Bioorganic & Medicinal Chemistry Letters 24 (2014) 630–635

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Structure–activity relationship studies and biological characterization of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase inhibitors Damien Y. Duveau, Adam Yasgar, Yuhong Wang, Xin Hu, Jennifer Kouznetsova, Kyle R. Brimacombe, Ajit Jadhav, Anton Simeonov, Craig J. Thomas, David J. Maloney ⇑ National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, United States

a r t i c l e

i n f o

Article history: Received 19 November 2013 Accepted 27 November 2013 Available online 4 December 2013 Keywords: HPGD 15-PGDH Prostaglandin PGE2 Inhibitor

a b s t r a c t The structure–activity relationship (SAR) study of two chemotypes identified as inhibitors of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD, 15-PGDH) was conducted. Top compounds from both series displayed potent inhibition (IC50 28 lM in phosphate buffered saline (PBS) at pH 7.4). Results from the PAMPA assay, which measures passive (artificial) membrane permeability, showed that halogen substituents seem to improve permeability (e.g., compounds 4a and 4b). On the other hand, substitution with a nitrile moiety led to a 10-fold drop in permeability (4i). Unfortunately, ML149 and its analogues degraded quickly in the presence of rat liver microsomes, as evidenced by their short half-life (47 lM), except

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D. Y. Duveau et al. / Bioorg. Med. Chem. Lett. 24 (2014) 630–635

a O

NO2

HO

b, c

N

HO

d

N

NH

9

NO2

HO

O

O

O

10

R1

N

N R2

N

ML148, 11a-n

F

8

O

e

O

O NO2

N

f

NO2

N

F

12

13a-n

NH R3

b, c

N

N

N

ML148, 14a-n R 3

Scheme 4. Reagents and conditions: (a) o-toluidine, NMP, 60 °C; (b) hydrazine, Ra-Ni, MeOH, 23 °C; (EtO)3CH, TsOH, THF, MW, 120 °C; (d) R1R2NH or R3NH2, HATU, DIPEA, DMF, 23 °C; (e) (COCl)2, DMF, DCM, 23 °C, then piperidine–HCl, DCM, 23 °C; (f) R3NH2, DIPEA, NMP, 120 °C.

Table 2 SAR of ML148 and related analogues IC50a (nM)

R1

Compound

Compound

IC50a (nM)

R2

O

O N

R1

N

N

N 14a-n

ML148, 11a-n N

ML148

N

11a

N R2

19

14a

Phenyl

22

22

14b

2-OMe-phenyl

27

271

14c

3-Cl-phenyl

12

48

14d

3-OMe-phenyl

19

68

14e

3-CF3-phenyl

48

68

14f

3-iPr-phenyl

68

76

14g

3-tBu-phenyl

171

1524

14h

4-Me-phenyl

19

383

14i

4-Cl-phenyl

22

383

14j

4-OMe-phenyl

19

429

14k

4-CF -phenyl

86

N 11b

N

11c

N

11d

F 3C

N

11e

N 11f

Br

11g

F3C

N N

11h

N

11i

11j

Ph

3

11k

N H

961

14l

4-tBu-phenyl

54

11l

NH

1358

14m

tBu

34

2710

14n

nBu

68

11m 11n a

N

N O

N N

3040

Determined using the HTS enzymatic assay and tested in triplicate.

for compound 11d (5.6 lM). PAMPA permeability of ML148 and related analogues was similar to the ML149 series. As with ML149, the rat liver microsomal stability remained undesirable, except for compound 14j (T1/2 = 23 min). Selectivity toward HPGD: In order to evaluate the selectivity of the prepared compounds toward HPGD, analogues of ML148 and of ML149 were tested for their inhibitory activities versus a panel

of five other dehydrogenases (Supplemental Table 1).18 Encouragingly, none of the compounds tested showed any activity against GAPDH, LDHA, LDHB and HSD17b4. Some inhibition was observed against ALDH1A1, though all compounds displayed considerable selectivity toward HPGD (greater than 28-fold selectivity). Analogues 4b (142-fold selectivity), 14c (160-fold selectivity), and 14e (142-fold selectivity) were the most selective HPGD inhibitors.

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Table 3 In vitro ADME properties of selected analogues of ML149 and ML148

a b c d

Compound

IC50a (nM)

ML149 4a 4b 4f 4h 4i ML148 11d 14c 14e 14j

112 22 34 48 34 76 19 68 12 48 19

Represents Represents Represents Represents

the the the the

Permeabilityb (10

6

cm/s)

599 1646 1100 1512 1330 229 1196 2265 895 1346 624

Kinetic solubilityc (lM)

RLM T1/2d (min)

>45 32 37 33 28 >49 >47 5.6 >50 >55 >50

Nd 1.9 2.0 1.9 1.8 8.1 7.3 1.8 6.4 4.4 23

IC50 against HPGD in the HTS enzymatic assay. PAMPA permeability at pH 7.4. kinetic aqueous solubility in PBS buffer (pH 7.4) as measured by UV quantification. T1/2 in rat liver microsomes (RLM) in the presence of NADPH.

