Aqueous Physiological Conditions Research Articles

Development of Novel Immobilized Copper-Ligand Complex for Click Chemistry of Biomolecules.

Copper-catalyzed azide-alkyne cycloaddition click (CuAAC) reaction is widely used to synthesize drug candidates and other biomolecule classes. Homogeneous catalysts, which consist of copper coordinated to a ligand framework, have been optimized for high yield and specificity of the CuAAC reaction, but CuAAC reaction with these catalysts requires the addition of a reducing agent and basic conditions, which can complicate some of the desired syntheses. Additionally, removing copper from the synthesized CuAAC-containing biomolecule is necessary for biological applications but inconvenient and requires additional purification steps. We describe here the design and synthesis of a PNN-type pincer ligand complex with copper (I) that stabilizes the copper (I) and, therefore, can act as a CuAAC catalyst without a reducing agent and base under physiologically relevant conditions. This complex was immobilized on two types of resin, and one of the immobilized catalyst forms worked well under aqueous physiological conditions. Minimal copper leaching was observed from the immobilized catalyst, which allowed its use in multiple reaction cycles without the addition of any reducing agent or base and without recharging with copper ion. The mechanism of the catalytic cycle was rationalized by density functional theory (DFT). This catalyst's utility was demonstrated by synthesizing coumarin derivatives of small molecules such as ferrocene and sugar.

Revisiting the Chemistry and Photophysics of 3‐(N‐Methylpyridinium‐4‐yl)Coumarins for Designing “Covalent‐Assembly” and “Molecular Disassembly” Fluorescent Probes**

AbstractThe constant need for high‐performance aniline‐ or phenol‐based fluorophores suitable for the construction of activity‐based fluorescent probes, led us to study both synthesis and photophysics of C3‐N‐methylpyridinium‐4‐yl substituted 7‐(dialkylamino)/7‐hydroxycoumarins. Indeed, in the field of photoactive organic molecules, the positively charged N‐alkylpyridinium‐4‐yl groups are often used as acceptor units to dramatically impact spectral features through promoting intramolecular charge transfer (ICT) processes. They are also known as effective water‐solubilizing and mitochondria targeting moieties. The poor fluorescence efficiency of cationic 7‐hydroxycoumarin derivatives in aqueous physiological conditions was highlighted and rationalized by the predominance of a neutral quinonoid form in such buffer medium. The ability of the excited singlet state (S1) of this neutral species to undergo intersystem crossing (ISC) to triplet state (T1) was partly supported by phosphorescence measurements of singlet oxygen. We also took advantage of green‐emissive properties of 7‐(diethylamino)‐3‐(N‐methylpyridinium‐4‐yl)coumarin to successfully design and validate a novel small‐molecule fluorescent probe for the detection of alkaline phosphatase (ALP), based on the “covalent‐assembly” principle. A practical use of ortho‐formylated 7‐hydroxy‐3‐(N‐methylpyridinium‐4‐yl)coumarin was next considered with the synthesis of a Fe(III)‐salen complex whose the potential as a “molecular disassembly” probe for fluorogenic sensing of pyrophosphate (PPi) anion was assessed.

Emerging risk from “environmentally-friendly” solvents: Interaction of methylimidazolium ionic liquids with the mitochondrial electron transport chain is a key initiation event in their mammalian toxicity

