A concise review on application of squaric acid derivatives and their metal complexes in medicine

In this review


Introduction
Over the years, researchers have been synthesizing and characterizing many metal complexes with squaric acid and its derivatives. All these complexes were studied for different applications like chemosensors, inhibitors, etc. But a few investigations on the application of squaric acid derivatives and their metal complexes were prepared and published. In 2020 Chasák et al. 1 published a review about the biological activity of squaric acid and its derivatives.
The focus of our review is on application of the squaric acid, squaramides and their metal complexes in medicine. Recently many researchers published synthesis and investigation of metal complexes of squaric acid and its derivatives. Furthermore, biological activity of the obtained metal complexes has been studied.

Squaric Acid and Oxocarbones -Characteristics
In 1960, West and co-workers identified a new class of organic compounds with cyclic structure and general formula (CnOn) 2-. These compounds were named oxocarbons. [2][3][4][5] The oxocarbon dianions (see Figure (1)) present a strong absorption in UV and visible regions with the exception of the deltate, which absorbs at wavelength lower than 200 nm. That raised the question "Are the oxocarbon dianions really aromatic?" The general conclusion is that the aromaticity decreases with the ring size. This conclusion is based not only on the symmetry shown from vibrational spectra, but also on the great stability of the anions, shown from the large dissociation constants of the corresponding acids. In crystal engineering, oxocarbon ions have been currently employed as building blocks, photoreceptors and semiconductor materials having non-linear optical properties ( Figure 1). [6][7][8][9][10][11]  One representative of the group of oxocarbons is squaric acid (3,4-dihydroxycyclobut-3-ene-1,2-dione). In 1959 Cohen et al. 12 have obtained it for the first time and called quadratic acid because its four carbon atoms are forming square ( Figure 2).  13 , showed by X-ray crystallography that the nonionized compound of squaric acid in reality is asymmetric, so in fact it forms trapezium. Samuelsen et al. 14 , in 1975, examined the structure of the squaric acid and proved that it consists of planar layers of hydrogen bonded molecules. They determined that each molecule participates in four asymmetric hydrogen bonds, and thus there are two hydrogen atoms located nearest to anyone (C4O4) 2unit. The structure of the squaric acid allows the formation of intermolecular hydrogen bonds with carbonyl groups of proton acceptors and also formation of complex compounds with metal ions. This, in combination with its inherent high structural rigidity, allows squaric acid and its derivatives to have a variety of applications in organic chemistry, biomedical chemistry and material science. Squaric acid has a remarkable acidity, which is the highest among all cyclic oxocarbon acids and is explained by resonance stabilization of the dianion. It is a strong hydrogen-bonded and remarkably stable solid and forms clear crystals. [15][16][17][18] Squaric acid has a four-membered cyclic ring that exhibits two acidic hydroxyl groups, as well as two highly polarized carbonyl groups. [18][19][20] Squaric acid can form mono-and dianions ( Figure 3). It has two donor O-H groups along with two carbonyl acceptors, while its monoanion has one donor O-H group and three proton acceptors. All three species possess a certain degree of delocalization, but it is most pronounced in the dianion which is considered to be aromatic. [18][19][20][21][22][23]  Squaric acid versatility as a ligand is attributable to the fact that all four of its oxygen atoms are potential coordination sites, because they are chemically equivalent. This is due to resonance stabilization, leading to a fully delocalized aromatic structure. After losing two protons a squarate dianion (C4O4) 2is formed. It is a resonance-stabilized dianion owing to the delocalization of the negative charge over the C4 ring and the four appended oxygen atoms. This delocalization is evidenced by the near equivalence of the respective C-C and C-O bond lengths of the squarate ion, except where hydrogen bonding causes a distortion resulting in a lowering of the symmetry from D4h. [21][22][23][24][25] The squarate dianion [C4O4] 2is water soluble and exhibits very unusual electronic and vibrational properties. It is a remarkable ligand in different coordination complexes, because all four oxygen atoms are potential coordination sites ( Figure 4). It has also been shown to participate in a range of different coordination modes. The squarate dianion can be both a chelate and a bridge ligand, such as a 1,2-bidentate chelate, 1,2-bis (monodentate) and 1,3-bis (monodentate) linking bridge. It has also been frequently used as a polyfunctional (μ1 to μ6 bridges) ligand that forms hydrogen bonding and π-π stacking interactions to form more extended networks. 26-33 X-ray diffraction studies by Oliveira et al. 33 have revealed all possible coordination modes of the squarate dianion with transition metals and lanthanides ( Figure 5).

