CHAPTER 3
Combinatorial Chemistiy
DOUGLAS R. HENRY
The term f)arath/,'m s/si/i is an overused one. hut in the mid-
1980s a true paradigm shift occurred in the way new drugs
are synthesized and screened for activity. Prior to then, most
drug compounds were synthesized in milligram quantities
in a serial one-at-a-time fashion. After synthesis, the com-
pound was sent to a biologist, who tested it in several in
vitro assays and returned the results to the chemist. Based
on the assay results, the chemist would apply sonic struc-
ture—activity relationship (SARI or use chemical intuition
to decide what changes to make in future versions of the
molecule to improve activity. Using this iterative process, a
chemist would be able to synthesize only a handful of struc-
tures per week. Since the yield of marketable drugs from
compounds synthesized and tested is only about I in 10.0(X).
the road to success has been a long and expensive one, taking
6 to 12 years and costing S5(X) to $800 million per drug.
In the mid- 1980s. this approach to drug synthesis changed
dramatically with the introduction ol combinatorial chemis-
try. The drug discovery process becanic a highly parallel
one, in which hundreds or even thousands of structures could
be synthesized at one time. Interestingly, biologists had for
some lime been using high-throughput screening HTS) to
perform their in vitro assays, running assays in 96-well mi-
crotiter plates and even using laboratory robotics for pi-
petting and analysis. The bottleneck had become the synthe-
sis of the compounds to test. Chemists realized that syntheses
could also be conducted by using a parallel approach. The
term conrb,,satoru,l chemistry was coined to refer to the par—
aDd generation of all possible co,ubinaiions of substituents
uc components in a synthetic experiment. Whereas the yield
fmm a serial synthesis is a single compound. the yield from a
combinatorial synthesis is a chemical liltrar. Figure 3-I
shows two common types of chemical libraries—a generic
library, based on a single parent or scaffold structure and mul-
tiplesubstituentsorresidues. and a mixture library, containing
a variety of structure types. The total number of structures in
alibrary iseitherthe product of the various nunibersofsubstit-
nents (for a generic library) or the total number of structures
in a mixture. The goal of conihinatonal chemistry is to be able
to synthesize, purify, chemically analyze. and biologically
test all the structures in the library, using a.s few synthetic cx-
perimenisas possible. This chapterdescrihes how combinato-
rial chemistry and HTS are being used in drug design and dis-
cosery to find new lead structures in a sluwter time.
HOW IT BEGAN: PEPTIDES AND OTHER
LINEAR STRUCTURES
Combinatorial chemistry was first applied to the synthesis
of peptides, since a convenient method for the automated
synthesis of these compounds already in svidesprcad
use. In 1963. Merrifield introduced the efficient synthesis
of peptides on a solid support or resin (Fig. 3-2).' This made
the rapid, automated synthesis of peptides possible, and
earned Merrilield a Nobel Prize in 1984. A key feature of
his approach is the attachment of a growing peptide chain
loan inert polymer bead. tisually about 100 4um in diameter.
composed of polystyrene cross-linked with divinyl bcnzcne.
Such beads were originally designed for size exclusion chro-
matography. The beads can be immersed in solvents.
washed, heated. etc.. and when the synthesis is complete.
the beads can be filtered l'rorn solution, and the reaction
products can be cleaved front the polymer. yielding pure
products. A Hungarian chemist. Arpad Furka, realized that
Merrifield's approach could be extended to allow the sytithe-
civ of all possible combinations of a given set of amino acids
in a limited number of steps. He accomplished this by split-
ting and remixing portions of the peptide-bound resin at each
step in the synthesis (Fig. 3-3). His description of the use
of combinatorial chemistry to synthesize polypeptides ap-
peared in the Hungarian patent literature in 1982. Appar-
ently, it is the lirst literature reference to a combinatorial
chemistry experiment.2
As seen in Figure 3-3. the advantage of split-and-mix syn-
thesis is that all 27 tripeptides can be synthesized in just
three steps, instead of 27 steps. The disadvantage of this
approach is that in the end, one obtains three mixtures of
beads with tripcptides attached, rather than the pure com-
pounds themselves. If activity is detected in one of the mix-
tures, it becomes necessary to go back and resynthesize some
or all of the structures in that mixture, to see which tripeptide
is responsible for the activity. As we shall see, various meth-
ods for tagging and deconvoluting combinatorial libraries
have been devised that reduce or eliminate the need for re-
synthesis.
The first combinatorial chemistry experiments were ap-
plied to the study of epitopes—the short sequences of amino
acids responsible for antibody recognition and binding to
proteins. Early researchers used solid-phase resin beads in
vials. microtiter plates. colutnns, and porous plastic mesh
"tea bags" They also used brush-like arrays of plastic pins.
at the ends of which compounds could be synthesized. Other
media that have been used include paper and polymer sheets
and glass chips—basically anything that can immobilize a
structure for the purpose of exposing it to reagents and sol-
vents (Fig. 3-4).
Peptides. of course, make poor oral drug molecules be-
cause they hydrolyze in the acidity of the stomach. As com-
binatorial methods were applied to the synthesis of drugs, a
need developed for methods of generating small (molecular
weight. <500) nonpeptide molecules as potential drug leads.
Among the first alternatives that were studied were Chiron's
"peptoids' '—molecules in which the variation occurs in
43
44 and Tt-',ibaok of Organic Medicinal and Pharn,areuziial C!wn,,.szrv

A1 = H, 4-OH

A2 = 5-Cl, 4-COOH+5C1

A3 = CH3. CH(CH3)2. CH2Ph(4-OH). CH2Ph,

A4 = H, CH3, CH2CH3. CH2CH=CH2, CH2Ph

Figure 3—1 • Generic (a) and mixture

(b) combinatorial libraries. The size of the
generic library is the product of the various
numbers of substituenis (here, 2 x 2 x 4
x 5. or 80). The size of the mixture library
is simply the total number of structures.

R2

R2
BOCNH
N
NH2 NI-IBOC
0
DCC 0
Figure 3—2 • Merrifield synthe-
sis on a polymer bead support.
The growing peptide chain is at-
tached to a polymer support, usu-
ally in the form of small beads.
The next amino acid (bearing R2)
is attached, and its protecting
group (BOC) is removed with CF3COOH Repeat
N
acid/base treatment. BOC. butyl- NH2
oxycarbonyl. DCC, dicyclohexyl- NEt3
carbodiimide.

Split & Mix

OAA Q-AB 0-AC

0-BA 0-BB 0-BC

& Mix
0-AAA Q-AAC
0-BAA 0-BAB 0-BAC
0-CAA 0-CAB 0-CAC
0-ABA 0-ABB 0-ABC
0-BBA 0-BBB 0-BBC
Q-CBA Q-CBB Q-CBC Figure 3—3 • Split-and-mix synthesis of tripeptides In the first step, all the
Q-ACc beads in a given container have a single monopeptide. These are all mixed
Q.ACA Q-ACB
together, then split into three aliquots and re-treated, attaching a second
Q-BCA 0-BCB 0-BCC peptide. After just one more step, all 27 possible combinations exist, spread
0-CCA Q-CCB Q-CCC among the three containers.
 Chapter 3 • Comb jmug,riul 45

Seal

Polypropylene

Resin

Label
b

•IeIIOSIS .•••••••
'OIl,.,.
.I,.,.,., ,,,ll•..
...,l,l• It
"I'.,
•IIIIIOSIS
d
FIgure 3—4 • Various approaches to immobilizing and separating chemical compounds during combinato-
rial synthesis have been devised, a. Tea bag synthesis. b. Pins and "lollypops. c. Dots on cellulose. d.
Spatial arrays on microchips

molecular <500) the attachment to the aunide nitro- in this way limited the total number of compounds to just
gee (Fig. Although these structures could potentially 204. Nevertheless, several potent ligands were found, includ-
place side chain functional groups in positions similar to ing a nanomolar a-adrencrgic inhibitor (Fig. 3-6a) and a
those on the corresponding peptides. they differ significantly similarly active ia-opiate receptor ligand (Fig. 3-6b).6 Be-
in that they lack peptide hydrogen bonds and chiral centeN. cause of the ease of synthesis, other classes of linear chain
They also show more rotational flexibility than the cone-
pcptides. since the peptoid amide bonds show less
0
double-bond character than those in peptides. Miller demon-
strated the stability of peptoids to a number of enzymes. 0
including chymotrypsin. papain. pepsin, and carboxypepti-
dase Zuckerman et demonstrated in 1994 that biolog-
ically active peptoids could be obtained by using combinato-
dat chemistry. He used 24 monomers to generate tripeptoids.
each of which had one hydroxylic. one aromatic, and one
diverse side chain. Limiting the composition of ihe peptoids

Peptide

b
Figure 3—6 • Biologically active peptoids. Compound a is an
Peptoid
a-adrenergic inhibitor, while compound b is a receptor
Figure 3—5 • Comparison of peptide and peptoid. ligand.
 _____

46 Wilson and Giscolds Textbook of Organic Medicinal and Pharmaceutical Chemisir

NH2

R3
R2 Attach R2 Fmoc P2 I

to solid amino Cycllze

support acid
— __O — __o
0

Ri RI

Figure 3—7 • Synthesis of t ,4-benzodiazepines on a solid support. Fmoc 9-fluorenylmethoxycarbonyl, a

common organic protecting group.

molecules have been investigated. These include oligonucle-
otides (DNA and RNA). oligoureas. and carbohydrates.
DRUG-likE MOLECULES
The real advance in combinatorial chemistry for drug discov-
ery purposes was the introduction of synthetic methodology
to yield true drug-like structures. Bunin and Ellman7 in 1992
demonstrated the synthesis of I .4-benzodiazepine com-
pounds. using three components: a 2-aminobenzophenone,
a protected amino acid, and an alkyl halide (Fig. 3-7). Al-
though we normally think of benzodiazepines as muscle re-
laxants and tranquilizers, a search for drug structures con-
taining the benzodiazepine scaffold returns antiviral,
alcohol-deterrent, uterine-relaxant, antineoplastic, anticon-
vulsant. antiulcerative. analgesic. antiarthritic. and sedative
structures, among many others. For this reason, the demon-
stration of the solid-phase synthesis of these structures vir-
tually opened the door to the use of combinatorial chemistry
in drug discovery.
In the decade since the first drug molecules were gener-
ated by using combinatorial chemistry, solid-phase synthe-
ses have been discovered for most common classes of drug
structure. Some examples of these are shown in Figure 3-8.
Of necessity, the reactions that can be performed in combina-
torial chemistry are simpler than many reactions that a chem-
ist using standard synthetic procedures can perform. Ex-
tremes of temperature and pressure, the use of highly caustic
reagents, inert atmospheres, and multistep reactions are gen-

P4

HO

P2

Figure 3—8 • Examples of drug-like

structures that can be generated by solid-
phase synthesis. (Structures drawn from
Balkenhohi, F., et al.: Combinatorial syn-
thesis of small organic molecules. Angew.
P3 Chem. Int. Ed. EngI. 35:2288—2337,
1996.)
 Chapter 3 • Co,,,bi,nuorial C'Iie,nis:rv 47

avoided in combinatorial chemistry. Also, the reac-
should not yicld alternative products. The yield of reac-
tions in combinatorial chemistry should be high (80% or
hut this can often be achieved by using an excess of
Ivagent and then washing the beads afterward to remove
the excess. An important recent advance in combinatorial
chemistry is the use of microwave heating in place of stan-
dard heating methods.5
Other classes of therapeutically important compounds that
have been synthesized successfully by using solid-phase
combinatorial chemistry include carbohydrdtcs and naturdl
products.9 Polysaccharides are important For various carbo-
hydrate—protein interactions. Carbohydrate antibiotics, in-
cluding vancomycin and aminoglycosides. have been the tar-
gets of combinatorial chemistry, as well as complex
oligosaccharides like the one shown in Figure 3-9.'° A van-

a HO b

B2O
B2
NHFmec
o 0

— R2

e R3

FIgure 3—9 • Carbohydrate and natural product synthetic targets. a. Bauhinia purpurea lectin ligand
analogues. b. Vitamin D3 analogues. c. Erythromycin analogues. d. Neocarzinostatin anticancer agents. a.
Galanthamine cholinesterase inhibitors. In most cases, the molecules are assembled by connecting large
subfragments in a small number of synthetic steps rather than by attempting total synthesis. Fmoc, 9-
fluorenytmethoxycarbonyl, a common organic protecting group.
48 Wilsoti and Gj.cvo/r/'x Textbook of Medicinal and Pharnzareuiieal Che,nixgr,

ely of natural products arc being studied, mainly in the areas
at' infectious disease and cancer but also as scaffolds for
many other therapeutic categories. Figure 3-9 shows exam-
ples of natural products that have been studied. including
vitamin D analogues. crythromycin-like antibiotics, antican-
cer neocarzinostatlns. and galanthamine. a cholinestcrasc in-
hibitor.°
SUPPORTS AND LINKERS
Most solid-state combinatorial chemistry is conducted by
using polymer beads IC) to 750 in diameter. These heads
swell in organic solvents, allowing the free diffusion of sol-
vent and reagent into the interior of the bead and greatly
expanding the available area for the attachment of product.
The polymers are inert, except the functional groups to
which the molecules arc attached. In general, the compounds
to he synthesized arc not attached directly to the polymer
molecules. They are usually attached using a "linker"
moiety that (a) enables attachment in a way that can be
easily reversed without destroying the molecule that is being
synthesized and (b) allows some room for rotational freedom
of the molecules attached to the polymer. Sometimes, the
molecules attached to the polymer are used directly as sub-
strates in in vitro assays without removing them from the

