Stereochemistry-activity relationship of orally active tetralin S1P agonist prodrugs

Article abstract of DOI:10.1016/j.bmcl.2010.02.006

Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (-)-(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydrona

Full text of DOI:10.1016/j.bmcl.2010.02.006

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2264–2269
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Stereochemistry–activity relationship of orally active tetralin S1P agonist
prodrugs
a
a
a
a
Bin Ma a, , Kevin M. Guckian , Edward Yin-Shiang Lin , Wen-Cherng Lee , Daniel Scott ,
Gnanasambandam Kumaravel a, Timothy L. Macdonald b, Kevin R. Lynch c, Cheryl Black a, Sowmya Chollate a,
Kyungmin Hahm a, Gregg Hetu a, Ping Jin a, Yi Luo a, Ellen Rohde a, Anthony Rossomando a,
Robert Scannevin a, Joy Wang a, Chunhua Yang a
*
a BiogenIdec Inc., 14 Cambridge Center, Cambridge, MA 02142, USA
b Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, VA 22904-4319, USA
c Department of Biochemistry and Molecular Genetics, and Department of Pharmacology, University of Virginia, McCormick Road, Charlottesville, VA 22904-4319, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin
Received 29 December 2009
Revised 31 January 2010
Accepted 2 February 2010
Available online 6 February 2010
analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (ꢀ)-
(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydronaphthalen-2-yl)propan-1-ol was identified as
a good
SphK2 substrate and potent S1P1 agonist with good oral bioavailability.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
S1P
FTY720
Multiple sclerosis
SphK2
Prodrug
Tetralin
X-ray
Multiple sclerosis (MS) is a chronic de-myelinating autoim-
mune disease that progressively worsens over time, affecting the
nerves in the brain, spinal cord, and other parts of the central ner-
vous system.1 MS affects two to three times as many women as
men with over 400,000 people in the United States having MS
and as many as 2,500,000 people affected worldwide.
Among many oral MS therapeutics under development, FTY720
(1, fingolimod) is interesting as it is the first in a new class of dis-
ease-modifying treatments called sphingosine 1-phosphate recep-
tor (S1P-R) modulators and has a novel mode of action.2 FTY720 is
a synthetic analog of myriocin, an antifungal antibiotic isolated
from entomopathogenic fungus Isaria sinclairii.3–5 Both myriocin
and FTY720 are sphingosine analogs that modulate immune re-
sponses in animals studies. Initial results from the two-year Phase
III FREEDOMS study show that oral FTY720 was superior to placebo
in reducing both relapses and disability progression in patients
with relapsing–remitting MS (RRMS), and some adverse effects
including bradycardia, skin cancer, liver injury, infections, and in-
creased blood pressure were observed during the clinic trials.6–9
FTY720 is a prodrug that is phosphorylated in vivo by sphingo-
sine kinase 2 (SphK2) to monophosphate FTY720-P (2)10 which is
an agonist of 4 of the 5 S1P receptors (S1P1, 3, 4, 5) but not
S1P2.11,12 Interaction of FTY720-P with S1P1 causes lymphopenia
by sequestering lymphocytes in secondary lymphoid organs.
Depletion of lymphocytes from the periphery is thought to be
the primary mechanism of action for FTY720.13 S1P3 activation
of FTY720-P is thought to be connected with the adverse effects,
such as bradycardia and bronchoconstriction in rodents.14,15
Here we report our effort to further define the molecular pharma-
cology of the S1P receptor family and SphK2 enzyme. By restricting
the two rotatable bonds between the phenyl ring and the aminodiol
head portion of FTY720 (1) with an sp3 hybridized backbone, a con-
formational restricted tetralin analog 3 (Fig. 1) was identified, which
evokes a profound, long lasting lymphopenia.16 However, com-
pound3 was amixtureof twostereoisomers, and aclearunderstand-
ing of biological activity of each isomer was necessary. In addition, it
is reported that FTY720 is phosphorylated stereo specifically by
SphK2 to produce the (S)-phosphate 2.10 This interesting observa-
tion prompted us to design the des-OH analog 5. This report presents
the synthesis of the two isomers of 3, all four isomers of 5, and the
effect of stereochemistry on their in vitro and in vivo activities.