Recent studies have identified the 8C alkyl chain methylimidazolium ionic liquid 1-octyl-3-methylimidazolium in the environment and its potential to trigger the auto-immune liver disease primary biliary cholangitis. The toxicity of a range of methylimidazolium ionic liquids were therefore examined. Oxygen consumption was rapidly inhibited, with potency increasing with alkyl chain length. This preceded caspase 3/7 induction and DNA fragmentation. Time- and dose-dependent loss of dye reduction capacities reflected these effects, with a >700 fold difference in potency between 2C and 10C alkyl chain liquids. None of the ionic liquids directly inhibited mitochondrial complexes I-IV or complex V (F0F1-ATPase). However, dithionite reduction and ESR spectroscopy studies indicate a one electron reduction of oxygen in the presence of a methylimidazolium ionic liquid, suggesting methylimidazolium ionic liquids function as mitochondrial electron acceptors. However, only longer chain ionic liquids form a non-aqueous phase or micelle under aqueous physiological conditions and lead to increases in reactive oxygen species in intact cells. These data therefore suggest that the longer chain methylimidazolium liquids are toxic in sensitive liver progenitor cells because they both readily integrate within the inner mitochondrial membrane and accept electrons from the electron chain, leading to oxidative stress.

Investigating the Structure‐Toxicity Relationship of Methylimidazolium Ionic Liquids in Isolated Primary Human Hepatocytes

Ionic liquids are man‐made non‐volatile chemicals proposed to be “environmentally‐friendly” alternatives to current volatile solvents. However, they persist in the environment. In this respect, an 8 carbon alkyl chain derivative methylimidazolium ionic liquid (M8OI) has recently been found to be present at high levels in soils near a landfill waste site in the UK in an area characterized by a high incidence of the autoimmune liver disease “primary biliary cholangitis” (PBC). Recent work by this laboratory also demonstrated that M8OI was toxic to the liver progenitor (B‐13) cell line (Probert et al., J. Hepatol 69:1123–1135). To gain insight into its mechanism(s) of toxicity, the effects of several structurally‐related methylimidazolium liquids (differing in their alkyl chain length from 2C to 10C) have been examined in primary human hepatocytes. The ionic liquids inhibited human hepatocytes oxygen consumption within minutes of exposure, with potency increased with increasing alkyl chain length, an effect associated primarily with inhibition of mitochondrial respiration and ATP production. These effects were accompanied by an increase in extracellular acidification rate indicative of compensatory enhanced glycolysis, confirmed by dose‐dependent increases in glucose consumption, lactate production and AMPK phosphorylation. These metabolic changes preceded induction of caspase 3/7 activities and genomic DNA fragmentation, indicative of an initial apoptotic mode of cell death. Time‐ and dose‐dependent loss of MTT reduction capacities reflected these effects. Similar mechanistic changes were seen with B‐13 cells although sensitivities were increased (with regards to MTT reduction capacity). Despite their effects on mitochondrial oxygen consumption, none of the ionic solvents directly inhibited the function of the electron transport chain complexes at concentrations relevant to toxicity. However, chemical reduction and electron paramagnetic resonance (EPR) spectroscopy studies suggest that all the ionic solvents function as electron acceptors under biologically‐relevant reducing environments, suggesting that the methylimidazolium ionic liquids have the potential capacity to accept electrons from the mitochondrial electron transport chain. However, nile red fluorescence measurements indicated that only longer chain ionic solvents form a non‐aqueous phase or micelle under aqueous physiological conditions. These data, therefore, suggest that the longer chain methylimidazolium solvents are most toxic because they both readily integrate within the inner mitochondrial membrane and accept electrons from the electron chain. This reduction leads to an inhibition of oxidative phosphorylation and a compensatory increase in glycolysis before the induction of apoptosis.Support or Funding InformationThis work was funded by the Newton‐Mosharafa Fund (in the form of a studentship supporting T.M.A) and by a research grant from the LIVErNORTH charity (to A.C.L and M.C.W.).

