Cefiderocol (S-649266), A new siderophore cephalosporin exhibiting potent activities against Pseudomonas aeruginosa and other gram-negative pathogens including multi-drug resistant bacteria: Structure activity relationship

Article abstract of DOI:10.1016/j.ejmech.2018.06.014

The structure-activity relationship (SAR) for a novel series of catechol conjugated siderophore cephalosporins is described with their in vitro activities against multi-drug resistant Gram-negative pathogens including Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia and Enterobacteriaceae. Cefiderocol (3) was one of the best molecules which displayed well-balanced and potent activities against multi-drug resistant Gram-negative pathogens including carbapenem resistant bacteria among the prepared compounds with the modified C-7 side chain and the modified C-3 side chain. Cefiderocol (3) is a highly promising parenteral cephalosporin for the treatment of multi-drug resistant Gram-negative infection.

Full text of DOI:10.1016/j.ejmech.2018.06.014

Accepted Manuscript
Cefiderocol (S-649266), A new siderophore cephalosporin exhibiting potent activities
against Pseudomonas aeruginosa and other gram-negative pathogens including
multi-drug resistant bacteria: Structure activity relationship
Toshiaki Aoki, Hidenori Yoshizawa, Kenji Yamawaki, Katsuki Yokoo, Jun Sato,
Shinya Hisakawa, Yasushi Hasegawa, Hiroki Kusano, Masayuki Sano, Hideki
Sugimoto, Yasuhiro Nishitani, Takafumi Sato, Masakatsu Tsuji, Rio Nakamura, Toru
Nishikawa, Yoshinori Yamano
PII:
S0223-5234(18)30505-1
10.1016/j.ejmech.2018.06.014
EJMECH 10484
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 February 2018
Revised Date: 21 May 2018
Accepted Date: 4 June 2018
Please cite this article as: T. Aoki, H. Yoshizawa, K. Yamawaki, K. Yokoo, J. Sato, S. Hisakawa,
Y. Hasegawa, H. Kusano, M. Sano, H. Sugimoto, Y. Nishitani, T. Sato, M. Tsuji, R. Nakamura, T.
Nishikawa, Y. Yamano, Cefiderocol (S-649266), A new siderophore cephalosporin exhibiting potent
activities against Pseudomonas aeruginosa and other gram-negative pathogens including multi-drug
resistant bacteria: Structure activity relationship, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.06.014.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Title
Cefiderocol (S-649266), A New Siderophore Cephalosporin Exhibiting Potent Activities Against
Pseudomonas aeruginosa and Other Gram-Negative Pathogens Including Multi-drug Resistant
Bacteria: Structure Activity Relationship
Affiliations
1
Medicinal Chemistry Research Laboratory, Shionogi & Co., Ltd., 1-1, Futabacho, 3-chome,
Toyonaka, 561-0825, Osaka, Japan.
2
Drug Discovery & Disease Research Laboratory, Shionogi & Co., Ltd., 1-1, Futabacho, 3-chome,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
Toyonaka, 561-0825, Osaka, Japan.
3
Pharmaceutical Research Division, Shionogi & Co., Ltd., 1-1, Futabacho, 3-chome, Toyonaka,
561-0825, Osaka, Japan.
Authors
1
1
1
1
1
Toshiaki Aoki , Hidenori Yoshizawa , Kenji Yamawaki , Katsuki Yokoo , Jun Sato , Shinya
1
1
1
1
1
Hisakawa , Yasushi Hasegawa , Hiroki Kusano , Masayuki Sano , Hideki Sugimoto , Yasuhiro
1
2
2
2
2
3
Nishitani , Takafumi Sato , Masakatsu Tsuji , Rio Nakamura , Toru Nishikawa , Yoshinori Yamano
Corresponding Author
Toshiaki Aoki
Email: toshiaki.aoki@shionogi.co.jp
Tel: +81-6-6331-6334
Fax: +81-6-6332-6385
Keywords
cefiderocol
siderophore
siderophore cephalosporin
multi-drug resistant Gram-negative pathogens
Abbreviations used
ESBL, extended-spectrum β-lactamase; MDR, multi-drug resistant; PG, protecting group;
ACLE·HCl, (7-amino-3-chloromethyl-3-cephem-4-carboxylate p-methoxybenzyl ester
hydrochloride); ACLH·HCl, (7-amino-3-chloromethyl-3-cephem-4-carboxylate benzhydryl ester
hydrochloride); Boc, tert-butoxycarbonyl; Tr, trityl; PMBCl, p-methoxy benzyl chloride; DMF,
N,N-dimethylformamide;
THF,
tetrahydrofuran;
EDC·HCl,

ACCEPTED MANUSCRIPT
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole;
MsCl, methanesulfonyl chloride; DPPA, diphenylphosphoryl azide; BH, benzhydryl; NMM,
N-methylmorphiline; m-CPBA, m-chloroperoxybenzoic acid; DMA, N,N-Dimethylacetamide; TFA,
trifluoroacetic acid; MIC, minimum inhibitory concentration; ED , 50% effective dose
5
0
Abstract
The structure-activity relationship (SAR) for a novel series of catechol conjugated siderophore
cephalosporins is described with their in vitro activities against multi-drug resistant Gram-negative
pathogens including Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas
maltophilia and Enterobacteriaceae. Cefiderocol (3) was one of the best molecules which displayed
well-balanced and potent activities against multi-drug resistant Gram-negative pathogens including
carbapenem resistant bacteria among the prepared compounds with the modified C-7 side chain and
the modified C-3 side chain. Cefiderocol (3) is a highly promising parenteral cephalosporin for the
treatment of multi-drug resistant Gram-negative infection.
1. Introduction
In recent years, the threat of multidrug-resistant Gram-negative pathogens e.g. Pseudomonas
1
,2
aeruginosa, Acinetobacter baumannii and Enterobacteriaceae is expanding all over the world. The
World Health Organization recently identified carbapenem resistant Acinetobacter baumannii,
Pseudomonas aeruginosa and Enterobacteriaceae as the number 1 priority (critical) for which new
3
antibiotics are needed. The Review on Antimicrobial Resistance estimated that the number of deaths
attributable to antimicrobial resistance could increase to 10 million each year by 2050, unless action
4
is taken now. Nevertheless, the therapeutic treatment options for infections caused by multi-drug
5
resistant Gram-negative pathogens are severely limited. Therefore, there is an urgent need for
6
developing new and potent antibiotics targeting multi-drug resistant Gram-negative pathogens.
Microorganisms naturally secrete siderophores that are high affinity iron chelating compounds to
3
+
7
acquire iron (Fe ) effectively under an iron limited environment. The major groups of siderophores
include catechols, hydroxamates and carboxylates. For instance, enterobactin and pyoverdine
secreted by E. coli and P. aeruginosa (1a and 1b, Figure 1.) contain catechol groups as an iron
8
chelating moiety.

ACCEPTED MANUSCRIPT
6
7
8
6
Figure 1. Structures of enterobactin (1a) and pyoverdine (1b); catechol groups highlighted.
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
One strategy to discover an antibiotic capable of eradicating multidrug-resistant Gram-negative
3
+
pathogens is to utilize the bacterial iron (Fe ) acquisition system to enhance the outer membrane
penetration of Gram-negative bacteria through the attachment of an iron chelating moiety such as a
9
catechol to the parent molecule. This strategy was the focus of a number of research teams in
1980-90s and they reported that catechol or 4-pyridone conjugated β-lactam analogues had shown
1
0-16
potent antibacterial activities against Gram-negative pathogens including P. aeruginosa.
However, none of those compounds reached clinical stage development. Our team identified in
1
7
1990s a catechol conjugated cephalosporin A-2 (2, Figure 2), which displayed potent activity
against P. aeruginosa (see Table 1 for the in vitro activity), however compound 2 did not progress to
clinical stage development due to poor physicochemical properties. We restarted in 2008 the drug
discovery research of siderophore cephalosporins based on the structure and the activity of
compound 2 to identify a new candidate with potent activities against several multi-drug resistant
1
-3
pathogens including new varieties of carbapenemase
(carbapenem-hydrolyzing enzyme)
producing carbapenem resistant pathogens that had become a global health issue. Several analogues
modified at the C-3 side chains (quaternary ammonium, amide linker and catechol moiety) and the
C-7 side chains (aminothiazole and oxime moiety) were synthesized and evaluated in vitro and in
vivo for antibacterial activities against Gram-negative pathogens (Figure 2). Here, we report the
SAR results of several siderophore cephalosporins that led to the selection of cefiderocol (3).
8
9
9
0
Figure 2. Structures of A-2 (2) and cefiderocol (3, S-649266), and a condeptual SAR plan that led to
3.
91

ACCEPTED MANUSCRIPT
9
9
9
9
9
9
9
9
2
3
4
5
6
7
8
9
2. Chemistry
Figure 3 outlines the two synthetic pathways of novel siderophore cephalosporins. An
aminothiazolylacetic acid derivative as a C-7 side chain was coupled with a known cephalosporin
nucleus ACLE·HCl (7-amino-3-chloromethyl-3-cephem-4-carboxylate p-methoxybenzyl ester
hydrochloride) or ACLH·HCl (7-amino-3-chloromethyl-3-cephem-4-carboxylate benzhydryl ester
hydrochloride) to introduce a C-7 side chain, followed by quaternization with a tertiary amine as a
C-3 side chain to give a precursor of a new cephalosporin. Removal of all protective groups with
Lewis acid or Brønsted acid afforded a novel cephalosporin derivative (Method A). A C-3 side chain
was introduced to a C-7 protected cephalosporin, then a C-7 amino protecting group was selectively
cleaved, followed by coupling with a C-7 side chain then removal of all the protecting groups
produced a novel cephalosporin derivative (Method B). The details for the synthesis of all
intermediates can be found in Supplementary Information.
1
1
1
1
1
00
01
02
03
04
1
1
05
06
Figure 3. Generalized synthetic approaches toward a novel siderophore cephalosporin.
107
108
109
110
111
Scheme 1 shows the synthesis of the catechol moiety. The oxidation under Pinnick oxidation
condition of the benzaldehyde 4 provided the corresponding benzoic acid 5. After cleavage of the
two methyl ethers with boron tribromide, the resulting benzoic acid analogue was esterified in acidic
methanol to give the methyl ester 6. Protection of the dihydroxy group and hydrolysis of the methyl

ACCEPTED MANUSCRIPT
112
113
114
ester 7 produced the key benzoic acid 8.
a
Scheme 1. Synthesis of the catechol 8
115
116
117
118
119
120
121
122
As shown in Scheme 2 and 3, amidation of benzoic acid 8 and the other benzoic acid derivatives
1
8-21
22-28
(see Supplementary Information)
with several known amines
was achieved by using a
condensation reagent or methane sulfonyl chloride to give the amide type C-3 side chains 9a-s and
11a-n.
a
Scheme 2. Synthesis of the amide type C-3 side chains 9a-s
1
23
24
a
1
Scheme 3. Synthesis of the amide type C-3 side chains 11a-n

