Sustainability by design: Automated nanoscale 2,3,4-trisubstituted quinazoline diversity

Article abstract of DOI:10.1039/d0gc00363h

Small molecule synthesis is equally important for materials and pharmaceuticals. However, the traditional approach performed in pharmaceutical R&D including maintenance of million-sized libraries and optimization synthesis of hundreds or even thousands of chemicals on a mmol or larger scale lacks sustainability. Here, we exemplify the synthetic execution of a newly designed quinoxaline reaction towards a transformative sustainability in chemistry. This includes nanoscale synthesis, deep chemical space exploration, scalability over 6 orders of magnitude from milligram up to 10-gram resynthesis of quinazolines enabled by the simultaneous variation of four classes of building blocks. Benefits of our approach include a simple to perform, one-step procedure, mild reaction conditions and access to a very large chemical space through accessing many available building blocks. More than thousand derivatives were produced in an automated fashion on a nanoscale using positive pressure facilitated dispensing. Along with these advantages, there is a considerable reduction in synthetic effort, reagents, solvent, glass and plastic consumables and power consumption to decrease the footprint of synthetic chemistry.

Full text of DOI:10.1039/d0gc00363h

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Sustainability by Design: Automated Nanoscale 2,3,4-Trisubstituted Quinazoline Diversity
Mojgan Hadian,1‡ Shabnam Shaabani,1‡ Pravin Patil,1‡ Svitlana V. Shishkina,2 Harry Boeltz,3 Alexander
Dömling1*
1 Pharmacy Department, Drug Design group, University of Groningen, Netherlands.
2 SSI “Institute for Single Crystals,” National Academy of Science of Ukraine, 60 Lenina Ave., Kharkiv
61001, Ukraine.
3 Dispendix GmbH, Heßbrühlstraße 7, 70565 Stuttgart, Germany.
‡ These authors contributed equally to this work.
*Correspondence: a.s.s.domling@rug.nl
ABSTRACT – Small molecule synthesis is equally important for materials and pharmaceuticals. However,
the traditional approach performed in pharmaceutical R&D including maintenance of million-sized
libraries and optimization synthesis of hundreds or even thousands of chemicals on a mmol or larger scale
lacks sustainability. Here, we exemplify the synthetic execution of a newly designed quinoxaline reaction
towards a transformative sustainability in chemistry. This includes nanoscale synthesis, deep chemical
space exploration, scalability over 6 orders of magnitude from milligram up to 10-gram resynthesis of
quinazolines enabled by the simultaneous variation of four classes of building blocks. Benefits of our
approach include the simple to perform, one-step procedure, the mild reaction conditions and the access
to a very large chemical space through access to many available building blocks. More than thousand
derivatives were produced in an automated fashion on a nanoscale using positive pressure facilitated
dispensing. Along with these advantages, there is a considerable reduction in synthetic effort, reagents,
solvent, glass and plastic consumables and power consumption to decrease the footprint of synthetic
chemistry.