HF

Product

Memiteld resin (peplide products)

Produci

P AM resin (peptide or carboxylic acid products)

cl-c
Product

1% TFA

Trityl resin (carboxylic acid products)

-N Product

Nucleophile

NM BA resin (peptide products)

Figure 3—10 • Common linker functional

H2 I-I 1%TFA groups and the reagents that cleave the product.
Note that different tinkers provide different spac-
ing and steric freedom between the product anc
the support. HF, hydrofluoric acid; TFA, trifluoro
ADCC resin (amine products) acetic acid.

polymer. In such cases. if the molecules are too tightly
packed on the polymer. the enzyme molecules cannot gain
access to the substrates. A similar situation exists br access
by sonic of the reagents and catalysts used to synthesize the
molecules. lit general. about I mmnol of linker is attached
per gram of solid support. The types of solid supports that
are used include
• Polystyrene resins. Polystyrene cross-linked with divinyl
bcnzcnc ZibOLII I cross-linking>. These are common resins
used in site exclusion chromatography.
• TentaGel resins. Polystyrene in which some of the phenyl
groups have polyethylene glycol mPEG) groups attached in
position. Thc free OH groups of the PEG allow the
attachment of compounds to be synthesited.
• Polyacrylamide resins. Like "simper glue.' these resins swell
betmer in polar solvents and, since they contain amide bonds.
more closely resemble biological materials.
• Glass and ceramic heads. Not a type of organic resin hut
sometimes used when high-temperature or high-pressure reac-
tions are needed.
To support the attachment of a synthetic target, the poly-
mer is usually modified by equipping it with a linker or
anchor group. Such groups must be stable under the reaction
conditions, but they must be susceptible to a "cleavage"
reaction that allows removal of the product. Sonic common
linkers arc shown in Figure 3-la. along with the reagents
that cleave the prraiuct from the resin.
Some specialized linkers have been developed to meet
particular reaction or product conditions. So-called Iraceless
linkers can be cleaved from the resin with no residual func-
tionality left. This allows the attachment of aryl and alkyl
products that do not have OH or NH functionality. These
linkers usually include a silyl group that is sen-
sitive to acids amid can be cleaved to give unsubstituted
phenyl or alkyl products. A class of linkers known as
"safety-catch'' linkers are inert to the synthesis conditions
hut have to be chemically transformed to allow final libera-
lion of the product fromn the resin. Typically, two reactions
are required to break the linker (hence the name). A rather
elegant approach to linker chemistry is to use linkers that
are sensitive to ultraviolet WV) light, The Affymux group
has used these in the synthesis of carboxylic acid and carbox-
amide products,L Finally, some groups have used linkers
that can only he cleaved by cnzymues)'
SOLUTION-PHASE COMBINATORIAL
CHEMISTRY
Most ordinary synthetic chemistry takes place in solution.
When a reaction must be modified to accommodate a solid
support, it takes time and resources to develop and optimize
the reaction conditions. Indeed, a combinatorial chemist may
spend months designing a solid-phase reaction and gathering
the necessary materials but then conduct the entire synthesis
in a matter of hours or days! Many reactions cannot ever be
run on solid supports because of poor yields or failed reac-
tions. For these reasons, there has been much interest in
using solution-phase chemistry for the preparation of combi-
natorial libraries. Unlike one-bead one-compound synthesis
Chapter 3 • ('mnhinc,torial CIu'n,isirv 49
described above, solution-phase combinatorial chemistry
often leads to a mixture of products. Imagine reacting a set
of 10 amincs with 10 acid chlorides, all in one flask, and
with the reactants and conditions chosen so that no reaction
of amines with amines or chlorides with chlorides occurs.
only reactions between amines and chlorides. The result
would be a mixture of 1(X) amides, one for each possible
combination of amine and acid chloride. The resulting mix-
ture could then be tested for activity, under the assumption
that the inactive amides did not interfere with binding of
active molecules (not always a valid assumption). If activity
is found, smaller subsets of amines and chlorides can he
tested to eventually find the structure(s) responsible fur ac-
tivity.
Researchers have gone one step further by reacting multi-
plc kinds of reactants together to produce some rather aniaz-
ing mixtures. Figure 3-Il shows an example of a four-com-
ponent Ugi reaction that yields, alter appropriate further
transformation of the intermediate product, a mixture of car-
boxylic acids, esters and thioesters. pyrroles. I .4-benzodi-
azepine-2.5-diones. and even a monosaccharide.'4 Despite
the diversity of the chemistry. the yields of products in such
mixture-based experiments are often linind to he about 90%
or better. Although this is an extreme example of a multi-
component reaction, it illustrates the utility of solution-phase
chemistry forgenerating great diversity in chemical libraries.
An approach that is intermediate between solid-phase
chemistry and solution-phase chemistry is to use soluble
polymers as a support for the product. PEG is a common
vehicle in many pharmaceutical preparations. Depending on
the degree of polymerization. PEG can he liquid or solid at
room temperature and show varying degrees of soluhility in
aqueous and organic solvents. Each molecule of PEG has
an OH group at either end:
By converting one OH group to a methyl ether
(MeO—PEG—OH), it is possible to attach a carboxylic acid
functionality to the free OH and use solution-phase comuhina-
tonal chemistry to synthesize, for example. iV-aryl-sulfon-
amide structures.° The resulting mixture of PEG-bound sul-
fonamides can be separated by use of chromnatography.
Another type of soluble support is dendnimers. These are
large, highly branched molecttles with terminal amino
groups that can be used like the OH groups of PEG fur the
attachment of products. Finally, a class of molecules known
as fluorous phases are a form of "liquid Teflon," consisting
mainly of long chains of (—CF2-) groups attached to a sili-
con atom. When these phases are used as a soluble support
for synthesis, the resulting product can be readily separated
from any organic solvents and reaction by-products by ex-
tracting the reaction mixture with fluorocarbon solvents."
A unique application using complementary DNA as a "sup-
port" has been reported by Harvard To "en-
courage" pairs of molecules in solution to react under mild
conditions, they attach short strands of complementary DNA
or RNA to the structures to "zip" the structures together
and promote reaction. The DNA is then removed, yielding
product that would not otherwise be synthesized. Using this
method makes it possible to prevent reaction of certain pairs
of structures ax well.
50 Wilson wul Textbook of. Mediiinul and
R1—COOH + R2—NH2 t NC
0 R3
R2 0
0
/
R3
Figure 3—11 u Four-component Ugi reaction reported by Keating and Armstrong."1 A combinatorial mix-
ture of the intermediate cyclohexenyl amide can be split into several portions, and each can be further
reacted to give a variety of products, all of which will be combinatorial. (Reaction redrawn from description
from Keating, T. A., and Armstrong. R. W: Molecular diversity via a convertible isocyanide in the Ugi four-
component condensation J. Am. Chem. Soc. 117:7842—7843, 1995.)
POOLING STRATEGIES
Although some solid-phase combinatorial chemistry is con-
ducted by use of the one-bead one-compound strategy.
chemists have devised numerous other approaches to pool-
ing reactants and inlemsediales to getlerate libraries. The
goal is generally to achieve a balance between the simplicity
of mixing everything together in one step but then having
to 'deconvolute" the resulting mixture and working with
more, but smaller, mixtures. II has been likened to someone
giving you a rake and a magnet and telling you to go find
and describe a needle in a field of hay. You can make one
big haystack you know contains the needle, then have to
deal with ever-smaller "subhaystacks." or you can use more
clever approaches, such as dividing the field into regions.
using overlapping regions. etc. The major approaches that
have been used include the following:
• One-bead one-compound strategy. With this strategy, a spe-
cific quantity of beads is allocated for cacti possible structure
in the library: those beads Contain only molecules of the given
library member. The beads may be tagged in various ways
Isec the next section) to help identify the synthetic compound.
The advantage of one-bead one-compound strategy is the viol-
plicity of analysis and screening. The disadvantage is keeping
the beads separate and having to deal with a large number of
syntheses in parallel. As advances were made in robotics and
automation. the problems were reduced, and today, probably
most combinatorial experiments involve a one-head one-corn-
pound strategy.
• Iterative deconvolution. This is the strategy ftrst described
20 years ago when combinatorial chemistry was started. Reex-
amine Figure 3-3 and imagine starting at the bottom nI the
Iigam with three groups of beads. Each group has beads bear-
ins a variety olcompounds. but a given structure only appears
in one of the groups. Suppose lhc active structure is ABC (we
pretend here there is only one—in reality there will probably
-
H
0
R2
H
/ 0
he several) in the third group. Since it is in the third group.
know aC in position 3 iv needed for activity. We synthesii.e
a smaller library of the structures, in three groups: AAC +
RAC + ('AC. ARC + BI3C ± CRC. and ACC + BCC +
CCC. We do this by skipping the last set of pooling shown
in Figure 3-3. Now when we screen these we find
activity in the middle group of beads. This tells us that a It in
position 2 is required activity. The final step is to synthesise
ABC. BI3C. and ('BC. keeping iheiti separate, and screen each,
to find ABC a.s active structure.
• SubtractIve deconvolution. This is similar to iterative decon-
solution but uses negative logic, namely, leave out a functional
group, and if activity is absent, the functional group that is
missing must be needed for activity. This is particularly useful
for quantitative structure—activity relationship (QSAR)-type
studies in which. say. a —Cl group is placed at several posi.
tions on a phenyl ring. The entire library is screened as a
mixture to get the activity level. If activity is detected.
a set of sublibraries is prepared, with each missing one build-
ing block (subtr.ictiott ofa Functional group). Sublibraries that
are missing functional groups front the active compound(s)
will be less active thait the parent library. The least active
subhibraries the most important functional groups. A
reduced library containing only these luncticinal groups is then
prepared, and the most active compounds are identified by
either one.compcnind synthesis or iterative dcconvolution.
• Bogus-coin detection. This begins with generating and
screening the ctitire library as a single mixture. If activity is
detected. the building blocks are divided into three group,. (a,
fI. atid yt, and additional sublibrarics arc prepared. In these
subsets. the nuttther of building blocks From the a group is
decreased, the tiumber front (./ group is increased, and the
number front the y group is unchanged. The resulting effect
on acttvcty tup. down. ccnehanged suggests which group of
building blocks was conlributing most to activity. This ap.
proach is applied iteratively to zoom in on the groups that are
most active.
• Orthogonal pxling. The tenhl orthrim,.o,tal means perpendicti-
lar or uncorrelatcd. In this type of pooling, we the

