2-Aminoadipic Acid-C(O)-Glutamate Based Prostate-Specific Membrane Antigen Ligands for Potential Use as Theranostics

Article abstract of DOI:10.1021/acsmedchemlett.8b00318

The design and synthesis of prostate specific membrane antigen (PSMA) ligands derived from 2-aminoadipic acid, a building block that has not previously been used to construct PSMA ligands, are reported. The effects of both the linker length and of an N-substituent of our PSMA ligands were probed, and X-ray structures of five of these ligands bound to PSMA were obtained. Among the ligands disclosed herein, 13b showed the highest inhibitory activity for PSMA. As ligand 13b can readily be radiolabeled since its fluorine atom is adjacent to the nitrogen atom of its pyridine ring, the use of this and related compounds as theranostics can be pursued.

Full text of DOI:10.1021/acsmedchemlett.8b00318

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Letter
2-Aminoadipic Acid-C(O)-Glutamate Based Prostate-Specific
Membrane Antigen Ligands for Potential Use as Theranostics
Ryo Nakajima, Zora Novakova, Werner Tueckmantel, Lucia Motlova, Cyril Barinka, and Alan P. Kozikowski
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00318 • Publication Date (Web): 24 Oct 2018
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2-Aminoadipic Acid-C(O)-Glutamate Based Prostate-Specific
Membrane Antigen Ligands for Potential Use as Theranostics
Ryo Nakajima,†, ⊥ Zora Nováková,‡, ⊥ Werner Tueckmantel,* Lucia Motlová,‡ Cyril Bařinka ‡
and Alan P. Kozikowski,*
† Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, IL, 60612.
‡ Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, 252 50 Vestec, Czech
Republic.
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*StarWise Therapeutics LLC, 505 S Rosa Road, Suite 27, Madison, Wisconsin 53719-1235.
KEYWORDS: theranostic, PSMA ligands, prostate cancer, PET imaging, X-ray structure, arginine patch
ABSTRACT: The design and synthesis of PSMA ligands derived from 2-aminoadipic acid, a building block that has not
previously been used to construct PSMA ligands, are reported. The effects of both the linker length and of an N-substituent
of our PSMA ligands were probed, and X-ray structures of five of these ligands bound to PSMA were obtained. Among the
ligands disclosed herein, 13b showed the highest inhibitory activity for PSMA. As ligand 13b can readily be radiolabeled
since its fluorine atom is adjacent to the nitrogen atom of its pyridine ring, the use of this and related compounds as
theranostics can be pursued.
Prostate Cancer (PCa) is the most frequently diagnosed
cancer and the second leading cause of death in men in the
US after lung cancer, with an estimated 180,890 new cases
and 26,120 deaths in 2016. The aggregate cost of treating
PCa patients has been estimated at $8-10 billion/year in the
US, thus making it a significant disease with a heavy
societal cost-burden1, 2. The tissue-specific protein, prostate
specific membrane antigen (PSMA), is an excellent target
for imaging and therapy because it is a cell surface protein
which presents a large extracellular target. Most PCa cells
overexpress PSMA compared to the limited expression
pattern observed in normal prostate cells, and the
overexpression is significantly correlated to poor disease
prognosis3-7. Therefore, several PSMA ligands including
affinity of the length of the second amino acid moiety (in
addition to the obligatory P1’ glutamate) has not been
elucidated yet since almost all of the PSMA ligands for PET
imaging were synthesized starting from lysine or another
molecule of glutamic acid16.
The internal substrate/inhibitor-binding cavity of PSMA
can be divided into the prime (S1’) and non-prime sections
separated by the active site harboring two Zn2+ ions (Fig.
