Fluorine walk: The impact of fluorine in quinolone amides on their activity against African sleeping sickness

Article abstract of DOI:10.1016/j.ejmech.2018.04.055

Human African Trypanosomiasis, also known as African sleeping sickness, is caused by the parasitic protozoa of the genus Trypanosoma. If there is no pharmacological intervention, the parasites can cross the blood-brain barrier (BBB), inevitably leading to death of the patients. Previous investigation identified the quinolone amide GHQ168 as a promising lead compound having a nanomolar activity against T. b. brucei. Here, the role of a fluorine substitution at different positions was investigated in regard to toxicity, pharmacokinetics, and antitrypanosomal activity. This ‘fluorine walk’ led to new compounds with improved metabolic stability and consistent activity against T. b. brucei. The ability of the new quinolone amides to cross the BBB was confirmed using an ¹⁸F-labelled quinolone amide derivative by means of ex vivo autoradiography of a murine brain.

Full text of DOI:10.1016/j.ejmech.2018.04.055

Accepted Manuscript
Fluorine walk: The impact of fluorine in quinolone amides on their activity against
African sleeping sickness
Michael Berninger, Christine Erk, Antje Fuß, Joseph Skaf, Ehab Al-Momani, Ina
Israel, Martina Raschig, Paul Güntzel, Samuel Samnick, Ulrike Holzgrabe
PII:
S0223-5234(18)30397-0
10.1016/j.ejmech.2018.04.055
EJMECH 10404
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 November 2017
Revised Date: 12 March 2018
Accepted Date: 26 April 2018
Please cite this article as: M. Berninger, C. Erk, A. Fuß, J. Skaf, E. Al-Momani, I. Israel, M. Raschig,
P. Güntzel, S. Samnick, U. Holzgrabe, Fluorine walk: The impact of fluorine in quinolone amides on
their activity against African sleeping sickness, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.04.055.
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ACCEPTED MANUSCRIPT
Fluorine Walk: The Impact of Fluorine in Quinolone Amides on their
Activity Against African Sleeping Sickness
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Berninger, Michael ; Erk, Christine ; Fuß, Antje ; Skaf, Joseph ; Al-Momani, Ehab ; Israel,
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Ina ; Raschig, Martina ; Güntzel, Paul ; Samnick, Samuel ; Holzgrabe, Ulrike
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Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074
Würzburg, Germany
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Medical Mission Institute Würzburg, Hermann-Schell-Str. 7, 97074 Würzburg, Germany
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Department of Nuclear Medicine, University Hospital of Würzburg, Oberduerrbacher Str. 6,
97080 Würzburg, Germany
Corresponding author:
Prof. Dr. Ulrike Holzgrabe
Phone: +49-931-3185460
Email: ulrike.holzgrabe@uni-wuerzburg.de
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Abstract
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Human African Trypanosomiasis, also known as African sleeping sickness, is caused by the
parasitic protozoa of the genus Trypanosoma. If there is no pharmacological intervention, the
parasites can cross the blood-brain barrier (BBB), inevitably leading to death of the patients.
Previous investigation identified the quinolone amide GHQ168 as a promising lead
compound having a nanomolar activity against T. b. brucei. Here, the role of a fluorine
substitution at different positions was investigated in regard to toxicity, pharmacokinetics, and
antitrypanosomal activity. This ‘fluorine walk’ led to new compounds with improved
metabolic stability and consistent activity against T. b. brucei. The ability of the new
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quinolone amides to cross the BBB was confirmed using an F-labelled quinolone amide
derivative by means of ex vivo autoradiography of a murine brain.
Keywords: quinolone amides, Trypanosoma brucei brucei, structure-activity relationship,
fluorine walk, metabolism, blood-brain barrier, autoradiography
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1 Introduction
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Human African Trypanosomiasis (HAT) is a serious life threatening disease occurring in 36
African countries and is caused by the vector-borne parasites Trypanosoma brucei
rhodesiense (T. b. rhodesiense) or Trypanosoma brucei gambiense (T. b. gambiense),
respectively [1]. HAT can be differentiated into two clinically relevant stages: stage 1 is
characterized by unspecific headache, fever, and pruritus [2]. In stage 2, parasites have
crossed the blood-brain barrier (BBB) and invaded the central nervous system (CNS), causing
confusion, sensory impairments, and the eponymous sleep disturbances. Untreated patients
finally progress to coma, systemic organic failure, and inevitable to death [3]. Among
available drugs, there are only three applicable compounds, i.e. melarsoprol, eflornithine, and
nifurtimox which is also active against stage 2 of HAT [4]. Additionally, the current
chemotherapy suffers from severe side effects, requires an intravenous or intramuscular
administration, and has a limited efficacy targeting only one of the Trypanosoma subtypes.
E.g., an intravenous infusion of melarsoprol being administered over a long time period is the
only effective drug against T. b. rhodesiense being able to pass the BBB [5]. Moreover, the
accompanying side effects of the toxic arsenic are severe, resulting in an encephalic reaction
in approximately 10% of the treated patients and being lethal in 50% of the cases [6]. Taken
together, there is an urgent need for novel and safe antitrypanosomal drug candidates with
CNS permeability that allow application in both stages 1 and 2 of HAT. Hence, we recently
established quinolone amides that are active against T. b. brucei in the nanomolar
concentration range and possess in vivo efficacy [7, 8].
Generally, in medicinal chemistry the incorporation of a fluorine atom in molecules can
influence conformation, pK value, intrinsic potency, membrane permeability, metabolic
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stability, as well as pharmacokinetics [9-11]. The systematic ‘walk’ of fluorine around the
quinolone scaffold was already applied successfully for quinolones addressing the M1
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acetylcholine receptor [12]. Additionally, the F isotope of fluorine can be deployed for
labelling, and subsequent positron-emission tomography (PET) and autoradiography,
respectively [9]. The impact of different substitution patterns, particularly of fluorine, on the
quinolone amide skeleton is explored herein. The aim of this ‘fluorine walk’ was to enhance
the antitrypanosomal potency and to expand the structure-activity relationship. As
lipophilicity and metabolic stability are strongly affected by fluorine, these parameters were
studied simultaneously. The most potent drug candidate GHQ168 was used for optimization
with regard to activity, cytotoxicity, metabolism, and lipophilicity and solubility.
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The ability of the quinolone amide to pass the BBB was investigated by means of ex vivo
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autoradiography. Therefore, the quinolone amide GHQ168 was radiofluorinated with F and
injected intravenously into mice for the respective studies.
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2 Results and Discussions
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2.1 Chemistry
Starting off with the corresponding aniline derivatives, the quinolone scaffolds 1a-h were
formed via Gould-Jacobs procedure (cf. Scheme 1) [13]. Subsequently, the N-1 position was
deprotonated utilizing potassium carbonate followed by alkylation using alkyl halides (n-
bromobutane (A) and benzyl-protected 3-bromopropan-1-ol (B)) in the presence of catalytic
amounts of potassium iodine; compounds 2a-j were obtained [14]. Compound 2j was
synthesized using non-freshly distilled N,N-dimethylformamide which contains residual
amounts of dimethyl amine as a degradation product. The amination of position 5 via a
nucleophilic aromatic substitution proceeded due to stabilisation effects of the C-4 oxo group
[15, 16]. Without preceding characterisation of the ethyl esters 2a-j, the products 2a-j were
hydrolysed to yield the carboxylic acid derivatives 3a-j. Depending on the respective
substitution pattern and leaving group, either 2 M HCl (compounds 2c-f, 2h, 2j) or 3 M KOH
(compounds 2a, 2b, 2g, 2i) was used for hydrolysis of the ethyl esters. Position 5 of
compound 3d was further utilized for introducing a methoxy group into the quinolone
scaffold, obtaining compound 3k.
Position 7 of the quinolone scaffold was substituted with morpholine, resulting in compounds
4a-k [7]. In order to favour a substitution in position 7 over position 5, position 7 of
compounds 3d and 3f was activated by means of a borate complex according to ref. [15, 17]
(cf. Scheme 1). To this end, compounds 3d, 3f, and boron triflouride diethyl etherate were
dissolved in CH Cl and heated to 50 °C yielding the boron-chelated intermediate which was
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subsequently refluxed in morpholine for 4 h at 80 °C to give C1 and C2. The ensuing
hydrolysis of the boron ester with 2 M NaOH afforded compounds 4d and 4f with a
regioselective substitution in position 7.
Finally, the amidation step was carried out after generating the anhydride derivatives from the
corresponding carboxylic acids in-situ which were subsequently reacted with the benzyl
amines to generate the quinolone amides 5-18 [7].
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The benzyl protected alcohol group of 12 was cleaved using microwave irradiation and
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catalytical amounts of Pd/C suspended in CHCl (cf. Scheme 2). The resulting compound 19
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was treated with N,N-diethylaminosulfur trifluoride (DAST) to give the monofluoroalkyl
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derivative 21 as a reference standard for the corresponding labelled [ F]21. For the synthesis
of the precursor 20, compound 19 was treated with methanesulfonyl chloride, attaching a
mesylate ester to the terminal alcohol residue. The mesylated precursor 20 was
radiofluorinated via nucleophilic substitution. The crude labelled product was purified by
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means of radio-HPLC and a tC cartridge to give [ F]21 in 60 ± 5% radiochemical yield
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(RCY). The total synthesis time was 50 min including purification and formulation with 10%
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EtOH/saline solution. The identity and radiochemical purity of the radiotracer [ F]21 were
confirmed by co-injection with the corresponding standard 21 using radio-HPLC (cf. Fig. S7).
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2.2 Structure-activity relationship – the fluorine walk
The in vitro antitrypanosomal activities were demonstrated by means of the trypomastigote
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forms of T. b. brucei laboratory strain TC 221 using the AlarmarBlue assay and photometric
measurement of the viability [7, 18-20]. The cytotoxicity was evaluated on the viability of
macrophage cell line J774.1 [8, 20, 21]. The in vitro results are summarized in Table 1.
The quinolone amide GHQ168 carrying a fluorine in position 6 showed a high activity
against T. b. brucei (IC = 47 nM), T. b. rhodesiense (IC = 9 nM), and a moderate
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cytotoxicity (CC = 57 µM) [7]. Here, the chemical strategy and the SAR analysis targeted
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the antitrypanosomal improvement particularly by varying the fluorine substitution pattern.
Quinolone Core. Desfluoroquinolone. The development of commercially available
antibacterial quinolones started with non-fluorinated quinolones (e.g., nalidixic acid) and
proceeded with 6-fluoro substituted compounds (e.g., ciprofloxacin), but none of them
showed any antitrypanosomal activity [22]. Fluorine is generally used as a bioisosteric
interchange for hydrogen, as demonstrated herein. However, size and electronic effects of the
two atoms are fairly different. The fluorine atom is approximately 20% larger than hydrogen
(van der Waals radius 1.20 Å vs 1.47 Å) and their electronegativities differ in 1.78 resulting
in a highly polarised C-F bond [23]. Since the target site of the quinolone amides is not yet
elucidated, the impact of fluorine towards this compound class is not understood to date.
Hence, despite of the marked differences between hydrogen and fluorine, it was worthwhile
to investigate a quinolone core that lacks this particular electron withdrawing element. The
antitrypanosomal activity of the des-fluoroquinolone 5 was decreased by a factor of five
(IC = 230 nM for 5) indicating the considerable impact of fluorine on the activity. However,
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the CC was higher than 100 µM.
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Shifting Fluorine. Relocation of the fluorine atom from position 6 to 8 (cf. compound 6)
resulted in a decreased antitrypanosomal activity (IC = 790 nM). Fluorine in position 8
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apparently could not compensate the missing fluorine atom in position 6 and additionally
negatively influenced the activity against trypanosomes comparing to the desfluorquinolone
5. When shifting fluorine from position 6 to 5 (cf. compound 7), the trypanocidal activity was
comparable to GHQ168 (IC = 50 nM for 7) and interestingly, no cytotoxic effects were
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observed up to concentration levels of 100 µM. Thus, 7 surpassed the selectivity index (SI =
CC /IC ) of the lead compound and was 2000 times more selective towards T. b. brucei
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parasites. Hence, the 5-fluoro quinolone core was a superior scaffold possessing promising
biological properties.
Additional Fluorine. Inserting an additional fluorine in position 8 of some approved
fluoroquinolones results in a very high antibacterial activity, whereas the corresponding
amide 8 did not benefit from a double fluorination with the antitrypanosomal activity
remaining the same (IC = 60 nM). This supported the initially assertion that the fluorine in
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position 8 did not positively influence the antitrypanosomal activity. Introducing an extra
fluorine in position 5 slightly affected the trypanocidal activity of compound 9 (IC50
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40 nM). Thus, fluorine in position 5 was assigned a more important role since it was at least
well-tolerated or even advantageous (cf. compounds 7, 9).
Fluorine Replacement. The methoxy moiety demonstrated bioisosteric properties to fluorine
in thrombin inhibitors [11, 24] and in the drug ezetimibe [25, 26]. Beside the difference in
size, both groups (C-F and C-O) are hydrogen bond acceptors, even though fluorine possesses
considerably less proton affinity [11]. Accordingly, we explored a methoxy group in position
6 (cf. compound 10) leading to a loss in activity by a factor of nearly seven (IC = 310 nM
