University of Groningen

Target-based drug discovery: from protein structure to small-molecules by MCR chemistryWang, Yuanze

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Chapter 1

General Introduction and Scope of this Thesis

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1. Phenotypic-based Drug Discovery

Drug discovery can generally be viewed as a challenging process that includes the

identification of active substances with a desirable therapeutic effect on certain

diseases and optimization of those substances to increase their efficacy,

pharmacokinetics and safety. Historically, phenotypic-based screening strategies,

also known as classical pharmacology or forward pharmacology, were the main

drug discovery paradigms used in both academic research centers and the

pharmaceutical industry (Figure 1.1). The phenotypic approach could be

specifically defined as the screening of a large number of randomly selected

molecules in a system-based and target-agnostic approach that monitors the

desirable change in phenotype. Thus, the active ingredient was obtained without

any knowledge of the biological target. More often, an effort was made to identify

the target after the active substance was identified. 1-3

Figure 1.1 Evolution of drug screening and lead discovery (adapted from reference 1).

2. Target-based Drug Discovery

Since the late 1980s, technological advances in molecular biology and sequencing

of the human genome, which allowed rapid production of large quantities of

biological targets initiated a new era of molecular target-based screening approach

for modern drug discovery.

Target-based or hypothesis-based drug discovery is an approach in which small

molecules are synthesized to target a known pathological/physiological pathway,

which is hypothesized to be involved in the treatment of a particular disease. This

idea came from the conclusion that a drug has to interact with biological

macromolecules of the human body (enzymes, receptors, channels, etc.). Thus,

biological macromolecules are used by the scientist in the lead discovery

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screening, rational design and optimization. After that, the selected lead

compounds were tested in cell and animal experiments to prove their efficacy. It

was believed that this approach would result in an increased productivity and

improved efficiency toward development of novel treatments for a validated

target as it avoids the mass screening of banks of stored compounds. Moreover,

according to a recent analysis, 78 of 113 first-in-class drugs approved by FDA

from 1999 to 2013 were discovered through the target-based approach.4

2.1 High Throughput Screening

The drug-discovery process usually involves high throughput screening (HTS),

wherein a large number of compound libraries are tested for identifying a few

‘active hits’ to modify the target. These ‘hits’ provide starting points for drug

design and are then followed up to undergo several iterative screening runs to

evaluate their properties, including off-target toxicity. The ‘validated hits’ are

then used as ‘lead compounds’ for further studies. HTS has become one of the

dominant paradigms for lead identification and has gained widespread popularity

among both the academic researchers and industrial scientists over the last three

decades.5 The main goal of the HTS technique is to accelerate the drug discovery

process and reduce the costs of drug development. Meanwhile, it is of vital

importance that HTS also takes advantage of the changes in chemical synthesis

strategy. It is obvious that combinatorial chemistry and parallel synthesis enable

the efficient generation of a vast number of novel compounds.

With the increasing popularity of HTS, the technology required for HTS has also

evolved rapidly. Using robotics, liquid handling devices, data processing software

and high content imaging, an Ultra High-Throughput Screening process allows

the researcher to conduct 100,000 assays per day. Initially, the libraries were

arrayed into 96 micro-well plates, but now these have been replaced by high-

density microplates with up to 3456 wells per plate.

The quality of the compounds in the HTS library can be assessed by several

parameters. One of the most important parameters is the Lipinski's Rule of Five:

the cLogP not greater than 5, molecular mass less than 500 Daltons, no more than

5 hydrogen bond donors and no more than 10 hydrogen bond acceptors. Some

other parameters, such as lipophilicity and ligand efficiency are also used to assess

the drug-likeness of the compounds.6

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2.2 Fragment based drug discovery

HTS has considerably expanded the number of high quality lead molecules that

could be evaluated for many drug discovery programs. However, the drug

development process still suffered from high attrition rates as optimization of the

screening hits could be problematic due to large size and inappropriate physical

properties. Additionally, hit rates were generally low when some intractable

biological targets were screened. In the context of the great demand to find small

molecule drugs more efficiently, Fragment based drug discovery (FBDD) has

emerged as a complementary strategy for our drug discovery arsenal.7-11As a new

promising approach, FBDD has several advantages. In FBDD, each atom of the

fragment is involved in the desired binding interactions with the target. In other

words, although the binding affinity of the fragment is much weaker than the HTS

hit, it actually forms interactions with higher quality. Thus, the optimization of

the fragment into a higher affinity lead is much easier than optimizing the HTS

hit which contains some mismatched groups. In addition, the size and other

physical properties such as lipophilicity and polar surface area can be better

controlled.

Obviously, the successful design of a fragment library is the basis of the fragment-

based approach.12 The library should be designed on the basis of several

considerations such as sample relevant chemical space, appropriate complexity,

synthetic tractability, fragment hit rate and knowledge of binding target. More

importantly, fragments in the library typically obey a rule of three (RO3):

molecular mass < 300 Da, the number of hydrogen bond donors < 3, the number

of hydrogen bond acceptors < 3 and cLogP ≤ 3. Another consideration is the

number of the fragments included in the library, normally on the order of 103,

because these fragments cover better chemical space than the compounds

included in the HTS libraries.

As the fragment normally forms relatively weak interaction with the target

macromolecules (100 µM to 10 mM range), sensitive biophysical techniques need

to be used. According to a report by S. D. Pickett and coworkers, surface plasmon

resonance (SPR), NMR spectroscopy and fluorescence-based thermal shift assay

are the most popular techniques used for fragment-based drug discovery, followed

by X-ray crystallography, mass spectrometry (MS) and isothermal titration

calorimetry (ITC). Other useful techniques include capillary electrophoresis (CE),

biolayer interferometry (BLI) and microscale thermophoresis (MST). In our lab,

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the fragment screening strategy involves a three-stage cascade of biophysical

screens: 1) preliminary screening using medium throughput technique:

differential scanning fluorimetry (DSF), 2) validation of the binding of fragment

hits with target protein by ITC or MST, 3) characterization of the fragment-target

interaction by X-ray crystallography (Figure 1.2).

