Scale up synthesis of trypanocidal sulfonamides and in vitro ADME studies

DA SILVA, R.B.*1, DOS SANTOS, T.C.F1, DE SOUSA, I.L1. ROCCO, S.A., CORDEIRO, A.T.1

1. CENTRO NACIONAL DE PESQUISA EM ENERGIA E MATERIAIS (CNPEM)

E-mail: [email protected]

Introduction Results

The treatment of Chagas disease is a challenge and requires new

pH_1.7 pH_7.4 pH_8.9
chemotherapies. Screening of the Chagas Box compounds identified

Normalized solubility (%)

Normalized solubility (%)

Normalized solubility (%)
120 120 120
sulfonamides as potent inhibitors of the parasitic malic enzyme, which is 100 100 100
80 80 80
important for parasite growth and survival. We synthesized four new 60 60 60
40 40 40
sulfonamides for in vitro ADME and in vivo efficacy studies, in the order 20 20 20
of 0.5-1.2 grams. 0
lol 61 83 41 01
0
lol 61 83 41 01
0
lol 61 83 41 01
n o C C0 C1 R n o C C0 C1 R n o C C0 C1 R
E Y E Y E Y
p re M ME ME P p re M ME ME P p re M ME ME P
l l l
A A A

Methodologies Figure 1: Sulfonamides normalized solubility after incubation for 90

minutes at 25°C, in three different pH´s.
Chemistry: Sulfonamide synthesis route.
pH_1.7 pH_7.4 pH_8.9

Normalized stability (%)

Normalized stability (%)

Normalized stability (%)

120 120 120

100 100 100

80 80 80

60 60 60

40 40 40

20 20 20

0 0 0

01
l
61

01
l
61

1
01
l
61

lo

lo
lo

08

14

08

14
08

14

R
EC
no

R
EC
R
EC

no
no

EC

EC

EC

EC
EC

EC

PY

PY
PY

re

re
M
re

M
M

M
M

lp

lp
lp

A
A
Figure 2: Sulfonamides normalized stability after incubation for 24 hours
at 37°C, in different pH´s.

40
5

Papp.10-6(cm/s)
4 30
Log D 3 20
2
10
1
0 0
i l 61 83 41 01

EC l
Ve low

1
01
i
m C C 1 R

14
a

R
p E E EC PY

pa
el

PY
a M M M

ry
r

ra
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Figure 3: Log D. Figure 4: Caco2 Permeability.

100 Verapamil
concentration

Elimination parameters
normalized

MEC141
PYR01
50 T1/2 CL int. mic. CL int. hep.
(min) (µL· min-1·mg-1) (µL· min-1·Kg-1)

Verapamil 10 142 574

in vitro ADME 0 MEC141 7 185 751

0 20 40 60
PYR01 11 128 518
minutes
Solubility and Stability Assays Lipophilicity – logD
Experimental conditions:
Solubility: 5 µM / 0 min and 90 min / 25°C Experimental conditions: 30 µM / 90 min / HPLC analysis Figure 5: Metabolic stability assay using mouse microsome liver.
Stability: 5 µM / 0 min, 90 min and 24 hours / 37°C

Conclusion

• A methodology was developed to synthesize four novel sulfonamides.

Sloubility: Ideal Values > 85% Stability: Ideal Values > 90%
% of AUC ≥ 85%: Excellent % of AUC ≥ 90%: stable
Ideal Values = 1-3
• Except for MEC061, compounds showed moderate solubility and
% of e AUC 60-85%: Aceptable % of e AUC 70-90%: moderately stable
% of AUC < 60%: Poor % of AUC < 70%: unstable chemical stability, suggesting feasibility for formulation and oral
administration.
General setup of Caco-2 permeability assay In vitro metabolic stability assay • MEC061 and MEC083 were not assayed for Caco2 permeability neither
Experimental conditions: 100 µM, 2 h, pH 7.4
Experimental conditions: Sample 1 µM, 0.25 mg Microsome
enriched fraction, 0, 5, 15, 30, 45 e 60 minutes
microsome stability due to poor solubility in assay conditions.
• From these results we recommend to progress with MEC141 and PYR01
https://dda.creative-bioarray.com/

?? ? for in vivo DMPK and Chagas animal models.

Apical

Caco-2 cell monolayer

Basolateral Financial Support

Half life time: Hepatic elimination values:
Semipermeable membrane t½ > 60 min: High < 8 ml/min/kg: low
Ideal Values: Papp > 10 x 10-6 cm/s t½ 30-60 min: moderate 8 to 24 mL/min/kg: moderate This study was supported by the Financiadora de Estudos e Projetos – FINEP n° 01.22.0473.00
t½ < 30 min: low >24 mL/min/kg: high
KERNS, E. H.; DI, L. Drug Like Proprieties: Concept, Structure Design and Methods from Toxicity
Optimization Press: Elsevier, London, UK, 2008.
and Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP n° 2021/14741-9.