Stem Cell Mobilizers Targeting Chemokine Receptor CXCR4

Subscriber access provided by MAHIDOL UNIVERSITY (UniNet)

Article

Stem Cell Mobilizers Targeting Chemokine Receptor CXCR4 : Renoprotective Application in Acute Kidney Injury Chien-Huang Wu, Jen-Shin Song, Kuei-Hua Chang, Jiing-Jyh Jan, Chiung-Tong Chen, Ming-Chen Chou, Kai-Chia Yeh, Ying-Chieh Wong, Chen-Tso Tseng, Szu-Huei Wu, Ching-Fang Yeh, Chung-Yu Huang, Min-Hsien Wang, Amit A. Sadani, Chun-Ping Chang, Chia-Yi Cheng, Lun Kelvin Tsou, and Kak-Shan Shia J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 17, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 52

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Stem Cell Mobilizers Targeting Chemokine Receptor CXCR4 : Renoprotective Application in Acute Kidney Injury

Chien-Huang Wu,† Jen-Shin Song,† Kuei-Hua Chang, Jiing-Jyh Jan, Chiung-Tong Chen, Ming-Chen Chou, Kai-Chia Yeh, Ying-Chieh Wong, Chen-Tso Tseng, Szu-Huei Wu, Ching-Fang Yeh, Chung-Yu Huang, Min-Hsien Wang, Amit A. Sadani, Chun-Ping Chang, Chia-Yi Cheng, Lun K. Tsou, and Kak-Shan Shia*

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli County 35053, Taiwan, R.O.C.

1 ACS Paragon Plus Environment

Page 2 of 52

Page 3 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Abstract: We have discovered a novel series of quinazoline-based CXCR4 antagonists. Of these, compound 19 mobilized CXCR4+ cell types, including hematopoietic stem cells and endothelial progenitor cells, more efficiently than the marketed 1 (AMD3100) with subcutaneous administration at the same dose (6 mg/kg) in mice. This series of compounds thus provides a set of valuable tools to study diseases mediated by the CXCR4/SDF-1 axis, including myocardial infarction, ischemic stroke and cancer metastasis. More importantly, treatment with compound 19 significantly lowered levels of blood urea nitrogen and serum creatinine in rats with renal ischemia-reperfusion injury, providing evidence for its therapeutic potential in preventing ischemic acute kidney injury. CXCR4 antagonists such as 19 might also be useful to increase circulating levels of adult stem cells, thereby exerting beneficial effects on damaged and/or inflamed tissues in diseases that currently are not treated by standard approaches.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION Acute kidney injury (AKI) is the rapid loss of renal function taking place within 48 hours, typically characterized by oliguria, nephritis, and renal vein thrombosis due to the abrupt decline of glomerular filtration.1,2 Affecting 3~7% of patients, AKI is one of the most common complications in hospitals, especially in intensive care units, representing a huge financial burden of about $4.7 billion on the US healthcare system each year. In addition, the glomerular filtration dysfunction could trigger other severe complications, such as uremia, metabolic acidosis and hyperkalemia, which collectively contribute to the high mortality of AKI. Ischemia-reperfusion injury (IRI), intrinsic kidney damage and urinary tract obstruction are common causes for the pathogenesis of AKI. Although many patients could recover from AKI, some sustained persistent renal impairment over several months and resulted in renal failure, which may require kidney transplantation or lifelong dialysis as the last resort. To date, no effective chemical agents have been developed to prevent or alleviate AKI in the clinic. However, increasing evidence from AKI animal models have demonstrated that exogenous stem cell transplantation could significantly attenuate inflammation, promote angiogenesis, mitigate apoptosis, and thereby restore kidney function.3-8 These results implied that cell-based therapy could be a promising approach for AKI. As a member of the CXC chemokine family, stromal cell-derived factor-1 (SDF-1; CXCL12) is a sequence consisting of 67 amino acid


Page 4 of 52

Page 5 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


residues, highly expressed by bone marrow stromal cells and immobilized in the bone marrow. SDF-1 serves as a specific ligand to CXCR4, a G protein-coupled receptor with seven transmembrane domains on the surface of many stem cell types.9,10 The CXCR4/ SDF-1 axis has been well known to regulate cancer metastasis, stem cell homing, trafficking, and mobilization.11-16 Thus, CXCR4 antagonists that mobilize various stem cells, including hematopoietic stem cells (HSCs), endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs), out of the bone marrow into the peripheral blood, might potentially serve as therapeutic agents to treat AKI either via autologous transplantation or simply by raising stem cell levels in blood.17 Indeed, such cell-based therapy concept has been validated by a selective CXCR4 antagonist, a small molecule known as AMD3100 (Plerixafor, compound 1).18-21 Approved by the US FDA in December 2008, a regimen combining compound 1 with granulocyte colony-stimulating factor (G-CSF, Filgrastim) that synergistically augments stem cell mobilization has been used for autologous transplantation of CD34+ cells (HSCs) in patients suffering multiple myeloma or non-Hodgkin’s lymphoma.22-26 Such procedures allowed rapid restoration of immune system after undergoing destructive radio- or chemotherapy.22-26 Moreover, other findings revealed that compound 1 could effectively ameliorate the injured regions caused by myocardial infarction or ischemic stroke, suggesting that CXCR4 antagonists might have great therapeutic potential in anti-inflammation and/or tissue repair.27-29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Though many small molecules and peptidomimetics have been successfully developed to target CXCR4 receptors,30,31 only a fraction of them (Figure 1), including TG-0054 (Phase II),32 AMD070 (Phase I/II),33 MSX-122 (Phase I),34 BL8040 (T140) (Phase I/II),35 LY2510924 (Phase II),36 CTCE-9908 (Phase I/II)37 and POL6326 (Phase I/II),38 have entered clinical development. These clinical candidates mainly act as HIV entry inhibitors or stem cell mobilizers for autologous transplantation.

Figure 1. CXCR4 antagonists under clinical development In continuation of our studies on stem cell mobilizers targeting CXCR4 receptors,39 herein, we wish to disclose the design and synthesis of a novel series of quinazolinebased CXCR4 antagonists, leading to the identification of a highly potent and selective CXCR4 antagonist 19 with better in vivo efficacy than compound 1 in preventing ischemia-reperfusion-induced AKI. Detailed description is presented as follows.


Page 6 of 52

Page 7 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS AND DISCUSSION In continuation of our studies on stem cell mobilizers targeting CXCR4 receptors for potential cell therapy and regenerative medicine, through screening a diverse compound collection and subsequent medicinal chemistry efforts, we have developed generic structure I-based compounds (Chart 1) with good binding affinities (IC50 = 36~132 nM) towards CXCR4, particularly when the terminal R group is a four- to seven-membered hydrocarbon ring.39 Among this series of quinazoline analogues, compound 2 mobilized CD34+ and CD133+ stem cells in mice as efficiently as the marketed compound 1, and was thus considered a model compound for further structural modifications to generate more potent stem cell mobilizers.

Chart 1. Generic I and Structures of Compounds 1 and 2

Following a similar synthetic route previously reported by our laboratories,39 a class of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

novel derivatives 3-12 with various substituents on the phenyl ring of the quinazoline core was synthesized. Their binding affinities toward CXCR4 receptors are compiled in Table 1. According to the SAR analysis, compounds 3-7 (IC50 = 4.7~31.5 nM) containing an electron-donating group (e.g. OMe or Me) either at C-6 or C-7 position are 4- to 7-fold more potent than compound 2 (IC50 = 34.2 ± 3.1 nM). In contrast, compounds 10-12 (IC50 = 39.1~211.6 nM) modified with an electron-withdrawing group (e.g. CF3 or NO2) at the corresponding positions exhibited 3- to 6-fold lower binding affinities than 2. These findings suggest that higher electron density on the phenyl ring is favorable for the quinazoline-based compounds, presumably due to the enhancement of a weak but essential π-π hydrophobic interaction between the receptor and ligand. These stereoelectronic effects appeared more susceptible to the C-7 substituent as demonstrated by compounds 4 (IC50 = 6.8 ± 1.8 nM) and 6 (IC50 = 5.1 ± 1.3 nM) relative to those of compounds 10 (IC50 = 211.6 ± 24.0 nM) and 12 (IC50 = 111.3 ± 10.9 nM). Indeed, a dramatic decrease in binding affinities by 20- to 40-fold was observed when the electron-rich groups (i.e. OMe and Me) were replaced with electron-deficient ones (i.e. CF3 and NO2). Conversely, substitutions at the C-6 position of the quinazoline appeared to be more tolerated as evidenced by compounds 3 (IC50 = 8.7 ± 1.1 nM) and 5 (IC50 = 4.7 ± 1.3 nM) with an electron-donating group relative to compound 11 (IC50 = 39.1 ± 2.9 nM) with an electron-withdrawing group, wherein only a margin of


Page 8 of 52

Page 9 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Binding Affinity of Benzyl Polyamines against CXCR4 Receptors

Compd

R1

R2

IC50 [nM]a

2

H

H

34.2 ± 3.1

3

OMe

H

8.7 ± 1.1

4

H

OMe

6.8 ± 1.8

5

Me

H

4.7 ± 1.3

6

H

Me

5.1 ± 1.3

7

OMe

OMe

31.5 ± 3.0

8

Cl

H

18.1 ± 4.0

9

H

Cl

8.6 ± 1.7

10

H

CF3

211.6 ± 24.0

11

NO2

H

39.1 ± 2.9

12

H

NO2

111.3 ± 10.9

1

-

-

213.1 ± 26.2

a

Determined by 50% inhibition of radioligand [125I]SDF-1 binding to hCXCR4-transfected HEK293 membrane; values represent the mean ± SD of at least three independent experiments.

