Synthesis and Evaluation of
Substituted 1,6-Dihydro-1,3,5triazine-2,4-diamines as Potential
Antimicrobial Compounds.
Bruce A. Hathaway
Department of Chemistry,
Southeast Missouri State University,
Cape Girardeau, MO, 63701.
E-mail: bahathaway@semo.edu
1
Introduction
The first clinically useful antimicrobial
compounds (sulfa drugs and penicillins) were
developed in the 1930’s and 40’s. While these
compounds were effective against a variety of
bacterial diseases, over time, a number of
bacteria became resistant to them. By 1990, it
was estimated that 80% of both hospital- and
community-acquired strains of Staphylococcus
aureus were resistant to Penicillin G. Some
strains of Staphylococci, Tuberculosis, and
Pneumococci are resistant to six or more drugs,
while Enterococcus faecium and Pneumococcus
cepacia are not sensitive to any currently
approved drugs. The need for new drugs that
are effective against resistant organisms is
evident.
2
Resistance of Plasmodium
falciparum to Antifolates,
such as Cycloguanil
The resistance of P. falciparum to antifolates is an
important problem in treatment of malaria. This
resistance is associated with mutations in the
enzyme dihydrofolate reductase (DHFR). Several
mutants of P. falciparum DHFR have been isolated,
which have one or more amino acids replaced.
These mutations significantly decrease the
effectiveness of cycloguanil and related compounds
as inhibitors of P. falciparum DHFR (1). Some
examples are shown in the following table.
Cl
NH2
N
H2N
N
N
CH3 Cycloguanil
CH3
3
Reduction of Dihydrofolate
by DHFR and NADPH
O
OH
COOH
NH
COOH
N
N
H2N
NH
N
NADPH + H+
N
H
Dihydrofolate Reductase
Dihydrofolate
NADP+
O
OH
H
COOH
NH
COOH
H
N
N
H2N
Cl
NH
N
NH2
N
N
H
Tetrahydrofolate
H2N
N
N
CH3 Cycloguanil
CH3
4
Inhibition Constants (Ki) to PfDHFR and
Antiplasmodial Activities (I50) to Wild Type and
Mutant P. falciparum of Cycloguanil (2)
DHFR or Organism Ki (nM)
Wild type
I50 (μM)
1.5
0.04
A16V+S108T mutant
1314
3.7
N51I+C59R+S108N
mutant
C59R+S108N+I164L
mutant
N51I+C59R+S108N
+I164L mutant
31.4
5.8
285
37.9
385
>100
5
Effects of Structural
Modifications to Cycloguanil
on P. falciparum
Kamchonwongpaisan, S., et al (2), prepared
several analogs of Cycloguanil, and tested them
against P. falciparum in vitro, and against P.
falciparum DHFR. One of their compounds, the 6heptyltriazine shown below, showed good activity
against mutant P. falciparum and mutant P.
falciparum DHFR. Some of their results are
shown on the next slide. This was the only
triazine with a single alkyl group in the 6-position
that they reported on.
NH2
N
H2N
N
Cl
(CH2)6CH3
N
H
HCl
6
Inhibition Constants (Ki) to PfDHFR and
Antiplasmodial Activities (IC50) to Wild Type and
Mutant P. falciparum of the 6-Heptyltriazine (2)
DHFR or Organism
Ki
(nM)
2.7
IC50 (μM)
A16V+S108T mutant
3.8
0.004
N51I+C59R+S108N
mutant
C59R+S108N+I164L
mutant
N51I+C59R+S108N
+I164L mutant
1.0
0.33
0.3
0.14
0.8
2.5
Wild type
0.04
7
Introduction to Our Research:
6-Alkyltriazines
We were interested to find out if 6alkyltriazines would inhibit the growth of
bacteria. Coats, et al (3) had reported that
hydrophobic groups on the benzene ring
imparted increased antimicrobial activity
against Lactobacillus casei in cell culture.
