Synthesis of GN8 derivatives and evaluation of their antiprion activity in TSE-infected cells

Article abstract of DOI:10.1016/j.bmcl.2010.12.132

A series of GN8 derivatives were synthesized from various diamines, carboxylic acid derivatives, and nitrogen nucleophiles, and their antiprion activity was tested in TSE-infected mouse neuronal cells. We found that two ethylenediamine units, hydrophobic substituents on the nitrogen atoms, and the diphenylmethane scaffold were essential structural features responsible for the activity. Seven derivatives bearing substituents at the benzylic position exhibited an improved antiprion activity with the IC₅₀ values of 0.51-0.83 μM. Conformational analysis of model compounds suggested that the introduction of the substituent at the benzylic position restricted the conformational variability of the diphenylmethane unit.

Full text of DOI:10.1016/j.bmcl.2010.12.132

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1502–1507
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of GN8 derivatives and evaluation of their antiprion activity
in TSE-infected cells
Tsutomu Kimura a, Junji Hosokawa-Muto a, Yuji O. Kamatari a, Kazuo Kuwata a,b,
⇑
a Center for Emerging Infectious Diseases, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan
b CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of GN8 derivatives were synthesized from various diamines, carboxylic acid derivatives, and
nitrogen nucleophiles, and their antiprion activity was tested in TSE-infected mouse neuronal cells. We
found that two ethylenediamine units, hydrophobic substituents on the nitrogen atoms, and the
diphenylmethane scaffold were essential structural features responsible for the activity. Seven deriva-
tives bearing substituents at the benzylic position exhibited an improved antiprion activity with the
Received 6 December 2010
Revised 27 December 2010
Accepted 29 December 2010
Available online 6 January 2011
IC50 values of 0.51–0.83 lM. Conformational analysis of model compounds suggested that the introduc-
tion of the substituent at the benzylic position restricted the conformational variability of the
diphenylmethane unit.
Keywords:
Prion diseases
Transmissible spongiform encephalopathies
Antiprion compound
Conformational analysis
Ó 2011 Elsevier Ltd. All rights reserved.
Prion diseases, also known as transmissible spongiform enceph-
alopathies (TSEs), are invariably fatal neurodegenerative disorders
of mammals characterized by the accumulation of a pathogenic
isoform (PrPSc) of cellular prion protein (PrPC) in the central ner-
vous system.1,2 These disorders include Creutzfeldt–Jakob disease,
Gerstmann–Sträussler–Scheinker syndrome (GSS), fatal familial
insomnia, and kuru in humans, scrapie in sheep and goat, bovine
spongiform encephalopathy in cattle, and chronic wasting disease
in deer and elk. There is no cure for prion diseases to date.
Since the appearance of a new variant Creutzfeldt–Jakob dis-
ease, considerable effort has been devoted to developing a thera-
peutic treatment for the disease.3,4 Consequently, a number of
compounds have been identified that inhibit PrPSc accumulation
in prion-infected cells.5 However, most of these compounds are
inadequate for use as therapeutic agents for the following reasons:
(i) their antiprion activity is not sufficient and the chemical struc-
ture is unsuitable for the appropriate derivatization necessary for
lead compound optimization; (ii) low blood–brain barrier perme-
ability results in a limited antiprion effect and serious adverse ef-
fects in vivo; and (iii) strain-dependent activity restricts the
drug’s use to a small range of prion diseases.6 Recently, we discov-
ered a novel antiprion compound, N,N0-(methylenedi-4,1-pheny-
lene)bis[2-(1-pyrrolidinyl)acetamide] (GN8),7–10 through a virtual
screening based on the structure of PrPC (Fig. 1).11,12 GN8 is a
promising antiprion lead compound because it is strain-indepen-
dent, and when administered subcutaneously it prolonged the sur-
vival time of TSE-infected mice. Herein, we report the synthesis of
GN8 derivatives and evaluation of their antiprion activity in TSE-
infected cells.
GN8 (1) is composed of a diphenylmethane scaffold and two
2-(1-pyrrolidinyl)acetylamino groups at the 4,40-positions of
diphenylmethane (Fig. 1). To identify the chemical structure
responsible for the antiprion activity and to optimize lead
compound 1, a series of GN8 derivatives 2–7 were prepared. GN8
fragments 2b–f and derivatives 2g–i were synthesized from 4,40-
diaminodiphenylmethane 2a as a starting material (Scheme 1).
