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BACKGROUND: Acetaminophen (APAP) is a widely self-prescribed analgesic for mild to moderate pain, but overdose or repeat doses can lead to liver injury and death. Kalyra Pharmaceuticals has developed a novel APAP analog, KP-1199, currently in Phase 1 clinical studies, which lacks hepatotoxicity. In this study, the authors evaluated the antinociceptive effect of KP-1199 on thermal injury-induced nociceptive behaviors as well as hemostatic parameters using human blood samples. METHODS: Full-thickness thermal injury was induced in anesthetized adult male Sprague-Dawley rats. On day 7 post-injury, KP-1199 (30 and 60 mg/kg) or APAP (60 mg/kg) was administered orally. Antinociception of KP-1199 and APAP were assessed at multiple time points using Hargreaves' test. In separate experiments, human whole blood was collected and treated with either KP-1199, APAP, or Vehicle (citrate buffer) at 1× (214 µg/ml) and 10× (2140 µg/ml) concentrations. The treated blood samples were assessed for: clotting function, thrombin generation, and platelet activation. RESULTS: APAP did not produce antinociceptive activity. KP-1199 treatment significantly increased the nociceptive threshold, and the antinociceptive activity persisted up to 3 h post-treatment. In human samples, 10× APAP caused significantly prolonged clotting times and increased platelet activation, whereas KP-1199 had caused no negative effects on either parameter tested. CONCLUSION: These results suggest that KP-1199 possesses antinociceptive activity in a rat model of thermal injury. Since KP-1199 does not induce platelet activation or inhibit coagulation, it presents an attractive alternative to APAP for analgesia, especially for battlefield or surgical scenarios where blood loss and blood clotting are of concern.

Background: Wee1 is a crucial cell cycle checkpoint kinase that regulates the G2/M checkpoint in response to DNA damage, and its inhibition can cause mitotic catastrophe and apoptosis in tumor cells. Inhibitors of BCL-2 have been shown to also induce apoptosis. ZN-d5 and ZN-c3, currently in clinical development for the treatment of cancer, are highly selective and potent inhibitors of Bcl-2 and Wee1, respectively. The combination of ZN-d5 with ZN-c3 induced robust tumor regressions in preclinical models and the mechanism of action of this activity is presented. Methods: Cell proliferation in tumor cell lines was measured using CellTiter-Glo® (Promega). Cleaved caspase-3, cleaved PARP, phosphorylated CDK1 (p-CDK1), γH2AX, Wee1, RRM2 and other markers were measured by Western blotting, IHC and/or flow cytometry. Anti-tumor efficacy was determined in several xenograft models. Results: The combination of ZN-c3 and ZN-d5 resulted in additive or synergistic anti-proliferative activity in several tumor cell lines. This enhancement of anti-proliferative activity was also observed at concentrations of ZN-d5 that induced caspase activation, but minimal apoptosis. Treatment of tumor cell lines with ZN-d5 alone at these sublethal concentrations caused induction of γH2AX, a marker of double-stranded DNA breaks, and degradation of caspase-sensitive proteins relevant to the DNA Damage Response (DDR), such as RRM2 (a ribonucleotide reductase subunit) and Wee1. Degradation of Wee1 correlated with decreases in p-CDK1, a marker of Wee1 inhibition. These effects were mainly reversible and were inhibited by pre-treatment with caspase inhibitors. Mechanistically, the combination of ZN-c3 and ZN-d5 resulted in increased levels of γH2AX, enhanced inhibition of p-CDK1, and increased levels of apoptotic markers compared to single agent treatment. Similar results were obtained when ZN-c3 was combined with inhibitors of other BCL-2 family proteins such as inhibitors of MCL-1 and BCL-xL. Finally, the combination of ZN-c3 with ZN-d5 or other BH3 mimetics showed synergistic anti-tumor activity in several xenograft models further supporting the rationale for combining these agents. Conclusions: ZN-d5 or other BH3 mimetics caused caspase-mediated DNA damage and degraded relevant proteins such as Wee1 and RRM2 resulting in additive or synergistic anti-tumor activity when combined with ZN-c3. These results support a novel use of BH3 mimetics to activate caspase activity, independently of apoptosis induction, to significantly enhance the activity of DDR inhibitors such as ZN-c3 Citation Format: Hooman Izadi, Noah Ibrahim, Tiffany Hoang, Jianhui Ma, Petrus R. de Jong, Joseph Pinchman, Kevin D. Bunker, Ahmed A. Samatar, Fernando Doñate. BH3 mimetics synergize with the Wee1 inhibitor ZN-c3 by activating caspases which induce DNA damage and degrade key proteins [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 2605.

