Exploration of the nitrogen heterocyclic periphery around the core of the advanced FFA1 agonist fasiglifam (TAK-875)

Article abstract of DOI:10.1002/ardp.202000275

Three types of heterocyclic moieties—piperidines fused to a heteroaromatic moiety—were explored as potential periphery motifs for the pharmacophoric core of fasiglifam (TAK-875), with fasiglifam being the most advanced agonist of free fatty acid receptor 1, a promising target for therapeutic intervention in type 2 diabetes. Several observed structure–activity relationship trends were corroborated by in silico docking results. Balanced selection based on potency and Caco-2 permeability advanced six compounds to cellular efficacy tests (glucose-stimulated insulin secretion in rat insulinoma INS1E cells). This led to the nomination of compound 16a (LK1408, 3-[4-({4-[(3-{[(2-fluorobenzyl)oxy]methyl}-1-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl]benzyl}oxy)phenyl]propanoic acid hydrochloride) as the lead for further development.

Full text of DOI:10.1002/ardp.202000275

Received: 30 July 2020
Revised: 6 November 2020
Accepted: 13 November 2020
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DOI: 10.1002/ardp.202000275
F U L L P A P E R
Explorationofthenitrogenheterocyclicperipheryaroundthe
core of the advanced FFA1 agonist fasiglifam (TAK‐875)
Alexey Lukin1 | Anna Bakholdina1 | Nikolay Zhurilo1
Oleksandra Onopchenko2 | Elena Zhuravel2 | Sergey Zozulya2,3
Maxim Gureev4 | Alexander Safrygin5 | Mikhail Krasavin5
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1Lomonosov Institute of Fine Chemical
Technologies, MIREA–Russian Technological
University, Moscow, Russian Federation
Abstract
Three types of heterocyclic moieties—piperidines fused to a heteroaromatic
moiety—were explored as potential periphery motifs for the pharmacophoric core
of fasiglifam (TAK‐875), with fasiglifam being the most advanced agonist of free
fatty acid receptor 1, a promising target for therapeutic intervention in type 2
diabetes. Several observed structure–activity relationship trends were corrobo-
rated by in silico docking results. Balanced selection based on potency and Caco‐2
permeability advanced six compounds to cellular efficacy tests (glucose‐stimulated
insulin secretion in rat insulinoma INS1E cells). This led to the nomination of
compound 16a (LK1408, 3‐[4‐({4‐[(3‐{[(2‐fluorobenzyl)oxy]methyl}‐1‐methyl‐1,4,6,
7‐tetrahydro‐5H‐pyrazolo[4,3‐c]pyridin‐5‐yl)methyl]benzyl}oxy)phenyl]propanoic
acid hydrochloride) as the lead for further development.
2Enamine Ltd., Kyiv, Ukraine
3Taras Shevchenko National University, Kyiv,
Ukraine
4I. M. Sechenov First Moscow State Medical
University, Moscow, Russian Federation
5Institute of Chemistry, Saint Petersburg
State University, Saint Petersburg, Russian
Federation
Correspondence
Mikhail Krasavin, Institute of Chemistry, Saint
Petersburg State University, Saint Petersburg,
Russian Federation, 199034.
Email: m.krasavin@spbu.ru
K E Y W O R D S
Funding information
FFA1 receptor, free fatty acids, glucose‐stimulated insulin secretion, GPR40, heterocyclic
periphery, hyperglycemia, type 2 diabetes mellitus
Russian Foundation for Basic Research,
Grant/Award Number: 19‐33‐90169
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1
INTRODUCTION
as activation of FFA1 by exogenous agonists carries little risk of
leading to hypoglycemia, even if the drug remains in circulation after
The G‐protein‐coupled receptor GPR40 was de‐orphaned in 2003
and renamed as free fatty acid receptor 1 (FFA1), owing to its acti-
vation by medium‐ to long‐chain free fatty acids as endogenous
ligands.[1] The physiological significance of this activation was es-
tablished shortly thereafter by demonstrating that binding of a free
fatty acid to FFA1 is crucial for regulating secretion of insulin in
pancreatic β‐cells and, thus, to maintain glucose homeostasis. The
uniqueness of this regulatory mechanism was also revealed, which
lies in the generally low expression levels of FFA1 in the normogly-
cemic state. However, once the glucose levels rise (as in diabetic
hyperglycemia), there is also an increase in the expression of FFA1.
Moreover, as soon as insulin excretion normalizes the glucose levels,
the expression levels of FFA1 also revert to the normal low. This
makes FFA1 an ideal target for therapeutic intervention in patho-
logical hyperglycemic states such as type 2 diabetes mellitus (T2DM),
normalization of the glucose levels.[2]
These observations about FFA1 prompted numerous industry
and academic research teams to focus on the validation of this re-
ceptor as a principally new antidiabetic target as well as on the
discovery of a number of agonists that were reported in the litera-
ture during the decade that followed after FFA1 de‐orphaning.[3]
However, this area of drug research was adversely impacted in
December 2013 by the discontinuation of phase III clinical trials of
Takeda's fasiglifam (TAK‐875), the most advanced drug candidate at
the time, due to reports of idiosyncratic liver toxicity observed
within an extended population of patients. Although there is no
evidence that this adverse effect was target‐related, the fasiglifam
setback (and substantial financial losses are inevitable if a drug fails
in such an advanced development phase) led to a sharp decrease in
the research efforts worldwide aimed at bringing new FFA1 agonists
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Arch Pharm. 2020;e2000275.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202000275

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to the market as a treatment for T2DM.[4] The cumulative discovery
results in this area have been summarized in several recent com-
prehensive reviews.[5] However, at the time of preparing this
manuscript, only one phase I clinical trial was underway—compound
P11187 of the undisclosed structure by Piramal.[5a] The apparent
inactivity in this area is disappointing, as the proof of principle of
activating FFA1 to achieve a positive clinical outcome in T2DM pa-
tients was, in fact, achieved in the course of clinical investigation of
fasiglifam.[6] The hepatotoxicity of the latter is almost unanimously
linked to the highly lipophilic[7] character of the molecule rather than
its action at the receptor, which is primarily expressed in the pan-
creas and in the brain.[8] Thus, the principal focus of medicinal
chemistry research toward new FFA1 agonists, which continues
predominantly in academic laboratories, is on the development of
less lipophilic compounds that possibly do not exhibit adverse effects
on the liver.[9]
Our efforts toward the development of less lipophilic FFA1 ago-
nists have recently focused on the former approach, that is, varying the
polar periphery without changing fasiglifam's basic 3‐(4‐benzyloxy)
phenylpropionic acid scaffold.[15–17] In some cases, however, total
elimination of centroids of lipophilicity from the peripheral motifs led to
a complete ablation of potency (as was the case with compound 5). This
loss of affinity to the receptor could be drastically reversed by bringing
back lipophilic aromatic (as in compounds 6) or even adding polar
heteroaromatic groups (as in compound 7). This restoration of potency
was shown to be only partly due to new hydrophobic interactions with
L542.51, L1354.54, and V813.27 of the receptor; more considerable
(particularly in the case of 7) was the additional π stacking interaction
with W1314.50 (Figure 2).[15–17]
In this study, we aimed to continue exploiting the strategy of fine‐
tuning the periphery around the basic 3‐(4‐benzyloxy)phenylpropionic
acid scaffold of fasiglifam (TAK‐875) to arrive at novel agonists that could
be considered as the next generation of FFA1 agonists devoid of overly
high lipophilicity and thus carrying less potential risk of hepatotoxicity
in vivo, which was the likely cause for fasiglifam's failure in the clinical
development. Mindful of the importance of counterbalancing the addi-
tion of polar heterocyclic motifs with aromatic appendages capable of
building a network of hydrophobic (free fatty acid‐like) and π stacking
interactions with the protein target, we designed three types of appen-
dages. Capitalizing upon the successful use of piperidine peripheral
motifs,[15–17] we set off to explore piperidine‐fused pyrazole 8, 2‐
pyridone 9, and 2‐alkoxypyridine 10, which could be elaborated into
potential FFA1 agonists via reductive amination reaction with aldehyde
The pursuit of less lipophilic FFA1 agonists may seem like a sort of
“tug of war,” as a ligand's lipophilicity is known to drive its affinity to the
target whose endogenous ligand (free fatty acid) is itself quite
lipophilic.[10] Hence, the FFA1 agonist structure should be modified to-
ward decreasing log P with care, so as not to lose the on‐target activity
altogether. Typically, this is achieved via either employing more polar
appendages around the 3‐phenylpropionic acid pharmacophore (as in
compounds 1[11] and 2[12] where fasiglifam's benzyloxy group is replaced
with a thiazole and an oxazole congener, respectively) or by replacing the
said core altogether with higher polarity isosteres: such a “scaffold‐
hopping” exercise is exemplified by compounds 3[13] and 4[14] (Figure 1).
FIGURE 1 Polar appendage (1, 2) and scaffold‐hopping (3, 4) approaches toward less lipophilic FFA1 agonists

LUKIN ET AL.
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FIGURE 2 Polar periphery ablates the affinity of ligands to FFA1 (in 5), which is promptly restored by the addition of aromatic groups
(lipophilic, in 6, as well as hydrophilic ones, in 7) due to a network of additional hydrophobic and π stacking contacts (shown here for
compound 6)[16]
11 whose synthesis on a multigram scale had been performed earlier
(Figure 3).[18] Herein, we report the results of this investigation.
with LiAlH4 to give 3‐hydroxymethyl pyrazole 14. The latter was
alkylated using benzyl halides or arylated via the Mitsunobu protocol
to give, after removal of the Boc protecting group, pyrazole‐fused
piperidines 8a–d (Scheme 1).
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2
RESULTS AND DISCUSSION
Chemistry
2‐Pyridone 15, which served as a starting material for the pre-
paration of two groups of heterocyclic amines (9 and 10), was also
prepared according to the literature protocol.[20] This key building block
was alkylated with various benzyl chlorides (9(10)a,c–f) as well as
alkyl bromides (9(10)b,g) in the presence of cesium carbonate in
dimethylformamide (DMF). In all cases (except ethyl bromide and
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2.1
Pyrazole 12 prepared according to the literature procedure[19] was
selectively alkylated at N1 (to give 13) and subjected to reduction
FIGURE 3 Fused piperidines 8–10 and the key building block 11 employed in the synthesis of FFA1 agonists in this study

