Synthesis and bio-properties of 4-piperidone containing compounds as curcumin mimics

The broad spectrum of curcumin's beneficial properties has encouraged medicinal researchers to investigate its therapeutic efficacy against diverse diseases. The clinical potential of curcumin is, however limited due to its poor pharmacodynamic/pharmacokinetic properties (such as low solubility, pH instability, poor absorption in circulation, rapid elimination from the body and photochemical degradation). 3,5-Bis(ylidene)-4-piperidone scaffolds are considered a curcumin mimic that exhibit diverse bio-properties. The current review provides a brief overview of these mimics and highlights biological activities relevant to drug development.


Introduction
Natural products remain one of the main resources for a variety of diverse human needs. Many natural compounds are used directly as drugs or have inspired the development of potent biologically active agents for clinically use. Artemisinin 1 (Fig. 1) is a classic example which was initially extracted from Artemisia annua and subsequently approved as an anti-malarial (Plasmodium falciparum) drug. The discoverer of artemisinin was granted the Nobel Prize in 2015 in recognition of contribution to medicine. 1 Curcumin 2 is another famous example of a natural compound with clinical potential. Curcumin was extracted from roots/rhizomes of Curcuma longa and has earned a high reputation among medicinal chemists due to its usage in Ayurvedic medicine, as a food additive and as a dye in many Asian countries. Historically, it was rst described over 4000 years ago in ancient India where its usage was associated with religious practice. 2 The broad spectrum of curcumin's benecial properties has encouraged medicinal researchers into its therapeutic efficacy against diverse diseases. It has been reported to possess anti-inammatory properties and its sodium salt derivative modulates iNOS and COX-2 (cyclooxygenase-2) gene expression in cultured RAW 264.7 cells. 3  bis(3-ethylbenzthiazoline-6-sulfonic acid)], ROOc (TRAP) and O 2 c (NET) assays have also shown a free radical scavenging effect of curcumin. 4 Antiproliferative properties of curcumin against many human cancer cell lines [SMMC-7721 (hepatoma), MCF-7 (breast), PC-3 (prostate), NCI-H460 (non-small cell lung) and K562 (chronic myeloid leukemia)] have been reported. 4,5 In addition curcumin has been shown to display anti-malarial properties against Plasmodium falciparum. 6 Although an enormous amount of research effort has been invested in bringing curcumin and its analogs towards clinical use, thus far no curcumin based product has been approved for use. Over 120 clinical trials on curcumin have failed and consumed federal funds equivalent to 150 million dollars as stated in NIH reports from the last 25 years. This is in part attributed to the promising in vitro results obtained in preclinical studies, but poor in vivo activities. Thus far curcumin has only been approved as a dietary supplement. 1 The next section highlights some of the important ndings of the biological properties of curcumin reported within the last decades with focus on the promising bio-properties of curcumin and its analogs and the potential in identifying more effective hits/leads.

