Synthesis, Structure–Activity Relationship Studies, and ADMET Properties of 3-Aminocyclohex-2-en-1-ones as Chemokine Receptor 2 (CXCR2) Antagonists

Article abstract of DOI:10.1002/cmdc.201800027

Herein we describe the synthesis and structure–activity relationships of 3-aminocyclohex-2-en-1-one derivatives as novel chemokine receptor 2 (CXCR2) antagonists. Thirteen out of 44 derivatives were found to inhibit CXCR2 downstream signaling in a Tango a

Full text of DOI:10.1002/cmdc.201800027

Accepted Article
Title: Synthesis, Structure-activity Relationship Studies and ADMET
Properties of 3-aminocyclohex-2-en-1-ones as Chemokine
Receptor 2 (CXCR2) Antagonists
Authors: Weiyang Dai, Wenmin Chen, Bikash Debnath, Wu Yong, and
Nouri Neamati
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To be cited as: ChemMedChem 10.1002/cmdc.201800027
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10.1002/cmdc.201800027
ChemMedChem
FULL PAPER
Synthesis, Structure-activity Relationship Studies and ADMET
Properties of 3-aminocyclohex-2-en-1-ones as Chemokine
Receptor 2 (CXCR2) Antagonists
Weiyang Dai, [a,b] Wenmin Chen, [a] Bikash Debnath, [a] Yong Wu, *[b] and Nouri Neamati*[a]
[a] W. Dai, Dr. W. Chen, Dr. B. Debnath and Prof. N. Neamati*
Department of Medicinal Chemistry, College of Pharmacy
University of Michigan
1600 Huron Parkway, Ann Arbor, MI, USA
E-mail: neamati@med.umich.edu
[b]
W. Dai and Prof. Y. Wu*
Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education, West China School of Pharmacy
Sichuan University
No.17 People's South Road, Chengdu, 610041, P. R. of China
Supporting information for this article is given via a link at the end of the document.
Abstract: Herein, we describe the synthesis and structure-activity
relationship of 3-aminocyclohex-2-en-1-one derivatives as novel
CXCR2 antagonists. Thirteen out of 44 derivatives inhibit CXCR2
down-stream signaling in a Tango assay specific for CXCR2 with
IC50 values less than 10 µM. In silico ADMET prediction suggests
that all active compounds possess drug-like properties. None of
these compounds show cytotoxicity, suggesting their potential
application in inflammatory mediated diseases. A structure-activity
relationship (SAR) map has been generated to gain better
understanding of their binding mechanism to guide further
optimization of these new CXCR2 antagonists.
inhibitors with different scaffolds have been reported, including
diaryl ureas, boronic acids, squaramides, pyrimidines and
ketoprofen derivatives (Figure 1). Some CXCR2 inhibitors have
advanced to clinical trials. Reparixin is a ketoprofen derivative
being investigated in prevention and treatment of delayed graft
function and pancreatic islet transplantation[9]. Additionally, a
combination of ketoprofen and paclitaxel inhibited brain tumor
metastasis[10]. Reparixin is now in Phase II for both breast
cancer and ischemia-reperfusion injury. A potent and selective
CXCR2 diarylurea antagonist SB225002 inhibits CXCL8-induced
neutrophil migration[11], and its analog SB656933, has advanced
into clinical trials for COPD and cystic fibrosis[12]. Results from
these trials demonstrated that CXCR2 inhibitors can effectively
suppress ozone and LPS- mediated lung inflammation in healthy
subjects by reducing sputum neutrophils. Another CXCR2
inhibitor, SCH527123[13], was tested in a phase II clinical trial for
Introduction
the oral treatment of moderate to severe COPD and asthma[14]
.
The CXC chemokine receptor CXCR1 and CXCR2 are members
of class A (rhodopsin-like) family of seven transmembrane G-
protein-coupled receptors[1] expressed on inflammatory cells
such as neutrophils, monocytes, T-lymphocytes, and basophils
as well as on keratinocytes, endothelial, fibroblasts, central
nervous system (CNS) neurons and melanoma cells[2]. These
receptors are activated by chemokines CXCL8 (Interleukin-8, IL-
8), NAP2, CXCL1 (GRO-α), GRO-β, GRO-γ, ENA-78, γIP-10, I-
TAC[3]. Chemokine CXCL1 is selective for CXCR2, whereas,
CXCL8 can activate both CXCR1 and CXCR2[4]. Upon
chemokine binding, CXCR1/2 couples to pertussis toxin-
sensitive G-protein via Gαi subunit to regulate signaling
cascades that mediate neutrophil activation and chemotaxis.
The G-protein signaling is tightly regulated by rapid
desensitization of the receptor. Receptor internalization is one of
the desensitization processes. CXCR2 is more rapidly
internalized and at low ligand concentrations than the CXCR1[5].
CXCR2 plays a significant role in the activation and recruitment
of neutrophils at the sites of inflammation in several
inflammatory diseases including chronic obstructive pulmonary
disease (COPD), arthritis, asthma, psoriasis as well as CNS
demylinating disorders[6]. CXCR2 signaling is also implicated in
tumorigenesis and metastasis[7]. Therefore, discovery of novel
peptides and small molecule inhibitors of CXCR2 antagonists
emerges as a promising therapy for the treatment of various
inflammatory disorders[8]. Serveral small molecule CXCR2
Bicyclic thiazolopyrimidine compounds exemplified by AZD-8309
effectively inhibited the increase of LPS-induced neutrophil
recruitment in the nasal lavage of healthy patients[15]. Its analog
AZ13381758
decreased
metastases
and
augmented
immunotherapy response in a mouse model of pancreatic
cancer[16]. Another bicyclic thiazolopyrimidine small molecule
AZD5069 is also in clinical trials for COPD, asthma and
advanced solid tumors. SX-517 is another small molecule
CXCR2 antagonist with unique boronic acid moiety that
effectively inhibited CXCL1-induced Ca2+ flux in human
PMNs[17]
augmented immunotherapy in a mouse model of prostate cancer
.
Its analog SX-682 decreased metastases and
[18]
and has advanced to Phase I clinical trial for melanoma
.
However, there is not yet an approved CXCR2 antagonist on the
market. We previously reported the discovery of a novel
phenylcyclohex-1-enecarbothioamide derivative CX4338 that
inhibits CXCL8-mediated chemotaxis through selective
regulation of CXCR2-mediated signaling[19]. It selectively blocks
CXCR2/β-arrestin-2 signaling and receptor internalization.
CX4338 also showed inhibition of CXCL8-induced chemotaxis in
CXCR2-overexpressing cells and human neutrophils and
significantly reduced neutrophils in bronchoalveolar lavage
induced by LPS in murine studies. Therefore, CX4338 is a good
hit compound for further investigation and optimization. Herein,
we describe our effort
1
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10.1002/cmdc.201800027
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FULL PAPER
OH
B
Cl
Cl
H
O
O
O
N
S
O
S
OH
O
S
N
N
F
N
N
N
F
S
O
O
H
H
F3CO
N
H
H
O
OH
O
H
OH
Cl
N
N
HN
N
H
O
F
Danirixin
SB656933
SX-682
Repertaxin
OH
HN
N
O
O
S
S
N
N
NH
NH
O
O
N
N
H
H
HN
S
O
OH
O
F
F
CX4338
AZD-8309
SCH527123
(Nevarixin)
Figure 1. CXCR2 receptor antagonists
towards modifications of CX4338 to establish a SAR for CXCR2
inhibition.
OH
O
Cl
O
O
O
a
N
N
O
NH2
b
H
H
N
H
O
N
H
4
Results and Discussion
5
6
O
O
HN
N
Chemistry
H
N
H
Different synthetic methods were applied to prepare
compounds 1-3 with various core structures (Scheme 1). The
1
synthesis of 1 started from methyl 3-amino-3-methylbutanoate
[20]
4 in three steps to generate 5 using an established method
.
O
OH
O
OH
O
Chlorination of 5 occurred by exposure to POCl3 at room
temperature to yield mono chloro product 6. Compound 1 was
obtained by coupling the chlorinated product 6 with benzyl
amine in DCM. Quinolinone 2 was prepared from isatoic
anhydride 7 treating with NaH and diethyl malonate in DMF to
provide the intermediate 8, which was heated with aniline to
yield 9. The chlorination of 9 was non-selective yielding the
bichloro intermediate 10 by refluxing of 9 with POCl3. Then
chlorine at the 2-position of compound 10 was subsequently
converted to keto in AcOH with AcONa to give 11. 4-
Chloroquinolinone intermediate 11 was then heated with benzyl
amine and triethylamine in acetonitrile to provide 2 in a 66%
yield. Synthesis of 3 started from the cyclization of 2-
animothiozole with excess triethyl methanetricarboxylate to
afford thiazolopyrimidine intermediate 13. Then 13 was heated
under reflux with TsCl and followed by coupling with benzyl
amine to provide 14. Compound 14 was hydrolyzed with
aqueous LiOH for 20 h. Amidation with aniline by EDCI and
HOBt in DCM under reflux condition furnished 3 in a 51% yield.
