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Why Do Drugs Look the Way they Do?

By Wolfgang K.-D. Brill

Discovery Research Oncology, Pharmacia Corp.,

Viale Pasteur 10, I-20014 Nerviano, Italy; e-mail: wolfgang.brill@pharmacia.com

I. Introduction Heterocycles are very common among drugs. According to the CMC2001.1 database, 56.8% of

the current drugs contain heterocyclic entities.1 Why are heterocycles so frequent among drug-like

molecules? Cyclic molecules provide the highest density of atoms per surface, heterocycles the highest

density of chemical functionalities with well-defined orientation in space per surface. In this paper I

will address why certain features, such as being a heterocycle, are determining whether a molecule is

drug-like. According to the FDA drugs are2….(B) articles intended for use in the diagnosis, cure,

mitigation, treatment, or prevention of disease in man or other animals; and (C) articles (other than

food) intended to affect the structure or any function of the body of man or other animals; and….

animals; and…. Thus in order to alter metabolic pathways in a favorable way, a drug has to interact

with adequate targets. The interaction of a drug to its target, whatever it may be, must be sustained by

specific interactions, which can only be provided between chemical functionalities of a drug and those

of its target. If cyclic structures provide the highest clustering of atoms and, in organic molecules,

heteroatoms provide most functional groups, then the greatest density of functionality can only be a

heterocycle.

II. Biologically Relevant Targets Among the biopolymers involved in all crucial cellular processes proteins and nucleic acids

clearly stand out as potential targets for chemotherapeutic agents. Paul Ehrlich has already proposed

receptor proteins as drug targets in the late 19th century. The concept in which the receptor serves as a

"switch" that receives and generates specific signals and can be either blocked by antagonists or turned

on by agonists was recognized by J. N. Langley in 1905.3 The pharmacological characterization of

receptors in almost all organs, including the brain, provided the basis for a large number of very diverse

drugs: β-blockers4; β-agonists5; benzodiazepines, which enhance the effects of γ-aminobutyric acid and

chloride flux by way of the benzodiazepine receptor6; and monoclonal antibodies, which block

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receptors of growth or differentiation factors on tumor cells.7 A comprehensive analysis of the drug

targets underlying current drug therapy undertaken in 1996 showed that present-day therapy addresses

only about 500 molecular targets. According to this analysis, cell membrane receptors, largely

heterotrimeric GTP-binding protein (G protein)-coupled receptors, constitute the largest subgroup

with 45% of all targets, and enzymes account for 28% of all current drug targets.8 The number of

potential targets has been exploding as a result of the sequencing of the human genome. However, the

disease processes have to be considered at the molecular (genetic) level to determine the optimal

molecular targets for drug intervention. Not every product of "disease gene" may in itself be a suitable

target. However, its function will likely be linked to that of other proteins in physiological or

pathophysiological circuits. Based on the assumption that the number of such "linked" proteins that

constitute suitable targets for drug intervention is between 5 and 10 per disease gene, J. Drews

estimated the number of potential drug targets to lie between 5,000 and 10,000, with 10 times as many

still to be exploited for future drug therapy.9c

The other part of the story is to find a chemical entity, which is able to penetrate various organs

such as the digestive system, body fluids, such as the blood and in many cases cellular membranes to

reach its target. Among ADME (absorption, distribution, metabolism and excretion), the oral

absorption is highly desired in pharmaceutical industry and poor absorption characteristics constitute a

bottleneck in drug development.9 Statistical analysis of properties of drugs has lead to the “Rule of

Five“10 and other physicochemical constraints characterizing molecules, that are most probably orally

absorbed.11 These possibility schemes of properties are invariant, as they are determined by the

physiology of the patient. Computational methods have recently been designed to estimate these

properties in silco prior to synthesis of a drug with good accuracy.12

Table 1. Some features of compounds, which have a high probability of absorption. Entries 1-4 are the original “Rules of 5”10

No Properties Value 1 Number of hydrogen bond

donors:(NHs and OHs) 0-5

2 Number of hydrogen bond acceptors: (Ns and Os)

0-10

3 LogP -2 -+5 4 Molecular weight 200-500 5 Number of rotatable bonds 0-8 6 Formal charge -2 +2 7 Number of heavy atoms 20-50 8 Polar surface area (TPSA)11b <90 A2

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Of course, not all biologically active compounds have to comply with those constraints. Whenever it is

possible for a drug to use special uptake or distribution mechanisms or vehicles, dramatic variation

from the above property constraints can be tolerated in active compounds.10

III: The “Drug-likeness” of a Small Molecule Determines which Target is

Drugable. Molecules that are orally bioavailable are found to be restricted to specific property ranges (Table

1)10-12 Generally these drug-like molecules are small compared to their targets. Yet, to bind to an

appropriate target, they must bind with as many of their surface features to as many of those of the

target protein epitopes. However the protein surfaces are generally covered with water, which has to be

displaced by a drug as described in equation (1)13 Can we, looking at the thermodynamics of drug-

target interactions, identify epitopes which are more likely to be addressed by drug-like compounds

than others?

Daq + Raq DRaq + mH2O

(1)

In this equation Daq is the drug solvated by water, Raq is the hydrated receptor, DRaq is the

receptor complex and mH2O is the amount of water released during the binding process.

Mechanistically such a binding event may be viewed as in Figure 1. according to Andrews et al.13a

Here, the binding interactions of a trifunctional drug with an optimal receptor are shown. The cyan

colored circles represent water molecules, the enthalpies of hydration of the drug and the receptor

being ∆HDW and ∆HRW, respectively. The free drug has an overall rotational and translational enthropy

of ∆Srt and an internal enthropy of ∆Sint. If a drug is dissolved in water, not all of its surface

functionalities can form hydrogen bonds with the solvent. Around the hydrophobic portions of the drug

molecule, the water molecules cluster to adopt an “iceberg-like” structure reminiscent of ice chlathrates

and lose enthropy.14 On binding, the drug is fixed on its receptor and both terms, ∆Srt and ∆Sint are

lost. This unfavorable contribution may be compensated for by an increase in enthropy (∆Sw ) due to

the loss of structured water which was formerly clustered around the drug and the receptor. Another

increase in enthropy (∆Svib) is caused by new low frequency vibrational modes associated with non-

covalent with drug-receptor interactions (Figure 1)

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Figure 1: The Drug receptor binding event.

