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Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus
G Model
CCLET 3258 1–5 Chinese Chemical Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet 1 2 3 4 5 6 7 8
Original article
Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus Ding a,1, Wen-Ke Wang b,1, Qiao Cao a, Wen-Jing Chu a, Le-Fu Lan a, Wen-Hao Hu b, Yu-She Yang a,*
Q1 Shi
a b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, East China Normal University, Shanghai 200062, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 December 2014 Received in revised form 2 February 2015 Accepted 17 March 2015 Available online xxx
In order to develop novel LpxC inhibitors with good activities and metabolic stability, two series of compounds with hydrophilic terminus have been synthesized and their in vitro antibacterial activities against Escherichial coli and Pseudomonas aeruginosa were evaluated. Especially, compounds 22b and c exhibited comparable antibacterial activities to CHIR-090 and better metabolic stability than CHIR-090 and LPC-011 in liver microsomes (rat and mouse), which indicated the terminal methylsulfone may be a preferred structure in the design of LpxC inhibitors and worthy of further investigations. ß 2015 Yu-She Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. All rights reserved.
Keywords: LpxC CHIR-090 Kojic acid derivatives Methylsulfone derivatives Metabolic stability
9 10
1. Introduction
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Drug resistant Gram-negative infections have been a serious threat to public health, especially for hospitalized patients. Unfortunately, while resistance to current therapies is spreading rapidly, new antibacterial agents to treat these infections are few in number [1]. Thus, there is a great need to develop novel antibacterial agents with new mechanisms of action. The outer membrane of the Gram-negative bacteria is a very efficient permeability barrier, obstructing the development of new antibiotics severely. Lipid A is a critical component of lipopolysaccharide (LPS), which mainly consists of the outer leaflet of the outer membrane [2]. The UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second-step of biosynthesis of lipid A in Gram-negative bacteria and is a validated antibiotic target [3,4]. Thus, inhibition of LpxC can obstruct the biosynthesis of lipid A, and sequentially kill most Gram-negative bacteria [2]. The X-ray crystal structure of LpxC from Aquifex aeolicus (aaLpxC) reveals a catalytic zinc ion and a hydrophobic tunnel accommodating a myristate fatty acid side chain [5,6]. Thus, most of the LpxC
* Corresponding author. E-mail address: [email protected] (Y.-S. Yang). 1 These authors contributed equally to this work.
inhibitors contain a zinc-binding group (e.g. hydroxamic acid) and a hydrophobic tail which mimics the myristate fatty acid chain. These lipophilic moieties make important van der Waals interactions with the enzyme in the hydrophobic tunnel, which is critical for the inhibition of LpxC [1]. Since the discovery of L-573,655 and L-161,240 (Fig. 1) in 1996 [4], LpxC inhibitors have been developed for two decades. The inhibitors in the early days mainly exhibited strong antibacterial activities against Escherichia coli (E. coli), but rarely inhibited the growth of Pseudomonas aeruginosa (P. aeruginosa) [4,7–11]. In 2004, Anderson et al. disclosed a new series of LpxC inhibitors, exemplified by CHIR-090 (Fig. 1), which displayed high affinity to both E. coli and P. aeruginosa LpxC and remarkable antibacterial activity against a wide range of Gram-negative pathogens [12]. On the basis of CHIR-090, Lee et al. [13] synthesized a series of compounds with linear diphenylacetylene scaffold which exhibited better antibacterial activities. In order to improve the watersolubility of these compounds, NH2 substituent was introduced in their distal phenyl ring, exemplified by LPC-011 (Fig. 1) [14]. LPC011 exhibited better water-solubility and antibacterial activity, while the metabolic data was not optimistic (the data will be mentioned later). In 2012, Pfizer reported a series of novel LpxC inhibitors with methylsulfone, exemplified by 1a (Fig. 1). 1a exhibited excellent antibacterial activity against P. aeruginosa but suffered from the problems of low water-solubility and high protein binding [1]. Then the central phenyl ring of 1a was replaced
http://dx.doi.org/10.1016/j.cclet.2015.03.029 1001-8417/ß 2015 Yu-She Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. All rights reserved.
Please cite this article in press as: S. Ding, et al., Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.029
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
G Model
CCLET 3258 1–5 S. Ding et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
2
HO O
n1 = 0, 1, 2; n2 = 0, 1; n 3 = 1, 2, 3
H N
N H
OH N
OH
O
n1 R
HO
n2
O
H N
R= ,
HO
,
O
O
O S O
n3
Series 2
Series 1
Fig. 2. Two series of novel LpxC inhibitors.
Fig. 1. The structures of classic LpxC inhibitors.
