Synthesis and Structure-Activity Relationships of5-Amino-6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-

8-methylquinolonecarboxylic Acid Antibacterials Having Fluorinated 7-[(3R)-3-(1-Aminocyclopropan-1-yl)pyrrolidin-1-yl] Substituents

Hiroaki Inagaki etc.J. Med. Chem. 2003, 46, 1005-1015

분자생명과학부김미금

2003-06-03

1. Introduction

(1) Multidrug-resistant Gram-positive pathogens

a. Acqusition of resistance to vancomycin(VRE and vancomycin-

intermediate-resistant staphylococcal strains; VISA), an antibac-

terial agents generally regarded as the agent of last resort

for serious infection

b. Quinolone antibacterial agents 의 영역에서는 내성을 가진 Gram-

positive 박테리아에 효능이 있는 약품을 얻기 위한 다양한 시도 및 travafloxacin(TVFX), moxifloxacin(MFLX), gemifloxacin(GMFX),

gatifloxacin(GFLX) 등의 계발

c. Driving force for the development of new antibacterial agents

- Antibacterial agents 에 대한 Gram-positive bacteria 의 내성 범위의 증가 - 임상에서 사용되고 있는 Quinolone 이 아닌 몇몇의 물질이 보이는 내성 돌연변이와 부작용

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(2) Target molecule a. 선행연구

- C-7 위치에 3-(aminocycleopropal-1-yl)pyrrolidin-1-yl 치환기 (R2= c-C3H5) 를

포함하는 3-(1-amino-1-substituted-methyl)pyrrolidin-1-yl

groups 를 갖는 여러 quinolone 유도체의 Gram-positive bacteria 에 대한 antibacterial activity

- 7-{(3R)-3-(1-amino-1-substituted-methyl)pyrrolidin-1-yl}

quinolone 유도체 : 좋은 활성을 보였으나 높은 genotoxicity 가짐

- Genotoxicity 를 줄이기 위한 N-1 위치에 cyclopropyl 치환기 (R1= H) 대신에

(1R, 2S)-2-fluorocyclopropan-1-yl 치환기 ((R1= F) 의 도입의 유용함

- 5-amino-8-methylquinolone 유도체 (R3= NH2) 의 Gram-positive bacteria 에

대해 antibacterial activity 및 8-methylquinolone 유도체 (R3= H) 에 비해 적은

chromosomal toxicity 의 보고 3/19

N

F

O

COOH

N

H2N

R2

CH3

R1

R3

- Genotoxicity 를 줄이면서 Gram-positive bacteria 에 대해 높은 효능을 보이는 화합물

- C-7 위치에 (3R)-3-(1-aminocyclo-propan-1-yl)pyrrolidin-1-yl 치환기를 갖는 5-amino-1-[(1R,2S)-2-fluorocyclopropan-1-yl]- 8-methylquinolone(1, Figure 1)

- 화합물 1 : 높은 antibacterial 활성을 보였지만 , micronucleus test 에서 양성 반응을 보이고 chromosome injuring test 에서 독성 - C-7 pyrrolidine 치환기로의 fluorine atom 의 도입의 genotoxicity 감소에 효과를 화합물 1 에 도입

N

ONH2

CH3

COOHF

N

R1 R2

H

FH2N

Figure 1.

1: R1 = H, R2 = H2: R1 = H, R2 = F 3 (= DQ113): R1 = F, R2 = H4: R1 = F, R2 = F

b. Target molecule

4/19

2. Chemistry

(1) Retrosynthesis

N

ONH2

CH3

COOHF

N

R1 R2

H

FH2N

NH

R1 R2

H

NHBoc

5: R1 = H, R2 = H6: R1 = H, R2 = F 7: R1 = F, R2 = H8: R1 = F, R2 = F

N

ONH2

CH3

COOHF

F

F

+

1-4 9

Scheme 1

5/19

(2) Synthesis of compound 9

F

F

NO2

CH3

F

COOEt

O

10

a, b, c

N

ONO2

CH3

COOEtF

F

F

d

N

ONH2

CH3

COOR3F

F

F

11 12: R3 = Et 9: R3 = H

e

Scheme 2a

a Reagents: (a) Ac2O, CH(OEt)3, reflux; (b) (1R, 2S)2-2-fluoro-1-cyclopropylamine p-toluenesulfonic acid salt, Et3N/CH2CL2; (c) NaH/1,4-dioxane;(d) H2, Raney Ni (W-6)/MeOH, 1,4-dioxane; (e) concentrated aqueous HCl, AcOH, reflux

6/19

F

F

NO2

CH3

F

COEt

O

10

H H CH

OEt

EtO OEt+

O

F

F

NO2

CH3

F

COEt

O O-

CH

O+Et

EtO OEt+

H

CEtO+OEt

F

F

NO2

CH3

F

COEt

O O-

+

HF

F

NO2

CH3

F

COEt

O O

CHOEtEtO

H F

F

NO2

CH3

F

COEt

O O-

CHOEtEtO+

H

-----------------------------------------------Mechanism(1)