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SCHEME 1 Preparation of the pyrazole‐fused piperidines 8a–d
AB
2‐methoxyethyl bromide, both of which gave a sole N‐alkylation product
in a low yield), there was the formation of both N‐ and O‐alkylation
products that were separated chromatographically, whereupon the Boc
group was removed by treatment with 4 M HCl solution in 1,4‐dioxane
to give compounds 9 and 10 as hydrochloride salts (Scheme 2). The
regiochemistry of alkylation was confirmed by comparing the correlation
spectra (COSY) of both regioisomers (see electrospray ionization [ESI]
for such comparison made for the 9g/10g pair).
propranol, quinidine, and atenolol[23] that displayed high (Papp
,
14.9 1.4 cm·s−1·10−6), medium (PappAB, 11.6 0.8 cm·s−1·10−6), and low
(PappAB, 0.2 0.04 cm·s−1·10−6) permeability in this assay, respectively
(Table 1).
To our delight, all compounds prepared and investigated in this
study demonstrated potent agonism with respect to FFA1 receptor,
with nine compounds residing in the sub‐micromolar range. At the
same time, some generalizations with respect to structure–activity
relationships (SAR) could be drawn. In the N‐alkylated 5,6,7,8‐
tetrahydro‐1,6‐naphthyridin‐2(1H)‐one series (obtainable from
building blocks 9), bulky benzyl groups (as in 16e,g,h) reduced the
affinity to the receptor (only a small ethyl group in 16f was toler-
ated). Although they were somewhat similar in topology to each
other, pyrazole compounds (16a–d) demonstrated generally lower
potency as compared with 2‐alkoxypyridines (16i–l). The reasons for
both SAR trends have been investigated with the aid of in silico
modeling (vide infra).
All fused piperidines, 9a–g, 10a–b, and 10e–g, thus obtained
were employed in the preparation of final, fully elaborated FFA1
agonist 16, as shown in Scheme 3. The yields of the final products are
provided in Table 1 (Section 2.2). Unfortunately, attempts to prepare
compound 16 from piperidines 9e–g and 10b of purity that would be
adequate for biological testing failed.
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2.2
Biological activity
On the basis of the combination of potency and predicted cell
membrane permeability, six compounds (16a–c, 16f, and 16i,j) were
selected for further profiling. Specifically, we aimed to establish if
these compounds are capable of stimulating insulin secretion from
rat insulinoma INS1E cells[24] in the presence of a fixed concentra-
tion of glucose. To be able to compare the effects of our compounds
with those produced by the reference FFA1 agonist GW9805,[21] we
determined the glucose concentration at which the addition of
10 μM of GW9805 would lead to a significant increase in insulin
levels. As shown in Figure 4a, at 0.5 mM glucose concentration, the
addition of GW9805 had no effect; however, the increase in the
Potential FFA1 agonists 16a–l, synthesized as described above, were
tested for their ability to activate FFA1 using the calcium flux assay
employing Chinese hamster ovary (CHO) cells engineered to
stably express human FFA1. All compounds were tested in
a
concentration–response mode to calculate the respective EC50 values
and determine the percentage of maximum efficacy achieved for active
compounds, relative to the commercially available reference FFA1
agonist GW9508.[21] Additionally, compounds' cell membrane perme-
ability was assessed in human epithelial colorectal adenocarcinoma
(Caco‐2) cells[22] in comparison with the reference compounds
SCHEME 2 Preparation of the 2‐pyridone‐ and 2‐alkoxypyridine‐fused piperidines 9 and 10

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SCHEME 3 Preparation of final FFA1 agonist 16. DCM, dichloromethane; STAB, sodium triacetoxyborohydride; TEA, triethylamine, TFA,
trifluoroacetic acid
TABLE 1 FFA1 agonists 16a–l studied in this study
Compound
Rʹ
8–10 Yield (%) FFA1; EC50 SD (μM)a % Efficacyb Caco‐2; Papp (cm·s−1·10−6 c
AB
)
cLog Pd
16a (LK1408)
8a
8b
8d
8c
9a
56
25
74
87
33
0.65 0.12
0.60 0.08
0.61 0.10
0.43 0.03
1.74 0.18
95.0
89.2
93.4
90.2
88.4
10.4 2.2
9.2 0.1
7.0 0.3
ND
5.40
16b
16c
16d
16e
5.42
5.45
4.78
5.27
3.4 0.2
16f
9b
9c
42
20
0.69 0.07
93.5
70.5
4.6 0.3
3.16
4.38
16g
2.97 0.43
5.7 1.2
(Continues)

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TABLE 1 (Continued)
Compound
16h
Rʹ
8–10 Yield (%) FFA1; EC50 SD (μM)a % Efficacyb Caco‐2; Papp (cm·s−1·10−6 c
AB
)
cLog Pd
9d
32
1.13 0.11
100.3
5.8 0.3
4.51
16i
16j
16k
16l
10a
10e
10f
10g
56
16
27
14
0.32 0.09
0.33 0.04
0.37 0.09
0.15 0.05
90.3
64.5
94.9
68.5
<0.1
5.22
5.01
4.49
3.34
11.4 0.3
0.9 0.5
<0.1
Abbreviation: ND, not determined.
aEach value is an average of n = 4.
bRelative to GW9508[21] (5 μM).
cEach value is an average of n = 2, measured at c = 10 μM.
dCalculated using www.molinspiration.com.
FIGURE 4 Insulin secretion in INS1E cells (a) in the presence of 10 μM GW9805 at 0.5 mM and 16.7 mM concentration of glucose and (b) in
the presence of 10 μM GW9805 and 20 μM nateglinide at 16.7 mM concentration of glucose. Data shown are the mean SD of values from
three replicate samples. *p < .05; **p < .01, compared with dimethyl sulfoxide (DMSO) control

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All six frontrunner compounds (16a–c, 16f, and 16i,j) were tes-
ted in INS1E cells in the presence of 16.7 mM glucose (Figure 5). To
our delight, all six compounds appeared to reproduce the effects of
both GW9805 and nateglinide on insulin secretion. Moreover,
compound 16a (LK1408), whose profile most closely resembled that
of the two reference drugs with a high statistical significance
(p < .01), was tested in the same assay in a dose‐dependent manner
and its EC50 was determined to be in the range of 0.3–0.7 nM in
three independent experiments.
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2.3
In silico modeling
We employed in silico docking to rationalize some of the most pro-
nounced SAR trends observed within the set of FFA1 agonists 16a–l.
In particular, we were curious to understand the structural reasons
for (i) the more than fourfold decrease in potency on going from
N‐ethyl to N‐benzyl substitution pattern in compounds 16f and 16g,
(ii) the even more pronounced increase in potency on relocating the
4‐(trifluoromethyl)benzyl group from the nitrogen atom in 16e to the
oxygen atom in 16i, and (iii) the similar difference in potency
between N‐alkyl derivative 16f and its closest O‐alkyl congener 16l.
FIGURE 5 Insulin secretion in INS1E cells treated with six
frontrunner compounds (10 μM), GW9805 (10 μM), and nateglinide
(20 μM) in the presence of 16.7 mM glucose. Data shown are the
mean SD of values from three replicate samples. The experiment
was repeated twice with similar results (data not shown). *p < .05;
**p < .01, compared with dimethyl sulfoxide (DMSO) control
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Comparative docking of 16f and 16g
concentration to 16.7 mM produced a significant elevation of the
agonist‐induced insulin levels. Hence, our agonists were subse-
quently tested in comparison to GW9805 (10 μM) as well as to na-
teglinide (20 mM), a clinically used glucose‐lowering agent operating
via the blockage of ATP‐dependent potassium channels in the
membrane of pancreatic β cells.[25] The latter drug also produced a
significant elevation of insulin levels in INS1E cells in the presence of
16.7 mM glucose (Figure 4b).
2.3.1
The introduction of the lipophilic phenyl substituent (16g) in lieu of
the methyl group in 16f appears to change the profile of hydrophobic
contacts by the molecular periphery of FFA1 agonists. For instance,
the benzyl substituent of 16g is involved in the network of interac-
tions with the hydrophobic surface delineated by Leu140, Leu144,
Leu151, and Trp150. The total incremental contribution of this hy-
drophobic interaction to the binding energy was calculated as
320.87 kcal/mol. In contrast, the hydrophobic interactions by the
ethyl substituent of 16f contributed only 40.04 kcal/mol (Figure 6).
These observations are consistent with the generally more fa-
vorable component analysis performed for both ligands using the
MM‐GBSA method (Table 2) where the most significant differences
in Coulomb energy and energy of strained interactions are evident.
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2.3.2
Comparative docking of 16e and 16i
A comparison of the activity profile of groups of agonists 16a–d and
16i–l, both of which have a “linear,” extended tail topology, with that
of N‐alkyl derivatives 16e–h having more of an “angular” tail dis-
position indicates the preferred character of the former ligand shape.
However, we were interested in obtaining more accurate docking
results, so we corroborated this observation with calculated binding
energy values and various components thereof.
Docking of compounds 16e and 16i, allowing for a direct com-
parison of the N‐ and O‐alkylated series, respectively, revealed
that a more effective network of hydrophobic contacts of the
FIGURE 6 Binding poses of compounds 16f (green) and 16g
(yellow) within the hydrophobic region of FFA1 endogenous ligand
binding (partial view of the tail binding)

