A shortened, protecting group free, synthesis of the anti-wrinkle venom analogue Syn-Ake exploiting an optimized Hofmann-type rearrangement

Article abstract of DOI:10.1016/j.tetlet.2014.08.117

An unusual five-step synthesis of H-β-Ala-Pro-DabNHBz diacetate (a muscular nicotinic acetylcholine receptor antagonist) has been delineated through Hofmann-type rearrangement as a final step to build the target skeleton. The synthesis has been carried out using a protecting group free strategy and employed readily available reagents as the starting materials.

Full text of DOI:10.1016/j.tetlet.2014.08.117

Tetrahedron Letters 55 (2014) 5745–5747
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Tetrahedron Letters
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A shortened, protecting group free, synthesis of the anti-wrinkle
Ò
venom analogue Syn-Ake exploiting an optimized Hofmann-type
rearrangement
⇑
A. N. Balaev , K. A. Okhmanovich, V. N. Osipov
Pharm-Sintez, Vtoroy Roschinsky blvd., 8, Moscow 115419, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
An unusual five-step synthesis of H-b-Ala-Pro-DabNHBz diacetate (a muscular nicotinic acetylcholine
receptor antagonist) has been delineated through Hofmann-type rearrangement as a final step to build
the target skeleton. The synthesis has been carried out using a protecting group free strategy and
employed readily available reagents as the starting materials.
Received 29 May 2014
Revised 11 August 2014
Accepted 28 August 2014
Available online 6 September 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Peptides
Tripeptide
Syn-AkeÒ
Hofmann-type rearrangement
mnAChR antagonist
Ò
H-b-Ala-Pro-DabNHBz diacetate (Syn-Ake ) (1) is a synthetic
could be obtained from readily available and inexpensive starting
reagents. Our synthetic approach for the synthesis of 1 was
envisioned via an unusual route, without the use of protecting
groups/deprotection strategies, and relies on a Hofmann-type
rearrangement as the final step as shown in Scheme 1.
tripeptide (Fig. 1), with a mode of action similar to that of Waglerin
, a polypeptide that is found in the venom of the Temple viper
1
(
Tropidolaemus wagleri).1
The peptide 1 is an antagonist of the muscular nicotinic acetyl-
choline membrane receptor (mnAChR). When mnAChR is blocked,
the ion channel remains closed. There is no uptake of Na and the
muscle cells stay relaxed.
A traditional peptide approach to the synthesis of 1 from the
corresponding amino acids (b-alanine, L-proline, and L-2,4-diamin-
No information on the acylation of L-proline with 3-chloropro-
+
pionyl chloride has been reported in the literature up to now.
Unfortunately, direct acylation provided not only N-(3-chloropro-
pionyl)proline (6), but also significant quantities of a dehydrochlo-
rination by-product—N-acryloylproline (7) (20–50% according to
LSMS data). After several attempts using both organic and inor-
obutyric acid) involves a 10-step synthesis including the installa-
2
tion of protecting groups, condensation, and finally deprotection.
ganic bases, we found that bases such as Et
CO gave poor yields of 6. The use of an additional one equiva-
lent of -proline was chosen as an optimum base—the acylation
3
N, pyridine, and
Also, in the case of a
L-2,4-diaminobutyric acid (
L-DAB), selective
K
2
3
protection of the amino groups is required and this amino acid is
not a commercially available low cost reagent.
As part of our ongoing research aimed at developing a new, fac-
ile, and short synthesis of 1, we found that the target tripeptide
L
product was recovered without any dehydrochlorination occur-
ring. Thus, the reaction of 3-chloropropionyl chloride with two
equivalents of
L
-proline in methylene chloride at À10 °C gave 6
in 74% yield. Although there was no need to isolate 6, the pure
product could be obtained as white crystals by removal of methy-
lene chloride under low pressure and crystallization of the residue
H
N
NH
2
N
CONHBz
O
3
from EtOAc.
H N
O
2
AcOH
AcOH
A solution 6 in methylene chloride was cooled to À5 °C and one
equivalent of pentafluorophenol (PfpOH) was added, followed by
1.1 equiv of DCC in methylene chloride. The resulting mixture
was stirred at 0 °C for one hour and target 2 was isolated as a mix-
ture with N-(3-pentafluorophenoxypropanoyl)proline (8) (92:8).
