Isosteres of ester derived glucose uptake inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127406

Glucose transporters (GLUTs) facilitate glucose uptake and are overexpressed in most cancer cells. Inhibition of glucose transport has been shown to be an effective method to slow the growth of cancer cells both in vitro and in vivo. We have previously reported on the anticancer activity of an ester derived glucose uptake inhibitor. Due to the hydrolytic instability of the ester linkage we have prepared a series of isosteres of the ester moiety. Of all of the isosteres prepared, the amine linkage showed the most promise. Several additional analogues of the amine-linked compounds were also prepared to improve the overall activity.

Full text of DOI:10.1016/j.bmcl.2020.127406

Bioorganic&MedicinalChemistryLetters30(2020)127406
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isosteres of ester derived glucose uptake inhibitors
Dennis A. Robertsa, Liyi Wanga, Weihe Zhanga, Yi Liuc,d, Pratik Shriwasd, Yanrong Qiana,b,d
,
⁎
Xiaozhuo Chena,b,c,d, Stephen C. Bergmeiera,d,
a Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA
b Edison Biotechnology Institute, Ohio University, Athens, OH 45701, USA
c Department of Biomedical Science, Ohio University, Athens, OH 45701, USA
d Program of Molecular and Cellular Biology, Ohio University, Athens, OH 45701, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Glucose uptake
Cancer
Glucose transporters (GLUTs) facilitate glucose uptake and are overexpressed in most cancer cells. Inhibition of
glucose transport has been shown to be an effective method to slow the growth of cancer cells both in vitro and
in vivo. We have previously reported on the anticancer activity of an ester derived glucose uptake inhibitor. Due
to the hydrolytic instability of the ester linkage we have prepared a series of isosteres of the ester moiety. Of all
of the isosteres prepared, the amine linkage showed the most promise. Several additional analogues of the
amine-linked compounds were also prepared to improve the overall activity.
Transporters
Bioisostere
GLUT
An estimated 1.8 million new cancer cases are expected to be di-
agnosed in 2020 in US alone, and this will result in an estimated
606,520 deaths in the same year. 1 out of every 2 American males and 1
in every 3 females is expected to be diagnosed with cancer once in their
lifetimes. The overall five-year survival rate for all types of cancers has
increased from 3% in 1970s to 18% today.1 Cancers tend to favor
glycolysis over oxidative phosphorylation for biomass synthesis and
ATP production. Cancer cells proliferate faster than normal human cells
and outpace the diffusion limits of local blood supply. Hypoxic condi-
tions in human tumors favor aerobic glycolysis over oxidative phos-
phorylation.2 Glucose based metabolism in cancer can be targeted in
several ways such as by inhibiting glycolytic enzymes or by inhibiting
glucose uptake itself.3,4 Glucose transporters (GLUTs) are proteins that
facilitate uptake of glucose inside cancer cells. GLUT1-4 are found to be
the most important in tumor growth, survival and metastasis. However,
GLUT1 is the most upregulated transporter in a wide variety of human
cancer cells.5 Human tumors are addicted to glucose and glucose de-
privation is toxic to cancer cells. Inhibiting GLUT1 and other GLUTs
could be an efficient way of preventing glucose from entering the cell
and undergoing glycolysis, and eventually leading to cell death by
starvation. There are several natural and synthetic small organic com-
pounds that have shown potency to inhibit GLUT-mediated glucose
transport.4 We have previously reported on a class of phenolic esters
(e.g. 1 (WZB117)) that have shown high GLUT1 inhibitory activity.6–8
Compound 1 has been found to suppress cancer cell growth in vitro as
well as in vivo. Human A549 lung tumors were xenografted onto male
nude mice and the mice were treated daily by IP injection of compound
1 at a dose of 10 mg/kg of body weight for 10 weeks. A 70% reduction
in tumor volume was observed after treatment.8
Since our initial reports several additional GLUT inhibitors have
been reported.9,10 Some of these new GLUT inhibitors include the
pyrazole BAY-876,11,12 analogs of the macrocyclic peptide rapafucin,13
and a series of salicylketoximes.14,15 In addition, the use of computa-
tional methods for the discovery of GLUT inhibitors have also been
reported.16,17
Despite the excellent activity observed with 1, this compound could
be problematic because it can be readily hydrolyzed into compounds 2
and 3 (Scheme 1), neither of which are biologically active.6 Only about
25% of 1 remained undegraded after it was introduced in human serum
for 1 h.9
We report herein the synthesis and evaluation of several ester iso-
steres that were prepared to address the poor stability of 1. Some of the
more stable isosteres of esters are amides, sulfoxides, and sulfones, and
other nontraditional isosteres include amines, ethers, and sulfides.18–21
Figure 1 shows a general structure of the compounds that were chosen
to prepare in order to determine both stability issues and functional
group tolerance.
