Design, Synthesis, and Biological Evaluation of Novel DNA Gyrase

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Design, Synthesis and Biological Evaluation of Novel DNA GyraseInhibiting Spiropyrimidinetriones as Potent Antibiotics for Treatment of Infections Caused by Multidrug-Resistant Gram-Positive Bacteria Chenghui Shi, Yinyong Zhang, Ting Wang, wenchao lu, Shuhua Zhang, Bin Guo, Qian Chen, Cheng Luo, Xian-Li Zhou, and Yushe Yang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01750 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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Journal of Medicinal Chemistry

Design, Synthesis and Biological Evaluation of Novel DNA Gyrase-Inhibiting Spiropyrimidinetriones as Potent Antibiotics for Treatment of Infections Caused by Multidrug-Resistant Gram-Positive Bacteria Chenghui Shi, †,‡,|| Yinyong Zhang, †,‡,|| Ting Wang, § Wenchao Lu, †,‡ Shuhua Zhang, § Bin Guo, †,‡ Qian Chen,†, ‡ Cheng Luo, †,‡ Xianli Zhou*,‡ and Yushe Yang*,†,‡ †State

Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences, Shanghai 201203, China. ‡University ‡School

of Chinese Academy of Sciences, Beijing 100049, China.

of Life Science and Engineering, Southwest Jiaotong University, Chengdu,

Sichuan Province 610031, China. §Department

of Microbiology, Sichuan Primed Bio-Tech Group Co., Ltd., Chengdu,

Sichuan Province 610041, China. ABSTRACT: Spiropyrimidinetriones are a novel class of antibacterial agents that target the bacterial type II topoisomerase via a new mode of action. Compound ETX0914 is thus far the only drug from this class that is being evaluated in clinical trials. To improve the antibacterial activity and pharmacokinetic properties of ETX0914, we carried out systematic structural modification of this compound, and a number of compounds with increased potency were obtained. The most promising compound 33e, with incorporation of a spirocyclopropane at the oxazolidinone 5 position reduced metabolism, exhibited excellent antibacterial activity against Gram-positive pathogens and a good

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pharmacokinetic profile combined with high aqueous solubility. In addition, compound 33e exhibited good selectivity for Staphylococcus aureus gyrase over human Topo IIα. In a murine model of systemic MRSA infection, 33e exhibited superior in vivo efficacy (ED50 = 3.87 mg/kg) compared to ETX0914 (ED50 = 11.51 mg/kg). 

INTRODUCTION Antibacterial drugs, including β-lactams, glycopeptides, aminoglycosides, macrolides

and fluoroquinolones, have saved innumerable human lives over the past 80 years, but the continuing emergence of drug-resistant bacteria has limited the use of many antibiotic drugs.1,

2

In contrast to the growing antibacterial resistance, few new antibacterial

compounds have entered the drug market during the last two decades.3, 4 Recently, the World

Health

Organization

(WHO)

published

the

global

priority

list

of

antibiotic-resistant bacteria to guide the research, discovery and development of new antibiotics.5 In addition to some worrisome Gram-negative pathogens, Gram-positive pathogens,

including

vancomycin-resistant

methicillin-resistant Enterococci

(VRE)

Staphylococcus and

aureus

(MRSA),

penicillin-resistant Streptococcus

pneumoniae (PRSP) are also causes for concern. Among these pathogens, MRSA is one of the main causes of hospital-acquired infections and community-acquired infection.6 One of the most effective ways to address bacterial resistance is to develop antibacterial agents that have new targets or new modes of action to avoid cross-resistance to the drugs currently in clinical use. Bacterial DNA type II topoisomerases, including DNA gyrase and topoisomerase IV,


