Flavocillin is a new beta lactam derivative of Penicillin where the R substituent is modified by a flavone group. Flavocillin ammonium salt was synthesized and tested in vitro on certain Gram + and negative bacteria for the purpose of this study. The activity of the flavocillin ammonium salt standard substance against S. aureus ATCC 6538 (1 µg/mL), S. epidermidis ATCC 12228 (8 µg/mL), M. catarrhalis ATCC 23245 (0,5 µg/mL), S. Mutans ATCC 25175 (0,5 µg/mL) and C. stiratum ATCC BAA 1293 (1 µg/mL) was determined. Interestingly the tested E. coli ATCC 8439 and S. aureus BAA44 were resistant to flavocillin. The results evidenced that Flavocillin mainly targeted the types of bacteria that infect the lungs and cause pneumonia, bronchitis and sepsis.

Keywords: penicillin, flavonoid, flavone, drug, discovery, development, S. aureus, MRSA, ınfection, ınfectious, disease

Mix 40mg of Flavone in 50 mL of Ethanol then dry and wash it twice with Diethyl Ether and distilled water for complete purification. Prepare an Ice Bath reaction and Dissolve the purified Flavone substance in 150mL of DCC/DMAP (250:250) in an aqeous solution in that Ice Bath by using a magnetic stirrer for 160 minutes. Remove and dissolve with Ethanol and wash with Diethyl Ether by using Distilled water for complete Flavone purification. Once purified, dissolve 40mg of purified Flavone in 150mL of aqeous Ethanol and then separate purified Flavone from Ethanol by using a Reflux reaction with the involvement of evaporation with Methanol, apply TLC once in every one hour until 4 hours of reflux process is complete. Remove, evaporate the solution by using a Rotaror at 40 ⁰ C low temperature. Remove the round flask pour the flavone inside 250 mL Beaker, and dry in 40 ⁰ C oven for 3 days. Remove it on the fourth day, and dissolve it in the initially prepared and preserved stock solution of 250 mL DCC to 250 mL DMAP mixture. Slowly add 40mg of 6-APA (6-Amino Penicillanic Acid) while mixing and spreading the powder inside the solution.

Mix for 4 hours at a speed of 200 Hz speed by using a mixing device. Remove and by using a separating funnel after complete separation of the 80 mg of produced Flavocillin (Figure 1) by crystallization with Ethanol by heating at 100 ⁰ C for 40 minutes (Take precaution by covering covering the beaker by using a wide 500 mL normal flask during this experimental procedure, Isolate Flavocillin by using rotaror evaporation at 100 ⁰ C for 130 minutes.

Figure 1 Chemical structure of flavocillin.

Once durationis over, remove and place it into a 500 mL Beaker, let it dry for half an hour and twice wash with Diethyl ether and distilled water, re-crystallize from Methanol by rapid heating with 100 mL of Ethanol for 40 minutes at 40 ⁰ C at while mixing by using a magnetic stirrer and the same amount of Methanol should have been prepared previously to be cooled at 20 ⁰ C for 40 minutes.

By using a rotator, remove Ethanol at 40 ⁰ C evaroparation for 60 minutes. Remove the powder, put it in a test tube and place 80 mg of column chromatography free purified Flavocillininto 8 different eppendorfs in 10 milligrams. Pack each 10 milligrams with special sheets be well covered and sealed in containers labelled as Flavocillin (10 milligrams).

Resistance to antibiotics and Penicillins in general is the growing problem of the Century and it is defined by World Health Organization (WHO) as a “global health threat’’ (Antimicrobial Resistance, WHO, 17 November, 2021).¹ Antibiotic resistance happens when the bacteria survive and continue to multiply which results in more harm to the patient suffering from the infection. Bacteria can do this through several mechanisms. Resistance to antibiotics may occur through decreased uptake, where the bacterial cell wall does not allow the antibiotic to penetrate the cell wall and as a result, the antibiotic can not enter into the cell to show its effects. The resistance inside the bacterial cell can also happen through target alteration mechanisms where the genetic code of the bacterial DNA changes in response to antibiotic and the antibiotic can not be effective enough. There is also alternative enzyme mechanism where instead a certain bacterial enzyme A is converted into enzyme B by using a certain catalyst, a different catalyst is used for this conversion to mistaken the antibiotic trying to target bacterial enzymes. In other words, the bacteria camouflages the target and as a result the antibiotic becomes ineffective. There are also inactivating enzymes which inactivates the antibiotic by destroying or separating the structure into compartments, and that results in structure inactivity because the drug is split in structure. Finally, there are efflux pumps which pump the antibiotic to outside of the cell after its entry into the bacterial cell.²

Because of these antibiotic resistance mechanisms, the development of new antibiotics are needed where it would be more difficult for the bacteria to adapt and resist to a new antibiotic structure, either because of the main structure of the derived antibiotic or due to the originality and effectiveness of its functional group.

Because Flavonoids are known as antimicrobial compounds which makes the immune system stronger when taken into the body, it was initially considered that a Flavonoid can possibly be attached to 6-APA (6-Aminopenicillanic acid) compound, which is found in the common structure of Penicillins.³ Both the compounds 6-APA and Flavonoid have antimicrobial activities. The purpose was to create a strong antibiotic by combining them. Beginning from certain antimicrobial Chalcones, the synthesis of Flavocillin was made as a result of 3 months work and the exact methodologies of Chemical synthesis were derived. Flavocillin is indexed in the United States National Library of Medicine as a new drug molecule.⁴

Synthesis of flavocillin

Initially, flavocillin acid free form were synthesized by using the following chemical synthesis methods (Figure 2):

Figure 2 Synthetic route for the synthesis of flavocillin.

Synthesis of flavocillin

Synthesis of (E)-4-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl) Benzoic Acid

Ethanolic NaOH, room temperature

2-Hydroxy Acetaphenone (MW= 136.144 g/mol, d= 1.133 g/mL) and 4-Formyl Benzoic Acid (MW=150.13 g/mol) (1:1) were reacted by using the amount of 2-Hydroxy Acetaphenone as 0.5 grams to obtain the reaction products in sufficient amounts. Based on this, the required volume of 2-HA and the required weight of 4-FBA was calculated as follows, for the reactants to be in 1:1 ratio:

2-HA:

Moles (n) = m/MW = 0,5 / 136,144 =0,0037 mol

Density (d) = mass (m) / volume (V)

Therefore;

Volume (V) = mass(m) / density (d) = 0,5 / 1,133 = 0.441 mL required

4-FBA:

Reaction is 1:1 so 0,0037 mol 2-HA is required to react with 0,0037 mol 4-FBA.

Moles = Mass/Molecular Weight

Therefore;

Mass = Moles*Molecular Weight = 0.0037 * 150,13 = 0,5555 g required

9,8 grams of KOH was dissolved in 100 mL of Ethanol. 100 mL Ethanol is required for 30 mmol. From here, the volume of Ethanol required for 3,7 mmol was calculated as follows:

30 mmol requires 100 mL Ethanol

3,7 mmol requires ? mL Ethanol

From here, for 3,7 mmol, the calculated amount of Ethanol to be used was 12,33 mL.

The reactants were weighed and dissolved in 12,33 mL Ethanol and at 50⁰ C the reaction was carried out for 2 hours.