Acetylation of 2a with acetic anhydride gave bis(acetamide) 2b.
Dehydrative condensation of 2a with N-Boc-glycine followed by
deprotection with HCl provided bis(2-aminoacetamide) 2c. As a
key precursor for the synthesis of unsymmetrical derivatives, com-
pound 2d was prepared via mono-N-protection of diamine 2a.
Acetamide 2e and 2-aminoacetamide 2f were prepared from 2d
in a similar manner as that for the syntheses of 2b and 2c, respec-
tively. N,N0-Dimethyl-4,40-diaminodiphenylmethane was prepared
by formylation of 2a with ethyl formate and a subsequent reduc-
tion of the resulting bisformamide with LiAlH4. The reaction of
the diamine with bromoacetyl bromide and pyrrolidine gave
Abbreviations: PrPC, cellular form of prion protein; PrPSc, infectious isoform of
prion protein; PrPres, proteinase K-resistant prion protein.
⇑
Corresponding author. Tel.: +81 58 230 6143; fax: +81 58 230 6144.
E-mail address: kuwata@gifu-u.ac.jp (K. Kuwata).
Figure 1. Chemical structure of 1 (GN8).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.132

T. Kimura et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1502–1507
1503
Scheme 1. Synthesis of GN8 derivatives 2b–i. Reagents and conditions: (a) Ac2O, pyridine, DMAP, CH2Cl2; (b) (i) N-Boc-glycine, EDCI, CH2Cl2; (ii) HCl, MeOH/AcOEt; (c) (i)
(Boc)2O, Et3N, THF; (ii) BrCH2C(O)Br, pyridine, DMAP, CH2Cl2; (iii) pyrrolidine, K2CO3, THF; (iv) HCl, MeOH/AcOEt; (d) (i) HCO2Et, THF; (ii) LiAlH4, THF; (e) (i) BrCH2C(O)Br,
pyridine, DMAP, CH2Cl2; (ii) pyrrolidine, K2CO3, THF; (f) c-PenCO2H, EDCI, CH2Cl2; (g) LiAlH4, THF.
N,N0-dimethyl derivative 2g. Condensation of 2a with cyclopentan-
ecarboxylic acid gave bis(cyclopentanecarboxamide) 2h. Reduction
of 1 with LiAlH4 afforded bis(ethylenediamine) 2i.
The synthesis of derivatives 3 and 4, in which one or two pyrro-
lidinyl groups in 1 were replaced with other N-substituted amino
groups, is illustrated in Scheme 2. Symmetrical derivatives 3 were
prepared by the substitution reaction of bis(2-bromoacetamide) 10
with primary and secondary amines in 39–97% yield. For the syn-
thesis of unsymmetrical derivatives 4, a key precursor 2d was used
as the starting material. The reaction of 2d with bromoacetyl bro-
mide and secondary amines provided unsymmetrical derivatives 4
in 40–87% yield. The reaction of 2a with 2-pyridinecarboxylic acid
in the presence of EDCI gave bis(2-pyridinecarboxamide) 5a, and
condensation of 2a with N-Boc-L-proline followed by deprotection
with HCl provided bis(2-pyrrolidinecarboxamide) 5b. Bis[2-(1-pyr-
rolidinyl)-2-methylpropanamide] 5c was prepared by the reaction
of bis(2-bromo-2-methylpropanamide) with pyrrolidine in the
presence of Ag2O.13
Scheme 2. Synthesis of GN8 derivatives 3–5. Reagents and conditions: (a) HNRR0, K2CO3, THF or HNRR0 NaH, DMF; (b) (i) BrCH2C(O)Br, pyridine, DMAP, CH2Cl2; (ii) HNRR0,
K2CO3, THF; (c) 2-PyCO2H, EDCI, CH2Cl2; (d) (i) N-Boc-L-proline, EDCI, CH2Cl2; (ii) HCl, MeOH/AcOEt; (e) (i) BrCMe2C(O)Br, pyridine, DMAP, CH2Cl2; (ii) pyrrolidine, Ag2O,
MeCN/H2O.