Figure 1.2 Three-stage cascade of biophysical screens for fragment based drug discovery.

To improve the potency of the validated fragment hits, three main approaches

have been successfully established with the knowledge of the fragment binding

mode: fragment linking, fragment merging and fragment growing.13Fragment

linking requires covalently joining two or more fragments that are known to bind

in different but close pockets to form a novel-scaffold ligand on the same target.

Fragment merging involves the incorporation of the structural features of other

overlapping ligand or substrates into the fragment. Fragment growing refers to the

modification of the fragment by chemical synthesis to incorporate more

interactions. More frequently, fragment growing was used as the preferred

fragment-optimization strategy, as fragment linking and merging are more

challenging.

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3. Proteins as drug target

Target-based drug discovery normally starts with the identification and validation

of a drug target which can be used for lead screening. It is very important to have

enough evidence to support the drug target selected before it is used for the drug

discovery. A good drug target includes three core factors: 1) it participates in a

crucial biological process linked to diseases; 2) it contains binding sites which can

interact with drug-like molecules; 3) its structure and function has been clearly

characterized and is different with known targets. The vast majority of the

approved drugs use proteins as their targets. In a recent survey, the number of

protein targets for all classes of available marketed drugs was found to be 324,

including both human proteins and proteins from pathogenic organisms.14 In

terms of potential drug targets, it was estimated that there are 2000-3000

druggable proteins in humans.

3.1 Protein expression and purification

The structural biology studies, such as the ligand-protein binding assay and the

X-ray crystallization experiment, need large amount of proteins. In general, for

the production of recombinant protein, Escherichia coli (E. coli) is used as the

preferred host because it has several advantages: 1) simple and convenient; 2)

rapid and cheap; 3) labeling methods for NMR are well established; 4) a wide

variety of commercial products are available. The protein expression starts with

introducing the plasmid that encodes the protein of interest into the E. coli cell,

then the expression is induced and the presence of the protein is finally checked

by SDS-PAGE analysis. It is important to establish the optimal growth and

expression conditions with small-scale cultures before the protein is produced in

large-scale. Most recombinant proteins can be expressed at high levels in E. coli

when the host strain and vectors are carefully chosen, and the culture conditions

are properly controlled.

If the produced protein is stable and soluble, the purification can be easily

achieved by the affinity tag column, ion-exchange chromatography and size

exclusion chromatography. However, many polypeptide gene products are

expressed in an insoluble form that lack functional activity. This happens

especially when the protein is expressed at high levels, leading to aggregation and

formation of inclusion bodies. The formation of inclusion bodies can also be

influenced by the vector and host cell selected, the nature of the protein, as well

as the growth and induction conditions. The inclusion bodies need to be purified

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under denaturing conditions instead of standard purification methods, which are

based on the protein’s native solubility. It is usually necessary to renature the

inclusion bodies to reconstruct its three dimensional structure before it is used for

structural studies. In this case, the refolding experiments need to be designed

empirically for each individual protein.15-18

3.2 Protein crystallization

The understanding of the molecular mechanism of the protein function by its

three-dimensional structure is a very important tool for the modern

biotechnological research.19 Protein crystallization is one of the most powerful

ways for structure determination, which have significant impact on the rational

drug design. Therefore, the field of protein crystallography has undergone an

enormous expansion in recent decades, which was indicated by the rapid growth

of protein structures deposited in the Protein Data Bank (PDB).

To crystallize a protein, we first need to purify large quantities of proteins (5-50

mg) with high purity and homogeneity. There are several methods to check the

quality of the protein. The CD spectroscopy can help us to detect if the protein is

correctly folded. The presence of aggregates could be determined by techniques

such as dynamic light scattering (DLS). These techniques are crucial because

sometimes the unfolded protein might also exist in a soluble form. In addition,

additives like salts, metals and co-factors are sometimes necessary for the protein

stabilization, which can be identified by the established DSF buffer screening

methods. It is vital to keep in mind that protein quality is one of the key factors

for the success of the crystallization.

Even when soluble protein with good quality is available, finding crystallization

conditions for a new protein is still sometimes like searching for a needle in a

haystack, because it is a complex and multi-parametric process. The most basic

factors for crystallization include protein concentration, the presence of salt,

precipitate and the pH of the buffer. Among these factors, the role of the

precipitate is to bind with water molecules in order to bring the solution into

supersaturation. Ammonium sulfate and polyethylene glycol are two commonly

used precipitates.

From the crystallization phase diagram, we can see that there are four areas

distinguished (Figure 1.3). The precipitation zone, where the protein is super

saturated and precipitates; the nucleation zone is less supersaturated, where

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.

Figure 1.3 Protein crystallization phase diagram (adapted from reference 19).

nucleation will not take plate but it is good for crystals to grow; the metastable

zone is the best area, where large and well-ordered crystals might form and the

under-saturated zone is an area, where the protein will never crystallize, because

the concentration is too low. The process of the protein crystallization proceeds

in two steps: the formation of nuclei and crystal growth. In an ideal experiment,

once a critical nucleus has formed, the system might go into the metastable zone

naturally as the protein concentration drops. If this is not the case, there are several

methods like (i) microbatch, (ii) vapor diffusion, (iii) dialysis and (iv) free

interface diffusion which can help to reach the nucleation zone and the metastable

zone.

