Research and Development of Quinolones in Daiichi Sankyo Co., Ltd

Special Feature Research and Development of Quinolones in Daiichi Sankyo Co., Ltd.

Shogo Atarashi, PhD Senior Advisor Marketing Department ASCA Company Daiichi Sankyo Co., Ltd. Tokyo, Japan

Quinolones, which are antimicrobials first launched in the 1960s, have exhibited expanded significance in the clinical setting roughly every two decades. The first-generation quinolones were used for the treatment of intestinal and urinary tract infections because of their antimicrobial efficacy against Gram-negative bacteria. During the 1980s, quinolones emerged as broad-spectrum "new quinolones." During the 2000s, quinolones were used for the treatment of a wide variety of infectious diseases, mainly respiratory tract infections, as "respiratory quinolones." During these periods, innovative quinolones representa- tive of each period were produced. Daiichi Pharmaceutical Co., Ltd. (currently Daiichi Sankyo Co., Ltd.) has continued the discovery and research and development of quinolones for more than 40 years, and has released four quinolones, including three in-house development products and one licensed-in product, that have been highly acknowledged by healthcare professionals who treat bacterial infections all over the world. This document describes the basic concept of drug discovery research of Daiichi's three original quinolones, i.e., ofloxacin, levofloxacin, and sitafloxacin, particularly from the perspective of medicinal chemistry.

Introduction ever, as early as the middle of the 20th century, In 1910, the German bacteriologist Paul Ehrlich the development of resistant bacteria that reduce and his student Sahachiro Hata developed sal- the efficacy of these antibiotics became a signifi- varsan, an agent effective in the treatment of cant concern. syphilis, and this was the world’s first synthetic At that time, Sterling-Winthrop Inc. in the chemotherapeutic agent. Humans thus became USA found that a chloroquinoline derivative pro- able to create treatments for bacterial infections. duced during the manufacturing of chloroquine, In 1929, Alexander Fleming isolated penicil- an antimalarial agent, had antimicrobial activity. lin, the world’s first antibiotic, from Penicillium no- Through research on this derivative, they found Address for correspondence tatum. Howard Florey and other physicians began nalidixic acid (NA), the first quinolone type com- Shogo Atarashi, PhD to use penicillin in the clinical setting, and in this pound, in 1962 (Figure 1) (1). Since nalidixic acid Marketing Department, ASCA way completely changed clinical treatment of has a naphthyridine ring in its basic structure, ex- Company, Daiichi Sankyo Co., bacterial infections. Around the same time, the erts antimicrobial activity mainly against Gram- Ltd. first sulfa drug was synthesized, and streptomycin negative bacteria, and is effective against bacteria 1-6-5, Nihonbashi Honcho, Chuo- (an antituberculosis agent), tetracycline, and other resistant to sulfa drugs and other antibiotics, it at- ku, Tokyo 103-8234, Japan Phone: 03-3246-7031 antibiotics with excellent antimicrobial efficacy tracted attention as a new synthetic antimicrobial Fax: 03-3246-6916 were found one after another. Treatment of infec- agent with a unique chemical structure. E-mail: atarashi.shogo.f7@ tious diseases has thus advanced significantly, how- Daiichi Pharmaceutical Co., Ltd. (currently daiichisankyo.co.jp

47 Special Feature

Figure 1. Chemical structures of nalidixic acid, pipemidic acid, and norfloxacin O O O COOH COOH F COOH N N N N H3C NN N N

HN HN 2 5 C2H5 C2H5 C H

Nalidixic acid (NA) Pipemidic acid (PPA) Norfloxacin (NFLX)

Acidic type Zwitter type

Daiichi Sankyo Co., Ltd.) concluded a contract Although compounds with better antimicrobial with Sterling-Winthrop Inc. to introduce nalidix- activities were obtained, they were only effective ic acid into Japan, and launched it with the trade against Gram-negative bacteria and metabolically name of Wintomylon® for the treatment of enter- unstable. No compounds with physicochemical ic infection and urinary tract infection (UTI) in characteristics sufficient for oral administration 1964. were obtained. Pipemidic acid (PPA), discovered in 1972, From acidic quinolones to zwitterionic quino- and norfloxacin (NFLX), in 1978 (Figure 1), are lones—launching ofloxacin zwitter type compounds containing an amino sub- After the release of nalidixic acid in Japan, Daiichi stituent with excellent tissue penetration and uri- Pharmaceutical began to develop its own acidic nary excretion not achievable with acidic quino- quinolones with higher antimicrobial activities lones (“old quinolones”). In particular, norfloxacin, and broader antimicrobial spectra using nalidixic a compound containing fluorine that exhibits good acid as the prototype. During a 15-year period, antimicrobial activity against Gram-positive bac- over 1,000 acidic compounds were synthesized. teria, was expected to be beneficial in the treat-

