Accepted Manuscript Synthesis and SAR study of potent and selective PI3Kδ inhibitors Minna Bui, Xiaolin Hao, Youngsook Shin, Mario Cardozo, Xiao He, Kirk Henne, Julia Suchomel, John McCarter, Lawrence R. McGee, Tisha San Miguel, Julio C. Medina, Deanna Mohn, Thuy Tran, Sharon Wannberg, Jamie Wong, Simon Wong, Leeanne Zalameda, Daniela Metz, Timothy D. Cushing PII: DOI: Reference:

S0960-894X(15)00003-7 http://dx.doi.org/10.1016/j.bmcl.2015.01.001 BMCL 22341

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

17 October 2014 31 December 2014 2 January 2015

Please cite this article as: Bui, M., Hao, X., Shin, Y., Cardozo, M., He, X., Henne, K., Suchomel, J., McCarter, J., McGee, L.R., Miguel, T.S., Medina, J.C., Mohn, D., Tran, T., Wannberg, S., Wong, J., Wong, S., Zalameda, L., Metz, D., Cushing, T.D., Synthesis and SAR study of potent and selective PI3Kδ inhibitors, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.01.001

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Synthesis and SAR study of Potent and Selective PI3Kδ Inhibitors Minna Bui*a, Xiaolin Hao*a, Youngsook Shina, Mario Cardozoa, Xiao Hea, Kirk Henneb, Julia Suchomela, John McCarterc, Lawrence R. McGeea, Tisha San Migueld, Julio C. Medinaa, Deanna Mohnd, Thuy Tranb, Sharon Wannbergd, Jamie Wongb, Simon Wongb, Leeanne Zalamedad, Daniela Metzd, Timothy D. Cushinga Department of Therapeutic Discovery and bDepartment of Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States cDepartment of Therapeutic Discovery and dDepartment of Inflammation Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States a

ABSTRACT: 2, 3, 4-Substituted quinolines such as (10a) were found to be potent inhibitors of PI3Kδ in both biochemical and cellular assays with good selectivity over three other class I PI3K isoforms. Some of those analogs showed favorable pharmacokinetic properties.

There has been intense interest in the class I phosphoinositide 3-kinases (PI3Ks) that regulate phosphatidylinositol 4,5-bisphosphate (PIP2) phosphorylation. PI3K Converts PIP2 to the scaffolding binding element Phosphatidylinositol (3,4,5)trisphosphate (PIP3). PIP3 plays a key regulatory role in cell survival, signal transduction, control of membrane trafficking and other functions.1,2 Its dysregulation leads to various disease states such as cancer, inflammatory and auto-immune disorders. The Class I PI3Ks consist of four kinases further delineated into 2 subclasses. Class 1A PI3Ks consist of three closely related kinases, PI3Kα, β, and δ existing as heterodimers composed of a catalytic subunit (p110α, β or δ) and one of several regulatory subunits. They generally respond to signaling through receptor tyrosine kinases (RTKs). PI3Kγ single class 1B isoform, responds mainly to G-protein coupled receptors (GPCRs), and is composed of a p110γ catalytic subunit and one of two distinct regulatory subunits. PI3Kα and PI3Kβ are ubiquitously expressed throughout a wide variety of tissue and organ types. PI3Kγ is found mainly in leukocytes, but also in skeletal muscle, liver, pancreas, and heart.3 The expression pattern of PI3Kδ is restricted, to spleen, thymus, and peripheral blood leukocytes.4 Because of this expression pattern and evidence accumulated with genetically modified mice, PI3Kδ has been implicated as a major player in the adaptive immune system. The recent success of the B-cell inhibiting biologics rituximab (Rituxan) and later belimumab (Benlysta) targeting rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), respectively, helped trigger our interest in developing a small molecule targeting PI3Kδ.

idelalisib (GS 1101)

