to an embodiment as disclosed herein;
[0057] FIG. 11(a) illustrates inhibition curve of AAPT inhibitor 1 against PSMA protein isolated from prostate cancer cell LNCaP SD (n=1), according to an embodiment as disclosed herein;
[0058] FIG. 11(b) illustrates inhibition curve of PSMA inhibitors 2 against PSMA protein isolated from prostate cancer, LNCaP cells SD (n=3), according to an embodiment as disclosed herein;
[0059] FIG. 11(c) illustrates Inhibition curve of PSMA inhibitors 3 against PSMA protein isolated from prostate cancer, LNCaP cells SD (n=3), according to an embodiment as disclosed herein;
[0060] FIG. 12 illustrates dose-response curve of standard inhibitor, 2-PMPA, against PSMA protein isolated from prostate cancer cells, LNCaP; error bars represent SD (n=3), according to an embodiment as disclosed herein;
[0061] FIG. 13 illustrates solid phase synthetic strategy for preparation of AAPT rhodamine B conjugate 22 AAPT-C17201 reagents and conditions, according to an embodiment as disclosed herein;
[0062] FIG. 14 illustrates laser scanning confocal microscopy uptake study of PSMA targeted AAPT-C17201 fluorescent conjugate 22 in prostate cancer cell LNCaP at various concentrations, according to an embodiment as disclosed herein;
[0063] FIG. 15 illustrates binding affinity constant K.sub.D, determination of fluorescent conjugate 22 AAPT-C17201 in prostate cancer cell LNCaP SD (n=3), according to an embodiment as disclosed herein;
[0064] FIG. 16 illustrates solid phase synthetic strategy for preparation of AAPT rhodamine B conjugate 25 reagents and conditions, according to an embodiment as disclosed herein;
[0065] FIG. 17 illustrates binding of AAPT-arene rhodamine conjugate 25 in PSMA+ LNCaP cells for a range of concentrations plotted against the mean fluorescence intensity to yield a dissociation constant K.sub.D of 130 nM, according to an embodiment as disclosed herein;
[0066] FIG. 18 illustrates synthesis of AAPT ligand conjugated chelating linker 31 reagents and conditions, according to an embodiment as disclosed herein; and
[0067] FIG. 19 illustrates synthesis of AAPT ligand conjugated arene chelating linker 34 reagents and conditions, according to an embodiment as disclosed herein;
[0068] FIG. 20 illustrates structures of the PSMA scaffolds, glutamate urea heterodimers, glutamate phosphoramidates, 2 (phosphinylmethyl) pentanedioic acid and acetamide derivatives, according to an embodiment as disclosed herein.
DETAILED DESCRIPTION OF INVENTION
[0069] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. Also, the various embodiments described herein are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments. The term or as used herein, refers to a non-exclusive or, unless otherwise indicated. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein can be practiced and to further enable those skilled in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
[0070] The accompanying drawings are used to help easily understand various technical features and it should be understood that the embodiments presented herein are not limited by the accompanying drawings. As such, the present disclosure should be construed to extend to any alterations, equivalents and substitutes in addition to those which are particularly set out in the accompanying drawings. Although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are generally only used to distinguish one element from another.
[0071] Accordingly, embodiments describes a compound and method for diagnosis and treatment of malignancy arising out of prostate, brain, breast, bladder tissues and few of the neurodegenerative diseases like schizophrenia and ALS.
[0072] In an embodiment, the present disclosure describes a small molecule inhibitors or ligands for diagnosis and treatment of cancers such as prostate, brain, breast, etc., and other neurodegenerative diseases.
[0073] The term alkyl as used herein includes a chain of carbon atoms, which is optionally branched.
[0074] The term aryl as used herein includes molecular fragments or radicals comprising an aromatic mono or polycyclic ring of carbon atoms, such as phenyl, naphthyl, and the like.
[0075] The term substituted aryl as used herein includes molecular fragments or radicals comprising aryl with one or more substituents, such as alkyl, heteroalkyl, halo, hydroxy, amino, alkyl or dialkylamino, alkoxy, alkylsulfonyl, aminosulfonyl, carboxylate, alkoxycarbonyl, aminocarbonyl, cyano, nitro, and the like. It is to be understood that the alkyl groups in such substituents may be optionally substituted with halo.
[0076] The term amino acid as used herein includes molecular fragments or radicals comprising an aminoalkylcarboxylate, where the alkyl radical is optionally substituted with alkyl, hydroxy alkyl, sulfhydrylalkyl, aminoalkyl, carboxyalkyl, and the like, including groups corresponding to the naturally occurring amino acids, such as serine, cysteine, methionine, aspartic acid, glutamic acid, and the like.
[0077] Referring now to the drawings and more particularly to FIGS. 1a through 19 is shown preferred embodiments.
[0078] FIG. 1a illustrates four components or modules, according to an embodiment as disclosed herein.
[0079] In an embodiment in FIG. 1a, the four components or modules include rational structural design of several small molecule inhibitors based on binding with PSMA protein using in silico study, design and execution of simple, mild and high yielding chemical synthetic strategy for the preparation of small molecules ligands or inhibitors, strategy for introduction of fluorescent imaging moieties or cargos (anticancer drugs) using differentially protected lysine amino acid in a continuous process through solid phase peptide synthesis during the preparation of ligand conjugates and sequential introduction of hydrophilic and hydrophobic amino acids by targeting first, second and third hydrophobic pockets present in the PSMA protein along with chelation moieties based on molecular docking studies and linker length calculations. Selective targeting of PSMA protein expressed on several cancers and neurodegenerative diseases. Preparation of targeting ligands in an affordable manner by a cost-effective process. Tagging of near-infra red fluorescent molecules or cargos for diagnosis, intra-operative surgery and therapy of PCa Enhanced binding affinity to PSMA protein and tagging of radioisotopes for treatment of metastatic castration resistant prostate cancer (mCRPC) for which no cure is known.
[0080] FIG. 1b illustrates molecular modelling method for the inhibitors, according to an embodiment as disclosed herein.
[0081] FIG. 1c illustrates chemical synthesis of designed inhibitor (L-glutamic acid based amino acetamide inhibitors), according to an embodiment as disclosed herein.
[0082] FIG. 1d illustrates design of bioconjugate handle in the linker for attaching cargos such as near-infrared fluorescent agents or anticancer drugs, according to an embodiment as disclosed herein. The peptide spacer as can be observed in this figure comprises of 2 phenylalanine residues, and the fluorescent molecule herewith attached to the peptide spacer is rhodamine.
[0083] FIG. 1e illustrates design of hydrophobic peptidic spacer for enhanced binding affinity to PSMA protein along with chelating moieties for tagging nuclear radioisotopes or nanoparticles that can be used for imaging and therapy, according to an embodiment as disclosed herein. The peptide spacer as can be observed in this figure comprises of 3 phenylalanine residues.
[0084] In an embodiment in FIG. 1b, FIG. 1c, FIG. 1d and FIG. 1e, the molecules or inhibitors have been designed after carrying out extensive in silico molecular docking and quantitative structure activity relationship (QSAR) studies. The effect of substituents and structural activity relationship of the newly designed molecules with the binding cavity of the PSMA protein of interest has been thoroughly studied by modeling studies. After the rational design of the molecules or inhibitors or ligands, using computational methods a new, mild and simple chemical synthetic strategy is developed to synthesize a small library of new PSMA inhibitors. All the inhibitors or ligands or molecules were characterized by various spectroscopic techniques for structural confirmation. Finally, inhibitors which had shown the most promising theoretical binding affinity to PSMA protein labeled as AAPT were carried forward for in vitro and in vivo biological evaluations. The high binding affinity inhibitor AAPT is also conjugated with fluorescent tag such as rhodamine B through a peptidic spacer and the resultant fluorescent conjugate was evaluated for its uptake in malignant cell lines expressing the targeted protein PSMA of interest. The binding affinity constant K.sub.D of the fluorescent peptide conjugate to PSMA protein is determined to be in nano-molar concentration. In vitro evaluation studies have confirmed the selectivity and specificity of the molecules to bind to cancer cell lines over-expressing PSMA protein. The work now awaits in vivo evaluation in small animals such as mice, rat or guinea pigs which will be carried out in near future by tagging the inhibitors via chelating linkers to radioisotopes such as .sup.99mTc, .sup.68Ga, .sup.18F for diagnostic and therapeutic (.sup.17Lu) applications.
[0085] FIG. 2 illustrates design of lead aminoacetamide peptidomimetic 1 for PSMA enzyme inhibition, according to an embodiment as disclosed herein.
[0086] In an embodiment in FIG. 2, the initial design of the small molecule inhibitor is based on the understanding of the catalytic active site of metalloprotease enzyme, PSMA. The site of cleavage of the substrates has been shown for both NAAG and folyl -glutamate by PSMA enzyme. Keeping the architecture of endogenous PSMA substrates e.g. NAAG as well as folyl--Glu constant, at P1 pocket, an extra carbon has been inserted after the scission of amide bond, in the newly designed inhibitors, to enhance the number of new interactions in the binding pocket of PSMA. In our initial model, P1 pocket contains another L-glutamic acid residue which is strategically attached with an extra carbon atom to form the aminoacetamide moiety. The carbonyl oxygen of amino-acetamide moiety coordinates with the Zn atom in the active site of PSMA.
[0087] FIG. 3 illustrates design of library of peptidomimetics based on amino acetamide scaffold, according to an embodiment as disclosed herein.
[0088] In an embodiment in FIG. 3, further a small library of L-glutamic acid based amino acetamide inhibitors 1-10 which closely resembles NAAG as well as folyl--Glu has been designed to examine the structural and functional requirements necessary for binding in the active site of the PSMA protein.