O
O
O O
a
O
HO O OH
b
c
N H
I Si
1
3
2
O
e
d
HO
PMBO
OH
Ph 2HCO
h
7 O
Ph 2HCO
OH
O
i
Ph 2HCO
OH
N OH 9
8
OH
N
O
O g
f N H 6
5
OH
O
OPMB
PMBO
OH
O
Kojic acid
4
O
O PMBO
O O
N OCHPh2 10
OH
Scheme 1. Conditions and reagents: (a) Trimethylsilylacetylene, PdCl2(PPh3)2, CuI, Et3N, THF, r.t., 98%; (b) NaOH, MeOH, r.t., 80%; (c) L-Threonine methyl ester hydrochloride, EDCI, HOBt, DIEA, DMF, r.t., 70%; (d) Kojic acid, PMBCl, K2CO3, DMF, 80 8C, 80%; (e) NH3H2O, EtOH, 70 8C, 70%; (f) PMBCl, K2CO3, DMF, 80 8C, 56.2%; (g) Ph2CN2, EtOH, 40 8C, 77%; (h) NH2OHHCl, CH3COONa3H2O, EtOH/H2O, 70 8C, 44%; (i) Ph2CHCl, K2CO3, NaI, DMSO, r.t., 98%.
HO O
HO O R
OH
N H
+ n
HO
O
a
O
N H R
n = 1, 12 n = 2, 13
11a-d
n
O
HO O O
b
N H
O
R
n
O
14a-h
H N
HO O OH
O
R:
O S O G1
,
O S O G2
, PMBO
, Ph2HCO O G3
H N ;
, HO
, HO OPMB
O
O
G4
G5
G6
11a 11b 11c 11d
G1 G2 G3 G4
n
O
1 5a -h
OH N
N
N H R
H N
OH
O
1 6a -d
R OCHPh 2 N
c
14a, 14b, 14c, 14d, 14e, 14f , 14g, 14h,
15a 15b 15c 15d 15e 15f 15g 15h
n
R
1 2 1 2 1 2 1 2
G1 G1 G2 G2 G3 G3 G4 G4
16a 16b 16c 16d
n
R
1 2 1 2
G5 G5 G6 G6
Scheme 2. Conditions and reagents: (a) DEAD, PPh3, THF, r.t., 40–60%; (b) NH2OHH2O, DCM/MeOH, r.t., 40–80%; (c) TFA, DCM, r.t., 70–90%.
Scheme 3. Conditions and reagents: (a) DEAD, PPh3, THF, r.t., 40–60%; (b) 4, PdCl2(PPh3)2, CuI, Et3N, THF, r.t., 80–90%; (c) Trimethylsilylacetylene, PdCl2(PPh3)2, CuI, Et3N, THF, r.t., 80–90%; (d) TBAF, THF, r.t., 40–90%; (e) 4, Cu(Ac)2, Pyridine/MeOH, r.t., 20–30%; (f) NH2OHH2O, DCM/MeOH, r.t., 40–80%; (g) TFA, DCM, r.t., 70–90%.
Please cite this article in press as: S. Ding, et al., Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.029
G Model
CCLET 3258 1–5 S. Ding et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
3
Table 1 In vitro antibacterial activities of synthetic compounds. HO O N H
H N
OH
O
n1 R
O
n2
n3
Compound
MIC (mg/mL)
Structure n1
n2
n3
R
P. aeruginosa PAO1
P. aeruginosa PA14
E. coli DH5a
E. coli AB1157
15a
0
0
3
O S O
>1000
>1000
>1000
>1000
15b
0
1
3
O S O
15c
0
0
2
O S O
>1000
>1000
>1000
>1000
15d
0
1
2
O S O
250
125
50
25
16a
0
0
1
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
3.125
OH N
3.125
2.100
2.100
HO O
16b
0
1
1
OH N HO O
16c
0
0
1
H N HO O
16d
0
1
1
H N HO O
22a
2
1
3
O S O
22b
1
1
3
O S O
1.560
1.200
0.195
0.195
22c
1
1
2
O S O
0.780
1.560
0.195
0.195
23a
1
1
1
OH N
100
30
25
25
>1000
>1000
50
50
50
50
50
50
HO O
23b
2
1
1
H N HO O
23c
1
1
1
H N HO O
CHIR-090
–
–
–
–
1.250
1.250
0.300
0.200
LPC-011
–
–
–
–
0.310
0.310
0.040
0.040
Please cite this article in press as: S. Ding, et al., Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.029
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CCLET 3258 1–5 S. Ding et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
4
Table 2 In vitro antibacterial activities of CHIR-090, LPC-011, 23a and c under standard condition and low-iron condition. Compound
23a 23c CHIR-090 LPC-011 a b
P. aeruginosa PAO1
P. aeruginosa PA14
E. coli DH5a
E. coli AB1157
Standarda
Low-ironb
Standard
Low-iron
Standard
Low-iron
Standard
Low-iron
100 50 1.250 0.310
50 25 0.400 0.200
50 50 1.250 0.310
25 25 0.400 0.200
25 40 0.156 0.019
10 10 0.080 0.010
25 40 0.156 0.019
10 10 0.080 0.010
The compounds were tested in the normal LB media by the standard process. The compounds were tested in the low-iron LB media by the standard process.