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for 10), while the cytotoxicity remained in a range comparable to GHQ168. Thus, a more
stronger hydrogen bond acceptor in position 6 was not beneficial. Afterwards, evaluating the
influence of an even more voluminous residue the fluorine atom was replaced by a
trifluoromethyl group (van der Waal radius 2.12 Å; cf. compound 11) which is apparently
similar to an isopropyl and an ethyl group, respectively [11, 23]. The antitrypanosomal
activity of 6-CF -substituted 11 was diminished to an IC value of 540 nM and a CC value
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of 78.6 µM. Hence, this particular bulky and rather lipophilic substituent did not enhance the
antitrypanosomal activity.
Since the 5-fluorine quinolone core (cf. compound 7) exhibited high antitrypanosomal activity
the compatibility of further substituents in position 5 was investigated. Compounds 13 and 14,
having a dimethylamine and a methoxy group in position 5, respectively, possessed a strongly
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reduced antitrypanosomal activity (IC = 1.85 µM and 0.76 µM, respectively). The decreased
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biological activity might be attributed to the poor tolerance towards sterically demanding
residues in position 5. Nevertheless, 14 was superior to 13 because of its slightly reduced
cytotoxicity (CC = 51.8 µM vs. 44.0 µM), and thus higher selectivity (SI = 68 vs. 24).
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Benzylamide Residue. Compound 15 having a p-fluorine substituent exhibited a high activity
against T. b. brucei (IC = 50 nM). Additionally, compound 16 bearing a fluorine in ortho
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position exhibited only one-eighth of the antitrypanosomal activity of GHQ168 (IC50
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410 nM for 16), and a moderate cytotoxicity (CC = 37.0 µM). When occupying both para
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and ortho positions with fluorine (cf. compound 17), the activity could be restored again and
was even slightly improved (IC = 30 nM, CC = 59.6 µM).
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Compound 18 combined the superior fluorine substitution pattern of the quinolone core in
position 5 and the fluorination of the benzylamide residue in para position, subsequently
leading to the most active substance with an IC value of 20 nM and a CC value higher than
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25 µM (SI = > 1250).
N-1 Alkyl Residue. Compound 19, having a terminal alkyl hydroxyl group, merely possessed
approximately one fifth of the trypanocidal activity of GHQ168. Neither the hydrogen bond
acceptor nor the donor properties of the hydroxyl moiety positively affect the biological
activity. Compound 21 carrying a terminal fluorine was only as half effective as the lead
compound. The respective activities of 270 nM and 120 nM were still in the submicromolar
concentration range. Additionally, both analogues exhibited a moderate cytotoxicity (CC =
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43.1 µM and 42.4 µM, respectively).
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2.3 Metabolism
2.3.1 Metabolites of 7, 15, 18
For newly developed drugs the metabolism and the metabolites are important to know,
because these compounds can be harmful to the organism, and may influence the
pharmacokinetics. Therefore, the phase-I-metabolism of GHQ168 was investigated [23]
using microsomes from rat male liver (induced by phenobarbital and ß-naphthoflavon) and
cytosol from rat male liver (induced by aroclor 1254). These studies were carried out as
described by Gareis [23] and were applied here, accordingly.
For compound 7, eight metabolites could be identified by means of LC/MSD ion trap (cf.
Scheme 3 and Fig. S3). The m/z value of 454 hints to two different kinds of hydroxylation:
the first one at the N-benzyl substituent, and the second one at the N-alkyl chain. The
structures of both metabolites could be confirmed by LC-MS/MS fragmentation due to the
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corresponding N-debenzylation resulting in m/z values of 348 and 364, respectively. The
metabolite with m/z = 442 can be explained by a double hydroxylation of the benzyl moiety
and an oxidative N-desalkylation, the biotransformation to m/z = 349 indicates an amide
hydrolysis, and m/z = 306 hints at an oxidative N-desalkylation in combination with an amide
hydrolysis. Finally, two more metabolites (m/z = 426 and 412) were identified where
hydroxylation and desalkylation are very likely. In conclusion, the metabolites were mainly
produced by aromatic and aliphatic hydroxylation as well as oxidative N-desalkylation and
amide hydrolysis combined with hydroxylation. Mainly, metabolites by hydroxylation are
formed.
The metabolites of compounds 15 and 18 are similar; they were predominantly metabolized
via an N-desalkylation reaction. As expected, a hydroxylation of the benzyl substituent was
not observed due to the fluorine at the phenyl ring, but a hydroxylation at the N-alkyl chain,
N-desalkylation, amide hydrolysis, and a combination of hydroxylation and N-desalkylation
were found for both compounds. The assignment of the metabolites was achieved in analogy
to references [8][20]. Details can be found in Figures S4 and S5.
2.3.2 Metabolic turnover
Blocking metabolically reactive sites by fluorine substituents is a well-established method [1].
Since fluorination enhances the metabolic stability, the benzyl amide moiety of GHQ168 (t1/2
= 5.8 h [20]), which was prone to metabolism (hydroxylation), was fluorinated. The electron
withdrawing effect of fluorine should inactivate the benzyl residue for oxidative metabolic
processes and enhance the compound’s stability. Indeed, 15 was less prone to metabolism (t1/2
= 6.4 h). Interestingly, compound 7 (t = 7.2 h) with fluorine in position 5 exhibited an even
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more metabolic stability than 15. Consequently, the combined feature of fluorine in position 5
and a para fluorinated benzyl residue (18, t = 7.7 h) is the most stabile substitution pattern.
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The highest proportion of metabolites was formed in the cytosol, indicating that aldehyde
dehydrogenases and monoamine oxidases might be mainly responsible for the metabolic
transformation. Furthermore, in-depth investigations of the quinolone amides turnover in the
cytosol revealed compound 18 (cf. Table 2) to exhibit the lowest turnover in this environment
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(3.89 ± 0.29 pmol×min ×mg×proteine ). These findings substantiated the outcome of an
enhanced metabolic stability of the quinolone amides in the order GHQ168 < 15 < 7 < 18.
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2.4 Lipophilicity and solubility
The lipophilicity, represented as logP value, was determined by means of an HPLC method.
The logarithmized capacity factor of the calibration substances was correlated with the
experimental octanol/water logP values (cf. Fig. S2) [7, 27]. Since the lipophilicity strongly
influences permeation and solubility processes, it can be regarded as a surrogate parameter for
predicting oral bioavailability; a logP value lower than 5 is considered more desirable [28].
In general, additional fluorine substituents at an aromatic ring system increase lipophilicity
due to good overlapping orbitals which holds true for 8 and 15 (logP = 4.57 and 4.13) (cf.
Table 1) [29]. Shifting the fluorine atom from position 6 to 5 of the quinolone scaffold (cf.
compound 7) significantly and unexpectedly reduced the lipophilicity (logP = 3.36). This
effect could possibly emerged due to the polarization of the carbonyl oxygen atom in position
4 by the fluorine atom in close proximity [30]. Consequently, the surrounding water
molecules might form more stronger hydrogen bonds with this oxygen atom. Additionally, the
fluorine in vicinity to the oxygen could increase the overall polarity of the quinolone molecule
[30]. Hence, in contrary to the general rule the double fluorinated compounds 9 and 18
possessed a lower logP value (logP = 3.97 and 3.41, respectively) than the mono fluorinated
GHQ168. Adding a fluorine substituent to a saturated alkyl chain could rather lead to a
reduced logP, consequently compound 21 has a lower logP value of 3.34 [29]. Additionally,
compared to the lead compound GHQ168 (logP = 4.10) the logP could be decreased to 3.04
by introducing a hydrophilic hydroxyl group at the N-1 residue (cf. compound 19).
Compound GHQ168 possessed a low thermodynamic solubility of 0.005 µg/mL in PBS
buffer [8]. Therefore, the solubility of certain quinolone amides (5-7, 10, 19, 21) was
examined applying the shake flask method in accordance to reference [8]. In general,
solubility could be moderately improved for compounds 5-7, and 21 (S = 0.12-1.36 µg/mL;
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cf. Fig. S6). However, the solubility of compounds 10 and 19 was remarkably enhanced (S =
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2.73 µg/mL and 18.34 µg/mL, respectively; cf. Fig. S6). As indicated by the logP value of 19,
water solubility could be substantially improved by the introduction of polar groups, e.g. a
hydroxy group (cf. compound 19, logP = 3.04).
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2.5 Radiochemistry and Autoradiography
The positron emitter radionuclide F-18 exhibits good radionuclear properties with low
positron energy (Eβmax = 0.635 MeV) and a suitable half-life time (t ) of 109 min. Since most
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commercially available antibacterial quinolones originally possess a fluorine atom, a direct
nucleophilic displacement of F-19 to F-18 for lemofloxacin and trovafloxacin via isotopic
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exchange was reported [31, 32]. However, a direct nucleophilic exchange in position 6
through a nucleophilic aromatic substitution was described not to occur [33]. Thus, an
appropriate leaving group, i.e., a mesylate group, was attached to the quinolone amide at the
N-hydroxyalkyl substituent (19). F-18 labelling was conveniently applied as the very last step
(cf. Scheme 2), resulting in compound 21.
Since the delivery to the brain is crucial for the treatment of stage 2 of HAT, the permeation
of the quinolone amide was evaluated by means of autoradiography. The labelled compound
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[ F]21 was injected into a mouse tail vein and the mouse was sacrificed 60 min afterwards.
The murine brain was dissected and the ex vivo autoradiography illustrated the distribution of
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compound [ F]21 within the brain tissue (cf. Figure 1 and Fig S8). The compound
accumulated within the entire brain in medium concentration levels (indicated by green),
combined with areas of high concentration levels in the inner brain sections (indicated by
red). The autographic images confirmed the uptake of the quinolone amides in healthy murine
brain.
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The systematic walk of fluorine around the scaffold of the lead compound GHQ168 was
successfully implemented. Compound 18 with a striking antitrypanosomal activity and
moderate cytotoxicity (IC = 20 nM against T. b. brucei, CC = >25 µM) was revealed.
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Hence, the biological activity against trypanosomes was enhanced and the 5-fluoro-
substituted quinolone is considered superior to the lead compound. Its logP value suggested a
moderate water solubility, but the aqueous solubility was determined at 0.12 µg/mL, though.
Additionally, the influence of fluorine substitution patterns on metabolic stability was
examined. The most potent compound 18 (t = 7.7 h) showed an extension of the metabolic
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half-live of 25% in comparison to GHQ168 (t1/2 = 5.8 h[20]), resulting in a longer drug
exposure to the trypanosomes.
Moreover, the permeation of quinolone amides through the murine BBB could be proved
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using the [ F]-labelled derivative [ F]21. Therefore, this compound class could be suitable
for treatment of HAT stage 2, when parasites have affected CNS.
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99 4 Experimental Section
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4.1 Chemistry
Microwave reactions were performed using synthWAVE and rotaPREP systems (both MLS,
Leutkirch, Germany). Melting points were determined with a capillary melting point
apparatus (Sanyo Gallenkamp, Leicestershire, UK) and were not corrected. IR spectra were
recorded on a JASCO FT-IR-6100 spectrometer (Jasco, Groß-Umstadt, Germany). Thin layer
chromatography (TLC) was performed on pre-coated silica gel (UV ) glass plates
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(Macherey-Nagel, Düren, Germany). Column chromatography was performed using silica gel
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with a particle size of 0.063-0.200 mm (Merck, Darmstadt, Germany). H (400.131 MHz) and
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C (100.623 MHz) Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker
AV 400 NMR spectrometer (Bruker Biospin, Ettlingen, Germany). The signals of the
1
13
deuterated solvents were used as an internal standard (DMSO-d : H 2.50 ppm, C 39.43;
6
1
13
19
CDCl : H 7.26 ppm, C 77.00). F NMR spectra were recorded on a Bruker 400 NMR
3
spectrometer. NMR data are presented with chemicals shifts in ppm, the multiplicity (s,
singlet; d, doublet; t, triplet; q, quartet; quint, quintet; sext, sextet; m, multiplet; dd, doublet of
doublet), and coupling constants J given in Hz. The electron spray mass spectra were acquired
on a Shimadzu LCMS 2020 instrument. Reagents were purchased with a minimum purity of
95% from conventional commercial suppliers and were used without further purification. The
purity of target compounds was ≥95% and was confirmed by means of HPLC analysis using a
Synergi 4µ fusion-RP column (150 mm × 4.6 mm) (Phenomenex, Aschaffenburg, Germany),
a Shimadzu instrument (Kyoto, Japan) equipped with an SPD-20A UV/Vis detector (λ =
254 nm), and a mobile phase being a mixture of water (A) and methanol (B); gradient elution
programme: 5% B → 90% B from 0 to 8 min; 90% B from 8 to 13 min; 90% B → 5% B from
1
8
13 to 15 min; 5% B from 15 to 18 min. The flow rate was adjusted to 1 mL/min. [ F]Fluoride
®
was produced on the PETtrace cyclotron (GE Medical Systems, Uppsala, Finland) at the
1
8
18
interdisciplinary PET centre of the University of Würzburg via an O(p,n) F reaction by
1
8
irradiating 3.0 mL of 95% enriched [ O]water with 16.5 MeV protons.
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4.1.1 General synthesis of 1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acids 3a-i
according to [7, 14]. The appropriate ethyl-4-hydroxyquinoline-3-carboxylate 1a-h (1 eq) (cf.
Fig. S1, synthesised according to ref [7, 14, 34-38]) and potassium carbonate (4 eq) were
suspended in N,N-dimethylformamide under Ar atmosphere. The reaction was heated 30 min
at 60 °C, followed by adding a catalytic amount of potassium iodide and the appropriate alkyl
halide (1.5-5 eq). After 20-48 h of heating at 75-90 °C, the solvent was removed in vacuo and
1
1

