Pestic. Sci. 1990, 29, 221-240

Acidic Isostere Design: Synthetic Strategies and Recent Progress in Understanding Electronic Properties and

Metabolic Stability *

Christopher A. Lipinski & Bert L. Chenard

Pfizer Central Research, Groton, Connecticut 06340, U S A

(Manuscript received 8 December 1989; accepted 9 December 1989)

ABSTRACT

The efficient synthesis of a family of twelve acidic heterocycles of varying acidity f iom a single common intermediate facilitates the search for new acidic bioisosteres. An extension of this chemical approach led to a new family of phosphonate replacements in prototypes related to the N-methyl D- asparate ( N M D A ) antagonist 2-amino-7-phosphonoheptanoic acid (AP7). Acidic isostere design may be facilitated by grouping hydroxylic heterocyclic carboxylic isosteres into one of two electronic classes based on the Gandour hypothesis. The limitations of normal hydroxamic acids as carboxylic acid surrogates suggest that the excellent metabolic stability of reverse hydroxamic acids may be useful in prospective acidic isostere design.

1 INTRODUCTION

Bioisosteres are groups or molecules having chemical and physical similarities which produce broadly similar biological properties. The many types of isostere such as acidic, basic, amide carbonyl, phosphate and catechol have been reviewed.’,* Acidic bioisosteres have chemical and/or physical properties mimicking acidic groups such as carboxylic acids, phosphates or phosphonates and phenols. While these functional groups are structurally different, it was hypothesized that it might be possible to design a series of acidic heterocycles with appropriate structural features to probe for new acidic bioisosteric relationships to one or more of these groups.

* Based on a paper presented at the Symposium ‘Isosteric replacements in drug and pesticide chemistry’ organised by the Pesticides Group of the Society of Chemical Industry and held in London on 6 June 1989.

227

Pestic. Sci. 0031-613>(/90/$03.50 0 1990 Society of Chemical Industry. Printed in Great Britain

228 C. A . Lipinski, B. L. Chenard

This paper focuses on experimental work on the design of a series of acidic heterocycles as probes for new acidic bioisosteric relationships and describes biological results testing the suitability of these heterocycles as phosphonate isosteres. Recent literature reports that may be applicable to the discovery of new acidic bioisosteres complement these experimental studies and are discussed.

2 MERCAPTOAZOLES-NEW ACIDIC HETEROCYCLES

2.1 Introduction

The known carboxylic acid isosteres-tetrazoles, various five-membered hydroxylic heterocycles and hydroxamic acids are each made by a different synthetic sequence. This is a major impediment to systematically exploring a series of potential acidic bioisosteres attached to a common framework. A series of acidic heterocycles that could be prepared by simple chemistry from a single synthetic intermediate in high yield would facilitate the synthesis of prototypes probing for new acidic isosteric relationships. The likelihood of discovering new acidic bioisosteric relationships could be increased if the acidic heterocycles encompassed a broad acidity range and contained structural features mimicking a variety of acidic groups. These considerations led to the design of a series of acidic mercaptoazoles.

2.2 Medicinal design

Acidity can be varied over a wide range in five-membered heterocyclic azoles such as imidazole, 1,2,4-triazole and 1,2,3-triazole since the pKa values of these rings can vary between 4-8 (carboxylic-acid-like) to 14.5 (less acidic than a phenol) depending on the nature of the azole substitution. Table 1 shows that the acidity of five- membered heterocyclic azoles increases (numerically lower pKa) in the sequence imidazole (either proton) < 1,2,4-triazole < 1,2,3-triazole. As a ring substituent in Table 1 becomes more electron-withdrawing (numerically higher sigma meta value) the ring becomes more acidic. This is the same pattern as is found in carboxylic acids and phenols and is the basis for the definitions of substituent sigma value^.^

The various oxidation states of a mercaptoalkyl moiety, e.g. alkylthio (as exemplified by SCH,), sulfoxide (SOCH,) and sulfone (SO,CH,) span a large sigma meta range and hence a large electron-withdrawing range.3 It was expected that these substituents, appended to heterocyclic azoles, would lead to acidity in the phenol (pKa 10) to carboxylic acid (pKa 4.5) range. A similar strategy using sulfide, sulfoxide and sulfone substitution could also modulate the acidity of the related 1,2,4-triazo1-3-one ring system.