Similar results were reported with Escherichia
coli (4) and leukemia cells (5). Therefore, we
prepared and tested a number of 6substituted triazines for inhibition of growth
of L. casei in cell culture. We used the wellknown “two-component synthesis” of Modest
and Levine (6) to prepare the triazines.
8
Synthesis of
6-Alkyltriazines
Cl
NH
N
Cl
C
N
H2N
NH
reflux
H2N
C
N
HCl, ethanol
+
H2N
NH2
NH2
HCl
Cl
NH2
O
H
C
R
N
N
6
ethanol, reflux
H2N
N
H
R
HCl
9
Proton NMR Spectrum
of 6-Hexyltriazine
Cl
NH2
N
H2N
Cl
N
(CH2)5CH3
N
+
H
-
H
10
Inhibition Assay
(L. casei)
• Prepared decade
dilutions of
compounds in
standard folic acid
assay medium.
• Measured OD660
after 18-24 hours.
• Repeated in
triplicate when the
linear range was
deduced.
• Calculate I50.
Determine
concentration that
gives 50% inhibition of
growth
11
% Max. Growth
I50 Determination of
Selected Compounds in L. casei
100
90
80
70
60
50
40
30
20
10
0
Cycloguanil
n-Hexyl
0
0.02
0.04
0.06
0.08
0.1
Conc (mM)
12
Inhibition Assay
(Mycobacterium tuberculosis )
• Screening was performed by the Tuberculosis
Antimicrobial Acquisition and Coordinating
Facility (TAACF), http://www.taacf.org/.
• All compounds are initially screened against
M. tuberculosis strain H37Rv at the single
concentration, 6.25 µg/mL (≈ 20 µM).
• Compounds are considered active in the
primary screen if, at this concentration,
inhibition ≥ 90%.
• Active compounds are screened by serial
dilution beginning at 6.25 µg/mL to determine
I50.
13
I50’s (L. casei) & % Inhibition @ 6.25
µg/mL (M. tub.) of 6-Alkyltriazines
R1
R2
I50 (µM)
% Inh.
CH3 CH3
44
38
H
Ethyl
300
46
H
n-Propyl
>1000
51
H
n-Butyl
900
57
H
3-Pentyl
>1000
46
H
Phenyl
>1000
49
14
I50’s (L. casei) & % Inhibition @ 6.25
µg/mL (M. tub.) of 6-Alkyltriazines
R1
R2
I50 (µM)
% Inh.
CH3 CH3
44
38
H
n-Hexyl
17
84
H
n-Nonyl
4.1
H
n-Undecyl
3.4
99
(I50=1.7µM)
Not tested
H
2-Phenethyl
300
77
15
Summary of Results for
6-Alkyltriazines
• A long, lipophilic chain seems to
enhance antimicrobial activity.
• Small chains have reduced
activity.
• Bulky groups have reduced activity
• But triazines with long lipophilic
chains often had solubility
problems in the media.
16
1-(Substituted Phenyl)
Triazines
NH2
N
H2N
R
1N
N
CH3
CH3
Coats, et al (3) had reported that hydrophobic
groups on the benzene ring imparted
increased antimicrobial activity against
Lactobacillus casei in cell culture. We also
prepared a number of 1-(substituted phenyl)
triazines. To do this, we used the well-known
“three-component synthesis” of Modest (7) to
prepare these triazines. We varied the
location, the size and shape of the group
attached to the benzene ring, and made some
different derivatives than those previously
17
reported.