4. Small molecules synthesized by MCR chemistry

4.1 Ugi reaction

Multicomponent reactions (MCRs) are generally defined as a one-pot process

during which three or more starting materials react in a single chemical step to

form a product that incorporates all or most of the atoms of the reactants. Through

the years, MCRs have attracted considerable interest in organic chemistry due to

their convergence, atom economy, efficiency, shortened reaction time and

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simplicity. Meanwhile, MCRs have also met renewed interest for medicinal

chemists, because they are also powerful synthetic strategies for the construction

of biologically interesting scaffolds with huge chemical diversity and molecular

complexity. Among MCRs, the Ugi four-component reaction (Ugi-4CR) is one of

the most widely used reactions which based on the peculiar reactivity of

isocyanides. The Ugi-4CR involves a condensation of a carbonyl component

(aldehyde or ketone), an amine, a carboxylic acid and an isonitrile to afford the

peptide-like α-acylaminoamides with a newly created stereogenic center.

4.2 Mechanism of Ugi reaction

After the pioneer work of Ivar Ugi on the discovery of the Ugi reaction in 1959,

the first mechanistic proposal was postulated by Ugi himself.20 Almost all the

synthetic developments around this reaction rely on this mechanistic assumption

as depicted in Scheme 1.1, path A. The initial step of the reaction is the formation

of an imine 3 by condensation of the amine 1 with the oxo-component

(aldehydes/ketones) 2 with loss of one equivalent of water. The imine 3 is then

activated by proton exchanging with carboxylic acid 4. Subsequent nucleophilic

addition of the isocyanide 6 with its terminal carbon to the iminium ion 5 forms

intermediate nitrilium ion 7. This intermediate then undergoes a second

nucleophilic addition by the carboxylate anion gives intermediate imidate 9. The

last step is an irreversible Mumm rearrangement with transfer of the acyl imidate

to generate the final product 10. In this mechanism, all the reaction sequence are

in equilibrium except for the last step, which is considered as the driving force for

the total reaction sequence.

Scheme 1.1 Two possible mechanisms for the Ugi reaction

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Recently, an alternative mechanism raised the debate by proposing the formation

of hemiaminal as a key intermediate (See Scheme 1.1, path B). The first step of

this pathway is still the formation of imine which is activated by acid component.

However, instead of isocyanide addition, the carboxylate anion first attacks the

iminium ion to form intermediate hemiaminal 8, which is followed by isocyanide

insertion to furnish the same imidate intermediate 9.

Scheme 1.2 The Ugi reaction started form electrophilic iminium species

(Structure adapted form reference 21-24)

The formation of the imine as the first step was generally admitted in both

mechanisms. This was also demonstrated by the direct use of electrophilic

iminium species in the Ugi reaction (Scheme 1.2). In 1982, Nutt and co-workers

first reported the use of substituted 1-pyrrolines instead of the amine and aldehyde

components to produce substituted prolyl peptides by an Ugi-type three-

component reaction (U-3CR).21In 2004, N-acylazinium salt formation in situ was

reported by Lavilla and co-workers as a new source of iminium ion equivalents.22

In 2007, the use of the 3,4-dihydroisoquioline dehydrogenated by in situ oxidation

of the corresponding secondary amine tetrahydroisoquinoline in U-3CR was

explored by Zhu and co-workers.23 Recently, Orru’s group reported

stereoselective synthesis of highly functionalized, optically pure 3,4-substituted

prolyl peptides by Ugi reaction starting from optically active 1-pyrrolines.24

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Scheme 1.3 Imidate intermediate isolated from Ugi reaction

(Structure adapted form reference 25-27)

In fact, the existence of imidate intermediate was also proved by several studies

(Scheme 1.3). The first successful isolation of the imidate 11 and 12 has been

reported by Ugi in 1971.25 In a research to explore thiols in the Ugi–Smiles

reaction, Barthelon and co-workers surprisingly obtained thioimidate 13 in good

yields when using methyl mercaptosalicylate as acid component.26In 2009, Faggi

and co-workers isolated a stable imidate 14 in the tautomeric enediamine form,

which was further demonstrated by X-ray crystallography.27

Figure 1.4 Energy profile of intermediate in Ugi reaction

(Structure adapted form reference 28)

In 2012, Cheron and co-workers reported the first theoretical calculation of Ugi

reaction, which was performed at the M06-2X/6-31+G (d, p) level of theory

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including ZPE corrections, based on Ugi’s proposal presented in Scheme 1.1, path

A.28 Both Ugi-Mumm and Ugi-smiles reaction were studied with a realistic model

and the calculation was computed separately in two solvents: methanol and

toluene. According to the energy profile, optimization of the transition state (TS)

for the insertion of the isocyanide in the hemiaminal leads to the TS of subsequent

isocyanide addition to the iminium. Thus, path B proceeds through hemiaminal

first fragmentation into the iminium and carboxylate anion and then the

isocyanide addition. This was also confirmed by the intrinsic reaction coordinate

(IRC) approach, which is a valuable tool to check if the given transition state is

the expected transition state for the reaction of interest. Therefore, path A in

Scheme 1.1 was considered as the privileged mechanistic pathway for the Ugi

reaction. More importantly, two commonly accepted features in the Ugi

mechanistic pathway were challenged by this theoretical research. Firstly,

although the formation of imine as the first step is obviously confirmed, the

involvement of iminium during isocyanide insertion was questioned by their

calculation. Instead of activation of imine by a proton transfer, their computed

results suggested that the formation of a hydrogen bonded complex between imine

and acid substrate is more favorable. This conclusion was also in accordance with

a recent NMR experimental study in imine activation reported by Fleischmann

and co-workers. Secondly, according to the energy profile, the imidate solvated

by a methanol dimer 9a lies at -33.6 kcal mol-1, whereas the highest TS 15 for

Mumm rearrangement lies at -32.5 kcal mol-1, which means this final

rearrangement can evolve easily with a barrier of only 0.9 kcal mol-1 (Figure 1.4).

