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FimH Antagonists - Research Paper in Medicine

7 pages
1726 words
Harvey Mudd College
Type of paper: 
Research paper
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Urinary tract infections are known to affect millions of people yearly. The main cause of UTIs is the Uropathogenic Escherichia Coli (UPEC) which is responsible for 70% of the infections. Treatment of urinary tract infections requires administration of antibiotics. The problem rendered is that repetitive use of antibiotics results in the development of microbial resistance. This implies that the effectiveness of the treatment is lessened. There is, therefore, need to develop efficient ways of preventing and treating UTIs.

The symptomatic urinary tract infections exhibit various steps before the symptoms are seen. Initially, the UPEC attaches itself to urothelial cells. For this to occur, it requires the aid of bacterial adhesion FimH. FimH has two domains: N-terminal lectin domain and a C-terminal pilin domain. The Lectin domain further has a Carbohydrate Recognition Domain (CRD). The pilin domain, on the other hand, serves the purpose of enabling switching between various states of the CRD. In this case, a potential solution for prevention and stopping of the infection lies with preventing bacterial adhesion by inhibiting the FimH-CRD.

The possible antagonists for prevention of type 1 pili adhesion are mannosides and oligomannosides in the presence of an aglycone. For control where daily doses are required, it is advisable to use oral administration. The most critical factors for the use of the antibiotic depend on its oral absorption, metabolic stability, and renal absorption. The oral absorption depends on solubility and permeability.

The membrane permeability and the potential for oral absorption in biphenyl a-D-mannoside, 1a is affected by the carboxylic acid moiety. This moiety refers to the electron withdrawal potential during chemical reactions. For this reason, the polar carboxylate was improved by esterification (1b8h) to improve passive permeability. The ester, 1b, however, retained almost similar properties for pulling away electrons. Another advantage of this process is that it enhanced renal absorption. The ester 1b brought about drawbacks due to its low aqueous solubility, limited absorptive flux through the mucosa, fast metabolic activity, and faster renal excretion which implies that the required concentration could not be achieved in the bladder for the recommended amount of time. This problem forced Scientists to come up with structural changes to the aglycone as shown in the figure below.


Figure 1

The aglycone variations are obtained through 3 approaches (a, b and c) as shown above.

Alteration of Substitution of the pattern

Introduction of heteroaryl aglycones

Replacement of carboxylate moiety with a bioisosteric cyano group

The research was done with the aim of establishing the pharmacokinetic characteristics of various possible FimH antagonists which could be used to offer a solution to the problem of antibiotic resistance to antibiotics due to frequent administration of the drugs. The first thing to note is that UTIs are associated with the kidney as an excretory organ. It is therefore important to understand the principle of operation ranging from urine storage in the bladder to the manner in which waste substances are excreted, and other components are reabsorbed into the bloodstream. For the case of the antibiotics, once ingested, they are absorbed in the intestines and end up in the kidney. For optimum effectiveness, it is a requirement for the FimH antagonist to be able to undergo slow renal excretion to dwell in the bladder for a long time and hence fight the UPEC. In this research, the pyrrolylphenyl mannoside 42f met this conditions better than any other compound. It displayed therapeutic urine concentration for up to 6 h and is, therefore, a promising oral candidate for UTI prevention and treatment.


The aim was to improve three therapeutic factors of orally administered antibiotics for either the treatment or prevention of Urinary tract infections. The conditions are oral absorption, metabolism and renal absorption of the FimH antagonists.

Synthesis of FimH antagonists

Synthesis of various FimH antagonists was done following procedures based on chemical composition. The objective was to improve the pharmacokinetic procedures which include oral absorption, metabolic stability, and renal absorption. The methods of improvement used are those illustrated in figure 1 above. It is important to note that modifying the substitution pattern is done to disrupt molecular planarity and symmetry. The heteroaryl aglycone used to improve hydrophobicity is the heterocyclic biaryl aglycone. For Biphenyl Mannosides, the synthesis was done as described above for the compounds 1a and 1b.

Synthesis of Heteroaromatic building blocks is illustrated in the scheme shown below. To obtain azidophenols 13a, b from aminophenols, a diazo transfer reaction is performed in the presence of freshly prepared triflyl azide in pyridine and Copper (II) Sulphate. The next step is a copper(I) catalyzed Huisgen cycloaddition to give triazolylphenols 14a,b. To obtain 16a, a pyrazole-4-carboxylate was reacted with 4-iodoanisole. When carried out in a solvent-free environment, the process yields 16b. The synthesis for cyano-substituted pyrroles 23a, b, involves treatment of benzotriazole-1-methyl isocyanides with the electron-deficient alkenes 22a,b under basic heterocyclization conditions.


Figure 2

Steps a to g from the figure are explained below

TfN3, CuSO4, triethylamine, pyridine, 0 1C to rt, 2 h;

ethyl propiolate, CuSO4,5H2O, sodium ascorbate, tBuOH/H2O (1:1), rt, 30 min (yield for two steps: 77% for 14a, 48% for 14b);

ethyl 1H-pyrazole-4-carboxylate or ethyl 3-trifluoromethyl-1Hpyrazole-4-carboxylate, CuI, trans-N,N0-dimethyl-1,2-cyclohexanediamine, K2CO3, NMP as solvent for 16a and solvent free for 16b, 110 1C, 24 h (80% for 16a, quant. for 16b);

AlCl3, cat. nBu4NI, DCE (for 17a), or AlCl3 in 1-dodecanethiol (for 17b), 0 1C to rt (60% for 17a, 26% for 17b);

i. nBuLi, hexane, toluene, 78 1C, 1 h; ii. CO2 (g), 78 1C to rt, 7 h;

conc. H2SO4 (0.8 equiv.), MeOH, reflux, overnight (37% for two steps);

nitrile 22a,b, tBuOK, THF, 0 1C to reflux, 2 h (60% for 23a, 54% for 23b).