56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73
with pyridine (e.g. 2a in Fig. 1), and this series of compounds exhibited excellent Gram-negative antibacterial activity, improved solubility and increased free fraction. However, the half-life of these compounds in rats was shortened [15]. As discussed above, the balance of antibacterial activities and metabolic properties is a major challenge to the development of LpxC inhibitors. Thus, we designed and synthesized two series of compounds with hydrophilic terminus mainly aimed to achieve novel LpxC inhibitors with improved activity, metabolic stability and water solubility. The terminus of series 1 are kojic acid derivatives, which can deliver the compound through the outer membrane of the Gram-negative bacteria as a siderophore by the Trojan Horse approach [16]. Meanwhile, Kojic acid derivatives with NH3 and NH2OH substitution are proved effective siderophores [17,18]. The terminus of series 2 is methylsulfone, which is a steady group for metabolism. Hydrophobic tails with different length have also been synthesized to match the hydrophobic tunnel (Fig. 2).
74
2. Experimental
75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106
The synthetic route of intermediates 4, 7 and 10 was outlined in Scheme 1. Compound 2 was obtained by the coupling reaction of compound 1 and trimethylsilyacetyene in THF. The hydrolyzation and deprotection of compound 2 under alkaline condition afforded the acid compound 3, and then treated with l-threonine methyl ester hydrochloride in the condition of condensation reaction to afford compound 4. Protection of Kojic acid with 4-methoxybenzylchloride gave compound 5 and then treated with ammonium hydroxide to give compound 6. The reaction of compound 6 with 4-methoxybenzylchloride afforded the aromatization product compound 7. Compound 8 was obtained from the Kojic acid and diphenyldiazomethane under alkaline condition, then treated with alkalized hydroxylamine hydrochloride to yield compound 9. The reaction of compound 9 with diphenyldiazomethane afforded compound 10. pt?>The synthetic route of compounds 15a–d and 16a–d was outlined in Scheme 2. Compounds 14a–h were obtained by the Mitsunobu reaction of RCH2OH and compounds 12 and 13, then treated with NH2OH (50% in water) and yielded compounds 15a–h. Deprotection of compounds 15e–h in trifluoroacetic acid yielded compounds 16a–d. The synthetic route of compounds 22a–c and 23a–c was outlined in Scheme 3. Compounds 18a–d were obtained by the Mitsunobu reaction of RCH2OH and 4-iodoaniline. Compounds 19a and b were obtained by the coupling reaction of compounds 18a and d with trimethylsilyacetyene in THF, respectively. Then 19a and b were treated with tetrabutylammonium fluoride to yield compounds 20a and b. Compounds 21a–f were obtained by the coupling reaction of compound 4 with compounds 18a–d and 20a and b, then treated with NH2OH (50% in water) to yield compounds 22a–f. Deprotection of compounds 22d–f in trifluoroacetic acid yielded compounds 23a–c.
3. Results and discussion
107
Minimum inhibitory concentration (MIC) of each compound was tested by the modified National Committee for Clinical Laboratory Standards, which is adapted to 96-well plates and LB media in the presence of 5% DMSO [19]. Tested bacterial strains were classic Gram-negative pathogens, including P. aeruginosa (PAO1, PA14) and E. coli (AB1157, DH5a). The MIC values of all tested compounds were listed in Table 1. Unfortunately, most compounds of series 1 lost their antibacterial activities. Only the compounds 23a and c with diphenylacetylene scaffold exhibited weak antibacterial activities against E. coli and P. aeruginosa. In series 2, compounds with biphenyl scaffold (15b) and diphenylacetylene scaffold (22b and c) showed remarkable antibacterial activities against both E. coli and P. aeruginosa. Especially, compounds 22b and c exhibited comparable antibacterial activities to CHIR-090. In both series, the length of the hydrophobic tails also played an important role: too long (diacetylene scaffold) or too short (phenyl scaffold) would lead to a loss of antibacterial activity. Moreover, we compared the antibacterial activities of compounds 23a and c, CHIR-090 and LPC-011 both under standard condition and low-iron condition. As shown in Table 2, all of the tested compounds exhibited better antibacterial activities under low-iron condition. Unexpectedly, similar improvements were observed in the antibacterial activities of tested compounds. It is speculated that the hydroxamic acid group can serve as a potent siderophore to help the inhibitors entering the outer membrane of the bacteria and the extra siderophore (kojic acid derivative) does not influence this process obviously. The detailed mechanism is not clear and needs further biological investigations. In addition, we evaluated the metabolic stability of compounds 22b and c, CHIR-090 and LPC-011 in liver microsomes (rat and mouse). As shown in Table 3, compounds 22b and c exhibited better metabolic stability than CHIR-090 and LPC-011, which
108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139
Table 3 Half-lives and intrinsic clearances of CHIR-090, LPC-011, 22b and c in liver microsomesa Compound
Species
Microsomal t1/2 b (min)
Clint c (mL min protein 1)
22b
Rat Mouse Rat Mouse Rat Mouse Rat Mouse
2226.3 314.2 –d 328 171.2 88.9 75.8 80.9
1 7 0 6 12 24 28 26
22c CHIR-090 LPC-011
1
mg
a 0.33 mg/mL microsomal protein, NADP+-regenerating system, [inhibitor], 0.1 mmol/L, incubation at 37 8C, samples taken at 0, 7, 17, 30, and 60 min, determination of parent compound by MS. b t1/2: elimination half-life in rat liver microsomes. c Clint: intrinsic body clearance. d t1/2 was not calculated, because compound 22b was not metabolized at 60 min.
