Ophiocordyceps, which include species able to manipulate the behaviour of ants, are known as the “zombie-ant fungi” and have attracted much attention over the last decade. They are widespread within tropical, subtropical and even temperate forests worldwide, with relatively few reports from subtropical monsoon evergreen broad-leaved forest. Fungal specimens have been collected from China, occurring on ants and producing hirsutella-like anamorphs. Based on a combination of morphological characters, phylogenetic analyses  and ecological data, two new species, Ophiocordycepstortuosa and O.ansiformis, are identified and proposed herein. Ophiocordycepstortuosa and O.ansiformis are recorded on the same species of Colobopsis ant, based on phylogenetic analyses (COI), which may be sharing the same host. Ophiocordycepstortuosa and O.ansiformis share the morphological character of producing lanceolate ascospores. They have typical characteristics distinguished from other species. The ascospore of O.tortuosa are tortuously arranged in the ascus and the ascospore of O.ansiformis have a structure like a handle-shape in the middle. Our molecular data also indicate that O.tortuosa and O.ansiformis are clearly distinct from other species.Species of the genus  Fungi associated with insects, morphologically similar, but genetically distinct cryptic closely-related species, have given rise to spectacular diversity across a wide range of taxa in the kingdom of fungi. Molecular studies have routinely unmasked several cryptic species and have revealed this as a common phenomenon for the entomogenous fungi . It was established originally by Petch . Hirsutella Pat., Hymenostilbe Petch and Paraisaria Samson & B.L. Brady are commonly asexual morphs within Ophiocordyceps. Species of Hirsutella typically produced one to several conidia in a limited number of mucus droplets borne on basally subulate phialides that tapered into slender necks  worldwide  stalk(s) arising from the dorsal pronotum of dead ants, with one or multiple lateral cushions from the base to the top along the stroma attached unilaterally (hence the epithet), exhibiting hirsutella-like anamorphs, whole and septate ascospores that do not disarticulate into part-spores that often exhibit secondary germination (capilliconidiophore) (COI) gene as molecular marker was used for the ant’s phylogenetic studies, for exploring the diversity of the ants and distinguishing species and subspecies in the ant complex  . In addi complex . There a complex , b. In potus sp. . Evans eotus sp. , 2018 haFormicinae (Formicidae) exhibiting similar behavioural manipulation have been reported in previous studies  of Yunnan University.The specimens were collected from south-western China. Collections took place in subtropical monsoon evergreen broad-leaved forest. The ant’s death location from above the ground and the ants attached (biting) to substrate types  were measured and recorded in the field, then all specimens were collected in sterilised plastic containers, transported to the laboratory and examined within the same day if possible or stored at 4 °C. The specimens were deposited in the Yunnan Herbal Herbarium . The stromata and the legs from the same ant host were moved for morphological studies. A cryosectioning of the ascoma was performed using a Freezing Microtome HM525NX . Samples were mounted on a slide with sterile water or lactophenol cotton blue solution for light microscopy examination using an Olympus BX53 . Micro-morphological characteristics  were examined. The naturally released ascospores and germination events were examined using an Olympus BX53 and the detailed method was based on the research of PCR) was used to amplify genetic markers using the following primer pairs: NS1/NS4 for small subunit nuclear ribosomal DNA (SSU) (TEF1a) (RPB1) (DNA templates (contains the host and fungus from the same specimen) were obtained directly from fresh specimens using the Plant DNA Isolation Kit  according to the manufacturer’s protocols. Polymerase chain reaction (NA (SSU) , 2218R/9 (TEF1a) , CRPB1/R) (RPB1)  and LCO1) (RPB1) .PCR reaction contained 2.5 µl of PCR 10× Buffer (2 mmol/l Mg2+) , 17.25 µl of sterile water, 2 µl of dNTP (2.5 mmol/l), 1 µl of each forward and reverse primer (10 µmol/l), 0.25 µl of Taq DNA polymerase  and 1 µl of DNA template (500 ng/µl). The PCR reactions were placed in a Bio-Rad T100 thermocycler  under the following conditions: For SSU, (1) 4 min at 95 °C, (2) 22 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C for 1 min, and extension at 72 °C for 1.3 min, followed by (3) 12 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 72 °C for 1.35 min and (4) 8 min at 72 °C  4 min at 95 °C, (2) 8 cycles of denaturation at 94 °C for 50 s, annealing at 52 °C for 50 s and extension at 72 °C for 1 min, followed by (3) 30 cycles of denaturation at 94 °C for 50 s, annealing at 51 °C for 50 s and extension at 72 °C for 1 min and (4) 10 min at 72 °C  4 min at 95 °C, (2) 30 cycles of denaturation at 94 °C for 50 s, annealing at 52 °C for 50 s and extension at 72 °C for 1 min, followed by (3) 8 cycles of denaturation at 94 °C for 50 s, annealing at 51 °C for 50 s and extension at 72 °C for 1 min and (4) 10 min at 72 °C  1 min at 95 °C, (2) 5 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1.5 min and extension at 72 °C for 1.5 min, followed by (3) 35 cycles of denaturation at 94 °C for 1 min, annealing at 54 °C for 1.5 min and extension at 72 °C for 1 min and (4) 5 min at 72 °C  and sequenced by Beijing Genomics Institute . All LSU and RPB2 sequences were downloaded from GenBank.Each 25 µl-at 72 °C . For TEFat 72 °C . For RPBat 72 °C . For COIat 72 °C . PCR proOphiocordyceps, most of the DNA sequences used in this work were based on previous phylogenetic studies . Then the nucleotide sequences were combined with those generated in our study (Table ML) and Bayesian Inference (BI) analyses according to the corrected Akaike Information Criterion (AIC). For ML analyses, tree searches were performed in IQ-tree v.2.1.3  were run, each beginning with a random tree and sampling one tree every 100 generations of 2,000,000 generations and the first 25% of samples were discarded as burn-in. The tree was visualised with its Maximum-Likelihood bootstrap proportions (ML-BS) and Bayesian posterior probability (BI-BPP) in Figtree v.1.4.3. Adobe Illustrator CS6 was used for editing.To construct a phylogeny of major lineages in  studies . Phylogedy Table , adjustedy Table . ModelFidy Table  was empl v.2.1.3 , based o v.2.1.3  in a sin v.3.2.7 . The BI COI gene sequences. Most of the DNA sequences used in this work were based on previous phylogenetic studies  in a concatenated tree, using morphological features for comparison, enabled identification of two new species (O.tropiosa and O.ansiformis). We have inferred the phylogeny, based on each single gene  and present the details below. Ophiocordycepstortuosa was recovered as sister to O.lilacina (BS = 88%) , based on TEF1a or RPB1 , based on SSU , based on TEF1a or RPB1 , the alignment comprised 143 taxa , O.oecophyllae branched as its sister taxon with BS = 98%, BPP = 80%. The subclade O.kniphofioides was sister to the core clade O.unilateralis + O.oecophyllae clade with strong support . The phylogenetic analysis indicated that the two species in this study were clustered together in the O.unilateralis core clade within the Southeast Asian clade and that the two new taxa formed distinct lineages from the other species, respectively. The sister relationships between O.tortuosa and O.contiispora were recovered with strong support  and obtained the same topological structure as the single gene (TEF1a and RPB1) tree  and also obtained the same topological structure as the single gene (TEF1a and RPB1) tree  and Polyrhachis  within Formicinae formed each a monophyletic clade with strong supports and statistical topology. The phylogenetic analysis indicated that the hosts Colonopsis sp. (YHH 2210006 and YHH 2210007) formed a clade and were infected by both O.tortuosa and O.ansiformis  in Tang et al. studies (2023b). The results in this work suggest that the fungal pathogen infects the same host as Colonopsis sp. and that the two species may share the same niche.Phylogenetic analyses showed that the genera Taxon classificationFungiHypocrealesOphiocordycipitaceaeHong Yu bis, D.X. Tang & J. Zhaosp. nov.6C6D9418-4ECB-5993-B59B-AA3E2E670754849060Tortuosa = tortuous, the epithet referred to the “tortuous” arrangement of ascospores in the asci.Ophiocordycepstortuosa and related species is that Ophiocordycepstortuosa produces lanceolate and obvious separate ascospores, while O.contiispora produces fusiform and no obvious separate ascospores.The difference between Colobopsis sp. (Formicinae) biting into a leaf of Lauraceae Juss., 22°42'40"N, 100°57'28"E, alt. 1345 m, 03 October 2022, Hong Yu bis .China, Yunnan Province, Puer City, Simao District. Infected Sexual morph: External mycelia produced from all orifices and sutures, often covering the host body, initially white turning brown. Stromata single to multiple, produced from dorsal pronotum, part branched, 16–24 mm in length, cylindrical, pale white to light brown, becoming pinkish at the apical part. Fertile region of lateral cushions, 1–3, commonly 2 per stroma, hemispherical, chocolate brown at maturity, 1–1.9 × 0.8–1.3 mm. Perithecia immersed to partially erumpent, flask-shaped, (211–) 218–298 (–305) × (94–) 99–142 (–158) μm, with short, exposed neck or ostiole. Asci 8-spored, hyaline, cylindrical, (92–) 96–132 (–134) × 7–11 (–13) μm. Ascus caps slightly prominent, hemispherical, 4–5 × (2–) 3–4 μm. Ascospores hyaline, thin-walled, lanceolate, tortuous arrangement in the ascus, 47–64 × 5–7 μm, 6–7-septate, gently curved at round apex, tapered end shorter than round apex.Asexual morph: Hirsutella-A type associated with the apical part of stromata. Hirsutella-C type produced from the leg and antennal joints. Phialides lageniform, 54–99 μm long, 4–6 μm width at base, tapering to a long neck, 1–2 μm in width. Conidia fusiform to limoniform, 6–8 × 3–5 μm, slightly narrowing at the top.Germination process: The released ascospores germinated within 48 h to produce 1–2 long and extremely narrow hair-like capilliconidiophores, (27–) 44–65 (–69) × 1–2 μm, bearing a single terminal capilliconidium, (5–) 6–9 × 3–4 (–5) μm, hyaline, smooth-walled, limoniform to fusiform, slightly narrowing and curved at the top.Host: Colobopsis sp. (Formicinae).Colobopsis sp. biting into a leaf of Lauraceae Juss., from 1.2 to 2.4 m above the ground.Subtropical monsoon evergreen broad-leaved forest. Infected China, Yunnan Province, Puer City.Colobopsis sp. biting into a leaf of Lauraceae Juss., 22°42'40"N, 100°57'28"E, alt. 1,345 m, 03 October 2022, D.X. Tang .China, Yunnan, Puer City, Simao District. Infected O.tortuosa was sister to O.contiispora  biting into a leaf of Rubiaceae Juss., 22°31'24"N, 100°58'57"E, alt. 1,029 m, 02 October 2022, Hong Yu bis .China, Yunnan Province, Jinghong City, Puwen Town. Infected Sexual morph: External mycelia produced from all orifices and sutures, brown at maturity. Stroma single, produced from dorsal pronotum, never branched, 25–28 mm in length, cylindrical, dark brown at maturity, light brown at the apical part. Fertile region of lateral cushions, 1–3, hemispherical, 1–1.3 × 0.7–1 mm. Perithecia immersed to partially erumpent, flask-shaped, (174–) 189–290 × 99–126 (–128) μm, with short, exposed neck or ostiole. Asci 8-spored, hyaline, cylindrical, (88–) 92–108 (–112) × 7–10 (–11) μm. Ascus caps prominent, hemispherical, 4–6 (–7) × 2–3 (–4) μm. Ascospores hyaline, thin-walled, lanceolate, having a handle-shape in the middle, 45–59 × 5–6 (–7) μm, 6–9-septate, tapering at apex.Asexual morph: Hirsutella-A type present along stromata. Phialides lageniform, 15–24 × 3–4 μm, tapering to a short neck, 6–8 μm in length. Conidia were not observed.Germination process: Ascospores released on agar germinated after 48 h to produce 1–2 