Tetrahydroimidazo[1,2-a]pyrazine Derivatives: Synthesis and Evaluation as Gαq-Protein Ligands

Article abstract of DOI:10.1002/chem.202001446

The 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine derivative BIM-46174 and its dimeric form BIM-46187 (1) are heterocyclized dipeptides that belong to the very few cell-permeable compounds known to preferentially silence Gα_q proteins. To explore the chemical space of Gα_q inhibitors of the BIM chemotype, a combinatorial approach was conducted towards a library of BIM molecules. This library was evaluated in a second messenger-based fluorescence assay to analyze the activity of Gα_q proteins through the determination of intracellular myo-inositol 1-phosphate. Structure–activity relationships were deduced and structural requirements for biological activity obtained, which were (i) a redox reactive thiol/disulfane substructure, (ii) an N-terminal basic amino group, (iii) a cyclohexylalanine moiety, and (iv) a bicyclic skeleton. Active compounds exhibited cellular toxicity, which was investigated in detail for the prototypical inhibitor 1. This compound affects the structural cytoskeletal dynamics in a Gα_q/11-independent manner.

Full text of DOI:10.1002/chem.202001446

Accepted Article
Accepted Article
Title: Tetrahydroimidazo[1,2-a]pyrazine Derivatives: Synthesis and
Evaluation As Gαq-Protein Ligands
Authors: Michael Gütschow, Jim Küppers, Tobias Benkel, Suvi Annala,
Kenichi Kimura, Lisa Reinelt, Bernd K. Fleischmann, and Evi
Kostenis
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.202001446
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01/2020

10.1002/chem.202001446
Chemistry - A European Journal
FULL PAPER
Tetrahydroimidazo[1,2-a]pyrazine Derivatives: Synthesis and
Evaluation As Gαq-Protein Ligands
Jim Küppers+,[a] Tobias Benkel+,[b,c] Suvi Annala,[b] Kenichi Kimura,[d] Lisa Reinelt,[a] Bernd K.
Fleischmann,[d] Evi Kostenis,*[b] and Michael Gütschow*[a]
[a]
[b]
J. Küppers,+ L. Reinelt, Prof. Dr. M. Gütschow
Pharmaceutical Institute, Department of Pharmaceutical & Medicinal Chemistry
University of Bonn
An der Immenburg 4, 53121 Bonn (Germany)
E-mail: guetschow@uni-bonn.de
T. Benkel,+ S. Annala, Prof. Dr. E. Kostenis
Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology
University of Bonn
Nussallee 6, 53115 Bonn (Germany)
E-mail: kostenis@uni-bonn.de
[c]
[d]
Research Training Group 1873, University of Bonn, Germany
K. Kimura, Prof. Dr. B. K. Fleischmann
Institute of Physiology I, Life and Brain Center, Medical Faculty
University of Bonn
Sigmund-Freud-Str. 25, 53105 Bonn (Germany)
[+]
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the document.
Abstract: The 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine derivative
BIM-46174 and its dimeric form BIM-46187 (1) are heterocyclized
dipeptides that belong to the very few cell-permeable compounds
known to preferentially silence Gαq proteins. To explore the chemical
space of Gαq inhibitors of the BIM chemotype, a combinatorial
approach was conducted towards a library of BIM molecules. This
library was evaluated in a second messenger-based fluorescence
assay to analyze the activity of Gαq proteins through the
determination of intracellular myo-inositol 1-phosphate. Structure-
activity relationships were deduced and structural requirements for
one specific receptor, the manipulation of an individual GPCR is
suitable. However, in case of complex disorders, such as cancer
or pain, which involve multiple receptors and their associated
pathways, the interference at the post-receptor level appears to
be particularly promising and the intracellular G proteins can
thus be envisaged to serve as potential drug targets.[6,7]
Furthermore, the direct influence on specific G proteins may
constitute a useful pharmacological strategy to unravel their role
under physiological and pathophysiological conditions.
According to the amino acid sequence homology of the Gα
subunits, heterotrimeric G proteins are subdivided into four
families, Gαs, Gαi/o, Gαq and Gα12/13. The Gαq family comprises
four members, i.e. Gαq, Gα11, Gα14 and Gα15/16, among which the
isoforms Gαq and Gα11 are most crucial and ubiquitously
expressed.[8,9] In their customary role, Gαq family members
activate their major downstream effector, the enzyme
phospholipase C-β (PLCβ), leading to hydrolysis of membrane-
biological activity obtained, which were (i)
a redoxreactive
thiol/disulfane substructure, (ii) an N-terminal basic amino group, (iii)
a cyclohexylalanine moiety, and (iv) a bicyclic skeleton. Active
compounds exhibited cellular toxicity, which was investigated in
detail for the prototypical inhibitor 1. This compound affects the
structural cytoskeletal dynamics in a Gαq/11-independent manner.
