Design and synthesis of conformationally constrained 3-(N-alkylamino) propylphosphonic acids as potent agonists of sphingosine-1-phosphate (S1P) receptors

Article abstract of DOI:10.1016/j.bmcl.2004.07.049

A series of conformationally constrained analogs of 3 were synthesized and evaluated as S1P receptor agonists. Several novel scaffolds were identified as suitable for further investigation. A series of conformationally constrained 3-(N-alkylamino)propylphosphonic acids were systematically synthesized and their activities as S1P receptor agonists were evaluated. Several pyrrolidine and cyclohexane analogs had S1P receptor profiles comparable to the acyclic lead compound, 3-(N-tetradecylamino)propylphosphonic acid (3), lowered circulating lymphocytes in mice after iv administration and were thus identified as being suitable for further investigations.

Full text of DOI:10.1016/j.bmcl.2004.07.049

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4861–4866
Design and synthesis of conformationally constrained
3-(N-alkylamino)propylphosphonic acids as potent agonists
of sphingosine-1-phosphate (S1P) receptors
a
a,ꢀ
Lin Yan,a, Jeffrey J. Hale, Christopher L. Lynch, Richard Budhu,a Amy Gentry,a
Sander G. Mills,a Richard Hajdu,b Carol Ann Keohane,b Mark J. Rosenbach,b
James A. Milligan,b Gan-Ju Shei,b Gary Chrebet,b James Bergstrom,b Deborah Card,b
Hugh Rosenb,ꢁ and Suzanne M. Mandalab
*
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Immunology and Rheumatology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 3 June 2004; accepted 21 July 2004
Available online 20 August 2004
Abstract—A series of conformationally constrained 3-(N-alkylamino)propylphosphonic acids were systematically synthesized and
their activities as S1P receptor agonists were evaluated. Several pyrrolidine and cyclohexane analogs had S1P receptor profiles com-
parable to the acyclic lead compound, 3-(N-tetradecylamino)propylphosphonic acid (3), lowered circulating lymphocytes in mice
after iv administration and were thus identified as being suitable for further investigations.
Ó 2004 Elsevier Ltd. All rights reserved.
FTY720 (1, Fig. 1) is a novel immunosuppressant that
prolongs the survival of organ allografts in animals; it
has progressed through the clinic and is currently in
Phase III trials for the prevention of kidney transplant
rejection.1 The mechanism of FTY720-mediated
immunosuppression, while not being fully understood,
appears to be different from those of existing immuno-
suppressants. Recent studies have shown that FTY720
is phosphorylated in vivo by sphingosine kinase and that
FTY720-phosphate (2) is a potent agonist of a family of
Previous studies from these laboratories5 have shown
that 3-(N-tetradecylamino)propylphosphonic acid (3) is
R
O
NH2
OH
1, R = -H, FTY720
2, R = -PO3H2, FTY720 phosphate
sphingosine-1-phosphate (S1P)
G
protein-coupled
a
b
d
receptors.2,3 The activation of S1P1 receptor by 2 is
presumably responsible for the observed immunosup-
pressive efficacy due to the subsequent sequestration of
lymphocytes into secondary lymphoid organs, which
reduces and/or inhibits infiltration of lymphocytes into
graft sites and inflamed tissues.4
OH
OH
N
R
P
O
c
3, R = -H, S1P1 IC50 = 3.0 nM
4, R = -CH3, S1P1 IC50 = 16 nM
OH
OH
N
H
P
O
*
Corresponding author. Tel.: +1-732-594-5419; fax: +1-732-594-
2210; e-mail: lin_yan@merck.com
5, S1P1 IC50 = 0.2 nM
ꢀ Present address: Abbott Laboratories, Dept. R4N6, Bldg. AP-10,
Abbott Park, IL 6006-6101, USA.
ꢁ Present address: The Scripps Research Institute, ICND-118, 10550 N.
Torry Pines Road, La Jolla, CA 92037, USA.