The same sulfur moiety that can fine-tune the ring acidity of a heterocyclic azole can also serve to anchor the heterocylic azole to an atomic framework. The long bonds of a sulfur linkage and the tetrahedral nature of the sulfoxide and sulfone moieties may mimic the long phosphorus-oxygen bonds of a tetrahedral phosphonate or phosphate. Azoles substituted by sulfide, sulfoxide or sulfone groups contain an acidic tautomeric N-H moiety with pKa range that could vary from carboxylic-acid-like to phenol-like. Carboxylic acid isosteres such as tetrazole and phenolic isosteres such as pyrrole and pyrazole also contain an acidic

Acidic isostere design 229

TABLE 1 Azole PKa Values from the Literature

2-PKa" 4-PKab PKa PKa

Substituent

H SCH COCH, Br SOCH, SOZCH, NO2

Sigma meta -

H 0.00 14.52'; 14.52' 10.26d 0.15 11.6/ 0.376 0.391 1 1.03d; 12.32' 0.52 0.60 0.71 7.1Sh; 9-20' 6.05'

9.42'

6.63g

4.79k

PKa of imidazole substituted at the 2 position. PKa of imidazole substituted at the 4 position. Reference 27. Reference 28. Reference 29. Reference 28. Based on 2-mercaptoimidazole. Reference 30. Value based on CHO substituent.

Reference 28. PKa in 1 :2 dimethylformamide-water; Reference 32. *Reference 28. PKa in 1:l methanol-water. See also reference 31.

j Reference 33. ' Reference 34.

tautomeric azole N-H moiety.* This combination of tetrahedral sulfur4xygen bonds and acidic tautomeric N-H moieties could reveal bioisosteric relationships to phosphates or phosphonates, carboxylic acids or phenols (Fig. 1).

2.3 Synthesis

A mercapto heterocycle can be selectively alkylated on sulfur with an alkyl halide (or equivalent). The alkyl halide is chosen so as to yield an appropriate backbone. The benzyl group was chosen as the smallest (and hence least lipophilic) aralkyl backbone group so as to maximize aqueous solubility for pKa studies. The very simple synthetic route to the target compounds is shown in Figs 2 and 3. The alkyl halide starting material is reacted with either a mercaptoazole to give the sulfide of

R-CH2COOH or R-PO,H, = Y=Z Y=Z

\

YNH SNa ;YkH- x' ,S(O),

+ R

X . Y . 2 = N.C.CO n = 0.1.2

RX

Fig. 1. Strategy for acidic group replacement.

230 C . A . Lipinski, B. L. Chenard

H .. H

4 TEA, Ethanol

H 9 X=CH,Y:CH,Z=N Irnidazolcs cg.1. Table 2 X = N .Y=CH . Z = N 1.2.4-Tr iaroles e g . l V , Table 2 X: N .Y = N ,Z=CH 1.2.3-Triazoles eg. VII .Table 2

Sulfoxidcs eg.11,V.VIII Table 2

Sulf ones eg. I l l . V I , I X Table 2

Fig. 2. Synthesis of imidazoles and triazoles.

an imidazole, 1,2,4- or 1,2,3-triazoIe or with carboethoxy thiosemicarbazide to give the sulfide of a 1,2,4-triazol-3-one. These four sulfide series can then be oxidized further to the sulfoxide or sulfone giving a total of twelve heterocycles.

Starting materials for the four series of heterocyclic azoles are the single alkyl halide precursor and the commercially available 2-mercapto-imidazole, mercapto- 1,2,4-triazole and mercapto-l,2,3-triazole (Fig. 2). A fourth series is prepared from carboethoxy thio~emicarbazide~ which in turn is available from thiosemicarbazide (Fig. 3). In all four series, sulfoxides and sulfones are easily prepared from sulfides using simple standard oxidation reaction conditions, e.g. for sulfoxides, m- chloroperbenzoic acid, dichloromethane, 23°C or 'Oxone', methanol, water, 23"C, and for sulfones: 30% hydrogen peroxide, acetic acid or oxone, methanol, water, 50°C.

