Synthesis and evaluation of (E)-2-(5-phenylpent-2-en-4-ynamido)cyclohex-1-ene-1-carboxylate derivatives as HCA2 receptor agonists

Article abstract of DOI:10.1016/j.bmc.2017.06.028

Novel series of compounds consisting of 2-amidocyclohex-1-ene carboxylate and phenyl parts which are connected by enyne (compounds 2a–f), but-1-yne (compounds 4a–j), and phenylethylene (compounds 5a–f) linkers as HCA2 full agonists were designed and their

Full text of DOI:10.1016/j.bmc.2017.06.028

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of (E)-2-(5-phenylpent-2-en-4-
ynamido)cyclohex-1-ene-1-carboxylate derivatives as HCA2 receptor
agonists
Olga Bobileva a, Martins Ikaunieks a, Gunars Duburs a, Ilona Mandrika b,
Ramona Petrovska b, Janis Klovins b, Einars Loza a,
⇑
a Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV-1006, Latvia
b Latvian Biomedical Research and Study Centre, Ratsupites 1, Riga LV-1067, Latvia
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel series of compounds consisting of 2-amidocyclohex-1-ene carboxylate and phenyl parts which are
connected by enyne (compounds 2a–f), but-1-yne (compounds 4a–j), and phenylethylene (compounds
5a–f) linkers as HCA2 full agonists were designed and their functional activity using cAMP assay and
binding affinity using radioligand (3H-niacin) binding assay were evaluated. In general, compounds of
all three series exhibit similar HCA2 binding and activation profile. However, the activity is strongly
dependent on the substituent at the aromatic part of the structure. Among the structures evaluated,
the highest affinity and potency in all series were exhibited by compounds containing 4-hydroxy and/
or 2-chloro or 2-fluoro substituents. The most active compounds in the enyne and but-1-yne series in
the cAMP assay are 2-fluoro,4-hydroxy and 2-chloro,4-hydroxy phenyl derivatives 2f, 4f, and 4g showing
potency similar to the previously described 4-hydroxy-biphenyl analogue 5c.
Received 26 April 2017
Revised 5 June 2017
Accepted 9 June 2017
Available online xxxx
Keywords:
HCA2
Niacin receptor
CAMP
Radioligand binding
Agonist
Ó 2017 Elsevier Ltd. All rights reserved.
2-Amidocyclohex-1-ene carboxylate
1. Introduction
HCA2 receptor and the clinical trial of HCA2 agonists MK-19033
and SCH9002714 (Fig. 1) demonstrated that the niacin antilipolytic
Hydroxycarboxylic acid receptor 2 (HCA2) is a G-protein cou-
pled receptor, highly expressed in adipocytes, immune cells such
as macrophages, monocytes, neutrophils, and epidermal cells.
The endogenous ligand of HCA2 is hydroxylated short chain fatty
acid 3-hydroxybutyrate, which is the product of metabolic b-oxi-
dation process in liver.1
The most well known HCA2 agonist is niacin, which is used in
the treatment of lipid disorders since the 1950s. It was believed
that antidyslipidemic properties of niacin were based on HCA2
activation in adipocytes with subsequent decrease of the plasma
levels of low-density lipoproteins (LDL) and increase of high-den-
sity lipoproteins (HDL).2 However, the study on mice lacking the
effect is independent of the HCA2 activation.5 HCA2 mediates the
decrease of the levels of free fatty acids in plasma, but has no effect
on the decrease of LDL levels or increase of the HDL.
Atherosclerosis is chronic inflammatory disease in different
stages of which macrophages and monocytes are involved.6 Using
the mouse model of atherosclerosis, it is shown that niacin inhibits
disease progression through the HCA2 expressed by macrophages
without an effect on HDL cholesterol plasma level.7 The study on
human monocytes showed that niacin and HCA2 agonist acipimox
(Fig. 1) have anti-inflammatory activity, modulating function of
immune cells that leads to antiatherogenic effect.8
While HCA2 agonists were mostly studied for the treatment of
dyslipidemia and atherosclerosis, the beneficial effect of HCA2 ago-
nists are found in some other conditions related to inflammation.
For example, HCA2 agonists dimethyl fumarate and monomethyl
fumarate are used in the treatment of psoriasis.9 Dimethyl fuma-
rate also showed protective effect in the mouse model of autoim-
mune disease multiple sclerosis with data suggesting that
dimethyl fumarate acted through HCA2.10 The HCA2 activation
by endogenous and exogenous ligands has also been associated
Abbreviations: LDL, low-density lipoprotein; HDL, high-density lipoprotein;
cAMP, 30-50-cyclic adenosine monophosphate; SAR, structure-activity relationship;
RLB, radioligand binding; GPCR, G-protein coupled receptor; BSA, bovine serum
albumin; DCM, dichloromethane; TEA, triethylamine; DIPEA, diisopropylethy-
lamine; TBDMS, tert-butyldimethylsilyl; Pd(dppf)Cl2, [1,10-bis(diphenylphosphino)-
ferrocene]dichloropalladium(II).
⇑
Corresponding author.
E-mail address: einars@osi.lv (E. Loza).
http://dx.doi.org/10.1016/j.bmc.2017.06.028
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 1. Structures of selected HCA2 receptor agonists.
with anti-inflammatory effects in other diseases. In particular,
studies report that niacin has the ability to reduce inflammation
in sepsis, diabetic retinopathy, and renal disease.11 These data sug-
gest that HCA2 may be an important target for treating inflamma-
tory processes, thus the design of new potent and selective HCA2
ligands may be important for determining other HCA2 functions
as well as for the development of new drugs.
Merck chemists developed cyclohex-1-ene carboxylic acid
derivatives I (Fig. 2) as potent and selective high affinity HCA2 full
agonists.12–14 They have shown that Ar in the molecules I preferen-
tially should be aromatic bicycles or other higher fused systems
which are beneficial for affinity.15
In our previous work we described the studies of cyclohex-1-
ene carboxylic acid derivatives as HCA2 ligands and demonstrated
that a change of a saturated ethylene linker to such rigidity ele-
ments as E-double bond, triple bond, or trans-substituted cyclo-
propane ring into the amide part of appropriate aryl substituted
2-amidocyclohex-1-enes II allowed modulating the potency and
selectivity of these compounds towards the HCA2 receptor activa-
tion.16 During these studies we confirmed that a presence of such
aromatic bicycles as naphthalene and quinoline in molecules II is a
prerequisite to construct active compounds as well. However, we
found that simple (E)-2-acrylamidocyclohex-1-enecarboxylic acid
derivative 1 containing a phenylethynyl group attached to the E
double bond exhibits comparable activation of HCA2 with aromatic
bicycle based compounds (Fig. 2).
In this paper, we report the synthesis and SAR of a series of novel
cyclohex-1-ene carboxylic acid derivatives 2a–f, 3, 4a–h, and 5a–f
(Table 1) whose structure design is based on hit structure 1. All
compounds were evaluated in a functional assay and a binding
assay. In these assays, Flp-In-293 cells, which express the recombi-
nant human HCA2, were utilized. For the functional assay, forsko-
lin-stimulated cAMP accumulation assay (cAMP) was arranged.
For the radioligand binding assay (RLB), displacement of radiola-
beled niacin from the membrane preparation was measured.
Almost all active analogues described in this paper had a maximum
level of response Emax to niacin at least 90% in the cAMP assay and
could be, therefore, characterized as full agonists (Table 1).
Arylhalides 6a–f in the Sonogashira reaction with propargyl
alcohol gave 3-phenylprop-2-yn-1-ols 7a–f which were oxidized
to aldehydes 8a–f with MnO2 or Dess-Martin periodinane. The
aldehydes 8a–f were utilized in the subsequent Wittig reaction
with phosphorane, generated from previously described triph-
enylphosphonium bromide 9,16 to give E-alkenes 10a–f contami-
nated with a minor amount of the corresponding Z-isomer. It is
necessary to mention that the employed triphenylphosphonium
bromide ethyl ester 9 contained some amount (ca 5%) of the
related methyl ester impurity unavoidably coming from the syn-
thesis method of the compound 9. Compounds 10a–c, f were con-
verted into free phenols 11a–c, f and the esters 10d, e and 11a–c, f
were hydrolyzed into desired carboxylic acids 2a–f.
The synthesis of compound 3 with the but-1-ene linker was
performed as follows (Scheme 2). 3-Phenylpropanal (12) was sub-
jected to the Wadsworth-Emmons reaction with methyl 2-
(dimethoxyphosphoryl)acetate followed by the hydrolysis provid-
ing (E)-5-phenylpent-2-enoic acid (13).17 The acid 13 was used for
the preparation of the corresponding acid chloride which in turn
was condensed with ethyl 2-aminocyclohex-1-ene-1-carboxylate
to give ethyl ester of the acid 13. The hydrolysis of the latter gave
the desired compound 3.
To obtain the necessary core of compounds 4a–j containing the
but-1-yne linker, we tried a strategy consisting of the synthesis of
appropriate 5-arylpent-4-ynoic acid which was employed to acy-
late ethyl 2-aminocyclohex-1-ene-1-carboxylate. Thus, following
this approach the Sonogashira reaction of 1-chloro-2-iodobenzene
with pent-4-ynoic acid (15) afforded 5-(2-chlorophenyl)pent-4-
ynoic acid (16) (Scheme 3). The acid 16 was converted into the cor-
responding acid chloride which in turn was treated with ethyl 2-
aminocyclohex-1-ene-1-carboxylate to give desired amidoester
17d. Unfortunately, the yield of the acid 16 was low due to a com-
peting aryl-c-methylene-c
-butyrolactone formation18 and difficul-
ties in the isolation of the pure product. All attempts to increase
the yield of the acid 16 by changing catalysts, bases, and/or sol-
vents failed. However, a prior formation of the propynyl ami-
doester 18 by acylation of ethyl 2-aminocyclohex-1-ene-1-
carboxylate with the acid 15 chloride eliminated the above men-
tioned drawbacks connected with the preparation of 5-arylpent-
4-ynoic acid 16. The Sonogashira reaction of the amidoester 18
and appropriate aryl iodides proceeded as expected allowing effi-
cient preparation of desired amidoesters 17a–c, e–j. The hydrolysis
of the amidoesters 17a–j finished the preparation of the corre-
sponding target compounds 4a–j (Scheme 3).
2. Synthesis
Compounds 2a–f containing the enyne linker were synthesized
using the Sonogashira reaction
(Scheme 1).
– Wittig reaction sequence
Fig. 2. Structures of known HCA2 receptor agonists I, II, and 1.
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O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Table 1
Affinity and potency of the synthesized compounds in the HCA2 receptor RLB and cAMP assays.
En-try
1
No.
R
RLB IC50 SD, nMa
327 71
cAMP EC50 SD, nMa
1010 590
Emax (%)b
1
79
4
2
2a
37 11
250 110
78
6
3
4
2b
2c
383 68
390 160
130 20
92
97
3
3
25
4
5
6
7
2d
2e
2f
88 24
270 140
450 150
110 60
88
84
96
6
6
5
16
1
331 81
8
9
3
2828 927
229 89
19600 3100
720 100
70
93
3
5
4a
10
11
4b
4c
138
13
9
2980 490
160 60
89
91
7
6
6
12
13
4d
4e
56 17
190 80
36 12
95
90
4
4
67
9
(continued on next page)
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O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1 (continued)
En-try
14
No.
R
RLB IC50 SD, nMa
cAMP EC50 SD, nMa
80 10
Emax (%)b
4f
28
2
93
94
91
74
99
90
4
7
6
5
4
8
15
16
17
18
19
4g
4h
4i
76 19
9
2
665 13
3620 542
291 78
3370 1390
5610 1100
630 90
4j
5a
40
5
410 140
20
21
5b
5c
32
21
8
4
230 120
96
92
2
5
70 10
22
23
24
5d
5e
5f
75
7
130 60
190 40
260 90
92
97
99
7
2
3
282 49
58
9
a
Each value represents the mean SD calculated from at least three experiments, each performed in duplicate.
Maximal inhibition (%) of forskolin-stimulated cAMP.
b
A series of compounds 5a–f containing phenylethyl linker was
bond of biphenyl acid 21. After the amide bond formation and
basic hydrolysis the product 5d was obtained. To minimize the
number of the required synthetic steps, a modified synthetic
route20 was used for the synthesis of other biphenyl amidoesters
22a–c, e–f. At first, ethyl 2-aminocyclohex-1-ene-1-carboxylate
was condensed with the acid 19 chloride to give aryliodide 23.
The iodide 23 was coupled with appropriate aryl pinacolyl boro-
synthesized from 3-(4-iodophenyl)propanoic acid19 (19) by two
similar protocols differing by the sequence of the employed chem-
ical conversions (Scheme 4). According to the first approach, the
iododerivative 19 was converted into boronate 20 using the
Miyaura borylation reaction which was coupled with 1-chloro-2-
iodobenzene by the Suzuki reaction forming the necessary CAC
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O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Scheme 1. Synthesis of compounds 2a–f. Conditions: (a) propargyl alcohol, Pd(PPh3)4 (3 mol%), CuI (6 mol%), TEA, DMF, rt or 80 °C, 15–18 h; (b) Dess-Martin periodinane,
DCM, rt, 2 h; (c) MnO2, DCM, rt, 48–72 h; (d) phosphonium bromide 9, t-BuOK, DMSO, rt, 1 h; (e) TBAF, THF, rt, 2 h; (f) 6 mol% pTSA, DCM, MeOH, rt, 6 h; (g) 2 N NaOH, THF/
MeOH, 6–18 h.
Scheme 2. Synthesis of compound 3. Conditions: (a) methyl 2-(dimethoxyphosphoryl)-acetate, t-BuOK, DMSO, rt, 1 h; (b) NaOH, MeOH, H2O, rt; (c) oxalyl chloride, DMF
(cat.), DCM, rt, 1 h; (d) ethyl 2-aminocyclohex-1-ene-1-carboxylate, TEA, DCM, 0 °C, 1 h; (e) 2 N NaOH, THF/MeOH.
Scheme 3. Synthesis of compounds 4a–j. Conditions: (a) Pd(PPh3)4 (3 mol%), CuI (6 mol%), TEA, DMF, rt, 18 h; (b) oxalyl chloride, DMF, DCM, rt, 1 h; (c) ethyl 2-
aminocyclohex-1-ene-1-carboxylate, TEA, DCM, 0 °C, 1 h; (d) appropriate aryl iodide, PdCl2(PPh3)2 (5 mol%), CuI (5 mol%), TEA, MeCN, 2–18 h, rt; (e) 2 N NaOH, EtOH or THF/
MeOH, rt, 15–24 h.
nates or boronic acids by the Suzuki protocol to give amidoesters
22a–c, e–f which were converted into desired target compounds
5a–c, e–f by the basic hydrolysis (Scheme 4).
The
non-substituted
compound
1
exhibited
EC50
3600 1600 nM in the cAMP assay in the presence of 2% bovine
serum albumin (BSA) as it was determined earlier.16 Albumin is a
transport protein in plasma and is known to bind drugs, especially
lipophilic molecules and carboxylic acids.21 Since our target com-
pounds were carboxylic acids, we were interested to establish
the activity of the compound 1 without the presence of BSA, which
could interfere with the assessment of EC50. Indeed, the cAMP
assay for the compound 1 without the BSA afforded a 3-fold lower
EC50 value equal to 1010 590 nM (Table 1, entry 1). Thus, in the
following experiments the binding and functional assays were per-
formed without the BSA additive.
3. Biological activity and structure-activity relationships (SAR)
The pharmacophoric description of compound 1 and analogues,
synthesized in this paper, can be represented as consisting of
cyclohexene and aromatic parts which are connected by more or
less flexible linker. In the case of the compound 1, the linker con-
tains a combination of the triple and double bonds – enyne linker,
which is completely planar and conformationally stable. The
restriction of the conformation of the molecule could allow evalu-
ation of an appropriate conformation of the ligand for binding with
the receptor.
To test the possibility to increase the activity of the compound 1
by adding substituents at the phenyl ring of the molecule, a series
of analogues 2a–f was prepared.