Figure 1. FTY720 (1), FTY720-phosphate (2), and analogs 3–5 are all
potent agonists of S1P receptors. The letters shown by structure 3
mark the positions that were identified for tethering using short alkyl
chains.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.049

4862
L. Yan et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4861–4866
a potent agonist of S1P receptors and that the proper
incorporation of a phenyl ring into the long alkyl chain
of this compound gives 5, which has 15-fold greater
affinity for S1P1 than does 3. N-Methylation of 3, giving
4, results in a 5-fold loss of affinity for S1P1. To extend
the scope of this work, efforts were undertaken to sys-
tematically synthesize analogs of 3 in which the nitrogen
and a neighboring carbon atom or a pair of adjacent
carbons of this compound were tethered with short alkyl
chains, with the goal of identifying novel scaffolds for
S1P receptor agonists. This paper describes the synthesis
and evaluation of these conformationally constrained
amino phosphonic acids and amino a-hydroxyl phos-
phonic acids.6 Scaffolds identified here as being suitable
for S1P receptor agonists were elaborated into analogs
of 5 to determine whether S1P1 receptor affinity was en-
hanced similarly to that observed on going from 3 to 5.
with n-dodecylphosphorane and then hydrogenated to
supply 11. Piperidine 12 was then obtained using proce-
dures analogous to those used to convert 7 to 8. To link
the nitrogen and Cb, Swern oxidation of N-Boc-pyrroli-
dinemethanol (13) followed by Horner–Emmons olefina-
tion gave 14. This was hydrogenated and then treated
with anhydrous HCl in EtOH to furnish 15. Reductive
amination of 15 with n-tetradecylaldehyde followed by
ester cleavage delivered pyrrolidine 16. Connecting the
nitrogen atom and Cc with an ethylene chain, featured
the use of 1,3-dipole cyclization chemistry.9 Reacting
17 and methyl acrylate in the presence of catalytic TFA
followed by hydrogenation gave 18. Reductive amina-
tion and DIBAL-H reduction of the ester provided alde-
hyde 19, which was coupled with diethyl phosphite under
Pudovik conditions10 followed by ester cleavage to give
20.11 Tethering the nitrogen and Cd with ethyl and pro-
pyl chains was accomplished starting from 21a and
21b, respectively, and also featured the Pudovik reaction.
The syntheses of analogs of 3 in which the nitrogen and
an adjacent carbon are linked are described in Scheme 1.
To tether the nitrogen to Ca with a propyl chain, treat-
ment of 6 with s-BuLi in the presence of TMEDA7 fol-
lowed by tetradecyl bromide gave pyrrolidine 7.
Removal of Boc followed by N-alkylation of the result-
ing pyrrolidine with diethyl 3-bromopropylphosphonate
and ester cleavage led to pyrrolidine 8. For the cor-
responding piperidine analog 12, reduction of N-Boc-
piperidine-2-carboxylic acid (9) followed by TPAP
oxidation8 provided aldehyde 10. This was condensed
The syntheses of analogs in which a pair of adjacent car-
bons are tethered are shown in Scheme 2. To connect Ca
and Cb with a propyl chain, formylation of 11 using
