E.J. Rastelli et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128167
the crude photooxygenation material through a short plug of SiO
2
, the
O
a,b
c,d
filtrate was stirred in a methanolic suspension of NH
4
OAc in a sealed
2
, pure imine 7a
3
N
NHBoc
◦
89%
H
3
70%
tube at 60 C. After re-filtration through a bed of SiO
could be isolated in moderate yield over 3 steps.
13
The corresponding ester analog (8) was prepared following a similar
protocol but starting with a DCC-mediated esterification between 3 and
O
O
,4-butanediol.20 Notably, the synthesis of 8 did not require the NH
e
1
4
OAc
O
N
H
N
H
3
55%
imination step, as chromatographic separation of the crude imine/trione
mixture after the in-flow photooxygenation provided pure imine 8.
Additionally, a zone 3 N-alkylated analog (9) was synthesized in which
the tethered adamantyl group was attached to the imide nitrogen of
JMS-053.20
1
4
Bpin
O
O
O
N
H
N
H
In addition to the ether linkages in 7 and 8, we were also interested in
conjugating JMS-053 with a more polar 2-hydroxyacetamide to the
adamantane residue. This modification broadened the scope of our
bifunctional analogs by: (1) allowing access to the wide variety of po-
tential diamine linkers, and (2) avoiding the Mitsunobu reaction con-
ditions which required multiple rounds of chromatography to purify the
desired products from the excess of DIAD and its hydrazodicarboxylate
byproduct. Therefore, phenol 10 was first alkylated with ethyl bro-
moacetate. While this reaction was high yielding, generating the car-
boxylic acid without hydrolyzing the boronic ester proved to be difficult.
Instead, phenol 10 was alkylated with tert-butyl bromoacetate. The tert-
3
f,g,h
1%
O
NH
15
4
S
2
O N
O
O
O
N
N
3
H
H
O
1
6
S
NH
O
2 2
butyl group was then cleaved by stirring in a solution of TFA in CH Cl ,
providing acid 12 in 71% yield over 2 steps.20
HN
Starting with N-Boc protected 1,3-diaminopropane, amide coupling
with the acid chloride derived from 3 provided compound 13 in excel-
lent yield (Scheme 2). The Boc group in 13 was removed with TFA, and
the resulting primary amine was acylated with acid 12. Using our
optimized Suzuki conditions, cross coupling of boronic ester 14 and
thiophene bromide 11 cleanly generated intermediate 15 without the
need for chromatography. Finally, nitro compound 15 was transformed
into conjugate 16 by catalytic hydrogenation of the nitro group, in-flow
photooxygenation of the resulting enamine, and imination of the imine/
2
HO C
O
Bpin
12
O
O
O
O
N
N
H
2
H
O
NH
17
S
HN
O
4
trione mixture with NH OAc. Following the same synthetic sequence,
conjugate 17 was generated using 1,2-bis(2-aminoethoxy)ethane as the
◦
2
, 0 C
Scheme 2. a) SOCl
to rt; c) TFA/CH Cl
PPh , K CO , dioxane/H
2
, reflux; b) N-Boc-1,3-diaminopropane, Et
3
N, CH
2
Cl
diamine starting material.
◦
◦
2
2
, 50 C; d) 12, HATU, i-Pr
O (5:1), reflux; f) H
2
NEt, THF, 0 C to rt; e) 11, Pd
We next examined the bifunctional PTP4A3 inhibitors in
a
(
3
)
4
2
3
2
2
, Pd/C, dioxane/MeOH (2:1);
biochemical enzyme assay (Table 1). While less potent than the parent
monofunctional compound, JMS-053 (Entry 7, IC50 = 84 nM) in these
assays,7 the conjugates retained different abilities to inhibit PTP4A3
enzymatic activity in vitro, with 2-hydroxyacetamide analogs 16 and 17
◦
g) air, 425 nm-LED, MeOH, in flow; h) NH
4
OAc, MeOH, 60 C.
b
Table 1
a
(
Entries 5 and 6, IC50 = 100–200 nM) showing substantially higher
potency than the aliphatic ether linked analogs 7 and 8 (Entries 1–3,
M). These significant differences in potency support our
In vitro inhibition of PTP4A3 phosphatase activity.
Entry
Compound
IC50
(μM)
±SEM
N
IC50 = 1–7
μ
1
2
3
4
5
6
7
7a (EJR-876-35)
7b (EJR-925-45)
8 (EJR-876-34)
9 (EJR-980-67)
16 (EJR-887-24)
17 (EJR-887-35)
1 (JMS-053)
1.16
6.59
4.98
1.92
0.51
1.99
0.31
6
3
3
1
3
3
6
assumption that linker length and composition are critical features of
bifunctional conjugates. Interestingly, conjugate 7b containing a trione
motif instead of the imine functionality was ~ 6-fold less potent than 7a.
This result confirms that the imino-thienopyridinedione motif remains
important for the PTP4A3 inhibitory interaction in the bifunctional
compounds. In agreement with the data for JMS-053 analogs with large
0.206
0.107
0.084
0.011
0.016
0.041
7
b
a
substituents at the imide nitrogen in zone 3, conjugate 9 also lost
substantial inhibitory activity (Entry 4).
Recombinant human PTP4A3 phosphatase was used and the enzymatic assay
7
was performed as previously described. See SI for additional details.
Although Ser/Thr kinases and phosphatases are more commonly
associated with the UPR,21 a mechanistic understanding of the links
transcription factor that initiates BiP synthesis25 in MDA-MB-231 breast
cancer cells is consistent with known links between PTP inhibition and
1
0
between PTPs and ER stress and cell death has also been emerging. For
2
2
example, PTP1B regulates the UPR, and PTPN2 mediates ER stress
response-dependent neuronal damage.23 While the adamantyl moiety in
bifunctional compounds should induce a basal level of ER stress, we
were most interested in PTP4A inhibitors that potentiated this effect.
Therefore, we evaluated the ability of the most active bifunctional in-
hibitors 7a, 16, and 17 to induce ER stress, as well as diminish cancer
cell viability. Dithiothreitol (DTT), a potent UPR activator, served as a
positive control in the ER stress measurements (Fig. 2). Notably, com-
pound 7a-dependent induction of the canonical UPR reporter, BiP
2
6
cancer cell death (Fig. 2). In fact, MDA-MB-231 cells also express high
2
7
levels of p53, a key survival enzyme which in turn regulates PTP4A3
levels.28 BiP induction, concomitant with a strongly enhanced expres-
sion of the pro-apoptotic gene, CHOP (DDIT3),29 provides a mechanism
to explain the prominent effect of 7a on MDA-MB-231 viability (Fig. 3
and Table 2, Entry 3; EC50 ~ 8.6 M). Interestingly, analog 7a was
μ
significantly more potent in these cell-based assays than 16 and 17, even
though it was 6–10 times less potent in the biochemical PTP4A3 enzyme
assay. The monofunctional adamantyl linker 4 served as a negative
(
HSPS5A),24 along with a moderate accumulation of the spliced Xbp1
3