Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth

Full text of DOI:10.1016/j.chembiol.2019.06.005

Article
Inhibition of Glucose Transporters and Glutaminase
Synergistically Impairs Tumor Cell Growth
Graphical Abstract
Authors
Elena S. Reckzeh, George Karageorgis,
Melanie Schwalfenberg, ...,
Carsten Strohmann, Slava Ziegler,
Herbert Waldmann
Correspondence
herbert.waldmann@
mpi-dortmund.mpg.de
In Brief
Cell-based screening identified the
piperazin-2-one Glutor as a nanomolar
inhibitor of glucose uptake that targets
the glucose transporters GLUT-1/-3.
Glutor inhibits glycolysis and effectively
incudes cell death of monolayer- and
spheroid-cultured cancer cells. Glutor-
mediated glucose starvation upregulates
GLUT-1/-3 expression. Glutor and
glutaminase inhibitor CB-839
synergistically inhibit cancer cell growth.
Highlights
d
Development of the GLUT-1-3-selective inhibitor Glutor to
suppress glucose uptake
d
Glutor potently induces cell death in 2D and 3D cancer cell
culture
d
d
Glutor-induced hypoglycemia upregulates GLUT-1/-3
Glutor and GLS inhibitor CB-839 synergistically inhibit cell
growth
Reckzeh et al., 2019, Cell Chemical Biology 26, 1–15
September 19, 2019 ª 2019 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2019.06.005

Please cite this article in press as: Reckzeh et al., Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth, Cell
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Cell Chemical Biology
Article
Inhibition of Glucose Transporters
and Glutaminase Synergistically
Impairs Tumor Cell Growth
Elena S. Reckzeh,1 George Karageorgis, Melanie Schwalfenberg, Javier Ceballos, Jessica Nowacki,
,2
1,4
1
1,5
1,6
Marcus C.M. Stroet,1 Aylin Binici, Lena Knauer, Silke Brand, Axel Choidas, Carsten Strohmann, Slava Ziegler,
and Herbert Waldmann
Max Planck Institute of Molecular Physiology, Department of Chemical Biology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
2Technical University Dortmund, Faculty of Chemistry and Chemical Biology, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany
3Lead Discovery Center GmbH, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany
,7
1
2
1
3
2
1
1,2,8,
*
1
4Present address: School of Chemistry, University of Leeds, LS2 9JT Leeds, UK
5Present address: Laboratory of Catalysis and Organic Synthesis, EPFL SB ISIC LCSO, BCH 4221, CH-1015 Lausanne, Switzerland
6Present address: Chemical Genomics Center, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany
7Present address: Optical Molecular Imaging/SPECTRIM/Radiochemistry, Radiology Department, Erasmus Medical Center,
Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
8Lead Contact
*Correspondence: herbert.waldmann@mpi-dortmund.mpg.de
https://doi.org/10.1016/j.chembiol.2019.06.005
SUMMARY
and breast cancer (Krzeslak et al., 2012), colon- and pancreatic
cancer (Yamamoto et al., 1990), head and neck tumors (Ayala
Cancer cells sustain growth by altering their meta- et al., 2010; Mellanen et al., 1994), and non-small-cell lung
bolism to accelerated aerobic glycolysis accompa- cancer (Younes et al., 1997). Glucose transporters are involved
in central cellular processes, such as insulin secretion in the
pancreas (GLUT-2) and insulin-regulated glucose transport
nied by increased glucose demand and employ
glutamine as additional nutrient source. This meta-
(GLUT-4), and are expressed tissue specifically, i.e., GLUT-1 is
bolic adaptation induces upregulation of glucose
transporters GLUT-1 and -3, and simultaneous tar-
geting of both transporters and of glutamine meta-
bolism may offer a promising approach to inhibit
cancer cell growth. We describe the discovery of
the very potent glucose uptake inhibitor Glutor, which
ubiquitously expressed in normal tissue, whereas GLUT-3 is pri-
marily expressed in neurons (Zhao and Keating, 2007). These
findings have led to the notion that selective inhibition of the ma-
jor facilitative transporter GLUT-1 may open up a promising
approach for the treatment of cancer (Shi et al., 2018), and
recently the first GLUT-1-selective inhibitor has been reported
targets glucose transporters GLUT-1, -2, and -3, at- (Siebeneicher et al., 2016a, 2016b). However, in the cancers
tenuates glycolytic flux and potently and selectively mentioned above, both GLUT-1 and GLUT-3 are upregulated,
suppresses growth of a variety of cancer cell lines. and in esophageal adenocarcinoma GLUT-3 expression was
increased in patients undergoing radiotherapy or surgery, which
Co-treatment of colon cancer cells with Glutor and
glutaminase inhibitor CB-839 very potently and
synergistically inhibits cancer cell growth. Such a
dual inhibition promises to be particularly effective
because it targets the metabolic plasticity as well as
metabolic rescue mechanisms in cancer cells.
indicates clonal selection (Fonteyne et al., 2009). Thus, dual-
specific GLUT-1/-3 inhibitors may be required for cancer target-
ing, and we recently identified such a compound, albeit with
moderate potency (Karageorgis et al., 2018).
In addition, rapidly dividing (e.g., cancer) cells rely on gluta-
mine as C- and N-source, and interference with glutamine meta-
bolism is considered a promising approach for cancer drug
discovery (Martinez-Outschoorn et al., 2017). Reduction of
INTRODUCTION
GLUT-1 expression sensitizes lung cancer cells to inhibition of
glutamine utilization and causes apoptosis and growth reduction
Proliferating cells, such as cancer cells, activated T cells, and and effective combinatory inhibition of glutaminolysis and glycol-
endothelial cells ensure rapid growth and proliferation by upre- ysis was described for ovarian cancer (Lee et al., 2016; Sun et al.,
gulating glycolysis, even under aerobic conditions (this phenom- 2017). Hence, combination of a GLUT-1/-3 inhibitor with an in-
enon is termed the Warburg effect) (Koppenol et al., 2011; hibitor of glutamine metabolism might synergistically inhibit
Vander Heiden et al., 2009). Aerobic glycolysis is characteristic cancer cell growth and, therefore, target the metabolic plasticity
for several diseases, including cancer and inflammatory dis- and overcome cellular rescue mechanisms of cancer cells.
eases such as psoriasis (Hanahan and Weinberg, 2011; Zhang
Here we describe the identification of the GLUT inhibitor
et al., 2018). In particular, various cancers upregulate facilitative Glutor (glucose uptake inhibitor), which defines an unprece-
glucose transporters (GLUTs) to increase nutrient uptake, for dented glucose uptake inhibitor chemotype. Glutor selectively
example, thyroid carcinomas (J o´ ꢀz wiak et al., 2012), endometrial targets GLUT-1, -2, and -3 with low nanomolar potency, induces
Cell Chemical Biology 26, 1–15, September 19, 2019 ª 2019 Elsevier Ltd.
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(
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upregulation of GLUT-1 and -3 in cancer cells, inhibits glycolysis Replacement of the 4-ethyl group in the N-benzyl amide (A) of
and efficiently suppresses the growth of various cancer cell lines. 3-phenylpyrazole 2h by disubstituted amines (2b, 2c) or an ethyl
Co-treatment with Glutor and the glutaminase inhibitor CB-839 ether (2d) did not alter the activity substantially (Figure 1B, cen-
(
Gross et al., 2014) synergistically inhibits cancer cell growth.
tral panel; compare entries 11–13 with entry 17). However, bulky
substituents in the 4-position of N-benzylamide A led to an in-
crease in potency (Figure 1B, central and right panel; compare
entries 14 and 26 with entry 17) to 0.19 ± 0.06 mM (2e) and
0.16 ± 0.09 mM (3f). The highest potency was recorded for com-
RESULTS
Identification of Glucose Uptake Inhibitors
Glucose uptake inhibitors were identified by means of a semi- pound 3f which embodies a morpholino group as 4-substituent
automated HCT116 colon cancer cell-based screen that moni- of N-benzyl amide A. Activity was also lower when larger un-
tors the uptake of 2-deoxy-D-glucose (2-DG) (Yamamoto et al., branched substituents were introduced in the 4-position (see,
2
006) (for details see Figure S1A). The screen employed a library e.g., 2a and 2f, entries 8 and 15). Thus, for further optimization
of over 150,000 compounds from different commercial and the 4-morpholino group was kept.
academic sources that were selected according to chemical Methylation of the exocyclic secondary amide of 2a to yield 2i
2
diversity and drug likeness. In addition, the collection included (R = Me) rendered the compound inactive (Figure 1B, central
3
natural products and sp -rich compounds from Edelris. The panel, compare entries 8 and 18). To improve the solubility of
screen identified piperazin-2-one-based hit compound 1a as 3f, a nitrogen was introduced in the phenyl ring of benzyl amide
2
-DG uptake inhibitor with an half maximal inhibitory concentra- C (3a). Notably, the 3-pyridine group (3f) enhanced the inhibitory
tion (IC50) value of 3.29 ± 2.04 mM in the semi-automated screen potency by nearly one order of magnitude (Figure 1B, right panel;
and 588.0 ± 143.3 nM in a separate manually performed assay compare entries 19 and 26) to yield the most potent glucose
(
Figures S1B and S1C).
import inhibitor 3a. If the 3-pyridyl (3a) group was replaced by
For delineation of a structure-activity relationship (SAR) a total a 2-pyridyl (3d) the biological activity was lowered by 30-fold
of 95 analogs was synthesized by means of a modified Ugi four- and replacement by a 4-pyridyl group (3c) reduced the activity
component reaction as described by Ivachtchenko and col- by 4-fold to 0.12 ± 0.01 mM (Figure 1B, right panel, compare entry
leagues (Ilyn et al., 2005) (for details see the STAR Methods) or 19 with entries 23 and 24). Introduction of additional substituents
obtained from commercial sources (ChemDiv, UK). Investigation such as fluorine or a methoxy group reduced activity as well (Fig-
of the compounds in the semi-automated 2-DG uptake assay ure 1B; right panel). Separation of the enantiomers of the most
revealed a coherent SAR. General trends are illustrated in Fig- active compound 3a revealed that the (+)-enantiomer inhibits
ure 1A. Thus, replacement of the 4-ethyl substituent in the glucose uptake with IC50 = 19 ± 2 nM, whereas the (À)-enan-
N-benzyl amide (A) of the hit compound 1a (IC50 = 3.29 ± tiomer was inactive at 30 mM (Figure 1B, compare entries
2.04 mM) by a 4-methoxy group (1b) did not alter the activity of 20 and 21). Crystal structure analysis (Figure S1D, CCDC:
the compound (Figure 1B, left panel, compare entries 1 and 2). 1883523) unambiguously proved that the inactive enantiomer
However, introduction of a methoxy group in the 3-position was R-configured. By analogy the (S)-configuration was as-
(
1c) reduced the activity ca. 7-fold (Figure 1B, left panel, signed to the active (+)-enantiomer of 3a, which we termed
compare entries 1 and 3). If the methoxy group was placed in Glutor ((S)-3a, Figure 1C). Irrespective of the originating tissue,
the 2-position (1d) inhibiting activity was lost (Figure 1B, left Glutor reduced the uptake of 2-DG with similar potency in
panel, compare entry 2 with entries 3 and 4). Replacement of different cancer cell lines such as HCT116 (IC50 = 10.8 ±
the p-ethyl substituent in the benzyl amide A by a fluorine (Fig- 5.3 nM), UM-UC-3 (IC50 = 8.3 ± 2.0 nM, urinary bladder), UO-
ure 1B, left panel, compare entries 1 and 5) or other halogens 31 (IC50 = 3.6 ± 1.5 nM, kidney), and MIA PaCa-2 (IC50 = 1.1 ±
(
Table S1, entry 17) led to loss of activity. Replacement of the 0.3 nM, pancreas) (Figures 1D and 1E). Furthermore, Glutor
-methoxy benzyl amide C by a benzyl group (1g) did not alter does not interfere with cellular hexokinase activity (Figure S1E)
2
the potency (Figure 1B, left panel, compare entries 1 and 7). and potently reduces glycolytic flux in HCT116 cells (Fig-
However, if in 1g (IC50 = 3.64 ± 1.73 mM) the indole heterocycle ure 1F), which prevents a decrease in oxidative phosphorylation
(
B) was replaced by a 3-phenylpyrazole scaffold (2h), the IC50 (OXPHOS) rate upon glucose addition (Figure 1G). The com-
value was lowered by one order of magnitude to 0.23 ± pound does not induce mitochondrial toxicity (Marroquin et al.,
.11 mM (Figure 1B, left and central panel, compare entries 7 2007) in HCT116 cells (Figures S1F and S1G).
and 17). Therefore, further compound optimization was per- Glutor is an unprecedented GLUT inhibitor substance class
0
formed with the 3-phenylpyrazole scaffold as core structure. (Figure 1C) with distant structural similarity (N-heterocycle
Figure 1. Glutor Inhibits 2-DG Uptake and Thereby Reduces Glycolytic Flux in HCT116 Cells
(A) Structure-activity relationship for the piperazin-2-one class (see also Tables S1 and S2).
(B) IC50 values for 2-DG uptake inhibition by selected Glutor derivatives (see also Tables S1 and S2). Data are mean values ± SD of n = 3–21. cmpd, compound;
n.a., not active at 30 mM.
(
(
C) Structural comparison of selected GLUT inhibitors.
D and E) Dose-response curve for 2-DG uptake inhibition by Glutor (see also Figure S1A). The graphs show mean values ± SD. n = 3, N = 3 for HCT116 (D), MIA
PaCa-2 (E), and UM-UC-3 (E); n = 4, N = 3 for UO-31 (E).
F and G) Glycolytic stress test of Glutor-treated HCT116 cells. The extracellular acidification rate (ECAR) (F) and the oxygen consumption rate (OCR) (G) were
(
measured for 2 h. Data are mean values ± SD (n = 3, N = 3). Cmpd, Glutor/DMSO; Glc, 10 mM glucose; Oligo, 2.5 mM oligomycin; 2-DG, 50 mM 2-DG. See also
Figures S1F and S1G for mitochondrial toxicity.
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Figure 2. GLUT Isoform Selectivity of Glutor
(A–C) CHO cells were transfected with a plasmid coding for the different isoforms (see also Figures S2A–S2D) GLUT-1 (A), GLUT-2 (B), GLUT-3 (C), or GLUT-4
before performing the 2-DG uptake assay (see also Figure S2E). Mock transfected CHO cells served as control. Data are mean values ± SD (n = 3, N = 3).
(
legend continued on next page)
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containing medium-sized small molecules) to the GLUT-1/-3 bars). Among the nine most sensitive cell lines (upper 10%,
selective Chromopynone-1 (Karageorgis et al., 2018) and the IC50 % 11 nM, Figure 3A), three cell lines originate from urinary
GLUT-1 selective inhibitor BAY-876 (Siebeneicher et al., bladder carcinoma and are known to be glucose addicted, i.e.,
2
016b) (Figure 1C). The GLUT-1 selective inhibitor WZB117 UM-UC-3 (Lea et al., 2015; Massari et al., 2016) (IC50 = 4 nM),
(
Liu et al., 2012) is structurally distinct from the aforementioned 5637 (Lea et al., 2015) (IC50 = 6 nM), and T-24 (Lea et al., 2015)
small molecules (Figure 1C).
