Column chromatography-free solution-phase synthesis of a natural piper-amide-like compound library

Article abstract of DOI:10.1021/co400003d

We have achieved an efficient solution-phase parallel synthesis of a library of natural piper-amide-like compounds from the bifunctional β-phosphono-N-hydroxy-succinimidyl ester intermediate. The primary important feature in our study is the construction of natural-product-like molecules through the adaptation of sophisticated organic reactions that create water-soluble byproducts for a chromatography-free purification. This simple and efficient method rapidly provides a combinatorial library of high yield and purity. The library was evaluated against GPCR targets to demonstrate its potential use as a tool for drug discovery and in chemical biology.

Full text of DOI:10.1021/co400003d

Research Article
pubs.acs.org/acscombsci
Column Chromatography-Free Solution-Phase Synthesis of a Natural
Piper-Amide-like Compound Library
Sumin Kim,†,+ Chaemin Lim,†,+ Sukjin Lee,† Seokwoo Lee,† Hyunkyung Cho,† Joo-Youn Lee,†,‡
Dong Sup Shim,‡ Hee Dong Park,‡ and Sanghee Kim*,†
†College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea
‡Drug Discovery Department, LG Life Sciences, Ltd., R&D Park, 104-1, Munji-dong, Yuseong-gu, Daejeon, 305-380, Korea
S
* Supporting Information
ABSTRACT: We have achieved an efficient solution-phase
parallel synthesis of a library of natural piper-amide-like
compounds from the bifunctional β-phosphono-N-hydroxy-
succinimidyl ester intermediate. The primary important feature
in our study is the construction of natural-product-like molecules
through the adaptation of sophisticated organic reactions that
create water-soluble byproducts for a chromatography-free
purification. This simple and efficient method rapidly provides
a combinatorial library of high yield and purity. The library was evaluated against GPCR targets to demonstrate its potential use
as a tool for drug discovery and in chemical biology.
KEYWORDS: natural products, combinatorial chemistry, chromatography-free, solution-phase synthesis
INTRODUCTION
■
Chemical libraries are important tools in the preliminary phase
of drug discovery and chemical biology. Despite the advent of
modern high-throughput synthetic technologies, the generation
of chemical libraries still consumes considerable amounts of
time and energy because of the inherently large number of
synthetic operations required. Thus, the development of an
efficient strategy for library synthesis is highly desirable to
reduce the time and effort.
As part of our ongoing research focused into the synthesis of
chemical libraries, we became interested in the structure of
natural piper amides, the most common constituents of the
genus Piper,1 as a scaffold for these libraries. This became our
focus because piper amides have a wide range of interesting
biological activities including anti-inflammatory,2 antifungal,3
insecticidal,4 analgesic,5 antidepressant,6 and antitumor activ-
Figure 1. Structures of representative natural piper amides.
ities.7 Some representative natural piper amides are shown in
Figure 1. Piper amides are structurally characterized by the
amines, and ketenylidenetriphenylphosphorane (Ph3PC
presence of an α,β-unsaturated amide group. The α,β-
unsaturated amide moiety is also found in many biologically
CO).11 However, this one-pot process suffers from low
chemical yields and requires chromatographic purification,
which render this process unsuitable for library generation.
Another notable strategy is the solid-phase synthesis of N-
(phenylalkyl)cinnamides via the formation of the polymer-
bound Horner−Wadsworth−Emmons (HWE) reagent.12 This
method is suitable for generating a library, however only one
point is available for structural diversification on the polymer-
active synthetic compounds and pharmaceuticals. For instance,
AMG-9810 and SB-366791 (Figure 2) are very potent vanilloid
receptor TRPV1 antagonists,8 and tranilast is a clinical drug
used in the treatment of allergic diseases and keloids.9
Because the α,β-unsaturated amide moiety is an attractive
scaffold in medicinal chemistry and is found in natural products,
a number of synthetic strategies for its assembly have been
devised.10 Most syntheses focus on the amidation of α,β-
unsaturated carboxylic acid or on the olefination reaction for
unsaturated double bonds. One notable strategy is the
intermolecular three-component reaction between aldehydes,
Received: January 10, 2013
Revised: February 13, 2013
© XXXX American Chemical Society
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Research Article
hydroxysuccinimide (NHS) and dialkylphosphate salt, thus
allowing for an aqueous workup purification. The reaction of 1
with a subequivalent amount of the amine would produce β-
phosphono amide 2 and water-soluble N-hydroxysuccinimide.