[0089] Table 1 shows results of docking study are reported in the form of docking score which depends on various parameters like hydrogen bonding, lipophilic and - interactions. Molecular docking study used in the present disclosure to examine ligand-protein interactions of designed ligands responsible for inhibition of PSMA enzyme. In the present disclosure, various amino-acetamide based ligands were designed and their protein binding affinity were analysed through molecular docking. After validation using standard ligand JB7, same docking protocol was applied for designed ligands 1-10 and the docking scores of ligands 1-10 and JB7 are shown in table 1. Surflex Dock module of Sybyl X 2.1.1. program was utilized to find the binding conformations of JB7 and newly designed amino-acetamide derivatives 1-10 at the active site of GCPII protein. It is well documented that the active site of the GCPII protein contains hydrophobic (S1 pocket) and hydrophilic pocket (S1 pocket) and the interactions at S1 site is believed to be more critical for better binding affinity. Literature report suggest that JB7 interact with several amino acids residues such as Tyr 700, Arg 210, Lys 699, Asn 257, Gly 518, Tyr 552, Glu 424, Asn 519, Arg 536 and Arg 534 through hydrogen bonds that are critical for better binding of ligand with the protein. The aforementioned aminoacid interactions should be taken into consideration while performing docking studies of newly designed ligands 1-10 with GCPII protein.
TABLE-US-00001 TABLE 1 Molecular docking scores of ligands 1-10 and JB7 with GCPII protein (PDB 4NGM) Rank Ligand Docking score 1 JB7 16.38 2 1 13.96 3 3 13.63 4 2 12.51 5 6 12.01 6 9 11.99 7 8 11.45 8 4 11.35 9 7 10.94 10 10 10.79 11 5 10.67
[0090] FIG. 4 illustrates molecular docking study of JB7 with GCPII protein (PDB id-4NGM), according to an embodiment as disclosed herein.
[0091] Table 2 shows a correlation between the amino acid residues of S1 and S1 pockets present in PSMA protein and the newly designed amino-acetamide derivatives 1-10 interacting through hydrogen bonds. During the re-docking study of JB7 with the protein, similar amino acids interactions were observed as reported for JB7 with native protein (PDB 4NGM). In addition, an extra hydrogen bonding interaction with Lys 699 and Arg 536 residues were also observed. Moreover, in Surflex dock module of SYBYL, hydrogen bond distance more than 3 length are considered as weak and are not visible during docking study. As a result, hydrogen bonding interaction of JB7 with aminoacid residue Glu 424 of the protein was not observed which is of the order of 3.1 in length. After successful validation of docking procedure of JB7 with GCPII, similar protocol was applied for studying the docking interactions of newly designed amino-acetamide derivatives 1-10 with GCPII.
TABLE-US-00002 TABLE 2 Hydrogen bonding interactions between GCPII protein and ligands 1-10 along with bond distance of interaction in . JB7 H-bonding JB7 Post or interaction re docking Amino with GCPD in interactions acid from with GCPII H-bonding interactions of ligands 1-10 with GCPII protein in Site residue PDB 4NGM in 1 3 2 6 9 8 4 7 10 5 S1 site Arg 210 2.8 2.3 2.72 1.89 1.87 1.74 2.03 2.12 2.34 2.18 2.03 1.86 of GCPII 2.05 2.47 2.41 2.49 (Hydrophilic Asn 257 2.89 2.03 1.90 1.94 1.89 1.92 2.72 2.32 2.00 1.78 1.93 pocket) 2.40 2.70 1.91 2.73 2.41 Lys 699 2.7 2.47, 2.06 1.83 1.87 2.08 1.98 2.59 2.25, 2.55 1.88 1.9 2.52 1.79 Tyr 552 2.63 2.65 1.72 1.89 2.66 2.33 2.64 2.27 1.7 2.35 2.45 2.16 1.79 2.54 Tyr 700 2.53 1.8 2.73 1.84 2.2 2.32 2.27 2.69 2.72 2.06 Glu 424 3.01 2.06 2.04 2.79 2.67 2.41 2.1 1.91 2.03 1.85 1.85 2.23 2.01 Glu 425 2.01 2.13 2 2.55 2.55 2.13 2.74 2.44 S1 site Gly 51S 3.04, 3.05 1.88 2.4 2.15 1.76 2.44 1.94 1.9 of GCPII 1.85 (Hydrophobic Asn 519 2.98 2.16 2.10 2.47 1.99 1.81 2.04 2.28 2.08 pocket) 2.07 Arg 534 2.84 2.03 1.91 2.09 2.21 2.03 2.03 1.83 2.04 2.53 1.92 2.06 Arg 536 2.99, 3.0 2.32, 1.99, 1.96 2.21 2.68 2.61 2.21 2.57 2.08 1.90 2.31 Asp 453 2.51 2.57 Asp 387 2.56 2.37 2.72 Ser 454 2.29 1.72 2.30 1.89 Tyr 549 2.34 1.8 2 Ser 517 2.03 2.23 2.05 Arg 463 2.4 Total number of 6/6 6/6 9/7 10/4 7/6 11/2 7/4 8/7 9/6 4/8 5/5 9/0 hydrogen bonds at S1/S1
[0092] In an embodiment in FIG. 4, among all the newly designed amino-acetamide ligands, the docking score of ligand 1 is found to be highest followed by ligands 3 (second most active) and 2 (third most active) in the series (Table 1). The docking conformation of the most active ligand, 1, in the series show nine hydrogen bonding interactions at hydrophilic S1 pocket with Tyr 700, Arg 210, Lys 699, Asn 257, Tyr 552, Glu 424 and Glu 425 (new hydrogen bonding interactions) residues. This fact suggested that the glutamate moiety of 1 interacts strongly with the protein active site compared to JB7. However, ligand 1 exhibits similar hydrogen bonding interaction as JB7 with hydrophobic S1 pocket (FIG. 5). Insertion of an extra methylene group after the amide bond leads to an increase in the number of hydrogen bond interactions with aminoacid residues such as Arg 210, Asn 257, Glu 424, Asn 519 and Arg 534. The carbonyl oxygen of amino acetamide ligand in 1 also interacts with the hydroxy group of Tyr 552 in S1 pocket of PSMA with a bond length of 1.72 which is considerably less than the bonding interaction (2.65 ) of urea carbonyl oxygen moiety of JB7 with OH group of Tyr 552. It is important to note that Tyr 552 is positioned near Zn atoms of GCPII protein, which is important for catalytic activity of PSMA.
[0093] FIG. 5 illustrates molecular docking study of ligand 1 designated as AAPT at GCPII active cavity (PDB id-4NGM), according to an embodiment as disclosed herein.
[0094] In an embodiment in FIG. 5, Strong interaction of 1 with Tyr 552 residue at the active site of the protein might be helpful in inhibiting the catalytic activity of GCPII. This strong hydrogen bonding interaction of 1 with GCPII could be responsible for higher binding affinity of 1 among other ligands in the series. Though ligand 1 forms more number of hydrogen bonds due to the presence of polar glutamate scaffold, the overall docking score of JB7 was found to be higher as compared to 1. This is because the efficacy of inhibitory activity of ligands not only depends on polar interactions but also on other non-polar interactions such as lipophilic and - stacking interactions due to the presence of benzyl group in the lysine moiety of JB7. Further, to analyze the effect of polar and non-polar substituents on the efficiency of ligands to inhibit activity of GCPII enzyme, several derivatives of amino acetamide ligands such as 2-10 have been designed and synthesized for evaluation. After glutamate ligand 1, phenylalanine (3) and tyrosine (2) derivatives were observed to have better docking score than the other designed analogs. The docking conformation of the second most active ligand (3) in series show ten hydrogen bonding interaction with the active site residues such as Arg 210, Asn 257, Lys 699, Tyr 552 and Glu 424 at the hydrophilic pocket of the enzyme. Slightly less activity of 3 as compared to 1 is due to less number of hydrogen bonding interactions (Table 2) of 3 at the hydrophobic pocket of GCPII enzyme.
[0095] FIG. 6 illustrates computational docking of aminoacetamide inhibitor 3 in the active site of GCPII (PDB=4NGM), according to an embodiment as disclosed herein.
[0096] In an embodiment in FIG. 6, because of the presence of hydroxyl group of tyrosine moiety in 2, there is a change in the orientation of tyrosine moiety leading to change in the total number of amino acid interactions of 2 as compared to 3 with GCPII. At 51 site (hydrophilic pocket), 3 forms seven hydrogen bonding interactions with Arg 210, Asn 257, Lys 699 and Glu 425 aminoacid residues, whereas 2 forms only two hydrogen bonding interactions with Arg 210 and Lys 699 and no interaction with Asn 257 and Glu 425 residues when compared to 3. However, 2 forms an additional H-bonding interaction with Tyr 552 along with a new interaction with Tyr 700 in the active site of GCPII in comparison to 3. Including two interactions of both 2 and 3 with Glu 424 residue, the total number interactions of 2 and 3 at hydrophilic pocket are seven and ten respectively.
[0097] FIG. 7 illustrates hydrogen bonding interactions of aminoacetamide inhibitor 2 in the active site of GCPII (PDB=4NGM) in comparison to 3, according to an embodiment as disclosed herein.
[0098] In an embodiment in FIG. 7, at S1 site (hydrophobic pocket), 3 forms four hydrogen bonding interactions with Gly 518, Asn 519 and Arg 534 which were absent in 2. Due to change in the orientation of 2, it forms six new interactions with Asp 453, Asp 387, Ser 454, Tyr 549 and Ser 517 aminoacid residues that were absent in 3. This study infers that at GCPII active site, 3 forms fourteen hydrogen bonding interactions while 2 forms only thirteen interactions. This may be the plausible reason for less docking score of ligand 2 as compared to 3. In summary, we have designed and developed amino acetamide derivatives as a new class of GCPII inhibitor through extensive molecular docking studies. Ten amino acetamide based ligands/inhibitors (1-10) have been rationally designed and through molecular docking study top three rank derivatives (1-3) were selected for detailed analysis of amino acid-ligand interactions. However, derivatives 4-10 were also studied for various interactions at the active site of GCPII without detailed interpretation of the theoretical data presented in table 2.
[0099] In an embodiment in FIG. 4, FIG. 5, FIG. 6 and FIG. 7, silico study and hydrogen bonding interactions between the designed inhibitors or ligands and different amino acids (bond length in A) present in the binding pockets of prostate specific membrane antigen (PSMA) enzyme. In the drug discovery, for prediction of binding mode of an active ligand over a protein, docking studies are usually performed. In the present disclosure, Glutamate Carboxy Peptidase II (GCPII) receptor or prostate specific membrane antigen (PSMA), complexed with urea-based inhibitor JB7 (PDB code 4NGM) was retrieved from protein data bank and docking studies were performed by surflex dock method using sybyl X2.1.1 software.