(IC50 = 11 nM). Aerobic glycolysis is the prevalent metabolic
pathway in most urinary bladder cancers which renders this can-
cer highly glucose dependent (Lea et al., 2015). Other cell lines
Glutor Targets GLUT-1, -2, and -3
To determine the isoform-selectivity of Glutor by means of a within the most sensitive 10% of the cell panel are MIA PaCa-2
rescue experiment, the different GLUT isoforms were overex- (Daemen et al., 2015) (IC50 = 4 nM, pancreas), SK-N-AS (Huang
pressed (9- to 15-fold) in CHO cells (Figures S2A–S2D) (Kara- et al., 2015) (IC50 = 6 nM, brain), and SU-DHL-6 (Liu et al., 2018)
georgis et al., 2018). Determination of 2-DG uptake revealed (IC50 = 11 nM, hematologic) cells, which are also known to exhibit
that overexpression of GLUT-1, GLUT-2, and GLUT-3 led to a high glucose dependence or sensitivity toward glycolytic inhibi-
shift to higher IC50 values for treatment with Glutor and, therefore, tors. To the best of our knowledge, for the remaining three cell
a partial rescue (Figures 2A–2C, 2E, and S2E). GLUT-1 overex- lines within the upper 10% of the panel (TE671, WSU-NHL,
pression increased the IC50 from 6.2 ± 1.2 to 34 ± 3.5 nM (6.5- and A431) no records about their metabolic phenotypes are
fold) (Figures 2A and 2E) and GLUT-2 overexpression to 26 ± available. Pancreatic cancer cell lines exhibited a similar
2
.9 nM (4.2-fold) (Figures 2B and 2E). GLUT-3 overexpression sensitivity toward glucose starvation by means of Glutor treat-
>
shifted the IC50 value from 5.9 ± 1.6 to 24 ± 2.6 nM (4.1-fold) (Fig- ment as urinary bladder cancer, except for the BxPC-3 (IC50
ures 2C and 2E). In contrast, overexpression of GLUT-4 did not 30 mM) cell line that was resistant (Figure 3A). In agreement
rescue the 2-DG uptake inhibition by Glutor (IC50 = 6.4 ± with these results, BxPC-3 cells exhibit a lipogenic and non-
1
.7 nM compared with 6.2 ± 1.2 nM in mock transfected CHO glycolytic phenotype (Daemen et al., 2015). Growth of the model
cells; Figures 2E and S2E). These data suggest that Glutor in- cell line HCT116 employed in the 2-DG import assay was
hibits 2-DG uptake by GLUT isoforms 1, 2, and 3, but most likely impeded with an IC50 value of 40 nM (Figure 3A). The observed
not by GLUT-4. Targeting of GLUT-1 and GLUT-3 was further sensitivity tendencies in UM-UC-3, MIA PaCa-2, and HCT116
confirmed by comparing inhibition of 2-DG uptake in DLD-1 cells, cells could be confirmed by means of kinetic live-cell analysis
which mainly express GLUT-1, and the isogenic DLD-1 GLUT1 (Figures S3A–S3C). Growth rate determination revealed 50%
(
À/À) cell line which mainly expresses GLUT-3 (Figures S2F of maximal growth inhibition (GI50) values of 10.2 ± 1.1 nM for
and S2G) (Karageorgis et al., 2018). In DLD-1 cells, 2-DG uptake UM-UC-3, 58.4 ± 30.5 nM for MIA PaCa-2, and 428.1 ±
was potently inhibited with an IC50 value of 35.5 ± 4.8 nM, and in 157.3 nM for HCT116 cells (Figures S3D and S3E). The sensitiv-
DLD-1 GLUT1 (À/À) cells uptake was inhibited with IC50 of 9.8 ± ities apparent from the cell line panel may provide guidance for
1
.4 nM (Figures 2D and 2E). In addition, Glutor increased the ther- possible applications of glucose import inhibition in particular
mal stability of GLUT-3 in SW480 whole-cell lysates, which cancers, such as urinary bladder carcinomas or pancreatic can-
exhibit high GLUT-1 and GLUT-3 content (Schmidt et al., 2018) cers, which are strongly dependent on glucose for proliferation.
in a cellular thermal shift assay with a shift in melting temperature
ꢀ
Investigation of small-molecule activity in 2D culture systems
> 10 C (Figure 2F), which further proves that the compound is considered to be of limited significance (Moffat et al., 2017),
DT
m
targets GLUT-3. The thermal shift of GLUT-1 in the presence of and 3D cell culture systems are regarded as more physiologically
Glutor was smaller (Figure 2F). Overall, these results demonstrate relevant. Therefore, we generated spheroids of HCT116 cells of
engagement of GLUT-1 and GLUT-3, and suggest a more potent ca. 800 and 380 mm diameter as suggested by Kunz-Schughart
inhibition of GLUT-3 than GLUT-1 by Glutor.
and colleagues (Friedrich et al., 2009). Spheroid size determines
gradients of, for example, O and nutrients and formation of a
2
Glutor Selectively Impairs Cancer Cell Growth and
Inhibits Glycolytic Flux
necrotic core and may influence compound activity (Friedrich
et al., 2009). Under conditions of glucose reduction (0 and
Since inhibition of glucose uptake is expected to impair cell 5 mM glucose), cytotoxic events (monitored by propidium iodide
viability and proliferation, we investigated growth of 94, mostly [PI] staining) were observed in 800-mm spheroids after 24 h and
malignant cell lines of different origin by means of sulforhod- resulted in the enlargement of the necrotic spheroid core area
amine B assay after 72 h treatment with Glutor (Figure 3A; Table and, finally, in spheroid bursting under glucose-free conditions
S3). The observed IC50 values ranged from low nanomolar (nM) (Figures S4A and S4B). Treatment with Glutor mirrored the effect
to micromolar (mM) activity, and cell lines with IC50 > 30 mM of glucose starvation in a concentration-dependent manner (Fig-
were regarded as resistant to Glutor treatment. Gratifyingly, ures S4C and S4D). Glucose starvation or treatment of HCT116
the growth of the non-malignant peripheral blood mononuclear spheroids with a diameter of approximately 380 mm with
cells (PBMCs) and IMR-90 embryonic lung cells was not increasing concentrations of Glutor revealed a similar behavior
impaired. The growth of 44 cancer cell lines of diverse tissue without bursting of the spheroid (Figure 3B). In cells cultured in
origin was suppressed with IC50 < 100 nM (Figure 3A, white monolayer with equal seeding density, onset of cytotoxicity
(
(
(
D) 2-DG uptake assay was performed with DLD-1 and DLD-1 GLUT1 (À/À) cells (see also Figures S2F and S2G). Data are mean values ± SD (n = 3, N = 3).
E) Summary of IC50 values and fold change in IC50 values (A–D) (see also Figure S2E).
F) Cellular thermal shift assay in SW480 whole-cell lysates by means of immunoblot-based detection of GLUT-1 and GLUT-3 (representative immunoblots) after
compound or DMSO treatment. Data are mean values ± SD (n = 3).
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Figure 3. Glutor Impairs Cancer Growth and Enhances Formation of Necrotic Center in Spheroids
(A) Growth inhibition (IC50) of 94 different cell lines by means of Glutor treatment was assessed using a sulforhodamine B assay (Vichai and Kirtikara, 2006) after
72 h (see also Figure S3).
(
legend continued on next page)
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Figure 4. Glutor Inhibits Glycolytic Flux
(
A–D) The impact of Glutor on glycolysis (extracel-
lular acidification rate [ECAR]) and oxidative phos-
phorylation (oxygen consumption rate [OCR]) was
assessed for UM-UC-3 cells (A and B) and BxPC-3
cells (C and D) using a glycolysis stress test. Data
are mean values ± SD (n = 3, N = 3). Cmpd, com-
pound; Glc, 10 mM glucose; Oligo, 2.5 mM oligo-
mycin; 2-DG, 50 mM 2-deoxy-D-glucose.
(
(
(
E and F) Glycolytic capacity, glycolytic reserve
E), and reduction of OCR after glucose addition
OXPHOS reduction) (F) of UM-UC-3, HCT116, and
BxPC-3 cells were determined during the glycolysis
stress test depicted in Figures 1E and 1F (HCT116)
and (A–D) (UM-UC-3, BxPC-3).
2
(
G) Linear regression and correlation coefficient R
of the glycolytic reserve and OXPHOS reduction as
shown in (E and F) with IC50 values from the sulfo-
rhodamine B assay were determined (Figure 3A;
Table S3).
Glutor-sensitive cell lines HCT116 (Figures
1
F and 1G) and UM-UC-3 (Figures 4A and
B) switched from OXPHOS to glycolysis
4
upon glucose addition, which reduced the
oxygen consumption rate by 34% (Figures
4F and 4G). In contrast, the Glutor-resistant
BxPC-3 cell line (Figures 4C and 4D)
increased glycolysis upon glucose addition
(Glc), but respiration remained at a con-
stant high level (OXPHOS reduction 11%,
Figures 4F and 4G). Impairment of mito-
chondrial ATP production by oligomycin
(
Figure 4D) induced a strong shift from OX-
was observed after 6 h of treatment. Quantification of PI staining PHOS to glycolysis in BxPC-3 cells such that these cells dis-
after 72 h revealed that Glutor was 2-fold more effective in played the highest glycolytic capacity (545% ECAR, Figure 4E)
inducing cytotoxic events in spheroids (half maximal effective and reserve (213% ECAR, Figures 4E and 4G). As mentioned
concentration [EC50] = 81.7 ± 13.1 nM) than in cells cultured in before, BxPC-3 cells possess a lipogenic and non-glycolytic
monolayer (EC50 = 192.9 ± 34.4 nM) (Figure 3C). Of note, irre- phenotype, which matches with our observations (Daemen
spective of the spheroid size, the cells at the outer border of et al., 2015). The Glutor-sensitive cell lines HCT116 and UM-
the spheroid stayed viable during the experiment. These results UC-3 accelerated the rate of glycolysis after OXPHOS blockage
prove that the potent growth inhibitory effect of GLUT inhibition by 109% and 45%, respectively (glycolytic reserve, Figure 4E).
observed in 2D cell culture can be recapitulated under 3D cell The cellular growth behavior of HCT116 and UM-UC-3 cells is
culture conditions.
known to depend on glucose (Ma et al., 2014; Massari et al.,
To gain further insight into the different cell line responses to 2016). Notably, the switch from respiration to glycolysis upon
Glutor treatment, the metabolic profiles of the most sensitive glucose addition, as well as the glycolytic reserve of the investi-
cell line UM-UC-3 (Figures 4A and 4B), the Glutor-sensitive gated cell lines, correlated with the IC50 for growth inhibition (Ta-
HCT116 cell line (Figures 1F and 1G), and the resistant BxPC-3 ble S3; Figure 3A) and, therefore, the cellular sensitivity toward
cell line (Figures 4C and 4D) were analyzed in a glycolytic stress compound treatment (Figure 4G). Similar observation was
test after 1.5 h glucose deprivation. Addition of 250 nM Glutor described by Liu et al. (2018), whereby glucose-dependent B
blocked glycolytic flux (ECAR) in all investigated cell lines (Figures cell lymphoma possessed a lower glycolytic reserve than lym-
1
F, 4A, and 4C). However, glycolytic stress responses in phoma cell lines that are dependent on other nutrients (in this
untreated cell lines revealed differences (Figures 4E–4G). The case glutamine).
(B) HCT116 cells were seeded in monolayer (2D) or as spheroids (3D, 380 mm diameter) and treated with varying Glutor or glucose concentrations and propidium
iodide (PI) to detect dead cells. Cell growth and toxic events were monitored by means of live-cell imaging. Representative images are shown for 72 h of
compound treatment (see also Figures S4A and S4C).
(C) Toxicity was analyzed by means of PI intensity (3D) or red confluence (2D) (see also Figures S4B and S4D). Data are representative values ± SD (n = 3, N = 8).
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Figure 5. Glutor and Glucose Starvation Lead to GLUT-1 Upregulation
(
A–C) DLD-1 cells were treated with the indicated glucose concentrations for 24 h (A and D) or 48 h (B and E) or with 0.5 mM Glutor at 25 mM glucose for 24 or 48 h
C and F). (A–C) qRT-PCR was performed using specific primers for GLUT1 or ATP1A1, TUBB, and ACTB as reference genes. All values were normalized to values
(
of cells treated with 25 mM glucose (A and B) (dotted line) or DMSO (C) (dotted line), respectively. Data shown are mean values ± SD of n = 3 (A and C) or n = 4 (B)
+
see also Figures S4E and S4F). (D–F) Cells were lysed and GLUT-1 and Na -K -ATPase were detected using immunoblotting. Representative immunoblots of six
+
(
biological replicates are depicted. A dashed line indicates cropped immunoblot images from the same blot. The band intensities for GLUT-1 were normalized to
+
+
the values for Na -K -ATPase and the respective control (D and E) (25 mM glucose, dotted line); (F) (DMSO, dotted line). Data are median values of n = 6 with the
interquartile range. Statistical analysis was performed using unpaired two-tailed t test with Welch’s correction. *p < 0.05, **p < 0.01, ***p < 0.001. See also
Figure S4G for qRT-PCR data on HCT116 cells.
Inhibition of Glucose Uptake by Glutor Induces
Upregulation of GLUT-1 and GLUT-3 Expression
press GLUT-1, were treated with 0.5 mM Glutor in the presence of
high glucose concentration (25 mM) for 24 and 48 h, and mRNA
In cancer cells (Mar ´ı n-Hern a´ ndez et al., 2014) and in neuronal rat (GLUT1-4) and protein (only GLUT-1 and -3) levels were quanti-
cells (Nagamatsu et al., 1994) GLUT-1 and GLUT-3 expression is fied by means of qRT-PCR and immunoblotting, respectively. By
upregulated under glucose deprivation to increase nutrient analogy, the cells were cultured in the absence of the inhibitor,
uptake. To investigate whether this rescue mechanism is also employing 25 mM glucose, physiological glucose concentration
operative upon inhibition of glucose import, we investigated (5 mM) (G u€ emes et al., 2016), or under glucose deprivation (1 mM
GLUT1-4 (also known as SLC2A1-4) expression after treatment glucose or no glucose) for 24 and 48 h (Figures 5 and 6).
with Glutor in comparison with glucose depletion. To this end, Under hypoglycemic conditions (1 mM glucose or no glucose)
the colorectal carcinoma-derived DLD-1 cells, which mainly ex- after 24 and 48 h GLUT1 and GLUT3 mRNA levels had increased
8
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Figure 6. Glutor and Hypoglycemia Lead to GLUT-3 Upregulation
(A–C) DLD-1 cells were treated with the indicated glucose concentrations for 24 h (A and D) or 48 h (B and E) or were treated with 0.5 mM Glutor at 25 mM glucose
for 24 or 48 h (C and F). (A–C) qRT-PCR was performed using specific primers for GLUT3 or ATP1A1, TUBB and ACTB as reference genes All values were
normalized to values of cells treated with 25 mM glucose (A and B) (dotted line) or DMSO (C) (dotted line), respectively. Data shown are mean values ± SD of n = 3
(A and C) or n = 4 (B) (see also Figures S4E and S4F). (D–F) Cells were lysed and GLUT-3 and Vinculin were detected using immunoblotting. Representative
immunoblots of n = 6 (D), n = 8 (E), or n = 7 (F) are depicted. A dashed line indicates cropped immunoblot images from the same blot. The band intensities for
GLUT-3 were normalized to the values for Vinculin and the respective control (D and E) (25 mM glucose, dotted line); (F) (DMSO, dotted line). Data are
median values of n = 6 (D), n = 8 (E), or n = 7 (F) with the interquartile range. Statistical analysis was performed using unpaired two-tailed t test with Welch’s
correction. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S4G for qRT-PCR data on HCT116 cells.
ca. 1.6-fold (Figures 5A, 5B, 6A, and 6B). After treatment increased more than GLUT-1 expression (4.3- versus 3.2-fold
with Glutor the level of GLUT1 (Figure 5C) and GLUT3 (Fig- in the absence of glucose; 5.5- versus 2.8-fold in the presence
ure 6C) mRNA already increased significantly after 24 h by 1.8- of 0.5 mM Glutor; compare Figures 5E, 5F, 6E, and 6F). Expres-
and 1.4-fold, respectively. Under hypoglycemic conditions sion of GLUT4 was not increased under glucose starvation con-
(no glucose or in the presence of 0.5 mM Glutor) the level of the ditions upon treatment with Glutor after 24 and 48 h (see Figures
GLUT-1 protein already increased by ca. 2-fold after 24 h (Fig- S4E and S4F). GLUT2 mRNA could not be detected for all tested
ures 5D and 5F), and, after 48 h, a ca. 3-fold increase was conditions.
detected (Figures 5E and 5F). Under hypoglycemic conditions
A significant upregulation of GLUT3 mRNA by 1.5-fold could
and in the presence of Glutor, a significant increase in GLUT-3 be also observed for HCT116 cells after 48 h of treatment with
protein level was observed after 48 h (4.3- and 5.5-fold, respec- 0.5 mM Glutor or under glucose reduction (0 or 1 mM) (Fig-
tively, Figures 6E and 6F). Notably, GLUT-3 protein expression ure S4G). However, the GLUT1 mRNA level was unaltered under
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Figure 7. Disruption of Glutamine Metabolism Sensitizes HCT116 Cells to Glucose Deprivation
(A) HCT116 cells were cultured over 120 h under different nutrient concentrations (see also Figure S4H). Cell growth was monitored via live-cell imaging over a
period of 5 days. Cellular confluence was analyzed as a measure of growth. Data show representative values ± SD (n = 3, N = 3).
(
legend continued on next page)
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hypoglycemic conditions (Figure S4G). These findings confirm which inhibits growth of triple-negative breast cancer with low
the observations that a hypoglycemic environment induces nanomolar activity. We treated HCT116 cells, which are less
overexpression of GLUT-1, and especially GLUT-3 (Mar ı´ n-Her- sensitive toward CB-839 treatment (GI50 = 16.2 mM) (National
n a´ ndez et al., 2014) and proves that the same effect is achieved Cancer Institute, 2019; NCI-60 panel, CAS(CB-839) 1439399-
by inhibition of glucose import through Glutor. As observed in 58-2), with CB-839 at different concentrations together with
adipose tissue (Khayat et al., 1998), cancer cells only increase varying concentrations of Glutor in the presence of 4 mM gluta-
GLUT-1 and GLUT-3 mRNA and protein level, but not GLUT4 mine and 25 mM glucose (Figures 7C–7E and S5). The co-treat-
(
and GLUT2) mRNA, under glucose deprivation. Thereby cancer ment led to a maximal 40-fold improvement of the GI50 from
cells employ a rescue mechanism to increase glucose uptake in 428.1 ± 157.3 nM (0 mM CB-839) to 10.4 ± 0.8 nM in the presence
times of glucose shortage. Thus, inhibition of GLUT-1 alone may of 5 mM CB-839 (Figure 7D). The GI50 of CB-839 then decreased
not be sufficient to efficiently inhibit cancer cell growth, and the from 11.8 mM (0 mM Glutor), which is in line with the reported GI50
GLUT-2 inhibiting activity of Glutor is at least less important for value from the NCI-60 panel, to GI50 < 50 nM (1 mM Glutor)
reduction of cancer cell growth in the presence of the inhibitor.