We expected that the remaining β-phosphono ester 1 could be
removed from the reaction mixture as the water-soluble
hydrazine derivative 4 by treatment with the carbonyl
compound scavenger Girard’s reagent T (3), followed by
aqueous workup.13,14 An HWE-type reaction between the
obtained β-phosphono amide 2 and an excess of aldehyde
would provide the desired α,β-unsaturated amide 5 with the
release of water-soluble dialkylphosphate. We envisioned that
the removal of the remaining aldehyde from the product could
be achieved also by using Girard’s reagent T as a scavenger.
As shown in Scheme 2, the requisite bifunctional
intermediate 1a was readily prepared in two steps from the
Figure 2. Structures of bioactive non-natural piper amides.
bound HWE reagent because the amine of the amide group is
directly bound to the resin.
Piper amides are structurally simpler than other types of
natural products and their syntheses require relatively fewer
steps. We decided that the application of solid-phase synthetic
techniques for generating a library of these natural-product-like
compounds was not economically attractive because nearly half
of the synthetic processes were wasted on linker manipulation,
attachment to and detachment from the resin. Thus, we
planned to generate the piper-amide-like library using solution-
phase chemistry.
Scheme 2. Synthesis of Bifunctional Intermediate 1a
Herein, we wish to report an efficient solution-phase parallel
synthesis of a combinatorial library of natural piper-amide-like
compounds. Our strategy is characterized by the creation of
water-soluble byproducts for chromatography-free purification
of the reaction products. This approach offers the benefits of
both time and cost savings in that the designed library was
rapidly accessed without any laborious chromatographic
separations or expensive solid-supported purification steps.
commercially available β-phosphono ester 6 without chromato-
graphic purification. It is notable that intermediate 1a contains
a bulky diisopropyl phosphonate group because the steric bulk
of the phosphonate group is crucial for enhancing the E-alkene
selectivity in the HWE reaction.15 The hydrolysis of 6 in EtOH
with NaOH gave phosphoryl acetic acid 7. After aqueous
workup without further purification, the obtained acid 7 was
treated with EDCI and NHS in CH2Cl2 to give the active
succinimidyl ester 1a. The reaction mixture was purified only
RESULTS AND DISCUSSION
■
Our chromatography-free solution-phase synthetic strategy is
shown in Scheme 1. The key intermediate in our synthetic
sequence was bifunctional β-phosphono-N-hydroxysuccinimid-
yl ester 1. The distinct feature of this intermediate is that, after
the reaction, it releases the water-soluble byproducts N-
Scheme 1. Outline of a Chromatography-Free Solution-Phase Piper-Amide-like Library Synthesis
B
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by aqueous extraction. The product thus obtained was
essentially pure and could be used for further reactions.
The acquired bifunctional intermediate 1a was coupled with
a subequivalent amount (0.85 equiv) of eleven different amines
8{1−11} in CH2Cl2 to give eleven amides 2 (Figure 3). The
Figure 4. Synthesis of α,β-unsaturated amide 5 and structures of
employed aldehydes 9.
the case of library member 5{11,9}, which has two phenolic
hydroxyl groups, only starting β-phosphono amide was
recovered without any desired condensation product. There-
fore, we were able to obtain 153 natural piper-amide-like
compounds stereoselectively out of the planned 154 (>99%
success rate) with high purity by using only aqueous extractive
workup.
