ACS Medicinal Chemistry Letters
Letter
a
of HD. The result of this profiling suggests that the compounds
may be better suited as treatments for Celiac disease, where
BBB permeability would be a disadvantage and 24 h systemic
coverage may not be required for efficacy.
Table 3. Phenethylamide Derivatives
In summary, we have developed a series of covalent TG2
inhibitors having selectivity against closely related trans-
glutaminase isoforms exhibiting low GSH reactivity and
excellent plasma stability. In vitro ADME profiling indicated
rapid rates of oxidative metabolism and high P-gp efflux,
suggesting that further optimization of ADME profiles will be
required to achieve brain exposure in vivo.
compd
R
TG2 IC50 SD (μM)
9a
9o
9p
9q
9r
C6H5
0.18 0.029
0.55 0.049
0.28 0.016
0.11 0.0028
22 15
4-CF3-C6H4
4-Cl-C6H4
4-CH3O-C6H4
4-t-Bu-C6H4
ASSOCIATED CONTENT
■
9s
4-Ph-C6H4
57 8.8
S
* Supporting Information
9t
2-naphthyl
0.28 0.0071
0.31 0.016
0.23 0.016
0.46 0.093
0.28 0.052
0.26 0.081
0.17 0.021
0.18 0.044
Synthetic disclosures including schemes and full experimental
procedures and characterization of key compounds, supporting
figures, details of the ADME profiling assays, and trans-
glutaminase selectivity profiling data. This material is available
9u
9v
9w
9x
9y
9z
9aa
7-quinolinyl
2-CH3O-C6H4
3,4-(CH3O)2-C6H3
benzo[d][1,3]dioxol-4-yl
2,3-dihydrobenzofuran-7-yl
3-Cl-C6H4
AUTHOR INFORMATION
3,4-(CH3)2-C6H3
■
a
Values accompanied by standard deviations were averaged from at
least two independent experiments.
Corresponding Author
Notes
0.11 μM. It is important to note that all of the compounds
tested against mTG2 had activity comparable to their human
TG2 values, confirming the species-independent nature of this
activity. Typical selectivity against TG1 was 3−10-fold, and
>100-fold selectivity was routinely observed against TG6, while
10−20-fold selectivity was typical against FXIIIa. The TG3
activity was not tested since the compounds were expected to
be inactive, this based on a large body of testing.19 The
selectivity profiling revealed that further effort might be needed
to improve selectivity against TG1 and FXIIIa; selectivity
profiles would need to be factored into any decision to consider
these compounds for in vivo studies.
Several compounds were profiled in in vitro metabolism
assays of kinetic solubility, plasma stability, liver microsomal
stability, and permeability/efflux. The results for compound 9a
were typical; it showed aqueous solubility of <0.01 mg/mL and
mouse and human liver microsomal intrinsic clearance values of
99 and 595 μL/min/mg, respectively. The high rate of
metabolism was likely due to the phenethyl and amino-
piperidinyl moieties, which are susceptible to oxidative
metabolism at multiple sites. All compounds tested were stable
in both mouse and human plasma, with a half-life >6 h and
several showing a half-life >24 h. In addition, 8a and 9z showed
no evidence of conjugation to the prototypical biological
nucleophile glutathione (GSH) when tested in vitro over a
period of 120 h.21
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are indebted to Ina Sternberger for outstanding technical
assistance in conducting the transglutaminase biochemical
assays. We also thank Andreas Ebneth for valuable discussions.
REFERENCES
■
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To be effective agents for neurodegenerative disorders, it is
advantageous for compounds to possess a high rate of
permeability and a low efflux rate. P-glycoprotein (P-gp) is
one of the main efflux transporters in brain; the potential for
compounds of interest to be effluxed by P-gp was assessed in an
MDCK-MDR1 transfected cell line. Compound 9a showed a
P
app A-B of 83 nm/s in MDCK-WT and an effective efflux ratio
(EER) of 44, indicating moderate permeability but high active
efflux via P-gp. These results were typical of all compounds
tested. Combining this outcome with the results of microsomal
stability testing indicated that no compound was identified as
being a suitable candidate for in vivo evaluation in the context
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dx.doi.org/10.1021/ml3001352 | ACS Med. Chem. Lett. 2012, 3, 731−735