1A). Within the non-prime section, one of the most
prominent structural features is the so-called arginine patch
comprising Arg463, Arg534, and Arg536. Ionic
interactions between the positively charged patch and the
P1 carboxylate of PSMA-selective inhibitors are critical for
the design of high affinity urea inhibitors. Furthermore, the
flexibility of the arginine side chains allows for the
formation of an S1 accessory hydrophobic pocket upon
inhibitor binding that can be in turn exploited for the design
of high affinity inhibitors as reported herein20-22. Our efforts
focused on using 2-aminoadipic acid as a novel building
block and to investigate the best chain length for interaction
with the S1 hydrophobic pocket to enhance the compounds’
affinity for PSMA, with the goal of possibly reducing their
dose of administration and achieving a higher image
resolution.
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11
antibodies8,
,
peptides10,
,
aptamers12, and small
14
molecules13,
have been developed to deliver imaging
agents for the diagnosis of prostate cancer. Among them,
small molecules exhibit favorable features: reproducible
chemical synthesis, non-immunogenicity, and in general
fast clearance from normal tissues. In 2001, Kozikowski et
al. first developed urea-based PSMA inhibitors as GCPII
(glutamate carboxypeptidase II) inhibitors15. Since then,
many researchers have been using this urea-based scaffold
for targeting PSMA due to the high affinity of these ureas
for PSMA as well as their ease of synthesis. Recently, these
urea-based PSMA ligands labeled with 18F or 68Ga have
been investigated as PET imaging agents in Phase III
clinical trials16, 17. However, such studies have revealed that
uptake of these radioligands also takes place in off-target
tissues including the kidneys and salivary glands, which
might perhaps be avoided through proper design features.
It is possible, for example, that such drawbacks can be
We have chosen to concentrate on fluorinated PSMA
ligands since it is well known that the radionuclide 18F has
yielded improved imaging resolution compared to 68Ga23.
Fluorinated PSMA ligands incorporating 2-aminoadipic
acid were synthesized as shown in Scheme 1. First, the
distal carboxyl group of L-2-aminoadipic acid
(enantiopurity: 98%) was protected by benzylation, and the
monobenzyl ester was directly coupled with the
enantiopure requisite isocyanate24. The free carboxyl group
of 4 was protected as tert-butyl ester, and then the benzyl
ester was deprotected by hydrogenation to form the
carboxylic acid 6. The desired amides 7‒15 were obtained
overcome by enhancing the compounds’ hydrophilicity,
19
resulting in faster clearance from off-target tissues18,
.
Even though hundreds of PSMA ligands have been
explored in the past decades, the influence on PSMA
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DMF, r.t., 16 h; ii. TFA, rt, 30 min, 7a: 76%, 7b: 89%, 7c: 76%,
8a: 89%, 8b: 85%, 9a: 72%, 9b: 83%, 10: 83%, 11a: 79%, 11b:
77%, 12: 72%, 13a: 62%, 13b: 24%, 14: 93%, 15: 68%.
by amidation of 6, followed by deprotection of the tri-tert-
butyl esters. All final compounds 7‒15 were purified by
preparative high-performance liquid chromatography
(preparative HPLC), and their chemical structures were
confirmed by NMR and LCMS-IT-TOF. The purities of all
tested compounds were >95%, as determined by analytical
HPLC. The inhibition constants (IC50 values) of these
compounds for the enzyme PSMA were determined using
the radioenzymatic assay with [3H]NAAG as a substrate
(Table 1). The IC50 values of the benzylamide derivatives
7a‒7c were almost identical, demonstrating that the
position of the fluorine atom does not influence their
potency. Introduction of a methyl group on the amide
nitrogen of 7a and 7b, on the other hand, increased their
potency for PSMA (compounds 8a and 8b). At the same
time, however, compounds 9a and 9b that have a methyl
group attached to the benzylic position showed
approximately 2- and 10-fold lower inhibitory affinity for
PSMA compared to 7a and 8a, respectively. The decrease
in the inhibitory affinity of 9a and 9b is likely due to the
steric hindrance of the methyl group clashing with the side
chain of Arg463 and interfering thus with the full insertion
of the inhibitor ring into the S1 hydrophobic accessory
pocket. Additionally, it is plausible that the presence of the
methyl group would also elicit a steric strain with the ring
moiety within the inhibitor backbone at the rotation angle
observed in the crystal structure of the PSMA/7a complex
(see below). Compound 10, in which the benzene ring is
replaced with a pyridine ring, showed marginally lower
affinity compared to 7a.
