CD4 mimics targeting the mechanism of HIV entry

Article abstract of DOI:10.1016/j.bmcl.2009.10.098

A structure-activity relationship study was conducted of several CD4 mimicking small molecules which block the interaction between HIV-1 gp120 and CD4. These CD4 mimics induce a conformational change in gp120, exposing its co-receptor-binding site. This induces a highly synergistic interaction in the use in combination with a co-receptor CXCR4 antagonist and reveals a pronounced effect on the dynamic supramolecular mechanism of HIV-1 entry.

Full text of DOI:10.1016/j.bmcl.2009.10.098

Bioorganic & Medicinal Chemistry Letters 20 (2010) 354–358
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
CD4 mimics targeting the mechanism of HIV entry
Yuko Yamada a, Chihiro Ochiai a, Kazuhisa Yoshimura b, Tomohiro Tanaka a, Nami Ohashi a, Tetsuo Narumi a,
Wataru Nomura a, Shigeyoshi Harada b, Shuzo Matsushita b, Hirokazu Tamamura a,
*
a Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan
b Center for AIDS Research, Kumamoto University, Kumamoto 860-0811, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A structure–activity relationship study was conducted of several CD4 mimicking small molecules which
block the interaction between HIV-1 gp120 and CD4. These CD4 mimics induce a conformational change
in gp120, exposing its co-receptor-binding site. This induces a highly synergistic interaction in the use in
combination with a co-receptor CXCR4 antagonist and reveals a pronounced effect on the dynamic supra-
molecular mechanism of HIV-1 entry.
Received 19 September 2009
Revised 20 October 2009
Accepted 22 October 2009
Available online 4 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
CD4 mimic
HIV entry
Synergistic effect
CXCR4
Recently, remarkable success has attended the clinical treat-
ment of HIV-infected and AIDS patients, with ‘highly active anti-
retroviral therapy (HAART)’. This approach involves a combination
of two or three agents from two categories: reverse transcriptase
inhibitors and protease inhibitors.1 In addition, the molecular
mechanism involved in HIV-entry and -fusion into host cells has
been described in detail.2 The complex interactions of surface pro-
teins on cellular and viral membranes, which are designated as a
dynamic supramolecular mechanism of HIV entry, are reported
to be crucial to the viral infection. In a first step, an HIV envelope
protein, gp120 interacts with a cell surface protein, CD4, leading
to a conformational change in gp120 followed by subsequent bind-
ing of gp120 to a co-receptor CCR53 or CXCR4.4 CCR5 and CXCR4
are the major co-receptors for the entry of macrophage-tropic
(R5-) and T cell line-tropic (X4-) HIV-1, respectively. The interac-
tion of gp120 with CCR5 or CXCR4 triggers entry of another enve-
lope protein, gp41 to the cell membrane and formation of a gp41
trimer-of-hairpins structure, which causes fusion of HIV/cell-mem-
branes and completes the infection.
Informed by this mechanism, a fusion inhibitor, enfuvirtide (fuz-
eon, Trimers & Roche)5 and a CCR5 antagonist, maraviroc (Pfizer)6 in
addition to an integrase inhibitor, raltegravir (Merck)7 have been
used clinically. However, serious problems with chemotherapy still
persist, including the emergence of viral strains with multi-drug
resistance (MDR), considerable adverse effects and high costs. Con-
sequently, development of novel drugs possessing mechanisms of
action different from those of the above inhibitors is currently re-
quired. We have previously developed selective CXCR4 antagonists8
and fusion inhibitors.9 Furthermore, N-(4-Bromophenyl)-N0-
(2,2,6,6-tetramethylpiperidin-4-yl)-oxalamide (1) and N-(4-chloro-
phenyl)-N0-(2,2,6,6-tetramethylpiperidin-4-yl)-oxalamide (2) were
previously found using chemical library screening to inhibit syncy-
tium formation by other researchers.10 1 and 2 bind to gp120 with
binding affinities of Kd = 2.2 lM and 3.7 lM, respectively, blocking
the interaction of gp120 with CD4 in the first step of an HIV-1 entry.
Thus, in the present study we focus on the development of CD4 mim-
ics that can block the interaction between gp120 and CD4. We have
investigated the effect of CD4 mimics on conformational changes of
gp120 and on their use in combination use with a CXCR4 antagonist.