Scheme 2 describes the synthesis of 16-24, 26-38, 55-56
(structures in Table 2-4 and Figure 3). Starting with
commercially available 5,5-dimethylcyclohexane-1,3-dione 57,
which was stirred with benzylamine in toluene at 80 °C to afford
5-(benzylimino)-3,3-dimethylcyclohexanone 58. To facilitate the
reaction progress, anhydrous sodium sulfate was added to
abosorb generated water and removed by filtration upon
completion of the reaction. The crude product of 58 was further
purified by recrystallization with hexane/toluene 1:1. The
resultant pure product 58 was exposed to various commercially
available isocyanates to generate diverse β-enamioketone(16-
25, 27, 28, 35, 40-42). Corresponding isocyanates 29, 37, 38,
c
O
d
O
N
H
e
N
O
N
H
O
N
H
O
H
9
7
8
Cl
O
Cl
O
N
f
HN
O
O
g
N
H
H
N
Cl
N
N
H
O
H
10
N
H
11
2
HO
N
O
S
h
i
S
j
HN
N
S
O
N
N
H2N
O
N
O
O
O
12
13
14
k
HN
N
O
HN
HO
N
S
S
H
N
N
N
O
O
O
3
15
Scheme 1. Reagents and conditions: (a) Phosphorus oxychloride, DIEA, rt,
18h, 56%; (b) benzylamine, TEA, DCM, rt, 3h, 52%. (c) diethyl malonate,
NaH, DMF, reflux, 5 h,70%; (d) 160°C,10 min; (e) phosphorus oxychloride,
110 °C, 2 h, 89%; (f) AcONa, AcOH, 120°C, 20 h, 57%; (g) benzylamine,
TEA, DCM, rt, 4 h, 66%. (h) triethyl methanetricarboxylate, xylene, reflux, 5 h,
60%; (i) p-toluenesulfonyl chloride, TEA, acetonitrile, reflux, 3 h, benzyl
amine, reflux, 2 h, 69%. (j) LiOH, MeOH, H2O, 40°C, 20 h, 70%. (k) aniline,
EDCI, HOBt, TEA, DCM, 40°C, 24 h, 51%.
2
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10.1002/cmdc.201800027
ChemMedChem
FULL PAPER
products 26. Optical isomers 55 and 56 were prepared by the
same scheme using commercialy available R(+)- and S-(-)1-
phenylethylamine as starting material.
O
O
R1
NH
N
a
b
O
NH
R3
R3
O
O
Another efficient parallel synthetic approach was used for the
installation of R2 groups to generate compounds 25, 31-34, 43-
54 (Scheme 3). Starting material 5,5-dimethyl- cyclohexane-
1,3-dione 58 and corresponding isocyanates were stirred with 3
equiv of TEA in acetone overnight to give intermediate 59, 60
and 61 which were subsequently chlorinated with oxalyl
chloride and catalytic amounts of DMF. The resulting
intermediates (62, 63, and 64) were coupled with the
corresponding substituted amines or thioalcohol to obtain final
products 43-54. Compounds 47 were treated with boron
tribromide to give products 44. Compound 31 was obtained
from 33 using LiOH and then conjugated with aniline or
methylamine by EDCI/HOBt to provide 32 and 34.
57
R3=H=
58,
=
16-24,27-29,31-35,37,38
R3= H
65,
66
R3=R-(+)-Me=
R3=S-(-)-Me=
55,
56
R3=R-(+)-Me=
R3=S-(-)-Me=
c
d
e
NH2
O
S
NH
NH OH
O
N
O
O
NH
NH
O
NH
NH
O
26
36
30
Scheme 2. Reagents and conditions: (a) Sodium sulfate anhydrous, toluene,
80°C, 5 h, 86~97%; (b) R1NCO, 125°C, 1.5 h, 44%~81%. (c) BBr3, DCM, -
78°C, 30 min, rt, 2h, 52%; (d) Tin(II) dichloride, EtOH, HCl,78°C,3 h, 43%; (e)
2-Isocyanatobenzothiazole, EtOH, microwave, 120°C, 30 min, 12%;
In vitro biological evaluation and SAR studies
O
R1
NH
O
R1
NH
To establish a robust SAR, CX4338 was conceptualized as of
having three structural units: (1) cyclohexanone core; (2)
phenylthioamide moiety; and (3) benzyl amino functionality. All
compounds were initially tested at 10 μM in a CXCR2 specific
Tango assay as previously described[19]. Initial SAR study was
based on a scaffold hopping strategy by keeping two side
chains as well as the carbonyl group on the ring (Table 1).
Bicyclic core including thiazolopyrimidinone and quinolinone (2,
3) resulted in a loss of activity. Similarly, simple replacement of
one carbon of cyclohexanone with a heteroatom (e.g.
O
O
HO
b
Cl
S
NH
O
a
d
O
O
O
43
=
R1=60
=
R1=63
57
O
O
=
R1=64
=
R1=61
O
O
R1=62=Ph
R1=59=Ph
O
HO
NH
NH
O
O
R1
NH
e
R2 NH
c
O
Table 1. IC50 values of CXCR2 of compounds 1, 2, 3 and 16 in CXCR2
44
Tango assay
NHR
OH
=
R1= 25
f
O
O
O
O
O
Cpd
Structure
aIC50 (µM)a
4.4±1.1
g
=
R1= 31
NH
NH
NH
NH
O
O
S
O
=
R1= 45-54 Ph
NH
NH
O
CX4338
33
=
R= 32 Ph
=
R= 34 Me
O
NH
NH
O
Scheme 3. Reagents and conditions: (a) Acetone/acetonitrile, TEA, rt or
reflux, overnight, 31~85%; (b) oxalyl chloride, DMF, rt, overnight. 4 h,76%; (c)
R2NH2, rt, TEA, DCM, 4 h, 29~78%; (d) benzyl mercaptan, 40°C, TEA,
acetonitrile, 31%; (e) BBr3, DCM, -78°C, 30 min, rt, 2h, 30%; (f) LiOH, MeOH,
H2O, rt, 12 h, 89%; (g) aniline/methylamine hydrochloride, EDCI, HOBt, TEA,
DCM, rt, 12 h, 43~57%
1
2
>10
>10
NH
O
NH
NH
O
NH
39 were prepared with substituted anilines and triphosgene in
refluxing toluene. Heating the mixture of 2 equiv of isocyanate
and 1 equiv of 58 under neat condition gave the desired
products. The yields are dependent on the reactivity of the
isocyanates (44%~81%) and were decreased when longer
reaction time was applied. Substrates containing hydroxyl or
carboxylic ester cannot react under this condition. For most
compounds, ethanol was added after the reaction was
completed to precipitate the crude products as white solids in
moderate to excellent purity by recrystallization. Microwave
irradiation of 2-isocyanatobenzothiazole with 58 at 130 °C in
ethanol was conducted to give 30 (12%). Compound 27 was
treated with boron tribromide to give corresponding hydroxyl
O
N
NH
N
NH
O
3
>10
S
O
NH
NH
O
16
4.2±1.1
[a]CXCR2 inhibition was determined using the CXCR2 Tango assay. Values
are the mean ± SD of at least three independent determinations.
3
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10.1002/cmdc.201800027
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compound 1)，caused a significant decrease in activity. Based
on these results, we postulate that the cyclohexanone moiety
possibly occupies a hydrophobic pocket with a moderate size,
which couldn’t accommodate groups with polar properties or
bulky substituent. The thiocarboxamide of CX4338 was
replaced with bioisosteres because of its high chemical
reactivity and potential in vivo toxicity[21]. Replacement of the
thiocarboxamide moiety with carboxamide resulted in 16, that
maintained CXCR2-inhibiory potency. Because of its improved
chemical stability and synthetic accessibility, the cyclohexene-
1-carboxamide can serve as a new CXCR2 antagonist template.
Compound 16 and CX4338 are not very soluble in protic
solvent during synthesis. It is likely that an intramolecular H-
bond is formed, as observed in many β-ketoamides[22] (Figure
2). Compared with 16, an analog CX1142 with a methylene
connection lacking intramolecular H-bond, showed 8 fold
decreased potency ( IC50 = 32.0 ± 5.5 μM). This suggested
that the intramolecular H-bond may restrict the compound in a
favorable conformation and it is important for binding.
Considering intramolecular H-bond may improve membrane
and blood-brain barrier (BBB) permeability[23], such inhibitors
can be optimized to treat CNS tumors and inflammatory
diseases.