1
Figure 1. Structure of Syn-AkeÒ.
⇑
Corresponding author. Tel.: +7 916 682 5299; fax: +7 495 796 9434.
E-mail address: balaev.alexandr@yandex.ru (A.N. Balaev).
2
Crude 2 was crystallized from Et O–hexane to give pure (>98%) 2
http://dx.doi.org/10.1016/j.tetlet.2014.08.117
040-4039/Ó 2014 Elsevier Ltd. All rights reserved.
0

5
746
A. N. Balaev et al. / Tetrahedron Letters 55 (2014) 5745–5747
COOH
N
O
7
a
COOH
COOH
COPfp
COOH
N
H
N
N
N
+
PfpO
Cl
O
Cl
O
O
6
2
8
H N
COOMe
HCl
2
COOMe
H
N
COOMe
CONHBz
H
N
COOH
b
c
N
COOH
N
O
O
Cl
O
Cl
O
3
4
H
CONH
2
d
N
e
N
1
CONHBz
O
HCl
H N
O
2
5
Scheme 1. Reagents and conditions: (a) 3-chloropropionyl chloride, CH
2
Cl
2
, À10 °C, 0.5 h; À10 °C to 0 °C; 10% aq NaCl; Na
2
SO
4
. PfpOH, DCC, À5 °C, then 0 °C, 1 h; (b) NMM,
O, Py, 5 °C, 5 h.
CH Cl , rt, 48 h; (c) BzNH , HOBt, DCC, DMF, 0 °C, then 5 °C, 18 h; (d) NH
2
2
2
3
, MeOH, 10 °C, then rt, 48 h; (e) PhI(OAc)
2
, 1,4-dioxane–H
2
benzylamide 4. Under the optimum conditions: HOBt (1.07 equiv),
benzylamine (1.23 equiv) and DCC (1.03 equiv), 18 h, the yield of 4
7
was 69%.
In the next step, direct ammonolysis of the methyl ester and
alkyl chloride resulted in tripeptide 5. This procedure is conven-
tionally carried out in the presence of methanol as the reaction
medium and requires a large excess of ammonia to ensure that fur-
ther substitution does not take place. Peptide 5 was recovered in
5
1% yield after purification by flash chromatography on silica gel
8
(
MeOH/CHCl
3
, 1:1).
2 2 2 2
In the final step, conversion of –CH C(O)NH into –CH NH was
achieved by the aid of a Hofmann-type rearrangement. So far, to the
best of our knowledge, there have been no reports on the synthetic
application of selective Hofmann-type degradation of bisamides of
glutamic residues in peptides. However, Rodionov et al., described
Figure 2. X-ray crystal structure of compound 2.
3 2
the utility of PhI[OC(O)CF ] (PIFA) for the degradation of protected
linear dipeptides containing glutamine. Reactions were performed
4
5
9
in 56% yield as large colorless crystals. The X-ray crystal structure
of 2 is shown in Figure 2.
with PIFA (1.5 equiv) in DMF–H O (1:1) for three hours at room
2
The reaction of 5-methyl ester of
L
-glutamic acid with pentaflu-
temperature in the presence of pyridine (2.0 equiv). However, we
orophenyl ester 2 produced acylated dipeptide 3. The reaction was
carried out under standard peptide synthesis conditions in the
presence of NMM at rt for 48 h; methylene chloride was used as
the solvent. The yield of pure (96%) 3 was 81%.6
Treatment of dipeptide 3 with benzylamine in the presence of
the coupling reagents DCC and HOBt at 0–5 °C in DMF afforded
encountered difficulties which can be ascribed mainly to the
presence of two different amide groups (CONHBz and CONH2).
A series of compounds including NaOBr, (diacetoxyiodo)benzene
[PhI(OAc)2, PIDA], PIFA, and [hydroxy(tosyloxy)iodo]benzene
(HTIB) were investigated as reagents for this rearrangement and
the results are shown in Table 1.
