Molecular design of small organic molecules based on structural information for a conformationally constrained peptide that binds to G-CSF receptor

Article abstract of DOI:10.1016/j.bmcl.2009.12.010

Based on structural information for a peptide (P8-2KAQ) that binds to granulocyte-colony stimulating factor receptor (G-CSFR), small ligands with a biaryl scaffold were designed and their binding affinities were evaluated.

Full text of DOI:10.1016/j.bmcl.2009.12.010

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1169–1172
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Molecular design of small organic molecules based on structural
information for a conformationally constrained peptide that binds to G-CSF
receptor
b
a
a
Radwan El-Haggar a, Ken Kamikawa b, , Kazuya Machi , Zhengmao Ye , Yuko Ishino ,
*
Takeshi Tsumuraya a, Ikuo Fujii a,
*
a Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
b Department of Chemistry, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on structural information for a peptide (P8-2KAQ) that binds to granulocyte-colony stimulating
factor receptor (G-CSFR), small ligands with a biaryl scaffold were designed and their binding affinities
were evaluated.
Received 7 November 2009
Revised 27 November 2009
Accepted 2 December 2009
Available online 4 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Helix-loop-helix peptides
Peptidomimetics
G-CSF receptor
N-Benzyl aniline ligand
Biphenyl ligand
Despite intense research into the design of small ligands that
target protein–protein interaction interfaces, a novel methodology
for the rational design of such ligands remains an elusive goal. To
design such ligands, our group has examined the directed evolu-
tion of peptides in a phage-displayed library of conformationally
constrained peptides. Screening of the library against targeted pro-
teins should provide bioactive peptides whose rigid structures
indicate the required spatial orientation of the pharmacophores,
thus facilitating structure-based design of peptidomimetics. In a
previous study, we constructed a library of de novo designed he-
lix-loop-helix peptides (35 amino acids).1 This structural motif al-
account the spatial orientations of these amino acid residues sug-
gested that N-benzyl aniline and biphenyl frameworks would be
suitable scaffolds for peptidomimetics of this helix-loop-helix pep-
tide. Thus, we designed and synthesized the four ligands listed in
Figure 1. The design of these ligands took into account simulations
of all the amino acid side chains involved in the binding of the ac-
tive peptide with G-CSF receptor. N-Benzyl aniline derivative li-
gand 2 was synthesized first (Scheme 1). Commercially available
3-nitrocinnamic acid 6 was treated with water soluble carbodiim-
ide (WSCI), 1-hydroxybenzotriazole (HOBT) and aqueous ammonia
to give amide 7 in 55% yield. Both the double bond and the nitro
group of 7 were reduced by hydrogenation to give arylamine 8 in
91% yield. Reductive amination of 8 with aldehyde 9 using
NaBH3CN gave N-benzyl aniline 10. N-Benzyl aniline 10 was sub-
jected to a second reductive amination and subsequent hydrolysis
to afford acid 11. Finally, 11 was hydrolyzed using TFA to give N-
benzyl aniline ligand 2 in 88% yield.
Synthesis of another N-benzyl aniline derivative ligand 3 is de-
picted in Scheme 2. Reductive amination of 3-bromoaniline with
aldehyde 9 afforded the secondary amine 13 in 91% yield. Next, a
second reductive amination of 13 with methyl 3-oxopropanoate
and subsequent hydrolysis by NaOH furnished acid 14 in 85% yield.
N-Benzyl aniline 14 was treated with WSCI, HOBT and aqueous
ammonia to give amide 15 in 87% yield. Amide 15 was subjected
to the Mizoroki–Heck reaction5 with methyl acrylate, affording
16 in 55% yield. Next, the reduction of the double bond in 16 gave
lows polypeptides to form stable
a
-helices2 that often present
recognition sequences in biological processes.3
We have applied our method to ligand design of granulocyte-
colony stimulating factor (G-CSF) receptor that an important cyto-
kine receptor stimulating bone marrow to release granulocytes and
stem cells into the blood.4 The phage-displayed library was
screened to give a binding peptide, P8-2KAQ (1). Here, we report
the design and synthesis of small molecule ligands for the receptor,
based on structural information of the helix-loop-helix peptide.
The binding affinity of P8-2KAQ (1) was found to be dependent
on the amino acids leucine, lysine, glutamic acid and glutamine on
the
a-helix (i, i+2, i+4 and i+5). Virtual screening which took into
* Corresponding authors. Tel./fax: +81 72 254 9834 (I.F.).
E-mail address: fujii@b.s.osakafu-u.ac.jp (I. Fujii).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.010

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R. El-Haggar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1169–1172
H2N
O
O
H2N
O
O
O
O
HO
N
H2N
N
NH2
OH
O
O
2
3
H2N
O
O
H2N
O
O
NH2
OH
O
O
HO
O
H2N
O
4
5
Figure 1. Design of tetrasubstituted ligands.
O
O
WSCI, NH4OH aq.
NO2
NO2
H2N
HO
HOBT, CH2Cl2
55%
6
7
BocHN
O
O
O
NH2
CHO
Pd/C, H2, MeOH
91%
9
H2N
NaBH3CN, MeOH, AcOH
91%
8
BocHN
O
O
BocHN
O
O
O
O
O
1.
H
OMe
HN
HO
N
NaBH3CN, MeOH, AcOH
2. NaOH, MeOH
NH2
NH2
68%, 2 steps
O
O
10
11
TFA, CH2Cl2
88%
2
Scheme 1. Synthesis of N-benzyl aniline ligand 2.