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TABLE 2 Binding free energy
component calculated for ligands 16f and
16g using the MM‐GBSA method
Glide
ΔG H‐bond
(kcal/mol)
ΔG Coulomb
ΔG Lipo
ΔG Strain
Compound score
(kcal/mol)
(kcal/mol)
(kcal/mol)
16f
−9.03
−9.49
−2.12
−0.36
−24.54
−13.28
−38.90
−39.73
4.71
16g
7.95
improvement of potency at the FFA1 receptor, we were curious to see
what structural reasons could be responsible for a similar potency trend
on changing the substitution pattern between 16f and 16l (where there
was no additional π stacking with Trp150, as was the case with
compounds 16i).
The docking results (Figure 8) indicated that more efficient
contacts of the tetrahydro‐1,6‐naphthyridin‐2‐one moiety with the
hydrophilic area of the receptor is the likely reason for the greater
than fourfold higher affinity of 16l as compared with 16f.
As was observed previously, the component analysis of the
associated binding energy performed using the MM‐GBSA
method demonstrated a significantly more preferable contribu-
tion of the Coulomb and lipophilic components for compound 16l
(Table 4), which is quite in line with the visual inspection of the
biding pose.
FIGURE 7 Binding poses of compounds 16i (green) and 16e
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(yellow); a partial view of the tail binding
3
CONCLUSION
We have widened the range of heterocyclic peripheral motifs
that could be employed in combination with the known 3‐(4‐
benzyloxy)phenylpropionic acid scaffold to produce potent
(EC50 ~10−7 to 10−6 M) and efficacious agonists (as measured in
glucose‐stimulated insulin excretion assay performed in rat
insulinoma INS1E cells). These efforts resulted in the identifica-
tion of the new lead compound 16a (LK1408) for further devel-
opment as potential therapy for type 2 diabetes that is devoid of
hypoglycemia risk.
O‐(4‐trifluoro)benzyl tail of 16i is involved. In contrast to 16e whose
hydrophobic tail only makes hydrophobic contacts with Leu140,
Leu144, Leu151, and Trp150, 16i also forms a π stacking interaction
with the latter amino acid residue. Additionally, a greater flexibility
of the benzyloxy tails in 16i allows it to reach into the hydrophobic
area, whereas its fused piperidine moiety is an effectively hydrophilic
area (Figure 7).
The component analysis of the associated binding energy per-
formed using the MM‐GBSA method (Table 3) showed a significantly
preferable contribution from the three components—Coulomb,
lipophilic, and strained—whereas the hydrogen bonding component
remained the same.
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4
EXPERIMENTAL
Chemistry
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4.1
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General
2.3.3
Comparative docking of 16f and 16l
4.1.1
Considering that moving bulky benzylic substituents from the nitrogen
atom to the oxygen atom of the tetrahydro‐1,6‐naphthyridin‐2‐one
heterocyclic periphery (16e → 16i, vide supra) led to a substantial
All reactions were conducted in oven‐dried glassware in atmosphere
of nitrogen. Melting points were measured with a Buchi В‐520
melting point apparatus and were not corrected. Analytical thin‐layer
TABLE 3 Binding free energy
component calculated for ligands 16e and
Glide
ΔG H‐bond
(kcal/mol)
ΔG Coulomb
ΔG Lipo
ΔG Strain
Compound score
(kcal/mol)
(kcal/mol)
(kcal/mol)
16i using the MM‐GBSA method
16e
16i
−9.03
−9.49
−2.12
−0.36
−24.54
−13.28
−38.90
−39.73
4.71
7.95

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FIGURE 8 Binding poses of compounds 16l (green) and 16f (yellow); a partial view of the tail binding
chromatography was carried out on Silufol UV‐254 silica gel plates
using appropriate mixtures of ethyl acetate and hexane. Compounds
were visualized with short‐wavelength UV light. The 1H nuclear
magnetic resonance (NMR) and 13C NMR spectra (see Supporting
Information) were recorded on Bruker MSL‐300 spectrometers in
dimethyl sulfoxide (DMSO)‐d6 or CDCl3 using tetramethylsilane as
an internal standard. Mass spectra were recorded using Shimadzu
LCMS‐2020 system with ESI. All reagents and solvents were ob-
tained from commercial sources and used without purification.
All mass spectroscopic measurements required for the de-
termination of ADME properties were performed using the Shi-
madzu VP HPLC system including vacuum degasser, gradient pumps,
reverse phase high‐performance liquid chromatography (HPLC) col-
umn, column oven, and autosampler. The HPLC system was coupled
with a tandem mass spectrometer API 3000 (PE Sciex). The Tur-
boIonSpray ion source was used in both positive and negative ion
modes. Acquisition and analysis of the data were performed using
Analyst 1.5.2 software (PE Sciex).
[4,3‐c]pyridine‐3,5‐dicarboxylate (12)[19] (33.9 mmol, 10 g) was added
with stirring. Iodomethane (42.4 mmol, 2.64 ml) was then added drop-
wise and stirring continued for 6 h at room temperature. The progress of
the reaction was monitored using thin‐layer chromatography and 3%
methanol in chloroform as an eluent. The reaction mixture was washed
with 5% aq. K2CO3 (25 ml), 5% aq. citric acid (25 ml), and water (25 ml).
The organic phase was dried over anhydrous sodium sulfate, filtered,
and concentrated in vacuo to give the title compound. Yield, 10 g (92%);
white solid; m.p. 124.0–124.5°C. 1H NMR (300 MHz, CDCl3) δ 4.58 (s,
2H), 4.37 (q, J = 7.1 Hz, 2H), 3.80 (s, 3H), 3.70 (t, J = 5.7 Hz, 2H), 2.66
(t, J = 5.6 Hz, 2H), 1.46 (s, 9H), 1.37 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 162.5, 155.1, 138.7, 138.1, 117.8, 80.3, 60.8, 41.5, 40.1, 36.5,
28.5, 22.0, 14.5. High‐resolution mass spectrometry (HRMS)
(ESI) m/z calcd for
С
15H24N3O4 [M+H+] 310.1761 Da, found
310.1767 0.0015 Da.
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4.1.3
Synthesis of tert‐butyl 3‐(hydroxymethyl)‐1‐
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
methyl‐1,4,6,7‐tetrahydro‐5H‐pyrazolo[4,3‐c]‐pyridine‐5‐
carboxylate (14)
5‐tert‐Butyl 3‐ethyl‐1‐methyl‐1,4,6,7‐tetrahydro‐5H‐pyrazolo[4,3‐c]pyr
idine‐3,5‐dicarboxylate (13; 3.1 mmol, 10 g) was dissolved in anhydrous
tetrahydrofuran (THF; 50 ml), and LiAlH4 (26.2 mmol, 1 g) was added in
small portions at 0°C. The progress of the reaction was monitored using
thin‐layer chromatography and 2% methanol in chloroform as an eluent.
After the completion of the reaction, water (1 ml), 15% aq. NaOH (1 ml),
and again water (3 ml) were successively added, and the resulting
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4.1.2
Synthesis of 5‐tert‐butyl 3‐ethyl‐1‐methyl‐
1,4,6,7‐tetrahydro‐5H‐pyrazolo[4,3‐c]pyridine‐3,
5‐dicarboxylate (13)
To a suspension of sodium hydride (62.5 mmol, 1.5 g) in anhydrous to-
luene (25 ml), solid 5‐tert‐butyl 3‐ethyl‐1,4,6,7‐tetrahydro‐5H‐pyrazolo
TABLE 4 Binding free energy component calculated for ligands 16e and 16i using the MM‐GBSA method
Glide
score
ΔG H‐bond
(kcal/mol)
ΔG Coulomb
ΔG Lipo
ΔG Strain
Compound
16f
(kcal/mol)
(kcal/mol)
(kcal/mol)
−9.03
−9.24
−2.12
−2.31
−24.54
−26.30
−38.90
−39.16
4.71
4.34
16l