The thermodynamics of drug-receptor interactions may be expressed by the Gibbs free energy ∆G

which is directly correlated with the association constant Ka (equation 2)

aKRTGG ln+°∆=∆ (2)

[ ] [ ][ ] [ ]aqaq

maq

RDOHDRRTGG

⋅⋅+°∆=∆ 2ln (3)

and consists of the binding enthalpy and enthropy as indicated in Figure 1.

For equilibrium conditions

0=∆G (4) and the enthalpic and enthropic contributions may be shown as:

STHG ∆−∆=°∆ (5)

It is difficult to measure the amount of water, which is displaced when a drug binds to a receptor

according to Figure 1. In turn, the effect of the solvent was shown in the case of a number of different

drugs, binding to various (G protein)-coupled receptors and ligand–gated ion channels. The ∆S° versus

∆H° scatter plot of all those measured binding events produced a straight regression line. This means

that any decease of binding enthalpy was compensated by a parallel decrease of binding enthropy and

vice versa. Tight binding to receptor can be achieved either enthalpy or enthropy-driven, depending on

which interactions the drug establishes with the receptor. For example, the entropy driven binding of

agonists to adenosine A1 receptor was attributed to the displacement of water from a pocket of the

receptor by a ribose residue of the agonists. The binding of antagonists of the same receptor, which do

not have a ribose residue to fill this pocket, and replace the water is enthalpy driven. The scatter plot

also reveals affinity constant values (Ka) cannot be greater than 0.01 nM-1.13b It is intriguing, that this

value is completely independent of the chemical entities that are involved in the reversible drug–

protein interactions, or whether the drug binds in a more enthalpy or more enthropy driven mode.13b

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IV. Which Forces Make Drugs Bind to their Targets? Hydrogen bonding, though very significant for molecular recognition, cannot be the key player

for drug receptor interactions unless hydrogen bonds with water are significantly weaker than those in

a drug-receptor complex. However hydrogen bonds do not have a well-defined length, strength and

orientation. They are generally 20 to 30 times weaker than covalent bonds and extremely susceptible to

stretching and bending. Exchange of hydrogen bonds with water or other polar residues is isoenthalpic,

if there are no geometric constraints.15

Unpolar residues of molecules will appear to attract and combine via hydrophobic interactions

due to a favorable increase in entropy due to release of solvent from the highly ordered cluster around

the unpolar surface.16 Van der Waals forces17 are caused by induction of the polarization of a molecule

in an electric field. Thus at any given instant, the electronic distribution within atomic groups is

asymmetric due to electron fluctuations. Therefore, dipoles in one group of atoms polarize the

electronic system of neighboring atoms or molecules, thus inducing dipoles which attract each other.18

The binding energy of hydrophobic interactions falls off approximately by the sixth power of the

molecular separation. Thus, tight contact between hydrophobic residues of protein and drug as in an

organic liquid18j or organic solid18e are prerequisite for binding. The π-interactions between aromatic

residues are common within proteins and aromatic residues19 resembling contact patterns in benzene

crystals and the rearrangement of aromatic rings in aromatic host guest complexes.20 In hydrophobic

interactions with amide bonds, the N-H bond dipole is oriented along the normal of the plane through

the phenyl ring, however perpendicular to the amide plane.21 Interactions with hydroxyl functions22,

cations and aromatic residues are of importance as shown in various biological systems.23

Drug binding can be approximated as the sum of all the above-mentioned interactions13a, which

can be attributed to the functional groups of the drug, which is making the interaction (equation 6).

Thus the free energy of drug-receptor binding may be ∆G may be written in the following way (if one

neglects coupling terms between functional groups: compare also with calculations of TPSA11b):

ΧΧ∑++∆=∆ EnEnTG dofdofrtS (6)

Herein ∆Srt is the loss of overall rotational and translational enthropy of the bound drug-molecule

(Scheme 1). ndof are the number of internal degrees of conformational freedom in the drug molecule

and Edof is the change in energy associated with the loss of each such degree of conformational

freedom. EΧ is the intrinsic binding energy of a functional group Χ.

EΧ consists of the enthalpy of interaction between functional groups with the receptor and enthropy

associated with the displacement of water by the functional group and subsequent integration into the

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solvent. The examination of various compound data sets leads to the following average intrinsic

binding energies. These may vary upon the alignment of functional groups. (Table 2)

Table 2: Intrinsic binding energies:

No. group Energy kcalmol-1

rangea

1 DOFb -0.7 -0.7- -1.0 2 C(sp2) 0.7 0.6- 0.8 3 C(sp3) 0.8 0.1- 1.0 4 N+ 11.5 11.4-15.0 5 N 1.2 0.8- 1.8 6 CO2

- 8.2 7.3- 10.3 7 OPO3

- 10.0 7.7- 10.6 8 OH 2.5 2.5- 4.0 9 C=O 3.4 3.2- 4.0

10 O,S 1.1 0.7- 2.0 11 halogen 1.3 0.2- 2.0

a)Range of binding energies for six random 100-compound data sets. b)Degrees of internal conformational freedom.

For small drug-like molecules the value of ∆Srt is mainly determined by physical constants and

calculated to be –14 kcalmol-1. The potential of a drug molecule to bind a receptor, the expected

binding free energy ∆G, may be calculated. It appears that, in order to have sufficient binding to a

target, a drug has to have enough surface area to provide a sufficient number of functionalities to

interact with its target in an optimal way. Considering the limits of molecular weight (Table 1) implied

by the bioavailability only structures can be drugs, which provide the maximal surface per molecular

weight.

V. How Must a Protein Surface Look like to Allow Tight Binding with Small

Hydrophobic Molecules? Hypothetical polar, shallow protein domains, which provide many hydrogen bonds and few

hydrophobic contact-surfaces, can only bind polar drugs, which complement the hydrogen bonds.

However the isoenergetic trans-hydration of a hypothetical very polar drug with a protein epitope does

render this type of interaction unlikely to provide tight drug-target binding, especially considering the

physicochemical constraints implied by the bioavailability.

