Identification of novel non-peptide CXCR4 antagonists by ligand-based design approach

Article abstract of DOI:10.1016/j.bmcl.2008.05.092

The design and synthesis of novel non-peptide CXCR4 antagonists is described. The peptide backbone of highly potent cyclic peptide-based CXCR4 antagonists was entirely replaced by an indole framework, which was expected to reproduce the disposition of the

Full text of DOI:10.1016/j.bmcl.2008.05.092

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4124–4129
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of novel non-peptide CXCR4 antagonists by ligand-based
design approach
Satoshi Ueda a, Manabu Kato a, Shinsuke Inuki a, Hiroaki Ohno a, Barry Evans b, Zi-xuan Wang b,
Stephen C. Peiper b, Kazuki Izumi c, Eiichi Kodama c, Masao Matsuoka c, Hideko Nagasawa d,
a,
Shinya Oishi a, , Nobutaka Fujii
*
*
a Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
b Department of Pathology, Medical College of Georgia, GA 30912, USA
c Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
d Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and synthesis of novel non-peptide CXCR4 antagonists is described. The peptide backbone of
highly potent cyclic peptide-based CXCR4 antagonists was entirely replaced by an indole framework,
which was expected to reproduce the disposition of the key pharmacophores consistent with those of
potential bioactive conformations of the original peptides. A structure–activity relationship study on a
series of modified indoles identified novel small-molecule antagonists having three pharmacophore func-
tional groups through the appropriate linkers.
Received 6 April 2008
Revised 21 May 2008
Accepted 22 May 2008
Available online 29 May 2008
Keywords:
Chemokine
CXCR4
Ó 2008 Elsevier Ltd. All rights reserved.
SDF-1
Anti-HIV
Indole
H-Arg1-Arg2-Nal3-Cys4-Tyr5-Arg6-Lys7
-Lys8
Pro9
Chemokines are a family of small proteins with chemotactic and
proactivatory effects on leukocytes. Chemokines mediate their bio-
logical effects by binding to the specific G-protein coupled receptor
subtypes that are differentially and widely expressed in blood cells.
Among these chemokine receptors, CXCR4 has a broad tissue dis-
tribution and the activation by its endogenous ligand CXCL12
(SDF-1, stromal cell-derived factor 1) leads to chemotaxis, immu-
nomodulation, and other regulatory functions including progenitor
cell migration during embryologic development of the cardiovas-
cular, hematopoietic, and central nervous systems. In addition to
its physiological roles, CXCR4 also plays important roles in patho-
logical conditions. These include tumor growth and metastasis1
and rheumatoid arthritis (RA).2 CXCR4 has also been reported to
act as a major co-receptor involved in the entry of T-cell-line-tropic
human immunodeficiency virus type 1 (HIV-1) strains into target
cells.3 Thus, CXCR4 is considered as an important therapeutic tar-
get for multiple diseases. Inhibitory compounds of CXCL12 or
HIV-1 binding to CXCR4 could be novel classes of anti-cancer,
anti-RA, and anti-HIV-1 drugs. Previously, we found highly potent
peptide-based CXCR4 antagonists such as 1, 2, and 3 (Fig. 1).4,5
D
S
1
S
HO-Arg14-Cys13-Cit12-Arg11-Tyr10
2
cyclo(-
cyclo(-
D
-Tyr1-
L
-Arg2-
L
-Arg3-
L
-Nal4-
L
-Gly5-)
-Gly5-)
3
D
-Tyr1-
D
-Arg2-
L
-Arg3-
L
-Nal4-
L
Figure 1. Structures of 1 and its downsized peptides 2 and 3. Bold residues are the
indispensable residues for the potent CXCR4-antagonistic activity. Nal, -3-(2-
naphthyl)alanine; Cit, -citrulline.
L
L
Peptide 1 and its derivatives effectively blocked X4-HIV-1 entry
to the cell by specifically binding to CXCR4,6 and also showed an
anti-metastatic effect against breast cancer7 and anti-RA activity8
in mouse models.