Table 1
Results of the Hofmann-type rearrangement using various reagents and different conditions
Entry
Reagent (equiv)
Reaction conditions
Yield % (HPLC)
1
2
3
4
5
6
7
8
9
NaOBr (1.5)
PIDA (1.5)
PIFA (1.5)
HTIB (1.5)
PIDA (2.0)
PIDA (3.0)
PIDA (4.0)
PIDA (5.0)
PIDA (4.0)
PIDA (4.0)
PIDA (4.0)
PIDA (4.0)
1,4-Dioxane–H
1,4-Dioxane–H
1,4-Dioxane–H
1,4-Dioxane–H
1,4-Dioxane–H
1,4-Dioxane–H
1,4-Dioxane–H
1,4-Dioxane–H
1,4-Dioxane–H
1,4-Dioxane–H
1,4-Dioxane–H
1,4-Dioxane–H
2
2
2
2
2
2
2
2
2
2
2
2
O, Py, 5 °C, 2 h
O, Py, 5 °C, 2 h
O, Py, 5 °C, 2 h
O, Py, 5 °C, 2 h
O, Py, 5 °C, 2 h
O, Py, 5 °C, 2 h
O, Py, 5 °C, 2 h
O, Py, 5 °C, 2 h
O, Py, 5 °C, 3 h
O, Py, 5 °C, 5 h
O, Py, 5 °C, 8 h
O, Py, 5 °C, 5 h (2.4 equiv PIDA, then 1.6 equiv after 3 h)
<3
30
29
24
36
42
46
48
49
53
51
1
1
1
0
1
2
a
59 (54 )
a
Isolated yield.

A. N. Balaev et al. / Tetrahedron Letters 55 (2014) 5745–5747
5747
Among the reagents used (entries 1–4), PIDA gave a slightly bet-
ter yield than PIFA and HTIB, and subsequent experiments were car-
ried out with PIDA. During the next optimization (entries 5–12), it
was found that the significant factors for the success of this reaction
were the amount of PIDA, the time of exposure, as well as the frac-
tional addition of the reagent. Thus, with the optimum conditions
the isolated yield of target 1 was 54%. The reaction procedure is very
structure in this Letter have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication CCDC 1016617.
. Compound 3: Pentafluorophenyl ester 2 (9.29 g, 25 mmol) was dissolved in
CH Cl (80 ml). To this, 5-methyl ester L-glutamic acid hydrochloride (5.93 g,
2 2
30 mmol) and NMM (3.14 g, 31 mmol) were added at rt, and the reaction
mixture was stirred for 48 h and washed with 15% citric acid solution (40 ml).
6
2 4
The organic layer was dried over Na SO and concentrated under reduced
pressure. The residue was dissolved in MeCN (10 ml) and the contents were
allowed to stand at 5 °C for 1 d. The resulting precipitate was filtered and dried
under reduced pressure. Yield 7.09 g (81%). White amorphous powder; purity
1
0
simple. A mixture of pyridine and 1,4-dioxane was added to a solu-
tion of tripeptide 5 in H O. The solution was cooled in an ice bath and
2
5
1
9
1
6.2% (HPLC); [
a
]
D
À66.80 (c 5, MeOH); H NMR (360.13 MHz, d
6
-DMSO): d
2
.70–1.94 (m, 4H), 1.96–2.12 (m, 2H), 2.33–2.41 (m, 2H), 2.66–2.88 (m, 2H),
then PIDA (2.4 equiv) in 1,4-dioxane was added at 5 °C. After three
hours, an additional quantity of PIDA (1.6 equiv) was added and
the mixture was stirred for a further two hours. The mixture was
diluted with ice water, washed twice with ethyl acetate, and once
with ethyl acetate/butanol (4:1). The aqueous layer was evaporated
and the residue was chromatographed (HPLC) to give target 1.
In summary, we have reported a useful and short method for
the synthesis of H-b-Ala-Pro-DabNHBz (1). The synthesis has been
carried out without the use of protecting groups/deprotection
3.35–3.56 (m, 2H), 3.59 (s, 3H), 3.68–3.81 (m, 2H), 4.14–4.30 (m, 1H), 4.32–
.45 (m, 1H), 8.05 (d, J = 8.3 Hz, 0.6H) and 8.33 (d, J = 8.3 Hz, 0.4H) (due to
rotamers), 12.58 (br s, 1H); MS (ESI), m/z (%): 349.3 [M+H] (100).
Compound 4: Acid 3 (6.98 g, 20 mmol) was dissolved in DMF (80 ml) and cooled
to 0 °C. BzNH2 (2.46 g, 23 mmol), HOBt monohydrate (3.52 g, 23 mmol), and
DCC (4.33 g, 21 mmol) were added successively. After stirring at 5 °C for 18 h,
the DCU by-product was filtered off and the filtrate diluted with EtOAc
4
+
7
.
