Conjugate Additions of Amines to Maleimides via Cooperative Catalysis

Article abstract of DOI:10.1002/adsc.201800160

A cooperative system comprising of a lithium Lewis acid and amine base significantly enhances the rate of the conjugate addition of a wide array of amines to maleimides. This operationally simple, scalable method provides mono-addition products in high yields and purity. This conjugation was successfully applied to the kinase inhibitor crizotinib in a chemoselective ligation to create novel fluorescent probe. (Figure presented.).

Full text of DOI:10.1002/adsc.201800160

Title: Conjugate Additions of Amines to Maleimides via Cooperative
Catalysis
Authors: Karl Scheidt, Brice Uno, Kristine Deibler, Carlos Villa, and
Arjun Raghuraman
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201800160
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10.1002/adsc.201800160
Advanced Synthesis & Catalysis
UPDATE
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Conjugate Additions of Amines to Maleimides via Cooperative
Catalysis
Brice E. Uno,a Kristine K. Deibler,a Carlos Villa,b Arjun Raghuraman,b and Karl A.
Scheidta*
a
Department of Chemistry, Center for Molecular Innovation and Drug Discovery, Chemistry of Life Processes Institute,
Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA E-mail: scheidt@northwestern.edu; Tel: 847-
491-6659
Dow Chemical Co., Freeport, TX 77541, USA.
b
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.
the conjugate addition of amines to maleimides. A
Lewis acid and amine base significantly enhances the rate rate acceleration of this amino-conjugate addition
Abstract. A cooperative system comprising of a lithium
of the conjugate addition of a wide array of amines to
maleimides. This operationally simple, scalable method
provides mono-addition products in high yields and purity.
This conjugation was successfully applied to the kinase
inhibitor crizotinib in a chemoselective ligation to create
novel fluorescent probe.
could broaden access to a more diverse array of
biologically active small molecule probes. Although
reactions with nitrogen nucleophiles with various
conjugate acceptors have been extensively studied,[7]
very few systems involve maleimides.[8] With this
specific electrophile, reactions are typically
performed at elevated temperatures and/or extended
times, potentially curtailing application in complex
settings.[9] In many cases, mixtures of 1,2- and 1,4-
addition products are also observed (vide infra). As a
result of these reactivity and chemoselectivity
limitations, amines are currently underutilized as
nucleophiles with maleimides.
Keywords: Amino-conjugate addition; bioconjugation;
cooperative catalysis; crizotinib; maleimide; synergistic
catalysis
Bioactive small molecules are the foundation of
modern pharmaceutical sciences.[1] They can also
serve as critical probes for elucidating complex
biological pathways. Understanding how these
important small molecules interact with biological
systems has been aided through covalent conjugation
of fluorophores and affinity labels.[2] Such conjugated
small molecules are employed to label specific
cellular components including tubulin, actin, as well
as innumerable enzymes and proteins.[3] This strategy
is effective when the covalent linkages are stable in
vivo and do not adversely affect the overall
pharmacology through perturbations in steric
interactions, charge and/or solubility. The selective
formation of N–C bonds fulfills most of the above
criteria.[4] Highly useful systems, such as
perfluorosulfones, isothiocyanates and N-hydroxy-
succinimide esters provide stable conjugated
products.[5] However, the latter two reagents generate
thiourea and amide linkages possessing altered
charge states to the native amines, which could
potentially alter the pharmacology of the resulting
bioconjugated products. In addition to the
specifications of the structural components, the
preparation of the conjugate must be high yielding,
mild, atom economical, and chemoselective. The
reaction between maleimides and 1° and 2° amines
produces stable conjugates, but the rates for these
additions are much slower by comparison, thereby
potentially limiting their deployment and utility.[6]
To broaden the chemical toolbox, we sought to
develop a rapid and selective conjugation strategy for
Maleimide conjugation strategies
O
O
O
H
NH2
SH
S
N
R1
R1
R1
R1
N
R2
N
R2
N
R2
(Eq. 1)
(Eq. 2)
O
O
O
•rapid bond formation
•reversible
•slow bond formation
•stable C-N bond
This work: Rapid and chemoselective amino-conjugate addition
O
Cat
H
O
A) Previous work:
N
H
R1
R1
N
R2
+
N
R2
N
O
H
O
O
NH2
R2
+
N
R1
R1
H
O
•high yielding
NH
Et
Li+
O
R2
N
•single addition
•scalable
B) This work:
Cooperative catalysis
N
Et3N
Et
O
Li+
Et
Figure. 1. General reaction design.