Squaramides and Their Application in Medicine
One group of amino derivatives of squaric acid is represented by squaramides. They have been known since 1950s, but have only recently emerged as particularly useful chemical entities in a variety of applications.
Squaramides are known to possess remarkable ring systems and structural rigidity 34 ( Figure 6). They are ditopic hydrogen-bonding synthons that can self-associate through two acceptors (C=O groups) and two donors (N−H groups) of hydrogen bond directly opposite one another on a cyclobutenedione ring. [34][35] Squaramides are used as coupling units for chelator and targeting vectors in radiopharmaceuticals for diagnostic and therapeutic purposes in nuclear medicine. 36 Chiral squaramides are powerful bifunctional hydrogen-bonding catalysts, and promoted numerous catalytic asymmetric transformations. 37 The squaramide-functional group has recently been exploited in supramolecular chemistry for the design of anion receptors. Elmes and co-workers 38 reported the synthesis of eight amino-acid based squaramide-anion receptors. They proved that the molecular properties of these receptors attenuated without affecting their anion recognition properties and this maybe will affect their anion recognition properties in biological or environmental applications. Busschaert et al. 39 reported for the anion transport properties of a series of squaramides and compared their transport abilities with analogous ureas and thioureas. This can be explained by the enhanced anion-binding properties of the squaramide-based receptors. In the last years very interesting results have been obtained for the realizing of asymmetric catalytic synthesis of nitro compounds in presence of small chiral organic molecules (organocatalysts). 40 Among the most efficient organocatalysts for enantioselective reactions of nitro alkanes and nitro alkenes are bifunctional chiral tertiary amines containing squaramide units. 41 Squaramides have also found wide applications in different annulation reactions. 42 Annulation reactions represent a powerful strategy for the construction of cyclic molecular frameworks. The asymmetric organocatalytic annulation reactions were used in total synthesis of natural products. Some novel squaramides have been prepared by Peng Li et al. 43 for the potential treatment of drug-resistant tuberculosis. The compounds displayed good to excellent in vitro antituberculosis activity and low cytotoxicity.

Application of Squaric Acid and Squaramides as Bioisosteres
Bioisosteric replacement is an important strategy to modify and optimize the properties of potential drugs. It may improve stability, optimize activity and minimize the side effects. 1,[44][45][46] In 1950 Harris Friedman introduced the term "bioisosteres" and defined it as compounds eliciting a similar biological effect. 45 Squaric acid and its derivatives can serve as a bioisostere of several functional groups, physiological important for medical chemistry and pharmacology. The sqaurate and sqauramide group can replace the pharmacologically important groups such as phosphate, carboxylate, sulphonate groups ( Figure 7) because of the similar structural, electronic and physical properties like acidity, size of molecule, polarity, H-bonding. [48][49][50][51][52][53][54]  Squaramides have rigid planar structures, so they exhibit 10-50 times greater affinity for halides than thiourea. 61 Urbahns et al. 62 have synthesized a variety of arginine bioisosteres, with one of them squaric acid amide, which mimicked urea fragments. The 3,4-diaminocyclobut-3-ene-1,2-dione fragment can replace the urea functionality since researchers at Wyetht, while working on the potassium channel openers (KCOs), have demonstrated that it is an effective replacement of a N-cyanoguanidine. Bioisosteric replacement of the Ncyanoguanidine moiety of the drug pinacidil with diaminocyclobutenedione template affords a novel series of bladder-selective potassium channel openers (KCOs). 56,63 Monosquaramide and disquaramide derivatives can serve as mimetics (isosteres) of amino acids based on structural homology, on the inherent strong dipole, and on electron density residing on the oxygen atoms. The 3,4-diamino-3-cyclobutene-1,2-dione group was described as a useful α-amino acid bioisostere. 46,56,64