2.4 Chemical stability

The four series of mercaptoazoles are chemically stable. No sign of hydrolysis at 23°C in the pH range 3 to 10 to the hydroxy heterocycle and corresponding benzyl sulfide, sulfinic, or sulfonic acid was observed in the UV assay' used to measure compound pKa. This result is quite consistent with the known stability of sulfinyl and sulfonyl 1,2,4-triazoles6 and is probably due to two factors. Firstly, five- membered heterocycles are more electron-rich than their six-membered counterparts having the same number of nitrogens, and hence are less susceptible to

Acidic isostere design 23 1

I pi NaOH, H,O

1.2.4-Triazolon -3-ones eg.X ,Table 2

H

o + N T s

rn-CPBA &oz, cH3c02H

eg. X I I , Table 2 Su If oxides Sulfones eg. X I , Table 2

Fig. 3. Synthesis of 1,2,4-triazol-3-ones.

nucleophilic attack. Secondly, the mercaptoazoles are protected from attack by base because of their anionic state at basic pH. This stability contrasts with the chemistry that is observed with the electrondeficient six-membered heterocyclic rings such as pyridines or pyrimidines, where oxidized alkyl mercapto groups (sulfones) at the 2 or 4 positions are employed as leaving groups in nucleophilic aromatic substitution reactions.

2.5 Acidity range

Apparent pKa values in water at 23°C for the mercaptoazoles in Table 2 were determined by a UV te~hnique.~ Values in parentheses are standard deviations for triplicate determinations. The pKa values uniformly span the range from weak acid (phenol, pKa 10) to carboxylic acid (pKa 4) and can be used to search for a bioisosteric relationship in a particular pKa range. For example, entries I, 11,111, IV and VII have phenol-like acidities while IX, XI and XI1 might be explored as carboxylic acid replacements. Entries with a pKa numerically close to physiological pH (7.4), such as V, VI, VII and X, are particularly interesting. These azoles exist in both ionized and neutral form at physiological pH and might be useful in situations

232 C. A . Lipinski, B . L . Chenard

TABLE 2 Benzylsubstituted Mercaptoazoles

No. n X Y 2 PKa (S.0.')

I 0 I1 1 111 2

IV 0 v 1 v 1 2

VII 0 VIII 1 IX 2

x o XI 1 XI1 2

CH CH CH N N N N N N

c=o c=o c=o

CH N CH N CH N CH N CH N CH N N CH N CH N CH NH N NH N NH N

11.6 9.80 (01 1) 8.67 (003)

9.50 (007) 7.38 (0.04) 6.23 (010)

8.20 (010) 6-09 (0.14)

5.15

7.85 (010) 5.2

4.79 (018)

a Standard deviation of triplicate measurements of apparent pKa at 23°C in H,O measured by ultraviolet spectroscopy. See Ref. 5.

where the biological receptor recognizes an anion but where a considerable fraction of neutral compound is advantageous for transport properties.'

3 MERCAPTOAZOLES AND ARYLS AS PHOSPHONATE ISOSTERES

As previously described, the mercaptoazoles might function as weak acid (phenol) or strong acid (carboxylic acid) replacements by analogy with weakly acidic (pyrazole or pyrrole) and strongly acidic (tetrazole) heterocycles. In addition to these possibilities, the tetrahedral oxidized sulfur feature of the mercaptoazoles makes them suitable as potential phosphonate or phosphate replacements.

3.1 Medicinal design

The pKa values of the azoles substituted by sulfoxide or sulfone groups can be adjusted to mimic those of phosphonic acids. Methylphosphonic acid can be used as a model. The pKa values of 2.3 and 7.9 for this acid' predict that at physiological pH the molecule wi!l exist as 24 % of the mono-anion and 76 % of the di-anion. Thus, a single delocalized anionic site on a heterocycle in close proximity to an additional electronegative atom (the sulfoxide or sulfone oxygen) could function as a mimic for either a phosphonate mono-anion or di-anion. This is illustrated in Fig. 4 which

Acidic isostere design

n

233

a b Y

Fig. 4. Computer images of (a) methylphosphonic acid and (b) overlapping structure of methylphosphonic acid (shaded) and S-methylsulfonyl-l,2,4-triazole.

compares structures of methylphosphonic acid with 5-methylsulfonyl-l,2,4- triazole.