Stability of antibacterial Te(IV) compounds: A combined experimental and computational study

Inorganic Te(IV) compounds are important cysteine protease inhibitors and antimicrobial agents; AS-101 [ammonium trichloro (dioxoethylene-O,O′)tellurate] is the first compound of a family with formula NH4[C2H4Cl3O2Te], where a Te(IV) centre is bound to a chelate ethylene glycol, and showed several protective therapeutic applications. This compound is lacking in stability performance and is subjected to hydrolysis reaction with displacement of the diol ligand. In this paper, we report the stability trend of a series of analogues complexes of AS-101 with generic formula NH4[(RC2H3O2)Cl3Te], where R is an alkyl group with different chain length and different electronic properties, in order to find a correlation between structure and stability in aqueous-physiological conditions. The stability was studied in solution via multinuclear NMR spectroscopy (1H, 13C, 125Te) and computationally at the Density Functional Theory level with an explicit micro solvation model. The combined experimental and theoretical work highlights the essential role of the solvating environment and provides mechanistic insights into the complex decomposition reaction. Antimicrobial activity of the compounds was assessed against different bacterial strains.

Synthesis, stability and spectral behavior of fluorogenic sulfone-pyronin and sulfone-rosamine dyes

The first synthesis of sulfone-pyronin and sulfone-rosamine dyes bearing optically tunable primary amino groups (acting as fluorogenic centers) is presented. Sulfone analogs of xanthene-based fluorophores have recently been highlighted as a new class of near-infrared (NIR) fluorescent dyes (Liu et al. ACS Appl Mater Interfaces 2016; 8(35):22953-62), and the availability of fluorogenic derivatives is essential for the rapid construction of “turn-on” reactive probes for chemoselective bioimaging. However, these fluorescent anilines have been found to be unstable in aqueous physiological conditions due to the marked electrophilicity of their meso-position and hence its propensity to undergo nucleophilic attack by a water molecule or a hydroxyde anion. Further investigations have helped us to determine the acidic pH range at which the sulfone-rosamine 2 is fully-stable and a complete assessment of the photophysical properties could be performed. Its pro-fluorescent character and utility were then proved by the preparation and in vitro enzymatic activation of a penicillin G acylase (PGA) sensitive fluorogenic probe.

UCST‐type phase transition driven by protein‐derived polypeptide employing gelatin and chitosan

Here, we describe the thermosensitive reversible phase transition behaviors of polyelectrolyte complex composed of gelatin and chitosan (G/C complex). An aqueous dispersion of the G/C complexes showed a clear upper critical solution temperature (UCST) at around 30°C. The thermosensitive phase transition behavior showed excellent reversibility and large thermal hysteresis as a usual phenomenon based on the intra‐ and inter‐molecular interaction change. A high correlation was observed between the UCST of the G/C complex and the helix‐melting temperature of gelatin by circular dichroism, which suggested that the phase transition of the G/C complex corresponded to the secondary structure (helix‐coil) transition of gelatin. Notably, the UCST of the G/C complex shifted to lower temperatures in the presence of urea, which is well known to destabilize gelatin, whereas the addition of salt led to the dissolution of the G/C complex. It is envisaged that the results of this study will have a significant impact on the fabrication of UCST‐type thermosensitive materials, which can be utilized under aqueous physiological conditions using well‐known biopolymers. This protein‐derived functional material, which responds to the secondary structure transition, could also be used for the development of novel UCST‐type thermosensitive biomaterials. Copyright © 2017 John Wiley & Sons, Ltd.

Dinuclear Zinc(II) Macrocyclic Complex as Receptor for Selective Fluorescence Sensing of Pyrophosphate.

A new diethylenetriamine-derived macrocycle known as L, bearing 2-methylquinoline arms and containing m-xylyl spacers, was prepared in good yield by a one-pot [2 + 2] Schiff base condensation procedure, followed by reduction with sodium borohydride. Up to now this is the first hexaazamacrocycle with appended fluorophore units. Single-crystal X-ray diffraction determination of the dinuclear zinc(II) complex of L showed that metal centers are located at about 7.20(2) Å from one another. This complex exhibits only weak fluorescence in aqueous solution, but the addition of 1 equiv of pyrophosphate (PPi) caused a 21-fold enhancement of the fluorescence intensity. The sensor response is linear up to a value of 10 μM HPPi(3-) and has a detection limit of 300 nM. The receptor behaves as a highly selective sensor for pyrophosphate as other anions, including phosphate, phenylphosphate (PhP), adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP), failed to induce any fluorescence change and practically do not affect the fluorescence intensity of the sensor in the presence of HPPi(3-). Competition titrations carried out in aqueous solution at pH 7.4 [in 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer] by spectrofluorometry revealed a high association constant value of 6.22 log units for binding of PPi by the dinuclear zinc(II) receptor, one of the highest reported values for colorimetric/fluorometric sensors able to work under real aqueous physiological conditions, while association constant values for binding of the other phosphorylated substrates are in the 5.51-4.03 log unit range.

