Self-Assembly Dual-Responsive NO Donor Nanoparticles for Effective Cancer Therapy.

Drug resistance and the serious side effects caused by classical chemotherapy drugs necessitate the development of novel targeted drug delivery systems. The high lipophilicity and short half-life of nitric oxide (NO), a gas with strong antitumor activity, make it difficult to reach the tumor site and result in a poor therapeutic effect in vivo. In order to overcome the deficiencies of the existing NO donors and NO delivery vehicles, a novel strategy was proposed to deliver NO for cancer chemotherapy by the prodrug dimer self-assembly nanoparticles of NO donors. Specifically, phenylsulfonylfuroxan (FZ) was chosen as the NO donor to synthesize the prodrug dimer precursor (FZ-SS-FZ) by disulfide linkages and ester bonds. The insertion of disulfide linkages promotes the self-assembly of FZ-SS-FZ in water. After this, the dual-responsive and tumor-targeting NO delivery system (FZ-SS-FZ@FA NPs) will finally be fabricated by further introducing folic acid on the surface of nanoparticles. FZ-SS-FZ can self-assemble to form uniform nanoparticles in water, which can effectively deliver NO to the tumor site and be uptaken by tumor cells, thus resulting in specific NO release in tumor cells and inducing tumor cell apoptosis. FZ-SS-FZ@FA NPs significantly improve the drug loading and delivery efficiencies of NO for chemotherapy, while enhancing its efficacy, providing a novel strategy for the tumor-targeted delivery of NO and at the same time laying a theoretical basis for the clinical translation of NO-based gas chemotherapy, opening up a new approach for cancer chemotherapy.

Identifying urotropine derivatives as co-donors of formaldehyde and nitric oxide for improving antitumor therapy.

A pharmacophore integration strategy was utilized to develop the first co-donor of formaldehyde and nitric oxide (FANO), composed of urotropine derived nitramine/nitrosamine. FANO simultaneously generated formaldehyde and nitric oxide on-demand, resulting in synergistic anticancer effects. Importantly, liposomal formulation of FANO effectively inhibited tumor growth with minimal side-effects, providing a potent combined nitric oxide therapy for malignancy.

Nitric Oxide Photo-Donor Hybrids of Ciprofloxacin and Norfloxacin: A Shift in Activity from Antimicrobial to Anticancer Agents.

The potential anticancer effect of fluoroquinolone antibiotics has been recently unveiled and related to their ability to interfere with DNA topoisomerase II. We herein envisioned the design and synthesis of novel Ciprofloxacin and Norfloxacin nitric oxide (NO) photo-donor hybrids to explore the potential synergistic antitumor effect exerted by the fluoroquinolone scaffold and NO eventually produced upon light irradiation. Anticancer activity, evaluated on a panel of tumor cell lines, showed encouraging results with IC50 values in the low micromolar range. Some compounds displayed intense antiproliferative activity on triple-negative and doxorubicin-resistant breast cancer cell lines, paving the way for their potential use to treat aggressive, refractory and multidrug-resistant breast cancer. No significant additive effect was observed on PC3 and DU145 cells following NO release. Conversely, antimicrobial photodynamic experiments on both Gram-negative and Gram-positive microorganisms displayed a significant killing rate in Staphylococcus aureus, accounting for their potential effectiveness as selective antimicrobial photosensitizers.

Tumor-Penetrable Nitric Oxide-Releasing Nanoparticles Potentiate Local Antimelanoma Therapy.

Although nitric oxide (NO) has been emerging as a novel local anticancer agent because of its potent cytotoxic effects and lack of off-target side effects, its clinical applications remain a challenge because of the short effective diffusion distance of NO that limits its anticancer activity. In this study, we synthesized albumin-coated poly(lactic-co-glycolic acid) (PLGA)-conjugated linear polyethylenimine diazeniumdiolate (LP/NO) nanoparticles (Alb-PLP/NO NPs) that possess tumor-penetrating and NO-releasing properties for an effective local treatment of melanoma. Sufficient NO-loading and prolonged NO-releasing characteristics of Alb-PLP/NO NPs were acquired through PLGA-conjugated LP/NO copolymer (PLP/NO) synthesis, followed by nanoparticle fabrication. In addition, tumor penetration ability was rendered by the electrostatic adsorption of the albumin on the surface of the nanoparticles. The Alb-PLP/NO NPs showed enhanced intracellular NO delivery efficiency and cytotoxicity to B16F10 murine melanoma cells. In B16F10-tumor-bearing mice, the Alb-PLP/NO NPs showed improved extracellular matrix penetration and spatial distribution in the tumor tissue after intratumoral injection, resulting in enhanced antitumor activity. Taken together, the results suggest that Alb-PLP/NO NPs represent a promising new modality for the local treatment of melanoma.