ACCEPTED MANUSCRIPT
1
1
25
26
127
128
129
130
131
132
133
134
135
136
137
The synthesis of the analogues (12, 14 and 18) with the modified linker moiety such as a urea, a
flipped amide and a 1,3,4-Oxadiazole is depicted in Scheme 4. Compound 8 was treated with
diphenylphosphoryl azide to produce the corresponding isocyanate in situ which was trapped by the
amine to the urea analogue 12. The isocyanate was also converted to the corresponding amine 13
with (9H-fluoren-9-yl)methanol and piperidine. The amidation of 13 provided the flipped amide
analogue 14. Compound 8 was converted to the hydrazide 15, which was treated with chloroacethyl
chloride to give the compound 16. The treatment of the compound 16 with Burgess reagent
furnished the desired 1,3,4-oxadiazole 17 followed by alkylation with pyrrolidine to give the
oxadiazole analogue 18.
a
Scheme 4. Synthesis of the compound 12, 14 and 18

ACCEPTED MANUSCRIPT
138
139
140
141
142
143
144
The preparation of C-7 side chain was depicted in Scheme 5. Condensation of several alkoxyamines
(see Supplementary Information for the synthesis) with the keto acid 19a-c provided the oxime acids
2
2,29-32
20a-j, 21 and 22.
a
Scheme 5. Synthesis of the C-7 side chains 20a-j, 21 and 22
1
45
46
1

ACCEPTED MANUSCRIPT
147
148
149
150
151
152
Scheme 6 summarizes the synthesis of the cephalosporin nucleus (25a-d, 26a-d, 27a and 27b).
Compound 23a (ACLE·HCl) or 23b (ACLH·HCl) was condensed with the C-7 side chain analogues
described above (20a-e, 20i, 20j, 21 and 22) to afford the intermediate compound 24, which was
29
oxidized with m-CPBA to give the intermediate sulfoxide 25a-d, 26a-d, 27a and 27b.
a
Scheme 6. Synthesis of the cephalosporin nucleus
1
53
54
1
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
Scheme 7 shows the synthesis of the novel siderophore cephalosporins. After halogen exchange
from chloride of the cephalosporin nucleus (25a-d, 26a-d, 27a and 27b) to the corresponding iodide,
the quaternization between the resulting iodide and the C-3 side chain followed by the reduction of
the 1-sulfoxide gave the quaternary ammonium salts, which were treated with Lewis acid such as
aluminum trichloride in the presence of anisole to produce the new siderophore cephalosporins (2, 3,
28a-r, 29a-p, 29r, 30a-d, 30h, 30i, and 31a-d; see Table 1, 2 ,3 and 4 for the structure).
We were unable to quaternize the hydroxypyriodone analogue 11m with 25a or 26a. This might
be attributed to the acidity of the hydroxyl group of the 4-hydroxy pyridone moiety. Therefore, the
quaternary ammonium salt 28o possessing the terminal amine was coupled with the benzoic acid
analogue 32, followed by deprotection produced compound 29q. Some C-7 side chains (20f, 20g,
and 20h) were introduced to the cephalosporin nucleus after the quaternization step. ACLH·HCl
(23b) was treated with trityl chloride to produce the compound 33, which was oxidized to give the
compound 34. After the quaternization between compound 34 and the amine 9b, the reduction of the
1-sulfoxide, followed by deprotection of trityl group gave the compound 35, which was coupled with
the C-7 side chain (20f-h), then the removal of the protecting groups gave the analogues with the

ACCEPTED MANUSCRIPT
170
171
172
173
modified C-7 side chain 30e-g.
a
Scheme 7. Synthesis of the new siderophore cephalosporins
Metod A
S
S
N
X
O
X
O
O
S
H N
H
BocHN
H
2
N
S
N
N
a
b
N
N
Q
R6
N
N
Cl
Y
O
O
O
O
COO-
R4
COOR5
R4
2
2
2
5a-d
6a-d
7a, 27b
2, 3
28a-r
29a-p, 29r
Metod B
30a-d, 30h, 30i, 31a-d
S
S
O
O
H2N
H
N
O
OH
H2N
H
S
N
S
N
c, d
N
O
OH
N
N
+
HO
N
N
N
N
N
N
NH2
O
O
N
H
O
O
OPMB
COO
COO
OH
COOH
O
COOH
O
28m
32
29q
Metod C
O
S
HCl H2N
S
TrHN
S
TrHN
e
f
g, h, i
N
Cl
N
Cl
N
Cl
O
O
O
COOBH
COOBH
33
COOBH
34
23b
S
O
H2N
H
HCl H2N
S
N
S
N
O
Cl
j, k
O
Cl
I-
N
N
OPMB
OPMB
N
N
N
OH
OH
N
N
O
O
O
H
COO-
0e-g
H
COOBH
R4
3
35
aReagents and conditions: (a) (i) NaI, DMA, 10°C; (ii) C-3 side chain, DMA, 10°C, (iii) AcCl, KI, DMF, 0°C or
PBr , DMF, -40°C; (b) 2 mol/L AlCl /CH NO , anisole, CH Cl , -30°C; (c) (i) 32, PCl , Et N, THF, 15°C; (ii)
3
3
3
2
2
2
5
3
2
8m, Et N, 0°C; (d) TFA, anisole, CH Cl , 0°C; (e) TrCl, 2,6-lutidine, CH Cl , rt; (f) m-CPBA, CH Cl , 0°C; (g)
3 2 2 2 2 2 2
(i) NaI, DMA, rt; (ii) 9b, DMA, rt; (h) AcCl, KI, DMF, 0°C; (i) aq. 6 mol/L HCl, acetone, rt; (j) 20f-h, EDC·HCl,
pyridine, 10°C; (k) 2 mol/L AlCl /CH NO , anisole, CH Cl , -30°C.
1
1
74
75
3
3
2
2
2
176
177
178
179
180
181
182
183
184
185
3. Results and discussion
The minimum inhibitory concentrations (MICs) of the new siderophore cephalosporins against drug
resistant Gram-negative pathogens were determined by broth microdilution method in
cation-adjusted Mueller-Hinton broth supplemented with 20 µM human apo-transferrin to mimic the
3+
33,34
iron (Fe ) deficiency condition present in acute infections.
The MIC values (µg/mL) of cefepime
and meropenem were determined by broth microdilution method according to the Clinical and
Laboratory Standards Institute. The tested strains include both metallo-type carbapenemase
producing strains of P. aeruginosa (IMP-1 or VIM-2) and S. maltophilia (L-1) and serine-type
carbapenemase producing strains of A. baumannii (OXA-23 and OXA-58) and K. pneumoniae
(KPC-2) as well as extended-spectrum β-lactamase (ESBL) producing E. coli (SHV-4) (see footnote

ACCEPTED MANUSCRIPT
1
-3
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
in Table 1).
Meropenem, which is a carbapenem, exihbited low activities against these
carbapenemase producing strains (see Table 1 for the activites).
As shown in Table 1, compound 2 (A-2) showed potent activities against tested Gram-negative
pathogens except for IMP-1 producing P. aeruginosa (SR27001 and SR27060) and SHV-4
producing Escherichia coli. The difference between the MIC value against P. aeruginosa SR27060
and that against IMP-1 deficient P. aeruginosa 27060∆R1 implies stability against IMP-1. The MIC
difference of A-2 (P. aeruginosa SR27060/27060∆R1) was 32-fold higher and suggests A-2 was not
stable against IMP-1. Therefore, several modifications exemplified by the replacement of the
piperazine moiety of compound 2 to other quaternary ammoniums were achieved to increase the
activities against not only P. aeruginosa SR27060 but also other Gram-negative pathogens.
Compound 3 (cefiderocol) having a pyrrolidine group at the C-3 position exhibited good balanced
and potent activities against the strains tested and the 2-fold difference was observed between the
MIC value against P. aeruginosa SR27060 and that against P. aeruginosa 27060∆R1. The
introduction of methyl group on the methylene moiety (28a) and the homologation of the methylene
moiety (28b) provided no additional benefit compared to compound 3, while dimethyl ammonium
analogue 28c that did not contain a cyclic amine displayed significantly loss of activities against
AmpC, IMP-1, L-1 or KPC-2 producing strains except for susceptible P. aeruginosa and OXA
producing A. baumannii. The 6-membered ring analogue 28d showed the comparable activities
against the tested strains compared to compound 3. The other dimethyl ammonium analogue (28e
and 28f) did not exhibit the improved antibacterial activities. Compound 3 and 28d showed a 2-fold
difference between the MIC value against P. aeruginosa SR27060 and that against P. aeruginosa
27060∆R1. The replacement of the pyrrolidine moiety (3) with the bicyclic amine (28h-l) did not
lead to notably improved antibacterial activities. Some antibiotics such as ceftazidime and cefpirome
3
5-38
contain a quaternary pyridinium moiety at the C-3 position.
Subsequently, we evaluated the
effect of the introduction of a catechol group on the pyridinium moiety. The activities of the
compound 28n-r were comparable or inferior to that of compound 3 having the sterically more
bulky ammonium moiety as the C-3 side chain. In addition, the biaryl analogue 28p lacking of an
amide moiety showed significantly decreased activities against IMP-1 or L-1 producing strains. As
we anticipated, the removal of the catechol moiety (28m) led to dramatically decreased activities.
The compound 3 obviously exhibited more potent activities than meropenem against the tested
strains execpt for susceptible P. aeruginosa and SHV-4 producing E. coli. We next turned our
attention to replacement of the amide moiety as the linker and the catechol moiety.
Table 1. In vitro antibacterial activity of the analogues modified with the quaternary ammonium
moiety against Gram-negative pathogens.