Please cite this article in press as: S. Ding, et al., Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.029
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CCLET 3258 1–5 S. Ding et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
140 141
indicated that the terminal methylsulfone may be a preferred structure in the design of LpxC inhibitors.
142
4. Conclusion
143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158
In conclusion, two series of novel LpxC inhibitors containing the terminal kojic acid derivatives and methylsulfone were synthesized and evaluated. The MIC values indicated that the length of the hydrophobic tail played an important role and the compounds with diphenylacetylene scaffold exhibited best antibacterial activities. In series 1, most of the compounds lost their antibacterial activities and the introduction of kojic acid derivatives did not provide extra improvement of antibacterial activities obviously compared to CHIR-090 and LPC-011 under low-iron condition. In series 2, some compounds, especially 22b and c, exhibited comparable antibacterial activities to CHIR-090 against E. coli and P. aeruginosa and better metabolic stability than CHIR-090 and LPC-011 in liver microsomes (rat and mouse). These results manifested the terminal methylsulfone may be a preferred structure in the design of LpxC inhibitors and worthy of further investigations.
159
Acknowledgments
160 161 162 163
We are grateful to National Science and Technology Major Project for the support of this research. The project described was Q2 supported by Key New Drug Creation and Manufacturing Program, China (No. 2014ZX09507009-016).
164
Appendix A. Supplementary data
165 166
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2015.03.029.
167
References
168 169
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5
[2] T.J.O. Wyckoff, C.R.H. Raetz, J.E. Jackman, Antibacterial and anti-inflammatory agents that target endotoxin, Trends Microbiol. 6 (1998) 154–159. [3] A.W. Barb, A.L. McClerren, K. Snehelatha, et al., Inhibition of lipid A biosynthesis as the primary mechanism of CHIR-090 antibiotic activity in Escherichia coli, Biochemistry 46 (2007) 3793–3802. [4] H.R. Onishi, B.A. Pelak, L.S. Gerckens, et al., Antibacterial agents that inhibit lipid A biosynthesis, Science 274 (1996) 980–982. [5] D.A. Whittington, K.M. Rusche, H. Shin, C.A. Fierke, D.W. Christianson, Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 8146–8150. [6] B.E. Coggins, X.C. Li, A.L. McClerren, et al., Structure of the LpxC deacetylase with a bound substrate-analog inhibitor, Nat. Struct. Mol. Biol. 10 (2003) 645–651. [7] M.H. Chen, M.G. Steiner, S.E. de Laszlo, et al., Carbohydroxamido-oxazolidines: antibacterial agents that target lipid A biosynthesis, Bioorg. Med. Chem. Lett. 9 (1999) 313–318. [8] J.E. Jackman, C.A. Fierke, L.N. Tumey, et al., Antibacterial agents that target lipid A biosynthesis in gram-negative bacteria, J. Biol. Chem. 275 (2000) 11002–11009. [9] M.C. Pirrung, L.N. Tumey, C.R.H. Raetz, et al., Inhibition of the antibacterial target UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC): isoxazoline zinc amidase inhibitors bearing diverse metal binding groups, J. Med. Chem. 45 (2002) 4359–4370. [10] P. Calı´, L. Nærum, S. Mukhija, A. Hjelmencrantz, Isoxazole-3-hydroxamic acid derivatives as peptide deformylase inhibitors and potential antibacterial agents, Bioorg. Med. Chem. Lett. 14 (2004) 5997–6000. [11] J.M. Clements, F. Coignard, I. Johnson, et al., Antibacterial activities and characterization of novel inhibitors of LpxC, Antimicrob. Agents Chemother. 