Discovery of ß-carboline-(phenylsulfonyl)furoxan hybrids as potential anti-breast cancer agents.

The cytotoxicity properties of the ß-carboline alkaloids have been broadly investigated. However, the potential application of ß-carbolines was hindered due to the moderate activity in cancer. In the present study, thirty ß-carboline-(phenylsulfonyl)furoxan hybrids (11a-j, 12a-j and 13a-j) were designed and synthesized through esterification and amidation reaction strategy, and their inhibitory activities against the human breast cancer cell lines MCF-7 and MDA-MB-231 were evaluated by CCK-8 assay. Biological evaluation presented that the most promising amide derivative 13h, substituted with p-methoxyphenyl group at position 1, generated high concentration of NO and evidently depressed the MCF-7 (IC50 = 0.89 µM) and MDA-MB-231 (IC50 = 0.62 µM) cells proliferation. Particularly, the wound healing and transwell assays demonstrated that 13h significantly inhibited the migration and invasion of MDA-MB-231cells. Furthermore, the preliminary mechanisms studies indicated that 13h induced G2/M phase arrest and apoptosis possibly causing by ROS accumulation and ROS-mediated DNA damage. Based on these considerations, 13h may be a promising antimetastatic agent for breast cancer, which is noteworthy for further exploration.

A divergent mode of activation of a nitrosyl iron complex with unusual antiangiogenic activity.

Nitric oxide (NO) and nitroxyl (HNO) have gained broad attention due to their roles in several physiological and pathophysiological processes. Remarkably, these sibling species can exhibit opposing effects including the promotion of angiogenic activity by NO compared to HNO, which blocks neovascularization. While many NO donors have been developed over the years, interest in HNO has led to the recent emergence of new donors. However, in both cases there is an expressive lack of iron-based compounds. Herein, we explored the novel chemical reactivity and stability of the trans-[Fe(cyclam)(NO)Cl]Cl2 (cyclam = 1,4,8,11-tetraazacyclotetradecane) complex. Interestingly, the half-life (t1/2) for NO release was 1.8 min upon light irradiation, vs 5.4 h upon thermal activation at 37 °C. Importantly, spectroscopic evidence supported the generation of HNO rather than NO induced by glutathione. Moreover, we observed significant inhibition of NO donor- or hypoxia-induced HIF-1α (hypoxia-inducible factor 1α) accumulation in breast cancer cells, as well as reduced vascular tube formation by endothelial cells pretreated with the trans-[Fe(cyclam)(NO)Cl]Cl2 complex. Together, these studies provide the first example of an iron-nitrosyl complex with anti-angiogenic activity as well as the potential dual activity of this compound as a NO/HNO releasing agent, which warrants further pharmacological investigation.

Novel nitric oxide-releasing derivatives of triptolide as antitumor and anti-inflammatory agents: Design, synthesis, biological evaluation, and nitric oxide release studies.

A series of novel triptolide/furoxans hybrids were designed and synthesized as analogues of triptolide, which is a naturally derived compound isolated from the thunder god vine (Tripterygium wilfordii Hook. F). Some of these synthesized compounds exhibited antiproliferative activities in the nanomolar range. Among them, compound 33 exhibited both good antiproliferative activity and NO-releasing ability and the acute toxicity of compound 33 decreased more than 160 times (LD50 = 160.9 mg/kg) than triptolide. Moreover, compound 33 significantly inhibited the growth of melanoma at a low dose (0.3 mg/kg) and showed strong anti-inflammatory activity in vitro and in vivo. These results indicate that compound 33 could be a promising candidate for further study.

Enhancing doxorubicin anticancer activity with a novel polymeric platform photoreleasing nitric oxide.

