7 Comparative in vitro and in vivo StructureActivity Studies of Antiparasitic Downloaded via TUFTS UNIV on July 23, 2018 at 03:06:47 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
2-Methyleneamino-5-nitrothiazoles EBERHARD KUTTER, HANS MACHLEIDT, WOLFGANG REUTER, ROBERT SAUTER, and ALEXANDER WILDFEUER Karl Thomae GmbH, Chemisch-pharmazeutische Fabrik, 795 Biberach an der Riss, West Germany Eighteen nitrothiazoles have been prepared and tested for their trichomonicidal activity. Regression analysis and physicochemical parameters were used to correlate tricho monicidal in vivo and in vitro activity against T. foetus with physicochemical properties of the nitrothiazole derivatives. A good correlation of in vitro trichomonicidal activity was obtained with log Ρ and the oxidation-reduction potential E as the most important variables. This correlation, to gether with other findings, indicates that the 5-nitrothiazoles interfere with a normal metabolic redox process of the para site. A more qualitative correlation was found between the oxidation-reduction potentials of different 5-nitroheterocycles and their antimicrobial activity against several types of microorganisms. Correlation of in vivo trichomonicidal activities in mice was much less significant. Experimental evidence is presented indicating that the kinetics of metabo lism is the most important factor for in vivo activity. A study using optical isomers showed that metabolism proceeds highly stereospecifically. h
* T p h e m a i n interest of scientists i n t h e p h a r m a c e u t i c a l i n d u s t r y is not o n l y to p r o d u c e b i o l o g i c a l l y active c o m p o u n d s a n d to r a t i o n a l i z e their effects f r o m t h e p h y s i c o c h e m i c a l p o i n t of v i e w b u t also to d e v e l o p d r u g s w h i c h m i g h t b e u s e d i n h u m a n t h e r a p y .
effectively T h i s paper
gives a specific e x a m p l e of h o w this a p p r o a c h c a n increase t h e effective ness a n d r a t i o n a l i t y o f s u c h d r u g d e v e l o p m e n t .
F i g u r e 1 shows, i n a
98
Van Valkenburg; Biological Correlations—The Hansch Approach Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
7.
2-Methyleneamino-5-Nitrothiazoles
KUTTER E T A L .
99
Requirement o f a drug w i t h special therapeutic properties
Team o f s c i e n t i s t s
performing stage 2
s y n t h e s i s and p h a r m a c o l o g i c a l t e s t i n g . S e l e c t i o n of a few compounds f o r extended cological
stage 1
["drug d e s i g n "
pharma-
investigation
I Extended
p h a r m a c o l o g i c a l and
t o x i c o l o g i c a l s t u d i e s and selection
stage 3
o f one compound f o r
a preliminary c l i n i c a l
Clinical
trial.
trials
stage 5
Marketing
Figure 1.
stage 4
Several stages of drug
development
s i m p l i f i e d w a y , the stages of d e v e l o p m e n t a d r u g m u s t go t h r o u g h b e f o r e it is c o m m e r c i a l l y u s e f u l . A s is i m m e d i a t e l y evident, t h e H a n s c h a p p r o a c h c a n o n l y b e h e l p f u l at t h e s e c o n d stage of d e v e l o p m e n t .
M a n y different c o m p o u n d s a r e p r e -
Van Valkenburg; Biological Correlations—The Hansch Approach Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
100
BIOLOGICAL CORRELATIONS
T H E HANSCH A P P R O A C H
p a r e d a n d tested for t h e i r b i o l o g i c a l a c t i v i t y at this stage, a n d c o r r e l a t i o n s b e t w e e n p h y s i c o c h e m i c a l p r o p e r t i e s a n d activities are o n l y p o s s i b l e here. H o w e v e r , because of t h e t r e m e n d o u s a m o u n t of w o r k w h i c h u s u a l l y must be performed before a c o m p o u n d
c a n be selected for
extended
i n v e s t i g a t i o n , stage 2 is one of the r a t e - l i m i t i n g steps i n the d e v e l o p m e n t of d r u g s to treat h u m a n diseases. A n y a p p r o a c h w h i c h h e l p s to accelerate d e v e l o p m e n t at this stage a n d to r e d u c e t h e w o r k necessary s h o u l d l o w e r o v e r a l l r e s e a r c h expenses.
0
N
2
D M
0
S ^
A
H
N
1. C S
SO
2
/
KO-t-C H 4
9
2 . C H 3 I
N " ^ S ^ N = C
2
2
N
SCH. R
- 2
C H S H
[1 CH 2
3
η
ΗΝ" R III
" ^ e ^ ^
0 X = N Figure
Our
2.
N ^ S ^ N = C
2
/
0
/
S .