ACCEPTED MANUSCRIPT
222
a
MIC (µ g/mL)
P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa S. maltophilia A. baumannii K. pneumoniae
E. coli
compound
SR-24
0.125
SR-24-12
0.125
SR27001
4
SBRKM-28
N.T.
SR27060
8
27060∆R1
SR21970
1
SR27323
1
ATCC BAA-1705 ATCC BAA-200
2
0.25
0.125
0.063
N.T.
N.T.
0.5
32
1
3
0.25
0.25
0.5
0.5
0.25
0.5
4
1
1
0.125
0.125
0.125
0.25
0.125
0.25
0.5
2
2
1
0.25
0.25
2
0.5
0.25
0.125
>32
0.25
1
1
N.T.
N.T.
0.5
1
2
8a
8b
8c
N.T.
N.T.
>32
0.5
2
2
1
1
2
0.063
0.25
0.25
0.5
>32
1
32
2
2
2
8d
8e
0.25
0.5
1
1
0.031
0.25
0.125
0.063
N.T.
0.063
0.063
N.T.
N.T.
8
2
2
1
1
2
8f
8g
8h
4
4
1
1
2
2
2
2
0.25
0.25
0.25
0.25
0.25
0.25
2
0.5
0.25
0.5
0.5
0.25
0.5
8
2
0.125
0.125
0.25
0.063
0.125
0.25
N.T
4
1
0.25
0.125
0.5
1
1
2
1
4
1
N.T.
2
N.T.
2
2
8i
8j
0.5
0.5
4
4
1
2
1
0.5
0.5
2
1
2
2
8k
4
0.5
0.5
>32
1
N.T.
N.T.
8
N.T.
N.T.
16
2
8l
2
4
2
8m
32
2
16
2
4
2
2
2
2
8n
8o
8p
8q
0.063
N.T.
N.T.
0.25
0.25
0.5
0.25
N.T.
N.T.
2
0.125
0.125
0.25
N.T
0.5
N.T.
N.T.
4
2
0.25
N.T.
N.T.
0.25
0.125
32
2
4
4
0.5
1
N.T.
N.T.
0.5
0.5
16
N.T.
N.T.
4
>64
>32
16
>64
>64
16
>32
16
>64
>64
>32
>16
64
2
8r
0.5
16
0.5
0.5
16
16
1
FEP
16
4
MEM
≤0.063
0.125
64
>64
16
16
≤0.063
a
P. aeruginosa SR24: susceptible strain; P. aeruginosa SR24-12: AmpC overproducing strain; P. aeruginosa SR27001: IMP-1 producing strain (MDRP); P. aeruginosa
SBRKM-28: VIM-2 producing strain; P. aeruginosa SR27060: IMP-1 producing strain; P. aeruginosa 27060∆R1: deletion of R1 plasmid; S. maltophilia SR21970: L-1
producing strain; A. baumannii SR27323: OXA-23 and OXA-58 producing strain; K. pneumoniae ATCC BAA-1705: KPC-2 producing strain; E. coli ATCC BAA-200: SHV-4
producing strain; FEP: cefepime; MEM: meropenem.
2
2
23
24
Tabled 2 summarizes the antibacterial activities of the analogues modified with the linker moieties
(29a-d) and the catechol moieties (29e-r). The urea analogue (29a) displayed slightly decreased
activities. The replacement of secondary amide of the compoound 3 with a flipped amide (29b),
tertiary amide (29c) or 1,3,4-oxadiazole (29d) led to no benefit of activities against the starins tested.
These resuls implied that the electron withdrawing group attached to the benzen ring was important
for the antibacterial potency of the compound.
225
226
227
228
229
230
3
+
As is mentioned above, the catechol moiety plays an important role of chelating an iron (Fe ). Thus,

ACCEPTED MANUSCRIPT
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
we anticipated the antibacterial activities should change depending on the substitution pattern of the
catechol moiety at the C-3 side chain. Compound 29e-g showed potent activities against A.
baumannii and AmpC producing P. aeruginosa. The dichloro analogue 29h exhibited slightly
improved activities against most of strains tested. In comparison with the analogues possessing a
halogenated catechol moiety, the non-substituted catechol analogue 29i exhibited significantly
decreased activity against OXA producing A. baumannii and 29j exhibited slightly decreased
activities. The replacement of the chloro group of 3 with a methyl group (29k) or hydroxyl group
(29l) resulted in slightly decreased antibacterial activities. The activities of the catechol analogues
with the electron withdrawing group (29e-h) were superior to that of catechol analogues with the
electron donating group (29k, 29l, and 29m). The methyl ether analogue (29n) lacking one of the
hydroxyl groups of the catechol moiety exhibited significantly decreased antibacterial activities
against all tested strains.
Previous research reported that a 4-pyridone conjugated compound could also play a role of a
siderophore resulting in an increase of antibacterial activities against Gram-negative pathogens. To
1
3-16,39-41
explore the 4-pyridone conjugated cephalosporins, compounds 29o-r were prepared.
The
4-pyridone and the hydroxyl pyridone analogues (29o and 29q) exhibited 4-fold decrease in activity
against P. aeruginosa SR27001. The introduction of a chloride to the pyridone moiety to potentially
increase the activity led to significantly decreased potency against the all tested strains. The
2-pyridone analogue (29r) also displayed significantly decreased potency. Attempts to increase
activities replacing a catechol moiety with a 4-pyridone moiety were also unsuccessful. The
pyrrolidine analogues amidated with a halogenated catechol as the C-3 side chain (3, 29e-h)
displayed the most potent and broad-spectrum antibacterial activities.
Our chemical modification of the C-3 side chain revealed that the presence of a cyclic quaternary
ammonium moiety was important to produce potent activities against AmpC and carbapenemase,
such as IMP-1, L-1 or KPC-2, producing strains (3 vs 28c) and the substitution of a halogen on a
catechol moiety led to the increased activities against OXA producing A. baumannii (3 vs 29i).
Table 2. In vitro antibacterial activity of the analogues modified with the linker moiety and the
analogues modified with the catechol moiety against Gram-negative pathogens.

ACCEPTED MANUSCRIPT
260
a
MIC (µ g/mL)
P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa S. maltophilia A. baumannii K. pneumoniae
E. coli
compound
SR-24
0.25
SR-24-12
0.5
SR27001
1
SBRKM-28
0.125
SR27060
2
27060∆R1
SR21970
0.5
SR27323
1
ATCC BAA-1705 ATCC BAA-200
3
1
0.063
N.T.
0.25
N.T.
N.T.
≤0.031
≤0.031
≤0.031
0.031
0.5
1
N.T.
2
2
9a
9b
9c
1
0.25
0.5
1
0.5
0.5
0.5
0.125
0.125
0.125
0.25
1
4
2
0.5
0.25
0.125
0.125
N.T.
N.T.
N.T.
0.063
0.25
N.T.
N.T.
N.T.
N.T.
16
8
2
2
1
4
1
N.T.
2
2
2
1
1
0.5
0.5
0.25
1
0.25
0.5
0.25
0.25
0.25
0.125
2
N.T.
N.T.
0.25
1
N.T.
N.T.
0.5
2
2
2
9d
9e
0.5
2
2
0.063
0.063
0.125
0.125
0.125
0.063
0.5
1
1
2
9f
9g
9h
2
2
2
2
2
1
1
0.5
0.25
16
0.5
0.5
2
0.5
2
2
0.5
2
2
9i
9j
2
2
0.25
1
4
8
0.5
2
1
1
0.063
0.25
1
2
2
9k
8
8
1
8
8
2
9l
1
2
32
4
16
4
4
8
32
32
8
2
9m
0.125
2
0.125
8
2
2
1
0.25
4
2
2
2
2
9n
9o
9p
9q
32
4
32
4
32
1
8
>32
1
16
1
0.25
2
0.25
4
0.25
4
0.25
4
0.063
2
32
4
>32
4
8
32
16
1
0.125
2
0.5
2
0.063
N.T.
16
1
0.125
4
0.5
32
0.063
2
2
9r
16
>64
>64
8
4
8
FEP
0.5
16
>64
>64
16
16
64
16
32
4
MEM
≤0.063
0.125
64
>64
16
16
≤0.063
a
see footnote in Table 1.
261
262
263
264
265
266
267
268
269
270
As is shown in Table 3, attempts to increase the antibacterial activities by modifying the cis-oxime
substituent of gem-dimethyl group or replacement of the amino thiazole moiety were achieved.
Compound 30a-c lacking one or two methyl groups of the gem-dimethyl group at the C-7 side chain
showed lower activities. The compounds having a substituent at the α-position of the carboxylic acid
of the oxime moiety (3, 30b-e) showed potent activities compared to the compounds 30a and 30g.
The activity of the compound 30f against P. aeruginosa SR27060 was 16-fold decreased compared
to that of compound 3. The activity of the thiadiazole analogue 30h were comparable to that of
compound 3 and the chloro substituted aminothiazole analogue 30i showed the 4-fold decrease of
potency against L-1 producing S. maltophilia. The C-7 side chain analogues with the modified

ACCEPTED MANUSCRIPT
271
272
273
274
275
oxime moiety (30a-i) displayed no significant improvement of the activities against the strains
tested.
Table 3. In vitro antibacterial activity of the analogues modified with the C-7 side chain catechol
moiety against Gram-negative pathogens.
276
a
MIC (µ g/mL)
P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa S. maltophilia A. baumannii K. pneumoniae
E. coli
compound
SR-24
0.25
SR-24-12
0.5
SR27001
1
SBRKM-28
0.125
SR27060
2
27060∆R1
SR21970
0.5
SR27323
1
ATCC BAA-1705 ATCC BAA-200
3
1
0.063
0.25
0.063
0.063
0.063
0.031
N.T.
2
1
32
3
0a
0b
0c
0.125
0.25
0.25
0.25
0.25
N.T.
0.25
0.25
0.25
0.5
0.5
0.25
0.5
2
1
0.125
0.125
0.125
0.063
0.125
0.5
4
2
2
1
1
0.5
0.5
0.25
0.25
2
0.5
1
3
4
3
1
2
1
1
1
3
3
0d
0e
0.25
0.25
N.T.
0.5
1
1
0.5
0.5
N.T.
2
1
1
0.5
4
4
0.25
N.T.
0.5
N.T.
2
0.5
N.T.
16
3
0f
0g
0h
32
8
3
3
1
0.125
0.25
0.125
16
2
0.25
0.5
1
1
0.25
1
0.25
2
N.T.
0.063
32
N.T.
1
3
0i
1
1
FEP
16
>64
>64
>64
>64
16
16
64
16
4
MEM
≤0.063
0.125
64
>64
16
16
≤0.063
a
see footnote in Table 1.
2
77
78
2
Finally, we investigated the effect of the introduction of the catechol moiety to the C-7 side chain
instead of the C-3 side chain (Table 4). The analogues bearing the catechol moiety at the C-7 side
chain (31b and 31d) showed more potent activities against IMP-1 producing P. aeruginosa SR27001
than compound 3. The terminal amino group at the C-3 side chain was important to perform potent
activities (31a vs 31b, 31c vs 31d) against AmpC or IMP-1 producing P. aeruginosa. However, the
activities of the compound 31b and 31d against other pathogens such as SHV-4 producing E. coli
and L-1 producing S. maltophilia were significantly decreased. As a result, compounds having a
catechol at the C-3 side chain exhibited more balanced activities compared to compounds having a
catechol moiety at the C-7 side chain.
279
280
281
282
283
284
285
286
287
288
Table 4. In vitro antibacterial activities of the analogues having a catechol conjugated C-7 side chain