Low molecular weight compounds may only bind to predominantly hydrophobic pockets on

proteins. Though hydrogen bonds or charge-charge interaction within a hydrophobic environment

enhance binding dramatically, if being complemented by the drug, the consequence of the Lennard

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Jones potential is that the hydrophobic contacts between drug and receptor have to be maximized. This

can only be the case, if the drug molecule has a shape complementing that of its binding site on a

protein.23b,c Thus a tight bound drug molecule is likely to be buried deeply in a hole or a fold of its

receptor. Only proteins, having deep hydrophobic folds or grooves, are likely to be drugable

targets for small molecules. This principle has been recognized in computer-assisted identification of

binding sites for drug-like molecules on proteins with known structure.24 Of course shallow epitopes,

such as minor groves in DNA, are also known to bind drugs. However these drugs will likely have a

much larger molecular weight, in many cases a greater chemical complexity and must plug into certain

active uptake mechanisms. The shape of the binding site must be unique to allow selectivity. Often

more than one adjacent hydrophobic fold may have to be used to allow differentiation between

different target protein subtypes.25 It is very welcome, if some adjacent hydrophobic pockets have

derived from different evolutionary predecessors.

VI. Protein Kinases as Example for a Drug Target

OOPO

OO P

O

OP OO O

OHOH

N

NN

NO

NH2OH

Protein

OOPO

OP OO O

OHOH

N

NN

NO

NH2O

OPO

O

Protein

+

kinaseMg2+

+

Figure 2. The kinase reaction.

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Figure 3. Human receptor protein-tyrosine kinases

Figure 3. The symbols α− and β denote distinct RPTK subunits. RPTK members in bold and italic type

are implicated in human malignancies The horizontal double line represents the cell membrane. The

receptor binding sites are in the extracellular domains of the receptor (below the double line). The

kinase activity is associated with the intracellular domains (above the double line, red rectangles) of

most receptors and represents the drugable target. Some receptors are associated with kinases, which

are not covalently bound to the receptor. An asterisk indicates that the member is devoid of intrinsic

kinase activity.26

Protein kinase activities are often associated with receptors, which can bind to specific effectors,

often other proteins. Upon binding to the effector, they become activated and can catalyze the transfer of

a phosphate group from an ATP molecule onto a tyrosine, serine or threonine of another protein or onto

another domain of themselves.26 (Figure 2)

The resulting phosphorylated proteins are enabled to interact with other proteins differently than in their

unphosphorylated form. Thus phosphorylation of certain enzymes (among them other receptor kinases)

alters their catalytic functions, which leads to build up or depletion of their substrates or products. The

types and concentrations of phosphorylated proteins, which are empowered by external or internal

signals, have a profound impact on every aspect of the cell life. These signal transuduction pathways

regulate a number of cellular functions, such as cell growth, differentiation, and cell death. Figure 3

shows a schematic representation of some membrane bond receptor kinases27

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A variety of tumor types have dysfunctional growth factor receptor tyrosine kinases, resulting in

inappropriate mitogenic signaling. Protein tyrosine kinases (PTKs) are therefore attractive targets for

therapeutic agents, not only against cancer, but also against many other diseases.18d Protein kinases also

posses with their ATP-binding site a structural feature which renders them drugable targets. The

possibility of competitive displacement of the ATP-cofactor by filling up hydrophobic pockets

associated with the adenine portion of the cofactor (and neither phosphate nor substrate binding sites)

was first recognized by Pascal Furet.27

The ATP-binding site is composed of deep hydrophobic folds or grooves. The natural cofactor

ATP is not very tightly bound to the catalytic site28, since, after phosphorylation of the substrate, its

reaction product ADP and the phosphorylated substrate have to leave their binding site to make room

for new cofactor and substrate. Despite the fact, that the catalytic domains of kinases share significant

amino acid homology and conserved core structures29 the structural diversity between ATP-binding sites

is sufficient to allow the development of selective inhibitors.30 The development of many potent

inhibitors in recent years supports the significance for this binding site.31,32

The binding pocket consists of various regions (Figure 4), which are favorable for a drug target.8, 33

1) Adenine region

2) Sugar rocket

3) Hydrophobic region I

4) Hydrophobic region II

5) Phosphate binding region

Figure 4. The ATP binding site*

Figure 4: The enumeration of amino acid residues is based on c-AMP dependent protein kinase.

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VII. How Can Drugs Fill Hydrophobic Pockets?

Binding to one or a few adjacent hydrophobic pockets requires the following: 1) The shape of the drug has to complement that of its binding site on a protein.23c,d 2) The geometries of optimal hydrogen bonding between polar residues have to be fulfilled. 3) The various functionalities that interact upon binding have to be pre-oriented so that

binding results in minimal conformational strain on drug and target. 4) Electric fields within the binding pocket should be compensated. 5) The conformational flexibility should be as low as possible. (see DOF Table 2) The conformation of the drug molecule bound to its target relative to that in solution has to be considered, especially if rotational barriers are high. For example, many amide bonds do not rotate at physiological temperature. In turn, the Gibbs free energy associated with the rotational barrier of some carbamates was found to be between 15-20 kcal/mol34 that of some anilides and toluamides 12-14 kcal/mol35 which accounts for up to 5-10 uncharged average hydrophobic contact interactions according to Table 2! The preference for more rigid structures with in drugs is desired in order to minimize enthropic loss due to fixation on its target (Figure 1). Thus small aliphatic rings with 3 and 4 members are often preferred over linear alkyl chains. 6-membered aliphatic rings are mainly used, if ring inversion barriers are high, which can be implemented by appropriate ring substituents. (This may explain why N, N disubstituted piperazines are very frequent within drugs.)

