Synthesis of 1-boraadamantaneamine derivatives with selective astrocyte vs C6 glioma antiproliferative activity. A novel class of anti-hepatitis C agents with potential to bind CD81

Article abstract of DOI:10.1021/jm020326d

A variety of amine complexes with 1-boraadamatane were synthesized and subsequently evaluated for an antiproliferative effect on CD81-enriched cell lines to provide evidence for binding and activation of CD81. CD81 is a member of the tetraspanin family of

Full text of DOI:10.1021/jm020326d

J . Med. Chem. 2003, 46, 2823-2833
2823
Articles
Syn th esis of 1-Bor a a d a m a n ta n ea m in e Der iva tives w ith Selective Astr ocyte vs
C6 Gliom a An tip r olifer a tive Activity. A Novel Cla ss of An ti-Hep a titis C Agen ts
w ith P oten tia l to Bin d CD81
Carl E. Wagner,‡ Michael L. Mohler,§ Gyong Suk Kang,§ Duane D. Miller,§ Eldon E. Geisert,# Yu-An Chang,†
Everly B. Fleischer,‡ and Kenneth J . Shea*,‡
Department of Chemistry, University of CaliforniasIrvine, Irvine, California 92697, College of Pharmacy and College of
Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38163, and RIC Biotech & Trading, Inc.,
Orange County, California 92614
Received J uly 24, 2002
A variety of amine complexes with 1-boraadamatane were synthesized and subsequently
evaluated for an antiproliferative effect on CD81-enriched cell lines to provide evidence for
binding and activation of CD81. CD81 is a member of the tetraspanin family of membrane
proteins found in all cell lineages in the liver. CD81 signals for antiproliferation when bound
by antibodies. It is known that the HCV-E2 envelope glycoprotein binds to the CD81 protein.
While it is unclear whether virus entry into host cells is directly linked to virus attachment
via CD81 for HCV, this step in the viral life cycle has recently proven to be an effective point
of attack for other viruses including HIV and rhinoviruses. The aim of the current study
concerns the synthesis of amantidine analogues by appending primary amines to 1-boraada-
mantane to evaluate such compounds for CD81-dependent antiproliferation of CD81-enriched
cell lines (astrocyte) vs CD81-deficient cell lines (C6 glioma). If the antiproliferative effect of
these amantidine analogues proves to be an effect of binding and activating CD81, then these
compounds may have the potential to prevent or treat HCV infections. Each compound’s
potential for preventive and therapeutic activity stems from the compound’s potential to block
viral attachment, virus-cell fusion, or virus entry into host cells or to counter potential
mechanisms of HCV immune evasion. Out of a library of over 500 compounds, including
randomly selected small molecules and rationally designed small molecules, only the 1-bo-
raadamantaneamine compounds and structurally similar analogues display a significant
antiproliferative effect on the CD81-enriched astrocytes relative to the CD81-deficient cell lines.
In fact, 1-boraadamantane‚L-phenylalanine methyl ester complex (5), 1-boraadamantane‚
ethanolamine complex (8), and (S)-2-[(adamantane-1-carbonyl)amino]-3-phenylpropionic acid
(15) show a dose-dependent, astrocyte-selective antiproliferative activity in the concentration
range 0.1-10 µM. This is consistent with the binding and activation of CD81 and represents
a 2-fold improvement compared to the clinically prescribed anti-HCV agent, amantidine, in
the same concentration range. Consequently, the 1-boraadamantaneamine derivatives present
a promising lead in the development of small molecules with potential to bind to CD81 and
treat HCV infections.
In tr od u ction
the treatment of hepatitis C with amantidine alone and
in combination with R-interferon and ribavirin have
shown promise in the reduction of alanine aminotrans-
ferase (ALT), the enzyme released by the lysis of
damaged liver cells, and HCV RNA levels.3 Other
methods of treatment currently under investigation
involve viral protease inhibitors, but those developed
thus far include only a small variety of peptide sub-
strates and analogues.4 With the difficulty of adminis-
tering small peptides as therapeutic drugs and the
promise shown by drugs such as amantidine, there is
ample motivation to synthesize small-molecule ana-
logues of 1-aminoadamantane to be tested for activity
against hepatitis C.
The hepatitis C virus (HCV) affects a substantial part
of the world’s population, from 1% to 2%, and complica-
tions arising from the progression of the chronic infec-
tion of the liver include cirrhosis and liver cancer.1
Currently, treatment of HCV with R-interferons and
ribavirin affects a continued remittance of viral RNA
in less than 50% of treated patients.2 Recent studies for
* To whom correspondence should be addressed. Phone: (949) 824-
5844. Fax: (949) 824-2210. E-mail: kjshea@uci.edu.
‡ University of CaliforniasIrvine.
§ College of Pharmacy, University of Tennessee Health Science
Center.
#
College of Medicine, University of Tennessee Health Science
Center.
† RIC Biotech & Trading, Inc.
10.1021/jm020326d CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/24/2003

2824 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Wagner et al.
Sch em e 1
Sch em e 2
While the mechanism by which small molecules such
as amantidine effect a remittance of ALT and HCV RNA
levels is not well understood, it may involve blocking
viral adhesion to liver cells and subsequent infection.
Recently, the large extracellular loop of the CD81
transmembrane protein (CD81-LEL), a protein common
to all cell lineages in the liver, has been shown to bind
to the HCV-E2 envelope glycoprotein.5 The CD81-LEL
has been crystallized, and its crystal structure has been
reported at 1.6 Å resolution.6 In fact, the HCV-E2 binds
to CD81-LEL with a Kd of 1.8 nM at 25 °C,7 and the
region in the CD81-LEL that participates in E2 binding
is composed of largely hydrophobic amino acid residues.
While it is not known whether the CD81 protein serves
as a receptor for HCV entry into the cell, it arguably
serves as a receptor for virus attachment. Indeed, the
pronounced anti-HCV activity of 1-aminoadamantane
hydrochloride could possibly be attributed to a binding
interaction with the CD81-LEL. Molecular modeling of
this region may help to elucidate the key binding
interactions of molecules incorporating the 1-aminoada-
mantane core motif and aid in the development of new
small-molecule drugs to bind to CD81 and treat hepa-
titis C.
ing to the method of Zakharkin and co-workers.10 After
purification by sublimation, 1-boraadamantane‚THF
was procured in 46% overall yield from triallylborane.
The first 1-boraadamantaneamine complexes to be
synthesized and tested included complexes with 1-ami-
noadamantane (4) and L-phenylalanine methyl ester (5)
(Scheme 2). Complex 4 was assembled in 38% yield by
addition of a stoichiometric amount of 1-aminoadaman-
tane to 1-boraadamantane‚THF and purification by
recrystallization in ethanol, according to the protocol of
Mikhailov and co-workers.9a Complex 5 was synthesized
in quantitative yield by stoichiometric addition of L-
phenylalanine methyl ester to 1-boraadamantane‚THF.
