Structure-activity relationship (SAR) study and design strategies of nitrogen-containing heterocyclic moieties for their anticancer activities

Rational approaches, design strategies, structure activity relationship and mechanistic insights for anticancer hybrids

Review: 534 refs.

In this work, the design and synthesis of a new chalcone-trimethoxycinnamide hybrid (7) based on the combination of subunits of two promising antiproliferative compounds (CM-M345 (1) and BP-M345 (2)), previously obtained by our research group, are reported. In order to expand the structure-activity relationship (SAR) knowledge, a new series of 7-analogues was also designed and synthetized. All the compounds were evaluated for their antitumor activity against melanoma (A375-C5), breast adenocarcinoma (MCF-7), and colorectal carcinoma (HCT116) cell lines, as well as non-tumor HPAEpiC cells. Three of the newly synthesized compounds (6, 7, and 13) exhibited potent antiproliferative activity, mainly on colorectal tumor cells (GI50 = 2.66-3.26 μM), showing hybrid 7 selectivity for tumor cells. We performed molecular mechanism studies to evaluate the potential interference of compounds with the p53 pathway, namely, p53-MDM2 interaction and mitosis in HCT116 cells. The antiproliferative activities of compounds were shown to be p53-independent. Compound 7 emerged as an antimitotic agent by inducing the mitotic arrest of colorectal tumor cells, and subsequently, cell death.

TRAIL is an endogenous protein that initiates apoptosis selectively in cancer without toxic side effects, prompting interest in therapeutic modulation. We previously screened for small molecules that could upregulate the endogenous TRAIL gene to trigger apoptosis and restore anti-tumor immunity within tumor cells in a p53-independent manner. We showed that ONC201 (previously referred to as TIC10) is a dual inactivator of Akt and ERK, leading to nuclear translocation of Foxo3a and TRAIL gene activation (Allen et al., Science Translational Medicine, 2013). We recently found that ONC201 causes an early-stage upregulation of the integrated stress response through ATF4/CHOP/DR5 (Kline et al., Science Signaling, in press) and can inhibit cancer stem cell self-renewal (Prabhu et al., Cancer Research, 2015). ONC201 recently completed its first-in-man phase I clinical trial and several other trials in select advanced cancers are ongoing (NCT02250781, NCT02324621, NCT02420795, NCT02392572, NCT02609230, NCT02525692, NCT02038699). Leveraging the unique pharmacophore of ONC201, we synthesized ONC201 analogues in search for compounds with distinct therapeutic properties. After establishing the importance of the pyrido[3,4-e]pyrimidinone core structure of ONC201 in its anti-tumor activity (Wagner et al., Oncotarget, 2014), we performed detailed structure activity relationship (SAR) studies. We evaluated several ONC201 analogues with differing N-substituents around the core structure. Certain ONC201 analogues exhibit more rapid kinetics of activity and lowered IC50 values in some human cancer cell lines in vitro. Interim results of ONC212 sensitivity profiling in >100 genetically annotated cell lines from the Genomic of Drug Sensitivity in Cancer collection have corroborated this improvement in potency. One analogue, ONC212, has demonstrated compelling efficacy against several tumor types in vivo with no evidence of toxicity at therapeutic doses. Furthermore, in vitro mechanism studies have demonstrated overlap between ONC201- and ONC212-mediated signaling in tumor cells that includes activation of the integrated stress response. With a wide safety margin, distinct pharmacokinetics (PK), and robust potency, ONC212 is being developed as the next drug candidate from the new class of compounds defined by the novel pharmacophore of ONC201 in indications that complement the parent compound's use spectrum. Citation Format: Jessica Wagner, Gary Olson, Nallaganchu Rao Bhaskara, Richard S. Pottorf, Garnett J. Mathew, Cyril H. Benes, Rohinton Tarapore, Martin Stogniew, Lee Schalop, Wolfgang Oster, Josh E. Allen, Wafik S. El-Deiry. Structure-activity relationships and mechanistic analysis of analogues of the clinical-stage anti-cancer small molecule ONC201. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 349.