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LUKIN ET AL.
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biphasic mixture was stirred for 2 h at room temperature. The precipitate
formed was filtered off and the filtrate was collected and concentrated in
vacuo. The residue was dissolved in ethyl acetate (50 ml) and washed
with water (20 ml), 5% aq. citric acid (20 ml), and water (20 ml). The
organic phase was dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo to give the title compound. Yield, 6.3 g (79%); m.p.
96.0–96.5°C. 1H NMR (300 MHz, CDCl3) δ 4.59 (s, 2H), 4.42 (s, 2H), 3.69
(s, 3H), 3.68 (t, J = 5.6 Hz, 2H), 2.62 (t, J = 5.7 Hz, 2H), 1.46 (s, 9H); 13C
NMR (75 MHz, CDCl3) δ 155.1, 146.5, 138.1, 112.1, 80.2, 57.7, 40.3,
40.3, 35.5, 28.6, 22.0. HRMS (ESI) m/z calcd for С13H22N3O3 [M+H+]
268.1655 Da, found 268.1651 0.0020 Da.
(42%); m.p. 139–140°C. 1H NMR (300 MHz, DMSO‐d6) δ 9.86 (s, 2H),
7.43–7.34 (m, 1H), 7.20–7.02 (m, 3H), 4.48 (s, 2H), 4.46 (s, 2H), 4.03
(t, J = 4.1 Hz, 2H), 3.68 (s, 3H), 3.37–3.28 (m, 2H), 2.93 (t, J = 6.0 Hz, 2H);
13C NMR (75 MHz, DMSO‐d6) δ 162.2 (d, J = 243.4 Hz), 143.5, 141.3
(d, J = 7.2 Hz), 135.7, 130.3 (d, J = 8.3 Hz), 123.4 (d, J = 2.6 Hz), 114.2
(d, J = 20.9 Hz), 114.1 (d, J = 21.5 Hz), 107.8, 70.50, 6.75, 41.5, 38.8,
35.6, 18.4. HRMS (ESI) m/z calcd for С15H19FN3O [M+H+] 276.1506 Da,
found 276.1504 0.0020 Da.
|
4.1.6
Synthesis of 1‐methyl‐3‐{[(4‐methylpyridin‐2‐
yl)oxy]methyl}‐4,5,6,7‐tetrahydro‐1H‐pyrazolo
[4,3‐c]pyridine trifluoroacetate (8c)
|
4.1.4
Synthesis of 3‐{[(2‐fluorobenzyl)oxy]‐
methyl}‐1‐methyl‐4,5,6,7‐tetrahydro‐1H‐pyrazolo[4,3‐
c]pyridine hydrochloride (8a)
The title compound was synthesized analogously to 8a using
2‐chloro‐4‐methylpyridine as an arylating agent. The Boc group
was removed using TFA (3 equiv.) in dichloromethane. Yield, 2 g
(51%); m.p. 139–140°C. 1H NMR (300 MHz, DMSO‐d6) δ 9.12
(s, 2H), 8.06 (d, J = 5.3 Hz, 1H), 6.88 (d, J = 5.2 Hz, 1H), 6.71 (s, 1H),
5.24 (s, 2H), 4.16 (br.s, 2H), 3.71 (s, 3H), 3.47–3.34 (m, 2H), 2.91
(t, J = 5.9 Hz, 2H), 2.28 (s, 3H); 13C NMR (75 MHz, DMSO‐d6) δ
162.8, 158.7 (q, J = 37.3 Hz), 151.4, 145.7, 142.5, 135.5, 119.0,
115.6 (q, J = 289.9 Hz), 110.8, 108.2, 60.4, 40.5, 39.6, 35.7, 20.6,
18.5. HRMS (ESI) m/z calcd for С14H19N4O [M+H+] 259.1553 Da,
found 259.1551 0.0015 Da.
tert‐Butyl
3‐(hydroxymethyl)‐1‐methyl‐1,4,6,7‐tetrahydro‐5H‐
pyrazolo[4,3‐c]pyridine‐5‐carboxylate (14; 7.12 mmol, 2 g) was
dissolved in anhydrous THF (25 ml), and sodium hydride
(8.54 mmol, 0.342 g) was added in small portions. After 15 min
stirring at room temperature, 1‐(chloromethyl)‐2‐fluorobenzene
(8.54 mmol, 1.23 g) was added and the mixture was heated at re-
flux for 7 h. The progress of the reaction was monitored using thin‐
layer chromatography and 50% ethyl acetate in petroleum ether
as an eluent. The reaction mixture was washed with 5% aq. K2CO3
(25 ml), 5% aq. citric acid (25 ml), and water (25 ml). The organic
phase was dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo to give the crude product. The latter was
purified by column chromatography. Fractions containing the
target material were pooled and concentrated in vacuo, and the
residue was dissolved in anhydrous 1,4‐dioxane. A 4 M solution of
HCl in 1,4‐dioxane (2 ml) was added and the mixture was soni-
cated until a well‐formed hydrochloride salt precipitate separated.
The latter was filtered off, washed with ether, and air‐dried to give
the title compound. Yield, 1.5 g (45%); m.p. 145–146°C. 1H NMR
|
4.1.7
Synthesis of 3‐[(4‐fluorophenoxy)methyl]‐1‐
methyl‐4,5,6,7‐tetrahydro‐1H‐pyrazolo[4,3‐c]pyridine
hydrochloride (8d)
To
a
solution of tert‐butyl 3‐(hydroxymethyl)‐1‐methyl‐1
,4,6,7‐tetrahydro‐5H‐pyrazolo[4,3‐c]pyridine‐5‐carboxylate (14;
7.12 mmol, 2 g), 4‐fluorophenol (8.54 mmol, 0.95 g), and triphenyl
phosphine (7.83 mmol, 2.05 g) in dry THF (50 ml), diisopropylaza-
dicarboxylate (9.40 mmol, 1.64 g) was added at 0°C. The reaction
mixture was stirred at room temperature for 18 h and con-
centrated in vacuo. The residue was purified by column chroma-
tography on silica gel using chloroform as an eluent. Fractions
containing the target material were pooled and concentrated in
vacuo, and the residue was dissolved in anhydrous 1,4‐dioxane. A
4 M solution of HCl in 1,4‐dioxane (2 ml) was added and the
mixture was sonicated until a well‐formed hydrochloride salt
precipitate separated. The latter was filtered off, washed with
ether, and air‐dried to give the title compound. Yield, 2 g (57%);
m.p. 139–140°C. 1H NMR (300 MHz, DMSO‐d6) δ 9.77 (br.s, 2H),
7.15–7.07 (m, 2H), 7.05–6.98 (m, 2H), 4.99 (s, 2H), 4.07 (t,
J = 4.2 Hz, 2H), 3.70 (s, 3H), 3.37–3.28 (m, 2H), 2.94 (t, J = 6.0 Hz,
2H); 13C NMR (75 MHz, DMSO‐d6) δ 156.7 (d, J = 236.1 Hz), 154.5,
142.3, 135.7, 116.0, 115.9 (d, J = 32.3 Hz), 107.9, 63.4, 40.6, 38.8,
(300 MHz, DMSO‐d6)
δ 9.79 (br.s, 2H), 7.49–7.31 (m, 2H),
7.26–7.12 (m, 2H), 4.52 (s, 2H), 4.47 (s, 2H), 4.01 (s, 2H), 3.67
(s, 3H), 3.35–3.28 (m, 2H), 2.92 (t, J = 5.9 Hz, 3H); 13C NMR
(75 MHz, DMSO‐d6) δ 160.4 (d, J = 245.3 Hz), 143.5, 135.7, 130.5
(d, J = 4.4 Hz), 130.0 (d, J = 8.2 Hz), 125.0 (d, J = 14.8 Hz), 124.5
(d, J = 3.4 Hz), 115.3 (d, J = 21.2 Hz), 107.8, 65.3 (d, J = 3.4 Hz),
41.0, 38.9, 35.6, 18.4. HRMS (ESI) m/z calcd for С15H19FN3O
[M+H+] 276.1506 Da, found 276.1503 0.0010 Da.
|
4.1.5
Synthesis of 3‐{[(3‐fluorobenzyl)oxy]‐
methyl}‐1‐methyl‐4,5,6,7‐tetrahydro‐1H‐pyrazolo[4,3‐
c]pyridine hydrochloride (8b)
The title compound was synthesized analogously to 8a using
1‐(chloromethyl)‐3‐fluorobenzene as the alkylating agent. Yield, 1.4 g
35.6, 18.3. HRMS (ESI) m/z calcd for С
14H17FN3O [M+H+]
262.1305 Da, found 262.1305 0.0020 Da.

LUKIN ET AL.
11 of 16
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|
4.1.8
Synthesis of 1‐[4‐(trifluoromethyl)benzyl]‐
HRMS (ESI) m/z calcd for С10H15N2O [M+H+] 179.1178 Da, found
179.1180 0.0015 Da.
5,6,7,8‐tetrahydro‐1,6‐naphthyridin‐2(1H)‐one
hydrochloride (9a) and 2‐{[4‐(trifluoromethyl)benzyl]‐
oxy}‐5,6,7,8‐tetrahydro‐1,6‐naphthyridine
hydrochloride (10a)
10b: Yield, (9.2) 0.12 g (56%); white solid; m.p. 90–91°C. 1H NMR
(300 MHz, DMSO‐d6) δ 9.87 (s, 1H), 7.58 (d, J = 8.5 Hz, 1H), 6.72
(d, J = 8.5 Hz, 1H), 5.61 (s, 2H), 4.27 (q, J = 7.0 Hz, 1H), 4.16
(t, J = 4.3 Hz, 1H), 3.56 (s, 1H), 3.38 (s, J = 5.6 Hz, 1H), 2.98 (t,
J = 6.1 Hz, 1H), 1.29 (t, J = 7.0 Hz, 1H); 13C NMR (75 MHz, DMSO‐d6)
δ 162.05, 148.99, 138.61, 117.45, 109.24, 61.60, 42.57, 40.57, 27.82,
14.54. HRMS (ESI) m/z calcd for С10H15N2O [M+H+] 179.1178 Da,
found 179.1177 0.0010 Da.
tert‐Butyl 2‐oxo‐1,5,7,8‐tetrahydro‐1,6‐naphthyridine‐6(2H)‐carbox
ylate[20] (0.8 mmol, 0.2 g; 15) was dissolved in anhydrous DMF
(5 ml). 1‐(Chloromethyl)‐4‐(trifluoromethyl)benzene (0.96 mmol,
0.187 g) was added, followed by cesium carbonate (3.2 mmol,
1.041 g). The reaction mixture was stirred for 24 h. The progress of
the reaction was monitored using thin‐layer chromatography and 1%
methanol in chloroform as an eluent. After the completion of the
reaction, the mixture was diluted with water (100 ml) and extracted
with ethyl acetate (3 × 20 ml). The organic phase was dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo. The
residue was fractionated on silica gel column using 0–3% methanol in
chloroform as an eluent. Fractions eluting with Rf 0.5 and Rf 0.2 were
pooled and concentrated. Each residue was dissolved in anhydrous
1,4‐dioxane. A 4 M solution of HCl in 1,4‐dioxane (2 ml) was added
and the mixtures were sonicated until well‐formed hydrochloride
salt precipitates separated. The latter were filtered off, washed with
ether, and air‐dried to give 9a and 10a, respectively.
9a: Yield, 0.048 g (15%); white solid; m.p. 110–111 °C. 1H NMR
(300 MHz, DMSO‐d6) δ 9.62 (br.s, 2H), 7.71 (d, J = 8.1 Hz, 2H),
7.46–7.30 (m, 3H), 6.51 (d, J = 9.4 Hz, 1H), 5.38 (br.s, 2H), 4.00
(s, 2H), 3.27 (br.s, 2H), 2.87 (br.s, 2H); 13C NMR (75 MHz, DMSO‐d6)
δ 161.7, 141.6 (q, J = 1.1 Hz), 140.5, 139.2, 127.9 (q, J = 31.8 Hz),
127.3, 125.7 (q, J = 3.6 Hz), 124.3 (q, J = 272.0 Hz), 118.0, 107.6, 45.3,
41.9, 39.4, 30.8. HRMS (ESI) m/z calcd for С16H16F3N2O [M+H+]
309.1209 Da, found 309.1208 0.0020 Da.
|
4.1.10
Synthesis of 1‐benzyl‐5,6,7,8‐tetrahydro‐
1,6‐naphthyridin‐2(1H)‐one hydrochloride (9c)
It was prepared in full analogy to 9a/10a, except that only N‐
alkylation product 9c was obtained. Yield, 0.08 g (33%); white solid;
m.p. 80–81°C. 1H NMR (300 MHz, DMSO‐d6) δ 9.76 (br.s, 2H),
7.40–7.29 (m, 3H), 7.26 (d, J = 7.0 Hz, 1H), 7.14 (d, J = 7.1 Hz, 2H),
6.49 (d, J = 9.3 Hz, 1H), 5.28 (s, 2H), 3.99 (br.s, 2H), 3.32–3.21 (m,
3H), 2.90 (br.s, 2H); 13C NMR (75 MHz, DMSO‐d6) δ 161.73, 140.58,
138.86, 136.64, 128.72, 127.23, 126.43, 117.98, 107.30, 45.41,
41.89, 39.36, 23.06. HRMS (ESI) m/z calcd for С15H17N2O [M+H+]
241.1335 Da, found 241.1336 0.0020 Da.
|
4.1.11
Synthesis of 1‐(3‐fluorobenzyl)‐5,6,7,8‐
tetrahydro‐1,6‐naphthyridin‐2(1H)‐one
hydrochloride (9d)
It was prepared in full analogy with 9a/10a, except that only
N‐alkylation product 9d was obtained. Yield, 0.03 g (14%); white
solid; m.p. 89–90°C. 1H NMR (300 MHz, DMSO‐d6) δ 9.86 (br.s, 2H),
7.38 (d, J = 9.4 Hz, 1H), 7.39–7.31 (m, 1H), 7.14–7.03 (m, 1H),
7.03–6.89 (m, 2H), 6.49 (d, J = 9.3 Hz, 1H), 5.28 (s, 2H), 3.99 (br.s,
2H), 3.33–3.22 (m, 2H), 2.91 (t, J = 5.1 Hz, 2H); 13C NMR (75 MHz,
10a: Yield, 0.128 g (40%); white solid; m.p. 101–102°C. 1H NMR
(300 MHz, DMSO‐d6) δ 9.85 (br.s, 2H), 7.75–7.65 (m, 4H), 7.62
(d, J = 8.5 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 5.43 (s, 2H), 4.17
(t, J = 3.9 Hz, 2H), 3.45–3.34 (m, 2H), 2.99 (t, J = 6.1 Hz, 2H); 13C NMR
(75 MHz, DMSO‐d6)
δ
162.0, 149.2, 138.7, 129.7, 128.3
(d, J = 31.6 Hz), 126.9, 125.3 (q, J = 3.8 Hz), 124.3 (d, J = 272.0 Hz),
DMSO‐d6) δ 162.38 (d, J = 243.9 Hz), 161.75, 140.66, 139.65
118.1, 109.4, 45.0, 42.6, 40.6, 23.0. HRMS (ESI) m/z calcd for
(d, J = 7.4 Hz), 139.19, 130.81 (d, J = 8.4 Hz), 122.57 (d, J = 2.6 Hz),
117.94, 114.15 (d, J = 20.9 Hz), 113.36 (d, J = 22.1 Hz), 107.65, 45.20,
41.80, 39.32, 23.13. HRMS (ESI) m/z calcd for С15H16FN2O [M+H+]
259.1241 Da, found 259.1240 0.0020 Da.
С
16H16F3N2O [M+H+] 309.1209 Da, found 309.1209 0.0020 Da.
|
4.1.9
Synthesis of 1‐ethyl‐5,6,7,8‐tetrahydro‐1,6‐
naphthyridin‐2(1H)‐one hydrochloride (9b) and
2‐ethoxy‐5,6,7,8‐tetrahydro‐1,6‐naphthyridine
hydrochloride (10b)
|
4.1.12
Synthesis of 1‐(4‐chlorobenzyl)‐5,6,7,8‐
tetrahydro‐1,6‐naphthyridin‐2(1H)‐one hydrochloride
(9e) and 2‐[(4‐chlorobenzyl)oxy]‐5,6,7,8‐tetrahydro‐
1,6‐naphthyridine hydrochloride (10e)
They were prepared in full analogy to 9a/10a, except for the use of
ethyl bromide as an alkylating agent.
9b: Yield, 0.03 g (14%); white solid; m.p. 93–94°C. 1H NMR
(300 MHz, DMSO‐d6) δ 9.66 (br.s, 2H), 7.27 (d, J = 9.4 Hz, 1H), 6.37
(d, J = 9.3 Hz, 1H), 4.03–3.91 (m, 4H), 3.42–3.30 (m, 2H), 3.03
(t, J = 5.8 Hz, 2H), 1.15 (t, J = 7.0 Hz, 3H); 13C NMR (75 MHz, DMSO‐
d6) δ 161.3, 140.2, 138.4, 117.9, 107.0, 79.3, 42.1, 38.1, 22.8, 13.4.
They were prepared in full analogy with 9a/10a.
9e: Yield, 0.026 g (9%); white solid; m.p. 107–108°C. 1H NMR
(300 MHz, DMSO‐d6) δ 9.58 (br.s, 2H), 7.38 (dd, J = 10.7, 9.0 Hz, 3H),
7.18 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 9.3 Hz, 1H), 5.27 (s, 2H), 3.99 (s,
2H), 3.27 (s, 2H), 2.89 (d, J = 5.5 Hz, 2H); 13C NMR (75 MHz, DMSO‐