By use of the same protocol to synthesize amine
complex 5 (Scheme 2), complexes of 1-boraadamantane
with L-cysteine methyl ester (6) and L-leucine methyl
ester (7) were synthesized in quantitative yield. Ad-
ditionally, a complex of 1-boraadamantane with ethan-
olamine (8) was also synthesized in quantitative yield
for testing. The amine complexes 6-8 are air-stable,
Owing to the versatility with which functionalized
primary amines can be appended to the 1-boraadaman-
tane core, as well as the stability of the resulting
complexes, 1-boraadamantane represents a promising
substrate for modification and pharmacology studies en
route to 1-aminoadamantane analogues. In fact, several
1-boraadamantane complexes with amines and other
nitrogen-containing compounds display a pronounced
antiviral effect against the influenza A and B viruses.8
On the basis of the evidence for the potent antiviral
pharmacology of 1-boraadamantaneamine complexes
and the structural similarities to 1-aminoadamantane,
a number of 1-boraadamantaneamine complexes were
synthesized to be evaluated for a CD81-dependent
antiproliferative activity in astrocytes vs C6 glioma.
crystalline complexes. X-ray diffraction was performed
on single crystals of complexes 5, 6, and 8 (Figure 1).
The X-ray diffraction study for complex 5 reveals that
the amine group of L-phenylalanine methyl ester coor-
dinates to boron in boraadamantane. The crystal struc-
ture of 6 establishes that the amine group coordinates
to boron rather than the thiol group of L-cysteine methyl
ester, and the X-ray crystal structure of 8 reveals that
the amine group coordinates to boron rather than the
hydroxyl group of ethanolamine. The polarity of the
L-cysteine methyl ester as well as the free thiol group
improves the solubility of the amino acid methyl ester
complex 6 in polar, protic solvents. The compact size of
ethanolamine and its free hydroxyl group also improves
the solubility of complex 8 in polar, protic solvents. In
fact, the solubility of complexes 4-8 in water ranged
from 3.8 mg/mL for complex 8 to 0.6 mg/mL for complex
5. With the rare exceptions of triallylboranes, the
Ch em istr y
The 1-boraadamantaneamine complexes synthesized
for testing were conveniently derived from 1-boraadaman-
tane‚THF (3) in high yield by stoichiometric addition.
Thus, 1-boraadamantane‚THF (3) was synthesized by
the hydroboration of 3-methoxy-7-(methoxymethyl)-3-
borabicyclo[3.3.1]non-6-ene (2). This compound was
prepared by the reaction of triallylborane (1) with
methyl propargyl ether followed by methanolysis, ac-
cording to the procedure of Mikhailov and co-workers
(Scheme 1).9 Triallylborane (1) was synthesized accord-

1-Boraadamantaneamine Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 2825
F igu r e 1. X-ray crystal structures of 1-boraadamantane‚L-phenylalanine methyl ester (5), 1-boraadamantane‚L-cysteine methyl
ester (6), and 1-boraadamantane‚ethanolamine (8).
Sch em e 3
(C-sp3)-B bonds of trialkylboranes including trimeth-
ylborane are stable in water to 100 °C.11 The stability
of complex 4 in refluxing ethanol during recrystalliza-
tion attests to the stability of these complexes in polar,
protic solvents.
(2 equiv) to give amide 13 in 96% yield. Amide 14 was
Several diadamantyl compounds structurally similar
to complex 4 were prepared. 1-Adamantylcarbonyl
chloride was prepared from the reaction of 1-adaman-
tylcarboxylic acid with thionyl chloride, and the acid
chloride was subsequently reacted with 1-aminoada-
mantane to give 1,1-diadamantylamide (9) in 19%
yield.12 Additionally, di-1-adamantylamine (11) and its
hydrochloride salt (12) were prepared from 1-bromoada-
mantane (10) according to the procedure of Dervan and
McIntyre13 (Scheme 3). The reaction of 1-bromoada-
mantane with 1-aminoadamantane in a thick-walled
sealed glass tube (220 °C, 40 h) furnishes 11 after
workup with base in a simple, direct procedure. Di-1-
adamantylamine (11) can be protonated with HCl to
furnish the hydrochloride salt 12.
similarly prepared in 68% yield. Acids 15 and 16 were
prepared from the corresponding amides by saponifica-
tion with lithium hydroxide in 63% and 87% yield,
respectively.
Biologica l Assa y Ra tion a le
CD81 is a tetraspanin membrane protein that signals
for antiproliferation when bound by antibodies. CD81
has been extensively studied in the immune system14
where it is implicated in cellular activation,15 adhe-
sion,16 proliferation,17 and differentiation.18 Tetraspa-
nin-pathogen interactions have been previously re-
ported;19 however, the significance of these interactions
is unknown. Until recently, no ligands that bind CD81
Several compounds structurally similar to complexes
5 and 7 were also prepared. 1-Adamantylcarbonyl
chloride was reacted with L-phenylalanine methyl ester
hydrochloride in methylene chloride and triethylamine

2826 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Wagner et al.
mediated antiproliferation model. However, subsequent
experiments to provide direct evidence of CD81 binding
are necessary for the promising candidates identified
in this assay.
Da ta An a lysis
The screening data were collected in three separate
experiments of four wells per concentration. Also, the
average growth of eight wells containing no test com-
pound was used as a negative control for each microtiter
plate. The antiproliferative character of each compound
was reported as the percent survival (% survival),
calculated as the average A560 ratio for treated cells
divided by negative control cells, expressed as a per-
centage. Values less than 100% indicate some degree
of antiproliferation. Promising compounds in this assay
will display selective antiproliferation in a CD81-
enriched cell line (primary astrocyte culture), while the
proliferation of CD81-deficient cells (C6 glioma) will not
be affected in the presence of an equal concentration of
a given compound. The results for compounds 5, 7, 8,
13, 15, and 16, in addition to the reference anti-HCV
drug, amantidine, are summarized in Figure 3.
F igu r e 2. Anti-CD81 Western blot analysis of astrocyte and
C6 glioma cell lines.
have been described except for antibodies, many of them
antiproliferative, and the HCV-E2 glycoprotein. The
importance of the binding of the HCV-E2 glycoprotein
to CD81-LEL is not completely understood; however, it
is believed to be involved in mediating viral processes
of cell entry20 and immune evasion.21
Discu ssion
In the process of screening over 500 compounds on
proliferation of astrocyte cells over C6 glioma cells, the
1-boraadamantaneamine complexes 5, 7, and 8 and
amantidine analogues 13, 15, and 16 proved to be the
only compounds that demonstrate astrocyte selective
activity. The activity of these compounds was observed
in a limited concentration range with nonspecific cyto-
toxicity at higher concentrations. Among them, com-
pound 15 shows the least nonspecific cytotoxicity and
the strongest astrocyte selective activity.
In contrast, many compounds from several structural
classes have been observed to be C6 selective.24 This
suggests that C6 glioma antiproliferation is easier to
achieve because of its clonal, highly mitotic character.25
Amantidine analogues 4, 6, 9, 11, and 14 show a non-
astrocyte selective antiproliferative effect in the 0.1-
10 µM concentration range (Supporting Information).
The neonatal astrocytes by comparison are a hetero-
geneous collection of glial cell types whose common
denominator is the overexpression of CD81 to detect
cell-cell contact and signal a developmentally pro-
grammed contact inhibition. In accordance with the
experimental evidence of the present study, this body
of research strongly supports the model that the 1-bo-
raaadamantaneamine complexes and amantidine ana-
logues are antiproliferative because of CD81 activation.