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DOI: 10.1039/D0GC00363H
INTRO – Chemistry, the central science ameliorates human existence by producing compounds of great
use in energy, agriculture, electronics, pharmaceuticals, personal care, cosmetics, and flavors and
fragrances. However, with an ever-growing number of planetary inhabitants, sustainability is the only
guaranty of a livable future. Achieving a sustainable future in chemistry is the major goal of green
chemistry.1 Often chemists use the 12 Principles of Green Chemistry as a blueprint for chemical design for
sustainability throughout the life cycle of chemicals.2 Efforts to increase the green footprint of organic
synthetic chemistry (e.g. non chlorinated solvents, catalysis, non-toxic reagents) are therefore major
transformative drivers of a sustainable future in industry and society.3 Despite these honorable ideas, the
reality of contemporary chemistry in wide areas looks quite different: energy intensive processes, toxic
reagents and solvents, expensive, wasteful and time-consuming sequential target syntheses resulting in
overall low yields, clearly far from sustainability. Particularly, medicinal chemistry can be highlighted as a
chemistry area where inclusion of sustainable methods and processes would have a tremendous impact.4
Pharmaceutical R&D is a highly compound intensive chemistry area. Large and medium sized pharma
companies keep millions of compounds in screening libraries, traditionally on a gram scale, more recently
on a multi milligram scale. These have to be regularly renewed to stay competitive and cope with ‘novelty
erosion’.5 These screening compounds are mostly produced via multistep synthetic routes that are neither
optimal nor green. Next, the hits from high throughput screening of the million compounds have to be
optimized for ‘drug-like’ properties in tedious, lengthy and expensive process involving the synthesis of
thousands of intermediates and target compounds. Despite the exploratory scale, waste generation in
early drug discovery is considerable. Conservatively, it has been estimated that up to 2 million kilograms
of waste are produced per year in drug discovery with up to 1.5 million kilograms of additional waste
produced per year in preclinical studies.6 Inevitably, drug compound optimization involves multiple low-
yielding steps, requires often several solvent-intensive chromatographic purifications, and generates a
large amount of plastic, glass and paper consumable waste. Key to sustainability in synthetic chemistry is
waste reduction. The principle of prevention has been recently highlighted to overshadow all other
principles of green chemistry.7 The emerging field of reaction miniaturization is therefore offering an
outstanding opportunity in the ultra-conservation of advanced pharmaceutical intermediates and
additionally in the overall reduction of waste achieved by performing reactions on a smaller scale.4
Moreover, multicomponent reaction chemistry is based on the principles of atom economy, step
prevention, convergency and has been recognized since a long time to come close to ‘perfect’ green
chemistry and has also been recently reviewed in this context.8 It has been repeatedly shown that by
introducing a multicomponent reaction (MCR) into a target synthesis the overall number of steps can be

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considerably reduced while yields are increased.9-11 A perfect example is the Teleprevir HCV inhibitor
synthesis by Orru et al.10 Introducing two MCR steps in the synthesis of this complex peptidomimetic
compound reduced the lengthy >20 steps industrial synthesis by ~50%. Here we exemplary showcase the
discovery and scope and limitation study of a new reaction leading to the small molecule quinazoline
scaffold based on several major sustainability principles.
Figure 1. Overall research design. A) Design of medium cycle synthesis and surprising formation of highly
substituted quinazolines instead; B) crystal structure of compound H17; C) the quinazoline moiety in drugs
and natural products; D) sustainability principles used in the design of the present research.

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DESIGN – SYNTHESIS Initially, we designed a one-step general synthesis of medium cycles from readily
accessible starting materials employing the archetypical Ugi-4CR.12-14 We hypothesized that suitable -
formyl--carboxylic acids (C) will react to a medium cycle ring upon addition of a primary amine (D) and
an isocyanide (E) (Fig. 1A). To gain access to a diversity of -formyl--carboxylic acids (C) we planned to
react o-formyl anilines and heteroaromates (A) with cyclic carboxylic acid anhydrides (B). Both building
blocks are commercially available in larger numbers with different substituents and ring sizes. Surprisingly
and proven by i.a. a small molecule crystal structure we found that the formation of a 2,3,4-trisubstituted
quinazoline instead of the expected medium cycle (Fig. 1A and B, Fig. 2). Opportunistically we figured that
the quinazoline scaffold shares key pharmacophoric and structural features of a large number of
biologically relevant compounds (Fig. 1C).15
CHO
NH2
O
O
Z
O
Y
+
Y
X
Y
O
O
Y
X
Z
N
OH
n
n
H
CHO
X= Cl
Y= N
Z = CH2, O, S, N
n = 0-2
C
B
A
Y
X
Y
O
O
X
Y
N
Z
CO2H
1 + CN R2
n
Z
n
H2N R
+
N
OH
Y
N
R1
H
CHO
P
E
C
D
HN
R2
O
Figure 2. New 2-step quinazoline synthesis.
We commenced our study by the synthesis of -formyl--carboxylic acids as building blocks via a simple
ring opening reaction of cyclic anhydride with various derivatives of 2-aminobenzaldehyde, both
commercially available in great numbers. To optimize the reaction conditions of the model system 2-
aminobenzaldehyde/glutaric anhydride, we tried different solvents and mixtures, temperatures and ratios
of reactants (SI Table S1). The best conditions were 1:1.1 ratio of 2-aminobenzaldehyde/glutaric anhydride
and refluxing in dioxane to afford the desired product in 95% yield. Fifteen -formyl--carboxylic acids of
different lengths were synthesized in good purity and yields, each on a gram scale (Fig. 3). This included
benzene as well as pyridine derivatives (C-3, C-10). The diversity of the included anhydride ranges from 5
to 7-membered simple aliphatic chains (C-1, C-2, C-15), to 3,3-dimethyl substituents (C-4), to cis-alkene
(C-11, C-12), phenyl (C-13), spiro cycle (C-5) to a conformationally constrained cyclopropyl annulated ring
(C-14). It also includes basic nitrogen (C-7), oxygen (C-9) or sulfur (C-8). The aminobenzaldehyde