Table 2. IC50 values of CXCR2 of compounds 16-28 in CXCR2 Tango assay.
O
R1
NH
O
Cpd
R1
aIC50 (µM)
Cpd
R1
aIC50 (µM)
16
17
18
4.2±1.1
>50
23
24
25
5.0±2.1
5.7±0.8
>10
Cl
Cl
6.7±2.3
Cl
19
20
7.0±2.7
3.3±0.7
26
27
28
>10
>50
>30
Cl
Cl
OH
O
Cl
21
22
>50
F
O
5.0±0.1
O
O
[a] CXCR2 inhibition was determined using the CXCR2 Tango assay. Values
are the mean ± SD of at least three independent determinations
NH
N
O
NH
H
O
Table 3. IC50 values of CXCR2 of compounds 29-42 in CXCR2 Tango assay
O
R1
NH
16
CXCR2 IC50=4.2 ±1.1µM
CX1142
CXCR2 IC50=32.0 ±5.5 µM
O
Figure 2. Intramolecular H-bond is important for CXCR2 inhibition
Cpd
R1
aIC50 (µM)
>10
Cpd
R1
aIC50(µM)
>10
NH2
O
N
36
29
Our initial SAR efforts focused on phenylamide moiety (Table 2).
Elaboration of benzene ring with chlorine atoms showed
preference for the substituents at 4-position (18, IC50 = 6.7 µM)
over the 3-position (17, IC50 > 50 µM) and bi-substitution (19,
IC50 = 7.0 µM; 20, IC50 = 3.3 µM) favored over mono-
substitution. Thus, we synthesized additional analogs with 4-
position substitutions. Interestingly, The classic isosteric
replacement of 4-choloro with 4-methyl resulted in a totally
inactive analog 21 but 4-methoxyl analog retained potency (22,
IC50 = 5.0 µM). The extended benzyl analogues represented by
24 also retained potency (IC50 = 5.7 µM). This suggests that
there is some space to accommodate large groups in the
O
N
S
>10
>10
37
38
27.7±9.0
>10
30
31
N
O
S
O
O
N
O
N
O
H
N
>10
39
>10
32
O
OCF3
CF3
OH
>10
>10
>10
40
41
42
5.2±0.2
2.9±0.4
2.5±0.9
33
34
35
O
O
H
binding pocket. For example, the 4-tert-butyl analog 23 (IC50
=
N
5.0 µM) may bind to this pocket. Adding an extra methyl group
on 24 to give α-benzylmethyl analog 25 resulted in loss of
activity. It is possible that the decrease in potency is due to α-
Cl
NO2
CF3
benzylmethyl group forming
a non-coplanar conformation
whereas the binding pocket can only accommodate flat groups.
Further decorations at 2-position (21, 27, 28) to increase out-of-
plane steric hindrance resulted in loss of activity.
[a] CXCR2 inhibition was determined using the CXCR2 Tango assay. Values
are the mean ± SD of at least three independent determinations
In order to extend our SAR efforts, we examined heterocyclic
derivatives 29, 30 as well as glycine derivatives 31, 32, 33, 34
to explore potential H-bonding. However, these modifications
led to inactive compounds (Table 3), suggesting that
thebenzene ring contributes to potency. Therefore, changing
4
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10.1002/cmdc.201800027
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electron density of the ring may improve potency. Thus, a
series of derivatives with lipophilic/hydrophilic, electron-
withdrawing/electron-donating groups were synthesized (35-42).
The results showed that lipophilic substitutions produced active
inhibitions (40, 41, 42). Improved potency was shown in
compounds with trifluoromethyl group on either 3-position (41,
IC50 = 2.9 µM) or 4-position (42, IC50 = 2.5 µM). We postulate
that a electron-deficient group positioned in a hydrophobic
pocket may contribute to improved potency.
and benzyl amine moieties would be additive. We combined the
most favorable 3-trifluoromethyl-4-chloro phenylamide moiety
and the privileged α-benzylmethyl amine moiety to obtain
isomers 55 and 56. The resulting hybrid compounds did not
show improved potency over 42. Although the cLogP of 55 is
slightly higher than 42, its out-of-plane methyl substitution
reduces crystal packing, and to some extend can increase
solubility.
Cl
Cl
Next, our SAR efforts involved the modification of the benzyl
amino moiety. The sulfur ether replacement of NH moiety was
inactive, indicating that the NH is also important for retaining
potency (Table 4). Considering that the phenylamide moiety is
well tolerated with lipophilic groups and possibly sit in a
hydrophobic pocket, we hypothesized that benzyl amine moiety
is more likely to be solvent exposed. Many CXCR2 clinical
candidates possess hydroxyl near free NHs (SB656933,
SCH527123, AZD-8309, Figure 1). Therefore, we synthesized a
series of derivatives with hydroxyl groups on different positions
(44-46). Unfortunately, none of these derivatives were active.
Interestingly, the 2-methoxyl derivative 47 retained potency.
Since 2-methoxyl could cause the restricted rotation of the
benzene ring, we designed and examined analogs with other
bulky groups. Carboxylate derivative 48 lost activity while α-
benzylmethyl was active (49, IC50 = 4.2 µM). Installing a
hydroxyl on the methyl of 49 reduced potency (50, IC50 = 15.2
µM). That means moderate size groups can be well
accommodated and bulky groups (e.g. 48) were not tolerated.
All efforts to improve aqueous solubility by replacing phenyl
moiety with pyridine 51-52, 3-benzamide 53 and isoxozole 54
led to reduced potency, reinforcing the narrow requirement of
allowable lipophilicity.
CF3
CF3
O
O
NH
NH
O
NH
NH
O
56
55
CXCR2 IC50=2.9 0.5 µM
±
±
M
CXCR2 IC50=3.5 1.0 µ
Figure 3. IC50 values of CXCR2 of compounds 55-56 in CXCR2 Tango assay
Since there is no crystal structure for CXCR2 and establishing a
robust docking model for the ligands bound with GPCR
receptor is challenging, ligand-based drug design guided by
SAR is a potential strategy for designing new CXCR2 inhibitors.
In this study, we made extensive modification of the structure of
3-aminocyclohex-2-en-1-ones to develop a SAR model. As
shown in Figure 4, three hydrophobic sites are identified that
makes the molecule rather lipophilic, indicating its binding site
is dominantly hydrophobic. Incorporating nitrogen atom into the
hexane ring or replacing it with fused aromatic rings is
detrimental to potency and may not be well tolerated. EWG on
ring A enhances activity and a lipophilic benzene ring is
preferred on ring B. The NH group on the right side is probably
involving in the formation of intramolecular H-bond to minimize
the energy of active conformation, and consequently serves as
an essential moiety for activity. The NH group on the left side
may form H-bond with the receptor and is also important for
activity.
Table 3. IC50 values of CXCR2 of compounds 43-54 in CXCR2 Tango assay
O
R2
NH
O
Cpd
R2
aIC50 (µM)
>10
Cpd
R2
aIC50 (µM)
4.2±1.9
H
N
S
43
49
50
OH
H
N
H
N
44
11.1 ±6.4
15.2 ±4.8
HO
OH
H
H
N
N
45
46
>10
>10
51
52
>10
>10
N
OH
H
H
N
N
O
N
O
N
H
N
47
48
6.1±1.6
>10
53
54
>10
>10
H
N
H
N
H
N
N
O
O
O
Figure 4. The SAR map of 3-aminocyclohex-2-en-1-ones.
[a] CXCR2 inhibition was determined using the CXCR2 Tango assay. Values
are the mean ± SD of at least three independent determinations
To assess selectivity, all compounds were evaluated in the
CXCR4 Tango assay. All compounds were inactive at 10 μM in
a Tango assay specific for CXCR4 (Supplementary Table 1),
On the basis of above preliminary SAR results, we examined
whether the potency gains from modifications of phenylamide
5
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10.1002/cmdc.201800027
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FULL PAPER
indicating good selectivity for CXCR2 over CXCR4. Additionally,
these compounds are tested in a MTT assay and a colony
formation assay to evaluate their cytotoxicity (Supplementary
Table 2, Supplementary Table 3, Supplementary Figure 1).
These compounds show low toxicity on OVCAR8 cells (MTT
IC50 > 50 µM) and CXCR2-U2OS cells (MTT IC50 > 30 µM)
suggesting that cytotoxicty did not contribute to CXCR2
inhibition.