The identification of these astrocyte-selective antipro-
liferative compounds provides the stimulus to search for
even more potent compounds. At present, analogues 5,
7, 8, 13, 15, and 16 are the only known compounds that
selectively inhibit astrocyte proliferation without affect-
ing C6 glioma proliferation.
The choice of cells for this assay for CD81 binding and
activation was based on evidence that rat pup astrocytes
up-regulate the expression of CD81 during postnatal
days 4-8.12 This is a contact inhibition mechanism that
signals the end of the growth phase in rat glial develop-
ment. Primary cultures of astrocytes prepared prior to
the contact inhibition in vivo provide cell culture. The
CD81-deficient cell line, C6 glioma, is a commercially
available transformed cell line that expresses CD81 at
levels that are undetectable by Western blot analysis.22
C6 glioma cells are a transformed cell line derived from
glia that has retained many of the properties of astro-
cytes.23 The rat glial origin of both these cell lines
suggests that the cellular receptors present should be
very similar with the notable exception of CD81 (Figure
2). Thus, astrocyte-selective antiproliferative activity
provides compelling experimental evidence for a CD81
binding mediated mechanism.
Immunoblots of protein samples were taken from rat
astrocytes and rat C6 glioma. The total load of proteins
from both samples was balanced. The 110 kDa antigen
is recognized in both samples immunoblotted with
AMP1 (anti-CD81 antibody). The 26 kDa CD81 band is
stained only in the astrocyte band. This indicates CD81
is expressed at significantly lower levels in the C6 cells
relative to astrocytes.
An observed reduction in the percent survival of
astrocyte versus C6 glioma cells in cell lines treated with
a given compound at concentrations varying from 0.1
to 10 µM constitutes an efficient assay to identify small
molecules with strong potential for CD81-mediated
antiproliferation. Those compounds showing the most
promise in this assay serve as lead compounds in the
design of structurally similar analogues for testing. An
observed dose-dependence on the percent survival from
such a test provides further evidence for the CD81-
According to the assay, compound 15 displayed the
greatest astrocyte-selective antiproliferative character
(82% ( 2% cell survival at 5 µM). In contrast, the
reference anti-HCV drug, amantidine, displayed anti-
proliferative character at higher concentrations than
other analogues of this group. Unfortunately, analogues
5, 7, 8, 13, 15, and 16 display nonspecific cytotoxicity

1-Boraadamantaneamine Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 2827
F igu r e 3. Astrocyte-selective antiproliferative activity of amantidine analogues 5, 7, 8, 13, 15, and 16 in addition to amantidine.
Three separate dose-response experiments were performed at each concentration. Each experiment consisted of four wells per
concentration. The percent survival plotted is the average of all 12 wells of the three separate assays. Error bars represent standard
deviation. The graphs were generated using Microsoft Excel.

2828 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Wagner et al.
F igu r e 4. Three perspectives of the minimized structure showing the CD81-LEL protein binding complex 5. The Thr163 and
Phe186 residues of the CD81-LEL are shown in purple, and the 1-boraadamantane‚L-phenylalanine (5) is yellow for reference.
at high concentrations (>10 µM). We are currently
investigating the design of derivatives that lack the
nonspecific cytotoxicity based on the lead compounds
identified in this paper. The experiments demonstrate
the dose-dependent nature of the astrocyte-selective
activity of 15 compared to amantidine. This dose-
dependent, astrocyte-selective antiproliferative activity
is consistent with binding and activation of CD81. By
comparison, the standard anti-HCV drug amantidine
displayed only a slight reduction in astrocyte prolifera-
tion and only at higher concentration. The most promis-
ing lead compound, amantidine analogue 15, is cur-
rently being used as the basis for the design of new
derivatives in our laboratory for further characterization
of this astrocyte-selective antiproliferation.
Mod elin g
Because of the promising results indicating that
complexes 5 and 15 promote a CD81-dependent anti-
proliferation of astrocyte cell lines, a modeling study was
undertaken in an attempt to elucidate a possible site
on the CD81-LEL for the binding of complex 5 or other
molecules with a 1-aminoadamantane core motif. The
atomic coordinates of the human CD81-LEL are avail-
able at the Protein Data Bank (PDB) with the accession
code 1g8q. The PDB file for the CD81-LEL comprises
F igu r e 5. Hydrogen bonding and van der Waals interactions
as illustrated by modeling for complex 5 in the active site of
176 amino acids and 194 water molecules, and the two
chains of the CD81-LEL connected by two disulfide
bridges are numbered Phe113-His202 and Phe213-
His302 (the C-terminal His residue in both chains is
simply a His-tag).
CD81.
walls of the cleft and the adamantane framework, we
reasoned that other 1-aminoadamantane analogues
would bind in a similar fashion. Thus, complex 5 was
built in place of the 1-aminoadamantane simply by
changing the carbon of the 1-aminoadamantane to boron
and building the L-phenylalanine methyl ester off the
amine. The docking algorithm was reapplied to mini-
mize interactions between complex 5 and the residues
surrounding the cleft. Three perspectives of the result-
ing minimized structure showing the CD81-LEL protein
binding to complex 5 are shown in Figure 4. The Thr163
and Phe186 residues are purple and the 1-boraadaman-
tane‚L-phenylalanine (5) is yellow for reference.
In the minimized CD81-LEL complex 5 structure, one
of the amine hydrogen atoms of complex 5 is almost
within hydrogen-bonding distance (2.33 Å) of the oxygen
of the Thr163 hydroxyl group. Also, the carbonyl oxygen
of the L-phenylalanine methyl ester of 5 is within
hydrogen-bonding distance (1.84 Å) of the hydroxyl
The Thr163 and Phe186, both reported to participate
in HCV-E2 binding,26 are positioned on the C-helix and
D-helix, respectively. In fact, there is a cleft between
the C-helix and D-helix that appears suitable for a
competitive binding association of a small molecule with
a 1-aminoadamantane core motif. Thus, by use of the
Insight II modeling program, 1-aminoadamantane was
positioned in the cleft between the C and D helices of
the CD81-LEL. The interactions between the cleft and
1-aminoadamantane were minimized by applying a
suitable docking algorithm, and a minimized conforma-
tion was quickly obtained in which the adamantyl group
was buried inside the cleft and the amine group was
positioned next to the Thr163 free hydroxyl group. Since
the adamantane core fit into the cleft like a ball into a
socket, with marginal dead space between the interior

1-Boraadamantaneamine Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 2829
reference. All 11B NMR spectra were acquired at 237 MHz on
a Bruker spectrometer. All chemical shifts (δ) are listed in ppm
against boron trifluoride etherate as an external reference.
Infrared spectra (IR) were assayed on a Perkin-Elmer 1600
series FTIR spectrometer. High-resolution mass spectra (EI,
proton of the Thr163 residue. Together with these
hydrogen-bonding interactions and the van der Waals
interactions of the 1-boraadamantyl core in the cleft
formed by the C and D helices, we suggest that this is
the most probable mode of binding for the 1-boraada-
mantaneamine complexes to CD81-LEL (Figure 5).
hexanes) were recorded using either
a VG 7070e high-
resolution mass spectrometer or a Fisons Autospec mass
spectrometer. Melting points were assayed on a Thomas-
Hoover capillary melting point apparatus.