Our group has recently developed the first practical
asymmetric amino-conjugate addition reaction
between simple, unprotected alkyl amines and
maleimides.[10] Our studies on this reaction were
motivated by two overarching goals; the need for new
and strategically simple methods to access chiral
amines, as well as the potential to expand the existing
toolbox of maleimide-tagged biological probes that
are
already
commercially
available
and
1
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10.1002/adsc.201800160
Advanced Synthesis & Catalysis
predominantly marketed for use with more reactive
sulfhydryl nucleophiles.[11] With these challenges in
mind, we sought a general catalytic method for the
efficient and rapid addition of amines to maleimides
at ambient temperatures.[12] Given our interest in
cooperative catalysis[13] and processes that involve
dual activation of both electrophilic and nucleophilic
reactants,[14] we initiated an investigation to engage
maleimides with amines to afford conjugate addition
products through a Lewis acid/Brønsted base strategy,
in the context of enhancing the chemoselectivity of
this process for general and operationally simple
deployment with existing maleimide electrophiles
when enantioselectivity or the use of chiral catalysts
is not the primary goal of the application (Figure 1).
We began our studies with a reaction between
equimolar quantities of N-tolylamine (1) and N-
benzylmaleimide (2) focused on enhancing the
reaction rate (Table 1). The rate constant of the
uncatalyzed conjugate addition in THF at 0.1 M was
Table 1. Optimization of the title reaction.
a) NMR yields with 1,3,5-trimethoxybenzene as an internal
standard to calculate rate constants. b) t1/2 values derived
from rate constants c) Isolated yield after 4 h on 1 mmol
scale.
Figure 2. Observation of cooperativity via detai analysis.
Plot of 1/[starting material] vs. time. Lines with slopes
greater than the calculated “additive” plot are considered
cooperative (see SI).
approximately 4.40 x 10-4 M-1s-1 (entry 1).[15]
A
theoretical study by Yamabe on the conjugate
addition of dimethylamine with maleimide
derivatives suggested multiple molecules of reagent
amine must be invoked in the reaction mechanism in
order
to
rationalize
the
formation
of
aminosuccinimide products via an energetically
feasible pathway.[16] The additional amine molecules
are postulated to provide stabilization to the
zwitterionic adduct formed after initial attack of the
amine on maleimide via extended hydrogen-bonding
interactions, enabling the subsequent rate-limiting
proton transfer to generate product. We first
investigated the possibility of employing an
additional amine as a catalyst in an effort to improve
the rate of conjugate addition. Using 10 mol %
triethylamine (Et3N) as a sole additive produced a
negligible impact on the rate (entry 2). Based on the
understanding that lithium ions can be activators of
1,4-conjugated systems,[17] we added 10 mol%
lithium chloride (LiCl) and observed a significantly
increased rate of the reaction (entry 3). Strikingly,
when catalytic amounts of both Et3N and LiCl were
present, the rate significantly increased further (entry
4). While cooperative or synergistic effects of lithium
salts and amines have been invoked previously for
related transformations, the use of these descriptors
has been widely accepted to mean “working
together”.[18] Based on our detailed kinetic analysis,
we observed that the optimized conditions above are
indeed cooperative. The combined effect of the two
catalysts as depicted in Figure 2 (cooperative plot, red
) demonstrates that the rate enhancement
observed is greater than the sum of the individual
contributions of the catalysts depicted by the additive
plot (blue , Figure 2 and Supporting Information).
In the presence of triethylamine, other lithium salts
with weakly coordinating counter anions were
surveyed. LiOTf and LiBF4 were observed to
moderately improve the reaction rate beyond that of
LiCl (entries 5 and 6). Although LiPF6 was observed
to have the shortest t1/2 (entry 7), we mainly focused
on LiCl for our purposes due to its low cost/mol,
handling constraints, and reduced toxicity relative to
LiPF6. LiOH was investigated as a salt possessing
both Lewis acidic and Brønsted basic properties, and
we observed a similar profile to Et3N alone (entry 8).
Lastly, 10 mol% Et3N•HCl was examined as the
potential species responsible for the rate increase,
however we observed a similar profile to Et3N (entry
9). LiCl was selected as the Lewis acid to be paired
with Et3N moving forward due to its low molecular
weight as well as its cost and ease of manipulation.[19]
We next investigated a range of primary amines for
this cooperative process (Table 2). By employing a
slight excess of the amine, we ensured the full
consumption of maleimide 2 and improved the
isolated yields. We also simplified the purification by
filtering the excess amine and catalyst through a thin
plug of silica gel, yielding pure conjugated product
after concentration. Using this simplified protocol,
aminosuccinimide products were furnished in
excellent yields with primary amines (4—7).