Development of antibacterial and high light transmittance bulk materials: Incorporation and sustained release of hydrophobic or hydrophilic antibiotics

Infection associated with medical devices is one of the most frequent complications of modern medical biomaterials. Bacteria have a strong ability to attach on solid surfaces, forming colonies and subsequently biofilms. In this work, a novel antibacterial bulk material was prepared through combining poly(dimethyl siloxane) (PDMS) with either hydrophobic or hydrophilic antibiotics (0.1–0.2wt%). Scanning electron microscopy, water contact angle and UV–vis spectrophotometer were used to measure the changes of surface topography, wettability and optical transmission. For both gentamicin sulfate (GS) and triclosan (TCA), the optical transmission of the PDMS-GS and PDMS-TCA blend films was higher than 90%. Drug release studies showed initial rapid release and later sustained release of GS or TCA under aqueous physiological conditions. The blend films demonstrated excellent bactericidal and sufficient biofilm inhibition functions against Gram-positive bacteria (Staphylococcus aureus, S. aureus) measured by LIVE/DEAD bacterial viability kit staining method. Kirby-Bauer method showed that there was obvious zone of inhibition (7.5–12.5mm). Cytocompatibility assessment against human lens epithelial cells (HLECs) revealed that the PDMS-GS blend films had good cytocompatibility. However, the PDMS-TCA blend films showed certain cytotoxicity against HLECs. The PDMS-0.2wt% GS blend films were compared to native PDMS in the rabbit subcutaneous S. aureus infection model. The blend films yielded a significantly lower degree of infection than native PDMS at day 7. The achievement of the PDMS-drug bulk materials with high light transmittance, excellent bactericidal function and good cytocompatibility can potentially be widely used as bio-optical materials.

In vitro and in vivo evaluation of xanthan gum-succinic anhydride hydrogels for the ionic strength-sensitive release of antibacterial agents.

In this work, we report a new approach to prepare high gel performance hydrogels which are used as ionic strength-sensitive drug release systems. Succinic anhydride (SA)-modified xanthan (XG-SA) derivatives were prepared and confirmed by Fourier transform-infrared spectroscopy and proton nuclear magnetic resonance spectroscopy. Rheological measurements showed that the storage moduli (G') and loss moduli (G'') of XG-SA were much higher than native XG suggesting a higher stability of the hydrogels. XG-SA could form stable hydrogels when the content of a dry gel was 1.4 wt%. Drug release studies showed the ionic strength-sensitive and sustained release of gentamicin (GS) for 9 days under aqueous physiological conditions. Biofilm inhibition assay revealed that the XG-SA/GS hydrogels were sufficient to inhibit biofilm formation. The Kirby-Bauer method showed that there was a zone of inhibition at around 8.2 mm indicating the excellent bactericidal function of the hydrogels. Cytocompatibility assessment against human lens epithelial cells revealed that the hydrogels supported cell adhesion, proliferation and migration when the loading dosage of GS was 1 mg g-1. XG-SA/GS hydrogels were compared to native XG-SA in the rabbit subcutaneous S. aureus infection model. XG-SA/GS hydrogels yielded a significantly lower degree of infection than XG-SA hydrogels at day 7. In this way, XG-SA hydrogels are promising drug delivery materials for antibacterial applications.

Human Serum Albumin Increases the Stability of Green Tea Catechins in Aqueous Physiological Conditions.

