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Purpose: Pro-angiogenic and immune cells expressing chemokine receptor CXCR4 traffic along concentration gradients of the chemokine ligand CXCL12, which disseminates from stromal niches. The CXCR4/CXCL12 axis is hijacked by various cancer types characterized by dramatic CXCR4 and/or CXCL12 upregulation. This chemokine network misregulation causes hyperactivation of CXCR4-mediated processes, leading to (1) excessive stimulation of protective tumor-stromal interactions, (2) metastatic expansion of CXCR4+ cancer cells to CXCL12-rich sites, and (3) intratumoral infiltration of CXCR4+ immunosuppressive cells. Accordingly, CXCR4 antagonists have significant therapeutic potential against cancer. While clinical CXCR4 inhibitors are severely limited by suboptimal pharmacokinetic (PK) properties and off-target activities, several of our tetrahydroisoquinoline-based CXCR4 antagonists exhibit superior selectivity and PK profiles to these clinical comparators (e.g., X4P-001). Methods: To test the hypothesis that improved PK profiles would translate to enhanced efficacy, our lead CXCR4 antagonist EMU-116 was compared to X4P-001 head-to-head in three mouse models of genitourinary cancers. First, human 786.0 renal cell carcinoma (RCC) xenograft-bearing female nude mice were treated p.o., q.d. with vehicle, axitinib (30 mg/kg), X4P-001 (100 mg/kg), EMU-116 (3, 10, or 30 mg/kg), X4P + axitinib, or EMU-116 + axitinib. Subcutaneous tumor volume was monitored using calipers. Second, nude male mice bearing intratibial, luciferase-expressing human PC-3 prostate cancer xenografts were treated with vehicle (p.o., q.d.), docetaxel (10 mg/kg i.p. weekly), X4P-001 (10 or 30 mg/kg p.o., q.d.), EMU-116 (10 or 30 mg/kg p.o., q.d.), X4P + axitinib, or EMU + axitinib. Intratibial tumor volume was monitored via luminescence imaging. Third, syngeneic RENCA RCC tumor-bearing female Balb/c mice were treated p.o., q.d. with vehicle, X4P-001 (30 mg/kg), or EMU-116 (30 mg/kg). Subcutaneous tumor volume was monitored using calipers, and immune cell subsets in bone marrow, blood, tumor, and tumor-draining lymph nodes were quantified via flow cytometry. Results: In the RCC xenograft model in combination with axitinib, EMU-116 (3 or 10 mg/kg) was equally effective at decreasing tumor burden compared to X4P-001 (100 mg/kg), whereas EMU-116 (30 mg/kg) was more effective. In the bone metastatic prostate cancer xenograft model, EMU-116 was as or more effective than X4P-001 when paired with docetaxel. In the syngeneic RCC model, EMU-116 mobilized T cells more effectively than X4P-001. Conclusion: EMU-116, at the same or lower dose, was more efficacious than X4P-001 in these models. These results highlight EMU-116 as a clinical candidate with significant therapeutic potential to synergize with chemotherapeutics, targeted therapies, and immuno-oncology agents. Citation Format: Eric J. Miller, Carrie Q. Sun, Petra Gregorova, Edgars Jecs, Yesim Altas Tahirovic, Robert J. Wilson, Huy H. Nguyen, Savita K. Sharma, Perry Bartsch, Zachary Sticher, Levi Moellering, Priscilla Davidson, Ryan Jajosky, Michael D'Erasmo, Manohar Saindane, Zafer Sahin, Nicholas S. Akins, Alexander A. Kolykhalov, Lawrence Wilson, Rebecca S. Arnold, John A. Petros, Haydn Kissick, Lingjie Xu, Yi Jiang, Dennis C. Liotta. Orally bioavailable small molecule CXCR4 antagonists with enhanced efficacy in mouse models of genitourinary cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2649.

Establishing redox and thermodynamic relationships between metal-ion-bound O(2) and its reduced (and protonated) derivatives is critically important for a full understanding of (bio)chemical processes involving dioxygen processing. Here, a ferric heme peroxide complex, [(F(8))Fe(III)-(O(2)(2−))](−) (P) (F(8) = tetrakis(2,6-difluorophenyl)porphyrinate), and a superoxide complex, [(F(8))Fe(III)-(O(2)(•−))] (S), are shown to be redox interconvertible. Using Cr(η-C(6)H(6))(2), an equilibrium state where S and P are present is established in tetrahydrofuran (THF) at −80 °C, allowing determination of the reduction potential of S as −1.17 V vs Fc(+/0). P could be protonated with 2,6-lutidinium triflate, yielding the low-spin ferric hydroperoxide species, [(F(8))Fe(III)-(OOH)] (HP). Partial conversion of HP back to P using a derivatized phosphazene base gave a P/HP equilibrium mixture, leading to the determination of pK(a) = 28.8 for HP (THF, −80 °C). With the measured reduction potential and pK(a), the O–H bond dissociation free energy (BDFE) of hydroperoxide species HP was calculated to be 73.5 kcal/mol, employing the thermodynamic square scheme and Bordwell relationship. This calculated O–H BDFE of HP, in fact, lines up with an experimental demonstration of the oxidizing ability of S via hydrogen atom transfer (HAT) from TEMPO-H (2,2,6,6-tetramethylpiperdine-N-hydroxide, BDFE = 66.5 kcal/mol in THF), forming the hydroperoxide species HP and TEMPO radical. Kinetic studies carried out with TEMPO-H(D) reveal second-order behavior, k(H) = 0.5, k(D) = 0.08 M(−1) s(−1) (THF, −80 °C); thus, the hydrogen/deuterium kinetic isotope effect (KIE) = 6, consistent with H-atom abstraction by S being the rate-determining step. This appears to be the first case where experimentally derived thermodynamics lead to a ferric heme hydroperoxide OO–H BDFE determination, that Fe(III)-OOH species being formed via HAT reactivity of the partner ferric heme superoxide complex.