46 (2002) 1793–1799. [12] J. Zhang, L. Zhang, X. Li, W. Xu, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) inhibitors: a new class of antibacterial agents, Curr. Med. Chem. 19 (2012) 2038–2050. [13] C.J. Lee, X.F. Liang, X. Chen, et al., Species-specific and inhibitor-dependent conformations of LpxC: implications for antibiotic design, Chem. Biol. 18 (2011) 38–47. [14] X.F. Liang, C.J. Lee, X. Chen, et al., Syntheses, structures and antibiotic activities of LpxC inhibitors based on the diacetylene scaffold, Bioorg. Med. Chem. 19 (2011) 852–860. [15] J.I. Montgomery, M.F. Brown, U. Reilly, et al., Pyridone methylsulfone hydroxamate LpxC inhibitors for the treatment of serious gram-negative infections, J. Med. Chem. 55 (2012) 1662–1670. [16] U. Mo¨llmann, L. Heinisch, A. Bauernfeind, T. Ko¨hler, D. Ankel-Fuchs, Siderophores as drug delivery agents: application of the ‘‘Trojan Horse’’ strategy, Biometals 22 (2009) 615–624. [17] M.G.P. Page, C. Dantier, E. Desarbre, In vitro properties of BAL30072, a novel siderophore sulfactam with activity against multiresistant Gram-negative bacilli, Antimicrob. Agents Chemother. 54 (2010) 2291–2302. [18] M.E. Flanagan, S.J. Brickner, M. Lall, et al., Preparation, gram-negative antibacterial activity, and hydrolytic stability of novel siderophore-conjugated monocarbam diols, ACS Med. Chem. Lett. 2 (2011) 385–390. [19] X.F. Liang, C.J. Lee, J.S. Zhao, E.J. Toone, P. Zhou, Synthesis, structure, and antibiotic activity of aryl-substituted LpxC inhibitors, J. Med. Chem. 56 (2013) 6954–6966.
Please cite this article in press as: S. Ding, et al., Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.029
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CCLET 3258 1–5 Chinese Chemical Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet 1 2 3 4 5 6 7 8
Original article
Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus Ding a,1, Wen-Ke Wang b,1, Qiao Cao a, Wen-Jing Chu a, Le-Fu Lan a, Wen-Hao Hu b, Yu-She Yang a,*
Q1 Shi
a b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, East China Normal University, Shanghai 200062, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 December 2014 Received in revised form 2 February 2015 Accepted 17 March 2015 Available online xxx
In order to develop novel LpxC inhibitors with good activities and metabolic stability, two series of compounds with hydrophilic terminus have been synthesized and their in vitro antibacterial activities against Escherichial coli and Pseudomonas aeruginosa were evaluated. Especially, compounds 22b and c exhibited comparable antibacterial activities to CHIR-090 and better metabolic stability than CHIR-090 and LPC-011 in liver microsomes (rat and mouse), which indicated the terminal methylsulfone may be a preferred structure in the design of LpxC inhibitors and worthy of further investigations. ß 2015 Yu-She Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. All rights reserved.
Keywords: LpxC CHIR-090 Kojic acid derivatives Methylsulfone derivatives Metabolic stability
9 10
1. Introduction
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Drug resistant Gram-negative infections have been a serious threat to public health, especially for hospitalized patients. Unfortunately, while resistance to current therapies is spreading rapidly, new antibacterial agents to treat these infections are few in number [1]. Thus, there is a great need to develop novel antibacterial agents with new mechanisms of action. The outer membrane of the Gram-negative bacteria is a very efficient permeability barrier, obstructing the development of new antibiotics severely. Lipid A is a critical component of lipopolysaccharide (LPS), which mainly consists of the outer leaflet of the outer membrane [2]. The UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second-step of biosynthesis of lipid A in Gram-negative bacteria and is a validated antibiotic target [3,4]. Thus, inhibition of LpxC can obstruct the biosynthesis of lipid A, and sequentially kill most Gram-negative bacteria [2]. The X-ray crystal structure of LpxC from Aquifex aeolicus (aaLpxC) reveals a catalytic zinc ion and a hydrophobic tunnel accommodating a myristate fatty acid side chain [5,6]. Thus, most of the LpxC
* Corresponding author. E-mail address: [email protected] (Y.-S. Yang). 1 These authors contributed equally to this work.