(Figures S5A and S5B). Analysis of the data employing three
independent classical synergy models, namely Loewe (1953)
(Figures 7E and S5D), highest single agent (Tan et al., 2012) (Fig-
ures S5E and S5F), and Bliss (1939) (Figures S5G and S5H),
Glucose Uptake Inhibition by Glutor and Glutaminase
GLS Inhibition Synergistically Inhibits Cell Growth
Nutrient uptake, utilization, and dependence of cancer cells may confirmed the synergistic effect with regard to growth inhibition
differ and vary depending on cancer cell type and growth condi- in a concentration range for Glutor between 8 nM to 1 mM and
tions; for example, nutrient availability and microenvironment 50 nM to 5 mM CB-839 (Figures 7E and S5, see Supplemental In-
(
Davidson et al., 2016; Stock et al., 2016). In particular, cancer formation for model definitions). The analysis clearly indicates
cells use glutamine for amino acid synthesis and redox balance that dual targeting of glutamine metabolism and glucose uptake
maintenance to enable cell growth, and some cancers are gluta- may offer a promising approach for synergistic inhibition of tu-
mine addicted (Eagle, 1955). Thus, combination of glucose up- mor cell growth. Moreover, Glutor acts not only synergistically
take inhibition with inhibition of glutamine metabolism may syn- with CB-839 but may sensitize cell lines to GLS inhibition.
ergistically suppress cell growth.
Investigation of HCT116 revealed that, under glutamine deple- DISCUSSION
tion (0 mM glutamine and 25 mM glucose), cell growth is more
reduced than under glucose withdrawal (0 mM glucose and Cancer, immune and stem cells adapt their metabolism to meet
4
mM glutamine) (Figure S4H), suggesting that reduction of the requirements of sustained proliferation by increasing aerobic
glutamine concentration might sensitize the cells toward glucose glycolysis (Warburg effect) and glucose uptake (Cliff and Dalton,
starvation. Thus, we examined the growth of HCT116 cells in the 2017; Jones and Bianchi, 2015). To meet this demand, cancer
presence of different glucose and glutamine concentrations (Fig- cells upregulate facilitative GLUTs, in particular GLUT-1, and
ure 7A). At low glutamine concentration (0.05 and 0 mM) cell small-molecule inhibition of glucose uptake has been proposed
growth was impeded, regardless of the prevalent glucose con- as a promising approach to cancer drug discovery. Because the
centration (25, 10, or 5 mM) (Figure 7A). At physiological gluta- relevant glucose transporter isoforms GLUT-1-4 are expressed
mine concentration (0.5 mM) (Scriver and Rosenberg, 1973), in a tissue-specific manner and are involved in various central
reduced glucose (5 mM) led to strong growth reduction that processes, selective inhibition of GLUT-1 was deemed neces-
could only be circumvented in the presence of higher glucose sary for a wide therapeutic window. However, both GLUT-1
levels (10 or 25 mM). On the contrary, at physiological glucose and GLUT-3 are upregulated in various cancers, suggesting
concentration (5 mM) (G u€ emes et al., 2016), the growth of that dual inhibition of both transporters may be required for
HCT116 cells was dependent on the presence of sufficient successful glucose deprivation. A potent and selective GLUT-1
amounts of glutamine (Figure 7A). This finding suggests that inhibitor has been developed, but data on its application have
impairment of HCT116 cell growth by means of glucose import not been reported (Siebeneicher et al., 2016a, 2016b). We
inhibition should be more efficient at reduced glutamine concen- recently described a dual-selective GLUT-1/-3 inhibitor, which,
trations. Indeed, in the presence of 4 mM glutamine, Glutor in- however, suffers from low potency (Karageorgis et al., 2018).
hibited HCT116 growth with a GI 50 value of 604.1 ± 21.0 nM,
whereas in the presence of 0.5 mM glutamine the GI 50 value glucose uptake inhibitor. Glutor targets glucose transporters
was lowered to 210.3 ± 43.7 nM (Figure 7B). GLUT-1, -2, and -3. Inhibition of glucose uptake by Glutor
We discovered the piperazin-one Glutor ((S)-3a) as a potent
In light of this finding, we investigated co-treatment with Glutor attenuates glycolytic flux and increases the expression of
and the glutaminase (GLS) inhibitor CB-839 (Gross et al., 2014), GLUT-1 and GLUT-3. Glutor potently suppresses growth of a
(B) HCT116 cells were cultured over 96 h under high (4 mM) or physiological (0.5 mM) glutamine concentration and in presence of 25 mM glucose. The growth
behavior was assessed by means of live-cell imaging. Cellular confluence was analyzed as a measure of cell growth. The slopes of the exponential growth phase
were used for GI50 determination. Data show mean values ± SD (n = 3, N = 3).
(
C–E) HCT116 cells were cultured under 25 mM glucose and 4 mM glutamine and treated with different concentrations of CB-839 and Glutor. Cell growth was
monitored via live-cell imaging over 5 days. Cellular confluence was analyzed as a measure of growth (C). GI50 values for Glutor in the presence of different CB-
39 concentrations were determined using the growth rates between 24 and 72 h (D). For better visualization all values were normalized to the 0 mM CB-839
8
control. Synergism and antagonism scores were determined with the software combenefit (Di Veroli et al., 2016) (see also Figure S5) employing the Loewe model
and the score distribution is depicted in the dose-response surface within the concentration space. Data represent mean values ± SD (n = 3, N = 3).
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variety of cancer cell lines of different origin in 2D (monolayer) example, upregulating glutamine metabolism during glycolysis
and 3D (spheroids) culture systems, but not of non-malignant suppression (Lukey et al., 2018). In this context, reduction of
IMR-90 and PBMCs, which were employed to assess unspe- GLUT-1 expression sensitizes lung cancer cells to inhibition of
cific cytotoxicity. Nearly half of the examined cancer cell lines glutamine utilization (Lee et al., 2016). Naturally, the concomi-
(
44) were inhibited with an IC50 below 100 nM, thus demon- tant targeting of glutamine (unspecific transaminase inhibitor
strating the broad applicability for treatment against diverse aminooxyacetate) and glucose metabolism (with 2-DG) has a
cancer types. Furthermore, urinary bladder and pancreatic cumulative effect on ovarian cancer cell growth suppression
cancer cell lines were among the most sensitive cell lines (Sun et al., 2017).
within the conducted cell panel. Glucose dependence is a
We demonstrated that co-treatment of HCT116 cells with
metabolic phenotype observed in both tissue-derived cancer Glutor and glutaminase inhibitor CB-839 very potently and
types, and interference of the glycolytic flux alone or in combi- synergistically inhibited cancer cell growth at high glucose
nation with other cancer targeting agents were proposed as and glutamine levels. Thus, simultaneous inhibition of the two
therapeutic approaches (Daemen et al., 2015; Lea et al., major nutrient supply mechanisms employed by cancer cells
2
015; Massari et al., 2016). can synergistically reduce cancer cell growth. Such a dual
Our results indicate that dual inhibition of GLUT-1 and -3 inhibition may enable particularly effective suppression of
should be particularly effective for inhibition of tumor cell growth. cancer cell growth to avoid therapy resistance through
We demonstrate that tumor cells upregulate mainly GLUT3 metabolic plasticity. Overall, our findings suggest that both
and GLUT1 in a cell-dependent manner (but not GLUT2) cellular rescue mechanisms (i.e., upregulation of glucose
upon glucose starvation or treatment with Glutor. Hence, a import) and the metabolic plasticity of cancer to increase
dual-specific inhibitor will be more efficacious than a mono- alternative use of glutamine as nutrient may be synergistically
selective compound, and may overcome this cellular rescue overcome by simultaneous inhibition of glucose transporters
mechanism (Mar ı´ n-Hern a´ ndez et al., 2014; Nagamatsu et al., GLUT-1 and -3 and glutamine metabolism, for example, by in-
1
994). In fact, targeting of upregulated GLUT-3 may hibition of glutaminase GLS.
be particularly relevant, because GLUT-3 has a lower K value Finally, we note that, beyond the potential application in the
for glucose (K for 2-DG = 1.4 mM) than GLUT-1 (K for 2- discovery of anti-cancer agents, inhibition of glucose uptake
m
m
m
DG = 5 mM) (Zhao and Keating, 2007), thereby assuring efficient via GLUTs may offer promising opportunities for the treatment
glucose uptake also at low glucose concentrations. By analogy, of diseases related to inflammation, immunity, and infection,
GLUT-3 may have a protecting function from inhibitor influence such as graft-versus-host disease (Macintyre et al., 2014), co-
for neurons, which express mainly GLUT-3 and rely on glucose. litis (Macintyre et al., 2014), systemic lupus erythematosus
The low K
competitive advantage with respect to glucose at environmental (Gualdoni et al., 2018) and bacterial infections (Escoll and
glucose concentrations (1–2 mM) (Simpson et al., 2008). Buchrieser, 2018), and HIV persistence (Palmer et al., 2016),
m
and high capacity of GLUT-3 should give neurons a (Yin et al., 2015), psoriasis (Zhang et al., 2018), rhinoviral
Apart from glucose, cells metabolize glutamine, which is the because all these diseases are connected to an increased
most abundant amino acid in plasma (Daye and Wellen, 2012). glucose utilization.
An intricate interplay between glucose and glutamine meta-
bolism exists in cells that coordinate ATP supply and availability SIGNIFICANCE
of biosynthetic precursors, which are required for proliferation,
as well as redox balance maintenance. While in normal cells Cancer cell growth is sustained by increased glucose de-
glucose is converted to citrate, which fuels fatty acid synthesis, mand, which frequently induces upregulation of glucose
glutamine is used as an anaplerotic precursor for the tricarbox- transporters GLUT-1 and -3. Therefore, interference with
ylic acid cycle and supports mitochondrial respiration, ATP tumor metabolism by means of GLUT inhibition is consid-
production, and synthesis of nucleotides and some amino acids. ered a relevant strategy to impair cancer growth. However,
Cells can synthesize glutamine, and glutamine is non-essential currently available inhibitors of the glucose transporters
in normal, non-dividing tissue. However, under conditions of (GLUTs) suffer from low potency or mainly target GLUT-1.
stress or proliferation (e.g., T cell activation) (Ardawi, 1988; We discovered
a GLUT-1/-2/-3-selective inhibitor that
Carr et al., 2010), the glutamine consumption outpaces the potently inhibits glucose entry, glycolysis, and cancer cell
glutamine supply, and glutamine becomes an essential amino growth in monolayer and spheroids. The use of GLUT-1/-3
acid (Still and Yuneva, 2017). Thus, small-molecule inhibitors targeting—rather than isoform-specific inhibitors—enables
of glutamine metabolism are actively being developed as anti- efficient suppression of glucose uptake because cells upre-
cancer drugs (Akins et al., 2018). Various genetic alterations gulate both GLUT-1 and GLUT-3 upon glucose starvation
are known to drive glutamine metabolism, for example, in caused by small-molecule-mediated inhibition of glucose
MYC, KRAS, HIF1, HER2, p53, JAK/STAT, mTOR, and NRF2 uptake. Cancer cell growth and survival may also be sup-
activation, PKCz loss, PTEN loss, and RB1 loss (Altman et al., ported by glutamine as an additional nutrient source, which
2
016), and can confer glutamine/glutaminolysis dependence. confers metabolic flexibility. We demonstrate that simulta-
Thus, cancer cells rely on both glucose and glutamine, and neous targeting of glucose uptake by means of GLUT-1/-3
despite fulfilling separate roles in cellular metabolism both meta- inhibition and glutaminolysis synergistically inhibits cancer
bolic pathways may compensate for one another (Daye and cell growth. This dual strategy may open up unprecedented
Wellen, 2012). Due to this metabolic plasticity, cancer cells opportunities to overcome rescue mechanisms and meta-
may adapt to glucose- or glutamine-deprived conditions, for bolic plasticity in cancer, and may inspire chemical biology
12 Cell Chemical Biology 26, 1–15, September 19, 2019

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and medicinal chemistry programs aimed at the discovery of
alternative strategies and compound classes for the treat-
ment of cancer.
Altman, B.J., Stine, Z.E., and Dang, C.V. (2016). From Krebs to clinic: gluta-
mine metabolism to cancer therapy. Nat. Rev. Cancer 16, 619–634.
Ardawi, M.S. (1988). Glutamine and glucose metabolism in human peripheral
lymphocytes. Metabolism 37, 99–103.
Ayala, F.R.R., Rocha, R.M., Carvalho, K.C., Carvalho, A.L., da Cunha, I.W.,
Louren c¸ o, S.V., and Soares, F.A. (2010). GLUT1 and GLUT3 as potential prog-
nostic markers for oral squamous cell carcinoma. Molecules 15, 2374–2387.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
Bliss, C.I. (1939). The toxicity of poisons applied jointly. Ann. Appl. Biol. 26,
5
85–615.
d KEY RESOURCES TABLE
Carr, E.L., Kelman, A., Wu, G.S., Gopaul, R., Senkevitch, E., Aghvanyan, A.,
Turay, A.M., and Frauwirth, K.A. (2010). Glutamine uptake and metabolism
are coordinately regulated by ERK/MAPK during T lymphocyte activation.
J. Immunol. 185, 1037–1044.
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Cell Lines
Chrostowska, A., Matrane, A., Maki, D., Khayar, S., Ushiki, H., Graciaa, A.,
Belachemi, L., and Guillemin, J.-C. (2012). Are unsaturated isocyanides so
different from the corresponding nitriles? Chemphyschem 13, 226–236.
d METHOD DETAILS
B Biological Experiments
B Synthetic Procedure
Cliff, T.S., and Dalton, S. (2017). Metabolic switching and cell fate decisions:
implications for pluripotency, reprogramming and development. Curr. Opin.
Genet. Dev. 46, 44–49.
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND CODE AVAILABILITY
Creedon, S.M., Crowley, H.K., and McCarthy, D.G. (1998). Dehydration of
SUPPLEMENTAL INFORMATION
formamides using the Burgess Reagent:
a new route to isocyanides.
J. Chem. Soc. Perkin Trans. 1, 1015–1018.
Supplemental Information can be found online at https://doi.org/10.1016/j.
chembiol.2019.06.005.
Daemen, A., Peterson, D., Sahu, N., McCord, R., Du, X., Liu, B., Kowanetz, K.,
Hong, R., Moffat, J., Gao, M., et al. (2015). Metabolite profiling stratifies
pancreatic ductal adenocarcinomas into subtypes with distinct sensitivities
to metabolic inhibitors. Proc. Natl. Acad. Sci. U S A 112, E4410–E4417.
ACKNOWLEDGMENTS
Davidson, S.M., Papagiannakopoulos, T., Olenchock, B.A., Heyman, J.E.,
Keibler, M.A., Luengo, A., Bauer, M.R., Jha, A.K., O’Brien, J.P., Pierce, K.A.,
et al. (2016). Environment impacts the metabolic dependencies of Ras-driven
non-small cell lung cancer. Cell Metab. 23, 517–528.
Research at the Max Planck Institute of Molecular Physiology was supported
by the Max Planck Society and the European Research Council under the
European Union’s Seventh Framework Program (FP7/2007–2013, ERC Grant
agreement no. 268309). J.C. and E.S.R. acknowledge the International Max-
Planck Research School for a doctoral scholarship. G.K. received funding
from the Alexander von Humboldt-Stiftung for a post-doctoral fellowship.
The compound management and screening center (COMAS) in Dortmund is
acknowledged for performing the high-throughput screening and the orthog-
onal and counter screens. The Lead Discovery Center is acknowledged for
sharing the cell panel data. We acknowledge Beate Sch o¨ lermann for the tech-
nical support.
Daye, D., and Wellen, K.E. (2012). Metabolic reprogramming in cancer: unrav-
eling the role of glutamine in tumorigenesis. Semin. Cell Dev. Biol. 23, 362–369.
Di Veroli, G.Y., Fornari, C., Wang, D., Mollard, S., Bramhall, J.L., Richards,
F.M., and Jodrell, D.I. (2016). Combenefit: an interactive platform for the anal-
ysis and visualization of drug combinations. Bioinformatics 32, 2866–2868.
Dolomanov, O.V., Bourhis, L.J., Gildea, R.J., Howard, J.A.K., and Puschmann,
H. (2009). OLEX2: a complete structure solution, refinement and analysis
program. J. Appl. Crystallogr. 42, 339–341.
AUTHOR CONTRIBUTIONS
Eagle, H. (1955). Nutrition needs of mammalian cells in tissue culture. Science
S.Z. and H.W. designed the research. E.S.R., G.K., M.S., J.N., S.B., A.C., and
A.B. performed the biological research. E.S.R., J.N., and M.C.M.S. performed
the compound synthesis. E.S.R., G.K., M.S., J.N., M.C.M.S., A.B., and S.Z.
analyzed the data. J.C. contributed to compound development. L.K. and
C.S. performed the X-ray structure analysis. E.S.R., S.Z., and H.W. wrote
the manuscript. All authors discussed the results and commented on the
manuscript.
1
22, 501–514.
Escoll, P., and Buchrieser, C. (2018). Metabolic reprogramming of host cells
upon bacterial infection: why shift to a Warburg-like metabolism? FEBS J.
2
85, 2146–2160.
Fonteyne, P., Casneuf, V., Pauwels, P., van Damme, N., Peeters, M., Dierckx,
R., and van de Wiele, C. (2009). Expression of hexokinases and glucose trans-
porters in treated and untreated oesophageal adenocarcinoma. Histol.
Histopathol. 24, 971–977.
DECLARATION OF INTERESTS
Friedrich, J., Seidel, C., Ebner, R., and Kunz-Schughart, L.A. (2009). Spheroid-
H.W. is sponsor of a drug discovery program at the Lead Discovery Center of
the Max-Planck-Gesellschaft aimed at the development of GLUT inhibitors.
based drug screen: considerations and practical approach. Nat. Protoc. 4,
3
09–324.
