Glioblastoma (GBM) is a highly malignantbrain tumor characterizedby a heterogeneous population of genetically unstable and highly infiltrativecells that are resistant to chemotherapy. Although substantial effortshave been invested in the field of anti-GBM drug discovery in thepast decade, success has primarily been confined to the preclinicallevel, and clinical studies have often been hampered due to efficacy-,selectivity-, or physicochemical property-related issues. Thus, expansionof the list of molecular targets coupled with a pragmatic design ofnew small-molecule inhibitors with central nervous system (CNS)-penetratingability is required to steer the wheels of anti-GBM drug discoveryendeavors. This Perspective presents various aspects of drug discovery. The comprehensively coveredsections include the recent medicinal chemistry campaigns embarkedupon to validate the potential of numerous enzymes/proteins/receptorsas therapeutic targets in GBM. Surgery alone is usuallyinsufficient to treat GBM, and complete surgical resection is notpossible because the whole tumor is challenging to remove withoutdamaging normal brain tissue. Considering the cytological heterogeneityof GBM, a commonly employed methodology known as optimal multi-modalitytreatment involves surgery flanked by chemotherapy and radiotherapy.Despite numerous efforts directed toward establishing optimum treatmentprograms, GBM patients generally show a poor prognosis and experiencetumor progression with high mortality and a median survival of only12–15 months.10Glioblastoma (GBM), definedas a grade IV astrocytoma, is a highlymalignant brain tumor12 The literature indicates that methylguanine-DNA methyl transferase(MGMT)-mediated innate resistance to TMZ (a first-line chemotherapeuticGBM drug) is the primary reason for the failure of this GBM treatment.20 GSCs represent a small subset of cells within a malignant tumor,known as cancer stem-like cells (CSCs), that demonstrate ability similarto that of normal stem cells and are more resistant to anti-cancertherapeutics than bulk tumor cells.22 These revelationsindicate that CSCs can survive after therapy and become an underlyingcause of tumor recurrence.20 In this context, a search for potential anti-cancer interventionsthat exert simultaneous disruption of GBM and brain tumor stem cellhomeostasis is needed. In addition to TMZ, bevacizumab is approvedby the U.S. FDA for the treatment of primary and recurrent GBM; however,the outcome of some studies demonstrates the failure of bevacizumabto prolong overall survival.23 Along withoverall survival failure, it was found that the administration ofbevacizumab led to the overexpression of the receptor tyrosine kinase(RTK) c-Met, thereby causing tumor relapse.25The notorious nature of GBM in thecontext of resistance to chemotherapyhas been a major obstacle during the development stages of efficacioustherapy for its treatment. Presently, the anti-GBM drug armory mainlyrelies on temozolomide (TMZ), an oral alkylating agent, as the first-linechemotherapeutic drug in GBM treatment. TMZ kills cancer cells viaguanine/adenine methylation-mediated DNA base pair mismatches andsubsequent DNA damage-induced reactive oxygen species (ROS) accumulation1A.11,12 reatment1B.13 How27 Additionally, because the central nervous system (CNS) is considereda region for active immunosurveillance, immunotherapy is alsobeing exhaustively explored as a potential strategy for GBM. However,the complex state of a patient’s immune dysfunction in GBMalso poses several challenges for immunotherapy.28In addition to the resistanceissue, the obstacles that must beapproached involve issues related to delivery to the brain becausethe existence of the blood–brain barrier (BBB) lowers the efficiencyof systemic drug delivery to the target tumor in the brain. Attemptsat using dose escalation of drugs to enhance their therapeutic efficiencyhave often culminated in increased toxicity to normal cells and haveelevated the risk of adverse effects. Many efforts toward optimizingdrug cocktails (combination therapy) to counter the high genetic heterogeneityof GBM in patients have also proven fruitless, and the limitationswere again attributed to the enhanced risk of adverse effects.32 However, there is an opportunityto assemble a compilation that mainly focuses on robust drug designstrategies employed by the medicinal chemist to furnish anti-GBM adducts.Thus, we embarked on the task of compiling a comprehensive reviewof GBM that primarily focuses on recently conducted medicinal chemistrycampaigns and briefly presents various aspects of drug discovery relatedto GBM . The scientific literature covered inthis Perspective indicates that kinases (phosphoinositide 3-kinases(PI3K),38 focal adhesion kinase (FAK),41 DYRK,42 and 3-phosphoinositide-dependent kinase 1 (PDK1)44) have been extensively targeted through the pragmatic design ofheterocyclic compounds . Notably, these research groupshave conducted a series of studies on kinase inhibitors as anti-GBMagents, and their efforts have led to the identification of potentadducts worthy of detailed investigation.53 Another target that has been reasonably utilized for the constructionof anti-GBM agents in the recent past is histone deacetylase (HDAC).61 The drug design strategies for HDAC inhibitors discussed in thisPerspective clearly depict the flexibility of the three-componentHDAC inhibitory model. Specifically, the structural alteration ofthe surface recognition part of the HDAC inhibitory pharmacophorehas been the main focus of the medicinal chemist to extract anti-GBMeffects through the inhibition of several HDAC isoforms. Several structure–activityrelationship (SAR) studies were performed to design inhibitors ofisocitrate dehydrogenase (IDH)65 as well as translocator protein (TSPO).70 Notably, optimized IDH and TSPO inhibitory scaffolds were exhaustivelyexamined for modification at various sites to attain a clear-cut understandingof the impact of such alterations on the activity. In light of thepromising outcomes, it is anticipated that these aforementioned endeavorsmight emerge as model studies to further numerous future pursuitson IDH and TSPO inhibitors as anti-GBM agents. Protein disulfide isomerase(PDI),72 tubulin,83 and hypoxia-inducible factor (HIF)85 have alsogarnered significant attention as potential targets and spurred researchersto furnish inhibitors in the pursuit of anti-GBM efficacy. Additionally,the researchers have capitalized on the concept of balanced modulationof two targets as well as the degradation of the targets to outwitthe notoriety of GBM cells. Studies discussed in this Perspectiveon dual MDM-2–TSPO inhibitors,86 dual HDAC1–LSD inhibitors,87 dualPDK1–aurora kinase inhibitors,88 dual RGD integrin–MDM protein inhibitors,89 and others92 are expected to pave the way for the initiation ofsimilar programs to expand the size of the anti-GBM pipeline. Notably,at the preliminary and preclinical levels, medicinal chemists haveexplored synthetic adducts as well as natural product libraries tofurnish new chemical architectures for the treatment of GBM.100 Imaging tools and chemical probes for GBM  havealso been generated. Moreover, many preliminary studies merely focusingon the cellular effects of the new scaffolds have also been includedin this work.150 Although mechanistic studies were not performed, the study resultsappear to be promising, and the pinpointed potent scaffolds can besubjects of future investigation. Importantly, the literature coveredin this Perspective validates the potential of numerous enzymes/proteins/receptorsas therapeutic targets in GBM. Several interesting scaffold constructionapproaches, such as fragment stitching, scaffold installation, regiovariation,bioisosteric replacement, structure simplification, structure rigidification,and molecular hybridization leveraged by the medicinal chemist todesign new small-molecule inhibitors with anti-GBM potential, alongwith SAR, bioactivity, molecular modeling, and other studies conductedto elucidate the mechanisms, are comprehensively discussed in thiscompilation. These approaches have culminated in generating a voluminouslibrary of CNS-penetrating scaffolds capable of tackling the shield(BBB), and we are quite hopeful that some of the candidates mightemerge as potential anti-GBM agents for the clinic.Previous literature has indicated that, despite demonstratingstrikingefficacy and selectivity, the progress of small-molecule inhibitorsas anti-GBM agents has often been halted by their poor BBB permeabilityas well as drug resistance issues. To overcome the above obstacles,the prudent design of libraries of mechanistically diverse small-moleculeinhibitors comprising lipophilic structural components appears tobe a practical step forward. Accordingly, an increasing number ofstudies have investigated this direction for construction of new assemblagesas therapeutic options in GBM. Notably, the experience and intuitionof the medicinal chemist play key roles in the design of chemicaltools with ideal physicochemical properties to emerge as CNS drugs.Recently, some reviews have been published that presented therapeuticstrategies and recent advances in GBM therapy.2The presence of several barriers, includingthe BBB, blood–brain–tumorbarrier (BBTB), intra-brain tissue diffusion, and drug resistance,has often hindered the drug discovery and delivery process for GBM.Prior to commencement of the task of designing new anti-GBM scaffolds,a thorough understanding of these factors is imperative to amplifythe translational rate of preclinical studies to clinical explorations.These physiological barriers restrict the entry of drugs into thebrain and make GBM treatment more challenging.2.1166 Additionally,anatomical features such as the presence of multi-drug-resistant proteinsfurther restrict the entry of drugs into the brain. These anatomicalfeatures prevent the accumulation of administered drug molecules insidethe brain, resulting in the failure of the administered drugs to achievethe desired pharmacological impact.168 Additionally,when drugs are transferred through transcellular diffusion, they aremetabolized by several metabolic enzymes. For example, decarboxylationof 3-alanine to dopamine occurs during transit.170The CNS is vascularized with uniquely architecturedblood vesselsknown as the BBB. These blood vessels strictly regulate the movementof ions, molecules, and cells between the blood and the brain. TheBBB is designed for proper neuronal function and to protect neuraltissue from toxins and pathogens. The BBB is a major obstacle forefficient chemotherapy because it reduces the effective penetrationof drugs into the brain and spinal cord due to its highly selectivepermeability for oxygen and nutrients.More than 98% of small drug molecules cannot cross the BBB.The BBB halts more than 95% of drug molecules at the drug developmentstage. Thus, targeted drug delivery to the brain is not a prime focusarea for most pharmaceutical giants.173Previous studies have alsorevealed that the BBB is a dynamic interfacethat keeps changing its morphology and physiology under certain pathologicalconditions. In the presence of such stringent barriers, GBM cellscan aggressively infiltrate the surrounding tissues and progress exponentially.Single GBM cells can aggressively develop tumors by infiltration intosurrounding tissues and eventually can breach the tight BBB followinga multi-step process. GBM cells migrate and accumulate around theexisting blood vessels. This causes displacement of the astrocyticend feet processes from vessels. The involvement of TGF-β2,caveolin-1, ROS, and pro-inflammatory peptides in the induction ofmatrix metalloproteinase (MMP) degradation of tight junctions contributessignificantly to the breach of GBM cells through the BBB.176 Additionally, a disrupted BBB leads to major clinical complicationssuch as vasogenic brain edema and a significant increase in intra-cranialpressure (leaky BBB).173Unfortunately, even a disrupted BBB does not allow the permeationof drug molecules to tumor cells because different inhibitory mechanisms,such as drug resistance, poor blood perfusion, and high intra-tumoralinterstitial pressure, are still active.2.2177The progression of GBM from low-grade tumorsto high-grade tumorsalters the structure, function, and organization of the BBB. Thistransformation of tumors leads to the invasion of surrounding healthybrain tissue, including BBB disruption, resulting in the formationof neoplastic lesions. These neoplastic lesions have a network