* Corresponding author. Tel.: +1 617 914 4955; fax +1 617 679 3635.
E-mail address: bin.ma@biogenidec.com (B. Ma).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.006

B. Ma et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2264–2269
2265
NH2
OH
OH
Sphingosine
O
OH NH2
OH
CO2H
OH
Myriocin (ISP-1)
NH2
OR
OH
1. R = H, FTY720
2. R = PO
3H2, FTY720-P
Restricted
Conformation
NH2
NH2
Removal
nonphosphorylated OH
OR
OR
OH
5. R = H
6. R = PO
3. R = H,
4. R = PO
3H2
3H2
Figure 1. Further optimization strategy.
To obtain all isomers of 3 and 5, a convergent route was de-
signed to use a common intermediate 11 (Scheme 1). Starting from
commercially available 6-methoxy-3,4-dihydronaphthalen-1(2H)-
one (7), demethylation under HBr gave free phenol 8. The phenol
was converted to triflate 9 under mild condition. The C8 tail was
installed by Suzuki coupling of 9 with n-octyl-BBN to yield 10.
Selective bromination of 10 with CuBr2 in EtOAc/CHCl3 yielded
intermediate 11 smoothly.
The diol analog 3 was synthesized as follows (Scheme 2). Dis-
placement of bromide 11 with sodium acetylamino diethylmalo-
nate, followed by reduction gave diester 12. Chiral separation of
12 with ChiralPAK AZ gave (R)-13 and its enantiomer (S) -13.
LAH reduction followed by hydrolysis yielded pure (+)-(20R)-3
and (ꢀ)-(20S)-3. Crystallization of (20S)-3ꢁHBr in aqueous methanol
yielded a single-crystal, and its X-ray diffraction established the
structure unambiguously (Fig. 2).17
The methyl analog 5 was synthesized as illustrated in Scheme 3.
Starting from bromide 11, displacement of bromide with sodium
methyl diethylmalonate gave ketone 14. Reduction of the arylke-
tone in the presence of the diester was achieved under TiCl4/Et3-
SiH/DCM condition16 to yield 15 with moderate yield. Other
conditions, including hydrogenation (H2/Pd/C with AcOH/MeOH,
HCl/EtOH or H2SO4/EtOAc), Zn reduction (with HCl or AcOH), and
Et3SiH reduction (with BF3ꢁEt2O or TFA), failed to give acceptable
yield of 15, due to either lack of reactivity of the ketone or forma-
tion of lactone intermediates. Monohydrolysis of diester 15 under
KOH/EtOH yielded acid 16. A very mild Curtius reaction18 was ap-
plied to acid 16 to yield free aminoester 17 directly in one pot.
Although 5 could be obtained by direct reduction of aminoester
17, the 4 compounds mixture of 5 couldn’t be separated in our
hands, the acid 16 also proved to be problematic. To our delight,
a two stages chiral separation can separate all four isomers of 17.
Separation with chiral AD-H gave (2R,20R)-17, (2R,20S)-17 and a
mixture of the other two isomers which was further separated
with chiral AY to give (2S,20R)-17 and (2S,20S)-17. (Scheme 4).
LAH reduction of pure isomers of 17 yielded (+)-(2R,20R)-5, (ꢀ)-
(2R,20S)-5, (+)-(2S,20R)-5 and (ꢀ)-(2S,20S)-5, respectively.20
The phosphate of the diol analog 3 was synthesized as illus-
trated in Scheme 5. The amine (20R)-3 was protected with Cbz to
afford diol 18, which upon phosphorylation under Ag2O/[(BnO)2-
PO]2O gave the oxazoline 19. Chiral separation of 19 with Chir-
alPAK AZ gave 20 and its diastereoisomer. Hydrogenation
followed by hydrolysis yielded both isomers of (20R)-3-P.
The phosphate 6 was synthesized as exemplified in Scheme 6.