IPI-145 (INK-1197) Figure 1

Several PI3Kδ selective inhibitors, such as, idelalisib (GS-1101), IPI-1455 and AMG 3196 have entered the clinic targeting hematological malignancies, (Fig. 1). The most advanced idelalisib, is approved for relapsed follicular B-cell non-Hodgkin’s lymphoma (FL) and for relapsed chronic lymphocytic leukemia (CLL) in combination with rituximab. In spite of the success of B-cell modulating drugs like rituximab and belimumab, few PI3Kδ inhibitors have been reported to have advanced further than phase 1 for inflammation, with the exception of IPI-145 (dual inhibitor of PI3Kδ, γ) which has advanced into phase 2 for rheumatoid arthritis7 and asthma.8

quinoline, A

reversed quinoline,B

Figure 2 As part of our PI3Kδ drug discovery program for inflammation, we recently reported a series of potent and selective inhibitors with a quinoline core structure.9,10,11,12 Herein, we describe our efforts to expand the chemistry series, identifying an alternative core structure, its synthesis, SAR studies and in vivo PK properties. We knew from previous SAR that small polar groups such as aza-nitrogen substitution at the 4-position of quinoline A were tolerated, but structural studies indicated that the 1-quinoline position would be amenable to larger polar groups, providing an opportunity for further enhancement of other physico-chemical parameters.14 Additionally, molecular modeling indicated that these so-called reverse quinolines B, would not deviate much from the overall confirmation of A (Fig 2).

Scheme 1a H2N

H N

O

a F

Ph

EtO2C

N

d 43%, 3 steps

F

F OH

11

r

12 N

N

Cl

HO

b, c

N

e F

f F

70%

Cl 13

F 58%

Cl 14

S 17

m

N

o

47%

g Br

N

F

N

OMe

F

15

F

S g, h

S

16

18

OH NPhth N

p, g, h, q F

NPhth N

NPhth N

OR n

h, 71% 2 steps

F

R= Me: 20a, 60% R= H: 20b, 80%

S

F O

j, k, l

19

S

21

OH

NH2 CN

N N

NH2

NH2

j, k, l 25%

CN

N

NH

N

NH

N

F

OR

2b: R= H, 33% a

NH

N

F

2a: R= Me, 24%

CN

N N

N

i, j, k, l 27%

F O

S

OH

S O O

1

3

Reagents and conditions: (a) diethyl 2-phenylmalonate, pyr. 130 oC, 16 h, 44%; (b) LiOH, THF, 2 h; (c) PPA, 130 oC, 2 h; (d) POCl3, 100 oC, 2 h, 43% 3 steps; (e) ZnEt2, K2CO3, PdCl2(dppf)2.DCM, THF, reflux, 6 h, 70-92%; (f) NaSMe, DMF, 60 oC, 30 min, 58%; (g) NBS, benzoyl peroxide, CCl4, reflux, 3 h; (h) phthalimide potassium salt, DMF, 60 oC, 16 h, 71% 2 steps; (i) oxone, THF/water (1:1), rt, 16 h, 83%; (j) hydrazine, EtOH, 60 oC, 30 min; (k) 4-amino-6-chloropyrimidine-5-carbonitrile, n-BuOH, 100 oC, 16 h; (l) chiral separation AD column; (m) NaOMe, DMF, 80 °C, 5 h, 47%; (n) BBr3, DCM, 0 °C-rt, 16 h, 80%; (o) HSCH2CH2OH, DMF,100 °C, 16 h, 44%; (p) TBS-Cl, imidazole, DMF, 2 h, 97%; (q) oxone, THF/water (3:1), rt, 16 h, 75%; (r) 2-phenylmalonic acid, POCl3, 60oC, 60%.

Initial preparation of the 2,3,4-reversed quinoline analog 1 was carried out as shown in Scheme 1. 4-Fluoroaniline was condensed with diethyl 2-phenylmalonate to provide 11. Hydrolysis of ethyl ester 11 with LiOH in THF provided the corresponding acid, which was then cyclized in polyphosphoric acid to give 12. Intermediate 12 underwent chlorination by heating in phosphorus oxychloride providing 13, which was treated with diethyl zinc to give the key chloro-quinoline 14. Alternatively, we found that 13 could be accessed directly by condensation and cyclization of 4-fluoroaniline and 2-phenylmalonic acid in hot POCl3. Chloro-quinoline 14 was treated with NaOMe, 2mercaptoethanol, or NaSMe to give 15, 16 and 17 respectively. Bromination of 17 with NBS provided adduct 18, which was treated with phthalimide potassium salt to give 19. Subsequent oxone oxidation, phthalamide deprotection and treatment with 4-amino-6chloropyrimidine-5-carbonitrile gave the desired product as a racemic mixture. Enantiomerically pure 1 was obtained after chiral chromatography. The assignment of chirality was based on the biochemical potency in comparison with known analogs.17 Analogs 2a-b and 3 were obtained in the similar manner where as 3 was the racemic sulfoxide. Analogs synthesized other than by direct displacement at the 4-position required slightly modified reaction conditions (Scheme 2). Bromination of 14 with NBS provided 22, which was treated with phthalimide potassium salt to give 23. Compounds 24a-e were obtained by nucleophilic displacement of the chloride 23 with various amines followed by phthalimide deprotection with hydrazine to provide 25a-e. Finally, treatment with 4-amino-6-chloropyrimidine-5-carbonitrile and chiral chromatography separation gave 4a-e. Scheme 2a