After the reaction, the formed reaction product was treated with diluted Sulphuric Acid and there was huge amount of precipitation even with a few drops of the acid. (5mL of Sulphuric acid were diluted with 5mL of distilled water-of course, caution were taken, for the purpose of lab safety and for the correct procedure of mixing solutions, the acid were slowly added into water, and not water into acid).

The solution was then filtered to obtain the powder and the powder was allowed to dry. 1.98 grams of product was obtained. A very small sample was taken from the powder and an attempt to dissolve it in water was made and it was not soluble in water which indicated the product was not NaSO₄ but it was likely the Chalcon compound. Melting point was checked and it was in the range of 251.2-252.8 which is the same as the melting point in that of the literature for (E)-4-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl) Benzoic Acid. The compound was given the code M1.

Synthesis of 3-(4-oxo-4H-Benzopyran-2-yl) Benzoic Acid (M2) from (E)-4-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl) Benzoic Acid (M1)

The obtained compound was mixed with Iodine by following exactly the same procedure defined in the previously made synthesis procedures in the literature (Imran, 2015- Synthesis of Novel Flavone Hydrazones: In vitro Evaluation of Alpha-Glucosidase Inhibition, QSAR analysis and Docking Studies-European Journal of Medicinal Chemistry 105 (2015), 156-170, section 4.3)⁵ as follows:

“Compound 1 (22.4 mmol) was mixed with Iodine (0.23 mmol). The mixture was dissolved in 50 mL of DMSO and refluxed at 170⁰ C. After 3 hours, sodium thiosulfate was added to the reaction mixture followed by excess amount of water to allow precipitation. The product was rinsed and allowed to dry at room temperature to afford pure product.’’

Light yellow solid was obtained as stated in this literature. The compound was given another code, M2. There was crystallization that occured in solution part of M2 and this was also filtrated and the obtained powder was given the code M3.

In addition, a very small sample of the obtained compounds were dissolved in DMSO for the purpose of being followed by TLC towards the end of reaction and it was understood that reaction was complete and reaction products were obtained (Solvent system used was Ethyl Acetate: Hexane – 7:3).

Reactions were repeated in higher amounts.

Reactions to obtain flavones

0,4 grams of the previously obtained chalcone was reacted with 0,15 grams of Iodine in 20 mL DMSO where the reaction was refluxed at 170⁰ C for 3 hours. At the end of the reaction, again 0,240 grams (roughly one mole equivalent) of Sodium Thiosulfate was added into the obtained solution and then the solution was treated with excess distilled water to allow precipitation. Flavones that were obtained were kept at vacuum oven in bottles at 40⁰ C for 2 days for drying.

Reactions to obtain flavocillin

0,230 grams of Flavone was mixed with 0,188 grams of 6-APA inside a round bottom flask and they were dissolved in 13 mL of Acetonitrile. Volume equivalent to 0,05 grams of Triethylamine was added, DMAP at the tip of a spatula was added and 0,213 grams of DCC was added. By using a magnet and stirrer, the reaction was mixed inside an ice bath (at around 4 ⁰C) for 24 hours. (For the purpose of continuing with stirring at the times we were not present in lab, a thick and closed carton with foam was taken and the beaker containing the round bottom reaction flask when covered by sufficient amount of ice was placed inside the first half of this carton box and inside the carton was balanced and the stirring was allowed to continue during the whole night).

Following ice bath reaction, the round bottom reaction flask was placed inside an ice bath with Acetone (for the reaction to be at – (minus) 20⁰ C)for 30 minutes, the solution was then filtered. The filtrate was evaporatedby using the rotaror. The solid remaining from this evaporation was extracted with Ethyl Acetate and then washed with icy water, and then with 0,05 moles of KHSO4 (Potasium Bisulphate), then with saturated 2% NaHCO₃ (2 grams of Sodium bicarbonate in 100 mL distilled water) solution and finally it was dried on Sodium Thiosulfate (Rute Madeira Lau et. al., Synthesis of Penicillin N and Isopenicillin N, Tetrahedron 56 (2000) 7601-7606)⁶

(The amount of potasium bisulfate: 0,680 grams of Potasium Bisulphate were dissolved in 100 mL of solution.

The required amount of Potasium Bisulphate were calculated as follows):

MW of Potasium Bisulphate = 136,169 g /mol

n (mol) = mass (m) / Molecular Weight (MW)

0,05 = m / 136,169

m = 0,05 * 136,169 = 6,80 grams required for 1 Liters (1000 mL)

6,80 g is required for 1000 mL

? g is required for 100 mL

(100*6,80)/1000 = 0, 680 g required)

The obtained brown powder considered to be Flavocillin, which was inside the 250 mL round bottom flask, which was made by 24-hour icy water reaction (Tetrahedron procedure) and extracted with Ethyl acetate, was treated with some acetone and then it was evaporated by using the rotaror and when solid remained, the flask was removed and the honey like semi viscous hard substance inside the round bottom flask was given the code FLC-2.

NMR results confirmed FLC-2 contained Flavocillin compound but it was not so pure. For that reason, it was decided to purify FLC-2 to obtain the target compound in more pure form by using column chromatography. Roughly 120-150 mg of the compound was taken and the column chromatography was made.

Column chromatography for FLC-2 was made according to the well known methods (mixing the appropriate solvent Ethyl Acetate Hexane 6:3 with silica and using a cotton etc.). 46 tubes were used to get all the liquid and the following TLC results, where each of the numbered tube contents on chromatogram appeared as single leision, showed that test tubes 19-27 contained the target substance Flavocillin.

For that reason, these tubes were combined into a round bottom reaction flask, the liquid content was then evaporated and the remaining 80 mg solid was obtained. Because this amount was low enough to be taken into an eppendorf for NMR analysis, a very thiny amount was collected and then the remaining substance was taken by addition of 1 mL Acetone inside the round bottom flask and it was collected by using a pasteur pipette and added into the eppendorf. In addition, all the liquid inside the test tubes 28-46 were also mixed inside another round bottom reaction flask, and then the contents of this second flask was evaporated by using the rotaror until 1 mL remained. When 1 mL solution remained, the flask was removed from the rotaror and the 1 mL yellow liquid that remained was taken into another eppendorf.

The lids of both eppendorfs were left open as shown below so that acetone inside the first eppendorf containing purified Flavocillin evaporates and solid remains, and so that the yellow liquid inside the second eppendorf dries as much as possible (because Ethyl acetate and hexane were expected to evaporate). Next day, these samples were placed inside vacuum oven to be dried at 40 ⁰ C for two days when their lids were open but still covered with paraffin sheets, where dot sized holes were opened on paraffin sheets covering the lids of eppendorfs. Then they were removed and were sent to H-NMR analysis.

In addition, the rest of FLC-2 compound was also purified with column chromatography (solvent system : Ethyl Acetate Hexane 600:300). TLC results showed that out of 58 test tubes, test tubes numbered 22-33 contained the target compound. The solutions contained by test tubes 22-33, 19-21 and 15-18 were separately combined inside 3 round bottom reaction flasks. The flask that contained 22-33 numbered solutions were evaporated until only yellow solid powders remained, and the others (flasks containing 19-21 and 15-18) were evaporated until 1 mL yellow liquid remained. Then, the remaining 1 mL of the of 19-21 and 15-18 were separately poured inside two separate eppendorfs, and the lids of eppendorfs were once again left for solvent to evaporate and solid to remain. Also, a little amount of sample from the 22-33 flask were taken and were put inside another eppendorf.