Boc protection of amine 5 followed by phosphorylation and hydro-
genation gave Boc-protected phosphoronic acid 24, and acid work-
up gave phosphate 6.
The diol 3 and methyl analog 5 show distinct activity when
measuring phosphorylation in vitro on SphK2 (mouse and human)
(Table 1). FTY720 is well phosphorylated into (S)-FTY720-P (2). In
O
O
O
a
b
H3CO
HO
TfO
8
9
7
c
O
O
Br
d
11
10
Scheme 1. Synthetic scheme for preparation of 11. Reagents and conditions: (a) 48% HBr, 120 °C, 7.5 h. 93%; (b) Tf2O, DMAP, 2,6-lutidine, CH2Cl2, ꢀ30 °C to 0 °C, 2 h. 96%; (c)
(i) 9-BBN, 1-octene, THF; (ii) 9, KBr, Pd(PPh3)4, K3PO4, H2O, THF, reflux, 3 h. 80%; (d) CuBr2, EtOAc, CHCl3, reflux, 2 h. 87%.

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B. Ma et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2264–2269
NHAc
CO2Et
O
NHAc
CO2Et
Br
a,b
c
CO2Et
CO2Et
n-C8H17
n-C8H17
13
d,e
12
11
NH2
CH2OH
NH2.HBr
CH2OH
CH2OH
CH2OH
n-C8H17
(R)-3
(S)-3*HBr
Scheme 2. Synthetic scheme for preparation of 3. Reagents and conditions: (a) AcNHCH(CO2Et)2, NaH, DMF, 0 °C to rt, overnight, 78%; (b) TiCl4, Et3SiH, CH2Cl2, 0 °C to rt,
overnight, 73%; (c) ChiralPAK AZ, hexane/i-PrOH, 92%, 99% ee; (d) LAH, THF, 0 °C to rt, 2 h, 59%; (e) LiOH, MeOH/THF/Water, reflux, 5 h, 59%.
Figure 2. X-Ray crystal structure of (20S)-3ꢁHBr salt.
O
CO2Et
O
CO2Et
Br
CO2Et
a
CO2Et
b
Me
Me
n-C8H17
n-C8H17
11
15
14
c
CO2H
NH2
H
d
CO2Et
Me
CO2Et
Me
n-C8H17
n-C8H17
17
16
Scheme 3. Synthetic scheme for preparation of 17. Reagents and conditions: (a) MeCH(CO2Et)2, NaH, DMF, 0 °C to rt, overnight. 83%; (b) TiCl4, Et3SiH, CH2Cl2, 0 °C to rt. 57%;
(c) KOH, EtOH, 60 °C, overnight, 70%; (d) (i) DPPA, Et3N, PhMe, reflux, 2 h; (ii) NaOTMS, THF, 0 °C to rt, overnight. 71%.
contrast, both diols (20R)-3 and (20S)-3 are poor SphK2 (human)
substrates with <10% phosphorylation (9% and 3%, respectively).
Phosphorylation of the methyl analogs, however, is much better
with (2R,20S)-5 showing approximately equal phosphorylation
(77% in human and 90% in mouse) to that seen with FTY720. It is
interesting to note that (2R,20R)-5, the exact methyl analog of bet-
ter phosphorylated diol (20R)-3, shows only moderate 38% phos-
phorylation by the human kinase. Both (2S,20R)-5 and (2S,20S)-5
are not substrates for SphK2, this implies that the R configuration
of the aminoalcohol head portion is critical for in vivo phosphory-
lation to convert prodrug (2R,20S)-5 into its phosphate (2R,20S)-6.
