Synthesis and evaluation of thiomannosides, potent and orally active FimH inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.06.017

FimH is a type I fimbrial lectin located at the tip of type-1 pili of Gram-negative uropathogenic Escherichia coli (UPEC) guiding its ability to adhere and infect urothelial cells. Accordingly, blocking FimH with small molecule inhibitor is considered as a promising new therapeutic alternative to treat urinary tract infections caused by UPEC. Herein, we report that compounds having the S-glycosidic bond (thiomannosides) had improved metabolic stability and plasma exposures when dosed orally. Especially compound 5h showed the potential to inhibit biofilm formation and also to disrupt the preformed biofilm. And compound 5h showed prophylactic effect in UTI model in mice.

Full text of DOI:10.1016/j.bmcl.2018.06.017

Accepted Manuscript
Synthesis and evaluation of thiomannosides, potent and orally active FimH in-
hibitor
Jitendra A. Sattigeri, Malvika Garg, Pragya Bhateja, Ajay Soni, Abdul Rehman
Abdul Rauf, Mahendrakumar Gupta, Mahesh S. Deshmukh, Tarun Jain, Nidhi
Alekar, Tarani Kanta Barman, Paras Jha, Tridib Chaira, Ramesh B. Bambal,
Dilip J. Upadhyay, Takahide Nishi
PII:
S0960-894X(18)30505-5
https://doi.org/10.1016/j.bmcl.2018.06.017
BMCL 25898
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 April 2018
5 June 2018
11 June 2018
Please cite this article as: Sattigeri, J.A., Garg, M., Bhateja, P., Soni, A., Rauf, A.R.A., Gupta, M., Deshmukh, M.S.,
Jain, T., Alekar, N., Barman, T.K., Jha, P., Chaira, T., Bambal, R.B., Upadhyay, D.J., Nishi, T., Synthesis and
evaluation of thiomannosides, potent and orally active FimH inhibitor, Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.06.017
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Synthesis and evaluation of thiomannosides, potent and orally active FimH inhibitor
Jitendra A. Sattigeria,*, Malvika Garga, Pragya Bhatejaa, Ajay Sonia, Abdul Rehman Abdul Raufa,
Mahendrakumar Guptaa, Mahesh S. Deshmukha, Tarun Jaina, Nidhi Alekara, Tarani Kanta Barmana,
Paras Jhaa, Tridib Chairaa, Ramesh B. Bambala, Dilip J. Upadhyaya, and Takahide Nishib,*
aDaiichi Sankyo India Pharma Pvt Ltd., Village Sarhaul, Sector 18, Udyog Vihar Industrial Area,
Gurugram 122015 Haryana, India
bDaiichi Sankyo RD Novare Co., Ltd., 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
Corresponding author email address: jitendra.sattigeri@gmail.com. (J. Sattigeri),
nishi.takahide.su@rdn.daiichisankyo.co.jp (T. Nishi)
Key words: Thiomannoside, FimH inhibitor, Urinary tract infection
Abstract – FimH is a type I fimbrial lectin located at the tip of type-1 pili of Gram-negative
uropathogenic Escherichia coli (UPEC) guiding its ability to adhere and infect urothelial cells.
Accordingly, blocking FimH with small molecule inhibitor is considered as a promising new therapeutic
alternative to treat urinary tract infections caused by UPEC. Herein, we report that compounds having
the S-glycosidic bond (thiomannosides) had improved metabolic stability and plasma exposures when
dosed orally. Especially compound 5h showed the potential to inhibit biofilm formation and also to
disrupt the preformed biofilm. And compound 5h showed prophylactic effect in UTI model in mice.
Urinary tract infection (UTI) presents a significant burden for women with nearly 20 million cases
reported annually. Recurrent UTIs are infections that follow resolution of an earlier episode, usually
after an appropriate treatment. It has been estimated that 20-50% women will experience recurrent
episode within 6 months of their initial diagnosis.1 Long term (~6 months) antimicrobial prophylaxis has
proved effective in reducing the risk of recurrent UTIs in women with at least two episodes of infection
in previous years.1 Because of the increasing prevalence of recurrent infections, as well as the increasing
emergence of antibiotic resistant strains, including multidrug resistant UPEC such as the ESBL
(extended spectrum β-lactamase),2 the desire for new therapeutics and non-antimicrobial prophylaxis of
UTI has escalated rapidly in recent years.3
The majority of UTIs (85−95%) are caused by members of the Enterobacteriaceae family;
uropathogenic Escherichia coli (UPEC) are isolated in approximately 80−85% of community-acquired
UTI. Escherichia coli (E. coli) is the predominant uropathogen responsible for recurrence of UTIs. It has
now been established that the first step in establishing infection involves bacterial adhesion to the
uroepithelium. This initial adhesion is mediated by mannose specific lectins FimH which is located at
the tip of type 1 pilli.4 Thus, several research groups have been involved in discovering potent FimH