Con clu sion
Gen er a l P r oced u r es. Tetrahydrofuran, methylene chlo-
ride, diethyl ether, and benzene were dried by filtration
through alumina according to the procedure described by
Grubbs.29 All other solvents were distilled from CaH2 prior to
use. Boron trifluoride etherate (BF3‚Et2O, Aldrich) was dis-
tilled over CaH2 under reduced pressure (20 mmHg, 65 °C).
Allyl bromide (Aldrich) was distilled over CaH2. All reactions
were run in flame-dried glassware under a positive N2 or argon
atmosphere. Removal of volatile solvents transpired under
reduced pressure using a Bu¨chi rotary evaporator and is
referred to as removing solvents in vacuo. Thin-layer chroma-
tography was conducted on precoated (0.25 mm thickness)
silica gel plates with 60F-254 indicator (Merck). Column
chromatography was conducted using 230-400 mesh silica gel
(E. Merck reagent silica gel 60).
A variety of 1-boraadamataneamine complexes were
synthesized by stoichiometric addition of a primary
amine to 1-boraadamantane‚THF. These complexes
were subsequently evaluated for an antiproliferative
effect on CD81-enriched cell lines to provide evidence
for binding and activation of the CD81 receptor. Ad-
ditional amantidine analogues structurally similar to
the 1-boraadamantaneamine complexes were also syn-
thesized. Compounds 5, 7, 8, 13, 15, and 16 display an
antiproliferative effect on the CD81-enriched astrocytes
relative to the CD81-deficient cell lines, suggesting that
the compounds bind and activate the CD81 receptor
over 0.1-10 µM. However, analogue 15 displayed the
greatest astrocyte-selective antiproliferative character
in this range of concentration.
These studies demonstrate that a reproducible dif-
ference between the percent survival of astrocyte and
that of C6 glioma cell lines for a small molecule in
micromolar concentrations is consistent with CD81-
mediated antiproliferation. The astrocyte-selective an-
tiproliferation, albeit in a limited concentration range
of 0.1-10 µM of analogues 5, 7, 8, 13, 15, and 16,
suggests CD81-dependent antiproliferation and estab-
lishes the potential of 1-boraadamantaneamine deriva-
tives and analogues derived therefrom as anti-HCV
agents. If this finding proves to be true, such 1-boraada-
mantaneamine derivatives and structurally similar
analogues will have the potential to prevent or treat
HCV infections by blocking viral attachment, virus-
cell fusion, or virus entry into host cells or by countering
potential mechanisms of HCV immune evasion. While
much remains to be learned about HCV viral attach-
ment and entry, this step in the viral life cycle has
proven to be an effective point of attack for combating
other viruses including HIV27 and rhinoviruses.28 Com-
pounds 5, 7, 8, 13, 15, and 16 are currently being
evaluated in a cell-based HCV viral assay to determine
the extent to which they inhibit HCV RNA synthesis.
Finally, molecular modeling provides an illustration
for how the adamantyl framework of 1-boraadamanta-
neamine compounds might bind to CD81. Indeed, the
ease with which amines can be appended to the 1-bo-
raadamantane framework makes it an ideal substrate
for the quick and convenient synthesis of 1-aminoada-
mantane analogues for evaluation of potential binding
to CD81.
Tr ia llylbor a n e (1). The method of Zakharkin and co-
workers was followed to make 1.10 To a flame-dried 250 mL
round-bottom flask sealed with a rubber septum and equipped
with a magnetic stir bar, reflux condenser, and thermometer
was transferred solid aluminum granules (12.03 g, 446 mmol)
and HgCl2(s) (88 mg, 0.32 mmol). Dry, N2-purged diethyl ether
(60 mL) was added, and the solution was stirred and heated
to 35 °C. Allyl bromide (52.1 mL, 602 mmol) was added
dropwise under a nitrogen atmosphere. Care must be taken
to add the allyl bromide slowly, since the reaction with
aluminum is exothermic. The reaction mixture was stirred at
reflux in an oil bath set at 67 °C for 3 h. The reaction mixture
was then transferred via syringe into a septum-sealed flame-
dried 250 mL round-bottom flask containing BF3‚Et2O (20.0
mL, 142 mmol) under nitrogen. This solution was refluxed at
70 °C for 3 h. The ether was removed by short-path distillation
under nitrogen. The residue was then distilled under vacuum
(20 mmHg) with a short-path distillation head at a head
temperature of 65 °C to give crude triallylborane as a clear,
colorless liquid (17.00 g, 78%). 11B NMR (237 MHz, CDCl3) of
this distillate showed a major peak, δ 80.7 (br s), 83%, and
minor peaks at δ 50.6 (br s), 11%, and δ 32.0 (br s), 6%. The
peak at δ 80.7 (br s) has been assigned to triallylborane. On
the basis of chemical shift, the peak at δ 50.6 has been
tentatively assigned to a borinic ester impurity, most likely
the product of triallylborane’s reaction with oxygen. A second
vacuum distillation (20 mmHg) yielded pure triallylborane as
a clear, colorless liquid (13.07 g, 60%): 1H NMR (500 MHz,
CDCl3) δ 2.2 (br s, 6H), 4.9 (br s, 6H), 5.89-5.98 (p, J ) 10.6,
3H); 13C NMR (125.8 MHz, CDCl3) δ 134.8, 114.7 (br s), 34.4
(br s); 11B NMR (237 MHz, CDCl3) δ 80.6 (br s); IR (neat) 3076,
2999, 2973, 2914, 1807, 1635, 1420, 1370, 1270, 1170, 993, 900
cm-1
.
3-Meth oxy-7-(m eth oxym eth yl)-3-bor abicyclo[3,3,1]n on -
6-en e (2). The basic procedure of Mikhailov and co-workers
was followed to synthesize 2.9b To a flame-dried, two-neck 50
mL flask with stir bar and condenser was added distilled
triallylborane (8.5 g, 63 mmol) under a nitrogen atmosphere.
The flask was heated with stirring under nitrogen at 130 °C
in a preheated oil bath (10 min.). Methyl propargyl ether (4.5
mL, 64 mmol) was added via syringe over the course of 10 min
to the stirring triallylborane. The reaction mixture was heated
at 130 °C for 1 h. The reaction solution was cooled to room
temperature, and methanol (2.9 mL, 72 mmol) was added
dropwise under nitrogen with stirring. The reaction was
exothermic and accompanied by the evolution of propylene gas.
After an additional 5 min of stirring, the reaction mixture was
distilled under vacuum (0.10 mmHg) with a short-path distil-
lation head at a head temperature of 79 °C to give NMR-pure
3-methoxy-7-(methoxymethyl)-3-borabicyclo[3,3,1]non-6-ene 6
Exp er im en ta l Section
In str u m en ta tion . All 1H NMR spectra were acquired at
400 or 500 MHz on Bruker spectrometers. Chemical shifts (δ)
are listed in ppm against deuterated solvent peaks as an
internal reference. Coupling constants (J ) are reported in
hertz, and the abbreviations for splitting include the fol-
lowng: s, single; d, doublet; t, triplet; q, quartet; p, pentet; m,
multiplet; br, broad. All 13C NMR spectra were acquired on
Bruker instruments at 125.8 MHz. Chemical shifts (δ) are
listed in ppm against solvent carbon peaks as an internal

2830 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Wagner et al.
