T.S. Mansour, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127288
Table 3
Table 4
Comparative inhibition (in nM) of 4 relative to reference compounds.
Cellular activity of 4 and 5 (nM) in select cells in the NCI-60 Panela.
b
Entry
Kinase
RFBa
CaPBAa
KINOMEscan™
Cell Line
Origin/Disease
4
5
1
2
3
4
5
EGFRWT
EGFRL858R
Her2
2.4/125
419/58.7
121/104
NA
12.3/50.5
18.1/12.6
112/131
NA
NT
GI50
42
TGI
146
208
175
246
263
883
GI50
112
148
93
TGI
283
454
209
608
312
1830
9.2/2.8
NT
CCRF-CEM
K-562
Leukemia/ALLb
Leukemia/CMLc
94
Her3
380
MOLT-4
SR
Leukemia/ALLb
74
Her4
0.03/535
12.2/29.4
6/54
Leukemia/Large cell
NSCLC/Carcinoma
NSCLC/Adenocarcinoma
66
132
128
690
NCI-H460
103
198
aIC50 4/ IC50 staurosporine, Kd 4/Kd lapatinib, NA not available, NT not
b
NCI-H226
tested.
MCF7
Breast/Adenocarcinoma
Breast/Adenocarcinoma
Breast/Adenocarcinoma
Breast/Carcinoma
86
182
237
155
282
204
424
204
251
136
778
91
193
722
MDA-MB-231
MDA-MB-468
HS 578 T
103
70
208
120
512
174
637
98
that quantitatively measures the ability of a compound to compete with
an immobilized, active-site directed ligand. Despite the good potency of
4 against Her4 in these two assays relative to reference compounds, the
potent picomolar inhibition of Her4 seen in the RFB assay was reduced
in these assays (Table 3). Moreover, variations in the degree of
EGFRL858R inhibition between the RFB and the other two assays were
noted (IC50 419 vs 18.1, 9.2 nM) while inhibition studies of EGFRWT
and Her2 (ErbB2) kinases revealed no appreciable differences in the
inhibition profile. Furthermore, 4 inhibited Her3 (ErbB3) with a Kd
value of 380 nM (Table 3). These data highlight the need for care in
comparing kinase assay data between different platforms due to re-
combinant kinases used, inhibition of active versus inactive kinases,
assay conditions, ATP concentrations and format.
385
105
89
1740
455
PC-3
Prostate/Adenocarcinoma
Prostate/Carcinoma
DU-145
HCT-116
M14
134
88
1365
209
Colon/Adenocarcinoma
Malignant melanoma
Malignant melanoma
Anaplastic astrocytoma
83
141
129
404
482
SK-MEL-28
63
350
SF-268
83
1325
aFive dose response curve, each value is an average of 2 experiments; Acute
b
c
interacting with residues in the front pocket, gate area, and back
pocket.18 The difference between the binding mode of lapatinib be-
tween EGFR and Her4 is a flip in the fluorophenyl ring, which alters the
position of the fluorine but does not introduce or break contacts with
residues not found in both EGFR and Her4.18 Likewise, the structure of
neratinib bound to a mutant EGFR (PDB code: 3W2Q, 2.2 Å) shows that
it binds to an inactive form of the enzyme spanning the front pocket,
gate area and back pocket.19 It is possible, based on these pre-
cedents18,19 that 4 engages the inactive form of EGFR/Her4 but the
specific amino acids residues that contact the novel C6 and C7 sub-
stituents remain to be determined.20,21
The clinical benefits of inhibiting both wild type and mutant EGFR
and Her2 are now clearly established.1 Disease prognosis associated
these receptors which impact signaling pathways.9 In several cancers,
the role of Her4 inhibition is now being recognized. In breast cancer
cells, Her4 signaling promotes differentiation and growth inhibition
and loss of Her4 expression is a marker for resistance to tamoxifen.9
Molecular alterations in esophageal and head-and-neck squamous cell
carcinomas cell lines overexpressing Her4 are hypersensitive to afa-
tinib.10 These cell lines harbor an Her4 G1109C mutation which is an
activating oncogenic mutation with a transformational ability and tu-
morigenicity.10 The addiction of cancer cells to activating mutations in
EGFR for proliferation suggests that targeted therapy with Her4 in-
hibitors may have future value in certain breast, esophageal and head-
and-neck squamous cell carcinomas.
