A double prodrug system for colon targeting of benzenesulfonamide COX-2 inhibitors

The design, synthesis and delivery potential of a new type of benzenesulfonamide cyclo-oxygenase-2 (COX-2) inhibitor prodrug is investigated using celecoxib. The approach involves a double prodrug that is activated first by azoreductases and then by cyclization triggering drug release. We studied the intramolecular aminolysis of the acylsulfonamide. The cyclization was surprisingly rapid at physiological pH and very fast at pH 5. The prodrug is activated specifically under conditions found in the colon but highly stable in the presence of human and rodent intestinal extracts. Finally, the prototype with celecoxib was transported much more slowly in the Caco-2 transepithelial model than the parent. The design therefore shows significant promise for the site specific delivery of benzenesulfonamide COX-2 inhibitors to the colon.     

The effect of substitution patterns on the release rates of opioid peptides DADLE and [Leu(5)]-enkephalin from coumarin prodrug moieties

A coumarin-based prodrug system has been developed in our laboratory for the preparation of esterase-sensitive prodrugs of amines, peptides, and peptidomimetics. The drug release rates from this prodrug system were found to be dependent on the structural features of the drug moiety. The effect of the phenyl ring substitutions on the release kinetics of such prodrugs of model amines was examined recently and it was found that appropriately positioned alkyl substituents on the phenyl ring could help to facilitate the release. Aimed at further understanding the structure-release rate relationship of the coumarin-based cyclic prodrugs, we synthesized and examined a series of substituted coumarinic acid derivatives of opioid peptides, DADLE, and [Leu(5)]-enkephalin.     

Modulating paclitaxel bioavailability for targeting prostate cancer

Four novel water-soluble peptide-paclitaxel conjugates were designed and synthesized as prostate-specific antigen (PSA)-activated prodrugs for prostate cancer therapy. These prodrugs were composed of a peptide, HSSKLQ or SSKYQ, each of which is selectively cleavable by PSA; a self-immolative linker, either para-aminobenzyl alcohol (PABS) or ethylene diamine (EDA); and the parent drug, paclitaxel. Introduction of a PABA or EDA linker between the peptide and paclitaxel in prodrugs 2-5 resulted in products with an increased rate of hydrolysis by PSA. The stability of prodrugs 2 and 3, with the PABA linker, was poor in the serum-containing medium because of the weak carbonate bond between the PABA and paclitaxel; however, this disadvantage was overcome by introducing a carbamate bond using an EDA linker in prodrugs 4 and 5. Thus, the incorporation of an EDA linker increased both the stability and PSA-mediated activation of these prodrugs. The cytotoxicity of each prodrug, as compared to paclitaxel, was determined against a variety of cell lines, including the PSA-secreting CWR22Rv1 prostate cancer cell line. The EDA-derived prodrug of paclitaxel 5 was stable and capable of being efficiently converted to an active drug that killed cells specifically in the presence of PSA, suggesting that this prodrug and similarly designed PSA-cleavable prodrugs may have potential as prostate cancer-specific therapeutic agents.     

聚苹果酸- 羟喜树碱前药的合成和性质初探

目的:化学全合成聚苹果酸(poly(β-malic acid),PMLA),将其作为高分子药物载体,制备聚苹果酸-羟喜树碱前药(PMLA-HCPT)。研究其体外释药特点和体外细胞毒性。方法:以L-天冬氨酸为原料,通过化学方法全合成PMLA,通过酰胺键键合羟基喜树碱(HCPT)。通过红外光谱、核磁共振光谱表征该前药的结构,利用体外动态透析的方法模拟体外释药特点,用高效液相色谱法测定不同pH值聚合物药物中前喜树碱的释药特性。采用人卵巢癌HO-8910细胞系研究该前药的体外毒性。结果:①经核磁共振表征PMLA-HCPT前药合成完成。②在pH 5.6、pH 6.8及pH 7.4的PBS缓冲体系16 h中,羟喜树碱药物累积释放率分别为76.8%,47.2%和18.1%,证实PMLA-HCPT中羟喜树碱的释放具有pH依赖性。③细胞实验证实PMLA-HCPT的细胞毒性和游离的HCPT相比没有降低。结论:PMLA是一种良好的药物载体材料,PMLA-HCPT有望成为具有pH敏感性的聚合物前药。     