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are the traditional targets for both Gram-positive and Gram-negative bacteria.7-9 Spiropyrimidinetriones (SPTs) are a new class of antibacterials that inhibit bacterial DNA gyrase and topoisomerase IV and exhibit no cross-resistance with fluoroquinolones because of differences in the modes of action of these compounds.10-12 QPT-1 (1) (Figure 1) was the first compound of this class, which was discovered via high-throughput screening at Pharmacia Upjohn and determined to be an inhibitor of DNA gyrase by reverse genomics.13 Then, scientists at Pfizer explored the structure–activity relationship (SAR) of 1 including via placement of various substituents at different positions on the benzene ring or modification of the spirocyclic pyrimidinetrione pharmacophore and the morpholine ring.14, 15 Scientists at AstraZeneca synthesized a series of compounds using a benzisoxazole scaffold to replace the benzene ring and identified active compound 2. However, compound 2 did not enter clinical trials due to bone marrow toxicity and genotoxicity.16 After further examination of the SARs, compound 3 (zolifodacin, ETX0914, AZD0914), which has an oxazolidinone substituent on the benzisoxazole ring, was developed; this compound did not exhibit bone marrow toxicity and genotoxicity and had improved antibacterial activity.17, 18 Compound 3 was shown to be effective against bacteria that were resistant to other topoisomerase inhibitors as well as against fastidious Gram-negative bacteria, including Haemophilus influenzae and Neisseria gonorrhoeae. Based on these factors in addition to a clean toxicity profile and efficacy in a murine Staphylococcus aureus infection model, compound 3 became the first compound of this class that entered clinical trials for anti-gonorrhea therapy and one phase II clinical trial



Page 4 of 83

has been completed. However, the moderate antibacterial activity and suboptimal pharmacokinetic (PK) properties (high plasma protein binding and high clearance rate) of 3 resulted in high doses (2 or 3 g) in clinical trials. High doses in usually lead to some problems in humans, including low patient compliance and idiosyncratic drug toxicity.19 Hence, the identification of a compound with high antibacterial activity and improved PK properties is necessary. H N

O O2 N

O NH

N

H

1 (QPT-1)

O O

O

H3 C

O NH

N O

N F

O H

5

O

H N

O

2

O

4

H N

O

N

O NH

N O

N F

O H

O

3 (zolifodacin, ETX0914, AZD0914)

Figure 1. SPTs. Co-crystal structures of DNA-cleavage complexes for compound 1 has been reported to reveal the main interactions between spirocyclic pyrimidinetrione pharmacophore and DNA gyrase.20 However, there is a lack of clear understanding regarding the interactions between other moieties on SPTs and gyrase. Herein, we used traditional medicinal chemistry strategies such as isostere replacement and structural fine-tuning to design a series of SPTs and tested the antibacterial activities of these compounds by phenotypic screening in different strains. Optimized compounds with superior antibacterial activity were selected for further testing of their PK properties in rodent models. A number of novel compounds with superior antibacterial activities and PK properties have been discovered, including the preclinical candidate 33e, which qualifies for further evaluation.


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

CHEMISTRY The key reaction in the synthesis of this class of compounds was the tertiary amino

effect reaction (T-reaction)21-23 using aromatic aldehyde with ortho-dialkylamine and barbituric acid, which has been previously described. As shown in Figure 2, 2,6-dimethylmorpholine with different configurations yielded various mixtures with different diastereomeric ratios, wherein the (2R,4S,4aS) isomer was the active ingredient.16 R1

H

R2

N R3

O

H

H N

O

O R1

NH

4aS

R2

N R3

H N

O

O R1

4aR

O

R2

N R3

O 4S

H

O NH O

O 4R

2R

2R

major

O R1

H

R2

R2 O

N R3

H

O NH

4aS

N R3

H N

O R1

O O

4S

2R

H N

O R1

NH

4aR

R2

N R3

H

O

O O

H N

O R1

NH

4aR

R2

4R

2S

N R3

H

2R

O O

4R

H N

O

O R1

4aS

R2

N R3

O NH O

H

O 4S

2S

major

major

Figure 2. T-reaction for the synthesis of SPTs. Considering the high cost of (2R,6R)-2,6-dimethylmorpholine, compounds (±)-16a–d, (±)-20,

(±)-24

and

(±)-33a–e

were

synthesized

as

racemates

using

cis-2,6-dimethylmorpholine. After acquiring active racemic (±)-33e, enantiomer 33e was synthesized using (2R,6R)-2,6-dimethylmorpholine, and the other final compounds with the active configuration (2R,4S,4aS) were obtained using this method. The absolute configuration of the final compounds was confirmed by a crystal structure of 39e (Figure 3).



