Bioorganic & Medicinal Chemistry Letters 24 (2014) 3142–3145

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Discovery of novel 2-[2-(3-hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide derivatives as HIF prolyl 4-hydroxylase inhibitors; SAR,synthesis and modeling evaluation

http://dx.doi.org/10.1016/j.bmcl.2014.05.0030960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +82 31 628 2801; fax: +82 31 628 2701 (S.R.);tel.: +82 31 290 7711; fax: +82 31 292 8800 (Y.H.J.).

E-mail addresses: sgro@cgxinc.com (S. Ro), yhjung@skku.edu (Y.H. Jung).

Yong Rae Hong a,b, Hyun Tae Kim a, Seonggu Ro a,⇑, Joong Myung Cho a, Sang Hwi Lee b, In Su Kim b,Young Hoon Jung b,⇑a CrystalGenomics, Inc., 5F, Tower A, Korea Bio Park 694-1, Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Koreab School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 November 2013Revised 22 April 2014Accepted 2 May 2014Available online 15 May 2014

Keywords:HIF-PHs (PHDs)StabilizerInhibitorEPO

The design, synthesis, and capacity to inhibit HIF prolyl 4-hydroxylases (PHDs) are described for 2-[2-(3-hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide analogs. These analogs revealed two kinds of novel scaf-folds as PHD2 inhibitors. Synthetic routes were developed for the preparation of their analogs containingthe new scaffolds. In addition, the structure–activity relationship (SAR) of the 2-[2-(3-hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide derivatives and their biological activities were reported. The complex struc-ture of compound 18 with PHD2 was also obtained for the purpose of more efficient lead optimization.

� 2014 Elsevier Ltd. All rights reserved.

Hypoxia-inducible factor (HIF) is a transcription factor and keyregulator for the body’s systemic and local responses to oxygendeprivation (hypoxia). It was identified in 1992 by Wang and Sem-enza and found to bind to the hypoxia-responsive element (HRE) inthe enhancer region of the erythropoietin gene. Through transcrip-tional activation, HIF regulates various physiological processessuch as angiogenesis, erythropoiesis, energy utilization, vasculartone, apoptosis, and cellular proliferation.1,2

HIF itself is primarily regulated by prolyl hydroxylases (PHD1, 2,3) as well as asparginyl hydroxylase (FIH). The HIF transcriptionfactor consists of an HIF-a/b heterodimer that binds to hypoxia-response elements within the HIF target genes to controltranscription.3

HIF-a is a constitutively produced protein whose stability is pri-marily regulated by cellular pO2. Under normoxic conditions, thePHDs specifically hydroxylate converse proline residues (Pro402and Pro564 on human HIF-1a, Pro405 and Pro531 on humanHIF-2a), creating recognition sites for the von Hippel–Lindau pro-tein (VHL) for the purpose of proteasomal degradation. Thus, underadequate oxygen levels, the body continually expresses anddegrades HIF-a.4

Proline hydroxylation of HIF-a is catalyzed by PHDs in a reac-tion involving Fe(II), 2-Oxoglutarate (2-OG) and O2. As pO2 falls,the prolyl group of HIF-a is poorly hydroxylated and protein deg-radation is slowed; consequently, HIF-a mediated transcriptionoccurs. Therefore, stabilization of HIF-a by inhibition of PHDscould be a potential therapy for anemia, neuro-protection, andischemic diseases including stroke.5–8 For this reason, manyresearch programs are at various stages in the investigation ofsmall molecule inhibitors of PHDs, as listed in Table 1.9,10

One of the pioneers in commercial research on prolyl hydroxy-lase inhibition is Fibrogen, which is running some of the mostadvanced stage clinical trials for the therapeutic use of the HIF sta-bilizers FG-2216 and FG-4592. The second generation FG-4592 iscurrently enrolling for Phase IIa and IIb trials in the USA.11–13

GlaxoSmithKline has been testing its investigational HIF prolylhydroxylase inhibitor GSK1278863 for the treatment of anemiasince 2008. It has completed several Phase I studies in the USAand is currently running Phase II studies in several countries.14–16

GSK360A has also entered the pre-clinical development stage.17

Akebia is also currently recruiting CKD (Chronic Kidney Disease)patients for Phase II trials on AKB-6548 administered once daily totest changes in Hb levels.18

In the present paper, we present novel scaffolds for PHD inhib-itors that differ from PN-3602. The initial hits were designed basedon the binding moiety of 2-OG with PHD2 as a substrate mimic.

