Synthesis of pacidamycin analogues via an Ugi-multicomponent reaction

Article abstract of DOI:10.1016/j.bmcl.2012.05.050

The second-generation synthesis of 3′-hydroxypacidamycin D (2) has been accomplished via an Ugi-four component reaction at a late stage of the synthesis. This approach provided ready access to a range of analogues including diastereomers of the diaminobut

Full text of DOI:10.1016/j.bmcl.2012.05.050

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4810–4815
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Bioorganic & Medicinal Chemistry Letters
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Synthesis of pacidamycin analogues via an Ugi-multicomponent reaction
Kazuya Okamoto a, Masahiro Sakagami a, Fei Feng b, Fumiyo Takahashi a, Kouichi Uotani c, Hiroko Togame a,
d
Hiroshi Takemoto a, Satoshi Ichikawa d, , Akira Matsuda
⇑
a Shionogi Innovation Center for Drug Discovery, Shionogi & Co., Ltd, Kita-21, Nishi-11, Kita-ku, Sapporo 001-0021, Japan
b Faculty of Advanced Life Science, Hokkaido University, Kita-21, Nishi-11, Kita-ku, Sapporo 001-0021, Japan
c Medicinal Research Laboratories, Shionogi & Co., Ltd, 3-1-1, Futaba-cho, Toyonaka, Osaka 561-0825, Japan
d Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
a r t i c l e i n f o
a b s t r a c t
The second-generation synthesis of 30-hydroxypacidamycin D (2) has been accomplished via an Ugi-four
component reaction at a late stage of the synthesis. This approach provided ready access to a range of
analogues including diastereomers of the diaminobutylic acid residue and hybrid-type analogues of
mureidomycins. Biological evaluations of these analogues indicated that the stereochemistry at the diam-
inobutylic acid residue has a crucial impact on both the MraY biochemical inhibition and whole-cell anti-
bacterial activity.
Article history:
Received 18 April 2012
Revised 9 May 2012
Accepted 11 May 2012
Available online 18 May 2012
Keywords:
Antibacterial
Ó 2012 Elsevier Ltd. All rights reserved.
Ugi-four component reaction
Natural product
Nucleoside
The pacidamycins (Fig. 1, 1),1 first isolated from Streptomyces
coeruleorubidus AB 1183F-64 in 1989, are members of a class of
uridylpeptide antibiotics,2 that are composed of the mureidomyc-
ins,3 the napsamycins,4 and the recently identified sansanmycins.5
Sharing a characteristic structural feature, namely a 30-deoxyuri-
dine which is attached to a tetrapeptide moiety by an enamide
linkage, these compounds exhibit selective antibacterial activity
against Pseudomonas aeruginosa, a common nosocomial pathogen
that is intrinsically resistant to a variety of drugs currently used
in the clinic. Consequently, immuno-compromised patients are at
greater risk of infection. Moreover, the increased rate of acquired
multi-drug resistance in P. aeruginosa complicates anti-pseudomo-
nas chemotherapy, thereby making the development of novel anti-
bacterial agents that are active against multi-drug resistance P.
aeruginosa all the more urgent.6–8 These uridylpeptide antibiotics
are sub-nanomolar inhibitors of their biological target, the phos-
pho-MurNAc-pentapeptide transferase (MraY),2,9 which is respon-
sible for the formation of lipid I in the peptidoglycan biosynthesis
pathway.2,10 Inhibition of the MraY by these antibiotics causes a
malfunction of the peptidoglycan production in a manner different
from b-lactams such as the carbapenems. Since MraY is an essen-
Figure 1. Structure of pacidamycin D and its 30-hydroxy analogue 2.
tial enzyme in bacteria2 and a novel target, uridylpeptide antibiot-
ics are expected to be highly useful anti-Pseudomonal agents.
Intrigued by the promising biological activity, several groups have
conducted structure–activity relationship studies.11–15
Abbreviations: DABA, a,b-diaminobutylic acid; HATU, 2-(1H-7-azabenzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; MraY, phospho-MurNAc-
pentapeptide transferase; NMM, N-methylmorpholine; SAR, structure–activity
relationship; U-4CR, Ugi-four component reaction.
We have recently accomplished the total synthesis of pacida-
⇑
Corresponding author. Tel.: +81 11 706 3229; fax: +81 11 706 4980.
E-mail address: ichikawa@pharm.hokudai.ac.jp (S. Ichikawa).
mycin D (1) and its 30-hydroxyl analogue 2, which features a ster-
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.050

K. Okamoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4810–4815
4811
efficiently is preferable. A multi-component reaction would be ide-
ally suited for structure–activity relationship (SAR) studies since it
allows for diversification of accessible analogs simply by altering
each component. We have successfully applied an Ugi-four compo-
nent reaction (U-4CR) in pursuing the SAR of the muraymycins,
which are antibacterial nucleoside natural products.17 Herein, we
describe the second-generation synthesis of 2 via a U-4CR in order
to establish a convergent synthetic strategy appropriate for the
SAR study of uridylpeptide antibiotics. The biological evaluation
of several analogues is also described.
Our second-generation retrosynthetic analysis of 2 is summa-
rized in Scheme 1. We planned to install the N-terminal amino acid
residue 3 on 4 in the final stage of the synthesis considering the ini-
tial efforts to prepare 2 as described later. We retrosynthetically di-
vided 4 into the urea dipeptide 5, 2,4-dimethoxybenzylamine 6,
the 2-N-methylaminopropionaldehyde derivative 7, and the
a,b-
unsaturated isonitrile derivative of uridine 8 for the U-4CR. This
strategy allowed us to construct 4 with the non-proteinogenic ami-
no acid
a,b-diaminobutylic acid (DABA) residue, with linkages to
the urea dipeptide and uridine moieties at the C- and N-termini.
Moreover, the U-4CR strategy has the advantage of efficiently fur-
nishing diastereomers that are useful for the SAR. Considering the
nature of the multi-component assemblage, this strategy allows
easy access to diversified analogues.
The aldehyde unit 7 was prepared as shown in Scheme 2.
Namely, N-formyl-L-alanine 9 was reduced by LiAlH4, and the
resulting N-methylaminoalcohol was selectively protected by a
Boc group to provide 10 in 98% yield over 2 steps. The primary hy-
droxyl group of 10 was oxidized by Dess-Martin periodinane to
give 7 in 84% yield. The isonitrile 8 was prepared from the Z-ena-
mide 12 by the copper-catalyzed C–N cross-coupling of the Z-vinyl
iodide 1116 and formamide as describe in Scheme 3. The iodide 11
was reacted with formamide using 0.2 equiv of CuI, 0.4 equiv of
MeNHCH2CH2NHMe, Cs2CO3 in THF at 70 °C to give the desired
Z-enamide 12, which was obtained selectively in 90% yield. The
corresponding E-isomer was not observed at all during the course
of the reaction. Dehydration of 12 was achieved by a treatment
with triphosgene and Et3N in CH2Cl2 at ꢀ78 °C to provide the iso-
nitrile 8 in 88% yield.
Scheme 1. Retrosynthetic analysis of 2 disconnected by an Ugi-four component
reaction.
With 7 and 8 in hand, we then examined the assemblage of the
four components by the U-4CR (Scheme 4). The assemblage pro-
ceeded simply by mixing 5, 6, 7, and 8 in EtOH at room tempera-
ture for 48 h, and the desired 13 and its diastereomer at the
newly formed stereogenic center were obtained in acceptable
yields (33% for 13 and 30% for 14). These were separated by silica
gel column chromatography. Initially we planned to use an alde-
hyde of the dipeptide derivative such as 21, which was prepared
by reduction of the Weinreb amide derivative of the dipeptide
20, as the aldehyde component in the U-4CR assemblage
(Scheme 5). However, it turned out that the resulting aldehyde
21 easily cyclized to form a rather stable cyclic aminal 22, and
the U-4CR did not proceed at all. Therefore we turned our attention
to the stepwise synthetic route toward the target compounds as
shown in Scheme 4 in order to avoid the undesired cyclization.
Scheme 2. Preparation of the aldehyde unit 7.
eoselective construction of the Z-oxy-acyl enamide architecture by
a copper-catalyzed C–N cross-coupling.16 These compounds exhib-
ited strong MraY inhibitory activity (IC50 = 22 nM for 1, 42 nM for
2) and potent antibacterial activity against P. aeruginosa (MIC 8–
64 l
g/mL). The existence of the hydroxyl group at the 30-position
of the uridine moiety does not have any impact on either MraY
inhibition or antibacterial activity. This first generation strategy,
however, is a linear synthesis, where the vinyl iodide derivative
of uridine was coupled with the tetrapeptide carboxamide moiety.
Nonetheless, from a medicinal point of view, the development of a
convergent synthetic strategy that would provide analogues more
Scheme 3. Preparation of the isonitrile unit 8.

4812
K. Okamoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4810–4815
Scheme 4. Total synthesis of 30-hydroxypacidamycin D (2) and its epimer 19.
Scheme 5. Attempt to use the dipeptide aldehyde 21 in the U-4CR.
The final assemblage of the remaining amino acid and global
deprotection were then investigated. Upon selective removal of
the Boc group of 13 (10% TFA, CH2Cl2, 0 °C), the liberated secondary
amine of 15 was condensed with N-Boc-L-Ala 3 (HATU, N-methyl-
morpholine (NMM), CH2Cl2) to give the fully protected 30-hydroxyl
pacidamycin D 17 in 79% yield over 2 steps. Finally removal of all
the protecting groups (BCl3, CH2Cl2, 0 °C, 5HFꢁNEt3, 22% over 2
steps) successfully afforded 2. The analytical data for 2 in this sec-
ond-generation synthesis were identical to those of the material
previously synthesized.16a The deprotection step gave poor yield
Figure 2. Structure of the mureidomycins.
of the desired 2 because BCl3 treatment resulted in decomposition
of the acid sensitive enamide moiety as well as migration of the
2,4-dimethoxy group to the indole moiety of the Trp residue.

K. Okamoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4810–4815
4813
Scheme 6. Synthesis of analogues 27a–c, 30 and 31.
Similar problems were also observed for the formation of 19, 27,
30, and 31, which were described later. The epimer at the DABA
residue 19 was also prepared from 14 in a manner similar to that
of the synthesis of 2.

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K. Okamoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4810–4815
Table 1
stead retained the activity. However, moving the hydroxyl group to
the para-position at the N-terminal phenyl ring resulted in a large
decrease in inhibitory activity with an IC50 value of 4000 nM for
27b. Therefore the position of the hydroxyl group on the phenyl
ring plays a crucial role. Analogue 27c showed a slight decrease
in MraY inhibitory activity. The antibacterial activity of 2, 19,
27a–c, 30, and 31 was then evaluated against a range of clinically
isolated P. aeruginosa strains (Table 2).20 The overall antibacterial
activity was well correlated to the MraY inhibitory activity. Thus,
27a showed a similar anti-Pseudomonal activity to that of 1 and
2, whereas 19, 27b, 27c, 30, and 31 exhibited no activity up to
MraY inhibitory activity of 30-hydroxypacidamycin analogues
1
2
19
27a
27b
27c
30
31
a
IC50 (nM)
22
42
4000
22
4000
65
650
8900
a
The inhibitory activity of compounds on the purified MraY enzyme (Staphylo-
coccus aureus) was then examined by fluorescence based MraY assay using UDP-
MurNAc-dansylpentapeptide, where the formation of dansylated lipid I was mon-
itored by fluorescence enhancement (excitation at 355 nm, emission at 535 nm).
Table 2
32 lg/mL. The exception was the Gly analogue 27c, which showed
no anti-Pseudomonal activity in spite of potent MraY inhibitory
activity (Table 1, IC50 65 nM).
anti-Pseudomonal activity of 30-hydroxypacidamycin analogues
Compound
MICa
(lg/mL)
In summary, the second-generation synthesis of 2 has been
accomplished via a U-4CR at a late stage of the synthesis. This ap-
proach provided ready access to a range of analogues including the
diastereomers of the DABA residue and hybrid-type analogues of
the mureidomycins. Biological evaluations of these analogues indi-
cated that the stereochemistry at the DABA residue has a signifi-
cant impact on both the MraY biochemical inhibition and whole-
cell antibacterial activity.
P. aeruginosa P. aeruginosa
P. aeruginosa P. aeruginosa
PAOl
YYI65 (4mexB) ATCC 25619
SR 27156
1
2
19
27a
27b
27c
30
64
16
16
16
32
8
16
16
>32
>32
>32
>32
>32
>32
>32
16
>32
>32
>32
>32
>32
32
>32
16
>32
>32
>32
>32
>32
>32
>32
>32
31
a
MICs were determined by a microdilution broth method as recommended by
Supplementary data
the NCCLS with cation-adjusted Mueller-Hinton broth (CA-MHB). Serial twofold
dilutions of each compound were made in appropriate broth, and the plates were
inoculated with 5 ꢂ 104 CPU of each strain in a volume of 0.1 mL. Plates were
incubated at 35 °C for 20 h and then MICs were scored.
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.bmcl.2012.05.050.
References and notes
The mureidomycins (Fig. 2)3 were first isolated from Strepto-
myces flavidoviridens SANK 60486 and belong to the same class
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1.56 l
g/mL.3b We therefore planned to prepare an analogue 27a,
which is a hybrid-type of pacidamycin D and mureidomycin C,
by introducing the Gly-L-m-Tyr moiety into the intermediates
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the L-m-Tyr residue of 30.
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on the purified MraY enzyme (Staphylococcus aureus) was next
examined by fluorescence based MraY assay using UDP-MurNAc-
dansylpentapeptide, where the formation of dansylated lipid I
was monitored by fluorescence enhancement (excitation at
355 nm, emission at 535 nm),19 and the results are summarized
in Table 1. It turned out that the epimer 19 was much weaker than
the MraY inhibitors 1 or 2 with an IC50 value of 4000 nM. The ste-
reochemistry at the DABA residue was very important for enzyme
inhibitory activity. This was also true for the analogues 30 and 31,
which possess the same stereochemistry at the DABA residue (IC50
650 and 8900 nM, respectively). Analogue 27a showed similar
inhibitory activity (IC50 22 nM) to that of 1 and 2, and introducing
the Gly-L-m-Tyr moiety at the N-terminus did not improve but in-
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