Metaverse technology is spurring a transformation in healthcare and has the potential to cause a disruptive shift in rehabilitation interventions. The technology will surely be a promising field offering new resources to improve clinical outcomes, compliance, sustainability, and patients' interest in rehabilitation. Despite the growing interest in technologies for rehabilitation, various barriers to using digital services may continue to perpetuate a digital divide. This article proposes a framework with five domains and elements to consider when designing and implementing Metaverse-based rehabilitation services to reduce potential inequalities and provide best patient care.The framework was developed in two phases and was informed by previous frameworks in digital health, the Metaverse, and health equity. The main elements were extracted and synthesized via consultation with an interdisciplinary team, including a knowledge user.The proposed framework discusses equity issues relevant to assessing progress in moving toward and implementing the Metaverse in rehabilitation services. The five domains of the framework were identified as equity, health services integration, interoperability, global governance, and humanization.This article is a call for all rehabilitation professionals, along with other important stakeholders, to engage in developing an equitable, decentralized, and sustainable Metaverse service and not just be a spectator as it develops. Challenges and opportunities and their implications for future directions are highlighted. HealthcSnow Crash by Neal Stephenson, the roots of Metaverse date back further in the 20th century  Augmentation: technologies that layer information onto our perception of the physical environment; (2) Simulation: technologies that model reality; (3) Intimate: technologies that are focused inwardly on the identity and actions of the individual or object, and (4) External: technologies that are focused outwardly towards the world at large . A MetavDespite the concept of the Metaverse being proposed for decades, a unified architecture still lacks. In their respective works, Duan et al.  and Jon While it is not the purpose of this article to explore each component's specifics, it is important to highlight that the Metaverse relies on two essential resources, hardware and software, which hold potential applications in the field of physical rehabilitation. Hardware encompasses critical components such as chips, display screens, interactive devices, and terminals. On the other hand, software includes operating systems, rendering tools, computing capabilities, memory storage, and file data . Proper In the context of physical rehabilitation, VR has gained popularity as a valuable tool for treating various conditions. It has been employed in balance training to enhance balance and mobility for individuals with Parkinson's disease , post-toExamples of specific devices applicable to the virtual rehabilitation world include the 3D Oculus Rift, one of the pioneers in consumer-based VR headsets developed by Oculus in 2014. This type of headset may be useful in making repetitive rehabilitation exercises more immersive and engaging and can simulate movements used in everyday life for people with various physical and neurological impairments. For instance, patients recovering from stroke can use the Oculus device to virtually practice reaching for objects , which mThe rapid digitization of healthcare must be approached cautiously to avoid increasing health inequities. Virtual care (VC) will be compromised if solutions are not developed to address current limitations and barriers. Many studies have pointed out the limitations of poor access to the internet, privacy and security issues, and technological and digital literacy as barriers to VC in several population groups –23. SomeThis paper embodies the latest and most comprehensive discussion in the field of Metaverse development. Its novelty lies in its selection of the crucial domains within Metaverse development and its effective adaptation to the field of rehabilitation. Particularly, this work stands as the pioneering work in proposing a Metaverse framework tailored specifically for the rehabilitation domain, encompassing various health professions like physical therapy, occupational therapy, and speech therapy, which collectively contribute to the advancement of rehabilitation practices. To the best of our knowledge, no previous effort has been undertaken to establish a framework that facilitates the development of the Metaverse specifically dedicated to systematizing and formalizing the state-of-the-art advancements in Metaverse technology for rehabilitation purposes. Subsequently, this paper aims to present a framework to ensure equitable and accessible rehabilitation services and research using the Metaverse for delivering care or designing research studies.2.(1) identifying data to inform the methodological framework; (2) developing the methodological framework; and (3) validating, testing and refining the methodological framework”  framework was thus conducted in two phases. The first phase identified evidence from the literature to inform this framework by searching for previous frameworks or guidance pertaining to the equitable aspects of the Metaverse. Our search strategy was based on a review of literature on equity frameworks, supplemented by additional searches for specific terms associated with the Metaverse . Searches were performed on PubMed, Google Scholar, and grey literature , Microsoft, Descentraland (Metaverse Platform), and NVIDIA) and Metaverse Standards Forum. The main studies used for this first phase were: The Health Equity Framework by Peterson et al. (2021) , A FrameThe second phase consisted of developing a framework by extracting and synthesizing the main elements of equity, digital health, and Metaverse frameworks. This involved an interdisciplinary team of clinicians and researchers within the fields of rehabilitation, computer sciences, equity, and global health, as well as a knowledge user. After the synthesis, the results were returned to the study team for refinement. This iterative approach was followed until consensus was reached by the study team on the domains and subdomains to be included.3.3.1.The proposed framework is composed of five domains, each with several subdomains, to guide and support both researchers and rehabilitation clinicians who use the Metaverse, as outlined in 3.1.1.Health equity is a priority in public health and also in rehabilitation services . Canada,Haptics and haptic technology are crucial to those deafblind individuals who depend on touch to interact with the world and present a revolutionary intervention to improve accessibility. Haptics technology facilitates interaction with tools and other users by transmitting the sense of touch over distance . It enabFurther, to increase the accessibility of Metaverse technology in rural and remote areas, the development of “Metaverse cafes” where Metaverse technology such as headsets, interaction and sensory feedback devices, and sufficiently fast internet connection could be publicly accessed would be beneficial. These would allow communities to have greater public access to this technology and would minimize the number of technological resources needed as they could be shared among community members. However, there would be other considerations with this, such as ensuring the sustainability of these cafes and providing transportation support to community members living in these remote areas.As we strive to enhance accessibility, it is essential to guarantee that the services maintain their quality and effectiveness. In a recent randomized controlled trial investigating the therapeutic effects of Metaverse rehabilitation for cerebral palsy, researchers compared the impact of Metaverse physical therapy and conventional physical therapy on different outcomes  . The stup < 0.001) as measured by the Modified Oswestry Low Back Pain Disability Index, and Neck Disability Index improved by 23.2% (p = 0.02). These findings highlight the potential benefits of incorporating VR in pain management and rehabilitation, offering a comprehensive approach to address various aspects of patients' pain  compared to other populations. In addition, Metaverse technology should be piloted within these communities to ensure that they are appropriate, as we recognize there is cultural diversity across communities even within a single population and cannot assume a “one-size-fits-all” for these technological resources. Features of physical appearance, such as body shape and size, hair color/style, clothing, and accessories that include racial preferences, should have similar costs to most choices.one of the fields of computer science with a mathematical process that has the potential to enhance the healthcare system via novel delivery strategies, informed decision making, and facilitation of patient engagement” . Opportunities, for example, can include enhanced communication between health professionals and aiding them in performing their clinical tasks such as education, reminder, etc. The landscape of the rehabilitation field is changing with the introduction of AI, defined here as “agement” . AI can agement” .Furthermore, outside of clinical practice, at an educational level, the Metaverse could also improve training opportunities for health professionals to participate in simulations and VR training exercises to practice their rehabilitation skills . Rehabilitation in the Metaverse also has the potential to deliver rehabilitation services for long-term risk factor management, consequently improving fairness in the access to rehabilitation services for patients.3.1.2.Integration and continuity of care are essential for a health system to provide effective rehabilitation services. Health systems worldwide face challenges in responding to the complexity of the needs of aging populations with chronic diseases . IntegraWhen recommending VC, we acknowledge the significance of neuroplasticity, which involves repetition in the process of rehabilitation. Utilizing a virtual world for rehabilitation can offer distinct advantages, such as increased motivation and the ability to facilitate repetitive exercises crucial for promoting neuroplasticity . HoweverSeveral studies have indicated that adopting a less engaging lifestyle over time can accelerate cognitive decline due to reduced cognitive reserve, impacting the brain's ability to withstand age-related changes and pathology . Similarhealth system strategy in which many figures are involved, in which the patient should play the leading role together with the collaboration with a care coordinator through self-management, active collaboration and sharing of information”  while maintaining experience and assets such as taking your avatar and medical records with you . Another3.1.4.a system of norms governing the creation and enforcement of content policies, as well as the distribution of powers among entities responsible for each task”  but not supported yet by Health Canada? Will the regulatory agencies worldwide work together to regulate devices/technologies used in the Metaverse? There are more questions than answers when considering the global perspective. The Metaverse has the potential to remove many of the geographic boundaries and improve access to rehabilitation services worldwide, but therein lies a problem when considering different health regulations, including those for professionals in other provinces, states, and countries. Should the Metaverse follow the same real-world laws as a digital version of the analog  world? Which laws will be different, or which ones will extend to the healthcare Metaverse? These are examples of foreseen issues to be discussed and evaluated from equitable, accountability and ethical perspectives. According to Fernandez and Hui, the Metaverse should be inclusive and outside of local regulations that diminish users' freedom . TherefoMost publications on the Metaverse advocate for a decentralized Metaverse using blockchain technology and a distributed network with a decentralized ownership structure where no single entity can own a Metaverse . The ownRehabilitation professionals must encourage the participation of underrepresented groups and advocate for their voting rights, their agency and participation. A system of governance must protect populations such as children, older adults, visible minorities, Indigenous peoples, and all people with disabilities and, at the same time, allow innovations. Health technology is advancing, but access needs to be more equitable. As rehabilitation professionals continue to rely on technological innovations to improve patient engagement, access, and outcomes, a new social determinant of health has been identified: the digital divide . AlthougOther important governance questions to consider are: what are the environmental impacts of the Metaverse, and what is the role of rehabilitation professionals regarding the effects of climate change on health? Many experts believe blockchain technology establishes a transparent, immutable, decentralized network for the Metaverse. It operates autonomously with a decentralized content ledger and authorizes several parties to transact between themselves and record those transactions on the blockchain without a centralized authority . The tecWhile blockchain technology has the potential to make rehabilitation services in the Metaverse more secure and private, it raises climate concerns due to the streaming of data and its analogy to the carbon footprint of computational demands of mining and subsequent substantial contribution to global carbon emissions. These concerns about carbon emissions have been previously mentioned, and many companies such as Google, Amazon, and Microsoft have expressed their commitment to carbon-free energy . In the In addition to reducing patient (even therapist) travel for services, especially a concern for remote communities, it is possible that the Metaverse could positively impact the environment over the long term by substantially reducing carbon emissions through the substitution of physical efforts for digital ones, reducing the number of commuting employees and physical plant considerations resulting in fewer transport and provider-related emissions, replacing real-world presence with virtual interactions .The World Health Organization emphasizes that the risks to human health due to climate change undermine many social determinants for good health, such as access to health care and social support structures, equality, etc. . In 20193.1.5.a process of communication and caring among people that leads to self-transformation, an understanding of the fundamental spirit of life, and a sense of compassion and unity”  in the Metaverse's development and implementation. While the primary focus of this paper centers on the rehabilitation field of practice, it is worth briefly noting the potential impact of the Metaverse in the field of education. Specifically, the Metaverse is anticipated to bridge the gap between abstract concepts and tangible experiences for rehabilitation professionals. In the field of medicine, discussions have arisen regarding the Metaverse's potential in medical education, emphasizing the importance of incorporating digital health approaches such as virtual reality and the Metaverse in medical and rehabilitation training. To effectively pursue a successful integration of the Metaverse into rehabilitation, we will be called upon to break down the silos and work efficiently and effectively in a shared space with other sectors, where we can collectively address the already known gaps in VC/telerehabilitation that contribute to the digital divide.The domains and subdomains described in this article are a sine qua non to have accountable, transparent, effective, efficient and inclusive rehabilitation interventions in the Metaverse. The proposed framework can now be evaluated and refined using an intervention or case study to test the recommended domains.