Epicatechin (EC), epigallocatechin (EGC), epicatechingallate (ECG) and epigallocatechingallate (EGCG) are antioxidants present in the green tea, a widely used beverage whose health benefits are largely recognized. Nevertheless, major physicochemical limitations, such as the high instability of catechins, pose important questions concerning their potential pharmacological use. Recent studies indicate that binding of catechins with plasmatic proteins may modulate their plasma concentration, tissue delivery and biological activity. After 5 minutes of incubation with HSA both ECG and EGCG were fully bound to HSA, while after 48h incubation only 41% of EC and 70% of EGC resulted linked. HSA had a strong stabilizing effect on all catechins, which could be found in solution between 29 and 85% even after 48h of incubation. In the absence of HSA, EGC and EGCG disappeared in less than 24h, while ECG and EC were found after 48h at 5 and 50%, respectively. The stabilizing effect of HSA toward EGCG, obtained in aqueous physiological conditions, resulted stronger in comparison to cysteine and HCl, previously reported to stabilize this polyphenol. Because of the multitude of contradictory data concerning in vivo and in vitro antioxidant-based experimentations, we believe our work may shed some light on this debated field of research.

Preliminary Evidence That Bioactive Cements Occlude Artificial Marginal Gaps.

The objective of this preliminary laboratory study was to explore whether bioactive dental cements have the ability to seal marginal gaps as compared with other classes of dental cements. Tooth discs (dentin/enamel) were positioned adjacent to a second disc of one of five possible dental cements (two bioactive and three conventional) and separated by a 50-μm Mylar film, creating a controlled gap between the two discs. The edges of the tooth segments were abraded to expose dentin such that the dentin interface was positioned opposite the cement. The artificial gap assembly was stabilized with adhesive and composite resin. Artificial gap assemblies for each cement type were immersed in a phosphate-containing, simulated body fluid (phosphate buffered saline) for up to 8 months. Occlusion of artificial marginal gaps (including deposition of crystal-like, mineralized deposits within the gaps) was observed and scored categorically during the observation period. Deposition of radiodense material within the artificial marginal gap space was confirmed by micro-computerized tomography analysis. There was no evidence of marginal gap occlusion for the three conventional control cements, whereas both bioactive, surface apatite-forming cements demonstrated occlusion of the artificial marginal gaps. The preliminary findings of this initial study suggest the possible capability of surface apatite-forming, calcium-based, bioactive dental cements to seal or reseal artificial marginal gaps in simulated aqueous physiological conditions. The results of this laboratory study, although preliminary in nature, suggest a new functional property for bioactive dental materials, namely the ability to significantly improve marginal stability with tooth/restorative material interface. Such behavior in a restorative material could improve the survival and serviceability of dental restorations.

ChemInform Abstract: Nucleic Acid Ion Structures in the Gas Phase

AbstractReview: 160 refs.

Nucleic acid ion structures in the gas phase.

Nucleic acids are diverse polymeric macromolecules that are essential for all life forms. These biomolecules possess a functional three-dimensional structure under aqueous physiological conditions. Mass spectrometry-based approaches have on the other hand opened the possibility to gain structural information on nucleic acids from gas-phase measurements. To correlate gas-phase structural probing results with solution structures, it is therefore important to grasp the extent to which nucleic acid structures are preserved, or altered, when transferred from the solution to a fully anhydrous environment. We will review here experimental and theoretical approaches available to characterize the structure of nucleic acids in the gas phase (with a focus on oligonucleotides and higher-order structures), and will summarize the structural features of nucleic acids that can be preserved in the gas phase on the experiment time scale.