12 of 16
LUKIN ET AL.
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d6) δ 161.7, 140.5, 138.9, 135.7, 131.8, 128.6, 128.5, 118.0, 107.4,
44.9, 41.9, 39.4, 23.1. HRMS (ESI) m/z calcd for С15H16ClN2O
[M+H+] 275.7527 Da, found 275.7525 0.0010 Da.
43.1, 41.9, 30.8, 23.2. HRMS (ESI) m/z calcd for С11H17N2O2 [M+H+]
209.1284 Da, found 209.1284 0.0020 Da.
10e: Yield, 0.12 g (41%); white solid; m.p. 105–106°C. 1H NMR
(300 MHz, DMSO‐d6) δ 9.72 (br.s, 2H), 7.60 (d, J = 8.5 Hz, 1H), 7.45
(q, J = 8.6 Hz, 4H), 6.79 (d, J = 8.5 Hz, 1H), 5.31 (s, 2H), 4.18
(t, J = 4.2 Hz, 2H), 3.46–3.36 (m, 2H), 2.99 (t, J = 6.2 Hz, 2H); 13C NMR
(75 MHz, DMSO‐d6) δ 161.7, 149.1, 140.2, 138.6, 136.3, 130.0,
128.5, 117.9, 109.4, 42.7, 41.4, 39.4, 23.0. HRMS (ESI) m/z calcd for
|
4.1.15
Synthesis of 3‐[4‐({4‐[(3‐{[(2‐fluorobenzyl)‐
oxy]methyl}‐1‐methyl‐1,4,6,7‐tetrahydro‐5H‐
pyrazolo‐[4,3‐c]pyridin‐5‐yl)methyl]benzyl}oxy)
phenyl]‐propanoic acid hydrochloride (16a)
(LK01408)
С
15H16ClN2O [M+H+] 275.7527 Da, found 275.7526 0.0020 Da.
8a (0.29 mmol, 0.077 g) was dissolved in dichloromethane (3 ml).
Triethylamine (0.58 mmol, 0.08 ml) and tert‐butyl 3‐{4‐[(4‐
formylbenzyl)oxy]phenyl}propanoate (11; 0.29 mmol, 0.1 g) were
added. After 15 min of stirring, sodium triacetoxyborohydride (STAB;
1.17 mmol, 0.248 g) was added and the stirring continued for 36 h.
The progress of the reaction was monitored using thin‐layer chro-
matography and 10% methanol in chloroform as an eluent. The re-
action mixture was washed with 5% aq. K2CO3 (5 ml) and water
(2 × 5 ml), dried over anhydrous Na2SO4, filtered, and concentrated
in vacuo. The crude reduction amination product was fractionated on
silica gel using chloroform as an eluent. Fractions containing the
target material were pooled and concentrated to dryness. The re-
sidue was dissolved in anhydrous 1,4‐dioxane. A 4 M solution of HCl
in 1,4‐dioxane (2 ml) was added and the mixture was sonicated until
a well‐formed hydrochloride salt precipitate separated (18 h). The
latter was filtered off, washed with ether, and air‐dried to give the
title compound. Yield, 0.09 g (53%); white solid; m.p. 123–124°C. 1H
NMR (300 MHz, DMSO‐d6) δ 11.73 (br.s, 1H), 7.68 (d, J = 7.8 Hz, 2H),
7.48 (d, J = 7.8 Hz, 2H), 7.37–7.28 (m, 2H), 7.21–7.15 (m, 2H), 7.13 (d,
J = 8.6 Hz, 2H), 6.91 (d, J = 8.3 Hz, 2H), 5.09 (s, 2H), 4.54–4.32 (m,
2H), 4.45 (s, 2H), 4.41 (s, 2H), 4.04 (br.s, 2H), 3.67 (s, 3H), 3.66–3.57
(m, 1H), 3.34 (br.s, 1H), 3.20–2.95 (m, 2H), 2.74 (t, J = 7.4 Hz, 2H),
2.48 (t, J = 7.4 Hz, 2H); 13C NMR (75 MHz, DMSO‐d6) δ 173.6, 160.2
(d, J = 245.5 Hz), 156.6, 143.4, 138.6, 135.3, 133.2, 131.4, 130.3
(d, J = 4.4 Hz), 129.7 (d, J = 8.2 Hz), 129.2, 129.2, 127.7, 124.8
(d, J = 14.8 Hz), 124.3 (d, J = 3.5 Hz), 115.1 (d, J = 21.3 Hz), 114.6,
107.01, 68.8, 65.2 (d, J = 3.5 Hz), 64.9, 57.0, 48.2, 46.1, 35.6, 35.5,
|
4.1.13
Synthesis of 1‐(4‐fluorobenzyl)‐5,6,7,8‐
tetrahydro‐1,6‐naphthyridin‐2(1H)‐one hydrochloride
(9f) and 2‐[(4‐fluorobenzyl)oxy]‐5,6,7,8‐tetrahydro‐
1,6‐naphthyridine hydrochloride (10f)
They were prepared in full analogy with 9a/10a.
9f: Yield, 0.082 g (29%); white solid; m.p. 109–110°C. 1H NMR
(300 MHz, DMSO‐d6)
δ 9.54 (s, 2H), 7.35 (d, J = 9.4 Hz, 1H),
7.25–7.12 (m, 4H), 6.49 (d, J = 9.3 Hz, 1H), 5.26 (s, 2H), 3.99 (s, 2H),
3.28 (s, 2H), 2.90 (t, J = 5.7 Hz, 2H). HRMS (ESI) m/z calcd for
15H16FN2O [M+H+] 259.1241 Da, found 259.1239 0.0020 Da.
С
10f: Yield, 0.082 g (29%); white solid; m.p. 100–101°C. 1H NMR
(300 MHz, DMSO‐d6) δ 9.83 (br.s, 2H), 7.59 (d, J = 8.5 Hz, 1H),
7.54–7.46 (m, 2H), 7.24–7.14 (m, 2H), 6.77 (d, J = 8.5 Hz, 1H), 5.30 (s,
2H), 4.17 (t, J = 4.2 Hz, 2H), 3.45–3.36 (m, 2H), 3.01 (t, J = 6.2 Hz, 2H);
13C NMR (75 MHz, DMSO‐d6) δ 161.7, 161.3 (d, J = 242.9 Hz), 140.5,
138.9, 132.79 (d, J = 3.1 Hz), 128.6 (d, J = 8.2 Hz), 118.0, 115.4 (d,
J = 21.5 Hz), 107.3, 44.8, 41.9, 39.4, 23.0. HRMS (ESI) m/z calcd for
С
15H16FN2O [M+H+] 259.1241 Da, found 259.1240 0.0015 Da.
|
4.1.14
Synthesis of 1‐(2‐methoxyethyl)‐5,6,7,8‐
tetrahydro‐1,6‐naphthyridin‐2(1H)‐one hydrochloride
(9g) and 2‐(2‐methoxyethoxy)‐5,6,7,8‐tetrahydro‐1,6‐
naphthyridine hydrochloride (10g)
29.5, 18.2. HRMS (ESI) m/z calcd for
С
32H35FN3O4 [M+H+]
They were prepared in full analogy with 9a/9b, except for the use of
2‐methoxyethyl bromide as an alkylating agent.
544.2606 Da, found 544.2602 0.0010 Da.
10g: Yield, 0.138 g (57%); white solid; m.p. 62−63°C. 1H NMR
(300 MHz, DMSO‐d6) δ 9.91 (br.s, 2H), 7.58 (d, J = 8.5 Hz, 1H), 6.74
(d, J = 8.5 Hz, 1H), 4.34 (t, J = 4.6 Hz, 2H), 4.19–4.12 (m, 2H), 3.63 (t,
J = 4.6 Hz, 2H), 3.43–3.33 (m, 2H), 3.28 (s, 3H), 2.99 (t, J = 6.1 Hz, 2H);
13C NMR (75 MHz, DMSO‐d6) δ 161.9, 149.0, 138.6, 117.7, 109.3,
70.2, 64.8, 58.2, 42.4, 40.4, 27.8. HRMS (ESI) m/z calcd for
|
4.1.16
Synthesis of 3‐[4‐({4‐[(3‐{[(3‐fluorobenzyl)‐
oxy]methyl}‐1‐methyl‐1,4,6,7‐tetrahydro‐5H‐
pyrazolo‐[4,3‐c]pyridin‐5‐yl)methyl]benzyl}oxy)
phenyl]‐propanoic acid hydrochloride (16b)
С
11H17N2O2 [M+H+] 209.1284 Da, found 209.1282 0.0020 Da.
9g: Yield, 0.05 g (21%); white solid; m.p. 66–67°C. 1H NMR
It was synthesized analogously to 16a using 8b (0.29 mmol, 0.081 g),
triethylamine (0.58 mmol, 0.08 ml), tert‐butyl 3‐{4‐[(4‐formylbenzyl)
oxy]phenyl}propanoate (11, 0.29 mmol, 0.1 g), and STAB (1.17 mmol,
0.248 g). Yield, 0.04 g (24%); white solid; m.p. 124–125°C. 1H NMR
(300 MHz, DMSO‐d6) δ 11.41 (br.s, 1H), 7.65 (d, J = 7.8 Hz, 2H), 7.49
(d, J = 8.0 Hz, 2H), 7.43–7.34 (m, 1H), 7.13 (d, J = 8.6 Hz, 2H),
(300 MHz, DMSO‐d6) δ 9.59 (s, 2H), 7.29 (d, J = 9.4 Hz, 1H), 6.39 (d,
J = 9.3 Hz, 1H), 4.10 (t, J = 5.3 Hz, 2H), 3.99 (s, 2H), 3.54 (t, J = 5.3 Hz,
1H), 3.39–3.28 (m, 1H), 3.22 (s, 2H), 3.07 (t, J = 5.8 Hz, 1H); 13C NMR
(75 MHz, DMSO‐d6) δ 161.4, 140.9, 138.7, 117.8, 106.8, 69.6, 58.5,