[0100] FIG. 8 illustrates synthesis of precursors 15a-j for acetamide based GCPII ligands 1-10, according to an embodiment as disclosed herein. The process for synthesis of the acetamide based GCPII inhibitors 1-3 from precursors 15a-c is provided in the examples section of the specification. The acetamide based GCPII inhibitor precursors 15a-15j synthesized by said process are illustrated in FIG. 9.
[0101] FIG. 10 illustrates synthesis of acetamide based GCPII inhibitors 1-3 from precursors 15a-c, according to an embodiment as disclosed herein. The process comprises catalytic reduction (PdC/H.sub.2) in the presence of methanol at room temperature for a period of 24 hours, followed by de-protection of t-butyl group on the carboxy terminus using CF.sub.3COOH in CH.sub.2Cl.sub.2 (1:1) at room temperature for 2 hours to obtain the acetamide based GCPII inhibitors 1-3.
[0102] In an embodiment in FIG. 8, FIG. 9 and FIG. 10, chemical synthetic strategy for the preparation of small molecule inhibitors and PSMA enzyme inhibition assay, chemical synthesis of inhibitors 1-3. After the completion of molecular docking study, inhibitors 1-3 were chemically synthesized by deprotection of carboxy protecting groups (benzyloxy and tertiary-butyl) from their precursors 15a-c and selected for further in vitro biological evaluation to inhibit GCPII enzyme activity. Carboxy protected acetamide precursors 15d-j of acetamide based GCPII inhibitors 4-10 were also synthesized and completely characterized for future studies if required. As per molecular docking studies GCPII inhibitors 4-10 (table 2) had poor docking scores when compared to inhibitors 1-3 and hence not synthesized chemically in the lab though their precursors 15d-j are synthesized, characterized using various spectroscopic techniques and readily available for further experimental studies. In vitro PSMA enzyme inhibition NAALADase assay for AAPT inhibitor 1. The new PSMA inhibitor 1, called as AAPT, was next analyzed for its ability to inhibit PSMA protein expressed on LNCaP cancer cell to hydrolyze a natural substrate N-acetylaspartylglutamate (NAAG) using fluorescence based Amplex Glutamate kit by competitive inhibition. The results of this study are presented in FIG. 11a-11c. Briefly, PSMA enzyme was extracted from membrane lysates of LNCaP cell lines after separating the soluble cytosolic PSM portion by ultracentrifugation at 100,000g. The PSMA enzyme (8.3027 ng, 100 L) free from cytosolic portion was incubated with various concentrations of the AAPT inhibitor (0.1 nM to 1000 nM, 100 L) in the presence of NAAG (30 nM, 50 L) for 60 min in a 24-well plate. The amount of glutamic acid released by the hydrolysis of NAAG during competitive inhibition by AAPT inhibitor was measured by incubating each well with a working solution of Amplex Red reagent (100 uM, 50 L) for 30 min at 37 C. The fluorescence emission from each well, proportional to the amount of released glutamic acid, was measured using Synergy H1 multimode plate reader (BioTek Instruments, Inc., Winooski, Vt., USA) at an excitation and emission wavelength of 530 nm and 590 nm respectively.
[0103] Further dose vs response curves were obtained from a plot of semi-log[conc] vs intensity of fluorescence emission and IC.sub.50 (concentration at which 50% of the enzymatic activity is inhibited) was calculated for AAPT inhibitor against PSMA enzyme using GraphPad Prism, version 7.02 for Windows (GraphPad Software, San Diego, Calif.). In the first trial, the enzyme inhibition analysis was performed by incubating PSMA enzyme with increasing concentrations of inhibitor, AAPT. The half maximal inhibitory concentration (IC.sub.50) of the inhibitor, AAPT, was determined to be 38.5 or 95 or 78 nM (FIG. 11a-11c).
[0104] FIG. 12 illustrates dose-response curve of standard inhibitor, 2-PMPA, against PSMA protein isolated from prostate cancer cells, LNCaP; Error bars represent SD (n=3). For this purpose, GCPII enzyme inhibition assay was performed with a standard PSMA inhibitor, 2-(phosphonomethyl) pentanedioic acid (PMPA) whose IC.sub.50 is reported to be 0.28 nM and experimentally we have observed a value of IC.sub.50=0.40 nM (FIG. 12) validating our assay for inhibiting GCPII with PSMA inhibitors 1-3.
[0105] FIG. 13 illustrates solid phase synthetic strategy for preparation of AAPT rhodamine B conjugate 22 AAPT-C17201 reagents and conditions, according to an embodiment as disclosed herein.
[0106] In an embodiment in FIG. 13, Strategy for introduction of fluorescent imaging moiety or cargo (anticancer drugs) using differentially protected lysine amino acid in a continuous process through solid phase peptide synthesis (SPPS). Solid phase peptide synthesis of AAPT rhodamine B conjugate 22. 1,2-diaminoethanetrityl resin (0.050 g, 0.0525 mmol) was swelled initially with CH.sub.2Cl.sub.2 (5 mL) for 30 minutes by bubbling nitrogen and after draining CH.sub.2Cl.sub.2, the resin is swelled once again with DMF (5 mL) thrice for 15 minutes each. Fmoc-Asp(OtBu)-OH (0.054 g, 0.1312 mmol), PyBOP (0.068 g, 0.1312 mmol) and DIPEA (0.091 mL, 0.525 mmol) in DMF (0.5 mL) was added to the peptide vessel and the coupling reaction was continued for 6 h. The resin was washed with DMF (3.0 mL3) followed by isopropanol (3.0 mL3). The completion of reaction was confirmed by performing the Kaiser test. A solution of 20% piperidine in DMF (4 mL) was added to the peptide vessel and the resin beads were bubbled for 10 minutes. The procedure was repeated twice (3 mL2) to ensure complete deprotection of Fmoc protecting group from the coupled amino acid. The resin beads were washed with DMF (3.0 mL3) and isopropanol (3.0 mL3) and the formation of free amine was confirmed by performing the Kaiser test. Consecutively, Fmoc-Lys(Tfa)-OH, Fmoc-8-aminocaprylic acid, Fmoc-Phe-OH, Fmoc-Phe-OH and Fmoc-8-aminocaprylic acid were attached to the growing peptide chain in sequence as mentioned before. After deprotection of Fmoc group from the last amino acid, Fmoc-8-aminocaprylic acid, tris-tert butyl protected AAPT ligand 16a (0.040 g, 0.079 mmol), PyBOP (0.068 g, 0.1312 mmol) and DIPEA (0.091 mL, 0.525 mmol) in DMF (0.5 mL) was added to the resin beads and mixed for 6 h. The completion of coupling reaction was confirmed by performing the Kaiser test. Finally, the trifluoroacetyl (Tfa) protecting group of lysine amino acid was cleaved with 2M aqueous piperidine (10 mL) at room temperature for 6-12 h (depending on completion of the reaction) and the deprotection of Tfa group was confirmed by the Kaiser test. Rhodamine B dye (0.038 g, 0.079 mmol), PyBOP (0.068 g, 0.1312 mmol) and DIPEA (0.091 mL, 0.525 mmol) in DMF (0.5 mL) was added to the peptide vessel and reacted for 6 h at room temperature. The completion of the coupling reaction was confirmed by performing the Kaiser test. A mixture of 9.5 mL trifluoroacetic acid (TFA), 0.25 mL triisopropylsilane (TIPS), and 0.25 mL H.sub.2O was prepared in a 15 mL centrifuge tube, and 5 mL of this cocktail solution was added to the resin beads and nitrogen gas was bubbled through the solution for 30 minutes. The cocktail with cleaved rhodamine peptide conjugate in peptide vessel was collected to a round bottom flask (25 mL). The resin beads were treated again with the cocktail solution twice (2.5 mL2) for 15 minutes each and the mother liquor was collected in the same round bottom flask (25 mL). The pooled cocktail mixture with cleaved peptide conjugate 22 was transferred to a 15 mL centrifuge tube, fitted with a septum and concentrated under reduced pressure to obtain a viscous liquid. Ice cold ether (5 mL) was added to the concentrated viscous mixture to precipitate rhodamine conjugate 22 as a bright red solid and the solid was washed thrice with ice cold ether (5 mL3). The crude product 22 was purified by RP-HPLC using pentafluorophenyl preparative column (5 m, 10 mm150 mm) [=555 nm; solvent gradient 1% B to 70% B in 25 min, 80% B wash 15 min; A=0.1% TFA, pH=2; B=acetonitrile (ACN)]. Acetonitrile was removed reduced pressure from HPLC fractions, and the pure fractions were freeze-dried to yield peptide rhodamine conjugate 22 as pink solid. The purity of rhodamine conjugate 22 was confirmed by analytical RP-HPLC and the molecular weight is determined by HRMS (+ESI) calcd for [MCl].sup.+ (C86H118N13O18)+: 1620.8718 found 1620.8727.
[0107] In an embodiment in FIG. 13, Solid phase synthetic strategy for preparation of .sub.AAPT rhodamine B conjugate 22, AAPT-C17201, Reagents and conditions (a) Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA, DMF, 6 h (b) (i) 20% Piperidine in DMF, rt, 30 min (ii) Fmoc-Lys(Tfa)-OH, PyBOP, DIPEA, DMF, 6 h (c) (i) 20% Piperidine in DMF, rt, 30 min (ii) Fmoc-8-aminocaprylic acid, PyBOP, DIPEA, DMF, 6 h (d) (i) 20% Piperidine in DMF, rt, 30 min (ii) Fmoc-Phe-OH, PyBOP, DIPEA, DMF, 6 h (e) (i) 20% Piperidine in DMF, rt, 30 min (ii) Fmoc-Phe-OH, PyBOP, DIPEA, DMF, 6 h (f) (i) 20% Piperidine in DMF, rt, 30 min (ii) Fmoc-8-aminocaprylic acid, PyBOP, DIPEA, DMF, 6 h (g) (i) 20% Piperidine in DMF, rt, 30 min (ii) 16a, PyBOP, DIPEA, DMF, 6 h (h) (i) 2M Piperidine in water, rt, 6-12 h (ii) Rhodamine B, PyBOP, DIPEA, DMF, 6 h (iii) TFA/TIS/H2O (95.0:2.5:2.5) (15 mL, 30 min; 22.5 mL, 15 min each) (iv) Evaporate TFA (v) Precipitate in ice cold diethylether.