Figure 2. Derivatization of pyridobenzoxazine molecule

O O O O COOH F COOH F COOH N N N R N HN O O CH3 C2H5 R’

DJ-6779 Norfloxacin

Pyridobenzoxazine molecule

Figure 3. Structure-activity relationship of ofloxacin structure

F > CI > CN ≃ H H ≃ NH2 > NO2 > OCH3 H O F COOH

Ring size Ring size 6 > 7 N N N 6 > 7 > 5 H3C O CH3

CH3 ≥ C2H5 > H > O > N-CH3 ≥ S >> NH CH3 ≃ = CH2 > CH2F > C3H7 > CH2C6H5 > C2H5 > H > C3H7 > C6H5 Ofloxacin (OFLX)

48 Research and Development of Quinolones in Daiichi Sankyo Co., Ltd.

Figure 4. Modification of ofloxacin chemical structure O F COOH

N 7 N Asymmetric carbon N 3 O * H C 3 CH3 Two optical isomers

Structure modification Optical resolution ment of respiratory tract infections (RTIs). Daiichi came fierce. In Daiichi Pharmaceutical, calls for Pharmaceutical, which had reached the limits next-generation quinolones with a pyridoben- of R&D of acidic quinolones, became aware of zoxazine structure increased. the importance of physicochemical characteristics, At that time, we knew that the ofloxacin especially the tissue penetration of compounds in molecule had two possible modifications (Figure the body, and changed its targets from acidic to 4). The first is further modification of the amino zwitter quinolones (“new quinolones” or “fluoro- substituent at position 7. The second, the carbon quinolones”). The company incorporated its abundant findings on structure-activity relationships of acidic quinolones in R&D on zwitter Figure 5. HPLC chart of optical active 3,5- quinolones. From among several candidate struc- dinitrobenzoyl derivatives tures, the company selected a pyridobenzoxazine skeleton, the basic structure of DJ-6779, which O was poorly absorbed following oral administration F COOEt but exhibited the most potent antimicrobial activities with the widest antimicrobial spectrum F N

O NO2 among the acidic compounds tested. Daiichi H Pharmaceutical developed a lead compound by OC adding characteristic structures of norfloxacin (flu- (1) orine plus amino substituent) to the pyridoben- O NO2 zoxazine skeleton, and produced various analogues O called fluoroquinolones (Figure 2 and Figure 3). F COOEt Among them was ofloxacin (OFLX), which ex- NO2 ceeds norfloxacin in all microbiologically signifi- F N cant respects and exhibits high blood concentra- O OC tion, extensive urinary excretion, good tissue R penetration, and a broad antimicrobial spectrum H O NO2 covering Gram-positive bacteria (Figure 3) (2). (2) Ofloxacin was released in Japan with the trade name of Tarivid® in 1985, and was then S launched in the USA and Europe. This drug is the first export product of Daiichi Pharmaceutical. Although ofloxacin was not the world’s first new quinolone, it was approved as a drug for the treat- SUMIPAX OA-4200 ment of various infectious diseases including low- 4.6 x 250 mm er RTIs, and became Daiichi’s first successful new quinolone to achieve almost all goals of research.

Introduction of stereochemistry and optical resolution—development of levofloxacin 0 4 8 12 16 20 24 28 Before and after the release of ofloxacin, other Time (min) companies released new quinolones such as norfloxacin, enoxacin, and ciprofloxacin, and the Abbreviation: HPLC = high-performance liquid chromatography. global competition in the quinolone market be-

49 Special Feature

Figure 6. Optical resolution and synthetic pathway to two enantiomers of ofloxacin O O O O COOEt COOEt COOEt F F COOEt F F Optical F NO2 F NO2 resolution N N F N F N O O NO2 O OCO OCO H O OH H OCO NO2 NO2 (6 mg) (2) (1) 600 mg NO2 (2.5 mg) (2.5 mg) 250 mg 250 mg

