Transcript Bioassay of Cycloguanil Derivatives for Potential Anti

Bioassay of Cycloguanil Derivatives for
Potential Anti-tumor and Antimicrobial
Activities: An Interdisciplinary Project.
A. VANWEELDEN, C. CASTAÑEDA, B. A.
HATHAWAY, and JAMES E. CHAMPINE
Southeast Missouri State University, Cape
Girardeau, MO.
Dihydrofolate Reductase Activity
Folate metabolism is
essential in rapidly
growing systems, i.e.
– Bacteria
– Parasites
– Tumors
Polshakov. 2001. Russian Chemical Bulletin
50(10):1733-1751
Some Inhibitors of DHFR
Methothrexate (4)
– Tumor diseases
Trimethoprim (6)
– Bacterial Infections
Pyrimethamine (7)
– Protozoan infections
(Plasmodium)
Cycloguanil (not
pictured)
Polshakov. 2001. Russian Chemical Bulletin
50(10):1733-1751
Project Description
Synthesize triazines similar to cycloguanil
– Use various aldehydes in the synthesis to modify
substituents at number 2 carbon
– Known to provide stearic hinderance
Use Lactobacillus as model organism
– Fastidous: growth dependent on folic acid
– Used in in bioassay for folic concentration in food
and vitamins
– Previoulsy used to study this type of drug
Preparation of 1-(Chlorophenyl)2-Substituted Triazines
Cl
Cl
NH2
N
C
N
H-Cl
N
N
ethanol
C
H2N
H2N
NH2
O
reflux
C
R
R
H2N
N
+
H
Cl
-
H
H
1-(Chlorophenyl)-1,2-dihydro-s-triazine; R=H
Cycloguanil; 2,2-CH3
Derivatives tested: R= Ethyl, Butyl, Propyl, Hexyl, or Phenyl
Inhibition Assay
Prepared decade dilutions of
compounds in Standard folic
acid assay medium
Used DMSO as carrier to
dissolve
Measured OD660 after 18-24
hours
Repeated in triplicate when
the linear range was
deduced
Goal - I50
Determine concentration that
gives 50% Inhibition of growth
Results – Propyl Derivative Example
120%
100%
% Growth
80%
60%
40%
20%
0%
0.001
0.01
0.1
mM [Propyl Derivative]
1
10
Results – Multiple Derivatives
120
Phenyl
% Growth
100
Parent
Compound
Cycloguanil
80
60
Hexyl
40
Butyl
20
Ethyl
0
0.001
0.01
0.1
mM [Derivative]
1
I50 Determination of Effective Compounds
% Growth
100
Hexyl
Parent
Compound
Cycloguanil
50
0
0
0.02
0.04
0.06
mM [Compound]
0.08
0.1
Summary and Discussion
1-(Chlorophenyl)-1,2-dihydro-2-hexyl-striazine was shown to have a lower I50 (0.024
mM) than cycloguanil (0.048 mM), but not the
base compound (0.013 mM)
This suggests investigation into other
derivatives is warranted
Future studies include trying the hexyl
derivative with purified DHFR and against
methotrexate strains of Lactobacillus