All three samples were waited inside vacuum oven at 40⁰ C for two days, same as the previously made procedure.

NMR results for the purified compound (it was coded as FLC-MP) confirmed that all the peaks that the target compound Flavocillin should have contained were there and therefore it was decided to do the activity testing for the purified compound. In addition, IR data for the compound Flavocillin was obtained.

Because the obtained flavocillin powder was not pure as indicated by TLC results for the sample, the product was purified by column chromatography and then another TLC was taken for the finalized product, which confirmed it was more pure after column chromatography.

Antibacterial testing for flavocillin

In order to find out whether the microorganisms are resistant (R) or susceptible (S) to antibiotics, inhibition zones around the discs at the end of the incubation period are measured with a ruler and the microorganism was evaluated as (S) or resistant (R) by comparing with the EUCAST Clinical Limit Chart. The Limit Value is different for the microorganism and each antibiotic. It is even updated and changed periodically.

For Enterobacteriaceae family (bacterial family) and ampicillin, the zone diameter is set as> 14 for S and <14 for R. In other words, if the zone diameter is greater than 14, it is considered bacteria sensitive (S), if it is small, it is considered bacteria resistant (R). The sensitivity of the microorganism means that the antibiotic will be effective against the microorganism. The resistance of the microorganism means that the antibiotic will not affect the microorganism.

For the studied flavocillin compound; for all the microorganisms it exceeded the Breakpoint in the bacteria tested. In other words, the microorganism is sensitive to this dose, the antibiotic will be effective at this dose.

While the Limit Value for E. hirae was 10 mm, the compound made a 12mm zone diameter.

While the Limit Value for E. faecalis was 10 mm, the compound made 14 mm.

While S. aureus Breakpoint was 18 mm, the compound made 24 mm.

While the Limit Value for E. coli was 14 mm, the compound made a zone diameter of 17 mm. (Table 1)

There has been purification problems during experiments and the compound Flavocillin was synthesized in low purities and in low yields.

Disk diffusion method

Preparation of the medium: Mueller-Hinton Agar information for Enterococcus hirae ATCC 10541, Enterococcus faecalis ATCC 29212, Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 11229). Plates were stored at 4-8 ° C under laboratory conditions, with a thickness of 4 mm ± 0.5 mm (25 mL in a 90 mm circular plate).

Preparation of the inoculum: Method of Direct Colony Suspension, sterile saline value of the organism was prepared 0.5 McFarland turbidity standard concentrated suspension. (Approximately 1-2 x 108 CFU / mL for Escherichia coli). The suspension was prepared from fresh colonies grown after an overnight incubation on a non-selective medium (sheep blood Agar) with sterile loop and sterile saline personal. The inoculum suspension was standardized to the Densitometer signal 0.5 McFarland standard density. The turbidity standard was mixed before use and mixed for 15 minutes after preparation.

Cultivation of agar plates: Agar plates were kept at room temperature before inoculation. The sterile cotton-tipped swab was dipped into the suspension and the swab was rotated on the inner wall of the tube to remove excess fluid. The inoculum was spread over the surface of the plate such that the automatic plate rotator was evenly distributed over the entire agar surface.

Placing antimicrobial discs: Flavocillin was prepared as 100 µg / disc and absorbed into 6 mm diameter Blank Discs. Ampicillin (Oxoid-10 µg) standard discs as Positive Control. DMSO and H2O assist as Negative Control. The discs kept to room temperature were placed on the seeded and dried agar plate (15 minutes after sowing) in the contact area of ​​the agar and uniformly.

Placing antimicrobial discs: Flavocillin was prepared as 100 µg / disc and absorbed into 6 mm diameter Blank Discs. Ampicillin (Oxoid-10 µg) standard discs as Positive Control. DMSO and H2O assist as Negative Control. The discs kept to room temperature were placed on the seeded and dried agar plate (15 minutes after sowing) in the contact area of ​​the agar and evenly.

Incubation of plates: The incubation was started after the discs were inside for 15 minutes, after the plates were inverted and it was made sure that the discs did not fall off the agar surface. Plates containing Enterococcus hirae (ATCC 10541), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 11229) were incubated at 35 ± 1 ° C, normal atmosphere, 18-20 incubations.

Measuring zones and ınterpreting sensitivity: The zone of inhibition was assessed as the point at which growth was completely inhibited when viewed with the naked eye, held 30 cm from the eye. The diameter of the inhibition zone was measured with a ruler. Zone diameters were evaluated according to the EUCAST (European Committee for Antimicrobial Susceptibility Tests - European Antimicrobial Susceptibility Testing) Breakpoint Table. Zone Diameters for Flavocillin; Enterococcus hirae (12 mm), Enterococcus faecalis (14 mm), Staphylococcus aureus (24 mm), Escherichia coli (17 mm) were measured.

Quality control: Testing control strains specified on the EUCAST (European Committee for Antimicrobial Susceptibility Testing - European Committee for Antimicrobial Susceptibility Testing) to monitor the test.^7,8

Testing of 10 milligrams of Flavocillin provided the results that Flavocillin passed the resistance limits in all types of bacteria it was tested on and it was active in the bacteria Enterecoccus Hirae (bacteria causing Urinary Tract Infections, UTI, hepatobiliary infection, endocarditis, surgical wound infection, bacteremia, and neonatal sepsis), Enterococcus faecalis (bacteria causing endocarditis, urinary tract infections, prostatitis, intra-abdominal infection, cellulitis, and wound infection), Staphylococcus aureus (bacteria causing skin infections and also life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, and sepsis) and also Eschericia coli (bacteria causing cholecystitis, bacteremia, cholangitis, urinary tract infection (UTI), traveler's diarrhea and neonatal meningitis ).⁹

The very initial hypothesis about Flavocillin synthesis was that due to the combination of the weak antibiotic acitivty of 6-APA as a Penicillin nucleus with Flavonoid as an antimicrobial and immune supporting substance would create a strong antibiotic which could work as a generalized Chemical substance against multiple types of heavy and resistant infectious diseases, and there have been certain studies where drug resistant Staphylococcus Aureus was not cured with Penicillin alone but it was cured with the combination of Penicillin and flavonoids together. For the Patent, namely “Flavocillin: A New Penicillin Derivative’’¹⁰ (Pehlivan M., EP3752509, December 2020), search reports were provided by patent examiners who cited the study in relation to this statement. (ANA CRISTINA ABREU ET AL, "Combinatorial Activity of Flavonoids with Antibiotics Against Drug-Resistant Staphylococcus aureus", MICROBIAL DRUG RESISTANCE., US, (20151201), vol. 21, no. 6, doi:10.1089/mdr.2014.0252, ISSN 1076-6294, pages 600 - 609, XP055474797 [A] 1-4 * the whole document.¹¹

Synthesis of flavocillin ammonium salt

At the final step of Flavocillin re-synthesis, an additional synthetic pathway to the described method for the initially determined Flavocillin synthesis was developed for the last step because the final product was not stable under acidic conditions. Shift was noticed to some other compound with precisely the same molecular mass after C18 chromatography with 0.1% HCl. However, this was avoided by purification on the C18 column with 0.1% ammonium acetate as a buffer and over 97% (detected at 210 nm) as the impurity was mainly this shifted side product with the same mass. This way, via the modified synthesis method, Flavocillin ammonium salt was obtained in 97% purity prior to the defined in vitro tests.

