Targeting cyclic nucleotide phosphodiesterase 5 (PDE5) in brain: Toward the development of a PET radioligand labeled with fluorine-18

Targeting cyclic nucleotide phosphodiesterase 5 (PDE5) in brain: Toward the

development of a PET radioligand labeled with fluorine-18

1

§#

2#

1

Barbara Wenzel , Jianrong Liu , Sladjana Dukic-Stefanovic , Winnie Deuther-Conrad ,

1

2

Rodrigo Teodoro , Friedrich-Alexander Ludwig , Jean-Michel Chezal , Emmanuel Moreau ,

1

2

Peter Brust , Aurelie Maisonial-Besset

1

Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research,

Department of Neuroradiopharmaceuticals, Leipzig, Germany;

UMR 1240 INSERM IMOST, Université Clermont-Auvergne, Clermont-Ferrand, France

2

#

These authors contributed equally to the work.

corresponding author

§

Abstract

With the aim to develop a specific radioligand for imaging the cyclic nucleotide

phosphodiesterase 5 (PDE5) in brain by positron emission tomography (PET), seven new

fluorinated inhibitors (3 – 9) were synthesized on the basis of a quinoline core. The inhibitory

activity for PDE5 together with a panel of other PDEs was determined in vitro and two

derivatives were selected for IC50 value determination. The most promising compound 7 (IC50

=

5.92 nM for PDE5A), containing a 3-fluoroazetidine moiety, was further radiolabeled by

aliphatic nucleophilic substitution of two different leaving groups (nosylate and tosylate)

1

8

using [ F]fluoride. The use of the nosylate precursor and tetra-n-butyl ammonium

1

8

18

[

F]fluoride ([ F]TBAF) in 3-methyl-3-pentanol combined with the addition of a small

amount of water proved to be the best radiolabeling conditions achieving a RCY of 4.9 ±

.5% in an automated procedure. Preliminary biological investigations in vitro and in vivo

1

8

were performed to characterize this new PDE5 radioligand. Metabolism studies of [ F]7 in

mice revealed a fast metabolic degradation with the formation of radiometabolites which

have been detected in the brain.

1

8

18

Keywords: PDE5, F-radiolabeling, nosylate, tosylate, F-fluoroazetidine

1

1. Introduction

The cyclic nucleotide phosphodiesterases (PDEs) are a large family of enzymes that regulate

intracellular levels of the two important second messengers cyclic adenosine 3’,5’-

monophosphate (cAMP) and cyclic guanosine 3’,5’-monophosphate (cGMP) by cleavage of

their phosphodiester bond. To date, 11 subtypes of these enzymes have been identified and

classified mainly according to their amino acid sequences. Phosphodiesterase 5 (PDE5) is

specific for cGMP which is generated by the nitric oxide (NO)-dependent soluble guanylate

cyclase and activates the protein kinase G (PKG). PKG activation triggers phosphorylation of

numerous intracellular proteins that in turn regulate many primary physiological functions

such as modulation of vascular tone and vasorelaxation in vascular smooth muscle. This was

the basis for the breakthrough of one of the most celebrated PDE5 inhibitors namely

sildenafil (Viagra®) which is used as treatment for erectile dysfunction and chronic

pulmonary hypertension. Moreover, currently this enzyme arouses increasing interest of

clinical researchers as a multifunctional biomarker and drug target because PDE5 inhibition

also affects neurodegenerative [1] and cancerous processes [2]. For example, increased

PDE5 levels have been reported in several carcinomas including colon adenocarcinoma,

bladder squamous carcinoma, thyroid tumors, metastatic breast, prostate, pancreatic,

bladder and lung cancers [3-9]. As it was demonstrated, that cGMP can reduce cell growth

and induce apoptosis [10, 11], and that PDE5 catalyzes the degradation of cGMP, this

enzyme has been suggested to be involved in tu mor progression because its elevation goes

along with increasing tumor grade and stage [4, 12]. Moreover, PDE5 inhibitors are able to

enhance the chemotherapeutic efficacy of anticancer drugs in different tumors and cancer

cell types [9, 13-15] and inhibition of PDE5 has an impact on the attenuatio n of multidrug

resistance, one of the major causes of unsuccessful anticancer treatments [16, 17].

Despite the tremendous growth of preclinical and clinical studies on the use of PDE5

inhibitors and their promising results, the mechanisms of action are in most cases not fully

understood and detailed insights are needed. For example, the currently available data are

insufficient to make any co nclusive statement regarding the exact role of the PDE5 enzyme

in cancer pathogenesis [12, 18].

2

The aim of our research is the development of a PDE5-specific radioligand to support in vitro

and in vivo studies on the investigation of pathology-specific changes in the availability of

the enzyme using PET. Regarding tumor imaging, those studies would enable the assessment

of the spatiotemporal pattern of PDE5 expression. Thereby, our particular interest is

dedicated to a future investigation of brain tumors by characterizing the expression of PDE5

in healthy and diseased brain.

In recent years, several attempts have been made to develop PET radioligands targeting the

PDE5 enzyme as reviewed elsewhere [19, 20]. Most of the radiotracers are labeled with

carbon-11 and suffer from a lack of specificity [21, 22]. Chekol et al. reported on the

development of fluorine-18 labeled PDE5 radioligands [23, 24]. In particular a vardenafil-

based derivative showed a high retention in the lungs which was interpreted to be PDE5-

specific because of the high expression of PDE5 in this organ [23]. Biodistribution studies in

mice showed that none of the ligands investigated in this study demonstrated significant

brain uptake. By contrast, a recent report by the same group revealed that a

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8

pyridopyrazinone based F-labeled derivative has shown accumulation in rat brain.

Although the radiotracer readily entered the brain, the radioactivity uptake was not specific

toward PDE5. On the other hand, this compound demonstrated specific binding in transgenic

mice with cardiomyocyte-specific PDE5 overexpression [24].

Searching for a suitable lead compound for the development of a brain penetrating PDE5

radioligand, we decided to focus our attention on the heterocyclic quinoline based

compounds 1 and 2 (Figure 1). These derivatives were d escribed with a high PDE5 potency

and the potential to cross the blood-brain barrier [25, 26]. Structure-activity relation ship

studies (SAR) revealed important structural features of this family of PDE5 inhibitors [25, 26].

The quinoline scaffold bearing a hydroxymethyl group at position C-3 (“east region”)

improves the inhibitory activity on PDE5 and the solubility compared to the other core ring

systems tested. The binding to the protein is highly dependent on the presence of the

secondary amino functionality at position C-4 (“north region”) [27]. Moreover, the influence

of a combination of different substituents on positions C-6 and C-8 of the quinoline scaffold

was studied resulting in the so far most potential candidates 1 [25] and 2 [26]. Based on

these results, we started our attempts to introduce a fluorine containing structural moiety at

the "east region" of 1 and 2 by substituting the hydroxymethyl group on C-3 by a fluoroethyl

and fluoroethoxymethyl group [28], resulting in the development of the new PDE5

3

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8

radioligand [ F]ICF24027 [29]. However, due to the fast formation of a brain-penetrable

radiometabolite, this radiotracer was not suitable for PDE5 imaging in brain. The aim of the

present study was to investigate the possibility of introducing fluorine containing moieties at

the "north" or "south region" (position 4 or 8) of the quinoline scaffold (Figure 1) in order to

1

8

develop a specific and metabolically more stable F-labeled radioligand for imaging of the

PDE5 enzyme in brain.

2. Results and discussion

2

.1. Ligand development

.1.1. Concept

2

For introduction of the fluorine atom at the "north region" of the lead structures 1 and 2,

modification of the chlorobenzene scaffold was intended (Figure 1). As the secondary amine

substituted at the C-4 position of the quinoline core is known to be crucial for the PDE5

inhibitory activity [27], it was not considered for an alkylation with a fluorinated alkyl side

chain. In order to enable a straightforward radiofluorination, the substitution of a 2-fluoro-3-

methoxypyridine ring was favored (3 and 4). This strategy allows nucleophilic

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8

heteroaromatic substitution with [ F]fluoride using synthetic easily accessible halogen or

nitro substituted precursor compounds [30].

Structural modifications performed at the "south region" of the lead compound 1 usually

have a minor impact on the PDE5 inhibitory activity [26]. Therefore, we selected four

structurally diverse moieties to introduce the fluorine at the C-8 position of the quinoline

core by substituting the ethyl group of 1 as follows (Figure 1): (i) fluoropropyl alkyl chain (5),

(ii) 4-fluoropiperidine (6) and (iii) 3-fluoroazetidine (7) as aliphatic rings and (iv) 2-

fluoropyridines (8 and 9) as aromatic rings.

4

Figure 1. Design of fluorinated compounds with introduction of the fluorine atom on substituents located at C-

4

or C-8 position of the quinoline core of the lead compounds 1 and 2.

2.1.2. Organic Chemistry

The synthesis of derivatives 3 and 4 bearing modifications in the "north region" of the

quinoline scaffold, started with the coupling of amine 10a with the 4-chloroquinoline 11 via

aromatic nucleophilic substitution at the C-4 position of the quinoline ring (Scheme 1). 4-

Chloroquinoline 11 was prepared as previously described by our group [28]. The necessary

amine 10a was synthesized in 5 steps according to known procedures (see supporting

information). The resulting derivative 12 was further functionalized at the C-8 position via a

Suzuki-Miyaura cross-coupling reaction with commercially available ethylboronic or

cyclopropylboronic acid to generate 13a and 13b with a yield of 68% and 86%, respectively.

The desired fluorinated compounds 3 and 4 were obtained by a selective reduction of the

ester function with lithium tri-tert-butoxyaluminum hydride (LTBA).

5

Scheme 1. Synthesis of "north region" compounds 3 and 4.

Reagents and conditions: (a) 10a, N,N-diisopropylethylamine (DIPEA), n-propanol, reflux, 2 h; (b) ethylboronic

3 4 2 3

acid for 13a and cyclopropylboronic acid for 13b, Pd(PPh ) , Cs CO , toluene, reflux, 15-22 h; (c) LTBA, THF, 50

°C, 30 h.

For the synthesis of compounds 5-9 modified at the C-8 position of the quinoline core, two

different palladium-catalyzed reaction pathways were developed in order to form the C-C

bond in 5 and the C-N bond in 6-9 starting from compound 14 [28]. The synthesis of 5 started

with the reaction of allyl alcohol with 9-borabicyclo[3.3.1]nonane (9-BBN) in tetrahydrofuran

to afford the corresponding boronic ester, generated in situ at the α-position of the alkene

reactant. This complex was used directly for the subsequent Suzuki-Miyaura cross-coupling

reaction with 14 (Scheme 2, strategy 1) to afford the alcohol 15 with a yield of 26%. It is

worth mentioning, that the concomitant formation of the debrominated analog of 14 was

observed in a ratio of about 1/1.

To further convert the alcohol function into fluorine, 15 was treated by diethylaminosulfur

trifluoride (DAST) under classical conditions. However, the formation of the desired

fluorinated product 16 could not be observed and only a by-product, identified as compound

17, was obtained with a yield of 57%. We assume that the formation of this stable six-

membered ring in 17 is a result of an intramolecular cyclization caused by the electron donor

effects of the free secondary amine located at the C-4 position of the quinoline, as previously

observed during the chemical modifications at the "east region" of this compound class [28].

Therefore, an alternative strategy (Scheme 2, strategy 2) was developed, in which the amine

at the C-4 position of 14 was beforehand protected using di-tert-butyl dicarbonate (Boc O) in

2

the presence of catalytic amounts of N,N-4-dimethylaminopyridine (DMAP) to obtain 18 with

6

a yield of 74%. For the following Suzuki-Miyaura coupling at C-8 position, a fluorinated

boronic ester was used, generated from the reaction of allyl bromide with tetra-n-

butylammonium fluoride (TBAF) and 9-BBN. Thus, the fluoroalkylated compound 19 was

successfully synthesized and used in the next synthesis step without further purification.

Finally, the ester group was converted into the corresponding alcohol to obtain 20 which was

treated with trifluoroacetic acid (TFA) to remove the Boc protecting group resulting in the

desired derivative 5 with a yield of 31%.

Scheme 2. Synthesis of "south region" compound 5.

Reagents and conditions: (a) 1) Allyl alcohol, 9-borabicyclo[3.3.1]nonane (9-BBN), THF, 0 °C → rt, 7 h; 2)

Pd(PPh

3

)

4

, K

2

CO

3

, H

2

O, DMF, 80 °C, 22 h; (b) Diethylaminosulfur trifluoride (DAST), CH

2 2

Cl , -60 °C → -30 °C, 2 h;

(

c) (Boc)

2

O, DMAP, Et

3

N, THF, rt, 2.5 h; (d) 1) Allyl bromide (reagent and solvent), TBAF, rt, 30 min; 2) 9-BBN,

3 4 2 3 2 2 2

THF, -10 °C → rt, 7 h; 3) Pd(PPh ) , K CO , H O, DMF, 80 °C, 16 h; (e)LTBA, THF, 60 °C, 20 h; (f) TFA/CH Cl , 1/2,

v/v, rt, 3 h.