Combinations of conventional chemotherapeutics with unconventional anticancer agents such as reactive oxygen and nitrogen species may offer treatment benefits for cancer therapies. Here we report a novel polymeric platform combining the delivery of Doxorubicin (DOXO) with the light-regulated release of nitric oxide (NO). An amphiphilic block-copolymer (P1) was designed and synthesized as the drug carrier, with pendant amine groups to attach DOXO via a urea linkage and a NO photodonor (NOPD) activable by visible light. The two grafted-copolymers (P1-DOXO and P1-NOPD) self-assembled via solvent displacement methods into nanoparticles (NPs), containing both therapeutic components (NP1) and, for comparison, the individual NOPD (NP2) and DOXO (NP3). All the NPs were fully characterized in terms of physicochemical, photochemical and photophysical properties. These experiments demonstrated that integration of the NOPD within the polymeric scaffold enhanced the NO photoreleasing efficiency when compared with the free NOPD, and that the proximity to DOXO on the polymer chains did not significantly affect the enhanced photochemical performance. Internalization of the NPs into lung, intestine, and skin cancer cell lines was investigated after co-formulation with Cy5 fluorescent tagged polymers, and cytotoxicity of the NPs against the same panel of cell lines was assessed under dark and light conditions. The overall results demonstrate effective cell internalization of the NPs and a notable enhancement in killing activity of the dual-action therapeutic NP1 when compared with NP2, NP3 and the free DOXO, respectively. This suggests that the combination of DOXO with photoregulated NO release, achieved through the mixed formulation strategy of tailored polymer conjugate NPs, may open new treatment modalities based on the use of NO to improve cancer therapies.

Real-time redox monitoring of a nitrosyl ruthenium complex acting as NO-donor agent in a single A549 cancer cell with multiplex Fourier-transform infrared microscopy.

Multiplex Fourier-transform infrared microscopy (µFT-IR) helped to monitor trans-[Ru(NO) (NH3)4 (isn)]3+(I), uptake by A549 lung carcinoma cell, as well as the generation of its product, nitric oxide (NO), inside the cell. Chronoamperometry with NO-sensor and µFT-IR showed that exogenous NADH and the A549 cell induced the NO release redox mechanism. Chemical imaging confirmed that (I) was taken up by the cell, and that its localization coincided with its consumption in the cellular environment within 15 min of exposure. The Ru-NO absorption band in the IR spectrum shifted from 1932 cm-1, when NO was coordinated to Ru as {RuII-NO+}3+, to 1876 cm-1, due the formation of reduced species {RuII-NO0}2+, a precursor of NO release. Futhermore, the µFT-IR spectral profile demonstrated that, as a result of the NO action on the target, NO interacted with nucleic acids, which provided a biochemical response that is detectable in living cells.

Synthesis and Antitumor Evaluation in Vitro of NO-Donating Ursolic Acid-Benzylidene Derivatives.

Antitumor activity of triterpenoid and its derivatives has attracted great attention recently. Our previous efforts led to the discovery of a series of NO-donor betulin derivatives with potent antitumor activity. Herein, we prepared eight compounds derived from ursolic acid (UA). All the compounds were evaluated for their in vitro cytotoxicity against four human cancer cell lines (HepG-2, MCF-7, HT-29 and A549). Among the compounds tested, compound 4a was found to be most active against HT-29 (IC50 =4.28 µm). Further biological assays demonstrated that compound 4a could induce cell cycle arrest at G1 phase and apoptosis in a dose-dependent manner. In addition, compound 4a was found to upregulate pro-apoptotic Bax, p53 and downregulate anti-apoptotic Bcl-2. All these results suggested that compound 4a is a potential candidate drug for the therapy of colon cancer.

Design, synthesis and biological study of hybrid drug candidates of nitric oxide releasing cucurbitacin-inspired estrone analogs for treatment of hepatocellular carcinoma.

