Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity.

T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell-mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti-PD-1, to improve cancer patient outcomes.

Discovery of Potent and Selective Methylenephosphonic Acid CD73 Inhibitors.

Solid tumors are often associated with high levels of extracellular ATP. Ectonucleotidases catalyze the sequential hydrolysis of ATP to adenosine, which potently suppresses T-cell and NK-cell functions via the adenosine receptors (A2a and A2b). The ectonucleotidase CD73 catalyzes the conversion of AMP to adenosine. Thus, increased CD73 enzymatic activity in the tumor microenvironment is a potential mechanism for tumor immune evasion and has been associated with poor prognosis in the clinic. CD73 inhibition is anticipated to restore immune function by skirting this major mechanism of adenosine generation. We have developed a series of potent and selective methylenephosphonic acid CD73 inhibitors via a structure-based design. Key binding interactions of the known inhibitor adenosine-5'-(α,ß-methylene)diphosphate (AMPCP) with hCD73 provided the foundation for our early designs. The structure-activity relationship study guided by this structure-based design led to the discovery of 4a, which exhibits excellent potency against CD73, exquisite selectivity against related ectonucleotidases, and a favorable pharmacokinetic profile.

Discovery of AB680: A Potent and Selective Inhibitor of CD73.

Extracellular adenosine (ADO), present in high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T cell and NK cell activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases, CD39 (ATP → AMP) and CD73 (AMP → ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Extensive interrogation of structure-activity relationships (SARs), structure-based drug design, and optimization of pharmacokinetic properties culminated in the discovery of AB680, a highly potent (Ki = 5 pM), reversible, and selective inhibitor of CD73. AB680 is further characterized by very low clearance and long half-lives across preclinical species, resulting in a PK profile suitable for long-acting parenteral administration. AB680 is currently being evaluated in phase 1 clinical trials. Initial data show AB680 is well tolerated and exhibits a pharmacokinetic profile suitable for biweekly (Q2W) iv-administration in human.

Click chemistry-facilitated comprehensive identification of proteins adducted by antimicrobial 5-nitroimidazoles for discovery of alternative drug targets against giardiasis.

Giardiasis and other protozoan infections are major worldwide causes of morbidity and mortality, yet development of new antimicrobial agents with improved efficacy and ability to override increasingly common drug resistance remains a major challenge. Antimicrobial drug development typically proceeds by broad functional screens of large chemical libraries or hypothesis-driven exploration of single microbial targets, but both strategies have challenges that have limited the introduction of new antimicrobials. Here, we describe an alternative drug development strategy that identifies a sufficient but manageable number of promising targets, while reducing the risk of pursuing targets of unproven value. The strategy is based on defining and exploiting the incompletely understood adduction targets of 5-nitroimidazoles, which are proven antimicrobials against a wide range of anaerobic protozoan and bacterial pathogens. Comprehensive adductome analysis by modified click chemistry and multi-dimensional proteomics were applied to the model pathogen Giardia lamblia to identify dozens of adducted protein targets common to both 5'-nitroimidazole-sensitive and -resistant cells. The list was highly enriched for known targets in G. lamblia, including arginine deiminase, α-tubulin, carbamate kinase, and heat shock protein 90, demonstrating the utility of the approach. Importantly, over twenty potential novel drug targets were identified. Inhibitors of two representative new targets, NADP-specific glutamate dehydrogenase and peroxiredoxin, were found to have significant antigiardial activity. Furthermore, all the identified targets remained available in resistant cells, since giardicidal activity of the respective inhibitors was not impacted by resistance to 5'-nitroimidazoles. These results demonstrate that the combined use of click chemistry and proteomics has the potential to reveal alternative drug targets for overcoming antimicrobial drug resistance in protozoan parasites.

Evaluation of high-affinity phenyltetrahydroisoquinoline aldoximes, linked through anti-triazoles, as reactivators of phosphylated cholinesterases.

