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Synthesis of highly strained fused substituted dihydrobenzopyran cyclopropyl lactones derived from coumarin carboxylates are reported. Substrate scope tolerates a variety of 6- and 8-substituents on the coumarin ring. Substitution at the 5- or 7-position is resistant to tricyclic lactone formation except with 7-methyl substitution. Benzamide-containing coumarins afford the tricyclic ketal. A plausible mechanism is proposed for the formation of the fused lactone; intramolecular rearrangement of trans cyclopropyl methyl ketones with phenolic acetate via the formation of a hemiacetal.

Inhibition of specific carbonic anhydrase (CA) enzymes is a validated strategy for the development of agents to target cancer. The CA isoforms IX and XII are overexpressed in various human solid tumors wherein they play a critical role in regulating extracellular tumor acidification, proliferation, and progression. A series of novel sulfonamides based on the coumarin scaffold were designed, synthesized and characterized as potent and selective CA inhibitors. Selected compounds show significant activity and selectivity over CA I and CA II to target the tumor-associated CA IX and CA XII with high inhibition activity at the single digit nanomolar level. Twelve compounds were identified to be more potent compared with acetazolamide (AAZ) control to inhibit CA IX while one was also more potent than AAZ to inhibit CA XII. Compound 18f (Ki’s = 955 nM, 515 nM, 21 nM and 5 nM for CA’s I, II, IX and XII respectively) is highlighted as a novel CA IX and XII inhibitor for further development.

A novel nitrogen mustard CBISC has been synthesized and evaluated as an anticancer agent. CBISC has been shown to exhibit enhanced cell proliferation inhibition properties against mutant p53 cell lines colorectal cancer WiDr, pancreatic cancer (MIAPaCa-2 and PANC-1), and triple negative breast cancer (MDA-MB-231 and MDA-MB-468). In vitro mechanism of action studies revealed perturbations in the p53 pathway and increased cell death as evidenced by western blotting, immunofluorescent microscopy and MTT assay. Further, in vivo studies revealed that CBISC is well tolerated in healthy mice and exhibited significant in vivo tumor growth inhibition properties in WiDr and MIAPaCa-2 xenograft models. These studies illustrate the potential utility of CBISC as an anticancer agent.

The human papilloma virus (HPV) high-risk variants (HPV-HR) such as HPV16 and HPV18 are responsible for most HPV related cancers, including anogenital and head and neck cancers. Here, we present two patients with HPV-HR-associated gynecological malignancies who, after failing radiation therapy, were treated with experimental salvage immunotherapy regimen resulting in complete, durable responses in both patients. Each patient was diagnosed with recurrent, radiation-refractory, HPV-HR positive, squamous cell carcinoma of the lower genital tract. Patient A was a 90-year-old, African American, with metastatic vulvar cancer to the right inguinal-femoral triangle and pulmonary metastases. Patient B was a 41-year-old, Caucasian, with a central-recurrence of cervix cancer. Each patient received at least two intratumoral quadrivalent HPV-L1 vaccine (Gardasil™) injections and daily topical TLR-7 agonist (imiquimod) to the tumor surface 2 weeks apart. This combination of intratumoral vaccinations and topical TLR-7 agonist produced unexpected complete resolution of disease in both patients. The importance of radiation therapy, despite being considered a treatment failure by current definitions, cannot be understated. Radiation therapy appears to have offered a therapeutic immune advantage by modifying the tumor microenvironment. This immune protocol has potential to help patients with advanced HPV-HR-related malignancies previously considered incurable.

A series of nitric oxide (NO) donor furoxan conjugates of N, N-dialkylcarboxy coumarins have been synthesized as potential anticancer agents. The synthesized compounds have been tested for their in vitro antiproliferative activities on various cancer and noncancerous cell lines. The candidate derivatives exhibit selectivity towards cancer cells with excellent activities in low nM to µM concentrations. In vitro mechanistic studies indicate that the candidate compounds generate substantial NO, inhibit colony formation, and cause apoptosis in cancer cells. A preliminary in vivo tolerance study of the lead candidate 10 in mice indicates that it is well-tolerated, evidenced by zero mortality and normal body weight gains in treated mice. Further translation of the lead derivative 10 using MDA-MB-231 based tumor xenograft model shows good tumor growth reduction.