bound
phosphatidylinositol-4,5-bisphosphate
(PIP2)
into
diacylglycerol (DAG), which in turn activates protein kinase C,
and into myo-inositol 1,4,5-trisphosphate (IP3), which initiates
the release of calcium ions from the endoplasmic or
sarcoplasmic reticulum into the cytosol by opening IP3-sensitive
calcium channels.[7,8]
Introduction
G protein-coupled receptors (GPCRs), also known as seven-
transmembrane receptors, represent the largest family of cell-
surface receptors in eukaroytes.[1] GPCR activation initiates a
plethora of intracellular multistep signaling events.[2] About one
third of prescription drugs on the market target GPCRs either as
agonists or antagonists.[3] Upon agonist binding, GPCRs
undergo conformational changes resulting in the activation of
receptor-associated heterotrimeric guanine nucleotide-binding
proteins (G proteins), which consist of three different subunits
referred to as α, β and γ.[4] The capability of acting as molecular
switches, thus transducing extracellular signals via GPCRs into
intracellular signal cascades, makes heterotrimeric G proteins
vitally important.[5] So far, GPCRs rather than their associated G
proteins have been targeted in contemporary drug development.
For pathologies, which are characterized by the dysregulation of
The modulation of the Gαq protein subfamily is of particular
relevance, since its members account for a wide variety of
cellular responses,[7,10] leading to important physiological
functions, such as platelet aggregation,[11] insulin-stimulated
glucose
transport,[12]
as
well
as
pathophysiological
consequences, such as heart failure,[13] and cancer.[6,9,14] Hence,
selective and potent modulators are highly required as tool
compounds to investigate G protein-mediated signaling or as
feasible drug candidates. Prominent modulators of Gαq proteins
include, on the one hand, isolated natural products, YM-254890
and FR900359, and on the other hand, molecules gained from
organic syntheses, the BIM molecules (BIM-46174 and BIM-
1
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10.1002/chem.202001446
Chemistry - A European Journal
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46187) and 27-mer(I860A), all of which represent selective Results and Discussion
inhibitors for Gαq.[7] Among these, the cyclic depsipeptides YM-
254890 and FR900359, obtained from the fermentation broth of
Chromobacterium sp. QS3666 or from the leaves of the plant
Ardisia crenata sims, respectively, exhibit extraordinary
selectivity and receive exceptional attention.[6-8,15-16] The linear
peptide 27-mer(I860A), derived from the phospholipase C-β
isoform PLC-β3, has also been identified as a selective Gαq
inhibitor, however its molecular mechanism of action still
remains unclear.[7,17] The two BIM molecules (Chart 1) have
been previously reported as pan-G protein inhibitors,
corresponding to their ability to likewise inhibit all of the four G-
protein families.[15,18] This conclusion relied on the compounds’
inhibitory interference with two second messenger pathways, the
cyclic adenosine-3',5'-monophosphate (cAMP) and the myo-
inositol 1-phosphate (IP1) pathway, as evaluated in human
breast cancer MCF7 and melanoma A2058 cell lines,
respectively.[15,18] Furthermore, both molecules potently inhibited
critical functions in cancer progression, in particular cell
proliferation and cell survival.[18,19] A detailed evaluation of BIM
action on various GPCR/G protein pairs, co-expressed in
different cellular backgrounds such as Chinese hamster ovary
(CHO), human embryonic kidney (HEK) 293, and CV-1 in origin
with SV40 genes (COS) 7 cells revealed preferential silencing of
Gαq signaling in a cellular context-dependent manner.[19] Along
the activation pathway, BIM allows GDP release, but prevents
GTP entry, thus trapping Gαq in the nucleotide-free, empty
pocket conformation.[19] The dimeric BIM-46187 has often been
experimentally employed,[20] particularly in order to take
advantage of its preferred Gαq inhibition.[21]
Chemistry. The structure of the BIM monomer can be
rationalized as a heterocyclized dipeptide emerged from L-
cysteine and L-cyclohexylalanine. The C-terminal carboxyl group
is replaced by a 4-phenyl-substituted imidazole in which the N-1
nitrogen is bridged to the peptidic N atom via an ethylene linker,
resulting in a 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine core. This
scaffold enables a combinatorial approach by either modifying
the heterobicyclic structure or introducing various substituents at
different points of diversity. The synthesis (Scheme 1) started
with the esterification of different D- or L-configured, N-protected
amino acids
(bromoacetyl)pyridine. The resulting α-acyloxy ketones B were
subjected to Davidson-type heterocondensation upon
treatment with ammonia in refluxing toluene. 2,4-Disubstituted
imidazoles were reacted with ethyl bromoacetate; the
A
with either phenacyl bromide or 3-
a
C
regioselectivity of this alkylation to occur at N-1 has been
recently confirmed by X-ray crystallography.[22] Cbz-protected
intermediates D, after hydrogenolytic deprotection, underwent
an instantaneous entropy-driven lactamization to E. The Boc
protecting group of other representatives of type D was removed
under acidic conditions and ring closure to E occurred at room
temperature upon deprotonation of the ammonium group. The
further conversion was accomplished by a borane-promoted
reduction, resulting in relatively stable amine-borane adducts,
which could not completely be decomplexed with methanol
alone, but in the presence of palladium,[23] leading to the desired
set of 10 secondary amines F.