NaHCO3

O O O O F COOH F COOEt F COOEt F COOH Hydrolysis N N F N F N F N N H3C O O O OH X O CH3 CH3 H H H H 7–8 mg X = I [DR-3355] Reduction Levofloxacin X = H (LVFX) O F COOH

N DR-3355: [α]D = −76.9° N DR-3354: [α]D = +76.7° N H3C O H

CH3 7–8 mg [DR-3354]

at position 3, is a chiral carbon, meaning that two arately eluted based on differences in affinity. We enantiomers are present in terms of the spatial attempted to separate several ofloxacin analogues position of the methyl group. Ofloxacin is a race- using some commercially available columns, and mic compound of two enantiomers that would found that a newly synthesized 3,5-dinitrobenzoyl subsequently be separated. None of the acidic and derivative could be clearly separated into two en- zwitter quinolones available for human use at that antiomers (1), (2) when a specific chiral column is time had asymmetric carbons. Ofloxacin was the used (Figure 5). However, the column was de- first racemic quinolone for which stereochemical signed specifically for analytical use, and chiral research would prove fruitful. columns for fractionation able to yield ample Following successful development of ofloxa- amounts (about 10 mg) of enantiomers for analy- cin, Daiichi Pharmaceutical attempted to separate sis of physicochemical characteristics and antimi- enantiomers using various techniques including crobial activities were not available at that time. diastereomer methods, but could not separate We asked the manufacturer of the column to pro- them. At that time, the development of chiral col- duce fraction columns containing the same sta- umns for high-performance liquid chromatogra- tionary phase, while we specified suitable eluting phy (HPLC) advanced (3), and various columns conditions in terms of solvent, flow rate, and tem- were introduced. These columns use optically ac- perature among other parameters to ensure good tive polymers as the stationary phase, in which ra- separation of enantiomers by HPLC. With this cemic compounds travel differently through the technique, however, we could only run about optically active environment and can then be sep- 6 mg of a 3,5-dinitrobenzoyl derivative to sepa-

50 Research and Development of Quinolones in Daiichi Sankyo Co., Ltd.

Table 1. Characteristics of ofloxacin and its two enantiomers, DR-3355 and DR-3354

O O O F COOH F COOH F COOH N N N N N N 3 N N H3C N O H C O H3C O CH3 CH3 CH3 Ofloxacin (racemate) DR-3355 (levofloxacin) DR-3354 Antibacterial activity (standard strains) MIC (μg/ml) Staphylococcus aureus 0.39 0.20 25.0 Staphylococcus epidermidis 0.39 0.39 25.0 Streptococcus pyogenes 3.13 1.56 >100 Streptococcus faecalis 3.13 1.56 >100 Escherichia coli ≤0.05 ≤0.05 0.39 Proteus vulgaris ≤0.05 ≤0.05 0.39 Klebsiella pneumoniae 0.10 ≤0.05 1.56 Enterobacter cloacae ≤0.05 ≤0.05 0.78 Serratia marcescens 0.10 ≤0.05 1.56 Pseudomonas aeruginosa 0.20 0.10 6.25 Toxicity in mice (IV, single) Mortality (200 mg/kg) 2/5 0/5 3/5

LD50 (mg/kg) 208 248 163 Physicochemical properties

23 Optical rotation [a]D (0.05N NaOH) − –76.9° +76.7° Melting point (℃) 260–270 225–229 226–230 Partition coefficient (P’) a 4.95 5.1 5.1