1504
T. Kimura et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1502–1507
The derivatives 6 and 7, in which the diphenylmethane unit of 1
was replaced with other scaffolds, were synthesized by the reac-
tion of the appropriate diamines with bromoacetyl bromide and
subsequent substitution of the bromo groups with pyrrolidine
(Schemes 3–5). Monosubstituted and disubstituted 4,40-diam-
inodiphenylmethanes for the synthesis of 7h–l were prepared by
the reaction of carbonyl compounds with aniline in the presence
of aniline hydrochloride.14 Reduction of ketone 7c with NaBH4
gave benzhydryl alcohol 7d.
To evaluate the antiprion activity of GN8 derivatives 2–7, a
mouse neuronal cell culture (GT1-7) persistently infected with
mouse-adapted human GSS agent (Fukuoka-1 strain), designated
GT + FK, was used as a cell culture model of prion disease.15 PrPC
is overexpressed in the GT1-7 cells, and the PrPC level in the
GT1-7 cells is eight times that in N2a cells.16 The amount of pro-
teinase K-resistant prion protein (PrPres) was quantified and com-
pared to that in the negative control. Percentages of PrPres level
relative to the negative control at a concentration of 10 lM and
effective concentrations for 50% inhibition of PrPres (IC50) are
Scheme 4. Synthesis of GN8 derivatives 7a–g. Reagents and conditions: (a) (i)
BrCH2C(O)Br, pyridine, DMAP, CH2Cl2; (ii) pyrrolidine, K2CO3, THF; (b) NaBH4,
MeOH.
shown in Figures 2–5, respectively. The IC50 value of 1 was
1.4
l
M, and the percentage of PrPres at 10
lM was 25%.
Initially, antiprion activity of a series of GN8 derivatives 2 were
tested in GT + FK cells (Scheme 1, Fig. 2). Truncation of one or two
tetramethylene units, pyrrolidine units, or 2-(1-pyrrolidinyl)acetyl
units resulted in a significant decrease in activity compared to that
of 1. Introduction of methyl groups to the amide nitrogen atoms
and replacement of the nitrogen atoms of two pyrrolidine rings
with methine units led to a decrease in activity, whereas use of
bis(ethylenediamine) 2i led to retained activity. These results sug-
gest that the overall structure of 1 contributes to the activity and
that two 2-(1-pyrrolidinyl)ethylamino groups are essential along
with the amide N–H and nitrogen atoms of the pyrrolidine rings
that act as H-bond donors and acceptors, whereas the carbonyl
groups are less important.
Second, the GN8 derivatives bearing a variety of monosubsti-
tuted and disubstituted amino groups at the a-position of carbonyl
Scheme 5. Synthesis of GN8 derivatives 7h–l. Reagents and conditions: (a) PhNH2,
PhNH3Cl; (b) (i) BrCH2C(O)Br, pyridine, DMAP, CH2Cl2; (ii) pyrrolidine, K2CO3, THF.
groups 3–5 were prepared to investigate the effects of the substit-
uents on the nitrogen atoms of the amino groups on antiprion
activity (Scheme 2 and Figs. 3 and 4). Most of the compounds de-
rived from acyclic and cyclic alkyl amines (3a–d, 3f–i, 4a, and 5b)
Figure 2. Antiprion activities of 1 and 2 at 10 lM. Bars represent means SD.
had comparable antiprion activity to 1. In contrast, the derivatives
having hydrophilic substituents on the pyrrolidine rings showed
weak or no antiprion activity at 10 lM. For example, the deriva-
tives bearing a hydroxymethyl group (3n, 3o, 4c, and 4d) or a
methoxycarbonyl group (3p and 3q) at the 2-position of the pyrrol-
idine ring and those bearing a hydroxy group at the 3-position of
the pyrrolidine ring (3s, 3t, 4e, and 4f) were less active than 1.