ADMET properties
ADMET Predictor 8.0[24] was used to caculat select ADMET
properties. We calculated various PK properties like topological
polar surface area (T_PSA), LogP (S+logP)，molecular weight
Table 5. ADMET risk descriptors of CX4338 analogs and clinical trial
compound
Absn_
Risk[a]
ADMET_
Risk[b]
CYP_
Risk[c]
TOX_
Risk[d]
Rule
Identifier
Of 5[e]
Desired
values[f]
<3.5
<7.5
<2.5
<3.3
≤1
CX4338
1.59
0.45
1.82
1.94
2.00
2.00
0.55
1.91
0.33
1.27
1.94
2.00
2.01
0.57
0.69
1.03
2.15
2.15
0.71
0.00
0.00
0.00
0.00
1.59
2.79
5.14
2.13
3.77
3.62
3.62
3.63
1.78
3.91
2.44
3.02
4.00
5.22
5.77
0.74
2.25
2.90
6.14
6.14
1.23
0.68
1.21
6.00
2.00
1.81
6.75
3.55
1.67
1.95
1.68
1.62
1.63
1.22
2.00
2.11
1.75
1.48
2.00
2.00
0.17
1.56
1.87
2.00
2.00
0.52
0.68
0.21
3.00
0.00
0.22
1.96
0
0
0
0
0
0
1
1
0
1
0
0
0
0
1
0
0
0
1
1
0
0
0
0
0
0
0
16
17
0
18
0
18
0
20
0
22
0
23
0
24
28
0
0
40
0.17
0
41
42
0
44
0
47
0
Figure 5. ADMET properties of CX4338 analogs. Frequency distribution plots
for HBA (A), HBD (B), molecular weight (C), LogP (D), number of rotatable
bonds (E) and topological polar surface area (F).
49
0
55
0.09
0.09
0
56
SB656933
Repertaxin
SCH527123
AZD-8309
DF2156A
Danirixin
AZD5069
0
1
3
2
0
2
[a] Absorption risk model: eight rules based on descriptors and predicted
properties that contribute directly to the absorption of drugs. [b] ADMET risk
model: all of the risk models combined and two others such as low unbound
fraction and high steady-state volume of distribution. [c] CYP risk model:
eight rules based on predicted enzymatic clearances and CYP inhibition. [d]
Toxicity risk model: seven rules based on calculated toxicities including
mutational risk model. [e] Lipinski’s Rule of 5 violation count [i] Desired
values are reported in the tutorial of the ADMET Predictor.
Figure 6. Topological polar surface area in Å2 for each of CX4338 (green), its
analogs (red) and CXCR2 antagonists in clinical trials (blue) is plotted against
their corresponding calculated LogP.
6
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10.1002/cmdc.201800027
ChemMedChem
FULL PAPER
polar surface area (T_PSA), LogP (S+logP)，molecular weight
(MW), hydrogen bond acceptor (HBA), hydrogen bond donor
(HBD), number of rotatable bonds (N_FrRotB), Lipinski’s rule of
5 violation (Ruleof5), risks descriptors including abosoption risk
(Absn_Risk), AMET_risk, cytochrome P450 metabolism risk
(CYP_Risk) and toxicity risk (Tox_risk). Figure 5 presents
frequency distribution plots MW, LogP (S+ LogP), HBA, HBD,
T_PSA in A°2, Rota_bonds of CX4338 analogs. Number of
rotatable bonds represents conformational space of a molecule.
The conformational behaviors directly or indirectly affect
pharmacodynamics as well as pharmacokinetic properties. Too
many rotatable bonds (optimal value ≤ 7) are not desirable for
molecule to be drug-like[25]. Similar frequency plots of CXCR2
antagonists in clinical trials are presented in Supplementary
Figure 2. CX4338 analogs showed similar ADMET properties to
CXCR2 antagonists in clinical trials and obey Lipinski’s rule.
Figure 6 shows a T_PSA vs LogP plot (Egan plot) suggesting
over 90% of the compounds are within desirable range of
T_PSA (<140) as well as S+ logP (< 5) values. Egan plot
represents prediction of drug absorption and considers
correlations between these two parameters to oral absorption.
On the basis of Egan et al absorption model[26], the outer ellipse
represents a 99% confidence, whereas the inner ellipse a 95%
confidence. T_PSA vs S+logP plots for optimized analogs (red
points) are shifted from CXC4338 (green point) towards clinical
trial antagonists (blue points) indicating that our lead
optimization strategy is reasonable. Table 5 summarizes risk
descriptors for absorption, CYP inhibition, toxicity and overall
ADMET properties of the active compounds. Most of the
compounds are predicted to be well tolerated as values are well
below the threshold level of the risk descriptors.、
Column chromatography was performed using a Biotage
Isolera chromatography system by normal phase silica gel
columns. NMR spectra were recorded on a Bruker Ultrashield
300 MHz or a Bruker Ascend 400 MHz NMR spectrometer.
Chemical shifts (δ) are reported in parts per million (ppm) units
relative to residual undeuterated solvent. Mass spectra were
obtained on a Shimadzu LCMS-2020 liquid chromatography
mass spectrometer using the electron spray ionization (ESI)
method. HPLC was used to determine purity of biologically
tested compounds by Shimadzu HPLC Test Kit C18 column (3
μm, 4.6 × 50 mm) under the following gradient elution condition:
acetonitrile/water (10-95%), gradient time: 15 min. The purity
was established by integration of the areas of major peaks
detected at 254 nm and all final products are >95% pure as
determined by the Shimadzu LCMS-2020. All spectral
information is listed in the Supporting Information.
4-Chloro-6,6-dimethyl-2-oxo-N-phenyl-1,2,5,6-
tetrahydropyridine-3-carboxamide 6
To 10 mL phosphorus oxychloride was added 4-hydroxy-6,6-
dimethyl-2-oxo-N-phenyl-1,2,5,6-tetrahydropyridine-3-
carboxamide
5
(200 mg, 0.769 mmol) and N,N-
diisopropylethylamine (2 mL). The solution immediately turned
red. The reaction mixture was stirred at room temperature for
18 h and solvent was removed under reduced pressure. Then,
the resulting crude was extracted with EtOAc and 1N aqueous
sodium bicarbonate for 3 times. The combined organics layer
was washed with brine, dried over sodium sulfate anhydrous,
filtered, concentrated in vacuo to afford the crude compound.
The crude was purified via silica gel chromatography to afford 6
(120 mg, 56% yield) as colorless oil.
4-(Benzylamino)-6,6-dimethyl-2-oxo-N-phenyl-1,2,5,6-
tetrahydropyridine-3-carboxamide 1
Conclusions
To a solution of 6 (50 mg, 0.179 mmol) in DCM (10 mL) was
added triethylamine (0.074 mL, 0.538 mmol) and benzyl amine
(23 µL 0.215 mmol). The reaction mixture was stirred at room
temperature for 3 h. Then, 10 mL 1N aqueous HCl was added
and the solution was extracted with EtOAc for 3 times. The
combined organics layer was washed with brine, dried over
sodium sulfate anhydrous, filtered, and concentrated in vacuo
and the crude was purified via silica gel chromatography to
afford 1 (33 mg, 52% yield).
In the present study, we describe our initial SAR studies of a
novel scaffold of CXCR2 inhibitor CX4338. Scaffold hopping
was conducted to optimize the core structure. Compound 16
was synthesized by replacing sulfur with oxygen. Then, a series
of 3-aminocyclohex-2-en-one derivatives were synthesized to
select optimized ring substituents. Thirteen compounds showed
IC50 values < 10 µM. We observed that both of the benzene
rings as well as two NH groups are critical moieties for potency.
Trifluromethylphenyl substituent on carboxamide moiety and α-
benzylmethyl amino moiety improve the activity. All active
compounds show selectivity against CXCR2 over CXCR4 and
do not exhibit cytotoxicity. Compound 55 (IC50 = 2.9 ± 0.4 µM)
and 42 (IC50 = 2.5 ± 0.9 µM) are new antagonists with desirable
properties. In silico ADMET predicted properties suggest that
lead optimization is reasonable according to Egan plot, where
properties of new analogs are improved towards the CXCR2
antagonists in clinical trials. Since this scaffold is new, our SAR
studies provided valuable data for further optimization and of
new CXCR2 antagonist for treating inflammatory diseases.
Ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate
8
To a solution of isatoic anhydride 7 (1.0 g, 6.13 mmol) in
anhydrous DMF (20 mL) under argon was added sodium
hydride (0.49 g, 60% in mineral oil, 12.3 mmol) at -5 °C. The
suspension was stirred for 20 min and then diethyl malonate
(1.12 mL, 7.36 mmol) was added. The mixture was heated to
reflux for 5 h. Then the mixture was cooled to 0 °C and was
carefully added 1N aqueous hydrochloric (50 mL). The resulting
solid was collected by filtration, washed with water and
diethylether and then dried in vacuo to give the desired
compound 8 (1.0 g, 70% yield) .