(10.75 g, 87%) as a clear, viscous liquid: 1H NMR (500 MHz,
CDCl3) δ 5.66 (br s, 1H), 3.74 (d, J ) 11.7, 1H), 3.64 (d, 11.8),
3.58 (s, 3H), 3.19 (s, 3H), 2.49 (br s, 1H), 2.43 (br s, 1H), 2.25
(d, J ) 17.9, 1H), 1.74 (d, J ) 12.7, 1H), 1.70 (d, J ) 17.8,
1H), 1.60 (d, J ) 12.7, 1H), 1.04 (d, J ) 17.4, 1H), 0.96 (dd, J
) 17, 7.5, 1H), 0.86 (d, J ) 17.4, 1H), 0.83 (dd, J ) 17, 5.3,
1H); 13C NMR (125.8 MHz, CDCl3) δ 131.7, 130.8, 77.0, 56.9,
53.0, 34.9, 32.5, 29.0, 26.9, 25.4 (br s), 24.1 (br s); 11B NMR
(237 MHz, CDCl3) δ 55.4 (br s); IR (thin film) 3397, 2909, 1465,
1431, 1371, 1298, 1167, 1101, 1038, 1019, 996, 955, 914, 880,
32.5, 25.6 (br s); 11B NMR (237 MHz, CDCl3) δ -7.0; IR (neat)
3282, 3220, 2850, 1738, 1563, 1497, 1445, 1405, 1254, 1078,
989, 941, 855, 838, 779, 747, 701 cm-1; HRMS (CI) m/ z calcd
for C19H29BO2N (M + H)+ 314.2291, found 314.2266. Anal.
Calcd for C19H28BO2N: C, 72.85; H, 9.01; N, 4.47. Found: C,
72.62; H, 8.99; N, 4.48.
1-Bor a a d a m a n ta n te‚L-Cystein e Meth yl Ester (6). L-
Cysteine methyl ester hydrochloride (5.62 g, 32.6 mmol) was
dissolved in deionized water (43.0 mL) containing sodium
bicarbonate (4.86 g, 57.7 mmol), and the solution was satu-
rated in sodium chloride. The aqueous solution was extracted
with methylene chloride, and the organic layer was washed
with saturated sodium chloride and dried over sodium sulfate.
The methylene chloride was removed in vacuo to afford
l-cysteine methyl ester (4.04 g, 29.9 mmol) as a clear, colorless
liquid in 91% yield. To a solution of L-cysteine methyl ester
(1. 0 g, 7.4 mmol) in methylene chloride (10.0 mL) was added
a solution of 1-boraadamantane‚THF 3 (1.4694 g, 7.1 mmol)
in methylene chloride (10.0 mL) with stirring. The methylene
chloride was removed in vacuo to afford 1-boraadamantane‚
L-cysteine methyl ester 6 (1.9 g, 7.1 mmol) as a colorless
crystalline solid: mp 109-111 °C; [R]24D -11.2 (c 0.79, CHCl3);
1H NMR (500 MHz, CDCl3) δ 3.99 (m, 1H), 3.88 (m, 1H), 3.84
(s, 3H), 3.76 (br s, 1H), 3.04 (dd, J ) 9.1, 4.6, 2H), 2.13 (br s,
3H), 1.59 (m, 3H), 1.48 (m, 3H), 1.29 (t, J ) 9.1, 1H), 0.44 (br
s, 6H); 13C NMR (125.8 MHz, CDCl3) δ 53.7, 40.1, 32.7, 30.4,
26.7; 11B NMR (237 MHz, CDCl3) δ -8.4 (br, s); IR (KBr) 3422,
3280, 3235, 2856, 1734, 1552, 1439, 1388, 1281, 1232, 1205,
1177, 1076, 988 cm-1; HRMS (CI) m/ z calcd for C13H25BO2NS
847, 831, 678 cm-1; HRMS (EI/hexanes) m/ z calcd for C11H19
-
BO2 (M+) 194.1478, found 194.1481.
1-Bor a a d a m a n ta n e‚THF (3). The protocol of Mikhailov
and co-workers was followed to synthesize 3.9a To a flame-
dried, two-neck 50 mL flask with a stir bar and condenser was
added 3-methoxy-7-(methoxymethyl)-3-borabicyclo[3,3,1]non-
6-ene (2.8698 g, 14.8 mmol). A solution of borane THF (14.80
mL, 1 M, 14.8 mmol) was slowly added with stirring. The
reaction was exothermic, and the mixture came to reflux
during addition of the borane‚THF. The reaction solution was
then lowered into an oil bath preheated to 80 °C and refluxed
for 1 h. The THF was removed by distillation at atmospheric
pressure under nitrogen, followed by vacuum distillation (20
mmHg). The white residue was then sublimed under vacuum
(0.100 mmHg, 70 °C) onto an ice/water chilled coldfinger to
yield 1-boraadamantane‚THF 3 (1.6140 g, 53%) as a solid,
white, crystalline compound: mp 89-91 °C; 1H NMR (500
MHz, C6D6) δ 3.34 (t, J ) 6.8, 4H), 2.74 (br s, 3H), 2.00 (m,
6H), 1.01 (m, 10H); 13C NMR (125.8 MHz, C6D6) δ 68.4, 40.7,
34.6, 29.7 (br s), 24.3; 11B NMR (237 MHz, CDCl3) δ 5.10 (br
s); IR (KBr) 2868, 1438, 1350, 1299, 1252, 1226, 1192, 1119,
1078, 1025, 982, 930, 918, 855, 728, 614 cm-1; HRMS (CI) m/ z
calcd for C13H24BO (M + H)+ 207.1923, found 207.1929.
(M + H)+ 270.1702, found 270.1692. Anal. Calcd for C13H24
-
BO2SN: C, 58.00; H, 8.99; N, 5.20. Found: C, 58.21; H, 9.05;
N, 5.27.