Page 6 of 83

S O

H N

O

N

O NH

N O

N F

H

O O

39e

Figure 3. Single-crystal X-ray structure of 39e. The synthesis of the fluoroquinazoline series (±)-16a–d is shown in Scheme 1. 2,3,4-Trifluorobenzoic acid 4 was treated with lithium diisopropylamide (LDA) followed by the addition of DMF to afford product 5, which formed ester 6 upon reaction with iodomethane. Protection of the aldehyde group on 6 with ethylene glycol gave compound 7. Compound 8 was prepared by a selective SNAr reaction of the fluorine adjacent to the methyl ester with ammonia. Hydrolysis of the ester by treatment of 8 with a 2 M NaOH aqueous solution followed by ethylene glycol deprotection using a 2 M HCl aqueous solution gave compound 9. cis-2,6-Dimethylmorpholine substituted the fluorine to afford compound 10. The aldehyde on 10 was again protected with O-methylhydroxylamine hydrochloride to afford compound 11, which was subsequently converted to fluoroquinazoline 12 with formamidine acetate. Chlorination of fluoroquinazoline with phosphorus oxychloride afforded the key intermediate 13. Compounds 14a, 14b and 14d were prepared via SNAr reactions or Buchwald-Hartwig cross-coupling of compound 13 with corresponding nucleophiles. Subsequent O-methylhydroxylamine deprotection using dilute hydrochloric acid gave compounds 15a, 15b and 15d. Compound 15c was


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prepared by SNAr reaction of 13 with thiomorpholine, and then, O-methylhydroxylamine was deprotected with dilute hydrochloric acid directly. Finally, compounds 15a–d were converted to compounds (±)-16a–d via T-reactions with barbituric acid. Scheme 1. Synthesis of Compounds (±)-16a–da O

O HO

a F

O

F

F

O

O

H2 N

F

HO

F

O

O

H2 N

N 10

O

R

N

k F 13

N

OH i

R m

N

c

b

N

O

O

O

H N

O NH

N

F 15a-d

O

N F 12

O

N

N

N O

N

O

F

N

N

R

N

a

H2 N

O

F 11

O

F 14a-d

O

F 8

HO

l N

O

d

F

N

H2 N

N

N

N

O

O

O

F 7

O

9

N

F

F

h F

O O

F 6

F

Cl j

F

O

O

c

HO

g

O

O

O

F 5

F 4

e,f

O b

HO

O N H F O (+)-16a-d

d O

R:

αReagents

CH3

N

O

N

S

N

O

and conditions: (a) LDA, DMF, THF, –78 °C, 77%; (b) CH3I, K2CO3, DMF, rt,

59%; (c) ethylene glycol, p-toluenesulfonic acid, toluene, 140 °C, 80%; (d) 7 M NH3 in MeOH, sealed tube, 90 °C, 42%; (e) 2 M NaOH aqueous solution, EtOH, 40 °C; (f) 2 M HCl aqueous solution, EtOH, rt, 90% (for two steps); (g) cis-2,6-dimethylmorpholine, DIPEA, CH3CN, 90 °C, 85%; (h) methoxyammonium chloride, Et3N, MeOH, rt, 87%; (i) formamidine acetate, 1,4-dioxane, MeOH, microwave at 90 °C, 70%; (j) POCl3, Et3N, toluene, 105 °C, 84%; (k) for 14a: CH3MgBr, THF, Ar, 0 °C to rt, 64%; for 14b: morpholine, THF, rt, 78%; for 14c: thiomorpholine, THF, rt; for 14d: 3-ketomorpholine,



Page 8 of 83

dimethylbisdiphenylphosphinoxanthene, Pd2(dba)3, Cs2CO3, 1,4-dioxane, 100 °C, 45%; (l) 2 M HCl aqueous solution, formaldehyde, CH3COOH, acetone, rt, 79% for 15a, 80% for 15b, 66% for 15c (for two steps), 46% for 15d; (m) barbituric acid, CH3COOH, toluene, 100 °C, 50–87%. Originally we attempted to use this procedure to synthesize compound (±)-20. Unfortunately, the 1,2,4-triazole was removed under the conditions used for O-methylhydroxylamine

deprotection.

Therefore,

we

deprotected

the

O-methylhydroxylamine before chlorination of intermediate 12 to obtain compound 17 (Scheme 2). Then, chlorination of 17 afforded compound 18. Subsequently, 1,2,4-triazole substituted the chlorine to give compound 19, which was then converted to compound (±)-20 by T-reaction with barbituric acid. Scheme 2. Synthesis of Compound (±)-20a N OH