Chemotherapeutic bone-targeted bisphosphonate prodrugs with hydrolytic mode of activation

Osseous tissues are considered to be limited as therapeutic target sites due to their biological properties. We have designed and synthesized two kinds of hydrolytically activated chemotherapeutic prodrugs containing bisphosphonate, a bone-targeting moiety. The first can be conjugated to drug molecules with an available hydroxy group; the drug is attached to the bisphosphonate component through an ester-labile linkage. The second is for use with drug molecules with amine functional group. In this case, a self-immolative linker is used to attach the drug to the bisphosphonate component through a carbonate-labile linkage. The concept was demonstrated using the drugs camptothecin, which has a hydroxy functional group, and tryptophan, which is a model molecule for a drug with amine functionality. Both prodrugs showed significant binding capability to hydroxyapatite, the major component of bone, and were hydrolytically activated under physiological conditions.     

Conformational analysis by NMR and molecular dynamics of adamantane-doxorubicin prodrugs and their assemblies with β-cyclodextrin: A focus on the design of platforms for controlled drug delivery

Nanoscale design and construction of affinity-based drug delivery systems (ADDS) is an active research area with enormous potential for the improvement of cancer treatment. For the therapeutic load of these ADDS, a promising strategy is the design of pH-sensitive prodrugs based on the construction of conjugates between adamantane and doxorubicin (Ad-Dox), which stands out as an excellent model system to obtain novel supramolecular materials. Construction of these prodrugs involves a modification of three zones of doxorubicin which in principle does not affect the action mechanism: the carbonyl group C13 (hydrazone linker), the primary alcohol neighboring the carbonyl (ester linker) and the 3′ amino group of daunosamine sugar (amide linker). These modifications are aimed to improve the efficacy and reduce the systemic toxicity of the drug chemotherapy by controlling its release in cancer cells. In this work, we performed 2D NMR experiments and molecular dynamics simulations to characterize the conformational changes of three constructed prodrugs. Our results demonstrated that ring A and the daunsamine sugar of the hydrazone and amide linkers conserve the half-chair state ⁹H₈, while the ester linker disrupts this conformation. Our study also showed that the hydrazone-linked compound (Ad-h-Dox) does not modify the conformation of the original drug and maintains cytotoxic activity. Moreover, the inclusion complex (IC) of Ad-h-Dox with β-cyclodextrin (βCD) generated a highly soluble platform in water, whereas the ester-linked compound (Ad-e-Dox) causes the loss of biological activity. This study proves that Ad-h-Dox prodrug can be an optimum prodrug and act as a building block for a more complex drug transport system.     

A safety catch linker for Fmoc-based assembly of constrained cyclic peptides.

A new safety-catch linker for Fmoc solid-phase peptide synthesis of cyclic peptides is reported. The linear precursors were assembled on a tert-butyl protected catechol derivative using optimized conditions for Fmoc-removal. After activation of the linker using TFA, neutralization of the N-terminal amine induced cyclization with concomitant cleavage from the resin yielding the cyclic peptides in DMF solution. Several constrained cyclic peptides were synthesized in excellent yields and purities.     

Novel nonviral vectors for gene delivery: Synthesis and applications

Summary We have synthesized novel cationic lipids for gene delivery bearing an ester bond between the lipid moiety and the polyamine head. We have found that an intramolecular rearrangement occurs during purification of one of the products. The rearrangement led to a cyclic lipopolyamine which was active for DNA gene transfer. The formation of cyclization products depends on the spacer found between the lipid and the polyamine. The introduction of arginine in the linker position prevents the formation of cyclic products. Linear as well as cyclic analogues showed high-efficiency gene transfer when tested in vitro for luciferase gene expression as compared to dioctadecylamidoglycyl spermine or lipofectamine and also in vivo in the Lewis lung carcinoma model. The introduction of arginine in the linker position promoted increased transfection activity, demonstrating that a diversity of linkers, such as short peptides or glycosides, can be introduced into cationic lipids for targeted gene transfer.     