Antibacterial testing for flavocillin ammonium salt

Summary for the in vitro Bioactivity results for Flavocillin ammonium salt is expressed in table 2. The results showed that Flavocillin is more potent and had higher activity when formulated as ammonium salt. This was probably because the solubility of Flavocillin increased in ammonium salt form. According to the results at Badebio lab of Eskişehir Anadolu University of Turkey, the activity of Flavocillin ammonium salt were compared to the activity of other combination of antibiotics such as Amoxicillin, Ampicillin and Amoxicllin plus Clavulanic acid. The results showed that Flavocillin ammonium salt had MIC values twice better than those MIC values of Ampicillin on S. Mutans and S. Aureus. In addition, Ampicillin was not active on C. Stiratum at all whereas Flavocillin ammonium salt had the highest activity with an MIC value of 1. Neither of the tested antibiotics were effective on S. Aureus BAA44. The activity of Flavocillin ammonium salt were equal to that of Ampicillin on M. Catarrhalis and higher than the rest of the tested antibiotics (Table 2).

Bacterial cultures stored at -85⁰ C and -20⁰ C were incubated for 24/48 hours at 37 ⁰ C using Mueller Hinton Agar (MHA) and Brain Heart Infusion (BHI) agar for resuscitation. Escheria coli ATCC 8439, Staphylococcus aureus ATCC 6538, S. aureus BAA44(MRSA), Staphylococcus epidermidis ATCC 12228, Moraxella catarrhalis ATCC 23245, Streptococcus mutans ATCC 25175 and Corynebacterium S tiratum ATCC BAA 1293 were incubated in aerobic media. Culture densities of microorganisms were adjusted acoording to Mcfarland No: 0.5 standard with turbidimeter.

Microtitration Petri dishes with 96 U type wells were used in the study. 100 µL of the pre-diluted test sample was transferred to microliter plates and serial dilutions were prepared at 32-0.25 µg/mL for Flavocillin ammonium salt. Ampicillin, amoxicillin and amoxicillin-clavulanic acid were used as positive controls for bacteria in the concentration range of 32-0.25 µg/mL. Density adjustment is mad efor each well and 100 µL of 1:100 diluted bacterial solution was added, and then incubated at 37⁰ C for 24 hours.

At the end of the incubation period, 20 µL of 0.01% (w/v) resazurin solution was added to the wells for diagnosis and incubated for 3 more hours at 37 ⁰ C. In the result Table 2, the colorless wells at the end of the incubation period are given as the minimum inhibition concentrations (MIC) mg/mL with no growth. All experiments were performed in 3 replicates at different times.

The activity of the flavocillin ammonium salt standard substance against S. aureus ATCC 6538 (1 µg/mL), S. epidermidis ATCC 12228 (8 µg/mL), M. catarrhalis ATCC 23245 (0,5 µg/mL), S. mutans ATCC 25175 (0,5 µg/mL) and C. stiratum ATCC BAA 1293 (1 µg/mL)was determined. No effect ahas been determined gainst E. coli ATCC 8439 and S. aureus BAA44.

In Vitro Biological activity tests confirmed that Flavocillin ammonium salt was more potent and had higher MIC values when compared to that of Flavocillin alone. Flavocillin specifically targeted certain lung infecting antibiotic resistant pathogenic bacteria with high MIC scores which prooved good activities. However, same results also showed that Flavocillin and Flavocillin ammonium salt did not have any anti fungal activity. The activity of Flavocillin ammonium salt when compared to the combinations of other antibiotics were highest on C. stiratum, a bacteria which penetrates human skin and infects the lungs resulting in bronchitis and sepsis. Flavocillin specifically targeted the bacteria that mainly infects the lungs and the activity was very good when compared to other few Penicillins. While Flavocillin is 100% synthetic in origin, both 6−APA and flavone groups of the Flavocillin compound are natural in origin. A combination of the two were made by Chemical synthesis. The naturality of the flavone group of Flavocillin is expected to result in less side effects when compared to many other Penicillins. This will probably one of the main reasons for Flavocillin antibiotics (Figure 3) to be successful new drug candidates following clinical development.

Figure 3 Flavocillin antibiotics.

Concluding remarks to improve the efficiencies of experimental procedures to obtain the best yields based on the analytical method validation: KOH instead of NaOH, and standardized solvent system 7 to 3 Ethyl acetate: Hexane ratio, 99.7% pure Methanol instead of 96% in certain cases were used as our standards in many trials, after we learnt based on our observations from TLC and solubility of compounds that these should have been the standards.

Literature for Chalcon preparation played very important roles in determination of our way for obtaining compound 1 and adjust our standard values to be our standard values.

The experiments are recently continuing by the application of these standards in many situations and by repeating some experiments where appropriate, where we are deriving and substituting many variables with others, such as using 4-Formyl Benzoic Acid instead of 3-Formyl Benzoic Acid, and vice versa, depending on the experiment type so that it fits for purpose.

It was observed that both 3-FBA and 4-FBA can be used to close the ring for obtaining the target compounds, but experiments wth 3-FBA proceeded much better because more precipitation and crystallization occured with 3-FBA.

Mechanism of action as hypothesized based on the collected data and ın silico computational drug target prediction studies: Recent studies on Flavocillin, a new Penicillin derivative that has activity against S. Aureus and the types of bacteria that infect the lungs and cause Pneumonia have outlined various targets In vitro and In silico for Flavocillin. In this article, a possible in detail mechanism of action of Flavocillin is outlined based on the combination of all the Biological data to date is being provided in relation to and with the comparisions of these data with studies about Penicillins, Flavones and Flavonoids in Scientific literature. The softwares and online resources such as PASS, Pharmaexpert and way2drug were used to aid and support with obtaining additional datas for the determination of a mechanism of action for Flavocillin compound. It is expected that this study will also be useful for other early stage drug development projects or proposals for researchers who would like to use similar pathways for proividng stronger and useful levels of evidence and in detail Pharmacological propoerties of their original drug compounds.

Chromone derivatives prevent allergic symptoms of asthma and bronchoconstriction by preventing the release of inflammatory mediators such as histamine and leukotrienes.¹² Flavonoids are compounds present in various natural sources such as plants and herbal teas and certain Flavonoids have anti-inflammatory and anti-oxidant Pharmacological activities¹³ whereas other Flavonoid derivatives show anti-cancer, anti-viral, neuro-protective and cardio-protective properties.¹⁴

Flavocillin, a recent Penicillin derivative which was first protected by PCT patent application (2018) carries 6-Amino Penicillanic Acid (6-APA) which acts as main core structure of Penicillin and a flavone group attached to variable R side chain. Flavocillin showed Pharmacological activities agains a wide range of Staphylococcus Aureus species which cause infection of the lung and Pneumonia¹⁵ and also against certain strain of MRSA, Streptococcus Mutans, Moraxella Catarrhalis and Corynebacterium Striatum,¹⁵ but most importantly and interestingly, Flavocillin has suppressing effects on MCF-7 and therefore it would be useful in future following drug development for it to suppress the growth of human breast tumours.¹⁶