ofnewly built blood vessels that is often referred to as the BBTB. Comparedwith the BBB, the BBTB is considered more permeable. However, theBBTB is still significantly less permeable than any other tumor neovasculaturedeveloped in any other organ of the body. Therefore, the BBTB is alsoa major challenge for brain drug delivery.178 Notably, the GBM neovasculatureand its heterogeneity determine the permeability of the drug. TheGBM neovasculature demonstrates variable vessel diameter and densityand can be classified into three different types: (i) continuous,non-fenestrated endothelial vasculature; (ii) continuous, fenestratedendothelial vasculature; and (iii) discontinuous endothelial vasculature.179 The neovessels commonly show abnormal endothelialhyperplasia, pinocytic vesicles, fenestration, and opening or lossof tight junctions between endothelial cells. Although the permeabilityof the BBTB is enhanced by these abnormalities, the cranial microenvironmentand specificity of glioma reduce the permeability, thereby hinderingthe delivery of most anti-tumor agents.182Collectively, GBM is associated with the formation of a highlyabnormal lymphatic vasculature and is the most vascularized amonghuman tumors.2.3182 High infiltration of GBM cellsinto the brain parenchyma or neighboring brain tissues is anotherchallenge. Because most drugs cover only a few millimeters aroundthe delivery site, a larger area must be targeted to counter the problemof infiltration.183 Under such conditions,targeting signaling events and regulatory pathways involved in themigration and invasion of GBM cells appears to be an effective approach.184Oncethey pass through the BBB, the drugs reach the cerebrospinalfluid (CSF) and brain extracellular space (ECS). From there, theyeventually reach the targeted lesion. The diffusion efficiency ofdrugs in the ECS is limited by several factors, including the structuraland physicochemical properties of the drugs and the physiologicalproperties of the ECS.2.4187 Several cellular factors enable CSCs to possess chemotherapy andradiation resistance, such as an increased DNA damage repair capacity,increased survival signaling, and upregulated ROS scavengers.191 Notably, TMZ resistance, a troubling issue, is primarily drivenby GSCs. Revelations in this context indicate that enriched populationsof stem-like CD133+ cells mediated via upregulation inDNA repair mechanisms are produced by radiation and chemotherapy regimens.Recently, studies have identified reliable GSC markers, includingCD133, CD44, CD15, CD70, S100A4, ALDH1A3, Nanog, SOX-2, and Nestin.Outcomes of fate mapping studies using genetic barcoding have indicatedthat chemotherapy leads to evolutionary selective pressure that causesthe expansion of drug-resistant GSCs. Although GSCs comprise a verylow percentage of cells in GBM tumors, their ability to regeneratetumor heterogeneity makes them a potential target for emerging anti-neoplastictherapeutic approaches.195CSCs are a sub-populationof cells within a tumor mass that reproduce tumors and drive malignantprogression after treatment. Strong experimental and clinical evidencesuggests that CSCs can resist ionizing radiation and chemotherapy.2.5197 Treatment strategies shouldbe designed to overcome the cell cycle dependence and lack of specificityof chemotherapeutic agents.178The potential of drug molecules to cross theBBB and treat GBM isaffected by several factors, including the physicochemical propertiesof the drug molecule, its pharmacokinetic (PK) profile, characteristicsof the drug delivery system (DDS), and the pathophysiological conditionof the patient. Most of the drug molecules used to treat GBM are non-specificagents that target actively dividing cells. Thus, these therapeuticagents not only kill cancerous cells but also destroy actively dividinghealthy cells, and physiological aberrations, including immunologicalsuppression, mental depression, and neurological degeneration, havebeen reported.199 It has been reported that tertiary nitrogen-bearing compounds  exhibit a higher degree of brainpermeation.200 As such, the partitioningof the drugs into membrane lipids occurs as neutral species and dependson the concentration of the neutral species and its lipophilic properties.Acids and bases that are too strong are usually precluded from BBBpenetration, such as carboxylic acids, which demonstrate difficultyin penetrating the CNS.201 Thus, the pKa limits for BBB penetration defined by Fischeret al.202 are between 4 and 10.Notably,the physiochemical properties of the pharmaceutical agent, such asthe size, flexibility, chemical conformation, ionization, and lipophilicityof the drug molecule, play critical roles in determining the abilityof the drugs to reach the targeted site in the brain. Generally, drugsthat are moderately lipophilic tend to cross the BBB through passivediffusion, while polar molecules act as better drug molecules if takenthrough active transport across the CNS. Key disclosures indicatethat CNS drugs (basic) exist in an equilibrium between their chargedand neutral states under physiological conditions or are amphiphilicif they also possess an acidic group. Additionally, brain permeationis favored by possessing a positive charge at pH 7–8.2,pKa = 8.4, RB = 4.5, and MW = 305.3 Da.204Arelative comparison of CNS with non-CNS drugs indicates thatdrugs belonging to the former category are smaller and more lipophilicand have fewer hydrogen-bond donors and lower polar surface area (PSA).The profile of a desirable CNS candidate depicts the following values:cLogP = 2.8, cLogD = 1.7, HBD = 1, TPSA = 44.8 Å153 Notably, the lack of an appropriate PK profileof both developmental and marketed drugs leads to failure in advanceddevelopment stages and market withdrawal.205 Additionally, substantial potency plus selectivity combined withthe ability to achieve target tissue concentrations above a certainthreshold value is desired to achieve the optimum therapeutic efficacyof a drug candidate. To attain the above-mentioned features, structuraloptimization of the chemical architectures has become an imperativetask of drug discovery campaigns, and the implementation of logicalstrategies by the medicinal chemist can favorably modulate the PKproperties of an agent.In addition to the above-mentioned, an appropriate PK profile, which plays a key role indefining the disposition of a drug candidate and ultimately its developmentas a suitable marketable drug candidate, is equally important.179 Thepotential of other therapeutic approaches, such as gene targeting184 and the use of aptamers as delivery agents,182 has also been explored. Despite the significantprogress in this field, the present scenario necessitates the introductionof potentially effective DDSs that specifically target GBM cells withoutaffecting healthy cells.Similarly, the selection of a suitabledosage form is also equallycritical. As mentioned previously, the physicochemical properties,such as the particle size, zeta potential, lipophilicity, permeability,and dissolution rate, of the drug delivery tool directly influencethe potential of drug molecules to cross the BBB. Interestingly, thesecharacteristics can be customized by selecting a suitable carriersystem and a suitable composition using suitable formulation methodology,modification of the surface chemistry, and grafting of the surfacewith specific ligands. In the past few years, extensive work has beenconducted in this area, and various approaches have been exploredto improve the specific biodistribution, surface characteristics,and targeting of anti-cancer drugs.207 Factors such as systemic enzymatic stability,mode of absorption, clearance rate, and site of administration arealso of considerable importance.Additionally, existing pathologicalconditions and drug affinitiesfor efflux mechanisms influence the pharmacological outcome of thedrug.3Significant explorationshave been conducted to identify immunotherapeuticand chemotherapeutic targets to treat GBM. This section presents abrief overview of potential targets for GBM 2 along w326 Notably, Asc1, Brn2,and Ngn2 (ABN) were found to be predominant transcription factorsthat abruptly reduced the growth of GBM cells in vitro and in vivo and promoted the conversion of GBMcells to non-divisible neurons.327 Recently,a study revealed the potential of small molecules to reprogram GBMcells. Lee et al. identified a cocktail of forskolin, ISX9, CHIR99021I-BET 151, and DAPT that successfully reprogrammed malignant cellsinto neurons.328 The involvement of smallmolecules in GBM cell reprogramming promotes the applicability ofsmall molecules and opens the door for medicinal chemists to designsynthetically relevant reprogrammable scaffolds for GBM.Apart from diverse chemotherapeutictargets, reprogramming of GBMcells has also emerged as a potential approach that promotes the differentiationof GBM cells to neuron-like cells through transcription factor-mediatedreprogramming.4in vitro and in vivo)revealed during the biological evaluation of the new anti-GBM constructs.Medicinal chemistshave exerted numerous efforts to capitalizeon the imperative revelations made by biologists regarding the involvementof factors/targets in the initiation and progression of glioma. Manyof the logically constructed assemblages are currently being investigatedin preliminary/preclinical explorations. This section covers the drugdesign strategies employed to furnish rationally assembled scaffolds,along with a discussion of the results of the cellular and enzymaticassays coupled with SAR studies, molecular modeling studies, and mechanisticinsights  or mTOR  are activated by RTKs to generate phosphatidylinositol-3,4,5-trisphosphate(PIP3).486 The subsequent activation ofPI3K activates the mTOR complex, namely, complex 1  is activated by PI3K signaling along with growthfactors through unknown processes, causing organization of the cytoskeleton,lipid metabolism, cell survival, and Akt kinase phosphorylation.490 The involvement of the PI3K/Akt/mTOR (PAM) pathway has been reportedin GBM patients, where various signaling proteins, such as the lossof function of tensin homolog (PTEN), affect the pathogenesis of GBMalong with PI3K.491 PTEN, a tumor suppressor gene,negatively regulates PIP3 levels and the PI3K/Akt pathway througha protein phosphatase that triggers mTOR activity, resulting in theproliferation and survival of the cells. Additionally, RTK/PI3K/Aktsignaling pathway activation results in the stabilization of HIF1α,which leads to the development of cancer.492The PI3K/mTOR signalingpathway is important for the survival, growth, motility, and metabolismof cells.33 The group utilized a previously reported dual PI3K/mTOR inhibitor(1) as a chemical probe to understand the binding modeusing different isoforms of PI3K. The bidentate hydrogen-bonding interactionof the triazine ring of 1 with Val882 and the hydrogen-bondinginteraction of the phenolic −OH with Asp841 and Tyr867 arenecessary for binding to the PI3Kγ pocket. Despite demonstratingsubstantial efficacy, poor PK properties and extensive metabolismof benzimidazole 1 were some of the shortcomings associatedwith its use, and this disclosure rendered the scope of structurallyrefining its chemical architecture to the authors. Given this clearunderstanding, a novel structure comprising a monocyclic or bicyclichinge binder linked to a central 2-aminopyridine core was designed.The SAR study was focused on establishing well-defined propertiesrequired to inhibit PI3Kα because of its involvement in GBM.Overall, the structural optimization as depicted in 2). The crystalstructure of 2 bound to PI3Kα also suggested thatthe compound displayed affinity toward the binding pocket and interactedwith the major amino acids Tyr867, Asp841, Ala805, and Lys802. Furthermore,an in vitro PK study of 2 was performed,and the results were intriguing because 2 demonstrateda mean residual time of 1.6 h, a clearance (CL) of 1.7 L/(h·kg),and a VSS value of 2.6 L/kg. Additionally,the hepatocyte growth factor (HGF)-stimulated PI3K signaling inhibitionability of 2 was assessed in a mouse liver pharmacodynamic(PD) assay where a lower dose of 25 mg/kg exhibited near-completetarget coverage for 8 h, while a higher dose of 75 mg/kg maintainedsufficient plasma concentrations for 24 h. The tumor growth inhibitionpotential of 2 was evaluated in a U-87MG xenograft modelin CD1 nude mice at oral doses of 3, 10, 25, and 75 mg/kg q.d. Additionally, 2 exerted a dose-dependent inhibition of tumor growth withED50 = 6.0 mg/kg. Furthermore, tumor