The alignment of the functional groups in their optimal binding positions is essential as seen in rate acceleration of enzymic and intramolecular reactions.35 In narrow protein folds the optimal orientation of clustered functional groups may only be achieved upon fixation onto or integration into cyclic structures.36 Aromatic heterocycles provide great specifically functionalized surface provided by a minimum of atoms. Figure 5. Different H-bondpatterns of kinase inhibitors with the “hinge region” of the ATP-binding site.a

A

HN N

H

NN

NNO

rib

B

HN N

N

NN

NHO

R1

R2

R3

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C

N

N

NOH

NH

Cl

HN NN

O N

NH

H

Traxler et al ref. 31(EGFR Kinase, 3nM)

R1

R2

R3

Eb

N

O H

NN

H

R1

R2

R3

NNH

CH2 NHN

CH2

D

HN O

O NH

A

NH

O

NH

SU 5416(Sugen VEGFR Kinase)

NH

O

NNH

Br

SNH2

OO

Bramson et al. ref.CDK2 IC50 60nM

a The animated structures are taken from Noble et al.18j The ATP-binding site in Figure 4 is an excellent example how a hydrophobic pocket in an enzyme can

be filled by a drug. The adenine binding region itself is very narrow and of mostly hydrophobic

character. For example, in the c-AMP dependent protein kinase the N1 and N6 of the adenine ring are

engaged in hydrogen bonds with the carbonyl of Glu121 and the NH of the amide of Val 123. The

hydrogen bonds surrounded by a hydrophobic environment are extremely attractive to promote tight

drug-target interaction and are therefore also used by other inhibitors. Scheme 4 indicates in how many

different ways various heterocyclic inhibitors align to the hinge region to make the contacts. While ATP

displays a hydrogen-bonding pattern similar to that in DNA, roscovitine interacts with CDK2 in a

Hoogsteen type hydrogen-bond pattern. In turn, pyrrolopyrimidines (7-deazapurines)31 have been shown

to bind via N3 and H9 to the hinge region. The natural product staurosporine, Sugen 5416 and also an

isatine hydrazone38 bind via alignment of an oxopyrrole moiety. In case of the pyrazole binding the X-

ray structure of the phenyl methyl pyrazole is reported, however only the IC50 of the methylene-bridged

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compound is reported. It is interesting to note that the tautomerism of the pyrazoles allows displaying

two inverse hydrogen bond donor-acceptor patterns.

It is likely that the IC50 of the unbridged compound (Figure 5, E, CH2 in gray) is much lower, due

to unfavorable entropic contributions caused by fixation of the aryl rings on the same plane. In one

model for the binding of a quinazoline with EGFR kinase, the inhibitor is interacting with the backbone

NH of Met-769 and with Tyr 766. In this model the aminoaryl substituent fills up the hydrophobic

region 1 (Figure 4), while the ribose pocket is not being used. (Figure 6, A) If, further hydrogen bonds

with the hinge region are established, either by alkoxy substituents in position 7 of the quinazoline or by

condensation with a pyrrole or pyrazole the IC50 is still dramatically lowered.39 Alternatively the

quinazolines, which probably lack the central hydrogen-bonding interaction, may however be

Figure 6. Superimposititon of a quinazoline and ATP binding to EGFR.

The gray rods represent the peptide backbone.

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O O

O

O

OOH

HN

HN

H

H

H

N

N

NH

O

OBr

N

N

NH Cl

F

O

ONO

ZD1839 (Astra-Zeneca, EGFR kinase)

HN NN

H

OHN

NR

O

R2R1

O

OOH

OH

OH

N

N

NH

O

OOO

genistein

CP 358774 (Pfizer, EGFR Kinase)

HN O

HN

NN

H

NR

HN

O

R2

R1

SU 5271/PD 133035 (Sugen, EGFR/Psoriasis)

A B

The animated pictures are taken from Palmer et al.39 engaged in another hydrogen bond in the “hydrophobic region 1 (Figure 4). A similar hydrogen-bonding pattern is also proposed for genistein.18d (Figure 6, B) The sugar pocket is of hydrophilic character in most kinases. However within the EGFR family a

cysteine residue is present in this region. Aromatic residues, such as chlorophenyl, have been found to

be effective replacements for the much more polar ribosyl moiety in that case.31 It is assumed that

halogen substituted aromatics are engaged in an interaction with the cysteine residue also present in that

pocket in some kinases. In the EGF-receptor kinases, R-methylbenzyl has been demonstrated to be very

effective. Some more hydrophilic groups have been found very effective32a,33, however they cause the

drug to be more solvated by water, which might overcompensate the gain of making a new hydrogen

bond. Figure 7 shows how aryl groups of various inhibitors may act as bioisostere for the ribosyl

moiety. In a special case acetylene acts as a lipophilic spacer, bypasses most of the sugar pocket and

allows polar residues to bind directly to residues in the phosphate region.40 (Figure 8)

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Figure 7. Superposition of dianilinophthalimide (gray), 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine (EGF IC50: 1.9µµµµM) (cyan), and ATP (blue).

Source: Traxler et al. 31

Figure 8. An acetylene spacer allows the binding of an OH-group (red) with polar residues in the phosphate region.

Source: Ducrot et al. 40

The hydrophobic region I (Figure 4) is not occupied by adenine residues, but can be used by

inhibitors such to gain activity and selectivity. Various purine derivatives such as olomoucine32c,41,

purvalanol B33 (Figure 5) but also the flavonoid L 86827642 and roscovitine33 (Figure 5) fill this pocket

and establish π-interactions with the peptide side chains and Van der Waals interactions with alkyl

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residues. Thus purvalanol B bearing a 3-chloro-4-carboxyanilino group has an IC50 against

CDK2/cyclin A, which is 1000 fold lower than that of olomoucine.33 Among different kinases there is

variability of amino acids involved in generating this pocket, which is advantageous for the

development of selective inhibitors.43 The hydrophobic region II (Figure 4) is a hydrophobic slot open to the solvent. This region is also

not used by ATP and may be used by inhibitors. The phosphate binding pocket is very solvent exposed

and very polar. Inhibitors addressing this region consequently also have

Figure 9. Two inhibitors filling hydrophobic pocket of CDK2.

A

B

IC50= 60nM

Compound A (5-aryl-1H-pyrazole) is reported by Furet et al.37, compound B by Bramson et al.38

to bear polar, hydrated groups. As a consequence, considering the physicochemical constraints implied

by bioavailability and the competition with hydration this binding pocket is not of primary importance.