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Table 1Development status of the known PHDs inhibitors

CompdNo.

Name Company Clinicalstage

Chemicalstructure

1 DFO — Prototype Disclosed2 NOG — Prototype Disclosed3 FG-0041 Fibrogen Prototype Disclosed4 FG-2216 Fibrogen Phase II Disclosed5 FG-4592 Fibrogen Phase II Undisclosed6 AKB-6548 Akebia Phase II Undisclosed7 PN-3602 Palkyon

(CrystalGenomics)Pre-clinical

Undisclosed

8 GSK360A GSK Pre-clinical

Disclosed

9 GSK1278863 GSK Phase II Undisclosed10 BAY85-3934 Bayer Phase I Undisclosed

Table 2Inhibitory activities of designed compounds to increase additional interactionsagainst the PHD2 enzyme

Compounds Structure IC50(lM)

Stdev.

FG-2216N

OH

Cl

NH

O

OH

O3.9 0.1

11N N

HNO

OHO

OH

27.5 0.7

12

N

OH

S

NOH

O195.0 7.1

13

N

OH

S

N

HN

O

81.0 1.4

14N N

HNO

OHO

OH

NC 13.5 2.1

15

N

OH

S

NOH

ONC6.0 0.7

16

N

OH

S

N

HN

ONC5.4 0.6

Y. R. Hong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3142–3145 3143

Our studies were initiated by overlapping the designed hits to the2-OG complex structure [PDB: 3OUJ], as shown in Figure 1.

In overlapping studies, the designed initial hits of compound 11and compound 12 in Figure 1 had similar fits to the active site asseen with 2-OG, but enzymatic activities of these compounds forPHD2 were no better than those of FG-2216 of Fibrogen (Table 2).The binding showed an additional interaction point where charge-charge interactions could occur with Y329 and R383, similar to2-OG. A lead generation study based on the hits was designed toprepare new analogs of compounds 14–16 by the introduction ofa CN (nitrile) group in the 5-position of the pyridine ring forthe interaction with Y329 and R383. After these compounds weresynthesized, we tested these predictions using the fluorescencepolarization method to determine the capacity of the compoundsto inhibit PHD2.19 The structure activity relationship (SAR) fromhit to lead is highlighted in Table 2. The introduction of CN to inter-act with Y329 and R383 substantially increased the inhibition ofPHD2 activity. Compound 16 showed similar activity to FG-2216due to these additional interactions, as might be expected.

EPO expression due to PHD2 inhibition was determined inHep3B cells using the EPO ELISA method20 EPO expression resultsfor each compound is presented in Figure 2.

The initial structure–activity relationship (from Table 2 andFig. 2) of the synthesized analogs revealed that compounds 11and 16 at 50 lM caused the greatest EPO secretion; however, com-pound 11 is an undesirable candidate since it showed significantly

Fe2+

Y329

R383

Y303

R322

R252D254

H2O

Figure 1. Overlap of the stick form of compound 11 (yellow), compound 12 (blue),and 2-OG (cyan) showing hydrogen bonding interactions with amino acids in theactive site of PHD2. [PDB: 3OUJ] Black dotted lines indicate interactions between 2-OG and PHD2 in complex with 2-OG. Red dotted lines indicate expected interac-tions between each compound and PHD2. Pink circle shows that these charge-charge interactions of 2-OG are absent in compounds 11 and 12. PDB code 3OUJ.

decreased EPO secretion at 100 lM. An SAR exploration of thePHD2 inhibitor for novel two classes was enabled by the successfuldevelopment of optimal synthetic routes. Compound 11 analogswere prepared for various ‘R1’ according to Scheme 1.

Compound 16 analogs for various ‘R2 and R3’ were also pro-duced by the synthetic route shown in Scheme 2.

With the preliminary SAR established, our strategy was tomaintain compound 16 as our core scaffold to expand its deriva-tives and examine the substitution effects on the amide side chainand aromatic rings. As shown in Table 3, replacement of the hydro-gen with a methyl at the R4 position and introduction of abranched acetamide chain at the R6 position increased the inhibi-tory activity, whereas R5 substitutions had no influence on theactivity. Compound 33 showed the best activity of these analogs,with an IC50 of 0.4 lM.