Development of hybrid drug candidates is well known strategy for designing antitumor agents. Herein, a novel class of nitric oxide donating cucurbitacin inspired estrone analogs (NO-CIEAs) were designed and synthesized as multitarget agents. Synthesized analogs were initially evaluated for their anti-hepatocellular carcinoma activities. Among the tested analogs, NO-CIEAs 17 and 20a exhibited more potent activity against HepG2 cells (IC50 = 4.69 and 12.5 µM, respectively) than the reference drug Erlotinib (IC50 = 25 µM). Interestingly, NO-CIEA 17 exerted also a high potent activity against Erlotinib-resistant HepG2 cell line (HepG2-R) (IC50 = 8.21 µM) giving insight about its importance in drug resistance therapy. Intracellular measurements of NO revealed that NO-CIEAs 17 and 20a showed a significant increase in NO production in tumor cells after 1 h of incubation comparable to the reference prodrug JS-K. Flow cytometric analysis showed that both NO-CIEAs 17 and 20a mainly arrested the HepG2 cells in the G0/G1 phase. Also, In-Cell Based ELISA screening showed that NO-CIEA 17 resulted in a potential inhibitory activity towards the EGFR and MAPK (25% and 29% inhibition compared to untreated control cells, respectively). This data suggests the binding ability of NO-CIEA 17 to the EGFR and ERK to be well correlated along with the docking and cellular studies. Also, treatment of HepG2-R cells with NO-CIEA 17 showed a potential reduction of MRP2 expression in a dose dependent manner providing a significant impact on the chemotherapeutic resistance. Overall, the current study provides a potential new approach for the discovery of a novel antitumor agent against HCC.

Cytotoxicity, cellular uptake, and subcellular localization of a nitrogen oxide and aminopropyl-ß-lactose derivative ruthenium complex used as nitric oxide delivery agent.

This work investigates how the luminescent ruthenium-nitrite complexes cis-[Ru(py-bodipy)(dcbpy)2(NO2)](PF6) (I) and cis-[Ru(py-bodipy)(dcbpy-aminopropyl-ß-lactose)2(NO2)](PF6) (II) behave toward the melanoma cancer cell line B16F10. The chemical structure and purity of the synthesized complexes were analyzed by UV-Visible and FTIR spectroscopy, MALDI, HPLC, and 1H NMR. Spectrofluorescence helped to determine the fluorescence quantum yields and lifetimes of each of these complexes. In vitro MTT cell viability assay on B16F10 cancer cells revealed that the complexes possibly have a tumoricidal role. The metal-nitrite complexes evidenced the dichotomous NO nature: at high concentration, NO exerted a tumoricidal effect, whereas cancer cells grew at low NO concentration. Flow cytometry or fluorescence microscopy aided cellular uptake calculation. Cell staining followed by fluorescence microscopy associated with organelle markers such as DAPI and Rhodamine 123 detected preferential intracellular localization of the ruthenium-nitrite py-bodipy and aminopropyl lactose derivative ruthenium complex in mitochondria. Thus, the cytotoxicity of compounds (I) and (II) against B16F10 cancer cell line show concentration-dependent results. The present studies suggest that nitric oxide ruthenium derivative compounds could be new potential chemotherapeutic agents against cytotoxic cells.

Effects of nitric oxide donor N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine on the expression of interferon-gamma in tumor infiltrating lymphocytes.

Nitric oxide (NO) has been proven to be a key regulator in the mammalian immune response, such as the innate and adaptive immune responses to tumors. The messenger NO involves T helper cell differentiation and lymphocyte biofunctions. In this study, we employed N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine as NO donor and released NO around tumor infiltrating lymphocytes in vitro by short-time blue light irradiation. The interferon-γ secretion of tumor infiltrating lymphocytes was investigated to study the functional changes caused by the accurate spatio-temporal delivery of NO. The downregulation of interferon-γ in tumor infiltrating lymphocytes after NO treatment indicates promising biological applications to potentially play a role in the treatment of autoimmune diseases. The study was approved by the Medical Ethics Committee of the Shenzhen Second People's Hospital, the First Affiliated Hospital of Shenzhen University, China (approved No. 065) on February 12, 2018.

Incorporation of nitric oxide donor into 1,3-dioxyxanthones leads to synergistic anticancer activity.

Fifty 1,3-dioxyxanthone nitrates (4a ∼ i-n, n = 1-6) were designed and synthesized based on molecular similarity strategy. Incorporation of nitrate into 1,3-dioxyxanthones with electron-donating groups at 6-8 position brought about synergistic anticancer effect. Among them, compound 4g-4 was confirmed the most active agent against HepG-2 cells growth with an IC50 of 0.33 ±â€¯0.06 µM. It dose-dependently increased intramolecular NO levels. This activity was attenuated by either NO scavenger PTIO or mitochondrial aldehyde dehydrogenase (mtADH) inhibitor PCDA. Apoptosis analysis indicated different contributions of early/late apoptosis and necrosis to cell death for different dose of 4g-4. 4g-4 arrested more cells on S phase. Results from Western Blot implied that 4g-4 regulated p53/MDM2 to promote cancer cell apoptosis. All the evidences support that 4g-4 is a promising anti-cancer agent.