stasis was achievedat 25 mg/kg q.d. At the lower dose, no tumor reduction was observed,while a higher dose reduced the tumor weight by 15% after dosing for14 days. Given the above-mentioned findings, a daily dose of 2 for at least 8 h per day over 14 days might attain tumorinhibition >60%. Collectively, the results culminated in identifying 2 as a selective and potent PI3Kα inhibitor requiringfurther optimization to emerge as a drug candidate.Considering the activation of the PAM signaling network inGBM, Smith et al. designed a novel series of potent and selectiveclass-I PI3K inhibitors that demonstrated striking tumor growth inhibitorypotential against the U-87MG human GBM cell line 2.33 The designed2.33 The 34 Initially, 2 was investigated comprehensively by administeringan intravenous dose to bile-duct-cannulated rats, and the drug wasquantified in excreta  for up to 24 h. Quantificationof the drug in urine, bile, and feces showed that only 2.5% of thedrug was excreted in its parent form, which suggested that metabolismwas the major clearance pathway. Therefore, metabolite studies wereperformed to identify the metabolites by incubating 2 with rat and human liver microsomes or hepatocytes. LC-MS analysisrevealed that most of the metabolites were formed due to oxidativemetabolism. Additionally, two metabolic pathways were identified atthe methoxypyridine and benzylic piperazine regions that led to metabolites 3 and 4. Based on these revelations, compoundswith improved PK properties needed to be developed. Thus, a seriesof compounds was synthesized by modifying the metabolic spots (vulnerablesites) of the structure. A total of 21 compounds were synthesizedand evaluated against the PI3Kα, PI3Kβ, PI3Kγ, PI3Kδ,and U-87MG human GBM cell lines. Among the synthesized compounds, 5 was the most active, with IC50 = 4 nM (PI3Kα),6 nM (PI3Kβ), 2 nM (PI3Kγ), 1 nM (PI3Kδ), and 4nM (U-87MG). Additionally, a rat and human liver microsomal (RLM andHLM) study was performed. Compound 5 displayed excellentresults with RLM and HLM values of 20 and 22 μL/min/mg, respectively.The SAR was evaluated for two different metabolic spots (oxidationregions) of the compound, as shown in 5 showed an attractive PK profile because it displayed the lowest in vivo clearance (0.4 L/kg/h), a high volume of distribution(1.7 L/kg), and a moderate mean residual time (3.9 h). Furthermore,a PD study of 5 was performed in a mouse liver PD model.Compound 5 was administered orally at doses of 3, 10,and 30 mg/kg, and HGF was administered after 6 h to activate PI3K-dependentAkt phosphorylation in the liver. The results revealed that 5 suppressed PI3K signaling in a dose-dependent manner, andthe plasma EC50 was 228 ng/mL. Tumor inhibition activitywas evaluated in a mouse U-87MG glioblastoma xenograft model in which 5 was administered at doses of 1, 3, and 10 mg/kg for 12 successivedays. After treatment, a significant reduction in tumor growth (approximately70%) was observed at a dose of 1 mg/kg q.d., and the ED50 was deduced to be 0.6 mg/kg. Overall, the study led to the identificationof a new PI3Kα inhibitor that was selected for further clinicalevaluation in the treatment of cancer and was named AMG 511.Later, the group continuedthis work and published a series ofcompounds with improved potency and PK properties 3.34 Init6)and dual PI3K/mTOR inhibitors GNE-493 (7) and GDC-0980(8)  along withimproved B-A/A-B (MDR1) B-A/A-B (Bcrp1) mouse CI, mouse t1/2, and mouse F% values  4.35 Amon% values 4. Furthe36 The group fused an anti-PDGFRβ aptamer with biodegradablepolymeric nanoparticles (PNPs) to deliver a promising chemotherapeuticagent, dactolisib  (11) is a potentdual PI3K-mTOR inhibitor under investigation for the treatment ofsolid tumors and was recently proven to be an efficacious radiosensitizerand chemosensitizer in a preclinical mouse GBM model. Despite itspromising activity profile, the poor water solubility of 11 affects its bioavailability, and a high dose is required to achievea therapeutic effect. Therefore, the water-in-oil-in-water double-emulsionsonication method was used to entrap the drug, followed by amino-terminatedconjugation of the anti-PDGFRβ aptamer Gint4.T to a COOH groupof the nanosystem. The resultant formulation of 11-PNPs-Gint4.Twas characterized by dynamic light scattering (DLS), where the diameterof the particles was 52 ± 1 nm, with a polydispersity index of0.169. The amount of aptamer conjugated to the PNPs was evaluatedby RT-qPCR analysis in which the concentration of Gint4.T and theconjugation efficiency were 1.4 nM and 5.4%, respectively, with anoverall 11-PNPs-Gint4.T concentration of 18.4 mg/mL.To check the targeting efficiency of the formulation, in vitro internalization studies were performed by fusing Gint4.T or scrambled(SCR) aptamers on U-87MG cells. Gint4.T aptamer-loaded formulationswith PNPs specifically targeted GBM cells and actively enhanced intracellularuptake. Furthermore, in vitro cytotoxicity studieswere performed against the GBM cell line, and 11-PNPs-Gint4.Tdisplayed 1000-fold higher cytotoxicity than free drug 11  5. Dactol drug 11 5. The Gi12) for anti-GBM activity.37 The structural alteration program led to theidentification of a potent compound bearing a 6-amino-4-methoxy substitutionat the benzimidazole ring (13 significantly inhibited the growth of tumors up to 81%at a dose of 50 mg/kg (i.p. injection) for 10 (q.d.) days; however,the solubility profile of the compound was unfavorable.In 2011, Rewcastle et al. published a SAR studyof a class 1 PI3Kinhibitor  score of 0.015. Additionally, 16 and 17 displayed significant tumor growthinhibition of >90% in the U-87MG brain xenograft model and showedacceptable safety profiles.In 2018, Lin et al. reported a series of 2-amino-4-methylquinazolinederivatives as potential PI3K inhibitors furnished through scaffoldhybridization and hopping strategies.ll lines 6B. Furth498 Tamura et al. revealed the involvement of phosphatase and tensinhomolog (PTEN) in the dephosphorylation of active FAK at Y397 in GBMcell lines.498 In the active and phosphorylatedstate, it increases the expression of CCND1/cyclin-D1 and decreasesthe levels of p21/CDKN1A cyclin-dependent kinase (CDK) inhibitor,causing enhanced proliferation of cells through accelerated transitionfrom the G1-S phase.499 Mamillapalli etal. found that PTEN negatively regulated the G1/S phase transitionby obstructing S-phase kinase-associated protein-2 expression (SKP2)and ultimately alleviating the levels of p27/CDKN1B.500 In GBM, loss of PTEN causes the activation of FAK and apoptoticresistance due to the absence of contact (cell–matrix). Accordingto a study conducted by Alza et al., PF-573228 (an FAK inhibitor)arrests cell proliferation, increases the size of cells, and diminishesneurosphere growth in GBM due to an increase in the levels of β-galactosidaseand p27/CDKN1B activity.501 The inhibitionof FAK also reduces p62/SQSTM-1 expression (autophagy cargo receptor),stimulating p27 transcriptional upregulation (senescent-like phenotype)and leading to proliferation arrest and cell death. Based on the evidence,in 2014, Dao et al. designed a novel series of imidazotriazine derivatives via structural modificationof the previously reported potent FAK inhibitory compound 18(PHM16), which showed striking anti-tumor activity . The binding poses showed that the compound fit well in the bindingpocket and interacted with the major amino acids of the binding pocket,Met499, Asp564, Leu567, and Ile428. Furthermore, the selected compoundswere evaluated for FAK autophosphorylation ability and growth inhibitionpotential toward U-87MG cell lines. All the tested compounds showedpromising anti-proliferative activity in the mentioned cell lines,with IC50 values in the low micromolar range. Additionally, 21 and 22 delayed the progression of the cellcycle and arrested the cell cycle at the G2/M phase in the U-87MGglioma cell line. Furthermore, 19–22 inhibited the cell matrix adhesion, migration, and invasion of U-87MGcells. Collectively, these findings underscore the magnificent activityprofile of the compounds against human GBM.39Focal adhesion kinase (FAK/PTK2)is a tyrosine kinase that is presentin the cytoplasm and is responsible for focal adhesions involvingthe dynamics of cellular migration by linking the actin cytoskeletonwith integrin. FAK regulates the survival, proliferation, migration,invasion, and microenvironment of tumor cells like angiogenesis.activity 7. In totactivity 7. Furthe40 In their study, 23, a previously reported FAK inhibitor, was employed as thelead compound, and two different series of compounds  demonstrated the ability of compound 28 to penetrate into cells and bind to DYRK1A. As such, compound 28 was endowed with excellent DYRK1A inhibitory activity (IC 50 = 43 nM) and appears to be a suitable chemical tool forfuture campaigns.42Reports investigatingthe upregulated expression of the dual specificitytyrosine phosphorylation regulated kinases (DYRK) in some malignanciesprompted a research group to employ a lead modification strategy anddesign a series of novel 7-azaindole derivatives as DYRK inhibitors9. All co503 PDK is essential to producing lactate from pyruvate and can be usedas a therapeutic target along with standard therapy. Various studieshave shown the association of PDK1 with the progression of cancer.The overexpression of PDK1 has been observed in gastric cancer dueto HIF-1α and the maintenance of melanoma cells.505 Additionally, the elevated expression of PDK1 has been observedin head and neck carcinoma and non-small-cell lung cancer and in specimensof human GBM, and silencing PDK1 produced significant anti-proliferativeand apoptotic effects on 5310 and U251 cells.507 Due to the role of PDK1 in theprogression of GBM, Sestito et al. revealed a series of 2-oxindolederivatives as putative PDK1 inhibitors.43 A total of 16 compounds were synthesized, where 30 wasthe most potent in the series, with IC50 = 112 nM  and 998 nM (at 25 μM),respectively, and induced differentiation among CSCs. The low efficacyof heptamethine cyanine dyes (HMCDs) toward brain cancer cells isan uphill battle. In order to confront the low efficacy, Choi et al.introduced a conjugate of Crizotinib and heptamethine cyanine dyeIR-786 which showed potential cytotoxicity against T141, T146, andT84 GBM cell lines.45 In an EdU cell proliferationassay, 32 displayed promising anti-proliferative activity,with IC50 = 4.7 nM. Interestingly, 32 showedthe synergism with TMZ which enhance the potency of compound by 4-fold.Overall, the introduction of 32 intensifies the applicabilityof heptamethine cyanine dyes (HMCDs) for the GBM. In 2018, Bertuzziet al. published an efficient synthesis of quinone-fused pyrazolesthrough 1,3-dipolar cycloadditions as prudent anti-GBM agents.46 Various substituted quinone-fused pyrazoleswere synthesized with a good yield and evaluated over a panel of U251,DBTRG, and U-87MG GBM cell lines. Among all, 33 displayedpotential anti-GBM activity, with IC50 = 2.5 μM.Further, the docking studies and biological studies revealed thatthe 33 possibly inhibits the PI3K/mTOR kinase which isa responsible cofactor of the cancer development. The chemical structuresof other potent kinase inhibitors with anti-glioma potential are alsopresented in 53PDK1-a Ser/Thr kinaseinhibits the formation of acetyl-CoA frompyruvate to stimulate the progression and formation of GBM.