P. Mougenot et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
O
N
O
heterocycle
replacement
a, b
c
O,N-introduction
S
O
HO
O
O
O
8
9
O
N
N
solubilizing moieties
salt formation
N
OH
O
ether moieties
N-introduction
O
H
d
S
O
R
O
S
N
S
N
OH
N
H
R
O
N
H
O
O
N
H
N
H
R
12
11
Figure 4. Strategy to increase solubility of the thiadiazole series.
Scheme 1. Synthesis of compound 12. Reagents and conditions: (a) (COCl)2, DCM,
(b) KSCN, CH3CN; (c) RCONHNH2 10, CH3CN, reflux; (d) TFA, four steps 30–65%.
The synthesis15 of compounds 4-cis and 5-cis is depicted in
Scheme 2 starting from the carboxylic acid 6 which is transformed
into the acid chloride with oxalyl chloride. Subsequent coupling
reactions with 2-amino-1,3,4-thiadiazoles led to the amides 7.
We also used PyBroPÒ for the synthesis of amides 7 from car-
boxylic acid 6 and 2-amino-[1,3,4]thiadiazoles. Finally, the corre-
sponding carboxylic acids 4-cis and 5-cis were obtained after
basic hydrolysis.
As seen in Table 3, cis derivatives were at least ten fold more
potent that their trans counterparts which could be explained by
a better alignment when superimposed on 1-trans as shown in
Figure 5.
ligand based methods. Identification of the causal metabolic ‘hot
spots’ in compound 3 would allow the design of improved candi-
dates. We first applied the MetaSite13 prediction tool that includes
3D models of the most important human cytochromes, including
CYP3A4, and allows recognition of labile sites of metabolism
(SOM). Since CYP3A4 was the main contributor to its metabolism
we assessed compound 3 in a CYP3A4 model from a ketoconazole
co-crystal, with built-in corrections for reactivity of the enzyme
and ligand. The predicted primary sites of metabolism were
located on the cyclopentylethyl fragment (Fig. 3).
In accordance with MetaSite predictions, we introduced oxygen
atoms at different positions of the cyclopentylethyl moiety to block
oxidative metabolism and to assess the effect on solubility in
parallel.
Unfortunately, the aqueous solubility at pH = 7.4 of compounds
4-cis and 5-cis was not dramatically improved compared to 1-trans
and 2. On the other hand, these ether analogues were found to be
very potent in enzymatic and cell-based assays. Overall, compound
5-cis displayed a favourable profile with very good potency, good
metabolic stability and intestinal permeability but only a limited
gain in solubility, which made it a good candidate for salt
screening.
In order to assess a possible synergistic effect between the
incremental improvements in solubility seen in 12a and 5-cis, we
introduced a second oxygen in compound 12a, in the hope of fur-
ther improving solubility. The synthesis15 of compound 16 is
depicted in Scheme 3 starting from the carboxylic acid 6. The suc-
cessive reactions with oxalyl chloride and potassium isothio-
cyanate gave the benzoylisothiocyanate 13. Addition of
cyclopentyloxymethylhydrazide followed by trifluoroacetic acid
treatment gave the thiadiazole 15. Finally, basic hydrolysis led to
the carboxylic acid 16 (see Table 4).
As expected, solubility of compound 16 was improved com-
pared to 12a and 5-cis but still remained too low.
Due to the insufficient improvement in solubility by the intro-
duction of oxygen atoms in the above compounds, we decided to
assess the effect of nitrogen insertion.
We first tried the replacement of the phenyl by a pyridyl in
compound 4-cis. The general synthesis15 is depicted in Scheme 4.
Starting from the ethyl hydroxynicotinic ester 19 and cis-tert-butyl
4-hydroxycyclohexanecarboxylic ester 20 in a Mitsunobu reaction
gave, after hydrolysis, the carboxylic acid 22. The subsequent
The synthesis15 of corresponding compounds 12a–c is depicted
in Scheme 1 starting from the carboxylic acid 8. The successive
reactions with oxalyl chloride and potassium isothiocyanate gave
the acylisothiocyanate 9. Addition of the hydrazide 10 followed
by trifluoroacetic acid treatment gave the thiadiazole 12.
As shown in Table 2, the introduction of an ether function on
the cyclopentylethyl group led to an increase in metabolic stability
and solubility but also to a drop in activity compared to compound
3. Compounds 12b and 12c displayed acceptable solubility up to
90 lM at pH = 7.4 but at the expense of potency. On the other
hand, compound 12a retained good potency but its solubility
was too low.
Encouraged by the favourable effect on solubility obtained with
compounds 12a–c, we considered a more aggressive strategy to
address this issue: (i) introduction of heteroatoms at other sites
of the scaffold; (ii) thiadiazole replacement by more soluble five-
membered heterocycles; (iii) introduction of solubilising groups
and (iv) salt formation (Fig. 4).
As had been shown earlier,6 the aliphatic acid appears to be
essential for maintaining the enzymatic activity. Therefore, we first
envisaged the introduction of an oxygen atom between the phenyl
and the cyclohexyl moieties whilst keeping the distance between
the thiadiazole and the carboxylic acid constant as in compounds
4-cis/trans and 5-cis/trans.
Table 2
Modulation of cyclopentyl derivatives 3, 12a–c
O
N
N
N
OH
H
3, 12
S
R
O
Compd
R
Enz. DGAT-1 IC50
(l
M)
Cell. DGAT-1 IC50
(lM)
% met. h/m/r
Caco2 (10ꢀ7 cm/s)
Sol. pH = 7.4 (lM)
3
0.019
0.038
0.012
0.066
36:5:10
3:0:0
90
<2
9
O
12a
103
O
12b
12c
0.144
0.233
ND
ND
9:8:3
ND
72
90
95
O
ND