Fulmer, G.R., Miller, A.J.M., Sherden, N.H., Gottlieb, H.E., Nudelman, A.,
Stoltz, B.M., Bercaw, J.E., and Goldberg, K.I. (2010). NMR chemical shifts of
trace impurities: common laboratory solvents, organics, and gases in deuter-
ated solvents relevant to the organometallic chemist. Organometallics 29,
Received: February 22, 2019
Revised: May 31, 2019
Accepted: June 17, 2019
Published: July 11, 2019
2
176–2179.
REFERENCES
Gross, M.I., Demo, S.D., Dennison, J.B., Chen, L., Chernov-Rogan, T., Goyal,
B., Janes, J.R., Laidig, G.J., Lewis, E.R., Li, J., et al. (2014). Antitumor activity
of the glutaminase inhibitor CB-839 in triple-negative breast cancer. Mol.
Cancer Ther. 13, 890–901.
Akins, N.S., Nielson, T.C., and Le, H.V. (2018). Inhibition of glycolysis and glu-
taminolysis: an emerging drug discovery approach to combat cancer. Curr.
Top. Med. Chem. 18, 494–504.
Gualdoni, G.A., Mayer, K.A., Kapsch, A.-M., Kreuzberg, K., Puck, A., Kienzl, P.,
Oberndorfer, F., Fr u€ hwirth, K., Winkler, S., Blaas, D., et al. (2018). Rhinovirus
Allen, C.L., Atkinson, B.N., and Williams, J.M.J. (2012). Transamidation of pri-
mary amides with amines using hydroxylamine hydrochloride as an inorganic
catalyst. Angew. Chem. Int. Ed. 51, 1383–1386.
induces an anabolic reprogramming in host cell metabolism essential for viral
replication. Proc. Natl. Acad. Sci. U S A 115, E7158–E7165.
Cell Chemical Biology 26, 1–15, September 19, 2019 13

Please cite this article in press as: Reckzeh et al., Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.06.005
G u€ emes, M., Rahman, S.A., and Hussain, K. (2016). What is a normal blood
glucose? Arch. Dis. Child. 101, 569–574.
(2014). The glucose transporter Glut1 is selectively essential for CD4 T cell acti-
vation and effector function. Cell Metab. 20, 61–72.
Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of cancer: the next gener-
Mar ı´ n-Hern a´ ndez, A., L o´ pez-Ram ı´ rez, S.Y., Del Mazo-Monsalvo, I.,
Gallardo-P e´ rez, J.C., Rodr ı´ guez-Enr ı´ quez, S., Moreno-S a´ nchez, R., and
Saavedra, E. (2014). Modeling cancer glycolysis under hypoglycemia, and
the role played by the differential expression of glycolytic isoforms. FEBS
J. 281, 3325–3345.
ation. Cell 144, 646–674.
Huang, C.-C., Wang, S.-Y., Lin, L.-L., Wang, P.-W., Chen, T.-Y., Hsu, W.-M.,
Lin, T.-K., Liou, C.-W., and Chuang, J.-H. (2015). Glycolytic inhibitor 2-deoxy-
glucose simultaneously targets cancer and endothelial cells to suppress
neuroblastoma growth in mice. Dis. Model. Mech. 8, 1247–1254.
Marroquin, L.D., Hynes, J., Dykens, J.A., Jamieson, J.D., and Will, Y. (2007).
Circumventing the Crabtree effect: replacing media glucose with galactose in-
creases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci.
97, 539–547.
Ilyn, A.P., Trifilenkov, A.S., Tsirulnikov, S.A., Kurashvily, I.D., and Ivachtchenko,
A.V. (2005). Synthesis of 4-oxo-4,5,6,7-tetrahydropyrazolo1,5-apyrazine-6-
carboxamides using a modification of Ugi condensation. J. Comb. Chem. 7,
8
06–808.
Martinez-Outschoorn, U.E., Peiris-Pag e´ s, M., Pestell, R.G., Sotgia, F., and
Lisanti, M.P. (2017). Cancer metabolism: a therapeutic perspective. Nat.
Rev. Clin. Oncol. 14, 11–31.
Jones, W., and Bianchi, K. (2015). Aerobic glycolysis: beyond proliferation.
Front. Immunol. 6, 227.
J o´ ꢀz wiak, P., Krze ꢀs lak, A., Pomorski, L., and Lipi nꢀ ska, A. (2012). Expression of
hypoxia-related glucose transporters GLUT1 and GLUT3 in benign, malignant
and non-neoplastic thyroid lesions. Mol. Med. Rep. 6, 601–606.
Massari, F., Ciccarese, C., Santoni, M., Iacovelli, R., Mazzucchelli, R., Piva, F.,
Scarpelli, M., Berardi, R., Tortora, G., Lopez-Beltran, A., et al. (2016).
Metabolic phenotype of bladder cancer. Cancer Treat. Rev. 45, 46–57.
˚
Matsushita, K., Uchida, K., Saigusa, S., Ide, S., Hashimoto, K., Koike, Y.,
Otake, K., Inoue, M., Tanaka, K., and Kusunoki, M. (2012). Glycolysis inhibitors
as a potential therapeutic option to treat aggressive neuroblastoma express-
ing GLUT1. J. Pediatr. Surg. 47, 1323–1330.
Kajanus, J., Jacobson, I., Astrand, A., Olsson, R.I., Gran, U., Bj o¨ re, A.,
Fjellstr o¨ m, O., Davidsson, O., Emtenas, H., Dahlen, A., et al. (2016).
Isoindolinone compounds active as Kv1.5 blockers identified using a multi-
component reaction approach. Bioorg. Med. Chem. Lett. 26, 2023–2029.
¨
€
´
Karageorgis, G., Reckzeh, E.S., Ceballos, J., Schwalfenberg, M., Sievers, S.,
Ostermann, C., Pahl, A., Ziegler, S., and Waldmann, H. (2018).
Chromopynones are pseudo natural product glucose uptake inhibitors target-
ing glucose transporters GLUT-1 and -3. Nat. Chem. 10, 1103–1111.
Mellanen, P., Minn, H., Gr e´ nman, R., and H a€ rk o¨ nen, P. (1994). Expression of
glucose transporters in head-and-neck tumors. Int. J. Cancer 56, 622–629.
Moffat, J.G., Vincent, F., Lee, J.A., Eder, J., and Prunotto, M. (2017).
Opportunities and challenges in phenotypic drug discovery: an industry
perspective. Nat. Rev. Drug Discov. 16, 531–543.
Khayat, Z.A., McCall, A.L., and Klip, A. (1998). Unique mechanism of GLUT3
glucose transporter regulation by prolonged energy demand: increased pro-
tein half-life. Biochem. J. 333, 713–718.
Nagamatsu, S., Sawa, H., Inoue, N., Nakamichi, Y., Takeshima, H., and
Hoshino, T. (1994). Gene expression of GLUT3 glucose transporter regulated
by glucose in vivo in mouse brain and in vitro in neuronal cell cultures from rat
embryos. Biochem. J. 300 (Pt 1), 125–131.
Koppenol, W.H., Bounds, P.L., and Dang, C.V. (2011). Otto Warburg’s contri-
butions to current concepts of cancer metabolism. Nat. Rev. Cancer 11,
3
25–337.
National Cancer Institute (2019). Developmental therapeutics program. http://
dtp.cancer.gov.
Krzeslak, A., Wojcik-Krowiranda, K., Forma, E., Jozwiak, P., Romanowicz, H.,
Bienkiewicz, A., and Brys, M. (2012). Expression of GLUT1 and GLUT3 glucose
transporters in endometrial and breast cancers. Pathol. Oncol. Res. 18,
Neochoritis, C.G., Stotani, S., Mishra, B., and D o¨ mling, A. (2015). Efficient iso-
cyanide-less isocyanide-based multicomponent reactions. Org. Lett. 17,
721–728.
2
002–2005.
Lea, M.A., Altayyar, M., and desBordes, C. (2015). Inhibition of growth of
bladder cancer cells by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one in com-
bination with other compounds affecting glucose metabolism. Anticancer Res.
Palmer, C.S., Cherry, C.L., Sada-Ovalle, I., Singh, A., and Crowe, S.M. (2016).
Glucose metabolism in T cells and monocytes: new perspectives in HIV path-
ogenesis. EBioMedicine 6, 31–41.
35, 5889–5899.
Reckzeh, E.S., Brockmeyer, A., Metz, M., Waldmann, H., and Janning, P.
Lee, Y.-M., Lee, G., Oh, T.-I., Kim, B.M., Shim, D.-W., Lee, K.-H., Kim, Y.J.,
Lim, B.O., and Lim, J.-H. (2016). Inhibition of glutamine utilization sensitizes
lung cancer cells to apigenin-induced apoptosis resulting from metabolic
and oxidative stress. Int. J. Oncol. 48, 399–408.
(
2019). Target engagement of small molecules: thermal profiling approaches
on different levels. Methods Mol. Biol. 1888, 73–98.
Schmidt, T., Samaras, P., Frejno, M., Gessulat, S., Barnert, M., Kienegger, H.,
Krcmar, H., Schlegl, J., Ehrlich, H.-C., Aiche, S., et al. (2018). ProteomicsDB.
Nucleic Acids Res. 46, D1271–D1281.
Liu, X., Wang, L., Jiang, W., Lu, W., Yang, J., and Yang, W. (2018). B cell lym-
phoma with different metabolic characteristics show distinct sensitivities to
metabolic inhibitors. J. Cancer 9, 1582–1591.
Scriver, C.R., and Rosenberg, L.E. (1973). Amino acid metabolism and its dis-
orders. Major Probl. Clin. Pediatr. 10, 1–478.
Liu, Y., Cao, Y., Zhang, W., Bergmeier, S., Qian, Y., Akbar, H., Colvin, R.,
Ding, J., Tong, L., Wu, S., et al. (2012). A small-molecule inhibitor of glucose
transporter 1 downregulates glycolysis, induces cell-cycle arrest, and in-
hibits cancer cell growth in vitro and in vivo. Mol. Cancer Ther. 11,
Sheldrick, G.M. (2008). A short history of SHELX. Acta Crystallogr. A 64 (Pt 1),
1
12–122.
Sheldrick, G.M. (2015). SHELXT - integrated space-group and crystal-struc-
1
672–1682.
ture determination. Acta Crystallogr. A 71 (Pt 1), 3–8.
Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression
data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.
Methods 25, 402–408.
Shi, Y., Liu, S., Ahmad, S., and Gao, Q. (2018). Targeting key transporters in
tumor glycolysis as a novel anticancer strategy. Curr. Top. Med. Chem. 18,
454–466.
Loewe, S. (1953). The problem of synergism and antagonism of combined
Siebeneicher, H., Bauser, M., Buchmann, B., Heisler, I., M u€ ller, T., Neuhaus,
R., Rehwinkel, H., Telser, J., and Zorn, L. (2016a). Identification of novel
GLUT inhibitors. Bioorg. Med. Chem. Lett. 26, 1732–1737.
drugs. Arzneimittelforschung 3, 285–290.
Lukey, M.J., Katt, W.P., and Cerione, R.A. (2018). Targeting therapy resis-
tance: when glutamine catabolism becomes essential. Cancer Cell 33,
€
Siebeneicher, H., Cleve, A., Rehwinkel, H., Neuhaus, R., Heisler, I., Muller, T.,
Bauser, M., and Buchmann, B. (2016b). Identification and optimization of the
first highly selective GLUT1 inhibitor BAY-876. ChemMedChem 11,
2261–2271.
7
95–797.
Ma, Y.-S., Yang, I.-P., Tsai, H.-L., Huang, C.-W., Juo, S.-H.H., and Wang, J.-Y.
2014). High glucose modulates antiproliferative effect and cytotoxicity of
-fluorouracil in human colon cancer cells. DNA Cell Biol. 33, 64–72.
(
5
Simpson, I.A., Dwyer, D., Malide, D., Moley, K.H., Travis, A., and Vannucci,
S.J. (2008). The facilitative glucose transporter GLUT3: 20 years of distinction.
Am. J. Physiol. Endocrinol. Metab. 295, E242–E253.
Macintyre, A.N., Gerriets, V.A., Nichols, A.G., Michalek, R.D., Rudolph, M.C.,
Deoliveira, D., Anderson, S.M., Abel, E.D., Chen, B.J., Hale, L.P., et al.
14 Cell Chemical Biology 26, 1–15, September 19, 2019

Please cite this article in press as: Reckzeh et al., Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.06.005
Still, E.R., and Yuneva, M.O. (2017). Hopefully devoted to Q: targeting gluta-
Yamamoto, N., Sato, T., Kawasaki, K., Murosaki, S., and Yamamoto, Y. (2006).
A nonradioisotope, enzymatic assay for 2-deoxyglucose uptake in L6 skeletal
muscle cells cultured in a 96-well microplate. Anal. Biochem. 351, 139–145.
mine addiction in cancer. Br. J. Cancer 116, 1375–1381.
Stock, K., Estrada, M.F., Vidic, S., Gjerde, K., Rudisch, A., Santo, V.E., Barbier,
M., Blom, S., Arundkar, S.C., Selvam, I., et al. (2016). Capturing tumor
complexity in vitro: comparative analysis of 2D and 3D tumor models for
drug discovery. Sci. Rep. 6, 28951.
Yamamoto, T., Seino, Y., Fukumoto, H., Koh, G., Yano, H., Inagaki, N.,
Yamada, Y., Inoue, K., Manabe, T., and Imura, H. (1990). Over-expression of
facilitative glucose transporter genes in human cancer. Biochem. Biophys.
Res. Commun. 170, 223–230.
Sun, L., Yin, Y., Clark, L.H., Sun, W., Sullivan, S.A., Tran, A.-Q., Han, J., Zhang,
L., Guo, H., Madugu, E., et al. (2017). Dual inhibition of glycolysis and glutami-
Yin, Y., Choi, S.-C., Xu, Z., Perry, D.J., Seay, H., Croker, B.P., Sobel, E.S.,
Brusko, T.M., and Morel, L. (2015). Normalization of CD4+ T cell metabolism
reverses lupus. Sci. Transl. Med. 7, 274ra18.
nolysis as
a therapeutic strategy in the treatment of ovarian cancer.
Oncotarget 8, 63551–63561.
Younes, M., Brown, R.W., Stephenson, M., Gondo, M., and Cagle, P.T. (1997).
Overexpression of Glut1 and Glut3 in stage I nonsmall cell lung carcinoma is
associated with poor survival. Cancer 80, 1046–1051.
Tan, X., Hu, L., Luquette, L.J., Gao, G., Liu, Y., Qu, H., Xi, R., Lu, Z.J., Park, P.J.,
and Elledge, S.J. (2012). Systematic identification of synergistic drug pairs tar-
geting HIV. Nat. Biotechnol. 30, 1125–1130.
Zhang, Z., Zi, Z., Lee, E.E., Zhao, J., Contreras, D.C., South, A.P., Abel, E.D.,
Chong, B.F., Vandergriff, T., Hosler, G.A., et al. (2018). Differential glucose
requirement in skin homeostasis and injury identifies a therapeutic target for
psoriasis. Nat. Med. 24, 617–627.
Vander Heiden, M.G., Cantley, L.C., and Thompson, C.B. (2009).
Understanding the Warburg effect: the metabolic requirements of cell prolifer-
ation. Science 324, 1029–1033.
Vichai, V., and Kirtikara, K. (2006). Sulforhodamine B colorimetric assay for
Zhao, F.-Q., and Keating, A.F. (2007). Functional properties and genomics of
cytotoxicity screening. Nat. Protoc. 1, 1112–1116.
glucose transporters. Curr. Genomics 8, 113–128.