N

O

OH

N

a F 12

αReagents

O

Cl

N

b N

N

N

O

17

O

N

N

c N

N F

O

O

N

N

N F 18

N N

d N

O

O

H N

O

N

O NH

N

N F 19

N

N F (+)-20

H

O O

and conditions: (a) 2 M HCl aqueous solution, formaldehyde, CH3COOH,

acetone, rt, 90%; (b) POCl3, Et3N, toluene, reflux, 33%; (c) 1,2,4-triazole, THF, 80 °C, 89%; (d) barbituric acid, CH3COOH, toluene, 100 °C, 51%. Compound (±)-24 was prepared from compound 13, which reacted with ethanolamine to afford compound 21 (Scheme 3). Then, compound 21 was treated with CDI to give compound 22, which was then converted to (±)-24 in the same manner as described for


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compounds (±)-16a–d. Scheme 3. Synthesis of Compound (±)-24a HO Cl

N

O

N

N

a

αReagents

O

N

O

O

N

b F 21

O

N

N

N O

F 13

O

N

N

N

O

HN

c

O

F 22

O

O

N

N

N

O

N

d

N

N

N O

F 23

H N

O

N

O NH

N

N O

F (+)-24

H

O O

and conditions: (a) ethanolamine, THF, rt, 93%; (b) CDI, DMF, 105 °C, 66%;

(c) HCl aqueous solution, formaldehyde, CH3COOH, acetone, rt, 48%; (d) barbituric acid, CH3COOH, toluene, 100 °C, 73%. The procedures for the synthesis of compounds 26a–c were initiated from compound 25, as described by Gregory S. Basarab and coworkers17, by using different isosteres or analogs of barbituric acid in the T-reaction (Scheme 4). Scheme 4. Synthesis of Compounds 26a–ca O O

H N

O

N

S NH

N O

N F

O H

O

26b

b O O

H O N S O NH

O

N N O

N F 26a

αReagents

O H

O

O a

O

O O

N

c N

O

O

N

N

N

O

O

N F

O

25

O

N

N F

O H

O

26c

and conditions: (a) 1,2,6-thiadiazinane-3,5-dione 1,1-dioxide, CH3COOH, H2O,

110 °C, 74%; (b) 2-thioxodihydropyrimidine-4,6(1H,5H)-dione, CH3COOH, H2O, 110 °C, 55%; (c) 1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione, CH3COOH, H2O, 110 °C, 80%.



Page 10 of 83

General procedures for the synthesis of compounds 33a–h are depicted in Scheme 5. Compound 2717 was chlorinated with NCS, and then, appropriate amines were added to substitute the chlorine atom, followed by the addition of Cs2CO3 to form the benzisoxazoles 28a–h, which were treated with CDI or 1,1'-thiocarbonyldiimidazole to obtain compounds 29a–h. Subsequent removal of the ethylene glycol in the acid gave compounds

30a–h.

Nucleophilic

displacement

of

the

fluorine

with

cis-2,6-dimethylmorpholine or (2R,6R)-2,6-dimethylmorpholine afforded compounds 31a–e or compounds 32e–h. Finally, the T-reaction of compounds 31a–e and 32e–h with barbituric acid yielded compounds (±)-33a–e and 33e–h. Scheme 5. Synthesis of Compounds 33a–ha O HO

F

O

R O

N

a,b,c

N O

F F 27

O

R O

d

O

N O

F F 28a-h

F F 29a-h

e H N

O

R

O NH

N O

N F 33e-h

h

g O

O

O

28b

28c

28d

αReagents

N H

N H

N H

N H

N F 30a-h

OH

28e

F 31a-e

OH

28f

OH

28g

OH

28h

OH

N H

29-31a,33a

29-31b,33b O

29-33e N O N

OH

N H

OH

N H O

29-31c,33c

O

O N

29-31d,33d

S

O

O

O N

29f,30f, 32f,33f

O

29g,30g, 32g,33g

O

29h,30h, 32h,33h

O

O N O N

O N

O H

R:

O O

N F (+)-33a-e

O

R: OH

N H

O

N

O NH

N

N O

H N

O

R h

F

R:

R:

O

R f

O

N F 32e-h

28a

O

R

N

O H

O

R

O

O

N

and conditions: (a) NCS, DMF, 40 °C; (b) appropriate amines, DMF, 0 °C to rt;

(c) Cs2CO3, DMF, rt to 40 °C, 53–76% (for three steps); (d) for 29a–c and 29e–h: CDI,