Solubility in H2O (mg/ml) 2,400 24,500 25,800

a CHCl3/0.1 M of phosphate buffer (pH 7.4). Abbreviations: MIC = minimum inhibitory concentration, IV = intravenous. rate the two enantiomers, into about 2.5 mg each, strength of effects of ofloxacin was intermediate over 1.5 to 2 hours of elution. We repeated the between the two enantiomers. The water solubili- elution over about one month to obtain 250 mg ties of DR-3354 and DR-3355 are as high as of each enantiomer (1) and (2). Using these enan- 25 mg/ml, about 10-fold that of ofloxacin. Inject- tiomers, we finally succeeded in obtaining the en- able DR-3355 was therefore considered feasible antiomers of ofloxacin, DR-3354 and DR-3355, (Table 1). with optical purities of nearly 100% (Figure 6) (4, DR-3354 and DR-3355 in amounts suffi- 5). X-ray crystal structure analysis of an interme- cient for these non-clinical studies could not be diate compound obtained through a different obtained with the chiral column technique. Tech- route revealed that DR-3355 is (S)-ofloxacin (4), niques to obtain several to several hundred grams which was later termed levofloxacin (LVFX). of DR-3355 such as the proline method (4), the Evaluation of the antimicrobial activities of enzyme method (6), and asymmetric synthesis (7) ofloxacin, DR-3354, and DR-3355 (levofloxacin) were investigated to ensure rapid progress of non- revealed that DR-3355 exhibited antimicrobial clinical studies. activity twice that of ofloxacin in most strains Regarding the other point at which modifi- used, while the antimicrobial activity of DR-3354 cation is possible, the amino substituent at posi- was 1/10 to 1/100 that of DR-3355 (Table 1), tion 7, other companies reported that a five- suggesting that the active enantiomer of ofloxacin membered aminopyrrolidine ring improves is DR-3355. In an acute toxicity study in mice, antimicrobial activities against Gram-positive bac- the LD50 of DR-3354 was lower than that of teria (8, 9). Based on these reports, we synthesized DR-3355, suggesting that DR-3354 was a major about 300 compounds using various aminopyrro- cause of adverse drug reactions to ofloxacin. Fur- lidine groups. However, though antimicrobial ac- ther studies on effects of these substances on tivities were increased by inserting aminopyrro- arousal of rats and cats and convulsions in mice lidine groups, we could not obtain any substances also revealed that the effects were smallest with acceptable in terms of physicochemical properties DR-3355 and largest with DR-3354, and the and toxicity (Figure 7). As a result of the above-

51 Special Feature

Figure 7. Modification at 7C -position O F COOH Asymmetric N 3 7 N carbon CH N * O CH3 CH3 H3C

Modification Optical resolution Synthesized ca. 300 samples

O O F COOH F COOH

N N N N H H2N O * N O CH3 H3C CH3 Levofloxacin (LVFX)

H3C R’H2C N : N N N N etc. RN RHN H2N H2N RHN

CH2R’

described investigations, DR-3355 (levofloxacin) Challenge to severe and intractable infectious was finally selected as a post-ofloxacin quinolone diseases—selection of sitafloxacin to be investigated in clinical studies. Although levofloxacin was acknowledged to be Clinical research was initiated in 1987 and an ultimate quinolone, other pharmaceutical proceeded smoothly. In December 1993, levo- companies were working hard to develop and floxacin was launched in Japan with the trade launch quinolones with higher antimicrobial ac- name of Cravit®. As the world’s first optically activities against Gram-positive bacteria and excel- tive quinolone, Daiichi Pharmaceutical launched lent efficacy in the treatment of severe RTIs. levofloxacin in Asian countries (from 1994) and Daiichi Pharmaceutical also needed to take a further licensed it out in the USA and European coun- step in creating completely new quinolones not tries (launched from 1997 to 1998). As of 2010, containing the pyridobenzoxazine structure used levofloxacin is available in more than 120 coun- as the basis of its quinolone development. tries due to expansion of its sales network The PK of quinolones would depend on the throughout the world (Figure 8). In Japan, levo- combination of their basic structure and types of floxacin was approved at a dose of 100 to 200 mg substituents, and differences in PK affect the inci- three times daily, while in the USA clinical devel- dence and types of adverse drug reactions. Selec- opment of levofloxacin was started at 500 mg tion of suitable chemical structures is facilitated if once daily according to pharmacokinetic/phar- there are specific criteria for selection of promis- macodynamic (PK/PD) modeling, which was ing compounds. From the launch of ofloxacin, new at that time (10). The regimen of 500 mg Daiichi Pharmaceutical measured the apparent once daily has become the world standard for le- partition coefficients (P’) of quinolones in

vofloxacin therapy (in Japan, the 500 mg once- CHCl3/0.1 M phosphate buffer as a parameter of daily dose was approved in 2009). Because of its lipophilicity, and found that quinolones with a P’ broad safety margin, levofloxacin has been ap- of 2–5 would be well absorbed after oral adminis- proved for use at a dose of 750 mg once daily in tration and rarely cause adverse drug reactions in the USA and Asian countries* and at 500 mg the central nervous system (CNS) (compounds twice daily in Europe. with P’ < 2 are poorly absorbed after oral admin- Levofloxacin is being used as a blockbuster istration, and compounds with P’ > 5 may fre- drug with the best sales in the world market of quently cause adverse CNS effects). The company all original antimicrobial agents in the treatment used this as a criterion for selection in the discov- of infectious diseases throughout the world. ery of new-generation quinolones. Levofloxacin, a compound with P’ = 5, is at the upper limit of

* In some Asian countries, a dose of 500 mg twice daily has been approved.