capilliconidiophores, (54–) 60–79 (–84) × 0.8–1.4 μm, bearing a terminal capilliconidium, hyaline, smooth-walled, limoniform, 6–10 × 3–4 μm, slightly narrowing apically.Host: Colobopsis sp. (Formicinae).Colobopsis sp. biting into a leaf of Rubiaceae Juss., from 0.8 to 1 m above the ground.Subtropical monsoon evergreen broad-leaved forest. Infected China, Yunnan Province, Jinghong City.Colobopsis sp. biting into a leaf of Rubiaceae Juss., 22°31'24"N, 100°58'57"E, alt. 1,029 m, 02 October 2022, D.X. Tang (YHH 2210007).China, Yunnan Province, Jinghong City, Puwen Town. Infected O.ansiformis formed a sister lineage with O.tortuosa and O.contiispora, was clustered in the O.unilateralis core clade, with statistical support from bootstrap proportions (BS = 85%) and Bayesian posterior probabilities (BPP = 95%)  Fig. . Ophiocosp. Fig. . Howeverle Table .Hymenoptera are considered cryptic species within the genus Ophiocordyceps. These species are distinguished by morphological features and molecular phylogenetic studies. Known examples of these fungi were found occurring on adult ants in the O.myrmecophila species complex, for example, O.megacuculla and O.granospora  and ecological data. The O.unilateralis complex species was sister to O.oecophyllae and both are sister to the O.kniphofioides sub-clade. The species within O.unilateralis clade infects exclusively Camponotini ants   infected by O.nooreniae , was infected by ant pathogenic fungi, including O.basiasca, O.contiispora, O.acroasca, O.subtiliphialida, O.tortuosa and O.ansiformis  . Entomopcificity . However O.satoi  and two ooreniae . The samsiformis . The antO.unilateralis core clade showed slightly micro-morphological characteristics  that made them recognised from other species. Ophiocordycepansiformis differed from O.contiispora by producing lanceolate ascospores with a handle-shape in the middle, while O.tortuosa differed from O.contiispora by producing lanceolate ascospore with obvious separation and tortuous arrangement in the ascus. In addition, O.tortuosa and O.ansiformis differed in the size of perithecia (211–305 × 94–158 µm vs. 174–290 × 99–128 µm), asci (92–134 × 7–13 µm vs. 88–112 × 7–11 µm) and ascospores , this work supporting the idea of cryptic species . We conducted a two-year tracking survey on one of the O.unilateralis complex species at the same location and season in subtropical monsoon evergreen broad-leaved forest. It was found that the death location of the host ant on the underside of leaves above the ground seems to be influenced by environmental factors such as rainfall, humidity, temperature etc. We found that there were differences in the location of death from the ground in the O.unilateralis complex at different years in the same species. In addition, The two novel species within the  species . The spem to 2 m , 2023b. Therefore, future studies are recommended to examine the impact of changes in environmental conditions on the height at which host ants die.Ophiocordycepstortuosa was recovered sister to O.contiispora with strong support and consistent topology, based on the concatenated and single gene (TEF1a and RPB1) tree. The species O.ansiformis was also recovered sister to O.tortuosa + O.contiispora with weak to strong support and consistent topology, based on the concatenated and single gene (TEF1a and RPB1) tree  for the two species proposed in this work. Other species , based on SSU, TEF1a and RPB1 trees have similar topological structures in this work. SSU phylogenies indicate their utility as a well marker to infer phylogenetic relationships at the subclass level  and ecological data.We had inferred the phylogeny, based on each single gene and also used a concatenated dataset in this study. ss level , but it


Dermanyssus gallinae) infestation have reduced efficacy or exhibit hazardous effects on chickens. Considering the economic importance of chickens, development of a safe and effective method for exterminating PRMs is necessary. Ivermectin and allicin are effective against some ectoparasites; however, their acaricidal efficacies against PRMs remain unknown.Existing treatments against poultry red mite  of ivermectin (1 mL) were applied via dropping method in different insect culture dishes (ICDs), prior to transferring PRMs. For the spraying method, PRMs were transferred to ICDs, before spraying ivermectin (1 mg/mL) solution (1 mL). Further, the acaricidal effect of allicin on PRMs was evaluated by applying different concentrations (0.25–1.0 mg/mL) of allicin (1 mL). The combined acaricidal effects of ivermectin and allicin were analysed using four concentration combinations. PRM death rates were determined after 2 h, 24 h, 2 days, 5 days and 7 days of drug application.Ivermectin application (1 mg/mL) exterminated 64% and 100% of PRMs on 1 and 5 days, respectively, and prevented their revival. Further, 0.5 mg/mL ivermectin and 1 mg/mL allicin individually exterminated 98% and 44% of PRMs, respectively, within 7 days of treatment. In combination, 0.5 mg/mL ivermectin and 0.5 mg/mL allicin exterminated 100% of PRMs within 5 d of treatment. The most effective combination was 0.25 mg/mL ivermectin + 1.00 mg/mL allicin.The efficacy of ivermectin–allicin combination in exterminating PRMs was demonstrated. This novel approach could be optimised for industrial applications. Ivermectin and allicin synergistically exterminated poultry red mite  during 7 days of treatment, and this combination treatment may influence the physiological processes of PRMs.  Approximately 1 mL (5 puffs) of ivermectin solution (1000 ppm) was sprayed onto the PRMs in the ICDs. The percentage of dead PRMs was determined after 2 h, 24 h, 2 days, 5 days and 7 days of ivermectin application.Furthermore, the acaricidal effect of allicin on PRMs was evaluated by applying 1 mL of allicin  with a micropipette. The percentage of dead PRMs after allicin treatment was determined using similar methods to ivermectin treatment. The combined acaricidal effects of ivermectin and allicin on PRMs were also analysed. Four combinations  were applied to 20 PRMs in ICDs by using a micropipette, and their effects were examined in accordance with the procedure described earlier in this section.3Chemicals used to control PRMs may adversely affect workers through direct exposure and indirect consumption of pesticide residue‐containing eggs  were inhibited within 1 day of 1 mg/mL ivermectin application. The remaining PRMs were inhibited (100%) within 5 days of treatment (1 mg/mL). Ivermectin treatment also prevented PRM revival. Even 0.5 mg/mL ivermectin exterminated 32%, 72%, 93% and 98% of the PRMs after 1, 2, 5 and 7 days of treatment, respectively. Compared with the untreated control, lower ivermectin concentrations (0.25 and 0.1 mg/mL) showed considerable and concentration‐dependent extermination for different durations. Allicin (1 mg/mL) alone exterminated 23%, 29%, 35% and 44% of the PRMs after 1, 2, 5 and 7 days of treatment, respectively. However, lower allicin concentrations (0.5 and 0.25 mg/mL) exhibited relatively low PRM extermination rates. The number of PRMs remained stable until 2 days of observation although 11.67% and 13.33% of PRMs died on the fifth and seventh days of observation, respectively, under usual environmental conditions without any treatment. The diluent used to prepare the drug solution also elicited no remarkable effect on the mortality of PRMs. Within 24 h of treatment with the diluent, 7.50% of PRMs were exterminated, and 17.50% and 20.00% were exterminated on the fifth and seventh days of treatment, respectively.The application of the ivermectin–allicin combination increased the mortality of PRMs. Different concentrations of the ivermectin–allicin combination were applied, and the acaricidal effects of some combinations, which showed synergistic or additive effects are stated in this report. The acaricidal effect of the combination of 0.5 mg/mL ivermectin and 0.5 mg/mL allicin was greater than that of individual drugs used at the same concentrations and other drug combinations. The PRMs were completely exterminated on the fifth day of treatment with 0.5 mg/mL ivermectin and 0.5 mg/mL allicin. The effects of both drugs were concentration and exposure‐duration dependent, and ivermectin was more potent than allicin. However, a high ivermectin concentration can be retained in poultry eggs. Therefore, it is prohibited in many countries for food safety. In this study, we evaluated the effects of low concentrations of ivermectin, which may have no adverse effects to nontargets and consumers if it retains in poultry eggs and meat. Although high allicin concentration (30–59 mg) may have mild adverse effect such as stomach irritation, but the application of allicin table (5 mg), and injection (1 mg) could not show adverse effect to human suggesting that allicin is safe (Salehi et al., Our findings demonstrated that the combination of 0.25 mg/mL ivermectin and 1.00 mg/mL allicin could exterminate PRMs and could be optimised for clinical use. Nevertheless, the development of a suitable dosage of 0.25 mg/mL ivermectin and 1.00 mg/mL allicin combination would be effective for its rapid application and transport.Conceptualisation, J.W.K., M.A.H. Methodology, J.W.K., M.A.H. Investigation, J.W.K., M.A.H. Resources, J.W.K. Data curation, H.Y.C, M.A.H. Writing – original draft preparation, J.W.K. Writing, review and editing, J.W.K., M.A.H. Supervision, J.W.K. Project administration, J.W.K. Funding acquisition, J.W.K. All authors have read and agreed to the published version of the manuscript.This study was supported by veterinary science research project grants from the Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.The authors declare no conflicts of interest.https://publons.com/publon/10.1002/vms3.1136.The peer review history for this article is available at


Cdk2-/- and Spdya-/- phenotypes.Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception. However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs. In this paper, we describe the development and mechanism of action of type III inhibitors that bind CDK2 with nanomolar affinity. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as contraceptive agents is seen by incubation with spermatocyte chromosome spreads from mouse testicular explants, where they recapitulate  Despite the therapeutic interest in targeting CDK2, developing a selective CDK2 inhibitor has been challenging. Here, the authors describe a potent and selective CDK2 inhibitor that binds an allosteric pocket, preventing activating protein partners from binding and showing potential as a contraceptive. In addition, allosteric type III inhibitors, compounds that bind the kinase outside of the active site, have been a promising avenue to selectively inhibit kinases as these distal sites often have reduced structural similarity. Some allosteric kinase inhibitors even demonstrate selectivity for oncologic mutant isoforms of the same kinase5. To date, four allosteric kinase inhibitors have been approved by the U.S. Food and Drug Administration to treat BRAF-mutated cancers and recently asciminib was approved as a treatment for chronic myeloid leukemia targeting an allosteric pocket of ABL kinase10. Despite this success, few high affinity and well-characterized allosteric kinase inhibitors have been reported in the literature12. In this paper, we will showcase a series of anthranilic acid-derived inhibitors that bind an allosteric pocket of cyclin-dependent kinase 2 (CDK2) with nanomolar affinity.Kinases remain attractive therapeutic targets to treat a variety of diseases, but with over 500 kinases in the human proteome, the selective targeting of a particular kinase remains challenging. This is in large part due to the structural similarity among kinases, especially at the ATP-site where most kinase inhibitors bind. To meet this challenge, a variety of strategies have emerged including developing inhibitors that either bind unique inactive conformations, target unique residues including