EPO secretion following PHD2 inhibition by the new analogswas determined in Hep3B cells using the EPO ELISA method. EPOsecretion results for each compound are presented in Figure 3.

The secondary SAR (Table 3 and Fig. 3) of the newly synthesizedanalogs revealed that compounds of 21 and 22 generated superiorEPO secretion than did FG-2216 at 50 lM. However, neither com-pound showed a continuous increase in EPO secretion until theywere added at a high concentration, and EPO secretion somewhatdecreased at 100 lM. Although the reason for this is still not clear,EPO secretion seen at 50 lM may be sufficient to cure a disease likeanemia because the blood concentration of the inhibitor would notreach 100 lM in the real body system. Otherwise, some derivatives

EPO Immunoassay

0

5

10

15

20

25

30

35

40

45

50

DMSO FG-2216 11 12 13 14 15 16

Compounds

EP

O F

old

50uM

100uM

Figure 2. EPO secretion in Hep3B cells due to PHD2 inhibition 24 h after treatmentwith each compound.

a

HN

P1 P2

b

c

d

N

FR1

CN N

R1

N

OHR1

NH

NH2

OOH

NO

NH2

OEtEtOO

EtO

O

N

OH

R1N

NOH

O

OEt N

OH

R1N

NOH

O

OH

P3 P4

(R1=H or CN)

Scheme 1. Reagents and conditions of the synthetic route for compound 11analogs: (a) t-BuOK/DMF, rt, 62–71%; (b) 5% Pd-C/MeOH, under H2 gas, rt, 92–98%;(c) NaOEt, NaBH4, NiCl2/ EtOH, 70 �C, 20–33%; (d) LiOH/H2O, MeOH, THF, 70 �C, 52–63%.

aO

P

P5 P6

P7 P8

P9 P10

bc

d

e

f

N

NO2Br

CN N

OBr

CN

SHS

OEtOEt

N

OHBr

NH2S

OOCl

N

OHBr

SN

OO

N

OHNC

S

NO

O

N

OHNC

S

NOH

O

HNR2

R3N

OHNC

SN

NO R3

R2

(R2, R3= H or Alkyl)

Scheme 2. Reagents and conditions of the synthetic route for compound 16analogs: (a) NaOMe/MeOH, reflux, 76–82%; (b) H2O, 90 �C, 35–48%; (c) LiI/n-BuOH,140 �C, 55–64%; (d) Zn(CN)2, Pd(PPh3)4/DMF, microwave (160 �C, 150 W, 25 min.),63–69%; (e) NaOH/THF, EtOH, H2O, rt, 87–93%; (f) TBTU, Et3N/DMF, rt, 68–76%.

Table 3Inhibitory activities of compound 16 analogs against the PHD2 enzyme

N

OH

S

NR6

ONC

R4

R5

Analogs R4 R5 R6 IC50 (lM) Stdev.

16 H H NHCH2Ph 3.9 0.617 H H NHCH2COOH 2.2 0.0

18 H H HNO

OH 7.3 0.4

19 H H

HNO

OH2.8 0.0

20 H H

HNO

OH2.6 0.4

21 H HHN

OH

2.1 0.0

22 H HHN

OH

2.4 0.2

23 H H NHCH2CH2Ph 3.4 0.3

24 H H

HN

N3.0 0.0

25 H CH3

HN

N1.2 0.0

26 H Ph

HN

N24.0 1.4

27 CH3 H

HN

N0.7 0.1

28 CH3 CH3

HN

N0.8 0.1

29 CH3 Ph

HN

N5.7 0.2

30 CH3 CH3 NHCH2CH3 1.4 0.1

31 H HHN

2.0 0.1

32 CH3 H

HNN

3.8 0.3

33 CH3 CH3

HNN

0.4 0.1

3144 Y. R. Hong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3142–3145

(compounds 23, 24, 25, 27, 28 and 29) that R6 position is replacedwith phenethyl amine or pyridine ethylene amine showed similarEPO secretion than did FG-2216 at 50 lM and showed a continu-ous increased EPO secretion until high concentration treatment.But they did not show superior EPO secretion than did FG-2216at 100 lM. Also, according to PHD2 inhibitory activities from com-pound 24 to compound 29 in Table 3, we know that methylshowed better the inhibitory activity than hydrogen in R5 positionand bulky group like phenyl decreased the inhibitory activity in R4