The phosphates were also studied in whole cell assay to deter-
mine their effect on S1P receptors. It’s known that S1P1 couples to
Gi solely and S1P3 couples to Gi and Gq, therefore, both Gi and Gq
dependent cellular assays were used to determine agonist/antago-
nist activity of 3-P and 6 at the human S1P1 or S1P3 receptors
(Tables 2 and 3). In the Gi assay, (20R)-3-P isomer 1 is similar to
(S)-FTY720-P, and is sixfold less potent at S1P1 (EC50 = 0.077 nM)
and twofold less potent at S1P3 (EC50 = 0.215 nM). Interestingly,
its diastereoisomer (20R)-3-P isomer 2 is quite active on S1P1
(EC50 = 0.344 nM), while (R)-FTY720-P is less active (EC50 = 23.25
nM). Both (R)-FTY720-P and (20R)-3-P isomer 2 are not active on
S1P3. It should be noted that only (S)-FTY720-P is made in vivo
from FTY720. For the methyl analogs at S1P1, compound (2R,20S)-
6 and (S)-FTY720-P have approximately equal EC50s (0.018 nM vs
0.012 nM, respectively). (2R,20R)-6 is also similar to (S)-FTY720-P
but less potent. Both (2S,20S)-6 and (2S, 20R)-6 were significantly
less active at S1P1 and showed no activity at S1P3. The correspond-
ing aminoalcohols of these two are not substrates for the kinase
SphK2. This observation is consistent with FTY720 phosphates. It
is interesting that those in vivo disfavored phosphates, (R)-
FTY720-P, (2S,20S)-6 and (2S,20R)-6 show great S1P1/S1P3 selectiv-
ity. Clearly, stereochemistry can influence S1P1 and S1P3 activity
significantly.

B. Ma et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2264–2269
2267
NH2
H
NH2
H
CO2Et
CH2OH
Me
c
Me
n-C8H17
n-C8H17
n-C8H17
n-C8H17
(2R,2'R)-17
(2R,2'R)-5
a
a
NH2
NH2
H
H
CO2Et
CH2OH
Me
Me
c
NH2
H
n-C8H17
CO2Et
Me
(2R,2'S)-17
(2R,2'S)-5
NH2
NH2
n-C8H17
H
H
rac-17
CO2Et
CH2OH
b
b
Me
a
Me
c
n-C8H17
(2S,2'R)-17
(2S,2'R)-5
NH2
NH2
H
H
CO2Et
Me
CH2OH
Me
c
n-C8H17
n-C8H17
(2S,2'S)-17
(2S,2'S)-5
Scheme 4. Synthetic scheme for preparation of 5. Reagents and conditions: (a) CHIRALPAK AD-H, ACN/MeOH, 93–98%, 97.6–98% ee; (b) CHIRALPAK AY, hexane/i-PrOH, 79–
88%, 96.4–99% ee; (c) LiAlH4, THF, reflux, 3 h, 41–85%.
O
O
HN
OBn
OH
NH2
HN
H
H
H
OH
O
a
b
OH
OH
OPO(OBn)2
n-C8H17
n-C8H17
n-C8H17
19
(2'R)-3
18
c
O
O
NH2
*
HN
*
HN
*
H
H
H
e
d
OH
O
O
OPO(OH)2
(2'R)-3-P isomer 1
OPO(OH)2
OPO(OBn)2
n-C8H17
n-C8H17
n-C8H17
21
20
Scheme 5. Synthetic scheme for preparation of (20R)-3-P. Reagents and conditions: (a) Cbz-Cl, KHCO3, EtOAc, H2O, rt, 1.5 h, 92%; (b) [(BnO)2PO]2O, Ag2O, hex4NI, CH2Cl2, rt,
3d, 44%; (c) ChiralPAK AZ, ACN/MeOH, 42%, >99.9% de; (d) H2, Pd-C, MeOH, rt, 2 h, 91%; (e) LiOH, EtOH/Water, reflux, overnight, 49%.
O
Boc
HN
O
OH
NH2
HN
HN
H
H
H
O
P
O
OH
O
a
b
O
n-C8H17
n-C8H17
n-C8H17
23
(2R,2'S)-5
22
c
O
NH2
O
O
H
H
d
O
PO(OH)2
PO(OH)2
n-C8H17
n-C8H17
24
(2R,2'S)-6
Scheme 6. Synthetic scheme for preparation of 6. Reagents and conditions: (a) Boc2O, CHCl3, aq NaHCO3, rt, overnight, 86%; (b) (i) N,N-diethyl-1,5-dihydro-
benzo[e][1,3,2]dioxaphosphepin-3-amine, tetrazole, THF, rt, overnight; (ii) HOOH, rt, 1 h, 74%; (c) Pd-C, H2, MeOH, rt, 1 h. 59%; (d) HOAc, HCl, rt, 6 h. 86%.