Initially, molecular modeling of compound 2 docked into gp120
was carried out using docking simulations performed by the Flex-
SIS module of SYBYL 7.1 (Tripos, St. Louis) (Fig. 1).11 The atomic
coordinates of the crystal structure of gp120 with soluble CD4
(sCD4) were retrieved from Protein Data Bank (PDB) (entry 1RZJ)
(Fig. 1a) and it was observed that Phe43 and Arg59 of the CD4 have
multiple contacts with Asp368, Glu370 and Trp427 of gp120, which
are all conserved residues. An inspection of the environment of
compound 2 docked in gp120 revealed the presence of a large cav-
ity around the p-position of the phenyl ring of compound 2, which
could interact with the viral surface protein gp120 (Fig. 1b and c).
Several analogs of 2 with substituents on the phenyl ring were
therefore synthesized.
All compounds except 12 were synthesized by previously
published methods (Scheme 1).10b,12,13 Aniline derivatives (3) were
coupled with ethyl oxalyl chloride to yield the corresponding ethyl
oxalamates 4. Saponification of the above oxalamates to the
corresponding free acids and the subsequent coupling with 4-ami-
* Corresponding author.
E-mail address: tamamura.mr@tmd.ac.jp (H. Tamamura).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.098

Y. Yamada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 354–358
355
Scheme 2. Reagents and conditions: (i) oxalyl chloride, Et3N; (ii) 4-amino-2,2,6,6-
tetramethylpiperidine, Et3N.
The anti-HIV activity of the synthetic compounds was evaluated
against various viral strains including both laboratory and primary
isolates (Table 1). IC50 values were determined by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method15 as the concentrations of the compounds which conferred
50% protection against HIV-1-induced cytopathogenicity in PM1/
CCR5 cells. Cytotoxicity of the compounds based on the viability
of mock-infected PM1/CCR5 cells was also evaluated using the
MTT method. CC50 values were determined as the concentrations
achieving 50% reduction of the viability of mock-infected cells.
Compounds 1 and 2 showed potent anti-HIV activity against labo-
ratory isolates, IIIB (X4, Sub B) and 89.6 (dual, Sub B) strains, and
compound 2 also possessed potent activity against a primary iso-
late, an fTOI strain (R5, Sub B). All of the IC50 values were between
4
lM and 10
lM. Compound 1 was not tested against primary iso-
lates. The potencies of compounds 1 and 2 are comparable to the
reported binding affinities for gp120 (Kd = 2.2 and 3.7 lM, respec-
tively).10 Several of the new analogs of compounds 1 and 2 showed
significant anti-HIV activity. Compound 5, which has a phenyl
group in place of the p-chlorophenyl group of compound 2, did
not show significant anti-HIV activity at concentrations below
100 lM against all strains tested except for an fTOI strain (R5,
Sub B). This result suggests that a substituent at the p-position of
the phenyl ring is critical for potent activity. Compound 6, which
has a fluorine atom at the p-position of the phenyl ring, showed
moderate anti-HIV activity against laboratory isolates, IIIB (X4,
Sub B) and 89.6 (dual, Sub B) strains (IC50 = 61 and 81
lM, respec-
tively), but, at concentrations below 100 M, failed to show signif-
l
icant anti-HIV activity against a primary isolate, a KYAG strain (R5,
Sub B). Among halogen atoms, fluorine is less suitable than bro-
mine or chlorine as a substituent at the p-position of the phenyl
ring, as evidenced by compound 6, which is 8–15-fold less potent
than compounds 1 and 2 against IIIB (X4, Sub B) and 89.6 (dual,
Sub B) strains. Compound 7, which has a methyl group at the p-po-
sition of the phenyl ring, showed relatively more potent activity
against IIIB (X4, Sub B) and 89.6 (dual, Sub B) strains (IC50 = 23
Figure 1. (a) The crystal structure of gp120 with soluble CD4 (sCD4) retrieved from
the PDB (entry 1RZJ); (b) docking structure of compound 2 and gp120; (c) a focused
figure of (b) shown by space-filling model.
