Design, synthesis and biological evaluation of 2,5-dimethylfuran-3-carboxylic acid derivatives as potential IDO1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.03.005

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a vital role in tumor immune escape and has emerged as a promising target for cancer immunotherapy. In this study, a novel series of 2,5-dimethylfuran-3-carboxylic acid derivatives were designed, synthesized and evaluated for inhibitory activities against IDO1, and their structure-activity relationship was investigated. Among these, compound 19a exhibited excellent IDO1 inhibitory activity (HeLa cellular IC₅₀ = 4.0 nM, THP-1 cellular IC₅₀ = 4.6 nM). Further molecular docking studies revealed that the compound 19a formed a coordinate bond with the heme iron through the carboxylic acid moiety. These results indicate that compound 19a is a potential IDO1 inhibitor for further investigation.

Full text of DOI:10.1016/j.bmc.2019.03.005

Accepted Manuscript
Design, synthesis and biological evaluation of 2,5-dimethylfuran-3-carboxylic
acid derivatives as potential IDO1 inhibitors
Xiaojun Yang, Shi Cai, Xueting Liu, Pan Chen, Jinpei Zhou, Huibin Zhang
PII:
S0968-0896(19)30102-6
https://doi.org/10.1016/j.bmc.2019.03.005
BMC 14797
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
20 January 2019
25 February 2019
2 March 2019
Please cite this article as: Yang, X., Cai, S., Liu, X., Chen, P., Zhou, J., Zhang, H., Design, synthesis and biological
evaluation of 2,5-dimethylfuran-3-carboxylic acid derivatives as potential IDO1 inhibitors, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.03.005
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Design,
synthesis
and
biological
evaluation
of
2
,5-dimethylfuran-3-carboxylic acid derivatives as potential
IDO1 inhibitors
a
a
b
b
b,*
a,*
Xiaojun Yang , Shi Cai , Xueting Liu , Pan Chen , Jinpei Zhou , Huibin Zhang
a
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b
Department of Medicinal Chemistry, China Pharmaceutical University, TongjiaXiang 24,
2
10009 Nanjing, PR China
*
Corresponding author. E-mail address: jpzhou668@163.com (J. Zhou),
zhanghb80@cpu.edu.cn (H. Zhang).
Abstract: Indoleamine 2,3-dioxygenase 1 (IDO1) plays a vital role in tumor
immune escape and has emerged as a promising target for cancer immunotherapy. In
this study, a novel series of 2,5-dimethylfuran-3-carboxylic acid derivatives were
designed, synthesized and evaluated for inhibitory activities against IDO1, and their
structure-activity relationship was investigated. Among these, compound 19a
exhibited excellent IDO1 inhibitory activity (HeLa cellular IC =4.0 nM, THP-1
5
0
cellular IC =4.6 nM). Further molecular docking studies revealed that the compound
5
0
1
9a formed a coordinate bond with the heme iron through the carboxylic acid moiety.
These results indicate that compound 19a is a potential IDO1 inhibitor for further
investigation.
Keywords: IDO1 inhibitor, immunotherapy, HeLa assay, THP-1 assay
1. Introduction
Cancer research is a rapidly evolving field with new discoveries emerging daily
on its biology, diagnosis, treatment and prevention. In particular, several key
immunotherapy milestones have been achieved recently and dramatically changed the
1
, 2
landscape of treatment of cancer.
Since the first regulatory approval of Yervoy
(which blocks cytotoxic T lymphocyte-associated protein 4 (CTLA-4)) in 2011, the
US Food and Drug Administration (FDA) has approved 5 monoclonal antibodies
targeting programmed cell death 1 (PD-1) receptor (including Opdivo and Keytruda)
or programmed cell death ligand 1 (PD-L1) (including Tecentriq, Bavencio and
3
, 4
Imfinzi) for multiple types of cancer.
In 2017, the FDA has also approved two
chimeric antigen receptor (CAR) T-cell therapies, Kymriah (for B-cell precursor acute
lymphoblastic leukemia that is refractory or in second or later relapse) and Yescarta
5
(
for relapsed or refractory large B-cell lymphoma). Both the immune-checkpoint

blockers and CAR T-cell therapies have demonstrated unprecedented therapeutic
benefits for solid and blood cancers. However, they have drawbacks, including failure
to show a response in most patients, administration by intravenous injection, and
6
severe immune-related adverse effects.
Preclinical and clinical studies have demonstrated a trend of immunotherapeutic
combination strategies to confer a greater survival benefit over single-agent
7
, 8
approaches.
In this area, indoleamine-2,3-dioxygenase 1 (IDO1) has received
significant attention from both industry and academia. IDO1 is an intracellular
heme-containing enzyme that catalyzes the initial and rate limiting step in the
catabolism of L-tryptophan to N-formyl-kynurenine via the kynurenine pathway,
9
, 10
leading to the formation of a series of biologically active metabolites.
Depletion of
local tryptophan and accumulation of kynurenine and other metabolites assists IDO1
enzyme to suppress the local immune response by blocking the activation of
T-lymphocytes and facilitating the differentiation of regulatory T cells (Tregs) ,
1
1
resulting in an immunosuppressive microenvironment. What's more, IDO1 promotes
1
2, 13
immune escape via down-regulating natural killer (NK) receptors on NK cells.
IDO1 is interferon-inducible and found to be up-regulated in several types of cancer
1
4-17
cells and tumor environmental cells.
Overexpression of IDO1 in the tumor
microenvironment is often associated with both tumor progression and poor
1
8-20
prognosis.
All these findings validate IDO1 as a therapeutic molecular target for
cancer immunotherapy.
To date, several IDO1 inhibitors are currently being evaluated in various stages of
2
1
clinical trials (Fig.1). The D-enantiomer of 1-Methyltryptophan (1-MT, indoximod)
from NewLink Genetics was the first reported inhibitor of IDO1 and is the
N-methylated analog of tryptophan. Despite an inhibitory activity of 34 μM,
indoximod has shown efficacy in several phase II clinical trials for the treatment of
2
2
solid tumors in combination with various antibody. Another IDO1 inhibitor, the
imidazole Navoximod (GDC-0919) is in phase I clinical trials at NewLink Genetics in
23, 24
collaboration with Genentech in subjects with recurrent advanced solid tumors.
Epacadostat (INCB024360) developed by Incyte Corporation has also shown
25, 26
promising efficacy and is currently being evaluated in phase III clinical trials.
Recently, two novel IDO1 inhibitors, PF-0684003 and Linrodostat (BMS-986205),
have also entered into clinical trials within the last few months. PF-06840003
developed by iTeos Therapeutics is currently under evaluation in phase I clinical trials
2
7
for the treatment of glioma. Linrodostat (BMS-986205) is an IDO1 inhibitor
licensed by Flexus Inc. to Bristol-Myers Squibb for clinical development and has
28
shown promise in phase III clinical trials in combination with nivolumab.

Fig. 1. Representative inhibitors of IDO1.
There is currently no FDA-approved IDO1 inhibitor for treating human diseases.
Novel IDO1 inhibitors with different structural scaffolds are still necessary to explore
their therapeutic potential in different cancers. Recently, Bristol-Myers Squibb
described a new series of o-phenylenediamine-based inhibitors (e.g., compound 1) of
2
9, 30
IDO1 identified by high-throughput screening.
Although those new inhibitors
showed low nanomolar cellular IC values (e.g., compound 1 IC =13 ± 4 nM), little
5
0
50
data are available about their specificity and mode of action. To understand the
binding conformation of this series, a molecular docking simulation was performed
with Glide docking in Schrodinger 2018. Compound 1 was docked into IDO1
cocrystal structure (PDB id: 5EK3). Analysis of the docking poses of compound 1
showed that the biphenyl carboxylic acid moiety formed the six-coordination with the
heme iron via the carboxyl oxygen atom, the phenyl ring occupied the Pocket A while
the diisobutylamino group and p-tolyl urea moiety reached out to an expanded Pocket
B, and the proximal NH of the urea formed a hydrogen bond with the propionic acid
of the heme (Fig.2). These IDO1 inhibitors bearing a biphenyl carboxylic acid
scaffold have high lipophilicity, which likely correlated with poor water-solubility,
3
1, 32
metabolic toxicity and associated with a higher risk of attrition.
Based on these
modeling and findings, we envisioned that replacement of the terminal benzene ring
of compound 1 with a heterocycle was a worthy approach to hunt a new scaffold with
desired drug-like properties. In addition, we also investigated the effect of substitution
of R , R and R on the activity of the compounds. On the other hand, the urea moiety
1
2
3
could be replaced by substituted amino-heterocyclic derivatives to improve their
lipophilicity. As a result, a novel series of 2,5-dimethylfuran-3-carboxylic acid
derivatives were designed (Fig.3), synthesized and evaluated for their IDO1 inhibitory
activities.

Fig. 2. Molecular docking of compound 1 (green) into IDO1 (PDB id: 5EK3). The procedure was
performed in Maestro 11.5 implemented in the Schrodinger 2018.
Fig. 3. Design of the novel IDO1 inhibitors.
2
. Results and discussion
2
.1. Chemistry
The synthetic procedure for desired compounds 8a-u is shown in Scheme 1. The
commercially available 4-fluorobenzaldehyde reacted with nitric acid to afford
nitrated intermediate 2. Heating intermediate 2 with potassium carbonate and various
°
substituted secondary amines in DMF at 100 C cleanly afforded compounds 3a-d.
The intermediates 4a-d was formed by the Henry reaction of compounds 3a-d with
nitroethane in the presence of ammonium acetate. Then the obtained 4a-d were
further converted to intermediates 5a-d by the addition and cyclization reaction of
3
3
methyl acetoacetate and potassium acetate in acetonitrile at reflux. Reduction to
intermediates 6a-d was accomplished by treatment of 5a-d with zinc and ammonium
chloride in ethanol-water. The intermediates 6a-d was treated with a variety of
isocyanates ArNCO or substituted anilines with triphosgene to afford intermediates
7
7
a-u. Finally, the target compounds 8a-u were obtained by hydrolysis of compounds
a-u with LiOH in tetrahydrofuran- methanol-water.

°
Scheme 1. General synthesis of 8a~u. Reagents and conditions：(a) HNO , H SO , 0 C~r.t.；(b)
3
2
4
°
°
R R NH or 11, K CO , DMF, 100 C; (c) NH OAc, Nitroethane, 80 C; (d) Methyl acetoacetate,
1
2
2
3
4
°
°
KOAc, MeCN, reflux; (e) Zn, NH Cl, EtOH/H O, 80 C; (f) i) R PhNCO, THF, 50 C; ii)
4
2
3
°
R PhNH , triphosgene, DIPEA, DCM, r.t.; (g) LiOH, THF/MeOH/H O, 60 C; (h) Isobutyryl
3
2
2
°
chloride, TEA, DCM, 0 C ~r.t.; (i) LiAlH , THF, reflux.
4
The compound 17 was synthesized as outlined in Schemes 2. The commercially
available tert-butyl acetoacetate and ammonium acetate were refluxed in methanol to
afford intermediate 13. Then the obtained 13 was further converted to intermediate 14
by the FeCl -catalyzed addition and cyclization reaction of intermediate 4c in glycol
3
33
°
at 100 C. The intermediate 16 was prepared by reduction of intermediate 14,
followed by reaction with p-tolyl isocyanate. The desired compound 17 was obtained
by hydrolysis of tert-butyl ester 16 with trifluoroacetic acid.
Scheme 2. General synthesis of 17. Reagents and conditions：(a) NH OAc, MeOH, reflux; (b) 4c,
4
°
°
FeCl , ethylene glycol, 100 C; (c) Zn, NH Cl, EtOH/H O, 80 C; (d) p-tolyl isocyanate, THF,
3
4
2
°
5
0 C; (e) CF COOH, DCM, r.t..
3