Kaempferia parviflora rhizomes, its leaves are wasted as by-products. The foliage could be an alternative source to rhizome, but its VOCs composition has not been explored previously. In this study, the VOCs in the leaves and rhizomes of K. parviflora plants grown in a growth room and in the field were analyzed using the headspace solid-phase microextraction (HS-SPME) method coupled with gas chromatography and time-of-flight mass spectrometry (GC-TOF-MS). The results showed a total of 75 and 78 VOCs identified from the leaves and rhizomes, respectively, of plants grown in the growth room. In the field samples, 96 VOCs were detected from the leaves and 98 from the rhizomes. These numbers are higher compared to the previous reports, which can be attributed to the analytical techniques used. It was also observed that monoterpenes were dominant in leaves, whereas sesquiterpenes were more abundant in rhizomes. Principal component analysis (PCA) revealed significantly higher abundance and diversity of VOCs in plants grown in the field than in the growth room. A high level of similarity of identified VOCs between the two tissues was also observed, as they shared 68 and 94 VOCs in the growth room and field samples, respectively. The difference lies in the relative abundance of VOCs, as most of them are abundant in rhizomes. Overall, the current study showed that the leaves of K. parviflora, grown in any growth conditions, can be further utilized as an alternative source of VOCs for rhizomes.Volatile organic compounds (VOCs) play an important role in the biological activities of the medicinal Zingiberaceae species. In commercial preparations of VOCs from  In addition, it was shown that 21 volatiles were significantly more abundant in the leaves than in the rhizomes. In contrast, 68 compounds were significantly higher in the rhizomes. Overall, it is evident that there is wide variation in the relative abundance of VOCs in K. parviflora tissues according to the change in growth environment.As indicated in 2-transformed fold-change values of normalized responses of each annotated compound, and the extent of any significant difference was assessed with a p value< 0.05  were recorded based on logue< 0.05 .In the case of the rhizomes, the relative abundance of almost all volatiles were significantly higher in the field conditions compared to the growth room, except for three compounds: 2 hexenal, octanal, and hexadecenal. A similar trend was observed in leaf volatiles. Only neophytadiene and borneol showed no significant differences with the change of growth environment. These results suggest that in both tissues, the field condition contributed a significant change in the VOC profile of the plant in terms of quality and relative quantity. This is consistent with the PCA results that the Field_ Rhizome and Field_Leaf groups were clearly separated from the growth room samples.® and the log2-transformed values explained that tissues from the field contain significantly higher numbers and relative abundances of VOCs. Moreover, K. parviflora plants are normally cultivated in field conditions for commercial purposes.In line with the objective of the study, to evaluate whether leaves can be used as substitutes for rhizomes, field data were prioritized and used to identify the volatiles with similar compositions in both tissues. This is because the previously reported information from SIMCA2-transformed values, it was already mentioned that a total of 11 leaf volatiles had similar relative abundances of VOCs with those of the rhizomes in the field conditions . These are humulene, caryophyllene, 1,4-dichlorobenzene, m-mentha-1(7),8-diene, carveol, nerolidol, bicyclo [4.2.0]-oct-1-ene, 7-endo-ethenyl-, methyl palmitate, β-myrcene, allo-aromadendrene epoxide, and caryophyllene oxide. Box plot diagrams for these compounds were created based on the normalized peak intensities  were the major group in rhizomes. This result was consistent with the results of other studies on K. parviflora rhizomes [K. galanga, phenylpropanoids constitute the largest proportion of their rhizomes [10H16) are the dominant compounds in K. parviflora leaves, which is first time that this has been reported for the species. Rhizomes are grown underground and exposed to pathogenic attacks by fungi and bacteria. Sesquiterpenes are known to have antifungal and antibacterial properties [K. parviflora rhizomes and leaves are both potential sources of several active pharmacological compounds with potential commercial applications.In this research, a non-targeted approach coupled with HS-SPME-GC-TOF-MS was applied for the identification of VOCs from the rhizomes and leaves of erpenes CH24 were rhizomes ,49,50. Irhizomes ,91. On toperties ,92,93. OK. parviflora plants grown in two different environments . This may be related to the large VOC pool synthesized in the secretory tissues of rhizomes. VOCs are synthesized in oil and resin ducts, or glandular trichomes, and easily released from storage pools due to high temperature or herbivore feeding or movements on surface [K. parviflora, the rhizome has many oil ducts, while the leaves have few and no trichomes. On the contrary, there were also VOCs which were significantly abundant in the leaves compared to the rhizomes . These compounds have the potential to substitute the same compounds from rhizomes, while also taking into consideration their economic values.However, the main difference in the VOCs of the tissues lies in the relative abundance of these compounds. This is estimated in the study by calculating the logompounds . The res surface . In the K. parviflora rhizomes and leaves collected from plants grown in the growth room and open field were explored for the first time using the HS-SPME approach coupled with non-targeted GC-TOF-MS. This study affirmed that the utilized technique used is more efficient for analyzing the VOCs of the samples. It also confirmed that the composition of VOCs is highly affected by the growth conditions. The field-grown plants showed higher numbers and abundances of volatiles compared to plants grown in the growth room. In terms of the VOC composition of the tissues, monoterpenes were found to be the major VOCs of leaves, whereas the rhizomes were rich in sesquiterpenes. The majority of the VOCs present in the rhizomes were also found in the leaves. However, the concentrations of most of the rhizome VOCs were higher than those of the leaves. Nevertheless, the findings confirmed that K. parviflora leaves are also a rich reservoir of volatile organic compounds. Therefore, for economic purposes, fresh rhizomes of field-grown plants, as well as their leaves, can be utilized as sources of valuable volatiles.The VOC profiles of


Aberrant androgen receptor (AR) signaling drives prostate cancer (PC), and it is a key therapeutic target. Although initially effective, the generation of alternatively spliced AR variants (AR-Vs) compromises efficacy of treatments. In contrast to full-length AR (AR-FL), AR-Vs constitutively activate androgenic signaling and are refractory to the current repertoire of AR-targeting therapies, which together drive disease progression. There is an unmet clinical need, therefore, to develop more durable PC therapies that can attenuate AR-V function. Exploiting the requirement of coregulatory proteins for AR-V function has the capacity to furnish tractable routes for attenuating persistent oncogenic AR signaling in advanced PC. DNA-PKcs regulates AR-FL transcriptional activity and is upregulated in both early and advanced PC. We hypothesized that DNA-PKcs is critical for AR-V function. Using a proximity biotinylation approach, we demonstrated that the DNA-PK holoenzyme is part of the AR-V7 interactome and is a key regulator of AR-V–mediated transcription and cell growth in models of advanced PC. Crucially, we provide evidence that DNA-PKcs controls global splicing and, via RBMX, regulates the maturation of AR-V and AR-FL transcripts. Ultimately, our data indicate that targeting DNA-PKcs attenuates AR-V signaling and provide evidence that DNA-PKcs blockade is an effective therapeutic option in advanced AR-V–positive patients with PC.  