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The Grohe method and quinolone

Antibiotics are medicines that are used to treat bacterial for modern fluoroquinolones. The Grohe process and the . They contain active ingredients belonging to var- synthesis of sparked AG’s extensive ious substance classes, with modern fluoroquinolones one research on fluoroquinolones and the global competition of the most important and an indispensable part of both that produced additional potent antibiotics. human and veterinary medicine. It is largely thanks to Klaus Grohe – the “father of Bayer quinolones” – that this entirely In chemical terms, the antibiotics referred to for simplicity synthetic class of antibiotics now plays such a vital role for as quinolones are derived from 1,4-dihydro-4-oxo-3-quin- medical practitioners. From 1965 to 1997, Grohe worked oline carboxylic acid (1) substituted in position 1. as a chemist, carrying out basic research at Bayer AG’s ­Fluoroquinolones possess a atom in position 6. In main research laboratory (WHL) in Leverkusen. During this addition, ciprofloxacin (2) has a cyclopropyl group in posi- period, in 1975, he developed the Grohe process – a new tion 1 and also a group in position 7 (Figure A). multi-stage synthesis method for quinolones. It was this This substituent pattern plays a key role in its excellent achievement that first enabled him to synthesize active an- antibacterial efficacy. tibacterial substances such as ciprofloxacin – the prototype

O 5 O 4 3 6 COOH F COOH

7 2 N N N 8 1 H N R

(1) (2)

Figure A: Basic structure of quinolone (1) (R = various substituents) and ciprofloxacin (2)

Quinolones owe their antibacterial efficacy to their inhibition This unique mode of action also makes fluoroquinolones of essential bacterial enzymes – DNA gyrase (topoisomer- highly effective against a large number of pathogenic ase II) and IV. The active substances prevent ­bacteria that areCl resistant to , , ami- CCl2 HC COOR2 the bacterial DNA fromN supercoiling, meaning that the BasenoglycosideN and .COOR2 1 can no longerCCl3 multiply and theyC ultimatelyR die. In3HCl 1 the medical context, quinolones areH2 Ntherefore also referred Cl N R to as gyrase inhibitors. (3) (4) (5)

The Grohe method and quinolone antibiotics 1

O O HC COOR2 2 COCl Base I C C COOR2 Base II COOR R R R C R1 1 1 Hal HN HCl Hal C R HHal N R R3 HN R3 R3 (6) (7) (8) (9)

O O O COOH N COOH F COOH N CH3 N N HN N N N R R CH3

(10) (11) (12)

O F COCl HC COOEt F C Base Ⅰ C COOEt Base Ⅱ Cl Cl CH HCl Cl Cl CH HCl HN HN

(13) (14) (15)

O O F COOEt a) Verseifung F COOH Cl b) R N NH N R N N N

(16) (2)

O

Mg(OEt)2 F C (+) (a) CH(COOEt)2 H3O 13 CH2 (COOEt)2 HCl Cl Cl

O O C CH COOEt C F 2 HC(OEt)3 F C COOEt NH2 15 Cl Cl Ac2O Cl Cl CH ETOH OEt

O (b) 13 HC COOEt Base F C C COOEt NH2 CH HCl 15 Cl Cl (CH3)2NH (CH3)2N CH (CH3)2N

O O H H F COOH F COOH N H N H N N N N OCH CN H 3 H

(17) (18) O 5 O 4 3 6 COOH F COOH

7 2 N N N 8 1 H N When Grohe started working as aR chemist at Bayer AG’s as cycloacylation. The cyclocondensation of polyfunctional O WHL in 51965, he initially focused on the synthesis of acylatingO agents with tautomerizable enamines and en- 4 3 (1) (2) organochlorine6 andCOOH organofluorine intermediates. InF 1969, hydrazinesCOOH enabled him to synthesize compounds such as at his own request, he transferred to the WHL’s pharma- pyrimidines, thiazolones, uracils and pyrazolium betaines. 7 2 ceutical researchN division. In the early 1970s, Grohe’sN basic CycloacylationN of trichloromethyl isocyanide dichloride (3) 8 1 H N research work led to the discovery of a general synthesis with enamino esters (4), for example, produced pyrimidine R principle for 5- and 6-membered N-heterocycles known carbon esters (5) (Figure B). (1) (2) Cl CCl2 HC COOR2 N Base N COOR2 1 CCl3 C R 3HCl 1 H2N Cl N R Cl CCl2 HC COOR2 N (3) Base(4) N COOR2 (5) 1 CCl3 C R 3HCl 1 Figure B: SynthesisH2 ofN pyrimidine carbon esters (5) using cycloacylationCl N R