Structures of
1-(Substituted Phenyl) Triazines
Previous workers
NH2
4
3
N
N
1
Variable Bridged Phenyl
N
(atom)1-4
Z
G
N
CH3
H2N
3
N
CH3
H2N
4
NH2
Y
CH3
N
CH3
Y = O or S and Z = CH2, or Y = CH2 and Z = O or S;
G = various groups
Bridged Alkyl
4
NH2
N
H2N
3
N
H2N
Alkyl
3
N
CH3
CH3
4
NH2
CH2 S Alkyl
N
N
Alkyl
N
CH3
CH3
18
Synthesis of 1-(Substituted Phenyl)
Triazines: Nitro Compounds
X-CH2R, K2CO3
O 2N
OH
CH2
O 2N
acetone, reflux
X
O 2N
HS-R, K2CO3
acetone, reflux
O
R
CH2
S
O 2N
19
R
Synthesis of 1-(Substituted
Phenyl) Triazines: Anilines
3 H2, Pd-C
O 2N
O
CH2
O 2N
R
S
R
or Fe, H+
H2N
Sn, HCl
O
CH2
R
S
H2N
20
R
Synthesis of 1-(Substituted
Phenyl) Triazines: 3-Alkyl Anilines
HNO3
R
H2SO4 (-10 - 0o)
R
O 2N
O
O
> 5 NH2NH2
Cat. KOH, Heat
R
H2N
21
Synthesis of 1-(Substituted
Phenyl) Triazines: ThreeComponent Synthesis
R
N
C
N
NH2
H2N
H-Cl
C
H2N
NH2
N
N
reflux
O
C
CH3
H2N
R
N
CH3
CH3
HCl
CH3
22
I50’s (L. casei) & % Inhibition @
6.25 µg/mL (M. tub.) of 1-(3Substituted Phenyl) Triazines
R
I50 (µM)
% Inh.
-H
18
Not tested
-CH2-CH3
11
57
-O-(CH2)2-Ph
2.5
67
-O-(CH2)3-Ph
0.2
78
-CH2-S-CH2-Ph
0.22
72
23
I50’s (L. casei) & % Inhibition
@ 6.25 µg/mL (M. tub.) of 1-(4Substituted Phenyl) Triazines
R
I50 (µM)
% Inh.
-CH2-S-n-C4H9
2.0
61
-CH2-S-CH2Ph
1.5
72
-CH2-SCyclohexyl
-Cl (Cycloguanil)
2.0
74
44
38
24
Summary of Results for
1-(Substituted Phenyl)
Triazines
• Maximum potency for inhibition of
cell growth was by a bridge of 3-4
atoms between the benzene rings
in the 3-position.
• Sulfur-bridged groups in the 4position produce effective
inhibitors versus M. tub., but these
were not as good versus L. casei.
25
Inhibition of Human DHFR
• Dihydrofolate reductase (DHFR) enzyme
assay kit from Sigma-Aldrich© was used.
• Inhibitors dissolved 40% ethanol in assay
buffer. After dilutions, a 0.1% final ethanol
concentration had no effect on the enzyme.
• Analysis were done using a Beckman
DU530 UV-Vis spectrophotometer.
• Decrease in absorbance at 340 nm (due to
NADPH) was measured every 15 seconds
for a total of 150 seconds at room
temperature as per the manufacturer’s
suggestion.
• Ki = concentration of inhibitor which reduces
rate of reaction by 50%.
26
I50’s and Ki’s of
Substituted Triazines
Compound
I50 (L.
Ki (Human
casei), µM DHFR), µM
6-nonyl
4.1
0.8
6-undecyl
3.4
1.6
Cycloguanil
(4’-Cl)
3’-CH2SCH2Ph
44
10
0.22
0.6
3’-O(CH2)3Ph
0.2
4.0
Methotrexate
0.00025
0.02
27
Isolation Of E. coli DHFR
• Cells were solubilized with lysozyme and then
detergent
• Cell debris was centrifuged away
• The protein was precipitated with ammonium
sulfate
• The sample was dialyzed to remove salt
• The sample was applied to a DEAE-cellulose
anion-exchange column, and fractions with
activity were pooled
• The sample was applied to a Sephadex G-100
size-exclusion column, and fractions with
activity were pooled
28
Partial Purification of E. coli
DHFR
Fraction
[Protein]
(mg/mL)
Specific
Activity
(units/mg
protein)
Fold
Purified
Cell Free
Extract
NH4(SO4)2
Precipitate
25
0.0024
1.00
11
0.0045
1.88
DEAE 20+21
0.6
0.0118
4.90
Sephadex
Fraction 8
0.04
0.1301
54.20
29
E. coli I50’s and Ki’s of
Substituted Triazines
Compound
I50, µM
Ki, µM
Cycloguanil (4’-Cl)
68
9
H
4.5
9
3’-CH2SCH2Ph
6.2
9
3’-O(CH2)3Ph
1.8
10
Methotrexate
<0.01
<0.01
30
Other Work
• Biology graduate student Wes
Sanders set up a malaria assay
based on an enzyme-linked
immunosorbent assay, or ELISA.