LUKIN ET AL.
13 of 16
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7.12–7.05 (m, 3H), 6.91 (d, J = 8.6 Hz, 2H), 5.09 (s, 2H), 4.53–4.34
(m, 2H), 4.42 (br.s, 4H), 4.11–3.99 (m, 2H), 3.69 (s, 3H), 3.67–3.61
(m, 1H), 3.40–3.26 (m, 1H), 3.12–2.97 (m, 2H), 2.74 (t, J = 7.6 Hz, 2H),
2.47 (t, J = 7.6 Hz, 2H); 13C NMR (75 MHz, DMSO‐d6) δ 173.6, 162.1
(d, J = 243.6 Hz), 156.5, 143.4, 141.0 (d, J = 7.3 Hz), 138.7, 135.2,
133.2, 131.3, 130.2 (d, J = 8.3 Hz), 129.2, 129.1, 127.7, 123.3 (d,
J = 2.7 Hz), 114.6, 114.1 (d, J = 21.0 Hz), 113.9 (d, J = 21.5 Hz), 107.0,
70.5 (d, J = 1.8 Hz), 68.7, 64.8, 57.1, 48.2, 46.5, 35.6, 35.5, 29.4, 18.2.
HRMS (ESI) m/z calcd for С32H35FN3O4 [M+H+] 544.2606 Da, found
544.2637 0.0010 Da.
4.1.19
Synthesis of 3‐{4‐[(4‐{[1‐methyl‐3‐({[4‐
(trifluoromethyl)benzyl]oxy}methyl)‐1,4,6,7‐
tetrahydro‐5H‐pyrazolo[4,3‐c]pyridin‐5‐yl]methyl}‐
benzyl)oxy]phenyl}propanoic acid hydrochloride (16e)
It was synthesized analogously to 16a using 9a (0.14 mmol, 0.048 g),
triethylamine (0.29 mmol, 0.04 ml), tert‐butyl 3‐{4‐[(4‐formylbenzyl)
oxy]phenyl}propanoate (11, 0.145 mmol, 0.05 g), and STAB
(0.585 mmol, 0.124 g). Yield, 0.03 g (33%); white solid; m.p.
123–124°C. 1H NMR (300 MHz, DMSO‐d6) δ 11.31 (s, 1H), 7.69 (d,
J = 8.2 Hz, 2H), 7.62 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H),
7.42–7.34 (m, 3H), 7.14 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H),
6.48 (d, J = 9.4 Hz, 1H), 5.35 (dd, J = 59.2, 15.6 Hz, 2H), 5.10 (s, 2H),
4.40 (br.s, 2H), 4.05 (br.s, 2H), 3.75–3.58 (m, 1H), 3.23–3.11 (m, 1H),
3.01 (br.s, 2H), 2.75 (t, J = 7.5 Hz, 2H), 2.48 (t, J = 7.5 Hz, 2H); 13C
NMR (75 MHz, DMSO‐d6) δ 173.7, 161.6, 156.6, 141.4, 140.4, 138.9,
138.8, 132.5, 131.4, 129.2, 127.9, 127.8 (d, J = 31.6 Hz), 127.2, 125.5
(d, J = 2.9 Hz), 124.2 (d, J = 271.8 Hz), 118.0, 114.6, 106.7, 68.7, 57.5,
49.4, 46.8, 45.4, 35.5, 29.5, 23.6. HRMS (ESI) m/z calcd for
|
4.1.17
Synthesis 3‐(4‐{[4‐({3‐[(4‐fluorophenoxy)‐
methyl]‐1‐methyl‐1,4,6,7‐tetrahydro‐5H‐pyrazolo[4,3‐
c]pyridin‐5‐yl}methyl)benzyl]oxy}phenyl)propanoic
acid hydrochloride (16c)
It was synthesized analogously to 16a using 8d (0.29 mmol, 0.081 g),
triethylamine (0.58 mmol, 0.08 ml), tert‐butyl 3‐{4‐[(4‐formylbenzyl)
oxy]phenyl}propanoate (11, 0.29 mmol, 0.1 g), and STAB (1.17 mmol,
0.248 g). Yield, 0.084 g (51%); white solid; m.p. 110–111°C. 1H NMR
(300 MHz, DMSO‐d6) δ 11.84 (br.s, 1H), 7.72 (d, J = 7.9 Hz, 2H), 7.51
(d, J = 7.9 Hz, 2H), 7.15–7.04 (m, 4H), 6.95–6.85 (m, 4H), 5.09 (s, 2H),
4.94 (s, 2H), 4.54–4.34 (m, 2H), 4.08 (br.s, 2H), 3.69 (s, 3H), 3.67–3.58
(m, 1H), 3.41–3.26 (m, 1H), 3.24–2.98 (m, 2H), 2.74 (t, J = 7.5 Hz, 2H),
2.47 (t, J = 7.5 Hz, 2H); 13C NMR (75 MHz, DMSO‐d6) δ 173.8, 156.7
(d, J = 236.3 Hz), 156.6, 154.4 (d, J = 1.8 Hz), 142.4, 138.7, 135.6,
133.2, 131.5, 129.5, 129.3, 127.9, 116.0 (d, J = 1.4 Hz), 115.8 (d,
J = 16.6 Hz), 114.6, 107.3, 68.8, 63.8, 56.9, 48.1, 46.1, 35.8, 35.6, 29.5,
18.2. HRMS (ESI) m/z calcd for С31H33FN3O4 [M+H+] 530.2450 Da,
found 530.2446 0.0020 Da.
С
33H32F3N2O4 [M+H+] 577.2309 Da, found 577.2309 0.0015 Da.
|
4.1.20
Synthesis of 3‐[4‐({4‐[(1‐ethyl‐2‐oxo‐
1,5,7,8‐tetrahydro‐1,6‐naphthyridin‐6(2H)‐yl)methyl]‐
benzyl}oxy)phenyl]propanoic acid hydrochloride (16f)
It was synthesized analogously to 16a using 9b (0.14 mmol, 0.03 g),
triethylamine (0.29 mmol, 0.04 ml), tert‐butyl 3‐{4‐[(4‐formylbenzyl)oxy]
phenyl}propanoate (11, 0.145 mmol, 0.5 g), and STAB (0.585 mmol,
0.124).
Yield, 0.03 g (42%); white solid; m.p. 122–123°C. 1H NMR
(300 MHz, DMSO‐d6) δ 11.33 (br.s, 1H), 7.66 (d, J = 7.9 Hz, 2H), 7.54
(d, J = 8.0 Hz, 2H), 7.25 (d, J = 9.4 Hz, 1H), 7.14 (d, J = 8.5 Hz, 2H), 6.93
(d, J = 8.5 Hz, 2H), 6.34 (d, J = 9.3 Hz, 1H), 5.13 (s, 2H), 4.50–4.38 (m,
2H), 4.03 (br.s, 2H), 3.96 (q, J = 7.0 Hz, 2H), 3.71–3.62 (m, 1H),
3.37–3.24 (m, 1H), 3.22–3.09 (m, 2H), 2.77 (t, J = 7.5 Hz, 2H), 2.48 (t,
J = 7.5 Hz, 2H), 1.15 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, DMSO‐d6)
δ 173.8, 161.1, 156.6, 140.0, 138.8, 138.2, 133.2, 131.5, 129.3, 129.1,
128.0, 117.8, 114.6, 106.0, 79.2, 68.7, 57.3, 49.4, 47.2, 35.6, 29.5,
|
4.1.18
Synthesis of 3‐[4‐({4‐[(1‐methyl‐3‐{[(4‐
methylpyridin‐2‐yl)oxy]methyl}‐1,4,6,7‐tetrahydro‐
5H‐pyrazolo[4,3‐c]pyridin‐5‐yl)methyl]benzyl}oxy)‐
phenyl]propanoic acid hydrochloride (16d)
It was synthesized analogously to 16a using 8c (0.29 mmol, 0.07 g),
triethylamine (0.58 mmol, 0.08 ml), tert‐butyl 3‐{4‐[(4‐formylbenzyl)oxy]
phenyl}propanoate (11, 0.29 mmol, 0.1 g), and STAB (1.17 mmol, 0.248 g).
Yield, 0.06 g (34%); white solid; m.p. 63–64°C. 1H NMR (300 MHz,
DMSO‐d6) δ 11.74 (br.s, 1H), 7.71 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 7.8 Hz,
2H), 7.37 (d, J = 6.6 Hz, 1H), 7.14 (d, J = 8.3 Hz, 2H), 6.92 (d, J = 8.4 Hz,
2H), 6.28 (s, 1H), 6.18 (d, J = 6.6 Hz, 1H), 5.11 (s, 2H), 4.66 (d, J = 6.3 Hz,
2H), 4.49–4.36 (m, 2H), 4.21–4.05 (m, 2H), 3.70 (s, 3H), 3.68–3.61 (m,
1H), 3.39–3.22 (m, 1H), 3.14–3.02 (m, 2H), 2.75 (t, J = 7.4 Hz, 2H),
2.49–2.44 (m, 2H), 2.15 (s, 3H); 13C NMR (75 MHz, DMSO‐d6) δ 173.5,
156.5, 154.3, 147.0, 138.6, 134.9, 134.8, 133.1, 131.3, 129.3, 129.1,
127.7, 116.7, 114.6, 110.3, 106.4, 79.1, 68.7, 57.0, 48.1, 46.7, 35.4, 35.3,
29.4, 21.0, 18.1. HRMS (ESI) m/z calcd for С31H35N4O4 [M+H+]
527.2652 Da, found 527.2659 0.0020 Da.
23.1, 13.3. HRMS (ESI) m/z calcd for
С
27H31N2O4 [M+H+]
447.2278 Da, found 447.2274 0.0010 Da.
|
4.1.21
Synthesis of 3‐[4‐({4‐[(1‐benzyl‐2‐oxo‐
1,5,7,8‐tetrahydro‐1,6‐naphthyridin‐6(2H)‐yl)methyl]‐
benzyl}oxy)phenyl]propanoic acid
hydrochloride (16 g)
It was synthesized analogously to 16a using 9c (0.29 mmol, 0.08 g),
triethylamine (0.29 mmol, 0.08 ml), tert‐butyl 3‐{4‐[(4‐formylbenzyl)oxy]
phenyl}propanoate (11, 10; 0.29 mmol, 0.1 g), and STAB (1.17 mmol,
0.248 g). Yield, 0.03 g (20%); white solid; m.p. 106–107°C. 1H NMR