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component showed variation in halogen (C-6, C-7, C-12, C-14) as well as two positional pyridine isomers
(C-3, C-10).
CHO
NH2
O
O
Z
O
Y
X
Y
O
O
Y
1,4-dioxane (0.16 M)
reflux, 18 h
+
Z
N
OH
Y
n
X
H
CHO
n
X= Cl
Y= N
Z = CH2, O, S, N
n = 0-2
A
B
C
O
N
O
O
CO2H
CO2H
N
H
CO2H
N
N
H
H
CHO
CHO
CHO
C-2
C-3
C-1
, 47%
, 86%
, 95%
Cl
O
O
O
CO2H
CO2H
CO2H
N
H
N
H
N
H
CHO
CHO
CHO
C-6
, 56%
C-4
C-5
, 60%
, 70%
Cl
O
O
O
N
CO2H
O
CO2H
S
CO2H
N
H
N
N
H
H
CHO
CHO
CHO
C-9
, 91%
C-7
, 75%
C-8
, 89%
Cl
N
O
O
CO2H
O
CO2H
CO2H
N
N
N
H
H
H
CHO
CHO
C-12
CHO
C-10
, 70%
, 52%
C-11
, 85%
Cl
O
O
O
CO2H
CO2H
N
H
N
CO2H
N
H
H
CHO
CHO
CHO
C-15
, 45%
C-14
C-13
, 80%
, 83%
Figure 3. Synthesis of -formyl--carboxyl building blocks C with isolated yields and selected crystal
structures.

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In the next step, we optimized the conditions of the quinazoline ring closure via Ugi reaction (Fig. 4). The
optimization was performed by using 5-((2-formylphenyl)amino)-5-oxopentanoic acid (C-2), 2-(4-
methoxyphenyl)ethan-1-amine (D-22), and benzyl isocyanide (E-25) as a model reaction (SI Table S2). We
probed various parameters including solvents, acid additives, ratios of reactants, microwave, sonification.
Polar aprotic solvents such as THF and CH3CN gave the product in moderate yields of 30% and 22%,
respectively, at room temperature, but methanol and trifluoroethanol showed better results. As
trifluoroethanol (TFE, 76% yield) was slightly superior to MeOH (71% yield), we chose TFE for further scope
and limitation studies. Next, different acids such as ZnCl2, Sc(OTf)3, Al(OTf)3, and p-TSA were screened. It
was found that the addition of Lewis or Broenstedt acids does not make a great difference on percentage
yield of products. Additionally, we investigated different stoichiometries of -formyl--carboxylic acids,
amines and isocyanides. Using less than 2 equivalents of amine and isocyanide does not have any effect
on the percentage yield, but it reduces the amount of unreacted starting materials. Moreover, we
investigated sonication and microwave conditions, however the reaction works best at room
temperature.
O
CHO
NH2
O
O
O
1,4-dioxane (0.16 M)
reflux, 18 h
CO2H
+
N
H
CHO
C-2
, 95%
A-1
1 eq
B-1
1.1 eq
CO2H
CN
H2N
N
O
TFE (0.2 M)
rt, 18 h
+
+
N
CO2H
N
OMe
H
CHO
HN
O
OMe
C-2
1 eq
D-22
1.1 eq
E-25
P2
, 84%
1.1 eq
Figure 4. Optimized conditions for the anhydride ring opening (6 mmol scale) and U-4CR (1 mmol scale).
With the optimized reaction conditions in hand, the scope and limitations of the Ugi-quinazoline reaction
were further investigated by synthesizing large libraries of compounds using nano scale automated
chemistry (Fig. 5).

