Joseph Kim

902494525
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 Used

Material Formula MW (g/mol) Volume (mL)
E. coli TG1 1/100 --------- --------- 5.0
2-nitrobenzaldehyde C7H5NO3 151.12 ---------
3-nitrobenzaldehyde C7H5NO3 151.12 ---------
5-nitro-2-furaldehyde C5H3NO4 141.08 ---------
4-bromophenylhydrazine BrC6H4NHNH2 * HCl 223.50 ---------
hydrochloride
4-cyanophenylhydrazine C7H7N3 * HCl 169.61 ---------
hydrochloride
Aminoguanidine bicarbonate CH6N4 * H2CO3 136.11 ---------

Table II. Volume of Each Compound in Eppendorf Tubes

Compound Volume (drops)
M1 M2 M3 M4 M5 M6
2-nitrobenzaldehyde 15 ------ ------ 5 5 5
3-nitrobenzaldehyde ----- 15 ------ 5 5 5
5-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
 Joseph Kim
902494525
Table III. Structure of Aldehydes and Hydrazines
Compound Structure
2-nitrobenzaldehyde
O

H NO2

3-nitrobenzaldehyde
O

NO2

5-nitro-2-furaldehyde

O-

N+
O

O
H

4-bromophenylhydrazine
hydrochloride Br

NH2
N
H

4-cyanophenylhydrazine
hydrochloride
N

HCl

NH2
N
H
 Joseph Kim
902494525
Aminoguanidine bicarbonate
NH
H HO
N

H2N
NH2 HO O

Table IV. Observations

Solution 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 Product

Compound 4-bromophenylhydrazine 4-cyanophenylhydrazine Aminoguanidine
hydrochloride hydrochloride bicarbonate
2-
nitrobenzaldehyde
NO2 H NO2 H NO2 H

H H NH
N N H
N N N N

NH2
Br N

3-
nitrobenzaldehyde
H H H

H H H NH
O2N N N O2N N N O2N N N

NH2
Br N

5-nitro-2-
furaldehyde
H H H
H H NH
N N N N H
-O -O
-O N N
O O +
N+ N+ N O

Br
NH2
O O
N O
 Joseph Kim
902494525
Table VI. Antibiotic Effect Result
Section M4 M5 M6
M1 Neg Neg Pos
M2 Neg Neg Pos
M3 Pos Pos Pos
* Orange = the hit compound *

Table VII. Structure of Hit Compound

Compound (Aldehyde + Hydrazine) Structure
(2E)-2-[(5-nitro-2-furyl)methylene]
hydrazinecarboximidamide H
NH
H
-O N N
N+ O
NH2
O

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 Kim
902494525
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

H
N R2
O
N
H + H2O
R1
+ H2 N N R2

H R1
H
 Joseph Kim
902494525

Figure IV. Reaction Mechanism for the Hit Compound

O- NH

N+ O-
HN
O
N+ NH
+ H N NH2 O
O
H HN
H O
H N+ NH2
O
-O H
H

O-
O-
N+ NH
O N+ NH
HN O
O H HN
N NH2 O
H N NH2
H
O+
H O
H H
H

H3O+
O-

N+ NH
O O-
HN
O N+ NH
N+ NH2 O
HN
O
H N NH2
H
H2O
H
 Joseph Kim
902494525
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.