52 Research and Development of Quinolones in Daiichi Sankyo Co., Ltd.

Figure 8. Global development of levofloxacina

500–750 mg q.d. (500 mg b.i.d.)

EU USA Asia Japan

500–750 mg q.d.

500 mg q.d. or b.i.d. 100–200 mg 500 mg t.i.d q.d. (From June 2009) a Marketed in more than 120 countries in the world. Abbreviations: q.d. = once daily, b.i.d. = twice daily, t.i.d. = three times daily. favorable lipophilicity. more hydrogen atoms of the aliphatic chain are It was generally believed that inclusion of replaced by a fluorine atom (11). We first investi- fluorine would increase the lipophilicity of a gated the usefulness of a new fluorine-containing compound, though Daiichi Pharmaceutical substituent created by substitution with fluorine found, through its abundant data on P’ of many of the cyclopropyl substituent at position 1 of quinolones, that quinolones become more lipo- ciprofloxacin (CPFX), which exhibits excellent philic when one or more hydrogen atoms of the antimicrobial activity against Gram-negative bac- aromatic ring are substituted with a fluorine teria including Pseudomonas aeruginosa. Two pairs atom, but became less lipophilic when one or of quinolone analogues containing cis- and trans-

Table 2. Influence of fluorine atom at2’ C -position of cyclopropyl substituent on antibacterial activity and lipophilicity of the molecule O F COOH N N N R2 R1 Configuration of fluorine atom

FFFFF FFFFF R1 FFFFF FFFFF cisciscisciscis transtranstranstranstranstrans cisciscisciscis trans trans trans trans trans

R2 H H H CH3 CH3 CH3 CPFX Antibacterial activity (standard strains) MIC (μg/ml) Escherichia coli ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 Pseudomonas aeruginosa ≤0.05 ≤0.05 0.1 ≤0.05 ≤0.05 0.39 Staphylococcus aureus 0.1 0.1 0.78 0.2 0.1 0.78 Enterococcus faecalis 0.78 1.56 12.5 0.78 0.78 12.5 Partition coefficient (P’) a 0.89 0.16 − 20.0 7.10 10.7

a CHCl3/0.1 M of phosphate buffer (pH 7.4). Abbreviations: MIC = minimum inhibitory concentration, CPFX = Ciprofloxacin.

53 Special Feature

Table 3. Influence of the stereochemistry of N1- and C7-substituents on antibacterial activity among four optical isomers of (3) O N N F COOH N N 1 H H N N F H H2N 7 F 1’ NH2 H 7’ Cl F H H NH2 (7S) (7R) (3) 2’ (1R, 2S) (1S, 2R) Configuration In vitro antibacterial activity: MIC (μg/ml) Compound 1’, 2’ 7’ S. a. S. e. S. p. E. f. E. c. S. m. K. p. P. a. DU-6859aa cis (1R, 2S) (S) 0.008 0.063 0.031 0.25 0.008 0.031 0.031 0.125 DU-6856 cis (1S, 2R) (S) 0.016 0.063 0.063 0.25 0.016 0.063 0.031 0.25 DU-6857 cis (1R, 2S) (R) 0.063 0.25 0.5 1 0.031 0.063 0.063 0.25 DU-6858b cis (1S, 2R) (R) 0.063 0.25 0.25 1 0.063 0.125 0.125 0.5 LVFX 0.25 0.5 1 2 0.031 0.125 0.063 0.5 CPFX 0.25 0.5 1 2 0.008 0.063 0.031 0.125

a Sitafloxacin. b Enantiomer of DU-6859a. Abbreviations: MIC = minimum inhibitory concentration, S. a. = Staphylococcus aureus 209P, S. e. = Staphylococcus epidermidis 56500, S. p. = Streptococcus pneumoniae J24, E. f. = Enterococcus faecalis ATCC 19443, E. c. = Escherichia coli KL16, S. m. = Serratia marcescens 10100, K. p. = Klebsiella pneumoniae Type 1, P. a. = Pseudomonas aeruginosa PAO1, LVFX = Levofloxacin, CPFX = Ciprofloxacin.