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O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Scheme 4. Synthesis of compounds 5a–f. Conditions: (a) bis(pinacolato)diboron, Pd(dppf)Cl2 (5 mol%), KOAc, DMF, 80 °C, 2.5 h; (b) 1-chloro-2-iodobenzene, Pd(dppf)Cl2
(5 mol%), K2CO3, dioxane/H2O, 80 °C, 2 h; (c) oxalyl chloride, DMF (cat.), DCM, rt, 1 h; (d) ethyl 2-aminocyclohex-1-ene-1-carboxylate, TEA, DCM, 0 °C, 1 h; (e) appropriate
arylboronic acid or arylboronic acid pinacolate ester, Pd(dppf)Cl2 (5 mol%), K2CO3, dioxane/H2O, 80 °C, 1–4 h; (f) 2 N NaOH, THF/MeOH, rt, 16–18 h.
In general, the introduction of a hydroxyl group or halide (F or Cl)
in the aromatic part of the molecule improved activity both in the
RLB and cAMP assays. The SAR studies by RLB method revealed that
affinities of the analogues 2a–f towards HCA2 decreased in the fol-
lowing order [substitution pattern (compound number, IC50)]: 2-F
(2e, 16 nM) > 4-OH (2c, 25 nM) > 2-OH (2a, 37 nM) > 2-Cl (2d,
88 nM) > H (1, 327 nM) ꢀ 2-F, 4-OH (2f, 331 nM) ꢀ 3-OH (2b,
383 nM).
(4f, 28 nM) > 2-Cl (4d, 56 nM) > 2-F (4e, 67 nM) > 2-Cl, 4-OH (4g,
76 nM) ꢀ 3-OH (4b, 138 nM) ꢀ 2-OH (4a, 229 nM) ꢀ 4-F (4j,
291 nM) > 4-OH, N(2) (4h, 665 nM) > 4-COOH (4i, 3620 nM).
The highest potencies in this series in the functional assay was
exhibited by a group of compounds 4c, 4f, 4g containing 4-hydroxy
group at the benzene ring. In this case, the presence of an addi-
tional 2-fluoro or 2-chloro atom at the benzene ring for the struc-
tures 4f and 4g, accordingly, seems to be beneficial for the potency
(4f, R = 2-F, 4-OH, EC50 = 80 10 nM; 4g, R = 2-Cl, 4-OH,
EC50 = 90 20 nM) if compared with the monosubstituted 4-
hydroxy derivative 4c (EC50 = 160 60 nM). 2-Chloro and 2-fluoro
monosubstituted derivatives 4d and 4e exhibited remarkable
activities both in the RLB and cAMP assays with a tendency of pos-
sessing slightly reduced potency (EC50 = 190 80 nM and
360 120 nM, accordingly) in functional assay if compared with
the 4-hydroxy substituted derivatives 4c, 4g, 4h, nevertheless
being superior to the 2-hydroxy analogue 4a. It is noteworthy that
4-fluorobenzene 4j exhibited distinctly lower activity in binding
and functional assays than the corresponding 4-hydroxy analogue
4c. Despite quite notable affinity of 3-hydroxy derivative 4b to
HCA2 receptor (IC50 = 138 9 nM), the potency of this compound
was only in a low micromolar range (EC50 = 2980 490 nM). Quite
surprisingly, compound 4h with 4-hydroxy-2-pyridine in the aro-
matic part of the molecule, the analogue of the previously
described Merck HCA2 agonist MK-689213, has lost the activity
comparing to the 4-hydroxybenzene 4c. 4-Carboxybenzene 4i
exhibited only weak activity both in RLB and cAMP tests. In addi-
tion, it seems to be a partial agonist of HCA2 (Emax = 74 5%).
IJzerman et.al. recently described novel 4-hydroxybiphenyl
cyclohexene derivative 5c which showed remarkable affinity
(IC50 = 108 4 nM) towards HCA2 receptor and longer residence
time profile than niacin.20 Since the 4-hydroxyphenyl derivatives
2c and 4c of the series 2a–f and 4a–j were among the most active
compounds both in the binding and functional assays, we decided
to extend our study so that to cover biphenyl derivatives 5a–f
which formally can be regarded as consisting of the cyclohexene
and benzene parts joined by a phenylethyl linker. The model calcu-
lations of the superimposed structures 2c, 4c, and 5c revealed that
the biphenyl derivative 5c is about 2Å longer than the analogues 2c
and 4c. The affinities of the compounds 5a–f towards HCA2 recep-
tor in the binding assay decreased in the following order [substitu-
tion pattern (compound number, IC50]: 4-OH (5c, 21 nM) > 3-OH
(5b, 32 nM) > 2-OH (5a, 40 nM) > 2-F, 4-OH (5f, 58 nM) > 2-Cl
(5d, 75 nM) ꢀ 2-F (5e, 282 nM). Thus, the higher affinity in the ser-
ies 5a–f was exhibited by 4-hydroxy, 3-hydroxy, and 2-hydroxy
However, in the cAMP assay 2-fluoro-4-hydroxy derivative 2f
exhibited the highest potency (EC50 = 110 60 nM), showing ca
9-fold increase of the activity compared to the parent substance
1 (EC50 = 1010 590 nM). Similar potencies of 8 to 4-fold increase
in the cAMP assay were shown by 4-hydroxy derivative 2c
(EC50 = 130 20 nM), 2-hydroxy derivative 2a (EC50 = 250 110 -
nM), and 2-chloro analogue 2d (EC50 = 27 140 nM). 3-Hydroxy
and 2-fluoro analogues 2b and 2e exhibited slightly lower poten-
cies (EC50 = 390 160 nM and 450 150 nM, accordingly) than
the above mentioned compounds 2a, 2c, 2d, and 2f, however, still
being 2–3-fold more active than the parent compound 1. It is inter-
esting to note, that the 2-fluoro, 4-hydroxy analogue 2f having the
highest cAMP activity exhibited quite modest affinity to HCA2 in
the RLB assay comparable with the affinity of the compound 1.
On the contrary, the 2-fluoro analogue 2e with 20-fold higher
RLB affinity than the unsubstituted structure 1 did not exhibit such
remarkable improvement in the potency in cAMP assay
(EC50 = 450 150 nM for 2e versus EC50 = 1010 590 nM for 1).
To establish which bond (double or triple) of the enyne linker is
more important for the activity of compounds of the series 2a–f, we
synthesized derivative 3 containing the double bond in a combina-
tion with a fully saturated triple bond (i.e., the ethylene group) in
the linker part of the molecule. This modification resulted in a sig-
nificant loss of the binding and functional activity comparing to
the parent structure 1 (Table 1, entries 1 and 8). We assumed that
the conformationally restricted linker part with the triple bond
flanked by the ethylene group and the benzene ring is apparently
essential for the activity of the compound 1 and likely for the
analogues 2a–f as well.
The saturation of the double bond of the enyne linker leaving the
triple bond intact led to a series of compounds 4a–j being on the
average just as active as the series 2a–f with the fully unsaturated
and rigid enyne linker. Since the compounds 4a–j do not have acti-
vated unsaturated bonds, they are more appropriate for biological
systems than compounds 2a–f. The affinities of the analogues 4a–j
towards HCA2 in RLB assay decreased as follows [substitution
pattern (compound number, IC50)]: 4-OH (4c, 13 nM) > 2-F, 4-OH
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O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
derivatives 5c, 5b, and 5a. Then followed 2-fluoro-4-hydroxy, 2-
chloro, and, finally, 2-fluoro analogues 5f, 5d, and 5e, respectively.
Although in the functional assay, similarly as in the case of the ser-
ies 4a–j, 4-hydroxy derivative 5c showed the best potency
(EC50 = 70 10 nM, 2-chloro and 2-fluoro analogues 5d and 5e
with EC50 = 130 60 nM and 260 90 nM exhibited the tendency
to be more potent than the compounds 5b and 5a with 3-hydroxy
(1.7
l
m, 2.1 ꢁ 50 mm) on a gradient of 10–95% MeCN in 0.1%
aqueous HCOOH. HRMS (ESI) was performed on a Waters Synapt
G2-Si Mass Spectrometer. Mass is reported in positive ionization
mode unless stated otherwise. GCMS was performed on Agilent
Series
5975C
GC/MSD
using
Agilent
Technologies
30 m ꢁ 0.250 mm column. The purity of the final compounds was
confirmed to be ꢂ 95% with Waters Alliance LC systems equipped
with 2695 separation module with quaternary pump, degasser,
autosampler, column thermostat, and LiChrospher PR Select
4.0 ꢁ 250 mm column. Waters 2489 dual absorbance detector
was used for the analysis. Elemental analyses were obtained with
Carlo Erba EA 1108 instrument. Chromatographical purifications
of compounds were performed by column chromatography using
silica gel, 0.035–0.070 mm or prepacked columns on a Biotage Isol-
era One Purification system. Reaction conditions and yields were
not optimized.
and
2-hydroxy
substituents
(EC50 = 230 120 nM
and
410 140 nM, accordingly). However, unlike the series with the
enyne linker 2a–f and the but-1-yne linker 4a–j, an additional 2-
fluoro substituent did not increase the potency of 4-hydroxy
derivative and the corresponding 2-fluoro-4-hydroxy analogue 5f
showed moderate EC50 value of 260 90 nM.
4. Conclusions
We have designed and synthesized three series of novel aryl
derivatives of 2-amidocyclohex-1-ene carboxylate as HCA2 full
agonists which can be described as consisting of cyclohexene and
aromatic parts which are connected by a more or less flexible lin-
ker. The linker part of the first series of compounds 2a–f contained
a combination of the triple and double bond (enyne linker), the
second series of compounds 4a–j it was a combination of the triple
bond and the ethylene group (pent-1-yne linker), and the third ser-
ies included biphenyl derivatives 5a–f which can formally be
regarded as consisting of the cyclohexene and benzene parts joined
by a 4-substituted phenylethyl linker.
In the case of enyne series 2a–f, it was possible to evaluate the
impact of a particular substitution pattern of the benzene ring on
the activity towards HCA2 receptor by direct comparison with
the unsubstituted structure 1. Thus, the incorporation of a hydroxy
group at the 2-, 3-, or 4-position or a halogen atom (Cl or F) at the
2-position of the phenyl group of the structure 1 increased the
potency in all studied cases.
The presence of the 4-hydroxy group at the benzene ring
boosted both the affinities and potencies of compounds belonging
to all three series (structures 2c, 4c, 5c). In the case of the series
with enyne and pent-1-yne linkers, a tendency of further improve-
ment of the potency of 4-hydroxyphenyl derivatives was observed
by adding an additional 2-fluoro or 2-chloro atom at the benzene
ring (compounds 2f, 4f, 4g).
The presence of a rigid triple bond or phenyl group next to the
benzene ring according to the employed pharmacophoric model
seems to be a prerequisite to design active molecules towards
HCA2 receptor. Compound 3 containing the double bond in a
combination with a fully saturated triple bond (i.e., the ethyl
group) in the linker part of the molecule exhibited a significant loss
of the binding and functional activity comparing to the parent
structure 1.
5.1.1. tert-Butyl(2-iodophenoxy)dimethylsilane (6a)
2-Iodophenol (1.00 g, 4.54 mmol) and imidazole (0.62 g,
9.09 mmol) were dissolved in DMF (10 ml) under argon atmo-
sphere. A solution of TBDMSCl (1.03 g, 6.81 mmol) in DMF (2 mL)
was added dropwise at rt over 5 min and the obtained mixture
was stirred for 2 h. The mixture was diluted with ice water,
extracted with diethyl ether, the organic layer was washed 3 times
with water, brine, and dried over Na2SO4. The solvent is removed
under reduced pressure and the residue was chromatographed
on silica gel (EtOAc in petroleum ether, 1:10) to give compound
6a (1.50 g, 99%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d
7.76 (dd, J = 7.9, 1.7 Hz, 1H), 7.20 (ddd, J = 8.1, 7.3, 1.7 Hz, 1H),
6.83 (dd, J = 8.1, 1.4 Hz, 1H), 6.68 (td, J = 7.6, 1.4 Hz, 1H), 1.06 (s,
9H), 0.28 (s, 6H). GCMS m/z: 277.0 [M-C4H9]+.
5.1.1.1. tert-Butyl(4-iodophenoxy)dimethylsilane (6c). Compound 2c
was prepared in the same manner as compound 6a using 4-
iodophenol (2.00 g, 9.08 mmol), imidazole (0.62 g, 9.11 mmol),
and TBDMSCl (1.37 g, 9.09 mmol). The product 6c (2.85 g, 92%)
was obtained as a colorless oil. 1H NMR (300 MHz, CDCl3) d 7.50
(d, J = 8.9, 2H), 6.61 (d, J = 8.9, 2H), 0.97 (s, 9H), 0.18 (s, 6H). GCMS
m/z: 334 M+.
5.1.1.2. tert-Butyl(3-fluoro-4-iodophenoxy)dimethylsilane (6g). Com-
pound 2g was prepared in the same manner as compound 6a using
3-fluoro-4-iodophenol (1.05 g, 4.41 mmol), imidazole (0.60 g,
8.82 mmol), and TBDMSCl (1.00 g, 6.62 mmol). The product 6g
(1.51 g, 97%) was obtained as a colorless oil. 1H NMR (300 MHz,
CDCl3) d 7.53 (dd, J = 8.6, 7.7 Hz, 1H), 6.59 (dd, J = 9.5, 2.6 Hz, 1H),
6.45 (ddd, J = 8.6, 2.6, 0.7 Hz, 1H), 0.97 (s, 9H), 0.20 (s, 6H). GCMS
m/z: 352.0 M+.
5.1.1.3. tert-Butyl(3-chloro-4-iodophenoxy)dimethylsilane (6h). Com-
pound 2h was prepared in the same manner as compound 6a using
3-chloro-4-iodophenol (1.30 g, 5.11 mmol), imidazole (0.70 g,
10.2 mmol), and TBDMSCl (1.16 g, 7.66 mmol). The product 6h
(1.78 g, 95%) was obtained as a colorless oil. 1H NMR (300 MHz,
CDCl3) d 7.64 (d, J = 8.6 Hz, 1H), 6.97 (d, J = 2.7 Hz, 1H), 6.49 (dd,
J = 8.6, 2.7 Hz, 1H), 0.97 (s, 9H), 0.20 (s, 6H). GCMS m/z: 368.0 M+.
5. Experimental
5.1. Chemistry
Chemicals and reagents were obtained from commercial suppli-
ers and were used without further purification. TLC analyses were
performed with Merck F254 Alumina Silica Plates using UV visual-
ization or staining. 1H NMR and 13C NMR spectra were recorded at
ambient temperature with Bruker (300 MHz) or Varian (400 MHz)
spectrometers. Chemical shifts are reported in parts per million
and referenced to the residual solvent signal. LCMS was performed
on a Waters Acquity UPLC system connected to the Micromass Q-
TOF micro hybrid quadrupole time-of-flight mass spectrometer
operating in the electrospray ionization (ESI) positive and negative
ion mode and using reverse-phase Acquity UPLC BEH C18 column
5.1.1.4. 5-((tert-Butyldimethylsilyl)oxy)-2-iodopyridine (6i). Com-
pound 6i was prepared in the same manner as compound 6a using
6-iodopyridin-3-ol (0.39 g, 1.76 mmol), imidazole (0.24 g,
3.53 mmol), and TBDMSCl (0.40 g, 2.65 mmol). The product 6i
(0.59 g, 99%) was obtained as a colorless oil. 1H NMR (300 MHz,
CDCl3) d: 7.99 (dd, J = 4.5, 1.6 Hz, 1H), 7.11 (dd, J = 8.0, 4.5 Hz,
1H), 7.00 (dd, J = 8.0, 1.6 Hz, 1H), 1.07 (s, 9H), 0.29 (s, 6H). GCMS
m/z: 277.9 [M-C4H9]+.
Please cite this article in press as: Bobileva O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.028

8
O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5.1.1.5. tert-Butyl(3-iodophenoxy)dimethylsilane (6j). Compound 6j
was prepared in the same manner as compound 6a using 3-
iodophenol (2.00 g, 9.08 mmol), imidazole (0.62 g, 9.11 mmol),
and TBDMSCl (1.37 g, 9.09 mmol). The product 6j (2.91 g, 94%)
was obtained as a colorless oil. 1H NMR (400 MHz, CDCl3) d 7.29
(ddd, J = 7.8, 1.6, 1.0, 1H), 7.21 (dd, J = 2.3, 1.6, 1H), 6.94 (t, J = 8.0,
1H), 6.79 (ddd, J = 8.0, 2.4, 1.0, 1H), 0.98 (s, 9H), 0.20 (s, 6H). GCMS
m/z: 334 M+.