[0108] FIG. 14 illustrates laser scanning confocal microscopy uptake study of PSMA targeted AAPT-C17201 fluorescent conjugate 22 in prostate cancer cell LNCaP at various concentrations, according to an embodiment as disclosed herein.
[0109] In an embodiment in FIG. 14, Uptake study of ligand conjugated fluorescent conjugate 22, AAPT-C17201, in prostate cancer cells, LNCaP, using laser scanning confocal microscopy. The selective uptake of the newly synthesized PSMA targeted near-infra red fluorescent conjugate, AAPT-C17201, was evaluated by studying the ability of AAPT inhibitor or ligand to deliver diagnostics or fluorescent cargos to PSMA protein expressing cancer cell lines such as LNCaP cells. The fluorescent conjugate, AAPT-C17201, was synthesized by tethering a high affinity AAPT inhibitor via a peptidic spacer to Rhodamine B using standard solid phase peptide synthesis methodology. The purified PSMA targeted ligand conjugate, AAPT-C17201, was evaluated in prostate cancer cells (LNCaP) expressing PSMA protein using laser scanning confocal microscopy for wide range of low nanomolar concentrations-5, 10, 25 nM. Laser scanning confocal microscopy uptake study of PSMA targeted AAPT-C17201 fluorescent conjugate 22 in prostate cancer cells, LNCaP, at various concentrations (i) 5 nM, (ii) 10 nM, (iii) 25 nM for 1 h incubation and (iv-vi) differential interference contrast (DIC) images of PCa cells.
[0110] FIG. 15 illustrates binding affinity constant KD, determination of fluorescent conjugate 22 AAPT-C17201 in prostate cancer cell LNCaP SD (n=3), according to an embodiment as disclosed herein.
[0111] In an embodiment in FIG. 15, Evaluation of binding affinity of PSMA targeted fluorescent conjugate 22, AAPT-C17201, using flow cytometry analysis. The binding affinity constant of PSMA targeted fluorescent conjugate, AAPT-C17201, to target PSMA was determined in vitro in LNCaP cells by measuring the mean fluorescence intensity per cell for different concentrations of the fluorescent conjugate in triplicate using Fluorescence Activated Cell Sorting (FACS) study. A hyperbolic curve of different concentrations of fluorescent conjugates 22 or 25 against the mean fluorescence intensity in the PSMA+ LNCaP cells yields a dissociation constant (KD) of 85 nM or 130 nM, respectively. The high dissociation constant value (85 nM) gives us undisputable evidence for the high affinity of AAPT-C17201 fluorescent conjugate to PSMA protein and its perfect fit to the 20 protein tunnel present in PSMA.
[0112] FIG. 16 illustrates solid phase synthetic strategy for preparation of AAPT rhodamine B conjugate 25 reagents and conditions, according to an embodiment as disclosed herein.
[0113] In an embodiment in FIG. 16, Solid phase peptide synthesis of AAPT rhodamine B conjugate 25. The arene rhodamine B conjugate 25 is synthesized using SPPS by introduction of 4-carboxylic acid benzylamine in the peptide spacer to enhance the binding affinity to PSMA protein. 1,2-diaminoethanetrityl resin (0.050 g, 0.0525 mmol) was swelled initially with CH2Cl2 (5 mL) for 30 minutes by bubbling nitrogen and after draining CH2Cl2, the resin is swelled once again with DMF (5 mL) thrice for 15 minutes each. Fmoc-Asp(OtBu)-OH (0.054 g, 0.1312 mmol), PyBOP (0.068 g, 0.1312 mmol) and DIPEA (0.091 mL, 0.525 mmol) in DMF (0.5 mL) was added to the peptide vessel and the coupling reaction was continued for 6 h. The resin was washed with DMF (3.0 mL3) followed by isopropanol (3.0 mL3). The completion of reaction was confirmed by performing the Kaiser test. A solution of 20% piperidine in DMF (4 mL) was added to the peptide vessel and the resin beads were bubbled for 10 minutes. The procedure was repeated twice (3 mL2) to ensure complete deprotection of Fmoc protecting group from the coupled amino acid. The resin beads were washed with DMF (3.0 mL3) and isopropanol (3.0 mL3) and the formation of free amine was confirmed by performing the Kaiser test. Consecutively, Fmoc-Lys(Tfa)-OH, Fmoc-8-aminocaprylic acid, Fmoc benzylamine-4-carboxylic acid, Fmoc-Phe-OH, Fmoc-Phe-OH and Fmoc-8-aminocaprylic acid were attached to the growing peptide chain in sequence as mentioned before. After deprotection of Fmoc group from the last amino acid, Fmoc-8-aminocaprylic acid, tris-tert butyl protected AAPT ligand 16a (0.040 g, 0.079 mmol), PyBOP (0.068 g, 0.1312 mmol) and DIPEA (0.091 mL, 0.525 mmol) in DMF (0.5 mL) was added to the resin beads and mixed for 6 h. The completion of coupling reaction was confirmed by performing the Kaiser test. Finally, the trifluoroacetyl (Tfa) protecting group of lysine amino acid was cleaved with 2M aqueous piperidine (10 mL) at room temperature for 6-12 h (depending on completion of the reaction) and the deprotection of Tfa group was confirmed by the Kaiser test. Rhodamine B dye (0.038 g, 0.079 mmol), PyBOP (0.068 g, 0.1312 mmol) and DIPEA (0.091 mL, 0.525 mmol) in DMF (0.5 mL) was added to the peptide vessel and reacted for 6 h at room temperature. The completion of the coupling reaction was confirmed by performing the Kaiser test. A mixture of 9.5 mL trifluoroacetic acid (TFA), 0.25 mL triisopropylsilane (TIPS), and 0.25 mL H.sub.2O was prepared in a 15 mL centrifuge tube, and 5 mL of this cocktail solution was added to the resin beads and nitrogen gas was bubbled through the solution for 30 minutes. The cocktail with cleaved rhodamine peptide conjugate in peptide vessel was collected to a round bottom flask (25 mL). The resin beads were treated again with the cocktail solution twice (2.5 mL2) for 15 minutes each and the mother liquor was collected in the same round bottom flask (25 mL). The pooled cocktail mixture with cleaved peptide arene rhodamine B conjugate 25 was transferred to a 15 mL centrifuge tube, fitted with a septum and concentrated under reduced pressure to obtain a viscous liquid. Ice cold ether (5 mL) was added to the concentrated viscous mixture to precipitate arene rhodamine B conjugate 25 as a bright red solid and the solid was washed thrice with ice cold ether (5 mL3). The crude product 25 was purified by RP-HPLC using pentafluorophenyl preparative column (5 m, 10 mm150 mm) [=555 nm; solvent gradient 1% B to 70% B in 25 min, 80% B wash 15 min; A=0.1% TFA, pH=2; B=acetonitrile (ACN)]. Acetonitrile was removed reduced pressure from HPLC fractions, and the pure fractions were freeze-dried to yield peptide arene rhodamine B conjugate 25 as pink solid. The purity of arene rhodamine B conjugate 25 was confirmed by analytical RP-HPLC and the molecular weight is determined by HRMS (+ESI) calcd for [MCl].sup.+ (C94H125N14O19)+: 1753.9245 found 1753.9239.
[0114] In an embodiment in FIG. 16, Solid phase synthetic strategy for preparation of AAPT rhodamine B conjugate 25, Reagents and conditions (a) Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA, DMF, 6 h (b) (i) 20% Piperidine in DMF, rt, 30 min (ii) Fmoc-Lys(Tfa)-OH, PyBOP, DIPEA, DMF, 6 h (c) (i) 20% Piperidine in DMF, rt, 30 min (ii) Fmoc-8-aminocaprylic acid, PyBOP, DIPEA, DMF, 6 h (d) (i) 20% Piperidine in DMF, rt, 30 min (ii) Fmoc benzylamine-4-carboxylic acid, PyBOP, DIPEA, DMF, 6 h (e) (i) 20% Piperidine in DMF, rt, 30 min (ii) Fmoc-Phe-OH, PyBOP, DIPEA, DMF, 6 h (f) (i) 20% Piperidine in DMF, rt, 30 min (ii) Fmoc-Phe-OH, PyBOP, DIPEA, DMF, 6 h (g) (i) 20% Piperidine in DMF, rt, 30 min (ii) Fmoc-8-aminocaprylic acid, PyBOP, DIPEA, DMF, 6 h (h) (i) 20% Piperidine in DMF, rt, 30 min (ii) 16a, PyBOP, DIPEA, DMF, 6 h (i) (i) 2M Piperidine in water, rt, 6-12 h (ii) Rhodamine B, PyBOP, DIPEA, DMF, 6 h (iii) TFA/TIS/H2O (95.0:2.5:2.5) (15 mL, 30 min 22.5 mL, 15 min each) (iv) Evaporate TFA (v) Precipitate in ice cold diethylether.
[0115] FIG. 18 illustrates synthesis of AAPT ligand conjugated chelating linker 31 reagents and conditions, according to an embodiment as disclosed herein.
[0116] In an embodiment in FIG. 18, Solid phase synthesis of AAPT ligand conjugated chelating linker 31.H-Cys-2-ClTrt resin (0.100 g, 0.09 mmol) was swelled in CH.sub.2Cl.sub.2 (5 mL) for 30 minutes by bubbling nitrogen and after draining CH.sub.2Cl.sub.2, the resin is swelled once again with DMF (5 mL3) thrice for 15 minutes each. Fmoc-Asp(OtBu)-OH (93 mg, 0.225 mmol), PyBOP (116 mg, 0.225 mmol) and DIPEA (0.16 mL, 0.90 mmol) in DMF (0.5 mL) was added to the peptide vessel and the coupling reaction was continued for 6 h. The resin was washed with DMF (3.0 mL3) followed by isopropanol (3.0 mL3). The completion of reaction was confirmed by performing the Kaiser test. A solution of 20% piperidine in DMF (4 mL) was added to the peptide vessel and the resin beads were bubbled for 10 minutes. The procedure was repeated twice (3 mL2) to ensure complete deprotection of Fmoc .sub.protecting group from the coupled amino acid. The resin beads were washed with DMF (3.0 mL3) and isopropanol (3.0 mL3) and the formation of free amine was confirmed by performing the Kaiser test. Consecutively, Boc-Dap(Fmoc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH and Fmoc-8-aminocaprylic acid were attached to the growing peptide chain in sequence as mentioned before. After deprotection of NHFmoc group from the last amino acid, Fmoc-8-aminocaprylic acid, tris-tertbutyl protected compound 16a (68 mg, 0.135 mmol), PyBOP (116 mg, 0.225 mmol) and DIPEA (0.16 mL, 0.90 mmol) in DMF (0.5 mL) were added to the vessel and mixed for 6 h. The completion of reaction was confirmed by the Kaiser test.