Br

N

N

a

b

O

O

N

N

F

71%, 2 steps

F

Cl Cl

14

F Cl

22

23 NH2

c

O

N

NH2

O

d

N

e, f

N 30-60%

85-94% F HN 24a-e

R

CN

N

F HN 25a-e

34-40%

N

NH N

R

F HN 4a-e

R

a

Reagents and conditions: (a) NBS, benzoyl peroxide, CCl4, reflux, 3 h; (b) phthalimide potassium salt, DMF, 60 oC, 16 h, 71%, 2 steps; (c) amines, Pd2(dba)3, Xantphos, NaOtBu, 100 oC, 3 h, 30-60%; (d) hydrazine, EtOH, 60 oC, 30min; (e) 4-amino-6chloropyrimidine-5-carbonitrile, n-BuOH, 100 oC, 16 h; (f) chiral separation AD column, 34-40% for 2 steps

Pyridine analog 26 was synthesized from (s)-alanine derived Weinreb amide13 and isopropyl magnesium bromide followed by (pyridin-2-yl-methyl)lithium. Employing the Friedlander synthesis,2-amino-5-fluorobenzaldehyde14 was condensed and cyclized with 26 to form racemic 27 as rapid epimerization of the starting material recurred under these basic conditions. This was followed by Boc-deprotection and displacement of the chloride with 4-amino-6-chloropyrimidine-5-carbonitrile to give the desired product as a racemic mixture 5 (Scheme 3). Scheme 3a NH2

Boc Boc NH O N O

NHBoc

58%

NH

O

N 26

N N

b

a

CN

N

52%

F N

27

NH

c, d

N

63%

F N

5

a

Reagents and conditions: (a) isopropylmagnesium chloride, THF, -60 °C, (pyridin-2-yl-methyl) lithium, 58%; (b) 2-amino-5fluorobenzaldehyde, K2CO3, EtOH, reflux, 1 h, 52%; (c) TFA, DCM; (d) 4-amino-6-chlorophyridine-5-carbonitrile, n-BuOH, 100 oC, 16 h, 63% for 2 steps.

Another synthetic route was developed to introduce different groups at the 3position. 4-Fluoroaniline was treated with methyl-3-oxopentanoate to give cyclized product 28. Iodination of 28 delivered 29, which was subjected to Stille coupling conditions to provide 30. Treatment of 30 with POCl3 provided 31, which upon bromination, phthalimide formation and deprotection gave amine 32. This led directly to 6 upon treatment with 4-amino-6-chloropyrimidine-5-carbonitrile followed by a chiral purification. As the intermediate phthalimide was not stable under the conditions required to install the 4-amino substituents found in 7a-c, a protecting group exchange was employed. Hence, Boc-protected amine 33 was prepared under standard conditions from amine 32 and analogs 7a-c could be synthesized via a 3-step sequence followed by chiral chromatography (Scheme 4).

