Solution-phase synthesis of a muramyl dipeptide analogue MDA

Article abstract of DOI:10.1016/j.cclet.2011.07.020

The solution-phase synthesis of a muramyl dipeptide (MDP) analogue of N^α-[4-chlorocinnamoyl-l-alanyl-d-isoglutaminyl]-l-lysine (MDA, 2) is reported that possesses the features of easy feasibility, safety and low cost in large scale of synthesis.

Full text of DOI:10.1016/j.cclet.2011.07.020

Available online at www.sciencedirect.com
Chinese Chemical Letters 22 (2011) 1443–1446
www.elsevier.com/locate/cclet
Solution-phase synthesis of a muramyl dipeptide analogue MDA
*
Nan Zhao, Yao Ma, Gang Liu
Department of Synthetic Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100050, China
Received 25 May 2011
Available online 10 October 2011
Abstract
The solution-phase synthesis of a muramyl dipeptide (MDP) analogue of Na-[4-chlorocinnamoyl-L-alanyl-D-isoglutaminyl]-L-
lysine (MDA, 2) is reported that possesses the features of easy feasibility, safety and low cost in large scale of synthesis.
# 2011 Gang Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Muramyl dipeptide analogue; Solution-phase synthesis; MDA
Muramyl dipeptide (MDP, 1, Fig. 1) is the minimum structure of the cell wall of Gram-positive bacteria that is
recognized by human immunological response. It non-specifically stimulates human macrophages to become active
against virus, bacteria, and tumors [1–3]. In view of its activities, we embarked on the structural modification of MDP
for many years [4–8]. A MDP analogue of Na-[4-chlorocinnamoyl-L-alanyl-D-isoglutaminyl]-L-lysine (MDA, 2,
Fig. 1) has been conjugated with Paclitaxel in our group to synthesize a novel compound termed as MTC-220 [8]. The
unique pharmacological property allows MTC-220 to effectively inhibit tumor growth in nude mice and significantly
prevent tumor metastasis in Lewis lung carcinoma and 4T1-tumor-bearing mice through suppressing myeloid derived
suppressor cell (MDSC) accumulation in the spleen and bone marrow of tumor-bearing mice and repressing
inflammatory cytokines in tumor tissue [8]. MTC-220 is currently under research for its preclinical studies as a dual
function drug candidate to treat breast and lung cancers. Therefore, it is important to find a feasible method for
production of MTC-220 as well as its intermediate MDA in large scale.
We previously reported that MDA was prepared through a solid-phase synthetic route with the total yield of 96%
[8]. Solid-phase synthesis is a common strategy of peptide chemistry. The advantages of this method mainly include
highly coupling efficiency and yield. However, drawbacks of solid-phase synthesis often limit the preparation of aim
product in large scale including the necessary and expensive solid resin, costly washing and cleavage solvents,
incomplete/truncated peptides and/or resin anchored impurities that are produced in the process of synthesis.
Especially, the building blocks of non-natural amino acids need to be pre-prepared and pre-protected in solution-phase
where addresses additional chemical processes. Therefore, alternative solution-phase synthesis of certain peptide or
peptide analogue, especially bearing non-natural amino acids, is normally considered to facilitate the production in
* Corresponding author.
E-mail address: gliu@imm.ac.cn (G. Liu).
1001-8417/$ – see front matter # 2011 Gang Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2011.07.020