Application of Squaric Acid and its Derivatives in Medicine
It is known that various derivatives of squaric acid, substituted in a specific manner, have pharmacologically useful properties. The application of squaric acid and squaramide derivatives as anticancer agents has not been extensively studied, therefore no studies on various human tumor cell lines have been reported. [65][66] It has been shown that some squaramides selectively bind with protein kinases 66 or the CXCR2 receptor. 67 This shows that these compounds can selectively bind to cellular targets. Therefore it suggests that squaramides may serve as a good starting point for identifying the molecules which can specifically target cancer cells. Quintana et al. 68 studied a series of squaramides and squaramates for antitumor activity against different cancer cells that confirms the antitumor properties of this class of promising drug candidates. Two cancer cell lines were used -HeLa (cervical carcinoma) and HGC-27 (human gastric cell line). At first, preliminary screening was performed and then the most potent compounds were further evaluated. The data showed that HGC-27 cells seem to be more sensitive than HeLa cells to the effect of the tested squaramides.
Squaric acid and its derivatives have been studied for cytotoxic activity against a panel of human tumor cell lines especially against human leukemia cell lines. Liu et al. prepared series of novel 3,4-diaryl squaric acid analogues and studied their cytotoxic activity. Some of the new compounds exhibit strong cytotoxicity against human leukemia cells 57 (Figure 9). The cytotoxic activity may be due to the presence of three methoxy groups of A ring according to the SAR analysis results. For the design of structurally related tubulin inhibitors or combretastatin analogs the obtained results are very important. The new compounds were tested at a single dose of 10 μM and they did not exhibit significant growth inhibition. The same compounds were also tested for antiviral activity. The results showed that the compounds demonstrated reasonable antiviral and cytotoxicity profiles could be candidates for several additional follow-up analyses. 69 A series of bioavailable 3,4-diaminocyclobutenediones with various amide modifications and substitution on the left side phenyl ring were prepared and found to show significant inhibitory activities towards CXCR2 (Chemokine (C-X-C Motif) Receptor 2) and CXCR1 (Chemokine (C-X-C Motif) Receptor 1) receptors. 70 The same group of researchers has also investigated a series of the 3,4-diaminocyclobutenediones with amide modifications and substitution on the right side phenyl ring. If the benzylic amine is keeping on the right side as constant, a number of amides were prepared and evaluated in the membrane binding CXCR2 and CXCR1 assays. The mono substituted alkyl amides showed weaker binding affinity towards the CXCR2 receptor compared to N,N-dimethyl amide that showed excellent inhibitory activity towards both receptors. 71 The synthesis and testing of a series of substrate-mimic SNM1A (DNA cross-link repair 1A protein) inhibitors bearing squaramide or thiosquaramide ZBGs were reported by Berney et al. 72 Squaramides can chelate cations through their two carbonyl oxygen atoms. 73 Their derivatives as N-hydroxysquaramides have shown promise as ZBGs in inhibitors for metalloproteases. [74][75] It was shown that an oligonucleotide bearing a squaramide at the 5′-terminus is bound to SNM1A. 76 The use of squaramides as metal chelators in biological applications has not been fully explored yet. The compounds containing a squaramide group at the 5′-position proved to be ineffective, but some nucleoside derivatives with a squaramide moiety at the 3′-position demonstrated inhibition of SNM1A. The quantitative data showed that a thymidine derivative bearing a 5′thiosquaramide was the most potent inhibitor, followed by a thymidine derivative bearing a 3′-squaric acid.

