Joseph Kim902494525

Combinatorial Chemistry: Determination of a Possible Drug Structure

Abstract:The combinatorial chemistry technique was used to find an antibiotic against E. coli. Six

solutions using three hydrazines and aldehydes were prepared and tested on E. coli. to find the hit compound. A positive effect against E. coli was shown in the hit compound through the formation of a clear circle around the cups. The hit compound, based on the agar plates, was the compound produced from aminoguanidine bicarbonate and 5-nitro-2-furaldehyde. The structure of the hit compound was determined to be (2E)-2-[(5-nitro-2-furyl)methylene] hydrazinecarboximidamide using the structures of the reactants and mechanism of the reaction.

Using a marker, two agar plates were divided into three sections each, labeled M1-M6. The bacterial culture E. coli TG1 1/100 (5 mL) was used to cover two agar plates with E. coli. With a transfer pipette, the extra liquid was carefully removed into a waste tube. Using the extra third plate, the cupping procedure was practiced. After a few practices, a small cup was made in the middle of each section on the two prepared agar plates. Six Eppendorf tubes were taken and labeled M1-M6. Different volumes of hydrazines and aldehydes, which are listed in Table II, were added into six tubes to produce a series of structurally similar drugs. All six tubes were shaken rigorously for 10-15 seconds. The observations of each tube were recorded. With a new transfer pipette, 2 drops of each solution were placed into the cups of the appropriate section. The finished agar plates were incubated at 37 °C for 24 hours. Images of the agar plates were taken after 24 hours in order to determine which solution had an antibiotic effect on E. coli.

Data and Observation:

Table I. Material UsedMaterial Formula MW (g/mol) Volume (mL)

E. coli TG1 1/100 --------- --------- 5.02-nitrobenzaldehyde C7H5NO3 151.12 ---------3-nitrobenzaldehyde C7H5NO3 151.12 ---------5-nitro-2-furaldehyde C5H3NO4 141.08 ---------

4-bromophenylhydrazine hydrochloride

BrC6H4NHNH2 * HCl 223.50 ---------

4-cyanophenylhydrazine hydrochloride

C7H7N3 * HCl 169.61 ---------

Aminoguanidine bicarbonate CH6N4 * H2CO3 136.11 ---------

Table II. Volume of Each Compound in Eppendorf TubesCompound Volume (drops)

M1 M2 M3 M4 M5 M62-nitrobenzaldehyde 15 ------ ------ 5 5 53-nitrobenzaldehyde ----- 15 ------ 5 5 55-nitro-2-furaldehyde ----- ------ 15 5 5 5

4-bromophenylhydrazine hydrochloride 5 5 5 15 ------ -------4-cyanophenylhydrazine hydrochloride 5 5 5 ------ 15 -------

Aminoguanidine bicarbonate 5 5 5 ------ ------ 15

Table III. Structure of Aldehydes and HydrazinesCompound Structure

2-nitrobenzaldehyde

O

H NO2

3-nitrobenzaldehyde

NO2

O

H

5-nitro-2-furaldehyde

O

N+

O

O-

O

H

4-bromophenylhydrazine hydrochloride

Br

NH

NH2

4-cyanophenylhydrazine hydrochloride

N

NH

NH2

HCl

Joseph Kim902494525

Aminoguanidine bicarbonate

H2N

HN

NH

NH2

HO

HO O

Table IV. ObservationsSolution Observations

M1 The color of the solution is orange.M2 The color of the solution is yellow.M3 The color of the solution is dark red.M4 The color of the solution is red.M5 The color of the solution is orange.M6 The solution is transparent.