Mechanism(2)

F

F

NO2

CH3

F

COEt

O O

HCOEt

N+H2

F

H

+

F

F

NO2

CH3

F

COEt

O O

HC+OHEt

Et3N

N-H

F

+

F

F

NO2

CH3

F

COEt

O O

CH

N

F

H

NaH

F

F

NO2

CH3

F

COEt

O O

CH

N-

F

F

F

NO2

CH3

NF

O

COOEt

F

11

7/19

(3) Synthesis of compound 17a

EtOOC

O

a

EtOOC

OH

COOEt

b

EtOOC

COOEt

13 14 15

----------------------------------------------Mechanism (13 → 14 ; The Reformatsky reaction)

EtOOC

O

Zn + BrCH2CO2Et H2C C

O- Zn2+

0Et

H2C C

O- Zn2+

0Et+

EtOOC

O

COOEt13 14

NH

EtOOC

O-

COOEt

SCl Cl

O

EtOOC

O

COOEt

S Cl

O

Cl-

EtOOC

Cl

COOEt

H

H

DBU

EtOOC

COOEt15

Reagents: (a) Zn, BrCH2CO2Et, cat. I2/THF, reflux; (b) (1) SOCl2/pyridine, (2) DBU/CH2Cl2

8/19

EtOOC

COOEt

EtOOC

N

O

Ph

c

15

16

EtOOC

N

O

Ph

H

d

17a(3S) : 17b(3r) = 3.5 : 1

EtOOC

N

O

Ph

H

HOOC

N

O

Ph

H

e f

17a 18

aReagents: (c) (1) NBS, AIBN/CCl4, (2) (S)-1-phenylethylamine, NaHCO3/EtOH, reflux; (d) H2, PtO2/MeOH; (e) silica gel column chromatography;

(f) aqueous NaOH/EtOH.

----------------------------------------------Mechanism

EtOOC

COOEt

15

AIBN, NBSEtOOC

COOEt

BrH2N Ph , NaHCO3

EtOOC

N

O

Ph

16AIBN(2,2'-Azobisisobutyronitrile) :

NC

N N

CN

NBS(N-bromosuccinimide) :N

O O

Br 9/19

(4) Incorporation of fluorine atom(s) into the pyrrolidinone ring

EtOOC

N

O

Ph

H

EtOOC

N

O

Ph

H

R1 R2

NH

N

O

Ph

H

F H

Boc

17a

a

19: R1 = H, R2 = F

20: R1 = F, R2 = H

21: R1 = F, R2 = Fa b

22

aReagents: (a) LDA, then (PhSO2)2NF/THF; (b) LDA, then 2,6-di-tert-butylphenol/THF; (c) aqueous NaOH/EtOH;

(d) diphenylphosphoryl azide, Et3N/toluene, then tert-BuOH.

c,d

LDA(Lithium diisopropylamide) : hindered non-nucleophilic strong

[(CH3)2CH]2NLi

(PhSO2)2NF(N-fluorobenzenesulfonimide) : transfer F+ to enolates

and carbanions

-----------------------------------------------

10/19

Mechanism (20 → 22 : subsequent Curtius rearrangement reaction)

Et3N/tolueneC

O

R OH

C

O

R O-

+(PhO)2P

O

N3

C

O

R O

P(OPh)2

O

N3-+ C

O

R-

N

+

N N

R N C O+OC(CH3)3+

H+

R

HN

Boc

DPPA

11/19

(5) Synthesis of compound 27a-d

EtOOC

N

O

Ph

H

R1 R2

EtOOC

N

S

Ph

H

R1 R2

EtOOC

N

Ph

H

R1 R2

EtOOC

N

H

R1 R2

CbzHOOC

N

H

R1 R2

Cbz

NH

N

H

R1 R2

Cbz

Boc

17a: R1 = H, R2 = H19: R1 = H, R2 = F

20: R1 = F, R2 = H

21: R1 = F, R2 = F

a

23a: R1 = H, R2 = H23b: R1 = H, R2 = F

23c: R1 = F, R2 = H

23d: R1 = F, R2 = F

24a: R1 = H, R2 = H24b: R1 = H, R2 = F

24c: R1 = F, R2 = H

24d: R1 = F, R2 = F

25a: R1 = H, R2 = H25b: R1 = H, R2 = F

25c: R1 = F, R2 = H

25d: R1 = F, R2 = F

26a: R1 = H, R2 = H26b: R1 = H, R2 = F

26c: R1 = F, R2 = H

26d: R1 = F, R2 = F

27a: R1 = H, R2 = H27b: R1 = H, R2 = F

27c: R1 = F, R2 = H

27d: R1 = F, R2 = F

b

c d e

Reagents: (a) Lawesson's reagent/benzene; (b) Raney Ni (W-6)/EtOH; (c) Cbz-Cl/CH2Cl2; (d) aqueous NaOH/EtOH; (e) diphenylphosphoryl azide, Et3N/tert-BuOH.