5.1.3.2.
3-(4-((tert-Butyldimethylsilyl)oxy)phenyl)prop-2-yn-1-ol
(7c). Compound 7c was prepared in the same manner as com-
pound 7a using iodide 6c (0.50 g, 1.50 mmol), triethylamine
(2.2 ml, 15.7 mmol), propargyl alcohol (0.11 ml, 1.79 mmol), [Pd
(PPh3)4] (5 mg, 0.01 mmol), and CuI (3 mg, 0.02 mmol). The pro-
duct 7c (0.35 g, 88%) was obtained as an oil. 1H NMR (400 MHz,
CDCl3) 7.36–7.29 (m, 2H), 6.81–6.74 (m, 2H), 4.48 (d, J = 6.2 Hz,
2H), 1.58 (t, J = 6.2 Hz, 1H), 0.97 (s, 9H), 0.20 (s, 6H). LCMS (ESI)
m/z 263 [M+H]+.
5.1.2. 2-(3-Bromophenoxy)tetrahydro-2H-pyran (6b)
p-Toluenesulfonic acid (10 mg, 0.06 mmol) was added to DCM
(5 ml), the mixture was stirred for 10 min, and the solid was fil-
tered off. 3-Bromophenol (1.00 g, 5.78 mmol) was dissolved in
the filtrate and to the obtained solution dihydropyrane (0.53 ml,
5.78 mmol) was added dropwise. The mixture was stirred for
1.5 h at rt and quenched by the addition of saturated NaHCO3 solu-
tion. The phases were separated and the aqueous phase was
extracted with DCM. The combined organic phases were dried over
Na2SO4 and the solvent was removed under reduced pressure. The
residue was purified by column chromatography on silica gel
(EtOAc in petroleum ether, 1:5, with 0.2% triethylamine) to give
the product 6b (0.97 g, 65%) as a colorless oil. 1H NMR (300 MHz,
CDCl3) d: 7.25–7.20 (m, 1H), 7.17–7.07 (m, 2H), 6.98 (dt, J = 7.0,
2.4 Hz, 1H), 5.40 (t, J = 3.2 Hz, 1H), 3.87 (ddd, J = 11.3, 9.6, 3.2 Hz,
1H), 3.66–3.57 (m, 1H), 2.07–1.89 (m, 1H), 1.89–1.79 (m, 2H),
1.77–1.55 (m, 3H). GCMS m/z: 172.0 [M-C5H8O]+.
5.1.3.3. 3-(2-Chlorophenyl)prop-2-yn-1-ol (7d). Compound 7d was
prepared in the same manner as compound 7a using 1-chloro-2-
iodobenzene (0.80 g, 3.35 mmol), triethylamine (4.70 ml,
33.5 mmol), propargyl alcohol (0.40 ml, 6.71 mmol), [Pd(PPh3)4]
(116 mg, 0.10 mmol), and CuI (60 mg, 0.20 mmol). The reaction
mixture was stirred for 18 h at rt. The product 7d (0.55 g, 98%)
was obtained as a brown oil. 1H NMR (300 MHz, CDCl3) d:
7.51–7.44 (m, 1H), 7.44–7.36 (m, 1H), 7.30–7.17 (m, 2H), 4.56
(d, J = 6.2 Hz, 2H), 1.69 (t, J = 6.2 Hz, 1H). GCMS m/z: 166.0 M+.
5.1.3.4. 3-(2-Fluorophenyl)prop-2-yn-1-ol (7e). Compound 7e was
prepared in the same manner as compound 7a using 1-fluoro-2-
iodobenzene (0.39 g, 1.76 mmol), triethylamine (2.46 ml,
17.57 mmol), propargyl alcohol (0.21 ml, 3.51 mmol), [Pd(PPh3)4]
(61 mg, 0.05 mmol), and CuI (20 mg, 0.11 mmol). The reaction
was stirred for 18 h at rt. The product 7e (0.24 g, 91%) was obtained
as a yellow oil. 1H NMR (300 MHz, CDCl3) d: 7.43 (td, J = 7.6, 1.9 Hz,
1H), 7.36–7.26 (m, 1H), 7.15–7.01 (m, 2H), 4.54 (d, J = 6.2 Hz, 1H),
1.70 (t, J = 6.2 Hz, 1H). LCMS (ESI): no ionization.
5.1.2.1. 2-(4-Bromo-3-fluorophenoxy)tetrahydro-2H-pyran (6f). Com-
pound 6f was prepared in the same manner as compound 6b using
4-bromo-3-fluorophenol (0.80 g, 4.19 mmol), p-toluenesulfonic
acid (39 mg, 0.21 mmol), and dihydropyrane (0.57 ml, 6.28 mmol).
The product 6f (0.50 g, 43%) was obtained as a colorless oil. 1H
NMR (300 MHz, CDCl3) d: 7.40 (t, J = 8.4 Hz, 1H), 6.88 (dd,
J = 10.5, 2.6 Hz, 1H), 6.82–6.68 (m, 1H), 5.37 (unresolved t, 1H),
3.91–3.77 (m, 1H), 3.67–3.55 (m, 1H), 2.07–1.89 (m, 1H), 2.06–
1.50 (m, 7H). LCMS (ESI): no ionization.
5.1.3.5. 3-(2-Fluoro-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-
2-yn-1-ol (7f). Compound 7f was prepared in the same manner
as compound 7a using bromide 6f (0.49 g, 1.78 mmol), triethy-
lamine (1.24 ml, 8.91 mmol), propargyl alcohol (0.21 ml,
3.56 mmol), [Pd(PPh3)4] (103 mg, 0.09 mmol), and CuI (10 mg,
0.05 mmol). The reaction mixture was stirred for 24 h at 70 °C.
The product 6f (0.11 g, 25%) was obtained as a yellow oil. 1H
NMR (300 MHz, CDCl3) d: 7.32 (t, J = 8.6 Hz, 1H), 6.93–6.72 (m,
2H), 5.40 (t, J = 3.2 Hz, 1H), 4.51 (d, J = 6.0 Hz, 2H), 3.91–3.77 (m,
1H), 3.67–3.57 (m, 1H), 2.06–1.51 (m, 7 H). LCMS (ESI) m/z:
251.18 [M+H]+.
5.1.3. 3-(2-((tert-Butyldimethylsilyl)oxy)phenyl)prop-2-yn-1-ol (7a)
A
solution of tert-butyl(2-iodophenoxy)dimethylsilane (6a)
(0.80 g, 2.39 mmol) and triethylamine (3.35 ml, 23.92 mmol) in
DMF (3 ml) was degassed by bubbling argon for 10 min, then
propargyl alcohol (0.28 ml, 4.79 mmol) followed by [Pd(PPh3)4]
(83 mg, 0.07 mmol) and CuI (27 mg, 0.14 mmol) were added. The
reaction mixture was stirred for 7 h at rt, concentrated, diluted
with water, and extracted with EtOAc. The organic extract was
concentrated under reduced pressure and purified by column chro-
matography on silica gel (EtOAc in petroleum ether, 1:4) to give
the product 7a (0.36 g, 45%) as a yellow oil 1H NMR (300 MHz,
CDCl3) d: 7.37 (dd, J = 7.6, 1.7 Hz, 1H), 7.20 (td, J = 8.1, 1.7 Hz,
1H), 6.90 (td, J = 7.6, 1.1 Hz, 1H), 6.82 (dd, J = 8.1, 1.1 Hz, 1H),
4.50 (d, J = 6.2 Hz, 2H), 1.53 (t, J = 6.2 Hz, 1H), 1.04 (s, 9H), 0.23
(s, 6H). LCMS (ESI) m/z: 263.10 [M+H]+.
5.1.4. 3-(2-((tert-Butyldimethylsilyl)oxy)phenyl)propiolaldehyde (8a)
To a solution of alcohol 7a (0.28 g, 1.06 mmol) in DCM (1 ml)
was added MnO2 (0.93 g, 10.7 mmol), and the slurry was stirred
at rt for 7 days. The mixture was filtered through a pad of silica,
washed with DCM, the filtrate was concentrated and the residue
was chromatographed on silica gel (EtOAc in petroleum ether,
1:10) to give the product 8a (0.18 g, 43%) as a brown oil. 1H NMR
(300 MHz, CDCl3) d: 9.42 (s, 1H), 7.50 (dd, J = 7.6, 1.8 Hz, 1H),
7.36 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 6.97 (td, J = 7.6, 1.0 Hz, 1H),
6.90–6.84 (m, 1H), 1.05 (s, 9H), 0.26 (s, 6H). GCMS m/z: 203.1
[M-C4H9]+.
5.1.3.1. 3-(3-((Tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-2-yn-1-ol
(7b). Compound 7b was prepared in the same manner as com-
pound 7a using bromide 6b (0.60 g, 2.33 mmol), triethylamine
(3.27 ml, 23.33 mmol), propargyl alcohol (0.28 ml, 4.67 mmol),
[Pd(PPh3)4] (81 mg, 0.07 mmol), and CuI (27 mg, 0.14 mmol). The
reaction mixture was stirred for 9 h at 70 °C. The product 7b
(0.30 g, 55%) was obtained as a yellow oil. 1H NMR (300 MHz,
CDCl3) d: 7.21 (t, J = 7.9 Hz, 1H), 7.15 (dd, J = 2.5, 1.3 Hz, 1H), 7.06
(dt, J = 7.7, 1.0 Hz, 1H), 7.02 (ddd, J = 8.2, 2.5, 1.1 Hz, 1H), 5.40 (t,
J = 3.2 Hz, 1H), 4.49 (d, J = 6.2 Hz, 2H), 3.89 (ddd, J = 11.3, 9.4,
3.2 Hz, 1H), 3.65–3.66 (m, 1H), 2.05–1.89 (m, 1H), 1.91–1.76 (m,
1H), 1.73–1.55 (m, 3H), 1.61 (t, J = 6.2 Hz, 1H). LCMS (ESI) m/z:
233.06 [M+H]+.
5.1.4.1.
3-(3-((Tetrahydro-2H-pyran-2-yl)oxy)phenyl)propiolalde-
hyde (8b). Compound 8b was prepared in the same manner as
compound 8a using propynol 7b (0.30 g, 1.29 mmol) and MnO2
(1.12 g, 12.9 mmol). The reaction mixture was stirred at rt for
4 days. The product 8b (0.14 g, 46%) was obtained as a brown oil.
1H NMR (300 MHz, CDCl3) d: 9.42 (s, 1H), 7.36–7.21 (m, 3H), 7.18
(ddd, J = 8.2, 2.5, 1.4 Hz, 1H), 5.42 (t, J = 3.2 Hz, 1H), 3.87 (ddd,
J = 11.4, 9.5, 3.2 Hz, 1H), 3.62 (dtd, J = 11.4, 4.1, 1.5 Hz, 1H), 2.09–
1.91 (m, 1H), 1.93–1.81 (m, 1H), 1.77–1.55 (m, 4H). LCMS (ESI)
m/z: 231.13 [M+H]+.
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O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
5.1.5. 3-(4-Hydroxyphenyl)propiolaldehyde (8c)
410.28 [M+H]+). 1H NMR (300 MHz, CDCl3) d: 11.88 (s, 1H), 7.24
(t, J = 7.9 Hz, 1H), 7.20–7.16 (m, 1H), 7.10 (dt, J = 7.6, 1.1 Hz, 1H),
7.05 (ddd, J = 8.2, 2.5, 1.1 Hz, 1H), 6.93 (d, J = 15.4 Hz, 1H), 6.44
(d, J = 15.4 Hz, 1H), 5.42 (t, J = 3.3 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H),
3.90 (ddd, J = 11.3, 9.3, 3.3 Hz, 1H), 3.69–3.56 (m, 1H), 3.10–2.97
(m, 2H), 2.39–2.29 (m, 2H), 2.08–1.87 (m, 1H), 1.90–1.82 (m,
2H), 1.73–1.57 (m, 7H), 1.31 (t, J = 7.1 Hz, 3H). LCMS (ESI) m/z:
424.30 [M+H]+. In addition, the corresponding Z-isomer of com-
pound 10b (17 mg, 6%) after column chromatography was isolated.
(Z)–10b, 1H NMR (300 MHz, CDCl3) d: 11.92 (s, 1H), 7.21 (t,
J = 7.9 Hz, 1H), 7.17 (dd, J = 2.5, 1.5 Hz, 1H), 7.11 (dt, J = 7.6,
1.3 Hz, 1H), 7.03 (ddd, J = 8.2, 2.5, 1.1 Hz, 1H), 6.25 (d, J = 11.5 Hz,
1H), 6.17 (d, J = 11.5 Hz, 1H), 5.39 (t, J = 3.2 Hz, 1H), 4.09 (q,
J = 7.1 Hz, 2H), 3.87 (ddd, J = 11.3, 9.4, 3.1 Hz, 1H), 3.65–3.54 (m,
1H), 3.13–2.99 (m, 2H), 2.39–2.27 (m, 2H), 2.07–1.90 (m, 1H),
1.90–1.79 (m, 2H), 1.76–1.49 (m, 7H), 1.23 (t, J = 7.1 Hz, 3H). LCMS
(ESI) m/z: 424.30 [M+H]+.
To a solution of alcohol 7c (0.35 g, 1.31 mmol) in DCM (5 ml)
Dess-Martin periodinane (15% solution in DCM, 5.0 ml, 1.80 mmol)
was added. After stirring at rt for 1 h, the reaction mixture was
diluted with DCM, washed with 10% aqueous sodium thiosulfate
solution, saturated NaHCO3 solution, brine, dried over Na2SO4,
and concentrated. The residue was chromatographed on silica gel
(EtOAc in petroleum ether, 1:9) to give the product 8c (0.32 g,
93%) as a brown oil. 1H NMR (400 MHz, CDCl3) 9.40 (s, 1H),
7.55–7.47 (m, 2H), 6.89–6.80 (m, 2H), 0.98 (s, 9H), 0.23 (s, 6H).
GCMS m/z: 260.0 M+.
5.1.5.1. 3-(2-Chlorophenyl)propiolaldehyde (8d). Compound 8d was
prepared in the same manner as compound 8c using alcohol 7d
(0.55 g, 3.30 mmol) and Dess-Martin periodinane (15% solution in
DCM, 12.1 ml, 4.29 mmol). The product 8d (0.51 g, 94%) was
obtained as a brown oil. 1H NMR (300 MHz, CDCl3) d: 9.49 (s,
1H), 7.63 (dd, J = 7.7, 1.6 Hz, 1H), 7.48 (dd, J = 8.1, 1.5 Hz, 1H),
7.42 (ddd, J = 8.1, 7.7, 1.6 Hz, 1H), 7.30 (td, J = 7.5, 1.5 Hz, 1H).
GCMS m/z: 164.0 M+.
5.1.6.2. Ethyl (E)-2-(5-(2-chlorophenyl)pent-2-en-4-ynamido)cyclo-
hex-1-ene-1-carboxylate (10d). Compound 10d was prepared in
the same manner as compound 10a using aldehyde 8d (0.105 g,
0.64 mmol), phosphonium bromide 9 (0.39 g, 0.71 mmol), and t-
BuOK (72 mg, 0.64 mmol). The product 10d (0.18 g, 79%) was
obtained as a yellow solid which contained the corresponding
methyl carboxylate (ca 4%, LCMS (ESI) m/z: 344.24 [M+H]+, 1H
NMR (300 MHz, CDCl3) d: 3.75 (s)) and the Z-isomer (ca 6%, LCMS
(ESI) m/z: 358.26 [M+H]+, 1H NMR (300 MHz, CDCl3) d: 6.31 (d,
J = 11.5 Hz), 6.23 (d, J = 11.5 Hz)) as impurities.1H NMR (300 MHz,
CDCl3) d: 11.89 (s, 1H), 7.55–7.45 (m, 1H), 7.47–7.37 (m, 1H),
7.35–7.18 (m, 2H), 6.98 (d, J = 15.4 Hz, 1H), 6.50 (d, J = 15.4 Hz,
1H), 4.20 (q, J = 7.1 Hz, 2H), 3.14–2.97 (m, 2H), 2.40–2.28 (m,
2H), 1.72–1.56 (m, 4H), 1.31 (t, J = 7.1 Hz, 3H). LCMS (ESI) m/z:
358.24 [M+H]+.