Nucleic acid triggered catalytic drug and probe release: a new concept for the design of chemotherapeutic and diagnostic agents.

Recently, we described a new concept for the design of highly selective antiviral and anticancer chemotherapeutic agents that makes use of a disease-specific nucleic acid sequence to template the association of a prodrug with a catalyst which catalyzes the release of the drug. Herein, we report on the effect of mismatches, sterics, and electronics on the rate and specificity of drug and probe release in both two and three component model systems, and on the stability of the prodrug linker in human serum.     

Docetaxel prodrug self-assembled nanosystem: Synthesis,formulation and cytotoxicity

Conventional drug delivery systems of docetaxel (DTX) are challenged with low drug loading efficiency and potential carriers-induced toxicity. In this work, a docetaxel prodrug self-assembled nanosystem was designed and synthesized by conjugating docetaxel with oleic acid (OA) exploring a thioether as the linker, which is redox-sensitive to the redox environment within tumor cells. Notably, the carrier-free nanomedicine which does not need any carrier has obviously high drug loading that reaches 58%. Moreover, the cytotoxicity of DTX-S-OA maintains an equal level with DTX. The novel prodrug conjugate therefore has a promising perspective as carrier-free nanomedicine for cancer therapy due to its high drug loading property, redox-sensitive release and long circulation mechanism.     

Synthesis and stability study of a modified phenylpropionic acid linker-based esterase-sensitive prodrug

An esterase-sensitive amide prodrug 1 with a modified phenylpropionic acid linker was synthesized. The prodrug can be converted to the drug using isolated porcine esterase and human plasma. Paraoxon, an esterase inhibitor, can inhibit prodrug-to-drug conversion. The conversion of prodrug 1 was via phenol intermediate 9 followed by a lactonization reaction to give lactone 2 and the drug.     

Synthesis, photophysical, electrochemical, tumor-imaging, and phototherapeutic properties of purpurinimide-N-substituted cyanine dyes joined with variable lengths of linkers

Purpurinimide methyl esters, bearing variable lengths of N-substitutions, were conjugated individually to a cyanine dye with a carboxylic acid functionality. The results obtained from in vitro and in vivo studies showed a significant impact of the linkers joining the phototherapeutic and fluorescence imaging moieties. The photosensitizer-fluorophore conjugate with a PEG linker showed the highest uptake in the liver, whereas the conjugate linked with two carbon units showed excellent tumor-imaging and PDT efficacy at 24 h postinjection. Whole body imaging and biodistribution studies at variable time points portrayed enhanced fluorescent uptake of the conjugates in the tumor compared to that in the skin. Interestingly, the conjugate with the shortest linker and the one joining with two carbon units showed faster clearance from normal organs, e.g., the liver, kidney, spleen, and lung, compared to that in tumors. Both imaging and PDT efficacy of the conjugates were performed in BALB/c mice bearing Colon26 tumors. Compared to the others, the short linker conjugate showed poor tumor fluorescent properties and as a corollary does not exhibit the dual functionality of the photosensitizer-fluorophore conjugate. For this reason, it was not evaluated for in vivo PDT efficacy. However, in Colon26 tumor cells (in vitro), the short linker was highly effective. Among the conjugates with variable linkers, the rate of energy transfer from the purpurinimide moiety to the cyanine moiety increased with deceasing linker length, as examined by femtosecond laser flash photolysis measurements. No electron transfer from the purpurinimide moiety to the singlet excited state of the cyanine moiety or from the singlet excited state of the cyanine moiety to the purpurinimide moiety occurred as indicated by a comparison of transient absorption spectra with spectra of the one-electron oxidized and one-electron reduced species of the conjugate obtained by spectroelectrochemical measurements.     

