Rapid microwave-assisted syntheses of derivatives of HIV-1 entry inhibitors

Article abstract of DOI:10.1055/s-2006-926339

The direct amidation of esters with 4-amino-2,2,6,6-tetramethylpiperidine was achieved by computer-controlled microwave irradiation in toluene. The microwave protocol allowed the new amides to be prepared in three hours rather than the three days required by traditional thermal methods. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2006-926339

PAPER
807
Rapid Microwave-Assisted Syntheses of Derivatives of HIV-1 Entry Inhibitors
R
apid Microwave
h
-Assisted Sy
r
ntheses
i
of
D
eri
s
vatives of
H
IV-
M
1
Entry Inhibitors cFarland,a David A. Vicic,*a Asim Kumar Debnath*b
a
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA
E-mail: dvicic@uark.edu
Laboratory of Molecular Modeling and Drug Design of the Lindsley F. Kimball Research Institute of the New York Blood Center,
310 E. 67th St., New York, NY 10021, USA
b
Received 20 September 2005
The X-ray structure of an HIV-1 envelope glycoprotein
gp120 core domain complexed to a two-domain fragment
(D1D2) of the cellular receptor CD4 and to the Fab frag-
ment of the human neutralizing antibody 17b (gp120-
CD4-17b)14,18 and a recently published structure of an un-
liganded simian immunodeficiency virus (SIV) gp120
core, indicated distinct conformational differences in the
unliganded and the CD4-bound structures.19,20 These
structures also revealed a narrow cavity in the gp120 core
structure near the CD4 binding site, and that cavity has
been suggested as the putative binding site of BMS-
378806, a potent entry inhibitor reported by a group at
Bristol-Meyers Squib.21,22 In order to identify other small
molecule compounds that block gp120-CD4 interactions,
we initiated a screening program of commercially avail-
able chemical libraries. Recently, the two N-phenyl-N¢-
(2,2,6,6-tetramethylpiperidin-4-yl)oxalamide analogues 1
and 2 were identified as novel human immunodeficiency
virus type 1 (HIV-1) entry inhibitors that block the gp120-
CD4 interaction (Figure 2).23 These small organic mole-
cules showed potent cell fusion and virus-cell fusion in-
hibitory activity at low micromolecular levels.23 Inspired
by these initial findings, we set out to develop a new pro-
tocol that would allow the rapid synthesis of a variety of
derivatives of these new entry inhibitors.
Abstract: The direct amidation of esters with 4-amino-2,2,6,6-tet-
ramethylpiperidine was achieved by computer-controlled micro-
wave irradiation in toluene. The microwave protocol allowed the
new amides to be prepared in three hours rather than the three days
required by traditional thermal methods.
Key words: microwave-assisted synthesis, HIV, amidation, CCR5,
gp120
Knowledge of both the mechanism of action of the HIV
virus and the structures of the many protein–protein inter-
actions that are involved in HIV entry into cells is central
to developing possible treatments and cures for those in-
fected. The entry of HIV-1 into a host is mediated by the
interaction between the viral envelope glycoprotein
gp120 and the host cell receptor CD4. This entry triggers
a cascade of conformational changes in the viral envelope
glycoprotein resulting in the exposure of the co-receptor
(CXCR4 or CCR5) binding site on gp120.1–4 The binding
of gp120 to the coreceptor leads to the destabilization of
the gp120/gp41 complex.5–8 As a result, gp41 undergoes
conformational change and exposure of the hydrophobic
fusion peptide, which inserts into the target cell mem-
brane and initiates the HIV-1 entry process (Figure 1).9–12
Therefore, the binding between gp120 and CD4 is a key
step for HIV-1 entry into cells and has been suggested as
a potential novel target for anti-HIV-1 therapy.9,13–17
Our synthetic strategy to prepare derivatives of 1 and 2
was influenced by the large number of substituted anilines
Figure 1 Schematic diagram of the HIV-1 entry process.
SYNTHESIS 2006, No. 5, pp 0807–0812
x
x
.
x
x
.2
0
0
6
Advanced online publication: 07.02.2006
DOI: 10.1055/s-2006-926339; Art ID: M06205SS
© Georg Thieme Verlag Stuttgart · New York