Acetylcholinesterase (AChE) is a pivotal enzyme in neurotransmission. Its inhibition leads to cholinergic crises and could ultimately result in death. A related enzyme, butyrylcholinesterase (BChE), may act in the CNS as a co-regulator in terminating nerve impulses and is a natural plasma scavenger upon exposure to organophosphate (OP) nerve agents that irreversibly inhibit both enzymes. With the aim of improving reactivation of cholinesterases phosphylated by nerve agents sarin, VX, cyclosarin, and tabun, ten phenyltetrahydroisoquinoline (PIQ) aldoximes were synthesized by Huisgen 1,3 dipolar cycloaddition between alkyne- and azide-building blocks. The PIQ moiety may serve as a peripheral site anchor positioning the aldoxime moiety at the AChE active site. In terms of evaluated dissociation inhibition constants, the aldoximes could be characterized as high-affinity ligands. Nevertheless, high binding affinity of these oximes to AChE or its phosphylated conjugates did not assure rapid and selective AChE reactivation. Rather, potential reactivators of phosphylated BChE, with its enlarged acyl pocket, were identified, especially in case of cyclosarin, where the reactivation rates of the lead reactivator was 100- and 6-times that of 2-PAM and HI-6, respectively. Nevertheless, the return of the enzyme activity was affected by the nerve agent conjugated to catalytic serine, which highlights the lack of the universality of reactivators with respect to both the target enzyme and OP structure.

Counteracting tabun inhibition by reactivation by pyridinium aldoximes that interact with active center gorge mutants of acetylcholinesterase.

Tabun represents the phosphoramidate class of organophosphates that are covalent inhibitors of acetylcholinesterase (AChE), an essential enzyme in neurotransmission. Currently used therapy in counteracting excessive cholinergic stimulation consists of a muscarinic antagonist (atropine) and an oxime reactivator of inhibited AChE, but the classical oximes are particularly ineffective in counteracting tabun exposure. In a recent publication (Kovarik et al., 2019), we showed that several oximes prepared by the Huisgen 1,3 dipolar cycloaddition and related precursors efficiently reactivate the tabun-AChE conjugate. Herein, we pursue the antidotal question further and examine a series of lead precursor molecules, along with triazole compounds, as reactivators of two AChE mutant enzymes. Such studies should reveal structural subtleties that reside within the architecture of the active center gorge of AChE and uncover intimate mechanisms of reactivation of alkylphosphate conjugates of AChE. The designated mutations appear to minimize steric constraints of the reactivating oximes within the impacted active center gorge. Indeed, after initial screening of the triazole oxime library and its precursors for the reactivation efficacy on Y337A and Y337A/F338A human AChE mutants, we found potentially active oxime-mutant enzyme pairs capable of degrading tabun in cycles of inhibition and reactivation. Surprisingly, the most sensitive ex vivo reactivation of mutant AChEs occurred with the alkylpyridinium aldoximes. Hence, although the use of mutant enzyme bio-scavengers in humans may be limited in practicality, bioscavenging and efficient neutralization of tabun itself or phosphoramidate mixtures of organophosphates might be achieved efficiently in vitro or ex vivo with these mutant AChE combinations.

Reversal of Tabun Toxicity Enabled by a Triazole-Annulated Oxime Library-Reactivators of Acetylcholinesterase.

Acetylcholinesterase (AChE), an enzyme that degrades the neurotransmitter acetylcholine, when covalently inhibited by organophosphorus compounds (OPs), such as nerve agents and pesticides, can be reactivated by oximes. However, tabun remains among the most dangerous nerve agents due to the low reactivation efficacy of standard pyridinium aldoxime antidotes. Therefore, finding an optimal reactivator for prophylaxis against tabun toxicity and for post-exposure treatment is a continued challenge. In this study, we analyzed the reactivation potency of 111 novel nucleophilic oximes mostly synthesized using the CuAAC triazole ligation between alkyne and azide building blocks. We identified several oximes with significantly improved in vitro reactivating potential for tabun-inhibited human AChE, and in vivo antidotal efficacies in tabun-exposed mice. Our findings offer a significantly improved platform for further development of antidotes and scavengers directed against tabun and related phosphoramidate exposures, such as the Novichok compounds.