nucleophilic residues for covalent attachmentCdk2−/− mice are sterile but otherwise healthy14, making CDK2 a validated target for non-hormonal contraceptive development15. In meiosis, the protein Speedy1 (SPY1) binds unphosphorylated CDK2 at the same site where cyclins bind16 and appears to be the crucial binding partner of CDK2 in the pachytene stage of prophase I17. While the Cdk2−/− phenotype suggests a selective CDK2 inhibitor will have a favorable safety profile, further investigation reveals that selective inhibition of CDK2/cyclin complexes can be relatively toxic for healthy cells due to the sequestration of cyclins and thus the loss of compensatory kinase activation18. However, a compound that disrupts the protein-protein interaction (PPI) between CDK2 and its protein partners like cyclin or SPY1 has the potential to be an alternative, less toxic CDK2 inhibitor. In this manner, an ideal inhibitor would free cyclins normally bound to CDK2, leading to A) the activation of compensatory kinases by displaced cyclins in somatic cells and B) disruption of the CDK2/SPY1 interaction crucial for meiosis in spermatocytes, providing a safe and efficacious contraceptive option.CDK2 is a member of the CDK family of kinases that control the cell cycle and is activated by binding E-type cyclins at the G1-S transition, then by A-type cyclins in S phase. In addition, CDK2 must be phosphorylated on the activation loop to become fully activated. CDK2 also plays a critical role in meiosis since 20. Despite this promise, ANS is known to be a promiscuous protein binder25 and contains a sulfonic acid moiety that confers poor pharmacokinetic properties. However, we exploited the environmentally sensitive fluorescent nature of ANS to develop a high-throughput assay and used it to discover new chemical scaffolds with improved physicochemical properties that bind the allosteric pocket with higher affinity27. From those efforts, we herein reveal the highest affinity allosteric CDK inhibitors reported, all containing an anthranilic acid scaffold. We confirm their type III allosteric interaction by X-ray crystallography and use other biophysical techniques to demonstrate negative cooperativity with cyclin binding. This cooperative relationship continues to hold promise as an underexplored mechanism of CDK2 inhibition, only observed with ANS prior19. Furthermore, the challenge of developing selectivity for CDK2 over structurally similar kinases like CDK1 has been overcome, as seen in both biophysical and the appropriate cellular contexts. Finally, we show that mouse testes incubated with our allosteric CDK2 inhibitors recapitulate features of a Cdk2−/− phenotype.We previously discovered that the dye 8-anilino-1-naphthalene sulfonic acid (ANS) bound an unrecognized allosteric pocket within CDK2 with moderate affinity with a 5–10-fold enhancement in affinity in the presence of certain orthosteric inhibitors27. The first compound crystallized of this series was 1, which binds CDK2 with low micromolar affinity as measured by isothermal titration calorimetry (ITC) and by surface plasmon resonance (SPR) using the steady-state approximation (SSA) affinity fit  and type I inhibitors  or larger halogens like chlorine (4) or bromine (5) at this position afford binding with less enthalpic benefit and greater entropic contribution .To explore the inhibitory potential of these compounds, lix Fig. . It is l29. As cyclin A is titrated into CDK2 and the activation loop is stabilized in its active outward conformation, the donor:acceptor ratio of the dyes increases  of CDK2 with corresponding KD values of 280 nM and 200 nM, respectively  assay developed to indirectly track the position of the activation loop on CDK2 and thus various conformational states of the kinase. In this assay, CDK2 is modified by attaching an Alexa Fluor® donor/acceptor dye pair split between a relatively stable region of the kinase and the mobile activation loopses Fig. . In the ely Fig. –8. Howevold Fig. . This ch5 rather than 4 due to its stronger affinity observed in the FRET experiments in the presence of cyclin A. Because traditional activity assays where the CDK2/cyclin complex is preformed do not demonstrate inhibition with our allosteric CDK2 inhibitors, we preincubated p-CDK2 with our allosteric inhibitor before adding varying amounts of cyclin A. At a 1:1 p-CDK2:cyclin molar ratio, the IC50 of 5 is ~8-fold more potent than at a 1:10 ratio and >100-fold more potent than at a 1:100 ratio, showing a negative cooperative relationship with cyclin  but also has a 10 + -fold slower dissociation rate compared to the allosteric inhibitors tested 34, making it possible to observe binding of our compounds to free, noncomplexed CDK2. Compound 5 is cell permeable, as shown by the intracellular engagement and thermal stabilization of CDK2 (∆Tagg = 8.3 °C). No stabilization of CDK1 is observed (∆Tagg = 0.4 °C), suggesting that 5 is selective for CDK2 over the very similar CDK1 in this cellular context ext Fig. .Fig. 4TaD (SSA) = 78 ± 16 nM for CDK2 and a KD (SSA) = 1.8 ± 0.2 µM for CDK1 (KD = mean ± standard deviation from two independent repeats)30. In contrast, we find compound 5 does not bind CDK1 while it binds CDK2 with a KD (SSA) = 15 nM and KD (SSA) = 17 nM . We confirmed the validity of our CDK1 protein by testing dinaciclib, which binds CDK1 similarly to what was discovered previously with a KD (SSA) = 1.2 µM and KD (SSA) = 1.8 µM . Therefore, both the CETSA and SPR data suggest that allosteric inhibitor 5 has exquisite selectivity for CDK2 over CDK1. In addition, a kinome screen of compound 5 did not show >80% inhibition of any kinase at a 10 µM concentration, and only 4% of the 371 kinases tested showed >50% inhibition  show significantly more abnormal RAD51 staining at or near the telomeres in synapsed regions compared to control treated explants, indicating arrested and unrepaired DNA double strand breaks . In addition, more meiotic cells show abnormal CDK2 staining on chromosomes within the nuclei in the presence of 5 (50% of samples) compared to control samples (17%), indicating disruption of CDK2 meiotic function via improper localization of CDK2 to telomeres, as the microscopic CDK2 signal fades into the cytoplasmic background but robust expression of CDK2 can still be seen by Western blot  in CDK2, a residue conserved across kinases. An alternative explanation may be the divergent energy landscapes of inactive kinases. Prior work suggests that despite the structural similarity between CDK2 and CDK1, the energy landscapes of these kinases without cyclin bound differ greatly compared to their respective cyclin-bound states30. Because we optimized our ligands to bind an inactive, non-cyclin bound state of CDK2, we observe selectivity in the Jurkat cell CETSA and SPR experiments, where our compounds bind CDK2 but not CDK1. While the ligand-bound inactive states of CDK2 and CDK1 are likely nearly identical due to the structural similarity of these kinases, the binding of these ligands may be biased toward CDK2 over CDK1 due to CDK2’s propensity to sample this inactive state or an enhanced flexibility to accommodate allosteric ligand binding, although the precise mechanism remains to be determined. This latter explanation would be consistent with the large degree of conformational sampling observed for inactive, monomeric CDK229. In addition, it has been inferred that inactive CDK1 is more stable than inactive CDK2 and binds cyclin A less readily as a result41. The exquisite selectivity of this series beyond CDK1 is further supported by the kinome screen where compound 5 did not significantly inhibit any kinase >80% and only inhibited a handful of kinases >50% at the high concentration of 10 µM.One of the most striking findings of this series is the marked selectivity for CDK2 over CDK1, a protein with 65% overall sequence identity and 89% sequence similarity in the ATP-site where most CDK2 inhibitors bind29. This is confirmed by the structural FRET assay, where the KD of compound 5 toward CDK2 weakens as more cyclin A is added, and by the kinase activity assay, where the IC50 shows less potency as the p-CDK2:cyclin molar ratio decreases. Furthermore, CETSA data with 5 shows stabilization of CDK2 in Jurkat cells, where cyclins are not overexpressed, but not in OVCAR-3 cells, where cyclin E1 drives cell proliferation and survival. While our compounds are not cytotoxic in cyclin overexpressing cancer contexts, disruption of the CDK2/cyclin PPI remains a viable anticancer strategy. Recent work has shown that the complex natural product homoharringtonine (HHT) disrupts the PPI between CDK2 and cyclin A to act as an efficacious anticancer agent, although the allosteric binding mode of HHT has not been experimentally confirmed outside of computational models and its selectivity amongst kinases not explored42. In addition, disrupting the CDK2/cyclin interaction may be less toxic than traditional ATP-site inhibitors that sequester cyclin via their positive cooperativity with cyclin binding18. Further development of our allosteric inhibitors might confer an advantage in this regard compared to other promising compounds, such as recently developed CDK2/cyclin E1 inhibitor PF-07104091, a selective ATP-site inhibitor targeting the cyclin-bound activated state of the kinase43. Future iterations of our allosteric inhibitors with improved affinity and slower off-rates may very well be selective and efficacious in inhibiting a CDK2/cyclin-driven disease state. Similar success was recently found with allosteric EGFR inhibitors optimized to overcome undesirable EGFR homodimerization, analogous to our goal of overcoming the strength of CDK2/cyclin heterodimerization by increasing the interaction strength between our allosteric inhibitor and CDK25. In addition, the observed compatibility of our allosteric inhibitors with type I inhibitors (but not ATP) demonstrates the potential for higher efficacy if type I and our type III CDK2 inhibitors were administered in combination.Whereas most previously developed ATP-site inhibitors are often positively cooperative with cyclin and bind the CDK2/cyclin complex, our allosteric inhibitors are negatively cooperative with cyclin5 potently affects meiotic CDK2 function, most likely by interfering with SPY1 binding, although the exact mechanism of meiotic disruption by 5 is yet to be confirmed. Prior work has shown that when the scaffolding and catalytic functions of the CDK2/SPY1 complex are disrupted, prophase I is arrested17. Our inhibitors similarly arrest prophase I, as evidenced by the significantly increased localization of double-strand break repair protein RAD51 on synapses. In addition, spermatocytes from testis explants treated with 5 show more abnormal CDK2 localization compared to the control while retention of SPY1 localization to telomeric ends. While our compounds arrest meiosis, their potential as effective but safe non-hormonal contraceptive agents requires further exploration. If our hypothesis that these allosteric inhibitors selectively disrupt the meiotic CDK2/SPY1 PPI while leaving the mitotic CDK2/cyclin PPI intact is true, they would be a rare example of PPI inhibitors that derive selectivity by exploiting the different affinities of protein binding partners engaging at the same binding site. Aside from the impact of this work on CDK2 inhibitor development, we hope as the conformational landscapes of other kinases are further elucidated and more allosteric pockets are discovered, this work can serve as a model for allosteric inhibitor development to target other therapeutically important kinases.Compound 1–5 were determined to be >95% pure by qNMR45 and described further in the Supplementary Information.Dinaciclib was purchased from Cayman Chemicals with >98% purity and roscovitine from Selleck Chemicals with >99% purity. The production and purity assessment (>95%) of GST-CDK1 was made possible via NIH HHSN27520180007I. The purity of compounds The Animal Care and Use Proposal (ACUP) ACUP Number: 2019 -2508 has been reviewed and approved by the University of Kansas Institutional Animal Care and Use Committee.E. coli BL21 DE3 RIPL cells. Cells were grown at 37 °C for 3 h to OD600 = 0.6, then grown further until OD600 = 0.9 at 16 °C, induced with 0.5 mM IPTG and allowed to grow further overnight. Cells were harvested by centrifugation at 6000 × g for 20 min, the lysate was subjected to GST-affinity chromatography (GE Life Sciences), the GST tag was removed by PreScission protease, and cleaved CDK2 was subjected to a second GST-affinity chromatography step as described previously46. Flow-through fractions containing CDK2 were combined, concentrated and further purified by size exclusion chromatography using a Superdex 75 26/60 column (GE Life Sciences) in 50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 0.01 mM ADP, 2 mM DTT. For ITC experiments, purified CDK2 was concentrated to 50–100 µM and flash frozen in appropriate ITC volume aliquots for –80 °C storage.The gene encoding human CDK2 (1-298) was custom-synthesized (GenScript), subcloned into pGEX6P1 vector providing an N-terminal GST-tag and expressed in 29. Briefly, to generate CDK2 phosphorylated on T160 (p-CDK2), a 6His-TEV-CDK2 construct (pCDFduet) was coexpressed with yeast CAK (pGEX) overnight in Terrific broth at 18 °C. Cells were harvested by centrifugation at 5000 × g for 20 min and lysed by homogenization (Avestin Emulsiflex C3). Lysates were cleared by centrifugation at 20,000 × g for 1 h, subjected to Ni purification (GE HisTrap HP), and p-CDK2 was eluted with 1× PBS, 500 mM imidazole, 10% glycerol. Imidazole was removed by desalting the sample into 1× PBS, 10% glycerol (GE HiPrep 26/10), and the 6His tag was cleaved overnight with TEV protease. The protein was further purified by size exclusion chromatography (GE Superdex S75 10/300 GL), flash frozen and stored at –80 °C.Expression and purification of p-CDK2 and cyclin A have been described previously2, 10% glycerol using a 0–500 mM imidazole gradient. Cyclin A was further purified by size exclusion chromatography (GE Superdex 200 16/600) into 50 mM Tris pH 8.25, 100 mM MgCl2, 5 mM 2-mercaptoethanol.Bovine cyclin A  was expressed overnight in BL21(DE3)pLysS in Terrific broth at 20 °C. Cells were lysed and lysates cleared as above, and the protein was isolated through Ni purification into 50 mM Tris pH 8.25, 300 mM NaCl, 100 mM MgCl1 complex was obtained by co-crystallization (15% (v/v) Jeffamine ED2001, 50 mM HEPES, pH 7.5, 4.5 mg/mL CDK2, 5 mM 1) at 19 °C. The structures of CDK2 with compound 1 and dinaciclib, compound 1 and roscovitine, MgATP, and compound 2 were obtained by in-diffusion of apo CDK2 crystals with the ligands (1–5 mM) at 19 °C for 24 h (10%(v/v) PEG 3350, 50 mM HEPES, pH 7.5, 10% (v/v) ethylene glycol, 4.5 mg/mL CDK2).Purified CDK2 was buffer exchanged into 100 mM Na/K phosphate, pH 6.2, 2 mM DTT using PD10 columns (GE Life Sciences) and concentrated to 9 mg/mL. The CDK2/compound 1 or Mg-ATP were collected on a Rigaku Micro-Max 007-HF X-ray generator, equipped with a CCD Saturn 944 system located in the Chemical Biology Core of the Moffitt Cancer Center. Data of CDK2 liganded with compound 2, compound 1 and dinaciclib, compound 1 and roscovitine were collected at the GM/CA beamline  at Argonne National Laboratory. All data were collected at 93 K, processed and scaled with XDS47. The structures were solved by molecular replacement using PDB 4KD1 as the start model in PHASER48. Ligand geometrical restraints were generated with Phenix Elbow49. All structures were refined with Phenix50, and model building was performed with Coot51. Data collection and refinement statistics are shown in Supplementary Table X-ray diffraction data of CDK2 liganded with compound The NMR spectra and HRMS data for the compounds described below are shown in Supplementary Figs. nAr followed by hydrolysis to yield anthranilic acids 1–5: Commercial methyl 2-bromo or 2-fluoro-5-(nitro) or 5-(trifluoromethyl)benzoates (0.30 mmol) were dissolved in DMF (5 mL) with 1.1 equivalents of commercial tryptamine derivatives  and sodium bicarbonate (3-4 equiv). An exception was that for 3, the synthesis of the nitrile tryptamine precursor was accomplished using a previously described method52. The reaction temperature employed was room temperature for compound 1 and 70 °C for compounds 2–5. The solution was stirred for 12 h. After, 1 M HCl (aq) was added until pH <7 the solution was extracted into DCM (2×). The DCM solvent was removed via rotary evaporation and the crude product was dissolved in MeOH to proceed to the hydrolysis step without purification. Water was added to the MeOH mixture until a 1:1 ratio of solvents was reached. A solution of 5 M NaOH (aq) (1 mL) was added, and the hydrolysis proceeded at 50 °C for 12 h and was monitored by TLC for completion. The mixture was cooled to room temperature and the solvent was removed by rotary evaporation. 1 M HCl (aq) was added until pH < 4 and then the solution was extracted into EtOAc (2×). The EtOAc layer was dried with MgSO4 and loaded onto Celite. Subsequent purification was accomplished by silica gel flash column chromatography with a gradient of EtOAc in hexanes of 0–100% over 7 min, eluting around 60% EtOAc as the free carboxylic acid. The solvent was removed by rotary evaporation and the acid was isolated as the final product.General method of SH-Indol-3-yl)ethyl)amino)−5-nitrobenzoic Acid (1). 78% yield. Yellow solid. m.p. 216–218 °C. 1H NMR  δ 13.35 , 10.90 , 8.85 , 8.63 , 8.16 , 7.60 , 7.35 , 7.25 , 7.08 , 6.99 , 6.93 , 3.64 , 3.07 . 13C NMR  δ 168.6, 154.5, 136.3, 134.5, 129.5, 128.6, 127.0, 123.3, 121.1, 118.4, 118.3, 111.7, 111.4, 110.9, 109.0, 43.0, 24.3. HRMS (m/z): [M]- calcd. for C17H15N3O4, 324.0990; found, 324.0985.2-((2-(1H-Indol-3-yl)ethyl)amino)−5-(trifluoromethyl)benzoic Acid (2). 33% yield. Light yellow solid. m.p. 201–202 °C. 1H NMR  δ 13.05 , 10.88 , 8.34 , 8.02 , 7.63 , 7.59 , 7.35 , 7.23 , 7.08 , 7.01–6.92 , 3.55 , 3.05 . 13C NMR  δ 168.8, 152.6, 136.2, 130.6, 128.7, 126.9, 124.6 , 123.0, 120.9, 118.18, 118.18, 113.8 , 111.8, 111.3, 111.0, 109.1, 42.6, 24.2. HRMS (m/z): [M]- calcd. for C18H15F3N2O2, 347.1013; found, 347.1006.2-((2-(1H-indol-3-yl)ethyl)amino)−5-(trifluoromethyl)benzoic Acid (3). 9% yield over 5 steps52. Colorless solid. m.p. 190–191 °C. 1H NMR  δ 13.13–13.08 , 11.50 , 8.37 , 8.01 , 7.85 , 7.78 , 7.63 , 7.57 , 7.32 , 6.96 , 3.56 , 3.07 . 13C NMR  δ 169.0, 152.7, 135.0, 130.7, 130.2, 128.9, 128.0, 124.8 , 121.0, 120.8, 119.6, 116.3, 114.0 , 112.5, 112.0, 109.3, 102.3, 42.7, 24.0. HRMS (m/z): [M]- calcd. for C19H14F3N3O2, 372.0965; found, 372.0959.2-((2-(6-Cyano-1H-indol-3-yl)ethyl)amino)−5-(trifluoromethyl)benzoic Acid (4). 70% yield. Colorless solid. m.p. 207–210 °C. 1H NMR  δ 13.09 , 11.03 , 8.33 , 8.01 , 7.67–7.57 , 7.39 , 7.29 , 6.99 , 6.96 , 3.57–3.53 , 3.03 . 13C NMR  δ 168.9, 152.7, 136.6, 130.7, 128.8, 125.9, 125.8, 124.7 , 124.3, 119.8, 118.6, 113.9 , 111.9, 111.6, 111.0, 109.2, 42.7, 24.2. HRMS (m/z): [M]- calcd. for C18H14ClF3N2O2, 381.0623; found, 381.0637.2-((2-(6-Chloro-1H-indol-3-yl)ethyl)amino)−5-(trifluoromethyl)benzoic Acid (5). 44% yield. Colorless solid. m.p. 213–214 °C. 1H NMR  δ 13.10 , 11.04 , 8.33 , 8.01 , 7.63 , 7.60 – 7.51 , 7.28 , 7.11 , 6.96 , 3.57–3.52 , 3.03 . 13C NMR  δ 169.0, 152.7, 137.1, 130.7, 128.9, 126.2, 125.7, 124.3, 123.9, 121.2, 120.2, 113.9, 113.8, 111.9, 111.7, 109.3, 42.7, 24.2. HRMS (m/z): [M]- calcd. for C18H14BrF3N2O2, 425.0118; found, 425.0133.2-, with 5% DMSO added after buffer exchange into the protein solution and from a 1:20 concentrated DMSO stock for the ligand solution. CDK2 concentration was determined using a NanoDrop™ spectrophotometer and calculated using absorption at 280 nm . Into a 96-well plate for automatic injection for the MicroCal iTC200 , a final volume of 400 µL protein solution for the sample cell and 200 µL inhibitor solution for the injecting syringe were placed into the appropriate wells. The final inhibitor concentration in the syringe was 500–1000 µM depending on compound solubility. For experiments with compound 2 in the presence of ATP-site inhibitors roscovitine or dinaciclib, 100 μM ATP-site inhibitor was present in both the syringe and sample cell. For compounds 3–5, to get more accurate data on the ITC with appropriate c values53, the CDK2 concentration was lowered ~10-fold to 5 µM and the syringe concentration lowered to 75 µM. The ITC experiments were conducted at 25 °C and 750 rpm stirring, with a discarded initial 0.4 µL injection and 20 subsequent 4 µL injections with 180 s between injections. A one-site binding model was used to fit the ITC data after adjusting the baseline to account for slight buffer mismatch between the cell and syringe samples.CDK2 was thawed at room temperature and the buffer was exchanged using Zeba™ spin desalting columns. The final ITC buffer was 1× PBS, 10 mM MgCl30. A Biacore S200 (Cytiva) at 20 °C and CM5 chips were used for binding analysis. Multi-cycle runs were used for small molecule binding analysis, whereas a single-cycle run was used to measure cyclin binding. The buffer used to prepare the protein samples was 20 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 0.01% Tween 20, pH = 7.4). For runs with small molecules, an additional 1% DMSO was added for solubility. GST capture kit conditions  were used to capture anti-GST antibody on both the sample and reference cells . Both surfaces had high affinity sites capped with an additional 3 min of GST flowed over  followed by regeneration . On the subtractive reference surface, GST was immobilized . On the sample surface, GST-tagged CDK1 or GST-tagged CDK2 (with the GST cleavage stepped skipped during the purification process) was immobilized in a similar fashion . For GST-tagged unphosphorylated CDK1, a filtration through a FisherbrandTM syringe filter: PTFE Membrane  was performed to lessen aggregation.The SPR method used was largely adapted from a published method for CDK1 and CDK2 SPR studiesD at the highest and <0.1*KD at the lowest. To evaluate the binding of 5 against both CDK1 and CDK2, the concentrations employed were based on its KD for CDK2, as 5 did not appear to bind CDK1. For 1, the highest concentration used was 25 µM due to nonspecific binding to the chip surface at higher concentrations. Zero concentrations were performed at the beginning and end of the dose response as a baseline and to ensure internal reproducibility. An additional maximum concentration of compound was injected after the second zero concentration as an additional internal control. Lastly, a five-point DMSO solvent correction was performed for each compound before analysis with 0.6%, 0.8%, 1.0%, 1.2%, and 1.4% DMSO in buffer. To determine the KD and kinetic binding parameters of each compound against their respective protein, the Biacore S200 Evaluation Software (Cytiva) using affinity fit via the steady-state approximation (SSA), and when possible kinetic fit (koff/kon), was used.For small molecule binding analysis, two startup cycles of running buffer were carried out on both surfaces after protein immobilization. Depending on observed binding kinetics, a 60–180 s association time, and a 180–360 s dissociation time at 30 µL/min for each concentration of a particular compound over both surfaces was injected. An eight-point dose response was conducted with increasing concentrations for each compound with concentrations that were >10*KD. For both small molecule and cyclin affinity experiments, two independent replicates were conducted.For protein-protein affinity analysis with cyclin, the same buffer was used but without DMSO. A single-cycle run with increasing concentrations of cyclin A was used to determine the affinity of the CDK2/cyclin A interaction due to the slow off-rate of cyclin binding. After protein immobilization, one startup run of buffer was flowed over both the reference and sample surface to equilibrate the system. In an eight-point dose response, each concentration of