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Figure 10. Purines have received great attention as PTK-inhibitors.40,44

N

NN

N

NH

NH

N

NN

N

NH

NH

Cl

R1

R2

Olomoucine (R1= H, R2=Me): IC50 =7000 nM

R/S Roscovitine (R1= Et, R2=iPr): IC50 = 650 nM

R1

Novartis series: R1 = H; R2 = Et; R3 = IC50 = 25-40 nM

Purvalanol A: R1 = H; R2 = iPr; R3 = IC50 = 4 nM

Purvalanol B: R1 = CO2H; R2 = iPr; R3 = " IC50 = 6 nM

R2

R4: 3 or 4 NH2, OH

R3

R4

Figure 10. Some purines and their inhibition of CDK1/2-Cyclin B according to reference 44.

The choice of core structures to fit the adenine pocket is of great importance, since it determines

the orientation and concomitantly the topological alignment of the groups, which address the adjacent

hydrophobic pockets. Figure 9 shows how two very different inhibitors are embedded within the ATP-

binding pocket of a kinase. The pyrazole A is probably only a weak inhibitor, since it barely fills the

adenine and the adjacent hydrophobic pocket II, the latter by its phenyl group. In turn the more

optimized structure on the right (B) fills two pockets surrounding the adenine pocket and performs three

hydrogen bonds with the hinge region and an adjacent hydrophobic region II. Note that the oxindole

core of the isatin hydrazone (B) extents into the hydrophobic pocket I, while the pyrazole addresses that

pocket only with a methyl group.

Equally important is the variability of high yielding chemical alterations of the structure, which

centers around the question, whether such a structure is readily derivatized. Aromatic heterocycles such

as purines (Figure 10) allow to fine-tune hydrophobic interactions and dipole interactions by electronic

alterations of their π-systems. The chemistry of many types of heterocycles is well known and various

versatile methodologies exist to attach a wide variety of functional groups. These features are very

favorable when large numbers of analogs of a certain type have to be synthesized using multiparallel

synthesis.

VIII. Heterocycles as Bioisosteres Many functional groups or their attachment within a molecule may cause problems related to

ADME or toxicity. In many cases substitution of those functional groups by isomorphic heterocycles

renders a safer drug. Thus, bioisoteres are isomorphes, with approximately the same distribution of

electrons and similar physico chemical properties.45 The type of bioisostere to be used is determined by

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its ability to reconstitute the desirable interactions with the target, its effect on ADME and toxicity of a

drug. Isoelectronic properties and the (later so called) “isollobality”46 were recognized early by

Langmuir47, Grimm (Grimm´s Hydride Displacement Law)48 and Erlenmeyer.49 Some features of

bioisosterism implied by heterocycles involve the orientation of hydrogen bonds within a hydrophobic

environment within a hydrophobic pocket are often considered sticky points24, which have to be bound

by a drug. Heterocycles are an excellent way to arrange hydrogen donors or acceptors appropriately in

the binding pocket. The tautomerization37,50 found in several heteroromatic compounds allows them to

adapt to their environment. Thus a C-OH moiety of a heterocycle will tautomerize to C=O, and a

C=NH to C-NH- functions.51 Heterocycles with similar topological and electronic features may be

bioisosters of each other. Ring equivalents are another frequent type of bioisosterism. In many cases

pyridyl (2) and phenyl (1) have been demonstrated to be bioisosteres however, the metabolism and

solubility of pyridyl functions are very different from that of phenyl moieties.

Certain nitrogen heterocycles can serve as bioisosteres for phenols (3), which may cause ADME and

toxicological problems (Figure 11). Heterocycles such as indoles (4) and benzimidazoles (6) have been

shown to be particularly effective.52 The replacement of a phenol-residue by a pyrrolo ring has been

ascribed to the ability of both groups to hydrogen bond.53 The, very polar catecholes (5) could be

replaced by benzimidazoles (6)54, 3-hydroxypyrid-4-ones (7a), 3-hydroxypyranones (7b) 1-

hydroxypyrid-2-ones (8) and 3-hydroxypyridines (9).55

Figure 11. Bioisosteres of phenol and catechol.

N

OH NH

OHOH N

NH

Y

OOH N

OOH

N

OH

1 2

3 4

5 6 7a Y: O7b Y: NH

8 9

A common bioisostere for the carboxylic acid function (10), is the tetrazole moiety (11).

Comparison between carboxylic acids and tetrazoles at physiological pH reveals that the tetrazole

group is almost 10 times more lipophilic, while having similar acidity. (pKa tetrazole= 4.9, pKaRCOOH 4.2-

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4.4).56 In the case of model complexes with an amidine the tetrazolate anion with its four N-atoms was

found to be a flexible hydrogen bond acceptor, which adapts quite easily to different binding

(coordination) modes. However, the tetrazole is smaller in size than the carboxylate anion and

therefore cannot bind an amidine as tightly.57 Other monocyclic bioisosteres for carboxylates are

isoxazoles (12)58 oxadiazolones (13).59

Several biosteres have been used for esters (15) to increase hydrolytic stability and

bioavailability.60 Some heterocycles employed are piperazine diones (16), oxazolidinones (17, 18)60,

1,2,4-oxadiazoles (19a), 1,3,4-thiadiazoles (19b)61 isoxazoles and isothiazoles (20a, 21a), furans

(22)61, 1,2,5–oxadiazoles, (23a) 1,2,5-thiadiazoles (23b)62, 1,2,3-triazol-4-yl (24a), tetrazol-5-yl groups

(24b)63. In some bicyclic isosteres for carboxylic acids or esters are isoxazolidines (14a, 25a) and

isothiazole (14b, 25b) fused with saturated rings64 which may be favorable for functional group

orientation.