Click Chemistry-Facilitated Structural Diversification of Nitrothiazoles, Nitrofurans, and Nitropyrroles Enhances Antimicrobial Activity against Giardia lamblia.

Giardia lamblia is an important and ubiquitous cause of diarrheal disease. The primary agents in the treatment of giardiasis are nitroheterocyclic drugs, particularly the imidazoles metronidazole and tinidazole and the thiazole nitazoxanide. Although these drugs are generally effective, treatment failures occur in up to 20% of cases, and resistance has been demonstrated in vivo and in vitro Prior work had suggested that side chain modifications of the imidazole core can lead to new effective 5-nitroimidazole drugs that can combat nitro drug resistance, but the full potential of nitroheterocycles other than imidazole to yield effective new antigiardial agents has not been explored. Here, we generated derivatives of two clinically utilized nitroheterocycles, nitrothiazole and nitrofuran, as well as a third heterocycle, nitropyrrole, which is related to nitroimidazole but has not been systematically investigated as an antimicrobial drug scaffold. Click chemistry was employed to synthesize 442 novel nitroheterocyclic compounds with extensive side chain modifications. Screening of this library against representative G. lamblia strains showed a wide spectrum of in vitro activities, with many of the compounds exhibiting superior activity relative to reference drugs and several showing >100-fold increase in potency and the ability to overcome existing forms of metronidazole resistance. The majority of new compounds displayed no cytotoxicity against human cells, and several compounds were orally active against murine giardiasis in vivo These findings provide additional impetus for the systematic development of nitroheterocyclic compounds with nonimidazole cores as alternative and improved agents for the treatment of giardiasis and potentially other infectious agents.

Expanded therapeutic potential in activity space of next-generation 5-nitroimidazole antimicrobials with broad structural diversity.

Metronidazole and other 5-nitroimidazoles (5-NI) are among the most effective antimicrobials available against many important anaerobic pathogens, but evolving resistance is threatening their long-term clinical utility. The common 5-NIs were developed decades ago, yet little 5-NI drug development has since taken place, leaving the true potential of this important drug class unexplored. Here we report on a unique approach to the modular synthesis of diversified 5-NIs for broad exploration of their antimicrobial potential. Many of the more than 650 synthesized compounds, carrying structurally diverse functional groups, have vastly improved activity against a range of microbes, including the pathogenic protozoa Giardia lamblia and Trichomonas vaginalis, and the bacterial pathogens Helicobacter pylori, Clostridium difficile, and Bacteroides fragilis. Furthermore, they can overcome different forms of drug resistance, and are active and nontoxic in animal infection models. These findings provide impetus to the development of structurally diverse, next-generation 5-NI drugs as agents in the antimicrobial armamentarium, thus ensuring their future viability as primary therapeutic agents against many clinically important infections.

Wnt inhibition correlates with human embryonic stem cell cardiomyogenesis: a structure-activity relationship study based on inhibitors for the Wnt response.

Human embryonic stem cell-based high-content screening of 550 known signal transduction modulators showed that one "lead" (1, a recently described inhibitor of the proteolytic degradation of Axin) stimulated cardiomyogenesis. Because Axin controls canonical Wnt signaling, we conducted an investigation to determine whether the cardiogenic activity of 1 is Wnt-dependent, and we developed a structure-activity relationship to optimize the cardiogenic properties of 1. We prepared analogues with a range of potencies (low nanomolar to inactive) for Wnt/ß-catenin inhibition and for cardiogenic induction. Both functional activities correlated positively (r(2) = 0.72). The optimal compounds induced cardiogenesis 1.5-fold greater than 1 at 30-fold lower concentrations. In contrast, no correlation was observed for cardiogenesis and modulation of transforming growth factor ß (TGFß)/Smad signaling that prominently influences cardiogenesis. Taken together, these data show that Wnt signaling inhibition is essential for cardiogenic activity and that the pathway can be targeted for the design of druglike cardiogenic molecules.

Nonquaternary reactivators for organophosphate-inhibited cholinesterases.