5- to 8-fold decrease in binding affinities was found. However, we observed that the binding affinities of compounds 8 (IC50 = 18.1 ± 4.0 nM) and 9 (IC50 = 8.6 ± 1.7 nM) with a weak electron-withdrawing group (i.e. Cl) were comparable to those of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

compounds 3 and 4 with a strong electron-donating group (i.e. OMe). Compound 11 (IC50 = 39.1 ± 2.9 nM) with a strong electron-withdrawing group (i.e. NO2) was comparable to compound 7 (IC50 = 31.5 ± 3.0 nM) with a strong electron-donating group (i.e. OMe) at the C-6 position. These results indicate that it is difficult to correlate the electron-donating or withdrawing properties with binding affinities between the R1 substituents. The quinazoline series shown above, along with many reported cases,40 again suggested that CXCR4-targeting antagonists appeared to have a preference for polyamine features. A plausible explanation could be that polyamine-containing compounds might provide essential nitrogen elements to mimic the highly positively charged SDF-1 enriched with Lysine and Arginine residues at the interaction interface.41 With more potent in vitro activities toward CXCR4 than 2, compounds 3-7 were selected for stem-cell mobilizing test in mice. To our surprise, these compounds were inferior to compounds 2 and 1 in the mobilization of CXCR4+ stem cells out of bone marrow and the treatment of AKI in rats. This is presumably due to rapid oxidative metabolism of these compounds in vivo as the electron density on the phenyl ring is enhanced. Though the above series of compounds failed to show in vivo efficacy in the AKI model, compound 2 remained to be a good starting point for further structural modifications in light of its comparable ability with compound 1 in mobilizing CXCR4+ stem cells.39 As such, a new synthetic strategy was then adopted by which the bisbenzyl moiety of 2 was


Page 10 of 52

Page 11 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


replaced with a triazole ring to generate a novel class of polyamines 13-25. A general synthetic procedure is illustrated in Scheme 1 using compound 13 as a typical example. Starting from 1,4-dichlroquinazoline, a chemoselective C4-substitution of 4-amino-1Boc-piperidine at room temperature was carried out, which was followed by a second C2-substitution of a protected linker 1 under microwave irradiation at 120 oC. Finally, acidic deprotection of all Boc groups afforded the desired compound 13 in three steps with an overall yield of 41%. Scheme 1. The Series of Quinoline Polyamines Containing a Triazole Ringa

a

Reagents and conditions: a) 4-amino-1-Boc-piperidine, TEA, CH2Cl2, 5 oC to rt, 16 h, 78%; b)

linker 1, 1-pentanol, microwave, 120 oC, 15 min, 63%; c) 1N HCl in diethylether, CH2Cl2, rt, 16 h, 92%.

Linker 1 can also be readily obtained using propargylamine as starting material as illustrated in Scheme 2. Propargylamine was first protected with benzyl chloroformate to form a Cbz-protected intermediate in a quantitative yield (95%), which in turn was coupled with 2-azido-ethanol to yield triazole alcohol (83%) by making use of the click chemistry as a key operation. Triazole alcohol thus obtained could undergo mesylation to afford mesylate (78%), which was subsequently coupled with ethane-1,2-diamine



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

followed by reductive amination (cyclohexanone) and protection (Boc2O) to afford a polyamine precursor in 50% yield over three steps. Finally, the Cbz protecting group was removed under hydrogenolysis to provide the desired linker 1 in 89% yield. With linker 1 in hand, compounds 13 (IC50 = 156.0 ± 19.8 nM), 14 (IC50 = 250.4 ± 65.1 nM) and 15 (IC50 = 185.3 ± 11.4 nM) in Table 2 were first synthesized and evaluated for their binding affinities toward CXCR4 receptor.

Scheme 2. Synthesizing Linker 1 Using Propargylamine as Starting Materiala

a

Reagents and conditions: (a) benzyl chloroformate, K2CO3, THF/H2O = 1/2, 5 oC to rt, 15 h,

95%; (b) 2-azido-ethanol, CuSO4, (+) sodium L-asorbate, K2CO3, EtOH/H2O = 4/1, rt, 15 h, 83%; (c) MsCl, TEA, CH2Cl2, 5 oC to rt, 15 h, 78%; (d) ethane-1,2-diamine, THF, 65 oC, 15 h, 82%; (e) cyclohexanone, MeOH, 60 oC, 6 h, 2. NaBH4, 5 oC, 1 h; (f) Boc2O, CH2Cl2, rt, 15 h, 61% over two steps; g) H2, Pd/C, MeOH, rt, 6 h, 89%.

As a result, binding affinities of compounds 13, 14 and 15 were dramatically reduced by 5- to 33-fold as compared to their respective counterparts 2, 4 and 7 in Table 1. To improve the binding affinity, we varied the linker length by adjusting the number of the methylene units, as indicated by the integers m and n, to optimize spatial orientations of


Page 12 of 52

Page 13 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Binding Affinity of Triazole Polyamines against CXCR4 Receptors

Compd

R1

R2

n

m

IC50 [nM]a

13

H

H

2

2

156.0 ± 19.8

14

H

OMe

2

2

250.4 ± 65.1

15

OMe

OMe

2

2

185.3 ± 11.4

16

H

H

2

3

39.7 ± 5.1

17

H

H

3

2

30.2 ± 6.4

18

H

OMe

3

2

35.9 ± 7.0

19

H

H

3

3

12.6 ± 0.9

20

H

OMe

3

3

31.4 ± 7.9

21

OMe

H

3

3

22.6 ± 1.2

22

OMe

OMe

3

3

18.1 ± 3.2

23

H

Cl

3

3

44.7 ± 11.9

24

H

CF3

3

3

56.3 ± 4.9

25

H

H

4

3

84.5 ± 10.6

1

-

-

-

-

245.1 ± 22.2

a

Determined by 50% inhibition of radioligand [125I]SDF-1 binding to hCXCR4-transfected HEK293 membrane; values represent the mean ± SD of at least three independent experiments



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 52

two essential secondary nitrogen elements. Encouragingly, compounds 16-18 (IC50 = 30~39 nM), containing a C2 linker with five methylene units (m + n = 5), exhibited a significant improvement of CXCR4 binding affinities compared to those of 13-15 (IC50 = 156~250 nM) with a C2 linker of four methylene units (m + n = 4), suggesting that the C2 polyamine linker could be flexible in length while the terminal cyclohexyl group was projected into a hydrophobic pocket on CXCR4. However, as m + n was increased to 6 or 7, compounds 19-25 possessed IC50 values ranging from 12.6 to 84.5 nM, indicating that a ceiling effect on the C2 linker was supposed to be m + n = 6. Considering its remarkable binding affinity, the specificity of compound 19 was evaluated against a panel of closely related chemokine G protein-coupled receptors, including CXCR2, CCR2, CCR4, and CCR5. As shown in Table 3, compound 19’s selectivity index was

Table 3. Specificity of Compound 19 against Several Related Chemokine Receptors

a

Parameter

CXCR4

CXCR2

CCR2

CCR4

CCR5

Inhibition [%]a

100

13

31

22

22

IC50 [nM]

12.6

>10000

>10000

>10000

>10000

Selectivity Index

–

>793

>793

>793

>793

Percent inhibition was determined at 10 µM; weak inhibition was observed for all tested

chemokine receptors except CXCR4.


Page 15 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


found to be more than 793-fold in all cases examined, demonstrating that compound 19 is a highly selective CXCR4 antagonist. The related functional study was then conducted to evaluate 19’s role in cellular migration of CXCR4+ cells. As seen in the SDF-1-induced chemotaxis assay (Figure 2), compound 19 blocked SDF-1-guided movement of CCRF-CEM cells, a human cell line with high endogenous expression of CXCR4, at a low concentration (IC50 = 108.9 ± 24.2 nM). This cellular functional study strongly supports that compound 19 is a highly potent and selective CXCR4 antagonist, thus prompting us to further investigate its in vivo efficacy on the stem cell mobilization and the AKI disease model. Meanwhile, toxicity analysis showed that the CC50 values of compounds 19 and 1 in the Detroit 551 cell line (human normal skin fibroblast cells) were more than 10 µM, and their maximum tolerated doses (MTD) were determined to be 6 mg kg-1 in C57BL/6 mice, respectively, following subcutaneous administration.

Figure 2. SDF-1α-induced chemotaxis assay was performed for compound 19. Data were collected from three independent experiments and represent the mean ± SD.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Leading compound 19 and positive control 1 were subcutaneously administered at a dose of 6 mg/kg in 8- to 10-week-old C57BL/6 male mice followed by peripheral blood collection and flow cytometry analysis. As illustrated in Figure 3A, compounds 19 and 1 significantly increased the amount of CXCR4+ cells in the circulating blood by 11.7- and 9.0-fold (p < 0.001) relative to the vehicle control, respectively. We are particularly interested in the CD34+ and CD133+ cell types, as they have widely been recognized as biomarkers of HSCs and EPCs, respectively.

Figure 3. Stem/progenitor cell mobilization. (A) CXCR4+ cells; (B) CXCR4+CD34+ cells; (C) c-Kit+Sca-1+Lin- cells; (D) CXCR4+CD133+ cells; (E) CXCR4+KDR+ cells. Cell levels were measured at a time point of 2 h after subcutaneous administration with compound 1 (6 mg/kg) or compound 19 (6 mg/kg) in C57BL/6 mice. Data represent the mean ± SEM (n = 5 per group). Statistical analysis was performed by t-test: *p < 0.05, **p < 0.01, and ***p < 0.001 between control and the indicated test group.


Page 16 of 52

Page 17 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Thus, cell populations from the double staining of CXCR4+CD34+ and CXCR4+CD133+ were harvested, and results were depicted in Figures 3B and 3D. Gratifyingly, compound 19 was more effective in mobilizing both CXCR4+CD34+ (8.3-fold vs 5.8fold) and CXCR4+CD133+ (4.0-fold vs 2.4-fold) cells than compound 1. In addition, cKit+Sca-1+Lin- and CXCR4+KDR+ were also reported as “specific” biomarkers for HSCs and EPCs, respectively.42,43 Therefore, we were set out to measure cell populations with these biomarkers. As shown in Figures 3C and 3E, treatment of compound 19 could increase the number of c-Kit+Sca-1+Lin- and CXCR4+KDR+ cells in the peripheral blood by 5.3-fold and 4.2-fold, respectively, as compared to the vehicle control. As a benchmark, compound 1 merely yielded a 4.0-fold and 2.9-fold increase in the

mobilization

of

c-Kit+Sca-1+Lin-

and

CXCR4+KDR+

cells,

respectively.

Interestingly, as shown in Figures 3B vs. 3C and 3D vs. 3E, the trend of increasing cKit+Sca-1+Lin- and CXCR4+KDR+ cells is found to be consistent with that of the corresponding CXCR4+CD34+ and CXCR4+CD133+ cells, indicating that they might represent cell types of high similarity. Notably, the above cell populations contributed to about 3% of mobilized CXCR4+ cells (Figure 3A). Thus, identification of the remaining CXCR4-expressing cell types and their physiological functions will be important for the advancement of cell-based therapy. The observed efficacy of compound 19 in stem cell mobilization also translates to the treatment of ischemic AKI in rats.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Effects of CXCR4 antagonists in ischemic AKI. (A) BUN and (B) Scr levels were measured at a time point of 24 h after subcutaneous administration with compound 1 (6 mg/kg) or compound 19 (6 mg/kg) in ischemic AKI rats induced by IRI. Data represent the mean ± SEM with eight to nine rats per group. Statistical analysis was performed by t-test: *p < 0.05, **p < 0.01, and ***p < 0.001 between control and the indicated test group.