ACCEPTED MANUSCRIPT
289
a
MIC (µ g/mL)
P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa S. maltophilia A. baumannii K. pneumoniae
E. coli
compound
SR-24
0.25
SR-24-12
0.5
SR27001
1
SBRKM-28
0.125
SR27060
2
27060∆R1
SR21970
0.5
SR27323
1
ATCC BAA-1705 ATCC BAA-200
3
1
0.063
N.T.
0.25
0.25
0.125
32
1
N.T.
>32
>32
8
3
1a
1b
1c
1d
0.125
0.125
0.125
0.125
0.5
1
4
0.125
N.T.
N.T.
N.T.
16
4
2
0.063
0.5
8
N.T.
4
3
0.25
0.5
0.25
8
0.125
4
>32
16
3
1
3
0.063
16
0.5
>64
>64
0.125
>64
>64
0.063
16
16
0.5
16
16
FEP
64
4
MEM
≤0.063
0.125
64
16
>64
16
≤0.063
a
see footnote in Table 1.
2
2
90
91
3
+
Ito et al. reported the chelating activity of cefiderocol (3) with iron (Fe ) determined by color
4
2,43
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
change of chrome azurol B (CAB) in the presence of cetyltrimethylammonium bromide
and they
concluded that cefiderocol (3) utilized the iron transport system of P. aeruginosa by the binding of
the catechol moiety with extracellular iron. However, the molecular stoichiometry how cefiderocol
3
+
42
(3) chelates with iron (Fe ) was not revealed. We evaluated the chelating activity of the catechol
3
+
analogues (3, 29h-l and 29o) with iron (Fe ) to investigate the relationship between the chelating
activity and the antibacterial activities of the siderophore cephalosporin. Figure 4 and Figure 5
3
+
summarize the iron (Fe ) chelating activity of the catechol analogues including cefiderocol (3) and
3
+
the pKa values calculated by ACD/Percepta. The chelating activity with iron (Fe ) decreased the
following order: 29h > 29l and 29o > 29j and 3 > 29i and 29k. The chelating activity with iron
3+
(Fe ) increased with decreasing calculated pKa values between 29i (pKa = 8.1 ± 0.8), 29k (pKa =
8.1 ± 0.8), 3 (pKa = 7.2 ± 0.9), and 29h (pKa = 5.9 ± 0.9). However, this correlation between the
chelating activity and the pKa value was not observed between the catechol analogue 29j (pKa = 7.8
± 0.5), the pyrogallol analogue 29l (pKa = 7.6 ± 0.9) and the 4-pyridone analogue 29o (pKa = 8.0 ±
0.9). We anticipated that the antibacterial activity of a siderophore cephalosporin would increase
with increasing iron chelating activity due to more effective utilization of the bacterial iron
acquisition system. However, the chelating activity of the tested compounds (3, 29h-l and 29o) with
3
+
iron (Fe ) did not correlate with the antibacterial activities against the susceptible strain P.
aeruginosa SR-24 or other β-lactamase producing Gram-negative pathogens (see Table 2 for the
activities). This result implies that the process of outer membrane penetration of siderophore molecule is
not simple, and other factors impeding the siderophore molecular recognition by siderophore receptors
might be present in other than direct chelating activity of the compounds for determining the process.
Otherwise, there might be a difference in affinity to penicillin-binding proteins, β-lactam target enzymes,
among the series of compounds. To better understand the siderophore conjugate strategy for antibiotics,
further investigations such as comparison of iron-binding stoichiometry of the compounds as well as
pharmacological evaluations are needed.

ACCEPTED MANUSCRIPT
0
.4
.35
.3
.25
.2
.15
.1
0
0
0
0
0
3
2
2
2
9h
9i
0
9j
0
29k
2
2
9l
.05
0
9o
0
.1
1
10
100
1000
Concentration (µmol/L)
317
318
319
320
Figure 4. Chelating activity of cefiderocol (3) and the analogues modified with the catechol moiety
3
+
with iron (Fe ).
S
N
O
H2N
H
N
S
O
N
N
N
N
R6
O
O
H
COO
COOH
pKa = 7.8 ± 0.5
OH
R6=
Cl
Cl
OH pKa = 7.2 ± 0.9
OH pKa = 5.9 ± 0.9
OH pKa = 9.6 ± 0.9
OH pKa = 8.1 ± 0.8
OH pKa = 11.9 ± 1.0
OH pKa = 11.8 ± 0.5
OH pKa = 10.3 ± 1.0
Cl
29h
3
29i
29j
pKa=7.6±0.9
OH
H
N
Me
OH pKa = 8.1 ± 0.8
OH pKa = 11.9 ± 1.0
OH pKa = 15.0 ± 1.2
OH pKa = 8.0 ± 0.9
OH pKa = 10.0 ± 0.9
O
2
9k
29l
29o
3
3
21
22
Figure 5. Calculated pKa values of cefiderocol (3) and the analogues modified with the catechol
323
324
325
326
327
328
329
330
moiety.
Some compounds with the modified C-3 side chain and C-7 side chain were selected and evaluated
in vivo efficacy against multi-drug resistant P. aeruginosa SR27001 using a murine systemic
4
4
infection model. As shown in Table 5, compound 3 (cefiderocol) showed potent in vivo efficacy
(ED50 = 5.48 mg/kg) against MDR P. aeruginosa. The analogues with the modified quaternary
ammonium moiety (28e, 28g and 28j) exhibited comparable or slightly decreased in vivo efficacy
(ED50 = 5.48, 9.42 and 10.2 mg/kg, respectively). For analogues with the modified catechol moiety,

ACCEPTED MANUSCRIPT
331
332
333
334
335
336
337
29e, 29f, and 29g, which included halogenated catechol moiety, they exhibited slightly decreased or
comparable efficacy. In comparison with compound 3, the pyridone analogue 29o exhibited slightly
decreased in vivo efficacy. In addition, the 4-pyridone analogue 29q did not exhibit the clear
correlation between in vitro and in vivo efficacy. Compounds 30a and 30c with the modified C-7
side chain exhibited slightly decreased or comparable efficacy compared to that of compound 3.
a
Table 5. In vivo efficacy of selected compounds .
compound
3
28e
28g
28j
29e
29f
MIC (µ g/mL)
1
2
2
0.5
1
2
P.aeruginosa SR27001
ED50 (mg/kg)
5.48
5.48
9.42
10.5
10.2
10.2
compound
29g
29o
29q
30a
30c
FEP
MIC (µ g/mL)
2
4
4
2
1
>64
P.aeruginosa SR27001
ED50 (mg/kg)
5.48
10.2
>30
9.84
5.48
>100
a
The in vivo potency was determined by using a systemic mouse infection model. The mice (Jcl:ICR, male 5-weeek-old) were injected
4
intraperitoneally with ca .5×10 CFU of bacterial suspension. The compounds were administered intravenously 1 and 5 h after infection.
Survival rate was recorded over 7 days to estimate the 50% effective dose (ED50) which were determined by the logit method.
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
4. Conclusion
In summary, we have described our structure activity relationship results of the catechol conjugated
siderophore cephalosporin based on the structure of 2. Many compounds modified with the C-3 and
C-7 side chain exhibited potent antibacterial activities against multi-drug resistant Gram-negative
pathogens such as P. aeruginosa and A. baumannii. The presence of a cyclic quaternary ammonium
moiety and a halogenated catechol group at the C-3 position of a siderophore cephalosporin
dramatically affected the in vitro potency against IMP-1, L-1, KPC-2 and OXA producing strains.
The C-3 position was suitable to attach a catechol moiety which provided good balanced of
antibacterial activities. Importantly, the specific markers of carbapenem resistance, namely both
serine and metallo-carbapenemases did not impair the in vitro activity of cefiderocol. We have
4
5
subsequently shown that cefiderocol is stable when exposed in vitro to purified enzyme extracts.
3
+
We also investigated the relationship between the chelating activity with iron (Fe ), the calculated
pKa value and the antibacterial activities, however obvious correlations were not identified among
those factors. Cefiderocol (3) was one of the best molecules which displayed well-balanced and
potent activities against multi-drug resistant Gram-negative pathogens including carbapenemase
producing strains. We selected cefiderocol (3) as a candidate because cefiderocol displayed not only
potent in vivo efficacy also satisfactory safety and physicochemical properties. These results indicate
that cefiderocol (3) is a highly promising parenteral cephalosporin for the treatment of
multidrug-resistant including carbapenem resistant Gram-negative infection. Cefiderocol (3) is now
4
6
in late clinical development.

ACCEPTED MANUSCRIPT
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
5. Experimental
5.1 Chemistry
Reagents and solvents were purchased from commercial sources and used without purification.
Reactions were carried out under a nitrogen atmosphere with anhydrous solvents. Reaction mixtures
were monitored using thin-layer chromatography (TLC) on silica gel F254 TLC plates. Column
chromatography was carried out using silica gel. Reverse phase column chromatography was carried
1
out using HP20SS and octadecylsilyl Silica Gel (Yamazen ULTRA PACK). H-NMR spectra were
recorded on a Varian Gemini-300 spectrometer (300 MHz) or Bruker AV400 spectrometer (400
MHz). Chemical shifts (δ) are reported in parts per million (ppm) from trimethylsilane (in CDCl3
and DMSO-d ) or 2,2-dimethyl-2-silapentane-5-sulfonate (DSS in D O) as internal standard.
6
2
Low-resolution mass spectra were collected on a Waters ZQ mass detector.
5.1.1 2-chloro-3,4-dimethoxybenzoic acid (5). To a solution of 4 (20.06 g, 100 mmol) in 1,4-dioxane
(300 mL) were added a solution of sulfamic acid (21.36 g, 220 mmol) in water (200 mL) and
NaClO (19.9 g, 220 mmol) in water (100mL) at 0°C. After stirring for 30 min at 0°C, the mixture
2
was quenched by a solution of sodium bisulfite (45.8 g, 440 mmol) in water (200 mL). The aqueous
layer was extracted with EtOAc and the combined extracts were washed with water and brine, dried
1
over anhydrous MgSO , filtered, and concentrated in vacuo to afford 5 (20.74 g, 96%). H-NMR
4
(DMSO-d ) δ: 13.04 (br s, 1H), 7.63 (d, J = 8.9 Hz, 1H), 7.11 (d, J = 8.9 Hz, 1H), 3.89 (s, 3H), 3.74
6
(s, 3H).
5.1.2 methyl 2-chloro-3,4-dihydroxybenzoate (6). To a solution of 5 (28.1 g, 130 mmol) in CH Cl2
2
(180 mL) was added dropwise BBr (30.7 mL, 324 mmol) at 0°C. After stirring for 2 h at room
3
temperature, the resulting mixture was poured into MeOH (270 mL) slowly. After stirring for 2 days
at room temperature, to this mixture was added ice water then MeOH was evaporated in vacuo. The
resulting precipitated solid was collected by filtration, washed with water and dried in vacuo to give
1
6 (25.41 g, 97%). H-NMR (DMSO-d ) δ: 10.50 (br s, 1H), 9.39 (br s, 1H), 7.22 (d, J = 8.5 Hz, 1H),
6
6.78 (d, J = 8.5 Hz, 1H), 3.76 (s, 3H).
5.1.3 2-Chloro-3,4-bis[(4-methoxybenzyl)oxy]benzoic acid (8). To a solution of 6 (25.3 g, 125
mmol) in DMF (180 mL) were added K CO (43.1 g, 312 mmol), NaI (18.72 g, 125 mmol) and
2
3
PMBCl (39.1 mL, 287 mmol). After stirring for 3.5 h at 50°C, the resulting mixture was poured into
a mixture of H O (500 mL) and IPE (200mL). The resulting precipitated solid was collected by
2
filtration and dissolved in THF (230 mL) and MeOH (180 mL). To this mixture was added aq. 2
mol/L NaOH (125 mL, 250 mmol) and the resulting mixture was stirred at reflux for 1.5 h. After

