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Article history: Adenoviral infections are associated with a wide range of acute diseases, among which ocular viral con-
Received 14 November 2014 junctivitis (EKC) and disseminated disease in immunocompromised patients. To date, no approved spe-
Revised 15 December 2014 cific anti-adenoviral drug is available, but there is a growing need for an effective treatment of such
Accepted 16 December 2014
infections. The adenoviral protease, adenain, plays a crucial role for the viral lifecycle and thus represents
Available online 24 December 2014
an attractive therapeutic target. Structure-guided design with the objective to depeptidize tetrapeptide
nitrile 1 led to the novel chemotype 2. Optimization of scaffold 2 resulted in picomolar adenain inhibitors
Keywords:
3a and 3b. In addition, a complementary series of irreversible vinyl sulfone containing inhibitors were
Adenoviral protease inhibitor
Irreversible inhibitor
rationally designed, prepared and evaluated against adenoviral protease. High resolution X-ray co-crystal
Peptidomimetic structures of representatives of each series proves the successful design of these inhibitors and provides
X-ray co-crystal structure an excellent basis for future medicinal chemistry optimization of these compounds.
Epidemic keratoconjunctivitis (EKC) Ó 2014 Elsevier Ltd. All rights reserved.
Adenoviruses are non-enveloped, double-stranded DNA viruses human homologues. Thus, selective inhibitors of the adenoviral
with more than 55 serotypes identified that have been classified protease may offer efficacious treatments for adenoviral infections.
into seven subgroups (A–G).1 Specific serotypes are linked to a The increasing need for specific and effective anti-adenoviral
number of acute, highly contagious infections such as gastrointes- therapies prompted us to engage in the development of inhibitors
tinal illness, disseminated adenovirus disease in immunocompro- of adenain, in particular for the treatment of EKC caused by sero-
mised patients and ocular conjunctivitis such as epidemic types 8, 37, and 64. We recently disclosed our initial efforts in this
keratoconjunctivitis (EKC).2 The occurrence of severe adenoviral area, which led to the discovery and structure-based optimization
infections is increasing due to the growing numbers of transplan- of glycine- and pyrimidine nitrile inhibitors of the adenoviral pro-
tations. To date, there are no approved anti-adenoviral therapeu- tease. Among other structures our previous hit discovery campaign
tics available, which contrasts with a growing unmet medical identified tetrapeptide nitrile 1 as a potent inhibitor of adenain.13
need for specific anti-adenoviral drugs, in particular for immuno- As part of our ongoing efforts to optimize this compound we aimed
suppressed patients or for an effective treatment of EKC.3–5 at further reducing the peptidic character of 1 (Fig. 1) by designing
Adenoviruses, as all viruses, encode their own protease and a novel replacement of the P4–P3 amide bond and by investigating
depend on its activity for the development of infectivity. The pro- irreversible inhibitors.
tease of adenovirus, the cysteine protease adenain6,7 plays a key While tetrapeptide nitrile 1 proved to be a low nM inhibitor of
role in several steps of the viral life cycle.8 Adenain is involved in adenain in vitro, it was inactive in a viral replication assay. It was
the uncoating of virus particles during viral entry,9 it is essential thus the primary objective of our work to develop compounds with
for the cleavage of seven core precursor proteins that are required increased permeability and, hence, improved cellular potency.
for the formation of mature virions10 and it is also implicated in the Guided by the X-ray co-crystal structure of adenain and bound 1
cleavage of cytokeratins, which leads to host cell lysis.11,12 In addi- we designed a novel peptidomimetic scaffold, where the NH–
tion, adenain represents an attractive therapeutic target as it lacks CH(CH2Ph) unit of the central Phe residue is replaced by a meta-
substituted phenyl ring. Molecular modelling suggested that the
original P4–P3 binding mode could be conserved by introducing
⇑ Corresponding author. Tel.: +41 (0)61 696 1227. an ethanone linker between this phenyl moiety and the P4 phenyl
E-mail address: eva.altmann@novartis.com (E. Altmann). group together with an ortho-substituent on the central phenyl
http://dx.doi.org/10.1016/j.bmcl.2014.12.057
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
P. Grosche et al. / Bioorg. Med. Chem. Lett. 25 (2015) 438–443 439
Table 1
SAR and profiling data for inhibitors 2a–d
R3
O
R2 O
R1 H
Cl N
N
H N
O O
Cl
1 2 3
Compd R R R IC50 AVP8a [lM] IC50 AVP5a [lM] Log PAMPA [10 6
cm s 1
] Solb [mM] PSA [Å2]
c c
2a H H CH3 2.74 1.1 nd nd 108
2b H CH3 CH3 0.95 0.31 ndc ndc 108
2c CH3 CH3 CH3 0.04 0.03 5.0 0.05 108
2d CH3 CH3 (CH3)2N(CH3)2 0.01 0.007 5.5 >1 111
a
Inhibition of AVP8 and AVP514 in an FLT assay using Ac-WLRGG*ARC(PT14)-NH2 as substrate. Data represent mean of at least 2 experiments performed in duplicate.