On the contrary, in the isocyanide addition step, it requires 19.8 kcal mol-1 of

activation energy to reach a stable nitrilium-acetate ion pair. Therefore, they

proposed that Ugi reaction should not be considered as an equilibratory reaction

sequence driven by a final irreversible step as previously stated. Instead, the

isocyanide addition step where a new stereogenic center was formed was the only

rate-determining step.

In 2014, the mechanism of the Ugi reaction was investigated by Eberlin and co-

workers using electrospray ionization mass spectrometry (ESI-MS) with

alternatively two imidazolium charge-tagged reagents (a carboxylic acid or an

amine).29 The Ugi’s original mechanistic proposal (Scheme1.1, path A) was

consolidated as the key intermediate 7 has been isolated and characterized. In

addition, the energetics of the final Mumm rearrangement were calculated by

Density Functional Theory (DFT) studies. It predicted a very low energy barrier

from transient imidate 9 to the final product 10, which is consistent with both ESI-

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MS/MS and TWIM-MS data. In the same year, Angelis and co-workers developed

finely selected reaction conditions for ESI-MS characterization of Ugi reaction

mechanism which can avoid the impact of ion tagged reactants on the reaction

pathway. Remarkably, their data demonstrated that the formation of nitrilium ion

7 is kinetically favored and its formation is the rate-determining step. This

conclusion not only strongly supported the original hypothesis of Ugi, but also

agreed with the theoretical findings predicted by Fleurat-Lessard and co-

workers30

4.3 Ugi reaction and its post-cyclizations

Although Ugi reaction stands out as a powerful method for the construction of

compounds with great diversity, the backbone of these compounds are normally

linear which thus lack the conformational constriction. In this context, the

combination of Ugi reaction with a subsequent secondary transformation,

typically a ring-forming process, has been proven to be an extremely powerful

strategy for the synthesis of structurally diverse complex molecules, especially

highly functionalized heterocyclic compounds. Inevitably, a large variety of

reactions has been introduced for the post-Ugi transformations strategy such as

acid/base-catalyzed cyclizations, cycloadditions, condensations, Ugi-

deprotection-cyclization (UDC), SNAr reactions, macrolactonizations, SN2

reactions, aryl couplings, ring closing metathesis, radical cyclization etc.

Therefore, all kinds of functionalized skeletons have been successfully

constructed by this two-step procedure, as concluded in Scheme 1.4.

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Scheme 1.4 Functionalized skeletons obtained by Ugi reaction and its post-cyclization.

(Structure adapted form reference 31-81)

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5. Aim and scope of this thesis

Refolding of proteins derived from inclusion bodies is very promising as it can

provide a reliable source of target proteins of high purity. However, inclusion

body-based protein production is often limited by the lack of techniques for the

detection of correctly refolded protein. Thus, the selection of the refolding

conditions is mostly achieved using trial and error approaches and is thus a time-

consuming process. In chapter 2, we use the latest developments in the

differential scanning fluorimetry guided refolding approach as an analytical

method to detect correctly refolded protein. We describe a systematic buffer

screen that contains a 96-well primary pH-refolding screen in conjunction with a

secondary additive screen. Our research demonstrates that this approach could be

applied for determining refolding conditions for several proteins. In addition, it

revealed which “helper” molecules, such as arginine and additives are essential.

In chapter 3, we describe the fast and efficient synthesis of libraries of positional

isomeric 1H-tetrazoles and 5H-tetrazoles for the purpose of testing binding

hypothesis of isomeric tetrazoles in fragment-based drug discovery.

Isocyanide-based multicomponent reactions (IMCR) are by far the most versatile

reactions that can construct relatively complex molecules by one-pot synthesis.

More importantly, the development of post IMCR modifications significantly

improves the scaffold’s diversity. In chapter 4, we describe the use of N-Boc

protected hydrazine together with -amino acid derived isocyanides in the Ugi-

tetrazole reaction and its post-cyclization under both acidic and basic conditions.

The cyclization in acidic conditions was conducted in a one pot fashion, which

give 7-aminotetrazolopyrazinone and tetrazolotriazepinone cyclic products. The

post cyclization under basic conditions could selectively afford Boc-protected 7-

aminotetrazolopyrazinone products in yield from 38 to 87%.

In chapter 5, the Pomeranz-Fritsch reaction was for the first time successfully

applied in the Ugi post-cyclization strategy by using aminoacetaldehyde diethyl

acetal and electron rich aldehydes as starting materials. Isoquinoline derivatives

and benzo[d]azepinone scaffolds with great diversity were constructed in

moderate to good yield. In addition, the isoquinoline-tetrazoles and an alkaloid-

like tetrazole-fused tetracyclic compound were synthesized by this method in a

very efficient manner.

Encouraged by the results from chapter 5, the Schlittler-Müller modification was

successfully used in the Ugi post-cyclization strategy in chapter 6. The acoustic

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dispensing-enabled scouting enabled a pipeline of fast and efficient nL scale

scouting to mg to gram scale synthesis. Isoquinoline derivatives and heterocyclic

compounds were constructed by this method in moderate to excellent yield. These

synthetic approaches were unprecedented, simple and efficient. Meanwhile, the

substrate scope and functional group tolerance were proved to be exceptional.

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References

[1]Zheng, W., Thorne, N., & McKew, J. C. (2013). Phenotypic screens as a renewed approach

for drug discovery.Drug discovery today,18(21-22), 1067-1073.