Metal Catalyzed Click Chemistry for Molecular Imaging Probes

Cu-catalyzed azide-alkyne cycloaddition has been developed as a new, simple, efficient, and generally applicable synthetic strategy, known as ‘Click Chemistry’. The reaction utilizes two bioorthogonal functional groups, azide and alkyne, which can be readily incorporated into biological molecules with straightforward chemical modification. The reaction can be performed in low substrate concentration under a wide range of conditions including aqueous physiological conditions, which finds a span of application ranging from material science, chemical biology/bio-conjugation, and pharmaceutical research and development. In this review, we focused on the new progress in developing the metal catalyzed, bioorthogonal azide-alkyne cycloaddition reaction (‘Click chemistry’) about catalyst species, the ligands and substrates and its application in the bio-imaging field from four aspects: (a) labeling of live cell surface; (b) imaging small molecules inside of living cells; (c) modifying cells with nanomaterials; and (d) vivo imaging.

Conducting polymers with immobilised fibrillar collagen for enhanced neural interfacing

Conducting polymers with pendant functionality are advantageous in various bionic and organic bioelectronic applications, as they allow facile incorporation of bio-regulative cues to provide bio-mimicry and conductive environments for cell growth, differentiation and function. In this work, polypyrrole substrates doped with chondroitin sulfate (CS), an extracellular matrix molecule bearing carboxylic acid moieties, were electrochemically synthesized and conjugated with type I collagen. During the coupling process, the conjugated collagen formed a 3-dimensional fibrillar matrix in situ at the conducting polymer interface, as evidenced by atomic force microscopy (AFM) and fluorescence microscopy under aqueous physiological conditions. Cyclic voltammetry (CV) and impedance measurement confirmed no significant reduction in the electroactivity of the fibrillar collagen-modified conducting polymer substrates. Rat pheochromocytoma (nerve) cells showed increased differentiation and neurite outgrowth on the fibrillar collagen, which was further enhanced through electrical stimulation of the underlying conducting polymer substrate. Our study demonstrates that the direct coupling of ECM components such as collagen, followed by their further self-assembly into 3-dimensional matrices, has the potential to improve the neural-electrode interface of implant electrodes by encouraging nerve cell attachment and differentiation.

Polyelectrolyte Multilayers for Tunable Release of Antibiotics

Polyelectrolyte multilayers incorporating gentamicin were fabricated using layer-by-layer deposition. The multilayers could be precisely tuned with regard to dosage, and release rate under aqueous physiological conditions could be controlled. The films were demonstrated efficacious against Staphylococcus aureus and nontoxic toward murine osteoblasts MC3T3. The films were made in a layer-by-layer process with a heterostructural architecture that represents a general strategy for incorporating charged small molecular species into polyelectrolyte multilayers without the need for any physical or chemical premodification, expanding the range of active species that can be delivered from these films while maintaining the ease of layer-by-layer fabrication process.

Alkylation Activity and Molecular Properties of Two Antineoplastic Agents that Utilise Indometacin and a Conjugate of Aspirin with 2-Aminonicotinic Acid to Transport Nitrogen Mustard Groups

Nitrogen mustard (N-mustard) compounds are considered important anticancer drugs. Various transporting agents have been utilised to carry N-mustard groups including coumarins, amides, polyaromatic molecules and cycloalkyl structures. N-mustards act as bifunctional alkylating agents that induce cross-linking within DNA strands and cytotoxic activity. Compounds that transport the N-mustard group in vivo can also express drug-likeness that can have advantages in clinical application. This study presents data on two anticancer drugs with N-mustard groups covalently attached to NSAIDs. Two alkylating compounds were synthesised by covalently attaching a single N-mustard group to 2-2-acetoxybenzoylaminonicotinic acid (for compound I) and indometacin (for compound II). Molecular properties such as aqueous solubility, 1-octanol/water partitioning coefficient (log Kow), molar volume, polar surface area, 1-octanol/water partitioning at pH values other than human blood (log D) and the dermal permeability coefficient (Kp) were determined. The rate-order of reaction and rate constant of alkylation were determined by reacting compound I and compound II with a target compound having a primary amine group in buffered aqueous solution at blood pH 7.4 and 37 masculineC, and monitoring absorbance at 400nm. RESULTS AND C onclusion: Compounds I and II were stable at room temperature, soluble in water and effectively alkylated a nucleophilic primary amine target at physiological temperature and pH. The water solubility of compound I was considerably greater than that of compound II. Both compounds showed second-order rate order of alkylation and effectively alkylated a nucleophilic target under aqueous physiological conditions of pH 7.4 and 37 masculineC. Kp values for compounds I and II were determined to be 0.000786 cm/h and 0.024 cm/h, respectively. Both compound I and compound II had zero percent ionisation at pH 7.4, and compound I showed zero violations of the Rule of 5. Log P values for compounds I and II were 3.27 and 5.08, respectively. This study describes the benefits of antineoplastic agents with NSAID substituents that provide favourable pharmacological properties.

