1. Substituents and Bio-isosteres in Medicinal Chemistry
Scott Jarvis
Prof. Charette’s Laboratories
Based largely on “The Practice of Medicinal Chemistry”,
Elsevier Ltd, 2003
April 14, 2009

2. What is an Isostere?
Defined by Langmuir in 1919 (JACS, 1919, ):
“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.)

3. 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.

4. 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)

5. Taxol Taxol – IV Drug 4 H-Donors 15 H-Acceptors MW 853.9 Log P >99*
*Cancer Chemotherapy and Pharmacology, 1997, 40,

6. Properties that can be modified by changing substituents/functional groups
Activity
Solubility (Log P)
Electronic Density
H-Bonding (donor/acceptor)
π-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

7. Properties that can be modified by changing substituents/functional groups
Activity
Solubility (Log P)
Electronic Density
H-Bonding (donor/acceptor)
π-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

8. 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
Saturation level

9. Small Unit Biosteres Atom interchange (C, N, O, S, etc) Methyl Vinyl
Allyl
Acetylene
Halogen

10. 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.

11. 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)

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

17. 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

18. 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.

19. 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.
“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,
Bond
Bond Length (picometers)
Group
Van der Waals radius
C-C H
1.2
C-H F
1.35
C-F
134 Cl
1.8
C-Cl
176
CH3 ~2
CF3

20. 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.

21. Hansch and Hammet constants
Group π σ H
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
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
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 John-Wiley, New York

22. Functional Group Biosteres
Acid
Ester
Amide
Ketone (Sulfone/Sulphoxide)
Phenol
Amine
Urea

23. 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).
Talk about changing acid to biostere should always be investigated (may improve profile) but it is not necessary as many acids are drugs and can always make it an ester prodrug.

24. 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.

25. Drugs on the market with an “acid”

26. A case where the acid couldn’t be replaced (statins)

27. 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!).

28. 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

29. 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).

30. 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.

31. 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.

32. Biosteres for a peptidic amide

33. Amide reversal example
Enzyme
Ki Value (µM)
NEP 24.11
0.0019
Themolysin
1.8
ACE
0.14
0.0023
2.3
>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.

34. 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.

35. 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.

36. Phenols

37. 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.

38. Model of a GABA type C receptor with GABA
Biophysical Journal, 2008, 4115.

39. 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.

40. Rings Compound BP (°C) Benzene 80 Thiophene 84 Methylbenzene 110
Benzene 80
Thiophene 84
Methylbenzene
110
2-Methyl-thiophene
113
Chlorobenzene
132
2-Chloro-thiophene
130
Acetylbenzene
200
2-Acetyl-thiophene
214

41. Rings: Example

42. 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.

43. 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.

44. 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.

45. Rings

46. 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 hetero-atoms being oxidized and/or ring opening reactions possible, see case-by-case.

47. In drug design, the use of biosteres is still very intuitive and empirical.