J. Wen, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
In parallel to the analogues described above, we sought to replace
the triazole with an amine, which led to the synthesis of analogue 8.
The purpose of preparing this analogue was two-fold. First, our pre-
viously prepared ITZ analogues demonstrate poor solubility and we
hypothesized that addition of the primary amine could enhance this
essential pharmacokinetic property. Second, we thought that the amine
would be a promising chemical handle to attach additional function-
ality to the scaffold. Interestingly, analogue 8 was significantly less
active than the other analogues with methyl mimics in the triazole
position (IC50 = 0.55 μM). Amide intermediate 7 was less active
(
IC50 = 0.94 μM), suggesting a polar group in the dioxolane 2-position
Scheme 1. Reagents and conditions. (a) Imidazole/Phthalimide,
K
2
CO ,
3
is unfavorable for potent anti-Hh activity. Based on our initial results
showing increased bulk at this position is detrimental to anti-Hh ac-
tivity, we did not make additional analogues with triazole mimics
linked through the amine.
CH CN, reflux, 4 h, 96–98%; (b) TsOH (cat.), toluene, reflux/RT, overnight,
3
2
3–88% (a:b in 1:1 ratio); (c) (R)-3-Chloro-1,2-propanediol, K
overnight, 91%; (d) TsCl, pyridine, CH Cl , RT, overnight, 93% (a:b in 1:1
ratio); (e) Cs CO , DMF, 80 °C/RT, 4 h, 32–87%.
2
CO
3
, DMF, RT,
2
2
2
3
We utilized topological polar surface area (TPSA) in an attempt to
quantify the influence of polarity in the triazole region on anti-Hh ac-
tivity. Based on the calculation, a correlation between TPSA and Hh
inhibition is summarized in Fig. S1. Our results demonstrate the TPSA is
2
preferred to be approximately 70 Å , as represented by analogues 1 and
2
2
5
. Analogues with TPSA above 70.1 Å (up to 113.2 Å ) were less active
suggesting the hydrophobic nature of the binding sub-pocket for this
2
region. In addition, a TPSA value below 90 Å also correlates with en-
2
3
hanced cell permeability.
In conclusion, a series of stereochemically defined ITZ analogues
focused on triazole modifications was synthesized and evaluated to
continue our SAR studies of ITZ analogue inhibition of Hh signaling.
These modifications ranged in size from proton to phthalimide. Based
on our results, a single methyl group represents the most favorable
substituent on dioxolane C-2 position. In addition, there is no sig-
nificant difference in Hh inhibition between 2R,4R and 2S,4R diaster-
eomers, suggesting the stereochemistry on dioxolane C-2 position, is
less important for potent Hh inhibition. Finally, a nonpolar substituent
is better than a polar substituent, suggesting the hydrophobic nature of
the binding pocket. This study would be useful for the understanding of
the chemical space of the dioxolane region, as well as developing next
generation ITZ analogues as Hh-pathway antagonists.
Fig. 2. Elucidating the absolute stereochemistry by 1H NMR.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Scheme 2. Reagents and conditions. (a) Diethyl oxalate, Mg, I
9%; (b) (R)-3-Chloro-1,2-propanediol, Cs CO , DMF, RT, overnight, 95%; (c)
Ammonium hydroxide, 25% in water, 50 °C, 4 h, quant.; (d) TsCl, pyridine,
CH Cl , RT, overnight, 72%; (e) 19, Cs CO , DMF, 80 °C, 4 h, 71%; (f) LiAlH
THF, 0 °C, 30 min, 28%.
2
, Et
2
O, RT, 4 h,
8
2
3
2
2
2
3
4
,
Acknowledgments
The authors gratefully acknowledge support of this work by the
National Institutes of Health/National Cancer Institute (CA190617).
ASZ001 cells were provided by Dr. Ervin Epstein (Children’s Hospital
Oakland Research Institute).
Increasing the substituent size to a fused-cycle phthalimide (3a and 3b)
lead to a sharp decrease in activity (IC50 values of approximately
1
.0 μM); while completely removing the triazole group (4a and 4b,
R = H) lead to a moderate drop in activity (IC50 values of approxi-
mately 0.3 μM).
Appendix A. Supplementary data
Taking these initial results into consideration, we thought that a
single methyl group might represent the optimal size substituent at
the C-2 dioxolane position to fit into the sub-pocket. Analogues with
bulkier (ITZ, 2, and 3) or no substituent (4) were significantly less
active than the analogues containing methyl groups (1a and 1b).
With this in mind, we further designed and synthesized ITZ analo-
gues bearing methyl bioisosteres. Not surprisingly, when a chlor-
omethyl replaces the methyl group, these analogues (5a and 5b)
showed potent activity against the Hh pathway (with IC50 va-
References
2
3
4
.
.
.
lues = 0.025 and 0.02 μM, respectively). Introducing
a
tri-
6
7
8
.
.
.
fluoromethyl group (6a and 6b) resulted in a slight decrease in ac-
tivity compared to 1a and 1b. These results provide further evidence
that moieties maintaining the approximate size of a methyl group are
optimal for activity of our scaffold.
1
3