Individual data points in each experiment were within 2-fold range of each other.
b
High throughput equilibrium solubility at pH 6.8.
c
Not determined.
440 P. Grosche et al. / Bioorg. Med. Chem. Lett. 25 (2015) 438–443
Ser3 Ser 3
Glu5 N-term Glu 5
N-term
Gln 115
Cys122
Arg 48
Arg48 Asn44
Asn 44
Table 2
SAR and profiling data for pyrimidine nitrile inhibitors 3a–b
R
O
N
H
Cl N
N
N
O O
Cl
a
Compd R IC50AVP8 [lM] IC50 AVP5a [lM] EC50 CPEb [lM] Log PAMPA [10 6
cm s 1
] Solc [mM] PSA [Å2]
d d
3a CH3 0.0006 0.0002 >50 nd nd 105
3b (CH3)2N(CH3)2 0.0001 0.0001 3.9 4.2 0.49 108
a
Inhibition of AVP8 and AVP5 in an FLT assay using Ac-WLRGG*ARC(PT14)-NH2 as substrate. Data represent mean of at least 2 experiments performed in duplicate.
Individual data points in each experiment were within 2-fold range of each other.
b
HEK293 cells.
c
High throughput equilibrium solubility at pH 6.8.
d
Not determined.
P. Grosche et al. / Bioorg. Med. Chem. Lett. 25 (2015) 438–443 441
Table 3
SAR and profiling data for vinyl sulfone inhibitors 4–7
Compd Structures IC50 AVP8a [lM] IC50 AVP5a [lM] EC50 CPEb [lM] Log PAMPA [10 6
cm s 1
] PSA [Å2]
Cl
O O H
4 N S O 0.001 0.001 ndc 4.1 118
Cl N
H O
O
O
O H O
N O
N N S
5 H H O 0.01 0.01 4.5 5.6 121
O
Cl Cl
H O H O
N N O
N N S
6 H H O <0.001 <0.001 31 ndd 133
O
Cl Cl
O O
N O
N N S
7 H O H O 0.004 0.002 8.9 5.5 112
Cl Cl
a
Inhibition of AVP8 and AVP5 in an FLT assay using Ac-WLRGG*ARC(PT14)-NH2 as substrate. Compounds were pre-incubated with AVP8 and AVP5, respectively, for 1 h at
23 °C, then the reaction was started by the addition of substrate solution and the IC50 values were determined after 1 h reaction time. Complete experimental details are given
in the Supplementary material. Data represent mean of at least 2 experiments performed in duplicate. Individual data points in each experiment were within 2-fold range of
each other.
b
HEK293 cells.
c
Compound displayed cytotoxicity in HEK293 cells.
d
Not determined.
9500 Ser4
substrate control Ser3
enzyme control
Glu5
Fluorescence life time [ps]
9000 compound 5
Cys122
8500
Gln115
Arg 48
8000
Gly51 Val53
7500
Tyr84
7000
0 50 100 150 200
time (min) Figure 7. X-ray co-crystal structure of vinyl sulfone inhibitor 6 in AVP8 (PDB code
4WX6). The picture was produced using PyMol.
Figure 6. Rapid dilution assay. Time course of substrate cleavage: compound 5
(blue triangles, n = 4); substrate control (black circles, n = 16). Enzyme control
shows the uninhibited reaction (red squares, n = 16). Error bars indicate standard with CH3I and KOtBu. Acid hydrolysis of the nitrile moiety (HCl)
error of the mean.
and subsequent TBTU-mediated coupling of the resulting acid 12
with 2-amino-N-(cyanomethyl)-acetamide yielded the target mol-
The X-ray co-crystal structure of 6 captured in Figure 7 confirms ecule 2c.
the covalent nature of the complex (PDB code 4WX6). The synthesis of the vinyl sulfone-based inhibitors listed in
The active site Cys122 is irreversibly tethered via a thioether to Table 3 followed the route exemplified in Scheme 2 for compound 5.
the carbon distal to the SO2 group of the vinyl sulfone moiety. In Starting from commercially available Boc-L-a-tert-butyl-glycine
addition, the inhibitor has extensive non-covalent interactions 13 and glycine methyl ester 14 we prepared intermediate 15 by
with the substrate binding site. Specifically, both oxygen atoms HBTU-mediated coupling. Treatment of 15 with 4 M HCl in dioxane
of the SO2 group are involved in hydrogen bonding interactions afforded amine 16. Subsequent HBTU-mediated coupling of 3,5-
with the side chain of Gln115 and with the phenolic hydroxyl dichlorophenylacetic acid 17 and amine 16 yielded intermediate
group of Tyr 84. 18. Methyl ester hydrolysis with LiOH followed by coupling of
The preparation of compounds of type 2 and 3 is exemplified in the resulting acid with amine 20 then gave target molecule 5.