Scheme 4a

a

Reagents and conditions: (a) p-toluene sulfonic acid, methyl-3-oxapentanoate cyclohexane, 95 oC, 16 h, 43%; (b) Na2CO3, I2, THF, rt, overnight, 90%; (c) 2-tributylstannyl)pyridine, Pd(PPh3)4, dioxane, reflux, overnight, 13%; (d) POCl3, 100 oC, 2 h, 92%; (e) NBS, benzoyl peroxide, CCl4, reflux, 3 h; (f) phthalimide potassium salt, DMF, 60 oC, (g) hydrazine, EtOH, 60oC, 30min; 16 h, 41% for 3 steps; (h) Boc2O, DCM; (i) amines, Pd2(dba)3, Xantphos, NaOtBu, 100 oC, 3 h; (j) TFA, DCM, 40-74% 2 for steps; (k) 4-amino-6chloropyrimidine-5-carbonitrile, n-BuOH, 100oC, 16 h; (l) chiral separation AD column.

Still other routes were developed for specific compounds. Friedlander condensation and cyclization of 26 with 1-(2-amino-5-fluorophenyl)-2-chloroethanone15 gave 34. Treatment of 34 with methanolic sodium methoxide followed by BOC deprotection with TFA provided 35, which was treated with 4-amino-6-chloropyrimidine5-carbonitrile and a final chiral separation to afford 8. Attempted SNAr displacement of the chlorine in 31with sodium cyanide failed to provide compound 37. Instead, Pfitzinger condensation of 1-(pyridin-2-yl)-butan-2-one16 and 5-fluoroisatin provided the expected carboxylic acid in 80% yield but also fortuitously amide 36 as a minor product (9%). Because there was a sufficient quantity of 36, the carboxylic acid was set aside. Amide 36 was then converted to nitrile 37 using trifluoroacetic anhydride. Subsequently, following the steps previously described in Scheme 4 for the synthesis of 6, nitrile analog 9 was obtained in 61% yield from 37. 2-amino-5-fluoro-N-methoxy-N-methylbenzamide17 was treated with (pyridin-2yl) magnesium bromide or (6-methylpyridin-2-yl) magnesium bromide to give ketones 38a-b, respectively, albeit in low yield. Condensation and cyclization of 38a-b with 26 provided intermediates 39a-b, followed by displacement of the chloride with 4-amino-6chloropyrimidine-5-carbonitrile and chiral purification to give 10a-b (Scheme 5).

Scheme 5a

a

Reagents and conditions: (a) 1-(2-amino-5-fluorophenyl)-2-chloroethanone, neodymium(iii) nitrate hexahydrate, EtOH, 80 oC, overnight, 50%; (b) NaOMe, MeOH, 60 °C,4 h; (c) TFA, DCM, 40%; (d) 4-amino-6-chloropyrimidine-5-carbonitrile, nBuOH, 100 o C, 16 h; (e) chiral separation AD column; (f) 5-fluoroisatin, KOH, EtOH, reflux, 3 h; (g) pyridine, TFAA, dioxane, 0 oC to rt, overnight, 85%.(h) NBS, benzoyl peroxide, CCl4, reflux, 3 h; (i) phthalimide potassium salt, DMF, 60 oC, (j) hydrazine, EtOH, 60 oC, 30 min; 16 h, (k) (pyridin-2-yl)magnesium bromide, isopropylmagnesium chloride, THF, -78 °C to rt, overnight, 4%; (l) 26, sodium tetrachloroaurate(III) dihydrate, 2-propanol, reflux, 3 days, 59%.

As envisioned 1 showed sub-nanomolar potency in biochemical and cellular assays and reasonable selectivity against the isoforms as the modeling of quinoline A and quinoline B had indicated (Table 1).15 However, compound 1 was rapidly cleared in the rat (CL = 10 L/h/kg). Significant amount of glutathione adduct of the SO2Me was observed after incubation of 1 with glutathione for 30 minutes. Replacing the reactive sulfone with a more stable group was hypothesized to improve the in vivo PK properties. The methoxy compound 2a had sub-nanomolar potency in the biochemical and cellular assays but lost selectivity over other isoforms. Interestingly, the hydroxy compound 2b not only showed good biochemical and cellular activity, but also had moderate selectivity over the isoforms. Furthermore, it also had a reasonable rat PK profile with good clearance, 0.7 (L/h/Kg), and low %F (15%)18, while analogs 3 and 4a-e still retained excellent biochemical and cell potency. However, several 3-phenyl analogs, 2a and 4a-e had high hPXR activation that roughly correlated with high clogP. Replacing the 3-phenyl moiety with the more polar 3-pyridine could lower the clogP and perhaps effect the hPXR liability. Table 1: SAR of 3-phenyl analogs