[2]Swinney, D. C. (2013). Phenotypic vs. target-based drug discovery for first-in-class

medicines. Clinical Pharmacology & Therapeutics,93(4), 299-301.

[3]Brown, D. (2007). Unfinished business: target-based drug discovery.Drug Discovery Today,

12(23-24), 1007-1012.

[4]Eder, J., Sedrani, R., & Wiesmann, C. (2014). The discovery of first-in-class drugs: origins

and evolution.Nature Reviews Drug Discovery,13(8), 577.

[5]Hajare, A. A., Salunkhe, S. S., Mali, S. S., Gorde, S. S., Nadaf, S. J., & Pishawikar, S. A.

(2013). Review on: high-throughput screening is an approach to drug discovery.Am. J.

PharmTech. Res,4, 112-129.

[6]Lipinski, C. A. (2004). Lead-and drug-like compounds: the rule-of-five revolution.Drug

Discovery Today: Technologies,1(4), 337-341.

[7]Chessari, G., & Woodhead, A. J. (2009). From fragment to clinical candidate - a historical

perspective. Drug discovery today,14(13-14), 668-675.

[8]Murray, C. W., & Rees, D. C. (2009). The rise of fragment-based drug discovery.Nature

chemistry, 1(3), 187.

[9]Sledz, P., Silvestre, H. L., Hung, A. W., Ciulli, A., Blundell, T. L., & Abell, C. (2010).

Optimization of the interligand Overhauser effect for fragment linking: application to

inhibitor discovery against Mycobacterium tuberculosis pantothenate synthetase. Journal

of the American Chemical Society, 132(13), 4544-4545.

[10]Scott, D. E., Coyne, A. G., Hudson, S. A., & Abell, C. (2012). Fragment-based approaches

in drug discovery and chemical biology. Biochemistry, 51(25), 4990-5003.

[11]Mashalidis, E. H., Śledź, P., Lang, S., & Abell, C. (2013). A three-stage biophysical

screening cascade for fragment-based drug discovery.Nature protocols,8(11), 2309.

[12]Keseru, G. M., Erlanson, D. A., Ferenczy, G. G., Hann, M. M., Murray, C. W., & Pickett,

S. D. (2016). Design principles for fragment libraries: maximizing the value of learnings

from pharma fragment-based drug discovery (FBDD) programs for use in academia.Journal

of medicinal chemistry,59(18), 8189-8206.

[13]Hung, A. W., Silvestre, H. L., Wen, S., Ciulli, A., Blundell, T. L., & Abell, C. (2009).

Application of fragment growing and fragment linking to the discovery of inhibitors of

Mycobacterium tuberculosis pantothenate synthetase. Angewandte Chemie International

Edition, 48(45), 8452-8456.

[14] Bakheet, T. M., & Doig, A. J. (2009). Properties and identification of human protein drug

targets. Bioinformatics,25(4), 451-457.

[15]Yang, Z., Zhang, L., Zhang, Y., Zhang, T., Feng, Y., Lu, X., ... & Wang, X. (2011). Highly

efficient production of soluble proteins from insoluble inclusion bodies by a two-step-

denaturing and refolding method. PloS one, 6(7), e22981.

[16]Baneyx, F., & Mujacic, M. (2004). Recombinant protein folding and misfolding in

Escherichia coli. Nature biotechnology, 22(11), 1399.

[17]Hartl, F. U., & Hayer-Hartl, M. (2009). Converging concepts of protein folding in vitro and

in vivo. Nature Structural and Molecular Biology, 16(6), 574.

[18]Biter, A. B., Andres, H., Thapar, R., Lin, J. Z., & Phillips, K. J. (2016). DSF guided

refolding as a novel method of protein production. Scientific reports, 6, 18906.

[19]Chayen, N. E., & Saridakis, E. (2008). Protein crystallization: from purified protein to

diffraction-quality crystal. Nature methods, 5(2), 147.

Page | 24

[20] Ugi, I.; Meyr, R.; Fetzer, U.; Steinbruckner, C. (1959). Angewandte Chemie International

Edition, 71, 386-386.

[21]Nutt, R. F., & Joullie, M. M. (1982). Four-component condensation: a new versatile method

for the synthesis of substituted prolyl peptides. Journal of the American Chemical Society,

104(21), 5852-5853.

[22]Díaz, J. L., Miguel, M., & Lavilla, R. (2004). N-Acylazinium salts: A new source of

iminium ions for Ugi-type processes. The Journal of organic chemistry, 69(10), 3550-3553.

[23]Ngouansavanh, T., & Zhu, J. (2007). IBX‐Mediated Oxidative Ugi‐Type Multicomponent

Reactions: Application to the N and C1 Functionalization of Tetrahydroisoquinoline.

Angewandte Chemie International Edition, 46(30), 5775-5778.

[24]Znabet, A., Zonneveld, J., Janssen, E., De Kanter, F. J., Helliwell, M., Turner, N. J., ... &

Orru, R. V. (2010). Asymmetric synthesis of synthetic alkaloids by a tandem

biocatalysis/Ugi/Pictet-Spengler-type cyclization sequence. Chemical Communications,

46(41), 7706-7708.

[25]Gokel, G., & Ludke, G. (1971). I. Ugi in Isonitrile Chemistry.

[26]Barthelon, A., El Kaim, L., Gizolme, M., & Grimaud, L. (2008). Thiols in Ugi‐and

Passerini–Smiles‐Type Couplings. European Journal of Organic Chemistry, 35, 5974-5987.

[27]Faggi, C., Garcia-Valverde, M., Marcaccini, S., & Menchi, G. (2010). Isolation of Ugi four-

component condensation primary adducts: A straightforward route to isocoumarins.

Organic letters, 12(4), 788-791.