and 41
showed significant anti-HIV activity against primary isolates, fTOI
(R5, Sub B) and KYAG (R5, Sub B) strains (IC50 = 16 and 51 M,
lM, respectively) than compound 6. Compound 7 also
l
respectively). Compound 8, with a methoxy group at the p-position
of the phenyl ring, did not show significant anti-HIV activity
against all strains tested until a concentration of 100 lM was
reached. In the biological assays, derivatives having electron-with-
drawing substituents such as bromine, chlorine and fluorine at the
p-position of the phenyl ring are relatively potent, whereas deriv-
atives having electron-donating groups such as methoxy at this po-
sition are not potent. Furthermore, the steric effect of a substituent
at the p-position of the phenyl ring appears to be critical to anti-
Scheme 1. Reagents and conditions: (i) ethyl oxalyl chloride, Et3N; (ii) 1 M NaOH;
4-amino-2,2,6,6-tetramethylpiperidine, 1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide hydrochloride, 1-hydroxybenzotriazole, Et3N.
no-2,2,6,6-tetramethylpiperidine using 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide hydrochloride (EDC) and 1-hydroxybenzotri-
azole (HOBt) yielded compounds 5–9. In the case of compound 12,
whose amide bond is not stable during the reaction of the saponifica-
tion of the corresponding oxalamates, an alternative synthetic
scheme was used (Scheme 2).14 The reaction of p-nitroaniline (10)
with oxalyl chloride gave the corresponding oxoacetamide 11, which
was subsequently coupled with 4-amino-2,2,6,6-tetramethylpiperi-
dine to yield the desired compound 12.
HIV activity. The sum of Hammett constants (r) of benzoic acid
substituents16 shown in Table 1 can be used to evaluate the elec-
tron-withdrawing or -donating effect of the substituents on the
aromatic ring. The Taft Es values16a,17 were used as steric
parameters for substituents at the p-position of the phenyl ring.
The order of potency found for the halogen-containing derivatives
in anti-HIV activity against laboratory isolates, IIIB (X4, Sub B) and
89.6 (dual, Sub B), is: compound 1 (R = Br) (
r
= 0.23, Es = ꢀ1.16), 2

356
Y. Yamada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 354–358
Table 1
Hammett constants (r) and steric effects (Es) of substituted aromatic rings and anti-HIV activity and cytotoxicity of synthetic compounds
Ra
rb
Es
IC50
(
lM)
CC50 (lM)
c
e
e
Compd
Lab. isolates
89.6 (dual)
Primary isolates
fTOI (R5) KYAG (R5)
IIIB (X4)
1
2
5
6
7
8
9
Br
Cl
H
F
CH3
OCH3
CF3
NO2
0.23
0.23
0
0.06
ꢀ0.17
ꢀ0.27
0.54
0.78
ꢀ1.16
ꢀ0.97
0
ꢀ0.46
ꢀ1.24
ꢀ0.55
ꢀ2.40
ꢀ1.77d
4
8
>100
61
23
>100
ND
ND
0.010
9
10
>100
81
41
>100
27
42
ND
5
81
ND
16
ND
ND
ND
0.0044
ND
150
170
350
320
210
340
72
>30
>100
>100
51
>100
ND
12
sCD4
ND
ND
230
ND
0.021
a
See Schemes 1 and 2.
b
c
r
= Hammett constant of a substitutent on a benzoic acid derivative.16
Es = steric effect of a substituent at the para position on the aromatic ring.16a,17
d
e
The average value of ꢀ1.01 and ꢀ2.52, which are Es values of the NO2 group, ꢀ1.77, was used.
Values are means of at least three experiments (ND = not determined).