For the Gq dependent cellular assay, the trend was similar. (S)-
FTY720-P and the (2R) methyl compounds (2R,20S)-6, (2R,20R)-6
are very active, while their enantiomers (R)-FTY720-P, (2S,20S)-6
are not active. Similarly, the diol phosphate (20R)-3-P isomer 1
(EC50 = 1.95 nM), is as active as (S)-FTY720-P (EC50 = 2.02 nM),
and its diastereomer (20R)-3-P isomer 2 (EC50 = 133 nM) is also
active but less so. None of the compounds are active on S1P2
and all are active on S1P3, S1P4, and S1P5. Compared to

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B. Ma et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2264–2269
phorylation. Clearly, (2R,20S)-5 is very efficacious with very good
oral bioavailability and good phosphorylation.
Table 1
Lymphopenia and phosphorylation of 3 and 5a
In summary, a potent, well phosphorylated, orally bioavailable
tetralin analog of FTY720 was identified. Stereochemistry on the
tetralin ring can significantly influence the phosphorylation and
lymphopenia activity, as well as receptor activity. The S1P1 over
S1P3 selectivity of this tetralin series was further improved and
will be described at a later date.
Compounds Mouse
Mouse PK
Phosphorylation Phosphorylation
lymphopenia phosphorylation (mSphK2) %
ED50, mg/kg
(hSphK2) %
%
FTY720
0.03
0.2
3.8
0.1
0.8
>5
—
3
87
4
76
9
(20R)-3
(20S)-3
—
63
—
—
—
—
90
—
—
—
3
(2R,20S)-5
(2R,20R)-5
(2S,20R)-5
(2S,20S)-5
77
38
0
Acknowledgments
>5
0
a
These studies were supported in part by a Grant from the NIH
(R01 GM067958 to K.R.L. and T.L.M.). The authors thank Dr. Doug-
las M. Ho for performing single-crystal X-ray diffraction.
Assay performed as described in the Ref. 19.
Table 2
hS1P1 and S1P3 receptors activation on calcium mobilization Gi assaya
Supplementary data
EC50 (nM)
S1P1
0.027
S1P3
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.02.006.
S1P
0.449
0.134
>5000
0.215
>5000
0.286
0.150
>5000
>5000
(S)-FTY720-P
(R)-FTY720-P
(20R)-3-P isomer 1
(20R)-3-P isomer 2
(2R,20S)-6
0.012
23.25
0.077
0.344
0.018
0.088
19.72
13.65
References and notes
1. Compston, A.; Coles, A. Lancet 2008, 372, 1502.
2. Hla, T. Pharmacol. Res. 2003, 47, 401.
3. Fujita, T.; Inoue, K.; Yamamoto, S.; Ikumoto, T.; Sasaki, S.; Toyama, R.; Chiba, K.;
Hoshino, Y.; Okumoto, T. J. Antibiot. 1994, 47, 208.
(2R,20R)-6
(2S,20R)-6
(2S,20S)-6
4. Adachi, K.; Kohara, T.; Nakao, N.; Arita, M.; Chiba, K.; Mishina, T.; Sasaki, S.;
Fujita, T. Bioorg. Med. Chem. Lett. 1995, 5, 853.
a
Assay performed as described in the Ref. 19.
5. Kiuchi, M.; Adachi, K.; Kohara, T.; Minoguchi, M.; Hanano, T.; Aoki, Y.; Mishina,
T.; Arita, M.; Nakao, N.; Ohtsuki, M.; Hoshino, Y.; Teshima, K.; Chiba, K.; Sasaki,
S.; Fujita, T. J. Med. Chem. 2000, 43, 2946.