Recently, via the improvement of computational drug modelling softwares, it is becoming more possible to see the possible effects, Pharmacological properties and solubility characteristics of new drug candidates to further support drug like properties and drug safety. In this study, we are reporting and comparing the In Silico results of Flavocillin on acute rat toxicity with the real laboratory results of Ampicillin on acute rat toxicity. We are also providing the In Silico results of what other bacterial targets Flavocillin would be effective on and in what ranges. We have used PASS and way2drug for our modelling and results for Flavocillin. Earlier computation modellings had provided that Flavocillin might be a useful and strong new drug candidate additionally against Malariabecause of its inhibitory actions on Trxr and OATP. In addition, computational results of the same study provided the result that Flavocillin drug safety profile is high because toxic effects of Flavocillin acid free form would begin at very high doses, around 5,6 grams per kilogram (LD50).¹⁷

Materials and methods: Flavocillin was mostly compared with Ampicillin during In vitro Biological activity tests which confirmed its activities. According to the mice and rat toxicity experiments of Ampicillin, it was found that “Ampicillin is non-toxic to mice and rats when administered either orally or subcutaneously in doses of 5 g/kg. As regards intravenous dosage for mice, 2 g/kg has been administered without lethal effects, although muscle tremors, slowed respiration and mild clonic con- vulsions have sometimes occurred.’’¹⁸

For the analysis of additional targets and comparision with Ampicillin rat toxicity data, Chemical SMILES ID of Flavocillin was generated based on its Chemical structure

(O=c(cc(o1)c(cc(c2)C(=O)N[C@H](C3=O)[C@@H](SC4(C)C)N3C4C(O)=O)cc2)c(ccc2)c1c2)

and this SMILES ID was typed and searched for potential targets and possible acute rat toxicity.^19–21

Antibacterial targets for Flavocillin was docked In Silico and the results were provided (Data 1).

Results: Results of In Silico Acute Rat Toxicity Prediction for Flavocillin by GUSAR showed that Flavocillin does not have Intraperitoneal and Intrevenous toxicity in LD50 classifications and it is in applicability domain whereas it can have little acute toxicity (class 5) for oral and subcutaneous routes of administration.

This indicated that Flavocillin might not be as safe as Ampicillin for subcutaneous administrations but also indicated the likelihood of Flavocillin acid free forms to be more safe for Intravenous IV administrations when compared to Ampicillin IV injections. Given that there are more than one possible ways for the synthesis of Flavocillin derivatives and there are still clinical studies that need to be made, future studies on Flavocillin are expected to provide evidence for what derivatives or formulations would be more safe than others in IV routes or for oral administrations, especially in comparision to already existing Penicillins such as Ampicillin.

Discussion: Data about substrate and metabolite based prediction result In Silico for Flavocillin was also outlined. Most interactions regarding human substrates might not be feasible because all antibiotics offer high selectivity towards bacterial targets, thus those interactions are insignificant and may even be not possible.

The accuracy of the used softwares in predictions for Flavocillin and the results seem accurate because anti-bacterial target results for Flavocillin showed that it has the strongest action mostly on wide range of Staphylococcus Aureus species because Flavocillin In vitro Biological activity results are confirmed agains S. aureus strains.²²

It is usually a single functional group in Chemical structure that distinguishes or specifies one compound with chromone based structure from another resulting in the variety, effectiveness or specificity in Pharmacological activities.

The suggested possible mechanism of action of Flavocillin according to all of the collected data to date would be as follows; Flavocillin will inhibit transpeptidase of the bacterial cell wall like Ampicillin does²³ because of Flavone acting as an activity enhancer group for the bacterial Beta lactam inhibitory action of 6-APA. Due to carrying a Flavone group, Flavocillin is expected to do its action against Pneumonia or related antibiotic resistant lung infections more specifically by targeting lungs and at the same time will fight S. Aureus originated antibiotic resistant lung infections due to combined Chemical effects of a Flavone with 6-APA.²⁴ An interesting and important conclusion from the evidence of various Flavone related studies were that certain Flavones such as flavanols and cathecins had therapeutic effects for reducing or treating the symptoms of Chronic Obstructive Pulmonary Disease (COPD).²⁵ It is also expected that Flavocillin will suppress the effects of MCF-7 for arresting the chest tumour growth at low doses in milligrams following entry into the body by interacting with intercellular membranes of chest tumours.²⁶

Flavocillin is likely to interact with Oxidative pentose phosphate pathway and may act as a TrxR inhibitor through prevention of binding of the oxidizing substrate (Trx) in NADPH pathway.^26,27 This inhibitory action of Flavocillin will enable it potential uses in future against malaria and tuberclosis in addition to its potential success for targeting antibiotic resistant Staphylocuccus Aureus strains.

Results of PASS and way2drug modellings provided that Flavocillin has low Ki value and the lower the Ki, the better the drug compound works.

The affinity of Flavocillin for certain strains is associated with that specific strain, and the bigger the value, the more selective the drug towards that strain. Mostly it's the first few strains that the antibiotic is potent against in such molecular predictions (Figures 4–8).

Figure 4 In silico predicted LD50 of flavocillin.

Figure 5 In silico acute rat toxicity prediction for flavocillin by GUSAR.

Figure 6 Substrate and metabolite based Pa and Pi prediction results for flavocillin.

Figure 7 In silico predicted protein targets for flavocillin.

Figure 8 Chemical structures of chromone, flavone, flavonoid and flavocillin.

Direct interactions (possible direct targets):

Target name Confidence ChEMBL id

Tyrosyl-DNA phosphodiesterase 1  0.2834 CHEMBL1075138 Cytochrome P450 3A4

0.2464 CHEMBL340

Tankyrase-1 0.0699 CHEMBL6164

Low molecular weight phosphotyrosine protein phosphatase 0.0616 CHEMBL4903

Hematopoietic cell protein-tyrosine phosphatase 70Z-PEP 0.0340 CHEMBL2889 DNA-dependent protein kinase0.0164 CHEMBL3142

Prolyl endopeptidase 0.0096 CHEMBL3202

Baculoviral IAP repeat-containing protein 2 0.0094 CHEMBL5462 Cyclin-dependent kinase 5

0.0063 CHEMBL4036

Inhibitor of apoptosis protein 3 0.0053 CHEMBL4198

Histone-lysine N-methyltransferase, H3 lysine-9 specific 3 0.0052 CHEMBL6032 Baculoviral IAP repeat-containing protein 3 0.0031 CHEMBL5335

Neprilysin 0.0018 CHEMBL1944

Mediated interactions (Possible targets):

Target name Confidence ChEMBL id

Voltage-gated N-type calcium channel alpha-1B subunit/Amyloid beta A4 precursor protein-binding family A member 1 0.4462 CHEMBL2097170

Solute carrier family 22 member 6  0.2053 CHEMBL1641347

Solute carrier family 22 member 8  0.1247 CHEMBL1641348

G-protein coupled receptor 35 0.1073 CHEMBL1293267

Solute carrier family 15 member 2  0.0982 CHEMBL1743125 Tyrosine-protein kinase FYN

0.0547 CHEMBL1841

Casein kinase II 0.0363 CHEMBL2095191 Thromboxane-A synthase                 0.0256