As illustrated in Figures 4A and 4B, compound 19 successfully reduced the levels of blood urea nitrogen (BUN) and serum creatinine (Scr) by 70% and 79%, respectively, in rats suffering renal IRI. The greater reduction in BUN and Scr levels observed with 19 compared to compound 1 suggested that it might possess better therapeutic potential in preventing AKI.44 Overall, compound 19 exhibited better ability than 1 in mobilizing different CXCR4+ cells into the peripheral blood and improved renal function in the AKI model. As inflammation occurs earlier than tissue damage at injured sites with AKI progression, we cannot rule out the improvement of renal function as a result of potential antiinflammation properties of 19. Moreover, it is hard to justify that tissue repair could


Page 18 of 52

Page 19 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


occur so rapidly during such a short experimental time frame. 44,45

CONCLUSION In conclusion, blockade of CXCR4/SDF-1 axis mobilizes not only HSCs and EPCs, but also many other types of stem cells from the bone marrow into the peripheral blood. Currently, CXCR4 antagonists are mainly used in autologous stem cell transplantation but increasing evidence indicates that these compounds possess considerable therapeutic potential in many disease indications, such as tissue repair/regeneration, inflammation and oncology. Herein, we have discovered a novel series of quinazoline-based CXCR4 antagonists, in which the representative compound 19 could mobilize various CXCR4+ cells, including HSCs and EPCs, more efficiently than the marketed 1. Our SAR of the C2 polyamine linker in combination with the quinazoline-core should guide the design of many more structurally diverse CXCR4 antagonists. Detailed mechanism on the rapid improvement of renal IRI by CXCR4 antagonist 19 remained to be determined. Nevertheless, BUN and Scr data are significantly reduced in 24 h after ischemia/reperfusion injury, suggesting that blocking CXCR4 receptors may serve as a potential strategy to prevent ischemic AKI.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

EXPERIMENTAL SECTION General. Unless otherwise stated, all materials used were commercially available and used as supplied. Reactions requiring anhydrous conditions were performed in flame-dried glassware and cooled under an argon or nitrogen atmosphere. Unless otherwise stated, reactions were carried out under argon or nitrogen and monitored by analytical thin layer chromatography performed on glass-backed plates (5 × 10 cm) precoated with silica gel 60 F254 as supplied by Merck. Visualization of the resulting chromatograms was performed by looking under an ultraviolet lamp (λ = 254 nm) followed by dipping in an ethanol solution of vanillin (5% w/v) containing sulfuric acid (3% v/v) or phosphomolybdic acid (2.5% w/v) and charring with a heat gun. Solvents for reactions were dried and distilled under an argon or nitrogen atmosphere prior to use as follows: THF and diethyl ether (ether)from a dark blue solution of sodium benzophenone ketyl; toluene, dichromethane, and pyridine from calcium hydride. Flash chromatography was used routinely for purification and separation of product mixtures using silica gel 60 of 230−400 mesh size as supplied by Merck. Eluent systems are given in volume/volume concentrations. Melting points were determined using a KRUSS KIP1N melting point meter. 1H and

13

C NMR spectra were recorded on a

Varian Mercury-300 (300 MHz) and a Varian Mercury-400 (400 MHz). Chloroform-d, methanol-d4 or deuterium oxide-d2 was used as the solvent and TMS (δ 0.00 ppm) as an


Page 20 of 52

Page 21 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


internal standard. Chemical shift values are reported in ppm relative to the TMS in delta (δ) units. Multiplicities are recorded as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), and m (multiplet). Coupling constants (J) are expressed in hertz. Electrospray mass spectra (ESMS) were recorded as m/z values using an Agilent 1100 MSD mass spectrometer. All test compounds displayed more than 95% purity as determined by an Agilent 1100 series HPLC system using a C18 column (Thermo Golden, 4.6 mm × 250 mm). The gradient system for HPLC separation was composed of MeOH (mobile phase A) and H2O solution containing 0.1% trifluoro-acetic acid (mobile phase B). The starting flow rate was 0.5 mL/min and the injection volume was 10 µL. During first 2 min the percentage of phase A was 10%. At 6 min, the percentage of phase A was increased to 50%. At 16 min, the percentage of phase A was increased to 90% over 9 min. The system was operated at 25 oC. Peaks were detected at 254 nm. IUPAC nomenclature of compounds was determined with ACD/Name Pro software. N2-{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-6-methoxy-N4piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (3). Following a similar synthetic procedure for compound 2,39 products 3-25 were readily prepared in a sequence of three steps using various 2,4-dichloroquinazoline derivatives as starting material. As typified by product 3, 2,4-dichloro-6-methoxyquinazoline (0.89 g, 3.87



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mmol) was first coupled with 4-amino-1-Boc-piperidine (1.03 g, 5.14 mmol) in dichloromethane (30 mL) in the presence of triethylamine (0.91 g, 9.01 mmol) at room temperature for 16 h to afford C4-substituted intermediate (1.23 g) in 81% yield after usual workup and chromatographic purification. The above C4-substituted intermediate (403.7 mg, 1.03 mmol) was further coupled with bisbenzyl linker (501.1 mg, 1.05 mmol) in 1-pentanol (3 mL) under microwave at 120 oC to give Boc-protected 3, which after simple chromatographic purification, was dissolved in dichloromethane (10 mL) and subsequently deprotected by addition of 1N HCl in deiethylether (20 mL, 20 mmol,) to afford the desired product 3 (385 mg) in 58% yield over two steps as a white solid. 1H NMR (300 MHz, D2O) δ 7.56−7.50 (m, 4H), 7.15−6.93 (m, 3H), 4.71 (s, 2H), 4.26 (m, 2H), 4.24 (m, 1H), 3.73 (s, 3H), 3.51 (m, 2H), 3.20−3.05 (m, 6H), 2.13−1.70 (m, 10H), 1.56 (m, 1H), 1.33−1.11 (m, 6H); 13C NMR (75 MHz, D2O) δ 158.69, 155.42, 151.48, 140.27, 132.41, 130.21, 129.52, 127.50, 124.55, 117.57, 108.96, 103.77, 57.33, 56.12, 50.61, 46.92, 44.02, 43.79, 43.18, 41.13, 28.72, 27.19, 24.32, 23.77, 22.75; ESMS m/z: 532.3 (M+1); HPLC purity = 95.35 %, tR = 14.35 min. N2-{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-7-methoxy-N4piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (4). Starting from 2,4dichloro-7-methoxyquinazoline (334.2 mg, 1.46 mmol), compound 4 (362 mg) was obtained in 39% yield over three steps: 1H NMR (300 MHz, D2O) δ 7.65 (d, J = 9.0 Hz,


Page 22 of 52

Page 23 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1H), 7.55−7.48 (m, 4H), 6.50 (dd, J = 9.0, 2.1 Hz, 1H), 6.38 (d, J = 2.1 Hz, 1H), 4.67 (s, 2H), 4.26 (s, 2H), 4.21 (m, 1H), 3.74 (s, 3H), 3.53 (m, 2H), 3.17−3.01 (m, 6H), 2.13−1.71 (m, 10H), 1.56 (m, 1H), 1.33−1.13 (m, 6H);

13

C NMR (75 MHz, D2O) δ

164.03, 158.85, 152.18, 140.29, 140.11, 130.20, 129.53, 127.53, 125.11, 113.62, 102.28, 97.72, 57.34, 55.96, 50.60, 46.78, 44.05, 43.75, 43.15, 41.15, 28.62, 27.33, 24.34, 23.77, 22.74; ESMS m/z: 532.3 (M+1); HPLC purity = 96.25 %, tR = 14.26 min. N2-{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-6-methyl-N4piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (5). Starting from 2,4dichloro-6-methylquinazoline (300.6 mg, 1.41 mmol), compound 5 (339 mg) was obtained in 38% yield over three steps: 1H NMR (300 MHz, D2O) δ 7.76 (d, J = 2.1 Hz, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.53−7.48 (m, 4H), 7.29 (dd, J = 9.0, 2.1 Hz, 1H), 4.77 (s, 2H), 4.33 (m, 1H), 4.27 (s, 2H), 3.49 (m, 2H), 3.19−3.01 (m, 6H), 2.60 (s, 3H), 2.17−1.96 (m, 6H), 1.93−1.76 (m, 4H), 1.66 (m, 1H), 1.35−1.13 (m, 6H); 13C NMR (75 MHz, D2O) δ 161.45, 154.23, 142.66, 138.61, 138.14, 137.22, 132.82, 132.03, 130.14, 124.98, 118.30, 110.70, 59.90, 53.22, 49.41, 46.66, 46.53, 45.76, 43.79, 31.21, 29.87, 26.91, 26.35, 25.33, 22.81; ESMS m/z: 516.3 (M+1); HPLC purity = 98.49 %, tR = 14.79 min. N2-{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-7-methyl-N4piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (6). Starting from 2,4-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

dichloro-7-methylquinazoline (289.2 mg, 1.36 mmol), compound 6 (402 mg) was obtained in 47% yield over three steps: 1H NMR (300 MHz, D2O) δ 7.72 (d, J = 8.1 Hz, 1H), 7.53−7.48 (m, 4H), 7.08−7.05 (m, 2H ), 4.74 (s, 2H), 4.27 (s, 2H), 4.25 (m, 1H), 3.47 (m, 2H), 3.25−3.05 (m, 6H), 2.35 (s, 3H), 2.13−1.96 (m, 6H), 1.93−1.73 (m, 4H), 1.61 (m, 1H), 1.38−1.11 (m, 6H); 13C NMR (75 MHz, D2O) δ 159.07, 151.92, 146.95, 140.06, 137.91, 130.29, 129.52, 127.62, 125.89, 122.96, 115.54, 106.14, 57.38, 50.69, 46.89, 44.18, 44.05, 43.22, 41.27, 28.69, 27.38, 24.40, 23.82, 22.81, 21.21; ESMS m/z: 516.3 (M+1); HPLC purity = 96.35 %, tR = 14.47 min. N2-{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-6,7-dimethoxy-N4piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (7). Starting from 2,4dichloro-6,7-dimethoxyquinazoline (348.6 mg, 1.35 mmol), compound 7 (374 mg) was obtained in 41% yield over three steps: 1H NMR (300 MHz, D2O) δ 7.57−7.48 (m, 4H), 7.26 (s, 1H), 6.65 (s, 1H), 4.75 (s, 2H), 4.26 (s, 2H), 4.23 (m, 1H), 3.83 (s, 6H), 3.48 (m, 2H), 3.19−3.00 (m, 6H), 2.13−1.96 (m, 6H), 1.93−1.71 (m, 4H), 1.65 (m, 1H), 1.33−1.13 (m, 6H); 13C NMR (75 MHz, D2O) δ 158.55, 154.62, 151.75, 146.03, 140.18, 134.66, 130.15, 129.39, 127.30, 103.61, 101.54, 97.33, 57.38, 56.40, 56.22, 50.67, 46.89, 44.03, 43.92, 43.19, 41.18, 28.69, 27.34, 24.33, 23.78, 22.78; ESMS m/z: 562.3 (M+1); HPLC purity = 95.27 %, tR = 14.23 min. N2-{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-6-chloro-N4-