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N. Zhao et al. / Chinese Chemical Letters 22 (2011) 1443–1446
O
O
OH
H2N
HO
HO
NHAc
O
O
O
NH2
H
N
O
H
N
N
H
N
H
CONH2
OH
N
H
O
CONH2
Cl
O
O
1
2
Fig. 1. Chemical structure of the natural MDP 1 and its analogue MDA 2.
COOBzl
CONH2
COOBzl
COOH
a
COOBzl
COOH
b
c, d
COOH
COOH
Boc
N
H
Boc
N
H
H2N
H2N
3
4
5
6
Scheme 1. Synthetic route of t-butoxycarbonyl-D-isoglutaminyl benzyl ester 6. Reagents and conditions: (a) benzyl alcohol, BF3ꢀEt2O, r.t., 15 h; (b)
(Boc)2O, NaHCO3, 50% dioxane/H2O, r.t., 20 h; (c) HOSu, DIC, 0 8C, 5 h, r.t., 20 h; (d) dry ammonia gas, THF, ꢁ10 8C, 1.5 h.
N
H
Z
N
H
Z
a, b
Boc
N
H
COOH
Boc
N
H
CONH2
7
8
Scheme 2. Synthetic route of Na-t-butoxycarbonyl-Ne-carbobenzyloxy-L-lysinyl amide 8. Reagents and conditions: (a) HOSu, DIC, 0 8C, 5 h, r.t.,
20 h; (b) dry ammonia gas, THF, ꢁ10 8C, 1.5 h.
large scale. In this letter, we reported a solution-phase synthetic method of MDA that can be used to prepare MDA in
kilogram scale (Schemes 1–3).
t-Butoxycarbonyl-D-isoglutaminyl benzyl ester 6 and Na-t-butoxycarbonyl-Ne-carbobenzyloxy-L-lysinyl amide 8
were prepared from commercial available D-glutamic acid 3 and Na-t-butoxycarbonyl-Ne-carbobenzyloxy-L-lysine 7
in Schemes 1 and 2, respectively. g-Carboxyl group of 3 was firstly esterified in benzyl alcohol to gain D-glutaminyl g-
benzyl ester 4 in the presence of Lewis acid BF3ꢀEt2O [9]. The amino group of 4 was then protected by (Boc)2O to
yield t-butoxycarbonyl-D-glutaminyl g-benzyl ester 5 under a weakly basic condition [10]. Compound 5 was further
activated by HOSu and DIC, then reacted with dry ammonia gas to finally form compound 6 in a yield of 53% (Scheme
1) [4,5]. Similarly, compound 8 was obtained by the method in a yield of 92% shown in Scheme 2.
The peptide assembly in solution-phase was carried out as described in Scheme 3. All Boc groups in various
building blocks were generally removed under an acidic condition of 50% TFA/DCM (v/v). And all coupling steps
were performed by HOSu and DIC activation of carboxyl groups. Building blocks of t-butoxycarbonyl-L-alanine 9 and
t-butoxycarbonyl-D-isoglutaminyl benzyl ester 6 were firstly coupled to yield a protected dipeptide of t-
butoxycarbonyl-L-alaninyl-D-isoglutaminyl benzyl ester 10 in 81% yield. After removal of Boc group and
performance of coupling reaction again, a protected tripeptide of 4-chlorocinnamoyl-L-alanyl-D-isoglutaminyl benzyl
ester 12 was obtained in a total yield 79% of two reaction steps. Considering the sensitivity of chlorine atom and a
double bond of 4-chlorocinnamoyl group, palladium–charcoal/hydrogenation was avoided to deprotect Bzl group in
this letter. Various basic conditions were investigated to completely cleave Bzl group such as K2CO3, NaHCO3 and
LiOH aqueous solution. Unexpectedly, two major products (molar ratio = 4:6) with the same molecular weight were
gained at all the time. Results of elucidation by 1H NMR indicated that they were the aim compound 4-
chlorocinnamoyl-L-alanyl-D-isoglutamine 13 and its structural isomer 4-chlorocinnamoyl-L-alanyl-D-glutamine 13a
that very likely were produced through a cyclic intermediate 3(R)-[4-chlorocinnamoyl-L-alanyl]-amino-2,6-
dioxopiperidine 15 (Scheme 4). We then switch our attention to acidic conditions to deprotect Bzl group. A solution of
HBr/AcOH was optimally selected to afford 9 in a high yield of 85%. Final iteration of the deprotection of Boc group
and coupling procedure led Na-[4-chlorocinnamoyl-L-alanyl-D-isoglutaminyl]-Ne-carbobenzyloxy-L-lysine 14
produced in a yield of 74%. With the assistance of a mixture of BF3ꢀEt2O, TFA and EtSH (v:v:v = 9:9:2), Z