For the synthesis of compounds 6-9 via C-N bond formation at the C-8 position of compound

4 (Scheme 3), the Buchwald-Hartwig coupling reaction was used. Therefore, 14 was treated

1

with commercially available 4-fluoropiperidine, 3-fluoroazetidine and the synthesized

7

pyridines 10a, b (see supporting information) to afford 21a-d in 56-93% yields. Further

reduction of the ester function to the corresponding alcohol with LTBA provided the desired

fluorinated compounds 6-9.

Scheme 3. Synthesis of "south region" compounds 6-9.

Reagents and conditions: (a) 4-fluoropiperidine hydrochloride for 21a, 3-fluoroazetidine hydrochloride for 21b,

10a for 21a and 10b for 21b, Pd(OAc)

2 2 3

, BINAP, Cs CO , toluene, 120 °C, 3 h for 21a and b, 16 h for 21c and 4 h

for 21d; (b) LTBA, THF, 60 °C, 20 h.

2.2. Determination of inhibitory activity

The inhibitory potential of the new fluorinated derivatives 3-9 on the human recombinant

PDE5A1 protein and selected other human PDEs were evaluated by using an enzyme assay

[31] with sildenafil as standard reference compound. The tests were performed at two

different concentrations of the compounds; 100 nM for PDE5A1 and 1.0 µM for the other

PDEs. The results are summarized in Table 1. Analyses of the data revealed a considerable

decrease of PDE5A1 potency for derivatives 3 and 4 (34.6 and 20.9% of inhibition,

respectively) compared to the lead compounds 1 and 2 (90.3 and 84.2%, respectively).

Hence, the substitution of the 3-chloro-4-methoxy phenyl ring by a 2-fluoro-3-methoxy

pyridine ring in the "north region" of the molecule is unfavorable for binding to the PDE5A1

protein. By contrast, derivatives 5–9 are characterized by structural modifications in the

"

south region" at C8 of the quinoline core. Although the introduced motifs are structurally

quite diverse, their influence on the inhibitory activity toward the PDE5A1 is rather low and

the obtained values do not differ considerably compared to 1 and 2. This is in accordance

8

with the results reported by Fiorito et al. [26], who also modified the functionalities at the

C8-position. All new derivatives 3-9 demonstrated a high selectivity for the PDE5 enzyme as

shown by the weak inhibitory potential against PDE2A3, PDE3A, PDE4A1 and C2, PDE6AB,

PDE9A1, PDE10A1, and PDE11A1 at 1 µM concentrations.

As derivatives 6 and 7 showed the highest inhibition values for PDE5A1, they were selected

for determination of IC50 values by measuring the inhibition at seven different

concentrations (0.25 – 1000 nM) [31]. With an IC50 value of 5.92 nM compound 7 showed

the highest inhibitory potential which is comparable to sildenafil (IC50 = 6.23 nM). Although

the inhibitory acti vity of 7 is slightly lower compared to our first developed radiotracer

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[

F]ICF24027 [28, 29], this compound demonstrated a good selectivity toward the other

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PDEs and was therefore selected for F-labeling to generate a potential radiotracer for PET

imaging of PDE5A.

Table 1. Percentage inhibition values of synthesized compounds against selected PDEs

Percentage of inhibition (%)

IC50(PDE5A1)

(nM)

Comp.

a

b

a

PDE2A3 PDE3A PDE4A1 PDE4C2 PDE5A1 PDE6AB PDE9A1 PDE10A1 PDE11A1

c

1

22.4

15.3

10.4

14.1

16.0

17.0

18.9

76.6

39.5

NI

45.4

36.9

33.4

22.2

37.3

36.8

18.3

28.6

24.5

40.9

22.4

4.52

15.9

18.6

35.3

16.1

15.3

18.7

90.3

84.2

34.6

20.9

80.7

87.8

90.3

64.2

80.5

49.8

32.4

35.9

23.2

NI

40.0

22.9

27.9

15.8

46.8

17.4

23.5

17.2

21.0

27.2

24.1

11.8

8.80

16.4

23.7

13.2

8.82

11.2

c

2

3

2.87

NI

n.d.

25.6

5.92

n.d.

1.86

6.23

4

5

NI

6

24.7

10.8

13.9

5.79

20.6

8.71

23.4

NI

7

8

9

c

ICF24027

7

.49

18.2

63.9

9.03

65.8

32.4

55.4

88.4

86.7

60.4

65.7

NI

14.1

74.6

5.21

78.5

Sildenafil

67.7

74.0

a

The compounds were tested at 1 µM against PDE2A3, PDE3A, PDE4A1, PDE4C2, PDE6AB, PDE9A1,

b

PDE10A1 and PDE11A1 and at 100 nM against human PDE5A1. NI means no inhibition; n.d. means not

c

determined. Own reported data [28].

2.3. Precursor synthesis and radiochemistry

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For the synthesis of the new radioligand [ F]7, the aliphatic nucleophilic substitution

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18

reaction of a precursor with a suitable leaving group by [ F]fluoride was intended. As F-

9

labeling of an azetidine ring was only described in a single report [32], and the radiolabeling

at a secondary carbon atom is usually more challenging than on a primary carbon atom [33],

we investigated the use of two sulfonate leaving groups with different reactivity. Therefore,

the precursor molecules 27 and 28 with methylbenzenesulfonate (tosylate) and 4-

nitrobenzenesulfonate (nosylate), respectively, were synthesized. Acco rding to the

literature, the nosylate is known to be slightly more reactive than tosylate [34, 35].

Based on former experiments with this compound class (data not shown), the alcohol

functionality was not protected as its acidity is expected to be too low to considerably

influence the radiolabeling reaction.

2.3.1. Synthesis of the precursor compounds

The precursor compounds 27 and 28 were prepared as shown in Scheme 4. In a first step, the

ester function of 14 was reduced to the corresponding alcohol 22, which was further

protected as a silyl ether group to give 23. As described above for compound 21b, the

azetidine moiety was then coupled on the C-8 position of the quinoline core of derivative 23

using freshly prepared azetidin-3-ol trifluoroacetate [36] to produce compound 24 with a

yield of 51%. Afterwards, the tosylation and nosylation reactions were performed under

classical conditions to afford 25 and 26, respectively. Finally, the triisopropyl silyl (TIPS)

protecting group was cleaved in the presence of TBAF to afford the desired tosylate 27 and

nosylate 28 precursors with yields in the range of 60-70%.

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Scheme 4. Synthesis of precursor compounds 27 and 28 for F-radiolabeling.

10

Reagents and conditions: (a) LTBA, THF, 60 °C, 20 h, (b) TIPSCl, Et

trifluoroacetate, Cs CO , 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP), Pd(OAc)

TsCl for 25 or NsCl for 26, Et N, DMAP, DCM, rt, 19 h, (e) TBAF, THF, rt, 30 min.

3

N, DMAP, DMF, rt, 36 h, (c) azetin-3-ol

2

3

2

, toluene, reflux, 3 h, (d)

3

2

.3.2. Radiosyntheses

.3.2.1. Methylbenzenesulfonate (OTs) as leaving group

2

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The F-labeling of the tosylate precursor 27 was investigated with the conventional

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potassium carbonate (K

2

CO ) - kryptofix-222 system (K[ F]F/K222 complex) changing

3

different reaction parameters (solvents, temperatures and reaction times). When the

reaction was performed in acetonitrile (ACN) at 90 °C and in DMF at 130 or 150 °C, only the

formation of by-products could be observed. Using DMSO at 130 °C resulted in the

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generation of [ F]7 with a radiochemical yield of 2 - 3% after 5 min (determined by radio

TLC of a sample of the reaction mixture). However, the RCY decreased with increasing

reaction time indicating a decomposition of the radiotracer. The same effect was observed

for the precursor 27 itself, which was entirely decomposed after 15 min reaction time

according to HPLC analysis, probably due to a competing ß-elimination reaction. Further

investigations revealed that 100 °C was the optimal temperature for this radiolabeling

system resulting in RCYs of 3 - 4% after 15 min (Scheme 5). The reactions were performed

with a precursor amount of 2.0 - 2.5 mg; higher concentrations of 27 did not result in an

increase of RCY. Due to the presumed base sensitivity of both, the precursor and the

radiotracer, potassium oxalate as less basic counter ion was tested in combination with K222

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in DMSO. However, these conditions did not lead to the formation of [ F]7. Moreover, we

1

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18

tested [ F]TBAF as a mild nucleophilic F-fluorination agent in tert-amyl alcohol, as

previously used by Zhang et al. for radiolabeling of the same azetidine motif in a PDE2A

radioligand [32]. However, in contrast to the reported results (RCY of 7.3% at the end of the

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synthesis) we could not obtain the desired radiotracer [ F]7 under these conditions.

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Finally, it turned out that using the K[ F]F/K222 complex in DMSO was most useful for the

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synthesis of [ F]7 on the basis of a tosylate precursor and was therefore translated to an

automated procedure using the TRACERlab FX2 N synthesis module (GE Healthcare). The

synthesizer setup is described in the experimental part. Briefly, after trapping and elution of

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[

F]fluoride by an aqueous potassium carbonate solution from an anion exchange cartridge,

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the labeling reaction of the azeotropically dried K[ F]F/K222 complex with the precursor 27

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was performed in DMSO for 15 min at 100 °C. For isolation of [ F]7, the crude reaction

11

mixture was diluted with aqueous acetonitrile and directly applied to a semi-preparative

HPLC system (chromatogram in supporting information). The radiotracer fraction was

collected at a retention time of 22 - 24 min and purified by solid phase extraction (SPE) using

a C18-cartridge. The obtained radiotracer eluate was transferred out of the hot cell,

concentrated and formulated in sterile isotonic saline containing 10% of ethanol. In a total

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synthesis time of 80 min, [ F]7 could be reproducibly produced with a high radiochemical

purity of ≥ 99%, a radiochemical yield of 2.5 ± 0.1% (n = 3) and molar activities between 14

and 28 GBq/µmol (n = 3, end of synthesis EOS) with starting activities of 4 - 5 GBq. Co-

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injection of the corresponding reference compound 7 confirmed the identity of [ F]7 by

analytical radio-HPLC (chromatogram in supporting information).

2.3.2.2. 4-Nitrobenzenesulfonate (ONs) as leaving group

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For F-labeling of the nosylate precursor 28 we started as for the tosylate by using the

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K[ F]F/K222 complex. Reactions were performed in ACN, DMF and DMSO at different

temperatures (90 °C for ACN, 130 and 150 °C for DMF and DMSO, resp.) resulting in RCYs

lower than 1%. Moreover, the tertiary alcohols tert-BuOH and 3-methyl-3-pentanol (3M3P)

were tested, because nonpo lar protic solvents are described to b e beneficial for aliphatic

1

8

18

nucleophilic substitution [37, 38] as e.g. reported for [ F]FLT [39, 40] and [ F]FES [41]. 3-

Methyl-3-pentanol (3M3P) was recently used by Marchand et al. [40] because of its high

1

8

boiling point of 123 °C avoiding an excessive pressure in the reactor. For the [ F]FLT

synthesis, the ratio o f nosylate precursor to base was also shown to be crucial for the

labeling process [40, 42]. In the presence of an excess amount of base, the precursor is

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8

undergoing an E2 elimination mechanism, thus reducing the desired nucleophilic F-

fluorination [42]. Therefore, we started with a constant precursor to base ratio of 1.5 using 4

mg of precursor 28 and 0.6 mg of K

2

CO as base. While reaction in tert-BuOH at 95 °C

3

resulted in an RCY of 2%, a higher RCY of 7% could be achieved in 3M3P at 125 °C. With the

aim to minimize the amount of precursor used, we further investigated the 3M3P system

and found that a minimal ratio of 0.75 (2 mg precursor and 0.6 mg base) is necessary to

achieve this RCY. However, when transferring this procedure to an automated process, we

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8

observed an insufficient elution of [ F]fluoride from the anion exchange cartridge with this

low amount of base. To reduce the loss of activity on the cartridge to a minimum of 10%, 1.2

12

mg of K

2

CO has been found to be necessary in our automated system, which would result in

3

the undesired use of higher precursor amounts.

1

8

Because of the observed base sensitivity of the nosylate precursor 28, [ F]TBAF was also

investigated as fluorination agent. While in DMSO no radiolabeling could be obtained, an

RCY of up to 10% was found in 3M3P at 125 °C with 2 mg of 28. By chance we observed that

1

8

small amounts of water favored this radiolabeling reaction. The presence of water in F-

fluorination reactions can positively influence the RCY as already described by different

groups [43, 44] and has been ex plained with the dependence of the nucleophilicity of

fluoride by its hydration state [45, 46]. Indeed, in our system the addition of 50 – 100 µL

water to the reaction mixture (total volume 1 mL) increased the RCY up to 20% (Scheme 5).