808
C. McFarland et al.
PAPER
O
microwave protocols provide rapid, ‘green’, and predict-
able chemical functionalizations.25–36 The dramatic rate
enhancements that have also been observed using micro-
wave technology28 make it a promising method for the
current study. Indeed, repeating the amidation reactions
with 5 under microwave conditions produced 6 in only
three hours in yields that were adequate for biochemical
inhibition studies. The reactions were not run to comple-
tion, but rather optimized for rapid throughput, hence
moderate yields were typical. Figure 4 shows the new en-
try inhibitor analogues that were prepared via the micro-
wave procedure.
H
N
HN
O
X
N
H
1, X = Cl
2, X = Br
Figure 2
that are commercially available. An attractive route is the
addition of a substituted aniline to ethyl chlorooxoacetate
in the presence of base (Scheme 1)24 to provide access to
a large number of building blocks of the general structure
4, which could then be further derivatized. Despite the
poor nucleophilicity of electron deficient anilines, we
found that their additions to ethyl chlorooxoacetate pro-
ceeded smoothly and the yields of the resulting esters 4
were generally in the range 80–90%. Figure 3 shows the
variety of esters that have been prepared by this method.
With this mild procedure, even aminopyridines could be
condensed leading to the nitrogen-rich products 7, 22, and
25. The silyl-protected ethers 27 and 28 were also pre-
pared, potentially providing access to two new hydroxyl-
containing derivatives.
Compounds 1 and 2 were also re-synthesized by the mi-
crowave protocol, and X-ray quality crystals of 2 were
grown in the hope of providing a low-energy model of this
class of active HIV-1 entry inhibitors for future docking
studies. The ORTEP diagram of 2 is shown in Figure 5.37
Interestingly, the carbonyl groups of the amide linkages
were found to lie out of the plane of the aromatic ring.
This structural feature can perhaps be rationalized by the
intermolecular hydrogen bonding of the amide groups ob-
served in the packing diagram of 2 (Figure 6). The hydro-
gen of the alkyl amide portion of 2 forms a close contact
(2.310 Å) with the aryl amide oxygen O2 of another mol-
ecule and no hydrogen bonding was observed for the aryl
amide hydrogen of N1. The solid-state interactions pro-
vide insight into some hydrogen-bonding capabilities of 2,
but because of the intermolecular interactions, it appears
that computational methods are best suited to providing a
low-energy model appropriate for solution-phase simula-
tions.
With esters of the general structure 4 in hand, we then ex-
amined whether esters of type 4 could be directly convert-
ed to amides 6 without having to convert the ester
functionality to an acid chloride or whether peptide cou-
pling procedures would be necessary using the carboxylic
acid derivative. Unfortunately, we found that the thermal
amidation reactions with 4-amino-2,2,6,6-tetramethylpip-
eridine (5) to afford 6 (Scheme 1) proceeded quite slowly,
requiring three days at reflux in toluene solution. These
thermal reactions were clearly not suitable to rapidly pre-
pare derivatives of 1 and 2.
The microwave-assisted chemical synthesis of novel hu-
man immunodeficiency virus type 1 (HIV-1) entry inhib-
itors that block the gp120-CD4 interaction has been
achieved. The microwave methods allow the derivatives
to be synthesized in a fraction of the time compared to
normal thermal methods, and also circumvent the need to
convert esters 4 to acid chlorides or carboxylic acids be-
fore further derivatization.
We therefore turned our attention to the use of microwave
technology to aid in the amidation reactions with 5. Mi-
crowave-assisted chemical synthesis has enjoyed explo-
sive growth in recent years as developments in the
H2N
NH
5
Et3N
toluene
O
reflux, 3 d
O
OEt
O
O
H
NH2
HN
N
N H
O
HN
OEt
Cl
O
Et3N
R
R
R
5
Et3N
3
4
6
toluene
microwave
3 h, 150 °C
Scheme 1
Synthesis 2006, No. 5, 807–812 © Thieme Stuttgart · New York