Next, the cellular activity of 4 and 5 was evaluated in the NCI-60
panel and reported in Table 4 as GI50 (the concentration of the drug
causing 50% growth inhibition) and TGI (the total growth inhibition)
values in nM. Compound 4 has consistently, albeit marginally better
activity over 5 particularly in a select set of cell lines from the leukemia,
breast, NSCLC and prostate panels. This trend was also noted in colon,
melanoma and CNS lines. The GI50 values range for 4 are between 42
and 198 nM for the 17 cell lines shown in Table 4 and the TGI data
are < 300 nM in all cell lines shown in Table 4 except for three lines
(NCI-H226, DU-145 and SF-268). The LC50 values measuring the cy-
totoxic concentration are > 375 nM resulting in therapeutic indices
(TI = LC50/GI50) of > 4 as listed in Supplementary Table 4.
understanding many aspects of their functional signaling.1,11 Active
ErbB kinases, due to phosphorylation of tyrosine residues, bind ATP
which results in several conformational changes including 1) relative
orientation of N- and C-terminal lobes, 2) disposition of the activation
loop (DFG) 3) orientation of the αC helix and 4) electrostatic switch
where the β3 lysine salt bridge with the DFG-D residue in the inactive
form switches to a salt bridge between the β3-lysine and the αC-glu-
tamate with the concomitant formation of the αC-in conformation. This
knowledge has aided in the design of small molecule inhibitors of ErbB
family targeting certain protein sites. Thus, inhibitors are classified as
type I when they bind within the adenine-binding pocket of an active
protein kinase,12,13 type II inhibitors bind to an inactive kinase with the
DFG-D out motif12,13; type III inhibitors12–14 bind in an allosteric site
between the N-terminal and C-terminal lobes; type IV bind in an al-
losteric site outside of the adenine-binding pocket12,14; type V are bi-
valent inhibitors that span two distinct parts of the protein kinase do-
main15 and type VI are covalent inhibitors.16
Furthermore, based on the data in Table 4, we choose to profile 4 in
assay22 using the ProQinase protocol (ProQinase Reaction Biology,
GmbH, Freiburg, Germany) in female NMRI nude mice with the ob-
jective of selecting one or more cell lines for future xenograft studies.
The study design used 12 animals for vehicle (5% DMSO, 5% DMA,
20% PEG400, 40% PG, 30% PBS) and testing with dosing over 14 days
at 10 mpk. All 12 animals (vehicle and test) exhibited no signs of
toxicity or body weight loss during the 14 day period. Inhibitor 4
(SAB402, NSC code: D-787238) reduced cell viability using CellTiter-
Glo® in the intraperitoneally implanted hollow fibers which have been
loaded with MOLT-4 cells in a statistically very significant manner
(p = 0.0062) when compared to vehicle (Fig 4). Similarly, as shown in
cells (p = 0.0116), whereas the HCT-116 cells group showed no sig-
nificant reduction of cell viability. No substantial effects were observed
in the sc compartment in all three cell lines.
To date there are no structural data of 4 bound to either Her4 or
EGFR. However, since 4 has an anilino substituent similar to that of
lapatinib and a core 3-cyanoquinoline similar to neratinib but different
C6 and C7 substituents, the reported x-ray crystallographic structural
information from lapatinib and neratinib bound to EGFR and Her4 are
relevant to 4. Lapatinib inhibits Her4 with an IC50 of 36717 or
1400 nM.18 It binds to the inactive form of Her4 (PDB code: 3BBT,
2.8 Å) and EGFR (PDB code: 1XKK, 2.4 Å) in the same binding site
In conclusion, we have developed a synthetic protocol to 2-(1,2-
dithiolan-4-yl)acetic acid
8 for use to prepare target novel 3-
4