To add an additional point of diversity on the piper amide
scaffold, we also designed a bifunctional intermediate that
contained the substituent at the α-position. α-Methyl β-
phosphono-N-hydroxysuccinimidyl ester 1b was chosen as an
example of such a bifunctional intermediate (Scheme 3).
Employing the same procedure as for the column chromatog-
raphy-free preparation of 1a, the α-branched intermediate 1b
was easily prepared from the known α-methyl β-phosphono
ester 10.19 For the library reported here, a total of six amines
(8{1}, 8{3}, 8{5}, 8{6}, 8{8}, and 8{10}) were used to give six
β-phosphono amides 2b by reaction with 1b. The selected
fourteen aldehydes 9{1−14} were condensed with 2b in a
combinatorial manner to give 11.
Figure 3. Synthesis of β-phosphono amide 2a and structures of the
employed amines 8.
coupling reaction of 1a with alkyl amines 8{1−3} and 8{6−11}
was completed within 4 h at room temperature without any
additives. However, reaction with the less nucleophilic aryl
amines 8{4} and 8{5} required triethylamine and a catalytic
amount of DMAP to be completed in the same amount of time
and at the same temperature. Without base additives, the
reaction with these aryl amines required a longer reaction time
(12 h) and higher temperature (40 °C).16 When complete
consumption of amines 8 was observed by LC or TLC, the
reaction mixture was treated with a solution of Girard’s reagent
T (3) in MeOH to convert the remaining succinimidyl ester 1a
to water-soluble derivative 4a. The condensation reaction of
Girard’s reagent T with 1a was somewhat slow, but the reaction
was completed within 12 h at room temperature. After standard
aqueous extractive workup, essentially pure β-phosphono amide
2a{1−11} was obtained in high yield.17
The HWE reaction between β-phosphono amide 2a and a
moderate excess (1.5 equiv) of aldehyde was investigated
(Figure 4). After considerable effort, it was recognized that t-
BuOK was an appropriate base for this type of reaction.18 Most
aldehydes reacted with β-phosphono amide 2a in the presence
of t-BuOK in THF to give the desired α,β-unsaturated amide 5
in high yield with exclusive E-selectivity. However, in the case
of hydroxy benzaldehyde, such as 9{9}, NaH was a more
appropriate reagent to complete the reaction.
The selected fourteen aldehydes 9{1−14}, shown in Figure
4, were reacted with the obtained eleven β-phosphono amides
2a{1−11} in a combinatorial manner. After completion of the
reaction, excess aldehyde 9 was removed from the mixture by
treatment with a solution of Girard’s reagent T (3) in MeOH
and a standard aqueous workup. Table 1 presents the obtained
yields and purities. In all cases except one, the products could
be obtained with an average HPLC purity of 95%. However, in
As shown in Table 2, the planned 84 α-methyl piper amide
compounds were successfully obtained. In most cases, the
desired α,β-unsaturated amide was obtained in high yield with
complete or very high E-selectivity. However, in the case of the
HWE reaction of β-phosphono amide 2b{3}, derived from the
coupling of 1b with the cyclic secondary amine pyrrolidine
8{3}, the stereoselectivity was low or even reversed to afford a
cis isomer as the major product. This low stereoselectivity of β-
phosphono secondary amide 2b{3} was somewhat surprising,
and further studies were required to elucidate its origin.
Although low stereoselectivity was found in some particular
cases, the above results implied that our column chromatog-
raphy-free protocol could be applicable to synthesizing a library
of piper-amide-like compounds with a substituent at the α-
position.