(
250 ml). The organic layer was washed with brine (2 Â 100 ml), 5% citric acid
solution (2 Â 70 ml), 1 M NaHCO (2 Â 70 ml), and H O (2 Â 70 ml) and dried
3
2
over Na
solidified on triturating with Et
69%). White amorphous powder; purity 95.9% (HPLC); [
2
SO
4
. On evaporating the EtOAc, an oily residue was obtained, which
2
O (50 ml) and slight warming. Yield 6.05 g
25
(
a
]
D
À62.63 (c 5,
): d 1.94–2.18 (m, 6H), 2.35–2.51 (m, 2H),
2.67-2.88 (m, 2H), 3.45-3.76 (m, 4H), 3.62 (s, 3H), 4.24–4.32 (m, 1H), 4.40–4.58
strategies, uses readily available reagents, and relies on
a
1
MeOH); H NMR (360.13 MHz, CDCl
3
Hofmann-type rearrangement as the final step. Hopefully, this
new synthetic procedure will receive further attention for the
preparation of DAB-containing peptides.
(
[
m, 3H), 7.15–7.36 (m, 6H), 7.56 (d, J = 7.7 Hz, 1H); MS (ESI), m/z (%): 438.4
M+H] (100), 460.0 [M+Na] (1.6).
+
+
8.
Compound 5: Dipeptide 4 (5.69 g, 13 mmol) was dissolved in 20% methanolic
NH (200 ml) at 10 °C and kept for 48 h at rt. MeOH was distilled in vacuo and
3
the oily residue triturated with THF (3 Â 40 ml) until solidification occurred.
References and notes
Crude 5 was filtered, dissolved in MeOH, and purified by flash chromatography
on silica gel (MeOH/CHCl
3
, 1:1). Yield 2.93 g (51%). White amorphous powder;
1
.
(a) Namjoshi, S.; Caccetta, R.; Benson, H. A. E. J. Pharm. Sci. 2008, 97, 2524–
542; (b) Chhipa, N. M. R.; Chaudhari, B. G. J. Curr. Pharm. Res. 2012, 9, 11–18;
c) Fields, K.; Rodan, K.; Bush, L. J. Cosmet. Dermatol. 2009, 8, 8–13; (d) Ziegler,
H.; Heidl, M. Fragr. J. 2006, 34, 93–98; (e) Kozma, J.; Siko, G. Kozmetika 2006, 55,
6–22.
Pentapharm A. -G. WO 20,060,447,900, 2006; Chem. Abstr. 2006, 144, 456033.
23
1
purity 96.8% (HPLC); [
a
]
D
À40.70 (c 5, MeOH); H NMR (360.13 MHz, d
6
-
2
(
DMSO): d 1.72–2.38 (m, 8H), 2.53–2.75 (m, 2H), 2.91–3.05 (m, 2H), 3.36–3.61
(
m, 2H), 4.09–4.44 (m, 4H), 4.87 (br s, 3H), 7.19–7.33 (m, 5H), 7.73 (br s, 2H);
13
C NMR (90.56 MHz, D
2
O): d 23.60, 25.94, 28.99, 30.21, 30.54, 34.60, 42.33,
1
4
1
8
7.18, 53.51, 59.83, 126.60, 126.86, 128.12, 137.73, 170.27, 172.31, 173.72,
2
3
.
.
+
+
77.18; MS (ESI), m/z (%): 404.3 [MÀCl] (100), 426.2 [MÀHCl+Na] (12.5),
2
5
White crystals; purity 97.8% (HPLC); mp 82–84 °C; [
a
]
D
À74.95 (c 5, MeOH);
): d 1.84–2.27 (m, 4H), 2.76–2.84 (m, 2H), 3.46–
.65 (m, 2H), 3.66–3.83 (m, 2H), 4.42–4.54 (m, 1H), 10.25 (s, 1H); MS (ESI), m/z
+
+
07.5 [2MÀHClÀCl] (10.2), 829.4 [2MÀ2HCl+Na] (1.6).
1
H NMR (360.13 MHz, CDCl
3
9
.