5.1.5.2. 3-(2-Fluorophenyl)propiolaldehyde (8e). Compound 8e was
prepared in the same manner as compound 8c using alcohol 7e
(0.24 g, 1.60 mmol) and Dess-Martin periodinane (15% solution in
DCM, 4.97 ml, 1.76 mmol). The product 8e (0.19 g, 80%) was
obtained as a yellow oil. 1H NMR (300 MHz, CDCl3) d: 9.45 (s,
1H), 7.58 (td, J = 7.2, 1.8 Hz, 1H), 7.54–7.44 (m, 1H), 7.19 (td,
J = 7.6, 1.0 Hz, 1H), 7.15 (t, J = 8.8 Hz, 1H). LCMS (ESI): no
ionization.
5.1.5.3. 3-(2-Fluoro-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)propi-
olaldehyde (8f). Compound 8f was prepared in the same manner as
compound 8c using alcohol 7f (106 mg, 0.42 mmol) and Dess-Mar-
tin periodinane (15% solution in DCM, 1.38 ml, 0.55 mmol). The
product 8f (88 mg, 84%) was obtained as a brown oil. 1H NMR
(300 MHz, CDCl3) d: 9.42 (s, 1H), 7.54–7.41 (m, 1H), 6.92–6.81
(m, 2H), 5.46 (t, J = 3.0 Hz, 1H), 3.81 (ddd, J = 11.2, 10.0, 3.0 Hz,
1H), 3.63 (dtd, J = 11.2, 4.1, 1.4 Hz, 1H), 2.04–1.80 (m, 2H), 1.82–
1.55 (m, 4H). LCMS (ESI) m/z: 249.11 [M+H]+.
5.1.6.3. Ethyl (E)-2-(5-(2-fluorophenyl)pent-2-en-4-ynamido)cyclo-
hex-1-ene-1-carboxylate (10e). Compound 10e was prepared in
the same manner as compound 10a using aldehyde 8e (0.10 g,
0.68 mmol), phosphonium bromide 9 (0.45 g, 0.81 mmol), and t-
BuOK (76 mg, 0.68 mmol). The product 10e (0.20 g, 87%) was
obtained as a yellow solid which contained the corresponding
methyl carboxylate (ca 5%, LCMS (ESI) m/z: 328.21 [M+H]+, 1H
NMR (300 MHz, CDCl3) d: 3.75 (s)) and the Z-isomer (ca 8%, LCMS
(ESI) m/z: 342.24 [M+H]+, 1H NMR (300 MHz, CDCl3) d: 6.29 (d,
J = 11.5 Hz), 6.22 (d, J = 11.5 Hz)) as impurities. 1H NMR
(300 MHz, CDCl3) d: 11.90 (s, 1H), 7.46 (td, J = 7.4, 1.8 Hz, 1H),
7.35 (tdd, J = 7.8, 5.3, 1.8 Hz, 1H), 7.17–7.06 (m, 2H), 6.96 (d,
J = 15.4 Hz, 1H), 6.49 (d, J = 15.4 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H),
3.12–2.98 (m, 2H), 2.41–2.29 (m, 2H), 1.72–1.56 (m, 4H), 1.31 (t,
J = 7.1 Hz, 3H). LCMS m/z: 342.37 [M+H]+.
5.1.6. Ethyl (E)-2-(5-(2-((tert-butyldimethylsilyl)oxy)phenyl)pent-2-
en-4-ynamido)cyclohex-1-ene-1-carboxylate (10a)
To a solution of (2-((2-(ethoxycarbonyl)cyclohex-1-en-1-yl)
amino)-2-oxoethyl)triphenylphosphonium bromide (9) (0.36 g,
0.65 mmol) and t-BuOK (60 mg, 0.54 mmol) in DMSO (4 ml) was
added a solution of aldehyde 8a (0.14 g, 0.54 mmol) in DMSO
(1.5 ml) and the resulting mixture was stirred at rt for 2 h. The
reaction mixture was diluted with water and extracted with EtOAc.
The organic extract was washed successively with water, brine,
dried over Na2SO4, concentrated, and purified by column chro-
matography on silica gel (EtOAc in petroleum ether, 1:10) to afford
the product 10a (0.21 g, 65%) as a brown oil. 1H NMR (300 MHz,
CDCl3) d: 11.87 (s, 1H), 7.39 (dd, J = 7.7, 1.7 Hz, 1H), 7.25–7.18
(m, 1H), 6.96 (d, J = 15.4 Hz, 1H), 6.96–6.90 (m, 1H), 6.83 (d,
J = 7.8 Hz, 1H), 6.40 (d, J = 15.4 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H),
3.12–2.97 (m, 2H), 2.40–2.27 (m, 2H), 1.69–1.57 (m, 4H), 1.31 (t,
J = 7.1 Hz, 3H), 1.05 (s, 9H), 0.23 (s, 6H). LCMS (ESI) m/z: 454.31
[M+H]+.
5.1.6.4. Ethyl (E)-2-(5-(2-fluoro-4-((tetrahydro-2H-pyran-2-yl)oxy)
phenyl)pent-2-en-4-ynamido)cyclohex-1-ene-1-carboxylate
(10f). Compound 10f was prepared in the same manner as com-
pound 10a using aldehyde 8f (85 mg, 0.34 mmol), phosphonium
bromide 9 (0.23 g, 0.41 mmol), and t-BuOK (42 mg, 0.38 mmol).
The product 10f (0.14 g, 93%) was obtained as a yellow solid, which
contained the corresponding methyl carboxylate (ca 3%, LCMS (ESI)
m/z: 428.32 [M+H]+, 1H NMR (300 MHz, CDCl3) d: 3.75 (s)) and the
Z-isomer (ca 7%, LCMS (ESI) m/z: 442.32 [M+H]+, 1H NMR
(300 MHz, CDCl3) d: 6.27 (d, J = 11.5 Hz), 6.16 (d, J = 11.5 Hz)) as
impurities. 1H NMR (300 MHz, CDCl3) d: 11.87 (s, 1H), 7.39–7.31
(m, 1H), 6.95 (d, J = 15.4 Hz, 1H), 6.88–6.75 (m, 2H), 6.44 (d,
J = 15.4 Hz, 1H), 5.42 (t, J = 3.1 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H),
3.84 (ddd, J = 11.1, 9.6, 3.1 Hz, 1H), 3.67–3.56 (m, 1H), 3.09–2.98
(m, 2H), 2.40–2.27 (m, 2H), 2.07–1.80 (m, 1H), 1.88–1.83 (m,
5.1.6.1. Ethyl (E)-2-(5-(3-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)
pent-2-en-4-ynamido)cyclohex-1-ene-1-carboxylate
(10b). Com-
pound 10b was prepared in the same manner as compound 10a
using aldehyde 8b (0.16 g, 0.70 mmol), phosphonium bromide 9
(0.46 g, 0.83 mmol), and t-BuOK (78 mg, 0.70 mmol). The product
10b (0.24 g, 87%) was obtained as a yellow oil which contained
the corresponding methyl carboxylate (ca 6%, LCMS (ESI) m/z:
Please cite this article in press as: Bobileva O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.028

10
O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
2H), 1.77–1.56 (m, 7H), 1.31 (t, J = 7.1 Hz, 3H). LCMS (ESI) m/z:
d: 3.76 (s)) and the Z-isomer (ca 4%, LCMS (ESI) m/z: 358.21 [M+H]+,
1H NMR (300 MHz, CDCl3) d: 6.29 (d, J = 11.5 Hz), 6.17 (d,
J = 11.5 Hz)) as impurities. 1H NMR (300 MHz, CDCl3) d: 11.89 (s,
1H), 7.31 (t, J = 8.1 Hz, 1H), 6.94 (d, J = 15.4 Hz, 1H), 6.66–6.69
(m, 2H), 6.42 (d, J = 15.4 Hz, 1H), 6.06 (s, 1H), 4.21 (q, J = 7.1 Hz,
2H), 3.09–2.99 (m, 2H), 2.40–2.29 (m, 2H), 1.71–1.55 (m, 4H),
1.32 (t, J = 7.1 Hz, 3H). Rf 0.4 (EtOAc in petroleum ether, 1:4). LCMS
(ESI) m/z: 358.21 [M+H]+.
442.31 [M+H]+.
5.1.7. Ethyl (E)-2-(5-(4-((tert-butyldimethylsilyl)oxy)phenyl)pent-2-
en-4-ynamido)cyclohex-1-ene-1-carboxylate (10c)
A mixture of aldehyde 10c (0.17 g, 0.64 mmol), phosphonium
bromide 9 (0.42 g, 0.76 mmol), K2CO3 (0.11 g 0.76 mmol), and
18-crown-6 (6 mg, 0.02 mmol) in DCM (8 ml) was stirred for
16 h at rt. The mixture was diluted with DCM (20 ml) and washed
successively with saturated NH4Cl solution, water, brine, dried
over Na2SO4, and concentrated. The residue was purified by col-
umn chromatography on silica gel (EtOAc in petroleum ether, 0–
20%) to give the product 10c (0.24 g, 83%) as an oil. 1H NMR
(400 MHz, CDCl3) d: 11.85 (s, 1H), 7.41–7.31 (m, 2H), 6.93 (d,
J = 15.4 Hz, 1H), 6.84–6.75 (m, 2H), 6.40 (d, J = 15.4 Hz, 1H), 4.20
(q, J = 7.1 Hz, 2H), 3.07–3.02 (m, 2H), 2.37–2.31 (m, 2H), 1.72–
1.56 (m, 4H), 1.31 (t, J = 7.1 Hz, 3H), 0.98 (s, 9H), 0.21 (s, 6H). 13C
NMR (100 MHz, CDCl3) d: 170.2, 163.2, 157.0, 152.3, 133.7, 133.2,
123.2, 120.5, 115.3, 105.7, 98.3, 86.3, 60.5, 28.7, 25.8, 24.5, 22.0,
21.8, 18.4, 14.4, -4.3. LCMS (ESI) m/z: 454.32 [M+H]+.
5.1.10. (E)-2-(5-(2-Hydroxyphenyl)pent-2-en-4-ynamido)cyclohex-1-
ene-1-carboxylic acid (2a)
To a solution of ethyl carboxylate 11a (0.12 g, 0.53 mmol) in a
mixture of THF (2 ml) and MeOH (1 ml) 2 N NaOH solution
(0.53 ml, 1.06 mmol) was added and the mixture was stirred for
6 h at rt. The mixture was acidified with aqueous 1 N HCl to pH
4, concentrated in vacuo to 1 ml, the precipitated solid was filtered,
and dried in vacuo over P2O5. This residue was chromatographed
on silica gel (MeOH in DCM, 1:20 to 1:10) to give the product 2a
(59 mg, 54%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) d:
12.66 (b s, 1H), 11.70 (s, 1H), 10.12 (b s, 1H), 7. 35 (dd, J = 7.6,
1.6 Hz, 1H), 7.25 (ddd, J = 8.3, 7.4, 1.6 Hz, 1H), 6.91 (dd, J = 8.3,
1.0 Hz, 1H), 6.84 (d, J = 15.5 Hz, 1H), 6.82 (td, J = 7.5, 1.0 Hz, 1H),
6.49 (d, J = 15.5 Hz, 1H), 2.90–2.84 (m, 2H), 2.29–2.23 (m, 2H),
1.62–1.50 (m, 4H). 13C NMR (100 MHz, DMSO-d6) d: 171.0, 161.9,
158.8, 149.9, 133.5, 133.2, 131.2, 121.9, 119.2, 115.7, 108.9,
107.0, 95.1, 90.3, 28.1, 24.5, 21.5, 21.3. LCMS (ESI) m/z: 310.12
[MꢃH]ꢃ. Anal. Calcd for C18H17NO4 ꢁ 0.13 CHCl3 (4.7%): C 66.62,
H 5.28, N 4.29. Found C 66.54, H 5.25, N 4.26.
5.1.8. Ethyl (E)-2-(5-(2-hydroxyphenyl)pent-2-en-4-
ynamido)cyclohex-1-ene-1-carboxylate (11a)
To a solution of TBDMS-ether 10a (0.19 g, 0.42 mmol) in THF
(4 ml) was added 1 M solution of tetra-n-butylammonium fluoride
in THF (0.46 ml, 0.46 mmol) by cooling the reaction flask in a cold
water bath and the reaction mixture was stirred at rt for 10 min.
The reaction mixture was diluted with ethyl acetate, successively
washed with water, saturated NH4Cl solution, brine, and dried over
Na2SO4. The solvents were removed and the residue was purified
by column chromatography on silica gel (EtOAc in petroleum
ether, 1:4) to give the product 11a (0.12 g, 84%) as a yellow solid
which contained the corresponding methyl carboxylate (ca 4%,
LCMS (ESI) m/z: 326.33 [M+H]+, 1H NMR (300 MHz, CDCl3) d:
3.76 (s)) as an impurity. 1H NMR (300 MHz, CDCl3) d: 11.94 (s,
1H), 7.38 (dd, J = 7.7, 1.3 Hz, 1H), 7.36–7.26 (m, 1H), 7.06–6.86
(m, 3H), 6.49 (d, J = 15.5 Hz, 1H), 5.71 (s, 1H), 4.22 (q, J = 7.1 Hz,
2H), 3.13–3.00 (m, 2H), 2.42–2.31 (m, 2H), 1.75–1.58 (m, 4H),
1.33 (t, J = 7.1 Hz, 3H). LCMS (ESI) m/z: 340.22 [M+H]+.
5.1.10.1. (E)-2-(5-(3-Hydroxyphenyl)pent-2-en-4-ynamido)cyclohex-
1-ene-1-carboxylic acid (2b). Compound 2b was prepared in the
same manner as compound 2a using ethyl carboxylate 11b
(0.17 g, 0.50 mmol) and 2 N NaOH solution (0.75 ml, 1.50 mmol)
in water. The product 2b (79 mg, 51%) was obtained as a yellow
solid. 1H NMR (400 MHz, DMSO-d6) d: 11.75 (b s, 1H), 9.80 (b s,
1H), 7.22 (t, J = 7.9 Hz, 1H), 6.95 (dt, J = 7.7, 1.1 Hz, 1H), 6.89–6.83
(m, 2H), 6.82 (d, J = 15.5 Hz, 1H), 6.55 (d, J = 15.5 Hz, 1H), 2.89–
2.82 (m, 2H), 2.30–2.22 (m, 2H), 1.63–1.50 (m, 4H). 13C NMR
(100 MHz, DMSO-d6) d: 171.0, 161.8, 157.4, 149.6, 134.7, 130.1,
122.6, 122.3, 121.3, 117.9, 117.2, 107.3, 97.2, 86.4, 28.1, 24.6,
5.1.9. Ethyl (E)-2-(5-(3-hydroxyphenyl)pent-2-en-4-
21.5, 21.3. HRMS (ESI) m/z: calcd. for C
18H18NO4 [M+H]+
ynamido)cyclohex-1-ene-1-carboxylate (11b)
312.1230, found 312.1242.
p-Toluenesulfonic acid (5 mg, 0.03 mmol) was added to a sus-
pension of compound 10b (0.23 g, 0.54 mmol) in a mixture of
DCM (5 mL) and methanol (2.5 mL). After stirring at rt for 6 h,
the solvent was evaporated. The residue was diluted with water,
extracted with EtOAc, the organic the extract was washed with
water, brine, dried over Na2SO4, and concentrated to give the pro-
duct 11b (0.18 g, 98%) as a yellow solid. 1H NMR (300 MHz, CDCl3)
d: 11.90 (s, 1H), 7.22 (t, J = 7.9 Hz, 1H), 7.05 (dt, J = 7.6, 1.1 Hz, 1H),
6.95 (dd, J = 2.5, 1.4 Hz, 1H), 6.94 (d, J = 15.4 Hz, 1H), 6.85 (ddd,
J = 8.1, 2.5, 0.9 Hz, 1H), 6.44 (d, J = 15.4 Hz, 1H), 5.07 (s, 1H), 4.20
(q, J = 7.1 Hz, 2H), 3.10–2.99 (m, 2H), 2.40–2.30 (m, 2H), 1.72–
1.56 (m, 4H), 1.31 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d: 170.2, 163.3, 156.0, 152.0, 134.0, 129.9, 124.5, 123.5, 123.2,
118.7, 117.0, 106.5, 97.8, 86.7, 60.7, 28.7, 24.5, 22.0, 21.7, 14.4. Rf
0.4 (EtOAc in petroleum ether, 1:4). LCMS (ESI) m/z: 340.23 [M
+H]+.