functional groups to be considered into sets of libraries. A. B.
C. etc.. which can contain mixtures of the same compounds.
However, the functional groups are distributed such that any
subset in A and B shares only one functional group. For exam-
ple, if we have a very small library of structures—au. ab. and
ac—we might put na and ab into group A, an and ac into
group B, and ab and ac into group C. If ab is the active sttuc-
tare, screening A. B. and C would show activity in A and C,
but not in B. telling us that ub (the only structure in common)
is the active one.
• PositIonal scanning. This is a noniterative screening strategy
in which a subset library is created with a single building
block fixed at one position and all building blocks in the other
positions. In principle, by selecting the lunctional group from
the most active subset at each position. the most active com-
pound overall is discovered. This ignores interactions between
building blocks, which complicate the results.
Certain problems with mixtures must be considered when
pooling. Complex mixtures with only one or a few active
stniclures can have solubility problems, especially if the
compounds are poorly soluble. The inactive compounds con-
tribute to the total ionic concentration but not to the activity.
Sometimes, compounds that have a common scaffold will
have many active species, arising from the scaffold and not
the substiluents. Thus, many poorly active structures may
show additivity of activity, leading us to think the mixture
contains a single active structure (false-positive results). Fi-
nally. partial binding of inactive structures can sometimes
prevent an active structure from showing full activity (false-
negative results).
DETECTION. PURIFICATION. AND ANALYSIS
Detection, analysis, and purification of combinatorial librar-
ies places high demands on existing analytical techniques
because (a) the quantities to be analyzed are very small,
sometimes picomoks of material, (b) the analysis should
be nondestructive, to allow recovery of the compound if
possible, and (c) the methods must be suitable for rapid,
parallel analysis—analysis cannot be the rate-limiting step
in the procedure. No single analytical technique can lit all
lIre requirements. so usually some "hyphenated" analytical
techniques are used, for example. high-performance liquid
chromatography with a mass spectrometer detection system
IHPLC-MS). We describe this and other techniques in this
section,
Chromatography is usually the first step in the analysis
of a combinatorial mixture. If we start with solid-phase
chemistry, we chemically cleave the compounds from the
support and filter off the beads, giving a solution containing
the compounds we synthesized. If the solution contains just
a single compound. we might use a spectrophotometer, to
measure infrared (IR) and ultraviolet (UV) absorbance or
fluorescence directly, or even nuclear magnetic resonance
INMR) spectroscopy. to determine the structure of the com-
pound in solution. If the solution contains a mixture of com-
pounds, one must separate them before determining their
structures. HPLC is a standard approach. A sample of the
mixture is injected into the flow of solvent entering a chro-
matographic column. The components in the mixture travel
down the column at different rates, depending on their affin-
Chapter 3 • Combinatorial Clwmis;n 51
ity for the stationary phase in the column, and they exit or
elate from the column at difl'erent times. They are detected
by some optical method (UV absorption. fluorescence, re-
fractive index. etc.) that gives rise to peaks on a graphical
readout. Sometimes, the output from the column is passed
into a spectrophototneter or mass spectrometer to generate
a spectrum for each fraction of the output. These spectra can
be interpreted to determine the structure of the compound
that caused a given peak. It is also possible to use much
larger chromatographic columns and run preparative HPLC
to separate up to several milligrams of material for further
analysis or biological assay.
Chromatographic separations and analyses can be fully
automated. Thus, a chemist can place all the reaction vessels.
microtiter plates, etc. from a combinatorial experiment into
racks and use a robotic system to draw samples. inject them
into the HPLC, and collect the data output into computer
file.s or databases—all without further intervention from the
chemist (except to wash the dishes!). For this reason, speed
and solvent handling are special concerns with combinatorial
experiments. One approach that has been adopted to speed
up analyses and reduce the amount of solvent that must he
consumed is .supercrilical fluid (SFC),
Here. the solvent is not a common organic solvent such as
acetone or ethanol. Instead, it is a pressurized gas like CO2
that evaporates from the output. leaving pure compound be-
hind. Another advantage of SFC is speed: since the solvent
molecules are small, diffusion is rapid, and separations take
place in about half the time of ordinary HPLC separations
or less. Finally, the amount of "solvent" that is consumed is
significantly lower with SFC. A disadvantage is that certain
compounds may not separate as well under SFC as under
i-IPLC.'8
JR spectroscopy is often applied in combinatorial chemis-
try. Since IR light can be reflected from materials, one can
analyze resin beads directly, without cleaving the products
from them. Since the loading of product on any given bead is
very small, usually computer-enhanced methods like Fourier
transform JR (FTIR) are needed to enhance the very small
spectral signal from one or a few beads. Interestingly, the
shape of the beads has been found to affect the IR spectra
results, and flattened rather than spherical beads give
stronger IR signals)9 NMR spectroscopy gives more struc-
tural information than IR or UV spectroscopy. but it has
traditionally not been nearly as sensitive. Compounds arc
normally cleaved from solid support before analysis by
NMR, since NMR on solid resin or on resin swollen by
solvent gives broadened peaks and low resolution. A type
of NMR called magic angle spinning NMR, in which the
sample is inserted into the magnetic field at an angle of
about 550, reduces the peak broadening and has been used
to analyze swollen polymer beads directly. Recent improve-
ments and the use of "nanoprobcs" have allowed NMR
analysis of I00-mjz beads bearing less than 80() pniol of
compound. Other NMR techniques that have been used to
analyze combinatorial mixtures include various "two-di-
mensional" (2D) NMR techniques that use multiple bag-
netic fields. HPLC-NMR, capillary ekctrophoresis coupled
to NMR (CE-NMR), and even NMR to detect the binding
of drugs to receptors to identify active agents. This latter
technique has been termed SAR mm'iI!m NMR.24t
Mass spectrometry (MS) is the technique most widely
52 tViIxo,i alul Gi.cvold s jeribr,ok of !ile'diri,,aI and PI,ar,nace,aical ('he,njsiry

used for combinatorial library analysis. The measurements 0 H

can he made on resin beads directly, a wide range of com-
pounds can be analyzed, and MS analyses can be highly
automated. Included among a number of MS techniques in
use arc
Electrospray ionization. A solution containing the corn-
pounds to be analy,.ed is passed into a macs spcctronteter
through an electrically charged capillaty. The droplets that
emerge from lime capillary hear strong electric charges theft-
selves, and they literally "exphxlc" into smaller and smaller
droplets and eventually into singly charged ions that are de-
tected by the mass spcctromemer.
• Matrlx.as,sisted laser desorptionllonlzation tlmc.of-flight
(MALI)l-TOF). Quite a mouthful, ii simply means the sample
is embedded in some solid matris (e.g.. 2.5.dihydroxybenzoic
acid) and then bonibanied with a laser. Sample ntokcules axe
vaporized and ionized in a 'gentle' fashion that allows
whole-molecule ions of the sample to be analyzed. The analy-
sis is dune with use of a time-of-flight analyzer, in which ions
of different mass travel different distances in a given amount
of lime.
• Other less-used MS techniques. These include secondary.
ion MS (SIMS) in which the sample is hit by a metal ion
rather than tIme electron beam itself, and Fourier translorm MS.
A very important use of MS in combinatorial chemistry
is in quality control of combinatorial libraries. As much as
possible. we would like to have pure compounds generated
in high yield, with no side reactions or by-products. We
also need to verify that every component actually exists in
a library (i.e.. that no reactions failed). Only MS provides
the sensitivity and versatility to perform this checking with
both solid-phase and solution-phase libraries
ENCODING COMBINATORIAL LIBRARIES
Once we have found a mixture or sublibrary that shows bio-
logical activity, how do we determine exactly which struc-
ture or structures are responsible for the activity? We can
purify and analyze as described in the previous section. but
if no direct analysis is available, we need to encode or tag
the support or the molecules themselves. using physical or
molecular "barcodes." An obvious approach that can be
used only with small libraries is to physically label each vial
of one-bead one-compound resin. This may be practical for
a few lens of compounds, hut what if we have a library
of 32.(X)0 compounds, or even I .000,000 compounds. in a
mixture? Clearly. there is a need for a more automated means
of identifying the structures that are in the library.
The most common approach to encoding solid-phase li-
braries is to attach a chemical tag to the resin beads as the
target molecule gets synthesized. 'rypically. at each step in
the reaction, a tag is attached that is unique for the given
step. For example, if we are creating a tripeptide and we
have 10 possible amino acids at each position, we need to
attach either a single tag that says tripeptide on this
bead has amino acid Ala at position I, Phc al position 2,
Peplide library
C)— Linker — N"-'
H
a Primer-DNA tag.Primer
Linker2 — N — Peptide library
H
Linker, — Primer-DNA lag.Primer
linker2 — N — Peptide library
H
(
Linker2— N — Peplicte library
H
Figure 3—12 • Two ways of attaching DNA tags to solid sup-
ports. a. A DNA tag is attached with each peptide molecule via
a bifunctional serine residue. b. The DNA and peptide groups
have separate linkers
to beads on which peptides or pcptoids were being built.2'
Since there are 20 possible amino acids and only four nuelco-
tide bases, enough bases must be attached at each amino
acid addition step to identify properly the amino acid being
attached. Although three bases are used in the DNA genetic
code, it is customary to use up to six bases for library tagging.
For decoding. the DNA tag is amplilied by use of the poly-
,nerase ihau, reunion (PCR). the same reaction that is used
in forensic DNA analysis. For this reason, the chemical tag
must also bear PCR primer sequences.
TWO types of anchors have been used to connect the DNA
tags to the solid support (Fig. 3-12). (none type, the growing
DNA chain is attached to the a carbon of a serine group
that is anchored to the solid support by a linker molecule.
The growing peptide chain is attached to the serine atnino
group, possibly through a spacer. In the second type of an-
chor, the DNA chain has its own anchor to the solid support.
in this case, fewer DNA tags are attached than the number
of polypeptide molecules.22
If DNA tags cannot be used, one can label beads by using
a "binary" approach. Suppose we are bttilding a tripeplide
with four possible amino acids at each position. We can use
binary digits to encode which amino acid is at a given posi-
tion us follows (each binary "number" is read from the
right) (Table 3-I): Thus, using 1K different tags (3 x 6) we
can encode br any of the (4' = 64) members of the library.
For example, if the product is Ala-Gly-Lys. the encoding
TABLE 3-1 Binary Encoding of a Tripeptide.
UsIng 18 PossIble Tags
Amino Add Position I PosItion 2 Position 3
Ala 000000 (N) 1*) OIl IX) (10 (Ni

and Gly at position 3." or we need to attach three different Plie 000001 1)00100 010000
tags, one for each position. Cay 000010 11010(5) 100015)
One of the earliest types of chemical encoding was the l.ys (1000 II 00 11(51 110000
attachment of oligonucleotides (usually single-strand DNA)

would be 00000000100011 0000, and 3 of the 18
possible tags would be attached to the support, along with
the tripeptide. It is common to use polyhalogenated aromatic
compounds as lags, such as
where X represents some combination of halogen atoms. The
halogens make the tags show up clearly in MS analysis of
the mixture, and by varying the chain length a, the tag can
be made flexible enough not to interfere with attachment of
the product. Other chemical tags that have been used include
isotopically labeled peptides and dyes.
When it is not possible to use chemical tags, one must
physically label the solid or liquid support itself. One alterna-
tive is to use radiofrequency encoding, in which tiny mi-
crochips are added to the resin or to the solution phase. As
various reactions are conducted to generate the products, at
each step a radiofrequency signal is stored in the microchip.
This signal can be recalled to identify the sequence of reac-
tions that generated the product (a similar principle is used
when your dog or cat gets a small identification (ID) pellet
implanted under the skin of the neck). Laser optical encoding
is yet another approach, in which the solid support consists
of a ceramic chip covered with a polypropylene—polystyrene
polymer solid phase. The barcode pattern is actually burned
into the ceramic at each step in the reaction and is decoded
visually with use of a microscope. Finally, one can embed
semiconductor particles into the solid phase that fluoresce
at different wavelengths. These are called "quantum dots"
by their manufacturer.23
HIGH-THROUGHPUT SCREENING (HTS)
Without the ability to screen libraries rapidly for activity,
there would be no combinatorial chemistry. Fortunately, the
biologists are just as adept at developing rapid high-through-
pat assays as the chemists are at generating structures. HTS
is an extremely broad topic. encompassing enzymes. organ-
elks, cells, various tissues, whole organs, and even whole-
animal testing, via cassette dosing. This section briefly dis-
cusses only a part of the role of HTS in drug discovery, with
emphasis on a few recent
Successful HTS programs integrate several activities, in-
cluding target identification (genomics and molecular biol-
ogy groups), reagent preparation (protein expression and Pu-
rification groups), compound management (information
management group), assay development (biologist and phar-
macologist), and high-throughput library screening (biolo-
gists and chemists). Formerly, these activities were handled
separately, and multiple handoffs of samples were involved.
It is becoming more common to integrate the activities and
share expertise. This increases efficiency of the screening
process. Another route to increasing efficiency is a move to
Chapter 3 • C€nnbinumria! Oiemisirv 53
higher-density screening platforms. The standard layout for
HTS has been a 96-well microtiter plate (12 x 8). Denser
formats, up to 1,536 wells per plate, are increasingly being
used. This requires advances in liquid handling, precision
of detection, and laboratory automation.
One of the first activities in developing a HTS assay is
selecting the target. About 500 targets are currently being
used by drug companies. Of these, cell membrane receptors.
mostly G-protein—coupled receptors, make up the largest
group (about 45% of the total). Enzymes make up the next
largest group (28%), followed by hormones (lIck), un-
knowns (7%). ion channels (5%), nuclear receptors (2%).
and finally DNA (2%).25 It is expected that the annotation
of the human genome will add additional targets. although
the rate of this addition is not known. New targets must he
part of some regulatory pathway in the cell and should be
sensitive to some disease state, not be expressed all the time
and everywhere in the cell.
The next concern in I-IFS is the library to be screened.
Throughout our discussion, we have perhaps offered the
impression that a given library for a particular project was
the only set of compounds that were ever screened for activ-
ity. In fact, much HTS involves screening compounds that
are part of the corporate storehouse of compounds synthe-
sized in the past, or they may be a library purchased from
a vendor. Such libraries usually Consist of microtiter plates
containing frozen or dried samples of compound—perhaps
only micrograms per well. The size of such libraries may
range from a few thousand compounds to nearly a million.
The cost of completely screening such a library against just
a single assay may amount to over $300,000, so such large-
scale screens are conducted rather infrequently, compared
with routine day-to-day screens. It has been estimated that
one must screen at least 120,000 "quality" compounds (i.e..
diverse drug-like structures) to discover a single-lead
for a therapeutically sound target.25'
As discussed above in the section on pooling strategies.
one can reduce the screening effort by pooling groups of
structures and running assays on mixtures of compounds.
This also conserves reagents and biological material, has
smaller storage requirements, and requires fewer personnel.
There are potential problems with pooling. A number of
factors limit the number of different compounds we can test
in a given well, including ionization, reactivity, and solubil-
ity. Compounds can enter a screening program in a nonran-
dom order, such that a given assay plate may have com-
pounds that are highly similar structurally. This may give
rise to false-positive hits. False-negative hits are less likely
to arise from pooling. Another concern is the use of repli-
cates—compounds from the same series—in a given assay.
If only one representative of a given series is present. the
chance of missing that series as a possible lead series is
greater than if multiple members are present. Therefore, it
is common to include several members of each series in a
given assay when possible.
To be effective, a given compound must dissolve com-
pletely in the assay medium. It is common to add a small
amount (1%) of dimethyl sulfoxide (DMSO) to the assay to
assist solvation. The best concentration of compound to use
is somewhat debatable. High concentrations (10 jaM and
above) often lead to more false positives than screening at
54 Wilson and Gi.;wld's of Organic Medicinal and P/,arnzaceutieal C/wn,is:rv