Prostate cancer (PC) is the third most common malignancy worldwide. The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that regulates a canonical gene expression program involved in prostate homeostasis and, upon deregulation, cancer development , 2. The AR gene  is a serine/threonine protein kinase complex that consists of a Ku heterodimer (Ku70/Ku80) and a catalytic subunit (DNA-PKcs). It has a critical role in the DNA damage response (DDR) through facilitation of double-strand break repair via the nonhomologous end joining pathway –29. Outsignaling ; (b) supignaling ; and (c)ignaling . Given tIn contrast to our understanding of the functional relationship between AR-FL and DNA-PKcs, interplay between AR-Vs and DNA-PKcs remains poorly characterized. Outside of the demonstration that the AR isoforms AR-V7 and ARv567es interacted with DNA-PKcs in CWR22Rv1  and R1-DTo that end, we developed and utilized an APEX2 peroxidase-mediated biotinylation pipeline , which hcis-regulatory elements of canonical AR-target genes in CWR22Rv1-AR-EK and CWR22Rv1 cells to levels equivalent to untagged endogenous AR-V7 (https://doi.org/10.1172/JCI169200DS1)  have helped define the interactome of the AR variant ARv567es in R1-D567 PC cells in steady-state and in response to DNA damage , 41. To d in 2O2 , the magd in 2O2 . Importad in 2O2 , we chos2O2) compared with the control (–H2O2)  value that represents individual protein abundance. The protein lists were then processed to omit common contaminants identified in the control experiments and to enrich for proteins that were identified by 2 or more unique peptides. As expected, we identified considerably more proteins in the –IR and +IR experimental arms (+H (–H2O2) , and bot (–H2O2) , B and CReassuringly, AR was the fourth most abundant protein in both the –IR and +IR arms, and known AR interacting proteins were identified, including PARP1  and FUS PRKDC (DNA-PKcs) expression in primary and metastatic disease has been previously reported, which we confirmed by analyzing 2 additional data sets from the The Cancer Genome Atlas (TCGA)   and Grass (TCGA)   . Given tas found . Importaas found  to levelas found , suggestas found , furtheras found . Two addas found , with siompounds . Subsequ S phase .PSA, KLK2, UBE2C, and CCNA2 in CWR22Rv1-AR-EK cells  identified 3 shared DNA-PKI–altered pathways among NU7441, NU5455, and AZD7648 gene sets, “p53,” “cell cycle,” and “DNA replication”  or treated with vehicle, 1 μM NU7441, NU5455, or AZD7648 for 24 hours . Validatication” . We nextication” , 47. Surication”  and RNA-ication” .AR-V7, as well as AR-V1, -V6 and -V9 and FL-AR in VCaP, CWR22Rv1-AR-EK and CWR22Rv1 cells  to determine whether DNA-PKcs (PRKDC) mRNA expression was associated with AR/AR-V7 mRNA and AR/AR-V7 activity scores  and with AR-V7 mRNA expression in the SU2C/PCF cohort  but not the ICR/RMH cohort   and AR-V7  activity scores in both cohorts studied  . Interes studied . TogetheAR-V (and FL-AR) mRNA levels in PC cells, we next sought to define the mechanism(s) of DNA-PKcs–mediated regulation of AR-V expression. First, we examined whether DNA-PKcs controlled deposition of AR at the downstream repressive element (DRE) in intron 2 of the AR gene, which has been shown to negatively regulate AR expression in PC cells  and siDNA-PKcs  treatment , 1,167 of which were statistically significant, with alternative first exon and exon skipping being the most predominant changes . Crucialdetected . Althougdetected , reassurnd AR-FL . It is tnt AR-Vs .RBMX mRNA expression significantly positively associated with AR mRNA expression in the ICR/RMH cohort , but not the SU2C/PCF cohort , and with AR-V7 mRNA expression in the ICR/RMH cohort , but not the SU2C/PCF cohort   and AR-V7  activity scores in both cohorts  CWR22Rv1-AR-EK cells was reduced upon RBMX knockdown, which, we speculate is, in part, a consequence of reduced AR isoform expression  . Interes cohorts , supportpression . Togetheactivity , deregulAR gene mutations and amplification remains largely sensitive to these therapies AR-Vs in PC cell lines. We subsequently identified RBMX as a suitable candidate for regulating pathogenic splicing in advanced PC. By qRT-PCR and ChIP analyses, we showed that RBMX is a direct target gene of DNA-PKcs and detected a significant correlation of DNA-PKcs and RBMX transcript levels in several large PC patient data sets. Partial rescue of AR-V transcript levels by RBMX overexpression in cells treated with DNA-PK inhibitors supports DNA-PKcs-RBMX interplay in controlling AR-V expression. Importantly, RBMX knockdown reduced transcripts of multiple AR-Vs, and AR-FL, without affecting AR pre-mRNA abundance, suggesting that RBMX controls AR mRNA processing downstream of transcription. This notion is supported by the demonstration that (a) RBMX interacts with the phosphorylated C-terminal domain of RNA polymerase II to cotranscriptionally regulate splicing  RBMX depletion does not affect AR isoform turnover or ectopic expression of prespliced AR-V7 cDNA; and (d) RBMX and AR-V7 transcript abundance correlate in PC. Mechanistically, we provide evidence that RBMX enhances inclusion of spliced exon 2–exon 3–containing mRNAs, which encode many clinically relevant AR-Vs, including AR-V1, AR-V6, AR-V7, and AR-V9 and AR-FL, explaining the observed reduction in these transcripts when RBMX is depleted. All together, we have uncovered a regulatory pathway involving DNA-PKcs and RBMX that controls generation of AR-Vs.One consequence of these splicing alterations in response to compromised DNA-PKcs activity was the detection of markedly reduced expression of multiple splicing ; (b) RBMPRKDC and RBMX mRNA expression associate with AR/AR-V7 mRNA expression and AR/AR-V7 activity scores in one or more cohort studied, suggesting a role in AR RNA processing. Despite these interesting findings, it is important to consider the limitations of these studies, which include potential discordance between mRNA and protein expression or activity, and heterogenous patient cohorts in terms of both treatments received and timing of the CRPC biopsies. Consistent with this, these interesting findings will need to be further validated using orthogonal methods, in larger patient cohorts with homogenous treatments, preferably in prospective clinical studies with multivariate analyses to confirm both the prognostic and predictive nature of DNA-PK (PRKDC) and RBMX in CRPC.When considering the clinical relevance of these findings we have used two independent CRPC patient transcriptome cohorts to demonstrate that both Our findings provide a rationale for applying DNA-PKIs in AR-V–positive patients with PC by demonstrating they partially inhibit AR-V signaling and decrease AR-V synthesis, which together decrease variant-driven PC cell growth. Of note, however, is that, although each of the tested DNA-PKIs demonstrated comparative phenotypic impact, there was considerable discrepancy in the magnitude of gene expression changes among NU5455, NU7441, and AZD7648, with the former demonstrating considerably more DEGs than the other 2 compounds. NU5455 is more selective than the first-generation DNA-PKI NU7441 . OutsideIn all, our data have provided robust evidence that DNA-PKcs is a key regulator of AR-Vs at multiple levels and suggest a tractable route for AR-V inactivation using clinically relevant DNA-PKIs.The siRNA screen of 315 genes related to the spliceosome in 22Rv1 cells was performed previously . AR, AR-P values of less than 0.05 were considered significant.Unless otherwise stated, all graphical data represent the mean of 3 individual experiments, and data are shown as the mean ± SEM. For analysis of DNA-PKcs inhibition on AR-mediated gene expression, ChIP, and cell viability in CWR22Rv1-AR-EK cells, 1-way ANOVA was conducted. For cell viability in CWR22Rv1 and VCaP cells, a 2-way ANOVA was conducted using Prism 8 software. Approval for patient involvement in this study was granted by the Royal Marsden Hospital Ethics Review committee (reference 04/Q0801/60), as described in Fenor de la Maza et al .RNA-Seq data generated in this study are publicly available at GEO (accession GSE242255). Additional information can be found in the BA performed most of the experiments and, together with NB, developed the biotinylation pipeline. LW, RD, SL, JMJV, AP, JW, and LG performed some experiments. SM and RJSB provided guidance on biotinylation sample preparation and conducted all proteomics. BA, NB, SL, DB, WY, and GS performed all bioinformatics. SRM, CNR, EW, RH, PR, IH, AS, JSDB, and LG devised the project. BA, AS, and LG wrote the paper.