= 112 nM 10. Furth4.2508 The most noticeable epigenetic changes in tumorcells are hyperacetylation/hypomethylation of oncogenes andhypoacetylation/hypermethylation of tumor suppressor genes.509 Bezecny et al. reported a mutation in 60% ofpediatric glioma cases (Lys 27-to-methionine (K27M)) at one alleleof H3F3A and one of the two genes encoding histone H3 variant H3.3,signifying the role of modifications on histone and DNA in GBM throughtumor initiation, progression, and resistance to treatment.510 Under normal circumstances, histone proteinsare responsible for the modulation of chromatin structure/functionand the expression of genes. The modifications of histone tails afterthe translation process include acetylation, ubiquitination, phosphorylation,and methylation and regulate the remodeling of chromatin.512 Histone acetyl transferases (HATs) transfer acetyl moieties to lysineresidues, and HDACs remove them. HATs promote gene transcription andexpression, whereas HDACs suppress them and regulate gene expressionby directly interacting with transcription factors, such as protein53, E2f, activator and signal transducer of transcription 3 (Stat3),transcription factor IIE (TFIIE), nuclear factor kappa B (NF-κB),and retinoblastoma protein. In addition, HDACs deacetylate non-histoneproteins that are responsible for maintaining homeostasis in cells andbecome abnormal in tumor cells.514 Lucio-Eterovicet al. revealed that H3 histones are hyperacetylated in GBM and onthe progression of astrocytomas to GBM; however, class II and IV HDACswere not found to be expressed, indicating that class II and IV HDACsare amenable to the progression of astrocytoma to GBM.515 Moreover, the differential/dysregulated expressionof HDAC4, 6, and 8 has been associated with resistance to standardtreatment in GBM CSCs due to distorted signaling mechanisms, includingthe sonic hedgehog (SHH) pathway  and correlates with glioma progression.517GBM is the most lethaland malignant brain tumor (grade IV) due to the presence of CSCs ortumor-initiating cells, epigenetic mechanisms, and cellular pathways.519 Sathornsumetee et al. also reported increased expression of HDACs in GBM. HDAC inhibitors are used to re-establishthe balance of HAT to HDAC activity and sensitize tumor cells to HDACinhibitors as monotherapeutic agents and in combination with radiationtherapy. HDAC inhibitors are reported to be valuable in GBM therapyin preclinical phases, as they enhance tumor cell sensitivity to DNAalkylating chemotherapeutic agents through open chromatin conformationin tumor cells and help reverse abnormal genetic silencing in GBM,leading to the enhanced arrest of the cell cycle and apoptosis.520Furthermore, enhanced levels of class III (NAD-dependent)HDACs, SIRT1/2, have been reported in CSCs of GBM. SIRT1 knockdownenhances the radiosensitivity of GSCs and reduces tumor volume witha positive therapeutic outcome on CD133-positive GBM tumors.54 The implementation and execution of appropriateactions based on the above-mentioned concept led to the identificationof a series of compounds involving the stapling of the memantine core with the zinc bindinggroup via chemically diverse linkers. With this background, Nepaliet al. reported some memantine-based HDAC inhibitors as potentialanti-GBM agents. All the synthesized compounds were initially evaluatedfor anti-proliferative activity in the U-87MG glioma cell line. In vitro cytotoxicity studies led to the establishment ofa structure-cytotoxicity relationship, and several properties weregenerated that were critical in conferring cell growth inhibitoryeffects to the designed compounds. The N-benzyl linker used to tetherthe memantine skeleton with hydroxamic acid functionality was notfavorable in terms of inducing anti-glioma effects; however, the incorporationof a vinyl bond and long alkyl chain between the N-benzyl and zincbinding motifs was beneficial, and compounds bearing acrylamide moietiesshowed promising anti-proliferative effects. Among the synthesizedcompounds, 43–46 exhibited promisinganti-proliferative effects  cyclin B levels, indicating the apoptosis-promotingability of the adduct. Compound 45 was also found tobe active against TMZ-resistant glioma cells and inhibited the growthof TMZ-resistant U-87MG glioma cells in a dose-dependent manner. Toelucidate the mechanism responsible for these striking anti-gliomaeffects of 45, all the synthesized compounds were screenedagainst a panel of HDAC isoforms where the compounds displayed moderateinhibitory potential toward HDAC1, HDAC3, and HDAC8 isoforms in thelow micromolar range; however, they were substantially selective towardthe HDAC6 isoform. Notably, 45 demonstrated a strikinglyselective inhibitory potential toward HDAC6, with IC50 =5.42 nM. These results agreed with previous findings of the elevatedexpression of HDAC6 in GBM. Furthermore, to rationalize the experimentalstudies using computational studies, docking studies were performed,revealing that 45 displayed good binding affinity withthe HDAC6 isoform and interacted with major amino acids W496, H500,H611, F620, and H615 of the enzyme isoforms. The permeability potentialof 45 was also evaluated using a parallel artificialmembrane permeability assay. This assay rationalized the strategyof incorporating a stress-free bulky hydrocarbon, memantine, as asurface recognition part of the HDAC inhibitory model because 45 demonstrated remarkable CNS-penetrating ability with apermeability value of 33.9. Furthermore, the in vivo evaluation results revealed that treatment with hydroxamic acid 45 could prolong the survival of TMZ-resistant U-87MG-inoculatedorthotopic mice. In summary, 45 has an impressive anti-GBMprofile and warrants further investigation.Intrigued by the unsuccessful journeyof HDAC inhibitors in thecontext of clinical advancement in GBM, possibly due to a lack ofCNS-penetrating ability, Nepali et al. conceived that compensatingfor the enhanced hydrophilicity conferred by hydroxamic acid functionalityvia logical installation of CNS drugs (FDA-approved), as the surfacerecognition part of HDAC inhibitory pharmacophores would be a prudentapproach to furnish CNS-penetrating tractable anti-glioma drugs. effects 11. FurthR/S-N-3-cyanophenyl-N′-urea, as a potent anti-glioma agent.55 Previously, the authors disclosed some compoundsderived from cromakalim  channel opener) containing an arylurea or arylthiourea moietyat the 4-position  that showed anti-gliomaactivity potential. Given the above, the authors further investigatedthe amplified benefits in GBM and accordingly synthesized a new seriesof compounds. The furnished adducts were evaluated against three humanhigh-grade glioma cell lines, U373, T98G, and Hs683. All the synthesizedcompounds displayed promising activity profiles, and 50 was strikingly potent, with IC50 = 6 ± 1, 14 ±1, and 4 ± 1 μM toward the U373, T98G, and HS683 cell lines,respectively  and SIRT2 complex using Auto DockVina followed by an in vitro assay. Compound 50 fit well in the binding sites with average dock scoresof −9.0 against SIRT-1 and −9.2 against SIRT-2. In the in vitro studies, 50 inhibited both SIRT1 andSIRT2, with IC50 = 6.2 ± 1.7 and 4.2 ± 1.6 μM,respectively, while no inhibition was observed against HDAC1, 2, 3,8, 6, 10, and 11 and SIRT-3 activities. Additionally, computer-assistedphase contrast microscopy (quantitative video microscopy) suggestedthat 50 exerted cytostatic effects rather than cytotoxiceffects on both Hs683 and U373 glioma cell lines. Furthermore, 50 induced accumulation in the G1 phase and promoted senescence-associatedβ-galactosidase  activity. The impact of compound 50 on the spheroid-forming capacity of GBM cells was alsomonitored, which showed that the compound reduced the surface areaof tumor spheroids from glioma Hs683 and U373 cell lines. Additionally, 50 abrogated tumor development in the zebrafish xenotransplantationmodel. Continued evaluation of 50 in the presence ofmutated p53 and overexpressed MDR efflux pumps ABCB1 and ABCC1 ledthe authors to deduce that the aberrant behavior of both did not affectthe activity of 50. Additionally, 50 wastested in the NCI-60 cell line panel, where it displayed a mean GI50 value of −5.5 (∼3 μM); however, it didnot exhibit effects on peripheral blood mononuclear cells. Overall,compound 50 was found to be a potent inhibitor of classIII HDAC that can be used as a lead in the development of potent anti-GBMagents.In 2017, Schnekenburgeret al. identified a new class III HDACinhibitor, ectively 12. Furth56 The hydroxamicgroup of vorinostat binds to the catalytic site of the HDAC enzymeand forms a chelate complex with Zn2+ metal. Based on therevealed binding modes, O-alkylated derivatives of vorinostat weresynthesized, and their chelating capacity was evaluated ,and displayed potential anti-proliferative activity. The outcome ofthe study led to the identification of an effective strategy to overcomethe poor pharmacokinetics of HDAC inhibitors using an approach ofuncaging an inactive precursor of vorinostat by heterogeneous Pd catalysisin glioma cells.In 2016, Rubio-Ruiz et al. reported an efficientmethod for therelease of vorinostat , triggered by palladium-functionalized resins, toenhance its PK properties by modulating the metal chelating effectof hydroxamic groups.54) with vorinostat (LII) as a selective HDAC6inhibitor.57 The design strategy to constructthe hybrid was based on the anticipation that the inclusion of a corefragment of primaquine will confer the ability to the resulting adductto interfere with endosomal trafficking to the plasma membrane andinhibit the multi-drug-resistance transporter P-glycoprotein and autophagy.With the above-mentioned design rationale, the compound was synthesizedby fusing the active pharmacophore of varinostat with primaquine andwas evaluated in the U251N glioma cell line. The evaluation resultsrevealed that 55 inhibited the U251N cell line with IC50 = 10 μM. Enlightened by its promising cell growthinhibitory effects, 55 was evaluated for a cell invasionassay, and 55 did not inhibit cell migration. However,a combination of 55 with quercetin efficiently inhibitedcell migration by 42%. Additionally, the hybrid of primaquine (54) and sahaquine (55) inhibited p-glycoproteinactivity at a 10 μM dose. Continued evaluations revealed that 55 selectively inhibited the HDAC-6 isoform and reduced thelevels of EGFR, ERK1/2, and Akt alone as well as in combination withquercetin  (uercetin 14.56) ,largazole thiol, in the mouse brain. Additionally, treatment withlargazole led to Pax6 upregulation, which suppressed GBM proliferation.Collectively, the results highlight the need for a comprehensive evaluationof largazole in GBM.Al-Awadhi et al. investigated largazole (56) 14B to as59 Eleven peptide-based HDACi weresynthesized and screened over CHP-134, IMR-32, SK-N-AS, and NB-1 (neuroblastoma)and G55T2 (glioblastoma) cell lines, where 57 was foundto be most potent in the series, with the IC50 values shownin 57 was found to be non-selective againstHDAC1 and HDAC6. To identify a new scaffold with potential anti-GBMactivity against resistant cell lines, Ellert-Miklaszewska et al.screened three diverse groups of scaffolds with 4-vinylbiphenyl skeleton,3-arylidene-oxindole, and isothiazolonaphthoquinone core substitutions.60 The identification of the scaffold was carriedout by screening the compounds from the various series over LN18 andT98 GBM cell lines. Among all the compounds, 58, witha HDAC inhibitor architecture, showed significant cell growth inhibitionof more than 70% against the LN18 and T98 cell lines. In addition,it was revealed that 58 potentially inhibited all formsof HDAC with the prudential inhibition of HDAC6 and 8. Overall, thesefindings suggest that the compounds with HDAC frameworks can serveas potential inhibitors against resistant GBM cell lines .ll lines 14D.61 Theresults revealed that GBM samples contained high expression of HDAC6,and HDAC6 overexpression correlated with advanced glioma grade andpoor patient survival. HDAC6 was also enriched in glioma stem cells,and its expression positively correlated with several GSC markers. In addition to the above-mentionedfindings, the study also identified JOC 1 (59) . In the transcriptomic analysis, 59 decreased the cell cycle pathways and elevated neural differentiationand cell death in glioma stem cells.In additionto scaffold assembly studies, some efforts have alsobeen directed toward the determination of HDAC6 expression in GBM.Auzmendi-Iriarte et al. conducted HDAC6 expression analysis in GBMusing the Rembrandt cohort (28 control and 219 GBM samples), TCGAcohort (4 control and 156 GBM samples), Gravendol cohort ,vital cohort, and Donson cohort.C 1 (59) 14E as a 4.32+ and NADP+ (or NAD+) ascofactors. Reports have suggested that because of mutations, IDH convertsα-ketoglutaric acid to d-2-hydroxyglutaric acid, whichis an unfavorable factor of cancer initiation in glioma. Additionally,it has been found that IDH1 inactivation causes reduced biosynthesisof deoxynucleotide and lipid and enhanced congregation of ROS, whichdecreases the growth of GBM through RNA obstruction. Moreover, IDHinactivation promotes the sensitivity of tumor cell to both senescenceinduced by radiation and erlotinib, increasing the survival of micewith xenografts derived from patient.522 In addition,a decrease in cell growth in orthotopic GBM mouse models was observedafter genetic inhibition of IDH3α due to enhanced total NADPH/NADP+ ratio, metabolites of pyrimidine pathway, nucleotide biosynthesis,and epigenetic alterations (DNA methylation) of potent growth factorsin highly proliferative GBM cells, generating metabolic vulnerability.523 To exploit these revelations, Liu et al. designedand synthesized a series of 61 compounds as IDH1 inhibitors.62 All the synthesized compounds displayed excellentinhibition within the nanomolar to micromolar range. Among the series, 60 and 61 displayed functional IDH1 inhibitionwith Ki = 0.15 and 0.34 μM, respectively.The SAR was investigated, and the properties generated are presentedin Ki) in the range of 0.14–9.5 μM. The bestcompounds, 60 and 61, inhibited IDH1 (R132C)and WT IDH1 with Ki = 0.26 and >30μMand 0.80 and 14 μM, respectively. Furthermore, the impact ofthe synthesized compounds on D2HG concentrations was evaluated inHT1080 human fibrosarcoma cells, revealing that 60 and 61 inhibited D2HG production, with IC50 = 1.1 μM.As part of the continued investigation, a blood–brain permeabilitystudy was performed in MDCK-MDR1 cells. 