A new class of amidine-oxime reactivators of organophosphate (OP)-inhibited cholinesterases (ChE) was synthesized and tested in vitro and in vivo. Compared with 2-PAM, the most promising cyclic amidine-oxime (i.e., 12e) showed comparable or greater reactivation of OP-inactivated AChE and OP-inactivated BChE. To the best of our knowledge, this is the first report of a nonquaternary oxime that has, comparable to 2-PAM, in vitro potency for reactivation of Sarin (GB)-inhibited AChE and BChE. Amidine-oximes were tested in vitro, and reactivation rates for OP-inactivated butyrylcholinesterase (BChE) were greater than those for 2-PAM or MINA. Amidine-oxime reactivation rates for OP-inactivated acetylcholinesterase (AChE) were lower compared to 2-PAM but greater compared with MINA. Amidine-oximes were tested in vivo for protection against the toxicity of nerve agent model compounds. (i.e., a model of Sarin). Post-treatment (i.e., 5 min after OP exposure, i.p,) with amidine oximes 7a-c and 12a, 12c, 12e, 12f, and 15b (145 µmol/kg, i.p.) protected 100% of the mice challenged with the sarin model compound. Even at 25% of the initial dose of amidine-oxime (i.e., a dose of 36 µmol/kg, i.p.), 7b and 12e protected 100% of the animals challenged with the sarin nerve agent model compound that caused lethality in 6/11 animals without amidine-oxime.

Oxime-assisted acetylcholinesterase catalytic scavengers of organophosphates that resist aging.

The cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase, are primary targets of organophosphates (OPs). Exposure to OPs can lead to serious cardiovascular complications, respiratory compromise, and death. Current therapy to combat OP poisoning involves an oxime reactivator (2-PAM, obidoxime, TMB4, or HI-6) combined with atropine and on occasion an anticonvulsant. Butyrylcholinesterase, administered in the plasma compartment as a bio-scavenger, has also shown efficacy but is limited by its strict stoichiometric scavenging, slow reactivation, and a propensity for aging. Here, we characterize 10 human (h) AChE mutants that, when coupled with an oxime, give rise to catalytic reactivation and aging resistance of the soman conjugate. With the most efficient human AChE mutant Y337A/F338A, we show enhanced reactivation rates for several OP-hAChE conjugates compared with wild-type hAChE when reactivated with HI-6 (1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium)). In addition, we interrogated an 840-member novel oxime library for reactivation of Y337A/F338A hAChE-OP conjugates to delineate the most efficient oxime-mutant enzyme pairs for catalytic bio-scavenging. Combining the increased accessibility of the Y337A mutation to oximes within the space-impacted active center gorge with the aging resistance of the F338A mutation provides increased substrate diversity in scavenging potential for aging-prone alkyl phosphate inhibitors.

Amidine-oximes: reactivators for organophosphate exposure.

A new class of amidine-oxime reactivators of organophosphate (OP)-inhibited cholinesterases (ChE) were designed, synthesized, and tested. These compounds represent a novel group of oximes with enhanced capabilities of crossing the blood-brain barrier. Lack of brain penetration is a major limitation for currently used oximes as antidotes of OP poisoning. The concept described herein relies on a combination of an amidine residue and oxime functionality whereby the amidine increases the binding affinity to the ChE and the oxime is responsible for reactivation. Amidine-oximes were tested in vitro and reactivation rates for OP-BuChE were greater than pralidoxime (2-PAM) or monoisonitrosoacetone (MINA). Amidine-oxime reactivation rates for OP-AChE were lower compared to 2-PAM but greater compared with MINA. After pretreatment for 30 min with oximes 15c and 15d (145 µmol/kg, ip) mice were challenged with a soman model compound. In addition, 15d was tested in a post-treatment experiment (145 µmol/kg, ip, administration 5 min after sarin model compound exposure). In both cases, amidine-oximes afforded 100% 24 h survival in an animal model of OP exposure.

Investigating the structural influence of surface mutations on acetylcholinesterase inhibition by organophosphorus compounds and oxime reactivation.