Page 24 of 52

Page 25 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (8). Starting from 2,4,6trichloroquinazoline (287.2 mg, 1.23 mmol), compound 8 (380 mg) was obtained in 48% yield over three steps: 1H NMR (400 MHz, D2O) δ 7.93 (d, J = 9.0 Hz, 1H), 7.53−7.49 (m, 4H), 7.41 (d, J = 2.1 Hz, 1H), 7.31 (dd, J = 9.0, 2.1 Hz, 1H), 4.80 (s, 2H), 4.35 (m, 1H), 4.27 (s, 2H), 3.48 (m, 2H), 3.21−3.01 (m, 6H), 2.11−2.05 (m, 6H), 1.86−1.81 (m, 4H), 1.66 (m, 1H), 1.31−1.15 (m, 6H);

13


161.20, 154.72, 143.10, 142.41, 141.37, 132.81, 131.97, 130.09, 127.68, 127.18, 117.98, 109.71, 59.90, 53.21, 49.67, 46.81, 46.48, 45.70, 43.76, 31.19, 29.70, 26.89, 26.32, 25.32; ESMS m/z: 536.3 (M+1); HPLC purity = 96.51 %, tR = 15.16 min. N2-{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-7-chloro-N4piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (9). Starting from 2,4,7trichloroquinazoline (322.3 mg, 1.38 mmol), compound 9 (357 mg) was obtained in 40% yield over three steps: 1H NMR (300 MHz, D2O) δ 7.94 (d, J = 2.1 Hz, 1H), 7.58−7.48 (m, 5H), 7.26 (dd, J = 8.7, 2.1 Hz, 1H), 4.79 (s, 2H), 4.31 (m, 1H), 4.26 (s, 2H), 3.48 (m, 2H), 3.21−3.06 (m, 6H), 2.14−1.99 (m, 6H), 1.98−1.78 (m, 4H), 1.63 (m, 1H), 1.41−1.16 (m, 6H);

13

C NMR (75 MHz, D2O) δ 158.17, 152.00, 139.91, 136.60,

134.81, 130.20, 129.36, 128.98, 127.43, 122.73, 117.82, 109.44, 57.28, 50.61, 47.10, 44.19, 43.85, 43.09, 41.15, 28.60, 27.07, 24.28, 23.71, 22.71; ESMS m/z: 536.3 (M+1); HPLC purity = 97.07 %, tR = 14.99 min.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 52

N2-{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-7-trifluoromethylN4-piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (10). Starting from 2,4-dichloro-7-trifluoromethylquinazoline (306.2 mg, 1.15 mmol), compound 10 (341 mg) was obtained in 44% yield over three steps: 1H NMR (300 MHz, D2O) δ 8.21 (d, J = 8.4 Hz, 1H), 7.74(d, J = 2.1 Hz, 1H), 7.68 (dd, J = 8.4, 2.1 Hz, 1H), 7.53−7.48 (m, 4H), 4.83 (s, 2H), 4.39 (m, 1H), 4.28 (s, 2H), 3.49 (m, 2H), 3.23−3.01 (m, 6H), 2.16−1.97 (m, 6H), 1.93−1.77 (m, 4H), 1.67 (m, 1H), 1.35−1.15 (m, 6H);

13

C NMR (75

MHz, D2O) δ 158.88, 152.62, 139.74, 138.37, 135.32 (q, J = 32.1 Hz), 130.26, 129.53, 127.30, 125.34, 122.58 (q, J = 271.4 Hz), 120.60, 113.53, 111.36, 57.51, 50.74, 47.32, 44.28, 44.16, 43.18, 41.33, 28.77, 27.03, 24.46, 23.88, 22.87; ESMS m/z: 570.3 (M+1); HPLC purity = 96.60 %, tR = 15.30 min. N2-{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-6-nitro-N4piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (11). Starting from 2,4dichloro-6-nitroquinazoline (286.0 mg, 1.17 mmol), compound 11 (325 mg) was obtained in 42% yield over three steps: 1H NMR (300 MHz, D2O) δ 9.09 (d, J = 2.1 Hz, 1H), 8.54 (dd, J = 9.3, 2.1 Hz, 1H), 7.58 (d, J = 9.3 Hz, 1H), 7.54−7.48 (m, 4H), 4.84 (s, 2H), 4.39 (m, 1H), 4.28 (s, 2H), 3.49 (m, 2H), 3.21−2.99 (m, 6H), 2.15−1.97 (m, 6H), 1.92−1.76 (m, 4H), 1.67 (m, 1H), 1.37−1.15 (m, 6H);

13


158.99, 152.84, 142.79, 142.71, 139.48, 130.31, 129.60, 129.36, 127.36, 120.92, 117.80,


Page 27 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


108.84, 57.42, 50.75, 47.51, 44.48, 44.13, 43.13, 41.32, 28.78, 26.95, 24.46, 24.00, 22.85; ESMS m/z: 547.3 (M+1); HPLC purity = 97.93 %, tR = 14.52 min. N2-{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-7-nitro-N4piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (12). Starting from 2,4dichloro-7-nitroquinazoline (273.5 mg, 1.12 mmol), compound 12 (343 mg) was obtained in 47% yield over three steps: 1H NMR (300 MHz, D2O) δ 8.36−8.23 (m, 2H), 8.16 (d, J = 8.7 Hz, 1H), 7.56−7.50 (m, 4H), 4.83 (s, 2H), 4.40 (m, 1H), 4.27 (s, 2H), 3.48 (m, 2H), 3.23−3.00 (m, 6H), 2.15−1.97 (m, 6H), 1.92−1.76 (m, 4H), 1.66 (m, 1H), 1.37−1.14 (m, 6H); 13C NMR (75 MHz, D2O) δ 158.59, 152.81, 150.44, 139.57, 138.92, 130.24, 129.51, 127.28, 126.12, 118.23, 113.16, 111.54, 57.39, 50.74, 47.47, 44.31, 44.05, 43.00, 41.25, 28.75, 26.89, 24.42, 23.84, 22.81; ESMS m/z: 547.3 (M+1); HPLC purity = 98.31 %, tR = 14.26 min. N2-{1-[2-(2-Cyclohexylamino-ethylamino)-ethyl]-1H-[1,2,3]triazol-4-ylmethyl}N4-piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (13). Starting from 2,4-dichloroquinazoline (285.2 mg, 1.43 mmol), compound 13 (372 mg) was obtained in 41% yield over three steps: 1H NMR (400 MHz, D2O) δ 8.15 (s, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.68 (dd, J = 8.0, 7.6 Hz, 1H), 7.31 (dd, J = 8.4, 7.6 Hz, 1H), 7.25 (m, 1H), 4.90 (m, 2H), 4.85 (s, 2H), 4.38 (m, 1H), 3.78 (t, J = 5.2 Hz, 2H), 3.61−3.44 (m, 5H), 3.24−3.16 (m, 3H), 2.19 (m, 2H), 2.07 (m, 2H), 1.94 (m, 2H), 1.83 (m, 2H), 1.66 (m,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1H), 1.41−1.18 (m, 6H);

Page 28 of 52

13

C NMR (75 MHz, D2O) δ 159.77, 152.40, 145.71, 138.17,

135.39, 124.84, 124.27, 123.31, 116.41, 109.13, 58.02, 47.19, 46.84, 46.32, 43.74, 43.10, 39.79, 36.37, 28.69, 27.21, 24.32, 23.77; ESMS m/z: 493.3 (M+1); HPLC purity = 97.4 %, tR = 13.75 min. N2-{1-[2-(2-Cyclohexylamino-ethylamino)-ethyl]-1H-[1,2,3]triazol-4-ylmethyl}7-methoxy-N4-piperidin-4-yl-quinazoline-2,4-diamine

hydrochloride

salt

(14).

Starting from 2,4-dichloro-7-methoxyquinazoline (308.0 mg, 1.34 mmol), compound 14 (333 mg) was obtained in 37% yield over three steps: 1H NMR (400 MHz, D2O) δ 8.15 (s, 1H), 7.75 (d, J = 9.0 Hz, 1H), 6.74 (dd, J = 9.0, 2.1 Hz, 1H), 6.53 (d, J = 2.1 Hz, 1H), 4.90 (m, 2H), 4.62 (s, 2H), 4.38 (m, 1H), 3.83 (s, 3H), 3.78 (t, J = 5.2 Hz, 2H), 3.62−3.45 (m, 5H), 3.24−3.16 (m, 3H), 2.18 (m, 2H), 2.06 (m, 2H), 1.92 (m, 2H), 1.82 (m, 2H), 1.63 (m, 1H), 1.38−1.17 (m, 6H); 13C NMR (75 MHz, D2O) δ 164.30, 159.19, 152.43, 145.60, 140.43, 125.30, 124.34, 113.85, 102.75, 98.28, 58.02, 56.03, 47.18, 46.78, 46.31, 43.76, 43.13, 39.80, 36.35, 28.69, 27.38, 24.32, 23.77; ESMS m/z: 523.3 (M+1); HPLC purity = 96.3 %, tR = 13.26 min. N2-{1-[2-(2-Cyclohexylamino-ethylamino)-ethyl]-1H-[1,2,3]triazol-4-ylmethyl}6,7-dimethoxy-N4-piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (15). Starting from 2,4-dichloro-6,7-dimethoxyquinazoline (328.0 mg, 1.27 mmol), compound 15 (294 mg) was obtained in 33% yield over three steps: 1H NMR (400 MHz,


Page 29 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


D2O) δ 8.15 (s, 1H), 7.20(s, 1H), 6.51 (s, 1H), 4.88 (t, J = 6.0 Hz, 2H), 4.83 (s, 2H), 4.38 (m, 1H), 3.78 (s, 6H), 3.76 (m, 2H), 3.61−3.43 (m, 5H), 3.24−3.15 (m, 3H), 2.20 (m, 2H), 2.06 (m, 2H), 1.94 (m, 2H), 1.83 (m, 2H), 1.67 (m, 1H), 1.41−1.19 (m, 6H);

13

C

NMR (75 MHz, D2O) δ 158.73, 154.76, 151.80, 146.23, 145.50, 134.69, 124.37, 103.33, 101.82, 97.56, 58.02, 56.49, 56.32, 47.18, 46.90, 46.34, 43.77, 43.21, 39.80, 36.38, 28.67, 27.42, 24.32, 23.77; ESMS m/z: 553.3 (M+1); HPLC purity = 99.3 %, tR = 13.13 min. N2-{1-[3-(2-Cyclohexylamino-ethylamino)-propyl]-1H-[1,2,3]triazol-4ylmethyl}-N4-piperidin-4-yl-quinazoline-2,4-diamine

hydrochloride

salt

(16).