The desired amides and corresponding amines were prepared as
outlined in Scheme 2. Commercially available analogs of 3-hydro-
xybenzoic acid (5) were protected as either the benzyl ether or acetate
Compound 1 reduces glucose-uptake with an IC50 of 9.6
1.0 µM and
cell-growth with an IC50 value of 25.7 6.8 µM in H1299 cells.
⁎ Corresponding author at: Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA.
E-mail address: bergmeis@ohio.edu (S.C. Bergmeier).
https://doi.org/10.1016/j.bmcl.2020.127406
Received 21 May 2020; Received in revised form 7 July 2020; Accepted 9 July 2020
Availableonline15July2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

D.A. Roberts, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127406
Scheme 1. Hydrolysis of 1 (WZB117).
Scheme 3. Reagents and conditions: (a) PBr3, CH2Cl2, 0 °C – room temperature,
18 hr, 96%; (b) Et3N, 3-mercaptophenol, CH2Cl2, 0 °C – room temperature, 18
hr, 100%; (c) mCPBA, 0 °C, 30 min, 54%; (d) mCPBA, 0 °C, 30 min, 71%.
Fig. 1. Planned isosteres of diester 1.
The 4-fluoro and 4-chloro benzenediamines were used as those sub-
stitution patterns are more readily available. Deprotection of the
acetate or benzyl ether followed by reduction with LiAlH4 provided
amines 4m-4x.
Sulfoxides and sulfones have previously been used as carbonyl iso-
steres in a series of HCVNS5B polymerase inhibitors.22 To synthesize
these isosteres the known dibromide 923 was prepared from the diol 8
using PBr3. The alkylation of 3-mercaptophenol with dibromide 9 in the
presence of Et3N provided thioether 4y. By regulating the stoichiometry
of mCPBA, 4y was oxidized to the sulfoxide 4z or the sulfone 4aa
(Scheme 3).
Another carbonyl isostere prepared was the inverted ester of 1. As
an ester, it will be readily hydrolyzed, it will however provide in-
formation regarding the optimal location of the carbonyl. Along with
the amide and ester isosteres, inverted isosteres have been examined in
the studies of meperidine analogs.24 As shown in Scheme 4, 3-fluor-
ophthalic acid (10) was coupled with the mono benzylated resorcinol
11 that was prepared using the procedure from Zhang.25 The resulting
ester was then deprotected via hydrogenolysis to obtain ester 4ab.
The next set of isosteres involved the removal of the carbonyl group.
The resulting compounds will not undergo hydrolysis so they should be
much more stable in serum than their ester counterparts. As shown in
Scheme 5, 3-hydroxybenzoic acid (5a) was converted to the chloride 12
via a 4-step sequence of esterification, MOM-protection, reduction, and
chloride formation. Alkylation of catechols 13a-b with chloride 12
followed by deprotection provided ether derivatives 4ac and 4ad.
An inverted ether derivative was prepared as shown in Scheme 6.
Phenol 14 was prepared from resorcinol and further alkylated with
dibromide 9 followed by deprotection to provide target compound 4ae.
A final isostere was an all carbon linker between the central and
pendent aromatic rings (Scheme 7). Wittig salt 15 was prepared by the
addition of triphenylphosphine to dibromide 9 in methylene chloride.
n-BuLi was added to 15 to produce the Wittig reagent followed by the
addition of 16. The product was a mixture of cis and trans isomers.
Scheme 2. Reagents and conditions: (a) Ac2O, NaOH, H2O; (b) BnBr, K2CO3,
acetone, NaOH; (c) (COCl)2, DMF, CH2Cl2; (d) DIEA, 3-fluoro benzenediamine,
3-chloro-1,2-benzenediamine, or 4-chloro-1,2-benzenediamine, CH2Cl2; (e)
NaOH, H2O, THF; (f) H2, 10 wt% Pd/C, MeOH; (g) LiAlH4, THF, reflux.
prior to conversion to the corresponding acid chloride. The acid
chloride 6 was then treated with either 3-fluoro benzenediamine, 3-
chloro-1,2-benzenediamine, or 4-chloro-1,2-benzenediamine to provide
the corresponding amides 4a-4l. The use of 3-fluoro benzenediamine
(X = F) provides a direct analog to ester 1 with a 3-fluoro substituent.