Target compounds 19a–c, 21a-b, 23 and 24 were synthesized as outlined in
Schemes 3. As depicted in Scheme 3, the intermediate 6d was treated with
1
5
,1'-thiocarbonyldiimidazole
-amino-1,2,4-thiadiazole
to
get
isothiocyanate
19a-c were
18.
produced
The
desired
by the
compounds
3
4
Cu(OTf) -catalyzed reaction of amidine hydrochlorides with isothiocyanate 18,
2
followed by hydrolysis with LiOH. The thiourea 20 was prepared from the
isothiocyanate 18 by addition of ammonia. Then the desired 2-aminothiazole
compounds 21a-b was achieved by the condensation of thiourea 20 with
3
5
α-bromoketones, followed by hydrolysis with LiOH. The intermediate 22 was
prepared by addition reaction between 18 and acethydrazide. Then the
thiosemicarbazide 22 was reacted with p-TsCl, triethylamine in NMP or EDCI in
DMSO respectively, followed by hydrolysis to afford the desired compounds 23 and
3
6
2
4.
Scheme 3. General synthesis of 19a-c, 21a-b, 23 and 24. Reagents and conditions：(a)
°
1
,1'-Thiocarbonyldiimidazole, MeCN, r.t.; (b) R C(NH)NH , Cu(OTf) , Cs CO , THF, 50 C; (c)
1
2
2
2
3
°
LiOH, THF/MeOH/H O, 60 C; (d) NH OH, EtOH, r.t.; (e) bromoacetone or
2
4
2
-bromo-1-phenylethan-1-one, EtOH, reflux; (f) CH CONHNH , THF, r.t.; (g) p-TsCl, TEA, NMP,
3 2
°
r.t.; (h) EDCI, DMSO, 60 C.
2
.2. Biological evaluation
2
.2.1. IDO1 inhibitory activity and SAR study
As reported, the HeLa human cervical cancer line expressing native human IDO1
specifically induced by IFN-γ was chosen as the primary screen for investigating
3
7
IDO1 inhibitors, due to its greater biological relevance and reported correlation
issues between IDO1 enzymatic and cellular assays. All synthesized compounds
2
9

were tested for their abilities to inhibit tryptophan degradation and kynurenine
production in the HeLa kynurenine assay, and both INCB024360 and compound 1
were evaluated as the control compounds. The inhibition rate and IC value of IDO1
5
0
shown in Table 1 and Table 2 indicated that many of our compounds strongly
inhibited the catalytic activity of IDO1.
The importance of the scaffold was first investigated. Compound 8a (IC = 10.1 ±
5
0
2
.1 nM) proved equipotent to compound 1, when we initially replaced the terminal
benzene ring with 2,5-dimethylfuran. The methyl ester intermediate 7a (inhibition rate
8.9% at 1 μM), which showed a sharply decreased IDO1 inhibitory activity
=
compared with that of 8a, approved that the carboxylic acid on the furan ring was
necessary to metal binding interaction. However, substitution of 2,5-dimethylfuran
with 2,5-dimethyl-1H-pyrrole (8k and 17) resulted in loss in potency (compound 17,
inhibition rate =15.7% at 1 μM), suggesting that the lone pair electrons on pyrrole
ring could interfere the coordination of carboxyl oxygen atom with the heme iron.
Accordingly, the 2,5-dimethylfuran-3-carboxylic acid moiety was selected for further
modification.
Next, we evaluated the different substituted secondary amines which could
occupy the Pocket B. Compared with diisobutylamino derivatives (8a-8d, IC values
5
0
of 6.6-10.1 nM), the N-methylcyclohexanamino derivatives (8e, 8h-8j) exhibited
slightly lower inhibitory effects against IDO1 (IC values of 19.5-38.1 nM), while
50
the N-ethylcyclohexanamino derivatives (8k, 8n-8p) showed almost the same
inhibitory activities (IC50 values of 6.5-9.6 nM). Interestingly,
N-isobutylcyclohexanamino analogs (8q, 8t-8u) displayed increased potency (IC50
values of 3.3-5.7 nM) compared with the corresponding diisobutylamino derivatives.
Analysis of the docking conformation of compound 1 with IDO1 revealed that the
diisobutylamino moiety extended to pocket B without specific hydrogen-bonding
interactions.
We
assumed
that
the
potency
enhancement
of
N-isobutylcyclohexanamino derivatives was attributed to their high lipophilicity.
Then the substituent effect of the aryl urea moiety was explored. As for 8f, 8l and
r, the introduction of methyl into the ortho-position of the phenyl ring caused a
8
considerable loss of potency (IC50 values of 37.1-258 nM) compared with
corresponding para-methyl derivatives (8e, 8k and 8q, IC values of 4.2-38 nM).
5
0
When methyl was relocated from the ortho-position to the meta-position to give
compounds 8g, 8m and 8s, their cellular activities showed further reduction (IC50
values of 52.7-539 nM). As shown in Table 1, the halogen-substituted benzyl
derivatives were more potent than analogous methyl derivatives. Among these
derivatives, 2,4-difluoro substituted compounds showed more robust IDO1 inhibitory
activities than the 2-fluoro or 4-fluoro derivatives (e.g., 8d, IC values of 6.6 nM vs
5
0
8
b and 8c, IC values of 7.1 and 8.5 nM, respectively).
50

Table 1
Structures of the target compounds and their inhibitory activities for inhibiting IDO1.
HeLa
Inh%
Compound
X
R1
R2
R3
b
a
IC (nM)
5
0
(
at 1μM)
8
8
a
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
99.8
10.1 ± 2.1
7.1 ± 0.6
8.5 ± 1.1
6.6 ± 0.7
38.1 ± 1.3
258 ± 17
539 ± 25
29.5 ± 2.8
35.7 ± 1.4
19.5 ± 2.6
7.5 ± 0.9
151 ± 23
223 ± 19
7.4 ± 1.3
9.6 ± 0.5
7.8 ± 2.3
4.2 ± 0.6
b
99.5
100
8
c
8d
100
8
e
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
98.7
87.3
62.3
95.9
94.6
99.7
100
8
f
8
8
g
h
8
i
8
j
8k
CH
CH
CH
CH
CH
CH
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
3
3
3
3
3
3
8
l
93.6
84.4
100
8m
8
n
o
p
q
8
99.5
100
8
8
99.5

8
r
O
O
95.5
94.1
99.9
100
37.1 ± 1.5
52.7 ± 3.2
3.3 ± 0.2
5.7 ± 1.3
ND
8
s
8
t
O
8u
O
1
7
7
a
NH
CH
CH
2
3
15.7
8.9
ND
Compound 1
INCB024360
98.5
99.7
16.4 ± 2.1
10.3 ± 1.6
a
Inhibition rate mean values at a screening concentration of 1 μM were obtained from three
independent experiments.
b
IC values are the mean of at least three independent assays, presented as mean ± SD.
5
0
Then we replaced the urea moiety with substituted amino-heterocyclic derivatives
to obtain compounds 19a-24. As shown in Table 2, the inhibitory activity of 19a
3-methyl) > 19b (3-cyclopropyl) > 19c (3-phenyl) indicated that introduction of
(
lipophilic substituents on 1,2,4-thiadiazole caused the decrease of inhibitory effects
and the same phenomenon was also observed in compound 21a and 21b. Among
these methyl heterocyclic derivatives, 19a (5-amino-1,2,4-thiadiazole), 21a
(2-aminothiazole) and 23 (2-amino-1,3,4-thiadiazole) exhibited the similar inhibitory
activity (IC50 values of 4.0-5.5 nM), while 24 (2-amino-1,3,4-oxadiazole) showed
decreased inhibitory effect (IC values of 55.8 nM).
5
0
Table 2
Structures of the target compounds and their inhibitory activities for inhibiting IDO1.
HeLa
IC (nM)
Inh%
Compound
A
R
b
a
5
0
(
at 1μM)
1
9a
9b
9c
CH
3
99.6
100
4.0 ± 0.7
20.3 ± 1.9
306 ± 13
1
1
88.1

2
1a
CH
3
100
91.2
99.9
98.0
4.6 ± 0.8
341 ± 27
5.1 ± 1.1
55.8 ± 4.6
21b
2
3
CH
CH
3
3
2
4
Compound 1
INCB024360
98.5
99.7
16.4 ± 2.1
10.3 ± 1.6
a
Inhibition rate mean values at a screening concentration of 1 μM were obtained from three
independent experiments.
b
IC values are the mean of at least three independent assays, presented as mean ± SD.
5
0
To complement the classical HeLa cellular assay, an additional assay tested the
activity of IDO1 in the human peripheral blood-derived monocytic THP-1 cell line
2
7
stimulated by LPS and IFN-γ. Several compounds with single nanomolar HeLa
cellular IC values were tested and showed activities both in the HeLa assay and
5
0
THP-1 assay. According to the results, only 8t, 8u, 19a and 23 maintained strong
IDO1 inhibitory activities with lower cLogP values compared with compound 1.
Besides the inhibitory activity, the ligand efficiency was taken advantage of in the
3
8
drug-like evaluation of derivatives. The calculated LE based on the HeLa cell assay
suggested that compound 19a was the most potent inhibitor with better LE value for
further investigation.
Table 3
IDO1 inhibitory activities of compounds in HeLa and THP-1 cells
a
b
c
Compound
HeLa IC (nM) THP-1 IC (nM)
MW
cLogP
LE
50
50
8
q
4.2 ± 0.6
3.3 ± 0.2
5.7 ± 1.3
4.0 ± 0.7
4.6 ± 0.8
5.1 ± 1.1
16.4 ± 2.1
10.3 ± 1.6
5.8 ± 0.3
5.9 ± 0.9
8.1 ± 0.4
4.6 ± 1.3
7.4 ± 1.2
6.6 ± 0.8
10.7 ± 0.9
5.6 ± 0.6
517.29
521.27
521.27
482.24
481.24
482.24
473.27
-
7.65
7.10
7.55
6.78
7.44
6.78
7.49
-
0.302
0.305
0.297
0.338
0.332
0.334
0.305
-
8
t
8u
1
2
9a
1a
2
3
Compound 1
INCB024360
a
IC values are the mean of at least three independent assays, presented as mean ± SD.
5
0
b
cLogP values were calculated by the BioByte's algorithm as implemented in ChemBioDraw
Ultra 14.0.
c
LE values were calculated using HeLa pIC by LE=(1.37/HAC) × pIC
50.
5
0
2
.2.3. Western blot analysis
To figure out whether IDO1 inhibitors limit the degradation of tryptophan by the
way of inhibiting the enzymatic activity of IDO1 or suppressing the expression of

IDO1, Western blot analysis was subsequently performed in the HeLa cell-based assay.
The results suggested that compound 19a was incapable of suppressing IDO1
expression (Fig.4A), which suggesting it was a catalytic inhibitor just as
INCB024360.
Fig.4 (A) Western blot analysis of compound 19a tested in HeLa cells; (B) Cell viability of 19a
evaluated by the MTT assay.
2
.2.4. MTT assay
Since the inhibition of tryptophan degradation could simply be affected by
cytotoxicity, the compound 19a was evaluated against HeLa cells by MTT assay
Fig.4B). The results of this assay indicated that compound 19a had no effect on the
(
viability of HeLa cells at their effective concentration under the experimental
conditions.
2
.2.5. Molecular docking study of compound 19a
To understand the binding mode of this series, we performed docking
experiments with Glide docking in Schrodinger 2018. Compound 19a was docked
into IDO1 cocrystal structure (PDB id: 5EK3) and analysis of the docking
conformation of compound 1 and 19a with IDO1 revealed that the ligand-protein
interactions were similar for these two molecules (Fig.4). The metal binding
interaction was observed between heme iron and carboxylic acid moiety. Pocket A
was occupied by 2,5-dimethylfuran ring that formed hydrophobic interactions with
Tyr126, Phe163, Ser167, Gly262 and Ala264. The N-isobutylcyclohexanamino group
and 5-amino-1,2,4-thiadiazole reached out to an expanded Pocket B to form
interactions with Leu234, Ser235, Ser263, Ile354 and Leu384. The NH on the phenyl
ring formed a hydrogen bond with the propionic acid of the heme.
Fig.5 (A) Docking conformation of compound 19a (yellow) with IDO1 (PDB id: 5EK3). (B)
Superimposition of structures of compound 1 (green) and compound 19a (yellow) with IDO1.

3
. Conclusions
In summary, on the basis of the biphenyl carboxylic acid scaffold reported by
Bristol-Myers Squibb, we designed and synthesized a series of novel
,5-dimethylfuran-3-carboxylic acid derivatives as potential IDO1 inhibitors. All the
2
compounds were initially screened by HeLa assay and several compounds with single
nanomolar IC values were tested by complementary THP-1 assay. It was found that
5
0
compound 19a maintained robust potency of HeLa and THP-1 cellular IDO1
inhibition (IC = 4.0 nM, IC = 4.6 nM, respectively) with lower cLogP value and
5
0
50
better LE value compared with compound 1, and its unsatisfactory lipophilicity is still
needed to be improved. The tryptophan catabolism was terminated by inhibiting the
enzymatic activity of IDO1 indicated by both western blot and MTT assays. In
addition, docking studies demonstrated that carboxylic acid on the furan ring formed
the coordination with the heme iron. These results establish 19a as being worthy of
further studies and structural modification as well as the pharmacokinetic profile will
be carried out in the follow-up study.
4
. Experimental section
4
.1. Chemistry
All chemical reagents were commercially available and treated with standard
methods before use. Solvents were dried and redistilled before use. Column
chromatography (CC): silica gel 60 (200-300 mesh). Thin-layer chromatography
(TLC): silica gel 60 F254 plates (250 nm; Qingdao Ocean Chemical Company,
Qingdao, China). M.p.: capillary tube, RY-1 capillary apparatus (Tianjin Optical
1
13
Instrument Company, Tianjin, China), uncorrected. H and C NMR spectra: Bruker
1
13
ACF-300Q apparatus (300 MHz for H NMR and 75 MHz for C NMR), in
DMSO-d6 or CDCl unless otherwise indicated. Chemical shifts were given in ppm (δ)
3
downfield from tetramethylsilane. Proton coupling patterns were described as singlet
(s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Mass
spectrometry (MS): Hewlett-Packard 1100 LC/MSD spectrometer; elemental analyses:
CHNO-Rapid instrument.
4
.1.1. 4-fluoro-3-nitrobenzaldehyde (2)
A solution of 4-Fluorobenzaldehyde (1.25 g, 10 mmol) in conc. sulfuric acid (5
°
mL) was cooled to 0 C, and nitric acid (0.54 mL,12 mmol) was dissolved in 1 mL of
conc. sulfuric acid, then added dropwise to the reaction mixture. The mixture was
allowed to warm gradually to room temperature and stirred for 1 h. It was then poured
into 50 mL of ice-cold water. The produced precipitate was filtered and washed with
cold water. The solid was then dried under reduced pressure to give the title
1
compound (1.54 g, 90.4%) as a white solid. H NMR (300 MHz, CDCl ) δ 10.06 (s,
3
1
H), 8.62 (dd, J = 7.1, 2.1 Hz, 1H), 8.22 (ddd, J = 8.6, 4.2, 2.1 Hz, 1H), 7.52 (dd, J =