Starting from 2,4-dichloroquinazoline (278.6 mg, 1.40 mmol), compound 16 (399 mg) was obtained in 44% yield over three steps: 1H NMR (400 MHz, D2O) δ 8.12 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.73 (dd, J = 8.0, 7.6 Hz, 1H), 7.36 (dd, J = 8.4, 7.6 Hz, 1H), 7.32 (m, 1H), 4.86 (s, 2H), 4.84 (m, 2H), 4.42 (m, 1H), 3.70 (t, J = 6.0 Hz, 2H), 3.57 (m, 2H), 3.26−3.12 (m, 6H), 2.22−2.04 (m, 6H), 1.92 (m, 2H), 1.83 (m, 2H), 1.66 (m, 1H), 1.41−1.17 (m, 6H); 13C NMR (75 MHz, D2O) δ 159.98, 152.61, 145.86, 138.35, 135.43, 124.87, 124.20, 123.37, 116.53, 109.37, 57.47, 46.78, 46.60, 46.23, 44.92, 43.09, 41.15, 36.37, 28.78, 27.18, 24.40, 23.84, 22.69; ESMS m/z: 507.3 (M+1); HPLC purity = 97.3 %, tR = 13.61 min.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 52

N2-{1-[2-(3-Cyclohexylamino-propylamino)-ethyl]-1H-[1,2,3]triazol-4ylmethyl}-N4-piperidin-4-yl-quinazoline-2,4-diamine

hydrochloride

salt

(17).

Starting from 2,4-dichloroquinazoline (273.2 mg, 1.37 mmol), compound 17 (394 mg) was obtained in 44% yield over three steps: 1H NMR (400 MHz, D2O) δ 8.07 (s, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.70 (dd, J = 8.0, 7.6 Hz, 1H), 7.36−7.2 (m, 2H), 4.83 (s, 2H), 4.57 (t, J = 6.8 Hz, 2H), 4.36 (m, 1H), 3.57 (m, 2H), 3.44−3.41 (m, 4H), 3.22−3.16 (m, 4H), 2.38 (m, 2H), 1.98−2.04 (m, 4H), 1.92 (m, 2H), 1.82 (m, 2H), 1.66 (m, 1H), 1.41−1.18 (m, 6H); 13C NMR (75 MHz, D2O) δ 159.88, 152.50, 145.53, 138.29, 135.40, 124.85, 123.73, 123.36, 116.49, 109.24, 58.03, 47.29, 46.81, 45.24, 43.32, 43.01, 39.82, 36.48, 28.69, 27.16, 26.23, 24.32, 23.79; ESMS m/z: 507.3 (M+1); HPLC purity = 99.5 %, tR = 13.57 min. N2-{1-[3-(2-Cyclohexylamino-ethylamino)-propyl]-1H-[1,2,3]triazol-4ylmethyl}-7-methoxy-N4-piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (18). Starting from 2,4-dichloro-7-methoxyquinazoline (308.4 mg, 1.35 mmol), compound 18 (316 mg) was obtained in 34% yield over three steps: 1H NMR (300 MHz, D2O) δ 8.13 (s, 1H), 7.76 (d, J = 9.0 Hz, 1H), 6.75 (dd, J = 9.0, 2.1 Hz, 1H), 6.54 (d, J = 2.1 Hz, 1H), 4.83 (s, 2H), 4.59 (t, J = 6.8 Hz, 2H), 4.35 (m, 1H), 3.82 (s, 3H), 3.60 (m, 2H), 3.44−3.41 (m, 4H), 3.22−3.17 (m, 4H), 2.37 (m, 2H), 2.20−2.04 (m, 4H), 1.90 (m, 2H), 1.82 (m, 2H), 1.66 (m, 1H), 1.38−1.19 (m, 6H);

13



Page 31 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


164.29, 159.16, 152.40, 145.34, 140.40, 125.27, 123.85, 113.83, 102.69, 98.21, 58.00, 56.02, 47.32, 46.77, 45.21, 43.32, 43.13, 39.82, 36.40, 28.68, 27.35, 26.23, 24.32, 23.77; ESMS m/z: 537.3 (M+1); HPLC purity = 96.9 %, tR = 14.05 min. N2-{1-[3-(3-Cyclohexylamino-propylamino)-propyl]-1H-[1,2,3]triazol-4ylmethyl}-N4-piperidin-4-yl-quinazoline-2,4-diamine

hydrochloride

salt

(19).

Starting from 2,4-dichloroquinazoline (272.0 mg, 1.37 mmol), compound 19 (373 mg) was obtained in 41% yield over three steps: 1H NMR (400 MHz, D2O) δ 8.05 (s, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.72 (dd, J = 8.0, 7.6 Hz, 1H), 7.36−7.31 (m, 2H), 4.84 (s, 2H), 4.55 (t, J = 6.8 Hz, 2H), 4.40 (m, 1H), 3.56 (m, 2H), 3.20−3.14 (m, 8H), 2.34 (m, 2H), 2.20−2.02 (m, 6H), 1.92 (m, 2H), 1.82 (m, 2H), 1.66 (m, 1H), 1.41−1.18 (m, 6H); 13C NMR (75 MHz, D2O) δ 160.21, 152.85, 145.89, 138.67, 136.05, 125.50, 124.72, 124.00, 116.97, 109.50, 58.17, 48.35, 47.63, 45.52, 45.41, 43.83, 41.96, 37.07, 29.52, 27.94, 26.98, 25.17, 24.59, 23.61; ESMS m/z: 521.3 (M+1); HPLC purity = 96.2 %, tR = 13.54 min. N2-{1-[3-(3-Cyclohexylamino-propylamino)-propyl]-1H-[1,2,3]triazol-4ylmethyl}-7-methoxy-N4-piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (20). Starting from 2,4-dichloro-7-methoxyquinazoline (303.3 mg, 1.32 mmol), compound 20 (317 mg) was obtained in 34% yield over three steps: 1H NMR (400 MHz, D2O) δ 8.07 (s, 1H), 7.77 (d, J = 9.0 Hz, 1H), 6.80 (d, J = 9.0 Hz, 1H), 6.60 (s, 1H), 4.84



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(s, 2H), 4.57 (t, J = 6.9 Hz, 2H), 4.36 (m, 1H), 3.84 (s, 3H), 3.57 (m, 2H), 3.23−3.08 (m, 8H), 2.34 (m, 2H), 2.20−2.02 (m, 6H), 1.92 (m, 2H), 1.84 (m, 2H), 1.65 (m, 1H), 1.40−1.18 (m, 6H); 13C NMR (75 MHz, D2O) δ 164.36, 159.30, 152.53, 145.73, 140.53, 125.27, 123.65, 113.86, 102.84, 98.38, 57.44, 55.97, 47.30, 46.72, 44.77, 44.60, 43.10, 41.16, 36.46, 28.75, 27.30, 26.20, 24.39, 23.81, 22.80; ESMS m/z: 551.4 (M+1); HPLC purity = 95.6 %, tR = 13.91 min. N2-{1-[3-(3-Cyclohexylamino-propylamino)-propyl]-1H-[1,2,3]triazol-4ylmethyl}-6-methoxy-N4-piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (21). Starting from 2,4-dichloro-6-methoxyquinazoline (304.2 mg, 1.33 mmol), compound 21 (325 mg) was obtained in 35% yield over three steps: 1H NMR (400 MHz, D2O) δ 8.07 (s, 1H), 7.27 (s, 1H), 7.23−7.16 (m, 2H), 4.84 (s, 2H), 4.57 (t, J = 6.8 Hz, 2H), 4.38 (m, 1H), 3.81 (s, 3H), 3.57 (m, 2H), 3.23−3.09 (m, 8H), 2.33 (m, 2H), 2.20−2.02 (m, 6H), 1.92 (m, 2H), 1.82 (m, 2H), 1.65 (m, 1H), 1.38−1.16 (m, 6H); 13C NMR (75 MHz, D2O) δ 159.30, 155.85, 151.86, 145.60, 132.85, 124.69, 123.69, 118.00, 109.73, 104.47, 57.45, 56.14, 47.35, 46.89, 44.78, 44.61, 43.15, 41.18, 36.46, 28.77, 27.19, 26.22, 24.40, 23.84, 22.83; ESMS m/z: 551.4 (M+1); HPLC purity = 99.3 %, tR = 13.69 min. N2-{1-[3-(3-Cyclohexylamino-propylamino)-propyl]-1H-[1,2,3]triazol-4ylmethyl}-6,7-dimethoxy-N4-piperidin-4-yl-quinazoline-2,4-diamine hydrochloride


Page 32 of 52

Page 33 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


salt (22). Starting from 2,4-dichloro-6,7-dimethoxyquinazoline (329.7 mg, 1.27 mmol), compound 22 (314 mg) was obtained in 34% yield over three steps: 1H NMR (400 MHz, D2O) δ 8.06 (s, 1H), 7.27 (s, 1H), 6.61 (s, 1H), 4.83 (s, 2H), 4.56 (t, J = 6.8 Hz, 2H), 4.38 (m, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.57 (m, 2H), 3.23−3.08 (m, 8H), 2.34 (m, 2H), 2.18−2.00 (m, 6H), 1.94 (m, 2H), 1.82 (m, 2H), 1.64 (m, 1H), 1.38−1.18 (m, 6H); 13C NMR (75 MHz, D2O) δ 158.88, 154.93, 151.92, 146.30, 145.88, 134.89, 123.63, 103.45, 101.95, 97.76, 57.44, 56.38, 56.29, 47.32, 46.83, 44.77, 44.58, 43.16, 41.15, 36.46, 28.75, 27.35, 26.19, 24.39, 23.81, 22.80; ESMS m/z: 581.4 (M+1); HPLC purity = 97.5 %, tR = 13.91 min. 7-Chloro-N2-{1-[3-(3-cyclohexylamino-propylamino)-propyl]-1H-[1,2,3]triazol4-ylmethyl}-N4-piperidin-4-yl-quinazoline-2,4-diamine hydrochloride salt (23). Starting from 2,4,7-trichloroquinazoline (306.1 mg, 1.31 mmol), compound 23 (372 mg) was obtained in 40% yield over three steps: 1H NMR (400 MHz, D2O) δ 8.12 (s, 1H), 7.87 (d, J = 2.0 Hz, 1H), 7.47 (dd, J = 8.4, 2.0 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 4.83 (s, 2H), 4.57 (t, J = 6.8 Hz, 2H), 4.35 (m, 1H), 3.57 (m, 2H), 3.22−3.08 (m, 8H), 2.35 (m, 2H), 2.21−2.01 (m, 6H), 1.95 (m, 2H), 1.79 (m, 2H), 1.61 (m, 1H), 1.36−1.18 (m, 6H); 13

C NMR (75 MHz, D2O) δ 158.61, 152.26, 145.23, 136.82, 135.13, 129.34, 123.94,

122.90, 118.14, 109.92, 57.45, 47.47, 47.15, 44.80, 44.64, 43.10, 41.21, 36.55, 28.77,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 52