60 penetratedthe experimental BBB wall with permeability values of 5.39  and 8.88 , and the efflux ratiowas 1.7. Additionally, the activity of the selected inhibitor wasevaluated against the glioma cell lines BT-142 BXD-4687 and BXD-3752,where 60 and 61 inhibited cell growth withEC50 = 0.63, 1.2, and 2.5 μM and 0.26, 7.6, and 2.8μM, respectively. No growth inhibition was observed in normalfibroblast WI-38 cells, suggesting that the compounds selectivelyinhibited cell growth in glioma.IDH is an essential enzymein the tricarboxylic acid cycle thatconverts isocitric acid (ICT) to α-ketoglutaric acid (α-KG)using Mg62) as a cancer-associated mutant IDH-1 inhibitor.63 A total of 37 compounds were furnished withvarious substitutions, and their activity was evaluated against IDH1(R132H). Among the synthesized series, 63 and 64 were the most potent, with Ki valuesshown in 64 for α-KG and NADPH was performedand expressed as a Lineweaver–Michaelis–Burk or Mentenplot, where 64 was found to be competitive with α-KGand non-competitive with NADPH. Moreover, 63 and 64 inhibited BT142 glioma cells bearing IDH1 R132H mutations.In 2015, Wu et al. published a SAR study of a potent 2-thiohydantoinderivative  using the diaphorase assay.Among the screened compounds, quinolinone was the most suitable scaffold,and 68 was considered a hit compound for mIDH1 activity.Efforts were made to achieve potency, and a SAR was generated forhit 68  displayed moderatePK results for 69, and the results were as follows: MLMleft = 42% in 30 min, solubility = 0.43 μM, half-life (T1/2) = 1.27 h, Cmax = 1.06 μM, and AUC0–8h = 2.45 μM.Thus, the results of the PK studies presented a scope of improvementof the PK properties of 69. Subsequently, another structure-basedguided optimization was performed for 69, leading tothe identification of 70 with striking IDH1 inhibitoryactivity, as demonstrated by both biochemical and cellular assays.The PK/PD properties of 70 were studied, including theADME and safety profiles, and the compound showed excellent livermicrosomal and plasma stability. 70 displayed low activityfor P450 (CYP450) enzymes and exhibited a safety window of 200-fold, also named olutasidenib, as a potent IDH-1 inhibitor. Overall, 71 displayed substantial inhibitory potential against IDH-1R132H along with good HLM-MLM stability and an improved solubilityprofile. In biochemical and cellular assays, the compounds showedbalanced activity profiles, as shown in 71 was evaluated in male CD-1 mice at oral doses of5 and 100 mg/kg, where the blood/plasma ratios were 0.24 and 0.38,respectively. In the artificial membrane PAMPA (19.9 × 10–6 cm/s mean) and Caco-2 cell system pyrimidine (79) isconsidered a privileged structure endowed with high affinity towardTSPO. To exploit this finding, Narlawar et al. designed pyrazolopyrimidine analogs and explored scaffolds for allosteric-likemodulation of human wild-type TSPO.67 Atotal of five compounds with various cores were synthesized, and theirbinding interactions were investigated by competing with the radioligand[3H]PK 11195 in HEK 293T and T98G cell membranes. The resultsare presented as Hill slopes. A Hill slope of 1 represents the on-siteinteractions of the compound, while a Hill slope of −1 andshallower than −1 shows a negative modulation where the compoundis predicted to bind at another site. The compounds with nitrogendisplayed a Hill slope of 1 that depicted the one-site interactionof compounds at the same site of the radioligand. All the tested compounds(79–83) showed activity (Ki) in the low nanomolar range except 83, whichdid not demonstrate binding to TSPO  pharmacophore/topologicalmodel was used to design probes suggesting 4-phenylquinazoline asa promising pharmacophore of TSPO. Additionally, an in-house libraryof quinazoline was used, and a 3D model of interaction with TSPO wasstudied. The encouraging results prompted the authors to synthesizethe series of compounds that were evaluated for the TSPO binding assay,and the compounds demonstrated activity in the nanomolar range. Amongthe series, 84 was the most potent, with Ki = 0.47 nM. 84 was selected for furthermodifications, and fluorescent probes 85 and 86 were synthesized by inserting 7-nitro-2,1,3-benzoxadiazolyl (NBD)with tetramethylene and hexamethylene as spacers, respectively. Thecompounds were further evaluated for TSPO binding affinity, where 85 showed effective results with Ki = 19.2 nM and a retention time (tR)of 53 min. The fluorescent labeling of 85 and 86 was evaluated in the human GBM cell Line U343, in which both compoundsshowed uniform cytoplasmic labeling. Additionally, mitochondrial labelingwas investigated using MitoTracker Red, and 85 labeledTSPO at the mitochondrial level. Additionally, 85 completelydisplaced the non-fluorescent ligand PK11195 at a concentration of50 μM without cytotoxicity , mesenchymal (GBM-2), and classical (GBM-3)GBM. profile 23.4.5525 The relationshipbetween clinical–pathological outcomes of GBM and mRNA expressionof PDIs studied in the CGGA and TCGA database analysis indicated thatthe somatic alterations in the GBM (high PDI signature and risk score)are involved in the aberrations of driver oncogenes PIK3CA, MUC16,and TTN and amplification peaks of oncogenes . The additional involvement in the deletion peaks of tumorsuppressor genes  suggeststhe importance of PDI in the malignant processes of GBM, includingendoplasmic reticulum (ER)-associated degradation, unfolded proteinresponse, cell adhesion, endoplasmic reticulum stress (ERS), WNT signalingpathways, DNA sensing (cytosolic), and apoptosis. In addition, PDIsinteract with numerous signaling pathways, such as endoplasmic reticulum-associatedprotein degradation (ERAD), ERS, and unfolded proteins (UPR).526 Moreover, PDIs regulate the activation stepof integrins (SH to S-S conversion) and ERS by inhibiting misfoldedprotein accumulation as well as activate anti-apoptotic caspase-3and -7, which are responsible for the invasion and migration of gliomacells, their resistance to TMZ, and apoptosis inhibition.530 In addition to tumor progression and survival, PDIs regulate theimmune response in gastric cancer patients by involving PDIA3 complexationwith MCH class I through NKG2D ligands.532 The exhaustive engagement of PDI in tumor development and survivalmakes PDI a prudent target against GBM.The overexpression of PDI6 is involved inthe progression, invasion,and migration of GBM cells.71 The research group conducted a SAR study onthe previously identified PDI inhibitor BAP-2 (90), obtainedfrom a high-throughput screening (IC50 = 930 nM). A totalof 68 compounds were synthesized and evaluated against PDI using thePDI reductase assay. For the structural optimization program, thenitrile group was maintained at ring B, and various substitutionswere attempted at ring A. A structural scanning program demonstratedthat the replacement of hydroxy groups with bromine, methoxy, andamine groups reduced the PDI activity; however, introducing a sulfonamidemoiety instead of the hydroxyl group enhanced the PDI activity. Additionally,replacement of the nitrile group with carboxylic acid or carboxymethylester at the meta position of ring B led to favorable trends; theresulting compounds displayed better binding toward PDI than chalconescontaining electron-withdrawing groups. Furthermore, the replacementof nitrile with a trifluoromethyl group reduced the potency of compoundsby 2–4-fold. Overall, the SO2NHR substitution atR1 and hydroxy substitution at R2 are the key features for PDI activity.From the preliminary studies, 23 compounds with IC50 valueslower than 1.5 μM were selected for further evaluations. Theselected compounds were tested in a panel of brain cancer cell lines, where compounds showed moderate to strongcell growth inhibition in the U-87MG, A172, and NU04 cell lines. Amongthe evaluated compounds, 91 was the most potent in A172and NU04 cell lines, with IC50 = 5.6 and 9.0 μM,respectively. Additionally, 93 displayed promising results,with IC50 = 3.8 μM against NU04 cell lines. In thebiochemical thermal shift assay, an elevation of more than 1 °Cin melting temperature was observed, strongly indicating that thecompounds had good binding interactions with PDI. In contrast, 91 and 93 did not stabilize PDI in the thermalshift assay but still demonstrated good activity, indicating thatthe compounds might bind to the hydrophobic pocket of the b′domain. Further analysis revealed that the compounds promoted theERS response in GBM cells and inhibited cell migration in a dose-dependentmanner in the wound-healing assay in A172 cells. Furthermore, thesynthesized compounds displayed synergism with arsenic trichlorideand DNA damage-inducing radiation therapies, indicating the possibleusefulness of these compounds in combination , with IC50 = 300 and 90 nM, respectively. The leads were also evaluated againstU-87MG GBM cell lines, in which both compounds inhibited cell growth,with IC50 = 18.3 μM for 94 and 10.6μM for 95. To establish the SAR, a total of 89compounds from the Chemdiv library and NCI development program weretested using the PDI reductase assay. 5-Hydroxybenzo[d]dioxole substituted with various amines, such as morpholine,piperidine, piperazine, and pyrrolidine, were active and exerted PDIinhibition at an IC50 < 1 μM. In contrast, ureasubstitution was not active, and urea-substituted compounds exhibiteda complete loss of activity. For further studies, three series ofcompounds were synthesized and evaluated PDI activity. Among the synthesizedcompounds, 96–99 showed promisingPDI inhibition was found in undifferentiated GBM cells and glial fibrillary acidicprotein (GFAP)+ normal neural progenitors.539 Moreover, the expression of GFAP+/nestin+/βIII-tubulin+cells in GBMs and normal human fetal astrocytes has been reported in vitro, indicating the involvement of tubulin in the progressionand survival of GBM.541Tubulin is a diametricglobular protein that forms microtubules and is responsible for variouscellular processes. GBM shows major alterations in the cytoskeletonof microtubules, such as anomalous γ-tubulin and class III β-tubulinisotype (βIII-tubulin) expression, which are linked to anaplastictumor phenotypes.73 In the study, a previously reported methuosisinducing 103 was used as a lead. Well equipped with thestructural requisites for methuosis-inducing ability of the lead 103, such as para-pyridinyl, methoxy substitution (5-position),and small alkyl group (2-position), the authors first used the structuraloptimization program to determine the impact of various substitutionsof the indole ring, as shown in 104–107 displayed promisingresults with GI50 = 0.268, 0.272, 0.023, and 0.008 μM,respectively. Furthermore, 106 and 107 causedconsiderable accumulation of cells at the G2/M phase and increasedthe percentage of cells in the sub-G1/G0 phase. Additionally, theeffect of selected 104–107 on microtubulepolymerization was evaluated in U251 GBM cells using immunofluorescencemicroscopy. In this study, 107 distracted the stainingpattern at a dose of 0.1 μM. In the biochemical method, polymerizationdisruption was also observed when 104, 106, and 107 were analyzed by Western blot analysis. Collectively,the switch in the mechanism responsible for the cytotoxic effectsof the compounds from methuosis to microtubule disruption was accompaniedby a substantial increase in potency.No apoptotic cell death,or methuosis, involves the accumulation of macropinosomes, leadingto the loss of membrane integrity. The literature indicates that smallmolecules with methuosis-inducing ability outshine the candidatesfor apoptosis-inducing conventional anti-cancer drugs. To exploitthe above-mentioned information, Trabbic et al. initiated a medicinalchemistry campaign