14 of 16
LUKIN ET AL.
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(300 MHz, DMSO‐d6) δ 11.98 (br.s, 1H), 7.68 (d, J = 7.7 Hz, 2H), 7.49 (d,
J = 7.7 Hz, 2H), 7.37–7.20 (m, 4H), 7.19–7.08 (m, 4H), 6.91 (d, J = 8.3 Hz,
2H), 6.45 (d, J = 9.3 Hz, 1H), 5.25 (br.s, 2H), 5.10 (s, 2H), 4.42 (br.s, 2H),
4.13–3.95 (m, 2H), 3.56 (br.s, 1H), 3.32–2.89 (m, 3H), 2.74 (t, J = 7.4 Hz,
2H), 2.48 (t, J = 7.4 Hz, 2H); 13C NMR (75 MHz, DMSO‐d6) δ 173.7,
161.6, 156.6, 140.4, 138.7, 138.7, 136.5, 133.2, 131.5, 129.2, 129.0,
128.6, 127.8, 127.1, 126.4, 117.9, 114.6, 106.6, 68.7, 57.2, 49.1, 46.6,
45.7, 35.6, 29.5, 23.5. HRMS (ESI) m/z calcd for С32H33N2O4 [M+H+]
509.2435 Da, found 509.2447 0.0020 Da.
4.1.24
Synthesis of 3‐{4‐[(4‐{[2‐[(4‐chlorobenzyl)‐
oxy]‐7,8‐dihydro‐1,6‐naphthyridin‐6(5H)‐yl]methyl}‐
benzyl)oxy]phenyl}propanoic acid hydrochloride (16j)
It was synthesized analogously to 16a using 10e (0.58 mmol,
0.091 g), triethylamine (1.09 mmol, 0.16 ml), tert‐butyl 3‐{4‐[(4‐
formylbenzyl)oxy]phenyl}propanoate (11, 0.58 mmol, 0.2 g), and
STAB (2.34 mmol, 0.496 g). Yield, 0.05 g (16%); white solid; m.p.
166–167°C. 1H NMR (300 MHz, DMSO‐d6) δ 11.33 (br.s, 1H), 7.68
(d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.6 Hz, 1H), 7.54 (d, J = 8.0 Hz, 2H), 7.45
(q, J = 8.6 Hz, 4H), 7.15 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 6.78
(d, J = 8.5 Hz, 1H), 5.32 (s, 2H), 5.11 (s, 2H), 4.53–4.40 (m, 2H), 4.24
(br.s, 2H), 3.49–3.33 (m, 2H), 3.31–3.17 (m, 1H), 3.07–2.90 (m, 1H),
2.75 (t, J = 7.5 Hz, 2H), 2.48 (t, J = 7.5 Hz, 2H); 13C NMR (75 MHz,
DMSO‐d6) δ 174.0, 161.9, 156.6, 148.8, 138.8, 138.5, 136.2, 133.2,
132.4, 131.5, 130.0, 129.3, 129.2, 128.4, 128.0, 117.2, 114.7, 109.4,
68.7, 66.2, 58.0, 50.4, 48.4, 35.6, 29.5, 28.1. HRMS (ESI) m/z calcd for
|
4.1.22
Synthesis 3‐{4‐[(4‐{[1‐(3‐fluorobenzyl)‐2‐
oxo‐1,5,7,8‐tetrahydro‐1,6‐naphthyridin‐6(2H)‐yl]‐
methyl}benzyl)oxy]phenyl}propanoic acid
hydrochloride (16h)
It was synthesized analogously to 16a using 9d (0.58 mmol, 0.15 g),
triethylamine (1.09 mmol, 0.16 ml), tert‐butyl 3‐{4‐[(4‐formylbenzyl)oxy]
phenyl}propanoate (11, 0.58 mmol, 0.2 g), and STAB (2.34 mmol,
0.496 g). Yield, 0.098 g (32%); white solid, m.p. 146–147°C. 1H NMR
(300 MHz, DMSO‐d6) δ 11.37 (br.s, 1H), 7.63 (d, J = 7.8 Hz, 2H), 7.52 (d,
J = 7.8 Hz, 2H), 7.43–7.32 (m, 2H), 7.19–7.05 (m, 3H), 7.03–6.84 (m, 4H),
6.47 (d, J = 9.3 Hz, 1H), 5.43–5.14 (m, 2H), 5.10 (s, 2H), 4.41 (br.s, 2H),
4.04 (br.s, 2H), 3.67–3.53 (m, 1H), 3.31–3.17 (m, 1H), 3.03 (br.s, 2H),
2.74 (t, J = 7.3 Hz, 2H), 2.47 (t, J = 7.3 Hz, 2H); 13C NMR (75 MHz,
DMSO‐d6) δ 173.6, 162.2 (d, J = 243.8 Hz), 161.5, 156.5, 140.3, 139.4 (d,
J = 7.2 Hz), 138.7, 138.6, 133.1, 131.34, 130.6 (d, J = 8.2 Hz), 129.1,
128.9, 127.8, 122.4 (d, J = 1.8 Hz), 117.9, 114.5, 113.9 (d, J = 20.8 Hz),
113.3 (d, J = 22.1 Hz), 106.6, 68.6, 64.8, 57.3, 49.1, 46.6, 35.5, 29.49,
23.4. HRMS (ESI) m/z calcd for С32H32FN2O4 [M+H+] 527.2341 Da,
found 527.2352 0.0015 Da.
С
32H32ClN2O4 [M+H+] 543.2045 Da, found 543.2063 0.0015 Da.
|
4.1.25
Synthesis of 3‐{4‐[(4‐{[2‐[(4‐fluorobenzyl)‐
oxy]‐7,8‐dihydro‐1,6‐naphthyridin‐6(5H)‐yl]methyl}‐
benzyl)oxy]phenyl}propanoic acid hydrochloride (16k)
It was synthesized analogously to 16a using 10f (0.58 mmol, 0.091 g),
triethylamine (1.09 mmol, 0.16 ml), tert‐butyl 3‐{4‐[(4‐formylbenzyl)
oxy]phenyl}propanoate (11, 0.58 mmol, 0.2 g), and STAB (2.34 mmol,
0.496 g). Yield, 0.09 g (27%); white solid; m.p. 220–221°C. 1H NMR
(300 MHz, DMSO‐d6)
δ 10.74 (br.s, 1H), 7.66–7.60 (m, 2H),
7.59–7.46 (m, 5H), 7.21 (d, J = 8.9 Hz, 2H), 7.18–7.09 (m, 2H), 6.92 (d,
J = 8.7 Hz, 2H), 6.78 (d, J = 8.5 Hz, 1H), 5.30 (s, 2H), 5.12 (s, 2H), 4.48
(br.s, 2H), 4.26 (br.s, 2H), 3.76–3.59 (m, 1H), 3.25–3.12 (m, 1H),
3.08–2.95 (m, 2H), 2.75 (t, J = 7.5 Hz, 2H), 2.48 (t, J = 7.5 Hz, 2H); 13
C
|
4.1.23
Synthesis of 3‐{4‐[(4‐{[2‐{[4‐
NMR (75 MHz, DMSO‐d6) δ 173.8, 162.0, 161.7 (d, J = 243.5 Hz),
156.6, 148.7, 138.8, 138.4, 133.3 (d, J = 7.2 Hz), 131.4, 130.3 (d,
J = 8.3 Hz), 129.3, 129.2, 129.1, 127.9, 117.0, 115.1 (d, J = 21.3 Hz),
114.6, 109.4, 68.7, 66.3, 57.9, 50.4, 48.4, 35.5, 29.5, 28.0. HRMS (ESI)
(trifluoromethyl)benzyl]oxy}‐7,8‐dihydro‐1,6‐
naphthyridin‐6(5H)‐yl]methyl}benzyl)oxy]phenyl}‐
propanoic acid hydrochloride (16i)
m/z calcd for
С
32H32FN2O4 [M+H+] 527.2341 Da, found
It was synthesized analogously to 16a using 10a (0.29 mmol,
0.101 g), triethylamine (0.58 mmol, 0.08 ml), tert‐butyl 3‐{4‐[(4‐
formylbenzyl)oxy]phenyl}propanoate (11, 0.29 mmol, 0.1 g), and
STAB (1.17 mmol, 0.248 g). Yield, 0.1 g (56%); white solid; m.p.
128–129°C. 1H NMR (300 MHz, DMSO‐d6) δ 11.56 (br.s, 1H),
7.77–7.64 (m, 6H), 7.61 (d, J = 8.6 Hz, 1H), 7.54 (d, J = 8.0 Hz, 2H),
7.14 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 6.82 (d, J = 8.5 Hz, 1H),
5.43 (s, 2H), 5.12 (s, 2H), 4.51–4.42 (m, 2H), 4.23 (br.s, 2H), 3.73–3.62
(m, 1H), 3.46–3.31 (m, 1H), 3.30–3.20 (m, 1H), 3.04–2.90 (m, 1H),
2.75 (t, J = 7.4 Hz, 2H), 2.49 (t, J = 7.4 Hz, 2H); 13C NMR (75 MHz,
DMSO‐d6) δ 173.9, 161.8, 156.6, 148.9, 138.8, 138.6, 133.2, 131.6,
129.3, 129.2, 128.4, 128.3 (q, J = 31.8 Hz), 127.5, 125.3 (q, J = 3.7 Hz),
124.3 (q, J = 272.1 Hz), 117.3, 114.6, 109.4, 68.7, 66.1, 57.9, 50.3,
48.3, 35.6, 29.5, 28.1. HRMS (ESI) m/z calcd for С33H32F3N2O4
[M+H+] 577.2309 Da, found 577.2331 0.0020 Da.
527.2363 0.0020 Da.
|
4.1.26
Synthesis of 3‐{4‐[(4‐{[2‐(2‐
methoxyethoxy)‐7,8‐dihydro‐1,6‐naphthyridin‐6(5H)‐
yl]methyl}benzyl)oxy]phenyl}propanoic acid
hydrochloride (16l)
It was synthesized analogously to 16a using 10g (0.29 mmol, 0.071 g),
triethylamine (0.58 mmol, 0.08 ml), tert‐butyl 3‐{4‐[(4‐formylbenzyl)oxy]
phenyl}propanoate (11, 0.29 mmol, 0.1 g), and STAB (1.17 mmol,
0.248 g). Yield, 0.02 g (14%); white solid; m.p. 89–90°C. 1H NMR
(300 MHz, DMSO) δ 11.60 (br.s, 1H), 7.70 (d, J = 7.5 Hz, 2H), 7.60–7.48
(m, 3H), 7.14 (d, J = 7.9 Hz, 2H), 6.93 (d, J = 8.1 Hz, 2H), 6.71 (d,
J = 8.3 Hz, 1H), 5.11 (s, 2H), 4.45 (br.s, 2H), 4.35 (br.s, 2H), 4.22 (br.s,