5.1.10.2. (E)-2-(5-(4-Hydroxyphenyl)pent-2-en-4-ynamido)cyclohex-
1-ene-1-carboxylic acid (2c). Compound 2c was prepared in the
same manner as compound 2a using ethyl carboxylate 10c
(95 mg, 0.21 mmol) and NaOH (0.42 ml, 0.84 mmol). The product
2c (37 mg, 57%) was obtained as
a
yellow solid. 1H NMR
(400 MHz, DMSO-d6) d: 12.50 (b s, 1H), 11.71 (s, 1H), 10.10 (b s,
1H), 7.36 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 15.3 Hz, 1H), 6.80 (d,
J = 8.6 Hz, 2H), 6.45 (d, J = 15.3 Hz, 1H), 2.91–2.81 (m, 2H), 2.30–
2.21 (m, 2H), 1.63–1.48 (m, 4H). 13C NMR (100 MHz, DMSO-d6)
d: 171.0, 162.0, 158.9, 149.9, 133.6, 133.1, 121.9, 115.9, 111.6,
107.0, 98.5, 85.6, 28.1, 24.1, 21.5, 21.3. LCMS (ESI) m/z: 312.10
[M+H]+. Anal. Calcd for C18H17NO4: C 69.44, H 5.50, N 4.50. Found
C 68.97, H 5.49, N 4.37.
5.1.10.3. (E)-2-(5-(2-Chlorophenyl)pent-2-en-4-ynamido)cyclohex-1-
ene-1-carboxylic acid (2d). Compound 2d was prepared in the same
manner as compound 2a using ethyl carboxylate 10d (0.18 g,
0.50 mmol) and 2 N NaOH (0.75 ml, 1.51 mmol). The product 2d
(63 mg, 38%) was obtained as a yellow solid. 1H NMR (400 MHz,
DMSO-d6) d: 12.70 (b s, 1H), 11.67 (s, 1H), 7.66 (dd, J = 7.4,
1.7 Hz, 1H), 7.60 (dd, J = 7.8, 1.1 Hz, 1H), 7.48 (td, J = 7.8, 1.7 Hz,
1H), 7.41 (td, J = 7.4, 1.1 Hz, 1H), 6.88 (d, J = 15.5 Hz, 1H), 6.63 (d,
J = 15.5 Hz, 1H), 2.90–2.80 (m, 2H), 2.31–2.21 (m, 2H), 1.64–1.48
5.1.9.1.
Ethyl
(E)-2-(5-(2-fluoro-4-hydroxyphenyl)pent-2-en-4-
ynamido)cyclohex-1-ene-1-carboxylate (11f). Compound 11f was
prepared in the same manner as compound 11b using THP-ether
10f (0.14 g, 0.32 mmol) and p-toluenesulfonic acid (3 mg,
0.02 mmol). The product 11f (92 mg, 81%) was obtained as a yel-
low solid which contained the corresponding methyl carboxylate
(ca 5%, LCMS (ESI) m/z: 344.19 [M+H]+, 1H NMR (300 MHz, CDCl3)
Please cite this article in press as: Bobileva O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.028

O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
(m, 4H). 13C NMR (100 MHz, DMSO-d6) d: 170.9, 161.5, 149.5,
5.1.12. (E)-2-(5-Phenylpent-2-enamido)cyclohex-1-ene-1-carboxylic
acid (3)
135.6, 134.8, 133.8, 131.2, 129.5, 127.5, 121.3, 120.7, 107.5, 93.0,
91.6, 28.1, 24.6, 21.5, 21.3. LCMS (ESI) m/z: 328.22 [MꢃH]ꢃ. Anal.
Calcd for C18H16ClNO3: C 65.56, H 4.89, N 4.25. Found: C 65.04, H
4.91, N 4.22.
5-Phenylpent-2-enoic acid (13) (0.18 g, 1.02 mmol) in DCM
(2 ml) under argon atmosphere at rt was treated with a catalytic
amount of DMF (1 drop) and oxalyl chloride (0.26 ml, 3.06 mmol).
The mixture was stirred for 40 min and the volatiles were evapo-
rated. The residue was dissolved in DCM (2 ml) and to the obtained
solution at 0 °C a mixture of ethyl 2-aminocyclohex-1-ene-1-car-
boxylate (0.26 g, 1.54 mmol) and triethylamine (0.14 ml,
1.00 mmol) in DCM (1 ml) was added dropwise over 5 min. The
mixture was stirred for 40 min at rt, then the mixture was supple-
mented with DCM, washed with saturated NH4Cl solution, brine,
dried over Na2SO4, and concentrated. The residue was chro-
matographed on silica gel (EtOAc in petroleum ether, 1:10 to
1:6) to give crude ethyl (E)-2-(5-phenylpent-2-enamido)cyclo-
5.1.10.4. (E)-2-(5-(2-Fluorophenyl)pent-2-en-4-ynamido)cyclohex-1-
ene-1-carboxylic acid (2e). Compound 2e was prepared in the same
manner as compound 2a using ethyl carboxylate 10e (0.20 g,
0.59 mmol) and 2 N NaOH (0.88 ml, 1.76 mmol). The product 2e
(70 mg, 38%) was obtained as a yellow solid. 1H NMR (300 MHz,
CDCl3) d: 11.61 (s, 1H), 10.23 (b s, 1H), 7.52–7.44 (m, 1H), 7.40–
7.30 (m, 1H), 7.16–7.06 (m, 2H), 7.00 (d, J = 15.5 Hz, 1H), 6.45 (d,
J = 15.5 Hz, 1H), 3.13–3.04 (m, 2H), 2.45–2.36 (m, 2H), 1.75–1.58
(m, 4H). 13C NMR (100 MHz, CDCl3) d: 174.6, 162.9 (d, JCF = 253.6 -
Hz), 162.8, 155.1, 134.1, 133.9, 131.2 (d, JCF = 8.1 Hz), 124.3 (d,
JCF = 3.8 Hz), 123.2, 115.8 (d, JCF = 20.8 Hz), 111.2 (d, JCF = 15.6 Hz),
104.9, 91.6 (d, JCF = 3.2 Hz), 91.3, 29.1, 24.6, 21.9, 21.7. LCMS (ESI)
m/z: 312.14 [MꢃH]ꢃ. Anal. Calcd for C18H16FNO3: C 69.00, H 5.15,
N 4.47. Found: C 68.28, H 5.08, N 4.35.
hex-1-ene-1-carboxylate (0.21 g) as
a
colorless oil. 1H NMR
(300 MHz, CDCl3) d: 11.71 (s, 1H), 7.33–7.16 (m, 5H), 6.92 (dt,
J = 15.3, 6.8 Hz, 1H), 5.92 (dt, J = 15.3, 1.5 Hz, 1H), 4.19 (q,
J = 7.1 Hz, 1H), 3.09–2.98 (m, 2H), 2.85–2.69 (m, 2H), 2.59–2.45
(m, 2H), 2.39–2.25 (m, 2H), 1.73–1.56 (m, 4H), 1.30 (t, J = 7.1 Hz,
3H). LCMS (ESI) m/z: 328.37 [M+H]+. The oil was dissolved in a
mixture of THF (2 ml) and EtOH (1 ml) and to the obtained solution
2 N NaOH (0.92 ml, 1.84 mmol) was added. The mixture was stir-
red for 16 h at rt followed by stirring for 5 h at 50 °C. The reaction
mixture was cooled to rt and acidified to pH 4 with 1 N HCl. The
solvents were evaporated and the residue was partitioned between
DCM and water. The organic layer was dried over Na2SO4, evapo-
rated, and the residue was chromatographed on silica gel (MeOH
in DCM, 1:20) to give an oily solid. The solid was triturated with
EtOAc petroleum ether mixture (1:4), filtered, and dried in vacuo
to give the product 3 (44 mg, 14%, calculated with respect to 13)
as white crystals. 1H NMR (300 MHz, CDCl3) d: 11.47 (s, 1H),
10.40 (b s, 1H), 7.33–7.25 (m, 2H), 7.23–7.16 (m, 3H), 6.93 (dt,
J = 15.3, 6.9 Hz, 1H), 5.98 (dt, J = 15.3, 1.3 Hz, 1H), 3.10–3.01 (m,
2H), 2.83–2.74 (m, 2H), 2.59–2.49 (m, 2H), 2.42–2.33 (m, 2H),
1.73–1.56 (m, 4H). 13C NMR (100 MHz, CDCl3) d: 174.6, 164.2,
155.6, 146.1, 141.0, 128.6, 128.5, 126.3, 125.7, 103.7, 34.7, 34.1,
29.1, 24.6, 21.9, 21.8. HRMS (ESI) m/z: calcd. for C18H22NO3 [M
+H]+ 300.1594, found 300.1587.
5.1.10.5. (E)-2-(5-(2-Fluoro-4-hydroxyphenyl)pent-2-en-4-ynamido)-
cyclohex-1-ene-1-carboxylic acid (2f). Compound 2f was prepared
in the same manner as compound 2a using ethyl carboxylate 11f
(92 mg, 0.26 mmol) and 2 N NaOH (0.39 ml, 0.77 mmol). The pro-
duct 2f (32 mg, 38%) was obtained as a white solid. 1H NMR
(400 MHz, DMSO-d6) d: 12.67 (b s, 1H), 11.70 (s, 1H), 10.60 (b s,
1H), 7.43–7.38 (m, 1H), 6.83 (d, J = 15.5 Hz, 1H), 6.70–6.65 (m,
2H), 6.49 (d, J = 15.5 Hz, 1H), 2.89–2.83 (m, 2H), 2.29–2.23 (m,
2H), 1.62–1.50 (m, 4H). 13C NMR (100 MHz, DMSO-d6) d: 171.0,
163.1 (d, JCF = 249.8 Hz), 161.8, 160.8 (d, JCF = 11.8 Hz), 149.7,
134.6 (d, JCF = 2.9 Hz), 133.8, 121.4, 112.5 (d, JCF = 2.6 Hz), 107.2,
103.1 (d, JCF = 22.9 Hz), 100.0 (d, JCF = 15.8 Hz), 91.4, 90.1 (d,
JCF = 2.9 Hz), 28.2, 24.6, 21.5, 21.3. LCMS (ESI) m/z: 328.10 [MꢃH]ꢃ.
Anal. Calcd for C18H16FNO4: C 65.65, H 4.90, N 4.25. Found: C 65.29,
H 4.83, N 4.22.
5.1.11. (E)-5-Phenylpent-2-enoic acid (13)
To
a
solution of methyl 2-(dimethoxyphosphoryl)acetate
5.1.13. 5-(2-Chlorophenyl)pent-4-ynoic acid (16)
(0.63 ml, 4.45 mmol) in DMSO (2.5 ml) was added t-BuOK
(0.46 g, 4.10 mmol) and the mixture was stirred at rt for 10 min.
To this mixture 3-phenylpropanal (12) (0.50 g, 3.73 mmol) was
added and stirring was continued for 1 h. The reaction mixture
was diluted with water and extracted with EtOAc, the organic layer
was washed with brine, dried over Na2SO4, and concentrated. The
residue was purified by column chromatography on silica gel
(EtOAc in petroleum ether, 1:7) to give a mixture of E and Z iso-
mers of methyl 5-phenylpent-2-enoate (0.27 g, 38%) as a colorless
oil. Methyl (E)-5-phenylpent-2-enoate: 1H NMR (300 MHz, CDCl3)
d: 7.34–7.11 (m, 5H), 7.01 (dt, J = 15.7, 6.8 Hz, 1H), 5.85 (dt,
J = 15.7, 1.4 Hz, 2H), 3.72 (s, 3H), 2.84–2.71 (m, 2H), 2.60–2.46
(m, 2H). The oil was dissolved in a mixture of THF (1.5 ml) and
MeOH (1 ml), to the obtained solution was added 2 N NaOH
(2.5 ml, 5.00 mmol) and the resulting mixture was stirred at rt
for 4 h. The solvents were evaporated, the residue was dissolved
in water and acidified to pH 2 with 1 N HCl. The mixture was
extracted with DCM, the extract was dried over Na2SO4, and con-
centrated. The residue was purified by Biotage on reverse phase
column (MeCN in water, 20–100%) to give a mixture of E and Z iso-
mers of the product 13 (0.18 g, 61%) (NMR ratio 1:0.21) as a white
solid. 1H NMR (300 MHz, CDCl3) d: 11.00 (b s, 1H), 7.52–6.88 (m,
6H), 5.86 (d, J = 15.6 Hz, 1H), 2.87–2.72 (m, 2H), 2.66–2.49 (m,
2H). LCMS (ESI) m/z: 177.03 [M+H]+ (E isomer, content 65%),
177.05 [M+H]+ (Z isomer, content 23%).
Compound 16 was prepared in the same manner as compound
7a using 1-chloro-2-iodobenzene (14) (0.50 g, 2.10 mmol), triethy-
lamine (2.92 ml, 20.96 mmol), pent-4-ynoic acid (15) (0.21 ml,
3.51 mmol), [Pd(PPh3)4] (73 mg, 0.06 mmol), and CuI (24 mg,
0.13 mmol). The reaction was stirred for 18 h at rt. The product
16 (92 mg, 21%) was obtained as
a
brown solid. 1H NMR
(300 MHz, CDCl3) d: 10.15 (b s, 1H), 7.44–7.40 (m, 1H), 7.39–7.34
(m, 1H), 7.24–7.13 (m, 2H), 2.85–2.74 (m, 2H), 2.77–2.70 (m,
2H). LCMS (ESI) m/z: 207.03 [MꢃH]ꢃ.
5.1.14. Ethyl 2-(5-(2-chlorophenyl)pent-4-ynamido)cyclohex-1-ene-
1-carboxylate (17d)
Acid 16 (0.10 g, 0.48 mmol) in DCM (2 ml) under argon atmo-
sphere at rt was treated with a catalytic amount of DMF (1 drop)
and oxalyl chloride (0.12 ml, 1.44 mmol). The mixture was stirred
for 20 min and the volatiles were evaporated. The residue was dis-
solved in DCM (2 ml) and cooled to 0 °C, to this solution a mixture
of ethyl 2-aminocyclohex-1-ene-1-carboxylate (0.12 g, 0.72 mmol)
and triethylamine (67 ll, 0.48 mmol) in DCM (1 ml) was added
dropwise over 5 min. The mixture was stirred for 40 min at rt,
diluted with DCM, washed with NH4Cl, brine, dried over Na2SO4,
and concentrated. The residue was chromatographed on silica gel
(EtOAc in petroleum ether, 1:10 to 1:6) to give the product 17d
(92 mg, 53%) as a colorless solid. 1H NMR (300 MHz, CDCl3) d:
11.72 (s, 1H), 7.47–7.34 (m, 2H), 7.25–7.13 (m, 2H), 4.18 (q,
Please cite this article in press as: Bobileva O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.028

12
O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
J = 7.1 Hz, 2H), 3.05–2.96 (m, 2H), 2.88–2.79 (m, 2H), 2.72–2.64 (m,
2H), 2.37–2.30 (m, 2H), 1.72–1.57 (m, 4H), 1.31 (t, J = 7.1 Hz, 3H).
LCMS (ESI) m/z 360.28 [M+H]+.
obtained as a yellow oil. 1H NMR (300 MHz, CDCl3) d: 11.69 (s,
1H), 7.25 (d, J = 8.7 Hz, 2H), 6.73 (d, J = 8.7 Hz, 2H), 4.16 (q,
J = 7.1 Hz, 2H), 3.02–2.94 (m, 2H), 2.77–2.69 (m, 2H), 2.65–2.58
(m, 2H), 2.35–2.26 (m, 2H), 1.70–1.55 (m, 4H), 1.29 (t, J = 7.1 Hz,
3H), 0.97 (s, 9H), 0.18 (s, 6H). LCMS (ESI) m/z: 456.44 [M+H]+.