H)-yl)-acetohydrazide (4) has twenty-nine compounds. The starting material for their corresponding mono, dipeptides and reactions with active methylene compounds were produced by chemoselective N-alkylation of 4-Benzyl-2H-phthalazin-1-one (2) with ethyl chloroacetate to afford  methyl acetate (3). The ester 3 was hydrazinolyzed to give hydrazide 4, then azide 5 coupled with amino acid ester hydrochloride and/or amines to produce several monopeptides, then the methyl -yl) acetyl) glycinate (7a) was hydrazinolyzed to produce corresponding hydrazide 2--yl)-N-(2-hydrazineyl-2-oxo ethyl) acetamide (8a). The hydrazide 8a under azide coupling method was coupled with amino acid ester hydrochloride and/or amines to produce several dipeptides, and the hydrazide 8a was also condensed and/or cyclized with several carbonyl compounds. The cytotoxicity of the synthesized compounds was tested using MTT assay, as well as apoptosis-induction through EGFR inhibition. Compounds 11d, 12c and 12d exhibited potent cytotoxic activities with IC50 values of 0.92, 1.89 and 0.57 μM against MDA-MB-231 cells compared to Erlotinib (IC50 = 1.02 μM). Interestingly compound 12d exhibited promising potent EGFR inhibition with an IC50 value 21.4 nM compared to Erlotinib (IC50 = 80 nM). For apoptosis, compound 12d induced apoptosis in MDA-MB-231 cells by 64.4-fold (42.5% compared to 0.66 for the control); hence, this compound may serve as a potential target-oriented anti-breast cancer agent. These results agreed with the molecular docking studies that highlighted the binding disposition of compound 12d towards EGFR protein. Hence, compound 12d may serve as a potential and selective anti-breast cancer agent.The parent 2-(4-benzyl-1-oxophthalazin-2(1The online version contains supplementary material available at 10.1186/s13065-023-00995-2. Cancer has been considered one of the major issues of concern, most especially for the public health system globally, which has been a leading cause of death worldwide in the last decade . It is aAfter over half a century of chemotherapy research and despite the advancement in the knowledge of biochemical processes associated with carcinogenesis, the successful treatment of cancer remains a significant challenge because of the general toxicity associated with the clinical use of traditional cancer chemotherapeutic agents and because of some factors that include limitations of animal models, tumor diversity, drug resistance and the side effects assigned for therapy . TherefoEpidermal growth factor receptor (EGFR) is a type of membrane-bound tyrosine kinase receptor which addicted to the treatment of cancer . EGFR pl. Previous literacture alkanoate7a–c -phthalazinone 7a has the 1H-NMR spectrum of characteristic signals at δ 6.71 broad signal for NH molecule, 3.64 singlet peak of OCH3, 4.00–4.01 doublet peak of CH2NH, 4.23 and 4.90 ppm singlet peaks for CH2ph & CH2CO respectively and the 13C-NMR spectrum has signals at 169.99, 167.72 and 159.78 for three C=O groups, 54.80, 52.27, 41.29 and 38.91 ppm for CH2CO, OCH3, CH2NH and CH2ph groups respectively.The glycine methyl ester of 4-benzyl-1-yl)-N-(2-hydrazineyl-2-oxo ethyl)acetamide (8a) was elucidated by various analysis like 1H-NMR which give characteristic peaks for protons at δ 9.02 & 7.91–7.93 broad signals for 2 NH, 4.87 singlet peak for CH2CO, 4.32 singlet peak for CH2ph, 4.23 doublet peak of CH2NH and doublet peak at 3.74–3.75 for NH2 and the 13C-NMR spectrum has signals at 168.38, 167.73, 159.08 for three C=O groups and peaks at 53.98, 41.43, 38.13 ppm for CH2CO, CH2NH and CH2ph respectively.The structure of starting hydrazide 2--yl)-N-(2-hydrazineyl-2-oxoethyl)acetamide (8a) was treated with a mixture of sodium nitrite and HCl solution in water to give its corresponding azide solution which further reacted with different amines like benzyl, n-propyl, n-butyl, cyclohexyl, tetra decyl, allyl, pepridine and morphline amines to obtain N-substituted-2--yl)-2-oxoethyl) acetamides 10a–h, as in Scheme Under azide coupling condition, 2--yl)-N-(2-(butyl amino)-2-oxoethyl) acetamide (10c) was interpreted by 1H-NMR analysis including two broad singlet peaks at δ 6.76 and 6.50 for NH, 4.94 singlet peak of NCH2CO, 4.34 singlet peak of CH2ph, 3.99 doublet peak of NHCH2CO, 3.24–3.29 quartet peak for NHCH2CH2, 1.48–1.54 quintet peak for CH2CH2CH2, 1.32–1.37 sextet peak of CH2CH2CH3 and 0.90–0.93 triplet peak of CH3 and the 13C-NMR spectrum has signals at 168.34, 167.85 and 159.94 for three C=O groups, 55.80, 43.43, 39.41, 38.92, 31.41, 20.01 and 13.67 ppm for NCH2CO, NHCH2CO, NHCH2CH2, CH2ph CH2CH2CH2, CH2CH2CH3 and CH3 groups respectively.The chemical structure of 2--acetylamino]alkanoates 11a–d in reasonable yield -yl) acetamido acetamido) propanoate (11b) was interpreted by various analysis method including 1H-NMR analysis that noticed characteristic peaks: two broad singlet peaks at δ 6.88 for NH, 4.95 singlet peak of NCH2CO, 4.34 singlet peak of CH2ph, 3.97–3.98 doublet peak of NHCH2CO, 3.67 singlet peak of OCH3, 3.54–3.55 quartet peak for NHCH2CH2CO and 2.56–2.59 triplet peak for NHCH2CH2CO and the 13C-NMR spectrum has peaks at 172.50, 168.57, 167.88 and 159.93 of four C=O, 55.59, 51.73, 43.25, 38.91, 35.18 and 33.71 ppm for NCH2CO, OCH3, NHCH2CO, CH2ph, NHCH2CH2CO and NHCH2CH2CO respectively.The structure of methyl 3--yl)-N-(2-hydrazineyl-2-oxoethyl)acetamide (8a) with active methylene compounds such as malononitrile, ethyl cyano acetate and acetyl acetone in ethanol under reflux to obtain novel derivatives of phthalazinone 12a, 12d and 12e respectively in reasonable yield. Similarly, reaction of hydrazide 8a with ketones such as cyclohexanone and 2-furyl methyl ketone gave the corresponding hydrazones 12b and 12c respectively as shown in Scheme Condensation of the hydrazide 2--yl)-N-2--2-oxoethyl)acetamide (12a) was elucidated using various analysis methods for example 1H-NMR which gave signals at δ 9.02 broad signal for NHCH2CO, 7.10–7.12 singlet peak for the proton of pyrazole ring NH2-C=CH-C-NH2, 5.46–5.47 doublet peak for NHCH2CO, 4.86 singlet peak of NCH2CO, 4.26 singlet peak of CH2ph and 4.32 & 3.52 two broad singlet peaks for 2 NH2 molecules on pyrazole ring and the 13C-NMR spectrum has peaks at 171.42, 168.34 and 159.21 of three C=O, 148.14 & 148.36 peaks of carbons of pyrazole ring 2 C-NH2, 110.35 characteristic peak for carbon atom of NH2-C=CH-C-NH2, 55.28, 42.82 and 38.64 ppm for NCH2CO, NHCH2CO and CH2ph respectively.The chemical structure of 2-, and compounds 11a, 7c, 10f, and 10 h with second order activity of moderate cytotoxicity (IC50 ≤ 20 μM), while compounds 6 h, 6a and 6f with poor cytotoxicity (IC50 ≥ 20 μM). As summarized in Fig. 12d was worthy to be further investigated for the effective target and cell death mechanism.Based on the cytotoxicity results of the investigated compounds as summarized previously in Table 11d, 12c, and 12d were tested for their inhibition against VEGFR2; interestingly, as seen in Table 12d exhibited