To determine the target of the recently identified lead compound NSC130362 that is responsible for its selective anti-cancer efficacy and safety in normal cells, structure-activity relationship (SAR) studies were conducted. First, NSC13062 was validated as a starting compound for the described SAR studies in a variety of cell-based viability assays. Then, a small library of 1,4-naphthoquinines (1,4-NQs) and quinoline-5,8-diones was tested in cell viability assays using pancreatic cancer MIA PaCa-2 cells and normal human hepatocytes. The obtained data allowed us to select a set of both non-toxic compounds that preferentially induced apoptosis in cancer cells and toxic compounds that induced apoptosis in both cancer and normal cells. Anti-cancer activity of the selected non-toxic compounds was confirmed in viability assays using breast cancer HCC1187 cells. Consequently, the two sets of compounds were tested in multiple cell-based and in vitro activity assays to identify key factors responsible for the observed activity. Inhibition of the mitochondrial electron transfer chain (ETC) is a key distinguishing activity between the non-toxic and toxic compounds. Finally, we developed a mathematical model that was able to distinguish these two sets of compounds. The development of this model supports our conclusion that appropriate quantitative SAR (QSAR) models have the potential to be employed to develop anti-cancer compounds with improved potency while maintaining non-toxicity to normal cells.

According to the statistics from American Cancer Society, the 5-year survival rate for patients with advanced melanoma is as low as 5%. Treatment of advanced melanoma, therefore, represents an unmet medical need. In this dissertation, I will show the effort to develop new generations of bioavailable tubulin inhibitors targeting the colchicine binding site and selective small-molecule survivin inhibitors for treating advanced melanoma. Extensive structure-activity relationship (SAR) studies of lead molecules ABI-231 and UC-112 have been performed. Chapter 1 will introduce the current situation of advanced or metastatic melanoma, its clinical drug treatments, as well as problems in current drug treatments. Microtubule dynamics and survivin will be discussed as promising therapeutic targets for developing anticancer drugs. 20S-hydroxyvitamin D3 (20S-OH-D3) will be introduced as a promising anti-inflammatory scaffold. Chapter 2 will disclose the SAR study of ABI-231, a previously reported potent tubulin inhibitor from our lab. In this chapter, a new synthetic method was developed to enable the synthesis of ABI-231 analogues modifying the indole moiety. The novel synthetic method involved the synthesis of a key diamine intermediate and imidazoline formation. From the new synthetic method, thirty ABI-231 analogues were synthesized and tested for activities. Among all analogues, 10ab with a 4-methyl-3-indole moiety and 10bb with a 4-indole moiety showed the most potent antiproliferative activities against a panel of melanoma cell lines. 10ab and 10bb had IC50s of 2.2 and 3.0 nM, respectively. The SAR result revealed that modification of the indole moiety in ABI-231 was beneficial to activity. In Chapter 3, we will describe our effort to develop the SAR study of ABI-231 focusing on modification of the 3,4,5-TMP moiety. This is selected since it is one of the most common moieties in current tubulin inhibitors targeting the colchicine binding site. To circumvent the use of potentially explosive azide reported in Chapter 2, an alternative was established to efficiently generate ABI-231 analogues. This new synthetic method involved Suzuki coupling and Grignard reactions to modify the 3,4,5-TMP moiety and to produce target compounds in gram-scale. Among the eight analogues synthesized, the one containing an unique 3-methoxybenzo[4,5]-dioxene moiety had the strongest antiproliferative activity against a panel of melanoma cell lines with an average IC50 of 1.9 nM. To our best knowledge, it represents the most successful instance of isosterically modifying the 3,4,5-TMP moiety in CA-4 derivatives. Chapter 4 will highlight our effort to synthesize reverse ABI (RABI) analogues for SAR study. In this chapter, a novel and concise synthetic route was established toaccess RABI scaffold. RABI scaffold was constructed through a Grignard reaction/Suzuki-Miyaura coupling reaction strategy. From this new synthetic method, twelve novel RABI analogues were synthesized. Compared to MX-RABI (the previously reported most potent RABI), several new RABI analogues showed significantly improved cytotoxicities. In particular, analogue 15i with a 4-indazole moiety showed the most potent antiproliferative activity against a panel of melanoma cell lines and had an average IC50 of 0.8 nM. This is the first sub-nM anti-tubulin compound in the related scaffolds. Chapter 5 will reveal our latest SAR study of UC-112, a previously reported selective survivin inhibitor. Fourteen UC-112 analogues modifying the benzyloxy moiety of UC-112 were synthesized. Their corresponding SAR result demonstrated that indole moiety was the most favorable (analogue 12a). Subsequent structural optimization of 12a by introducing either mono-substituent or di-substituent to the indole moiety led to the synthesis of another twenty-four new UC-112 analogues. Several substituted indole analogues showed equipotency to that of UC-112 and MX-106. Importantly, new indole analogues exhibited significant abilities to overcome multidrug-resistance mediated by Pgp overexpression. Chapter 6 is characterized by the establishment of a total synthetic method of 20SOH-D3 which showed comparable antiproliferative activity to 1,25α-dihydroxyvitamin D3 without hypercalcemic toxic effect upto a concentration of 60 μg/kg in vivo. The total synthesis of 20S-OH-D3 involved parallel generation of key intermediates from ergocalciferol. The vitamin D3 core structure was constructed through Wittig-Horner coupling reaction. Deprotection of SEM and TBS was achieved in one step. 20S-OH-D3 was furnished in sixteen steps with an overall yield of 0.4%.