Scheme 4. Reagents and conditions: (a) DCC, DMAP, CH2Cl2, 12 hr, 59%; (b)
H2, 10 wt% Pd/C, MeOH, 18 hr, 87%.
2

D.A. Roberts, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127406
Table 1
Activity of initial isosteres of 1*.
Compound #26
Glucose uptake inhibition (%)27
Cell growth inhibition (%)28
1 (WZB117)
92.8
20.0
89.7
83.7
54.8
39.2
52.2
98.1
17.2
61.4
78.2
92.6
91.2
89.5
60.7
0.1
1.0
2.5
0.7
5.3
0.8
9.7
0.2
4.5
9.4
1.3
2.2
0.8
2.9
5.4
56.9
33.4
5.7
9.0
7.8
4a
4b
2.1
4c
5.9
1.4
4m
4n
5.0
16.9
8.4
37.7
56.6
−2.0
0.2
4o
11.2
4y
20.2
4.5
4z
4aa
4ab
4ac
4ad
4ae
4af
11.8
15.3
10.6
14.3
−1.4
−22.7
2.2
11.5
2.0
2.7
17.4
8.6
Scheme 5. Reagents and conditions: (a) H2SO4, MeOH, 5 hr, 91%; (b) MOMCl,
K2CO3, DMF, 0 °C, 4 hr, 85%; (c) LiAlH4, THF, 0 °C, 1 hr, 99%; (d) MsCl, Et3N,
CH2Cl2, 0 °C – room temperature, 18 hr, 84%; (e) K2CO3, DMF, 80 °C, 78–80%;
(f) HCl, MeOH, 50 °C, 79–92%.
*All compounds were tested at a concentration of 30 μM in H1299 cells.
Replacement of the ester functionality with a sulfide (4y) sulfoxide
(4z) or sulfone (4aa) all led to compounds with a poor activity profile.
While both the sulfide (4y) and sulfone (4aa) showed good to moderate
glucose uptake inhibition, neither had any significant cell growth in-
hibition. The sulfoxide had little activity in either assay.
The inverted ester 4ab showed moderate activity at inhibition of
glucose uptake but rather poor inhibition of cell growth. Similarly, the
ether 4ac, it’s 4-Cl variant 4ad, and the inverted ether 4ae showed good
levels of glucose uptake inhibition but poor inhibition of cell growth.
The last isostere, the all carbon chain 4af again showed reasonable
activity in glucose uptake inhibition but no activity inhibiting cell
growth.
Scheme 6. Reagents and conditions: (a) K2CO3, DMF, 80 °C, 64%; (b) HCl,
MeOH, 50 °C, 76%.
Relatively poor correlation was found between the glucose uptake
assay and the cell viability/proliferation assay. Several factors con-
tribute to the poor correlations. First, the glucose uptake assay is a
50 min acute assay while the cell viability/proliferation assay is a 24 hr
chronic assay. It is well known that H1299 cells express different GLUTs
at different levels and these GLUTs play somewhat different roles in the
overall glucose metabolism.29 These compounds are likely to inhibit
GLUT-mediated glucose transport with varying potency, resulting in
different cancer cell growth inhibitory activities.
Scheme 7. Reagents and conditions: (a) n-BuLi, 0 °C – room temperature, 18 hr,
79%; (b) H2, 10 wt% Pd/C, THF, 18 hr, 50%.
Additionally, most cancer cell lines, including H1299 cells used in
this study, exhibit macropinocytosis, through which glucose can be
taken into cancer cells via bypassing GLUTs, weakening the correlation.
Nevertheless, our ultimate goal of this study was to identify those
compounds that reduce cancer cell viability/proliferation through in-
hibiting GLUTs, justifying the sequential use of the two assays in the
study.
Reduction of the isomeric olefins along with the deprotection of the
benzyl ethers was accomplished via catalytic hydrogenation to obtain
compound 4af.
The first set of compounds synthesized were simple isosteres of 1.
For these compounds, the fluorine and pendent aryl groups are ar-
ranged in positions-2,3,4 (Fig. 1) about the central aromatic ring.