Cell Chemical Biology 26, 1–15, September 19, 2019 15

Please cite this article in press as: Reckzeh et al., Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.06.005
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
Antibodies
SOURCE
IDENTIFIER
Mouse monoclonal anti-GLUT-1 (1:5,000)
Abcam
Cat# ab40084;
RRID: AB_2190927
Rabbit polyclonal anti-GLUT-2 (1:200)
Rabbit monoclonal anti-GLUT-3 (1:5,000)
Rabbit monoclonal anti-GLUT-3 (1:5,000)
Rabbit polyclonal anti-GLUT-4 (1:500)
Santa Cruz
Abcam
Cat# sc-9117;
RRID: AB_641068
Cat# ab191071;
RRID: AB_2736916
Santa Cruz
Millipore
Cat# sc-74399;
RRID: AB_1124975
Cat# 07-1404;
RRID: AB_1587080
+
Rabbit monoclonal anti-Na /K -ATPase (1:10,000)
+
Abcam,
Cat# ab76020;
RRID: AB_1310695
Mouse monoclonal anti-Vinculin (1:10,000)
Santa Cruz
LI-COR
Cat# sc-59803;
RRID: AB_794011
Donkey anti-mouse (1:5,000),
infrared dye 800CW-labeled
Cat# 926-32212;
RRID: AB_621847
Donkey anti-rabbit (1:5,000),
infrared dye 680RD-labeled
LI-COR
Cat# 926-68073;
RRID: AB_10954442
Donkey anti-rabbit (1:5,000),
infrared dye 800CW-labeled
LI-COR
Cat# 926-32213;
RRID: AB_621848
Donkey anti-mouse (1:5,000),
infrared dye 680RD-labeled
LI-COR
Cat# 926-68072; RRID:AB_10953628
Goat polyclonal anti-rabbit (1:100,000),
horseradish peroxidase-labeled
Thermo Fisher Scientific
Thermo Fisher Scientific
Cat# 31430;
RRID: AB_228307
Goat polyclonal anti-mouse, (1:10,000)
horseradish peroxidase-labeled
Cat# 31460;
RRID: AB_228341
Chemicals, Peptides, and Recombinant Proteins
Chemical: 3-Bromopyruvate
Alfa Aesar
Cat# L00720
CAS: 1113-59-3
Chemical: 2-Deoxy-a-D-glucose (2-DG)
Protein: Diaphorase, Clostridium kluyvery
Santa Cruz
Cat# sc-202010
CAS: 154-17-6
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
AppliChem
Cat# D5540-500UN
CAS: 9001-18-7
Protein: Glucose-6-phosphate dehydrogenase,
Cat# G8404
Leuconostoc mesenteroides
CAS: 9001-40-5
Protein: Insulin, bovine pancreas
Cat# I6634
CAS: 11070-73-8
+
Chemical: NADP
Cat# A1394,0001
CAS: 1184-16-3
Chemical: NP-40 alternative
Chemical: Propidium iodide (PI)
Chemical: Resazurin
Merck-Calbiochem
Sigma-Aldrich
Acros Organics
Cat# 492016
CAS: 9016-45-9
Cat# P4864
CAS: 25535-16-4
Cat# AC418900050
CAS: 62758-13-8
Critical Commercial Assays
Sso Advanced SYBR Green Mix
SuperSignal West Pico Chemiluminescent
Bio-Rad
Cat# 1725270
Cat# 10177533
Thermo Fisher Scientific
(Continued on next page)
e1 Cell Chemical Biology 26, 1–15.e1–e25, September 19, 2019

Please cite this article in press as: Reckzeh et al., Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.06.005
Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
SuperSignal West Femto Maximum Sensitivity Substrate
QuantiTect Reverse transcription kit
Seahorse Glycolysis Stress test
Deposited Data
Thermo Fisher Scientific
Qiagen
Cat# 34095
Cat# 205313
Cat# 103020-100
Agilent
X-ray structure compound (R)-2a
Experimental Models: Cell Lines
Human: DLD-1 cells (male)
This paper
CCDC: 1883523
Horizon Discovery
Horizon Discovery
Cat# HD PAR-086; RRID: CVCL_0248
Human: DLD-1 GLUT1(-/-) cells (male)
Cat# HD R00-024;
RRID: CVCL_HD63
Human: UM-UC-3 cells (male)
Human: HCT116 cells (male)
Human: BxPC-3 cells (female)
Human: UO-31 cells (female)
Human: MIA PaCa-2 (male)
Human: SW480 cells (male)
Hamster: CHO cells (female)
ATCC
Cat# CRL-1749;
RRID: CVCL_1783
DSMZ
Cat# ACC581;
RRID: CVCL_0291
DSMZ
Cat# ACC760;
RRID: CVCL_0186
National Cancer Institute
MTA #1-4488-14;
RRID: CVCL_1911
ATCC
ATCC
ATCC
Cat# CRM-CRL-1420;
RRID: CVCL_0428
Cat# CCL-228;
RRID: CVCL_0546
Cat# CRL-9618; RRID: CVCL_0214
Oligonucleotides
Primers for GLUT1, see Table S5
Primers for GLUT2, see Table S5
Primers for GLUT3, see Table S5
Primers for GLUT4, see Table S5
Primers for ACTB, see Table S5
Primers for TUBB, see Table S5
Primers for ATP1A1, see Table S5
Eurofins
Eurofins
Eurofins
Eurofins
Eurofins
Eurofins
Eurofins
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Recombinant DNA
Plasmid: GLUT-1 (pTCN)
Plasmid: GLUT-2 (pTCN)
Plasmid: GLUT-3 (pCMV-SPORT 6)
Plasmid: GLUT-4 (pTCN)
Plasmid: pTCN
transOMIC
transOMIC
Dharmacon
transOMIC
transOMIC
Dharmacon
Cat# BC121804
Cat# BC060041
Cat# OHS1770-202314070
Cat# BC034387
Cat# BC060823
N/A
Plasmid: pCMV-SPORT 6
Software and Algorithms
CFX Manager
Bio-Rad
RRID: SCR_003375
http://www.bioconductor.org/packages/release/
bioc/html/HTqPCR.html
Prism 5
Prism 7
GraphPad
GraphPad
RRID:SCR_002798
http://www.graphpad.com/
RRID:SCR_002798
http://www.graphpad.com/
Combenefit
(Di Veroli et al., 2016)
LI-COR
N/A
Image Studio Ver. 5.2
RRID:SCR_015795
https://www.licor.com/bio/products/software/
image_studio/
(Continued on next page)
Cell Chemical Biology 26, 1–15.e1–e25, September 19, 2019 e2

Please cite this article in press as: Reckzeh et al., Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.06.005
Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
IncuCyte ZOOM 2015/2016A
(EssenBioscience)
Satorius
https://www.essenbioscience.com/en/
products/software/
IncuCyte S3 2017A
(EssenBioscience)
Satorius
https://www.essenbioscience.com/en/
products/software/
APEX 3 Suite (v.2017.3-0)
Bruker
https://www.bruker.com/products/
microtomography/micro-ct-software/3dsuite.html
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Herbert
Waldmann (herbert.waldmann@mpi-dortmund.mpg.de).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell Lines
HCT116 (human, male), UM-UC-3 (human, male), UO-31 (human, female), MIA PaCa-2 (human, male) were grown in 4.5 g/l glucose
Dulbecco’s modified Eagle’s medium (DMEM, PAN Biotech) supplemented with 10% v/v fetal calf serum (FCS, Invitrogen-Gibco)
with 1 mM sodium pyruvate (PAN Biotech) and 100 U/mL penicillin/ 0.1 mg/mL streptomycin (PAN Biotech). CHO cells (hamster, fe-
male) were cultured in in 4.5 g/l glucose Dulbecco’s modified Eagle’s medium (DMEM, PAN Biotech) supplemented with 10% v/v
fetal calf serum (FCS, Invitrogen-Gibco), 100 U/mL penicillin/ 0.1 mg/mL streptomycin (PAN Biotech) and non-essential amino acids
(PAN Biotech). DLD-1 (human, male) and DLD-1 GLUT1 (-/-) (human, male) as well as BxPC-3 (human, female) cells were cultured in
RPMI1640 medium supplemented with 10% fetal calf serum (FCS, Invitrogen-Gibco) and 1100 U/mL penicillin/ 0.1 mg/mL strepto-
mycin (PAN Biotech). SW480 cells (human, male) were cultured in Leibovitz L-15 medium supplemented with 10% fetal calf serum
ꢀ
(
FCS, Invitrogen-Gibco) and 1100 U/mL penicillin/ 0.1 mg/mL streptomycin (PAN Biotech). All cell lines were grown at 37 C with 5%
2
CO in humidified atmosphere and regularly tested negative for mycoplasma contamination.
METHOD DETAILS
Biological Experiments
Resazurin-coupled 2-DG Uptake Assay
Cells were seeded in a 96-well plate (black wall, clear bottom, Corning) in complete medium with a density of 40,000 cells/well. Cells
ꢀ
were grown for 24 h at 37 C with 5% CO
2
in humidified atmosphere. Adherent cells were washed three times with freshly prepared
PO , 1 mM MgSO , 1 mM CaCl , 136 mM NaCl, 4.7 mM KCl, pH
.4, 0.1% w/v BSA). 1 mM 2-DG was added to the cells together with the respective compound dilution and incubated for 30 min at
Krebs-Ringer bicarbonate (KRB) buffer (20 mM HEPES, 5 mM KH
2
4
4
2
7
room temperature. DMSO served as a control. The assay buffer was removed and the 2-DG uptake was stopped by washing the cells
thrice with ice-cold KRB buffer. 60 mL 0.06 M HCl supplemented with 1% w/v CHAPS was added to the cells and the cells were incu-
ꢀ
bated for 15 min at 65 C for cell lysis. Neutralization was performed with 20 mL 0.5 M Tris at room temperature. 60 mL enzyme mix
(
+
16 U/mL glucose-6-phosphate dehydrogenase (Sigma Aldrich), 0.2 U/mL diaphorase (Sigma-Aldrich), 0.1 mM NADP (Applichem),
2
.5 mg/mL resazurin (Acros Organics) in 0.125 M Tris-HCl (Carl Roth), pH 8.4 supplemented with 0.1% w/v BSA) was added and the
plate was incubated for 2 h in the dark. Fluorescence of resorufin (ex/em 535 nm/590 nm) was measured using the Tecan Infinite
M200 (Tecan Trading). The background (no addition of 2-DG) was subtracted from the raw values prior to normalization to the
DMSO control. IC50 values were calculated using a nonlinear regression fit with variable slope (four parameters) using GraphPad
Prims 5 or 7.
The semi-automated screening was performed as described above with slight modifications: 15,000 HCT116 cells/well were
seeded into 384- well black-walled, clear bottomed plates (CellBIND, Corning) and incubated overnight. Compounds were tested
at 30 mM concentration (for the screening) or in serial dilutions staring from 30 mM (IC50 determinations) from DMSO stocks with
an acoustic nanoliter dispenser (ECHO 520). Fluorescence intensity (ex/em 535 nm/590 nm) was measured with a SpectraMax Para-
digm Multi-Mode Microplate Reader (Molecular devices, bottom read-out) using the Rhodamine filter settings. For hit compounds,
cell viability was assessed using the CellTiter Glo reagent (Promega) after 30 min of treatment in HCT116 cells. Washing steps and
liquid additions were performed by means of automated cell washer or multidrop device, respectively.
Hexokinase Assay
HCT116 cells were grown to confluence, washed thrice with PBS and lysed with RIPA buffer (10 mM Tris-Cl (pH 8.0, 1 mM EDTA, 1%
Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF)) that was
supplemented with protease inhibitor cocktail as recommended by the manufacturer (Roche). Cells were lysed on ice for 5 min
ꢀ
and collected. The lysate was centrifuged (500 x g, 4 C, 10 min) and the supernatant transferred to a fresh tube. After protein
e3 Cell Chemical Biology 26, 1–15.e1–e25, September 19, 2019

Please cite this article in press as: Reckzeh et al., Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.06.005
ꢀ
concentration determination with Bradford reagent (Bio-Rad), the lysates were snap frozen and stored at -80 C until further usage.
The assay was performed in a black 96 well plate in 100 mL total volume. 10% v/v cell lysate (conc. 0.04 mg/mL) were supplemented
+
with 0.3 mM ATP, 10% v/v of 2-DG (concentration range 0-15.6 mM), 43% v/v enzyme mix (0.1 mM NADP , 16 U/mL G6PDH in assay
buffer) and 10% v/v compound (300 mM Glutor or 300 mM 3-bromopyruvate) (Matsushita et al., 2012) and filled up with assay buffer
+
ꢀ
(
100 mM Tris, 5 mM MgCl
40 nm/445 nm) was measured with the Tecan Infinite M200 (Tecan Trading). The background was subtracted and all values
2
, pH 8.5). Samples were incubated for 40 min at 37 C. Fluorescence intensity of NADPH+H (ex/em
3
were normalized to the maximal fluorescence intensity at 15.6 mM 2-DG.
Cell Panel Profiling
Cell panel profiling to assess the growth behavior of 94 cell lines in presence of Glutor was performed by Oncolead (Germany). Deter-
mination of cell growth was performed after 72 h of compound treatment using sulforhodamine B assay (Vichai and Kirtikara, 2006).
ꢀ
Cells were fixed by addition of 10% TCA (adherent cells) or 50% TCA (semi-adherent or suspension cells) for 1 h at 4 C followed by
two washing steps with 400 ml deionized water. After drying, cells were stained with 100 mL of 0.04% w/v sulforhodamine B and
incubated for at least 30 min at room temperature. Unbound dye was removed by washing six times with 1% acetic acid and drying
samples at room temperature. Bound sulforhodamine B was dissolved in 100 mL of 10 mM Tris. Absorbance was determined at 492,
5
20 and 560 nm using a Deelux-LED96 plate reader (Deelux Labortechnik GmbH, Germany).
Glycolysis Stress Test
Glycolysis Stress Test (Agilent) was performed using the Seahorse XFp analyzer (Agilent). Briefly, 24 h prior the experiment cells were
seeded with a density of 75,000 cells/well in the respective full medium. The next day, medium was exchanged to glucose-free assay
ꢀ
medium (Agilent) and cells were incubated at 37 C without CO
2
for 45 min. After compound addition (first injection, 30 min incubation)
the glycolysis stress test protocol was performed according to the manufacturer’s instructions. The data was analyzed using the
Wave software (Agilent) and all oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) values were normalized
rd
to the time point before compound addition (3 measurement). A second normalization was done to calculate the rate relative to the
DMSO control. For metabolic profiles, the second of three measurements from one experimental phase (e.g. after glucose addition)
was used to plot the glycolytic reserve, glycolytic capacity and the OXPHOS reduction.
Reverse Transcription-Quantitative PCR
DLD-1 or HCT116 cells were seeded at 63,000 cells/well in a 12-well plate and incubated for 24 h prior to treatment. Cells were incu-
bated with 0.5 mM Glutor (RPMI medium for DLD-1 cells; DMEM medium for HCT116 cells) or different glucose concentrations
(
(
DMEM medium) for the indicated time points. Total RNA was isolated by means of RNeasy Mini Kit as described by the manufacturer
Qiagen). The Bioanalyzer (Agilent) was used with the corresponding RNA 6000 nano Kit (Agilent) to perform integrity measurements
of the isolated RNA following the manufacturer’s instructions. Reverse transcription was done using QuantiTect Reverse transcrip-
tion kit (Qiagen) and according to the instructions of the manufacturer. RT-qPCR was performed with Sso Advanced SYBR Green Mix
(
Bio-Rad) using the iCycler iQ5 thermal cycler (Bio-Rad). Gene-specific primer sequences, their respective efficiencies and amplicon
sizes are depicted in Table S5. Data was analyzed using CFX Manager. GLUT1-4 values were normalized to the levels of ACTB, TUBB
and ATP1A1 using the DDC -method (Livak and Schmittgen, 2001). Efficiencies were measured in DLD-1 wt lysates (GLUT1, GLUT3,
t
ACTB, TUBB, ATP1A1) or with isoform-specific plasmids (GLUT2, GLUT4).
Immunoblotting
5
5
DLD-1 cells were seeded in a 6-well plate with 3.06*10 cells/well (24 h treatment) or 1.53*10 cells/well (48 h treatment) 24 h prior to
the experiment. Different glucose concentrations were tested in DMEM medium. Cells were treated with Glutor in RPMI1640
medium. DMSO served as a control. Cells were treated for 24 or 48 h prior to cell lysis. Briefly, cells were washed once with PBS,
ꢀ
detached with cell dissociation solution (Sigma Aldrich) and washed again with PBS. After centrifugation for 5 min (4 C,
1
,200 rpm) the pellet was lysed in PBS supplemented with 2% w/v dodecyl-b-D-maltoside (Carl Roth) on ice (15 min). After centri-
ꢀ
fugation at 14,000 rpm (4 C, 15 min) the supernatant was collected to determine the protein concentration by means of DC protein
assay (Bio-Rad) or with Bradford reagent (Bio-Rad). SDS-PAGE was carried out using 6% SDS gels followed by transfer on
polyvinyldiene difluoride membrane (PVDF, Merck) by means of semi-dry electrophoretic device (Bio-Rad). Membranes were
blocked using Odysseyꢀ blocking buffer (LI-COR) for 1 h at room temperature. The proteins GLUT-1 and GLUT-3 were detected
+
+
using the primary antibodies anti-GLUT-1 (mouse) and anti-GLUT-3 (rabbit, Abcam) in Odyssey blocking buffer (LI-COR). Na /K -
ATPase (rabbit) and vinculin (mouse) were detected for normalization (housekeeping proteins). The primary antibodies were
ꢀ
incubated over night at 4 C and washed three times with PBS. For detection, infrared dye-labeled 800CW anti-mouse (donkey)
+
+
and 680RD anti-rabbit (donkey) secondary antibody were used to detect GLUT-1 and Na /K -ATPase and 800CW donkey anti-rabbit
and 680RD anti-mouse secondary antibody to detect GLUT-3 and vinculin. The membranes were incubated with the secondary
antibody solutions for 1 h at room temperature followed by two washing steps with PBS-T (0.1% Tween-20) and one washing
step with PBS. The fluorescence detection was carried out using the Odyssey CLx imaging system (LI-COR). Densitometric analysis
was used for protein band quantification (Image Studio Ver. 5.2).
GLUT-1-4 Overexpression in CHO Cells
6
x10 CHO cells were seeded 10 cm (diameter) cell culture plates and incubated overnight. Cells were transiently transfected with the
1
respective GLUT-isoform containing plasmids using Lipofectamine 3000 according to the supplier’s instructions. Briefly, DNA-lipid
complexes (100 mg plasmid DNA, DNA:lipid ratio 1:2) were prepared in OptiMEM and added to the cells. GLUT-4-transfected cells
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were treated with insulin (100 mg/mL). After 48 h the cells were reseeded (40,000 cell/well) in 96-well plates for 2-DG uptake moni-
toring or lysed to assess protein expression level by means of immunoblotting. For immunoblotting, samples were lysed in 1.5x SDS
sample buffer without bromophenol blue. Protein concentrations were determined by means of DC protein assay (Bio-Rad). Proteins
were separated using SDS-PAGE and transferred to PVDF membrane via semi-dry transfer. Blocking was performed using 5% w/v
skimmed milk in 0.1% Tween-20 in Tris buffer (TBS-T) for 1 h at room temperature. The primary antibody GLUT-1, GLUT-2, GLUT-3
+
+
ꢀ
(
Santa Cruz), GLUT-4 or/and Na -K -ATPase (housekeeping protein) (TBS-T) was incubated at 4 C overnight. The respective
secondary horseradish peroxidase-labeled antibody (TBS-T) was incubated for 1 h at room temperature after washing with
TBS-T. Signals visualization was performed using SuperSignal West Pico Chemiluminescent or Femto Maximum Sensitivity
Substrate (Thermo Fischer) employing the Odyssey Fc imaging system (Li-COR). Band quantification was analyzed by means of
densitometric analysis.