Experimental Section
4-Hydroxy-2-oxo-N-phenyl-1,2-dihydroquinoline-3-
carboxamide 9
Compound 8 (500 mg, 2.13 mmol) and aniline (191 µL, 2.13
mmol ) were mixed in a round-bottom flask under argon. The
Chemistry
All commercial reagents and anhydrous solvents were
purchased and used without purification unless specified.
7
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10.1002/cmdc.201800027
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FULL PAPER
reaction mixture was stirred at 160 °C for 10 min. The mixture
was cooled to room temperature. 10 mL ethanol was added
and stirred for 30 min and filtered to give white solid 9 (480
mg,78% yield).
To a solution of 14 (50 mg, 0.152 mmol) in methanol (5 ml) was
added lithium hydroxide (19 mg, 0.456 mmol) monohydrate and
H2O (1 ml). The reaction mixture was stirred at 40°C for 20 h.
Then the mixture was cooled to room temperature, 20 ml 1N
aqueous HCl was added and extracted with EA for 3 times. The
combined organics layer was washed with brine, dried over
sodium sulfate anhydrous, filtered, and concentrated in vacuo
to afford 15 (32 mg, 70% yield ).
2,4-Dichloro-N-phenylquinoline-3-carboxamide 10
To 10 mL phosphorus oxychloride was added 9 (200 mg, 0.769
mmol) and the reaction mixture was stirred at 110 °C for 2 h
and solvent was removed under reduced pressure. The
resulting crude was directly used for next step without further
purification (200 mg, 89% yield)
7-(Benzylamino)-5-oxo-N-phenyl-5H-thiazolo[3,2-
a]pyrimidine-6-carboxamide 3
To a solution of 15 (20 mg, 0.0664 mmol) in DCM (10 mL) was
added EDCI(19 mg, 0.0997 mmol), HOBt (13 mg, 0.0997
mmol), triethylamine (18 µL, 0.199 mmol) and aniline(9.4
µL ,0.0997 mmol). The reaction mixture was stirred at 40 °C for
24 h. Then 10 ml 1N aqueous HCl was added and the solution
was extracted with DCM for 3 times. The combined organic
layer was washed with brine, dried over sodium sulfate
anhydrous, filtered, and concentrated in vacuo and the crude
was purified via silica gel chromatography to afford 3 (13 mg,
51% yield) as a white solid.
4-Chloro-2-oxo-N-phenyl-1,2-dihydroquinoline-3-
carboxamide 11
To a solution of 10 (50 mg, 0.158 mmol) in acetic acid (5 mL)
was added sodium acetate anhydrous (16 mg, 0.174 mmol).
The reaction mixture was stirred at 120 °C for 20 h. The mixture
was cooled to room temperature and poured into 20 ml water.
The resulting solid was filtered and washed with water and
recrystallized with hexane and ethyl acetate to provide white
solid 11 (27mg, 57% yield).
4-(Benzylamino)-2-oxo-N-phenyl-1,2-dihydroquinoline-3-
carboxamide 2
5-(Benzylimino)-3,3-dimethylcyclohexanone 58
To a solution of 5,5-dimethylcyclohexane-1,3-dione 57 (2.0 g,
14.2 mmol) in toluene (20 mL) was added 2.00 g sodium sulfate
anhydrous and benzylamine (1.86 mL, 16.9 mmol). The
reaction mixture was stirred at 80 °C for 5 h. The mixture was
filtered immediately at high temperature and washed with hot
toluene 2 times. The filtrate was concentrated in vacuo and
recrystallized with hexane/toluene 1:1 to afford 58 (3.16 g, 97%
yield) as a light-yellow needle crystal.
To a solution of 11 (20 mg, 0.0692mmol) in acetonitrile (10 mL)
was added triethylamine (19 µL, 0.138 mmol) and benzyl amine
(8 µL, 0.0761 mmol). The reaction mixture was heated to reflux
for 4h. The reaction was quenched by 10 ml 1N HCl solution
and extracted with EA for 3 times. The combined organics layer
was washed with brine, dried over sodium sulfate anhydrous,
filtered, and concentrated in vacuo to afford the crude
compound. The crude was purified via silica gel
chromatography to afford 2 (17 mg, 66% yield) as a white solid.
(R)-5,5-Dimethyl-3-((1-phenylethyl)amino)cyclohex-2-en-1-
one 65
Ethyl-7-hydroxy-5-oxo-5H-thiazolo[3,2-a]pyrimidine-6-
carboxylate 13
To a solution of 5,5-dimethylcyclohexane-1,3-dione 57 (1.0 g,
7.10 mmol) in toluene (20 mL) was added 1.00g sodium sulfate
anhydrous and (R)-(+)-1-phenylethylamine (1.03 mL ， 8.52
mmol). The reaction mixture was stirred at 80 °C for 5 h. The
mixture was filtered immediately at high temperature and
washed with hot toluene 2 times. The filtrate was concentrated
in vacuo and recrystallized with hexane and toluene to afford 65
(1.52 g, 88% yield) as a light-yellow needle crystal.
To a solution of 2-animothiozole 12 (1.0 g, 10.0 mmol) in xylene
(20 ml) was added triethyl methanetricarboxylate (2.32 g, 30.0
mmol). The mixture was heated to reflux for 5h. Then solvent
was removed under vacuum and the resulting brown solid was
recrystallized with isopropanol to afford yellow solid 13 ( 1.44g,
60% yield ).
Ethyl-7-(benzylamino)-5-oxo-5H-thiazolo[3,2-a]pyrimidine-
6-carboxylate 14
(S)-5,5-Dimethyl-3-((1-phenylethyl)amino)cyclohex-2-en-1-
one 66
To a solution of 13 (200 mg, 0.833 mmol) in acetonitrile (20 ml)
was added triethylamine (460 µL, 3.34 mmol) and p-
toluenesulfonyl chloride (158 mg, 0.833 mmol). The mixture
was heated to reflux for 5 h. The mixture was cooled to room
temperature and benzylamine was added (89 µL, 0.833 mmol).
The solution was refluxed for another 2h. The reaction was
quenched by 10 ml 1N HCl solution and extracted with EA for 3
times. The combined organics layer was washed with brine,
dried over sodium sulfate anhydrous, filtered, and concentrated
in vacuo to afford the crude compound. The crude was purified
via silica gel chromatography to afford 14 (189 mg, 69% yield)
as a white solid.
Compound 66 was prepared from 5,5-dimethylcyclohexane-1,3-
dione 57 and (S)-(-)-1-phenylethylamine according to the
same procedure as 65 to give light-yellow needle crystal, 1.49 g,
in 86% yield.
General procedure I of the preparation of compounds 17-
24,27-29,31-35,37,38, 55,56,
5-imino-3,3-dimethylcyclohexanone (1 eq.) and
2
eq.
isocyanates were mixed in a round-bottom flask under argon.
The reaction mixture was stirred at 125 °C for 2 h. The mixture
was cooled to room temperature. Ethanol (5mL) was added and
stirred for 30 min and filtered to give pure compounds.
7-(Benzylamino)-5-oxo-5H-thiazolo[3,2-a]pyrimidine-6-
carboxylic acid 15
2-(Benzylamino)-N-(3-chlorophenyl)-4,4-dimethyl-6-
oxocyclohex-1-enecarboxamide 17
8
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10.1002/cmdc.201800027
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FULL PAPER
Compound 17 was prepared from 58 and 4-chlorophenyl
isocyanate according to the general procedure I described
above as a white solid, in 70% yield.
anhydrous, filtered, and concentrated in vacuo to afford the
crude compound. This crude was purified via silica gel
chromatography to afford 36 (16 mg, 43% yield) as an off white
solid.
2-(Benzylamino)-4,4-dimethyl-6-oxo-N-phenylcyclohex-1-
enecarboxamide 16
Compound 16 was prepared from 58 and phenyl isocyanate
according to the general procedure I described above as a
white solid, in 61% yield.
2-(Benzylamino)-4,4-dimethyl-6-oxo-N-(p-tolyl)cyclohex-1-
enecarboxamide 21
Compound 21 was prepared from 58 and p-tolyl isocyanate
according to the general procedure I described above as a
white solid, in 43% yield.
2-(Benzylamino)-N-(4-chlorophenyl)-4,4-dimethyl-6-
oxocyclohex-1-enecarboxamide 18
Compound 18 was prepared from 58 and 4-chlorophenyl
isocyanate according to the general procedure I described
above as a white solid, in 66% yield.
2-(Benzylamino)-N-(4-methoxyphenyl)-4,4-dimethyl-6-
oxocyclohex-1-enecarboxamide 22
Compound 22 was prepared from 58 and 4-methoxyphenyl
isocyanate according to the general procedure I described
above, in 47% yield.
