Automated and accelerated synthesis of indole derivatives on a nano-scale

Article abstract of DOI:10.1039/c8gc03039a

Automated, miniaturized and accelerated synthesis for efficient property optimization is a formidable challenge in chemistry in the 21^st century as it helps to reduce resources and waste and can deliver products in shorter time frames. Here, we used for the first time acoustic droplet ejection (ADE) technology and fast quality control to screen the efficiency of synthetic reactions on a nanomole scale in an automated and miniaturized fashion. The interrupted Fischer indole combined with Ugi-type reactions yielded several attractive drug-like scaffolds. In 384-well plates, a diverse set of interrupted Fischer indole intermediates were produced and reacted with the tricyclic hydantoin backbone in a 2-step sequence. Similarly, preformed Fischer indole intermediates were used to produce diverse sets of Ugi products and the efficiency was compared with that of the in situ method. Multiple reactions were performed again on a preparative millimole scale, showing scalability from nano to mg and thus synthetic utility. An unprecedented large number of building blocks were used for fast scope and limitation studies (68 isocyanides, 72 carboxylic acids). Miniaturization and analysis of the generated big synthesis data enabled deeper exploration of the chemical space and permitted the gain of knowledge that was previously impractical or impossible, such as the rapid survey of reactions, and building block and functional group compatibility.

Full text of DOI:10.1039/c8gc03039a

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Automated and accelerated synthesis of indole
derivatives on a nano-scale†
Cite this: DOI: 10.1039/c8gc03039a
a
a
a
a
Shabnam Shaabani, ‡ Ruixue Xu,‡ Maryam Ahmadianmoghaddam, Li Gao,
Received 27th September 2018,
Accepted 7th December 2018
a
b
b
c
Martin Stahorsky, Joe Olechno, Richard Ellson, Michael Kossenjans,
Victoria Helan and Alexander Dömling
c
a
*
DOI: 10.1039/c8gc03039a
rsc.li/greenchem
Automated, miniaturized and accelerated synthesis for efficient prerequisite in modern science and technology, and is already
property optimization is a formidable challenge in chemistry in the well established in many areas of molecular and cell biology,
st
2–4
2
1
century as it helps to reduce resources and waste and can medicine, materials science, engineering and physics.
deliver products in shorter time frames. Here, we used for the first Organic synthesis of new matter is a central science and
time acoustic droplet ejection (ADE) technology and fast quality technology and it contributes in societal progress through
control to screen the efficiency of synthetic reactions on a nanomole novel medication, efficient and safe plant protection, new
5
–8
scale in an automated and miniaturized fashion. The interrupted materials, contributors to human health, food supply.
Fischer indole combined with Ugi-type reactions yielded several Nonetheless, small molecule synthesis laboratory technology
attractive drug-like scaffolds. In 384-well plates, a diverse set of inter- mostly is at a stage not much different from what it was 200
rupted Fischer indole intermediates were produced and reacted with years ago. Reactions are performed sequentially often
the tricyclic hydantoin backbone in a 2-step sequence. Similarly, pre- unnecessarily on a large scale by synthesizing small arrays of
formed Fischer indole intermediates were used to produce diverse compounds and are not targeted towards the goal of discovery
sets of Ugi products and the efficiency was compared with that of of novel properties (Fig. 1). While major progress has been
the in situ method. Multiple reactions were performed again on a pre- made in new catalysis, novel reactions, and stereoselective syn-
parative millimole scale, showing scalability from nano to mg and thesis, the pace of synthetic reactions is slow with typically
8
thus synthetic utility. An unprecedented large number of building ∼1–5 reactions per worker/FTE per week.
blocks were used for fast scope and limitation studies (68 isocya-
Although there exist very useful automated methods for the
nides, 72 carboxylic acids). Miniaturization and analysis of the gener- assembly of specialized molecule classes such as peptides, oli-
ated big synthesis data enabled deeper exploration of the chemical gonucleotides, and oligosaccharides, their synthetic scope is
space and permitted the gain of knowledge that was previously rather limited. Only a few and similar molecular building
9
,10
impractical or impossible, such as the rapid survey of reactions, and blocks and less than half a dozen reactions are used.
building block and functional group compatibility.
Attempts to automate the synthesis of certain natural products
using a few reliable coupling reactions and rather easily acces-
sible building blocks are also described. Automated synthesis
9
therefore recently became an important topic in synthetic
Automation is a global force that will
transform economies and the
workforce
1
1
engineering. Key to miniaturization and acceleration of
synthetic chemistry are reactors of small dimensions and fast,
precise and efficient building block dispensing methods.