Real-Time Live-Cell Imaging and Analysis
Cells were seeded 24 h prior to treatment at 5,000 cells/well in a 96-well plate. In case the nutrient concentrations were varied, DMEM
medium without glucose and glutamine was used. Glutor or DMSO was added to the cells and the growth behavior of the cells were
monitored for 48 h or 120 h, depending on the duration of the experiment using the IncuCyte ZOOM or IncuCyte S3. Cell confluence
was analyzed as a measure of cell growth using the respective software IncuCyte ZOOM 2016B or IncuCyte S3 2017A.
3
D vs. 2D Cell Culture
Cells were seeded at 750 cells/well (380 mm spheroids) or 10,000 cells/well (800 mm spheroids) in monolayer or as spheroids in ultra-
low attachment (ULA) plates (Corning) in full DMEM medium (200 mL/well). After 48 h 100 mL medium of the large spheroids (and
monolayer control) was exchanged by fresh medium. After 72 h the cells were treated with the compound or different glucose
concentrations in the presence of propidium iodide (PI) (0.02 mg/mL, Sigma Aldrich) to assess cell toxicity. For different glucose con-
centrations, 100 mL of medium was exchanged six times by the respective medium to achieve the desired concentration of glucose.
For compound addition, 100 mL medium were exchanged for 100 mL fresh medium containing 2x the desired compound concentra-
tion. Cell growth was monitored by means of live-cell imaging using IncuCyte S3. Analysis of PI intensity was performed using the
IncuCyte S3 2017A software. Spheroid toxicity was evaluated using all brightfield objects red mean intensities (RCU) whereas toxicity
of monolayer cultured cells was analyzed by red confluence (%). EC50 values were calculated using a nonlinear regression fit with
variable slope (four parameters) with GraphPad Prims 5 or 7.
Synergism/Antagonism Scores
Data generated during live-cell imaging (growth rates) in co-treatment experiments were normalized to DMSO-treated cells (%) and
analyzed using different models.
Definition of synergy models as implemented in the open source software combenefit (Di Veroli et al., 2016).
a=drug A, b=drug B, a
Loewe Model. The reference effect for the combination of drug A and drug b (RAB) is calculated by determining the two doses a
and b such that the efficacy of both drugs is equal (Loewe, 1953).
u u
=dose drug A, b =dose drug B, R=reference effect, E=efficacy
u
u
Formula to define the reference effect:
R
u u
AB_Loewe(a,b) = E(a ) = E_B (b )
With the extension:
RAB_Loewe(a,b) = E(b
u B
) = E (b)
The extension is included for cases when drug A and drug B have a different maximum effect.
Bliss Model. The reference effect for the combination of drug A and drug b (RAB) is calculated by multiplying the effects at the
concentrations of drug A and drug B (Bliss, 1939).
Formula to define the reference effect:
A B
RAB_Bliss(a,b) = E (a) x E (b)
With the definition:
The effect of drug A (or drug B) is the fraction of the cell population that is not affected by treatment.
Highest Single Agent (HAS) Model. The reference effect for the combination of drug A and drug b (RAB) is calculated by determining
the greatest difference between the drug A and drug B, when given as single agent (Tan et al., 2012).
Formula to define the reference effect:
A B
RAB_HSA(a,b) = MIN(E (a), E (b))
Cellular Thermal Shift Assay (CETSA)
For whole cell lysates, SW480 cells were grown to nearly confluence, washed with PBS and detached using cellular detachment so-
lution (Sigma Aldrich) according to Reckzeh et al. (Reckzeh et al., 2019). Cells were resuspended using PBS, centrifuged at 1,200 rpm
and washed with PBS. After a second centrifugation step, the pellet was resuspended in lysis buffer (PBS, 0.4% v/v NP40 alternative)
on ice. To lyse the cells, four freeze-and-thaw cycles were applied to the cell suspension, followed by short sonication step (10 s). The
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ꢀ
supernatant was collected after ultracentrifugation (4 C, 20 min, 100,000 x g) and the protein concentration was determined by using
ꢀ
a Bradford reagent (Bio-Rad). Samples were snap frozen and stored at -80 C. For cellular thermal shift assay (CETSA), lysates were
diluted to 2 mg/mL and divided into two reaction tubes. One fraction was treated with 10 mM Glutor, whereas the control fraction was
supplemented with 1% (v/v) DMSO. The lysates were incubated for 10 min at room temperature, followed by fractionation (10 tubes)
ꢀ
ꢀ
and subjection to a temperature gradient (36.9-67 C) for 3 min. After ultracentrifugation (4 C, 20 min, 100,000 x g), the soluble
fractions were collected and analyzed using immunoblotting with antibodies against GLUT-1 and GLUT-3.
Mitochondrial Toxicity
Mitochondrial toxicity was analyzed by assessing compound sensitivity of HCT116 under 25 mM glucose and 10 mM galactose (Mar-
roquin et al., 2007). Briefly, cell were seeded at 5,000 cells/well in 96-well plates and treated after 24 h with the inhibitor and in the
presence of glucose or galactose. Oligomycin was used as a control for mitochondrial toxicity. Cell growth was monitored by means
of live-cell imaging using IncuCyte ZOOM and analyzed as described above.
Synthetic Procedure
Materials
All chemicals were obtained from Acros Organics, Activate Scientific, Alfa Aesar or Sigma-Aldrich and used as provided, unless
otherwise indicated. Dry solvents over molecular sieves were purchased from Sigma-Aldrich or Acros Organics.
Instrumentation
1
13
Proton and carbon nuclear magnetic resonance ( H- and C-NMR) spectra were recorded on Varian Mercury 400 (400 MHz), Bruker
Avance DRX 500 (500 MHz), INOVA 500 (500 MHz), Bruker AV600 (600 MHz) and Bruker AV700 (700 MHz) at ambient temperature.
Proton chemical shifts are indicated as parts per million (ppm, d-scale) and are referenced to residual protium in the NMR solvent
(
3 2 2 3 2 3
CHCl , d7.26 ppm; CH Cl , d5.30 ppm; (CH ) SO, d2.50 ppm, CH OH, d3.31 ppm) (Fulmer et al., 2010). Data are represented as
follows: chemical shift, multiplicity (s = singlet, bs = broad singlet, d = doublet, dd= doublet of doublets, ddd= doublet of doublet
of doublets, dt = doublet of triplets, t = triplet, td = triplet of doublets, q = quartet, s = septet, m = multiplet), coupling constant (J)
in Hertz (Hz) and integration. Carbon chemical shifts are expressed in parts per million (ppm, d-scale) and are referenced to the car-
3 2 2 3 2 3
bon resonances of the NMR solvent (CDCl , d77.16 ppm; CD Cl , d53.84 ppm; (CH ) SO, d39.52 ppm; CD OD, d49.00 ppm). All NMR
spectra were analyzed using MastReNova Version 12.0.0 and are depicted in Data S1. Low resolution mass spectra were collected
via 7890a (Agilent) equipped with a 5975c mas spectrometer (Agilent) and a hp-5ms column (Agielnt) (MS-EI) or using uHPLC system
1
2
290 infinity system (Agilent) equipped with a 6150 mass spectrometer (Agilent) and zorbax eclipse plus C18 rapid resolution HD
.1x50mm 1.8 mm material column (Agilent). High resolution mass spectra (HR-MS) were recorded on a LTQ Orbitrap mass
spectrometer 5 coupled to an Acceka HPLC-System (HPLC column: Hypersyl GOLD, 0 m x 1 mm, particle size 1.9 mm, ionization
method: electron spray ionization). One exemplary spectrum of 3a is depicted in Data S1. Systematic names for molecules according
to IUPAC rules were generated using ChemDraw Professional version 16.0.
Synthetic Methods
ꢀ
Scheme of synthetic procedure to generate piperazin-2-one-based compound library. i) Triphosgene, ACN, 0 C, 0.5-2 h; ii)
ꢀ
ꢀ
Burgess reagent, ACN / MeOH (5:1), rt, 2 h; iii) Benzenesulfonyl chloride, trioctylamine, 80 C, 2 h; iv) HCONH
ꢀ
2
ꢀ
, HCO
2
H, 180 C, con-
1
NOH*HCl, toluene, 110 C, 24 h; R =heteroar-
ventional heating 0.5-2 h or microwave 3 min; v) HCO
2
2
Et, 60 C, 24 h; vi) HCONH
2
, H
2
1
2
omatic, R =alkyl, aromatic, heteroaromatic, R’=R ,R , R’’=aromatic. annul. Cy=annulated cyclohexane. 2x=2a, 2b, 2c, 2e, 2h;
x=3a, 3b, 3c, 3e, 3f, 3g, 3h, 3i, 3l, 3m, 3n, 3o, 3p, 3s. Late stage functionalization to generate 2i, 3d, 3k, 3q and 3r are described
in the compound characterization.
3
General Procedure 1A. Modified Leuckart-Wallach reaction (Neochoritis et al., 2015)
General Procedure for Conventional Heating. A microwave vial was charged with the corresponding aldehyde (3.6 mmol, 1 equiv.)
ꢀ
dissolved in formamide (36 mmol, 10 equiv.) and formic acid (18 mmol, 5 equiv.). The reaction mixture was vigorously stirred at 180 C
for 2-4 h. After cooling the mixture to room temperature, the reaction was extracted with DCM and the combined organic layers were
dried over Na
2 4
SO , filtered and concentrated under reduced pressure. The crude product was subsequently purified by flash silica
gel column chromatography (elute as indicated) to yield the corresponding formamide.
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General Procedure for Microwave Irradiation. A microwave vial was charged with the corresponding aldehyde (0.4 mmol, 1 equiv.)
dissolved in formamide (18.4 mmol, 50 equiv.) and formic acid (1.8 mmol, 5 equiv.). The reaction mixture was irradiated in a
ꢀ
microwave oven for 30 sec. at 180 C, quenched with 10 mL water and extracted with DCM. The combined organic layers were dried
2 4
over Na SO , filtered and concentrated under reduced pressure. Residual formamide was removed in vacuo. The crude residue was
purified by means of flash silica gel column chromatography (elute as indicated) to yield the corresponding formamide.
Formamide Formation from Amines
Formamides were synthesized from amines as described by Kajanus et al. (Kajanus et al., 2016) or Allen et al. (Allen et al., 2012).
All amines and isocyanides that were incorporated in the modified Ugi reaction are depicted in Table S4.
General Procedure 2: Modified Ugi Reaction. The bifunctional carboxylic acid/ketone (0.20 mmol, 1.0 equiv.) and the amine
(
0.20 mmol, 1.0 equiv. or 0.18 mmol, 0.9 equiv.) were dissolved in MeOH (1 mL), stirred for 30min at room temperature and the iso-
ꢀ
cyanide (0.20 mmol, 1.0 equiv.) was added. The mixture was vigorously stirred at 40 C until full conversion of the starting material was
indicated by TLC. The solvent was removed under reduced pressure and the product was extracted thrice with a mixture of water and
2 4
dichloromethane (15 mL). The combined organic layers were dried over Na SO , filtered and concentrated under reduced pressure.
The product was purified by flash silica gel column chromatography (eluate: petroleum ether / EtOAc or DCM / MeOH) (Ilyn
et al., 2005).
General Procedure 3A: Modified Ugi Reaction with In Situ Isocyanide Formation Using Phosgene. The reaction was performed as
described by Neochoritis et al. (2015). The corresponding formamide (0.06 mmol, 1.0 equiv.) was dissolved in DCM (1 mL) and cooled
ꢀ
to 0 C. Triethylamine (0.15 mmol, 2.5 equiv.) was added, the mixture was stirred for 10 min and triphosgene (0.02 mmol, 0.3 equiv.) in
DCM (1 mL) was added dropwise over a period of 30 min. After consumption of the formamide (monitored by TLC), the corresponding
bifunctional carboxylic acid/ketone (0.07 mmol, 1.2 equiv.) and amine (0.06 mmol, 1.0 equiv.) were added and the reaction mixture
was vigorously stirred for 5-72 h. The crude mixture was concentrated under reduced pressure and purified by means of silica gel
chromatography using the indicated elute.
General Procedure 3B: Modified Ugi Reaction with In Situ Isocyanide Formation Using Burgess Reagent. The reaction was per-
formed as described by Creedon et al. (Creedon et al., 1998). Briefly, an oven-dried Schlenck tube was charged with the formamide
derivative (220 mmol, 1 eq) and dry acetonitrile under argon atmosphere. Solid Burgess reagent (348 mmol, 1.5-2 eq) was added and
the solution was stirred for 0.5-2 h at room temperature. The reaction progress was checked by means of thin layer chromatography
(
TLC). The amine (198 mmol, 0.9 eq) and bifunctional compound (220 mmol, 1 eq) were combined in MeOH, stirred for approximately
0 min and added to the reaction mixture. The reaction mixture was vigorously stirred at room temperature for 1-5 days. Saturated
1
NaHCO
over Na
3
was added to the reaction mixture and the product was extracted thrice with DCM. The combined organic layers were dried
SO , filtered and concentrated under reduced pressure. Purification was performed over silica or by recrystallization from
2
4
MeOH as indicated.
Propargyl isocyanide was alternatively synthesized in situ from N-(propargyl)formamide as described by Chrostowska et al.
(
Chrostowska et al., 2012).
Preparative HPLC-MS
Separations were performed by means of mass-directed preparative HPLC (Agilent Series, 1100/LC/MSD VL) using reversed-phase
C18 column with a constant flow of 20.0 mL/min. Solvent A: water +0.1% v/v TFA; solvent B: acetonitrile +0.1% v/v TFA.
Separation of the Enantiomeric Mixture
Chiral separation was performed by means of solid phase separation via an Ultimate 3000 HPLC (Dionex, Thermo Fisher) employing
an IC column (CHIRALPAK IC, column no. IC00CG-MA004) column. An isocratic gradient was used to separate both enantiomers
(
elute: isohexane / DCM/MeOH(5%)).
Single Crystal X-ray Structure Analyses
Data collection was conducted on a Bruker D8 Venture four-circle diffractometer by Bruker AXS GmbH using a PHOTON100 CMOS
area detector by Bruker AXS GmbH. X-ray radiation was generated by microfocus sources ImS Cu or Mo by Incoatec GmbH with
HELIOS mirror optics and a single-hole collimator by Bruker AXS GmbH.
For the data collection, the programs APEX 3 Suite (v.2017.3-0) with the integrated programs SAINT (integration) and SADABS
adsorption correction) by Bruker AXS GmbH were used. Using Olex2 (Dolomanov et al., 2009), the structures were solved with
(
the ShelXT (Sheldrick, 2015) structure solution program using Intrinsic Phasing and refined with the XL (Sheldrick, 2008) refinement
package using Least Squares minimization.
Compound Characterization
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Methyl 1-(2-oxopropyl)-3-phenyl-1H-pyrazole-5-carboxylate (5a). Methyl 3-phenyl-1H-pyrazole-5-carboxylate 4a (30.0 mg,
0
2 3
.15 mmol, 1.0 equiv.), 3-chloroacetone (11.9 mL, 0.15 mmol, 1.0 equiv.), K CO (24.6 mg, 0.18 mmol, 1.2 equiv.) and
1
8-crown-6-ether (0.4 mg, 1.5 mmol, 0.01 equiv.) were suspended in 1,4-dioxane (1 mL) and vigorously stirred under reflux conditions
for 6 h. Afterwards, water (50 mL) and DCM (50 mL) were added to the reaction mixture and the layers were separated. The aqueous
phase was extracted two more times with DCM (2x 50 mL) and the combined organic layers were dried over MgSO , filtrated
and evaporated under reduced pressure. The residue was purified by means of silica gel column chromatography (elute:
4
1
cyclohexane / EtOAc) and obtained as white solid (23.0 mg, 0.15 mmol, quant.). R
400 MHz, CDCl ) d 7.79 (d, J = 7.3 Hz, 2H), 7.41 (t, J = 7.3 Hz, 2H), 7.33 (t, J = 7.3 Hz, 1H), 7.19 (s, 1H), 5.39 (s, 2H), 3.88 (s, 3H),
.23 ppm (s, 3H). C NMR (100 MHz, CDCl
f
= 0.24 (cyclohexane / EtOAc = 3:1). H NMR
(
3
1
3
2
3
) d 201.3, 162.9, 146.5, 143.9, 129.9, 129.4, 129.4, 129.1, 109.6, 59.8, 52.5, 27.4
+
15 2 3
ppm. HR-MS: calc. for [M+H] C14H N O 259.1077 found 259.1075.