2-(Benzylamino)-N-(3,5-dichlorophenyl)-4,4-dimethyl-6-
oxocyclohex-1-enecarboxamide 19
Compound 19 was prepared from 58 and 3,5-dichlorophenyl
isocyanate according to the general procedure I described
above as a white solid, in 45% yield.
2-(Benzylamino)-N-(4-(tert-butyl)phenyl)-4,4-dimethyl-6-
oxocyclohex-1-enecarboxamide 23
Compound 23 was prepared from 58 and 4-(tert-butyl)phenyl
isocyanate according to the general procedure I described
above as a white solid, in 30% yield.
2-(Benzylamino)-4,4-dimethyl-6-oxo-N-(4-
(trifluoromethyl)phenyl)cyclohex-1-enecarboxamide 40
N-benzyl-2-(Benzylamino)-4,4-dimethyl-6-oxocyclohex-1-
enecarboxamide 24
Compound
40
was
prepared
from
58
and
4-
(trifluoromethyl)phenyl isocyanate according to the general
procedure I described above as a white solid, in 76% yield.
Mixed 5-(benzylimino)-3,3-dimethylcyclohexanone 58 (50 mg,
0.218 mmol) was mixed with benzyl isocyanate (58 μL, 0.436
mmol) in a round-bottom flask under argon. The reaction
mixture was stirred at 125 °C for 3 h. The mixture was cooled to
room temperature and acidified to pH 3 with hydrochloric acid
(1 N). The acidic solution was extracted with EtOAc. The
combined organic layer was washed with brine, dried over
sodium sulfate anhydrous, filtered, and concentrated in vacuo
to afford the crude compound. This crude was purified via silica
gel chromatography to afford 24 (25 mg, 31% yield) as a white
solid.
2-(Benzylamino)-N-(4-chloro-3-(trifluoromethyl)phenyl)-4,4-
dimethyl-6-oxocyclohex-1-enecarboxamide 42
Compound 42 was prepared from 58 and 4-chloro-3-
(trifluoromethyl)phenyl isocyanate according to the general
procedure I described above as a white solid (yield 80%).
2-(Benzylamino)-N-(3,4-dichlorophenyl)-4,4-dimethyl-6-
oxocyclohex-1-enecarboxamide 20
Compound 20 was prepared from 58 and 3,4-dichlorophenyl
isocyanate according to the general procedure I described
above as a white solid (yield 65%).
2-(Benzylamino)-N-(2-methoxyphenyl)-4,4-dimethyl-6-
oxocyclohex-1-ene-1-carboxamide 27
Compound 27 was prepared from 58 and 4-methoxyphenyl
isocyanate according to the general procedure I described
above as a white solid (yield 51%).
2-(Benzylamino)-N-(2-fluorophenyl)-4,4-dimethyl-6-
oxocyclohex-1-enecarboxamide 28
Compound 28 was prepared from 58 and 2-fluorophenyl
isocyanate according to the general procedure I described
above as a white solid, in 72% yield.
2-(Benzylamino)-N-(2-hydroxyphenyl)-4,4-dimethyl-6-
oxocyclohex-1-ene-1-carboxamide 26
A solution of 26 (100 mg, 0.264 mmol) in anhydrous DCM (20
mL) was stirred at -78°C for 30 min, 1M BBr3 solution in THF
(1.32 ml, 1.32 mmol) was added dropwise. The reaction mixture
was slowly warmed to room temperature and stirred for 2 h.
Water(20 ml) was slowly added to quench the reaction. The
mixture was extracted with DCM for 3 times. The combined
organics layer was washed with brine, dried over sodium
sulfate anhydrous, filtered, and concentrated in vacuo to afford
a clear oil. The crude was refluxed in 1M HCl ethanol solution
for 2h to dissociate the boron complex. Solvent was then
removed and the crude was purified via silica gel
chromatography to afford 30 (50 mg, 52% yield) as a white
solid.
2-(Benzylamino)-4,4-dimethyl-N-(4-nitrophenyl)-6-
oxocyclohex-1-enecarboxamide 35
Compound 35 was prepared from 58 and (4-nitrophenyl
isocyanate according to the general procedure I described
above as a yellow solid. in 81% yield.
N-(4-Aminophenyl)-2-(benzylamino)-4,4-dimethyl-6-
oxocyclohex-1-enecarboxamide 36
To a solution of 35 (40 mg, 0.102 mmol) in EtOH (10 mL) was
added Tin (II) dichloride (114 mg, 0.508mmol) and 1 drop
hydrochloric acid (36%). The reaction mixture was stirred at
78 °C for 3 h. The mixture was cooled to room temperature and
pH was adjusted to 8 with sodium carbonate solution (1 N). The
water solution was extracted with EtOAc. The combined
organic layer was washed with brine, dried over sodium sulfate
General procedure II of the preparation of isocyanates 67-
70
9
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10.1002/cmdc.201800027
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FULL PAPER
A solution of triphosgene (0.35 eq.) in anhydrous toluene (10
mL) was added anilines (1eq.) in anhydrous DCM (10 mL)
dropwise at 0°C within 15 min. After addition, the reaction was
refluxed for 8 h. The solvent was removed in vacuo and the
resulting isocyanate was used directly without further
purification.
was removed and the crude was purified via silica gel
chromatography to afford 36 as a white solid (11mg, 12% yield).
2-(Benzylamino)-4,4-dimethyl-6-oxo-N-(4-
(trifluoromethoxy)phenyl)cyclohex-1-ene-1-carboxamide 40
Compound
40
was
prepared
from
58
and
4-
(trifluoromethoxyl)phenyl isocyanate according to the general
procedure I described above as a white solid. yield 44%.
3-Isocyanato-N,N-dimethylbenzenesulfonamide 67
Compound
67
was
prepared
from
3-amino-N,N-
dimethylbenzenesulfonamide according to the general
procedure II described above as a colorless oil.
(R)-N-(4-Chloro-3-(trifluoromethyl)phenyl)-4,4-dimethyl-6-
oxo-2-((1-phenylethyl)amino)cyclohex-1-ene-1-
carboxamide 55
3-Isocyanato-N,N-dimethylbenzamide 68
Compound 55 was prepared from 65 and 4-chloro-3-
(trifluoromethyl)phenyl isocyanate according to the general
procedure I described above as a white solid, in 78% yield.
Compound
68
was
prepared
from
3-amino-N,N-
dimethylbenzamide according to the general procedure II
described above as a colorless oil.
(S)-N-(4-Chloro-3-(trifluoromethyl)phenyl)-4,4-dimethyl-6-
oxo-2-((1-phenylethyl)amino)cyclohex-1-ene-1-
carboxamide 56
4-Isocyanato-N,N-dimethylbenzamide 69
Compound
69
was
prepared
from
4-amino-N,N-
dimethylbenzamide according to the general procedure II
described above as a colorless oil.
Compound 56 was prepared from 66 and 4-chloro-3-
(trifluoromethyl)phenyl isocyanate according to the general
procedure I described above as a white solid in 81%yield.
3-Isocyanato-5-methylisoxazole 70
Compound 70 was prepared from 3-amino-5-methylisoxazole
according to the general procedure II described above as a
colorless oil.
2-Hydroxy-4,4-dimethyl-6-oxo-N-(1-phenylethyl)cyclohex-1-
ene-1-carboxamide 59
To a solution of 5,5-dimethylcyclohexane-1,3-dione (1.5 g, 10.7
mmol) in DCM (30 mL) was added triethylamine (5.9 ml, 32.1
mmol) and stirred for 10 min at 0 °C . Then a solution of phenyl
isocyanate (1.91g, 16.50 mmol) in DCM (10 ml) was added
dropwise. The reaction mixture was stirred at room temperature
for 12 h. The reaction was quenched by 20 ml 1N HCl solution
and extracted with DCM for 3 times. The combined organics
layer was washed with brine, dried over sodium sulfate
anhydrous, filtered, and concentrated in vacuo to afford the
crude compound. The crude was purified via silica gel
chromatography to afford 59 (2.33 g, 85% yield) as a white
solid..
2-Isocyanatobenzothiazole 71
Compound 71 was prepared from 2-aminobenzothiazole
according to the general procedure II described above as a
yellow solid.
2-(Benzylamino)-N-(3-(N,N-dimethylsulfamoyl)phenyl)-4,4-
dimethyl-6-oxocyclohex-1-ene-1-carboxamide 37
Compound 37 was prepared from 58 and 67 according to the
general procedure I described above as a white solid, in 60%
yield.
3-(2-(Benzylamino)-4,4-dimethyl-6-oxocyclohex-1-ene-1-
carboxamido)-N,N-dimethylbenzamide 38
Compound 38 was prepared from 58 and 68 according to the
general procedure I described above as a white solid, in 43%
yield.