Although peptides are excellent lead molecules for develop-
ment of pharmaceutical agents, special drug delivery systems
are usually required for their clinical use because of the poor
bioavailability and instability against enzymes. Whereas several
peptide-based CXCR4 antagonists have been reported, only
small numbers of small-molecule CXCR4 antagonists have been
* Corresponding authors. Tel: +81 75 753 4551; fax: +81 75 753 4570 (N. Fujii).
E-mail addresses: soishi@pharm.kyoto-u.ac.jp (S. Oishi), nfujii@pharm.kyoto-u.
ac.jp (N. Fujii).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.092

S. Ueda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4124–4129
4125
reported.9 These prompted us to design novel non-peptide CXCR4
antagonists based on the SAR and conformational studies on pep-
tide ligands 1–3.
Cyclic pentapeptide-based CXCR4 antagonists 2 and 3 were
identified by screening of cyclic pentapeptide libraries, which were
designed based on SAR studies of peptide 1. The constrained back-
bone of the cyclic peptide was utilized as a template for positioning
the key functional groups in space as is found in parent peptide 1.
Subsequent conformational analysis of 2 permitted us to deter-
mine the topology of the four indispensable residues, then, rational
approach toward the de novo design of non-peptide antagonists
may be envisaged.10
Our previous SAR studies on 2 and its derivatives have shown
that at least three functional groups on the peptide side-chains
are required: (1) an aromatic ring such as 2-naphthyl- or 3-indolyl
group at position 4; (2) a guanidino group at position 3; (3) a gua-
nidino group at position 2 or a phenol group at position 1.11 How-
ever, it was difficult to determine the spatial relationships between
these functional groups due to the free rotation of the side-chain
torsion (
v
) angles. In our structural analyses, peptide 2 adopted a
a
OH
OH
(7.9-9.1)
(7.3-8.4)
HN
H
N
H
HN
N
O
O
O
O O
HN
HN
O
O
O
(4.7-5.4)
O
O
(4.7-5.4)
NH
NH
N
N
H
(8.0-9.2)
H
N
H
N
H
H2N
H2N
(4.5-5.3)
NH
NH
HN
NH
NH
NH2
NH2
HN
b
(8.1)
H
N
H
N
R2
R1
(4.5)
N
O
(6.7)
R3
Figure 2. Design of indole-based CXCR4 antagonists based on molecular dynamics calculation of 2. Distances (Å) between Cb atoms bearing three essential functional groups
during 1000 ps MD calculation of 2 (a) and between two key atoms of energy-minimized 5-acetamido-1-methylindole-2-carboxamide (b) are shown in parentheses. R1–R3
include naphthyl, indolyl, guanidino, and phenol groups.
1
O N
2
O N
2
O N
2
O N
2
HN R
O
a, b
d, e
c
CO Et
2
CO Et
2
NH
2
N
N
N
N
H
H
4
5
6
7
NH
Boc
NH
Boc
H
H
2
2
1
1
1
R
N
R
N
H N
2
HN R
O
HN R
O
HN R
g
O
O
f
h, i
N
N
N
O
NH
Boc
NH
HN
NH
Boc
8
9
10
NH
2
HN
NH
NH
Boc
NH
2
H
2
O N
R
N
O N
2
HN
2
HN
O
CO Et
2
O
N
j
N
O
d, k
N
f, g, h, i
5
11
12
13
Scheme 1. Reagents and conditions; (a) ethyl pyruvate, EtOH, reflux; (b) polyphosphoric acid, xylene, 130°C; (c) NaH, N-Boc-3-bromopropylamine, DMF, 70 °C; (d) 1 N NaOH
aq., EtOH-THF; (e) R1-NH2, HATU, Et3N, DMF; (f) NH4CO2H, Pd/C, EtOH-THF, reflux; (g) R2-CO2H, HATU, Et3N, DMF; (h) 95% TFA-H2O; (i) 1-H-pyrazolecarboxamidine
hydrochloride, Et3N, DMF; (j) 2-(2-naphthyl)ethyl bromide, NaH, DMF, 70 °C; (k) N-Boc-ethylenediamine, HATU, Et3N, DMF.