When one or two pyrrolidinyl groups in 1 was replaced with
hydrophilic morpholinyl groups, the gradual loss of activity was
observed in the order 1 (relative PrPres level = 25%) to the mono-
morpholinyl derivative 4b (relative PrPres level = 77%) and the
Scheme 3. Synthesis of GN8 derivatives 6. Reagents and conditions: (a) (i)
BrCH2C(O)Br, pyridine, DMAP, CH2Cl2; (ii) pyrrolidine, K2CO3, THF.

T. Kimura et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1502–1507
1505
Figure 3. Antiprion activities of 1 and 3 at 10 lM. Bars represent means SD.
Figure 4. Antiprion activities of 1 and 4–7 at 10 lM. Bars represent means SD.
Finally, the GN8 derivatives 7, in which the methylene tether of
two benzene rings was replaced with other linkers, were investi-
gated (Schemes 4 and 5 and Figs. 4 and 5). Replacement of the
central methylene tether with ether, sulfide, carbonyl, or sulfone
linkers did not change the level of activity, whereas the introduc-
tion of a hydroxy group led to a slight decrease in activity. Intrigu-
ingly, derivatives 7f–l bearing one or two alkyl or aryl substituents
on the methylene tether exhibited an improved antiprion activity
with IC50 values of 0.51–0.83 lM (Fig. 5, see Table S1 in
Supplementary data). Among them, the most active derivatives,
7h and 7l, were approximately three times more potent than 1.
No cytotoxicity was observed in the cellular assay with 7h and 7l
at 2 l
M. The PrPC expression level was not affected by the addition
of antiprion active derivatives, as evidenced by the bioassay
without proteinase K digestion (see Supplementary data).
As mentioned above, various compounds have been reported to
be active at low nanomolar concentrations in scrapie-infected cells.
However, these compounds showed weak or no antiprion activity
in our GT + FK cells (Fig. 6). For example, Cp-60,17 edaravone deriv-
Figure 5. IC50 values of 1 and 7f–l in GT + FK cells. Bars represent means SD. ⁄IC50
values of 7h–l were significantly different from that of 1 (P <0.01, Student’s t-test).
ative,18 chrysoidine,19
mides21 were inactive even at 10
red22 was 5.5
M and that of the most effective compound, quina-
crine,23 was 1.1
M. Therefore, the derivatives 7f–l constitute a
D
-penicillamine,20 and indole-3-glyoxyla-
bis-morpholinyl derivative 3j (relative PrPres level = 93%). The
above results indicate that the hydrophobic property of the sub-
stituents on the nitrogen atoms of both terminal amino groups
plays a crucial role in the ligand–protein interaction. PrPres accu-
mulation was facilitated by the addition of imidazolyl derivative
lM. The IC50 value of Congo
l
l
class of the most potent antiprion compounds in GT + FK cells re-
ported so far.
3m. The introduction of methyl groups to the
a-position of car-
bonyl groups did not improve antiprion activity.
Next, we turned our attention to the synthesis of derivatives 6
from various diamines rather than from 2a (Scheme 3 and
Fig. 4). The derivatives containing phenylene or naphthylene units
(6a–d) were inactive at 10 lM. Deletion or elongation of the
central methylene unit led to a slight decrease in activity. On the
other hand, the derivatives containing a diphenylmethane unit
such as the regioisomers of 1 (6g and 6h) and ortho-substituted
diphenylmethane derivatives (6j and 6k) showed a similar order
of activity to that of 1. These results suggest that the diphenylme-
thane scaffold is an essential component that acts as a core
structure to interact with PrPC and to maintain proper distance be-
tween two acylamino groups.
Figure 6. Western blotting of PrPres in GT + FK cells after treatment with 1, 7f–l, N-
(4-chlorophenyl)-2-(1H-indol-3-yl)-2-oxoacetamide (In), Congo red (Co), and quin-
acrine (Qu) at 2 lM.

1506
T. Kimura et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1502–1507
Thermal denaturation study using the circular dichroism indi-
cated that the cellular form of the prion protein is significantly sta-
bilized upon binding with 7l (Fig. 7). The degree of stabilization by
7l, DDH = 14.2 kcal/mol, was approximately twice compared with
that by GN8, DDH = 6.7 kcal/mol,7 essentially consistent with their
IC50 values, 0.51 and 1.40 lM, respectively. The increase in DDH
may be due to the additional interaction between PrPC and substi-
tuent at the benzylic position. Binding affinity of 7l for PrPC was
also elucidated by a surface plasmon resonance (SPR) binding as-
say.7 The derivative 7l had the ability to bind to PrPC with the RU
value of 32, which was larger than that of 1 (RU = 15). These results
indicate that the antiprion effect of these derivatives is attributed
to the binding with the prion protein and stabilization of its
conformation.
