Substituents and Bioisosteres in Medicinal
Chemistry
Scott Jarvis
Prof. Charette’s Laboratories
Based largely on “The Practice of Medicinal Chemistry”,
Elsevier Ltd, 2003
April 14, 2009
What is an Isostere?
 Defined by Langmuir in 1919 (JACS, 1919, 1543-1559):
 “Comolecules are thus isosteric if they contain the same number and
arrangement of electrons. The comolecules of isosteres must,
therefore, contain the same number of atoms. The essential
differences between isosteres are confined to the charges on the
nuclei of the constituent atoms.”
 ie: C=O and N=N, N=N=N- and N=C=O-
 This has concept progressed to include groups that have similar
properties but not necessarily the same number of atoms or electrons
(Erlenmeyer 1932, included thiophene and benzene as biosteres,
oxygen and sulphur, Cl being aprox. equivalent to cyanide, etc.)
What is a Bio-isostere?
 In medicinal chemistry, bio-isosteres (biostere) are substituents or
groups with similar physical or chemical properties that impart similar
biological properties to a chemical compound.
 Why do we need them?
 “A lead compound with a desired pharmacological activity may have
associated with it undesirable side effects, characteristics that limit its
bioavailability, or structural features which adversely influence its
metabolism and excretion from the body.”
Chem Rev. 1996, 3147.
 The purpose of exchanging one group for a biostere is to enhance the
desired biological or physical properties of a compound without
making “significant” changes in chemical structure.
Guidelines for Oral Bio-Availability
 Lipinski's rule of 5 says that, in general, an orally active drug has
no more than one violation of the following criteria:
 Not more than 5 hydrogen bond donors (nitrogen or oxygen atoms
with one or more hydrogen atoms)
 Not more than 10 hydrogen bond acceptors (nitrogen or oxygen
atoms)
 A molecular weight under 500 daltons
 An octanol-water partition coefficient (log P) of less than 5
 Veber’s Guidelines (based on oral rat data)
 ≤10 rotatable bonds
 ≤ 140 Å Polar surface area or ≤ 12 Hydrogen bonds (acceptors and
donors)
Taxol
 Taxol – IV Drug
 4 H-Donors
 15 H-Acceptors
 MW 853.9
 Log P >99*
 *Cancer Chemotherapy and Pharmacology, 1997, 40, 285-292.
Properties that can be modified by changing
substituents/functional groups
 Activity
 Solubility (Log P)
 Electronic Density
 H-Bonding (donor/acceptor)
 -Bonding
 Steric bulk affected
 Conformation
 Specificity (Interactions with other substrates )
 Bioavailability (ability to cross membranes, ie: active
transporters)
 Metabolism (life span of compound in-vivo)
 Toxicity
Properties that can be modified by changing
substituents/functional groups
 Activity
 Solubility (Log P)
 Electronic Density
 H-Bonding (donor/acceptor)
 -Bonding
 Steric bulk affected
 Conformation
 Specificity (Interactions with other substrates )
 Bioavailability (ability to cross membranes, ie: active
transporters)
 Metabolism (life span of compound in-vivo)
 Toxicity
Where Biosteres Fit Into Drug Design
(In No Particular Order)
 Things commonly changed in optimizing activity:
 Ring connectivity
 Closing of a ring (rigidity)
 Opening of a ring (other conformations available – usually done as a “me too” strategy)
 Size of ring (bigger, smaller)
 Reorganization of the rings (splitting fused rings)
 Homologues (Vinylogues)
 Spacer between two binding units
 Substituents/Functional Groups (H donor/acceptor, electronic affects, and steric
demands)
 Switch for other substituents
 Simplify the molecule by chopping off pieces that aren’t important
 Make it more complex by adding
 Modify with biosteres
 Conformation (affected by all) –increase active conformation population
 Chirality
 Affected by all other factors, ie: rings and substituents
Small Unit Biosteres
 Atom interchange (C, N, O, S, etc)
 Methyl
 Vinyl
 Allyl
 Acetylene
 Halogen
Methyl - Solubility
 Depending on the location within the
substrate, the hydrophobic interactions
can make it less soluble in water or
more soluble.
 This is due to an entropic effect: in
aqueous solution the compound is
encased in a network of water
molecules, if the cluster around the
molecule is more compact it is more
favourable and therefore the compound
is more soluble.
Methyl: Electronic Effect
 Alkyl groups are the only substituents acting by an inductive effect
solely, all others can also have a mesomeric effect (excluding
Hydrogen).