Many compounds containing a five-membered heterocyclic ring display exceptional chemical properties and versatile biological activities. The objective of the present study was to prepare the 5-substituted 2-amino-1,3,4- oxadiazole and 2-amino-1,3,4-thiadiazole derivatives and evaluate their potential anticancer, antibacterial and antifungal activities. Twenty-seven derivatives were synthesized by iodine-mediated cyclization of semicarbazones or thiosemicarbazones obtained from condensation of semicarbazide or thiosemicarbazide and aldehydes. The structures were confirmed by 1H-NMR, 13C-NMR and MS spectra. The antibacterial and antifungal activities were evaluated by diffusion method and the anticancer activities were evaluated by MTT assay. Twenty-seven derivatives have been synthesized in moderate to good yields. A number of derivatives exhibited potential antibacterial, antifungal and anticancer activities. Compounds (1b, 1e and 1g) showed antibacterial activity against Streptococcus faecalis, MSSA and MRSA with MIC value ranging between 4 to 64 μg/mL. Compound (2g) showed antifungal activity against Candida albicans (8 μg/mL) and Aspergillus niger (64 μg/mL). Compound (1o) exhibited high cytotoxic activity against HepG2 cell line (IC50 value 8.6 μM) which is comparable to the activity of paclitaxel, and is non-toxic on LLC-PK1 normal cell line. The structure activity relationship and molecular docking study of the synthesized compounds have also been reported.