R. El-Haggar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1169–1172
BocHN O
1171
BocHN
O
O
O
CHO
9
Br
NH2
HN
NaBH3CN, MeOH, AcOH
91%
12
Br
13
BocHN
O
O
O
O
1.
H
OMe
O
WSCI, NH4OH aq.
NaBH3CN, MeOH, AcOH
HO
N
HOBT, CH2Cl2
87%
2. NaOH, MeOH
85%, 2 steps
Br
BocHN
14
BocHN
O
O
O
O
O
OMe
O
O
Pd(PPh3)4, DMF, Et3N
55%
H2N
N
H2N
N
OMe
Br
BocHN
15
O
16
O
O
O
Pd/C, H2, MeOH
95%
1. NaOH, MeOH
2. TFA, CH2Cl2
77%, 2 steps
3
H2N
N
OMe
17
O
Scheme 2. Synthesis of N-benzyl aniline ligand 3.
17. Completion of the requisite N-benzyl aniline ligand 3 was
achieved by ester hydrolysis and then deprotection.
in 77% yield over two steps. A second biphenyl ligand 5 was also
synthesized using a similar procedure.9
We next examined the synthesis of biphenyl ligands. Hamilton
and co-workers reported that a terphenyl scaffold could reason-
ably mimic functionality in a spatial orientation, involving side
chains of the i, i+3 and i+7 residues on an
the important pharmacophores of the G-CSFR binding peptide
The binding affinities of ligands 2–5 for G-CSF receptor were
evaluated using surface plasmon resonance (SPR) biosensor tech-
niques, which directly provide information regarding the charac-
teristics of small molecule–protein interactions, and allow real-
time monitoring of the interactions (Table 1).10 Initially, N-benzyl
aniline ligand 2 showed promising binding affinity for G-CSF
a
-helix.6 In our case,
are enshrined on the amino acid residues of i, i+2, i+4 and i+5
on the
a
-helix. Although the positions of the targeted residues
receptor, with a dissociation constant (Kd) of 47 lM (entry 1). As
are different, it would be probable that the biphenyl framework
can also act as an effective scaffold for the design of small ligands.
Therefore, we designed and synthesized tetrasubstituted biaryl li-
gands 4 and 5 with four residues attached at the 3-, 30-, 40- and 5-
positions.
expected from the design, replacement of the functional groups
at positions R3 and R4 resulted in total loss of binding affinity (en-
try 2, ligand 3). Biphenyl ligand 4 bound to G-CSF receptor with a
Kd of 20 lM (entry 3). On the other hand, the replacement of func-
tional groups in the biphenyl scaffold resulted in loss of binding
affinity (entry 4, ligand 5), similar to that with the N-benzyl aniline
scaffold.
The synthesis of biphenyl ligand 4 is shown in Scheme 3. 2-Bro-
mo-5-hydroxybenzaldehyde 187 was first converted to an
a,b-
unsaturated ester by Horner–Wadsworth–Emmons olefination,
followed by treatment with NaOH/MeOH to afford acid 19. Treat-
ment of 19 with WSCI, HOBT, and aqueous ammonia provided
the corresponding amide 20 in 62% yield. Amide 20 was reacted
with t-butyl acrylate using the Mizoroki–Heck protocol to give
Thus, the position of the functional groups strictly affects affin-
ity in both N-benzyl aniline and biphenyl ligands. These observa-
tions suggest that the observed binding affinity is due to specific
interactions between the ligands and the receptor, and are not
due to nonspecific hydrophobic interactions.
a
,b-unsaturated ester 21. Next, two double bonds in 21 were re-
In conclusion, we designed and synthesized small molecules
binding to G-CSF receptor based on the structure of a conforma-
tionally constrained helix-loop-helix peptide. Both N-benzyl ani-
line and biphenyl scaffolds were found to be candidates for small
compounds binding to G-CSF receptor. The interaction of G-CSF
with its receptor stimulates survival, proliferation, differentiation
and function of neutrophil precursors and mature neutrophils.11
duced by hydrogenation to give phenol 22. Phenol 22 was trans-
formed into trifilate 23 in moderate yield (68%). Trifilate 23 was
coupled with boronic acid 24 under Suzuki–Miyaura cross-cou-
pling conditions8 to give the biphenyl coupling product. Subse-
quently, deprotection of the biphenyl compound using acetic acid
pre-saturated with hydrochloric acid afforded the target ligand 4