Background: The Bcl-2 inhibitor venetoclax is approved in combination with hypomethylating agents such as azacitidine for the treatment of newly diagnosed elderly patients with AML; however, most patients eventually relapse, especially those with TP53 mutations who have poor prognosis. Wee1 is a crucial cell cycle checkpoint kinase that regulates the G2/M checkpoint in response to DNA damage, and its inhibition can cause mitotic catastrophe and apoptosis. ZN-d5 and ZN-c3 are highly selective and potent inhibitors of Bcl-2 and Wee1, respectively. We show here that the combination is highly active in preclinical models of AML. Methods: Cell proliferation in tumor cell lines and AML patient’s samples were measured using CellTiter-Glo® (Promega). Relevant markers were measured by Western blotting. Anti-tumor efficacy was determined in cell line derived subcutaneous xenograft models of acute myeloid leukemia (AML) and in an AML patient-derived systemic xenograft (PDX) model. Flow cytometry was used for analysis of the PDX model to measure human AML blast populations. Results: The in vitro combination of ZN-d5 and ZN-c3 was tested in AML cell lines and showed synergistic or additive anti-proliferative activity which was independent of BCL-2 sensitivity in some cases. In addition, ZN-d5 in combination with ZN-c3 had a synergistic anti-tumor activity in vivo in cell line-derived AML models, such as MV4-11 and HL-60. Also, the triple combination of ZN-d5 + ZN-c3 + azacitidine in the HL-60 in vivo model resulted in synergistic anti-tumor efficacy and significant tumor growth inhibition compared to single- and double-agent treatments. The ZN-d5 + ZN-c3 combination was also tested in vitro in 29 AML patient-derived samples. Response to ZN-c3 alone was observed in 23/29 samples (IC50 < 450 nM), independent of TP53 mutation status. Response to ZN-d5 alone was observed in 17/29 samples (IC50 10 μM). Combination of ZN-d5 and ZN-c3 was also highly active in an AML patient-derived PDX model, resulting in a 99.5% decrease in human CD45+ blast population in the bone marrow. Finally, the combination of ZN-d5 and ZN-c3 was active in 3/3 ex vivo experiments of patients derived AML samples who had progressed on venetoclax. Conclusions: These results justify testing the ZN-d5 and ZN-c3 combination or possibly the triple combination including azacitidine in AML patients independently of TP53 status. Due to synergism observed with these combination(s) and activity in samples from patients resistant to venetoclax, activity may be seen in patients with low sensitivity to Bcl-2 inhibitors or even in patients who progressed on venetoclax plus azacitidine. Citation Format: Hooman Izadi, Tiffany Hoang, Noah Ibrahim, Joseph Pinchman, Brant C. Boren, Jianhui Ma, Petrus R. de Jong, Jiali Li, Kevin D. Bunker, Ahmed A. Samatar, Fernando Doñate. Combination of the BCL-2 inhibitor ZN-d5 with the WEE1 inhibitor ZN-c3 shows additive or synergistic anti-tumor activity in acute myeloid leukemia (AML) models [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 2591.