27.16, 26.22, 24.42, 23.84, 22.84; ESMS m/z: 555.3 (M+1); HPLC purity = 97.8 %, tR = 14.12 min. N2-{1-[3-(3-Cyclohexylamino-propylamino)-propyl]-1H-[1,2,3]triazol-4ylmethyl}-N4-piperidin-4-yl-7-trifluoromethyl-quinazoline-2,4-diamine hydrochloride salt (24). Starting from 2,4-dichloro-7-trifluoromethylquinazoline (336.9 mg, 1.26 mmol), compound 24 (381 mg) was obtained in 41% yield over three steps: 1H NMR (300 MHz, D2O) δ 8.04 (d, J = 9.0 Hz, 1H), 7.91 (s, 1H), 7.52 (s, 1H), 7.48 (d, J = 9.0 Hz, 1H), 4.83 (s, 2H), 4.57 (t, J = 6.8 Hz, 2H), 4.38 (m, 1H), 3.57 (m, 2H), 3.22−3.08 (m, 8H), 2.34 (m, 2H), 2.21−2.01 (m, 6H), 1.95 (m, 2H), 1.79 (m, 2H), 1.61 (m, 1H), 1.36−1.18 (m, 6H);

13

C NMR (75 MHz, D2O) δ 159.45, 152.99, 145.34,

138.58, 135.49 (q, J = 31.5 Hz), 128.50, 125.07, 122.80 (q, J = 271.5 Hz), 120.75, 114.03, 111.97, 57.45, 47.36, 47.06, 44.78, 44.61, 43.04, 41.18, 36.57, 28.77, 27.00, 26.19, 24.40, 23.82, 22.83; ESMS m/z: 589.3 (M+1); HPLC purity = 96.8 %, tR = 14.01 min. N2-{1-[4-(3-Cyclohexylamino-propylamino)-butyl]-1H-[1,2,3]triazol-4ylmethyl}-N4-piperidin-4-yl-quinazoline-2,4-diamine

hydrochloride

salt

(25).

Starting from 2,4-dichloroquinazoline (275.1 mg, 1.38 mmol), compound 25 (392 mg) was obtained in 42% yield over three steps: 1H NMR (400 MHz, D2O) δ 8.07 (s, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.63 (dd, J = 8.0, 7.6 Hz, 1H), 7.26−7.21 (m, 2H), 4.82 (s, 2H),


Page 35 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4.47 (t, J = 6.8 Hz, 2H), 4.33 (m, 1H), 3.57 (m, 2H), 3.20−3.04 (m, 8H), 2.15−1.96 (m, 8H), 1.84 (m, 2H), 1.78 (m, 2H), 1.75−1.60 (m, 3H), 1.39−1.17 (m, 6H); 13C NMR (75 MHz, D2O) δ 159.67, 152.32, 145.21, 138.11, 135.32, 124.76, 123.63, 123.27, 116.34, 109.03, 57.38, 49.77, 46.91, 46.77, 44.41, 43.02, 41.18, 36.37, 28.72, 27.09, 26.49, 24.35, 23.78, 22.77, 22.53; ESMS m/z: 535.4 (M+1); HPLC purity = 96.7 %, tR = 13.55 min. Animals. Eight to ten week-old male C57BL/6 mice were used in the study of stem/progenitor cell counting. Male Sprague–Dawley rats (weighing approximately 200250 g) were used in the study of AKI. All animals were purchased from National Laboratory Animals Center (Taipei, Taiwan, R.O.C.). The Institutional Animal Care and Use Committee (IACUC) of National Health Research Institutes (NHRI) approved all experimental protocols. Establishment of Human CXCR4 Stable Cell Line and Membrane Purification. The hCXCR4 cDNA was subcloned into pIRES2-EGFP vector (Clontech Laboratories, Inc., Mountain View, CA). Transfected HEK-293 cells stably expressed hCXCR4 (HEK-293 CXCR4) were selected by EGFP and 1 mg/ml G418 sulfate. The selected clone was maintained in DMEM supplemented with 10% fetal bovine serum and 0.5 mg/ml G418 sulfate with 5% CO2 at 37oC in the humidified incubator. For membrane purification, cells were homogenized in ice-cold buffer A (50 mM Tris-HCl, pH 7.4, 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mM MgCl2, 2.5 mM EDTA, 10% sucrose) with freshly prepared 1 mM PMSF. The homogenate was centrifuged at 3500 x g for 15 min at 4oC. The pellet was removed and the supernatant then was centrifuged at 43000 x g for additional 30 min at 4oC. The final pellet was resuspended in buffer A and stored at -80 oC. Radioligand Binding Assay. An amount of 2-4 µg of purified membrane with CXCR4 was incubated with 0.16 nM [125I]SDF-1 and compounds of interest in the incubation buffer (50 mM HEPES-NaOH, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) The nonspecific binding was defined in the presence of 50 µM AMD3100 (plerixafor). The reaction mixtures were incubated for 1.5 h at 30oC and then were transferred to a 96-well GF/B filter plate (Millipore Corp., Billerica, MA, USA). The reaction mixtures were terminated by maniford filtration and washed with ice-cold wash buffer (50 mM HEPES-NaOH, pH 7.4, 100mM NaCl) for four times. The radioactivity bound to the filter was measured by Topcount (PerkinElmer Inc., Waltham, MA, USA). IC50 values were determined by the concentration of compounds required to inhibit 50% of the specific binding of [125I]SDF-1 and calculated by nonlinear regression (GraphPad software, San Diego, CA, USA). Chemotaxis Assay. CCRF-CEM (T-cell acute lymphoblastic leukemia) cells were suspended in RPMI 1640 containing 10% FBS and then preincubated with indicated concentrations of compounds for 10 min at 37 oC. The assay was performed in Millicell


Page 36 of 52

Page 37 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hanging Cell Culture Inserts (pore size 5 µm; 24-well plate; Millipore, Bedford, MA, USA). Compounds containing 10 nM SDF-1 were plated in the lower chambers of inserts, and cells with compounds were plated in the upper chambers of inserts at a density of 2.5 × 105 cells/well. After 2.5 h incubation at 37 oC, cells in both chambers of inserts were measured by flow cytometer (Guava Technologies, Hayward, CA, USA). Flow Cytometry Analysis for Stem/progenitor cell counting. C57BL/6 male mice were treated with potential CXCR4 antagonist individually by subcutaneous injection, and then blood samples containing mobilized stem/progenitor cells were collected 2 hours later. After labeled with specific antibodies, including APC-conjugated anti-CXCR4 (clone 2B11; eBioscience), FITC-conjugated anti-CD34 (clone RAM34; eBioscience), PE-conjugated anti-CD133 (clone 13A4; eBioscience) and anti-KDR (clone Avsa12a1; eBioscience), anti-c-Kit (clone 2B8; eBioscience), anti-Sca-1 (clone D7; eBioscience), anti-linage (Mouse Hematopoietic Lineage Biotin Panel, eBioscience) and Streptavidin PE-Cy7 (eBioscience), cells were washed, characterized and quantified by flow cytometer (Guava Technologies, Hayward, CA, USA). Each data point included at least 300,000 events for measurement of c-Kit+Sca-1+Lin- cells and 60,000 events for analysis of other cell types. Animal Model for Acute Kidney Injury (AKI) induced by IschemiaReperfusion injury (IRI). Animals were treated with vehicle or potential CXCR4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

antagonist individually by subcutaneous injection 40 minutes before surgery. AKI was induced by clamping bilateral renal vein and artery for 1 hour followed by releasing vessel clips to allow 24 h of reperfusion. Rats were randomly assigned to different groups to evaluate the BUN and Scr levels of AKI induced by IRI as follows: group 1, sham (surgery without IRI, n = 9); group 2, Control (vehicle, surgery with IRI, n = 8 ); group 3, AMD3100 (6 mg/kg, surgery with IRI, n = 9); and group 4, compound 19 (6 mg/kg, surgery with IRI, n = 9). BUN and Scr were collected at 24 h after IRI and determined by biochemistry analyzer, FUJI DRI-CHEM 3500s (Fujifilm, Tokyo, Japan), using the original kits. Statistical Analysis. Apart from chemotaxis assay, which is presented as mean ± standard deviation (SD), other values are expressed as mean ± standard error of the mean (SEM). The results were analyzed by Student's t-test. For all comparisons between groups, p < 0.05 was considered statistically significant.

ASSOCIATED CONTENT Supporting Information Counter screening against a panel of chemokine G protein-coupled receptors, including CXCR2, CCR2, CCR4, and CCR5, has been conducted by Eurofins Panlabs Taiwan Ltd. for compound 19. As exemplified by linker 1, the synthetic protocols of all new linkers and their characterization data are described and elucidated on pages S11-S14.


Page 38 of 52

Page 39 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

H and

13

C NMR spectra of representative final products 13, 19, 20, 21 and 23 are

displayed on pages S15-S24. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Telephone:

+886-37-246-166

ext.

35709.

Fax:

+886-37-586-456.

E-mail:

[email protected] Author Contributions †

These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We are grateful to the National Health Research Institutes and Ministry of Science and Technology of the Republic of China (MOST 101-2325-B-400-016) for financial support.

ABBREVIATIONS USED SDF-1, stromal cell-derived factor-1; G-CSF, granulocyte colony-stimulating factor; AKI, acute kidney injury; IRI, ischemia-reperfusion injury; HSCs, hematopoietic stem cells; EPCs, endothelial progenitor cells; MSCs, mesenchymal stem cells; SAR,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

structure-activity relationship; BUN, blood urea nitrogen; Scr, serum creatinine; SD, standard deviation; SEM, standard error of the mean; ESMS, electrospray mass spectra

REFERENCES (1) Hilton, R. Acute renal failure. BMJ 2006, 333, 786-790. (2) Waikar, S. S.; Liu, K. D.; Chertow, G. M. Diagnosis, epidemiology, and outcomes of acute kidney injury. Clin. J. Am. Soc. Nephrol. 2008, 3, 844-861. (3) Liu, N.; Patzak, A.; Zhang, J. CXCR4-overexpressing bone marrow-derived mesenchymal stem cells improve repair of acute kidney injury. Am. J. Physiol.

Renal Physiol. 2013, 305, F1064-F1073. (4) Liu, N.; Tian, J.; Cheng, J.; Zhang, J. Migration of CXCR4 gene-modified bone marrow-derived mesenchymal stem cells to the acute injured kidney. J. Cell.