The focus of this study is in the description of synthetic heme/copper/O(2) chemistry employing a hemecontaining binucleating ligand which provides a tridentate chelate for copper ion binding. The addition of O(2) (−80 °C, tetrahydrofuran (THF) solvent) to the reduced heme compound (P(ImH))Fe(II) (1), gives the oxy-heme adduct, formally a heme–superoxide complex Fe(III)−(O(2)(•−)) (2) (resonance Raman spectroscopy (rR): ν(O−O), 1171 cm(−1) (Δ(18)O(2), −61 cm(−1)); ν(Fe−O), 575 cm(−1) (Δ(18)O(2), −24 cm(−1))). Simple warming of 2 to room temperature regenerates reduced complex 1; this reaction is reversible, as followed by UV–vis spectroscopy. Complex 2 is electron paramagnetic resonance (EPR)-silent and exhibits upfield-shifted pyrrole resonances (δ 9.12 ppm) in (2)H NMR spectroscopy, indicative of a six-coordinate low-spin heme. The coordination of the tethered imidazolyl arm to the heme–superoxide complex as an axial base ligand is suggested. We also report the new fully reduced heme–copper complex [(P(ImH))Fe(II)Cu(I)](+) (3), where the copper ion is bound to the tethered tridentate portion of P(ImH). This reacts with O(2) to give a distinctive low-temperature-stable, high-spin (S = 2, overall) peroxo-bridged complex [(P(ImH))Fe(III)−(O(2)(2−))−Cu(II)](+) (3a): λ(max), 420 (Soret), 545, 565 nm; δ(pyrr), 93 ppm; ν(O−O), 799 cm(−1) (Δ(18)O(2), −48 cm(−1)); ν(Fe−O), 524 cm(−1) (Δ(18)O(2), −23 cm(−1)). To 3a, the addition of dicyclohexylimidazole (DCHIm), which serves as a heme axial base, leads to low-spin (S = 0 overall) species complex [(DCHIm)(P(ImH))Fe(III)−(O(2)(2−))−Cu(II)](+) (3b): λ(max), 425 (Soret), 538 nm; δ(pyrr), 10.2 ppm; ν(O−O), 817 cm(−1) (Δ(18)O(2), −55 cm(−1)); ν(Fe−O), 610 cm(−1) (Δ(18)O(2), −26 cm(−1)). These investigations into the characterization of the O(2)-adducts from (P(ImH))Fe(II) (1) with/without additional copper chelation advance our understanding of the dioxygen reactivity of heme-only and heme/Cu-ligand heterobinuclear system, thus potentially relevant to O(2) reduction in heme–copper oxidases or fuel-cell chemistry.

Peroxynitrite (−OON═O, PN) is a reactive nitrogen species (RNS) which can effect deleterious nitrative or oxidative (bio)chemistry. It may derive from reaction of superoxide anion (O2•−) with nitric oxide (·NO) and has been suggested to form an as-yet unobserved bound heme-iron-PN intermediate in the catalytic cycle of nitric oxide dioxygenase (NOD) enzymes, which facilitate a ·NO homeostatic process, i.e., its oxidation to the nitrate anion. Here, a discrete six-coordinate low-spin porphyrinate-FeIII complex [(PIm)-FeIII(−OON═O)] (3) (PIm; a porphyrin moiety with a covalently tethered imidazole axial “base” donor ligand) has been identified and characterized by various spectroscopies (UV–vis, NMR, EPR, XAS, resonance Raman) and DFT calculations, following its formation at −80 °C by addition of ·NO(g) to the heme-superoxo species, [(PIm)FeIII(O2•−)] (2). DFT calculations confirm that 3 is a six-coordinate low-spin species with the PN ligand coordinated to iron via its terminal peroxidic anionic O atom with the overall geometry being in a cis-configuration. Complex 3 thermally transforms to its isomeric low-spin nitrato form [(PIm)FeIII(NO3−)] (4a). While previous (bio)chemical studies show that phenolic substrates undergo nitration in the presence of PN or PN-metal complexes, in the present system, addition of 2,4-di-tert-butylphenol (2,4DTBP) to complex 3 does not lead to nitrated phenol; the nitrate complex 4a still forms. DFT calculations reveal that the phenolic H atom approaches the terminal PN O atom (farthest from the metal center and ring core), effecting O–O cleavage, giving nitrogen dioxide (·NO2) plus a ferryl compound [(PIm)FeIV═O] (7); this rebounds to give [(PIm)FeIII(NO3−)] (4a).The generation and characterization of the long sought after ferriheme peroxynitrite complex has been accomplished.