Other FimH antagonists that were synthesized for the research include Triazolylphenyl and pyrazolylphenyl mannosides, Pyridinylphenyl, pyrazinylphenyl, and pyrimidinylphenyl mannosides, and Pyrrolylphenyl mannosides.

Physicochemical properties and in vitro pharmacokinetics of FimH antagonists

These are based on the assessment of potential intestinal absorptions using lipophilicity (log D7.4), aqueous solubility, and permeability. Permeability through an artificial membrane (PAMPA) is referred to as effective permeability and denoted by Log Pc. Apparent permeability, on the other hand, refers to Papp determined through a Caco-2 cell monolayer. The two parameters for permeability have their datum measuring points, and once data for both is obtained, a comparison is made to confirm the viability of the FimH antagonist.

Thresholds for effective permeability: Low - Log Pc < -6.3 cms-1; medium -5.7 cms-1; and high > -5.7 cms-1.

Thresholds for apparent permeability: Low Papp < 2 106 cms-1; Moderate 20 106 cms-1; and high > 20 106 cms-1.

Biphenyl Mannosides


Initially, they exhibit low aqueous solubility of 24 ug mL-1 due to symmetrical para substitution and hence stacking effects. The carboxylic acid moiety in 1a was therefore moved from the para to the Meta position thus improving aqueous solubility moderately to 41 ug mL-1 due to the dihedral angle remaining constant. Shifting of the chloro substituent from the Ortho to the Meta positions alters the dihedral angle from 39.61 to 60.31. The aqueous solubility also improves by triplicating to around 134 ug mL-1


All biphenyl mannosides have high lipophilicity (log D7.4 1.7). This implies high effective and moderate apparent permeability as per the thresholds indicated above.

Metabolic Stability

Incubation of prodrug 1a with rat liver microsomes shows a fast degradation with a half-life of 2.1 minutes. This is the same for other biphenyl mannosides other than 1a and implies that despite the elevated solubility and high aqueous permeability, these compounds do not meet the therapeutic potential. The fast metabolic ester hydrolysis means rapid renal excretion of the polar metabolite hence.


Heteroaryl Mannosides


Higher aqueous solubility than biphenyl a-D-mannoside 1a. With increased polarity, there is an increase in solubility. For the pyrazolylphenyl, the pyridinylphenyl and the pyrrolylphenyl mannosides high solubility was achieved despite having high lipophilicity.


Permeability reduces with increase in polarity and solubility, i.e., they are inversely proportional. Heteroaryl mannosides with log D7.4 > 1 have effective permeability ranging from moderate to high. It is, therefore, necessary to enhance lipophilicity to values greater than one. This is done by either introduction of an ortho-chloro substituent to Ring A of compound 25a (Table 1) or replacing the methyl ester by an ethyl ester.

Metabolic Stability

Five-membered heteroaryl mannosides were less susceptible to CES-mediated conversion and exhibited a half-life of more than 30 minutes than the parent biphenyl mannoside 1a. Six-membered heteroaryl mannosides on the other hand dissociated with a half-life of fewer than 15 minutes.

In vitro Binding Affinities

The affinities were determined in a cell-free competitive binding assay. The esters recorded higher affinities than the corresponding acids. This observation could be accredited to the fact that esterification reduces the dissolving costs.

Biphenyl mannosides

Affinity is reduced by changing the position of carboxylic acid on terminal ring B of the aglycone accompanied by alteration of the substitution pattern on ring A. The ortho-chloro substituent present in the antagonists 1b and 6b provide extra internal forces in addition to the covalent bonds. This is with references to the presence of Van der Waal's forces of attraction whose outcome is the reduction of binding affinity.

Heteroaryl Mannosides

The results indicate that all heteroaryl mannosides were weaker than the biphenyl mannoside 1b despite the values also being in the nano-molar range. With the help of the Glide Software Package, performing silicon studies on the FimH-CRD gives a similarity in the out-docking mode giving a consistent trend of p-p stacking mode with Tyrosine-48.

Triazolylphenyl and pyrrolylphenyl mannosides showed a higher affinity by being approximately almost five times than that of pyrazolylphenyl analogs. Substitution patterns were also influential on binding affinity with an ortho-chloro, or an ortho-trifluoromethyl substituent on ring A observed to improve anity approximately thrice. Position of the electron-withdrawing carboxylic acid substituent is also a factor that influences the binding affinity

In vivo Pharmacokinetic Study

The pharmacokinetic study was performed on antagonist 42f, Table 1. This is because it showcased the best pharmacokinetic profile. Due to low solubility for the 10 mg kg-1, a solubilizer was used. The solubilizer consisted of 5% DMSO and 1% surfactant Tween 80.

Plasma concentrations were low with a maximum of 0.04 ug mL-1 between 40 minutes and one and a half hours. Accumulation of urine results in flattening of the line graph relating the time with plasma concentration. The predicted permeability of both parallel artificial membrane permeability assay (PAMPA) and transport through Caco-2 cell monolayer was a high effective permeability and moderate apparent permeability. The 42f is a substrate of efflux transporters, however,...

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