To visualize the binding mode of one of our new PSMA
ligands within the internal PSMA pocket, we first
determined the crystal structure of the PSMA/7a complex
to a final resolution of 1.48 Å (PDB code 5OF0). As
expected, the position and interaction pattern of the urea-
glutamate moiety in the S1’ pocket is identical to
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PSMA/urea-based
inhibitor
complexes
published
previously20. Favorably, and in-line with our hypothesis,
the aromatic ring of 7a is inserted into the S1 hydrophobic
pocket, thereby increasing its inhibitor potency for PSMA
through interactions contributed by the distal aromatic ring.
It should be noted that the placement of the aromatic ring
of 7a is virtually identical to that observed in the complex
of PSMA with DCIBzL, a urea-based inhibitor bearing
lysine as the main moiety of the P1 linkers (Fig. 1B). These
findings suggest that when there are seven intervening
atoms between the urea function and the P1 aromatic
moiety, the distal aromatic ring is preferably inserted into
the hydrophobic accessory pocket within the arginine
patch.
Scheme 1. Synthesis of compounds 7 - 15.a
Figure 1. A: Schematic representation of the internal cavity of
PSMA (arginine patch – purple, zinc ions – light blue) in complex
with 7a (green sticks). B: Comparison of the binding modes of 7a
(green) and 16 (DCIBzL;pink).
We next evaluated a series of compounds 11‒13 having the
P1 linker shortened by one atom, i.e. having six intervening
atoms between the urea function and the P1 aromatic
moiety, as this linker length has not been explored before.
All compounds from this series showed sub-nanomolar
IC50 values (Table 1) revealing that aminoadipic as the main
moiety of the linker serves as an excellent substitute for the
traditional lysine used in “canonical” urea-based
compounds.
The affinities of the anilides 11‒13 were on average higher
than those of the corresponding benzylamides 7‒10, which
indicate that the distance between the aromatic ring and the
urea moiety in the former is more suitable for strong
interactions with residues in the internal pocket of PSMA,
an aspect of these urea-based ligands that was not explored
in detail in previous studies24-27. Furthermore, the N-
aReagents and conditions: a) 12 M HCl, BnOH, 100 °C, 1 h,
84%; b) i. triphosgene, DIPEA, DCM, -78 °C, 1 h, then rt, 15 min;
ii. 2, DMF, -78 °C to r.t., overnight, 56%; c) 2-tert-butyl-1,3-
diisopropylisourea, DCM, reflux, overnight, 71%; d) H2, 10%
Pd/C, MeOH, r.t., overnight, 80%; e) i. amine, HATU, DIPEA,
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methylated anilides 12 and 13b exhibited enhanced affinity
The data presented here together with prior studies20, 29 offer
a comprehensive picture of relation between the P1 linker
length and the position of the terminal aromatic moiety of
urea-based compounds (Fig. 3). For flexible linkers with
seven intervening atoms, the distal aromatic ring will most
likely be located within the S1 accessory pocket of the
arginine patch. In the case of shorter (6 intervening atoms)
and longer (>8 atoms) linkers the distal aromatic groups
will either interact with one of the defined hydrophobic
patches lining the entrance funnel or adopt several diverse
conformations inside the funnel. It should be noted that a
key aspect of the linker chain is its very flexibility. When
comparing the structures of 7a (reported here) and 16
(DCIBzL; PDB code 3D7H)20 bound to PSMA, the position
of their terminal halogenated rings inside the pocket is
virtually identical, and this particular placement is
facilitated by the linker‘s flexibility, as it can adopt several
(most favorable) conformations within the spacious
internal cavity.
compared with the corresponding N-H derivatives 11a and
13a, respectively. Particularly, 13b showed the highest
inhibitory activity (IC50 = 0.075 nM) for PSMA among all
of the PSMA ligands synthesized from 2-aminoadipic acid.