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DMAP, DMF, 100 °C, 63–91%; for 29d: 1,1'-thiocarbonyldiimidazole, DMAP, DMF, 100 °C, 40%; (e) for 30a–g: 6 M HCl aqueous solution, 1,4-dioxane, rt, 80–99%; for 30h: 4 M H2SO4 aqueous solution, 1,4-dioxane, rt, 75%; (f) cis-2,6-dimethylmorpholine, DIPEA, CH3CN, 90 °C, 41–69%; (g) (2R,6R)-2,6-dimethylmorpholine, DIPEA, CH3CN, 90 °C, 41–88%; (h) barbituric acid, CH3COOH, H2O, 110 °C, 43–86%. General procedures for the synthesis of compounds 39a–o are depicted in Scheme 6. Compounds 34a–m were prepared using compound 27 via similar procedures as those used for compounds 28a–h. Compounds 35a–o were obtained by the Mitsunobu reaction with thioacetic acid, 28c–d and 34a–m. Hydrolysis of the thioester in compounds 35a–o using NaOH yielded the corresponding thiol anions, which reacted with CDI to afford compounds 36a–o. The subsequent steps to obtain 39a–o from 36a–o were similar to the procedures described for 33e. Scheme 6. Synthesis of Compounds 39a–oa



Page 12 of 83

O HO O HO

F

a,b,c

g

O

HN O

N

R

S O

2

d

O

N

O

O

O

F F 36a-o

R

S

O

O

H N

O

N

O NH

N O

N F

F

O

N

F

O

O

O

i

37a-o

4

N

F 35a-o

N

F

e,f

O O

N

O h

N

2

R

S

R

S

N

F F 28c-d,34a-m

F

R

S

O

F 27

5

1

R

HN O

N

1

O

N F

38a-o

H

O O

39a-o

35-39a R=H 34a,35b 2S, R = 2-CH3

36-39b 4S, R = 4-CH3

34b,35c 2R, R = 2-CH3

36-39c 4R, R = 4-CH3

34c 1S, R = 1-CH3; 35d 1R, R = 1-CH3

36-39d 5R, R = 5-CH3

34d 1R, R = 1-CH3; 35e 1S, R = 1-CH3

36-39e 5S, R = 5-CH3

34e,35f 2S, R = 2-CH2CH3

36-39f 4S, R = 4-CH2CH3

34f,35g 2R, R = 2-CH2CH3

36-39g 4R, R = 4-CH2CH3

34g 1S, R = 1-CH2CH3; 35h 1R, R = 1-CH2CH3

36-39h 5R, R = 5-CH2CH3

34h 1R, R = 1-CH2CH3; 35i 1S, R = 1-CH2CH3

36-39i 5S, R = 5-CH2CH3

34i 1R, R = 1-phenyl; 35j 1S, R = 1-phenyl

36-39j 5S, R = 5-phenyl

34j,35k 1S, R = 2-benzyl

36-39k 4S, R = 4-benzyl

35l R = 2-CH2CH2-2

36-39l R = 4-CH2CH2-4

34k 1S,2S, R = 1-CH2CH2CH2-2; 35m 1R,2S, R = 1-CH2CH2CH2-2

36-39m 4S,5R, R = 4-CH2CH2CH2-5

34l 1S,2S, R = 1-CH2CH2CH2CH2-2; 35n 1R,2S, R = 1-CH2CH2CH2CH2-2 34m 1R,2R, R = 1-CH2CH2CH2-2; 35o 1S,2R, R = 1-CH2CH2CH2-2

36-39n 4S,5R, R = 4-CH2CH2CH2CH2-5 36-39o 4R,5S, R = 4-CH2CH2CH2-5

αReagents

and conditions: (a) NCS, DMF, 40 °C; (b) corresponding amine, DMF, 0 °C to

rt; (c) Cs2CO3, DMF, rt to 40 °C, 46–88% (for three steps); (d) PPh3, DIAD, thioacetic acid, THF, 0 °C, 49–99%; (e) 2 M NaOH aqueous solution, DL-1,4-dithiothreitol, MeOH, rt; (f) CDI, DMAP, DMF, 100 °C, 26–93% (for two steps); (g) 6 M HCl aqueous solution, 1,4-dioxane, rt, 56–100%; (h) (2R,6R)-2,6-dimethylmorpholine, DIPEA, CH3CN, 90 °C, 39–87%; (i) barbituric acid, CH3COOH, H2O, 110 °C, 59–84%. Synthesis of compound 50 was also performed via the key intermediate 27 by using the similar

procedures

as

described

for

compounds

39a–o

(Scheme

7).