The synthesis of cyclopropyl methyl ketones and highly strained fused substituted dihydrobenzopyran cyclopropyl lactones derived from coumarin carboxylates and chloroacetone in the presence of potassium carbonate is reported. One step synthetic access to the previously unknown dihydrobenzopyran cyclopropyl tricyclic ketals is achieved with wide substrate scope. Substituted coumarin carboxylates, phenylamides or a quinolin-2(1H)-one possessing alkyl electron donating (methyl, t-butyl) and electron withdrawing groups (F, Cl, Br, NO2) in the 4 or 6 positions formed the highly strained dihydrobenzopyran cyclopropyl tricyclic ketals in moderate yield alongside the expected coumarin carboxylate. Saturation or substitution at the 5-position or 6-OMe afforded no tricyclic ketal compound but solely coumarin carboxylates. The formation of both structures in selected derivatives was confirmed by X-ray crystallography. A plausible mechanism is proposed for the formation of the fused lactone; via intramolecular rearrangement of cis cyclopropyl methyl ketones with phenolic acetate via the formation of a hemiacetal.

Background Breast cancer remains as one of the most lethal types of cancer in women. Among various subtypes, triple-negative breast cancer (TNBC) is the most aggressive and hard to treat type of breast cancer. Mechanistically, increased DNA repair and cell cycle checkpoint activation remain as the foremost reasons behind TNBC tumor resistance to chemotherapy and disease recurrence. Methods We evaluated the mechanism of prexasertib-induced regulation of homologous recombination (HR) proteins using 20S proteasome inhibitors and RT-PCR. HR efficiency and DNA damages were evaluated using Dr-GFP and comet assays. DNA morphology and DNA repair focus studies were analyzed using immunofluorescence. UALCAN portal was used to evaluate the expression of RAD51 and survival probability based on tumor stage, subtype, and race in breast cancer patients. Results Our results show that prexasertib treatment promotes both post-translational and transcriptional mediated regulation of BRCA1 and RAD51 proteins. Additionally, prexasertib-treated TNBC cells revealed over 55% reduction in HR efficiency compared to control cells. Based on these results, we hypothesized that prexasertib treatment induced homologous recombination deficiency (HRD) and thus should synergize with PARP inhibitors (PARPi) in TNBC cells. As predicted, combined treatment of prexasertib and PARPi olaparib increased DNA strand breaks, γH2AX foci, and nuclear disintegration relative to single-agent treatment. Further, the prexasertib and olaparib combination was synergistic in multiple TNBC cell lines, as indicated by combination index (CI) values. Analysis of TCGA data revealed elevated RAD51 expression in breast tumors compared to normal breast tissues, especially in TNBC subtype. Interestingly, there was a discrepancy in RAD51 expression in racial groups, with African-American and Asian breast cancer patients showing elevated RAD51 expression compared to Caucasian breast cancer patients. Consistent with these observations, African-American and Asian TNBC patients show decreased survival. Conclusions Based on these data, RAD51 could be a biomarker for aggressive TNBC and for racial disparity in breast cancer. As positive correlation exists between RAD51 and CHEK1 expression in breast cancer, the in vitro preclinical data presented here provides additional mechanistic insights for further evaluation of the rational combination of prexasertib and olaparib for improved outcomes and reduced racial disparity in TNBC.

Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) are involved in tumor development and progression. Their expression levels are related to clinical disease prognosis. Accordingly, both MCTs are promising drug targets for treatment of a variety of human cancers. The noninvasive imaging of these MCTs in cancers is regarded to be advantageous for assessing MCT-mediated effects on chemotherapy and radiosensitization using specific MCT inhibitors. Herein, we describe a method for the radiosynthesis of [18 F]FACH ((E)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylic acid), as a novel radiolabeled MCT1/4 inhibitor for imaging with PET. A fluorinated analog of α-cyano-4-hydroxycinnamic acid (FACH) was synthesized, and the inhibition of MCT1 and MCT4 was measured via an L-[14 C]lactate uptake assay. Radiolabeling was performed by a two-step protocol comprising the radiosynthesis of the intermediate (E)/(Z)-[18 F]tert-Bu-FACH (tert-butyl (E)/(Z)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylate) followed by deprotection of the tert-butyl group. The radiofluorination was successfully implemented using either K[18 F]F-K2.2.2 -carbonate or [18 F]TBAF. The final deprotected product [18 F]FACH was only obtained when [18 F]tert-Bu-FACH was formed by the latter procedure. After optimization of the deprotection reaction, [18 F]FACH was obtained in high radiochemical yields (39.6 ± 8.3%, end of bombardment (EOB) and radiochemical purity (greater than 98%).