Curcumin bio-availability
It has been reported that the major clinical limitation of curcumin is due to poor pharmacological properties such as poor pharmacodynamics/pharmacokinetics, low solubility, 7 pH instability, 8 poor absorption in circulation, rapid elimination 9 and photochemical degradation (giving rise to vanillin, ferulic acid, and other small phenols). 10 The bioavailability of curcumin has been intensively studied. In a phase I clinical trial, Sharma et al. reported that the production of the prostaglandin E 2 (PGE 2 ) was (57-62%) decreased in blood samples aer 1 h oral administration in patients (with colorectal cancer). Mild diarrhea and discernible toxicity was detected at doses of 0.5-3.6 g daily administration up to four months. Curcumin and its conjugates were detected in plasma and urine and also noticed in patient feces. The entire study concluded that doses of 3.6 g curcumin are recommended for more systemic pharmacological antitumor studies. Additionally, low oral bioavailability (in both animal and human) is supported probably due to intestinal metabolism. The observed bio-properties of curcumin (anti-inammatory and anticancer) can be attributed to its antioxidant capacity in neutral and acidic pH. [11][12][13] Another study investigated the bioavailability of curcumin in rats (in vivo) utilizing high performance liquid chromatography. Disappearance of curcumin was noticed from the rat's plasma within one hour of dosing (i.v. 40 mg kg −1 ). However, it has   been detected in plasma upon p.o. 400 mg kg −1 administration suggesting that the gastrointestinal tract is more exposed to the unmetabolized curcumin compared to other tissues. 14 Low solubility is one of the major limiting parameters for curcumin's therapeutic use. Different approaches have been considered to overcome this problem and improve bioavailability. Piperine (Fig. 2) has been considered for concomitant administration with curcumin, due to the inhibitory properties in both hepatic and intestinal glucuronidation. Increased serum curcumin was noted upon oral administration (2 g kg −1 ) with piperine (20 mg kg −1 ) in rats, with a signicant reduction in the half time of clearance. Similar observations were noticed in human volunteers with slight differences. Time to reach curcumin maximum serum concentration was shorter (earlier) in humans than rats, presumably due to physiological differences. Also, the elimination (clearance) time was shorter in humans than rats. 15 Another approach considered the mechano-chemical grinding of curcumin with diverse agents (nicotinamide, ferulic acid, hydroquinone, p-hydroxybenzoic acid and L-tartaric acid) (Fig. 3) in different stoichiometry ratios in an attempt to optimize the physico-chemical properties accessible for solid state oral dosage application. Signicant enhancement in solubility and dissolution rates were noticed of the binary eutectics co-crystalline solids. 16 Another study mentioned the possibility of water solubility enhancement through nano-vehicles with curcumin encapsulated in liposomes, exosomes, dendrimers and micelles. 17 Curcumin encapsulation in liposomes modied with DDAB (didecyldimethylammonium bromide) was studied using cervical cancer cell lines (HeLa, SiHa). It was observed that the uptake of DDAB liposomes was better than the non-modied ones but more toxic. Additionally, curcumin was released at a faster rate from cationic DDAB liposomes presumably due to the decrease in interaction of the lipid chains as a result of cationic charges. 18 Sodium salt curcumin diacetate ( Fig. 4) was also mentioned as an improved water soluble bio-active agent. Enhancement was noticed relative to that of curcumin itself in aqueous conditions and ability to protect lipid membranes. However, further detailed studies were recommended to support the accessibility for application. 19 Other studies also adopted bioavailability enhancement of curcumin via combination with cyclodextrin, 20 conjugation with biopolymers 21 or composite nanoparticles. 22

Curcumin chemical structure modification
Many efforts have been directed towards designing novel bioactive agents of enhanced potency and better bioavailability to overcome the drawbacks of curcumin. Manipulation of curcumin chemical structure is usually focused on the aryl rings, carbonyl groups, active methylene or the carbon linker (Fig. 5). Most of the approaches of curcumin chemical structure alterations can be summarized in one of the following approaches. 9 -Modication of the main curcumin skeletal entities.
-Conjugation with other moieties.