2-Chloro-4,4-dimethyl-6-oxo-N-phenylcyclohex-1-ene-1-
carboxamide 62
To a solution of 4,4-dimethyl-2,6-dioxo-N-phenylcyclohexane-1-
carboxamide 59 (1.0 g, 3.86 mmol) in anhydrous DCM (20 mL)
was added oxalyl chloride (3.30ml, 38.6mmol) and 0.05 ml
DMF. The solution immediately became red. The reaction
mixture was stirred at room temperature for 18 h and was
quenched by carefully adding 2 ml water in an ice bath. Then
the mixture was extracted with EtOAc and 1N aqueous sodium
bicarbonate for 3 times. The combined organics layer was
washed with brine, dried over sodium sulfate anhydrous, filtered,
concentrated in vacuo to afford the crude compound. This
crude was purified via silica gel chromatography to afford 62
(0.82 g, 76% yield) as colorless oil.
4-(2-(Benzylamino)-4,4-dimethyl-6-oxocyclohex-1-ene-1-
carboxamido)-N,N-dimethylbenzamide 39
Compound 39 was prepared from 58 and 69 according to the
general procedure I described above as a white solid, in 51%
yield.
2-(Benzylamino)-4,4-dimethyl-N-(5-methylisoxazol-3-yl)-6-
oxocyclohex-1-ene-1-carboxamide 29
Compound 29 was prepared from 58 and 70 according to the
general procedure I described above as a white solid, in 44%
yield.
2-Hydroxy-4,4-dimethyl-6-oxo-N-(1-phenylethyl)cyclohex-1-
ene-1-carboxamide 60
To a solution of 5,5-dimethylcyclohexane-1,3-dione 57 (500 mg,
3.57 mmol) in acetonitrile (20 mL) was added triethylamine
(1.97 ml,10.7 mmol) and stirred for 10 min at 0 °C . Then a
solution of (1-isocyanatoethyl)benzene (786 g, 5.35 mmol) in
DCM (10 ml) was added dropwise. The reaction mixture was
heated to reflux for 12 h. The reaction was quenched by 20 ml
N-(Benzo[d]thiazol-2-yl)-2-(benzylamino)-4,4-dimethyl-6-
oxocyclohex-1-ene-1-carbothioamide 30
To a solution of 58 (76 mg, 0.436 mmol) in EtOH (5 mL) added
71 (50 mg, 0.218 mmol) in a microwave tube. The mixture was
reacted in microwave reactor at 120°C for 30 min, then solvent
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1N HCl solution and extracted with DCM for 3 times. The
combined organics layer was washed with brine, dried over
sodium sulfate anhydrous, filtered, and concentrated in vacuo
to afford the crude compound. The crude was purified via silica
gel chromatography to afford 60 (317 mg, 31% yield) as a white
solid.
Methyl-(2-(benzylamino)-4,4-dimethyl-6-oxocyclohex-1-ene-
1-carbonyl)glycinate 31
To a solution of 64 (50 mg, 0.183 mmol) in DCM (10 mL) was
added triethylamine (0.076 ml, 0.549 mmol) and benzyl
amine(24 µL, 0.219 mmol). The reaction mixture was stirred at
room temperature for 3 h. Then 10ml 1N aqueous HCl was
added and the solution was extracted with EtOAc for 3 times.
The combined organics layer was washed with brine, dried over
sodium sulfate anhydrous, filtered, and concentrated in vacuo
and the crude was purified via silica gel chromatography to
afford 31 (34 mg, 54% yield)as a white solid.
2-Chloro -4,4-dimethyl-6-oxo-N-(1-phenylethyl)cyclohex-1-
ene-1-carboxamide 63
To a solution of 60 (100 mg, 0.348 mmol) in anhydrous DCM
(10 mL) was added oxalyl chloride (0.297 ml, 3.48 mmol) and
0.01 ml DMF. The solution immediately became red. The
reaction mixture was stirred at room temperature for 18 h and
was quenched by carefully adding 2 ml water in an ice bath.
Then the mixture was extracted with EtOAc and 1N aqueous
sodium bicarbonate for 3 times. The combined organics layer
was washed with brine, dried over sodium sulfate anhydrous,
filtered, concentrated in vacuo to afford 105mg red oil. the
resulting crude was used directly without further purification.
(2-(Benzylamino)-4,4-dimethyl-6-oxocyclohex-1-ene-1-
carbonyl)glycine 33
To a solution of 31 (50 mg, 0.145 mmol)in methanol (5 ml)
added lithium hydroxide (18 mg, 0.436 mmol)monohydrate and
H2O (1 ml). The reaction mixture was stirred at room
temperature for 12 h. Then the mixture was cooled to room
temperature and 20 ml 1N aqueous HCl was added and the
suspension was stirred for 15 min. The precipitated white solid
was collected by filtration and dried in vacuo to afford 33 (43
mg, 89% yield).
2-(Benzylamino)-4,4-dimethyl-6-oxo-N-(1-
phenylethyl)cyclohex-1-ene-1-carboxamide 25
To a solution of 63 (50 mg, 0.163 mmol) in DCM (10 mL) was
added triethylamine (0.068 ml, 0.491 mmol) and benzyl amine
(21 µL, 0196 mmol). The reaction mixture was stirred at room
temperature for 3 h. Then 10ml 1N aqueous HCl was added
and the solution was extracted with EtOAc for 3 times. The
combined organics layer was washed with brine, dried over
sodium sulfate anhydrous, filtered, and concentrated in vacuo
and the crude was purified via silica gel chromatography to
afford 25 (21 mg, 34% yield) as a white solid.
2-(Benzylamino)-4,4-dimethyl-6-oxo-N-(2-oxo-2-
(phenylamino)ethyl)cyclohex-1-ene-1-carboxamide 32
To a solution of 33 (30 mg, 0.091 mmol) in DCM(10 ml) was
added EDCI (27 mg, 0.136 mmol), HOBt (18 mg, 0.136 mmol),
triethylamine (30 µL, 0.272 mmol) and aniline (10.5 µL ,0.109
mmol). The reaction mixture was stirred at room temperature
for 12 h. Then 10ml 1N aqueous HCl was added and the
solution was extracted with DCM for 3 times. The combined
organic layer was washed with brine, dried over sodium sulfate
anhydrous, filtered, and concentrated in vacuo and the crude
was purified via silica gel chromatography to afford 32 (21 mg,
57% yield) as a white solid.
Methyl-(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene-1-
carbonyl)glycinate 61
To a solution of 5,5-dimethylcyclohexane-1,3-dione 57(500 mg,
3.57 mmol) in acetonitrile (20 mL) was added triethylamine
(1.97 ml, 10.7 mmol) and stirred for 10 min at 0 °C. Then a
solution of methyl 2-isocyanatoacetate (615 mg, 5.35 mmol) in
DCM (10ml) was added dropwise. The reaction mixture was
heated to reflux for 12 h. The reaction was quenched by 20 ml
1N HCl solution and extracted with DCM for 3 times. The
combined organics layer was washed with brine, dried over
sodium sulfate anhydrous, filtered, and concentrated in vacuo
to afford the crude compound. The crude was purified via silica
gel chromatography to afford 61 (295 mg, 32% yield) as a white
solid.
2-(Benzylamino)-4,4-dimethyl-N-(2-(methylamino)-2-
oxoethyl)-6-oxocyclohex-1-ene-1-carboxamide 34
To a solution of 33 (30 mg, 0.091 mmol) in DCM(10ml) added
EDCI (27 mg, 0.136 mmol), HOBt (18 mg, 0.136 mmol),
triethylamine (30 µL, 0.272 mmol) and methylamine
hydrochloride (7.4 mg ,0.109 mmol). The reaction mixture was
stirred at room temperature for 12 h. Then 10 ml 1N aqueous
HCl was added and the solution was extracted with DCM for 3
times. The combined organics layer was washed with brine,
dried over sodium sulfate anhydrous, filtered, and concentrated
in vacuo and the crude was purified via silica gel
chromatography to afford 34 (16 mg, 43% yield) as a white
solid.
Methyl-(2-chloro-4,4-dimethyl-6-oxocyclohex-1-ene-1-
carbonyl)glycinate 64
To a solution of 61 (100 mg, 0.392 mmol) in anhydrous DCM
(10 mL) was added oxalyl chloride (0.335 ml, 3.92 mmol) and
0.01ml DMF. The solution immediately became red. The
reaction mixture was stirred at room temperature for 18 h and
was quenched by carefully adding 2 ml water in an ice bath.
Then the mixture was extracted with EtOAc and 1N aqueous
sodium bicarbonate for 3 times. The combined organics layer
was washed with brine, dried over sodium sulfate anhydrous,
filtered, concentrated in vacuo to afford 120 mg red oil. the
resulting crude was used directly without further purification.