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water was added. The aqueous layer was extracted with EtOAc and the combined organic
layers were dried over anhydrous Na SO . The solvent was removed in vacuo and the oily
2
4
residue was subsequently purified by column chromatography utilizing silica gel. Without
further characterization, the resulting products 2a-2i were hydrolysed using either 2 M HCl or
3 M KOH at 100 °C. If necessary, the solution was acidified to pH 2 and the precipitated
product was collected. Afterwards, the solid was washed with cold water and dried in vacuo
to give compounds 3a-i.
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344
4.1.1.1 1-Butyl-7-chloro-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3a).
The compound was synthesised according to the general procedure described in 4.1.1.
Spectroscopic data are in accordance with reference [7].
345
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356
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4.1.1.2 7-Bromo-1-butyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3b). According to the
general procedure 4.1.1, a solution of compound 1b (1 eq, 3.2 g, 10.8 mmol) and potassium
carbonate (4 eq, 6.0 g, 43.2 mmol) in N,N-dimethylformamide was treated with n-
bromobutane (5 eq, 5.8 mL, 54.0 mmol), and the reaction was heated at 85 °C for 24 h. The
crude product was purified by column chromatography (eluent: CHCl /i-PrOH = 75:1, R =
3
f
0.62) and then hydrolysed by refluxing compound 2b under basic conditions (3 M KOH).
After acidification with 2 M HCl under ice cooling, the precipitates were collected and dried
-1
in vacuo to yield 2.38 g of 3b. Yield: 68%; mp 224–225 °C. IR [cm ]: 3144, 3084, 2976,
1
2904, 1695, 1607, 1523, 1458, 1374, 1192, 1069. H-NMR (DMSO-d , δ [ppm], J [Hz]):
6
3
4
3
14.94 (s, 1H), 9.02 (s, 1H), 8.29 (m, 3H), 8.57 (dd, J = 8.8, J = 1.2, 1H), 4.57 (t, J = 7.2,
3
3
3
13
2H), 1.74 (quint, J = 7.2, 2H), 1.35 (sext, J = 7.6, 2H), 0.93 (t, J = 7.2, 3H). C-NMR
(DMSO-d , δ [ppm], J [Hz]): 177.7, 165.6, 150.0, 140.0, 129.4, 128.3, 127.8, 124.5, 120.5,
6
107.9, 53.6, 30.6, 18.9, 13.4.
3
3
3
3
3
3
3
3
59 4.1.1.3 1-Butyl-7-chloro-8-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid
(3c).
60
61
62
63
64
65
66
According to the general procedure 4.1.1, a solution of compound 1c (1 eq, 3.00 g,
11.1 mmol) and potassium carbonate (4 eq, 6.15 g, 44.5 mmol) in N,N-dimethylformamide
was treated with n-bromobutane (5 eq, 6.0 mL, 55.6 mmol), and the reaction was heated at
80 °C for 48 h. The crude product was purified by column chromatography (eluent:
CHCl /MeOH = 100:1, R = 0.72) and then hydrolysed by refluxing compound 2c under
3
f
acidic conditions (2 M HCl). The precipitates were collected and dried in vacuo to yield
-1
1.80 g of 3c. Yield: 55%; mp 211-213 °C. IR [cm ]: 3091, 3048, 2930, 2858, 1713, 1620,
1
2

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1602, 1541, 1440. H-NMR (DMSO-d , δ [ppm], J [Hz]): 14.85 (s, 1H), 9.01 (s, 1H), 8.20
6
3
5
3
4
(dd, J = 7.2, J = 1.6, 1H), 8.57 (dd, J = 8.8, J = 6.4, 1H), 4.60–4.56 (m, 2H), 1.82–1.77 (m,
3
13
2H), 1.38 –1.33 (m, 2H), 0.94 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ [ppm], J [Hz]): 177.7
6
4
1
2
(d, J = 2.4, 1C), 165.1, 152.1, 147.2 (d, J = 251.4, 1C), 129.7 (d, J = 7.1, 1C), 127.4,
C,F
C,F
C,F
2
4
4
126.8, 126.4 (d, J = 18.9, 1C), 122.5 (d, J = 4.9, 1C), 108.1, 58.0 (d, J = 14.4, 1C),
C,F
C,F
C,F
5
31.9 (d, J = 4.1, 1C), 18.9, 13.3.
C,F
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
4.1.1.4 1-Butyl-5,7-difluoro-4-oxo-1,4-diyhdroquinoline-3-carboxylic acid (3d). According to
the general procedure 4.1.1, a solution of compound 1d (1 eq, 10.0 g, 39.5 mmol) and
potassium carbonate (4 eq, 16.7 g, 158.0 mmol) in N,N-dimethylformamide was treated with
n-bromobutane (5 eq, 21.3 mL, 197.5 mmol), and the reaction was heated at 90 °C for 20 h.
The crude product was purified by column chromatography (eluent: CHCl /MeOH = 50:1, R
3
f
= 0.80) and then hydrolysed by refluxing compound 2d under acidic conditions (2 M HCl).
The precipitates were collected and dried in vacuo to yield 9.77 g of 3d. Yield: 88%; mp 208–
-1
210 °C. IR [cm ]: 3368, 3118, 2965, 2871, 1708, 1614, 1512, 1437, 1343, 1283, 1168, 1127,
1
1014. H-NMR (DMSO-d , δ [ppm], J [Hz]): 14.98 (s, 1H), 8.98 (s, 1H), 7.78–7.75 (m, 1H),
6
3
3
7.51–7.46 (m, 1H), 4.47 (t, J = 7.2, 2H), 1.76–1.68 (m, 2H), 1.34 (m, 2H), 0.89 (t, J = 7.2,
1
3
3
3H). C-NMR (DMSO-d , δ [ppm], J [Hz]): 176.8 (d, J = 1.6, 1C), 165.43, 164.6 (dd,
6
C,F
1
3
1
3
JC,F = 250.4, J = 15.2, 1C), 156.7 (dd, J = 248.7, J = 15.6, 1C), 150.1, 144.3 (dd,
C,F
C,F
C,F
3
3
2
4
2
JC,F = 15.0, J = 14.2, 1C), 113.7 (dd, J = 8.4, J = 2.4, 1C), 108.8, 100.1 (dd, J
=
C, F
C,F
C,F
C,F
2
2
4
25.1, J = 25.1, 1C), 92.6 (dd, J = 26.7, J = 4.5, 1C), 54.2, 30.3, 18.9, 13.5.
C,F
C,F
C,F
1
3

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4.1.1.5 1-Butyl-6,7,8-trifluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3e). According
to the general procedure 4.1.1, a solution of compound 1e (1 eq, 2.40 g, 8.85 mmol) and
potassium carbonate (4 eq, 4.90 g, 35.4 mmol) in N,N-dimethylformamide was treated with n-
bromobutane (5 eq, 5.75 mL, 44.3 mmol), and the reaction was heated at 90 °C for 48 h. The
crude product was purified by column chromatography (eluent: CHCl /i-PrOH = 50:1, R =
3
f
0.49) and then hydrolysed by refluxing compound 2e under acidic conditions (2 M HCl). The
precipitates were collected and dried in vacuo to yield 1.62 g of 3e. Yield: 62%; mp 216–218
-1
°C. IR [cm ]: 3056, 2963, 2934, 2875, 1713, 1614, 1560, 1519, 1482, 1455, 1412, 1390,
1
1284, 1110, 1056. H-NMR (DMSO-d , δ [ppm], J [Hz]): 14.48 (s, 1H), 9.04 (s, 1H), 8.24–
6
1
3
8.19 (m, 1H), 4.62–4.57 (m, 2H), 1.84–1.80 (m, 2H), 1.38–1.33 (m, 2H), 0.93 (t, 3H). C-
3
NMR (DMSO-d , δ [ppm], J [Hz]): 175.4 (d, J = 1.2, 1C), 164.6, 152.6 149.2 (m, 1C),
6
C,F
3
146.9 (m, 1C), 142.5 (m, 1C), 127.1, 122.6 (d, J = 6.8, 1C), 108.2 (m, 1C), 107.4, 58.1 (d,
C,F
4
5
19
JC,F = 13.5, 1C), 32.4 (d, J = 3.9, 1C), 19.4, 13.9. F-NMR (DMSO-d , δ [ppm], J [Hz]):
C,F
6
-134.44 (dd, J6,8 = 24.8, J6,7 = 6.9), -140.68 (dd, J6,8 = 6.9, J7,8 = 20.0), -149.05 (dd, J6,7
24.8, J = 20.0).
=
7
,8
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
4.1.1.6 1-Butyl-5,6,7-trifluoro-4-oxo-1,4-diyhroquinoline-3-carboxylic acid (3f). According to
the general procedure 4.1.1, a solution of compound 1f (1 eq, 9.12 g, 33.6 mmol) and
potassium carbonate (4 eq, 18.60 g, 134.4 mmol) in N,N-dimethylformamide was treated with
n-bromobutane (5 eq, 18.0 mL, 168.0 mmol), and the reaction was heated at 70 °C for 20 h.
The crude product was purified by column chromatography (eluent: CHCl /MeOH = 100:1,
3
R = 0.81) and then hydrolysed by refluxing compound 2f under acidic conditions (2 M HCl).
f
The precipitates were collected and dried in vacuo to yield 2.13 g of 3f. Yield: 28%; mp 233-
-1
1
236 °C. IR [cm ]: 3099, 2964, 2879, 1715, 1650, 1455, 1346, 1297, 1186. H-NMR (DMSO-
3
3
d , δ [ppm], J [Hz]): 9.00 (s, 1H), 8.12–8.07 (m, 1H), 4.49 (t, J = 7.2, 2H), 1.71 (quint, J =
6
3
3
13
7.2, 2H), 1.34 (sext, J = 7.2, 2H), 0.89 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ [ppm], J
6
1
2
3
[Hz]): 176.2, 165.3, 153.2 (ddd, J = 251.9, J = 11.0, J = 4.6, 1C), 150.0, 149.8 (ddd,
C,F
C,F
C,F
1
2
3
1
2
JC,F = 264.2, J = 10.8, J = 5.0, 1C), 136.9 (dt, J = 248.8, J = 15.3, 1C), 136.5 (d,
C,F
C,F
C,F
C,F
3
2
2
3
JC,F = 12.9, 1C), 113.9 (d, J = 5.3, 1C), 108.4, 102.4 (dd, J = 22.5, J = 4.5, 1C),
C,F
C,F
C,F
19
54.1, 30.3, 18.9, 13.4. F-NMR (DMSO-d , δ [ppm], J [Hz]): -123.76 (m), -133.87 (m), -
6
161.99 (m).
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4