inhibitors contain a zinc-binding group (e.g. hydroxamic acid) and a hydrophobic tail which mimics the myristate fatty acid chain. These lipophilic moieties make important van der Waals interactions with the enzyme in the hydrophobic tunnel, which is critical for the inhibition of LpxC [1]. Since the discovery of L-573,655 and L-161,240 (Fig. 1) in 1996 [4], LpxC inhibitors have been developed for two decades. The inhibitors in the early days mainly exhibited strong antibacterial activities against Escherichia coli (E. coli), but rarely inhibited the growth of Pseudomonas aeruginosa (P. aeruginosa) [4,7–11]. In 2004, Anderson et al. disclosed a new series of LpxC inhibitors, exemplified by CHIR-090 (Fig. 1), which displayed high affinity to both E. coli and P. aeruginosa LpxC and remarkable antibacterial activity against a wide range of Gram-negative pathogens [12]. On the basis of CHIR-090, Lee et al. [13] synthesized a series of compounds with linear diphenylacetylene scaffold which exhibited better antibacterial activities. In order to improve the watersolubility of these compounds, NH2 substituent was introduced in their distal phenyl ring, exemplified by LPC-011 (Fig. 1) [14]. LPC011 exhibited better water-solubility and antibacterial activity, while the metabolic data was not optimistic (the data will be mentioned later). In 2012, Pfizer reported a series of novel LpxC inhibitors with methylsulfone, exemplified by 1a (Fig. 1). 1a exhibited excellent antibacterial activity against P. aeruginosa but suffered from the problems of low water-solubility and high protein binding [1]. Then the central phenyl ring of 1a was replaced
http://dx.doi.org/10.1016/j.cclet.2015.03.029 1001-8417/ß 2015 Yu-She Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. All rights reserved.
Please cite this article in press as: S. Ding, et al., Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.029
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
G Model
CCLET 3258 1–5 S. Ding et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
2
HO O
n1 = 0, 1, 2; n2 = 0, 1; n 3 = 1, 2, 3
H N
N H
OH N
OH
O
n1 R
HO
n2
O
H N
R= ,
HO
,
O
O
O S O
n3
Series 2
Series 1
Fig. 2. Two series of novel LpxC inhibitors.
Fig. 1. The structures of classic LpxC inhibitors.
O
O
O O
a
O
HO O OH
b
c
N H
I Si
1
3
2
O
e
d
HO
PMBO
OH
Ph 2HCO
h
7 O
Ph 2HCO
OH
O
i
Ph 2HCO
OH
N OH 9
8
OH
N
O
O g
f N H 6
5
OH
O
OPMB
PMBO
OH
O
Kojic acid
4
O
O PMBO
O O
N OCHPh2 10
OH
Scheme 1. Conditions and reagents: (a) Trimethylsilylacetylene, PdCl2(PPh3)2, CuI, Et3N, THF, r.t., 98%; (b) NaOH, MeOH, r.t., 80%; (c) L-Threonine methyl ester hydrochloride, EDCI, HOBt, DIEA, DMF, r.t., 70%; (d) Kojic acid, PMBCl, K2CO3, DMF, 80 8C, 80%; (e) NH3H2O, EtOH, 70 8C, 70%; (f) PMBCl, K2CO3, DMF, 80 8C, 56.2%; (g) Ph2CN2, EtOH, 40 8C, 77%; (h) NH2OHHCl, CH3COONa3H2O, EtOH/H2O, 70 8C, 44%; (i) Ph2CHCl, K2CO3, NaI, DMSO, r.t., 98%.
HO O
HO O R
OH
N H
+ n
HO
O
a
O
N H R
n = 1, 12 n = 2, 13
11a-d
n
O
HO O O
b
N H
O
R
n
O
14a-h
H N
HO O OH
O
R:
O S O G1
,
O S O G2
, PMBO
, Ph2HCO O G3
H N ;
, HO
, HO OPMB
O
O
G4
G5
G6
11a 11b 11c 11d
G1 G2 G3 G4
n
O
1 5a -h
OH N
N
N H R
H N
OH
O
1 6a -d
R OCHPh 2 N
c
14a, 14b, 14c, 14d, 14e, 14f , 14g, 14h,
15a 15b 15c 15d 15e 15f 15g 15h
n
R
1 2 1 2 1 2 1 2
G1 G1 G2 G2 G3 G3 G4 G4
16a 16b 16c 16d
n
R
1 2 1 2
G5 G5 G6 G6
Scheme 2. Conditions and reagents: (a) DEAD, PPh3, THF, r.t., 40–60%; (b) NH2OHH2O, DCM/MeOH, r.t., 40–80%; (c) TFA, DCM, r.t., 70–90%.