Triterpenoids are nature-derived compounds, broadly distributed in plant kingdom in a free form as well as in a form of numerous glycosides. The complex structures of triterpenoids chiefly involve squalene derivatives, lanostanes, cycloartanes, dammaranes, euphanes, tirucallanes, tetranortriterpenoids, quassinoids, lupanes, oleananes, friedelanes, ursanes, hopanes, serratanes and another less known groups. These compounds present interesting and perspective subject for experiments, chemical and biological as well. As it is known from literature data, triterpenoids and their saponins with respect to their multitude pharmacological activities could be involved in several areas such as chemistry, medicine and pharmacology, biomedicine and agrochemical sciences. The special structure of triterpenic skeletons allows to perform numerous chemical transformations in order to obtain many new derivatives with pharmacological activity. Taking under consideration the above data, chemistry, biology and pharmacology of triterpenoids and their saponins appear to be riped for revisiting. Therefore, this special issue aims to review the advances in triterpenoids science during the last decade. Carcinomas are probably the second leading cause of death in humans, so scientists intense their research upon the development of new biological sources with antitumor activity. Medicinal plants are now the most promising sources of new potent substances in cytotoxic drug development. As Csuk presented in his work, such compound of anticancer activity is glycyrrhetinic acid, obtained e.g. from liquorice, known as sweetwood (Glycyrrhiza glabra) and some derivatives obtained from this triterpenic acid. Antitumor activity also exhibits, as presented by Paduch and Kandefer-Szerszeń, other triterpenes, such as: asiatic acid, betulinic acid, boswellic acid, lupeol, oleanolic acid or ursolic acid and their analogs and derivatives. These compounds exhibit a cytotoxicity against a variety of tumor cell lines comparable to some clinically used drugs. Most of them also have important antiviral properties, especially anti-HIV activity, which makes them potentially useful additives to current anti-HIV therapy. Another group of triterpenoids with anticancer and antiviral activity is limonoids family, belonging to tetranortriterpenoids. As Pękala and co-workers present, limonoids are a very valuable group of triterpenoids because of a wide spectrum of biological properties, such as cytotoxic, antimalarial, anti-inflammatory, antifeedant, antiviral, neuroprotective, antimicrobial, antibacterial, antifungal and other activities. Limonoids present rich variety of structures: until 2011, about 1300 limonoids with more than 35 carbon frameworks had been isolated from four families (Meliaceae, Rutaceae, Simaroubaceae, and Cneoraceae). The unique structural features of these terpenoids have attracted continuous attention of chemists and pharmacologists. Triterpenoids often occur as ingredients of daily diet or of herbal products used for therapeutic purposes. These phytochemicals occur in nature in free form or as glycosylated compounds, known as saponins, due to their characteristic foaming properties. Pharmacological activities of saponins are very broad and include numerous directions of action which were discussed in many works. Podolak and Janeczko summarize pharmacological properties of natural, non-glycosylated triterpenoids, that were reported over the last five years. The work discusses e.g. antimicrobial, cytotoxic, anti-inflammatory, antinociceptive, antidiabetic, hepatoprotective, wound healing and other activities of triterpenoids, focusing on less known compounds. Plants remain as the most viable source of triterpenoid saponins. As plants are the source of pharmacologically active saponins and free triterpenoids, it is obvious that the development of new methodologies to improve saponin yields is a significant issue. As Yendo et al. state in their work current challenges to improve triterpenoid saponin production including a better understanding of the signal transduction pathways leading to their accumulation, isolation and heterologous expression of biosynthetic genes, as well as structural and modeling studies of biosynthetic enzymes and their catalytic mechanisms. Some triterpenoids and their semi-synthetic derivatives can be identified as potential therapeutics against neurodegenerative diseases. Ruszkowski and Bobkiewicz-Kozlowska discuss the ability of celastrol, oleanolic acid, ursolic acid, asiatic acid, erythrodiol, and some triterpenoid saponins to protect the brain against neurodegeneration and neuroinflammation processes. The authors focus on triterpenoids as possible drugs to treat or slow the progression of neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis and multiple sclerosis (MS). Further, pharmacological activities of naturally-derived triterpenenoids and some of their synthetic derivatives are discussed by Jeong and Bae, Jaworska-Paszel et al. and by Luo et al. The anti-inflammatory effect is a common property of many triterpenoids. Jeong and Bae discuss the anti-inflammatory property of several triterpenoids derived from natural sources and from chemical synthesis. These triterpenoids include avicins, boswellic acid, celastrol, diosgenin, escin, ginsenosides, glycyrrhizin, lupeol, oleanolic acid, platycodon D, saikosaponins, ursolic acid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), and some of their derivatives. Jaworska-Paszel et al. describe anti-inflammatory, antioxidative, antitumor, antileukemic, hepatoprotective, gastroprotective, cardioprotective and many other important pharmacological activities of oleanolic acid. Luo et al. focus on recent advances in enone and NOreleasing derivatives of oleanolic acid with anti-cancer activity. Other compounds exhibiting numerous pharmacological activities are betulin and betulinic acid, both with pentacyclic lupane-type skeleton. Betulin, betulinic acid and their derivatives have been studied in the last decades for their diversified pharmacological activities like anti-HIV, anticancer, antibacterial, anti-inflammatory and many others. In the last few years, great progress had been made on the synthesis and biological activity of triterpenoids derived from betulin and betulinic acid. Shi and co-workers discuss recent research and development in chemistry of betulin and betulinic acid and present recent modification within A-ring, E-ring, both, at the C-3 and at the C-28 position as well as at the C-30 position of betulin and betulinic acid. The authors also present methods of conjunction of the above triterpenes with drugs. Supercritical fluid extraction has been a widely studied green technology to obtain triterpenoids enriched extracts from various vegetable matrices. De Melo and co-workers discuss research advances regarding the supercritical fluid extraction of triterpenoids in terms of biomass sources, operating conditions and their optimization, and modeling. The authors provide overview concerning the recent works (both publications and patents) upon supercritical fluid extraction of plant material leading to triterpenoids with the most important experimental and/or theoretical information. Many families of bioactive natural products are usually extracted from numerous plant sources in low yields. Triterpenic acids belong to a group of the most often studied compounds, mainly because of their abundance in a large number of food and medicinal plants. Domingues et al. in their work present overview of the biogenetic origin of some triterpenic acids, on the analytical extraction and hyphenated methodologies (mainly GC-MS) used on their identification and quantification. The authors also present an overview of the major sources of some triterpenic acids, indicating their potential large scale production pathways, with especial emphasis on abundant agroforestry biomass residues. The future perspectives towards improved therapeutic strategies using these triterpenic acids are also discussed herein. The root bark of the Chinese medicine Tripterygium wilfordii contains a quinone methide triterpenoid - celastrol. Literature data presents, that celastrol is one of the most potent antitumor activity compounds among the natural triterpenoids. Salvador et al. discuss different mechanisms of antitumor activity of celastrol, such as abilities of inhibing proliferation, inducing apoptosis and suppressing invasion and metastasis of tumor cells. The authors also describe the anticancer property of semi-synthetic celastrol derivatives. As they noticed, the quinone methide moiety is required for its antitumor activity. I thank all the contributors or this special issue and hope that the progresses highlighted in the topics covered will inspire more research in this challenging area of organic synthesis. I thank Prof. Atta-ur-Raman for the kind invitation to be the Guest Editor of this issue and all the reviewers for their time and valuable comments. I also thank Ms. Hina Wahaj (Publications Manager, Bentham Science Publishers) for the help in organizing this issue.