Table 1 shows the results of the assays performed. All compounds were
evaluated in H1299 cells at a concentration of 30 μM.27,28
Of all of the isosteres prepared amide 4a and amine 4n and 4o
showed the closest correlation between cell growth inhibition and
glucose uptake inhibition. While their overall activity was lower than
ester 1, we felt that additional modifications should yield improved
activity.
Amide 4a and the corresponding amine 4m are direct analogues of
ester 1. Amide 4a showed a significant decrease in both glucose uptake
inhibition and cell growth inhibition relative to ester 1. The corre-
sponding amine 4m showed a similar loss of activity. As previously
stated, modification of the halogen and its placement on the central ring
was varied due to availability of the starting materials. The 4-fluoro and
4-chloro analogues of amide 4a (4b and 4c) showed improved glucose
uptake inhibition but even more pronounced loss of cell growth in-
hibition. The corresponding amines 4n (replacement of 3-F with 4-F)
showed a moderate loss of activity in glucose uptake inhibition but a
substantial increase in cell growth inhibition. It is significant that the
glucose uptake and cell growth assays showed similar levels of inhibi-
tion indicative of glucose uptake inhibition causing the cell growth
inhibition. Replacement of the 4-F with a 4-Cl (4o) again improved
both the glucose uptake inhibition and cell growth inhibition.
Based on these initial results a series of additional amides and
amines were prepared to optimize the activity of 4n. Table 2 shows the
activity of the amides and amines synthesized. For both the amide and
amine derivatives analogs with a chloro-substitution at positions R1, R2,
and R3 were prepared in order to identify initial structure activity re-
lationships relative to position around the phenolic ring. Amide deri-
vatives 4d (R3 = Cl) showed a relatively similar inhibition of glucose
uptake and cell growth inhibition relative to the unsubstituted 4b.
Amide 4e (R2 = Cl) showed a decrease in glucose uptake and an in-
crease in cell growth inhibition. Significantly the levels of glucose up-
take inhibition and cell growth inhibition were relatively similar.
Amide 4f showed
a similar pattern as both 4b and 4d. The
3

D.A. Roberts, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127406
Table 2
reduced both glucose uptake inhibition and cell growth inhibition.
The simple isosteric replacement of the ester bond did not lead to
any compounds with similar activity in both glucose uptake inhibition
and cell growth inhibition. Modification of the phenolic ring of the
amide/amine series did lead to an improved set of analogs. The best
compound (4v) showed significantly better stability in human serum.
To determine the compound stability, 30 μM of 1 and 4v were in-
cubated in serum-containing cell culture medium DMEM at 37 °C for
different durations (0, 1, 2, 4, 6, 9, 12, 24, 48 and 72 h). After the
incubation, 100 μL of compound-containing media were used to treat
A549 cells at 37 °C. After incubation for 24 hrs, cell viability/pro-
liferation was measured by a Resazurin assay.30 The assay revealed that
1 started to lose its cancer cell inhibitory activity at 12 hrs while 4v
retained its full activity even after the 72 hr incubation, indicating that
4v is at least 5 times more stable than 1.
Activity of additional amide and amine derivatives*.
Compound #26
X
R1
R2
R3
Glucose uptake
inhibition (%)27
Cell growth
inhibition (%)28
In addition, this improved amine analog (4v) showed improved
activity in both glucose uptake inhibition and cell growth inhibition.
Compound 4v has IC50 values in H1299 cells for glucose uptake in-
4b
4o
4d
4e
4f
CO
CH2
CO
CO
CO
CO
CO
CO
CO
CO
CO
CH2
CH2
H
H
H
H
Cl
H
H
F
89.7
52.2
85.2
60.6
89.9
47.8
97.3
2.5
9.7
2.4
1.7
2.0
6.5
0.4
3.6
1.2
12.1
8.2
10.9
4.5
1.2
1.6
1.1
4.6
4.5
5.3
1.1
5.7
2.1
11.2
H
H
56.6
19.0
61.3
45.3
25.7
54.6
26.0
55.0
84.7
21.0
70.9
47.6
70.8
22.1
74.1
22.0
77.5
60.4
46.3
H
H
8.0
7.9
7.4
10.1
4.2
14.0
7.0
0.8
2.3
11.7
7.2
4.2
7.4
7.0
9.5
7.1
8.2
1.3
hibition of 1.9
7.0
0.3 µM and for cell growth inhibition an IC50 of
H
Cl
H
0.6 µM. Future work on optimization of this class of compounds
Cl
H
as well as determining their in vivo efficacy will be reported in due
course.