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4.1.1.7 1-Butyl-7-chloro-6-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid
(3g).
According to the general procedure 4.1.1, a solution of compound 1g (1 eq, 5.5 g, 19.6 mmol)
and potassium carbonate (4 eq, 10.83 g, 78.4 mmol) in N,N-dimethylformamide was treated
with n-bromobutane (5 eq, 10.5 mL, 98.0 mmol), and the reaction was heated at 85 °C for
24 h. The crude product was purified by column chromatography (eluent: CHCl /iPrOH =
3
150:1, R = 0.59) and then hydrolysed by refluxing compound 2g under basic conditions (3 M
f
KOH). After acidification with 2 M HCl under ice cooling, the precipitates were collected and
.1
dried in vacuo to yield 4.13 g of 3g. Yield: 68%; mp 232–233 °C. IR [cm ]: 3041, 2963,
1
2941, 2874, 1702, 1607, 1457, 1433, 1219, 1058. H-NMR (DMSO-d , δ [ppm], J [Hz]):
6
3
3
15.20 (s, 1H), 8.98, 8.27 (s, 1H), 7.86 (s, 1H), 4.61 (t, J = 7.2, 2H), 1.74 (quint, J = 7.6, 2H),
3
3
13
1.32 (sext, J = 7.6, 2H), 0.98 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ [ppm], J [Hz]): 176.3,
6
165.8, 152.8, 148.3, 133.6, 129.6, 125.8, 119.9, 107.7, 106.6, 56.7, 53.5, 30.8, 18.9, 13.4.
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
4.1.1.8 1-Butyl-7-fluoro-4-oxo-6-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic
acid
(3h). According to the general procedure 4.1.1, a solution of compound 1h (1 eq, 5.20 g,
17.1 mmol) and potassium carbonate (4 eq, 9.45 g, 68.4 mmol) in N,N-dimethylformamide
was treated with n-bromobutane (5 eq, 9.2 mL, 85.5 mmol), and the reaction was heated at
80 °C for 24 h. The crude product was purified by column chromatography (eluent:
CHCl /MeOH = 150:1, R = 0.43) and then hydrolysed by refluxing compound 2h under
3
f
acidic conditions (2 M HCl). The precipitates were collected and dried in vacuo to yield
-1
3.11 g of 3h. Yield: 55%; mp 198-200 °C. IR [cm ]: 3050, 2967, 2878, 1721, 1609 1455,
1
1388 1304, 1257, 1139. H-NMR (DMSO-d , δ [ppm], J [Hz]): 14.45 (s, 1H), 9.11 (s, 1H),
6
4
3
3
3
8.57 (d, J = 8.0, 1H), 8.30 (d, J = 12.8, 1H), 4.54 (t, J = 7.6, 2H), 1.76 (quint, J = 7.6 2H),
3
3
13
1.35 (sext, J = 7.2, 2H), 0.91 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ [ppm], J [Hz]): 177.4,
6
1
3
165.6, 161.2 (d, J = 257.0, 1C), 152.0, 144.0 (d, J = 6.8, 1C), 126.9 (m, 1C), 122.6 (d,
C,F
C,F
4
1
2
JC,F = 1.8, 1C), 122.3 (q, J = 269.9, 1C), 115.9–115.2 (m, 1C), 109.5, 107.6 (m, J
=
C,F
C,F
19
26.1, 1C), 54.3, 31.0, 19.4, 13.9. F-NMR (DMSO-d , δ [ppm], J [Hz]): -60.49 (d, J = 12.2,
6
3F), -107.92 (m).
449
450
451
452
453
4.1.1.9 1-(3-(Benzyloxy)propyl)-7-chloro-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3i). According to the general procedure 4.1.1, a solution of compound 1a (1 eq, 5.80 g,
21.5 mmol) and potassium carbonate (4 eq, 10.6 g, 86.0 mmol) in N,N-dimethylformamide
was treated with ((3-bromopropoxy)methyl)benzene (1.5 eq, 5.7 mL, 36.0 mmol), and the
reaction was heated at 85 °C for 48 h. The crude product was purified by column
1
5

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chromatography (eluent: CHCl /EtOAc = 20:1, R = 0.40) and then hydrolysed by refluxing
3
f
compound 2i under basic conditions (3 M KOH). After acidification with 2 M HCl under ice
cooling, the precipitates were collected and dried in vacuo to yield 7.48 g of 3i. Yield: 89%;
-1
1
mp 179–180 °C; IR [cm ]: 3046, 2864, 1715, 1613, 1557, 1508, 894. H-NMR (DMSO-d , δ
6
4
3
[ppm], J [Hz]): 9.01 (s, 1H), 8.40 (d, J = 6.0, 2H), 8.17 (d, J = 9.2, 1H), 7.31–7.23 (m, 5H),
3
3
3
13
4.66 (t, J = 6.0, 2H), 4.39 (s, 2H), 3.49 (t, J = 6.0, 2H), 2.08 (quint, J = 6.0, 2H). C-NMR
4
1
(DMSO-d , δ [ppm], J [Hz]): 176.4 (d, J = 2.5, 1C), 165.4, 155.6 (d, J = 247.8, 1C),
6
C,F
C,F
4
2
150.3, 137.9, 136.4 (d, JC,F = 1.7, 1C), 128.1 (2C), 127.4 (2C), 127.3, 127.1, 125.9 (d, JC,F
=
2
6.5, 1C), 121.0, 111.8 (d, JC,F = 22.7, 1C), 107.6, 72.0, 66.2, 51.6, 28.3.
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
4.1.1.10
1-Butyl-5-(dimethylamino)-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3j). A solution of ethyl 5,7-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate 1c (1 eq,
7.27 g, 29.25 mmol) in non-freshly distilled N,N-dimethylformamide (40 mL, containing the
degradation product dimethyl amine) was treated with potassium carbonate at 60 °C for
30 min. Afterwards, n-bromobutane (20.04 g, 146.25 mmol, 15.8 mL) and a catalytic amount
of potassium iodide was added to the reaction and was heated at 90 °C for 24 h. The solvent
was removed under reduced pressure and the crude product was mixed with water (60 mL).
The aqueous layer was extracted with EtOAc (3 x 50 mL), the organic layers were combined,
and the solvent was evaporated. The intermediate carboxylic ethyl ester 2c was hydrolysed in
2 M HCl by refluxing for 6 h. The mixture was consequently extracted with CHCl (3 x 25
3
mL) and the combined organic layers were dried over anhydrous sodium sulfate. After the
evaporization of the solvent, the product was recrystallized from EtOH to give 1.6 g of 3j.
-1
Yield: 18%; mp 205–208 °C. IR [cm ]: 3057, 2952, 2867, 1716, 1628, 1563, 1513, 1436,
1
1276, 1231, 1187, 1167, 1124, 1029. H-NMR (CDCl , δ [ppm], J [Hz]): 15.53 (s, 1H), 8.60
3
3
(s, 1H), 6.62–6.57 (m, 2H), 4.13 (t, J = 7.2, 2H), 2.97 (s, 6H), 1.91–1.83 (m, 2H), 1.76 (sext,
3
3
13
J = 7.2, 2H), 1.00 (t, J = 7.2, 3H). C-NMR (CDCl δ [ppm], J [Hz]): 177.1, 167.4, 165.4
3
1
3
3
(d, J = 249.0, 1C), 156.7 (d, J = 13.0, 1C), 147.4, 144.3 (d, J = 15.0, 1C), 113.7 (d,
C,F
C,F
C,F
4
2
2
JC,F = 1.2, 1C), 109.0, 100.1 (d, J = 25.0, 1C), 92.6 (d, J = 27.8, 1C), 55.3, 44.5 (2C),
C,F
C,F
1
9
+
30.4, 19.9, 13.5. F-NMR (DMSO-d , δ [ppm], J [Hz]): -102.56. Mass: [M + H] 307.2 m/z,
6
found 307.1 m/z.
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6

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4.1.1.11
1-Butyl-7-fluoro-5-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid
(3k). A cold suspension of compound 3d (1 eq, 1.25 g, 4.44 mmol) and methanol (30 mL)
was treated with sodium hydride (10 eq, 107 mg, 44.4 mmol) under ice cooling. Afterwards,
the reaction was heated at 90 °C for 10 h and then quenched with aqueous HCl (3 M). The
reaction solution was extracted with CHCl and the combined organic layers were dried over
3
Na SO with removing subsequently of the organic solvent under reduced pressure. Yield:
2
4
1
4
2
40%. H-NMR (CDCl , δ [ppm], J [Hz]): 15.19 (s, 1H), 8.60 (s, 1H), 6.66 (dd, J = 2.0, J =
3
4
2
3
3
8.0, 1H), 6.64 (dd, J = 2.0, J = 10.8, 1H), 4.12 (t, J = 7.2, 2H), 3.97 (s, 3H), 1.82 (quint, J =
3
3
13
7.2, 2H), 1.39 (sext, J = 7.2, 2H), 0.95 (t, J = 7.2, 3H). C-NMR (CDCl δ [ppm], J [Hz]):
3
1
3
3
176.4, 166.1, 165.8 (d, JC,F = 250.2, 1C), 159.7 (d, J = 14.4, 1C), 148.2, 145.3 (d, J
=
C,F
C,F
4
2
2
14.6, 1C), 113.3 (d, J = 1.2, 1C), 109.3, 96.3 (d, J = 24.0, 1C), 92.4 (d, J = 25.0,
C,F
C,F
C,F
1C), 55.9, 53.6, 30.3, 19.3, 13.5.
498
499
500
501
502
503
504
4.1.2 General synthesis of 1-alkyl-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxylic
acids 4a, 4f-i, 4k [7]. 1-Alkyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 3a, 3f-k (1 eq),
dissolved in 5-15 ml morpholine, was heated 4-10 h under microwave irradiation at 110 °C.
The reaction was acidified with 2 M HCl at 0 °C (pH 2) and the precipitate was collected. The
resulting yellow solid was dried subsequently in vacuo and recrystallized from
EtOH/EtOAc/CHCl3.
505
506
507
508
4.1.2.1 1-Butyl-6-fluoro-7-morpholino-4-oxo-1,4-diyhdroquinoline-3-carboxylic acid (4a).
The compound was synthesised according to the general procedure described 4.1.2.
Spectroscopic data are in accordance with reference [7].
509
510
511
512
513
514
515
516
517
518
4.1.2.2 1-Butyl-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4b). According
to the general procedure 4.1.2, compound 3b (400 mg, 1.34 mmol) was dissolved in 10.0 mL
morpholine and was heated 4.5 h under microwave irradiation at 110 °C. After acidification
(pH 2) and drying in vacuo, the yellow solid was recrystallized from EtOH to yield 210 mg of
-1
4b. Yield: 47%; mp 229–230 °C; IR [cm ]: 3066, 2956, 2862, 1714, 1615, 1519, 1444, 1243,
1
3
1103. H-NMR (CDCl , δ [ppm], J [Hz]): 15.33 (s, 1H), 8.62 (s, 1H), 8.34 (d, J = 9.4, 1H),
3
3
4
4
3
7.13 (dd, J = 9.2, J = 2.4, 1H), 6.66 (d, J = 2.4, 1H), 4.22 (t, J = 7.6, 2H), 3.92–3.90 (m,
3
3
3
4H), 3.40–3.38 (m, 4H), 1.91 (quint, J = 7.6, 2H), 1.47 (sext, J = 7.6, 2H), 1.02 (t, J = 7.2,
1
3
3H). C-NMR (CDCl , δ [ppm], J [Hz]): 177.48, 167.6, 154.9, 148.0, 141.3, 128.5, 118.4,
3
114.5, 107.9, 97.8, 66.4, 53.6, 47.6, 30.6, 18.9, 13.4.
1
7