To provide a mechanistic explanation for these SAR
findings, we determined the crystal structures of 11a (PDB
code 6H7Y), 13a (PDB code 6HKJ), and 13b (PDB code
6H7Z) in complex with PSMA at a final resolution of 1.81,
2.09, and 2.00 Å, respectively (Supplementary Table S1).
Compared to the structure of PSMA in complex with 7a,
which has the P1 aromatic ring inserted into the S1
hydrophobic pocket of the arginine patch, the position of
the P1 aromatic moiety of both 11a and 13a differs
substantially (Fig. 2). For 11a and 13a the distal ring is
inserted into a shallow pocket lined with the side chain of
Y700 and the backbone of G206, K207, and V208. Also
critical are F546, S547, G548, and W541, residues present
within the flexible entrance lid28 that form the opposite wall
of the binding pocket (Fig. S1).
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Table 1. PSMA inhibitory activity of compounds 7a-c,
8a-b, 9a-b, 10, 9a-b, 12, 13a-b, 14, 15, 21a-b and 22a-b.a
Compou
nd
IC50 (nM)
Compound
IC50 (nM)
7a
7b
7c
8a
8b
9a
9b
10
2.32 ± 0.55
2.91 ± 0.33
2.11 ± 1.01
0.64 ± 0.07
0.46 ± 0.10
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5.44 ± 2.54
4.46 ± 1.03
0.35 ± 0.01
0.28 ± 0.09
12
0.20 ± 0.11
0.99 ± 0.05
0.075 ± 0.005
1.31 ± 0.18
1.19 ± 0.73
0.94 ± 0.26
0.15 ± 0.02
1.11 ± 0.22
0.13 ± 0.01
13a
13b
14
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21a
21b
22a
22b
11a
11b
16 (DCIBzL) 0.021 ± 0.004
Figure 2. Comparison of binding modes of 7a (green), 11a (cyan),
and 13a (orange), zinc ions shown as blue spheres. Note that while
the positioning of the glutamate-urea moiety in the PSMA internal
cavity is indistinguishable for both complexes, the distal aromatic
rings occupy different portions of the entrance funnel due to
differences in the length of the linker connecting the urea nitrogen
and the distal aromatic ring (6 vs 7 intervening atoms).
Inhibition constants of studied compounds were determined using the
radioenzymatic assay with [3H]NAAG (radiolabeled at the terminal
glutamate) as described previously22.
Introduction of a N-methyl group into 13a, leading to 13b,
is linked to a ten-fold increase in potency for PSMA and a
slightly different interaction pattern. While the distal rings
of 13b and 13a partially overlap, the former is inserted
deeper into the pocket because of the cis conformation of
the linking amide bond. Additionally, the increased potency
of compounds with the pyridine ring in the cis
conformation (e.g., 13b) vs ones with either the phenyl
group (e.g., 22b) or pyridines in the trans conformation
(e.g., 13a) can likely be attributed to the water-mediated
contact between the ring nitrogen and the hydroxyl group
of Y234 (Fig. S2). It should be noted that the above-
mentioned hydrophobic patch is one of the interaction
hotspots in the PSMA internal pocket as it is exploited by
other inhibitory scaffolds such as hydroxamates or
phosphoramidates21.
The crystal structure of the PSMA/13b complex shows that
the fluorinated pyridine ring of 13b is constrained to adopt
the cis-amide conformation with the N-methyl group
pointing towards the unoccupied space of the entrance
funnel (Fig. 4A). This particular structural arrangement
brings about the possibility of the addition of a second,
larger substituent on the amide nitrogen. Such “branching”
can be exploited for fine-tuning of both physicochemical
and pharmacokinetic parameters to create the next
generation of refined analogs.
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Scheme 2. Synthesis of compounds 21a-b and 22a-b.a
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Figure 3. Chain length from the “left” nitrogen of the urea moiety
to the aromatic ring in compounds 7a, 11a, 16, 21a, and 21b.