3.2 Glutamate antagonist application

Testing whether the mercaptoazoles might be useful in discovering new phosphonate bioisosteric relationships requires a phosphonatecontaining drug which can be mimicked by a mercaptoazole. The presence of a phosphonate moiety in glutamate antagonists of the NMDA receptor subtype provided an opportunity to apply phosphonate bioisostere ideas to the generation of a new lead series of glutamate antagonists.

Glutamate is a mammalian excitatory amino acid neurotransmitter, and antagonists may be useful in the therapy of neurodegenerative diseases such as Alzheimer's disease, Huntington's chorea, and ischemic brain damage.' Glutamate is also a major insect neurotransmitter acting through a variety of glutamate receptor subtypes."

The compound AP7 (2-amino-7-phosphonoheptanoic acid) is an antagonist of the NMDA receptor and prevents cyclic-GMP increases in rat cerebellum." The phosphono group was replaced chemically by a mercaptoazole. As shown in Fig. 5, replacing the phosphono group in AP7 by the three members of the mercapto-1,2,4- triazole series did not lead to biologically active compounds. However, an active compound resulted when the acidic hydrogen which is tautomeric in the 1,2,4- triazoles was localized at an ortho position to the side chain by replacing the triazole by a mercapto ortho-phenol.

Sulfoxide and sulfone derivatives were prepared in the hope that they would be more active. Rather surprisingly, as shown in Table 3, sulfoxide and sulfone derivatives were all inactive. The ortho aniline derivative VIII was prepared in follow-up of the phenol and proved to be the most potent of ail with an IC,, corresponding to a potency one-third that of AP7. This series is currently being studied, and preliminary data suggest that VIII is a competitive antagonist.

234 C . A . Lipinski, B. L. Chenard

0 COOH AP-7 H o , l d Active antagonist

NH2 lead H 0’

COOH n -0 Inactive n-1 Inactive

NH2 n - 2 Inactive N-N

U

Fig. 5. Mercaptotriazole and phenol analogs of 2-amino-7-phosphonoheptanoic acid (AP-7).

3.3 Conclusions

The evolution of this series illustrates how the mercaptoazole acid surrogates can be used in prototype studies and in new lead generation. The results can be quite unexpected, since replacing a phosphonate moiety by an ortho-mercapto aniline is unprecedented, especially since a weakly basic aniline and an acidic phosphonate are so different in acidity. However precedent does exist among histamine H2- agonist-antagonist structure-activity relationships for replacement of a highly basic guanidine in an agonist by a neutral guanidine surrogate in the antagonist.I2 Interpreting the present results is further complicated, since AP7 is an antagonist and there is no guarantee that the ortho-amino thiophenyl or the phosphonate binds in the same way as the carboxylic acid moiety found in the glutamate agonist. Much remains to be learned about which structural features of mercaptoazole or mercaptoaryl groups are important as phosphonate replacements.

4 NARROWING THE CHOICE OF ACIDIC BIOISOSTERES

4.1 Introduction

Discovering new examples of acidic bioisosterism in a prospective sense, using the existing literature, can be very slow because each compound requires a new synthesis. Any factor that could improve the chances of an active compound early in a program is important. One way this can be accomplished is to narrow the choice of carboxylic acid bioisosteres to two electronic classes based on the Gandour hypothesis.

4.2 Nomenclature

Carboxylic acid anions possess two sp2 hybridized lone-pair oxygen orbitals that are particularly important in receptor interactions. The syn orbital of one anionic oxygen is directed syn (towards the same edge) of the carboxylate anion plane as

Acidic isostere design 235

TABLE 3 PKa and Potency in Inhibition of NMDA-Induced Cyclic GMP Increases

Compound Phenol pKir IC,o ( x I F 4 ) (f S D ) (n)

HO COOH 2.64 (k 003) 7.72 (kO.11)