(R = Cl) (
(R = H) (
withdrawing ability and also of their size. Methyl (
Es = ꢀ1.24) is an electron-donating group, but is almost as bulky
as a bromine atom. Thus, the p-methyl derivative 7 has relatively
potent anti-HIV activity against laboratory isolates, IIIB (X4, Sub
B) and 89.6 (dual, Sub B), higher than that of compound 6 (R = F)
but lower than that of compound 1 (R = Br) or 2 (R = Cl). The elec-
r
r
= 0.23, Es = ꢀ0.97), 6 (R = F) (
r
= 0.06, Es = ꢀ0.46) and 5
produced a remarkable increase in binding affinity for 4C11, simi-
lar to that observed in pretreatment with sCD4 (MFI = 37.90). This
is consistent with the results in the previous paper10 where it was
reported that compound 2 enhances the binding of gp120 to the
17b monoclonal antibody which recognizes the co-receptor bind-
ing site of gp120. Env-expressing cells, which were not pretreated
with sCD4 or a CD4 mimic compound, did not show significant
binding affinity for 4C11 (Fig. 2, blank). The increase in binding
affinity for monoclonal antibodies may be due to conformational
changes in gp120, which were caused by the interaction of sCD4
or a CD4 mimic with gp120. It is hypothesized that such conforma-
tional changes involve the exposure of the co-receptor binding site
of gp120 (the V3 loop), which is hidden internally, since the bind-
ing of gp120 to 17b is enhanced. Compound 5, which failed to
show significant anti-HIV activity, and compounds 7, 9 and 12,
which had significant anti-HIV activity, were assessed in the FACS
analysis. The profile of the binding of 4C11 to the Env-expressing
cell surface pretreated with compound 5 (MFI = 14.34) was similar
to that of the blank (MFI = 11.24), suggesting that compound 5 of-
fers no significant enhancement of binding affinity for 4C11. This
result is compatible with the anti-HIV activity of compound 5.
The profile of the binding of 4C11 to the Env-expressing cell sur-
face pretreated with compound 7 (MFI = 38.33) was entirely simi-
lar to that of compound 2 used as a pretreatment. Pretreatment of
the cell surface with compounds 9 and 12 (MFI = 29.09 and 30.01,
respectively) produced a slightly lower enhancement of binding
affinity for 4C11, compared to those of compounds 2 and 7 as pre-
treatments. However, in the ITC experiments reported else-
where,10c compound 9 (R = CF3) has a high affinity for gp120,
comparable to that of compound 2, but compound 12 (R = NO2)
does not have significant affinity for gp120, indicating that these
are not consistent with the current FACS studies, possibly due to
the difference in the assay systems. Although the anti-HIV activity
of 7 is weaker than that of compound 2, the level of compound 7
inducing an enhancement of binding affinity of gp120 for 4C11 is
comparable to that of compound 2. The concentration of com-
= 0, Es = 0). This is the order of substituents’ electron-
r
= ꢀ0.17,
tron-donating ability of a methoxy group is stronger (
r
= ꢀ0.27),
but the bulk size is smaller (Es = ꢀ0.55), than that of a methyl
group. Thus, the p-methoxy derivative 8 has no significant anti-
HIV activity against all strains tested at concentrations below
100
tron-withdrawing substituents such as trifluoromethyl (R = CF3)
= 0.54, Es = ꢀ2.40) and nitro (R = NO2) ( = 0.78, Es = ꢀ1.77) at
lM. Two derivatives containing bulkier and more potent elec-
(r
r
the p-position of the phenyl ring were evaluated. Compounds 9
(R = CF3) and 12 (R = NO2) showed significant anti-HIV activity
against an 89.6 (dual, Sub B) strain. These are less potent than com-
pounds 1 and 2 and this is perhaps due to the excessive size of the
substituents at the p-position. This suggests that a certain level of
the bulk size and a potent electron-withdrawing ability of the sub-
stituents are preferable for anti-HIV activity. It is estimated that a
cavity around the p-position of the phenyl ring of CD4 mimicking
compounds would be optimally filled by bromine (Es = ꢀ1.16) or
a methyl group (Es = ꢀ1.24) at p-position, and that an electron-
deficient aromatic ring might interact tightly with a negatively
charged group such as carboxy of Glu370. In isothermal titration
calorimetry (ITC) experiments reported elsewhere,10c compound
5 (R = H) does not have significant affinity for gp120, and com-
pound 6 (R = F) has less potent affinity for gp120 than compound
2, consistent with the present data. In all but one of the com-
pounds, no significant cytotoxicity was detected (CC50 >150
Table 1), the exception being compound 9 (R = CF3) (CC50 = 72
Compounds 7 and 12 have relatively low cytotoxicities, compared
lM,
lM).
to compounds 1 and 2.