R2
R2
O
a
b
PG
OH
PG
O
N
H
N
H
O
O
O
O
H2N
HS
H2N
N
N
Z
N
N
A
B
N
N
S
2
R2
R2
BIM-46174
BIM-46187 (1)
PG
PG
N
N
c
N
H
N
H
HN
N
O
Chart 1. Structures of the monomeric and dimeric BIM molecules.
Z
Z
C
D
O
R2
R2
Despite the apparent usability of BIM-46187, systematic
medicinal chemistry approaches are lacking. Just one recent
study on BIM fragments demonstrated that distinct structural
reductions were incompatible with a Gαq inhibitory activity.[22]
Therefore, we introduced several points of diversity into the full-
size BIM structure and envisaged a combinatorial approach
towards an extended series of derivatives of BIM-46187 (1). We
devised a variety of BIM dimer structural analogs, which are
expected to exhibit similar redox behavior. Hence, their activity
in a cellular environment should be comparable. Moreover, the
thiol function of the BIM monomer was exchanged for other
groups or converted to prodrug forms. This series of designed
compounds, none of which bearing a free thiol group, was
synthesized by means of combinatorial chemistry and subjected
to biological investigations on Gαq protein inhibition. We
attempted to draw structure-activity relationships, define the BIM
pharmacophore and explore the chemical space of Gαq
inhibitors of the BIM chemotype.
N
N
d
g,h
HN
HN
N
or e,f
N
Z
Z
O
E
F
Scheme 1. Synthesis of 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazines F.
Reagents and conditions: (a) 2-bromoacetophenone (Z CH) or
=
3-(bromoacetyl)pyridine hydrobromide (Z = N), K2CO3, abs. DMF, rt, 4 h,
66-98%; (b) NH4OAc, abs. toluene, reflux, 3 h, 69-91%; (c) ethyl bromoacetate,
Cs2CO3, abs. DMF, rt, 3.5 h, 60-96%; (d) Pd/C, H2 (1 atm), abs. MeOH, rt, 24
h (PG = Cbz), 18-99%; (e) TFA, abs. CH2Cl2, rt, 2 h; (f) Et3N, CH2Cl2, rt, 2 h
(PG = Boc); (g) BH3  THF, abs. THF, 90 °C, 48 h, Ar; (h) Pd/C, abs. MeOH, rt,
16 h, Ar; 53-93%. PG = protecting group.
Two representatives of this set, i.e. the (S)- and (R)-
configured cyclohexylalanine derivatives G (Scheme 2) were
employed for the synthesis of BIM-46187 (1),[19] its enantiomer 4
and its diastereomers 2 and 3. The carbodiimide-mediated
amide coupling introduced the cysteine unit in H. Both the tert-
butyloxycarbonyl
and
trityl
group
were
removed
2
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contemporaneously under acidic conditions in the presence of
excess triisopropylsilane, a hydride donor, acting as a scavenger
of the trityl, and also the tert-butyl cation.[24] The intermediate
thiols were oxidized with iodine, and stereoisomers 1-4 were
purified by column chromatography, then dissolved in ethyl
acetate and precipitated as hydrochlorides.
O
H
2
N
N
N
N
a
HN
Boc
N
Y
N
Trt-S
K
J
O
O
H
8'
8'
H2N
S
2
9
2R, Y = C(CH3)2
2
N
N
N
N
b, c
N
a
HN
Boc
N
10 2S, Y = C(CH3)2
11
Y
N
2S, Y = CH2CH2
N
N
Trt-S
2
G
H
Scheme 4. Synthesis of BIM analogs 9-11. Reagents and conditions: (a) Boc-
L-Pen(Trt)-OH or Boc-D-Pen(Trt)-OH or Boc-L-Hcy(Trt)-OH, DCC, DIPEA, abs.
CH2Cl2, rt, 48.5 h, Ar, 35-41%; (b) TFA, (i-Pr)3SiH, abs. CH2Cl2, rt, 18 h, Ar; (c)
I2, MeOH, H2O, rt, 2 h, 5-38%.
O
8'
H2N
S
2
N
1
2
3
4
2R, 8'S
b,c
N
2S, 8'S
2R, 8'R
2S, 8'R
N
2
The starting compound
J was also used to prepare
intermediates L which were subsequently Boc-deprotected to
the final compounds 12-18 (Scheme 5). To obtain 13 from the
Boc-Trt-protected precursor, we added triisopropylsilane to
facilitate the liberation of alcoholic group. In the other cases, the
same acidic conditions were applied in the final step. The route
to the 1,2-diaminopropionate (H-Dap-OH) derivative 14 involved
the -amino protection of Boc-L-Dap-OH, followed by the
coupling step and the simultaneous release of both amino
groups. To furnish 15, L-cysteic acid was initially N-Boc-
protected, then subjected to coupling conditions in which the
free sulfonyl group remained unaffected, and finally deprotected.