Rodionov, I. L.; Rodionova, L. N.; Baidakova, L. K.; Romashko, A. M.; Balashova,
3
T. A.; Ivanov, V. T. Tetrahedron 2002, 58, 8515–8523.
+
+
+
(
%): 206.5 [M+H] (100), 228.2 [M+Na] (28.4), 433.3 [2M+Na] (94.0).
1
0. H-b-Ala-Pro-DabNHBz (1): Tripeptide 5 (2.64 g, 6 mmol) was dissolved in H
60 ml). To this solution were added 1,4-dioxane (30 ml) and Py (8 ml). The
mixture was cooled to 5 °C, followed by the addition of PIDA (4.64 g,
4.4 mmol) in 1,4-dioxane (60 ml). After stirring at 5 °C for 3 h, another
portion of PIDA (3.09 g, 9.6 mmol) in 1,4-dioxane (40 ml) was added. Stirring
was continued for 2 h and the mixture was diluted with ice-H O (100 ml) and
2
O
4
.
Compound 2: -Proline (11.50 g, 100 mmol) was suspended in CH Cl (100 ml).
L
2
2
(
The suspension was cooled to À10 °C and 3-chloropropanoyl chloride (6.35 g,
5
0
0 mmol) was added in one portion. After 30 min, the mixture was heated to
°C, washed with brine (2 Â 100 ml), and dried over Na SO . After cooling the
1
2
4
organic phase to À5 °C, PfpOH (9.20 g, 50 mmol) and DCC (11.35 g, 55 mmol) in
CH Cl (50 ml) were added. The mixture was stirred at 0 °C for 1 h. It was then
allowed to warm to rt, filtered, and concentrated under reduced pressure. The
residue was stirred with Et O (10 ml) and hexane (30 ml) and left to stand
overnight at 5 °C. The solid residue thus obtained was separated from the
solution by filtration and recrystallized from Et O–hexane. Yield 10.44 g (56%).
Large colorless crystals; purity 98.1% (HPLC); mp 51–53 °C; [
2
2
2
EtOAc (300 ml). The aqueous layer was washed with EtOAc (2 Â 100 ml) and
EtOAc/BuOH (4:1) (100 ml) and evaporated under reduced pressure. The
residue was chromatographed (HPLC) to give target 1 as yellowish oil. Yield
2
24
1
1
.61 g (54%). Purity >98% (HPLC);
360.13 MHz, CD OD): 1.94–2.12 (m, 4H), 2.18–2.29 (m, 2H), 2.77 (t,
J = 6.2 Hz, 2H), 3.05 (t, J = 7.2 Hz, 2H), 3.15 (t, J = 6.3 Hz, 2H), 3.54–3.68 (m,
[
a
]
D
À81.32 (c 1, MeOH);
H NMR
2
(
3
d
2
4
a
]
D
À78.80 (c 5,
): d 1.98–2.29 (m, 3H), 2.33–2.54 (m, 1H),
.82 (t, J = 6.8 Hz, 2H), 3.57–3.93 (m, 4H), 4.77–4.86 (m, 1H); MS (ESI), m/z (%):
1
CHCl
2
3
3
); H NMR (360.13 MHz, CDCl
3
13
2
(
5
H), 4.38–4.43 (m, 3H), 4.48–4.54 (m, 1H), 7.21–7.34 (m, 5H); C NMR
90.56 MHz, D O): d 23.65, 28.04, 29.04, 30.34, 34.61, 35.81, 42.43, 47.23,
0.92, 59.47, 126.59, 126.92, 128.16, 137.06, 170.26, 171.43, 177.93; MS (ESI),
+
+
2
72.4 [M+H] (100), 394.1 [M+Na] (4.8).
5
.
Single crystal X-ray diffraction on a sample of 2 was performed by using a
Bruker SMART APEX2 diffractometer. Mercury software was used for the
crystal structure plot. Crystals at 150 K, orthorhombic, space group P 21 21 21,
a = 6.4269(2) Å, b = 9.9541(3) Å, c = 23.7321(6) Å. Crystallographic data for the
+
+
m/z (%): 376.2 [MÀAcOHÀAcO] (100), 751.1 [2MÀ3AcOHÀAcO] (33.2); IR
À1
(m
max, cm ): 3060, 2212, 1961, 1651, 1557, 1410, 1341, 1276, 1203, 1181,
1137, 1049, 1015, 924, 888, 837, 800, 723, 701, 659, 621.