The conformational flexibility of the compound is one of the
most important factors in the process of lead compound optimiza-
tion. The improved activity of 7f–l would be attributable to the
conformational rigidity provided by the substituents at the ben-
zylic position rather than additional interactions between PrPC
and the substituents, based upon the fact that all of these deriva-
tives showed increased activity regardless of the properties of their
substituents. To elucidate the effects of the substituents at the ben-
zylic position on the conformation of the diphenylmethane unit,
conformational analysis24,25 of model compounds diphenylme-
thane (10) and 2,2-dimethyl-1,1-diphenylpropane (7i0) was per-
formed using the 6-31G(d) basis set at the B3LYP level with the
GAUSSIAN 03 program (Fig. 8).26 The energy surface of 10 had a large
flat-bottomed area. In contrast, the conformational space of 7i0 was
confined to a narrow valley due to the steric repulsion between the
ortho hydrogen atoms of the phenyl rings and the tert-butyl group.
Therefore, the substituents at the benzylic position restricted the
dihedral angles of the phenyl groups to a relevant orientation
accessible to the binding site of PrPC. Reduction of the degree of
freedom of the compound in the unbound state would be advanta-
geous in terms of the binding affinity because of the resulting
smaller entropic loss upon binding to the biomolecule.
Figure 8. Three-dimensional conformational energy surfaces of model compounds
diphenylmethane [10, (a)] and 2,2-dimethyl-1,1-diphenylpropane [7i0, (b)] gener-
ated by the rotation of two phenyl rings (blue: 0–2 kcal/mol; orange: 2–4 kcal/mol;
In summary, a total of 64 derivatives were systematically
synthesized by the reaction of diamines with carboxylic acid deriv-
atives and nitrogen nucleophiles. It was found that two ethylenedi-
amine units, hydrophobic substituents on the nitrogen atoms, and
the diphenylmethane scaffold are the basic requirements for antip-
rion activity. Seven derivatives bearing one or two substituents at
the benzylic position were found to be more potent than the origi-
green: 4–6 kcal/mol; purple 6–8 kcal/mol). Dihedral angles
u1 and u2 were defined
as Cortho1–Cipso1–CHR–Cipso2 and Cortho2–Cipso2–CHR–Cipso1, respectively.
nal lead compound 1, and the most effective derivatives (7h and 7l)
in the series exhibited antiprion activity with the IC50 value of
0.51 lM. Introduction of the substituents at the benzylic position
restricted the conformational variability of the diphenylmethane
unit, increased the stability of the prion protein upon binding,
and led to an improvement of antiprion activity. Further investiga-
tions focused on the synthesis and evaluation of a series of GN8
derivatives with substituents at the benzylic position as well as
exhaustive conformational analyses of the derivatives are currently
ongoing and will be reported in due course.
Acknowledgments
This work was supported by the Program for the Promotion of
Fundamental Studies in Health Sciences of the National Institute
of Biomedical Innovation, the Molecular Imaging Research Pro-
gram of the Ministry of Education, Culture, Sports, Science and
Technology of Japan, and the CREST program. We thank Ms. Tom-
omi Saeki, Ms. Miku Yamada and Ms. Miki Horii for technical help.
Figure 7. Thermal unfolding profiles of recombinant mouse PrP (amino acids 121–
231, 10.7
using CD spectroscopy. Parameter sets obtained by a nonlinear fit, that is, melting
temperature (Tm) and enthalpy change ( H), without 7l were 68.8 0.4 °C and
34.7 1.7 kcal/mol, respectively, whereas those with 7l were 70.3 0.2 °C and
48.9 2.1 kcal/mol, respectively.
lM) without (red) or with (blue) 7l (100 lM) in acetate buffer pH 4.8
Supplementary data
D
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.132.