To test this assumption we synthesized compounds 21a,b
and 22a,b as shown in Scheme 2. The starting material 18a
was commercially available, while 18b was obtained from
N-phenyl-1,2-ethanediamine by monoprotection with a
Cbz group. Both compounds underwent HATU-mediated
amide formation with the building block 6 to give the N-H
and N-Ph amide derivatives, respectively. The Cbz groups
were removed by hydrogenation, and the obtained amines
were reacted with 4-fluorobenzoic acid or 6-fluoronicotinic
acid, followed by deprotection of the tri-tert-butyl esters to
obtain the desired PSMA ligands 21a,b and 22a,b. As
shown in Table 1, the derivatives 21b and 22b with an N-
phenyl group exhibited 6- and 8-fold higher inhibitory
activity for PSMA compared to the secondary amides 21a
and 22a, respectively, suggesting that our strategy of using
the phenyl ring as an anchor for these ligands is consistent
with our working hypothesis (Fig. 4B).
aReagents and conditions: a) Cbz-Cl, DIPEA, DCM, 0 °C to r.t.,
1 h, 93%; b) 18a or 18b, HATU, DIPEA, DMF, r.t., 16 h; c) H2,
10% Pd/C, MeOH, r.t., overnight, 20a: 81%, 20b: 59%; d) i.
carboxylic acid, HATU, DIPEA, DMF, r.t., 16 h; ii. TFA, r.t., 30
min, 21a: 34%, 21b: 52%, 22a: 34%, 22b: 57%.
In conclusion, we have designed and synthesized PSMA
ligands derived from 2-aminoadipic acid, a building block
that has not previously been used to construct PSMA
ligands. The effects of both the linker length and of an N-
substituent on our PSMA ligands were probed. Among
these ligands, 13b showed the highest inhibitory activity for
PSMA. Moreover, The labeling methods previously
described for [18F]DCFPyL and [18F]PSMA-100730, 31 offer
strong support that the preparation of [18F]13b could be
possible. Finally, a branching moiety can be attached to the
amide function of 13b, thus allowing the facile modulation
of physicochemical and pharmacokinetic properties of the
next generation of compounds while preserving the high
affinity of the original compound for PSMA.
ASSOCIATED CONTENT
Supporting Information
Figure 4. A: Superposition of binding modes of 13a (orange) and
13b (red). B: Detail of the amide methyl group (13b; red) and
pyridine group (22b; blue; PDB code 6KHZ) pointing towards the
unoccupied space of the internal funnel (dashed line).
Supporting Information that includes synthetic methods,
analytical characterization of compounds, methods of
crystallization, diffraction data collection, and structure
determination, data collection and refinement statistics, and
enzymatic assays is available free of charge on the ACS
Publications website (PDF).
A similar strategy of using the distal P1 phenyl group as an
anchor has been recently reported by Tykvart et al.
However, exploiting the S1 accessory pocket of the
arginine patch as an anchor point in their case led to mixed
results as the insertion of the phenyl group into the pocket
was unpredictable and strongly dependent on the
AUTHOR INFORMATION
Corresponding Author
physicochemical
parameters
of
the
branching
functionality29. To the contrary, we believe that the
structure-assisted branching strategy outlined in this report
will be mostly independent of the nature of the branching
functionality as it exploits the unoccupied space of the
entrance funnel free of obvious steric barriers.
A.P.K.: e-mail: akozikowski@starwisetrx.com. Phone: (+1)
773-793-5866. C.B.: e-mail: cyril.barinka@ibt.cas.cz. Phone:
(+420) 325-873-777
Author Contributions
⊥
R.N. and Z.N. contributed equally.
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6.
Ghosh, A.; Heston, W. D., Tumor Target Prostate
Funding Sources
Specific Membrane Antigen (PSMA) and its Regulation in
Prostate Cancer. J. Cell. Biochem. 2004, 91 (3), 528-539.
7.