centered on the privileged bicyclic heteroarylring, indole, and reported a series of indolyl-based pyridinyl propenones.74 The adducts were evaluated for anti-tumor effectsagainst glioma cell lines . The results were overwhelminglypositive, with the compounds demonstrating striking cell growth inhibitoryeffects in the two-digit nanomolar range , colchicine binding inhibition= 96%; 112, IC50 = 1.2 μM (tubulinassembly), colchicine binding inhibition = 92% , PD-GBM, and PD-GBM , and it potentially inhibitedcell growth, as shown in 115 binds to the colchicine bindingsite of tubulin and disrupts microtubule stability, which leads toGBM cell death.Diaz et al. revealed a series of modified carbazoles as microtubuledestabilizing agents that potentially inhibited the growth of GBMcell lines.78 In the developmentof pyrrole derivatives, structural engineering was carried out overlead compound 116, which resulted in a series of compoundsthat were further evaluated for tubulin activity. Among the synthesizedcompounds, 117 displayed promising activity against tubulin,with IC50 = 0.39 μM. Furthermore, the activity of 117 was evaluated for hTopoI and hTopo 2, where 117 selectively inhibited hTopo II expression at 100 μM, whereasno activity was observed over hTopo I. Moreover, compound 117 showed potential inhibition against U343MG and U-87MG GBM cells adhesion, integrin-linkedkinase (ILK) activation, a surge in HIF-1α, and VEGF productionin GBM.546 Moreover, it was found that HIF-1αrequires elevated concentrations of heat shock proteins 70 and 90,which induce tumor progression.547HIF is a key heterodimeric transcriptionfactor that activatesvarious transcription genes involved in tumor survival, invasion,angiogenesis, and glucose metabolism124.84 For the structure analysis, 124 was divided into four regions, and each was explored inthe context of substituent preference. Various substitutions wereattempted at each region, and a series of compounds was accomplishedthat were further evaluated against HIF-1-mediated transcription inthe LN229-HRE-Lux glioma cell line. Among the synthesized compounds, 125–129 were the most potent in series,with IC50 = 0.3, 0.8, 0.4, 0.3, and 0.2 μM, respectively.The SAR of the compounds is summarized in 125 and 126 suppressed the expression of HIF-1α,as demonstrated by Western blot analysis. In conclusion, the identifiedHIF pathway inhibitors exhibited beneficial effects against hypoxictumor resistance to chemotherapy and radiotherapy.To develop potent HIF pathway inhibitors, Mooring et al. conducteda structural optimization campaign of previously reported compound 85 The group used compound 130, whichwas previously identified through high-throughput screening as a lead.The lead modification study was initiated to overcome the limitationof the poor aqueous solubility of 130 that was pinpointedas an obstacle for the in vivo evaluations. The researchgroup furnished a series of 12 compounds with molecular weights rangingfrom 371 to 403 g/mol. All the synthesized compounds were evaluatedfor HIF transcriptional activity in LN229-V6R cell lines, and thecompounds showed promising results with IC50 values underor close to 5 μM  under normoxic and hypoxic conditions, with IC50 = 73 and 146 μM and 92 and 113 μM, respectively.Furthermore, the aqueous solubility of compounds was determined, andthe results confirmed the improved aqueous solubility of 131 and 132 (LogP = 3.1 and 1.3). Notably, N-[methyl]-N-(propan-2-yl)pyridine-2-sulfonamide (132)demonstrated a solubility improvement of ∼9000-fold comparedwith that of the lead. Additionally, both compounds showed an optimumPK profile, according to the results of the metabolic studies conductedin homogenized mouse liver, where 131 and 132 displayed t1/2 = 13 and 15 h, respectively.Overall, the designed compounds demonstrated promising efficacy coupledwith an optimum PK profile, and the findings can be leveraged as aninitiation point of several anti-glioma drug discovery campaigns.In 2012, Mun et al.reported a novel HIF-1 pathway inhibitor withimproved pharmacological properties as a potential anti-glioma agent. to 5 μM 33. The c4.889 The group considered the chemicalarchitecture of previously reported compounds 134 and 135 as a lead compound for the structural optimization program.Thus, 135 showed good integrin inhibitory potential coupledwith a magnificent activity profile toward MDM2 and MDM4 . The structural tuning program wasplanned to confer enhanced potency and a balanced activity profileto the new analogs of 135 toward both integrin and MDMs.In total, 14 compounds were synthesized and evaluated against integrinbinding fibronectin (α5β1) and vitronectin (αvβ3)and the human p53/MDM2 or p53/MDM4 complex. Among the synthesizedcompounds, 136 was identified as the most potent in theseries. Furthermore, the p53 protein-mediated activity of integrinsand MDM2/4 inhibitor 136 was investigated in U-87MG GBMcells , where it inhibited GBM cell growth with IC50 = 116 ± 10 nM, while efficacy against T98G cells was observedat micromolar concentrations. Additionally, a docking study of 136 with corresponding binding pockets was performed. In bothαvβ3 and α5β1, 136 was boundthrough the canonical RGD binding pattern and interacted with themajor amino acids Y122, S123, N215, Y133, S134, N224, Q221, D227,and D227. In MDM2, NMR and docking were performed, revealing thatthe biphenyl moiety of 136 reached the W23 and F19 pocketsand that the remainder of the molecule was oriented toward the L2loop or flipped into the N-terminal region. In the NMR study, a massivechemical shift was observed in the A13, S22, R29, K51, F55, Y56, G58,Y60, M62, F91, S92, V93, K94, and I103 amino acids, indicating thatthese residues might interact with 136. The overall studyled to the identification of multi-targeting RGD integrins and dualmurine double minute protein inhibitors as anti-glioma agents thatemerge as suitable alternatives to combination therapy.To extract the evidenced benefits attained from the simultaneousinhibition of multiple targets in cancer, Merlino et al. in 2018 reporteda series of RGD integrins and dual MDM protein inhibitors to treatGBM.BMcells 34.The c137, MDM/p53interaction IC50 = 11.65 nM, TSPO Ki = 438 nM), to treat gliomas.86 To optimize the structural features, six compounds were synthesizedand investigated as MDM2/p53 complex disruptors in U-87MG cells. Amongthe series, five compounds displayed promising inhibition in the IC50 range of 4.3–24.8 nM, and 138 was themost potent in the series, with IC50 = 4.3 nM. Furthermore,the binding affinity of selected compounds toward TSPO was evaluatedusing radioligand binding assays, in which 138 showedgood binding activity with Ki = 87.2 nM,which was better than that of lead 137 (Ki = 438 nM). Docking studies of the most potent 138 were performed (MDM2 protein), in which the phenyl ringattached to the indole interacted with the Trp23 pocket and showedhydrophobic interactions with the L57, I61, F86, F91, I99, and I103side chains, while the indole ring was involved in the interactionwith Phe19. The glyoxylamide-NH formed a hydrogen bond with L54, andthe side chain was involved in hydrophobic interactions with Leu26residues I19, Y100, L54, and M50, while the methyl ester moiety interactedwith Q24 residues of the binding pocket . Overall, dual MDM-2/TSPO inhibitors were effectiveagainst glioma cell lines and can be used as therapeutic agents againstcancer where p53 signaling is affected and TSPO is overexpressed.In 2016, Daniele et al. optimized 2-phenylindolylglyoxylyldipeptide,a lead dual inhibitor of MDM-2 and TSPO (g pocket 35. Cell 143) can yield conclusive benefits in the context of cancer treatment.90 To exploit these disclosures, the authors initiateda medicinal chemistry campaign and furnished 10 oxidosqualene cyclaseinhibitors. The inhibitors were evaluated in 11 cancer cell linesderived from various tissues and in one non-tumoral human brain-derivedendothelial cell line. All the synthesized compounds displayed goodcell proliferation inhibition with IC50 values in the micromolarrange. Among the series, 139–142 werethe most potent against LN18 and LN229 GBM cells and HCEC brain-derivedendothelial cells. Considering the above results and quest to amplifythe anti-tumor potential, the combination of the most potent compoundsand atorvastatin (143) was evaluated in human GBM LN18and LN229 cells and non-tumoral HCEC endothelial cells. Compounds 140 and 141 in combination with AT (143) displayed promising results in the context of synergistic anti-cancerefficacy. Overall, the combination of oxidosqualene cyclase inhibitorswith atorvastatin (143) could serve as a good combinationto treat GBM  is a synthetic hybrid agent that comprisesthe structural attributes of entinostat (144), a classI HDAC inhibitor, and tranylcypromine (145), an LSD1inhibitor (146) increased HDAC-targeted H3K27acand LSD1-targeted H3K4me1 at differentiation-associated genes. Theinduction of cell death, cell cycle arrest, and a cellular differentiationphenotype was observed with corin treatment along with transcriptionalchanges correlating with increased survival time in DIPG patients.The outcome of this study clearly shows that dual HDAC-LSD1 inhibitionis a logical strategy to treat DIPG.87Corin (nhibitor 37A. Rece147) to explore its activity in GBM. SB747651A(147) is a multi-targeted small molecule that inhibitsmultiple pathways, including the MAPK, PI3K-Akt-mTOR, and JNK pathways.The team investigated this molecule using three well-characterizedpatient-derived GBM spheroid cultures . The resultsof the cell viability and apoptosis assays suggested that SB747651A,when combined with TMZ , inducedapoptosis-mediated cell death. SB747651A  that inhibited the growth of U343MG andANGM-CSS , with IC50 = 49.7 and 44.0 nM, respectively . It was found that the nanoparticles ofTMZ in combination with GEM and DAC displayed a synergistic effectand inhibited U-87 cells more predominantly over the glycolysis pathwaythan TMZ alone. Moreover, it was found that the formulation is suitablefor the thermal destruction of cancer. Overall, the delivery of multipledrugs as a cocktail showed promising growth and opened the door todevelop more efficacious drug combinations.To improve the efficacy and reduce the drug resistanceof TMZ,Sahli et al. introduced hybrid drug nanoparticles of gold-TMZ combinedwith gemcitabine (GEM) and decitabine (DAC).4.9149 with PROTAC-like efficacy for the treatment of GBM.548 Compound 149 caused p62 accumulationand proteasomal degradation, leading to proteolysis of aberrantlyoverexpressed HDAC6 isoforms in GBM. Additionally, 149 demonstrated substantial cell growth inhibitory effects on the U-87MGglioma cell line (IC50 = 1.56 μM), decreased cellmigration, increased autophagic cancer cell death, and reduced theimmunosuppressive activity of PD-L1 , as a potent inhibitor of tumor progression and stemcell-likecells following Wnt/β-catenin signaling , revealing substantialdose-dependent cell growth inhibitory effects of 150.Western blot analysis further demonstrated that ZBC260 (150) downregulated the expression of BRD2/3/4 in glioma cell Lines U-87,U251, H4, and A172. Subsequent studies revealed that ZBC260 arrestedcell growth at the G2/M phase, promoted the expression of p21, p27Bax, cleaved caspase-3, and caspase-9 and suppressed cyclin D1, cyclinB1, BCl-2, and BCl-XL.550 Furthermore,the effect of ZBC 260 on cell invasion, migration, and EMT was evaluated,and 150 downregulated the expression of the epithelialmarkers N-cadherin, SNA12, CD44, and vimentin and inhibited cell invasionand migration. ZBC260 also demonstrated in vivo anti-tumorpotential and decreased the levels of Ki-67, Bcl-2, and PCNA. Notably,the stem cell-like markers ALDH-1, KLF4, SOX2, NANOG, and ABCG2 weresignificantly inhibited by ZBC 260, indicating that the compound canalso inhibit CSCs. The mechanism of stem-like cell inhibition wasstudied by analyzing the expression of the proteins/genes GLI1, NICD1,and β-catenin, which are involved in maintaining CSCs. The compoundreduced the expression of β-catenin, as confirmed by immunohistochemicaland Western blot analyses.Recently, Tian et al. reported a BET degrader, ZBC260  that targets and degrades CDK6 in cancer cells.551 POTAC CP10 (151) effectively degradedCDK6 in U-87 GBM cells with degradation rates of 72% and 89% at dosesof 10 and 100 nM, respectively  with a mean IC50 value of 0.7 μMin Hs683 and U373 glioma cell lines, suggesting that the compoundspossibly acted as protein synthesis inhibitors.94In 