cyclin (0–100 nM) had a 180 s association time and 180 s dissociation time. A kinetic fit was used to determine the K29. Briefly, CDK2 (C118A C177S A93C R157C) was labeled with FRET donor and acceptor dyes , purified by gel filtration, mixed with varying concentrations of bovine cyclin A and dispensed into a 384-well plate (Greiner Bio-One) into which 1 μL inhibitor had been pre-dispensed. The final concentration of CDK2 in the FRET assays was 5 nM in 1× PBS pH 7.4, 10 mM MgCl2, 5 mM DTT, 0.5 mg/mL bovine gamma globulins and 0.02 % Tween 20. Plates were incubated for 30 min, and measurements were performed on a fluorescence plate reader (Fluorescence Innovations). Emission peaks were spectrally unmixed54, and the ratios of the donor and acceptor peaks were used to calculate the apparent KD for each inhibitor.The CDK2 FRET assay between cyclin and small molecules has been described previously5 (10 µM) and Alexa 488 dye (5 nM) were dissolved in water (0.1% DMSO) and tested on the Horiba Fluoromax-4. The emission spectra of both compounds were obtained upon excitation at the 473 nm wavelength used for FRET. In addition, the emission spectrum of compound 5 was obtained upon excitation at 350 nm to examine its fluorescence from the indole core.For data generated in Supplementary Fig. 5) were measured using the ADP-Glo Kinase Assay (Promega) in a luminescence plate reader (Tecan Infinite M1000 PRO). Triplicate reactions were carried out in ADP Quest Assay buffer . Assays were performed with 5 nM phosphorylated CDK2, 200 μM ATP (Promega), 100 μM PKTPPKAKKL substrate (GenScript), and varying concentrations of cyclin A . Phosphorylated CDK2 was pre-incubated with inhibitor at 20 °C for 15 min. Reactions were initiated by simultaneously adding ATP and cyclin A and incubated at 20 °C for 15 min. Reactions were then quenched with ADP-Glo reagent following the manufacturer’s instruction. Luminescence was measured using a 1 s integration time and normalized to the no-inhibitor 5% DMSO controls. Kinase activity was determined by globally fitting relative luminescence data from all three cyclin A concentrations to a quadratic binding model using non-linear regression in GraphPad Prism 9. For fitting, CDK2 concentration was constrained to 5 nM with data sets from each p-CDK2:cyclin A ratio globally constrained to share the lower baseline parameter. IC50 values reported represent the 95% confidence intervals obtained from the fits (n = 3) using the symmetrical likelihood method in GraphPad Prism 9.Kinase activity of phosphorylated CDK2 with different cyclin A concentrations and varying amounts of inhibitor (dinaciclib or 5). Peptide substrate (100 µM) and cyclin A (5 nM) were added together to initiate the reaction and incubated at 20 °C for 30 min. Following the incubation, the reaction was quenched, and its activity measured and analyzed as above.For analogous assays with different concentrations of ATP, a similar setup was employed. Phosphorylated CDK2 (5 nM) was incubated at 20 °C for 15 min with either 83, 250, or 750 µM ATP and varying amounts of inhibitor  and 1% penicillin-streptomycin (Corning), and OVCAR-3 with 20% fetal bovine serum and 1% penicillin-streptomycin. The CETSA was performed following a reported protocol55. Briefly, in two separate T75 flasks for each cell line, Jurkat cells were grown to ~3 million/mL in 10 mL medium and OVCAR-3 cells to ~1 million/mL in 10 mL medium. Compound 5 was added to one flask for each cell line with a final concentration of 20 µM and DMSO (0.5%) was added to the other respective flasks. Cells were incubated with the indicated treatments for 1 h (Jurkat) or 2 h (OVCAR-3) at 37 °C, then collected through centrifugation (Jurkat) or trypsinization (Gibco) followed by centrifugation (OVCAR-3). The cell pellets were then resuspended in 1 mL PBS supplemented with complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche). For each treatment group, cells were aliquoted into ten different PCR tubes with 100 µL of cell suspension in each tube (~3 million cells per tube for Jurkat and ~1 million cells per tube for OVCAR-3). Cells in each tube were heated to their designated temperatures (40–67 °C) for 3 min in a thermal cycler, and then the tubes were incubated at room temperature for another 3 min before snap-freeze of the samples in liquid nitrogen. Cells were fully lysed by two more cycles of freeze-thaw. The lysates were cleared by centrifugation, and an equivalent amount (12 µL) of cell lysate from each sample was electrophoresed on a 4–12% NuPAGE gradient gel (Invitrogen) and transferred onto low fluorescent polyvinylidene difluoride membranes (Bio-Rad). Immunoblotting was performed with anti-CDK2 , anti-CDK1  and anti-β-actin  antibodies followed by secondary goat-anti-mouse  and goat-anti-mouse  Alexa680 antibodies. When using horseradish peroxidase-conjugated anti-mouse antibody for CDK2 and CDK1, West Femto Maximum Sensitivity Substrate (Thermo Scientific) was added to the membranes before imaging on an Odyssey Fc Imaging system (Li-Cor). Band intensity was quantified with ImageJ 1.52a, and data were then fitted to obtain apparent Tagg values using the Boltzmann Sigmoid equation within GraphPad Prism (v. 9.1.1) software. Densitometry data in the melting curves are mean ± SEM from three biological replicates.Jurkat and OVCAR-3 cell lines were purchased from the American Type Culture Collection. Both Jurkat and OVCAR-3 cells were cultured in RPMI 1640 (Corning) medium at 37 °C in a humidified chamber under 5% COg for 10 min at 4 °C. Cell debris was removed and the total protein concentration normalized by a BCA assay . The proteins were electrophoresed on a 4–12% Bis-Tris NuPage gradient gel (Invitrogen) and transferred to a low fluorescent polyvinylidene difluoride membrane (Bio-Rad). Immunoblots were conducted with anti-cyclin E1 , anti-CDK2 , and anti-β-actin  followed by secondary horseradish peroxidase-conjugated-goat-anti-mouse  and Alexa680 conjugated-goat-anti-mouse  antibodies. When using horseradish peroxidase-conjugated secondary antibody, West Femto Maximum Sensitivity Substrate (Thermo Scientific) was added to the membranes before imaging on an Odyssey Fc Imaging system (Li-Cor). Band intensity was quantified with ImageJ 1.53a and plotted in GraphPad Prism 9.3.0. Experiments were performed four times with two different cell amounts (0.5 and 1 million cells); data were aggregated to yield 8 replicates. Unpaired two-sided student’s t test without adjustments was conducted to determine significance in GraphPad Prism.For both Jurkat and OVCAR3 cell lines, 0.5 and 1 million cells were lysed in 100 mL RIPA buffer (Thermo Scientific) containing PhosSTOP phosphatase inhibitor cocktail (Roche) and complete protease inhibitor cocktail (Roche). Lysate was centrifugated at 11,000 × 5 (100 µM) in DMEM/F12. Following incubation, meiotic chromosome spreads were prepared as described previously56. Briefly, samples were placed in a hypotonic extraction buffer  for 10 min. Samples were then moved to a 100 mM sucrose solution (pH 8.2) and spermatocytes were detached by pipetting. Drops of the cell suspension were then placed onto a coverslip that had previously been soaked in a 1% paraformaldehyde (Sigma-Aldrich) and 0.15% Triton X-100 (Sigma-Aldrich) solution. Coverslips were dried at room temperature overnight and either used immediately for staining or stored at –80 °C.For spermatocyte chromosome spreads, nine 4-month-old adult WT C57BL/6 J mouse testes were dissected and incubated at 37 °C for 6 h in either Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12  or compound 17 (1:1) and anti-SYCP1 polyclonal antibody , or anti-RAD51 polyclonal antibody  and anti-SYCP-3 monoclonal antibody  overnight at 4 °C. Fluorescence was developed using Alexa Fluor 488 and 564 conjugated secondary antibodies . A Nikon Eclipse TiE inverted microscope with A1R-SHR confocal was used for imaging. RAD51 localization was quantitively assessed by determining the number of positive staining sites on the chromosome and telomeres per cell for 6 samples per group. An unpaired two-tail t-test with p < 0.05 was used to determine significance. CDK2 and SPY1 staining was qualitatively assessed through abnormal or normal scoring for 17 control samples and 6 samples treated with compound 5, where an abnormal score was given if 3+ telomeres within the sample did not stain positive for CDK2 or SPY1. The same anti-CDK2 antibody was used for Western blot with anti-β actin antibody  as an internal control.For immunofluorescent staining of chromosome spreads, slides were washed with PBS (Thermo Scientific) and 0.015% Triton X-100, blocked with 5% bovine serum albumin (Sigma-Aldrich) in PBS at room temperature for 1 h, and then incubated with anti-CDK2 monoclonal antibody  or anti-SPY1 R55A antibodyFurther information on research design is available in the Supplementary InformationReporting Summary


Introduction: The plant Patrinia villosa Juss. (PV) has long been used as a medicinal herb for treating intestinal disorders. Pharmacological activities such as anti-oxidation, anti-inflammation, and anti-cancer effects of compounds isolated from PV have been reported, but these bioactive compounds were not derived from PV water extract (PVW). Therefore, in the present study, we aimed to identify the active component(s) of PVW which exhibit inhibitory activities in colon cancer cells viability and migration.Methods: Human colon cancer HCT116 cells were treated with the isolated compounds of PVW and then subjected to MTT and transwell migration assays.Results: Our results showed that an active compound in PVW, 8,9-didehydro-7-hydroxydolichodial (DHD) inhibited cell viability of HCT116 cells, with IC50 value at 6.1 ± 2.2 μM. Interestingly, DHD was not detected in the herbal material of PV. Further investigation revealed that DHD is in fact a heat-generated compound derived from a natural compound present in PV, namely valerosidate. Valerosidate also reduced cell viability in HCT116 cells, with IC50 value at 22.2 ± 1.1 μM. Moreover, both DHD (2.75 μM) and valerosidate (10.81 μM) suppressed cell migration in HCT116 cells, with inhibitory rates at 74.8% and 74.6%, respectively. In addition, western blot results showed that DHD (5.5 μM) could significantly increase p53 expression by 34.8% and PTEN expression by 13.9%, while valerosidate (21.6 μM) could increase expressions of p53 and PTEN by 26.1% and 34.6%, respectively in HCT116 cells after 48 h treatment.Discussion: Taken together, this is the first report that a naturally-occurring valerosidate present in PV could actually transform to DHD by thermal hydrolysis, and both compounds exhibited inhibitory effects on cell viability and migration in HCT116 cells via increasing the expressions of tumor suppressors (p53 and PTEN). Our findings demonstrated that valerosidate is present in raw herb PV but not in PVW, while DHD is present in PVW rather than in raw herb PV. This difference in chemical profiles of raw herb and boiled water extract of PV may affect the anti-cancer activity, and hence further investigations are warranted. Patrinia villosa Juss. (PV) as a medicinal herb has been used over thousands of years in China . Therefore, in the present study, we aimed to identify the active component(s) of PVW which exhibit inhibitory activities in colon cancer cells viability and migration. The findings would provide scientific evidences for supporting the clinical use of PVW in colon cancer.For anti-cancer studies, the ethanol extract of PV has been proven with inhibitory effects on tumor growth in cervical cancer in mice, in which the inhibitory rates were 49.2% and 54.2% at 10 g/kg and 15 g/kg, respectively . Moreove254 (TLC) plates . MS-grade methanol (MeOH) and acetonitrile (ACN) were obtained from RCI Labscan, Ltd. . Formic acid was purchased from Thermo scientific, Inc. . Ultrapure water was prepared with a Milli-Q system .Two different ultra-performance liquid chromatography-mass spectrometers were used: Agilent 1290 