Scheme 3. Conditions and reagents: (a) DEAD, PPh3, THF, r.t., 40–60%; (b) 4, PdCl2(PPh3)2, CuI, Et3N, THF, r.t., 80–90%; (c) Trimethylsilylacetylene, PdCl2(PPh3)2, CuI, Et3N, THF, r.t., 80–90%; (d) TBAF, THF, r.t., 40–90%; (e) 4, Cu(Ac)2, Pyridine/MeOH, r.t., 20–30%; (f) NH2OHH2O, DCM/MeOH, r.t., 40–80%; (g) TFA, DCM, r.t., 70–90%.
Please cite this article in press as: S. Ding, et al., Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.029
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CCLET 3258 1–5 S. Ding et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
3
Table 1 In vitro antibacterial activities of synthetic compounds. HO O N H
H N
OH
O
n1 R
O
n2
n3
Compound
MIC (mg/mL)
Structure n1
n2
n3
R
P. aeruginosa PAO1
P. aeruginosa PA14
E. coli DH5a
E. coli AB1157
15a
0
0
3
O S O
>1000
>1000
>1000
>1000
15b
0
1
3
O S O
15c
0
0
2
O S O
>1000
>1000
>1000
>1000
15d
0
1
2
O S O
250
125
50
25
16a
0
0
1
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
3.125
OH N
3.125
2.100
2.100
HO O
16b
0
1
1
OH N HO O
16c
0
0
1
H N HO O
16d
0
1
1
H N HO O
22a
2
1
3
O S O
22b
1
1
3
O S O
1.560
1.200
0.195
0.195
22c
1
1
2
O S O
0.780
1.560
0.195
0.195
23a
1
1
1
OH N
100
30
25
25
>1000
>1000
50
50
50
50
50
50
HO O
23b
2
1
1
H N HO O
23c
1
1
1
H N HO O
CHIR-090
–
–
–
–
1.250
1.250
0.300
0.200
LPC-011
–
–
–
–
0.310
0.310
0.040
0.040
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Table 2 In vitro antibacterial activities of CHIR-090, LPC-011, 23a and c under standard condition and low-iron condition. Compound
23a 23c CHIR-090 LPC-011 a b
P. aeruginosa PAO1
P. aeruginosa PA14
E. coli DH5a
E. coli AB1157
Standarda
Low-ironb
Standard
Low-iron
Standard
Low-iron
Standard
Low-iron
100 50 1.250 0.310
50 25 0.400 0.200
50 50 1.250 0.310
25 25 0.400 0.200
25 40 0.156 0.019
10 10 0.080 0.010
25 40 0.156 0.019
10 10 0.080 0.010
The compounds were tested in the normal LB media by the standard process. The compounds were tested in the low-iron LB media by the standard process.
56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73
with pyridine (e.g. 2a in Fig. 1), and this series of compounds exhibited excellent Gram-negative antibacterial activity, improved solubility and increased free fraction. However, the half-life of these compounds in rats was shortened [15]. As discussed above, the balance of antibacterial activities and metabolic properties is a major challenge to the development of LpxC inhibitors. Thus, we designed and synthesized two series of compounds with hydrophilic terminus mainly aimed to achieve novel LpxC inhibitors with improved activity, metabolic stability and water solubility. The terminus of series 1 are kojic acid derivatives, which can deliver the compound through the outer membrane of the Gram-negative bacteria as a siderophore by the Trojan Horse approach [16]. Meanwhile, Kojic acid derivatives with NH3 and NH2OH substitution are proved effective siderophores [17,18]. The terminus of series 2 is methylsulfone, which is a steady group for metabolism. Hydrophobic tails with different length have also been synthesized to match the hydrophobic tunnel (Fig. 2).
74
2. Experimental
75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106
The synthetic route of intermediates 4, 7 and 10 was outlined in Scheme 1. Compound 2 was obtained by the coupling reaction of compound 1 and trimethylsilyacetyene in THF. The hydrolyzation and deprotection of compound 2 under alkaline condition afforded the acid compound 3, and then treated with l-threonine methyl ester hydrochloride in the condition of condensation reaction to afford compound 4. Protection of Kojic acid with 4-methoxybenzylchloride gave compound 5 and then treated with ammonium hydroxide to give compound 6. The reaction of compound 6 with 4-methoxybenzylchloride afforded the aromatization product compound 7. Compound 8 was obtained from the Kojic acid and diphenyldiazomethane under alkaline condition, then treated with alkalized hydroxylamine hydrochloride to yield compound 9. The reaction of compound 9 with diphenyldiazomethane afforded compound 10. pt?>The synthetic route of compounds 15a–d and 16a–d was outlined in Scheme 2. Compounds 14a–h were obtained by the Mitsunobu reaction of RCH2OH and compounds 12 and 13, then treated with NH2OH (50% in water) and yielded compounds 15a–h. Deprotection of compounds 15e–h in trifluoroacetic acid yielded compounds 16a–d. The synthetic route of compounds 22a–c and 23a–c was outlined in Scheme 3. Compounds 18a–d were obtained by the Mitsunobu reaction of RCH2OH and 4-iodoaniline. Compounds 19a and b were obtained by the coupling reaction of compounds 18a and d with trimethylsilyacetyene in THF, respectively. Then 19a and b were treated with tetrabutylammonium fluoride to yield compounds 20a and b. Compounds 21a–f were obtained by the coupling reaction of compound 4 with compounds 18a–d and 20a and b, then treated with NH2OH (50% in water) to yield compounds 22a–f. Deprotection of compounds 22d–f in trifluoroacetic acid yielded compounds 23a–c.