Products generated from branched amines were also
well tolerated (8—10). When the steric size of the
2
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10.1002/adsc.201800160
Advanced Synthesis & Catalysis
nucleophile was further increased to 1-adamantyl
(11), the rate of the reaction slowed as judged by the
yield after 90 min (20% yield). Other protic
functional groups were well accommodated under
these conditions, (12—14). Not surprisingly,
nucleophiles containing two nitrogen atoms (15—18)
selectively reacted at the more reactive amine
position over others. For example, protected amino
acid Cbz-Lys-OMe (19) was an excellent substrate.
The reactivity of deactivated amines was increased
with higher amounts of the cooperative Lewis
acid/Brønsted base mixture (20—23).
conditions, which is consistent with the observation
that aniline is unreactive (not shown).
The scope of conjugate acceptors through variation
of the maleimide’s nitrogen substituent was also
investigated.
More
electron-deficient
N-
arylmaleimides react much faster with amines
compared to the corresponding reaction with N-
benzylmaleimide 2. However, N-arylmaleimides
suffer from competitive 1,2-additions.[12a,
N-
12b]
phenylmaleimide (44) in the absence of the
cooperative catalysis conditions reacted with N-
tolylamine 1 with a t1/2 of 94 min, producing a
reaction mixture that contained a ~3:1 ratio of 1,4- to
1,2-addition products (45:46, Scheme 1). In the
presence of the cooperative catalysts, the observed t1/2
was 21 min with an improved product ratio of 10:1.
Table 2. Scope of primary amine nucleophiles
Table 3. Scope of secondary amine nucleophiles
a) isolated yield. b) NMR yield. c) LiPF6 (10 mol %), Et3N
(10 mol %). d) LiPF6 (100 mol %), Et3N (100 mol %).
a) Isolated yield after 90 min on 1 mmol scale. b) Isolated
yield after 15 h. c) As observed by NMR after 90 min. d)
LiPF6 (10 mol %),
Secondary amines were also successful
nucleophiles under the optimized set of reaction
conditions (Table 3). Various cyclic secondary
amines were excellent substrates (24—31). Proline
methyl ester reacts rapidly under cooperative
conditions with LiPF6 (10 mol %) (32). Acyclic and
secondary amines were not as successful nucleophiles
since they required extended reaction times to react
with maleimide 2 (33—38). Bulky acyclic secondary
alkyl amines were inert under these conditions as
well (39—41). These data are consistent with the
observation that over-addition products are not
generated under the reaction conditions. For example,
secondary amine product 3 of the title reaction was
re-subjected to the optimized conditions and did not
produce 41. Furthermore, secondary amines with aryl
substituents (42 and 43) were unreactive under these
Scheme 1. Improvement of 1,4-addition selectivity.
3
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10.1002/adsc.201800160
Advanced Synthesis & Catalysis
Other maleimides were also explored as a platform
for future potential translation to
After exploring the scope of our conjugate addition
with a variety of amines and maleimides, we
extended this method to a more complex reaction
partner with potential biological/medical application.
Crizotinib (55) was approved in 2011 for the
treatment of late-stage lung cancer, anaplastic large
cell lymphoma, and neuroblastoma.[23] Although 55
strongly binds ALK, it has also been found to bind
ROS proto-oncogene 1-encoded kinase (ROS1), c-
MET, and proto-oncogene-encoded kinase of the
hepatocyte growth factor receptor (HGFR).[24] Based
on the ALK-57 bound X-ray structure, 55 is observed
to bind to the ATP pocket through the aminopyridine
portion and to possess a hydrophobic interaction
through its halogenated benzyloxy moiety. The
piperidine moiety was observed to protrude out of the
binding pocket, into the solvent exposed region,
making this moiety an excellent handle for
conjugation. We predicted that conjugation to the
fluorescein-maleimide tag 56 would selectively occur
at the piperidine nitrogen of 55 based on the
materials/polymerization studies. For example, 44
underwent efficient 1,4-addition with N-benzylamine
to afford the corresponding aminosuccinimide 45 in
86% yield. Other nitrogen-functionalized maleimides
were suitable conjugate acceptors, providing the
corresponding aminosuccinimide products of N-
ethylmaleimide (47) and maleimide (48), in 97% and
93% yields, respectively (Table 4). As a potential
entry into maleimide containing materials,[20][21] N-
alkyl and N-aryl-bismaleimides were also
investigated as electrophiles. The bismaleimides
shown in Table 4 are commonly employed monomers
in the synthesis of poly(imido) succinimides.[22] N,N-
diaryl-bis-maleimide 49 provided its corresponding
product in 85% yield, and was easily separated from
the remaining 12% of 1,2-addition product formed.