Design and synthesis of a folate-receptor targeted diazepine-ring-opened pyrrolobenzodiazepine prodrug conjugate

Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds and are currently under development as novel payloads in antibody-drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed and synthesized a diazepine-ring-opened bis-PBD prodrug (pro-PBD-PBD) folate conjugate lacking the one of the two imine moieties found in the corresponding free bis-PBD. Upon entering a targeted cell, cleavage of the linker system, including the hydrolysis of an oxazolidine moiety, results in the formation of a reactive intermediate which possesses a newly formed aldehyde as well as an aromatic amine. A fast and spontaneous intramolecular ring-closing reaction subsequently takes place as the aromatic amine adds to the aldehyde with the loss of water to give the imine, and as a result, the diazepine ring, thereby delivering the bis-PBD to the targeted cell. The in vitro and in vivo activity of this conjugate has been evaluated on folate receptor positive KB cells. Sub-nanomolar activity with good specificity and high cure rates with minimal toxicity have been observed.     

Biocatalytic route to sugar-PEG-based polymers for drug delivery applications

Sugar-PEG-based polymers were synthesized by enzymatic copolymerization of 4-C-hydroxymethyl-1,2-O-isopropylidene-β-L-threo-pentofuranose/4-C-hydroxymethyl-1,2-O-benzylidene-β-L-threo-pentofuranose/4-C-hydroxymethyl-1,2-O-isopropylidene-3-O-pentyl-β-L-threo-pentofuranose with PEG-600 dimethyl ester using Novozyme-435 (Candida antarctica lipase immobilized on polyacrylate). Carbohydrate monomers were obtained by the multistep synthesis starting from diacetone-D-glucose and PEG-600 dimethyl ester, which was in turn obtained by the esterification of the commercially available PEG-600 diacid. Aggregation studies on the copolymers revealed that in aqueous solution those polymers bearing the hydrophobic pentyl/benzylidene moiety spontaneously self-assembled into supramolecular aggregates. The critical aggregation concentration (CAC) of polymers was determined by surface tension measurements, and the precise size of the aggregates was obtained by dynamic light scattering. The polymeric aggregates were further explored for their drug encapsulation properties in buffered aqueous solution of pH 7.4 (37 °C) using nile red as a hydrophobic model compound by means of UV/vis and fluorescence spectroscopy. There was no significant encapsulation in polymer synthesized from 4-C-hydroxymethyl-1,2-O-isopropylidene-β-L-threo-pentofuranose because this sugar monomer does not contain a big hydrophobic moiety as the pentyl or the benzylidene moiety. Nile red release study was performed at pH 5.0 and 7.4 using fluorescence spectroscopy. The release of nile red from the polymer bearing benzylidene moiety and pentyl moiety was observed with a half life of 3.4 and 2.0 h, respectively at pH 5.0, whereas no release was found at pH 7.4.     

N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide as a potential bioreductively activated prodrug of phosphoramide mustard

N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide isomers (DMNA-NH-CPA, 4) were synthesized stereospecifically from Boc-l-Hse(OBn)-OH and the degradation of the corresponding reduced amine 5a was investigated by UV/vis spectroscopy and LC/MS. The rate of cyclization of 5a was found to increase with decreasing pH, with half-lives ranging from 3.2 to 54 min at pH 4–7.4, suggesting that the cyclization is catalyzed by the hydronium ions. LC/MS analysis of the degradation products of 5a indicates that 4-aminocyclophosphamide is rapidly released from 4 upon reductive activation under acidic conditions and further decomposes into the cytotoxic phosphoramide mustard. These results validated 4-aminocyclophosphamide as a prodrug form of phosphoramide mustard and suggest that compound 4 can potentially be used as a prodrug of phosphoramide mustard for bioreductive activation.     