 They are therefore electron donors regardless of the environment
(acidic, basic, neutral)
Methyl
 H-Bonding
(not biostere, more SAR like)
 Can act as a block on hetero-atoms, preventing them from being
being H-donors (A quick way of determining whether that H-donor is
necessary for activity) Though this has a caveat! With amides this
also affects the conformation.
 Steric/Conformational Constraint
 Substitution on a heterocyclic ring causes ortho substituents to be out
of plane.
 Amide –methylating the amide can cause an increase in the
population of the cis form (active conformation population change). ie:
peptides: ~1% of peptide bonds are cis and ~99% trans except proline
(n-alkyl amide) which is ~30/70 cis/trans which is why they are key for
beta-turns.
Methyl - Metabolism
 Phenyl methyl – clearance pathway available (cytochrome P450), this
is a way of reducing toxicity since the compound cannot build up to
dangerous levels
 When R is activating or an enzyme is specifically oxidizing a
methylene, can block oxidation by adding methyls.
Methyl - Metabolism
 Amide – methylation significantly slows peptidases from cleaving the amide
bond as it is “unnatural”, also N-terminal alkylation is “unnatural” so it slows
metabolism
 Methyls can activate other positions to oxidation due to electron donating
effect.
Vinyl
 Vinyl is not extensively used in medicinal chemistry, as it is easily oxidized to
the epoxide in-vivo but is sometimes used as a “me too” strategy or as a
masked epoxide however, it can be found in some drugs.
 Cyclopropane, and Phenyl (for cis C=C) are both biosteres of a vinyl. Neither
of them can have spontaneous conversion from cis to trans, where-as with
vinyl this can be a problem in-vivo and are not oxidized to epoxides.
Allyl
 Allyl are generally hepatotoxic (cause liver damage), and are oxidized quite
quickly in-vivo. When substituted with a good leaving group becomes an
alkylating agent.
•
Used as a fast acting analgesic, which had rapid onset and short
duration of action. Useful for surgery for example, and though mostly
replaced by compounds with better safety profiles still used in some
eastern european countries like Poland.
 Ragwort (plant) produces a toxin that kills the animals that eat it, one of the
toxins (shown below) causes liver cancer by acting as an alkylating agent.
Acetylene
 Electronic effect: electron attracting, can be reinforced by substituting the
acetylenic hydrogen. Can increase the acidity of an alpha-alcohol for
example.
 Spacer: 4 in line carbons, can act as a rigid spacer.
 Due to the pi system, can act as a biostere of a phenyl as they give similar
donor-acceptor interactions. However, can be metabolized quite quickly by
hydrolysis to the ketone.

Metabolism example
Halogens (One third of all current drugs are halogenated)
 Most used in Medicinal chemistry are Fluorine and Chlorine. Bromine is used
almost exclusively as a phenyl substituent. Iodine is used almost exclusively
for thyroid disorders.
 Inductive effect: strong for chlorine and bromine, less for iodine.
 Mesomeric effect: the donor effect is usually not involved in biological media.
 Fluorine is a biostere for a Hydrogen bonded to a carbon but is more
lipophilic, and not typically metabolized (since the C-F bond is so strong).
 Organic Fluorines rarely accept hydrogen bonds.
 Chlorine can be a biostere of Fluorine for aromatic carbons (and vice-versa),
but Cl has d-orbitals which can have additional interactions.
Fluorine: Sterics/Conformation
 Every additional F on a carbon shortens the other bonds attached to that
carbon, and therefore depending on how it is attached can affect the
conformation of the compound (ie: ‘a’ value of cyclohexanes) making it appear
bigger than it actually is.
Bond
Bond Length
(picometers)
Group
Van der Waals
radius
C-C
C-H
C-F
120-154
106-112
134
H
F
Cl
1.2
1.35
1.8
C-Cl
176
CH3
~2
CF3
~2
 “The A value of the trifluoromethyl group is greater than that of the isopropyl
group (2.37 versus 2.21), but smaller than that of the tert-butyl group (4.87).”
New J Chem, 2006, 442-446.
Fluorine
 Fluorine on an alkyl chain usually decreases lipophilicity due to polarization,
however on an aromatic ring it increases lipophilicity. Chem. Soc. Rev, 2008,
237.
 CF3 on a benzene is aproximately as sterically demanding as an ethyl though
of a different shape. OCH3 on a benzene has a preferred planar
conformation, where-as a OCF3 is out of plane in biological media.