promising EGFR enzyme inhibition with IC50 values of 21.4 nM with 97.6% inhibition compared to erlotinib with standard EGFR inhibition with IC50 value of 80 nM (inhibition 93.9%). Additionally, compounds 11d and 12c exhibited promising EGFR inhibitory activities with IC50 values 79.6 and 65.4 nM, with enzyme inhibition by 92.9% and 96.2%, respectively.Three compounds with potent cytotoxicity 12d  and were investigated for their apoptosis-inducing activity using Annexin V/PI staining. As seen in Fig. 12d significantly stimulated total apoptotic breast cancer cell death by 64.4-fold (42.5% compared to 0.66% for the control). It induced early apoptosis by 24.2% and late apoptosis by 18.3% compared to 0.66% and 0.15%, respectively, for the control. Moreover, it stimulated cell death by necrosis by 9.25-fold .Deregulation of apoptosis is a hallmark of all cancer cells, and the agents that activate apoptosis in cancer cells could be valuable anticancer therapeutics; breast cancer cell lines that hyper express the EGFR have been documented to undergo receptor-mediated apoptosis. MDA-MB-231 cancer cells were treated with compound These results of apoptosis-induction of phthalazine-based derivatives agreed with previous studies that exhibited promising cytotoxic activities as apoptotic agents through EGFR inhibition.12d, it was screened for virtual binding towards EGFR protein using the molecular docking approach. As shown in Fig. 12d was docked inside EGFR protein with a binding energy of − 18.4 kcal/mol and formed one H-bond with Met 769, one H-bond with Lys 721, besides it formed the lipophilic interactions through phenyl groups with the lipophilic amino acids of Ala 719 and Leu 694. Hence, docking results indicated highlighted the virtual mechanism of binding of compound 12d through the phthalazine moiety for interactions toward EGFR protein, which agreed with its promising experimental activity. Physiochemical properties and ADME pharmacokinetics revealed the drug-likeness score of 1.09, which obeys the Lipinsiki’s rule of five, having molecular weight = 432 g/mol, topological polar surface area (TPSA) = 144.8 A2, log (P) = 1.56, H-bond donor = 4, and H-bond acceptor = 5.Based on the promising EGFR inhibition activity of compound 254 aluminum sheets  in the following solvent system ethyl acetate/ petroleum ether (1:1) and ethyl acetate/ petroleum ether (2:1), the spots on thin layer plates were detected by UV lamp. The melting points were determined using a Buchi 510 melting-point system and are uncorrected. At the Micro Analytical Laboratory, Faculty of Science, Cairo University, Cairo, Egypt, element analyses were performed on a Flash EA-1112 apparatus. The nuclear magnetic resonance laboratory, Faculty of Science, Sohag University, Egypt, used a Bruker spectrometer running at 400 MHz to estimate 1H-NMR spectra.The purity of the synthesized compounds was checked by thin layer chromatography (TLC) technique was carried out on silica gel 60 FH -phthalazin-2-yl) methyl acetate (3) was prepared from 4-Benzyl-2H-phthalazin-1-one (2) according to the method described in Marzouk et al. [H)-yl)-acetohydrazide (4) [The precursor  .4  in acetic acid (60 mL) and hydrochloric acid  was added portion wise under stirring to a cold solution (0 °C) of sodium nitrite  in water (30 mL). After stirring at the same temperature for 30 min, the in situ generated azide was extracted with cold ethyl acetate and washed successively with cold water and 5% Na2CO3.A cold solution at (− 5 °C) of acetohydrazide 2CO3 and water then dried over anhydrous sodium sulphate, the solvent was evaporated in vacuum and the residue was crystallized from ethyl acetate-petroleum ether to give products 6a–h.After drying over anhydrous sodium sulphate, the azide was used without further purification in the next step. Amines (12 mmol) were added to the previously prepared cold dried solution of the azide. Afterwards, the mixture was kept 12 h in the refrigerator and then at room temperature for another 12 h. The reaction mixture was filtered and the filtrated solution washed with 0.1N HCl, 5% Na1H-NMR , , : 8.42–8.44 ; 7.70–7.76 ; 7.27–7.30 ; 7.22–7.25 ; 6.62 ; 4.99 ; 4.50–4.51 ; 4.32 .13C-NMR: 167.47 (C=O); 159.75 (C=O); 146.39 (C-Ar); 138.02 (C-Ar); 137.51 (C-Ar); 133.22 (CH-Ar); 131.45 (CH-Ar); 129.45 (C-Ar); 128.74 (2 CH-Ar); 128.61 (2 CH-Ar); 128.39 (2 CH-Ar); 128.08 (C-Ar); 127.57 (2 CH-Ar);127.37 (2 CH-Ar); 126.78 (CH-Ar); 125.39 (CH-Ar); 55.16 (CH2CO); 43.56 (CH2NH); 38.88 (CH2ph).White crystals (84%), m.p. 174–176 °C, +. Elemental analysis: calculated for C24H21N3O2 (383.45): % C, 75.18; % H, 5.52; % N, 10.96. Found: % C, 75.20; % H, 5.53; % N, 10.92.MS  m/z: 406.48 (M + Na)1H-NMR , , : 8.36–8.38 ; 7.63–7.68 ; 7.19–7.21 ; 7.12–7.15 ; 6.16 ; 4.83 ; 4.23 ; 3.14–3.19 ; 1.41–1.46 ; 0.79–0.83 .13C-NMR: 167.46 (C=O); 159.73 (C=O); 146.32 (C-Ar); 137.56 (C-Ar); 133.21 (CH-Ar); 131.44 (CH-Ar); 129.44 (C-Ar); 128.72 (2 CH-Ar); 128.39 (2 CH-Ar); 128.11 (C-Ar); 127.38 (CH-Ar);126.79 (CH-Ar); 125.38 (CH-Ar); 55.33 (CH2CO); 41.30 (CH2NH); 38.83 (CH2ph); 22.70 (CH2);11.17 (CH3).White crystals (82%), m.p. 162–164 °C, +. Elemental analysis: calculated for C20H21N3O2 (335.41): % C, 71.62; % H, 6.31; % N, 12.53. Found: % C, 71.63; % H, 6.33; % N, 12.51.MS  m/z: 358.43 (M + Na)1H-NMR , , : 8.34–8.37 ; 7.61–7.68 ; 7.19–7.20 ; 7.11–7.14 ; 6.17 ; 4.82 ; 4.22 ; 3.17–3.21 ; 1.35–1.42 ; 1.20–1.28 ; 0.78–0.82 .13C-NMR: 167.49 (C=O); 159.79 (C=O); 146.43 (C-Ar); 137.55 (C-Ar); 133.34 (CH-Ar); 131.54 (CH-Ar); 129.39 (C-Ar); 128.78 (2 CH-Ar); 128.40 (2 CH-Ar); 128.02 (C-Ar); 127.36 (CH-Ar);126.84 (CH-Ar); 125.47 (CH-Ar); 55.33 (CH2CO); 39.39 (CH2NH); 38.89 (CH2ph); 31.54 (CH2); 20.02 (CH2);13.73 (CH3).White crystals (85%), m.p. 170–172 °C, +. Elemental analysis: calculated for C21H23N3O2 (349.43): % C, 72.18; % H, 6.63; % N, 12.03. Found: % C, 72.21; % H, 6.61; % N, 12.07.MS  m/z: 372.46 (M + Na)1H-NMR , , : 8.44–8.47 ; 7.71–7.76 ; 7.27–7.30 ; 7.21–7.24 ; 6.12 ; 4.90 ; 4.32 ; 3.82 ; 1.11–1.89 .13C-NMR: 166.58 (C=O); 159.73 (C=O); 146.35 (C-Ar); 137.58 (C-Ar); 133.31 (CH-Ar); 131.52 (CH-Ar); 129.37 (C-Ar); 128.78 (2 CH-Ar); 128.39 (2 CH-Ar); 128.04 (C-Ar); 127.41 (CH-Ar);126.83 (CH-Ar); 125.45 (CH-Ar); 55.25 (CH2CO); 48.40 (CHNH); 38.87 (CH2ph); 32.92 (2 CH2); 25.49 (CH2);24.72 (2 CH2).White crystals (86%), m.p. 158–160 °C, +. Elemental analysis: calculated for C23H25N3O2 (375.47): % C, 73.57; % H, 6.71; % N, 11.19. Found: % C, 73.55; % H, 6.75; % N, 11.24.MS  m/z: 398.50 (M + Na)1H-NMR , , : 8.45–8.47 ; 7.71–7.76 ; 7.26–7.30 ; 7.22–7.24 ; 6.22 ; 4.92 ; 4.32 ; 3.27 ; 1.22–1.66 ; 0.87–0.91 . 13C-NMR: 167.50 (C=O); 159.78 (C=O); 146.42 (C-Ar); 137.54 (C-Ar); 133.33 (CH-Ar); 131.53 (CH-Ar); 129.38 (C-Ar); 128.78 (2 CH-Ar); 128.40 (2 CH-Ar); 128.02 (C-Ar); 127.35 (CH-Ar); 126.84 (CH-Ar); 125.46 (CH-Ar); 55.32 (CH2CO); 39.71 (CH2NH); 38.90 (CH2ph); 31.93 (CH2); 29.66 (2 CH2);29.56 (2 CH2); 29.46 (2 CH2); 29.36 (2 CH2); 29.26 (CH2); 26.86 (CH2); 22.70 (CH2); 14.13 (CH3).White crystals (82%), m.p. 126–128 °C, +. Elemental analysis: calculated for C31H43N3O2 (489.70): % C, 76.03; % H, 8.85; % N, 8.58. Found: % C, 76.09; % H, 8.80; % N, 8.60.MS  m/z: 512.68 (M + Na)1H-NMR , , : 8.36–8.39 ; 7.63–7.68 ; 7.18–7.21 ; 7.13–7.16 ; 6.21 ; 5.70–5.79 ; 5.01–5.12 ; 4.87 ; 4.24 ; 3.83–3.85 . 13C-NMR: 167.47 (C=O); 159.82 (C=O); 146.57 (C-Ar); 137.49 (C-Ar); 133.78 (CH-Ar); 133.40 (CH=CH2); 131.60 (CH-Ar); 129.40 (C-Ar); 128.79 (2 CH-Ar); 128.42 (2 CH-Ar); 127.99 (C-Ar); 127.38 (CH-Ar);126.86 (CH-Ar); 125.49 (CH-Ar); 116.43 (CH=CH2); 55.25 (CH2CO); 41.92 (CH2NH); 38.90 (CH2ph).White crystals (87%), m.p. 164–166 °C, +. Elemental analysis: calculated for C20H19N3O2 (333.39): % C, 72.05; % H, 5.74; % N, 12.60. Found: % C, 72.01; % H, 5.77; % N, 12.54.MS  m/z: 356.41 (M + Na)1H-NMR , , : 8.36–8.37 ; 7.60 ; 7.20–7.21 ; 7.12 ; 5.01 ; 4.23 ; 3.53 ; 3.41 ; 1.53–1.60 . 