4126
S. Ueda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4124–4129
variety of global conformations, in which the distances between
indispensable functional groups were variable. On the other hand,
relatively rigid cyclic peptide backbone and fixed distances
between Cb atoms, which append key functional groups, were
observed.5 Hence, we envisioned that introduction of crucial
functional moieties for receptor binding onto a bicyclic heterocycle
scaffold, which mimics the relatively fixed cyclic pentapeptide
backbone of 2, would provide non-peptide CXCR4 ligands. In this
letter, we report a part of our ongoing research to develop novel
non-peptide small molecule CXCR4 antagonists.
molecular modeling suggested that it met the spatial requirements
for displaying the three key substituents (Fig. 2);12 (2) accessible
synthetic approaches were available for attachment of the three
substituents; (3) indoles represent an important class of bioactive
compounds and the physicochemical properties in terms of medic-
inal chemistry are well-documented.
Syntheses of indole-based compounds were achieved as
shown in Scheme 1. (4-Nitrophenyl)hydrazine 4 was converted
to the corresponding hydrazone, which was subjected to
Fischer ring closure reaction to produce an indole 5. Alkylation
of N1 position of the indole 5 with N-Boc-3-bromopropylamine
gave 6. This was hydrolyzed using 1 N aqueous sodium
Among several molecular scaffold candidates, we first selected
5-aminoindole-2-carboxylic acid for the following reasons: (1)
Table 1
Inhibitory activities of indole derivatives 10a–j and 13a–b against binding of 125I-SDF-1
a
to CXCR4
H
N
HN R2
R3
N
O
R1
Compound
R1
R2
R3
% inhibition at 10
23
lM
O
O
O
O
NH
NH2
H
N
OH
NH2
NH
10a
N
H
NH
NH2
H
N
NH2
NH
10b
10c
10d
10e
10f
63
61
88
70
86
77
72
62
55
51
53
N
H
NH
NH2
H
N
NH2
NH
N
H
NH
NH
NH
NH
NH
NH
NH2
H
N
NH2
NH
N
H
NH
NH2
O
O
NH
NH2
N
N
H
H
NH
NH2
N
H
N
NH2
NH
O
NH
NH2
H
N
10ga
10ha
10i
NH2
N
H
i
-Bu NH
O
H
NH
NH2
N
NH2
NH
N
H
Ph
NH
NH2
OH
O
O
N
H
NH
NH2
10j
N
H
OH
OH
H
N
NH2
NH
O
O
13a
H
N
NH2
NH
13b
OH
a
Evaluated as a racemic mixture.

S. Ueda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4124–4129
4127
Table 2
hydroxide in EtOH-THF and the resulting free carboxylic acid
IC50 values of selected indole derivatives
was coupled with amines using O-(7-azabenzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) as
coupling reagent to give 7. The nitro group of 7 was reduced
to amine 8 upon treatment with Pd-C and ammonium formate
in EtOH. The aminoindole 8 was then coupled with carboxylic
acids to give 9. Deprotection of Boc group(s) by 95% TFA and
guanylation of the free amino group(s) produced the target
compounds 10. Another series of compounds 13 were synthe-
sized from 5 using similar reaction sequences described for
10.
Compound
Structure
IC50 (lM)
NH
NH
H
N
HN
O
H2N
N
H
O
N
10d
3.0
NH
NH2
HN
NH
All indole-based compounds listed in Tables 1 and 2 were
purified by preparative reverse-phase HPLC (purity >95%) and
characterized by MALDI-TOF-MS. These compounds were tested
for competitive binding inhibition in human CXCR4 transfected
NH
H2N
N
H
N
HN
O
10f
3.0
1.2
2.2
1.7
3.7
4.8
N
O
Chinese hamster ovary (CHO) cells using
radioligand, with the results given as percentage inhibition
at 10 M. IC50 values of selected compounds are shown in
Table 2.