a low concentration (3 suM). The reason for this may be
nonspecific binding at the higher concentration.
Just as there are several ways to dctcct and identify mem-
bers of a combinatorial library, there are many ways so mea-
sure activity in I-ITS assays. Any such method must be accu-
rate. reproducible. and have a high signal.to-noise ratio (SI
N). Typically, the result of FITS is a qualitative (yes/no)
or semiquantitative one (high-medium-low), rather than a
precise value (e.g.. K180 or The methods for detection
in FITS fall into the categories of nonradiometric and radio.
metric.
Nonradiometric methods include absorbance, fluores-
cence, and luminescence spectroscopy. Enzyme assays are
a common example. The assay is usually run at or below
the value of substrate. svith only about 5% of the
substrate consumed during the assay, and multiple enzyme
turnovers occur during the assay. Sometimes enzyme reac-
tions arc coupled, especially if the target reaction does not
produce a product that can be detected directly in the assay.
An example is carboxypeptidase. which is coupled to the
reduction of NADP to NADPH. giving rise to absorbance
at 340 nm.
Radiometric methods include filtration and scintillation
proximity assay (SPA). These assays use radioisotopes. so
safe storage and handling are of concern. In filtration assay.
a radioactive substrate bound to a capture group is cleaved
by its enzyme. removing the radioactivity from the capture
group. The mixture is filtered through special filter paper
that the capture group sticks to. but everything else passes
through. A scintillation fluid is added, and the radioactivity
of the filter is measured. The degree to which the radioactiv-
ity is retained measures the strength of the inhibition (Fig.
3-l3a).
SPA is a newer, simpler method (Fig. 3-I3b). We start
with the same radioactive substrate, which may not necessar-
ily need a capture group. The enzyme and potential drug are
added, causing the cleavage of the substrate to some degree.
Now, instead of filtering, a special resin bead coated with a
.cc-in:illant—a compound that fluoresces in the near presence
of the radioactive substrate (near being about 20 zrn)—is
added to the mixture. The lysed and unlysed substrate binds
to the beads, and if the radioactive part of the substrate is
still attached, the bead will fluoresce. If not, the radioactive
parts of the substrate floating in the solution will be too far
from the beads to cause any fluorescence. The presence of
fluorescence implies that the test compound inhibited the
enzyme. The advantage of SPA over filtration is that no
filtering of the solution is needed, so beads can be added
directly to the assay mixture in wells or test tubes. Also.
special scintillation fluid is not needed. The beads for SPA
can be engineered to attach a variety of substrate types.
Other HTS assay advances include the use of microorga-
nisms such as bacteria and yeast, the cloning and expression
of mammalian receptors in microorganisms, probing pro-
tein—protein interactions, and very importantly. DNA and
protein arrays. These are too involved to discuss here, but
excellent reviews exist.24 V The increasing use of HTS to
screen for a molecule's absorption, distribution, metabolism.
excretion, and toxicity (ADMET) properties has been cov-
ered as well.28
VIRTUAL (IN SILICO) SCREENING
Virtual, or in silico, screening refers to the use of computers
to predict whether a compound will show desired properties
or activity on the basis of its two-dimensional (2D) or three-
dimensional (3D) chemical structure or its physicochemical
properties. The motivation for using virtual screening arises
from the flood of new structures coming from combinatorial
chemistry. the expense and time required to run conventional
HTS. the ethical concerns about using animal tissue instead
of predictive models, and an increasing failure rate for struc-
tures coming out of combinatorial programs. In general, a
virtual screening program attempts to answer one or both of
these questions:

Enzyme CO—Substrate, Substrate(i ).—__ CC—Substrate,

CO—Substrate, * Substrate(')

Enzyme inhibitor CO—Substrate, — SubsIrate(')

CO—Substrate, - Substrate(')

FILTER
a

Enzyme CG—Substiale, Subslrate(') CO—Substrate,

Substrate(')
CO—Substrate,
CG—Substrale, — Substrate(')
Enzyme I nhlbltor CO—Substrate, — Substrate(')

b SPA Effect

Figure 3—13 • Comparison of filtration and scintiUation proximity assays. a. In filtration assay, the enzyme.
substrate, and inhibitor are mixed; the uninhibited enzyme splits the radioactive portion (*) off the substrate,
and filtering the mixture, followed by measuring the radioactivity of the filter, tells how much inhibition
has occurred. b. In SPA, the same mixture is treated with resin beads containing a scintillant that fluoresces
only in close proximity to the radioactive source. Any radioactivity that was split off by the enzyme does
not need to be filtered in SPA.

I. Will i particulur compound show sufficient binding iou known
rL'ccpisr?
2 Will a particular compound possess any undesirable ADMET
properties?
To answer these questions, we must build computer
utodds of the interaction of drugs with receptors (docking,
molecukir modeling, quantum mechanics) and models for
predicting ADMET properties on the basis of chemical struc-
ture (QSAR models). Like much of drug discovery, the vir-
tual screening process is a cyclic otie. We stan with a collec-
tion of structures and run predictive models on them.
generating some subset of "best" structures. We then test
these slnictUres in real assays or screens to see if the predic-
tions were accurate. Finally, we incorporate information
learned From the real assays back into our predictive models
to improve them for future useY1
Models tor the prediction of binding arise from the field
of molecular modeling. This includes molecular mechanics
predicting the 3D structure olmolecules from the standpoint
of the atondc nuclei) and quantum mechanics (predicting
the 3D structure of molecules from the standpoint of their
electrons). We can generate fairly accurate 3D structures of
stand-alone molecules with either of these approaches. We
can also model the interaction of tWo molecules and predict
whether they will show hydrogen bonding or electrostatic
or lipophilie interactions, If we know (he 3D structure of a
receptor, we can predict whether a given compound will
"fit'' into the receptor with sufficiently tight binding to pre-
sent normal substrates from binding. i.e.. an enzyme inhibi-
tor (or drug). Typically, this does not consider any effects
of transport, metabolism, interaction with solvent. etc. Con-
sequently. the existing models for the binding of drugs to
receptors are rather crude, with errors of 50% or more.
Nevertheless, for the purpose of screening, this is often ade-
quate. II a structure is predicted to show tight binding, it
will probably be synthesiied and testcd in a real HTS assay.
loobtain a more accurate estimate of its activity.30 An inter-
esting application of high-throughput docking is the United
Devices screensuver.'5t This is a PC program that can be
downloaded to run in the background and participate in
worldwide projects to screen large databases of structures
again.ct such targets as cancer and anthrax.
We can combine the prediction of binding to a receptor
with the design ofomolecules. lfwe start with a set of building
blocks and a known receptor, we can align complementary
building blocks with pockets in the receptor, in conforma-
tions that masintize hydrogen bonding and other interac-
lions, When the fragments are aligned, we can then connect
the fragtiients with appropriately sited spacer fragments to
"build" a drug molecule within the confines of the receptor.
lids approach is sometimes called de not'u or structure-
hosed drug design. As our models for binding to receptors
inuiprove. this approach is becoming more popular. '
In 1997. researchers at Ptizcr looked at the previous 10
scars' worth of drug design. including all of their combinato-
rial chemistry efforts.33 They found that since the introduc-
tion of combinatorial chemistry, chemists had tended to de-
sign larger. more complex, and more lipophilic structures
thutu in mite past. .Siiice the structures were SUpposed to be
desioned as lead structures rather than optimized drug mole-
euks, it was becoming timore diflicult to optimize them into
Chapter 3 U Cwnbi,wrorial CIw,nistrv 55
drugs. This led Lipinski to enumerate some rules for the
rejection of structures. He proposed rejecting any structures
that fail two or more of the following criteria:
• Molecular weight should be <5(X).
• Number of hydrogen bond donors (NH. OH should be fewer
than 5.
• Number of hydrogen bond acceptors t—N—. —0—.
—S-—) should be fewer than 10.
• Calculated log P value should be less than 5.
Since the number 5 shows up in many of these criteria.
they have become known as the "rule of five." These and
similar rules have become widely adopted by drug compa-
nies in their discovery programs. They ball under the general
category of "business rules' '—guidelines for the proper
conduct of business and research. One should be cautiouS
though and use them as guidelines and not strict rules, to
avoid missing important drug structures that happen to fail
the rules but still show high activity and acceptable ADMET
properties.34 The "rule of five" is designed to be a yes/no
filter for the rejection of structures. More quantitative
models designed to predict some value or level of property
have also been developed. Examples include predicting
Caco-2 cell permeability (Caco-2 cells are human intestinal
epithelial cells that can be layered and studied in vitro)33
and predicting binding to cytochrume P-450 (a major liver
enzyme involved in metabolism),35
CHEMICAL DIVERSITY AND LIBRARY
DESIGN
The universe of drug-molecule--sized compounds that could
be synthesized is enormous. For example, a polypcptidc with
100 residues could regenerate I 005° or 1040 possible struc-
tures. If just I g of each were synthesized, it would he a
mass comparable to the mass of the known universe (about
1050 kgh) (Peterson. J,: Universe in the Balance. jVeii' Scien-
tist 168:26. 2000), Clearly, a combinatorial chemist nced.s
to select carefully the scaffolds and building blocks that will
make up the library to be synthesized. Since the 1980s.
chemists have adopted many computational techniques to
aid in the design and selection of library structures. These
techniques have been borrowed largely from the fields of
QSAR and molecular modeling. This section describes snore
of the most common computational approaches to library
design.
Typically in drug design there are lour possible scenarios
for the amount of information we are starting with37
I. We do not know the structure of the receptor, and we do not
have any known ligands or inhibitors. This is mainly a case for
high-throughput in vitro screening nt evisting drug databa.scs
and libraries, in the hope thai sonic structure may hind to the
receptor and serve as a sturtittg point for lead development.
Often it is straightforward to (md existing structures with micro.
molar potency. The problem then becomes one ot deciding how
to modify the structures to yield ones that arc novel from a
patent standpoint and show suitable hiophannaceutical proper.
ties.
2. We do not know the structure of the receptor. hut we do have
known ligands or inhibitors. This was once the most commotu
56 Wilson and Gisvold'x Textbook of Medicinal and Plior,nacriaical Clu'n,,srrv
situation in drug design. The usual approach to selecting since-
lures br Further testing is to lind structures in chemical data-
h;ises that are similar in structure and properties to the known
inhibitors. A common approach is to develop a phannacophscre
model (icr the receptor, based on supenmposing structures with
known activity. This can then be used as a scan!i query in a
chemical structure database to find structures that have the same
functional groups in the same relative positions.
3. We do know the structure of the receptor and we base known
ligands or inhibitors. This is increasingly becoming the state of
affairs br known receptors. There are about 100 known recep-
tor—ligand complexes published in the Protein Data Bank that
have relevance to existing human illness. Pharmaceutical com-
panics have many more examples. As soon as one drug company
patents a drug with activity for a known receptor, other compa-
nies are qtmick to use this information to try to develop novel
structures with greaier potency, longer-lasting activity, fewer
side effects. etc. This is c.spccially (rue if the market for the
agent is a large one. The techniques for finding new drugs in this
situation combine (a) pharmacophore discovery using known
ligands with (b) docking and molecular modeling of new struc-
tures to see how well they fit into the receptor. This is knowit
as virtual and it is becoming more important all the
time. The quality of virtual screening results depends on the
quality of the protein and drug molecule structures we start with
and the accuracy of the mathematical functions that predict the
degree of binding from such factors as steric. electrostatic, hy-
drogen bonding, and lipophilic interactions between the drug
and the receptor.
4. In the final scenario, we know the structure of the receptor, but
we do not have known ligands or inhibitors. This is a less. likely
case than scenario 3, but the decoding of the hutnan genome
may change that. 01 the 3(1.0(X) or more genes that arc being
annotuted front the genome. it is estimated that perhaps
may have some relevance to human disease. This would repre-
sent 3.000 new enzyme targets for drug designers to work with.
Researchers expect 10 or more new targets to be elucidated each
year. so this promises to be a long-term project. In many cases,
the enzyme or structural protein that a gene encodes may be
dirncult to crystallize for x-ray analysis or to study in solution
using NMR. In such cases, protein molecular modeling may be
used to predict the structure of the enzyme. A common approach
is lmonu,logy modeling, in which sequences of the unknown pro.
1cm lime assigned secondary and tertiary structttrcs based on
those oh' known proteins with similar sequences. IBM has em-
barked on a project known as "Blue Gene" to try to predict
the structures and receptor Sites of all the protein products of
the human genome. over a period of a few years, using super-
computers. Once the enzyme structures are known or predicted.
virtual screening can be used to find drug molecules that might
tit into the receptor.
Assuming that one of (he above scenarios holds for a given
drug discovery project. one commonly proceeds through a
sequence of chemical libraries on the way to lead discovery
and optimization. This process has several phases, and the
libraries that are designed at any given phase show varying
levels of diversity. size, and specificity for the given recep-
tor. The goal of library design is to select a subsel of mole-
cules from some larger collection such that we are taking
samples from various regions of chemical space. If the li-
brary is an initial or exploratory library, we want to sample
as much chemical space as possible, so we seek structures
with high diversity, If the library is a morefo<-u.sed library.
we want to sample a smaller region of chemical space in
the neighborhood of the most active structure discovered in
the exploratory library. If the library is an oplimization Ii-
brury. we want to look for small changes in the httsic struc-
ture, to find the best comnhintttion of suhstituents that will
yield the highest activity.
The first task is to define and qttantify chetnical dit'er.sit.
This requires a delinition oichemicul .thnilariit. since diver-
sity is essentially the opposite of similarity. There are several
ways to quantify tuolecular sitnilarity. Two common ap-
proaches in combinatorial chemistry are (a) to define simi-
larity as the closeness of structttres to each other in the space
of some physicochensical or topological elcscnptoni (e.g..
log P. solubility. or polar molecular surface area) and (hj to
delitie similarity as the nutnber olsimple structural features
the compounds have in common (e.g.. carbonyl groups.
phenyl rings. etc.).
Consider the molecules in Table 3-2. If the first molecule,
diethylstilbestrol. is used as a similarity probe in a database
of drug structures. several other structures can be found
(some of which are estrogens, hut many of which have other
activities). The tuost similar structures do have estrogenic
activity, and they have values similar to those of some of
the other descriptors in the table. Sonie descriptors by them-
selves would be poor predictors of molecular similarity. For
example. t)OC can bind many structures with sitnilar log P
values but with different structural characteristics. For this
reason, there has been some controversy about what the
''best'' approach to similarity and diversity is.38 In the de-
sign of exploratory libraries, the goal is to sample a wide
variety of chemical structures, so it is reasonable to use di-
versity measures that focus on structural differences, such
as 2D molecttlar similarity. We select molecules that are
dissimilar to each tither. In the design of optimization librar-
ies, the gnal is often twolold: to increase potency and to
optimize ADMET properties.Minor changes in the structure
arc usually made at this point, so all the structures show
high 21) similarity to each other. But tninor changes in sub-
stituents can change physicochemical properties and modify
the interaction of the drug with the receptor. so the other
descriptors in Table 3-2 are <then more useful in the lead
optimization stage of a project. An example of a simple
change that causes a large decrease in binding affinity is
seen in Figure 3-14. where the replacement of a H atom by
a methyl group reduces activity 80-IbId.39
CH3
R - H (6.7 nM)
R Me (470 nM)
Figure 3—14 • Changing a H atom to a methyl group has a
large effect on binding to the ru-aminobutyric acid (GABAA}
receptor.
 Chapter 3 U ChL',ni.r:r% 57