Synthesis
[CH ]
fru
M — r x
2
1 n
Π=2,3.
of
2-methyleneamino-5-nitrothiazoles
e x a m p l e concerns a c e r t a i n k i n d of a n t i m i c r o b i a l agent a n d m a y
i l l u s t r a t e h o w u s e f u l the H a n s c h a p p r o a c h a c t u a l l y c a n b e i n this second stage of d r u g d e v e l o p m e n t .
W e b e c a m e interested i n c h e m o t h e r a p e u t i c
agents effective against t r i c h o m o n i a s i s for s e v e r a l reasons. T r i c h o m o n i a s i s is
a c e r t a i n k i n d of v e n e r e a l disease a n d is c a u s e d b y the p a r a s i t e T .
vaginalis.
I n a n i m a l s , infections c a n also b e c a u s e d b y another strain of
parasite c a l l e d T. foetus.
We
b e g a n synthesis i n this
field
by using
2 - a m i n o - 5 - n i t r o t h i a z o l e as starting m a t e r i a l ( F i g u r e 2 ) .
Van Valkenburg; Biological Correlations—The Hansch Approach Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
ο
Η-»
I—»
Figure 3.
Chemical structures of 18 new nitrothiazole
derivatives
s*
I*
ι
?
ο
1s*
ι
to
>
»
H
M H
Van Valkenburg; Biological Correlations—The Hansch Approach Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
102
BIOLOGICAL CORRELATIONS
2 - A m i n o - 5 - n i t r o t h i a z o l e reacts w i t h C S
2
T H E HANSCH APPROACH
and subsequently C H I 3
to
y i e l d the d i m e t h y l t h i o d e r i v a t i v e I I . A m b i f u n c t i o n a l n u c l e o p h i l i c amines react w i t h this k e y c o m p o u n d I I , w i t h e l i m i n a t i o n of C H S H , a n d y i e l d 3
as the m a i n p r o d u c t the 2 - m e t h y l e n e a m i n o - 5 - n i t r o t h i a z o l e s , I I I . 2 - A m i n o 5 - n i t r o t h i a z o l e itself has a w e a k t r i c h o m o n i c i d a l effect. It was h o p e d that changes i n the p h y s i c o c h e m i c a l p r o p e r t i e s w h i c h are b r o u g h t a b o u t b y t h e synthesis of the m e t h y l e n e a m i n o n i t r o t h i a z o l e s , I I I , w o u l d l e a d to more p o w e r f u l drugs. Eighteen compounds w h i c h were synthesized b y this route a n d tested in vitro a n d in vivo for their a n t i t r i c h o m o n a l a c t i v i t y are s h o w n i n F i g u r e 3. F r o m F i g u r e 3 i t is o b v i o u s that the m e t h y l e n e a m i n o n i t r o t h i a z o l e s c a n b e d i v i d e d i n t o three e l e c t r o n i c a l l y different g r o u p s : those c o n t a i n i n g a n a m i d i n e f u n c t i o n , those c o n t a i n i n g a n i s o u r e a f u n c t i o n , a n d those c o n taining an isothiourea function.
F o r c o n v e n i e n c e w e s h a l l c a l l t h e m the
N - t y p e , the O - t y p e , a n d the S-type c o m p o u n d s , r e s p e c t i v e l y . Table I. Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
a
logV
h
1.38 1.23 0.74 0.16 0.92 1.80 1.82 2.28 2.02 1.95 2.35 1.88 1.34 0.43 0.57 2.40 1.28 1.88
E
h
c
(volt)
-0.262 -0.262 -0.262 -0.262 -0.262 -0.262 -0.262 -0.262 -0.262 -0.262 -0.262 -0.232 -0.232 -0.232 -0.232 -0.232 -0.212 -0.212
MICT.foetus [ig/ml 2.50 2.50 10.00 100.00 5.00 2.50 1.00 1.66 2.50 1.66 2.50 1.00 1.66 5.00 1.25 1.25 0.25 0.125
Structure-Activity d
log 1/C* T.foetus (obs.) 1.93 2.01 1.39 0.43 1.68 1.96 2.36 2.16 1.99 2.16 2.01 2.41 2.14 1.71 2.23 2.33 3.04 3.40
° Coding according to Figure 3. Partition coefficients in the octanol-pH 7.4-phosphate-buffer system. Nitrothiazole oxidation-reduction potentials (volts) as calculated from their half-wave potentials, as determined using a Polarecord Ε 261 polarograph (Metrohm A G , Herisau, Switzerland) and a saturated A g / A g C l reference electrode. Measurements were performed at 20°C and a drop time of 1 drop/2.8 sec. The compounds were dis solved in 1 ml dimethyl formamide and added to 24 ml of a borax-potassium biphosphate buffer of p H 7.3 [prepared according to J . M . Kolthoff, J. Biol. Chem. (1925) 68, 135]. A p H of 7.4 resulted. Standard error of determination ± 3 mv. Test organism : Trichomonas foetus. Minimal inhibition concentrations ^ g / m l ) were determined microscopically after 48 hr-incubation at 37°C. The two-fold serial b
c
d
Van Valkenburg; Biological Correlations—The Hansch Approach Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
7.