1-Bor a a d a m a n ta n e‚L-Leu cin e Meth yl Ester (7). L-Leu-
cine methyl ester hydrochloride (5.95 g, 32.8 mmol) was
dissolved in deionized water (43.0 mL) containing sodium
bircarbonate (4.86 g, 57.7 mmol), and the solution was
saturated in sodium chloride. The aqueous solution was
extraced with methylene chloride, and the organic layer was
washed with saturated sodium chloride and dried over sodium
sulfate. The methylene chloride was removed in vacuo to afford
l-leucine methyl ester (4.26 g, 29.3 mmol) as a clear, colorless
liquid in 90% yield. To a solution of L-leucine methyl ester (1.06
g, 7.36 mmol) in methylene chloride (10.0 mL) was added a
solution of 1-boraadamantane‚THF 3 (1.50 g, 7.29 mmol) in
methylene chloride (10 mL) with stirring. The methylene
chloride was removed in vacuo to afford 1-boraadamantane‚
L-leucine methyl ester 7 (1.96 g, 7.02 mmol) as a white
crystalline solid in 96% yield: mp 91-93 °C; [R]24D -2.0 (c 0.88,
CHCl3); 1H NMR (500 MHz, CDCl3) δ 4.14 (m, 1H), 3.80 (s,
3H), 3.74 (dd, J ) 8.5, 11.7, 1H), 2.83 (d, J ) 11.7, 1H), 2.11
(br s, 3H), 1.62 (m, 2H), 1.58 (d, J ) 12.3, 3H), 1.52 (d, J )
8.5, 1H), 1.48 (d, J ) 11.2, 3H), 0.98 (t, J ) 5.7, 6H), 0.42 (d,
J ) 10.5, 3H), 0.35 (d, J ) 10.5, 3H); 13C NMR (125.8 MHz,
1-Bor a a d a m a n ta n e‚1-a m in oa d a m a n ta n e (4). The pro-
cedure of Mikhailov and co-workers was followed to make
complex 4.9a In a 100 mL flask, 1-aminoadamantane (1.05 g,
6.9 mmol) was dissolved in methylene chloride (15.0 mL). In
a 25 mL flask, sublimed 1-boraadamantane THF (1.43 g, 6.9
mmol) was dissolved in methylene chloride (10.0 mL), and this
solution was added to the 1-aminoadamantane solution with
stirring. The solvent was removed in vacuo to yield crude
1-boraadamantane‚1-aminoadamantane 4 (1.78 g, 90%) as a
fine off-white powder. The 1-boraadamantane‚1-aminoada-
mantane was recrystallized in a minimum amount of ethanol
to yield 4 (0.76 g, 38%) as clear, white crystals: mp 190-194
1
°C; H NMR (500 MHz, CDCl3) δ 2.63 (br s, 1H), 2.11 (br s,
6H), 1.98 (br s, 1H), 1.83 (br s, 6H), 1.63 (m, 10H), 1.52 (m,
3H), 0.57 (m, 6H); 13C NMR (125.8 MHz, CDCl3) δ 54.9, 43.4,
40.2, 35.8, 33.4 (br), 33.0, 29.4; IR (KBr) 3275, 3228, 2905,
1567, 1455, 1303, 1231, 1072, 1013, 989, 938, 783, 763 cm-1
;
HRMS (FAB) m/ z calcd for C19H32BN (M+) 285.2628, found
285.2609. Anal. Calcd for C19H32BN: C, 79.99; H, 11.31; N,
4.91. Found: C, 79.84; H, 11.38; N, 4.91.
CDCl3) δ 53.2, 51.1, 42.8, 40.2, 32.7, 30.1, 25.1, 22.8, 22.0; 11
B
1-Bor a a d a m a n ta n e‚L-P h en yla la n in e Meth yl Ester (5).
L-Phenylalanine methyl ester hydrochloride (1.80 g, 8.35
mmol) was dissolved in methylene chloride (25 mL), and this
solution was extracted twice with an equal volume of saturated
aqueous sodium bicarbonate. The organic layer was dried over
sodium sulfate, the solution was filtered, and solvent was
removed in vacuo to give L-phenylalanine methyl ester (1.00
g, 67%). L-Phenylalanine methyl ester (0.9079 g, 5.1 mmol)
was dissolved in methylene chloride (10.0 mL) in a 100 mL
flask. In a 25 mL flask, sublimed 1-boraadamantane‚THF
(1.0442 g, 5.1 mmol) was dissolved in methylene chloride (10
mL), and this solution was added with stirring to the L-
phenylalanine solution. The solvents were removed in vacuo
to give 1-boraadmanatane‚L-phenylalanine methyl ester 5 (1.6
NMR (237 MHz, CDCl3) δ -4.7; IR (KBr) 3308, 3242, 2865,
1733, 1560, 1438, 1388, 1288, 1252, 1219, 1148, 1071, 989, 942,
789 cm-1; HRMS (CI) m/ z calcd for C16H31BO2N (M + H)+
280.2451, found 280.2450. Anal. Calcd for C16H30BO2N: C,
68.82; H, 10.83; N, 5.02. Found: C, 68.99; H, 11.2; N, 5.06.
1-Bor a a d a m a n ta n e‚Eth a n ola m in e (8). To a solution of
ethanolamine (0.356 g, 5.8 mmol) in methylene chloride (10.0
mL) was added a solution of 1-boraadamantane‚THF 3 (1.20
g, 5.8 mmol) in methylene chloride (10.0 mL) with stirring.
The methylene chloride was removed in vacuo to afford
1-boraadamantane‚ethanolamine 8 (1.14 g, 5.8 mmol) as a
white, crystalline solid in quantitative yield: mp 131-133 °C;
1H NMR (500 MHz, CDCl3) δ 3.78 (t, J ) 4.9, 2H), 3.15 (br s,
2H), 2.87 (m, 2H), 2.14 (br s, 3H), 1.62 (d, J ) 11.6, 3H), 1.51
(d, J ) 11.9, 3H), 0.44 (br s, 6H); 13C NMR (125.8 MHz, CDCl3)
δ 60.8, 41.9, 40.8, 32.8, 30.5 (br s); 11B NMR (237 MHz, CDCl3)
δ -7.0; IR (KBr) 3610, 3425, 3289, 3247, 2861, 1587, 1231,
1068 cm-1; HRMS (CI) m/ z calcd for C11H23BNO (M + H)+
195.1797, found 195.1791. Anal. Calcd for C11H22BNO: C,
67.72; H, 11.37; N, 7.18. Found: C, 67.47; H, 11.43; N, 7.32.
g, 5.1 mmol) as a white crystalline solid: mp 53-59 °C; [R]24
D
1
+5.7 (c 0.92, CHCl3); H NMR (500 MHz, CDCl3) δ 7.38 (m,
3H), 7.13 (d, J ) 6.9, 2H), 3.99 (m, 1H), 3.89 (m, 1H), 3.85 (s,
3H), 3.17 (dd, J ) 14.3, 5, 1H), 2.97 (dd, J ) 14.3, 7.95, 1H),
2.91 (m, 1H), 2.09 (br s, 3H), 1.57 (d, J ) 12.0, 3H), 1.44 (d, J
) 11.1, 3H), 0.35 (m, 3H), 0.27 (m, 3H); 13C NMR (125.8 MHz,
CDCl3) δ 172.1, 133.6, 129.5, 129.3, 128.2, 53.2, 53.0, 40.0, 38.3,

1-Boraadamantaneamine Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 2831
Di-1-a d a m a n tyla m id e (9). To a solution of 1-adamantyl-
carbonyl chloride (3.31 g, 16.6 mmol) in CH2Cl2 (16.0 mL) at
0 °C were added 1-aminoadamantane (2.52 g, 16.7 mmol) and
pyridine (4.0 mL, 49.5 mmol). The reaction mixture was
warmed to room temperature and stirred (3 h). The reaction
was quenched by washing with 10% HCl (25.0 mL) and
saturated NaCl (25.0 mL). The organic layer was separated,
dried over sodium sulfate, and filtered, and excess CH2Cl2 was
removed in vacuo. The crude product was chromatographed
(SiO2, 2:1 hexane/EtOAc) to give 9 as a white solid (1.0 g,
19%): mp 314-315 °C; 1H NMR (500 MHz, CDCl3) δ 5.22 (br
s, 1H), 2.06 (m, 6H), 1.98 (d, J ) 3.0, 6H), 1.80 (d, J ) 2.6,
6H), 1.68 (m, 12H); 13C NMR (125.8 MHz, CDCl3) δ 177.2, 51.2,
41.7, 41.6, 40.9, 39.4, 36.6, 36.4, 29.5, 28.2; IR (KBr) 3328,
acid (1.09 g, 6.0 mmol) was dissolved in thionyl chloride (15.0
mL, 206 mmol) and heated to reflux (1 h) under nitrogen.