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Figure 5. Nanoscale synthesis of a library of quinazolines. A) I-DOT nano dispenser instrument. B) The
“Immediate Drop on Demand Technology” (I-DOT) uses a non-contact, pressure-based dispensing
technology. Applying a well-defined pressure pulse on top of a 96-well microliter plate with holes in the
bottom of each well forms a highly precise nanoliter droplet that is released into any target plate. C) A
source 96-well plate with isocyanide and -formyl--carboxylic acid building blocks. D) A 384-well
polypropylene target plate. E) A heat plot of a representative 384-well plate based on MS analysis. F) A
pie chart of the overall synthesis success of the 384-well plate.
NANOSCALE SYNTHESIS Recently we reported nanoscale synthesis of libraries of different scaffolds using
acoustic dispensing ejection technology (ADE).16-18 A disadvantage of ADE is the limited solvent

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compatibility which somehow restricts organic chemical reactions. Currently, only solvents are compatible
with a rather high boiling point and a low volatility such as DMSO, water, ethylene glycol and similar.
However, Ugi reactions prefer to be performed in low boiling alcohols and in the current reaction we
found trifluoroethanol to be favored over other solvents. Therefore, we were evaluating a range of other
nano dispensing technologies which are compatible with a broader range of volatile solvents. The
“Immediate Drop on Demand Technology” (I-DOT) is such a nano dispensing technology and has been
mostly applied to analytical and biological questions (Fig. 5A-D).19-22 A well-defined pressure pulse on top
of a 96-well microliter plate housing the building block stock solutions with holes in the bottom of each
well forms a highly precise nanoliter droplet that is released into any target plate (96-, 384-well or higher
formats). The I-DOT source plate is located above the target plate that moves underneath. Each individual
source well can dispense into each well in the target plate with a different and precise volume. Up to 96
different liquids can be used within one dispensing run. This approach enables highly efficient
combinatorial dispensing useful in chemistry. Larger volumes are achieved by applying up to 400 pulses
per second. As the ADE system, the platform works contact-less and is sustainable as it avoids the use of
plastic tips. Using in-house written software, we synthesized non-combinatorial libraries in 384-well plates
by combining three building blocks in each target plate well.

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Figure 6. Representative 384-well plate with and compounds resynthesized on a mmol scale with isolated
yields and an exemplary X-ray structure.1
The reactions were performed at 0.16 M concentration with a total volume of 500 nL per well by
dispensing TFE stock solutions of the building blocks into the target plate wells. 42 Primary amines and 41
isocyanides were used to build the libraries (Fig. 7). The time to fill a 384-well plate with the three starting
material classes of building blocks was less than 20 min. The reactions were run at room temperature for
24 h by sealing the plates and leaving them on the shaker. Then the plates were desealed and analysis of
the wells was performed by injecting the crude mixtures into the mass spectrometer. Using an in-house
written software, the raw MS data was analyzed for the presence of the reaction’s products. A qualitative
three-color classifier was used: blue for no reaction product, yellow for minor product formation and
green for major product formation. A typical analysis outcome is shown in Fig. 6 resulting in 39% major
product and 34% minor product formations and 27% failed reactions (Fig. 5F). A total of three 384-well
plates was filled resulting in 1.152 reactions. The used building blocks go much beyond what is usually
employed during the report of a new reaction and scaffold. However, it is important to challenge new
reactions with many different sterical and electronical building block variants to get a ‘real-world’ insight
into scope and limitations.
1 K7 is shown blue because of I-DOT reagent transfer failure, but their synthesis worked nicely.

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Figure 7. 96-well source plate and the color-coded structures of the amine (blue) and isocyanide (red)
building blocks used. The -formyl--carboxyl building blocks are shown in Figure 3.
Of note is the great functional group tolerance of the new quinazoline synthesis: dimethoxyacetale (D-23,
E-14), heterocycles such as tetrahydrofuran (E-29), indole (D-35, E-22), thiophene (D-24, E-38),
morpholine (D-39, E-6), piperidine (D-40), azetidine (D-25), pyridine (D-3, D-21, D-33, E-3), furan (D-28),
alkyne (D-14), alkene (D-41), primary alcohol (D-7, D-15), ether (D-10, D-17, E-31), Boc-protected amine
(D-11, D-25, D-27, E-19), ester (E-15, E-22, E-41), nitrile (D-12), sulfonamide (D-31), and tertiary amine (D-

