Quantum Chemical-Based Protocol for the Rational Design of Covalent Inhibitors

Article abstract of DOI:10.1021/jacs.6b03052

We propose a structure-based protocol for the development of customized covalent inhibitors. Starting from a known inhibitor, in the first and second steps appropriate substituents of the warhead are selected on the basis of quantum mechanical (QM) computations and hybrid approaches combining QM with molecular mechanics (QM/MM). In the third step the recognition unit is optimized using docking approaches for the noncovalent complex. These predictions are finally verified by QM/MM or molecular dynamic simulations. The applicability of our approach is successfully demonstrated by the design of reversible covalent vinylsulfone-based inhibitors for rhodesain. The examples show that our approach is sufficiently accurate to identify compounds with the desired properties but also to exclude nonpromising ones.

Full text of DOI:10.1021/jacs.6b03052

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Communication
Quantum chemical-based Protocol for
the rational Design of covalent Inhibitors
Tanja Schirmeister, Jochen Kesselring, Sascha Jung, Thomas Schneider,
Anastasia Weickert, Johannes Becker, Wook Lee, Denise Bamberger, Peter R.
Wich, Ute Distler, Stefan Tenzer, Patrick Johe, Ute A. Hellmich, and Bernd Engels
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b03052 • Publication Date (Web): 27 Jun 2016
Downloaded from http://pubs.acs.org on June 28, 2016
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Quantum chemical-based Protocol for the
rational Design of covalent Inhibitors
Tanja Schirmeister,a Jochen Kesselring,a Sascha Jung,a Thomas H. Schneider,b Anastasia Weickertb, Jo-
hannes Becker,b Wook Lee,b Denise Bamberger,a Peter R. Wich,a Ute Distler,c Stefan Tenzer,c Patrick
Johé,a Ute A. Hellmich,d Bernd Engelsb
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a Institut für Pharmazie und Biochemie, Johannes Gutenberg Universität Mainz, Staudinger Weg 5, 55128 Mainz.
b Institut für Phys. und Theor. Chemie, Universität Würzburg, Emil-Fischer-Straße 42, 97074 Würzburg.
c Institut für Immunologie, Universitätsmedizin der Johannes-Gutenberg Universität Mainz, Langenbeckstr. 1, 55131 Mainz.
d Institut für Pharmazie und Biochemie, Johannes Gutenberg Universität Mainz, J.-J. Becherweg 30, 55128 Mainz and Cen-
ter for Biomolecular Magnetic Resonance (BMRZ), Goethe University, Frankfurt, Germany.
Supporting Information Placeholder
inhibitors comprise at least two very different steps (Figure 1).9
ABSTRACT: We propose a structure-based protocol for the
In the first step a non-covalent enzyme inhibitor complex (E⋅⋅⋅I,
Figure 1) is formed. Its stability (ΔGB) and its geometrical
arrangement which are mainly influenced by the interactions
between the recognition unit of the inhibitor and the enzyme
environment determine if the subsequent chemical reaction
leading to the covalent complex E–I can take place. The free
reaction energy ΔGR of this subsequent chemical reaction which
strongly depends on the chemical properties of the warhead is
mainly responsible whether the covalent inhibition step is
reversible or irreversible. Consequently, in the design of
covalent drugs recognition unit and the warhead have to be
optimized concomitantly.
development of customized covalent inhibitors. Starting from a
known inhibitor, in the first and second steps appropriate sub-
stituents of the warhead are selected on the basis of quantum
mechanical (QM) computations and hybrid approaches combin-
ing QM with molecular mechanics (QM/MM). In the third step
the recognition unit is optimized using docking approaches for
the non-covalent complex. These predictions are finally verified
by QM/MM or molecular dynamic simulations. The applicabil-
ity of our approach is successfully demonstrated by the design
of reversible covalent vinylsulfone-based inhibitors for
rhodesain. The examples show that our approach is sufficiently
accurate to identify compounds with the desired properties but
also to exclude non-promising ones.