position. In case of compound 33 which showed highest inhibitoryactivity for PHD2, it did not show increased EPO secretion untilhigh concentration treatment. This disagreement between enzy-matic and cellular activities can be explained by its physicochem-ical properties. In Table 4, the permeability of compound 33through PAMPA assay is very low therefore compound 33 seems

hard to transmit a cell wall. Low EPO secretions of compound 17,19, 20 and 32 are also explained with the same reason.

We attempted to confirm the binding elements responsible forthe potent inhibition of the target protein with the analogs of theseries shown in Table 3 by obtaining a co-crystal structure ofPHD2 with compound 18 from this series. The co-crystal structureof compound 18 with PHD2 [PDB: 4KBZ] is presented in Figure 4and is overlapped with FG-2216 in [PDB: 2HBT] to determine any

EPO Immunoassay

0

5

10

15

20

25

30

35

40

45

50D

MSO

FG-2

216 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Compounds

EP

O F

old

50µM

100µM

Figure 3. EPO secretion by PHD2 inhibition after 24 h from each compoundtreatment in Hep3B cells.

Table 4Physicochemical properties for the compounds of Table 3

Analogs Solubility (lg/mL at pH7.4) Permeability in PAMPA (�logPe)

16 24.1 Medium (5.6)17 105.8 Low (10.0)18 28.9 Medium (5.7)19 >195 Low (10.0)20 >195 Low (10.0)21 >188 Medium (5.1)22 >188 Medium (5.0)23 13.6 Medium (5.4)24 >181 Medium (5.8)25 78.4 Medium (5.8)26 117 High (4.3)27 11.2 Low (10.0)28 6.4 High (3.0)29 0.74 High (3.0)30 13.9 High (4.5)31 107.5 Medium (5.7)32 >185 Low (10.0)33 85.9 Low (10.0)

Fe2+

Y329

R383

Y303

R322H374

H313

D315

Figure 4. Superimposition of FG-2216 (magenta) in [PDB: 2HBT] over PHD2 (cyan)in complex with compound 20 (grey) [PDB: 4KBZ].

Y. R. Hong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3142–3145 3145

similarities or differences in the interactions with the PHD2protein.

Compound 20 binds to the Fe(II) by bidentate coordinationthrough nitrogen of its hydroxyl pyridine ring and nitrogen ofthe thiazole ring. This mode of binding enables the formation ofa hydrogen bond between the hydroxyl moiety of compound 20and the conserved Y303. The charge–charge interaction betweenY329 and R383 and the CN group of compound 20, which is similarto the interaction shown by the carboxylate of FG-2216, is impor-tant for increasing the inhibitory action. This novel interactioncould explain the improved potency of the 2-[2-(3-hydroxy-pyri-din-2-yl)-thiazol-4-yl]-acetamide derivatives relative to the com-pounds lacking a CN group at the 5-position of the pyridine ring.

In summary, we established the structure–activity relationshipof the 2-[2-(3-hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide ana-logs, which were designed based on the 2-OG of the PHD substrate.The use of these analogs revealed two kinds of novel scaffolds asPHD2 inhibitors and synthetic routes were also developed for thepreparation of analogs based on the new scaffolds. In addition,their biological activities were reported; some compounds showedsuperior inhibition of enzymatic activities and promoted greaterEPO secretion than did the FG-2216 lead compound of Fibrogen.The complex structure of compound 18 with PHD2 was obtainedto permit more efficient lead optimization. Further studies on thetherapeutic potential of selected analogs are in progress.

Acknowledgments

The authors would like to acknowledge the assistance of Dr.Dongkyu Shin for the help in preparation of co-crystal figures forthis work. We also wish to express our gratitude for the initial sup-port and efforts from CrystalGenomics in in vitro studies.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2014.05.003.

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Discovery of novel 2-[2-(3-hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide derivatives as HIF prolyl 4-hydroxylase inhibitors; SAR, synthesis and modeling evaluationAcknowledgmentsSupplementary dataReferences and notes