Infinity UHPLC system coupled to an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight Mass Spectrometer with Dual Electrospray Ionization Source ; and Waters ACQUITY UPLC I-Class PLUS System coupled to a Waters SQ Detector 2 with Electrospray Ionization Source . The Nuclear Magnetic Resonance (NMR) spectra were performed using a Bruker AVANCE-500 , and the chemical shifts were referenced to tetramethylsilane (TMS). Column chromatography (CC) was run on silica gel 60 , and Sephadex LH-20 . Thin layer chromatography (TLC) analysis was carried out using Silica gel 60 FDried herb of PV  was purchased from Zisun Hong Kong Limited (China) and was morphologically identified by Dr. David Tai-Wai Lau of Shiu-Ying Hu Herbarium, The Chinese University of Hong Kong. A voucher specimen (No. 3704) was deposited at the museum of Institute of Chinese Medicine at The Chinese University of Hong Kong.Dried herb of PV (400 g) were soaked in distilled water at room temperature for 1 h, then boiled at 100°C for 1 h. After cooling, the extract solution was filtered using filter paper, and then added with the same volume of distilled water to extract once more .On the other hand, the dried herb of PV (600 g) were also extracted with 75% ethanol under reflux twice  0.1% formic acid in deionized water; and (B) 0.1% formic acid in acetonitrile, with gradient: 0–2 min, 5% B; 2–2.5 min, 5%–13% B; 2.5–9 min 13% B; 9–10 min 13%–100% B. The column was flushed with 100% mobile phase B for 2 min and re-equilibrium for another 2.5 min after each injection. Data were collected between 0–9 min. As the curtain and collision gas, 10 L/min of high-purity nitrogen was utilized. The temperature of the dehydrating gas was set to 350°C, and the pressure of the nebulizer was set to 35 psi. At a discharge voltage of 4000 V, spectra were captured in negative ion mode. The mass scan range was between 100–1,000 m/z. Agilent MassHunter Workstation Qualitative Analysis Software  was used to analyze the data. Valerosidate was determined at 485.1998 m/z [M + Na]+ and DHD was determined at 163.0754 m/z [M-H2O + H]+.UPLC-QTOF analysis was used for qualitatively detecting DHD and valerosidate in the extract of PV. The analysis was conducted using an Agilent 1290 UHPLC coupled to 6530 Accurate-Mass Quadrupole Time-of-Flight Mass Spectrometer with Dual Electrospray Ionization Source . The column used was Agilent ZORBAX Eclipse Plus C® BEH C18 column . Gradient elution was utilized in the chromatographic separation method under the following conditions: column temperature, 30°C; injection volume, 1 μL; flow rate, 0.5 mL/min. Mobile phases A and B were 0.1% v/v formic acid (FA) in water and 0.1% v/v FA in acetonitrile, respectively. Elution in UPLC used the following linear gradient: 0–2 min, 5% B; 2–2.5 min, 5%–13% B; 2.5–6.5 min, 13% B; 6.5–7 min, 13%–100% B; 7–9 min, 100% B; and 9–11 min, 5% B. The mass spectrometric parameters were set as below: the ion source was electrospray ionization (ESI); the mass spectra were recorded in positive mode; nitrogen nebulizing gas was used for desolvation at a flow rate of 700 L h−1 at 400°C; the capillary voltage was 3000 V; the temperature of the ionization source was 150°C; and the cone voltage was 50 V. Data were acquired in single ion recording (SIR) mode with unit resolution. Data were generated and analyzed on the Masslynx v 4.2 software.UPLC-MS was used for quantitative analysis of DHD and valerosidate present in the extract of PV. The equipment used was a Waters ACQUITY UPLC I-Class PLUS System consisting of a binary high-pressure pump with vacuum degasser, a single quadrupole mass detector (SQD2), an autosampler and the Acquity UPLC2 in an incubator , the 96-well plate was centrifuged at 2000 g for 5 min. Absorbance of the supernatant was measured at 540 nm using a μQuant microplate spectrophotometer . The percentage of hemolysis was calculated using the formula as below:Hemolytic activity was determined using mouse red blood cells with serial concentrations of each tested compounds. Whole blood was collected from Balb/c mice under anesthesia and the mice were sacrificed by cervical dislocation as described in the previous study . The aniWhere O.D. is optical density.2.Human colon cells HCT116 and the human fibroblast cells Hs27 were purchased from ATCC . HCT116 cells were grown in McCoy’s 5A medium supplemented 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. Hs27 cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. All cell culture media and supplements were obtained from Thermo Fisher Scientific . All cells were incubated in an incubator  at 37°C and 5% CO3 cells per well were seeded and incubated overnight in a 96-well microplate. The cells were then incubated for 48 h with 100 μL of PV extracts or compounds at indicated concentrations. Then, MTT solution (30 μL of 5 mg/mL) was added to each well, and the cells were incubated for an additional 4 h. MTT crystals were dissolved in dimethyl sulfoxide, and the absorbance at 540 nm (OD540) was measured using a μQuant microplate spectrophotometer . The half maximal cytotoxic concentration (CC50) was the concentration of a compound that can kill 50% of the cell viability on normal cells (Hs27 in the present study). The half maximal inhibitory concentration (IC50) is the concentration of a compound that can inhibit 50% of the cell viability on cancer cells (HCT116 in the present study). The CC50 and IC50 were determined by plotting a dose-response curve, where the concentration of the compound was plotted against the percentage of surviving cells. To calculate the CC50 and IC50 value, the dose-response curve was analyzed to determine the concentration of the compound that produces a 50% reduction in cell viability. This is typically performed using a mathematical model such as a sigmoidal dose-response curve or a linear regression analysis -2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 5×10analysis . The cely-axis) was plotted against the log drug concentration using GraphPad Prism v 8.0 software, and the dose-response curve was obtained to determine the CC50 or IC50 value. The selectivity index (SI) was calculated, with formula SI = 50% cytotoxic concentration (CC50)/50% inhibitory concentration (IC50).Then, the cell viability  after washing, fixing, and reacting cells with anti-BrdU reagent.The cell proliferation was determined using the 5-bromo-2′-deoxyuridine  assay according to the manufacturer’s instructions. Briefly, 5 × 104 cells in serum-free medium with or without tested compounds were added to the upper chamber of a transwell , while 500 μL of a chemoattractant medium containing 10% FBS was added to the lower chamber. After 24 h of incubation, the cells were fixated for 3 min with methanol and stained for 5 min with hematoxylin. The cells on the top surface of the membrane were removed using a cotton swab, and the residual adherent cells were photographed using an Olympus IX-71 microscope equipped with a digital camera . Using Image J software, the number of migrated cells was determined, which reflected their ability to migrate.The cell migration on colon cancer cells was evaluated using transwell migration method. In brief, 100 μL of 5×10HCT116 cells were inoculated overnight in 6-well plates. The cells were then exposed to test compounds for 48 h. After that, the cells were lysed on ice with a lysis buffer . The concentration of protein was measured using a BCA kit . 30–50 μg of protein were deposited onto 10% SDS-PAGE gels before being transferred to polyvinylidene fluoride (PVDF) membranes. An hour was spent blocking membranes with 5% non-fat milk in Tris-buffered saline Tween 20 (TBST). The blots were incubated overnight at 4°C with primary antibodies , including rabbit antibodies p53 and PTEN , and mouse antibodies β-actin  as listed in post-hoc Dunnett’s multiple comparison test, p values < 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism v 8.0 software .Data from the cell culture experiments were expressed as mean ± standard deviation (S.D.). Differences among groups were tested using one-way ANOVA, followed by 50 value of PVW was 806.0 ± 8.7 μg/mL, which was lower than that of PVE  in HCT116 cells  in order to extract the active compounds . This ap16 cells , indicat50 at 198.4 ± 10.9 μg/mL) exhibited stronger inhibitory activity on cell viability in HCT116 cells than residual extract  (50 at 8.95 ± 1.3 μg/mL) was much stronger than that of the combined fraction  in HCT116 cells  and residual extract. The effects of PVB and residual extract on cell viability was compared using MTT assay. Results showed that PVB (IC0 μg/mL) , indicat16 cells , suggest10H12O3 based on HR-ESI-MS at m/z 163.0754 [M- H2O + H]+ . 1H-NMR  δH 9.93 , 9.48 , 5.94 , 3.92 , 4.71 , 2.20 , 1.95 , 1.89 . 13C{1H}-NMR  δC 195.7 (C-3), 190.3 (C-1), 166.7 (C-8), 152.8 (C-4), 138.8 (C-9), 133.9 (C-11), 80.0 (C-7), 40.6 (C-5), 40.3 (C-6), 11.6 (C-10). The compound was identified by 1H and 13C-NMR analysis  . Besides, DHD at 500 µM showed hemolytic activity  . Furtherigration . As showSince DHD was shown to be an active compound in PVW, we then tried to measure its content in the PV raw herb. Since ethanol is a commonly used solvent to extract naturally occurring components from herbal medicines in chemical analysis , we trie21H34O11 based on HR-ESI-MS at m/z 485.1998 [M + Na]+ . 1H-NMR  δH 6.12 , 6.28 , 2.99 , 1.94 , 3.70 , 2.20 , 1.31 , 4.04 , 4.21, 2.16 , 2.04 , 0.92 , 4.25 , 3.14 , 3.31 , 3.21 , 3.22 , 3.61 , 3.82 . 13C{1H}-NMR  δC 91.9 (C-1), 139.5 (C-3), 117.0 (C-4), 32.1 (C-5), 38.1 (C-6), 80.9 (C-7), 82.3 (C-8), 48.3 (C-9), 22.9 (C-10), 70.0 (C-11), 173.2 (C-1′), 44.3 (C-2′), 26.8 (C-3′), 22.7 , 103.6 (C-1″), 75.2 (C-2″), 78.1 (C-3″), 71.7 (C-4″), 77.9 (C-5″), 62.8 (C-6″). The structure and stereochemical assignments of this natural iridoid glycoside were also confirmed by HMBC  and ROESY .Valerosidate  was isolOur UPLC-MS results showed that valerosidate was only detected in either maceration method  or refluxed in 75% ethanol extract, which is at low heat (below 80 °C) condition . In addi50 value of 22.2 ± 1.1 μM for 48 h treatment, while it exhibited mild inhibitory activity on cell viability in Hs27 cells . The SI index was 3.21, which was higher than that of DHD, indicating valerosidate has selective inhibitory activities in cancer cells than DHD (p < 0.01)  . Besides < 0.01) . Hemolyt < 0.01) . Hemolys < 0.01) . TherefoSince both DHD and valerosidate exhibited cell viability and anti-migratory effects on HCT116 cells, their regulatory effects on cancer-related proteins expressions were further explored. P53 and PTEN are crucial tumor suppressors in colorectal cancer . In otheIn order to develop DHD or valerosidate as anti-cancer agents for colon cancer, further preclinical experiments and clinical trials need to be conducted. For example, tumor-bearing animal studies will be required to confirm the efficacy of both compounds in colon cancer. Besides, the pharmacokinetic study and drug safety of these two compounds should also be performed and evaluated.This study has proven that DHD in PVW is in fact a heat-generated product of valerosidate present in the raw herb PV. This is the first report that a naturally-occurring valerosidate present in PV could actually transform to DHD by thermal hydrolysis, and both compounds exhibited potent inhibitory effects on cells viability and migration in colon cancer HCT116 cells. Besides, both DHD and valerosidate could increase the expressions of tumor suppressor proteins p53 and PTEN. Our findings demonstrated that different ways of preparing the water extract of PV (by maceration or boiling) may result in differences in the chemical constituents, which may in turn affect the anti-cancer activity, and hence further investigations are warranted. This study raises an important point for further consideration: how the raw herb PV should be prepared for patients in order to achieve better anti-cancer effect in colon cancer, pending further clinical investigations.