Curcumin derivatives with potential biological properties
Curcumin connected to amino acid sodium salts 3 were reported as water-soluble agents. Scheme 1 depicted the synthetic pathway via alkylation of the appropriate aldehyde with chloroacetic acid in the presence of NaOH followed by reaction with glycine ethyl ester hydrochloride. Reaction with 2,4-pentandione followed by hydrolysis with methanolic NaOH afforded the targeted agents 3. The synthesized water-soluble agents displayed enhanced antiproliferative properties (MTT assay) against HeLa (cervical cancer) cells (IC 50 = 0.5 mM for both the synthesized agents) relative to curcumin (IC 50 = 4.33 mM) with induction of p53 activity, p21 expression and mediated apoptosis. The p53 is the tumor suppressor protein capable for induction of genes controlling cell cycle and apoptosis. It usually arrests the cell cycle at G2/M phase/transition affecting cyclin dependent kinase (Cdc2) necessary for mitosis. 23  A series of curcumin-amino acid conjugates 4 were synthesized through reaction of curcumin 2 with the corresponding protected amino acid (benzyloxycarbonyl "Cbz" and uorenylmethyloxycarbonyl "Fmoc") in the presence of EDAC [1ethyl-3-(3-dimethylaminopropyl)carbodiimide] and DMAP (4dimethylaminopyridine) at −5 to 0°C. The unprotected curcumin-amino acid conjugates 5 were obtained from the Boc (ter-butyloxycarbonyl) protected analogs (Scheme 2). Some of the synthesized conjugates revealed anti-inammatory properties (acute carrageenan-induced paw edema in rats) with potency higher than curcumin itself and the standard references used (indomethacin and ibuprofen, clinically used non-steroidal anti-inammatory drugs). These agents also displayed minor or no ulcerations or lesions on the gastric mucosa of the animals tested, supporting the enhanced bio-properties of the synthesized agents. Additionally, enhanced peripheral (acetic acid-induced abdominal writhing methodology) and central (hot plate technique) analgesic properties were also revealed by some of the synthesized conjugates comparable to curcumin, indomethacin and ibuprofen. The anti-inammatory properties observed were correlated with the nitric oxide production by lipopolysaccharide-stimulated peritoneal macrophages. Most of the synthesized conjugates showed high antibacterial properties against S. aureus, S. pyogenes (Grampositive) and S. typhi, P. aeruginosa (Gram-negative) bacteria with potency higher than noroxacin and ciprooxacin (standard reference, antibiotic useable drugs). 24 Dimethoxycurcumin 6 ( Fig. 6) revealed antiproliferative properties with efficacy comparable to that of curcumin 2 capable to arrest the cell cycle at S-phase. Dimethoxycurcumin is about three times more metabolically stable relative to curcumin in mice. The mode of action was mentioned through oxidative stress and mitochondrial dysfunction. 25 Curcumin analogs 7 and 8 synthesized through basecatalyzed (NaOH, EtOH) condensation of the appropriate aldehyde with acetone were found to be potent inhibitors (MTT assay) on a variety of human pancreatic cancers (PANC-1, BXPC-3, MIA-PACA-2, ASPC-1, HPAC and HPDE) and apoptosis inducers relative to curcumin 26 (Scheme 3).
Scheme 7 Synthetic route towards curcumin mimics conjugated chromen-4-one analogs 20.  Most of the synthesized agents exhibited cytostatic properties against human Molt4/C8, CEM (T-lymphocytes) and L1210 (leukemic) cell lines with higher potency than curcumin and melphalan (standard reference, used in chemotherapeutical combination for chronic leukemia and wide range of malignancies). Safety prole was achieved in vitro (WI-38, human broblasts cells) and in vivo (mice) testing. Some of the synthesized agents showed potent inhibitory properties of topoisomerase IIa. This enzyme facilitates DNA replication by preventing the buildup of supercoils during replication fork progression. Inhibitors of this enzyme result in the generation of multiple DNA strand breaks, arrest cell division and eventually lead to apoptosis. As such numerous topoisomerase inhibitors are currently used in clinics as chemotherapy for numerous malignancies. The synthesized agents also revealed antioxidant properties. 36 A series of N-arylsulfonyl-3,5-bis(arylidene)-4-piperidones 25 were synthesized through base-catalyzed arylsulfonation (catalytic amount of pyridine in CH 2 Cl 2 at room temperature) of the corresponding N-unsubstituted piperidones (Scheme 9). The synthesized agents showed anti-inammatory properties supported by the inhibition of IL-6 and TNF-a in RAW264.7 cells induced by lipopolysaccharide (LPS of Gram-negative bacteria). Promising antiproliferation properties were also mentioned by some of the synthesized agents against liver (HepG2, SMMC-7721, QGY-7703) cancer cell lines (MTT assay) with induction of apoptosis. Association of chronic inammation with cancer progression especially, hepatic cancer is the rational for investigation of anti-inammatory and antiproliferation properties of the synthesized agents. 37 Other sets of dissymmetric pyridine-containing 3,5bis(arylidene)-4-piperidones 26-29 were also reported ( Fig. 11) with anti-inammatory and anti-hepatoma properties similar to compounds 25. 38 Pyrido[4,3-d]pyrimidines 30 were synthesized through reaction of the corresponding uoro-containing N-arylsulfonyl-3,5-Scheme 9 Synthetic route towards N-arylsufonyl-3,5-bis(arylidene)-4-piperidones 25.  bis(arylidene)-4-piperidones with guanidine hydrochloride in ethanolic KOH (Scheme 10). Antiproliferative properties were observed by the targeted agents against a variety of hepatocellular carcinoma cells (HepG2, SMMC-7721) using MTT assay. Inhibition of the nuclear translocation of NF-kB induced by TNF-a or LPS supports the anti-inammatory properties of these compounds, considering that NF-kB is the signal pathway connecting the chronic inammation and hepatocellular carcinoma. 39 Potent antiproliferative agents against HCT116 (colon) and A431 (skin/squamous) cancer cell lines were exhibited by 1-(alkylsulfonyl)-3,5-bis(ylidene)-4-piperidinones 31 (Scheme 11) relative to 5-uorouracil (approved drug for colon, breast and skin cancers). Some of the synthesized agents also showed high potency against MCF7 (breast) and A549 (lung) cancer cell lines (relative to 5-uorouracil and doxorubicin) with minimal cytotoxicity against RPE1 (non-cancer, retinal pigment epithelial) cell line. The synthesized agents exerted their mode of action via inhibitory properties of topoisomerase IIa which is the enzyme responsible for breaking double strand DNA helix during DNA replication, transcription and repairing. 40 A set of 4-piperidone-1-carboxamides 32 were synthesized via reaction of isocyanate with the appropriate N-unsubstituted 3,5diylidene-4-piperidine in DMF in the presence of TEA (Scheme 12). Most of the synthesized agents revealed high potency against HCT116 (colon), MCF7 (breast) and A431 (skin/ squamous) cancer cell lines with higher efficacy than that of 5-uorouracil and safe behavior against non-cancer (RPE1) cell line. The synthesized agents revealed topoisomerase II-a inhibitory properties supporting their mode of action. 41