5.1.15. Ethyl 2-(pent-4-ynamido)cyclohex-1-ene-1-carboxylate (18)
4-Pentynoic acid (15) (1.00 g, 10.19 mmol) in DCM (20 ml)
under argon atmosphere at rt was treated with DMF (39 ll,
5.1.16.3. Ethyl 2-(5-(2-fluorophenyl)pent-4-ynamido)cyclohex-1-ene-
1-carboxylate (17e). Compound 17e was prepared in the same
manner as compound 17a using 1-fluoro-2-iodobenzene (56 mg,
0.25 mmol), acetylene 18 (55 mg, 0.22 mmol), triethylamine
(0.15 ml, 1.07 mmol), PdCl2[PPh3]2 (8 mg, 0.01 mmol), and CuI
(2 mg, 0.01 mmol). The product 17e (54 mg, 71%) was obtained
as a brown solid. 1H NMR (300 MHz, CDCl3) d: 11.70 (s, 1H), 7.37
(td, J = 7.6, 1.9 Hz, 2H), 7.29–7.19 (m, 1H), 7.09–6.98 (m, 2H),
4.16 (q, J = 7.1 Hz, 2H), 3.01–2.95 (m, 2H), 2.85–2.76 (m, 2H),
2.71–2.60 (m, 2H), 2.35–2.27 (m, 2H), 1.68–1.53 (m, 4H), 1.28 (t,
J = 7.1 Hz, 4H). LCMS (ESI) m/z: 344.23 [M+H]+.
0.51 mmol) and oxalyl chloride (2.22 ml, 25.48 mmol). The mix-
ture was stirred for 40 min and the volatiles were evaporated.
The residue was dissolved in DCM (3 ml) and the obtained solution
was added to a mixture of ethyl 2-aminocyclohex-1-ene-1-car-
boxylate (0.10 g, 0.59 mmol) and DIPEA (1.76 ml, 10.2 mmol) in
DCM (15 ml) at ꢃ5 °C to 2 °C under argon atmosphere. The mixture
was stirred for 1 h at 0 °C and warmed to rt. The mixture was
diluted with DCM, washed with saturated NH4Cl, brine, and dried
over Na2SO4. The solvent was evaporated and the residue was
chromatographed on silica gel (EtOAc in petroleum ether, 1:20 to
1:10) to afford a white solid. Recrystallization from n-heptane gave
the product 18 (1.20 g). The filtrate after crystallization was con-
centrated and the residue was chromatographed on Biotage (EtOAc
in petroleum ether, 2–20%) to give the product (0.25 g); the com-
bined yield was 1.45 g, 57%. 1H NMR (300 MHz, CDCl3) d: 11.66
(s, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.03–2.92 (m, 2H), 2.65–2.49 (m,
4H), 2.36–2.25 (m, 2H), 1.97 (t, J = 2.3 Hz, 1H), 1.70–1.54 (m, 4H),
1.30 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) d: 170.1, 169.6,
152.1, 104.9, 82.8, 69.2, 60.4, 37.2, 28.7, 24.3, 22.0, 21.8, 14.6,
14.4. LCMS (ESI) m/z 250.18 [M+H]+.
5.1.16.4. Ethyl 2-(5-(4-((tert-butyldimethylsilyl)oxy)-2-fluorophenyl)
pent-4-ynamido)cyclohex-1-ene-1-carboxylate (17f). Compound 17f
was prepared in the same manner as compound 17a using iodide
6g (0.22 g, 0.63 mmol), acetylene 18 (0.13 g, 0.52 mmol), triethy-
lamine (0.36 ml, 2.61 mmol), PdCl2[PPh3]2 (18 mg, 0.03 mmol),
and CuI (5 mg, 0.03 mmol). The product 17f (0.15 g, 62%) was
obtained as a colorless oil. 1H NMR (400 MHz, CDCl3) d: 11.68 (s,
1H), 7.21 (t, J = 8.5 Hz, 1H), 6.56–6.49 (m, 2H), 4.16 (q, J = 7.1 Hz,
2H), 3.01–2.95 (m, 2H), 2.78 (t, J = 7.3 Hz, 2H), 2.67–2.60 (m, 2H),
2.35–2.27 (m, 2H), 1.67–1.55 (m, 4H), 1.29 (t, J = 7.1 Hz, 3H), 0.96
(s, 9H), 0.19 (s, 6H). LCMS (ESI) m/z: 358.00 [M-C6H15Si]+.
5.1.16. Ethyl 2-(5-(2-((tert-butyldimethylsilyl)oxy)phenyl)pent-4-
ynamido)cyclohex-1-ene-1-carboxylate (17a)
To a degassed solution of tert-butyl(2-iodophenoxy)dimethylsi-
lane (6a) (0.21 g, 0.63 mmol), ethyl 2-(pent-4-ynamido)cyclohex-
1-ene-1-carboxylate (18) (0.13 g, 0.52 mmol), and triethylamine
(0.36 ml, 2.61 mmol) in MeCN (1 ml) under argon atmosphere
PdCl2[PPh3]2 (18 mg, 0.03 mmol) and CuI (5 mg, 0.03 mmol) were
added. The mixture was stirred for 15 h at rt. The mixture was con-
centrated, diluted with saturated NH4Cl solution, extracted with
DCM (20 ml), dried over Na2SO4, and concentrated. After a silica
gel chromatography on Biotage (EtOAc in petroleum ether, 3–
30%) the product 17a (0.12 g, 52%) was obtained as a yellow oil.
1H NMR (300 MHz, CDCl3) d: 11.69 (s, 1H), 7.32 (dd, J = 7.6,
1.8 Hz, 1H), 7.14 (ddd, J = 8.1, 7.6, 1.8 Hz, 1H), 6.86 (td, J = 7.6,
1.1 Hz, 1H), 6.78 (dd, J = 8.2, 1.1 Hz, 1H), 4.16 (q, J = 7.1 Hz, 2H),
3.05–2.88 (m, 2H), 2.86–2.70 (m, 2H), 2.70–2.54 (m, 2H), 2.37–
2.20 (m, 2H), 1.68–1.54 (m, 4H), 1.29 (t, J = 7.1 Hz, 3H), 1.03 (s,
9H), 0.21 (s, 6H). LCMS (ESI) m/z: 456.46 [M+H]+.
5.1.16.5. Ethyl 2-(5-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)
pent-4-ynamido)cyclohex-1-ene-1-carboxylate
(17g). Compound
17g was prepared in the same manner as compound 17a using
iodide 6h (0.23 g, 0.63 mmol), acetylene 18 (0.13 g, 0.52 mmol),
triethylamine (0.36 ml, 2.61 mmol), PdCl2[PPh3]2 (18 mg,
0.03 mmol), and CuI (5 mg, 0.03 mmol). The product 17g (94 mg,
37%) was obtained as a colorless oil. 1H NMR (300 MHz, CDCl3) d:
11.69 (s, 1H), 7.27 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 2.4 Hz, 1H), 6.64
(dd, J = 8.4, 2.4 Hz, 1H), 4.16 (q, J = 7.1 Hz, 2H), 3.04–2.93 (m, 2H),
2.85–2.73 (m, 2H), 2.68–2.61 (m, 2H), 2.37–2.25 (m, 2H), 1.68–
1.54 (m, 4H), 1.29 (t, J = 7.1 Hz, 2H), 0.96 (s, 9H), 0.19 (s, 6H). LCMS
(ESI) m/z: 490.39 [M+H]+.
5.1.16.6. Ethyl 2-(5-(5-hydroxypyridin-2-yl)pent-4-ynamido)cyclo-
hex-1-ene-1-carboxylate (17h). Compound 17h was prepared in
the same manner as compound 17a using iodide 6i (0.23 g,
0.68 mmol), acetylene 18 (0.13 g, 0.52 mmol), triethylamine
(0.36 ml, 2.61 mmol), PdCl2[PPh3]2 (18 mg, 0.03 mmol), and CuI
(5 mg, 0.03 mmol). The product 17h (74 mg, 41%) was obtained
as a yellow solid. 1H NMR (300 MHz, CDCl3) d: 11.74 (s, 1H), 8.47
(dd, J = 4.8, 1.3 Hz, 1H), 7.65 (dt, J = 8.3, 1.1 Hz, 1H), 7.13 (dd,
J = 8.3, 4.8 Hz, 1H), 6.67 (d, J = 0.8 Hz, 1H), 4.16 (q, J = 7.1 Hz, 1H),
3.20 (t, J = 7.5 Hz, 2H), 3.00–2.91 (m, 2H), 2.80 (t, J = 7.5 Hz, 1H),
2.36–2.24 (m, 2H), 1.69–1.53 (m, 4H), 1.29 (t, J = 7.1 Hz, 2H). LCMS
(ESI) m/z: 343.16 [M+H]+.
5.1.16.1. Ethyl 2-(5-(3-((tert-butyldimethylsilyl)oxy)phenyl)pent-4-
ynamido)cyclohex-1-ene-1-carboxylate (17b). Compound (17b)
was prepared in the same manner as compound 17a using iodide
6j (0.21 g, 0.63 mmol), acetylene 18 (0.13 g, 0.52 mmol), triethy-
lamine (0.36 ml, 2.61 mmol), PdCl2[PPh3]2 (18 mg, 0.03 mmol),
and CuI (5 mg, 0.03 mmol). The product 17b (0.19 g, 79%) was
obtained as a yellow oil. 1H NMR (300 MHz, CDCl3) d: 11.70 (s,
1H), 7.11 (t, J = 7.9 Hz, 1H), 6.97 (dt, J = 7.6, 1.2 Hz, 1H), 6.85 (d,
J = 2.2 Hz, 1H), 6.75 (ddd, J = 8.1, 2.5, 1.0 Hz, 1H), 4.16 (q,
J = 7.1 Hz, 2H), 3.03–2.93 (m, 2H), 2.80–2.70 (m, 2H), 2.68–2.57
(m, 2H), 2.36–2.26 (m, 2H), 1.69–1.54 (m, 4H), 1.28 (t, J = 7.1 Hz,
3H), 0.97 (s, 9H), 0.18 (s, 6H). LCMS (ESI) m/z: 456.41 [M+H]+.
5.1.16.7.
Methyl
4-(5-((2-(ethoxycarbonyl)cyclohex-1-en-1-yl)
amino)-5-oxopent-1-yn-1-yl)benzoate (17i). Compound 17i was
prepared in the same manner as compound 17a using methyl 4-
iodobenzoate (0.16 g, 0.63 mmol), acetylene 18 (0.13 g,
0.52 mmol), triethylamine (0.36 ml, 2.61 mmol), PdCl2[PPh3]2
(18 mg, 0.03 mmol), and CuI (5 mg, 0.03 mmol). The product 17i
(0.18 g, 90%) was obtained as a yellow solid. 1H NMR (300 MHz,
CDCl3) d: 11.72 (s, 1H), 7.94 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 8.6 Hz,
1H), 4.15 (q, J = 7.1 Hz, 2H), 3.91 (s, 3H), 3.03–2.93 (m, 2H), 2.84–
5.1.16.2. Ethyl 2-(5-(4-((tert-butyldimethylsilyl)oxy)phenyl)pent-4-
ynamido)cyclohex-1-ene-1-carboxylate (17c). Compound 17c was
prepared in the same manner as compound 17a using iodide 6c
(0.16 g, 0.48 mmol), acetylene 18 (0.10 g, 0.40 mmol), triethy-
lamine (0.28 ml, 2.00 mmol), PdCl2[PPh3]2 (14 mg, 0.02 mmol),
and CuI (4 mg, 0.02 mmol). The product 17c (94 mg, 51%) was
Please cite this article in press as: Bobileva O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.028

O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
13
2.72 (m, 2H), 2.70–2.58 (m, 2H), 2.36–2.26 (m, 2H), 1.70–1.55 (m,
CDCl3) d: 11.45 (s, 1H), 10.10 (pl s, 1H), 7.43–7.39 (m, 1H), 7.37–
7.33 (m, 1H), 7.20 (td, J = 7.5, 2.1 Hz, 1H), 7.16 (td, J = 7.5, 1.7 Hz,
1H), 3.06–2.99 (m, 2H), 2.83 (t, J = 7.0 Hz, 2H), 2.66 (t, J = 7.0 Hz,
2H), 2.39–2.32 (m, 2H), 1.70–1.55 (m, 4H). 13C NMR (100 MHz,
CDCl3) d: 174.4, 169.8, 155.0, 135.9, 133.5, 129.2, 128.9, 126.4,
123.5, 103.9, 93.8, 78.5, 37.5, 29.0, 24.4, 21.9, 21.7, 15.9. HRMS
(ESI) m/z: calcd. for C18H19ClNO3 [M+H]+ 332.1048 [M+H]+, found
332.1049.
4H), 1.28 (t, J = 7.1 Hz, 3H). LCMS (ESI) m/z: 384.39 [M+H]+.
5.1.16.8. Ethyl 2-(5-(4-fluorophenyl)pent-4-ynamido)cyclohex-1-ene-
1-carboxylate (17j). Compound 17j was prepared in the same man-
ner as compound 17a using 1-fluoro-4-iodobenzene (0.14 g,
0.63 mmol), acetylene 18 (0.13 g, 0.52 mmol), triethylamine
(0.36 ml, 2.61 mmol), PdCl2[PPh3]2 (18 mg, 0.03 mmol), and CuI
(5 mg, 0.03 mmol). The product 17j (0.14 g, 78%) was obtained as
a white solid. 1H NMR (300 MHz, CDCl3) d: 11.70 (s, 1H), 7.40–
7.30 (m, 2H), 7.02–6.91 (m, 2H), 4.16 (q, J = 7.1 Hz, 2H), 3.04–
2.92 (m, 2H), 2.81–2.68 (m, 2H), 2.68–2.56 (m, 2H), 2.37–2.26
(m, 2H), 1.71–1.55 (m, 4H), 1.29 (t, J = 7.1 Hz, 3H). LCMS (ESI) m/
z: 344.29 [M+H]+.
5.1.17.4.
2-(5-(2-Fluorophenyl)pent-4-ynamido)cyclohex-1-ene-1-
carboxylic acid (4e). Compound 4e was prepared in the same man-
ner as compound 4a using ethyl carboxylate 17e (95 mg,
0.28 mmol) and 2 N NaOH (0.42 ml, 0.84 mmol). The product 4e
(26 mg, 30%) was obtained as a white solid. 1H NMR (300 MHz,
CDCl3) d: 11.44 (s, 1H), 10.33 (b s, 1H), 7.37 (td, J = 7.6, 1.9 Hz,
1H), 7.29–7.20 (m, 1H), 7.08–7.04 (m, 1H), 7.04–6.99 (m, 1H),
3.06–2.95 (m, 2H), 2.85–2.75 (m, 2H), 2.70–2.60 (m, 2H), 2.42–
2.26 (m, 2H), 1.72–1.51 (m, 4H). 13C NMR (100 MHz, CDCl3) d:
174.4, 169.8, 162.9 (d, JCF = 250.7 Hz), 154.9, 133.7, 129.6 (d,
JCF = 8.0 Hz), 123.9 (d, JCF = 3.7 Hz), 115.5 (d, JCF = 21.0 Hz), 112.1
(d, JCF = 15.9 Hz), 103.8, 93.6 (d, JCF = 3.4 Hz), 75.0, 37.5, 29.0,
24.4, 21.9, 21.7, 15.8. HRMS (ESI) m/z: calcd. for C18H19FNO3 [M
+H]+ 316.1343, found 316.1344.
5.1.17. 2-(5-(2-Hydroxyphenyl)pent-4-ynamido)cyclohex-1-ene-1-
carboxylic acid (4a)
To a solution of ethyl carboxylate 17a (0.12 g, 0.26 mmol) in
THF (1 ml) and EtOH (1 ml) 2 N NaOH solution (0.40 ml,
0.80 mmol) was added and the mixture was stirred for 70 h at rt.