N. Zhao et al. / Chinese Chemical Letters 22 (2011) 1443–1446
1445
H
H
N
a
N
COOBzl
COOBzl
COOBzl
CONH2
COOBzl
CONH2
a
Boc
N
H
H2N
c
O
CONH2
10
O
CONH2
Boc
N
H
H2N
Boc
c
6
COOH
COOSu
b
b
Boc
N
H
COOH
O
N
H
COOSu
Cl
Cl
9
11
O
H
N
H
N
d
COOBzl
b
COOH
N
H
N
H
O
CONH2
O
CONH2
Cl
Cl
12
13
O
H
N
COOSu
N
H
H
O
CONH2
Z
N
Cl
c
e
O
O
H
N
Z
N
a
N
H
Z
H
N
N
H
CONH2
H
Boc
N
H
CONH2
O
CONH2
H2N
CONH2
Cl
14
8
H2N
O
O
H
N
N
N
H
CONH2
H
O
CONH2
Cl
2
Scheme 3. Solution-phase synthesis of MDA. Reagents and conditions: (a) 50% TFA/DCM, r.t., 1 h; (b) HOSu, DIC, THF, 0 8C, 5 h, then, r.t., 20 h;
(c) THF, 0 8C, 5 h, r.t., 24 h; (d) HBr/AcOH, r.t., 3 h; (e) BF3ꢀEt2O, TFA, EtSH (9:9:2), r.t., 2 h.
group of 14 was completely removed with a yield of 72%. The aimed MDA 2 was obtained eventually after
purification by ODS column chromatography and fully characterized by 1H NMR, 13C NMR, IR and HR MS (TOF)
[11].
In conclusion, we have developed a solution-phase synthesis of MDA. This strategy can be employed to synthesize
a novel antitumor agent of MTC-220 in kilogram scale. After all steps, targeted compound MDAwas obtained with the
total yield of 14%.
O
O
H
N
N
H
O
12
O
Cl
O
NH2
under basic conditions
O
H
N
N
15
H
O
Cl
O
N
H
O
O
O
O
O
H
N
H
N
N
H
OH
N
NH2
H
O
O
Cl
O
NH2
Cl
O
OH
13
13a
Scheme 4. Structural isomers of 13 produced under basic conditions.

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N. Zhao et al. / Chinese Chemical Letters 22 (2011) 1443–1446
Acknowledgment
This research is supported financially by the National Natural Science Foundation of China (No. 90713045).
References
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25
[11] Chemical data of (2) list as following: mp: 215–217 8C, ½aꢂD +37.7 (c 11.0 mg/mL, DMF). 1H NMR (600 MHz, DMSO-d6): d lysine’s part:
7.90 (d, 1H, J = 8.4 Hz, NH), 7.10 (s, 1H, CONHa), 7.30 (s, 1H, CONHb), 4.11 (m, 1H, a-H), 1.46 (m, 1H, b-Ha), 1.63 (m, 1H, b-Hb), 1.27 (m,
2H, g-H), 1.53 (m, 2H, d-H), 2.73 (m, 2H, e-H), 7.75 (br. s, 2H, NH2); D-isoglutamine’s part: 8.21 (d, 1H, J = 8.4 Hz, NH), 6.98 (s, 1H, CONHa),
7.41 (s, 1H, CONHb), 4.14 (m, 1H, a-H), 1.71 (m, 1H, b-Ha), 1.97 (m, 1H, b-Hb), 2.15 (t, 2H, J = 7.2 Hz, g-H); alanine’s part: 8.39 (d, 1H,
J = 6.6 Hz, NH), 4.38 (m, 1H, a-H), 1.26 (m, 3H, b-H); 4-chloro-cinnamoyl part: 6.75 (d, J = 15.9 Hz, 1H, trans-a-H), 7.39 (d, 1H,
J = 15.9 Hz, trans-b-H), 7.57 (d, 2H, J = 8.4 Hz, ph-o-H), 7.47 (d, 2H, J = 8.4 Hz, ph-m-H). 13C NMR (150 MHz, DMSO-d6): d lysine’s part:
173.3 (CONH2), 52.1 (a-C), 31.3 (b-C), 22.4 (g-C), 26.8 (d-C), 38.7 (e-C); D-isoglutamine’s part: 173.8 (CONH2), 52.2 (a-C), 27.7 (b-C), 31.7
(g-C), 171.6 (CONH); alanine’s part: 172.4 (CONH), 48.8 (a-C), 18.1 (b-C); 4-chloro-cinnamoyl part: 164.7 (CONH), 122.7 (trans-a-C),
137.6 (trans-b-C), 133.8 (ph-q-C), 129.2 (ph-o-C), 129.0 (ph-m-C), 134.0 (ph-p-C). IR (KBr, cmꢁ1): 3281.5, 3198.9, 3063.4, 2935.2, 1609.8,
1539.0, 1452.2, 1200.2, 1134.1, 973.6, 821.6, 799.8, 720.2. HR MS (TOF): found 509.2270 [M+H]+; calcd. for C23H34ClN6O5 509.2279.