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4.1.2.3 1-Butyl-8-fluoro-7-morpholino-1,4-dihydroquinoline-3-carboxylic
acid
(4c).
Compound 3c (400 mg, 1.34 mmol) was dissolved in N,N-dimethylformamide (7.5 mL) and
the reaction was treated with morpholine (0.5 mL), and was heated for 20 h at 130 °C.
Afterwards, the solvent was reduced under reduced pressure, the mixture was acidified to
pH 2, and the yellow solid was collected. The crude product was recrystallized from EtOH to
-1
yield 290 mg of 4c. Yield: 62%; mp 272-273 °C; IR [cm ]: 3046, 2955, 2857, 1719, 1614,
1
3
5
1444, 1247, 1119, 926. H-NMR (CDCl , δ [ppm], J [Hz]): 8.58 (s, 1H), 8.26 (dd, J = 8.8, J
3
3
= 1.6, 1H), 7.19 (m, 1H), 4.41 (m, 2H), 3.92–3.91 (m, 4H,), 3.30–3.27 (m, 4H), 1.88 (quint, J
3
3
13
= 7.2, 2H), 1.34 (sext, J = 7.2, 2H), 0.92 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ [ppm], J
6
4
2
1
[Hz]): 177.2 (d, J = 2.5, 1C), 166.8, 150.9, 144.7 (d, J = 8.5, 1C), 143.2 (d, J =
C,F
C,F
C,F
2
4
3
246.9, 1C), 129.9 (d, JC,F = 6.3, 1C), 123.4 (d, J = 3.9, 1C), 122.1, 117.3 (d, J = 3.1,
C,F
C,F
4
4
5
1C), 108.1, 66.7 (2C), 59.4 (d, JC,F = 16.0, 1C), 50.7 (d, J = 4.2, 2C), 32.8 (d, J = 4.0,
C,F
C,F
1C), 19.7, 13.6.
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
4.1.2.4 1-Butyl-5-fluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4d).
Compound 3d (1 eq, 1.0 g, 3.78 mmol), triethylamine (1.5 eq, 750 µL, 5.36 mmol) and boron
trifluoride diethyl etherate (1.5 eq, 675 µL, 5.36 mmol) were dissolved in CH Cl and
2
2
refluxed for 2 h. The solvent was removed under reduced pressure and the crude product was
washed with water/MeOH (1:1), and dried in vacuo. Afterwards, the borate complex (1 eq,
980 mg, 2.98 mmol) was dissolved in 25 mL ethanol and the reaction mixture was treated
with triethylamine (2 eq, 830 µL, 5.97 mmol) and morpholine (1 eq, 260 mL, 2.98 mmol),
and heated 4 h at 60 °C. The solvent was removed under reduced pressure and the
intermediate C1 was refluxed in 2 M NaOH for 2 h. Finally, the compound 4d precipitated
-1
after the addition of 2 M HCl. Yield: 25%; mp 257–260 °C. IR [cm ]: 3389, 3049, 2975,
1
1701, 1630, 1539, 1519, 1448, 1365, 1265, 1216, 1160, 1118, 1051. H-NMR (CDCl , δ
3
3
3
[ppm], J [Hz]): 8.54 (s, 1H), 6.71 (d, J = 14.4, 1H), 6.45 (s, 1H), 4.18 (t, J = 6.8, 2H), 3.92–
3
3
3.90 (m, 4H), 3.40–3.38 (m, 4H), 1.90–1.87 (m, 2H), 1.42 (sext, J = 7.2, 2H), 1.09 (t, J =
1
3
3
1
7.2, 3H). C-NMR (CDCl , δ [ppm], J [Hz]): 177.1 (d, J = 1.7, 1C), 167.2, 163.4 (d, J
C,F
3
C,F
3
3
= 260.8, 1C), 154.5 (d, J = 12.9, 1C), 148.3, 142.6 (d, J = 5.7, 1C), 108.6, 108.3 (d,
C,F
C,F
2
2
JC,F = 9.9), 100.4 (d, J = 25.6, 1C), 94.0, 66.1 (2C), 55.0, 47.1 (2C), 30.3, 19.9, 13.6.
C,F
5
51
52
4.1.2.5 1-Butyl-6,8-difluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4e).
In accordance to 4.1.2.3, compound 3e (600 mg, 1.81 mmol) was dissolved in N,N-
5
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dimethylformamide (10.0 mL) and the reaction mixture was treated with morpholine
(0.5 mL), and was heated 20 h at 130 °C. Afterwards, the solvent was removed under reduced
pressure, the mixture was acidified, and the yellow precipitate was collected. The crude
product was purified by means of column chromatography on silica gel (eluent:
CHCl /MeOH/FA = 100:2:1, R = 0.31) and subsequent recrystallization from EtOH yielding
3
f
-1
240 mg of 4d. Yield: 36%; mp 210–212 °C. IR [cm ]: 3051, 2953, 2850, 1715, 1615, 1539,
1
1464, 1378, 1279, 1207, 1113, 1051, 1016. H-NMR (DMSO-d , δ [ppm], J [Hz]): 14.73 (s,
6
3
5
1H), 8.91 (s, 1H), 7.86 (dd, J = 11.2, J = 2.4, 1H), 4.59–4.55 (m, 2H), 3.75–3.72 (m, 4H),
3
3
3
13
3.34 (br, 4H), 1.80 (quint, J = 7.2, 2H), 1.30 (sext, J = 7.2, 2H), 0.91 (t, J = 7.2, 3H). C-
4
1
NMR (DMSO-d , δ [ppm], J [Hz]): 175.6 (d, J = 2.4, 1C), 165.4, 154.3 (dd, J = 248.4,
6
C,F
C,F
3
1
3
JC,F = 6.2, 1C), 151.3, 146.3 (dd, JC,F = 249.7, J = 6.6, 1C), 133.4 (m, 1C), 127.2 (dd,
C,F
2
4
2
4
JC,F = 7.1, J = 2.0, 1C), 120.6 (m, 1C), 107.2 (dd, J = 22.8, J = 2.7, 1C), 106.7, 66.6
C,F
C,F
C,F
4
4
5
(2C), 57.9 (d, J = 15.6, 1C), 47.1 (t, J = 3.8, 1C), 31.9 (d, J = 4.1, 1C), 18.9, 13.3.
C,F
C,F
C,F
1
9
F-NMR (DMSO-d , δ [ppm], J [Hz]): -119.37 (d, J = 11.4), -129.13 (d, J = 11.4).
6
6,8
6,8
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
4.1.2.6 1-Butyl-5,6-difluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4f).
In accordance to 4.1.2.4, compound 3f (1 eq, 650 mg, 2.17 mmol), triethylamine (1.5 eq, 451
µL, 3.26 mmol) and boron trifluoride diethyl etherate (1.5 eq, 465 µL, 3.26 mmol) were
dissolved in CH Cl and refluxed for 2 h. The solvent was removed under reduced pressure
2
2
and the product was washed with water/MeOH (1:1), and dried in vacuo. Afterwards, the
borate complex (1 eq) was dissolved in 25 mL ethanol and was treated with triethylamine (2
eq), and morpholine (1 eq), and was heated 4 h at 60 °C. The solvent was removed under
reduced pressure and the intermediate C2 was refluxed in 2 M NaOH for 2 h. Finally, the
1
compound 4f precipitated after addition of 2 M HCl. Yield: 25%; H-NMR (DMSO-d , δ
6
4
3
[ppm], J [Hz]): 8.63 (s, 1H), 6.56 (d, J = 5.6, 1H), 4.21 (t, J = 7.2, 3H), 3.93-3.90 (m, 4H),
3
3
3
3.34-3.32 (m, 4H), 1.88 (quint, J =7.2, 2H), 1.45 (sext, J = 7.2, 2H), 1.02 (t, J = 7.2, 3H).
1
3
1
2
C-NMR (DMSO-d , δ [ppm], J [Hz]): 175.0, 165.3, 153.2 (dd, J = 249.1, J = 12.4,
6
C,F
C,F
3
1
2
1C), 149.8, 144.8 (m, 1C), 139.8 (d, J = 10.4, 1C), 138.6 (dd, J = 250.0, J = 14.5,
C,F
C,F
C,F
2
3
4
1C), 112.9 (d, J = 6.8, 1C), 110.1, 103.4 (d, J = 4.5, 1C), 66.3 (2C), 53.5, 48.9 (d, J
C,F
C,F
C,F
= 4.2, 2C), 29.3, 18.9, 13.4.
584
585
586
4.1.2.7 1-Butyl-6-methoxy-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4g).
According to the general procedure 4.1.2, compound 3g (330 mg, 1.07 mmol) was dissolved
in 15.0 mL morpholine and was heated 9 h under microwave irradiation at 110 °C. After
1
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acidification and drying in vacuo, the yellow solid was recrystallized from EtOH to yield
-1
110 mg of 4d. Yield: 29%; mp 248–250 °C. IR [cm ]: 3045, 2952, 1711, 1616, 1470, 1446,
1
1256, 1229, 1111, 1041, 1006. H-NMR (DMSO-d , δ [ppm], J [Hz]): 15.77 (s, 1H), 8.87 (s,
6
3
1H), 7.67 (s, 1H), 7.09 (s, 1H), 4.58 (t, J = 7.2, 2H), 3.95 (s, 3H), 3.79–3.75 (m, 4H), 3.26–
3
3
3
13
3.23 (m, 4H), 1.79 (quint, J = 7.2, 2H), 1.33 (sext, J = 7.2, 2H), 0.92 (t, J = 7.2, 3H). C-
NMR (DMSO-d , δ [ppm], J [Hz]): 175.8, 166.5, 150.7, 147.2, 147.1, 135.0, 120.0, 106.5,
6
104.9, 104.8, 66.0 (2C), 55.8, 53.2, 49.9 (2C), 30.3, 19.0, 13.4.
595
596
597
598
599
600
601
602
603
604
605
606
4.1.2.8 1-Butyl-morpholino-4-oxo-6-(trifluoromethyl)-1,4-diyhdroquinoline-3-carboxylic acid
(4h). According to the general procedure 4.1.2, compound 3h (600 mg, 1.81 mmol) was
dissolved in 10.0 mL morpholine and was heated 6 h under microwave irradiation at 110 °C.
After acidification and drying in vacuo, the yellow solid was recrystallized from EtOH to
-1
yield 500 mg of 4h. Yield: 69%; mp 182-185 °C. IR [cm ]: 3046, 2952, 2857, 1725, 1610,
1
1455, 1393, 1303, 1244, 1102. H-NMR (CDCl , δ [ppm], J [Hz]): 14.52 (s, 1H), 8.68 (s,
3
3
1H), 8.67 (s, 1H), 7.18 (s, 1H), 4.25 (t, J = 7.6, 2H), 3.84-3.82 (m, 4H), 3.07-3.05 (m, 4H),
3
3
3
13
1.85 (quint, J = 7.2, 2H), 1.40 (sext, J = 7.6, 2H), 0.91 (t, J = 7.2, 3H). C-NMR (CDCl , δ
3
3
2
[ppm], J [Hz]): 177.4, 165.4, 156.2, 149.4, 142.2, 128.6 (q, J = 5.5, 1C), 124.6 (q, 1C, J
C,F
C,F
1
= 30.6, 1C), 123.2 (q, J = 278.5, 1C), 121.7, 109.4, 109.2, 66.8 (2C), 54.3, 53.4 (2C), 30.7,
C,F
19.8, 13.5.
607
608
609
610
611
612
613
614
615
616
617
618
619
620
4.1.2.9 1-(3-(Benzyloxy)propyl)-6-fluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (4i). According to the general procedure 4.1.2, compound 3i (4.00 g, 10.3
mmol) was dissolved in 10.0 mL morpholine and was heated 6 h under microwave irradiation
at 110 °C. After acidification and drying in vacuo, the yellow solid was recrystallized from
-1
EtOAc/CHCl (10:1) to yield 2.61 g of 4i. Yield: 58%; mp 191–192 °C. IR [cm ]: 2858,
3
1
1713, 1626, 1508, 1453, 1406, 1354, 1302, 1265, 1206, 1101, 1025. H-NMR (DMSO-d , δ
6
3
[ppm], J [Hz]): 14.7 (s, 1H), 8.91 (s, 1H), 7.91 (d, J = 13.6, 2H), 7.33–7.28 (m, 5H), 7.19 (d,
3
3
3
J = 7.2, 1H), 4.64 (t, J = 6.8, 2H), 4.44 (s, 2H), 3.72–3.70 (m, 4H), 3.49 (t, J = 5.6, 2H),
3
13
3.23-3.22 (m, 4H), 2.11 (quint, J = 6.0, 2H). C-NMR (DMSO-d , δ [ppm], J [Hz]): 176.1
6
4
1
2
(d, J = 2.5, 1C), 166.0, 152.4 (d, J = 247.8, 1C), 149.1, 145.1 (d, J = 10.4, 1C),
C,F
C,F
C,F
3
2
138.1, 128.2 (2C), 128.1 (2C), 127.5, 119.3 (d, J = 10.4, 1C), 111.1 (d, J = 23.2, 1C),
C,F
C,F
3
4
106.8, 105.7 (d, J = 4.8, 1C), 107.6, 72.1, 66.4, 65.7 (2C), 51.6, 49.6 (d, J = 4.7, 2C),
C,F
C,F
28.1.
2
0