s-BuLi in the presence of TMEDA12 gave 24 as a 2:3
mixture of cis and trans isomers. The mixture could be
enriched in the cis isomer (6:1 cis/trans) by treating it
with silica gel. Condensation of 24 with tetraethyl
methylenediphosphonate provided a mixture of phos-
phonate esters, which were readily separated by silica
A. N-Ca:
a
b, c, d
PO3H2
N
n-C14H29
N
N
Boc
Boc
n-C14H29
8
6
7
e, f
i, j
g, h
b, c, d
N
PO3H2
N
Boc
9
CO2H
OH
N
Boc
10
CHO
N
Boc
n-C13H27
n-C13H27
12
11
h, k
l, d
B. N-Cb:
PO(OEt)2
PO(OEt)2
PO3H2
N
Boc
N
Boc
N
H
N
n-C14H27
16
HO
13
14
15
CO2CH3
CHO
PO3H2
C. N-Cc:
D. N-Cd:
m, n
l, o
p, d
TMS
N
Bn
OCH3
N
H
N
N
n-C14H29
n-C14H29
17
18
19
20
HO
HO
O
P(O)(OEt)2
n
PO3H2
p
k, l, d
n
n
N
N
N
Boc
Boc
n-C14H29
n = 1 21a
n = 2 21b
22
23
Scheme 1. Reagents and conditions: (a) s-BuLi, TMEDA, THF, ꢀ78°C, then n-C14H29Br, ꢀ78°C to rt (11%); (b) 9:1 CH2Cl2/TFA, rt; (c) diethyl
3-bromopropylphosphonate, K2CO3, DMF, 100°C (32–100%, two steps); (d) TMSI, CH2Cl2, rt (55–83%); (e) BH3ÆTHF, THF, rt (100%); (f) NMO,
˚
cat. TPAP, 4A molecular sieves, CH2Cl2, rt (68%); (g) n-C12H25P(Ph)3Br, n-BuLi, THF, ꢀ78°C to rt (100%); (h) H2, 10% Pd/C, EtOH, rt (97%);
(i) (COCl)2, DMSO, DIEA, CH2Cl2, ꢀ78 to 0°C; (j) tetraethyl methylenediphosphonate, NaHMDS, THF, (95%, two steps); (k) HCl, EtOH (100%);
(l) n-C13H27CHO, DIEA, NaB(OAc)3H, CH2Cl2 (92%); (m) methyl acrylate, cat. TFA, CH2Cl2, 0°C to rt (100%); (n) Pd/C, NHþCHOꢀ, CH3OH,
4
2
40°C (64%); (o) DIBAL, CH2Cl2, ꢀ78 to ꢀ65°C (50%); (p) diethyl phosphite, Et3N, 100°C (24–71%). All final analogs are racemic or equal mixtures
of isomers.

L. Yan et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4861–4866
4863
A. Ca-Cb:
a
e
b
c, d
N
Boc
n-C13H27
n-C13H27
N
Boc
24
CHO
n-C13H27
N
P(O)(OEt2)2
n-C13H27
N
H
PO3H2
PO3H2
Boc
25-cis
26-cis
11
25-trans
26-trans
f
b, c, d
OH
HO
n-C14H29
OHC
n-C14H29
N
N
n-C14H29
N
N
H
Boc
Boc
27
Boc
28
29
13
O
N
CBz
31
HO
P(O)(OEt2)2
HO
PO3H2
OCH3
B. Ca-Cd:
g
h, i
c, j
n-C14H29
N
n-C14H29
N
H
n-C14H29
N
CBz
32
30
33
n-C14H29
HN
OH
O
C. Cb-Cc:
O
k
l
m, d
PO3H2
P(O)(OEt2)2
P(O)(OEt2)2
35
36
37
34
n-C14H29
D. Cb-Cd:
O
O
n
HN
n
m, d
40a (1:1 mix of cis and trans)
40b-cis
40b-trans
n
n
P(O)(OEt2)2
PO3H2
39
n = 1 38a
n = 2 38b
Scheme 2. Reagents and conditions: (a) (1) s-BuLi, TMEDA, THF, ꢀ78 to ꢀ30°C, (2) DMF, ꢀ78°C (83%); (b) tetraethyl methylenediphosphonate,
NaN(TMS)2, THF, 0°C to rt (67–85%); (c) H2, Pd/C, EtOH, rt (66–100%); (d) TMSBr, CH3CN, 70°C (44–100%); (e) (1) s-BuLi, TMEDA, THF,
˚
ꢀ78°C, then n-C14H29Br, ꢀ78°C to rt (4–9%); (f) NMO, cat. TPAP, 4A molecular sieves, CH2Cl2, rt (60–77%); (g) (1) CBZ-Cl, THF, ꢀ23°C, (2)
n-C14H29MgCl, THF, ꢀ23 to ꢀ16°C, (3) 10% HCl, rt (100%); (h) L-Selectride, THF, ꢀ20°C to rt (56%); (i) diethyl phosphite, Et3N, 100°C (15%);
(j) TMSI, CH2Cl2, rt; (k) n-BuLi, diethyl methylphosphonate, BF3ÆEt2O, THF, ꢀ78°C (100%); (l) (COCl)2, DMSO, Et3N, CH2Cl2, ꢀ78 to 0°C
(87%); (m) n-C14H29NH2, NaB(OAc)3H, CH2Cl2, rt (38%); (n) (1) diethyl phosphite, N,N-bis(trimethylsilyl)acetamide, TMSOTf, CH2Cl2, 0°C to rt,
(2) 1.0N HCl, rt (70–91%). All final analogs are racemic or equal mixtures of isomers.