No.

a

-R

Biochemicala

Cellularb

Potency PI3K IC50 (µM)

potency PI3Kδ pAKT

hPXRc

Safety

clogP

δ

γ

β

α

IC50 (µM)

POC 2µM

1

0.0007

0.22

0.51

0.92

0.0001

6

2.42

2a

0.0012

0.011

0.0082

0.42

0.0008

30

3.30

2b

0.016

0.21

>20

>20

0.0028

1.9

3.50

3

0.0025

0.38

0.16

0.62

-

-

2.03

4a

0.0013

0.062

0.28

5.08

0.007

58

3.06

4b

0.0004

0.12

0.46

1.20

0.0031

48

3.22

4c

0.0006

0.013

0.42

1.08

0.0024

86

3.98

4d

0.0017

1.60

5.0

1.60

-

24

4.19

4e

0.0002

0.019

0.50

0.90

0.0005

71

2.87

PI3K Alphascreen® assay; bHTRF® assays, phosphorylated-AKT; chuman Pregnane X Receptor, .data are shown as Percent Of Control, control, rifampicin, 10 µM.

As expected, analogs 5 and 6 were potent and even the hPXR activation of 5 was improved. (Table 2). Encouraged by this result, we conducted further SAR studies to understand the impact of the 3-pyridyl moiety. Certain substitution such as in 8 and 9 showed improved selectivity against other isoforms compared to 6. Substituting the 4position with a 2-pyridyl group was also examined. Analogs 10a-b significantly improved selectivity against the other isoforms. Additionally, 10a showed low clearance (CL = 0.057 L/h/kg) and good rat oral bioavailability (%F = 51).22 Table 2: SAR of 3-pyridyl analogs

No.

-R

Biochemicala

Cellularb

Potency PI3K IC50 (µM)

potency PI3Kδ pAKT

hPXRc

Safety

clogP

δ

γ

β

α

IC50 (µM)

POC 2µM

5

0.0088

1.10

>20

>20

-

-0.5

2.26

6

0.0040

0.25

0.23

1.37

0.011

34

2.76

7a

0.0014

0.29

0.71

1.15

0.0036

32

2.99

7b

0.018

0.51

0.28

1.21

0.0014

8

1.97

7c

0.0012

0.20

0.08

0.83

0.0012

5

2.28

8

0.016

4.95

4.03

-

0.0031

31

1.76

9

0.0083

1.42

0.24

4.4

0.012

5

1.84

10a

0.0024

0.85

2.8

7.9

0.0046

6

2.32

10b

0.015

>20

>20

>20

-

35

2.52

idelalisib

0.024

0.58

1.4

9.7

0.0027

3.7

3.62

a

PI3K Alphascreen® assay; bHTRF® assays, phosphorylated-AKT; chuman Pregnane X Receptor. Data are shown as Percent Of Control;

Moreover, the corresponding 3-methyl 10b had a superior selective profile; although, the rat clearance (CL = 0.21 L/h/kg) was inferior to that of 10a. Overall, the 3pyridyl analogs showed a superior PK profile, PXR activation levels and in the case of 10b improved selectivity. In summary, a series of potent and selective PI3Kδ inhibitors were synthesized. 3-Pyridyl substitution at the quinoline 3-position was critical for the overall improvement

of the analogs. The most promising compound, 10a, has good selectivity against other isoforms, good rat PK profile and reduced hPXR liability. References and Notes