Calcium/calmodulin‐stimulated adenylyl cyclase (AC) isoforms AC1 and AC8 are potential targets for treating chronic pain, opioid dependence, and anxiety. The objective of our study was to carry out a series of structure activity relationship (SAR) studies on selective inhibitors of AC1 and/or AC8 activity. Our lab has recently identified a selective inhibitor of AC1 activity, W001, that displayed anti‐allodynic properties in a mouse model of inflammatory pain. Moreover, we revealed that the effect of this compound was prevented by co‐administration of forskolin consistent with an in vivo effect on cAMP signaling. Despite these promising results, W001 also potentiated the activity of AC2 suggesting the potential for unwanted side effects. In an effort to achieve greater overall AC selectivity we undertook two approaches. In the first approach, we performed SAR studies using both commercially available and novel synthesized analogs of W001. The ability of these compounds to inhibit A23187 (calcium ionophore)‐stimulated AC1 activity was examined in HEK cells stably expressing AC1. Active compounds were subsequently evaluated on the closely related AC8 isoform and other representative ACs (i.e. AC2 and AC5). The SAR studies for W001's analogs revealed important structural elements that are important for its inhibitory activity as well as for its selectivity for AC1. Several W001 analogs showed inhibitory activity on both AC1 and AC8, and to date we identified one analog with modest selectivity for AC8 over AC1. This new tool compound may allow us to probe for the role of AC8 in mouse behavioral models. In the second approach toward identifying AC1 inhibitors, we completed a 10,000‐compound screen. We identified two promising scaffolds that have robust AC1 inhibitory activity with IC50 values in the low micromolar range (2–10 μM). These novel and diverse scaffolds revealed both AC1 selective and dual AC1/AC8 inhibition. Additionally, we have identified structural features that diminish the activity at other AC isoforms (e.g. potentiation of AC2). In addition to the SAR studies, an AC1 homology model was developed to predict the binding mode of W001, its analogs, and our newly identified scaffolds. The comparison of computational homology models of AC1 and AC8, in combination with the AC1 and AC8 SAR analyses, provided information about the inhibitor‐residue interactions responsible for the AC1 selectivity of W001. Lastly, we are evaluating our lead compounds in cellular models with the ultimate goal of testing them in animal models of neuropathic pain and opioid dependence. In conclusion, the SARs and molecular docking results indicate that AC1 and AC8 selective inhibition has a structural relationship; this information can be applied to the design and synthesis of more potent and selective AC inhibitors with drug‐like properties.Support or Funding InformationPurdue EVPRP, Indiana Clinical and Translational Sciences Institute (CTSI) and R21 MH101673/NIMH