ACCEPTED MANUSCRIPT
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
evaporation of the solvent in vacuo, IPE and aq. 2 mol/L HCl (135 mL, 270 mmol) were added to
the residue. The resulting precipitated solid was collected by filtration to give 8 (51.12 g, 95%).
1
H-NMR (DMSO-d ) δ: 7.59 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 8.7 Hz, 2H),
6
7.22 (d, J = 8.8 Hz, 1H), 6.98 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.5 Hz, 2H), 5.17 (s, 2H), 4.87 (s, 2H),
3.78 (s, 3H), 3.74 (s, 3H).
5.2 General procedure for the synthesis of 9a-s and 11a-n.
[Method A] To a solution of 1.0 equivalent of the acid derivative and 1.1 equivalent of amine in
DMF were added 1.1 equivalent of HOBt and EDC·HCl, and stirred at room temperature for 1 h.
The solvent was evaporated in vacuo, the resulting residue was diluted with a mixture of aq. 2 mol/L
NaOH and EtOAc. The organic layer was separated and the aqueous layer was extracted with EtOAc
and the combined extracts were washed with water and brine, dried over anhydrous MgSO , filtered,
4
and concentrated in vacuo. The resulting residue was subjected to silica gel chromatography, eluting
with EtOAc (containing 5% of Et N)-MeOH. The eluted fractions containing the desired compound
3
were concentrated in vacuo to afford a target amide derivative.
[Method B] To a solution of 1.0 equivalent of the acid derivative in DMF were added 1.1 equivalent
of Et N. To this solution was added 1.1 equivalent of MsCl at one portion at -10°C and stirred for 30
3
min. To this resulting mixture were added 1.1 equivalent of Et N and 1.1 equivalent of amine, and
3
stirred for 1 h at 0°C. The solvent was evaporated in vacuo, the resulting residue was diluted with a
mixture of aq. 2 mol/L NaOH and EtOAc. The organic layer was separated and the aqueous layer
was extracted with EtOAc and the combined extracts were washed with water and brine, dried over
anhydrous MgSO , filtered, and concentrated in vacuo. The resulting residue was subjected to silica
4
gel chromatography, eluting with EtOAc (containing 5% of Et N)-MeOH. The eluted fractions
3
containing the desired compound were concentrated in vacuo to afford a target amide derivative.
5.2.1
(2-chloro-3,4-bis((4-methoxybenzyl)oxy)phenyl)(4-methylpiperazin-1-yl)methanone
(9a).
1
7
Compound 9a was prepared according to the known procedure.
5.2.2 2-chloro-3,4-bis((4-methoxybenzyl)oxy)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (9b). [Method
1
B] 89% yield. H-NMR (CDCl ) δ: 7.43 (d, J = 8.7 Hz, 1H), 7.35 (d, J = 8.5 Hz, 4H), 6.94-6.90 (m,
3
4H), 6.83 (d, J = 8.7 Hz, 2H), 5.08 (s, 2H), 4.95 (s, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.59-3.52 (m, 2H),
2.70 (t, J = 6.1 Hz, 2H), 2.58-2.51 (m, 4H), 1.81-1.74 (m, 4H).
5.2.3 (S)-2-chloro-3,4-bis((4-methoxybenzyl)oxy)-N-(1-(pyrrolidin-1-yl)propan-2-yl)benzamide (9c).
1
[Method A] 83% yield. H-NMR (CDCl ) δ: 7.50-7.25 (m, 4H), 7.00-6.80 (m, 6H), 5.07 (s, 2H),
3

ACCEPTED MANUSCRIPT
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
4.94 (s, 2H), 4.25-4.10 (m, 1H), 3.83 (s, 3H), 3.80 (s, 3H), 2.80-2.40 (m, 6H), 1.80-1.60 (m, 4H),
1.32 (d, J = 6.3 Hz, 3H).
5.2.4 2-chloro-3,4-bis((4-methoxybenzyl)oxy)-N-(3-(pyrrolidin-1-yl)propyl)benzamide (9d). [Method
1
B] 69% yield. H-NMR (CDCl ) δ: 7.76 (m, 1H), 7.37-7.34 (m, 5H), 6.93-6.90 (m, 3H), 6.84 (d, J =
3
8.4 Hz, 2H), 5.07 (s, 2H), 4.94 (s, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.55 (q, J = 6.0 Hz, 2H), 2.62 (t, J
= 6.3 Hz, 2H), 2.48 (br, 4H), 1.81-1.77 (m, 2H),1.67 (br, 4H).
5.2.5 2-chloro-N-(2-(dimethylamino)ethyl)-3,4-bis((4-methoxybenzyl)oxy)benzamide (9e). [Method
1
A] 76% yield. H-NMR (DMSO-d ) δ: 8.19 (t, J = 5.6 Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.32 (d, J =
6
8.7 Hz, 2H), 7.18 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 6.98 (d, J = 8.7 Hz, 2H), 6.87 (d, J =
8.5 Hz, 2H), 5.15 (s, 2H), 4.87 (s, 2H), 3.77 (s, 3H), 3.75 (s, 3H), 3.31-3.25 (m, 2H), 2.37 (t, J = 7.0
Hz, 2H).
5.2.6 2-chloro-3,4-bis((4-methoxybenzyl)oxy)-N-(2-(piperidin-1-yl)ethyl)benzamide (9f). [Method B]
1
73% yield. H-NMR (CDCl ) δ: 7.49 (d, J = 8.5 Hz, 1H), 7.39-7.31 (m, 4H), 7.13 (br s, 1H),
3
6.97-6.90 (m, 3H), 6.86-6.81 (m, 2H), 5.09 (s, 2H), 4.96 (s, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.53 (dd,
J = 11.0, 5.8 Hz, 2H), 2.53 (t, J = 6.0 Hz, 2H), 2.42 (s, 4H), 1.70-1.41 (m, 6H).
5.2.7
(R)-(2-chloro-3,4-bis((4-methoxybenzyl)oxy)phenyl)(3-(dimethylamino)pyrrolidin-1-yl)methanone
(9g). [Method B] 88% yield. 1H-NMR (CDCl ) δ: 7.36-7.31 (m, 4H), 6.99-6.89 (m, 4H), 6.81 (d, J =
3
6.8 Hz, 2H), 5.06 (s, 2H), 4.97 (s, 2H), 4.16-3.87 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.61-3.51 (m,
1H), 3.38-3.16 (m, 2H), 3.01 (s, 3H), 2.94 (s, 3H), 22.78-2.71 (m, 1H), 1.86-1.69 (m, 2H).
5.2.8 (2-chloro-3,4-bis((4-methoxybenzyl)oxy)phenyl)(4-(dimethylamino)piperidin-1-yl)methanone
(9h). [Method A] 29% yield. 1H-NMR (CDCl ) δ: 7.37-7.29 (4H, m, 4H), 6.98-6.08 (m, 4H), 6.80 (d,
3
J = 7.8 Hz, 2H), 5.06 (s, 2H), 5.00-4.92 (m, 2H), 4.73 (brt, J = 9.2 Hz, 1H), 3.83 (s, 3H), 3.79 (s, 3H),
3.38 (brt, J = 7.8 Hz, 1H), 3.03-2.73 (m, 2H), 2.27 (s, 6H), 2.34-2.22 (m, 1H), 1.93 (brt, J = 7.8 Hz,
1H), 1.74-1.46 (3H, m, 3H).
5.2.9 2-chloro-3,4-bis((4-methoxybenzyl)oxy)-N-(quinuclidin-4-ylmethyl)benzamide (9i). [Method A]
1
90% yield. H-NMR (DMSO-d ) δ: 7.47 (d, J = 8.7 Hz, 1H), 7.36-7.31 (m, 4H), 7.25 (s, 1H),
6
6.96-6.82 (m, 5H), 6.36 (t, J = 6.3 Hz, 1H), 5.09 (s, 2H), 4.95 (s, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.26
(d, J = 6.3 Hz, 2H), 2.95 (t, J = 7.8 Hz, 6H), 1.49 (t, J = 7.8 Hz, 6H).

ACCEPTED MANUSCRIPT
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
5.2.10
(1,4-diazabicyclo[3.2.2]nonan-4-yl)(2-chloro-3,4-bis((4-methoxybenzyl)oxy)phenyl)methanone (9j).
1
[Method A] 51% yield. H-NMR (CDCl ) δ: 7.34 (t, J = 9.3 Hz, 4H), 6.98-6.87 (m, 4H), 6.81 (d, J =
3
8.4 Hz, 2H), 5.07 (s, 2H), 4.99 (dd, J = 26.2, 10.3 Hz, 2H), 4.79 (m, 0.8H), 4.34-4.26 (m, 0.2H), 3.84
(s, 3H), 3.79 (s, 3H), 3.67-2.80 (m, 8H), 2.23-2.00 (m, 2H), 1.86-1.70 (2H, m, 2H).
5.2.11
(S)-(2-chloro-3,4-bis((4-methoxybenzyl)oxy)phenyl)(hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl)meth
1
anone (9k). [Method A] 58% yield. H-NMR (CDCl ) δ: 7.39-7.28 (m, 4H), 7.01-6.89 (m, 4H),
3
6.85-6.76 (m, 2H), 5.06-4.61 (m, 4H), 3.83 (s, 3H), 3.79 (s, 3H), 3.44-2.51 (m, 5H), 2.31-1.41 (m,
8H).
5.2.12
2-chloro-3,4-bis((4-methoxybenzyl)oxy)-N-((1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)benz
1
amide (9l). [Method A] 58% yield. H-NMR (CDCl3) δ: 8.00 (d, J = 4.80 Hz, 1H), 7.42 (d, J = 8.10
Hz, 2H), 7.32 (d, J = 8.40 Hz, 2H), 7.07 (d, J = 9.00 Hz, 1H), 6.97 (d, J = 8.40 Hz, 2H), 6.87 (d, J =
8.10 Hz, 2H), 5.15 (s, 2H), 4.87 (s, 2H), 3.83 (m, 1H), 3.76 (s, 3H), 3.75 (s, 3H), 2.97 (brs, 2H), 2.14
(s, 3H), 2.04-1.91 (m, 5H), 1.68 (d, J = 15.0 Hz, 2H).
5.2.13
(2-chloro-3,4-bis((4-methoxybenzyl)oxy)phenyl)((3aR,6aS)-5-methylhexahydropyrrolo[3,4-c]pyrrol-
1
2(1H)-yl)methanone (9m). [Method B] 52% yield. H-NMR (CDCl ) δ: 7.38-7.30 (m, 4H), 6.98-6.89
3
(m, 4H), 6.83-6.79 (m, 2H), 5.06 (s, 2H), 4.98 (s, 2H), 3.91-3.87 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H),
3.58 (dd, J = 4.3, 12.8 Hz, 1H), 3.37-3.30 (m, 1H), 3.00 (dd, J = 4.4, 11.6 Hz, 1H), 2.94-2.85 (m,
1H), 2.82-2.74 (m, 1H), 2.68 (t, J = 7.2 Hz, 1H), 2.57 (t, J = 7.2 Hz, 1H), 2.42 (dd, J = 4.6, 9.5 Hz,
1H), 2.31 (s, 3H), 2.25 (dd, J = 4.6, 9.5 Hz, 1H).
5.2.14
2-chloro-3,4-bis((4-methoxybenzyl)oxy)-N-(((1S,4S)-2-methyl-2-azabicyclo[2.2.1]heptan-5-yl)methy
1
l)benzamide (9n). [Method A] 76% yield. H-NMR (CDCl ) δ: 7.38-7.31 (m, 4H), 7.00-6.90 (m, 4H),
3
6.82 (d, J = 8.7 Hz, 2H), 5.07 (d, J = 2.0 Hz, 2H), 4.99 (d, J = 3.8 Hz, 2H), 4.87 (s, 0.6H), 3.84 (s,
3H), 3.79 (s, 3H), 3.77-3.25 (m, 3.4H), 3.02-2.93 (m), 1H, 2.81-2.76 (m, 1H), 2.44, 2.41 (s×2, 3H),
1.94-1.78 (m, 2H).
5.2.15 2-chloro-3,4-bis((4-methoxybenzyl)oxy)-N-methyl-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (9o).
1
[Method B] 84% yield. H-NMR (CDCl ) δ: 7.36-7.31 (m, 4H), 6.97-6.89 (m, 4H), 6.84-6.80 (m,
3