CO,old and kCO,new), where Mn-HbpaBr (1) had the greatest kCO by both methods . Carbon monoxide release was also evaluated using the myoglobin assay, indicating the release of 1.248 to 1.827 carbon monoxides upon light irradiation.Carbonyl compounds are widely explored in medicinal inorganic chemistry and have drawn attention due to their signaling functions in homeostasis. Carbon-monoxide-releasing molecules (CORMs) were developed with the purpose of keeping the CO inactive until its release in the intracellular environment, considering its biological relevance. However, for therapeutic applications, the mechanisms of photorelease and which electronic and structural variations influence its rates must be fully understood. In this work, four ligands containing a pyridine, a secondary amine, and a phenolic group with different substituents were used to prepare new Mn(I) carbonyl compounds. Structural and physicochemical characterization of these complexes was carried out and confirmed the proposed structures. X-ray diffractometry structures obtained for the four organometallic compounds revealed that the substituents in the phenolic ring promote only negligible distortions in their geometry. Furthermore, UV-Vis and IR kinetics showed the direct dependence of the electron-withdrawing or donating ability of the substituent group, indicating an influence of the phenol ring on the CO release mechanism. These differences in properties were also supported by theoretical studies at the DFT, TD-DFT, and bonding situation analyses (EDA-NOCV). Two methods were used to determine the CO release constants (k Such considerable variation, especially on the asymmetric stretches, is a result of the ligand’s nature, indicating that TPYOH has lower π-acidity compared to compounds (1)–(4) from this work, resulting in an increase in the Mn–CO π-backbonding energy.The Ford group (2019)  develope1)–(4) in solution. The compounds were analyzed through UV-Vis, cyclic voltammetry, square wave voltammetry –(4) are presented in Electronic spectra of compounds –(4) lack the necessary π-acidity coming from high electronic delocalization, making them UV active.Since the development of the first photoCORM, much effort has been put into the development of visible and near-infrared photoactivated compounds. The CO-releasing mechanism depends on the photoexcitation on the MLCT region, which weakens M–C bonds and promotes CO release. It has been established that this bathochromic shift on similar manganese(I) compounds can be achieved through the combined effect of the π-donor character of the ancillary ligand and the electronic delocalization of the bidentate ligand ,20,30. CThe absorption band at around 323 nm is not affected by the electronic influence of the substituent group, which indicates that it may not be associated with the phenolic ring. Hence, TD-DFT calculations were performed to identify the molecular orbitals involved in that electronic transition . Those b1)–(4) were studied through cyclic voltammetry (CV) and square wave voltammetry (SWV)  . Cyclic ry (SWV)  reveal an sphere ,32.CO stretching bands in IR are identical between the compounds, like MLCT electronic transitions have the same wavelength in UV-Vis. If the phenolic rings were coordinated with the manganese(I) center, the substituent group would have a direct influence over the electronic transitions and stretching bands.Oxidation potentials share the same tendency with UV-Vis electronic transitions below 295 nm. The necessary potential to reach the anodic peak decreases as the electron-donating ability of the substituent group increases, indicating that the phenolic ring could have an influence over the electron density of the metal center. On the other hand, solid-state characterization and UV-Vis spectroscopy demonstrate that the phenolic ring does not influence the metal center’s electron density, once ν4) has the least energetic electronic transition, just like it has the lower oxidation potential in CV. So, this behavior could be associated with the stability of Mn(II) species formed in this process.UV-Vis bands below 295 nm, which are associated with the phenolic ring orbitals, have a specific tendency, where (4) the most thermodynamically favored during Mn(I) oxidation.We hypothesize that, after the Mn(I) oxidation, carbonyl ligands could be released from the coordination sphere, making the phenolic ring susceptible to coordinate with the electron-deficient Mn(II) center. In this case, the π-donating ability of the substituent group would influence the stability of the formed species, which would make compound (1) deviates from this tendency. Although bromide is characterized as an electron-withdrawing substituent, it can donate electron density through π orbitals, which results in a similar behavior to that of compound (3).Once again, compound –(4) (summarized in White and collaborators (2021)  develope2) at 0.972 V, as a reduction process is observable for (4) at −0.081 V. Both processes can be associated with the organic moiety, given that the phenol ring has its own redox behavior +), a fragmentation at 242.18 m/z, and another fragmentation at 456.00 m/z. In compound (4), the base peak appears at 382.96 m/z ([Mn(HbpaOMe)(CO)3]+), and two other fragmentations of lower intensity at 472.00 m/z and 763.07 m/z. The 16 m/z difference between the 456 m/z in compound (3) and the 472 m/z in compound (4) indicates that identical species are formed in this fragmentation.Compound (1H NMR spectra of compounds (1)–(4) in deuterated acetone are shown in Besides the presence of the methylene hydrogens’ signals, the aromatic hydrogens from the pyridine and the phenol group were present and the integration of the signals corresponded with the number of aromatic hydrogens. The NH signal was also observed in all cases above 9.0 ppm, with minimal shift between the complexes, while the OH signal presented greater variation, once the substituent groups on the phenol ring had a direct impact on the electronic shielding of the hydroxyl group.2) and the isomers of compound (4) observed in X-ray diffraction, the relative difference in the final Gibbs free energy was 0.11 kcal mol−1 and 0.02 kcal mol−1 for (2) and (4), respectively. In the solid-state and solution characterizations as well, they showed no significant variation.To perform the computational study, the structures obtained through X-ray were used as the starting point, considering the obtained isomers. Initially, the structures were optimized in a vacuum and the analytical frequencies were obtained, showing a good correspondence between the calculated and experimental results, validating the parameters used in the calculations . In Figu2) and isomers of compound (4) do not show any substantial difference. The molecular orbitals HOMO-1 of all compounds have the same profile, with contributions from the zp and yp orbitals of the bromide, z2d and xyd of the Mn(I) and a small contribution from the π orbitals of the carbonyls. The HOMO orbitals from the isomers (4) have the electronic density on the uncoordinated phenol. Compounds (1)–(3) have contributions from the xp and yp orbitals of the bromide, xd2−y2 and yzd of the Mn(I), and π orbitals of the CO. The LUMO orbitals are composed of the π orbitals of the pyridyl group coordinated to the manganese(I), as well as the LUMO+1 orbitals of compounds (2), (3), and (4), which, in turn, are composed of the π* orbitals of the pyridyl group. Differently, compound (1) has its LUMO + 1 centered on the phenolic group with a small contribution from the manganese(I) and carbonyls.A new optimization was carried out considering the solvent dichloromethane, used in the characterizations and CO photorelease, to obtain the electronic spectra and the frontier orbitals of the studied compounds . As obse1)–(4). It is interesting to note that compounds (2), (3), and (4) also show a contribution of a charge transfer between the HOMO − 1 orbitals located on the Br–Mn–CO to the LUMO + 2 orbitals located on the π* orbitals of the phenolic moiety, which does not occur in compound (1), where this orbital is the LUMO + 1. The transition located in the region of ≈327 nm is characteristic of an MLCT, being a transition from the HOMO → LUMO orbitals of compounds (1) to (3) and HOMO − 1 → LUMO of (4). At higher energies, a transition from the π orbitals of the phenolic moiety to the π* orbital is observed with a small contribution from the Mn–CO orbitals.Electronic spectra of all complexes were calculated considering a TD-DFT approach. trans to bromide, 2-CO is trans to pyridyl group, and 3-CO is trans to the amino group, as shown in int) for the 1-CO fragment is the most stable  compared to the 2-CO and 3-CO fragments , indicating that the 1-CO interacts more strongly with the metal center than the other two fragments. This is related to the fact that the bromide is trans to this fragment, as it is a σ-donor and a better donor than the pyridyl and amino groups. Consequently, it will donate a greater electron density to the metal center, making the trans Mn–CO bond more stable compared to the other two carbonyl groups. Comparing the 2-CO and 3-CO fragments shows that the one trans to the amino group is slightly more stable than the CO trans to the pyridyl group.Calculations regarding the energy decomposition analysis (EDA) were carried out to obtain information about the bonding strength between each carbonyl and the rest of the molecule, in addition to the effects of substituent groups used in the phenolic portion in this interaction. The results are summarized in Pauli), which showed a small variation between the three analyzed fragments . The stabilization of the Mn–CO interactions is composed of the attractive forces, which are the sum of the electrostatic interaction (ΔEelstat), the orbital interaction (ΔEoi), and the dispersion energy (ΔEdisp). On average, the ΔEelstat contribution is 54.7%, the ΔEoi is 44.2%, and the ΔEdisp is 1.1%, showing a variation of ±1.1%, ±1.0%, and ±0.1%, respectively. Therefore, the substituent groups present in the phenolic portion do not directly influence the energy values between the three carbonyl groups and the metal center. The difference between the values is directly influenced by the groups/atom present in the position trans to the carbonyl ligand, as can be observed for the values of ΔEPauli, ΔEelstat, and ΔEoi, which were higher (ΔEPauli) and lower (ΔEelestat and ΔEoi) for the fragment trans to the bromide (1-CO) compared to the 2-CO and 3-CO fragments trans to the Npy and Naliph, respectively.The destabilization of the Mn–CO interaction is represented by the Pauli repulsion (ΔEoi-σ and ΔEoi-π in 2a), 1-CO, 2-CO, and 3-CO, respectively. The other manganese carbonyl compounds showed the same behavior, as observed in 1) describes the formation of the σ-bond between the manganese and the carbonyl, with a charge transfer (Δq1) estimated at 0.66, 0.62, and 0.62 for 1-CO, 2-CO, and 3-CO fragments, respectively. The second and third density flow channels describe a π-bond interaction, with Δq2 = 0.56, 0.52, and 0.52 and Δq3 = 0.46, 0.48, and 0.47 for 1-CO, 2-CO, and 3-CO, respectively. The values of ΔEoi show that the energy of the π-bond for the 1-CO fragment is more stable compared to the 2-CO and 3-CO, a consequence of the trans effect, as the bromide will further strengthen the π-backbonding compared to the pyridyl and amino group. The main associated symmetrized fragment orbitals (SFO) that contribute to the formation of the three density flow channels are shown in s and p orbitals and manganese d orbitals.The orbital interactions can be separated into the contributions σ and π (ΔE1)–(4) in solution were performed through UV-Vis, IR spectroscopy, and molar