[28]Cheron, N., Ramozzi, R., Kaim, L. E., Grimaud, L., & Fleurat-Lessard, P. (2012).

Challenging 50 years of established views on Ugi reaction: A theoretical approach. The

Journal of organic chemistry, 77(3), 1361-1366.

[29]Medeiros, G. A., da Silva, W. A., Bataglion, G. A., Ferreira, D. A., de Oliveira, H. C.,

Eberlin, M. N., & Neto, B. A. (2014). Probing the mechanism of the Ugi four-component

reaction with charge-tagged reagents by ESI-MS (/MS). Chemical Communications, 50(3),

338-340.

[30]Ramozzi, R., Chéron, N., El Kaim, L., Grimaud, L., & Fleurat‐Lessard, P. (2014).

Predicting New Ugi–Smiles Couplings: A Combined Experimental and Theoretical Study.

Chemistry-A European Journal, 20(29), 9094-9099.

[31]Zohreh, N., Alizadeh, A., Bijanzadeh, H. R., & Zhu, L. G. (2010). Novel approach to 1, 5-

benzodiazepine-2-ones containing peptoid backbone via one-pot diketene-based ugi-

4CR.Journal of combinatorial chemistry,12(4), 497-502.

[32]Banfi, L., Basso, A., Guanti, G., Kielland, N., Repetto, C., & Riva, R. (2007). Ugi

multicomponent reaction followed by an intramolecular nucleophilic substitution:

convergent multicomponent synthesis of 1-sulfonyl 1, 4-diazepan-5-ones and of their

benzo-fused derivatives.The Journal of organic chemistry,72(6), 2151-2160.

[33] Azuaje, J., Pérez-Rubio, J. M., Yaziji, V., El Maatougui, A., Gonzalez-Gomez, J. C.,

Sanchez-Pedregal, V. M., ... & Sotelo, E. (2015). Integrated Ugi-based assembly of

functionally, skeletally, and stereochemically diverse 1, 4-benzodiazepin-2-ones.The

Journal of organic chemistry,80(3), 1533-1549.

[34] Gunawan, S., Ayaz, M., De Moliner, F., Frett, B., Kaiser, C., Patrick, N., ...& Hulme, C.

(2012). Synthesis of tetrazolo-fused benzodiazepines and benzodiazepinones by a two-step

protocol using an Ugi-azide reaction for initial diversity generation.Tetrahedron,68(27-28),

5606-5611.

[35]Mossetti, R., Saggiorato, D., & Tron, G. C. (2011). Exploiting the Acylating Nature of the

Imide-Ugi Intermediate: A Straightforward Synthesis of Tetrahydro-1, 4-benzodiazepin-2-

ones. The Journal of organic chemistry, 76(24), 10258-10262.

Page | 25

[36] Keating, T. A., & Armstrong, R. W. (1996). A remarkable two-step synthesis of diverse 1,

4-benzodiazepine-2, 5-diones using the Ugi four-component condensation.The Journal of

organic chemistry,61(25), 8935-8939.

[37] Sanudo, M., Garcia-Valverde, M., Marcaccini, S., Delgado, J. J., Rojo, J., & Torroba, T.

(2009). Synthesis of benzodiazepine β-turn mimetics by an Ugi 4CC/Staudinger/Aza-Wittig

sequence. Solving the conformational behavior of the Ugi 4CC adducts.The Journal of

organic chemistry,74(5), 2189-2192.

[38] Xiang, Z., Luo, T., Lu, K., Cui, J., Shi, X., Fathi, R., ...& Yang, Z. (2004). Concise

synthesis of isoquinoline via the Ugi and Heck reactions.Organic Letters,6(18), 3155-3158.

[39] Che, C., Li, S., Yu, Z., Li, F., Xin, S., Zhou, L., ...& Yang, Z. (2013). One-pot syntheses

of isoquinolin-3-ones and benzo-1, 4-diazepin-2, 5-diones utilizing Ugi-4CR post-

transformation strategy.ACS combinatorial science,15(4), 202-207.

[40] Tyagi, V., Khan, S., Giri, A., Gauniyal, H. M., Sridhar, B., & Chauhan, P. M. (2012). A

Ligand-Free Pd-Catalyzed Cascade Reaction: An Access to the Highly Diverse Isoquinolin-

1 (2 H)-one Derivatives via Isocyanide and Ugi-MCR Synthesized Amide

Precursors.Organic letters,14(12), 3126-3129.

[41]Ma, Z., Xiang, Z., Luo, T., Lu, K., Xu, Z., Chen, J., & Yang, Z. (2006). Synthesis of

functionalized quinolines via Ugi and Pd-catalyzed intramolecular arylation

reactions.Journal of combinatorial chemistry,8(5), 696-704.

[42] Li, Y., Meng, J. P., Lei, J., Chen, Z. Z., Tang, D. Y., Zhu, J., ... & Xu, Z. G. (2017).

Efficient synthesis of fused oxazepino-isoquinoline scaffolds via an Ugi, followed by an

intramolecular cyclization.ACS combinatorial science,19(5), 324-330.

[43] Ji, F., Yi, W. B., Sun, M., Lv, M. F., &Cai, C. (2013). Synthesis of novel isoquinolinone

and 1, 2-dihydroisoquinoline scaffolds via Ugi reaction and ring opening reaction of

furans.Molecular diversity,17(2), 295-305.

[44] Pandey, S., Khan, S., Singh, A., Gauniyal, H. M., Kumar, B., & Chauhan, P. M. (2012).

Access to Indole-And Pyrrole-Fused Diketopiperazines via Tandem Ugi-

4CR/Intramolecular Cyclization and Its Regioselective Ring-Opening by Intermolecular

Transamidation.The Journal of organic chemistry,77(22), 10211-10227.