Tert-butyldimethylsilyl chloride (TBSCl) was used to protect the primary alcohol to


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afford compound 41. Mitsunobu reaction of compound 41 with thioacetic acid yielded compound 42. Hydrolysis of the thioester and treatment with CDI yielded compound 43. Deprotection of ethylene glycol and TBS under acidic conditions yielded compound 44. The subsequent procedures were similar to the synthesis of 33e. The synthesis of compound 51 was initiated by methylation of intermediate 44 with trimethyloxonium tetrafluoroborate

to

yield

compound

46,

which

was

reacted

with

(2R,6R)-2,6-dimethylmorpholine to afford compound 47 (Scheme 7). The T-reaction of compound 47 with barbituric acid yielded compound 51. Methyl sulfonylation of intermediate 45 to compound 48 followed by displacement with the azide group afforded compound 49, which reacted with barbituric acid to yield compound 52 (Scheme 7). Compound 69 was obtained by reduction of 52 to 68 followed by acetylation of the amine group with acetic anhydride. Scheme 7. Synthesis of Compounds 50–52 and 69a



OH O

F

a,b,c

O

O

O

N O

O

N O

h

O

O l

O

O

o

O

52

NH2

69

NH

O

46

N F

F

O H

50,51,52,68,69 i

O NH

O

F

S

H N

N O

N3

68 p

N3

N

O

N 51

m

S

N

O

48

R

O

OH

N F

O

j

S

50

O

45

k

R:

N

N F

F 44

O

N

O

N

F

F 43

OMs S

N

O i

O

O

F F 42

OH

N

F

O

S

N

O

O

N

41

S O

e

F F

OH

S

O

HN O

F 40 OTBS

f,g

d

F

F 27

O

S

HN O

N

F

N

OTBS

HO

HN O

N

O

OTBS

HO HO

Page 14 of 83

O

O n

O

N N O

N F

O

49

n

O

O S O

O

N N O

N F 47

αReagents

O

and conditions: (a) NCS, DMF, 40 °C; (b) (S)-3-aminopropane-1,2-diol, DMF,

0 °C to rt; (c) Cs2CO3, DMF, rt to 40 °C, 77% (for three steps); (d) TBSCl, imidazole, DMF, rt, 74%; (e) PPh3, DIAD, thioacetic acid, THF, 0 °C, 86%; (f) 2 M NaOH, DL-1,4-dithiothreitol, MeOH, rt; (g) CDI, DMAP, DMF, 100 °C, 73% (for two steps); (h) 6 M HCl aqueous solution, 1,4-dioxane, rt, 74%; (i) (2R,6R)-2,6-dimethylmorpholine, DIPEA, CH3CN, 90 °C, 49–66%; (j) barbituric acid, EtOH, 6 M HCl aqueous solution, 100 °C, 87%; (k) trimethyloxonium tetrafluoroborate, 2,6-bis(1,1-dimethylethyl)pyridine, DCM, rt, 99%; (l) MsCl, DIPEA, DCM, 0 °C to rt, 87%; (m) NaN3, DMF, 70 °C, 90%;


Page 15 of 83 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 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 56 57 58 59 60


(n) barbituric acid, CH3COOH, H2O, 110 °C, 40–85%; (o) PPh3, THF, H2O, rt, 62%; (p) acetic anhydride, DIPEA, DCM, EtOAc, rt, 86%. Compounds 57, 61 and 67 were also prepared via the same intermediate 43 (Scheme 8). Removal

of

TBS

from

43

yielded

compound

53.

Then,

fluorination

or

trifluoromethylation of the hydroxyl group afforded compound 54 or 58, respectively, which was then converted to 57 or 61 in the same manner as that described for 33e. Methyl sulfonylation of compound 53 afforded compound 62. Sodium thiomethoxide replaced the methanesulfonate to form compound 63, which was converted to sulfone 64 by oxidation of the thioether. The subsequent procedures were the same as those described for the synthesis of 33e, yielding compounds 67. Scheme 8. Synthesis of Compounds 57, 61 and 67a R

