Spectroscopic,computational modeling and cytotoxicity of a series of meso-phenyl and meso-thienyl-BODIPYs

A series of twenty-two BODIPY compounds were synthesized, containing various meso-phenyl and meso-thienyl groups, and their spectroscopic and structural properties were investigated using both experimental and computational methods. Further functionalization of the BODIPY framework via iodination at the 2,6-pyrrolic positions was explored in order to determine the effect of these heavy atoms on the photophysical and cytotoxicity of the meso-aryl-BODIPYs. BODIPYs bearing meso-thienyl substituents showed the largest red-shifted absorptions and emissions and reduced fluorescence quantum yields. The phototoxicity of the BODIPYs in human carcinoma HEp2 cells depends on both the presence of iodines and the nature of the meso-aryl groups. Six of the eleven 2,6-diiodo-BODIPYs investigated showed at least a sevenfold enhancement in phototoxicity (IC₅₀ = 3.5–28 μM at 1.5 J/cm²) compared with the non-iodinated BODIPYs, while the others showed no cytotoxicity, while their singlet oxygen quantum yields ranged from 0.02 to 0.76. Among the series investigated, BODIPYs 2a and 4a bearing electron-donating meso-dimethoxyphenyl substituents showed the highest phototoxicity and dark/phototoxicity ratio, and are therefore the most promising for application in PDT.     

Exploring new near-infrared fluorescent disulfide-based cyclic RGD peptide analogs for potential integrin-targeted optical imaging

We synthesized disulfide-based cyclic RGD pentapeptides bearing a near-infrared fluorescent dye (cypate), represented by cypate-c(CRGDC) (1) for integrin-targeted optical imaging. These compounds were compared with the traditional lactam-based cyclic RGD counterpart, cypate-c(RGDfK) (2). Molecular modeling suggests that the binding affinity of 2 to integrin α_vβ₃ is an order of magnitude higher than that of 1. This was confirmed experimentally, which further showed that substitution of Gly with Pro, Val and Tyr in 1 remarkably hampered the α_vβ₃ binding. Interestingly, cell microscopy with A549 cells showed that 1 exhibited higher cellular staining than 2. These results indicate that factors other than receptor binding affinity to α_vβ₃ dimeric proteins mediate cellular uptake. Consequently, 1 and its analogs may serve as valuable molecular probes for investigating the selectivity and specificity of integrin targeting by optical imaging.     

Design,synthesis and evaluation of anti-CD38 antibody drug conjugate based on Daratumumab and maytansinoid

Daratumumab, an FDA approved antibody drug, displays specific targeting ability to abnormal white blood cells overexpressing CD38 and provides efficacious therapy for multiple myeloma. Here, in order to achieve enhanced remission of multiple myeloma, we designed Dara-DM4, antibody drug conjugates (ADCs) by conjugating Daratumumab and DM4 via a disulfide linker. Dara-DM4 showed significantly higher cellular uptake and inhibitory efficacy on MM1S cells that overexpressing CD38 with an IC₅₀ of 0.88?µg/mL post 72?hr treatment. These results support a promising ADCs strategy for multiple myeloma treatment.     

Synthesis,photophysical properties,and photodynamic activity of positional isomers of TFPP-glucose conjugates

The synthesis and characterization of a ‘complete set’ of positional isomers of tetrakis(perfluorophenyl)porphyrins (TFPP)-glucose conjugates (1OH, 2OH, 3OH, 4OH, and 6OH) are reported herein. The cellular uptake and photocytotoxicity of these conjugates were examined in order to investigate the influence of location of the TFPP moiety on the d-glucose molecule on the biological activity of the conjugates. An In vitro biological evaluation revealed that the certain of these isomers have a greater effect on cellular uptake and cytotoxicity than others. The TFPP-glucose conjugates 1OH, 3OH, and 4OH were found to exert exceptional photocytotoxicity in several types of cancer cells compared to 2OH and 6OH substituted isomers.     