1,4-bismethylenecylohexyl
bis-maleimide
50
afforded its corresponding product in 95% yield as a
1:1 mixture of cis- and trans- diastereomers. Equally
suitable bismaleimides were ethylenediamine (51)
and hexamethylenediamine-derived bismaleimide
(52), generating their corresponding conjugated
products excellent yields.
chemoselectivity
parameters
we
previously
established (see Tables 2 and 3).
Using a full equivalent of LiCl (5 wt %) and Et3N
(10 wt %), we were able to selectively synthesize 57
in 87% yield in less than 10 min (Scheme 3). A
higher catalyst loading was employed to expedite the
reaction and highlight the expendability of these
simple catalysts without detriment to yield or
selectivity. Gratifyingly, 57 maintained activity
against A549, human non-small cell lung cancer cells
in vitro with an IC50 value of 9.0 M. Although less
potent compared to 57 (IC50= 1.0 M), 57 produced
high quality confocal fluorescent images indicating
the conjugate material localizing on the periphery of
the cell and outside the nucleus (Figure 4). Further
studies to understand this observation with our new
crizotinib-fluorophore probe are ongoing.
Table 4. Differentially substituted maleimides as
electrophiles
a) isolated yields after 90 min
To further highlight the ease and utility of these
conditions, we conducted the reaction on 100 mmol
scale with N-ethylmaleimide 53 (the same starting
material that provided 47) and N-benzylamine. At 10
mol % loading of each catalyst, the reaction reached
full conversion in 90 min with no observed exotherm.
The large-scale reaction was purified by filtration
through a diatomaceous earth-supported pad of silica
gel to yield 22.4 g of pure conjugated product 54 in
96% yield.
Scheme 3. Conjugation of Crizotinib to a Maleimide-
Scheme 2. Scale-up of amine conjugate addition
4
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10.1002/adsc.201800160
Advanced Synthesis & Catalysis
Experimental Section
All reactions were carried out under ambient atmosphere in
air-dried glassware with magnetic stirring. THF, toluene,
and DMF were purified by passage through a bed of
activated alumina. Reagents were purified prior to use
unless otherwise stated following the guidelines of Perrin
and Armarego.[26] N-tolylamine was distilled from CaH2.
Purification of reaction products was carried out by flash
chromatography using EM Reagent silica gel 60 (230-400
mesh). See the Supporting Information for details
regarding characterization of all new products.
Preparation of stock solution A:
Figure 3. Preliminary characterization of crizotinib-
fluorophore probe. A) 3-day viability assay data validated
effect of fluorophore/malemide on drug, crizotinib results
were comparable to previous literature values.[25]. B) IC50
values indicate that (56) and fluorescein methyl ester do
not greatly effect the activity of the original drugs, mostly
likely due to loss of solubility seen to the naked eye. C)
Example images of condition optimization of probe
incubation with A549 cells with and without probe 57
[24hr, 10uM probe] collected via ImageXpress. D)
Fluorophore incorporation into cell to ensure limited
autofluorescence background signal of A549 cell line in
the FTIC range.
A 20 mL vial was charged with THF (10 mL), followed by
LiCl (21.2 mg, 0.2 mmol) and triethylamine (69.6 uL, 0.2
mmol). The resulting mixture was stirred until all LiCl
crystals were fully dissolved and the stock solution became
homogenous.
Preparation of stock solution B:
In a 20 mL vial, N-benzylmaleimide (940 mg, 2 mmol)
was dissolved in THF (10 mL). The stock solution was
stirred until homogenous.
General
procedure
for
the
synthesis
of
aminosuccinimides:
To a 20 mL screw-cap vial equipped with a magnetic stir
bar was added 500 L of stock solution A, followed by
0.11 mmol (1.1 equivalents) of the primary or secondary
amine nucleophile at 23 ºC. 500 L of stock solution B
was added to the reaction mixture, and the resulting
solution was stirred at ambient temperature for 90 minutes.