OH O

N

O

N

O

N

b or f

O

c

O

O

O O

N O

O

F F 43

αReagents

S O

N

O O

N O

F F 62

h

N

O O

O

F

i

N N F 57,61,67

O H

O

R: O

N

54,55,56,57 O

N O

F 63

O NH

O S O

S O

N

O

F 56,60,66

H N

O

N

O

N

S

S

S

O

N

F

c OMs

OTBS N

O e

F 55,59 ,65

a

O

S N

O d

F 54,58

g

O

N

F

R

S N

O O

N

F F 53

R

S

O O

O

R

S

S

58,59,60,61

F F 64

65,66,67

F O CF3 O S O

and conditions: (a) TBAF, DMF, 0 °C, 72%; (b) diethylaminosulfur

trifluoride, THF, 35 °C, 74%; (c) 6 M HCl aqueous solution, 1,4-dioxane, rt, 80–99%; (d) (2R,6R)-2,6-dimethylmorpholine, DIPEA, CH3CN, 90 °C, 59–91%; (e) barbituric acid, CH3COOH, H2O, 110 °C, 34–65%; (f) AgOTf, selectfluor, KF, 2-fluoropyridine,



Page 16 of 83

CF3TMS, EtOAc, rt, 11%; (g) MsCl, DIPEA, DCM, 0 °C, 92%; (h) CH3SNa, DIPEA, 0 °C, 43%; (i) H2O2, Na2WO4·2H2O, MeOH/H2O, rt, 41%. General procedures for the synthesis of compounds 79a–c are depicted in Scheme 9. 2,6-Difluorobenzonitrile 70 was treated with LDA followed by the addition of DMF to yield product 71, which was protected with ethylene glycol to yield compound 72. Compound 73 was obtained by introducing an aldehyde group in compound 72 and was then converted to oxime 74 using hydroxylamine hydrochloride. Compound 74 was chlorinated with NCS and then treated with the corresponding amines to yield compounds 75a–c. Subsequently, the ethylene glycol was removed with 6 M HCl aqueous solution, and the pH of the mixture was adjusted to 7–8 with NaHCO3 to construct

the

benzisoxazole,

affording

compounds

76a–c.

(2R,6R)-2,6-dimethylmorpholine was reacted with 76a–c to yield compounds 77a–c. Compounds 78a–c were prepared by reaction of 77a–c and CDI, and these compounds were reacted with barbituric acid to yield compounds 79a–c. Compound 82 was synthesized using 77c as the starting material, which reacted with thioacetic acid via a Mitsunobu reaction to yield 80. The thioester of compound 80 was hydrolyzed under basic conditions and then reacted with CDI to afford compound 81. Finally, the SPT architecture was constructed to afford compound 82 (Scheme 10). Scheme 9. Synthesis of Compounds 79a–ca


Page 17 of 83 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 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 56 57 58 59 60


F

F

F

F

O

O HO

O

c F

F

d

F

O

N F

CN

CN

CN

CN

CN

71

72

73

74

O O

N F

O

R g,h

i

N O

F 75a-c

76a-c

77a-c

OH

HN

HN

O

CN

78a,79a O

OH

N

O

CN

O

N

O H

O

79a-c

78b,79b

O

O NH

N

N

78a-c

75c-77c

OH

HN

O O

CN

75b-77b

k

H N

O

R

N

N

CN

75a-77a

R:

j O

O

R

N

F

CN

O

R

e,f

F

70 R HO

O

b

a F

O

O

O

78c,79c

O

O

N

O N

and conditions: (a) LDA, DMF, THF, –78 °C, 48%; (b) ethylene glycol,

αReagents

p-toluenesulfonic acid, toluene, 140 °C, 76%; (c) LDA, DMF, THF, –78 °C, 64%; (d) hydroxylamine hydrochloride, pyridine, MeOH, rt, 96%; (e) NCS, DMF, 40 °C; (f) the corresponding amine, DMF, 0 °C to rt, 27–77% (for two steps); (g) 6 M HCl aqueous solution, 1,4-dioxane, 35 °C; (h) NaHCO3, DMAP, DMF, rt, 18–44% (for two steps); (i) (2R,6R)-2,6-dimethylmorpholine, DIPEA, CH3CN, 40 °C, 70–88%; (j) CDI, DMAP, DMF, 100 °C, 50–70%; (k) barbituric acid, CH3COOH, H2O, 110 °C, 32–54%. Scheme 10. Synthesis of Compound 82a O S

HO O

HN

a N O 77c

αReagents

b,c N

O

O

S O

N

d N

O

N CN

S O

HN

80

O

H N

O

N

O

N CN 81

O

O NH

N O

N CN

O

N CN

O H

O

82

and conditions: (a) PPh3, DIAD, thioacetic acid, THF, 0 °C, 35%; (b) 2 M

NaOH aqueous solution, DL-1,4-dithiothreitol, MeOH, rt; (c) CDI, DMAP, DMF, 90 °C,



Page 18 of 83

62% (for two steps); (d) barbituric acid, CH3COOH, H2O, 110 °C, 62%. 