Computer-assisted design for paracetamol masking bitter taste prodrugs

It is believed that the bitter taste of paracetamol, a pain killer drug, is due to its hydroxyl group. Hence, it is expected that blocking the hydroxy group with a suitable linker could inhibit the interaction of paracetamol with its bitter taste receptor/s and hence masking its bitterness. Using DFT theoretical calculations we calculated proton transfers in ten different Kirby’s enzyme models, 1–10. The calculation results revealed that the reaction rate is linearly correlated with the distance between the two reactive centers (r_GM) and the angle of the hydrogen bonding (α) formed along the reaction pathway. Based on these results three novel tasteless paracetamol prodrugs were designed and the thermodynamic and kinetic parameters for their proton transfers were calculated. Based on the experimental t_1/2 (the time needed for the conversion of 50% of the reactants to products) and EM (effective molarity) values for processes 1–10 we have calculated the t_1/2 values for the conversion of the three prodrugs to the parental drug, paracetamol. The calculated t_1/2 values for ProD 1–3 were found to be 21.3 hours, 4.7 hours and 8 minutes, respectively. Thus, the rate by which the paracetamol prodrug undergoes cleavage to release paracetamol can be determined according to the nature of the linker of the prodrug (Kirby’s enzyme model 1–10). Further, blocking the phenolic hydroxyl group by a linker moiety is believed to hinder the paracetamol bitterness.     

A doxorubicin prodrug activated by the staudinger reaction

Much effort has been devoted to prodrug systems that effect drug release at the tumor through enzymatic action. To widen the scope of prodrug therapy, the use of the selective Staudinger reaction as prodrug activator, instead of relying on enzymatic action, was investigated. Doxorubicin was conjugated to a p-azidobenzyl trigger that is cleaved after reacting with the chemical activator, triphenylphosphine. The prodrug activation was confirmed in water, cell growth medium, and serum, using HPLC and LCMS. Next, this approach was tested in a cell proliferation assay with A431 human vulvar skin squamous carcinoma cells. The doxorubicin prodrug was shown to exhibit a 176-fold higher IC50 of 15.1 microM vs 0.086 microM for the parent drug, doxorubicin. Addition of triphenylphosphine (5 x 60 microM in 72 h) to the prodrug in cell culture effected the complete recovery of the activity of the parent drug as evidenced by an IC50 value of 0.074 microM. Furthermore, high levels of triphenylphosphine were tolerated well by the cells. The demonstrated usefulness of the Staudinger reaction in cell culture and its in vivo potential opens up new avenues for prodrug therapy.     

5'-(2-Nitrophenylalkanoyl)-2'-deoxy-5-fluorouridines as potential prodrugs of FUDR for reductive activation

Four 5'-(2-nitrophenylalkanoyl)-2'-deoxy-5-fluorouridines (1a-d) were designed and synthesized as potential prodrugs of FUDR for reductive activation. Two methyl groups were introduced alpha to the ester carbonyl to increase both the rate of cyclization activation and the stability of the conjugates towards serum esterases. Chemical reduction of the nitro group into an amino leads to cyclization and release of the active FUDR. Kinetic analysis of the cyclization activation process indicates that the two methyl groups alpha to the ester carbonyl restrict the rotational freedom of ground state molecule and promote the cyclization reaction. However, the two methyl groups also were found to render the conjugates as poor substrates of E. coli B nitroreductase. Conjugate 1c, without the two methyl groups, was reduced by E. coli B nitroreductase (t(1/2)=8 h) to give two products, a N-hydroxyl lactam and the drug FUDR, suggesting that the enzymatic reduction and subsequent cyclization activation proceeded through the hydroxylamine intermediate. These results indicate that cyclization activation will occur once the nitro group is reduced either to an amino or to a hydroxylamino group. The fact that the amino intermediates cyclized easily to release the incorporated drug FUDR suggests the feasibility of using peptide-linked acyl 2-aminophenylalkanoic acid esters as potential prodrugs for proteolytic activation.