Fluorescence activated cell sorting (FACS) analysis was per-
formed15 to investigate whether these synthetic compounds inter-
act with gp120 inducing the conformational change necessary for
the approach of an anti-envelope antibody or a co-receptor to
the gp120. The profile of binding of an anti-envelope CD4-induced
monoclonal antibody, 4C11, to the Env-expressing cell surface (an
R5-HIV-1 strain, JR-FL,-infected PM1 cells) pretreated with the
above CD4 mimic analogs was examined. Comparison of the bind-
ing of 4C11 to the cell surface was measured in terms of the mean
fluorescence intensity (MFI), and is shown in Figure 2. Pretreat-
ment of the Env-expressing cells with compound 2 (MFI = 38.42)
pounds used in the FACS analysis was 100
lM, much beyond the
IC50 values of compounds 2 and 7. A concentration of 100
l
M
would be also sufficient for the expression of anti-HIV activity
caused by compounds 2 and 7.
An effect on the use of compound 2 combined with another entry
inhibitor was investigated. Analysis of the synergistic effects of anti-
HIV agents was performed according to the median effect principle
using the CalcuSyn version 2 computer program18 to estimate IC50
values of compounds in different combinations. Combination indi-
ces (CI) were estimated from the data evaluated using the MTT assay

Y. Yamada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 354–358
357
Figure 2. JR-FL (R5, Sub B) chronically infected PM1 cells were preincubated with 100 lM of a CD4 mimic or sCD4 (11 nM) for 15 min, and then incubated with an anti-HIV-1
mAb, 4C11, at 4 °C for 15 min. The cells were washed with PBS, and fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG antibody was used for antibody-
staining. Flow cytometry data for the binding of 4C11 (green lines) to the Env-expressing cell surface in the presence of sCD4 or a CD4 mimic are shown among gated PM1
cells along with a control antibody (anti-human CD19: black lines). Data are representative of the results from a minimum of two independent experiments. The number at
the top of each graph shows the mean fluorescence intensity (MFI) of the antibody 4C11.
Health and Labour Sciences Research Grants from Japanese Minis-
try of Health, Labor, and Welfare.
Table 2
Combination indices (CI) for compound 2 or sCD4 and a CXCR4 antagonist, T140,
against an HIV IIIB strain
CI values at different ICa
References and notes
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Acknowledgments
This work was supported by Mitsui Life Social Welfare Founda-
tion, Grant-in-Aid for Scientific Research from the Ministry of Edu-
cation, Culture, Sports, Science, and Technology of Japan, and

358
Y. Yamada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 354–358
module, into the crystal structure of gp120 (PDB, entry 1RZJ). The binding site
n-hexane gave the title compound 7 (363 mg, 1.14 mmol, 39.8%) as colorless
crystals, mp = 176 °C; dH (400 MHz; CDCl3) 1.07 (1H, m, NH), 1.16 (6H, s, CH3),
1.29 (6H, s, CH3), 1.44 (2H, m, CH2), 1.91 (1H, d, J 3.7, CHH), 1.94 (1H, d, J 3.7,
CHH), 2.34 (3H, s, CH3), 4.25 (1H, m, CH), 7.17 (2H, d, J 8.3, ArH), 7.33 (1H, m,
NH), 7.50 (2H, d, J 8.4, ArH), 9.18 (1H, s, NH); HRMS (FAB), m/z calcd for
C18H28N3O2 (MH)+ 318.2182, found 318.2173.
was defined as residues Val255, Asp368, Glu370, Ser375, Ile424, Trp427, Val430 and
Val475, and included residues located within a radius 4.4 Å. The ligand was
considered to be flexible, and all other options were set to their default values.
Figures were generated with ViewerLite version 5.0 (Accelrys Inc., San Diego,
CA).
12. For example, the synthesis of compound 7: To a solution of ethyl oxalyl chloride
(0.400 mL, 3.48 mmol) in THF (20 mL) were added triethylamine (Et3N)
(0.480 mL, 3.48 mmol) and p-toluidine (373 mg, 3.48 mmol) with stirring at
0 °C. The reaction mixture was allowed to warm to room temperature, and
then stirred for 6 h. After removal by filtration of the resulting salts, the filtrate
was concentrated under reduced pressure. The residue was extracted with
EtOAc (50 mL), and the extract was washed successively with brine (20 mL),
1 M HCl (20 mL ꢁ 2), brine (20 mL), saturated NaHCO3 (20 mL ꢁ 2) and brine
(20 mL ꢁ 3), then dried over MgSO4. Concentration under reduced pressure
gave the crude ethyl oxalamate, which was used without further purification.