Products 12-16 can be considered as analogs of the BIM
monomer, in which the thiol function is exchanged for a methyl,
hydroxy, amino, sulfo, and methylthiomethyl group. Analogs 17
and 18 are BIM-prodrugs; the acetamidomethyl (Acm) group of
the former might undergo intracellular hydrolytic deacetylation
and a subsequent decay to the BIM monomer; the latter is
expected to undergo fragmentation in the reducing environment
of the cell to release the corresponding thiol.
Scheme 2. Synthesis of BIM-46187 (1) and its stereoisomers (2-4). Reagents
and conditions: (a) Boc-L-Cys(Trt)-OH or Boc-D-Cys(Trt)-OH, DCC, DIPEA,
abs. CH2Cl2, rt, 48.5 h, Ar, 63-79%; (b) TFA, (i-Pr)3SiH, abs. CH2Cl2, rt, 18 h,
Ar; (c) I2, MeOH, H2O, rt, 2 h, 44-72%.
The subseries 5-8 was prepared in order to examine the
effect of the basic primary amino group on the bioactivity of the
analogs. The NH2 group was either removed or acetylated, or
equipped with a bulky carbamoyl substituent. The first three
intermediates I (Scheme 3) were synthesized by means of DCC,
while N-acetyl-S-trityl-L-cysteine was introduced with HATU.
Prior to oxidation, the detritylation to 5 and 6 and the orthogonal
deprotection to 7 and 8 were carried out as before.
G
a or b
O
O
R4
8'
8'
R4
S
N
N
N
c,d
N
J
N
N
Trt-S
a
2
I
8'S, R4 = H
5
6
7
8
8'R, R4 = H
8'S, R4 = NH-Fmoc
8'S, R4 = NH-Ac
O
O
H
H2N
N
N
N
b or c
N
Boc
N
R3
R3
N
Scheme 3. Synthesis of BIM analogs 5-8. Reagents and conditions: (a)
3-(tritylthio)propanoic acid or Fmoc-L-Cys(Trt)-OH, DCC, DIPEA, abs. CH2Cl2,
rt, 48.5 h, Ar (R4 = H or NH-Fmoc); (b) Ac-L-Cys(Trt)-OH, HATU, DIPEA, abs.
DMF, rt, 48.5 h, Ar (R4 = NH-Ac), 29-68%; (c) TFA, (i-Pr)3SiH, abs. CH2Cl2, rt,
18 h, Ar; (d) I2, MeOH, H2O, rt, 2 h, 8-13%.
N
R3 = CH2CH3
R3 = CH2OH
R3 = CH2NH2
L
12
13
14
15 R3 = CH2SO3H
16 R3 = CH2CH2SCH3
R3 = CH2SCH2NH-Ac
17
18
R3 = CH2S-SC(CH3)3
Moreover, products with a modified cystine methylene unit were
devised whose syntheses are depicted in Scheme 4. The
dimeric penicillamine (H-Pen-OH) derivatives 9 and 10 are
dimethylated analogs of 1 and 2, respectively. In compound 10,
the structure of the drug D-penicillamine was embedded. We
employed homocysteine (H-Hcy-OH) in its double-protected
form for the methylene-ethylene replacement to provide the
homocystine derivative 11. Its absolute geometry did not change,
but, owing to CIP priority rules, the configuration at the C-2
carbon is (S).
Scheme 5. Synthesis of BIM analogs 12-18. Reagents and conditions: (a)
Boc-L-Abu-OH or Boc-L-Ser(Trt)-OH or Boc-L-Dap(Boc)-OH or Boc-L-cysteic
acid or Boc-L-Met-OH or Boc-L-Cys(Acm)-OH or Boc-L-Cys(StBu)-OH, DCC,
DIPEA, abs. CH2Cl2, rt, 48.5 h, Ar, 21-82%; (b) TFA, abs. CH2Cl2, rt, 2 h (to 12,
14-18), 5-95%; (c) TFA, (i-Pr)3SiH, abs. CH2Cl2, rt, 18 h (to 13), 66%.
3
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The synthetic route in Scheme
6
followed the
assay which we have employed takes advantages of the
inhibition of IP1 degradation by lithium chloride, allowing IP1 to
accumulate in the cell, where it can be quantified as a substitute
for IP3 and a measure of Gαq inhibition. IP1 was determined in a
competitive immunoassay after 2 hours incubation of test
compounds (for structures, see Table 1) in a concentration of
100 µM followed by 35 min CCh stimulation. The results are
shown in Figure 1.
aforementioned
coupling-deprotection-oxidation sequence.