Table 4. Influence of the stereochemistry of 1N - and C7-substituents on product properties

O O O O F COOH F COOH F COOH F COOH N N N N N N N N Cl Cl Cl F Cl F NH2 NH2 NH2 NH2

DU-6859a DU-6668 DU-6611 Clinafloxacin (Sitafloxacin) (S-form) Antibacterial activity (standard strains) MIC (μg/ml) Escherichia coli ≤0.003 0.006 0.006 ≤0.003 Pseudomonas aeruginosa 0.05 0.1 0.025 0.1 Staphylococcus aureus 0.006 0.006 0.012 0.025 Enterococcus faecalis 0.1 0.1 0.2 0.2 Micronucleus test Negative Positive Negative Positive Partition coefficient (P’) a 3.1 11.1 0.58 2.3

a CHCl3/0.1 M of phosphate buffer (pH 7.4). Abbreviation: MIC = minimum inhibitory concentration.

1-amino-2-fluorocyclopropane groups were syn- antimicrobial activities. P’ were 0.89 and 20 in thesized and compared with the corresponding non-fluorine compounds, 0.16 and 7.10 in cis- analogue not substituted with fluorine in terms of compounds, and not determined and 10.7 in antimicrobial activities and lipophilicity (Table 2). trans-compounds. Introduction of fluorine in the The cis-compounds exhibited antimicrobial activ- cyclopropyl group resulted in a substantial de- ities similar to the non-fluorine compounds, crease in lipophilicity. It was therefore concluded while the trans-compounds exhibited poor activi- that cis-1-amino-2-fluorocyclopropane was a ties against Gram-positive bacteria. As in the case promising substituent at position 1 for maintain- of levofloxacin, it was speculated that the stereo- ing favorable antimicrobial activity, although this chemical environment around position 1 affected required careful stereochemical consideration

54 Research and Development of Quinolones in Daiichi Sankyo Co., Ltd.

Figure 9. The effects of spirocyclopropane ring on quinolone-induced convulsions

Incidence of chronic convulsions (%) 100 (quinolone [100 mg/kg in mice, IV] + BPAA [400 mg/kg, PO]) 90 100 98 100 Inhibition of GABA receptor binding (%) (quinolone [10-5 M; 3–4 mg/ml] + 80 BPAA [10-4 M; 22 mg/ml])

70 70 60

10 16 14 0 0 0

O O O O F COOH F COOH F COOH F COOH

N N N N N N N N N HN Cl Cl Cl C2H5 H H F NH2 NH2 NH2 Enoxacin Clinafloxacin DU-6668 Sitafloxacin

Abbreviations: IV = intravenous, PO = orally, BPAA = biphenyl acetic acid, GABA = γ -aminobutyric acid.

(11). tivities similar to DU-6859a), DU-6611 (substitu- Using the quinolone structure with the cis- ent at position 7 not a spiro type), clinafloxacin fluorocyclopropyl group, compounds containing (development of it at Warner-Lambert Co. was different substituents at position 7 were synthe- discontinued later), and sitafloxacin with a P’ of sized. Among five-membered aminopyrrolidine 3.1 and no abnormal findings in micronucleus groups investigated, 3-amino-4-spirocyclopropyl- tests was selected as a compound for clinical de- pyrrolidine, which contains a spirocyclic structure velopment (Table 4). The results indicated that consisting of two carbon rings linked by a com- inclusion of a fluorine atom in the substituent at mon carbon atom, was the best substituent in position 1 affected lipophilicity and prevented the terms of yielding a substantial increase in antimi- induction of micronuclei. Quinolones such as na- crobial activity against Gram-positive bacteria and lidixic acid and enoxacin may induce convulsions, appropriate lipophilicity, and compound (3) was and this had been considered a class effect. Com- selected as a candidate molecule (Table 3). Since parison of enoxacin, clinafloxacin, DU-6668, and the substituent at position 1 has two asymmetric sitafloxacin in terms of convulsion-inducing ac- carbon atoms and that at position 7 has one tivity revealed that the presence of a cyclopropane asymmetric carbon atom, compound (3) has 8 ring in the substituent at position 7 significantly (23=8) optical isomers. Because the cis-position decreased the incidence of convulsions. It ap- had already been selected for the substituent at peared that the cyclopropane ring of sitafloxacin position 1, four optical isomers of the cis-com- and DU-6668 caused steric hindrance with the pound were synthesized and compared. Antimi- binding of these quinolones to γ-aminobutyric crobial activities were most favorable for DU- acid (GABA) receptors, and thereby decreased 6859a (sitafloxacin [STFX]) (Table 3) (12). CNS activation (Figure 9). Comparisons in terms of lipophilicity and induc- The results of clinical studies of sitafloxacin tion of micronuclei were made among DU- in various infectious diseases in Japan demonstrat- 6859a, DU-6668 (a compound with no fluorine ed that it exerted excellent antimicrobial activity atom at position 1 and excellent antimicrobial ac- against Gram-positive bacteria and also exhibited