gel chromatography. The individual diastereomers, 25-
cis and 25-trans, were hydrogenated then treated with
iodotrimethylsilane to deliver the final piperidines, 26-
cis and 26-trans. For the corresponding 2,5-trans pyrrol-
idine analog 29, treatment of 13 with s-BuLi in the
presence of TMEDA followed by n-tetradecyl bromide
led to 27.13 Alcohol 27 was oxidized to aldehyde 28,
which was readily converted to pyrrolidine 29.14 To
tether Ca and Cd, acylation of 4-methoxypyridine (30) with
benzyl chloroformate followed by alkylation with n-tet-
radecylmagnesium chloride15 gave 1,2,3,4-tetrahydropy-
ridine 31. Conjugate reduction of 31 with L-Selectride
followed by a Pudovik reaction afforded a-hydroxyl
phosphonate 32, which was readily converted to piperi-
dine 33. Epoxy ring opening of cyclohexene oxide (34)
by diethyl methylphosphonate16 was the first step in a
route used to link Cb and Cc. Swern oxidation of alcohol
35 followed by reductive amination and ester cleavage
resulted in cyclohexane 37. To tether Cb and Cd, Mi-
chael addition of diethyl phosphate to either cyclopente-
none or cyclohexenone17 was featured in the syntheses
of cyclopentane 40a (obtained as a mixture of cis and
trans isomers) and cyclohexanes 40b-cis and 40b-trans.
agonists, so analogs of these with a phenyl ring incorpo-
rated into their long alkyl chains5 were prepared
(Scheme 3). For the cis and trans analogs of 29,
Friedel–Crafts acylation of 1-phenylnonane (38) with
succinic anhydride followed by esterification of the
resulting carboxylic acid gave a c-keto ester 43. The ke-
tone carbonyl group was converted into an oxime,
which was subsequently reduced and cyclized to furnish
lactam 44. Treatment of 44 with Meerweinꢂs salt led to
an imino ether, which was condensed with Meldrumꢂs
acid to afford isopropylidene malonate adduct 45.18
Methanolysis of 45 provided an enamine ester, which
was subsequently reduced by sodium cyanoborohydride
to give a separable 2:1 cis/trans mixture of b-amino es-
ters 46. The relative stereochemistry of substituents on
the pyrrolidine rings were determined with NOE NMR
experiments. Reduction of 46-cis with LAH followed by
N-Boc protection and TPAP oxidation delivered an
aldehyde, which was condensed with diethyl phosphite
to provide a-hydroxyl phosphonate 47. The hydroxyl
group was reduced using the Barton–McCombie proto-
col;19 this was followed by global deprotection to
furnish 48-cis. 48-Trans was obtained analogously from
46-trans. To stereospecifically prepare the analog of
cyclohexane 37, ketone 21b was reduced to 1,2-cis
alcohol with L-Selectride. The hydroxyl group was con-
verted to inverted amine 50 with in three steps; this was
Three different scaffolds, exemplified by pyrrolidine 23,
pyrrolidine 29 and cyclohexane 40b-trans, were among
those found to be tolerated as scaffolds for S1P receptor

4864
L. Yan et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4861–4866
O
A.
OCH3
a, b
c, d, e
+
O
O
N
H
O
O
O
n-C9H19
n-C9H19
n-C9H19
41
42
43
44
f, g
OH
O
h, i
j, k, l
CO2CH3
O
P(O)(OEt)2
N
N
H
N
H
n-C9H19
n-C9H19
n-C9H19
BOC
O
O
47
45
46
m, n, o
n.o.e.
n.o.e.
X
H
Ar
H
H
Ar
H
CO2CH3
CO2CH3
N
H
N
H
PO3H2
N
H
n-C9H19
46-cis
46-trans
48-cis
48-trans
B.