13C-NMR: 164.92 (C=O); 159.70 (C=O); 145.24 (C-Ar); 138.00 (C-Ar); 132.79 (CH-Ar); 130.98 (CH-Ar); 129.68 (C-Ar); 128.66 (2 CH-Ar); 128.38 (2 CH-Ar); 128.38 (C-Ar); 127.37 (CH-Ar);126.58 (CH-Ar); 125.24 (CH-Ar); 52.54 (CH2CO); 45.96 (CH2N); 43.29 (CH2N); 39.00 (CH2ph); 26.25 (CH2CH2N);25.38 (CH2CH2N); 24.47 (CH2CH2CH2N).White crystals (80%), m.p. 192–194 °C, +. Elemental analysis: calculated for C22H23N3O2 (361.45): % C, 73.11; % H, 6.41; % N, 11.63. Found: % C, 73.08; % H, 6.39; % N, 11.60.MS  m/z: 384.44 (M + Na)1H-NMR , , : 8.44–8.46 ; 7.68–7.73 ; 7.29–7.30 ; 7.20–7.23 ; 5.10 ; 4.32 ; 3.59–3.75 . 13C-NMR: 165.45 (C=O); 159.71 (C=O); 145.60 (C-Ar); 137.83 (C-Ar); 133.01 (CH-Ar); 131.19 (CH-Ar); 129.62 (C-Ar); 128.71 (2 CH-Ar); 128.36 (2 CH-Ar); 128.10 (C-Ar); 127.33 (CH-Ar);126.68 (CH-Ar); 125.36 (CH-Ar); 66.78 (CH2O); 66.40 (CH2O); 52.31 (CH2CO); 45.37 (CH2N); 42.41 (CH2N); 38.97 (CH2ph).Off-white crystals (81%), m.p. 210–212 °C, +. Elemental analysis: calculated for C21H21N3O3 (363.42): % C, 69.41; % H, 5.82; % N, 11.56. Found: % C, 69.46; % H, 5.88; % N, 11.59.MS  m/z: 386.46 (M + Na)4  in acetic acid (60 mL) and hydrochloric acid  was added portion wise under stirring to a cold solution (0 °C) of sodium nitrite  in water (30 mL). After stirring at the same temperature for 30 min, the in situ generated azide was extracted with cold ethyl acetate and washed successively with cold water and 5% Na2CO3.A cold solution at (− 5 °C) of acetohydrazide 2CO3 and water then dried over anhydrous sodium sulphate, the solvent was evaporated in vacuum and the residue was crystallized from ethyl acetate-petroleum ether to give products 7a–c.After drying over anhydrous sodium sulphate, the azide was used without further purification in the next step. Amino acids methyl ester hydrochloride (15 mmol); glycine, methionine and valine which were placed with triethyl amine  in ethyl acetate solution at (− 5 °C) for 20 min. Then the amino acid methyl ester hydrochloride solution was added to the previously prepared cold dried solution of the azide. Afterwards, the mixture was kept 12 h in the refrigerator and then at room temperature for another 12 h. The reaction mixture was filtered and the filtrated solution washed with 0.1N HCl, 5% Na1H-NMR , , : 8.35–8.38 ; 7.60–7.68 ; 7.18–7.21 ; 7.11–7.15 ; 6.71 ; 4.90 ; 4.23 ; 4.00–4.01 ; 3.64 .13C-NMR: 169.99 (C=O); 167.72 (C=O); 159.78 (C=O); 146.45 (C-Ar); 137.55 (C-Ar); 133.28 (CH-Ar); 131.50 (CH-Ar); 129.44 (C-Ar); 128.75 (2 CH-Ar); 128.44 (2 CH-Ar); 128.03 (C-Ar); 127.37 (CH-Ar);126.78 (CH-Ar); 125.46 (CH-Ar); 54.80 (CH2CO); 52.27 (OCH3); 41.29 (CH2NH); 38.91 (CH2ph).White crystals (85%), m.p. 166–168 °C, +. Elemental analysis: calculated for C20H19N3O4 (365.39): % C, 65.74; % H, 5.24; % N, 11.50. Found: % C, 65.66; % H, 5.18; % N, 11.43.MS  m/z: 388.41 (M + Na)1H-NMR , , : 8.37–8.39 ; 7.62–7.68 ; 7.19–7.22 ; 7.12–7.15 ; 6.80–6.82 ; 4.84–4.94 ; 4.66–4.71 ; 4.24 ; 3.64 ; 2.43 ; 2.08–2.13 ; 1.88–1.97 ; 1.55 . 13C-NMR: 172.00 (C=O); 167.34 (C=O); 159.72 (C=O); 146.45 (C-Ar); 137.53 (C-Ar); 133.28 (CH-Ar); 131.51 (CH-Ar); 129.44 (C-Ar); 128.76 (2 CH-Ar); 128.40 (2 CH-Ar); 128.07 (C-Ar); 127.39 (CH-Ar);126.79 (CH-Ar); 125.49 (CH-Ar); 54.90 (CH2CO); 52.46 (OCH3); 51.75 (CHNH); 38.94 (CH2ph); 31.53 (CH2CH2S); 29.90 (CH2S); 15.35 (SCH3).White crystals (81%), m.p. 232–234 °C, +. Elemental analysis: calculated for C23H25N3O4S (439.53): % C, 62.85; % H, 5.73; % N, 9.56; % S, 7.29. Found: % C, 62.81; % H, 5.70; % N, 9.52; % S, 7.23.MS  m/z: 462.56 (M + Na)J, Hz): 8.47–8.49 ; 7.70–7.75 ; 7.27–7.30 ; 7.20–7.25 ; 6.71–6.74 ; 4.98 ; 4.59–4.63 ; 4.32 ; 3.70 ; 2.16–2.21 ; 0.87–0.94 . 13C-NMR: 172.12 (C=O); 167.45 (C=O); 159.79 (C=O); 146.42 (C-Ar); 137.56 (C-Ar); 133.33 (CH-Ar); 131.55 (CH-Ar); 129.40 (C-Ar); 128.77 (2 CH-Ar); 128.39 (2 CH-Ar); 128.02 (C-Ar); 127.41 (CH-Ar); 126.80 (CH-Ar); 125.54 (CH-Ar); 57.20 (CHNH); 55.00 (CH2CO); 52.15 (OCH3); 38.97 (CH2ph); 31.38 (CH3CHCH3); 18.90 (CH3CH); 17.74 (CH3CH).White crystals (86%), m.p. 150–152 °C, 1H-NMR , , : % C, 67.80; % H, 6.18; % N, 10.31. Found: % C, 67.86; % H, 6.26; % N, 10.34.MS  m/z: 430.49 (M + Na)7a  in ethyl alcohol (30 mL) was added hydrazine hydrate . The reaction mixture was refluxed for 6 h, cooled and the white precipitate filtered and recrystallized from ethanol to obtain the corresponding hydrazide 2--yl)-N-(2-hydrazineyl-2-oxoethyl) acetamide 8a.To a solution of ester 1H-NMR , , : 9.02 ; 8.38–8.41 ; 8.27–8.29 ; 7.91–7.93 ; 7.80–7.88 ; 7.34–7.36 ; 7.27–7.31 ; 7.18–7.21 ; 4.87 ; 4.32 ; 4.23 ; 3.74–3.75 . 13C-NMR: 168.38 (C=O); 167.73 (C=O); 159.08 (C=O); 145.48 (C-Ar); 138.56 (C-Ar); 133.82 (CH-Ar); 132.14 (CH-Ar); 129.38 (C-Ar); 129.01 (2 CH-Ar); 128.84 (2 CH-Ar); 128.08 (C-Ar); 126.95 (CH-Ar);126.85 (CH-Ar); 126.27 (CH-Ar); 53.98 (CH2CO); 41.43 (CH2NH); 38.13 (CH2ph).White crystals (84%), m.p. 202–204 °C, +. Elemental analysis: calculated for C19H19N5O3 (365.39): % C, 62.46; % H, 5.24; % N, 19.17. Found: % C, 62.44; % H, 5.21; % N, 19.12.MS  m/z: 388.42 (M + Na)H)-yl)-N-(2-hydrazineyl-2-oxoethyl) acetamide (8a)  in acetic acid (60 mL) and hydrochloric acid  was added portion wise under stirring to a cold solution (0 °C) of sodium nitrite  in water (30 mL). After stirring at the same temperature for 30 min, the in situ generated azide was extracted with cold ethyl acetate and washed successively with cold water and 5% Na2CO3.Under azide coupling method as previewed before, A cold solution at (− 5 °C) of 2- were added to the previously prepared cold dried solution of the azide. Afterwards, the mixture was kept 12 h in the refrigerator and then at room temperature for another 12 h. The reaction mixture was filtered and the filtrated solution washed with 0.1N HCl, 5% Na1H-NMR , , : 8.23–8.25 ; 7.68–7.76 ; 7.27–7.33 ; 7.22–7.25 ; 7.04 ; 4.92 ; 4.43–4.45 ; 4.31 ; 4.01–4.03 . 13C-NMR: 168.65 (C=O); 168.02 (C=O); 159.93 (C=O); 146.66 (C-Ar); 138.00 (C-Ar); 137.44 (C-Ar); 133.37 (CH-Ar); 131.51 (CH-Ar); 129.52 (C-Ar); 128.78 (2 CH-Ar); 128.57 (2 CH-Ar); 128.41 (2 CH-Ar); 127.83 (C-Ar); 127.72 (2 CH-Ar); 127.32 (CH-Ar); 127.19 (CH-Ar);126.84 (CH-Ar); 125.47 (CH-Ar); 55.76 (NCH2CO); 43.51 (NHCH2CO); 43.35 (NHCH2ph); 38.88 (CH2ph).Off-white crystals (83%), m.p. 186–188 °C, +. Elemental analysis: calculated for C26H24N4O3 (440.50): % C, 70.89; % H, 5.49; % N, 12.72. Found: % C, 70.93; % H, 5.56; % N, 12.77.MS  m/z: 463.52 (M + Na)1H-NMR , , : 8.41–8.44 ; 7.72–7.79 ; 7.28–7.31 ; 7.22–7.25 ; 6.88 ; 6.62 ; 4.94 ; 4.34 ; 3.98–3.99 ; 3.19–3.24 ; 1.52–1.57 ; 0.89–0.92 . 13C-NMR: 168.46 (C=O); 167.89 (C=O); 159.93 (C=O); 146.62 (C-Ar); 137.44 (C-Ar); 133.41 (CH-Ar); 131.57 (CH-Ar); 129.57 (C-Ar); 128.78 (2 CH-Ar); 128.41 (2 CH-Ar); 127.94 (C-Ar); 127.17 (CH-Ar);126.84 (CH-Ar); 125.55 (CH-Ar); 55.71 (NCH2CO); 43.42 (NHCH2CO); 41.37 (NHCH2CH2); 38.91 (CH2ph); 22.57 (CH2CH2CH3);11.31 (CH3).White crystals (86%), m.p. 152–154 °C, +. Elemental analysis: calculated for C22H24N4O3 (392.46): % C, 67.33; % H, 6.16; % N, 14.28. Found: % C, 67.36; % H, 6.22; % N, 14.32.MS  m/z: 415.47 (M + Na)1H-NMR , , : 8.43–8.45 ; 7.73–7.80 ; 7.29–7.32 ; 7.23–7.27 ; 6.76 ; 6.50 ; 4.94 ; 4.34 ; 3.99 ; 3.24–3.29 ; 1.48–1.54 ; 1.32–1.37 ; 0.90–0.93 . 13C-NMR: 168.34 (C=O); 167.85 (C=O); 159.94 (C=O); 146.67 (C-Ar); 137.42 (C-Ar); 133.44 (CH-Ar); 131.59 (CH-Ar); 129.58 (C-Ar); 128.79 (2 CH-Ar); 128.42 (2 CH-Ar); 127.93 (C-Ar); 127.21 (CH-Ar);126.86 (CH-Ar); 125.56 (CH-Ar); 55.80 (NCH2CO); 43.43 (NHCH2CO); 39.41 (NHCH2CH2); 38.92 (CH2ph); 31.41 (CH2CH2CH2); 20.01 (CH2CH2CH3);13.67 (CH3).Off-white crystals (85%), m.p. 156–158 °C, +. Elemental analysis: calculated for C23H26N4O3 (406.49): % C, 67.96; % H, 6.45; % N, 13.78. Found: % C, 67.99; % H, 6.55; % N, 13.86.MS  m/z: 429.53 (M + Na)1H-NMR , , : 8.43–8.45 ; 7.73–7.79 ; 7.28–7.32 ; 7.22–7.25 ; 6.81 ; 6.37–6.38 ; 4.94 ; 4.34 ; 3.97–3.98 ; 3.75–3.76 ; 1.86–1.89 ; 1.69–1.72 ; 1.60–1.63 ; 1.25–1.36 ; 1.15–1.22 . 