However, the addition of more water lead to a reduction of the RCY. Attempts to skip the

1

8

azeotropic drying step by eluting the [ F]fluoride with tetra-n-butylammonium hydrogen

carbonate (TBAHCO ) dissolved in the radiolabeling solvent 3M3P and 100 µL water failed

3

due to the immiscibility of this tertiary alcohol and water.

Finally, the conditions were applied to an automated procedure using the TRACERlab FX2 N

module. The detailed procedure is described in the experimental part. Briefly, after trapping

1

8

on an anion exchange cartridge, the [ F]fluoride was eluted with TBAHCO dissolved in an

3

1

8

ACN/water solution and the resulting [ F]TBAF was dried by azeotropic distillation. The

radiolabeling reaction was performed after addition of the nosylate precursor 28 dissolved in

1

8

3

M3P/ACN/water (700/200/80, v/v/v) for 10 min at 125 °C. For isolation of [ F]7 with semi-

preparative HPLC, the solvent was removed under reduced pressure because a strong peak

broadening was observed when the tertiary alcohol remained in the injection solution. After

evaporation, the crude reaction mixture was diluted with aqueous acetonitrile and applied

to a semi-preparative HPLC system (chromatogram in supporting information). The

radiotracer fraction was collected at a retention time of 26 - 29 min and purified by solid-

phase extraction (SPE) using a C18 cartridge. The obtained radiotracer eluate was

transferred out of the hot cell, concentrated and formulated in sterile isotonic saline

containing 10% of ethanol. The entire process for this radiosynthesis lasted about 85 min.

1

8

Finally, [ F]7 could be reproducibly produced with a high radiochemical purity of ≥ 99%, a

radiochemical yield of 4.9 ± 1.5% (n = 3) and molar activities between 28 and 58 GBq/µmol

1

8

(

n = 3, EOS) with starting activities of 4 - 5 GBq. The identity of [ F]7 was confirmed by

analytical radio-HPLC (chromatogram in supporting information).

13

1

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Scheme 5. F-Labeling procedures developed with the tosylate and nosylate precursors (27 and 28)

1

8

to radiosynthesize the new PDE5 radioligand [ F]7.

The stability of the radiotracer was investigated by incubation at 40 °C in phosphate-

1

8

buffered saline (PBS) and pig plasma samples. [ F]7 proved to be stable in these media, and

no defluorination or degradation was observed within 60 min of incubation time.

1

8

2

.4. In vitro autoradiographic studies of [ F]7 on porcine brain

1

8

To investigate the distribution of binding sites of [ F]7 in brain, in vitro autoradiographic

studies on porcine brain slices were performed. The most dense binding was detected in the

fornix (Fx), followed by corpus callosum (CC) and thalamus (Th). Although in human, PDE5

expression was demonstrated in cerebellum (Cb), cortex (Cx), and hippocampus (Hip) [47],

1

8

only modest levels of binding were observed for [ F]7 on these structures of the porcine

brain.

1

8

To investigate the specificity of [ F]7 binding, blocking studies with co-incubation of 1 µM of

1

8

7

or 1 µM of sildenafil were performed (data in Figure 2). Only a slight decrease of [ F]7

binding in the range of 10-25% for all of the investigated brain regions was observed

indicating a high non-specific binding of the radiotracer. This high non-specific binding has

1

8

also been observed for our previously reported PDE5 radioligand [ F]ICF24027 and we

assumed it could be related to its high lipophilicity (logD of 3.9 calculated using ACD/Labs)

1

8

18

[

29]. However, in comparison to [ F]ICF24027, the hydroxyl group of [ F]7 is non-alkylated

resulting in a calculated logD value of 1.56. Thus, the high non-specific binding might be

caused by other properties. Probably with an IC50 value of 5.92 nM as a measure of the

14

1

8

inhibitory potential, the actual affinity of [ F]7 is still too low for a specific imaging of PDE5

as recently discussed by P. Cumming [48]. Based on an estimation of Bmax values in certain

brain regions, the author proposed the need of ligands with sub-nanomolar affinity to

achieve a binding potential suitable for PET imaging.

1

8

18

Figure 2. Binding of [ F]7 (2.02 ± 0.75 nM) in porcine brain in vitro (n=3). Remaining binding of [ F]7 in the

presence of 1 µM 7 or 1 µM sildenafil is presented as percentage of total binding (= 100%). Abbreviations: Acb

=

Nucleus accumbens, Cb = Cerebellum, CC = Corpus callosum, Cd = Nucleus caudatus, CS = Colliculus superior,

Cx = Cortex, Fx = Fornix, Hip = Hippocampus, Th = Thalamus.

1

8

2

.5 In vivo studies of [ F]7 in mouse

.5.1. Organ uptake

2

For mouse brain, we determined standard uptake values (SUV) of 1.10 (n=1) and 0.08 (n=2;

1

8

single values: 0.10 and 0.05) at 5 and 30 min, respectively, after injection of [ F]7, which

indicate perfusion-mediated uptake of the radioligand in the brain followed by a relatively

fast washout of the activity. In periphery, we could detect the high initial uptake in organs

with comparatively high expression of PDE5 such as lung (SUV = 8.70 and 0.98 at 5 and 30

min p.i.) and heart (SUV = 5.00 and 0.48 at 5 and 30 min p.i.) [49]. Although it might be

assumed that the herein observed order of late-stage SUV with lung > heart > brain (0.98,

0.48, 0.08 at 30 min p.i.) in CD1 mouse indeed correlates with the reported mRNA levels in

rabbit [49], this assumption remains to be investigated.

2.5.2. Metabolism studies

15

1

8

In vivo metabolism of [ F]7 was studied in CD-1 mice (n = 2) by investigation of plasma and

brain samples obtained from at 30 min after injection of the radioligand. Analysis of the

samples was performed by the use of micellar chromatography (MLC) and RP-HPLC. MLC

allows the direct injection of samples into the HPLC system without eliminating the tissue

matrix due to the ability of micellar aggregates to dissolve the proteins and other

components. For RP-HPLC analysis proteins were precipitated by treating aliquots of plasma

and brain homogenates with a mixture of acetone/water, resulting in recoveries of about

97% of activity in the supernatants.

The results obtained with both methods correlated very well. According to the

chromatograms (Figure 3), a high fraction of radiometabolites was detected in plasma with

1

8

only 13% of total radioactivity representing the nonmetabolized radioligand [ F]7. Several

radioactive metabolites were observed and we assume that some of them are brain

penetrable as they were also observed in the corresponding brain samples. Thus, only 24%

1

8

of total activity was represented by intact [ F]7 in brain (Figure 3). In particular, the more

1

8

lipophilic radiometabolite [ F]M4 seems to easily penetrate the blood-brain barrier and

1

8

accumulates in the brain. By contrast, the metabolites group [ F]M1 is very polar as the

species are eluting without significant retention on the hydrophobic HPLC column. Despite

this polarity, they were also detected in the brain samples to a considerable amount (Figure

3). We assume that this group of radiometabolites consists of small molecular weight species

containing alcohol, aldehyde or acid functions which might be formed due to N-dealkylation

of the azetidine ring. However, as the radioligand described herein doesn't show the

potential for future use, we didn't characterize the structure of the radiometabolites.

16

Figure 3. Exemplary MLC (A) and RP-HPLC (B) radio-chromatograms of a mouse plasma and brain sample at 30

1

8

minutes p.i. of [ F]7 (conditions MLC: Reprosil-Pur C18-AQ, 250 x 4.6 mm, gradient with an eluent mixture of

THF/100 mM SDS/10 mM Na HPO aq., 1.0 mL/min; conditions RP-HPLC: Reprosil-Pur C18-AQ, 250 x 4.6 mm,

gradient with an eluent mixture of ACN/20 mM NH OAc aq., 1.0 mL/min)

2

4

3. Summary and conclusion

Continuing our efforts in the development of PET imaging probes for characterization of the

expression pattern of the cyclic nucleotide phosphodiesterase PDE5 in brain, we synthesized

a series of fluorinated derivatives based on a quinoline lead compound (1). While structural

modification in the "east and west regions" of 1 have been already investigated by our group

[28], the present study was dedicated to modifications in the "north and south regions" of 1.

Substitution of a 2-fluoro-3-methoxypyridine ring in the "north part" of 1 caused a loss of

inhibitory potential on PDE5. By contrast, the "south part" of 1 is less restricted for structural

modifications resulting in the potential 3-fluoroazetidine containing derivative 7 with an IC50

value of 5.92 nM for PDE5 and high selectivity regarding other PDEs.

1

8

As F-labeling of a secondary carbon is challenging, two different leaving groups were

1

8

investigated for a nucleophilic aliphatic substitution with [ F]fluoride. Interestingly, highest

1

8

RCY of 4.9 ± 1.5% (EOS) was achieved for automated radiosynthesis of [ F]7 due to the

17

addition of small amounts of water to the reaction mixture of the nosylate precursor in 3-

1

8

18

methyl-3-pentanol and [ F]TBAF. By contrast, using the tosylate precursor and K[ F]F/K222

1

8

complex in DMSO resulted in lower RCY of 2.5 ± 0.1% (EOS). First in vivo studies of [ F]7 in

mice indicated perfusion-mediated uptake of the radioligand in the brain followed by a

1

8

relatively fast washout of activity. However, investigating the in vivo metabolism of [ F]7 in

mice disclosed the fast formation of several radiometabolites of which a considerable

amount has been also detected in the brain. Moreover, in vitro autoradiographic studies in

1

8

porcine brain revealed a high nonspecific binding of [ F]7 which might be related to

insufficient affinity toward PDE5.

In conclusion, the results of this study support the assumption that for PET imaging of the

PDE5 enzyme, specific radioligands with an affinity in the sub-nanomolar range seem to be

crucial.

4. Experimental section

4

.1 Organic chemistry

.1.1 General

All commercially available reagents and solvents were purchased from the following

commercial suppliers: Sigma Aldrich, Acros Organics, Carlo Erba, and Alfa Aesar and were

used without further purification. Room temperature (rt) refers to 20-25 °C. All solvents were

dried using common techniques. Air and moisture sensitive reactions were carried out under

anhydrous argon atmosphere. Magnesium sulfate (MgSO

4

) and sodium sulfate (Na

2

SO ) were

4

used as drying agents. Monitoring of the reaction progress was performed using thin layer

chromatography (TLC) on silica 60 F254 or alumina gel 60A + F254 plates and visualized with

UV light (UV lamp Fisher Bioblock Scientific, 365 nm or 254 nm) or with an appropriate

staining agent. Column chromatography was performed on silica gel (Chromagel 60 ACC, 40-

63 µm, Carlo Erba Reagents) or on alumina gel (Merck, column chromatographic adsorption

analysis ACC. to Brockmann, neutral aluminium oxide 90, standardised, 63-200 µm).

Uncorrected melting points (mp) were measured on an IA9100 Digital Melting Point

-1

Apparatus. Infrared spectra (IR) were recorded in the range of 4000-600 cm on a Nicolet

IS10 with attenuated total reflectance (ATR) accessory (ν: stretch, as: asymmetric, s:

symmetric, δ: deformation; op: out of plane). Nuclear magnetic resonance (NMR) spectra

1

13

were recorded on a Bruker Avance 500 instrument (500 MHz for H, 125 MHz for C), a

1

13

Bruker Avance 400 instrument (400 MHz for H, 100 MHz for C) or a Bruker Avance 200

1

13

instrument (200 MHz for H, 50 MHz for C). All chemical shifts (δ) are reported in parts per

million (ppm). Coupling constants (J) are given in Hertz (Hz). Spectral coupling patterns are

indicated as follow: s: singlet; d: doublet; t: triplet; q: quartet; m: multiplet, brt: broad triplet

and brs: broad singlet. All new compounds were analyzed by HRMS (High-Resolution Mass

Spectrometry, Waters® Micromass® Q-Tof micro™ Mass Spectrometer, UCA-START, Clermont-

18

Auvergne University, Clermont-Ferrand, France). The isotope peaks for chlorine and bromine

atoms are given with their relative intensities.