ACCEPTED MANUSCRIPT
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4.1.2.10
1-Butyl-5-(dimethylamino)-7-morpholino-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (4j). In accordance to 4.1.2.3, compound 3j (500 mg, 1.78 mmol) was
dissolved in N,N-dimethylformamide (20 mL) and was treated with morpholine (0.5 mL), and
the mixture was heated 2 h at 130 °C. The solvent was removed under reduced pressure and
the crude product was purified by means of column chromatography (eluent:
CHCl /MeOH/FA, R = 0.61). Recrystallization from EtOAc yielded 160 mg of 4j. Yield
3
f
-1
56%; mp 179–180 °C. IR [cm ]: 2945, 2833, 1696, 1595, 1526, 1434, 1359, 1233, 1191,
1
1110, 1009. H-NMR (DMSO-d , δ [ppm], J [Hz]): 8.67 (s, 1H), 6.44–6.42 (m, 2H), 4.39 (t,
6
3
J = 7.2, 2H), 3.77–3.74 (m, 4H), 3.41–3.38 (m, 4H), 2.80 (s, 6H), 1.76–1.71 (m, 2H), 1.32
3
3
13
(sext, J = 7.2, 2H), 0.92 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ [ppm], J [Hz]): 175.9,
6
167.0, 154.7, 153.9, 147.6, 143.9, 108.7, 106.5, 99.7, 91.4, 65.8 (2C), 53.5, 46.6 (2C), 44.1
+
(2C), 29.7, 19.0, 13.4. Mass: [M + H] 374.2 m/z, found 374.5 m/z.
634
635
636
637
638
639
640
641
642
643
644
4.1.2.11
1-Butyl-5-methoxy-7-morpholino-4-oxo-1,3-dihydroquinoline-3-carboxylic
acid (4k). According to the general procedure 4.1.2, compound 3k (4.00 g, 10.3 mmol) was
dissolved in 10.0 mL morpholine and was heated 6 h under microwave irradiation at 110 °C.
After acidification and drying in vacuo, the yellow solid was recrystallized from
-1
EtOAc/CHCl (10:1) to yield 2.61 g of 4k. Yield: 34%; mp 258-259 °C. IR [cm ]: 2961,
3
1
2863, 1704, 1624, 1600, 1545, 1421, 1375, 1263, 1115. H-NMR (CDCl , δ [ppm], J [Hz]):
3
4
3
3
8.54 (s, 1H), 6.44 (d, J = 1.6, 1H), 6.43 (d, J = 1.6, 1H), 4.15 (t, J = 7.6, 2H), 4.00 (s, 3H),
3
3
3.93–3.91 (m, 4H), 3.41–3.39 (m, 4H), 1.87 (quint, J = 7.2, 2H), 1.44 (sext, J = 7.2, 2H),
3
13
0.99 (t, J = 7.2 3H). C-NMR (CDCl , δ [ppm], J [Hz]): 177.8, 167.9, 162.6, 154.8, 147.6,
3
143.5, 109.9, 108.7, 95.3, 91.6, 65.8 (2C), 56.3, 55.1, 47.6 (2C), 30.3, 19.9, 13.6.
645
646
647
648
649
650
651
652
653
4.1.3 General synthesis of N-benzyl-1-alkyl-7-morpholino-4-oxo-1,4-dihydroquinoline-3-
carboxamide GHQ168, 5-18 [7]. 1-Alkyl-7-morpholino-4-oxo-1,4-dihydroquinoline-3-
carboxylic acids 4a-k (1 eq) and N-methylmorpholine (NMM, 5 eq) were dissolved in N,N-
dimethylformamide under Ar atmosphere and the reaction was stirred 1 h at 0 °C. Then, i-
butyl chloroformate (4 eq) was added and stirred for 1 h at 0 °C, until the benzylamine
derivative (4 eq) was added. After 45 min of stirring at room temperature, the solvent was
removed in vacuo and residue was purified by column chromatography on silica gel. The
crude solid was recrystallized to give GHQ168, and 5-18, respectively as white crystals.
2
1

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4.1.3.1 N-Benzyl-1-butyl-6-fluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxamide
(GHQ168). The compound was synthesised according to the general procedure described in
4.1.3. Spectroscopic data are in accordance with reference [7].
658
659
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661
662
663
664
665
666
667
668
669
670
671
672
4.1.3.2 N-Benzyl-1-butyl-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxamide (5).
A
solution of compound 4b (460 mg, 1.39 mmol) in N,N-dimethylformamide was treated with
NMM (764 µL, 6.95 mmol), i-butyl chloroformate (723 µL, 5.56 mmol), and benzylamine
(608 µL, 5.56 mmol) as depicted in the general procedure 4.1.3. The crude product was
purified by column chromatography (eluent: CHCl /MeOH = 100:1, R = 0.57) and
3
f
-1
recrystallized from EtOAc to produce 99 mg of 5. Yield: 17%; mp 172 °C. IR [cm ]: 3171,
1
3039, 2965, 2937, 2877, 2840, 1652, 1597, 1542, 1529, 1466, 1451, 1237, 1126. H-NMR
3
3
(DMSO-d , δ [ppm], J [Hz]): 10.5 (t, J = 6.0, 1H), 8.71 (s, 1H), 8.12 (d, J = 9.2, 1H), 7.35–
6
3
4
4
3
7.33 (m, 5H), 7.20 (dd, J = 9.2, J = 0.8, 1H), 6.90 (d, 1H, J = 0.8), 4.53 (d, J = 5.6, 2H),
3
3
4.42 (t, J = 7.2, 2H), 3.77-3.79 (m, 4H), 3.34 (m, 4H), 1.75 (quint, J = 7.2, 2H), 1.32 (sext,
3
3
13
J = 7.6, 2H), 0.91 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ [ppm], J [Hz]): 174.7, 164.4,
6
153.9, 147.8, 140.5, 139.4, 128.3 (2C), 127.3 (3C), 126.8, 119.0, 113.4, 110.0, 98.4, 65.8
+
(2C), 52.4, 47.0 (2C), 42.0, 30.2, 19.1, 13.5. Mass: [M + H] 420.2 m/z, found 420.3 m/z.
HPLC purity 97%.
673
674
675
676
677
678
679
680
681
682
683
684
685
686
4.1.3.3 N-Benzyl-1-butyl-8-fluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxamide
(6). A solution of compound 4c (230 mg, 0.66 mmol) in N,N-dimethylformamide was treated
with NMM (362 µL, 3.3 mmol), i-butyl chloroformate (343 µL, 2.64 mmol), and
benzylamine (289 µL, 2.64 mmol) as depicted in the general procedure 4.1.3. The crude
product was purified by column chromatography (eluent: CHCl /MeOH = 100:3, R = 0.28)
3
f
-1
and recrystallized from EtOH to produce 180 mg of 6. Yield: 62%; mp 170 °C; IR [cm ]:
1
3039, 2951, 2854, 1654, 1590, 1555, 1549, 1447, 1243, 1121. H-NMR (DMSO-d , δ [ppm],
6
3
3
5
J [Hz]): 10.25 (t, J = 6.0, 1H), 8.72 (s, 1H), 8.08 (dd, J = 8.8, J = 0.8, 1H), 7.34–7.31 (m,
3
3
4H), 7.27–7.24 (m, 2H), 4.54 (d, J = 6.0, 2H), 4.48-4.46 (m, J = 7.2, 2H), 3.78–3.76 (m,
3
3
3
4H), 3.20–3.17 (m, 4H), 1.75 (quint, J = 7.2, 2H), 1.31 (sext, J = 7.2, 2H), 0.90 (t, J = 7.2,
1
3
4
3H). C-NMR (DMSO-d , δ [ppm], J [Hz]): 174.2 (d, J = 1.7, 1C), 163.8, 150.5, 143.5 (d,
6
C,F
2
1
2
JC,F = 8.4, 1C), 143.1 (d, JC,F = 246.1, 1C), 139.3, 129.1 (d, J = 5.9, 1C), 128.3 (2C),
C,F
4
2
127.3 (2C), 126.8, 122.9, 122.3 (d, J = 3.6, 1C), 116.6 (d, J = 2.4, 1C), 110.1, 66.0
C,F
C,F
4
4
5
(2C), 57.5 (d, J = 15.8), 50.3 (d, J = 4.0, 2C), 42.1, 32.0 (d, J = 3.8, 1C), 18.9, 13.4.
C,F
C,F
C,F
2
2

ACCEPTED MANUSCRIPT
1
9
+
6
6
6
87
88
89
F-NMR (DMSO-d , δ [ppm], J [Hz]): -134.80. Mass: [M + H] 438.2 m/z, found 438.2 m/z.
6
HPLC purity 96%.
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
4.1.3.4 N-Benzyl-1-butyl-5-fluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxamide
(7). A solution of compound 4d (260 mg, 0.75 mmol) in N,N-dimethylformamide was treated
with NMM (412 µL, 3.75 mmol), i-butyl chloroformate (390 µL, 3.00 mmol), and
benzylamine (330 µL, 3.00 mmol) as depicted in the general procedure 4.1.3. The crude
product was purified by column chromatography (eluent: CHCl /MeOH = 100:1, R = 0.35)
3
f
-1
and recrystallized from EtOAc to produce 130 mg of 7. Yield: 40%; mp 200 °C. IR [cm ]:
3030, 2964, 2945, 2983, 2856, 1662, 1624, 1573, 1530, 1497, 1467, 1264, 1215, 1117, 1003.
1
3
H-NMR (DMSO-d , δ [ppm], J [Hz]): 10.39 (t, J = 6.0, 1H), 8.65 (s, 1H), 7.34–7.31 (m,
6
3
4
4
3
4H), 7.27–7.24 (m, 1H), 6.91 (dd, J = 15.6, J = 1.6, 1H), 6.65 (d, J = 1.6, 1H), 4.52 (d, J =
3
3
6.0, 2H), 4.37 (t, J = 7.2, 2H), 3.76–3.73 (m, 4H), 3.39–3.37 (m, 4H), 1.72 (quint, J = 7.2,
3
3
13
2H), 1.31 (sext, J = 7.2, 2H), 0.90 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ [ppm], J [Hz]):
6
3
1
3
174.3 (d, J = 1.7, 1C), 164.1, 162.7 (d, J = 256.1, 1C), 153.5 (d, J = 13.1, 1C),
C,F
C,F
C,F
3
3
147.9, 141.9 (d, J = 5.7, 1C), 139.5, 128.3 (2C), 127.3 (2C), 126.8, 110.7, 108.6 (d, J
=
C,F
C,F
2
8.5, 1C), 99.2 (d, J = 25.6, 1C), 94.3, 65.6 (2C), 52.9, 46.5 (2C), 42.5, 29.8, 19.1, 13.4.
C,F
1
9
+
F-NMR (DMSO-d , δ [ppm]): -109.862. Mass: [M + H] 438.2 m/z, found 438.2 m/z. HPLC
6
purity: 97%.
707
708
709
710
711
712
713
714
715
716
717
718
719
720
4.1.3.5 N-Benzyl-1-butyl-6,8-difluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-3-
carboxamide (8). A solution of compound 4e (190 mg, 0.52 mmol) in N,N-
dimethylformamide was treated with NMM (316 µL, 2.6 mmol), i-butyl chloroformate (270
µL, 2.1 mmol), and benzylamine (227 µL, 2.1 mmol) as depicted in the general procedure
4.1.3. The crude product was purified by column chromatography (eluent: CHCl /MeOH =
3
50:1, R = 0.77) and recrystallized from EtOAc to produce 60 mg of 8. Yield: 25%; mp 165–
f
-1
167 °C. IR [cm ]: 3176, 3064, 3033, 2861, 1651, 1596, 1536, 1472, 1450, 1377, 1281, 1213,
1
3
1109, 1026. H-NMR (DMSO-d , δ [ppm], J [Hz]): 10.16 (t, J = 6.0, 1H), 8.74 (s, 1H), 7.80
6
3
5
3
(dd, J = 10.8, J = 1.6, 1H), 7.35–7.32 (m, 4H), 7.27–7.24 (m, 1H), 4.55 (d, J = 6.0, 2H),
4.48–4.44 (br, 2H), 3.73–3.71 (m, 4H), 3.29 (br, 4H), 1.80–1.73 (m, 2H), 1.38–1.31 (m, 2H),
3
13
4
0.91 (t, J = 7.2, 3H). C NMR (DMSO-d , δ [ppm], J [Hz]): 173.1 (d, J = 1.4, 1C), 163.5,
6
C,F
1
3
1
3
153.6 (dd, J = 246.0, J = 6.0, 1C), 150.3, 146.3 (dd, J = 249.0, J = 7.0, 1C),
C,F
C,F
C,F
C,F
2
3
139.2, 132.3 (t, J = 14.0, 1C), 128.3 (2C), 127.3 (2C), 126.8, 126.6 (m, 1C), 122.7 (d, J
C,F
C,F
2
4
4
= 8.0, 1C), 109.7, 107.0 (dd, JC,F = 19.0, J = 2.0, 1C), 66.6 (2C), 57.7 (d, J = 15.0,
C,F
C,F
2
3

ACCEPTED MANUSCRIPT
4
5
19
721
722
723
724
1C), 50.7 (t, J = 6.0, 2C), 42.1, 32.0 (d, J = 4.0, 1C), 18.9, 13.4. F-NMR (DMSO-d , δ
C,F
C,F
6
+
[ppm], J [Hz]): -121.44 (d, J = 9.6), -130.03 (d, J = 9.6). Mass: [M + H] 456.2 m/z, found
6
,8
6,8
456.2 m/z. HPLC purity: 99%.
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
4.1.3.6 N-Benzyl-1-butyl-5,6-difluoro-7-morpholino-4-oxo-1,4-diyhdroquinoline-3-
carboxamide (9). A solution of compound 4f (200 mg, 0.55 mmol) in N,N-
dimethylformamide was treated with NMM (300 µL, 2.75 mmol), i-butyl chloroformate (286
µL, 2.20 mmol), and benzylamine (240 µL, 2.20 mmol) as depicted in the general procedure
4.1.3. The crude product was purified by column chromatography (eluent: CHCl /MeOH =
3
100:1, R = 0.33) and recrystallized from EtOAc to produce 110 mg of 9. Yield: 44%; mp
f
-1
179-180 °C. IR [cm ]: 3035, 2958, 2867, 1655, 1629, 1602, 1484 1377, 1274, 1115, 1009.
1
3
H-NMR (DMSO-d , δ [ppm], J [Hz]): 10.26 (t, 1H, J = 6.0), 8.74 (s, 1H), 7.35–7.32 (m,
6
4
3
3
4H), 7.27–7.23 (m, 1H), 6.82 (d, J = 6.4, 1H), 4.54 (d, J = 6.0, 2H), 4.43 (t, J = 7.2, 2H),
3
3
3.79–3.77 (m, 4H), 3.30–3.27 (m, 4H), 1.74 (quint, J = 7.2, 2H), 1.31 (sext, J = 7.2, 2H),
3
13
1
0.91 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ [ppm], J [Hz]): 174.2, 163.7, 149.4 (dd, J
=
6
C,F
2
1
2
257.7, J = 13.2, 1C), 147.0, 144.1 (m, 1C), 139.5 (dd, J = 245.6, J = 13.7, 1C),
C,F
C,F
C,F
3
2
139.4, 136.3 (d, J = 3.9, 1C), 128.3 (2C), 127.3 (2C), 126.7, 111.5 (d, J = 5.4, 1C),
C,F
C,F
4
19
110.9, 99.5, 65.7 (2C), 53.0, 49.3 (d, J = 4.4, 2C) 42.0, 29.8, 19.1, 13.4. F-NMR
C,F
(DMSO-d , δ [ppm], J [Hz]): -140.03 (d, J = 18.0), -151.62 (dd, J5,6 = 18.0, J6,8 = 8.0).
6
5,6
+
Mass: [M + H] 456.2 m/z, found 456.1 m/z. HPLC purity: 99%.
2
4