12/19Lawesson's reagent

P

S

S S

P

S

H3CO

OCH3

(6) Synthesis of final product 1-4

N

ONH2

CH3

COOHF

F

N

R1 R2

H

H2N

NH

NHH

R1

R2

Boc27a-d

1: R1 = H, R2 = H

2: R1 = H, R2 = F (HCl salt)

3 (= DQ113): R1 = F, R2 = H

4: R1 = F, R2 = F (HCl salt)

5: R1 = H, R2 = H6: R1 = H, R2 = F

7: R1 = F, R2 = H

8: R1 = F, R2 = F

a

Reagents: (a) H2, Pd-C/EtOH; (b) 9, Et3N/DMSO, heat; (c) concentrted aqueous HCl.

b, c

(7) Synthesis of Reference compounds 30, 31

N

ONH2

CH3

COOHF

F

F

+

NHR4R5

NHBocN

ONH2

CH3

COOHF

F

NR4R5H2N

H

9

28: R4 = H, R5 = H

29: R4 = Me, R5 = H

30: R4 = H, R5 = H

31: R4 = Me, R5 = H

Reagents: (a) Et3N/DMSO, heat; (b) concentrted aqueous HCl.

13/19

3. Results and Discussion

(1) 대표적인 Gram-positive 와 Gram-negative bacteria 에 대한 1-4, 30, 31 의 minimum inhibitory concentrations(MICs) (Table 1)

- 화합물 1-4, 30, 31 은 Gram-positive bacteria 에 대해

reference quinolones TVFX, MFLX, GFLX, CPFX 보다 2-128 배 활성

-C-7 위치에 fluorinated pyrrolidine 을 갖고있는 화합물 2-4 와

non-fluorinated 화합물 1 은 거의 동일한 antibacterial 활성

→pyrrolidine substituents 에 fluorine atom(s) 를 도입하는 것은

antibacterial 활성에 크게 영향을 미치지 않음 .

14/19

(2) Result of intravenous single-does toxicity study and micronucleus test (Table 2)

a. Intravenous single-does toxicity study

- 1 ((3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-yl substituent) < 30-31

- 2 (trans-oriented) > 1 (non-fluorinated) = 4 (difluorinated) > 3 (cis-oriented)

b. Micronucleus test

- Non-fluorinated 화합물 1, 31 : 양성반응 - Fluorinated 화합물 2-4 : 음성반응

15/19

(3) Solubility in water (Table 3)

: 3 (fluorinated 화합물 ) > 1 (non-fluorinated 화합물 ) → fluorine atom 이 물에서의 용해도를 향상시키는데 기여

16/19

(4) Antibacterial activities of 3 (DQ- 113) (Table 4)

a. clinically 분리된 내성을 갖는 Gram-positive bacteria 에 대한 MICs 가 결정 b. 다른 quinoline antibacterial agents, VCM, TEIC, LNZ 보다 더 효과적 c. levofloxacin-resistant MRSA 에 대항하는 QPR/DPR 과 비슷한 활성d. PRSP 에 대항해서는 , 3 이 열거된 화합물들 중에서 가장 효능을 보였다 .

e. VRE strains 모두에서 가장 효능을 보였다 .

17/19

(5) pharmacokinetic profiles of 3 (DQ- 113) (Table 5)

a. time-concentration curve 에서 높은 plasma concentration 과 area

b. 간 , 신장 , 폐에서 좋은 분포 경향

18/19

4. Conclusion

(1) C-7 위치에 (3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-yl 치환기를 갖고있는 5-amino-6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methylquinolones

시리즈 합성

(2) Fluorinated 화합물 2-4 는 non-fluorinated 화합물 1 과 비교해서 효능을 보였고 ,

Gram-positive bacteria 에 대해 reference quinolone 보다는 적어도 4 배의 효능을 보였다 .

(3) Fluorinated 화합물들 중에서는 cis-oriented 유도체 3(DQ-113) 이 1 에 비해서 줄어든 정맥내로의 single-does toxicity 를 보였고 , 쥐에서 좋은 pharmacokinetic

profiles 를 보였으며 , 시험된 다른 quinolines 와 비교했을 때 clinically 분리된 내성을 갖는 Gram-positive bacteria 에 대해 더 좋은 antibacterial 활성을 보였다 .

(4) 화합물 3 은 이 논문에서 언급한 것들에 더불어 다른 clinically 분리된 bacteria 에 대한 완벽한 antibacterial 활성을 보였고 , further preclinical evaluation 을 위해 선택되었다 .

19/19