[0117] In an embodiment in FIG. 18, A mixture of 9.25 mL trifluoroacetic acid (TFA), 0.25 mL ethane dithiol, 0.25 mL triisopropylsilane (TIPS), and 0.25 mL H.sub.2O was prepared in a 15 mL centrifuge tube, and 5 mL of this cocktail solution was added to the resin beads and nitrogen gas was bubbled through the solution for 30 minutes. The cocktail with cleaved ligand targeted cysteine chelating linker 31 in peptide vessel was collected to a round bottom flask (25 mL). The resin beads were treated again with the cocktail solution twice (2.5 mL2) for 15 minutes each and the mother liquor was collected in the same round bottom flask (25 mL). The pooled cocktail mixture with cleaved peptide conjugate 31 was transferred to a 15 mL centrifuge tube, fitted with a septum and concentrated under reduced pressure to obtain a viscous liquid. Ice cold ether (5 mL) was added to the concentrated viscous mixture to precipitate ligand targeted cysteine chelating linker 31 as a white solid and the solid was washed thrice with ice cold ether (5 mL3). The crude product 31 was purified by RP-HPLC using pentafluorophenyl preparative column (5 m, 10 mm150 mm) [=254 nm; solvent gradient 0% B to 100% B in 45 min, 100% B wash 5 min; A=0.1% TFA, pH=2; B=0.1% TFA in acetonitrile (ACN)]. Acetonitrile was removed under reduced pressure from HPLC fractions, and the pure fractions were freeze-dried to yield ligand targeted cysteine chelating linker 31 as white solid. The purity of 31 was confirmed by analytical RP-HPLC and ESI-HRMS (m/z): [M+H].sup.+ calcd. for C48H67N9017S, 1073.4463; found, 1073.4480. UV/vis: max=254 nm. Synthesis of AAPT ligand conjugated chelating linker 31; Reagents and conditions (a) Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA, DMF, 6 h (b) (i) 20% piperidine/DMF, rt, 30 min (ii) Fmoc-diaminopropionic (DAP) acid, PyBOP, DIPEA, DMF, 6 h (c) (i) 20% Piperidine/DMF, rt, 30 min (ii) Fmoc-Phe-OH, PyBOP, DIPEA, DMF, 6 h (d) (i) 20% Piperidine/DMF, rt, 30 min (ii) Fmoc-Phe-OH, PyBOP, DIPEA, DMF, 6 h (e) (i) 20% Piperidine/DMF, rt, 30 min (ii) Fmoc-8-aminooctanoic (EAO) acid, PyBOP, DIPEA, DMF, 6 h (f) (i) 20% Piperidine/DMF, rt, 30 min (ii) 16a, PyBOP, DIPEA, DMF, 6 h (g) TFA/H2O/TIPS/EDT (92.5:2.5:2.5:2.5) (15 mL, 30 min 22.5 mL, 15 min) (iv) Evaporate TFA (v) Precipitate in ice cold diethylether.
[0118] FIG. 19 illustrates synthesis of AAPT ligand conjugated arene chelating linker 34 reagents and conditions, according to an embodiment as disclosed herein.
[0119] In an embodiment in FIG. 19, Solid phase synthetic procedure for preparation of AAPT ligand conjugated arene chelating linker 34. H-Cys-2-ClTrt resin (0.100 g, 0.09 mmol) was swelled in CH2Cl2 (5 mL) for 30 minutes by bubbling nitrogen and after draining CH2Cl2, the resin is swelled once again with DMF (5 mL3) thrice for 15 minutes each. Fmoc-Asp(OtBu)-OH (93 mg, 0.225 mmol), PyBOP (116 mg, 0.225 mmol) and DIPEA (0.16 mL, 0.90 mmol) in DMF (0.5 mL) was added to the peptide vessel and the coupling reaction was continued for 6 h. The resin was washed with DMF (3.0 mL3) followed by isopropanol (3.0 mL3). The completion of reaction was confirmed by performing the Kaiser test. A solution of 20% piperidine in DMF (4 mL) was added to the peptide vessel and the resin beads were bubbled for 10 minutes. The procedure was repeated twice (3 mL2) to ensure complete deprotection of Fmoc protecting group from the coupled amino acid. The resin beads were washed with DMF (3.0 mL3) and isopropanol (3.0 mL3) and the formation of free amine was confirmed by performing the Kaiser test. Consecutively, Boc-Dap(Fmoc)-OH, Fmoc-8-aminocaprylic acid, 4-(Fmoc-aminomethyl) benzoic acid Fmoc-Phe-OH, Fmoc-Phe-OH and Fmoc-8-aminocaprylic acid were attached to the growing peptide chain in sequence as mentioned before. After deprotection of NHFmoc group from the last amino acid, Fmoc-8-aminocaprylic acid, tris-tertbutyl protected compound 16a (68 mg, 0.135 mmol), PyBOP (116 mg, 0.225 mmol) and DIPEA (0.16 mL, 0.90 mmol) in DMF (0.5 mL) were added to the vessel and mixed for 6 h. The completion of reaction was confirmed by the Kaiser test.
[0120] In an embodiment in FIG. 19, A mixture of 9.25 mL trifluoroacetic acid (TFA), 0.25 mL ethane dithiol, 0.25 mL triisopropylsilane (TIPS), and 0.25 mL H.sub.2O was prepared in a 15 mL centrifuge tube, and 5 mL of this cocktail solution was added to the resin beads and nitrogen gas was bubbled through the solution for 30 minutes. The cocktail with cleaved AAPT ligand conjugated arene chelating linker 34 in peptide vessel was collected to a round bottom flask (25 mL). The resin beads were treated again with the cocktail solution twice (2.5 mL2) for 15 minutes each and the mother liquor was collected in the same round bottom flask (25 mL). The pooled cocktail mixture with cleaved AAPT ligand conjugated arene chelating linker 34 was transferred to a 15 mL centrifuge tube, fitted with a septum and concentrated under reduced pressure to obtain a viscous liquid. Ice cold ether (5 mL) was added to the concentrated viscous mixture to precipitate arene chelating linker 34 as a white solid and the solid was washed thrice with ice cold ether (5 mL3). The crude product 34 was purified by RP-HPLC using pentafluorophenyl preparative column (5 m, 10 mm150 mm) [=254 nm; solvent gradient 0% B to 100% B in 45 min, 100% B wash 5 min; A=0.1% TFA, pH=2; B=0.1% TFA in acetonitrile (ACN)]. Acetonitrile was removed under reduced pressure from HPLC fractions, and the pure fractions were freeze-dried to yield AAPT ligand conjugated arene chelating linker 34 as white solid. The purity of 34 was confirmed by analytical RP-HPLC and ESI-HRMS (m/z): [M+H].sup.+ calcd. for C64H89N110195, 1347.6057; found, 1348.6048. UV/vis: max=254 nm. Synthesis of AAPT ligand conjugated arene chelating linker 34; Reagents and conditions (a) Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA, DMF, 6 h (b) (i) 20% Piperidine/DMF, rt, 30 min (ii) Fmoc-diaminopropionic (DAP) acid, PyBOP, DIPEA, DMF, 6 h (c) (i) 20% Piperidine/DMF, rt, 30 min (ii) Fmoc-8-aminooctanoic (EAO) acid, PyBOP, DIPEA, DMF, 6 h (d) (i) 20% Piperidine/DMF, rt, 30 min (ii) 4-(Fmoc-aminomethyl)benzoic acid, PyBOP, DIPEA, DMF, 6 h (e) 20% Piperidine/DMF, rt, 30 min (ii) Fmoc-Phe-OH, PyBOP, DIPEA, DMF, 6 h (f) (i) 20% Piperidine/DMF, rt, 30 min (ii) Fmoc-Phe-OH, PyBOP, DIPEA, DMF, 6 h (g) (i) 20% Piperidine/DMF, rt, 30 min (ii) Fmoc-8-aminooctanoic (EAO) acid, PyBOP, DIPEA, DMF, 6 h (h) (i) 20% Piperidine/DMF, rt, 30 min (ii) 16a, PyBOP, DIPEA, DMF, 6 h (h) TFA/H2O/TIPS/EDT (92.5:2.5:2.5:2.5) (15 mL, 30 min; 22.5 mL, 15 min) (iv) Evaporate TFA (v) Precipitate in ice cold diethylether.
[0121] FIG. 20 illustrates structures of the PSMA scaffolds, glutamate urea heterodimers, glutamate phosphoramidates, 2-(phosphinylmethyl) pentanedioic acid and acetamide derivatives, according to an embodiment as disclosed herein.
[0122] In an embodiment in FIG. 20, based on the NAAG interaction in the binding pockets of PSMA three kinds of PSMA scaffold have been documented in literature which can inhibit the hydrolysis of NAAG, a substrate for PSMA. These molecules are named as (i) glutamate-urea heterodimers a, (ii) glutamate phosphoramidates b and (iii) 2-(phosphinylmethyl)pentanedioic acid derivatives c. Structures of the PSMA scaffolds: glutamate-urea heterodimers a, glutamate phosphoramidates b, 2-(phosphinylmethyl)pentanedioic acid c and acetamide derivatives 4.
[0123] Literature reports show that except these three classes of small molecule inhibitors, no other new small molecule inhibitors are known till date for PSMA. Research group has taken a step forward to invent a new class of ligands called as aminoacetamide 4 based PSMA inhibitors. This class of inhibitor 4 has been designed by extensive in silico studies. After optimizing docking studies with PSMA protein, we have discovered a novel, simple, mild and high yielding synthetic strategy for the preparation of 4 via retrosynthetic analysis. The newly synthesized inhibitors 4 were screened for PSMA enzyme inhibition by in vitro experiments using PSMA enzyme isolate from human prostate cancer lines. Experimental analysis shows that the newly designed inhibitors has similar inhibitory activity when compared to reported inhibitors. Fluorescent agent was tethered to 4 through a peptidic spacer to perform uptake study of the fluorescent conjugate in prostate cancer cell lines and the selectivity for PSMA+ cells was unequivocally proved.