3. Results and discussion
107
Minimum inhibitory concentration (MIC) of each compound was tested by the modified National Committee for Clinical Laboratory Standards, which is adapted to 96-well plates and LB media in the presence of 5% DMSO [19]. Tested bacterial strains were classic Gram-negative pathogens, including P. aeruginosa (PAO1, PA14) and E. coli (AB1157, DH5a). The MIC values of all tested compounds were listed in Table 1. Unfortunately, most compounds of series 1 lost their antibacterial activities. Only the compounds 23a and c with diphenylacetylene scaffold exhibited weak antibacterial activities against E. coli and P. aeruginosa. In series 2, compounds with biphenyl scaffold (15b) and diphenylacetylene scaffold (22b and c) showed remarkable antibacterial activities against both E. coli and P. aeruginosa. Especially, compounds 22b and c exhibited comparable antibacterial activities to CHIR-090. In both series, the length of the hydrophobic tails also played an important role: too long (diacetylene scaffold) or too short (phenyl scaffold) would lead to a loss of antibacterial activity. Moreover, we compared the antibacterial activities of compounds 23a and c, CHIR-090 and LPC-011 both under standard condition and low-iron condition. As shown in Table 2, all of the tested compounds exhibited better antibacterial activities under low-iron condition. Unexpectedly, similar improvements were observed in the antibacterial activities of tested compounds. It is speculated that the hydroxamic acid group can serve as a potent siderophore to help the inhibitors entering the outer membrane of the bacteria and the extra siderophore (kojic acid derivative) does not influence this process obviously. The detailed mechanism is not clear and needs further biological investigations. In addition, we evaluated the metabolic stability of compounds 22b and c, CHIR-090 and LPC-011 in liver microsomes (rat and mouse). As shown in Table 3, compounds 22b and c exhibited better metabolic stability than CHIR-090 and LPC-011, which
108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139
Table 3 Half-lives and intrinsic clearances of CHIR-090, LPC-011, 22b and c in liver microsomesa Compound
Species
Microsomal t1/2 b (min)
Clint c (mL min protein 1)
22b
Rat Mouse Rat Mouse Rat Mouse Rat Mouse
2226.3 314.2 –d 328 171.2 88.9 75.8 80.9
1 7 0 6 12 24 28 26
22c CHIR-090 LPC-011
1
mg
a 0.33 mg/mL microsomal protein, NADP+-regenerating system, [inhibitor], 0.1 mmol/L, incubation at 37 8C, samples taken at 0, 7, 17, 30, and 60 min, determination of parent compound by MS. b t1/2: elimination half-life in rat liver microsomes. c Clint: intrinsic body clearance. d t1/2 was not calculated, because compound 22b was not metabolized at 60 min.
Please cite this article in press as: S. Ding, et al., Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.029
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140 141
indicated that the terminal methylsulfone may be a preferred structure in the design of LpxC inhibitors.
142
4. Conclusion
143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158
In conclusion, two series of novel LpxC inhibitors containing the terminal kojic acid derivatives and methylsulfone were synthesized and evaluated. The MIC values indicated that the length of the hydrophobic tail played an important role and the compounds with diphenylacetylene scaffold exhibited best antibacterial activities. In series 1, most of the compounds lost their antibacterial activities and the introduction of kojic acid derivatives did not provide extra improvement of antibacterial activities obviously compared to CHIR-090 and LPC-011 under low-iron condition. In series 2, some compounds, especially 22b and c, exhibited comparable antibacterial activities to CHIR-090 against E. coli and P. aeruginosa and better metabolic stability than CHIR-090 and LPC-011 in liver microsomes (rat and mouse). These results manifested the terminal methylsulfone may be a preferred structure in the design of LpxC inhibitors and worthy of further investigations.
159
Acknowledgments
160 161 162 163
We are grateful to National Science and Technology Major Project for the support of this research. The project described was Q2 supported by Key New Drug Creation and Manufacturing Program, China (No. 2014ZX09507009-016).
164
Appendix A. Supplementary data
165 166
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2015.03.029.
167
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168 169
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Please cite this article in press as: S. Ding, et al., Design, synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.029
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