CHEMBL1835 DNA-dependent protein kinase0.0132 CHEMBL3142

Cysteinyl leukotriene receptor 1 0.0114 CHEMBL1798 Integrin alpha2/beta1 0.0081

CHEMBL3137268

Troponin, cardiac muscle  0.0068 CHEMBL2095202

ATP-binding cassette sub-family G member 2 0.0020 CHEMBL5393

Name Confidence ChEMBL ID

RESISTANT Staphylococcus simulans 0.8947 CHEMBL612425 Streptococcus oralis

0.8870 CHEMBL613305

Dialister propionicifaciens 0.8585 CHEMBL615040 Dialister invisus 0.8566

CHEMBL615037

RESISTANT Helicobacter pylori 0.8489 CHEMBL612600

RESISTANT Staphylococcus aureus subsp. aureus RN4220    0.8414 CHEMBL2366906

Staphylococcus lugdunensis 0.8381 CHEMBL613303

Porphyromonas asaccharolytica 0.8318 CHEMBL615058 Staphylococcus sciuri 0.8229

CHEMBL613150 Actinomyces meyeri 0.8228 CHEMBL612289

Prevotella oralis 0.7994 CHEMBL612687 Staphylococcus simulans   0.7514

CHEMBL612425 Streptococcus constellatus 0.7261 CHEMBL613151

RESISTANT Aeromonas punctata  0.7107 CHEMBL613786 Staphylococcus lentus 0.7064

CHEMBL613149

RESISTANT Streptococcus agalactiae 0.7031 CHEMBL614622 RESISTANT Streptococcus sp. group B 0.6998 CHEMBL613186 Yersinia enterocolitica 0.6951 CHEMBL614398

Finegoldia magna 0.6944 CHEMBL614615

RESISTANT Clostridium perfringens 0.6774 CHEMBL614967 Streptococcus intermedius 0.6755 CHEMBL3301402

RESISTANT Finegoldia magna 0.6557 CHEMBL614615 Peptostreptococcus anaerobius 0.6533 CHEMBL614420

RESISTANT Clostridium paraputrificum 0.6390 CHEMBL615027 RESISTANT Clostridium septicum 0.6390 CHEMBL614968 RESISTANT Clostridium tertium 0.6289 CHEMBL613074

Streptococcus sp. 0.6249 CHEMBL614621

RESISTANT Yersinia enterocolitica 0.6100 CHEMBL614398 Streptococcus sp. group B

0.5976 CHEMBL613186

Prevotella bivia 0.5908 CHEMBL612321

Peptostreptococcus micros 0.5875 CHEMBL612380 Veillonella parvula 0.5866

CHEMBL612244

Burkholderia pseudomallei 0.5779 CHEMBL3140323

RESISTANT Peptoniphilus asaccharolyticus 0.5744 CHEMBL614419 RESISTANT Peptostreptococcus micros 0.5744 CHEMBL612380 Fusobacterium varium 0.5630 CHEMBL614416

Fusobacterium mortiferum 0.5569 CHEMBL614415 RESISTANT Bacillus anthracis 0.5432

CHEMBL613904 Carnobacterium divergens 0.5392 CHEMBL614757 RESISTANT Escherichia coli 0.5355 CHEMBL354 Clostridium ramosum 0.5349

CHEMBL614971 Shigella sp. 0.5313 CHEMBL614397

RESISTANT Streptococcus pneumoniae R6 0.5288 CHEMBL2366794 Dialister micraerophilus 0.5104 CHEMBL615038

Dialister pneumosintes 0.5104 CHEMBL615039 Anaerococcus prevotii 0.5061 CHEMBL612351 Eggerthella lenta 0.4865 CHEMBL612337 Actinomyces israelii 0.4858 CHEMBL614976 Listeria monocytogenes 0.4849 CHEMBL614974 Prevotella buccae 0.4823 CHEMBL612234

Pseudomonas fluorescens 0.4680 CHEMBL612500 Neisseria meningitidis 0.4561 CHEMBL614431 Campylobacter jejuni 0.4443 CHEMBL612492

RESISTANT Shigella sonnei 0.4358 CHEMBL614595 Vibrio fluvialis 0.4058 CHEMBL614602

Pectobacterium atrosepticum 0.3990 CHEMBL613241 Actinomyces naeslundii 0.3951 CHEMBL614975 Fusobacterium nucleatum             0.3938 CHEMBL614609

Kocuria rhizophila 0.3910 CHEMBL1075349 Staphylococcus haemolyticus     0.3850 CHEMBL612507

RESISTANT Peptostreptococcus anaerobius 0.3735 CHEMBL614420 Bacillus sphaericus 0.3567 CHEMBL613863

Escherichia coli K12 0.3550 CHEMBL612336

Streptococcus pneumoniae R6 0.3510 CHEMBL2366794 Bacteroides stercoris                0.3507 CHEMBL614750

Streptococcus mutans 0.3505 CHEMBL612426 Listeria innocua0.3495 CHEMBL612636

RESISTANT Bacillus subtilis 0.3490 CHEMBL359 Staphylococcus saprophyticus 0.3490 CHEMBL613764 Stenotrophomonas maltophilia 0.3460 CHEMBL612366 Sarcina 0.3310 CHEMBL614617

Staphylococcus hominis    0.3250 CHEMBL614423 RESISTANT Proteus mirabilis 0.3120 CHEMBL614444

RESISTANT Prevotella intermedia 0.3031 CHEMBL613261 Bacteroides distasonis 0.3018 CHEMBL614413

Escherichia coli DH5[alpha] 0.2926 CHEMBL2366740 Streptococcus mitis      0.2910 CHEMBL614952

Bacillus subtilis subsp. subtilis str. 168 0.2735 CHEMBL613315 Clostridium cadaveris   0.2731 CHEMBL614970

Bacteroides uniformis 0.2727 CHEMBL612622 Staphylococcus intermedius     0.2688 CHEMBL614422

RESISTANT Staphylococcus hominis 0.2668 CHEMBL614423 RESISTANT Klebsiella pneumoniae 0.2661 CHEMBL350 Prevotella intermedia 0.2644 CHEMBL613261

Lactococcus lactis 0.2605 CHEMBL613069

RESISTANT Neisseria gonorrhoeae 0.2406 CHEMBL614430 Streptococcus salivarius 0.2388 CHEMBL614620

RESISTANT Salmonella typhimurium 0.2375 CHEMBL351 RESISTANT Bacteroides fragilis 0.2364 CHEMBL614411

Vibrio parahaemolyticus   0.2313 CHEMBL614403 Streptococcus sanguinis    0.2181CHEMBL612314 Clostridium innocuum 0.2051 CHEMBL614761

Corynebacterium jeikeium 0.2027 CHEMBL613351

RESISTANT Staphylococcus haemolyticus  0.1989 CHEMBL612507 Enterococcus sp.  0.1966 CHEMBL613770

Mycobacterium mageritense 0.1960 CHEMBL612959

RESISTANT Fusobacterium nucleatum        0.1959 CHEMBL614609 Staphylococcus aureus 0.1955 CHEMBL352

RESISTANT Salmonella enterica 0.1854 CHEMBL613762 Bacteroides caccae               0.1842 CHEMBL614749

Escherichia coli ATCC 8739 0.1803 CHEMBL2366847 Prevotella disiens 0.1800 CHEMBL612235

Prevotella corporis 0.1757 CHEMBL615061 Bacillus anthracis 0.1729 CHEMBL613904 Bacteroides ovatus 0.1640 CHEMBL612686