LUKIN ET AL.
15 of 16
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2H), 3.64 (s, 3H), 3.42–3.31 (m, 2H), 3.28 (s, 3H), 3.04–2.91 (m, 1H),
2.75 (t, J = 6.9 Hz, 2H), 2.48 (t, J = 6.9 Hz, 2H); 13C NMR (75 MHz,
DMSO) δ 174.0, 162.3, 156.7, 148.8, 138.8, 138.4, 133.3, 131.6, 129.4,
129.3, 128.1, 116.8, 114.7, 109.4, 70.2, 68.8, 64.8, 58.2, 57.9, 50.4, 48.4,
35.7, 29.6, 28.1. HRMS (ESI) m/z calcd for С28H33N2O5 [M+H+]
477.2384 Da, found 477.2401 0.0010 Da.
an autosampler (Shimadzu SIL‐HTc), a switching valve (FCV‐14AH),
and a degasser (Shimadzu DGU‐14A). The mass spectrometric analysis
was performed using API 3000 (triple‐quadrupole) instrument from PE
Sciex with electrospray interface. The data acquisition and system con-
trol were performed using Analyst 1.5.2 software from PE Sciex.
The formula for calculating Papp (expressed in 10−6 cm·s−1) is as
follows:
Papp = (VA/((Area) × (Time)) × ([drug]acc/[drug]init,d),
|
4.2
Biological assays
|
4.2.1
In vitro FFA1 activation assay
where VA is the volume of transport buffer in the acceptor well;
Area, the surface area of the insert (equal to effective growth area of
the insert); Time, the time of the assay; [drug]acc, the concentration
of the test compound in the acceptor well; and [drug]init,d, the initial
concentration of the test compound in the donor well.
CHO cells stably expressing human FFA1 (stable CHO‐GPR40 line
created at Enamine Ltd.) were seeded (12,500 cells/well) into 384‐well
black‐wall, clear‐bottom microtiter plates 24 h before assay. Cells were
loaded for 1 h with fluorescent calcium dye (Fluo‐8 Calcium Assay Kit,
ab112129; Abcam) and tested using a fluorometric imaging plate reader
(FLIPR Tetra® High‐Throughput Cellular Screening System; Molecular
Devices Corp.). The maximum change in fluorescence over the baseline
was used to determine the agonist response. A potent and selective
agonist for FFA1 GW9508 (S8014; Selleckchem) was tested with
the test compounds as a positive control. The concentration–response
curve data were fitted using Molecular Devices ScreenWorks® System
Control Software (Molecular Devices).
|
4.2.3
Insulin secretion assay in INS1E cells
Cell culture
Rat insulinoma (clonal INS‐1E cells isolated from the parental INS‐1
line, Cat. C0018009; AddexBio) was cultured in RPMI‐1640 medium
containing 11 mM glucose, supplemented with 10% fetal bovine
serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 mM
HEPES, 1 mM sodium pyruvate, and 50 μM β‐mercaptoethanol. Cells
were maintained in the logarithmic growth phase at 37°C in a hu-
midified atmosphere containing 5% CO2.
|
4.2.2
Caco‐2 permeability assay
Caco‐2 cells (human colorectal adenocarcinoma line, Cat. HTB‐37; ATCC)
were cultivated in a Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum (FBS), 1% nonessential amino acids solution,
and 0.1% penicillin/streptomycin in a humidified atmosphere at 37°C in
5% CO2 to 70–80% confluence, which were then seeded at 1 × 105 cells/
well on 24‐well semipermeable insert plates (Millicell Multiwell PCF
0.4 µm or similar). The medium was changed every 2 days. After 10 days
of cell growth, the integrity of differentiated Caco‐2 monolayers was
verified by transepithelial electrical resistance (TEER) measurements
using Millicell‐ERS Voltohmmeter (Millipore EMD). Caco‐2 cell mono-
layers were considered acceptable for transport studies if the final values
of TEER were greater than 1000 ohm·cm−2. For the permeability studies,
the 24‐well insert plate was removed from its feeder plate and placed in
a new sterile 24‐well receiver plate. The cell layer was washed twice with
phosphate‐buffered saline. Aliquots (300 µl) of the test compound solu-
tion (in duplicates, at 10 µM, in Hanks' balanced salt solution with
5.6 mM glucose buffered with 10 mM HEPES, pH 7.4) were added into
the apical compartments of the trans‐well insert and 1000 µl of the same
buffer was added to the basolateral compartments. The plates were then
incubated for 2 h at 37°C. High, low, and intermediate permeability
controls (atenolol, propranolol, quinidine) were run with every experi-
mental batch to verify assay validity. The concentrations of the com-
pounds tested in the A–B permeability assay were determined using the
HPLC‐MS method. The LC system comprised a Shimadzu liquid chro-
matograph equipped with isocratic pumps (Shimadzu LC‐10ADvp),
Insulin secretion assay
INS1E cells were seeded in 96‐well plates at the density of 5 × 104 cells/
well and cultured at 37°C and 5% CO2 for 72 h. The cell medium was
replaced with glucose‐free RPMI 1640 containing 1% FBS for 1 h. Next,
cells were washed with Krebs–Ringer bicarbonate HEPES buffer
(KRBH; 129 mmol/l NaCl, 4.8 mmol/l KCl, 2.5 mmol/l CaCl2, 1.2 mmol/l
MgSO4, 1.2 mmol/l KH2PO4, 5 mmol/l NaHCO3, and 10 mmol/l HEPES,
pH 7.4) containing 0.1% BSA (fatty acid‐free) with 0.5 mM glucose and
then stimulated with a test or reference compounds in KRBH with 0.1%
BSA for 2 h in the presence of 0.5 or 16.7 mM glucose. DMSO was used
as a negative control and GW9508 (10 μM) as a positive control (final
DMSO concentration, 0.5%). The test compounds were added at 10 μM
concentration. Supernatants (100 μl) were harvested from all wells to
determine insulin levels.
The secreted insulin in the cell culture medium of INS1E cells was
measured using the HTRF Assay Kit (Cat. 62IN1PEG; Cisbio Bioassays)
according to manufacturer's recommendations. Briefly, 5 μl of the test or
control sample was added into the respective wells of a black 384‐well
microplate and supplemented with 25 μl of anti‐insulin‐Eu3+ Cryptate
donor antibody and 50 μl of anti‐insulin‐XL665 antibody. The plate was
incubated overnight at room temperature and the HTRF signal was read
with the excitation at 317 nm wavelength and emissions at 620 nm (for
donor) and 665 nm (for acceptor). The results were expressed as the
ratio of 665 nm/620 nm. For insulin quantification, a calibration curve
was constructed using the insulin standard included in the kit.

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4.3
Docking studies
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ACKNOWLEDGMENTS
This study was supported by the Russian Foundation for Basic
Research (project grant 19‐33‐90169). Alexander Safrygin is thank-
ful for the postdoctoral fellowship from Saint Petersburg State
University. The authors are grateful to the Center for Chemical
Analysis and Materials Research of Saint Petersburg State University
for providing high‐resolution mass spectrometry data.
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CONFLICTS OF INTERESTS
[20] A. C.‐M., Daugan, PCT Int. Pat. Appl. WO 2009056556; Chem. Abstr.
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The authors declare that there are no conflicts of interests.
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ORCID
Sergey Zozulya
Maxim Gureev
http://orcid.org/0000-0002-1179-2993
http://orcid.org/0000-0002-0385-922X
http://orcid.org/0000-0002-2436-9019
http://orcid.org/0000-0002-0200-4772
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Alexander Safrygin
Mikhail Krasavin
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F. Saudek, P. Ježeka, Int. J. Biochem. Cell Biol. 2008, 40, 1522.
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https://doi.org/10.1002/ardp.202000275