This paper present a quantum chemical based protocol for the
rational design of covalent ligand with desired properties. As a
proof of principle we apply it to derive reversible covalent
inhibitors of rhodesain. Such covalent inhibitors currently
experience an intensive renaissance not only in academic1 but
also in industrial drug development due to their various
advantages, including prolonged residence times, lower
sensitivity against pharmacokinetic aspects, and high efficacy.2
Examples are kinase inhibitors, e.g. afatinib or ibrutinib,3 or
proteasome inhibitors, e.g. carfilzomib4 or marizomib.5,6 In the
first case non-covalent kinase inhibitors have been converted
into covalent ones by introducing an α,β-unsaturated amide
moiety which covalently and irreversibly reacts with a Cys
residue nearby the active site. The same approach has been
applied to the GTPase K-Ras which opened a door for targeting
drug targets which were thought to be undruggable.7 In case of
the two proteasome inhibitors the ligands stem from natural
products which have been found serendipitously. Also many
Chart 1: Lewis structures of the inhibitors. K11777: X=Y=H,
R2=4-Me-piperazinyl; 1: X=Y=H, R2=4-pyridinyl; 2: X=Cl, Y=H,
R2=4-Me-piperazinyl; 3: X=F, Y=H, R2=4-Me-piperazinyl; 4:
X=Br, Y=H, R2=4-Me-piperazinyl; 5: X=F, Y=H, R2=4-pyridinyl;
6: X=Cl, Y=H, R2=4-pyridinyl; for compounds with X=CN,
Y=NHR1, R=SCH3 see Supplementary Information.
Various tools to design covalent inhibitors are available,10 but
the so called covalent docking is not as well established and
elaborated as the corresponding methods for the design of non-
covalent ones.11 Some problems arise because the approaches
mainly focus on the final covalent enzyme-inhibitor complex
but neglect the properties of the initially formed non-covalent
complex. Drawbacks may also result because reaction barrier
and reaction energy of the covalent step are neglected in most
covalent docking approaches.
other
marketed
covalent
drugs
were
discovered
serendipitously.1,8
This is due to the fact that the design of covalent drugs is more
complicate than the development of their non-covalent
counterparts because the reaction mechanisms of covalent
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calculations were performed with the CHEMSHELL package
in combination with the TURBOMOLE program.21 Interaction
between QM and MM parts were treated by an electrostatic em-
bedding scheme.12 These QM/MM computations predict a reac-
tion barrier of about 6 kcal/mol and a reaction energy of about
-23 kcal/mol. Both values are in good agreement with experi-
ments which find an efficient and irreversible inhibition of
rhodesain by K11777. The corresponding potential energy sur-
face (PES) is given in the Supporting Information.
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Figure 1. Energy diagram of the inhibition mechanism of a cova-
lently reacting ligand.
Here, we present a new protocol, which can give valuable in-
formation for the rational design of covalent inhibitors. It com-
putes the reaction course via QM/MM hybrid approaches12 that
are also used in other multiscale-modeling areas.13 In our ap-
proach, the warhead and the involved residues were included
into the QM-part while the influence of the enzyme environ-
ment on the reaction profile was taken into account via force
field approaches (MM). As input, our protocol requires an X-
ray structure of an appropriate enzyme-inhibitor complex. For
the present example we use the well-known vinylsulfone (VS)
K11777 (Chart 1, X=Y=H, R2=4-Me-piperazinyl) which irre-
versibly inhibits rhodesain.14 The formation of the covalent
complex E–I out of the initially formed non-covalent complex
E⋅⋅⋅I starts with an addition of the deprotonated Cys25 residue
of rhodesain to C1 of the vinylsulfone double bond (Chart 1).
The inhibition reaction is completed by a transfer of the proton
from the protonated His162 residue to C2.15
Figure 3. Computed potential energy surface of the covalent bond
formation between rhodesain and the compound with X=F, Y=H
(cpd. 3). The numbers gives the relative energies with respect to the
covalent enzyme-inhibitor complex. The corresponding PES for
X=Cl and Br (cpds. 2, 4) are provided in the Supporting Infor-
mation.