Enterococcus faecalis ATCC 29212 0.1555 CHEMBL2366997 Pseudomonas aeruginosa 0.1493 CHEMBL348

Bacillus licheniformis 0.1474 CHEMBL612626 Vibrio cholerae 0.1431 CHEMBL614402

Escherichia coli 0.1394 CHEMBL354

Staphylococcus aureus subsp. aureus RN4220 0.1382 CHEMBL2366906 Helicobacter pylori SS1 0.1371 CHEMBL613197

Clostridium sordellii 0.1346 CHEMBL613072

RESISTANT Enterobacter cloacae 0.1293 CHEMBL349 Neisseria gonorrhoeae 0.1292 CHEMBL614430 Propionibacterium acnes      0.1290 CHEMBL612639

Bacillus megaterium 0.1170 CHEMBL613071 Clostridium septicum 0.1152 CHEMBL614968 Propionibacterium 0.1146 CHEMBL614980 Mycobacterium aurum 0.1137 CHEMBL612952

Lactobacillus fermentum   0.1050 CHEMBL613076 Micrococcus luteus 0.1020 CHEMBL614421

Streptococcus 0.0990 CHEMBL612313

Peptoniphilus asaccharolyticus 0.0930 CHEMBL614419 Parabacteroides merdae 0.0902 CHEMBL615057 Clostridium difficile                0.0887 CHEMBL614965 Nocardia otitidiscaviarum 0.0867 CHEMBL613233

Bacillus subtilis subsp. spizizenii ATCC 6633 0.0849 CHEMBL2367178 RESISTANT Providencia stuartii 0.0842 CHEMBL614445

Salmonella enterica 0.0840 CHEMBL613762 Bacillus subtilis 0.0770 CHEMBL359

Pseudomonas aeruginosa PAO10.0749 CHEMBL613264 Enterococcus faecium 0.0740 CHEMBL357

RESISTANT Enterobacter aerogenes 0.0726 CHEMBL612508 RESISTANT Serratia marcescens 0.0718 CHEMBL614593

RESISTANT Klebsiella     0.0703 CHEMBL612535

Clostridium tetani 0.0656 CHEMBL613073 Fusobacterium necrophorum 0.0648 CHEMBL614417 Streptococcus pyogenes                0.0556 CHEMBL356 Lactobacillus plantarum 0.0524 CHEMBL614973 Salmonella typhi 0.0521 CHEMBL614448

RESISTANT Bacteroides ovatus 0.0490 CHEMBL612686 Serratia marcescens                0.0445 CHEMBL614593

RESISTANT Enterobacter 0.0413 CHEMBL614439

RESISTANT Pseudomonas aeruginosa PAO1 0.0410 CHEMBL613264 Peptostreptococcus sp. 0.0310 CHEMBL614616

Providencia stuartii 0.0302 CHEMBL614445 Salmonella paratyphi   0.0297 CHEMBL612293

RESISTANT Anaerococcus prevotii 0.0256 CHEMBL612351 RESISTANT Bacteroides vulgatus 0.0218 CHEMBL612287 Enterococcus hirae 0.0205 CHEMBL614623

Enterobacteriaceae 0.0141 CHEMBL614436 Clostridium perfringens 0.0139 CHEMBL614967 Shigella flexneri0.0103 CHEMBL614396 Providencia 0.0097 CHEMBL612617

Proteus mirabilis 0.0091 CHEMBL614444

Prevotella melaninogenica 0.0087 CHEMBL612236 Streptococcus viridans 0.0072 CHEMBL612332

RESISTANT Klebsiella oxytoca 0.0013 CHEMBL614441 Citrobacter freundii 0.0009 CHEMBL612615 Serratia 0.0009 CHEMBL614592

Data 1 In silico results for additional possible activity of flavocillin on various range of bacterial targets

Anticancer activity of flavocillin ammonium salt

Flavocıllın suppresses the growth of MCF-7 human breast cancer cells

Breast cancer is one of the most common types of cancers in women and middle aged women is more frequently diagnosed with breast cancer. Various recent studies showed that Flavones and Flavonoids have antimicrobial, antioxidant and anticancer activities. Flavocillin, a recently discovered new Penicillin derivative antibiotic that carries a flavone group in its structure attached to the aryl group of 6-Amino Penicillanic Acid (6-APA), has Pharmacological activities on the types of bacteria that infect the lungs and causes pneumonia, bronchitis and sepsis. The purpose of this article is to outline a very recent data where the tumour cytotoxicity studies were performed for Flavocillin. The results indicated that Flavocillin suppressed the tumour growth of MCF-7 human breast cancer cells.

MCF-7, namely derived from “Michigan Cancer Foundation‘’ which is the institution where the cell line was isolated from a 69 year old woman in 1970,²⁸ is a very important indicator for studying Chemotherapeutic drug resistance in vitro.²⁹ An important property of MCF-7 is that it needs the formation of estradiol for tumour formation in vivo.³⁰ As a consequence of the inhibition of apoptosis by certain Chemotherapeutic agents such as cisplatin, resistance to MCF-7 occurs.³¹ Further, certain hormonal therapies or bile acids have enhancing factors which trigger the growth of MCF-7 human breast cancer cells by acting on oestrogen receptors or oestrogen-regulated proteins.³²

Certain flavones carrying Isopentyl group showed inhibitory effects against DNA synthesis of estrogen-dependent ER(+) MCF-7. It was therefore concluded that by inducing authophagy, Flavones had antiproliferative effect on certain human breast cancer cells including MCF-7.³³

Flavocillin, a recently discovered Penicillin antibiotic which carries a flavone as a subgroup of flavonoids has promising MIC values on various bacterial species that infect the lungs and cause antibiotic resistant Pneumonia, such as S. Aureus, MRSA, Moraxella Catarrhalis, Streptococcus Mutans and most importantly, Corynebacterium Stiratum where Ampicillin did not have any activity in vitro.³⁴ In addition, In Silico docking experiments by using Mcule docking software evidenced that the inhibitory activity of Flavocillin against Trxr and OATP would be promising in possible treatment of leprosy and tuberclosis.³⁵

Methodology: In the most recent study for Flavocillin, approximately 900 milligrams of Flavocillin were tested against various cancer cell lines for measuring tumour growth inhibition rates in vitro.

Results: One dose data results indicated that Flavocillin in low concentrations (1.00E-5 Molar) suppressed tumour growth of MCF-7 in vitro by 14%. There were certain other cancer cell lines where the inhibitory effects of Flavocillin against the growth of certain tumours in low amounts were noticed. However, the most promising effects were obtained on MCF-7.