RESULTS AND DISCUSSION SARs of the Newly Synthesized SPTs. To improve the antibacterial activity as well as

the PK properties of the SPTs, we carried out structural modification of compound 3 via empirical medicinal chemistry approaches. All the new compounds were evaluated for in vitro antibacterial activity against a panel of susceptible and resistant Gram-positive strains, including fluoroquinolone-resistant MRSA, and the results are summarized in the form of minimal inhibitory concentrations (MICs). Previously, we described a series of SPTs containing various benzoheterocycle scaffolds, such as benzoxazolinone and benzimidazolone (Figure 4)24, but all these compounds showed very weak potency against the tested strains. The newly designed quinazoline compounds (±)-16a–d, (±)-20 and (±)-24, which retain the aromatic nature as well as a hydrogen bond acceptor, also exhibited distinct loss of activity against these strains (Table 1S). Taken together with previous results from our laboratory, these results suggested that the benzisoxazole scaffold may be optimal for antibacterial activity. 5

R N

O

X

N

1O

H N

O NH

O F

O H

O

nonaromatic heterocycle

O previous study

2

4

H N

O

N3

NH

N O

N F

O H

R

O

O

ETX0914

H N

O

N

O NH

N

N F

O H

O

aromatic heterocycle

X = O, NH

Figure 4. Modification of the benzisoxazole scaffold. The barbituric acid moiety on SPT is critical for antibacterial activity because of a


Page 19 of 83 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 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 56 57 58 59 60


hydrogen bond between the central keto oxygen of the barbituric acid moiety and the N–H of Asp437 on gyrase.20 To further explore the SARs of this pharmacophore, compounds 26a–c which retained this important hydrogen bond acceptor, were synthesized via isostere replacement of barbituric acid or its analogs. However, these compounds exhibited no activity against the strains mentioned above (Table 2S). These results again confirmed that spirocyclic pyrimidinetrione is a critical pharmacophore, and even slight modification can lead to significant loss of activity. It was reported that the substituent at the benzisoxazole 3 position is tolerant to structural modification and that degradation of oxazolidinone is one of the main metabolic pathways of 3 in mice.18, 25 Therefore, we decided to fine-tune the substituent at the benzisoxazole 3 position to improve potency and reduce the metabolism of 3 (Table 1). Using a 3-ketomorpholine to replace the oxazolidinone led to compound (±)-33a, which exhibited very weak antibacterial activity, indicating that ring expansion was harmful to potency. Compound (±)-33b, with a gem-dimethyl at the oxazolidinone 4 position, lost almost all its potency. Compound (±)-33c, in which the gem-dimethyl was cyclized into a cyclopropane, exhibited higher antibacterial activity than compound (±)-33b but was 2- to 30-fold less potent than compound 3. Moving the cyclopropane from position 4 to position 5 yielded compound (±)-33e. Surprisingly, (±)-33e exhibited great improvement in potency, with MIC values ≤ 0.03 mg/L against most tested strains. An optically pure form of this compound, 33e, exhibited improved potency than the racemate. Compound (±)-33d, with an oxazolidine-2-thione moiety in place of the



Page 20 of 83

oxazolidinone in 3, exhibited weaker of antibacterial activity. Unexpectedly, compound 39a, with a 2-thiazolidinone moiety, exhibited significantly improved antibacterial activity against all strains (MIC ≤ 0.03 mg/L). Both compounds 33e and 39a are 2- to 32-fold more potent than their prototype compound 3 respectively, indicating that subtle structural modification could greatly improve the antibacterial activity of 3. Table 1. Antibacterial Activity of SPTs 33a–e and 39a R

H N

O

O NH

N O

N F

O H

O

MIC (mg/L)a Compound

R MSSA

MRSA

MSSE

MRSE

PRSP

Spy

4–8

1–64

4

1–8

4–8

4

16

16

16

16

16

16

1

1–4

1–2

0.5–2

2

1–2

0.5–1

1–2

0.5–2

1

1–2

2

Design, Synthesis, and Biological Evaluation of Novel DNA Gyrase

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