inhibitors with a view to find non-antimicrobial alternate approach towards prevention of recurrent UTIs.
Several potent FimH inhibitors have been disclosed in recent literature.5-7 A few representative examples
(1-4) are shown in Fig. 1. Extensive efforts have been made not only to improve the affinity and
selectivity of FimH inhibitors, but also to ameliorate their pharmacokinetic properties.5,6 However, these
compounds have low plasma exposures and low bioavailability. Thus, from oral administration and for
drug development perspective their profile needs further optimization. While O-mannosides 1-2 have
good efficacy in animal models of UTI, they have low bioavailability and short half-life, which can
possibly be attributed to the metabolic instability of the O-glycosidic bond to either hydrolysis in the
stomach or intestinal tract or, alternately, from the enzymatic action of mannosidases. C-mannoside 4
has significantly increased compound exposure and, as a result, compound 4 shows enhanced efficacy in
mouse models of acute and chronic UTI.
Fig. 1 Examples of FimH inhibitor
We report herein our efforts towards identifying druggable FimH inhibitors. We specifically address
and eliminate the potential metabolic liability of O-mannosides with new stable thiomannosides. Our
prime focus while designing compounds was to maintain the LogD values between 0 - 2 for a moderate
lipophilicity which would be favorable for optimal oral absorption and renal excretion.8 First, we
focused our attention on design and synthesis of substituted pyridone and pyridine derivatives as X
shown in 5a-j (Fig. 2). These ring systems offer great flexibility and ease for varying substitutions and
hence modulate LogD over the desired range of 0-2.

Fig. 2 Structures of designed FimH inhibitors
General synthetic procedures are shown in Scheme 1. Thiomannosides were synthesized starting
with thioglycosylation of mannonse pentacetate (6) with 4-bromo-2-methylthiophenol in presence of
BF3·Et2O to yield thiomannoside as a mixture of α- and β-isomers in 85:15 ratio. Both isomers were
separated by flash column chromatography to obtain desired α-isomer 7 in 54% yield. Intermediates
15a-f and 17a were coupled with 7 under Suzuki coupling conditions to yield 9a-f and 9h in good to
excellent yields (42 - >95%). As 9g, 9i and 9j could not be obtained in good enough yields to proceed
further by this sequence, thiomannoside 7 was converted to boronate 8 using Miyaura conditions9 in
>95% yield. Intermediates 17b and 17c were then coupled with 8 under Suzuki coupling conditions to
afford 9i and 9j. Base mediated hydrolysis of acetyl group eventually gave 5a-f, 5h-j in unoptimised low
to good yields. Unfortunately, compound 9g could not be obtained using either of these sequences.
Hence, an alternate route was devised wherein the acetyl group on 7 was replaced with
methoxymethyl (MOM) protecting group. Thus, 7 on treatment with NaOMe gave 10 which was
protected with MOM group to afford 11 in 50% yield over two steps. Compound 11 was converted to
boronate 12 under Miyaura conditions in 92% yield. Coupling of 12 with 16 afforded 13 in 58% yield
followed by hydrolysis under acidic conditions gave 5g in 59% yield (Scheme 2).
Synthetic procedure of key intermediates of boronates 15a-f, 16 and 17c are shown in Scheme 3.
Alkylation or arylation of commercially available 5-bromopyridone afforded corresponding N-
substituted pyridones 14a-f, these pyridone derivatives were then converted into boronates 15a-f.
Compound 16 was obtained by n-butylation of 4-bromopyridone. Compound 17c was synthesized by
reacting azetidine with 5-bromo-2-chloropyridine in presence of Et3N. Intermediates 17a and 17b for the
synthesis of 5h, i procured commercially.