ACCEPTED MANUSCRIPT
742
743
744
745
746
747
748
749
750
751
752
753
754
755
4.1.3.7 N-Benzyl-1-butyl-6-methoxy-7-morpholino-4-oxo-1,4-dihydroquinoline-3-
carboxamide (10). A solution of compound 4g (230 mg, 0.63 mmol) in N,N-
dimethylformamide was treated with NMM (346 µL, 3.2 mmol), i-butyl chloroformate (327
µL, 2.52 mmol), and benzylamine (275 µL, 2.52 mmol) as depicted in the general procedure
4.1.3. The crude product was purified by column chromatography (eluent: CHCl /MeOH =
3
50:1, R = 0.73) and recrystallized from acetonitrile to produce 100 mg of 10. Yield: 34%; mp
f
-1
1
156 °C. IR [cm ]: 3045, 2952, 1711, 1616, 1470, 1446, 1256, 1229, 1111, 1041, 1006. H-
3
NMR (CDCl , δ [ppm], J [Hz]): 10.57 (t, J = 5.2, 1H), 8.70 (s, 1H), 7.85 (s, 1H), 7.42–7.21
3
3
3
(m, 5H), 6.82 (s, 1H), 4.71 (d, J = 5.6, 2H), 4.23 (t, J = 7.6, 2H), 3.99 (s, 3H), 3.94–3.92 (m,
3
3
3
4H), 3.24–3.22 (m, 4H), 1.90 (quint, J = 7.2, 2H), 1.4 (sext, J = 7.2, 2H), 1.0 (t, J = 7.2,
1
3
3H). C-NMR (CDCl , δ [ppm], J [Hz]): 175.4, 165.4, 150.4, 146.6, 146.2, 138.9, 134.5,
3
128.8 (2C), 127.7 (2C), 127.0, 123.42, 111.0, 106.6, 103.7, 66.8 (2C), 55.5, 54.0, 50.7 (2C),
+
43.3, 31.0, 20.0, 13.6. Mass: [M + H] 450.2 m/z, found 450.3 m/z. HPLC purity: 96%.
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
4.1.3.8 N-Benzyl-1-butyl-7-morpholino-4-oxo-6-(trifluoromethyl)-1,4-dihydroquinoline-3-
carboxamide (11). A solution of compound 4h (220 mg, 0.56 mmol) in N,N-
dimethylformamide was treated with NMM (307 µL, 2.8 mmol), i-butyl chloroformate
(231 µL, 2.24 mmol), and benzylamine (214 µL, 2.56 mmol) as depicted in the general
procedure 4.1.3. The crude product was purified by column chromatography (eluent:
CHCl /MeOH = 100:1, R = 0.67) and recrystallized from EtOAc to produce 40 mg of 11.
3
f
-1
Yield: 15%; mp 168 °C. IR [cm ]: 3232, 2958, 2935, 1659, 1626, 1598, 1486, 1452, 1267
1
3
1240 1108. H-NMR (DMSO-d , δ [ppm], J [Hz]): 10.18 (t, J = 7.6, 1H), 8.90 (s, 1H), 8.54
6
(s, 1H), 7.68 (s, 1H), 7.35-7.33 (m, 4H), 7.28-7.25 (m, 1H), 4.57-4.54 (m, 4H), 3.77-3.75 (m,
3
3
3
4H), 3.09-3.05 (m, 4H), 1.75 (quint, J = 7.2, 2H), 1.35 (sext, J = 7.6, 2H), 0.92 (t, J = 7.2,
1
3
3H). C-NMR (DMSO-d , δ [ppm], J [Hz]): 174.6, 163.6, 154.9, 149.5, 142.0, 139.1, 128.4
6
(2C), 127.3 (2C), 126.6, 126.5 (m, 1C), 124.5 (m, 1C), 122.7, 121.8 (m, 1C), 112.0, 111.5,
+
66.3 (2C), 52.9 (2C), 52.7, 42.1, 30.4, 19.0, 13.4. Mass: [M + H] 488.2 m/z, found 488.1 m/z.
HPLC purity: 97%.
771
772
773
774
775
4.1.3.9 N-Benzyl-1-(3-(benzyloxy)propyl)-6-fluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-
3-carboxamide (12). A solution of compound 4i (400 mg, 0.90 mmol) in N,N-
dimethylformamide was treated with NMM (463 µL, 4.50 mmol), i-butyl chloroformate (468
µL, 3.60 mmol), and benzylamine (393 µL, 3.60 mmol) as depicted in the general procedure
4.1.3. The crude product was purified by column chromatography (eluent: CHCl /MeOH =
3
2
5

ACCEPTED MANUSCRIPT
776
777
778
779
780
781
782
783
784
785
786
100:3, R = 0.56) and recrystallized from EtOAc to produce 360 mg of 12. Yield: 76%; mp
f
-1
1
174–175 °C. IR [cm ]: 3181, 3038, 2939, 2856, 1652, 1536, 1586, 1358, 1266. H-NMR
3
3
(DMSO-d , δ [ppm], J [Hz]): 10.37 (t, J = 6.8, 1H), 8.81 (s, 1H), 7.87 (d, J = 13.6, 2H),
6
4
3
7.36–7.25 (m, 10H ), 7.13 (d, J = 7.2, 1H), 4.58–4.55 (m, 5H), 4.44 (s, J = 6.0, 2H), 3.75–
.
3
3
13
3.70 (m, 4H), 3.49 (t, J = 5.6, 2H), 3.20–3.18 (m, 4H), 2.10 (quint, J = 6.0, 2H). C-NMR
4
1
(DMSO-d , δ [ppm], J [Hz]): 174.7 (d, J = 2.5), 164.6, 153.2 (d, J = 247.8), 148.6,
6
C,F
C,F
2
144.8 (d, J = 10.4), 139.9, 138.1, 137.2, 128.9 (2C), 128.7 (2C), 128.7, 128.1 (2C), 127.8,
C,F
4
2
3
127.9 (2C), 122.0 (d, J = 7.0), 112.1 (d, J = 22.9, 1C), 110.6, 106.0 (d, J = 4.8, 1C),
C,F
C,F
C,F
4
+
72.1, 66.8, 66.7 (2C), 51.6, 50.3 (d, J = 4.7, 2C), 42.6, 28.8. [M + H] 530.3 m/z, found
C,F
530.1 m/z. HPLC purity: 100%.
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
4.1.3.10
N-Benzyl-1-butyl-5-(dimethylamino)-7-morpholino-4-oxo-1,4-
dihydroquinoline-3-carboxamide (13). A solution of compound 4j (350 mg, 0.94 mmol) in
N,N-dimethylformamide was treated with NMM (517 µL, 4.70 mmol), i-butyl chloroformate
(490 µL, 3.78 mmol), and benzylamine (410 µL, 3.78 mmol) as depicted in the general
procedure 4.1.3. The crude product was purified by column chromatography (eluent:
CHCl /MeOH = 100:3, R = 0.82) and recrystallized from EtOH to produce 180 mg of 13.
3
f
-1
Yield: 42%; mp 168–170 °C. IR [cm ]: 3154, 3030, 2954, 2861, 2823, 1653, 1594, 1564,
1
1526, 1485, 1452, 1373, 1284, 1230, 1120, 1013, 741. H-NMR (DMSO-d , δ [ppm], J [Hz]):
6
3
10.69 (t, J = 6.0, 1H), 8.54 (s, 1H), 7.34–7.33 (m, 4H ), 7.27–7.25 (m, 1H), 6.36–6.34 (m,
.
3
3
2H), 4.52 (d, J = 6.0, 2H), 4.29 (t, J = 7.2, 2H), 3.76–3.74 (m, 4H), 3.34–3.31 (m, 4H), 2.75
3
3
13
(s, 6H), 1.74–1.70 (m, 2H), 1.33 (sext, 2H, J = 7.2), 0.91 (t, J = 7.2, 3H). C-NMR (DMSO-
d6, δ [ppm], J [Hz]): 174.8, 164.7, 154.5, 153.2, 146.3, 143.5, 139.6, 128.3 (2C), 127.2 (2C),
126.7, 111.1, 110.5, 99.1, 91.2, 65.9 (2C), 53.0, 47.0 (2C), 42.0 (2C), 40.1, 29.9, 19.1, 13.5.
+
Mass: [M + H] 463.3 m/z, found 463.3 m/z. HPLC purity: 99%.
802
803
804
805
806
807
808
809
4.1.3.11
N-Benzyl-1-butyl-5-methoxy-7-morpholino-4-oxo-1,4-dihydroquinoline-3-
carboxamide (14). A solution of compound 4k (220 mg, 0.61 mmol) in N,N-
dimethylformamide was treated with NMM (335 µL, 3.05 mmol), i-butyl chloroformate (317
µL, 2.44 mmol), and benzylamine (305 µL, 2.44 mmol) as depicted in the general procedure
4.1.3. The crude product was purified by column chromatography (eluent: CHCl /MeOH =
3
50:1, R = 0.63) and recrystallized from EtOH to produce 140 mg of 14. Yield: 51%; mp 159-
f
-1
1
161 °C. IR [cm ]: 2987, 2954, 2924, 2894, 1661, 1611, 1523, 1470, 1356, 1258, 1237. H-
3
NMR (DMSO-d , δ [ppm], J [Hz]): 10.67 (t, J = 6.0, 1H), 8.57 (s, 1H), 7.35–7.32 (m, 4H),
6
2
6