4.1.2 Syntheses

4.1.2.1. Ethyl 8-bromo-6-cyano-4-[((6-fluoro-5-methoxypyridin-2-

yl)methyl)amino]quinoline-3-carboxylate (12):

N,N-Diisopropylethylamine (293 mg, 2.32 mmol, 4.0 eq.) and the crude product 10a (550 mg,

2.85 mmol, 5.0 eq., synthesis in supporting information) were added under stirring to a

solution of 11 [28] (193 mg, 0.57 mmol, 1.0 eq.) in n-propanol (15 mL). The resulting mixture

was refluxed for 2 hours. After cooling to rt, water (30 mL) was added. The formed

precipitate was collected by filtration, washed with ethanol (10 mL) and dried to give the

desired product (160 mg, 0.35 mmol, 61%) as a white solid. R

acetate, 7/3, v/v): 0.15; Mp: 186 ± 1 C; IR (cm )  2241 (νC≡N), 1463 (δCH ), 1173 (νC-O); H

f

(SiO

2

, cyclohexane/ethyl

o

-1

1

3

NMR (400 MHz, DMSO-d

6

) δ 9.49 (brs, 1H, NH), 8.94 (m, 2H, H

2

, H

5

), 8.49 (d, 1H, JH7-H5 = 1.5

3

4

3

Hz, H

7

), 7.65 (dd, 1H, JH5’-H6’ = 8.1 Hz, JH5’-F = 10.5 Hz, H5’), 7.32 (d, 1H, JH6’-H5’ = 8.1 Hz, H6’),

3

4

J

.92 (d, 2H, JHd-NH = 5.2 Hz, H

d

), 4.30 (q, 2H, JHb-Hc = 7.1 Hz, H

b

), 3.86 (s, 3H, H ), 1.29 (t, 3H,

), 153.9 (C

e

3

13

Hc-Hb = 7.1 Hz, H

c

); C NMR (100 MHz, DMSO-d

6

) δ 166.7 (C

-F = 11 Hz, C1’), 141.5 (d, J

a

2

), 153.5 (C

4

), 152.0

1

3

2

(d, J

C

-F= 234 Hz, C3’), 148.5 (C8a), 143.8 (d, J

C

₄_’-F = 25 Hz, C4’),

3

’

1’

3

4

135.1 (C

7

), 131.7 (C

5

), 125.3 (C

8

), 123.7 (d, J

C

-F = 3 Hz, C5’), 120.9 (d, J

C

₆_’-F = 4 Hz, C6’), 119.8

5

’

(

C

4a), 117.8 (CN), 107.3 (C

6

), 105.4 (C

3

), 61.2 (C

b

), 56.3 (C

e

), 50.4 (C

d

), 14.0 (C

c

); HRMS (ESI)

7

9

+

79

+

m/z calculated for C20

4

H

17 BrFN

4

O

3

[M+H] : 459.0468, found C20

H17 BrFN

4

O

3

[M+H] :

8

1

+

59.0474 (100%); C20

H

17 BrFN

4

O

3

[M+H] : 461.0457 (98%).

4.1.2.2. General procedure for 13a and 13b:

Under stirring and argon atmosphere, cyclopropylboronic acid (3.0 eq.) or ethylboronic acid

16 eq.), tetrakis(triphenylphosphine)palladium(0) (15 mg, 12.9 µmol, 0.06 eq.) and caesium

(

carbonate (2.5 eq.) were added to a solution of 12 (1.0 eq.) in anhydrous toluene (10 mL).

The resulting mixture was refluxed until the TLC monitoring revealed the complete

consumption of 12 (15 h for 13a and 22 h for 13b). After cooling to room temperature, the

solvent was evaporated under reduced pressure. The crude product was purified by column

chromatography to give 13a or 13b as white solid.

4

.1.2.2.1. Ethyl 6-cyano-8-ethyl-4-[((6-fluoro-5-methoxypyridin-2-

yl)methyl)amino]quinoline-3-carboxylate (13a):

Column chromatography conditions: SiO , ethyl acetate/cyclohexane, 5/5, v/v; yield: 68% (60

2

o

-1

mg, 0.15 mmol); R



CDCl

=

f

(SiO

2

, cyclohexane/ethyl acetate, 6/4, v/v): 0.65; Mp: 152 ± 1 C; IR (cm )

1

2229 (νC≡N), 1687 (νC=O), 1570 (δN-H), 1358 (δCH3), 1283 (νC-O), 1187 (νC-O); H NMR (400 MHz,

4

3

) δ 9.80 (brs, 1H, NH), 9.21 (s, 1H, H

2

), 8.39 (d, 1H, JH5-H7= 1.7 Hz, H ), 7.64 (d, 1H, JH7-H5

5

3

1.7 Hz, H

7

), 7.31 (dd, 1H, JH5’-H6’ = 8.0 Hz, JH5’-HF = 9.8 Hz, H5’), 7.23 (d, 1H, JH6’-H5’ = 8.0 Hz,

3

H

6’), 4.92 (d, 2H, JHd-NH = 5.5 Hz, H

d

), 4.39 (q, 2H, JHb-Hc = 7.1 Hz, H

b

), 3.91 (s, 3H, H ), 3.19 (q,

e

19

3

13

2

H, JHf-Hg = 7.5 Hz, H

f

), 1.40 (t, 3H, JHc-Hb = 7.1 Hz, H

c

), 1.32 (t, 3H, JHg-Hf = 7.5 Hz, H

g

); C

1

NMR (100 MHz, CDCl

3

) δ 168.0 (C

a

), 156.6 (C

4

), 154.1 (d, JC3’-F = 241 Hz, C3’), 152.6 (C

2

), 151.0

), 129.7

),

); HRMS (ESI) m/z

3

2

(

C

8a), 144.9 (C

8

3

), 144.1 (d, JC1’-F = 11 Hz, C1’), 142.4 (d, JC4’-F = 25 Hz, C4’), 130.1 (C

7

4

5

), 122.3 (d, JC5’-F = 4 Hz, C5’), 119.4 (d, JC6’-F = 4 Hz, C6’), 119.1 (CN), 118.7 (C4a), 107.0 (C

04.8 (C

calculated for C22

6

1

3

), 61.1 (C

b

), 56.3 (C

e

), 52.5 (C

[M+H] : 409.1676, found 409.1657.

d

), 24.9 (C

f

), 14.3 (C

g

), 14.1 (C

c

+

H22FN

4

O

3

4.1.2.2.2. Ethyl 6-cyano-8-cyclopropyl-4-[((6-fluoro-5-methoxypyridin-2-yl)methyl)amino]

quinoline-3-carboxylate (13b):

Column chromatography conditions: SiO , ethyl acetate/cyclohexane, 4/6, v/v; yield: 86% (80

2

o

-

mg, 0.19 mmol); R

f

(Al

2

O

3

, cyclohexane/ ethyl acetate, 5/5, v/v): 0.74; Mp: 155 ± 1 C; IR (cm

1

)

 2226 (νC≡N), 1667(νC=O), 1473 (δCH3), 1198 (νC-O); H NMR (400 MHz, CDCl

3

) δ 9.98 (brs, 1H,

, H5’), 7.22 (d, 1H, JH6’-H5’

), 3.91 (s, 3H,

or Hg’), 0.79 (m, 2H, H

4

3

NH), 9.27 (s, 1H, H

2

), 8.36 (d, 1H, JH5-H7 = 1.4 Hz, H

5

), 7.29 (m, 2H, H

), 4.40 (q, 2H, JHb-Hc = 7.1 Hz, H

7

3

=

8.1 Hz, H6’), 4.93 (d, 2H, JHd-NH = 5.5 Hz, H

d

b

3

H

e

), 3.04 (m, 1H, H

f

), 1.39 (t, 3H, JHc-Hb = 7.1 Hz, H

b

), 1.23 (m, 2H, H

g

1

3

1

or Hg’); C NMR (100 MHz, CDCl

3

) δ 167.7 (C

, C1’), 142.5 (d, JC4’-F = 25 Hz, C4’), 128.8 (C

a

), 156.8 (C

4

), 153.1 (d, JC3’-F = 241 Hz, C3’), 152.1

2

(

C

2

), 144.3 (C8a), 143.7 (2C, C

8

5

), 126.4 (C ), 122.3

7

3

d, JC5’-F = 4 Hz, C5’), 119.4 (d, JC6’-F = 4 Hz, C6’), 118.9 (CN or C4a), 118.5 (CN or C4a), 107.4

C

6

), 104.8 (C

3

), 61.3 (C

b

), 56.3 (C

e

), 52.4 (C

[M+H] : 421.1676, found 421.1688.

d

), 14.1 (C

c

), 11.3 (C

f

), 9.7 (2C, C , Cg’); HRMS (ESI)

g

+

m/z calculated for C23

H

22FN

4

O

3

4.1.2.3. General procedure for 3 and 4:

A 1.1 M solution of lithium tri-tert-butoxyaluminum hydride in anhydrous tetrahydrofuran

(2.7 mL, 2.97 mmol, 6.0 eq.) was added to a solution of 13a or 13b (1.0 eq.) in anhydrous

tetrahydrofuran (15 mL) under stirring and argon atmosphere. The resulting mixture was

o

heated at 50 C for 30 hours. After cooling to room temperature, an 0.1 M aqueous sodium

hydroxide solution (40 mL) was slowly added under stirring (pH 10-11). The aqueous layer

was extracted with ethyl acetate (4 x 40 mL). The combined organic layers were washed with

brine (60 mL), dried over anhydrous magnesium sulfate, filtered and then concentrated

under reduced pressure. The crude product was purified by chromatography (SiO ,

2

ethanol/dichloromethane, 1/9, v/v) to give the desired products 3 (white solid) or 4 (light

yellow solid).

4.1.2.3.1. 8-Ethyl-4-[((6-fluoro-5-methoxypyridin-2-yl)methyl)amino]-3-

(

hydroxymethyl)quinoline-6-carbonitrile (3):

Yield: 36% (65 mg, 0.18 mmol); R

f

(SiO

2

, ethanol/dichloromethane, 1/9, v/v): 0.29; Mp: 168 ±

o

-1

1

C; IR (cm )  3420 (νN-H), 2228 (νC≡N), 1563 (δN-H), 1479 (δCH3), 1010 (νC-O); H NMR (400

4

MHz, DMSO-d

Hz, H

6

) δ 8.81 (d, 1H, JH5-H7 = 1.5 Hz, H

), 7.59 (dd, 1H, JH5’-H6’ = 8.1 Hz, JH5’-F = 10.6 Hz, H5’), 7.53 (t, 1H, JNH-Hb = 6.7 Hz, NH),

5

), 8.48 (s, 1H, H

2

), 7.75 (d, 1H, JH7-H5 = 1.5

3

4

3

7

3

7.21 (d, 1H, JH6’-H5’ = 8.1 Hz, H6’), 5.36 (t, 1H, JOH-Ha = 5.1 Hz, OH), 4.84 (d, 2H, JHb-NH = 6.7 Hz,

3

H

b

), 4.47 (d, 2H, JHa-OH = 5.1 Hz, H

a

), 3.83 (s, 3H, H

c

), 3.13 (q, 2H, JHd-He = 7.5 Hz, H

d

), 1.25 (t,

3

13

1

3H, JHe-Hd = 7.5 Hz, H

e

); C NMR (100 MHz, DMSO-d

6

) δ 155.2 (C

2

), 151.9 (d, JC3’-F = 236 Hz,

3

2

C

3’), 149.9 (C

4

), 148.1 (C8a), 147.5 (d, JC1’-F = 11 Hz, C1’), 144.1 (C

8

), 140.9 (d, JC4’-F = 25 Hz,

20

3

4

C

4’), 127.6 (C

Hz, C6’), 115.5 (C

m/z calculated for C20

7

), 127.4 (C

5

), 123.6 (d, JC5’-F = 4 Hz, C5’), 119.6 (2C, CN, C4a), 119.2 (d, JC6’-F = 4

3

), 106.1 (C

6

), 59.7 (C

a

+

), 56.2 (C

c

), 48.8 (C

b

), 24.2 (C

d

), 14.8 (C ); HRMS (ESI)

e

H20FN

4

O

2

[M+H] : 367.1570; found 367.1528.

4.1.2.3.2. 8-Cyclopropyl-4-[((6-fluoro-5-methoxypyridin-2-yl)methyl)amino]-3-

(

hydroxymethyl)quinoline-6-carbonitrile (4):

Yield: 45% (120 mg, 0.32 mmol); R

f

(SiO

2

, ethanol/dichloromethane, 1/9, v/v): 0.40; Mp: 183

o

-1

1

±

1 C; IR (cm )  2226 (νC≡N), 1542 (δN-H), 1478 (δCH3), 1260 (νC-O), 1036 (νC-O); H NMR (400

4

3

MHz, DMSO-d

6

) δ 8.73 (d, 1H, JH5-H7 = 1.4 Hz, H

5

), 8.51 (s, 1H, H

2

), 7.58 (dd, 1H, JH5’-H6’ = 8.2

4

3

4

Hz, JH5’-F = 10.5 Hz, H5’), 7.50 (t, 1H, JNH-Hb = 6.6 Hz, NH), 7.35 (d, 1H, JH7-H5 = 1.4 Hz, H

7

), 7.21

),

3

(d, 1H, JH6’-H5’ = 8.2 Hz, H6’), 5.39 (t, 1H, JOH-Ha = 5.1 Hz, OH), 4.84 (d, 2H, JHb-NH = 6.6 Hz, H

b

3

4

.49 (d, 2H, JHa-OH = 5.1 Hz, H

a

), 3.83 (s, 3H, H

c

), 3.13 (m, 1H, H

) δ 155.1 (C

d

), 1.11 (m, 2H, H

e

or He’), 0.85

1

3

1

(m, 2H, H

e

or He’); C NMR (100 MHz, DMSO-d

), 148.7 (C8a), 147.5 (d, JC1’-F = 11 Hz, C1’), 144.4 (C

), 123.5 (d, JC5’-F = 4 Hz, C5’), 122.5 (C

6

2

), 151.9 (d, JC3’-F = 236 Hz, C3’),

3

2

1

49.9 (C

26.2 (C

4

8

), 140.9 (d, JC4’-F = 25 Hz, C4’),

3

4

5

7

), 119.6 (CN), 119.5 (C

), 48.9 (C ), 10.6 (C ), 10.1 (2C, C

3

), 119.2 (d, JC6’-F = 3 Hz,

C

6’), 115.7 (C4a), 106.4 (C

6

), 59.7 (C

a

), 56.1 (C

c

b

d

e

, Ce’); HRMS

+

(ESI) m/z calculated for C21

H20FN

4

O

2

[M+H] : 379.1570, found 379.1535.