The resemblance to the natural products was one of the basic
criteria in the selection of building blocks for the library. The
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Table 1. Library Data for α,β-Unsaturated Amides 5
product 5 {amine 8,
aldehyde 9}
yield
a
purity
b
product 5 {amine 8,
aldehyde 9}
yield
a
purity
b
product 5 {amine 8,
aldehyde 9}
yield
a
purity
b
entry
(%)
(%)
entry
(%)
(%)
entry
(%)
(%)
1
5{1,1}
5{1,2}
5{1,3}
5{1,4}
5{1,5}
5{1,6}
5{1,7}
5{1,8}
5{1,9}
5{1,10}
5{1,11}
5{1,12}
5{1,13}
5{1,14}
5{2,1}
5{2,2}
5{2,3}
5{2,4}
5{2,5}
5{2,6}
5{2,7}
5{2,8}
5{2,9}
5{2,10}
5{2,11}
5{2,12}
5{2,13}
5{2,14}
5{3,1}
5{3,2}
5{3,3}
5{3,4}
5{3,5}
5{3,6}
5{3,7}
5{3,8}
5{3,9}
5{3,10}
5{3,11}
5{3,12}
5{3,13}
5{3,14}
5{4,1}
5{4,2}
5{4,3}
5{4,4}
5{4,5}
5{4,6}
5{4,7}
5{4,8}
5{4,9}
5{4,10}
99
99
62
98
74
80
69
94
74
62
78
71
91
99
98
98
89
98
99
99
99
98
90
96
90
100
94
99
97
98
67
99
90
97
89
99
97
58
83
89
94
100
100
99
88
99
76
47
62
99
96
75
98
99
97
100
99
99
99
100
93
96
99
92
97
99
97
98
98
97
87
98
97
99
99
93
85
97
98
82
95
97
99
97
96
99
100
99
99
98
95
92
93
95
91
81
79
97
88
92
82
86
85
93
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
5{4,11}
5{4,12}
5{4,13}
5{4,14}
5{5,1}
5{5,2}
5{5,3}
5{5,4}
5{5,5}
5{5,6}
5{5,7}
5{5,8}
5{5,9}
5{5,10}
5{5,11}
5{5,12}
5{5,13}
5{5,14}
5{6,1}
5{6,2}
5{6,3}
5{6,4}
5{6,5}
5{6,6}
5{6,7}
5{6,8}
5{6,9}
5{6,10}
5{6,11}
5{6,12}
5{6,13}
5{6,14}
5{7,1}
5{7,2}
5{7,3}
5{7,4}
5{7,5}
5{7,6}
5{7,7}
5{7,8}
5{7,9}
5{7,10}
5{7,11}
5{7,12}
5{7,13}
5{7,14}
5{8,1}
5{8,2}
5{8,3}
5{8,4}
5{8,5}
5{8,6}
81
71
57
69
89
90
70
100
99
99
100
97
58
98
77
92
100
79
95
100
85
67
75
78
82
97
75
81
82
88
87
97
93
94
66
94
83
96
90
89
78
85
69
80
78
78
97
68
84
93
100
88
92
84
89
77
90
92
95
96
83
82
89
93
99
87
99
86
85
99
98
99
97
99
94
86
100
84
99
92
83
86
99
92
98
99
99
100
88
99
99
90
100
95
95
95
97
96
99
99
97
100
100
99
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
5{8,7}
99
100
98
99
91
100
99
100
96
98
73
99
58
76
54
100
76
82
52
74
70
75
99
91
87
96
87
90
100
96
86
85
62
92
93
94
89
87
62
57
99
68
66
99
-
98
97
98
93
99
88
97
99
95
100
99
96
95
99
98
95
98
90
96
91
93
99
97
99
97
98
98
98
99
98
95
92
97
95
95
97
100
99
96
95
85
91
99
99
-
2
5{8,8}
3
5{8,9}
4
5{8,10}
5{8,11}
5{8,12}
5{8,13}
5{8,14}
5{9,1}
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
5{9,2}
5{9,3}
5{9,4}
5{9,5}
5{9,6}
5{9,7}
5{9,8}
5{9,9}
5{9,10}
5{9,11}
5{9,12}
5{9,13}
5{9,14}
5{10,1}
5{10,2}
5{10,3}
5{10,4}
5{10,5}
5{10,6}
5{10,7}
5{10,8}
5{10,9}
5{10,10}
5{10,11}
5{10,12}
5{10,13}
5{10,14}
5{11,1}
5{11,2}
5{11,3}
5{11,4}
5{11,5}
5{11,6}
5{11,7}
5{11,8}
c
5{11,9}
5{11,10}
5{11,11}
5{11,12}
5{11,13}
5{11,14}
99
60
79
79
100
77
95
90
93
89
a
b
Yields (%) were obtained after aqueous workup. Purity was determined by HPLC (ZORBAX Eclipse plus C18 column, 4.6 mm × 150 mm, 5 μm;
c
ultraviolet absorption detector at 250 nm; gradient, 20−100% MeOH/H2O, 40 or 60 min). No reaction. Recovered starting material.