The mixture was acidified with 1 N HCl to pH 2, concentrated in
vacuo to ꢄ 2 ml, extracted with DCM, dried over Na2SO4, and con-
centrated in vacuo. The residue was chromatographed on Biotage
reverse phase column (MeCN in water, 10–100%) to give the pro-
duct 4a (23 mg, 28%) as a white solid. 1H NMR (300 MHz, DMSO-
d6) d: 12.46 (b s, 1H), 11.66 (s, 1H), 9.65 (b s, 1H), 7.19 (dd,
J = 7.6, 1.7 Hz, 1H), 7.12 (ddd, J = 8.2, 7.3, 1.7 Hz, 1H), 6.83 (dd,
J = 8.2, 1.1 Hz, 1H), 6.72 (td, J = 7.5, 1.1 Hz, 1H), 2.87–2.78 (m,
2H), 2.66 (t, J = 6.9 Hz, 2H), 2.58–2.52 (m, 2H) 2.26–2.19 (m, 2H),
1.59–1.47 (m, 4H). 13C NMR (100 MHz, DMSO-d6) d: 171.2, 169.1,
158.1, 150.4, 132.9, 129.2, 118.9, 115.4, 110.3, 105.1, 92.2, 77.7,
36.8, 28.1, 24.4, 21.5, 21.4, 15.2. HRMS (ESI) m/z: calcd. for
5.1.17.5. 2-(5-(2-Fluoro-4-hydroxyphenyl)pent-4-ynamido)cyclohex-
1-ene-1-carboxylic acid (4f). Compound 4f was prepared in the
same manner as compound 4a using ethyl carboxylate 17f
(0.15 g, 0.32 mmol) and 2 N NaOH (0.48 ml, 0.96 mmol). The pro-
duct 4f (46 mg, 44%) was obtained as a white solid. 1H NMR
(400 MHz, DMSO-d6) d: 12.51 (b s, 1H), 11.63 (s, 1H), 10.24 (b s,
1H), 7.21 (t, J = 8.7 Hz, 1H), 6.62–6.64 (m, 2H), 2.87–2.78 (m, 2H),
2.65 (t, J = 7.1 Hz, 2H), 2.58–2.50 (m, 2H, overlapped with DMSO),
2.27–2.18 (m, 2H), 1.61–1.46 (m, 4H). 13C NMR (100 MHz,
DMSO-d6) d: 171.1, 169.0, 162.9 (d, JCF = 247.5 Hz), 159.0 (d,
JCF = 11.7 Hz), 150.3, 134.0 (d, JCF = 3.4 Hz), 111.9 (d, JCF = 2.8 Hz),
105.0, 102.8 (d, JCF = 23.2 Hz), 101.3 (d, JCF = 15.8 Hz), 91.9, 74.4,
C
18H20NO4 [M+H]+ 314.1387, found 314.1393.
5.1.17.1. 2-(5-(3-Hydroxyphenyl)pent-4-ynamido)cyclohex-1-ene-1-
carboxylic acid (4b). Compound 4b was prepared in the same man-
ner as compound 4a using ethyl carboxylate 17b (0.17 g,
0.37 mmol) and 2 N NaOH (0.55 ml, 1.10 mmol). The product 4b
(55 mg, 47%) was obtained as a white solid. 1H NMR (300 MHz,
DMSO-d6) d: 11.67 (s, 1H), 9.61 (pl s, 1H), 7.11 (td, J = 7.5, 1.2 Hz,
1H), 6.77 (dt, J = 7.5, 1.2 Hz, 1H), 6.75–6.71 (m, 1H), 6.72 (d,
J = 1.2 Hz, 1H), 2.86–2.80 (m, 2H), 2.64 (t, J = 6.8 Hz, 2H), 2.54 (t,
J = 6.8 Hz, 2H), 2.27 – 2.19 (m, 2H), 1.60–1.46 (m, 4H). 13C NMR
(100 MHz, DMSO-d6) d: 171.2, 169.0, 157.2, 150.2, 129.6, 123.9,
122.1, 117.8, 115.5, 105.2, 88.6, 81.0, 36.6, 28.1, 24.4, 21.5, 21.4,
14.9. HRMS (ESI) m/z: calcd. for C18H20NO4 [M+H]+ 314.1387, found
314.1388.
36.6, 28.1, 24.4, 21.5, 21.4, 15.0. HRMS (ESI) m/z: calcd. for C18H19
-
FNO4 [M+H]+ 332.1293, found 332.1303.
5.1.17.6. 2-(5-(2-Chloro-4-hydroxyphenyl)pent-4-ynamido)cyclohex-
1-ene-1-carboxylic acid (4g). Compound 4g was prepared in the
same manner as compound 4a using ethyl carboxylate 17g
(94 mg, 0.19 mmol) and 2 N NaOH (0.29 ml, 0.58 mmol). The pro-
duct 4g (29 mg, 44%) was obtained as a white solid. 1H NMR
(400 MHz, DMSO-d6) d: 12.55 (b s, 1H), 11.67 (s, 1H), 10.21 (b s,
1H), 7.27 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 2.4 Hz, 1H), 6.69 (dd,
J = 8.5, 2.4 Hz, 1H), 2.90–2.78 (m, 2H), 2.67 (t, J = 7.0 Hz, 2H), 2.53
(t, J = 7.0 Hz, 2H, overlapped with DMSO), 2.29–2.16 (m, 2H),
1.62–1.43 (m, 4H). 13C NMR (100 MHz, DMSO-d6) d: 171.1, 169.0,
158.1, 150.4, 135.2, 134.2, 115.9, 114.7, 112.9, 105.0, 91.8, 77.8,
5.1.17.2. 2-(5-(4-Hydroxyphenyl)pent-4-ynamido)cyclohex-1-ene-1-
carboxylic acid (4c). Compound 4c was prepared in the same man-
ner as compound 4a using ethyl carboxylate 17c (90 mg,
0.20 mmol) and 2 N NaOH (0.31 ml, 0.62 mmol). The product 4c
(26 mg, 41%) was obtained as a white solid. 1H NMR (400 MHz,
DMSO-d6) d: 12.55 (b s, 1H), 11.65 (s, 1H), 9.72 (b s, 1H), 7.23–
7.12 (m, 2H), 6.74–6.65 (mz, 2H), 2.87–2.80 (m, 2H), 2.61 (t,
J = 6.8 Hz, 2H), 2.54–2.49 (m, 2H, overlapped with DMSO), 2.26–
2.18 (m, 2H), 1.62–1.44 (m, 4H). 13C NMR (100 MHz, DMSO-d6)
d: 171.1, 169.1, 157.3, 150.4, 132.7, 115.5, 113.2, 105.0, 86.5,
81.2, 36.9, 28.1, 24.4, 21.5, 21.4, 15.0. HRMS (ESI) m/z: calcd. for
36.8, 28.1, 24.4, 21.5, 21.4, 15.2. HRMS (ESI) m/z: calcd. for C18H19
-
ClNO4 [M+H]+ 348.0997, found 348.0988.
5.1.17.7. 2-(5-(5-Hydroxypyridin-2-yl)pent-4-ynamido)cyclohex-1-
ene-1-carboxylic acid (4h). Compound 4h was prepared in the same
manner as compound 4a using ethyl carboxylate 17h (73 mg,
0.21 mmol) and 2 N NaOH (0.32 ml, 0.64 mmol). The product 4h
(18 mg, 27%) was obtained as a white solid. 1H NMR (300 MHz,
DMSO-d6) d: 12.57 (b s, 1H), 11.65 (s, 1H), 8.49 (b s, 1H). 7.91 (d,
J = 8.3 Hz, 1H), 7.25 (dd, J = 8.3, 4.6 Hz, 1H), 6.80 (s, 1H), 3.10 (t,
J = 7.3 Hz, 2H), 2.86–2.70 (m, 4H), 2.26–2.15 (m, 2H), 1.62–1.45
(m, 4H). 13C NMR (100 MHz, DMSO-d6) d: 171.1, 169.1, 162.5,
150.3, 148.3, 146.9, 145.4, 118.4, 117.5, 105.2, 103.4, 34.6, 28.1,
24.4, 23.7, 21.4, 21.3. HRMS (ESI) m/z: calcd. for C17H19N2O4 [M
+H]+ 315.1339, found 315.1340.
C
18H20NO4 [M+H]+ 314.1387, found 314.1402.
5.1.17.3. 2-(5-(2-Chlorophenyl)pent-4-ynamido)cyclohex-1-ene-1-
carboxylic acid (4d). Compound 4d was prepared in the same man-
ner as compound 4a using ethyl carboxylate 17d (90 mg,
0.25 mmol) and 2 N NaOH (0.38 ml, 0.75 mmol). The product 4d
(38 mg, 46%) was obtained as white solid. 1H NMR (300 MHz,
Please cite this article in press as: Bobileva O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.028

14
O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5.1.17.8. 4-(5-((2-Carboxycyclohex-1-en-1-yl)amino)-5-oxopent-1-
yn-1-yl)benzoic acid (4i). Compound 4i was prepared in the same
manner as compound 4a using ethyl carboxylate 17i (0.18 g,
0.47 mmol) and 2 N NaOH (0.70 ml, 1.40 mmoil). The product 4i
(86 mg, 54%) was obtained as a white solid. 1H NMR (400 MHz,
DMSO-d6) d: 12.81 (s, 2H), 11.65 (s, 1H), 7.88 (d, J = 8.4 Hz, 2H),
7.46 (d, J = 8.4 Hz, 2H), 2.87–2.79 (m, 2H), 2.74–2.66 (m, 2H),
2.62–2.54 (m, 2H), 2.27–2.19 (m, 2H), 1.61–1.45 (m, 4H). 13C
NMR (100 MHz, DMSO-d6) d: 171.1, 169.0, 166.7, 150.3, 131.4,
130.0, 129.4, 127.4, 105.1, 92.5, 80.3, 36.4, 28.1, 24.4, 21.5, 21.4,
15.0. HRMS (ESI) m/z: calcd. for C19H20NO5 [M+H]+ 342.1336, found
342.1314.
without further purification. 1H NMR (300 MHz, CDCl3) d: 11.67
(s, 1H), 7.51–7.27 (m, 8H), 4.19 (q, J = 7.1 Hz, 3H), 3.11–3.03 (m,
2H), 3.03–2.96 (m, 2H), 2.74–2.65 (m, 2H), 2.36–2.29 (m, 2H),
1.74–1.53 (m, 4H), 1.31 (t, J = 7.1 Hz, 3H). LCMS (ESI) m/z: 412.21
[M+H]+.
5.1.21. Ethyl 2-(3-(4-iodophenyl)propanamido)cyclohex-1-ene-1-
carboxylate (23)
Compound 23 was prepared in the same manner as compound
18 using 3-(4-iodophenyl)propanoic acid (19) (0.79 g, 2.86 mmol),
oxalyl chloride (0.62 ml, 7.15 mmol), ethyl 2-aminocyclohex-1-
ene-1-carboxylate (0.73 g, 4.29 mmol), and triethylamine
(0.40 ml, 2.86 mmol). The product 23 (0.53 g, 43%) was obtained
as a colorless solid. 1H NMR (300 MHz, CDCl3) d: 11.60 (s, 1H),
7.62–7.56 (m, 2H), 7.06–6.89 (m, 2H), 4.16 (q, J = 7.1 Hz, 2H),
3.00–2.85 (m, 4H), 2.60 (t, J = 7.7 Hz, 2H), 2.35–2.26 (m, 2H),
1.66–1.52 (m, 4H), 1.29 (t, J = 7.1 Hz, 2H). LCMS (ESI) m/z: 428.14
[M+H]+.
5.1.17.9.
2-(5-(4-Fluorophenyl)pent-4-ynamido)cyclohex-1-ene-1-
carboxylic acid (4j). Compound 4j was prepared in the same man-
ner as compound 4a using ethyl carboxylate 17j (0.13 g,
0.38 mmol) and 2 N NaOH (0.57 ml, 1.14 mmol). The product 4j
(63 mg, 53%) was obtained as a white solid. 1H NMR (400 MHz,
CDCl3) d: 11.50 (b s, 1H), 11.43 (s, 1H), 7.38–7.31 (m, 2H), 7.00–
6.90 (m, 2H), 3.06–2.97 (m, 2H), 2.79–2.70 (m, 2H), 2.67–2.58
(m, 2H), 2.40–2.27 (m, 2H), 1.70–1.56 (m, 4H). 13C NMR
(100 MHz, CDCl3) d: 174.5, 169.9, 162.3 (d, JCF = 248.5 Hz), 155.1,
133.5 (d, JCF = 8.3 Hz), 119.7 (d, JCF = 3.4 Hz), 115.5 (d, JCF = 21.8 Hz),
103.7, 87.8, 80.6, 37.6, 29.0, 24.4, 21.9, 21.7, 15.6. HRMS (ESI) m/z:
calcd. for C18H19FNO3 [M+H]+ 316.1343, found 316.1349.
5.1.22.1. Ethyl 2-(3-(20-hydroxy-[1,10-biphenyl]-4-yl)propanamido)-
cyclohex-1-ene-1-carboxylate (22a). Compound 22a was prepared
in the same manner as compound 21 using iodide 23 (0.16 g,
0.37 mmol), 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phe-
nol (0.11 g, 0.49 mmol), 2 M K2CO3 (0.56 ml, 1.12 mmol), and Pd
(dppf)Cl2 (14 mg, 0.02 mmol). The product 28a (0.11 g, 77%) was
obtained as a colorless solid. 1H NMR (300 MHz, CDCl3) d: 11.62
(s, 1H), 7.39 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 7.28–7.23
(m, 1H), 7.22 (dd, J = 7.9, 1.8 Hz, 1H), 6.98 (td, J = 7.5, 1.2 Hz, 1H),
6.98 (dd, J = 8.0, 1.2 Hz, 1H), 5.22 (s, 1H), 4.17 (q, J = 7.1 Hz, 2H),
3.11–3.00 (m, 2H), 3.01–2.93 (m, 2H), 2.73–2.64 (m, 2H), 2.35–
2.26 (m, 2H), 1.67–1.55 (m, 4H), 1.29 (t, J = 7.1 Hz, 3H). LCMS
(ESI) m/z: 394.37 [M+H]+.
5.1.18. 3-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)
propanoic acid (20)
A suspension of 3-(4-iodophenyl)propanoic acid (19) (0.50 g,
1.81 mmol), bis(pinacolato)diboron (0.69 g, 2.72 mmol), and KOAc
(0.71 g, 7.24 mmol) in DMF (1 ml) was degassed by bubbling argon
through the mixture for 15 min, then Pd(dppf)Cl2 (66 mg,
0.09 mmol) was added to this mixture. The reaction was heated
to 80 °C for 2.5 h. The reaction mixture was cooled to rt, filtered
through a pad of silica gel, and washed with EtOAc. The filtrate
was concentrated under reduced pressure and purified by column
chromatography on silica gel (EtOAc in petroleum ether, 1:2) to
give the product 20 (0.32 g, 63%) as a white solid. 1H NMR
(300 MHz, CDCl3) d: 10.43 (b s, 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.22
(d, J = 8.0 Hz, 2H), 2.98 (t, J = 7.8 Hz, 2H), 2.68 (t, J = 7.8 Hz, 2H),
1.34 (s, 12H). LCMS (ESI) m/z: 277.09 [M+H]+.
5.1.22.2. Ethyl 2-(3-(30-hydroxy-[1,10-biphenyl]-4-yl)propanamido)-
cyclohex-1-ene-1-carboxylate (22b). Compound 22b was prepared
in the same manner as compound 21 using iodide 23 (0.14 g,
0.33 mmol), (3-hydroxyphenyl)boronic acid (59 mg, 0.43 mmol),
2 M K2CO3 (0.49 ml, 0.98 mmol), and Pd(dppf)Cl2 (12 mg,
0.02 mmol). The product 22b (96 mg, 75%) was obtained as a color-
less solid. 1H NMR (300 MHz, CDCl3) d: 11.65 (s, 1H), 7.48 (d,
J = 8.1 Hz, 2H), 7.34–7.22 (m, 3H), 7.14 (ddd, J = 7.8, 1.5, 0.9 Hz,
1H), 7.04 (dd, J = 2.4, 1.5 Hz, 1H), 6.80 (ddd, J = 8.0, 2.4, 0.9 Hz,
1H), 5.02 (s, 1H), 4.16 (q, J = 7.1 Hz, 2H), 3.07–2.92 (m, 4H), 2.67
(t, J = 7.8 Hz, 2H), 2.34–2.25 (m, 2H), 1.70–1.52 (m, 4H), 1.28 (t,
J = 7.1 Hz, 3H). LCMS (ESI) m/z: 394.38 [M+H]+.