Compounds 19-26 differ with respect to two points of diversity,
the amino acid-derived residue R2 and the aromatic substituent,
phenyl or 3-pyridyl, at the imidazole. The structural variability
was already inherent in the substitution pattern of 8 secondary
amines M. The achiral precursor for 23 bears two methyl groups
at position 8', the other intermediates M were obtained from (S)-
configured amino acids, whereupon (S)-serine led to the (R)-
configured final product 24.
R2
8'
O
R2
8'
Cbz
Cbz
H
N
N
N
a
N
H
b,c
N
N
N
a
H
HN
Boc
N
HN
N
N
N
Z
Z
Trt-S
O
M
N
O
P
O
R2 = CH2C6H5, Z = CH
R2 = C6H11, Z = CH
19
20
21
O
R2
8'
H2N
N
b, c
N
R2 = CH2CH(CH3)2, Z = CH
HN
HN
22 R2 = CH2CH3, Z = CH
23 R2 = (CH3)2, Z = CH
24
25
N
N
N
d,e
f
Z
S
O
2
N
N
R2 = CH2OH, Z = CH
R2 = CH2CO2H, Z = CH
Q
S
R2 = CH2C6H11, Z = N
26
Scheme 6. Synthesis of BIM analogs 19-26. Reagents and conditions: (a)
Boc-L-Cys(Trt)-OH, DCC, DIPEA, abs. CH2Cl2, rt, 48.5 h, Ar, 40-80%; (b) TFA,
(i-Pr)3SiH, abs. CH2Cl2, rt, 18 h, Ar; (c) I2, MeOH, H2O, rt, 2 h, 4-38%.
O
O
Boc-HN
Trt-S
H2N
S
g,h
N
N
N
N
2
N
N
It was intended to expand the fused 6-membered ring in 1 to
a 1,4-diazepane substructure in 27 (Scheme 7). Somewhat
different reaction conditions were needed to obtain compound Q,
since the increased ring strain of the 7-membered ring and the
reduced electrophilicity of the ester carbonyl in P, when
compared with D (Scheme 1), exacerbated the lactamization.
The propylene linker was formed by the borane-promoted
reduction of Q. Subsequent coupling, deprotection and oxidation
yielded the envisaged product 27, in which the relative
orientation of the phenyl group and the half-cystinyl residue are
slightly shifted.
T
27
Scheme 7. Synthesis of the imidazo[1,2-a][1,4]diazepine derivative 27.
Reagents and conditions: (a) ethyl 3-bromopropionate, Cs2CO3, abs. DMF, rt,
3.5 h, 88%; (b) Pd/C, H2 (1 atm), abs. MeOH, rt, 24 h; (c) abs. EtOAc, TsOH,
reflux, 16 h, 55%; (d) BH3  THF, abs. THF, 90 °C, 48 h, Ar; (e) Pd/C, abs.
MeOH, rt, 16 h, Ar, 51%; (f) Boc-L-Cys(Trt)-OH, DCC, DIPEA, abs. CH2Cl2, rt,
48.5 h, Ar, 79%; (g) TFA, (i-Pr)3SiH, abs. CH2Cl2, rt, 18 h, Ar; (h) I2, MeOH,
H2O, rt, 2 h, 12%.
O
Analogs with
a
higher degree of conformational
a
b
heterogeneity were prepared as shown in Scheme 8. We either
removed the ethylene linker of 1 in the target compound 28 or
cut one N-C bond in 29. These two monocyclic imidazole
derivatives possess a higher flexibility owing to the free rotation
about the Cα-C and Cα-N cyclohexylalanine backbone bonds.
As in Scheme 7, the synthesis started with compound O which
was ethylated at position N-1 to intermediate U. Both O and U
were subjected to hydrogenolytic N-deprotection and the
resulting two primary amines V were converted via intermediates
W to products 28 and 29.
G Protein Inhibition. We used carbachol (CCh), an agonist
of muscarinic acetylcholine receptors (mAChRs) to induce G
protein signaling in HEK293 cells. The M3 mAChR is expressed
in HEK293 wild-type (wt) cells and couples to Gαq and Gα12
proteins thereby stimulating phospholipases (PLs) C and D in a
pertussis toxin-insensitive manner. In particular, the molecular
interaction of Gαq and PLC-β effectuates the cleavage of a
membrane phospholipid, i.e. PIP2 into DAG and IP3, both acting
as important second messengers. IP3 is further hydrolyzed
through sequential dephosphorylation to its downstream
metabolites myo-inositol 1,4-bisphosphate (IP2) and IP1. The
Cbz
N
N
N
H
b
H2N
c
N
O
N
R
U
V
O
H
N
H2N
S
N
N
Boc
N
d,e
N
H
H
N
N
Trt-S
R
R
2
W
28
R = H
29 R = CH2CH3
Scheme 8. Synthesis of monocyclic BIM analogs 28 and 29. Reagents and
conditions: (a) bromoethane, Cs2CO3, abs. DMF, rt, 3.5 h, 88%; (b) Pd/C, H2
(1 atm), abs. MeOH, rt, 24 h, 94-95%; (c) Boc-L-Cys(Trt)-OH, DCC, DIPEA,
abs. CH2Cl2, rt, 48.5 h, Ar, 80-94%; (d) TFA, (i-Pr)3SiH, abs. CH2Cl2, rt, 18 h,
Ar; (e) I2, MeOH, H2O, rt, 2 h, 12-32%.