Compound 10d with 2-(3-indolyl)ethyl group at the R2 posi-
tion showed 88% inhibition at 10 M (IC50 = 3.0 M) and was
[ a
125I]SDF-1 as
NH
l
NH2
HN
i
-Bu
NH
NH
NH
NH
NH
H
N
HN
l
l
H2N
H2N
H2N
H2N
N
H
more potent than compounds having (4-hydroxyphenyl)-, (1-
O
N
N
N
N
O
naphthyl)- or (2-naphthyl)-alkyl group at the R2 position
(S)-10g
(R)-10g
(S)-10h
(R)-10h
10i
(compounds 10a–c, 23–63% inhibition at 10 lM). Further SAR
NH
studies based on 10d were undertaken. Chain elongation of
the guanidinoacetyl group (R3) of 10d caused slight decrease
in the affinity (10e). The use of N-amidinopiperidine-4-carbonyl
NH2
NH2
NH2
NH2
HN
i
-Bu
NH
H
N
was also acceptable for high potency [IC50 (10f) = 3.0
lM].
HN
O
N
Introduction of an isobutyl or benzyl group into the -carbon
a
H
O
of guanidinomethyl carbonyl group of 10d did not cause signif-
icant drop in binding affinity (compounds 10g and 10h). Com-
pounds with S-configuration at the chiral center showed more
potent CXCR4 antagonistic activity as compared with the corre-
sponding R-isomers. (S)-10g was identified as the most potent
NH
HN
l
M].13 Compound (S)-10g also
Ph
compound [IC50 ((S)-10g) = 1.2
showed potent anti-HIV-1 activity (IIIB strain, inhibition of
HIV-1 induced cytopathogenicity: EC50 = 5.4 M). The IC50 value
NH
H
N
HN
O
N
l
H
O
of (S)-10g is 34-fold lower as compared with parent peptide 2.
This is probably due to the absence of phenol functionality in
(S)-10g which corresponds to D-Tyr side-chain of peptide 2. De-
creased number of amide bond in (S)-10g might also lead to
the lower affinity. We have previously showed the importance
of backbone amide functionalities of 2 for CXCR4 antagonistic
activity by using reduced-amide isosteres or (E)-alkene dipep-
tide isosteres.14
NH
HN
Ph
NH
H
N
HN
O
N
H
O
Indole-based compounds having a phenol group at R3 position
showed moderate CXCR4-binding affinity (10i, 10j, 13a, and
13b). Interestingly, these compounds did not show complete inhi-
bition even at higher concentrations in the binding inhibition
experiments, while compounds having a guanidino group at R3 po-
sition (10c, 10d, and (S)-10g) achieved complete inhibition (Fig. 3).
These results suggest that 10c, 10d and (S)-10g are inhibitors that
competitively bind to the SDF-1 binding site of CXCR4, while 10i,
10j, 13a, and 13b may bind to an allosteric site of CXCR4 and par-
tially antagonize the SDF-1 binding.
Comparison of energy-minimized structures of (S)-10g and pre-
viously reported solution conformation of 2 revealed that three
functional groups on the indole template well overlapped the three
pharmacophore residues of 2 as expected. In this model, indole
scaffold favorably mimicked the backbone of Arg–Arg–Nal se-
quence of 2 (see Fig. 4).
In summary, a series of indole-based compounds were de-
signed, synthesized, and characterized as a novel class of CXCR4
antagonists. Although their IC50 values are in the lM range, these
NH
HN
H
N
HN
O
N
O
HO
HO
NH
NH2
HN
NH
HN
NH2
H
N
HN
O
N
O
13b
2
2.7
indole derivatives could serve as a useful lead for further medicinal
Cyclo(-
D-Tyr–Arg–Arg–Nal–Gly–)
0.035
chemistry programs.

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S. Ueda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4124–4129
Figure 3. Ligand binding dose response of the compounds (a) having two guanidino pharmacophores and (b) having a phenol pharmacophore, and (c) the comparison of the
two subsets with the parent peptide 2.
2. Nanki, T.; Hayashida, K.; EI-Gabalawy, H. S.; Suson, S.; Shi, K.; Girschick, H. J.;
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M.; Baggiolini, M.; Moser, B. Nature 1996, 382, 833.