To a solution of the crude ethyl oxalamate (640 mg, 3.09 mmol) in THF (30 mL)
were added aqueous 1 M NaOH (3.40 mL, 3.40 mmol), water (50 mL) and
MeOH (20 mL) with stirring at 0 °C. The reaction mixture was allowed to warm
to room temperature, and then stirred for 20 h. After the addition of aqueous
1 M HCl (5 mL), MeOH and THF were evaporated under reduced pressure. The
13. McFarland, C.; Vicic, D. A.; Debnath, A. K. Synthesis 2006, 807.
14. The synthesis of compound 12: To a solution of Et3N (417
nitroaniline (138 mg, 1.00 mmol) in THF (1.3 mL) was added oxalyl dichloride
(85.8 L, 1.00 mmol) with stirring at 0 °C. After being stirred for 30 min at 0 °C,
Et3N (167 L, 1.20 mmol) and 4-amino-2,2,6,6-tetramethylpiperidine (156 L,
lL, 3.00 mmol) and 4-
l
l
l
0.90 mmol) were added. The reaction mixture was stirred for 6 h at 0 °C. After
removal by filtration of the resulting salts, the filtrate was concentrated under
reduced pressure. The residue was dissolved in CHCl3 (20 mL), and the mixture
was washed successively with brine (10 mL), saturated NaHCO3 (10 mL ꢁ 2)
and brine (10 mL ꢁ 3), and dried over MgSO4. Concentration under reduced
pressure followed by flash chromatography over silica gel with CHCl3–MeOH
(9:1) gave 42.4 mg (0.122 mmol, 13.5%) of the title compound 12 as colorless
crystals, mp = 190 °C; dH (400 MHz; CDCl3) 1.09 (1H, m, NH), 1.17 (6H, s, CH3),
1.29 (6H, s, CH3), 1.43 (2H, m, CH2), 1.92 (1H, d, J 3.8, CHH), 1.95 (1H, d, J 3.8,
CHH), 4.28 (1H, m, CH), 7.29 (1H, m, NH), 7.82 (2H, d, J 9.1, ArH), 8.28 (2H, d, J
9.1, ArH), 9.55 (1H, s, NH); HRMS (FAB), m/z calcd for C17H25N4O4 (MH)+
349.1876, found 349.1871.
residue was acidified to pH
2 with 1 M HCl, and extracted with EtOAc
(50 mL ꢁ 2). The combined organic layer was washed with brine (20 mL ꢁ 3),
and dried over MgSO4. Concentration under reduced pressure gave the crude
acid, which was used for the next reaction without further purification. To a
solution of the above crude acid (514 mg, 2.87 mmol) in THF (10 mL) were
added 1-hydoxybenzotriazole (484 mg, 3.16 mmol), 4-amino-2,2,6,6-
15. Yoshimura, K.; Shibata, J.; Kimura, T.; Honda, A.; Maeda, Y.; Koito, A.;
Murakami, T.; Mitsuya, H.; Matsushita, S. AIDS 2006, 20, 2065.
16. (a) Chapman, N. B.; Shorter, J. Advances in Linear Free Energy Relationship;
Plenum Press: London, 1972; (b) Chapman, N. B.; Shorter, J. Correlation Analysis
in Chemistry; Plenum Press: London, 1978; (c) Hansch, C.; Leo, A. J.; Hoekman,
D. Exploring QSAR, Hydrophobic, Electronic, and Steric Constants; American
Chemical Society: Washington, DC, 1995.
17. Taft, R. W. In Steric Effects in Organic Chemistry; Newman, M. S., Ed.; John Wiley:
New York, 1956; p 556.
18. (a) Chou, T. C.; Talalay, P. J. Biol. Chem. 1977, 252, 6438; (b) Chou, T. C.; Hayball,
M. P. CalcuSyn, 2nd ed.; Biosoft: Cambridge, UK, 1996.
tetramethylpiperidine
(446
lL,
2.58 mmol),
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (606 mg, 3.16 mmol) and
Et3N (0.439 mL, 3.16 mmol) with stirring at 0 °C. The reaction mixture was
allowed to warm to room temperature, and then stirred for 20 h. After
evaporation of THF, the residue was dissolved in CHCl3 (50 mL). The mixture
was washed with saturated NaHCO3 (20 mL ꢁ 2) and brine (20 mL ꢁ 3), and
dried over MgSO4. Concentration under reduced pressure gave the crude
crystalline mass. The usual work-up followed by recrystallization from EtOAc–