Biochem. 2013, 114, 2677-2689. (5) Chen, Y. T.; Yang, C. C.; Zhen, Y. Y.; Wallace, C. G.; Yang, J. L.; Sun, C. K.; Tsai, T. H.; Sheu, J. J.; Chua, S.; Chang, C. L.; Cho, C. L.; Leu, S.; Yip, H. K. Cyclosporine-assisted adipose-derived mesenchymal stem cell therapy to mitigate acute kidney ischemia-reperfusion injury. Stem Cell Res. Ther. 2013, 4, 62-82. (6) Chen, B.; Bo, C. J.; Jia, R. P.; Liu, H.; Wu, R.; Wu, J.; Ge, Y. Z.; Teng, G. J. The renoprotective effect of bone marrow-derived endothelial progenitor cell


Page 40 of 52

Page 41 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


transplantation on acute ischemia-reperfusion injury in rats. Tansplant. Proc. 2013,

45, 2034-2039. (7) Humphreys, B. D.; Bonventre, J. V. Mesenchymal stem cells in acute kidney injury. Annu. Rev. Med. 2008, 59, 311-325. (8) Wise, A. F.; Ricardo, S. D. Mesenchymal stem cells in kidney inflammation and repair. Nephrology 2012, 17, 1-10. (9) Crump, M. P.; Gong, J. H.; Loetscher, P.; Rajarathnam, K.; Amara, A.; ArenzanaSeisdedos, F.; Virelizier, J. L.; Baggiolini, M.; Sykes, B. D.; Clark-Lewis, I. Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO J. 1997, 16, 6996-7007. (10) Anders, H. J.; Romagnani, P.; Mantovani, A. Pathomechanisms: homeostatic chemokines in health, tissue regeneration, and progressive diseases. Trends Mol.

Med. 2014, 20, 154-165. (11) Scotton, C. J.; Wilson, J. L.; Scott, K.; Stamp, G.; Wilbanks, G. D.; Fricker, S.; Bridger, G.; Balkwill, F. R. Multiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer. Cancer Res. 2002, 62, 5930-5938. (12) Balkwill, F. The significance of cancer cell expression of the chemokine receptor CXCR4. Semin. Cancer Biol. 2004, 14, 171-179.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(13) Hwang, J. H.; Hwang, J. H.; Chung, H. K.; Kim, D. W.; Hwang, E. S.; Suh, J. M.; Kim, H.; You, K. H.; Kwon, O. Y.; Ro, H. K.; Jo, D. Y.; Shong, M. CXC chemokine receptor 4 expression and function in human anaplastic thyroid cancer cells. J. Clin. Endocrinol. Metab. 2003, 88, 408-416. (14) Peng, S. B.; Peek, V.; Zhai, Y.; Paul, D. C.; Lou, Q.; Xia, X.; Eessalu, T.; Kohn, W.; Tang, S. Akt activation, but not extracellular signal-regulated kinase activation, is required for SDF-1α/CXCR4-mediated migration of epitheloid carcinoma cells.

Mol. Cancer Res. 2005, 3, 227-236. (15) Leng, Q.; Nie, Y.; Zou, Y.; Chen, J. Elevated CXCL12 expression in the bone marrow of NOD mice is associated with altered T cell and stem cell trafficking and diabetes development. BMC Immunol. 2008, 9, 51-62. (16) Clark, B. R.; Keating, A. Biology of bone marrow stroma. Ann. NY. Acad. Sci. 1995, 770, 70-78. (17) Lattanzio, M. R.; Kopyt, N. P. Acute kidney injury: new concepts in definition, diagnosis, pathophysiology, and treatment. J. Am. Osteopath. Assoc. 2009, 109, 1319. (18) De Clercq, E. The AMD3100 story: the path to the discovery of a stem cell mobilizer (Mozobil). Biochem. Pharmacol. 2009, 77, 1655-1664.


Page 42 of 52

Page 43 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(19) Liles, W. C.; Broxmeyer, H. E.; Rodger, E.; Wood, B.; Hübel, K.; Cooper, S.; Hangoc, G.; Bridger, G. J.; Henson, G. W.; Calandra, G.; Dale, D. C. Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD3100, a CXCR4 antagonist. Blood 2003, 102, 2728-2730. (20) Devine, S. M.; Flomenberg, N.; Vesole, D. H.; Liesveld, J.; Weisdorf, D.; Badel, K.; Calandra, G.; DiPersio, J. F. Rapid mobilization of CD34+cells following administration of the CXCR4 antagonist AMD3100 to patients with multiple myeloma and non-Hodgkin’s lymphoma. J. Clin. Oncol. 2004, 22, 1095-1102. (21) Hübel, K.; Liles, W. C.; Broxmeyer, H. E.; Rodger, E.; Wood, B.; Cooper, S.; Hangoc, G.; MacFarland, R.; Bridger, G. J.; Henson, G. W.; Calandra, G.; Dale, D. C. Leukocytosis and mobilization of CD34+ hematopoietic progenitor cells by AMD3100, a CXCR4 antagonist. Support. Cancer Ther. 2004, 1, 165-172. (22) Holtan, S. G.; Porrata, L. F.; Micallef, I. N. M.; Padley, D. J.; Inwards, D. J.; Ansell, S. A.; Johnston, P. B.; Gastineau, D. A.; Markovic, S. N. AMD3100 affects autograft lymphocyte collection and progression-free survival after autologous stem cell transplantion in non-Hodgkin lymphoma. Clin. Lymphoma Myeloma 2007, 7, 315-318. (23) Cashen, A.; Lopez, S.; Gao, F.; Calandra, G.; MacFarland, R.; Badel, K.; DiPersio, J. A phase II study of plerixafor (AMD3100) plus G-CSF for autologous



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

hematopoietic progenitor cell mobilization in patients with Hodgkin lymphoma.

Biol. Blood Marrow Transplant. 2008, 14, 1253-1261. (24) Brave, M.; Farrell, A.; Lin, S. C.; Ocheltree, T.; Miksinski, S. P.; Lee, S. L.; Saber, H.; Fourie, J.; Tornoe, C.; Booth, B.; Yuan, W.; He, K.; Justice, R.; Pazdur, R. FDA review summary: Mozobil in combination with granulocyte colonystimulating factor to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation. Oncology 2010, 78, 282-288. (25) DiPersio, J. F.; Uy, G. L.; Yasothan, U.; Kirkpatrick, P. Plerixafor. Nature Rev.

Drug Discov. 2009, 8, 105-106. (26) Calandra, G.; McCarty, J.; McGuirk, J.; Tricot, G.; Crocker, S. A.; Badel, K.; Grove, B.; Dye, A.; Bridger, G. AMD3100 plus G-CSF can successfully mobilize CD34+ cells from non-Hodgkin's lymphoma, Hodgkin's disease and multiple myeloma patients previously failing mobilization with chemotherapy and/or cytokine treatment: compassionate use data. Bone Marrow Transplant. 2008, 41, 331-338. (27) Shepherd, R. M.; Capoccia, B. J.; Devine, S. M.; DiPersio, J.; Trinkaus, K. M.; Ingram, D.; Link, D. C. Angiogenic cells can be rapidly mobilized and efficiently harvested from the blood following treatment with AMD3100. Blood 2006, 108, 3662-3667.


Page 44 of 52

Page 45 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(28) Xia, X. M.; Wang, F. Y.; Xu, W. A.; Wang, Z. K.; Liu, J.; Lu, Y. K.; Jin, X. X.; Lu, H.; Shen, Y. Z. CXCR4 antagonist AMD3100 attenuates colonic damage in mice with experimental colitis. World J. Gastroenterol. 2010, 16, 2873-2880. (29) Chen, J.; Chen, J.; Chen, S.; Zhang, C.; Zhang, L.; Xiao, X.; Das, A.; Zhao, Y.; Yuan, B.; Morris, M.; Zhao, B.; Chen, Y. Transfusion of CXCR4-primed endothelial progenitor cells reduces cerebral ischemic damage and promotes repair in db/db diabetic mice. PloS One 2012, 7, e50105. (30) For recent reviews, see: (a) Längle, D.; Halver, J.; Rathmer, B.; Willems, E.; Schade, D. Small molecules targeting in vivo tissue regeneration. ACS Chem. Biol. 2014, 9, 57-71. (b) Debnath, B.; Xu, S.; Grande, F.; Garofalo, A.; Neamati, N. Small molecule inhibitors of CXCR4. Theranostics 2013, 3, 47-75. (c) Choi, W. T.;

Duggineni, S.; Xu, Y.; Huang, Z.; An, J. Drug discovery research targeting the CXC chemokine receptor 4 (CXCR4). J. Med. Chem. 2012, 55, 977-994. (d) Allsopp, T. E.; Bunnage, M. E.; Fish, P. V. Small molecule modulation of stem cells in regenerative medicine: recent applications and future direction. Med. Chem.

Commun. 2010, 1, 16-29. (31) Tamamura, H.; Xu, Y.; Hattori, T.; Zhang, X.; Arakaki, R.; Kanbara, K.; Omagari, A.; Otaka, A.; Ibuka, T.; Yamamoto, N.; Nakashima, H.; Fujii, N. A low-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 52

molecular-weight inhibitor against the chemokine receptor CXCR4: a strong antiHIV peptide T140. Biochem. Biophys. Res. Commun. 1998, 253, 877-882. (32) (a) Hsu, W. T.; Jui, H. Y.; Huang, Y. H.; Su, M. Y. M.; Wu, Y. W.; Tseng, W. Y. I.; Hsu, M. C.; Chiang, B. L.; Wu, K. K.; Lee, C. M. CXCR4 antagonist TG-0054 mobilizes mesenchymal stem cells, attenuates inflammation, and preserves cardiac systolic function in a porcine model of myocardial infarction. Cell Transplant. DOI: 10.3727/096368914X681739. (b) Chung, D. T.; Chang, L. W.; Huang, Y. H.; Tsai, C. Y.; Hsu, C. H.; King, C. H. R.; Yuan, J. H.; Yen, C. F.; Chen, Y. M.; Lu, Y. C.; Hsu, M. C. TG-0054, a novel and potent stem cell mobilizer, displays excellent PK/PD and safety profile in phase I trial. Blood (ASH Annual Meeting

Abstracts), 2009, 114:866. (c) Huang, Y. H.; Liu, Y. C.; Yen, C. F.; Chen, H. C.; Chen, S, J.; King, C. H. R.; Hsu, M. C. Rapid mobilization of murine hematopoietic stem and progenitor cells with TG-0054, a novel CXCR4 antagonist.

Blood (ASH Annual Meeting Abstracts), 2009, 114:3542. (d) Yen, C. F.; Hu, C. K.; Chou, M. C.; Tseng, C. T.; Wu, C. H.; Huang, Y. H.; Chen, S. J.; King, C. H. R. Pyrimidine compounds. US7589197 B2. (e) Yen, C. F.; Hu, C. K.; Huang, C. P.; Huang, Y. H.; Hakimelahi, G. H.; King, C. H. R. Heterocyclic compounds. US8372849

B2.

(f)

(http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=44479007,


Burixafor. news

Page 47 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


released on December 15, 2009). (g) PD and safety of TG-0054 combined with GCSF in multiple myeloma, non-Hodgkin lymphoma and Hodgkin disease patients (http://clinicaltralsgov). (33) (a) Stone, N. D.; Dunaway, S. B.; Flexner, C.; Tierney, C.; Calandra, G. B.; Becker, S.; Cao, Y. J.; Wiggins, I. P.; Conley, J.; MacFarland, R. T.; Park, J. G.; Lalama, C.; Snyder, S.; Kallungal, B.; Klingman, K. L.; Hendrix, C. W. Multipledose escalation study of the safety, pharmacokinetics, and biologic activity of oral AMD070, a selective CXCR4 receptor inhibitor, in human subjects. Antimicrob.