Organophosphates (OPs) exert their toxicity by inhibiting primarily acetylcholinesterase (AChE) and to a lesser extent butyrylcholinesterase (BChE). Binary mixtures of mammalian AChE and oximes of varying structure have been recently considered for treatment of OP poisoning as catalytic bioscavengers. In this study wild type human AChE and human AChE with residue mutations D134H, D134H_E202Q and D134H_F338A were characterized and investigated for inhibition by OPs and consequent oxime reactivation of phosphylated enzymes. The rationale for selecting these substitution positions was based on D134H being a naturally occurring single nucleotide polymorphism (SNP) in humans and that E202Q and F338A mutations slow aging of OP inhibited AChEs. Inhibition of D134H by paraoxon and analogues of cyclosarin was 2-8 times slower than inhibition of wild type (wt), while reactivation of the paraoxon inhibited enzyme by 2PAM was 6 times faster. Both inhibition and reactivation of D134H_E202Q and D134H_F338A double mutants were up to two orders of magnitude slower than the wt indicating that introduction of the active center substitutions abolished fully the effect of the peripherally located D134H. These results indicate that selected residues outside the active center influence inhibition, reactivation and catalysis rates through longer range interactions.

Interaction kinetics of oximes with native, phosphylated and aged human acetylcholinesterase.

Oximes are commonly used nucleophilic reactivators of alkyl phosphorylated and alkyl methylphosphonylated acetylcholinesterase (AChE) and butyrylcholinesterase. Covalent inhibition of these enzymes by organophosphate (OP) pesticides results typically in phosphorylated enzymes, while covalent inhibition by nerve agent OPs results in methyl phosphonylated cholinesterases. In this study we determined kinetic constants for interaction of three triazole containing oximes with native human AChE, enzyme diethylphosphorylated by paraoxon, enzyme phosphonylated by VX and cyclosarin as well as enzyme aged upon phosphonylation by soman. Stopped-flow kinetics of oxime interaction was monitored using quenching of intrinsic tryptophan fluorescence of AChE as an indicator of oxime binding. Triazole oximes were efficiently synthesized using copper catalyzed cycloaddition between azide and alkyne building blocks ("Click chemistry"). Equilibrium dissociation constants determined for both native enzymes were in low micromolar range for all three oximes, while dissociation constants for phosphylated (phosphorylated and phosphonylated) enzymes were typically one to two orders of magnitude larger. Dissociation constants for interaction with aged enzymes were similar or smaller than those determined for native enzymes. Similar results were obtained with reference oximes, 2PAM and HI6. Association rate constants for formation of oxime complexes were similar for both native, phosphylated and aged enzymes. In summary our data suggest that modification of active site gorge in AChEs by phosphylation of the active serine compromises oxime binding. Dealkylation of phosphonylated enzyme, however opens space in the gorge allowing oximes to bind tighter.

Synthesis and electrochemistry of 2-ethenyl and 2-ethanyl derivatives of 5-nitroimidazole and antimicrobial activity against Giardia lamblia.

Infections with the diarrheagenic pathogen, Giardia lamblia, are commonly treated with the 5-nitroimidazole (5-NI) metronidazole (Mz), and yet treatment failures and Mz resistance occur. Using a panel of new 2-ethenyl and 2-ethanyl 5-NI derivatives, we found that compounds with a saturated bridge between the 5-NI core and a pendant ring system exhibited only modestly increased antigiardial activity and could not overcome Mz resistance. By contrast, olefins with a conjugated bridge connecting the core and a substituted phenyl or heterocyclic ring showed greatly increased antigiardial activity without toxicity, and several overcame Mz resistance and were more effective than Mz in a murine giardiasis model. Determination of the half-wave potential of the initial one-electron transfer by cyclic voltammetry revealed that easier redox activation correlated with greater antigiardial activity and capacity to overcome Mz resistance. These studies show the potential of combining systematic synthetic approaches with biological and electrochemical evaluations in developing improved 5-NI drugs.

Identification of a new endogenous metabolite and the characterization of its protein interactions through an immobilization approach.