O
OH
NH2
HN
p
q, r, s
t, o
n-C9H19
P(O)(OEt)2
OCH3
P(O)(OEt)2
P(O)(OEt)2
PO3H2
21b
49
50
51
C.
n-C9H19
NH
N
u, d
t, o
TMS
N
Bn
P(O)(OEt)2
PO3H2
17
52
53
Scheme 3. Reagents and conditions: (a) AlCl3, CH2Cl2, 0°C to rt; (b) CH3OH, toluene, c.H2SO4, 80°C (77%, two steps); (c) H2NOHÆHCl, NaOAc,
EtOH, reflux; (d) H2 (50psi), Pd/C, AcOH; (e) pyridine, 80°C (79–83%, three steps); (f) (CH3)3OÆBF4, CH2Cl2, rt; (g) Meldrumꢂs acid, Et3N, benzene,
reflux (35%, two steps); (h) NaOCH3, CH3OH, reflux (58–61%); (i) Na(CN)BH3, pH3–4, CH3OH, rt (96–99%); (j) (1) LAH, THF, rt, then (BOC)2O,
rt (65–86%); (k) cat. TPAP, NMO, CH2Cl2, 0°C to rt (60–74%); (l) diethyl phosphite, NaHMDS, 0°C to rt (63–100%); (m) NaH, CS2, imidazole,
CH3I, THF, 0°C (39–41%); (n) Bu3 SnH, cat. AIBN, toluene, reflux (80–100%); (o) TMSBr, CH3CN, 65°C (66%); (p) L-Selectride, THF, ꢀ78°C
(54%); (q) MsCl, DIEA, CH2Cl2, 0°C (65%); (r) NaN3, DMF, 60°C (55%); (s) H2 (40psi), 10% Pd/C, EtOH (100%); (t) 4-nonylbenzaldehyde,
NaB(OAc)3H, CH2Cl2, rt (32%); (u) diethyl vinylphosphonate, cat. TFA, CH2Cl2, rt (100%). All final analogs are racemic or equal mixtures of
isomers.
followed by reductive amination and ester cleavage to
provide 51. For the des-hydroxy analog of pyrrolidine
23, 1,3-dipole cyclization of diethyl vinylphosphonate
and 17 was featured in the sequence that gave pyrrol-
idine 53.
the tethering position, while most compounds had lower
affinities for S1P2 and S1P4. Three n-alkyl analogs that
maintained the secondary amine group of 3, piperidine
26-trans, pyrrolidine 29 and cyclohexane 40b-trans, were
found to have single digit nanomolar S1P1 affinities
comparable to that of 3. Comparing the receptor data
for the diastereomers of piperidine 26 and cyclohexane
40 indicates that the trans stereochemistry is preferred
for both of these scaffolds. Of the other scaffolds, pyrr-
olidines 20 and 23a also appeared promising since their
S1P1 affinities were both found to be within a factor of
three to that of tertiary amine 4. Unfortunately, none
of the new scaffolds appeared to offer advantages over
3 or 4 regarding S1P receptor subtype selectivity.
The S1P receptor binding affinities (IC50s) of test com-
pounds were determined in competitive binding assays
with human S1P receptors transfected in Chinese ham-
ster ovary (CHO) cell membranes using [33P]-labeled
S1P as the ligand.2 Agonism of S1P receptors was
determined by measuring the uptake of [35S]-GTPcS by
transfected CHO cell membranes expressing S1P recep-
tors. All compounds were found to be agonists of S1P
receptors while calculated IC50 and EC50 values were
generally found to agree within a factor of 10 (only
40b has a factor of 26). The binding affinities of lead
compounds 3 and 4 and those of the conformationally
constrained analogs can be compared in Table 1. The
binding affinities of all of the new analogs for S1P1,
S1P3, and S1P5 receptors were significantly affected by
From the new scaffolds, three were chosen for further
investigation and analogs with a phenyl ring in their
long alkyl chains were prepared. Pyrrolidine analogs
48-cis and 48-trans and cyclohexane analog 51 were all
found to have greatly enhanced affinity for S1P1 as com-
pared to their n-alkyl counterparts (Table 2). A similar

L. Yan et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4861–4866
4865
Table 1. Inhibition (IC50, nM) of [33P]-S1P binding to S1P receptorsa
by conformationally constrained analogs of 3
ists.20a This precluded the determination of ED50 values
for these compounds. Pyrrolidine 53 was determined to
have an ED50 value of 2.2mg/kg iv in this assay. No
attempts were made to administer any of these
compounds orally after it was determined that the max-
imum plasma concentrations in the rat after a 2mg/kg
po dose of 48-cis were found to be less than 10ng/mL
at all time points.