4.1.2.4. Ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-(3-hydroxypropyl)quinoline-

3-carboxylate (15):

A solution of 0.5 M 9-borabicyclo[3.3.1]nonane (9-BBN) in anhydrous tetrahydrofuran (25

o

mL, 12.5 mmol, 6.0 eq.) was slowly added at 0 C and under argon atmosphere to a solution

of allyl alcohol (0.86 mL, 12.6 mmol, 6.0 eq.) in anhydrous and previously degassed

tetrahydrofuran (3.0 mL). The resulting mixture was stirred at rt for 7 hours. This solution

was then added, under argon atmosphere, to a mixture of 14 [28] (1.0 g, 2.11 mmol, 1.0 eq.),

tetrakis(triphenylphosphine)palladium(0) (0.24 g, 0.21 mmol, 0.1 eq.), potassium carbonate

(

1.16 g, 8.39 mmol, 4.0 eq.), and water (1.0 mL) in anhydrous N,N-dimethylformamide (40

o

mL). The resulting mixture was stirred at 80 C for 22 hours. After cooling to room

temperature, water (40 mL) was added and the aqueous layer was extracted with ethyl

acetate (3 × 50 mL). The combined organic layers were washed with brine (3 × 100 mL), dried

with anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The

crude residue was purified by column chromatography (SiO , ethyl acetate/cyclohexane, 5/5,

2

v/v) to give by order of elution the desired product 15 (0.25 g, 0.55 mmol, 26%) as a yellow

solid, and ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyanoquinoline-3-carboxylate (0.24

o

g, 0.61 mmol, 29%). R

f

(SiO

2

, cyclohexane/ethyl acetate, 5/5, v/v): 0.17; Mp: 118 ± 1 C; IR

-1

(

cm )  2231 (νC≡N), 1673 (νC=O), 1595 (νC=C), 1505 (δN-H), 1262 (νC-O), 1190 (νC-O), 1068 (νC-O);

1

4

H NMR (400 MHz, CDCl

3

) δ 9.80 (brt, 1H, NH), 9.18 (s, 1H, H

2

), 8.40 (d, 1H, JH5-H7 = 1.7 Hz,

), 7.39 (d, 1H, JH2’-H6’ = 2.2 Hz, H2’), 7.26 (dd, 1H, JH6’-H5’ = 8.4

Hz, JH6’-H2’ = 2.2 Hz, H6’), 6.97 (d, 1H, JH5’-H6’ = 8.4 Hz, H5’), 4.88 (d, 2H, JHd-NH = 5.5 Hz, H

4

3

H

5

), 7.70 (d, JH7-H5 = 1.7 Hz, H

7

4

3

d

),

3

4

2

1

.36 (q, 2H, JHb-Hc = 7.1 Hz, H

b

), 3.91 (s, 3H, H

e

), 3.39 (m, 2H, H

h

), 3.29 (m, 2H, H

f

), 1.94 (m,

) δ 168.2 (C ), 156.8 (C

), 130.5 (C ), 130.2 (C1’), 129.4 (C2’),

3

13

H, H

g

), 1.40 (t, 3H, JHc-Hb = 7.1 Hz, H

55.1 (C4’), 153.0 (C

c

); C NMR (100 MHz, CDCl

3

a

4

),

2

), 151.5 (C8a), 142.8 (C

8

), 132.7 (C

7

5

21

1

5

C

26.9 (C6’), 123.5 (C3’), 118.8 (CN), 118.6 (C4a), 112.6 (C5’), 107.4 (C

6

), 104.3 (C

3

), 61.5 (C ),

b

9.5 (C

h

5

), 56.3 (C

e

), 52.0 (C

d

), 33.9 (C

g

), 27.0 (C

f

), 14.3 (C

c

); HRMS (ESI) m/z calculated for

3

+

35

+

24

H25 ClN

3

O

4

[M+H] : 454.1534, found C24

H

25 ClN

3

O

4

[M+H] : 454.1559 (100%);

3

7

+

24

H

25 ClN

3

4

[M+H] : 456.1554 (33%).

4.1.2.5. Ethyl 1-[(3-chloro-4-methoxybenzyl)imino]-9-cyano-6,7-dihydro-1H,5H-

pyrido[3,2,1-ij]quinoline-2-carboxylate (17):

Diethylaminosulfur trifluoride (DAST, 41 µL, 0.31 mmol, 2.0 eq.) was added dropwise, at -60

°

C and under stirring, to a solution of 15 (70 mg, 0.15 mmol, 1.0 eq.) in anhydrous

o

dichloromethane (5.0 mL). The temperature was slowly increased to -30 C and the mixture

o

was stirred between -30 C and -20 C for 2 hours before addition of an aqueous saturated

sodium bicarbonate solution (10 mL) at -20 °C (pH = 7-8). This mixture was extracted with

dichloromethane (2 × 15 mL). The combined organic layers were washed with brine (50 mL),

dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure.

The residue was purified by column chromatography (Al

v/v) to give 17 (30 mg, 84.8 µmol, 57%) as a yellow solid. R

2

O

3

, ethyl acetate/cyclohexane, 7/3,

(SiO , cyclohexane/ethyl acetate,

f

2

o

-1

3

1

H

/7, v/v): 0.25; Mp: 152 ± 1 C; IR (cm )  2224 (νC≡N), 1702 (νC=O), 1574 (νC=N), 1502 (νC=C),

1

271 (νC-O), 1170 (νC-O), 1062 (νC-O); H NMR (400 MHz, CDCl

3

) δ 8.53 (s, 1H, H

5

), 7.73 (s, 1H,

3

4

3

2

), 7.38 (m, 2H, H2’, H

7

), 7.29 (dd, 1H, JH6’-H5’ = 8.4 Hz, JH6’-H2’ = 2.1 Hz, H6’), 6.90 (d, 1H, JH5’-

3

H6’ = 8.4 Hz, H5’), 4.33 (s, 2H, H

), 2.95 (m, 2H, H

CDCl ) δ 166.3 (C ), 153.6 (C4’), 149.2 (C

), 129.6 (C2’), 129.1 (C ), 127.1 (C6’), 126.4 (C4a), 122.2 (C3’), 119.0 (CN), 122.1 (C5’), 107.5

), 106.0 (C ), 61.1 (C ), 56.4 (C

d

), 4.31 (q, 2H, JHb-Hc = 7.1 Hz, H

b

), 3.96 (m, 2H, H

h

), 3.88 (s, 3H,

3

13

H

e

f

), 2.14 (m, 2H, H

g

), 1.35 (t, 3H, JHc-Hb = 7.1 Hz, H

c

); C NMR (100 MHz,

3

a

4

), 145.4 (C

2

), 137.5 (C8a), 135.7 (C1’), 131.8 (C ), 129.7

7

(C

5

6

8

(C

3

b

e

), 55.5 (C

d

), 51.7 (C

[M+H] : 436.1428, found C24

h

), 26.8 (C

f

), 20.9 (C

g

), 14.5 (C

c

); HRMS

[M+H] :

3

5

+

35

+

(ESI) m/z calculated for C24

H

23 ClN

3

O

3

+

H23 ClN

3

O

3

7

436.1423 (100%); C24

H

23 ClN

3

O [M+H] : 438.1433 (33%).

3

4.1.2.6. Ethyl 8-bromo-4-[(tert-butoxycarbonyl)(3-chloro-4-methoxybenzyl)amino] -6-

cyanoquinoline-3-carboxylate (18):

Di-tert-butyl dicarbonate (0.92 g, 4.22 mmol, 2.0 eq.), triethylamine (59 µL, 4.22 mmol, 2.0

eq.) and N,N-4-dimethylaminopyridine (51 mg, 0.42 mmol, 0.2 eq.) were added under argon

to a solution of 14 [28] (1.0 g, 2.11 mmol, 1.0 eq.) in anhydrous tetrahydrofuran (40 mL). The

resulting mixture was stirred at rt for 2.5 hours before addition of water (40 mL). The

aqueous layer was extracted with dichloromethane (3 × 50 mL). The combined organic layers

were washed with brine (100 mL), dried over magnesium sulfate, filtered and concentrated

under reduced pressure. The crude residue was purified by column chromatography (SiO

2

,

ethyl acetate/cyclohexane, 3/7, v/v) to give the desired product (0.90 g, 1.57 mmol, 74%) as

o

-1

a white solid. R

234 (νC≡N), 1708 (νC=O), 1366 (δCH3), 1255 (νC-O), 1151 (νC-O); Analyses of NMR spectra

f

(SiO , ethyl acetate/cyclohexane, 3/7, v/v): 0.48; mp: 139 ± 1 C; IR (cm ) ν

2

revealed that this product was a mixture of two rotamers, named A and B with a ratio A/B =

1

4

1

/0.3; H NMR (400 MHz, CDCl

3

) δ 9.41 (s, 1H, H2(B)), 9.40 (s, 1H, H2(A)), 8.03 (d, 1H, JH5(A)-H7(A)

22

4

=

1.7 Hz, H5(A)), 8.02 (d, 1H, JH5(B)-H7(B) = 1.6 Hz, H5(B)), 7.77 (d, 1H, JH7(B)-H5(B) = 1.6 Hz, H7(B)),

4

7

.75 (d, 1H, JH7(A)-H5(A) = 1.7 Hz, H7(A)), 6.98 (d, 1H, JH2’(B)-H6’(B) = 1.9 Hz, H2’(B)), 6.91 (d, 1H,

H2’(A)-H6’(A) = 2.0 Hz, H2’(A)), 6.89 (dd, 1H, JH6’(A)-H2’(A) = 2.0 Hz, JH6’(A)-H5’(A) = 8.4 Hz, H6’(A)), 6.81

dd, 1H, JH6’(B)-H2’(B) = 1.9 Hz, JH6’(B)-H5’(B) = 8.4 Hz, H6’(B)), 6.67 (m, 1H, H5’(A/B)), 5.00 (d, 1H,

Hd(A)-Hd’(A) = 14.6 Hz, Hd(A)), 4.89 (d, 1H, JHd(B)-Hd’(B) = 14.9 Hz, Hd(B)), 4.25 (m, 3H, Hb(A/B), Hd’(B)),

.14 (d, 1H, JHd’(A)-Hd(A) = 14.6 Hz, Hd’(A)), 3.71 (s, 3H, Hh(B)), 3.69 (s, 3H, Hh(A)), 1.50 (s, 9H,

g(B)), 1.29 (t, 3H, JHc(A/B)-Hb(A/B) = 7.1 Hz, Hc(A/B)), 1.08 (s, 9H, Hg(A)); C NMR (100 MHz, CDCl )

4

3

J

4

3

(

2

J

2

4

H

3

13

3

δ 163.2 (Ca(A)), 162.9 (Ca(B)), 154.8 (C2(A/B)), 154.6 (C4’(A/B)), 153.5 (C4(B)), 153.0 (C4(A)), 149.4

(C

e(B)), 148.8 (Ce(A)), 148.5 (C8a(B)), 148.4 (C8a(A)), 134.6 (C5(A/B)), 131.1 (C2’(A/B)), 130.8 (C7(B)),

1

(

1

30.7 (C7(A)), 129.0 (C6’(A/B)), 128.9 (C1’(A/B)), 127.0 (C4a(B)), 126.9 (C4a(A)), 126.2 (C8(B)), 126.1

C

8(A)), 123.4 (C3(B)), 122.8 (C3(A)), 121.9 (C3’(A/B)), 116.4 (CN(B)), 116.3 (CN(A)), 111.8 (C5’(A)),

11.7 (C5’(B)), 111.4 (C6(B)), 111.3 (C6(A)), 82.5 (Cf(B)), 81.6 (Cf(A)), 62.2 (Cb(A)), 62.0 (Cb(B)), 55.8

h(A/B)), 54.3 (Cd(B)), 53.2 (Cd(A)), 28.1 (3C, Cg(B)), 27.7 (3C, Cg(A)), 13.8 (Cc(A/B)); HRMS (ESI) m/z

(C

3

5

79

+

35 79

+

calculated for C26

H

26 Cl BrN

3

O

5

[M+H] : 574.0744, found C26

H

26 Cl BrN

3

O

5

[M+H] :

3

7

79

35 81

+

574.0799 (77%); C26

H

26 Cl BrN

3

O

5

or C26

H

26 Cl BrN

3

O

5

[M+H] : 576.0721 (100%);

3

7

81

+

C

26

H

26 Cl BrN

3

O

5

[M+H] : 578.0730 (24%).