(3) (4) (5) Despite all kinds of further pharmaceutical and crop In 1975, Grohe modified the general reaction principle, protection research on numerous derivatives of these using o-halogenated benzoyl chlorides (6) as a cyclocon- heterocycles, there was no breakthrough that resulted in a densation agent for the first time. Based on combined marketable product. acylation and arylation with secondary enamino esters (7) by way of intermediates (8), he thus obtained the quinolone carboxylic acid esters (9) inO a completely new way. O HC COOR2 2 COCl Base I C C COOR2 Base II COOR R R R C R1 1 1 Hal HN O HCl Hal C R O HHal N R COCl HC COOR2 C 2 Base3 I C COOR2 BaseHN II COOR 3 R R R R R C R1 1 1 Hal HN HCl Hal C R HHal R3 N R R3(6) (7) HN (8) R3 (9) R3 (6) (7) (8) (9)

Figure C: Grohe process (Hal = Cl, Br)

Known as cycloaracylation (Figure C), this approach was Grohe was keen to continue his work on quinolones, so subsequently referred to in literature Oas the Grohe process that same yearO the new head of research transferredO him or Grohe method. from the WHL’s pharmaceutical research division to the O COOHO cropN protection team,OCOOH where he Fwas able to pursueCOOH this COOH COOHresearchF alongside hisCOOH other activities. N N CH3 N N HN N N N N CH3 N N HN N N N R R CH3 R R CH3

(10) (11) (12) (10) (11) (12)

The Grohe method and quinolone antibiotics O O 2 F COCl HC COOEt F C F COCl BaseHC Ⅰ COOEt C COOEtF Base ⅡC Base Ⅰ C COOEt Base Ⅱ Cl Cl CH HCl Cl Cl HCl CH Cl Cl ClHN Cl CH HNHCl CH HCl HN HN

(13) (14) (15) (13) (14) (15) O O F COOEt a) OVerseifung F COOH O Cl b) R N NH F N F COOEt R a)N VerseifungN N COOH Cl b) R N NH N R N N N (16) (2)

(16) (2)

O

Mg(OEt)2 F C (+) (a) CH(COOEt)2 H3O 13 CH2 (COOEt)2 HCl Cl Cl O

Mg(OEt)2 F C (+) O (a) O CH(COOEt)2 H3O 13 CH2 (COOEt)2 C CH COOEt HClC Cl Cl F 2 HC(OEt)3 F C COOEt NH2 15 Cl Cl Ac2O Cl Cl CH ETOH O O OEt C CH COOEt C F 2 HC(OEt)3 F C COOEt NH2 O 15 (b) 13 HC COOEtCl Cl Ac2O Cl Cl CH ETOH Base F C C COOEt NH2 OEt CH HCl 15 Cl Cl (CH3)2NH (CH3)2N CH (CH3)2N O (b) 13 HC COOEt Base F C C COOEt NH2 CH HCl 15 Cl Cl (CH3)2NH (CH3)2N CH (CH3)2N

O O H H F COOH F COOH N H N H N N N N OCH CN H 3 H

(17) O (18) O H H F COOH F COOH N H N H N N N N OCH CN H 3 H

(17) (18) O 5 O 4 3 6 COOH F COOH

7 2 N N N 8 1 H N R

(1) (2)

Cl CCl2 HC COOR2 N Base N COOR2 1 CCl3 C R 3HCl 1 H2N Cl N R

(3) (4) (5)

O O HC COOR2 2 COCl Base I C C COOR2 Base II COOR R R R C R1 1 1 Hal HN HCl Hal C R HHal N R R3 HN R3 R3 (6) (7) (8) (9) As someone who was passionate about ­experimenting, came across (10), (11) and Grohe was spurred on by the many different new ­chemical (12) (Figure D), also led him to believe that the possibilities of his method when compared with the quinolones he had produced offered huge medical and traditional Gould-Jacobs reaction. Associated docu- pharmaceutical potential. mentary ­research carried out by Grohe, during which he

O O O COOH N COOH F COOH N CH3 N N HN N N N R R CH3

(10) (11) (12)