1
4
0.1, 8.6 Hz, 1H).
.1.2. General procedure for preparation of intermediates (3a-d)
To a solution of compound 2 (2.00 g, 11.8 mmol) and K CO (2.45 g, 17.7 mmol)
2
3
in DMF (8 mL), corresponding substituted secondary amine (17.7 mmol) was added.
°
The reaction mixture was stirred at 100 C for 1 h. After cooling to room temperature,
the mixture was poured into 100 mL of ice-cold water. The precipitate formed was
filtered and washed with cold water. The solid was then dried under reduced pressure
to afford the title compound 3a-d.
4
.1.2.1. 4-(diisobutylamino)-3-nitrobenzaldehyde (3a)
1
Orange solid, yield 91.1%. H NMR (300 MHz, CDCl ) δ 9.80 (s, 1H), 8.28 (d, J
3
=
2.1 Hz, 1H), 7.88 (dd, J = 9.0, 2.1 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 3.07 (d, J = 7.3
Hz, 4H), 2.02 – 1.82 (m, 2H), 0.79 (d, J = 6.6 Hz, 12H).
4
.1.2.2. 4-(cyclohexyl(methyl)amino)-3-nitrobenzaldehyde (3b)
1
Orange solid, yield 88.3%. H NMR (300 MHz, DMSO-d ) δ 9.81 – 9.72 (m, 1H),
6
8
2
.32 – 8.23 (m, 1H), 7.90 – 7.78 (m, 1H), 7.34 (t, J = 7.7 Hz, 1H), 3.60 (s, 1H), 2.69 –
.60 (m, 3H), 1.73 (s, 4H), 1.58 (d, J = 12.9 Hz, 3H), 1.34 (d, J = 14.3 Hz, 2H), 1.13
(s, 1H).
4
8
.1.2.3. 4-(cyclohexyl(ethyl)amino)-3-nitrobenzaldehyde (3c)
Orange solid, yield 84.4%. H NMR (300 MHz, DMSO-d ) δ 9.84 – 9.74 (m, 1H),
.29 – 8.18 (m, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.46 – 7.35 (m, 1H), 3.17 (s, 1H), 1.73
1
6
(s, 4H), 1.56 (d, J = 11.7 Hz, 3H), 1.21 (d, J = 16.2 Hz, 3H), 1.01 – 0.91 (m, 3H).
4
.1.2.4. 4-(cyclohexyl(isobutyl)amino)-3-nitrobenzaldehyde (3d)
1
Orange solid, yield 84.4%. H NMR (300 MHz, CDCl ) δ 9.85 (d, J = 2.3 Hz,
3
1
1
1
H), 8.22 (t, J = 2.2 Hz, 1H), 7.90 (dt, J = 8.8, 2.1 Hz, 1H), 7.17 (dd, J = 8.8, 2.4 Hz,
H), 3.21 – 3.09 (m, 1H), 3.05 (dd, J = 7.4, 2.3 Hz, 2H), 1.98 – 1.76 (m, 5H), 1.70 (s,
H), 1.52 (d, J = 37.2 Hz, 2H), 1.36 – 1.22 (m, 2H), 1.13 (t, J = 13.1 Hz, 1H), 0.94
(dd, J = 6.6, 2.3 Hz, 6H).
4
.1.3. General procedure for preparation of intermediates (4a-d)
A stirred mixture of compound 3a-d (1.8 mmol), ammonium acetate (0.208 g, 2.7
mmol) and nitroethane (5 mL) was heated at reflux for 4 h. After cooling to room
temperature, the reaction mixture was concentrated in vacuo and the residue was
dissolved in DCM (20 mL) and washed with water (20 mL), followed by brine (20
mL). The organic layer was concentrated under reduced pressure and the residue was
purified by silica gel flash chromatography to afford the title compound 4a-d.
4
7
.1.3.1. N, N-diisobutyl-2-nitro-4-(2-nitroprop-1-en-1-yl) aniline (4a)
1
Orange oily product, yield 79.7%. H NMR (300 MHz, CDCl ) δ 8.01 (s, 1H),
3
.91 (d, J = 2.3 Hz, 1H), 7.48 (dd, J = 9.0, 2.3 Hz, 1H), 7.15 (d, J = 8.9 Hz, 1H), 3.02

(d, J = 7.3 Hz, 4H), 2.52 (d, J = 1.1 Hz, 3H), 1.99 (dt, J = 13.5, 6.8 Hz, 2H), 0.87 (dd,
J = 6.6, 1.2 Hz, 12H).
4
.1.3.2. N-cyclohexyl-N-methyl-2-nitro-4-(2-nitroprop-1-en-1-yl) aniline (4b)
1
Orange oily product, yield 62.1%. H NMR (300 MHz, DMSO-d ) δ 8.04 (dd, J =
6
1
2
3.6, 5.2 Hz, 2H), 7.70 (dd, J = 8.7, 6.0 Hz, 1H), 7.29 (t, J = 8.2 Hz, 1H), 3.45 (s, 1H),
.68 – 2.59 (m, 3H), 2.44 – 2.36 (m, 3H), 1.70 (d, J = 13.0 Hz, 4H), 1.57 (s, 3H), 1.30
(d, J = 10.9 Hz, 2H), 1.11 (s, 1H).
4
7
.1.3.3. N-cyclohexyl-N-ethyl-2-nitro-4-(2-nitroprop-1-en-1-yl) aniline (4c)
1
Orange oily product, yield 56.5%. H NMR (300 MHz, CDCl ) δ 8.02 (s, 1H),
3
.82 (d, J = 2.5 Hz, 1H), 7.48 (d, J = 8.9 Hz, 1H), 7.17 (d, J = 8.6 Hz, 1H), 3.29 (q, J
=
7.0, 6.5 Hz, 2H), 3.12 (t, J = 12.0 Hz, 1H), 2.57 – 2.47 (m, 3H), 1.85 (d, J = 31.0 Hz,
5
1
H), 1.66 (d, J = 13.4 Hz, 1H), 1.50 (d, J = 12.3 Hz, 2H), 1.28 (d, J = 12.8 Hz, 2H),
.10 – 1.04 (m, 3H).
4
.1.3.4. N-cyclohexyl-N-isobutyl-2-nitro-4-(2-nitroprop-1-en-1-yl) aniline (4d)
1
Orange oily product, yield 54.1%. H NMR (300 MHz, CDCl ) δ 7.98 (d, J = 7.5
3
Hz, 1H), 7.85 – 7.76 (m, 1H), 7.45 (dt, J = 8.2, 4.1 Hz, 1H), 7.13 (t, J = 8.3 Hz, 1H),
2
2
.95 (t, J = 7.4 Hz, 3H), 2.55 – 2.44 (m, 3H), 1.88 – 1.72 (m, 5H), 1.59 (d, J = 8.3 Hz,
H), 1.43 (t, J = 11.1 Hz, 2H), 1.21 (s, 1H), 1.06 (d, J = 12.2 Hz, 1H), 0.89 (t, J = 7.1
Hz, 6H).
4
.1.4. General procedure for preparation of intermediates (5a-d)
To a solution of compound 4a-d (1.1 mmol) and KOAc (0.216 g, 2.2 mmol) in
acetonitrile (8 mL) was added methyl acetoacetate (0.256 g, 2.2 mmol), and the
reaction mixture was stirred at reflux overnight. After completion of the reaction
detected by TLC, the mixture was cooled to room temperature. The reaction mixture
was diluted with EtOAc (15 mL) and then washed with H O (25 mL). The organic
2
layer was concentrated in vacuo and the residue was purified by chromatography on
silica gel to afford the title compound 5a-d.
4
4
.1.4.1.
methyl
-(4-(diisobutylamino)-3-nitrophenyl)-2,5-dimethylfuran-3-carboxylate (5a)
1
Orange oily product, yield 54.1%. H NMR (300 MHz, CDCl ) δ 7.64 (d, J = 2.2
3
Hz, 1H), 7.34 – 7.28 (m, 1H), 7.11 (d, J = 8.7 Hz, 1H), 3.69 (s, 3H), 2.96 (d, J = 7.2
Hz, 4H), 2.58 (s, 3H), 2.23 (s, 3H), 1.96 (dt, J = 13.5, 6.8 Hz, 2H), 0.87 (d, J = 6.5 Hz,
1
2H).
4
4
.1.4.2.
methyl
-(4-(cyclohexyl(methyl)amino)-3-nitrophenyl)-2,5-dimethylfuran-3-carboxylate (5b)
1
Orange oily product, yield 51.8%. H NMR (300 MHz, DMSO-d ) δ 7.65 – 7.51
6
(m, 1H), 7.35 (d, J = 2.1 Hz, 1H), 7.29 – 7.15 (m, 1H), 3.69 – 3.55 (m, 3H), 3.23 (s,
1
H), 2.69 – 2.56 (m, 3H), 2.25 – 2.13 (m, 3H), 1.74 (d, J = 12.9 Hz, 4H), 1.54 (d, J =

1
4.1 Hz, 3H), 1.26 (d, J = 13.8 Hz, 2H), 1.11 (d, J = 16.0 Hz, 1H).
4
4
.1.4.3.
methyl
-(4-(cyclohexyl(ethyl)amino)-3-nitrophenyl)-2,5-dimethylfuran-3-carboxylate (5c)
1
Orange oily product, yield 49.2%. H NMR (300 MHz, CDCl ) δ 7.56 (d, J = 2.4
3
Hz, 1H), 7.38 – 7.30 (m, 1H), 7.20 (d, J = 8.5 Hz, 1H), 3.71 (t, J = 2.1 Hz, 3H), 3.19
d, J = 7.3 Hz, 2H), 3.02 (s, 1H), 2.58 (d, J = 2.5 Hz, 3H), 2.24 (d, J = 2.5 Hz, 3H),
(
1
2
.89 (d, J = 12.1 Hz, 2H), 1.79 (d, J = 12.5 Hz, 2H), 1.64 (s, 1H), 1.42 (q, J = 12.2 Hz,
H), 1.25 (d, J = 13.1 Hz, 3H), 1.01 (td, J = 7.4, 2.4 Hz, 3H).
4
4
.1.4.4.
methyl
-(4-(cyclohexyl(isobutyl)amino)-3-nitrophenyl)-2,5-dimethylfuran-3-carboxylate
(5d)
1
Orange oily product, yield 57%. H NMR (300 MHz, CDCl ) δ 7.58 (s, 1H), 7.27
3
(
3
s, 1H), 7.15 (s, 1H), 3.75 – 3.65 (m, 3H), 2.90 (d, J = 5.9 Hz, 2H), 2.57 (d, J = 4.3 Hz,
H), 2.24 (d, J = 4.4 Hz, 3H), 1.86 (s, 2H), 1.59 (d, J = 4.1 Hz, 3H), 1.41 (d, J = 12.0
Hz, 2H), 1.23 (d, J = 16.2 Hz, 3H), 1.07 (s, 1H), 0.92 – 0.83 (m, 6H).
4
.1.5. General procedure for preparation of intermediates (6a-d)
To a stirred solution of compound 5a-d (1.44 mmol) in ethanol (10 mL) was
added 4 mL of ammonium chloride (0.565 g, 8.65 mmol) aqueous solution and zinc
powder (0.462 g, 8.65 mmol). The mixture was stirred at reflux for 1 h, cooling to
room temperature then diluted with DCM (15 mL) and filtered through a celite pad.
The filtrate was washed with water (30 mL), and brine (30 mL), dried over Na SO ,
2
4
and concentrated in vacuo. The residue was purified by column chromatography on
silica gel to afford the title compound 6a-d.
4
4
.1.5.1.
methyl
-(3-amino-4-(diisobutylamino)phenyl)-2,5-dimethylfuran-3-carboxylate (6a)
1
Light yellow oily product, yield 74.5%. H NMR (300 MHz, CDCl ) δ 7.06 (d, J
3
=
8.0 Hz, 1H), 6.66 – 6.57 (m, 2H), 4.12 (s, 2H), 3.68 (s, 3H), 2.63 (d, J = 7.2 Hz, 4H),
2
.56 (s, 3H), 2.22 (s, 3H), 1.80 (dt, J = 13.5, 6.8 Hz, 2H), 0.93 (d, J = 6.6 Hz, 12H).
4
4
.1.5.2.
methyl
-(3-amino-4-(cyclohexyl(methyl)amino)phenyl)-2,5-dimethylfuran-3-carboxylate
(6b)
1
Light yellow oily product, yield 63.7%. H NMR (300 MHz, CDCl ) δ 7.03 (d, J
3
=
8.1 Hz, 1H), 6.68 – 6.54 (m, 2H), 4.07 (s, 2H), 3.69 (s, 3H), 2.98 (s, 3H), 2.68 (s,
1
1
H), 2.57 (s, 3H), 2.24 (s, 3H), 1.70 (d, J = 13.0 Hz, 4H), 1.57 (s, 3H), 1.30 (d, J =
0.9 Hz, 2H), 1.11 (s, 1H).
4
4
.1.5.3.
methyl
-(3-amino-4-(cyclohexyl(ethyl)amino)phenyl)-2,5-dimethylfuran-3-carboxylate (6c)