For amides (27) several heterocycles such as 1,2,4 oxadiazoles (28)65, 1,3,4-oxadiazoles (29)66,

1,2,4-triazoles (30)67, isoxazolines (31)68 and imidazoline (32)69 have been used. (Figure 12)

Figure 12. Bioisosteres for carboxylic acids, esters and amides

YN

R

YN

OR

YNN

O R NN

NY

RO

R

NH

NYOR

N

NOOR

23a Y: O23b Y: S

20a Y: O20b Y: S

25a Y: O25b Y: S

21a Y: O21b Y: S

22 24a Y: CH24b Y: N

26

OH

O

NN

NNH

NO

NHO

NHN

O

O

R

YN R

O

OO

N

O

R

OR' Y

NN

R

N

NYOH

NO

O

OR

10 11 12 13 14a Y: O14b Y: S

1617a Y: O17b Y: CH217c Y: NH17d Y: NR'

18 19a Y: O19b Y: S

15

175

NH2

O

ON

N

NNH

N

NN

O

N

NH

NO

27 28 29 30

31 32

IX. Toxic structures Many chemically feasible heterocycles, which are tight binders to a target, fail to become drugs

due to toxicity. In most cases transition metals, and functionalities, which could modify biopolymers

are discarded such as alkylating and acylating agents etc.70

Expert systems such as TOPDCAT, DEREK, StAR, OncoLocic and MULTICASE have been

developed to predict toxic substructures, toxicophores, mainly based on training sets of existing

structures.71 Though, these methods have become more frequently used to predict toxicity, however the

relevance of the prediction is difficult to assess. The improvement of expert systems remains an

ongoing effort.72

Some toxic substructures are72b:

1-alkylamino benzene

4-heteroiminomethyl

N-heteroarylimine

Phenylhydrazone

Aryldimethyl amine

1,2 dihydroxybenzene

Some toxic heterocycles are often tricyclic aromatics such as dibenzodioxines 73, acridines74,

dibenzofuranes75 and the food toxin 8MeQX76 (Figure 13), the latter can both intercalate and

aminoarylate DNA.

176

Figure 13. Formation of a 8MeGX adduct with DNAa.

OOPOO

O

N

NHN

NO

O

NH2

N

N NN

NH2

N

N NN

NH

OSO3-

OOPOO

O

N

NHN

NO

O

NH2

NH

NN

NN

8-MeIQx

Metabolism

35 (DNA)

mutagenic adduct

3334

36

Source: Gooderham et al76a and Gauvin et al.76b

1. The synthesis in solution phase: Here the desired reaction product is separated from side

products upon precipitation, liquid-liquid extraction, solid phase-extraction or derivatization of the side

product or the desired one to an easily extractable form. During the synthesis often polymer-supported

reagents81 are used, which can readily be removed by filtration. 2. The solid phase synthesis: Here the product is bound to the solid phase via a covalent linker.

The linker must allow selective removal of the final product from the support, but must be stable under

the reaction conditions throughout the synthesis. The advantage of a solid phase approach is that

reagents can be used in large excess to drive reactions to completion and most side products are just

washed off from the solid phase. However, the solid phase implies steric constraints onto the reactions

performed. Many reactions, which proceed well in solution, proceed on a solid support with a lower

rate or not at all. The choice of synthesis method depends on the synthetic problem, is often not

obvious and the result of a reaction optimization.

177

Often neither solution nor solid phase approaches lead to sufficient compound purity so that high

throughput MS or UV-triggerd HPLC purification has to be performed.80

In the following section substituted purines may serve as representative examples for the

derivatization of aromatic heterocycles, which are very common in medicinal chemistry. The

characteristic reactivities are nucleophilic aromatic substitution (A), functional group interchanges (B)

cyclo condensation reactions (C) and oxidations (D). The fact that many heteroatoms are present

within a ring provides a wide variety of opportunities for synthetic disconnections.(Figure 14)

The problem with application of toxicophor predictions is that the definition of toxicity is very much

dependant on the cost/benefit ratio for any specific compound in any particular pharmacological or

clinical area or endpoint. Oncology, for instance provides many illustrations of the need for good

judgment following post-efficacy testing in deciding what constitutes a toxicophore. Nucleoside and

nucleotide analogs, nitrogen mustards, folic acid antagonists, etc. are definitely toxic classes of

chemicals, but they still provide drugs for use in treating disease such as cancer.

X. Combinatorial Synthesis of Heterocycles The suitability of a certain molecule type, to become a drug is very much determined by the

complexity of its synthesis. In fact the art of medicinal chemistry is to find the structure, which, being

assessable with the most facile synthesis, obtains the best biological activity. The variability of high

yielding chemical alterations of a drug is highly desirable.77 Whether a lead-structure is readily

derivatizable by high throughput multiparallel or combinatorial synthesis schemes is a key factor in the

competitive industrial environment. Many heterocycles meet these criteria. In recent years many

combinatorial methods have been developed to provide either large numbers of compounds for lead

finding or smaller compound collections focused on a specific target for lead optimization. Originally,

combinatorial chemistry set out to provide mostly peptide like molecules, often in mixtures78 out of

which the active ingredient, the lead, would be obtained after an often lengthy deconvolution process.79

However, many times an intrinsic activity in a screen of a compound mixture could not be attributed to

a single compound. Such “false positives” caused an enormous work, since often all compounds of a

large mixture would have to be re-synthesized. The current trend is therefore to synthesize single

compounds and, if necessary, purify them.80 In this way compounds from combinatorial schemes are

indistinguishable from traditionally synthesized compounds and the amount of false positives is

reduced. Various combinatorial schemes exist, many of which are frequently applied in the

derivatization of heterocycles.

178

Figure 14. Some characteristic reactions used for derivatizations of aromatic heterocycles

N

(N,C)

OH N

(N,C)

X

N

(N,C)

NH2 N

(N,C)

N+

N N

(N,C)

Y

POX3

diazotationNuc: Y-

or radical Y.

N

(N,C)

X N

(N,C)

Y

NH2

NH2

(RO,O R'(RO,O)

(O,OR)

NH

NR'

NH

NH

R'NH

NR'

NH

N

NH

NX

oxidation:

oxidation:

A: B:

C:

Y- Nuclephile

Y-

condensation+ H2O or ROH+

D:

The ability of purines to allow nucleophilic aromatic substitution under relatively mild conditions,

which are compatible with many functional groups was exploited in many syntheses of combinatorial

libraries.33,82 To increase the hit-rate of these libraries certain filters for bioavailability prediction12,

toxicity71,72 and solubility are often applied.83

Figure 15. Solution phase synthesis of 2, 6, 9 triamino purines.

N

NN

NH

X

Y N

NN

NH

NH

Y N

NN

NH

NH

NH

N

NN

N

NH

NH

R1

R2

R3

R1 R1

R2

i ii iii1

23

4

56

7

8

937 38 39 40

Figure 15. X, Y: Cl; i) amine R1NH2 (1.05eq.), DMF, 80°C, 2-24 h; ii) amine R2NH2 (5eq.) DMF, Et3N (1.1eq.), 150°C, 30h, then addition of formylpolystyrene beads; iii) R3-Cl, K2CO3, DMF, rt, 24 h or R3OH (1.05eq), DEAD (3eq.), PPh3 (3eq.), dioxane, rt, 6h, chromatography.