Scheme 1. Reagents: (a) BF3·Et2O (3.3 eq), CH2Cl2, reflux, 48h, 54% (-isomer); (b) bis(pinacolato)diboron (2 eq),
KOAc (4 eq), Pd(dppf)Cl2 (0.1 eq), dioxane, 85oC, 18h, >95%; (c) Cs2CO3 (3 eq), Pd(PPh3)4 (0.1 eq), dioxane, 85oC;
(d) NaHCO3 (3 eq), Pd(dppf)Cl2 (0.1 eq), dioxane, 90oC, 42 - >95%; (e) NaHCO3 (3 eq), Pd(dppf)Cl2 (0.1 eq), dioxane,
85 oC, 16h, 14 - 63%; (f) NaOMe, MeOH, r.t., 3 - 81%
Scheme 2. Reagents: (a) NaOMe, MeOH, 16h; (b) MOMCl, i-Pr2NEt, t-Bu4NI, CH2Cl2, 18h, 50% (2 steps) ; (c)
bis(pinacolato)diboron (2 eq), KOAc (4 eq), Pd(dppf)Cl2 (0.1 eq), DMF, 80oC, 12h, 92%; (d) 16, K3PO4 (3 eq),
Pd(PPh3)4 (0.05 eq), dioxane, 85 oC, 3h, 58%; (e) 3N HCl, r.t., 16h, 59%

Scheme 3. Reagents: (a) MeI or EtI or n-BuBr or i-BuBr or isobutylene oxide, Cs2CO3, DMF, r.t., 16h, 52 - 82%;
(b) bis(pinacolato)diboron (1.2 eq), KOAc (2 eq), Pd(dppf)Cl2 (0.05 eq), DMF, 80oC, 6h, 27 - 97%; (c) PhB(OH)2,
CuOAc, Et3N, pyridine, 45oC, 16h, 50%; (d) n-BuBr, Cs2CO3, DMF, 67%; (e) Et3N, NMP, 70 oC, 6h, 21%
Physicochemical properties and profile of thiomannosides are shown in Table 1. Different alkyl and
aryl substitution on pyridone and pyridine rings (5a-j) allowed variation of LogD in the range of 0.3 to
2.3. Being mannnosides, as expected all the compounds had high PSAs and are distributed over a range
of 131 – 156. It is possible that high PSAs of these compounds may become the cause of low PAMPA
permeability’s. Most of compounds possess moderate – good aqueous solubility (142 - 863 g/ml) in
JP2 buffer. All the compounds were found to show high metabolic stability in mouse liver microsomes
(MLM) (>80% stable for 30 min). The FimH inhibitory potential of these compounds was measured in
terms of their potential to inhibit hemaglutination using compound 1 as a standard positive control

(EC90; 1.25 M).5 These compounds exhibited hemaglutination inhibition (HAI) titer values between 0.3
– 2.5 g/mL, the most potent compound being 5h. Similar potencies for these compounds are not
surprising as these compounds structurally vary only at the terminal end of the molecule which does not
directly fit into the binding pocket of FimH.
Compounds 5c-g when dosed orally (10 mg/kg) to mice, exhibited low bioavailability (< 20 %).
These compounds were however excreted in urine to a considerable extent (5.3 - 24 %). Interestingly
compound 5h, a pyridine derivative obtained by O-alkylation of tautomeric form of pyridone, exhibited
high oral bioavailability in mice (86%). This improvement was understandable as this molecule also
exhibited moderate passive permeability. Following oral dosing, compound 5h also showed a simulated
concentration in urine above its HAI titer for up to 24 h (Fig. 3). S- and N- substituted pyridyl
derivatives 5i and 5j were similarly evaluated. The bioavailability of these compounds remained low (23
and 14% respectively), however both compounds showed good excretion in urine (11.7 and 7 %
respectively).
Table 1 Physicochemical properties and profile of thiomannosides
Urine
Excretion
(%)
HAI titer¥ BA§
(EC90 µM) (%)
Solubility
(g/mL)*
LogD
(pH 7.4)
Compound
PSA
PAMPA$ MLM#
0.3
0.7
1.7
1.5
0.5
1.2
1.8
2.1
2.3
2.1
136
136
136
136
156
136
136
138
153
131
719
Low
Low
72
100
95
1.25
2.5
ND
ND
3
ND
ND
7.0
8.1
5.3
24
5a
5b
5c
5d
5e
5f
668
740
Low
0.6
863
Low
95
2.5
1
729
Low
97
1.25
1.25
0.6
0
142
Low
93
15
1
582
Low
60
ND
2.8
11.7
7.0
5g
5h
5i
663
Moderate
Low
100
61
0.31
0.6
86
23
14
150
217
ND
91
1.25
5j
* JP2: pH 6.8 buffer; $PAMPA (X10-6): 0 - 12 Low, 12 - 30 Moderate, >30 High. # % remaining after 30 min; ¥E.
coli strain UTI89; §PO: 10 mg/kg in PEG 400:(Milli-Q or 5% w/v glucose) (3:97), pH 5.15 - 6.15; IV: 2 mg/kg in
PEG 400: (Milli-Q or 5% w/v glucose) (3:97), pH 5.68 – 5.96; ND Not Determined.