2-(Benzylthio)-4,4-dimethyl-6-oxo-N-phenylcyclohex-1-ene-
1-carboxamide 43
To a solution of 62 (50 mg, 0.180 mmol) in acetonitrile (10 mL)
was added triethylamine (0.075 ml, 0.540 mmol) and benzyl
mercaptan (26 µL, 0.216 mmol). The reaction mixture was
stirred at 40°C temperature for 4 h. Then the mixture was
cooled to room temperature and 10 ml 1N aqueous HCl was
added. The solution was extracted with EtOAc for 3 times. The
combined organic layer was washed with brine, dried over
sodium sulfate anhydrous, filtered, and concentrated in vacuo.
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The crude was purified via silica gel chromatography to afford
43 as a yellow solid (20 mg, 31% yield).
General procedure III of the preparation of 45-54
Compound 51 was prepared from 62 and methyl pyridin-2-
ylmethanamine according to the general procedure III
described above as a white solid in 60% yield..
To a solution of 62 (50 mg, 0.180 mmol) in DCM (10 mL) was
added triethylamine (0.075 ml, 0.540 mmol) and amines (0.216
mmol). The reaction mixture was stirred at room temperature
for 3 h. Then 10 ml 1N aqueous HCl was added and the
solution was extracted with EtOAc for 3 times. The combined
organics layer was washed with brine, dried over sodium
sulfate anhydrous, filtered, and concentrated in vacuo and the
crude was purified via silica gel chromatography.
4,4-Dimethyl-6-oxo-N-phenyl-2-((pyridin-3-
ylmethyl)amino)cyclohex-1-ene-1-carboxamide 52
Compound 52 was prepared from 62 and methyl pyridin-3-
ylmethanamine according to the general procedure III
described above as a white solid in 29% yield.
3-(((5,5-Dimethyl-3-oxo-2-(phenylcarbamoyl)cyclohex-1-en-
1-yl)amino)methyl)-N,N-dimethylbenzamide 53
Compound 53 was prepared from 62 and methyl 2-
(aminomethyl)-N,N-dimethylbenzamide according to the
general procedure III described above as a white solid in 42%
yield.
2-((2-Hydroxyphenyl)amino)-4,4-dimethyl-6-oxo-N-
phenylcyclohex-1-ene-1-carboxamide 45
Compound 45 was prepared from 62 and 2-hydroxyaniline
according to the general procedure III described above as a
white solid. yield 44%.
4,4-Dimethyl-6-oxo-N-phenyl-2-((1-
2-((1-Hydroxypropan-2-yl)amino)-4,4-dimethyl-6-oxo-N-
phenylcyclohex-1-ene-1-carboxamide 46
Compound 46 was prepared from 62 and 2-amino-1-propanol
according to the general procedure III described above as a
white solid in 44% yield.
phenylethyl)amino)cyclohex-1-ene-1-carboxamide 49
Compound 49 was prepared from 62 and methyl 1-
phenylethan-1-amine according to the general procedure III
described above as a white solid in 78% yield.
4,4-Dimethyl-2-(((3-methylisoxazol-5-yl)methyl)amino)-6-
oxo-N-phenylcyclohex-1-ene-1-carboxamide 54
Compound 54 was prepared from 62 and methyl (5-
methylisoxazol-3-yl)methanamine according to the general
procedure III described above as a white solid in 67% yield.
2-(((5,5-Dimethyl-3-oxo-2-(phenylcarbamoyl)cyclohex-1-en-
1-yl)amino)methyl)benzoate 48
Compound 48 was prepared from 62 and methyl 2-
(aminomethyl)benzoate according to the general procedure III
described above as a white solid in 56% yield.
ADMET properties calculation
2-((2-Hydroxy-1-phenylethyl)amino)-4,4-dimethyl-6-oxo-N-
phenylcyclohex-1-ene-1-carboxamide 50
Compound 50 was prepared from 62 and DL-2-Phenylglycinol
according to the general procedure III described above as a
white solid in 33% yield.
ADMET properties and associated risk values were calculated
using ADMET Predictor 8.0 (Simulation Plus, Inc.)[28]. The
absorption risk model includes eight rules, each contributes a
value of 1, based on descriptors and predicted properties that
directly contributes to the absorption of drugs, such as
molecular weight, H-bond donor, H-bond acceptor, polar
surface area, number of rotatable bonds, etc. The CYP risk
model is composed of eight rules, each contributes a value of 1,
based on predicted enzymatic clearances and CYP inhibitions.
The toxicity risk model comprises seven rules, each contributes
a value of 1, based on calculated toxicities, such as hERG
toxicity, acute toxicity in rats including the mutational risk model.
The ADMET risk model comprises a total of 23 rules, each
contributes a value of 1, which include all of these risk models
and two others, such as low unbound fraction and high steady
state volume of distribution. Descriptors BBB Filter and LogBB
were presented by two blood-brain barrier models (a qualitative
permeability model and a blood-brain partition coefficient
model). LogP and topological polar surface area were also
calculated.
2-((2-Methoxybenzyl)amino)-4,4-dimethyl-6-oxo-N-
phenylcyclohex-1-ene-1-carboxamide 47
Compound 47 was prepared from 62 and methyl 2-
methoxybenzylamine according to the general procedure III
described above as a white solid in 70% yield.
2-((2-Hydroxybenzyl)amino)-4,4-dimethyl-6-oxo-N-
phenylcyclohex-1-ene-1-carboxamide 44
A solution of 44 (50 mg, 0.132 mmol) in anhydrous DCM (10
mL) was stirred at -78 °C for 30 min, 1M BBr3 solution in THF
(0.67 ml, 0.66 mmol) was added dropwise. The reaction mixture
was slowly warmed to room temperature and stirred for 2 h.
20ml water was slowly added to quench the reaction. The
mixture was extracted with DCM for 3 times. The combined
organics layer was washed with brine, dried over sodium
sulfate anhydrous, filtered, and concentrated in vacuo to afford
a clear oil. The crude was refluxed in 1M HCl ethanol solution
for 2h to dissociate the boron complex. Solvent was then
removed and the crude was purified via silica gel
chromatography to afford 19 (14 mg, 30% yield) as a white
solid.
Biology
Cell culture: Tango TM CXCR2-bla and CXCR4-bla U2OS cells
were purchased from Invitrogen (Carlsbad, CA, USA) and
grown in McCoy5A supplemented with 10% dialized FBS,
zeocin (200 μg·mL−1), hygromycin (50 μg·mL−1), geneticin
(100 μg·mL−1), 1 mM sodium pyruvate, 0.1 mM non-essential
amino acids and 25 mM HEPES. OVCAR8 cells were cultured
in RPMI 1640 medium (Gibco) supplemented with 10% FBS
4,4-Dimethyl-6-oxo-N-phenyl-2-((pyridin-2-
ylmethyl)amino)cyclohex-1-ene-1-carboxamide 51
12
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(Gibco). All of the cells were grown at 37 °C in a humidified
atmosphere of 5% CO2. All cell lines used were maintained in
culture under 35 passages and tested regularly for Mycoplasma
contamination using Plasmo Test (InvivoGen, San Diego, CA).
three times. The colonies were imaged by iBRIGHT imaging
system.
CXCR2/4 Tango assay: The compounds inhibition potency for
stimulus mediated CXCR2/4 β-arrestin-2 recruitment was
assayed by TangoTM assay (Thermo Fisher) as described
previously[17]. CXCR2/4-bla (beta-lactamase) U2OS cells were
genetically modified to stably overexpress CXCR2 or CXCR4
linked to a TEV protease site and a GAL4-VP16 transcription
factor, via a reporter-gene system. These cells also stably
express a β-arrestin-2/TEV protease fusion protein and a β-
lactamase reporter gene. Upon corresponding stimulus (IL8 or
SDF1-α) binding and resulting in CXCR2 or CXCR4 activation,
the β-arrestin-2/TEV fusion protein is recruited to the receptor
and cleaves the peptide linker that links CXCR2/4 to the GAL4-
VP16 transcription factor. GAL4-VP16 now can enter the
nucleus and promote the transcription of the β-lactamase gene.
Acknowledgements
This study was funded by the Department of Defense Lung
Cancer Research Program, Grant No. LC090363 (N. N.). W.
Dai was supported by China Scholarship Council
201606240054
Keywords: chemokine receptors • CXCR2 antagonist • SAR •
ADMET
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Entry for the Table of Contents
New derivatives of 3-aminocyclohex-2-en-1-ones were synthesized and evaluated for their CXCR2 inhibition. Structure-activity
relationship of these compounds was discussed. Several compounds display CXCR2 IC50 values less than 10 µM. they also show
selectivity against CXCR2 and low cytotoxicty. In silico ADMET prediction suggests most active compounds possess good drug-like
properties.
15
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