Tuning the Hydrophobicity of a Mitochondria-Targeted NO Photodonor.

A few compounds in which the nitric oxide (NO) photodonor N-[4-nitro-3-(trifluoromethyl)phenyl]propane-1,3-diamine is joined to the mitochondria-targeting alkyltriphenylphosphonium moiety via flexible spacers of variable length were synthesized. The lipophilicity of the products was evaluated by measuring their partition coefficients in n-octanol/water. The obtained values, markedly lower than those calculated, are consistent with the likely collapsed conformation assumed by the compounds in solution, as suggested by molecular dynamics simulations. The capacity of the compounds to release NO under visible light irradiation was evaluated by measuring nitrite production by means of the Griess reaction. The accumulation of compounds in the mitochondria of human lung adenocarcinoma A549 cells was assessed by UPLC-MS. Interestingly, compound 13 [(9-((3-((4-nitro-3-(trifluoromethyl)phenyl)amino)propyl)amino)-9-oxononyl) triphenylphosphonium bromide] displayed both the highest accumulation value and high toxicity toward A549 cells upon irradiation-mediated NO release in mitochondria.

Synthesis and Biological Evaluations of NO-Donating Oxa- and Aza-Pentacycloundecane Derivatives as Potential Neuroprotective Candidates.

In order to utilize the neuroprotective properties of polycyclic cage compounds, and explore the NO-donating ability of nitrophenyl groups, an array of compounds was synthesized where the different nitrophenyl groups were appended on oxa and aza-bridged cage derivatives. Biological evaluations of the compounds were done for cytotoxicity, neuroprotective abilities, the inhibition of N-methyl-d-aspartate (NMDA)-mediated Ca2+ influx, the inhibition of voltage-mediated Ca2+ influx, and S-nitrosylation abilities. All of the compounds showed low toxicity. With a few exceptions, most of the compounds displayed good neuroprotection and showed inhibitory activity for NMDA-mediated and voltage-gated calcium influx, ranging from high (>70%) to low (20-39%) inhibition. In the S-nitrosylation assay, the compounds with the nitro moiety as the NO-donating group exhibited low to good nitrosylation potency compared to the positive controls. From the biological evaluation of the tested compounds, it was not possible to obtain a simple correlation that could explain the results across all of the biological study domains. This can be ascribed to the independent processes evaluated in the different assays, which reiterate that neuroprotection is a result of multifactorial biochemical mechanisms and interactions. However, these results signify the important aspects of the pentacylcoundecylamine neuroprotectants across different biological study realms.

Synthesis and Characterization of Nitric Oxide-Releasing Platinum(IV) Prodrug and Polymeric Micelle Triggered by Light.

Herein, we report the proof of concept of photoresponsive chemotherapeutics comprising nitric oxide-releasing platinum prodrugs and polymeric micelles. Photoactivatable nitric oxide-releasing donors were integrated into the axial positions of a platinum(IV) prodrug, and the photolabile hydrophobic groups were grafted in the block copolymers. The hydrophobic interaction between nitric oxide donors and the photolabile groups allowed for the loading of platinum drugs and nitric oxide-releasing donors in the photolabile polymeric micelles. After cellular uptake of micelles, light irradiation induced the release of nitric oxide, which sensitized the cancer cells. Simultaneously, photolabile hydrophobic groups were cleaved from micelles, and the nitric oxide-releasing donor was altered to be more hydrophilic, resulting in the rapid release of platinum(IV) prodrugs. The strategy of using platinum(IV) prodrugs and nitric oxide led to enhanced anticancer effects.

Molecular Design and Medicinal Applications of Nano-Nitric Oxide Delivery Systems.

