Flavocillin: A New Penicillin Derivative

Pharm. Mustafa Pehlivan

Abstract
This invention is about the discovery of a new novel compound as an antibiotic
to stop this potential resistance problem completely. With Organic Chemistry
studies and methods and other modified antibiotics, adding a flavonoid group to
varibale side R instead which will result in flavocilin formation will be the most
effective and revolutionary antibiotic that the bacteria will not easily be able to
resist. Evidentful pathways have been conculded stating that the Flavocilin use
in Medicine for heavy infectious conditions or where no other antibiotic works
will outweigh all its risks.
Specification
Resistance to antibiotics is a complicated growing problem of the centruy.
Presently, this problem has been so much getting the attention of Medical
Research Institutions and health organizations that there is a huge alert in
preventing antibiotic resistance. There are reports estimating that many people
will die in 2050’s due to antibiotics not working anymore as a result of this
resistance. The simplest evidence that Flavocillin will be an effective antibiotic
is that many stronger antibiotics were obtained by modifying the variable group
(R) in Penicilin Chemical structure.
For obtaining Flavocilin, the R group must be replaced with a basic flavonoid
and there may be more ways than what is being described at the claims part of
this invention. Fortunately, obtaining Flavocilin is a simple Chemical process as
a conclusion drawn from its theoretical studies.
Flavonoids are very useful antimicrobial compounds of herbal origin found
naturally in certain teas and Propolis, a solution available to be used in Medicine
which is what the honey bee uses to prevent his honey comb from microbes
while producing honey. Propolis alone, readily available from Holland and
Barret Stores, is full of flavonoids and as such it is effective in treating certain
respiratory tract infections and also persistent flue, as a natural remedy.
In drug discovery and development process, adding effective Bioactive
compounds to R groups of already active Chemical compounds as drug
molecules is important to obtain much stronger effects than that of those drug
molecules being used alone in treatment.

1
 Possible advantages and disadvantages of Flavocilin as a new drug
development
Advantages:
Due to dual destructive mechanism effects (Cell wall inhibition and microbial
destruction by bioactive flavonoid group as R (variable) group of Penicillin), the
bacteria will not easily have the ability to resist to Flavocilin and therefore it will
be the most powerful antibiotic via this defined dual strength of bioactivity.
As suggested in Chemical synthesis route part of claims, Flavocilin can easily be
obtained with simple reactions.
Non-competitive advantage for the developer industry or for the drug company
as a company holding the license rights of production during 20 years of patent
protection.
Flavocilin can always be used as a final choice of antibiotic where patient
responds to no other antibiotic treatment, and it is impossible not to respond to
Flavocilin due to dual mechanism of action stated previously.
Flavocilin will be the solution for the problem of the century, antibiotic
resistance, even after centuries due to penicilin-flavonoid dual strength effects.
Flavocilin needs to be tested against virus effectiveness also. Due to its
flavonoid group and Penicilin group, it can have a wider range of
Pharmacological use and tests can determine if it will cancel, prevent, treat or
minimise viral resistance, and if so, to what levels? The likelihood of finding out
that it is very powerful against virus also will add benefit to its revolutionary
importance and non-competitive unique advantages.
Disadvantages:
Stronger effects may bring further side effects (but as flavonids are naturally
found in tea and little or moderate tea consumption is known to have no
noticable toxic effects, this risk is not likely).
Clinical testing to fully determine Pharmacological effects have not yet been
determined.
Easy making of flavocilin can result in easy illegal flavocilin production (This is
a probability but yet it is not very likely as per strict regulationbs and restrictions
of patent law and also because a laboratory is still needed for its production
although the method is not quite complicated.
The figure below illustrates the structure of Flavocillin

2
Figure 1-Chemical Structure of Flavocillin ( IUPAC name : (2S,5R)-6-[2-(3-
(4-oxo-benzopyran-2-yl)-1-phenylamido]-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo-[3.2.0]-heptane-2-carboxylic acid ).

Synthesis of Flavocillin
A theoretical synthesis strategy was considered for Flavocillin compound .
Synthesis strategy and work beginning from 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 made.

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:

3
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 were 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 NaSO 4 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 .

4
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
(Ref: 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) 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 170 0 C. After 3 h, 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 170 0 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

5
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) 20 0 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
Na2SO4 . ( Reference : Rute Madeira Lau et. al., Synthesis of Penicillin N and
Isopenicillin N, Tetrahedron 56 (2000) 7601-7606 )

(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 )

6
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.

7
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 40 0C 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.

Tests which confirmed that Flavocillin is actually active in treating drug

resistant bacterial infections

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 our studied flavocillin compound ; for all the microorganisms it
exceeded the Breakpoint in the bacteria tested. In other words, the

8
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.
Microorganism Ampicilin Flavocillin *EUCAST limit DMSO H2O
value
10 100
µg/Disk µg/Disk
Enterococcus hirae 20 12 ≥ 10 - -
Zon Çapı (mm)
Enterococcus 22 14 ≥ 10 - -
faecalis
Zon Çapı (mm)
Staphylococcus 30 24 ≥ 18 - -
aureus
Zon Çapı (mm)
Escherichia coli 25 17 ≥ 14 - -
Zon Çapı (mm)

Figure 2-Bioactivity data for Flavocillin

Measuring of Antimicrobial Activity

1. Disk Diffusion Method

9
1.1. 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).

1.2. 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.
1.3. 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.
1.4. 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.

1.4. 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.

10
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.

1.5. 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 (1).
1.6. Measuring Zones and Interpreting 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 (2). Zone Diameters for Flavocillin; Enterococcus
hirae (12 mm), Enterococcus faecalis (14 mm), Staphylococcus aureus (24 mm),
Escherichia coli (17 mm) were measured. It is shown in Table 1.
1.7. 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.
1.8. References
1) EUCAST Disk Diffusion Method for Antimicrobial Susceptibility Test –
Version 8.0 (January 2020)
2) EUCAST Clinical Breakpoint Table Version 10.0 (January 2020)

References:
1. Nature volume 179, pages 892–893 (04 May 1957)
doi:10.1038/179892a0

11
2. J. Am. Chem. Soc. , 1957, 79 (5), pp 1262–1263. DOI:
10.1021/ja01562a063

3. Protein Sci. 2009 Mar; 18(3): 595–605. doi: 10.1002/pro.67 PMCID:

PMC2760365

12