Excess thionyl chloride was removed in vacuo, and the crude
1-adamantanecarbonyl chloride was dissolved in benzene (10
mL). The solution of 1-adamantanecarbonyl chloride was
added to a solution of L-leucine methyl ester hydrochloride
(1.09 g, 6.0 mmol) and triethylamine (1.68 mL, 12.0 mmol) in
benzene (40 mL) at 0 °C with stirring. The reaction mixture
was warmed to room temperature and stirred overnight. The
reaction mixture was filtered to remove triethylamine hydro-
chloride, and excess solvents were removed in vacuo. The crude
product was chromatographed (SiO2, 2:1 hexane/EtOAc) to give
14 (1.26 g, 68%) as a white solid: mp 137-138 °C; [R]24D -2.2
(c 0.38, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.96 (m, 1H),
4.63 (m, 1H), 3.72 (s, 3H), 2.05 (s, 3H), 1.87 (m, 6H), 1.61 (m,
6H), 1.54 (m, 3H), 0.94 (s, 3H), 0.91 (s, 3H); 13C NMR (125.8
MHz, CDCl3) δ 177.7, 173.9, 52.2, 50.4, 41.8, 40.6, 39.1, 36.5,
28.1, 24.9, 22.8, 22.1; IR (KBr) 3342, 2913, 1747, 1635, 1533
cm-1; HRMS (CI) m/ z calcd for C18H30NO3 (M + H)+ 308.2226,
found 308.2221. Anal. Calcd for C18H29NO3: C, 70.32; H, 9.51;
N, 4.56. Found: C, 70.04; H, 9.49; N, 4.69.
2902, 1637, 1540, 1449 cm-1; HRMS (CI) m/ z calcd for C21H32
-
-
NO (M)+ 313.2405, found 313.2406. Anal. Calcd for C21H31
NO: C, 80.46; H, 9.97; N, 4.47. Found: C, 80.59; H, 9.97; N,
4.49.
Di-1-a d a m a n tyla m in e (11). Di-1-adamantylamine was
synthesized according to the protocol of Dervan and McIntyre.
A thick-walled glass tube, closed at one end and narrowed at
the other, was filled with 1-adamantylamine (7.4 g, 49 mmol)
and 1-adamantyl bromide (5.52 g, 26 mmol). After the narrow
neck was sealed under vacuum, the tube was placed in a
ceramic heating oven and heated to 220 °C for 40 h. The solid
melted and then solidified after 1 day. Upon cooling to room
temperature, the crude product was crushed with a mortar
and pestle and extracted with diethyl ether (250 mL) and 25%
NaOH (250 mL) by shaking vigorously in a separatory funnel.
The ether layer was separated and shaken with 2.4 N HCl
(250 mL) to precipitate di-1-adamantylamine as the hydro-
chloride salt. The precipitate was filtered and washed with
water (50 mL). The resulting white solid was shaken with a
two-phase layer of ether (200 mL) and 25% NaOH (250 mL)
to regenerate di-1-adamantylamine in ether. The ether layer
was separated and dried over anhydrous K2CO3. After filtra-
tion and removal of the solvent in vacuo, 1.87 g (26%) of di-
1-adamantylamine (11) was obtained as a fluffy white solid:
mp 194-195 °C (lit. 196-197 °C); 1H NMR (500 MHz, CDCl3)
δ 2.01 (br s, 6H), 1.76 (br d, 12H), 1.61 (br s, 12H); 13C NMR
(125.8 MHz, CDCl3) δ 52.6, 46.5, 36.6, 30.0; IR (KBr) 2898,
(S)-2-[(Ad a m a n ta n e-1-ca r bon yl)a m in o]-3-p h en ylp r op i-
on ic Acid (15). To a solution of amide 14 (0.43 g, 1.3 mmol)
dissolved in THF (1.2 mL) was added a solution of lithium
hydroxide (0.076 g, 1.8 mmol) in water (1.2 mL). The reaction
mixture was stirred for 1 h and acidified with HCl, and excess
solvents were removed in vacuo. The residue was chromato-
graphed (SiO2, 95:5 chloroform/MeOH) to give 15 (0.26 g, 63%)
as a solid white foam: mp 65-67 °C; [R]24 +46.8 (c 0.47,
D
CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.29 (m, 3H), 7.15 (d, J
) 7.1, 2H), 6.06 (d, J ) 6.3, 1H), 4.81 (q, J ) 6.1, 1H), 3.20
(m, 2H), 2.0 (s, 3H), 1.76 (m, 6H), 1.7 (m, 6H); 13C NMR (125.8
MHz, CDCl3) δ 178.7, 174.5, 135.7, 129.4, 128.6, 127.2, 53.1,
40.6, 38.9, 37.0, 36.3, 27.9; IR (KBr) 3423, 2906, 1735, 1624,
1522 cm-1; HRMS (CI) m/ z calcd for C20H25NO3 (M)+ 327.1834,
found 327.1833. Anal. Calcd for C20H25NO3: C, 73.37; H, 7.70;
N, 4.28. Found: C, 73.61; H, 7.97; N, 4.15.
(S)-2-[(Ad a m a n t a n e-1-ca r bon yl)a m in o]-4-m et h ylp en -
ta n oic Acid (16). To a solution of amide 13 (0.24 g, 0.78 mmol)
dissolved in THF (1.2 mL) was added a solution of lithium
hydroxide (0.052 g, 1.24 mmol) in water (1.2 mL). The reaction
mixture was stirred for 1 h and acidified with HCl, and excess
solvents were removed in vacuo. The residue was chromato-
graphed (SiO2, 95:5 chloroform/MeOH) to give 16 (0.20 g, 87%)
1448, 1342, 1304, 1160 cm-1; HRMS (CI) m/ z calcd for C20H32
N
(M)+ 285.2456, found 285.2453. Anal. Calcd for C20H31N: C,
84.15; H, 10.95; N, 4.91. Found: C, 83.92; H, 10.98; N, 4.95.