[45] Golebiowski, A., Jozwik, J., Klopfenstein, S. R., Colson, A. O., Grieb, A. L., Russell, A.

F., ... & Chen, J. J. (2002). Solid-supported synthesis of putative peptide β-turn mimetics

via Ugi reaction for diketopiperazine formation.Journal of combinatorial chemistry,4(6),

584-590.

[46] Kennedy, A. L., Fryer, A. M., & Josey, J. A. (2002). A new resin-bound universal isonitrile

for the Ugi 4CC reaction: preparation and applications to the synthesis of 2, 5-

diketopiperazines and 1, 4-benzodiazepine-2, 5-diones.Organic letters,4(7), 1167-1170.

[47] Cho, S., Keum, G., Kang, S. B., Han, S. Y., & Kim, Y. (2003). An efficient synthesis of 2,

5-diketopiperazine derivatives by the Ugi four-center three-component reaction.Molecular

diversity,6(3-4), 283-286.

[48] Stucchi, M., Cairati, S., Cetin-Atalay, R., Christodoulou, M. S., Grazioso, G., Pescitelli,

G., ...& Lesma, G. (2015). Application of the Ugi reaction with multiple amino acid-derived

components: synthesis and conformational evaluation of piperazine-based minimalist

peptidomimetics.Organic & biomolecular chemistry,13(17), 4993-5005.

[49] Santra, S., & Andreana, P. R. (2011). A rapid, one-pot, microwave-influenced synthesis of

spiro-2, 5-diketopiperazines via a cascade Ugi/6-exo-trig aza-Michael reaction.The Journal

of organic chemistry,76(7), 2261-2264.

[50] Abdessalem, A. B., Abderrahim, R., Agrebie, A., Dos Santos, A., El Kaïm, L., & Komesky,

A. (2014). Formal [3+2] cycloaddition of Ugi adducts towards pyrrolines.Chemical

Communications,51(6), 1116-1119.

Page | 26

[51] Moni, L., Banfi, L., Basso, A., Carcone, L., Rasparini, M., & Riva, R. (2015). Ugi and

Passerini reactions of biocatalytically derived chiral aldehydes: application to the synthesis

of bicyclic pyrrolidines and of antiviral agent telaprevir.The Journal of organic

chemistry,80(7), 3411-3428.

[52] Banfi, L., Basso, A., Cerulli, V., Guanti, G., & Riva, R. (2008). Polyfunctionalized

Pyrrolidines by Ugi Multicomponent Reaction Followed by Palladium-Mediated SN2

‘Cyclizations.The Journal of organic chemistry,73(4), 1608-1611.

[53] Polindara-Garcia, L. A., & Miranda, L. D. (2012). Two-step synthesis of 2, 3-

dihydropyrroles via a formal 5-endo cycloisomerization of Ugi 4-CR/propargyl

adducts.Organic letters,14(21), 5408-5411.

[54] C Polindara-García, L. A., & Vazquez, A. (2014). Combinatorial synthesis of nicotine

analogs using an Ugi 4-CR/cyclization-reduction strategy.Organic & biomolecular

chemistry,12(36), 7068-7082.

[55]Znabet, A., Blanken, S., Janssen, E., de Kanter, F. J., Helliwell, M., Turner, N. J., ... & Orru,

R. V. (2012). Stereoselective synthesis of N-aryl proline amides by biotransformation-Ugi-

Smiles sequence.Organic & biomolecular chemistry,10(5), 941-944.

[56] Gulevich, A. V., Balenkova, E. S., & Nenajdenko, V. G. (2007). The first example of a

diastereoselective thio-Ugi reaction: A new synthetic approach to chiral imidazole

derivatives.The Journal of organic chemistry,72(21), 7878-7885.

[57] Yan, Y. M., Rao, Y., & Ding, M. W. (2016). One-Pot Synthesis of Multisubstituted

Benzimidazoles via Sequential Ugi and Catalytic Aza-Wittig Reaction Starting from 2-

Aminobenzoyl Azides.The Journal of organic chemistry,81(3), 1263-1268.

[58] Guchhait, S. K., & Chaudhary, V. (2014). Desilylative activation of TMSCN in

chemoselective Strecker-Ugi type reaction: functional fused imidazoles as building blocks

as an entry route to annulated purines.Organic & biomolecular chemistry,12(34), 6694-

6705.

[59] Tyagi, V., Khan, S., Bajpai, V., Gauniyal, H. M., Kumar, B., & Chauhan, P. M. (2012).

Skeletal diverse synthesis of N-fused polycyclic heterocycles via the sequence of Ugi-type

MCR and cui-catalyzed coupling/tandem Pictet–Spengler reaction.The Journal of organic

chemistry,77(3), 1414-1421.

[60] Guchhait, S. K., Chaudhary, V., & Madaan, C. (2012). A chemoselective Ugi-type reaction

in water using TMSCN as a functional isonitrile equivalent: generation of heteroaromatic

molecular diversity.Organic & biomolecular chemistry,10(46), 9271-9277.

[61] Song, G. T., Xu, Z. G., Tang, D. Y., Li, S. Q., Xie, Z. G., Zhong, H. L., ... & Chen, Z. Z.

(2016). Facile microwave-assisted synthesis of benzimidazole scaffolds via Ugi-type three-

component condensation (3CC) reactions.Molecular diversity,20(2), 575-580.

[62] Garrido, M., Corredor, M., Orzáez, M., Alfonso, I., & Messeguer, A. (2016).

Regioselective Synthesis of a Family of β-Lactams Bearing a Triazole Moiety as Potential

Apoptosis Inhibitors.ChemistryOpen,5(5), 485-494.