Introduction and Aim: As a timely need for potent anticancer drugs, we attempted to synthesize analogues of podophyllotoxin which are related to etoposide and tenoposide presently in the market. Materials and Methods: Compounds 8-13 were synthesized at standard condition by modifying the ring C structure of the parent podophyllotoxin and characterized by IR, NMR, Mass spectra and elemental analysis. Anticancer (MTT assay) activity for synthetic compounds was carried out on B16F10 mouse melanoma cell lines and antimitotic activity (cytotoxic) assay was on mitotic cells of root tips of Allium cepa L. Results: Analogues 8 and 9 exhibited greater anticancer activity with the IC50 values of 1.6 and 1.75mM respectively, and strong inhibition of mitosis with the ID50 values of 1.85mM and 2.10mM respectively, whereas the analogues 10, 11, 12 and 13 showed moderate anticancer activity. Conclusion: Analogues 8 and 9 would become the novel anticancer drugs in future for cancer chemotherapy after further investigations. Keywords: Synthesis; podophyllotoxin; anticancer; antimitotic.

Betulinic acid, a pentacyclic triterpene, is widely distributed throughout the tropics. It possesses several biological properties such as anticancer, anti-inflammatory, antiviral, antiseptic, antimalarial, spermicidal, antimicrobial, antileshmanial, antihelmentic and antifeedent activities. However, betulinic acid was highly regarded for its anticancer and anti-HIV activities. Anticancer role of betulinic acid appeared by inducing apoptosis in cells irrespective of their p53 status. Due to high order safety in betulinic acid, a number of structural modifications carried out to improve its potency and efficacy. The C-1, C-2, C-3, C-4, C-20 and C-28 positions are the diversity centers in betulinic acid, and the derivatives resulted on various structural modifications at these positions screened for their anticancer activity. This review presents the structure activity relationship carried out on C-1, C-2, C-3, C-4, C-20, C-28, A-ring, D-ring and E-ring modified betulinic acid derivatives. We have compiled the most active betulinic acid derivatives along with their activity profile in each series. Structure activity relationship studies revealed that C-28 carboxylic acid was essential for the cytotoxicity. The halo substituent at C-2 position in betulinic acid enhanced the cytotoxicity. Though the relation of the cytotoxicity with the nature of substituents at C-3 position could not be generalized but the ester functionality appeared to be a better substituent for enhancing the cytotoxicity. An interesting observation is that the three rings skeleton (A, B and C rings) had played an important role in eliciting anticancer activity, which could be a new molecular skeleton to design new anticancer drugs.