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40); aliphatic cycles from 3-6 membered (D-6, D-34, D-29, D-4), highly substituted benzene (E-23) and
very bulky adamantly group (D-37).
MMOL AND GRAM SCALE SYNTHESIS – Scalability of a given reaction is important. For example, during
the optimization process of a lead, gram amount of high-quality material is needed for performing many
different biological assays including pharmacokinetic studies in animals. Scalability is more complex than
just making larger quantities of a particular compound. It is well known that many reactions are difficult
to reproduce (yield, impurity profile, reaction setup, course, and workup), pose safety concerns
(exotherms, decomposition profiles), exhibit issues in waste handling (limited number of acceptable
solvents), cost (yields, expensive stoichiometric reagents), sourcing (suppliers), and ease of operation
(avoiding chromatography, fractional vacuum distillation, etc.). Thus, we investigated scalability from
nano scale to gram scale of our novel quinazoline synthesis. Resynthesis of several compounds on a mmol
scale confirmed the excellent performance of the new MCR quinazoline synthesis. 15 Compounds could
be isolated in 40 to 88% yield with an average of 66% and fully characterized by NMR, HRMS, IR and one
X-ray structure (SI page 76), (Fig. 6).
Figure 8. Multicomponent quinazoline synthesis on a 10 g scale.
Next, we investigated the scalability of the reaction beyond 1 mmol (Figure 8). The first acylation step was
performed in refluxing dioxane to isolate 90% of -formyl--carboxylic acid (C-2) on a 7 g scale as a pale-
yellow powder. The Ugi reaction in trifluoroethanol as a solvent at room temperature gave C11 in >80%
yield and >10g of a yellow solid. Thus, we could show that our newly discovered quinazoline synthesis is
easily scalable over 6 orders of magnitude from nmol (81 nmol) to mmol (30 mmol).

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A mechanism of this unusual Ugi reaction is proposed in Figure 9. Accordingly, a Schiff base (I1) is formed
from the primary amine (D) and the -formyl--carboxylic acid (C) which is protonated (I2) and thereby
activated towards the nucleophilic attack of the isocyanide forming the nitrilium ion (I3). Nucleophilic
attack of the carboxylate to the nitrilium carbon (I4), followed by a transannular attack of the secondary
amine to the benzamide carbonyl (I5) followed by dehydration yields the tricyclic intermediate (I6).
Unusual in this mechanistic proposal is the avoidance of the common Mumm rearrangement (I4) which
would give a strained medium cycle intermediate (ring size between 9 and 11, dependent on the ring size
of the anhydride). We hypothesize that the medium cycle is very unstable based on the considerable
antiperiplanar and transannular ring stains. The ring strain in medium sized carbocycles has been
intensively investigated and described.23, 24 Water instead is hydrolyzing the N-hetero anhydride structure
(I6) and the tricylic bridgehead structure forms a favorable 6-membered bicyclic product (P).
OH
Z
n
O
O
N
O
Ar
Z
Ar
+
OH
H2N R1
+
C
N R2
N
n
N
O
H
R1
CHO
HN
R2
P
C
D
E
O
O
O
OH
O
OH
Z
n
Z
Z
n
n
NH
N
O
NH
O
NH
N
O
Ar
NH2
Ar
+
Ar
C
N
R2
H
protonation
of imine
-H2O
R1
E
O
H
R1
R1
C
D
I1
I2
H
N
O
OH
O
O
Z
HN
n
Z
n
Ar
Z
NH
O
N
n
Ar
R1
Ar
O
R2
O
N
C
O
N
O
HN
R1
R2
NH
R1
N
R2
I5
I4
I3
-H2O
OH
N
Z
n
Ar
Z
N
O
N
n
R1
Ar
N
R1
O
N
O
O
H
R2
H
HN
R2
O
P
I6
Figure 9. Hypothesized reaction mechanism of this Ugi MCR.
CHEMOINFORMATICS - A simplified pharmacophore model and some calculated properties for the novel
quinazoline scaffold is shown in figure 10. The compounds are zwitterions with a positive and negative