Application of Metal Complexes of Squaric Acid and its Derivatives as Cytotoxic Agents
Over the years, many articles about the synthesis and characterization of metal complexes with squaric acid and its derivatives were published but there is little data about their application in medicine. Squaric acid participates in the formation of large variety of complexes with transition metals because of the presence of four oxygen atoms.
The mode of coordination of squaric acid and its derivatives can be monodentate, bismonodentate, bidentate, trismonodentate and tetrakismonodentate. When the coordination is bismonodentate, it is often achieved by bridging either μ-1,2 or μ-1,3. 21 Studies on the structures of alkaline earth squarates are very limited. K. T. Vadhana et al. 77  H2O}n. All complexes were tested in vitro for cytotoxicity against human breast cancer cell line MCF-7. The significant cytotoxic activity against the MCF-7 cell line is due to the carbonyl group, planarity and chelation of the organic ligand present in the metal complexes. [78][79] Recently a great attention has been paid to the complexes containing 1,2-diaminocyclohexane (dach) as a ligand. Many researchers have used it and synthesized the complexes with squaric acid analogous as a second ligand. Zhang et al. 80 have prepared series of estradiol-derived Ni(II), Zn(II) and Pd(II) complexes containing a unique squaramide moiety ( Figure 10). These authors have also examined the binding affinities of these compounds to the estrogen receptorligand binding domain (ER-LBD). The results showed effective binding of the compounds to the estrogen receptors. The compounds were also tested for transcriptional activity in human embryonic kidney 293T (HEK-293T) cells by a Luciferase reporter gene assays. All compounds synthesized were agonists on Erα in HEK-293T cells. In conclusion the tested compounds have showed low efficacy acting as antagonist on ERα.
Since the squarate ion is chemically correlated to the oxalate ion, some platinum complexes with squaric acid instead of oxalate ion as a ligand were synthesized. Dioxycyclobutenedione-(1,2-cyclohexanediamine)-platinum(II), (cis-[Pt(dach)(SA)], where SA is a dianion of squaric acid was reported by Yang and coworkers 81 (Figure 11). The complex has very close structure to that of oxaliplatin. It was studied for cytotoxic activity in vitro on six human tumor cell lines. The complex shows stronger cytotoxicity than cisplatin against human immature granulocyte leukemia (HL-60), erythroleukemia (K-562), human gastric carcinoma (BGC), human nasopharyngeal carcinoma (KB), human colon carcinoma (HCT) and human hepatocellular carcinoma (Bel-7402). The complex binds to DNA covalently, at the same way as that of cisplatin. The stronger cytotoxicity of cis-[Pt(dach)(SA)] than that of cisplatin is caused by its greater destruction to DNA than cisplatin.  Lialiaris and co-workers 84 synthesized and studied for biological activity binuclear platinum complexes with squaric acid that have biologically active squaric moiety and platinum active center in view to compare them with cisplatin. The goal was to prepare active anticancer agents by modification of the prototype cisplatin. In structures of the compounds there are ammonia molecule as a carrier ligand and chloride ion as a leaving group. The effect of the complexes on Sister Chromatid Exchange (SCE) rates and human lymphocyte proliferation kinetics was studied. SCEs have been proposed as a very sensitive method for detecting mutagens and/or carcinogens and lately as a method of evaluating chemotherapy in vitro 85 and in vivo 86 .
Studies have also shown that the determination of proliferation rates in lymphocyte cultures should be a useful and sensitive indicator of the cellular toxicity of chemotherapeutic agents. 87 . The complexes tested were [Pt2(NH3)2Cl2(SA)] and [Pt2(NH3)4Cl2(SA)]. The results showed that the first compound is the most effective, on a molar basis in causing the cell division delays in comparison with the second one and cisplatin.
Squaramide motifs are class of interesting photoresponsive species. In 2018 Morales and co-workers 88 synthesized two squaramide-based Pt(II) complexes. They were evaluated for antiproliferative activity on HeLa cell line and compared with carboplatin. Complex Pt(C12H19N2O2S2)Cl is the first example of a platinum complex directly coordinated to a squaramide motif. It showed moderated cytotoxicity, whereas its irradiated form could not be evaluated because of its poor solubility. The Pt(C12H20N2O2S2)Cl2 complex is inactive on HeLa cells, but under hypoxic conditions, C2′H revealed remarkable enhancement of the antiproliferative activity that is in the same as of carboplatin ( Figure 13).   The squaramide complex has a neuroprotective effect and it could be gut mimic of the carboxylic acid and pyridine groups in this case. 89,90

Conclusions
In this review, the literature data about the application of squaric acid, its derivatives and their metal complexes in medicine were described. A large number of squaric acid analogous were synthesized and studied for different applications. Squaric acid, squaramides, squaramates and other analogues have more than two coordination modes to bind with metals and formed complex compounds. Also, these compounds can be used as monodentate, bidentate, bridge ligands to form monuclear and binuclear metal complexes. However, little data about their application in medicine was published. In recent years the efforts of researchers were focused on studies of this class of organic compounds for cytotoxic activity, antiviral activity etc. The authors believe that this review will be very useful for researchers studying the application of the squaric acid analogues and their metal complexes in medicine.