179

Thus 2,6,9-trisubstituted purines could be synthesized upon selective displacement of the chloro

functions of 2,6-dichloro-purine, followed by N7-alkylation82a,82d However the aminations are restricted

to nucleophilic amines R2NH2 in step ii (Figure 10) only. The alkylation of N9, is often incomplete.

When excess of alkylating agent is used the desired product is contaminated with N6- and N7alkylated

product. In order to widen the scope 2-fluoro 6-chloropurine was being used as starting material with a

Y=F being more readily displaced. The syntheses of some mixed halopurines is also performed via

Schiemann reaction on 42.84(Figure 16).

N

NHN

NH

O

NH2 N

NN

NH

Cl

NH2

N

NN

NH

Cl

N+

N

N

NN

NH

Cl

F

i

ii

41 42

43 44

Figure 16. i) POCl3; ii) a) aq. NaNO3, b) 48% aq. HF

In a solution phase approach, “overalkylation” of N6 was avoided by performing the alkylation

step on N9 first, prior to substituting the halides on the heterocycle. Though the fluoro-atom on C2 is

more readily displaced than the chloro-atom, the substitution on C2 is still limited to very nucleophilic

primary amines. However, if N6 was acylated the substition on C2 is dramatically facilitated.

Subsequent N6-protection also enhances the nucleophilicity of N2 to allow N-alkylation under

Mitsunobu conditions.85 The choice of Boc as “N6 protecting/N2 activating group” was governed by its

ability to give volatile cleavage products.82f (Figure 17)

180

N

NN

N

Cl

F N

NN

N

NH

F N

NN

N

N

F

O

O

N

NN

N

N

NH

O

O

N

NN

N

N

N

O

O

N

NN

N

NH

N

R1

i ii iii44

45 46

R2

R1

iv

47

48

R3

R1

R2

R1

49R2'

R3 R3

R3R3

vvi

R2

R1

50R2'R3

Figure 17. i)R3OH (1.2 eq.), DEAD (1.3 eq.), PPh3 (1.3 eq.), THF –10°C-rt.; ii) R1NH2 (R1=aryl) (1.0

eq) DIPEA, nBuOH, 140°C; iii) Boc2O, DIEA, DMAP, THF, rt.; iv) R3NH2 (1.2 eq.) DIEA,

DMSO, 70-80°C; v) R2’Br, Bu4NI, NaH, DMF; vi) pTSA, MeOH/DCM, rt.

In order to avoid purification steps in between the reactions several solid phase approach were

performed. These begin with the immobilization of an amine R1NH2 onto an appropriate linker via

reductive amination. Subsequently the polymer bound amine is allowed to react with C6 of 44.

Subsequent steps are N9-alkylation, C2-substitution and cleavage from the resin. Drawback of this

approach is the low reactivity of a polymer bound amine. This problem could be addressed even upon

activation of the heterocycle with ammonium salts86 and by the choice of linker. Mainly acid labile

indole87 53b and PAL-linkers82a,88 53a had been employed. The activation of C2 by acylation of N6 was

not exploited.82f,89(Figure.18)

181

O

OMe

OMe

O

NH

NO O

P O P NH

N

NN

NH

N

F N

NN

N

N

F

N

NN

N NH

N

N

NN

N NH

NH

P P

P

NoR1i

AMS resin

R1 R1

ii iii

iv

54 55

R2R2

v

56 57

R3

R3

R1 R1

R3

51

52

44

53a

53b

Crowns

Figure 18. i) R1NH2, [Me4N]+[HB(OAc)3]-, then NaBH3CN; ii) 53b, DIEA, THF, 60°C, 16h; iii)

R2OH (10 eq.) P(Ph)3 (10 eq.), DEAD (10 eq.), THF; iv) R3NH2, n-BuOH/DMSO, 120°C; v) 5%

TFA/DCM.

Another approach to overcome the lack of reactivity at the C2-position is the use of Pd-catalyzed

C-N and also C-C formations.90 Some combinatorial approaches lead to other substitution patterns of

the purine. The 2,6,8- trisubstituted purines are synthesized from dichloropurine bound to a polymeric

support via N9. After synthesis of 2,6-diaminopurines via selective substitution reactions, the C8-

position was oxidized with a bromine lutidine complex to give compound 72.91 The latter could

undergo Stille couplings to afford the trisubstituted purine.90a(Figure 19)

182

N

NN

N

Cl

Cl

OH

N

NN

N N

N

N

NN

N N

N

BrN

NN

N N

N

N

NN

NH N

N

NBr

Br

i iii

69

iv

R2

70

R1 R1'

R2

R2

72

R3

R1 R1'

R2

R2

73

R1 R1'

R2

R3R2

R1 R1'

R2

v

ii

68

74

71

Figure 19. i) TFAA, 2,6-lutidine, then 37, NMP, 2,6-lutidine; ii) aminations; iii) 71(5x) NMP, 3h, r.t.;

iv) R3-Sn(Bu)3, Cu2O, Pd(OAc)2 (0.2 eq.), dppp (=1,3-bis(diphenylphosphino)propane) (0.2eq.).

In many cases, high yields (>75% based on polymer loading) of the final, pure product were

obtained. Drawback of this approach is some dehalogenation of 72 to 70 which is accompanying the

Stille coupling and Pd impurities in some products and structural constraints imposed by the

bromination reaction.