Figure D: Nalidixic acid (10, R = ethyl), pipemidic acid (11, R = ethyl) and flumequine (12)

The antibacterially effective azaquinolone carboxylic acids It was, however, perfectly possible to do so with the Grohe (10, 11) and tricyclic quinolone carboxylic acid (12) all method, as he was able to demonstrate using cyclopropyl have a similar structure to the compounds Grohe syn- variants of nalidixicO acid (10) and pipemidic acid (11) (R = thesized. Since they have only a relatively weak cyclopropyl) as an example. As he had hoped, the antibac- F COCl HC COOEt F C effect against gram-negative bacteria, exhibit poor oralBase Ⅰ terial efficacy of bothC variantsCOOEt wasBase far superior Ⅱ to that of the absorption and lead to a rapid development of resistance, comparable compounds. Unfortunately, this clear progress Cl Cl CH HCl Cl Cl CH HCl how­ever, they were primarily usedHN to treat urinary tract was not sufficientHN to justify further development at the time. infections. Discovering that a cyclopropyl group in position 1 can Grohe wanted to use his new process, which synthesized significantly improve antibacterial efficacy did, however, (13) (14) (15) quinolones with a completely new kind of substituent pat- convince Grohe he was on the right track – and that en- tern, to improve these properties and obtain a quinolone couraged him to continue his research work. antibiotic that was suitableO for systemic use. The experi- O enced chemist was particularly interested in new chemical His breakthrough came in the late 1970s when – initially F COOEt F COOH groups in position 1, that is to say on the nitrogena) Verseifung atom. on paper – he combined the basic quinolone structure Having an ethylCl group in thisN position, as in b)nalidixic R N acidNH with the cyclopropylN groupN in position 1, the fluorine atom (10) or pipemidic acid (11), had been regarded as optimum inR ­position 6N and the piperazine group in position 7. On for the antibacterial effect up to that point. November 20, 1979 – long before the arrival of from Japanese company Kyorin – he thus became the Due to their size and also(16) for sterical and energy reasons, “father of cyclopropyl(2) fluoroquinolones” by devising the however, Grohe felt a cyclopropyl group was a more structural formula of ciprofloxacin. promising alternative. It also occurred to him that he had not come across any mention of 1-cyclopropyl quinolone carboxylic acids in his documentary research. He soon dis- covered why – it was impossible to produce compounds of this kind using methods such as the Gould-Jacobs O reaction. Mg(OEt)2 F C (+) (a) CH(COOEt)2 H3O 13 CH2 (COOEt)2 HCl Cl Cl

O O C CH COOEt C F 2 HC(OEt)3 F C COOEt NH2 15 Cl Cl Ac2O Cl Cl CH ETOH OEt

O The Grohe method(b) and quinolone13 antibiotics HC COOEt 3 Base F C C COOEt NH2 CH HCl 15 Cl Cl (CH3)2NH (CH3)2N CH (CH3)2N

O O H H F COOH F COOH N H N H N N N N OCH CN H 3 H

(17) (18) O 5 O 4 3 6 COOH F COOH 7 O2 O 5 4 N N 1 3 H N N 6 8 COOH F COOH

7 R 2 N N N 8 1 H N (1) R (2)

(1) (2)

Cl CCl2 2 N HC COOR 2 Base N Cl COOR CCl2 1 2 CClN3 CHC R COOR 3HCl 2 H N Base ClN N COORR1 2 1 CCl3 C R 3HCl 1 H2N Cl N R (3) (4) (5) (3) (4) (5)

OO O O 2 COCl HCHC COOR COOR2 C 2 2 COCl BaseBase I C CCCOORCOOR2 2 BaseBase II II COORCOOR R R RR R R CC RR1 1 HClHCl 1 1 1 1 HalHal HNHN HalHalCCR R HHalHHal N RN R HN R3R3 HN R3 R3 RR3 3 (6) (6) (7)(7) (8)(8) (9) (9)

O O O O O COOH N O COOH F COOH F COOH COOH N COOH N CH3 N N HN N N N N CH3 N N R HN N N RN CH3 R R CH3 (10) (11) (12)

(10) (11) (12) Neither a serious illness nor a decision by the compa- synthesize ciprofloxacin (2, R = H) on April 15, 1981 and ny’s Pharma Division in September 1980 to halt work on (2, R = ethyl) three days later(Figure E) . quinolones prevented him from using the Grohe process to