For irreversible inhibitors the risk for potential toxic effects is
higher.22 In order to identify covalent, but reversible inhibitors,
in Step I of our protocol we screened VSs to find those substi-
tution patterns for which the addition reaction is only slightly
exothermic because such compounds should act reversibly. Be-
cause we only need rough estimates at that point we computed
reaction energies and used the model reaction of the given VS
with methylthiol in a polar medium. The reaction energies were
obtained from B3LYP17 calculations17 in combination with the
TZVP18 basis set. In all computations, the COSMO approach
with e=78.39 was employed.23 The calculations predicted that
the substitution pattern X=Hal, Y=H should be appropriate be-
cause the reaction is less exothermic than the corresponding re-
action of the warhead of K11777.24 For X=Br or Cl we com-
puted reaction energies of about -6 kcal/mol while for X=F -10
kcal/mol were predicted. We also tested more bulky groups for
the position of Y but the reactions became endothermic. These
findings are in line with previous investigations about the influ-
ence of substituents on the reaction energies of VSs.25
Figure 2. Protocol for the development of covalent reversible in-
hibitors starting from an irreversible inhibitor.
In Step II of the protocol the influences of the enzyme environ-
ment on the inhibition reaction with these promising patterns
were calculated. We started with the non-covalent enzyme-in-
hibitor complex of K11777 (X=Y=H), substituted X=H by Br,
Cl, or F and computed the reaction profiles using the same
QM/MM approach as described above. Figure 3 shows the re-
action profile for X=F (3). The distance d(S-C) between the S-
atom of Cys25 of rhodesain and C1 of the inhibitor describes
the attack of the thiolate at the double bond while d(N-H) mim-
ics the proton transfer from His162 of rhodesain to C2 of the
Prior to the investigations we used the X-ray structure of the
covalent complex E–I of K11777 and rhodesain (PDB ID
2P7U)16 and computed the reaction course backwards to the
corresponding non-covalent complex E⋅⋅⋅I. The computations
were performed with QM/MM. For the QM-part we used
BLYP17/TZVP18 for energy computations. The protein environ-
ment was modelled at the force field level using the AMBER
parameterization19 in combination with the DL Poly code.20 All
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inhibitor. All other geometrical parameters are optimized to ob-
tain the minimum energy path (MEP) of the reaction course.
The reaction starts at the non-covalent enzyme-inhibitor com-
plex which is predicted to be about 16 kcal/mol higher in energy
than the covalent enzyme inhibitor complex, the end point of
the inhibition. The inhibition reaction has a barrier of about 7
kcal/mol. For X=Cl and Br (2, 4) we computed barriers of about
12 and 13 kcal/mol and reaction energies of -11 and -10
kcal/mol.15 The reactions of irreversible inhibitors were com-
puted to be more exothermic than -22 kcal/mol.9c,12d With -10
and -11 kcal/mol the inhibition reactions are clearly reversible
for X=Cl and Br while X=F seems to represent a border case..
DOCKTITE10c indicated that the replacement of the N-methyl
piperazine moiety by a pyridine ring should increase the affin-
ity. The corresponding score values are discussed in the SI. In
Step IV of our protocol MD simulations showed that the cova-
lent reaction step is still possible (see SI). They also supported
the orientations predicted by the employed docking approach.
The syntheses and testing of the respective reversible inhibitors
with X=F (5) and Cl (6) and also with X=H (1), an irreversible
inhibitor like K11777, indeed showed much higher affinities: Ki
values [nM]: 3.7 (H, 1, k2nd=1.9 • 106 M-1s-1), 32 (F, 5), 190 (Cl,
6), i.e. the predictions were again fully confirmed. The new re-
versible halogenated compounds (5, 6) also show slightly better
anti-trypanosomal activity compared to the N-methy piperazi-
nyl derivatives (2, 3), but exhibit less cytotoxicity than their ir-
reversible counterpart (1): (EC50 [µM] (T. brucei) / CC50 [µM]
(J774.1 macrophages) / CC50 [µM] (HELA cells): (5): 3.0 / >
100 / > 500; (6): 3.1 / > 100 / > 500; (1): 1.7 / 8.6 / 11; (3): 12.5
/ 38 / 10; (2): 13 / 23 / 19.