ACCEPTED MANUSCRIPT
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
2H), 5.05 (s, 2H), 4.97 (s, 2H), 3.83 (s, 3H), 3.79 (s, 3H), 3.12 (s, 1.5H), 2.81 (s, 1.5H), 2.75 (t, J =
7.2 Hz, 1H), 2.60-2.53 (m, 3H), 2.48-2.30 (m, 2H), 1.80-1.77 (m, 2H), 1.68 (s, 2H).
5.2.16 2-chloro-3,4-bis((4-methoxybenzyl)oxy)-N-(pyridin-4-yl)benzamide (9p). [Method B] 94%
1
yield. H-NMR (DMSO-d ) δ: 10.78 (s, 1H), 8.47 (d, J = 6.3 Hz, 2H), 7.67 (d, J = 6.3 Hz, 2H), 7.45
6
(d, J = 8.7 Hz, 2H), 7.37-7.31 (m, 3H), 7.27 (d, J = 8.7 Hz, 1H), 6.99 (d, J = 8.5 Hz, 2H), 6.89 (d, J
= 8.7 Hz, 2H), 5.21 (s, 2H), 4.92 (s, 2H), 3.78 (s, 3H), 3.75 (s, 3H).
5.2.17
(2-chloro-3,4-bis((4-methoxybenzyl)oxy)phenyl)(3,4-dihydro-1,5-naphthyridin-1(2H)-yl)methanone
1
(9q). [Method B] 30% yield. H-NMR (DMSO-d ) δ: 8.20 (s, 1H), 7.44 (d, J = 8.6 Hz, 2H),
6
7.27-7.17 (m, 4H), 6.98 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 9.1 Hz, 2H), 5.14 (s, 2H), 4.87 (s, 2H), 3.78
(s, 3H), 3.73 (s, 3H), 2.97-2.88 (m, 3H), 1.95 (t, J = 12.0 Hz, 3H). LC/MS (ESI): m/z 545.
5.2.18
2-chloro-N-(imidazo[1,5-a]pyridin-7-yl)-3,4-bis((4-methoxybenzyl)oxy)benzamide
(9r).
1
[Method ] 30% yield. H-NMR (DMSO-d ) δ: 10.45 (s, 1H), 8.27 (s, 1H), 7.48-7.41 (m, 2H),
6
7.35-7.22 (m, 4H), 7.00-6.97 (m, 2H), 6.91-6.86 (m, 2H), 5.20 (s, 2H), 4.92 (s, 2H), 3.78 (s, 3H),
3.75 (s, 3H).
5.2.19
2-chloro-N-(2,3-dihydro-1H-imidazo[1,2-b]pyrazol-7-yl)-3,4-bis((4-methoxybenzyl)oxy)benzamide
1
(9s). [Method A] 76% yield. 24%. H-NMR (CDCl ) δ: 7.85 (s, 1H), 7.53 (d, J = 8.7 Hz, 1H),
3
7.38-7.31 (m, 4H), 7.26-7.23 (m, 1H), 6.96 (d, J = 8.7 Hz, 1H), 6.92 (d, J = 8.7 Hz, 2H), 6.83 (d, J =
8.2 Hz, 2H), 5.10 (s, 2H), 4.98 (d, J = 12.4 Hz, 2H), 4.22 (t, J = 7.6 Hz, 2H), 4.02 (t, J = 7.8 Hz, 2H),
3.83 (s, 3H), 3.80 (s, 3H).
5.2.20
3-chloro-4,5-bis((4-methoxybenzyl)oxy)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide
(11a).
1
[Method A] 79% yield. H-NMR (DMSO-d ) δ: 7.61 (s, 1H), 7.53 (s, 1H), 7.44 (d, J = 8.16 Hz, 2H),
6
7.27 (d, J = 8.16 Hz, 2H), 6.98 (d, J = 8.16 Hz, 2H), 6.85 (d, J = 8.16 Hz, 2H), 5.17 (s, 2H), 5.00 (s,
2H), 3.77 (s, 3H), 3.73 (s, 3H).
5.2.21 2-chloro-4,5-bis((4-methoxybenzyl)oxy)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (11b).
1
[Method A] 88% yield. H-NMR (DMSO-d ) δ: 8.16 (t, J = 5.64 Hz, 1H), 7.35-7.31 (m, 4H), 7.12 (s,
6
1H), 7.09 (s, 1H), 6.94-6.91 (m, 4H), 5.08 (s, 2H), 5.02 (s, 2H), 3.74 (s, 6H), 2.56-2.54 (m, 2H),
2.48-2.44 (m, 4H), 1.69-1.65 (4H, m, 4H).

ACCEPTED MANUSCRIPT
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
5.2.22 2-fluoro-3,4-bis((4-methoxybenzyl)oxy)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (11c). [Method
1
A] 83% yield. H-NMR (CDCl ) δ: 7.74 (t, J = 8.7 Hz, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.6
3
Hz, 2H), 7.19-7.08 (m, 1H), 6.91 (d, J = 8.4 Hz, 2H), 6.85-6.78 (m, 3H), 5.08 (s, 2H), 4.99 (s, 2H),
3.83 (s, 3H), 3.79 (s, 3H), 3.56 (dd, J = 11.3, 5.3 Hz, 2H), 2.69 (t, J = 6.2 Hz, 2H), 2.59-2.52 (m, 4H),
1.84-1.74 (m, 4H).
5.2.23 2,5-dichloro-3,4-bis((4-methoxybenzyl)oxy)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (11d).
1
[Method B] 60% yield. H-NMR (CDCl ) δ: 7.47 (s, 1H), 7.47 (1H, s), 7.40-7.36 (m, 4H), 6.92-6.87
3
(m, 4H), 5.01 (s, 2H), 4.98 (s, 2H), 3.828 (s, 3H), 3.823 (s, 3H), 3.56-3.58 (m, 2H), 2.75 (m, 2H),
2.61 (br, 4H), 1.81 (br, 4H).
5.2.24 3,4-bis((4-methoxybenzyl)oxy)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (11e). [Method A] 83%
1
yield. H-NMR (CDCl ) δ: 7.51-7.49 (m, 1H), 7.38-7.32 (m, 4H), 7.26-7.23 (m, 1H), 6.93-6.86 (m,
3
4H), 6.69 (br s, 1H), 5.12 (s, 4H), 3.81 (s, 6H), 3.51 (q, J = 5.6 Hz, 2H), 2.68 (t, J = 6.0 Hz, 2H),
2.57-2.52 (br m, 4H), 1.83-1.76 (br m, 4H).
5.2.25 2,3-bis((4-methoxybenzyl)oxy)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (11f). [Method A] 99%
1
yield. H-NMR (DMSO-d ) δ: 8.22 (t, J = 5.34 Hz, 1H), 7.42 (d, J = 8.39 Hz, 2H), 7.29-7.22 (m,
6
4H), 7.11 (t, J = 7.78 Hz, 1H), 6.96 (d, J = 8.39 Hz, 2H), 6.83 (d, J = 8.39 Hz, 2H), 5.10 (s, 2H),
4.93 (s, 2H), 3.76 (s, 3H), 3.73 (s, 3H), 3.30 (q, J = 5.80 Hz, 2H), 2.44 (t, J = 6.71 Hz, 2H), 2.38 (br
s, 4H), 1.60 (br s, 4H).
5.2.26
3,4-bis((4-methoxybenzyl)oxy)-2-methyl-N-(2-(pyrrolidin-1-yl)ethyl)benzamide
(11g).
1
[Method A] 96% yield. H-NMR (DMSO-d ) δ: 8.02 (t, J = 5.57 Hz, 1H), 7.42 (d, J = 8.54 Hz, 2H),
6
7.27 (d, J = 8.54 Hz, 2H), 7.01 (s, 2H), 6.96 (d, J = 8.54 Hz, 2H), 6.86 (d, J = 8.54 Hz, 2H), 5.11 (s,
2H), 4.80 (s, 2H), 3.76 (s, 3H), 3.74 (s, 3H), 3.30-3.25 (m, 2H), 2.17 (s, 3H), 1.69-1.64 (4H, m, 4H).
5.2.27 2,3,4-tris((4-methoxybenzyl)oxy)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (11h). [Method A]
1
quantative yield. H-NMR (DMSO-d ) δ: 8.10 (t, J = 5.26 Hz, 1H), 7.51 (d, J = 8.77 Hz, 1H), 7.41
6
(d, J = 8.31 Hz, 2H), 7.30 (d, J = 8.31 Hz, 2H), 7.25 (d, J = 8.31 Hz, 2H), 7.02 (d, J = 8.77 Hz, 1H),
6.97 (d, J = 8.31 Hz, 2H), 6.90 (d, J = 8.31 Hz, 2H), 6.84 (d, J = 8.31 Hz, 2H), 5.11 (s, 2H), 5.01 (s,
2H), 4.89 (s, 2H), 3.77 (s, 3H), 3.74 (s, 6H), 3.30-3.26 (m, 2H), 2.42-2.35 (m, 6H), 1.63-1.58 (m,
4H).
5.2.28 3,4-bis(benzyloxy)-2-methoxy-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (11i). [Method A]
1
quantative yield. H-NMR (DMSO-d ) δ: 8.31 (t, J = 4.9 Hz, 1H), 7.57-7.31 (m, 11H), 7.03 (1H, d, J
6