1
Light yellow oily product, yield 73.1%. H NMR (300 MHz, DMSO-d ) δ 6.92 (d,
6
J = 8.0 Hz, 1H), 6.54 (d, J = 1.9 Hz, 1H), 6.37 (d, J = 8.2 Hz, 1H), 4.73 (s, 2H), 3.65
–
3.54 (m, 3H), 2.98 (q, J = 7.0 Hz, 2H), 2.70 (s, 1H), 2.18 (s, 3H), 1.81 (d, J = 12.1
Hz, 2H), 1.70 (d, J = 11.3 Hz, 2H), 1.54 (d, J = 9.8 Hz, 1H), 1.32 – 1.11 (m, 5H), 0.83
t, J = 6.9 Hz, 3H).
(
4
4
.1.5.4.
methyl
-(3-amino-4-(cyclohexyl(isobutyl)amino)phenyl)-2,5-dimethylfuran-3-carboxylate
(6d)
1
Light yellow oily product, yield 79.6%. H NMR (300 MHz, CDCl ) δ 7.02 (dd, J
3
=
8.1, 3.4 Hz, 1H), 6.67 – 6.52 (m, 2H), 4.01 (s, 2H), 3.65 (d, J = 3.2 Hz, 3H), 2.69 (d,
J = 12.8 Hz, 2H), 2.54 (d, J = 3.4 Hz, 3H), 2.21 (d, J = 3.4 Hz, 3H), 1.81 (dd, J = 29.7,
1.1 Hz, 4H), 1.55 – 1.31 (m, 3H), 1.29 – 1.17 (m, 2H), 1.14 – 1.02 (m, 1H), 0.83 (dd,
J = 6.8, 3.5 Hz, 6H).
1
4
4
.1.6.
methyl
-(4-(diisobutylamino)-3-(3-(p-tolyl)ureido)phenyl)-2,5-dimethylfuran-3-carboxylate
(7a)
To a stirred solution of compound 6a (0.08 g, 0.215 mmol) in THF (4 mL) was
added 1-isocyanato-4-methylbenzene (0.043 g, 0.322 mmol). The solution was stirred
°
at 50 C for 4 h. Then the mixture was concentrated under reduced pressure and the
residue was purified by column chromatography on silica gel to afford the title
1
compound 7a (0.08 g, 73.7%) as a light-yellow solid. H NMR (300 MHz, CDCl ) δ
3
7
.92 (d, J = 2.0 Hz, 1H), 7.81 (s, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.14 (dd, J = 8.2, 6.6
Hz, 3H), 6.91 (dd, J = 8.1, 2.0 Hz, 1H), 6.67 (s, 1H), 3.67 (s, 3H), 2.61 (d, J = 7.2 Hz,
4
1
H), 2.56 (s, 3H), 2.33 (s, 3H), 2.27 (s, 3H), 1.81 – 1.69 (m, 2H), 0.87 (d, J = 6.6 Hz,
2H).
4
.1.7. General procedure for preparation of intermediates (7b-v)
A: To a stirred solution of compound 6a-d (0.268 mmol) in THF (5 mL) was
added corresponding substituted phenyl isocyanates (0.403 mmol). The solution was
°
stirred at 50 C for 4 h. Then the mixture was concentrated under reduced pressure.
The residue was directly used for the next step without further purification. This
procedure was used to afford intermediates 7b, 7e-7h, 7k-7n, 7r-7u.
B: To a solution of appropriate substituted anilines (0.59 mmol) in DCM (5 mL)
°
at 0 C was added DIPEA (0.152 g, 1.18 mmol). The solution was stirred for 10 min
and triphosgene (0.053 g, 0.177 mmol) in DCM (2 mL) was slowly added to the
reaction mixture that was warmed up to room temperature and left under stirring for 1
°
h. The mixture was cooled to 0 C and the compound 6a-d (0.295 mmol) dissolved in
THF (4 mL) was added. The solution was stirred at room temperature for 3 h. Then
the mixture was concentrated in vacuo and dissolved in EtOAc (20 mL) and washed
with 1N HCl (20 mL), followed by brine (20 mL). The organic layer was concentrated
under reduced pressure and the residue was directly used for the next step without
further purification. This procedure was used to afford intermediates 7c-7d, 7i-7j,

7
o-7q,7v.
4
.1.8. General procedure for preparation of compounds (8a-v)
To a solution of compound 7a-v (0.158 mmol) in THF/MeOH (1:1, 6 mL) was
°
added 2 mL of LiOH (0.038g, 1.58 mmol) aqueous solution and stirred at 50 C
overnight. The solvent was evaporated in vacuo and the residue was acidified with 1N
HCl. The mixture was extracted with EtOAc (20 mL) and washed with water (20 mL)
and brine (20 mL), dried over Na SO , and concentrated in vacuo. The residue was
2
4
purified by column chromatography to afford the title compound.
4
4
.1.8.1.
-(4-(diisobutylamino)-3-(3-(p-tolyl)ureido)phenyl)-2,5-dimethylfuran-3-carboxylic
acid (8a)
°
1
White solid, yield 77.1%; HPLC purity: 96.6%; m.p.: 134-136 C; H NMR (300
MHz, DMSO-d ) δ 9.50 (s, 1H), 7.84 (s, 1H), 7.74 (d, J = 2.0 Hz, 1H), 7.36 (d, J =
6
8
.1 Hz, 2H), 7.07 (t, J = 8.0 Hz, 3H), 6.93 (dd, J = 8.3, 2.0 Hz, 1H), 2.64 (d, J = 7.0
Hz, 4H), 2.40 (s, 3H), 2.23 (s, 3H), 2.19 (s, 3H), 1.69 (p, J = 6.5 Hz, 2H), 0.86 (d, J =
13
6
.5 Hz, 12H); C NMR (75 MHz, DMSO-d ) δ 152.78, 145.45, 139.06, 137.41,
6
1
2
33.84, 130.47, 129.05, 123.93, 122.14, 121.12, 120.99, 118.68, 62.70, 25.82, 21.07,
-
0.30, 13.46, 11.93; ESIMS m/z [M-H] 490.1; Anal. calcd. For C H N O : C, 70.85;
2
9
37
3
4
H, 7.59; N, 8.55; Found: C, 70.83; H, 7.62; N, 8.51.
4
4
.1.8.2.
-(4-(diisobutylamino)-3-(3-(2-fluorophenyl)ureido)phenyl)-2,5-dimethylfuran-3-carb
oxylic acid (8b)
°
1
White solid, yield 68.3%; HPLC purity: 98.1%; m.p.: 119-121 C; H NMR (300
MHz, DMSO-d ) δ 12.23 (s, 1H), 9.33 (s, 1H), 8.09 (s, 1H), 7.99 (s, 1H), 7.26 – 7.07
6
(
m, 3H), 7.02 (s, 1H), 6.87 (d, J = 7.9 Hz, 1H), 2.71 (d, J = 7.0 Hz, 4H), 2.22 – 2.13
1
3
(m, 3H), 1.71 (s, 2H), 0.87 (dd, J = 7.6, 3.2 Hz, 12H); C NMR (75 MHz, DMSO-d )
6
δ 164.74, 156.29, 154.48, 152.61, 151.27, 146.60, 139.94, 133.36, 127.69, 127.36,
1
2
27.22, 124.30, 122.91, 122.42, 121.12, 120.82, 115.24, 114.99, 113.54, 62.32, 25.81,
+
1.04, 20.71, 13.69, 11.63; ESIMS m/z [M+H] 496.2; Anal. calcd. For C H FN O :
2
8
34
3
4
C, 67.86; H, 6.92; N, 8.48; Found: C, 67.83; H, 6.90; N, 8.46.
4
4
.1.8.3.
-(4-(diisobutylamino)-3-(3-(4-fluorophenyl)ureido)phenyl)-2,5-dimethylfuran-3-carb
oxylic acid (8c)
°
1
Light yellow solid, yield 81.1%; HPLC purity: 97.2%; m.p.: 135-138 C; H
NMR (300 MHz, DMSO-d ) δ 9.50 (s, 1H), 7.90 – 7.78 (m, 2H), 7.53 – 7.42 (m, 2H),
6
7
3
.23 – 7.05 (m, 3H), 6.86 (dd, J = 8.2, 2.0 Hz, 1H), 2.67 (d, J = 6.9 Hz, 4H), 2.49 (s,
H), 2.18 (s, 3H), 1.68 (dq, J = 13.7, 6.8 Hz, 2H), 0.88 (d, J = 6.6 Hz, 12H); C NMR
1
3
(
75 MHz, DMSO-d ) δ 164.77, 158.92, 156.27, 155.77, 152.59, 146.60, 139.18,
6
1
36.09, 134.02, 128.23, 123.81, 121.50, 121.36, 120.89, 120.39, 120.29, 120.13,
+
115.35, 115.06, 62.74, 25.89, 21.07, 20.80, 13.68, 11.64; ESIMS m/z [M+H] 496.3;

Anal. calcd. For C H FN O : C, 67.86; H, 6.92; N, 8.48; Found: C, 67.84; H, 6.93;
2
8
34
3
4
N, 8.47.
4
4
.1.8.4.
-(3-(3-(2,4-difluorophenyl)ureido)-4-(diisobutylamino)phenyl)-2,5-dimethylfuran-3-
carboxylic acid (8d)
°
1
Light yellow solid, yield 72.8%; HPLC purity: 98.7%; m.p.: 177-179 C; H
NMR (300 MHz, CDCl ) δ 8.21 (s, 1H), 8.01 (d, J = 1.9 Hz, 1H), 7.99 – 7.87 (m, 1H),
3
7
2
.16 (d, J = 8.2 Hz, 1H), 7.00 – 6.80 (m, 3H), 6.61 (s, 1H), 2.62 (d, J = 7.2 Hz, 4H),
.56 (s, 3H), 2.25 (s, 3H), 1.76 (dt, J = 13.5, 6.9 Hz, 2H), 0.95 – 0.87 (m, 12H); C
1
3
NMR (75 MHz, CDCl ) δ 168.26, 158.60, 152.02, 147.25, 139.16, 133.61, 129.28,
3
1
6
24.41, 123.65, 123.53, 121.17, 120.48, 120.27, 112.15, 110.93, 110.64, 103.21,
2.84, 25.66, 20.41, 13.86, 11.46; ESIMS m/z [M+H] 514.2; Anal. calcd. For
+
C H F N O : C, 65.48; H, 6.48; N, 8.18; Found: C, 65.50; H, 6.47; N, 8.19.
28
33
2
3
4
4
4
.1.8.5.
-(4-(cyclohexyl(methyl)amino)-3-(3-(p-tolyl)ureido)phenyl)-2,5-dimethylfuran-3-car
boxylic acid (8e)
°
1
White solid, yield 76.3%; HPLC purity: 95.8%; m.p.: 150-153 C; H NMR (300
MHz, DMSO-d ) δ 12.10 (s, 1H), 9.40 (s, 1H), 8.26 (s, 1H), 7.99 (s, 1H), 7.34 (d, J =
6
8
2
1
1
1
2
7
.2 Hz, 2H), 7.20 – 7.11 (m, 1H), 7.07 (d, J = 8.0 Hz, 2H), 6.80 (d, J = 8.0 Hz, 1H),
.58 (d, J = 4.9 Hz, 4H), 2.19 (dd, J = 16.9, 5.5 Hz, 6H), 1.84 (s, 2H), 1.68 (s, 2H),
1
3
.52 (s, 1H), 1.26 – 1.06 (m, 5H); C NMR (75 MHz, DMSO-d ) δ 164.76, 156.29,
6
52.65, 146.56, 139.19, 137.17, 135.07, 130.71, 129.14, 128.77 (d, J = 4.5 Hz),
22.97, 122.37, 121.03, 119.79, 118.47, 113.60, 61.66, 37.71, 29.33, 25.47, 24.98,
+
0.25, 13.65, 11.61; ESIMS m/z [M+H] 476.2; Anal. calcd. For C H N O : C,
2
8
33
3
4
0.71; H, 6.99; N, 8.84; Found: C, 70.73; H, 6.97; N, 8.83.
4
4
.1.8.6.
-(4-(cyclohexyl(methyl)amino)-3-(3-(o-tolyl)ureido)phenyl)-2,5-dimethylfuran-3-car
boxylic acid (8f)
°
1
White solid, yield 73.7%; HPLC purity: 97.4%; m.p.: 205-207 C; H NMR (300
MHz, DMSO-d ) δ 12.21 (s, 1H), 8.62 (s, 1H), 8.31 (s, 1H), 7.87 (s, 1H), 7.56 (d, J =
6
8
4
.1 Hz, 1H), 7.21 – 7.09 (m, 3H), 6.99 (s, 1H), 2.58 (s, 4H), 2.46 (s, 3H), 2.22 (q, J =
.5, 3.4 Hz, 3H), 2.16 (t, J = 3.2 Hz, 3H), 1.77 (s, 2H), 1.67 (s, 2H), 1.51 (s, 1H), 1.16
1
3
(dt, J = 35.3, 10.7 Hz, 5H); C NMR (75 MHz, DMSO-d ) δ 164.75, 156.24, 153.10,
6
1
1
46.53, 139.79, 137.00, 134.58, 130.26, 128.31, 126.10, 123.85, 123.18, 121.91,
21.01, 120.75, 113.62, 61.56, 36.63, 29.00, 25.51, 25.10, 17.90, 13.68, 11.65;
+
ESIMS m/z [M+H] 476.2; Anal. calcd. For C H N O : C, 70.71; H, 6.99; N, 8.84;
2
8
33
3
4
Found: C, 70.72; H, 6.96; N, 8.85.
4
4
.1.8.7.
-(4-(cyclohexyl(methyl)amino)-3-(3-(m-tolyl)ureido)phenyl)-2,5-dimethylfuran-3-ca
rboxylic acid (8g)