2012, Frédérick et al. designed and synthesizeda series of trisubstituted harmine derivatives as anti-GBM agents.f 0.7 μM39. Conti154 as a promisinganti-GBM agent, with IC50 = 12 and 16 nM against the T98Gand Hs683 oligodendroglioma cell lines, respectively.95 Further explorations indicated that 154 inhibitedthe activity of the proteasome at a concentration (IC50) of 7 μM in U373GBM cells  and neuroblastomacell lines (SK-N-SH and SK-N-F1). Compounds 155 and 156 demonstrated promising cell growth inhibitory effects proliferation, inducing cell apoptosis, and inhibitingmetastasis in a PKM2 expression-dependent manner by obstructing theSTAT3 signaling pathway. Furthermore, in vivo studieswere performed in the U118 mouse xenograft tumor model. Owing to thelow water solubility of 158, its prodrug (159) was synthesized and administered to the mouse at a dose of 50 mg/kgi.p. for 3 weeks. Compound 159 demonstrated remarkabletumor growth inhibitory potential  showed potential cell growth inhibition over theT98 GBM cell line, and the IC50 dose was found to be 9.85μL/mL. Additionally, 161 inhibited cancer cellmigration and induced apoptosis by inhibiting the PAM signaling pathway.Overall, PA (161) showed impactful results against GBMcell lines and can be used in combination with other anti-GBM drugs(162), a marine natural product, against GBM cell lines.100 Grincamycin-B (162) showed potentialcell inhibition against the U251 and 091214 GBM cell lines, with IC50 = 2.04 ± 0.24 and 3.11 ± 0.25 μM, respectively.Further biological evaluation revealed that grincamycin-B (162) targets CSCs in GBM by targeting the RHOA and PI3K/Akt signalingpathways , a clinical PARP-1 inhibitor, was usedas a lead, and a series of olaparib analogs were synthesized bearingvarious substitutions. Initially, all the synthesized compounds wereevaluated using the PARP-1 inhibition assay, lipophilicity (LogPoct), and percentage plasma protein binding (%PPB) by high-performanceliquid chromatography; all the synthesized compounds exhibited promisingresults. Among the synthesized compounds, 164 showedthe highest activity, with a cell-free PARP-1 inhibition IC50 value of 3.3 nM, lipophilic properties (LogPoct) of 3.0,and a percentage of plasma protein binding (%PPB) of 96%  of 85 μL min–1 mg–1, respectively. The lead compoundwas next labeled with 125I (165), and the ex vivo biodistribution was evaluated in nude mice in ahuman GBM xenograft model. Ex vivo studies revealedthat radiotracer 165 specifically binds to PARP-1 andis retained at the tumor site. The overall results indicated that 165 displayed good in vivo properties toserve as a promising imaging tool in GBM surgeries.In 2015, Zmuda et al. introducednovel radio-iodinated tracers withspecificity to PARP-1 for GBM imaging.) of 96% 42. The l11C]MPC-6827, withbrain-penetrating ability properties to detect GBM.102 Compound 166 (MPC-6827), a microtubule inhibitor(IC50 = 1.5 nM), was first evaluated over various braintargets, where the compound demonstrated targeting against the histamine-4receptor and sigma-1 receptor with Ki =155 and 426 nM, respectively (167 ([11C]MPC-6827) was synthesized, and in vivo bindingwas evaluated in white male mice. The compound retained its peak inthe brain for 5 min and was gradually washed out, indicating desirablekinetics. Additionally, 167 showed 70% brain accumulationfollowed by accumulation in the muscles, spleen, and lungs with specificbinding of 60%, 42%, and 30% at a dose of 5 mg/kg i.v., respectively.Notably, the in vivo binding of [11C]MPC-6827(167) was evaluated with unlabeled MPC-6827, and thecompound showed specific binding and retention in the brain, confirmingits potential as a bioimaging tool.In 2018, Kumar et al. introduced the first microtubule positronemission tomography (PET) radioligand, ,[18F]-labeled acetamido benzoxazoloneanalogs using 168 as a lead compound (in vitro binding affinity (Ki) of the synthesized compounds was evaluated for TSPO in the ratbrain by assaying competitive binding, where 169, 171, and 172 showed a high binding affinity with Ki = 20.1, 15.5, and 13.4 nM, respectively. Thelipophilicity values of the compounds were in the range of 2.35–3.00.The in vitro and in vivo specificbinding of [11C]-labeled 168–171 and [18F]-labeled 172 for TSPOwas investigated in the ischemic rat brain, where the compounds displayedradioactivity on the ipsilateral side compared with the contralateralside with average binding concentrations of 12.0, 1.2, 21.6, and 29.8,respectively. PET imaging displayed a higher uptake of radioactivityon the ipsilateral sides of the brain with standard uptake valuesas follows: 169, 1.72; 171, 0.98; and 172, 1.70. Additionally, displacement studies using unlabeledand labeled 169–172 revealed thatthe labeled compounds were highly specific for TSPO in the ischemicbrain. Furthermore, the biodistribution of 172 was investigatedin the bones and whole body of mice, and the results indicated highuptake of radioactivity in the lungs, heart, and kidneys, moderateuptake in the small intestine, muscle, liver, spleen, and testis,and no significant activity in the bones. The radiolabeled metaboliteaccommodation in the brain was investigated by HPLC, indicating that 172 was metabolized into a single metabolite that was notaccommodated in the brain for a long time. Finally, PET imaging studiesof 172 were performed in a rat model bearing C6 gliomacells in the brain. The images displayed good accommodation of thecompound at the tumor site, while the radioactivity was rapidly clearedfrom the contralateral side, suggesting that the radiotracer specificallytargets the GBM. Overall, 172 demonstrated sufficientpromise to emerge as a potential chemical tool to trace inflammationand GBM.In 2017, Fujinaga etal. developed compound 44. Initi104 A total of seven compoundswith molecular weights of 366–527 Da and LogP = 1.5–2.7were synthesized from the corresponding 2,6-diiodo-substituted compoundsusing Suzuki and Sonogashira coupling reactions. All the synthesizedcompounds showed no dark cytotoxicity in T98G GBM cells; however, 173 and 174 displayed good phototoxicity, withIC50 = 80 and 40 μM, respectively. The cell uptakevalues for 173 and 174 were 0.11 and 1.5nM/cell with LogP = 1.73 and 1.95, respectively. The subcellular localizationsites of the compounds were determined in the Hep2 cell line usingfluorescence microscopy, revealing that the compounds were localizedprimarily in the endoplasmic reticulum  showed promising results in initialevaluations, and further modifications and evaluations are requiredto establish them as useful candidate probes for boron neutron capturetherapy. In 2019, He et al. incorporated m-carboraneinto the amino acid cystine (175), and its activity wasobserved in the U-87 GBM cell line.105 Itwas found that 175 was rapidly taken up by U-87 cellsand showed a reduction in cell viability in a dose-dependent manner.Moreover, 175 targeted CDKs and other genes associatedwith the cell cycle triggered cell death at S phase, which was furthervalidated with qPCR studies  as probes for boron neutron capturetherapy.eticulum 45. Furth studies 45.106 Gemcitabine is a well-established drug againsta wide range of solid tumors and has been approved for the treatmentof breast, non-small-cell lung, ovarian, and pancreatic cancers. Gemcitabinehas also been investigated for GBM; however, the therapeutic responsewas not promising, and it was deduced that the low BBB permeability,short half-life because of enzyme metabolism, and selectivity of gemcitabinetoward tumor versus healthy tissues must be improved. The literatureprecedents reveal the ability of heptamethine dyes (cyanine-7 or Cy7)to preferentially accumulate and retain tumors and their use in in vivo tumor models to deliver therapeutic and toxic cargoes.To overcome the limitations of gemcitabine, the group designed fourcyanine–gemcitabine conjugates, 176–179, which locally aggregate to the tumor and achieve thedesired therapeutic value. Structural engineering attempts (50) within the range of 0.01–0.02μM. The effects of 177 and gemcitabine were alsoevaluated in LN18, LN229, and HEK293 cell lines, and the results wereoverwhelmingly positive because adduct 177 displayeda magnificent anti-tumor profile  of halopemide (181), a classical atypical anti-psychotic agent, which showed a directand potent dual PLD1/2 inhibitory effect . The above approach identifieda PLD inhibitor (182) that elicited pronounced selectivity(75-fold higher selective inhibition) toward PLD2. The 1,3,8-triazaspiro[4.5]decanecore was identified as a PLD2-preferring motif. Keeping the 3-fluorophenylmoiety of 182 constant, different amides were exploredto improve the PLD2 selectivity of the compounds. Compound 183 (ML298), bearing a 3,4-difluorophenyl moiety, was identified with>53-fold selectivity toward PLD2. Furthermore, as a part of structuraloptimization, the team introduced a chiral methyl group at the αposition to the amide group, leading to the synthesis of 184 and 185. The (S)-enantiomer (184) was found to be the most potent compound of the series,showing dual potency toward PLD1 (IC50 = 6 nM) and PLD2(IC50 = 20 nM). Notably, the results of the cell-basedassays using U-87-MG cells revealed that both 183 and 184 cells dose-dependently decreased invasive migration inU-87-MG GBM cells  selective inhibitors.BM cells 47.vβ3 antagonists as anti-gliomaagents (186), a conjugate of tetraiodothyroaceticacid and polyethylene glycol (PEG) 4000, as an efficient agent ina GBM mouse model. They synthesized a smaller and monodisperse PEG36derivative 187 that exhibited integrin αvβ3 binding affinity; however, its detailed investigationwas blocked due to low aqueous solubility. To circumvent this issue, 188 was designed and demonstrated remarkably higher aqueoussolubility (>120 mg/mL) than 187 (1.4 mg/mL). Furtherinvestigation (PK studies) revealed that 188 could notcross the BBB, likely due to its deliberately decreased lipophilicityto improve the aqueous solubility. Considering the above findings,another compound (189) bearing a fluorobenzyl group wasfurnished that demonstrated significant αvβ3 binding ability (0.23 nM) coupled with good aqueous solubility(120 mg/mL) and BBB permeability. 189 displayed substantial in vivo anti-tumor potential because it led to a reducedGBM tumor size with a maximum loss of 98% of the tumor following 21days of administration at a dose of 10 mg/kg. Fluorescence dye labelingstudies also indicated that 189 easily crosses the BBBand localizes to GBM brain tumor tissue compared with normal braintissue  was involved in hydrogen bonding interactionswith serine residue S73. Additionally, 190 suppressedandrogen receptor transcription, induced degradation of androgen receptorsin tumor tissues, and elicited remarkable tumor growth inhibitoryeffects in a U-87 xenograft model.In 2021, Li et al. investigated four nimesulide analogs to treatGBM.8G cells 49. Becau194) and antagonistof the α7 and α9α10 nAChR agent MG624 (195).110 The structural template of the designedhybrids comprised three structural elements: a stilbene core, an alkylenelinker, and a terminal onium. All the compounds were evaluated fortheir in vitro functional activity on α7 andα9α10, nAChR subtypes, demonstrating the potency of allthe ammonium-based compounds in inhibiting 10 μM or 200 μMacetylcholine (ACh)-induced currents in oocytes expressing the humanα7 and α9α10 subtypes. Furthermore, the authorsevaluated the cell growth inhibitory effects of the compounds towardU-87MG glioblastoma, A549 adenocarcinoma, SH-SY5Y neuroblastoma, andwild-type mouse astrocytes on all nine compounds . The MTT assay demonstrated that 197 and 198 were selectively toxic against GBM cells. Notably, theenhanced anti-GBM activity of ammonium-based hybrid 199 coincided with greater antagonism against the α7 and α9α10subtypes. Additionally, the ability of all nine compounds to interferewith ATP production was evaluated by incubating them with U-87MG cellsfor 1 or 72 h, where 194, 200, 208, and 209 significantly reduced ATP production afteronly 1 h of incubation  are highly expressed inGBM cell lines. Considering these revelations, Pallavicini et al.synthesized potent anti-GBM hybrid scaffolds via fusion of the pro-oxidantmitocan agent RDM-4′ BTPI  as proliferation,migration, and invasion inhibitors of GSCs.112 Among the synthesized compounds, 10 compounds showed adequate inhibitoryefficacy with IC50 < 10 μM toward GSCs (Gli4 andGli7) and GBM cell lines (SNB75 and C6). Additionally, two compounds, 214 and 215, were exhaustively