 1,2-Difluoroethane prefers the gauche conformation, not anti.
Hansch and Hammet constants




H
0.00
0.00
SCN
0.41
0.52
F
0.14
0.06
NO2
-0.28
0.78
Cl
0.71
0.23
CO2H
-0.32
0.45
Br
0.86
0.23
COCH3
-0.55
0.50
I
1.12
0.18
CF3
0.88
0.54
OH
-0.67
-0.37
CH3
0.56
-0.17
OCH3
-0.02
-0.27
CN
-0.57
0.66
NH2
-1.23
-0.66
SO2CF3
0.55
0.93
NH3+
-
0.60
SCF3
1.44
0.50
Group
Group
Chu, K.C (1980), The quantitative analysis of structure-activity relationships. In Wolf, M.E. (ed.) The
basis of Medicinal Chemistry/Burger’s Medicinal Chemistry, pp 393-418. John-Wiley, New York
Functional Group Biosteres
 Acid
 Ester
 Amide
 Ketone (Sulfone/Sulphoxide)
 Phenol
 Amine
 Urea
Functional Groups: Acid
 Carboxylic acids are obviously proton donors for hydrogen bonding to the
target, for example H-bonding with basic amino acids such as Arginine, Lysine
or Histidine in a protein.
 The pka of the biostere is one criteria, but also the steric requirements of your
target, lipophilicity and bio-availability are very important.
 An acid is solubilizing (easier to formulate), but to be bio-available it’s often
changed to be a prodrug (ester).
Acids
 As a general rule: Strong and highly ionized acids cannot cross the biological
membranes which are permeable only to non-dissociated molecules.
 They are therefore subject to rapid clearance from the body.
 Once absorbed they can establish strong ionic bonds with the basic amino
acid residues in proteins.
 Solubilizing, which can be enhanced through salt formation. For small
molecules the presence of a carboxylic acid can fundamentally change the
biological activity (activity and toxicity are reduced typically). In larger drugs
(ie: penicillins) the effect of whether the carboxylic acid is present or not is
smaller.
 Hydroxamic acids are very good at binding metals (ie: Zn) but can be
metabolized to the acid so it can act as a prodrug also.
Drugs on the market with an “acid”
A case where the acid couldn’t be
replaced (statins)
Tetrazoles
 Unknown in nature (therefore stable in-vivo)
 pka: 4.90 (Acetic acid is 4.76)
 Slightly larger than an acid
 Can be alkylated or acylated at either the 2 or 3 position (difficult to
control).
 Process chemists generally consider it undesirable due to the danger
of synthesis (explosive!).
Hydroxy-Isoxazole
 pka: 5.3 (can be modified by substituting the ring)
 Sterically very similar to the carboxylic acid if the two carbons
attached to the carbonyl are included.
 The hydrogen is localized to two atoms (N and O)

J. Med. Chem. 1981, 1377.
 Synthesis
Functional Group: Ester
 Hydrogen bond acceptor (Carbonyl oxygen)
 It can serve as a masked acid (prodrug), and occasionally acts as a
reactive functional group to acylate the target (ie: Aspirin acylates a
Serine in the active site of the COX enzyme that is involved in
prostaglandin synthesis)
 Can be cleaved in-vivo to the acid easily, so if not acting as a prodrug
or acylating reagent it is typically altered to a biostere of the ester:
Amide is most common (easy to do), ketone, or other hydrogen bond
acceptors such as sulphone or sulfoxide or heterocycles (pyridine like
nitrogens).
Functional Group: Amide
 Both a Hydrogen donor and acceptor so they are capable of binding
two separate sites simultaneously.
 Amide bonds are quite prevalent in nature, and yet are typically stable
enough for an in-vivo response (see ‘Methyl’ section for example)
 The major conformation of secondary amides is trans, tertiary is a mix
of the two possible conformers.
 Amides are typically considered to be regarded as having low water
solubility since they are non-ionic, but the bonus is they can therefore
pass membranes and are bio-available.
 Most typical replacement is sulfonamide even though they are more
acidic, however for peptides there are other biosteres than
sulfonamides since alpha-amino sulfonamides are unstable.
Amide vs Sulfonamide
 Similarities in Metabolism:
 N-Acylation (mainly in the liver), important since N-Acyl sulfonamides
are typically less soluble so can cause renal toxicity by precipitating in
the kidneys.