Table 4 PGE2 produced in A549 and LNCaP cells Compound

ML149 4a 4b 4f 4h 4i ML148 11d 14c 14e 14j

A549 cells

LNCaP cells

IC50a (nM)

AC50b (lM)

% Inductionc

AC50b (lM)

% Inductionc

54 22 34 48 34 76 19 68 12 48 19

0.743 0.296 0.590 2.09 0.166 2.64 0.469 0.743 0.372 2.64 0.662

106 110 114 145 107 350* 129 127 170 173 174

2.80 0.352 0.558 2.22 0.703 1.77 0.884 4.43 0.222 2.80 0.884

73 105 68 44 71 63 62 53 85 70 69

a

Represents the IC50 against HPGD in the HTS enzymatic assay. Represents the half-maximal concentration in the PGE2 production assay. c Represents the percentage induction of PGE2 production. The titration curve was incomplete.

b

*

PGE2 production: A selection of top actives around the ML148 and ML149 chemotypes were tested for their ability to increase PGE2 levels. Lung cancer A549 cells and androgen-sensitive human prostate adenocarcinoma LNCaP cells were titrated with the selected HPGD inhibitors; the half-maximal activation concentration (AC50) and percentage induction (% induction) of PGE2 production are reported in Table 4. All the compounds tested increased PGE2 production levels when compared to a DMSO control, in both A549 cells and LNCaP cells. The percent induction was consistently higher in A549 cells (from 106% for ML149 to 174% for 14j) than in LNCaP cells (from 44% for 4f to 105% for 4a). The majority of the compounds tested displayed sub-micromolar activity in both cell lines, and a good correlation in AC50 was observed between the two cell lines: low AC50 values measured in A549 cells correlated with low AC50 values in LNCaP cells, with the exception of ML149 [AC50 = 0.743 lM (A549) and AC50 = 2.80 lM (LNCaP)] and of 11d [AC50 = 0.743 lM (A549) and AC50 = 4.43 lM (LNCaP)]. Unfortunately, no tractable SAR emerged from the PGE2 production assay, a likely consequence of the often diverging enzymatic potency and cell permeability properties of the inhibitors. Indeed, compound 4i displayed particularly low cell permeability in the PAMPA assay (229  10 6 cm/s), probably accounting for the low activity measured in the A549 (AC50 = 2.64 lM) and LNCaP (AC50 = 1.77 lM) cell lines. It is also possible that enzyme occupancy of different agents and the kinetics of the sequential enzymatic events adds a level of complexity that may account for these differences. Overall, the

data obtained in the PGE2 production assay showed that treatment with our inhibitors leads to increased PGE2 production in A549 lung cancer and in LNCaP prostate cancer cells, consistent with inhibition of HPGD in the cell. Conclusions: The structure–activity relationship (SAR) study of two small molecule inhibitors of the human HPGD, ML148 and ML149, was carried out. These studies revealed that while the fused seven-membered ring and the presence of a 2,5-dimethylpyrrole were required for activity of ML149, a certain tolerance in the substitution pattern of the phenyl ring was observed. As a result compound 4h was found to have improved potency toward HPGD, improved cell permeability and induced PGE2 production both in A549 and in LNCaP cells, when compared to ML149. For the benzimidazole chemotype (ML148), the cyclohexylamide group was found to be optimal for enzymatic activity, although small substituents at the 3-position of the cyclohexylamine group were tolerated. Moreover, substitution at the meta-position and para-position of the phenyl ring was also well-tolerated, leading to analogs 14c and 14j. Both compounds displayed similar ADME properties, although 14j was more stable in the RLM assay, and both analogues had comparable effects on PGE2 levels in A549 and in LNCaP cells. Unfortunately, none of the HPGD inhibitors tested led to any observable down regulation of NF-jB, as measured in our assay (Supplemental Fig. 1), perhaps due to compensatory mechanisms counteracting the effect of modulation of HPGD activity on NF-jB regulation. In conclusion, we have identified potent and selective inhibitors of HPGD (e.g., 4h and

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14j) and hope these compounds help better define the role of HPGD in a variety of diseases. As such, we make these compounds freely available to the research community upon request. Acknowledgements We thank Jim Bougie, Thomas Daniel and William Leister for compound purification, as well as Danielle van Leer, Misha Itkin, Crystal Mcknight, Christopher LeClair and Paul Shinn for assistance with compound management. We also thank Udo Oppermann at the Structural Genomics Consortium (SGC), Oxford, UK for providing the ALDH1A1 and HSD17b4 enzymes. This research was supported by the Molecular Libraries Initiative of the National Institutes of Health Roadmap for Medical Research Grant U54MH084681 and the Intramural Research Program of the National Center for Advancing Translational Sciences at the National Institutes of Health. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 11.081. References and notes 1. Tai, H.-H.; Cho, H.; Tong, M.; Ding, Y. Curr. Pharm. Des. 2006, 12, 955.

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