Compound
S1P1
S1P2
S1P3
S1P4
S1P5
2
0.28
3.7
16
1100
580
6.3
3.6
9.8
1200
410
190
17
15
140
0.77
13
3
4
2200
––
24
8
12
1100
200
89
>1000
>1000
>1000
>1000
>1000
>1000
250
>10000
>10000
870
680
230
660
40
16
20
50
49
150
920
In conclusion, conformationally constrained 3-(N-alkyl-
amino)propylphosphonic acids were systematically syn-
thesized and evaluated as S1P receptor agonists, which
led to the identification of several suitable scaffolds for
further investigations. The proper incorporation of a
phenyl ring into the long alkyl chains of these com-
pounds significantly enhanced their binding affinities
and some of these new compounds were shown to in-
duce a lowering of peripheral lymphocytes in the mouse.
While none of the constrained compounds prepared
here had improved selectivity for S1P1 over S1P3 (or
other S1P receptor subtypes) as compared to acyclic
compounds such as 3 or 4, they do provide a set of novel
structure classes of S1P1 receptor agonists. Further elab-
oration of these leads with the objective of identifying
potent, selective, and orally bioavailable S1P receptor
agonists is currently underway and will be reported in
the future.
23a
23b
26-cis
26-trans
29
19
51
34
220
16
>10000
310
180
270
15
8.2
7.0
5.2
220
370
30
4.9
6.9
260
360
82
760
330
61
17
120
33
37
>1000
>1000
>1000
>1000
320
>10000
6300
760
>1000
>1000
130
110
8.2
40a
40b-cis
40b-trans
23
3.1
27
3.1
2000
260
a Displacement of [33P]-labeled sphingosine-1-phosphate (S1P) by test
compounds from human S1P receptors expressed on CHO cell
membranes. Data are reported as mean for n = 3 determinations. SD
were generally 20% of the average. See Ref. 2 for assay protocol.
Table 2. Inhibition (IC50, nM) of [33P]-S1P binding to S1P receptorsa
and mouse peripheral lymphocyte loweringb (PLL) for selected analogs
Compound S1P1 S1P2
S1P3 S1P4 S1P5 PPL ED50
(mg/kg iv)
References and notes
5
0.16
0.10
750
422
2.7
8.4 0.73 87% @ 0.25c
48-cis
48-trans
51
2.8 31
4.7 32
0.58 89% @ 0.25c
82% @ 0.25c
45% @ 0.1c
2.2
1. (a) Budde, K.; Schmouder, R. L.; Brunkhorst, R.;
Nashan, B.; Lucker, P. W.; Mayer, T.; Choudhury, S.;
Skerjanec, A.; Kraus, G.; Neumayer, H. H. J. Am. Soc.
Nephrol. 2002, 13, 1073–1083; (b) Budde, K.; Schmouder,
R. L.; Nashan, B.; Brunkhorst, R.; Lucker, P. W.; Mayer,
T.; Brookman, L.; Nedelman, J.; Skerjanec, A.; Bohler, T.;
Neumayer, H.-H. Am. J. Transplant. 2003, 3, 846–854; (c)
Kahan, B. D.; Karlix, J. L.; Ferguson, R. M.; Leichtman,
A. B.; Mulgaonkar, S.; Gonwa, T. A.; Skerjanec, A.;
Schmouder, R. L.; Chodoff, L. Transplantation 2003, 7,
1079–1084; (d) Ebeling, T.; Fishman, M. Novartisꢂ 2003
ꢃR&D Dayꢂ, 19 November 2003.
0.16 >1000
0.33 >1000
6.9
1.2
2.2
6.3
18
>1000 220
86
45
53
a Displacement of [33P]-labeled sphingosine-1-phosphate (S1P) by test
compounds from human S1P receptors expressed on CHO cell
membranes. Data are reported as mean for n = 3 determinations. SD
were generally 20% of the average. See Ref. 2 for assay protocol.
b See Ref. 6 for assay protocol.
c Percentage decrease of peripheral blood lymphocyte counts in test
animals at the dose indicated (n = 3) versus control.
2. Mandala, S.; 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,
346–349.
3. (a) 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.
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