The crude reaction mixture was subsequently filtered
through a 1 cm pad of SiO2 supported on a 0.5 cm plug of
diatomaceous earth. The filter cake was flushed with
EtOAc (3 x 1 mL). The combined organic filtrates were
concentrated in vacuo on a rotary evaporator. The
concentrated material was triturated with 1 mL of CH2Cl2
and re-concentrated in vacuo three additional times to yield
the product aminosuccinimides in the reported yields (See
Supporting Information for details).
Figure 4. Confocal images (86 x 86 mm) of 10 M (57).
IC50 values: 55: 1.0 M, 57
Cell assay procedures
A549 cells were obtained from Northwestern University’s
Developmental Therapeutics Core. Cells were grown in
Roswell Park Memorial Institute (RPMI) 1640 media
(GIBCO, Life Technologies, Grand Island, NY) and
supplemented with 10% FBS (Whittaker, Walkersville,
In conclusion, the combination of a Lewis acid and
Brønsted base is not unproductive based on canonical
interactions, but instead catalyzes the conjugate
addition reaction of amines and maleimides.
Significant improvements in the rate and selectivity
of the reaction were observed under these conditions.
For electronically and sterically deactivated amines,
greater amounts of cooperative catalysts can be
employed in to increase reactivity. These mild
conditions have been successfully deployed to
generate biologically active chemical probes from an
FDA approved drug. With this cooperative catalysis
platform, the current array of commercial
functionalized-maleimide tags (exclusively marketed
toward sulfhydryl systems) could potentially be
employed with amine-containing pharmacophores to
shed light on biological effects such as cell
permeability, trafficking, and localization. Additional
investigations into further exploring this late stage
pharmaceutical conjugation approach are underway.
MD),
1%
HEPES
(4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid) buffer and 1% penicillin-
streptomycin.[25c] Cells were maintained at 37 °C in a
humidified atmosphere of 5% carbon dioxide, with media
changes occurring three times per week. Cells were also
maintained in the exponential growth phase, and routinely
certified as mycoplasma free.
Cell viability assay:
MTT
(dimethylthiazol-diphenyltetrazolium
bromide)
growth inhibition assays: Three-day growth inhibition
assays were performed in clear Greiner TC microtiter
plates as previously described.[25c] First, 3000 A549 cells
per 100 μL of cell culture media were plated into each well
and were incubated for 24 hours. The synthetic analogs
and controls, as well as crizotinib (obtained from Combi-
Blocks, Inc.; positive control), were suspended in culture
media and added to the wells to give a final volume of 200
μL per well. Crizotinib and all of the synthetic probes were
suspended in DMSO and stored at −20 °C prior to use. The
final DMSO concentration did not exceed 0.5% in any
experiment. The treated cells were then incubated for an
5
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10.1002/adsc.201800160
Advanced Synthesis & Catalysis
additional 3 days, at which time MTT was added to each
well (20 μL of a stock solution containing 5 mg/mL MTT
in PBS) and the cells were incubated at 37 °C. Four hours
later, the cells were lysed by addition of 200 μL of DMSO
to each well, and the optical density at 540 nm was
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measured on
a
EnSpire multimode plate reader
(PerkinElmer, Waltham, MA, USA). The absorbance
values were expressed as a ratio of treated to untreated
cells. The concentration required to inhibit the growth of
tumor cells by 50% (IC50) was used to evaluate the effect
of the drug and analogs. Assays were performed in
triplicate and repeated (n = 2). Mean and standard error
(SE) were calculated. (See Figures 3A and B and the
Supporting Information for detailed procedures for the
fluorescence microscopy imaging of treated cells).
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Financial support for this work was generously provided by the
DOW Chemical Company.
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7
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10.1002/adsc.201800160
Advanced Synthesis & Catalysis
UPDATE
rapid, mild, selective conjugation
Conjugate Additions of Amines to Maleimides via
Cooperative Catalysis
R1
O
O
•high yielding
NH
Li+
H
R2
N
•scalable
NH2
R2
+
N
R1
N
Et3N
Et
Et
•bioconjugation
compatible
Adv. Synth. Catal. Year, Volume, Page – Page
O
Li+
Et
O
Brice E. Uno, Kristine K. Deibler, Carlos Villa,
Arjun Raghuraman, and Karl A. Scheidt*
inhibitor-bound ALK cells
tagged with fluorescent
maleimide probe
8
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