While pico-, femto- and attoliter reactor sizes have been
described, implementation of this scale of synthesis in a
flexible and high-throughput manner has been largely
1
In the age of artificial intelligence, miniaturization, auto-
mation and acceleration of experimental data collection is a
1
2–14
unexplored.
a
University of Groningen, Department of Drug Design, A. Deusinglaan 1, 9713 AV
Groningen, The Netherlands. E-mail: a.s.s.domling@rug.nl
b
Labcyte Inc., 170 Rose Orchard Way, San Jose, CA 95134, USA
c
Nano one-pot reactions
Hit Discovery, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg,
SE-43183 Mölndal, Sweden
Here, we use ADE-enabled fast, automated and nanomole
scale organic synthesis exemplified by scouting the reaction
spaces of the interrupted Fischer indole and different Ugi
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8gc03039a
These authors contributed equally to this work.
‡
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Fig. 1 Traditional and ADE-enabled synthetic chemistry. ADE-enabled synthetic chemistry needs a fraction of the materials compared to traditional
chemistry. Many more building block combinations can be evaluated and rich datasets are produced which would be too time-consuming and
expensive to generate traditionally.
multicomponent reaction modifications. ADE technology uses
sound waves applied to liquids to generate and precisely trans-
1
5
port nanoliter droplets. Isocyanide-based multicomponent
reactions (IMCRs) are a highly relevant reaction class and span
a relevant chemical space in drug discovery as they enable easy
discovery and/or access to multiple commercial and experi-
1
6,17
mental drugs, e.g. anti-pain carfentanil and xylocaine,
1
8
19
anti-seizure lacosamide, anti-depressant olanzepine, blood
2
0,21
coagulant rivaroxaban and clopidogrel,
HCV drug
2
2
23
telaprevir, birth-controlling epelsiban, or cancer-starvation
2
4
ivosidenib, just to mention a few. In particular, IMCR is
robust, scalable, safe, and green and fulfils the important
requirement of tolerating nitrogen heteroatoms and (un-
protected) polar functional groups common in drug molecules
Thus, novel modifications of IMCR
scaffolds are an intensive area of research and innovation.
2
5–27
for syntheses.
2
8
Scheme 1 Investigated interrupted Fischer indole/IMCR scaffolds and
theoretically accessible chemical space based on the used building
blocks.
Schiff bases are key intermediates in the majority of the very
versatile IMCRs. Cyclic Schiff bases 3 of high diversity can
be formed by an interrupted Fischer indole synthesis,
whereby α,α′-disubstituted carbaldehydes react under acidic
2
9,30
conditions with (hetero)aromatic hydrazines.
Per se this
is a highly versatile synthetic transformation, since many
Next, we synthesized 192 random combinations of 9 cyclic
aldehyde and hydrazine building blocks can be potentially Schiff base building blocks with 68 different isocyanides and
combined resulting in a large number of low molecular 72 carboxylic acids to yield 48 imino hydantoin and 48 tetra-
weight key intermediates for further derivatizations in an zole and 96 U-3CR products, respectively (Fig. 2). The reagents
3
1
overall short sequence of steps. To better understand the were dissolved in ethylene glycol or 2-methoxyethanol (car-
scope and limitations of the combination of different build- boxylic acids) as 0.5 M stock solutions and kept in a 384-well
ing blocks, we planned to investigate the interrupted Fischer source plate and transferred into a corresponding 384-well des-
indole reaction in combination with several IMCRs 4–6 tination plate using an Echo555 acoustic dispenser. The
(
Scheme 1).
The three reactions we combined with the interrupted the often-used methanol for IMCRs and to account for a
Fischer reaction 3 were the Ugi three-component reaction viscous not volatile transport solvent required in ADE. The
U-3CR) 4, the Ugi tetrazole reaction (UT-3CR) 5 and the Ugi scale of each discrete reaction per well was 375 or 500 nano-
solvent ethylene glycol was chosen because of its similarity to
(
imino hydantoin reaction (UH-3CR) 6. First, we synthesized mole, for UH-3CR and U-3CR and UT-3CR, respectively. 750 or
and isolated a number of interrupted Fischer building blocks 1000 nL of each building block was transferred into the desti-
2
9,30
on a gram scale (Scheme 1, Fig. 2).
nation plate which was accomplished in less than 1.5 h (ESI†).
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Fig. 2 Diversity of cyclic Schiff base, isocyanide, carboxylic acid and other building blocks used.
The plate was left sealed for 12 h to react at room temperature.