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Y.; Tamamura, H.; Ibuka, T.; Murakami, T.; Koyanagi, Y.; Waki, M.; Matsumoto,
A.; Yamamoto, N.; Funakoshi, S.; Fujii, N. Biochem. Biophys. Res. Commun. 1992,
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5. Fujii, N.; Oishi, S.; Hiramatsu, K.; Araki, T.; Ueda, S.; Tamamura, H.; Otaka, A.;
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Peiper, S. C. Angew. Chem. Int. Ed. 2003, 42, 3251.
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7. (a) Tamamura, H.; Hori, A.; Kanzaki, N.; Hiramatsu, K.; Mizumoto, M.;
Nakashima, H.; Yamamoto, N.; Otaka, A.; Fujii, N. FEBS Lett. 2003, 550, 79; (b)
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Kitagawa, A.; Kawai, S.; Nakashima, H.; Fujii, N.; Igarashi, R. Biochem. Biophys.
Res. Commun. 2004, 320, 226.
Figure 4. Overlay of a low-energy structure of (S)-10g (green) and 2 (gray). The
molecular modeling of (S)-10g was performed using MacroModel-Program (Ver.
8.1) with ‘MMFF’ force field.
8. Tamamura, H.; Fujisawa, M.; Hiramatsu, K.; Mizumoto, M.; Nakashima, H.;
Yamamoto, N.; Otaka, A.; Fujii, N. FEBS Lett. 2004, 569, 99.
9. (a) Donzella, G. A.; Schols, D.; Lin, S. W.; Este, J. A.; Nagashima, K. A.; Maddon, P.
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Kurosaki, N.; Takaku, H.; Koyanagi, Y.; Yamamoto, N. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 4185; (c) Tamamura, H.; Ojida, A.; Ogawa, T.; Tsutsumi, H.; Masuno,
H.; Nakashima, H.; Yamamoto, N.; Hamachi, I.; Fujii, N. J. Med. Chem. 2006, 49,
3412; (d) Zhan, W.; Liang, Z.; Zhu, A.; Kurtkaya, S.; Shim, H.; Snyder, J. P.; Liotta,
D. C. J. Med. Chem. 2007, 50, 5655.
10. Conversion of peptide to nonpeptide by scaffolding strategy, see: (a)
Hirschmann, R.; Nicolaou, K. C.; Pietranico, S.; Salvino, J.; Leahy, E. M.;
Sprengeler, P. A.; Furst, G.; Strader, C. D.; Cascieri, M. A.; Candelore, M. R.;
Donaldson; Cindy; Vale, W.; Maechler, L. J. Am. Chem. Soc. 1992, 114, 9217; (b)
Kawato, H. C.; Nakayama, K.; Inagaki, H.; Ohta, T. Org. Lett. 2001, 3, 3451; (c)
Nakayama, K.; Kawato, H. C.; Inagaki, H.; Ohta, T. Org. Lett. 2001, 3, 3447.
11. (a) Ueda, S.; Oishi, S.; Wang, Z.-x.; Araki, T.; Tamamura, H.; Cluzeau, J.; Ohno,
H.; Kusano, S.; Nakashima, H.; Trent, J. O.; Peiper, S. C.; Fujii, N. J. Med. Chem.
2007, 50, 192; (b) Tamamura, H.; Araki, T.; Ueda, S.; Wang, Z.; Oishi, S.; Esaka,
A.; Trent, J. O.; Nakashima, H.; Yamamoto, N.; Peiper, S. C.; Otaka, A.; Fujii, N. J.
Med. Chem. 2005, 48, 3280.
Acknowledgments
This work was supported by Grant-in-Aid for Scientific Re-
search and Targeted Proteins Research Program from the Ministry
of Education, Culture, Sports, Science, and Technology of Japan, and
Health and Labour Sciences Research Grants (Research on HIV/
AIDS). Computation time was provided by the Supercomputer Lab-
oratory, Institute for Chemical Research, Kyoto University. S.U. and
S.I. are grateful to the JSPS Research Fellowships for Young
Scientists.