°
1
White solid, yield 79.4%; HPLC purity: 96.5%; m.p.: 148-155 C; H NMR (300
MHz, DMSO-d ) δ 9.48 (s, 1H), 7.94 (s, 1H), 7.35 – 7.20 (m, 3H), 7.14 (t, J = 7.7 Hz,
6
2
3
4
1
1
H), 6.78 (d, J = 7.4 Hz, 1H), 2.63 (s, 4H), 2.48 (s, 3H), 2.26 (s, 3H), 2.20 – 2.16 (m,
H), 1.86 (s, 2H), 1.69 (s, 2H), 1.54 (d, J = 10.9 Hz, 1H), 1.26 (s, 1H), 1.22 – 1.00 (m,
13
H); C NMR (75 MHz, DMSO-d ) δ 164.73, 156.36, 152.70, 146.65, 139.68,
6
37.91, 134.77, 128.59, 122.57, 122.37, 120.88, 118.79, 115.44, 113.57, 110.23,
06.85, 37.94, 29.14, 26.38, 25.39, 24.94, 21.15, 13.67, 11.63, 11.48; ESIMS m/z
+
[
M+H] 476.2; Anal. calcd. For C H N O : C, 70.71; H, 6.99; N, 8.84; Found: C,
2
8
33
3
4
7
0.73; H, 6.96; N, 8.86.
4
4
.1.8.8.
-(4-(cyclohexyl(methyl)amino)-3-(3-(2-fluorophenyl)ureido)phenyl)-2,5-dimethylfur
an-3-carboxylic acid (8h)
°
1
Light yellow solid, yield 68.7%; HPLC purity: 96.9%; m.p.: 155-158 C; H
NMR (300 MHz, DMSO-d ) δ 12.22 (s, 1H), 9.37 (s, 1H), 8.67 (s, 1H), 8.05 (t, J =
6
8
=
.2 Hz, 1H), 7.93 (s, 1H), 7.16 (dq, J = 17.3, 8.9, 7.9 Hz, 3H), 7.00 (s, 1H), 6.82 (d, J
8.2 Hz, 1H), 2.67 (s, 1H), 2.60 (s, 3H), 2.48 (s, 3H), 2.17 (s, 3H), 1.86 (d, J = 11.5
1
3
Hz, 2H), 1.68 (s, 2H), 1.53 (s, 1H), 1.33 – 1.18 (m, 3H), 1.16 – 1.07 (m, 2H); C
NMR (75 MHz, DMSO- d ) δ 164.76, 156.16, 154.11, 150.91, 146.49, 139.77, 134.75,
6
1
2
28.58, 127.43, 124.29, 122.73, 122.30, 121.81, 120.71, 115.14, 113.73, 61.67, 37.47,
+
9.28, 25.55, 13.68, 11.68; ESIMS m/z [M+H] 480.2; Anal. calcd. For C H FN O :
2
7
30
3
4
C, 67.62; H, 6.31; N, 8.76; Found: C, 67.65; H, 6.33; N, 8.75.
4
4
.1.8.9.
-(4-(cyclohexyl(methyl)amino)-3-(3-(4-fluorophenyl)ureido)phenyl)-2,5-dimethylfur
an-3-carboxylic acid (8i)
°
1
Light yellow solid, yield 65.1%; HPLC purity: 97.4%; m.p.: 170-173 C; H
NMR (300 MHz, DMSO-d ) δ 9.60 (s, 1H), 8.32 (s, 1H), 7.99 (s, 1H), 7.48 (dd, J =
6
9
.0, 4.9 Hz, 2H), 7.12 (t, J = 8.7 Hz, 3H), 6.83 (s, 1H), 2.63 (s, 4H), 2.50 (s, 3H), 2.19
1
3
(s, 3H), 1.87 (s, 2H), 1.71 (s, 2H), 1.55 (d, J = 10.5 Hz, 1H), 1.30 – 1.07 (m, 5H); C
NMR (75 MHz, DMSO-d ) δ 164.78 (d, J = 8.3 Hz), 158.87, 156.36, 155.72, 152.76,
6
1
46.67, 136.04, 134.70, 122.41, 120.02 (d, J = 7.4 Hz), 115.40, 115.10, 113.55,
1
10.25, 106.88, 37.96, 29.18, 26.39, 25.38, 24.93, 13.66, 11.54 (d, J = 11.6 Hz);
-
ESIMS m/z [M-H] 478.2; Anal. calcd. For C H FN O : C, 67.62; H, 6.31; N, 8.76;
2
7
30
3
4
Found: C, 67.61; H, 6.33; N, 8.77.
4
4
.1.8.10.
-(4-(cyclohexyl(methyl)amino)-3-(3-(2,4-difluorophenyl)ureido)phenyl)-2,5-dimeth
ylfuran-3-carboxylic acid (8j)
°
1
Light yellow solid, yield 58.6%; HPLC purity: 96.7%; m.p.: 171-174 C; H
NMR (300 MHz, CDCl ) δ 7.84 (s, 2H), 7.16 (d, J = 33.5 Hz, 3H), 6.93 – 6.78 (m,
3
3
1
1
H), 3.06 (s, 4H), 2.57 (s, 3H), 2.25 (s, 3H), 1.84 (s, 3H), 1.64 (d, J = 11.2 Hz, 2H),
1
3
.22 (d, J = 30.0 Hz, 5H); C NMR (75 MHz, CDCl ) δ 167.83, 160.14, 158.89,
3
56.74, 155.32, 153.98, 152.17, 152.00, 147.75, 132.36, 123.98, 121.91, 120.68,

1
19.32, 111.99, 110.64, 110.35, 103.73, 103.40, 103.07, 26.40, 24.53, 22.14, 13.82,
+
11.48, 10.94; ESIMS m/z [M+H] 498.3; Anal. calcd. For C H F N O : C, 65.18; H,
27 29 2 3 4
5
.88; N, 8.45; Found: C, 65.19; H, 5.86; N, 8.44.
4
4
.1.8.11.
-(4-(cyclohexyl(ethyl)amino)-3-(3-(p-tolyl)ureido)phenyl)-2,5-dimethylfuran-3-carb
oxylic acid (8k)
°
1
White solid, yield 81.2%; HPLC purity: 98.5%; m.p.: 180-183 C; H NMR (300
MHz, DMSO-d ) δ 12.22 (s, 1H), 9.43 (s, 1H), 8.44 (s, 1H), 8.06 (t, J = 2.0 Hz, 1H),
6
7
2
2
.41 – 7.28 (m, 2H), 7.22 – 7.03 (m, 3H), 6.88 – 6.72 (m, 1H), 3.03 (d, J = 6.4 Hz,
H), 2.73 (s, 1H), 2.51 (d, J = 1.9 Hz, 3H), 2.32 – 2.11 (m, 6H), 1.92 (s, 2H), 1.69 (s,
H), 1.54 (d, J = 11.7 Hz, 1H), 1.10 (d, J = 41.6 Hz, 5H), 0.91 – 0.79 (m, 3H); C
1
3
NMR (75 MHz, DMSO-d ) δ 164.76, 156.13, 152.55, 146.48, 137.49, 137.43, 135.47,
6
1
2
30.45, 129.24, 129.07, 124.06, 122.61, 121.21, 119.41, 118.47, 113.80, 61.49, 43.64,
9.77, 25.61, 25.16, 20.30, 13.67, 12.73, 11.71; ESIMS m/z [M-H] 488.3; Anal. calcd.
-
For C H N O : C, 71.14; H, 7.21; N, 8.58; Found: C, 71.15; H, 7.22; N, 8.57.
2
9
35
3
4
4
4
.1.8.12.
-(4-(cyclohexyl(ethyl)amino)-3-(3-(o-tolyl)ureido)phenyl)-2,5-dimethylfuran-3-carb
oxylic acid (8l)
°
1
White solid, yield 76.3%; HPLC purity: 97.6%; m.p.: 192-194 C; H NMR (300
MHz, DMSO-d ) δ 12.24 (s, 1H), 8.66 (s, 1H), 8.39 (s, 1H), 7.95 (s, 1H), 7.47 (d, J =
6
7
2
2
.9 Hz, 1H), 7.23 – 7.10 (m, 3H), 7.03 (t, J = 7.3 Hz, 1H), 6.79 (d, J = 8.2 Hz, 1H),
.96 (d, J = 6.2 Hz, 2H), 2.64 (s, 1H), 2.48 (s, 3H), 2.23 (s, 3H), 2.16 (s, 3H), 1.80 (s,
H), 1.65 (s, 2H), 1.51 (d, J = 11.0 Hz, 1H), 1.11 (s, 5H), 0.81 (t, J = 6.9 Hz, 3H); C
1
3
NMR (75 MHz, DMSO-d ) δ 164.72, 156.21, 153.16, 146.52, 137.01, 136.83, 135.96,
6
1
2
31.24, 130.31, 128.91, 126.14, 124.85, 124.37, 123.62, 122.81, 120.02, 61.47, 42.76,
9.59, 25.57, 25.15, 17.88, 13.67, 12.92, 11.67; ESIMS m/z [M+H] 490.2; Anal.
+
calcd. For C H N O : C, 71.14; H, 7.21; N, 8.58; Found: C, 71.16; H, 7.22; N, 8.59.
2
9
35
3
4
4
4
.1.8.13.
-(4-(cyclohexyl(ethyl)amino)-3-(3-(m-tolyl)ureido)phenyl)-2,5-dimethylfuran-3-carb
oxylic acid (8m)
°
1
White solid, yield 79.5%; HPLC purity: 97.8%; m.p.: 145-147 C; H NMR (300
MHz, DMSO-d ) δ 12.18 (s, 1H), 9.45 (s, 1H), 8.45 (s, 1H), 8.05 (d, J = 2.7 Hz, 1H),
6
7
2
.33 – 7.21 (m, 2H), 7.15 (s, 2H), 6.78 (dd, J = 10.8, 7.9 Hz, 2H), 3.02 (d, J = 7.4 Hz,
H), 2.71 (s, 1H), 2.48 (s, 3H), 2.26 (d, J = 2.4 Hz, 3H), 2.17 (d, J = 3.4 Hz, 3H), 1.90
(
s, 2H), 1.68 (s, 2H), 1.52 (d, J = 11.8 Hz, 1H), 1.16 (s, 4H), 1.02 (s, 1H), 0.86 – 0.79
1
3
(m, 3H); C NMR (75 MHz, DMSO-d ) δ 164.71, 156.27, 152.49, 146.56, 139.81,
6
1
1
37.87, 128.57, 124.07, 122.70, 122.49, 119.40, 118.87, 115.49, 113.66, 110.33,
06.82, 105.77, 61.58, 43.64, 29.70, 25.55, 25.12, 21.16, 13.67, 12.68, 11.68; ESIMS
+
m/z [M+H] 490.2; Anal. calcd. For C H N O : C, 71.14; H, 7.21; N, 8.58; Found:
2
9
35
3
4
C, 71.15; H, 7.23; N, 8.59.

4
4
.1.8.14.
-(4-(cyclohexyl(ethyl)amino)-3-(3-(2-fluorophenyl)ureido)phenyl)-2,5-dimethylfura
n-3-carboxylic acid (8n)
°
1
White solid, yield 71.8%; HPLC purity: 97.1%; m.p.: 127-128 C; H NMR (300
MHz, DMSO-d ) δ 12.20 (s, 1H), 9.42 (s, 1H), 8.79 (s, 1H), 8.12 – 7.97 (m, 2H), 7.28
6
–
7.08 (m, 3H), 7.06 – 6.97 (m, 1H), 6.84 (dd, J = 8.1, 2.1 Hz, 1H), 3.04 (q, J = 6.9
Hz, 2H), 2.73 (s, 1H), 2.50 (d, J = 1.7 Hz, 3H), 2.19 (s, 3H), 1.69 (s, 2H), 1.54 (d, J =
13
1
1.6 Hz, 1H), 1.22 – 1.01 (m, 5H), 0.85 (t, J = 6.9 Hz, 3H); C NMR (75 MHz,
DMSO-d ) δ 164.72, 156.26, 154.26, 152.54, 146.56, 137.04, 136.05, 129.00, 127.36
6
(d, J = 10.9 Hz), 124.36, 124.04, 123.11, 122.97, 122.10, 120.18, 115.21, 114.96,
1
13.64, 61.59, 43.30, 29.65, 25.59, 25.18, 13.67, 12.69, 12.64, 11.67; ESIMS m/z
+
[
M+H] 494.2; Anal. calcd. For C H FN O : C, 68.14; H, 6.54; N, 8.51; Found: C,
28 32 3 4
6
8.15; H, 6.53; N, 8.52.
4
4
.1.8.15.
-(4-(cyclohexyl(ethyl)amino)-3-(3-(4-fluorophenyl)ureido)phenyl)-2,5-dimethylfura
n-3-carboxylic acid (8o)
°
1
Light yellow solid, yield 65.1%; HPLC purity: 96.8%; m.p.: 187-189 C; H
NMR (300 MHz, DMSO-d ) δ 12.22 (s, 1H), 9.57 (s, 1H), 8.45 (s, 1H), 8.04 (d, J =
6
2
1
.2 Hz, 1H), 7.47 (dd, J = 8.9, 5.0 Hz, 2H), 7.17 – 7.06 (m, 3H), 6.80 (d, J = 8.2 Hz,
H), 3.02 (d, J = 7.4 Hz, 2H), 2.71 (s, 1H), 2.48 (s, 3H), 2.17 (d, J = 3.3 Hz, 3H), 1.90
(
6
1
s, 2H), 1.67 (s, 2H), 1.52 (d, J = 11.1 Hz, 1H), 1.15 (s, 4H), 1.01 (s, 1H), 0.83 (t, J =
1
3
.7 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 164.83, 156.38, 152.66, 146.63,
6
37.12, 135.92, 135.71, 129.18, 124.15, 122.85, 121.01, 120.32, 120.23, 119.26,
1
15.40, 115.10, 113.53, 61.48, 43.61, 29.74, 25.49, 25.05, 13.60, 12.64, 11.54; ESIMS
+
m/z [M+H] 494.2; Anal. calcd. For C H FN O : C, 68.14; H, 6.54; N, 8.51; Found:
2
8
32
3
4
C, 68.14; H, 6.55; N, 8.53.
4
4
.1.8.16.
-(4-(cyclohexyl(ethyl)amino)-3-(3-(2,4-difluorophenyl)ureido)phenyl)-2,5-dimethylf
uran-3-carboxylic acid (8p)
°
1
Light yellow solid, yield 67.3%; HPLC purity: 97.3%; m.p.: 119-123 C; H
NMR (300 MHz, DMSO-d ) δ 12.20 (s, 1H), 9.39 (s, 1H), 8.74 (s, 1H), 8.00 (t, J =
6
3
6
2
.6 Hz, 2H), 7.30 (s, 1H), 7.17 (d, J = 8.3 Hz, 1H), 7.04 (t, J = 8.9 Hz, 1H), 6.89 –
.79 (m, 1H), 3.03 (d, J = 7.4 Hz, 2H), 2.73 (s, 1H), 2.50 (s, 3H), 2.18 (s, 3H), 1.91 (s,
H), 1.70 (s, 2H), 1.54 (d, J = 11.5 Hz, 1H), 1.10 (d, J = 38.3 Hz, 5H), 0.85 (t, J = 6.9
1
3
Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 164.68, 156.20, 154.52, 152.57, 146.52,
6
1
37.08, 135.92, 129.11, 124.05, 123.53, 123.42, 123.09, 121.10, 120.06, 113.70,
1
1
6
11.03, 110.75, 104.09, 103.75, 103.41, 61.57, 43.41, 29.70, 25.61, 25.17, 13.67,
2.71, 11.69; ESIMS m/z [M+H] 512.2; Anal. calcd. For C H F N O : C, 65.74; H,
28 31 2 3 4
.11; N, 8.21; Found: C, 65.75; H, 6.12; N, 8.23.
+
4
4
.1.8.17.
-(4-(cyclohexyl(isobutyl)amino)-3-(3-(p-tolyl)ureido)phenyl)-2,5-dimethylfuran-3-c