conductivity measurements. To characterize the solvent effect on the manganese coordination sphere, studies were performed in non-coordinating (dichloromethane) and coordinating (acetonitrile) solvents.Stability tests of compounds , the energy band shift is evident, making an isosbestic point observable (CO) stretching bands, a shoulder of lower intensity can be observed at a 20 cm−1 higher energy. This indicates that there is a mixture between the original species, [Mn(κ2-Hbpa-R)(CO)3Br], and the solvent substituted species, [Mn(κ2-Hbpa-R)(CO)3(CH3CN)]+. IR measurements corroborate the idea that CO is not released from the coordination sphere after the bromide substitution.For compound (servable . The pre1)–(4) in acetonitrile and dichloromethane at 0 h and 24 h relative to the preparation of the solutions. Results corroborate UV-Vis and IR implications –(4) were performed qualitatively via IR spectroscopy and quantitatively via UV-Vis spectroscopy. In acetonitrile, the original species reach equilibrium states with solvent-substituted species, which could have a direct impact on the CO release mechanism. To characterize the effect of the phenolic ring, we opted to perform the studies on dichloromethane, which does not alter compound structures.After the characterization of the formed species in solution, CO release studies of compounds (em = 395 ± 5 nm) over the solutions, ν(CO) stretching bands gradually decreased until they completely disappeared.A qualitative analysis of CO release is presented in pseudo-Cv2 group point, which results in two stretching bands of A1 and B1 symmetry. The B1 stretching band usually appears at around 1850 cm−1. The absence of biscarbonyl stretching bands indicates that this species is unstable, and the other two carbonyl ligands should be quickly released from the coordination sphere, making this intermediate undetectable  species formed by (1) would be the least stable among all compounds, resulting in a faster release of the remaining carbonyl.Compound  → Mn(II) oxidation potentials are directly influenced by the substituent groups. The dependency of the basicity of the phenol ring, as the coordination of the hydroxyl group itself, could be associated with an oxidation of the manganese(I) center after the release of the first carbonyl group, resulting in the coordination of the phenolic ring in order to decrease the electron deficiency of the manganese(II) center, and the consequent release of the remaining carbonyl groups due to π-backbonding weakening. Evidence of the spontaneous oxidation of the metal center was found in similar works ,34,35.CO,old) have a direct association with the Hammett sigma parameters (σp). This is another indication that the phenol group could be coordinating the metal center after the CO release, once the greater the electron-withdrawing ability of the substituent group, the greater the CO release constant, because the coordination of the phenol group would directly influence the release of the other carbonyl ligands. The carbon monoxide releasing constants obtained from the pseudo-first-order kinetics (k1) remains as the compound with the highest kCO,new, while the other compounds deviate from the Hammett parameters. Compound (2) shows a lower kCO than expected, which could be associated with the behavior of the compound during the whole CO release process. On the other hand, a different tendency is observed when a new approach to determine CO release is used. It is observable that  and with the quantum yield (Φ), since the CO release occurs in the excited state. It is important to note that MLCT transitions appear as shoulders in the UV-Vis, which means that ε is more of a range than a precise value itself. Regarding the quantum yield, it could be observed that the direct association is valid only for (1), while the other compounds presented a deviation from the tendency, indicating the possibility of side reactions and different photoproducts.Furthermore, the k1)–(4) were diluted in PBS buffer solutions of hemoglobin in its reduced form (Mb). Upon exposure to light, the formation of MbCO was observed at 540 nm and 570 nm. The solutions were exposed to light for 210 min, until no further variation was observed. The spectral variation of myoglobin solutions is presented in CO release was also confirmed through the myoglobin interaction assay. Compounds  for 72 h and degassed by argon flux or freeze pump using the Schlenk technique. The solutions containing the organometallic compounds were kept in the dark and wrapped in aluminum foil whenever possible.−1 to 450 cm−1. UV-Vis spectra were recorded on a PerkinElmer Lambda 750 in quartz cuvettes, using spectroscopic-grade dichloromethane as solvent for the compounds. Molar conductivity of the organometallic compounds was performed on an MS Tecnopon mCA150 in spectroscopic-grade dichloromethane and acetonitrile. Cyclic voltammetry and square wave voltammetry were investigated on a BAS Epsilon potentiostat, using spectroscopic-grade dichloromethane as solvent under argon atmosphere. Tetrabutylammonium hexafluorophosphate (TBAPF6) with a concentration of 0.1 mol L−1 was used as supporting electrolyte, and the electrodes were Ag/Ag+ (reference) and platinum (work and auxiliary). The electrochemical potentials were referenced versus the ferrocene/ferrocenium (Fc/Fc+) redox pair. Elemental analysis was performed on a PerkinElmer 2400 Series II. ESI-MS positive ion mode measurements were performed on an Amazon spectrometer Ion Trap MS in spectroscopic-grade acetonitrile with an approximate concentration of 500 ppb and estimated flow of 180 μL min−1, with a capillary voltage of −400 V. 1H NMR spectra of the ligands were obtained on a Bruker AC-200 NMR spectrometer in CDCl3 solutions. For the organometallic compounds, 1H NMR spectra were carried out on a Bruker Ascend400 Varian FT-NMR 400 MHz using acetone-d6 (CD3)2CO as the solvent, at 25 °C. Chemical shifts were referenced to tetramethylsilane (TMS = 0.00 ppm), and J values are given in Hz. Stability of the complexes in solution was observed by UV-Vis and IR in spectroscopic dichloromethane and acetonitrile in the absence of light, where UV-Vis spectra were acquired at each hour for 24 h, and IR spectra were collected at the end of the experiment.Infrared spectra  were recorded on a PerkinElmer Spectrum 100 FT-IR in the range from 4000 cm2  . After reaching room temperature, the solution was refluxed for 12 h under argon atmosphere in the dark. The four compounds synthesized resulted in a yellow solution. The solution was then dried at 30 °C in a laboratory kiln, leading to the formation of yellow precipitates. The precipitates were then washed with cold hexane three times and recrystallized in a 1:1 CH2Cl2/hexane, resulting in yellow crystals.The photoCORMs  ν/cm−1 3400 (OH), 3222 (NH), 2025 (COsym), 1940 (COasym), 1907 (COasym), 1607–1495 (CC and CN), 1261 (CO), 771 (CHar); UV-Vis (CH2Cl2) λmax/nm (ε/L mol−1 cm−1) 284 (4127), 321sh (2128), 379sh (1600); ESI-MS m/z 430.86 ; Elemental Analysis Found: C, 42.23; H, 3.59; N, 5.70%. Calc. for C16H12Br2MnN2O4·1 CH2Cl2: C, 42.21; H, 3.63; N, 5.79%. 1H NMR δH/ppm 2CO; TMS): 3.90 , 4.10-4.26 , 4.31 , 4.78 , 6.96 , 7.37–7.67 , 7.93 , 8.99 , 9.65 (1HNH). Crystal data for (2) (M = 512.04 g mol−1): triclinic, space group P-1, a = 7.8714(7) Å, b = 8.5609(7) Å, c = 14.1438(12) Å, α = 97.177(2)°, β = 105.699(2)°, γ = 97.734(2)°, V = 896.10(13) Å3, Z = 2, Dcalc = 1.898 Mg/m3, 15003 reflections measured (1.517° ≤ 2Θ ≤ 30.577°), 5509 unique (Rint = 0.0258). The final R1 was 0.0279 (I > 2σ(I)), and wR2 was 0.0587 .Yellow crystals, 159.44 mg, 78%, 511.03 g mol−1; IR  ν/cm−1 3413 (OH), 3231 (NH), 2025 (COsym), 1930 (COasym), 1914 (COasym), 1610–1458 (CC and CN), 1260 (CO), 760 (CHar); UV-Vis (CH2Cl2) λmax/nm (ε/L mol−1 cm−1) 273 (6524), 323sh (3716), 379sh (2900); ESI-MS m/z 352.93 ; Elemental Analysis Found: C, 44.43; H, 3.33; N, 6.32%. Calc. For C16H13BrMnN2O4: C44.47; H, 3.03; N, 6.48%. 1H NMR δH/ppm 2CO; TMS): 4.07–4.15 , 4.26 , 4.76–4.82 , 6.91 , 6.99 , 7.27 , 7.40 , 7.50–7.54 , 7.92 , 8.98 , 9.25 (1HNH); Crystal data for (1) (M = 433.14 g mol−1): monoclinic, space group P21/n, a = 13.5543(8) Å, b = 9.7453(6) Å, c = 26.0146(16) Å, β = 93.5850(10)°, V = 3429.6(4) Å3, Z = 8, Dcalc = 1.678 Mg/m3, 38127 reflections measured (1.569° ≤ 2Θ ≤ 30.145°), 10119 unique (Rint = 0.0186). The final R1 was 0.0272 (I > 2σ(I)), and wR2 was 0.0624 .Yellow crystals, 153.83 mg, 89%, 432.13 g mol−1; IR  ν/cm−1 3420 (OH), 3224 (NH), 2027 (COsym), 1940 (COasym), 1907 (Coasym), 1608–1447 (CC and CN), 1261 (CO), 768 (CHar); UV-Vis (CH2Cl2) λmax/nm (ε/L mol−1 cm−1) 281 (6021), 323sh (3030), 379sh (2400); ESI-MS m/z 366.96 ; Elemental Analysis Found: C, 44.33; H, 3.64; N, 5.95%. Calc. for C17H15BrMnN2O4·0.8 H2O: C, 44.33; H, 3.63; N, 6.08%. 1H NMR δH/ppm 2CO; TMS): 2.27 , 4.0 , 4.02–4.14 , 4.23–4.30 , 4.75 , 4.82 , 6.87 , 7.07 , 7.20 , 7.49–7.54 , 7.92 , 8.91–9.00 . Crystal data for (3) (M = 447.17 g mol−1): triclinic, space group P-1, a = 7.7748(10) Å, b = 8.4882(11) Å, c = 14.1772(18) Å, α = 77.426(2)°, β = 75.239(2)°, γ = 84.486(2)°, V = 882.2(2) Å3, Z = 2, Dcalc = 1.683 Mg/m3, 20422 reflections measured (2.461° ≤ 2Θ ≤ 33.229°), 6734 unique (Rint = 0.0205). The final R1 was 0.0299 (I > 2σ(I)), and wR2 was 0.0735 .Yellow crystals, 162.40 mg, 91%, 446.16 g mol−1; IR  ν/cm−1 3434 (OH), 3222 (NH), 2025 (COsym), 1928 (COasym), 1911 (COasym), 1611–1434 (CC and CN), 1265 (C-O), 764 (C-Har); UV-Vis (CH2Cl2) λmax/nm (ε/L mol−1 cm−1) 295 (5926), 329sh (~2300), 379sh (~1800); ESI-MS m/z 382.96 ; Elemental Analysis Found: C, 42.23; H, 3.59; N, 5.70%. Calc. for C17H15BrMnN2O5·1.2 H2O: C, 42.21; H, 3.63; N, 5.79%. 1H NMR δH/ppm 2CO; TMS): 3.76 , 4.04–4.19 , 4.23–4.33 , 4.75 , 6.05 , 6.80–6.94 , 7.04 , 7.53 , 7.92 , 8.82 (1HNH), 8.98 . Crystal data for (4) (M = 463.17 g mol−1): triclinic, space group P-1, a = 7.5412(9) Å, b = 13.8743(16) Å, c = 18.566(2) Å, α = 79.549(2)°, β = 87.930(2)°, γ = 77.108(2)°, V = 1862.1(4) Å3, Z = 4, Dcalc = 1.652 Mg/m3, 40852 reflections measured (1.726° ≤ 2Θ ≤ 32.822°), 13728 unique (Rint = 0.0470). The final R1 was 0.0491 (I > 2σ(I)), and wR2 was 0.1187 .Yellow crystals, 164.52 mg, 89%, 462.16 g molN,O-donor set and p-substituted phenols were synthesized and fully characterized through structural and physicochemical methods. All complexes presented an octahedral geometry where the carbonyl ligands were coordinated in a facial arrangement.A series of mononuclear Mn(I) complexes containing bioinspired tridentate ligands with a A thorough description of the electronic structure of all complexes were carried out considering the DFT approach through which frontier orbitals were calculated, as well as the energy decomposition analysis through natural orbitals for chemical valence (EDA-NOCV), which allowed us to rationalize all the contributions of each moiety present  in the formed chemical bonds.- by solvent) when acetonitrile was employed. CO release rates were quantitatively determined through UV-Vis, in which complex [MnBr(HbpaBr)(CO)3] (1) presented the highest value (2.36 ± 0.11 × 10−3 s−1) and quantum yield (0.031 ± 0.0002). The CO release was also confirmed using myoglobin assays, with all complexes presenting similar results.The stability of these compounds was observed using UV-Vis and molar conductivity in dichloromethane, showing a partial ligand substitution (Br1)–(4) to assess their cytotoxicity.Finally, a common viable CO-release mechanism for all complexes was proposed, according to which, after light irradiation, a CO group was promptly released, followed by the metal center’s oxidation, suggesting the consequent coordination of the phenol group. Further studies in vitro will be performed with compounds (