TABLE 3-2 SimIlarity of Sthactures to Diethyistilbestrol (DES). Using Various Descriptors

Polar No. of No. of No. of 20 Molecular
Surface N-Bond H-Bond Rotatable Similarity
Molecule Log P Area Acceptors Donors Bonds to DES

40.5 2 2 2 100

7.91 29.5 I 2 5 76

6.7K 52.6 0 4 8 56

5.68 40.5 2 2 5 4))

5.34 20.2 I I I) 36

5.58 40.5 2 2 4 33
58 Wilson (i.srold'.s Te.ttl,ook of Organic Medicinal and I'han,uus'iaica! Che,nistrv
Having decided on some measure(s) of diversity. the next
step is to actually select structures from the starting collec-
tion. Typically, we select either whole compounds for HTS
or reagents to be used in a particular synthetic step. The
selection process consists of using similarity/diversity mea-
sures to pick compounds that sample chemical space in a
manner appropriate for the library being generated. Thus.
for an exploratory library, we would pick diverse structures
with as much structural variation as possible. perhaps subject
to some rules about size. lipophilicity. etc. For an optimiza-
tion library, we would pick substituents for a given scaffold
that span traditional QSAR space. which includes steric,
electronic, and lipophilic properties of the substituents.
The methods of selecting structures come mainly from
statistics and QSAR.4° They include the following:
• Random selection.Here we let the computer pick structures
at random from the initial collection. We may "bias" the
r.mdom selection by littering the structures as they are picked
and rejecting ones that we know we do not want or that are
too similar to structures already picked.
• Visual selection. If we have selected, for example, 10 descrip-
tors for our chemical space, it is difficult to lind display meth-
ods that can display data in high dimensions. There are projec-
tion methods in statistics that can make a "shadow" of the
points in high-dimensional space onto two or three dimen-
sions, at which point a standard two-dimensional (2D) or
three-dimensional (3D) scaflerplot can be used to see how the
points are distributed and to select compounds. A standard
method for this projection is principal campollenix onalssis
(PCA). In this approach. we generate a couple of new descrip-
tors, the principal components. that are linear combinations
of the original descriptors (pe, = w,x1 + + --) with
each descriptor (v) weighted 1w) according to how much it
contributes to the overall variation of the data.
• Binning. If we partition chemical space into regions, like
squares on a checkerboard, we can pick molecules from each
region to build our library. Probably some regions will havc
many structures in them, and we may pick several from such
areas. Some regions will be empty. These are termed hok.s in
our collection, anti we may want to design structures with
property values that would help fill these holes.
• Cluster analysis. This method involves using statistical pro-
cedures that try to discover natural groupings of compounds
on the basis of similarity or distance between them. Cluster
methods function either by partitioning space on the basis of
the density of the compounds (e.g.. K-means clustering) or by
linking the compounds in a tree-like structure (hierarchical
clustering). The result is to assign each compound to a group
or clustcr. Compounds within a cluster are more similar or
closer to each other, on average, than to compounds in other
clusters.
• Experimental design. If we have selected a subset of struc-
tures from our collection, we can use any of several statistical
measures to quantify the diversity of our subset. Further, there
are statistical selection procedures that, if followed, will make
it more likely (but not guarantee) that we pick more and more
diverse subsets. These are optimization procedures, and they
are widely used in the design of experiments, statistical sur-
veys. etc. An example of a common procedure is one called
0-optimal selec:io,,. which is designed to pick a subset of
poiuits that are as widely separated from each other as possible.
• Genetic algorithms. This optimitation procedure is inspired
by the way genetics and natural selection work. To use a ge-
netic algorithm, we pretend that our collection is an artificial
'genome." Each structure in the collection is a base or string
of bases in the gcnome (instead of A. T, 6, and C, the base
designation is just I or (I). We start by generating random
strings of Is and Os to represent the genome. Each string is
measured for the diversity the structures in the string represent.
This is called the fitswss of the string. Then, we apply the
genetic operations of mutation. crossover, and reconihination.
to generate new populations of Is and Os.Some of these will
have higher fitness values (more diversity) than others. Over
time, as we repeat the genetic operations, the overall genome
will tend It) improve toward more diverse collections of struc-
tures.
Most of these methods, including calculating descriptors
and measuring molecular similarity arc part of combinatorial
chemistry software systems. These systems are provided by
molecular modeling and by chemical information companies
such as Accelrys. Daylight. MDL. and Tripos. They usually
function in the context of a chemical structure database. Such
databases can store 2D structures. 3D models, reactions, ge-
neric structures, building blocks, and all the physicochemical
and inventory data in a single repository. sometimes called
a duo, ;t'are/rou.se. Chemical data are indexed and accessed
by structure or structure ID. Biological data are typically
indexed and accessed by assay or test ID. For this reason.
most drug companies have traditionally stored chemical and
biologicitl data separately and used different systems for ac-
cess. In the past few years. it has become common to store
both chemical structures and biology data in relational data-
bases such as Oracle. This trend toward integration of the
two types of data is motivated in large part by the flood
of information that combinatorial chemistry and HTS are
generating. Most large pharmaceutical companies are syn-
thesizing and testing times as many structures today
as they were 15 to 20 years ago. with a similar increase in
the amount of information that must be collected, organized.
stored, utid interpreted. The drug companies are taking a cue
from large retailers and financial vendors and adopting "data
mining" techniques to search for hidden associations, clus-
ters, and predictive relationships in the mountains of data
they arc collecting itt their databases.4'
REPORT CARD ON COMBINATORIAL
CHEMISTRY: HAS IT WORKED?
A report published in 1998 showed that virtually all of time
major pharmaceutical firms had adopted combinatorial
chemistry to some extent in their drug discovery process.42
The degree of adoption ranged from 16 to 100% of new
drug synthesis, with an average of 66%. Clearly, the phanna-
ceutical firms have a big stake in combinatorial chemistry,
but has it really worked out? As of this writing, the phamia-
ceutical firms are suffering large increases in research and
development expenses, but with a decline in the number of
new drugs in the pipeline. Many finns have a large fraction
of their blockbuster drugs (>$l billion in sales per year)
going off patent in the next few years. Part of the problem has
been the pursuit of only a few, highly profitable, therapeutic
targets. For example, there are at least seven Stalin
cholesterol drugs on the market: the most prolitable one.
Lipitor, currently collects about 57 billion in sales per year
for its developer. Another problem has been the marketing
of drugs that appeared to be safe, even throughout clinical
 Chapter 3 a Combinatorial Chemistry 59

riuls, hut were later found to cause serious and even fatal
side effects (e'., Seldane and Baycol).
There is tithe question that combinatorial chemistry has
been effective in generating large numbers of lead structures.
pharmaceutical companies began using combinatorial
chemistry to build up their in-house libraries of structures
that could be "mined" for activity against newly discovered
receptors. A typical pharmaceutical firm has access to infor-
mation on 10 to 20 million structures from commercial
sources (various chemical software vendors and the Amen-