T. Kimura et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1502–1507
1507
18. Kimata, A.; Nakagawa, H.; Ohyama, R.; Fukuuchi, T.; Ohta, S.; Doh-ura, K.;
Suzuki, T.; Miyata, N. J. Med. Chem. 2007, 50, 5053.
References and notes
19. Doh-ura, K.; Tamura, K.; Karube, Y.; Naito, M.; Tsuruo, T.; Kataoka, Y. Cell. Mol.
Neurobiol. 2007, 27, 303.
20. Fukuuchi, T.; Doh-ura, K.; Yoshihara, S.; Ohta, S. Bioorg. Med. Chem. Lett. 2006,
16, 5982.
21. Thompson, M. J.; Borsenberger, V.; Louth, J. C.; Judd, K. E.; Chen, B. J. Med. Chem.
2009, 52, 7503.
22. Caughey, B.; Race, R. E. J. Neurochem. 1992, 59, 768.
23. Doh-Ura, K.; Iwaki, T.; Caughey, B. J. Virol. 2000, 74, 4894.
24. Feigel, M. J. Mol. Struct. 1996, 366, 83.
25. Strabner, T. Can. J. Chem. 1997, 75, 1011.
1. Prusiner, S. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13363.
2. Prusiner, S. B.; Scott, M. R.; DeArmond, S. J.; Cohen, F. E. Cell 1998, 93, 337.
3. Cashman, N. R.; Caughey, B. Nat. Rev. Drug Disc. 2004, 3, 874.
4. Mallucci, G.; Collinge, J. Nat. Rev. Neurosci. 2005, 6, 23.
5. Sim, V. L.; Caughey, B. Infect. Disord.: Drug Targets 2009, 9, 81.
6. Zerr, I. Infect. Disord.: Drug Targets 2009, 9, 92.
7. Kuwata, K.; Nishida, N.; Matsumoto, T.; Kamatari, Y. O.; Hosokawa-Muto, J.;
Kodama, K.; Nakamura, H. K.; Kimura, K.; Kawasaki, M.; Takakura, Y.; Shirabe,
S.; Takata, J.; Kataoka, Y.; Katamine, S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
11921.
26. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.;
Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.;
Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.;
Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,
O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken,
V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A.
J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G.
A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.;
Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe,
M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.
GAUSSIAN 03, Revision D.02; Gaussian, Inc.: Wallingford, CT, 2004.
8. Yamamoto, N.; Kuwata, K. J. Phys. Chem. B 2009, 113, 12853.
9. Ishikawa, T.; Ishikura, T.; Kuwata, K. J. Comput. Chem. 2009, 30, 2594.
10. Kranjc, A.; Bongarzone, S.; Rossetti, G.; Biarnés, X.; Cavalli, A.; Bolognesi, M. L.;
Roberti, M.; Legname, G.; Carloni, P. J. Chem. Theory Comput. 2009, 5, 2565.
11. Nicoll, A. J.; Collinge, J. Infect. Disord.: Drug Targets 2009, 9, 48.
12. Hosokawa-Muto, J.; Kamatari, Y. O.; Nakamura, H. K.; Kuwata, K. Antimicrob.
Agents Chemother. 2009, 53, 765.
13. Vachal, P.; Fletcher, J. M.; Hagmann, W. K. Tetrahedron Lett. 2007, 48, 5761.
14. Guzmán-Lucero, D.; Guzmán, J.; Likhatchev, D.; Martínez-Palou, R. Tetrahedron
Lett. 2005, 46, 1119.
15. Milhavet, O.; McMahon, H. E. M.; Rachidi, W.; Nishida, N.; Katamine, S.; Mangé,
A.; Arlotto, M.; Casanova, D.; Riondel, J.; Favier, A.; Lehmann, S. Proc. Natl. Acad.
Sci. U.S.A. 2000, 97, 13937.
16. Nishida, N.; Harris, D. A.; Vilette, D.; Laude, H.; Frobert, Y.; Grassi, J.; Casanova,
D.; Milhavet, O.; Lehmann, S. J. Virol. 2000, 74, 320.
17. Perrier, V.; Wallace, A. C.; Kaneko, K.; Safar, J.; Prusiner, S. B.; Cohen, F. E. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 6073.