Lithospermic acid derivatives from Lithospermum erythrorhizon increased expression of serine palmitoyltransferase in human HaCaT cells

A MeOH extract of the dry root of Lithospermum erythrorhizon showed strong increasing effect on serine palmitoyltransferase (SPT) in normal human keratinocyte cells (HaCaT cells). Bioassay-guided separation on this extract using repeated chromatography resulted in the isolation of lithospermic acid (1) and two derivative esters, 9″-methyl lithospermate (2) and 9′-methyl lithospermate (3). Compounds 1–3 significantly increased SPT expressions in the relative quantity (%) of SPT1 mRNA as well as SPT2 mRNA. These constituents also raised the level of SPT protein in HaCaT cells in a dose-dependent manner, with the increased level of SPT protein in HaCaT cells of 55%, 23%, and 81% at the concentration of 100 μg/ml, respectively. This finding suggests that lithospermic acid and its derivatives from L. erythrorhizon might improve the permeability barrier by stimulating the protein level of SPT.     

Sulfenamide derivatives can improve transporter-mediated cellular uptake of metformin and induce cytotoxicity in human breast adenocarcinoma cell lines

Metformin, the most frequently administered oral anti-diabetic drug, is a substrate for organic cation transporters (OCTs). This determines not only its pharmacokinetic properties but also its biochemical effects in humans, including its recently-discovered antiproliferative properties. The aim of the study was to verify the hypothesis whether chemical modification of its biguanide backbone may increase the cellular uptake and antiproliferative efficacy of metformin.The study examines five sulfenamide derivatives of metformin with differing lengths of alkyl chains. It determines their cellular uptake and the role of OCTs in their transport in human breast adenocarcinoma cells (epithelial-like MCF-7, and MDA-MB-231). It also evaluates whether increased cellular uptake of metformin derivatives is associated with their cytotoxic properties.Sulfenamide derivatives were characterized by a greater ability to bind to OCTs than metformin. Compound 2 with n-octyl alkyl chain was found to possess the greatest affinity towards OCTs, as measured by determination of [¹⁴C]choline uptake inhibition (IC₅₀ = 236.1 ± 1.28 μmol/L, and 217.4 ± 1.33 μmol/L, for MCF-7 and MDA-MB-231 respectively). Sulfenamides were also found to exhibit better cellular uptake in comparison with the parent drug, metformin. For instance, the uptake of cyclohexyl derivative 1 was 1.28 ± 0.19 nmol/min/mg of proteins and thus was 12-fold higher than the metformin in MCF-7 cells. Furthermore, higher uptake was associated with the greatest antiproliferative properties expressed as the lowest IC₅₀ value i.e. inhibiting the growth of 50% of the cells (IC₅₀ = 0.72 ± 1.31 μmol/L).Collectively, chemical modification of metformin into sulfenamides with different alkyl substituents obtains better substrates for OCTs, and subsequently higher cellular uptake in MCF-7 and MDA-MB-231 cells. Additionally, the length of alkyl chain introduced to the sulfenamides was found to influence selectivity and transport efficiency via OCT1 compared to other possible transporters, as well as potential intracellular activity and cytotoxicity.     

Synthesis,photophysical properties and photocytotoxicity of mono-, di-, tri- and tetra-glucosylated fluorophenylporphyrins

In order to explore the effect of substitution patterns on the photocytotoxicity of glycoconjugated porphyrins, we synthesized and characterized a ‘complete set’ of tetrakis(perfluorophenyl)porphyrins having β-d-glucopyranosylthio groups on the phenyl ring. Among five possible derivatives, trans-substituted S-glucosylated porphyrin trans-2_OH exerted outstanding photocytotoxicity (EC₅₀ value was <5 nM) in HeLa cells. The excellent photocytotoxicity of trans-2_OH was found to be attributable to several factors, namely high optical transition probability in aqueous media, efficient type I photoreactions and enhanced cellular uptake.     