Novel N-phenylindazole based diarylureas have been designed, synthesized and evaluated as potential anticancer agents. In vitro cell viability studies of these derivatives illustrate good potency with IC50 values in the range of 0.4–50 μM in several cancer cell lines including murine metastatic breast cancer 4T1, murine glioblastoma GL261, human triple negative breast cancer MDA-MB-231, human pancreatic cancer MIAPaCa-2, and human colorectal cancer cell line WiDr. The ester group in the lead compound 8i was modified to incorporate amino-amides to increase solubility and stability while retaining biological activity. Further in vitro studies reveal that lead candidates inhibit tube length in HUVEC cells. In vivo systemic toxicity studies indicate that these candidate compounds are well tolerated in mice without any significant side effects. Anticancer efficacy studies in WiDr tumor xenograft and 4T1 tumor syngraft models demonstrate that the lead candidate 11 exhibits significant antitumor properties as a single agent in these tumor models.

Arylphosphonium-benzoxaborole conjugates have been synthesized as potential mitochondria targeting anticancer agents. The synthesized compounds have been tested for their effects on cell viability in various solid tumor cell lines including breast cancer 4T1 and MCF-7, pancreatic cancer MIAPaCa-2 and colorectal adenocarcinoma WiDr. Compound 6c is designated as a lead compound for further studies due to its enhanced effects on cell viability in the above-mentioned cell lines. Seahorse Xfe96 based metabolic assays reveal that the lead candidate 6c inhibits mitochondrial respiration in 4T1 and WiDr cell lines as evidenced by the reduction of mitochondrial ATP production and increase in proton leak. Epiflourescent microscopy experiments also illustrate that 6c causes significant mitochondrial fragmentation in 4T1 and WiDr cells, morphologically consistent with programmed cell death. Our current studies illustrate that arylphosphonium-benzoxaborole conjugates have potential to be further developed as anticancer agents.

Objective: MCT1-4 are involved in several diseases, particularly in cancer. [18F]FACH has recently been developed as a novel MCT-targeting imaging agent (1), which could be used for monitoring MCT-based treatment approaches. With the aim to develop a similar potent radiotracer with higher brain permeability for future brain tumor studies, we designed structurally modified analogs of FACH possessing increased lipophilicity. Methods: Two analogs of FACH (I and II) were synthesized by introduction of a 2-fluoropyridinyl moiety via Buchwald-Hartwig cross coupling reaction. Inhibition of MCT1 was measured by [14C]lactate uptake assay using rat brain endothelial cells and analog I with higher inhibition was selected to synthesize corresponding precursor used for radiofluorination by aromatic nucleophilic substitution. LogD7.4 of [18F]I was experimentally determined in the n-octanol-PBS system. In vitro autoradiography and dynamic PET studies of [18F]I were performed in CD-1 mice. Results: The analogs I and II showed a moderate MCT1 inhibition with IC50 values of 118 and 274 nM, respectively. [18F]I was obtained with radiochemical yields of 73±12% (n=4, non-isolated) and a high radiochemical purity of > 98%. A logD7.4 value of 0.816 was achieved for [18F]I, which was 2-fold higher than that for [18F]FACH. By in vitro autoradiography in cryosections of the mouse kidney, nearly complete displacement of [18F]I by 10-5 M CHC-Na was observed. In vivo, similar to [18F]FACH, a low uptake of [18F]I in the brain without significant washout was found with an almost constant SUV of 0.15 between 15 and 60 min p.i. Conclusion: Despite a higher lipophilicity of [18F]I compared to [18F]FACH, the brain uptake of [18F]I was in a similar low range. However, the high and specific uptake of the new radiotracer in the kidneys suggests suitability of [18F]I for detecting MCTs’ expression in vivo. References: (1) Sadeghzadeh M, et al. J Label Compd Radiopharm.2019; 62: 411-424.