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Rapid Microwave-Assisted Syntheses of Derivatives of HIV-1 Entry Inhibitors
809
O
O
O
O
O
OEt
OEt
OEt
OEt
OEt
HN
HN
HN
HN
HN
Me
F3C
Me
O
Me
O
O
Br
O
F
O
94%
92%
96%
99%
62%
N
Br
Cl
Cl
Br
10
Cl
7
8
11
9
O
O
OEt
O
O
O
HN
OEt
OEt
OEt
OEt
HN
O
F
HN
HN
HN
O
F
O
F
O
F3C
O
98%
99%
97%
99%
97%
Br
Br
CF3
Cl
Br
13
12
14
15
16
O
OEt
O
O
O
O
HN
OEt
OEt
OEt
OEt
O
HN
17
HN
HN
HN
O
O
Cl
O
Cl
O
85%
99%
85%
90%
94%
N
Br
Br
20
Cl
18
19
21
O
O
OEt
O
O
O
HN
OEt
OEt
OEt
OEt
HN
O
Me
HN
HN
HN
Cl
O
Me
O
NC
O
O
88%
84%
39%
76%
95%
N
N
Br
OMe
Cl
CN
22
23
24
25
26
O
O
O
OEt
OEt
OEt
HN
HN
HN
O
TBSO
O
O
TBSO
79%
80%
92%
Cl
Cl
Cl
28
27
29
Figure 3
Unless otherwise noted, all reactions were carried out under a nitro-
gen atmosphere. All commercially available compounds were pur-
chased from Aldrich Chemical Company, GFS Chemicals, or Alfa
Aesar Organics and used as received unless otherwise specified.
THF, Et2O, toluene, and CH2Cl2 were distilled over a drying agent
prior to use. All 1H and 13C NMR spectra were obtained on a JEOL
270 MHz or a Bruker 300 MHz (for 1H) instrument (67 MHz or 75
MHz for 13C, respectively). LRMS were conducted on an Agilent
system. Mps were acquired on a Mel-Temp II melting point appara-
tus by Laboratory Devices. Microwave reactions were carried out
using a CEM Discovery System with an Explorer auto-sampler at-
tachment.
Esters 7-28; Typical Procedure
Ethyl chlorooxoacetate (620 mL, 5.58 mmol) was added dropwise to
a cooled (0 °C) mixture of 4-chloro-2-fluoroaniline (5.58 mmol)
and Et3N (660 mL, 5.58 mmol) in THF (50 mL). The reaction mix-
ture was allowed to warm slowly to r.t. and stirred for 12 h. The
mixture was then filtered to remove the ammonium salts, and the
filtrate was washed with HCl (2 N; 1 × 50 mL). The organic layer
was then dried over MgSO4, filtered, and concentrated under vacu-
um yielding N-(2-fluoro-4-chlorophenyl)oxalamic acid ethyl ester
(9), an off-white crystalline solid, in 92% yield.
1H NMR (300 MHz, CDCl3): d = 1.40 (t, J = 7.1 Hz, 3 H), 4.40 (q,
J = 7.1 Hz, 2 H), 7.14 (dd, J = 2.2, 8.8 Hz, 2 H), 8.33 (t, J = 8.5 Hz,
1 H), 9.06 (br s, 1 H).
Synthesis 2006, No. 5, 807–812 © Thieme Stuttgart · New York