13C-NMR: 167.75 (C=O); 167.40 (C=O); 159.87 (C=O); 146.59 (C-Ar); 137.44 (C-Ar); 133.40 (CH-Ar); 131.56 (CH-Ar); 129.59 (C-Ar); 128.78 (2 CH-Ar); 128.42 (2 CH-Ar); 127.95 (C-Ar); 127.21 (CH-Ar); 126.84 (CH-Ar); 125.55 (CH-Ar); 55.73 (NCH2CO); 48.44 (NHCH2CO); 43.45 (NHCHCH2); 38.93 (CH2ph); 32.83 (2 CH2);25.53 (CH2); 24.81 (2 CH2).White crystals (87%), m.p. 157–158 °C, +. Elemental analysis: calculated for C25H28N4O3 (432.52): % C, 69.42; % H, 6.53; % N, 12.95. Found: % C, 69.48; % H, 6.61; % N, 12.94.MS  m/z: 455.54 (M + Na)1H-NMR , , : 8.40–8.42 ; 7.71–7.77 ; 7.29–7.30 ; 7.21–7.24 ; 6.70 ; 6.62 ; 4.94 ; 4.33 ; 3.96–3.97 ; 3.20–3.25 ; 2.70–2.74 ; 2.35 ; 1.48 ; 1.28 ; 0.88–0.91 . 13C-NMR: 168.64 (C=O); 167.96 (C=O); 159.88 (C=O); 146.49 (C-Ar); 137.47 (C-Ar); 133.33 (CH-Ar); 131.47 (CH-Ar); 129.57 (C-Ar); 128.76 (2 CH-Ar); 128.39 (2 CH-Ar); 127.93 (C-Ar); 127.14 (CH-Ar); 126.81 (CH-Ar); 125.52 (CH-Ar); 55.64 (NCH2CO); 43.34 (NHCH2CO); 42.06 (CH2); 39.74 (NHCH2CH2); 38.91 (CH2ph); 33.38 (CH2); 31.89 (2 CH2); 29.31–29.63 (4 CH2); 26.85 (2 CH2); 22.64 (2 CH2); 14.04 (CH3).Off-white crystals (82%), m.p. 149–150 °C, +. Elemental analysis: calculated for C33H46N4O3 (546.76): % C, 72.49; % H, 8.48; % N, 10.25. Found: % C, 72.53; % H, 8.45; % N, 10.36.MS  m/z: 569.80 (M + Na)1H-NMR , , : 8.42–8.44 ; 7.73–7.77 ; 7.28–7.32 ; 7.24–7.25 ; 6.83 ; 6.63 ; 5.81–5.88 ; 5.11–5.22 ; 4.95 ; 4.34 ; 4.02 ; 3.90 . 13C-NMR: 168.39 (C=O); 167.98 (C=O); 159.97 (C=O); 146.73 (C-Ar); 137.41 (C-Ar); 133.77 (CH = CH2); 133.46 (CH-Ar); 131.60 (CH-Ar); 129.57 (C-Ar); 128.79 (2 CH-Ar); 128.41 (2 CH-Ar); 127.90 (C-Ar); 127.26 (CH-Ar);126.86 (CH-Ar); 125.54 (CH-Ar); 116.50 (CH2=CH); 55.80 (NCH2CO); 43.43 (NHCH2CO); 41.98 (NHCH2CH); 38.92 (CH2ph).White crystals (88%), m.p. 188–190 °C, +. Elemental analysis: calculated for C22H22N4O3 (390.44): % C, 67.68; % H, 5.68; % N, 14.35. Found: % C, 67.66; % H, 5.61; % N, 14.23.MS  m/z: 413.47 (M + Na)1H-NMR , , : 8.44–8.46 ; 7.70–7.71 ; 7.30–7.32 ; 7.22–7.23 ; 7.07 ; 5.00 ; 4.34 ; 4.10–4.11 ; 3.56 ; 3.40 ; 1.56–1.65 . 13C-NMR: 168.34 (C=O); 167.46 (C=O); 159.89 (C=O); 146.43 (C-Ar); 137.56 (C-Ar); 133.34 (CH-Ar); 131.56 (CH-Ar); 129.57 (C-Ar); 128.76 (2 CH-Ar); 128.42 (2 CH-Ar); 127.92 (C-Ar); 127.21 (CH-Ar);126.67 (CH-Ar); 125.53 (CH-Ar); 55.74 (NCH2CO);45.86 (CH2N); 43.64 (CH2N); 43.34 (NHCH2CO); 39.08 (CH2ph); 26.55 (CH2CH2N);25.41 (CH2CH2N); 23.94 (CH2CH2CH2N).White crystals (79%), m.p. 138–140 °C, +. Elemental analysis: calculated for C24H26N4O3 (418.50): % C, 68.88; % H, 6.26; % N, 13.39. Found: % C, 68.92; % H, 6.31; % N, 13.48.MS  m/z: 441.54 (M + Na)1H-NMR , , : 8.47–8.48 ; 7.72–7.73 ; 7.28–7.32 ; 7.23–7.24 ; 7.10 ; 5.01 ; 4.34 ; 4.13 ; 3.64–3.69 ; 3.43 . 13C-NMR: 167.44 (C=O); 166.34 (C=O); 159.67 (C=O); 146.34 (C-Ar); 137.62 (C-Ar); 133.20 (CH-Ar); 131.47 (CH-Ar); 129.49 (C-Ar); 128.74 (2 CH-Ar); 128.44 (2 CH-Ar); 128.11 (C-Ar); 127.46 (CH-Ar);126.74 (CH-Ar); 125.50 (CH-Ar); 66.67 (CH2O); 66.33 (CH2O); 54.60 (NCH2CO);44.86 (NHCH2CO); 42.36 (CH2N); 41.25 (CH2N); 39.01 (CH2ph).White crystals (81%), m.p. 148–150 °C, +. Elemental analysis: calculated for C23H24N4O4 (420.47): % C, 65.70; % H, 5.75; % N, 13.33. Found: % C, 65.64; % H, 5.65; % N, 13.26.MS  m/z: 443.50 (M + Na)H)-yl)-N-(2-hydrazineyl-2-oxoethyl) acetamide (8a)  in acetic acid (60 mL) and hydrochloric acid  was added portion wise under stirring to a cold solution (0 °C) of sodium nitrite  in water (30 mL). After stirring at the same temperature for 30 min, the in situ generated azide was extracted with cold ethyl acetate and washed successively with cold water and 5% Na2CO3.A cold solution at (− 5 °C) of 2-; glycine, β-alanine, methionine and valine which were placed with triethyl amine  in ethyl acetate solution at (− 5 °C) for 20 min. Then the amino acid methyl ester hydrochloride solution was added to the previously prepared cold dried solution of the azide. Afterwards, the mixture was kept 12 h in the refrigerator and then at room temperature for another 12 h. The reaction mixture was filtered, and the filtrated solution washed with 0.1N HCl, 5% Na1H-NMR , , : 8.42 ; 7.73–7.75 ; 7.29–7.31 ; 7.23–7.24 ; 7.08 ; 4.97 ; 4.33 ; 4.04–4.05 ; 3.72 .White crystals (88%), m.p. 162–164 °C, 13C-NMR: 172.11 (C=O); 168.08 (C=O); 167.63 (C=O); 159.94 (C=O); 146.70 (C-Ar); 137.46 (C-Ar); 133.39 (CH-Ar); 131.56 (CH-Ar); 129.55 (C-Ar); 128.78 (2 CH-Ar); 128.42 (2 CH-Ar); 127.93 (C-Ar); 127.26 (CH-Ar);126.83 (CH-Ar); 125.49 (CH-Ar); 55.63 (NCH2CO); 52.27 (OCH3); 43.13 (NHCH2COO); 41.17 (NHCH2CO); 38.89 (CH2ph).+. Elemental analysis: calculated for C22H22N4O5 (422.44): % C, 62.55; % H, 5.25; % N, 13.26. Found: % C, 62.49; % H, 5.22; % N, 13.30.MS  m/z: 445.46 (M + Na)1H-NMR , , : 8.44–8.46 ; 7.72–7.78 ; 7.28–7.31 ; 7.22–7.25 ; 6.88 ; 4.95 ; 4.34 ; 3.97–3.98 ; 3.67 ; 3.54–3.55 ; 2.56–2.59 . 13C-NMR: 172.50 (C=O); 168.57 (C=O); 167.88 (C=O); 159.93 (C=O); 146.62 (C-Ar); 137.47 (C-Ar); 133.38 (CH-Ar); 131.58 (CH-Ar); 129.55 (C-Ar); 128.77 (2 CH-Ar); 128.42 (2 CH-Ar); 127.98 (C-Ar); 127.28 (CH-Ar); 126.83 (CH-Ar); 125.51 (CH-Ar); 55.59 (NCH2CO); 51.73 (OCH3);43.25 (NHCH2CO); 38.91 (CH2ph); 35.18 (NHCH2CH2CO); 33.71 (NHCH2CH2CO).White crystals (85%), m.p. 108–110 °C, +. Elemental analysis: calculated for C23H24N4O5 (436.47): % C, 63.29; % H, 5.54; % N, 12.84. Found: % C, 63.35; % H, 5.63; % N, 12.82.MS  m/z: 459.48 (M + Na)1H-NMR , , : 8.48 ; 7.73–7.74 ; 7.55–7.56 ; 7.28 ; 7.19–7.21 ; 7.14–7.17 ; 4.99 ; 4.32 ; 4.23–4.26 ; 4.18 ; 3.76 ; 3.03–3.07 ; 2.25 ; 1.71 . 13C-NMR: 172.18 (C=O); 168.60 (C=O); 167.79 (C=O); 159.84 (C=O); 146.45 (C-Ar); 137.47 (C-Ar); 133.34 (CH-Ar); 131.54 (CH-Ar); 130.80 (C-Ar); 128.78 (2 CH-Ar); 128.42 (2 CH-Ar); 127.86 (C-Ar); 127.43 (CH-Ar);126.81 (CH-Ar); 125.53 (CH-Ar); 57.41 (NHCHCO); 55.56 (NCH2CO); 52.08 (OCH3);51.67 (NHCH2CO); 43.34 (CH2CH2S); 38.92 (CH2ph); 31.49 (CH2S); 16.85 (SCH3).Off-white crystals (84%), m.p. 130–132 °C, +. Elemental analysis: calculated for C25H28N4O5S (496.58): % C, 60.47; % H, 5.68; % N, 11.28; % S, 6.46. Found: % C, 60.33; % H, 5.57; % N, 11.15; % S, 6.40.MS  m/z: 519.61 (M + Na)1H-NMR , , : 8.45 ; 7.72–7.76 ; 7.28–7.32 ; 7.22–7.24 ; 6.88 ; 6.65 ; 4.98 ; 4.34 ; 4.25 ; 3.71 ; 3.51 ; 1.45 ; 0.92–0.95 . 13C-NMR: 172.48 (C=O); 168.52 (C=O); 167.82 (C=O); 159.91 (C=O); 146.44 (C-Ar); 137.36 (C-Ar); 133.38 (CH-Ar); 131.56 (CH-Ar); 130.82 (C-Ar); 128.79 (2 CH-Ar); 128.45 (2 CH-Ar); 127.87 (C-Ar); 127.40 (CH-Ar);126.84 (CH-Ar); 125.54 (CH-Ar); 57.43 (NHCHCO); 55.51 (NCH2CO); 52.08 (OCH3);43.30 (NHCH2CO); 38.94 (CH2ph); 29.68 (CH3CHCH3); 18.93 (CH3CH); 17.88 (CH3CH).White crystals (82%), m.p. 190–192 °C, +. Elemental analysis: calculated for C25H28N4O5 (464.52): % C, 64.64; % H, 6.08; % N, 12.06. Found: % C, 64.76; % H, 6.15; % N, 12.16.MS  m/z: 487.56 (M + Na)8a  and malononitrile  in ethanol (30 mL) was refluxed for 8 h. By cooling the solid product formed, filtered off and recrystallized from ethanol solvent gave compound 12a.A mixture of hydrazide 1H-NMR , , : 9.02 ; 8.27–8.28 ; 7.90–7.92 ; 7.81–7.85 ; 7.31–7.32 ; 7.18 ; 7.10–7.12 ; 5.46–5.47 ; 4.86 ; 4.32 ; 4.26 ; 3.52 . 13C-NMR: 171.42 (C=O); 168.34 (C=O); 159.21 (C=O); 148.14 (C-NH2); 148.36 (C-NH2); 146.38 (C-Ar); 137.32 (C-Ar); 133.30 (CH-Ar); 131.64 (CH-Ar); 130.85 (C-Ar); 128.78 (2 CH-Ar); 128.43 (2 CH-Ar); 128.09 (C-Ar); 127.49 (CH-Ar);126.76 (CH-Ar); 125.34 (CH-Ar); 110.35 (NH2-C=CH-C-NH2); 55.28 (NCH2CO); 42.82 (NHCH2CO); 38.64 (CH2ph).White crystals (85%), m.p. 236–238 °C, +. Elemental analysis: calculated for C22H21N7O3 (431.46): % C, 61.24; % H, 4.91; % N, 22.73. Found: % C, 61.32; % H, 5.02; % N, 22.80.MS  m/z: 454.49 (M + Na)8a  and cyclohexanone  in ethanol (30 mL) was refluxed for 8 h. By cooling the solid product formed, filtered off and recrystallized from ethanol solvent gave compound 12b.A mixture of hydrazide molecule 1H-NMR , , : 9.01 ; 8.40 ; 8.26–8.28 ; 7.90–7.91 ; 7.78–7.86 ; 7.30–7.34 ; 7.16–7.20 ; 7.16–7.20 ; 7.08–7.12 ; 5.46–5.47 ; 4.85 ; 4.32 ; 4.25 ; 2.37 ; 1.65 ; 1.14 . 