Approximately 90% of ovarian cancer (OC) is Epithelial ovarian Cancer (EOC) subtype and claims ~15,000 lives in the United States annually, making it the deadliest reproductive cancer in women. There are few treatment options for EOC, amongst them include surgical resection of tumors or debulking, and chemotherapy alone or combination. In general, the survival rate of patients with EOC is about 48% and this has not changed in last few decades. According to recent reports from American Cancer Society and SEER, racial disparity in ovarian cancer not only exists for African American (AA) patients, but is worsening over the past few years. African American (AA) patients presents with more advanced disease and develop chemoresistance frequently, and as such, they experience worse survival. Thus identifying the cause of this discrepancy, or more importantly, describing which AA patients are at the highest risk of therapeutic relapse would alter our current treatment strategies and improve overall disease free survival rate. Therefore, the central focus of this proposal is to delineate the molecular and genetic mechanisms contributing to racial disparity of AA patients. Therefore, the overall objective of our studies are to identify the etiology of racial disparity in ovarian cancer and define the molecular networks that contribute this discrepancy in outcomes. Lymphoblastic Leukemia-Derived Sequence 1 (LYL1) is a polypeptide that harbors basic helix-loop-helix transcription factor, a DNA binding motif and dysregulated in many cancers including EOC. Analysis of TCGA data for EOC revealed that LYL1 gene amplification in about 12% patients and associated with poor prognosis. Interestingly, further analysis of LYL1 copy number alteration in different ethnicities disclosed LYL1 amplification in about 36% of the African American (AA) EOC patients. Importantly, EOC patients with low LYL1 (n=1640) expression has better survival probability compared to patients with overexpressed LYL1 (n=202). This discrepancy in survival probability is much more prominent in AA EOC patients. As this is an intriguing observation, we evaluated the levels of LYL1 expression in different EOC cell lines in comparison with fallopian tube epithelial cells. Our data shows, upregulation of LYL1 in most of the EOC cell lines compared to normal fallopian tube epithelial cells. To examine the upregulated LYL1 in EOC cell lines, we performed siRNA mediated downregulation, and evaluated their clonogenic, migration invasion potential. Consistent with the TCGA data, knocking down LYL1 in EOC cells significantly attenuated their clongenic, migration and invasion potential. Furthermore, ectopic overexpression of LYL1 in EOC cells that shows deep deletion of the gene, exhibited increased clonogenicity, invasion and migration. Collectively, our studies indicate an important role for LYL1 in EOC tumor progression and metastatic phenotypes, and could be a biomarker for disparities in EOC outcomes. Citation Format: Damieanus Ochola, Shirisha Jonnalagadda, Swetha Peddibhotla, Tasmin Omy, Mark Reedy, Palle Komaraiah. Upregulated LYL1 promotes epithelial ovarian cancer (EOC) cell growth and metastasis [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-132.

Allylic acetates derived from Baylis-Hillman reaction undergo efficient nucleophilic isomerization with imidazoles and triazoles to provide imidazolylmethyl and triazolylmethyl cinnamates stereoselectively. Antifungal evaluation of these derivatives against Cryptococcus neoformans exhibits good minimum inhibitory concentration values. These compounds exhibit low toxicity in proliferating MCF-7 breast cancer cell line. Structure activity relationship studies indicate that halogenated aromatic derivatives provide better antifungal activity.

Epithelial Ovarian Cancer (EOC) is the fifth leading cause of cancer-related deaths in women in the USA, gets diagnosed at an advanced stage (stage III-IV) with local or distant metastasis. The primary therapeutic regimen of EOC involves surgical removal of all the visible tumor mass followed by platinum-based chemotherapy either as a single agent or in combination with a taxane. Despite initial responses, over 70% of EOC patients develop recurrent disease and they are often resistant to available chemotherapeutic drugs, contributing to poor survival. This appalling situation emphasizes the critical need to identify the molecular mechanisms involving recurrence of these tumors' aggressive growth and developing therapeutic resistance. Our analysis from chemo naïve and recurrent tumors from the same patients' samples, and isogenic ovarian cancer cell lines data showed recurrent tumors and resistant cell lines several upregulated cancer stem cell markers including ALDH1A1, SOX2 and altered expression of DNA damage response and repair genes that are involved in the processing of platinum adducts and crosslink repair. Prominently, RAD6, an E2ubiquitin-conjugating enzyme, is significantly (> 6 folds) overexpressed in recurrent ovarian tumors and associated with aggressive tumor cell growth, stemness, chemoresistance to platinum drugs, and poor prognosis. Our studies validated that, chemotherapy induced upregulated RAD6 reprograms epigenetic milieu in tumor cells through ubiquitination of histone variants H2A, H2AX, and H2B, which further recruits additional epigenetic modifiers to these regions and regulates genes involved in DNArepair, cell survival, stemness, and chemoresistance. Additionally, our mechanistic studies demonstrate RAD6 dependent recruitment of several epigenetic modifiers such as histone methylases and demethylases to regions of ubiquitylated histones and their crosstalk. Collectively, our study presents novel chemotherapy induced epigenetic modulator contributing to therapeutic recurrence of EOC and could be an important therapeutic target to treat chemo-resistant EOC and improve the progression-free survival of patients suffering from this deadly disease. Formatted: Right, Indent: Left: 1" Citation Format: Tasmin Rahman Omy, Shirisha Jonnalagadda, Mark Reedy, Komaraiah Palle. RAD6-mediated epigenetic reprogramming contributes to therapy-induced chemo-resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2111.