[0124] In the present disclosure, small molecule aminoacetamide PSMA inhibitors has tremendous applications in healthcare sector to treat cancer and neurodegenerative diseases. The new inhibitors and their NIR fluorescent conjugates, radionuclear conjugates, targeted nanomaterials will be used for early diagnosis, intraoperative guided-surgery, MRI contrast imaging and treatment of prostate cancers that are resistant to hormone therapy such as metastatic castration resistant prostate cancers (mCRPC) at hospitals in India and other countries. Moreover, the inhibitors can be used to treat neurodegenerative diseases such as amyotrophic lateral sclerosis. The inhibitors or ligands and their conjugates have high potentiality to become commercial products after the preclinical evaluation.
EXAMPLES
Example 1: General Procedure for the Synthesis of 2-Bromoacetamide Intermediates 13a-c
[0125] Bromoacetic acid 12 (0.208 mg, 1.5 mmol), dicylohexylcarbodiimide (0.619, 3.0 mmol) were dissolved in freshly distilled dichloromethane (8 mL), and the resulting mixture was stirred at 0 C. for 30 min. A solution of 11a-c (1.0 mmol) in dichloromethane (5 mL) was added to the reaction mixture. The reaction mixture was stirred for 12 h at room temperature. The progress of the reaction was monitored by thin layer chromatography (TLC). After completion of the reaction, dichloromethane was evaporated under reduced pressure and ethyl acetate was added to the residue of the crude reaction mixture. Dicyclohexyl urea (DCU) was filtered off from the reaction mixture through glass funnel by using Whatman filter paper. The ethyl acetate layer was concentrated under reduced pressure and the crude products 13a-c were purified through column chromatography using distilled 15-25% ethyl acetate in hexane.
Example 2: (S)-5-Benzyl 1-tert-butyl 2-(2-bromoacetamido) pentanedioate (13a)
[0126] Yellowish gummy liquid (yield=60%), Rf=0.56 (EtOAc:hexane=1:4); IR (CH2Cl2): 3322 (NH), 3032, 2975 (CH), 2928 (CH), 1729 (CO), 1652 (NH), 1537 (CC), 1454 (CH), 1166 (CO), 750, 699 (CH) cm-1. .sup.1H NMR (400 MHz, CDCl3): 7.37-7.31 (m, 5H), 7.05 (d, J=7.28 Hz, 1H), 5.13, 5.10 (ABquartet, J=13.28 Hz, 2H), 4.49 (ddd, J=7.28, 5.24, 5.14 Hz, 1H), 3.83 (s, 2H), 2.49-2.38 (m, 2H), 2.27-2.20 (m, 1H), 2.07-1.99 (m, 1H), 1.46 (s, 9H). .sup.13C NMR (100 MHz, CDCl3): 172.5, 170.3, 165.8, 135.6, 128.6, 128.3, 128.2, 83.0, 66.6, 52.8, 30.1, 28.6, 27.9, 27.3. HRMS (ESI) m/z [M+Na].sup.+ calcd. for C.sub.18H.sub.24BrNO.sub.5, 436.0730, found, 436.0766.
Example 3: (S)-tert-Butyl-2-(2-bromoacetamido)-3-(4-hydroxyphenyl) propanoate (13b)
[0127] Colourless gummy liquid (yield=70%), Rf=0.4 (EtOAc:hexane=1:4); IR (CH2Cl2): 3341 (OH), 3275 (NH), 2979 (CH), 2933 (CH), 1733 (CO), 1657 (NH), 1518 (CC), 1456 (CH), 1155 (CO), 750, 698 (CH) cm-1. .sup.1H NMR (400 MHz, CDCl3): 7.03 (d, J=8.44 Hz, 2H), 6.93 (d, J=7.32 Hz, 1H), 6.73 (d, J=8.44 Hz, 2H), 5.96 (brs, 1H), 4.71-4.63 (m, 1H), 3.85, 3.81 (ABquartet, J=13.80 Hz, 2H), 3.09-2.96 (m, 2H), 1.43 (s, 9H). .sup.13C NMR (100 MHz, CDCl3): 170.1, 165.3, 155.2, 130.6, 127.2, 115.4, 82.9, 54.3, 37.1, 28.7, 27.9. HRMS (ESI) m/z [M+Na].sup.+ calcd. for C.sub.15H.sub.20BrNO.sub.4, 380.0468, found, 380.0479.
Example 4: (S)-tert-Butyl 2-(2-bromoacetamido)-3-phenylpropanoate (13c)
[0128] Colourless gummy liquid (yield=65%), Rf=0.52 (EtOAc:hexane=1:4); IR (CH.sub.2Cl.sub.2): 3298 (NH), 2979 (CH), 2933 (CH), 1734 (CO), 1657 (NH), 1528 (CC), 1456 (CH), 1155 (CO), 740 (CH) cm-1. .sup.1H NMR (400 MHz, CDCl.sub.3): 7.31-7.26 (m, 3H), 7.17-7.15 (m, 2H), 6.89 (d, J=6.52 Hz, 1H), 4.72 (ddd, J=6.52, 6.0, 4.52 Hz, 1H), 3.87, 3.83 (ABquartet, J=13.80 Hz, 2H), 3.12 (d, J=6.04 Hz, 2H), 1.42 (s, 9H). .sup.13C NMR (100 MHz, CDCl.sub.3): 169.9, 164.9, 135.7, 129.5, 128.4, 127.1, 82.8, 54.1, 37.9, 28.8, 27.9. HRMS (ESI) m/z [M+Na].sup.+ calcd. for C.sub.15H.sub.20BrNO.sub.3, 364.0519, found, 364.0522.
Example 5: General Procedure for the Synthesis of Protected Amino Acetamide Derivatives 15a-j
[0129] Compounds 13a-c (1.0 mmol) and 14a-j (1.0 mmol) were dissolved in dry THF (5 mL) and DIPEA (0.52 mL, 3.0 mmol) was added in the reaction mixture. The reaction mixture was refluxed at 80 C. for 18-20 h. The reaction progress was monitored through TLC. After the completion of the reaction, THF was evaporated under reduced pressure and 30 mL of ethyl acetate was added to the crude reaction mixture. The organic layer was washed with distilled water (215 mL) and the resultant organic layer was dried over anhydrous sodium sulphate. The organic layer was concentrated under reduced pressure and the crude products 15a-j were purified through column chromatography using distilled 33% ethyl acetate and hexane mixture as eluent.
Example 6: (S)-5-Benzyl-1-tert-butyl-2-(2-(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)amino)acetamido)pentanedioate (15a)
[0130] Yellowish gummy liquid (yield=75%), Rf=0.3 (EtOAc:hexane=1:2), IR (CH.sub.2Cl.sub.2): 3349 (NH), 2978 (CH), 2928 (CH), 1729 (CO), 1682 (NH), 1517 (CC), 1456 (CH), 1155 (CO), 750, 699 (CH) cm-1. .sup.1H NMR (400 MHz, CDCl.sub.3): 7.70 (d, J=8.4 Hz, 1H), 7.40-7.26 (m, 5H), 5.10 (s, 2H), 4.52 (ddd, J=7.99, 5.66, 4.76, 1H), 3.39 (d, J=17.2 Hz, 1H), 3.13-3.05 (m, 1H), 3.00 (d, J=17.2 Hz, 1H), 2.48-2.32 (m, 4H), 2.28-2.18 (m, 1H), 2.04-1.91 (m, 3H), 1.87-1.75 (m, 1H), 1.45 (s, 9H), 1.44 (s, 9H), 1.42 (s, 9H). .sup.13C NMR (100 MHz, CDCl.sub.3): 173.7, 172.6, 171.3, 170.7, 135.9, 128.6, 128.3, 82.2, 81.9, 80.5, 66.4, 61.9, 51.7, 51.0, 32.4, 30.5, 28.7, 28.1, 28.0, 27.6. HRMS (ESI) m/z [M+Na].sup.+ calcd. for C.sub.31R.sub.48N.sub.2O.sub.9, 615.3252, found, 615.3308.
Example 7: (S)-5-Benzyl-1-tert-butyl-2-(2-(((S)-1-(tert-butoxy)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)amino)acetamido)pentanedioate (15b)
[0131] Gummy liquid (yield=75%), Rf=0.25 (EtOAc:hexane=1:2); IR (CH2Cl2): 3321 (OH), 3279 (NH), 3067, 2979 (CH), 2929, 2851 (CH), 1731 (CO), 1650 (NH), 1537, 1517 (CC), 1448 (CH), 1154 (CO), 750, 699 (CH) cm-1. .sup.1H NMR (400 MHz, CDCl.sub.3): 7.40-7.32 (m, 5H), 7.07 (d, J=7.52 Hz, 3H), 6.70 (d, J=7.52 Hz, 2H), 5.15, 5.10 (ABquartet, J=12.28 Hz, 2H), 4.31-4.26 (m, 1H), 3.36 (d, J=17.56 Hz, 1H), 3.33-3.25 (m, 1H), 3.00 (d, J=17.56 Hz, 1H), 2.93 (dd, J=13.8, 4.52 Hz, 1H), 2.68 (dd, J=12.80, 9.28 Hz, 1H), 2.40-2.21 (m, 2H), 2.10-1.95 (m, 1H), 1.87-1.58 (m, 2H), 1.44 (s, 9H), 1.41 (s, 9H). .sup.13C NMR (100 MHz, CDCl.sub.3): 173.4, 172.4, 171.3, 170.6, 137.2, 135.8, 129.6, 128.6, 128.4, 128.3, 126.7, 82.1, 81.8, 66.4, 63.2, 51.5, 50.6, 39.9, 30.5, 28.0, 27.9, 27.3. HRMS (ESI) m/z [M+H].sup.+ calcd. for C.sub.31H.sub.42N.sub.2O.sub.8, 571.3014, found, 571.3007.