Synthesis of (aminoalkyl)cycleanine analogues: cytotoxicity,cellular uptake,and apoptosis induction in ovarian cancer cells

Our previous studies demonstrated that cycleanine, a macrocyclic bisbenzylisoquinoline (BBIQ) alkaloid, showed potent anti-ovarian cancer activity via apoptosis induction. Here, we synthesized two novel (aminoalkyl)cycleanine analogues (2 and 3) through a simple and efficient two-step reaction starting from cycleanine isolated from Triclisia subcordata Oliv. These analogues showed greater potency than the unmodified cycleanine in three human ovarian cancer cell lines. Both 2 and 3 induced apoptosis in ovarian cancer cells by activations of caspases 3/7, cleavage of PARP, increase in subG₁ cell cycle phase and in the percentage of apoptotic cells. Further confocal fluorescence microscopy analysis confirmed the cellular uptake of alkaloids in ovarian cancer cells by using the unique (alkynyl)cycleanine (3) via click chemistry reaction. Our results suggest that cycleanine could be a hit compound for the future development in attacking ovarian cancer.     

Discovery of new potent protein arginine methyltransferase 5 (PRMT5) inhibitors by assembly of key pharmacophores from known inhibitors

Protein arginine methyltransferase 5 (PRMT5) is an epigenetics related enzyme that has been validated as a promising therapeutic target for human cancer. Up to now, two small molecule PRMT5 inhibitors has been put into phase I clinical trial. In the present study, a series of candidate molecules were designed by combining key pharmacophores of formerly reported PRMT5 inhibitors. The in vitro PRMT5 inhibitory testing of compound 4b14 revealed an IC₅₀ of 2.71?μM, exhibiting high selectivity over PRMT1 and PRMT4 (>70-fold selective). As expected, 4b14 exhibited potent anti-proliferative activity against a panel of leukemia and lymphoma cells, including MV4-11, Pfeiffer, SU-DHL-4 and KARPAS-422. Besides, 4b14 showed significant cell cycle arrest and apoptosis-inducing effects, as well as reduced the cellular symmetric arginine dimethylation level of SmD3 protein. Finally, affinity profiling analysis indicated that hydrophobic interactions, π-π stacking and cation-π actions made the major contributions to the overall binding affinity. This scaffold provides a new chemical template for further development of better lead compounds targeting PRMT5.     

New acrylamide-sulfisoxazole conjugates as dihydropteroate synthase inhibitors

New functionalized acrylamide derivatives bearing sulfisoxazole moiety were designed to target bacterial dihydropteroate synthase (DHPS). The in vitro antimicrobial activities of these compounds were assessed. The E-configuration of compound 5b was proved by single crystal X-ray analysis. Compounds 5g and 5h displayed double the activity of ampicillin against B. subtilis. Also, 5h was two times more active than gentamycin against E. coli. Interestingly, compounds 5f-g, 7c, 8a, 8c exhibited two folds the potency of amphotericin B against S. racemosum while 5h displayed three folds the activity of amphotericin B against S. racemosum. Most of the synthesized compounds showed superior activities to the parent sulfisoxazole and were non-toxic to normal cells. DHPS is confirmed to be a putative target for our compounds via antagonizing their antibacterial activity by the folate precursor (p-aminobenzoic acid) and product (methionine) on E. coli ATCC 25922. Docking experiments against DHPS rationalized the observed antibacterial activity. Additionally, compound 5g was evaluated as a selective targeting vector for ^99mTc that showed a remarkable uptake and targeting ability towards the infection site that was induced in mice.     