O F COCl HC COOEt F C Base Ⅰ C COOEt Base Ⅱ O Cl Cl CH HCl Cl Cl CH HCl F COCl HC COOEt F C HN Base Ⅰ HN C COOEt Base Ⅱ Cl Cl CH HCl Cl Cl CH HCl (13) HN (14) (15)HN

O O (13) (14) (15) F COOEt a) Saponification F COOH Cl b) R N NH O N R N N ON F COOEt a) Verseifung F COOH

Cl (16) b) R N NH (2) N R N N N

Figure E: Synthesis of ciprofloxacin (2, R = H) and enrofloxacin (2, R = ethyl) using the Grohe (cycloaracylation) process (16) (2) Since the enamino ester (14) was not easy to produce and production of (15) (Figure F). This significantly broadened only provided moderate yields of the intermediate (15) with the scope for varyingO his method on both a laboratory and (13), Grohe invented two further reaction sequencesMg(OEt) for the2 anF engineeringC scale. (+) (a) CH(COOEt)2 H3O 13 CH2 (COOEt)2 HCl Cl Cl O O O F C (+) F C CH2COOEt Mg(OEt)2 F C CH(COOEt)2 H3O (a) 13 CH (COOEt) HC(OEt)3 C COOEt NH2 2 2 HCl 15 Cl Cl Ac2O Cl ClCl CH ETOH OEt O O C CH COOEt C F 2 HC(OEt)3 F O C COOEt NH2 (b) 13 HC COOEt Base F C 15 Cl Cl Ac2O Cl Cl CCHCOOEt NHETOH2 CH HCl 15 Cl Cl OEt (CH3)2NH (CH3)2N CH (CH3)2N O (b) 13 HC COOEt Base F C C COOEt NH2 CH HCl 15 Cl Cl (CH3)2NH (CH3)2N CH (CH3)2N

Figure F: Malonic ester (a) and dimethylamino acrylester (b) variants of the Grohe process for producing the central intermediate (15)

The Grohe method and quinolone antibiotics 4 O O H H F COOH F COOH N H N H N N N N OCH CN H 3 H

(17) (18) O O H H F COOH F COOH N H N H N N N N OCH CN H 3 H

(17) (18) O 5 O 4 3 6 COOH F COOH

7 2 N N N 8 1 H N R

(1) (2)

Cl CCl2 HC COOR2 N Base N COOR2 1 CCl3 C R 3HCl 1 H2N Cl N R

(3) (4) (5)

O O HC COOR2 2 COCl Base I C C COOR2 Base II COOR R R R C R1 1 1 Hal HN HCl Hal C R HHal N R R3 HN R3 R3 (6) (7) (8) (9)

O O O COOH N COOH F COOH N CH3 N N HN N N N R R CH3

(10) (11) (12)

O F COCl HC COOEt F C Base Ⅰ C COOEt Base Ⅱ Cl Cl CH HCl Cl Cl CH HCl HN HN

(13) (14) (15)

O O F COOEt a) Verseifung F COOH Cl b) R N NH N R N N N

(16) (2)

O

Mg(OEt)2 F C (+) (a) CH(COOEt)2 H3O 13 CH2 (COOEt)2 HCl Cl Cl Ciprofloxacin is an exceptionally broad-spectrum antibi- The fight against never stops, though. otic. In addition to gram-negative bacteria – including the Following the synthesis of ciprofloxacin in 1981, the next aeruginosa ,O which is difficult to step was to make Oquinolones even more effective, espe- treat – it is also effective againstC CH gram-positiveCOOEt bacteria. cially in the gram-positiveC range. With Grohe acting as F 2 HC(OEt)3 F C COOEt NH2 Excellent , rapid absorption from the gas- project manager, providing guidance and assisting with 15 trointestinal tractCl and outstandingCl tissue penetrationAc 2meanO experiments,Cl a slowlyCl growingCH team set aboutETOH systemat- ciprofloxacin can be used to treat a whole host of systemic ically modifying the ciprofloxacin molecule’s substituents. infections. It was also the first that Functionalizing position 7OEt with cyclic diamines was a could be administered both orally and parenterally. ­particular focal point. O (b) In 1987, Bayer launched13 ciprofloxacin HC COOEt as a broad-spectrum In 1988, following severalC development products that Base F NH2 antibiotic in Germany, the UnitedCH Kingdom and the United raised high hopes but failedC COOEtas a result of toxicological 15 States. Enrofloxacin followed in 1988 for veterinary use.HCl By problems, the team produced (17), which Cl Cl (CH3)2NH 1992, ciprofloxacin’s(CH exceptional3)2N antibacterial properties met the above-mentionedCH objectives and was launched had already made it the world’s best-selling antibiotic and by Bayer in(CH 19993 )to2N treat bacterial respiratory diseases the gold standard in fluoroquinolones. It has now been (Figure G). Moxifloxacin is particularly effective against used to successfully treat many hundreds of millions of gram-positive bacteria such as streptococci and pneu- people. The antibiotic garnered international attention in mococci, but also against atypical pathogens including 2001 when pathogens were used as a biological chlamydia, mycoplasma and legionella. In addition to weapon in the United States. Ciprofloxacin was the only successfully treating community-acquired , approved to combat this dangerous disease at the and , this antibiotic is also prescribed for time, and it was successfully used for both prevention and patients. treatment.