A set of 1-[3-(2-methoxyethyloxy)propionyl]-4-piperidones 33
and their thio-analogs 34 were synthesized through reaction of the acyl chloride with the appropriate 3,5-bis(ylidene)-4piperidone in CH 2 Cl 2 containing triethylamine. The sulnyl-35 and sulfonyl-36 derivatives were obtained through oxidation of compounds 34 with peracetic acid and 3-chloroperoxybenzoic acid in CH 2 Cl 2 , respectively (Scheme 13). Antiproliferative properties were observed by the synthesized agents against human Molt 4/C8, CEM (T-lymphocyte) and L1210 (murine leukemia) cell lines with safer behavior towards nonmalignant cells. Some of the synthesized agents demonstrated PARP1 [poly(ADP-ribose)polymerase 1] cleavage, a characteristic hallmark of apoptosis. PARP1 is capable of repairing DNA single-stranded breaks, thus compounds that induce PARP1 cleavage can prevent DNA replication and are useful in cancer chemotherapy. 42 The piperidone-salicylate conjugates 37 were synthesized through dehydrohalogentation of acetylsalicylic acid chloride with the appropriate piperidone in DMF containing TEA as basic catalyst (Scheme 14). Potent antiproliferation properties were noticed by the synthesized conjugates against A431 (squamous skin), HCT116 (colon) and MCF7 (breast) cancer cell lines (MTT assay) with comparable efficacies to that of 5-uorouracil and sunitinib (standard references). Multi-targeted inhibitory properties were observed against VEGFR-2 (vascular endothelial growth factor receptor-2) and EGFR (epidermal growth factor receptor) in both MCF7 (breast) and HCT116 (colon) cancer cells. Enhanced COX-1 and COX-2 (cyclooxygenase-1 and -2) inhibitory properties were also revealed by the synthesized agents than that of aspirin supporting their anti-inammatory properties. Selective inhibition was noticed towards COX-2 compared to COX-1. Additionally, some of the synthesized agents revealed antiviral properties against SARS-CoV-2 (respiratory syndrome coronavirus 2) which is the responsible infectious microorganism of COVID-19 (corona virus disease 2019) pandemic. 43 Scheme 14 Synthetic route towards piperidone-salicylate conjugates 37.