Flavocillin at very low concentrations suppressed the tumour growth for MCF-7 as growth percent of MCF-7 remained 86% after treatment with Flavocillin (Figure 9). It was interesting that Flavocillin was initially synthesized in ammonium salt form 1 year before this testing, however, because the synthesized Flavocillin ammonium salt were unstable at room temperature and probably lost its stability when not protected at -200 C, after 1 year passed and after the synthesized Flavocillin ammonium salt remained at normal and higher temperatures prior to this testing, Flavocillin ammonium salt should either shifted to another isomeric form of the originally synthesized compound long before testing because the compound may exist in various resonance forms,^36,37 or the compound might have lost its ammonium salt and resulted in acid free form. The acid free powder form of Flavocillin looks yellow whereas its the ammonium salt form looks white.³⁸

Conclusion: Although Flavocillin loses stability when left at room temperatures or when exposed to heat or may undergo shifting to other resonance forms or degrade, even that unstable or shifted form of the originally synthesized Flavocillin compound after 1 year had high inhibitory effects against MCF-7. Therefore, Flavocillin derivatives would be promising agents that would be very promising for the treatment of infectious and also MCF-7 induced tumour growth. It is considered that one additional important advantage of possible drug development of Flavocillin for such treatment purposes would be the naturality of the flavone it carries, as flavones are natural substances present in green tea and certain plants. Therefore, while providing treatment effects for the mentioned diseases, it will cause less side effects with almost no harm to the patients. It is expected that the safety and efficacy studies for various Flavocillin derivatives will show what Flavocillin derivatives would be more stable with least side effects over the time. (Supplementary data)

None.

The authors declare that there are no conflicts of interest.

1. Antimicrobial resistance. 2023.
2. Lambert PA. Bacterial resistance to antibiotics: modified target sites. Adv Drug Deliv Rev. 2005;57(10):1471–1485.
3. Hosseinzade A, Sadeghi O, Biregani AN, et al. Immunomodulatory effects of flavonoids: possible induction of T CD4+ regulatory cells through suppression of mTOR pathway signaling activity. Front Immunol. 2019;10:51.
4. Flavocillin: a new penicillin derivative. PubChem. 2018.
5. Imran S, Taha M, Ismail NH, et al. Synthesis of novel flavone hydrazones: in-vitro evaluation of α-glucosidase inhibition, QSAR analysis and docking studies. Eur J Med Chem. 2015;105:156–170.
6. Lau RM, van Eupen JTH, Schipper D, et al. Synthesis of Penicillin N and Isopenicillin N. Tetrahedron. 2000;56(38):7601–7606.
7. EUCAST disk diffusion method for antimicrobial susceptibility test - Version 8.0. 2020.
8. EUCAST Clinical breakpoint table- Version 10.0. 2020.
9. Nakamura T, Ishikawa K, Matsuo T, et al. Enterococcus hirae bacteremia associated with acute pyelonephritis in a patient with alcoholic cirrhosis: a case report and literature review. BMC Infect Dis. 2021;21(1):999.
10. Pehlivan M. Flavocillin: a new penicillin derivative. EP3752509. 2024.
11. Abreu AC, Serra SC, Borges A, et al. Combinatorial activity of flavonoids with antibiotics against drug-resistant Staphylococcus aureus. Microb Drug Resist. 2025; 21(6):600–609.
12. Semwal RB, Semwal DK, Combrinck S, et al. Health benefits of chromones: common ingredients of our daily diet. Phytochem Rev. 2020;19(6):761–785.
13. Dias MC, Pinto DCGA, Silva AMS. Plant flavonoids: chemical characteristics and biological activity. Molecules. 2021;26(17):5377.
14. Ullah A, Munir S, Badshah SL, et al. Important flavonoids and their role as a therapeutic agent. Molecules. 2020;25(22):5243.
15. Ujayli BA, Nafziger DA, Saravolatz L. Pneumonia due to Staphylococcus aureus Clin Chest Med. 1995;16(1):111–120.
16. Pehlivan M. Official biological activity reports for flavocillin and flavocillin ammonium salt confirming the effectiveness of flavocillin antibiotics on MRSA, aureus, E. faecalis, E. faecium, M. catarrhalis, S. mutans, E. hirae, S. mutans, C. stiratum and S. epidermidis. 2023.
17. Pehlivan M. Flavocillin suppresses the growth of MCF-7 human breast cancer cells. Researchgate. 2023.
18. Pehlivan M. And Labrou D. Flavocillin: a potent TrxR and OATP inhibitor. Qeios. 2023.
19. Acred P, Brown DM, Turner DH, et al. Pharmacology and chemotherapy of ampicillin. Brit J Pharmacol. 1962;18:256–369.
20. Demos/smiles.
21. Ivanov SM, Lagunin AA, Rudik AV, et al. ADVERPred -web service for prediction of adverse effects of drugs. J Chem Inform Model. 2018;58(1):8–11.
22. Way2drug understanding chemical-biological interactions.
23. Petri WA. Penicillins, cephalosporins, and other beta-lactam antibiotics. In Brunton L, Chabner B, Knollman B (eds.). Goodman and Gilman's the Pharmacological Basis of Therapeutics (Twelfth Ed.). New York: McGraw Hill Professional. 2023;1477–1504.
24. Beigh S, Rehman MU, Khan A, et al, Therapeutic role of flavonoids in lung inflammatory disorders, Phytomed Plus. 2022;2(1).
25. Moon SJ, Dong W, Stephanopoulos GN, et al. Oxidative pentose phosphate pathway and glucose anaplerosis support maintenance of mitochondrial NADPH pool under mitochondrial oxidative stress. Bioeng Transl Med. 2020;5(3):e10184.
26. Saccoccia F, Angelucci F, Boumis G, et al. Thioredoxin reductase and its inhibitors. Curr Protein Pept Sci. 2014;15(6):621–646.
27. Lee AV, Oesterreich S, Davidson NE. MCF-7 Cells-Changing the Course of Breast Cancer Cancer Research and Care for 45 years. J Natl Cancer Inst. 2015;107(7):djv073.
28. ECACC, MCF-7. Cell line profile catalogue no: 86012803.
29. Kasid A, Lippman ME, Papageorge AG, et al. Transfection of v-rasH DNA into MCF-7 human breast cancer cells bypasses dependence on estrogen for tumorigenicity. Science. 1985;228(4700):725–728.
30. Kouhi, TK, Safarian S, Arnaiz B, et al. Enhancement of cisplatin sensitivity in human breast cancer MCF-7 cell line through BiP and 14-3-3ζ co-knockdown. Oncol Rep. 2021;45:665-679.
31. Baker P, Wilton J, Jones C, et al. Bile acids influence the growth, oestrogen receptor and oestrogen-regulated proteins of MCF-7 human breast cancer cells. Br J Cancer. 1992;65(4):566–572.
32. Pedro M, Lourenço CF, Cidade H, et al. Effects of natural prenylated flavones in the phenotypical ER (+) MCF-7 and ER (-) MDA-MB-231 human breast cancer cells. Toxicol Lett. 2006;164(1):24–36.
33. Official biological activity reports for flavocillin and flavocillin ammonium salt confirming the effectiveness of flavocillin antibiotics on MRSA, Aureus, E. Faecalis, E. Faecium, M. Catarrhalıs, S. Mutans, E. Hırae, S. Mutans, C. Stiratum and S. Epidermidis. 2022.
34. Labrou D, Pehlivan M. Flavocillin: a potent TrxR and OATP inhibitor. Qeios. 2023.
35. National center for biotechnology ınformation. PubChem substance record for SID 85393251, flavocillin. ChEBI. 2023.
36. National center for biotechnology ınformation. PubChem compound summary for CID 168007555, CID 168007555. 2023.
37. Pehlivan M. Certificate of structural analysis of flavocillin ammonium salt. 2022.
38. Sakamoto Y, Flavones in green tea: part I. Isolation and structures of flavones occurring in green tea ınfusion, agricultural and biological chemistry. Agri Biol Chem. 1967;31(9):1029–1034.