[63] Zidan, A., Garrec, J., Cordier, M., El-Naggar, A. M., Abd El‐Sattar, N. E., Ali, A. K., ...

& El Kaim, L. (2017). β‐Lactam Synthesis through Diodomethane Addition to Amide

Dianions.Angewandte Chemie International Edition,56(40), 12179-12183.

[64] Santra, S., & Andreana, P. R. (2007). A one-pot, microwave-influenced synthesis of

diverse small molecules by multicomponent reaction cascades.Organic letters,9(24), 5035-

5038.

[65] Hulme, C., Ma, L., Cherrier, M. P., Romano, J. J., Morton, G., Duquenne, C., ...&

Labaudiniere, R. (2000). Novel applications of convertible isonitriles for the synthesis of

mono and bicyclic γ-lactams via a UDC strategy.Tetrahedron Letters,41(12), 1883-1887.

Page | 27

[66] Tye, H., & Whittaker, M. (2004). Use of a Design of Experiments approach for the

optimisation of a microwave assisted Ugi reaction.Organic & biomolecular chemistry,2(6),

813-815.

[67] Polindara-García, L. A., Montesinos-Miguel, D., & Vazquez, A. (2015). An efficient

microwave-assisted synthesis of cotinine and iso-cotinine analogs from an Ugi-4CR

approach.Organic & biomolecular chemistry,13(34), 9065-9071.

[68] El Kaïm, L., Grimaud, L., Miranda, L. D., Vieu, E., Cano-Herrera, M. A., & Perez-Labrada,

K. (2010). New xanthate-based radical cyclization onto alkynes.Chemical

Communications,46(14), 2489-2491.

[69] Schneekloth, J. S., & Sorensen, E. J. (2009). An interrupted Ugi reaction enables the

preparation of substituted indoxyls and aminoindoles. Tetrahedron, 65(16), 3096-3101.

[70] Lu, K., Luo, T., Xiang, Z., You, Z., Fathi, R., Chen, J., & Yang, Z. (2005). A Concise and

Diversity-Oriented Strategy for the Synthesis of Benzofurans and Indoles via Ugi and Diels-

Alder Reactions. Journal of combinatorial chemistry, 7(6), 958-967.

[71] Gordillo-Cruz, R. E., Rentería-Gómez, A., Islas-Jácome, A., Cortes-García, C. J., Díaz-

Cervantes, E., Robles, J., & Gámez-Montaño, R. (2013). Synthesis of 3-tetrazolylmethyl-

azepino [4, 5-b] indol-4-ones in two reaction steps:(Ugi-azide/N-acylation/SN2)/free radical

cyclization and docking studies to a 5-Ht 6 model. Organic & biomolecular chemistry,

11(38), 6470-6476.

[72] Kumar, A., Li, Z., Sharma, S. K., Parmar, V. S., & Van der Eycken, E. V. (2013).

Switching the regioselectivity via indium (III) and gold (I) catalysis: a post-Ugi

intramolecular hydroarylation to azepino-and azocino-[c, d] indolones. Chemical

Communications, 49(60), 6803-6805.

[73] Zhang, L., Zhao, F., Zheng, M., Zhai, Y., & Liu, H. (2013). Rapid and selective access to

three distinct sets of indole-based heterocycles from a single set of Ugi-adducts under

microwave heating. Chemical Communications, 49(28), 2894-2896.

[74]Modha, S. G., Vachhani, D. D., Jacobs, J., Van Meervelt, L., & Van der Eycken, E. V.

(2012). A concise route to indoloazocines via a sequential Ugi-gold-catalyzed

intramolecular hydroarylation. Chemical Communications, 48(52), 6550-6552.

[75] Lesma, G., Cecchi, R., Crippa, S., Giovanelli, P., Meneghetti, F., Musolino, M., ...&

Silvani, A. (2012). Ugi 4-CR/Pictet-Spengler reaction as a short route to tryptophan-derived

peptidomimetics. Organic & biomolecular chemistry, 10(45), 9004-9012.

[76] Sinha, M. K., Khoury, K., Herdtweck, E., & Dömling, A. (2013). Various cyclization

scaffolds by a truly Ugi 4-CR. Organic & biomolecular chemistry, 11(29), 4792-4796.

[77] Cano-Herrera, M. A., & Miranda, L. D. (2011). Expedient entry to the

piperazinohydroisoquinoline ring system using a sequential Ugi/Pictet-Spengler/reductive

methylation reaction protocol. Chemical Communications, 47(38), 10770-10772.

[78] Yugandhar, D., Kuriakose, S., Nanubolu, J. B., & Srivastava, A. K. (2016). Synthesis of

alkaloid-mimicking tricyclic skeletons by diastereo-and regioselective Ugi/ipso-

cyclization/aza-Michael cascade reaction in one-pot. Organic letters, 18(5), 1040-1043.

[79] Cheng, G., He, X., Tian, L., Chen, J., Li, C., Jia, X., & Li, J. (2015). Ugi/Himbert

arene/allene Diels–Alder cycloaddition to synthesize strained polycyclic skeleton. The

Journal of organic chemistry, 80(21), 11100-11107.

[80] Kumar, A., Vachhani, D. D., Modha, S. G., Sharma, S. K., Parmar, V. S., & Van der

Eycken, E. V. (2013). Post-Ugi gold-catalyzed diastereoselective domino cyclization for

the synthesis of diversely substituted spiroindolines. Beilstein journal of organic chemistry,

9, 2097.

[81] Wang, W., Ollio, S., Herdtweck, E., & Domling, A. (2010). Polycyclic Compounds by

Ugi-Pictet-Spengler Sequence. The Journal of organic chemistry, 76(2), 637-644.

Page | 28