4
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Table 1. BIM analogs prepared.
O
R2
R4
N
N
R1
R3
R3
N
X
Compound
R1
C6H5
R2
R4
X
( )-CH S
R
1
2
(S)-CH2C6H11
(S)-CH2C6H11
(R)-CH2C6H11
(R)-CH2C6H11
(S)-CH2C6H11
(R)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
NH2
NH2
NH2
NH2
H
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
2
2
2
2
2
( )-CH S
S
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
2
( )-CH S
R
3
2
( )-CH S
S
4
2
CH2S
5
2
CH2S
6
H
2
( )-CH S
R
7
NH-Fmoc
NH-Ac
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
2
2
2
( )-CH S
R
8
2
(
)-C(CH ) S
3 2
R
9
2
2
2
( )-C(CH ) S
S
10
11
12
13
14
15
16
17
18
3 2
( )-CH CH S
S
2
2
(S)-CH2CH3
(S)-CH2OH
(S)-CH2NH2
(R)-CH2SO3H
(S)-CH2CH2SCH3
(R)-CH2SCH2NH-Ac
(R)-CH2S-SC(CH3)3
( )-CH S
R
19
20
21
22
23
24
25
26
27
28
29
C6H5
C6H5
(S)-CH2C6H5
(S)-C6H11
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2CH2
H, H
2
2
2
2
2
2
2
2
2
2
2
2
( )-CH S
R
2
( )-CH S
R
C6H5
(S)-CH2CH(CH3)2
(S)-CH2CH3
(CH3)2
2
( )-CH S
R
C6H5
2
( )-CH S
R
C6H5
2
( )-CH S
R
C6H5
(R)-CH2OH
2
( )-CH S
R
C6H5
(S)-CH2CO2H
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
(S)-CH2C6H11
2
( )-CH S
R
3-pyridyl
C6H5
2
( )-CH S
R
2
( )-CH S
R
C6H5
2
( )-CH S
R
C6H5
H, CH2CH3
2
In accordance with previous investigations,[19] the BIM dimer
(1) showed a strong inhibition of the Gαq-dependent IP1
formation and exhibited an IC50 value of 31.9 µM (see the
Supporting Information, Fig. S1). The results of the present
study revealed, for the first time, the effect of the
stereochemistry of isomers on the biological activity. The (R)-
configuration of the cystine substructure was required for Gαq
inhibition, but inversion of the cyclohexylalanine configuration
was tolerated. Hence, out of the four stereoisomers, only 1 and
3 (IC50 = 56.3 µM; see the Supporting Information, Fig. S1) were
potent inhibitors. We assume that 1-4 with highly similar
physicochemical properties share the cellular uptake capability
and specific intracellular drug-protein interactions account for
different IP1 accumulation. The positively charged terminal
ammonium group was essential for Gαq inhibitory activity, since
chain of 1 by one methylene group (in 11) led to an unforeseen
enhancement of IP1, both in CCh-stimulated cells (Fig. 1) and in
HEK293 cells without CCh pretreatment (see the Supporting
Information, Fig. S2). The increased level of cellular inositol
phosphates caused by 11 in a CCh-independent manner
potentially indicates a direct activation of Gαq or its downstream
signaling pathway.
Compound 10, with the substructure of the drug
D-penicillamine incorporated, was also inactive. IP1
accumulation was maintained after treatment with compounds
12-16. In contrast to 1 and 3, compounds 12-16 are incapable of
exhibiting redox reactivity. Under the assumption that BIM
dimers undergo an intracellular reductive cleavage, the thus
formed thiol group might be requisite for further transformations
or direct Gαq inhibition. Such a mode of action is not possible in
neither compounds 5 and 6, the desamino derivatives of 1 and 3, case of 12-16. Compounds 17 and 18 did not serve as
nor compounds 7 and 8, the N-protected analogs of 1, were
active. Subtle changes in the cystine side chain of 1 caused
inactivity. This was the case, when it was equipped with two
methyl groups (in 9). In contrast, elongation of the cystine side
successful prodrugs for the BIM monomer. Since 18 represents
a disulfane, albeit of unsymmetrical structure, we expected a
bioactivation resulting in an efficacy comparable to that of 1.
However, Gαq inhibition was not observed.