810
C. McFarland et al.
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O
O
O
O
Br
O
H
N
O
F
H
N
H
H
N
N
HN
HN
HN
HN
O
O
O
O
O
O
Br
Br
Br
Br
Cl
Cl
N
H
54%
N
H
N
H
N
H
39%
35%
45%
2
31
1
30
O
Me
F
O
O
F
H
H
N
H
H
N
N
N
HN
HN
HN
HN
O
O
O
Br
F
CN
Br
N
H
35%
N
H
N
H
N
H
46%
56%
55%
32
35
33
34
O
O
O
CF3
H
H
H
N
H
N
N
N
HN
HN
HN
HN
O
O
O
CF3
N
N
H
N
H
40%
32%
N
H
N
H
29%
58%
38
36
37
39
O
Me
O
Cl
O
Me
O
Cl
H
H
N
H
N
H
N
N
HN
HN
N
HN
HN
O
O
O
O
Cl
Cl
Cl
Br
36%
52%
N
H
N
H
N
H
N
H
46%
43%
42
43
41
40
Me
Me
O
CF3
O
CN
O
O
H
N
H
N
H
N
H
N
Br
HN
HN
HN
HN
N
O
O
O
O
Cl
Cl
OMe
61%
N
H
10%
N
H
N
H
38%
N
H
48%
46
47
44
45
Figure 4
13C NMR (75 MHz, CDCl3): d = 14.0, 64.0, 116.0 (d, J = 22.2 Hz),
122.0, 123.7 (d, J = 10.3 Hz), 125.1, 130.4 (d, J = 9.8 Hz), 150.6,
153.9, 160.2.
Microwave-Assisted Amide Bond Formation; Typical Proce-
dure
4-Amino-2,2,6,6-tetramethylpiperidine (80 mL, 0.46 mmol) was
added to a solution of N-(2-cyano-4-chlorophenyl)oxalamic acid
ethyl ester (23; 0.46 mmol) and Et3N (108 mL, 0.92 mmol) in tolu-
ene (5 mL). The reaction vial was sealed with a crimp-top and
placed in a microwave where the temperature was raised to 150 °C
in 10 min and held at that temperature for 3 h. Then HCl (2 N; 10
LRMS: m/z calcd for C10H9ClFNO3 (M+): 245.6; found: (M+) 245.
Synthesis 2006, No. 5, 807–812 © Thieme Stuttgart · New York

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Rapid Microwave-Assisted Syntheses of Derivatives of HIV-1 Entry Inhibitors
811
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Figure 6 Packing diagram of 2. All hydrogens except those at-
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mL) was added to the reaction mixture and extracted with EtOAc
(3 × 10 mL). Following the initial extraction, the remaining aque-
ous layer was basified with 2 N NaOH and re-extracted with CH2Cl2
(3 × 10 mL). The CH2Cl2 layer was dried over MgSO4, filtered, and
concentrated under vacuum providing N-(2-cyano-4-chlorophen-
yl)-N¢-(2,2,6,6-tetramethylpiperidin-4-yl)oxalamide (46), as a yel-
low crystalline solid in 61% yield.
1H NMR (300 MHz, CDCl3): d = 1.13 (t, J = 12.4 Hz, 2 H), 1.16 (s,
6 H), 1.27 (s, 6 H), 1.91 (dd, J = 3.3, 12.4 Hz, 2 H), 4.28 (m, 1 H),
7.56 (m, 2 H), 8.31 (d, J = 8.8 Hz, 1 H).
13C NMR (75 MHz, CDCl3): d = 28.3, 34.6, 43.9, 44.3, 51.3, 104.9,
114.4, 122.2, 130.5, 132.1, 134.3, 137.4, 157.9, 158.1.
LRMS: m/z calcd for C18H23ClN4O2 (M+): 362.9; found: (M+) 362.
Acknowledgment
D.A.V. would like to acknowledge NIH (RR-015569-06) for partial
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Synthesis 2006, No. 5, 807–812 © Thieme Stuttgart · New York