1

(a) Di Paolo, G., De Camilli, P. Nature 2006, 443, 651. (b) Parker, P. J. Biochem. Soc. Trans.2004, 32, 893. (c) Hawkins, P. T., Anderson, K. E.; Stephens, L. R. Biochem. Soc. Trans.2006, 34, 647. 2 Schaeffer, E. M.; Schwartzberg, P. L.; Curr. Opin. Immnunol. 2000, 12, 282. 3 Cantly, C.; Science 2002, 1655. 4 Knight, Z.; Gonzalez, B.; Feldman, M.; Zunder, E.; Goldenberg, D.; Williams, O.; Loewith, R.; Stokoe, D.; Balla, A.; Toth, B.; Balla, T.; Weiss, W.; Williams, R. Shokat, K. Cell 2006, 125, 733-747. 5 Winkler, D. J.; Faia, K. L.; DiNitto, J. P.; Ali, J. A; White, K. F.; Brophy, E. E.; Pink, M. M.; Proctor, J. L.; Lussier, J.; Martin, C. M.; Hoyt, J. G.; Tillotson, B.; Murphy, E. L.; Lim, A. R.; Thomas, B. D.; MacDougall, J. R.; Ren, P.; Liu, Y.; Li, L.-S.; Jessen, K. A.; Fritz, C. C.; Dunbar, J. L.; Porter, J. R.; Rommel, C. , Palombella,V. J.; Changelian, P. S.; Kutok, J. L. Chemistry & Biology 2013, 20, 11, 1364. 6 Cushing, T. D.; Hao, X.; Shin, Y.; Andrews, K.; Brown, M.; Cardozo, M.; Chen, Y.; Duquette, J.; Fisher, B.; Gonzalez Lopez de Turiso, F.; He, X.; Henne, K. R.; Hu, Yi-L.; Hungate, R.; Johnson, M. G.; Kelly, R. C.; Lucas, B.; McCarter, J.; McGee, L. R.; Medina, J. C.; San Miguel, T.; Mohn, D.; Pattaropong, V.; Pettus, L. H.; Reichelt, A.; Rzasa, R. M.; Seganish, J.; Tasker, A. S.; Wahl, R. C.; Wannberg, S.; Whittington, D. A.; Whoriskey, J.; Yu, G.; Zalameda, L.; Zhang, D.; Metz, D. P. J. Med. Chem. Article ASAP DOI: /10.1021 / jm501624r. 7 National Institutes of Health. Identifier: NCT01851707, Clinical Trials.gov 8 National Institutes of Health. Identifier: NCT01653756, Clinical Trials.gov 9 Bui, M.; Fisher, B.; Hao, X.; Lucas, B.; WO2012003274(A1) 10 Chen, Y.; Cushing, T. D.; Duquette, J. A.; Gonzalez Lopez De Turiso, F.; Hao, X.; He, X.; Lucas, B.; McGee, L. R.; Reichelt, A.; Rzasa, R. M.; Seganish, J.; Shin, Y.; Zhang, D.; WO2008118468(A1) 11 Chen, Y.; Cushing, T. D.; Hao, X.; He, X.; Reichelt, A.; Rzasa, R.; Seganish, J.; Shin, Y.; Zhang, D.; US 20107705018(B2) 12 Gonzalez-Lopez de Turiso, F.; Shin, Y.; Brown, Y.; Cardozo, M.; Chen, Y.; Fong, D.; Hao, X.; He, X.; Henne, K.; Hu, Y.-L.; Johnson, M. G.; Kohn, T.; Lohman, J.; McBride, H. J.; McGee, L. R.; Medina, J. C.; Metz, D.; Miner, K.; Mohn, D.; Pattaropong, V.; Seganish, J.; Simard, J. L.; Wannberg, S.; Whittington, D. A.; Yu, G.; and Cushing, T. D. J. Med. Chem. 2012, 55, 17, 7667. 13 Morwick, T.; Hrapchak, M.; DeTuri, M.; Cambell, S. Org. Lett. 2002, 4, 16, 2665. 14 Easily prepared in two steps from 2-amino-fluorobenzoic acid: LAH, THF, 0 °C to reflux, 1.5 h, 99%; then MnO2, DCM, rt,16 h, 81%. 15 Varasi, M.; Torre, A. Delle; Heidempergher, F.; Pevarello, P.; Speciale, C.; Guidetti, P.; Wells, D. R.; Schwarcz, R. Eur. J. Med. Chem. 1996, 31, 11. 16 Konakahara, T.; Tagagi, Y. Hetercycles 1980, 14, 4, 393. 17 Barrow, J. C.; Cube, R. V.; Ngo, P. L.; Rittle, K. E.; Yang, Z.; Young S. D. WO2006098969(A2) 18 PK experiments were carried out using male Sprague-Dawley rats (n=3). Test compounds formulated at appropriate concentrations for either iv (0.5 mg/kg) or oral (2-10 mg/kg) administration.