Hyd r och lor id e (12). Anal. Calcd for C20H32NCl: C, 74.62;
H, 10.02; N, 4.35; Cl, 11.01. Found: C, 74.55; H, 10.10; N, 4.36;
Cl, 11.06.
as a white powder: mp 173-174 °C; [R]24 -14.5 (c 0.36,
D
CHCl3); 1H NMR (500 MHz, CDCl3) δ 9.15 (br s, 1H), 6.06 (br
s, 1H), 4.59 (q, J ) 5.6, 1H), 2.04 (s, 3H), 1.86 (m, 6H), 1.65
(m, 8H), 1.58 (m, 1H), 0.94 (m, 6H); 13C NMR (125.8 MHz,
CDCl3) δ 178.7, 176.6, 50.8, 41.0, 40.6, 38.9, 36.4, 28.0, 24.9,
22.8, 22.0; IR (KBr) 3402, 3100 (br), 2911, 1735, 1626, 1542
cm-1; HRMS (CI) m/z calcd for C17H27O3N (M)+ 293.1991, found
293.1987. Anal. Calcd for C17H27NO3: C, 69.59; H, 9.28; N,
4.77. Found: C, 69.93; H, 9.54; N, 4.52.
(S)-2-[(Ad a m a n ta n e-1-ca r bon yl)a m in o]-3-p h en ylp r op i-
on ic Acid Meth yl Ester (13). 1-Adamantanecarboxylic acid
(1.09 g, 6.0 mmol) was dissolved in thionyl chloride (15.0 mL,
206 mmol), and the mixture was heated to reflux (1 h) under
nitrogen. Excess thionyl chloride was removed in vacuo, and
the crude 1-adamantanecarbonyl chloride was dissolved in
benzene (10 mL). The solution of 1-adamantanecarbonyl
chloride was added to a solution of L-phenylalanine methyl
ester hydrochloride (1.30 g, 6.0 mmol) and triethylamine (1.68
mL, 12.0 mmol) in benzene (40 mL) at 0 °C with stirring. The
reaction mixture was warmed to room temperature and stirred
overnight. The reaction mixture was filtered to remove tri-
ethylamine hydrochloride, and excess solvents were removed
in vacuo. The crude product was chromatographed (SiO2, 2:1
hexane/EtOAc) to give 13 (1.99 g, 96%) as a white solid: mp
98-98.5 °C; [R]24 +69.9 (c 0.74, CHCl3), lit. [R]25 +63.7;30
Molecu la r Mod elin g. Gen er a l Meth od ology. All molec-
ular modeling studies were done on
a Silicon Graphics
computer running the Insight II (version 97)31 and Discover
(version 2.98) software (Molecular Simulations, Inc., San
Diego, CA). Minimization methods were employed and refined
to the local minima of the structures as described below. The
docking between the adamantane derivatives and the enzyme
was carried out manually and then Assembly refined in the
Discover III mode employing an all multipole summation
method with a dielectric constant of 1.0. The potentials
employed were the ESFF (Extensible Systematic Force Field)
in the Insight package. Conjugate (Polak-Ribiere) was used
in energy minimizations. The maximum derivative was 0.899
with a total potential energy of -4095.7 kcal/mol.
D
D
1H NMR (500 MHz, CDCl3) δ 7.25 (m, 3H), 7.08 (d, J ) 6.9,
2H), 6.02 (br d, J ) 7.2, 1H), 4.87 (q, J ) 5.7, 1H), 3.73 (s,
3H), 3.12 (dq, J ) 13.8, 5.7, 2H), 2.02 (br s, 3H), 1.79 (m, 6H),
1.72 (m, 6H); 13C NMR (125.8 MHz, CDCl3) δ 177.3, 172.3,
135.9, 129.3, 128.5, 127.1, 52.7, 52.3, 40.6, 39.0, 37.8, 36.4, 28.0;
IR (KBr) 3318, 2901, 1751, 1639, 1534 cm-1; HRMS (CI) m/ z
calcd for C21H27NO3 (M)+ 341.1991, found 341.1993. Anal.
Calcd for C21H27NO3: C, 73.87; H, 7.97; N, 4.10. Found: C,
73.57; H, 7.94; N, 4.24.
Cell Cu ltu r e. The CD81-enriched astrocytes as described
by Geisert et al. were derived from rat pup glia primary culture
collected 4-8 days postpartum.32 The primary cultures were
prepared as previously described by McCarthy et al.33 C6
glioma is a CD81-deficient cell line that was derived from a
frozen rat cell culture bought from ATCC. The cells were
maintained in 10% fetal calf serum (FCS, Hyclone) in Basal
(S)-2-[(Ad a m a n t a n e-1-ca r bon yl)a m in o]-4-m et h ylp en -
ta n oic Acid Meth yl Ester (14). 1-Adamantanecarboxylic

2832 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Wagner et al.
Medium Eagle (BME, Gibco) in 75 cm2 flasks. The cells were
grown for several weeks before use in screening assays.
Scr een in g Assa ys. All samples were dissolved completely
to make a 100 µM stock solution in a 1% DMSO HBSS and
diluted to produce a series of concentrations. A 10 µL aliquot
of these initial solutions was added to 190 µL of 2% FCS-
BME to produce the test concentration. The final concentration
of DMSO was 0.05%, and it showed no effect on the cell
proliferation. The vehicle solution was tested as a control.
Dilutions were performed such that cosolvent concentrations
did not vary for a particular experiment. The assays were
performed with parallel manipulations of astrocyte and C6
glioma cells. The cells were trypsinized and transferred to 96-
well microtiter plates at a cell density of 103 C6 cells per well
and 3 × 103 astrocytes per well. The optimal cell seeding
density on cell growth was determined by doing identical
experiments at different cell densities. The range of cell
densities used in screening assays did not cause variability in
the growth rates of C6 or primary astrocyte cultures. The
experimental treatment of the cells was incubation with test
compound for 4 days at 37 °C, 5% CO2. The growth kinetics
did not show variability for the 4-day culture period for C6
and astrocytes.
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After the incubation period, cells were fixed with 4%
formaldehyde for 30 min. Fixative was removed by three
isotonic saline washes of 5 min duration. Cells were quanti-
tated spectrophotometrically using 0.1% cresylecth violet stain
prepared in a sodium acetate buffer, pH 3.7, as described
previously by Kueng et al.34 Briefly, the cells were dyed with
100 µL of the dye for 30 min. The excess dye was rinsed from
the wells with two 200 µL portions of normal saline. The dye
was released from the cells using a 100 µL aliquot of 10%
glacial acetic acid for 20 min. The absorbance of the resulting
supernatant was measured using a Titertech Pro Plus micro-
titer plate reader using a 560 µm wavelength filter.
Im m u n oblot Meth od . Samples containing equal amounts
of protein were dissolved in nonreducing sample buffer (2%
SDS, 10% glycerol in 0.05 M Tris-HCl buffer, pH 6.8) and run
on 10% SDS-PAGE. After electrophoresis, the gels were
transferred to nitrocellulose for immunoblot analysis. Then the
blots were blocked in nonfat dry milk, probed with AMP1
(CD81 antibody) as a primary antibody, rinsed in borate buffer,
pH 8.5, incubated in HRP-labeled secondary antibody, and
reacted with diaminobenzidine and hydrogen peroxide. The
level of immunoreaction product was determined by scanning
the blots and analyzing these scans with the National Insti-
tutes of Health image program.
Ack n ow led gm en t. This research was supported by
grants from the National Science Foundation (Grant
CHE-9617475) and PHS (Grant R01EY12369). C.E.W.
thanks Allergan, Inc. for a graduate fellowship.
Su p p or tin g In for m a tion Ava ila ble: X-ray data and
other data for compounds 4, 6, 9, 11, and 14. This material is
available free of charge via the Internet at http://pubs.acs.org.
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