Example 8: (S)-5-Benzyl-1-tert-butyl-2-(2-(((S)-1-(tert-butoxy)-1-oxo-3-phenylpropan-2-yl)amino)acetamido)pentanedioate (15c)
[0132] White solid (yield=70%), Rf=0.35 (EtOAc:hexane=1:2); IR (CH2Cl2): 3326 (NH), 2979 (CH), 2929 (CH), 1723 (CO), 1668 (NH), 1520 (CC), 1455 (CH), 1154 (CO), 735, 698 (CH) cm-1. 1H NMR (400 MHz, CDCl.sub.3): 7.40-7.34 (m, 5H), 7.28-7.20 (m, 5H), 5.13, 5.09 (ABquartet, J=14.04 Hz, 2H), 4.45-4.35 (m, 1H), 3.41-3.34 (m, 2H), 3.07-2.84 (m, 3H), 2.43-2.24 (m, 2H), 2.18-2.06 (m, 1H), 2.04-1.85 (m, 1H), 1.82-1.70 (m, 1H), 1.46 (s, 9H), 1.37 (s, 9H), 1.25-1.21 (m, 1H). 13C NMR (100 MHz, CDCl.sub.3): 173.7, 172.9, 171.9, 170.4, 155.0, 135.7, 130.6, 128.9, 128.6, 128.4, 128.3, 115.7, 82.1, 81.8, 66.6, 63.5, 51.7, 50.5, 38.9, 30.6, 28.0, 27.9, 26.9. HRMS (ESI) m/z [M+Na].sup.+ calcd. for C.sub.31R.sub.42N.sub.2O.sub.7, 577.2884, found, 577.2888.
Example 9: (S)-5-Benzyl-1-tert-butyl-2-(2-(((S)-1-(tert-butoxy)-1-oxopropan-2-yl)amino) acetamido)pentanedioate (15d)
[0133] White solid (yield=72%), Rf=0.32 (EtOAc:hexane=1:2); IR (CH2Cl2): 3338 (NH), 2974 (CH), 2927 (CH), 1730 (CO), 1666 (NH), 1523 (CC), 1455 (CH), 1152, 1064 (CO), 734, 697 (CH) cm-1. 1H NMR (400 MHz, CDCl.sub.3): 7.77 (d, J=8.52 Hz, 1H), 7.36-7.30 (m, 5H), 5.12, 5.08 (ABquartet, J=13.04 Hz, 2H), 4.53 (ddd, J=8.52, 5.78, 5.0 Hz, 1H), 3.37 (d, J=17.32 Hz, 1H), 3.16 (q, J=7.04 Hz, 1H), 3.04 (d, J=17.32 Hz, 1H), 2.52-2.32 (m, 2H), 2.27-2.15 (m, 1H), 2.04-1.92 (m, 1H), 1.45 (s, 9H), 1.44 (s, 9H), 1.28 (d, J=7.04 Hz, 3H), 1.24 (brs, 1H). 13C NMR (100 MHz, CDCl.sub.3): 174.5, 172.6, 171.4, 170.8, 135.8, 128.6, 128.3, 128.2, 82.3, 81.5, 66.5, 57.5, 51.6, 50.8, 30.4, 28.0, 27.9, 27.8, 19.3. HRMS (ESI) m/z [M+Na].sup.+ calcd. for C.sub.25H.sub.38N.sub.2O.sub.7, 501.2571, found, 501.2572.
Example 10: (S)-5-Benzyl-1-tert-butyl-2-(2-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutan-2-yl)amino)acetamido)pentanedioate (15e)
[0134] Yellowish gummy liquid (yield=75%), Rf=0.36 (EtOAc:hexane=1:2); IR (CH2Cl2): 3363 (NH), 2975 (CH), 2933 (CH), 1733 (CO), 1681 (NH), 1513 (CC), 1457 (CH), 1157 (CO), 746, 699 (CH) cm-1.1H NMR (400 MHz, CDCl3): 7.73 (d, J=8.28 Hz, 1H), 7.38-7.32 (m, 5H), 5.12, 5.08 (ABquartet, J=12.56 Hz, 2H), 4.51 (ddd, J=8.28, 5.66, 5.24, 1H), 3.40 (d, J=17.32 Hz, 1H), 2.98-2.93 (m, 2H), 2.51-2.33 (m, 2H), 2.25-2.15 (m, 1H), 2.05-1.93 (m, 1H), 1.44 (s, 18H), 1.30-1.13 (m, 2H), 0.97-0.86 (m, 6H). .sup.13C NMR (100 MHz, CDCl.sub.3): 173.9, 172.6, 171.6, 170.7, 135.9, 128.6, *128.4, 82.3, 81.6, 68.0, 66.5, 51.8, 51.3, 31.6, 30.5, 28.2, 28.1, 27.9, 19.5, 18.4. HRMS (ESI) m/z [M+Na].sup.+ calcd. for C.sub.27H.sub.42N.sub.2O.sub.7, 529.2884, found, 529.2882. (* indicates higher intensity carbon)
Example 11: (S)-5-Benzyl-1-tert-butyl-2-(2-(((S)-1-(tert-butoxy)-4-methyl-1-oxopentan-2-yl)amino)acetamido)pentanedioate (15f)
[0135] Yellowish liquid (yield=82%), Rf=0.4 (EtOAc:hexane=1:2); IR (CH2Cl2): 3354 (NH), 2959 (CH), 2934 (CH), 1734 (CO), 1681 (NH), 1511 (CC), 1456 (CH), 1155, 1081 (CO), 749, 699 (CH) cm-1.1H NMR (400 MHz, CDCl.sub.3): 7.74 (d, J=8.28 Hz, 1H), 7.40-7.30 (m, 5H), 5.11, 5.08 (ABquartet, J=12.80 Hz, 2H), 4.51 (ddd, J=8.28, 5.40, 5.28, 1H), 3.37 (d, J=17.32 Hz, 1H), 3.09 (t, J=7.0 Hz, 1H), 2.99 (d, J=17.32 Hz, 1H), 2.51-2.32 (m, 2H), 2.26-2.15 (m, 1H), 2.04-1.92 (m, 1H), 1.83-1.71 (m, 3H), 1.44 (s, 9H), 1.43 (s, 9H) 1.29-1.22 (m, 1H), 0.92 (d, J=6.52 Hz, 3H), 0.90 (d, J=6.52 Hz, 3H). .sup.13C NMR (100 MHz, CDCl.sub.3): 174.7, 172.5, 171.4, 170.6, 135.8, 128.6, 128.3, 128.2, 82.2, 81.5, 66.4, 60.9, 51.7, 50.8, 42.9, 30.4, 28.1, 27.9, 27.8, 24.9, 22.6, 22.5. HRMS (ESI) m/z [M+Na].sup.+ calcd. for C.sub.28H.sub.44N.sub.2O.sub.7, 521.3221, found, 521.3222.
Example 12: (2S)-5-Benzyl-1-tert-butyl-2-(2-(((2S)-1-(tert-butoxy)-3-methyl-1-oxopentan-2-yl)amino)acetamido)pentanedioate (15 g)
[0136] Yellowish solid (yield=86%), Rf=0.34 (EtOAc:hexane=1:2); IR (CH2Cl2): 3347 (NH), 2977 (CH), 2933 (CH), 1727 (CO), 1668 (NH), 1515 (CC), 1458 (CH), 1153 (CO), 734, 698 (CH) cm-1.1H NMR (400 MHz, CDCl.sub.3): 7.72 (d, J=8.52 Hz, 1H), 7.42-7.27 (m, 5H), 5.11, 5.08 (ABquartet, J=12.52 Hz, 2H), 4.51 (ddd, J=8.52, 5.38, 5.24, 1H), 3.40 (d, J=17.32 Hz, 1H), 2.95 (d, 1H, J=17.32 Hz), 2.86 (d, J=5.76 Hz, 1H), 2.51-2.32 (m, 2H), 2.26-2.14 (m, 1H), 2.04-1.87 (m, 4H), 1.44 (s, 18H), 1.28-1.23 (m, 1H), 0.99 (d, J=6.80 Hz, 3H), 0.95 (d, J=7.04 Hz, 3H). .sup.13C NMR (100 MHz, CDCl.sub.3): 173.6, 172.6, 171.5, 170.6, 135.8, 128.6, 128.3, 128.2, 82.2, 81.5, 66.8, 66.5, 51.7, 51.3, 38.5, 30.4, 28.2, 27.9, 27.8, 25.5, 15.7, 11.7. HRMS (ESI) m/z [M+Na].sup.+ calcd. for C.sub.25H.sub.44N.sub.2O.sub.7, 521.3221, found, 521.3223.
Example 13: (S)-5-Benzyl-1-tert-butyl-2-(2-(((S)-1,4-di-tert-butoxy-1,4-dioxobutan-2-yl)amino)acetamido)pentanedioate (15h)
[0137] Yellowish liquid (yield=85%), Rf=0.32 (EtOAc:hexane=1:2); IR (CH2Cl2): 3327 (NH), 2975 (CH), 2926 (CH), 1723 (CO), 1672 (NH), 1519 (CC), 1456 (CH), 1149 (CO), 750, 698 (CH) cm-1.1H NMR (400 MHz, CDCl.sub.3): 8.00 (d, J=8.52 Hz, 1H), 7.40-7.27 (m, 5H), 5.11, 5.08 (ABquartet, J=12.80 Hz, 2H), 4.50 (ddd, J=8.52, 6.14, 4.76 Hz, 1H), 3.48-3.40 (m, 2H), 3.18-3.14 (d, J=17.32 Hz, 1H), 2.66 (dd, J=16.7, 4.24, Hz, 1H), 2.57-2.37 (m, 3H), 2.30-2.17 (m, 1H), 2.10-1.96 (m, 1H), 1.44 (s, 9H), 1.43 (s, 9H), 1.41 (s, 9H), 1.27-1.24 (m, 1H). .sup.13C NMR (100 MHz, CDCl.sub.3): 172.6, 172.5, 171.6, 170.7, 170.4, 135.9, 128.5, 128.3, 128.2, 82.0, 81.9, 81.5, 66.4, 58.1, 51.8, 50.9, 38.8, 30.6, 28.1, *27.9, 27.2. HRMS (ESI) m/z [M+Na].sup.+ calcd. for C.sub.30H.sub.46N.sub.2O.sub.9, 578.3203, found, 579.3275. (* indicates higher intensity carbon).