5.1.19. 3-(20-Chloro-[1,10-biphenyl]-4-yl)propanoic acid (21)
A solution of boronate 20 (0.16 g, 0.58 mmol) and 1-chloro-2-
iodobenzene (0.14 g, 0.59 mmol) in dioxane (2.4 ml) was degassed
by bubbling argon through the mixture for 15 min, then 2 M K2CO3
(0.87 ml, 1.74 mmol) and Pd(dppf)Cl2 (21 mg, 0.03 mmol) under
argon atmosphere were added. The reaction flask was sealed and
stirred for 2 h at 80 °C. The mixture was cooled to rt, diluted with
saturated NH4Cl solution, extracted with EtOAc, and the organic
phase was dried over Na2SO4. The extract was concentrated and
the residue was purified by column chromatography on silica gel
(MeOH in DCM, 1:20) to give the product 21 (0.14 g, 91%) as a
white solid. 1H NMR (300 MHz, CDCl3) d: 10.70 (b s, 1H), 7.53–
7.23 (m, 8H), 3.03 (t, J = 7.8 Hz, 2H), 2.76 (t, J = 7.8 Hz, 2H). LCMS
(ESI) m/z: 259.04 [M+H]+.
5.1.22.3. Ethyl 2-(3-(40-hydroxy-[1,10-biphenyl]-4-yl)propanamido)-
cyclohex-1-ene-1-carboxylate (22c). Compound 22c was prepared
in the same manner as compound 21 using iodide 23 (0.14 g,
0.33 mmol), (4-hydroxyphenyl)boronic acid (59 mg, 0.43 mmol),
2 M K2CO3 (0.49 ml, 0.98 mmol), and Pd(dppf)Cl2 (12 mg,
0.02 mmol). The product 22c (0.11 g, 82%) was obtained as a color-
less solid. 1H NMR (300 MHz, CDCl3) d: 11.65 (s, 1H), 7.48–7.41 (m,
4H), 7.28–7.22 (m, 2H overlapped with CHCl3), 6.91–6.85 (m, 2H),
5.10 (s, 1H), 4.17 (q, J = 7.1 Hz, 2H), 3.08–2.93 (m, 4H), 2.73–2.61
(m, 2H), 2.35–7.26 (m, 2H), 1.71–1.52 (m, 4H), 1.29 (t, J = 7.1 Hz,
3H). LCMS (ESI) m/z: 394.36 [M+H]+.
5.1.20. Ethyl 2-(3-(20-chloro-[1,10-biphenyl]-4-yl)
propanamido)cyclohex-1-ene-1-carboxylate (22d)
5.1.22.4. Ethyl 2-(3-(20-fluoro-[1,10-biphenyl]-4-yl)propanamido)cy-
clohex-1-ene-1-carboxylate (22e). Compound 22e was prepared in
the same manner as compound 21 using iodide 23 (0.14 g,
Compound 22d was prepared in the same manner as compound
18 using acid 21 (0.13 g, 0.50 mmol), oxalyl chloride (0.13 ml,
1.50 mmol), ethyl 2-aminocyclohex-1-ene-1-carboxylate (0.10 g,
0.59 mmol), and triethylamine (0.07 ml, 0.50 mmol). The product
22d (0.14 g) was obtained as a colorless oil which contained start-
ing amine (1H NMR ratio 22d:amine = ca 1:0.3) as an impurity,
calc. yield 58%. The crude product 22d was used in the next step
0.33 mmol),
2-(2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-diox-
aborolane (0.10 g, 0.45 mmol), 2 M K2CO3 (0.49 ml, 0.98 mmol),
and Pd(dppf)Cl2 (19 mg, 0.03 mmol). The product 22e (0.10 g)
was obtained as a colorless oil, which contained unidentified impu-
rities and was used in the next step without further purification. 1H
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O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
15
NMR (300 MHz, CDCl3) d: 11.65 (s, 1H), 7.52–7.10 (m, 8H), 4.17 (q,
J = 7.1 Hz, 2H), 3.10–2.90 (m, 4H), 2.72–2.63 (m, 2H), 2.36–2.24 (m,
2H), 1.70–1.55 (m, 4H), 1.29 (t, J = 7.1 Hz, 3H). LCMS (ESI) m/z:
396.43 [M+H]+.
(ESI) m/z: 364.23 [MꢃH]ꢃ. Anal. Calcd for C22H23NO4 ꢁ 0.15 H2O
(0.7%): C 71.78, H 6.38, N 3.80. Found: C 71.71, H 6.20, N 3.75.
5.1.23.3. 2-(3-(40-Hydroxy-[1,10-biphenyl]-4-yl)propanamido)cyclo-
hex-1-ene-1-carboxylic acid (5c). Compound 5c was prepared in
the same manner as 5d using ethyl carboxylate 22c (0.11 g,
0.28 mmol) and 2 N NaOH (0.40 ml, 0.80 mmol). The product 5c
(60 mg, 62%) was obtained as a white solid. 1H NMR (300 MHz,
DMSO-d6) d: 12.53 (b s, 1H), 11.66 (b s, 1H), 9.48 (s, 1H), 7.51–
7.41 (m, 4H), 7.29–7.22 (m, 2H), 6.85–6.79 (m, 2H), 2.87 (t,
J = 7.5 Hz, 2H), 2.85–2.77 (m, 2H), 2.64–2.56 (m, 2H), 2.25–2.17
(m, 2H), 1.61–1.45 (m, 4H). 13C NMR (100 MHz, DMSO-d6) d:
171.2, 169.9, 156.9, 150.5, 138.8, 138.0, 130.8, 128.7, 127.5,
125.9, 115.7, 104.8, 39.0, 30.0, 28.1, 24.4, 21.5, 21.4. LCMS (ESI)
m/z: 364.20 [MꢃH]ꢃ. Anal. Calcd for C22H23NO4: C 72.31, H 6.34,
N 3.83. Found: C 71.62, H 6.28, N 3.64.
5.1.22.5.
Ethyl
2-(3-(20-fluoro-40-hydroxy-[1,10-biphenyl]-4-yl)
propanamido)cyclohex-1-ene-1-carboxylate (22f). Compound 22f
was prepared in the same manner as compound 21 using iodide
23 (0.18 g, 0.42 mmol), 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-diox-
aborolan-2-yl)phenol (0.13 g, 0.55 mmol), 2 M K2CO3 (0.63 ml,
1.26 mmol), and Pd(dppf)Cl2 (25 mg, 0.03 mmol). The product
22f (0.15 g, 87%) was obtained as a colorless solid. 1H NMR
(300 MHz, CDCl3) d: 11.66 (s, 1H), 7.40 (dd, J = 8.3, 1.7 Hz, 2H),
7.30–7.21 (m, 3H), 6.70–6.62 (m, 2H), 5.34 (s, 1H), 4.17 (q,
J = 7.1 Hz, 2H), 3.06–2.94 (m, 4H), 2.73–2.62 (m, 2H), 2.36–2.26
(m, 2H), 1.69–1.54 (m, 4H), 1.29 (t, J = 7.1 Hz, 3H). LCMS (ESI) m/
z: 412.29 [M+H]+.
5.1.23.4. 2-(3-(20-Fluoro-[1,10-biphenyl]-4-yl)propanamido)cyclohex-
1-ene-1- carboxylic acid (5e). Compound 5e was prepared in the
same manner as compound 5d using ethyl carboxylate 22e
(0.12 g, 0.30 mmol) and 2 N NaOH (0.46 ml, 0.92 mmol). The pro-
duct 5e (59 mg, 53%) was obtained as a white solid. 1H NMR
(300 MHz, CDCl3) d: 11.37 (s, 1H), 10.37 (pl s, 1H), 7.51–7.45 (m,
2H), 7.41 (td, J = 7.7, 1.9 Hz, 1H), 7.33–7.25 (m, 3H), 7.23–7.15
(m, 1H), 7.13 (ddd, J = 10.8, 8.0, 1.3 Hz, 1H), 3.07–2.95 (m, 4H),
2.72–2.63 (m, 2H), 2.38–2.28 (m, 2H), 1.70–1.54 (m, 4H). 13C
5.1.23. 2-(3-(20-Chloro-[1,10-biphenyl]-4-yl)propanamido)cyclohex-1-
ene-1-carboxylic acid (5d)
To a solution of crude ethyl carboxylate 22d (0.13 g, 0.32 mmol)
in THF (1 ml) and EtOH (1 ml) 2 N NaOH solution (0.47 ml,
0.95 mmol) was added and the mixture was stirred for 8 h at
50 °C. The mixture was acidified with 1 N HCl to pH 5, concen-
trated to ꢄ 2 ml, and extracted with CHCl3. The extract was dried
over Na2SO4, concentrated and the residue was chromatographed
on Biotage reverse phase column (MeCN in water, 10–100%) to give
the product 5d (71 mg, 40%) as a white solid. 1H NMR (300 MHz,
CDCl3) d: 11.38 (s, 1H), 10.38 (b s, 1H), 7.48–7.43 (m, 1H), 7.40–
7.35 (m, 2H), 7.33–7.23 (m, 5H), 3.08–2.96 (m, 4H), 2.73–2.63
(m, 2H), 2.38–2.30 (m, 2H), 1.70–1.54 (m, 4H). 13C NMR
(100 MHz, CDCl3) d: 174.6, 171.0, 155.1, 140.4 140.0, 137.5,
132.6, 131.5, 130.1, 129.8, 128.54, 128.2, 126.9, 103.6, 40.1, 31.1,
29.0, 24.4, 21.9, 21.7. LCMS (ESI) m/z: 382.10 [MꢃH]ꢃ. Anal. Calcd
for C22H22ClNO3: C 68.84, H 5.78, N 3.65. Found: C 68.82, H 5.78, N
3.62.
1
NMR (100 MHz, CDCl3) d: 174.4, 170.9, 159.9 (d, JCF = 247.6 Hz),
155.1, 140.2, 133.9 (JCF = 1.0 Hz), 130.8 (d, JCF = 3.5 Hz), 129.3 (d,
JCF = 3.0 Hz), 128.9 (d, JCF = 8.5 Hz), 128.6, 128.1 (d, JCF = 1.8 Hz),
124.4 (d, JCF = 3.7 Hz), 116.2 (d, JCF = 22.6 Hz), 103.6, 40.2, 31.0,
29.0, 24.4, 21.9, 21.7. LCMS (ESI) m/z: 366.22 [MꢃH]ꢃ. Anal. Calcd
for C22H23FNO3: C 71.95, H 6.04, N 3.81. Found: C 71.95, H 6.06, N
3.67.
5.1.23.5. 2-(3-(20-Fluoro-40-hydroxy-[1,10-biphenyl]-4-yl)propanamido)
cyclohex-1-ene-1-carboxylic acid (5f). Compound 5f was prepared
in the same manner as compound 5d using ethyl carboxylate 22f
(0.15 g, 0.36 mmol) and 2 N NaOH (0.55 ml, 1.10 mmol). The pro-
duct 5f (80 mg, 57%) was obtained as a white solid. 1H NMR
(300 MHz, DMSO-d6) d: 12.54 (b s, 1H), 11.65 (s, 1H), 9.97 (b s,
1H), 7.41–7.23 (m, 5H), 6.69 (dd, J = 8.3, 2.5 Hz, 1H), 6.64 (dd,
J = 12.8, 2.2 Hz, 1H), 2.88 (t, J = 7.6 Hz, 2H). 2.84–2.78 (m, 2H),
2.61 (t, J = 7.6 Hz, 2H), 2.26–2.17 (m, 2H), 1.60–1.46 (m, 4H). 13C
NMR (100 MHz, DMSO-d6) d: 171.0, 169.8, 159.6 (d, JCF = 245.0 Hz),
158.3 (d, JCF = 11.7 Hz), 150.4, 139.5, 133.2 (d, JCF = 1.9 Hz), 130.9
(d, JCF = 5.6 Hz), 128.4, 128.3 (d, JCF = 3.0 Hz), 118.7 (d, JCF = 13.4 -
Hz), 112.1 (d, JCF = 2.7 Hz), 104.8, 103.0 (d, JCF = 25.1 Hz), 38.9,
30.1, 28.1, 24.4, 21.5, 21.4. LCMS (ESI) m/z: 382.21 [MꢃH]ꢃ. Anal.
Calcd for C22H22FNO4: C 68.92, H 5.78, N 3.65. Found: C 68.51, H
5.71, N 3.52.
5.1.23.1. 2-(3-(20-Hydroxy-[1,10-biphenyl]-4-yl)propanamido)cyclo-
hex-1-ene-1-carboxylic acid (5a). Compound 5a was prepared in
the same manner as compound 5d using ethyl carboxylate 22a
(0.11 g, 0.28 mmol) and 2 N NaOH (0.42 ml, 0.84 mmol). The pro-
duct 5a (38 mg, 37%) was obtained as a white solid. 1H NMR
(300 MHz, DMSO-d6) d: 12.54 (b s, 1H), 11.65 (s, 1H), 9.45 (b s,
1H), 7.45 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 7.22 (dd,
J = 7.7, 1.7 Hz, 1H), 7.03 (ddd, J = 8.1, 7.3, 1.7 Hz, 1H), 6.92 (dd,
J = 8.1, 1.2 Hz, 1H), 6.85 (td, J = 7.4, 1.2 Hz, 1H), 2.88 (t, J = 7.6 Hz,
2H), 2.87–2.78 (m, 2H), 2.62 (t, J = 7.6 Hz, 2H), 2.26–2.18 (m, 2H),
1.60–1.45 (m, 4H). 13C NMR (100 MHz, DMSO-d6) d: 171.2, 169.9,
154.3, 150.6, 138.9, 136.3, 130.2, 129.0, 128.2, 127.8, 127.5,
119.4, 116.0, 104.7, 39.0, 30.1, 28.2, 24.4, 21.5, 21.4. LCMS (ESI)
m/z: 364.18 [MꢃH]ꢃ. Anal. Calcd for C22H23NO4: C 72.31, H 6.34,
N 3.83. Found: C 72.04, H 6.37, N 4.08.
5.2. Biology
5.1.23.2. 2-(3-(30-Hydroxy-[1,10-biphenyl]-4-yl)propanamido)cyclo-
hex-1-ene-1-carboxylic acid (5b). Compound 5b was prepared in
the same manner as compound 5d using ethyl carboxylate 22b
(95 mg, 0.24 mmol) and 2 N NaOH (0.36 ml, 0.72 mmol). The pro-
duct 5b (32 mg, 36%) was obtained as a white solid. 1H NMR
(300 MHz, DMSO-d6) d: 12.53 (b s, 1H), 11.63 (s, 1H), 9.48 (s,
1H), 7.50 (d, J = 8.2 Hz, 2H), 7.29 (d, J = 8.3 Hz, 2H), 7.23 (t,
J = 7.9 Hz, 1H), 7.03 (ddd, J = 7.7, 1.6, 0.9 Hz, 1H), 6.98 (t,
J = 2.0 Hz, 1H), 6.74 (ddd, J = 8.0, 2.5, 0.9 Hz, 1H), 2.89 (t,
J = 7.5 Hz, 2H), 2.85–2.78 (m, 2H), 2.61 (t, J = 7.5 Hz, 2H), 2.25–
2.17 (m, 2H), 1.61–1.45 (m, 4H). 13C NMR (100 MHz, DMSO-d6)
d: 171.2, 169.9, 157.8, 150.6, 141.4, 139.9, 138.1, 129.9, 128.8,
126.5, 117.3, 114.2, 113.3, 104.7, 30.0, 28.2, 24.3, 21.6, 21.4. LCMS
5.2.1. Intracellular cAMP assay
Flp-In-293 cells (Invitrogen) stably expressing human HCA2
receptor were grown in DMEM medium supplemented with 10%
fetal bovine serum, penicillin, streptomycin and hygromycin
(100 mg/ml each). For cAMP assay cells were distributed into a
384-well white microplate at a density 12,000 cells /well and stim-
ulated with the indicated compounds in the presence of 3 lM for-
skolin for 30 min. Cells and the compounds were diluted in 1X
phosphate-buffered saline (PBS) supplemented with 0.5 mM 3-iso-
butyl-1-methylxanthine (Sigma-Aldrich). Intracellular cAMP levels
were measured using a Lance cAMP kit (Perkin Elmer) according to
the manufacturer’s instructions. Plates were read on Victor3VTM
multilabel reader (Perkin-Elmer). The cAMP was quantitated for
Please cite this article in press as: Bobileva O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.028

16
O. Bobileva et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
each sample by comparison to a standard curve of known amounts
of cAMP provided in the kit. Data was analyzed using the GraphPad
Prism version 5.00 for Windows (GraphPad Software, La Jolla, CA).
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Acknowledgements
A part of this work was supported by Latvian State Research
Program Biomedicine. Olga Bobileva thanks Latvian Institute of
Organic Synthesis for funding her research (LIOS grant number
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.06.028.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
Please cite this article in press as: Bobileva O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.028