Small molecule tyrosine kinase inhibitors against the epidermal growth factor (EGFR) have made significant impact in the treatment of advanced non-small-cell lung cancer (NSCLC) with activating EGFR mutations. Although the first and second-generation EGFR inhibitors have remarkably improved survival in advanced EGFR mutant NSCLC patients, most of the patients develop acquired resistance. The gatekeeper mutation (T790M) is the most common acquired resistance mechanism to the first and second-generation EGFR inhibitors. To overcome the T790M acquired resistance, third-generation EGFR inhibitors, such as osimertinib, were developed. However, additional mutant selective EGFR inhibitors with improved toxicity profile are still needed as osimertinib treatment produced incidence of adverse events, such as diarrhea and skin rush. Here, we describe the identification and characterization of ZN-e4, an orally bioavailable, selective, irreversible third generation EGFR inhibitor. ZN-e4 selectively inhibited the kinase activity of several mutated forms of EGFR, including L858R, T790M/L858R, and Exon19 del in biochemical assay. In cell-based assays, ZN-e4 demonstrated a 20-40-fold selectivity ratio for mutant EGFR forms over the wild-type form thus minimizing the potential for on target toxicity. ZN-e4 potently inhibited the proliferation of NSCLC cell lines harboring EGFR activating and T790M mutation and induced tumor regression in small and large tumors with activating EGFR mutations in preclinical human NSCLC xenograft models. Taken together, our data suggest that ZN-e4 is a potent and selective third generation EGFR inhibitor. Currently, ZN-e4 is in a Phase I clinical trial in patients with EGFR mutant NSCLC showing clinical activity and encouraging toxicity profile. Citation Format: Jiali Li, Sunny Abraham, Jianhui Ma, Peter Q. Huang, Kevin D. Bunker, Fernando Doñate, Ahmed A. Samatar. Discovery of ZN-e4, an irreversible EGFR-TKI with potent anti-tumor activity in EGFR mutant non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2423.

Genome instability initiated by DNA damage or DNA replication stress is a driver of tumor development. To ensure the accuracy of DNA replication, DNA damage response (DDR) mediated by various cell cycle checkpoints activate the DNA repair system. One such checkpoint system is the G2-M checkpoint which is involved in the repair and prevention of excessive DNA damage. A key regulator of the G2-M checkpoint is the Wee1 kinase which causes cell cycle arrest to prevent damaged cells from entering mitosis. Thus, inhibition of Wee1 should prevent G2-M checkpoint activation which would lead to unscheduled mitotic entry and induction of apoptosis. Small-molecule inhibitors of Wee1 are currently being tested in several tumor types. Preliminary results show promising clinical activity of AZD-1775, a potent inhibitor of Wee1, however, achieving an optimal dose and a schedule that is well-tolerated has been a challenge. Here, we describe the characterization of ZN-c3, a potent, selective and orally bioavailable small-molecule inhibitor of Wee1. ZN-c3 exhibits potent anti-proliferative activity across multiple tumor cell lines. Compared to AZD-1775, ZN-c3 has superior selectivity profile against a panel of kinases. In vivo, ZN-c3 induced tumor regression as a single agent in several tumor models including non-small cell lung cancer, ovarian cancer, and uterine sarcoma. In combination studies, ZN-c3 potentiates the antitumor efficacies of carboplatin and gemcitabine. The pharmacokinetic and efficacy profiles of ZN-c3 and AZD-1775 were compared in tumor xenograft models. ZN-c3 showed higher plasma and tumor exposure than AZD-1775, which correlated with more potent tumor growth inhibition than AZD-1775. In summary, ZN-c3, with its key differentiated pharmacologic and kinase selectivity profile, is a promising new generation of DDR inhibitor. ZN-c3 is currently in clinical development for the treatment of advanced solid cancers. Citation Format: Jiali Li, Brant Boren, Peter Q. Huang, Kevin D. Bunker, Fernando Doñate, Ahmed A. Samatar. Discovery of ZN-c3, a potent Wee-1 inhibitor with a differentiated pharmacologic and kinase selectivity profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1965.