other criterion was the drug-like properties. To verify the drug
likeness, the structures of the library members were saved as SD
file format and submitted to the analysis of Lipinski20 and
Veber rules.21 According to the results, 96% of the library
members passed the rules (see Table S2 in Supporting
Information).22 To assess and demonstrate the value of the
piper amide library generated, 60 randomly selected library
compounds were tested in a new drug screening program at LG
Life Sciences. Many of the tested compounds showed moderate
to good activity against the G-protein coupled receptor
D
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Scheme 3. Synthesis of α-Methyl Piper Amide 11
(GPCR) and kinase targets. Noteworthy test results showed
that several compounds exhibited agonist activity toward
recombinant bombesin receptor subtype-3 (BRS-3) expressed
in CHO-K1 cells. These compounds had EC50 values in the
nanomolar to micromolar range. Most of the active compounds
had a phenethylamine group in common. The structures of
three representatives and their EC50 values are shown in Figure
5.
BRS-3 is an orphan GPCR which is implicated in the
regulation of energy homeostasis.23 This receptor has been
recently validated as a potential new target for treating
obesity.24 Recent extensive medicinal chemistry efforts have
yielded several classes of nonpeptidic small-molecule BRS-3
agonists.25 Our identified compounds are structurally distinct
Figure 5. Structures of hit compounds and their EC50 values for the
BRS-3 receptor.
from these small-molecules. Although a limited number of
library members has been examined, the most potent hit
compound exhibited an EC50 value of 53 nM, which is
comparable to that of the leading small-molecule agonist.26
These biological results have important implications in the
potential use of a piper amide library in drug discovery and
chemical biology.
Table 2. Library Data for α-Methyl Piper Amides 11
b
b
b
product 11 {amine 8, yield purity (%)
product 11 {amine 8, yield purity (%)
product 11 {amine 8, yield purity (%)
a c
a
c
a
c
entry
aldehyde 9}
(%)
(E:Z)
entry
aldehyde 9}
(%)
(E:Z)
entry
aldehyde 9}
(%)
(E:Z)
1
11{1,1}
11{1,2}
11{1,3}
11{1,4}
11{1,5}
11{1,6}
11{1,7}
11{1,8}
11{1,9}
11{1,10}
11{1,11}
11{1,12}
11{1,13}
11{1,14}
11{3,1}
11{3,2}
11{3,3}
11{3,4}
11{3,5}
11{3,6}
11{3,7}
11{3,8}
11{3,9}
11{3,10}
11{3,11}
11{3,12}
11{3,13}
11{3,14}
99
93
78
83
98
82
76
93
92
99
83
87
94
93
99
99
88
87
78
96
95
98
26
85
99
93
96
80
99
93
93
99
95
96
99
98
98
65
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
11{5,1}
11{5,2}
11{5,3}
11{5,4}
11{5,5}
11{5,6}
11{5,7}
11{5,8}
11{5,9}
11{5,10}
11{5,11}
11{5,12}
11{5,13}
11{5,14}
11{6,1}
11{6,2}
11{6,3}
11{6,4}
11{6,5}
11{6,6}
11{6,7}
11{6,8}
11{6,9}
11{6,10}
11{6,11}
11{6,12}
11{6,13}
11{6,14}
99
73
89
93
84
89
84
99
79
86
94
93
99
92
99
99
97
90
90
79
99
99
95
92
66
84
99
84
80
89
77
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
11{8,1}
99
99
85
90
95
80
97
98
86
99
72
86
97
92
99
99
95
84
86
96
94
96
88
77
71
92
93
76
99 (9:1)
97
2
11{8,2}
3
11{8,3}
97
4
99 (7:1)
11{8,4}
99
5
96
11{8,5}
99
6
87
11{8,6}
99
7
98
11{8,7}
99
8
94
11{8,8}
96
9
95
11{8,9}
97
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
94
11{8,10}
11{8,11}
11{8,12}
11{8,13}
11{8,14}
11{10,1}
11{10,2}
11{10,3}
11{10,4}
11{10,5}
11{10,6}
11{10,7}
11{10,8}
11{10,9}
11{10,10}
11{10,11}
11{10,12}
11{10,13}
11{10,14}
68
100
93
90
100
99
93
95
89
96
98
96
96
75 (3:1)
85 (2:1)
96 (2:1)
99 (4:1)