In vitro anti-proliferative,and in silico ribonucleotide reductase and pharmacokinetics studies of heteroleptic silver(I), nickel(II) and copper(II) complexes of 4-methyl-3-thiosemicarbazones and ibuprofen

ObjectivesThe present research focuses on the in vitro anti-proliferative, and in silico ribonucleotide reductase and pharmacokinetics studies of twelve heteroleptic metal complexes of the general formulae [Ag(L¹⁻⁴)(ibu)] (1–4) and [M(L¹⁻⁴)(ibu)₂] (5–12), where L¹⁻⁴ = 2-(1-(4-substitutedphenyl)ethylidene)-N-methylhydrazinecarbothioamide, ibu = non-steroidal anti-inflammatory drug (ibuprofen), and M = Cu(II) and Ni(II).MethodsVarious spectroscopic techniques were used to authenticate the structure of the synthesized complexes. UV-Vis and cyclic voltammetry techniques were used to analyse the stability and the reducing ability of the complexes. In vitro anti-proliferative studies by MTT assay, apoptotic behaviour and cellular uptake studies were investigated followed by the in silico interaction with ribonucleotide reductase (RNR) enzyme.ResultsThe spectral studies predicted distorted tetrahedral geometry around silver(I) ion and distorted octahedral geometry around nickel(II) and copper(II) ions. The reducing ability of the copper(II) complexes was analysed using ascorbic acid by UV-Vis and cyclic voltammetry techniques, which authenticate the reducing ability of the complexes and the possible interactions within the cells. The in vitro anti-proliferative activity of the synthesized complexes against three cancerous (estrogen positive (MCF-7), estrogen negative (MDA-MB-231) and pancreatic (PANC-1)) and one normal (MCF-10a) cell lines by MTT assay showed enhanced activity for copper(II) complexes 11 and 12 containing the hydrophobic substituents. The apoptotic and cellular uptake studies showed that the complex 12 is readily taken up by PANC-1 cell lines and induces ROS-mediated mitochondrial and caspase-dependent apoptosis. The in silico studies indicated hydrogen bonding, hydrophobic and π-pair (π–π, π–σ and π–cation) interactions between the complexes and the ribonucleotide reductase (RNR) enzyme. The in silico pharmacokinetics studies of the complexes predicted the drug-likeness characteristics of the complexes.ConclusionThe synthesized complexes are found to be less toxic to normal cells and inhibit the growth of cancerous cells by inducing mitochondrial-mediated and caspase dependent apoptotic pathway in PANC-1 cells.     

Design,synthesis and anti-inflammatory evaluation of novel pyrrolo[2,3-d]pyrimidin derivatives as potent JAK inhibitors

Aiming to develop potent JAK inhibitors, two series of 4-(1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine derivatives (8a–8p and 11a–11i) were designed and synthesized by coalescing various N-acylpiperidine motifs with baricitinib. The pharmacological results based on enzymatic and cellular assays identified the optimized compound 11e, which exerted over 90% inhibition rates against JAK1 and JAK2, and displayed the most compelling anti-inflammatory efficacy superior to baricitinib by inhibiting NO generation from LPS-induced RAW264.7 macrophages. Importantly, low cytotoxity of 11e was revealed by the IC₅₀ value of 88.2 μM against normal RAW264.7 cells. The binding mode of 11e with JAK1 and JAK2 identified the essential structural bases in accord with SARs analysis. Furthermore, cellular morphology observation and western blot analysis disclosed the ability of 11e to relieve cells inflammatory damage by significantly down-regulating LPS-induced high expression of JAK1, JAK2, as well as pro cytokine IL-1β. Together, 11e was verified as a promising lead for JAK inhibitors for the treatment of inflammatory diseases.     