13C-NMR: 170.62 (C=O); 168.32 (C=O); 159.18 (C=O); 150.11 (NH-C=CH-CH2); 145.83 (C-Ar); 138.34 (C-Ar); 133.77 (CH-Ar); 131.93 (CH-Ar); 129.52 (C-Ar); 129.06 (2 CH-Ar); 128.78 (2 CH-Ar); 128.07 (C-Ar); 127.04 (CH-Ar);126.87 (CH-Ar); 125.96 (CH-Ar); 102.45 (NH-C=CH-CH2); 54.86 (NCH2CO); 41.03 (NHCH2CO); 38.25 (CH2ph); 35.19 (NH-C-CH2); 34.32 (NH-C=CH-CH2); 26.50 (CH2); 22.71 (CH2).White crystals (89%), m.p. 233–234 °C, +. Elemental analysis: calculated for C25H27N5O3 (445.52): % C, 67.40; % H, 6.11; % N, 15.72. Found: % C, 67.33; % H, 6.01; % N, 15.63.MS  m/z: 468.55 (M + Na)8a  and 2-furyl methyl ketone  in ethanol (30 mL) was refluxed for 8 h. By cooling the solid product formed, filtered off and recrystallized from ethanol solvent gave compound 12c.A mixture of hydrazide molecule 1H-NMR , , : 10.64 ; 8.50 ; 8.27–8.29 ; 7.91–7.93 ; 7.81–7.88 ; 7.75 ; 7.35–7.37 ; 7.27–7.31 ; 7.17–7.21 ; 6.89 ; 6.58 ; 4.89 ; 4.32 ; 4.32 ; 2.18 . 13C-NMR: 172.73 (C=O); 168.82 (C=O); 159.30 (C=O); 152.64 (N=C-CH3); 146.11 (C-Ar); 144.58 (CH=C-O); 144.18 (CH=CH-O); 140.32 (C-Ar); 138.39 (CH-Ar); 133.81 (CH-Ar); 131.92 (CH-Ar); 129.75 (C-Ar); 128.98 (2 CH-Ar); 128.83 (2 CH-Ar); 128.07 (C-Ar); 127.12 (CH-Ar);126.40 (CH-Ar); 112.19 (CH=CH-O); 110.85 (CH=C-O); 55.16 (NCH2CO); 42.88 (NHCH2CO); 38.54 (CH2ph); 13.32 (CH3).Faint brown crystals (91%), m.p. 209–210 °C, +. Elemental analysis: calculated for C25H23N5O4 (457.49): % C, 65.64; % H, 5.07; % N, 15.31. Found: % C, 65.67; % H, 5.11; % N, 15.25.MS  m/z: 480.50 (M + Na)8a  and ethyl cyano acetate  in ethanol (30 mL) was refluxed for 8 h. By cooling the solid product formed, filtered off and recrystallized from ethanol solvent gave compound 12d.A mixture of hydrazide molecule 1H-NMR , , : 9.02 ; 8.39 ; 8.26–8.28 ; 7.90–7.92 ; 7.78–7.86 ; 7.30–7.34 ; 7.17–7.20 ; 7.17–7.20 ; 7.08–7.12 ; 5.46–5.47 ; 4.86 ; 4.32 ; 4.25 . 13C-NMR: 171.78 (C=O); 167.36 (C=O); 166.29 (C=O); 158.89 (C=O); 151.93 (NH2-C=CH-CO); 145.33 (C-Ar); 138.45 (C-Ar); 133.97 (CH-Ar); 132.18 (CH-Ar); 129.18 (C-Ar); 128.91 (2 CH-Ar); 128.76 (2 CH-Ar); 128.09 (C-Ar); 126.88 (CH-Ar);126.21 (CH-Ar); 125.32 (CH-Ar); 112.19 (NH2-C=CH-CO); 56.34 (NCH2CO); 43.24 (NHCH2CO); 38.11 (CH2ph).White crystals (84%), m.p. 237–238 °C, +. Elemental analysis: calculated for C22H20N6O4 (432.44): % C, 61.10; % H, 4.66; % N, 19.43. Found: % C, 61.04; % H, 4.61; % N, 19.50.MS  m/z: 455.48 (M + Na)8a  and acetyl acetone  in ethanol (30 mL) was refluxed for 8 h. By cooling the solid product formed, filtered off and recrystallized from ethanol solvent gave compound 12e.A mixture of hydrazide molecule 1H-NMR , , : 9.01 ; 8.26–8.28 ; 7.89–7.91 ; 7.78–7.86 ; 7.30–7.35 ; 7.16–7.20 ; 7.08–7.12 ; 5.46–5.48 ; 4.89 ; 4.25 ; 2.46 ; 2.43 . 13C-NMR: 171.28 (C=O); 168.37 (C=O); 159.72 (C=O); 148.84 (C-CH3); 146.35 (C-Ar); 144.33 (C-CH3); 137.22 (C-Ar); 133.29 (CH-Ar); 131.67 (CH-Ar); 130.45 (C-Ar); 128.75 (2 CH-Ar); 128.40 (2 CH-Ar); 128.07 (C-Ar); 127.46 (CH-Ar);126.74 (CH-Ar); 125.31 (CH-Ar); 116.25 (CH3-C=CH-C-CH3); 55.25 (NCH2CO); 42.65 (NHCH2CO); 38.41 (CH2ph); 16.21 (CH3); 13.74 (CH3).White crystals (87%), m.p. 237–238 °C, +. Elemental analysis: calculated for C24H23N5O3 (429.48): % C, 67.12; % H, 5.40; % N, 16.31. Found: % C, 67.07; % H, 5.47; % N, 16.34.MS  m/z: 452.50 (M + Na)50 value using GraphPad prism software [MCF-7, HepG2 cancer cells and WISH normal cells were obtained from the National Cancer Institute in Cairo, Egypt, they were cultured in complete media of RPMI and DMEM, respectively at 5% carbon dioxide and 37 °C following standard tissue culture work. The cells were grown in “10% fetal bovine serum (FBS) and 1% penicillin–streptomycin” in 96-multiwell plate. All the synthesized compounds were screened for their cytotoxicity using 20 µL of MTT solution  for 48 hours  using unsoftware .50 was determined using GraphPad prism7 using inhibition curves at five different concentrations of each compound.The most promising cytotoxic compounds were subjected to EGFR enzyme assay  using ELISA kit (Enzyme-Linked Immunosorbent Assay) following manufacturer information . The lum5 cells/well) and then treated with the IC50 values for 48 h with compound 12d. After that, the cells were incubated in a 100 µL solution of Annexin binding buffer "25 mM CaCl2, 1.4 M NaCl, and 0.1 M Hepes/NaOH, pH 7.4" in the dark for 30 min with "Annexin V-FITC solution (1:100) and propidium iodide (PI) at a concentration equivalent to 10 g/mL." The labeled cells were then extracted using the Cytoflex FACS machine. CytExpert software was used to analyze the data [MDA-MB-231 cells were incubated overnight in 6-well culture plates  using AutoDock Vina software following routine work , 52. Vin(2) and their chemical structure were elucidated via different analytical and spectroscopic methods. The cytotoxicity of the synthesized compounds was tested using MTT assay, as well as apoptosis-induction through EGFR inhibition. Compounds 11d, 12c and 12d exhibited potent cytotoxic activities with IC50 values of 0.92, 1.89 and 0.57 μM against MDA-MB-231 cells compared to Erlotinib (IC50 = 1.02 μM). Interestingly compound 12d exhibited promising potent EGFR inhbition with an IC50 value 21.4 nM compared to Erlotinib (IC50 = 80 nM). For apoptosis, compounds 12d induced apoptosis in MDA-MB-231 cells by 64.4-fold (42.5% compared to 0.66 for the control), Hence, this compound may serve as a potential target-oriented anti-breast cancer agent (Additional file In this study, we synthesized twenty-nine new phthalazinone derivatives starting from 4-Benzyl-2H-phthalazin-1-one Additional file 1. Characterization analyses for the synthesized compounds are provided as a Additional file. Figure S1. The 1H-NMR spectrum of compound 6a. Figure S2. The 13C-NMR spectrum of compound 6a.Figure S3. The 1H-NMR spectrum of compound 6b. Figure S4. The 13C-NMR spectrum of compound 6b. Figure S5. The 1H-NMR spectrum of compound 6c. Figure S6. The 13C-NMR spectrum of compound 6c. Figure S7. The 1H-NMR spectrum of compound 6d. Figure S8. The 13C-NMR spectrum of compound 6d. Figure S9. The 1H-NMR spectrum of compound 6e. Figure S10. The 13C-NMR spectrum of compound 6e. Figure S11. The 1H-NMR spectrum of compound 6f. Figure S12. The 13C-NMR spectrum of compound 6f. Figure S13. The 1H-NMR spectrum of compound 6g. Figure S14. The 13C-NMR spectrum of compound 6g. Figure S15. The 1H-NMR spectrum of compound 6h. Figure S16. The 13C-NMR spectrum of compound 6h. Figure S17. The 1H-NMR spectrum of compound 7a. Figure S18. The 13C-NMR spectrum of compound 7a. Figure S19. The 1H-NMR spectrum of compound 7c. Figure S20. The 13C-NMR spectrum of compound 7c. Figure S21.. The 1H-NMR spectrum of compound 7d. Figure S22. The 13C-NMR spectrum of compound 7d. Figure S23. The 1H-NMR spectrum of compound 8a. Figure S24. The 13C-NMR spectrum of compound 8a. Figure S25. The 1H-NMR spectrum of compound 10a. Figure S26. The 13C-NMR spectrum of compound 10a. Figure S27. The 1H-NMR spectrum of compound 10b. Figure S28. The 13C-NMR spectrum of compound 10b. Figure S29. The 1H-NMR spectrum of compound 10c. Figure S30. The 13C-NMR spectrum of compound 10c . Figure S31. The 1H-NMR spectrum of compound 10d. Figure S32. The 13C-NMR spectrum of compound 10d. Figure S33. The 1H-NMR spectrum of compound 10e. Figure S34.. The 13C-NMR spectrum of compound 10e. Figure S35. The 1H-NMR spectrum of compound 10f . Figure S36. The 13C-NMR spectrum of compound 10f. Figure S37. The 1H-NMR spectrum of compound 10h. Figure S38. The 13C-NMR spectrum of compound 10h. Figure S39. The 1H-NMR spectrum of compound 11a. Figure S40. The 13C-NMR spectrum of compound 11a. Figure S41. The 1H-NMR spectrum of compound 11b. Figure S42.. The 13C-NMR spectrum of compound 11b. Figure S43. The 1H-NMR spectrum of compound 12a.Figure S44. The 1H-NMR spectrum of compound 12b. Figure S45. The 1H-NMR spectrum of compound 12c. Figure S46. The 1H-NMR spectrum of compound 12d. Figure S47. The 13C-NMR spectrum of compound 12d. Figure S48. The 1H-NMR spectrum of compound 12e.