investigated,revealing overwhelmingly positive results. Specifically, 214 was the most promising because it manifested significant effectsagainst GLI4 and GLI7 cell lines, inhibited cell invasion, and targetedCNS cancer cells without affecting normal astrocyte and cortical neuronsurvival  cell lines alongwith other human cancer cell lines. The results led to the identificationof 221 as a potent anti-proliferative compound becauseit exerted significant inhibitory effects on the U-87MG cell lines,with IC50 = 1.7 μM. Additionally, in the Matrigelinvasion assay, 221 was substantially active againstU-87MG cells in the context of the inhibitory potential.In 2018, Madia et al. reported novel benzazole derivatives as potentanti-heparanase agents.ompounds 52. All t114 CXCR-4 is a transmembrane receptorthat regulates various cell types, including CSCs. The group usedtheir previously synthesized compound 222 as a lead thatwas reported as a potent HIV-1 entry CXCR4 co-receptor inhibitor targetingCXCR4 co-receptors without cytotoxicity. A total of three compoundswere synthesized, and affinity toward the CXCR4 receptors employinga conventional K+ channel patch-clamp assay was evaluated.Compounds 223–225 displayed goodaffinity, with IC50 = 0.35, 1.1, and 0.79 μM. Inthe toxicity studies, the maximum non-lethal doses for 223–225 were 2.0, 1.5, and 2.5 mg/kg, while theminimum lethal doses were 2.5, 2.0, and 3.0 mg/kg, respectively. Thecompounds were then evaluated against glioma-initiating cells by monitoringthe level of CD44+; all the compounds decreased the level of CD44+.The results were further confirmed by in vivo experimentsin the brains of NOD-SCID mice, where compounds decreased the numberof glioma-initiating cells  as a lead andoptimized the second and sixth positions of the phenyl ring of theheterocyclic core. A series of compounds was synthesized and evaluatedagainst various aldehyde dehydrogenases, where all the synthesizedcompounds displayed inhibition activity in the low micromolar range.Among the synthesized compounds, 228 displayed selectivitytoward ALDH1A3, with IC50 = 22.8 μM, while 227 was the most potent in the series, with IC50 = 3.5 μM  as a lead and designed a series of A3 adenosine receptorantagonists. The anti-proliferative properties of the compounds wereinvestigated in the U-87MG cell line, and 230 and 231 exhibited striking anti-proliferative activity, with IC50 = 0.74 and 0.061 nM, respectively. Additionally, dockingstudies were performed for the A3 receptor using AudoDock-4,revealing that 231 binds to the outer portion surroundedby TMs III, V, VI, and VII helices. The methyl group at R2 interacted with the L246 residue, and the pyrazolopyrimidine formedπ-stacking with F168 with additional H-bonding with N250. The6-phenyl ring interacted with residues I186, L91, W243, L246, andS247, where W243 was considered a crucial amino acid for the antagonisticproperty : GIN8, 11.2 μM; GIN28,7.7 μM; and GCE28, 7.2 μM. Furthermore, 239 was formulated in polymers using 2D inkjet printing, where a dispersionof 4 in Pluronic F-68, Tween 80, or PVPVA was found to be an efficientmethod; however, it showed comparable cytotoxicity to 238 in DMSO.118 Overall, pyrazolopyrimidines have shown magnificent results against GBMcell lines, and further developments might result in more efficienttherapeutics against GBM  with improvedCSF delivery to treat GBM.119 DON (240) is a glutamine mimicnon-natural amino acid that inhibits in vitro glutamine-dependenthuman cancer cells, reduces the tumor size, and improves the survivalrates. However, dose limitation and systemic toxicity are some ofthe obstacles in its development. Hence, the group explored the structureof DON (217), revealing that the furnished prodrugs 248 and 249 showed high plasma stability in monkeysand humans  and delineated its oncolytic efficacyand in vivo PK properties.120 First, the erythro was separated from the threo racemates by HPLC using a standard-phase Kromasilsilica column and was divided into two fractions. The first fraction(I) was separated again using semi-preparative HPLC and a ChiralcelOD-H column, while the second (II) fraction was isolated using a ChiralpakAD-H column, resulting in four enantiomerically pure fractions. Allthe fractions were evaluated in human patient-derived U3013 GBM cellsusing an ATP-based in vitro viability assay.The enantiomersobtained from the second fraction were more active than those fromthe first fraction, with IC50 = 3.5 and 3.8 μM (erythro enantiomers) and 9.9 and 10.5 μM (threo enantiomers), respectively. To evaluate the absoluteconfiguration of compounds from the second fraction, single-crystalX-ray diffraction and Flack’s X chirality parameters were used,leading to the identification of 255 and 256. Furthermore, the PK properties of the compounds were evaluatedin male NMRI mice treated with a single dose of 20 mg/kg )or 2 mg/kg (intravenously (i.v.)). The data suggested that 255 endowed good BBB crossing ability and was free from systemic orCNS toxicity. Furthermore, 255 was evaluated in a zebrafishmodel where no toxic effect was observed on zebrafish developmenteven at the highest tested concentration of 50 μM. Additionally, 255 reduced tumor growth over U3013 human glioma cells labeledwith cell tracker green zebrafish larvae, suggesting that 255 can inhibit human glioma cells without severe toxicity quinolin-4-yl](piperidin-2-yl)methanol(Vacquinol-1) acetohydroxamicacids that were active against multi-drug-resistant, GBM neurospherestem-like, and apoptosis-resistant cells.BMcells 59.122 Based on the scaffold, the hits were categorizedinto four classes: indolinones/imidazolinones, isoflavone, dihydroquinolinones,and miscellaneous. The hits were screened over D456 GBM PDX cells,where six compounds inhibited cell growth with IC50 valuesin the range of 1.69–41.22 μM  267 ligand that inhibited the growthof the LN229 glioma cell line with IC50 = 85.33 μM.124 In 2016, da Silva et al. reported thiazolidin-4-one-basedcompounds 268–271 as potential anti-GBMagents that reduced the viability of C6 cells by 61.2%, 52.2%, 48.0%,and 47.2%, respectively prodrug conjugate and evaluated its cell growth inhibitory effectsagainst glioma stem-like cells using a CellTiter-Glo luminescent assay.127 The results of the assay indicated that thePt(IV)-M13 conjugate (274) exhibited activity similarto that of cisplatin, with IC50 = 5 μM. Furthermore,the conjugates were evaluated for cell uptake and localization ofG9 glioma stem cells, where the Pt(IV)-M13 conjugate (274) displayed significant improvement (drug uptake) and was accommodatedin the cytosol. Additionally, pharmacokinetics and biodistributionstudies were performed, and the results revealed that the conjugatewas more stable in albumin and that more brain uptake was reported.Notably, a 15-fold higher amount of platinum was found in the brainafter treatment (5 h) with the conjugate compared with that aftercisplatin treatment (274) is an effective strategy to overcome the limitations of cisplatin.In 2016, Panayides et al. synthesized silyl- and trityl-substitutednucleosides that displayed promising U373 and Hs683 glioma cell growthinhibition with GI50 values in the range of 25–100μM.128 Among the evaluated compounds, 276 was the most abundant in the series, with GI50 = 25 and 27 μM, respectively. Additionally, the lipophilicityvalue for 275–277 was evaluated,and the ALogP values were 4.6, 2.6, and 5.0, respectively. In 2016,Hron et al. synthesized a series of 1,3-diazinane-5-carboxamide analogsof 1,3-diazinane-5-carboxamide , and their anti-canceractivity was evaluated in the LN-229 GBM cell line.129 Among the synthesized compounds, 278 was themost potent in the series and showed activity at 2.48 μM , a natural product obtained from the plant Helleborus odorus subsp. cyclophyllus (family Ranunculaceae),over three GBM cell lines.131 In studies,DHT significantly inhibited GBM cell viability, with IC50 = 7 × 10–5, 5 × 10–5, and 4 × 10–5 M over U251MG, T98G, and U-87GGBM cell lines, respectively. Further studies illustrated that DGHarrested the cell cycle at the G2/M phase, induced apoptosis, anddid not show any cytotoxicity in the zebrafish model -basednanovehicles (281), which ameliorated the half-life ofTMZ 3–4 times (5–7 h) compared with free TMZ (1.8 h)and significantly inhibited the growth of the GBM cell lines U-87MGand T98G  (PEG)-chitosanas a prodrug (282).135 Thedeveloped prodrug prudently elongated the half-life 7-fold at physiologicalpH and inhibited the growth of the GBM cell lines U118, SF767, andGBM6, with IC50 = 86.5, 66.0, and 119.8 μM, respectively via free radical copolymerizationwith enhanced high drug loading (50 mol% or greater).136 Moreover, the prodrug showed excellent watersolubility (>25 mg/mL) and provided stability to TMZ in aqueousmedium and inhibits the growthof the U-87EGFRvIII cell line, with IC50 = 1.78 μM.Moreover, 285 displayed significant anti-tumor activityover the in vivo xenograft model and showed promisingPK properties (polyMPC) polymer , and a series of 27 compoundswas introduced by utilizing alkylglycol polyethylene as linkers. Allthe synthesized compounds were evaluated over U251 EphA2 overexpressedand U251 wild-type GBM cell lines, where 295 and 296 were found to be the promising compounds in series whichactivity is shown in the figure. Moreover, both 295 and 296 induced the EphA2 phosphorylation which suggesting theEphA2 mediated anti-proliferative properties and make EphA2 as aneffective target against GBM . Some specific studies covered in this compilationexemplify the above-mentioned efforts of medicinal chemists, suchas the following: (i) the accommodation of memantine in the HDAC inhibitorystructural template to attain CNS-penetrating hydroxamic acids; (ii)the design of selective isoform inhibitors of PI3K to extract amplifiedanti-glioma efficacy; (iii) the aptamer functionalization of nanosystemsto target GBM through the BBB; (iv) the pragmatic design of HDAC6biased inhibitors based on the enhanced expression of HDAC6 isoformsin GBM; (v) the identification and modification of metabolic spots(vulnerable sites) of the potent anti-GBM agent to confer suitablePK properties; (vi) uncaging an inactive precursor of vorinostat byheterogeneous Pd catalysis in glioma cells; (vii) exploiting the prodrugstrategy to design adducts of 6-diazo-5-oxo-L-norleucine (DON) withimproved CSF delivery; (viii) the preliminary exploration of the rationallyconstructed dual HDAC/LSD1 inhibitor Corin for the treatment of DIPG,an incurable pediatric cancer; (ix) conventional structure-based virtualscreening to design selective anti-glioma effects of GULT inhibitors;(x) establishing the BET degrader ZBC260 as a potent inhibitor oftumor progression and stem-cell-like cells (GBM); and (xi) investigatingradiolabeled olaparib as a bio-imaging tool for glioma detection.The findings of the above-mentioned studies combined with other approachesencompassed in this Perspectove represent valuable information thatcan be leveraged to further numerous pursuits in this direction.Owing to the large number of scaffold furnishment programs conductedrecently, the pre-clinical pipeline of anti-GBM drugs comprises numerouscandidates that appear to be suitable chemical tools capable of overcomingthe obstacles of the anti-GBM drug discovery process and should beexhaustively explored to develop a therapeutic for GBM in the nearfuture. Some of the agents identified through the above-mentionedscaffold construction approaches have already entered clinical trials,and some of them are expected to emerge as effective therapeuticsfor GBM. Overall, this Perspective highlights the significant advancementsin the field of anti-GBM drug discovery with clear-cut knowledge ofthe challenges associated with the development of CNS drugs, particularlythe ideal physicochemical properties required by chemotherapeuticsfor GBM. Although the stage appears to be set for the near future,efforts must be precisely channeled toward exploiting the promisedemonstrated by the numerous studies covered in this Perspective.Given the historical failure associated with the clinical advancementof GBM drugs, the challenge is relatively more stringent comparedwith that encountered in other malignancies. Thus, the expertise ofinterdisciplinary teams composed of medicinal chemists, organic chemists,biologists, and formulation chemists along with researchers’well-versed computational aspects of drug design would be requiredto steer the clinical progress of the candidates covered in this Perspective.