/'-NH2 9.84 (k0.07) HO 0.35 (k 0.06) ( 3 ) \ 11 AP-7

COOH

I

I1

111

IV

V

VI

VII

VIIl

COOH 0

0 0

7.38

COOH

> 1 mM

> 1 mM

> 1 rnM

COOH C1

8 5 5 (kO.01) 2.2 (f 0 4 ) (3) /

2.6 ( + 0 4 ) (2)

1.4 (f 0.3) (4)

236 C . A . Lipinski, B. L. Chenard

Ant i lone pair S y n l o n e pair

lo3 to lo5 m o r e basic

R A Fig. 6. Gandour hypothesis.

that of the other oxygen while the anti orbital of an anionic oxygen is directed anti (away from) its oxygen partner (Fig. 6).

4.3 The Gandour hypothesis

The hypothesis suggests that the syn lone pair may be lo3 to lo5 times more basic than the anti lone pair and that this is important because enzyme carboxylates probably use syn-disposed lone pairs to interact with small molecule substrate^.'^ Recent experimental and theoretical studies support this hyp~thesis.'~-''

4.4 SynlAnti orbital significance

It was hypothesized that the carboxylate anion in a small drug molecule might preferentially interact with the guanidinium group (as in an arginine residue) of a receptor by using the syn rather than anti lone pair because of the known complementarity in hydrogen bonding between guanidinium cations and carboxylate anions.I8 If this is correct, a carboxylic acid bioisostere binding to a receptor guanidine is likely to be more active if its anion is more like a syn than an anti carboxylate anion.

4.5 Calculating syn-unti preference

The syn or anti preference in carboxylic acid bioisosteres can be based on a thermodynamic cycle argument which suggests that protonation of the more basic syn anion gives the more stable lower dipole moment, syn carboxylic acid. Calculating dipole moments of syn and anti methyl esters is similar to calculating dipoles of the free acids and gives a measure of syn and anti preference. A large negative difference for the syn methyl ester minus anti methyl ester dipole moment corresponds to a more basic syn anion.

The methyl ether of an acidic hydroxylic heterocycle is the isostere of a carboxylic acid methyl ester. A small negative or even positive difference in the dipole moment of a heterocyclic syn methyl ether minus the dipole moment of an anti methyl ether indicates a hydroxy heterocycle that is quite unlike a carboxylic acid in that there is little or no difference in basicity between syn and anti lone pairs.

4.6 Syn-Anti heterocyclic acid bioisosteres

Table 4 shows a series of structurally closely related bioiso~teres'~ of 4- aminobutyric acid (GABA) that span the pKa range of 2-6 to 8.0. Methyl ethers of these heterocyclic carboxylic isosteres are related to carboxylic acid methyl esters. Their dipoles were calculated using the Quantum Chemistry Exchange Program

Acidic isostere design 231

TABLE 4 Syn - Anti Isostere Preference

Entry Structure P K a SYn Anti SYn Dipole" Dipole" Preference

syn - anti

4.0 1.434 3.915 - 2.481

2.6 1.515 3.921 - 2.406 83

80

83

83

83

83

8,

H 2 N d

" 2 " L ( $

H 2 N d C H 3 5.5 2,042

4.8 1.545 4.248 - 2.703

H 2 N d - J 5.8 1-983 4.737 - 2-754

H 2 N L 4 $ 6.1 1.259 2.1 17 - 0.858

H 2 N d H 6.2 2.349 2,098 + 0.251

"4 8.0 2.534 3.127 - 0593 CH3

~~ ~ ~

a Dipole moments of the methyl ethers of fragments lacking the (CH,NH2) moiety were calculated using the Quantum Chemistry Exchange Program AMPAC. Refs 20 and 21.

AMPACZo*Z1 for syn (ether alkyl on the same side as the ring heteroatom) and anti (ether alkyl directed away from the heteroatom) methyl ethers.

The rightmost column in Table 4 gives the syn-anti difference. The more negative the number, the greater the relative basicity of the syn anion. The syn carboxylate anion (entry 1) is more basic than the anti, as predicted by the Gandour hypothesis. The numbers found for the syn - anti difference for the five-membered heterocycles in entries 2,3 and 5 are very similar to those for the carboxylate group. By contrast, the acidic ring systems in entries 6,7 and 8 are not predicted to show the large basicity difference between syn and anti anion forms. Entry 4 shows a limitation to these calculations. A reliable dipole cannot be calculated for the syn methyl ether of 4 because there is a major steric interaction between the 0-methyl and ring N-methyl. The dipole calculations for all the other entries can be compared because the ring systems are sterically similar.