O O H H F COOH F COOH N H N H N N N N OCH CN H 3 H

(17) (18)

Figure G: Moxifloxacin (17) and (18)

In the early 1990s, the work on quinolones resulted in the ciprofloxacin (2nd generation) and moxifloxacin (3rd discovery of another active substance – pradofloxacin (18) generation) cyclopropyl fluoroquinolones, which cannot be – which is mainly used to treat cats and dogs with bacterial obtained using the traditional Gould-Jacobs reaction. infectious diseases (Figure G). By making it possible to introduce many other substituents Back in the 1970s and 1980s, Grohe brought about the besides the cyclopropyl group into the effect-sensitive evolution of fluoroquinolones, especially cyclopropyl fluo- position 1 of quinolone carboxylic acids (phenyl, 4-flu- roquinolones, with the Grohe process and the synthesis of orophenyl, 2,4-difluorophenyl, hetaryl, tert-butyl, etc.), both ciprofloxacin and enrofloxacin at Bayer AG’s WHL in the Grohe process provided huge impetus for quinolone Leverkusen. research around the world. This was reflected in a flood of publications, patents and presentations at international The variants of his innovative process could be used to congresses. produce not only the well-known nalidixic acid (1st gener- ation) quinolones, but also, for the first time, themodern ­

The Grohe method and quinolone antibiotics 5 Global competition gave rise to a large number of fluo- Furthermore, there are many clear signs in relevant lit- roquinolone development products based on the Grohe erature that quinolone carboxylic acids and quinolone ­process. Examples include , , ­carboxylic acid amides could play a key role in future Heli- , and in human med- cobacter pylori, , antiviral, antitumor and icine, and also , and antiplasmodial research. The Grohe method is definitely the in veterinary medicine. Just like ciprofloxacin, moxifloxa- “tool” of choice for this upcoming research work. cin, enrofloxacin and pradofloxacin, some of these active ­ingredients have become indispensable to doctors, vets and hospitals.

About Klaus Grohe: Born in the German city of Ludwigshafen in 1934, Profes- sor Klaus Grohe studied chemistry and medical chemistry at the University of Würzburg. Having obtained a doctorate in organic chemistry in 1964, he spent nearly two years as a research assistant before starting work at Bayer AG’s main research laboratory (WHL) in Leverkusen at the end of 1965. In 1975, he invented the Grohe method, which en- abled him to synthesize the active antibacterial substances ciprofloxacin and enrofloxacin for the first time. This was the catalyst for the quinolone research at Bayer that went on to produce the quinolone-based active substance moxifloxacin and, subsequently, pradofloxacin, also under his guidance.

In 1987, Grohe was presented with the Otto Bayer Medal in recognition of his work on ciprofloxacin. Even though he retired in 1997, the “father of Bayer quinolones” was awarded the Otto Bayer Medal again in 2001, this time in honor of his life’s work. No one else has as yet received this accolade twice. In 2005, Grohe was presented with the Order of Merit of the Federal Republic of ­Germany, 1st Class. In 2010, the State Government of North Rhine-Westphalia conferred upon him the title of professor in recognition of his outstanding scientific achievements.

Shortly after the start of the new millennium, Grohe and his wife set up two foundations to support young scientists working in the fields of medical active substance research and infectiology.

The Grohe method and quinolone antibiotics 6