Here, we report that trifluoroacetic acid (TFAH) induces demetallation and protodesilylation of the cyclopentadiene ligand in cobalt-η4-cyclopentadiene complexes of general formula [(η5-C5H5)Co(η4-exo-C(TMS)═C(SO2Ph)CH═CRCH(CO2Et))] (1-Ph, R = Ph; 1-ArtBu, R = p-C6H4tBu; 1-ArNMe2, R = p-C6H4NMe2; and 1-Me, R = Me). The trisubstituted cyclopentadiene products are isolated as a mixture of two tautomers, [(CH2C(SO2Ph)═CHC(CO2Et)═CR)] (8-R-A) and [(CH═C(SO2Ph)CH2C(CO2Et)═CR)] (8-R-B). The endo isomer, [(η5-C5H5)Co(η4-endo-C(TMS)═C(SO2Ph)CH═CPhCH(CO2Et))] (1-Ph-endo), also undergoes demetallation and protodesilylation to give 8-Ph-A and 8-Ph-B in excellent yield. The cobalt-cyclopentadiene complex, [(η5-C5H5)Co(η4-exo-C(TMS)═C(SO2Ph)CH═C(CO2Me)CH(CO2Et))] (1-CO2Me), undergoes demetallation and protodesilylation upon treatment with TFAH to give a hydrogen-bonded fulvenol (8-CO2Me). Liberation of the ethoxy-substituted cyclopentadiene ligand of [(η5-C5H5)Co(η4-exo-C(TMS)═C(SO2Ph)CH═C(OEt)CH(CO2Et))] (1-OEt) leads to formation of a cyclopentenone derivative (11). Thermolysis of 8-Ph-A/8-Ph-B in the presence of maleimide leads to a highly functionalized Diels-Alder adduct, whereas 8-Ph-A/8-Ph-B serves as precursors to trisubstituted ruthenocenes by in situ deprotonation and reaction with [(η5-C5R5)Ru(NCMe)3]PF6 (16-H, R = H; 16-Me, R = Me).

Three diastereomers of a highly functionalized tetrasubstituted cyclopentadienylcobalt-diene complex, (Cp)Co[η4-CH(CO2Et)=C(SO2Ph)C(SiMe3)=CH(CO2Et)] (4-ZE, 4-EZ, 4-ZZ; Cp = C5H5), undergo oxidative demetallation by ceric ammonium nitrate (CAN) to give the corresponding metal-free dienes, CH(CO2Et)=C(SO2Ph)C(SiMe3)=CH(CO2Et) (5-ZE, 5-EZ, 5-ZZ), with retention of configuration. The oxidation of 4-ZE by iodine occurs with a different stereoselectivity than that observed with CAN, to provide the fourth diastereomer 5-EE in high yield. B97D/Def2-TZVPP(CH2Cl2) calculations predict the diene free-energy ordering as: 5-EE < 5-ZE < 5-EZ < 5-ZZ.

Crizotinib (1), an anaplastic lymphoma kinase (ALK) receptor tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration in 2011, is efficacious in ALK and ROS positive patients. Under pressure of crizotinib treatment, point mutations arise in the kinase domain of ALK, resulting in resistance and progressive disease. The successful application of both structure-based and lipophilic-efficiency-focused drug design resulted in aminopyridine 8e, which was potent across a broad panel of engineered ALK mutant cell lines and showed suitable preclinical pharmacokinetics and robust tumor growth inhibition in a crizotinib-resistant cell line (H3122-L1196M).

The reaction of (η5-C5H5)Co(PPh 3)2 with TMSCC(CO2Et) leads to the formation of the cobalt-alkyne complex (η5-C5H5) (PPh3)Co{η2-(Me3Si)CC(CO2Et)} (6). In benzene-d6 solution containing PPh3, 6 undergoes slow conversion to the cobaltacyclopentadiene complex (η5-C 5H5)(PPh3)Co{κ2-(TMS) CC(CO2Et)C(TMS)C(CO2Et)} (7, TMS = SiMe3). Alternatively, reaction of 6 and PhCCPh forms the metallacyclopentadiene regioisomers (η5-C5H5)(PPh 3)Co{κ2-(R1)CC(R2)C(Ph) =C(Ph)} [9-major, R1 = SiMe3, R2 = CO2Et; 9-minor, R1 = CO2Et, R2 = SiMe3). The metallacycle substitution pattern in 9-major and 9-minor is readily deduced from the 1H NMR spectral resonances of the diastereotopic ethoxycarbonyl hydrogens. When the diastereotopic hydrogens of the ethoxycarbonyl have similar chemical shifts the ester is situated on the β-carbon of the metallacycle. When the methylene hydrogens give rise to well-separated resonances the ethoxycarbonyl is situated on the α-carbon of the metallacycle. The solid state structures of 7 and 9-major were determined by X-ray crystallography. © 2013 Elsevier B.V. All rights reserved.