1172
R. El-Haggar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1169–1172
OH
OH
1. (EtO)2POCH2CO2Et,
WSCI, NH4OH aq.,
NaH, THF, 90%
OH
HOBT, CH2Cl2
62%
2. NaOH, MeOH, 86%
CHO
Br
19
O
Br
18
OH
OH
O
tBuO
NH2
NH2
O
Pd(PPh3)4, DMF,
Br
O
Et3N, 98%
20
tBuO
O
21
OH
OTf
Pd/C, H2, MeOH
90%
Tf2O, pry.
64%
NH2
NH2
O
O
tBuO
O
tBuO
O
22
23
BocHN
O
O
1.
B(OH)2
24
Pd(PPh3)4, MeOH, H2O, Na2CO3
2. HCl, AcOH, CH2Cl2
77%, 2 steps
Scheme 3. Synthesis of biphenyl ligand 4.
4
2. Suzuki, N.; Fujii, I. Tetrahedron Lett. 1999, 40, 6013.
Table 1
3. (a) Bazan, J. F. Science 1992, 257, 410; (b) De Vos, A. M.; Ultsch, M.; Kossiakoff,
A. A. Science 1992, 255, 306; (c) McKay, D. B. Science 1992, 257, 412; (d)
Redfield, C.; Smith, L. J.; Boyd, J.; Lawrence, G. M. P.; Edwards, R. G.; Smith, R. A.
G.; Dobson, C. M. Biochemistry 1991, 30, 11029.
4. (a) Nicola, N. A.; Metcalf, D.; Matsumoto, M.; Johnson, G. R. J. Biol. Chem.
1983, 258, 9017; (b) Souza, L. M.; Boone, T. C.; Gabriloe, J.; Lai, P. H.;
Zaebo, K. M.; Murdock, D. C.; Chazin, V. R.; Bruszewski, J.; Lu, H.; Chen, K.
K.; Barendt, J.; Platzer, E.; Moore, M. A. S.; Mertelsmann, R.; Welte, K.
Science 1986, 232, 61; (c) Clark, S. C.; Kamen, R. Science 1987, 236, 1229;
(d) Nagata, S.; Fukunaga, R. Prog. Growth Factor Res. 1991, 3, 131; (e)
Roberts, A. W. Growth Factors 2005, 23, 33; (f) Koda, M.; Nishio, Y.;
Kamada, T.; Someya, Y.; Okawa, A.; Mori, C.; Yoshinaga, K.; Okada, S.;
Moriya, H.; Yamazaki, M. Brain Res. 2007, 1149, 223.
Dissociation constants of ligands 2–5 determined by SPR
Entry
Ligand
Kd
(lM)
1
2
3
4
2
3
4
5
47
a
—
20
a
—
a
No binding affinity.
Therefore, ligands inhibiting the interaction would be of interest as
cytotoxic agents for the treatment of cancer.12 Further design of
new ligands are ongoing in an effort to delineate the activity of this
class of small compound.
5. (a) Heck, R. F. Org. React. 1982, 27, 345; (b) Beletskaya, I. P.; Cheprakov, A. V.
Chem. Rev. 2000, 100, 3009.
6. (a) Orner, B. P.; Ernst, J. T.; Hamilton, A. D. J. Am. Chem. Soc. 2001, 123, 5382; (b)
Kutzki, O.; Park, S.; Ernst, J. T.; Orner, B. P.; Yin, H.; Hamilton, A. D. J. Am. Chem.
Soc. 2002, 124, 1838; (c) Yin, H.; Lee, G. I.; Sedey, K. A.; Rodriguez, J. M.; Wang,
H. G.; Sebti, S. M.; Hamilton, A. D. J. Am. Chem. Soc. 2005, 127, 5463; (d) Yin, H.;
Sedey, K. A.; Kutzki, O.; Park, H. S.; Orner, B. P.; Ernst, J. T.; Wang, H. G.; Sebti, S.
M.; Hamilton, A. D. J. Am. Chem. Soc. 2005, 127, 10191; (e) Davis, J. M.; Truong,
A.; Hamilton, A. D. Org. Lett. 2005, 7, 5405; (f) Yin, H.; Hamilton, A. D. Angew.
Chem., Int. Ed. 2005, 44, 4130; (g) Kim, I. C.; Hamilton, A. D. Org. Lett. 2006, 7,
1751; (h) Cummings, C. G.; Ross, N. T.; Katt, W. P.; Hamilton, A. D. Org. Lett.
2009, 11, 25.
Acknowledgment
We are grateful to the Egyptian Government for the post-grad-
uate scholarship provided to R. El-H.
7. Kaiser, F.; Schwink, L.; Velder, J.; Schmalz, H. G. J. Org. Chem. 2002, 67, 9248.
8. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
9. See Supplementary data for detail.
Supplementary data
10. (a) Myszka, D. G. Curr. Opin. Biotechnol. 1997, 8, 50; (b) Myszka, D. G.
Methods Enzymol. 2000, 323, 325; (c) Myszka, D. G. Anal. Biochem. 2004,
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Craig, L. S.; Fulu, L.; Alyssa, D. G.; Katherine, S.; Daniel, C. L. Immunity
2002, 17, 413.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.12.010.
References and notes
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