BACKGROUND: Nitric oxide (NO) plays important regulatory roles in a plethora of biological functions and thus holds tremendous potential to be exploited for clinical uses. However, the chemistries in the molecular design of nano-nitric oxide delivery systems is currently lacking. OBJECTIVE: The overarching aim of this review is to provide the readers with the fundamentals that relate to the design of NO release molecules (NORMs), loading and releasing mechanism, as well as delivery of NORMs for nanotherapeutics. METHODS: We conducted a thorough literature search on the design and synthesis of NORMs, as well as the current state-of-the-art NO compatible delivery platforms to address various clinical needs. RESULTS: N-diazeniumdiolate and S-nitrosothiol based NO molecules are among the most widely used NORMs for anti-cancer and anti-microbial applications. The innovative integration of these NORMs with cytocompatible organic and inorganic nanocarriers enabled controlled spatiotemporal delivery and release of NO at the targeted diseased sites. CONCLUSION: We have provided a comprehensive summary of the fundamental chemistries underpinning the molecular design of the NORMs and critically assessed the recent advancements of nano-NO delivery systems for advanced biomedical applications.

Oxidative stress enhances and modulates protein S-nitrosation in smooth muscle cells exposed to S-nitrosoglutathione.

Among S-nitrosothiols showing reversible binding between NO and -SH group, S-nitrosoglutathione (GSNO) represents potential therapeutics to treat cardiovascular diseases (CVD) associated with reduced nitric oxide (NO) availability. It also induces S-nitrosation of proteins, responsible for the main endogenous storage form of NO. Although oxidative stress parallels CVD development, little is known on the ability of GSNO to restore NO supply and storage in vascular tissues under oxidative stress conditions. Aortic rat smooth muscle cells (SMC) were stressed in vitro with a free radical generator (2,2'-azobis(2-amidinopropane) dihydrochloride, AAPH). The cellular thiol redox status was reflected through levels of reduced glutathione and protein sulfhydryl (SH) groups. The ability of GSNO to deliver NO to SMC and to induce protein S-nitrosation (investigated via mass spectrometry, MS), as well as the implication of two redox enzymes involved in GSNO metabolism (activity of gamma-glutamyltransferase, GGT, and expression of protein disulfide isomerase, PDI) were evaluated. Oxidative stress decreased both intracellular glutathione and protein -SH groups (53% and 32% respectively) and caused a 3.5-fold decrease of GGT activity, while PDI expression at the plasma membrane was 1.7-fold increased without any effect on extracellular GSNO catabolism. Addition of GSNO (50 µM) increased protein -SH groups and protein S-nitrosation (50%). Mass spectrometry analysis revealed a higher number of S-nitrosated proteins under oxidative stress (83 proteins, vs 68 in basal conditions) including a higher number of cytoskeletal proteins (15, vs 9 in basal conditions) related with cell contraction, morphogenesis and movement. Furthermore, proteins belonging to additional protein classes (cell adhesion, transfer/carrier, and transporter proteins) were S-nitrosated under oxidative stress. In conclusion, higher levels of GSNO-dependent S-nitrosation of proteins from the cytoskeleton and the contractile machinery were identified under oxidative stress conditions. The findings may prompt the identification of suitable biomarkers for the appraisal of GSNO bioactivity in the CVD treatment.

Design and synthesis of pyrazolo[3,4-d]pyrimidines: Nitric oxide releasing compounds targeting hepatocellular carcinoma.

A new series of pyrazolo[3,4-d]pyrimidines tethered with nitric oxide (NO) producing functionality was designed and synthesized. Sulforhodamine B (SRB) protein assay revealed that NO releasing moiety in the synthesized compounds significantly decreased the cell growth more than the des-NO analogues. Compounds 7C and 7G possessing N-para-substituted phenyl group, released the highest NO concentration of 4.6% and 4.7% respectively. Anti-proliferative activity of synthesized compounds on HepG2 cell line identified compounds 7h, 7p, 14a and 14b as the most cytotoxic compounds in the series of IC50=3, 5, 3 and 5µM, respectively, compared to erlotinib as a reference drug (IC50=25µM). Flow cytometry studies revealed that 7h arrested the cells in G0/G1 phase of cell cycle while 7p arrested the cells in S phase. Moreover, docking study of the synthesized compounds on EGFR (PDB code: 1M17) and cytotoxicity study indicated that N-1 phenyl para substitution, pyrazole C-3 alkyl substitution and tethering the nitrate moiety through butyl group had a significant impact on the activity.