The solid state structures of the cobalt–alkyne complexes (η5-C5H5)(PPh3)Co{η2-(R3Si)C≡C(SO2Ar)} (3-TMS, R = Me, Ar = C6H5; 3-TIPS, R = CH(CH3)2, Ar = p-C6H4CH3) and the noncoordinated alkyne (Me3Si)C≡C(SO2Ph) (6-TMS) have been characterized by X-ray crystallography and, in the case of 3-TMS, 6-TMS, and (η5-C5H5)Co{η2-(Me3Si)C≡C(SO2Ph)} (5-TMS-calc), by B97D/Def2-TZVPP computational analysis. The phosphine-dissociated complex 5-TMS-calc is determined to be a ground state singlet. Analysis of bond angle and distance metrics, calculated NMR chemical shift data, and molecular orbital analysis provide strong evidence for a four-electron-donor alkyne ligand in 5-TMS-calc. The degree of asymmetry in metal–alkyne bonding, as defined by the Gladysz alkyne-slippage parameter, is dramatically reduced in 5-TMS-calc relative to that in the precursor complex 3-TMS-calc.

A new method was developed to synthesize highly functionalized lactams via a one pot reductive amination/lactam formation reaction. This methodology is amenable for parallel synthesis and was used to prepare a large number of lactam analogs in a library format with good ee (de) retention.

The room-temperature reaction of(η 5 -C 5 H 5 )Co(PPh 3 ) 2 (5) with aliphatic nitro compounds, RNO 2 , where R = Me, Et, iPr, leads to formation of the corresponding η 1 -nitroso derivatives (h 5 -C 5 H 5 )Co(PPh 3 )[η 1 (N)-N(O)R](6-Me, R = Me; 6-Et, R = Et; 6-Pr, R = iPr). Aromatic nitro compounds, ArNO 2 , where Ar = -C 6 H 5 , -p-C 6 H 4 C≡CTMS, undergo reaction with 5 to give the μ-η 1 :η 2 -nitroso derivatives, [(η 5 -C 5 H 5 )Co] 2 [μ-η 1 (N):η 2 (N,O)-N(O)R] 2 (7-Ph, R = -C 6 H 5 ; 7-Ar, R = -p-C 6 H 4 C≡CTMS). Complexes 6-Me and 7-Ar were characterized by X-ray crystallographic analyses.

Thermolysis of the η 2 -alkyne complex (η 5 -C 5 H 5 )Co(PPh 3 )[η 2 -(MeO 2 C)CC(CO 2 Me)] ( 1 -DMAD) leads to formation of an ortho -metalated vinyl-phosphonium derivative (η 5 -C 5 H 5 )Co[η 2 -(MeO 2 C)HCC(CO 2 Me)PPh 2 (η 1 - o -C 6 H 4 )] ( 4 ) as well as a small amount of the tricobalt bis(carbyne) cluster [(η 5 -C 5 H 5 )Co] 3 [μ 3 ,η 3 -C(CO 2 Me)] 2 ( 3 -CO 2 Me). Thermolysis of 1 -DMAD in the presence of added triphenylphosphine gives 4 as the major product, with no evidence for formation of 3 -CO 2 Me. Complexes 4 and 3 -CO 2 Me were characterized by X-ray crystallographic analyses.