Scheme 1 for derivative 2c. In conclusion, we have developed a novel class of highly potent
A Claisen condensation and in situ decarboxylation reaction of and specific non-peptidic inhibitors of the adenoviral protease by
methyl 5-cyano-2-methoxy-benzoate 8 with 3,5-dichlorophenyl- depeptidization of tetrapeptide nitrile 1. Guided by the X-ray
acetic acid 9 in the presence of NaHDMS led to aryl ketone 10.18 co-crystal structure of adenain and 1, molecular modelling studies
The geminal dimethylation to obtain compound 11 was achieved led to the design of a novel peptidomimetic scaffold. Further
442 P. Grosche et al. / Bioorg. Med. Chem. Lett. 25 (2015) 438–443
O
O Cl OH
a Cl b
O
+ O CN
CN O
Cl
O
8 9 Cl 10
O O
Cl c Cl OH d
CN
O O O
Cl Cl
11 12
O
O
H
Cl N
N CN
H
O O
Cl
2c
Scheme 1. Reagents and conditions: (a) NaHDMS, DMF, toluene, 20 ? 10 °C, 62%; (b) CH3I, KOtBu, THF, rt; (c) 6 M aq HCl, AcOH, 100 °C, 27%; (d) 2-amino-N-
(cyanomethyl)-acetamide, TBTU, DIEA, DMF, rt, 75%.
O O O O
a H b
O + H N N
O N 2 O N O
O
H H
OH O
13 14 15
O O O
H
O OH N
H c N O d
N + H
H 2N O O
O
Cl Cl Cl Cl
16 17 18
O O O O
H H O
N O N
N OH e N N S
H + H 2N S H H
O O O
O
Cl Cl Cl Cl
19 20 5
Scheme 2. Reagents and conditions: (a) HBTU, DIEA, CH2Cl2, rt, 81%; (b) 4 M HCl in dioxane, rt, 99%; (c) HBTU, DIEA, CH2Cl2, rt, 56%; (d) LiOHH2O, THF/H2O (6:1) rt, 98%, (e)
HBTU, DIEA, CH2Cl2, rt, 67%.
8. Greber, U. F. Rev. Med. Virol. 1998, 8, 213. 14. These subtypes were selected for testing, because AVP8 is the relevant protease
9. Greber, U. F.; Webster, P.; Weber, J.; Helenius, A. EMBO J. 1996, 15, 1766. for EKC, while inhibition of AVP5 is required for assessment of in vivo activity
10. Pérez-Berná, A. J.; Mangel, W. F.; McGrath, W. J.; Graziano, V.; Flint, J.; San in a rabbit based disease model. Rabbits are not infected by AVP8, but only by
Martín, C. J. Virol. 2014, 88, 1513. AVP5. AVP8 and AVP5 have 76% sequence identity and 88% sequence similarity
11. Chen, P. H.; Ornelles, D. A.; Shenk, T. J. Virol. 1993, 67, 3507. (BLAST).
12. Mangel, W. F.; Banjecki, M. L.; McGrath, W. J. Cell. Mol. Life Sci. 2003, 60, 15. Abagyan, R.; Totrov, M.; Kuznetsov, D. J. Comp. Chem. 1994, 15, 488.
2347. 16. Jones, G.; Willett, R.; Glen, R. C. J. Mol. Biol. 1995, 245, 43.
13. Mac Sweeney, A.; Grosche, P.; Ellis, D.; Combrink, K.; Erbel, P.; Hughes, N.; 17. Copeland, R. A. In Evaluation of Enzyme Inhibitors in Drug Discovery; Copeland, R.
Sirockin, F.; Melkko, S.; Bernardi, A.; Jarousse, N.; Altmann, E. ACS Med. Chem. A., Ed.; John Wiley and Sons: Hoboken, NJ, 2005; p 125.
Lett. 2014, 5, 937. 18. Wu, G.; Yin, W.; Hong, C.; Shen, H. C.; Huang, Y. Green Chem. 2012, 41, 580.