Agents Chemother. 2007, 51, 2351−2358. (b) Safety and activity of the oral HIV entry inhibitor AMD11070 in HIV infected patients (http://clinicaltralsgov). (34) (a) Shu, H. K. G.; Yoon, Y.; Hong, S.; Xu, K.; Gao, H.; Hao, C.; Torres-Gonzalez, E.; Nayra, C.; Rojas, M.; Shim, H. Inhibition of the CXCL12/CXCR4-axis as preventive therapy for radiation-induced pulmonary fibrosis. PloS One 2013, 8, e79768. (b) MSX-122 administered orally in patients with refractory metastatic or locally advanced solid tumors (http://clinicaltralsgov). (35) (a) Beider, K.; Darash-Yahana, M.; Blaier, O.; Koren-Michowitz, M.; Abraham, M.; Wald, H.; Wald, O.; Galun, E.; Eizenberg, O.; Peled, A.; Nagler, A. Combination of imatinib with CXCR4 antagonist BKT140 overcomes the protective effect of stroma and targets CML in vitro and in vivo. Mol. Cancer Ther.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 52

2014, 13, 1155-1169. (b) Imatinib and BL-8040 (novel anti CXCR4 antagonist) for improving

molecular

response

in

chronic

myelogenous

leukemia

(http://clinicaltralsgov). (36) (a) Galsky, M. D.; Vogelzang, N. J.; Conkling, P.; Raddad, E.; Polzer, J.; Roberson, S.; Stille, J. R.; Saleh, M.; Thornton, D. A phase I trial of LY2510924, a CXCR4 peptide antagonist, in patients with advanced cancer. Clin. Cancer Res. 2014, 20, 3581-3588. (b) A study of LY2510924 in patients with extensive-stage small cell lung carcinoma (http://clinicaltralsgov). (37) Wong, D.; Korz, W. Translating an antagonist of chemokine receptor CXCR4: from bench to beside. Clin. Cancer Res. 2008, 14, 7975-7980. (38) (a) Wolfson, W. Grabbing for the ring: macrocycles tweak the conventions of drug making. Chem. Biol. 2012, 19, 1356-1357. (b) Schmitt, S.; Weinhold, N.; Dembowsky, K.; Neben, K.; Witzens-Harig, M.; Braun, M.; Klemmer, J.; Wuchter, P.; Ludin, C.; Ho, A. D.; Goldschmidt, H. First results of a phase-II study with the new CXCR4 antagonist POL6326 to mobilize hematopoietic stem cells (HSC) in multiple myeloma (MM). Blood (ASH Annual Meeting Abstracts), 2010, 116:824. (c) Chen, Y.; Zeng, Z.; Shi, Y.; Jacamo, R.; Ludin, C.; Dembowsky, K.; Frenette, P. S.; Konopleva, M.; Andreeff, M. Targeting CXCR4, SDF1 and beta-adrenergic receptors in the AML microenvironment by novel antagonist POL6326, G-CSF and


Page 49 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Isoproterenol. Blood (ASH Annual Meeting Abstracts), 2010, 116:2179. (d) Safety and efficacy of POL6326 for mobilization/transplant of sibling donor in patients with hematologic malignancies (http://clinicaltralsgov). (39) Wu, C. H.; Chang, C. P.; Song, J. S.; Jan, J. J.; Chou, M. C.; Wu, S. H.; Yeh, K. C.; Wong, Y. C.; Hsieh, C. J.; Chen, C. T.; Kao, T. T.; Wu, S. Y.; Yeh, C. F.; Tseng, C. T.; Chao, Y. S.; Shia, K. S. Discovery of novel stem cell mobilizers that target the CXCR4 receptor. ChemMedChem 2012, 7, 209-212. (40) (a) Ichiyama, K.; Yokoyama-Kumakura, S.; Tanaka, Y.; Tanaka, R.; Hirose, K.; Bannai, K.; Edamatsu, T.; Yanaka, M.; Niitani, Y.; Miyano-Kurosaki, N.; Takaku, H.; Koyanagi, Y.;

Yamamoto, N. A duodenally absorbable CXC chemokine

receptor 4 antagonist, KRH-1636, exhibits a potent and selective anti-HIV-1 activity. Proc. Natl. Acad. Sci. USA. 2003, 100, 4185-4190. (b) Tamamura, H.; Araki, T.; Ueda, S.; Wang, Z.; Oishi, S.; Esaka, A.; Trent, J. O.; Nakashima, H.; Yamamoto, N.; Peiper, S. C.; Otaka, A.; Fujii, N. Identification of novel low molecular weight CXCR4 antagonists by structural tuning of cyclic tetrapeptide scaffolds. J. Med. Chem. 2005, 48, 3280-3289. (c) Ueda, S.; Oishi, S.; Wang, Z. X.; Araki, T.; Tamamura, H.; Cluzeau, J.; Ohno, H.; Kusano, S.; Nakashima, H.; Trent, J. O.; Peiper, S. C.; Fujii, N. Structure-activity relationships of cyclic peptide-based chemokine receptor CXCR4 antagonists: disclosing the importance of side-chain



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 52

and backbone functionalities. J. Med. Chem. 2007, 50, 192-198. (d) Demmer, O.; Dijkgraaf, I.; Schottelius, M.; Wester, H. J.; Kessler, H. Introduction of functional groups into peptides via N-alkylation. Org. Lett. 2008, 10, 2015-2018. (e) Thoma, G.; Streiff, M. B.; Kovarik, J.; Glickman, F.; Wagner, T.; Beerli, C.; Zerwes, H. G. Orally bioavailable isothioureas block function of the chemokine receptor CXCR4

in vitro and in vivo. J. Med. Chem. 2008, 51, 7915-7920. (f) Zhu, A.; Zhan, W.; Liang, Z.; Yoon, Y.; Yang, H.; Grossniklaus, H. E.; Xu, J.; Rojas, M.; Lockwood, M.; Snyder, J. P.; Liotta, D. C.; Shim, H. Dipyrimidine amines: a novel class of chemokine receptor type 4 antagonists with high specificity. J. Med. Chem. 2010,

53, 8556-8568. (g) Miller, J. F.; Gudmundsson, K. S.; Richardson, L. D.; Jenkinson, S.; Spaltenstein, A.; Thomson, M.; Wheelan, P. Synthesis and SAR of novel isoquinoline CXCR4 antagonists with potent anti-HIV activity. Bioorg. Med.

Chem. Lett. 2010, 20, 3026-3030. (h) Catalano, J. G.; Gudmundsson, K. S.; Svolto, A.; Boggs, S. D.; Miller, J. F.; Spaltenstein, A.; Thomson, M.; Wheelan, P.; Minick, D. J.; Phelps, D. P.; Jenkinson, S. Synthesis of a novel tricyclic 1,2,3,4,4a,5,6,10b-octahydro-1,10-phenanthroline

ring

system

and

CXCR4

antagonists with potent activity against HIV-1. Bioorg. Med. Chem. Lett. 2010, 20, 2186-2190. (i) Miller, J. F.; Turner, E. M.; Gudmundsson, K. S.; Jenkinson, S.; Spaltenstein, A.; Thomson, M.; Wheelan, P. Novel N-substituted benzimidazole


Page 51 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CXCR4 antagonists as potential anti-HIV agents. Bioorg. Med. Chem. Lett. 2010,

20, 2125-2128. (j) Skerlj, R.; Bridger, G.; McEachern, E.; Harwig, C.; Smith, C.; Wilson, T.; Veale, D.; Yee, H.; Crawford, J.; Skupinska, K.; Wauthy, R.; Yang, W.; Zhu, Y.; Bogucki, D.; Di Fluri, M.; Langille, J.; Huskens, D.; De Clercq, E.; Schols, D. Synthesis and SAR of novel CXCR4 antagonists that are potent inhibitors of T tropic (X4) HIV-1 replication. Bioorg. Med. Chem. Lett. 2011, 21, 262-266. (k) Inokuchi, E.; Yamada, A.; Hozumi, K.; Tomita, K.; Oishi, S.; Ohno, H.; Nomizu, M.; Fujii, N. Design and synthesis of amidine-type peptide bond isosteres: application of nitrile oxide derivatives as active ester equivalents in peptide and peptidomimetics synthesis. Org. Biomol. Chem. 2011, 9, 3421-3427. (l) Inokuchi, E.; Oishi, S.; Kubo, T.; Ohno, H.; Shimura, K.; Matsuoka, M.; Fujii, N. Potent CXCR4 antagonists containing amidine type peptide bond isosteres. Med.

Chem. Lett. 2011, 2, 477-480. (m) Mooring, S. R.; Liu, J.; Liang, Z.; Ahn, J.; Hong, S.; Yoon, Y.; Snyder, J. P.; Shim, H. Benzenesulfonamides: a unique class of chemokine receptor type 4 inhibitors. ChemMedChem 2013, 8, 622-632. (41) (a) Wu, B.; Chien, E. Y. T.; Mol, C. D.; Fenalti, G.; Liu, W.; Katritch, V.; Abagyan, R.; Brooun, A.; Wells, P.; Bi, F. C.; Hamel, D. J.; Kuhn, P.; Handel, T. M.; Cherezov, V.; Stevens, R. C. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 2010, 330, 1066-1071. (b)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Zhu, L; Zhao, Q.; Wu, B. Structure-based studies of chemokine receptors. Curr.

Opin. Struct. Biol. 2013, 23, 539-546. (42) Westvik, T. S.; Fitzgerald, T. N.; Muto, A.; Maloney, S. P.; Pimiento, J. M.; Fancher, T. T.; Magri, D.; Westvik, H. H.; Nishibe, T.; Velazquez, O. C.; Dardik, A. Limb ischemia after iliac ligation in aged mice stimulates angiogenesis without arteriogenesis. J. Vasc. Surg. 2009, 49, 464-473. (43) An, N.; Lin, Y. W.; Mahajan, S.; Kellner, J. N.; Wang, Y.; Li, Z.; Kraft, A. S.; Kang, Y. Pim1 serine/threonine kinase regulates the number and functions of murine hematopoietic stem cells. Stem Cells 2013, 31, 1202-1212. (44) Zuk, A.; Gershenovich, M.; Ivanova, Y.; MacFarland, R. T.; Fricker, S. P.; Ledbetter, S. CXCR4 antagonism as a therapeutic approach to prevent acute kidney injury. Am. J. Physiol. Renal Physiol. 2014, 307, F783-F797. (45) Novel developments in stem cell mobilization focus on CXCR4. Fruehauf, S.; Zeller, W. J.; Calandra, G.; Eds; Springer: New York, 2012.


Page 52 of 52

Page 53 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents Graphic


Stem Cell Mobilizers Targeting Chemokine Receptor CXCR4

Recommend Documents