96 (1:2)
99 (7:1)
99 (1:1)
97 (1:2)
91 (1:49)
90 (2:1)
90 (1:3)
97 (9:1)
99 (7:1)
94 (1:2)
88 (10:1)
97
86 (8:1)
99
80
99
85
100
100
99
96
95
97
99 (9:1)
97
85
99
99
79
84
91
100
90
85
81
84
94
99
a
b
Yields (%) were obtained after aqueous workup. Purity was determined by HPLC (ZORBAX Eclipse plus C18 column, 4.6 mm × 150 mm, 5 μm;
c
1
ultraviolet absorption detector at 250 nm; gradient, 20−100% MeOH/H2O, 40 or 60 min). E/Z ratio was determined by HPLC and H NMR
spectroscopic analyses. Ratios above 10:1 were not included in this table. For detailed information, see Table S1 in Supporting Information.
E
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Research Article
for selected compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
CONCLUSION
■
In summary, we have produced an α,β-unsaturated amide
combinatorial library inspired by the structures and biological
activities of natural piper amides. The library members exhibit
three points of diversity. The scaffold was easily accessible in
only two steps from the bifunctional β-phosphono-N-
hydroxysuccinimidyl ester without chromatographic purifica-
tion. The column chromatography-free and efficient nature of
this library synthesis hinges, in part, on the creation of water-
soluble byproducts. The key bifunctional intermediate released
a water-soluble byproduct only after the reaction, and the
excess reactants were removed from the product as the water-
soluble hydrazone derivative. Because our approach employed
aqueous workup for purification, there might be some
limitations especially in the synthesis of highly water-soluble
library members. However, the ease of operation and the
excellent purity of the piper amide library obtained are
important features of this protocol. The randomly selected
library members were tested mainly against GPCR targets,
resulting in the discovery of several compounds with BRS-3
agonist activity. Our biological results imply that the biological
relevance of the library of natural piper-amide-like compounds
is high and that such libraries is useful for discovering hits or
probes for either unknown or discovered biological targets.
Finally, we believe this study provides a novel direction for
further preparation of a more diversified and expanded library.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 82-2-880-2487. Fax: 82-2-880-0649. E-mail: pennkim@
snu.ac.kr.
Author Contributions
+S.K. and C.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the WCU program (R32-2008-
000-10098-0) of the National Research Foundation of Korea
(NRF) grant founded by the Korean government (MEST), the
research program of the Korea Food Research Institute
(E0121702), and the project of Global Ph. D. Fellowship of
the NRF conducts from 2011.
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■
General methods and additional experimental details are given
in the Supporting Information.
General Procedure for the Synthesis of β-Phosphono
Amides 2a and 2b. Amine 8 (4.25 mmol) was added to a
solution of β-phosphono-N-hydroxysuccinimidyl ester 1a or 1b
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THF (0.3 mL, 0.30 mmol) was added to a solution of β-
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ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental methods and full characterization for
representative library compounds and 1H and 13C NMR spectra
F
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H
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