Example 14: (S)-Di-tert-butyl-2-(2-(((S)-1-(tert-butoxy)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)amino)-2-oxoethyl)amino) pentanedioate (15i)
[0138] White solid (yield=85%), Rf=0.25 (EtOAc:hexane=1:2); IR (CH2Cl2): 3349 (OH), 3275 (NH), 2979 (CH), 2933 (CH), 1730 (CO), 1660 (NH), 1517 (CC), 1456 (CH), 1155 (CO), 753 (CH) cm-1. .sup.1H NMR (400 MHz, CDCl3): 7.57 (d, J=8.44 Hz, 1H), 7.01 (d, J=8.08 Hz, 2H), 6.72 (d, J=8.08 Hz, 2H), 4.75-4.65 (m, 1H), 3.35 (d, J=16.88 Hz, 1H), 3.10-2.90 (m, 4H), 2.37-2.20 (m, 2H), 1.90-1.67 (m, 2H), 1.43 (s, 18H), 1.42 (s, 9H), 1.27-1.22 (m, 1H). .sup.13C NMR (100 MHz, CDCl.sub.3): 173.6, 172.8, 171.2, 170.6, 155.3, 130.4, 127.7, 115.7, 82.1, 81.9, 80.8, 61.6, 53.3, 50.8, 37.2, 32.3, 28.7, 28.7, 28.09, 28.06, 28.0. HRMS (ESI) m/z [M+Na]+ calcd. for C.sub.28H.sub.44N.sub.2O.sub.8, 559.2990, found, 559.2983.
Example 15: (S)-Di-tert-butyl-2-((2-(((S)-1-(tert-butoxy)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)pentanedioate (15j)
[0139] White solid (yield=80%), Rf=0.30 (EtOAc:hexane=1:2); IR (CH2Cl2): 3350 (NH), 2979 (CH), 2933 (CH), 1732 (CO), 1682 (NH), 1518 (CC), 1456 (CH), 1155, 1080 (CO), 741 (CH) cm-1. 1H NMR (400 MHz, CDCl.sub.3): 7.54 (d, J=8.44 Hz, 1H), 7.30-7.15 (m, 5H), 4.80-4.65 (m, 1H), 3.36 (d, J=17.24 Hz, 1H), 3.15-3.07 (m, 2H), 3.07-3.01 (m, 1H), 2.98 (d, J=17.24 Hz, 1H), 2.35-2.17 (m, 2H), 1.92-1.83 (m, 1H), 1.78-1.67 (m, 2H), 1.44 (s, 9H), 1.43 (s, 9H), 1.39 (s, 9H). .sup.13C NMR (100 MHz, CDCl.sub.3): 173.7, 172.5, 170.8, 170.5, 136.5, 129.4, 128.4, 126.9, 82.0, 81.8, 80.4, 61.6, 53.2, 50.9, 37.9, 32.3, 28.6, 28.1, 28.0, 27.9. HRMS (ESI) m/z [M+H].sup.+ calcd. for C.sub.28H.sub.44N.sub.2O.sub.7, 521.3221, found, 521.3226.
Example 16: Deprotection of Carboxylic Benzylester Precursors 15a-c to Afford Tert-Butylcarboxylic Acids 16a-c
[0140] Compound 15a-c (1.0 mmol) was dissolved in MeOH (5 mL) in a 50 mL two-neck round bottom flask, 10% Pd/C (0.106 g, 0.1 mmol) was added in the solution. The reaction mixture was hydrogenated at 1 atm for 24 h at room temperature. After the completion of the reaction, Pd/C was filtered through a celite pad (sintered glass filter was half-filled with celite powder) and washed with ethyl acetate (320 mL). The ethyl acetate layer was concentrated under reduced pressure and the crude products 16a-c were purified through column chromatography by using distilled ethyl acetate to obtain pure 16a-c.
Example 17: (S)-5-(Tert-butoxy)-4-(2-(((S)-1,5-di-tert-butoxy-1,5-dioxo-pentan-2-yl)amino)acetamido)-5-oxopentanoic acid (16a)
[0141] Colourless gummy liquid (yield=60%). R.sub.f=0.58 (EtOAc:Hexane=1:1); IR (CH2Cl2): 3350 (NH), 2979 (CH), 2933 (CH), 1732 (CO), 1682 (NH), 1518 (CC), 1456 (CH), 1155, 1080 (CO), 741 (CH) cm-1. .sup.1H NMR (400 MHz, CDCl3): 7.81 (d, J=8.44 Hz, 1H), 4.50 (ddd, J=7.79, 6.06, 4.76 Hz, 1H), 3.43 (d, J=16.84 Hz, 1H), 3.15-3.07 (m, 1H), 3.04 (d, J=16.84 Hz, 1H), 2.45-2.35 (m, 4H), 2.27-2.15 (m, 1H), 2.05-2.15 (m, 1H), 2.05-1.92 (m, 2H), 1.90-1.80 (m, 1H), 1.46 (s, 9H), 1.45 (s, 9H), 1.43 (s, 9H), 1.13-1.07 (brs, 1H). .sup.13C NMR (100 MHz, CDCl3): 175.9, 173.7, 172.9, 171.8, 170.6, 82.3, 82.0, 80.8, 61.7, 51.9, 50.8, 32.3, 30.5, 28.6, 28.1, 27.9, 27.6. HRMS (ESI) m/z calcd for C.sub.24H.sub.42N.sub.2O.sub.9 [M+H].sup.+: 503.3004, found. 503.3011.
Example 18: Procedure for the Deprotection of Tert-Butylcarboxylic Acids in 16a-c to Afford Inhibitors 1-3
[0142] Precursors 16a-c (1.0 mmol) was dissolved in CH2Cl2 (2 mL) in a 50 mL round bottomed flask. A mixture of trifluoroacetic acid (2.5 mL) and CH2Cl2 (2.5 mL) in the ratio 1:1 was added to the reaction mixture at room temperature and stirred for 2 h. After the completion of reaction, the mixture of trifluoroacetic acid and CH.sub.2Cl.sub.2 were removed under reduced pressure. The products 1-3 were precipitated by the addition of ice-cold ether (5 mL). The crude products 1-3 were washed (35 mL) with ether to remove excess trifluoroacetic acid and other non-polar impurities. The products 1-3 were purified through Buchi Reveleris preparative high-performance liquid chromatography using RP-PFP column (XSelect CSH Prep Fluorophenyl 5 m OBD, 19 mm150 mm). The purity of the products 1-3 was confirmed by analytical high-performance liquid chromatography and LC-MS. The purified inhibitors 1-3 were used for NAALADase or PSMA enzyme inhibition assay to determine the IC50.
Analytical HPLC Method
[0143] The purity of ligands 1-3 were analyzed using a Dionex HPLC-Ultimate 3000 system. Typically a solution of each ligand (20 L, 1.0 mg/1.0 mL) in a mixture of CH.sub.3CN:H.sub.2O (1:1) was injected via autosampler and eluted using Dionex Acclaim 120 C18, 5 m, 4.6 mm250 mm analytical column at a flow rate of 1 mL/min (mobile phase, A=0.1% trifluoro acetic acid/H.sub.2O and B=acetonitrile). An isocratic flow of 40% B (v/v) was used during the run for 0 to 4 min and gradually gradient of B was increased to 100% B (v/v) over a period of 40-min. The chromatogram of each ligand was recorded on the Ultimate 3000 RS variable wavelength detector at 225-280 nm.
Example 19: Preparative HPLC Method
[0144] The purification of ligands 1-3 was performed using Buchi Reveleris Preparative HPLC System. Crude ligand (20 mg) was dissolved in 1:1 ratio of CH.sub.3CN:H.sub.2O (1 mL) and injected into the sample injector for elution using RP-PFP (Reverse Phase pentafluorophenyl) preparative column (XSelect CSH Prep Fluorophenyl 5 m OBD, 19 mm150 mm). A flow rate of 10 mL/min (mobile phase, A=0.1% trifluoro acetic acid/H2O and B=acetonitrile) is maintained throughout the run and the mobile phase gradient was increased from 1% B (v/v) to 50% B (v/v) over a period of 40 min. The mobile phase gradient was further increased to 80% B (v/v) in the next 15 min and the chromatogram was recorded at =200-254 nm as well as by ELSD detector. Pure fractions of 1-3 were collected using automatic fraction collector, acetonitrile was evaporated under reduced pressure, lyophilized to afford pure ligands 1-3. The pure ligands were further used for GCPII enzyme inhibition assay.
Example 20: Procedure for PSMA or GCPII Enzyme Inhibition Assay
[0145] Fluorescent-based enzyme inhibition assay was performed to determine the IC.sub.50 value of the newly synthesized GCPII inhibitors 1-3 (AAPT ligands) Amplex Glutamate kit was purchased from Invitrogen, and a working solution of Amplex Red reagent (5 mL, 100 M) was prepared. Meanwhile, membrane portion of PSMA enzyme was extracted from PSMA+ LNCaP cell line by following a reported protocol. Briefly LNCaP cells (1 million) were harvested in HEPES buffer (1 mL) and lyzed twice using probe sonicator for 30 s. The lysate was ultracentrifuged at 100,000g for 30 min, the supernatant was discarded, and the cell pellet was homogenized by addition of HEPES buffer (1 mL) and used for PSMA enzyme inhibition assay. The isolated enzyme (100 L, 8.3027 ng) was incubated with different concentrations (1, 5, 10, 25, 50, 75, 90, 100, 200, 300, 500 and 1000 nM) of the inhibitor 1-3 (100 L) in the presence of N-acetylaspartylglutamate (NAAG) (50 L, 30 nM) for 60 min. The amount of glutamic acid released by the hydrolysis of NAAG was measured by incubating a working solution of Amplex Red reagent (50 L, 100 M) for 30 min at 37 C. The fluorescence emission after the oxidation of Amplex Red reagent was measured by using Synergy H1 multimode plate reader (BioTek Instruments, Inc., Winooski, Vt., USA). The excitation wavelength was fixed at 530 nm, and the fluorescence emission is measured at 590 nm. Dose v/s response inhibition curve was obtained using semi-log plot of concentration of inhibitors 1-3 versus fluorescence intensity emission to provide experimental IC50 values and compared with a known standard GCPII inhibitor, PMPA by following a similar procedure. The data analysis was performed using GraphPad Prism, version 6.00 for Windows (GraphPad Software, San Diego, Calif.).
[0146] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