4.1.2.7. Ethyl 4-[(tert-butoxycarbonyl)-(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-(3-

fluoropropyl)quinoline-3-carboxylate (19):

Tetra-n-butylammonium fluoride (TBAF) solution (1.0 M in THF, 10 mL) was placed into a

three-necked flask under argon atmosphere. The solvent was removed under reduced

pressure in order to obtain anhydrous and pure TBAF. 3-Bromopropene (1.39 mL, 16.1 mmol,

1.0 eq.) was added and the resulting mixture was stirred at room temperature for 30

minutes. The product was then collected in a weighed gas trap with an anhydrous argon

stream flow as carrier for three hours to produce allyl fluoride (310 mg, 5.16 mmol, 32%) as a

o

colorless liquid at -60 C (see Figure S1 in supporting information).

Under stirring an 0.5 M solution of 9-BBN in tetrahydrofuran (2.1 mL, 1.05 mmol, 2.0 eq.)

o

was added at -10 C to the allyl fluoride solution (10.0 eq.) in anhydrous and previously

degassed tetrahydrofuran (3 mL). The resulting mixture was stirred at room temperature for

7

hours. This solution was added to a mixture of 18 (300 mg, 0.54 mmol, 1.0 eq.),

tetrakis(triphenylphosphine)palladium(0) (60 mg, 51.9 µmol, 0.1 eq.), potassium carbonate

(

288 mg, 2.08 mmol, 4.0 eq.) and water (0.3 mL) in N,N-dimethylformamide (20 mL). The

o

resulting mixture was stirred at 80 C for 16 hours. After cooling to room temperature, water

(30 mL) was added and the aqueous layer was extracted with ethyl acetate (3 × 30 mL). The

combined organic layers were washed with brine (100 mL), dried over anhydrous magnesium

sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by

column chromatography (SiO

2

, ethyl acetate/cyclohexane, 1/9, v/v) to give a mixture of 18

and 19 which was used in the next step without further purification (200 mg).

4.1.2.8. Tert-butyl 3-chloro-4-methoxybenzyl[6-cyano-8-(3-fluoropropyl)-3-

(

hydroxymethyl)quinolin-4-yl]carbamate (20):

23

A 1.1 M solution of lithium tri-tert-butoxyaluminum hydride in tetrahydrofuran (0.8 mL, 0.88

mmol) was added to a mixture of crude 19 (100 mg) in anhydrous tetrahydrofuran (5 mL)

o

under stirring and argon atmosphere. The resulting mixture was stirred at 60 C for 20 hours.

After cooling to room temperature, water (20 mL) was added under stirring. The solution was

extracted with ethyl acetate (3 × 20 mL) and then with dichloromethane (2 × 20 mL). These

two organic layers were separately washed with brine (100 mL). The combined organic layers

were dried over anhydrous magnesium sulfate, filtered and concentrated under reduced

pressure. The crude residue was purified by column chromatography (SiO

ethanol/dichloromethane, 5/95, v/v) to give the desired product (20 mg, 38.9 µmol) as a

white solid. Yield: 7%; R (SiO , ethanol/dichloromethane, 5/95, v/v): 0.43; Analyses of NMR

2

,

f

2

spectra revealed that this product was a mixture of two rotamers, named A and B with a

1

ratio A/B = 1/0.7; H NMR (400 MHz, CDCl

3

) δ 9.18 (s, 1H, H2(B)), 9.15 (s, 1H, H2(A)), 8.08 (m,

1

H, H5(A)), 7.85 (m, 1H, H5(B)), 7.75 (m, 1H, H7(A)), 7.72 (m, 1H, H7(B)), 7.13 (m, 1H, H2’(A)), 7.07

2

(m, 1H, H2’(B)), 7.04 (m, 1H, H6’(B)), 6.82 (m, 3H, H6’(A), H5’(B), H5’(A)), 5.17 (d, 1H, JHe(A)-He’(A) =

2

3

2

1

=

3

9

C

4.7 Hz, He(A)), 5.08 (d, 1H, JHe(B)-He’(B) = 14.8 Hz, He(B)), 4.55 (dt, 2H, JHi(A)-Hh(A) = 5.6 Hz, JHi(A)-F

47.3 Hz, Hi(A)), 4.36-4.29 (m, 3H, Hi(B), He(A), He(B)), 4.17 (m, 4H, Ha(A), Ha(B)), 3.90 (s, 3H, Hf(B)),

.89 (s, 3H, Hf(A)), 3.44 (m, 4H, Hg(A), Hg(B)), 2.22 (m, 4H, Hh(A), Hh(B)), 1.65 (s, 9H, Hd(A)), 1.26 (s,

3

5

+

H, Hd(B)); HRMS (ESI) m/z calculated for C27

H

30 ClFN

3

O

4

[M+H] : 514.1909, found

3

5

+

37

+

27

H

30 ClFN

3

O

4

[M+H] : 514.1953 (100%); C27

H

30 ClFN

3

O

4

[M+H] : 516.1985 (33%).

4.1.2.9. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-(3-fluoropropyl)-3-

(

hydroxymethyl)quinoline-6-carbonitrile (5):

Trifluoroacetic acid (1.0 mL) was added to a solution of 20 (20 mg, 38.9 µmol, 1.0 eq.) in

anhydrous dichloromethane (2 mL) under argon atmosphere. The resulting mixture was

stirred at rt for 3 hours and the solvent was evaporated under reduced pressure. The crude

residue was purified by column chromatography (SiO

2

, ethanol/dichloromethane, 5/95, v/v)

(SiO ,

to give the desired product (5.0 mg, 12.1 µmol, 31%) as a white solid; R

f

2

-1

ethanol/dichloromethane, 5/95, v/v): 0.19; IR (cm )  3412 (νN-H), 2229(νC≡N), 1503 (δN-H),

1

J

(

H

259 (νC-O), 1064 (νC-O), 1020 (νC-F); H NMR (400 MHz, CDCl

3

) δ 8.53 (s, 1H, H

2

), 8.35 (d, 1H,

4

H5-H7 = 1.7 Hz, H

5

), 7.64 (d, 1H, JH7-H5 = 1.7 Hz, H

7

), 7.37 (d, 1H, JH2’-H6’ = 2.2 Hz, H2’), 7.21

4

3

dd, 1H, JH6’-H2’ = 2.2 Hz, JH6’-H5’ = 8.4 Hz, H6’), 6.94 (d, 1H, JH5’-H6’ = 8.4 Hz, H5’), 4.77 (m, 4H,

3

2

a

, H

b

), 4.53 (td, 2H, JHf-He = 5.9 Hz, JHf-F = 47.2 Hz, H

f

), 3.92 (s, 3H, H

c

), 3.32 (m, 2H, H

[M+H] : 414.1385, found

[M+H] : 416.1244 (33%).

d

), 2.16

3

5

+

(

C

m, 2H, H

e

); HRMS (ESI) m/z calculated for C22

H

22 ClFN

3

O

2

3

5

+

37

+

22

H22 ClFN

3

O

2

[M+H] : 414.1357 (100%); C22

H22 ClFN

3

O

2

4

.1.2.10. Ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-(4-fluoropiperidin-1-yl)

quinoline-3-carboxylate (21a):

-Fluoropiperidine hydrochloride (133 mg, 0.95 mmol, 3.0 eq.), palladium(II) acetate (7.1

mg, 31.6 µmol, 0.1 eq.), caesium carbonate (618 mg, 1.90 mmol, 6.0 eq.) and BINAP (39 mg,

2.6 µmol, 0.2 eq.) were successively added to a solution of 14 [28] (150 mg, 0.32 mmol, 1

eq.) in anhydrous toluene (10 mL) in a sealed flask under stirring and argon atmosphere. The

4

6

24

resulting mixture was refluxed for 3 hours. After cooling to room temperature, the mixture

®

was filtered thought Celite 545 and washed with dichloromethane (60 mL). The filtrate was

concentrated under reduced pressure and the crude product was purified by column

chromatography (SiO

mg, 0.18 mmol, 56%) as a light yellow solid. R

2

, ethyl acetate/cyclohexane, 4/6, v/v) to give the desired product (90

(SiO , ethyl acetate/cyclohexane, 4/6, v/v):

f

2

o

-1

0

1

=

H

.24; Mp: 110 ± 1 C; IR (cm )  2226 (νC≡N), 1669 (νC=O), 1502 (δN-H), 1261 (νC-O), 1183 (νC-O),

1

4

027 (νC-F); H NMR (400 MHz, CDCl

3

) δ 9.47 (brs, 1H, NH), 9.19 (s, 1H, H

2

), 8.07 (d, 1H, JH5-H7

4

3

4

1.2 Hz, H

5

), 7.38 (d, 1H, JH2’-H6’ = 2.2 Hz, H2’), 7.24 (dd, 1H, JH6’-H5’ = 8.4 Hz, JH6’-H2’ = 2.2 Hz,

3

6’), 7.21 (m, 1H, H

7

), 6.96 (d, 1H, JH5’-H6’ = 8.4 Hz, H5’), 4.93 (m, 1H, H

h

), 4.82 (d, 2H, JHd-NH

=

3

5

H

.6 Hz, H

d

), 4.37 (q, 2H, JHb-Hc = 7.1 Hz, H

b

13

), 3.91 (s, 3H, H

); C NMR (100 MHz, CDCl

), 147.5 (C8a), 130.7 (C1’), 129.3 (C2’), 126.8 (C6’), 125.0 (C

e

), 3.40 (m, 4H, H

) δ 168.2 (C

f

, Hf’), 2.15 (m, 4H,

3

g

, Hg’), 1.40 (t, 3H, JHc-Hb = 7.1 Hz, H

c

3

a

), 157.0 (C

4

),

154.9 (C4’), 151.7 (C

2

), 150.8 (C

8

5

1

23.1 (C3’), 120.0 (C4a), 119.3 (CN), 118.1 (C

170 Hz, C ), 61.2 (C ), 56.2 (C ), 52.1 (C

, Cg’), 14.3 (C

7

), 112.5 (C5’), 107.4 (C

6

), 104.8 (C

3

), 88.1 (d, JCh-F

3

=

h

b

e

d

), 47.9 (d, 2C, JCf/f’-F = 5 Hz, C

f

35

, Cf’), 31.5 (d, 2C,

2

+

J

Cg/g’-F = 20 Hz, C

g

c

); HRMS (ESI) m/z calculated for C26

H

27 ClFN

4

O

3

[M+H] :

3

5

+

37

+

4

97.1756, found C26

99.1850 (33%).

H27 ClFN

4

O

3

[M+H] : 497.1751 (100%); C26

H27 ClFN

4

O

3

[M+H] :

4.1.2.11. Ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-(3-fluoroazetidin-1-

yl)quinoline-3-carboxylate (21b):

In a sealed flask compound 14 [28] (0.26 g, 0.55 mmol, 1.0 eq.) was diluted in anhydrous

toluene (20 mL) and 3-fluoroazetidine hydrochloride (91 mg, 0.82 mmol, 1.5 eq.),

palladium(II) acetate (12 mg, 53.4 µmol, 0.1 eq.), caesium carbonate (0.71 g, 2.18 mmol, 4.0

eq.), and BINAP (68 mg, 0.11 mmol, 0.2 eq.) were successively added under argon

o

atmosphere. The resulting mixture was stirred at 120 C for 3 hours. After cooling to room

®

temperature, the mixture was filtered on Celite 545 and washed with ethyl acetate (40 mL).

The filtrate was concentrated under reduced pressure and the crude residue was purified by

column chromatography (SiO

2

, ethyl acetate/cyclohexane, 2/8, v/v) to give the desired

(SiO , cyclohexane/ethyl acetate, 7/3,

product (0.20 g, 0.43 mmol, 78%) as a yellow solid. R

f

2

o

-1

v/v): 0.41; Mp: 155 ± 1 C; IR (cm )  2227 (νC≡N), 1670 (νC=O), 1590 (δN-H), 1504 (νC=C), 1437

1

(δCH2), 1067 (νC-O), 1180 (νC-O), 1066 (νC-O), 1024 (νC-F); H NMR (400 MHz, CDCl

3

) δ 9.40 (brs,

), 7.36 (d, 1H, JH2’-H6’ = 2.2 Hz, H2’),

4

1

7

1

H

=

H, NH), 9.02 (s, 1H, H

2

), 7.79 (d, 1H, JH5-H7 = 1.4 Hz, H

5

3

4

.23 (dd, 1H, JH6’-H5’ = 8.4 Hz, JH6’-H2’ = 2.2 Hz, H6’), 6.94 (d, 1H, JH5’-H6’ = 8.4 Hz, H5’), 6.57 (d,

4

3

H, JH7-H5 = 1.4 Hz, H

7

), 5.44 (m, 1H, H

g

), 4.79 (d, 2H, JHd-NH = 5.5 Hz, H

d

), 4.61 (m, 2H, H

f

or

3

f’), 4.35 (q, 2H, JHb-Hc = 7.1 Hz, H

b

), 4.27 (m, 2H, H

) δ 168.4 (C

), 144.5 (C8a), 130.9 (C1’), 129.4 (C2’), 126.9 (C6’), 123.1 (C3’), 120.7 (C

f

or Hf’), 3.90 (s, 3H, H

), 156.3 (C ), 154.9 (C4’), 150.6 (C

), 119.5 (2C, CN, C4a),

), 62.1 (C or Cf’), 61.9

e

), 1.39 (t, 3H, JHc-Hb

1

3

7.1 Hz, H

c

); C NMR (100 MHz, CDCl

3

a

4

2

), 148.2

(C

8

5

1

112.5 (C5’), 110.9 (C

7

), 107.7 (C

6

), 104.9 (C

3

), 83.4 (d, JCg-F= 201 Hz, C

g

f

(

C

f

or Cf’), 61.2 (C

b

), 56.3 (C

e

), 51.9 (C

d

), 14.3 (C

c

); HRMS (ESI) m/z calculated for

3

5

+

35

+

24

H

23 ClFN

4

O

3

[M+H] : 469.1443, found C24

H23 ClFN

4

O

3

[M+H] : 469.1453 (100%);

3

7

+

24

H

23 ClFN

4

3

[M+H] : 471.1489 (33%).