arboxylic acid (8q)
°
1
White solid, yield 80.7%; HPLC purity: 98.2%; m.p.: 175-176 C; H NMR (300
MHz, DMSO-d ) δ 9.41 (s, 1H), 7.91 (d, J = 9.1 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H),
6
7
2
.13 (d, J = 7.9 Hz, 1H), 7.07 (d, J = 8.0 Hz, 2H), 6.80 (d, J = 7.9 Hz, 1H), 2.78 (s,
H), 2.56 (m, 1H), 2.48 (s, 3H), 2.20 (d, J = 13.1 Hz, 6H), 1.94 – 1.83 (m, 2H), 1.67
(
1
s, 2H), 1.50 (d, J = 9.8 Hz, 1H), 1.36 (d, J = 6.8 Hz, 1H), 1.21 (t, J = 11.1 Hz, 2H),
1
3
.06 (dd, J = 16.8, 8.0 Hz, 3H), 0.82 (d, J = 6.5 Hz, 6H);
C NMR (75 MHz,
DMSO-d ) δ 164.69, 156.17, 152.69, 146.55, 137.26, 137.16, 135.73, 130.76, 129.08,
6
1
2
28.33, 123.10, 121.04, 119.00, 113.65, 62.87, 56.01, 29.14, 26.23, 25.54, 24.50,
+
1.03, 20.31, 13.70, 11.75; ESIMS m/z [M+H] 518.3; Anal. calcd. For C H N O :
3
1
39
3
4
C, 71.93; H, 7.59; N, 8.12; Found: C, 71.95; H, 7.58; N, 8.13.
4
4
.1.8.18.
-(4-(cyclohexyl(isobutyl)amino)-3-(3-(o-tolyl)ureido)phenyl)-2,5-dimethylfuran-3-c
arboxylic acid (8r)
°
1
White solid, yield 76.6%; HPLC purity: 95.7%; m.p.: 217-220 C; H NMR (300
MHz, DMSO-d ) δ 12.20 (s, 1H), 7.96 – 7.79 (m, 2H), 7.38 (dd, J = 7.9, 1.4 Hz, 1H),
6
7
2
1
1
1
2
.26 – 7.00 (m, 4H), 6.79 (dd, J = 8.1, 2.1 Hz, 1H), 2.72 (s, 2H), 2.51 – 2.47 (m, 4H),
.20 (d, J = 16.7 Hz, 6H), 1.67 (s, 4H), 1.51 (d, J = 9.3 Hz, 1H), 1.31 (q, J = 6.5 Hz,
1
3
H), 1.23 – 0.96 (m, 5H), 0.78 (d, J = 6.5 Hz, 6H); C NMR (75 MHz, DMSO-d ) δ
6
64.73, 156.23, 153.33, 146.57, 137.40, 136.52, 135.28, 132.33, 130.42, 128.07,
26.30, 125.72, 124.99, 123.14, 122.72, 121.21, 120.96, 113.58, 62.58, 54.93, 26.12,
+
5.59, 25.49, 20.86, 17.77, 13.68, 11.70; ESIMS m/z [M+H] 518.3; Anal. calcd. For
C H N O : C, 71.93; H, 7.59; N, 8.12; Found: C, 71.94; H, 7.58; N, 8.13.
31
39
3
4
4
4
.1.8.19.
-(4-(cyclohexyl(isobutyl)amino)-3-(3-(m-tolyl)ureido)phenyl)-2,5-dimethylfuran-3-c
arboxylic acid (8s)
°
1
White solid, yield 77.9%; HPLC purity: 97.5%; m.p.: 139-143 C; H NMR (300
MHz, DMSO-d ) δ 12.23 (s, 1H), 9.44 (s, 1H), 8.04 – 7.84 (m, 2H), 7.27 (d, J = 13.8
6
Hz, 2H), 7.14 (dd, J = 8.0, 3.7 Hz, 2H), 6.79 (t, J = 8.5 Hz, 2H), 2.79 (s, 2H), 2.49 (m,
4
H), 2.25 (s, 3H), 2.18 (s, 3H), 1.90 (s, 2H), 1.67 (s, 2H), 1.51 (s, 1H), 1.24 (d, J =
1
3
11.9 Hz, 3H), 1.15 – 0.97 (m, 3H), 0.83 (d, J = 6.4 Hz, 6H); C NMR (75 MHz,
DMSO-d ) δ 164.72, 156.24, 152.62, 146.58, 139.70, 137.87, 137.19, 135.60, 128.57,
6
1
2
28.32, 123.13, 122.72, 121.00, 119.38, 115.99, 113.60, 62.90, 55.96, 29.11, 26.19,
5.52, 21.13, 20.99, 13.69, 11.72; ESIMS m/z [M+H] 518.3; Anal. calcd. For
+
C H N O : C, 71.93; H, 7.59; N, 8.12; Found: C, 71.94; H, 7.58; N, 8.13.
31
39
3
4
4
4
.1.8.20.
-(4-(cyclohexyl(isobutyl)amino)-3-(3-(2-fluorophenyl)ureido)phenyl)-2,5-dimethylf
uran-3-carboxylic acid (8t)
°
1
Light yellow solid, yield 71.9%; HPLC purity: 96.9%; m.p.: 141-143 C; H
NMR (300 MHz, DMSO-d ) δ 12.27 (s, 1H), 9.42 (s, 1H), 8.17 (s, 1H), 7.97 (t, J =
6
8
.3 Hz, 1H), 7.79 (d, J = 2.1 Hz, 1H), 7.24 (dd, J = 11.5, 8.1 Hz, 1H), 7.14 (dd, J =

8
1
1
6
.4, 5.5 Hz, 2H), 7.05 (q, J = 6.7 Hz, 1H), 6.90 – 6.82 (m, 1H), 2.80 (s, 2H), 2.60 (s,
H), 2.51 (s, 3H), 2.21 (s, 3H), 1.90 (d, J = 11.2 Hz, 2H), 1.69 (s, 2H), 1.53 (s, 1H),
.41 (d, J = 8.2 Hz, 1H), 1.27 (d, J = 12.8 Hz, 2H), 1.15 – 1.01 (m, 3H), 0.85 (d, J =
1
3
.5 Hz, 6H); C NMR (75 MHz, DMSO-d ) δ 164.71, 156.18, 152.78, 151.49,
6
1
1
2
46.56, 138.15, 134.94, 127.88, 127.32, 127.17, 124.31, 123.75, 123.32, 123.22,
22.93, 122.75, 122.46, 120.89, 115.28, 115.02, 113.63, 62.87, 55.43, 29.09, 26.11,
-
5.57, 21.00, 13.70, 11.75; ESIMS m/z [M-H] 520.3; Anal. calcd. For C H FN O :
3
0
36
3
4
C, 69.08; H, 6.96; N, 8.06; Found: C, 69.09; H, 6.98; N, 8.08.
4
4
.1.8.21.
-(4-(cyclohexyl(isobutyl)amino)-3-(3-(4-fluorophenyl)ureido)phenyl)-2,5-dimethylf
uran-3-carboxylic acid (8u)
°
1
Light yellow solid, yield 69.4%; HPLC purity: 98.7%; m.p.: 206-210 C; H
NMR (300 MHz, DMSO-d ) δ 9.56 (s, 1H), 7.98 – 7.86 (m, 2H), 7.47 (dt, J = 8.8, 2.9
6
Hz, 2H), 7.18 – 7.04 (m, 3H), 6.88 – 6.78 (m, 1H), 2.79 (s, 2H), 2.65 – 2.55 (m, 1H),
2
1
.48 (d, 3H), 2.18 (s, 3H), 1.91 (d, J = 11.7 Hz, 2H), 1.68 (s, 2H), 1.51 (d, J = 10.1 Hz,
H), 1.38 (s, 1H), 1.23 (s, 2H), 1.16 – 1.00 (m, 3H), 0.88 – 0.79 (m, 6H); C NMR
1
3
(
75 MHz, DMSO-d ) δ 164.74, 158.95, 156.21, 155.79, 152.68, 146.58, 137.20,
6
1
36.14, 135.54, 128.34, 123.23, 123.17, 121.01, 120.51, 120.40, 115.36, 115.07,
-
113.62, 62.92, 55.98, 29.13, 26.20, 25.52, 21.00, 13.70, 11.74; ESIMS m/z [M-H]
5
6
20.3; Anal. calcd. For C H FN O : C, 69.08; H, 6.96; N, 8.06; Found: C, 69.10; H,
3
0
36
3
4
.95; N, 8.08.
.1.8.22.
4
4
-(4-(cyclohexyl(isobutyl)amino)-3-(3-(2,4-difluorophenyl)ureido)phenyl)-2,5-dimeth
ylfuran-3-carboxylic acid (8v)
°
1
Light yellow solid, yield 65.7%; HPLC purity: 96.6%; m.p.: 218-220 C; H
NMR (300 MHz, DMSO-d ) δ 9.37 (s, 1H), 8.13 (s, 1H), 7.91 (s, 1H), 7.82 (s, 1H),
6
7
2
1
.32 (s, 1H), 7.16 (d, J = 8.2 Hz, 1H), 7.06 (s, 1H), 6.86 (d, J = 8.2 Hz, 1H), 2.81 (s,
H), 2.60 (s, 1H), 2.51 (s, 3H), 2.21 (s, 3H), 1.90 (s, 2H), 1.71 (s, 2H), 1.54 (s, 1H),
13
.41 (s, 1H), 1.26 (s, 2H), 1.08 (s, 3H), 0.86 (d, J = 6.0 Hz, 6H); C NMR (75 MHz,
DMSO-d ) δ 164.80, 156.35, 153.06, 146.65, 137.98, 134.86, 127.98, 125.11, 123.69,
6
1
5
23.29, 122.88, 121.86, 120.78, 113.45, 111.21, 110.92, 104.20, 103.87, 103.52, 62.88,
5.39, 30.44, 26.06, 25.48, 20.82, 19.74, 13.61, 11.57; ESIMS m/z [M+H] 540.3;
+
Anal. calcd. For C H F N O : C, 66.77; H, 6.54; N, 7.79; Found: C, 66.74; H, 6.56;
3
0
35
2
3
4
N, 7.78.
4
.1.9. N-cyclohexylisobutyramide (10)
To a solution of cyclohexylamine (1g, 9.88 mmol) in DCM (15 mL) at 0 C was
°
added TEA (1.5g, 14.82 mmol). The solution was stirred for 10 min and isobutyryl
chloride (1.26g, 11.85 mmol) in DCM (10 mL) was added dropwise to the reaction
mixture. The mixture was allowed to warm to room temperature. After 12 h, the
reaction was quenched with saturated sodium bicarbonate solution then extracted with
DCM (20 mL×3). The combined organic layers were washed with 1N HCl (50 mL)