L.; Sauter, G.; Chadhuri, A.; Herrmann, F. R.; Penetrante,
R., Prostate ‐ Specific Membrane Antigen (PSMA) Protein
Expression in Normal and Neoplastic Tissues and Its
Sensitivity and Specificity in Prostate Adenocarcinoma: an
Immunohistochemical Study Using Mutiple Tumour Tissue
Microarray Technique. Histopathology 2007, 50 (4), 472-483.
We acknowledge Helmholtz-Zentrum Berlin for the
allocation of synchrotron radiation beamtime at the
MX14.2 beamline and funding from the CALIPSOplus
under the Grant Agreement 730872 from the EU
Framework Programme for Research and Innovation
HORIZON 2020. Additionally, this work was in part
supported by the CAS (RVO: 86652036), MEYS CR
(LM2015043 CIISB), and projects CIISB4HEALTH
Mhawech‐Fauceglia, P.; Zhang, S.; Terracciano,
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8.
Elsasser-Beile, U.; Reischl, G.; Wiehr, S.; Buhler,
(CZ.02.1.01/0.0/0.0/16_013/0001776)
(CZ.1.05/1.1.00/02.0109) from the ERDF.
and
BIOCEV
P.; Wolf, P.; Alt, K.; Shively, J.; Judenhofer, M. S.;
Machulla, H. J.; Pichler, B. J., PET Imaging of Prostate Cancer
Xenografts with a Highly Specific Antibody against the
Prostate-Specific Membrane Antigen. J. Nucl. Med. 2009, 50
(4), 606-611.
Notes
The authors declare no competing financial interest.
9.
Henry, M. D.; Wen, S.; Silva, M. D.; Chandra, S.;
Milton, M.; Worland, P. J., A Prostate-Specific Membrane
Antigen-Targeted Monoclonal Antibody–Chemotherapeutic
Conjugate Designed for the Treatment of Prostate Cancer.
Cancer Res. 2004, 64 (21), 7995-8001.
ACKNOWLEDGMENT
The authors gratefully acknowledge I. Jelinkova and B.
Havlinova for excellent technical assistance.
10.
Aggarwal, S.; Singh, P.; Topaloglu, O.; Isaacs, J.
T.; Denmeade, S. R., A Dimeric Peptide That Binds
Selectively to Prostate-Specific Membrane Antigen and
Inhibits Its Enzymatic Activity. Cancer Res. 2006, 66 (18),
9171-9177.
ABBREVIATIONS
SAR, structure-activity relationship; ZBG, zinc-binding
11.
Rege, K.; Patel, S. J.; Megeed, Z.; Yarmush, M. L.,
Peptide-Based Fusion Peptides and
group;
dichloromethane; HATU, 1-[bis(dimethylamino)methylene]-
1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide
DIPEA,
N,N-diisopropylethylamine;
DCM,
Amphipathic
Immunoconjugates for the Targeted Ablation of Prostate
Cancer Cells. Cancer Res. 2007, 67 (13), 6368-6375.
12.
hexafluorophosphate, TFA, trifluoroacetic acid; PDB, Protein
Data Bank.
Ni, X.; Zhang, Y.; Ribas, J.; Chowdhury, W. H.;
Castanares, M.; Zhang, Z.; Laiho, M.; DeWeese, T. L.;
Lupold, S. E., Prostate-Targeted Radiosensitization via
Aptamer-shRNA Chimeras in Human Tumor Xenografts. J.
Clin. Invest. 2011, 121 (6), 2383-2390.
13.
Pullambahatla, M.; Fox, J. J.; Castanares, M.; Lupold, S. E.;
Babich, J. W.; Mease, R. C.; Pomper, M. G.,
Radiohalogenated Prostate-Specific Membrane Antigen
(PSMA)-Based Ureas as Imaging Agents for Prostate Cancer.
J. Med. Chem. 2008, 51 (24), 7933-7943.
Chen, Y.; Foss, C. A.; Byun, Y.; Nimmagadda, S.;
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