25

4.1.2.12. Ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-[((6-fluoro-5-

methoxypyridin-2-yl)methyl)amino]quinoline-3-carboxylate (21c):

The crude product 10a (2.0 g, synthesis in supporting information) was dissolved in water

(20 mL) before addition of a saturated aqueous sodium bicarbonate solution (20 mL, pH

7/8). This aqueous mixture was extracted with dichloromethane (5 × 30 mL). The combined

organic layers were washed with brine (100 mL), dried over magnesium sulfate, filtered and

concentrated under reduced pressure. The obtained residue (350 mg) was dissolved in

anhydrous toluene (3 mL) under argon atmosphere. The resulting solution was added to a

mixture of 14 [28] (0.30 g, 0.63 mmol, 1.0 eq.), palladium(II) acetate (14 mg, 62.4 µmol, 0.1

eq.), caesium carbonate (1.03 g, 3.16 mmol, 5.0 eq.) and BINAP (79 mg, 0.13 mmol, 0.2 eq.)

in anhydrous toluene (10 mL) under argon atmosphere. The resulting mixture was refluxed

®

for 6 hours. After cooling to room temperature, the mixture was filtered thought a Celite

5

45 pad and washed with dichloromethane (100 mL). The filtrate was concentrated under

reduced pressure and the crude product was purified by column chromatography (SiO

ethyl acetate/cyclohexane, 3/7, v/v) to give the desired product (200 mg, 0.36 mmol, 58%)

2

,

o

-1

as a yellow solid. R



f

(SiO

2

, ethyl acetate/cyclohexane, 3/7, v/v): 0.19; Mp: 175 ± 1 C; IR (cm )

1

2231 (νC≡N), 1670 (νC=O), 1505 (δN-H), 1265 (νC-O), 1181 (νC-O), 1065 (νC-O), 1022 (νC-F); H NMR

3

4

(400 MHz, CDCl

3

) δ 9.55 (t, 1H, JHd-He = 5.2 Hz, H

d

), 9.04 (s, 1H, H

2

), 7.71 (d, 1H, JH5-H7 = 1.1

), 6.94 (d,

), 4.84 (d, 2H, JHe-Hd = 5.4 Hz, H ),

), 3.88 (s, 3H,

4

Hz, H

5

), 7.37 (d, 1H, JH2’-H6’ = 2.1 Hz, H2’), 7.23 (m, 2H, H6’, H11’), 7.11 (m, 2H, H10’, H

g

3

4

3

1H, JH5’-H6’ = 8.5 Hz, H5’), 6.58 (d, 1H, JH7-H5 = 1.1 Hz, H

7

e

3

4.47 (d, 2H, JHh-Hg = 5.6 Hz, H

h

), 4.35 (q, 2H, JHb-Hc = 7.1 Hz, H

b

), 3.90 (s, 3H, H

f

3

13

H

i

), 1.39 (t, 3H, JHc-Hb = 7.1 Hz, H

4’), 153.2 (d, JC8’-F = 240 Hz, C8’), 151.1 (C

c

); C NMR (100 MHz, CDCl

3

) δ 168.5 (C

a

), 156.3 (C

), 145.6 (d, JC12’-F = 10 Hz, C12’), 145.1 (C

4

), 154.9

), 142.0

1

3

(

C

2

8

3

2C, C8a, C9’), 130.8 (C1’), 129.4 (C2’), 126.9 (C6’), 123.1 (C3’), 122.4 (d, JC10’-F = 4 Hz, C10’), 119.9

CN), 118.9 (d, JC11’-F = 4 Hz, C11’), 118.6 (C

4

5

), 118.3 (C4a), 112.5 (C5’), 108.1 (C

6

), 106.7 (C ),

7

1

04.9 (C

3

), 61.3 (C

b

), 56.4 (C

i

), 56.3 (C

f

), 51.7 (C

e

), 47.5 (C

h

), 14.3 (C

c

); HRMS (ESI) m/z

3

5

+

35

+

calculated for C28

H

26 ClFN

5

O

4

[M+H] : 550.1657, found C28

H

26 ClFN

5

O

4

[M+H] : 550.1661

3

7

+

(100%); C28

H

26 ClFN

5

O

4

[M+H] : 552.1703 (33%).

4.1.2.13. Ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-[((6-fluoropyridin-2-yl)

methyl)amino]quinoline-3-carboxylate (21d):

The crude product 10b (0.14 g, synthesis in supporting information) was dissolved in

dichloromethane (30 mL) before addition of a saturated aqueous sodium hydrogen

carbonate solution (50 mL, pH 8-9). After decantation, the aqueous layer was extracted with

dichloromethane (7 × 30 mL). The combined organic layers were washed with brine (40 mL),

dried over magnesium sulfate, filtered and concentrated under reduced pressure to obtain

(6-fluoropyridine-2-yl)methylamine (0.13 g, 1.27 mmol, 3.0 eq.) as a yellow oil. This residue

was dissolved, under argon atmosphere, in a mixture of 14[28] (160 mg, 0.34 mmol, 1.0 eq.),

palladium(II) acetate (7.6 mg, 33.9 µmol, 0.1 eq.), caesium carbonate (549 mg, 1.69 mmol,

5.0 eq.), and BINAP (42 mg, 67.5 µmol, 0.2 eq.) in anhydrous toluene (15 mL). The resulting

26

mixture was refluxed for 4 hours in a sealed flask. After cooling to room temperature, the

®

reaction mixture was filtered on Celite 545 and washed with dichloromethane (210 mL).

The filtrate was concentrated under reduced pressure and the crude residue was purified by

column chromatography (SiO

product 21d (163 mg, 0.31 mmol, 93%) as an orange solid. R

acetate, 7/3, v/v): 0.38; Mp: 152 ± 1 C; IR (cm )  2229 (νC≡N), 1669 (νC=O), 1594 (δN-H), 1531

2

, ethyl acetate/cyclohexane, 3/7, v/v) to give the desired

(SiO , cyclohexane/ethyl

f

2

o

-1

1

(δN-H), 1501 (νC=C), 1434 (δCH3), 1252 (νC-O), 1191 (νC-O), 1064 (νC-O), 1023 (νC-F); H NMR (400

4

MHz, CDCl

3

) δ 9.61 (brs, 1H, H

d

), 9.05 (s, 1H, H

2

), 7.73 (m, 2H, H

5

, H10’), 7.38 (d, 1H, JH2’-H6’

=

3

2

.2 Hz, H2’), 7.23 (m, 3H, H6’, H11’,

H

g

), 6.94 (d, 1H, JH5’-H6’ = 8.5 Hz, H5’), 6.84 (m, 1H, H9’), 6.59

4

3

(d, 1H, JH7-H5 = 1.1 Hz, H

7

), 4.85 (d, 2H, JHe-Hd = 5.4 Hz, H

e

), 4.57 (d, 2H, JHh-Hg = 6.0 Hz, H

h

),

3

13

4.36 (q, 2H, JHb-Hc = 7.1 Hz, H

b

), 3.90 (s, 3H, H

f

), 1.39 (t, 3H, JHc-Hb = 7.1 Hz, H

c

); C NMR (100

1

3

MHz, CDCl

3

) δ 168.4 (C

a

), 163.5 (d, JC8’-F = 239 Hz, C8’), 157.1 (d, JC12’-F = 13 Hz, C12’), 156.4

3

(C

4

), 155.0 (C4’), 150.9 (C

2

), 144.9 (C

8

), 141.9 (d, JC10’-F = 8 Hz, C10’), 141.8 (C8a), 130.6 (C1’),

4

1

5

29.4 (C2’), 126.9 (C6’), 123.1 (C3’), 119.8 (CN), 118.7 (2C, C

5

, C

3

), 118.3 (d, JC11’-F = 4 Hz, C11’),

2

12.6 (C5’), 108.3 (C

1.7 (C ), 48.2 (C

6

), 108.2 (d, JC9’-F = 37 Hz, C9’), 106.9 (C

7

), 104.9 (C4a), 61.3 (C

b

), 56.3 (C ),

f

+

3

5

e

h

), 14.3 (C

c

); HRMS (ESI) m/z calculated for C27

H

24 ClFN

5

O

3

[M+H] :

3

5

+

37

+

20.1552, found C27

22.1701 (33%).

H24 ClFN

5

O

3

[M+H] : 520.1584 (100%); C27

H24 ClFN

5

O

3

[M+H] :

4.1.2.14. General procedure for 6-9:

A 1.1 M solution of lithium tri-tert-butoxyaluminum hydride in tetrahydrofuran (0.99 mL, 1.1

mmol, 6.0 eq. for 6, 7 and 8, 10.0 eq. for 9) was added, under stirring and argon atmosphere,

to a solution of 21a-d (1.0 eq.) in anhydrous tetrahydrofuran (15 mL). The resulting mixture

o

was stirred at 60 C for 20 hours. After cooling to room temperature, water (20 mL) was

added under stirring. The aqueous layer was extracted with ethyl acetate (3 x 40 mL), and

dichloromethane (3 x 30 mL). These two organic layers were separately washed with brine

(

100 mL). The combined organic layers were dried over sodium sulfate, filtered and

concentrated under reduced pressure. The crude product was purified by column

chromatography (SiO , ethanol/dichloromethane, 5/95, v/v) to give the desired products.

2

4

(

.1.2.14.1. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-(4-fluoropiperidin-1-yl)-3-

hydroxymethyl)quinoline-6-carbonitrile (6):

Yellow solid; yield 31% (25 mg, 55.0 µmol); R , ethanol/dichloromethane, 5/95, v/v):

f

(SiO

.14; Mp: 215 ± 1 C; IR (cm )  2226 (νC≡N), 1504 (δN-H), 1256 (νC-O), 1064 (νC-O), 1015 (νC-F);

H NMR (400 MHz, DMSO-d

2

o

-1

0

1

6

) δ 8.47 (m, 1H, H

5

), 8.43 (s, 1H, H

2

), 7.42 (m, 1H, NH), 7.39 (d,

4

3

4

1

7

2

H, JH2’-H6’ = 2.1 Hz, H2’), 7.27 (m, 1H, H

.10 (d, 1H, JH5’-H6’ = 8.5 Hz, H5’), 5.41 (t, 1H, JOH-Ha = 4.9 Hz, OH), 4.88 (m, 1H, H

H, JHb-NH = 6.7 Hz, H

.02 (m, 4H, H

7

), 7.23 (dd, 1H, JH6’-H5’ = 8.5 Hz, JH6’-H2’ = 2.1 Hz, H6’),

3

f

), 4.79 (d,

), 3.35 (m, 4H, H

), 150.4 (C ), 149.8

), 121.0 (C3’), 120.7 (C4a), 119.7

3

b

), 4.44 (d, 2H, JHa-OH = 4.9 Hz, H

a

), 3.81 (s, 3H, H

) δ 153.4 (C4’), 153.1 (C

c

d

, Hd’),

1

3

e

, He’); C NMR (100 MHz, DMSO-d

), 143.9 (C8a), 134.1 (C1’), 127.8 (C2’), 126.2 (C6’), 122.0 (C

6

2

4

(C

8

5

1

(CN), 115.8 (C

7

), 115.7 (C

3

), 112.9 (C5’), 106.6 (C

6

), 88.5 (d, JCf-F = 168 Hz, C

f

), 59.6 (C ), 56.1

a

27

2

(

C

c

), 47.6 (C

b

5

), 47.5 (2C, C

d

, Cd’), 31.3 (d, 2C, JCe/e’-F = 19 Hz, C

e

, Ce’); HRMS (ESI) m/z calculated

3

+

35

+

for C24

C

H

25 ClFN

4

O

2

[M+H] : 455.1650, found C24

H25 ClFN

4

O

2

[M+H] : 455.1617 (100%);

3

7

+

24

H

25 ClFN

4

O

2

[M+H] : 457.1621 (33%).