Alkylation Activity and Multivariate Analysis of the Molecular Properties of Four Antineoplastic Agents that Utilize a D-Amino Acid, Citric Acid, 1-Octanol, or 4-Oxoazetidine-2-Carboxylic Acid to Transport Alkylating Groups

Nitrogen (N)-mustard agents are the most widely utilized anticancer drugs and are used for the treatment of leukemias and solid tumors. Compounds utilized to carry these alkylating groups include coumarins, amides, polyaromatic molecules, and cycloalkyl structures. These alkylating agents can induce monoalkylation of DNA strands, which causes single-strand breaks in DNA for cytotoxic action. N-mustards may act as bifunctional agents that can induce cross-linking within DNA strands as their cytotoxic activity. Novel structures that transport the N-mustard group can express enhanced drug-likeness, which has advantages in clinical application. Four alkylating compounds were synthesized utilizing 4-oxoazetidine-2-carboxylic acid (compound I), D-alanine (compound II), citric acid (compound III), or 1-octanol (compound IV) as the framework. The resulting products formed monofunctional (I and IV) or polyfunctional (II and III) alkylating compounds. Various important molecular properties, such as aqueous solubility, log octanol-water partition coefficient (log Kow), molar volume, polar surface area (PSA), dermal permeability coefficient (Kp), and rate-order of reaction and rate constant of reaction were determined. Multivariate methods, such as cluster analysis and principal coordinates analysis, were used to analyze the molecular properties of these compounds and determine similarities and dissimilarities. Multivariate methods were used to discern relationships among this complex group of compounds. All four compounds were stable at room temperature, soluble in water, and effectively alkylated a nucleophilic primary amine target at physiological temperature and pH. Compounds I, II, and IV are predicted to be >90% absorbed in the intestinal tract and compound III >60% absorbed. Analysis via Rule of 5 indicates that compounds I, II, III, and IV are expected to show effective bioavailability and permeation. Compounds I, II, and IV will effectively penetrate the blood-brain barrier (BBB) based upon PSA values. Water solubilities of compounds I, II, III, and IV decrease as the value of log Kow increases. Values of molar refractivity, molar volume, and parachor increase as molecular weight increases. Multivariate analysis showed that compounds II and IV are highly similar and indicated that these compounds could have analogous clinical applications. Compound I is predicted to have potential antibacterial activity due to a β-lactam ring, in addition to effective alkylation activity. Compound II resists hydrolyzing-enzyme esterases due to the D-amino-acid structure. Compound III is a polyfunctional-alkylating agent that would inhibit mitosis as an adjunct to DNA cross-linking. Compound IV has a single methanesulfonate group providing monoalkylation of suitable nucleophilic sites. PSA and log Kow values indicate effective penetration of the BBB by compounds I, II, and IV. Rule of 5 analysis showed that compounds I, II, III, and IV would have effective bioavailability. All compounds effectively alkylated a nucleophilic target under aqueous physiological conditions of pH 7.4 and 37°C. This work demonstrates the benefits of designing antineoplastic agents with novel molecular structures that provide favorable pharmacological properties.