Synthesis and biological evaluation of 11C-labeled β-galactosyl triazoles as potential PET tracers for in vivo LacZ reporter gene imaging

In our aim to develop LacZ reporter probes with a good retention in LacZ expressing cells, we report the synthesis and preliminary evaluation of two carbon-11 labeled β-galactosyl triazoles 1-(β-d-galactopyranosyl)-4-(p-[¹¹C]methoxyphenyl)-1,2,3-triazole ([¹¹C]-6) and 1-(β-d-galactopyranosyl)-4-(6-[¹¹C]methoxynaphthyl)-1,2,3-triazole ([¹¹C]-13). The precursors for the radiolabeling and the non-radioactive analogues (6 and 13) were synthesized using straightforward ‘click’ chemistry. In vitro incubation experiments of 6 with β-galactosidase in the presence of o-nitrophenyl β-d-galactopyranoside (ONPG) showed that the triazolic compound was an inhibitor of β-galactosidase activity. Radiolabeling of both precursors was performed using [¹¹C]methyl iodide as alkylating agent at 70 °C in DMF in the presence of a small amount of base. The log P values were ?0.1 and 1.4, respectively, for [¹¹C]-6 and [¹¹C]-13, the latter therefore being a good candidate for increased cellular uptake via passive diffusion. Biodistribution studies in normal mice showed a good clearance from blood for both tracers. [¹¹C]-6 was mainly cleared via the renal pathway, while the more lipophilic [¹¹C]-13 was excreted almost exclusively via the hepatobiliary system. Despite the lipophilicity of [¹¹C]-13, no brain uptake was observed. Reversed phase HPLC analysis of murine plasma and urine revealed high in vivo stability for both tracers. In vitro evaluation in HEK-293T cells showed an increased cell uptake for the more lipophilic [¹¹C]-13, however, there was no statistically higher uptake in LacZ expressing cells compared to control cells.     

Somatostatin Analogues for Receptor Targeted Photodynamic Therapy

Photodynamic therapy (PDT) is an established treatment modality, used mainly for anticancer therapy that relies on the interaction of photosensitizer, light and oxygen. For the treatment of pathologies in certain anatomical sites, improved targeting of the photosensitizer is necessary to prevent damage to healthy tissue. We report on a novel dual approach of targeted PDT (vascular and cellular targeting) utilizing the expression of neuropeptide somatostatin receptor (sst₂) on tumor and neovascular-endothelial cells. We synthesized two conjugates containing the somatostatin analogue [Tyr3]-octreotate and Chlorin e6 (Ce6): Ce6-K₃-[Tyr3]-octreotate (1) and Ce6-[Tyr3]-octreotate-K₃-[Tyr3]-octreotate (2). Investigation of the uptake and photodynamic activity of conjugates in-vitro in human erythroleukemic K562 cells showed that conjugation of [Tyr3]-octreotate with Ce6 in conjugate 1 enhances uptake (by a factor 2) in cells over-expressing sst₂ compared to wild-type cells. Co-treatment with excess free Octreotide abrogated the phototoxicity of conjugate 1 indicative of a specific sst₂-mediated effect. In contrast conjugate 2 showed no receptor-mediated effect due to its high hydrophobicity. When compared with un-conjugated Ce6, the PDT activity of conjugate 1 was lower. However, it showed higher photostability which may compensate for its lower phototoxicity. Intra-vital fluorescence pharmacokinetic studies of conjugate 1 in rat skin-fold observation chambers transplanted with sst₂ ⁺ AR42J acinar pancreas tumors showed significantly different uptake profiles compared to free Ce6. Co-treatment with free Octreotide significantly reduced conjugate uptake in tumor tissue (by a factor 4) as well as in the chamber neo-vasculature. These results show that conjugate 1 might have potential as an in-vivo sst₂ targeting photosensitizer conjugate.     