1
-(2-Oxopropyl)-3-phenyl-1H-pyrazole-5-carboxylic acid (6a). Methyl 1-(2-oxopropyl)-3-phenyl-1H-pyrazole-5-carboxylate 5a
30.0 mg, 0.12 mmol, 1.0 equiv.) was suspended in water (1 mL) and NaOH was added slowly (5.6 mg, 0.14 mmol, 1.2 equiv.) and
(
stirred thoroughly at 70 C for 3 h. Afterwards, HCl (0.5 mL, 1 M) was added to the crude reaction mixture. Water (40 mL) and EtOAc
ꢀ
(
40 mL) were added to the crude reaction and the phases were separated. The aqueous phase was extracted two more times with
EtOAc (2x 40 mL) and the combined organic layers were dried over Na SO , filtered and concentrated under reduced pressure. The
product was obtained as light yellow solid (28.3 mg, 0.12 mmol, quant.). R = 0.41 (dichloromethane / MeOH = 7:3). H NMR (400 MHz,
CDCl ) d 7.88 (d, J = 7.3 Hz, 2H), 7.42 (t, J = 7.3 Hz, 2H), 7.35 (t, J = 7.3 Hz, 1H), 7.31 (s, 1H), 5.41 (s, 2H), 2.23 ppm (s, 3H). C NMR
100 MHz, CDCl
245.0921 found 245.0929.
2
4
1
f
13
3
+
(
3
) d 201.2, 163.6, 151.4, 133.4, 132.0, 128.9, 128.6, 125.9, 110.0, 61.5, 27.0 ppm. HR-MS: calc. for [M+H]
13 13 2 3
C H N O
Methyl 1-(3-oxobutyl)-3-phenyl-1H-pyrazole-5-carboxylate (5b). Methyl 3-phenyl-1-pyrazole-5-carboxylate 4a (100 mg, 495 mmol,
1
5
1
.0 equiv.) was dissolved in 1,4-dioxane (1.5 mL) and potassium carbonate (82 mg, 593 mmol, 1.2 equiv.), 18-Crown-6 ether (1.5 mg,
mmol, 0.01 equiv.) and 4-chlorobutan-2-one (47.3 mL, 495 mmol, 1.0 equiv.) were added. The solution was stirred thoroughly at
ꢀ
40 C for 4 h. The product was purified by means of silica gel column chromatography (elute: cyclohexane / EtOAc) and obtained
1
= 0.30 (cyclohexane / EtOAc = 3:1). H NMR (700 MHz, CDCl
as white solid (55 mg, 202 mmol, 40%). R
f
3
) d 7.77 (dd, J = 8.0, 1.2 Hz,
2
H), 7.40 (t, J = 8.0 Hz, 2H), 7.34 – 7.30 (m, 1H), 7.11 (s, 1H), 4.86 (t, J = 7.2 Hz, 2H), 3.92 (s, 3H), 3.09 (t, J = 7.2 Hz, 2H), 2.21 ppm
1
3
s, 3H). C NMR (176 MHz, CDCl
(
3
) d 206.0, 160.3, 150.3, 133.4, 132.6, 128.9, 128.3, 125.7, 108.3, 52.2, 46.9, 43.5, 30.3 ppm. HR-
+
17 2 3
MS: calc. for [M+H] C15H N O 273.12337 found 273.12343.
1
-(3-Oxobutyl)-3-phenyl-1H-pyrazole-5-carboxylic acid (6b). Methyl 1-(3-oxobutyl)-3-phenyl-1H-pyrazole-5-carboxylate 5b
ꢀ
(
70 mg, 0.26 mmol, 1.0 equiv.) was subjected to NaOH (2% w/v in water, 617 mL, 0.31 mmol, 1.2 equiv.) and stirred at 70 C for
h. The crude was acidified to pH 2 using 1 M HCl and extracted with EtOAc. The combined organic layers were dried over
Na SO , filtered and concentrated under reduced pressure. The product was purified by means of silica gel column chromatography
elute: cyclohexane / EtOAc +2% v/v acetic acid) and obtained as crystalline white solid (58 mg, 0.22 mmol, 87%). R = 0.37
) d 7.80-7.76 (m, 2H), 7.41 (t, J = 7.7 Hz, 2H), 7.36 – 7.31 (m, 1H), 7.22
4
2
4
(
(
f
1
EtOAc +2% v/v acetic acid). H NMR (700 MHz, CDCl
3
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1
3
(
s, 1H), 4.87 (t, J = 7.2 Hz, 2H), 3.12 (t, J = 7.2 Hz, 2H), 2.23 ppm (s, 3H). C NMR (176 MHz, CDCl
+
3
) d 206.2, 162.8, 150.5, 132.7, 132.4,
1
15 2 3
28.9, 128.4, 125.7, 109.6, 46.9, 43.4, 30.3 ppm. HR-MS: calc. for [M+H] C14H N O 259.10772 found 259.10779.
Methyl 4,5,6,7-tetrahydro-2H-indazole-3-carboxylate (4b). 4,5,6,7-Tetrahydro-2H-indazole-3-carboxylate (324 mg, 2.13 mmol, 1.0
ꢀ
equiv.) was dissolved in 7.8 mL methanol and cooled to -10 C before thionyl chloride (278.3 mL, 3.84 mmol, 1.8 equiv.) was added
dropwise to the solution. The reaction was vigorously stirred at room temperature for 5 days. The crude reaction mixture was concen-
trated under reduced pressure. The product was extracted thrice with EtOAc (20 mL) from water (20 mL) and the combined organic
layers were dried over Na
means of silica gel column chromatography (elute: DCM / MeOH) and obtained as white solid (161.7 mg, 0.9 mmol, 42%). R
2
SO
4
and filtered. The filtrate was concentrated under reduced pressure and the residual was purified by
= 0.23
f
1
DCM +4% v/v MeOH). H NMR (500 MHz, CDCl
13
3
) d 3.89 (s, 3H), 2.72 (td, J = 25.9, 6.1 Hz, 4H), 1.84-1.71 ppm (m, 4H). C NMR
(
+
126 MHz, CDCl ) d 162.5, 119.9, 51.8, 22.9, 22.6, 22.2, 21.5 ppm. HR-MS: calc. for [M+H] C H N O 181.09715 found 181.09697.
(
3
9 13 2 2
Methyl 2-(2-oxopropyl)-4,5,6,7-tetrahydro-2H-indazole-3-carboxylate (5c). Methyl-4,5,6,7-tetrahydro-2H-indazol-3-carboxylate
4
b (90.4 mg, 0.5 mmol, 1.0 equiv.) and 1-chloroactone (161.44 mL, 2.01 mmol, 4.0 equiv.) were dissolved in 2.3 mL acetone and
ꢀ
K
2
CO
3
(153.22 mg, 1.1 mmol, 2.2 equiv.) was added. The reaction mixture was vigorously stirred at 60 C for 2 days. The crude re-
action mixture was adjusted to pH 7 with 0.5 M HCl. The solvent was evaporated under reduced pressure. The crude reaction was
extracted thrice with EtOAc (3x 50 mL) from water (50 mL) and the combined organic layers were dried over Na SO and filtered. The
filtrate was evaporated under reduced pressure and purified by means of silica gel column chromatography (elute: DCM / MeOH).
2
4
The isomers were separated using preparative HPLC. The 2-alkylated product was obtained as white solid (24 mg, 0.1 mmol,
1
= 0.66 (DCM +5% v/v MeOH). H NMR (500 MHz, CDCl
2
0%). R
f
3
) d 4.88 (s, 2H), 3.90 (s, 3H), 2.77 (t, J = 6.0 Hz, 2H), 2.45
) d 201.0, 163.1, 141.0, 139.8, 120.7, 58.7,
237.12547 found 237.12337.
13
(
t, J = 6.2 Hz, 2H), 2.14 (s, 3H), 1.84 – 1.74 ppm (m, 4H). C NMR (126 MHz, CDCl
3
+
5
2.0, 26.9, 22.3, 22.0, 21.3, 21.1 ppm. HR-MS: calc. for [M+H] C12
H
17
N
2
O
3
2
-(2-Oxopropyl)-4,5,6,7-tetrahydro-2H-indazole-3-carboxylate (6c). Methyl 2-(2-oxopropyl)-4,5,6,7-tetrahydro-2H-indazole-3-
carboxylate 5c (24 mg, 0.1 mmol, 1.0 equiv.) was solved in 0.8 mL water and NaOH (4.9 mg, 0.12 mmol, 1.2 equiv.) was added
ꢀ
subsequently to the solution. The reaction mixture was stirred at 70 C for 5 h. The crude was acidified to pH 4-5 using 1 M HCl
and extracted thrice with EtOAc (3x 50 mL) from water (50 mL). The combined organic layers were dried over Na
2
SO
4
, filtered
= 0.56
CN) d 5.23 (s, 2H), 4.44 (s, 1H), 2.71 (t, J= 6.1 Hz, 2H), 2.62 – 2.57 (m, 2H), 2.13
s, 3H), 1.82 – 1.70 ppm (m, 4H). C NMR (126 MHz, CD CN) d 203.4, 161.9, 149.5, 129.7, 62.0, 27.3, 23.9, 23.8, 23.8, 23.1 ppm.
223.10772 found 223.1080.
and concentrated under reduced pressure. The product was obtained as white solid (12.2 mg, 0.05 mmol, 54%). R
f
1
(
(
DCM +35% v/v MeOH). H NMR (500 MHz, CD
3
1
3
3
+
15 2 3
HR-MS: calc. for [M+H] C11H N O
3
-(Azidomethyl)-5-methylpyridine. 3-(Chloromethyl)-5-methylpyridine (300 mg, 1.7 mmol, 1.0 equiv.) and sodium azide (470 mg,
.2 mmol, 4.2 equiv.) were dissolved in ACN (6.2 mL). N,N-Diisopropylethylamide (287 mL, 1.7 mmol, 1.0 equiv.), tetrabutylammonium
7
iodide (6 mg, 20 mmol, 0.1 equiv.) and 18-crown-6 (4 mg, 20 mmol, 0.1 equiv.) were added. The reaction mixture was stirred at room
temperature overnight. EtOAc was added to the crude product and the mixture was extracted with saturated NaHCO . The aqueous
layer was extracted twice with EtOAc and the combined organic layers were concentrated under reduced pressure. The product was
obtained as white crystalline solid (238 mg, 1.61 mmol, 95%) and used in the following step without further purification. R = 0.71
) d 8.43 (d, J = 2.0 Hz, 1H), 8.38 (d, J = 2.0 Hz, 1H), 7.47 (m, 1H), 4.35
3
f
1
(
EtOAc / MeOH / TEA = 9:1:0.1). H NMR (500 MHz, CDCl
3
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Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.06.005
1
3
+
(
s, 2H), 2.37 ppm (s, 3H). C NMR (126 MHz, CDCl
149.08217 found 149.08198.
3
) d 150.5, 146.6, 136.4, 133.5, 130.7, 52.2, 18.5 ppm. HR-MS: calc. for [M+H]
7 9 4
C H N
(
5-Methylpyridin-3-yl)methanamine (14a). 3-(Azidomethyl)-5-methylpyridine (244 mg, 1.7 mmol, 1.0 equiv.) was dissolved in 55 mL
EtOAc / MeOH (2:1) and Pd/C (175 mg, 0.17 mmol, 0.1 equiv.) was added. The solution was stirred under inert hydrogen atmosphere
ꢀ
for 1 h. The reaction mixture was filtered over Celite and the filtrate was concentrated under reduced pressure. The residual was
purified by means of silica gel column chromatography (elute: EtOAc / MeOH +1% v/v TEA) and the product was obtained as
1
OH = 50:8:1). H NMR (500 MHz, CDCl
pale yellow oil (108.6 mg, 0.89 mmol, 54%). R
f
= 0.23 (DCM / EtOH / sat.NH
4
3
) d 8.35
13
(
3
d, J = 1.5 Hz, 1H), 8.33 (d, J = 1.5 Hz, 1H), 7.49 – 7.47 (m, 1H), 3.87 (s, 2H), 2.33 ppm (s, 3H). C NMR (126 MHz, CDCl )
+
7 11 2
d 149.0, 146.2, 137.7, 135.6, 133.1, 43.9, 18.5 ppm. HR-MS: m/z calculated for [M+H] C H N 123.09167 found 123.09139.
N-((5-methylpyridin-3-yl)methyl)formamide (13a). (5-Methylpyridin-3-yl)methanamine 14a (100 mg, 0.82 mmol, 1.0 equiv.) was
ꢀ
dissolved in ethyl formate (200 mL, 2.5 mmol, 3.0 equiv.) and the solution was vigorously stirred at 60 C for 24 h. The solvent
was removed under reduced pressure and the residual was purified by means of silica gel column chromatography
1
elute: EtOAc / MeOH +1% v/v TEA). The product was obtained as yellow oil (111 mg, 0.74 mmol, 90%). H NMR (500 MHz,
(
1
) d 8.34 (d, J = 14.4 Hz, 2H), 8.29 (s, 1H), 7.46 (s, 1H), 4.47 (d, J = 6.1 Hz, 2H), 2.33 ppm (s, 3H). C NMR (126 MHz,
3
CDCl
CDCl
3
3
+
) d 161.2, 149.7, 146.3, 136.4, 133.5, 133.0, 39.6, 18.5 ppm. HR-MS: m/z calculated for [M+H]
8 11 2
C H N O 151.08659
found 151.08616.
N-(4-Picolyl)formamide (13b). 4-Picolylamine 14b (305 mL, 3.0 mmol, 1.0 equiv.) was dissolved in ethyl formate (724 mL, 9.0 mmol,
ꢀ
3
equiv.) and the solution was vigorously stirred at 60 C for 24 h. The reaction mixture was evaporated under reduced pressure and
1
the product was obtained as yellow oil (398 mg, 2.9 mmol, 97%). R
DMSO-d ) mixture of rotamers is observed, major rotamer is given; d 8.62 (bs, 1H), 8.50 (d, J = 6.0 Hz, 2H), 8.19 (s, 1H), 7.26
d, J = 6.0 Hz, 2H), 4.33 ppm (d, J = 6.2 Hz, 2H). C NMR (126 MHz, DMSO-d
f
= 0.21 (EtOAc / MeOH / TEA = 90:10:1). H NMR (500 MHz,
6
1
3
(
6
) d 161.5, 149.6, 148.0, 122.2, 39.8 ppm (under solvent
+
7 9 2
signal, determined by HSQC). LC-MS: m/z calculated for [M+H] C H N O 137 found 137.
N-(Pyrimidine-5-ylmethyl)formamide (13c). Prepared according to general procedure 1A with pyrimidine-5-carbaldehyde 12a
(300 mg, 2.8 mmol, 1.0 equiv.), formic acid (838 mL, 22 mmol, 7.9 equiv.) and formamide (1.6 mL, 33 mmol, 11.8 equiv.) under con-
ꢀ
ventional heating at 180 C for 30 min. The product was purified by means of silica gel column chromatography (elute: EtOAc /
MeOH +1% v/v TEA) to obtain the product as colorless oil (41 mg, 0.3 mmol, 10%). R
1
f
= 0.17 (EtOAc / MeOH / TEA = 90:10:1).
H NMR (600 MHz, CDCl ) mixture of rotamers is observed, major rotamer is given; d 9.15 (s, 1H), 8.71 (s, 2H), 8.32 (s, 1H), 6.16
1
bs, 1H), 4.51 ppm (d, J = 6.2 Hz, 2H). C NMR (151 MHz, CDCl ) d 161.3, 158.3, 156.6, 131.5, 37.6 ppm. HR-MS: m/z calculated
3
3
3
(
+
6 8 3
for [M+H] C H N O 138.0662 found 138.06579.
N-(3-Fluorobenzyl)formamide (13d). Prepared according to general procedure 1A with 3-fluorobenzaldehyde 12b (300 mL,
.8 mmol, 1.0 equiv.), formic acid (530 mL, 14 mmol, 5.0 equiv.) and formamide (1.3 mL, 28 mmol, 10.0 equiv.) under conventional
heating. The product was obtained by means of flash silica gel column chromatography (elute: cyclohexane / EtOAc) as pale yellow
2
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1
= 0.27 (cyclohexane / EtOAc = 1:1). H NMR (400 MHz, DMSO-d
oil (244 mg, 1.6 mmol, 56%). R
f
6
) mixture of rotamers is observed,
13
major rotamer is given; d 8.54 (bs, 1H), 8.15 (d, J = 0.8 Hz, 1H), 7.41-7.33 (m, 1H), 7.14 – 7.03 (m, 3H), 4.32 ppm (d, J = 6.2 Hz, 2H).
NMR (101 MHz, DMSO-d ) mixture of rotamers; d 165.0, 163.4, 161.2, 142.1, 142.0, 130.3, 130.3, 123.3, 123.2, 114.0, 113.8, 113.7,
13.5, 40.2 ppm. LC-MS: m/z calculated for [M+H] C
C
6
+
1
8 9
H FNO 154.0 found 154.0.
N-(4-Fluorobenzyl)formamide (13e). Prepared according to general procedure 1A with 4-fluorobenzaldehyde 12c (300 mL,
.8 mmol, 1.0 equiv.), formic acid (530 mL, 14 mmol, 5.0 equiv.) and formamide (1.3 mL, 28 mmol, 10.0 equiv.) under conventional
heating. The product was obtained by means of flash silica gel column chromatography (elute: cyclohexane / EtOAc) as white
2
1
crystalline solid (254 mg, 1.7 mmol, 59%). R
f 6
= 0.23 (cyclohexane / EtOAc = 1:1). H NMR (400 MHz, DMSO-d ) mixture of rotamers
13
is observed, major rotamer is given; d 8.50 (bs, 1H), 8.12 (s, 1H), 7.34-7.25 (m, 2H), 7.21-7.10 (m, 2H), 4.28 ppm (d, J = 6.2 Hz, 2H).
+
C
NMR (101 MHz, DMSO-d
54.0.