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4000
3000
DMSO control
cpd. (X=F)
3
We also used the covalent docking program DOCKTITE10c to
predict the inhibition potencies. DOCKTITE could indeed be
trained to recognize halogenated vinylsulfones as covalent in-
hibitors. It is also able to reproduce the structure of the final
covalent enzyme-K11777 complex. However, the computed
scores do not reflect the experimental trends in Ki values and do
not distinguish between irreversible or reversible inhibition.15
2000
1000
0
cpd. (X=Cl)
2
cpd. (X=Br)
4
K11777 (X=H)
0
500
1000
1500
2000
2500
This successful example indicates that the employed theoretical
approaches are very helpful for the design of covalent inhibitors
with desired properties. This is underlined by a second investi-
gation that was unsuccessful in terms of new reversible inhibi-
tors but proved the accuracy of our theoretical approaches. QM
computations in solution (Step I) indicated that inhibitors with
X=CN, R=SCH3, and Y=NHCH3 can reversibly block the en-
zyme via an SNV-mechanism. The results were confirmed by
NMR measurements in solution24 but QM/MM computations in
Step II predicted that the corresponding SNV reaction within the
enzyme is strongly endothermic (ΔEreac>+30 kcal/mol), i.e. no
inhibition is expected. However, first test measurements indi-
cated weak inhibition.15 The contradiction could finally be re-
solved by additional assays. They showed that a non-competi-
tive inhibition takes place, i.e. the inhibitors do not react with
the active site for which the computations were performed. This
example also shows that computations for the solvent situation
alone (only Step 1) might lead to wrong conclusions because
the enzyme environment strongly influences the reaction
course.
Time [s]
Figure 4: Experimental verification of the (ir)reversibility of the in-
hibition via dilution assays. The enzyme was incubated with an in-
hibitor concentration corresponding to 10 x the Ki to assure com-
plete inhibition; then the incubation mixture was diluted by a factor
of 100 yielding an inhibitor concentration of 0.1 x the Ki. The en-
zyme activity was then measured by adding the substrate.
To prove our predictions we synthesized the compounds
(K11777, 2-4) and tested their inhibition potencies by fluoro-
metric enzyme assays.24 The reversibility of the inhibition was
proven by dilution assays (Figure 4) and by dialysis assays (see
Supporting Information). For X=Br (4) mass spectrometry
could prove that the compound reacts covalently with the active
site Cys residue (see SI part).15 For X=F (3, 5), stable interac-
tions between compound and protein could additionally be
shown by 19F NMR spectroscopy.15 In case of the Br derivative
(4) the recovery of the enzyme activity was found to proceed
very slowly with only 20% activity after 60 min (Figure 4). The
MS data (see SI part) indicate that the inhibition by cpd. 4 be-
comes irreversible due to slow elimination of HBr. This is sup-
ported by QM/MM computations.15 Compared to K11777 the
Ki values for the halogenated derivatives increase: 20 nM
(K11777; k2nd =6.6 • 105 M-1s-1); 190 nM (F, 3), 1.01 µM (Cl,
2), 0.86 µM (Br, 4), i.e. the halogenated inhibitors exhibit lower
affinities.
We have proposed a new protocol which is very helpful for the
development of covalent inhibitors with desired properties. Its
applicability was successfully demonstrated by the design of re-
versible covalent vinylsulfone-based inhibitors for rhodesain.
Our results can directly be used for other vinylsulfone-based in-
hibitors.9,14,27 It should also be applicable to other compound
classes because it is based on highly reliable quantum chemical
approaches. Our approach could be combined with the faster
covalent docking approaches. While the latter is used for
screening our approach could be used to investigate the most
promising examples in more detail.
So far, we only changed the chemical properties of the warhead
but variations of the properties of the recognition unit are also
important. In our protocol promising variations in the recogni-
tion unit are investigated in Step III using standard docking rou-
tines. In our approach the docking can be performed directly for
the targeted non-covalent complex because we computed it in
the previous QM/MM computations. The docking studies per-
formed with the FlexX program package (version 2.1.3)15,26 and
ASSOCIATED CONTENT
Supporting Information
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Further figures as well as the complete experimental details for syn-
†Wook Lee: Department of Chemistry, Temple University, Phila-
theses and testing. This material is available free of charge via the
Internet at http://pubs.acs.org.
delphia, Pennsylvania, 19122, United States
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ACKNOWLEDGMENT
Financial support by the DFG (Deutsche Forschungsgemeinschaft)
(SFB630) is gratefully acknowledged. UAH acknowledges support
by the Carl-Zeiss foundation as well as the Center of Biomolecular
Magnetic Resonance (BMRZ) funded by the state of Hesse.
AUTHOR INFORMATION
Corresponding Authors
*schirmei@uni-mainz.de; *bernd.engels@uni-wuerzburg.de
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Present Addresses
REFERENCE
1(a) Singh, J.; Petter, R.C.; Baillie, T.A.; Whitty, A. Nat. Rev. Drug
Discovery 2011, 10, 307 (b) Groll, M.; Huber, R.; Potts, B.C. J.