TABLE 3-3 Examples of Lead Structures Obtained by Combinatorial Chemistry

Structure Source Target Mechanism

Merek III I intcgruse Block Strut integration

ci SinhkKiine Human AntagoniM: cognithe

leeeliam Scrotonin receptor

CF3 Abbou intcrlcukin-2 Cytokine Inititsitirm

N—N

CF3 Plizer Earnesyl trunufetase a Inhibition

Cl

Parkc Davis KDO-8-P synthetasc Inhibition; antibiictorijl

60 lViI.con and Gici'old's Textbook of Organic Medicinal and Pharmaceutical Chemistry
can Chemical Society Chemical Abstracts Service). In addi-
tion. large companies have their own multimillion-com-
pound databases. Golehiowski et describe how lead
structures with a wide variety of activity have been obtained
with use of combinatorial chemistry. Sonic examples are
shown in Table 3-3, demonstrating the variety of structural
types that have been generated. An industry perspective pub-
lished in 2001 reported 46 compounds in human clinical
trials that originated from HTS of libraries that were identi-
fled between 1992 and
What can be argued is whether the goal of generating lead
structures is sufficient, in light of an increasing rejection rate
of candidate drugs in clinical trials, caused by side effects
and other ADMEI'-related failures. Most researchers would
agree that we need to predict the "drugability" of a lead
better before much testing, if any, is done. As mentioned in
the section on virtual screening, much work is being devoted
to the development of better in vitro and computational
methods for predicting ADMET properties. Alternatives to
combinatorial chemistry arc appearing in the literature. An
examfle is the "non-combinatorial" approach of Everett
et al. These authors argue that the goal of combinatorial
chemistry should be the quality, not the quantity, of leads.
Some trends that are appearing in the literature include (a)
smaller libraries, a few thousand carefully selected structures
rather than 250,000 hastily designed ones; (b) more attention
to ADMET properties in the early phases of drug discovery;
(c) miniaturization of syntheses and assays, using ,nicroflu-
idics and nanoiechnologv. both for speed and to conserve
resources; and (d) an integration of genomic and combinato-
rial chemistry technology for better use human genome in-
formation in the design of new drugs.46 Most chemists agree
that combinatorial chemistry, after 20 years of evaluation.
is a vital, but not the only, implement in the drug discovery
toolkit that should be used. Like other tools, it can be applied
intelligently to great benefit, or it can be misused,
RESOURCES FOR COMBINATORIAL
CHEMISTRY
Beck-Sicklinger. A.. and Weber. P.: Combinatorial Strategies in
Biology and Chemistry. New York. John Wilcy & Sons. 2002.
(The finest short introduction available)
Bunin. B. A.: The Combinatorial Index. New York, Academic
Press, 1998 (a comprehensive, chemistry-oriented reference).
Crarnik. A. W.. and DeWitt. S. H. (eds.): A Practical Guide to
Combinatorial Chemistry. Washington. DC. American Chem-
ical Society, 1997.
Fennin. H.. Combinatorial Chemistry—A Practical Approach.
Oxford. UK. Oxford University Press. 2000. (Laboratory ex-
periments)
(Those. A. K.. and Viswanadhan. V. N.: Combinatorial Library
Design and Evaluation. Principles. Software Tools, and Appli-
cations in Drug Discovery. New York, Marcel Dekkcr, 200!.
Gordon, E. M., and Kerwin, J. J. F. (eds.): Combinatorial Chem-
istry and Molecular Diversity in Drug Discovery. New York,
Wiley-Lies, 1998.
Terrett. N.: Combinatorial chemistry. In Compton, R. G.. Davies.
S. 0.. and Evans. J. (edt.). Oxford Chemistry Masters. Oxford.
UK. Oxford University Press. 1998. (A brief, highly readable
introduction)
Journals
Combinatorial Chemist re and High-Tl,rou.c.hput Screen
ing—Bentham Publishers
Drug Discovery Today—Reed Elsevier
Journal of Chemical Information and Computer Sti
ences—American Chemical Society
Journal of Combinatorial Chen,isirs—American Cheinica
Society
Modern Drug Discovery—American Chemical Society
Molecular Diversitv—Kluwer
Nature Reviews Drug Discovery—Nature Publishing Group
Trends in Biotechnology (T1BT&'H)—Elscvicr
Videos
Che,nicai Diversity: Applications of Computational Approaches
Washington, DC. American Chemical Society. 1995.
Chemical Diversity: Synthetic Techniques of Co,nbinatoria,
Chemi,csry. Washington. DC. Anerican Chemical Society
1995.
Web Sites
lutp://lvsvw.comhi.web.com—Corporate-sponsored web portal.
Accessed Dec. 3, 2002.
http://www.combkhent.neilhonte/login.a.sp—Recent develop.
menu.. Accessed Dec. 3. 2002.
hsrp://www.contbinarorial.com—Weh site for The Combinato-
rial Index text. Accessed Dcc. 3, 2002.
hup://www.geocities.eo,n/ResearchTriangle/Lab/4688/
combinatorial chen,istrv.l,tm—llnofficial Combinatorial
Chemistry Web site. Updated Mar. 5. 2002.
http://wwiv.microarrays.org—tjnivcrsity of Califomia at San
Francisco site. Accessed Dcc. 3, 2002.
COMBINATORIAL CHEMISTRY
TERMINOLOGY
The following terms are some of the most common used in
combinatorial chemistry and HIS. More complete glossaries
can be found in Beck-Sicklinger. A., and Weber. P.: Combi.
natorial Strategies in Biology and Chemistry. New York.
John Wiley & Sons. 2002, and in MacLean. D., et al.: Glos-
sary of terms used in combinatorial chemistry. I. Comb.
Chem. 2:562—578. 2000.
ADMET (also ADME. ADMET-FK): The collection of a mole.
cule's properties related to absorption, distribution. meiabo-
lism. excretion, toxicity, and phartniscokinetics, These facwrs
are being increasingly considered in combinatorial library de-
sign, to yield molecules that will be more suitable as
Aptasner: RNA molecule that displays specific binding to a
target, usually a protein. Aptamers arc often used in
rays in place of antibodies, to bind peptide ligands.
Array synthesIs: The form of parallel synthesis in which the
reaction vessels are maintained in a particular spatial arrange.
ment, such as a grid in a microtiter plate. Such arrays generated
on a microscopic basis are termed a spatially addressable
library.
Backbone: A linear scaffold to which suhstituents are attached.
Common backbones include the a carbon backbones of pep.
tides and peptoids.
Bead: A spherical particle of solid support. Typically 50 to (K)

par or dianreter. they swell in solvent. allowing access by
synthetic reagents for reaction, washing. etc. The loading on
head is the amount of synthetic target that can be attached
To ii sinnlc head, which is in the nanomolar range.
Binary encoding: Encoding technique of a library based on the
presence or absence of tags on a bead. Thus, the sequence
01 ItCh would encode thc presence of three of six possible
tugs. The number of combinations that can he encoded is 2'.
where it is the number of positions in the string.
Binning: A computational procedure to allow selecting chciiiicul
structures across a wide range of diversity. The structures are
into bins on the basis of common physical or chemical
structures.
Building block: One of a set of interchangeable reagents that
can be used in the synthesis of a generic library.
Capacity: Theoretical amount of material that could be atiached
toa bead. Because of steric hindruncc of the synthetic target.
it may he greater than the actual amount.
Capillary electrophoresis: Method of separating components
of a nrixture by placing the mixture at one end of a capillary
Oiled with gel. A continuous gradient of electronic charge
across the capillary causes the components to separate. much
like a chmmatograpliic separation but based on charge, size.
and shape of the molecules.
Cleavage: The process of releasing a compound from a solid
support, allowing assay or analysis in solution. Special re-
agents or even enzymes may he used to release the compound
without reacting with or ahienng it.
Clinter analysis: Statistical or pattern recognition technique to
group a set of structures into ' natural" groupings or clusters
tin the ha.sis of physicochemical or structural properties. It is
similar to binning in its result, and both methods are corn-
manly used to select a representative sample of structures,
either for screening or as building blocks for combinatorial
synthesis.
CombInatorial: Relating to combinations of objects.
Combinatorial chemistry: Using a combinatorial process to
prepare sets of compounds from building blocks.
Combinatorial lIbrary: A set olcompounds prepared by combi-
natorial chemistry.
Cross-linking: The property of a polymer used in a solid SUpport
such that long strands of polymer are interconnected at various
points by relatively short sequences—much like rungs on a
flexible ladder. Cross-linking affects the properties of the
polymer, including its ability It) swell in different solvents.
Decode: To "read'' a chemical or electronic tag attached to a
bead or other solid support, for the purpose of determining
the of reaction steps that were applied to the given
head. This allows determining the composition of the synthetic
target on the head,
l)econvolute: To make the results of a combinatorial experiment
less complex, usually by backtracking and reanalyzing or re-
synthesizing a subset of the structures iii the library. The goal
of deconvolution is to determine which of a mixture of com-
pounds in actually responsible for activity.
Dendrimer: A polymer having a very highly branched structure.
Dendrimers can be used in place ol' solid supports for attach-
ment of synthetic targets, and then they can be separated by
ming size exclusion chromatography.
Descriptor: A numerical representation of a molecular property,
cithera bulk property (like log P) ora two-dimensional (2D) or
three-dimensional (3D) structural property. When descriptors
encode the presence or absence of a property, they are usually
represented by Is and Os. and the collection of descriptors is
called a fingerprint of the molecule,
Directed (focused) library: A library that uses a limited number
of building blocks chosen on the basis of information or some
hypothesis that defines the functionalities needed for activity.
Chapter 3 . ('o,nbi,,awrio! Chcn,i.un 61
A directed library lies midway between an initial e.qth;ra:i.-nr
library and a final libr,irv in its size and overall
diversity.
Diversity: The "unrelatedness" of a set of, for example. build-
ing blocks or members of a combinatorial library, Measured
using physicochemical orctructural descriptors, a set with high
diversity spans a larger fraction of "chemical space." Cluster
analysis is one technique used to quantify diversity.
Dynamic library: A mixture of compounds in a dynamic cqui.
lihriutn with, for example, a synthetic process. If a receptor
is introduced into the system, the equilibrium will shift to
produce mote of the compounds that bind tightly with the
receptor.
Encoding: The process of adding a chemical or electronic tag
to a bead for the purpose of "recording" the sequence of
reaction steps to which the bead has been exposed. By th'-od-
ing the resulting tag, perhaps by treating a DNA tag with
polymcr-.tse chain reaction and analyzing the oligonucietrtide.
the exact nature of the synthetic target on the bead can be
determined.
Enumeration: The process of explicitly describing all of the
specific structures that a generic structure or library contains.
Epitope: The region of a protein strand that is rccogtiized by
an antibody.
Fingerprint: An army of numbers In1, nj.. -. ) that numerically
represents a given structure as values of physicochemical or
structural descriptors. Commonly, the ttutnbers are binary It)
or I). but they may also be counts (whole numbers) or values.
Flow cytometry: Technique characterizing or separating par.
tides such as beads or cells, often on the basis of their tluores-
cence. Used to separate beads that have biologically active
molecules attached.
Fluorous synthesis: An approach to solution-phase synthesis
that uses highly fitiorinated compounds as soluble supports
for combinatorial chemistry. The addition of water or organic
solvents causes a phase separation of the fluorinated support
for subsequent cleavage of the synthetic target structure.
Generic structure: General structural formula of a library. con-
sisting of a .waffold(parent structure) pius rt'.cidue's (K groups).
A simple example is
Genetic algorithm: Method of library design by selecting sub.
stituents for a library in a stepwisc fashion, bused on the fitness
of the resulting library for some purpose (e.g.. biological activ-
ity). At each step, the substituents arc modified by use of the
genetic principles of recombination. crossover, mutation. etc.
Selection of the "fittest" conrbinatiot,s of substituenis yields
a library thai is locally optimal for the given purpose.
Green fluorescent protein (GFP): A protein isolated fromjelly-
Itch that has its own fluorescence. It can be modified at various
positions to generate molecules that fluoresce at different
wavelengths. The DNA for this protein can be inserted into
the genomes of cells to give them a fluorescent label.
High-throughput screening (HTS): The process for rapidly
assessing the activity of samples from a combinatorial library
or other compound coliection. usuaily done by running parallel
assays in plates of 96 or more wells. A screening rate of
100.000 assays per day is termed ul:ra/sigh-throughpui
screening.
Hit: A compound that has some required level of activity.
HPLC: High-performance liquid chromatography. Solvent is
pumped under high pressure through a chromatographic col-
umis containing a very finely divided support. The compounds
in the mixture separate according to their affinity for the sup-
port and elate from the column at different times, to be de-
tected by use of sonic optical or even mass spectronietric de-
tector (HPLC-MS),
In sillco screening: See i'iriual screening.
Lead compound: First compound in the development of a drug
62 Wilson and Gisi'ald's Textbook of Organic Medicinal and Pharmaceutical Chemistry
that has the desired biological and physicochemical properties.
It typically has micromolar potency, and by optimizing various
positions of the molecule, the potency can be increased to
nanomolar. at which point it would be considered for drug
candidacy.
Library: A collection of structures, either a generic library
(based on some scaffold plus multiple residues) or a mixture
library (containing diverse scaffolds). The number of specific
structures in a library is either the product of the numbers of
residues possible at each variable position (for a generic Ii-
brary) or simply the sum of the number of structures (rot a
mixture library).
Linker: A chemical chain that connects the solid or soluble
support to the synthetic target in a combinatorial experiment.
The linker is decomposed when the desired compound is
cleaved from the support.
Llplnski "rule of live": A set of criteria for predicting the oral
bioavailability of a compound on the basis of simple molecular
properties (molecular weight. <500: log P. <5; number of
hydrogen-bond donors. <5; and number of hydrogen-bond
acceptors. <10). Typically, the criteria are applied to a library
to filter structures from the library before any synthesis takes
place. Any structure exceeding two or more criteria is rejected.
LiquId-phase chemistry: The process of using a large, soluble
molecule at. the support for a combinatorial chemistry experi-
ment.
Loading: Characteristic property 01 a solid support that de.
scribes the amount of a specific chemical species that can be
attached synthetically to a unit mass of support.
Mapping: Analyzing the sequence of a protein with regard to
a desired property to identify the residues involved in binding
or activity. Typically, this involves generating short, overlap-
ping sequences of the protein, perhaps on a microarray. and
testing for activity.
Markush structure: A type of structure representation in which
very general terms, such us alkyl or alcohol, can be used to
describe the substituents in a generic structure. Used in the
patent literature and adapted for combinatorial chemistry pub-
lications.
Member: Either (a) a particular substituent at a given position in
a generic structure (an k-group member) or (h) an enumerated
structure of a generic library, which corresponds to a given
selection of substituents (a member of a library).
Mesh size: The density of wires in a sieve; also, a term In de-
scribe the size of particles. A 1(10- to 200-mesh particle will
pass through a 100-mesh filter but be trapped by a 200-mesh
filter and consist of particles 75 to 150 in diameter.
Microarray: Masks can be used in the same way that stencils
ate used to make printed circuit boards, to either allow or
block UV light from causing chemical reactions in a small
defined area. In this way. a library of hundreds or thousands
of compounds can be synthesi,.ed in a grid layout, over a very
small area, perhaps a fraction of a square inch. The resulting
microarray can be exposed to a given receptor, and examining
the chip under UV light can reveal which structures have
bound to the receptor.
Mimetics: Compounds that share the desired properties of other
molecules (e.g.. the affinity for a given receptor) but do not
share the undesirable properties, such a.'. susceptibility to pro-
teases. An important cla.ss of mimetics is peptide mimelics.
Mimotope: A compound that imitates an cpitopc; typically, a
nonpeptide sequence that can bind to a particular antibody.
Mimotopet. were an important class of compounds studied in
early combinatorial chemistry experiments.
Monomer: A member of a set of building blocks that can be
repeatedly incorporated into a library leg., amino acids in a
peptide library).
Neural network: Computational procedure to generate a predie-
tive model for some property or response. The model consists
of input (the input data), a set of "hidden" nodes.
and one or more output nodes (the predictions). Each node
behaves like a neuron, with a threshold value of input below
which it will not "fire" any output. The interconnection of
nodes allows the network to deal with complex nonlinear rela-
tionships. The network is trained by iteratively adjusting the
weights at the nodes on the basis of the difference between
the observed and the predicted output.
OmIssion library: Strategy for identifying active library
bers by the systematic omission of building blocks from ntis-
tures. Observation of reduced activity in a certain pool sug-
gests that the building block that was omitted in that poo1
contributes to activity.
One-bead one-compound strategy: The earliest strategy for
solid-phase combinatorial chemistry, in which each bead has
molecules of only a single structure attached rather than a
mixture of structures.
Orthogonal design: (a) Using protecting groups or linkers in a
combinatorial experiment that do not interfere with each other
chemically; or (b) a pooling strategy in which a given library
member appears in more than one pool, mixed with other
members. Pools have only one structure in common, so a hit
in several pools implies that a given member is responsible
for the activity.
Peptold: Oligomer of repenting N-substituted glycines that can
emulate a peptide. without being susceptible to acid degrada-
tion in the gastrointestinal tract.
Phage display: Use of bacteriophage viruses as vessels for pre-
senting short peptide segments of their native surface proteins.
By varying the gene sequences of the phages in a combinato-
rial manner, libraries of peptides can be generated and tested.
Pharmacophore: The ensemble of steric. electronic, and lipo-
philic factors needed to ensure interaction of a drug molecule
with a given receptor. Pharmacophores are most useful in
searching a three-dimensional (3D) structure database and in
filtering structures for virtual screening.
Photolithography: The process by which successive masking
generates light patterns that direct chemical transformations
in certain areas of a photosensitive surface. Coupling different
building blocks to discrete sites on the surface gives rise to
spatially addressable microarrays of compounds.
Pin: An elongated device in which the tip acts as a solid support.
An army of pins, fitting into wells of a microtiter plate. cmi
be used for parallel synthesis of a combinatorial library.
Polyethylene glycol (PEG): Polymer widely used in pharmscy
as an ointment base. It has been used as a soluble support and
u.s a linker in combinatorial synthesis. Its structure is
Polymerase chain reaction(PCR): Technique for amplification
of small amounts of DNA. starting with a few molecules and
yielding sufficient material to analyse the sequence. Also
widely used in forensic DNA analysis.
Pool: (a) A subset of a given combinatorial library: or (h) the
process of combining and mixing library components.
Pool/split (spilt and mix, split and pool): Strategy for assenl•
bling a combinatorial library. The solid support is divided into
portions, each of which is subjected to a given reaction with
a single building block. Pooling the portions gives a single
hatch with a mixture of components. Repeating the split. react,
and mix sequence results in a library in which each discrete
bead of solid support carries a single library member (one'
bead one-compound strategy). The number of members equals
tine product of the number of building blocks incorporated at
each step (i.e.. fully combinatorial).
Positional scan: Strategy for identifying individual compoundi
of interest in a library. A collection of sublibranes is prepared,