The data in Table 4 could be used to simplify the choice of synthetic targets to only two compounds. A first isosteric replacement might come from those heterocyclic anions that exhibit the greatest relative syn basicity as in entries 2,3 and 5 and a second from the electronically quite different 6, 7 and 8.

238 C . A . Lipinski, B. L . Chenard

5 IN-VIVO ACTIVE ACID BIOISOSTERES

5.1 Introduction

Acidic moieties which have good metabolic stability are attractive synthetic targets in prospective searches for new acidic isosteres because obtaining in-vivo activity is often the major medicinal challenge in a drug discovery project. Reverse hydroxamic acids have excellent metabolic stability and their structural features suggest they will be useful tools in the search for new acid bioisosteric relationships.

5.2 Reverse hydroxamic acids

Hydroxamic acids have been used as replacements for the carboxylic acid moiety in anti-inflammatory analogs of ibuprofen and indomethacin.’ One problem with this replacement is that in-vivo rat half-lives are short with the ‘normal’ type of hydroxamic acid in which an NHOH group replaces a carboxylic acid OH group because this type of hydroxamic acid is biologically readily converted back to the parent carboxylic acid. 2z,2

‘Reverse’ hydroxamic acids (Fig. 7) in which the hydroxamic acid acyl group is small relative to the group appended to the NOH have been studied as inhibitors of leukotriene b i o s y n t h e s i ~ . ~ ~ * ~ ~ Following oral administration, the reverse hydroxamic acids exhibited unusually long half-lives in rats in comparison to their normal hydroxamic acid counterparts. The nature of the small acyl group in the reverse hydroxamic acid is important, since formyl in contrast to acetyl analogs do not exhibit long rat half-livesz4

5.3 Structural features

Reverse hydroxamic acids can exist in the neutral state in two interconvertible geometrical forms.25 the cis (2) form in which the NOH is hydrogen bonded to the carbonyl oxygen (and is on the same side as the carbonyl; Fig. 7) is favored by larger acyl groups and by non-polar solvents. Reverse hydroxamates have pKa values about 3 units less acidic than their carboxylic acid counterpartsz6 and therefore exist in both neutral and anionic forms at the physiological pH of 7.4. The conformational behavior of reverse hydroxamates combined with the desirable pKa range suggests that this moiety could be a useful new tool in the search for new acidic bioisosteric relationships.

~ i s ~ ~ ~ & . *Lila11 Trans“% R;rnilll

large 0 R-N HO-N

‘4 N ,OH

\ R large

\ \ large

OH

Reverse hydroxamic acid R’ is small relative t o R

smaii

Nor ma I hyd roxam ic acid

Fig. 7. Hydroxamates and reverse hydroxamates.

Acidic isostere design 239

6 CONCLUSIONS

Three approaches to the design and discovery of new acidic bioisosteric relationships have been described in this paper. The first approach is the design and synthesis of a family of twelve easily prepared heterocycles that contain structural features that might mimic carboxylic acids, phosphates or phosphonates and phenols. This approach led to the discovery of a new series of NMDA antagonists in which a phosphonate in an antagonist is replaced by an ortho-mercapto phenol or aniline. The second approach is theoretical and narrows the choice of hydroxylic heterocyclic carboxylic acid isosteres by describing these as syn or anti carboxylate anion mimics. The third approach is a focus on the importance of in-vivo activity and suggests that the excellent metabolic stability of the reverse hydroxamic acid moiety may be an important tool in the search for new acidic bioisosteric relationships.

ACKNOWLEDGEMENTS

The authors thank Dr Ed. Mena and Mr Marty Pagnozzi for biological data, Professor Dan Kemp for discussion on the Gandour hypothesis, Mr Matt Teague for pKa data and Mr Todd Butler, Mr Garth Butterfield, Ms Lilian Marinovic and Mr Robert Ronau for experimental assistance.

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