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charge area. The isocyanide-derived amide group exits orthogonal to the quinazoline ring plane, thus
featuring a strong 3D character of the scaffold. The density and orientation of the scaffold-intrinsic
pharmacophores provides ample opportunities to interact with biological receptors. The ‘escape from
flatness’ 3D property (Fsp3) has recently be proposed to contribute to drug-likeliness of small molecules
(Fig. 10 B).26 Calculation of some physicochemical parameter features additionally drug-like behavior. The
lipophilicity and molecular weight are two other important determinants. The molecular weight and the
lipophilicity of a considerable part of the synthesized library is in a ‘preferred’ region (Fig. 10 C). Due to
the scaffold intrinsic charge it can be expected that the molecules will not easily pass the cell membrane.
However, many known charged bioactive compounds can pass the membrane if a transporter mechanism
applies.
Figure 10. Some chemoinformatic features of the quinazoline scaffold. A) Pharmacophore model featuring
positive and negative charges, an extended aromatic pi system, and an exocyclic secondary amide group
with hydrogen bond donor and acceptor. B) Moment of inertia analysis of the synthesized library as a
measure of complexity (Fsp3) with three explicit compounds (G23, K8, L14) shown. C)
Lipophilicity/molecular weight analysis indicating a considerable portion of the quinazoline chemical
space in the ‘preferred’ region MW<500 and cLogP<3.

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The accessibility and availability of small organic molecules with a high functional group density as seen
in the structural complexity of pharmaceuticals presents a significant challenge to modern synthetic
reactions. Many published methods that work well on simple substrates often fail when attempts are
made to apply them to complex drug intermediates. Despite the number of compounds synthesized in
the industry is steadily increasing over the years, a heavy population bias towards only a few chemical
reactions can be observed. It was noted that a limited arsenal of medicinal chemistry reactions may
narrow the available chemistry space to structurally similar compounds which spill over to perhaps
(unwanted) biased screening collections. Therefore, novel synthetic methods, e.g. multicomponent
reactions, may not only unlock access to previously unattainable chemical matter, but also inspire new
concepts as to how to design and build chemical matter.27 Our herein introduced quinazoline synthesis
comprise an example of high throughput experimentation on a nano scale allowing for the rapid screening
of scope and limitations of a unique reaction towards a novel scaffold.28 The two-step synthesis involves
in the first step a macroscopic gram-scale acylation of o-amino benzaldehydes or corresponding
heterocycles with cyclic carboxylic acid anhydride to afford -formyl--carboxylic acid building blocks,
followed by a microscopic nano-scale Ugi reaction to form highly substituted quinazolines according to an
unprecedented mechanism. The minute reagent and solvent consumption of ~50 mg of building blocks
and ~0.6 ml of solvent is a feature of this work contributing to the sustainability of the nano synthesis
approach described herein. The nano scale synthesis was facilitated by a positive pressure dispenser (I-
DOT) and >1000 reactions could be processed in several 384-well plates in a short time and in an
automated fashion. The nano scale synthesis and the tip-less feature of I-DOT additionally contributes to
lower the synthetic chemistry footprint. MS analysis of the plates revealed that 1/3 of the expected
products were formed well, while 1/3 where not formed at all. The automation aspect increases safety as
chemists are less in contact with hazardous chemicals. The scalability of the nano reactions was shown by
the resynthesis of 15 quinazolines on a mmol scale in generally good to very good yields. One quinazoline
was even prepared on a 10 g scale in 73% overall yield (over two steps). Moreover, the scope of the
building blocks was shown to be great and many functional groups were tolerated. The compounds of the
library were found have all over benign properties (MW, clogP and Fsp3) and the compounds where found
to be stable at room temperature and in aqueous buffer at 37 oC. We are currently testing the compound
libraries against therapeutic targets and screening results will be reported in due course.
ACKNOWLEDGEMENTS - This research has been supported through ITN “Accelerated Early stage drug
dIScovery” (AEGIS, grant agreement No 675555). Moreover, funding was received by the National

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Institute of Health (NIH) (2R01GM097082-05), the European Lead Factory (IMI) (grant agreement number
115489), the Qatar National Research Foundation (NPRP6-065-3-012) and COFUNDs ALERT (grant
agreement No 665250) and Prominent (grant agreement No 754425). SS is supported by a KWF
Kankerbestrijding grant (grant agreement No 10504).
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