5
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10.1002/chem.202001446
Chemistry - A European Journal
FULL PAPER
150
100
50
***
200
150
100
*
*
50
****
****
****
0
***
**
**
**
****
**
0
w/o 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
w/o
1
2
3
4
5
7
6
9
8 10 11 12 13 14 15
150
100
50
200
150
100
50
**
****
**
**
*
0
*
***
****
**
***
**
****
w/o 1 16 17 18 19 20 21 22 23 24 25 26 27 28 29
0
w/o
1 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Figure 1. Gαq-dependent IP1 formation in CCh-stimulated HEK293 cells
pretreated with 100 µM of BIM-46187 (1) and its analogs 2-15 (top) and 16-29
Figure 2. Viability of HEK293 cells treated with 100 μM BIM-46187 (1) and its
analogs 2-15 (top) and 16-29 (bottom). Data are presented as means ± s.e.m.
of two independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P <
0.0001 compared to w/o using Dunnett´s multiple comparisons after one-way
ANOVA.
(bottom). Data are means
± s.e.m. of two independent experiments.
** P < 0.01, *** P < 0.001, **** P < 0.0001 compared to w/o using Dunnett´s
multiple comparisons after one-way ANOVA.
We next examined whether the desired bioactivity could be
achieved in the course of structural modifications at the
cyclohexylmethyl substructure (in compounds 19-25). For
example, we exchanged the cyclohexane chair for the flat
benzene ring (in 19), removed a methylene (in 20) or propylene
(in 21) or pentylene unit (in 22), or introduced polar groups (in 24
and 25). Our data indicated that the presence of the
cyclohexylmethyl moiety (1 versus 19-25) as well as the phenyl
group (1 versus 26) was beneficial. A further active inhibitor of
the Gαq protein was achieved when we implemented the ring
molecules, specifically disrupt high-affinity agonist binding of
Gαq-selective GPCRs, as they impair formation of the
nucleotide-free ‚empty pocket state‘, the G protein species
required to stabilize this high-affinity interaction.
Cellular toxicity. The toxicity of all compounds was
determined by applying a CellTiter-Blue viability assay (Fig. 2). It
is well-established that Gαq inhibition per se is not linked to
cellular toxicity.[6] This finding offered the opportunity to separate
efficient Gαq inhibition from adverse toxic effects. On the one
hand, an indication for the possible independency of both
properties is apparent from the distinct cellular toxicity of several
BIM analogs which did not inhibit Gαq (e.g. 10-12, 14, 19, 28 and
29; Fig. 1 and Fig. 2). On the other hand, significant inhibition
was only observed for toxic BIM compounds (1, 3 and 27; Fig. 1
and Fig. 2). Among the derivatives without a free N-terminal
amino group, some were not toxic (5, 6 and 8, Fig. 2). Moreover,
representatives bearing polar groups as part of R2 (24 and 25) or
the acidic sulfonate moiety in R3 (15) did not affect cell viability
either. Hence, a zwitterionic structure (15, 25) might prevent
cellular toxicity, an assumption to be considered in future
attempts towards molecular separation of desired (Gαq
inhibition) and unwanted features (cellular toxicity) of BIM
analogs.
expansion of
1 as realized in the propylene-containing
compound 27 (IC50 = 50.2 µM; see the Supporting Information,
Fig. S1). An increase of conformational flexibility in case of the
monocyclic analogs 28 and 29 turned out to be disadvantageous.
Taken together, the following main structural requirements for
active BIM analogs were clarified (i) the redoxreactive
cystine/cysteine substructure, (ii) the N-terminal basic amino
group, (iii) the cyclohexylalanine moiety, (iv) a bicyclic skeleton.
These considerations provide a rather limited scope for the
future design of related Gαq inhibitors.
The extraordinary potency of YM-254890 and FR900359
(IC50 values of 1.06 µM and 0.77 µM, respectively, see the
Supporting Information, Fig. S1) was not attained with the BIM
analogs of this study. Furthermore, their mode of action is also
distinct. While BIM-46187 (1) and its monomer inhibit Gαq
signaling by precluding GTP entry while permitting GDP exit,[19]
YM-254890 and FR900359 function as inhibitors of GDP
dissociation, i.e. stabilize Gαq in its inactive GDP-bound
state.[6,25] Therefore, YM-254890 and FR900359, but not BIM
To investigate whether BIM type inhibitors require Gαq to
exert their cell cytotoxic effects, we took advantage of HEK293
cells, depleted by CRISPR/Cas9 of Gαq and Gα11 subunits of
heterotrimeric G proteins. Cells treated with the BIM dimer (1)
6
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10.1002/chem.202001446
Chemistry - A European Journal
FULL PAPER
were subjected to non-invasive live cell imaging. We studied the
response of wt and Gαq/11 KO HEK293 cells to a 30 min long
application of 1 (30 µM) versus DMSO (1:3000) as control.