103
2-Methyleneamino-5~Nitrothiazoles
KUTTER E T A L .
T h e in vitro b i o l o g i c a l a c t i v i t y w a s d e t e r m i n e d u s i n g t h e " s e r i a l d i l u t i o n m e t h o d " a c c o r d i n g to a s t a n d a r d i z e d test p r o c e d u r e .
T h e minimal
i n h i b i t i o n concentrations ( M I C values listed i n T a b l e I ) represent t h e lowest c o n c e n t r a t i o n of a p a r t i c u l a r n i t r o t h i a z o l e d e r i v a t i v e necessary to p r o d u c e t o t a l i n h i b i t i o n of t r i c h o m o n a d e g r o w t h after a fixed t i m e i n t e r v a l . T h e a d v a n t a g e of this test p r o c e d u r e c o m p a r e d w i t h other m e t h o d s a n d t h e usefulness of M I C values i n s t r u c t u r e - a c t i v i t y studies w e r e r e c e n t l y d e m o n s t r a t e d b y S e y d e l i n a n elegant s t u d y o n sulfa d r u g s ( I ) . I n starting c o r r e l a t i o n studies i n t h e in vitro
series of activities w e
first c a n c o m p a r e t h e M I C values ( T a b l e I ) i n a m o r e q u a l i t a t i v e sense. A t a first glance t h e S-type c o m p o u n d s seem to b e the most active i n this series, f o l l o w e d b y t h e O - t y p e .
T h e N - t y p e c o m p o u n d s , o n t h e other
h a n d , a p p e a r to b e t h e least active in vitro f r o m this p o i n t o f v i e w ; h o w ever, there are c e r t a i n exceptions to this r u l e , a n d therefore o n l y a q u a n t i tative c o r r e l a t i o n c a n r e v e a l u s e f u l structure a c t i v i t y i n f o r m a t i o n . Parameters for Nitrothiazoles log 1/C T.foetus (pred.)
Δ log 1/C
1.98 1.90 1.46 0.62 1.65 2.09 2.09 1.98 2.07 2.08 1.94 2.70 2.59 1.67 1.87 2.53 2.96 3.12
0.05 0.11 0.07 0.19 0.03 0.13 0.27 0.18 0.08 0.08 0.07 0.29 0.45 0.04 0.36 0.20 0.08 0.28
ED
5Od=17
T.foetus mg/kg >250 75 75 >100 37.5 37.5 >100 37.5 50.0 37.5 100.0 37.5 75.0 100.0 75.0 25.0 37.5 37.5
0
Acute Toxicit Mice p.o. LD g/kg b0
>4.0 5.64 — —
1.0 7.6 >4.0 >4.0 >10.0 >10.0 >4.0 >25.8 1.26 2.89 1.94 0.05 1.00 2.18
dilution method in fluid thioglycollate medium (with 10% horse serum) was used, and 20,000 protozoa were inoculated per ml medium. The compounds were dissolved in Titrisol buffer p H 7 (Merck Co., Darmstadt, West Germany) and tested at the following concentrations (^g/ml): 10, 5, 2.5, 1.66, 1.25, 1.0, 0.5, 0.25. In vitro activities against T. foetus. C is represented in μg/ml X mw units. Calculated using Equation 5. ' E D 60 ± π = 50 db 17% survival rate, of mice ( N M R I , 18-20 g, female) infected with 1-2 Χ 10 trichomonades. Dosage schedule: Orally twice daily for 3 days. The first treatment followed 2 hr after infection. The untreated control mice died within 5 days. 14 days LDso in g/kg. Mice, N M R I , female and male, 18-20 g. The substances were suspended in tylose and given by gavage. e
f
6
Λ
Van Valkenburg; Biological Correlations—The Hansch Approach Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
104
BIOLOGICAL CORRELATIONS
Table II.
T H E HANSCH APPROACH
Antibacterial and Antiprotozoal A c t i v i t y of Several E
0
Van Valkenburg; Biological Correlations—The Hansch Approach Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
h
{mo)
7.
2-Methyleneamino-5-Nitrothiazoles
KUTTER E T A L .
5-Nitroheterocycles Compared with Their Oxidation Potentials Ε. coli ATCC 9637 in vitro
Sc. Aronson in vitro
inactive
inactive
moderate
inactive
inactive
moderate
inactive
inactive
good
inactive
inactive
high
moderate
moderate
high
inactive
high
good
weak
weak
moderate
T . foetus T . vaginalis in vitro
Bacteria inactive weak activity moderate activity good activity high activity
>80 80 25-5 5-1 10
μg/ml
10
[Lg/m\
10-1.0 μg/ml 1.0-0.2 μg/ml