4

(

.1.2.14.2. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-(3-fluoroazetidin-1-yl)-3-

hydroxymethyl)quinoline-6-carbonitrile (7):

Yellow solid; yield 55% (90 mg, 0.21 mmol); R (SiO , ethanol/dichloromethane, 5/95, v/v):

f

2

o

-1

0

1

7

.47; Mp: 162 ± 1 C; IR (cm )  3396 (νN-H), 2228 (νC≡N), 1503 (δN-H), 1281 (νC-O), 1260 (νC-O),

065 (νC-O); H NMR (400 MHz, DMSO-d

.36 (d, 1H, JH2’-H6’ = 2.1 Hz, H2’), 7.24 (m, 1H, NH), 7.21 (dd, 1H, JH6’-H2’ = 2.1 Hz, JH6’-H5’ = 8.4

1

4

6

) δ 8.33 (s, 1H, H

2

), 8.14 (d, 1H, JH5-H7 = 1.4 Hz, H

5

),

4

3

4

Hz, H6’), 7.10 (d, 1H, JH5’-H6’ = 8.4 Hz, H5’), 6.69 (d, 1H, JH7-H5 = 1.4 Hz, H

7

), 5.47 (m, 1H, H

e

),

3

5.36 (t, 1H, JOH-Ha = 5.2 Hz, OH), 4.76 (d, 2H, JHb-NH = 6.7 Hz, H

b

), 4.54 (m, 2H, H

d

or Hd’), 4.41

3

13

(d, 2H, JHa-OH = 5.2 Hz, H

a

), 4.25 (m, 2H, H

d

or Hd’), 3.81 (s, 3H, H

c

); C NMR (100 MHz,

DMSO-d

6

) δ 153.4 (C4’), 152.8 (C ), 149.4 (C

2

4

), 147.8 (C ), 141.6 (C8a), 134.3 (C1’), 127.8 (C2’),

8

1

4

C

26.1 (C6’), 121.0 (C3’), 120.4 (C4a), 119.9 (CN), 117.2 (C

5

), 115.9 (C

3

), 112.9 (C5’), 108.1 (C

), 56.0 (C

7

),

1

2

06.9 (C

7.5 (C

6

), 84.3 (d, JCe-F = 196 Hz, C

e

), 61.7 (d, 2C, JCd/d’-F = 5 Hz, Cd/d’), 59.7 (C

a

c

3

5

+

b

); HRMS (ESI) m/z calculated for C22

H21 ClFN

4

O

2

[M+H] : 427.1337, found

3

5

+

37

+

22

H

21 ClFN

4

O

2

[M+H] : 427.1350 (100%); C22

H

21 ClFN O [M+H] : 429.1253 (33%).

4

2

4

.1.2.14.3. 4-[(3-chloro-4-methoxybenzyl)amino]-8-[((6-fluoro-5-methoxypyridin-2-

yl)methyl)amino]-3-(hydroxymethyl)quinoline-6-carbonitrile (8):

Light yellow solid; yield 55% (80 mg, 0.16 mmol); R (SiO , ethanol/dichloromethane, 5/95,

f

2

o

-1

v/v): 0.40; Mp: 194 ± 1 C; IR (cm )  3409 (νN-H), 2227 (νC≡N), 1546 (δN-H), 1260 (νC-O), 1004

1

4

(ν

C-F); H NMR (400 MHz, DMSO-d

6

) δ 8.38 (s, 1H, H

2

), 8.01 (d, 1H, JH5-H7 = 1.2 Hz, H

5

), 7.59

4

3

(

dd, 1H, JH10’-F = 10.6 Hz, JH10’-H11’ = 8.2 Hz, H10’), 7.45 (t, 1H, JHe-Hf = 6.3 Hz, H

J

e

), 7.37 (d, 1H,

4

3

H2’-H6’ = 2.1 Hz, H2’), 7.33 (t, 1H, JHb-Hc = 6.8 Hz, H

b

), 7.25 (d, 1H, JH11’-H10’ = 8.2 Hz, H11’), 7.21

3

4

3

(

dd, 1H, JH6’-H5’ = 8.5 Hz, JH6’-H2’ = 2.1 Hz, H6’), 7.10 (d, 1H, JH5’-H6’ = 8.5 Hz, H5’), 6.65 (d, 1H,

J

4

3

H7-H5 = 1.2 Hz, H

7

), 5.39 (t, 1H, JOH-Ha = 5.1 Hz, OH), 4.79 (d, 2H, JHc-Hb = 6.8 Hz, H

c

), 4.49 (d,

3

13

2H, JHf-He = 6.3 Hz, H

f

), 4.44 (d, 2H, JHa-OH = 5.1 Hz, H

a

), 3.84 (s, 3H, H

), 151.9 (d, JC8’-F = 235 Hz, C8’), 149.9 (C

), 140.8 (d, JC9’-F = 15 Hz, C9’), 139.3 (C8a), 134.3 (C1’),

g

), 3.81 (s, 3H, H

d

); C

1

NMR (100 MHz, DMSO-d

6

) δ 153.4 (C4’), 152.8 (C

2

4

),

3

2

146.1 (d, JC12’-F = 11 Hz, C12’), 145.0 (C

3

4

127.7 (C2’), 126.1 (C6’), 123.5 (d, JC10’-F = 3 Hz, C10’), 121.0 (C3’), 120.3 (CN), 119.7 (d, JC11’-F = 3

Hz, C11’), 119.2 (C4a), 116.1 (C

3

), 114.8 (C

5

), 112.9 (C5’), 107.2 (C

6

), 103.6 (C

7

5

), 59.7 (C

a

), 56.1

3

+

(

5

C

g

), 56.0 (C

d

), 47.3 (C

c

), 46.2 (C

24 ClFN

f

); HRMS (ESI) m/z calculated for C26

H

24 ClFN

5

O

3

[M+H] :

3

5

+

37

+

08.1552, found C26

10.1570 (33%).

H

5

O

3

[M+H] : 508.1528 (100%); C26

H24 ClFN

5

O

3

[M+H] :

5

4.1.2.14.4. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-[((6-fluoropyridin-2-yl)methyl)amino]-

3-(hydroxymethyl)quinoline-6-carbonitrile (9):

28

Light yellow solid; yield 41% (15 mg, 31.4 µmol); R

v/v): 0.40; Mp: 186 ± 1 C; IR (cm )  3400 (νN-H), 2232 (νC≡N), 1550 (δN-H), 1504 (δN-H), 1436

f

(SiO , ethanol/dichloromethane, 1/9,

2

o

-1

1

(

H

δ

CH3), 1261 (νC-O), 1228 (νC-O); H NMR (400 MHz, DMSO-d

6

) δ 8.40 (s, 1H, H

2

), 8.03 (m, 1H,

3

4

5

), 7.93 (m, 1H, H10’), 7.55 (t, 1H, JHe-Hf = 6.2 Hz, H

), 7.29 (m, 1H, H11’), 7.23 (dd, 1H, JH6’-H5’ = 8.6 Hz, JH6’-H2’ = 2.1 Hz,

6’), 7.10 (d, 1H, JH5’-H6’ = 8.6 Hz, H5’), 7.05 (m, 1H, H9’), 6.66 (d, 1H, JH7-H5 = 1.2 Hz, H

e

), 7.38 (d, 1H, JH2’-H6’ = 2.1 Hz, H2’), 7.35

3

4

(t, 1H, JHb-Hc = 6.7 Hz, H

b

3

4

H

(

7

), 5.40

), 4.45 (d, 2H, JHa-

3

brs, 1H, OH), 4.80 (d, 2H, JHc-Hb = 6.7 Hz, H

c

), 4.59 (d, 2H, JHf-He = 6.2 Hz, H

); C NMR (100 MHz, DMSO-d

), 150.0 (C

f

1

3

1

OH = 3.7 Hz, H

C

a

3

), 3.81 (s, 3H, H

d

6

) δ 162.6 (d, JC8’-F = 235 Hz,

), 145.0 (C

3

8’), 158.0 (d, JC12’-F = 13 Hz, C12’), 153.4 (C4’), 152.8 (C

2

4

8

), 142.7 (d, JC10’-

F

= 8 Hz, C10’), 139.3 (C8a), 134.3 (C1’), 127.8 (C2’), 126.1 (C6’), 121.0 (C3’), 120.2 (CN), 119.2

4

2

(

C

4a), 118.9 (d, JC11’-F = 3 Hz, C11’), 116.1 (C

3

), 115.0 (C

), 47.4 (C

5

), 112.9 (C5’), 107.6 (d, JC9’-F = 37 Hz,

9’), 107.3 (C

for C25

6

), 103.7 (C

7

), 59.8 (C

a

), 56.0 (C

d

c

), 46.9 (C

f

); HRMS (ESI) m/z calculated

3

5

+

35

+

H

22 ClFN

5

O

2

[M+H] : 478.1446, found C25

H22 ClFN

5

O

2

[M+H] : 478.1437 (100%);

3

7

+

C

25

H

22 ClFN

5

O

2

[M+H] : 480.1476 (33%).

4

.1.2.15. Preparation of precursors for radiolabelling

.1.2.15.1. 8-Bromo-4-[(3-chloro-4-methoxybenzyl)amino]-3-(hydroxymethyl)quinoline-6-

4

carbonitrile (22):

A 1.1 M solution of lithium tri-tert-butoxyaluminum hydride in tetrahydrofuran (5.70 mL,

6.27 mmol, 6.0 eq.) was added, under stirring and argon atmosphere, to a solution of 14 [28]

(

0.5 g, 1.05 mmol, 1.0 eq.) in anhydrous tetrahydrofuran (30 mL). The resulting mixture was

o

stirred at 60 C for 20 hours. After cooling to room temperature, water (20 mL) was added

under stirring. The aqueous layer was extracted with ethyl acetate (3 × 30 mL) and

dichloromethane (3 × 30 mL). These two organic layers were separately washed with brine

(

100 mL), then combined, dried over magnesium sulfate, filtered and concentrated under

reduced pressure. The crude residue was purified by column chromatography (SiO

ethanol/dichloromethane, 5/95, v/v) to give the desired product 22 (0.31 g, 0.72 mmol, 69%)

2

,

o

-1

as a white solid. R



f

(SiO

2

, ethanol/dichloromethane, 5/95, v/v): 0.41; Mp: 209 ± 1 C; IR (cm )

1

3403 (νN-H), 2229 (νC≡N), 1505 (δN-H), 1261 (νC-O), 1015 (νC-O); H NMR (400 MHz, DMSO-d

.96 (d, 1H, JH5-H7 = 1.5 Hz, H

NH-Hb = 6.7 Hz, NH), 7.40 (d, 1H, JH2’-H6’ = 2.1 Hz, H2’), 7.24 (dd, 1H, JH6’-H2’ = 2.1 Hz, JH6’-H5’

.5 Hz, H6’), 7.10 (d, 1H, JH5’-H6’ = 8.5 Hz, H5’), 5.48 (t, 1H, JOH-Ha = 5.2 Hz, OH), 4.85 (d, 2H,

6

) δ

4

8

5

), 8.53 (s, 1H, H

2

), 8.35 (d, 1H, JH7-H5 = 1.5 Hz, H

7

), 7.67 (t, 1H,

3

4

3

J

=

3

8

3

13

J

Hb-NH = 6.7 Hz, H

b

), 4.46 (d, 2H, JHa-OH = 5.2 Hz, H

a

), 3.81 (s, 3H, H

c

); C NMR (100 MHz,

DMSO-d

6

) δ 156.7 (C

2

), 153.5 (C4’), 150.3 (C

4

), 146.8 (C8a), 133.8 (C1’), 132.6 (C

7

), 129.7 (C

), 113.0 (C5’),

5

),

1

27.9 (C2’), 126.2 (C6’), 125.5 (C

8

), 121.1 (C3’), 120.4 (C4a), 118.1 (CN), 116.2 (C

3

5

79

06.7 (C

6

), 59.6 (C

a

), 56.1 (C

c

), 47.5 (C

b

); HRMS (ESI) m/z calculated for C19

H

16 Cl BrN

3

O

2

+

35 79

+

37 79

[M+H] : 432.0114, found C19

H

16 Cl BrN

3

O

2

[M+H] : 432.0105 (77%); C19

H16 Cl BrN

3

2

+

37 81

+

[M+H] : 434.0087 (100%); C19

H16 Cl BrN

3

O

2

[M+H] : 436.0092 (24%).

4.1.2.15.2. 8-Bromo-4-[(3-chloro-4-methoxybenzyl)amino]-3-

[((triisopropylsilyl)oxy)methyl]quinoline-6-carbonitrile (23):

29

Article Doi

Targeting cyclic nucleotide phosphodiesterase 5 (PDE5) in brain: Toward the development of a PET radioligand labeled with fluorine-18

DOI: 10.1016/j.bioorg.2019.01.037

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Article abstract of DOI:10.1016/j.bioorg.2019.01.037

Full text of DOI:10.1016/j.bioorg.2019.01.037

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