in number to the total number of building blocks in the
whole library. In each pool, one substituent position is held
constant by incorporating a single building block while the
other positions use all possible building blocks. Activity in a
given pool implies that the given substituent at the given posi-
turn is essential for activity.
PrincIpal components analysis: Computational approach to re-
duce the dimensionality (i.e.. the number of variables) in a
data analysis, by weighting variables according to their contri-
bution to the overall variation. A plot of points in the first
two principal components is like a two-dimensional (2D)
"shadow" of the multidimensional data that can be used to
(lad clusters and relationships among the points (i.e.. coin-
pounds),
Property space: Multidimensional representation of a set of
compounds as points in space. Each axis of the space repre-
sents some descriptor, either whole property or computed from
the two-dimensional (2D) or three-dimensional (3D) chemical
stnictuge. Compounds that are similar to each other chemically
will cluster together in property space. A further assumption
used in library design is that structurally similar compounds
will share similar biological activity.
I'rotecting group: Chemical group that reversibly blocks func-
tional groups ott a synthetic target, to prevent them from enter-
ing into undesired side reactions. For example, an OH group
might be protected by converting it to an ester, then hydrolyz-
ing the ester buck to an alcohol when the synthesis is complete.
Radlofrequency encoding: The process of embedding into solid
supports the minute electronic devices that emit rudiotre-
quency signals upon stimulation with an electromagnetic
source. The signals can be used to track the reaction history
of the given bead and thus the makeup of the compounds
attached.
Ratio encoding: Strategy in which the quantities of tags on ii
bead give itttormation about the compound identity rather than
tile nature of the tags.
Residue: The portion of a chemical structure that can be identi-
fied us coining front a particular building block, such as the
alunine residue in a polypeplide. In a generic structure, the
rx'sulucs ore the substiluents that correspond to the R-groups
in the uructure.
Resin: Insoluble polymeric material to which linkers, synthetic
targets, and tags are attached. Soitietintes. resins arc simply
used to scavenge side of a reaction. In chromatogra-
phy. resin heads are used to separate compounds by size or
by the charge ott a molecule.
Resynthesis: Preparation of individual members or subsets of
combinatorial library, to l'ollow up on sonic property ut
interest.
Reverse transcriptase: An enzyme that can reverse transcribe
RN/s into its correspotmding DNA.
Ribozyme: RN/s molecule with enzyme catalytic activity.
Robotic system: An automated system, usually controlled by a
cimitipuler, to transfer materials by physical movement of a
delivery device or by movement of' the reaction vessels. Ro-
botic systems can be general purpose, and capable of being
reprogrammed to do a variety of different tasks, or may be
specialiied. as part of a turnkey system.
Safety-catch linker: A linker that is cleaved by performing two
reactions instead of the customary one. This provides greater
control over the timing of the release of the synthetic target.
Scaffold: The core portion of a generic struCture, common to
all the imenthets of the library. Note that in the ease of Ph-
thc seatThld consists two disconnected
Iraginents, separated by the R1 substituent.
Setnenger resin: A resin introduced into a combinatorial experi-
ment to react with undesirable materials (excess reagent or
side products) and remove them from the experiment.
Chapter 3 • Co,nbinawrial Chemistry 63
Selectivity: Measure of a compound's tendency to hind only to
a certain target receptor. Drug molecules should have high
selectivity us well as potency.
Solid support: insoluble, functionalized polymeric material to
which library members or reagents may be attached, often via
a linker, allowing them to be readily separated from solvent.
excess reagent. etc. Typically, the solid support swells in sol-
vent, allowing reactions to occur in the interior of the bead
or other form. This greatly increases the available surface area
for reaction.
Soluble support: Typically, a large molecule that is soluble in
some solvents and, upon the addition of other solvents, sepa-
rates into phases. The molecule can serve as a support for
attaching members of a combinatorial library. The advantage
over solid supports is that reactions are more coniplete in
liquid phase. Sometimes termed liquid-phase chemistry. An
example of a soluble support is PffG, which is soluble in polar
solvents but separates 1mm organic ones.
Spacer: Same as a linker.
Spatially addressable: Having the ability to identify part or all
of the structure of a library component or pool from its physi-
cal location in a grid or array.
Spot synthesis: Solid-phase synthesis at certain points (spots) on
a two.dimensiotial (21)) surface (e.g.. a cellulose membrane).
Recently, the techniques of ink-jet printing have been applied
to spot synthesis, yielding very dense arrays of compounds.
Sublibrary: A subset of a combinatorial library in which. For
example, the substituent at one position is held constant while
other positions are varied. Also called a pool.
Supercritical fluid chromatography (SFC): HPLC using a
''solvent" such as liquid CO2 under high pressure. The advan-
tage is that the carrier evaporates, simplifying the detection
of the compounds as they elute from the chromatographic
column.
Tag: A nonrcaciis'e chentical functionality attached to a solid
support thaI carries information about the reaction history of
the given support and titus can at least partially idetitify the
attached synthetic target. An exuniple is attaching various
DNA bases to the bead at each synthetic step. The resulting
oligonucleotide can be multiplied by using PCR and identified
analytically.
Tea bag: A type ol' reaction vessel consisting of a porous mesh
hag that encloses the resin but allows passage of reagents and
solvents. Several tea bags cult be immersed in a given reagent
and then manipulated from one reagent to the next to generate
combinatorial libraries.
Virtual library: A combinatorial library that has no physical
existence; rather, it exists in a computer or on paper. Such
libraries eon he generated automatically and screened against
physiochentical tillers like time "rule of five" or be docked
into receptors by use of niolecular modeling.
Virtual screening: The selection of compounds by evaluating
their fitness by use of contptttttionmtl model. Also called in
.vilit'o
REFERENCES
I. Merrifield. K. it.: Solid ptta'.c peptide synthesis. I. TIme synthesis of a
teirapeplide. J. Am. ('hem. 85:2t49'-2154. 1963.
1. Furka. A.: Combinatorial cheitmistry: 20 years... Drug Discovery
Today 7:1 2002.
3. tloughten. K A.: mimethod hmr mIme rapid so)id-phasc synthesis
oh large numbers ot pcpmides: spcrilieimy miIamumigcn—aiutitssiy interaction
at level of individual amino Proc. Nat). Acad. Sri. U. S. A.
82:5l31—5t35, 985,
4. Simimmumu. R. J.. Kania. R. S.. 7uckcrnmann. K. N,. em it).: t'cpmoids: a
niusiular approach to drug discovery. t'roc. Nail. Acad. Sci. U. S. A.
89:9367—937). 992.
5. Miller. S. M . em it).: Promeotytme sftslies of lummnolugous peptide and N—
64 Wilson and Tn'xgbor,k of Organic Medicinal and Pharrnaci'uik'al Chemistry
substituted glycine pcpioid uligomer.. Bioorg. Med. Chem. Leit. 4:
2657—2662. 1994.
Ii. Zuckertuan, R. N.. ci aL: Discovery of nanotiwlar ligands for 7.truns-
incnthratie G.pnrteiit-coupled receptors from a diver.e
glycine peptoid library. J. Med. Chcni.37:2fi78—2685. 1994.
7. Bunin, B. A and ElIman. 3. A.: A gener.il and expedient method liii
the solid-phase synthesis of I ,4trenrodiarepine derivatives. J. Ant.
Chem. Six. 14:10997-10998. 1992.
8, Lartred, M.. md Hallbcrg. A.: Micruwave.assisted high.speed chentis-
try: a new tcchnique in drug discovery. Drug Discovery Today 6:
.UJ6-416. 2001.
9. Balkcnhohl. P.. ci at.: Combinatorial synthesis at small organic mole-
cules. Angew. Chem. tnt. Eel. Engl.35:2288—2337. 1996.
TO. Murvaurelle. I.. A.. and Seeberger. P. H.: Combinatorial carbohydrate
chemistry. Curr. 0gm. Chein. Biol. 6:289—296. 2002,
II. Nielsen, 3.: Combinatorial synthesis of natural products. Cure. Opin.
Chent. Biol. 6:297—305, 2002.
12. holmes, C. P.: Model studies For nev. o.nitrobenzyl photolabile Tinkers:
suhstiitrcni effects on the rates of photochemical cleavage. J. Org.
Chem. 62:2370—2381). 1997.
13. Reents, R.. Jeyaraj. U. A., and Waldman. H.: Enrymatically cleavahic
linker groups in polymer-supported synthesis. Drug Discovery Today
7:71—75, 2(102.
14. Keatiog. T. A,. and Armstrong. K. W.: Molecular diversity via a con-
verttble isocyanide in the Ugi Four-component condensation. i. Am.
Chetti. Soc. 117:7842—7843. 1995.
15. Han. H., et il.: l.iquid.pha.sn combinatorial synthesis. Proc. NatI. Acud.
Sci. U, S. A. 92:6419—6423. 1995.
16. Siuder. A., ci at.: Iluornus synthesis: a fluorous.phu.sc strategy for