6. Fujino, M.; Funeshima, N.; Kitazawa, Y.; Kimura, H.; Amemiya, H.; Suzuki, S.; Li,
X. K. J. Pharmacol. Exp. Ther. 2003, 305, 70.
Table 3
hS1P1–S1P5 receptors activation on calcium mobilization Gq assaya
7. Kappos, L.; Antel, J.; Comi, G.; Montalban, X.; O’Connor, P.; Polman, C. H.; Haas,
T.; Korn, A. A.; Karlsson, G.; Radue, E. W. N. Eng. J. Med. 2006, 355, 1124.
8. O’Connor, P.; Comi, G.; Montalban, X.; Antel, J.; Radue, E. W.; de Vera, A.;
Pohlmann, H.; Kappos, L. Neurology 2009, 72, 73.
9. Novartis. FTY720 FREEDOMS study: initial results. 2009 [cited; Available from:
http://www.novartis.com/newsroom/news/2009-09-30_spotlight-fty720.shtml].
10. Albert, R.; Hinterding, K.; Brinkmann, V.; Guerini, D.; Hartwieg, C.-M.; Knecht,
H.; Simeon, C.; Streiff, M.; Wagner, T.; Welzenbach, K.; Zecri, F.; Zollinger, M.;
Cooke, N.; Francotte, E. J. Med. Chem. 2005, 48, 5373.
EC50 (nM)
S1P1
S1P2
S1P3
27.84
16.06
18.75
25.72
97.44
38.17
922.10
S1P4
22.16
136.70
5.39
41.45
15.98
—
S1P5
(S)-FTY720-P
(R)-FTY720-P
(20R)-3-P isomer 1
(20R)-3-P isomer 2
(2R,20S)-6
(2R,20R)-6
(2S,20S)-6
2.02
>5000
1.95
133.00
1.79
5.62
>5000
>5000
>5000
>5000
>5000
—
0.36
1988.00
0.33
32.96
1.49
—
—
>5000
—
—
11. Brinkmann, V.; Davis, M. D.; Heise, C. E.; Albert, R.; Cottens, S.; Hof, R.; Bruns,
C.; Prieschl, E.; Baumruker, T.; Hiestand, P.; Foster, C. A.; Zollinger, M.; Lynch, K.
R. J. Biol. Chem. 2002, 277, 21453.
a
Assay performed as described in the Ref. 19.
12. Mandala, S. M.; Hajdu, R.; Bergstrom, J.; Quackenbush, E.; Xie, J.; Milligan, J.;
Thornton, R.; Shei, G.-J.; Card, D.; Keohane, C.; Rosenbach, M.; Hale, J.; Lynch, C.
L.; Rupprecht, K.; Parsons, W.; Rosen, H. Science 2002, 296, 346.
13. Marsolais, D.; Rosen, H. Nat. Rev. Drug Disc. 2009, 8, 297.
14. Forrest, M.; Sun, S. Y.; Hajdu, R.; Bergstrom, J.; Card, D.; Doherty, G.; Hale, J.;
Keohane, C.; Meyers, C.; Milligan, J.; Mills, S.; Nomura, N.; Rosen, H.;
Rosenbach, M.; Shei, G.-J.; Singer, I. I.; Tian, M.; West, S.; White, V.; Xie, J.;
Proia, R. L.; Mandala, S. J. Pharmacol. Exp. Ther. 2004, 309, 758.
15. Gon, Y.; Wood, M. R.; Kiosses, W. B.; Jo, E.; Sanna, M. G.; Chun, J.; Rosen, H. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 9270.
16. Lynch, K. R.; Macdonald, T. L. W.O. Patent 2007092638, 2007; Chem. Abstr.
2007, 147, 235303.
17. Crystallographic details of (2’S)-3 have been deposited at the Cambridge
Crystallographic Data Center and allocated the deposition number CCDC
759847.
FTY720, moderate selectivity of S1P1 over S1P3 was observed for
(2R,20S)-6.