Structure activity relationships (SAR) study to design and synthesize new tubulin inhibitors with enhanced anti-tubulin activity: In silico and in vitro analysis

DD01-01: NMS-1116354: More than an inhibitor of Cdc 7 kinase in S-phase

Tubulysins, a family of natural products originally isolated from myxobacteria culture, inhibit tubulin polymerization, leading to apoptosis, thereby making them potential candidates for anticancer drug development. In this article, we report an advancement in the synthesis of third-generation tubulin inhibitors developed in our laboratory by leveraging the first application of solid-phase peptide synthesis (SPPS) to circumvent the limitations of conventional multistep solution-phase synthesis. The tubulin inhibitors have been synthesized in high yields without incorporating tubuvaline and tubuphenylalanine fragments present in the natural tubulysins, which are known to be essential for their anticancer activity. The most active inhibitor contains 4 fragments, i.e., N-methyl-d-pipecolic acid (N-Mep), l-isoleucine (l-Ile), valine-thiazole, and asparagine (Asn), arranged sequentially from the N-terminus to the C-terminus. The protocols enumerate an efficient synthesis of valine thiazole and N-methyl-d-pipecolic acid fragments, which are tethered using SPPS with a single purification step, resulting in high yields and enhancing their potential for scalable synthesis and therapeutic applications. The newly synthesized tubulin inhibitors exhibit anticancer activities with IC50 values in the low nanomolar range. The incorporation of a simplified thiazole ring structure preserves the biological activity and chiral integrity of the molecules. The structure-activity relationship studies highlight that small changes at the N-terminal fragment with various heterocyclic groups significantly reduce potency from nM to µM. In contrast, aliphatic C-terminal changes have little effect, though aromatic C-terminal modifications significantly impact the activity. This article describes an efficient method to synthesize simplified tubulin inhibitors, supporting further development of potent anticancer drugs. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of Fmoc-protected valine thiazole and N-methyl-d-pipecolic acid fragments Basic Protocol 2: Synthesis and purification of tubulin inhibitors (11a to 11l) using SPPS Basic Protocol 3: In vitro cytotoxicity assay of tubulin inhibitors (11a to 11l) against cancer cells derived from prostate, cervical, breast, lung, and skin using MTT assay.

In the present study, a series of 4H-chromene and 5H-chromeno[2,3-d]pyrimidine derivatives was synthesized and evaluated as potential cytotoxic agents. The cytotoxic activities of the target compounds were evaluated against four cancer cell lines MCF-7, HCT-116, HepG-2, and A549 in comparison with vinblastine and colchicine as reference drugs. We explored the structure–activity relationship of 4H-chromenes with modification at the 2-,4- or 7-position, and fused pyrimidine ring at 2,3-position. Most of the screened compounds showed marginal antitumor activity against the different cell lines in comparison to the standard drugs. The structure–activity relationship study revealed that the antitumor activity of the synthesized compounds was significantly affected by the lipophilicity of the substituent at the 2-,4- or 7-position for the 4H-chromenes, and 5,8-position or fused pyrimidine ring at 2,3-positions for 5H-chromeno[2,3-d]pyrimidines. Structure–activity relationship was elaborated with the help of molecular docking studies. The structures of the synthesized compounds were established on the basis of the spectral data, infrared, proton nuclear magnetic resonance, 13-Carbon nuclear magnetic resonance and mass spectroscopic data.