The assembly of a heterocyclic skeleton on polymeric supports often has low yield, due to the

steric constraints imposed by the support. The fact, that some heterocyclic synthesis intermediates have

very poor solubility in about every solvent may however be exploited to facilitate purification in

solution phase approaches. Unfortunately poor solubility often results in low reactivity. For example, a

solution phase synthesis approach based on the de novo synthesis of the purine ring from 4,5,6-

triamino pyrimidine and carboxylic acids: The resulting 4-acylated 4,5,6-triamino pyrimidines could be

readily purified by precipitation into ethyl acetate. Cyclyzation with NaOMe resulted in highly

insoluble purines, which could be further alkylated under Mitsunobu conditions85 using a parallel

synthesizer, giving mixtures of 1,8-dialkyl adenines and 8,9-dialkyl adenines.92(Figure 20)

183

N

NNH2

NH2

NH2

N

NN

NH

NH2

OH

O

N

NNH2

NH

NH2

O

N

NN

N

NH2

N

NN

N

NH2

R1

ii

75

iii

+

R1

R1 R1 R1

R2 R2

+

76 77

78 79 80

i

Figure 20. i) 77, cyanuric fluoride, pyridine, DCM, 1.5 h, then 78, DIEA, DMAP, r. t. 1h (60-80%); ii)

NaOMe/MeOH, reflux, 3.5h (80-90%); iii) R2OH, P(Ph)3, DEAD, toluene/DCM 6.5/1 (v/v); r. t., 0.2-

1h 79: (65-80%), 80: (14-33%).

2,6,9 tri-substituted purines were also synthesized using a cyclization route with orthoester.93 The

amino substituent at C2 was converted to an iodo substituent via a diazonium salt.94 The iodo

substituent at C2 allows further aminations and Sonogashira couplings.95 (Figure 21)

N

NNH2

Cl

Cl

NH2 N

NN

N

Cl

NH2N

NNH2

NH

Cl

NH2

N

NN

N

Cl

I N

NN

N

NH

NH

ii

iii

R1

R1 R1

R2

81 82 83

84 85

i

R1

R3

iv

Figure 21. i) R1NH2, nBuOH, 100°C; ii) CH(OEt)3, H+, DMA, r. t.; iii) a) isoamyle nitrite (3 eq.) 45

min.; b) I2 (1eq.) CH2I2 (10 eq.), THF, reflux; iv) thermal aminations or Sonogashira couplings at C2.

Another approach to assemble the purine skeleton begins with mono amination of the 5-amino-

4,6-dichloropyrimidine.33,96 The resulting heterocycle (88) is not electron deficient enough to allow

further amination. Oxidative cyclization had to be performed under anhydrous conditions using silica

supported FeCl3. In turn aqueous conditions would lead to hypoxanthines97, whereas the use of “free”

FeCl3 would lead to filtration problems during workup. (Figure 22).

184

N

NNH2

Cl

Cl

N

NN

NH

Cl

N

NNH2

NH

Cl

ii

R1

R1

87 88

i

89 Figure 22: i) R1NH2, nBuOH, 100°C; ii) R2CHO, 15% FeCl3-SiO2, dioxane, 100°C, 44h;

A solid phase approach to obtain 6,8,9-triaminopurines using a “cyclocondensation route“ begins

by reacting 4,6-dichloro 5-nitro pyrimidine less hindered amine-bound to an acid labile support.98

Subsequently the remaining, less reactive chloro function of the resulting polymer bound pyrimidine

was substituted by an amine. After reduction of the nitro group employing aq. sodium dithionate and

dioctyl viologen99 as electron phase transfer catalyst a polymer bound triamino pyrimidine was

obtained. The latter was cyclized with various isothiocyanates to give the trisubstituted purine of high

purity. Due to the fact that often only primary amines are sufficiently reactive in the pyrimidine

substitutions, some times mixtures of regioisomers are obtained. (Figure 23)

N

N

Cl

ClO2N

ONH2

O

N N

NH

ClNO2 O

N N

NH

NHNO2

O

N N

NH

NHNH2

O O

N N

NH N

NNH

N

N N

N

R2

90

91

i

R1

R1 R1ii

R2

R1

R2

R1

R2

R1R3 R3

92 93

94

95a 95b

iii

iv iv

Figure 23. i) DIEA, DMF; ii) R2NH2, DIPEA, NMP; iii) 1,1’-dioctyl viologen, DCM, K2CO3,

Na2S2O4, H2O, 20h, r. t.; iv)R3SCN, DMF, DIC, 20-25min., 80°C

In an extension of the combinatorial approaches a heterocyclic drug may be conjugated with long

hydrophilic chains. The conjugates may be attached to hydrophilic matrix for affinity chromatography,

which then may be used for profiling of the selectivity of a drug. In turn, an affinity column bearing a

2,6,9-trisubstituted purine was found to bind various kinases. The set of bound proteins was dependent

185

upon the cell lines or tissue the protein extract was obtained from. This approach, was suggested to be

a general method for identifying intracellular targets of a given ligand.100

XI. Conclusion Heterocycles provide the highest possible density of directed chemical functionality per

molecular surface. This high density of functional groups is the prerequisite for a drug to complement

shape and functionality of hydrophobic binding pockets in targeted proteins. In turn, hydrophobic

binding pockets are the only target structures, which allow small molecules (MW<700 daltons) to bind

with sufficient affinity (IC50 < 50nM). Only molecules, such as heterocycles, which, while being

mostly hydrophobic, allow precise positioning of hydrogen bonds within hydrophobic protein pockets

allow high affinity binding with great selectivity. Only small molecules bearing hydrophobic

functionalities can reach their molecular target via a passive uptake mechanism, which includes

passage through hydrophobic membranes. Heterocycles can be synthesized in many ways using

multiparallel synthesis schemes. This allows the synthesis of a large number of derivatives around a

core structure, which is necessary to establish a SAR. Their electronic properties, as well as the

topology of heterocycles can be influenced independently upon introduction of a wide variety of

substituents. An electronic and structural variation within similar structures can be used to obtain

bioisosteres for various functional groups to improve the ADME properties of drugs.

Obviously only few of the aspects associated with syntheses and applications of low molecular

weight drug-like heterocycles can be mentioned here. Many other types of heterocycles with biological

relevance, such as cyclopeptides and oligosaccharides, as well as saturated heterocycles have not been

covered here.

Future developments in the synthetic preparation may involve the development of more mild,

robust and oxygen insensitive functionalization of aromatics and heteroaromatics. More efficient and

environmentally friendly high throughput purification methods are of need. The development of better

predictive tools for their properties, such as receptor binding, solubility and toxicity will dramatically

increase the hit rate of compound libraries, which will lead to a reduction of library sizes. The

identification of rules for active uptake mechanisms together with will expand the compound classes

considered to be drug-like and certainly enlarge the pharmacopia in the future. However heterocycles

are and will continue to determine how most drugs look like.

186

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