Novel silyl cyanocinnamic acid derivatives have been synthesized and evaluated as potential anticancer agents. In vitro studies reveal that lead derivatives 2a and 2b have enhanced cancer cell proliferation inhibition properties when compared to the parent monocarboxylate transporter (MCT) inhibitor cyano-hydroxycinnamic acid (CHC). Further, candidate compounds exhibit several-fold more potent MCT1 inhibition properties as determined by lactate-uptake studies, and these studies are supported by MCT homology modeling and computational inhibitor-docking studies. In vitro effects on glycolysis and mitochondrial metabolism also illustrate that the lead derivatives 2a and 2b lead to significant effects on both metabolic pathways. In vivo systemic toxicity and efficacy studies in colorectal cancer cell WiDr tumor xenograft demonstrate that candidate compounds are well tolerated and exhibit good single agent anticancer efficacy properties.

Monocarboxylate transporters (MCTs) support tumour growth by regulating the transport of metabolites in the tumour microenvironment. High MCT1 or MCT4 expression is correlated with poor outcomes in human patients with head and neck squamous cell carcinoma (HNSCC). Recently, drugs targeting these transporters have been developed and may prove to be an effective treatment strategy for HNSCC. Feline oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy resembling advanced or recurrent HNSCC. The goals of this study were to investigate the effects of a previously characterized dual MCT1 and MCT4 inhibitor, MD-1, in OSCC as a novel treatment approach for feline oral cancer. We also sought to determine the potential of feline OSCC as a large animal model for the further development of MCT inhibitors to treat human HNSCC. In vitro, MD-1 reduced the viability of feline OSCC and human HNSCC cell lines, altered glycolytic and mitochondrial metabolism and synergized with platinum-based chemotherapies. While MD-1 treatment increased lactate concentrations in an HNSCC cell line, the inhibitor failed to alter lactate levels in feline OSCC cells, suggesting an MCT-independent activity. In vivo, MD-1 significantly inhibited tumour growth in a subcutaneous xenograft model and prolonged overall survival in an orthotopic model of feline OSCC. Our results show that MD-1 may be an effective therapy for the treatment of feline oral cancer. Our findings also support the further investigation of feline OSCC as a large animal model to inform the development of MCT inhibitors and future clinical studies in human HNSCC.

Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) are involved in tumor development and progression. Their expression levels are related to clinical disease prognosis. Accordingly, both MCTs are promising drug targets for treatment of a variety of human cancers. The noninvasive imaging of these MCTs in cancers is regarded to be advantageous for assessing MCT-mediated effects on chemotherapy and radiosensitization using specific MCT inhibitors. Herein, we describe a method for the radiosynthesis of [

Potent and dual monocarboxylate transporter (MCT) 1 and 4 inhibitors have been developed for the first time as potential anticancer agents based on α-cyanocinnamic acid structural template. Candidate inhibitors 1-9 have been evaluated for in vitro cell proliferation against MCT1 and MCT4 expressing cancer cell lines. Potential MCT1 and MCT4 binding interactions of the lead compound 9 have been studied through homology modeling and molecular docking prediction. In vitro effects on extracellular flux via glycolysis and mitochondrial stress tests suggest that candidate compounds 3 and 9 disrupt glycolysis and OxPhos efficiently in MCT1 expressing colorectal adenocarcinoma WiDr and MCT4 expressing triple negative breast cancer MDA-MB-231 cells. Fluorescence microscopy analyses in these cells also indicate that compound 9 is internalized and concentrated near mitochondria. In vivo tumor growth inhibition studies in WiDr and MDA-MB-231 xenograft tumor models in mice indicate that the candidate compound 9 exhibits a significant single agent activity.