4g
4h
4i
H
H
H
Br
H
H
OCH3 39.5
4j
H
H
CH3
H
84.8
61.4
21.0
52.4
81.7
80.7
24.1
69.1
Declaration of Competing Interest
4k
4l
H
CH3
H
CH3
H
H
4p
4q
4r
4s
4t
H
Cl
H
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
H
Cl
H
CH2 Cl
H
CH2
CH2
CH2
CH2
CH2
H
H
H
H
H
H
F
H
Br
Acknowledgement
4u
4v
4w
4x
H
OCH3 42.2
H
CH3
H
93.7
75.0
82.9
CH3
H
NMR and HRMS spectra were obtained on instrumentation sup-
ported by the National Science Foundation under Grant No. CHE-
1338000 and Grant No. CHE-1428787.
CH2 CH3
H
*All compounds were tested at a concentration of 30 μM in H1299 cells.
Appendix A. Supplementary data
corresponding amines showed a rather different pattern with amine 4p
(R3 = Cl) showing no change in glucose uptake inhibition and an in-
crease in cell growth inhibition relative to amine 4o. Amine 4q showed
an increase in glucose uptake inhibition and a small decrease in cell
growth inhibition with respect to its amide homolog and the un-
substituted amine. Amine 4r (R1 = Cl) showed increases in both glu-
cose uptake inhibition and cell growth inhibition. With these somewhat
confusing directions as to which additional analogs to prepare the
corresponding derivatives in which R1, R2, or R3 = CH3 were also
prepared and evaluated.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127406.
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26. The identity of all compounds were verified by 1H-NMR, 13C-NMR and HRMS. All
compounds were purified to > 95% as determined by HPLC.
and control. After incubation, MTT was added and incubated with cells for 4 hours.
Cell growth rates were measured using a Biotek Cytation3 microplate reader to
measure the absorbance of MTT. The wavelength were measured was 570 nm. Each
experimental condition was performed in triplicates, quadruplets or hexads (or as
mentioned otherwise), and the experiment was repeated at least once. Data is re-
ported as mean standard deviation and analyzed using Student’s t-test or one-way
ANOVA, whichever is appropriate. P ≤ 0.05 was considered significant. *, P ≤ 0.05,
**, P ≤ 0.01, and ***, P ≤ 0.001, and ****, P ≤ 0.0001.”.
27. The glucose uptake assay and cell growth inhibition assay were conducted as pre-
viously described.8 Before the glucose uptake assay, H1299 cancer cells were in-
cubated with serum-free cell culture media for 2 hours and then KRP buffer for 30
minutes. The incubated H1299 cancer cells were treated with or without the com-
pounds (30 µM) for 10 minutes followed by the addition of 37 MBq/L [3H]2-deox-
yglucose and 1 mmol/L regular glucose in KRP buffer for glucose uptake for 30
minutes. The cells were then washed twice with ice-cold PBS and lysed using 0.1 mM
NaOH. The radioactivity was measured using Beckman Coulter Scintillation counter
LS6500. Each experimental condition was performed in triplicates, quadruplets or
hexads (or as mentioned otherwise), and the experiment was repeated at least once.
Data is reported as mean standard deviation and analyzed using Student’s t-test or
one-way ANOVA, whichever is appropriate. P ≤ 0.05 was considered significant. *, P
≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001, and ****, P ≤ 0.0001.”.
29. O'Byrne KJ, Baird AM, Kilmartin L, Leonard J, Sacevich C, Gray SG. Cancers.
2011;3:1550–1565.
30. Compounds at 30 µM were pre-incubated in A549 cells; DMEM cell culture medium
containing 10% FBS and 1% Pen/Strp at 37°C for different duration (0,1, 2, 4, 6, 9,
12, 24, 48 and 72 hours). 5 x 103 A549 cells were grown in DMEM cell culture media.
After overnight incubation, cells were washed with PBS 2 times. Then, pre-incubated
compound containing DMEM was added to cells (100 µl per well) for 24 hours.
Resazurin was then added for 30 minutes. Absorbance values were measured at Ex
560 nM and Em 590 nM. Mock (DMSO) treated cells were used as controls to nor-
malize all of the data points.
28. The cell growth inhibitory assay was performed as follows. H1299 cancer cells were
seeded at a density of 5,000 per well in 96-well plates and incubated with or without
compounds (30 µM) in hexads for 48 hrs. DMSO (< 0.5%v/v) was used as solvent
5