Synthesis of a terphenyl substituted 4-aza-2,3-didehydropodophyllotoxin analogues as inhibitors of tubulin polymerization and apoptosis inducers

A series of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates (8a–r) were synthesized by a straightforward one-step multicomponent synthesis that demonstrated anticancer activity against five human cancer cell lines (lung, colon, renal, prostate and cervical). All the tested compounds showed potent anticancer activity with IC₅₀ values ranging from 0.87 to 16.59 μM. Among them compounds 8n and 8p showed significant anticancer activity in lung cancer cells with IC₅₀ values 0.91 and 0.87 μM, respectively. Flow cytometric analysis revealed that these compounds induced cell cycle arrest in G₂/M phase in A549 cell line leading to caspase-3 dependent apoptotic cell death. The tubulin polymerization assay and immunofluorescence analysis showed that these compounds effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further, Hoechst staining, DNA fragmentation analysis also suggested that these compounds induced cell death by apoptosis. Overall, the current study demonstrated that the synthesis of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates as promising anticancer agents with G₂/M cell cycle arrest and apoptotic-inducing activities via targeting tubulin.     

Synthesis and exploration of novel radiolabeled bombesin peptides for targeting receptor positive tumor

Increasing evidence of peptide receptor overexpression in various cancer cells, warrant the development of receptor specific radiolabeled peptides for molecular imaging and therapy in nuclear medicine. Gastrin-releasing-peptide (GRP) receptor, are overexpressed in a variety of human cancer cells. The present study report the synthesis and biological evaluation of new bombesin (BBN) analogs, HYNIC-Asp-[Phe¹³]BBN(7–13)-NH-CH₂-CH₂-CH3:BA1, HYNIC-Pro-[Tyr¹³Met¹⁴]BBN(7–14)NH₂:BA2 as prospective tumor imaging agent with compare to BBN(7–14)NH₂:BS as standard. The pharmacophores were radiolabeled in high yields with ^99mTc, characterized for their stability in serum and saline, cysteine/histidine and were found to be substantially stable. Internalization/externalization and receptor binding studies were assessed using MDA-MB-231 cells and showed high receptor binding-affinity and favourable internalization. Fluorescence studies revealed that BA1 changed the morphology of the cells and could localize in the nucleus more effectively than BA2/BS. Cell-viability studies displayed substantial antagonistic and nuclear-internalization effect of BA1. BA1 also exhibited antiproliferative effect on MDA-MB-231 cell by inducing apoptosis. In vivo behaviour of the radiopeptides was evaluated in GRP receptor positive tumor bearing mice. The ^99mTc-BA1/^99mTc-BA2 demonstrated rapid blood/urinary clearance through the renal pathway and comparatively more significant tumor uptake image and favourable tumor-to-non-target ratios provided by ^99mTc-BA1. The specificity of the in vivo uptake was confirmed by co-injection with BS. Moreover, ^99mTc-BA1 provided a much clearer tumor image in scintigraphic studies than others. Thus the combination of favourable in vitro and in vivo properties renders BA1 as more potential antagonist bombesin-peptide for targeting GRP-receptor positive tumor. These properties are encouraging to carry out further experiments for non-invasive receptor targeting potential diagnostinc and therapeutic agent for tumors.     

Synthesis of stable benzimidazole derivatives bearing pyrazole as anticancer and EGFR receptor inhibitors

A new series of benzimidazole linked pyrazole derivatives were synthesized by cyclocondensation reaction through one-pot multicomponent reaction in absolute ethanol. All the synthesized compounds were tested for their in vitro anticancer activities on five human cancer cell lines including MCF-7, HaCaT, MDA-MB231, A549 and HepG2. EGFR receptor inhibitory activities were carried out for all the compounds. Majority of the compounds showed potent antiproliferative activity against the tested cancer cell lines. Compound 5a showed the most effective activity against the lungs cancer cell lines (IC₅₀ = 2.2 µM) and EGFR binding (IC₅₀ = 0.97 µM) affinity as compared to other members of the series. Compound 5a inhibited growth of A549 cancer cells by inducing a strong G₂/M phase arrest. In addition, same compound inhibited growth of A549 cancer cells by inducing apoptosis. In molecular docking studies compound 5a was bound to the active pocket of the EGFR (PDB 1M17) with five key hydrogen bonds and two π-π interaction with binding energies ΔG = −34.581 Kcal/mol.