References and notes
12. Distances (Å) between b-carbons of
2 during 1000 ps MD calculation; D-
Tyr1–Arg3: 8.0–9.2, -Tyr1–Nal4: 7.9–9.1, Arg3–Nal4: 4.7–5.4, Arg2–Arg3: 4.5–
D
1. Müller, A.; Homey, B.; Soto, H.; Ge, N.; Catron, D.; Buchanan, M. E.; Mcclanahan,
T.; Murphy, E.; Yuan, W.; Wagner, S. M.; Barrera, J. L.; Mohar, A.; Verástegui, E.;
Zlotnik, A. Nature 2001, 410, 50.
5.3, Arg2-Nal4: 7.3–8.4, Arg3–Nal4: 4.7–5.4. Distances (Å) between two key
atoms of energy-minimized 5-acetamido-1-methylindole-2-carboxamide:

S. Ueda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4124–4129
4129
(acetamide N)-(N-methyl C), 6.7; (acetamide N)–(carboxamide N), 8.1;
(methyl C)-(carboxamide N), 4.5.
1H NMR (500 MHz, DMSO-d6): d = 1.91 (tt, J = 7.2, 7.4 Hz, 2H), 2.96 (t, J = 7.3 Hz,
2H), 2.97–3.04 (m, 1H), 3.13 (dt, J = 5.4, 7.2 Hz, 2H), 3.20–3.27 (m, 1H), 3.54 (dt,
J = 6.0, 7.3 Hz, 2H), 4.54 (t, J = 7.4 Hz, 2H), 4.56–4.62 (m, 1H), 6.78–7.64 (brm,
8H), 6.98 (m, 1H), 7.03–7.10 (m, 2H), 7.19 (m, 1H), 7.21–7.27 (m, 1H), 7.27-7.40
(m, 6H), 7.55 (d, J = 8.9 Hz, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.71 (t, J = 5.3 Hz, 1H),
7.85 (d, J = 8.9 Hz, 1H), 7.89–7.92, (br, 1H), 8.64 (t, J = 6.0 Hz, 1H), 10.12 (br,
1H), 10.82 (br, 1H); LRMS (FAB): 607 (MH+, base peak), 444; HRMS (FAB): calcd
for C33H39N10O2 (MH+) 607.3257; found 607.3251.
13. Compound (S)-10g: ½a D23
ꢀ
ꢁ 6:4ðc0:35; CH3OHÞ; 1H NMR (500 MHz, DMSO-d6):
d = 0.93 (d, J = 6.0 Hz, 3H), 0.95 (d, J = 5.9 Hz, 3H), 1.60–1.76 (m, 3H), 1.92 (tt,
J = 6.6, 7.2 Hz, 2H), 2.97 (t, J = 7.6 Hz, 2H), 3.14 (dt, J = 6.2, 6.6 Hz, 2H), 3.55 (dt,
J = 6.7, 7.0 Hz, 2H), 4.26–4.41 (m, 1H), 4.55 (t, J = 7.2 Hz, 2H), 6.86–7.53 (brm,
8H), 6.99 (m, 1H), 7.03–7.11 (m, 2H), 7.20 (br, 1H), 7.33–7.43 (m, 2H), 7.55 (d,
J = 9.0 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.73 (t, J = 5.4 Hz, 1H), 7.90 (d, J = 9.0 Hz,
1H), 7.95–7.99, (br, 1H), 8.65 (t, J = 5.7 Hz, 1H), 10.08 (s, 1H), 10.82 (s, 1H);
LRMS (FAB): 573 (MH+, base peak), 444; HRMS (FAB): calcd for C30H41N10O2
14. Tamamura, H.; Hiramatsu, K.; Ueda, S.; Wang, Z.; Kusano, S.; Terakubo, S.;
Trent, J. O.; Peiper, S. C.; Yamamoto, N.; Nakashima, H.; Otaka, A.; Fujii, N. J.
Med. Chem. 2005, 48, 380.
(MH+) 573.3414; found 573.3418. Compound (S)-10h: ½a 2D3
ꢀ 3:3ðc0:34; CH3OHÞ;