Am. Chem. Soc. 2006, 128, 5136 (c) Smith, A. J.; Zhang, X.; Leach,
A.G.; Houk, K.N. J. Med. Chem. 2009, 52, 225 (d) Weisner, J.;
Gontla, R.; Van der Westhuizen, L.; Oeck, S.; Ketzer, J.; Janning,
P.; Richters, A.; Mühlenberg, T.; Fang, Z.; Taher, A.; Jendorossek,
V.; Pelly, S.C.; Bauer, S.; van Otterlo, W. A., Rauh, D. Angew.
Chem. Int. Ed. Engl. 2015, 54, 10313.
Thiel, W. Angew Chem Int Ed Engl 2009, 48, 1198 (d) Mladenovic,
M.; Fink, R.F.; Thiel, W.; Schirmeister, T.; Engels B. J. Am. Chem.
Soc. 2008, 130, 8696.
13 (a) Robles, V.M.; Heinisch, T.; Lledos, A.; Schirmer, T.; Ward,
T.R.; Marechal, J.D. J. Am. Chem. Soc. 2014, 136, 15676 (b)
Wallrapp, F.; Masone, D.; Guallar V. J. Phys.Chem A 2008, 112,
12989 (c) Glowacki, D.R.; Harvey, J.N.; Mulholland, A.J. Nat.
Chem. 2012, 4, 169.
2
Evers, A.; Hessler, G.; Wang, L. H.; Werrel, S.; Monecke, P.;
14 (a) Yang, P.Y.; Wang, M.; He, C.Y.; Yao, S.Q. ChemCom 2012,
48, 835 (b) Roush, W.R.; Gwaltney, II S.L.; Cheng, J.; Scheidt,
K.A.; McKerrow, J.H.; Hansell, E. J. Am. Chem. Soc. 1998, 120,
10994 (c) Palmer, J. T.; Rasnick, D.; Klaus, J. L.; Bromme, D. J
Med. Chem. 1995, 38, 3193 (d) Ettari, R.; Tamborini, L.; Angelo,
I. C.; Micale, N.; Pinto, A.; De Micheli, C.; Conti, P. J. Med. Chem.
2013, 56, 5637.
Matter, H. J. Med. Chem. 2013, 56, 4656 (b) Flanagan, M. E.; Ab-
ramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert,
A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.;
Schuff, B. P.; Uccello, D. P.; Walker, G. S.; Wu, Y.; Brown, M. F.;
Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe,
L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che,
Y. J. Med. Chem. 2014 57, 10072.
15 Further details of the employed methods, extra graphs and further
discussions are provided in the SI.
3 Hirsh, V. BioDrugs 2015, 29, 167.
4 Muchtar, E.; Gertz, M.A.; Magen, H. Eur. J. Haematol 2016, 96
564.
16 Kerr, I.D., Lee, J.H., Farady, C.J., Marion, R., Rickert, M., Sajid,
M., Pandey, K.C., Caffrey, C.R., Legac, J., Hansell, E., McKerrow,
J.H., Craik, C.S., Rosenthal, P.J., Brinen, L.S. J. Biol. Chem. 2009,
284, 25697 .
5 Groll, M.; Potts, B.D. Curr.Top.Med.Chem. 2011, 11, 2850
6 (a) Miller, R. M.; Paavilainen, V. O.; Krishnan, S.; Serafimova,
I. M.; Taunton, J. J. Am. Chem. Soc. 2013, 135, 5298 (b) London,
N.; Miller, R. M.; Krishnan, S.; Uchida, K.; Irwin, J. J.; Eidam,
O.; Gibold, L.; Cimermančič, P.; Bonnet, R.; Shoichet, B. K.;
Taunton, J. Nat. Chem. Biol. 2014, 10, 1066.
17 (a) Becke, A. D. Phys. Rev. A 1988, 38, 3098 (b) A. D. Becke,
J. Chem. Phys., 1993, 98, 5648 (c) Lee, C. T.; Yang, W. T.; Parr,
R. G. Phys. Rev. B 1988, 37, 785.
18 Weigand F.; PCCP 2005, 7 3297.
7 (a) Cox, A.D.; Fesik, S.W.; Kimmelman, A.C.; Luo, J.; Der, C. J.