The reaction of [1.1.1]propellane with di-tert-butyl azodicarboxylate and phenylsilane in the presence of Mn(dpm)(3) to give di-tert-butyl 1-(bicyclo[1.1.1]pentan-1-yl)hydrazine-1,2-dicarboxylate is described. Subsequent deprotection gives 1-bicyclo[1.1.1]pentylhydrazine followed by reduction to give 1-bicyclo[1.1.1]pentylamine. The reported route marks a significant improvement over the previous syntheses of 1-bicyclo[1.1.1]pentylamine in terms of scalability, yield, safety, and cost.

The first productive reactions of a characterized metallacyclobutene complex with alkenes are reported. Thus, the metallacyclobutene complex (eta5-C5H5)(PPh3)Co[kappa2-(C,C)-C(SO2Ph) C(Si(CH3)3)CH(CO2CH2CH3)] (2) undergoes reaction with alkenes to give 1,4-diene complexes with a high degree of regio- and stereoselectivity. A mechanism is proposed in which the metallacyclobutene generates a cyclic vinylcarbene intermediate that undergoes [4 + 2]-cycloaddition reactions with activated alkenes. A model of the vinylcarbene intermediate has been examined using quantum mechanical methods.

The SNAr-reaction of N-benzylpantolactam (I) with various halides is shown to proceed with no or low amounts of racemization and can be used in library synthesis.

The metal-enediyne complexes [(eta 5-C5H5)Fe[eta 5-1,2-C5H3C identical to C(CH2)nC identical to]] (4, n = 4; 5, n = 5) and [(eta 5-C5H5)-Fe[eta 5-1,2-C5H3(C identical to C Me)2]] (6) were prepared from 1,2-diethynylferrocene (3). Complexes 4 and 5 were characterized in the solid state by X-ray crystallographic analysis. The structures of 4 and 6 were determined by computation using ab initio methods. A correlation was observed between ring-strain and increased ease of electrochemical oxidation along the series 6 (+0.164 V) to 5(+0.152 V) to 4 (+0.123 V). A similar trend in ionization potentials was identified in both the gas phase and in solution by computational methods.

The alkynylsulfoxide, TMSCCSO(p-tolyl) (TMS = trimethylsilyl, tolyl = C6H4Me), undergoes reaction with (eta5-C5H5)Co(PPh3)2 at room temperature to give the cobaltosulfoxide complex, (C5H5)Co(PPh3)(eta1-CCTMS)[eta1-(S)-SO(p-tolyl)], which was characterized by X-ray crystallography. Exposure of this cobaltosulfoxide complex to oxygen gas leads to the formation of the corresponding metallosulfone complex, (C5H5)Co(PPh3)(eta1-CCTMS)[eta1-(S)-SO2(p-tolyl)], which was characterized by X-ray crystallography. Alternatively, in solution at room temperature, the metallosulfoxide is converted to a 1:4 mixture of the equatorial-equatorial and equatorial-axial bridging cobalt-thiolato dimers, {(C5H5)Co[mu-S(p-tolyl)]}2, respectively. The equatorial-equatorial isomer was characterized by X-ray crystallography.

The reaction of 2,4-dichloro-5-(trifluoromethyl)pyrimidine with 1H-pyrazole gave two structural isomers in a 1:1 ratio that were separable by chromatography. The title compound, C8H4ClF3N4, was the first product to elute and was characterized in the present study to confirm that substitution by the pyrazolyl group had occurred at position 4. The molecule (with the exception of the F atoms) is essentially planar, with a mean deviation of 0.034 Å from the least-squares plane through all non-H and non-F atoms. The bond angles in the pyrimidine ring show a pronounced alternating pattern with three angles, including those at the two N atoms being narrower, and the remaining three wider than 120°.

The title compound, C9H7ClN2O2, was prepared by reaction of methyl 4-chloro-3-(prop-1-ynyl)picolinate with hydroxylamine in MeOH/KOH solution. The two essentially planar molecules which make up the asymmetric unit have almost identical geometries and and are linked into dimeric aggregates via pairs of O—H...O hydrogen bonds. These aggregates have almost perfect inversion symmetry; however, quite unusually, the inversion center of the dimer does not coincide with the crystallographic inversion center.