ACCEPTED MANUSCRIPT
4
4
3
810
811
812
813
814
815
816
7.28-7.25 (m, 1H ), 6.53 (d, J = 1.6, 1H), 6.43 (d, J = 1.6, 1H), 4.50 (d, J = 6.0, 2H), 4.32 (t,
.
3
3
J = 7.6, 2H), 3.82 (s, 3H), 3.78–3.76 (m, 4H), 3.39–3.37 (m, 4H), 1.72 (quint, J = 7.2, 2H),
3
3
13
1.31 (sext, J = 7.2, 2H), 0.91 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ [ppm], J [Hz]):
6
175.25, 164.5, 161.8, 153.8, 146.8, 142.6, 139.5, 128.3 (2C), 127.3 (2C), 126.7, 110.9, 110.2,
+
95.0, 91.3, 65.8 (2C), 55.8, 53.0, 46.9 (2C), 42.0, 29.8, 19.1, 13.5. Mass: [M + H] 450.2 m/z,
found 450.1 m/z. HPLC purity: 98%.
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
4.1.3.12
1-Butyl-6-fluoro-N-(4-fluorobenzyl)-7-morpholino-4-oxo-1,4-
dihydroquinoline-3-carboxamide (15). A solution of compound 4a (90 mg, 0.26 mmol) in
N,N-dimethylformamide was treated with NMM (141 µL, 1.29 mmol), i-butyl chloroformate
(134 µL, 1.02 mmol), and (4-fluorophenyl)methanamine (118 µL, 1.02 mmol) as depicted in
the general procedure 4.1.3. The crude product was purified by column chromatography
(eluent: CHCl /MeOH = 100:1, R = 0.75) and recrystallized from EtOAc to produce 70 mg
3
f
-1
of 15. Yield: 58%; mp 177–179 °C; IR [cm ]: 3032, 2954, 2871, 1654, 1625, 1603, 1536,
1
3
1485, 1468, 1478, 1257, 1213, 1113. H-NMR (DMSO-d , δ [ppm], J [Hz]): 10.36 (t, J =
6
3
6.0, 1H), 8.78 (s, 1H), 7.87 (d, J = 13.6, 1H), 7.40–7.36 (m, 2H), 7.18–7.09 (m, 2H), 7.09 (d,
4
3
3
J = 7.6, 1H), 4.53 (d, J = 6.0, 2H), 4.48 (t, J = 7.2, 2H), 3.80–3.78 (m, 4H), 3.26–3.24 (m,
3
3
3
13
4H), 1.77 (quint, J = 7.6, 2H), 1.32 (sext, J = 7.6, 2H), 0.92 (t, J = 7.2, 2H). C-NMR
4
1
(DMSO-d , δ [ppm], J [Hz]): 174.6 (d, J = 2.5, 1C), 164.0, 160.2 (d, J = 240.4, 1C),
6
C,F
C,F
1
2
4
152.4 (d, J = 245.6, 1C), 147.7, 144.3 (d, J = 10.3, 1C), 136.6, 135.6 (d, J = 3.0, 1C),
C,F
C,F
C,F
3
3
2
2
129.3 (d, J = 8.0, 2C), 121.4 (d, J = 6.9, 1C), 115.1 (d, J = 21.1, 2C), 111.0 (d, J
C,F
C,F
C,F
C,F
3
4
= 22.4, 1C), 109.9, 105.5 (d, J = 3.3, 1C), 65.8 (2C), 52.8, 49.8 (d, J = 4.4, 2C), 41.3,
C,F
C,F
1
9
30.2, 19.1, 13.4. F-NMR (DMSO-d , δ [ppm], J [Hz]): -116.04, -123.79. Mass:
6
+
[M + H] 456.2 m/z, found 456.2 m/z. HPLC purity 98%.
835
836
837
838
839
840
841
842
843
4.1.3.13
1-Butyl-6-fluoro-N-(2-fluorobenzyl)-7-morpholino-4-oxo-1,4-
dihydroquinoline-3-carboxamide (16). A solution of compound 4a (210 mg, 0.60 mmol) in
N,N-dimethylformamide was treated with NMM (329 µL, 3.00 mmol), i-butyl chloroformate
(328 µL, 2.40 mmol), and (2-fluorophenyl)methanamine (300 mg, 2.40 mmol) as depicted in
the general procedure 4.1.3. The crude product was purified by column chromatography
(eluent: CHCl /MeOH = 100:1, R = 0.34) and recrystallized from EtOAc to produce 70 mg
3
f
-1
of 16. Yield: 25%; mp 154-156 °C. IR [cm ]: 3053, 2963, 2848, 1651, 1604, 1482, 1452,
1
3
1305, 1267, 1247, 1122. H-NMR (DMSO-d , δ [ppm], J [Hz]): 10.37 (t, J = 6.0, 1H), 8.77
6
3
4
(s, 1H), 7.87 (d, J = 13.6, 1H), 7.41–7.32 (m, 2H), 7.22–7.16 (m, 2H), 7.10 (d, J = 7.2, 1H),
2
7

ACCEPTED MANUSCRIPT
3
3
844
845
846
847
848
849
850
851
852
853
4.58 (d, J = 6.0, 2H), 4.47 (t, J = 7.2, 2H), 3.80–3.78 (m, 4H), 3.26–3.24 (m, 4H), 1.76
3
3
3
13
(quint, J = 7.6, 2H), 1.31 (sext, J = 7.6, 2H), 0.92 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ
6
4
1
1
[ppm], J [Hz]): 174.1 (d, J = 2.5, 1C), 164.2, 160.1 (d, J = 242.9, 1C), 152.4 (d, J
=
=
C,F
C,F
C,F
2
3
3
246.1, 1C), 147.8, 144.3 (d, JC,F = 10.3), 136.6, 129.7 (d, J = 4.5, 1C), 129.0 (d, J
C,F
C,F
2
4
3
8.0, 1C), 125.9 (d, J = 14.9, 1C), 124.4 (d, J = 3.4, 1C), 121.4 (d, J = 6.9, 1C), 115.1
C,F
C,F
C,F
2
2
3
(d, J = 21.0, 1C), 111.4 (d, J = 22.4, 1C), 109.9, 105.5 (d, J = 3.3, 1C), 65.9 (2C),
C,F
C,F
C,F
2
3
19
52.8, 49.8 (d, J = 4.4, 2C), 36.1 (d, J = 4.2, 1C), 30.2, 19.1, 13.4. F-NMR (DMSO-d ,
C,F
C,F
6
+
δ [ppm], J [Hz]): -118.89 (m), -123.70 (dd, J5,6 = 14.4, J6,8 = 7.7). Mass: [M + H] 456.2 m/z,
found 456.1 m/z. HPLC purity: 95%.
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
4.1.3.14
1-Butyl-N-(2,4-difluorobenzyl)-6-fluoro-7-morpholino-4-oxo-1,4-
dihydroquinoline-3-carboxamide (17). A solution of compound 4a (200 mg, 0.57 mmol) in
N,N-dimethylformamide was treated with NMM (313 µL, 3.00 mmol), i-butyl chloroformate
(297 µL, 2.28 mmol), and (2-fluorophenyl)methanamine (326 mg, 2.28 mmol) as depicted in
the general procedure 4.1.3. The crude product was purified by column chromatography
(eluent: CHCl /MeOH = 100:1, R = 0.41) and recrystallized from EtOH to produce 80 mg of
3
f
-1
17. Yield: 30%; mp 160-161 °C. IR [cm ]: 3031, 2959, 2871, 1659, 1625, 1601, 1485, 1449,
1
3
1256, 1210, 1102. H NMR (DMSO-d , δ [ppm], J [Hz]): 10.36 (t, J = 6.0, 1H), 8.76 (s, 1H),
6
3
3
7.88 (d, J = 13.6, 1H), 7.46–7.40 (m, 1H), 7.26–7.21 (m, 1H), 7.10 (m, 2H), 4.56 (d, J = 6.0,
3
3
2H), 4.46 (t, J = 7.2, 2H), 3.80–3.78 (m, 4H), 3.26–3.24 (m, 4H), 1.76 (quint, J = 7.6, 2H),
3
3
13
1.32 (sext, J = 7.6, 2H), 0.91 (t, J = 7.2, 3H). C NMR (DMSO-d , δ [ppm], J [Hz]): 174.0
6
4
1
3
1
(d, J = 2.5, 1C), 164.2, 161.4 (dd, J = 243.4, J = 12.1, 1C), 160.2 (dd, J = 245.8,
C,F
C,F
C,F
C,F
3
1
2
JC,F = 12.1, 1C), 152.4 (d, J = 246.1, 1C), 147.8, 144.3 (d, J = 10.3, 1C), 136.6, 130.8
C,F
C,F
3
3
2
4
4
(dd, J = 9.8, J = 6.8, 1C), 122.4 (dd, J = 14.9, J = 3.6, 1C), 124.4 (d, J = 3.4,
C,F
C,F
C,F
C,F
C,F
3
2
2
4
1C), 121.4 (d, J = 6.9, 1C), 111.4 (d, J = 22.4, 1C), 111.3 (dd, J = 20.9, J = 3.5,
C,F
C,F
C,F
C,F
3
2
1C), 109.9, 105.5 (d, J = 3.3, 1C), 103.7 (t, J = 25.6, 1C), 65.8 (2C), 52.8, 49.8 (d, 2C,
C,F
C,F
2
3
19
JC,F = 4.4, 1C), 36.1 (d, J = 4.2, 1C), 30.2, 19.1, 13.4. F-NMR (DMSO-d , δ [ppm], J
C,F
6
[Hz]): -111.89 (m), -114.45 (m), -123.72 (dd, J5,6 = 14.4, J6,8 = 7.7). Mass:
+
[M + H] 474.2 m/z, found 474.1 m/z. HPLC purity: 98%.
874
875
876
877
4.1.3.15
1-Butyl-5-fluoro-N-(4-fluorobenzyl)-7-morpholino-4-oxo-1,4-
dihydroquinoline-3-carboxamide (18). A solution of compound 4d (100 mg, 0.29 mmol) in
N,N-dimethylformamide was treated with NMM (160 µL, 1.45 mmol), i-butyl chloroformate
(150 µL, 1.16 mmol), and (4-fluorophenyl)methanamine (145 mg, 1.16 mmol) as depicted in
2
8

ACCEPTED MANUSCRIPT
878
879
880
881
882
883
884
885
886
887
888
889
890
891
the general procedure 4.1.3. The crude product was purified by column chromatography
(eluent: CHCl /MeOH = 100:1, R = 0.53) and recrystallized from EtOH to produce 40 mg of
3
f
-1
18. Yield: 31%; mp 191 °C. IR [cm ]: 3063, 2958, 2860, 1662, 1629, 1603, 1583, 1550,
1
3
1534, 1508. H-NMR (DMSO-d , δ [ppm], J [Hz]): 10.38 (t, J = 6.0, 1H), 8.65 (s, 1H), 7.39–
6
3
4
4
7.35 (m, 2H), 7.18– 7.13 (m, 2H), 6.95 (dd, J = 15.6, J = 1.6, 1H), 6.65 (d, J = 1.6, 1H),
3
3
4.50 (d, J = 6.0, 2H), 4.37 (t, J = 7.2, 2H), 3.76–3.73 (m, 4H), 3.39–3.37 (m, 4H), 1.72
3
3
3
13
(quint, J = 7.2, 2H), 1.31 (sext, J = 7.2, 2H), 0.90 (t, J = 7.2, 3H). C-NMR (DMSO-d , δ
6
3
1
1
[ppm], J [Hz]): 174.2 (d, JC,F = 1.7, 1C), 164.1, 163.2 (d, J = 256.1, 1C), 160.8 (d, J
=
C,F
C,F
3
3
3
240.8, 1C), 153.5 (d, J = 13.1, 1C), 147.9, 142. (d, J = 5.7, 1C), 135.7 (d, J = 3.0,
C,F
C,F
C,F
3
2
3
1C), 129.2 (d, J = 8.1, 2C), 115.0 (d, J = 21.1, 2C), 110.7, 108.7 (d, J = 8.5, 1C),
C,F
C,F
C,F
2
19
99.2 (d, J = 25.6, 1C), 94.3, 65.6 (2C), 52.9, 46.5 (2C), 42.5, 29.8, 19.1, 13.4. F-NMR
+
(DMSO-d , δ [ppm], J [Hz]): -109.89 (d, J = 16.6), -116.08 (m). Mass: [M + H] 456.2 m/z,
6
5,6
found 456.0 m/z. HPLC purity: 97%.
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
4.1.4 N-benzyl-6-fluoro-1-(3-hydroxypropyl)-7-morpholino-4-oxo-1,4-dihydroquinoline-3-
carboxamide (19). Compound 12 (0.20 g, 0.377 mmol) and a catalytic amount of Pd/C were
suspended in chloroform (15 mL). The mixture was pressurized with hydrogen (25 bar) and
was heated under microwave irradiation (100 °C/ 500 W) for 12 h. The solvent was then
removed in vacuo and the residue was purified via column chromatography (eluent:
CHCl /MeOH = 20:1, R = 0.22). The white residue was recrystallized from EtOAc to give
3
f
-1
110 mg of 19 as pale-white crystals. Yield: 66%; mp 182 °C. IR [cm ]: 3384, 3070, 2858,
1
1651, 1628, 1600, 1536, 1488, 1448, 1364, 1259, 1170, 1036. H-NMR (DMSO-d , δ [ppm],
6
3
3
J [Hz]): 10.36 (t, J = 6.8, 1H), 8.78 (s, 1H), 7.87 (d, J = 13.6, 1H), 7.34–7.33 (m, 4H), 7.28–
4
3
7.24 (m, 1H), 7.20 (d, J = 7.2, 1H), 4.79 (t, J = 4.8, 1H), 4.44–4.49 (m, 4H), 3.79–3.77 (m,
3
13
4H), 3.49–3.42 (m, 2H), 3.26–3.23 (m, 4H), 1.94 (quint, J = 7.2, 2H). C-NMR (DMSO-d ,
6
4
1
δ [ppm], J [Hz]): 174.0 (d, J = 2.5, 1C), 164.0, 153.3 (d, J = 247.8, 1C), 147.9, 144.1
C,F
C,F
2
3
(d, J = 10.4, 1C), 139.2, 136.6, 128.3 (2C), 127.1 (2C), 126.7, 121.3 (d, J = 7.0, 1C),
C,F
C,F
2
3
111.2 (d, J = 22.9, 1C), 109.9, 105.5 (d, J = 4.8, 1C), 65.7 (2C), 57.0, 50.2, 49.7 (d,
C,F
C,F
4
+
JC,F = 4.7, 2C) 41.9, 31.1. Mass: [M + H] 440.2 m/z, found 440.1 m/z. HPLC purity: 99%.
908
909
910
911
4.1.5 Synthesis of 3-(3-(benzylcarbamoyl)-6-fluoro-7-morpholino-4-oxoquinolin-1(4H)-
yl)propyl methanesulfonate (20). Compound 19 (0.230 mg, 0.523 mmol), triethylamine
(507 µL, 3.66 mmol) and methanesulfonyl chlorid (80 µL, 1.05 mmol) were dissolved in
CH Cl under Ar atmosphere at 0 °C. After 12 h of stirring at room temperature, the solvent
2
2
2
9