and brine (50 mL), dried over Na SO , and evaporated in vacuo to afford title
2
4
1
compound (1.25 g, 74.7%) as an off white solid. H NMR (300 MHz, DMSO-d ) δ
6
7
1
6
.50 (d, J = 8.0 Hz, 1H), 3.46 (ddp, J = 10.7, 7.4, 4.0 Hz, 1H), 2.28 (h, J = 6.8 Hz,
H), 1.66 (td, J = 10.4, 8.1, 3.6 Hz, 4H), 1.52 (dd, J = 9.8, 6.3 Hz, 1H), 1.27 – 1.03 (m,
H), 0.95 (d, J = 6.8 Hz, 6H).
4
.1.10. N-isobutylcyclohexanamine (11)
To a solution of compound 10 (1.25g, 7.38 mmol) in THF (10 mL) was slowly
°
added LiAlH4 (0.56 g, 14.77 mmol). The resulting solution was heated at 70 C for 12
°
h. Then the mixture was diluted with EtOAc (10 mL) and cooled to 0 C, water (2 mL)
was slowly added to the reaction mixture. The mixture was filtered through a Celite
pad and the organic layer of the filtrate was washed with water (30 mL) and brine (30
mL), dried over Na SO , and evaporated in vacuo to afford title compound (0.87 g,
2
4
1
7
5.9%) as a clear oily product. H NMR (300 MHz, CDCl ) δ 2.44 (dd, J = 6.8, 2.6
3
Hz, 2H), 2.41 – 2.34 (m, 1H), 1.93 – 1.86 (m, 2H), 1.78 – 1.69 (m, 3H), 1.62 (d, J =
1.3 Hz, 2H), 1.21 – 1.12 (m, 4H), 0.92 (dd, J = 6.8, 2.7 Hz, 6H).
1
4
.1.11. tert-butyl (Z)-3-aminobut-2-enoate (13)
A mixture of tert-Butyl acetoacetate (3 g, 18.96 mmol) and ammonium acetate
(4.39 g, 56.89 mmol) in 20 mL MeOH was stirred at reflux for 6 h. The reaction
mixture was then cooled to room temperature and evaporated in vacuo, the residue
was dissolved in EtOAc (30 mL) and washed with water (30 mL), followed by brine
(
30 mL), dried over Na SO . The organic layer was concentrated under reduced
2 4
1
pressure to afford title compound (2.78 g, 93.3%) as a white solid. H NMR (300
MHz, DMSO-d ) δ 7.67 (s, 1H), 6.82 (s, 1H), 4.20 (s, 1H), 1.77 (s, 3H), 1.38 (s, 9H).
6
4
4
.1.12.
tert-butyl
-(4-(cyclohexyl(ethyl)amino)-3-nitrophenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate
(14)
A mixture of compound 4c (0.62 g, 1.72 mmol), compound 13 (0.809 g, 5.15
°
mmol) and FeCl (0.093 g, 0.343 mmol) in 8 mL glycol was stirred at 100 C for 2 h.
3
The reaction mixture was cooled to room temperature, EtOAc (30 mL) was added to
the mixture and then washed with brine (30 mL). The organic layer was concentrated
in vacuo and purified by column chromatography to afford the title compound (0.685
1
g, 85%) as an orange solid. H NMR (300 MHz, DMSO-d ) δ 11.37 – 10.94 (m, 1H),
6
7
2
8
.41 – 7.25 (m, 3H), 3.09 (q, J = 7.0 Hz, 2H), 2.84 (t, J = 11.2 Hz, 1H), 2.37 (s, 3H),
.03 (s, 3H), 1.71 (s, 4H), 1.53 (d, J = 9.8 Hz, 1H), 1.23 (s, 9H), 1.11 (dd, J = 12.6,
.6 Hz, 3H), 0.87 (t, J = 6.9 Hz, 3H).
4
4
.1.13.
tert-butyl
-(3-amino-4-(cyclohexyl(ethyl)amino)phenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylat
e (15)
The title compound was prepared from compound 14 by the procedure used for
1
the conversion of 5a to 6a. Light yellow oily product, yield 74.6%. H NMR (300

MHz, CDCl ) δ 7.92 (s, 1H), 7.00 (dd, J = 8.2, 4.0 Hz, 1H), 6.65 – 6.55 (m, 2H), 4.06
3
(
3
s, 2H), 3.06 (d, J = 8.7 Hz, 2H), 2.76 (s, 1H), 2.50 (d, J = 4.1 Hz, 3H), 2.18 – 2.09 (m,
H), 1.91 (s, 2H), 1.74 (s, 2H), 1.30 – 1.26 (m, 9H), 1.17 (d, J = 12.6 Hz, 2H), 0.93 (d,
J = 5.9 Hz, 3H).
4
4
3
.1.14.
tert-butyl
-(4-(cyclohexyl(ethyl)amino)-3-(3-(p-tolyl)ureido)phenyl)-2,5-dimethyl-1H-pyrrole-
-carboxylate (16)
The title compound was prepared from compound 15 by the procedure used for
1
the conversion of 6a to 7a. Light yellow solid, yield 63.4%. H NMR (300 MHz,
DMSO-d ) δ 10.98 (s, 1H), 9.38 (s, 1H), 8.44 (s, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.35 (d,
6
J = 8.3 Hz, 2H), 7.09 (dd, J = 10.6, 8.1 Hz, 3H), 6.70 (dd, J = 8.0, 2.0 Hz, 1H), 3.01 (t,
J = 7.1 Hz, 2H), 2.71 (s, 1H), 2.37 (s, 3H), 2.23 (s, 3H), 2.03 (s, 3H), 1.93 (s, 2H),
1
.68 (s, 2H), 1.53 (d, J = 11.9 Hz, 1H), 1.20 (s, 14H), 0.85 (t, J = 6.9 Hz, 3H).
4
4
3
.1.15.
-(4-(cyclohexyl(ethyl)amino)-3-(3-(p-tolyl)ureido)phenyl)-2,5-dimethyl-1H-pyrrole-
-carboxylic acid (17)
°
To a solution of compound 16 (0.1 g, 0.175 mmol) in DCM (4 mL) at 0 C was
added TFA (1 mL). Then the mixture was warmed gradually to room temperature and
stirred for 1 h. The reaction mixture was evaporated in vacuo, the residue was
dissolved in EtOAc (20 mL) and washed with saturated NaHCO solution (20 mL),
3
followed by brine (20 mL), dried over Na SO . The organic layer was concentrated
2
4
under reduced pressure to afford title compound (0.051 g, 56.5%) as a yellow solid.
°
1
HPLC purity: 97.1%; m.p.: 179-184 C; H NMR (300 MHz, DMSO-d ) δ 10.38 (s,
6
1
H), 9.42 (s, 1H), 8.44 (s, 1H), 8.24 (s, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.10 (t, J = 7.5
Hz, 3H), 6.91 (d, J = 8.1 Hz, 1H), 3.01 (d, J = 9.0 Hz, 2H), 2.71 (s, 1H), 2.31 (s, 3H),
2
5
1
.25 (s, 3H), 2.16 (s, 3H), 1.92 (s, 2H), 1.68 (s, 2H), 1.53 (d, J = 11.5 Hz, 1H), 1.15 (s,
H), 0.84 (t, J = 7.0 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 167.79, 159.08,
54.33, 153.31, 151.02, 147.87, 132.06, 126.85, 125.59, 125.45, 122.76, 120.44,
1
19.15, 118.35, 118.06, 114.73, 111.86, 61.59, 43.63, 29.72, 24.52, 24.24, 13.86,
-
11.50; ESIMS m/z [M-H] 487.3; Anal. calcd. For C H N O : C, 71.28; H, 7.43; N,
29 36 4 3
11.47; Found: C, 71.26; H, 7.42; N, 11.48.
4
4
.1.16.
methyl
-(4-(cyclohexyl(isobutyl)amino)-3-isothiocyanatophenyl)-2,5-dimethylfuran-3-carbo
xylate (18)
To a stirred solution of compound 6d (0.31 g, 0.778 mmol) in acetonitrile (8
mL) was added 1,1'-thiocarbonyldiimidazole (0.277 g, 1.56 mmol). The mixture was
stirred at room temperature for 1 h. The solution was evaporated in vacuo, the residue
was dissolved in EtOAc (20 mL) and washed with 1N HCl (20 mL), followed by
brine (20 mL), dried over Na SO , and evaporated in vacuo to afford title compound
2
4
1
(
0.33 g, 96.3%) as a yellow solid. H NMR (300 MHz, DMSO-d ) δ 7.31 – 7.24 (m,
6
2
H), 7.17 (dd, J = 8.4, 2.1 Hz, 1H), 3.65 (s, 3H), 2.92 (d, J = 6.9 Hz, 3H), 2.55 (s, 3H),

2
3
.22 (s, 3H), 1.89 (d, J = 11.7 Hz, 2H), 1.77 (d, J = 12.1 Hz, 2H), 1.45 (d, J = 11.9 Hz,
H), 1.29 – 1.17 (m, 3H), 0.95 (d, J = 6.7 Hz, 1H), 0.86 (d, J = 6.6 Hz, 6H).
4
.1.17. General procedure for preparation of intermediates (19a-c)
A: To a solution of compound 18 (0.15 g, 0.34 mmol) and Cs CO (0.333 g, 1.02
2
3
mmol) in THF (5 mL) was added acetamidine hydrochloride (0.375 mmol) and stirred
°
at 50 C for 4 h. The solution was evaporated in vacuo, the residue was dissolved in
EtOAc (20 mL) and washed with water (20 mL), followed by brine (20 mL), dried
over Na SO . The organic layer was concentrated in vacuo and purified by column
2
4
1
chromatography to afford the intermediate (0.065 g, 38.4%). H NMR (300 MHz,
DMSO-d ) δ 6.98 (d, J = 6.5 Hz, 2H), 6.85 (dd, J = 8.0, 1.6 Hz, 1H), 5.54 (d, J = 3.3
6
Hz, 1H), 3.57 (s, 3H), 3.14 (s, 1H), 2.42 (s, 3H), 2.22 (s, 3H), 1.83 – 1.67 (m, 3H),
1
3
.57 (s, 2H), 1.38 (dt, J = 25.2, 12.0 Hz, 4H), 1.25 – 1.08 (m, 4H), 0.96 (d, J = 6.8 Hz,
H), 0.75 (d, J = 6.6 Hz, 3H).
B: To a solution of intermediate (0.131 mmol) in THF/MeOH (1:1, 4 mL) was
°
added 1 mL of LiOH (0.11g, 2.62 mmol) aqueous solution and stirred at 60 C for 20
h. The solvent was evaporated in vacuo and the residue was acidified with 1N HCl.
The mixture was extracted with EtOAc (10 mL) and washed with water (10 mL) and
brine (10 mL), dried over Na SO , and concentrated in vacuo. The residue was
2
4
purified by column chromatography to afford the title compound 19a (0.035 g, 55.4%)
as a yellow solid.
4
4
.1.17.1.
-(4-(cyclohexyl(isobutyl)amino)-3-((3-methyl-1,2,4-thiadiazol-5-yl)amino)phenyl)-2
,
5-dimethylfuran-3-carboxylic acid (19a)
Yellow solid, yield 55.4%; HPLC purity: 97.1%; m.p.: 187-190 C; H NMR (300
MHz, DMSO-d ) δ 12.33 (s, 1H), 7.05 – 6.92 (m, 2H), 6.86 (d, J = 8.0 Hz, 1H), 5.54
°
1
6
(s, 1H), 3.13 (s, 1H), 2.41 (s, 3H), 2.21 (s, 3H), 1.82 – 1.68 (m, 3H), 1.57 (s, 2H), 1.38
(
dt, J = 25.7, 12.0 Hz, 4H), 1.25 – 1.06 (m, 4H), 0.95 (d, J = 6.8 Hz, 3H), 0.75 (d, J =
1
3
6
1
3
4
7
.6 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 177.28, 169.39, 164.78, 156.38,
6
46.59, 141.60, 134.57, 125.39, 124.81, 120.71, 113.14, 112.44, 84.88, 62.39, 32.82,
+
0.80, 29.16, 25.48, 25.07, 18.83, 17.17, 15.73, 13.74, 11.77; ESIMS m/z [M+H]
83.2; Anal. calcd. For C H N O S: C, 64.70; H, 7.10; N, 11.61; Found: C, 64.71; H,
2
6
34
4
3
.12; N, 11.60.
4
4
.1.17.2.
-(4-(cyclohexyl(isobutyl)amino)-3-((3-cyclopropyl-1,2,4-thiadiazol-5-yl)amino)phen
yl)-2,5-dimethylfuran-3-carboxylic acid (19b)
°
1
Light yellow solid, yield 48.4%; HPLC purity: 97.4%; m.p.: 108-110 C; H
NMR (300 MHz, DMSO-d ) δ 12.32 (s, 1H), 7.04 (s, 1H), 6.96 (d, J = 6.3 Hz, 1H),
6
6
=
.85 (d, J = 8.1 Hz, 1H), 5.49 (s, 1H), 3.12 (s, 1H), 2.81 (d, J = 6.7 Hz, 2H), 2.23 (d, J
10.3 Hz, 6H), 1.82 (s, 2H), 1.68 (s, 2H), 1.57 (s, 2H), 1.24 (d, J = 7.6 Hz, 4H), 1.00
1
3
(s, 4H), 0.79 (dd, J = 16.6, 6.5 Hz, 8H); C NMR (75 MHz, DMSO-d ) δ 179.83,
6
1
77.15, 174.04, 164.71, 156.35, 146.60, 141.37, 135.17, 125.38, 124.58, 122.96,