itttproving separation efficicncy in organic sytithesis. Science 275:
823—826. 1997.
17 Caldemne, C. let at.: Directing otherwise incompatible reactions in a
single solution using I)NA-templatedorganic synthesis Angew. Chem.
tnt. Ed. EngI. 41:4t04.—4lll%. 2(Xl2.
18. Hughes. I.. and Hunter. D.: Techniques for analysis and purification in
higlt-thrcrughput chemistry. Cure. 0gm. Chem, Biol. 5:243—247.2001.
19. Van. B.. and Kunaaeuvel. G.: Probing solid-phase reactions by monitor-
ing the IR bands of compounds on a single 'flattened' resin head.
Tetrahedron 52:843-848. 1996.
20. Shuker. S. B., ci at.: Discovering high'aflinity ligands for proteitis:
SAR by NMR. Science 274:1531—1534. 1996.
21. Brenner. S.. and Lerner. K. A.: Encoded combinatorial chemistry. Proc.
Nail. Acad. Sci, U.S. A. 89:5381—5383. 1992.
22. Nielsen. 1., Brenner. S.. and Janda. K. U.: Synthetic methods for the
impletnentatititi of encoded combinatorial chemistry. 3. Am. Chem.
Sire. 115:9812—9813. 1993.
23. http://www.qdots.csitn. Quantunt dots. 2002.
24. Limnitro, 3. A., ci al.: HTS in the new itiillenniuitt—thr role of pharma-
colirgy and flexibility. J. Pharmacol. Toxicol, Methods 44:273 -289,
2(881.
25. Drews. J.: Drug discovery: a historical perspective. Science 287:
1960—1964. 21)00.
26. Spencer. K. W.: High-throughput screening of historic collections: ob-
servations on file sire, biological targets, and file diversity Biotechnol,
}Siocng. 61:61—67. 1998.
27, Venton, U. L., and Woodbury, C. P.: Screening combinatorial libraries.
Chcmometr. Intell. Lab. Syst. 48:131 — ISO. 1999.
28. Tarbit. M. H.. and Berman, 3.: High.throughput approaches forevaluat-
ing absorption, distribution, metabolism and excretion properties of
lend compounds. Cure. Opin. Chem. Biol. 2:411—416. 1998.
29. Walters. W. P.. Stahl. M. T.. and Murcko. M. A.: Virtual screening—an
overview. Drug Discovery Today 3:160—178. 1998.
30. Shoichet. B. K.. et at.: Lead discovery using molecular docking. Cure.
Opin. Chem. Biol. 6:439—446. 2002.
31. htmp'J/www.grid.orgiprojeus/patnot,htm, United Devices agent soft.
ware download. 2002,
32. tOOhtn. 3.-i.. and Stahl, M.: Structure-based library design: molecular
modelling merges with combinatorial chemistry. Cwr. Opmn. Chem.
Biol. 4:283—286, 2000.
33. Lipmnshi. C. A.. et al: E,xpeilinental and coniputational approaches to
estimate solubility and permeability in drug discovery and development
settings. Adv. Drug Delis. Rev. 23:3—25. 1997.
34. Opera. T. I.: Virtual screening in lend discovery: a viewpoint. Mole.
cules 7:51—62, 2002.
35. Opera, T. I.. and Gotil'ries. J.: Toward minintalistie modeling of oral
drug absorption. J, Mol. Model. Graph. 17:261 —274. 1999.
36. Zuegge. 3., ci al.: A Fast virtual screening filter for cytochromr P450
3A4 inhibition liability of compound libraries. QSAR 21:249—256.
2002.
37. Adang. E. P.. and l'lmrrrnkens. P. H. H.: The contribution oFcombinato-
rial chemistry to lead generation: an interim analysis. Med. ('hem.
8:985—998, 2001.
38. Spellmeycr, 0. C.. and Gruotenhuis, p. o. 3.: Recent developments in
molecular diversity: computational approaches to combinatorial cheni-
istry. Ann. Rep. Med. 0mm. 34:287—296. 1999.
39. Jacobsen. E. 3.. et al.: imidarol I ,5-alquinoxnline ureas as
high affinity GABA—a ligand of dual functionality. 3. Med. Cheat.
42:1123. 1999.
40. Martin, E. 3.. and Cntchlow. R. 0.: Beyond mere diversity: tailoring
combinatorial libr,tries for drag discovery. 3. Comb. Chem. 1:32-45.
1999.
41. Opera. T. I.. ci al.: Chemical information managetncnt in drug discov-
ery: opiinmiring the cotimpulalional and combinatonal chemistry inter,
Faces. 3. Mol. Model. Gr.mph. 18:512—524. 2000.
42. Merritt. A. 1.: Uptake of new technology in lead oplimieation fur drag
discovery. Drag Discovery Today 3:505—510. 1998.
43. Golebiowski, A., Kloptenstein. S. R., and Portlock, 0. E.: Lead com-
pounds discovered from libranes. Curr, 0gm. Chent. Biol. 5:273—284.
2001.
44. Owens. 3.. et uI.: News in brieflmiscellaneous—Itrst H'l'S doug cantli.
dates reach clinical trials. Drag Discoxcry Today 6:230. 2001.
45. Everett. 3., ci al.: The application of non-combinatorial chemistry to
lead discovery. Drug Discovery Today 6:779—785. 2001.
46. Lear. 0. R.. Nash. H. M.. and Jindal. S.: Chetnical ligands.genomics,
and drag discovery. Drug Discovery Today 5: 145—156. 2000.
CHA PT ER 4
Metabolic Changes of Drugs and
Related Organic Compounds
STEPHEN J. CUTLER AND JOHN H. BLOCK
Metabolism plays a central role in the elimination of drugs
and other foreign compounds (xe,:ohiotk.s) from the body.
A solid understanding of drug metabolic pathways is an es-
sential tool for pharmacists in their role of selecting and
monitoring appropriate drug lherjpy for their patients. Most
organic compounds entering the body are relatively lipid
soluble (ilpoplillic). To be absorbed, they must traverse the
lipoprotein membranes of the lumen walls of the gastrointes-
tinal (GIl tract. Then, once in the bloodstream, these mole-
cults can diffuse passively through other mcinbrane.s and be
distributed effectively to reach various target organs to exert
their pharmacological actions. Because of reabsorption in
the renal tubules. lipophilic compounds are not excreted to
any substantial extent in the urine. Xenohiotics then meet
their metabolic fate through various enzyme systems that
change the parent compound to render it more water soluble
(hs'dropltilw). Once the metabolite is sufficiently waler solu-
ble, ii may be excreted from the body. The statements above
chow thai a working knowledge of the ADME (absorption,
distribution. metabolism, and excretion) principles is vital
for successful determination of drug regimens.
If lipophilic drugs. or xenohiotics, were not metabolized to
polar, readily excretable water-soluble products, they would
remain indefinitely in the body. eliciting their biological ci-
Thus, the formation of water-soluble metabolites not
only enhances drug elimination, but also leads to compounds
thai are generally pharmacologically inactive and relatively
nontoxie. Consequently, drug metabolism reactions have tra-
ditionally been regarded as (!ewx,calion (or detrixifkazion)
processes.' Unfortunately, it is incorrect to assume that drug
nietabolisin reactions are always detoxifying. Many drugs
are hiotransformed to pharmacologically active rnetabolites,
These metabolites may have significant activity that contrib-
utes substantially to the pharmacological or toxicological
effect(s) ascribed to the parent drug. Occasionally, the parent
compottnd is inactive when administered and must be meta-
helically converted to a biologically active drug (metabo-
lite).1 These types of compounds are referred to as pro-
In addition, it is becoming increasingly clear that not
all meinbolites are nontoxic. Indeed, many adverse effects
e.g.. tissue necrosis, carcinogenicity. teratogenicity) of
drugs and environmental contaminants can be attributed di-
reedy to the formation of chemically reactive metabolites
that are highly detrimental to the dy.4t' This concept is
more important when the patient has a disease state that
inhibits or expedites xenohiutic metabolism. Also, more and
more drug metaboliics are being tbund in our sewage sys-
rena. These compounds may be nontoxic to humans but
hanniul to other animals or the environment.
GENERAL PATHWAYS OF DRUG
METABOUSM
Drug metabolism reactions have been divided into two cate-
gories: phase! (fiinctionalization) and phase!! (coI,jugalion)
reacttons.' Phase I, or functionalization reactions, include
oxidative. reductive, and hydrolytic biotransformations
(Table 4_l).C The purpose of these reactions is to introduce
a functional polar group(s) (e.g.. OH. COOH, NHa. SH) into
the xenobiotic molecule to produce a more water soluble
compound. This can be achieved by direct introduction of
the functional group (e.g.. aromatic and aliphatic hydroxyla.
tion) or by modifying or "unmasking" existing tunctionali-
ties (e.g.. reduction of ketones and aldehydes to alcohols;
oxidation of alcohols to acids: hydrolysis of ester and amides
TABLE 4—1 General Summary of Phase I and Phase Il
Metabolic Pathways
Phase t or Functlonallzatlon Reactions
Oxidatinc reactions
Oxidation of aromatic ntolctios
Oxidation ot ok'tins
Oxidation at henzytic. allytie carbon atoms. mid curbon stems o to
earhonyt and iminca
Oxidation at atipharic and aticyctic carbon atoms
O*idation involving curbon—hcicroatomri systems:
Carbon—nitrogen systems and aromatic anuses: inctudrs
N..dcatkyiaiion. dcjminarion. N'oxitk formation.
N-hydroxytamiun)
Carbon—oxygen srtcms tO-draikytatimml
Carbon—sulfur systems IS-deatkyiiition. S-oxidaiion, and
dcsuifwauion)
Oxidation of alcohols mind aidehyden
Other misctliuncomts oxidalivc reactions
Reductive Reactions
Reduction til aidrhydcs and kctonet
ReductIon of ,iiuu and azo compounds
Miscellaneous reductive reurciion.s
Hydrolytic Reactions
Hydrolysis of esters and amudc.s
Hydration ot epoxidcs and arene oxides by epoxide hydruse
Phase H or Conjugation Reactions
Gtueurnnic acid conjugation
Sutfatc conjugation
Conjugation with glycine. glittamine, and other amino acids
Gtuiaihione or mtrvupturie acid conjugation
Acetylation
Methytation
65