The information density produced by ADE-enabled nano-
Then, the reactions were analyzed overnight using automatic scale synthesis permits reactivity explorations that were pre-
SFC-MS and TLC-MS (Fig. 3, ESI†). The analysis revealed that a viously impractical or impossible, such as the rapid survey of
total of 159 products (designated with green and yellow colors: building blocks and several scaffolds at once. Patterns of the
8
3%) out of 192 reactions were formed and 114 products were functional group compatibility of the carboxylic acids in
in good quantities (green). Amongst the three investigated U-3CR 4 include free boronic acid (C1), biphenyls (C2, C3);
reactions, the imino hydantoin synthesis (UH-3CR) worked free phenolic –OH in different positions (C8, C9, C11, C12,
best with 92% of the wells showing product formation, C29, C43); several heterocyclic carboxylic acids including
followed by U-3CR (81%) and tetrazole synthesis (77%) (ESI†).
indole (C31, C32, C34), benzofurane (C33), pyrrole (C36), thio-
Noteworthily, multiple polar building blocks in all func- phene (C38), thiophene cinnamic acid (C66) worked well;
tional group classes worked well, including morpholine (B68), several chiral N-protected amino acids (C47, C48, C49, C52)
pyridine (B37), tetrahydrofuran (B55), tetrahydropyran (A8) yielded the expected products and aliphatic hydroxy groups
and unprotected boronic acid (C1). This is important to keep were compatible in C45. Bulky and small aliphatic groups were
control over the lipophilicity of the final products. Subtle reac- well tolerated; alkenes in different positions (C66, C67, C68,
tivity trends were discovered in the data analysis which tra- C70, C71) and nitrile (C61) are also compatible; however, meta
ditionally would be much more cumbersome to obtain. For substituted cinnamic acid (C69) did not give any product.
instance, it was found that phenyl unsubstituted indoles (A1– Overall, scaffold 4 showed a very good tolerance to protected
A4) were more reactive (ESI†). This trend was visible for all and unprotected carboxylic acid groups. A great functional
three Ugi-type reactions (4, 5, and 6). Amongst the phenyl sub- group compatibility is the signature of a synthetically valuable
stituted indoles, the 5-Br substrate A9 surprisingly represented reaction. Similar findings could be observed in the other
an island of reactivity with 66% success rate in all reactions.
scaffolds, 3 and 5.
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Fig. 3 Heat plot of the 192-well array of interrupted Fischer/MCR chemistries based on SFC-MS analysis and exemplary TLC-UV-MS (above) and
SFC-MS (below) analyses of well A22.
The diversity of isocyanides used in this HT experiment is the 192-nanoscale reactions on a 1 mmol scale (ESI†):
unprecedented. An unprecedented high number of 68 highly 10 imino hydantoins 6, 7 U-3CR 4 and 6 tetrazoles 5 (Fig. 4).
diverse isocyanides were brought to reaction, ranging from aro- The isolated yields varied between 26 and 96% with an average
matic to aliphatic to bulky to linear aliphatic to heterocyclic of 60%.
ones (morpholine, furan, tetrahydrofuran, indole, pyridine).
Finally, we investigated the possibility of forming the inter-
Functional group compatibility was tested positive for example rupted Fischer indole products in situ on a nmol-scale and
with amides (B65–B67), acrylamides (B68), ethers (B53, B55), subsequently adding the reagents for the UH-3CR scaffolds, in
and esters (B48, B57, B58, B60, B62). Among the esters, ethyl order to avoid the time-consuming traditional synthesis of the
2
-isocyanobenzoate (B10) did not give any product.
key cyclic Schiff bases. A random array of 96 reactions was per-
Synthetically useful methods should be scalable to ensure formed based on 9 aromatic hydrazine hydrochlorides and 9-
rapid adaption by industry to utilize novel synthetic transform- α,α-disubstituted aldehydes which were first reacted, followed
ations. To show smooth scalability, we resynthesized and fully by addition of aqueous potassium cyanate, Py·HCl and isocya-
characterized 23 unprecedented representative molecules of nides. It was found that the in situ reaction performed worse
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Fig. 4 Resynthesized compounds based on the three scaffolds 4–6 and on their performance in the nano-synthesis (Fig. 3).
(
48%) than the stepwise procedure (92%) using dispensed pur- of classical and new synthetic organic reactions including tran-
3
4
ified interrupted Fischer indoles (Fig. 5). Still, the much sition-metal catalysis by ADE (Fig. 6).
greater variability of accessible cyclic Schiff bases points to the
High throughput experimentation methods empower che-
possibility of synthesizing a very large chemical space of tri- mists to run orders of magnitude more experiments and
cyclic hydantoins with 3 highly variable building blocks (aryl- enable “big data” informatics approaches to reaction design
2
6,35–37
hydrazines, aldehydes, and isocyanides).
and troubleshooting.
The herein introduced nanoscale
ADE is a technology which has already found application in and high throughput ADE-enabled platform provides unpre-
medicine, genetics, biotechnology, high throughput screening cedented insight into structure–reactivity relationships (SRR).