Within 30 min, the cell shape changed, as cells of both cell lines
strongly rounded up (Supporting Information, Fig. S3A). From
these results we concluded that BIM-induced cell rounding does
not require heterotrimeric Gαq family proteins. Since the
cytoskeleton is an essential cellular component responsible for
the maintenance of the morphology and several functions of the
cell, we reasoned that cytoskeletal changes might be the origin
for the observed toxicity. To eludidate the potential mechanisms
underlying the compound-induced changes of cell shape, the
cytoskeleton was analyzed. For this purpose, we stained against
microtubules and intermediate filaments with a direct fluorescent
cytoskeletal dynamics of treated cells. Moreover, our finding
provides evidence that BIM-type inhibitors display their cellular
toxicity in
a Gαq-independent manner. The usability of
compounds targeting cytoskeletal dynamics has been
considered to be limited because they affect a variety of
processes in both cancer and normal cells. However, there are
microtubule-binding compounds that became valuable drugs for
cancer treatment, in particular vinca domain-binding agents, e.g.
vincristine, and taxol domain-binding agents, e.g. paclitaxel.[26]
Compound 1 does not exhibit typical molecular features of
cytoskeleton-targeting compounds. Accordingly, future studies
might be focused on structure-activity relationships with respect
to the induction of cytoskeletal changes by BIM-type Gαq
inhibitors.
antibody
against
β-tubulin,
and
through
indirect
immunofluorescence against vimentin, respectively. To visualize
microfilaments, we used rhodamine phalloidin as a fluorescent
probe for F-actin. The microtubular network of wt and Gαq/11 KO
HEK293 cells was not affected by 1 (Supporting Information, Fig.
Experimental Section
Detailed descriptions of synthetic procedures and cellular
experiments as well as analytical properties of all prepared
compounds and biological data are given in the Supporting
Information.
S3B), whereas both wt and Gαq/11 KO cells showed
preferential perinuclear accumulation of vimentin and
intermediate filaments in response to treatment with
a
1
(Supporting Information, Fig. S3C). The most prominent
changes could be observed in phalloidin stainings, as both
DMSO-treated cell lines displayed prominent F-actin stress
fibers with small protrusions. In clear contrast, compound 1
induced strong rounding up of the cells (Supporting Information,
Fig. S3D) and this was accompanied by predominant cortical
actin re-localization, whereas almost neither stress fibers nor
actin surrounding the nucleus of the cells occurred. These data
demonstrate that the BIM dimer (1) has prominent effects on cell
shape and that these are due to its action on the cytoskeletal
components. Cytoskeletal changes in transformed cells are
involved in cell proliferating and metastatic capabilities.[26]
Noteworthy, our data on the Gαq/11-independent influence of the
BIM dimer (1) on components of the cytoskeleton illustrates an
additional mechanism by which BIM molecules act as agents
against tumor cells.
Acknowledgements
B.K.F., E.K. and M.G. were supported by the German Research
Foundation (DFG)-funded Research Unit FOR2372 with grants
FL 276/8-1 and -2 (to B.K.F.), KO 1582/10-1 and -2 (to E.K.) and
GU 345/3-1 (to M.G.). T.B. was funded by the DFG-
214362475/GRK1873/2. The authors thank Andreas J.
Schneider for support in compound purification and chiral HPLC
and Nina Heycke for support in biological assays.
Conflict of interest
The authors declare no conflict of interest.
Keywords: Davidson cyclization • G proteins • lactamization •
structure-activity relationships • toxicity
Conclusion
These investigations were carried out to explore the chemical
space of Gαq inhibitors with a heterocyclized dipeptide structure.
Although the structure of BIM offers numerous opportunities for
combinatorial modifications and several have been realized in
this study, only a narrow frame for tolerable structural alterations
was recognized. Hence, we defined substructures required for
Gαq inhibition and identified two novel bioactive compounds
which possess high similarity with the BIM dimer. Redox
reactivity was shown to be requisite for cellular activity indicating
the involvement of intracellular thiols, in particular glutathione,
for bioactivation of BIM-type disulfanes, probably catalyzed by
the thioredoxin-glutaredoxin system.[27] Two cysteine residues,
Cys330 or Cys144, are conserved in all Gαq proteins and might
constitute potential binding sites for 1, consistent with the
intradomain movement within the target protein.[19] Future
studies are needed to ascertain whether a covalent interaction of
Gαq with BIM-type homodimers, their reduced monomers or
BIM-glutathione heterodimers takes place. Herein we discovered
that the prototypical Gαq inhibitor 1 affects the structural
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8
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Chemistry - A European Journal
FULL PAPER
Entry for the Table of Contents
Insert graphic for Table of Contents here.
O
H2N
N
N
N
S
2
BIM-46187
Different points of diversity (see colors) were introduced into the full-size BIM structure to explore the chemical space of G protein
inhibitors of the BIM chemotype.
9
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