The emerging field of global mass-based metabolomics provides a platform for discovering unknown metabolites and their specific biochemical pathways. We report the identification of a new endogenous metabolite, N(4)-(N-acetylaminopropyl)spermidine and the use of a novel proteomics based method for the investigation of its protein interaction using metabolite immobilization on agarose beads. The metabolite was isolated from the organism Pyrococcus furiosus, and structurally characterized through an iterative process of synthesizing candidate molecules and comparative analysis using accurate mass LC-MS/MS. An approach developed for the selective preparation of N(1)-acetylthermospermine, one of the possible structures of the unknown metabolite, provides a convenient route to new polyamine derivatives through methylation on the N(8) and N(4) of the thermospermine scaffold. The biochemical role of the novel metabolite as well as that of two other polyamines: spermidine and agmatine is investigated through metabolite immobilization and incubation with native proteins. The identification of eleven proteins that uniquely bind with N(4)-(N-acetylaminopropyl)spermidine, provides information on the role of this novel metabolite in the native organism. Identified proteins included hypothetical ones such as PF0607 and PF1199, and those involved in translation, DNA synthesis and the urea cycle like translation initiation factor IF-2, 50S ribosomal protein L14e, DNA-directed RNA polymerase, and ornithine carbamoyltransferase. The immobilization approach demonstrated here has the potential for application to other newly discovered endogenous metabolites found through untargeted metabolomics, as a preliminary screen for generating a list of proteins that could be further investigated for specific activity.

Efficient synthesis of 2-substituted-1,2,3-triazoles.

In this three-component reaction, alkynes undergo a copper(I)-catalyzed cycloaddition with sodium azide and formaldehyde to yield 2-hydroxymethyl-2 H-1,2,3-triazoles, which are useful intermediates that can be readily converted to polyfunctional molecules. The hydroxymethyl group can also be removed, providing convenient access to N H-1,2,3-triazoles. The reaction is experimentally simple and readily scalable.

Correlating the transcriptome, proteome, and metabolome in the environmental adaptation of a hyperthermophile.

We have performed a comprehensive characterization of global molecular changes for a model organism Pyrococcus furiosus using transcriptomic (DNA microarray), proteomic, and metabolomic analysis as it undergoes a cold adaptation response from its optimal 95 to 72 degrees C. Metabolic profiling on the same set of samples shows the down-regulation of many metabolites. However, some metabolites are found to be strongly up-regulated. An approach using accurate mass, isotopic pattern, database searching, and retention time is used to putatively identify several metabolites of interest. Many of the up-regulated metabolites are part of an alternative polyamine biosynthesis pathway previously established in a thermophilic bacterium Thermus thermophilus. Arginine, agmatine, spermidine, and branched polyamines N4-aminopropylspermidine and N4-( N-acetylaminopropyl)spermidine were unambiguously identified based on their accurate mass, isotopic pattern, and matching of MS/MS data acquired under identical conditions for the natural metabolite and a high purity standard. Both DNA microarray and semiquantitative proteomic analysis using a label-free spectral counting approach indicate the down-regulation of a large majority of genes with diverse predicted functions related to growth such as transcription, amino acid biosynthesis, and translation. Some genes are, however, found to be up-regulated through the measurement of their relative mRNA and protein levels. The complimentary information obtained by the various "omics" techniques is used to catalogue and correlate the overall molecular changes.

Search of nature of planar chirality for pendent benzodiazacoronands in the solid state: NMR, X-ray, and DFT studies.

In this work, we report the structural studies on the solid state of two benzodiazacoronads that form chiral and achiral crystals. Crystals have to be considered as a two-component system consisting of an organic unit and a water molecule in 1:1 ratio. Both components play an important role in the crystal structure. The strong (O-H...O, N-H...O) and weak (C-H...O) intermolecular hydrogen bonds are responsible for phase organization and, in consequence, formation of chiral or achiral crystals. The alignment of the water molecule with respect to the macrocycle is different for samples 1 and 2. Removal of water from the crystal lattice of 1 is reversible. Formation of chiral cocrystals from two different achiral molecules by self-assembly is well-known. However, in this paper, we show that the water molecule can be an important achiral cofactor responsible for chiral crystallization.