Table V. Structures of Possible ProductCompound 4-bromophenylhydrazine

hydrochloride4-cyanophenylhydrazine

hydrochlorideAminoguanidine

bicarbonate2-

nitrobenzaldehyde NO2

NHN

Br

H NO2

NHN

H

N

NO2

NHN

H

NH

NH2

3-nitrobenzaldehyde

NHN

Br

H

O2N NHN

H

N

O2N NHN

H

NH

NH2

O2N

5-nitro-2-furaldehyde

NHN

Br

H

ON+

-O

O

NHN

H

ON+

-O

ON

N HN

H

ON+

-O

O

NH

NH2

Table VI. Antibiotic Effect ResultSection M4 M5 M6

M1 Neg Neg PosM2 Neg Neg PosM3 Pos Pos Pos

* Orange = the hit compound *

Table VII. Structure of Hit CompoundCompound (Aldehyde + Hydrazine) Structure(2E)-2-[(5-nitro-2-furyl)methylene]

hydrazinecarboximidamide

N HN

H

ON+

-O

O

NH

NH2

Figure I. Plate One (M1-M3) Figure II. Plate Two (M4-M6)

Discussion:Six soltuions containing different aldehydes and hydrazines were prepared in order to test

their effect on E. coli. The positive effect on E. coli would have a clear circle around a cup as can be seen in the figures above. The clear circle represents the inhibition of E. coli growth. The solutions that do not have an effect on E. coli would not have any change around the cups. Based on two agar plates that were treated with E. coli and six solutions, only two sections show a positive effect, indicating that there is exactly one solution that possesses an antibiotic effect against E. coli. The cups on sections labeled M3 and M6 have clear circles. In the M3 solution, three hydrazines and 5-nitro-2-furaldehyde were mixed. In M6 solution, all three aldehydes and aminoguanidine bicarbonate were mixed. Therefore, based on two sections, the compound that has an effect on E. coli as an antibiotic is the one produced from aminoguanidine bicarbonate

Joseph Kim902494525

and 5-nitro-2-furaldehyde. The name of the hit compound is (2E)-2-[(5-nitro-2-furyl)methylene]hydrazinecarboximidamide. This compound works as a bacteriostatic agent (Masui); it inhibits the growth of bacterial cells without killing them (Erfe). The bacteriostatic compounds usually hinders the protein synthesis, nucleic acid synthesis, and other cellular metabolism (Erfe).

The mechanism invovled in the reaction between 5-nitro-2-furaldehyde and aminoguanidine bicarbonate is simple. First, the carbon-oxygen double bond shifts its electron pair to the oxygen atom, due to the lone pair on the nitrogen atom from the aminoguanidine attacking the carbon atom. This creates a carbon-nitrogen single bond and puts a positive charge on the nitrogen atom. Then, the oxygen atom utilizes one of its lone pairs to take a hydrogen atom from the nitrogen, resulting in protonation. The protonated oxygen will then take another proton from a hydronium ion with one of its other lone pairs. The nitrogen atom then moves an electron pair to form a carbon-nitrogen double bond; the nitrogen again obtains a positive charge. The formation of a carbon-nitrogen double bond causes the protonated oxygen to leave readily as a water molecule. The water molecule lastly takes a hydrogen atom from the nitrogen atom using its electron pair, thereby recreating a hydronium ion for other reactions to take place. An electron pair from the bond between the nitrogen and hydrogen atom moves to the nitrogen atom, removing the positive charge. The shift of an electron pair concludes the reaction, and the final sturcture is produced.

Figure III. Gerneral Reaction

R1

H

O

+ H2NHN R2

R1H

N

HN R2

+ H2O

Figure IV. Reaction Mechanism for the Hit Compound

O

N+

O

O-

O

H

+ H N

H

HN

NH

NH2

O

N+

O-

O

-OH

N+

H

HN

NH

NH2

H

O

N+

O-

O

OH

N

HN

NH

NH2

H

H

H3O+

O

N+

O-

O

O+

H

N

H

HN

NH

NH2H

H

H2O

O

N+

O-

O

N+

H

HN

NH

NH2

H

O

N+

O-

O

N

HN

NH

NH2

H

Joseph Kim902494525

References:

Erfe, Jasmine, Ericka Senegar-Mitchell, and Sandra Slivka. Combinatorial Chemistry and Drug Discovery Lab [PDF document]. Retrieved from bridgestoliteracy.com/biotech/assignments/CombiChemLab.pdf

Masui, Masamiki, et al. (1955). Mechanism of bacteriostatic action of nitrofuran compounds. Osaka City Medical Journal, 1, 47-55.