ACCEPTED MANUSCRIPT
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
= 9.0 Hz), 5.19 (s, 2H), 4.98 (s, 2H), 3.86 (s, 3H), 3.38 (q, J = 6.00 Hz, 2H), 2.58 (t, J = 6.41 Hz,
2H), 1.68-1.72 (m, 4H).
5.2.29 2-chloro-3-methoxy-4-((4-methoxybenzyl)oxy)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (11j).
1
[Method B] 78% yield. H-NMR (CDCl ) δ: 7.40 (d, J = 8.8 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H),
3
6.94-6.88 (m, 4H), 5.08 (s, 2H), 3.87 (s, 3H), 3.80 (d, J = 9.0 Hz, 3H), 3.58-3.51 (m, 2H), 2.58-2.50
(m, 4H), 1.80-1.74 (m, 4H).
5.2.30
5-((4-methoxybenzyl)oxy)-4-oxo-N-(2-(pyrrolidin-1-yl)ethyl)-1,4-dihydropyridine-2-carboxamide
1
(11k). [Method A] 20% yield. H-NMR (DMSO-d ) δ: 8.44 (t, J = 5.4 Hz, 1H), 8.11 (br s, 1H),
6
7.46-7.35 (m, 3H), 6.94 (d, J = 9.0 Hz, 2H), 5.15 (s, 2H), 3.75 (s, 3H), 3.41-3.32 (m, 2H), 2.57 (t, J =
6.5 Hz, 2H), 2.50 (td, J = 3.7, 1.9 Hz, 4H), 1.71-1.65 (m, 4H).
5.2.31
3-chloro-5-((4-methoxybenzyl)oxy)-4-oxo-N-(2-(pyrrolidin-1-yl)ethyl)-1,4-dihydropyridine-2-carbox
1
amide (11l). [Method A] 47% yield. H-NMR (DMSO-d ) δ: 8.82-8.79 (br, 1H), 7.90 (s, 1H),
6
7.50-7.12 (m, 5H), 5.24 (s, 2H), 4.00-3.00 (m, 8H), 2.00-1.80 (m, 4H).
5.2.32
1-((4-methoxybenzyl)oxy)-6-oxo-N-(2-(pyrrolidin-1-yl)ethyl)-1,6-dihydropyridine-3-carboxamide
1
(11n). [Method A] 93% yield. H-NMR (CDCl ) δ: 7.95 (d, J = 2.6 Hz, 1H), 7.62 (dd, J = 9.6, 2.5
3
Hz, 1H), 7.35 (dt, J = 9.6, 2.5 Hz, 2H), 6.89 (dt, J = 8.7, 2.4 Hz, 2H), 6.67-6.64 (m, 2H), 5.23 (2H, s),
3.81 (s, 3H), 3.45 (dd, J = 4.7, 10.9 Hz, 2H), 2.65 (t, J = 6.4 Hz, 2H), 2.58-2.50 (m, 4H), 2.22 (br s,
2H), 1.84-1.77 (m, 4H).
5.3 Synthesis of the compound 12, 14 and 18
5.3.1 1-(2-chloro-3,4-bis((4-methoxybenzyl)oxy)phenyl)-3-(2-(pyrrolidin-1-yl)ethyl)urea (12). To a
suspension of 8 (2.14 g, 5.00 mmol) in toluene (25.0 mL) were added Et N (0.83 mL, 6.00 mmol)
3
and DPPA (1.29 mL, 6.00 mmol). After stirring for 1 h at reflux, to the resulting mixture was added
2-(pyrrolidin-1-yl)ethan-1-amine (0.86 g, 7.5 mmol), and stirred for 2 h at reflux. The resulting
mixture was poured into a mixture of EtOAc, THF and water. The aqueous layer was extracted with
EtOAc and the combined extracts were washed with water and brine, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. To the resulting residue was added IPE, then the resulting
1
precipitated solid was collected by filtration to give 12 (1.84 g, 68%). H-NMR (DMSO-d ) δ: 8.65
6
(br s, 1H), 7.91 (br s, 1H), 7.67 (dd, J = 14.9, 9.6 Hz, 1H), 7.39 (d, J = 7.3 Hz, 2H), 7.29 (d, J = 7.6

ACCEPTED MANUSCRIPT
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
Hz, 2H), 7.09 (dd, J = 18.4, 8.9 Hz, 1H), 6.95 (d, J = 7.6 Hz, 2H), 6.86 (d, J = 7.8 Hz, 2H), 5.06 (s,
2H), 4.89 (s, 2H), 3.76 (s, 3H), 3.74 (s, 3H), 3.29-3.10 (m, 2H), 2.48-2.42 (m, 6H), 1.70-1.67 (m,
4H).
5.3.2 2-chloro-3,4-bis((4-methoxybenzyl)oxy)aniline (13). To a suspension of 8 (42.9 g, 100 mmol)
and Et N (16.63 mL, 120 mmol) in Toluene (500 mL) was added DPPA (25.8 mL, 120 mmol) at rt
3
The mixture was stirred at reflux for
1 h. To the resulting mixture was added
(9H-fluoren-9-yl)methanol (29.4 g, 150 mmol) and stirred at reflux for 4 h. To the resulting mixture
was added piperidine (49.5 mL, 500 mmol) was added. The resulting mixture was stirred at rt
overnight. The resulting mixture was concentrated in vacuo and diluted with H O, then extracted
2
with CH Cl . The organic layers were combined and washed with aq. 2 mol/L HCl, sat. NaHCO ,
2
2
3
and brine. The organic layer was dried over anhydrous MgSO , filtered and concentrated in vacuo.
4
1
The resulting residue was purified by silica gel chromatography to afford 13 (28.6 g, 71%). H-NMR
(CDCl ) δ: 7.39 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 8.7 Hz, 2H), 6.91-6.84 (m, 4H), 6.75 (d, J = 8.8 Hz,
3
1H), 6.46 (d, J = 8.7 Hz, 1H), 4.99 (s, 2H), 4.96 (s, 2H), 3.85-3.81 (m, 8H).
5.3.3 N-(2-chloro-3,4-bis((4-methoxybenzyl)oxy)phenyl)-3-(pyrrolidin-1-yl)propanamide (14). To a
solution of 1-pyrrolidinepropanoic acid hydrochloride (647 mg, 3.60 mmol) in CH Cl (12.0 mL)
2
2
was added oxalyl chloride (0.315 mL, 3.60 mmol) and stirred for 30 min at rt. To a solurion of 50
(1.20 g, 3.00 mmol) and pyridine (0.872 mL, 10.8 mmol) in CH Cl (12.0 mL) was added dropwised
2
2
the above described mixture containing 2-(pyrrolidin-1-yl)acetyl chloride at 0°C. After stirring for 1
h at 0°C, the mixture was quenched by a solution of sodium hydrogen carbonate in water. The
aqueous layer was extracted with CH Cl and the combined extracts were washed with water and
2
2
brine, dried over anhydrous MgSO , filtered, and concentrated in vacuo. The resulting residue was
4
1
purified by silica gel chromatography to afford 14 (834 mg, 53%). H-NMR (CDCl ) δ: 11.36 (s,
3
1H), 8.07 (d, J = 9.0 Hz, 1H), 7.38-7.34 (m, 4H), 6.93-6.89 (m, 3H), 6.85 (d, J = 9.0 Hz, 2H), 5.03 (s,
2H), 4.97 (s, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 2.83 (t, J = 5.8 Hz, 2H), 2.66 (s, 4H), 2.57 (t, J = 5.8 Hz,
2H), 1.87-1.84 (m, 4H).
5.3.4 2-chloro-3,4-bis((4-methoxybenzyl)oxy)benzohydrazide (15). To a solution of 8 (21.4 g, 50.0
mmol) in CH Cl (300 mL) were added N-hydroxysuccinimide (8.63 g, 75.0 mmol) and EDC·HCl
2
2
(11.50 g, 60.0 mmol) at 0°C. After stirring overnight at rt, t the mixture was poured into water. The
aqueous layer was extracted with CH Cl and the combined extracts were washed with water and
2
2
brine, dried over anhydrous MgSO , filtered, and concentrated in vacuo. To the resulting residue was
4
added IPE, then the resulting precipitated solid was collected by filtration to give the activated ester
24.4 g, 93 %). This activated ester (5.26 g, 10 mmol) was suspended DMF (50.0 mL). Hydrazine

ACCEPTED MANUSCRIPT
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
monohydlate (0.73 mL, 15.0 mmol) was added thereto with stirring, and then stirred at rt for 14 h.
Water was added to the reaction solution, the resulting solid was filtrated, washed with IPA, then IPE,
1
and then dried in vacuo to yield Compound 15 (4.03 g, 91%). H-NMR (DMSO-d ) δ: 9.42 (br s,
6
1H), 7.43 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.5 Hz, 1H), 7.09 (d, J = 8.2 Hz,
1H), 6.97 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 5.14 (s, 2H), 4.88 (s, 2H), 4.43 (br s, 2H),
3.77 (s, 3H), 3.75 (s, 3H).
5.3.5 2-chloro-N'-(2-chloroacetyl)-3,4-bis((4-methoxybenzyl)oxy)benzohydrazide (16). To a solution
of 15 (5.00 g, 11.3 mmol) in DMF (50.0 mL) were added NaHCO and chloroacetyl chloride (1.09
3
mL, 13.6 mmol). After stirring at 0°C for 45 min, EtOAc and water were added to the reaction
solution, and then the organic layer was separated. The organic layer was washed with water, and
then concentrated in vacuo. After water was added to the resulting residue, the resulting solid was
filtered, washed with water, then isopropanol, and then dried in vacuo to yield 16 (5.61 g, 96%).
1
H-NMR (DMSO-d ) δ: 10.43 (br s, 1H), 10.34 (br s, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.5
6
Hz, 2H), 7.26-7.19 (m, 2H), 6.98 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 5.16 (s, 2H), 4.89 (s,
2H), 4.17 (s, 2H), 3.77 (s, 3H), 3.75 (s, 3H).
5.3.6 2-(2-chloro-3,4-bis((4-methoxybenzyl)oxy)phenyl)-5-(chloromethyl)-1,3,4-oxadiazole (17). To
a
suspension of 16 (4.8 g, 9.24 mmol) in 1,4-dioxane was added methyl
N-(triethylammoniumsulfonyl)carbamate (6.61 g, 27.7 mmol). While stirring at 120°C, methyl
N-(triethylammoniumsulfonyl)carbamate (6.61 g, 27.7 mmol) was added and then stirred at the same
temperature for 40 min. After the temperature in the reaction solution was cooled to rt, the solvent
was evaporated in vacuo. Ethyl acetate and water were added thereto, and then the organic layer was
separated. The organic layer was washed with water, then saturated brine, and then dried with
anhydrous MgSO . Magnesium sulfate was removed by filtration, followed by concentration in
4
vacuo. The resulting solid was filtered. The filtered residue was washed with water, isopropanol,
1
then diisopropyl ether, and then dried in vacuo to yield 17 (3.86 g, 78%). H-NMR (CDCl ) δ: 7.66
3
(d, J = 8.7 Hz, 1H), 7.35 (dd, J = 8.4, 5.2 Hz, 4H), 6.99 (d, J = 8.8 Hz, 1H), 6.93 (d, J = 8.5 Hz, 2H),
6.83 (d, J = 8.5 Hz, 2H), 5.12 (s, 1H), 4.99 (s, 2H), 4.01 (s, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 2.74-2.70
(m, 4H), 1.87-1.82 (m, 4H).
5.3.7 2-(2-chloro-3,4-bis((4-methoxybenzyl)oxy)phenyl)-5-(pyrrolidin-1-ylmethyl)-1,3,4-oxadiazole
(18). To a solution of 17 (1.57 g, 11.4 mmol) in DMF (40.0 mL) were added K CO (1.57 g, 11.4
2
3
mmol) and pyrrolidine (0.69 mL, 8.34 mmol) and stirred at rt for 1h. Pyrrolidine (0.19 mL, 2.27
mmol) was further added to the mixture, and then stirred at room temperature for 30 min. EtOAc and
water were added, and then the organic layer was separated. The combined organic layer was