1
1
20.64, 113.27, 112.05, 84.85, 63.04, 62.08, 33.06, 30.73, 28.96, 25.09, 20.87, 17.04,
6.02, 13.74, 13.43, 11.80, 8.81, 8.36; ESIMS m/z [M+H] 509.2; Anal. calcd. For
+
C H N O S: C, 66.11; H, 7.13; N, 11.01; Found: C, 66.13; H, 7.12; N, 11.03.
28
36
4
3
4
4
.1.17.3.
-(4-(cyclohexyl(isobutyl)amino)-3-((3-phenyl-1,2,4-thiadiazol-5-yl)amino)phenyl)-2
,
5-dimethylfuran-3-carboxylic acid (19c)
Yellow solid, yield 41.6%; HPLC purity: 95.5%; m.p.: 100-101 C; H NMR (300
MHz, DMSO-d ) δ 12.34 (s, 1H), 9.77 (s, 1H), 8.10 (s, 2H), 7.97 (s, 1H), 7.48 (s, 3H),
°
1
6
7
3
1
1
6
5
6
.23 (d, J = 7.9 Hz, 1H), 7.00 (d, J = 8.3 Hz, 1H), 2.84 (s, 2H), 2.70 (s, 1H), 2.51 (d,
H), 2.28 (s, 3H), 1.88 (s, 2H), 1.67 (s, 2H), 1.46 (s, 2H), 1.30 (s, 3H), 1.23 (s, 3H),
1
3
.01 (s, 3H), 0.88 – 0.80 (m, 6H); C NMR (75 MHz, DMSO-d ) δ 164.68, 162.96,
6
56.23, 146.72, 136.78, 136.57, 128.92, 123.44, 123.10, 120.78, 113.67, 102.57,
+
2.65, 55.79, 29.55, 26.13, 25.42, 20.75, 17.09, 13.69, 11.85; ESIMS m/z [M+H]
45.2; Anal. calcd. For C H N O S: C, 68.36; H, 6.66; N, 10.29; Found: C, 68.35; H,
3
1
36
4
3
.68; N, 10.27.
4
4
.1.18.
methyl
-(4-(cyclohexyl(isobutyl)amino)-3-thioureidophenyl)-2,5-dimethylfuran-3-carboxyla
te (20)
A mixture of compound 18 (0.45 g, 1.02 mmol) and NH OH (1 mL) in 10 mL
4
EtOH was stirred at room temperature for 2 h. The solvent was evaporated in vacuo
and the residue was purified by column chromatography to afford the title compound
1
(
0.26 g, 55.6%) as a yellow solid. H NMR (300 MHz, DMSO-d ) δ 8.37 (s, 1H), 7.75
6
(s, 2H), 7.56 (d, J = 2.2 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.95 (dd, J = 8.3, 2.1 Hz,
1
2
1
H), 3.63 (s, 3H), 2.80 (d, J = 6.8 Hz, 2H), 2.67 (t, J = 11.7 Hz, 1H), 2.50 (s, 3H),
.28 (s, 3H), 1.80 (d, J = 11.8 Hz, 2H), 1.71 (d, J = 11.2 Hz, 2H), 1.57 – 1.30 (m, 5H),
.08 (t, J = 10.9 Hz, 2H), 0.84 (d, J = 6.5 Hz, 6H).
4
0
.1.19. General procedure for preparation of intermediates (21a-b)
A mixture of compound 20 (0.15 g, 0.328 mmol) and bromoacetone (0.09 g,
.656 mmol) in 10 mL EtOH was stirred at reflux for 3 h. The solvent was evaporated
in vacuo and the residue was purified by column chromatography to afford the
1
intermediate (0.14 g, 86.2%) as a light-yellow solid. H NMR (300 MHz, DMSO-d )
6
δ 8.68 (s, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.24 (d, J = 8.2 Hz, 1H), 6.83 (dd, J = 8.2, 2.0
Hz, 1H), 6.52 – 6.43 (m, 1H), 3.59 (s, 3H), 2.84 (d, J = 6.9 Hz, 2H), 2.29 – 2.16 (m,
6
1
H), 1.85 (d, J = 12.0 Hz, 2H), 1.70 (d, J = 11.6 Hz, 2H), 1.52 (d, J = 10.5 Hz, 1H),
.42 – 1.23 (m, 4H), 1.08 (d, J = 10.4 Hz, 2H), 0.84 (d, J = 6.6 Hz, 6H).
Then the title compound was prepared from the intermediate by the procedure B
used for the conversion of 18 to 19a.
4
4
.1.19.1.
-(4-(cyclohexyl(isobutyl)amino)-3-((4-methylthiazol-2-yl)amino)phenyl)-2,5-dimeth
ylfuran-3-carboxylic acid (21a)

°
1
Yellow solid, yield 66.2%; HPLC purity: 97.8%; m.p.: 201-204 C; H NMR (300
MHz, DMSO-d ) δ 12.30 (s, 1H), 8.65 (s, 1H), 7.83 (d, J = 2.1 Hz, 1H), 7.23 (d, J =
6
8
2
.2 Hz, 1H), 6.85 (dd, J = 8.2, 2.1 Hz, 1H), 6.51 – 6.44 (m, 1H), 2.83 (d, J = 6.8 Hz,
H), 2.58 (s, 1H), 2.21 (d, J = 14.2 Hz, 6H), 1.84 (d, J = 11.9 Hz, 2H), 1.70 (d, J =
1
1.1 Hz, 2H), 1.51 (d, J = 9.5 Hz, 1H), 1.34 (dd, J = 18.6, 9.1 Hz, 3H), 1.08 (t, J =
1
3
11.2 Hz, 3H), 0.84 (d, J = 6.5 Hz, 6H); C NMR (75 MHz, DMSO-d ) δ 164.69,
6
1
62.82, 156.25, 148.15, 146.72, 136.69, 136.64, 128.84, 123.43, 122.93, 120.80,
1
17.91, 113.65, 102.60, 62.55, 55.71, 29.54, 26.13, 25.43, 20.76, 17.22, 13.71, 11.87;
+
ESIMS m/z [M+H] 482.2; Anal. calcd. For C H N O S: C, 67.33; H, 7.32; N, 8.72;
2
7
35
3
3
Found: C, 67.31; H, 7.33; N, 8.73.
4
4
.1.19.2.
-(4-(cyclohexyl(isobutyl)amino)-3-((4-phenylthiazol-2-yl)amino)phenyl)-2,5-dimeth
ylfuran-3-carboxylic acid (21b)
°
1
Yellow solid, yield 45.1%; HPLC purity: 97.3%; m.p.: 121-123 C; H NMR (300
MHz, DMSO-d ) δ 12.30 (s, 1H), 8.84 (s, 1H), 8.12 (d, J = 2.0 Hz, 1H), 7.90 (dd, J =
6
1
3.9, 7.6 Hz, 2H), 7.46 – 7.35 (m, 4H), 7.26 (d, J = 8.4 Hz, 1H), 6.90 (td, J = 6.2, 3.0
Hz, 1H), 2.85 (d, J = 6.9 Hz, 2H), 2.62 (d, J = 11.7 Hz, 1H), 2.53 (s, 3H), 2.25 (d, J =
.3 Hz, 3H), 1.95 – 1.83 (m, 2H), 1.70 (d, J = 11.4 Hz, 2H), 1.62 – 1.40 (m, 3H), 1.34
t, J = 14.0 Hz, 4H), 1.11 (dd, J = 24.4, 11.1 Hz, 4H), 0.98 (d, J = 6.6 Hz, 2H), 0.86 (d,
7
(
1
3
J = 6.4 Hz, 6H); C NMR (75 MHz, DMSO-d ) δ 164.71, 163.26, 156.35, 150.23,
6
1
6
46.67, 136.93, 133.34, 128.59, 127.48, 125.66, 123.46, 118.49, 111.75, 103.16,
5.28, 62.69, 55.83, 29.54, 28.95, 26.14, 25.43, 20.80, 13.74, 11.89; ESIMS m/z
+
[
M+H] 544.2; Anal. calcd. For C H N O S: C, 70.69; H, 6.86; N, 7.73; Found: C,
3
2
37
3
3
7
0.65; H, 6.88; N, 7.75.
4
4
.1.20.
methyl
-(3-(2-acetylhydrazine-1-carbothioamido)-4-(cyclohexyl(isobutyl)amino)phenyl)-2,5
-dimethylfuran-3-carboxylate (22)
A mixture of compound 18 (0.287g, 0.652 mmol) and acethydrazide (0.097 g, 1.3
mmol) in 10 mL THF was stirred at room temperature for 20 h. The solvent was
evaporated in vacuo and the residue was purified by column chromatography to afford
1
the title compound (0.32g, 95.2%) as a light-yellow solid. H NMR (300 MHz,
DMSO-d ) δ 10.11 (s, 1H), 9.72 (s, 1H), 9.48 (s, 1H), 8.85 (s, 1H), 7.22 (d, J = 8.3 Hz,
6
1
3
H), 6.98 – 6.88 (m, 1H), 3.59 (s, 3H), 2.81 (d, J = 6.8 Hz, 2H), 2.26 (s, 3H), 1.91 (s,
H), 1.73 (dd, J = 24.4, 10.7 Hz, 4H), 1.54 (s, 1H), 1.25 (d, J = 9.0 Hz, 3H), 1.08 (d, J
=
8.6 Hz, 3H), 0.79 (d, J = 6.5 Hz, 6H).
4
4
.1.21.
-(4-(cyclohexyl(isobutyl)amino)-3-((5-methyl-1,3,4-thiadiazol-2-yl)amino)phenyl)-2
,
5-dimethylfuran-3-carboxylic acid (23)
To a solution of compound 22 (0.156 g, 0.303 mmol) in NMP (3 mL) was added
p-Toluenesulfonyl chloride (0.069 g, 0.364 mmol) and TEA (0.061 g, 0.606 mmol).
The mixture was stirred at room temperature for 2 h. Then the mixture was poured

into ice water (20 mL) and extracted with EtOAc (20 mL). The organic layer was
concentrated and purified by column chromatography to yield the intermediate (0.081
1
g, 53.8%) as a white solid. H NMR (300 MHz, DMSO-d ) δ 8.56 (s, 1H), 7.87 (d,
6
1
H), 7.28 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 3.57 (s, 3H), 2.84 (d, J = 6.8
Hz, 2H), 2.62 (s, 1H), 2.40 (s, 3H), 2.21 (s, 3H), 1.85 (d, J = 11.2 Hz, 2H), 1.75 – 1.67
m, 2H), 1.53 (d, J = 11.5 Hz, 1H), 1.42 – 1.16 (m, 6H), 1.14 – 1.04 (m, 2H), 0.83 (d,
J = 6.6 Hz, 6H).
Then the title compound was prepared from the intermediate by the procedure B
used for the conversion of 18 to 19a. Yellow solid, yield 69.1%; HPLC purity: 98.3%;
(
°
1
m.p.: 94-95 C; H NMR (300 MHz, DMSO-d ) δ 12.28 (s, 1H), 8.66 (d, J = 80.5 Hz,
6
1
2
3
1
1
1
1
6
H), 7.85 (dd, J = 32.2, 1.9 Hz, 1H), 7.25 (dd, J = 20.2, 8.2 Hz, 1H), 6.89 (dd, J = 8.1,
.0 Hz, 1H), 2.82 (s, 2H), 2.62 (t, J = 13.1 Hz, 1H), 2.40 (s, 2H), 2.19 (d, J = 5.5 Hz,
H), 1.85 (d, J = 12.2 Hz, 2H), 1.69 (s, 2H), 1.52 (s, 1H), 1.31 (d, J = 11.3 Hz, 3H),
1
3
.13 – 1.00 (m, 3H), 0.83 (d, J = 6.6 Hz, 6H); C NMR (75 MHz, DMSO-d ) δ
6
65.22, 164.12, 159.24, 157.39, 156.41, 146.72, 137.72, 136.43, 134.47, 129.46,
28.68, 123.88, 120.77, 117.81, 113.61, 62.43, 56.11, 29.38, 25.49, 20.79, 15.14,
+
3.72, 11.80, 10.37; ESIMS m/z [M+H] 483.2; Anal. calcd. For C H N O S: C,
2
6
34
4
3
4.70; H, 7.10; N, 11.61; Found: C, 64.68; H, 7.12; N, 11.63.
4
4
.1.22.
-(4-(cyclohexyl(isobutyl)amino)-3-((5-methyl-1,3,4-oxadiazol-2-yl)amino)phenyl)-2
,
5-dimethylfuran-3-carboxylic acid (24)
To a solution of compound 22 (0.156 g, 0.303 mmol) in DMSO (3 mL) was
°
added EDCI (0.07 g, 0.364 mmol) and stirred at 60 C for 2 h. Then the mixture was
poured into ice water (20 mL) and extracted with EtOAc (20 mL). The organic layer
was concentrated and purified by column chromatography to yield the intermediate
1
(
0.086 g, 59%) as a white solid. H NMR (300 MHz, DMSO-d ) δ 8.55 (s, 1H), 7.88
6
(d, J = 1.9 Hz, 1H), 7.29 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 3.55 (s, 3H),
2
1
0
.84 (m, 2H), 2.62 (s, 1H), 2.40 (s, 3H), 2.22 (s, 3H), 1.84 (d, J = 11.1 Hz, 2H), 1.78 –
.69 (m, 2H), 1.52 (d, J = 11.5 Hz, 1H), 1.39 – 1.18 (m, 6H), 1.14 – 1.06 (m, 2H),
.83 (d, J = 6.6 Hz, 6H).
Then the title compound was prepared from the intermediate by the procedure B
used for the conversion of 18 to 19a. Yellow solid, yield 42.5%; HPLC purity: 96.9%;
°
1
m.p.: 133-136 C; H NMR (300 MHz, DMSO-d ) δ 12.27 (s, 1H), 8.53 (s, 1H), 7.90
6
(
=
d, J = 1.9 Hz, 1H), 7.28 (d, J = 8.2 Hz, 1H), 6.89 (dd, J = 8.5, 1.6 Hz, 1H), 2.83 (d, J
6.9 Hz, 2H), 2.40 (s, 3H), 2.18 (s, 3H), 1.84 (d, J = 12.2 Hz, 2H), 1.70 (d, J = 12.1
Hz, 2H), 1.53 (d, J = 11.2 Hz, 1H), 1.34 (d, J = 11.7 Hz, 3H), 1.14 – 1.01 (m, 3H),
1
3
0
1
6
.83 (d, J = 6.6 Hz, 6H); C NMR (75 MHz, DMSO-d ) δ 164.60, 159.25, 157.40,
6
56.42, 146.73, 136.48, 134.45, 129.45, 123.88, 123.42, 120.77, 117.81, 113.61,
2.42, 56.09, 29.52, 28.98, 26.22, 25.42, 25.35, 20.79, 13.72, 11.74, 10.37; ESIMS
+
m/z [M+H] 467.3; Anal. calcd. For C H N O : C, 66.93; H, 7.35; N, 12.01; Found:
2
6
34
4
4
C, 66.94; H, 7.37; N, 11.99.
4
.2. Docking studies