 Both can be N-glucuronidated (sugar) or sulfonated making them
more water soluble, therefore excretable
 Differences:
 Amides can be hydrolyzed by proteases, sulfonamides are stable.
 Sulfonamides are more acidic, and can be difficult to solubolize.
 Primary sulfonamides have active transporters (most drugs are
diffusion limited) making them more bio-available.
 Sulfonamides being more polar are less likely to pass the blood brain
barrier.
Biosteres for a peptidic amide
Amide reversal example
Enzyme
NEP 24.11
Themolysin
ACE
Enzyme
NEP 24.11
Ki Value (µM)
0.0019
1.8
0.14
Ki Value (µM)
0.0023
Themolysin
2.3
ACE
>10
Retro-amides are generally more resistant to enzymatic attacks (proteases)
since they are not recognized as well. In the example above, the first has a
Glycine as the left side, and the second is a malonate – completely different to
enzymes.
Functional Group: Ketone
 H-Bond acceptors: Pyridine like N, O=C, O=S, S=C, HO-CH.
 Sulfoxides can be reduced or oxidized
 Thio-carbonyls are easily oxidized, less interesting due to stability
issues in-vivo.
 Pyridine like Nitrogens can mimic ketones since they have a free lone
pair to H-bond, however the lone pair direction is not necessarily the
same as the ketone. Also, they can be oxidized in-vivo rendering
them inactive.
Phenols
 H-Bond donors
 For phenols “Bio-isosteres are unlikely to be suitable in those
instances where biological activity is adversely affected by increased
molecular size or is strongly dependent on electronic parameters.”

Chem. Rev. 1996, 3147.
 Generally the biostere is an ‘N-H’ with an electron withdrawing group
attached to the nitrogen.
Phenols
Functional Group: Amines
 The basic residues are H-bond acceptors, however N-H can also be
H-bond donors.
 If only acting as a H-bond acceptor, can make it a tertiary amine or
heterocyclic Nitrogen (ie: pyridine, imidazole).
 If replaced by an RO-H the H-bond donor effect is kept, but no longer
a basic residue to bind acidic (an example is GHB as a biostere for GABA, see next
slide)
 Typical replacements: Amidines, Guanidines and imidazoles.
 R2N-H like RO-H, and RS-H are the nucleophilic groups so they are
metabolized by acetylation, glucuronidation, sulfonation, and of course
also oxidation like anything else.
 Model of a GABA type C receptor with GABA
 Biophysical Journal, 2008, 4115.
Ring Biosteres
 Benzene is the most common ring in drugs.
 Rings are a vital part of any medicinal compound, since they give:





Rigid directional/conformational stability
Hydrophobic interactions
Pi stacking if aromatic
Act as a spacer between two binding units
Heterocycles are capable of H-bonding (donor/acceptor)
 A general rule for finding a Biostere of an aromatic ring is the other
ring should have a similar boiling point so long as no H-bonding is
involved. The boiling point is an indirect measure of the dipole of the
ring.
 Sterically most heterocyclic rings are close enough to each other they
can replace one another, and depending on the rings 5 and 6
membered rings can place substituents at similar angles.
Rings
Compound
Benzene
BP (C)
80
Compound
Thiophene
BP (C)
84
Methylbenzene
110
2-Methyl-thiophene
113
Chlorobenzene
132
2-Chloro-thiophene
130
Acetylbenzene
200
2-Acetyl-thiophene
214
Rings: Example
Rings
 Pseudocycles ‘ring opening’, can present a conformational analogy
that may be simpler to synthesize – though is likely less selective
towards the target receptor compared to others since it is not
conformationally locked.
Rings
 Analogy by ‘ring closure’: useful in search for biologically active
conformation as it is a constrained molecule. However, can create
additional chiral centers complicating the synthesis and cyclization
may give the incorrect conformation.
Rings
 Ring enlargement/contraction (Homologues) – usually part of any
good SAR but when omitted it is an easy ‘me-too’ strategy for
competitors.
 Re-organization of the rings, such as converting simple rings into their
spiro derivitives, splitting fused bicyclic systems, and ring dissociation.
Rings
Rings: Metabolism
 The more electron rich position of the ring and the benzyl positions are
the most likely to be oxidized in aromatic carbocycles. With
heterocycles the metabolism can be more complicated with heteroatoms being oxidized and/or ring opening reactions possible, see
case-by-case.
 In drug design, the use of biosteres is still very intuitive and empirical.