3 8 9
) d 161.1, 129.3, 129.3, 115.2, 115.0, 40.0 ppm. LC-MS: m/z calculated for [M+H] C H FNO 154.0 found
1
N-(4-Methoxybenzyl)formamide (13f). Prepared according to general procedure 1A using 4-methoxybenzaldehyde 12d (44.6 mL,
.37 mmol, 1.0 equiv.), formic acid (72.9 mL, 1.84 mmol, 5.0 equiv.) and formamide (731.9 mL, 18.36 mmol, 50.0 equiv.) using
microwave irradiation. The product was purified by means of flash silica gel column chromatography (elute: cyclohexane / EtOAc)
0
1
and obtained as off-white solid (29.9 mg, 0.18 mmol, 49%). R
f 3
= 0.24 (cyclohexane / EtOAc 1:3). H NMR (400 MHz, CDCl ) mixture
of rotamers, only major rotamer given; d 8.24 (s, 1H), 7.22 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 5.75 (bs, 1H), 4.42 (d, J = 5.8 Hz,
1
3
2
5
3
H), 3.80 ppm (s, 3H). C NMR (100 MHz, CDCl ) mixture of rotamers, only major rotamer given; d 160.9, 159.4, 129.4, 128.5, 114.3,
+
5.5, 41.9 ppm. MS-EI: m/z (%): 165 (100) [M] , 136 (56), 121 (85), 109 (20), 91 (18), 77 (26).
N-(Furan-2-ylmethyl)formamide (13g). Prepared according to general procedure 1A with furfural 12e (300 mL, 3.6 mmol, 1.0 equiv.),
formic acid (683 mL, 18 mmol, 5.0 equiv.) and formamide (1.7 mL, 36 mmol, 10.0 equiv.) under conventional heating. The product
was obtained by means of flash silica gel column chromatography (elute: cyclohexane / EtOAc) as brown oil (164 mg, 1.3 mmol,
1
3
6%). R
f
= 0.11 (cyclohexane / EtOAc = 1:1). H NMR (600 MHz, DMSO-d
6
) mixture of rotamers is observed, major rotamer is given;
13
d 8.47 (bs, 1H), 8.06 (s, 1H), 7.62-7.55 (m, 1H), 6.39 (dd, J = 3.0, 1.9 Hz, 1H), 6.25 (d, J = 3.2 Hz, 1H), 4.28 ppm (d, J = 5.8 Hz, 2H).
+
C
NMR (151 MHz, DMSO-d
20), 53 (20).
6
) d 160.9, 151.8, 142.2, 110.5, 107.0, 34.0 ppm. MS-EI: m/z (%): 125 (100) [M] , 108 (5), 96 (50), 81 (48), 68
(
N-(Propargyl)formamide (13h). Propargylamine 14c (500 mL, 9.1 mmol, 1.0 equiv.) and formamide (360 mL, 9.1 mmol, 1.0 equiv.)
were dissolved in toluene (8 mL) and hydroxylamine hydrochloride (63 mg, 0.9 mmol, 0.1 equiv.) was added. The reaction mixture
was stirred to reflux for 24 h. The reaction mixture was allowed to cool down to room temperature and concentrated under reduced
pressure. The crude reaction was extracted with DCM and the organic layer was washed twice with water. The combined
organic layers were dried over Na
2
SO
4
, filtered and concentrated under reduced pressure. The product was obtained by means
= 0.28
3
cyclohexane / EtOAc = 1:1). H NMR (500 MHz, CDCl ) mixture of rotamers is observed, major rotamer is given; d 8.19 (s, 1H),
of silica gel column chromatography (elute: cyclohexane / EtOAc) as white crystalline solid (367 mg, 4.4 mmol, 48%). R
f
1
(
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13
5
3
.86 (bs, 1H), 4.10 (ddd, J = 5.4, 2.6, 0.8 Hz, 2H), 2.26 ppm (t, J = 2.6 Hz, 1H). C NMR (126 MHz, CDCl ) d 160.7, 78.9, 72.1,
2
8.0 ppm.
N-((1-Methyl-1H-imidazol-5-yl)methyl)formamide (13i). (1-Methyl-1H-imidazol-5-yl)methanamine 14d (233 mg, 2.1 mmol, 1.0
equiv.) was dissolved in ethyl formate (505 mL, 6.3 mmol, 3.0 equiv.) and the solution was transferred into a sealed glass vial. The
ꢀ
reaction was vigorously stirred at 60 C for 24 h. After completion of the reaction, the reaction mixture was evaporated under reduced
pressure and purified by means of silica gel column chromatography (EtOAc / MeOH). The product was obtained as colorless oil
1
= 0.16 (EtOAc / MeOH / TEA = 90:10:1). H NMR (700 MHz, DMSO-d
(
252 mg, 1.81 mmol, 86%). R
f
6
) mixture of rotamers is observed,
13
major rotamer is given; d 8.36 (bs, 1H), 8.04 (s, 1H), 7.53 (s, 1H), 6.78 (s, 1H), 4.28 (d, J = 5.7 Hz, 2H), 3.55 ppm (s, 3H). C NMR
+
(
6 6 10 3
176 MHz, DMSO-d ) d 160.8, 138.4, 128.6, 127.6, 30.9, 30.5 ppm. LC-MS: m/z calculated for [M+H] C H N O 140; found, 140.
N-Benzyl-6-methyl-5-(4-(methylthio)benzyl)-4-oxo-2-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-6-carboxamide (2a). Pre-
pared according to general procedure 2 using 1-(2-oxopropyl)-3-phenyl-1H-pyrazole-5-carboxylic acid 6a (200.0 mg, 0.82 mmol,
1.0 equiv.), 4-(methylthio)benzylamine 9e (144.1 mL, 0.82 mmol, 1.0 equiv.) and benzylisocyanide 8k (99.7 mL, 0.82 mmol, 1.0 equiv.).
The product was obtained as white solid (382.4 mg, 0.77 mmol, 94%). Enantiomers were separated by means of chiral preparative
ꢀ
ꢀ
HPLC. Optical rotation after enantiomer separation: R -0.250 ; S +0.258 . Crystals for X-ray structure determination were grown in
1
Et
.35 (t, J = 7.4 Hz, 1H), 7.27 (d, J = 7.9 Hz, 1H), 7.16 (d, J = 8.3 Hz, 2H), 7.12 – 7.08 (m, 3H), 7.06 (s, 1H), 6.78 (d, J = 6.4 Hz, 2H), 6.31
s, 1H), 5.15 (d, J = 15.5 Hz, 1H), 5.06 (d, J = 13.0 Hz, 1H), 4.61 (d, J = 15.5 Hz, 1H), 4.37 (dd, J = 14.9, 6.7 Hz, 1H), 4.19 (d, J = 13.0 Hz,
2 f 3
O / DCM (5:1). R = 0.45 (cyclohexane / EtOAc = 1:1). H NMR (500 MHz, CDCl ) d 7.77 (d, J = 7.4 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H),
7
(
13
1
H), 4.01 (dd, J = 14.9, 5.0 Hz, 1H), 2.45 (s, 3H), 1.70 ppm (s, 3H). C NMR (126 MHz, CDCl
3
) d 170.7, 159.1, 152.4, 138.5, 137.1,
1
34.6, 134.1, 132.3, 128.9, 128.7, 128.5, 128.2, 127.6, 127.1, 127.0, 125.8, 105.4, 66.0, 55.8, 45.7, 44.1, 22.0, 15.8 ppm. HR-MS:
+
29 4 2
calc. for [M+H] C29H N O S 497.2006 found 497.2002.
N-Benzyl-5-(4-(diethylamino)benzyl)-6-methyl-4-oxo-2-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-6-carboxamide
Prepared according to general procedure 2 using 1-(2-oxopropyl)-3-phenyl-1H-pyrazole-5-carboxylic acid 6a (20.0 mg,
.08 mmol, 1.0 equiv.), (4-aminomethyl)-N,N-diethylaniline 9c (13.3 mL, 0.07 mmol, 0.9 equiv.) and benzyl isocyanide 8k (10.0 mL,
(2b).
0
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0
.08 mmol, 1.0 equiv.). The product was obtained as light yellow solid (11.5 mg, 0.02 mmol, 27%). R
1
:1). H NMR (500 MHz, CDCl
f
= 0.42 (cyclohexane / EtOAc =
1
3
) d 7.80 (d, J = 7.4 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.34 (t, J = 7.4 Hz, 1H), 7.28 (s, 1H), 7.13 – 7.02
(
m, 4H), 6.70 (d, J = 7.0 Hz, 2H), 6.55 – 6.56 (m, 2H), 6.20 – 6.13 (m, 1H), 5.10 (d, J = 12.8 Hz, 1H), 4.95 (d, J = 15.0 Hz, 1H), 4.70
d, J = 15.0 Hz, 1H), 4.32 (dd, J = 15.0, 7.0 Hz, 1H), 4.17 (d, J = 12.8 Hz, 1H), 3.80 (dd, J = 15.0 Hz, 1H), 3.31 (q, J = 7.0 Hz, 4H),
(
1
3
.80 (s, 3H), 1.13 ppm (t, J = 7.0 Hz, 6H). C NMR (126 MHz, CDCl
1
3
) d 171.2, 159.1, 152.1, 147.5, 137.2, 134.5, 132.5, 129.8,
1
28.9, 128.6, 128.4, 127.4, 127.0, 125.8, 123.8, 112.1, 105.2, 65.9, 55.9, 45.4, 44.4, 44.0, 21.8, 12.6 ppm. HR-MS: calc. for
+
+
[
36 5 2 35 5 2
M+H] C32H N O 522.2864 found 522.2873, calc. for [M+Na] C32H N O Na 544.2694 found 544.2694.
N-Benzyl-5-(4-(dimethylamino)benzyl)-6-methyl-4-oxo-2-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-6-carboxamide
(2c).
Prepared according to the general procedure 2 using 1-(2-oxopropyl)-3-phenyl-1H-pyrazole-5-carboxylic acid 6a (36.7 mg,
0
.15 mmol, 1.0 equiv.), (4-aminomethyl)-N,N-dimethylaniline 9b (22.2 mL, 0.15 mmol, 1.0 equiv.) and benzyl isocyanide
k (18.3 mL, 0.15 mmol, 1.0 equiv.). The product was obtained as light-yellow solid (44.4 mg, 0.09 mmol, 60%). R = 0.38 (cyclo-
) d 7.81 – 7.79 (m, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.29
d, J = 8.6 Hz, 2H), 7.14 – 7.03 (m, 5H), 6.72 (d, J = 7.1 Hz, 2H), 6.61 (bs, 1H), 6.14 (t, J = 5.4 Hz, 1H), 5.10 (d, J = 12.8 Hz, 1H),
8
f
1
hexane / EtOAc = 1:1). H NMR (500 MHz, CDCl
3
(
4
4
1
.91 (d, J = 14.7 Hz, 1H), 4.77 (d, J = 14.7 Hz, 1H), 4.28 (dd, J = 14.9, 7.0 Hz, 1H), 4.17 (d, J = 12.8 Hz, 1H), 3.88 (dd, J = 14.9,
1
.6 Hz, 1H), 2.92 (s, 6H), 1.78 ppm (s, 3H). C NMR (126 MHz, CDCl
3
3
) d 171.1, 159.1, 152.2, 137.2, 134.4, 132.4, 129.4, 128.9,
+
28.6, 128.4, 127.4, 127.1, 125.8, 112.9, 105.2, 77.4, 65.9, 60.6, 55.9, 45.4, 44.1, 40.6, 21.8 ppm. HR-MS: calc. for [M+H]
+
30 32 5 2 31 5 2
C H N O 494.2551 found 494.2561, calc. for [M+Na] C30H N O Na 516.2370 found 516.2382.
N-Benzyl-5-(4-isopropoxybenzyl)-6-methyl-4-oxo-2-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-6-carboxamide (2e). Pre-
pared according to general procedure 2 using 1-(2-oxopropyl)-3-phenyl-1H-pyrazole-5-carboxylic acid 6a (20.0 mg, 0.08 mmol,
1
.0 equiv.), (4-isopropoxyphenyl)-methanamine 9d (13.7 mL, 0.08 mmol, 1.0 equiv.) and benzyl isocyanide 8k (10.0 mL, 0.08 mmol,
1
1
.0 equiv.). The product was obtained as brown solid (24.9 mg, 0.05 mmol, 60%). R
) d 7.99 (d, J = 7.2 Hz, 2H), 7.71 – 7.45 (m, 5H), 7.31 (d, J = 7.2 Hz, 3H), 6.98 – 7.03 (m, 4H), 6.65 – 6.77 (m, 1H),
.55 – 5.23 (m, 2H), 4.88 (d, J = 15.0 Hz, 1H), 4.73 (hept, J = 6.0 Hz, 1H), 4.59 (d, J = 15.0 Hz, 1H), 4.39 – 4.44 (m, 1H), 4.22
f
= 0.55 (cyclohexane / EtOAc = 1:1). H NMR
(
500 MHz, CDCl
3
5
13
(
3
d, J = 15.0 Hz, 1H), 1.94 (s, 3H), 1.55 ppm (d, J = 6.0 Hz, 6H). C NMR (126 MHz, CDCl ) d 170.9, 159.1, 157.6, 152.2, 137.2,
e13 Cell Chemical Biology 26, 1–15.e1–e25, September 19, 2019

Please cite this article in press as: Reckzeh et al., Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.06.005
1
34.3, 132.3, 129.6, 129.2, 128.9, 128.6, 128.4, 127.5, 127.0, 125.8, 116.3, 105.3, 69.9, 65.9, 55.8, 45.5, 44.0, 22.1, 21.9 ppm.
+
+
33 4 3 32 4 3
HR-MS: calc. for [M+H] C31H N O 509.2547 found 509.2561, calc. for [M+Na] C31H N O Na 531.2367 found 531.2382.
N-Benzyl-5-(4-ethylbenzyl)-6-methyl-4-oxo-2-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-6-carboxamide (2h). Prepared ac-
cording to the general procedure 2 using 1-(2-oxopropyl)-3-phenyl-1H-pyrazole-5-carboxylic acid 6a (50.0 mg, 0.20 mmol,
1
.0 equiv.), (4-ethylphenyl)methanamine 9f (29.2 mL, 0.20 mmol, 1.0 equiv.) and benzyl isocyanide 8k (24.9 mL, 0.20 mmol, 1.0 equiv.).
1
The product was obtained as light-yellow solid (72.8 mg, 0.15 mmol, 74%). R
CDCl ) d 7.96 (d, J = 7.3 Hz, 2H), 7.58 (t, J = 7.3 Hz, 2H), 7.52 (d, J = 7.3 Hz, 1H), 7.49 – 7.41 (m, 2H), 7.33 – 7.23 (m, 5H), 6.93 (d, J =
.1 Hz, 2H), 6.38 (t, J = 5.6 Hz, 1H), 5.24 (dd, J = 15.0, 5.4 Hz, 2H), 4.89 (d, J = 15.0 Hz, 1H), 4.53 (dd, J = 15.0, 5.1 Hz, 1H), 4.37 (d, J =
f
= 0.56 (cyclohexane / EtOAc = 1:1). H NMR (400 MHz,
3
7
1
13
5.0 Hz, 1H), 4.10 (dd, J = 15.0, 5.1 Hz, 1H), 2.78 (q, J = 7.7 Hz, 2H), 1.89 (s, 3H), 1.38 ppm (t, J = 7.7 Hz, 3H). C NMR (101 MHz,
CDCl
5.9, 45.9, 44.1, 28.6, 21.9, 15.6 ppm. HR-MS: calc. for [M+H]
Na 501.2261 found 501.2273.
3
) d 170.9, 159.1, 152.4, 144.2, 137.2, 135.2, 134.3, 132.4, 128.9, 128.8, 128.7, 128.4, 127.8, 127.6, 127.1, 125.9, 105.4, 66.0,
+
+
+
5
C
30
H
31
N
4
O
2
479.2442 found 479.2454, calc. for [M+Na]
30 30 4 2
C H N O
N-Benzyl-N,6-dimethyl-5-(4-(methylthio)benzyl)-4-oxo-2-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-6-carboxamide
(2i).
N-Benzyl-6-methyl-5-(4-(methylthio)benzyl)-4-oxo-2-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-6-carboxamide 2a (11 mg,
ꢀ
2
added and the solution was stirred at 0 C for 10 min. Subsequently, methyl iodide (1.7 mL, 27 mmol, 1.2 equiv.) was added and
2 mmol, 1.0 equiv.) was dissolved in DMF (0.5 mL) and cooled to 0 C. Sodium hydride (1.8 mg, 44 mmol, 2.0 equiv.) was thereto
ꢀ
the solution was allowed to warm up to room temperature overnight. The excess of sodium hydride was quenched by addition of
ꢀ
1 mL water at 0 C. The product was extracted thrice with EtOAc and the combined organic layers were concentrated under reduced
pressure. The product was purified by means of silica gel column chromatography (elute: cyclohexane / EtOAc) and obtained as
1
= 0.67 (cyclohexane / EtOAc = 1:1). H NMR (600 MHz, CDCl
white solid (4 mg, 8 mmol, 35%). R
f
3
) d 7.81 – 7.77 (m, 2H), 7.42
(
(
d, J = 7.2 Hz, 2H), 7.36 – 7.33 (m, 1H), 7.31 – 7.27 (m, 5H), 7.23 (s, 1H), 7.21 – 7.18 (m, 2H), 7.10 (d, J = 7.2 Hz, 2H), 4.77 – 4.67
13
m, 2H), 4.61 (d, J = 15.4 Hz, 2H), 4.36 (d, J = 13.5 Hz, 2H), 2.79 (s, 3H), 2.48 (s, 3H), 1.65 ppm (s, 3H). C NMR (151 MHz,
Cell Chemical Biology 26, 1–15.e1–e25, September 19, 2019 e14