55 Special Feature

Figure 10. Characteristics of sitafloxacin

O Broad antimicrobial spectrum and potent antimicrobial F COOH activities against aerobic and anaerobic Gram-negative and -positive bacteria and atypical bacteria 1

N N 1 H2O H 2 Effective against fluoroquinolone-resistant bacteria Cl F H Appropriate lipophilicity ensuring good oral absorption and NH2 H low incidence of CNS adverse effects

Sitafloxacin hydrate (STFX) Excellent clinical efficacy in patients with severe infection, Gracevit® recurrent/recrudescent infection, or infection with bacteria (-)-7-[(7S)-7-Amino-5-azaspiro[2.4]heptan-5-yl]- resistant to other agents 8-chloro-6-fluoro-1-[(1R,2S)-2-fluoro-1- cyclopropyl]-1,4-dihydro-4-oxo-3-quinoline- A promising new-generation fluoroquinolone carboxylic acid sesquihydrate

Abbreviation: CNS = central nervous system.

more potent antimicrobial activity against Gram- decreased after warnings on serious adverse drug negative bacteria than ciprofloxacin, and yielded reactions were included in their package inserts. excellent bacterial eradication rates and clinical However, the risk of serious adverse drug reaction effects. Daiichi Pharmaceutical submitted an ap- is not high with ofloxacin, levofloxacin, and sita- plication for approval of oral sitafloxacin (50 to floxacin, and the safety profile of levofloxacin has 100 mg twice daily) in 2006 and obtained ap- been established internationally. The number of proval in January 2008 for the treatment of 14 in- patients prescribed sitafloxacin is still not large, fectious diseases including chronic RTIs and and patient data need to be accumulated further complicated UTIs. In June 2008, sitafloxacin was to evaluate the safety of this drug. launched with the trade name of Gracevit® in Ja- pan as a next-generation quinolone. In foreign Conclusions countries, Phase I studies of oral sitafloxacin (500 It is extremely difficult to create substances that mg) and injectable sitafloxacin (400 mg) as well as will pass screening and investigations and finally Phase II studies of injectable sitafloxacin in Cau- prove usable as drugs. During quinolone research casian participants demonstrated favorable PK of for nearly a half century, Daiichi Sankyo Co., Ltd. oral sitafloxacin and excellent clinical effects in has found many promising candidate substances patients with severe infections (13–16). for transfer to the process of clinical development, Since the minimum inhibitory concentra- and launched three quinolones, i.e., ofloxacin, le- tions (MICs) of sitafloxacin for anaerobes as well vofloxacin, and sitafloxacin. The largest, and in- as drug-resistant bacteria such as penicillin-resis- deed never-ending challenge in the development tant pneumococci, quinolone-resistant Escherichia of antimicrobial drugs is the emergence of drug- coli, and multidrug-resistant P. aeruginosa are suffi- resistant bacteria. Medicinal chemists thus have ciently low (17–19), sitafloxacin is expected to the responsibility to continuously seek not only function as a key drug in the treatment of severe next-generation quinolones, but also novel anti- or intractable infections due to these organisms in microbial chemotherapeutic agents with new Japan and the world (Figure 10) (20). mechanisms of action.

Safety evaluation Acknowledgements Several review articles have been published on the This document is based on the award lecture for safety of quinolones (21–23). Quinolones may in- The Pharmaceutical Society of Japan Award for duce various clinically significant adverse drug re- Drug Research and Development ’10 (March, actions such as prolongation of the QT interval, 2010). The author gratefully acknowledges the disturbances of blood glucose, liver toxicity and cooperation of Dr. I. Hayakawa, Dr. K. Sakano, Dr. skin rash, and many quinolones have been with- S. Yokohama, and Dr. H. Takahashi, the co-recipi- drawn from the market or had their market size ents of the award.

56 Research and Development of Quinolones in Daiichi Sankyo Co., Ltd.

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