Cholinesterase inhibitors
Alzheimer's disease is a neurodegenerative disease comprising the main cause of dementia particular in elder people. One of the most common methods for treatment is the elevation of acetylcholine levels in the brain. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are two enzymes that exist in the central nervous system capable of hydrolyzing acetylcholine (neurotransmitter at cholinergic synapses). For this reason compounds with inhibitory properties against these enzymes are valuable for controlling progress of this disease. A series of 3,5-bis(ylidene)-4-piperidones 43 (Fig. 13) showed promising inhibitory properties against AChE and BChE (Ellman's method) compared to Tacrine and Donepezil (standard references). 51,52 Pyrido  . Promising AChE and BChE inhibitory properties were observed by some of the synthesized agents (Ellman's method) compared with Galantamine (reference standard). 56 Cycloaddition reaction of azomethine ylide (formed from condensation of 5-chloroisatin and L-proline) with N-acryloyl-3,5-bis(ylidene)-4-piperidones 23 afforded the corresponding spiro-heterocycles 48 via reaction with the acryloyl linkage rather than the exocyclic ylidene olenic linkage (Scheme 21). Stereochemical structure of 48 was supported by single crystal X-ray studies. AChE and BChE properties were exhibited by the synthesized agents in comparison with Galantamine (standard reference). 57 Additionally, azomethine cycloaddition reaction (formed from isatin and L-proline) with N-acryloyl-3,5-bis(ylidene)-4piperidones 23 in reuxing MeOH in equimolar values afforded the mono-spiro-heterocycles 49 in a similar manner to that of the aforementioned formation of 48. However, reaction of azomethine ylide in double folds amount (two molar equivalents) to that of piperidones 23, the bis-spiro-heterocycles 50 were obtained due to double cycloaddition reactions with both acryloyl and ylidene linkages (Scheme 22). Both compounds 49 and 50 revealed AChE and BChE inhibitory properties (Ellman's method) and some of them showed potency comparable to that of Galantamine. 58 Meanwhile, ionic liquid "[bmim]Br" mediated cycloaddition reaction of azomethine ylide derived from isatin and sarcosine with N-acryloyl-3,5-bis(ylidene)-4-piperidones 23 afforded the mono-spiro-pyrrolidines 51 due to cycloaddition reaction with the exocyclic ylidene linkage. However, reaction of the azomethine ylide with 23 in 2 : 1 molar value equivalent afforded the bis-spiro-pyrrolidines 52. The difference in observations of this reaction to that mentioned in Scheme 22 is attributed to the different reactant azomethine ylide derived from diverse amino acid (sarcosine and L-proline). Additionally, bis-spiro-pyrrolidines 52 were obtained from mono-spiro-pyrrolidines 51 by reaction with another mol equivalent of the azomethine ylide (Scheme 23). Both mono-spiro-51 and bis-spiro-pyrrolidines 52 revealed noticeable AChE and BChE inhibitory properties and some of them exhibited potency comparable to that of Galantamine. 59 Similarly, reaction of N-acryloyl-3,5-bis(ylidene)-4piperidones 23 with azomethine ylide generated from phenylglycine and isatin (in 1 : 1 molar equivalent) in ionic liquid "[bmim]Br" medium afforded the mono-spiro-pyrrolidines 53 due to cycloaddition reaction with the exocyclic ylidene linkage.
However, reaction of the azomethine ylide with 23 in 2 : 1 molar value equivalent afforded the bis-spiro-pyrrolidines 54. Reaction of 53 with another mol equivalent of azomethine ylide also afforded the bis-spiro-pyrrolidines 54 (Scheme 24). AChE and BChE inhibitory properties were shown by the mono-spiro-53 and bis-spiro-pyrrolidines 54 and some of them exhibited potency comparable to that of Galantamine.

Antimycobacterial active agents
Infectious diseases are still one of the main human global health problems. Tuberculosis (TB) is one of the top ten human life threatening globally. It is the second cause of mortality aer HIV/AIDS due to single infectious pathogen. Various pathogenic agents (Mycobacterium sp.) have been identied causing TB. Many drugs have been discovered for treating patients with TB but due to the side effects and drug resistant TB strains, novel therapeutical agents are still needed. 66 A series of 3,5-bis(ylidene)-4-piperidones 63 were identied with noticeable antimycobacterial properties (M. tuberculosis H 37 Rv). Some of them revealed properties in the rat liver mitochondria respiration with swelling in mitochondria 67 (Fig. 15).

Antimalarial active agents
Malaria is one of the most widely distributed infectious diseases in tropical and subtropical regions. Plasmodium sp. which is a protozoan organism transmitted to humans due to mosquito bites. Although many drugs are known to combat malaria emerging drug resistance means that new effective agents are still needed. 70 Anti-plasmodial properties were exhibited by 3,5-bis(ylidene)-N-methyl-4-piperidones 69 against chloroquine-sensitive Pf3D7, chloroquine-resistant PfINDO, and artemisininresistant PfMRA-1240 strains 71 (Fig. 16).

Conclusion
Curcumin is an important natural compound with broad spectrum biological properties. Limitation of clinical application of curcumin is mainly due to its poor bio-availability in vivo. 3,5-Bis(ylidene)-4-piperidone scaffolds are considered a curcumin mimic with diverse promising bio-properties. The distinguished biological observations of curcumin mimics can be considered for optimizing high potent hits/leads accessible in drug discovery program. Many articles dealing with the biological properties of this scaffold have appeared. It has been also intensively utilized for construction of diverse potentially bioactive heterocycles.

Conflicts of interest
There is no conict to declare.