ACCEPTED MANUSCRIPT
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
washed with water, then saturated brine, and then concentrated in vacuo. IPA was then added to the
resulting residue. The resulting solid was filtered, and then washed with isopropanol and diisopropyl
ether. This solid was recrystallized by CH Cl isopropanol, and then dried in vacuo to yield 18 (2.77
2
2
1
g, 68%). H-NMR (CDCl ) δ: 7.66 (d, J = 8.7 Hz, 1H), 7.35 (dd, J = 8.5, 5.3 Hz, 4H), 6.99 (d, J =
3
8.8 Hz, 1H), 6.93 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 5.12 (s, 2H), 4.99 (s, 2H), 4.01 (s,
2H), 3.84 (3H, s), 3.80 (s, 3H), 2.76-2.70 (m, 4H), 1.82-1.87 (m, 4H).
5.4 Synthesis of the cephalosporin nucleus
5.4.1 Compound 26c. To suspension of 20i (31.4 mmol), ACLH·HCl (23b; 14.2 g, 31.4 mmol)
purchased from Otsuka Chemical Co., Ltd. and phenyl dichlorophosphate (7.04 mL g, 47.1 mmol) in
CH Cl (300 mL) was added dropwised N-methylmorpholine (15.5 mL g, 141 mmol) at -40°C. After
2
2
stirring at -40°C for 4 h, the reaction mixture was diluted with aq. 0.2 mol/L HCl (93 mL), and then
allowed to room temperature. The aqueous layer was extracted with CH Cl and the combined
2
2
extracts were washed with aq. saturated sodium hydrogen carbonate, water and brine, dried over
anhydrous MgSO , filtered, and concentrated in vacuo to afford the intermediate, which was used for
4
the next reaction without further purification. To a solution of the intermediate (27.3 g, 20.3 mmol)
in CH Cl was added dropwised a solution of m-CPBA (4.84 g, 18. 24 mmol) in CH Cl (25 mL) at
2
2
2
2
-40°C, then the reaction mixture was stirred at -40°C for 1 h. The reaction solution was diluted with
CH Cl , washed with a 5% aqueous sodium hydrogen sulfite solution, an aqueous saturated sodium
2
2
bicarbonate solution, and brine, and dried over magnesium sulfate. After magnesium sulfate was
filtered, the filtrate was concentrated under reduced pressure, subjected to silica gel column
chromatography, and eluted with hexane/ethyl acetate. Fractions containing the desired compound
1
were concentrated under reduced pressure to obtain compound 26c (17.84 g, 65%). H-NMR
(CDCl ) δ: 8.14 (d, J = 8.39 Hz, 1H), 7.95 (br s, 1H), 7.64 (d, J = 9.30 Hz, 1H), 7.44 (d, J = 6.71 Hz,
3
2H), 7.35-7.23 (m, 23H), 6.91-6.76 (m, 8H), 5.98 (dd, J = 9.30, 4.58 Hz, 1H), 5.24 (dt, J = 8.18, 3.13
Hz, 1H), 4.97-4.68 (m, 6H), 4.42 (d, J = 4.73 Hz, 1H), 4.01 (d, J = 12.20 Hz, 1H), 3.82-3.72 (m, 7H),
3.26 (d, J = 18.68 Hz, 1H), 3.10 (d, J = 18.68 Hz, 1H), 1.52 (s, 9H).
5.4.2 Compound 25a. Compound 25a was prepared from 23a (ACLE·HCl) purchased from Otsuka
1
Chemical Co., Ltd. and 20a by the similar procedure as that described for 26c. 86% yield. H-NMR
(CDCl ) δ: 8.38 (br s, 1H), 7.92 (d, J = 9.9 Hz, 1H), 7.38 (d, J = 8.7 Hz, 2H), 7.30 (s, 1H), 6.94 (d, J
3
= 8.7 Hz, 2H), 6.23 (dd, J = 9.8, 4.8 Hz, 1H), 5.32 (d, J = 11.8 Hz, 1H), 5.26 (d, J = 11.8 Hz, 1H),
5.06 (d, J = 12.6 Hz, 1H), 4.61 (d, J = 4.8 Hz, 1H), 4.25 (d, J = 12.6 Hz, 1H), 3.88-3.80 (m, 4H),
3.44 (d, J = 18.1 Hz, 1H), 1.62 (s, 3H), 1.60 (s, 3H), 1.55 (s, 9H), 1.44 (s, 9H).
1
5.4.3 Compound 25b. 88% yield. H-NMR (CDCl3) δ: 8.38 (d, J = 9.2 Hz, 1H), 7.34-7.37 (m, 2H),

ACCEPTED MANUSCRIPT
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
6.88-6.92 (m, 2H), 6.07 (dd, J = 4.5 , 9.2 Hz, 1H), 5.29 (d, J = 12.0 Hz, 1H), 5.24 (d, J = 12.0 Hz,
1H), 5.10 (d, J = 12.6 Hz, 1H), 4.74 (s, 2H), 4.57 (dd, J = 1.5, 4.8 Hz, 1H), 4.22 (d, J = 12.6 Hz, 1H),
3.80 (d, J = 18.3 Hz, 1H), 3.40 (dd, J = 1.5, 18.3 Hz, 1H), 1.54 (9H, s, 9H), 1.42 (9H, s),
2
9
5.4.4 Compound 25c and 25d were prepared accroding to the published procedure.
1
5.4.5 Compound 26a. 61% yield. H-NMR (CDCl ) δ: 8.43 (s, 1H), 7.89 (d, J = 9.8 Hz, 1H),
3
7.50-7.43 (m, 2H), 7.40-7.26 (m, 9H), 6.96 (s, 1H), 6.23 (dd, J = 9.8, 4.9 Hz, 1H), 4.94 (d, J = 12.0
Hz, 1H), 4.61 (dd, J = 4.9, 1.4 Hz, 1H), 4.18 (d, J = 13.4 Hz, 1H), 3.82 (d, J = 18.6 Hz, 1H), 3.44 (d,
J = 18.8 Hz, 1H), 1.60 (s, 3H), 1.58 (s, 3H), 1.52 (s, 9H), 1.42 (s, 9H).
1
5.4.6 Compound 26b. 58% yield. H-NMR (CDCl ) δ: 8.30 (br s, 1H), 7.69 (d, J = 10.1 Hz, 1H),
3
7.47-7.12 (m, 31H), 6.95 (s, 1H), 6.87 (s, 1H), 6.86 (s, 1H), 6.05 (dd, J = 9.7, 4.8 Hz, 1H), 5.45 (t, J
= 6.5 Hz, 1H), 5.27-5.16 (m, 1H), 4.92 (d, J = 12.4 Hz, 1H), 4.16 (ddt, J = 30.9, 19.8, 5.1 Hz, 7H),
3.42 (d, J = 18.6 Hz, 1H), 3.23-3.16 (m, 3H), 3.03 (d, J = 18.8 Hz, 1H), 1.80 (d, J = 10.6 Hz, 1H).
1
5.4.7 Compound 26d. 65% yield. H-NMR (CDCl ) δ: 8.22 (br s, 1H), 7.78 (d, J = 9.91 Hz, 1H),
3
7.41-7.12 (m, 26H), 6.93-6.76 (m, 7H), 6.11 (dd, J = 9.91, 4.88 Hz, 1H), 5.25-5.22 (m, 1H),
4.97-4.81 (m, 5H), 4.46 (d, J = 3.66 Hz, 1H), 4.19-3.97 (m, 3H), 3.82-3.74 (m, 7H), 3.53 (d, J =
18.53 Hz, 1H), 3.16 (d, J = 18.53 Hz, 1H), 1.53 (s, 9H).
2
6, 47
5.4.8 Compound 27a and 27b were prepared accroding to the published procedure.
5.5 Synthesis of the new siderophore cephalosporins
5.5.1
(6R,7R)-7-[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-{[(2-carboxypropan-2-yl)oxy]imino}
acetamido]-3-({1-[2-(2-chloro-3,4-dihydroxybenzamido)ethyl]pyrrolidin-1-ium-1-yl}methyl)-8-oxo-
5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (cefiderocol, 3). To a solution of 25a (19.91 g,
25.0 mmol) in THF (200 mL) was added NaI (11.2 g, 75.0 mmol) at 15°C. After stirring at room
temperature for 30 min, the reaction solution was poured into a mixture of EtOAc and aq. 5%
NaHSO solution in water. The organic layer was separated, then the aqueous layer was extracted
3
with EtOAc and the combined extracts were washed with water and brine, dried over anhydrous
MgSO , filtered, and concentrated in vacuo to afford the intermediate iodide (22.8 g, 95%). This
4
intermediate was used without further purification. To a solution of the intermediate iodide (8.21 g,
8.51mmol) in DMF (24.0 mL) was added and compound 9b (4.47 g, 8.51 mmol) then the resulting
mixture was stirred at room temperature for 3.5 h. To the resulting mixture were added DMF (48.0
mL), potassium iodide (9.89 g, 59.6 mmol) and acetyl chloride (2.43 mL, 34.1 mmol), then stirred at