• Unfortunately, the triazines
interfered with the assay to
cause a color change.
31
Cl
Other Work
NH2
N
N
(CH2)5CH3
H2N
N
H
HCl
• Twila Mason tested several triazines
for their ability to inhibit the growth of
17 Bacillus cereus strains.
• The DHFR in B. cereus is 98%
identical to that in B. anthracis
(causative agent in anthrax).
• The 6-hexyl triazine was the most
effective, with IC50’s of about 93 µM.
32
Future Work
• Make more analogues, to
optimize distance between the
two benzene rings, and the
overall lipophilicity.
• Test compounds on other
microorganisms and on
methotrexate-resistant strains.
• Further purify bacterial DHFR.
33
Acknowledgements
• Carlos Casteñada, Danny Grohmann, and
Mark McGill, undergraduate Chemistry
students.
• Dr. Jim Champine, Dr. Christina Frazier, Dr.
Walt Lilly, Twila Mason, Andrea Van
Weelden, Marcus Cuff, and Wes Sanders,
Department of Biology.
• Grants and Research Funding Committee,
Southeast Missouri State University.
• Antimycobacterial data were provided by the
Tuberculosis Antimicrobial Acquisition and
Coordinating Facility (TAACF) through a
research and development contract with the
U.S. National Institute of Allergy and
Infectious Diseases.
34
Southeast Missouri State
University
• Founded in 1873, Southeast Missouri State
University has a proud history of excellence
in education.
• First founded as a teacher’s college, the
institution has grown into a comprehensive
university with more than 150 academic
programs in five colleges, with an
enrollment of over 9000 students.
• Located in Cape Girardeau (population
36,000), on the banks of the Mississippi
River, about 100 miles south of St. Louis.
35
Location of Southeast Missouri
State University in the USA
State of Missouri
Cape Girardeau, Missouri
36
Academic Hall
37
Kent Library
38
Rhodes Hall of Science
39
Magill Hall of Science
40
Thank You…
• For the invitation to speak, and
• For your kind attention.
• Are there any questions?
41
References
1.
2.
3.
4.
5.
6.
7.
Peterson, D.S., et al, Proc. Natl. Acad. Sci.
USA 1990, 87, 3018-3022.
Kamchonwongpaisan, S., et al, J. Med.
Chem. 2004, 47, 673-680.
Coats, E.A., et al, J. Med. Chem. 1981, 24,
1422-9.
Coats, E.A., et al, J. Med. Chem. 1985, 28,
1910-6.
Selassie, C.D., et al, J. Med. Chem. 1982,
25, 157-161.
Modest, E.J. and P. Levine, J. Org. Chem.
1956, 21, 14-20.
Modest, E.J., J. Org. Chem. 1956, 21, 113.
42
Eliza Assay
HRPII
1. Well is coated
with anti-HRPII
antibody.
2. Sample is
added to the
well and any
HRPII present
binds to the
anti-HRPII
antibody.
43
Eliza Assay
HRPII
HRPII
3. Anti-HRPII
antibody with
conjugated
enzyme is added
to the well and
binds HRPII.
4. Added substrate
reacts with
enzyme to
produce a color
change.
44
Triazine Interference with
the Eliza Assay
triazine
• The triazines
replace HRPII in
the process,
which gives a
false positive
reading in the
assay.
45