Nat. Rev. Drug Disc. 2014, 13, 828 (b) Lim, S.M.; Westover, K.D.;
Ficarro, S.B.; Harrison, R.A.; Choi, H.G.; Pacold, M.E.; Carracso,
M.; Hunter, J. Kim, N.D.; Xie, T.; Sim, T.; Jänne, P.A.; Meyerson,
M.; Marto, J.A.; Engen, J.R. Gray, N.S. Angew. Chem. Int.Ed.Engl.
2014, 53, 199 (c) Rudolph, J.; Stokoe, D. Angew. Chem.
Int.Ed.Engl. 2014, 53, 3777.
19 Cornell, W. D.; Cieplak P.; Bayly, C.I.; Gould, I. R.; Merz, K.
M. Jr.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J.
W.; Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 5179.
20 Smith, W.; Forester, T. J. Mol. Graph., 1996, 14, 136.
21 (a) Sherwood, P.; de Vries, A. H.; Guest, M. F.; Schreckenbach,
G.; Catlow, C. R. A.; French, S. A.; Sokol, A. A.; Bromley, S. T.;
Thiel, W.; Turner, A. J. THEOCHEM 2003, 632, 1.; (b)
TURBOMOLE V6.1 2009; University of Karlsruhe and For-
schungszentrum Karlsruhe GmbH: Karlsruhe, Germany, 2007.
8
Singh, J.; Petter, R. C.; Kluge, A. F.; Curr. Opin. Chem. Biol.
2010, 14, 475.
9
22
(a) Otto, H. H.; Schirmeister, T. Chem. Rev. 1997, 97 133 (b)
Johnson, D. S. Weerapana, E. Cravatt, B. F. Fut. Med. Chem
Powers, J.C.; Asgian, J. L.; Ekici, O. D.; James, K. E.; Chem. Rev.
2002, 102, 4639; (c) Mladenovic, M.; Junold, K; Fink, R.F.; Thiel,
W.; Schirmeister, T.; Engels, B. J. Phys. Chem. B 2008, 17, 5458.
10 (a) Jones, G.; Willett, P.; Glen, R. C. J. Mol. Biol. 1995, 245, 43
(b) Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G.: J. Mol. Biol.
1996, 261, 470 (c) Mcmartin, C.; Bohacek, R. S. J. Comput. Aid.
Mol. Des. 1997, 11, 333 (c) Scholz, C.; Knorr, S.; Hamacher, K.
Schmidt, B. J. Chem. Inf. Model, 2015, 55, 398.
2010, 2, 949.
23 A. Klamt and G. Schüurmann J. Chem. Soc., Perkin Trans. 1993,
2, 799.
24 T. H. Schneider, M. Reiger, K. Ansorg, A. N. Sobolev, T. Schir-
meister, B. Engels, S. Grabowsky New J. Chem. 2015, 39, 5841.
25 (a) Meadows, D.; C.; Gervay-Hague, J. Med. Res. Rev. 2006,
26, 793 (b) Bernasconi, C. F.; Rappoport, Z Acc. Chem. Res.,
2009, 42, 993 (c) Shainyan, B. A. J. Phys. Org. Chem., 1993, 6,
59.
11 (a) Ouyang, X.; Zhou, S.; Su, C. T. T.; Ge, Z.; Li, R.; Kwoh, C.
K. J. Comput. Chem. 2013, 34, 326 (b) Schröder, J.; Klinger, A.;
Oellien, F.; Marhöfer, R. J.; Duszenko, M.; Selzer, P. M. J. Med.
Chem. 2013, 56, 1478.
26
LeadIT/FlexX; Version 2.1.3; BioSolveIT GmbH, An der
Ziegelei 79, St. Augustin, Germany, 2012.
27
Doherty, W.; James, J.; Evans, P.; Martin, L.; Adler, N.; Nolan,
12
(a) Peters, M. B.; Raha, K.; Merz, K. M. Curr. Op. Drug Disc.
D.; Knox, A. Org. & Biomol. Chem. 2014, 12, 7561.
2006, 3, 370. (b) Lawan, N.; Ranaghan, K. E.; Manby, F. R.; Mul-
holland, A. J. Chem. Phys. Lett. 2014, 608, 380 (c) Senn, H. M.;
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