3
2
and protein crystallography. Surprisingly, however, no appli- In traditional reaction discovery/screening, optimization is
cations in the area of miniaturization and acceleration of the done under a few conditions leading to the mmol-scale syn-
synthesis of small molecules have been published. ADE- thesis with a low-density array of novel compounds. ‘Real-
enabled synthesis introduced here has several distinct advan- world’ scope and limitation studies including drug-like hydro-
tages over classical tip-based dispensing. It is a precise, accu- philic fragments to test the level of tolerance of a reaction
rate and contactless dispensing technology suitable for rapid toward the polar functional groups and nitrogen heteroatoms
transport of nanolitre amounts of reagent stock solutions from found in biologically active molecules are impossible to be per-
1
5
a source to a destination plate.
With a frequency of formed with reasonable resources, costs and in time using a
>
100 hertz 2.5 nanolitre-sized droplets can be transported into traditional mmol-based approach. Another advantage is the
nano-reactors to perform thousands of discrete reactions in minute reagent consumption. The ADE-enabled chemistry at
only a few hours. The ‘touch-less’ feature comes with a ‘con- the nanoscale led to a total reagent consumption to scout 288
sumable-less’ feature as compared to other tip-based nano-dis- reactions of less than 4 mg. For comparison, in a traditional
3
3
pensing systems. Moreover, it avoids potential sources of 1 mmol scale per reaction, 150 g of starting material would be
impurities by leakage of plastic additives. Combined ADE- used in addition to 75 L of solvent just to perform the reac-
enabled synthesis and fast quality control using TLC-MS and tions. Consequently, precious starting materials, such as a
SFC-MS was used here to scout three different reactions and large number of complex isocyanides, can be used here.
enable the mmol resynthesis of 23 unprecedented exemplary Moreover, due to the high degree of automation of the ADE-
compounds in good-to-excellent yields. Recently introduced platform, chemists have less contact with potentially hazar-
3
84- and 1536-well high density and sealable glass plates or dous chemicals, solvent fumes and reactions, contributing cri-
classical polypropylene plates promise to enable a wide variety tically to safety aspects. On the other hand, it should be noted
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Fig. 5 Sequence of 2 reactions on a nanoscale in one pot. (A): In situ interrupted Fischer indole followed by Ugi hydantoin reaction. (B): Used phenyl
hydrazine and aldehyde building blocks. (C): Quality control heat map based on SFC-MS data (ESI†). (D): Exemplary structures of formed compounds.
that the reduced analytical characterization (MS instead of chemistry could be advantageously used to pre-screen the
NMR and IR) of nmol over mmol or higher scale reactions is a feasibility of the mg-scale synthesis of a large number of build-
clear trade-off of the herein proposed miniaturization and ing block combinations including unusual and expensive
acceleration of synthetic chemistry and might lead in a few reagents. In traditional mmol-scale chemistry, this would be
cases to misassignment of the structure.
impractical or impossible due to resource, time and cost
High throughput screening (HTS) is a mainstay to the constraints.
current drug discovery paradigm. Million-sized compound col-
Further potential applications of this technology include
lections of small molecules each on a mg scale are housed in high throughput synthesis and in situ screening for protein
the libraries of pharmaceutical companies and form the basis target binding. The feasibility and advantage of miniaturized
of the HTS. Renewal of the content of the compound libraries synthesis and at the same time the successful screening of
3
8
is a very resource and time-demanding process. ADE-enabled unpurified reaction mixtures have already been established.
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Fig. 6 Workflow of automated and accelerated ADE-enabled syntheses. Above: Preparation of stock solutions of building blocks (A) and transfer
into 386-well source plates (B); ADE workplace with an ECHO555 in a chemical hood and an external remote computer control (C); a 384-well des-
tination plate (D). Middle: Quality control with SFC-MS (E) and TLC-UV-MS (F). Bottom: Data analysis (G) and resynthesis of compounds on a
0
.5 mmol scale (H).
Thus, it is conceivable that ADE-enabled organic high through- ment number 115489, the Qatar National Research
put synthesis can be synergistically combined with miniatur- Foundation (NPRP6-065-3-012), the European Union’s Horizon
ized and fast ligand–receptor or even phenotypical screening.
2020 research and innovation programma under the Marie
Sklodowska-Curie (ITN “Accelerated Early stage drug
dIScovery”, grant agreement no 675555; Cofunds ALERT,
(
grant agreement no 665250) and PROMINENT (grant agree-
Conflicts of interest
ment no 754425)). S. S. was supported by a postdoctoral
research fellowship from Kankerbestrijding (KWF grant agree-
ment no 10504). R. X. and L. G. were supported by Chinese
Scholarship Council grants. M. A. M. was supported by a NIH
grant (2R01GM097082-05).
There are no conflicts to declare.
Acknowledgements
We thank Pravin Patil, Konstantinos Neochoritis, Dominik
Zahr, Joe Olechno, Richard Elson and Tryfonas Zarganis for
helpful discussions and Nicolo Tito for writing an extension of
the RandReact program. This project has received funding (to
A. D.) from the European Lead Factory (IMI) under grant agree-
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