These compounds were investigated further in vivo by measur-
ing their ability to induce mouse lymphopenia (Table 1). Both
(20R)-3 and (20S)-3 induce lymphopenia, the superior activity of
(20R)-3 (ED50 = 0.2 mg/kg) over (20S)-3 (ED50 = 3.8 mg/kg) implies
(20R) configuration is favored in the diol context. Interestingly,
(2S,20R)-5 and (2S,20S)-5 are not active, while (2R, 20S)-5 is very ac-
tive (ED50 = 0.1 mg/kg), and (2R,20R)-5 is less active (ED50 = 0.8 mg/
kg). The favored (20S) configuration in the methyl series is opposite
to the diols, the reason for this is not well understood. Clearly, R
configuration in the aminoalcohol head portion is necessary for
the lymphopenia activity, which is consistent with the observation
of FTY720 analogs AAL-R and AAL-S.11
18. Ma, B.; Lee, W.-C. Tetrahedron Lett. 2010, 51, 385.
19. Lynch, K. R.; Macdonald, T. L.; Guckian, K.; Lin, E. Y. -S.; Ma, B. W.O. Patent
2009023854, 2009; Chem. Abstr. 2009, 150, 259836.
20. Absolute configuration of (+)-(2R,20R)-5 was established by a new synthesis
listed in the following scheme from an intermediate (+)-(R)-4-((R)-6-hydroxy-
1,2,3,4-tetrahydronaphthal-en-2-yl)-4-methyloxazolidin-2-one (25), whose
Both (20R)-3 and (2R,20S)-5 was measured in mouse PK/PD stud-
ies at 10 ꢂ ED50 for both compounds. 2 mg/kg dose of (20R)-3
evokes 72 h sustained lymphopenia, with oral bioavailability of
structure was confirmed by X-ray crystal structure of
a brominated
derivative (unpublished results). The other isomers were assigned by careful
comparison of spectroscopic data.
O
O
55% and half life t = 16 h. Only 3% conversion of (20R)-3 to its phos-
HN
HN
½
b
O
a
O
(+)-(2R,2'R)-5
phate was observed in this study which is consistent with the low
in vitro phosphorylation (4% in mouse in vitro assay, Table 1). The
low conversion to phosphate for compound (20R)-3 and low ED50
(0.2 mg/kg) is unexpected, and we do not understand this discon-
nection. In contrast, 1 mg/kg dose of (2R,20S)-5 evokes one-week
sustained lymphopenia, with oral bioavailability F = 55%,
HO
n-C6H13
26
25
Conditions: (a) Tf2O, py, CH2Cl2, rt; Pd(dppf)Cl2, tBuNH2, i-PrOH, H2O, n-
C6H13CH@CHBF3K, 100 °C; (b). Pd/C, H2, EtOH, rt; LiOH, EtOH, H2O, reflux.
26: 1H NMR (400 MHz,CDCl3) d 7.12 (d, J = 7.8 Hz, 1H), 7.06 (s, 1H), 7.01 (d,
J = 7.9 Hz, 1H), 6.32 (d, J = 15.8 Hz, 1H), 6.18 (dd, J = 6.7, 15.8 Hz, 1H), 4.34
Vmax = 16 L/Kg, Ci = 5.5 ml/min/kg, half life t = 36 h, and 63% phos-
½

B. Ma et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2264–2269
2269
(d, J = 8.6 Hz, 1H), 4.09 (d, J = 8.6 Hz, 1H), 2.96–2.71 (m, 3H), 2.62–2.51 (m,
1H), 2.19 (q, J = 6.8 Hz, 2H), 2.00–1.84 (m, 2H), 1.52–1.43 (m, 2H), 1.41 (s,
3H), 1.31 (br s., 7 H), 0.93–0.85 (m, 3H). 13H NMR (100 MHz, CDCl3) d 158.8,
135.89, 135.87, 133.40, 130.7, 129.37, 129.28, 126.2, 123.6, 74.4, 60.0, 43.5,
33.0, 31.7, 30.0, 29.5, 29.4, 28.9, 24.1, 23.4, 22.6, 14.1. LC–MS: m/z = 342.20
([M+1], 100%).