Fig. 3 Simulated urine PK profile of 5h in mice (PO)
Molecular docking of compound 5h was performed in E. coli FimH crystal structure (PDB ID:
4AVJ).10 This is quite evident from the docking studies that the terminal rings of these molecules lie in
the solvent exposed region of FimH binding pocket (Fig. 4). Compound 5h binds to FimH pocket with
four H-bonds in the mannose binding region (anchoring point). Methyl group makes van der Waals
interaction with Ile13. S linker appears to push the molecule further outside and pyridine ring makes a 
stacking interaction with Tyr48.

Fig. 4 Predicted Binding Mode of 5h in FimH
And compound 5h was evaluated in vitro for its ability to inhibit the formation of biofilm and to
disrupt the already formed biofilm. Compound 5h inhibited biofilm formation with an IC50 of 0.3 g/mL
in 96 well plate assays. Further, this compound also showed the potential to disrupt the preformed
biofilm with an IC50 of 0.56 g/mL in 96 well plate assays.
Compound 5h showed only slight inhibition of hERG channels (10.8%) at 100 M suggesting that
this compound has low risk of QT prolongation. Further, no MBI liability was observed with this
compound.
Given the improved in vivo PK profile of 5h, we investigated in vivo efficacy of this compound in
24h prophylactic UTI model (E. Coli UTI89) (Fig. 5). Hence, 5h was administered prophylactically (1,
10 & 100 mg/kg doses) half an hour before infection and the bladder CFU counts were determined at 2
and 24 h. Trimethoprim-sulfamethoxazole (TMP-SMX) combination is a standard of care for treating
recurrent UTI and was hence was used as reference standard in this study. While the vehicle treated
animals showed bacterial growth from 2 to 24 h, mice treated with 10 and 100 mg/kg of 5h showed
inhibition of bacterial growth compared to the 24 h vehicle control. The similar efficacy was seen with
TMP-SMX. This data correlates well with urine PK profile. Compound 5h thus has properties suitable
for oral drug development.
8
7
6
5
4
3
2
ns
**
***
**
2 h
24 h
1
10
100
#TMP-SMX
7/35 mg/kg
Vehicle
Compound 5h (mg/kg)
#Human equivalent dose
ns p > 0.05, ** p ≤ 0.01, *** p ≤ 0.001
CFU: Colony forming units
Statistical analysis: Unpaired T-test with Welch correction

Fig. 5 Prophylactic Effect of 5h in 24 h UTI Model in Mice (E. coli UTI89)
Hultgren and Janetka et al.11 made an observation that biphenyl groups substituted with electron
donating groups at para position are likely to be less potent. Interestingly, we have discovered that para
substitution with an electron donating group, such as compound 5h, not only have acceptable potency
when the linker is S-glycosidic bond, but also brings in good PK profile.
In conclusion, we present here a FimH inhibitor exhibiting good physicochemical properties and PK
profile in mouse which is suitable for further development. Compound 5h showed the potential to inhibit
biofilm formation and also to disrupt the preformed biofilm. And compound 5h showed prophylactic
effect in UTI model in mice. Presently, detailed evaluation of 5h and other derivatives is ongoing for
clinical development.
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1
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Synthesis and evaluation of thiomannosides, potent
and orally active FimH inhibitor
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Jitendra A. Sattigeri, Malvika Garg, Pragya Bhateja, Ajay Soni, Abdul Rehman Abdul Rauf, Mahendrakumar
Gupta, Mahesh S. Deshmukh, Tarun Jain, Nidhi Alekar, Tarani Kanta Barman, Paras Jha, Tridib Chaira, Ramesh
B. Bambal, Dilip J. Upadhyay, and Takahide Nishi

The highlights of our manuscript “Synthesis and evaluation of thiomannosides, potent and orally
active FimH inhibitor” are below.
 SARs of thiomannoside derivatives as FimH inhibitor
 FimH inhibitory activity and potential to inhibit biofilm formation
 Prophylactic effect in urinary tract infection model in mice